Regulation of P-Glycoprotein by Nuclear Receptors at the Blood-Brain Barrier: Relevance to Human Immunodeficiency Virus - 1 Pharmacotherapy

Home , Androstenol, Pregnane X receptor

Regulation of P-glycoprotein by Nuclear Receptors at the Blood-Brain Barrier: Relevance to Human Immunodeficiency Virus - 1 Pharmacotherapy

Gary Ngai Yin Chan

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Graduate Department of Pharmaceutical Sciences

Leslie Dan Faculty of Pharmacy University of Toronto

Regulation of P-glycoprotein by Nuclear Receptors at the Blood-Brain Barrier: Relevance to Human Immunodeficiency Virus – 1 Pharmacotherapy

Gary Ngai Yin Chan

Doctor of Philosophy

Graduate Department of Pharmaceutical Sciences Leslie Dan Faculty of Pharmacy, University of Toronto

2013

Abstract ATP-binding cassette membrane-associated drug efflux transporters expressed at the blood- brain barrier (BBB) are important determinants of drug disposition in the central nervous system.

Targeting the regulatory pathways that govern the expression of drug transporters, such as P- glycoprotein (P-gp) could provide novel approaches to selectively alter drug permeability in the brain.

Pregnane X Receptor (PXR) and Constitutive Androstane Receptor (CAR) are ligand-activated nuclear receptors which regulate the gene expression of several drug transporters, including P-gp.

Currently, knowledge on their significance at the BBB is limited. The overall goal of this Ph.D. thesis

was to examine the role of these two nuclear receptors in the regulation of P-gp functional expression

in brain microvessel endothelial cells, an important compartment constituting the BBB, and in a mouse

model. We demonstrated hPXR and hCAR protein expression in two human brain microvessel endothelial cell culture models (i.e., hCMEC/D3 and primary cultures of human brain-derived microvascular endothelial cells). We further showed the in vitro role of hPXR and hCAR in regulating

P-gp functional expression in the hCMEC/D3 cells. In addition, results obtained from Luciferase reporter gene assays demonstrated that several antiretroviral drugs currently used in the clinic are ligands for hPXR (i.e., amprenavir, atazanavir, darunavir, efavirenz, ritonavir and lopinavir) and

hCAR (i.e., abacavir, efavirenz and nevirapine). These drugs significantly induced P-gp functional

expression in hCMEC/D3 cells at clinical plasma concentrations. Applying quantitative intracerebral

microdialysis in CD-1 mice, we observed that P-gp induction mediated by a mouse PXR ligand/P-gp

inducer (i.e., dexamethasone) at the BBB significantly decreased the ratios of quinidine (an established

P-gp substrate) concentrations in brain extracellular fluid to unbound plasma concentrations at steady

state (Kp,uu,ECF/Plasma) when compared to vehicle-treated animals. Taken together, our data provide

evidence that hPXR and hCAR could be potential xenobiotic targets for the regulation of P-gp at the

BBB. This work will guide future development of novel pharmacotherapy that could target these receptors to alter drug transporters functional expression at the BBB, resulting in either enhanced CNS drug efficacy or reduced drug-associated neurotoxicity.

iii

Acknowledgements

I gratefully thank my thesis supervisor, Dr. Reina Bendayan, for providing me with the opportunity to undertake this interesting research project that I thoroughly enjoyed working on. Thank you especially for your continuous support, encouragement, patient, mentorship and guidance over the course of my thesis. I am also very thankful for the numerous opportunities to showcase my work in conferences and to collaborate in research projects with scientists from academia, government and industry. These experiences have greatly contributed to my intellectual and professional development as a scientist.

I would also like to thank Drs. Carolyn L. Cummins, David R. Hampson, Shinya Ito and Inés de Lannoy for your support, constructive criticism and overall guidance that aided in the completion of this Ph.D. project. My special gratitude goes to Dr. Inés de Lannoy, who provided the opportunity for me to learn intracerebral microdialysis and to collaborate in research related to my thesis. Your expertise and guidance were invaluable to my thesis and career development. The collaborative work at NoAb BioDiscoveries Inc. also would not be possible without the assistance from Victor Saldivia, Dr. Henrianna Pang, Mr. Yingbo Yang and Mrs. Tennile Tavares. I would like to offer my special thanks to Dr. Carolyn L. Cummins, who provided the laboratory, equipment and tremendous assistance during our collaboration applying qPCR and Luciferase reporter gene assays. Your generosity in sharing your time, knowledge and expertise has greatly helped in the completion of this Ph.D. project. My sincere appreciation also goes to Rucha Patel for her expertise and work throughout our collaboration.

I thank the NSERC, the University of Toronto and the Graduate Department of Pharmaceutical Sciences for the approval of several studentship awards. I also would like to acknowledge Dr. David S. Miller for providing me with an opportunity of learning the technique of brain capillary isolation in his laboratory at the National Institute of Environmental Health Sciences (NIEHS). I also thank Dr. Christopher R. Campos for his patient in teaching me the technique.

My sincere appreciation goes to all members of the Bendayan lab, past and present: Dr. Jason Zastre, Dr. Patrick Ronaldson, Karlo Babakhanian, Manisha Ramaswamy, Tianna Huang, Dr. Niladri Chattopadhyay, Christopher Tran, Janica Chan, Amanda Otting, Zhi Feng, Kevin Robillard, Olena Kis, Tamima Ashraf, Anu Shah, Dr. Md Tozammel Hoque, Monika Zhang, Dr. Maria De Rosa, Nilasha Banerjee, Dr. Wen Lei Jiang, Dr. Chiping Wu, Arpit Shah, Donald Wang, Amy Kao, Billy Huang. I would like to offer a special thank you to Dr. Jason Zastre for his guidance during the initial phase of my project and Dr. Md Tozammel Hoque for his assistance and guidance throughout my project. I sincerely wish everyone the best in their future endeavours.

Lastly, but certainly not least, I would like to express my deepest gratitude and appreciation for the care and love from my parents, Din Kwan Chan and Kwai Ching Chan Yiu. My accomplishment would not be possible today without your sacrifices for immigrating to Canada and providing me with all the support.

Table of Contents Abstract ...... ii Acknowledgements ...... iv Table of Contents ...... v List of Figures ...... viii List of Tables ...... xi List of Appendices ...... xi List of Abbreviations ...... xii 1. INTRODUCTION...... 1 1.1 Blood-Brain Barrier: The Neurovascular Unit...... 1 1.1.1 Historical Background...... 1 1.1.2 Structure and Function ...... 1 1.2 ABC Superfamily of Transporters – P-glycoprotein (P-gp) ...... 10 1.2.1 Structure, Function and Expression of P-gp ...... 13 1.2.2 Alteration of P-gp Function at Plasma membranes ...... 21 1.2.3 Regulation of P-gp mRNA and Protein Expression ...... 25 1.3 Nuclear Receptors ...... 30 1.3.1 PXR ...... 34 1.3.1.1 Expression, Structure and Function ...... 34 1.3.1.2 Regulation of Target Genes...... 41 1.3.2 CAR ...... 46 1.3.2.1 Expression, Structure and Function ...... 46 1.3.2.2 Regulation of Target Genes...... 51 1.4 HIV ...... 56 1.4.1 Combination Antiretroviral Therapy ...... 57 1.4.2 HIV Neurological Complications and Brain Permeation of Antiretroviral Drugs...... 60 1.4.3 Mechanisms of Antiretroviral Drug Interactions ...... 63 1.4.3.1 Antiretroviral Drug Interactions with Drug Transporters ...... 63 1.4.3.2 Antiretroviral Drug Interactions with Drug Metabolizing Enzymes ...... 67 1.4.3.3 Antiretroviral Drug Interactions with Nuclear Receptors ...... 69 2. Goal ...... 71 3. Rationale ...... 71 4. Hypotheses ...... 72

5. Specific Objectives ...... 72 6. Up-regulation of p-glycoprotein by HIV protease inhibitors in a human brain microvessel endothelial cell line ...... 74 6.1 Abstract ...... 76 6.2 Introduction ...... 76 6.3 Material and Methods ...... 79 6.3.1 Reagents ...... 79 6.3.2 Cell Culture ...... 79 6.3.3 Cell Viability Assay ...... 80 6.3.4 Immunocytochemistry ...... 81 6.3.5 Cytosolic and Nuclear Cell Fractions...... 81 6.3.6 Drug Accumulation Assays Using P-glycoprotein and MRP1 Over-expressing Cells 82 6.3.7 Drug Accumulation Assays Using hCMEC/D3 Cells...... 83 6.3.8 Induction of Drug Efflux Transporters in hCMEC/D3 Cells ...... 84 6.3.9 Statistical Analysis ...... 86 6.4 Results ...... 86 6.4.1 P-gp and MRP1 Transport of two HIV-1 Protease Inhibitors: Atazanavir and Ritonavir 86 6.4.2 Localization and Expression of Efflux Transporters and hPXR in hCMEC/D3 Cells 88 6.4.3 Cell Viability after Long Term Exposure to Atazanavir, Ritonavir, Rifampin and SR12813 ...... 91 6.4.3.1 P-gp Mediated Transport of Atazanavir and Ritonavir in hCMEC/D3 Cells ...... 91 6.4.3.2 MRP Related Transport of Atazanavir and Ritonavir in hCMEC/D3 Cells ...... 93 6.4.4 P-gp Expression in hCMEC/D3 Cells after Exposure to Selective Human PXR Ligands, Rifampin and SR12813 ...... 95 6.4.5 Drug Transport Interactions between Atazanavir and Ritonavir ...... 97 6.4.6 P-gp and MRP1 Expression in hCMEC/D3 Cells after Exposure to Atazanavir and Ritonavir ...... 98 6.4.7 Changes in P-gp Transport Associated with hCMEC/D3 Exposure to Atazanavir and Ritonavir ...... 100 6.5 Discussion ...... 102 6.6 Acknowledgements ...... 107 7. Regulation of P-gp functional expression by orphan nuclear receptors, hPXR and hCAR, in hCMEC/D3 cell culture system...... 108 7.1 Abstract ...... 110 7.2 Introduction ...... 110

7.3 Materials and Methods ...... 113 7.3.1 Materials and Reagents ...... 113 7.3.2 Cell Culture Systems ...... 113 7.3.3 Cell Viability Assay ...... 114 7.3.4 Cell Treatment ...... 114 7.3.5 Immunoblot Analysis ...... 115 7.3.6 RNA Extraction, Reverse Transcription and Quantitative Real-time PCR (qPCR) .. 116 7.3.7 Immunofluorescence Studies ...... 118 7.3.8 siRNA Downregulation Studies ...... 119 7.3.9 Statistical Analysis ...... 119 7.4 Results ...... 120 7.4.1 P-gp, hPXR and hCAR Expression in hCMEC/D3 cells and Human BBB-ECs ...... 120 7.4.2 Ligand-mediated Nuclear Translocation of hPXR and hCAR in hCMEC/D3 Cells . 125 7.4.3 Ligand-mediated Upregulation of MDR1 mRNA Expression in hCMEC/D3 Cells . 128 7.4.4 Effect of hPXR and hCAR Inhibitors on Ligand-mediated P-gp Induction ...... 130 7.5 Discussion ...... 134 7.6 Acknowledgements ...... 139 8. Induction of P-glycoprotein by Antiretroviral Drugs in Human Brain Microvessel Endothelial Cells ...... 140 8.1 Abstract ...... 142 8.2 Introduction ...... 142 8.3 Methods ...... 145 8.3.1 Materials and Reagents ...... 145 8.3.2 Cell Culture and Luciferase Reporter Assays ...... 146 8.3.3 hCMEC/D3 Cell Culture System and Ligand Treatment...... 147 8.3.4 Immunoblot Analysis ...... 148 8.3.5 Rhodamine-6G Transport Assay ...... 149 8.3.6 Statistical Analysis ...... 149 8.4 Results ...... 150 8.5 Discussion ...... 157 8.6 Acknowledgements ...... 161 8.7 Conflicts of interest ...... 161 9. In vivo induction of P-Glycoprotein expression at the mouse blood-brain barrier: an intracerebral microdialysis study ...... 162 9.1 Abstract ...... 164

vii

9.2 Introduction ...... 164 9.3 Materials and Methods ...... 167 9.3.1 Chemicals and Reagents...... 167 9.3.2 Intracerebral Microdialysis Study ...... 168 9.3.3 In vitro Recovery/Loss from the Microdialysis Probes ...... 169 9.3.4 Determination of Quinidine Unbound Fractions in Mouse Plasma ...... 170 9.3.5 Quinidine Quantification in Blood, Plasma, Perfusate, Dialysate and Dosing Solution 171 9.3.6 Mouse Brain Capillary Isolation and Brain Homogenate Preparation ...... 172 9.3.7 Immunoblot Analysis ...... 173 9.3.8 Data Analysis ...... 174 9.4 Results ...... 175 9.5 Discussion ...... 184 9.6 Acknowledgements ...... 191 9.7 Conflicts of Interest ...... 191 10. Overall Discussion and Summary ...... 192 11. Limitation and Future Directions...... 205 11.1 Experimental Limitations ...... 205 11.1.1 In vitro cell culture systems of human brain microvessel endothelial cells ...... 205 11.1.2 Species differences ...... 209 11.1.3 Microdialysis ...... 213 11.2 Future Directions ...... 214 11.2.1 Nuclear receptor regulation of phase I/II metabolic enzymes and other drug transporters in brain microvessel endothelial cells ...... 214 11.2.2 Nuclear receptor regulation of drug transporters in other cellular compartments of the brain parenchyma ...... 216 12. References ...... 217 13. List of Relevant Publications ...... 278

List of Figures

Figure 1-1. Morphology of the BBB, the neurovascular unit. Figure 1-2. Scheme of endothelial tight junctional complexes. Figure 1-3. Domain arrangement of ABC membrane transporters. Figure 1-4. Current proposed model of P-gp transport.

viii

Figure 1-5. Transcriptional regulation of P-gp expression. Figure 1-6. Functional domains and DNA binding of nuclear receptors. Figure 6-1. P-gp (A) and MRP1 (B) substrate properties of atazanavir and ritonavir in MDA- 435/LCC6-MDR1 and HeLa-MRP1 overexpressing cells and the corresponding wild type cell lines. Figure 6-2.Electron micrograph of human microvessel endothelial hCMEC/D3 cells. Figure 6-3.P-gp, MRP1 and hPXR expression in hCMEC/D3 cells. Figure 6-4. Cellular accumulation of atazanavir, ritonavir, and the P-gp substrate R-6G by hCMEC/D3 cells. Figure 6-5. Cellular accumulation of atazanavir, ritonavir and the MRP substrate BCECF by hCMEC/D3 cells. Figure 6-6. Expression of P-gp following treatment of hCMEC/D3 cells with 1, 5 or 10 µM rifampin or SR12813 compound. Figure 6-7. Drug transport interactions between atazanavir and ritonavir in hCMEC/D3 cells. Figure 6-8. Expression of MRP1 and P-gp following treatment of hCMEC/D3 cells with 1, 5 and 10 µM atazanavir or ritonavir. Figure 6-9. P-gp functional activity after treatment of hCMEC/D3 cells with 5 µM atazanavir or 5 µM ritonavir. Figure 7-1. Immunoblot analysis of P-gp, hPXR and hCAR protein expression in hCMEC/D3 cells. Figure 7-2. Selectivity of anti-hPXR and anti-hCAR antibodies used in immunoblot analysis. Figure 7-3. Human PXR and CAR mRNA expression in HepG2 and hCMEC/D3 cells. Figure 7-4. Analyses of hPXR and hCAR protein expression in human brain-derived microvascular endothelial cells (BBB-ECs). Figure 7-5. Immunocytochemical localization of hPXR and hCAR in hCMEC/D3 cells. Figure 7-6. Inductive effect of hPXR or hCAR ligands on MDR1 mRNA expression in hCEMC/D3 cells. Figure 7-7. P-gp expression in hCMEC/D3 cells treated with SR12813 and A792611 or CITCO and meclizine. Figure 7-8. Effect of hPXR and hCAR downregulation on P-gp protein expression in hCMEC/D3 cells. Figure 8-1. Activation of hPXR by Antiretroviral Drugs. Figure 8-2. Activation of hCAR by Antiretroviral Drugs. Figure 8-3. P-gp Immunoblot and Densitometric Analysis. Figure 8-4. R-6G Cellular Accumulation by hCMEC/D3 Cells. Figure 9-1.Unbound quinidine concentrations (ng/mL) in (A) plasma and (B) brain ECF from dexamethasone (DEX)-treated or vehicle-treated CD1 mice.

Figure 9-2. Unbound quinidine ECF to plasma concentration ratios in dexamethasone (DEX)-treated or vehicle-treated CD-1 mice. Figure 9-3.Representative immunoblot (top) and densitometric analysis (bottom) of P-gp expression in mouse brain capillary fractions (50 μg) isolated from dexamethasone (DEX)-treated or vehicle (VEH)-treated CD-1 mice. Figure 9-4. Representative immunoblot (top) and densitometric analysis (bottom) of P-gp expression in mouse brain homogenate fractions (50 μg) from dexamethasone (DEX)-treated or vehicle (VEH)-treated CD-1 mice. Figure 9-5. Representative immunoblot (top) and densitometric analysis (bottom) of mPXR expression in mouse brain capillary fractions (50 μg) from dexamethasone (DEX)-treated or vehicle (VEH)-treated CD-1 mice. Figure 9-6. Representative immunoblot (top) and densitometric analysis (bottom) of mPXR expression in mouse brain homogenate fractions (50 μg) from dexamethasone (DEX)-treated or vehicle (VEH)-treated CD-1 mice. Figure 9-7. Representative immunoblot of Cyp3a11 expression in mouse brain capillary fractions (50 μg) in dexamethasone (DEX)-treated or vehicle (VEH)-treated CD-1 mice. Figure 9-8. Representative immunoblot (top) and densitometric analysis (bottom) of Cyp3a11 expression in mouse brain homogenate fractions (50 μg) from dexamethasone (DEX)- treated or vehicle (VEH)-treated CD-1 mice. Figure B-1. R-6G accumulation by hCMEC/D3 cells. Figure B-2. R-6G accumulation by P-gp over-expressing cells, MDA-MDR1, and its corresponding wild-type cells, MDA-WT. Figure C-1. Transmitted light image of a mouse brain capillary. Figure C-2. Protein expression of P-gp, Bcrp, mPXR, zona occludens-1 (ZO-1) and occludin determined by immunoblot analysis in lysates of mouse brain capillary, mouse liver and Caco2 cells. Figure D-1. RT-PCR analysis of MDR1 mRNA in the immortalized human brain endothelial cell line, hCMEC/D3. Figure D-2. Western blot analysis of P-gp in rat brain microvessel endothelial cells (RBE4), hCMEC/D3, primary culture of rat astrocytes and MDCK-MDR1 cells. Figure D-3. Western blot analysis showing P-gp expression in RBE4 cells. Figure D-4. Immunocytochemical localization of P-gp in fixed RBE4 cells. Figure D-5. Functional activity of P-gp in primary cultures of rat astrocytes. Figure D-6. Functional activity of P-gp in hCMEC/D3 cells. Figure E-1. Localization of selected ABC and SLC transport proteins in A) brain microvessel endothelial cells at the BBB and B) choroid plexus epithelial cells at the BCFB.

Figure E-2. Transcriptional regulation of drug transporters by nuclear receptors. List of Tables

Table 1-1. Function of selected members of the ABC transporter superfamily. Table 1-2. Function of P-gp isoforms in humans and rodents. Table 1-3. Peripheral tissue distribution of PXR and CAR. Table 1-4. Brain distribution of PXR and CAR. Table 1-5. Human and rodent PXR regulation of phase I/II enzymes. Table 1-6. Human and rodent PXR regulation of transporters. Table 1-7. Human and rodent CAR regulation of phase I/II enzymes. Table 1-8. Human and rodent CAR regulation of transporters. Table 1-9. Preferred and alternative antiretroviral regimens for antiretroviral therapy-naive patients. Table 1-10. Pharmacokinetic parameters of antiretroviral drugs in humans. Table 1-11. Antiretroviral drug interactions with P-gp. Table 1-12. Metabolisms patterns of antiretroviral drugs. Table 1-13. Antiretroviral drug interactions with hPXR and hCAR. Table 7-1. List of primers used for qPCR analysis. Table A-1. Viability of hCMEC/D3 cells following 72 h incubation with ligands or antiretroviral drugs Table D-1. RT conditions and primers used in semi-quantitative PCR analysis are shown. FP – forward primer, RP – reversed primer. Table D-2. Antibodies used in immunoblotting analysis of transport proteins. IF – Immunofluorescence studies, WB- Western blot. Table D-3. Selective inhibitors, fluorescence substrates and radiolabelled substrates for drug transport studies to characterize P-glycoprotein function. Table E-1. Regulation of Drug Transporters Mediated by Nuclear Receptors and Aryl Hydrocarbon Receptor.

List of Appendices

Appendix A. Cell Viability Studies in hCMEC/D3 cells ...... 279 Appendix B. Initial characterization of P-gp-mediated transport of R-6G ...... 280 Appendix C. Morphology and Protein Expression of Mouse Brain capillaries ...... 282

Appendix D. Chan G.N.Y. and Bendayan R. 2010. In Vitro Study of P-glycoprotein at the Blood-brain Barrier: Molecular Expression, Localization and Functional Activity. Methods in Molecular Biology 686:313-36...... 284 Appendix E. Chan G.N.Y., Hoque Md. T. and Bendayan R. 2013. The Role of Nuclear Receptors in Drug Transporters Regulation in the Brain. Trends in pharmacological sciences. 34(7):361-72...... 316

List of Abbreviations

ABC, ATP-Binding Cassette aCSF, Artificial CSF AhR, Aryl Hydrocarbon Receptor AIDS, Acquired Immune Deficiency Syndrome Aldh, Aldehyde Dehydrogenase ANOVA, One Way Analysis of Variance AP-1, Activator Protein 1 BBB, Blood-Brain Barrier BBB-ECs, Primary Cultures of Human Brain-Derived Microvascular Endothelial Cells BCECF, 2', 7’-Bis-(2-carboxyethyl)-5(6)-Carboxyfluorescein BCECF-AM, Acetoxymethyl Ester Derivative of BCECF BCRP, Breast Cancer Resistance Protein BCSFB, Blood-Cerebrospinal Fluid Barrier BSA, Bovine Serum Albumin BSEP, Bile Salt Export Pump cAMP, Cyclic Adenosine Monophosphate CAR, Constitutive Androstane Receptor cART, Combination Antiretroviral Therapy CITCO, 6-(4-Chlorophenyl)-Imidazo[2,1-b]Thiazole-5-Carbaldehyde CCR5, Chemokine Receptor 5 CCRP, Cytoplasmic Constitutive Androstane Receptor Retention Protein C/EBP, CCAAT/Enhancer-Binding Protein CMV, Cytomegalovirus CMX, Cytomegalovirus-Based CNT, Concentrative Nucleoside Transporter CNS, Central Nervous System COX-2, Cyclooxygenase-2 CSF, Cerebrospinal Fluid CXCR4, Chemokine Receptor Type 4 CYP, Cytochrome P450

xii

DAPI, 4',6-Diamidino-2-Phenylindole DBD, DNA-Binding Domain DEX, Dexamethasone DMSO, Dimethyl Sulfoxide DR(x), Direct Repeats (x) ECF, Extracellular Fluid ENT, Equilibrative Nucleoside Transporter ET-1, Endothelin-1

ETB, Endothelin B ER, Estrogen Receptor ER(x), Everted Repeats (x) FBS, Fetal Bovine Serum FoxO1, fox-head insulin-responsive transcription factor 1 FXR, Farnesoid X Receptor GSTA, Glutathione S-Transferase Alpha GSTM, Glutathione S-Transferase Mu GR, Glucocorticoid Receptor GRIP, Glucocorticoid Receptor-Interacting Protein 1 HAART, Highly Active Antiretroviral Therapy HAND, HIV-Associated Neurocognitive Disorders HBSS, Hank's Balanced Salt Solution hCMEC/D3, Human Cerebral Microvascular Endothelial Cells/ Clone D3 HIV, Human Immunodeficiency Virus HNF4 α, Hepatocyte Nuclear Factor 4α hPXR, Human Pregnane X Receptor HSP90, Heat Shock Protein 90 ICAM-1, Intracellular Adhesion Molecule 1 IL, Interleukin iNOS, Inducible Nitric Oxide Synthase IR(x), Inverted Repeats (x) JAM, Junctional Adhesion Molecules JNK, c-Jun N-Terminal kinase LBD, Ligand-Binding Domain LXR, Liver X Receptor MED1, Multiple start site Element Downstream 1 MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-Tetrazolium Bromide MRP, Multidrug Resistance Protein NBD, Nucleotide-Binding Domain NcoR, Nuclear Receptor Co-Repressor NF-κB, Nuclear Factor Kappa B

xiii

NNRTI, Non-Nucleoside Reverse Transcriptase Inhibitor NMDA, N-Methyl-D-Aspartate NTCP, Na+ Taurocholate Cotransporting Polypeptide NRTI, Nucleoside Reverse Transcriptase Inhibitor OATP, Organic Anion Transporting Polypeptide OAT, Organic Anion Transporter OCT, Organic Cation Transporter OCTN, Novel Organic Cation Transporter PBS, Dulbecco’s Phosphate Buffered Saline PBREM, Phenobarbital-responsive enhancer module PECAM, Platelet-Endothelial Cell Adhesion Molecule P-gp, P-Glycoprotein PGC-1, PPAR-γ coactivator-1 PI3, Phosphoinositide 3 PI, Protease Inhibitor PKC, protein kinase C PMSF, Phenylmethanesulfonylfluoride PPARs, Peroxisome Proliferator-Activated Receptors PSC-833, Valspodar PVDF Hybond-P Polyvinylidene Difluoride PXR, Pregnane X Receptor R-6G, Rhodamine-6G Rac, Ras-related C3 botulinum toxin substrate RAR, Retinoic Acid Receptor RhoA, Ras homolog gene family member A ROS, Reactive Oxygen Species RXR, Retinoid X Receptor SDS, Sodium Dodecylsulphate SDS-PAGE, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis SHP, Short Heterodimer Partner SiRNA, Small Interfering RNA SLC, Solute Carrier SMRT, Silencing Mediator of Retinoid and Thyroid Hormone Receptors SNPs, Single Nucleotide Polymorphisms SRC-1, Steroid Receptor Co-activator-1 SULT, Sulfotransferase TBS-T, Tris-Buffered Saline Containing 0.1 % Tween 20 TEER, Transepithelial Electric Resistance TEMED, N,N,N,N-Tetramethyl Ethylenediamine TMD, Transmembrane Domain

xiv

TNF-α, Tumour Necrosis Factor-α UAS, Upstream Activation Sequence UGT, UDP-Glucuronosyltransferase VCAM-1, Vascular Cell Adhesion Molecule 1 VE-Cadherin, Vascular Endothelial Cadherin VEGF, Vascular Endothelial Growth Factor VDR, Vitamin D Receptor XREM, Xenobiotic Responsive Enhancer Module

1. INTRODUCTION

1.1 Blood-Brain Barrier: The Neurovascular Unit

1.1.1 Historical Background

The first observation of the existence of a “blood-brain barrier (BBB)” was reported by P.

Ehrlich in 1885 who found that water soluble dyes injected peripherally to rats could not penetrate into the central nervous system (CNS) (Ehrlich 1885). Later, E.E. Goldmann and M.

Lewandowsky were the first to describe the BBB as being a static barrier between the CNS and systemic circulation (Lewandowsky 1900, Goldmann 1913). Over the next 50 years, there was much controversy on the actual existence of the BBB; this was finally resolved in 1968 when several investigators identified the presence of tight junctions between brain microvessel endothelial cells using electron microscopy, suggesting that the brain microvessel endothelium is the anatomical location of this barrier (Reese & Karnovsky 1967, Brightman & Reese 1969).

Since then, methods to isolate brain microvessels from animals or human brain tissues or to culture brain microvessel endothelial cells have become widely available allowing the implementation of various ex vivo and in vitro BBB models to investigate CNS drug delivery and

BBB function (Cardoso et al. 2010, Naik & Cucullo 2012). At present, the BBB is recognized as a dynamic interface regulating systemic blood to CNS exchange of molecules (e.g., nutrients, ions and xenobiotics) (Hawkins & Davis 2005, Abbott et al. 2010, Pardridge 2012).

1.1.2 Structure and Function

Brain microvessels or capillaries form the structural basis of the BBB, which constitutes the third largest organ barrier after the intestine and lung in humans (Pardridge 2003, Hawkins &

Davis 2005). This barrier is present in all regions of the brain except in areas of the autonomic

nervous system and endocrine glands, where a high degree of vascular perfusion is required

(Ballabh et al. 2004). There are approximately 100 billion microvessels in a human brain with a total length of more than 600 km which covers a surface area of approximately 10 to 30 m2

(Zlokovic & Apuzzo 1998, Pardridge 2003, Miller et al. 2008). In addition, a high density of microvessels per brain tissue mass allows each microvessel to be in close proximity of approximately 40 µm apart from each other, thus ensuring that each neuron is sufficiently perfused (Rodríguez-Baeza et al. 2003). Although BBB permeability is primarily regulated by microvessel endothelial cells, endothelial interactions with neighbouring cells (i.e., astrocytes, neurons, and pericytes) and structural components (i.e., basement membrane) play an important role in the development and maintenance of many BBB phenotypes (Hawkins & Davis 2005,

Abbott et al. 2010) (Figure 1-1). Recently, the term “neurovascular unit” has been introduced to describe how these different cellular constituents found in constant and intimate contact with the endothelium are essential for both health and function of the CNS (Hawkins & Davis 2005).

Morphologically, a brain microvessel endothelial cell typically presents 50 to 100 µm in length and 10 to 20 µm in width and encapsulates the lumen of the microvessel with a diameter of 7 to 8

µm (Pober 2008). These cells are highly polarized with an apical (i.e., luminal) membrane facing the blood and a basolateral (i.e., abluminal) membrane facing the brain parenchyma (Cardoso et al. 2010). Furthermore, they grow in a long, flat and spindle structure with approximately only

300 nm cytoplasmic spacing between the luminal and abluminal membranes (Pardridge 2003).

Several unique structural phenotypes allow these cells to be functionally different compared with other vascular endothelial cells found in the periphery. For instance, the brain microvessel endothelium exhibits a lack of fenestration and low pinocytotic activity, thus allowing a tight

Figure 1-1. Morphology of the BBB, the neurovascular unit. Anatomically, microvessels that constitute the BBB are composed of a monolayer of brain microvessel endothelial cells jointed by inter-endothelial tight junctions. The endothelium is surrounded by a basement membrane which provides structural support to the microvessel. Astrocyte end feet, pericytes and neuronal axonal processes share this basement membrane and are in constant and intimate contact with the endothelium. Together, these different cellular constituents form the neurovascular unit. Adapted from Abbott et al. 2006 (Pardridge 2012).

regulation of transcellular diffusion of molecules and immune cells (De Boer & Gaillard 2006).

In addition, all brain microvascular endothelial cells are joined by junctional complexes, which consist of inter-connected membrane integral proteins. These complexes serve as a seal to prevent the distribution of membrane constituents between the apical and basolateral plasma membranes and limit paracellular permeability of immune cells and molecules (Schulze & Firth

1993, Kniesel & Wolburg 2000, Wolburg & Lippoldt 2002, Vorbrodt & Dobrogowska 2003,

Hawkins & Davis 2005). The two major groups of protein identified to be important constituents of the junctional complexes are tight junction proteins and adherens junction proteins (Schulze &

Firth 1993, Kniesel & Wolburg 2000, Wolburg & Lippoldt 2002, Vorbrodt & Dobrogowska

2003, Hawkins & Davis 2005) (Figure 1-2). Tight junction proteins at the apical regions of inter- endothelial cells cleft are tightly attached together to the actin cytoskeleton and these interactions are more complex compared with those found in the peripheral vasculatures (Nagy et al. 1984).

Several transmembrane proteins, such as claudin 3 and 5, occludin and junctional adhesion molecule 1, are linked closely to accessory cytoplasmic tight junction proteins (i.e., zonula occluden 1, 2 and 3) forming the primary seal of the tight junctions (Vorbrodt & Dobrogowska

2003). In addition, intracellular calcium levels and protein kinase C (PKC) activity play an important role in the organization and function of these tight junction proteins (Bazzoni et al.

2005, Hawkins & Davis 2005). In contrast, adherens junction proteins, such as vascular endothelial-cadherin, α-catenin and β-catenin, are primarily localized at the basolateral region of inter-endothelial cells cleft and are known to mediate endothelial cell-cell adhesion through anchoring to actin cytoskeleton, playing an important role in maintaining microvascular integrity

(Cook et al. 2008). Disruption of these proteins can lead to increased paracellular permeability and reduced junction stability (Cook et al. 2008). However, it is generally accepted that tight junction proteins are the dominant endothelial phenotype in conferring high transendothelial electrical resistance and low paracellullar permeability observed at the BBB (Abbruscato &

Davis 1999). The in vivo transendothelial electrical resistance at the BBB is estimated to be higher than 1500 Ω cm2 in mammals, whereas peripheral microvessels typically generate only 2

– 20 Ω cm2 of resistance (Hawkins & Davis 2005, Abbott et al. 2006, Crone & Olesen 1982).

Figure 1-2. Scheme of endothelial tight junctional complexes. Tight junction proteins [e.g., claudin 3 & 5, occludin, junctional adhesion molecules (JAMs)] and adherens junction proteins [e.g., vascular endothelial cadherin (VE-cadherin), platelet-endothelial cell adhesion molecule (PECAM)] are inter-connected membrane integral proteins that serve as a seal to prevent distribution of membrane constituents between the apical and basolateral plasma membranes and to limit paracellular permeability. These integral proteins are connected to actin cytoskeleton w ithin the cytoplasm by accessory tight junction proteins (i.e., zonula occluden (ZO) 1, 2 and 3) or accessory adherens junction proteins (e.g., catenins). Adapted from Abbott et al. 2010 (Hawkins & Davis 2005).

In addition to these endothelial structural phenotypes, the brain microvessel endothelial cells also exhibit transport functions which tightly regulate permeability of ions, neurotransmitters, macromolecules, neurotoxins and nutrients, essential for maintaining homeostasis of neural micro-environment (Hawkins & Davis 2005, Abbott et al. 2010). Gaseous molecules (i.e., oxygen and carbon dioxide) and small lipophilic molecules usually can diffuse into the brain along their concentration gradients unaffected by the high transendothelial resistance at the BBB. Permeability of other molecules will occur through the transendothelial route, where it is tightly regulated by several ion channels, carrier-mediated transporters and

ATP-dependent transport proteins (Ohtsuki & Terasaki 2007, Zlokovic 2008). For example, ion channels (i.e., K+ gated channel) and ATP-dependent ion transporters (e.g., Na+/K+-ATPase,

Na+/K+/2Cl¯ cotransporter) are essential to maintain an optimal ionic composition for proper synaptic function (Vorbrodt 1988, O'Donnell et al. 2006). The permeability of nutrients (e.g., glucose and amino acids) and hormones across the BBB is mediated by several carrier-mediated transporters, such as glucose transporter 1, Na+-independent amino acid transporters, glutamate transporters, nucleosides and nucleotides transporters, monocarboxylate transporters and thyroid hormone transporters (Pardridge 2005). Several lipophilic and non-polar xenobiotics have relatively reasonable permeability across the BBB, however this permeability is tightly regulated by several members of the ATP-binding cassette (ABC) and solute carrier (SLC) transporter family at the luminal and abluminal membranes of brain microvessel endothelial cells (Pardridge

2007, Zlokovic 2008, Eyal et al. 2009). Localization of transporter proteins at the BBB has not been fully elucidated. Current knowledge on their localization is summarized in a recent published review from our laboratory (Ashraf et al. 2013). In addition, transport of neuroactive peptides (e.g., arginine-vasopressin and luteinizing-hormone releasing hormone) across the BBB is mediated by various peptide transporters (Zlokovic 1995, Banks 2006). Furthermore, permeability of macromolecules (e.g., transferrin, low-density lipoproteins and insulin) across the BBB is primarily mediated by specific membrane-associated receptors expressed at the endothelial luminal membrane (Pardridge 2005). The sequential internalization or endocytosis of these macromolecules is primarily regulated by caveolin-1, which is enriched in lipid rafts at the cell surface and intracellular membrane vesicles (Drab et al. 2001, Parton & Richards 2003).

Aside from these transport functions, brain microvessel endothelial cells also express a number of cytoplasmic enzymes important for energy production and drug metabolizing processes

(Abbott et al. 2010). For example, acid and alkaline phosphatases, monoamine oxidase and dehydrogenases are highly involved in a variety of biochemical processes (Cucullo et al. 2011).

Whereas, phase I Cytochrome P450 (CYP) enzymes (e.g., CYP1B1 and 2D6) and phase II enzymes [e.g., Glutathione S-Transferase (GST)] are believed to represent a metabolic barrier which further reduces xenobiotics availability in the brain parenchyma (Dauchy et al. 2008,

Bauer et al. 2008a, Woodland et al. 2008, Dauchy et al. 2009). In order to meet the high metabolic demand of these active transport and metabolic systems, brain endothelial cells are characterized by a very high content of mitochondria (Cornford et al. 1997). Aside from being both the structural and metabolic barriers, the BBB also plays an essential role in governing immune cell permeability into and out of the CNS (Greenwood et al. 2011). In general, mononuclear leukocytes, monocytes and macrophages can be recruited to the brain during CNS pathological conditions, such as inflammation or infection, playing a complementary role to the residing microglia (Bechmann et al. 2001, Davoust et al. 2008). The interactions between immune cells and endothelial immunoglobulin adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM-1) and intracellular adhesion molecule 1 (ICAM-1), play an important role during leukocyte extravasation across the BBB (Zameer & Hoffman 2003). These immune cells are believed to be attracted to the site of inflammation guided by chemokines and are able to penetrate endothelial cells by a process of diapedesis without disrupting endothelial tight junctions (Engelhardt & Wolburg 2004, Wolburg et al. 2005, Carman & Springer 2008,

Engelhardt & Coisne 2011). During initial Human Immunodeficiency Virus (HIV) infection, infected monocytes have been proposed to carry viral genetic material across the BBB (Koenig et al. 1986, Jones & Power 2006, Kramer-Hämmerle et al. 2005, Peluso et al. 1985, Persidsky &

Poluektova 2006). In addition, direct virons transmigration into the brain across the BBB could

occur through events related to microvessel endothelial endocytosis. However, microvessel endothelial cells have not been reported to be directly infected (Banks et al. 2001, Liu et al.

2002, Bobardt et al. 2004, Kramer-Hämmerle et al. 2005, Miller et al. 2012).

In addition to endothelial cells, the neurovascular unit also consists of other essential neighbouring cells and structural components (Hawkins & Davis 2005, Abbott et al. 2010). For instance, a layer of basement membrane surrounds the microvessel endothelium and pericytes, which provides structural anchoring to these cells (Carvey et al. 2009). The membrane is composed of several different classes of matrix proteins, such as collagen type IV, elastin, fibronectin, laminin and proteoglycan, which in turn are secreted by endothelial cells, pericytes and astrocytes (Wolburg & Lippoldt 2002, Adibhatla & Hatcher 2008). Disruption of the basement membrane can alter organization of cellular components constituting the neurovascular unit and increase BBB permeability (Carvey et al. 2009). Pericytes are surrounded by the basement membrane at the BBB and cover approximately 30 - 70 % of the total outer endothelium wall (Mathiisen et al. 2010). These cells can be found in pre-capillary arterioles, capillary-beds and post-capillary venules throughout the body and they are replaced by vascular smooth muscle cells in veins and arteries (Dalkara et al. 2011). Aside from their function to synthesize matrix proteins for the basement membrane, they also regulate microvessel blood flow by a cellular contractile mechanism, which is governed by vasoconstrictors and vasodilators released from adjacent neuronal axonal processes (Fernández-Klett et al. 2010, Hamilton et al.

2010). In addition, pericytes are associated with endothelial tight junction regulation, BBB development and microvessel angiogenesis (Gerhardt et al. 2000, Hamilton et al. 2010, Bell et al. 2010, Daneman et al. 2010, Kamouchi et al. 2011). Astrocyte end feet form a lamellae on the outer surface of endothelium and basement membrane (Kacem et al. 1998, Abbott et al. 2006).

Currently, there is strong in vitro evidence supporting their role in regulating BBB function

(Abbott et al. 2006). For example, astrocytic secretion of several growth factors (e.g., transforming growth factor β, glial-derived neurotrophic factor, fibroblast growth factor and aniopoetin-1) can decrease trans-endothelial permeability for small molecules (Dehouck et al.

1990, Igarashi et al. 1999, Lee et al. 2003). In addition, co-culturing of brain endothelial cells with astrocytes or in astrocytes-conditioned media can enhance BBB phenotypes (i.e., upregulation of endothelial tight junctions) (Siddharthan et al. 2007, Colgan et al. 2008). In contrast, high expression of membrane-associated transporters and channels (e.g., Na+/K+-

ATPase and glutamine uptake transporters) on astrocyte end feet can tightly regulate solutes concentration and fluid volume around the endothelium, which is crucial for creating a suitable micro-environment for BBB function (Price et al. 2002, Simard et al. 2003, Kofuji & Newman

2004, Ballabh et al. 2004, Abbott et al. 2006). Furthermore, astrocytic release of histamine and

ATP that is triggered by neuronal axonal signalling can induce endothelial glucose uptake across the BBB, which suggests astrocytes can serve as an intermediate messenger for neurons in regulating endothelial transport function (Braet et al. 2004, Leybaert 2005). As well, microvessel endothelium and associated astrocyte end feet are also in direct contact with cholinergic, serotonergic, GABAergic and noradrenergic neuronal axonal processes, which are believed to play a role in regulating permeability of metabolic waste and nutrients across the BBB and maintain the homeostasis of the neuronal micro-environment (Cardoso et al. 2010, Ben-

Menachem et al. 1982, Kobayashi et al. 1985, Vaucher & Hamel 1995, Cohen et al. 1996, Cohen et al. 1997, Tong & Hamel 1999).

1.2 ABC Superfamily of Transporters – P-glycoprotein (P-gp)

ABC transporters are a family of membrane-associated proteins that catalyze directional transport of a wide variety of substrates across cellular membranes (i.e., plasma membrane)

(Dean & Allikmets 2001). These transporters are found in all existing phyla, from prokaryotes to humans, and belong to one of the oldest and largest protein superfamily (Jones & George 2004).

Currently, there are 49 members of ABC genes in humans, which encode 44 functional transporters recognizing amino acids, lipids, metal ions, peptides and xenobiotics as substrates, four translation factors lacking transport function and one pseudogene (Higgins 1992, Dean &

Allikmets 2001, Moitra & Dean 2011). These transporters use the energy from ATP hydrolysis at the nucleotide-binding domain (NBD) to translocate their substrates across cellular membranes

(George & Jones 2012). The NBD in all ATP-binding transporters contains two highly conserved motifs which are designated as Walker A and Walker B and are separated by 100 to 200 amino acid residues (Leslie et al. 2005). These two motifs are essential for nucleotide binding, of which the Walker A region can bind to the phosphate group of ATP and the Walker B region binds to magnesium ion (Sharom et al. 1999, George & Jones 2012). In addition, the LSGGC amino acid sequence, as designated as ABC signature or C motif, can be found in all members of the ABC superfamily (Dean & Allikmets 2001). Together with other consensus sequences (e.g.,

EATSALD or the D-loops), these motifs constitute the signature ATP hydrolysis pocket of a typical ABC transporter. In particular, substrate specificity is primarily determined by the transmembrane domain (TMD) which contains 6 to 12 membrane-spanning α-helices (Moitra &

Dean 2011). Currently, several X-ray crystallography structures of ABC transporters (nine from prokaryote and two from eukaryote) have been identified at low resolution (2.2 – 5.5 Å) and only two of these images were obtained when the transporter was associated with a substrate or

inhibitor (Oldham et al. 2007, Aller et al. 2009, George & Jones 2012). Resolved structures for all phases of the transport cycle have yet to be reported, therefore it remains unclear whether all

ABC transporters have specific substrate binding sites in the TMD or the TMD with its interactive residues simply serves as a path for substrate entry into the cell (George & Jones

2012). In general, the basic architecture of ABC transporters comprises of two TMDs and two

NBDs, but some gene products can also be organized as a half transport only containing one of each domain (Dean & Allikmets 2001) (Figure 1-3).

Figure 1-3. Domain arrangement of ABC membrane transporters. The basic structure of a half transporter consists of one TMD and one NBD (contain Walker A (A), Signature C (C) and Walker B motifs), whereas a full transporter, consists of two TMDs and two NBDs (Moitra & Dean 2011).

Based on phylogenetic analysis and structural organization of these domains, human

ABC proteins are divided into seven subfamilies, from A to G (Moitra & Dean 2011). Several common members for each family and their general physiological function are summarized in

Table 1-1. A comprehensive list of mammalian ABC transporters currently known can be found at the website developed by Dr. Michael Müller

(Agrotechnology and Food Sciences, Wageningen University, The Netherlands). Members belonging to subfamilies of ABCB (e.g., ABCB1/P-gp), ABCC (e.g., ABCC1-5/ Multidrug

Resistance Protein (MRP) 1-5) and ABCG [e.g., ABCG2/ Breast Cancer Resistance Protein

(BCRP)] are considered to be the major transporters responsible for: i) multi-drug resistance phenomena in tumour cells, ii) alteration of tissue distribution of therapeutic agents and iii) protective transport mechanisms against xenobiotics in cellular compartments and tissue barriers

(i.e., the BBB). In the following sections, only P-gp is discussed in detail. A comprehensive summary on the physiological roles, substrate specificity and tissue expression of BCRP and

MRPs can be found in reviews published by our group and others (Eisenblätter et al. 2003,

Nicolazzo & Katneni 2009, Pick et al. 2010, Lee et al. 2007b, Dallas et al. 2006, Slot et al. 2011,

Ashraf et al. 2012).

Table 1-1. Function of selected members of the ABC transporter superfamily.

Number of Subfamily Examples Main Function Members ABCA1 Cholesterol and phospholipid efflux ABC-A 12 & ABCA10 in macrophages MDR1 (P-gp); Xenobiotic transport; bile salt ABC-B 11 ABCB11 (BSEP) transport ABCCs 1 - 5 Xenobiotic transport; Chloride ion ABC-C 13 (MRPs 1 - 5) ABCC 7 (CFTR) channel

ABC-D 4 ABCD1 (ALDP) Peroxisomal import of fatty acids

ABC-E 1 Inhibit activity of ribonuclease L

ABC-F 3 ABCF1 Protein synthesis (?) Macrophage cholesterol ABCG1; ABCG2 (BCRP); efflux; xenobiotic transport; hepatic ABC-G 6 ABCG5/8 cholesterol secretion and intestinal apical cholesterol transport Adapted from Dr. M. Müller, accessed on March 2013, http://nutrigene.4t.com/humanabc.htm and Dean et al. 2001.

1.2.1 Structure, Function and Expression of P-gp

In humans, P-gp is encoded by two genes, ABCB1 (MDR1) and ABCB4 (MDR2 or 3)

(Ueda et al. 1986, Chin et al. 1989). In rodents, three P-gp isoforms have been identified: Abcb1a

(mdr1a), Abcb1b (mdr1b) and Abcb4 (mdr2) (Hsu et al. 1990). The gene products from human

MDR1 and rodent mdr1a and b are involved in xenobiotics (i.e., drugs) transport, whereas human

MDR2 and rodent mdr2 gene products are generally involved in hepatic transport of phosphatidylcholine into bile at the biliary canalicular membrane (Smit et al. 1993) (Table 1-2).

P-gp from different species share approximately 70 % amino acid sequence identity, while the human and rodent genes share approximately 80 % nucleotide sequence identity (Van der Bliek et al. 1988, Jones & George 1998). Although, MDR1 gene was identified as the human ortholog of the mouse mdr1a gene, species differences have been reported in substrate (e.g., colchicines,

actinomycin D and digoxin) affinities (Tang-Wai et al. 1995, Shoshani et al. 1998, Takeuchi et al. 2006, Suzuyama et al. 2007). Using cell culture models transfected with either human MDR1 or mouse Mdr1a gene, these studies were able to demonstrate that protein encoded by mdr1a isoform may be more efficient in substrate transport and can generate a more superior drug resistance compared to its human counterpart. More comprehensively, Feng et al. from Pfizer tested 3300 compounds in human MDR1-transfected and mouse mdr1a-transfected cells (i.e.,

MDCK) and found a good correlation (R2 = 0.92) between substrate efflux ratios generated from the human and mouse P-gp transfected cell lines (Feng et al. 2008). These authors suggest that P- gp species differences between human and mouse are relatively rare, however they do exist.

Similarly, inhibitory potency of several P-gp inhibitors towards human and mouse P-gp have been reported to be different. For instance, in vitro IC50 value for verapamil to inhibit digoxin transport by human P-gp (i.e., 18.3 μM) was reported to be slightly higher than values generated from cell transfected with rat mdr1a (i.e., 13.9 μM) or mdr1b gene (i.e., 8.7 μM), which implies that verapamil could be more potent to inhibit digoxin transport mediated by rat P-gp compared to human protein (Suzuyama et al. 2007). In contrast, quinidine exhibited a smaller IC50 value to inhibit digoxin transport in human MDR1-transfected cells (i.e., 18.3 μM) compared to cell transfected with rat mdr1a (i.e., 32.3 μM) or mdr1b gene (i.e., >50 μM), suggesting a greater inhibitory potency of quinidine on human P-gp than rat P-gp (Suzuyama et al. 2007). Overall, these studies collectively suggest that there could be species differences in P-gp substrate/inhibitor affinity between humans and rodents. The understanding of these differences could help in translating in vivo findings into more reliable interpretation in the clinic.

Table 1-2. Function of P-gp isoforms in humans and rodents.

Alternative Species Genes Expression Function Names Pancreas, lung, heart, adrenal gland, MDR1, PGY1, Humans ABCB1 lymphocytes, intestine, hepatocytes, CLCS, GP170 kidney, astrocytes, pericytes, microglia, Xenobiotics Rodents Abcb1a mdr1a, mdr3 brain microvessel endothelial cells (BBB), transport & choroid plexus epithelial cells (BCSFB), tissue defence Rodents Abcb1b mdr1b, mdr1 blood-testis barrier, blood-placenta barrier, etc. MDR2/3, Biliary Humans ABCB4 MDR3, PFIC-3 Hepatocytes phosphatidylcho Rodents Abcb4 mdr2 -line transport

Human P-gp spans 1280 amino acid residues with a molecular weight of approximately

170 kDa. Structurally, it is a full transporter that shows a tandem duplicated structure with two homologous halves (approximately 640 amino acid each) connected by a hydrophilic linker region (Loo & Clarke 1996) (Figure 1-3). Amino acid sequence of the two homologous halves is similar (Loo & Clarke 1996). Each half is composed of an intracellular NBD that constitutes the

ATP binding pocket and a hydrophobic TMD that is characterized by six transmembrane α- helices connected by intracellular and extracellular amino acid loops (Gottesman & Pastan 1993,

Rosenberg et al. 2003). Each NBD contains Walker A and Walker B motifs which are two signature characteristics found in most ABC transporters (Moitra & Dean 2011). The hydrophobic linker region can be phosphorylated by PKC, but it does not appear to affect transport function (Chambers et al. 1993, Higgins et al. 1997). Three glycosylation sites were identified on the first extracellular loop of P-gp which are believed to play a role in proper protein folding and stability in cellular membranes (Gottesman & Pastan 1993). Recently, X-ray crystal structure of the mouse P-gp revealed a nucleotide-free cytosolic-facing conformation that spanned approximately 136 Å perpendicular to and 70 Å in the plane of membrane lipid-bilayer

(Aller et al. 2009). In addition, several novel cyclic peptide inhibitors of P-gp were shown to be sandwiched between drug-binding transmembrane helices six and twelve (Aller et al. 2009).

Electron density map of the protein revealed the presence of a large internal substrate portal facing the cytoplasm and inner membrane leaflet. These findings suggest that substrate can enter the internal drug binding pocket through this portal following permeability across membrane leaflet either from the extracellular or intracellular compartment. Sequentially, hydrolysis of ATP at the NBDs can induce P-gp conformational changes which allow the substrate binding pocket to face the outer membrane leaflet and release substrate into the extracellular space, thus providing an uni-directional efflux mechanism (Aller et al. 2009) (Figure 1-4).

Figure 1-4. Current proposed model of P-gp transport. 1) Substrate (◊) permeates into the membrane lipid leaflet from either the extracellular (OUT) or intracellular (IN) environment and can enter the internal drug binding pocket. 2) ATPs are hydrolyzed at the NBDs. (3) Conformational changes in P-gp TMDs result in an “outward-facing” conformation with the substrate binding pocket facing the outer leaflet (i.e., extracellular space). 4) Substrate can be released into the extracellular space (Aller et al. 2009).

The presence of multiple overlapping drug binding sites on P-gp was initially proposed to provide a mechanistic rationale for the observed interactions (i.e., allosteric, competitive and

non-competitive inhibition) between substrates and inhibitors (Litman et al. 1997). Two binding sites were discovered using affinity binding of fluorescent substrates. The R site was found to interact with Rhodamine123 and the H site was found to bind Hoechst33342 (Shapiro & Ling

1997). Fluorescent substrate binding studies later showed that R site substrate binding can stimulate H site substrate transport (Sharom 2006). It provides further evidence that different overlapping regions are present within the large flexible substrate binding pocket allowing complex allosteric interactions. In addition, drugs are believed to interact with more than one amino acid residues that line the drug binding pocket by an “induced-fit” model using multiple

Van der Waals and hydrophobic interactions (Loo & Clarke 2005). This model explains the observed broad substrate specificity for P-gp. In general, P-gp transports a wide structurally and biochemically unrelated substrates with linear, cyclic, basic, uncharged, zwitterionic, hydrophobic, aromatic or amphipathic structures (Choudhuri & Klaassen 2006). The majority of

P-gp substrates, ranging between 250 to 4000 Da , are amphipathic, hydrophobic and organic cations containing two planar rings (Hodges et al. 2011). Hydrophilicity and the number of hydrogen bonds on the substrate appear to be directly correlated with substrate affinity (Ecker et al. 1999). Since the discovery of P-gp, many xenobiotics (i.e., drugs), β-amyloids, steroid hormones, lipids, environmental toxins and dietary compounds have been demonstrated to interact with the transporter (Hodges et al. 2011). Pharmaceuticals agents that are substrates or inhibitors of P-gp include anticancer, antiretroviral, immunosuppressive, antihypertensive, antiarrhythmic, anti-depressant, antimicrobial, H2-antagonistic and antiepileptic agents as well as steroids, natural products, fluorescent dyes and many others (Choudhuri & Klaassen 2006,

Hodges et al. 2011). A comprehensive list of P-gp substrates and inhibitors can be found on the

US Food and Drug Administration (FDA) website,

ApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/default.htm> (Accessed on

March 2013). In HIV pharmacotherapy, most protease inhibitors (PIs, e.g., darunavir, atazanavir, saquinavir, ritonavir and amprenavir), entry inhibitor (i.e., maraviroc) and integrase inhibitor

(i.e., raltegravir) were demonstrated to be P-gp substrates, as summarized in our recent review

(Kis et al. 2010a). Our laboratory also demonstrated that PIs, such as indinavir, ritonavir and saquinavir, can serve as P-gp inhibitors and can inhibit digoxin transport in the RBE4 rat brain endothelial cell line, primary cultures of rat astrocytes and the MLS-9 rat microglial cell line

(Bendayan et al. 2002, Ronaldson et al. 2004b, Ronaldson et al. 2004a). Interactions between P- gp and antiretroviral agents are further discussed in section 1.4.

P-gp expression in the body is widely distributed and can fulfill multiple physiological roles (Hodges et al. 2011). Its highly polarized expression in several tissue barriers plays an essential protective role limiting entry of xenobiotics (i.e., drugs), whereas its expression in secretory organs regulates xenobiotics elimination out from the body (Hodges et al. 2011). For instance, it limits drug bioavailability at the brush-border membranes of enterocytes in the intestinal tract (Tran et al. 2002, Wang & Zhong 2003, Fakhoury et al. 2005). At the canalicular membranes of hepatocytes and kidney proximal tubular cells, it enhances drug elimination from the body (Kamath & Morris 1998, Suzuki & Sugiyama 2000, Demeule et al. 2001). In addition,

P-gp expression in the adrenal cortex is involved in hormone transport (Meijer et al. 2003). P-gp expression in lymphocytes (e.g., peripheral blood mononuclear cells, CD8 T-lymphocytes) is believed to prevent entry of drugs and cytokines (Aggarwal et al. 1997, Albermann et al. 2005,

Köck et al. 2007, Giraud et al. 2010). More importantly, it is expressed in all major blood-tissue barriers (i.e., BBB, blood-testis barrier and blood-placenta barrier) restricting entry of toxins and drugs into important organs (Fromm 2004, Behravan & Piquette-Miller 2007, Su et al. 2009,

Waterkotte et al. 2011, Robillard et al. 2012). In the brain, its high expression at the luminal side of brain microvessel endothelial cells, constituting the BBB, is a major mechanism that restricts brain entry of xenobiotics (Miller et al. 2000, Bendayan et al. 2002, Bauer et al. 2004, Bendayan et al. 2006, Bauer et al. 2006, Perloff et al. 2007, Lombardo et al. 2008, Narang et al. 2008,

Dauchy et al. 2008, Zastre et al. 2009, Ott et al. 2009, Chan et al. 2011). At the blood- cerebrospinal fluid barrier (BCSFB), P-gp is found at the apical membrane of choroid plexus epithelium, which faces the cerebrospinal fluid (CSF) compartment (Rao et al. 1999); its pharmacological role at this site remains unclear. Findings from our laboratory and others have demonstrated that P-gp is also localized at the abluminal membrane of brain microvessel endothelial cells, in astrocytes, adjacent pericytes and microglia (Lee et al. 2001a, Schlachetzki

& Pardridge 2003, Ronaldson et al. 2004a, Bendayan et al. 2006, Kubota et al. 2006). In neurons, P-gp expression can be very low or undetectable in normal physiological conditions

(Tishler et al. 1995, Hagenbuch & Meier 2004, Volk et al. 2004). Neuronal P-gp expression seems only to be induced in pathological conditions, such as epilepsy (Roth et al. 2012). At present, the pharmacological role of P-gp at these cellular compartments of the brain parenchyma remains unclear, however it is anticipated that P-gp expression at membranes of different brain compartments, in particular astrocytes, can play a significant role in regulating drug distribution in these compartments and brain extracellular space.

In addition to P-gp plasma membrane localization, the transporter is also expressed in other intracellular compartments. For instance, it is found at the luminal Golgi apparatus membranes, but is rarely localized in the endocytic vesicles and lysosomes (Molinari et al. 1994,

Shapiro et al. 1998, Molinari et al. 2002). In addition, applying immunogold labelling with electron microscopy in rat brain microvessel endothelial cells, microglia and astrocytes, our

group has demonstrated that P-gp is also localized in membrane caveolae, clathrin-coated membrane vesicles, cytoplasmic vesicles, rough endoplasmic reticulum, Golgi apparatus and nuclear envelope (Ronaldson et al. 2004a, Lee et al. 2001c, Bendayan et al. 2002, Bendayan et al. 2006, Babakhanian et al. 2007). Although the role of subcellular localization of P-gp remains unclear, its expression has been proposed to serve as a reservoir for rapid P-gp trafficking to the plasma membrane or a cytosolic protective mechanism by removing cellular xenobiotics into vesicles, thus limiting xenobiotics exposure to the cellular organelles (i.e., nucleus) (Miller et al.

2008, Babakhanian et al. 2007).

There are more than 250 synonymous and non-synonymous single nucleotide polymorphisms (SNPs) associated with human MDR1 gene currently cited in the National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?geneId=5243

&ctg=NT_007933.15&mrna=NM_000927.4&prot=NP_000918.2&orien=reverse and The 1000

Genomes Projects, http://www.1000genomes.org/home (Accessed on March 2013). Among many synonymous SNPs, polymorphism at 3435C>T located in exon 26 has received most attention and was first suggested to result in a reduction of P-gp transport activity in the duodenum

(Hoffmeyer et al. 2000). In addition, 2677 G>T and 1236 C>T are also synonymous SNPs that have been extensively studied and shown to be associated with in vitro changes in P-gp ATPase activity (Fung & Gottesman 2009). However, most associations between MDR1 genotypes and clinical P-gp transport phenotypes have been inconsistent (Hodges et al. 2011). It is possible that the combination of several MDR1 SNPs could produce a more pronounced clinical phenotype

(Fung & Gottesman 2009). At present, there are no reports on homozygous deletion of MDR1 genes or non-functional P-gp mutations observed clinically, and drug dosing adjustment has not yet been recommended for any MDR1 SNPs by the FDA. Examination on nucleotide residue

location associated with several SNPs revealed interesting aspects of P-gp protein structure

(Fung & Gottesman 2009). For instance, although SNPs can occur in any of the 29 exons of

MDR1, most SNPs are found in the intracellular regions of P-gp (Fung & Gottesman 2009). In contrast, SNPs that were identified in the TMD, glycosylation sites, Walker A and several other important structural amino acid loops (i.e., A, D, H and Q) are considerably rare suggesting the importance of these domains and motifs for P-gp structure and function (Fung & Gottesman

2009).

1.2.2 Alteration of P-gp Function at Plasma membranes

P-gp is a key drug efflux transporter at many tissue barriers and cellular compartments, therefore a better understanding of the regulatory pathways which govern P-gp functional expression can offer strategies to improve drug permeation to these sites or enhance protection against xenobiotics that are substrates of this transporter. P-gp transport function can be altered through direct inhibition using selective inhibitors or modulation of post-translational mechanisms. Transcriptional regulation of the MDR1 gene can also alter P-gp protein expression and hence affect the overall P-gp mediated transport activity at the plasma membrane. This section primarily focuses on known information of P-gp inhibitors and post-translational events that can alter P-gp transport activity or localization at the plasma membranes.

Initial goal in the development of selective P-gp inhibitors was to reverse multidrug resistance in P-gp over-expressing drug-resistant tumour cells (Eyal et al. 2009). Later, the use of these inhibitors was intended to improve drug permeability into different organs, such as the brain (Schinkel & Jonker 2003). Discovered or synthetic P-gp inhibitors are commonly classified chronologically into different generations. The first generation of P-gp inhibitors includes, verapamil, cyclosporine A, tamoxifen, quinidine, amiodarone and nifedipine (Schinkel & Jonker

2003, Su & Sinko 2006). Most of these inhibitors are also pharmacological active but the concentrations required to inhibit P-gp being much higher than their therapeutic ones, they present very limited clinical applications (Eyal et al. 2009). For those agents that were tested in clinical trials, no significant beneficial effect was observed (Dalton et al. 1995, Sonneveld et al.

2001). Later, several analogs derived from the first generation of inhibitors were developed to reduce toxicity and improve P-gp inhibitory potency. One of the well-known second generation

P-gp inhibitor, valspodar (PSC833), a derivative of cyclosporine A, was demonstrated to be a more effective P-gp inhibitor during pre-clinical testing (Kusunoki et al. 1998, Smith et al.

1998). Intravenous administration of PSC833 in mice was able to significantly enhance brain accumulation of a P-gp substrate (i.e., paclitaxel) by three- to six-fold (Kemper et al. 2003,

Kemper et al. 2004b). Unfortunately, several phase III trials did not identify significant therapeutic improvement in patients that received chemotherapy with PSC833 compared to patients that did not receive this inhibitor (Friedenberg et al. 2006, Lhommé et al. 2008). Years later, highly potent and selective P-gp inhibitors, such as elacridar (GF120918), zosuquidar (LY335979) and tariquidar (XR9576), were developed and these agents are termed third generation inhibitors (Schinkel & Jonker 2003, Pusztai et al. 2005, Su & Sinko 2006).

Compared to previous generations, these compounds exhibit higher selectivity towards P-gp and a much lower affinity towards other drug transporters (i.e., MRPs) and drug metabolizing enzymes (i.e., CYP3A4) (Hyafil et al. 1993, Dantzig et al. 1996, De Bruin et al. 1999, Newman et al. 2000, Malingré et al. 2001, Mistry et al. 2001, Perego et al. 2001). However, clinical administration of tariquidar with several cancer chemotherapeutic agents (e.g., vinorelbine, anthracycline and taxane) only showed partial or limited clinical improvement or needed to be discontinued due to toxic side effects (Lötsch et al. 2002, Pusztai et al. 2005, Nobili et al. 2006).

In rodents, these third generation inhibitors have been demonstrated to be effective for altering

BBB permeability of several P-gp substrates (Choo et al. 2000, Karssen et al. 2001, Karssen et al. 2002, Brandt et al. 2006, Marchi et al. 2006, Van Vliet et al. 2006, Fox & Bates 2007,

Kuntner et al. 2010). In rodents, elacridar and tariquidar were able to enhance significantly brain accumulation of several HIV PIs which are known P-gp substrates, i.e., amprenavir (10 to 13- fold enhancement), saquinavir (7-fold enhancement) and nelfinavir (2 to 14-fold enhancement)

(Polli et al. 1999, Edwards et al. 2002, Savolainen et al. 2002, Kemper et al. 2004a, Park &

Sinko 2005, Choo et al. 2006). Recent clinical studies utilizing positron emission tomography provided evidence that the uptake of the P-gp substrate, verapamil, into the human brain could be significantly enhanced following administration of a P-gp inhibitor (i.e., tariquidar) (Wagner et al. 2009, Bauer et al. 2010, Bauer et al. 2012). Interestingly, Bauer et al. reported a noticeable species difference in the tariquidar-mediated enhancement of verapamil brain uptake between humans and rats, in which the enhancement observed in rats was much more pronounced compared to that in humans (Bauer et al. 2012). Therefore, careful consideration regarding species differences must be taken into account when interpreting results from rodent studies. To date, none of the P-gp inhibitors has been approved by the FDA or European regulatory agencies for P-gp inhibition in the clinic.

Aside from the use of P-gp inhibitors, P-gp transport activity at the plasma membrane can also be altered by intracellular signalling pathways that mediate rapid and reversible effects within minutes (Miller et al. 2008). Currently, intracellular signalling pathways that are able to alter P-gp transport without modulating its protein expression include the vascular endothelial growth factor (VEGF) pathway, caveolin-1 pathway, endothelin-1 (ET-1) pathway, vesicular internalization and ubiquitination (Miller et al. 2008, Miller 2010, Potschka 2010, Fu & Arias

2012). Treatment of isolated rat brain capillaries with VEGF rapidly and reversibly reduced P-gp transport activity, leading to an enhanced brain permeability of P-gp substrates, i.e., morphine and verapamil (Hawkins et al. 2010). Interestingly, previous studies demonstrated that VEGF pathway was also associated with caveolin-1 phosphorylation (Barakat et al. 2007). In fact, caveolin-1, a 22 kDa transmembrane protein, was found to be co-localized with P-gp in rat and primate brain microvessel endothelial cells and primary cultures of rat astrocytes (Schlachetzki &

Pardridge 2003, Ronaldson et al. 2004a, McCaffrey et al. 2012). Membrane microdomains containing caveolin-1 were found to interact with the N-terminus of P-gp through a caveolin- binding motif and the phosphorylation of caveolin-1 led to a decrease in P-gp transport activity at the plasma membrane of human and rat brain microvessel endothelial cells (Jodoin et al. 2003,

Barakat et al. 2008). Therefore, it is believed that phosphorylation of caveolin-1 mediated by

VEGF can decrease P-gp transport activity at the plasma membranes through protein-protein interactions. Another distinct pathway that can alter P-gp transport at the plasma membrane involves ET-1, a 21 amino acid vasoconstrictor polypeptide hormone (Masereeuw et al. 2000,

Miller et al. 2002). ET-1 has been demonstrated to alter P-gp transport in fish renal proximal tubules and brain capillaries through its binding to a G-protein coupled receptor endothelin B

(ETB) (Masereeuw et al. 2000, Miller et al. 2002). Hartz et al. showed that treatment of rat brain microvessel endothelial cells with ET-1 could rapidly reduce P-gp transport function at the luminal membrane with no effect on intracellular transporter expression (Hartz et al. 2004). As well, these authors and others demonstrated that inducible nitric oxide synthase (iNOS) and PKC are downstream signalling pathways of ET-1 stimulation in isolated rat brain microvessels and brain microvessel endothelial cells of humans and rats (Hartz et al. 2004, Hembury & Mabondzo

2008, Harati et al. 2012). Moreover, PKC has been proposed to modify P-gp function through

direct phosphorylation of the transporter or other accessory proteins (i.e., caveolin-1), hence altering P-gp trafficking between the plasma membrane and vesicular compartment or possibly affecting substrate affinity (Hartz et al. 2004). The overall P-gp transport activity at the plasma membrane can also be altered through intracellular protein trafficking between vesicular compartments and the plasma membranes (Fu & Arias 2012). At the bile canalicular membrane of human hepatocytes, P-gp internalization from the plasma membrane to intracellular vesicle pools was demonstrated to involve microtubules and Phosphoinositide 3 (PI3) kinase in response to cyclic adenosine monophosphate (cAMP) (Sai et al. 1999, Kipp & Arias 2002, Fu & Arias

2012). It is expected that similar mechanisms are also present in brain microvessel endothelial cells and other cellular compartments of the brain parenchyma, thus providing a rapid mechanism which regulates P-gp localization at plasma membranes. Similarly, protein ubiquitination processes can also remove P-gp from the plasma membrane. During ubiquitination, a short (76 amino acids) protein sequence, ubiquitin, is first attached to target proteins and the ubiquitin-associated P-gp complex is relocated to proteasome for degradation, thus reducing P-gp localization at plasma membranes and in turn, its overall cellular transport activity (Ciechanover 1998, Zhang et al. 2004, Nawa et al. 2012, Fu & Arias 2012).

1.2.3 Regulation of P-gp mRNA and Protein Expression

Transcriptional regulation of MDR1 gene can alter intracellular mRNA expression, and as a result modulate P-gp availability at the plasma membrane, leading to a change in the overall transport activity at the cell surface (Figure 1-5). This regulation is generally recognized to take place over hours or days and involves binding of several transcription factors to the MDR1 gene promoter regions (Scotto 2003). In general, gene transcription is initiated by binding of RNA polymerase II and other transcription factors to the TATA box (Scotto 2003). Interestingly, the

human MDR1 promoter region lacks such element, however gene transcription can be initiated by several other elements, such as the transcription initiator site, GC-box, a Y-box (inverted

CCAAT element), a p53 element, an inverse multiple-start-site Element Downstream (MED)1 element, CCAAT/Enhancer-binding Protein (C/EBP) element, heat shock element, activator protein (AP) 1, locating upstream of the transcription initiation site (Scotto 2003, Labialle et al.

2002, Labialle et al. 2004). Currently, three ligand-activated orphan nuclear receptors: pregnane

X receptor (PXR), constitutive androstane receptor (CAR) and vitamin D receptor (VDR), and a ligand-activated transcriptional factor, Aryl hydrocarbon receptor (AhR), have been identified to bind to the MDR1 gene promoter region (Geick et al. 2001, Burk et al. 2005, Saeki et al. 2008,

Mathieu et al. 2001). In particular, it was demonstrated that the three nuclear receptors and their common heterodimerizing partner, retinoid X receptor α (RXRα), regulate MDR1 gene expression through binding to the DR4 motifs (i.e., AG(G/T)TCA direct repeat sequence with a spacer of four nucleotides in between) located at approximately -7800 Kb from transcription start site of MDR1 (Geick et al. 2001, Burk et al. 2005, Saeki et al. 2008). This region is commonly referred to as the distal xenobiotic responsive enhancer module (XREM) (Timsit & Negishi

2007). Modulation (i.e., inhibition or activation) of nuclear receptors transcription activity by their ligands can alter MDR1 mRNA expression and affect protein expression, thus indirectly altering the overall transport activity at the plasma membrane. The molecular mechanisms for

PXR and CAR which regulate MDR1 gene expression and their role in governing drug transporters expression in the brain, are further discussed in section 1.3.

Figure 1-5. Transcriptional regulation of P-gp expression. A change in the overall P-gp transport activity at the cell surface can be achieved by indirect alterations of P-gp availability at the plasma membranes. One mechanism to alter P-gp cellular availability is through the regulation of intracellular MDR1 mRNA expression, which is governed by transcriptional events at the MDR1 gene. Endothelin-1 (ET-1), inducible nitric oxide synthase (iNOS), protein kinase C

(PKC), chemokine receptor 5 (CCR5), Ras homolog gene family member A (RhoA), Ras-related C3 botulinum toxin substrate (Rac), mix lineage kinase (MLK), N-Methyl-D-aspartate (NMDA), cyclooxygenase-2 (COX-2), nuclear factor Kappa B (NF-κB), c-Jun N-terminal kinase (JNK) and activator protein-1 (AP-1). Adapted from Miller et al. 2010.

Aside from ligand-activated nuclear receptors, P-gp expression can be altered through regulatory pathways related to inflammatory and oxidative stress responses (Bertilsson et al.

2001, Hartmann et al. 2002, Goralski et al. 2003, Petrovic et al. 2007). In particular, chronic inflammation and oxidative stress events observed during HIV infection are known to alter P-gp expression in the brain (Persidsky et al. 2006). Namely using immunohistochemistry, P-gp expression was reported to be increased clinically in glial cells from HIV encephalitis brain autopsy tissues (Langford et al. 2004). In contrast, the transporter expression was reported to be decreased in microvessels from brain autopsy tissues isolated from HIV-infected patients with or without encephalitis (Persidsky et al. 2000, Langford et al. 2004, Persidsky & Poluektova 2006).

In order to understand the mechanisms involved in P-gp regulation in the brain during HIV infection, many groups including ours have examined the effect of pro-inflammatory cytokines

[i.e., tumour necrosis factor-α (TNFα), interleukin(IL)-1β, IL-6] and HIV viral envelope proteins

(i.e., gp120 and tat) on P-gp expression in the brain, as summarized in a published review from our group (Ronaldson et al. 2008). Our laboratory has previously demonstrated that gp120 and

IL-6 significantly decreased P-gp expression in primary cultures of rat astrocytes, whereas TNFα and IL-1β modestly increased its expression (Ronaldson & Bendayan 2006). As well, a transcription factor, NF-κB, was shown to be associated with P-gp downregulation following gp120 binding to chemokine receptor five (CCR5) in primary cultures of human fetal astrocytes

(Ashraf et al. 2011). NF-κB was in fact demonstrated earlier by our group to also play a role in

MRP1 regulation in primary cultures of rat astrocytes (Ronaldson et al. 2010). It was proposed that NF-κB signalling can lead to increased production and secretion of pro-inflammatory cytokines, such as TNFα. Once secreted, TNFα may bind to TNFα surface receptor and lead to the activation of c-Jun N-terminal kinase (JNK) isoforms, a component of the mitogen-activated protein kinase pathway, which ultimately induces MRP1 expression (Ronaldson et al. 2010). The

JNK pathways are also known to upregulate P-gp expression in hepatic tumour cell lines (Li et al. 2006, Zhou et al. 2006, Yan et al. 2010). At the rodent BBB, TNFα release following endothelial exposure to diesel exhaust particles is also believed to act through c-jun and JNK pathways, leading to P-gp protein induction (Hartz et al. 2008). These pathways are thought to signal via activator protein 1 (AP-1), a transcription factor known to induce MDR1 gene transcription (Yagüe et al. 2003, Hartz et al. 2008). NF-κB has also been suggested to directly alter P-gp expression. At the BBB, TNFα binding to its surface receptor in mouse brain microvessel endothelial cells can induce NF-κB nuclear translocation which is followed by the

direct binding of this transcription factor to mdr1a/b promoter regions (Yu et al. 2008).

Similarly, ET-1 binding to its surface receptors on brain microvessel endothelial cells can activate iNOS and PKC, which can also lead to enhanced NF-κB nuclear translocation and result in sequential P-gp induction (Didier et al. 2002, Hartz et al. 2006, Bauer et al. 2007, Chauhan et al. 2007). HIV viral protein tat exposure is also closely related to NF-κB regulation of P-gp expression in the brain. Following Tat binding to lipid rafts (i.e., glycolipoprotein microdomains on plasma membrane), several upstream regulators of NF-κB, such as Ras homolog gene family member A (RhoA), Ras-related C3 botulinum toxin substrate (Rac) (i.e., signalling G proteins) and myosin light chain kinase (MLK), have been demonstrated to be involved in mdr1 gene transcription in mouse brain microvessel endothelial cells and astrocytes (Nath et al. 1999,

Hayashi et al. 2005, Hayashi et al. 2006). Taken together, these findings suggest that c- jun/JNK/AP-1, RhoA/Rac/MLK/NF-κB and iNOS/PKC/NF-κB pathways, triggered by TNFα or viral proteins (i.e., gp120 and tat) exposure, can regulate MDR1 gene transcription and sequential protein expression at the BBB or astrocytes. Furthermore, a distinct pathway that involves neurotransmitter glutamate and N-Methyl-D-aspartate (NMDA) receptor activation is also known to induce P-gp expression in brain through NF-κB pathway (Miller et al. 2008). Following

NMDA receptor activation, prostaglandin E2 is believed to activate cyclooxygenase-2 (COX-2) which is known to be an upstream regulator of NF-κB (Bauer et al. 2008b, Zibell et al. 2009).

Furthermore, hydrogen peroxide and intracellular reactive oxygen species (ROS) have also been shown to induce P-gp expression in human and rat brain microvessel endothelial cells via the glycogen synthase kinase-3 and wnt/β catenin pathways (Felix & Barrand 2002, Nwaozuzu et al.

2003, Lim et al. 2008, Lim et al. 2009). In summary, regulation of P-gp expression in brain microvessel endothelial cells is highly complex and can involve a mosaic network of molecular

pathways such as, nuclear receptors, intracellular signalling molecules and other transcription factors (Figure 1-5). Understanding the role of these pathways in regulating P-gp expression at the BBB during disease states (i.e., HIV infection, inflammatory diseases and epilepsy) can guide the identification of novel therapeutic targets to either enhance P-gp substrates permeability across the BBB or prevent neurotoxicity of drugs acting in the periphery.

1.3 Nuclear Receptors

Nuclear receptor genes can be found in all animals, but are absent in fungi and plants

(Sladek 2011). These genes first appeared approximately 635 million years ago when simple and unicellular organisms diversified into more complex multicellular organisms, predating the appearances of endocrine systems (Love et al. 2009). Nuclear receptors are recognized as members of a DNA-binding transcriptional factors superfamily that regulate gene transcription in response to endogenous and exogenous chemicals (Moore et al. 2006b, Sladek 2011). They play a role in every aspect of developmental processes, cellular differentiation, metabolic homeostasis and protective mechanisms against harmful chemicals (Moore et al. 2006b, Benoit et al. 2006,

Sladek 2011). The number of amino acid residues for each nuclear receptors can vary considerably, however all nuclear receptors consist of an assembly of functional domains, as summarized in Figure 1-6A (Brélivet et al. 2012). The DNA-binding domain (DBD), the most highly conserved region among nuclear receptors, consists of two zinc-binding motifs and three helices that allow docking of the receptor to DNA strands (Miller et al. 1985, Helsen et al. 2012).

This domain in some receptors can form a dimer with DBD from an identical or different receptor, and binds selectively to DNA response elements which usually contain two short sequences of six nucleotides, as depicted in Figure 1-6B (Tsai et al. 1988, Klein-Hitpass et al.

1989, Helsen et al. 2012). The dimer complex produces different DNA binding affinities to

response elements, such as direct repeats x (DRx), everted repeats x (ERx) and inverted repeats x

(IRx), where x can be any number of nucleotide residues (0 – 10) between the two sets of six- nucleotide sequences (Cairns et al. 1991, Lee et al. 1993, Helsen et al. 2012) (Figure 1-6C). In contrast, some nuclear receptors (e.g., human estrogen related receptors) can bind to the response elements as a monomer (Helsen et al. 2012). The C-terminus ligand-binding domain (LBD), which is connected to the DBD by a hinge region, allows nuclear receptors to recognize small molecule ligands, such as drugs, and therefore it is considered the most important structural feature in drug discovery that uses nuclear receptors as a therapeutic target (Whitfield et al. 1999,

Helsen et al. 2012). The LBD is typically described as three stacked α-helical sheets creating an internal cavity with hydrophobic residues in the inner lining which can interact with ligands

(Watkins et al. 2001, Watkins et al. 2002, Helsen et al. 2012). An activation function-2 (AF-2) domain which is essential for receptor ligand activation was also identified within the LBD

(Benoit et al. 2004, Hilser & Thompson 2011). The N-terminal activation function 1 (AF-1) domain is also important for ligand-independent activation of nuclear receptors through phosphorylation or accessory protein interactions (Germain et al. 2006b, Hilser & Thompson

2011). Upon ligand activation, nuclear receptors typically bind selectively to their specific DNA response elements and regulate gene transcription (Rosenfeld et al. 2006, George et al. 2011).

The interactions of nuclear receptors to different accessory proteins, such as heterodimerizing partners, co-activators, co-repressors and transcriptional factors are essential in the regulation of transcription at the chromosome site (Whitfield et al. 1999, Khorasanizadeh & Rastinejad 2001,

Wang & LeCluyse 2003, Benoit et al. 2004, Hilser & Thompson 2011). Taken together, functional receptor domains and protein-protein interactions allow nuclear receptors to relay or

transduce many different endogenous and exogenous chemical stimuli into gene transcription, ultimately altering cellular responses.

Figure 1-6. Functional domains and DNA binding of nuclear receptors. A) Functional domains of nuclear receptors include the amino terminus, activation function-1 (AF-1), DNA- binding domain (DBD), hinge region, ligand-binding domain (LBD), activation function-2 (AF-

2) and the carboxyl-terminus. B) Nuclear receptors can bind to the DNA response element as: i) monomers [e.g., retinoic acid-related orphan receptor (ROR), vertebrate homologue of Drosophila tailless gene (TLX)], ii) heterodimers with RXR [e.g., pregnane X receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator activated receptor (PPAR), liver x receptor (LXR), farnesoid x receptor (FXR), vitamin D receptor (VDR) and retinoic acid receptor (RAR)] or iii) homodimers [e.g., glucocorticoid receptor (GR) and hepatocyte nuclear factor 4 (HNF4)]. C) Most nuclear receptors can bind to the promoter region of target genes at the DNA response elements: direct repeat, inverted repeat or everted repeat of 6 base pairs separated by a fixed number (x) of residues (DRx, IRx and ERx), where x can be any number of nucleotide residues (0 – 10) between the two sets of six-nucleotide sequence. Adapted from Wang et al. 2003.

Forty-eight known nuclear receptors of the human genome have been classified into six evolutionary groups (1-6) according to nuclear receptor amino acid alignment and phylogenetic tree construction (Germain et al. 2006b). Conventionally, these nuclear receptors are often divided into three classes based on their ligand-binding specificities: i) steroid and endocrine receptors; ii) true orphan receptors and iii) adopted orphan receptors (Germain et al. 2006b, Li &

Wang 2010). The steroid and endocrine receptors, such as glucocorticoid receptor (GR; NR3C1) and estrogen receptors (ERs; NR3A1 & NR3A2) are well documented mediators of cellular activities associated with their steroid hormones (Germain et al. 2006b). By contrast, orphan receptors are those identified through molecular sequencing without prior knowledge of their endogenous ligands or functions. Within this category, true orphan receptors are those whose endogenous ligands have yet to be found or may not exist (Germain et al. 2006b). Adopted orphan nuclear receptors, such as PXR (NR1I2) and CAR (NR1I3), are receptors that were cloned without prior knowledge of their endogenous ligand profiles or function but have recently been found to be high capacity and low affinity xenobiotic sensors (Benoit et al. 2006, Li & Wang

2010). At present, several adopted orphan nuclear receptors, including PXR, CAR and peroxisome proliferator-activated receptors (PPARs; NR1C1, NR1C2 & NR1C3) as well as a ligand-activated transcriptional factor AhR have been identified as key regulators of gene transcription of phase I (i.e., CYP450 enzymes) and phase II drug metabolizing enzymes [i.e.,

GSTs, SULTs and UDP-Glucuronosyltransferases (UGTs)] and efflux/influx drug transporters from the ABC and SLC superfamilies (Urquhart et al. 2007, Li & Wang 2010). Both natural and synthetic compounds, including many current therapeutic agents, can alter receptor activity.

Ligand-mediated activation of these receptors has been demonstrated to alter expression of these enzymes and transporters, which can sequentially affect detoxicification processes, as well as

drug pharmacokinetic properties (Urquhart et al. 2007, Li & Wang 2010). Current evidence suggests that some of these receptors, such as PXR, PPARα, ERs, vitamin D receptor, liver X receptor, are also present at the BBB in addition to their expression in the peripheral organs.

Moreover, these receptors can regulate the expression of drug transporters, such as P-gp, BCRP and MRP1, at the BBB, as discussed in our recent published review included in the appendix E

(Chan et al. 2013a). Therefore, these receptors could be drug-targeted targets to regulate functional expression of drug transporters at the BBB in the clinic, altering drug distribution into the brain.

1.3.1 PXR

1.3.1.1 Expression, Structure and Function

Mouse NR1I2 (PXR) gene was first identified from screening of a genomic mouse liver library and was named “pregnane” because it was shown to be activated by derivatives of pregnenolone (Kliewer et al. 1998). At the same time, a human steroid X receptor (SXR) was cloned and later established to be the human ortholog of the mouse gene (Blumberg et al. 1998,

Bertilsson et al. 1998). In our work, SXR will be termed as human PXR (hPXR). Human NR1I2 gene produces three PXR protein isoforms. The transcript originates from exon 1 results in the synthesis of hPXR isoform 1. This isoform is predominantly expressed in the liver and gastrointestinal tract, but can also be found in peripheral cellular compartments, such as peripheral blood mononuclear cells, tissues and organs, such as, adrenal gland, breast, bone marrow, heart, liver, intestine (Table 1-3) and brain (i.e., brain cortex and thalamus (Table 1-4)).

HPXR Isoform 2 is originated from exon 2 with an extension of 39 amino acid residues at the N terminus compared to isoform 1, whereas isoform 3 is originated from an in-frame deletion of

111 base pairs in exon 5, which results in a 37 residue shorter LBD (Bertilsson et al. 1998,

Dotzlaw et al. 1999). Relative expression of these three hPXR transcripts was reported in 73 human liver samples, in which isoform 1 accounts for more than 93 ± 3.7 % of total transcript while isoforms 2 and 3 account for only 6.7 ± 3.5 % and 0.3 ± 0.5 %, respectively (Lamba et al.

2004b). Approximately 28 hPXR polymorphic variants, such as SNPs, have been identified near the LBD and N terminus regions and are believed to affect ligand-dependent transcriptional activity of hPXR (Hustert et al. 2001). The most frequent SNP is the Pro27Ser found in 14.9 % of African population, whereas other variants have an allelic frequency below 3 % (Hustert et al.

2001). The relative few number of SNPs present in the hPXR gene suggests that the conserved hPXR sequence among individuals is important for receptor function (Stanley et al. 2006).

In the human brain, PXR transcript expression has been reported in specific regions of the human brain such as thalamus, pons and medulla (Nishimura et al. 2004, Miki et al. 2005). Its protein expression has also been clearly demonstrated in hCMEC/D3 cell culture system, primary cultures of human brain microvessel endothelial cells (Chan et al. 2011) and human fetal brain tissue (Chan et al. 2010). However, its transcript or protein expression has not been demonstrated in isolated human brain microvessels and brain cortical tissues (Shawahna et al. 2011, Dauchy et al. 2008). At present, the function of hPXR at the BBB and cellular compartments of the brain parenchyma in the clinic remains unclear. In vivo transcript and protein expression of rodent and porcine PXR have also been reported in studies using isolated brain capillaries or in vitro brain microvessel endothelial cell culture systems (Bauer et al. 2006, Bauer et al. 2004, Ott et al. 2009,

Narang et al. 2008).

Table 1-3. Peripheral tissue distribution of PXR and CAR.

pecies Bone Lung Liver Heart Testis Spleen S Kidney Placenta Stomach Pancreas References Breast Tissues Breast Ovary & Uterus Ovary Peripheral Blood Intestine & Colon & Intestine Mononuclear Cells

PXR (Zhang et al. 1999) R + + + + + - - - (Kawana et al. 2003) M + (Masuyama et al. 2001) M + + - - - - + (Miki et al. 2005) H + + + + + + + - + (Petrick & Klaassen M + + + + + + + + + 2007) (Khan et al. 2009) H, R + +

(Schote et al. 2007,

H + Siest et al. 2008) (Nishimura et al. 2004) H + + + + + + + + + + + + (Lamba et al. 2004b) H + + - + - + - + - - - (Ott et al. 2009) P + + (Meyer Zu

Schwabedissen et al. H + 2008) CAR (Petrick & Klaassen M + + + + + + + + + 2007) (Choi et al. 1997, Wei M + et al. 2000) (Baes et al. 1994) M + (Nishimura et al. 2004) H + + + + + + - - + + + + (Savkur et al. 2003) H + + + + + + + + + + + + (Lamba et al. 2004a) H + + + - - + + + - - + H: Humans, R: Rats, M: Mouse and P: Porcine. Light grey: only transcript level was reported. Black: Protein expression was reported. No colour/symbol: not determined in the study. +: expression was detected. - : expression could not be confirmed.

Table 1-4. Brain distribution of PXR and CAR.

pillaries Cells Species Thalamus ain C References Endothelial Microvessel Microvessel Spinal Cord Astroglioma Brain Cortex Br Caudate Nucleus

PXR (Zhang et al. 1999) R - (Masuyama et al. 2001) M - (Miki et al. 2005) H + (Petrick & Klaassen 2007) M + (Trousson et al. 2009) M +

(Nishimura et al. 2004) H +

(Lamba et al. 2004b) H - + +

(Bauer et al. 2004, Bauer et al. 2006, M, + Ott et al. 2009) R

(Narang et al. 2008) R + +

(Ott et al. 2009) P + +

(Dauchy et al. 2008) H - -

(Dauchy et al. 2009) H +

(Shawahna et al. 2011) H - CAR

(Dauchy et al. 2008) H + +

(Dauchy et al. 2009) H -

(Shawahna et al. 2011) H - (Petrick & Klaassen 2007) M +

(Malaplate-Armand et al. 2005) H + (Nishimura et al. 2004) H + (Savkur et al. 2003) H +

(Lamba et al. 2004a) H - - - +

H: Humans, R: Rats, M: Mouse and P: Porcine. Light grey: only transcript level was reported. Black: Protein expression was reported. No colour/symbol: not determined in the study. +: expression was detected. - : expression could not be confirmed.

The PXR DBD among different species is highly similar and the high (96 %) DNA sequence homology between hPXR and rodent PXR suggests that the two orthologs have similar

DNA-binding properties (Wang & LeCluyse 2003, Stanley et al. 2006). In fact, rodent and human PXR share many identical target genes (e.g., MDR1/mdr1a/b, CYPs/Cyps) and are known to form heterodimers with RXRα during binding to gene regulatory regions (Wang & LeCluyse

2003, Stanley et al. 2006). The preferred DNA sequence for PXR/RXRα binding include phenobarbital responsive enhancer module (PBREM), DR3, DR4, ER6 ER8 and IR0 motifs, which contain two AG(G/T)TCA half sites (Lehmann et al. 1998, Geick et al. 2001, Burk et al.

2004, Song et al. 2004, Nakata et al. 2006, Cui et al. 2010). In contrast, PXR exhibits a striking difference in LBD amino acid sequence among different species with only 76 to 83 % DNA sequence homology (Timsit & Negishi 2007). For instance, LBD of rodents and humans only share 76 % sequence homology (Wang & LeCluyse 2003). As a result, ligand activation profile between hPXR and rodents PXR is significantly different (Moore et al. 2002, Chang & Waxman

2006). For example, rifampin is a potent hPXR agonist, but a weak agonist for rodent PXR; dexamethasone is a potent rodent PXR agonist, but a weak agonist for hPXR (Moore et al. 2002,

Chang & Waxman 2006). Recently, the crystal structures of the hPXR LBD reveal a sandwich of three α helices and five β sheets forming an extensive hydrophobic ligand binding cavity lining with several polar amino acid residues (Watkins et al. 2001, Watkins et al. 2002). The presence of an unique flexible loop structure from residue 309 to 321 allows the receptor to accommodate many structurally unrelated ligands by inducing changes in the ligand binding pocket size

(Watkins et al. 2001, Watkins et al. 2002). Therefore, hPXR ligand binding volume can range from 1200 Å3 in the absence of ligand to 1600 Å3 in the presence of rifampin, which is substantially larger than all the other nuclear receptors, thus demonstrating PXR’s ligand binding

promiscuity (Watkins et al. 2001, Chrencik et al. 2005). Currently, most hPXR ligands exhibit micromolar EC50 values compared with the nanomolar range seen with ligands of steroid nuclear receptors (di Masi et al. 2009). To date, the growing list of hPXR ligands includes a remarkably diverse array of chemicals with molecular weights ranging from 250 Da to more than 800 Da, such as prescription drugs (e.g., rifampin, ritonavir, paclitaxel and taxol), pesticides (e.g., chlordane and transnonachlor), environmental toxins (e.g., phenols), natural herbal compounds

(e.g., colupulone and hyperforin), endogenous steroids (e.g., progesterone, 5α-pregnane-3,20- dione) and bile acids (i.e., cholic acid) (Moore et al. 2002, Chang & Waxman 2006, Lemaire et al. 2007, di Masi et al. 2009). Single site mutagenesis studies at the hPXR LBD suggest that alterations at several amino acid residues (e.g., Met243, Gln285 and His407) within the ligand binding pocket can alter ligand binding properties (Moore et al. 2002). Conversely, single site mutagenesis studies at the AF-2 region of LBD (e.g., Ser247, Trp299Ala and Asp205Ala) can alter interactions with co-repressors or co-activators and receptor transcriptional activity (Orans et al. 2005).

PXR plays a role in the regulation of target genes transcription, meanwhile its expression and activity are in turn regulated by other protein interactions and signalling pathways (Stanley et al. 2006, di Masi et al. 2009). PXR gene expression can be regulated by GR and HNF4α implying that their activation can induce PXR expression and enhance its ligand-dependent inductive response (Pascussi et al. 2001, Kamiya et al. 2003, Pascussi et al. 2000, Gibson et al.

2006). In particular, Tirona et al. highlighted the critical involvement of HNF4α in PXR binding to the promoter region of CYP3A4 (Tirona et al. 2003). Later it was confirmed that HNF4α can compete with co-repressor [i.e., short heterodimer partner (SHP)], which enhances recruitment of co-activator [i.e., steroid receptor coactivator 1 (SRC-1)] to hPXR (Bhalla et al. 2004, Li &

Chiang 2006). Inside the nucleus, PXR transcription activities at the promoter regions of target genes are regulated by interaction with its heterodimerizing partner RXRα (Mangelsdorf et al.

1991, Mader et al. 1993, Germain et al. 2006a). Aside from PXR ligand activation of

RXRα/PXR heterodimer, the complex can also be activated by RXRα agonists. Interestingly, the presence of both a RXR agonist and a PXR agonist can produce a synergic transcription activity

(Westin et al. 1998). This phenomenon is also observed with other RXR heterodimerizing partner, such as PPARs, LXRs, FXR, VDR, PXR and CAR (Westin et al. 1998). In addition to the regulation mediated by RXRα, PXR transcriptional activity can be regulated by co-activator and co-repressor accessory proteins (Moore et al. 2006a, Pascussi et al. 2007). It has been suggested that co-repressor proteins, such as SHP, silencing mediator of retinoid and thyroid hormone receptors (SMRT) and nuclear receptor co-repressor (NcoR), can bind to unliganded

PXR and stabilize chromatin, thus suppressing gene transcription (Synold et al. 2001, Takeshita et al. 2002, Ourlin et al. 2003). PXR ligand activation results in co-repressor proteins dissociation from PXR which in turn allows recruitment of several major co-activator proteins, such as SRC-1 and glucocorticoid receptor-interacting protein 1 (GRIP1), to loosen chromatin structure thus allowing transcription processes to occur (Kliewer et al. 1998, Huss & Kasper

2000, Ourlin et al. 2003, Sugatani et al. 2005, Johnson et al. 2006, Harmsen et al. 2007,

Hariparsad et al. 2009). In the cytoplasm, post-translational regulation involving PXR phosphorylation and de-phosphorylation can also play an important role in modulating receptor activity (di Masi et al. 2009). PXR phosphorylation mediated by protein kinase A has been shown to enhance co-activators, such as SRC-1, binding to PXR and increased receptor transcription activity (Ding & Staudinger 2005a). In contrast, PKC alters phosphorylation status of PXR which can lead to decreased transcription activity (Ding & Staudinger 2005b). As well,

inhibitors of protein phosphatases 1A and 2A, such as okadaic acid, which significantly suppresses PXR transcription activity (Ding & Staudinger 2005b). Furthermore, PXR nuclear translocation is a crucial process upstream of transcriptional events, thus regulatory pathways on receptor nuclear translocation can indirectly govern PXR activity in the nucleus. Cytoplasmic retention proteins, such as cytoplasmic constitutive androstane receptor retention protein (CCRP) and heat shock protein 90 (HSP90), are believed to be part of this regulatory pathway to control nuclear translocation of unliganded cytosolic mouse PXR (Squires et al. 2004). PXR ligand binding in the cytoplasm could lead to PXR dissociation from these retention proteins, which allows the nuclear localization signal region in PXR DBD to promote receptor translocation into the nucleus (Kawana et al. 2003).

1.3.1.2 Regulation of Target Genes

PXR is currently accepted as a master xenobiotic sensor that plays an important role in governing the expression of phase I/II drug-metabolizing enzymes (Table 1-5) and drug transporters (Table 1-6) that are known to affect biotransformation and distribution of endogenous and exogenous compounds (Stanley et al. 2006, Urquhart et al. 2007, di Masi et al.

2009, Li & Wang 2010, Klaassen & Aleksunes 2010, Chan et al. 2013a). Phase I metabolism can involve oxidation, reduction and hydrolysis of the parent compound resulting in addition of hydrophilic functional groups, such as –OH,-NH2, -COOH or -SH on the parent compound. PXR target genes that participate in phase I metabolism including CYP1A2, CYP2B6, CYP2C9,

CYP2C19, CYP3A4, CYP3A7 and CYP7A1 in humans and Cyp2b10, Cyp3a11, Cyp3a13 and

Cyp3a23 in rodents (Urquhart et al. 2007, di Masi et al. 2009, Klaassen & Aleksunes 2010).

Phase II metabolism involves primarily conjugation of endogenous substrates, such as glutathione, sulphates and glucuronic acid. PXR target genes that participate in phase II

metabolism including SULT2A1, and UGT1A1, UGT1A3 and UGT1A4 in humans and Sult2a1,

Ugt1a1, GST alpha (Gsta) 1, Gsta2, Gsta4, GST Mu (Gstm) 1 and Gstm2, Aldehyde dehydrogenase (Aldh)1a1 and Aldh1a7 in rodents (Urquhart et al. 2007, di Masi et al. 2009,

Klaassen & Aleksunes 2010). Members belonging to the ABC and SLC transport families that are regulated by PXR include P-gp, MRP2, BCRP, organic anion transporting polypeptide

(OATP)1A2, bile salt export pump (BSEP), organic anion transporter 2 (OAT2), organic cation transporter 1 (OCT1) and Na+ taurocholate cotransporting polypeptide (NTCP) in humans and P- gp, Mrp2, Mrp3 and Oatp1a4 in rodents (Urquhart et al. 2007, di Masi et al. 2009, Klaassen &

Aleksunes 2010).

Table 1-5. Human and rodent PXR regulation of phase I/II enzymes.

Organs/Tissues Target Genes Reference(s)

Aldh 1a1, Aldh1a7 (Alnouti & Klaassen 2008) CYP1A2 (Maglich et al. 2002) CYP2B6 (Maglich et al. 2002, Olinga et al. 2008) Hepatocytes (Goodwin et al. 2001, Wang et al. CYP2C9, CYP2C19 2003a) CYP3A4, CYP3A5, (Gerbal-Chaloin et al. 2001, Chen et al. CYP3A7 2004, Olinga et al. 2008) (Huss & Kasper 2000, Wei et al. 2002a,

Phase I Cyp7A1 Anakk et al. 2003, Rosenfeld et al. Liver 2003) Cyp2b10 (Goodwin et al. 1999, Wagner et al. 2005) Cyp3a11 (Maglich et al. 2002, Rosenfeld et al. Intestine and liver Cyp3a13 2003, Wagner et al. 2005, Aleksunes & Cyp3a23 Klaassen 2012)

HepG2 and Caco-2 UGT1A1, UGT1A3, (Sonoda et al. 2002) cell lines UGT1A4 Sult2A1 (Staudinger et al. 2001) (Gardner-Stephen et al. 2004, Sugatani Ugt1a1 et al. 2005)

Phase II Phase Liver GSTA1, GSTA2, GSTA4 (Maglich et al. 2002) (Falkner et al. 2001, Maglich et al. GSTM1, GSTM2 2002, Rosenfeld et al. 2003) Adapted from Urquhart et al. 2007, Di Masi et al. 2009, Klaassen and Aleksunes 2010 and Chan et al. 2013a. Only human genes are presented with capital letters.

Table 1-6. Human and rodent PXR regulation of transporters.

Organs/Tissues Target Genes Reference(s) (Geick et al. 2001, Bauer et al. 2004, Liver, small intestine, Albermann et al. 2005, Bauer et al. 2006, Perloff et al. 2007, Bauer et al. PBMCs, brain MDR1/Mdr1a/b (P-gp) 2008a, Lombardo et al. 2008, Narang et microvessels and al. 2008, Olinga et al. 2008, Ott et al. endothelial cells 2009, Richert et al. 2009, Chan et al. 2011, Chan et al. 2013c) PBMCs ABC1 (MRP1) (Albermann et al. 2005) Brain microvessels, (Albermann et al. 2005, Fardel et al. ABCC2/Abcc2 PBMCs and small 2005, Klaassen & Slitt 2005, Bauer et (MRP2/Mrp2) intestine al. 2008a, Olinga et al. 2008) (Teng et al. 2003, Wagner et al. 2005, Liver Abcc3 (Mrp3) Aleksunes & Klaassen 2012) PBMCs and small (Albermann et al. 2005, Wagner et al. ABCG2 (BCRP) intestine 2005) (Sahi et al. 2006, Meyer Zu Breast SLC21A3(OATP1A2) Schwabedissen et al. 2008, Gui et al. 2008) (Guo et al. 2002, Cheng et al. 2005, Liver Slc21a5 (Oatp1a4) Aleksunes & Klaassen 2012) ABCB11(BSEP), SLC22A1(OCT1), Hepatocytes (Jigorel et al. 2006, Olinga et al. 2008) SLC22A7(OAT2), SLC10A1(NTCP) Adapted from Urquhart et al. 2007, Di Masi et al. 2009, Klaassen and Aleksunes 2010 and Chan et al. 2013a. Only human genes are presented with capital letters.

In contrast to CYP3A4, the proximal promoter region for PXR binding is missing in

MDR1 gene. In fact, PXR is known to bind enhancer elements, such as DR4 motifs (i.e., I, II and

III), a DR3 motif and an ER6 motif, at about -8 Kb upstream of the MDR1 transcription start site

(Geick et al. 2001, Guo et al. 2002, Teng et al. 2003, Albermann et al. 2005, Meyer Zu

Schwabedissen et al. 2008). Among several motifs found in CYP3A4 XREM region, only one motif (i.e., DR3) is required for CYP3A4 induction (Goodwin et al. 1999). Similarly, one motif

(i.e., DR4 closest to the 5’ end) in the MDR1 enhancer element is primarily responsible for P-gp induction (Geick et al. 2001). PXR regulation of P-gp expression has been studied using in vitro cell culture models of intestine, liver and peripheral blood mononuclear cells and in vivo rodent models (Staudinger et al. 2001, Geick et al. 2001, Guo et al. 2002). In the brain, in particular at the BBB, Bauer et al. were the first to demonstrate that P-gp can be regulated by PXR in rats

(Bauer et al. 2004) and PXR-humanized mouse brain capillaries (Bauer et al. 2006). These findings were later confirmed by several groups using primary cultures of brain microvessel endothelial cells or immortalized brain microvessel endothelial cell culture system derived from porcine (Ott et al. 2009), bovine (Perloff et al. 2007) and rodent (Lombardo et al. 2008, Narang et al. 2008). In humans, work generated from this thesis demonstrated that P-gp is also regulated by hPXR in hCMEC/D3, a representative in vitro model of the human BBB (Chan et al. 2011), please see Chapters 6 and 7 of this thesis. In addition to P-gp, a few studies have suggested that

PXR can also regulate Mrp2 expression in rodent brain microvessel endothelial cell culture systems (Narang et al. 2008, Lombardo et al. 2008) and isolated rat brain capillaries (Bauer et al.

2008a). Furthermore, a hPXR response element in the promoter region of human OATP1A2 gene has been identified (Meyer Zu Schwabedissen et al. 2008). However, it remains to be demonstrated that OATP1A2 expressed at the luminal membrane of brain capillaries is also regulated by hPXR. Currently, there is no evidence on the regulation of drug transporters by hPXR in other cellular compartments of the brain parenchyma. As well, the presence of a functional hPXR-mediated regulatory pathway on P-gp expression at the human BBB has not been demonstrated clinically.

Aside from serving as a xenosensor, PXR is also recognized to regulate a number of physiological processes. For example, in vivo PXR activation in rodents has been demonstrated

to protect against hepatocytoxic bile acid accumulation by inducing PXR target genes CYP3a11 and Oatp1a4, which are known to promote bile acid clearance (Lamba et al. 2004b). In addition, homeostasis of steroid hormones (i.e., corticosterone, aldosterone, progesterone and 17ß- estradiol) can be affected by PXR regulation of adrenal steroidogenic enzymes (i.e., CYP11As and CYP11Bs) and CYP3A4 (Sonoda et al. 2005, Ma et al. 2008). Furthermore, PXR appears to repress transcriptional activity of forkhead insulin-responsive transcription factor 1 (FoxO1) on insulin-response element, thus reducing gluconeogenesis (Niwa et al. 1998, Badawi et al. 2001,

Yu et al. 2005, Zhai et al. 2007). Recently, PXR has been identified to be associated with inflammatory bowel diseases, however the exact molecular mechanisms involved during these disorders are unclear (Kodama et al. 2004).

1.3.2 CAR

1.3.2.1 Expression, Structure and Function

Human NR1I3 (hCAR) was cloned in 1994 and initially termed MB67 (Baes et al. 1994).

Soon after, its rodent counterpart was identified to regulate CYP2B enzymes expression mediated by phenobarbital (Choi et al. 1997, Honkakoski et al. 1998, Yoshinari et al. 2001).

Twenty-two unique hCAR splice variants can be generated from multiple splicing events throughout the NR1I3 gene (Savkur et al. 2003, Auerbach et al. 2003, Lamba et al. 2004a).

Although some of these variants were reported in different organs (Table 1-3), their role in affecting receptor function (i.e., ligand-dependent or ligand-independent activation) is not well understood. In addition, thirty-three SNPs have been identified in exon 1, 2, 4, 5 and 8 of NR1I3 gene in the Japanese population (Ikeda et al. 2003, Ikeda et al. 2005). Most of these SNPs are located in the 5’ flanking region of the gene or result in synonymous amino acid alterations, which are unlikely to exhibit distinct receptor activity (di Masi et al. 2009). Four non-

synonymous amino acid alterations at the LBD have been identified: Val133Gly, His246Arg,

Leu308Pro and Asn323Ser (Ikeda et al. 2005). Constitutive receptor activity and ligand- dependent activation were shown to be significantly reduced only in variants His246Arg and

Leu308Pro, whereas variants Val133Gly and Asn323Ser exhibit identical activity compared with wild-type receptor (Ikeda et al. 2005, Jyrkkärinne et al. 2005).

In rodent brain, both the protein and transcript expression of CAR have been detected in isolated brain capillaries (Wang et al. 2010). CAR transcript expression has been reported in human brain tissue samples (Nishimura et al. 2004), caudate nucleus (Lamba et al. 2004a) and brain glioma cells (Malaplate-Armand et al. 2005). As well, work from this thesis provides in vitro evidence for human CAR protein expression in the hCMEC/D3 cell culture system (Chan et al. 2011) and in human fetal brain tissue (Chan et al. 2010), please refer to Chapters 6 and 7.

In human brain microvessels, CAR mRNA expression was reported (Dauchy et al. 2008), however protein expression was not detected in these samples (Shawahna et al. 2011). Overall, the functional relevance of hCAR in the human brain remains unclear.

hCAR and hPXR share approximately 53 % amino acid sequence homology and are believed to originate from a common progenitor (Handschin et al. 2000, di Masi et al. 2009).

Structurally similar to the PXR, the CAR DBD can also heterodimerize with RXRα and bind to

DNA sequences containing DR3, DR4, DR5 and ER6 motifs (i.e., PBREM on CYP2B6 and

Cyp2b10 genes) (Honkakoski et al. 1998, Kawamoto et al. 1999, Sueyoshi et al. 1999). X-ray crystallographic structures revealed that hCAR ligand binding pocket is approximately 675 Å3, half the size of hPXR (Shan et al. 2004, Suino et al. 2004, Xu et al. 2004). Although CAR ligand pocket is larger than many other nuclear receptors, it lacks the unique flexible loop structure found in PXR, which allows the ligand pocket to flexibly expand (Ingraham & Redinbo 2005). In

addition, hCAR LBD shares approximately 70 % amino acid sequence homology with rodent

CAR LBD, which helps explaining the observed differences in ligand activation profiles between

CAR and PXR (Timsit & Negishi 2007). For example, clotrimazole is an agonist of hPXR, but is an antagonist of hCAR (Moore et al. 2000, Honkakoski et al. 2003). The hepatomitogen,

TCPOBOP, is a potent rodent CAR ligand, yet it does not activate hCAR (Moore et al. 2000,

Honkakoski et al. 2003). In contrast, the hCAR synthetic agonist, CITCO, does not activate mouse CAR (Maglich et al. 2002). In general, hCAR can recognize many structural diverse compounds, including endogenous chemicals and pharmaceutical agents (Timsit & Negishi

2007). Androstane metabolites (i.e., androstanol) can inhibit hCAR transcriptional activity and several bile acids, e.g., cholic acid, 6-ketolithocholic acid, and 7-ketodeoxycholic acid methyl ester, were shown to inhibit human and rodent CAR activity (Moore et al. 2002). To date, there is no reported example of a physiological agonist with high affinity towards human or rodent

CAR. However, two synthetic hydrocarbons, TCPOBOP and CITCO, were identified to bind selectively and with high affinity to rodent and hCAR, respectively (Maglich et al. 2003, Jackson et al. 2004). Although human and rodent CAR ligand profiles are relatively narrow compared with human and rodent PXRs, several synthetic drugs were identified to bind hCAR directly and induce receptor activity, such as cholesterol-lowering drugs (e.g., simvastatin, atorvastatin), anticholinergic drugs (e.g., orphenadrine), antimalarial drugs (e.g., artemisinin) and antihyperlipidemic drugs (e.g., clofibrate) (Mäkinen et al. 2003, Kobayashi et al. 2005, Burk et al. 2005). As well, anticonvulsants (e.g., phenobarbital), antiepileptic drugs (e.g., phenytoin) and analgesic agents (e.g., acetaminophen) were also identified to indirectly induce hCAR activity through post-translational processes (i.e., nuclear translocation and phosphorylation) rather than

through direct receptor binding (Moore et al. 2000, Zhang et al. 2002, Huang et al. 2003,

Faucette et al. 2004, Wang et al. 2004).

In contrast to most nuclear receptors, rodent and human CAR seem to exhibit a constitutive transcription activity in the absence of ligand (Timsit & Negishi 2007). This activity can be inhibited by endogenous androstanol, possibly through interference of co-activator (i.e.,

SRC-1) binding to hCAR (Forman et al. 1998). It was suggested that hCAR in the nucleus adopts an active structural conformation in the absence of ligand and is able to recruit co-activators independent of ligand presence (Stanley et al. 2006). In particular, receptor activity can also be regulated by several cellular pathways (Timsit & Negishi 2007). In the nucleus, human and rodent CAR transcriptional activity on target genes can be altered by interactions with several coactivator and co-repressor accessory proteins, such as GRIP1, PPAR-γ coactivator-1 (PGC-1) and SRC-1, which can interact with PXR as well (Kim et al. 1998a, Muangmoonchai et al. 2001,

Min et al. 2002b, Choi et al. 2005). Aside from being a transcriptional co-repressor protein,

GRIP1 has also been identified to retain mouse CAR in the nucleus, affecting CAR nuclear accumulation and function (Min et al. 2002b, Min et al. 2002a, Xia & Kemper 2005). In comparison with other nuclear receptors (e.g., hPXR), human and rodent CAR exhibit a unique nuclear translocation pattern that can be triggered by a ligand-dependent or –independent process, possibly due to a non-functional nuclear localization signal in the DBD (Swales &

Negishi 2004). Instead, a leucine-rich sequence, termed xenobiochemial response signal, is important for nuclear translocation of human and rodent CAR (Swales & Negishi 2004). In primary cultures of human hepatocytes and intact rat livers, unliganded human and rodent CAR have been shown to initially reside in the cytoplasm and translocate into the nucleus after exposure to agonists, i.e., CITCO and TCPOBOP (Li et al. 2009) or activators that do not bind

directly to the receptor LBD, i.e., phenobarbital (Kawamoto et al. 1999). However, hCAR nuclear translocation in a transformed cell line [i.e., human liver carcinoma cell line (HepG2)] was not observed in the presence of ligands and the receptor was constitutively sequestered in the nucleus (Kawamoto et al. 1999, Zelko et al. 2001, Kanno et al. 2005, Guo et al. 2007). These observations have suggested that several cellular pathways or structural features of CAR essential for its nuclear translocation could be missing for in vitro transformed cell lines (Li &

Wang 2010). For example, in vitro CCRP over-expression can restore unliganded mouse CAR ability to reside in the cytoplasm and translocate into the nucleus in the presence of agonist (i.e.,

TCPOBOP) (Kobayashi et al. 2003). As well, it is believed that CCRP and mouse CAR recruit

HSP90 forming a protein complex in order to interact with protein phosphatase A2 and the sequential de-phosphorylation plays a crucial role in receptor nuclear translocation (Sidhu &

Omiecinski 1997, Honkakoski et al. 1998). Furthermore, mutagenesis studies on a phosphorylation site (i.e., Ser202) at the mouse CAR LBD could prevent receptor nuclear translocation, suggesting the different phosphorylation states of the receptor could be essential for proper nuclear translocation (Hosseinpour et al. 2006). However, it is currently unclear whether these interactions or phosphorylation events also regulate hCAR nuclear translocation.

Other molecular mechanisms can also alter human and rodent CAR transcription activity, such as conformational changes to the hCAR AF-2 domain upon ligand binding and/or protein-protein interactions with co-activators, co-repressors and RXRα in the nucleus (Kim et al. 1998a,

Muangmoonchai et al. 2001, Min et al. 2002b, Choi et al. 2005). Therefore, CAR nuclear translocation simply serves as a first step for sequential transcriptional events or interactions to occur via ligand-dependent or constitutive (i.e., ligand-independent) mechanisms. Alterations in

CAR nuclear translocation may not necessarily translate into an increase in CAR target gene

transcription, as demonstrated by several in vitro and in vivo findings (Kawamoto et al. 1999,

Marc et al. 2000, Honkakoski et al. 2003).

1.3.2.2 Regulation of Target Genes

Functionally similar to PXR, CAR has also been termed as a xenobiotic sensor that regulates the expression of phase I/II enzymes (Table 1-7) and drug transporters (Table 1-8) that are known to affect tissue elimination and distribution of endogenous and exogenous compounds

(Stanley et al. 2006, Urquhart et al. 2007, di Masi et al. 2009, Klaassen & Aleksunes 2010, Li &

Wang 2010, Chan et al. 2013a). In fact, many CAR target genes are also co-regulated by PXR.

CAR target genes encoding phase I metabolic enzyme include CYP2A6, CYP2B1, CYP2B6,

CYP2C8, CYP2C9, CYP2C19, CYP3A1 and CYP3A4 in humans and Cyp3a11, Cyp2b2,

Cyp2b10 and Cyp2c29 in mice. Phase II metabolic enzymes that are regulated by CAR include

GSTA1, GSTA2 and UGT1A1 in humans and Aldh1a1, Aldh1a7, Sult1a1, Sult2a9 and Sult2a1 in rodents. Transporters that are regulated by CAR include P-gp, BCRP, MRP2, MRP3,

OATP2B1, OCT1 and NTCP in humans and P-gp, Bcrp, Mrp2, Mrp3, Mrp4, Oatp1a1, Oatp1a4 and Oatp1a6 in rodents. In particular, it was demonstrated that CAR/RXRα binds specifically to

DR4 and ER6 found in the response elements located at the -7.8 Kb upstream of the MDR1 gene encoding for P-gp (Burk et al. 2005). In the brain, Wang et al. have recently shown that CAR is expressed in isolated rat and mouse brain microvessels and mCAR activation was able to induce functional expression of P-gp, Bcrp and Mrp2 in both in vitro and in vivo models of the BBB

(Wang et al. 2010). Work presented in this thesis provides evidence that hCAR can regulate P-gp mRNA and protein expression in the hCMEC/D3 cell culture system (Chan et al. 2011).

Furthermore, Lemmen et al. showed P-gp and Bcrp regulation by CAR in primary cultures of porcine brain capillary endothelial cells (Lemmen et al. 2013). Although, hCAR was

demonstrated to regulate CYP2C8 and CYP2C9 expression in response to cocaine exposure to glioma cells, hCAR regulatory role on glial P-gp expression was not determined in the study

(Malaplate-Armand et al. 2005). Currently to the best of our knowledge, there are no data on the regulation of drug transporters by hCAR in glial cells or other cellular compartments of the brain parenchyma. As well, the presence of a functional hCAR-mediated regulatory pathway on P-gp expression at the human BBB has not been demonstrated clinically.

Table 1-7. Human and rodent CAR regulation of phase I/II enzymes.

Organs/Tissues Target Genes Reference(s)

Aldh1a1, Aldh1a7 (Aleksunes & Klaassen 2012) CYP2A6 (Richert et al. 2009) CYP2B1,CYP2B6, (Kawamoto et al. 1999, Kim et al. 2001, Goodwin et al. 2001, Xiong et al. 2002, Wang et al. 2003a, Richert et al. 2009) CYP2C8,CYP2C9, (Ferguson et al. 2002, Gerbal-Chaloin et CYP2C19 al. 2002, Chen et al. 2003, Malaplate-

Armand et al. 2005, Ferguson et al. Liver and intestine 2005, Richert et al. 2009) CYP3A1, CYP3A4 (Goodwin et al. 2002, Olinga et al. Phase I 2008, Richert et al. 2009) Cyp2b2, Cyp2b10 (Kawamoto et al. 2000, Huang et al. 2004a, Kawase et al. 2007, Aleksunes & Klaassen 2012) Cyp2c29 (Jackson et al. 2004) Cyp3a11 (Wei et al. 2002b, Huang et al. 2004a, Anakk et al. 2004, Down et al. 2007, Ohbuchi et al. 2013)

GSTA1 GSTA2 (Huang et al. 2003, Richert et al. 2009) UGT1A1 (Sugatani et al. 2005, Huang et al. 2003, Liver Olinga et al. 2008) (Kitada et al. 2003, Saini et al. 2004,

Phase II Phase Sult1a1, Sult2a9, Sult2a1 Assem et al. 2004, Lee et al. 2007a) Adapted from Urquhart et al. 2007, Di Masi et al., 2009, Klaassen and Aleksunes 2010 and Chan et al. 2013a. Only human genes are presented with capital letters.

Table 1-8. Human and rodent CAR regulation of transporters.

Organs/Tissues Target Genes Reference(s) Abcb1a/b, MDR1 (P- (Maglich et al. 2003, Burk et al. 2005, gp) Jigorel et al. 2006, Olinga et al. 2008, Wang et al. 2010, Lemmen et al. 2013)

Liver, brain ABCG2/Abcg2 (Jigorel et al. 2006, Olinga et al. 2008, capillaries (BCRP/Bcrp) Wang et al. 2010, Lemmen et al. 2013) ABCC2/Abcc2 (Kast et al. 2002, Huang et al. 2003, (MRP2/Mrp2) Jigorel et al. 2006, Olinga et al. 2008, Aleksunes et al. 2009, Wang et al. 2010, Aleksunes & Klaassen 2012) ABCC3/Abcc3 (Kiuchi et al. 1998, Xiong et al. 2002, (MRP3/Mrp3) Cherrington et al. 2003, Staudinger et al. 2003, Olinga et al. 2008, Aleksunes & Klaassen 2012) Abcc4(Mrp4) (Assem et al. 2004, Olinga et al. 2008, Liver and intestine Aleksunes & Klaassen 2012) SLC21A9(OATP2B1), (Jigorel et al. 2006, Olinga et al. 2008) SLC10A1(NTCP), SLC22A1(OCT1) Slc21a1(Oatp1a1), (Huang et al. 2003, Staudinger et al. Slc21a5(Oatp1a4), 2003) Slc21a13(Oatp1a6) Adapted from Urquhart et al. 2007, Di Masi et al., 2009, Klaassen and Aleksunes 2010 and Chan et al. 2013a. Only human genes are presented with capital letters.

CAR also regulates several physiological processes. For example, CAR plays an important role in the metabolism and elimination of bilirubin, which is a product in heme catabolism and its accumulation can lead to the development of jaundice (di Masi et al. 2009). It is known that hCAR agonists (i.e., phenobarbital) can enhance bilirubin elimination by its inductive effect on biliary transporters (e.g., OATP1A2 and MRP2) and metabolic enzymes (e.g.,

UGT1A1 and GSTA1) (Sugatani et al. 2001, Huang et al. 2003, Sugatani et al. 2005).

Furthermore, CAR appears to interact with other nuclear receptors (i.e., PXR and FXR) to

protect against hepatic bile acid toxicity. Although FXR is well established to play a key role in bile acid elimination, several enzymes (e.g., SULT2A1) and transporters (e.g., MRP3) involved in these processes are also targets of hCAR (Gao & Xie 2010, Fiorucci et al. 2012). In addition,

CAR activation can affect homeostasis of hormone steroids (i.e., androgen and estrogen), because they can be actively metabolized by CYP2B and UGT1A1 enzymes, which are known

CAR targets (Kawamoto et al. 1999, Sugatani et al. 2005, Huang et al. 2003, Olinga et al. 2008).

Similar to PXR, CAR can attenuate the ability of FoxO1 to stimulate gluconeogenic gene (i.e., phosphoenolpyruvate carboxykinase or PEPCK), thus possibly playing a role in the regulation of gluconeogenesis (Niwa et al. 1998, Badawi et al. 2001, Yu et al. 2005, Zhai et al. 2007).

1.4 HIV

HIV is a lentivirus of the Retroviridae family and two variants of HIV have been identified; HIV-1 and HIV-2 (Sharp & Hahn 2010). HIV-1 is the causative virus for the majority of HIV infection observed worldwide, whereas HIV-2 induces acquired immune deficiency syndrome (AIDS) at a much slower rate (Weiss 1993). According to the Joint United Nations

Program on HIV/AIDS (UNAIDS), 31.4 to 35.9 million people were living with HIV-1 worldwide and approximately 71,300 of those are present in Canada (UNAIDS 2012,

HealthCanada 2011). The virus can migrate to different tissues and organs, but primarily infects cells from the macrophage or monocyte lineage and subsets of CD4 positive T lymphocytes

(Kedzierska & Crowe 2002). In brief, viral entry is initiated by the binding of viral envelope spike, a protein complex comprised of gp120 and gp41, to the CD4 receptor that triggers a protein conformational change and allows the subsequent binding of gp120 to the HIV-1 co- receptor (i. e., CCR5 and CXCR4) (Deng et al. 1996, Moore et al. 1997). Binding of gp120 to the co-receptor causes the fusion of viral and cell membranes (Wyatt & Sodroski 1998).

Following the release of viral genetic material into the cell cytoplasm, the immediate transcription of the viral RNA into double-stranded DNA by the viral reverse transcriptase allows the integration of the viral genetic material into the host cell genome. The subsequent replication, transcription, translation, post-translational modification and maturation precede the release of the new virions from infected cells (Sundquist & Krausslich 2012). Sustained viral replication continues during infection and the virus migrates to different tissues and organs. Even with the implementation of antiretroviral therapy, total viral eradication remains difficult primary due to the presence of a viral reservoir found in resting memory CD4 positive T cells (Finzi et al. 1999,

Siliciano et al. 2003). Aside from viral reservoirs found in resting T cell populations, CNS, testis

and gut-associated lymphoid tissue are also currently identified to be viral sanctuaries (Persidsky

& Poluektova 2006, Bailey et al. 2004, Dahl et al. 2010, Lowe et al. 2004). The persistence of

HIV at these sites prevents the cure of the disease.

1.4.1 Combination Antiretroviral Therapy

The concurrent administration of three or more drugs from different classes of antiretrovirals, known as combination antiretroviral therapy (cART), has been efficient in reducing plasma viral load and ultimately, in prolonging the life expectancy of infected individuals (Margolis & Hazuda 2013). Currently, there are six different classes of antiretroviral drugs: entry inhibitor, fusion inhibitor, nucleoside reverse transcriptase inhibitor (NRTI), non- nucleoside reverse transcriptase inhibitor (NNRTI), integrase inhibitor and PI, targeting different phases of HIV-1 life cycle (Sierra-Aragon and Walter, 2012). cART consists of a NRTI backbone regimen given in combination with a NNRTI, PI or integrase inhibitor as currently recommended in the 2013 NIH Guidelines for the Use of Antiretroviral Agents in HIV-1 Infected

Adults and Adolescents (http://aidsinfo.nih.gov/guidelines). Table 1-9 summarizes preferred and alternative antiretroviral regimens for the initial therapy of HIV-infected patients.

Table 1-9. Preferred and alternative antiretroviral regimens for antiretroviral therapy- naive patients.

Preferred Regimens

One of the following NRTI Backbone Regimen Antiretroviral Regimens NNRTI- 1 Efavirenz based 2 Atazanavir + PLUS Ritonavir PI-based 3 or Tenofovir Disoproxil Fumarate & Emtricitabine Darunavir + Ritonavir Integrase 4 Inhibitor- Raltegravir based

For Pregnant Women Lopinavir + Ritonavir PLUS Zidovudine and Lamivudine

Alternative Regimens One of the following NRTI Backbone Regimen Antiretroviral Regimens Efavirenz 1 OR PLUS Abacavir & Lamivudine NNRTI- Rilpivirine based 2 Rilpivirine PLUS Tenofovir & Emtricitabine Atazanavir & 3 Ritonavir PLUS Abacavir & Lamivudine Darunavir & 4 Ritonavir PI-based Fosamprenavir & 1 Abacavir & Lamivudine 5 Ritonavir PLUS OR Lopinavir & Tenofovir Disoproxil Fumarate 6 2 Ritonavir & Emtricitabine 7 Integrase Raltegravir PLUS Abacavir & Lamivudine Inhibitor- Tenofovir Disoproxil Fumarate 8 Elvitegravir PLUS based & Emtricitabine Adapted from the Feb 2013 NIH Guidelines for the Use of Antiretroviral Agents in HIV-1 Infected Adults and Adolescents (http://aidsinfo.nih.gov/guidelines).

Entry inhibitors were newly developed as antagonists of CCR5 and CXCR4 co-receptors.

Co-receptors inhibition can prevent binding of viral gp120 to target immune cells and attenuate sequential HIV entry (Coakley et al. 2005). Maraviroc, the first approved CCR5 antagonist is only effective against R5-tropic viruses (Perry 2010). Although maraviroc does not belong to the preferred nor alternative regimens for treating naive patients, it is prescribed in combination with

NRTIs (http://aidsinfo.nih.gov/guidelines). Fusion inhibitors were developed recently to prevent initiation of gp120/gp41viral envelope complex binding to target cell plasma membrane (Yu et al. 2012). Enfuvirtide is a recently approved fusion inhibitor for the treatment of HIV infected patients who have failed to respond to current antiretroviral regimens (Yu et al. 2012) (Do Canto et al. 2011). HIV-1 reverse transcriptase, a RNA-dependent DNA polymerase, catalyzes the conversion of viral RNA into DNA following release of viral capsid into the host cell cytoplasm

(Sierra-Aragón & Walter 2012). NRTIs and NNRTs were designed to inhibit viral DNA synthesis mediated by viral reverse transcriptase. NRTIs: abacavir, tenofovir, emtricitabine, lamivudine and zidovudine, were the first generation of antiretroviral drugs in the treatment of

HIV-1. NRTIs are intracellularly phosphorylated to their active tri-phosphatemetabolites and compete with endogenous deoxynucleotides for binding to the nucleotide-binding site of reverse transcriptase. After their incorporation into viral DNA, these compounds lacking the 3’-hydroxyl group lead to a chain termination and stop viral DNA synthesis (Sierra-Aragón & Walter 2012).

NNRTIs; efavirenz, nevirapine, etravirine and rilpivirin, are non-competitive transcriptase inhibitors, which are not incorporated into viral DNA, but instead bind to the hydrophobic pocket near the substrate recognition site inducing conformational changes which lead to viral enzyme inactivation (Sierra-Aragón & Walter 2012). Currently, NRTIs are widely-used as a backbone regimen in combination with other HIV drug classes, including NNRTIs. Integrase inhibitors

were recently developed to inhibit viral genomic integration into host DNA (Quashie et al.

2013). This class of agents binds competitively to the active sites (two conserved carboxylate residues at position 64 and 116) of HIV integrase and prevents transfer of viral DNA strands into host genome. Raltegravir and Elvitegravir, two clinically approved integrase inhibitors, are currently prescribed in combination with NRTIs and is shown to be superior compared with other first line regimens (Messiaen et al. 2013). HIV protease is a dimer aspartase enzyme that recognizes and cleaves specifically nine different sequences found in viral gag and gag-pol precursor proteins, playing an important role in viral maturation (Huff & Kahn 2001). PIs are peptidomimetic (small synthetic peptide-chain mimicking natural peptides) compounds that can selectively and reversibly inhibit catalytic activity of HIV protease thus prevent maturation and assembly of infectious viral particles (Velazquez-Campoy et al. 2003). This class of agents: atazanavir, fosamprenavir, lopinavir, ritonavir, indinavir and nelfinavir, serves as a crucial component of initial suppressive therapy used in combination with NRTIs. In order to boost PIs bioavailability, they are administered with low doses of ritonavir which is a HIV PI and also a potent inhibitor of CYP3A4, drug metabolizing enzyme for most PIs (Hsu et al. 1998).

1.4.2 HIV Neurological Complications and Brain Permeation of Antiretroviral Drugs

At present, although severe forms of HIV-associated neurocognitive disorders (HAND), such as HIV-associated dementia, have almost disappeared from clinical practice, the prevalence of other mild neurocognitive disorders, such as mild neurocognitive disorder and asymptomatic neurocognitive impairment, remains unchanged or is on the rise (Letendre 2011). These disorders may not produce severe cognitive impairments that were seen earlier in patients such as HIV- associated dementia, however they can generate neurobehavioral and psychiatric conditions, including depression, anxiety, sleep disorders, mania, and psychosis (Mamidi et al. 2002). In

addition, secondary CNS disorders, such as meningitis, meningoencephalitis and peripheral neuropathy, brain opportunistic infections and cerebrovascular diseases could also lower survival rate and affect the patient’s quality of life (Mamidi et al. 2002, Vivithanaporn et al. 2010).

CNS is currently recognized to be a viral sanctuary in which HIV can replicate with suboptimal drug challenges and can possibly generate genetic variants distinct from those found in plasma (Cunningham et al. 2000, Smit et al. 2004). The presence of brain viral sanctuary and the sustained prevalence of HAND could possibly be related to the limited brain permeability of different classes of antiretroviral drugs (Letendre et al. 2004, Letendre et al. 2008, Letendre

2011). The limited drug permeability could be partly explained by properties of most antiretroviral drugs, such as high molecular weight and extensive plasma protein binding, which are known to significantly restrict drug permeation across the BBB (Ene et al. 2011). The CSF concentration of these drugs has been clinically reported to be very low, in particular for drugs belonging to the HIV PI class (Table 1-10). Limited drug concentration in the CSF has been demonstrated to be associated with a higher CSF viral load (Letendre et al. 2004, Letendre et al.

2008, Letendre 2011). Recent studies further suggest that antiretroviral regimens containing drugs with better permeability into the brain may improve the treatment of HAND (Cysique &

Brew 2009). These findings support the concept that utilization of antiretroviral regimens with effective brain permeability could decrease viral loads in the brain and improve HIV-associated neurological complications. However, CNS toxicity associated with these drugs can limit their use (Gupta et al. 2012). Therefore, achieving therapeutic window for effective antiretroviral therapy in the CNS is currently recognized to be essential in preventing or improving HIV- associated neurological complications (Letendre 2011).

Table 1-10. Pharmacokinetic parameters of antiretroviral drugs in humans. Plasma Therapeutic Molecular Half CSF Protein Plasma CSF: Plasma Drug Weight Life Concentration Binding Concentration Concentration Ratio (g/mol) (hr)* (µM) (%) (µM) PI 0.008 - 0.018 Amprenavir 505.6 90 8.5 10.6 – 19.2 0 – 0.36 (Croteau et al. 2012) 0.008 - 0.02 Atazanavir 704.9 99 5.2 0.18 – 8.8 0 – 0.067 (Best et al. 2009) 0.003 - 0.078 Darunavir 548.0 95 10 3.3 – 23.5 0.029 – 0.39 (Yilmaz et al. 2009b) 0.001 - 0.008 Lopinavir 628.8 99 5.6 8.7 - 15 0 – 0.12 (Capparelli et al. 2005) 0.001 - 0.002 Saquinavir 670.9 98 2.5 1.8 – 3.2 0 – 0.008 (Kravcik et al. 1999) Not detected (Antinori et Nelfinavir 567.8 99 4.3 5.6 – 8.5 0 – 0.012 al. 2005, Solas et al. 2003) 0.001 - 0.005 Ritonavir 721.0 99 4.0 10.5 - 15 0 – 0.32 (Kravcik et al. 1999) NRTI 0.31 - 0.44 Abacavir 286.3 49 1.5 5.2 – 13 0.50 – 1.8 (McDowell et al. 1999) 0.032 - 0.24 Didanosine 236.2 < 5.0 1.5 2.1 – 11 0.17 – 0.51 (Huang et al. 2004b) 0.050 - 0.41 Emtricitabine 247.2 < 4.0 9.0 0.36 - 7.4 0.018 – 3.0 (Calcagno et al. 2011) 0.054 - 1.14 Lamivudine 229.3 < 36 1.4 4.3 – 8.7 0.050 – 1.14 (Foudraine et al. 1998) 0.004 - 0.84 Tenofovir 289.2 0.7 14 C : 1.1 0.0044 – 0.92 MAX (Best et al. 2012) 0.01 - 12.3 Zidovudine 267.2 34 – 38 1.1 4.5 – 6.7 0.12 – 0.41 (Foudraine et al. 1998) NNRTI 0.004 - 0.011 Efavirenz 315.7 99 64 9.2 – 17 0.0060 – 0.090 (Tashima et al. 1999) 0.41 – 0.77 Nevirapine 266.3 60 45 7.5 – 21 1.3 – 11 (Antinori et al. 2005) 0.012 Rilpivirine 402.9 99 50 Mean: 0.13 Mean: 0.002 (Mora-Peris et al. 2013) Integrase Inhibitor 0.010 - 0.61 Raltegravir 444.0 83 9.0 0.083 – 12 0.0045 – 0.28 (Yilmaz et al. 2009a) Elvitegravir 447.9 99 12.9 1.0 – 3.8 Not reported Not reported Entry 0.004 - 0.17 Inhibitor: 514.0 76 16 0.042 – 0.93 0.0036 – 0.024 (Tiraboschi et al. 2010) Maraviroc * Molecular weight, protein plasma binding, plasma and CSF concentrations at steady state from HIV infected patients were obtained from The Immunodeficiency Clinic, www.hivclinic.ca. Assessed on March 2013.

1.4.3 Mechanisms of Antiretroviral Drug Interactions

1.4.3.1 Antiretroviral Drug Interactions with Drug Transporters

Members from the ABC and SLC transporter family are known to participate in transport of many antiretroviral drugs as summarized in a review from our group (Kis et al. 2010a). Their diverse expression in the intestine, liver, kidney, brain, testis and other blood-tissue barriers can significantly affect drug distribution in mammalian systems (Kis et al. 2010a, Ashraf et al.

2013). P-gp has been demonstrated to transport all PIs (Lee et al. 1998, Polli et al. 1999, Van der

Sandt et al. 2001, Edwards et al. 2002, Ronaldson et al. 2004b, Janneh et al. 2007, Zastre et al.

2009, Brown et al. 2009, Fujimoto et al. 2009), maraviroc (Walker et al. 2005), abacavir (Shaik et al. 2007) and raltegravir (Kassahun et al. 2007). In vitro studies using isolated lymphocytes demonstrated that P-gp efflux affects intracellular accumulation of several PIs and NRTIs

(Antonelli et al. 1992, Jones et al. 2001, Meaden et al. 2002, Ford et al. 2004, Janneh et al. 2005,

Janneh et al. 2009). As well, in vivo studies using rodents and macaques showed the role of P-gp at the BBB in limiting brain accumulation of these drugs (Kim et al. 1998b, Shaik et al. 2007,

Kaddoumi et al. 2007). In addition, P-gp transport can also be inhibited by several PIs (i.e., ritonavir, lopinavir and nelfinavir) and NNRTIs (i.e., efavirenz) at clinically relevant plasma concentrations (Perloff et al. 2002, Vishnuvardhan et al. 2003, Storch et al. 2007, Tong et al.

2007). Our laboratory also demonstrated that PIs, such as indinavir, ritonavir and saquinavir, can inhibit digoxin transport in RBE4 rat brain endothelial cell line, primary cultures of rat astrocytes and MLS-9 rat microglial cells line (Bendayan et al. 2002, Ronaldson et al. 2004b, Ronaldson et al. 2004a). As well, chronic exposure to several PIs (i.e., ritonavir, amprenavir, nelfinavir, lopinavir, atazanavir) is known to induce both in vitro and in vivo P-gp expression in human intestinal tissues (Perloff et al. 2000, Huang et al. 2001, Vishnuvardhan et al. 2003, Gupta et al.

2008, Konig et al. 2010). NNRTIs and NRTIs have been reported to also induce P-gp expression but only at high concentrations in a human intestinal cell line, LS180 (Weiss et al. 2008). Table

1-11 provides a summary of antiretroviral drugs acting as substrate, inhibitor and/or inducer of P- gp in the literature.

Table 1-11. Antiretroviral drug interactions with P-gp.

Antiretroviral Interactions with P-gp P-gp Induction Drugs PIs Substrate (Lee et al. 1998, Polli et al. 1999, Van der LS180 (Perloff et al. 2000, Gupta et Amprenavir Sandt et al. 2001, Edwards et al. 2002) al. 2008), rat intestine and liver Inhibitor (Storch et al. 2007) (Huang et al. 2001) Substrate (Zastre et al. 2009) LS180 (Perloff et al. 2005, Gupta et Atazanavir Inhibitor (Storch et al. 2007) al. 2008) Substrate (Brown et al. 2009, Fujimoto et al. 2009) Darunavir LS180 (Konig et al. 2010) Inhibitor (Tong et al. 2007) Substrate (Janneh et al. 2007) LS180 (Vishnuvardhan et al. 2003, Lopinavir Inhibitor (Vishnuvardhan et al. 2003, Storch et al. 2007) Gupta et al. 2008) BMVECs (Perloff et al. 2004, Substrate (Van der Sandt et al. 2001, Zastre et al. 2009) Ritonavir Perloff et al. 2007), LS180 (Perloff Inhibitor (Perloff et al. 2002) et al. 2001, Gupta et al. 2008) NRTIs Substrate (Shaik et al. 2007) Abacavir Not Known Weak Inhibitor (Storch et al. 2007) Substrate (Not Known) Emtricitabine LS180 (Weiss et al. 2008) Not an inhibitor (Storch et al. 2007) Substrate (Not Known) No Induction in LS180 (Weiss et Lamivudine Not an inhibitor (Storch et al. 2007) al. 2008) Substrate (Not Known) Tenofovir Not Known Not an inhibitor (Storch et al. 2007) Substrate (Not Known) Zidovudine Not Known Not an inhibitor (Storch et al. 2007, Shaik et al. 2007) NNRTIs Substrate (Not Known) LS180 (Weiss et al. 2008, Weiss et Efavirenz Weak Inhibitor (Storch et al. 2007) al. 2009) Substrate (Not Known) Nevirapine LS180 (Weiss et al. 2008) Not an inhibitor (Storch et al. 2007) Integrase Inhibitor Substrate (Kassahun et al. 2007) Raltegravir Not Known Inhibitor (Not Known) Entry Inhibitor Substrate (Walker et al. 2005) Maraviroc Not Known Inhibitor (Not Known) BMVECs: primary cultures of bovine brain microvessel endothelial cells; LS180: intestinal human colon adenocarcinoma cell line. Our work regarding the inductive properties of several antiretroviral drugs on P-gp functional expression in the hCMEC/D3 cells is now published and can be found in Chapters 6 and 8 of this thesis (Zastre et al. 2009, Chan et al. 2013b).

BCRP shares a similar tissue distribution as P-gp and can also transport several antiretroviral agents belonging to the NRTI pharmacological class, including zidovudine, lamivudine, abacavir and stavudine (Wang et al. 2003b, Kis et al. 2010a, Ashraf et al. 2013).

Furthermore, in vivo studies using Bcrp knockout mice showed enhanced brain distribution of

abacavir, suggesting the role of BCRP in limiting NRTIs brain entry (Giri et al. 2008). PIs and

NNRTIs are not transported by BCRP, but these agents have been identified to inhibit BCRP transport, implicating their potential role in drug-drug interactions (Gupta et al. 2004, Weiss et al. 2007a). MRP transporters found in the intestine, liver, kidney, lymphocytes and blood-tissue barriers (i.e., BBB) also play an important role in the distribution of antiretroviral drugs. Most

PIs (e.g. atazanavir, ritonavir, lopinavir) are substrates and inhibitors of MRP1 and MRP2 (Van der Sandt et al. 2001, Huisman et al. 2002, Dallas et al. 2004a, Janneh et al. 2005, Janneh et al.

2007, Zastre et al. 2009). MRP4 and MRP5, exhibiting unique ability of transporting cyclic nucleotides, are found to transport several NRTIs (i.e., abacavir, zidovudine, stavudine and tenofovir) (Schuetz et al. 1999, Reid et al. 2003, Borst et al. 2004, Mallants et al. 2005, Bousquet et al. 2008). Although MRP7, MRP8 and MRP9 have been identified to transport lopinavir, atazanavir, ritonavir and zidovudine, their contribution in antiretroviral drugs distribution remains unclear (Guo et al. 2003, Bierman et al. 2010). In addition, many NNRTIs and NRTIs were identified as inhibitors of MRP1, MRP2 and MRP3 in in vitro transport assays, yet only efavirenz and emtricitabine showed significant inhibitory effect at clinical concentrations (Weiss et al. 2007b). In vivo treatment of mice with the MRP inhibitor, MK571, led to an enhanced brain accumulation of saquinavir, suggesting MRPs (i.e., MRP1 and MRP2) can contribute in limiting brain permeability of PIs (Park & Sinko 2005). Recent in vitro findings suggest a potential role of SLC transporters (e.g., OATPs, OATs, OCTs, Concentrative Nucleoside Transporters and

Equilibrative Nucleoside Transporters) in drug-drug interactions of antiretroviral drugs

(Cvetkovic et al. 1999, Campbell et al. 2004, Su et al. 2004, Demby 2008, Zhang et al. 2000,

Takeda et al. 2002, Bleasby et al. 2005, Uwai et al. 2007, Jung et al. 2008, Ritzel et al. 1997,

Lostao et al. 2000, Ritzel et al. 2001, Yao et al. 2001, Cano-Soldado et al. 2004, Baldwin et al.

2005). However, the current role of these transporters in affecting (i.e., enhancing) clinical brain permeability of antiretroviral drugs is poorly defined (Kis et al. 2010a, Ashraf et al. 2013). A more comprehensive discussion on the interaction between antiretroviral drugs and SLC transporters can be found in our recent published reviews (Kis et al. 2010a, Ashraf et al. 2013).

1.4.3.2 Antiretroviral Drug Interactions with Drug Metabolizing Enzymes

Several drug-metabolizing enzymes are involved in phase I and II metabolism of antiretroviral drugs, playing an important role in antiretroviral drugs disposition (Michaud et al.

2012). Enzymes belonging to the superfamily of CYPs generally metabolize lipophilic compounds into more water-soluble ones. In HIV pharmacotherapy, members from the CYP2 and CYP3 families are mainly involved in the metabolism of NNRTIs, PIs and maraviroc (Table

1-12). In particular, hepatic CYP2B6 expression is involved in the metabolism of NNRTIs, such as efavirenz and nevirapine (Wang & Tompkins 2008). In addition to being a substrate of

CYP2B6, efavirenz has been shown to induce CYP2B6 expression (Robertson et al. 2008, Zhu et al. 2009). In particular, hepatic CYP2C19 expression is found to primarily metabolize NNRTIs, including nelfinavir and etravirine (Michaud et al. 2012). Furthermore, CYP3A4 is the major enzyme involved in hepatic clearance and intestinal elimination of many antiretroviral drugs including all members of PIs and NNRTIs and maraviroc (Michaud et al. 2012). In HIV pharmacotherapy, the major advantage regarding co-administration of ritonavir with other PIs is that ritonavir serves as a potent CYP3A4 inhibitor and significantly decreases hepatic CYP3A4 activity and significantly enhances the bioavailability (substrates of CYP3A4) (Ribera & Curran

2008). Aside from CYP enzymes, glucuronidation mediated by UGTs also plays an essential role in phase II metabolism of many antiretroviral drugs. For example, UGT1A1 is involved in the metabolism of the NRTI, abacavir and integrase inhibitor, raltegravir (Kassahun et al. 2007,

Yuen et al. 2008), whereas UGT2B7 metabolizes zidovudine and efavirenz (Barbier et al. 2000,

Bélanger et al. 2009).

Table 1-12. Metabolism patterns of antiretroviral drugs.

Antiretroviral Drugs Major Metabolism *

PIs Amprenavir CYP3A4 Atazanavir CYP3A4 Darunavir CYP3A4 Lopinavir CYP3A4 Saquinavir CYP3A4 Nelfinavir CYP2C19 & CYP3A4 Ritonavir CYP3A4

NRTIs Abacavir UGT1A1 & Alcohol Dehydrogenase Didanosine Endogenous Purines Metabolism Emtricitabine Thiol Oxidation & UGT 1A1 Lamivudine Negligible Tenofovir Negligible Zidovudine Oxidation & UGT2B7

NNRTIs Efavirenz CYP2B6, CYP 3A4 & UGT2B7 Nevirapine CYP 3A4 and 2B6 Integrase Inhibitor UGT1A1 Raltegravir Entry Inhibitor CYP 3A4 Maraviroc * Drug elimination half life from HIV infected patients and information on major metabolizing pathways was obtained from The Immunodeficiency Clinic, www.hivclinic.ca. Assessed in March 2013.

1.4.3.3 Antiretroviral Drug Interactions with Nuclear Receptors

Many nuclear receptors and transcription factors are involved in the regulation of the expression of drug transporters and drug metabolizing enzyme expression, which plays a crucial role in antiretroviral drug distribution and elimination (Urquhart et al. 2007, Kis et al. 2010a,

Michaud et al. 2012). Among these regulatory pathways, only two nuclear receptors, hPXR and hCAR, have been suggested to interact with a few antiretroviral drugs (Table 1-13). HIV PIs (i.e. ritonavir, amprenavir and lopinavir) were the first group of antiretroviral drugs identified to activate hPXR as determined by reporter-based assays (Dussault et al. 2001). Later, a NNRTI, efavirenz, was also identified to induce in vitro CYP3A4 promoter activity in the presence of a full-length hPXR (Hariparsad et al. 2004). Moreover, in vitro exposure to PIs (i.e., saquinavir, indinavir, amprenavir, lopinavir, tipranavir and atazanavir) was reported to induce CYP3A4 promoter activity mediated by hPXR (Gupta et al. 2008). Recently, Svärd et al. reported distinctive findings in which only amprenavir, lopinavir, tipranavir and nelfinavir from the PIs class could induce CYP3A4 promoter activity mediated by hPXR. Although hPXR is known to regulate CYP2B6, activation of CYP2B6 promoter was only observed with darunavir and lopinavir (Svärd et al. 2010). Nevertheless, these authors were able to demonstrate that efavirenz can activate hPXR activity, which is consistent with in vitro findings reported previously in the literature (Hariparsad et al. 2004). Interactions between antiretroviral drugs with hCAR have not been extensively examined. To date, only one piece of evidence suggests that abacavir, amprenavir and lopinavir may activate hCAR activity, however the low sensitivity of the in vitro assay used in the study was unable to conclusively confirm that these agents can activate hCAR

(Svärd et al. 2010). In addition, despite availability of hPXR and hCAR X-ray crystallography structures, structural information on how antiretroviral drugs interact with the receptor ligand

binding pocket is unclear (Xu et al. 2004, Xue et al. 2007). Interestingly, a recent study has successfully predicted ligand activation of hPXR by efavirenz using an in silico ligand-docking approach, however the specific interactions between chemical structures of antiretroviral drugs and hPXR or hCAR ligand binding pocket have not been investigated (Khandelwal et al. 2008).

Currently, interactions between antiretroviral drugs and nuclear receptors, in particular hPXR and hCAR, have not been fully addressed.

Table 1-13. Antiretroviral drug interactions with hPXR and hCAR.

Antiretroviral Interactions with hPXR Interactions with hCAR Drugs PIs Amprenavir Not Known Not Known Atazanavir Not a ligand (Svärd et al. 2010) Not Known Darunavir Not Known Not Known Lopinavir YES (Svärd et al. 2010) YES (Svärd et al. 2010) YES (Dussault et al. 2001) and Ritonavir Not Known Not a ligand (Svärd et al. 2010) NRTIs Abacavir Not Known YES (Svärd et al. 2010) Emtricitabine Not Known Not Known Lamivudine Not a ligand (Svärd et al. 2010) Not Known Tenofovir Not a ligand (Svärd et al. 2010) Not Known Zidovudine Not a ligand (Svärd et al. 2010) Not Known NNRTIs YES (Hariparsad et al. 2004, Svärd Efavirenz Not Known et al. 2010) Nevirapine Not a ligand (Svärd et al. 2010) Not Known Integrase Inhibitor Raltegravir Not Known Not Known Entry Inhibitor Maraviroc Not a ligand (Svärd et al. 2010) Not Known

Our recent work regarding the interaction between several antiretroviral drugs and hPXR and hCAR is now published and can be found in Chapter 8 of this thesis (Chan et al. 2013b).

2. Goal

ABC membrane-associated drug efflux transporters, in particular P-gp, expressed at the

BBB are important determinants of antiretroviral drug disposition in the brain. The role of regulatory pathways (e.g., nuclear receptors) in governing P-gp expression is currently unclear at the human BBB. The overall goal of this thesis is to examine the regulation of P-gp functional expression by two nuclear receptor, hPXR and hCAR, in vitro in cell culture model of human brain microvessel endothelial cells and in vivo in a mouse model.

3. Rationale

Efficacy of CNS-targeted drugs is often impaired by limited drug entry into the brain. It has long been recognized that drug permeability into the CNS can be restricted by the BBB. The functional expression of several ABC drug efflux transporters, in particular P-gp, is known to restrict antiretroviral agent permeability across the BBB. In hepatic and intestinal tissues, P-gp expression is tightly regulated by two nuclear receptors, PXR and CAR, whose transcriptional activity can be altered upon binding to a wide range of endogenous and exogenous compounds, including drugs. A complete understanding of PXR and CAR regulation of P-gp expression at the human BBB could assist in the identification of potential pharmacological approaches which could alter drug transporter expression at the BBB clinically, in order to improve efficacy of

CNS drugs and/or minimize drug-associated neurotoxicity. Investigating the regulatory processes of antiretroviral drug transport into and out of the brain is of direct relevance to the prevention and treatment of HIV-1 induced CNS injury. It is relevant both for guiding the search of new antiretroviral drugs that will be more effective at targeting the CNS and for elucidating mechanisms related to pharmacological response during HIV infection of the brain. Moreover,

results from this work could potentially be relevant to the pharmacotherapy of other CNS disorders.

4. Hypotheses

1. Orphan nuclear receptors, such as PXR and CAR, regulate the functional expression of

drug efflux transporters (i.e., P-gp) at the BBB and are important determinants of drug

distribution in the brain.

2. Several antiretroviral drugs currently used in the clinic are ligands of hPXR and hCAR,

and alter functional expression of drug efflux transporters (i.e., P-gp) at the BBB.

5. Specific Objectives

1. To examine the cellular localization and transport function of P-gp in human cerebral

microvascular endothelial cells/clone D3 (hCMEC/D3) cell culture system.

2. To characterize PXR and CAR expression in primary cultures of human brain

microvascular endothelial cells (BBB-ECs) and hCMEC/D3 cell culture system.

3. To investigate the role of hPXR and hCAR in the regulation of P-gp in hCMEC/D3

cell culture system.

4. To assess whether hPXR and hCAR can be activated by antiretroviral agents

currently used in the clinic.

5. To examine P-gp functional expression in hCMEC/D3 cell culture system following

treatment with antiretroviral drugs identified as ligands of hPXR and/or hCAR.

6. To investigate, in vivo (i.e., mouse), the effect of brain endothelial P-gp induction on

brain extracellular distribution of a P-gp substrate, quinidine, using quantitative

intracerebral microdialysis.

6. Up-regulation of p-glycoprotein by HIV protease inhibitors in a human brain

microvessel endothelial cell line

This work is published and reproduced in this thesis with permission from Wiley-Blackwell:

Zastre JA, Chan GNY, Ronaldson PT, Ramaswamy M, Couraud PO, Romero IA, Weksler B,

Bendayan M and Bendayan R. (2009) Up-regulation of P-glycoprotein by HIV protease inhibitors in a human brain microvessel endothelial cell line. Journal of Neuroscience Research.

87:1023–1036.

Chapter 6 describes work in a human cerebral microvascular endothelial cell culture system, hCMEC/D3, examining i) the functional expression of P-gp, ii) the cellular localization of hPXR, and iii) the inductive effects of two HIV PIs, atazanavir and ritonavir, and known hPXR potent agonists, rifampin and SR12813, on P-gp expression. We demonstrated P-gp protein expression in hCMEC/D3 whole cell lysates using immunoblot analysis and cellular localization of this transporter along the plasma cell membrane applying immunogold cytochemistry with electron microscopy. In a drug transport assay, P-gp functional activity in hCMEC/D3 cells was observed using a known fluorescent substrate specific to P-gp, rhodamine-

6G (R-6G) and a P-gp inhibitor, the cyclosporine analog PSC833. Furthermore, immunoblot analysis showed hPXR expression in both the cytosolic and nuclear extractions from non-treated control cells, whereas immunogold cytochemistry with electron microscopy demonstrated the cytosolic localization of hPXR in these cells. We reported for the first time in hCMEC/D3 cells that prolonged treatment (72 h) with atazanavir and ritonavir at clinically relevant plasma concentrations resulted in a significant induction of P-gp expression and function. Furthermore, cells treated with rifampin and SR12813 also showed a significant P-gp protein induction, suggesting the involvement of the hPXR pathway in regulating P-gp expression. This work

provided initial results which guided subsequent studies investigating the role of nuclear receptors in regulating P-gp expression in hCMEC/D3 cells.

Author Contributions: Research design: JA Zastre (first author), GNY Chan (second author) and R Bendayan (principal investigator) Conducted experiments and data analysis: JA Zastre (figures 6-1, 6-2, 6-4, 6-7 and 6-8), GNY Chan (figures 6-2, 6-3, 6-4, 6-6, 6-8 and 6-9), PT Ronaldson (figure 6-5), M Ramaswamy (figure 6-3) and M Bendayan (figure 6-2) Writing of the manuscript: JA Zastre (initial submission), GNY Chan (re-submission and responses to reviewers’ comments) and R Bendayan (overall conceptual and editorial review of the several manuscript drafts and responses to reviewers’ comments) Provided the hCMEC/D3 cells: PO Couraud, IA Romero and B Weksler

The specific experimental contribution of GNY Chan to this manuscript is provided in each figure legend. Cell viability data indicated as “not shown”, produced by GNY Chan, can be found in Appendix A and the initial characterization of P-gp-mediated transport of R-6G, performed by GNY Chan, can be found in Appendix B.

6.1 Abstract

A major concern regarding the chronic administration of antiretroviral drugs is the potential for induction of drug efflux transporter expression (i.e., P-gp) at tissue sites that can significantly affect drug distribution and treatment efficacy. Previous data have shown that the inductive effect of HIV PIs is mediated through the human orphan nuclear receptor, hPXR. The objectives of this study were to investigate transport and inductive properties on efflux drug transporters of two PIs, atazanavir and ritonavir, at the BBB using a human brain microvessel endothelial cell line, hCMEC/D3. Transport properties of PIs by the drug efflux transporters P- gp and MRP1 were assessed by measuring the cellular uptake of 3H-atazanavir or 3H-ritonavir in

P-gp and MRP1 overexpressing cells as well as hCMEC/D3. While the P-gp inhibitor, PSC833, increased atazanavir and ritonavir accumulation in hCMEC/D3 cells by 2 fold, the MRP inhibitor

MK571 had no effect. P-gp, MRP1 and hPXR expression and localization were examined applying immunoblot analysis and immunogold cytochemistry at the electron microscope level.

Treatment of hCMEC/D3 cells for 72h with rifampin or SR12813 (two well established hPXR ligands) or PIs (atazanavir or ritonavir) resulted in an increase in P-gp expression by 1.8, 6 and

2-fold respectively with no effect observed for MRP1 expression. In hCMEC/D3 cells, cellular accumulation of these PIs appears to be primarily limited by P-gp efflux activity. Long term exposure of atazanavir or ritonavir to brain microvessel endothelium may result in further limitations in brain drug permeability due to the upregulation of P-gp expression and function.

6.2 Introduction

The ability of HIV to enter and infect target cells in the CNS represents a significant barrier for the long term suppression of viral replication and has been linked to the development of several neurological complications (Kramer-Hämmerle et al. 2005). Although highly active

antiretroviral therapy or HAART has significantly reduced the incidence of HIV-associated dementia, the prevalence of neurological disorders appears to be increasing due to prolonged patient survival and poor antiretroviral drug penetration into the CNS (Sacktor 2002, Langford et al. 2006, Nath & Sacktor 2006). Furthermore, subtherapeutic drug concentrations in the CNS may facilitate the development of viral resistance (Smit et al. 2004).

One major reason for the low penetration of antiretroviral drugs into brain parenchyma appears to be due to the functional expression of membrane-associated drug efflux transporters belonging to the ABC family of transporters, such as P-gp, MRPs, and BCRP. The localization of efflux transporters at the luminal membrane of brain microvessel endothelia, an important component of the BBB, constitutes an effective biochemical barrier which significantly limits drug entry into the CNS (Beaulieu et al. 1997, Regina et al. 1998, Bendayan et al. 2002, Aronica et al. 2005, Bendayan et al. 2006). Antiretroviral drugs, such as the HIV PIs, are well recognized substrates and/or inhibitors of drug efflux transporters and interactions with these transporters at the BBB have been demonstrated to effectively limit CNS penetration (Ronaldson et al. 2004b,

Gupta et al. 2004, Choo et al. 2000, Lee et al. 1998, Park & Sinko 2005, Bachmeier et al. 2005).

Numerous PIs have been shown to induce the expression of drug efflux transporters, such as P-gp and MRP1 in a human intestinal epithelial cell line (Perloff et al., 2000; Perloff et al.,

2001). For example, it has been reported that nelfinavir and efavirenz can increase the expression of P-gp in isolated peripheral blood mononuclear cells (Chandler et al., 2003). Although a few groups have demonstrated an up-regulation of drug efflux transporters at the BBB using prototypical inducing agents (Bauer et al., 2004; Perloff et al., 2007; Zong and Pollack, 2003), limited studies have investigated the inductive effect of PIs on the expression of drug efflux transporters at the human BBB and the mechanisms by which this phenomenon occurs.

In the past few years, nuclear receptors such as rodent PXR and human steroid xenobiotic receptor, hPXR or SXR, have been recognized as species-specific xenosensors that may regulate the expression of metabolic enzymes and drug efflux transporters (Staudinger et al. 2003, Francis et al. 2003). Recent evidence has suggest that PXR plays a much greater role in the regulation of

P-gp expression as compared to other nuclear receptors, such as the GR and CAR (Stanley et al.

2006, Urquhart et al. 2007). Furthermore, it has been shown that HIV PIs, particularly ritonavir, are ligands for hPXR and can up-regulate P-gp expression (Dussault et al. 2001).

Atazanavir, a new azapeptide PI, is currently recommended in ritonavir boosted and unboosted HAART regimens for the treatment of HIV infection (Hammer et al. 2006). The major advantages of atazanavir over other PIs include its greater potency, distinct cross- resistance profile, once daily administration and lower incidence of adverse effects on blood lipid profiles compared to other PIs (Goldsmith & Perry 2003). Recently, atazanavir has been shown to inhibit and induce the functional expression of P-gp in intestinal epithelial cells (Perloff et al.

2005). In addition, this PI can also inhibit the efflux activity of MRP1 and P-gp in isolated human peripheral blood lymphocytes and hemopoietic stem cells (Lucia et al. 2005). At present, little information is known regarding atazanavir transport properties and potential inductive properties on drug efflux transporters at the human BBB. The objectives of this study were to investigate the transport and inductive properties of atazanavir and ritonavir and evaluate interactions between atazanavir and ritonavir using a newly established and well characterized human brain microvessel endothelial cell line, hCMEC/D3, as an in vitro model of the human

BBB.

6.3 Material and Methods

6.3.1 Reagents

Pure unlabeled PIs (i.e., atazanavir and ritonavir) were obtained through the National

Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, NIAID,

NIH (Bethesda, MD). The P-gp inhibitor PSC833 (i.e., valspodar) was generously donated by

Novartis Pharma. 3H-atazanavir (1 Ci/mmol), and 3H-ritonavir (2 Ci/mmol) were purchased from Moravek (Brea, CA). 2', 7’-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and the acetoxymethyl ester derivative (BCECF-AM) were obtained from Invitrogen (Grand Island,

NY). Rhodamine 6G (R-6G), dimethyl sulfoxide (DMSO), rifampin, and the murine monoclonal anti-actin antibody (AC40) were purchased from Sigma-Aldrich (Oakville, ON). The MRP inhibitor, MK571 and the hPXR ligand, SR12813 were purchased from Biomol (Plymouth, PA).

The murine monoclonal anti-P-gp antibody (C219) was purchased from ID Labs (London, ON) and the rat monoclonal anti-MRP1 antibody (MRPr1) was obtained from Kamiya Biomedical

(Seattle, WA). The rabbit polyclonal anti-hPXR antibody (H-160), the goat polyclonal anti- hPXR antibody (A-20) and the goat polyclonal anti-Lamin B antibody (M-20) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All tissue culture reagents were obtained from Invitrogen (Grand Island, NY) unless otherwise stated.

6.3.2 Cell Culture

The human brain microvessel endothelial cell line, hCMEC/D3, was generously provided by Dr. P.O. Couraud (Université René Descartes, Paris, France). This cell line provides an excellent long-lasting alternative to primary cultures of mammalian microvessel endothelial cells and displays many BBB markers and most properties of brain endothelial in vivo, such as tight junction formation. As well, it expresses a number of drug efflux transporters, P-gp, MRP1 and

BCRP (Weksler et al., 2005). Cells were maintained at 37°C, 5% CO2, and 95% humidified air in EGM-2 media (Cambrex, Walkersville, MD) supplemented with vascular endothelial growth factor, insulin-like growth factor 1, epidermal growth factor, fibroblast growth factors, hydrocortisone, ascorbate, gentamycin, and 2.5% fetal bovine serum and grown on rat tail collagen type-1 coated flasks and 24 well plates as previously described (Weksler et al., 2005).

The P-gp-overexpressing human breast carcinoma cell line MDA435/LCC6/MDR1 and parental cell line MDA435/LCC6/WT were generous gifts from Dr. Robert Clarke (Georgetown

University, Washington, DC). MDA435/LCC6-WT and MDR1 cells were grown in alpha- minimum essential media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The MRP1 overexpressing cell line and the parent HeLa-WT cells were maintained in Dulbecco’s Modified Eagle’s Media supplemented with 10% FBS and 400 µg/mL

G418 (Dallas et al., 2003; Ito et al., 2001). Cells grown to confluence (5–6 days for HeLa-MRP1 cell lines, 5–7 days for MDA435 cell lines after seeding approximately 0.1 million cells/dish) were subcultured with 0.05% trypsin-EDTA (Invitrogen Inc., Burlington, ON), diluted (1/10) in fresh growth medium, and reseeded.

6.3.3 Cell Viability Assay

Cell viability was assessed in hCMEC/D3 cells treated with atazanavir and ritonavir using an MTT assay as described previously with modification (Denizot & Lang 1986). Briefly, hCMEC/D3 cells were incubated with 1, 5 and 10 µM atazanavir, ritonavir, rifampin or SR12813 for 72h as described above. The cells were then incubated for 4 hours at 37 ºC with a 2.5 mg/mL, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) solution in

Dulbecco’s phosphate buffered saline (PBS). The MTT solution was then removed and the cells washed with PBS followed by the addition of DMSO. The formazan content of each well was

determined by UV analysis at 580 nm using a SpectraMax 384 microplate reader (Molecular

Devices, Sunnyvale, CA). Cell viability was expressed as the treatment absorbance / control absorbance.

6.3.4 Immunocytochemistry

For the ultrastructure detection of P-gp and hPXR, cells cultured under standard conditions were fixed with paraformaldehyde-lysine-periodate for 24 h and processed for embedding in Lowicryl K4M as described in details previously (Bendayan et al. 2006). Thin sections of the cells were first treated with a saturated solution of sodium metaperiodate for

10min. The grids were then blocked with 1% ovalbumin and then incubated with the MRK16 antibody (1:10), which recognizes either the first or fourth external epitope of human and rat P- gp, or the H-160 antibody (1:10), which recognizes amino acid 101-260 of human PXR, for 24 h at 4ºC. Grids were then washed with PBS and incubated with protein A-gold complex (P-gp)

(Sigma-Aldrich) or anti-rabbit-gold complex (hPXR) (British Biocell International, Cardiff, UK) for 30 min. The gold complexes were prepared with 10 nm gold particules as described previously (Bendayan 1995). Upon counterstaining with uranyl acetate the grids were examined with a Philips 410 electron microscope. Controls of specificity were carried out by incubating the grids in the absence of primary antibody.

6.3.5 Cytosolic and Nuclear Cell Fractions

Cytosolic and nuclear cell fractions were prepared according to a previously published method (Pascussi et al. 2000). Cells were washed, harvested in ice-cold PBS and pelleted by centrifugation at 1500 g for 5 min. The pellet was resuspended in cytoplasmic lysis buffer

(10mM HEPES, 10mM KCl, 0.1mM EDTA, 0.1mM EGTA, 1mM dithiothreitol, 0.5mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail) and put on ice for 15 min

to allow for cell swelling. After which NP-40 was added (0.15% v/v) and gently mixed for

1min. Cells were then centrifuged for 5 min to pellet intact nuclei and the supernatant was carefully collected (cytoplasmic fraction). The pelleted nuclei were washed with PBS to remove excess cytoplasmic fraction and were centrifuged for 5 min. Pelleted nuclei were resuspended in ice-cold nuclear lysis buffer (20mM HEPES, 1.2M NaCl, 1mM EDTA, 1mM EGTA, 1mM dithiothreitol, 0.5mM PMSF, 0.1 % (v/v) protease inhibitor cocktail and DNaseI) at 4°C for 30 min. The nuclear extract was centrifuged for 5 min and the supernatant was stored at -80°C.

6.3.6 Drug Accumulation Assays Using P-glycoprotein and MRP1 Over-expressing Cells

P-gp substrate properties were assessed by comparing the cellular accumulation of atazanavir or ritonavir in a P-gp overexpressing human breast cancer cell line; MDA435/LCC6-

MDR1 with the parent MDA435/LCC6-WT cells (Leonessa et al., 1996). Cells were seeded into

48 well plates and used for accumulation studies upon confluency. Throughout this work, the accumulation assay buffer was composed of HBSS, 10 mM HEPES and 0.01% bovine serum albumin (BSA), pH = 7.4. The incorporation of 0.01% BSA in the assay buffer reduces adherence of atazanavir and ritonavir to plastic or glass, and ensures mass balance at the conclusion of the experiments. Cells were pre-equilibrated with accumulation assay buffer for

15 min, followed by addition of either 1 µM 3H-atazanavir or 3H-ritonavir for 60 min at 370C.

Cells were then washed with ice cold PBS and harvested with 1M NaOH for 30 min, then neutralized with HCl prior to liquid scintillation counting (Beckman Coulter, Fullerton, CA).

Cell accumulation was normalized to total protein content determined by the Bradford colorimetric method using BSA (Sigma-Aldrich) as the standard.

To evaluate the MRP1 mediated transport properties of atazanavir and ritonavir, experiments were performed as described above using the MRP1 overexpressing cell line HeLa-

MRP1 and the parent HeLa-WT (Dallas et al. 2003, Ito et al. 2001).

6.3.7 Drug Accumulation Assays Using hCMEC/D3 Cells

In vitro BBB transport properties of atazanavir and ritonavir were determined using the immortalized human brain microvessel endothelial cell line, hCMEC/D3 (Weksler et al. 2005).

Cells were cultured as described above. The cellular accumulation of the well established P-gp substrate, R-6G, was measured to determine the functional activity of P-gp in hCMEC/D3 cells

(Lampidis et al. 1989). Confluent hCMEC/D3 cells were exposed to 1 µM R-6G for 60 min in the presence of the P-gp inhibitor PSC833 (1 μM) and 10 µM of atazanavir or ritonavir. Cells were then washed with ice cold PBS and harvested with 1% triton X-100. Cell associated fluorescence was measured at an excitation wavelength = 530nm and emission wavelength =

560nm (SPECTRAmax, Molecular Devices, Sunnyvale, CA). Results were normalized to total protein content using the colorimetric Bradford method.

The cellular retention of the MRP substrate, BCECF, was utilized as a measure for MRP related functional activity in hCMEC/D3 cells as previously described with a few modifications

(Bachmeier et al. 2004). Briefly, confluent cells were washed and pre-incubated at 37°C for 30 min with accumulation assay buffer followed by incubation with the cell permeable BCECF-AM

(5 µM) for 120 min in the presence of the MRP inhibitor MK571 and 10 µM atazanavir or ritonavir. Since BCECF-AM is a known P-gp substrate (Bachmeier et al. 2004), all incubations were performed in the presence of 1.0 µM PSC833. At the end of the incubation time, the medium was aspirated and the reaction was terminated with ice cold PBS. The cells were then solubilized using 1% triton-X-100 and the cellular retention of BCECF was measured at an

excitation wavelength of 505 nm and an emission wavelength of 535 nm using a fluorescent plate reader (SPECTRAmax, Molecular Devices, Sunnyvale, CA) and normalized to total protein.

P-gp or MRP mediated transport of 1 µM 3H-atazanavir or 1 µM 3H-ritonavir in hCMEC/D3 cells was assessed in the presence of increasing concentrations of the P-gp inhibitor

PSC833 or the MRP inhibitor MK571. The effect of ritonavir on atazanavir transport was determined by measuring 3H-atazanavir (1 µM) cellular accumulation with increasing concentrations of ritonavir (0.1 to 50 µM). A similar approach was used to assess the effect of atazanavir on ritonavir cellular accumulation. Cells were washed and harvested prior to liquid scintillation counting as described above.

6.3.8 Induction of Drug Efflux Transporters in hCMEC/D3 Cells

Changes in P-gp and MRP1 expression in hCMEC/D3 cells were measured after 72h treatment with 1, 5 and 10 µM atazanavir or ritonavir. Changes in P-gp expression were measured after 72h treatment with well established hPXR ligands, rifampin or SR12813 at concentrations of 1, 5 and 10 µM. SR12813 is an inhibitor of HMG-CoA reductase activity and is a potent hPXR ligand (EC50 200nM) (Moore et al. 2002). Cells were cultured in 10 cm collagen coated Petri dishes and treated with each compound once cells were approximately 75% confluent. Atazanavir or ritonavir was added to the cell culture media from stock solutions prepared in 100% DMSO. Stock solutions were designed so that the cells were exposed to a final

DMSO concentration of 0.1%. Control cells were exposed to 0.1% DMSO in the absence of atazanavir or ritonavir. After 72 h exposure, the cells were scrapped and subsequently washed three times with ice cold PBS. Cells were then lysed for 15 min at 40C using a lysis buffer composed of 1% (v/v) NP-40 in 20mM Tris, 150mM NaCl, 5mM EDTA at pH=7.5 containing 1 mM PMSF and 0.1% (v/v) protease inhibitor cocktail. The cell lysate was then sonicated for 10 s

followed by centrifugation (40C) at 20,000 g for 10 min. An aliquot containing 50 µg protein was resolved on a 12% SDS-polyacrylamide gel (SDS-PAGE) with MDA435/LCC6-MDR1 and

HeLa-MRP1 cell lysates as positive controls for P-gp and MRP1, respectively. The gel was electrotransferred onto a polyvinylidene difluoride (PVDF) membrane. For P-gp detection, the blot was incubated for 3 h with a 1:500 dilution of C219 monoclonal anti-P-gp antibody (ID

Labs, London, ON), which recognizes a conserved intracellular epitope (VQEALD) on all human

(i.e., MDR1, MDR2) isoforms of P-gp (Okochi et al. 1997), followed by 1.5 h incubation with

1:3000 anti-mouse horseradish peroxidase-conjugated secondary antibody (Serotec, Raleigh,

NC). MRP1 was detected by three hour incubation with 1:250 dilution of MRPr1 monoclonal antibody, which recognizes an internal epitope of human Multidrug Resistance Protein 1

(MRP1), followed by a 1.5 h incubation of 1:4000 anti-rat horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich). hPXR was detected after 3 h incubation with 1:100 dilution of A-20 polyclonal antibody, which recognizes PXR.1 unique domain, and the sequential

1.5h incubation with 1:5000 anti-goat horseradish peroxidase-conjugated secondary antibody

(Sigma-Aldrich). A goat polyclonal antibody (M-20) was used to detect Lamin B, a nuclear envelope marker.

Actin was used for loading control and detected using the monoclonal AC40 antibody at a

1:500 dilution. Protein bands were visualized by enhanced chemiluminescence (SuperSignal,

Perkin Elmer) and the change in P-gp or MRP1 expression was estimated from densitometry analysis (Alpha DigiDoc RT2 imaging software, Alpha Innotech, San Leandro, CA). Data is reported as fold change in P-gp or MRP1, which was determined by the treatment:actin ratio / control:actin ratio.

The time dependent cellular accumulation of 1 µM R-6G was used to measure P-gp function associated with changes in P-gp expression after hCMEC/D3 cells were incubated for

72 h with 5 µM atazanavir or ritonavir. In addition, accumulation of 1 µM R-6G with or without

5 µM PSC833 for 30 min was also determined in cells treated for 72 h with 5 µM atazanavir or ritonavir. Cells were washed every 30 min with cell culture media over a period of 2 h to washout cell-associated atazanavir and ritonavir prior to all accumulation assays involving induced hCMEC/D3 cells. R-6G accumulation assay was then performed as described above.

6.3.9 Statistical Analysis

All results are expressed as the mean +/- SD of a minimum of three (n = 3) independent experiments in cells pertaining to different passage numbers (passage 30 to 40). Comparisons between groups were performed using either two tailed Student t-test or one way analysis of variance (ANOVA) with Tukey posthoc comparisons determined using SigmaStat v2.0 software

(SPSS Inc, Chicago, IL). A value of p<0.05 was considered to be statistically significant.

6.4 Results

6.4.1 P-gp and MRP1 Transport of two HIV-1 Protease Inhibitors: Atazanavir and Ritonavir

Figure 6-1 demonstrates the P-gp and MRP1 transport of atazanavir and ritonavir using

MDA435/LCC6-MDR1 and HeLa-MRP1 overexpressing cell systems, respectively. 5.3-fold and 2.8-fold reductions in atazanavir and ritonavir cellular accumulation were found in MDR1 overexpressing cells compared to wild type, respectively (Figure 6-1A). A 1.6- and 1.7- fold reduction in atazanavir and ritonavir accumulation was observed in MRP1 overexpressing cells compared to wild type, respectively (Figure 6-1B). These data suggest that both P-gp and MRP1 are involved in the transport of atazanavir and ritonavir.

Figure 6-1. P-gp (A) and MRP1 (B) substrate properties of atazanavir and ritonavir in MDA-435/LCC6-MDR1 and HeLa-MRP1 overexpressing cells and the corresponding wild type cell lines. Confluent cell monolayers were exposed to either 1 µM 3H-atazanavir or 1 µM 3H-ritonavir for 60 min at 37 °C. Data represent mean +/- SD with n=3 independent experiments. () Statistically significant differences between overexpressing and wild type cellular accumulation using Student t-test with p<0.05. Transport assays were performed by Dr. JA Zastre.

6.4.2 Localization and Expression of Efflux Transporters and hPXR in hCMEC/D3 Cells

Ultrastructural examination of the cell culture system showed the existence of a thin tight monolayer of flattened squamous cells (Figure 6-2A). Labeling for P-gp showed the presence of this transporter primarily along the plasma membrane (Figure 6-2B). For hPXR, the labeling was primarily observed in the cell cytoplasm in non-treated cells (Figure 6-2C). For both P-gp and hPXR labelling experiments, mitochondria were devoid of labeling and omission of the primary antibodies resulted in negligible number of randomly distributed gold particles. Using specific monoclonal or polyclonal antibodies, immunoblot analysis of non treated control cells revealed the presence of P-gp at the expected molecular weight (i.e., approximately 170 kDa,

Figure 6-3A), MRP1 ( i.e., approximately 190 kDa, Figure 6-3B) and hPXR in cytosolic and nuclear cell extraction (i.e., approximately 50 kDa, Figure 6-3C). Pelleted nuclei were viewed under a Nikon-TMS microscope at 40X magnification to ensure intact nuclei. Lamin B, a nuclear envelope marker, was not detected in cytosolic fractions suggesting minimal nuclear lysis takes place.

Figure 6-2.Electron micrograph of human microvessel endothelial hCMEC/D3 cells. (A) Low magnification of the cell culture illustrating the flat squamous cells forming the monolayer, bar = 5µm. (B) Immunocytochemical localization of P-gp. The labeling by gold particles is particularly seen associated with the plasma membrane (PM), bar = 0.2µm. (C) Immunocytochemical localization of hPXR. The labeling by gold particles is predominantly seen in cell cytoplasm (cyt), bar = 1µm. Cell fixing was performed by GNY Chan and electron micrographs were provided by Dr. M Bendayan.

Figure 6-3.P-gp, MRP1 and hPXR expression in hCMEC/D3 cells. Immunoblot analysis on (A) P-gp expression in whole cell lysate of hCMEC/D3 cells and MDCK cells transfected with human MDR1 (+, positive control). (B) MRP1 expression in whole cell lysate of hCME C/D3 cells and MRP1 overexpressing human cervical carcinoma cell line (MRP1- HeLa) (+, positive control). (C) Cytosolic and nuclear hPXR expression in hCMEC/D3 cells and whole cell lysate of human colorectal adenocarcinoma cells (COLO320) (CE, cytosolic extract; NE, nuclear extract; +, hPXR positive control). A 50 µg load of protein sample was resolved on a 12% SDS-PAGE gel and subsequently transferred to PVDF membrane. P-gp was detected using the monoclonal antibody C219 (1:500 dilution), MRP1 was detected using the monoclonal antibody MRPr1 (1:250 dilution), hPXR was detected using the polyclonal antibody A-20 (1:100 dilution) and lamin B was detected using the polyclonal antibody M-20 (1:200). Immunoblot analysis was performed by GNY Chan and M Ramaswamy.

6.4.3 Cell Viability after Long Term Exposure to Atazanavir, Ritonavir, Rifampin and

SR12813

The MTT assay was applied to evaluate the viability of hCMEC/D3 cells after 72 h exposure to increasing concentrations of PIs (atazanavir, ritonavir) and two well established hPXR ligands, rifampin and SR12813. Cell viability of hCMEC/D3 cells was greater than 90% of a concentration range of 1 to 10 µM for all compounds used in the induction studies (data not shown).

6.4.3.1 P-gp Mediated Transport of Atazanavir and Ritonavir in hCMEC/D3 Cells

To confirm functional activity of P-gp in hCMEC/D3 cells, the accumulation of the P-gp substrate R-6G was evaluated in the presence and absence of PSC833, a non immunosuppressive cyclosporine A analog and known P-gp inhibitor. A significant increase in R-6G accumulation by hCMEC/D3 cells was observed in the presence of 1 µM PSC833 compared to control cells, confirming functional activity of P-gp (Figure 6-4A). These data suggest that P-gp is functional in the hCMEC/D3 model. Additionally, accumulation of R-6G was assessed in the presence of ritonavir or atazanavir to determine if these HIV-1 PIs can reduce P-gp mediated transport.

Thus, the retention or increase in cellular R-6G fluorescence suggest P-gp related transport inhibition. Figure 6-4A demonstrates that ritonavir (10 µM) increased R-6G accumulation while atazanavir had no significant effect on the P-gp transport of R-6G. Figure 6-4B shows the fold enhancement in hCMEC/D3 cellular accumulation of atazanavir and ritonavir in the presence of increasing concentrations of PSC833. Both atazanavir and ritonavir accumulation were enhanced by PSC833 up to a maximum of approximately 1.9 and 1.7 fold, respectively (Figure 6-4B).

Taken together, these results suggest cellular accumulation of atazanavir and ritonavir can be modified by the transport function of P-gp.

Figure 6-4. Cellular accumulation of atazanavir, ritonavir, and the P-gp substrate R-6G by hCMEC/D3 cells. (A) Cellular uptake of R-6G was determined by exposing confluent hCMEC/D3 cells to 1 µM R-6G in accumulation assay buffer with or without the P-gp inhibitor

PSC833 (1 µM) or 10 µM atazanavir or 10 µM ritonavir for 60 min at 37 °C. (B) Cellular accumulation of () 1 µM 3H-atazanavir or () 1 µM 3H-ritonavir, with increasing concentrations of the P-gp inhibitor PSC833 for 60 min at 370C. Data represent mean ± S.D. for n=3 independent experiments. () Statistically significant differences between treatment groups to control using ANOVA with Tukey pairwise comparisons at a significance level of p < 0.05.

Transport assays were performed by Dr. JA Zastre (atazanavir and ritonavir) and GNY Chan

(R-6G).

6.4.3.2 MRP Related Transport of Atazanavir and Ritonavir in hCMEC/D3 Cells

MRP mediated transport activity in hCMEC/D3 cells was assessed using BCECF, a well established fluorescent probe for MRP-mediated transport activity. Cells were exposed to the cell permeable ester derivative, BCECF-AM, which is converted by intracellular esterases to the fluorescent MRP substrate, BCECF (Bachmeier et al. 2004).

BCECF accumulation was significantly increased in the presence of the MRP inhibitor

MK571, confirming MRP related transport activity in hCMEC/D3 cells (Figure 6-5A). Similar to the results for P-gp transport inhibition, only ritonavir (10 µM) demonstrated a significant 2- fold increase in BCECF accumulation compared to control suggesting that ritonavir is an MRP inhibitor (Figure 6-5A). In addition, the cellular accumulation of atazanavir and ritonavir was assessed in the presence of MK571 to evaluate MRP related transport in hCMEC/D3 cells. No change in atazanavir or ritonavir accumulation was observed up to concentrations of 100 µM

MK571, suggesting that MRP does not play a significant role in the transport of atazanavir or ritonavir in this cell culture system (Figure 6-5B).

Figure 6-5. Cellular accumulation of atazanavir, ritonavir and the MRP substrate BCECF by hCMEC/D3 cells. (A) Cellular accumulation of BCECF was determined by exposing confluent hCMEC/D3 cells to 5 µM BCECF-AM in accumulation assay buffer with or without the MRP inhibitor MK571 (5 and 50 µM) or 10 µM atazanavir or 10 µM ritonavir for 120 min 3 at 37 °C, all in the presence of 1 µM PSC833. (B) Cellular accumulation of () 1 µM H- atazanavir or () 1 µM 3H-ritonavir, with increasing concentrations of the MRP inhibitor MK571 for 60 min at 370C. Data represent mean ± S.D. for n = 3 independent experiments. () Statistically significant differences between treatment groups to control using ANOVA with Tukey pairwise comparisons at a significance level of p < 0.05. Transport assays were performed by PT Ronaldson.

6.4.4 P-gp Expression in hCMEC/D3 Cells after Exposure to Selective Human PXR Ligands,

Rifampin and SR12813

In order to determine if P-gp can be regulated by nuclear receptors, such as hPXR, in the hCMEC/D3 cell line, rifampin and SR12813, two well established hPXR ligands known to induce P-gp expression in other cell culture systems and in vivo, were used (Bauer et al. 2006,

Geick et al. 2001, Hennessy et al. 2002, Masuyama et al. 2005, Stanley et al. 2006, Synold et al.

2001). A modest but significant increase (1.8-fold) in P-gp expression as determined by immunoblot analysis and subsequent densitometry analysis was observed in cells exposed to 10

µM rifampin for 72 hours. In addition, a significant and substantial increase (approximately 6 fold) in P-gp expression was observed in cells exposed for 72 h to 5 µM or 10 µM SR12813

(Figure 6-6). Thus, P-gp expression can also be induced in hCMEC/D3 cells in the presence of well utilized hPXR ligands.

Figure 6-6. Expression of P-gp following treatment of hCMEC/D3 cells with 1, 5 or 10 µM rifampin or SR12813 compound. (A) Representative immunoblot analysis of P-gp expression in untreated cells (CTL) or treated with rifampin or SR12813. Whole cell lysate of

MDCK cells transfected with human MDR1 was used as positive control (+). A 50 µg load of control and treated cell lysates were resolved on a 12% SDS-PAGE gel and subsequently transferred to PVDF membrane. (B) Semiquantitative densitometric analysis was performed to determine the relative P-gp expression changes in hCMEC/D3 cells following 72 h exposure to 1, 5 and 10 µM rifampin or SR12813. P-gp was detected using the monoclonal antibody C219 (1:500 dilution). Densitometric analysis represents n=4 independent experiments and reported as mean ± S.D. () Statistically significant differences between hCMEC/D3 cells treated with rifampin or SR12813 to control using ANOVA with Tukey pairwise comparisons at a significance level of p < 0.05. Cell treatment and immunoblot analysis were performed by GNY Chan.

6.4.5 Drug Transport Interactions between Atazanavir and Ritonavir

Since atazanavir and ritonavir are clinically co-administered together, we evaluated the drug transport interactions between these two agents using hCMEC/D3. Atazanavir or ritonavir did not appear to significantly enhance the cellular accumulation of each other, with only an approximately 1.2 fold increase observed at concentrations greater than 10 µM of either atazanavir or ritonavir (Figure 6-7). These data suggest a lack of interaction between atazanavir and ritonavir at the level of drug transporters in the hCMEC/D3 cell line.

Figure 6-7. Drug transport interactions between atazanavir and ritonavir in hCMEC/D3 3 3 cells. 1 µM [ H] ritonavir with increasing concentrations of atazanavir (), or 1 µM [ H] atazanavir with increasing concentrations of ritonavir (), for 60 min at 37 °C. Data represent mean ± S.D. for n= 3 independent experiments. Transport assays were performed by Dr. JA Zastre.

6.4.6 P-gp and MRP1 Expression in hCMEC/D3 Cells after Exposure to Atazanavir and

Ritonavir

To examine the induction properties of the two HIV-1 PIs, hCMEC/D3 cells were exposed to increasing concentrations of atazanavir or ritonavir for 72 h. This resulted in a significant increase in P-gp expression as determined by immunoblot analysis and subsequent densitometry analysis (Figure 6-8A). A 2 to 2.5 fold increase in P-gp expression was observed after 72 h treatment with 5 or 10 µM atazanavir (Figure 6-8B). Similar concentrations of ritonavir increased P-gp expression by 1.5 to 2 fold (Figure 6-8B). A single band at approximately 43 kDa corresponding to actin was detected in each lane, which verified appropriate sample loading. In our hands, we did not observe any change in actin protein expression in response to PI treatment after 72 h. In contrast, no change in MRP1 expression was observed in hCMEC/D3 cells after 72 h exposure of atazanavir or ritonavir suggesting chronic exposure of antiretrovirals, such as HIV PIs, can alter or increase the expression of P-gp, but not

MRP1, in hCMEC/D3 cells.

Figure 6-8. Expression of MRP1 and P-gp following treatment of hCMEC/D3 cells with 1, 5 and 10 µM atazanavir or ritonavir. (A) Representative immunoblot analysis of MRP1 and P- gp expression in cells treated with atazanavir or ritonavir to untreated hCMEC/D3 cells (CTL), which only received the same volume of drug solvent (final concentration of DMSO < 0.1%).

Whole cell lysate from MDCK cells transfected with human MDR1 and from HeLa-MRP1 cells

transfected with human MRP1 was used as positive control (+). A 50 µg load of control and treated cell lysates were resolved on a 12% SDS-PAGE gel and subsequently transferred to PVDF membrane. (B) Semiquantitative densitometric analysis was performed to determine the relative MRP1 and P-gp expression changes in hCMEC/D3 cells following 72 h exposure to 1, 5 and 10 µM atazanavir or ritonavir. Densitometric analysis represents n = 4 independent experiments and reported as mean ± S.D. () Statistically significant differences between

hCMEC/D3 cells treated with atazanavir and ritonavir to control using ANOVA with Tukey pairwise comparisons at a significance level of p<0.05. Cell treatment was performed by Dr. JA Zastre and immunoblot analysis was performed by GNY Chan.

6.4.7 Changes in P-gp Transport Associated with hCMEC/D3 Exposure to Atazanavir and

Ritonavir

The accumulation of R-6G was determined in hCMEC/D3 cells to assess if the increase in P-gp expression observed after 72 h exposure of atazanavir or ritonavir also translates to an increase in P-gp functional activity. An approximately 2-fold decrease in R-6G accumulation was observed after 72 h exposure of hCMEC/D3 cells to 5 µM atazanavir or ritonavir (Figure 6-9A).

Additionally, PSC833 significantly increased the accumulation of R-6G in hCMEC/D3 cells treated for 72h with 5 µM atazanavir or ritonavir to levels corresponding to control cells (Figure

6-9B). Thus, the increase in P-gp expression resulted in an increase in P-gp-mediated transport activity in the hCMEC/D3 cells.

100

Figure 6-9. P-gp functional activity after treatment of hCMEC/D3 cells with 5 µM atazanavir or 5 µM ritonavir. (A) Time dependent accumulation of the P-gp substrate R-6G (1 µM) by hCMEC/D3 control cells and cells treated with 5 µM atazanavir or 5 µM ritonavir for

72 h at 37 °C. Data represents mean ± S.D. with n = 3 independent experiments. () Statistically significant differences in R-6G accumulation between hCMEC/D3 cells treated with 5 µM atazanavir or ritonavir to control cells using Student t-test with p < 0.05. (B) Accumulation of R-6G (1 µM) after 30 min exposure with (+) or without (-) the P-gp inhibitor

PSC833 (5 µM) by hCMEC/D3 control cells or cells treated with 5 µM atazanavir or 5 µM 0 ritonavir for 72h at 37 C. Data represents mean ± S.E.M. with n = 3 independent experiments. ( ) Statistically significant differences in R-6G accumulation between hCMEC/D3 cells treated with 5 µM atazanavir or ritonavir to control cells using Student t-test with p < 0.05. () Statistically significant differences in R-6G accumulation with 5 µM PSC833 compared to R-6G accumulation without PSC833 using Student t-test with p < 0.05. Transport assays were performed by GNY Chan.

101

6.5 Discussion

Advances in HIV-1 PIs design have resulted in the development of compounds with improved potency, pharmacokinetics, and toxicity profiles. One such example is the PI atazanavir, which can be administered once daily and does not appear to alter lipid profiles of patients (Goldsmith and Perry, 2003). However, as a class of drugs, PIs exhibit many drug-drug interactions due to their effect on metabolic enzymes such as CYP450 enzymes and drug efflux transporters (De Maat et al. 2003). Therefore, understanding the substrate and inhibitor properties of newly developed antiretrovirals, such as atazanavir, with drug efflux transporters will assist in our understanding of the potential for drug-drug interactions and identify limitations to drug therapy.

Recently, it has been reported that atazanavir can inhibit P-gp mediated efflux of the P-gp substrate rhodamine 123 in human intestinal cells (Perloff et al. 2005). Furthermore, atazanavir was shown to inhibit the efflux activity of P-gp and MRP1 in lymphocyte and hemopoietic stem cells (Lucia et al. 2005). However, inhibition of P-gp and MRP1 substrate transport does not necessarily imply that atazanavir will serve as a substrate of P-gp or MRP1. One common approach to assess substrate properties of drug transporters is to utilize over-expressing cell systems of the transporter of interest and compare the cellular uptake to wild type cells. Using this method, we observed a 5 and 1.6 fold reduction in atazanavir cellular accumulation by P-gp and MRP1 overexpressing cells compared to wild type cells, respectively. Consistent with previous reports, we also demonstrated that ritonavir cellular accumulation by P-gp and MRP1 overexpressing cells was significantly reduced compared to wild type (Van der Sandt et al.

2001). Overall these results suggest that atazanavir and ritonavir cellular permeability may be, in part, limited by the effect of the drug efflux transporters P-gp and MRP1.

102

The immortalized human brain microvessel endothelial cell line, hCMEC/D3, represents an important tool for advancing our understanding of human brain homeostatic regulation by the

BBB. This system has been well characterized to maintain a BBB specific phenotype in culture

(Weksler et al. 2005). Using high-resolution immunogold cytochemistry at the electron microscope level and a monoclonal P-gp antibody which recognizes a highly conserved extracellular epitope on the transport protein, we primarily localized P-gp at the level of the plasma membrane of hCMEC/D3 cells (Figure 6-2B). Previously, we demonstrated localization of the transport protein at the level of both luminal and abluminal membranes of brain capillaries in rat and human bran tissue fixed in situ (Bendayan et al. 2006). In addition, we further confirmed protein expression and function of P-gp and MRP1 in this system. Our findings also show that while the uptake of atazanavir and ritonavir was increased approximately 2-fold in the presence of the P-gp inhibitor PSC833, no enhancement in atazanavir or ritonavir accumulation was observed in the presence of the MRP inhibitor MK571. It appears that even though atazanavir and ritonavir were characterized as substrates for MRP1 in over-expressing cell systems (Figure 6-1), this transporter is not involved in limiting drug uptake into hCMEC/D3 cells. Interestingly, inhibition of MRP1 by MK571 in mice was reported to increase brain uptake of the PI saquinavir by greater than 4-fold (Park & Sinko 2005). It is unclear if the lack of MRP mediated accumulation of atazanavir and ritonavir in hCMEC/D3 cells is the result of tissue or model system differences in transport activity. Overall, these results are consistent with the primary role of P-gp in limiting PI permeability across the BBB (Choo et al. 2000, Kim et al.

1998b, Ronaldson et al. 2004b).

Although acute exposure of ritonavir appears to be capable of inhibiting the transport activity of P-gp and MRP1 (Figures 6-4 & 6-5), no significant alteration of the transporters

103

expression was observed in cells that were exposed to ritonavir up to 24 hours. Therefore, the observed inhibitory effect of ritonavir on the transporters is unlikely to be caused by a decrease in protein expression, rather the interaction takes place at the level of the transport protein binding site. Since both atazanavir and ritonavir can be administered concurrently, it is of interest to evaluate if the co-administration of these two PIs would enhance the cellular accumulation of each other into hCMEC/D3 cells. In hCMEC/D3 cells, our data demonstrates a modest increase in accumulation (1.2 fold) for both atazanavir and ritonavir accumulation when the cultures are co-exposed to these PIs (Figure 6-7). This slight enhancement occurs at concentrations greater than 10 µM of both atazanavir and ritonavir, which may not be clinically relevant since boosted atazanavir regimens contain substantially lower dosages of ritonavir and the maximal therapeutic concentration (Cmax) of atazanavir is approximately 10 µM. Thus, in vivo, it could be predicted that the co-administration may not result in enhanced BBB penetration.

The shift of HIV infection into a chronic disease necessitates the study of the long term effects of HIV infection and antiretroviral treatment. Alterations in the pharmacokinetics of antiretroviral drugs have been described after chronic administration. This is possibly the result of an up-regulation of drug efflux transporters and/or metabolic pathways such as CYP450 enzymes (Gisolf et al. 2000, Huang et al. 2001). Chronic exposure to antiretrovirals such as the

PIs and NNRTIs have been shown to induce the expression of CYP3A enzymes, P-gp, and

MRP1 in intestinal and hepatic tissues (Stormer et al. 2002, Huang et al. 2001, Perloff et al.

2000, Perloff et al. 2001, Kageyama et al. 2005). In lymphocytes, P-gp may also be up-regulated due to chronic exposure to PIs or NNRTIs (Chandler et al. 2003). However, limited reports have described the impact chronic exposure of antiretroviral drugs, such as the PIs, can have on the expression of drug efflux transporters at the BBB. Perloff et al demonstrated that oral

104

administration of either dexamethasone or ritonavir for 3 days resulted in a modest (1.3 fold) increase in rat brain endothelial expression of P-gp (Perloff et al. 2004). In addition, a recent study showed that extensive exposure (120 h) of ritonavir to primary cultures of bovine brain microvessel endothelial cells resulted in an increased in P-gp expression (Perloff et al. 2007).

Our data show that long exposure (72 h) of concentrations greater than 5 µM atazanavir or ritonavir to hCMEC/D3 cells induced P-gp expression by approximately 2 fold (Figure 6-8B).

This increase in P-gp expression successfully translated into an increase in functional activity as shown by an approximately 2 fold reduction in R-6G accumulation. This effect could be reversed by PSC833 further suggesting the involvement of P-gp (Figure 6-9). Interestingly, no change in

MRP1 expression was observed in the presence of several concentrations of atazanavir or ritonavir. Since human plasma concentrations can range between 0.2 to 10 µM for atazanavir and 5 to 15 µM for ritonavir, up-regulation of P-gp expression and function with 5 or 10 µM atazanavir or ritonavir is within an attainable clinical plasma concentration (Goldsmith & Perry

2003, Hsu et al. 1998). Moreover, data from our MTT cell viability assay suggest the exposure with these concentrations of atazanavir and ritonavir for 72 h did not result in any substantial toxicity to hCMEC/D3 cells. Taken together, it appears that the upregulation of P-gp observed with long term PIs treatment can result in an increased P-gp functional expression at the BBB and can further contribute to reducing CNS drug penetration.

In the past few years, nuclear receptors such as rodent pregnane X receptor (rPXR) and human steroid xenobiotic receptor (hPXR or SXR) have been shown to regulate the expression of many metabolic enzymes and drug efflux transporters (Francis et al. 2003, Staudinger et al.

2003). Previously, PXR transcripts were detected in freshly isolated rat brain capillaries and in immortalized rat brain microvessel endothelial cell lines, GPNT and RBE4 (Bauer et al. 2004,

105

Lombardo et al. 2008). However, recently, Akanuma et al. have reported the lack of rodent PXR transcripts expression in rat brain capillaries and immortalized rat brain capillary endothelial cell line, TR-BBB13 (Akanuma et al. 2008). In the present study, high-resolution immunogold cytochemistry at the electron microscope level in conjunction with immunoblot analysis revealed the localization and expression of hPXR in hCMEC/D3 cells. To the best of our knowledge, this is the first report on the localization and expression of hPXR in a human in vitro system of the

BBB. Previously, induction of P-gp expression by rifampin and other hPXR ligands has been demonstrated in several cell culture systems (i.e., colon cell line, endometrial cancer cell line, primary cultures of hepatocytes), isolated lymphocytes, isolated brain capillaries of hPXR- transgenic mice and in clinical studies (Bauer et al. 2006, Geick et al. 2001, Masuyama et al.

2005, Owen et al. 2006, Stanley et al. 2006, Synold et al. 2001, Greiner et al. 1999). To examine whether hCMEC/D3 cell line is a good model to study hPXR-dependent induction of P-gp expression, cells were exposed to two hPXR ligands, rifampin and SR12813 compound, an inhibitor of HMG-CoA reductase activity and potent hPXR ligand (EC50 200nM) (Moore et al.,

2002). Our data show that 10 µM rifampin induced P-gp expression by approximately 1.8 fold when hCMEC/D3 cells were exposed for 72 h while an increase of approximately 6 fold in P-gp expression was observed in cells exposed 72 h to 5 µM or 10 µM of SR12813. These results suggest that hPXR may represent a potential pathway involved in the inductive properties of atazanavir and ritonavir on P-gp expression in hCMEC/D3 cells. Interestingly, ritonavir was found to be a poor substrate for the rodent homolog PXR, but a potent substrate for hPXR

(Dussault et al. 2001). Therefore, differences in the induction of P-gp expression may reflect PIs substrate specificity between rodent PXR and human PXR, and further confirm the advantages for using hCMEC/D3 cells as an in vitro human model for the BBB over animal derived models.

106

In conclusion, our data suggest that atazanavir and ritonavir are susceptible to P-gp mediated efflux activity at the BBB as assessed using a newly developed human brain microvessel endothelial cell line. Furthermore, chronic exposure of either atazanavir or ritonavir at clinically relevant plasma concentrations resulted in an increase in P-gp expression and function but had no effect on the expression of MRP1. Overall, these results suggest that the inductive properties of PIs on drug efflux transporter expression in the BBB may further contribute to limit and restrict CNS penetration of antiretroviral drugs.

6.6 Acknowledgements

This research was supported by grants from the Canadian Institutes of Health Research

(CIHR Grant #MOP56976) and the Ontario HIV Treatment Network, Ontario Ministry of Health awarded to Dr. Reina Bendayan. Dr. Jason Zastre was a recipient of a CIHR/RX&D postdoctoral fellowship award at the time this work was undertaken.

107

7. Regulation of P-gp functional expression by orphan nuclear receptors, hPXR and

hCAR, in hCMEC/D3 cell culture system

This work is published and reproduced in this thesis with permission from Wiley-Blackwell:

Chan GNY, Hoque MdT, Cummins CL and Bendayan R. (2011). Regulation of P-glycoprotein by orphan nuclear receptors in human brain microvessel endothelial cells. Journal of

Neurochemistry. 118(2): 163-75.

At the BBB, regulation of P-gp via PXR and CAR has only been investigated in animal models, such as rodents. To date, there is limited information on the regulation of P-gp by hPXR and hCAR at the human BBB. Chapter 7 describes such regulation of P-gp in the hCMEC/D3 cell culture model. First, we characterized both the transcript and protein expression of hPXR and hCAR in this cell culture system. Furthermore, we observed a wide range of inter-individual differences in hPXR and hCAR protein expression in human brain-derived microvascular endothelial cells (BBB-ECs). These findings provide evidence that the two receptors are expressed in in vitro cell culture systems of human brain microvessel endothelial cells. In addition, ligand-induced nuclear translocation of hPXR was observed, although such effect could not be demonstrated for hCAR. Ligands of hPXR and hCAR induced P-gp expression at both the transcript and protein levels, while pharmacological inhibitors of hPXR and hCAR prevented ligand-mediated P-gp induction. Furthermore, down-regulation of hPXR and hCAR proteins using small interfering RNAs decreased P-gp expression. Overall, our results demonstrated for the first time that orphan nuclear receptors, hPXR and hCAR, are actively involved in the regulation of P-gp in this system and that these receptors could be potential xenobiotic-targeted candidates to guide future studies in examining P-gp alteration in human brain microvessel endothelial cells.

108

Author Contributions:

Research design: GNY Chan (first author), MdT Hoque (second author) and R Bendayan (principal investigator)

Conducted experiments: GNY Chan (figures 7-1 to 7-8) and MdT Hoque (figures 7-5 and 7-8)

Data analysis: GNY Chan (figures 7-1 to 7-8) and MdT Hoque (figure 7-5)

Writing of the manuscript: GNY Chan (manuscript drafts and responses to reviewers’ comments), MdT Hoque (editorial review of the several manuscript drafts) and R Bendayan (overall conceptual and editorial review of the several manuscript drafts and responses to reviewers’ comments)

Provided equipment and expertise for qPCR experiments (figures 7-3 & 7-6): CL Cummins

109

7.1 Abstract

In mammalian systems, PXR and CAR have been recognized as xenobiotic-sensors which can upregulate the functional expression of drug transporters, such as P-gp. In the brain, an increase in P-gp expression can further limit drug permeability across the BBB and potentially reduce CNS pharmacotherapy efficacy. At present, the involvement of hPXR and hCAR in the regulation of P-gp expression at the human BBB is currently unknown. Here we investigate the role of hPXR and hCAR in the regulation of P-gp expression using a human cerebral microvessel endothelial cell culture system (hCMEC/D3). We demonstrate that activation of hPXR and hCAR by their respective ligands leads to P-gp induction at both mRNA and protein levels, while pharmacological inhibitors of hPXR and hCAR prevent ligand-mediated P-gp induction. Ligand- induced nuclear translocation of hPXR is observed, although such effect could not be demonstrated for hCAR. Furthermore, down-regulation of hPXR and hCAR proteins using small interfering RNA decreased P-gp expression. Our findings provide first evidence for P-gp regulation by hPXR and hCAR at the human BBB and suggest insights on how to achieve selective P-gp regulation at this site.

7.2 Introduction

It is well established that the BBB constitutes a remarkable physical and biochemical barrier between the brain and systemic circulation and can significantly limit brain permeability of many xenobiotics including pharmacological agents (Pardridge 2010). It is now well accepted that the physical barrier does not only comprise non-fenestrated endothelial cells jointed by tight junctions but also involves pericytes and perivascular astrocytes, which are in close contact with the endothelium and maintain overall BBB homeostasis (Abbott et al. 2010). In addition to

110

the physical barrier, there is a selective biochemical-driven barrier that largely reflects expression and function of several receptors, ion channels, metabolic enzymes and influx/efflux transport proteins found predominantly at the BBB (Abbott et al. 2010). In particular, P-gp expressed at high levels in brain microvessel endothelial cells has long been recognized to play a significant role in restricting the permeability of several drugs across the BBB (Bendayan et al. 2002,

Ronaldson et al. 2004b, Miller et al. 2000, Schinkel et al. 1996, Tsuji et al. 1992). Early studies have demonstrated that P-gp knockout mouse models exhibited elevated (3- to 100-fold) brain drug concentrations compared to wild-type animals (Kim et al. 1998b). As well, co- administration of the HIV PI ritonavir, known to be a P-gp and CYP3A4 inhibitor, with another

HIV PI, indinavir, in HIV patients has been reported to result in higher indinavir cerebral spinal fluid concentrations (Van Praag et al. 2000). One of the important pathways involved in P-gp regulation is the adopted “orphan” nuclear receptors, PXR and CAR (Miller 2010). Both nuclear receptors can be activated by endogenous ligands and a wide range of xenobiotics and hence are recognized as xenosensors (Urquhart et al. 2007). In human tissues, hPXR and hCAR play a major role in coordinating the expression of a large number of phase I and phase II metabolizing enzymes and membrane transporters that are involved in the detoxification and/or transport of exogenous and endogenous compounds. In addition, many of these can in turn modulate the activity of hPXR and hCAR (Urquhart et al. 2007).

PXR and CAR are known to form a heterodimer with the retinoid X receptor. The heterodimeric complex interacts with the XREM in the promoter regions of target genes. Ligand binding induces a conformational change that allows the recruitment of co-activators (i.e., SRC-

1) to increase gene transcription (Timsit & Negishi 2007). The induction of P-gp has been shown to require the interaction of hPXR or hCAR with the DR4 motif in the promoter region of MDR1

111

gene (Geick et al. 2001, Burk et al. 2005). In vitro studies, performed in cell culture models of intestine, liver and lymphocytes, have examined the modulation of P-gp expression using typical ligands of hPXR (i.e., rifampin and hyperforin), (Geick et al. 2001, Synold et al. 2001, Hennessy et al. 2002). At the BBB, the induction of P-gp and other drug efflux transporters via PXR has only been investigated in animal models. Bauer et al. demonstrated that treating rats with pregnenolone 16α-carbonitrile or dexamethasone (ligands of rodent PXR) results in increased P- gp protein expression in brain capillaries (Bauer et al. 2004). A follow up study from the same group reported that dosing hPXR-transgenic mice with rifampin (ligand of hPXR) also increased

P-gp protein expression in brain capillaries (Bauer et al. 2006). Although, a few groups demonstrated the inductive effect of several PXR ligands on P-gp expression in rat, porcine and bovine brain microvessel endothelial cells (Perloff et al. 2007, Ott et al. 2009, Narang et al.

2008), limited information exists on the regulation of P-gp by hPXR and in particular by hCAR at the human BBB.

Since species differences in ligand specificity and intracellular signaling pathways for hPXR and hCAR exist, animal models may not fully predict xenobiotic interactions in humans.

Therefore, human derived systems are critical to investigate the transcriptional regulation mediated by the two nuclear receptors. In this study, we utilized hCMEC/D3 to examine the role of orphan nuclear receptors, hPXR and hCAR, in the regulation of P-gp expression at the human

BBB. Our results demonstrate for the first time that orphan nuclear receptors hPXR and hCAR are actively involved in the regulation of P-gp in this system. Selective modulation of P-gp expression at the BBB can potentially be achieved by xenobiotics-hPXR/hCAR interactions to improve CNS drug delivery or to enhance neuroprotection.

112

7.3 Materials and Methods

7.3.1 Materials and Reagents

Sterile PBS, 0.25% Trypsin and TRIZOL reagent were purchased from Invitrogen (Grand

Island, NY). Type I collagen was purchased from Becton-Dickinson (Franklin Lakes, NJ).

DMSO and acrylamide solution were obtained from Bioshop Canada Inc., (Burlington, ON). 6-

(4-Chlorophenyl)-imidazo[2,1-b]thiazole-5-carbaldehyde (CITCO) and SR12813 were supplied from BIOMOL Research Laboratories (Plymouth Meeting, PA). Rifampin, MTT, Chloroform,

PMSF and protease inhibitor cocktail were purchased from Sigma-Aldrich (Oakville, ON).

A792611 was a generous gift from Abbott Laboratories (Abbot Park, IL). Meclizine was ordered from Toronto Research Chemicals (Toronto, ON). Immunoblot stripping solution and enhanced chemilumescent were purchased from Pierce Thermo Fisher Scientific Inc. (Waltham, MA).

Hybond-P PVDF membrane was supplied from (GE Healthcare Bio-Sciences, AB). Microscope cover glass slide (22x 22 mm, thickness #1) was obtained from Fisher Scientific (Pittsburgh,

PA).

7.3.2 Cell Culture Systems

The immortalized human brain microvessel endothelial cell line, hCMEC/D3, was kindly provided by Dr. P.O. Couraud (Institut Cochin, Departement Biologie Cellulaire and Inserm,

Paris, France). This cell line has been widely utilized as a potential in vitro model of human BBB and is known to display many morphological and biochemical properties of human brain microvascular endothelium in vivo, such as functional expression of tight junction proteins, endothelial cell markers and drug efflux transporters. Cells were used at passages 28-39 for all experiments and were maintained at 37 °C, 5 % CO2, and 95 % humidified air in Endothelial Cell

Growth Media – 2 media (Lonza, Walkersville, MD) supplemented with vascular endothelial

113

growth factor, insulin-like growth factor 1, epidermal growth factor, fibroblast growth factors, hydrocortisone, ascorbate, gentamycin (Lonza, Walkersville), and 2.5 % fetal bovine serum and grown on rat tail collagen type I (BD) coated flasks, 60 mm Petri dishes and 6 well plates as previously described (Weksler et al. 2005). Whole cell pellets of primary cultures of human brain-derived microvascular endothelial cells (BBB-ECs) were generously provided by Dr.

Alexandre Pratt (Neuroimmunology Research Laboratory, Center of Excellence in Neuromics,

Faculty of Medicine, Centre Hospitalier de l'Université de Montréal, Montréal, Québec). These cells were originated from young adults undergoing surgery for the treatment of intractable epilepsy and provide an alternative in vitro model of human brain endothelium. The P-gp overexpressing human breast carcinoma cell line (MDA435/LCC6/MDR1) was a gift from Dr.

Robert Clarke. The MDA435/LCC6/MDR1 and HepG2 cells were cultured in alpha-minimum essential media supplemented with 10% FBS and 1% penicillin/streptomycin.

7.3.3 Cell Viability Assay

Cell viability in the presence of ligands was assessed using an MTT assay in which cells were incubated for 2 h at 37 ºC with a 2.5 mg/mL MTT solution in PBS. The formazan content, as dissolved in DMSO, from each well was determined by UV analysis at 580 nm using a

SpectraMax 384 microplate reader (Molecular Devices, Sunnyvale, CA). Cell viability was expressed as the ratio between the absorbance of treated cells and the absorbance of non-treated

(control) cells.

7.3.4 Cell Treatment

Monolayers of hCMEC/D3 cells grown on 60 mm petri dishes (approximately 80% confluence) were treated with hPXR ligands (rifampin or SR12813), or hPXR antagonist

(A792611), or with a hCAR ligand (CITCO), or with an inverse agonist of human CAR

114

(meclizine) for a specific time as indicated in the figure legends. At the beginning of each experiment, culture medium was aspirated and fresh medium containing ligands was added.

DMSO 0.1 % (v/v) was used to dissolve ligands. Control cells were exposed to 0.1 % (v/v)

DMSO in the absence of ligands. To ensure cells remain viable during treatment, all ligand concentrations used were tested applying the MTT assay as described above.

7.3.5 Immunoblot Analysis

Immunoblot analysis was performed as described previously in my published book chapter (Appendix D) with minor modifications (Chan & Bendayan 2011, Zastre et al. 2009) .

Monolayers of hCMEC/D3 cells were washed with warm PBS once and collected by scraping in ice cold PBS. Following centrifugation, whole cell lysates were prepared by exposing cell pellets to lysis buffer (1 % (v/v) NP-40 in 20 mM Tris, 150 mM NaCl, 5 mM EDTA at pH 7.5 containing 1 mM PMSF and 0.1 % (v/v) protease inhibitor cocktail) for 15 min at 4 °C. Cell lysates were sonicated for 10 sec and centrifuged at 20,000 g for 10 min at 4 °C to remove cell debris. Whole cell lysates were mixed in Laemmli buffer and resolved on 10 % SDS- polyacrylamide gel. After electrophoresis, the gel was washed three times (5 min each) in transfer buffer (25 mM Tris-HCl, pH 8, 200 mM glycine) containing 20% (v/v) methanol and then electrotransferred onto a PVDF membrane. The membranes were blocked for at least 2 hours in Tris-Buffered Saline containing 0.1 % Tween 20 (TBS-T) containing 5 % (m/v) skim milk. The membranes were incubated with primary antibody overnight at 4 °C followed by a 1.5 h incubation with anti-mouse (Jackson immunoresearch Laboratories, Inc., West Grove, PA), anti-goat or anti-rabbit (Sigma-Aldrich) horseradish peroxidase-conjugated secondary antibody at 1:10,000 dilution. P-gp expression was detected using a 1:500 dilution of mouse monoclonal

C219 antibody (ID Labs, London, ON). MDA435/LCC6-MDR1 cell lysates was used as a

115

positive control for P-gp. hPXR expression was detected using a 1:200 dilution of goat polyclonal A-20 antibody (sc-7737) (Santa Cruz Biotechnology, Inc.), which recognizes hPXR.1

(protein accession # O75469) unique domain. hCAR expression was detected using a 1:200 dilution of rabbit polyclonal M-127 antibody (sc-13065) (Santa Cruz Biotechnology, Inc.), which recognizes an epitope corresponding to amino acids 60-187 of hCAR (protein accession #

Q14994). A-20 neutralizing peptide and rabbit IgG control were used to verify the selectivity for

A-20 antibody and M-127 antibody, respectively. HepG2 cell lysates were used as positive control for hPXR and hCAR. Actin expression was used as loading control and was detected using mouse monoclonal AC40 antibody (1:1000 dilution) (Sigma-Aldrich). Protein bands were visualized by enhanced chemiluminescence and protein expression was determined from densitometry analysis using Alpha DigiDoc RT2 imaging software (Alpha Innotech, San

Leandro, CA).

7.3.6 RNA Extraction, Reverse Transcription and Quantitative Real-time PCR (qPCR)

Total RNA was extracted from confluent monolayers of hCMEC/D3 cells using TRIzol reagent (Invitrogen) and treated with amplification grade DNase I (Invitrogen) as previously described with minor modifications (Ronaldson et al. 2010). Briefly, 2.0 µg of RNA was reverse transcribed using the high capacity cDNA reverse transcriptase kit (Applied Biosystems, Foster

City, CA). All cDNA stock solutions were stored at -80 °C. MDR1, hPXR and hCAR mRNA expression was quantified using qPCR on an ABI 7900HT Fast Real-time PCR System (Applied

Biosystems). All primer sets (Table 7-1) were validated for efficiency using cDNA amplified from Huh7 cells mRNA. Primer specificity was determined using BLAST and dissociation curve analysis. The final reaction mix contains diluted 0.4 µg cDNA, 1.25 µM of primer set mix, 1 X

SYBR Green Master Mix (Applied Biosystems) and DNase/RNase-free H2O (Invitrogen). The

116

qPCR protocol was at 50 °C for 2 min, 95 °C for 10 min and 40 cycles of 95 °C for 15 sec, 60 °C for 1 min and 95 °C for 15 sec. MDR1 mRNA expression was examined in hCMEC/D3 cells at

10 h, 16 h, 24 h and 48 h after treatment with hPXR or hCAR ligands. For these experiments, Ct values for MDR1 mRNA are normalized by housekeeping gene 18S mRNA. Results are expressed as percentage fold change ± standard deviation S.D., using comparative CT method

(ΔΔ CT). MDR1 expression levels were normalized to vehicle treated cells at each time point.

Table 7-1. List of primers used for qPCR analysis.

117

7.3.7 Immunofluorescence Studies

The subcellular localization of hPXR and hCAR was examined by confocal microscopy on cells treated with hPXR or hCAR ligands for 5 h. Cell monolayers grown on glass coverslips were fixed with 4 % paraformaldehyde at room temperature for 20 min. After washing in PBS, cells were permeabilized with 0.1 % Triton X-100 for 10 min, as described previously (Hoque &

Ishikawa 2001). Fixed cells were blocked with 0.1 % (m/v) bovine serum albumin and 0.1 %

(m/v) skim milk in PBS for at least 1 h prior to primary antibody incubation for 1 h at room temperature. The rabbit polyclonal H-160 (sc-25381) and M-127 (sc-13065) antibodies (1:200 dilution each) (Santa Cruz Biotechnology, Inc.) were used to detect hPXR and hCAR subcellular localization, respectively. The mouse monoclonal antibody (ab8980) was used to visualize lamin

A expression, a marker for nuclear envelope (Abcam, Cambridge, MA). Fixed cells were washed under gentle agitation three times (15 min each) and incubated with Alexa Fluor® 488 conjugated secondary antibody (dilution of 1:500) (Invitrogen) for 1 h at room temperature to stain anti-hPXR (H-160) or anti-hCAR (M-127) antibodies. Alexa Fluor® 568 conjugated secondary antibodies (dilution of 1:500) (Invitrogen) was used to detect anti-lamin-A (Ab8980) antibody. Staining in the absence of primary antibodies was used as negative control. Coverslips were mounted on a 76 x 26 mm microscope slide (VWR, West Chester, PA) using vectashield mounting solution with 4',6-diamidino-2-phenylindole, DAPI (Vector Laboratory Inc.,

Burlingame, CA). Samples were examined under a C-Apochromat 63X objective lens with the

Zeiss LSM510 META microscopy system (Carl Zeiss Canada Ltd., Toronto, ON). Measurement of fluorescence intensity was determined using ImageJ software (ver. 1.29; http://rsb.info.nih.gov/ij). The average fluorescence intensity for each treatment group was the mean of all measurements taken from 100 cells.

118

7.3.8 siRNA Downregulation Studies

Small interfering RNA (siRNA) duplex (5’-GGC-CAC-UGG-CUA-UCA-CUU-C-3’) against hPXR was custom synthesized by Applied Biosystems (Foster City, CA). Predesigned validated siRNA against hCAR (siRNA ID# 5535) and non-silencing negative control siRNA

(cat# AM4611) were obtained from Ambion (Carlsbad, CA). The BLOCK-iT™ Alexa Fluor®-

555 fluorescent oligo (Invitrogen) was used to assess transfection efficiency in hCMEC/D3 cells. Cells were plated in a 6-well plate with 0.3 x 106 cells / well. After 24 h, cell monolayers with approximately 80% confluence were subjected to siRNA transfection. Transfection mix was prepared in Opti-MEM GlutaMax (Invitrogen) media with siRNA and lipofectamine 2000

(Invitrogen) according to manufacture’s protocol. The final concentration of siRNA and lipofectamine when added to the cells were 100 nM and 2 µL/mL, respectively. Cells were incubated in Opti-MEM GlutaMax media with lipofectamine siRNA complex for 24 h at 37 °C, as described previously (Huang et al. 2009). The following day, cells were fed with fresh hCMEC medium and cultured for an additional 48 h. Cells were harvested at 72 h post transfection and analyzed by immunoblot analysis.

7.3.9 Statistical Analysis

All experiments were repeated at least three times in cells pertaining to different passages. Results are reported as a mean ± S.D. Comparisons between groups were performed using either two-tailed Student’s t test or ANOVA with Bonferroni multiple comparison post-hoc test. Data were analyzed by SPSS software (Chicago, IL) and a value of p < 0.05 was considered to be statistically significant.

119

7.4 Results

7.4.1 P-gp, hPXR and hCAR Expression in hCMEC/D3 cells and Human BBB-ECs

Using immunoblot analysis, we detected hPXR and hCAR proteins expression in hCMEC/D3 cells at the expected molecular weight of 50 and 60 kDa, respectively. HepG2 cells known to express hPXR and hCAR served as a positive control (Figure 7-1).

Figure 7-1. Immunoblot analysis of P-gp, hPXR and hCAR protein expression in hCMEC/D3 cells. Whole cell lysates prepared from P-gp-overexpressing human breast carcinoma cell culture system (MDA-MDR1, 2 µg), human hepatocellular carcinoma cell culture system (HepG2, 25 µg) and hCMEC/D3 cells (50 µg) were resolved on a 10% SDS-PAGE gel and subsequently transferred to PVDF membrane. MDA-MDR1 served as positive control for P- gp, while HepG2 was used as positive control for hPXR and hCAR. P-gp was detected using the monoclonal antibody C219 (1:500 dilution), hPXR was detected using the polyclonal antibody A -20 (1:200 dilution), hCAR was detected using the polyclonal antibody M-127 (1:200 dilution) and actin was detected using AC40 monoclonal antibody (1:1000 dilution). Immunoblot analysis was performed by GNY Chan.

120

The immunostaining identities for hPXR and hCAR cells were further confirmed using hPXR antibody neutralizing peptide and non-conjugated IgG, respectively. The immunostaining of hPXR was abolished when the antibody was pre-absorbed with its specific neutralizing peptide antigen (Figure 7-2A). Similarly, we observed the absence of immunostaining for hCAR in hCMEC/D3 cells when anti-hCAR antibody was replaced by a rabbit IgG control (Figure

7-2B). Furthermore, hPXR and hCAR mRNA expression was detected using qPCR in hCMEC/D3 cells. The hCMEC/D3 cells exhibited approximately 20 times lower expression of hPXR and 8 times lower expression of hCAR in comparison to HepG2 cells, as determined by standard ΔΔCt method (Figure 7-3).

121

Figure 7-2. Selectivity of anti-hPXR and anti-hCAR antibodies used in immunoblot analysis. Whole cell lysates from hCMEC/D3 (50 µg) and HepG2 cells (25 µg) were resolved on a 10% SDS-PAGE gel and subsequently transferred to PVDF membrane. HepG2 cell lysate served as positive control for hPXR and hCAR expression. A) Anti-hPXR (A-20) antibody detected a band with apparent molecular weight of ~50 kDa in both HepG2 and hCMEC/3 cells (left two lanes), and the hPXR signal was abolished when the antibody was pre-absorbed with its specific neutralizing peptide (right two lanes). B) Anti-hCAR antibody detected a band at a molecular weight of ~60 kDa in both HepG2 and hCMEC/D3 cells (left two lanes); the hCAR signal was abolished when the membrane was incubated with non-conjugated rabbit isotype control (right two lanes). Immunoblot analysis was performed by GNY Chan.

122

Figure 7-3. Human PXR and CAR mRNA expression in HepG2 and hCMEC/D3 cells. Transcript levels of the indicated genes were analyzed by quantitative real-time PCR with validated primer sets summarized in Table 7-1. Results are presented as fold differences of NR1I2 (hPXR) and NR1I3 (hCAR) mRNA expression normalized to the house keeping gene (human cyclophilin B) compared to the hCMEC/D3 cells. Average Ct values for the gene of interest (hPXR or hCAR) from triplicate readings pertaining to each cell culture system (hCMEC/D3 and HepG2) are shown as inset. Threshold of detection was set between 0.1-0.2. Real-time qPCR analysis was performed by GNY Chan in Dr. CL Cummins Laboratory.

123

Similarly, whole cell pellets of human BBB ECs from young adults also showed clear expression of hPXR and hCAR proteins (Figure 7-4A). We further observed a wide range of inter-individual differences as much as 10-fold and 4-fold for hPXR and hCAR expression, respectively (Figure 7-4B).

Figure 7-4. Analyses of hPXR and hCAR protein expression in human brain-derived microvascular endothelial cells (BBB-ECs). Whole cell lysates prepared from HepG2 cells (25 µg) and human BBB-ECs pellets obtained from six individuals (S1 – S6) (50 µg each) were resolved on a 10% SDS-PAGE gel and subsequently transferred to PVDF membrane. HepG2 was used as positive control for hPXR and hCAR expression. hPXR was detected using the goat polyclonal antibody A-20 (1:200 dilution). hCAR was detected using the rabbit polyclonal antibody M-127 (1:200 dilution) and actin was detected using AC40 mouse monoclonal antibody (1:1000 dilution). Data generated from densitometric analysis is presented as ratio of hPXR or hCAR expression normalized to actin (loading control). Immunoblot analysis was performed by GNY Chan.

124

7.4.2 Ligand-mediated Nuclear Translocation of hPXR and hCAR in hCMEC/D3 Cells

hPXR and hCAR are transcription factors which have been shown to translocate into the nucleus from the cytoplasm upon ligand activation (Timsit & Negishi 2007). To examine the ligand-mediated nuclear translocation of hPXR and hCAR in hCMEC/D3 cells, immunofluorescence analysis was performed. We observed approximately 50% increase in hPXR nuclear to cytosolic fluorescence intensity in cells treated with 10 µM SR12813 for 5 h compared to control (Figure 7-5A). The data suggest that the increase in hPXR nuclear accumulation could be mediated by an interaction with SR12813, an established hPXR ligand. In contrast, in the presence of the selective hCAR ligand CITCO (at the highest tolerable concentration of 7.5 µM), nuclear to cytosolic fluorescence intensity was not significantly altered after 5 h ligand exposure (Figure 7-5B).

125

126

Figure 7-5. Immunocytochemical localization of hPXR and hCAR in hCMEC/D3 cells. Cells cultured on glass coverslips were fixed in 4 % paraformaldehyde and permeabilized with 0.1 % TritonX-100. hPXR protein was localized with anti-hPXR (H-160) polyclonal antibody

(1:200) and Alexa Fluor® 488 – conjugated secondary antibody (1:500). hCAR protein was localized with anti-hCAR M-127 polyclonal antibody (1:200) and Alexa Fluor® 488 – conjugated secondary antibody (1:500). Nuclear envelope was visualized with anti-lamin-A (ab8980) antibody (1:200) and Alexa Fluor® 568 – conjugated secondary antibodies (1:500). For negative control, only the Alexa Fluor® – conjugated secondary antibody was used. A) hPXR signal in the nucleus was increased in hCMEC/D3 cells exposed to 10 µM SR12813 for 5 h (bottom row) compared to cells exposed to DMSO vehicle control (top row). B) Enhancement in nuclear immunoreactivity of hCAR is not observed in cells exposed to 7.5 µM CITCO for 5 h

(bottom row) compared to cells exposed to DMSO vehicle control (top row). Average hPXR and hCAR fluorescence ratios (nuclear to cytosolic) normalized to control were obtained from three independent experiments performed in cells pertaining to different passages (100 cells / treatment group). * Statistically significant differences in protein fluorescence ratio compared to control as determined by student t-test at a significance level of p < 0.05. Immunocytochemistry and data analysis were performed by GNY Chan and Dr. MdT Hoque.

127

7.4.3 Ligand-mediated Upregulation of MDR1 mRNA Expression in hCMEC/D3 Cells

qPCR was performed to examine MDR1 mRNA expression in hCMEC/D3 cells exposed to the hPXR ligand, rifampin and the hCAR ligand, CITCO. Our earliest observable induction of

MDR1 mRNA was at 24 h in the presence of 10 µM rifampin and at 48 h in the presence of 7.5

µM CITCO (Figure 7-6). The modest but significant MDR1 mRNA induction of approximately

30-40 % in ligand-treated cells was not due to cytotoxicity. Cell viability experiments revealed that cells were viable following 72 h ligand treatment (data not shown).

128

Figure 7-6. Inductive effect of hPXR or hCAR ligands on MDR1 mRNA expression in hCEMC/D3 cells. Monolayers of hCMEC/D3 cells were exposed to: A) 10 µM rifampin, a selective hPXR ligand or B) 7.5 µM CTICO, a selective hCAR ligand, for 10, 16, 24 or 48 h. MDR1 mRNA expression was measured applying qPCR. All treatment and vehicle groups were performed in triplicates in three independent experiments pertaining to different passages. Ct values for MDR1 mRNA are normalized by housekeeping gene 18S mRNA. Results are expressed as percentage fold change ± S.D., using comparative CT method (ΔΔ CT). MDR1 mRNA expression in vehicle treated cells (control) is set to 100% for each individual time points. * Statistically significant differences in MDR1 mRNA expression compared to control as determined by one way ANOVA with Bonferroni post-hoc tests at a significance level of p < 0.05. Real-time qPCR was performed by GNY Chan in Dr. CL Cummins Laboratory.

129

7.4.4 Effect of hPXR and hCAR Inhibitors on Ligand-mediated P-gp Induction

We further tested whether the observed P-gp induction is primarily mediated by hPXR and/or hCAR in hCEMC/D3 cells. Cells were exposed to selective inhibitors for hPXR

(A792611) and hCAR (meclizine) in conjunction with agonists for 72 h. These inhibitors have been reported to attenuate the ligand-activation of hPXR and hCAR in other in vitro cell culture systems (Healan-Greenberg et al. 2008, Huang et al. 2004a, Meyer Zu Schwabedissen et al.

2008). As expected, addition of A792611 (2.5 µM) significantly decreased P-gp induction mediated by SR12813 (hPXR agonist) from approximately 80 % to 50 % (Figure 7-7A). In the current study, we observed approximately 60 % P-gp protein induction in cells that were exposed to 7.5 µM CITCO for 72 h, addition of meclizine at 2.5 µM resulted in an approximately 30 % decrease in P-gp induction mediated by CITCO (Figure 7-7B). Only low concentrations (2.5 µM) of A792611 and meclizine could be used without causing cytotoxicity in our cell system as determined by cell viability experiments (data not shown).

130

Figure 7-7. P-gp expression in hCMEC/D3 cells treated with SR12813 and A792611 or CITCO and meclizine. A) Representative immunoblot (top) and densitometric analysis (bottom) of P-gp expression in hCMEC/D3 cells exposed to vehicle, 2.5 µM A792611 alone, 10 µM SR12813 alone or 10 µM SR12813 in conjunction with 2.5 µM A792611. B) Representative immunoblot (top) and densitometric analysis (bottom) of P-gp expression in hCMEC/D3 cells exposed to vehicle, 2.5 µM meclizine alone, 7.5 µM CITCO alone or 7.5 µM CITCO in conjunction with 2.5 µM meclizine. Whole cell lysates from hCMEC/D3 cells (50 µg) were resolved on a 10% SDS-PAGE gel and subsequently transferred to PVDF membrane. P-gp expression was detected using the monoclonal antibody C219 (1:500 dilution) and relative levels of P-gp expression were determined by densitometric analysis. Results are expressed as percentage fold change normalized to vehicle treated cells (control) and reported as mean +/-

S.D. obtained from three independent experiments performed in cells pertaining to different passages. * Statistically significant differences between treatment group as determined by one way ANOVA with Bonferroni post-hoc tests at a significant level of p < 0.05. Immunoblot analysis was performed by GNY Chan.

131

Downregulation of P-gp Expression by hPXR-targeting and hCAR-targeting siRNA

hPXR-targeting and hCAR-targeting siRNA were used to determine the direct involvement of hPXR and hCAR in P-gp regulation in hCMEC/D3 cells. When hPXR was downregulated by 20%, we observed a significant decrease in P-gp protein expression

(approximately 30%) (Figure 7-8A). In hCAR-siRNA transfected cells, hCAR expression was downregulated by approximately 40% and P-gp expression was reduced by nearly 30% (Figure

7-8B). A higher concentration of transfected siRNA could not be used due to cytotoxicity. The uptake of BLOCK-iT™ fluorescent oligo, which is a RNA duplex with the same length, charge, and configuration as standard siRNA, served as an indication of successful siRNA transfection in hCMEC/D3 cells using the current lipofectamine 2000 transfection protocol (Figure 8C).

132

133

Figure 7-8. Effect of hPXR and hCAR downregulation on P-gp protein expression in hCMEC/D3 cells. A and B Top Panels: Equal protein loading (50 µg/each) of homogenates derived from either control siRNA treated hCMEC/D3 cells or cells treated with siRNA directed against hPXR or hCAR were subjected to immunoblotting with anti-hPXR and anti-hCAR antibodies (middle row). The blots were subsequently stripped and probed with anti-P-gp (upper row) or anti-actin antibody as loading control (lower row). A and B Bottom Panels: Relative levels of P-gp, hPXR and hCAR expression were determined by densitometric analyses. Results are expressed as percentage fold change normalized to control siRNA and reported as mean +/- S .D. obtained from three independent experiments performed in cells pertaining to different passages. * Statistically significant differences in P-gp, hPXR or hCAR expression compared to control (control siRNA) were determined by two-tailed Student’s t-test with P < 0.05. C: To assess transfection efficiency in hCMEC/D3 cells, the BLOCK-iTTM Alexa Fluor red fluorescent siRNA oligo was transfected for 48 h with lipofectamine 2000 (Top panel). Prevalent red fluorescence of nucleus in transfected cells showed relatively high siRNA transfection efficiency in these cells (Top panel). Untransfected cells served as negative control (Bottom panel). Initial optimization of siRNA transfection and confocal microscopy work were performed by Dr. MdT Ho que. Sequential experiments on siRNA transfection, cell treatment and immunoblot analysis were performed by GNY Chan.

7.5 Discussion

The two orphan nuclear receptors, PXR and CAR, appear to play an important role in drug disposition by coordinating the expression of several drug-metabolizing enzymes and drug transporters (Urquhart et al. 2007). In addition, these receptors have been recognized to be master sensors for a wide array of endogenous metabolites and xenobiotics (Timsit & Negishi

2007). Furthermore, species differences in their pharmacological activation profiles exist, therefore the use of animal models is not always suitable to predict ligand interactions and examine the underlying transcriptional regulation by PXR and CAR in human tissues. Due to the challenge in obtaining healthy human brain samples, the present study primarily utilized the hCMEC/D3 cell culture system, an in vitro representative model of the human BBB.

134

In the brain, expression of PXR mRNA and protein have been detected in the cortex, brain capillaries and primary cultures of brain microvessel endothelial cells obtained from mice, rats and pigs (Bauer et al. 2004, Bauer et al. 2006, Nishimura et al. 2004, Bauer et al. 2008a,

Nannelli et al. 2010, Ott et al. 2009, Narang et al. 2008). In humans, most studies have only reported the transcript expression of hPXR in brain cortex and cerebellum (Miki et al. 2005,

Nishimura et al. 2004, Dauchy et al. 2009). CAR mRNA expression has been observed in different regions of the human brain and in brain capillaries isolated from pigs and mice (Lamba et al. 2004a, Nannelli et al. 2010, Nishimura et al. 2004, Dauchy et al. 2008, Savkur et al. 2003).

In our previous publication, hPXR protein expression was demonstrated in hCMEC/D3 cells, however the role of P-gp regulation was not investigated (Zastre et al. 2009). In the present study, we observed mRNA and protein expression of hPXR and hCAR in the same cell culture system. In addition, in whole cell pellets of primary cultures of human BBB-ECs, we show interindividual differences in the expression of hPXR and hCAR proteins. Similar results have been reported at the mRNA level in human liver for the two nuclear receptors (Chang et al. 2003).

Interestingly, previous publication did not detect hPXR and observed very low hCAR transcript expression in brain capillaries isolated from patients with epilepsy or glioma (Dauchy et al.

2008). This discrepancy can be due to possible mRNA degradation in isolated brain tissue, and/or limited number of samples. Our findings help clarify and provide evidence that these two receptors are expressed at the human BBB and can serve as potential sites for drug-receptor interactions and regulation of drug transporters and metabolic enzymes.

PXR has been reported to translocate into the nucleus upon ligand activation (Timsit &

Negishi 2007). Studies using ex vivo mouse liver slices, primary cultures of human hepatocytes and porcine brain capillary endothelial cells have demonstrated the ligand-dependent manner of

135

PXR nuclear translocation (Squires et al. 2004, Pascussi et al. 2000, Ott et al. 2009, Kawana et al. 2003). Our findings corroborate these data in hCMEC/D3 cells showing a ligand-dependent nuclear accumulation of hPXR confirming the proposed mechanism for hPXR/PXR activation and previous findings. Furthermore, these data provide the first evidence that hPXR is likely to be functionally active at the human BBB and its cellular localization suggests hPXR downstream effects, such as the induction of drug efflux transporters.

In contrast to PXR, nuclear translocation for human and rodent CAR is less well characterized. To explore hCAR nuclear translocation mechanisms, transient and stable expression of hCAR in cell culture systems has been attempted, however these systems are limited by spontaneous accumulation of CAR in the nucleus (Timsit & Negishi 2007). Recently,

Li et al. were able to demonstrate hCAR nuclear translocation using several known hCAR ligands (i.e., CITCO) in primary cultures of human hepatocytes transfected with fluorescent protein-tagged hCAR. From the same study, transfected HepG2 cells were unable to retain such features (Li et al. 2009). In our hands, hCAR is primarily localized in the nucleus of the hCMEC/D3 cells and we did not observe nuclear translocation of hCAR in the hCMEC/D3 cells treated with 7.5 µM CITCO for 5 h. These data are in agreement with previous findings suggesting that immortalized cell culture systems, showing spontaneous accumulation of CAR in the nucleus, may not exhibit significant nuclear movement of hCAR in response to ligands.

Previous in vitro and in vivo studies have shown that PXR ligands can induce P-gp at the

BBB using several animal models (Bauer et al. 2004, Perloff et al. 2007, Ott et al. 2009, Narang et al. 2008, Aquilante et al. 1999, Bauer et al. 2006). We have previously reported in the hCMEC/D3 cells that hPXR ligands, rifampin and SR12813, induced P-gp protein expression

(Zastre et al. 2009). These two ligands are the most utilized selective chemical agents available

136

to study hPXR pharmacological activation. Cell-based screening showed SR12813 and rifampin do not interact with hCAR and have high affinity towards hPXR (Moore et al. 2002). In the current study, we confirm our previous results and further demonstrate that the earliest observable induction of MDR1 mRNA takes place at 24 h in the same cell culture system treated with rifampin. Our data further provide evidence that P-gp protein induction is mediated by the hPXR pathway in hCEMC/D3 cells. To the best of our knowledge, this is the first evidence demonstrating P-gp mRNA induction by hPXR in an in vitro cell culture system representative of the human BBB.

In our study, CITCO was used as a selective ligand to activate hCAR (Maglich et al.

2003). Treatment with CITCO induced MDR1 mRNA and P-gp protein expression by 40 % and

60 %, respectively, suggesting the involvement of hCAR in P-gp regulation in our cell culture system. However, this induction appears to occur with no observable nuclear translocation. Our findings also suggest that hCAR is widely distributed in cytosol and nucleus in our cell culture system. Hence, it is possible that activation of hCAR by CITCO in the nucleus is sufficient to induce MDR1 gene transcription without any significant shift in the receptor subcellular localization. Furthermore, previous in vitro studies have demonstrated that nuclear translocation of hCAR may not necessarily result in hCAR transcriptional activity (Zelko et al. 2001,

Kawamoto et al. 1999). Therefore, our data suggest that CITCO-mediated transcriptional regulation of MDR1 gene by hCAR may not significantly depend upon its nuclear translocation.

Cellular downregulation of hPXR or hCAR using siRNA technique was performed to examine the regulation of P-gp expression by nuclear receptors. We observed that a decrease in hPXR and hCAR expression results in a significant dowregulation of P-gp protein expression in

137

hCMEC/D3 cells. These data further suggest that hPXR and hCAR are involved in P-gp regulation in our cell culture system.

A broad array of prescription drugs, herbal remedies, steroid hormones, bile salts and vitamins have been shown to modulate activities of hPXR and hCAR (Moore et al. 2002). This pharmacological modulation could be beneficial in some cases while in others it could lead to adverse effects. Drug transporters, i.e., P-gp and BCRP, can serve as a major pathway for CNS drug clearance at the BBB and brain parenchyma, where drug metabolizing enzymes appear to be expressed at very low levels (Dauchy et al. 2008, Woodland et al. 2008). Inhibition of hPXR and hCAR activities could reduce P-gp expression rendering the BBB more permeable and potentially increase the delivery of CNS drugs that are known P-gp substrates. In contrast, activation of hPXR and hCAR can induce P-gp expression resulting in a less permeable barrier with increased protection against neurotoxicants or drug-induced CNS toxicity. Here, we provide evidence that the activity of hPXR and hCAR can be pharmacological modified by selective inhibitors (i.e., A-792611 and meclizine) thus P-gp induction mediated by activators could be prevented. Currently, different combinations of antiretroviral drugs used in the treatment of HIV can result in significant clinical drug-drug interactions in a number of peripheral tissues (Kis et al. 2010a). Furthermore, several drugs used concurrence for the treatment of HIV and/or HIV- associated complications have been identified to activate hPXR or hCAR. For example, ritonavir

(a common booster for several anti-HIV drugs) is a hPXR agonist; some HMG-CoA reductase inhibitors, used for treatment of lipodystrophy in HIV patients, are both hPXR and hCAR agonists (Dussault et al. 2001, Kobayashi et al. 2005, Bertrand-Thiebault et al. 2007). The chronic co-administration of HIV PIs and HMG CoA-reductase inhibitors is anticipated to induce expression of P-gp at the BBB and further limit antiretroviral drug brain permeability. In this

138

regard, the use of a selective hPXR inhibitor, i.e., A-792611 (a HIV), or a selective hCAR inhibitor can serve as an adjunct therapy to prevent P-gp induction caused by known inducers.

In summary, in this study we demonstrate the expression of hPXR and hCAR in two in vitro representative systems of the human BBB: i) human cerebral microvessel endothelial cell culture system (hCMEC/D3) and ii) primary cultures of human brain-derived microvascular endothelial cells (BBB-ECs). We provide first evidence that pharmacological activation of hPXR and hCAR increase P-gp mRNA and protein expression in hCMEC/D3 cells and that xenobiotic- hPXR/hCAR interactions can be used to counteract pharmacological induction of the drug efflux transporter, P-gp. As more xenobiotics are identified as ligands of hPXR and hCAR, the selective tightening of the human BBB can be manipulated by a careful design of drug regimens or diet which could improve CNS drug delivery or enhance neuroprotection.

7.6 Acknowledgements

The authors thank Dr. Pierre-Olivier Couraud (Institut Cochin, INSERM, Paris France) and Dr. Alexandre Pratt (Centre Hospitalier de l'Université de Montréal, Montréal, Canada) for kindly providing the hCMEC/D3 cell line system and cell pellets from primary cultures of human brain microvessel endothelial cells, respectively. We thank the contribution of Dr. Md.

Tozammel Hoque with undertaking of the siRNA experiments and the confocal imaging studies.

This research is supported by grants from the Canadian Institutes of Health Research (CIHR

Grant # MOP56976) and the Ontario HIV Treatment Network Ministry of Health of Ontario awarded to Dr. Reina Bendayan. Gary Ngai Yin Chan is a recipient of an NSERC (Natural

Sciences and Engineering Research Council of Canada) Postgraduate Scholarship.

139

8. Induction of P-glycoprotein by Antiretroviral Drugs in Human Brain Microvessel

Endothelial Cells

Chemotherapy. 2013, doi: 10.1128/AAC.00486-13.]

Chan GNY, Patel R, Cummins CL and Bendayan R (2013). Induction of P-glycoprotein by

Antiretroviral Drugs in Human Brain Microvessel Endothelial Cells. Antimicrobial Agents and

Chemotherapy. Jul 8. [Epub ahead of print]

Administration of three or more antiretroviral drugs has proven to be an effective pharmacotherapy for the suppression of HIV. However, drug-drug interactions between these agents could lead to sub-therapeutic or toxic drug concentrations. These interactions could be the result of an induction of membrane drug transporter expression (i.e., P-gp), which is known to play an essential role in the transport of these drugs. P-gp induction by currently used antiretroviral drugs at the BBB has not been fully investigated. Since P-gp expression is regulated by ligand-activated nuclear receptors i.e., hPXR and hCAR, these receptors could be involved in contributing to P-gp induction by antiretroviral drugs. The aims of this Chapter 8 were: i) to determine whether antiretroviral drugs currently used in HIV pharmacotherapy are ligands for hPXR or hCAR and ii) to examine P-gp function and expression in human brain microvessel endothelial cells treated with antiretroviral drugs identified as ligands of hPXR and/or hCAR. Luciferase reporter gene assays were performed to examine the activation of hPXR and hCAR by antiretroviral drugs. The human brain microvessel endothelial cell culture system (hCMEC/D3) was utilized to examine P-gp induction following 72 h exposure to these agents. Amprenavir, atazanavir, darunavir, efavirenz, ritonavir and lopinavir were found to

140

activate hPXR, whereas, abacavir, efavirenz and nevirapine were found to activate hCAR. P-gp expression and function were significantly induced in hCMEC/D3 cells treated with these drugs at clinical plasma concentrations. Together, our data suggest that P-gp induction could occur at the BBB during chronic treatment with antiretroviral drugs identified to be ligands of hPXR and/or hCAR.

Author Contributions:

Research design:

GNY Chan (first author), R Patel, CL Cummins and R Bendayan

(principal investigator)

Conducted experiments and data analysis:

GNY Chan (figures 8-1, 8-2, 8-3 & 8-4) and R Patel (figures 8-1 & 8-2)

Writing of the manuscript:

GNY Chan (manuscript drafts and responses to reviewers’ comments), R Patel (editorial review of manuscript drafts), CL Cummins (conceptual and editorial review of the several manuscript drafts and responses to reviewers’ comments) and R Bendayan (overall conceptual and editorial review of the several manuscript drafts and responses to reviewers’ comments)

141

8.1 Abstract

The membrane-associated drug transporter, P-gp, plays an essential role in drug efflux from the brain. Induction of this protein at the BBB could further affect drug permeability into the brain. At present, P-gp induction at the BBB mediated by antiretroviral drugs has not been fully investigated. Since P-gp expression is regulated by ligand-activated nuclear receptors i.e., hPXR and hCAR, these receptors could represent potential pathways involved in P-gp induction by antiretroviral drugs. The aims of this study were: i) to determine whether antiretroviral drugs currently used in HIV pharmacotherapy are ligands for hPXR or hCAR and ii) to examine P-gp function and expression in human brain microvessel endothelial cells treated with antiretroviral drugs identified as ligands of hPXR and/or hCAR. Luciferase reporter gene assays were performed to examine the activation of hPXR and hCAR by antiretroviral drugs. The hCMEC/D3 cell line, known to display several morphological and biochemical properties of the

BBB in humans, was utilized to examine P-gp induction following 72 h exposure to these agents.

Amprenavir, atazanavir, darunavir, efavirenz, ritonavir and lopinavir were found to activate hPXR, whereas, abacavir, efavirenz and nevirapine were found to activate hCAR. P-gp expression and function were significantly induced in hCMEC/D3 cells treated with these drugs at clinical plasma concentrations. Together, our data suggest that P-gp induction could occur at the BBB during chronic treatment with antiretroviral drugs identified to be ligands of hPXR and/or hCAR.

8.2 Introduction

In the last decade, the use of combination antiretroviral therapy has led to a significant decline in the morbidity and mortality of people infected by HIV. At present, although severe forms of HIV Associated Neurocognitive Disorders (e.g. HIV Associated Dementia) have almost

142

disappeared from clinical practice, the prevalence of other mild neurocognitive disorders (e.g.

Asymptomatic Neurocognitive Impairment), remain unchanged, suggesting that viral suppression in the brain is suboptimal (Letendre 2011). These disorders have been associated with lower survival rate and can greatly affect the quality of life of individuals infected by HIV

(Vivithanaporn et al. 2010). One potential explanation for the high prevalence of these disorders is the limited brain permeability of several antiretroviral drugs. A ranking system for drug penetration effectiveness into the central nervous system has been proposed by Dr. S. Letendre to evaluate whether brain penetration of antiretroviral drugs is associated with cerebrospinal fluid viral loads, a clinical indication of brain viral control (Letendre 2011). This group reported that

HIV patients generally exhibited a lower viral load in the cerebrospinal fluid while receiving antiretroviral drugs with higher brain penetration (Letendre 2011). In addition, further evidence suggested that the use of antiretroviral drugs that display high brain permeability can lead to better neurocognitive outcomes (Cysique & Brew 2009). These findings support the concept that the use of antiretroviral regimens with improved brain permeability could reduce viral loads in the brain and ultimately prevent HIV-associated neurological complications.

The presence of an intact BBB has long been recognized to restrict antiretroviral drug entry into the brain (Ene et al. 2011). In addition to the presence of tight junctions, one mechanism for the BBB to restrict drug entry is through the expression of several membrane- associated drug efflux transporters in brain microvessel endothelial cells (Löscher & Potschka

2005b). In particular, P-gp expression at the luminal membrane of these endothelial cells can actively transport substrates (i.e. xenobiotics and drugs) back into the blood following the initial diffusion of these substrates across the luminal membrane (Tsuji et al. 1992, Schinkel et al. 1996,

Lee et al. 2010). Substrates of P-gp include most HIV PIs, the nucleoside reverse transcriptase

143

inhibitor (abacavir), the chemokine CCR5 co-receptor antagonist (maraviroc) and the HIV integrase inhibitor (raltegravir) (Kis et al. 2010a). Therefore, an induction of the functional expression of this protein at the BBB is expected to further restrict brain permeability of antiretroviral drugs known to serve as substrates of this transporter. Our group and others have previously demonstrated that the HIV PIs, ritonavir and atazanavir, can induce P-gp expression in human and rodent brain microvessel endothelial cells which constitute the BBB (Perloff et al.

2004, Perloff et al. 2007, Zastre et al. 2009). However, little is known on the ability of other antiretroviral drugs to induce P-gp expression at this site. Induction of P-gp and other drug transporters (e.g. MRP2) in peripheral organs, such as liver and intestine, has been demonstrated to be mediated through the activity of xenobiotic-activating nuclear receptors, PXR and CAR

(Urquhart et al. 2007). At the BBB, PXR and CAR have also been demonstrated to regulate P-gp expression in brain microvessel endothelial cells or isolated brain capillaries from porcines and rodents (Bauer et al. 2004, Bauer et al. 2006, Ott et al. 2009, Wang et al. 2010, Lemmen et al.

2013). Recently, our group has shown that hPXR and hCAR are actively involved in the regulation of P-gp in a human brain microvessel endothelial cell culture system, hCMEC/D3

(Chan et al. 2011). These two nuclear receptors have been identified as xenobiotic sensors that are capable to interact with a wide array of pharmaceutical agents (Chang & Waxman 2006). At present, a few HIV PIs (i.e. ritonavir, amprenavir and lopinavir) have been identified to serve as ligands of hPXR as determined by reporter-based assays (Dussault et al. 2001, Svärd et al. 2010).

In addition, efavirenz, a non-nucleotide reverse-transcriptase inhibitor, is capable of inducing the promoter activity of a phase I drug metabolizing enzyme Cytochrome P450 enzyme 3A4 when transfected with a full-length hPXR plasmid (Hariparsad et al. 2004). Moreover, Svärd et al. used a panel of antiretroviral drugs to screen for CYP3A4 and CYP2B6 promoter activation

144

mediated by hPXR and hCAR, however they reported several discrepancies in receptor activation by several HIV PIs (i.e. nelfinavir, ritonavir, atazanavir) compared to previous published reports

(Gupta et al. 2008, Svärd et al. 2010). The aims of this study were: i) to determine whether several antiretroviral drugs currently used in first-line and alternative regimens during HIV pharmacotherapy are ligands for hPXR or hCAR and ii) to examine P-gp function and expression in human brain microvessel endothelial cells following treatment with antiretroviral drugs identified as ligands of hPXR and hCAR. In this work, we used an in vitro cell culture system of brain microvessel endothelial cells (hCMEC/D3) which has been demonstrated to display several morphological and biochemical properties of human brain microvascular endothelium, such as the expression of tight junction and adhesion proteins, endothelial cell markers, drug efflux transporters (e.g. P-gp) and nuclear receptors (e.g. PXR and CAR) (Chan et al. 2011, Weksler et al. 2005, Zastre et al. 2009).

8.3 Methods

8.3.1 Materials and Reagents

Cell culture media (OPTIMEM and DMEM), FBS, penicillin/streptomycin, sterile PBS,

Hank's Balanced Salt Solution, 0.25% Trypsin and liver digest medium were purchased from

Invitrogen (Grand Island, NY). Rat tail Type I collagen was purchased from Becton-Dickinson

(San Jose, CA). Heat shock fraction V BSA, DMSO and acrylamide solution were obtained from

Bioshop Canada Inc., (Burlington, ON, Canada). CITCO and SR12813 compounds were supplied from BIOMOL Research Laboratories (Plymouth Meeting, PA). Rifampin, MTT, R-6G, phenylmethanesulfonylfluoride, protease inhibitor cocktail and anti-actin (mouse monoclonal) antibody were purchased from Sigma-Aldrich (Oakville, ON, Canada). Antiretroviral drugs were kindly provided by the National Institutes of Health, AIDS Research and Reference Reagent

145

Program (Germantown, MD). Anti-P-gp (mouse monoclonal) antibody was obtained from ID

Labs Inc. (London, ON, Canada). The goat anti-mouse horseradish peroxidase-conjugated secondary antibody was ordered from Jackson ImmunoResearch Laboratories Inc. (West Grove,

PA). pRL-CMV was purchased from Promega (Madison, WI). UAS-luciferase, GAL4-CMX,

GAL4-hPXR ligand binding domain (LBD), CMX and β-galatosidase plasmids were provided by

Dr. David Mangelsdorf (University of Texas Southwestern Medical Center, Dallas, TX). Gal4- hCAR LBD was a generous gift from Dr. David Moore (Baylor College of Medicine, Houston,

TX).

8.3.2 Cell Culture and Luciferase Reporter Assays

The monkey kidney fibroblast CV-1 cells, purchased from ATCC, were maintained in

DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin (Invitrogen).

Transfections were performed using fuGENE®HD transfection reagent (Roche Diagnostics

Corp., IN) in 48-well plates in DMEM medium supplemented with 10% charcoal-treated FBS

(Invitrogen). The total amount of plasmid DNA was kept constant at 400 ng per well. Cells were cotransfected with GAL4-responsive luciferase reporter (UAS-luc, 200 ng) and GAL4 DNA binding domain fused to the LBD of hPXR (residues 184 - 433; 100 ng). Data were normalized to the β-galactosidase control (100 ng) and expressed as relative light units. As a negative control for the receptor, the cytomegalovirus-based (CMX) expression plasmid containing GAL4 alone was used as a negative control (GAL4-CMX). Antiretroviral drugs were added in fresh medium 6 h post-transfection, and cells were harvested 14-16 h later and assayed for luciferase and β-galactosidase activities. Primary cultures of mouse hepatocytes were isolated and cultured as previously described (Patel et al. 2011). In brief, liver of wild-type 129/SvEv mouse was perfused with liver digestion media (3 mL/min) and viable hepatocytes purified by centrifugation

146

3 times at 50 x g. Freshly prepared hepatocytes were seeded in attachment medium (William’s E

Media, 10% charcoal-treated FBS, 1× penicillin/streptomycin and 10 nM insulin) at a final density of 1.2 × 104 cells per well in type I collagen coated 48-well plates. Transfections were performed with 250 ng DNA per well using Lipofectamine 2000 reagent (Invitrogen) in

OPTIMEM without FBS. Cells were cotransfected with GAL4-responsive luciferase reporter

(UAS-luc, 125 ng) and GAL4 DNA binding domain fused to the LBD of hCAR (residues 101 -

348; 25 ng) and pRL-CMV Renilla control (50 ng). Transfection efficiency in the primary cultures of mouse hepatocytes was determined to be approximately 80 %, as determined by transfection of a green fluorescent protein, GFP, control plasmid in some cultures. At 24 h post- transfection, cells were exposed to M199 medium without FBS containing antiretroviral drugs or positive control ligands. Cells were harvested after an additional 14–16 h and assayed for luciferase and Renilla activities. Luciferase values were normalized for transfection efficiency using Renilla and expressed as RLU.

8.3.3 hCMEC/D3 Cell Culture System and Ligand Treatment

The immortalized human brain microvessel endothelial cell line, hCMEC/D3, was kindly provided by Dr. P.O. Couraud (Institut Cochin, Département de Biologie Cellulaire and Inserm,

Paris, France) (Weksler et al. 2005). Cells were cultured on rat tail type I collagen (BD)-coated

75-mm flasks, 60-mm dishes or 24-well plates and maintained at 37 °C, 5 % CO2, and 95 % humidified air in EGM-2 medium supplemented with EGM-2 Single Quot growth factors kit, which includes vascular endothelial growth factor, insulin-like growth factor-1, epidermal growth factor, fibroblast growth factors, hydrocortisone, ascorbate, gentamycin, and 2.5 % FBS as recommended (Lonza, Walkersville, MD). Once monolayers of hCMEC/D3 cells reached an

80% confluence, culture medium was aspirated and fresh medium containing hPXR or hCAR

147

ligands was added to the cells for 72 h. All ligands were dissolved in DMSO. To ensure cells remain viable during treatment, all ligand concentrations used were tested applying the tetrazolium salts (MTT) assay as described previously (Chan et al. 2011). In brief, cells were incubated in PBS containing 2.5 mg/mL MTT at 37 ºC for 2 h following 72 h ligand treatment.

Cell viability was assessed by comparing the absorbance (580 nm) of cellular reduced MTT in ligand-treated cells to vehicle-treated cells using SpectraMax 384 microplate reader (Molecular

Devices, Sunnyvale, CA).

8.3.4 Immunoblot Analysis

Protein expression of P-gp and actin was determined by SDS-PAGE according to previously published protocols (Chan et al. 2011). In brief, monolayers of hCMEC/D3 cells were washed with warm PBS once and collected by scraping in ice-cold PBS. Following centrifugation at 1000 g, whole cell lysates were prepared by exposing cell pellets to lysis buffer

(1 % (v/v) NP-40, 20 mM Tris, 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethanesulfonylfluoride and 0.1 % (v/v) protease inhibitor cocktail) for 15 min at 4 °C.

Cell lysates were sonicated for 10 sec and centrifuged at 20,000 g for 5 min at 4 °C. Supernatants containing 50 µg of protein were resolved by SDS-PAGE and electrotransferred onto a PVDF membrane (GE Healthcare Life Sciences, Piscataway, NJ). Membranes were blocked for 2 h in

Tris-Buffer (15mM Tris, 0.1% Tween20 and 5 % skim milk) and incubated with primary antibody overnight at 4 °C prior to further incubation in anti-mouse secondary antibody (Jackson

ImmunoResearch Laboratories Inc.). P-gp expression was detected using a 1:500 dilution of mouse monoclonal C219 antibody (ID Labs, London), which recognizes an internal, highly conserved amino acid sequence VQEALD and VQAALD of P-gp. β-actin expression (protein accession # P60710) was detected using a 1:3000 dilution of mouse monoclonal C-4 antibody

148

(Santa Cruz Biotechnology, Inc.). Densitometric analysis was performed using AlphaDigiDoc

RT2 software (Alpha Innotech, San Leandro, CA) to quantify protein expression detected by an enhanced chemiluminescence kit (Pierce Thermo Fisher Scientific Inc., Waltham, MA). Whole cell lysates of MDA435/LCC6/MDR1 cells (cultured in α-MEM medium supplemented with

10% FBS and 1% penicillin/streptomycin) served as positive control for P-gp expression.

8.3.5 Rhodamine-6G Transport Assay

The cellular accumulation of a well-established fluorescence P-gp substrate, R-6G, was measured to determine the activity of P-gp in hCMEC/D3 cells according to previously published protocols (Zastre et al. 2009). Following 72 h ligand (i.e. antiretroviral drugs or hPXR or hCAR agonists) treatment of hCMEC/D3 cell monolayer grown on 24-well plates, cells were rinsed with fresh medium without ligands three times before being incubated with transport buffer (1X

Hank's Balanced Salt Solution, 10 mM HEPES and 0.01% BSA, pH at 7.4) for 30 min. Cells were exposed to transport buffer containing 1 µM R-6G in the presence or absence of P-gp inhibitor PSC-833 (5 μM) at 37 °C for 30 min and were washed with ice-cold PBS three times to stop the reaction. Triton X-100 (1%) was added to wells and cellular R-6G accumulation was measured at an excitation and emission wavelengths of 530 and 560 nm, respectively, using a

SPECTRAmax plate reader (Molecular Devices, Sunnyvale, CA). Data are reported as the ratio of relative R-6G emission signals normalized to the protein content from corresponding wells between ligand-treated group and vehicle control.

8.3.6 Statistical Analysis

All experiments were repeated at least three times in cells with different passages. Results are reported as a mean ± S.D. Comparisons between groups were performed using one-way

149

ANOVA with Dunnett post hoc t-test at a significance level of p < 0.05. Data were analyzed by

SPSS software (Chicago, IL).

8.4 Results

Luciferase reporter gene assays performed in CV1 cells and primary cultures of mouse hepatocytes were utilized to examine the ability of antiretroviral drugs to serve as ligands of hPXR and hCAR. GAL4-hPXR chimeric receptor activity was induced (fold induction normalized to DMSO) in the presence of positive controls i.e., rifampin (4.5 ± 0.5) and SR12813

(17 ± 4.7), and the following antiretroviral drugs; Lopinavir (8.1 ± 0.2), amprenavir (6.1 ± 0.4), efavirenz (3.2 ± 0.3), darunavir (3.2 ± 0.2), ritonavir (2.5 ± 0.1) and atazanavir (2.1 ± 0.2)

(Figure 8-1). GAL4-hCAR chimeric receptor activity was induced (fold induction normalized to

DMSO) in the presence of positive control; CITCO (7.9 ± 1.2), and the following antiretroviral drugs; Efavirenz (3.7 ± 0.5), abacavir (2.4 ± 0.1) and nevirapine (1.8 ± 0.3) (Figure 8-2). Data are presented from a representative experiment. Each screen was performed on three independent occasions with CV1 cells of different passage numbers and independent isolations of primary mouse hepatocytes.

150

Figure 8-1. Activation of hPXR by Antiretroviral Drugs. Representative set of data from three independent luciferase reporter gene assays performed in transfected CV-1 cells after 14-16 h treatment with antiretroviral drugs (10 µM) are presented as mean ± S.D. of triplicates.

Luciferase activity (representing ligand activation) was normalized by constitutive β- galactosidase activity (to control for transfection efficiency) and reported as Relative Light Units (RLU). Statistically significant differences in receptor activation (normalized to vehicle control) were determined by one-way ANOVA with Dunnett’s post hoc test at a significance level of * p < 0.05. Antiretroviral drug solutions were prepared by GNY Chan and reporter gene assays were performed by R Patel.

151

Figure 8-2. Activation of hCAR by Antiretroviral Drugs. Representative data from three independent dual-luciferase reporter gene assays performed in transfected primary cultures of mouse hepatocytes after 14-16 h treatment with antiretroviral drugs (10 µM) are presented as mean ± S.D. of triplicates. Luciferase activity (representing ligand activation) was normalized by constitutive Renilla activity (to control for transfection efficiency) and reported as Relative Light Units (RLU). Statistically significant differences in receptor activation (normalized to vehicle control) were determined by one-way ANOVA with Dunnett’s post hoc test at a significance level of * p < 0.05. Antiretroviral drug solutions were prepared by GNY Chan and reporter gene assays were performed by R Patel.

The human brain microvessel endothelial cell culture system, hCMEC/D3, was utilized to examine P-gp induction mediated by antiretroviral drugs that exhibited hPXR and/or hCAR ligand properties. Previously, in the same culture system, our group demonstrated that 72 h cell exposure to 10 μM atazanavir or 10 μM ritonavir could induce P-gp functional expression by approximately 2-fold compared to vehicle control (DMSO) (Zastre et al. 2009). In the current

152

study, hCMEC/D3 cells were exposed to antiretroviral drugs for 72 h at clinically relevant plasma concentrations (i.e., lopinavir: 8.7 – 15 μM (Capparelli et al. 2005), amprenavir: 11 – 19

μM (Croteau et al. 2012), darunavir: 3.3 – 24 μM (Yilmaz et al. 2009b), efavirenz: 9.2- 17 μM

(Tashima et al. 1999), abacavir: 5.2 – 15 μM (McDowell et al. 1999) and nevirapine: 7.5 – 21

μM (Antinori et al. 2005)). Lopinavir (10 μM), amprenavir (15 μM) and darunavir (10 μM), which served as ligands of hPXR, induced P-gp expression in hCMEC/D3 cells by approximately 2.3 ± 0.1, 2.3 ± 0.3 and 1.7 ± 0.1 fold, respectively, compared to vehicle control

(Figure 8-3). Abacavir (15 μM) and nevirapine (15 μM), identified as ligands of hCAR, induced

P-gp expression in hCMEC/D3 cells by approximately 1.5 ± 0.1 and 1.6 ± 0.1 fold, respectively, compared to vehicle control. Efavirenz (10 μM), the only drug which served as a ligand of both hPXR and hCAR, induced P-gp expression in hCMEC/D3 cells by 2.0 ± 0.2 fold compared to vehicle control. Rifampin and SR12813 (established agonists of hPXR) and CITCO (established agonist of hCAR) served as positive controls for P-gp induction. Consistent with previous findings, these ligands showed approximately 2-fold increase in P-gp expression (Chan et al.

2011). Drug accumulation assays utilizing R-6G (a fluorescent P-gp substrate) were performed to assess changes in P-gp transport function in hCMEC/D3 cells following 72 h treatment with antiretroviral drugs that mediated P-gp protein induction as depicted in Figure 8-3. We observed that treatment of hCMEC/D3 cells with antiretroviral drugs significantly reduced cellular accumulation of R-6G by approximately 20 – 30 % compared to vehicle control (Figure 8-4) suggesting an increase in R6G efflux process. Moreover, PSC833 (a potent inhibitor of P-gp) at 5

µM abolished the differences in R-6G accumulation between vehicle control and treatment groups, further confirming the involvement of P-gp in the efflux of R-6G. Together, these data

153

demonstrate that P-gp induction mediated by antiretroviral drugs can result in an increase in P-gp transport function in human brain microvessel endothelial cells.

154

Figure 8-3. P-gp Immunoblot and Densitometric Analysis. P-gp expression normalized to vehicle control (DMSO) in hCMEC/D3 cells after 72 h treatment with i) hPXR ligands: rifampin

(10 µM; hPXR agonist) and SR12813 (10 µM; potent hPXR agonist), amprenavir (15 µM; HIV

PI), darunavir (10 µM; HIV PI) and lopinavir (10 µM; HIV PI); ii) hCAR ligands: CITCO (7.5 µM; synthetic potent hCAR agonist), abacavir (15 µM; NRTI), nevirapine (15 µM; NNRTI); and iii) dual hPXR and hCAR ligand: efavirenz (10 µM; NNRTI). Representative immunoblot of P- gp expression is shown on the top panel. Whole cell lysates prepared from P-gp-overexpressing human breast carcinoma cell culture system (MDA-MDR1, 2 µg) served as P-gp positive control. A 50 µg load of whole cell lysates of hCMEC/D3 was resolved on a 10% SDS-PAGE gel and subsequently transferred to PVDF membrane. Densitometric analysis was performed to determine the relative P-gp expression. P-gp was detected using the monoclonal antibody C219 (1:500) and anti-mouse secondary antibody (1:3000). Data represent percentage fold change normalized to vehicle control and reported as mean ± S.D. obtained from three experiments from cells of different passages. *Statistically significant differences in P-gp expression compared to control as determined by one-way ANOVA with Dunnett pos hoc t-test at a significance level of * p < 0.05. Cell treatment and immunoblot analysis were performed by GNY Chan.

155

Figure 8-4. R-6G Cellular Accumulation by hCMEC/D3 Cells. R-6G accumulation by the cell monolayer was measured after 72 h cell treatment with i) hPXR ligands: rifampin, SR12813, amprenavir, darunavir, lopinavir; ii) hCAR ligands: CITCO, abacavir, nevirapine; and iii) dual hPXR and hCAR ligand: efavirenz. In the group without PSC833 treatment (open bars), results are expressed as mean percent change in R-6G accumulation versus control (vehicle without PSC -833) ± S.E.M. obtained from three separate experiments using different passages of cells. In the PSC-833 group (solid bars), results are expressed as mean percent change in R-6G accumulation normalized to its corresponding treatment without PSC-833 ± S.E.M.. In each experiment, each treatment and vehicle control groups were performed in triplicate. One-way ANOVA with Dunnett pos hoc t-test at a significance level of p < 0.05 was performed. * S tatistically significant differences in R-6G accumulation between cells treated with antiretroviral drugs and vehicle control. ** Statistically significant differences in R-6G accumulation between cells exposed to PSC-833 and cells that were not exposed to PSC-833. Cell treatment and R-6G transport assay were performed by GNY Chan.

156

8.5 Discussion

The use of combination antiretroviral therapy has significantly increased the survival rate among individuals infected with HIV. Most antiretroviral drugs currently used in first line and alternative regimens during HIV pharmacotherapy are known to be metabolized by phase I enzymes (i.e. cytochrome P450 enzymes) and/or transported by several drug efflux transporters

(e.g. P-gp) (Kis et al. 2010a, Pal et al. 2011). Induction of these systems during prolonged antiretroviral treatment may profoundly alter pharmacokinetic parameters and ultimately impair effectiveness of antiretroviral drugs at target organs, contributing to the development of sanctuary sites and cellular reservoirs (Pomerantz 2003). At the BBB, the functional expression of P-gp has been recognized to restrict brain entry of antiretroviral drugs, in particular for the

HIV PI (Ene et al. 2011). P-gp induction at the BBB is expected to further limit brain permeability of these agents, preventing therapeutic concentrations to be achieved in brain parenchyma. It has been proposed that antiretroviral regimens containing drugs with better brain permeability may improve clinical outcomes in patients living with HIV- associated neurocognitive disorders (Letendre 2011). Therefore, knowledge on the ability of antiretroviral drugs to induce P-gp expression at the BBB can be useful for the design of drug regimens with limited P-gp induction during HIV pharmacotherapy, ultimately improving brain permeability of antiretroviral drugs that are known to be P-gp substrates.

Our group has recently demonstrated that well-established ligands of hPXR and hCAR can induce P-gp expression in the hCMEC/D3 cells, an in vitro representative system of the human BBB (Chan et al. 2011, Zastre et al. 2009). Luciferase reporter gene assays were utilized in the current study to examine the ability of antiretroviral drugs to serve as ligands for hPXR and/or hCAR. Our results demonstrate that most members of the HIV PI pharmacotherapy class

157

and efavirenz, a NNRTI, can serve as ligands of hPXR. This is consistent with earlier publications which demonstrated that ritonavir, lopinavir and efavirenz were able to trigger hPXR activity (Dussault et al. 2001, Hariparsad et al. 2004, Svärd et al. 2010). In addition, we provide the first evidence that amprenavir, darunavir and atazanavir can also serve as ligands of hPXR (Figure 8-1). Furthermore, there is evidence suggesting that lopinavir and abacavir can serve as ligands of hCAR in transfected human liver hepatocellular carcinoma cell line (HepG2) cells (Svärd et al. 2010). In the current work, we performed a reporter gene assay in primary cultures of mouse hepatocytes, which has previously been identified to exhibit enhanced CAR- inducible signalling (i.e. nuclear translocation) when compared to HepG2 cultures (Maglich et al.

2003, Li et al. 2009). We found that abacavir, belonging to the nucleoside/nucleotide reverse- transcriptase inhibitor (NRTI) class, as well as efavirenz and nevirapine from the NNRTI class can serve as ligands of hCAR (Figure 8-2). In addition, none of the members from the HIV PI class were identified to serve as ligands of hCAR. It is also interesting to note that lopinavir and ritonavir consistently reduced the activity of hCAR below its basal level in three experimental replicates at drug concentrations that were not found toxic to the mouse hepatocytes. In the current study, the activation of hPXR and hCAR by antiretroviral drugs could be due to specific binding between ligands and the ligand binding pocket of the nuclear receptors. Several X-ray crystal structures of the ligand binding pocket of hPXR and hCAR have been shown to be large and flexible with hydrophobic sites consisting of multiple polar residues (Xue et al. 2007, Xu et al. 2004). At present, structural information on how antiretroviral drugs interact with the ligand binding pocket of hPXR and hCAR is unclear. Interestingly, a recent study has successfully predicted ligand activation of hPXR by efavirenz using an in silico ligand-docking approach,

158

however the specific interactions between chemical structures of other antiretroviral drugs and the ligand binding pocket of hPXR and hCAR have not been reported (Khandelwal et al. 2008).

The effect of prolonged treatment of antiretroviral drugs on P-gp expression at the human

BBB is unclear. Perloff et al. demonstrated that ritonavir can induce P-gp in brain microvessel endothelial cells isolated from rat and bovine (Perloff et al. 2004, Perloff et al. 2007). In addition, our group has previously shown that ritonavir and atazanavir can induce P-gp functional expression in the hCMEC/D3 cells (Zastre et al. 2009). To further examine this effect, we treated hCMEC/D3 cells with antiretroviral drugs that we identified as ligands of hPXR and hCAR. Our data show that prolonged exposure (72 h) to these drugs can induce functional expression of P-gp in the hCMEC/D3 cells (Figures 8-3 and 8-4). As well, results from MTT cell viability assays suggest that concentrations used for these agents do not affect cell viability (data not shown). In the current study, P-gp induction in the hCMEC/D3 cells mediated by treatment with ritonavir, atazanavir, lopinavir, darunavir, nevirapine and efavirenz supports previous findings demonstrating that these drugs were able to induce P-gp expression in lymphocytes, intestinal and hepatic tissues (Gupta et al. 2008, Perloff et al. 2005, Chandler et al. 2003, Weiss et al. 2008, Dixit et al. 2007, Perloff et al. 2000, Mader et al. 1993, Konig et al. 2010).

Interestingly, efavirenz is the only drug which appears to serve as a ligand of both hPXR and hCAR. However, the induction effect was not significantly higher compared to other antiretroviral drugs which only served as ligands for one of the two receptors (hPXR or hCAR), suggesting that there is a lack of synergistic effects on P-gp induction mediated by hPXR and hCAR at the efavirenz concentration we used. Our current study cannot exclude the possibility that other ligand-activated nuclear receptors (e.g., vitamin D receptor and/or peroxisome proliferator-activated receptors) could play a role in the regulation of P-gp in hCMEC/D3 cells.

159

Further studies are required to examine the interactions of antiretroviral drugs with these other nuclear receptor pathways which are also known to regulate P-gp at the BBB.

In conclusion, several antiretroviral drugs currently used as first line and alternative regimens in HIV pharmacotherapy can serve as ligands of the nuclear receptors, hPXR and hCAR. In particular, most PIs are hPXR ligands, while abacavir and nevirapine can serve as hCAR ligands and efavirenz is a ligand of both, hPXR and hCAR. In addition, an increase in P- gp functional expression mediated by these antiretroviral drugs in the hCMEC/D3 cells, an in vitro model of human brain microvessel endothelial cells, suggests that chronic antiretroviral pharmacotherapy utilizing these agents could potentially lead to P-gp induction at the human

BBB. This effect could further restrict brain permeability of antiretroviral drugs that are P-gp substrates. In our study, efavirenz, shown to be P-gp inducer in the hCMEC/D3 cells, is currently recommended to be administered with abacavir as an alternative NNRTI-based regimen in HIV pharmacotherapy-naive patients. Since abacavir is a known P-gp substrate (Shaik et al. 2007), the potential P-gp induction mediated by efavirenz could further reduce abacavir concentrations in the brain. Positron emission tomography (PET) imaging with the use of 11C-verapamil (a P-gp substrate) in humans could be applied to investigate P-gp transport activity at the BBB and further examine the clinical effect of chronic efavirenz administration on P-gp functional expression at the BBB (Muzi et al. 2009, Bauer et al. 2012, Syvänen & Eriksson 2013). The results from these studies could guide clinical recommendations of drug regimens that avoid potential P-gp induction at the BBB and improve brain entry of antiretroviral drugs that are known P-gp substrates (i.e., abacavir).

160

8.6 Acknowledgements

This research was funded in part by a grant from the Canadian Institutes of Health

Research (CIHR Grant # MOP56976) awarded to Dr. Reina Bendayan and in part by the Natural

Sciences and Research Council of Canada (NSERC RGPIN 356873-08) awarded to Dr. Carolyn

L. Cummins. Dr. Bendayan is the recipient of a Career Scientist Award from the Ontario HIV

Treatment Network, Ministry of Health of Ontario.

8.7 Conflicts of interest

None declared.

161

9. In vivo induction of P-Glycoprotein expression at the mouse blood-brain barrier: an

intracerebral microdialysis study

This work is published and reproduced in this thesis with permission from Wiley-Blackwell:

Chan GNY, Saldivia V, Yang Y, Pang H, de Lannoy I and Bendayan R. (2013). Journal of

Neurochemistry. Jun 18. doi: 10.1111/jnc.12344. [Epub ahead of print]

Drug permeability across the BBB can be significantly restricted by the functional expression of the drug efflux transporter, P-gp. Although, the effect of P-gp induction on BBB drug permeability has been examined in vitro and in vivo, drug concentrations in brain microvessel endothelial cells and/or whole brain homogenate may not accurately predict unbound drug concentrations in brain extracellular fluid (ECF). Chapter 9 describes the use of quantitative intracerebral microdialysis to investigate the effect of P-gp induction at the mouse

BBB on brain ECF concentrations of quinidine, an established P-gp substrate. Induction was achieved by treating male CD-1 mice for three days with 5 mg/kg/day dexamethasone (DEX), a ligand of the PXR, and a known P-gp inducer. The relative loss of deuterated quinidine was validated and used to correct for recovery of quinidine across the dialysis probe. An LC-MS/MS method was used to quantify the analytes in dialysate, blood and plasma. P-gp, PXR and

Cyp3a11 (metabolizing enzyme for quinidine) protein expression in capillaries and brain homogenate was measured by immunoblot analysis. Following quinidine i.v. administration, the ratio of unbound quinidine concentrations in brain ECF to plasma at steady state (Kp,uu,ECF/Plasma), in the DEX-treated animals (0.063±0.025) was 2.5-fold lower compared to vehicle-treated animals (0.149±0.031). In DEX-treated animals, P-gp expression in brain capillaries was 1.5-fold higher compared to vehicle-treated animals while Cyp3a11 expression in brain capillaries was not significantly different between the two groups. These data demonstrate that P-gp induction

162

mediated by DEX at the BBB can significantly reduce quinidine brain ECF concentrations by decreasing its brain permeability and further suggest that drug-drug interactions as a result of P- gp induction at the BBB are possible.

Author Contributions:

Research design: GNY Chan (first author), I de Lannoy (collaborator and co-

corresponding author) and R Bendayan (principal investigator)

Conducted experiments: GNY Chan (every aspect of the study except animal surgery and LC- MS/MS: figures 9-1 to 9-8), V Saldivia (animal surgery and treatment: figures 9-1 to 9-2), and Y Yang (LC-MS/MS: figures 9-1 to 9-2) and H Pang (LC-MS/MS: figures 9-1 to 9-2) In vivo intracerebral microdialysis experiments (animal housing, surgery and treatment) and LC-MS/MS analysis were all conducted at NoAb BioDiscoveries Inc., Mississauga, Ontario. Data analysis: GNY Chan (figures 9-1 to 9-8), H Pang (figures 9-1 to 9-2) and I de Lannoy (figures 9-1 to 9-2) Writing of the manuscript: GNY Chan (manuscript drafts and responses to reviewers’ comments), H Pang (LC-MS/MS section), I de Lannoy (overall conceptual and editorial review of the several manuscript drafts and responses to reviewers’ comments) and R Bendayan (overall conceptual and editorial review of the several manuscript drafts and responses to reviewers’ comments)

163

9.1 Abstract

Intracerebral microdialysis was utilized to investigate the effect of P-glycoprotein (a drug efflux transporter) induction at the mouse BBB on brain extracellular fluid concentrations of quinidine, an established substrate of P-glycoprotein. Induction was achieved by treating male

CD-1 mice for three days with 5 mg/kg/day dexamethasone (DEX), a ligand of the nuclear receptor, PXR, and a P-glycoprotein inducer. LC-MS/MS method was used to quantify analytes in dialysate, blood and plasma. P-glycoprotein, PXR and Cyp3a11 (metabolizing enzyme for quinidine) protein expression in capillaries and brain homogenate was measured by immunoblot analysis. Following quinidine i.v. administration, the average ratio of unbound quinidine concentrations in brain extracellular fluid (determined from dialysate samples) to plasma at steady state (375-495 min) or Kp, uu, ECF/Plasma in the DEX-treated animals was 2.5-fold lower compared to vehicle-treated animals. In DEX-treated animals, P-glycoprotein expression in brain capillaries was 1.5-fold higher compared to vehicle-treated animals while Cyp3a11 expression in brain capillaries was not significantly different between the two groups. These data demonstrate that P-gp induction mediated by DEX at the BBB can significantly reduce quinidine brain extracellular fluid concentrations by decreasing its brain permeability and further suggest that drug-drug interactions as a result of P-gp induction at the BBB are possible.

9.2 Introduction

The BBB physically and metabolically functions as a neurovascular interface between the brain parenchyma and the systemic circulation. Non-fenestrated capillary endothelial cells are the major component of the BBB that participate in regulating the permeability of several endogenous substrates and xenobiotics into and out of the CNS (Eyal et al. 2009). In addition, several membrane-associated drug efflux transporters have been characterized at the luminal

164

membrane of brain capillary endothelial cells and serve as efflux pumps to extrude substrates

(i.e., pharmacological agents) from the CNS back into the systemic circulation. In particular, P- gp, belonging to the ATP Binding Cassette membrane-associated transporter superfamily, is one of the most extensively studied drug transporters in brain capillary endothelial cells. This transporter has been well recognized to actively efflux many structurally diverse molecules including, steroids, environmental toxins and clinically prescribed drugs (e.g. anticancer, antiretroviral, antihypertensive, antiarrhythmic, antimicrobial agents and others) (Schinkel et al.

1996, Eyal et al. 2009) and its high expression in brain capillary endothelial cells is considered to be an essential mechanism which restricts brain entry of many pharmacological agents

(Bendayan et al. 2002, Eyal et al. 2009). In addition, its expression at the BBB is well- recognized to be upregulated during several neurological disorders [e.g., seizure activity (Tishler et al. 1995, Dombrowski et al. 2001)], pathological stimuli [e.g. pro-inflammatory cytokines

(Yu et al. 2008, Bauer et al. 2007) and HIV viral proteins (Hayashi et al. 2005)], as well as chronic exposure to xenobiotics including drugs [e.g. ritonavir (Perloff et al. 2004, Zastre et al.

2009) and rifampin (Bauer et al. 2006)]. Among many molecular pathways, one mechanism which explains P-gp induction at the BBB involves agonist-activation of nuclear receptors, such as the PXR (Bauer et al. 2006, Narang et al. 2008, Chan et al. 2011). Upon agonist activation,

PXR can serve as a factor which promotes transcription of MDR1 and mdr1a/1b genes which encode P-gp in humans and rodents, respectively (Geick et al. 2001, Cui et al. 2010). A wide array of structurally diverse molecules, such as steroid hormones, bile acids, herbal and dietary constituents as well as therapeutic agents, are known ligands of human and/or rodent PXR

(Chang & Waxman 2006). Potent agonists of rodent PXR have been used to study, in vivo, P-gp induction at the BBB. For example, dexamethasone (DEX), a potent agonist of rodent PXR, has

165

been utilized to induce P-gp expression in rodent brain capillaries (Bauer et al. 2004).

Determination of the in vivo disposition of a drug in rodent brain is commonly performed via the bioanalysis of brain homogenate and/or cerebrospinal fluid (CSF) obtained by the terminal collection of whole brain and CSF samples at various time intervals following dosing of the drug

(De Lange & Danhof 2002, Shen et al. 2004). However, drug concentrations from these samples may not always accurately predict the unbound drug concentrations in the brain extracellular fluid (ECF) which are primarily related to drug concentrations at the site of action (Lin 2008,

Westerhout et al. 2012, Westerhout et al. 2013). Intracerebral microdialysis is a powerful technique that offers a continuous in vivo monitoring of drug concentrations in the brain ECF (De

Lange et al. 1998, De Lange et al. 2000, Chaurasia et al. 2007, Hammarlund-Udenaes et al.

2009). With the use of selective drug transporter substrates such as the antiarrhythmic drug quinidine, this technique has been utilized in rats to examine the role of P-gp at the BBB in vivo

(Krajcsi et al. 2012, Syvänen et al. 2012, Westerhout et al. 2013). Quinidine has been identified as a P-gp substrate and inhibitor by many groups (Kusuhara et al. 1997, Emi et al. 1998,

Giacomini et al. 2010). For example, the use of P-gp inhibitors, such as PSC-833 and LY-

335979, resulted in an increase in quinidine brain accumulation by seven to ten-fold in rodents

(Wang et al. 1996, Starling et al. 1997, Kusuhara et al. 1997). Moreover, P-gp knockout mice showed a 27 to 50 times increase in quinidine total brain to plasma concentration ratios compared to wild-type control (Kusuhara et al. 1997, Uchida et al. 2011a, Kodaira et al. 2011). In addition, several in vitro and in vivo studies suggest that P-gp constitutes the primary efflux mechanism for quinidine, while other major efflux drug transporters found at the BBB, such as breast cancer resistance protein and multidrug resistance-associated proteins show no interactions with quinidine (Kalvass et al. 2007, Sziráki et al. 2011, Kodaira et al. 2011). Cytochrome P450 (CYP)

166

enzyme 3A4 in humans and its mouse ortholog Cyp3a11 can metabolize quinidine, while 10 to

50% of the drug is excreted unchanged in urine (De Lange 2013). To date, to the best of our knowledge, no information is available regarding the effect of ligand-mediated P-gp induction at the BBB on the brain distribution of quinidine. In the current study, we utilized quantitative intracerebral microdialysis to examine quinidine distribution in the brain ECF of mice following

P-gp induction at the BBB mediated by DEX treatment.

9.3 Materials and Methods

9.3.1 Chemicals and Reagents

Quinidine hydrochloride monohydrate, DEX, corn oil, PMSF and protease inhibitor cocktail were purchased from Sigma-Aldrich (St. Louis, MO). Deuterated quinidine (quinidine-

D3) was obtained from Toronto Research Chemicals (Toronto, ON, Canada). Lidocaine was provided as a generous gift from AB Sciex (Concord, ON, Canada). Bovine Fraction V Heat

Shock BSA and Ficoll 400 were acquired from Bioshop (Burlington, ON, Canada). Sterile PBS was purchased from Invitrogen (Grand Island, NY). Murine monoclonal C219 antibody against

P-gp was purchased from ID Laboratories (London, ON, Canada). Murine monoclonal C-4 (sc-

47778) antibody against β-actin, goat polyclonal A-20 (sc-7737) antibody against mouse PXR and goat polyclonal L-14 (sc-30621) antibody against Cyp3a were purchased from Santa Cruz

Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated anti-mouse and anti-goat antibodies were obtained from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA) and Sigma-Aldrich (Oakville, ON, Canada), respectively. Immunoblot stripping solution and enhanced chemilumescent solution were purchased from Pierce Thermo Fisher Scientific Inc.

(Waltham, MA). MilliQTM water and reagent grade salts from Sigma-Aldrich were used for the

167

preparation of artificial CSF (aCSF). All organic solvents used for the sample bioanalysis were of analytical grade.

9.3.2 Intracerebral Microdialysis Study

Adult male CD-1® mice (8-10 week-old, 25 to 35 g) were purchased from Charles River

Laboratories (St. Constant, QC, Canada). Mice were housed in the animal facility at NoAb

BioDiscoveries Inc. (Mississauga, Ontario, Canada) and maintained on a 12 h light-dark cycle.

Mice had access to water and to Lab Diet® 5015 Mouse Diet (Ren’s Feed, Milton, ON, Canada) ad libitum. All procedures were reviewed by the NoAb BioDiscoveries Inc. Animal Care

Committee and were performed in accordance with the principles of the Canadian Council on

Animal Care. Newly arrived animals were acclimatized to their environment for at least five days prior to the insertion of jugular vein and carotid artery catheters under isoflurane anaesthesia. The animals were allowed to recover for one to two days prior to the implantation of a microdialysis guide cannula and dummy probe (CMA Microdialysis AB, Stockholm, Sweden) in the striatum

(AP 0.38, ML -1.80, DV -2.25). Before the microdialysis experiment, animals were administered subcutaneously with either DEX (5 mg/kg/day) or corn oil (vehicle control) for three days (n = 5 per group). One day after initiation of DEX treatment, a microdialysis guide cannula with a dummy probe was inserted into the striatum. The next day, the animals were allowed to acclimatize to individual RATURN cages (Bioanalytical Systems Inc. (BASi), West Lafayette,

IN). On that evening, the dummy probe was replaced with a 2 mm CMA/7 microdialysis probe

(CMA Microdialysis AB) and the probe was equilibrated with aCSF (147 mM NaCl, 2.7 mM

KCl, 1.2 mM CaCl2, 0.85 mM MgCl2) overnight. On the following morning, animals were administered quinidine prepared in saline (4 mg/kg i.v. loading dose and constant rate infusion of

0.8 mg/mL at 6 µL/min) via the jugular vein catheter and the microdialysis probes were perfused

168

with aCSF containing 100 ng/mL quinidine-D3 at 0.4 μL/min using a 30 to 40 cm long

Fluorinated Ethylene Propylene tubing (CMA Microdialysis AB) connected to a syringe pump

(Harvard Apparatus, Holliston, MA). Dialysate samples were collected at 30 min intervals over

510 min using a refrigerated fraction collector (BASi) at 4 °C. Blood (15 µL) was serially collected using an automated sampling system (BASi) from the carotid artery catheter at 15, 45,

75, 165, 255, 375, 435 and 495 min following the start of quinidine infusion. Blood was diluted with heparinized saline (50 µL) after each sample collection and were then stored at -80 °C until

LC-MS/MS analysis. At the end of the experiment, animals were sacrificed to collect whole blood and plasma (obtained by centrifugation at 4 ºC) samples to determine quinidine concentrations. Whole brain tissues were harvested to evaluate protein expression in isolated brain capillaries and brain homogenates using immunoblot analysis. Probe perfusates were collected at the beginning and end of the experiment.

9.3.3 In vitro Recovery/Loss from the Microdialysis Probes

Prior to the day of the in vitro recovery experiment, three CMA/7 microdialysis probes were first immersed in and perfused with aCSF solution overnight. On the following day, probes were immersed in a 1.5 mL reservoir of aCSF containing 100 ng/mL of quinidine at 37 °C and perfused with aCSF containing 100 ng/mL of quinidine-D3 at 0.4 µL/min using Fluorinated

Ethylene Propylene tubing (CMA Microdialysis AB). The dialysate was collected at 30 min intervals for 4 h and then stored at -20 °C prior to analysis. The relative probe recovery and loss of quinidine and quinidine-D3, respectively, were calculated as described in Eqs. 1 and 2.

169

Analyte Concentration in Dialysate Relative Recovery = [1] Analyte Concentration in Reservoir

Analyte Concentration in Dialysate Relative Loss = 1 [2] Analyte Concentration in Perfusate − The responsiveness of the dialysis system to a rapid change in quinidine and quinidine-D3 concentrations was also subsequently ascertained during a 2 h washout period by perfusing aCSF solution through the probes that were immersed in aCSF solution.

In vivo loss of quinidine and quinidine-D3 were compared in some of the animals one day after the microdialysis experiment. Following perfusion of the probes with aCSF solution overnight, a dialysate sample was collected to confirm the washout of the analytes and then the probes were perfused with a combination of 100 ng/mL quinidine and quinidine-D3 in aCSF solution. Dialysate was collected over 30 min intervals for 4 h and stored at -20 °C prior to analysis.

9.3.4 Determination of Quinidine Unbound Fractions in Mouse Plasma

The unbound quinidine fractions in mouse plasma were determined using a 96-well

Teflon equilibrium dialysis apparatus utilizing dialysis membranes with a molecular cut-off at

12-14 kDa (HTDialysis LLC, Gales Ferry, CT). Plasma was obtained from drug-naïve adult male

CD-1 mice (8 – 10 week-old). The donor side of the dialysis chamber contained 300 µL of plasma containing 2100 ng/mL quinidine while the receiver side contained the same volume of blank PBS buffer (pH. 7.4). The dialysis apparatus was shaken gently (100 rpm) at 37 ˚C for 16 h and equilibrium was observed after 12 h. A volume of 100 µL was collected from the receiver side and mixed with 50 μL of blank plasma. Meanwhile, 50 μL of plasma was collected from

170

donor side and was mixed with 100 μL of blank PBS buffer. Samples were stored at -80 ºC prior to LC-MS/MS analysis.

9.3.5 Quinidine Quantification in Blood, Plasma, Perfusate, Dialysate and Dosing Solution

Sample analysis was conducted using an AB Sciex API4000 tandem liquid chromatography mass spectrometric (LC-MS/MS) system equipped with an Agilent 1100 series binary pump (Agilent Technologies, Santa Clara, CA, USA), solvent degasser, CTC autosampler and a Valco VICI divert valve. The Agilent 1100 mixer was replaced with an Upchurch® U466S mixer (Upchurch Scientific, Oak Harbor, WA, USA) to decrease the LC system volume.

Dialysate samples and calibration standards containing quinidine and quinidine-D3 were prepared by diluting an aliquot (5 µL) with mobile phase containing lidocaine as an internal standard. Samples of dosing solution were diluted with mouse plasma and were analyzed to confirm the infusion solution concentration and determine the infusion rate. Protein precipitation in plasma and diluted mouse blood samples (10 µL) was performed using 50/50 methanol/acetonitrile containing quinidine-D3 (an internal standard) to extract the analyte from these samples. For the diluted blood samples, the supernatant of the protein precipitated mixture was dried down and reconstituted in the mobile phase prior to the LC-MS/MS analysis, whereas for the plasma samples, the supernatant was diluted in mobile phase prior to analysis. A Zorbax

XDB-C18 column (2.1 x 30 mm) (Agilent Technologies) was utilized for the chromatographic separation and the analytes were eluted using a combination of 10 mM ammonium formate pH

3.0 in water (mobile phase A, MPA) and 95/5 (v/v) methanol/10 mM ammonium formate pH 3.0 in water (mobile phase B, MPB) at 0.7 mL/min flow rate. The analytes were trapped on the column with an initial isocratic condition of 10% MPB delivered over 0.4 min, and then eluted from the column using an increasing step gradient (0.1 min) to 40% MPB and then the latter

171

condition was held for 0.6 min. The column was subsequently washed with 95% MPB prior to equilibration with 10% MPB. The cycle time (from injection to injection) was 2.7 min. Analytes were monitored using selected reaction monitoring in positive ion electrospray mode and quantified using peak area ratio of analyte to internal standard. The selected reaction monitoring for quinidine, quinidine-D3 and lidocaine were m/z 325 to 172 and 79, m/z 328 to 175 and 79, and m/z 235 to 86, respectively. Calibration curves were generated using AnalystTM software

(AB SCIEX, Framingham, MA, USA) from at least six concentrations of standards prepared in aCSF (0.2 to 200 ng/mL), mouse plasma (10 to 10,000 ng/mL) and diluted mouse blood (10 to

10,000 ng/mL undiluted concentration).

9.3.6 Mouse Brain Capillary Isolation and Brain Homogenate Preparation

Procedures to isolate mouse brain capillaries were adapted from previously published methods with slight modifications (Bauer et al. 2004). Morphology of brain capillaries was shown in Appendix E, figure E-1. Protein expression of endothelial markers (i.e., tight junction proteins ZO-1 and occludin), drug transporters (e.g., P-gp and Bcrp) and mouse PXR in lysates of brain capillaries is shown in Appendix E, figure E-2. In brief, fresh brains collected at the end of the experiment were rinsed with ice-cold PBS and stored on dry ice. The cortical gray matter was later isolated and homogenized at 400 rpm in ice-cold PBS. The mixture was centrifuged at

5,800 x g for 20 min at 4 °C after the addition of ice-cold Ficoll 400 (final concentration 15%).

The resulting pellet was resuspended in ice-cold PBS containing 1% BSA and filtered through a

200 µm nylon mesh. The filtrate containing capillaries was passed over a glass bead column and washed with ice-cold PBS. The column effluent was collected to serve as capillary-depleted brain homogenate samples. Capillaries retained in the column were collected by agitation of the

172

glass beads, then centrifuged (5 min of 500 x g) and the pellet was washed three times in ice-cold

PBS to remove impurities before snap-freezing in liquid nitrogen and storing at -80 °C.

9.3.7 Immunoblot Analysis

Protein expression of P-gp, PXR, Cyp3a11 and β-actin in isolated tissues was determined by SDS-PAGE according to previously published protocols from our group (Chan et al. 2011).

Tissue lysates were prepared in lysis buffer (1 % (v/v) NP-40 in 20 mM Tris, 150 mM NaCl, 5 mM EDTA at pH 7.5 containing 1 mM PMSF and 0.1 % (v/v) protease inhibitor cocktail), sonicated for 10 s and centrifuged at 20,000 x g for 10 min at 4 °C. Lysates containing 50 µg of protein from brain capillary and brain homogenate samples were mixed in Laemmli buffer and resolved on 10 % SDS-polyacrylamide gel. Mouse liver lysate (isolated from control non- surgical animals) containing 25 – 30 µg of protein was used as positive control for P-gp, PXR and Cyp3a11 expression. After electrophoresis, gels were washed three times (5 min each) in transfer buffer (25 mM Tris-HCl, pH 8.0, 200 mM glycine) containing 20% (v/v) methanol and then electrotransferred onto PVDF membranes. The membranes were blocked for 2 h in TBST-T and with 5 % (w/v) skim milk. The membranes were incubated with the appropriate primary antibody overnight at 4 °C. On the next day, the membranes were washed three times (10 min each) in TBST-T and were incubated with anti-mouse (Jackson immunoresearch Laboratories) or anti-goat (Sigma-Aldrich) horseradish peroxidase-conjugated secondary antibody at 1:3000 and

1:4000 dilution, respectively. P-gp expression was detected using a 1:500 dilution of mouse monoclonal C219 antibody (ID Labs, London), which recognizes an internal, highly conserved amino acid sequence VQEALD and VQAALD of P-gp. PXR expression was detected using a

1:100 dilution of goat polyclonal A-20 antibody (Santa Cruz Biotechnology, Inc.), which recognizes a unique domain of the mouse PXR.1 (protein accession # O75469). Cyp3a11

173

expression was detected using a 1:100 dilution of goat polyclonal L-14 antibody (Santa Cruz

Biotechnology, Inc.), which recognizes the N-terminus of CYP3a11 (protein accession #

Q64459). β-actin expression (protein accession # P60710) was detected using a 1:3000 dilution of mouse monoclonal C-4 antibody (Santa Cruz Biotechnology, Inc.). Protein bands were detected using an enhanced chemiluminescence kit. Densitometric analysis was performed using

AlphaDigiDoc RT2 software (San Leandro, CA) to quantify relative protein expression.

9.3.8 Data Analysis

Brain ECF concentrations of quinidine were calculated by dividing each dialysate sample concentration of quinidine by the relative loss of quinidine-D3 determined for that sample.

Blood sample concentrations were converted to plasma concentrations using the blood to plasma concentration ratio determined at the end of the in vivo microdialysis experiment for each animal.

Quinidine unbound plasma concentration was estimated using experimentally determined unbound fraction in mouse plasma (0.08) as described above. In figure 2, averages of quinidine brain ECF concentration to unbound plasma concentration ratios ± S.D. at each time point were calculated from ratios obtained in five individual animals per treatment group. Each ratio per animal per time point was determined from the corresponding individual quinidine brain ECF concentration and unbound plasma concentration at the midpoint of the dialysate collection interval. The total plasma clearance of quinidine was estimated as the infusion rate divided by the steady state (375 - 495 min) plasma concentration. The Student’s two-tailed t test for unpaired data was used to determine statistical significance for the comparison of unbound quinidine concentrations in plasma and brain ECF, ratios of quinidine brain ECF concentration to unbound plasma concentration and protein expression in isolated brain capillaries and capillary- depleted brain homogenates between the vehicle-treated and DEX-treated groups. Data were

174

analyzed by SPSS software (Chicago, IL) and a p-value of < 0.05 was considered to be statistically significant.

9.4 Results

A reliable recovery of quinidine is absolutely essential to correctly estimate quinidine brain ECF concentrations from the dialysate samples. The relative recovery for quinidine in these experiments was estimated by the relative loss of deuterium labelled quinidine (quinidine-D3).

To estimate relative recovery using an internal calibrator, one must assume that relative drug recovery from the brain ECF surrounding the microdialysis probe is equal to the relative drug loss from the probe perfusate into the ECF. We first validated this assumption in vitro by confirming that the in vitro relative loss of quinidine-D3 (35.1 ± 3.1%) and relative recovery of quinidine (38.7 ± 5.3%) were essentially the same, with the use of three probes. During a 2 h wash-out period, dialysate concentrations of quinidine and quinidine-D3 decreased significantly within the first 30 min collection interval and then were essentially non-detectable over the remainder of the 2 h, indicating that rapid changes in quinidine dialysate concentrations would be measurable. As well, in vivo relative loss of quinidine-D3 was 45.0 ± 7.2% and was found to be essentially identical to that of quinidine (44.8 ± 7.4%) during equilibrium, which was attained

150 min after the start of the perfusion. This was estimated in three of the animals after an overnight washout period following the cessation of the constant rate i.v. infusion.

Following a bolus dose (4 mg/kg) and continuous constant i.v. infusion (5 µg/min) of quinidine, unbound quinidine concentrations in plasma reached a steady state by 6 h in vehicle- treated animals (159.3 ± 5.2 ng/mL, n=5) and DEX-treated animals (181.9 ± 3.0 ng/mL, n=5,

Figure 9-1A). Blood concentrations were similar to plasma concentrations and the plasma to blood concentration ratio averaged 0.92 ± 1.4 and 0.86 ± 0.06 in vehicle- and DEX-treated

175

animals, respectively. The estimated total plasma clearance of quinidine did not differ between the DEX- and vehicle-treated animals (DEX-treated group: 9.3 ± 1.8 L/kg/h and vehicle-treated group: 9.5 ± 1.5 L/kg/h). Brain ECF drug concentrations also reached a steady state by 6 h.

However, quinidine brain ECF concentrations at steady state in DEX-treated animals (10.6 ± 1.6 ng/mL) were significantly lower than those of the vehicle-treated animals (24.7 ± 2.9 ng/mL,

Figure 9-1B).

176

Figure 9-1.Unbound quinidine concentrations (ng/mL) in (A) plasma and (B) brain ECF from dexamethasone (DEX)-treated or vehicle-treated CD1 mice. Data are presented as means ± S.D. from five animals per group at each time point. * p < 0.05 (statistically significant difference compared to the vehicle-treated group). Experiments conducted by GNY Chan with assistance from V Saldivia (animal surgery and treatment), Y Yang (LC-MS/MS analysis) and H Pang (LC-MS/MS analysis) at NoAb BioDiscoveries Inc., Mississauga, Ontario.

177

The average ratio of the unbound quinidine concentrations in brain ECF to that in plasma at steady state (375 – 495 min) or Kp, uu, ECF/Plasma in DEX-treated animals (0.063 ± 0.025) was approximately 2.5-fold lower than the ratio observed in vehicle-treated animals (0.149 ± 0.031,

Figure 9-2).

Figure 9-2. Unbound quinidine ECF to plasma concentration ratios in dexamethasone (DEX)-treated or vehicle-treated CD-1 mice. Ratios are presented as means ± S.D. from five animals per group. * p < 0.05 (statistically significant difference compared to the vehicle-treated group). Experiments conducted by GNY Chan with assistance from V Saldivia (animal surgery and treatment), Y Yang (LC-MS/MS analysis) and H Pang (LC-MS/MS analysis) at NoAb BioDiscoveries Inc., Mississauga, Ontario.

Immunoblot analysis of the mouse brain capillary samples isolated from the animals treated with 5 mg/kg/day DEX for 3 days showed a significant induction (~1.5 fold) of P-gp protein expression when compared to vehicle-treated animals (Figure 9-3).

178

Figure 9-3. Representative immunoblot (top) and densitometric analysis (bottom) of P-gp expression in mouse brain capillary fractions (50 μg) isolated from dexamethasone (DEX)- treated or vehicle (VEH)-treated CD-1 mice. Mouse liver lysate (25 μg) was used as positive control for P-gp expression. Data represent the percentage change in P-gp expression normalized to vehicle control and are shown as mean ± S.D. obtained from three independent brain capillary isolations. * p < 0.05 (statistically significant difference compared to VEH-treated group). Brain capillary isolation and immunoblot analysis were performed by GNY Chan in the laboratory of Dr. R Bendayan.

P-gp expression in mouse brain homogenate samples depleted of brain capillaries was also examined to investigate whether P-gp was induced in brain parenchyma, and was not found to be significantly different between DEX- and vehicle-treated groups (Figure 9-4).

179

Figure 9-4. Representative immunoblot (top) and densitometric analysis (bottom) of P-gp expression in mouse brain homogenate fractions (50 μg) from dexamethasone (DEX)- treated or vehicle (VEH)-treated CD-1 mice. Mouse liver lysate (30 μg) was used as positive control for P-gp expression. Data represent the percentage change in P-gp expression normalized to vehicle control and are shown as mean ± S.D. obtained from three independent brain homogenates. A statistically significant difference was not observed between VEH-treated group and DEX-treated group (p = 0.74). Brain homogenate preparation and immunoblot analysis were performed by GNY Chan in the laboratory of Dr. R Bendayan.

In addition, expression of PXR protein in mouse brain capillaries and brain homogenates depleted of brain capillaries was not significantly different between the groups (Figures 9-5 and

9-6).

180

Figure 9-5. Representative immunoblot (top) and densitometric analysis (bottom) of mPXR expression in mouse brain capillary fractions (50 μg) from dexamethasone (DEX)-treated or vehicle (VEH)-treated CD-1 mice. Mouse liver lysate (30 μg) was used as positive control for mPXR expression. Data represent the percentage change in mPXR expression normalized to vehicle control and are shown as mean ± S.D. obtained from three independent brain capillary isolations. A statistically significant difference was not observed between VEH-treated group and DEX-treated group (p = 0.23). Brain capillary isolation and immunoblot analysis were performed by GNY Chan in the laboratory of Dr. R Bendayan.

181

Figure 9-6. Representative immunoblot (top) and densitometric analysis (bottom) of mPXR expression in mouse brain homogenate fractions (50 μg) from dexamethasone (DEX)- treated or vehicle (VEH)-treated CD-1 mice. Mouse liver lysate (30 μg) was used as positive control for mPXR expression. Data represent the percentage change in mPXR expression normalized to vehicle control and are shown as mean ± S.D. obtained from three independent brain homogenates. A statistically significant difference was not observed between VEH-treated group and DEX-treated group (p = 0.18). Brain homogenate preparation and immunoblot analysis were performed by GNY Chan in the laboratory of Dr. R Bendayan.

Since, quinidine can be metabolized by mouse Cyp3a11 (De Lange 2013), the induction of Cyp3a11 by the mouse PXR agonist, DEX, could potentially also decrease brain ECF concentrations of quinidine. Therefore, Cyp3a11 expression in brain capillaries and brain homogenates was investigated in the DEX- and vehicle-treated animals. In the current study, although Cyp3a11 expression was not detected in mouse brain capillary samples isolated from

182

the two groups of animals (Figure 9-7), Cyp3a11 was expressed in mouse brain homogenates, but no significant differences were observed between the two groups (Figure 9-8).

Figure 9-7. Representative immunoblot of Cyp3a11 expression in mouse brain capillary fractions (50 μg) in dexamethasone (DEX)-treated or vehicle (VEH)-treated CD-1 mice. Mouse liver lysate (30 μg) was used as the positive control for Cyp3a11 expression. Brain capillary isolation and immunoblot analysis were performed by GNY Chan in the laboratory of Dr. R Bendayan.

183

Figure 9-8. Representative immunoblot (top) and densitometric analysis (bottom) of Cyp3a11 expression in mouse brain homogenate fractions (50 μg) from dexamethasone (DEX)-treated or vehicle (VEH)-treated CD-1 mice. Mouse liver lysate (30 μg) was used as positive control for Cyp3a11 expression. Data represent the percentage change in Cyp3a11 expression normalized to vehicle control and are shown as mean ± S.D. obtained from three independent brain homogenates. A statistically significant difference was not observed between VEH-treated group and DEX-treated group (p = 0.13). Brain homogenate preparation and immunoblot analysis were performed by GNY Chan in the laboratory of Dr. R Bendayan.

9.5 Discussion

Pharmacological mechanisms of CNS drugs, i.e., those that bind to or modulate cellular membrane receptors as their therapeutic target, are primarily related to unbound drug concentrations in the brain ECF (Hammarlund-Udenaes 2010). Intracerebral microdialysis, the gold-standard approach to measure in vivo chemical concentrations in brain ECF, has been a useful tool to examine drug disposition in this compartment (De Lange et al. 1998, De Lange et

184

al. 2000, Chaurasia et al. 2007, Hammarlund-Udenaes et al. 2009). Since drug concentrations in the brain ECF are affected by transport processes at the BBB, intracerebral microdialysis following the administration of substrates of drug transporters, can be used to study the in vivo role of drug transporters at the BBB (De Lange et al. 1998, Sawchuk & Elmquist 2000,

Hammarlund-Udenaes et al. 2009, Krajcsi et al. 2012). For example, intracerebral microdialysis using quinidine as a selective P-gp substrate has been utilized to investigate P-gp function at the rat BBB (Liu et al. 2009, Sziráki et al. 2011, Syvänen et al. 2012). However, to our knowledge, quinidine distribution in the brain ECF of rodents following ligand-mediated P-gp induction at the BBB has not been previously examined. In the current study, we utilized quantitative intracerebral microdialysis to investigate quinidine distribution in the brain ECF of mice following P-gp induction at the BBB mediated by a three-day treatment with DEX, a potent rodent PXR agonist and P-gp inducer (Bauer et al. 2004, Chang & Waxman 2006). The average ratio of the unbound quinidine concentrations in brain ECF to that in plasma at steady state (375

– 495 min) or Kp, uu, ECF/Plasma in DEX-treated mice was significantly lower when compared to vehicle-treated animals (Figure 9-2), indicating that quinidine distribution in the brain ECF was reduced in DEX-treated animals. In addition, P-gp expression in brain capillaries isolated from

DEX-treated animals was significantly induced by 1.5-fold compared to vehicle-treated animals, while P-gp expression in brain homogenate samples depleted of brain capillaries remained unchanged between the two groups. As well, we did not observe a significant induction of

Cyp3a11, which is believed to metabolize quinidine in mouse (De Lange 2013), neither in brain capillary nor in brain homogenate samples in DEX-treated animals when compared to vehicle- treated animals. These findings suggest that the reduction in brain ECF concentrations of

185

quinidine as a result of DEX treatment is primarily due to the induction of P-gp at the mouse

BBB, which ultimately results in enhanced efflux of quinidine from brain endothelial cells.

The use of a well-established P-gp substrate which is not known to interact with other major efflux drug transporters at the BBB is important to detect changes in P-gp function following its induction. Among many other lipophilic P-gp substrates, quinidine was chosen in this study because it does not significantly bind to the microdialysis probe and tubing, which would otherwise make the use of relative loss as an estimate of its relative recovery difficult

(Chaurasia et al. 2007). Our in vitro and in vivo relative recovery/relative loss for quinidine and quinidine-D3 were essentially the same (approximately 45%), confirming that the deuterium labelled analog of quinidine can be utilized for the quantification of quinidine in the brain ECF.

Furthermore, the relative recovery/relative loss of quinidine from the short (2 mm) microdialysis probe used in mice is relatively high and brain ECF concentrations of quinidine were high enough in the DEX-treated animals (Css = 10.6 ± 1.6 ng/mL) to be accurately quantified (the lower level of quantification of quinidine in ECF was 0.2 ng/mL).

There was no difference in the in vivo relative recovery of quinidine between the vehicle- and DEX-treated groups (40.9 ± 4.5 and 46.9 ± 9.5, respectively). This finding is consistent with a previous report demonstrating that the relative recovery of the analyte was not affected by inhibition of P-gp in rats (Sun et al. 2001). In the current study, Kp,uu,ECF/Plasma (375- 495 min) of quinidine in vehicle-treated mice was 0.149 ± 0.031, which is approximately six times higher than the Kp,uu,Brain/Plasma obtained from rats in a study using the whole brain homogenate method

(Kodaira et al. 2011). Interestingly, Liu et al., reported that the unbound quinidine brain concentrations at steady state determined by the brain homogenate method can underestimate the unbound quinidine ECF concentrations by approximately three-fold (Liu et al. 2009).

186

PXR, a xenobiotic-activating nuclear receptor, has been demonstrated to regulate P-gp expression in intestinal and hepatic tissues (Geick et al. 2001). Recently, several publications including ours have reported that P-gp is regulated by PXR in in vitro and ex vivo models of the

BBB (e.g., human and rat brain microvessel endothelial cell culture systems and isolated rodent and porcine brain microvessels) (Bauer et al. 2004, Bauer et al. 2006, Narang et al. 2008, Zastre et al. 2009, Chan et al. 2011, Ott et al. 2009). Several xenobiotics such as DEX, identified to serve as ligands of PXR, have been utilized to examine the inductive role of this nuclear receptor in rodents (Jones et al. 2000). Although DEX is a known anti-inflammatory agent, it is utilized as a P-gp inducer and PXR ligand in our current study. The microdialysis probe-related brain injury is anticipated to be minimal in our current protocol and we do not anticipate that a significant inflammatory reaction is produced (Benveniste & Diemer 1987, De Lange et al. 1995). The DEX dosage of 5 mg/kg/day used to treat our animals was selected based on previous in vivo studies performed in mice which showed induction of PXR targets (e.g., P-gp and Cyp3a11) (Scheer et al. 2010, Bauer et al. 2004). In the current study, we confirmed the protein expression of P-gp and PXR in brain capillaries and demonstrated that treatment of mice with DEX (5mg/kg/day for

3 days) resulted in a 1.5-fold P-gp induction in mouse brain capillaries. This finding is in agreement with previous published work where the same dosing regimen of DEX induced P-gp expression by two-fold in rat brain capillaries (Bauer et al. 2004). It is important to note that although PXR is functionally expressed in in vitro and in vivo BBB models in rodents, both the transcript and protein expression of human PXR have not been previously detected in human brain capillaries (Shawahna et al. 2011, Dauchy et al. 2008). However, human PXR transcript expression was reported by another group in specific regions of the human brain such as thalamus, pons and medulla (Nishimura et al. 2004, Miki et al. 2005). As well, our laboratory

187

has demonstrated PXR protein expression in human fetal brain tissue (Chan et al. 2010). Our current findings provide in vivo evidence that a PXR pathway may regulate P-gp functional expression at the mouse BBB. However, since DEX is also a known glucocorticoid receptor ligand, this pathway cannot be excluded. Further studies are needed to fully elucidate, in vivo, the potential role of these nuclear receptors in the regulation of P-gp at the BBB

To date, no information is available regarding the effect of ligand-mediated P-gp induction at the BBB on the brain distribution of a P-gp substrate. However, a few studies have examined the effect of P-gp induction at the BBB on P-gp substrate distribution in the brain ECF in different rodent models of disease states. For instance, Bauer et al. reported very interesting data showing that the anti-nociceptive effect of methadone (a P-gp substrate) was reduced in mice exhibiting P-gp induction in brain capillaries, although methadone concentrations in the brain were not determined (Bauer et al. 2006). As well, Wu et al. showed that P-gp induction in the brain capillaries of an inbred type II diabetic mouse model could lead to significantly lower concentrations of Rhodamine 123, an established P-gp substrate, in brain ECF compared to wild- type mice (Wu et al. 2009). These observations along with our current findings support the concept that concentrations of P-gp substrates in brain ECF are expected to be reduced following

P-gp induction at the BBB and this could be explained by the increased clearance of substrates from the brain ECF back into the circulation. In contrast, Bankstahl and Löscher demonstrated that while pilocarpine-induced epileptic rats showed a higher P-gp expression in brain capillaries, these animals exhibited higher phenytoin (a weak P-gp substrate) concentrations in brain ECF from the hippocampus when compared to non-treated controls as determined by non-quantitative microdialysis (Bankstahl & Löscher 2008). As well, a kainate-induced epileptic rat model, which was previously demonstrated to have a higher P-gp expression in brain capillaries when

188

compared to non-treated animals, exhibited no significant change in quinidine permeability across the BBB but higher brain ECF concentrations of quinidine in hippocampus regions

(Syvänen et al. 2012). Interestingly, these authors also reported a decrease in total brain concentrations of quinidine following drug-induced epileptic condition, however, this effect did not reach significance. Although, this finding may appear to contradict our results, Syvänen et al. had also suggested that neuro-inflammation in brain parenchyma and disease-mediated BBB dysfunction could occur during epileptic states and that these factors, in addition to P-gp induction at the BBB, glial cells and neurons, could affect quinidine distribution in total brain and the ECF compartment (Syvänen et al. 2012). Therefore, these discrepancies may be explained by differences in the mechanism and cellular locations by which P-gp induction was mediated between the epileptic rat models and our wild type mouse model that was triggered with a P-gp inducer systemically.

Our group has previously demonstrated that, in addition to brain capillaries, P-gp is expressed in different cellular compartments of brain parenchyma such as astrocytes and microglia (Lee et al. 2001c, Ronaldson et al. 2004a, Bendayan et al. 2006). The function of P-gp in brain parenchyma could potentially result in the efflux of quinidine from these cellular compartments into brain ECF for further distribution in the brain or removal across the BBB.

Therefore, alterations of P-gp expression in these cellular compartments could potentially affect quinidine concentrations in the brain ECF. In the current study, samples of mouse brain homogenate depleted from brain capillaries were used for the measurement of P-gp protein expression in brain parenchyma. Expression of P-gp in brain homogenates did not significantly differ between DEX- and vehicle-treated mice suggesting that the lower quinidine concentrations

189

observed in brain ECF of DEX-treated animals was likely not a result of P-gp induction in brain parenchyma.

It is well recognized that the cellular expression and activity of CYP enzymes in brain parenchyma and microvessel endothelial cells is significantly lower relative to their functional expression in hepatocytes (Woodland et al. 2008). Low expression implies that CYP enzymes may not participate substantially in the overall brain drug clearance. However, the potential that drug disposition in brain ECF could be affected by these enzymes still exists. Furthermore, the expression of human CYP3A4 and mouse Cyp3a11 enzymes, which can metabolize quinidine in the liver (De Lange 2013), are known to be regulated by PXR and can be induced by DEX in rodents (Perloff et al. 2004). Although little is known on Cyp3a11 induction mediated by PXR in the brain, this potential effect at the BBB and in brain parenchyma could reduce quinidine concentrations in brain ECF. In the current study, Cyp3a11 expression was not detected in mouse brain capillaries; these data are consistent with previous findings demonstrating that CYP3A4 is not present in human brain capillaries (Dauchy et al. 2008). Furthermore, Cyp3a11 induction in brain homogenates of DEX-treated animals was not observed. Together, these data indicate that the lower quinidine ECF concentrations observed in the DEX-treated animals mice were not a result of Cyp3a11 induction at the BBB or in brain parenchyma.

In summary, this study shows that intracerebral microdialysis utilizing quinidine and an internal calibrator (quinidine-D3) can be used to assess P-gp induction mediated by a P-gp inducer/PXR agonist (DEX) at the mouse BBB. Our findings demonstrate that P-gp induction mediated by DEX at the BBB can reduce quinidine concentrations in the brain ECF. These data support the concept that P-gp induction can further restrict the brain permeability of its substrates and illustrate the major role of P-gp at the BBB in protecting the brain against xenobiotics

190

including therapeutic agents. These data further suggest that drug-drug interactions as a result of

P-gp induction at the BBB are possible.

9.6 Acknowledgements

The authors thank Mrs. Tennile Tavares (NoAb BioDiscoveries Inc., Mississauga,

Canada) for the preparation of the samples for LC-MS/MS analysis and Jianghong Fan for performing quinidine plasma binding experiments (NoAb BioDiscoveries Inc., Mississauga,

Ontario, Canada, present address: InterVivo Solutions, Toronto, Ontario, Canada). This research was funded in part by a grant from the Canadian Institutes of Health Research (CIHR Grant #

MOP56976) awarded to Dr. Reina Bendayan and in part by NoAb BioDiscoveries Inc. Dr.

Bendayan is a recipient of a Career Scientist Award from the Ontario HIV Treatment Network,

Ministry of Health of Ontario, Canada.

9.7 Conflicts of Interest

None.

191

10. Overall Discussion and Summary

ABC drug transporters, such as P-gp, at the BBB and in various cellular compartments of the brain parenchyma constitute an essential biochemical barrier to harmful chemicals and serve as a major determinant of drug distribution and elimination in the CNS (Lee et al. 2001b, Lee &

Bendayan 2004, Löscher & Potschka 2005a, Dallas et al. 2006, Eyal et al. 2009, Urquhart &

Kim 2009, Ashraf et al. 2012). In the context of HIV pharmacotherapy, P-gp is known to transport all HIV PIs, maraviroc, abacavir, tenofovir DF and raltegravir (Lee et al. 1998, Polli et al. 1999, Van der Sandt et al. 2001, Edwards et al. 2002, Ronaldson et al. 2004b, Walker et al.

2005, Janneh et al. 2007, Shaik et al. 2007, Kassahun et al. 2007, Zastre et al. 2009, Brown et al.

2009, Fujimoto et al. 2009, Kis et al. 2010a). Therefore, its expression at the BBB, in particular in the luminal membranes of the brain microvessel endothelial cells, is believed to limit brain permeability of several antiretroviral drugs used in the clinic (Kis et al. 2010a, Ene et al. 2011).

Limited drug concentrations in the brain can contribute to the development of drug-resistant viral strains and result in the formation of a reservoir for HIV-1 (Varatharajan & Thomas 2009).

Furthermore, sub-therapeutic viral control in the CNS has been proposed to contribute to the cumulative prevalence of several HIV associated neurocognitive disorders, which are known to adversely affect HIV patients’ quality of life (Letendre et al. 2009, Letendre 2011).

A full understanding of the regulatory mechanisms that govern expression of drug efflux transporters (i.e., P-gp) at the BBB could provide novel strategies to selectively alter brain permeability of therapeutic agents (e.g., antiretroviral drugs). A number of xenobiotic-activated nuclear receptors, including hPXR and hCAR, have been identified as key transcriptional regulators of P-gp (Tirona & Kim 2005, Stanley et al. 2006, Urquhart et al. 2007, di Masi et al.

2009). Although these pathways have been extensively investigated in human hepatic and

192

intestinal cell lines, at the time this thesis was initiated very little was known regarding their regulation of P-gp functional expression in the brain, in particular at the BBB. In this thesis, we examined the role of hPXR and hCAR in regulating P-gp functional expression in hCMEC/D3 cell culture system, known to display several morphological and biochemical properties of the human brain microvessel endothelial cells (Weksler et al. 2013). P-gp transport activity in hCMEC/D3 cell culture system was initially characterized by others using several P-gp substrates (e.g., rhodamine 123 and calcein-AM) and a P-gp inhibitor (i.e., PSC833) (Weksler et al. 2005). In the current thesis, high-resolution immunogold cytochemistry with electron microscopy was utilized to demonstrate P-gp localization in hCMEC/D3 cells. This finding is consistent with previously published data by our group that showed P-gp localization at the luminal membrane of brain microvessels in rat and human brain tissue fixed in situ (Bendayan et al. 2006). In addition, immunoblot analysis also showed P-gp expression in whole cell lysates at the expected molecular weight of approximately 170 kDa. To assess P-gp transport activity, we evaluated the cellular accumulation of a P-gp fluorescent substrate, R-6G, in the presence or absence of PSC833, a non-immunosuppressive cyclosporine A analog. This agent is a well- established second generation P-gp inhibitor previously used for the study of P-gp transport activity by our group and others (Chen et al. 1997, Desrayaud et al. 1997, Jetté et al. 1998,

Kusunoki et al. 1998, Egashira et al. 1999, Song et al. 1999, Cabot et al. 1999, Bendayan et al.

2002, Ronaldson et al. 2004a, Ronaldson et al. 2004b, Ronaldson & Bendayan 2006, Robillard et al. 2012). The significant enhancement in cellular R-6G accumulation observed in cells exposed to PSC833 compared to control cells (i.e., cells only exposed to vehicle) confirmed P-gp transport function in hCMEC/D3 cells (Zastre et al. 2009). In addition, this effect was observed in the presence of ritonavir, which is also a known P-gp inhibitor (Perloff et al. 2002). In

193

contrast, the presence of MRPs inhibitor (i.e., MK571) and BCRP inhibitor (i.e., KO143) was unable to enhance R-6G cellular accumulation in hCMEC/D3 cells, suggesting that these transporters do not affect R-6G cellular accumulation (Appendices, figure B-1). Moreover, we utilized a P-gp over-expressing cell line, MDA-MDR1, to confirm P-gp selectivity on R-6G accumulation. We observed that R-6G cellular accumulation was significantly reduced in P-gp over-expressing cells compared to corresponding wild-type cells, and this effect could be reversed by the presence of PSC833, which suggests P-gp is responsible for R-6G cellular accumulation (Appendices, figure B-2). Taken together, our findings confirm that this transport protein is highly expressed and functional in human brain microvessel endothelial cells, constituting the BBB (Zastre et al. 2009). These findings also confirm previous data on P-gp expression and function at this site in other species (Miller et al. 2000, Bauer et al. 2004, Bauer et al. 2006, Perloff et al. 2007, Lombardo et al. 2008, Narang et al. 2008, Dauchy et al. 2008, Ott et al. 2009).

The expression of nuclear receptors, such as PXR and CAR, has been extensively investigated in the liver and intestinal tract, however very little was known on their expression in human brain and at the BBB (Stanley et al. 2006, Li & Wang 2010). In the current thesis, we demonstrated hPXR cellular localization in hCMEC/D3 cells using high-resolution immunogold cytochemistry with electron microscopy and applying immunoblot analysis, we detected both the cytosolic and nuclear expression of hPXR in these cells (Zastre et al. 2009). In our follow-up publication, we reconfirmed hPXR protein expression and showed hCAR protein expression in these cells at the expected molecular weight of 50 and 60 kDa, respectively (Chan et al. 2011).

The identities for hPXR and hCAR protein bands in the immunoblots were further confirmed using hPXR antibody neutralizing peptide and non-conjugated IgG, respectively. As well,

194

mRNA expression of these two receptors was detected in these cells using qPCR technique. To investigate further hPXR and hCAR expression at the human BBB, we obtained whole cell pellets of human BBB-ECs originated from temporal lobe specimens obtained from young adults undergoing surgery for the treatment of intractable epilepsy. We demonstrated hPXR and hCAR protein expression in these cells and observed a wide range of inter-individual differences as much as 10-fold and 4-fold for hPXR and hCAR expression, respectively, suggesting receptors’ expression can vary significantly at the BBB similar to what has previously been observed in human liver tissues (Chang et al. 2003). Taken together, we were the first to report expression of hPXR and hCAR in these two in vitro cell culture systems of human brain microvessel endothelial cells (Chan et al. 2011). As mentioned in Chapter 1, hCAR mRNA expression was reported in human brain microvessels (i.e., capillaries), however hPXR mRNA expression was not (Dauchy et al. 2008). In addition, hPXR and hCAR protein expression in human brain microvessels was not detected, this could be possibly due to limitations of the analytical procedures that were used (Shawahna et al. 2011). Our current findings help clarify and provide evidence that these two receptors could be expressed in human brain microvessel endothelial cells. In the brain parenchyma, other groups have reported a low level of hCAR mRNA in human brain cortical tissues (Nishimura et al. 2004), caudate nucleus (Lamba et al. 2004a) and brain glioma cells (Malaplate-Armand et al. 2005). Transcript expression of hPXR was also detected in specific regions of the human brain such as thalamus, pons and medulla (Nishimura et al. 2004,

Miki et al. 2005). Our laboratory has also demonstrated hPXR and hCAR protein expression in tissue lysates of human fetal brain tissues (Chan et al. 2010). These findings suggest that hPXR and hCAR could also serve as potential xenobiotic-targeted sites to regulate target gene expression in the brain.

195

Unliganded cytosolic mouse PXR is associated with cytoplasmic retention proteins, such as CCRP and HSP90 (Squires et al. 2004). Ligand binding to the receptor in the cytoplasm is believed to trigger receptor dissociation from these retention proteins, which allows receptor translocation into the nucleus (Kawana et al. 2003). Nuclear translocation of rodent and human

PXR has been demonstrated to be a crucial step that occurs prior to transcription events in the nucleus (di Masi et al. 2009). Studies using mouse liver slices, primary cultures of human hepatocytes and porcine brain microvessel endothelial cells have demonstrated that PXR nuclear translocation is ligand-dependent (Pascussi et al. 2000, Kawana et al. 2003, Squires et al. 2004,

Ott et al. 2009). In the current work, we utilized immunofluorescence studies to examine hPXR nuclear translocation upon ligand exposure to hCMEC/D3 cells. We observed an increase in hPXR nuclear to cytosolic fluorescence intensity in the presence of a known hPXR ligand,

SR12813, suggesting a ligand-dependent nuclear accumulation of hPXR (Chan et al. 2011). This finding supports the proposed mechanism for hPXR activation. In contrast to PXR, nuclear translocation of hCAR is not well characterized. Human and rodent CAR exhibit a unique nuclear translocation pattern that can be triggered by both ligand-dependent and –independent mechanisms (Swales & Negishi 2004). In primary cultures of human hepatocytes and ex vivo rat liver slices, a significant amount of CAR has been shown to initially reside in the cytoplasm and translocate to the nucleus following receptor agonist treatment (Li et al. 2009, Kawamoto et al.

1999). However, hCAR in immortalized cell culture systems was shown to be constitutively sequestered in the nucleus regardless of the presence of an agonist (Kawamoto et al. 1999, Zelko et al. 2001, Kanno et al. 2005, Guo et al. 2007). In hCMEC/D3 cells, we did not observe a significant nuclear translocation of hCAR in the presence of a known hCAR agonist, CITCO

(Chan et al. 2011). As was alluded in Chapter 1, it is possible that several cellular pathways

196

essential for hCAR nuclear translocation, such as phosphorylation of hCAR, CCRP and HSP90, may not be fully functional in hCMEC/D3 cells. Our data are in agreement with previous findings suggesting that immortalized cell culture systems, showing spontaneous accumulation of

CAR in the nucleus, may not exhibit significant nuclear movement of hCAR in response to agonist exposure (Kawamoto et al. 1999, Zelko et al. 2001, Kanno et al. 2005, Guo et al. 2007,

Chan et al. 2011).

PXR and CAR agonists can induce in vitro and in vivo P-gp expression in brain microvessel endothelial cells and isolated brain microvessels (Aquilante et al. 1999, Bauer et al.

2004, Bauer et al. 2006, Perloff et al. 2007, Narang et al. 2008, Ott et al. 2009, Wang et al.

2010). In the current thesis, treatment with agonists of hPXR (i.e., SR12813 and rifampin) and hCAR (i.e., CITCO) significantly induced both the MDR1 mRNA and P-gp protein in hCMEC/D3 cells (Chan et al. 2011). To examine whether P-gp protein induction was mediated by hPXR and hCAR, we treated cells with these agonists for 72 h in conjunction with selective inhibitors for hPXR (i.e., A792611) and hCAR (i.e., meclizine), which had been previously reported to attenuate hPXR and hCAR ligand-activation in other in vitro cell culture systems

(Huang et al. 2004a, Meyer Zu Schwabedissen et al. 2008, Healan-Greenberg et al. 2008).

Treatment with these inhibitors resulted in a significant decrease in P-gp induction mediated by agonists of hPXR or hCAR. Furthermore, hPXR-targeting and hCAR-targeting siRNAs were used to confirm the involvement of hPXR and hCAR in P-gp regulation in non-treated hCMEC/D3 cells. Cellular downregulation of hPXR or hCAR proteins following siRNA transfection significantly decreased P-gp protein expression. Taken together, our findings provided evidence that hPXR and hCAR regulate P-gp expression in the in vitro human brain microvessel endothelial cell culture system (Chan et al. 2011). Our data are consistent with other

197

recent in vivo findings that demonstrated the presence of PXR and CAR regulatory pathways in rodent and porcine BBB (Bauer et al. 2004, Bauer et al. 2006, Ott et al. 2009, Wang et al. 2010).

Nevertheless, due to limitations associated with most in vitro cell culture systems our findings generated in hCMEC/D3 cells are by no means sufficient to conclude the presence of these pathways in the human BBB. Rather, our in vitro findings suggest that hPXR and hCAR could be two potential xenobiotic targets involved in the regulation of P-gp expression in human brain microvessel endothelial cells. Once the functional expression of these two receptors is clearly demonstrated clinically, we propose that pharmacological activation or inhibition of hPXR and hCAR could be potential approaches to alter P-gp functional expression at this site.

In the last decade, the use of combination antiretroviral therapy has led to a significant decline in the morbidity and mortality of people infected by HIV (Margolis & Hazuda 2013).

Most antiretroviral drugs currently used in first line and alternative regimens during HIV pharmacotherapy are known to be metabolized by phase I enzymes (i.e., CYPs) and/or transported by several drug efflux transporters (e.g., P-gp) (Kis et al. 2010a, Pal et al. 2011).

Induction of these systems during prolonged antiretroviral treatment may profoundly alter pharmacokinetic parameters and ultimately impair effectiveness of antiretroviral drugs at target organs, contributing to the development of sanctuary sites and cellular reservoirs (Pomerantz

2003). At the BBB, P-gp functional expression has been recognized to restrict brain entry of antiretroviral drugs, in particular HIV PIs (Ene et al. 2011). P-gp induction at the BBB is expected to further limit brain permeability of these agents and prevent therapeutic drug concentrations to be achieved in brain parenchyma. Currently, there is limited information on the ability of antiretroviral drugs to induce P-gp expression at this site. As discussed in Chapter 1, hPXR and hCAR have been identified as xenobiotic sensors that are capable of interacting with a

198

wide array of pharmacological agents (Chang & Waxman 2006). At present, a few antiretroviral agents (i.e. ritonavir, lopinavir, nelfinavir, tipranavir and efavirenz) have been identified to serve as ligands of hPXR as determined by reporter-based assays (Dussault et al. 2001, Hariparsad et al. 2004, Gupta et al. 2008, Svärd et al. 2010). In contrast, antiretroviral drug activation profile for hCAR has not been well characterized. Therefore, we examined whether antiretroviral drugs currently used in first-line and alternative regimens during HIV pharmacotherapy are ligands for hPXR or hCAR. As well, we investigated P-gp induction in hCMEC/D3 cells following long- term treatment with antiretroviral drugs that were identified as ligands of hPXR and/or hCAR.

Our results generated from reporter gene assays demonstrated that most members of the

HIV PI pharmacotherapy class (i.e., lopinavir, amprenavir, darunavir, atazanavir and ritonavir) and efavirenz, a NNRTI, can serve as ligands of hPXR (Chan et al. 2013b). These findings are consistent with reports demonstrating that ritonavir, lopinavir and efavirenz can induce hPXR activity (Dussault et al. 2001, Hariparsad et al. 2004, Svärd et al. 2010). In addition, we provided the first evidence that amprenavir, darunavir and atazanavir can also serve as ligands of hPXR. A previous study has shown that lopinavir and abacavir can serve as ligands of hCAR in transfected human hepatocellular carcinoma cell line, HepG2 (Svärd et al. 2010). Our reporter gene assay was able to provide evidence that abacavir, efavirenz and nevirapine are ligands of hCAR (Chan et al. 2013b). However, none of the members of the HIV PI class were identified to serve as a ligand of hCAR. At present, structural information on how antiretroviral drugs interact with the ligand-binding pocket of hPXR and hCAR is unclear. Interestingly, a recent study successfully predicted ligand activation of hPXR by efavirenz using an in silico ligand-docking approach, however the specific interactions between chemical structures of other antiretroviral drugs and the ligand-binding pocket of hPXR and hCAR have not been reported (Khandelwal et al. 2008).

199

Several antiretroviral drugs have been reported to induce P-gp expression in lymphocytes, intestinal and hepatic tissues (Perloff et al. 2000, Mader et al. 1993, Chandler et al. 2003, Perloff et al. 2005, Dixit et al. 2007, Weiss et al. 2008, Gupta et al. 2008, Konig et al. 2010). Although

Perloff et al. demonstrated that ritonavir can induce P-gp in brain microvessel endothelial cells isolated from rat and bovine brains (Perloff et al. 2004, Perloff et al. 2007), the effect of prolonged treatment with antiretroviral drugs on P-gp expression at the human BBB remains unclear. In the current work, we demonstrated that hCMEC/D3 cells treated for three days with antiretroviral drugs, which were identified as ligands of hPXR and hCAR in our reporter gene assay, significantly induced P-gp protein expression and function at clinical plasma concentrations (Chan et al. 2013b). Together, our data suggested that P-gp induction could occur at the human BBB during chronic treatment with antiretroviral drugs identified to be ligands of hPXR and/or hCAR (Chan et al. 2013b).

P-gp induction in brain microvessel endothelial cells has been extensively studied in diseased animal models [e.g. drug-induced seizure animals (Tishler et al. 1995, Dombrowski et al. 2001)] and in healthy animal models exposed to pathological stimuli [e.g. pro-inflammatory cytokines (Bauer et al. 2007, Yu et al. 2008) and HIV viral proteins (Hayashi et al. 2005)] or xenobiotics [e.g. ritonavir (Perloff et al. 2004) and rifampin (Bauer et al. 2006)]. It is believed that P-gp induction at the BBB could reduce CNS therapeutic efficacy for drugs that are P-gp substrates, however, at the same time this induction could also avoid neurotoxicity caused by these drugs (Eyal et al. 2009). In general, pharmacological effects or neurotoxicity of CNS drugs, i.e., those that bind to or modulate cellular membrane receptors as their therapeutic targets, are primarily related to unbound drug concentrations in the brain ECF (Hammarlund-Udenaes 2010).

Prior to the publication of my thesis work, the in vivo effect of P-gp induction at the BBB on its

200

substrate distribution in brain ECF was primarily examined in a few rodent models of disease states (e.g., epileptic models) (Bankstahl & Löscher 2008, Wu et al. 2009, Syvänen et al. 2012).

No information was available regarding this effect at the BBB on brain ECF distribution of P-gp substrates following P-gp induction mediated by a pharmacological inducer. In the current thesis, we utilized quantitative intracerebral microdialysis to examine, in vivo, P-gp induction on brain

ECF distribution of quinidine, a known P-gp substrate. Quinidine was chosen in this thesis because it is a well-established substrate of P-gp and permeates reasonably well across the BBB

(Wang et al. 1996, Kusuhara et al. 1997, Starling et al. 1997, Emi et al. 1998, Kalvass et al.

2007, Giacomini et al. 2010, Uchida et al. 2011a, Kodaira et al. 2011, Sziráki et al. 2011, Fan et al. 2012). As well, it does not significantly bind to microdialysis probes and microdialysis plastic tubings. Our work provided in vivo evidence and a “proof of concept” that pharmacological induction of P-gp possibly through a nuclear receptor (i.e., PXR) pathway at the BBB can reduce brain ECF concentrations of a P-gp substrate (Chan et al. 2013c).

Intracerebral microdialysis, the gold-standard approach to measure in vivo chemical concentrations in brain ECF, has been a useful tool to examine drug disposition in this compartment (De Lange et al. 1998, De Lange et al. 2000, Chaurasia et al. 2007, Hammarlund-

Udenaes et al. 2009). This technique offers a continuous in vivo monitoring of drug concentrations in the brain ECF, which other conventional methods may not always accurately predict (i.e., bioanalysis of brain homogenate and/or CSF obtained by terminal collection of whole brain and CSF samples at various time intervals following dosing of the drug) (De Lange et al. 1998, De Lange et al. 2000, Chaurasia et al. 2007, Hammarlund-Udenaes et al. 2009, Lin

2008, Westerhout et al. 2012, Westerhout et al. 2013). In this study, we demonstrated that treatment of mice with DEX (5mg/kg/day for 3 days) resulted in a 1.5-fold P-gp induction in

201

mouse brain capillaries (Chan et al. 2013c). This finding is in agreement with a previous report that showed that DEX could induce P-gp expression by two-fold in rat brain capillaries (Bauer et al. 2004). Furthermore, we showed that ratios of quinidine concentrations in brain ECF to unbound plasma concentrations at steady state (Kp, uu, ECF/Plasma) in DEX-treated mice were significantly lower when compared to vehicle-treated animals, indicating that quinidine distribution in the brain ECF was reduced in DEX-treated animals (Chan et al. 2013c). Our data are consistent with the literature and support the concept that concentrations of P-gp substrates in brain ECF are expected to be reduced following P-gp induction at the BBB and this could be explained by the increased clearance of substrates from the brain ECF back into the circulation

(Bauer et al. 2006, Wu et al. 2009, Eyal et al. 2009). Interestingly, a kainate-induced epileptic rat model, which was previously demonstrated to have a higher P-gp expression in brain capillaries when compared to non-treated animals, exhibited no significant change in quinidine permeability across the BBB but a higher brain ECF concentrations of quinidine in hippocampus regions

(Syvänen et al. 2012). In the same animal model, the authors also reported a decrease in total brain concentrations of quinidine following drug-induced epileptic conditions, however, this effect did not reach significance. Although these findings may appear to contradict our results,

Syvänen et al. also suggested that neuro-inflammation in brain parenchyma and disease-mediated

BBB dysfunction could occur during epileptic states and that these factors, in addition to P-gp induction at the BBB, glial cells and neurons, could affect quinidine distribution in brain and

ECF compartment (Syvänen et al. 2012). Therefore, these discrepancies may be explained by differences in the mechanism by which P-gp induction was mediated between the epileptic rat model and our wild type mouse model that was triggered with a P-gp inducer systemically. Our group has previously demonstrated that P-gp is also expressed in different cellular compartments

202

of brain parenchyma, such as astrocytes and microglia (Lee et al. 2001c, Ronaldson et al.

2004a, Bendayan et al. 2006). The function of P-gp in brain parenchyma could potentially result in the efflux of quinidine from these cellular compartments into brain ECF for further distribution in the brain or removal across the BBB (Chan et al. 2013c). Therefore, alterations of

P-gp expression in these cellular compartments that could potentially affect quinidine concentrations in brain ECF. In our study, lysates of mouse brain homogenate depleted from brain capillaries were used for the measurement of P-gp protein expression in brain parenchyma.

We did not observe significant P-gp induction in brain homogenates of DEX-treated mice, suggesting that the lower quinidine concentrations observed in brain ECF of DEX-treated animals was unlikely due to expression changes of P-gp in brain parenchyma (Chan et al. 2013c).

In addition, cytochrome P450 (CYP) enzyme 3A4 in humans and its mouse ortholog Cyp3a11 can extensively metabolize quinidine, while 10 to 50% of the drug is excreted unchanged in urine

(De Lange 2013). Therefore, cyp3a11 expression in brain parenchyma and microvessel endothelial cells could potentially affect quinidine disposition in brain ECF. In the current work,

Cyp3a11 expression was not detected in mouse brain capillaries. These data are consistent with previous findings demonstrating that CYP3A4, the human homolog of Cyp3a11, is not present in human brain capillaries (Dauchy et al. 2008). Furthermore, Cyp3a11 induction in brain homogenates of DEX-treated animals was not observed. Taken together, these data suggest that the lower quinidine ECF concentrations observed in the DEX-treated animals were not a result of

Cyp3a11 induction at the BBB or in brain parenchyma (Chan et al. 2013c). Our in vivo findings provide in vivo proof of concept that pharmacological P-gp induction at the BBB is possible and highlight the role of P-gp at the BBB in regulating concentrations of therapeutic agents in the

203

brain ECF compartment. Our results could potentially be relevant to the pharmacotherapy of other CNS disorders when the drugs are known to interact with P-gp.

In summary, our data demonstrating the role of hPXR and hCAR pathways in regulating

P-gp expression in human brain microvessel endothelial cells are consistent with other in vivo studies in rodents that have reported the presence of these pathways in brain microvessels.

Furthermore, our data provide the first evidence suggesting that hPXR and hCAR could be potential xenobiotic targets for the regulation of P-gp at the human BBB. Our current study could guide future investigations examining regulation of drug transporters at the BBB. This concept is supported by our in vivo work utilizing intracerebral microdialysis that demonstrated P-gp induction mediated by a PXR agonist at the mouse BBB can reduce the concentration of a P-gp substrate, quinidine, in the brain ECF. In the context of HIV pharmacotherapy, P-gp induction at the BBB could further limit antiretroviral drug entry into the brain. Hence, future work is required to investigate P-gp induction in brain microvessels from HIV patients who were chronically treated with antiretroviral drugs, in particular agents that were demonstrated in this thesis to be receptor ligands and P-gp inducers. The results of such work may direct the development of novel pharmacological approaches to utilize hPXR and hCAR as molecular targets to alter clinical P-gp induction at the BBB to either enhance drug permeability and efficacy in the CNS or restrict drug permeability and minimize drug-associated neurotoxicity during HIV infection or other CNS disorders.

204

11. Limitation and Future Directions

11.1 Experimental Limitations

11.1.1 In vitro cell culture systems of human brain microvessel endothelial cells

At present, several in vivo and in situ techniques have been utilized to evaluate drug transport function across the BBB, such as intracerebral microdialysis, positron emission tomography and in-situ brain perfusion (Feng 2002, Cecchelli et al. 2007, Chaurasia et al. 2007,

De Lange 2013). However, these methods can often be labour intensive and expensive (Cecchelli et al. 2007). In contrast, in vitro (i.e., brain microvessel endothelial cell culture models) and ex vivo (i.e., brain capillary isolation) approaches can be useful alternatives. The major advantages for utilizing cell culture models and isolated brain capillaries are that the investigator can exert control over experimental conditions in order to elucidate specific molecular mechanisms involved in drug transporter regulatory pathways with relatively limited interference (i.e., plasma protein binding and hepatic drug metabolism) (Cecchelli et al. 2007).

Over the past decade, our laboratory has been interested in examining in vitro drug transport processes at the BBB and other brain cellular compartments (i.e., astrocytes and microglia) relevant to HIV-1 infection utilizing in vitro cell culture systems (Lee et al. 2001c,

Dallas et al. 2003, Dallas et al. 2004a, Dallas et al. 2004b, Ronaldson et al. 2004a, Ronaldson et al. 2004b, Ronaldson & Bendayan 2006, Ronaldson et al. 2008, Ronaldson et al. 2010, Ashraf et al. 2011, Hoque et al. 2012). Recently, our interest has also expanded to examine several efflux and influx drug transport processes at the blood-testis barrier and the intestinal epithelium utilizing in vitro, in situ and in vivo approaches (Kis et al. 2010b, Robillard et al. 2012, Kis et al.

2013). In this thesis, the immortalized human brain microvascular endothelial cell culture system, hCMEC/D3, was utilized as a tool for elucidating P-gp regulation by nuclear receptors at the

205

human BBB. This cell culture system has been well characterized to maintain stable BBB- specific transport function and phenotypes (Weksler et al. 2005, Weksler et al. 2013). In particular, these cells are morphologically similar to primary cultures of brain microvessel endothelial cells in which elongated cells are tightly packed in a confluent monolayer. These cells constitutively express several brain microvessel endothelial markers [e.g., PECAM-1 (CD-

31) and VE-cadherin (CD144)] (Weksler et al. 2013), tight junction proteins (i.e., catenins, ZO-

1, JAM-A, occludin and claudin-5) (Afonso et al. 2008, Liebner et al. 2008, Luissint et al. 2012,

Urich et al. 2012), cell surface adhesion proteins (e.g., VCAM-1, ICAM-1 and ICAM-2)

(Weksler et al. 2005), cytokines receptors (e.g., TNF receptor 1/2) and chemokine receptors (e.g.,

CXCR1-5 and CCR3-6) (Subileau et al. 2009, Fasler-Kan et al. 2010, Lopez-Ramirez et al.

2012), all of which are expressed physiologically in brain microvessel endothelial cells (Weksler et al. 2005). In addition, this cell culture system exhibits a biochemical barrier function through the expression of several phase I drug metabolizing enzymes (e.g., CYP1A1, CYP1B1, CYP2B6 and CYP2E1), glucose transporter (GLUT-1), neutral amino acid transporter B (ATB), ABC drug transporters (e.g., P-gp, BCRP, MRP1, MRP3, MRP4 and MRP5) and SLC transporters (e.g.,

OCT1-3, MCT1 and MCT3) (Weksler et al. 2005, Dauchy et al. 2009, Carl et al. 2010, Dickens et al. 2012, Kooijmans et al. 2012, Ohtsuki et al. 2013). Furthermore, this cell line has been utilized to study the effect of various human pathogens (e.g., viral, fungal, bacterial and parasitic infections) (Coureuil et al. 2009, Vu et al. 2009, Coureuil et al. 2010, Jambou et al. 2010,

Zougbédé et al. 2011) and inflammatory stimuli (i.e., HIV-1 and viral protein tat) (András et al.

2008, Huang et al. 2009, András et al. 2010, Huang et al. 2010, András & Toborek 2011) on brain microvessel endothelial cells properties (e.g., substrate permeability and tight junction protein expression).

206

A few phenotypic and functional differences have been reported in hCMEC/D3 cells in comparison with human brain microvessel samples, making this cell culture an incomplete in vivo representation of the BBB. For instance, protein expression of drug transporters (i.e., P-gp and BCRP) in hCMEC/D3 cells are lower compared with human brain microvessels, whereas

ABCC (MRP) transcript expression is relatively identical (Dauchy et al. 2009). This finding implies that transport data generated in hCMEC/D3 could potentially underestimate the effect of

P-gp in comparison to the clinical situation. In regards to BBB functions, confluent monolayers of hCMEC/D3 cells show restricted permeability to both low and high molecular weight paracellular diffusion markers (i.e., lucifer yellow and dextrans) and to many hydrophobic and hydrophilic small molecules (e.g., diazepam, morphine-6-glucuronide, prazosin, colchicine and vincristine), suggesting this cell system has retained the barrier function of brain microvessel endothelial cells (Weksler et al. 2005, Poller et al. 2008). However, confluent monolayers of hCMEC/D3 cells only exhibit a low TEER value of approximately 100 Ω·cm2 and currently it is accepted that in vivo mammalian BBBs, such as the rodent, possess high TEER values of approximately 1500 Ω·cm2 (Crone & Olesen 1982, Abbott et al. 2010). Nonetheless, the low

TEER value observed in hCMEC/D3 cells is expected, and this finding is consistent with the literature that in vitro immortalized cell culture systems of brain microvessel endothelial cells typically cannot achieve TEER values comparable to in vivo values (Weksler et al. 2013). As mentioned in Chapter 1, the brain microvessel endothelium is in close proximity to astrocyte end feet, pericytes and axonal processes (Abbott et al. 2010). Hence, the limitation of hCMEC/D3 cells to generate high TEER values could be due to the absence of highly complex cellular interactions between different cellular compartments of the BBB and/or a lack of exposure to endogenous factors and signals (i.e., hormones and neuronal signals) secreted by these cellular

207

compartments (Weksler et al. 2013). In fact, tight junction proteins (e.g., occludin) can be induced in hCMEC/D3 cells exposed to vascular growth factors (e.g., basic fibroblast growth factor) and Wnt/β-catenin signalling activator (i.e., lithium chloride) (Liebner et al. 2008, Förster et al. 2008). Unfortunately, a significant improvement in TEER values in hCMEC/D3 cells exposed to these compounds was not observed (Liebner et al. 2008, Förster et al. 2008). In contrast, several reports demonstrated that addition of astrocyte-conditioned medium to endothelial cell cultures or co-culturing of endothelial cells with astrocytes can induce tight junction protein expression and reduce paracellular drug permeability (Santaguida et al. 2006). In addition, brain microvessel endothelial cells in physiological conditions are known to form three- dimensional tube structures that are constantly exposed to flow-base shear stress (Abbott et al.

2010). HCMEC/D3 cell monolayer grown on a capillary cartridge system and subjected to medium flow was reported to exhibit TEER values as high as 1000 Ω·cm2, however the wide- utilization of this sophisticated in vitro endothelial cell culture model remains technically challenging (Poller et al. 2008).

In our laboratory, hCMEC/D3 cells has been cultured on rat-tail collagen-coated surface as a monolayer in endothelial cell growth medium supplemented with vascular endothelial growth factor, insulin-like growth factor 1, epidermal growth factor, fibroblast growth factors, hydrocortisone, ascorbate, gentamycin and fetal bovine serum, according to the initial protocol published by Weksler et al. 2005. The addition of serum and other supplements, in particular fibroblast growth factors, hydrocortisone and ascorbate, are believed to help the hCMEC/D3 cells to maintain several BBB phenotypes mentioned above (Weksler et al. 2005). Since obtaining fresh human brain tissues is extremely challenging, the hCMEC/D3 cell culture system has provided a stable and easily-accessible in vitro model of the human brain microvessel

208

endothelial cells. Similar to the limitation associated with the hCMEC/D3 cells, data generated from whole cell pellets of primary cultures of human brain-derived microvascular endothelial cells or BBB-ECs should also be interpreted with caution. Although these cells have been well- characterized to have limited contamination from other cells in the brain parenchyma (e.g., astrocytes) and are known to express several human brain microvessel endothelial cell markers, such as factor VIII-related and HT-7 antigens (Prat et al. 2000). They present limitations similar to the hCMEC/D3 cells in that in vitro culturing conditions can be very different compared to in vivo physiological conditions in the brain. In addition, the BBB-ECs were isolated from young epileptic adult brains, and anti-epileptic medications and/or epileptic conditions could possibly affect protein (i.e., nuclear receptors) expression in the brain compared to non-epileptic healthy brain tissue. Therefore, although we have demonstrated expression of hPXR and hCAR protein expression in BBB-ECs, our data cannot be directly translated to the clinic. Similarly, although we have demonstrated the regulation of P-gp by hPXR and hCAR in the hCMEC/D3 cells, we cannot conclude that similar results will be observed in the clinic. Rather, our in vitro data suggest that hPXR and hCAR could be potential xenobiotic targets for the regulation of P-gp at the human brain microvessel endothelial cells and could be useful to guide future studies which will examine brain regulation of P-gp and other drug transporters in the clinic.

11.1.2 Species differences

In addition to in vitro cell culture models, in vivo approaches using animals are also an important component of evaluating drug uptake and efflux processes at the BBB. However, potential species differences between humans and other mammals (i.e., rodents) can be a major limitation for making clinical inferences on data obtained from in vivo animal models. Hence, a complete understanding of potential species differences is essential for making rigorous

209

interpretation of in vivo results. In this thesis, we utilized the CD1 mouse model for the intracerebral microdialysis study. Species differences in P-gp and PXR between humans and rodents (i.e., mouse) have been reported and the following section describes current knowledge on these differences with emphasis on protein expression at the BBB and substrate/ligand affinity.

P-gp in different human and rodent P-gp shares approximately 80 % amino acid sequence identity (Van der Bliek et al. 1988, Jones & George 1998). Currently, several in vitro and in vivo studies suggest that P-gp expression at the BBB and P-gp substrate affinity can be different between human and rodents (Tang-Wai et al. 1995, Takeuchi et al. 2006, Feng et al. 2008).

Using cell culture models transfected with either human MDR1 or mouse Mdr1a gene, it was demonstrated that cells transfected with mouse mdr1a exhibited a 27-fold greater drug resistance to P-gp substrates (i.e., colchicine and actinomycin D) than cells transfected with human MDR1

(Tang-Wai et al. 1995). Recently, it was also shown that digoxin transport was greater in cells transfected with mouse mdr1a and 1b genes compared to human MDR1 transfected cells

(Takeuchi et al. 2006). These findings suggest that protein encoded by mdr1a isoform may be more efficient in transporting these drugs and can generate a more effective drug resistance compared to its human counterpart. Recently, Feng et al. tested 3300 compounds in human

MDR1-transfected and mouse mdr1a-transfected cells (i.e., MDCK) and found a good correlation (R2 = 0.92) between substrate efflux ratios generated from the human and mouse P-gp transfected cell lines (Feng et al. 2008). Feng et al. suggested that the mouse model can be a useful tool to predict P-gp activity in humans and species differences in P-gp function may only apply to a limited number of substrates. Quinidine was used in the current thesis because it is a well-established P-gp substrate in several in vitro and in vivo BBB transport studies (Wang et al.

210

1996, Kusuhara et al. 1997, Starling et al. 1997, Emi et al. 1998, Giacomini et al. 2010, Uchida et al. 2011a, Kodaira et al. 2011, Sziráki et al. 2011). Studies using in vitro cell culture model

(i.e., MDCK) reported that transepithelial permeability of quinidine generated by cells transfected with human MDR1 was 7.4, whereas cells transfected with mouse mdr1a was 6.6

(Feng et al. 2008). It is difficult to translate these in vitro findings generated from P-gp overexpressing cell culture systems to our in vivo mouse model where P-gp is not physiologically overexpressed. However, we do not anticipate to observe significant species differences in quinidine transport mediated by human P-gp compared to its mouse counterpart, since the permeability values of quinidine are very similar. Nonetheless, P-gp protein expression at the human BBB (i.e., brain microvessels) determined quantitatively was found to be 2.3-fold lower than in mice (Uchida et al. 2011b). It is suggested that our in vivo mouse model, as well as many other studies that used mice, could overestimate the overall transport activity mediated by P-gp at the BBB when compared to humans. Therefore, we should interpret our in vivo quinidine brain

ECF concentrations with caution if one wishes to apply these data to predict quinidine (an antiarrhythmic agent) concentrations in the human brain ECF compartment. Nevertheless, our study in this thesis was not designed to examine the clinical relevance of quinidine concentrations in the brain ECF. In contrast, quinidine was used in this thesis as a probe to assess the effect of in vivo P-gp induction at the BBB on drug distribution in the brain ECF compartment.

In vitro and in vivo evidence from our laboratory and others clearly demonstrates the expression of PXR in brain microvessel endothelial cells (Bauer et al. 2004, Bauer et al. 2006,

Narang et al. 2008, Ott et al. 2009, Zastre et al. 2009, Chan et al. 2011, Chan et al. 2013b, Chan et al. 2013c), however both the transcript and protein expression of hPXR has yet to be detected in human brain microvessels (Dauchy et al. 2008, Shawahna et al. 2011). To date, species

211

differences of PXR expression between human and mouse BBB remain unclear. In contrast, species differences between rodents and human in regards to PXR ligand-activation have been well documented and discussed in Chapter 1 (Stanley et al. 2006, Chang & Waxman 2006). In the current in vivo study, DEX was used as a P-gp inducer and potent agonist of rodent PXR.

DEX is a weak hPXR agonist (Pascussi et al. 2001, Jones et al. 2000, Yueh et al. 2005).

Therefore, it is expected that under similar DEX concentrations in plasma and brain microvessel endothelial cells, transcriptional activation of mouse PXR should be greater than hPXR. DEX is also known as a glucocorticoid receptor agonist (Pascussi et al. 2001) and this receptor pathway has been reported to be functional in rat brain microvessel endothelial cells (Narang et al. 2008).

At present, functional glucocorticoid responsive elements in the promoter regions of drug transporter genes in humans and rodents have yet to be reported. Due to the presence of the glucocorticoid responsive element in the promoter region of hPXR gene, it is believed that glucocorticoid receptor activation can indirectly affect the expression of PXR-targeted transporters by its regulation on PXR expression (Pascussi et al. 2000, Pascussi et al. 2003). In the current study, we did not observe a significant alteration of PXR protein expression in mouse brain capillaries between DEX and VEH-treated animals, suggesting glucocorticoid receptor may not play a significant role in affecting PXR expression in our model following DEX treatment

(Chan et al. 2013c). Yet, our study cannot totally exclude the potential involvement of glucocorticoid receptor pathway in the regulation of P-gp mediated by PXR. Taken together, our in vivo findings provide a proof of concept that pharmacological P-gp induction mediated by nuclear receptor pathways at the BBB can alter brain ECF distribution of P-gp substrates.

212

11.1.3 Microdialysis

Determination of the in vivo disposition of a drug in rodent brain is commonly performed via the bioanalysis of brain homogenate and/or CSF obtained from the terminal collection of whole brain and CSF samples at various time intervals following drug administration (De Lange

& Danhof 2002, Shen et al. 2004). However, drug concentrations from these samples may not always accurately predict the unbound drug concentrations in the brain ECF which are primarily related to drug concentrations at the site of action (Lin 2008, Westerhout et al. 2012, Westerhout et al. 2013). Intracerebral microdialysis is known to be a “gold-standard” technique that offers a continuous in vivo monitoring of unbound drug concentrations in the brain ECF of a freely moving animal (De Lange et al. 1998, De Lange et al. 2000, Chaurasia et al. 2007, Hammarlund-

Udenaes et al. 2009). The technique involves the implantation of a small double-lumen probe into a specific region of the brain parenchyma. During perfusion of aCSF through the probe, passive diffusion drives the exchange of solute (i.e., drug) along its concentration gradient from the brain ECF to the perfusate across a semi-permeable membrane of the probe. The perfusate collected from the probe contains a fraction of the brain extracellular diffusible solute (i.e., unbound drug) concentration (De Lange et al. 1998, De Lange et al. 2000, Chaurasia et al. 2007,

Hammarlund-Udenaes et al. 2009). This fraction is referred to as the relative recovery and the validation of this parameter is important to generate accurate quantitative brain ECF drug concentrations (De Lange et al. 2000). An advantage of applying intracerebral microdialysis is that the combined use of other powerful analytical technique, such as LC-MS/MS, allows simultaneous and quantitative measurement of low solute concentrations in very small volume perfusate samples (De Lange et al. 2000). As well, the technique eliminates protein precipitation procedures for analysis and potential ex vivo enzymatic degradation of the solute, hence

213

increasing the sensitivity of the analytical technique (De Lange et al. 2000). However, intracerebral microdialysis has several limitations similar to other in vivo techniques used for the study of BBB function and transport, such as in situ brain perfusion and positron emission tomography (PET) (Feng 2002, Cecchelli et al. 2007, Chaurasia et al. 2007, De Lange 2013). In particular, intracerebral microdialysis requires intensive labour for several experimental procedures (i.e., animal surgery and probe insertion) and optimization steps (i.e., probe recovery). In addition, the technique does not allow high throughput approach because animal surgery and blood sampling in rodents, in particular mouse, is very technically demanding and time consuming. Furthermore, we have also noticed that the technique was difficult to be implemented in the C57B/6 mouse strain due to very poor animal viability in comparison to our current mouse strain, CD1. Therefore, our choice of animal model has been limited, making the utilization of other mouse strains, such as PXR/CAR humanized mouse model with a C57B/6 background, not feasible.

11.2 Future Directions

11.2.1 Nuclear receptor regulation of phase I/II metabolic enzymes and other drug transporters

in brain microvessel endothelial cells

Several phase I drug metabolizing enzymes (e.g., CYP1A1, CYP1B1, CYP2B6,

CYP2C8), phase II metabolic enzymes (e.g., UGTs and GSTs), ABC transporters (i.e., P-gp,

MRPs, BCRP) and SLC transporters (i.e., ENT1 and MCT1) have been reported either at the transcript or protein expression in hCMEC/D3 cells and human brain microvessels (Dauchy et al.

2008, Dauchy et al. 2009, Shawahna et al. 2011). In the human liver and intestine, many of these genes are known targets of PXR, CAR and other nuclear receptors (e.g., VDR and PPARs)

(Tirona & Kim 2005, Urquhart et al. 2007, Hoque et al. 2012). However, most of their regulation

214

mediated by nuclear receptor pathways, namely PXR, CAR, VDR and PPARα, which are known to be functional in hCMEC/D3 cells, have not been fully investigated at the human BBB.

Therefore, the first step to examine the existence of these regulatory pathways at this site is to characterize the transcript and protein expression of these receptors in isolated human brain microvessels. In particular, in vivo studies using rodent models have demonstrated the role of several nuclear receptors (e.g., PXR, CAR, VDR, PPARs, LXR and ERs) in regulating the expression of drug transporters (i.e., P-gp and Bcrp) in brain microvessel endothelial cells, as reviewed in our recent published manuscript in TIPS (Chan et al. 2013a). However, it is unclear if these receptors also play a role in the in vivo regulation of drug-metabolizing enzymes (i.e.,

Cyp enzymes) at the rodent BBB. Therefore, a complete understanding of the role of nuclear receptors in the regulation of their gene targets in rodent brain microvessels is required. The result of this work can identify potential receptor targets to predict clinical induction of drug metabolizing enzymes and drug transporters at the human BBB. In particular, positron emission tomography imaging with the use of P-gp substrates, such as 11C-verapamil and 11C-loperamide in humans could be applied to investigate P-gp activity at the BBB and further examine the clinical effect of transport regulation at the BBB (Muzi et al. 2009, Loscher & Langer 2010,

Bauer et al. 2012, Syvänen & Eriksson 2013). Ultimately, this work may potentially guide future development of novel pharmacotherapy that interacts with these receptor targets to alter the expression of drug transporters and/or drug metabolizing enzymes in the brain, resulting in either enhanced CNS drug efficacy or reduced drug-associated neurotoxicity.

215

11.2.2 Nuclear receptor regulation of drug transporters in other cellular compartments of the brain parenchyma

In the CNS, functional expression of several drug efflux and influx transporters, belonging to the ABC or SLC superfamilies, can alter drug permeability across the BBB and possibly affect drug distribution in different cellular compartments of the brain parenchyma (i.e., neurons and astrocytes) (Eyal et al. 2009, Ashraf et al. 2012, Ashraf et al. 2013). In particular, astrocytes and neurons can represent important compartments that may be involved in sequestration of drugs in the brain. In fact, it has been proposed by our laboratory that drug efflux transporters (e.g., P-gp, MRPs and BCRP) can constitute a secondary biochemical barrier at the membranes of cellular compartments of the parenchyma in restricting drug accumulation within the brain (Lee et al. 2001b, Lee et al. 2001c, Ronaldson et al. 2004a, Ronaldson et al. 2004b,

Ronaldson & Bendayan 2006, Ronaldson et al. 2007, Ronaldson et al. 2008, Ronaldson et al.

2010, Ashraf et al. 2012, Ashraf et al. 2013). It is well established that some of these transporters can be highly regulated by several nuclear receptors, such as PXR and CAR (Germain et al.

2006b, Urquhart et al. 2007), however, it is not known if this regulation is also functional in cellular compartments of the brain parenchyma. Therefore, it remains to be determined whether these nuclear receptors play a role in regulating transporters expression at these sites.

216

12. References

Abbott, N. J., Patabendige, A. A., Dolman, D. E., Yusof, S. R. and Begley, D. J. (2010) Structure and function of the blood-brain barrier. Neurobiology of Disease, 37(1), 13-25.

Abbott, N. J., Rönnbäck, L. and Hansson, E. (2006) Astrocyte-endothelial interactions at the blood-brain barrier. Nature Reviews Neuroscience, 7(1), 41-53.

Abbruscato, T. J. and Davis, T. P. (1999) Protein expression of brain endothelial cell E-cadherin after hypoxia/aglycemia: Influence of astrocyte contact. Brain Research, 842(2), 277-286.

Adibhatla, R. M. and Hatcher, J. F. (2008) Tissue Plasminogen Activator (tPA) and matrix metalloproteinases in the pathogenesis of stroke: Therapeutic strategies. CNS and Neurological Disorders - Drug Targets, 7(3), 243-253.

Afonso, P. V., Ozden, S., Cumont, M. C., Seilhean, D., Cartier, L., Rezaie, P., Mason, S., Lambert, S., Huerre, M., Gessain, A., Couraud, P. O., Pique, C., Ceccaldi, P. E. and Romero, I. A. (2008) Alteration of blood-brain barrier integrity by retroviral infection. PLoS Pathogens, 4(11).

Aggarwal, S., Tsuruo, T. and Gupta, S. (1997) Altered expression and function of P-glycoprotein (170 kDa), encoded by the MDR 1 gene, in T cell subsets from aging humans. Journal of Clinical Immunology, 17(6), 448-454.

Akanuma, S. i., Hori, S., Ohtsuki, S., Fujiyoshi, M. and Terasaki, T. (2008) Expression of nuclear receptor mRNA and liver X receptor-mediated regulation of ABC transporter A1 at rat blood-brain barrier. Neurochemistry International, 52(4-5), 669-674.

Albermann, N., Schmitz-Winnenthal, F. H., Z'Graggen, K., Volk, C., Hoffmann, M. M., Haefeli, W. E. and Weiss, J. (2005) Expression of the drug transporters MDR1/ABCB1, MRP1/ABCC1, MRP2/ABCC2, BCRP/ABCG2, and PXR in peripheral blood mononuclear cells and their relationship with the expression in intestine and liver. Biochemical Pharmacology, 70(6), 949- 958.

Aleksunes, L. M. and Klaassen, C. D. (2012) Coordinated regulation of hepatic phase I and II drug-metabolizing genes and transporters using AhR-, CAR-, PXR-, PPARα-, and Nrf2-null mice. Drug Metabolism and Disposition, 40(7), 1366-1379.

Aleksunes, L. M., Yeager, R. L. and Klaassen, C. D. (2009) Application of multivariate statistical procedures to identify transcription factors that correlate with MRP2, 3, and 4 mRNA in adult human livers. Xenobiotica, 39(7), 514-522.

Aller, S. G., Yu, J., Ward, A., Weng, Y., Chittaboina, S., Zhuo, R., Harrell, P. M., Trinh, Y. T., Zhang, Q., Urbatsch, I. L. and Chang, G. (2009) Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. Science, 323(5922), 1718-1722.

217

Alnouti, Y. and Klaassen, C. D. (2008) Tissue distribution, ontogeny, and regulation of aldehyde dehydrogenase (Aldh) enzymes mRNA by prototypical microsomal enzyme inducers in mice. Toxicological Sciences, 101(1), 51-64.

Anakk, S., Kalsotra, A., Kikuta, Y., Huang, W., Zhang, J., Staudinger, J. L., Moore, D. D. and Strobel, H. W. (2004) CAR/PXR provide directives for Cyp3a41 gene regulation differently from Cyp3a11. Pharmacogenomics J, 4(2), 91-101.

Anakk, S., Kalsotra, A., Shen, Q., Vu, M. T., Staudinger, J. L., Davies, P. J. and Strobel, H. W. (2003) Genomic characterization and regulation of CYP3a13: role of xenobiotics and nuclear receptors. The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 17(12), 1736-1738.

András, I. E., Eum, S. Y., Huang, W., Zhong, Y., Hennig, B. and Toborek, M. (2010) HIV-1- induced amyloid beta accumulation in brain endothelial cells is attenuated by simvastatin. Molecular and Cellular Neuroscience, 43(2), 232-243.

András, I. E., Rha, G., Huang, W., Eum, S., Couraud, P. O., Romero, I. A., Hennig, B. and Toborek, M. (2008) Simvastatin protects against amyloid β and HIV-1 Tat-induced promoter activities of inflammatory genes in brain endothelial cells. Molecular Pharmacology, 73(5), 1424-1433.

András, I. E. and Toborek, M. (2011) HIV-1-induced alterations of claudin-5 expression at the blood-brain barrier level. Methods in Molecular Biology, 762(355-370.

Antinori, A., Perno, C. F., Giancola, M. L., Forbici, F., Ippolito, G., Hoetelmans, R. M. and Piscitelli, S. C. (2005) Efficacy of cerebrospinal fluid (CSF)-penetrating antiretroviral drugs against HIV in the neurological compartment: Different patterns of phenotypic resistance in CSF and plasma. Clinical Infectious Diseases, 41(12), 1787-1793.

Antonelli, G., Turriziani, O., Cianfriglia, M., Riva, E., Dong, G., Fattorossi, A. and Dianzani, F. (1992) Resistance of HIV-1 to AZT might also involve the cellular expression of multidrug resistance P-glycoprotein. AIDS Research and Human Retroviruses, 8(10), 1839-1844.

Aquilante, C. L., Letrent, S. P., Pollack, G. M. and Brouwer, K. L. R. (1999) Increased brain P- glycoprotein in morphine tolerant rats. Life Sciences, 66(4), PL-47-PL-51.

Aronica, E., Gorter, J. A., Redeker, S., van Vliet, E. A., Ramkema, M., Scheffer, G. L., Scheper, R. J., van der Valk, P., Leenstra, S., Baayen, J. C., Spliet, W. G. and Troost, D. (2005) Localization of breast cancer resistance protein (BCRP) in microvessel endothelium of human control and epileptic brain. Epilepsia, 46(6), 849-857.

Ashraf, T., Kis, O., Banerjee, N. and Bendayan, R. (2012) Drug Transporters at Brain Barriers: Expression and Regulation by Neurological Disorders. Advances in Experimental Medicine and Biology, 763(0): 20-69.

218

Ashraf, T., Robillard, K., Chan, G. N. Y. and Bendayan, R. (2013) Role of CNS transporters in pharmacotherapy of brain HIV-1 infection. Current Pharmaceutical Design. 19 Jun. [Epub ahead of print].

Ashraf, T., Ronaldson, P. T., Persidsky, Y. and Bendayan, R. (2011) Regulation of P- glycoprotein by human immunodeficiency virus-1 in primary cultures of human fetal astrocytes. Journal of Neuroscience Research, 89(11), 1773-1782.

Assem, M., Schuetz, E. G., Leggas, M., Sun, D., Yasuda, K., Reid, G., Zelcer, N., Adachi, M., Strom, S., Evans, R. M., Moore, D. D., Borst, P. and Schuetz, J. D. (2004) Interactions between hepatic Mrp4 and Sult2a as revealed by the constitutive androstane receptor and Mrp4 knockout mice. Journal of Biological Chemistry, 279(21), 22250-22257.

Auerbach, S. S., Ramsden, R., Stoner, M. A., Verlinde, C., Hassett, C. and Omiecinski, C. J. (2003) Alternatively spliced isoforms of the human constitutive androstane receptor. Nucleic Acids Research, 31(12), 3194-3207.

Babakhanian, K., Bendayan, M. and Bendayan, R. (2007) Localization of P-glycoprotein at the nuclear envelope of rat brain cells. Biochemical and Biophysical Research Communications, 361(2), 301-306.

Bachmeier, C. J., Spitzenberger, T. J., Elmquist, W. F. and Miller, D. W. (2005) Quantitative assessment of HIV-1 protease inhibitor interactions with drug efflux transporters in the blood- brain barrier. Pharmaceutical Research, 22(8), 1259-1268.

Bachmeier, C. J., Trickler, W. J. and Miller, D. W. (2004) Drug efflux transport properties of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and its fluorescent free acid, BCECF. Journal of Pharmaceutical Sciences, 93(4), 932-942.

Badawi, A. F., Cavalieri, E. L. and Rogan, E. G. (2001) Role of human cytochrome P450 1A1, 1A2, 1B1, and 3A4 in the 2-, 4-, and 16α-hydroxylation of 17β-estradiol. Metabolism: Clinical and Experimental, 50(9), 1001-1003.

Baes, M., Gulick, T., Choi, H. S., Martinolu, M. G., Simha, D. and Moore, D. D. (1994) A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements. Molecular and Cellular Biology, 14(3), 1544-1552.

Bailey, J., Blankson, J. N., Wind-Rotolo, M. and Siliciano, R. F. (2004) Mechanisms of HIV-1 escape from immune responses and antiretroviral drugs. Current Opinion in Immunology, 16(4), 470-476.

Baldwin, S. A., Yao, S. Y. M., Hyde, R. J., Ng, A. M. L., Foppolo, S., Barnes, K., Ritzel, M. W. L., Cass, C. E. and Young, J. D. (2005) Functional characterization of novel human and mouse equilibrative nucleoside transporters (hENT3 and mENT3) located in intracellular membranes. Journal of Biological Chemistry, 280(16), 15880-15887.

Ballabh, P., Braun, A. and Nedergaard, M. (2004) The blood-brain barrier: An overview: Structure, regulation, and clinical implications. Neurobiology of Disease, 16(1), 1-13.

219

Banks, W. A. (2006) The CNS as a target for peptides and peptide-based drugs. Expert Opinion on Drug Delivery, 3(6), 707-712.

Banks, W. A., Freed, E. O., Wolf, K. M., Robinson, S. M., Franko, M. and Kumar, V. B. (2001) Transport of human immunodeficiency virus type 1 pseudoviruses across the blood-brain barrier: Role of envelope proteins and adsorptive endocytosis. Journal of Virology, 75(10), 4681-4691.

Bankstahl, J. P. and Löscher, W. (2008) Resistance to antiepileptic drugs and expression of P- glycoprotein in two rat models of status epilepticus. Epilepsy Research, 82(1), 70-85.

Barakat, S., Demeule, M., Pilorget, A., Régina, A., Gingras, D., Baggetto, L. G. and Béliveau, R. (2007) Modulation of p-glycoprotein function by caveolin-1 phosphorylation. Journal of Neurochemistry, 101(1), 1-8.

Barakat, S., Turcotte, S., Demeule, M., Lachambre, M. P., Régina, A., Baggetto, L. G. and Béliveau, R. (2008) Regulation of brain endothelial cells migration and angiogenesis by P- glycoprotein/caveolin-1 interaction. Biochemical and Biophysical Research Communications, 372(3), 440-446.

Barbier, O., Turgeon, D., Girard, C., Green, M. D., Tephly, T. R., Hum, D. W. and Bélanger, A. (2000) 3'-Azido-3'-deoxythimidine (AZT) is glucuronidated by human UDP- glucuronosyltransferase 2B7 (UGT2B7). Drug Metabolism and Disposition, 28(5), 497-502.

Bauer, B., Hartz, A. M. S., Fricker, G. and Miller, D. S. (2004) Pregnane X receptor upregulation of P-glycoprotein expression and transport function at the blood-brain barrier. Molecular Pharmacology, 66(3), 413-419.

Bauer, B., Hartz, A. M. S., Lucking, J. R., Yang, X., Pollack, G. M. and Miller, D. S. (2008a) Coordinated nuclear receptor regulation of the efflux transporter, Mrp2, and the phase-II metabolizing enzyme, GSTπ, at the blood-brain barrier. Journal of Cerebral Blood Flow and Metabolism, 28(6), 1222-1234.

Bauer, B., Hartz, A. M. S. and Miller, D. S. (2007) Tumor necrosis factor α and endothelin-1 increase p-glycoprotein expression and transport activity at the blood-brain barrier. Molecular Pharmacology, 71(3), 667-675.

Bauer, B., Hartz, A. M. S., Pekcec, A., Toellner, K., Miller, D. S. and Potschka, H. (2008b) Seizure-induced up-regulation of P-glycoprotein at the blood-brain barrier through glutamate and cyclooxygenase-2 signaling. Molecular Pharmacology, 73(5), 1444-1453.

Bauer, B., Yang, X., Hartz, A. M. S., Olson, E. R., Zhao, R., Kalvass, J. C., Pollack, G. M. and Miller, D. S. (2006) In vivo activation of human pregnane X receptor tightens the blood-brain barrier to methadone through p-glycoprotein up-regulation. Molecular Pharmacology, 70(4), 1212-1219.

Bauer, M., Karch, R., Neumann, F., Wagner, C. C., Kletter, K., Müller, M., Löscher, W., Zeitlinger, M. and Langer, O. (2010) Assessment of regional differences in tariquidar-induced P-

220

glycoprotein modulation at the human blood-brain barrier. Journal of Cerebral Blood Flow and Metabolism, 30(3), 510-515.

Bauer, M., Zeitlinger, M., Karch, R., Matzneller, P., Stanek, J., Jäger, W., Böhmdorfer, M., Wadsak, W., Mitterhauser, M., Bankstahl, J. P., Löscher, W., Koepp, M., Kuntner, C., Müller, M. and Langer, O. (2012) Pgp-mediated interaction between (R)- 11CVerapamil and tariquidar at the human blood-brain barrier: A comparison with rat data. Clinical Pharmacology and Therapeutics, 91(2), 227-233.

Bazzoni, G., Tonetti, P., Manzi, L., Cera, M. R., Balconi, G. and Dejana, E. (2005) Expression of junctional adhesion molecule-A prevents spontaneous and random motility. Journal of Cell Science, 118(3), 623-632.

Beaulieu, É., Demeule, M., Ghitescu, L. and Béliveau, R. (1997) P-glycoprotein is strongly expressed in the luminal membranes of the endothelium of blood vessels in the brain. Biochemical Journal, 326(2), 539-544.

Bechmann, I., Priller, J., Kovac, A., Böntert, M., Wehner, T., Klett, F. F., Bohsung, J., Stuschke, M., Dirnagl, U. and Nitsch, R. (2001) Immune surveillance of mouse brain perivascular spaces by blood-borne macrophages. European Journal of Neuroscience, 14(10), 1651-1658.

Behravan, J. and Piquette-Miller, M. (2007) Drug transport across the placenta, role of the ABC drug efflux transporters. Expert Opinion on Drug Metabolism and Toxicology, 3(6), 819-830.

Bélanger, A. S., Caron, P., Harvey, M., Zimmerman, P. A., Mehlotra, R. K. and Guillemette, C. (2009) Glucuronidation of the antiretroviral drug efavirenz by UGT2B7 and an in vitro investigation of drug-drug interaction with zidovudine. Drug Metabolism and Disposition, 37(9), 1793-1796.

Bell, R. D., Winkler, E. A., Sagare, A. P., Singh, I., LaRue, B., Deane, R. and Zlokovic, B. V. (2010) Pericytes Control Key Neurovascular Functions and Neuronal Phenotype in the Adult Brain and during Brain Aging. Neuron, 68(3), 409-427.

Ben-Menachem, E., Johansson, B. B. and Svensson, T. H. (1982) Increased vulnerability of the blood-brain barrier to acute hypertension following depletion of brain noradrenaline. Journal of Neural Transmission - General Section, 53(2-3), 159-167.

Bendayan, M. (1995) Colloidal gold post-embedding immunocytochemistry. Progress in Histochemistry and Cytochemistry, 29(4), 1-159.

Bendayan, R., Lee, G. and Bendayan, M. (2002) Functional expression and localization of P- glycoprotein at the blood brain barrier. Microscopy Research and Technique, 57(5), 365-380.

Bendayan, R., Ronaldson, P. T., Gingras, D. and Bendayan, M. (2006) In situ localization of P- glycoprotein (ABCB1) in human and rat brain. Journal of Histochemistry and Cytochemistry, 54(10), 1159-1167.

221

Benoit, G., Cooney, A., Giguere, V., Ingraham, H., Lazar, M., Muscat, G., Perlmann, T., Renaud, J. P., Schwabe, J., Sladek, F., Tsai, M. J. and Laudet, V. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacological Reviews, 58(4), 798-836.

Benoit, G., Malewicz, M. and Perlmann, T. (2004) Digging deep into the pockets of orphan nuclear receptors: Insights from structural studies. Trends in Cell Biology, 14(7), 369-376.

Benveniste, H. and Diemer, N. H. (1987) Cellular reactions to implantation of a microdialysis tube in the rat hippocampus. Acta Neuropathologica, 74(3), 234-238.

Bertilsson, G., Heidrich, J., Svensson, K., Asman, M., Jendeberg, L., Sydow-Bäckman, M., Ohlsson, R., Postlind, H., Blomquist, P. and Berkenstam, A. (1998) Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proceedings of the National Academy of Sciences of the United States of America, 95(21), 12208-12213.

Bertilsson, P. M., Olsson, P. and Magnusson, K. E. (2001) Cytokines influence MRNA expression of cytochrome p450 3A4 and MDRI in intestinal cells. Journal of Pharmaceutical Sciences, 90(5), 638-646.

Bertrand-Thiebault, C., Masson, C., Siest, G., Batt, A. M. and Visvikis-Siest, S. (2007) Effect of HMGCoA reductase inhibitors on cytochrome P450 expression in endothelial cell line. Journal of Cardiovascular Pharmacology, 49(5), 306-315.

Best, B. M., Letendre, S. L., Brigid, E., Clifford, D. B., Collier, A. C., Gelman, B. B., McArthur, J. C., McCutchan, J. A., Simpson, D. M., Ellis, R., Capparelli, E. V. and Grant, I. (2009) Low atazanavir concentrations in cerebrospinal fluid. AIDS, 23(1), 83-87.

Best, B. M., Letendre, S. L., Koopmans, P., Rossi, S. S., Clifford, D. B., Collier, A. C., Gelman, B. B., Marra, C. M., McArthur, J. C., McCutchan, J. A., Morgello, S., Simpson, D. M., Capparelli, E. V., Ellis, R. J. and Grant, I. (2012) Low cerebrospinal fluid concentrations of the nucleotide HIV reverse transcriptase inhibitor, Tenofovir. Journal of Acquired Immune Deficiency Syndromes, 59(4), 376-381.

Bhalla, S., Ozalp, C., Fang, S., Xiang, L. and Kemper, J. K. (2004) Ligand-activated pregnane X receptor interferes with HNF-4 signaling by targeting a common coactivator PGC-1α. Functional implications in hepatic cholesterol and glucose metabolism. Journal of Biological Chemistry, 279(43), 45139-45147.

Bierman, W. F. W., Scheffer, G. L., Schoonderwoerd, A., Jansen, G., van Agtmael, M. A., Danner, S. A. and Scheper, R. J. (2010) Protease inhibitors atazanavir, lopinavir and ritonavir are potent blockers, but poor substrates, of ABC transporters in a broad panel of ABC transporter- overexpressing cell lines. Journal of Antimicrobial Chemotherapy, 65(8), 1672-1680.

Bleasby, K., Hall, L. A., Perry, J. L., Mohrenweiser, H. W. and Pritchard, J. B. (2005) Functional consequences of single nucleotide polymorphisms in the human organic anion transporter hOAT1 (SLC22A6). Journal of Pharmacology and Experimental Therapeutics, 314(2), 923-931.

222

Blumberg, B., Sabbagh Jr, W., Juguilon, H., Bolado Jr, J., Van Meter, C. M., Ong, E. S. and Evans, R. M. (1998) SXR, a novel steroid and xenobiotic-sensing nuclear receptor. Genes and Development, 12(20), 3195-3205.

Bobardt, M. D., Salmon, P., Wang, L., Esko, J. D., Gabuzda, D., Fiala, M., Trono, D., Van Der Schueren, B., David, G. and Gallay, P. A. (2004) Contribution of proteoglycans to human immunodeficiency virus type 1 brain invasion. Journal of Virology, 78(12), 6567-6584.

Borst, P., Balzarini, J., Ono, N., Reid, G., De Vries, H., Wielinga, P., Wijnholds, J. and Zelcer, N. (2004) The potential impact of drug transporters on nucleoside-analog-based antiviral chemotherapy. Antiviral Research, 62(1), 1-7.

Bousquet, L., Pruvost, A., Didier, N., Farinotti, R. and Mabondzo, A. (2008) Emtricitabine: Inhibitor and substrate of multidrug resistance associated protein. European Journal of Pharmaceutical Sciences, 35(4), 247-256.

Braet, K., Cabooter, L., Paemeleire, K. and Leybaert, L. (2004) Calcium signal communication in the central nervous system. Biology of the Cell, 96(1), 79-91.

Brandt, C., Bethmann, K., Gastens, A. M. and Löscher, W. (2006) The multidrug transporter hypothesis of drug resistance in epilepsy: Proof-of-principle in a rat model of temporal lobe epilepsy. Neurobiology of Disease, 24(1), 202-211.

Brélivet, Y., Rochel, N. and Moras, D. (2012) Structural analysis of nuclear receptors: From isolated domains to integral proteins. Molecular and Cellular Endocrinology, 348(2), 466-473.

Brightman, M. W. and Reese, T. S. (1969) Junctions between intimately apposed cell membranes in the vertebrate brain. Journal of Cell Biology, 40(3), 648-677.

Brown, K. C., Paul, S. and Kashuba, A. D. M. (2009) Drug interactions with new and investigational antiretrovirals. Clinical Pharmacokinetics, 48(4), 211-241.

Burk, O., Arnold, K. A., Geick, A., Tegude, H. and Eichelbaum, M. (2005) A role for constitutive androstane receptor in the regulation of human intestinal MDR1 expression. Biological Chemistry, 386(6), 503-513.

Burk, O., Koch, I., Raucy, J., Hustert, E., Eichelbaum, M., Brockmöller, J., Zanger, U. M. and Wojnowski, L. (2004) The induction of cytochrome P450 3A5 (CYP3A5) in the human liver and intestine is mediated by the xenobiotic sensors pregnane X receptor (PXR) and constitutively activated receptor (CAR). Journal of Biological Chemistry, 279(37), 38379-38385.

Cabot, M. C., Giuliano, A. E., Han, T. V. and Liu, Y. Y. (1999) SDZ PSC 833, the cyclosporine a analogue and multidrug resistance modulator, activates ceramide synthesis and increases vinblastine sensitivity in drug-sensitive and drug-resistant cancer cells. Cancer Research, 59(4), 880-885.

223

Cairns, W., Cairns, C., Pongratz, I., Poellinger, L. and Okret, S. (1991) Assembly of a glucocorticoid receptor complex prior to DNA binding enhances its specific interaction with a glucocorticoid response element. Journal of Biological Chemistry, 266(17), 11221-11226.

Calcagno, A., Bonora, S., Simiele, M., Rostagno, R., Tettoni, M. C., Bonasso, M., Romito, A., Imperiale, D., D'Avolio, A. and Di Perri, G. (2011) Tenofovir and emtricitabine cerebrospinal fluid-to-plasma ratios correlate to the extent of blood-brainbarrier damage. AIDS, 25(11), 1437- 1439.

Campbell, S. D., De Morais, S. M. and Xu, J. J. (2004) Inhibition of human organic anion transporting polypeptide OATP 1B1 as a mechanism of drug-induced hyperbilirubinemia. Chemico-Biological Interactions, 150(2), 179-187.

Cano-Soldado, P., Larráyoz, I. M., Molina-Arcas, M., Casado, F. J., Martinez-Picado, J., Lostao, M. P. and Pastor-Anglada, M. (2004) Interaction of nucleoside inhibitors of HIV-1 reverse transcriptase with the concentrative nucleoside transporter-1 (SLC28A1). Antiviral Therapy, 9(6), 993-1002.

Capparelli, E. V., Holland, D., Okamoto, C., Gragg, B., Durelle, J., Marquie-Beck, J., Van Den Brande, G., Ellis, R. and Letendre, S. (2005) Lopinavir concentrations in cerebrospinal fluid exceed the 50% inhibitory concentration for HIV. AIDS, 19(9), 949-952.

Cardoso, F. L., Brites, D. and Brito, M. A. (2010) Looking at the blood-brain barrier: Molecular anatomy and possible investigation approaches. Brain Research Reviews, 64(2), 328-363.

Carl, S. M., Lindley, D. J., Couraud, P. O., Weksler, B. B., Romero, I., Mowery, S. A. and Knipp, G. T. (2010) ABC and SLC transporter expression and pot substrate characterization across the human CMEC/D3 blood-'brain barrier cell line. Molecular Pharmaceutics, 7(4), 1057- 1068.

Carman, C. V. and Springer, T. A. (2008) Trans-cellular migration: cell-cell contacts get intimate. Current Opinion in Cell Biology, 20(5), 533-540.

Carvey, P. M., Hendey, B. and Monahan, A. J. (2009) The blood-brain barrier in neurodegenerative disease: A rhetorical perspective. Journal of Neurochemistry, 111(2), 291- 314.

Cecchelli, R., Berezowski, V., Lundquist, S., Culot, M., Renftel, M., Dehouck, M. P. and Fenart, L. (2007) Modelling of the blood-brain barrier in drug discovery and development. Nature Reviews Drug Discovery, 6(8), 650-661.

Chambers, T. C., Pohl, J., Raynor, R. L. and Kuo, J. F. (1993) Identification of specific sites in human P-glycoprotein phosphorylated by protein kinase C. Journal of Biological Chemistry, 268(7), 4592-4595.

Chan, G. N. Y. and Bendayan, R. (2011) Molecular and functional characterization of P- glycoprotein in vitro. Methods in molecular biology (Clifton, N.J.), 686(313-336.

224

Chan, G. N. Y., Hoque, M. T. and Bendayan, R. (2010) Regulation of P-glycoprotein (P-gp) by Orphan Nuclear Receptors in Human Brain. Gordon Research Conference. Barriers of the CNS: Blood/Brain Interfaces in Health and Disease. New London, NH, USA.

Chan, G. N. Y., Hoque, M. T. and Bendayan, R. (2013a) Role of nuclear receptors in the regulation of drug transporters in the brain. Trends in Pharmacological Sciences, 34(7), 361-372.

Chan, G. N. Y., Hoque, M. T., Cummins, C. L. and Bendayan, R. (2011) Regulation of P- glycoprotein by orphan nuclear receptors in human brain microvessel endothelial cells. Journal of Neurochemistry, 118(2), 163-175.

Chan, G. N. Y., Patel, R., Cummins, C. L. and Bendayan, R. (2013b) Induction of P-glycoprotein by Antiretroviral Drugs in Human Brain Microvessel Endothelial Cells. Antimicrobial Agents and Chemotherapy. 8 July, doi: 10.1128/AAC.00486-13. [Epub ahead of print]

Chan, G. N. Y., Saldivia, V., Yang, Y., Pang, H., de Lannoy, I. and Bendayan, R. (2013c) In vivo induction of P-glycoprotein expression at the mouse blood-brain barrier: An intracerebral microdialysis study. Journal of Neurochemistry. 18 Jun. doi: 10.1111/jnc.12344. [Epub ahead of print].

Chandler, B., Almond, L., Ford, J., Owen, A., Hoggard, P., Khoo, S. and Back, D. (2003) The effects of protease inhibitors and nonnucleoside reverse transcriptase inhibitors on P- glycoprotein expression in peripheral blood mononuclear cells in vitro. Journal of Acquired Immune Deficiency Syndromes, 33(5), 551-556.

Chang, T. K. H., Bandiera, S. M. and Chen, J. (2003) Constitutive androstane receptor and pregnane X receptor gene expression in human liver: Interindividual variability and correlation with CYP2B6 mRNA levels. Drug Metabolism and Disposition, 31(1), 7-10.

Chang, T. K. H. and Waxman, D. J. (2006) Synthetic drugs and natural products as modulators of constitutive androstane receptor (CAR) and pregnane X receptor (PXR). Drug Metabolism Reviews, 38(1-2), 51-73.

Chauhan, A., Hahn, S., Gartner, S., Pardo, C. A., Netesan, S. K., McArthur, J. and Nath, A. (2007) Molecular programming of endothelin-1 in HIV-infected brain: Role of Tat in upregulation of ET-1 and its inhibition by statins. FASEB Journal, 21(3), 777-789.

Chaurasia, C. S., Müller, M., Bashaw, E. D., Benfeldt, E., Bolinder, J., Bullock, R., Bungay, P. M., DeLange, E. C. M., Derendorf, H., Elmquist, W. F., Hammarlund-Udenaes, M., Joukhadar, C., Kellogg Jr, D. L., Lunte, C. E., Nordstrom, C. H., Rollema, H., Sawchuk, R. J., Cheung, B. W. Y., Shah, V. P., Stahle, L., Ungerstedt, U., Welty, D. F. and Yeo, H. (2007) AAPS-FDA workshop white paper: Microdialysis principles, application, and regulatory perspectives. Journal of Clinical Pharmacology, 47(5), 589-603.

Chen, C. C., Meadows, B., Regis, J., Kalafsky, G., Fojo, T., Carrasquillo, J. A. and Bates, S. E. (1997) Detection of in vivo P-glycoprotein inhibition by PSC 833 using Tc-99m sestamibi. Clinical Cancer Research, 3(4), 545-552.

225

Chen, Y., Ferguson, S. S., Negishi, M. and Goldstein, J. A. (2003) Identification of constitutive androstane receptor and glucocorticoid receptor binding sites in the CYP2C19 promoter. Molecular Pharmacology, 64(2), 316-324.

Chen, Y., Ferguson, S. S., Negishi, M. and Goldstein, J. A. (2004) Induction of Human CYP2C9 by Rifampicin, Hyperforin, and Phenobarbital Is Mediated by the Pregnane X Receptor. Journal of Pharmacology and Experimental Therapeutics, 308(2), 495-501.

Cheng, X., Maher, J., Chen, C. and Klaassen, C. D. (2005) Tissue distribution and ontogeny of mouse organic anion transporting polypeptides (Oatps). Drug Metabolism and Disposition, 33(7), 1062-1073.

Cherrington, N. J., Slitt, A. L., Maher, J. M., Zhang, X. X., Zhang, J., Huang, W., Wan, Y. J. Y., Moore, D. D. and Klaassen, C. D. (2003) Induction of multidrug resistance protein 3 (MRP3) in vivo is independent of constitutive androstane receptor. Drug Metabolism and Disposition, 31(11), 1315-1319.

Chin, J. E., Soffir, R., Noonan, K. E., Choi, K. and Roninson, I. B. (1989) Structure and expression of the human MDR (P-glycoprotein) gene family. Molecular and Cellular Biology, 9(9), 3808-3820.

Choi, E., Lee, S., Yeom, S. Y., Geun, H. K., Jae, W. L. and Kim, S. W. (2005) Characterization of activating signal cointegrator-2 as a novel transcriptional coactivator of the xenobiotic nuclear receptor constitutive androstane receptor. Molecular Endocrinology, 19(7), 1711-1719.

Choi, H. S., Chung, M., Tzameli, I., Simha, D., Lee, Y. K., Seol, W. and Moore, D. D. (1997) Differential transactivation by two isoforms of the orphan nuclear hormone receptor CAR. Journal of Biological Chemistry, 272(38), 23565-23571.

Choo, E. F., Kurnik, D., Muszkat, M., Ohkubo, T., Shay, S. D., Higginbotham, J. N., Glaeser, H., Kim, R. B., Wood, A. J. J. and Wilkinson, G. R. (2006) Differential in vivo sensitivity to inhibition of P-glycoprotein located in lymphocytes, testes, and the blood-brain barrier. Journal of Pharmacology and Experimental Therapeutics, 317(3), 1012-1018.

Choo, E. F., Leake, B., Wandel, C., Imamura, H., Wood, A. J. J., Wilkinson, G. R. and Kim, R. B. (2000) Pharmacological inhibition of P-glycoprotein transport enhances the distribution of HIV-1 protease inhibitors into brain and testes. Drug Metabolism and Disposition, 28(6), 655- 660.

Choudhuri, S. and Klaassen, C. (2006) Structure, function, expression, genomic organization, and single nucleotide polymorphisms of human ABCB1 (MDR1), ABCC (MRP), and ABCG2 (BCRP) efflux transporters. International Journal of Toxicology, 25(4), 231-259.

Chrencik, J. E., Orans, J., Moore, L. B., Xue, Y., Peng, L., Collins, J. L., Wisely, G. B., Lambert, M. H., Kliewer, S. A. and Redinbo, M. R. (2005) Structural disorder in the complex of human pregnane X receptor and the macrolide antibiotic rifampicin. Molecular Endocrinology, 19(5), 1125-1134.

226

Ciechanover, A. (1998) The ubiquitin-proteasome pathway: On protein death and cell life. EMBO Journal, 17(24), 7151-7160.

Coakley, E., Petropoulos, C. J. and Whitcomb, J. M. (2005) Assessing chemokine co-receptor usage in HIV. Current Opinion in Infectious Diseases, 18(1), 9-15.

Cohen, Z., Molinatti, G. and Hamel, E. (1997) Astroglial and vascular interactions of noradrenaline terminals in the rat cerebral cortex. Journal of Cerebral Blood Flow and Metabolism, 17(8), 894-904.

Cohen, Z. V. I., Bonvento, G., Lacombe, P. and Hamel, E. (1996) Serotonin in the regulation of brain microcirculation. Progress in Neurobiology, 50(4), 335-362.

Colgan, O. C., Collins, N. T., Ferguson, G., Murphy, R. P., Birney, Y. A., Cahill, P. A. and Cummins, P. M. (2008) Influence of basolateral condition on the regulation of brain microvascular endothelial tight junction properties and barrier function. Brain Research, 1193(C), 84-92.

Cook, B. D., Ferrari, G., Pintucci, G. and Mignatti, P. (2008) TGF-β1 induces rearrangement of FLK-1-VE-cadherin-β-catenin complex at the adherens junction through VEGF-mediated signaling. Journal of Cellular Biochemistry, 105(6), 1367-1373.

Cornford, E. M., Varesi, J. B., Hyman, S., Damian, R. T. and Raleigh, M. J. (1997) Mitochondrial content of choroid plexus epithelium. Experimental Brain Research, 116(3), 399- 405.

Coureuil, M., Lécuyer, H., Scott, M. G. H., Boularan, C., Enslen, H., Soyer, M., Mikaty, G., Bourdoulous, S., Nassif, X. and Marullo, S. (2010) Meningococcus hijacks a β2-adrenoceptor/β- arrestin pathway to cross brain microvasculature endothelium. Cell, 143(7), 1149-1160.

Coureuil, M., Mikaty, G., Miller, F., Lécuyer, H., Bernard, C., Bourdoulous, S., Duménil, G., Mège, R. M., Weksler, B. B., Romero, I. A., Couraud, P. O. and Nassif, X. (2009) Meningococcal type IV pili recruit the polarity complex to cross the brain endothelium. Science, 325(5936), 83-87.

Crone, C. and Olesen, S. P. (1982) Electrical resistance of brain microvascular endothelium. Brain Res, 241(1), 49-55.

Croteau, D., Letendre, S., Best, B. M., Rossi, S. S., Ellis, R. J., Clifford, D. B., Collier, A. C., Gelman, B. B., Marra, C. M., McArthur, J., McCutchan, J. A., Morgello, S., Simpson, D. M., Way, L., Capparelli, E. and Grant, I. (2012) Therapeutic amprenavir concentrations in cerebrospinal fluid. Antimicrobial Agents and Chemotherapy, 56(4), 1985-1989.

Cucullo, L., Hossain, M., Puvenna, V., Marchi, N. and Janigro, D. (2011) The role of shear stress in Blood-Brain Barrier endothelial physiology. BMC Neuroscience, 12(40.

Cui, J. Y., Gunewardena, S. S., Rockwell, C. E. and Klaassen, C. D. (2010) ChIPing the cistrome of PXR in mouse liver. Nucleic Acids Research, 38(22), 7943-7963.

227

Cunningham, P. H., Smith, D. G., Satchell, C., Cooper, D. A. and Brew, B. (2000) Evidence for independent development of resistance to HIV-1 reverse transcriptase inhibitors in the cerebrospinal fluid. AIDS, 14(13), 1949-1954.

Cvetkovic, M., Leake, B., Fromm, M. F., Wilkinson, G. R. and Kim, R. B. (1999) OATP and P- glycoprotein transporters mediate the cellular uptake and excretion of fexofenadine. Drug Metabolism and Disposition, 27(8), 866-871.

Cysique, L. A. and Brew, B. J. (2009) Neuropsychological functioning and antiretroviral treatment in HIV/AIDS: A review. Neuropsychology Review, 19(2), 169-185.

Dahl, V., Josefsson, L. and Palmer, S. (2010) HIV reservoirs, latency, and reactivation: Prospects for eradication. Antiviral Research, 85(1), 286-294.

Dalkara, T., Gursoy-Ozdemir, Y. and Yemisci, M. (2011) Brain microvascular pericytes in health and disease. Acta Neuropathologica, 122(1), 1-9.

Dallas, S., Miller, D. S. and Bendayan, R. (2006) Multidrug resistance-associated proteins: Expression and function in the central nervous system. Pharmacological Reviews, 58(2), 140- 161.

Dallas, S., Ronaldson, P. T., Bendayan, M. and Bendayan, R. (2004a) Multidrug resistance protein 1-mediated transport of saquinavir by microglia. NeuroReport, 15(7), 1183-1186.

Dallas, S., Schlichter, L. and Bendayan, R. (2004b) Multidrug resistance protein (MRP) 4- and MRP 5-mediated efflux of 9-(2-phosphonylmethoxyethyl)adenine by microglia. Journal of Pharmacology and Experimental Therapeutics, 309(3), 1221-1229.

Dallas, S., Zhu, X., Baruchel, S., Schlichter, L. and Bendayan, R. (2003) Functional expression of the multidrug resistance protein 1 in microglia. Journal of Pharmacology and Experimental Therapeutics, 307(1), 282-290.

Dalton, W. S., Crowley, J. J., Salmon, S. S., Grogan, T. M., Laufman, L. R., Weiss, G. R. and Bonnet, J. D. (1995) A phase III randomized study of oral verapamil as a chemosensitizer to reverse drug resistance in patients with refractory myeloma: A Southwest Oncology Group study. Cancer, 75(3), 815-820.

Daneman, R., Zhou, L., Kebede, A. A. and Barres, B. A. (2010) Pericytes are required for blood- brain barrier integrity during embryogenesis. Nature, 468(7323), 562-566.

Dantzig, A. H., Shepard, R. L., Cao, J., Law, K. L., Ehlhardt, W. J., Baughman, T. M., Bumol, T. F. and Starling, J. J. (1996) Reversal of P-glycoprotein-mediated multidrug resistance by a potent cyclopropyldibenzosuberane modulator, LY335979. Cancer Research, 56(18), 4171-4179.

Dauchy, S., Dutheil, F., Weaver, R. J., Chassoux, F., Daumas-Duport, C., Couraud, P. O., Scherrmann, J. M., De Waziers, I. and Decleves, X. (2008) ABC transporters, cytochromes P450 and their main transcription factors: Expression at the human blood-brain barrier. Journal of Neurochemistry, 107(6), 1518-1528.

228

Dauchy, S., Miller, F., Couraud, P. O., Weaver, R. J., Weksler, B., Romero, I. A., Scherrmann, J. M., De Waziers, I. and Declèves, X. (2009) Expression and transcriptional regulation of ABC transporters and cytochromes P450 in hCMEC/D3 human cerebral microvascular endothelial cells. Biochemical Pharmacology, 77(5), 897-909.

Davoust, N., Vuaillat, C., Androdias, G. and Nataf, S. (2008) From bone marrow to microglia: barriers and avenues. Trends in Immunology, 29(5), 227-234.

De Boer, A. G. and Gaillard, P. J. (2006) Blood-brain barrier dysfunction and recovery. Journal of Neural Transmission, 113(4), 455-462.

De Bruin, M., Miyake, K., Litman, T., Robey, R. and Bates, S. E. (1999) Reversal of resistance by GF120918 in cell lines expressing the ABC half-transporter, MXR. Cancer Letters, 146(2), 117-126.

De Lange, E. C. (2013) The mastermind approach to CNS drug therapy: translational prediction of human brain distribution, target site kinetics, and therapeutic effects. Fluids and Barriers of the CNS, 10(1), Epub: Feburary 26.

De Lange, E. C., Danhof, M., Zurcher, C., de Boer, A. G. and Breimer, D. D. (1995) Repeated microdialysis perfusions: periprobe tissue reactions and BBB permeability. Brain Research, 702(1-2), 261-265.

De Lange, E. C., De Boer, A. G. and Breimer, D. D. (2000) Methodological issues in microdialysis sampling for pharmacokinetic studies. Advanced Drug Delivery Reviews, 45(2-3), 125-148.

De Lange, E. C. M. and Danhof, M. (2002) Considerations in the use of cerebrospinal fluid pharmacokinetics to predict brain target concentrations in the clinical setting: Implications of the barriers between blood and brain. Clinical Pharmacokinetics, 41(10), 691-703.

De Lange, E. C. M., De Bock, G., Schinkel, A. H., De Boer, A. G. and Breimer, D. D. (1998) BBB transport and P-glycoprotein functionality using MDR1A (-/-) and wild-type mice. Total brain versus microdialysis concentration profiles of rhodamine-123. Pharmaceutical Research, 15(11), 1657-1665.

De Maat, M. M. R., Corine Ekhart, G., Huitema, A. D. R., Koks, C. H. W., Mulder, J. W. and Beijnen, J. H. (2003) Drug interactions between antiretroviral drugs and comedicated agents. Clinical Pharmacokinetics, 42(3), 223-282.

Dean, M. and Allikmets, R. (2001) Complete characterization of the human ABC gene family. J Bioenerg Biomembr, 33(6), 475-479.

Dehouck, M. P., Meresse, S., Delorme, P., Fruchart, J. C. and Cecchelli, R. (1990) An easier, reproducible, and mass-production method to study the blood-brain barrier in vitro. Journal of Neurochemistry, 54(5), 1798-1801.

229

Demby, V. E. (2008) OATP1B1, OATP1B3 and BCRP transporter characterization for fosamprenavir, amprenavir and lopinavir [abstract]. AAPS Annual Meeting and Exposition, 2355, 2008.

Demeule, M., Labelle, M., Régina, A., Berthelet, F. and Béliveau, R. (2001) Isolation of endothelial cells from brain, lung, and kidney: Expression of the multidrug resistance p- glycoprotein isoforms. Biochemical and Biophysical Research Communications, 281(3), 827- 834.

Deng, H., Liu, R., Ellmeier, W., Choe, S., Unutmaz, D., Burkhart, M., Di Marzio, P., Marmon, S., Sutton, R. E., Mark Hill, C., Davis, C. B., Peiper, S. C., Schall, T. J., Littman, D. R. and Landau, N. R. (1996) Identification of a major co-receptor for primary isolates of HIV-1. Nature, 381(6584), 661-666.

Denizot, F. and Lang, R. (1986) Rapid colorimetric assay for cell growth and survival - Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. Journal of Immunological Methods, 89(2), 271-277.

Desrayaud, S., Guntz, P., Scherrmann, J. M. and Lemaire, M. (1997) Effect of the P-glycoprotein inhibitor, SDZ PSC 833, on the blood and brain pharmacokinetics of colchicine. Life Sciences, 61(2), 153-163. di Masi, A., Marinis, E. D., Ascenzi, P. and Marino, M. (2009) Nuclear receptors CAR and PXR: Molecular, functional, and biomedical aspects. Molecular Aspects of Medicine, 30(5), 297-343.

Dickens, D., Owen, A., Alfirevic, A., Giannoudis, A., Davies, A., Weksler, B., Romero, I. A., Couraud, P. O. and Pirmohamed, M. (2012) Lamotrigine is a substrate for OCT1 in brain endothelial cells. Biochemical Pharmacology, 83(6), 805-814.

Didier, N., Banks, W. A., Créminon, C., Dereuddre-Bosquet, N. and Mabondzo, A. (2002) HIV- I-induced production of endothelin-I in an in vitro model of the human blood-brain barrier. NeuroReport, 13(9), 1179-1183.

Ding, X. and Staudinger, J. L. (2005a) Induction of drug metabolism by forskolin: The role of the pregnane X receptor and the protein kinase A signal transduction pathway. Journal of Pharmacology and Experimental Therapeutics, 312(2), 849-856.

Ding, X. and Staudinger, J. L. (2005b) Repression of PXR-mediated induction of hepatic CYP3A gene expression by protein kinase C. Biochemical Pharmacology, 69(5), 867-873.

Dixit, V., Hariparsad, N., Li, F., Desai, P., Thummel, K. E. and Unadkat, J. D. (2007) Cytochrome P450 enzymes and transporters induced by anti-human immunodeficiency virus protease inhibitors in human hepatocytes: Implications for predicting clinical drug interactions. Drug Metabolism and Disposition, 35(10), 1853-1859.

Do Canto, A. M. T. M., Carvalho, A. J. P., Ramalho, J. P. P. and Loura, L. M. S. (2011) Molecular dynamics simulations of T-20 HIV fusion inhibitor interacting with model membranes. Biophysical Chemistry, 159(2-3), 275-286.

230

Dombrowski, S. M., Desai, S. Y., Marroni, M., Cucullo, L., Goodrich, K., Bingaman, W., Mayberg, M. R., Bengez, L. and Janigro, D. (2001) Overexpression of multiple drug resistance genes in endothelial cells from patients with refractory epilepsy. Epilepsia, 42(12), 1501-1506.

Dotzlaw, H., Leygue, E., Watson, P. and Murphy, L. C. (1999) The human orphan receptor PXR messenger RNA is expressed in both normal and neoplastic breast tissue. Clinical Cancer Research, 5(8), 2103-2107.

Down, M. J., Arkle, S. and Mills, J. J. (2007) Regulation and induction of CYP3A11, CYP3A13 and CYP3A25 in C57BL/6J mouse liver. Archives of Biochemistry and Biophysics, 457(1), 105- 110.

Drab, M., Verkade, P., Elger, M., Kasper, M., Lohn, M., Lauterbach, B., Menne, J., Lindschau, C., Mende, F., Luft, F. C., Schedl, A., Hailer, H. and Kurzchalia, T. V. (2001) Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science, 293(5539), 2449-2452.

Dussault, I., Lin, M., Hollister, K., Wang, E. H., Synold, T. W. and Forman, B. M. (2001) Peptide mimetic HIV protease inhibitors are ligands for the orphan receptor SXR. Journal of Biological Chemistry, 276(36), 33309-33312.

Ecker, G., Huber, M., Schmid, D. and Chiba, P. (1999) The importance of a nitrogen atom in modulators of multidrug resistance. Molecular Pharmacology, 56(4), 791-796.

Edwards, J. E., Brouwer, K. R. and McNamara, P. J. (2002) GF120918, a P-glycoprotein modulator, increases the concentration of unbound amprenavir in the central nervous system in rats. Antimicrobial Agents and Chemotherapy, 46(7), 2284-2286.

Egashira, M., Kawamata, N., Sugimoto, K., Kaneko, T. and Oshimi, K. (1999) P-glycoprotein expression on normal and abnormally expanded natural killer cells and inhibition of P- glycoprotein function by cyclosporin A and its analogue, PSC833. Blood, 93(2), 599-606.

Ehrlich, P. (1885) Das sauerstufbudurfnis des organismus. Eine Farbenanalytische Studie.

Eisenblätter, T., Hüwel, S. and Galla, H. J. (2003) Characterisation of the brain multidrug resistance protein (BMDP/ABCG2/BCRP) expressed at the blood-brain barrier. Brain Research, 971(2), 221-231.

Emi, Y., Tsunashima, D., Ogawara, K. I., Higaki, K. and Kimura, T. (1998) Role of P- glycoprotein as a secretory mechanism in quinidine absorption from rat small intestine. Journal of Pharmaceutical Sciences, 87(3), 295-299.

Ene, L., Duiculescu, D. and Ruta, S. M. (2011) How much do antiretroviral drugs penetrate into the central nervous system? Journal of medicine and life, 4(4), 432-439.

Engelhardt, B. and Coisne, C. (2011) Fluids and barriers of the CNS establish immune privilege by confining immune surveillance to a two-walled castle moat surrounding the CNS castle. Fluids and Barriers of the CNS, 8(1).

231

Engelhardt, B. and Wolburg, H. (2004) Mini review: Transendothelial migration of leukocytes: Through the front door or around the side of the house? European Journal of Immunology, 34(11), 2955-2963.

Eyal, S., Hsiao, P. and Unadkat, J. D. (2009) Drug interactions at the blood-brain barrier: Fact or fantasy? Pharmacology and Therapeutics, 123(1), 80-104.

Fakhoury, M., Litalien, C., Medard, Y., Cavé, H., Ezzahir, N., Peuchmaur, M. and Jacqz-Aigrain, E. (2005) Localization and mRNA expression of CYP3A and P-glycoprotein in human duodenum as a function of age. Drug Metabolism and Disposition, 33(11), 1603-1607.

Falkner, K. C., Pinaire, J. A., Xiao, G. H., Geoghegan, T. E. and Prough, R. A. (2001) Regulation of the rat glutathione S-transferase A2 gene by glucocorticoids: Involvement of both the glucocorticoid and pregnane X receptors. Molecular Pharmacology, 60(3), 611-619.

Fan, J., Saldivia, V., Pang, H., Yang, Y., Bettinger, K., Weinstein, E. and de Lannoy, I. (2012) Impact of P-glycoprotein (P-gp) on the Brain Disposition of Quinidine and 9-Hydroxy Risperidone: Comparison of Brain Extracellular Fluid Concentrations (CECF) and Cerebrospinal Fluid Concentrations (CCSF) Using the Mdr1a Knockout Rat. 18th North American Regional International Society for the Study of Xenobiotics (ISSX) Meeting, Dallas, TX, USA

Fardel, O., Jigorel, E., Le Vee, M. and Payen, L. (2005) Physiological, pharmacological and clinical features of the multidrug resistance protein 2. Biomedicine and Pharmacotherapy, 59(3), 104-114.

Fasler-Kan, E., Suenderhauf, C., Barteneva, N., Poller, B., Gygax, D. and Huwyler, J. (2010) Cytokine signaling in the human brain capillary endothelial cell line hCMEC/D3. Brain Research, 1354(15-22.

Faucette, S. R., Wang, H., Hamilton, G. A., Jolley, S. L., Gilbert, D., Lindley, C., Yan, B., Negishi, M. and LeCluyse, E. L. (2004) Regulation of CYP2B6 in primary human hepatocytes by prototypical inducers. Drug Metabolism and Disposition, 32(3), 348-358.

Felix, R. A. and Barrand, M. A. (2002) P-glycoprotein expression in rat brain endothelial cells: Evidence for regulation by transient oxidative stress. Journal of Neurochemistry, 80(1), 64-72.

Feng, B., Mills, J. B., Davidson, R. E., Mireles, R. J., Janiszewski, J. S., Troutman, M. D. and De Morais, S. M. (2008) In vitro P-glycoprotein assays to predict the in vivo interactions of P- glycoprotein with drugs in the central nervous system. Drug Metabolism and Disposition, 36(2), 268-275.

Feng, M. R. (2002) Assessment of blood-brain barrier penetration: in silico, in vitro and in vivo. Current Drug Metabolism, 3(6), 647-657.

Ferguson, S. S., Chen, Y., LeCluyse, E. L., Negishi, M. and Goldstein, J. A. (2005) Human CYP2C8 is transcriptionally regulated by the nuclear receptors constitutive androstane receptor, pregnane X receptor, glucocorticoid receptor, and hepatic nuclear factor 4α. Molecular Pharmacology, 68(3), 747-757.

232

Ferguson, S. S., Lecluyse, E. L., Negishi, M. and Goldstein, J. A. (2002) Regulation of human CYP2C9 by the constitutive androstane receptor: Discovery of a new distal binding site. Molecular Pharmacology, 62(3), 737-746.

Fernández-Klett, F., Offenhauser, N., Dirnagl, U., Priller, J. and Lindauer, U. (2010) Pericytes in capillaries are contractile in vivo, but arterioles mediate functional hyperemia in the mouse brain. Proceedings of the National Academy of Sciences of the United States of America, 107(51), 22290-22295.

Finzi, D., Blankson, J., Siliciano, J. D., Margolick, J. B., Chadwick, K., Pierson, T., Smith, K., Lisziewicz, J., Lori, F., Flexner, C., Quinn, T. C., Chaisson, R. E., Rosenberg, E., Walker, B., Gange, S., Gallant, J. and Siliciano, R. F. (1999) Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nature Medicine, 5(5), 512-517.

Fiorucci, S., Zampella, A. and Distrutti, E. (2012) Development of FXR, PXR and CAR agonists and antagonists for treatment of liver disorders. Current Topics in Medicinal Chemistry, 12(6), 605-624.

Ford, J., Cornforth, D., Hoggard, P. G., Cuthbertson, Z., Meaden, E. R., Williams, I., Johnson, M., Daniels, E., Hsyu, P., Back, D. J. and Khoo, S. H. (2004) Intracellular and plasma pharmacokinetics of nelfinavir and M8 in HIV-infected patients: Relationship with P- glycoprotein expression. Antiviral Therapy, 9(1), 77-84.

Forman, B. M., Tzameli, I., Choi, H. S., Chen, J., Simha, D., Seol, W., Evans, R. M. and Moore, D. D. (1998) Androstane metabolites bind to and deactivate the nuclear receptor CAR- β. Nature, 395(6702), 612-615.

Förster, C., Burek, M., Romero, I. A., Weksler, B., Couraud, P. O. and Drenckhahn, D. (2008) Differential effects of hydrocortisone and TNFα on tight junction proteins in an in vitro model of the human blood-brain barrier. Journal of Physiology, 586(7), 1937-1949.

Foudraine, N. A., Hoetelmans, R. M. W., Lange, J. M. A., De Wolf, F., Van Benthem, B. H. B., Maas, J. J., Keet, I. P. M. and Portegies, P. (1998) Cerebrospinal-fluid HIV-1 RNA and drug concentrations after treatment with lamivudine plus zidovudine or stavudine. Lancet, 351(9115), 1547-1551.

Fox, E. and Bates, S. E. (2007) Tariquidar (XR9576): A P-glycoprotein drug efflux pump inhibitor. Expert Review of Anticancer Therapy, 7(4), 447-459.

Francis, G. A., Fayard, E., Picard, F. and Auwerx, J. (2003) Nuclear receptors and the control of metabolism. Annual Review of Physiology, 65(261-311.

Friedenberg, W. R., Rue, M., Blood, E. A., Dalton, W. S., Shustik, C., Larson, R. A., Sonneveld, P. and Greipp, P. R. (2006) Phase III study of PSC-833 (valspodar) in combination with vincristine, doxorubicin, and dexamethasone (valspodar/VAD) versus VAD alone in patients with recurring or refractory multiple myeloma (E1A95): A trial of the Eastern Cooperative Oncology Group. Cancer, 106(4), 830-838.

233

Fromm, M. F. (2004) Importance of P-glycoprotein at blood-tissue barriers. Trends in Pharmacological Sciences, 25(8), 423-429.

Fu, D. and Arias, I. M. (2012) Intracellular trafficking of P-glycoprotein. The International Journal of Biochemistry & Cell Biology, 44(3), 461-464.

Fujimoto, H., Higuchi, M., Watanabe, H., Koh, Y., Ghosh, A. K., Mitsuya, H., Tanoue, N., Hamada, A. and Saito, H. (2009) P-glycoprotein mediates efflux transport of Darunavir in human intestinal Caco-2 and ABCB1 gene-transfected renal LLC-PK1 cell lines. Biological and Pharmaceutical Bulletin, 32(9), 1588-1593.

Fung, K. L. and Gottesman, M. M. (2009) A synonymous polymorphism in a common MDR1 (ABCB1) haplotype shapes protein function. Biochimica et Biophysica Acta, 1794(5), 860-871.

Gao, J. and Xie, W. (2010) Pregnane X receptor and constitutive androstane receptor at the crossroads of drug metabolism and energy metabolism. Drug Metabolism and Disposition, 38(12), 2091-2095.

Gardner-Stephen, D., Heydel, J. M., Goyal, A., Lu, Y., Xie, W., Lindblom, T., Mackenzie, P. and Radominska-Pandya, A. (2004) Human PXR variants and their differential effects on the regulation of human UDP-glucuronosyltransferase gene expression. Drug Metabolism and Disposition, 32(3), 340-347.

Geick, A., Eichelbaum, M. and Burk, O. (2001) Nuclear Receptor Response Elements Mediate Induction of Intestinal MDR1 by Rifampin. Journal of Biological Chemistry, 276(18), 14581- 14587.

George, A. M. and Jones, P. M. (2012) Perspectives on the structure-function of ABC transporters: The Switch and Constant Contact Models. Progress in Biophysics and Molecular Biology, 109(3), 95-107.

George, C. L., Lightman, S. L. and Biddie, S. C. (2011) Transcription factor interactions in genomic nuclear receptor function. Epigenomics, 3(4), 471-485.

Gerbal-Chaloin, S., Daujat, M., Pascussi, J. M., Pichard-Garcia, L., Vilarem, M. J. and Maurel, P. (2002) Transcriptional regulation of CYP2C9 gene. Role of glucocorticoid receptor and constitutive androstane receptor. Journal of Biological Chemistry, 277(1), 209-217.

Gerbal-Chaloin, S., Pascussi, J. M., Pichard-Garcia, L., Daujat, M., Waechter, F., Fabre, J. M., Carrère, N. and Maurel, P. (2001) Induction of CYP2C genes in human hepatocytes in primary culture. Drug Metabolism and Disposition, 29(3), 242-251.

Gerhardt, H., Wolburg, H. and Redies, C. (2000) N-cadherin mediates pericytic-endothelial interaction during brain angiogenesis in the chicken. Developmental Dynamics, 218(3), 472-479.

Germain, P., Chambon, P., Eichele, G., Evans, R. M., Lazar, M. A., Leid, M., De Lera, A. R., Lotan, R., Mangelsdorf, D. J. and Gronemeyer, H. (2006a) International union of pharmacology. LXIII. Retinoid X receptors. Pharmacological Reviews, 58(4), 760-772.

234

Germain, P., Staels, B., Dacquet, C., Spedding, M. and Laudet, V. (2006b) Overview of nomenclature of nuclear receptors. Pharmacological Reviews, 58(4), 685-704.

Giacomini, K. M., Huang, S. M., Tweedie, D. J., Benet, L. Z., Brouwer, K. L. R., Chu, X., Dahlin, A., Evers, R., Fischer, V., Hillgren, K. M., Hoffmaster, K. A., Ishikawa, T., Keppler, D., Kim, R. B., Lee, C. A., Niemi, M., Polli, J. W., Sugiyama, Y., Swaan, P. W., Ware, J. A., Wright, S. H., Wah Yee, S., Zamek-Gliszczynski, M. J. and Zhang, L. (2010) Membrane transporters in drug development. Nature Reviews Drug Discovery, 9(3), 215-236.

Gibson, G. G., Phillips, A., Aouabdi, S., Plant, K. and Plant, N. (2006) Transcriptional regulation of the human pregnane-X receptor. Drug Metabolism Reviews, 38(1-2), 31-49.

Giraud, C., Manceau, S. and Treluyer, J. M. (2010) ABC transporters in human lymphocytes: Expression, activity and role, modulating factors and consequences for antiretroviral therapies. Expert Opinion on Drug Metabolism and Toxicology, 6(5), 571-589.

Giri, N., Shaik, N., Pan, G., Terasaki, T., Mukai, C., Kitagaki, S., Miyakoshi, N. and Elmquist, W. F. (2008) Investigation of the role of breast cancer resistance protein (Bcrp/Abcg2) on pharmacokinetics and central nervous system penetration of abacavir and zidovudine in the mouse. Drug Metabolism and Disposition, 36(8), 1476-1484.

Gisolf, E. H., Jurriaans, S., Pelgrom, J., Van Wanzeele, F., Van der Ende, M. E., Brinkman, K., Borst, M. J., De Wolf, F., Japour, A. J. and Danner, S. A. (2000) The effect of treatment intensification in HIV-infection: A study comparing treatment with ritonavir/saquinavir and ritonavir/saquinavir/stavudine. AIDS, 14(4), 405-413.

Goldmann, E. E. (1913) Vitalfarbung am zentralnervensystem. Abh Preuss Akad Wiss Phys- Math, 1(1), 1-60.

Goldsmith, D. R. and Perry, C. M. (2003) Atazanavir. Drugs, 63(16), 1679-1693.

Goodwin, B., Hodgson, E., D'Costa, D. J., Robertson, G. R. and Liddle, C. (2002) Transcriptional regulation of the human CYP3A4 gene by the constitutive androstane receptor. Molecular Pharmacology, 62(2), 359-365.

Goodwin, B., Hodgson, E. and Liddle, C. (1999) The orphan human pregnane X receptor mediates the transcriptional activation of CYP3A4 by rifampicin through a distal enhancer module. Molecular Pharmacology, 56(6), 1329-1339.

Goodwin, B., Moore, L. B., Stoltz, C. M., McKee, D. D. and Kliewer, S. A. (2001) Regulation of the human CYP2B6 gene by the nuclear pregnane X receptor. Molecular Pharmacology, 60(3), 427-431.

Goralski, K. B., Hartmann, G., Piquette-Miller, M. and Renton, K. W. (2003) Downregulation of mdr1a expression in the brain and liver during CNS inflammation alters the in vivo disposition of digoxin. British Journal of Pharmacology, 139(1), 35-48.

235

Gottesman, M. M. and Pastan, I. (1993) Biochemistry of multidrug resistance mediated by the multidrug transporter. Annual Review of Biochemistry, 62(385-427.

Greenwood, J., Heasman, S. J., Alvarez, J. I., Prat, A., Lyck, R. and Engelhardt, B. (2011) Review: Leucocyte-endothelial cell crosstalk at the blood-brain barrier: A prerequisite for successful immune cell entry to the brain. Neuropathology and Applied Neurobiology, 37(1), 24- 39.

Greiner, B., Eichelbaum, M., Fritz, P., Kreichgauer, H. P., Von Richter, O., Zundler, J. and Kroemer, H. K. (1999) The role of intestinal P-glycoprotein in the interaction of digoxin and rifampin. Journal of Clinical Investigation, 104(2), 147-153.

Gui, C., Miao, Y., Thompson, L., Wahlgren, B., Mock, M., Stieger, B. and Hagenbuch, B. (2008) Effect of pregnane X receptor ligands on transport mediated by human OATP1B1 and OATP1B3. European Journal of Pharmacology, 584(1), 57-65.

Guo, D., Sarkar, J., Suino-Powell, K., Xu, Y., Matsumoto, K., Jia, Y., Yu, S., Khare, S., Haldar, K., Rao, M. S., Foreman, J. E., Monga, S. P., Peters, J. M., Xu, H. E. and Reddy, J. K. (2007) Induction of nuclear translocation of constitutive androstane receptor by peroxisome proliferator- activated receptor alpha synthetic ligands in mouse liver. The Journal of Biological Chemistry, 282(50), 36766-36776.

Guo, G. L., Staudinger, J., Ogura, K. and Klaassen, C. D. (2002) Induction of rat organic anion transporting polypeptide 2 by pregnenolone-16α-carbonitrile Is via interaction with pregnane X receptor. Molecular Pharmacology, 61(4), 832-839.

Guo, Y., Kotova, E., Chen, Z. S., Lee, K., Hopper-Borge, E., Belinsky, M. G. and Kruh, G. D. (2003) MRP8, ATP-binding cassette C11 (ABCC11), is a cyclic nucleotide efflux pump and a resistance factor for fluoropyrimidines′,3′ 2 -dideoxycytidine and′ 9 -(2′- phosphonylmethoxyethyl)adenine. Journal of Biological Chemistry, 278(32), 29509-29514.

Gupta, A., Mugundu, G. M., Desai, P. B., Thummel, K. E. and Unadkat, J. D. (2008) Intestinal human colon adenocarcinoma cell line LS180 is an excellent model to study pregnane X receptor, but not constitutive androstane receptor, mediated CYP3A4 and multidrug resistance transporter 1 induction: Studies with anti-human immunodeficiency virus protease inhibitors. Drug Metabolism and Disposition, 36(6), 1172-1180.

Gupta, A., Zhang, Y., Unadkat, J. D. and Mao, Q. (2004) HIV protease inhibitors are inhibitors but not substrates of the human breast cancer resistance protein (BCRP/ABCG2). Journal of Pharmacology and Experimental Therapeutics, 310(1), 334-341.

Gupta, S., Knight, A. G., Losso, B. Y., Ingram, D. K., Keller, J. N. and Bruce-Keller, A. J. (2012) Brain injury caused by HIV protease inhibitors: role of lipodystrophy and insulin resistance. Antiviral Research, 95(1), 19-29.

Hagenbuch, B. and Meier, P. J. (2004) Organic anion transporting polypeptides of the OATP/SLC21 family: Phylogenetic classification as OATP/SLCO super-family, new

236

nomenclature and molecular/functional properties. Pflugers Archiv European Journal of Physiology, 447(5), 653-665.

Hamilton, N. B., Attwell, D. and Hall, C. N. (2010) Pericyte-mediated regulation of capillary diameter: A component of neurovascular coupling in health and disease. Front Neuroenergetics, 2(5), 1-14.

Hammarlund-Udenaes, M. (2010) Active-site concentrations of chemicals - Are they a better predictor of effect than plasma/organ/tissue concentrations? Basic and Clinical Pharmacology and Toxicology, 106(3), 215-220.

Hammarlund-Udenaes, M., Bredberg, U. and Friden, M. (2009) Methodologies to assess brain drug delivery in lead optimization. Current Topics in Medicinal Chemistry, 9(2), 148-162.

Hammer, S. M., Saag, M. S., Schechter, M., Montaner, J. S., Schooley, R. T., Jacobsen, D. M., Thompson, M. A., Carpenter, C. C., Fischl, M. A., Gazzard, B. G., Gatell, J. M., Hirsch, M. S., Katzenstein, D. A., Richman, D. D., Vella, S., Yeni, P. G. and Volberding, P. A. (2006) Treatment for adult HIV infection: 2006 recommendations of the International AIDS Society- USA panel. JAMA, 296(7), 827-843.

Handschin, C., Podvinec, M. and Meyer, U. A. (2000) CXR, a chicken xenobiotic-sensing orphan nuclear receptor, is related to both mammalian pregnane X receptor (PXR) and constitutive androstane receptor (CAR). Proceedings of the National Academy of Sciences of the United States of America, 97(20), 10769-10774.

Harati, R., Benech, H., Villegier, A. S. and Mabondzo, A. (2012) P-Glycoprotein, Breast Cancer Resistance Protein, Organic Anion Transporter 3, and Transporting Peptide 1a4 during Blood- Brain Barrier Maturation: Involvement of Wnt/beta-Catenin and Endothelin-1 Signaling. Molecular Pharmacology, 10(5), 1566-1580.

Hariparsad, N., Chu, X., Yabut, J., Labhart, P., Hartley, D. P., Dai, X. and Evers, R. (2009) Identification of pregnane-X receptor target genes and coactivator and corepressor binding to promoter elements in human hepatocytes. Nucleic Acids Research, 37(4), 1160-1173.

Hariparsad, N., Nallani, S. C., Sane, R. S., Buckley, D. J., Buckley, A. R. and Desai, P. B. (2004) Induction of CYP3A4 by efavirenz in primary human hepatocytes: Comparison with rifampin and phenobarbital. Journal of Clinical Pharmacology, 44(11), 1273-1281.

Harmsen, S., Meijerman, I., Beijnen, J. H. and Schellens, J. H. M. (2007) The role of nuclear receptors in pharmacokinetic drug-drug interactions in oncology. Cancer Treatment Reviews, 33(4), 369-380.

Hartmann, G., Cheung, A. K. Y. and Piquette-Miller, M. (2002) Inflammatory cytokines, but not bile acids, regulate expression of murine hepatic anion transporters in endotoxemia. Journal of Pharmacology and Experimental Therapeutics, 303(1), 273-281.

237

Hartz, A. M. S., Bauer, B., Block, M. L., Hong, J. S. and Miller, D. S. (2008) Diesel exhaust particles induce oxidative stress, proinflammatory signaling, and P-glycoprotein up-regulation at the blood-brain barrier. FASEB Journal, 22(8), 2723-2733.

Hartz, A. M. S., Bauer, B., Fricker, G. and Miller, D. S. (2004) Rapid regulation of P- glycoprotein at the blood-brain barrier by endothelin-1. Molecular Pharmacology, 66(3), 387- 394.

Hartz, A. M. S., Bauer, B., Fricker, G. and Miller, D. S. (2006) Rapid modulation of P- glycoprotein-mediated transport at the blood-brain barrier by tumor necrosis factor-α and lipopolysaccharide. Molecular Pharmacology, 69(2), 462-470.

Hawkins, B. T. and Davis, T. P. (2005) The blood-brain barrier/neurovascular unit in health and disease. Pharmacological Reviews, 57(2), 173-185.

Hawkins, B. T., Sykes, D. B. and Miller, D. S. (2010) Rapid, reversible modulation of blood- brain barrier P-glycoprotein transport activity by vascular endothelial growth factor. Journal of Neuroscience, 30(4), 1417-1425.

Hayashi, K., Pu, H., Andras, I. E., Eum, S. Y., Yamauchi, A., Hennig, B. and Toborek, M. (2006) HIV-TAT protein upregulates expression of multidrug resistance protein 1 in the blood- brain barrier. Journal of Cerebral Blood Flow and Metabolism, 26(8), 1052-1065.

Hayashi, K., Pu, H., Tian, J., Andras, I. E., Lee, Y. W., Hennig, B. and Toborek, M. (2005) HIV- Tat protein induces P-glycoprotein expression in brain microvascular endothelial cells. Journal of Neurochemistry, 93(5), 1231-1241.

Healan-Greenberg, C., Waring, J. F., Kempf, D. J., Blomme, E. A. G., Tirona, R. G. and Kim, R. B. (2008) A human immunodeficiency virus protease inhibitor is a novel functional inhibitor of human pregnane X receptor. Drug Metabolism and Disposition, 36(3), 500-507.

HealthCanada (2011) At a Glance - HIV and AIDS in Canada: Surveillance Report to December 31st, 2011. (C. f. C. D. a. I. Control ed.). Public Health Agency of Canada, Ottawa.

Helsen, C., Kerkhofs, S., Clinckemalie, L., Spans, L., Laurent, M., Boonen, S., Vanderschueren, D. and Claessens, F. (2012) Structural basis for nuclear hormone receptor DNA binding. Molecular and Cellular Endocrinology, 348(2), 411-417.

Hembury, A. and Mabondzo, A. (2008) Endothelin-1 reduces P-glycoprotein transport activity in an in vitro model of human adult blood-brain barrier. Cellular and Molecular Neurobiology, 28(7), 915-921.

Hennessy, M., Kelleher, D., Spiers, J. P., Barry, M., Kavanagh, P., Back, O., Mulcahy, F. and Feely, J. (2002) St. John's Wort increases expression of P-glycoprotein: Implications for drug interactions. British Journal of Clinical Pharmacology, 53(1), 75-82.

Higgins, C. F. (1992) ABC Transporters: From microorganisms to man. Annual Review of Cell Biology, 8(67-113.

238

Higgins, C. F., Callaghan, R., Linton, K. J., Rosenberg, M. F. and Ford, R. C. (1997) Structure of the multidrug resistance P-glycoprotein. Semin Cancer Biol, 8(3), 135-142.

Hilser, V. J. and Thompson, E. B. (2011) Structural dynamics, intrinsic disorder, and allostery in nuclear receptors as transcription factors. Journal of Biological Chemistry, 286(46), 39675- 39682.

Hodges, L. M., Markova, S. M., Chinn, L. W., Gow, J. M., Kroetz, D. L., Klein, T. E. and Altman, R. B. (2011) Very important pharmacogene summary: ABCB1 (MDR1, P-glycoprotein). Pharmacogenetics and Genomics, 21(3), 152-161.

Hoffmeyer, S., Burk, O., Von Richter, O., Arnold, H. P., Brockmöller, J., Johne, A., Cascorbi, I., Gerloff, T., Roots, I., Eichelbaum, M. and Brinkmann, U. (2000) Functional polymorphisms of the human multidrug-resistance gene: Multiple sequence variations and correlation of one allele with P- glycoprotein expression and activity in vivo. Proceedings of the National Academy of Sciences of the United States of America, 97(7), 3473-3478.

Honkakoski, P., Sueyoshi, T. and Negishi, M. (2003) Drug-activated nuclear receptors CAR and PXR. Annals of Medicine, 35(3), 172-182.

Honkakoski, P., Zelko, I., Sueyoshi, T. and Negishi, M. (1998) The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the CYP2B gene. Molecular and Cellular Biology, 18(10), 5652-5658.

Hoque, M. T. and Ishikawa, F. (2001) Human Chromatid Cohesin Component hRad21 Is Phosphorylated in M Phase and Associated with Metaphase Centromeres. Journal of Biological Chemistry, 276(7), 5059-5067.

Hoque, M. T., Robillard, K. R. and Bendayan, R. (2012) Regulation of Breast Cancer Resistant Protein (BCRP) by Peroxisome Proliferator-Activated Receptor Alpha (PPARalpha) in Human Brain Microvessel Endothelial Cells. Molecular Pharmacology, 81(4), 598-609.

Hosseinpour, F., Moore, R., Negishi, M. and Sueyoshi, T. (2006) Serine 202 regulates the nuclear translocation of constitutive active/androstane receptor. Molecular Pharmacology, 69(4), 1095-1102.

Hsu, A., Granneman, G. R. and Bertz, R. J. (1998) Ritonavir: Clinical pharmacokinetics and interactions with other anti-HIV agents. Clinical Pharmacokinetics, 35(4), 275-291.

Hsu, S. I., Cohen, D., Kirschner, L. S., Lothstein, L., Hartstein, M. and Horwitz, S. B. (1990) Structural analysis of the mouse mdr1a (P-glycoprotein) promoter reveals the basis for differential transcript heterogeneity in multidrug-resistant J774.2 cells. molecular and Cellular Biology, 10(7), 3596-3606.

Huang, L., Wring, S. A., Woolley, J. L., Brouwer, K. R., Serabjit-Singh, C. and Polli, J. W. (2001) Induction of P-glycoprotein and cytochrome P450 3a by HIV protease inhibitors. Drug Metabolism and Disposition, 29(5), 754-760.

239

Huang, W., Rha, G. B., Chen, L., Seelbach, M. J., Zhang, B., András, I. E., Bruemmer, D., Hennig, B. and Toborek, M. (2010) Inhibition of telomerase activity alters tight junction protein expression and induces transendothelial migration of HIV-1-infected cells. American Journal of Physiology - Heart and Circulatory Physiology, 298(4), H1136-H1145.

Huang, W., Sung, Y. E., András, I. E., Hennig, B. and Toborek, M. (2009) PPARα and PPARγ attenuate HIV-induced dysregulation of tight junction proteins by modulations of matrix metalloproteinase and proteasome activities. FASEB Journal, 23(5), 1596-1606.

Huang, W., Zhang, J., Chua, S. S., Qatanani, M., Han, Y., Granata, R. and Moore, D. D. (2003) Induction of bilirubin clearance by the constitutive androstane receptor (CAR). Proceedings of the National Academy of Sciences of the United States of America, 100(7), 4156-4161.

Huang, W., Zhang, J., Wei, P., Schrader, W. T. and Moore, D. D. (2004a) Meclizine is an agonist ligand for mouse constitutive androstane receptor (CAR) and an inverse agonist for human CAR. Molecular Endocrinology, 18(10), 2402-2408.

Huang, Y., Zurlinden, E., Lin, E., Li, X., Tokumoto, J., Golden, J., Murr, A., Engstrom, J. and Conte Jr, J. (2004b) Liquid chromatographic-tandem mass spectrometric assay for the simultaneous determination of didanosine and stavudine in human plasma, bronchoalveolar lavage fluid, alveolar cells, peripheral blood mononuclear cells, seminal plasma, cerebrospinal fluid and tonsil tissue. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 799(1), 51-61.

Huff, J. R. and Kahn, J. (2001) Discovery and clinical development of HIV-1 protease inhibitors. Advances in Protein Chemistry, 56(213-251.

Huisman, M. T., Smit, J. W., Crommentuyn, K. M. L., Zelcer, N., Wiltshire, H. R., Beijnen, J. H. and Schinkel, A. H. (2002) Multidrug resistance protein 2 (MRP2) transports HIV protease inhibitors, and transport can be enhanced by other drugs. AIDS, 16(17), 2295-2301.

Huss, J. M. and Kasper, C. B. (2000) Two-stage glucocorticoid induction of CYP3A23 through both the glucocorticoid and pregnane X receptors. Molecular Pharmacology, 58(1), 48-57.

Hustert, E., Zibat, A., Presecan-Siedel, E., Eiselt, R., Mueller, R., Fuß, C., Brehm, I., Brinkmann, U., Eichelbaum, M., Wojnowski, L. and Burk, O. (2001) Natural protein variants of pregnane X receptor with altered transactivation activity toward CYP3A4. Drug Metabolism and Disposition, 29(11), 1454-1459.

Hyafil, F., Vergely, C., Du Vignaud, P. and Grand-Perret, T. (1993) In vitro and in vivo reversal of multidrug resistance by GF120918, an acridonecarboxamide derivative. Cancer Research, 53(18), 4595-4602.

Igarashi, Y., Utsumi, H., Chiba, H., Yamada-Sasamori, Y., Tobioka, H., Kamimura, Y., Furuuchi, K., Kokai, Y., Nakagawa, T., Mori, M. and Sawada, N. (1999) Glial cell line-derived neurotrophic factor induces barrier function of endothelial cells forming the blood-brain barrier. Biochemical and Biophysical Research Communications, 261(1), 108-112.

240

Ikeda, S., Kurose, K., Jinno, H., Sai, K., Ozawa, S., Hasegawa, R., Komamura, K., Kotake, T., Morishita, H., Kamakura, S., Kitakaze, M., Tomoike, H., Tamura, T., Yamamoto, N., Kunitoh, H., Yamada, Y., Ohe, Y., Shimada, Y., Shirao, K., Kubota, K., Minami, H., Ohtsu, A., Yoshida, T., Saijo, N., Saito, Y. and Sawada, J. I. (2005) Functional analysis of four naturally occurring variants of human constitutive androstane receptor. Molecular Genetics and Metabolism, 86(1- 2), 314-319.

Ikeda, S., Tomoike, H., Nakajima, T., Matsumoto, K., Saito, H., Goto, Y., Kimura, H., Katoh, M., Sugai, K., Minami, N., Shirao, K., Kurose, K., Tamura, T., Yamamoto, N., Minami, H., Ohtsu, A., Yoshida, T., Saijo, N., Saito, Y., Sawada, J., Ozawa, S., Sai, K., Hasegawa, R., Komamura, K., Ueno, K., Kamakura, S. and Kitakaze, M. (2003) Twenty-six novel single nucleotide polymorphisms and their frequencies of the NR1I3 (CAR) gene in a Japanese population. Drug Metabolism and Pharmacokinetics, 18(6), 413-418.

Ingraham, H. A. and Redinbo, M. R. (2005) Orphan nuclear receptors adopted by crystallography. Current Opinion in Structural Biology, 15(6), 708-715.

Ito, K. I., Olsen, S. L., Qiu, W., Deeley, R. G. and Cole, S. P. C. (2001) Mutation of a Single Conserved Tryptophan in Multidrug Resistance Protein 1 (MRP1/ABCC1) Results in Loss of Drug Resistance and Selective Loss of Organic Anion Transport. Journal of Biological Chemistry, 276(19), 15616-15624.

Jackson, J. P., Ferguson, S. S., Moore, R., Negishi, M. and Goldstein, J. A. (2004) The constitutive active/androstane receptor regulates phenytoin induction of Cyp2c29. Molecular Pharmacology, 65(6), 1397-1404.

Jambou, R., Combes, V., Jambou, M. J., Weksler, B. B., Couraud, P. O. and Grau, G. E. (2010) Plasmodium falciparum adhesion on human brain microvascular endothelial cells involves transmigration-like cup formation and induces opening of intercellular junctions. PLoS Pathogens, 6(7), 1-13.

Janneh, O., Chandler, B., Hartkoorn, R., Kwan, W. S., Jenkinson, C., Evans, S., Back, D. J., Owen, A. and Khoo, S. H. (2009) Intracellular accumulation of efavirenz and nevirapine is independent of P-glycoprotein activity in cultured CD4 T cells and primary human lymphocytes. Journal of Antimicrobial Chemotherapy, 64(5).

Janneh, O., Jones, E., Chandler, B., Owen, A. and Khoo, S. H. (2007) Inhibition of P- glycoprotein and multidrug resistance-associated proteins modulates the intracellular concentration of lopinavir in cultured CD4 T cells and primary human lymphocytes. The Journal of antimicrobial chemotherapy.

Janneh, O., Owen, A., Chandler, B., Hartkoorn, R. C., Hart, C. A., Bray, P. G., Ward, S. A., Back, D. J. and Khoo, S. H. (2005) Modulation of the intracellular accumulation of saquinavir in peripheral blood mononuclear cells by inhibitors of MRP1, MRP2, P-gp and BCRP. AIDS, 19(18), 2097-2102.

241

Jetté, L., Murphy, G. F. and Béliveau, R. (1998) Drug binding to P-glycoprotein is inhibited in normal tissues following SDZ-PSC 833 treatment. International Journal of Cancer, 76(5), 729- 737.

Jigorel, E., Le Vee, M., Boursier-Neyret, C., Parmentier, Y. and Fardel, O. (2006) Differential regulation of sinusoidal and canalicular hepatic drug transporter expression by xenobiotics activating drug-sensing receptors in primary human hepatocytes. Drug Metabolism and Disposition, 34(10), 1756-1763.

Jodoin, J., Demeule, M., Fenart, L., Cecchelli, R., Farmer, S., Linton, K. J., Higgins, C. F. and Béliveau, R. (2003) P-glycoprotein in blood-brain barrier endothelial cells: Interaction and oligomerization with caveolins. Journal of Neurochemistry, 87(4), 1010-1023.

Johnson, D. R., Li, C. W., Chen, L. Y., Ghosh, J. C. and Chen, J. D. (2006) Regulation and binding of pregnane X receptor by nuclear receptor corepressor silencing mediator of retinoid and thyroid hormone receptors (SMRT). Molecular Pharmacology, 69(1), 99-108.

Jones, G. and Power, C. (2006) Regulation of neural cell survival by HIV-1 infection. Neurobiology of Disease, 21(1), 1-17.

Jones, K., Bray, P. G., Khoo, S. H., Davey, R. A., Meaden, E. R., Ward, S. A. and Back, D. J. (2001) P-glycoprotein and transporter MRP1 reduce HIV protease inhibitor uptake in CD4 cells: Potential for accelerated viral drug resistance? AIDS, 15(11), 1353-1358.

Jones, P. M. and George, A. M. (1998) A new structural model for P-glycoprotein. journal of Membrane Biology, 166(2), 133-147.

Jones, P. M. and George, A. M. (2004) The ABC transporter structure and mechanism: Perspectives on recent research. Cellular and Molecular Life Sciences, 61(6), 682-699.

Jones, S. A., Moore, L. B., Shenk, J. L., Wisely, G. B., Hamilton, G. A., McKee, D. D., Tomkinson, N. C. O., LeCluyse, E. L., Lambert, M. H., Willson, T. M., Kliewer, S. A. and Moore, J. T. (2000) The pregnane X receptor: A promiscuous xenobiotic receptor that has diverged during evolution. Molecular Endocrinology, 14(1), 27-39.

Jung, N., Lehmann, C., Rubbert, A., Knispel, M., Hartmann, P., Van Lunzen, J., Stellbrink, H. J., Faetkenheuer, G. and Taubert, D. (2008) Relevance of the organic cation transporters 1 and 2 for antiretroviral drug therapy in human immunodeficiency virus infection. Drug Metabolism and Disposition, 36(8), 1616-1623.

Jyrkkärinne, J., Windshügel, B., Mäkinen, J., Ylisirniö, M., Peräkylä, M., Poso, A., Sippl, W. and Honkakoski, P. (2005) Amino acids important for ligand specificity of the human constitutive androstane receptor. Journal of Biological Chemistry, 280(7), 5960-5971.

Kacem, K., Lacombe, P., Seylaz, J. and Bonvento, G. (1998) Structural organization of the perivascular astrocyte endfeet and their relationship with the endothelial glucose transporter: A confocal microscopy study. GLIA, 23(1), 1-10.

242

Kaddoumi, A., Choi, S. U., Kinman, L., Whittington, D., Tsai, C. C., Ho, R. J. Y., Anderson, B. D. and Unadkat, J. D. (2007) Inhibition of P-glycoprotein activity at the primate blood-brain barrier increases the distribution of nelfinavir into the brain but not into the cerebrospinal fluid. Drug Metabolism and Disposition, 35(9), 1459-1462.

Kageyama, M., Namiki, H., Fukushima, H., Terasaka, S., Togawa, T., Tanaka, A., Ito, Y., Shibata, N. and Takada, K. (2005) Effect of chronic administration of ritonavir on function of cytochrome P450 3A and P-glycoprotein in rats. Biological and Pharmaceutical Bulletin, 28(1), 130-137.

Kalvass, J. C., Maurer, T. S. and Pollack, G. M. (2007) Use of plasma and brain unbound fractions to assess the extent of brain distribution of 34 drugs: Comparison of unbound concentration ratios to in vivo P-glycoprotein efflux ratios. Drug Metabolism and Disposition, 35(4), 660-666.

Kamath, A. V. and Morris, M. E. (1998) Functional expression of P-glycoprotein in the hepatic canalicular membrane of developing rats. Journal of Pharmaceutical Sciences, 87(6), 785-786.

Kamiya, A., Inoue, Y. and Gonzalez, F. J. (2003) Role of the hepatocyte nuclear factor 4帢 in control of the pregnane X receptor during fetal liver development. Hepatology, 37(6), 1375- 1384.

Kamouchi, M., Ago, T. and Kitazono, T. (2011) Brain pericytes: Emerging concepts and functional roles in brain homeostasis. Cellular and Molecular Neurobiology, 31(2), 175-193.

Kanno, Y., Aoki, S., Mochizuki, M., Mori, E., Nakahama, T. and Inouye, Y. (2005) Expression of constitutive androstane receptor splice variants in rat liver and lung and their functional properties. Biological and Pharmaceutical Bulletin, 28(11), 2058-2062.

Karssen, A. M., Meijer, O. C., van der Sandt, I. C. J., De Boer, A. G., De Lange, E. C. M. and De Kloet, E. R. (2002) The role of the efflux transporter P-glycoprotein in brain penetration of prednisolone. Journal of Endocrinology, 175(1), 251-260.

Karssen, A. M., Meijer, O. C., Van Der Sandt, I. C. J., Lucassen, P. J., De Lange, E. C. M., De Boer, A. G. and De Kloet, E. R. (2001) Multidrug resistance P-glycoprotein hampers the access of cortisol but not of corticosterone to mouse and human brain. Endocrinology, 142(6), 2686- 2694.

Kassahun, K., McIntosh, I., Cui, D., Hreniuk, D., Merschman, S., Lasseter, K., Azrolan, N., Iwamoto, M., Wagner, J. A. and Wenning, L. A. (2007) Metabolism and disposition in humans of raltegravir (MK-0518), an anti-AIDS drug targeting the human immunodeficiency virus 1 integrase enzyme. Drug Metabolism and Disposition, 35(9), 1657-1663.

Kast, H. R., Goodwin, B., Tarr, P. T., Jones, S. A., Anisfeld, A. M., Stoltz, C. M., Tontonoz, P., Kliewer, S., Willson, T. M. and Edwards, P. A. (2002) Regulation of multidrug resistance- associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X- activated receptor, and constitutive androstane receptor. Journal of Biological Chemistry, 277(4), 2908-2915.

243

Kawamoto, T., Kakizaki, S., Yoshinari, K. and Negishi, M. (2000) Estrogen activation of the nuclear orphan receptor CAR (Constitutive Active Receptor) in induction of the mouse Cyp2b10 gene. Molecular Endocrinology, 14(11), 1897-1905.

Kawamoto, T., Sueyoshi, T., Zelko, I., Moore, R., Washburn, K. and Negishi, M. (1999) Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene. Molecular and Cellular Biology, 19(9), 6318-6322.

Kawana, K., Ikuta, T., Kobayashi, Y., Gotoh, O., Takeda, K. and Kawajiri, K. (2003) Molecular mechanism of nuclear translocation of an orphan nuclear receptor, SXR. Molecular Pharmacology, 63(3), 524-531.

Kawase, A., Yoshida, I., Tsunokuni, Y. and Iwaki, M. (2007) Decreased PXR and CAR inhibit transporter and CYP mRNA Levels in the liver and intestine of mice with collagen-induced arthritis. Xenobiotica, 37(4), 366-374.

Kedzierska, K. and Crowe, S. M. (2002) The role of monocytes and macrophages in the pathogenesis of HIV-1 infection. Current Medicinal Chemistry, 9(21), 1893-1903.

Kemper, E. M., Cleypool, C., Boogerd, W., Beijnen, J. H. and Van Tellingen, O. (2004a) The influence of the P-glycoprotein inhibitor zosuquidar trihydrochloride (LY335979) on the brain penetration of paclitaxel in mice. Cancer chemotherapy and pharmacology, 53(2), 173-178.

Kemper, E. M., Van Zandbergen, A. E., Cleypool, C., Mos, H. A., Boogerd, W., Beijnen, J. H. and Van Tellingen, O. (2003) Increased penetration of paclitaxel into the brain by inhibition of P- glycoprotein. Clinical Cancer Research, 9(7), 2849-2855.

Kemper, E. M., Verheij, M., Boogerd, W., Beijnen, J. H. and Van Tellingen, O. (2004b) Improved penetration of docetaxel into the brain by co-administration of inhibitors of P- glycoprotein. European Journal of Cancer, 40(8), 1269-1274.

Khan, A. A., Chow, E. C. Y., van Loenen-Weemaes, A. m. M. A., Porte, R. J., Pang, K. S. and Groothuis, G. M. M. (2009) Comparison of effects of VDR versus PXR, FXR and GR ligands on the regulation of CYP3A isozymes in rat and human intestine and liver. European Journal of Pharmaceutical Sciences, 37(2), 115-125.

Khandelwal, A., Krasowski, M. D., Reschly, E. J., Sinz, M. W., Swaan, P. W. and Ekins, S. (2008) Machine learning methods and docking for predicting human pregnane X receptor activation. Chemical Research in Toxicology, 21(7), 1457-1467.

Khorasanizadeh, S. and Rastinejad, F. (2001) Nuclear-receptor interactions on DNA-response elements. Trends in Biochemical Sciences, 26(6), 384-390.

Kim, H. J., Lee, S. K., Na, S. Y., Choi, H. S. and Jae Woon, L. (1998a) Molecular cloning of xSRC-3, a novel transcription coactivator from Xenopus, that is related to AIB1, p/CIP, and TIF2. Molecular Endocrinology, 12(7), 1038-1047.

244

Kim, J., Min, G. and Kemper, B. (2001) Chromatin Assembly Enhances Binding to the CYP2B1 Phenobarbital-responsive Unit (PBRU) of Nuclear Factor-1, which Binds Simultaneously with Constitutive Androstane Receptor (CAR)/Retinoid X Receptor (RXR) and Enhances CAR/RXR- mediated Activation of the PBRU. Journal of Biological Chemistry, 276(10), 7559-7567.

Kim, R. B., Fromm, M. F., Wandel, C., Leake, B., Wood, A. J. J., Roden, D. M. and Wilkinson, G. R. (1998b) The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV- 1 protease inhibitors. Journal of Clinical Investigation, 101(2), 289-294.

Kipp, H. and Arias, I. M. (2002) Trafficking of canalicular ABC transporters in hepatocytes. Annual Review of Physiology, 64(595-608.

Kis, O., Robillard, K., Chan, G. N. Y. and Bendayan, R. (2010a) The complexities of antiretroviral drug-drug interactions: role of ABC and SLC transporters. Trends in Pharmacological Sciences, 31(1), 22-35.

Kis, O., Zastre, J. A., Hoque, M. T., Walmsley, S. L. and Bendayan, R. (2013) Role of drug efflux and uptake transporters in atazanavir intestinal permeability and drug-drug interactions. Pharmaceutical Research, 30(4), 1050-1064.

Kis, O., Zastre, J. A., Ramaswamy, M. and Bendayan, R. (2010b) pH dependence of organic anion-transporting polypeptide 2B1 in Caco-2 cells: Potential role in antiretroviral drug oral bioavailability and drug-drug interactions. Journal of Pharmacology and Experimental Therapeutics, 334(3), 1009-1022.

Kitada, H., Miyata, M., Nakamura, T., Tozawa, A., Honma, W., Shimada, M., Nagata, K., Sinal, C. J., Guo, G. L., Gonzalez, F. J. and Yamazoe, Y. (2003) Protective role of hydroxysteroid sulfotransferase in lithocholic acid-induced liver toxicity. Journal of Biological Chemistry, 278(20), 17838-17844.

Kiuchi, Y., Suzuki, H., Hirohashi, T., Tyson, C. A. and Sugiyama, Y. (1998) cDNA cloning and inducible expression of human multidrug resistance associated protein 3 (MRP3). FEBS Letters, 433(1-2), 149-152.

Klaassen, C. D. and Aleksunes, L. M. (2010) Xenobiotic, bile acid, and cholesterol transporters: Function and regulation. Pharmacological Reviews, 62(1), 1-96.

Klaassen, C. D. and Slitt, A. L. (2005) Regulation of hepatic transporters by xenobiotic receptors. Current Drug Metabolism, 6(4), 309-328.

Klein-Hitpass, L., Tsai, S. Y., Greene, G. L., Clark, J. H., Tsai, M. J. and O'Malley, B. W. (1989) Specific binding of estrogen receptor to the estrogen response element. Molecular and Cellular Biology, 9(1), 43-49.

Kliewer, S. A., Moore, J. T., Wade, L., Staudinger, J. L., Watson, M. A., Jones, S. A., McKee, D. D., Oliver, B. B., Willson, T. M., Zetterstro?m, R. H., Perlmann, T. and Lehmann, J. M. (1998) An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell, 92(1), 73-82.

245

Kniesel, U. and Wolburg, H. (2000) Tight junctions of the blood-brain barrier. Cellular and Molecular Neurobiology, 20(1), 57-76.

Kobayashi, H., Magnoni, M. S., Govoni, S., Izumi, F., Wada, A. and Trabucchi, M. (1985) Neuronal control of brain microvessel function. Experientia, 41(4), 427-434.

Kobayashi, K., Sueyoshi, T., Inoue, K., Moore, R. and Negishi, M. (2003) Cytoplasmic Accumulation of the Nuclear Receptor CAR by a Tetratricopeptide Repeat Protein in HepG2 Cells. Molecular Pharmacology, 64(5), 1069-1075.

Kobayashi, K., Yamanaka, Y., Iwazaki, N., Nakajo, I., Hosokawa, M., Negishi, M. and Chiba, K. (2005) Identification of HMG-CoA reductase inhibitors as activators for human, mouse and rat constitutive androstane receptor. Drug Metabolism and Disposition, 33(7), 924-929.

Köck, K., Grube, M., Jedlitschky, G., Oevermann, L., Siegmund, W., Ritter, C. A. and Kroemer, H. K. (2007) Expression of adenosine triphosphate-binding cassette (ABC) drug transporters in peripheral blood cells: Relevance for physiology and pharmacotherapy. Clinical Pharmacokinetics, 46(6), 449-470.

Kodaira, H., Kusuhara, H., Fujita, T., Ushiki, J., Fuse, E. and Sugiyama, Y. (2011) Quantitative evaluation of the impact of active efflux by P-glycoprotein and breast cancer resistance protein at the blood-brain barrier on the predictability of the unbound concentrations of drugs in the brain using cerebrospinal fluid concentration as a surrogate. Journal of Pharmacology and Experimental Therapeutics, 339(3), 935-944.

Kodama, S., Koike, C., Negishi, M. and Yamamoto, Y. (2004) Nuclear receptors CAR and PXR cross talk with FOXO1 to regulate genes that encode drug-metabolizing and gluconeogenic enzymes. Molecular and Cellular Biology, 24(18), 7931-7940.

Koenig, S., Gendelman, H. E., Orenstein, J. M., Dal Canto, M. C., Pezeshkpour, G. H., Yungbluth, M., Janotta, F., Aksamit, A., Martin, M. A. and Fauci, A. S. (1986) Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encephalopathy. Science, 233(4768), 1089-1093.

Kofuji, P. and Newman, E. A. (2004) Potassium buffering in the central nervous system. Neuroscience, 129(4), 1045-1056.

Konig, S. K., Herzog, M., Theile, D., Zembruski, N., Haefeli, W. E. and Weiss, J. (2010) Impact of drug transporters on cellular resistance towards saquinavir and darunavir. J Antimicrob Chemother, 65(11), 2319-2328.

Kooijmans, S. A. A., Senyschyn, D., Mezhiselvam, M. M., Morizzi, J., Charman, S. A., Weksler, B., Romero, I. A., Couraud, P. O. and Nicolazzo, J. A. (2012) The involvement of a Na +- and Cl --dependent transporter in the brain uptake of amantadine and rimantadine. Molecular Pharmaceutics, 9(4), 883-893.

246

Krajcsi, P., Jani, M., Tóth, B., Erdo, F., Kis, E., Beéry, E. and Sziráki, I. (2012) Efflux transporters in the bloodbrain interfaces in vitro and in vivo methods and correlations. Expert Opinion on Drug Metabolism and Toxicology, 8(4), 419-431.

Kramer-Hämmerle, S., Rothenaigner, I., Wolff, H., Bell, J. E. and Brack-Werner, R. (2005) Cells of the central nervous system as targets and reservoirs of the human immunodeficiency virus. Virus Research, 111(2 SPEC. ISS.), 194-213.

Kravcik, S., Gallicano, K., Roth, V., Cassol, S., Hawley-Foss, N., Badley, A. and Cameron, D. W. (1999) Cerebrospinal fluid HIV RNA and drug levels with combination ritonavir and saquinavir. Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology, 21(5), 371-375.

Kubota, H., Ishihara, H., Langmann, T., Schmitz, G., Stieger, B., Wieser, H. G., Yonekawa, Y. and Frei, K. (2006) Distribution and functional activity of P-glycoprotein and multidrug resistance-associated proteins in human brain microvascular endothelial cells in hippocampal sclerosis. Epilepsy Research, 68(3), 213-228.

Kuntner, C., Bankstahl, J. P., Bankstahl, M., Stanek, J., Wanek, T., Stundner, G., Karch, R., Brauner, R., Meier, M., Ding, X., Müller, M., Löscher, W. and Langer, O. (2010) Dose-response assessment of tariquidar and elacridar and regional quantification of P-glycoprotein inhibition at the rat blood-brain barrier using (R)-[11C]verapamil PET. European Journal of Nuclear Medicine and Molecular Imaging, 37(5), 942-953.

Kusuhara, H., Suzuki, H., Terasaki, T., Kakee, A., Lemaire, M. and Sugiyama, Y. (1997) P- glycoprotein mediates the efflux of quinidine across the blood-brain barrier. Journal of Pharmacology and Experimental Therapeutics, 283(2), 574-580.

Kusunoki, N., Takara, K., Tanigawara, Y., Yamauchi, A., Ueda, K., Komada, F., Ku, Y., Kuroda, Y., Saitoh, Y. and Okumura, K. (1998) Inhibitory effects of a cyclosporin derivative, SDZ PSC 833, on transport of doxorubicin and vinblastine via human P-glycoprotein. Japanese Journal of Cancer Research, 89(11), 1220-1228.

Labialle, S., Dayan, G., Gayet, L., Rigal, D., Gambrelle, J. and Baggetto, L. G. (2004) New invMED1 element cis-activates human multidrug-related MDR1 and MVP genes, involving the LRP130 protein. Nucleic Acids Res, 32(13), 3864-3876.

Labialle, S., Gayet, L., Marthinet, E., Rigal, D. and Baggetto, L. G. (2002) Transcriptional regulators of the human multidrug resistance 1 gene: Recent views. Biochemical Pharmacology, 64(5-6), 943-948.

Lamba, J. K., Lamba, V., Yasuda, K., Lin, Y. S., Assem, M., Thompson, E., Strom, S. and Schuetz, E. G. (2004a) Expression of constitutive androstane receptor splice variants in human tissues and their functional consequences. Journal of Pharmacology and Experimental Therapeutics, 311(2), 811-821.

Lamba, V., Yasuda, K., Lamba, J. K., Assem, M., Davila, J., Strom, S. and Schuetz, E. G. (2004b) PXR (NR1I2): Splice variants in human tissues, including brain, and identification of

247

neurosteroids and nicotine as PXR activators. Toxicology and Applied Pharmacology, 199(3), 251-265.

Lampidis, T. J., Castello, C., del Giglio, A., Pressman, B. C., Viallet, P., Trevorrow, K. W., Valet, G. K., Tapiero, H. and Savaraj, N. (1989) Relevance of the chemical charge of rhodamine dyes to multiple drug resistance. Biochemical Pharmacology, 38(23), 4267-4271.

Langford, D., Grigorian, A., Hurford, R., Adame, A., Ellis, R. J., Hansen, L. and Masliah, E. (2004) Altered P-glycoprotein expression in AIDS patients with HIV encephalitis. Journal of Neuropathology and Experimental Neurology, 63(10), 1038-1047.

Langford, D., Marquie-Beck, J., de Almeida, S., Lazzaretto, D., Letendre, S., Grant, I., McCutchan, J. A., Masliah, E. and Ellis, R. J. (2006) Relationship of antiretroviral treatment to postmortem brain tissue viral load in human immunodeficiency virus-infected patients. Journal of NeuroVirology, 12(2), 100-107.

Lee, C. A., Cook, J. A., Reyner, E. L. and Smith, D. A. (2010) P-glycoprotein related drug interactions: clinical importance and a consideration of disease states. Expert Opinion on Drug Metabolism & Toxicology, 6(5), 603-619.

Lee, C. G. L., Gottesman, M. M., Cardarelli, C. O., Ramachandra, M., Jeang, K. T., Ambudkar, S. V., Pastan, I. and Dey, S. (1998) HIV-1 protease inhibitors are substrates for the MDR1 multidrug transporter. Biochemistry, 37(11), 3594-3601.

Lee, C. H., Ito, Y., Yanagiba, Y., Yamanoshita, O., Kim, H., Zhang, S. Y., Kamijima, M., Gonzalez, F. J. and Nakajima, T. (2007a) Pyrene-induced CYP1A2 and SULT1A1 may be regulated by CAR and not by AhR. Toxicology, 238(2-3), 147-156.

Lee, G., Babakhanian, K., Ramaswamy, M., Prat, A., Wosik, K. and Bendayan, R. (2007b) Expression of the ATP-binding cassette membrane transporter, ABCG2, in human and rodent brain microvessel endothelial and glial cell culture systems. Pharmaceutical Research, 24(7), 1262-1274.

Lee, G., Bendayan, M. and Bendayan, R. (2001a) Functional expression of P-glycoprotein (P-gp) in rat brain microglia. Clinical Pharmacology and Therapeutics, 69(2).

Lee, G. and Bendayan, R. (2004) Functional expression and localization of P-glycoprotein in the central nervous system: Relevance to the pathogenesis and treatment of neurological disorders. Pharmaceutical Research, 21(8), 1313-1330.

Lee, G., Dallas, S., Hong, M. and Bendayan, R. (2001b) Drug transporters in the central nervous system: Brain barriers and brain parenchyma considerations. Pharmacological Reviews, 53(4), 569-596.

Lee, G., Schlichter, L., Bendayan, M. and Bendayan, R. (2001c) Functional expression of p- glycoprotein in rat brain microglia. Journal of Pharmacology and Experimental Therapeutics, 299(1), 204-212.

248

Lee, M. S., Kliewer, S. A., Provencal, J., Wright, P. E. and Evans, R. M. (1993) Structure of the retinoid X receptor α DNA binding domain: A helix required for homodimeric DNA binding. Science, 260(5111), 1117-1121.

Lee, S. W., Kim, W. J., Choi, Y. K., Song, H. S., Son, M. J., Gelman, I. H., Kim, Y. J. and Kim, K. W. (2003) SSeCKS regulates angiogenesis and tight junction formation in blood-brain barrier. Nature Medicine, 9(7), 900-906.

Lehmann, J. M., McKee, D. D., Watson, M. A., Willson, T. M., Moore, J. T. and Kliewer, S. A. (1998) The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. Journal of Clinical Investigation, 102(5), 1016-1023.

Lemaire, G., Benod, C., Nahoum, V., Pillon, A., Boussioux, A. M., Guichou, J. F., Subra, G., Pascussi, J. M., Bourguet, W., Chavanieu, A. and Balaguer, P. (2007) Discovery of a highly active ligand of human pregnane X receptor: A case study from pharmacophore modeling and virtual screening to "in vivo" biological activity. Molecular Pharmacology, 72(3), 572-581.

Lemmen, J., Tozakidis, I. E. P., Bele, P. and Galla, H. J. (2013) Constitutive androstane receptor upregulates Abcb1 and Abcg2 at the blood-brain barrier after CITCO activation. Brain Research, 21(1501), 68-80.

Leslie, E. M., Deeley, R. G. and Cole, S. P. (2005) Multidrug resistance proteins: role of P- glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense. Toxicology and Applied Pharmacology, 204(3), 216-237.

Letendre, S. (2011) Central nervous system complications in HIV disease: HIV-associated neurocognitive disorder. Topics in Antiviral Medicine, 19(4), 137-142.

Letendre, S., Ellis, R. J., Best, B., Bhatt, A., Marquie-Beck, J., LeBlanc, S., Rossi, S., Capparelli, E. and Allen McCutchan, J. (2009) Penetration and effectiveness of antiretroviral therapy in the central nervous system. Anti-Inflammatory and Anti-Allergy Agents in Medicinal Chemistry, 8(2), 169-183.

Letendre, S., Marquie-Beck, J., Capparelli, E., Best, B., Clifford, D., Collier, A. C., Gelman, B. B., McArthur, J. C., McCutchan, J. A., Morgello, S., Simpson, D., Grant, I. and Ellis, R. J. (2008) Validation of the CNS penetration-effectiveness rank for quantifying antiretroviral penetration into the central nervous system. Archives of Neurology, 65(1), 65-70.

Letendre, S. L., McCutchan, J. A., Childers, M. E., Woods, S. P., Lazzaretto, D., Heaton, R. K., Grant, I. and Ellis, R. J. (2004) Enhancing antiretroviral therapy for human immunodeficiency virus cognitive disorders. Annals of Neurology, 56(3), 416-423.

Lewandowsky, M. (1900) Zur lehre von der cerebrospinalflüssigkeit. Z Klin Med, 40(480-494.

Leybaert, L. (2005) Neurobarrier coupling in the brain: A partner of neurovascular and neurometabolic coupling? Journal of Cerebral Blood Flow and Metabolism, 25(1), 2-16.

249

Lhommé, C., Joly, F., Walker, J. L., Lissoni, A. A., Nicoletto, M. O., Manikhas, G. M., Baekelandt, M. M. O., Gordon, A. N., Fracasso, P. M., Mietlowski, W. L., Jones, G. J. and Dugan, M. H. (2008) Phase III study of valspodar (PSC 833) combined with paclitaxel and carboplatin compared with paclitaxel and carboplatin alone in patients with stage IV or suboptimally debulked stage III epithelial ovarian cancer or primary peritoneal cancer. Journal of Clinical Oncology, 26(16), 2674-2682.

Li, H., Chen, T., Cottrell, J. and Wang, H. (2009) Nuclear translocation of adenoviral-enhanced yellow fluorescent protein-tagged-human constitutive androstane receptor (hCAR): A novel tool for screening hcar activators in human primary hepatocytes. Drug Metabolism and Disposition, 37(5), 1098-1106.

Li, H. and Wang, H. (2010) Activation of xenobiotic receptors: Driving into the nucleus. Expert Opinion on Drug Metabolism and Toxicology, 6(4), 409-426.

Li, L., Xu, J., Min, T. and Huang, W. (2006) Up-regulation of P-glycoprotein expression by catalase via JNK activation in HepG2 cells. Redox Report, 11(4), 173-178.

Li, T. and Chiang, J. Y. L. (2006) Rifampicin induction of CYP3A4 requires pregnane X receptor cross talk with hepatocyte nuclear factor 4α and coactivators, and suppression of small heterodimer partner gene expression. Drug Metabolism and Disposition, 34(5), 756-764.

Liebner, S., Corada, M., Bangsow, T., Babbage, J., Taddei, A., Czupalla, C. J., Reis, M., Felici, A., Wolburg, H., Fruttiger, M., Taketo, M. M., Von Melchner, H., Plate, K. H., Gerhardt, H. and Dejana, E. (2008) Wnt/β-catenin signaling controls development of the blood - brain barrier. Journal of Cell Biology, 183(3), 409-417.

Lim, J. C., Kania, K. D., Wijesuriya, H., Chawla, S., Sethi, J. K., Pulaski, L., Romero, I. A., Couraud, P. O., Weksler, B. B., Hladky, S. B. and Barrand, M. A. (2008) Activation of β-catenin signalling by GSK-3 inhibition increases p-glycoprotein expression in brain endothelial cells. Journal of Neurochemistry, 106(4), 1855-1865.

Lim, J. C., Mickute, Z., Zaman, M., Hopkins, S., Wijesuriya, H., Steckler, T., Moechars, D., Van Leuven, F., Sarnyai, Z., Hladky, S. B. and Barrand, M. A. (2009) Decreased expression of multidrug efflux transporters in the brains of GSK-3β transgenic mice. Brain Research, 1276(1- 10.

Lin, J. H. (2008) CSF as a surrogate for assessing CNS exposure: An industrial perspective. Current Drug Metabolism, 9(1), 46-59.

Litman, T., Zeuthen, T., Skovsgaard, T. and Stein, W. D. (1997) Competitive, non-competitive and cooperative interactions between substrates of P-glycoproteins as measured by ATPase activity. Biochimica et Biophysica Acta - Molecular Basis of Disease, 1361(2), 169-176.

Liu, N. Q., Lossinsky, A. S., Popik, W., Li, X., Gujuluva, C., Kriederman, B., Roberts, J., Pushkarsky, T., Bukrinsky, M., Witte, M., Weinand, M. and Fiala, M. (2002) Human immunodeficiency virus type 1 enters brain microvascular endothelia by macropinocytosis

250

dependent on lipid rafts and the mitogen-activated protein kinase signaling pathway. Journal of Virology, 76(13), 6689-6700.

Liu, X., Van Natta, K., Yeo, H., Vilenski, O., Weller, P. E., Worboys, P. D. and Monshouwer, M. (2009) Unbound drug concentration in brain homogenate and cerebral spinal fluid at steady state as a surrogate for unbound concentration in brain interstitial fluid. Drug Metabolism and Disposition, 37(4), 787-793.

Lombardo, L., Pellitteri, R., Balazy, M. and Cardile, V. (2008) Induction of nuclear receptors and drug resistance in the brain microvascular endothelial cells treated with antiepileptic drugs. Current Neurovascular Research, 5(2), 82-92.

Loo, T. W. and Clarke, D. M. (1996) Mutational analysis of the predicted first transmembrane segment of each homologous half of human P-glycoprotein suggests that they are symmetrically arranged in the membrane. Journal of Biological Chemistry, 271(26), 15414-15419.

Loo, T. W. and Clarke, D. M. (2005) Recent progress in understanding the mechanism of P- glycoprotein-mediated drug efflux. Journal of Membrane Biology, 206(3), 173-185.

Lopez-Ramirez, M. A., Fischer, R., Torres-Badillo, C. C., Davies, H. A., Logan, K., Pfizenmaier, K., Male, D. K., Sharrack, B. and Romero, I. A. (2012) Role of caspases in cytokine-induced barrier breakdown in human brain endothelial cells. Journal of Immunology, 189(6), 3130-3139.

Loscher, W. and Langer, O. (2010) Imaging of P-glycoprotein function and expression to elucidate mechanisms of pharmacoresistance in epilepsy. Current Topics in Medicinal Chemistry, 10(17), 1785-1791.

Löscher, W. and Potschka, H. (2005a) Blood-brain barrier active efflux transporters: ATP- binding cassette gene family. NeuroRx, 2(1), 86-98.

Löscher, W. and Potschka, H. (2005b) Role of drug efflux transporters in the brain for drug disposition and treatment of brain diseases. Progress in Neurobiology, 76(1), 22-76.

Lostao, M. P., Mata, J. F., Larrayoz, I. M., Inzillo, S. M., Casado, F. J. and Pastor-Anglada, M. (2000) Electrogenic uptake of nucleosides and nucleoside-derived drugs by the human nucleoside transporter 1 (hCNT1) expressed in Xenopus laevis oocytes. FEBS Letters, 481(2), 137-140.

Lötsch, J., Schmidt, R., Vetter, G., Schmidt, H., Niederberger, E., Geisslinger, G. and Tegeder, I. (2002) Increased CNS uptake and enhanced antinociception of morphine-6-glucuronide in rats after inhibition of P-glycoprotein. Journal of Neurochemistry, 83(2), 241-248.

Love, G. D., Grosjean, E., Stalvies, C., Fike, D. A., Grotzinger, J. P., Bradley, A. S., Kelly, A. E., Bhatia, M., Meredith, W., Snape, C. E., Bowring, S. A., Condon, D. J. and Summons, R. E. (2009) Fossil steroids record the appearance of Demospongiae during the Cryogenian period. Nature, 457(7230), 718-721.

251

Lowe, S. H., Sankatsing, S. U. C., Repping, S., Van Der Veen, F., Reiss, P., Lange, J. M. A. and Prins, J. M. (2004) Is the male genital tract really a sanctuary site for HIV? Arguments that it is not. AIDS, 18(10), 1353-1362.

Lucia, M. B., Golotta, C., Rutella, S., Rastrelli, E., Savarino, A. and Cauda, R. (2005) Atazanavir inhibits P-glycoprotein and multidrug resistance-associated protein efflux activity. Journal of Acquired Immune Deficiency Syndromes, 39(5), 635-637.

Luissint, A. C., Federici, C., Guillonneau, F., Chrétien, F., Camoin, L., Glacial, F., Ganeshamoorthy, K. and Couraud, P. O. (2012) Guanine nucleotide-binding protein Gαi2: A new partner of claudin-5 that regulates tight junction integrity in human brain endothelial cells. Journal of Cerebral Blood Flow and Metabolism, 32(5), 860-873.

Ma, X., Idle, J. R. and Gonzalez, F. J. (2008) The pregnane X receptor: From bench to bedside. Expert Opinion on Drug Metabolism and Toxicology, 4(7), 895-908.

Mader, S., Chen, J. Y., Chen, Z., White, J., Chambon, P. and Gronemeyer, H. (1993) The patterns of binding of RAR, RXR and TR homo- And heterodimers to direct repeats are dictated by the binding specificities of the DNA binding domains. EMBO Journal, 12(13), 5029-5041.

Maglich, J. M., Parks, D. J., Moore, L. B., Collins, J. L., Goodwin, B., Billin, A. N., Stoltz, C. A., Kliewer, S. A., Lambert, M. H., Willson, T. M. and Moore, J. T. (2003) Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes. Journal of Biological Chemistry, 278(19), 17277-17283.

Maglich, J. M., Stoltz, C. M., Goodwin, B., Hawkins-Brown, D., Moore, J. T. and Kliewer, S. A. (2002) Nuclear pregnane X receptor and constitutive androstane receptor regulate overlapping but distinct sets of genes involved in xenobiotic detoxification. Molecular Pharmacology, 62(3), 638-646.

Mäkinen, J., Reinisalo, M., Niemi, K., Viitala, P., Jyrkkärinne, J., Chung, H., Pelkonen, O. and Honkakoski, P. (2003) Dual action of oestrogens on the mouse constitutive androstane receptor. Biochemical Journal, 376(2), 465-472.

Malaplate-Armand, C., Ferrari, L., Masson, C., Visvikis-Siest, S., Lambert, H. and Batt, A. M. (2005) Down-regulation of astroglial CYP2C, glucocorticoid receptor and constitutive androstane receptor genes in response to cocaine in human U373 MG astrocytoma cells. Toxicology Letters, 159(3), 203-211.

Malingré, M. M., Beijnen, J. H., Rosing, H., Koopman, F. J., Jewell, R. C., Paul, E. M., Huinink, W. W. T. and Schellens, J. H. M. (2001) Co-administration of GF120918 significantly increases the systemic exposure to oral paclitaxel in cancer patients. British Journal of Cancer, 84(1), 42- 47.

Mallants, R., Van Oosterwyck, K., Van Vaeck, L., Mols, R., De Clercq, E. and Augustijns, P. (2005) Multidrug resistance-associated protein 2 (MRP2) affects hepatobiliary elimination but not the intestinal disposition of tenofovir disoproxil fumarate and its metabolites. Xenobiotica, 35(10-11), 1055-1066.

252

Mamidi, A., DeSimone, J. A. and Pomerantz, R. J. (2002) Central nervous system infections in individuals with HIV-1 infection. Journal of NeuroVirology, 8(3), 158-167.

Mangelsdorf, D. J., Umesono, K., Kliewer, S. A., Borgmeyer, U., Ong, E. S. and Evans, R. M. (1991) A direct repeat in the cellular retinol-binding protein type II gene confers differential regulation by RXR and RAR. Cell, 66(3), 555-561.

Marc, N., Galisteo, M., Lagadic-Gossmann, D., Fautrel, A., Joannard, F., Guillouzo, A. and Corcos, L. (2000) Regulation of phenobarbital induction of the cytochrome P450 269/10 genes in primary mouse hepatocyte culture: Involvement of calcium- and cAMP- dependent pathways. European Journal of Biochemistry, 267(4), 963-970.

Marchi, N., Guiso, G., Caccia, S., Rizzi, M., Gagliardi, B., Noé, F., Ravizza, T., Bassanini, S., Chimenti, S., Battaglia, G. and Vezzani, A. (2006) Determinants of drug brain uptake in a rat model of seizure-associated malformations of cortical development. Neurobiology of Disease, 24(3), 429-442.

Margolis, D. M. and Hazuda, D. J. (2013) Combined approaches for HIV cure. Current Opinion in HIV and AIDS, 8(3), 230-235.

Masereeuw, R., Terlouw, S. A., Van Aubel, R. A. M. H., Russel, F. G. M. and Miller, D. S. (2000) Endothelin B receptor-mediated regulation of ATP-driven drug secretion in renal proximal tubule. Molecular Pharmacology, 57(1), 59-67.

Masuyama, H., Hiramatsu, Y., Mizutani, Y., Inoshita, H. and Kudo, T. (2001) The expression of pregnane X receptor and its target gene, cytochrome P450 3A1, in perinatal mouse. Molecular and Cellular Endocrinology, 172(1-2), 47-56.

Masuyama, H., Suwaki, N., Tateishi, Y., Nakatsukasa, H., Segawa, T. and Hiramatsu, Y. (2005) The pregnane X receptor regulates gene expression in a ligand- and promoter-selective fashion. Molecular Endocrinology, 19(5), 1170-1180.

Mathieu, M. C., Lapierre, I., Brault, K. and Raymond, M. (2001) Aromatic Hydrocarbon Receptor (AhR)·AhR Nuclear Translocator- and p53-mediated Induction of the Murine Multidrug Resistance mdr1 Gene by 3-Methylcholanthrene and Benzo(a)pyrene in Hepatoma Cells. Journal of Biological Chemistry, 276(7), 4819-4827.

Mathiisen, T. M., Lehre, K. P., Danbolt, N. C. and Ottersen, O. P. (2010) The perivascular astroglial sheath provides a complete covering of the brain microvessels: An electron microscopic 3D reconstruction. GLIA, 58(9), 1094-1103.

McCaffrey, G., Staatz, W. D., Sanchez-Covarrubias, L., Finch, J. D., Demarco, K., Laracuente, M. L., Ronaldson, P. T. and Davis, T. P. (2012) P-glycoprotein trafficking at the blood-brain barrier altered by peripheral inflammatory hyperalgesia. Journal of Neurochemistry, 122(5), 962- 975.

McDowell, J. A., Chittick, G. E., Ravitch, J. R., Polk, R. E., Kerkering, T. M. and Stein, D. S. (1999) Pharmacokinetics of [ 14C]abacavir, a human immunodeficiency virus type 1 (HIV-1)

253

reverse transcriptase inhibitor, administered in a single oral dose to HIV-1-infected adults: A mass balance study. Antimicrobial Agents and Chemotherapy, 43(12), 2855-2861.

Meaden, E. R., Hoggard, P. G., Newton, P., Tjia, J. F., Aldam, D., Cornforth, D., Lloyd, J., Williams, I., Back, D. J. and Khoo, S. H. (2002) P-glycoprotein and MRP1 expression and reduced ritonavir and saquinavir accumulation in HIV-infected individuals. Journal of Antimicrobial Chemotherapy, 50(4), 583-588.

Meijer, O. C., Karssen, A. M. and de Kloet, E. R. (2003) Cell- and tissue-specific effects of corticosteroids in relation to glucocorticoid resistance: Examples from the brain. Journal of Endocrinology, 178(1), 13-18.

Messiaen, P., Wensing, A. M. J., Fun, A., Nijhuis, M., Brusselaers, N. and Vandekerckhove, L. (2013) Clinical Use of HIV Integrase Inhibitors: A Systematic Review and Meta-Analysis. PLoS ONE, 8(1).

Meyer Zu Schwabedissen, H. E., Tirona, R. G., Yip, C. S., Ho, R. H. and Kim, R. B. (2008) Interplay between the nuclear receptor pregnane X receptor and the uptake transporter organic anion transporter polypeptide 1A2 selectively enhances estrogen effects in breast cancer. Cancer Research, 68(22), 9338-9347.

Michaud, V., Bar-Magen, T., Turgeon, J., Flockhart, D., Desta, Z. and Wainberg, M. A. (2012) The dual role of pharmacogenetics in HIV treatment: Mutations and polymorphisms regulating antiretroviral drug resistance and disposition. Pharmacological Reviews, 64(3), 803-833.

Miki, Y., Suzuki, T., Tazawa, C., Blumberg, B. and Sasano, H. (2005) Steroid and xenobiotic receptor (SXR), cytochrome P450 3A4 and multidrug resistance gene 1 in human adult and fetal tissues. Molecular and Cellular Endocrinology, 231(1-2), 75-85.

Miller, D. S. (2010) Regulation of P-glycoprotein and other ABC drug transporters at the blood- brain barrier. Trends in Pharmacological Sciences, 31(6), 246-254.

Miller, D. S., Bauer, B. and Hartz, A. M. S. (2008) Modulation of P-glycoprotein at the blood- brain barrier: Opportunities to improve central nervous system pharmacotherapy. Pharmacological Reviews, 60(2), 196-209.

Miller, D. S., Graeff, C., Droulle, L., Fricker, S. and Fricker, G. (2002) Xenobiotic efflux pumps in isolated fish brain capillaries. American Journal of Physiology - Regulatory Integrative and Comparative Physiology, 282(1 51-1), R191-R198.

Miller, D. S., Nobmann, S. N., Gutmann, H., Toeroek, M., Drewe, J. and Fricker, G. (2000) Xenobiotic transport across isolated brain microvessels studied by confocal microscopy. Molecular Pharmacology, 58(6), 1357-1367.

Miller, F., Afonso, P. V., Gessain, A. and Ceccaldi, P. E. (2012) Blood-brain barrier and retroviral infections. Virulence, 3(2), 222-229.

254

Miller, J., McLachlan, A. D. and Klug, A. (1985) Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO Journal, 4(6), 1609-1614.

Min, G., Kim, H., Bae, Y., Petz, L. and Kemper, J. K. (2002a) Inhibitory cross-talk between estrogen receptor (ER) and constitutively activated androstane receptor (CAR): CAR inhibits ER-mediated signaling pathway by squelching p160 coactivators. Journal of Biological Chemistry, 277(37), 34626-34633.

Min, G., Kim Kemper, J. and Kemper, B. (2002b) Glucocorticoid receptor-interacting protein 1 mediates ligand-independent nuclear translocation and activation of constitutive androstane receptor in vivo. Journal of Biological Chemistry, 277(29), 26356-26363.

Mistry, P., Stewart, A. J., Dangerfield, W., Okiji, S., Liddle, C., Bootle, D., Plumb, J. A., Templeton, D. and Charlton, P. (2001) In vitro and in vivo reversal of P-glycoprotein-mediated multidrug resistance by a novel potent modulator, XR9576. Cancer Research, 61(2), 749-758.

Moitra, K. and Dean, M. (2011) Evolution of ABC transporters by gene duplication and their role in human disease. Biological Chemistry, 392(1-2), 29-37.

Molinari, A., Calcabrini, A., Meschini, S., Stringaro, A., Crateri, P., Toccacieli, L., Marra, M., Colone, M., Cianfriglia, M. and Arancia, G. (2002) Subcellular detection and localization of the drug transporter P-glycoprotein in cultured tumor cells. Current Protein and Peptide Science, 3(6), 653-670.

Molinari, A., Cianfriglia, M., Meschini, S., Calcabrini, A. and Arancia, G. (1994) P-glycoprotein expression in the Golgi apparatus of multidrug-resistant cells. International Journal of Cancer, 59(6), 789-795.

Moore, D. D., Kato, S., Xie, W., Mangelsdorf, D. J., Schmidt, D. R., Xiao, R. and Kliewer, S. A. (2006a) International union of pharmacology. LXII. The NR1H and NR1I receptors: Constitutive androstane receptor, pregnene X receptor, farnesoid X receptor α, farnesoid X receptor β, liver X receptor α, liver X receptor β, and vitamin D receptor. Pharmacological Reviews, 58(4), 742- 759.

Moore, J. P., Trkola, A. and Dragic, T. (1997) Co-receptors for HIV-1 entry. Current Opinion in Immunology, 9(4), 551-562.

Moore, J. T., Collins, J. L. and Pearce, K. H. (2006b) The nuclear receptor superfamily and drug discovery. ChemMedChem, 1(5), 504-523.

Moore, L. B., Maglich, J. M., McKee, D. D., Wisely, B., Willson, T. M., Kliewer, S. A., Lambert, M. H. and Moore, J. T. (2002) Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and benzoate X receptor (BXR) define three pharmacologically distinct classes of nuclear receptors. Molecular Endocrinology, 16(5), 977-986.

Moore, L. B., Parks, D. J., Jones, S. A., Bledsoe, R. K., Consler, T. G., Stimmel, J. B., Goodwin, B., Liddle, C., Blanchard, S. G., Willson, T. M., Collins, J. L. and Kliewer, S. A. (2000) Orphan

255

nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands. Journal of Biological Chemistry, 275(20), 15122-15127.

Mora-Peris, B., Watson, V., Vera, J. H., Weston, R., Khoo, S. and Mackie, N. E. (2013) Rilpivirine concentrations in plasma and cerebrospinal fluid after switching from nevirapine- containing cART. 14th International Workshop on Clinical Pharmacology of HIV Therapy, [abstract PP_04](Amsterdam), April 22-24.

Muangmoonchai, R., Smirlis, D., Wong, S. C., Edwards, M., Phillips, I. R. and Shephard, E. A. (2001) Xenobiotic induction of cytochrome P450 2B1 (CYP2B1) is mediated by the orphan nuclear receptor constitutive androstane receptor (CAR) and requires steroid co-activator 1 (SRC-1) and the transcription factor Sp1. Biochemical Journal, 355(1), 71-78.

Muzi, M., Mankoff, D. A., Link, J. M., Shoner, S., Collier, A. C., Sasongko, L. and Unadkat, J. D. (2009) Imaging of cyclosporine inhibition of P-glycoprotein activity using 11C-verapamil in the brain: Studies of healthy humans. Journal of Nuclear Medicine, 50(8), 1267-1275.

Nagy, Z., Peters, H. and Huttner, I. (1984) Fracture faces of cell junctions in cerebral endothelium during normal and hyperosmotic conditions. Laboratory Investigation, 50(3), 313- 322.

Naik, P. and Cucullo, L. (2012) In vitro blood-brain barrier models: Current and perspective technologies. Journal of Pharmaceutical Sciences, 101(4), 1337-1354.

Nakata, K., Tanaka, Y., Nakano, T., Adachi, T., Tanaka, H., Kaminuma, T. and Ishikawa, T. (2006) Nuclear receptor-mediated transcriptional regulation in Phase I, II, and III xenobiotic metabolizing systems. Drug metabolism and pharmacokinetics, 21(6), 437-457.

Nannelli, A., Rossignolo, F., Tolando, R., Rossato, P., Pellegatti, M., Longo, V. and Giovanni Gervasi, P. (2010) Expression and distribution of CYP3A genes, CYP2B22, and MDR1, MRP1, MRP2, LRP efflux transporters in brain of control and rifampicin-treated pigs. Molecular and Cellular Biochemistry, 337(1-2), 133-143.

Narang, V. S., Fraga, C., Kumar, N., Shen, J., Throm, S., Stewart, C. F. and Waters, C. M. (2008) Dexamethasone increases expression and activity of multidrug resistance transporters at the rat blood-brain barrier. American Journal of Physiology - Cell Physiology, 295(2), C440- C450.

Nath, A., Conant, K., Chen, P., Scott, C. and Major, E. O. (1999) Transient exposure to HIV-1 Tat protein results in cytokine production in macrophages and astrocytes: A hit and run phenomenon. Journal of Biological Chemistry, 274(24), 17098-17102.

Nath, A. and Sacktor, N. (2006) Influence of highly active antiretroviral therapy on persistence of HIV in the central nervous system. Current Opinion in Neurology, 19(4), 358-361.

Nawa, A., Fujita-Hamabe, W. and Tokuyama, S. (2012) Involvement of ubiquitination in the decrease of intestinal P-Glycoprotein in a streptozotocin-induced diabetic mouse model. Drug metabolism and pharmacokinetics, 27(5), 548-552.

256

Newman, M. J., Rodarte, J. C., Benbatoul, K. D., Romano, S. J., Zhang, C., Krane, S., Moran, E. J., Uyeda, R. T., Dixon, R., Guns, E. S. and Mayer, L. D. (2000) Discovery and characterization of OC144-093, a novel inhibitor of P- glycoprotein-mediated multidrug resistance. Cancer Research, 60(11), 2964-2972.

Nicolazzo, J. A. and Katneni, K. (2009) Drug transport across the blood-brain barrier and the impact of breast cancer resistance protein (ABCG2). Current Topics in Medicinal Chemistry, 9(2), 130-147.

Nishimura, M., Naito, S. and Yokoi, T. (2004) Tissue-specific mRNA expression profiles of human nuclear receptor subfamilies. Drug metabolism and pharmacokinetics, 19(2), 135-149.

Niwa, T., Yabusaki, Y., Honma, K., Matsuo, N., Tatsuta, K., Ishibashi, F. and Katagiri, M. (1998) Contribution of human hepatic cytochrome P450 isoforms to regioselective hydroxylation of steroid hormones. Xenobiotica, 28(6), 539-547.

Nobili, S., Landini, B., Giglioni, B. and Mini, E. (2006) Pharmacological strategies for overcoming multidrug resistance. Current Drug Targets, 7(7), 861-879.

Nwaozuzu, O. M., Sellers, L. A. and Barrand, M. A. (2003) Signalling pathways influencing basal and H2O 2-induced P-glycoprotein expression in endothelial cells derived from the blood- brain barrier. Journal of Neurochemistry, 87(4), 1043-1051.

O'Donnell, M. E., Lam, T. I., Tran, L. Q., Foroutan, S. and Anderson, S. E. (2006) Estradiol reduces activity of the blood-brain barrier Na-K-Cl cotransporter and decreases edema formation in permanent middle cerebral artery occlusion. Journal of Cerebral Blood Flow and Metabolism, 26(10), 1234-1249.

Ohbuchi, M., Yoshinari, K., Kaneko, H., Matsumoto, S., Inoue, A., Kawamura, A., Usui, T. and Yamazoe, Y. (2013) Coordinated roles of pregnane x receptor and constitutive androstane receptor in autoinduction of voriconazole metabolism in mice. Antimicrobial Agents and Chemotherapy, 57(3), 1332-1338.

Ohtsuki, S., Ikeda, C., Uchida, Y., Sakamoto, Y., Miller, F., Glacial, F., Decleves, X., Scherrmann, J. M., Couraud, P. O., Kubo, Y., Tachikawa, M. and Terasaki, T. (2013) Quantitative targeted absolute proteomic analysis of transporters, receptors and junction proteins for validation of human cerebral microvascular endothelial cell line hCMEC/D3 as a human blood-brain barrier model. Molecular Pharmaceutics, 10(1), 289-296.

Ohtsuki, S. and Terasaki, T. (2007) Contribution of carrier-mediated transport systems to the blood-brain barrier as a supporting and protecting interface for the brain; importance for CNS drug discovery and development. Pharmaceutical Research, 24(9), 1745-1758.

Okochi, E., Iwahashi, T. and Tsuruo, T. (1997) Monoclonal antibodies specific for P- glycoprotein. Leukemia, 11(7), 1119-1123.

Oldham, M. L., Khare, D., Quiocho, F. A., Davidson, A. L. and Chen, J. (2007) Crystal structure of a catalytic intermediate of the maltose transporter. Nature, 450(7169), 515-521.

257

Olinga, P., Elferink, M. G. L., Draaisma, A. L., Merema, M. T., Castell, J. V., Pérez, G. and Groothuis, G. M. M. (2008) Coordinated induction of drug transporters and phase I and II metabolism in human liver slices. European Journal of Pharmaceutical Sciences, 33(4-5), 380- 389.

Orans, J., Teotico, D. G. and Redinbo, M. R. (2005) The nuclear xenobiotic receptor pregnane X receptor: Recent insights and new challenges. Molecular Endocrinology, 19(12), 2891-2900.

Ott, M., Fricker, G. and Bauer, B. (2009) Pregnane X receptor (PXR) regulates P-glycoprotein at the blood-brain barrier: Functional similarities between pig and human PXR. Journal of Pharmacology and Experimental Therapeutics, 329(1), 141-149.

Ourlin, J. C., Lasserre, F., Pineau, T., Fabre, J. M., Sa-Cunha, A., Maurel, P., Vilarem, M. J. and Pascussi, J. M. (2003) The small heterodimer partner interacts with the pregnane X receptor and represses its transcriptional activity. Molecular Endocrinology, 17(9), 1693-1703.

Owen, A., Goldring, C., Morgan, P., Park, B. K. and Pirmohamed, M. (2006) Induction of P- glycoprotein in lymphocytes by carbamazepine and rifampicin: The role of nuclear hormone response elements. British Journal of Clinical Pharmacology, 62(2), 237-242.

Pal, D., Kwatra, D., Minocha, M., Paturi, D. K., Budda, B. and Mitra, A. K. (2011) Efflux transporters- and cytochrome P-450-mediated interactions between drugs of abuse and antiretrovirals. Life Sciences, 88(21-22), 959-971.

Pardridge, W. M. (2003) Blood-brain barrier drug targeting: the future of brain drug development. Molecular Interventions, 3(2), 90-105, 151.

Pardridge, W. M. (2005) Molecular biology of the blood-brain barrier. Molecular Biotechnology, 30(1), 57-70.

Pardridge, W. M. (2007) Blood-brain barrier delivery. Drug Discovery Today, 12(1-2), 54-61.

Pardridge, W. M. (2010) Biopharmaceutical drug targeting to the brain. Journal of Drug Targeting, 18(3), 157-167.

Pardridge, W. M. (2012) Drug transport across the blood-brain barrier. Journal of Cerebral Blood Flow and Metabolism, 32(11), 1959-1972.

Park, S. and Sinko, P. J. (2005) P-glycoprotein and mutlidrug resistance-associated proteins limit the brain uptake of saquinavir in mice. Journal of Pharmacology and Experimental Therapeutics, 312(3), 1249-1256.

Parton, R. G. and Richards, A. A. (2003) Lipid rafts and caveolae as portals for endocytosis: New insights and common mechanisms. Traffic, 4(11), 724-738.

Pascussi, J. M., Busson-Le Coniat, M., Maurel, P. and Vilarem, M. J. (2003) Transcriptional analysis of the orphan nuclear receptor constitutive androstane receptor (NR1I3) gene promoter:

258

Identification of a distal glucocorticoid response element. Molecular Endocrinology, 17(1), 42- 55.

Pascussi, J. M., Drocourt, L., Fabre, J. M., Maurel, P. and Vilarem, M. J. (2000) Dexamethasone induces pregnane X receptor and retinoid X receptor-α expression in human hepatocytes: Synergistic increase of CYP3A4 induction by pregnane X receptor activators. Molecular Pharmacology, 58(2), 361-372.

Pascussi, J. M., Drocourt, L., Gerbal-Chaloin, S., Fabre, J. M., Maurel, P. and Vilarem, M. J. (2001) Dual effect of dexamethasone on CYP3A4 gene expression in human hepatocytes. Sequential role of glucocorticoid receptor and pregnane X receptor. European Journal of Biochemistry, 268(24), 6346-6357.

Pascussi, J. M., Gerbal-Chaloin, S., Duret, C., Daujat-Chavanieu, M., Vilarem, M. J. and Maurel, P. (2007) The Tangle of Nuclear Receptors that Controls Xenobiotic Metabolism and Transport: Crosstalk and Consequences. Annual Review of Pharmacology and Toxicology.

Patel, R., Patel, M., Tsai, R., Lin, V., Bookout, A. L., Zhang, Y., Magomedova, L., Li, T., Chan, J. F., Budd, C., Mangelsdorf, D. J. and Cummins, C. L. (2011) LXRβ is required for glucocorticoid-induced hyperglycemia and hepatosteatosis in mice. Journal of Clinical Investigation, 121(1), 431-441.

Peluso, R., Haase, A. and Stowring, L. (1985) A Trojan horse mechanism for the spread of visna virus in monocytes. Virology, 147(1), 231-236.

Perego, P., Corna, E., De Cesare, M., Gatti, L., Polizzi, D., Pratesi, G., Supino, R. and Zunino, F. (2001) Reversal of multidrug resistance by the P-glycoprotein modulator, LY335979, from the bench to the clinic. Current Medicinal Chemistry, 8(1), 39-50.

Perloff, E. S., Duan, S. X., Skolnik, P. R., Greenblatt, D. J. and von Moltke, L. L. (2005) Atazanavir: effects on P-glycoprotein transport and CYP3A metabolism in vitro. drug Metabolism and Disposition, 33(6), 764-770.

Perloff, M. D., Von Moltke, L. L., Fahey, J. M., Daily, J. P. and Greenblatt, D. J. (2000) Induction of P-glycoprotein expression by HIV protease inhibitors in cell culture. AIDS, 14(9), 1287-1289.

Perloff, M. D., von Moltke, L. L. and Greenblatt, D. J. (2002) Fexofenadine transport in Caco-2 cells: inhibition with verapamil and ritonavir. Journal of Clinical Pharmacology, 42(11), 1269- 1274.

Perloff, M. D., VonMoltke, L. L., Fahey, J. M. and Greenblatt, D. J. (2007) Induction of P- glycoprotein expression and activity by ritonavir in bovine brain microvessel endothelial cells. Journal of Pharmacy and Pharmacology, 59(7), 947-953.

Perloff, M. D., VonMoltke, L. L. and Greenblatt, D. J. (2004) Ritonavir and dexamethasone induce expression of CYP3A and P-glycoprotein in rats. Xenobiotica, 34(2), 133-150.

259

Perloff, M. D., VonMoltke, L. L., Marchand, J. E. and Greenblatt, D. J. (2001) Ritonavir induces P-glycoprotein expression, multidrug resistance-associated protein (MRP1) expression, and drug transporter-mediated activity in a human intestinal cell line. Journal of Pharmaceutical Sciences, 90(11), 1829-1837.

Perry, C. M. (2010) Maraviroc: A review of its use in the management of CCR5-Tropic HIV-1 infection. Drugs, 70(9), 1189-1213.

Persidsky, Y. and Poluektova, L. (2006) Immune privilege and HIV-1 persistence in the CNS. Immunological Reviews, 213(1), 180-194.

Persidsky, Y., Ramirez, S. H., Haorah, J. and Kanmogne, G. D. (2006) Blood-brain barrier: Structural components and function under physiologic and pathologic conditions. Journal of NeuroImmune Pharmacology, 1(3), 223-236.

Persidsky, Y., Zheng, J., Miller, D. and Gendelman, H. E. (2000) Mononuclear phagocytes mediate blood-brain barrier compromise and neuronal injury during HIV-1-associated dementia. Journal of Leukocyte Biology, 68(3), 413-422.

Petrick, J. S. and Klaassen, C. D. (2007) Importance of hepatic induction of constitutive androstane receptor and other transcription factors that regulate xenobiotic metabolism and transport. Drug Metabolism and Disposition, 35(10), 1806-1815.

Petrovic, V., Teng, S. and Piquette-Miller, M. (2007) Regulation of drug transporters: During infection and inflammation. Molecular Interventions, 7(2), 99-111.

Pick, A., Klinkhammer, W. and Wiese, M. (2010) Specific inhibitors of the breast cancer resistance protein (BCRP). ChemMedChem, 5(9), 1498-1505.

Pober, J. S. (2008) Physiology and Pathobiology of Microvascular Endothelium. In: Microcirculation (Second Edition), (F. T. Ronald, N. D. Walter and L. Klaus eds.), pp. 37-55. Academic Press, San Diego.

Poller, B., Gutmann, H., Krähenbühl, S., Weksler, B., Romero, I., Couraud, P. O., Tuffin, G., Drewe, J. and Huwyler, J. (2008) The human brain endothelial cell line hCMEC/D3 as a human blood-brain barrier model for drug transport studies. Journal of Neurochemistry, 107(5), 1358- 1368.

Polli, J. W., Jarrett, J. L., Studenberg, S. D., Humphreys, J. E., Dennis, S. W., Brouwer, K. R. and Woolley, J. L. (1999) Role of P-glycoprotein on the CNS disposition of amprenavir (141W94), an HIV protease inhibitor. Pharmaceutical Research, 16(8), 1206-1212.

Pomerantz, R. J. (2003) Reservoirs, sanctuaries, and residual disease: the hiding spots of HIV-1. HIV Clinical Trials, 4(2), 137-143.

Potschka, H. (2010) Targeting regulation of ABC efflux transporters in brain diseases: a novel therapeutic approach. Pharmacology and Therapeutics, 125(1), 118-127.

260

Prat, A., Biernacki, K., Becher, B. and Antel, J. P. (2000) B7 expression and antigen presentation by human brain endothelial cells: requirement for proinflammatory cytokines. Journal of Neuropathology & Experimental Neurology, 59(2), 129-136.

Price, D. L., Ludwig, J. W., Mi, H., Schwarz, T. L. and Ellisman, M. H. (2002) Distribution of rSlo Ca2+-activated K+ channels in rat astrocyte perivascular endfeet. Brain Research, 956(2), 183-193.

Pusztai, L., Wagner, P., Ibrahim, N., Rivera, E., Theriault, R., Booser, D., Symmans, F. W., Wong, F., Blumenschein, G., Fleming, D. R., Rouzier, R., Boniface, G. and Hortobagyi, G. N. (2005) Phase II study of tariquidar, a selective P-glycoprotein inhibitor, in patients with chemotherapy-resistant, advanced breast carcinoma. Cancer, 104(4), 682-691.

Quashie, P. K., Mesplède, T. and Wainberg, M. A. (2013) HIV drug resistance and the advent of integrase inhibitors. Current Infectious Disease Reports, 15(1), 85-100.

Rao, V. V., Dahlheimer, J. L., Bardgett, M. E., Snyder, A. Z., Finch, R. A., Sartorelli, A. C. and Piwnica-Worms, D. (1999) Choroid plexus epithelial expression of MDR1 P glycoprotein and multidrug resistance-associated protein contribute to the blood-cerebrospinal-fluid drug- permeability barrier. Proceedings of the National Academy of Sciences of the United States of America, 96(7), 3900-3905.

Reese, T. S. and Karnovsky, M. J. (1967) Fine structural localization of a blood-brain barrier to exogenous peroxidase. Journal of Cell Biology, 34(1), 207-217.

Regina, A., Koman, A., Piciotti, M., El Hafny, B., Center, M. S., Bergmann, R., Couraud, P. O. and Roux, F. (1998) Mrp1 multidrug resistance-associated protein and P-glycoprotein expression in rat brain microvessel endothelial cells. Journal of Neurochemistry, 71(2), 705-715.

Reid, G., Wielinga, P., Zelcer, N., De Haas, M., Van Deemter, L., Wijnholds, J., Balzarini, J. and Borst, P. (2003) Characterization of the transport of nucleoside analog drugs by the human multidrug resistance proteins MRP4 and MRP5. Molecular Pharmacology, 63(5), 1094-1103.

Ribera, E. and Curran, A. (2008) Double-boosted protease inhibitor antiretroviral regimens: What role? Drugs, 68(16), 2257-2267.

Richert, L., Tuschl, G., Abadie, C., Blanchard, N., Pekthong, D., Mantion, G., Weber, J. C. and Mueller, S. O. (2009) Use of mRNA expression to detect the induction of drug metabolising enzymes in rat and human hepatocytes. Toxicology and Applied Pharmacology, 235(1), 86-96.

Ritzel, M. W. L., Ng, A. M. L., Yao, S. Y. M., Graham, K., Loewen, S. K., Smith, K. M., Ritzel, R. G., Mowles, D. A., Carpenter, P., Chen, X. Z., Karpinski, E., Hyde, R. J., Baldwin, S. A., Cass, C. E. and Young, J. D. (2001) Molecular Identification and Characterization of Novel Human and Mouse Concentrative Na+-Nucleoside Cotransporter Proteins (hCNT3 and mCNT3) Broadly Selective for Purine and Pyrimidine Nucleosides (System cib). Journal of Biological Chemistry, 276(4), 2914-2927.

261

Ritzel, M. W. L., Yao, S. Y. M., Huang, M. Y., Elliott, J. F., Cass, C. E. and Young, J. D. (1997) Molecular cloning and functional expression of cDNAs encoding a human Na+-nucleoside cotransporter (hCNT1). American Journal of Physiology - Cell Physiology, 272(2 41-2), C707- C714.

Robertson, S. M., Maldarelli, F., Natarajan, V., Formentini, E., Alfaro, R. M. and Penzak, S. R. (2008) Efavirenz induces CYP2B6-mediated hydroxylation of bupropion in healthy subjects. Journal of Acquired Immune Deficiency Syndromes, 49(5), 513-519.

Robillard, K. R., Hoque, M. T. and Bendayan, R. (2012) Expression of ATP-Binding cassette membrane transporters in rodent and human sertoli cells: Relevance to the permeability of antiretroviral therapy at the blood-testis barrier. Journal of Pharmacology and Experimental Therapeutics, 340(1), 96-108.

Rodríguez-Baeza, A., Reina-De La Torre, F., Poca, A., Martí, M. and Garnacho, A. (2003) Morphological Features in Human Cortical Brain Microvessels after Head Injury: A Three- Dimensional and Immunocytochemical Study. Anatomical Record - Part A Discoveries in Molecular, Cellular, and Evolutionary Biology, 273(1), 583-593.

Ronaldson, P. T., Ashraf, T. and Bendayan, R. (2010) Regulation of multidrug resistance protein 1 by tumor necrosis factor α in cultured glial cells: Involvement of nuclear factor-κB and c-Jun N-terminal kinase signaling pathways. Molecular Pharmacology, 77(4), 644-659.

Ronaldson, P. T., Babakhanian, K., Bendayan, R., You, G. and Morris, M. E. (2007) Drug Transporters: Molecular Characterization and Role in Drug Disposition. Chapter 14. Drug Transport in the brain. pg 411-461: Wiley Series in Drug Discovery and Development. A John Wiley & Son Inc., New Jersey.

Ronaldson, P. T., Bendayan, M., Gingras, D., Piquette-Miller, M. and Bendayan, R. (2004a) Cellular localization and functional expression of P-glycoprotein in rat astrocyte cultures. Journal of Neurochemistry, 89(3), 788-800.

Ronaldson, P. T. and Bendayan, R. (2006) HIV-1 viral envelope glycoprotein gp120 triggers an inflammatory response in cultured rat astrocytes and regulates the functional expression of P- glycoprotein. Molecular Pharmacology, 70(3), 1087-1098.

Ronaldson, P. T., Lee, G., Dallas, S. and Bendayan, R. (2004b) Involvement of P-glycoprotein in the transport of saquinavir and indinavir in rat brain microvessel endothelial and microglia cell lines. Pharmaceutical Research, 21(5), 811-818.

Ronaldson, P. T., Persidsky, Y. and Bendayan, R. (2008) Regulation of ABC membrane transporters in glial cells: relevance to the pharmacotherapy of brain HIV-1 infection. GLIA, 56(16), 1711-1735.

Rosenberg, M. F., Kamis, A. B., Callaghan, R., Higgins, C. F. and Ford, R. C. (2003) Three- dimensional structures of the mammalian multidrug resistance P-glycoprotein demonstrate major conformational changes in the transmembrane domains upon nucleotide binding. Journal of Biological Chemistry, 278(10), 8294-8299.

262

Rosenfeld, J. M., Vargas Jr, R., Xie, W. and Evans, R. M. (2003) Genetic profiling defines the xenobiotic gene network controlled by the nuclear receptor pregnane x receptor. Molecular Endocrinology, 17(7), 1268-1282.

Rosenfeld, M. G., Lunyak, V. V. and Glass, C. K. (2006) Sensors and signals: A coactivator/corepressor/epigenetic code for integrating signal-dependent programs of transcriptional response. Genes and Development, 20(11), 1405-1428.

Roth, M., Obaidat, A. and Hagenbuch, B. (2012) OATPs, OATs and OCTs: The organic anion and cation transporters of the SLCO and SLC22A gene superfamilies. British Journal of Pharmacology, 165(5), 1260-1287.

Sacktor, N. (2002) The epidemiology of human immunodeficiency virus-associated neurological disease in the era of highly active antiretroviral therapy. Journal of NeuroVirology, 8(Suppl 2), 115-121.

Saeki, M., Kurose, K., Tohkin, M. and Hasegawa, R. (2008) Identification of the functional vitamin D response elements in the human MDR1 gene. Biochemical Pharmacology, 76(4), 531- 542.

Sahi, J., Sinz, M. W., Campbell, S., Mireles, R., Zheng, X., Rose, K. A., Raeissi, S., Hashim, M. F., Ye, Y., De Morais, S. M., Black, C., Tugnait, M. and Keller, L. H. (2006) Metabolism and transporter-mediated drug-drug interactions of the endothelin-A receptor antagonist CI-1034. Chemico-Biological Interactions, 159(2), 156-168.

Sai, Y., Nies, A. T. and Arias, I. M. (1999) Bile acid secretion and direct targeting of mdr1-green fluorescent protein from Golgi to the canalicular membrane in polarized WIF-B cells. Journal of Cell Science, 112(24), 4535-4545.

Saini, S. P. S., Sonoda, J., Xu, L., Toma, D., Uppal, H., Mu, Y., Ren, S., Moore, D. D., Evans, R. M. and Xie, W. (2004) A Novel Constitutive Androstane Receptor-Mediated and CYP3A- Independent Pathway of Bile Acid Detoxification. Molecular Pharmacology, 65(2), 292-300.

Santaguida, S., Janigro, D., Hossain, M., Oby, E., Rapp, E. and Cucullo, L. (2006) Side by side comparison between dynamic versus static models of blood-brain barrier in vitro: A permeability study. Brain Research, 1109(1), 1-13.

Savkur, R. S., Wu, Y., Bramlett, K. S., Wang, M., Yao, S., Perkins, D., Totten, M., Searfoss Iii, G., Ryan, T. P., Su, E. W. and Burris, T. P. (2003) Alternative splicing within the ligand binding domain of the human constitutive androstane receptor. Molecular Genetics and Metabolism, 80(1-2), 216-226.

Savolainen, J., Edwards, J. E., Morgan, M. E., McNamara, P. J. and Anderson, B. D. (2002) Effects of a P-glycoprotein inhibitor on brain and plasma concentrations of anti-human immunodeficiency virus drugs administered in combination in rats. Drug Metabolism and Disposition, 30(5), 479-482.

263

Sawchuk, R. J. and Elmquist, W. F. (2000) Microdialysis in the study of drug transporters in the CNS. Advanced Drug Delivery Reviews, 45(2-3), 295-307.

Scheer, N., Ross, J., Kapelyukh, Y., Rode, A. and Wolf, C. R. (2010) In vivo responses of the human and murine pregnane X receptor to dexamethasone in mice. Drug Metabolism and Disposition, 38(7), 1046-1053.

Schinkel, A. H. and Jonker, J. W. (2003) Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: An overview. Advanced Drug Delivery Reviews, 55(1), 3-29.

Schinkel, A. H., Wagenaar, E., Mol, C. A. A. M. and Van Deemter, L. (1996) P-glycoprotein in the blood-brain barrier of mice influences the brain penetration and pharmacological activity of many drugs. Journal of Clinical Investigation, 97(11), 2517-2524.

Schlachetzki, F. and Pardridge, W. M. (2003) P-glycoprotein and caveolin-1alpha in endothelium and astrocytes of primate brain. NeuroReport, 14(16), 2041-2046.

Schote, A. B., Turner, J. D., Schiltz, J. and Muller, C. P. (2007) Nuclear receptors in human immune cells: Expression and correlations. Molecular Immunology, 44(6), 1436-1445.

Schuetz, J. D., Connelly, M. C., Sun, D., Paibir, S. G., Flynn, P. M., Srinivas, R. V., Kumar, A. and Fridland, A. (1999) MRP4: A previously unidentified factor in resistance to nucleoside- based antiviral drugs. Nature Medicine, 5(9), 1048-1051.

Schulze, C. and Firth, J. A. (1993) Immunohistochemical localization of adherens junction components in blood-brain barrier microvessels of the rat. Journal of Cell Science, 104(3), 773- 782.

Scotto, K. W. (2003) Transcriptional regulation of ABC drug transporters. Oncogene, 22(6), 7496-7511.

Shaik, N., Giri, N., Pan, G. and Elmquist, W. F. (2007) P-glycoprotein-mediated active efflux of the anti-HIV1 nucleoside abacavir limits cellular accumulation and brain distribution. Drug Metabolism and Disposition, 35(11), 2076-2085.

Shan, L., Vincent, J., Brunzelle, J. S., Dussault, I., Lin, M., Ianculescu, I., Sherman, M. A., Forman, B. M. and Fernandez, E. J. (2004) Structure of the murine constitutive androstane receptor complexed to androstenol: A molecular basis for inverse agonism. Molecular Cell, 16(6), 907-917.

Shapiro, A. B., Fox, K., Lee, P., Yang, Y. D. and Ling, V. (1998) Functional intracellular P- glycoprotein. International Journal of Cancer, 76(6), 857-864.

Shapiro, A. B. and Ling, V. (1997) Positively cooperative sites for drug transport by P- glycoprotein with distinct drug specificities. European Journal of Biochemistry, 250(1), 130-137.

Sharom, F. J. (2006) Shedding light on drug transport: structure and function of the P- glycoprotein multidrug transporter (ABCB1). Biochemistry and Cell Biology, 84(6), 979-992.

264

Sharom, F. J., Liu, R., Romsicki, Y. and Lu, P. (1999) Insights into the structure and substrate interactions of the P- glycoprotein multidrug transporter from spectroscopic studies. Biochimica et Biophysica Acta - Biomembranes, 1461(2), 327-345.

Sharp, P. M. and Hahn, B. H. (2010) The evolution of HIV-1 and the origin of AIDS. Philosophical Transactions of the Royal Society B: Biological Sciences, 365(1552), 2487-2494.

Shawahna, R., Uchida, Y., Declèves, X., Ohtsuki, S., Yousif, S., Dauchy, S., Jacob, A., Chassoux, F., Daumas-Duport, C., Couraud, P. O., Terasaki, T. and Scherrmann, J. M. (2011) Transcriptomic and quantitative proteomic analysis of transporters and drug metabolizing enzymes in freshly isolated human brain microvessels. Molecular Pharmaceutics, 8(4), 1332- 1341.

Shen, D. D., Artru, A. A. and Adkison, K. K. (2004) Principles and applicability of CSF sampling for the assessment of CNS drug delivery and pharmacodynamics. Advanced Drug Delivery Reviews, 56(12), 1825-1857.

Shoshani, T., Zhang, S., Dey, S., Pastan, I. and Gottesman, M. M. (1998) Analysis of random recombination between human MDR1 and mouse Mdr1a cDNA in a pHaMDR-dihydrofolate reductase bicistronic expression system. Molecular Pharmacology, 54(4), 623-630.

Siddharthan, V., Kim, Y. V., Liu, S. and Kim, K. S. (2007) Human astrocytes/astrocyte- conditioned medium and shear stress enhance the barrier properties of human brain microvascular endothelial cells. Brain Research, 1147(1), 39-50.

Sidhu, J. S. and Omiecinski, C. J. (1997) An okadaic acid-sensitive pathway involved in the phenobarbital-mediated induction of CYP2B gene expression in primary rat hepatocyte cultures. Journal of Pharmacology and Experimental Therapeutics, 282(2), 1122-1129.

Sierra-Aragón, S. and Walter, H. (2012) Targets for inhibition of HIV replication: Entry, enzyme action, release and maturation. Intervirology, 55(2), 84-97.

Siest, G., Jeannesson, E., Marteau, J. B., Samara, A., Marie, B., Pfister, M. and Visvikis-Siest, S. (2008) Transcription factor and drug-metabolizing enzyme gene expression in lymphocytes from healthy human subjects. Drug Metabolism and Disposition, 36(1), 182-189.

Siliciano, J. D., Kajdas, J., Finzi, D., Quinn, T. C., Chadwick, K., Margolick, J. B., Kovacs, C., Gange, S. J. and Siliciano, R. F. (2003) Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4 + T cells. Nature Medicine, 9(6), 727-728.

Simard, M., Arcuino, G., Takano, T., Liu, Q. S. and Nedergaard, M. (2003) Signaling at the gliovascular interface. Journal of Neuroscience, 23(27), 9254-9262.

Sladek, F. M. (2011) What are nuclear receptor ligands? Molecular and Cellular Endocrinology, 334(1-2), 3-13.

Slot, A. J., Molinski, S. V. and Cole, S. P. (2011) Mammalian multidrug-resistance proteins (MRPs). Essays in biochemistry, 50(1), 179-207.

265

Smit, J. J., Schinkel, A. H., Oude Elferink, R. P., Groen, A. K., Wagenaar, E., van Deemter, L., Mol, C. A., Ottenhoff, R., van der Lugt, N. M., van Roon, M. A. and et al. (1993) Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell, 75(3), 451-462.

Smit, T. K., Brew, B. J., Tourtellotte, W., Morgello, S., Gelman, B. B. and Saksena, N. K. (2004) Independent evolution of human immunodeficiency virus (HIV) drug resistance mutations in diverse areas of the brain in HIV-infected patients, with and without dementia, on antiretroviral treatment. Journal of Virology, 78(18), 10133-10148.

Smith, A. J., Mayer, U., Schinkel, A. H. and Borst, P. (1998) Availability of PSC833, a substrate and inhibitor of poglycoproteins, in various concentrations of serum. Journal of the National Cancer Institute, 90(15), 1161-1166.

Solas, C., Lafeuillade, A., Halfon, P., Chadapaud, S., Hittinger, G. and Lacarelle, B. (2003) Discrepancies between protease inhibitor concentrations and viral load in reservoirs and sanctuary sites in human immunodeficiency virus-infected patients. Antimicrobial Agents and Chemotherapy, 47(1), 238-243.

Song, S., Suzuki, H., Kawai, R. and Sugiyama, Y. (1999) Effect of PSC 833, a P-glycoprotein modulator, on the disposition of vincristine and digoxin in rats. Drug Metabolism and Disposition, 27(6), 689-694.

Song, X., Xie, M., Zhang, H., Li, Y., Sachdeva, K. and Yan, B. (2004) The pregnane X receptor binds to response elements in a genomic context-dependent manner, and PXR activator rifampicin selectively alters the binding among target genes. Drug Metabolism and Disposition, 32(1), 35-42.

Sonneveld, P., Suciu, S., Weijermans, P., Beksac, M., Neuwirtova, R., Solbu, G., Lokhorst, H., Van der Lelie, J., Dohner, H., Gerhartz, H., Segeren, C. M., Willemze, R. and Lowenberg, B. (2001) Cyclosporin A combined with vincristine, doxorubicin and dexamethasone (VAD) compared with VAD alone in patients with advanced refractory multiple myeloma: An EORTC- HOVON randomized phase III study (06914). British Journal of Haematology, 115(4), 895-902.

Sonoda, J., Chong, L. W., Downes, M., Barish, G. D., Coulter, S., Liddle, C., Lee, C. H. and Evans, R. M. (2005) Pregnane X receptor prevents hepatorenal toxicity from cholesterol metabolites. Proceedings of the National Academy of Sciences of the United States of America, 102(6), 2198-2203.

Sonoda, J., Xie, W., Rosenfeld, J. M., Barwick, J. L., Guzelian, P. S. and Evans, R. M. (2002) Regulation of a xenobiotic sulfonation cascade by nuclear pregnane X receptor (PXR). Proceedings of the National Academy of Sciences of the United States of America, 99(21), 13801-13806.

Squires, E. J., Sueyoshi, T. and Negishi, M. (2004) Cytoplasmic localization of pregnane X receptor and ligand-dependent nuclear translocation in mouse liver. Journal of Biological Chemistry, 279(47), 49307-49314.

266

Stanley, L., Horsburgh, B., Ross, J., Scheer, N. and Roland Wolf, C. (2006) PXR and CAR: Nuclear receptors which play a pivotal role in drug disposition and chemical toxicity. Drug Metabolism Reviews, 38(3), 515-597.

Starling, J. J., Shepard, R. L., Cao, J., Law, K. L., Norman, B. H., Kroin, J. S., Ehlhardt, W. J., Baughman, T. M., Winter, M. A., Bell, M. G., Shih, C., Gruber, J., Elmquist, W. F. and Dantzig, A. H. (1997) Pharmacological characterization of LY335979: A potent cyclopropyldibenzosuberane modulator of P-glycoprotein. Advances in Enzyme Regulation, 37(335-347.

Staudinger, J. L., Goodwin, B., Jones, S. A., Hawkins-Brown, D., Mackenzie, K. I., Latour, A., Liu, Y., Klaassen, C. D., Brown, K. K., Reinhard, J., Willson, T. M., Koller, B. H. and Kliewer, S. A. (2001) The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity. Proceedings of the National Academy of Sciences of the United States of America, 98(6), 3369-3374.

Staudinger, J. L., Madan, A., Carol, K. M. and Parkinson, A. (2003) Regulation of drug transporter gene expression by nuclear receptors. Drug Metabolism and Disposition, 31(5), 523- 527.

Storch, C. H., Theile, D., Lindenmaier, H., Haefeli, W. E. and Weiss, J. (2007) Comparison of the inhibitory activity of anti-HIV drugs on P-glycoprotein. Biochemical Pharmacology, 73(10), 1573-1581.

Stormer, E., von Moltke, L. L., Perloff, M. D. and Greenblatt, D. J. (2002) Differential modulation of P-glycoprotein expression and activity by non-nucleoside HIV-1 reverse transcriptase inhibitors in cell culture. Pharmaceutical Research, 19(7), 1038-1045.

Su, L., Cheng, C. Y. and Mruk, D. D. (2009) Drug transporter, P-glycoprotein (MDR1), is an integrated component of the mammalian blood-testis barrier. International Journal of Biochemistry and Cell Biology, 41(12), 2578-2587.

Su, Y. and Sinko, P. J. (2006) Drug delivery across the blodd-brain barrier: Why is it difficult? How to measure and improve it? Expert Opinion on Drug Delivery, 3(3), 419-435.

Su, Y., Zhang, X. and Sinko, P. J. (2004) Human organic anion-transporting polypeptide OATP- A (SLC21A3) acts in concert with P-glycoprotein and multidrug resistance protein 2 in the vectorial transport of Saquinavir in Hep G2 cells. Molecular Pharmacology, 1(1), 49-56.

Subileau, E. A., Rezaie, P., Davies, H. A., Colyer, F. M., Greenwood, J., Male, D. K. and Romero, I. A. (2009) Expression of chemokines and their receptors by human brain endothelium: Implications for multiple sclerosis. Journal of Neuropathology and Experimental Neurology, 68(3), 227-240.

Sueyoshi, T., Kawamoto, T., Zelko, I., Honkakoski, P. and Negishi, M. (1999) The repressed nuclear receptor CAR responds to phenobarbital in activating the human CYP2B6 gene. Journal of Biological Chemistry, 274(10), 6043-6046.

267

Sugatani, J., Kojima, H., Ueda, A., Kakizaki, S., Yoshinari, K., Gong, Q. H., Owens, I. S., Negishi, M. and Sueyoshi, T. (2001) The phenobarbital response enhancer module in the human bilirubin UDP-glucuronosyltransferase UGT1A1 gene and regulation by the nuclear receptor CAR. Hepatology, 33(5), 1232-1238.

Sugatani, J., Nishitani, S., Yamakawa, K., Yoshinari, K., Sueyoshi, T., Negishi, M. and Miwa, M. (2005) Transcriptional regulation of human UGT1A1 gene expression: Activated glucocorticoid receptor enhances constitutive androstane receptor/ pregnane X receptor-mediated UDP-glucuronosyltransferase 1A1 regulation with glucocorticoid receptor-interacting protein 1. Molecular Pharmacology, 67(3), 845-855.

Suino, K., Peng, L., Reynolds, R., Li, Y., Cha, J. Y., Repa, J. J., Kliewer, S. A. and Xu, H. E. (2004) The nuclear xenobiotic receptor CAR: Structural determinants of constitutive activation and heterodimerization. Molecular Cell, 16(6), 893-905.

Sun, H., Bungay, P. M. and Elmquist, W. F. (2001) Effect of capillary efflux transport inhibition on the determination of probe recovery during in vivo microdialysis in the brain. Journal of Pharmacology and Experimental Therapeutics, 297(3), 991-1000.

Sundquist, W. I. and Krausslich, H. G. (2012) HIV-1 assembly, budding, and maturation. Cold Spring Harbor Perspectives in Medicine, 2(7), a006924.

Suzuki, H. and Sugiyama, Y. (2000) Transport of drugs across the hepatic sinusoidal membrane: Sinusoidal drug influx and efflux in the liver. Seminars in Liver Disease, 20(3), 251-263.

Suzuyama, N., Katoh, M., Takeuchi, T., Yoshitomi, S., Higuchi, T., Asashi, S. and Yokoi, T. (2007) Species differences of inhibitory effects on P-glycoprotein-mediated drug transport. Journal of Pharmaceutical Sciences, 96(6), 1609-1618.

Svärd, J., Spiers, J. P., Mulcahy, F. and Hennessy, M. (2010) Nuclear receptor-mediated induction of CYP450 by antiretrovirals: Functional consequences of NR1I2 (PXR) polymorphisms and differential prevalence in whites and sub-Saharan Africans. Journal of Acquired Immune Deficiency Syndromes, 55(5), 536-549.

Swales, K. and Negishi, M. (2004) CAR, driving into the future. Molecular Endocrinology, 18(7), 1589-1598.

Synold, T. W., Dussault, I. and Forman, B. M. (2001) The orphan nuclear receptor SXR coordinately regulates drug metabolism and efflux. Nature Medicine, 7(5), 584-590.

Syvänen, S. and Eriksson, J. (2013) Advances in PET imaging of P-glycoprotein function at the blood-brain barrier. ACS Chemical Neuroscience, 4(2), 225-237.

Syvänen, S., Schenke, M., Van Den Berg, D. J., Voskuyl, R. A. and De Lange, E. C. (2012) Alteration in P-glycoprotein functionality affects intrabrain distribution of quinidine more than brain entry-A study in rats subjected to status epilepticus by kainate. AAPS Journal, 14(1), 87-96.

268

Sziráki, I., Erdo, F., Beéry, E., Molnár, P. M., Fazakas, C., Wilhelm, I., Makai, I., Kis, E., Herédi-Szabó, K., Abonyi, T., Krizbai, I., Tóth, G. K. and Krajcsi, P. (2011) Quinidine as an abcb1 probe for testing drug interactions at the blood-brain barrier: An in vitro in vivo correlation study. Journal of Biomolecular Screening, 16(8), 886-894.

Takeda, M., Khamdang, S., Narikawa, S., Kimura, H., Kobayashi, Y., Yamamoto, T., Cha, S. H. O., Sekine, T. and Endou, H. (2002) Human organic anion transporters and human organic cation transporters mediate renal antiviral transport. Journal of Pharmacology and Experimental Therapeutics, 300(3), 918-924.

Takeshita, A., Taguchi, M., Koibuchi, N. and Ozawa, Y. (2002) Putative role of the orphan nuclear receptor SXR (steroid and xenobiotic receptor) in the mechanism of CYP3A4 inhibition by xenobiotics. Journal of Biological Chemistry, 277(36), 32453-32458.

Takeuchi, T., Yoshitomi, S., Higuchi, T., Ikemoto, K., Niwa, S. I., Ebihara, T., Katoh, M., Yokoi, T. and Asahi, S. (2006) Establishment and characterization of the transformants stably- expressing MDR1 derived from various animal species in LLC-PK1. Pharmaceutical Research, 23(7), 1460-1472.

Tang-Wai, D. F., Kajiji, S., DiCapua, F., De Graaf, D., Roninson, I. B. and Gros, P. (1995) Human (MDR1) and mouse (mdr1,mdr3) P-glycoproteins can be distinguished by their respective drug resistance profiles and sensitivity to modulators. Biochemistry, 34(1), 32-39.

Tashima, K. T., Caliendo, A. M., Ahmad, M., Gormley, J. M., Fiske, W. D., Brennan, J. M. and Flanigan, T. P. (1999) Cerebrospinal fluid human immunodeficiency virus type 1 (HIV-1) suppression and efavirenz drug concentrations in HIV-1-infected patients receiving combination therapy. Journal of Infectious Diseases, 180(3), 862-864.

Teng, S., Jekerle, V. and Piquette-Miller, M. (2003) Induction of ABCC3 (MRP3) by pregnane X receptor activators. Drug Metabolism and Disposition, 31(11), 1296-1299.

Timsit, Y. E. and Negishi, M. (2007) CAR and PXR: The xenobiotic-sensing receptors. Steroids, 72(3), 231-246.

Tiraboschi, J. M., Niubo, J., Curto, J. and Podzamczer, D. (2010) Maraviroc concentrations in cerebrospinal fluid in HIV-infected patients. Journal of Acquired Immune Deficiency Syndromes, 55(5), 606-609.

Tirona, R. G. and Kim, R. B. (2005) Nuclear receptors and drug disposition gene regulation. Journal of Pharmaceutical Sciences, 94(6), 1169-1186.

Tirona, R. G., Lee, W., Leake, B. F., Lan, L. B., Brimer Cline, C., Lamba, V., Parviz, F., Duncan, S. A., Inoue, Y., Gonzalez, F. J., Schuetz, E. G. and Kim, R. B. (2003) The orphan nuclear receptor HNF4α determines PXR- and CAR-mediated xenobiotic induction of CYP3A4. Nature Medicine, 9(2), 220-224.

269

Tishler, D. M., Weinberg, K. I., Hinton, D. R., Barbaro, N., Annett, G. M. and Raffel, C. (1995) MDR1 gene expression in brain of patients with medically intractable epilepsy. Epilepsia, 36(1), 1-6.

Tong, L., Phan, T. K., Robinson, K. L., Babusis, D., Strab, R., Bhoopathy, S., Hidalgo, I. J., Rhodes, G. R. and Ray, A. S. (2007) Effects of human immunodeficiency virus protease inhibitors on the intestinal absorption of tenofovir disoproxil fumarate in vitro. Antimicrobial Agents and Chemotherapy, 51(10), 3498-3504.

Tong, X. K. and Hamel, E. (1999) Regional cholinergic denervation of cortical microvessels and nitric oxide synthase-containing neurons in Alzheimer's disease. Neuroscience, 92(1), 163-175.

Tran, C. D. H., Timmins, P., Conway, B. R. and Irwin, W. J. (2002) Investigation of the coordinated functional activities of cytochrome P450 3A4 and P-glycoprotein in limiting the absorption of xenobiotics in Caco-2 cells. Journal of Pharmaceutical Sciences, 91(1), 117-128.

Trousson, A., Bernard, S., Petit, P. X., Liere, P., Pianos, A., El Hadri, K., Lobaccaro, J. M. A., Said Ghandour, M., Raymondjean, M., Schumacher, M. and Massaad, C. (2009) 25- hydroxycholesterol provokes oligodendrocyte cell line apoptosis and stimulates the secreted phospholipase A2 type IIA via LXR beta and PXR. Journal of Neurochemistry, 109(4), 945-958.

Tsai, S. Y., Carlstedt-Duke, J., Weigel, N. L., Dahlman, K., Gustafsson, J. A., Tsai, M. J. and O'Malley, B. W. (1988) Molecular interactions of steroid hormone receptor with its enhancer element: Evidence for receptor dimer formation. Cell, 55(2), 361-369.

Tsuji, A., Terasaki, T., Takabatake, Y., Tenda, Y., Tamai, I., Yamashima, T., Moritani, S., Tsuruo, T. and Yamashita, J. (1992) P-Glycoprotein as the drug efflux pump in primary cultured bovine brain capillary endothelial cells. Life Sciences, 51(18), 1427-1437.

Uchida, Y., Ohtsuki, S., Kamiie, J. and Terasaki, T. (2011a) Blood-Brain Barrier (BBB) pharmacoproteomics: Reconstruction of in vivo brain distribution of 11 P-glycoprotein substrates based on the BBB transporter protein concentration, in vitro intrinsic transport activity, and unbound fraction in plasma and brain in mice. Journal of Pharmacology and Experimental Therapeutics, 339(2), 579-588.

Uchida, Y., Ohtsuki, S., Katsukura, Y., Ikeda, C., Suzuki, T., Kamiie, J. and Terasaki, T. (2011b) Quantitative targeted absolute proteomics of human blood-brain barrier transporters and receptors. Journal of Neurochemistry, 117(2), 333-345.

Ueda, K., Cornwell, M. M., Gottesman, M. M., Pastan, I., Roninson, I. B., Ling, V. and Riordan, J. R. (1986) The mdr1 gene, responsible for multidrug-resistance, codes for P-glycoprotein. Biochemical and Biophysical Research Communications, 141(3), 956-962.

UNAIDS (2012) Global report: UNAIDS report on the global AIDS epidemic 2012. (J. U. N. P. o. H. A. (UNAIDS) ed.). WHO Library Cataloguing-in-Publication Data.

270

Urich, E., Lazic, S. E., Molnos, J., Wells, I. and Freskgård, P. O. (2012) Transcriptional profiling of human brain endothelial cells reveals key properties crucial for predictive in vitro blood-brain barrier models. PLoS ONE, 7(5).

Urquhart, B. L. and Kim, R. B. (2009) Blood-brain barrier transporters and response to CNS- active drugs. European Journal of Clinical Pharmacology, 65(11), 1063-1070.

Urquhart, B. L., Tirona, R. G. and Kim, R. B. (2007) Nuclear receptors and the regulation of drug-metabolizing enzymes and drug transporters: Implications for interindividual variability in response to drugs. Journal of Clinical Pharmacology, 47(5), 566-578.

Uwai, Y., Ida, H., Tsuji, Y., Katsura, T. and Inui, K. I. (2007) Renal transport of adefovir, cidofovir, and tenofovir by SLC22A family members (hOAT1, hOAT3, and hOCT2). Pharmaceutical Research, 24(4), 811-815.

Van der Bliek, A. M., Kooiman, P. M., Schneider, C. and Borst, P. (1988) Sequence of mdr3 cDNA encoding a human P-glycoprotein. Gene, 71(2), 401-411.

Van der Sandt, I. C. J., Vos, C. M. P., Nabulsi, L., Blom-Roosemalen, M. C. M., Voorwinden, H. H., De Boer, A. G. and Breimer, D. D. (2001) Assessment of active transport of HIV protease inhibitors in various cell lines and the in vitro blood-brain barrier. AIDS, 15(4), 483-491.

Van Praag, R. M. E., Weverling, G. J., Portegies, P., Jurriaans, S., Zhou, X. J., Turner-Foisy, M. L., Sommadossi, J. P., Burger, D. M., Lange, J. M. A., Hoetelmans, R. M. W. and Prins, J. M. (2000) Enhanced penetration of indinavir in cerebrospinal fluid and semen after the addition of low-dose ritonavir. AIDS, 14(9), 1187-1194.

Van Vliet, E. A., Van Schaik, R., Edelbroek, P. M., Redeker, S., Aronica, E., Wadman, W. J., Marchi, N., Vezzani, A. and Gorter, J. A. (2006) Inhibition of the multidrug transporter P- glycoprotein improves seizure control in phenytoin-treated chronic epileptic rats. Epilepsia, 47(4), 672-680.

Varatharajan, L. and Thomas, S. A. (2009) The transport of anti-HIV drugs across blood-CNS interfaces: Summary of current knowledge and recommendations for further research. Antiviral Research, 82(2).

Vaucher, E. and Hamel, E. (1995) Cholinergic basal forebrain neurons project to cortical microvessels in the rat: Electron microscopic study with anterogradely transported Phaseolus vulgaris leucoagglutinin and choline acetyltransferase immunocytochemistry. Journal of Neuroscience, 15(11), 7427-7441.

Velazquez-Campoy, A., Muzammil, S., Ohtaka, H., Schön, A., Vega, S. and Freire, E. (2003) Structural and thermodynamic basis of resistance to HIV-1 protease inhibition: Implications for inhibitor design. Current Drug Targets - Infectious Disorders, 3(4), 311-328.

Vishnuvardhan, D., Von Moltke, L. L., Richert, C. and Greenblatt, D. J. (2003) Lopinavir: Acute exposure inhibits P-glycoprotein; extended exposure induces P-glycoprotein. AIDS, 17(7), 1092- 1094.

271

Vivithanaporn, P., Heo, G., Gamble, J., Krentz, H. B., Hoke, A., Gill, M. J. and Power, C. (2010) Neurologic disease burden in treated HIV/AIDS predicts survival: A population-based study. Neurology, 75(13), 1150-1158.

Volk, H. A., Burkhardt, K., Potschka, H., Chen, J., Becker, A. and Löscher, W. (2004) Neuronal expression of the drug efflux transporter P-glycoprotein in the rat hippocampus after limbic seizures. Neuroscience, 123(3), 751-759.

Vorbrodt, A. W. (1988) Ultrastructural cytochemistry of blood-brain barrier endothelia. Progress in Histochemistry and Cytochemistry, 18(3), 1-96.

Vorbrodt, A. W. and Dobrogowska, D. H. (2003) Molecular anatomy of intercellular junctions in brain endothelial and epithelial barriers: Electron microscopist's view. Brain Research Reviews, 42(3), 221-242.

Vu, K., Weksler, B., Romero, I., Couraud, P. O. and Gelli, A. (2009) Immortalized human brain endothelial cell line HCMEC/D3 as a model of the blood-brain barrier facilitates in vitro studies of central nervous system infection by cryptococcus neoformans. Eukaryotic Cell, 8(11), 1803- 1807.

Wagner, C. C., Bauer, M., Karch, R., Feurstein, T., Kopp, S., Chiba, P., Kletter, K., Löscher, W., Müller, M., Zeitlinger, M. and Langer, O. (2009) A pilot study to assess the efficacy of tariquidar to inhibit P-glycoprotein at the human blood-brain barrier with (R)-11C- verapamil and PET. Journal of Nuclear Medicine, 50(12), 1954-1961.

Wagner, M., Halilbasic, E., Marschall, H. U., Zollner, G., Fickert, P., Langner, C., Zatloukal, K., Denk, H. and Trauner, M. (2005) CAR and PXR agonists stimulate hepatic bile acid and bilirubin detoxification and elimination pathways in mice. Hepatology, 42(2), 420-430.

Walker, D. K., Abel, S., Comby, P., Muirhead, G. J., Nedderman, A. N. and Smith, D. A. (2005) Species differences in the disposition of the CCR5 antagonist, UK-427,857, a new potential treatment for HIV. Drug Metab Dispos, 33(4), 587-595.

Wang, H., Faucette, S., Moore, R., Sueyoshi, T., Negishi, M. and LeCluyse, E. (2004) Human constitutive androstane receptor mediates induction of CYP2B6 gene expression by phenytoin. Journal of Biological Chemistry, 279(28), 29295-29301.

Wang, H., Faucette, S., Sueyoshi, T., Moore, R., Ferguson, S., Negishi, M. and LeCluyse, E. L. (2003a) A novel distal enhancer module regulated by pregnane X receptor/constitutive androstane receptor is essential for the maximal induction of CYP2B6 gene expression. Journal of Biological Chemistry, 278(16), 14146-14152.

Wang, H. and LeCluyse, E. L. (2003) Role of Orphan Nuclear Receptors in the Regulation of Drug-Metabolising Enzymes. Clinical Pharmacokinetics, 42(15), 1331-1357.

Wang, H. and Tompkins, L. M. (2008) CYP2B6: New insights into a historically overlooked cytochrome P450 isozyme. Current Drug Metabolism, 9(7), 598-610.

272

Wang, K. and Zhong, L. F. (2003) The intestinal cytochrome P450 3A and P-glycoprotein: Barriers to drug absorption. Chinese Pharmacological Bulletin, 19(11), 1216-1219.

Wang, Q., Yang, H. and Elmquist, W. F. (1996) The effect of LY-335979 on the distribution kinetics of quinidine to the brain: An in vivo microdialysis study in freely-moving rats. Pharmaceutical Research, 13(S-456), 1.

Wang, X., Furukawa, T., Nitanda, T., Okamoto, M., Sugimoto, Y., Akiyama, S. I. and Baba, M. (2003b) Breast cancer resistance protein (BCRP/ABCG2) induces cellular resistance to HIV-1 nucleoside reverse transcriptase inhibitors. Molecular Pharmacology, 63(1), 65-72.

Wang, X., Sykes, D. B. and Miller, D. S. (2010) Constitutive androstane receptor-mediated upregulation of ATP-driven xenobiotic efflux transporters at the blood-brain barrier. molecular Pharmacology, 78(3), 376-383.

Waterkotte, B., Hambruch, N., Döring, B., Geyer, J., Tinneberg, H. R. and Pfarrer, C. (2011) P- glycoprotein is functionally expressed in the placenta-derived bovine caruncular epithelial cell line 1 (BCEC-1). Placenta, 32(2), 146-152.

Watkins, R. E., Noble, S. M. and Redinbo, M. R. (2002) Structural insights into the promiscuity and function of the human pregnane X receptor. Current Opinion in Drug Discovery and Development, 5(1), 150-158.

Watkins, R. E., Wisely, G. B., Moore, L. B., Collins, J. L., Lambert, M. H., Williams, S. P., Willson, T. M., Kliewer, S. A. and Redinbo, M. R. (2001) The human nuclear xenobiotic receptor PXR: Structural determinants of directed promiscuity. Science, 292(5525), 2329-2333.

Wei, P., Zhang, J., Dowhan, D. H., Han, Y. and Moore, D. D. (2002a) Specific and overlapping functions of the nuclear hormone receptors CAR and PXR in xenobiotic response. Pharmacogenomics Journal, 2(2), 117-126.

Wei, P., Zhang, J., Dowhan, D. H., Han, Y. and Moore, D. D. (2002b) Specific and overlapping functions of the nuclear hormone receptors CAR and PXR in xenobiotic response. Pharmacogenomics J, 2(2), 117-126.

Wei, P., Zhang, J., Egan-Haftey, M., Liang, S. and Moore, D. D. (2000) The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism. Nature, 407(6806), 920-923.

Weiss, J., Herzog, M., Konig, S., Storch, C. H., Ketabi-Kiyanvash, N. and Haefeli, W. E. (2009) Induction of multiple drug transporters by efavirenz. Journal of Pharmaceutical Sciences, 109(2), 242-250.

Weiss, J., Rose, J., Storch, C. H., Ketabi-Kiyanvash, N., Sauer, A., Haefeli, W. E. and Efferth, T. (2007a) Modulation of human BCRP (ABCG2) activity by anti-HIV drugs. The Journal of antimicrobial chemotherapy, 59(2), 238-245.

Weiss, J., Theile, D., Ketabi-Kiyanvash, N., Lindenmaier, H. and Haefeli, W. E. (2007b) Inhibition of MRP1/ABCC1, MRP2/ABCC2, and MRP3/ABCC3 by nucleoside, nucleotide, and

273

non-nucleoside reverse transcriptase inhibitors. Drug Metabolism and Disposition, 35(3), 340- 344.

Weiss, J., Weis, N., Ketabi-Kiyanvash, N., Storch, C. H. and Haefeli, W. E. (2008) Comparison of the induction of P-glycoprotein activity by nucleotide, nucleoside, and non-nucleoside reverse transcriptase inhibitors. European Journal of Pharmacology, 579(1-3), 104-109.

Weiss, R. A. (1993) How does HIV cause AIDS? Science, 260(5112), 1273-1279.

Weksler, B., Romero, I. A. and Couraud, P. O. (2013) The hCMEC/D3 cell line as a model of the human blood brain barrier. Fluids and Barriers of the CNS, 10(1), 1-10.

Weksler, B. B., Subileau, E. A., Perrière, N., Charneau, P., Holloway, K., Leveque, M., Tricoire- Leignel, H., Nicotra, A., Bourdoulous, S., Turowski, P., Male, D. K., Roux, F., Greenwood, J., Romero, I. A. and Couraud, P. O. (2005) Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J, 19(13), 1872-1874.

Westerhout, J., Ploeger, B., Smeets, J., Danhof, M. and De Lange, E. C. M. (2012) Physiologically based pharmacokinetic modeling to investigate regional brain distribution kinetics in rats. AAPS Journal, 14(3), 543-553.

Westerhout, J., Smeets, J., Danhof, M. and de Lange, E. C. M. (2013) The impact of P-gp functionality on non-steady state relationships between CSF and brain extracellular fluid. Journal of Pharmacokinetics and Pharmacodynamics, 1-16.

Westin, S., Kurokawa, R., Nolte, R. T., Wisely, G. B., McInerney, E. M., Rose, D. W., Milburn, M. V., Rosenfeld, M. G. and Glass, C. K. (1998) Interactions controlling the assembly of nuclear-receptor heterodimers and co-activators. Nature, 395(6698), 199-202.

Whitfield, G. K., Jurutka, P. W., Haussler, C. A. and Haussler, M. R. (1999) Steroid hormone receptors: Evolution, ligands, and molecular basis of biologic function. Journal of Cellular Biochemistry, 76(SUPPL. 32/33), 110-122.

Wolburg, H. and Lippoldt, A. (2002) Tight junctions of the blood-brain barrier: Development, composition and regulation. Vascular Pharmacology, 38(6), 323-337.

Wolburg, H., Wolburg-Buchholz, K. and Engelhardt, B. (2005) Diapedesis of mononuclear cells across cerebral venules during experimental autoimmune encephalomyelitis leaves tight junctions intact. Acta Neuropathologica, 109(2), 181-190.

Woodland, C., Huang, T. T., Gryz, E., Bendayan, R. and Fawcett, J. P. (2008) Expression, activity and regulation of CYP3A in human and rodent brain. Drug Metabolism Reviews, 40(1), 149-168.

Wu, K. C., Pan, H. J., Yin, H. S., Chen, M. R., Lu, S. C. and Lin, C. J. (2009) Change in P- glycoprotein and caveolin protein expression in brain striatum capillaries in New Zealand Obese mice with type 2 diabetes. Life Sciences, 85(23-26), 775-781.

274

Wyatt, R. and Sodroski, J. (1998) The HIV-1 envelope glycoproteins: Fusogens, antigens, and immunogens. Science, 280(5371), 1884-1888.

Xia, J. and Kemper, B. (2005) Structural determinants of constitutive androstane receptor required for its glucocorticoid receptor interacting protein-1-mediated nuclear accumulation. Journal of Biological Chemistry, 280(8), 7285-7293.

Xiong, H., Yoshinari, K., Brouwer, K. L. R. and Negishi, M. (2002) Role of constitutive androstane receptor in the in vivo induction of Mrp3 and CYP2B1/2 by phenobarbital. Drug Metabolism and Disposition, 30(8), 918-923.

Xu, R. X., Lambert, M. H., Wisely, B. B., Warren, E. N., Weinert, E. E., Waitt, G. M., Williams, J. D., Collins, J. L., Moore, L. B., Willson, T. M. and Moore, J. T. (2004) A structural basis for constitutive activity in the human CAR/RXRα heterodimer. Molecular Cell, 16(6), 919-928.

Xue, Y., Chao, E., Zuercher, W. J., Willson, T. M., Collins, J. L. and Redinbo, M. R. (2007) Crystal structure of the PXR-T1317 complex provides a scaffold to examine the potential for receptor antagonism. Bioorganic & Medicinal Chemistry, 15(5), 2156-2166.

Yagüe, E., Armesilla, A. L., Harrison, G., Elliott, J., Sardini, A., Higgins, C. F. and Raguz, S. (2003) P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation. Journal of Biological Chemistry, 278(12), 10344-10352.

Yan, F., Wang, X. M., Liu, Z. C., Pan, C., Yuan, S. B. and Ma, Q. M. (2010) JNK1, JNK2, and JNK3 are involved in P-glycoprotein-mediated multidrug resistance of hepatocellular carcinoma cells. Hepatobiliary & Pancreatic Diseases International, 9(3), 287-295.

Yao, S. Y. M., Ng, A. M. L., Sundaram, M., Cass, C. E., Baldwin, S. A. and Young, J. D. (2001) Transport of antiviral ′3 -deoxy-nucleoside drugs by recombinant human and rat equilibrative, nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced in Xenopus oocytes. Molecular Membrane Biology, 18(2), 161-167.

Yilmaz, A., Gisslén, M., Spudich, S., Lee, E., Jayewardene, A., Aweeka, F. and Price, R. W. (2009a) Raltegravir cerebrospinal fluid concentrations in HIV-1 infection. PLoS ONE, 4(9).

Yilmaz, A., Izadkhashti, A., Price, R. W., Mallon, P. W., De Meulder, M., Timmerman, P. and Gisslén, M. (2009b) Darunavir concentrations in cerebrospinal fluid and blood in HIV-1-infected individuals. AIDS Research and Human Retroviruses, 25(4), 457-461.

Yoshinari, K., Sueyoshi, T., Moore, R. and Negishi, M. (2001) Nuclear receptor CAR as a regulatory factor for the sexually dimorphic induction of CYP2B1 gene by phenobarbital in rat livers. Molecular Pharmacology, 59(2), 278-284.

Yu, A. M., Fukamachi, K., Krausz, K. W., Cheung, C. and Gonzalez, F. J. (2005) Potential role for human cytochrome P450 3A4 in estradiol homeostasis. Endocrinology, 146(7), 2911-2919.

275

Yu, C., Argyropoulos, G., Zhang, Y., Kastin, A. J., Hsuchou, H. and Pan, W. (2008) Neuroinflammation activates Mdr1b efflux transport through NFκB: Promoter analysis in BBB endothelia. Cellular Physiology and Biochemistry, 22(5-6), 745-756.

Yu, F., Lu, L., Du, L., Zhu, X., Debnath, A. K. and Jiang, S. (2012) Approaches for identification of HIV-1 entry inhibitors targeting gp41 pocket. Viruses, 5(1), 127-149.

Yueh, M. F., Kawahara, M. and Raucy, J. (2005) High volume bioassays to assess CYP3A4- mediated drug interactions: Induction and inhibition in a single cell line. Drug Metabolism and Disposition, 33(1), 38-48.

Yuen, G. J., Weller, S. and Pakes, G. E. (2008) A review of the pharmacokinetics of abacavir. Clinical Pharmacokinetics, 47(6), 351-371.

Zameer, A. and Hoffman, S. A. (2003) Increased ICAM-1 and VCAM-1 expression in the brains of autoimmune mice. Journal of Neuroimmunology, 142(1-2), 67-74.

Zastre, J. A., Chan, G. N. Y., Ronaldson, P. T., Ramaswamy, M., Couraud, P. O., Romero, I. A., Weksler, B., Bendayan, M. and Bendayan, R. (2009) Up-regulation of p-glycoprotein by HIV protease inhibitors in a human brain microvessel endothelial cell line. Journal of Neuroscience Research, 87(4), 1023-1036.

Zelko, I., Sueyoshi, T., Kawamoto, T., Moore, R. and Negishi, M. (2001) The peptide near the C terminus regulates receptor CAR nuclear translocation induced by xenochemicals in mouse liver. Molecular and Cellular Biology, 21(8), 2838-2846.

Zhai, Y., Pai, H. V., Zhou, J., Amico, J. A., Vollmer, R. R. and Xie, W. (2007) Activation of pregnane X receptor disrupts glucocorticoid and mineralocorticoid homeostasis. Molecular Endocrinology, 21(1), 138-147.

Zhang, H., Leculyse, E., Liu, L., Hu, M., Matoney, L., Zhu, W. and Yan, B. (1999) Rat pregnane X receptor: Molecular cloning, tissue distribution, and xenobiotic regulation. Archives of Biochemistry and Biophysics, 368(1), 14-22.

Zhang, J., Huang, W., Chua, S. S., Wei, P. and Moore, D. D. (2002) Modulation of acetaminophen-induced hepatotoxicity by the xenobiotic receptor CAR. Science, 298(5592), 422-424.

Zhang, L., Gorset, W., Washington, C. B., Blaschke, T. F., Kroetz, D. L. and Giacomini, K. M. (2000) Interactions of HIV protease inhibitors with a human organic cation transporter in a mammalian expression system. Drug Metabolism and Disposition, 28(3), 329-334.

Zhang, Z., Wu, J. Y., Hait, W. N. and Yang, J. M. (2004) Regulation of the stability of P- glycoprotein by ubiquitination. Molecular Pharmacology, 66(3), 395-403.

Zhou, J., Liu, M., Aneja, R., Chandra, R., Lage, H. and Joshi, H. C. (2006) Reversal of P- glycoprotein-mediated multidrug resistance in cancer cells by the c-Jun NH2-terminal kinase. Cancer Research, 66(1), 445-452.

276

Zhu, M., Kaul, S., Nandy, P., Grasela, D. M. and Pfister, M. (2009) Model-based approach to characterize efavirenz autoinduction and concurrent enzyme induction with carbamazepine. Antimicrobial Agents and Chemotherapy, 53(6), 2346-2353.

Zibell, G., Unkrüer, B., Pekcec, A., Hartz, A. M. S., Bauer, B., Miller, D. S. and Potschka, H. (2009) Prevention of seizure-induced up-regulation of endothelial P-glycoprotein by COX-2 inhibition. Neuropharmacology, 56(5), 849-855.

Zlokovic, B. V. (1995) Cerebrovascular permeability to peptides: Manipulations of transport systems at the blood-brain barrier. Pharmaceutical Research, 12(10), 1395-1406.

Zlokovic, B. V. (2008) The Blood-Brain Barrier in Health and Chronic Neurodegenerative Disorders. Neuron, 57(2), 178-201.

Zlokovic, B. V. and Apuzzo, M. L. J. (1998) Strategies to circumvent vascular barriers of the central nervous system. Neurosurgery, 43(4), 877-878.

Zougbédé, S., Miller, F., Ravassard, P., Rebollo, A., Cicéron, L., Couraud, P. O., Mazier, D. and Moreno, A. (2011) Metabolic acidosis induced by Plasmodium falciparum intraerythrocytic stages alters blood-brain barrier integrity. Journal of Cerebral Blood Flow and Metabolism, 31(2), 514-526.

277

13. List of Relevant Publications

1. Chan, G. N. Y., Patel, R., Cummins, C. L. and Bendayan, R. (2013) Induction of P- glycoprotein by Antiretroviral Drugs in Human Brain Microvessel Endothelial Cells. Antimicrobial Agents and Chemotherapy. 8 July, doi: 10.1128/AAC.00486-13. [Epub ahead of print]

2. Chan, G. N. Y., Saldivia, V., Yang, Y., Pang, H., de Lannoy, I. and Bendayan, R. (2013) In vivo induction of P-glycoprotein expression at the mouse blood-brain barrier: An intracerebral microdialysis study. Journal of Neurochemistry. 18 Jun. doi: 10.1111/jnc.12344. [Epub ahead of print].

3. Chan, G. N. Y., Hoque, M. T. and Bendayan, R. (2013) Role of nuclear receptors in the regulation of drug transporters in the brain. Trends in Pharmacological Sciences, 34(7), 361-372.

4. Ashraf, T., Robillard, K., Chan, G. N. Y., Bendayan, R. (2013) Role of CNS transporters in pharmacotherapy of brain HIV-1 infection. Current Pharmaceutical Design. 19 Jun. [Epub ahead of print]

5. Robillard K., Chan, G. N. Y., Zhang, G., La Porte, C., Cameron, W. and Bendayan, R. (2013) The Role of Drug transporters in Affecting Drug Permeability in Male Genital Tract. In preparation.

6. Durk, M. R., Chan, G. N. Y., Campos, C. R., Peart, J. C., Chow, E. C. Y., Lee, E., Cannon, R. E., Bendayan, R., Miller, D. S. and Pang, K. S. (2012) 1α,25- Dihydroxyvitamin D3-liganded vitamin D receptor increases expression and transport activity of P-glycoprotein in isolated rat brain capillaries and human and rat brain microvessel endothelial cells. Journal of Neurochemistry, 123(6), 944-953.

7. Chan, G. N. Y., Hoque, M. T., Cummins, C. L. and Bendayan, R. (2011) Regulation of P-glycoprotein by orphan nuclear receptors in human brain microvessel endothelial cells. Journal of Neurochemistry, 118(2), 163-175.

8. Chan, G. N. Y. and Bendayan, R. (2011) Molecular and functional characterization of P- glycoprotein in vitro. Methods in molecular biology (Clifton, N.J.), 686:313-336.

9. Kis, O., Robillard, K., Chan, G. N. Y. and Bendayan, R. (2010) The complexities of antiretroviral drug-drug interactions: role of ABC and SLC transporters. Trends in Pharmacological Sciences, 31(1), 22-35.

10. Zastre, J. A., Chan, G. N. Y., Ronaldson, P. T., Ramaswamy, M., Couraud, P. O., Romero, I. A., Weksler, B., Bendayan, M. and Bendayan, R. (2009) Up-regulation of p- glycoprotein by HIV protease inhibitors in a human brain microvessel endothelial cell line. Journal of Neuroscience Research, 87(4), 1023-1036.

278 Appendix A. Cell Viability Studies in hCMEC/D3 cells

Table A-1: Viability of hCMEC/D3 cells following 72 h incubation with ligands or antiretroviral drugs

Cell Viability ± Treatment S.E.M. (%)

SR12813 10 µM 87.3 ± 5.0

Rifampin 10 µM 89.5 ± 1.4

CITCO 7.5 µM 81.0 ± 0.60

Ligands A792611 2.5 µM 94.2 ± 4.1

Meclizine 2.5 µM 94.0 ± 3.9

SR12813 10 µM + A792611 2.5 µM 84.4 ± 1.2

CITCO 7.5 µM + Meclizine 2.5 µM 83.2 ± 3.1

Amprenavir 15 µM 109.7 ± 7.9

Atazanavir 10 µM 92.4 ± 3.1

Darunavir 10 M 104.9 ± 3.0

Lopinavir 10 µM 81.7 ± 4.3 Antiretroviral Drugs Ritonavir 10 µM 85.3 ± 0.20

Abacavir 15 µM 90.4 ± 1.1

Efavirenz 10 µM 86.8 ± 3.6

Nevirapine 15 µM 96.2 ± 5.3

Cell viability was calculated from normalizing the absorbance (580 nm) of reduced (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT, in drug- treated cells with that measured in vehicle-treated cells. MTT cell viability assays performed by GNY Chan in the laboratory of Dr. Reina Bendayan.

279 Appendix B. Initial characterization of P-gp-mediated transport of R-6G

3.0 * 2.5

2.0

1.5

1.0

0.5 R-6G Accumulation (pmol/mg protein) 0.0

M MK571 µM KO143 µM PSC833 µ 5 10 10 Control (DMSO)

Figure B-1: R-6G accumulation by hCMEC/D3 cells. Confluent cell monolayers were exposed to 1 µM of R-6G in the presence of DMSO (control), 5 µM P-gp inhibitor PSC833, 10 µM MRPs inhibitor MK571 or 10 µM BCRP inhibitor KO143 for 30 min at 37 °C. Data represent mean ± S.E.M. with n = 4 independent experiments. * Statistically significant difference in cellular accumulation of R-6G between control and PSC833-treated cells, determined by Student’s t test with p < 0.05. No statistically significant difference was observed between control and MK571 or KO143-treated cells (p = 0.67 and 0.56, respectively). (Transport assays performed by GNY Chan in the laboratory of Dr. Reina Bendayan)

280 MDA-WT 1.5 MDA-MDR1 ** * 1.0

0.5 R-6G Accumulation (pmol/mg protein) 0.0

No PSC833 µM PSC833 1

Figure B-2: R-6G accumulation by P-gp over-expressing cells, MDA-MDR1, and its corresponding wild-type cells, MDA-WT. Confluent cell monolayers were exposed to 5 µM of R-6G in the presence or absence of 1 µM selective P-gp inhibitor PSC833 for 30 min at 37 °C. Data represent mean ± S.E.M. with n = 4 independent experiments. * Statistically significant difference in cellular accumulation of R-6G without PSC833 between P-gp over-expressing and wild-type cells, determined by Student’s t test with p < 0.05. ** Statistically significant difference in cellular accumulation of R-6G between control and PSC833-treated P-gp overexpressing cells, determined by Student’s t test with p < 0.05. No statistically significant difference was observed between control and PSC833-treated wild-type cells (p = 0.99). (Transport assays performed by GNY Chan in the laboratory of Dr. Reina Bendayan)

281 Appendix C. Morphology and Protein Expression of Mouse Brain Capillaries

Figure C-1. Transmitted light image of a mouse brain capillary. Dark scale bar represents 20 μm. Brain capillaries were attached to glass cover slip and fixed in PBS containing 3 % paraformaldehyde and 0.25 % glutaraldehyde for 10 min. Image was taken using a Zeiss LSM510 META microscopy system under a C-Apochromat 63X lens (Carl Zeiss Canada, Ltd., Toronto, ON, Canada). (Brain capillary isolation and microscopy were performed by GNY Chan in the laboratory of Dr. Reina Bendayan and Princess Margaret Hospital, University Health Network, Toronto, ON, Canada, respectively)

282

Figure C-2. Protein expression of P-gp, Bcrp, mPXR, zona occludens-1 (ZO-1) and occludin determined by immunoblot analysis in lysates of mouse brain capillary, mouse liver and Caco2 cells. Whole cell lysates of Caco-2 (50 μg, positive control for ZO-1 and occludin), tissue lysates of mouse liver (25 μg, positive control for P-gp, Bcrp and mPXR) and mouse brain capillary (50 μg) were resolved on 10 % SDS-PAGE gels and subsequently transferred to PVDF membrane. P-gp was detected using the mouse monoclonal C219 anti-P-gp antibody at a dilution of 1:500 (ID Labs). Bcrp was detected using the rat monoclonal BXP-53 anti-Bcrp antibody at a dilution of 1:250 (Kamiya). Mouse PXR (mPXR) was detected using the goat polyclonal A-20 anti-PXR antibody at a dilution of 1:100 (Santa Cruz Biotechnology, Inc.). Actin was detected using the mouse monoclonal C4 anti-actin antibody at a dilution of 1:2000 (Santa Cruz Biotechnology, Inc.). ZO-1 was detected using the rabbit monoclonal anti-ZO-1 antibody at a dilution of 1:200 (Invitrogen, 40-2300). Occludin was detected using the rabbit monoclonal anti-occludin antibody at a dilution of 1:200 (Invitrogen, 71-1500). (Experiment performed by GNY Chan in the laboratory of Dr. Reina Bendayan)

283 Appendix D. Chan G.N.Y. and Bendayan R. 2010. In Vitro Study of P- glycoprotein at the Blood-brain Barrier: Molecular Expression, Localization and Functional Activity. Methods in Molecular Biology 686:313-36.

This manuscript is reproduced in this thesis with permission from Springer.

Author Contribution: Chan G.N.Y. (primary author; full manuscript and responses to reviewer

comments); Bendayan R (Editorial review of several drafts and responses to reviewer comments)

284 Abstract

The blood-brain barrier (BBB) physically and metabolically functions as a neurovascular

interface between the brain parenchyma and the systemic circulation, and primarily regulates the

permeability of several endogenous substrates and xenobiotics into and out of the central nervous

system (CNS). Several membrane-associated transport proteins, such as P-glycoprotein (P-gp),

Multidrug Resistance-Associated Proteins (MRPS), Breast Cancer Resistance Protein (BCRP) and Organic Anion Transporting Polypeptides (OATPs), have been characterized at the BBB and identified to play a major role in regulating drug brain permeability. This chapter reviews several well-established techniques for the study of the molecular expression, cellular localization and functional activity of transport proteins in primary and immortalized cell culture systems of the BBB. In particular, we describe the molecular characterization of P-gp/MDR1 transcript level using semi-quantitative Polymerase Chain Reaction (PCR), P-gp protein expression using immunoblotting analysis, P-gp localization using immunofluorescence studies and uptake/efflux and transepithelial flux studies which characterize P-gp transport activity.

Key Words: Blood-brain barrier, brain microvessel endothelial cells, astrocytes, ATP-binding

Cassette Membrane Transporter, P-glycoprotein, cell culture, PCR, immunoblotting, transport activity, transepithelial flux and drug accumulation studies.

1. Introduction

The blood-brain barrier (BBB) constitutes a remarkable physical and biochemical barrier between the brain and the systemic circulation. Structurally, it is primarily composed of non fenestrated microvessel endothelial cells characterized by the presence of very tight junctions.

These junctions form a continuous almost impermeable cellular barrier that dramatically limits paracellular flux and transport as well the influx of endogenous and exogenous substrates. The

285 high transendothelial electrical resistance further restricts the free flow of water and solutes. It is now well accepted that the functional unit of the BBB includes more than just capillary endothelial cells. Several other cell types, in particular pericytes and perivascular astrocytes are in constant and intimate contact with the endothelium and maintenance of the brain capillary phenotype seems to be critically dependent on interactions with these other cells (1,2). In

addition to the physical barrier, there is a selective metabolism-driven barrier that largely reflects

expression and function of several receptors, ion channels, metabolic enzymes and influx/efflux

transport proteins expressed prominently at the BBB. In particular, ATP-binding cassette (ABC)

membrane transporters such as P-glycoprotein (P-gp), Multidrug Resistance-associated Proteins

(MRPs) and Breast Cancer Resistance Protein (BCRP, ABCG2) play a significant role in restricting the permeability of several pharmacological agents including anti-cancer and anti-HIV

agents (3-6). They are expressed at the luminal membrane of the brain capillary and serve as

efflux pumps to extrude substrates from the brain tissue back into the circulation (5-11).

Together with the members of the organic anion transporting polypeptide family (OATPs), these

influx and efflux transport proteins are believed to ultimately regulate the overall

pharmacokinetic and pharmacodynamic profile of xenobiotics in the brain (3,6,9,12).

Functionally, brain transport proteins are similar to well-characterized systems in other tissues,

although the capacity and rate of transport can vary widely (13). Therefore, further investigation

is required to characterize and fully understand the clinical impact of these transport proteins in

the central nervous system (CNS).

Since the early 1990s, cell culture models have proven a useful approach to study the

membrane permeability of several substrates including therapeutic agents at several epithelial

barriers. In this chapter, we focus on the molecular characterization of P-gp/MDR1 transcript

286 level, protein expression, localization and transport activity using several cell systems which are

representative of the BBB, such as the human cerebral microvessel endothelial cells/D3

(hCMEC/D3), rat brain endothelial 4 cells (RBE4) and the primary cultures of rat and human

astrocyte. These cell systems are known to retain several properties of the neurovascular unit at

the BBB in vivo, including the functional expression of many transport proteins necessary for the

permeability of nutrients and pharmacological agents, adhesion molecules, tight junction

molecules and some of their respective regulatory pathways (1,2,7,8,14-21).

In vitro, in order to improve transepithelial electrical resistance (TEER) and reduce paracellular permeability, astrocytes can be co-cultured with brain microvessel endothelial cells

in a transwell culture system in the presence of cyclic adenosine monophosphate (CAMP) analog

and hydrocortisone (22,23). Co-culture of astrocytes and brain microvessl endothelial cells can provide a powerful in vitro tool which mimics the complexity of the in vivo BBB, for the study

of bidirectional flux (apical to basolateral and basolateral to apical compartments) of substrates

across the brain vasculature.

Reverse transcription Polymerase Chain Reaction (PCR) has been well-utilized to study

changes in mRNA or transcript expression which can directly affect downstream post-

transcriptional protein expression. The semi-quantitative PCR technique that is described in this

chapter can be used to detect expression of mRNA encoding transport proteins.

Transport proteins expression is commonly quantified using immunoblotting analysis in

whole cell lysate or crude membrane lysate. The use of antibodies and their working dilution as

described in table D-2 serve as a guideline, since sensitivity of antibodies can vary depending on

the manufacturer and lot. A positive control consists of purified transporter protein or lysate of

cells overexpressing the protein of interest and should always be included in the experiment. To

287 further confirm the identity of a band, transport proteins which are heavily glycoslated with

carbohydrates, such as P-gp, can be deglycoslated (24).This method can reduce the apparent

molecular weight of the transporter protein determined by immunoblotting analysis and produce

a shift in the location of the band.

The cellular localization of P-gp can be detected using immunofluorescence. This

technique requires cells to be attached on a surface. The glutaraldehyde fixation method we

describe and other alternatives generally cause proteins to cross link in a meshwork, preserving

the protein mass and cell morphology. The fixation step is required to expose antigenic sites of

the transport proteins allowing the binding of primary antibodies and subsequent binding of

fluorochrome-conjugated antibodies to the primary antibodies. However, concentrations of the

fixation solution and fixation period can affect the antigen recognition by antibodies. In this

chapter, we describe the glutaraldehyde fixation method which forms a looser matrix for

antibodies to diffuse compared to other methods such as ethanol fixation (25). Not all primary

antibodies are suitable for immunofluorescence and it is necessary to perform a careful selection

to optimize antigen recognition. As well, if two or more fluorochrome-conjugated antibodies are used, one must avoid overlapping of emission or absorbance spectra between different fluorochrome-conjugated antibodies. Spectra overlapping can produce high background making detection of transport proteins difficult.

The detection of both intracellular mRNA and protein expression do not necessarily predict the functional activity of the transport proteins. For example, we have previously observed that BCRP expressed in human and rat brain microvessel endothelial cells (primary human brain endothelial cells and RBE4) has a low transport activity in these in vitro cell culture systems (26). Several drug uptake/accumulation and flux studies can be performed using a

288 combination of substrates and inhibitors selective for the transport protein of interest.

Radioactive or fluorescence labeled substrates are often used to characterize the functional

activity of the transporter protein of interest. One should be cautious in the selection of the cell

harvesting buffer to minimize interference with the fluorescence substrate used. As well, pH,

temperature and albumin concentration in the transport buffer can affect physical and chemical

properties of substrates, such as charge and protein binding. To specifically investigate the

directional flux across the endothelial cells, transepithelial flux assay using transwell tissue

culture plates can be used. In brief, this can be achieved by measuring the basolateral to apical

flux and apical to basolateral flux of substrate in the presence or absence of inhibitors.

2. Materials

2.1. Cell Culture Models of the Blood-brain Barrier

1. Reagent, culture medium and container was optimized for hCMEC/D3, an immortalized

human brain microvessel endothelial cell line developed by the Couraud group (7,15).

2. Reagent, culture medium and container was optimized for RBE 4, an immortalized rat brain

microvessel endothelial cell line developed by Roux et al. (1994) (8,16,27).

2.2 Cell Culture Models of Astrocyets

1. Reagent, culture medium and container was optimized for primary culture of rat astrocytes

used in our laboratory (18-20).

2. Reagent, culture medium and container was optimized for primary culture of human fetal

astrocytes used in our laboratory (28).

2.3. Molecular Characterization of Transport Protein (P-gp) at the Transcript Level

2.3.1. Isolation of Total Cellular mRNA

TRIZOL reagent (Invitrogen).

289 Sterile DPBS (Invitrogen, Gibco).

Chloroform(Sigma).

Isoamyl alcohol (Sigma).

Isopropanol (Sigma).

75 % Ethanol (prepared with DNAase and RNAase free water).

DNAase and RNAase free water (Invitrogen).

Tris-HCl buffer (Sigma).

2.3.2. Reverse Transcription Assay

Tris-HCl buffer (sigma).

DNAase and RNAase free water (Invitrogen, CA).

Reaction Mix: Oligo(dt) primer, 10 X PCR buffer, 10mM dNTP mix, 25mM MgCl2, 0.1M DTT and 1 U/µl DNase I per 2 µg RNA (Invitrogen).

SuperScript II reverse transcriptase (Invitrogen).

2.3.3. Semi-quantitative PCR

Primers used in semi-quantitative PCR (see Table D-1).

SuperScript II reverse transcriptase (Invitrogen).

Agarose (preferably electrophoresis purity grade) (Sigma).

Ethidium bromide (1 mg/mL in DNase-RNase-free water).

1 x Tris Borate EDTA (TBE) buffer (Invitrogen, GIBCO).

100bp DNA ladder (Invitrogen).

PCR Mix Kit: 2.5 µl of 10 X PCR buffer, 1.5 mM of 25mM MgCl2, 400µM for each dNTP’s,

1.0 µM of forward primer, 1.0 µM of reversed primer, 2.5U of Platinum Taq DNA polymerase,

top volume up to 20 μL using DEPC water or DNAase and RNAase free water) (Invitrogen).

290 6 X Gel loading dye: 0.25% Bromophenol blue, 40% w/v sucrose in ddH2O.

Table D-1 RT conditions and primers used in semi-quantitative PCR analysis are shown. FP – forward primer, RP – reversed primer.

Accession Annealing Genes Primer Sequence Fragment Species No. Temperature (5’→ 3’) length (bp) (°C)

FP: GGT-GCT-GGT-TGC- MDR1 TGC-TTA-CA Human NM 000927.3 55 291 RP: TGG-CCA-AAA-TCA- CAA-GGG-T FP: GGA-CAG-AAA-CAG- mdr1a NM 133401 AGG-ATC-GC Rodent 55 440 RP: CCC-GTC-TTG-ATC- ATG-TGG-CC FP: GGA-CAG-AAA-CAG- mdr1b NM 012623 AGG-ATC-GC Rodent 55 355 RP: TCA-GAG-GCA-CCA- GTG-TCA-CT FP: GAC-TAT-GAC-TTA- NM 001101.2 GTT-GCG-TTA β-Actin human 55 504 RP:GCC-TTC-ATA-CAT- CTC-AAG-TTG 2.4. Molecular Characterization of Transporter (P-gp) at the Protein Level

2.4.1. Preparation of Whole Cell Lysates from Adherent cells

0.25% Trypsin/EDTA (Invitrogen, Gibco).

Whole cell lysis buffer: 150mM NaCl, 20 mM Tris pH 7.5, 5mM EDTA, 1% NP-40, 2µL of protease inhibitor cocktail / mL of buffer (Sigma), 1mM of Phenylmethylsulphonyl fluoride

(PMSF) (Sigma).

Bradford protein assay (Bio-Rad).

2.4.2. Preparation of Membrane Lysates

0.25% Trypsin/EDTA (Invitrogen, Gibco).

Crude-membrane isolation buffer: 250 mM sucrose buffer containing 1.0 mM EDTA and 0.1%

(v/v) protease inhibitor cocktail (Sigma).

291 Bradford protein assay (Bio-Rad).

2.4.3. Immunoblotting of Transport Protein (P-gp)

100% methanol .

Hybond-P PVDF membrane (GE Healthcare Bio-Sciences, AB).

Gel holder cassette with sponge set (Bio-Rad).

Filter paper (Bio-Rad).

Western blotting Enhanced Chemiluminescence (ECL) system (GE).

Hyper film ECL (GE).

Developer (Kodak, Rochester, New York, USA): working dilution is 1:4 with ddH2O.

Fixer (Kodak Rochester): working dilution is 1:4 with ddH2O.

Antibodies used (see Table D-2).

Tris – Cl pH 8.8: 15mM Trizma-base (Sigma) in ddH2O, pH 8.8.

Lamelli buffer: 350mM SDS (GE Healthcare Bio-Sciences AB, Piscataway, NJ, USA), 17%

(v/v) ddH2O, 40% (v/v) Tris-Cl pH 6.8, 33% (v/v) glycerol (GE), Piscataway, NJ, USA),

0.006%(w/v) bromophenol blue (Sigma) and 10% 2-Mercaptoethanol (Sigma).

Electrophoresis running buffer: 25mM Trizma-base, 200mM glycine (Sigma), 3.5 mM SDS

(Sigma) in ddH2O.

Transfer buffer: 25mM Tris-base, 200mM glycine, 20% (v/v) methanol, pH 8 in ddH2O.

Ponceau-S solution: 0.1% (w/v) Ponceau S (Sigma) in 5% (v/v) acetic acid in ddH2O.

Tris Buffered Saline pH 7.6 w/ Tween (TBS-T): 20 mM Trizma-base, 140 mM NaCl, 0.05%

(v/v) Tween-20 (Sigma), pH 7.6.

Blocking reagent: 5% (w/v) low fat milk powder in TBS-T.

292 Primary and secondary antibody staining solution: 2.5% to 5% (w/v) low fat milk powder in

TBS-T (see Note 2).

Table D-2 Antibodies used in immunoblotting analysis of transport proteins. IF –

Immunofluorescence studies, WB- Western blot.

Protein Antibody Epitope Species Dilution Secondary Source of region Specificity Range Antibody Interest Dilution P-gp C219 Intracellular human, WB 1:500 Anti-mouse ID Labs Biotechnology (MDR1, mouse and 1:3000 Inc. (London, ON, mdr1a/b) rat (WB) Canada)

IF 1:10 Alexa Fluor anti-mouse 1:100 (IF) P-gp JSB-1 Intracellular human, WB 1:50 - Anti-mouse Santa Cruz (MDR1, mouse and 1:200 1:3000 Biotechnology Inc.(Santa mdr1a/b) rat (WB) Cruz, CA, USA)

β-Actin Actin Intracellular human, WB 1:500 Anti-mouse ID Labs Biotechnology mouse and 1:3000 Inc. (London, ON, rat (WB) Canada)

2.4.4. Re-probing of PVDF membrane

Western blot stripping solution (Pierce, Thermo Fisher Scientific Inc, Waltham, MA).

2.4.5. Deglycosylation of Transport Proteins

SDS (Sigma).

β-mercaptoethanol (Sigma).

Peptide N-glycosidase F (PNGase F) (Biolabs, Lawrenceville, GA, USA).

Endoglycosidase H (Endo-H) (Biolabs).

2.5. Cellular Localization of Transport Proteins applying Immunofluorescence

2.5.1. Preparation of Adherent Culture Cells

Microscope cover glass slide (22x 22 mm) thickness #1 (Fisher Scientific, Pittsburgh, PA).

Tissue culture petri dish or 6-well plate (Sarstedt).

293 2.5.2. Glutaraldehyde Fixation

DPBS.

1% glutaraldehyde:

Dilute 50% glutaraldehyde (EM grade) (Canemco Inc., St. Laurent) using DPBS (0.1 M, pH 7.2)

(see Note 1).

2.5.3. Immunostaining and Mounting of Adherent Cells

Blocking buffer: 5% goat serum (Invitrogen, Gibco), 0.2% Triton X-100 dissolved in DPBS.

Primary and secondary antibody staining solution: Antibody, 5% goat serum, 0.2% Triton X-100

dissolved in DPBS.

Vectashield hard set mounting solution (Vector, Burlingame, CA).

Microscope cover glass slide (22x 22 mm) thickness #1 (Fisher Scientific, Pittsburgh, PA).

Microscope slide (Frosted 1end, 2 sides, 76 x 26 mm) (VWR, West Chester, PA, USA).

2.6. Functional Activity of Transport Proteins (P-gp, BCRP, OATPs and MRPs)

2.6.1. Drug Accumulation Studies using Radioactive Labeled and Fluorescence Substrates

Material used for cell culture can be found in sections 2.1. and 2.2.

Transport buffer: 10mM of HEPES (Sigma), 0.01% of BSA (Sigma) dissolved in Hanks balance solution (Invitrogen, Gibco), pH 7.4.

1 % Triton-X solution: Mix 5 mL of Triton-X solution in 500 mL of distilled water to create 1%

(v/v).

24-well or 48-well cell culture plate (Sarstedt).

DPBS (Invitrogen, Gibco).

Scintillation counting fluid (Picofluor 40) (Perkin Elmer, Waltham, Massachusetts, USA)

Bio-Rad Dc Protein Assay Kit (Bio-Rad).

294 PSC833 (P-gp inhibitor) (gift from Novartis Pharma (Basel, Switzerland)).

GF120918 (BCRP and P-gp inhibitor) (gift from GlaxoSmithKline (Research Triangle Park,

NC)).

Fluorescence substrates used (see Table D-3).

Table D-3. Selective inhibitors, fluorescence substrates and radiolabelled substrates for drug transport studies to characterize P-glycoprotein function.

Compounds Concentration used Source to characterize P- gp Radiolabelled P-gp Substrates [3H] Digoxin (37Ci/mmol) 100 nM Perkin Elmer Life Sciences (Boston, MA, USA.) Fluorescence P-gp Substrates Rhodamine123 5 µM Invitrogen Inc. (Excitation λ: 485nm, emission (Grand Island, NY, USA.) λ: 520) Rhodamine-6G 1 µM Invitrogen Inc. (Excitation λ: 530nm, emission (Grand Island, NY, USA) λ: 560) P-gp Inhibitors PSC 833 1 – 10 µM Novartis Pharmaceuticals Canada Inc. (Dorval, Québec,Canada) Cyclosporine A 10 – 50 µM Sigma-Aldrich (St. Louis, MO, USA) GF120918 10 µM GlaxoSmithKline (Research Triangle Park, NC, USA)

2.6.3. Transepithelial Flux Studies

Transport buffer: 10mM of HEPES (Sigma), 0.01% of BSA (Sigma) dissolved in Hanks balance solution (Invitrogen, Gibco), pH 7.4.

1 % Triton-X solution: Mix 5 mL of Triton-X solution in 500 mL of distilled water to create 1%

solution (v/v).

295 48-well tissue culture plate with transwell insert (Costar).

DPBS (Invitrogen, Gibco, CA).

Scintillation counting fluid (Picofluor 40) (Perkin Elmer).

Bio-Rad Dc Protein Assay Kit (Bio-Rad).

3. Methods

3.1. Cell Culture Models of the Blood-brain Barrier (BBB)

1. The protocols for cell culture i.e., culture maintenance, subculture and cryostorage and culture thawing Reagent, culture medium and container were optimized for hCMEC/D3 and were optimized for RBE 4 (7,8,15,16,27).

3.2. Cell Culture Models of Astrocytes

The protocols for the isolation of primary cultures of astrocytes, culture maintenance were optimized for primary culture of rat and human astrocytes used in our laboratory (18-20, 28).

3.3. Molecular Characterization of Transport Protein (P-gp) at the Transcript Level

This section describes the preparation for isolation of total cellular mRNA (see

Subheading 3.4.1.), reverse transcription assay (see Subheading 3.4.2.) and semi-quantitative

reverse transcriptase PCR (RT-PCR) (see Subheading 3.4.3.).

3.3.1. Isolation of Cellular mRNA

1. All RT-PCR steps should be performed using filter pipette tips.

2. Add TRIZOL to lyse cells and transfer to a sterile microcentrifuge.

3. Incubate sample for 5 min at room temperature to permit complete dissociation.

4. Add 200 µl of chloroform-isoamyl alcohol solution for every mL of TRIZOL used.

5. Vortex vigorously for ~ 1-3 min and incubate at room temperature for 2 ~ 3 min.

6. Centrifuge at 4°C for 15 min at 10,500 g.

296 7. Transfer as much of aqueous phase as possible to a clean, sterile microcentrifuge tube

without touching the organic interphase.

8. Add 0.5 mL ice-cold isopropanol to aqueous phase for every mL of TRIZOL used.

9. Mix vigorously and chill on wet ice for 5 min.

10. Centrifuge at 4°C for 10 min at 10,500 g to pellet RNA.

11. Remove supernatant carefully and wash RNA pellet by adding 1 mL ice-cold 75%

ethanol for every mL of TRIZOL used.

12. Centrifuge 4°C for 5 min at 6600 g and remove nearly every drop of 75% ethanol.

13. Air dry RNA pellet in fumehood for ~10-15 min.

14. Resuspend pellet in 30 - 50 µL DNAse- and RNAse-free water (see Note 3).

15. Record exact volume of water added and store sample at -80°C.

3.3.2. Reverse Transcription Assay

1. Quantitation of mRNA should be performed using Tris-HCl buffer and determine

absorbance at 260nm and 280nm and 260/280nm ratio using UV spectrometer.

2. Mix 6µL DNAase and RNAase free water with every 2 µg RNA and 1µL of oligo(dt)

primer in a sterile microcentrifuge tube.

3. Incubate at 65°C for 10 min and quick chill on ice.

4. Create reaction mix: 2µL of 10 X PCR buffer,1µL of 10mM dNTP mix, 4 µL of 25mM

MgCl2, 1 µL of 0.1M DTT and 2 µL of 1 U/µl DNase I per 2 µg RNA.

5. Mix gently and pulse centrifuge.

6. Incubate at 37°C for 30 min,and then incubate at 75°C for 5 min to denature DNase I.

7. Cool mixture on ice and add 1µl of Superscript II, mix gently and pulse centrifuge.

8. Incubate at 42°C for 20 min.

297 9. Store at -20°C until RT-PCR.

3.3.4. Semi-quantitative PCR (RT-PCR)

1. Prepare 20 μL of PCR mix solution for each sample (2.5 µl of 10 X PCR buffer, 1.5 mM

of 25mM MgCl2, 400µM for each dNTP’s, 1.0 µM of forward primer, 1.0 µM of

reversed primer, 2.5U of Platinum Taq, top volume up to 20 μL using DEPC water or

DNAase and RNAase free water).

2. Add 20 µl of PCR master mix to each template cDNA sample tube.

3. Add 5µl of template cDNA to each tube.

4. Start thermocycling using GeneAmp 2400 Thermocycler with PCR conditions of interest

(Perkin-Elmer) (see Note 4 and Table D-1).

5. If necessary, forward and reversed primers for a second gene of interest (i.e. loading

control, β-actin) can be added to reaction mix after 10 - 15 cycles.

6. Prepare 1.7% (w/v) of agarose using 1 x TBE buffer with 1mg/mL of ethidium bromide.

7. Heat agarose solution in a microwave oven to dissolve agarose completely.

8. Pour gel solution into gel tray avoiding entrapment of air bubbles.

9. Gently place comb into gel and allow it to solidify for at least 30 min.

10. Load 13µL of 100 bp DNA ladder and samples-loading dye mixture to respective wells.

11. Run at 75 V for 2 h in 1 x TBE buffer.

12. An exampleof MDR1 cDNA from hCMEC/D3 cells stained with 1.0 mg/ml ethidium

bromide and visualized by UV transilluminescence (Figure D-1).

13. Densitometric analysis of ethidium bromide stained gels is performed using ImageQuant

5.2 densitometric software (Molecular Dynamics, Sunnyvale, CA).

298 14. The ratio between the target mRNA and the appropriate housekeeping gene was

calculated to obtain the relative mRNA expression of the particular gene of interest.

Figure D-1. RT-PCR analysis of MDR1 mRNA in the immortalized human brain endothelial

cell line, hCMEC/D3. A representative ethidium bromide stained gel shows amplification of specific bands for MDR1 (291 bp) and loading control β actin (504 bp) from whole cell lysates of hCMEC/D3 cells in lane 2.

3.4. Molecular Characterization of Transport Protein (P-gp) at the Protein Level

This section describes the preparation of whole-cell lysates from adherent cells (see

Subheading 3.5.1.), preparation of membrane-rich lysates (see Subheading 3.5.2.), immunoblotting analysis of transport protein (P-gp) expression (see Subheading 3.5.3.), reprobing of the membrane (See Subheading 3.5.4.) and deglycosylation of transport proteins

(see Subheading 3.5.4.).

299 3.4.1. Preparation of Whole-Cell Lysates from Adherent cells

1. Harvest cells using trypsin/EDTA according to the subculture protocol (see Subheading

3.1.1a or 3.2.1b) (see Note 5).

2. Add ice-cold whole cell lysis buffer to cell pellet (see Note 6).

3. Homogenate cell pellet using with a Dounce homogenizer at 10 000 rev/min for 10 sec.

4. Allow cells to lyse at 4°C for 10 – 15 min.

5. Centrifuge at 17,000 rpm for 5 min at 4°C and collect supernatant (whole cell lysate).

6. Determine protein concentration using Bradford protein assay (Bio-Rad).

3.4.2. Preparation of Crude Membrane Lysates from Adherent Cells

1. Harvest cells using 0.25% Trypsin/EDTA.

2. Centrifuge cells suspension at 400 g for 10 min at 4°C.

3. Discard resulting supernatant.

4. Incubate pellet for 30 min at 4°C in Crude-membrane isolation buffer.

5. Homogenize cell suspension with a Dounce homogenizer at 10,000 rev/min for three

cycles of 10 s each.

6. Centrifuge homogenates at 3000 g for 10 min to eliminate cellular debris.

7. Collect and centrifuge supernatant at 10,000 g for 1h at 4°C.

8. Resuspend pellet in 10 mM Tris buffer, pH 8.8, and keep frozen at -20°C until further

use.

9. Protein concentration of crude membrane preparations is determined applying the

Bradford protein assay (Bio-Rad).

300 3.4.3. Immunoblotting of Transport Protein (P-gp)

1. Aliquot 50 μg of protein samples.

2. Add 4 μL of lamelli buffer (5x) and top sample volume up to 20 μL.

3. Mix by vortexing.

4. Load entire 20 μL of sample mix into each lane of SDS-PAGE gel.

5. Electrophorese samples at 60 V for 30 min to stack sample on top of resolving gel.

6. Increase voltage to 150 V and run gel until front dye reaches bottom.

7. Cut PVDF membrane of the size of gel and immerse membrane in 100% methanol for 15

s and rinse it in water before use.

8. Equilibrate membrane in transfer buffer for at least 10-15 min.

9. Sandwich gel and PVDF membrane together with pieces of sponge and filter papers in

Gel holder cassette (see Note 7).

10. Transfer proteins to membrane at 4°C at 200mA for 2 h, 3 h for 2 gels in transfer buffer.

11. Remove membrane from cassette using forceps.

12. Immediately pour Ponceau-S solution over membrane and incubate for 1 min at room

temperature (see Note 8).

13. Discard solution and wash membrane with water once.

14. Check for protein bands on membrane (unwanted lanes can be cut at this point).

15. Wash membrane with TBS-T for 10 min.

16. Incubate membrane in blocking reagent overnight at 4oC.

17. Incubate membrane in primary antibody solution for 3-4 h at room temperature under

constant rocking (see Table D-2).

18. Wash membrane with TBS-T 3 times for 10 min each (see Note 9).

301 19. Incubate membrane in secondary antibody solution for 1.5 h at room temperature under

constant rocking (see Table D-2).

20. Wash membrane with TBS-T three times for 10 min each (see Note 16).

21. Incubate membrane in ECL solution for 4 min.

22. In a dark room, place film over membrane and close cassette.

23. At desired time has passed, immerse in developer until bands appear (see Note 10).

24. Wash film in water to remove excess developer.

25. Transfer into tray containing fixer and wait until film becomes transparent.

26. Wash film with water and dry. An example of film showing bands of P-gp in RBE4,

hCMEC/D3 and primary culture of rat astrocytes (Figure D-2).

27. Save membrane for stripping and re-probing or dry it for future reference.

Figure D-2. Western blot analysis of P-gp in rat brain microvessel endothelial cells (RBE4), hCMEC/D3, primary culture of rat astrocytes and MDCK-MDR1 cells. Whole cell lysates were resolved in a 12% sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. P-gp (~170kDa) was detected using monoclonal C219 antibody (1:500 dilution) and secondary anti-mouse antibody (1:3000 dilution). The MDCK-MDR1 cells served as the positive control.

302 3.4.4. Re-probing of PVDF Membrane

1. Incubate membrane in western blot stripping solution with gentle rocking for 5 – 10 min.

2. Wash membrane in TBS-T three times for 10 min each.

3. Membrane can be probed with other appropriate antibody.

3.4.5. Deglycosylation of P-gp

1. Cell lysate is denatured in 0.5% SDS and 1% β-mercaptoethanol at 100°C for 10 min.

2. Incubate suspension with either 5 µL peptide N-glycosidase F (PNGase F) or

endoglycosidase H (Endo-H) for 60 min at 37°C.

3. Proteins can then be resolved by SDS-PAGE and probed with the appropriate antibody.

4. An example of film showing bands of de-glycosylated P-gp in RBE4 (Figure D-3).

Figure D-3. Western blot analysis showing P-gp expression in RBE4 cells. (A) Crude membrane preparations (50 μg protein) from RBE4 cells were treated with or without PNGase F endoglycosidase, resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. P-gp (~170kDa) was detected using the monoclonal C219 antibody (1:500 dilution) and secondary anti-mouse antibody (1:3000 dilution) (Lane 1). P-gp was de-glycosylated to ~140kDa (Lane 2). (Reproduced with permission from the University of Toronto.)

303 3.5. Cellular Localization of Transport Protein P-gp using Immunofluorescence

This section describes the preparation of adherent cells (see Subheading 3.5.1.), procedure of glutaraldehyde fixation (see Subheading 3.5.2.), immunostaining using antibodies and mounting of microscope cover slide (see Subheading 3.5.3.).

3.5.1. Preparation of Adherent Culture Cells

1. Grow cells according to appropriate conditions on microscope cover glass slide.

2. Cover glass can be placed in tissue culture petri dish or 6-well plate.

3.5.2. Glutaraldehyde Fixation of Adherent Culture Cells

1. Remove medium and gently rinse cells twice with ice-cold PBS.

2. Fix with 1% glutaraldehyde in phosphate-buffered saline (PBS) (0.1 M, pH 7.2) for 1 h at

room temperature (see Note 14).

3.5.3. Immunostaining and Mounting of Adherent Cells

1. Remove glutaraldehyde (or other fixing solution, see Note 14).

2. Rinse cells gently twice with DPBS.

3. Incubate cells in blocking buffer for 30 min at room temperature.

4. Remove blocking buffer and incubate cells in primary antibody staining solution for 1 h

at room temperature (see Table D-2).

5. Remove primary antibody staining solution and incubate cells in DPBS at room

temperature with gentle rocking for 15 min.

6. Replace with fresh DPBS and repeat rinsing-rocking cycle for 2 more times.

7. Remove all DPBS.

8. Incubate cells in secondary Alex Fluor fluorochrome-conjugated antibody staining

solution for 1 h in the dark to avoid photo bleaching (see Table D-2).

304 9. Incubate cells in fresh DPBS at room temperature with gentle rocking for 15 min.

10. Replace fresh DPBS and repeat step 12 and 13 three more times.

11. Let microscope cover glass slide dry at room temperature in the dark.

12. Add 1 drop of Vectashield mounting solution onto a new microscope slide.

13. Lay gently microscope cover glass slide on top of mounting solution droplet while

avoiding bubbles (cell growing side of cover slide should be in contact with mounting

solution).

14. Without applying any pressure, let mounting solution dry at room temperature for at least

1 h before viewing under microscope.

15. An example of fluorescent images of P-gp localization in RBE-4 cells (Figure D-4).

Figure D-4. Immunocytochemical localization of P-gp in fixed RBE4 cells. (A) P-gp protein was localized with monoclonal C219 antibody (1:10 Dilution) and Alexa-Fluor 594-conjugated secondary antibody (1:100 Dilution). Labeling is present at the nuclear (solid triangles) and plasma (arrowheads) membranes. (B) Images of cells incubated with only secondary antibody (negative control) show no immunostaining. Original magnification 100 x (Reproduced with permission from the University of Toronto).

305 3.6. Functional Activity of Transport Proteins (P-gp)

This section describes procedures used in drug uptake accumulation studies using radioactive labeled substrates (see Subheading 3.6.1a.), drug accumulation studies using fluorescence substrates (see Subheading 3.6.1b.) and transepithelial flux studies using in vitro models of the

BBB (see Subheading 3.6.2.)

3.6.1a. Drug Accumulation Studies using Radioactive Substrates

1. Culture cells in 24-well or 48-well plates at appropriate cell density (see Note 2).

2. Prepare required concentration of substrate solution with and without inhibitors in

transport buffer.

3. Warm all solutions to 37°C and place tissue culture plate in heating plate set at 37°C.

4. Replace medium in all the wells with blank transport buffer.

5. Let system equilibrate for 15 min.

6. Replace blank transport buffer with desired transport buffer containing substrates in the

absence or presence of inhibitors.

7. Leave some wells (3 wells for 24-well plate) for protein assay (see Note 11).

8. At the desired time, aspirate transport buffer and replace with ice-cold DPBS.

9. Rinse cells twice with fresh ice-cold DPBS.

10. spirate all DPBS before moving to the next well.

11. Leave wells empty until entire plate is finished.

12. Add 250 μL of 1 % Triton-X solution to harvest cells.

13. Incubate cells for 30 min at 37°C with gentle agitation.

14. Collect Triton-X solution.

15. Pipette 250 μL of 1 % Triton-X solution to all wells.

306 16. Collect Triton-X solution and combine with earlier fraction in step 14.

17. Transfer into separate labeled scintillation vials for scintillation counting.

18. Use fresh 1 % Triton-X solution as background measurement.

19. Use starting transport buffer containing radiolabelled substrates to determine specific

activity (amount of radioactive corresponding to a particular amount of substrate).

20. Measure radioactivity of all samples using scintillation counting.

21. Harvest wells designated for protein assay with 1 % Triton-X solution.

22. Determine protein content using Bio-Rad Dc Protein Assay Kit (Bio-Rad).

23. Substrate accumulation is expressed as mol of substrate per mg of protein (Figure D-5).

307

Figure D-5. Functional activity of P-gp in primary cultures of rat astrocytes. Cellular accumulation of [3H] Digoxin (100nM) was examined in the presence or absence of 1 µM PSC- 833 over 60 min. Inset: Cellular accumulation of [3H] Digoxin (100 nM) and [14C] Mannitol (100 nM) at 60 min. Results are expressed as mean +/- SD of three separate experiments, with each data point in an individual experiment representing quadruplicate measurements. Significant difference was observed between control and the PSC group (*p<0.05).

3.6.1b. Drug Accumulation Studies using Fluorescence Substrates

1 – 16. Identical to 3.7.1.

16. The volume of 1 % Triton-X solution used to harvest cells should be adjusted according

to the amount of fluorescence substrate.

17. Transfer each sample into three wells in the fluorescence reading plate to minimize

pipetting variability.

308 18. Harvest wells designated for protein assay with 1 % Triton-X solution.

19. Use NaOH to harest cells in wells and determine protein content using Bio-Rad Dc

Protein Assay Kit (Bio-Rad).

20. An example illustration of PSC-833-mediated enhancement of R-6G uptake in

hCMEC/D3 cells (Figure D-6).

Figure D-6. Functional activity of P-gp in hCMEC/D3 cells. Cellular accumulation of 1 µM Rhodamine-6G (R-6G) (pmol/mg protein) was examined over 30 min. Insert: Percent change in R-6G cellular accumulation in the presence of 5 µM PSC-833 compared to control at 30 min. Excitation and emission wavelengths for R-6G were 530nm and 560nm respectivley. Results are expressed as mean +/- SEM of three separate experiments, with each data point in an individual experiment representing triplicate measurements. (* p<0.05).

309 3.6.2. Transepithelial Flux Studies

3.6.2a. Cell adhesion to the outside of transwell insert

1. Invert transwell insert (outside of inserts are facing up) and place it on glass petri dish.

2. Add cells suspension in fresh medium with appropriate density.

3. Pour cell suspension onto insert. Make sure enough medium is added to cover insert.

4. Allow cells, i.e., astrocytes, to adhere for 1-2 days.

3.6.2b. Cell adhesion to the inside of transwell insert

1. Prepare cell suspension in fresh medium according to appropriate density (see Note 12).

2. Fill upper chamber (inside of insert) with cell suspension (250 µL medium).

3. Allow cells to adhere.

4. Monitor cell growth and change medium in upper and lower chamber according to the

optimized cell growth condition (see Note 13).

3.6.2c. Transepithelial Flux Assay (Apical-to-Basolateral Transport)

1. Transepithelial Electrical Resistant (TEER) Measurement should be performed prior to

any Transepithelial Flux Assay.

2. Prepare required concentration of substrate solution in transport buffer.

3. If an inhibitor for a particular transporter is used, first prepare transport buffer with the

inhibitor concentration of interest, then divide buffer into two; one transport buffer

without substrate (buffer A) and one buffer with substrate (buffer B).

4. Warm all solutions to 37°C and place transwell plate in a heating plate at 37°C.

5. Place buffer A into upper (0.25 mL) and lower (1 mL) compartments or chambers.

6. Let system equilibrate for 15 min.

7. Replace buffer A in top compartment with 0.25 mL of buffer B.

310 8. At the desired time has passed, transfer entire insert into an empty well with cold DPBS.

9. Remove solution in upper compartment.

10. Rinse upper compartment with ice-cold DPBS twice.

11. Cut membrane of insert and transfer into scintillation vial containing scintillation fluid.

12. Save buffer in bottom compartment for radioactive scintillation counting (this allows

quantification of substrate intracellular accumulation).

3.6.2d. Transepithelial Flux Assay (Basolateral-to-Apical Transport)

1 – 5. Procedures are identical to 3.6.3d.

6. Replace buffer A from bottom compartment with 1 mL of buffer B.

7. At the desired time, transfer entire insert into an empty well with cold DPBS.

8. Save buffer in top compartment for radioactive scintillation counting (amount substrate

flux from basolaterial to apical side).

9. Cut membrane of insert and transfer membrane into scintillation vial with scintillation

fluid (this allows quantification of substrate intracellular accumulation).

4. Notes

1. Glutaraldehyde solution must contain monomer and low polymers (oligomers) with

molecules small enough to penetrate membrane fairly quickly. "EM grade"

glutaraldehyde (25% or 50% solution) must be used, not "technical" grade which consists

largely of polymer molecules too large to fit between macromolecules.

2. Wells on the edge of the plate can be left blank to avoid “edge effect” due to condition

differences, e.g. humidity. Ensure an equal number of cells are transferred into each well.

Differences in cell numbers between wells can introduce significant variability.

3. If necessary, heat sample for 10 min at 55-60°C to completely dissolve mRNA pellet.

311 4. Each gene of interest may require unique experimental conditions. Modifications can be

made based on the following guideline for human MDR1 (P-gp): 95°C-5 min, (94°C-1

min, 55°C-1 min, 72°C-1.5 min)34, 72°C for 10 min.

5. Trypsin can decrease yield of transporter protein. An alternative method is to scrap off

cells from surface using cell scrapper and centrifuged at 1000g for 3 min to pellet cells.

6. Typically 40μL of whole cell lysis buffer is used in a cell pellet with approximately four

million cells.

7. Gas bubbles will restrict protein movement from gel to membrane. Roll with a glass test

tube over membrane to remove gas bubbles between membrane and gel.

8. We keep PVDF membrane wet throughout the entire immunoblotting procedure. Other

protocols involve drying the membrane completely and then reactivating it with 100%

methanol for 1 min before the blocking step. This strengthens the binding of protein to

the membrane.

9. The number and the time of T-BST rinse for each primary and secondary antibody should

be optimized to yield best intensity and minimal background. Longer and increase

number of rinses can remove non-selective background, however, it yields fainter signals.

10. Time of film exposure depends on the amount of antibody binding to the targets in

membrane, enzymatic activity of secondary antibody and strength of ECL. Exposure time

can ranged from 15 s to 10 min, but typically it is 1.5 – 4 min with C219 and anti-mouse

secondary antibody we have mentioned. In order to detect changes in intensity between

lanes, one should always avoid over-exposure.

11. Wells for protein assay are used to estimate the amount of protein in a typical well in a

given plate. It is important to keep the number of cells in each well identical.

312 12. 50,000 RBE4 cells or hCMEC/D3 cells can be plated in each insert and it is expected to

reach 100% confluency in 5-7 days.

13. It is difficult to observe cell growth either inside or outside of insert under light contrast

microscopy. As a reference one should culture cells separately in blank wells on the same

plate to avoid overgrowth and the formation of multi-cell layering.

14. Other cell fixation methods such as BD Cytofix fixation solution (BD, Franklin Lakes,

NJ) can be used according to manufacture instruction.

References

1. Abbott, NJ, Ronnback L, Hansson E (2006) Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci.7:41-53. 2. Hawkins BT, Davis TP (2005) The blood-brain barrier/neurovascular unit in health and disease. Pharmacol Rev 57(2):173-185. 3. Deeken JF, Löscher W (2007) The blood-brain barrier and cancer: Transporters, treatment, and trojan horses. Clin Cancer Res. 13(6):1663-1674. 4. Potschka H, Löscher W (2007) Handbook of Neurochemistry and Molecular Neurobiology. Springer, US. 5. Bendayan R, Ronaldson PT, Gingras D, Bendayan M (2006) In Situ Localization of P- glycoprotein (ABCB1) in Human and Rat Brain. J Histochem Cytochem. 54(10):1159– 1167. 6. Ronaldson PT, Babakhanian K, Bendayan R (2007) Drug Transporters: Molecular Characterization and Role in Drug Disposition. In: You and Morris (ed) Drug Transport in the brain, John Wiley & Sons, Inc, New Jersey. 7. Zastre J, Chan GNY, Ronaldson PT, Ramaswamy M, Couraud P, Romero IA, Weksler B, Bendayan R (2008) Up-regulation of P-glycoprotein by HIV protease inhibitors in a human brain microvessel endothelial cell line. J Neurosci Res. 87(4): 1023-36. 8. Bendayan R, Lee G, Bendayan M (2002) Functional expression and localization of P- glycoprotein at the blood brain barrier. Microsc Res Tech. 57:365-380. 9. Dallas S, Miller DS, Bendayan R (2006) Multidrug resistance-associated proteins: Expression and function in the central nervous system. Pharm Rev. 58: 140-161. 10. Kusuhara H, Sugiyama Y (2001a) Efflux transport systems for drugs at the blood-brain barrier and blood-cerebrospinal fluid barrier (Part 1). Drug Discov Today 6(3):150-156. 11. Lee G, Bendayan R (2004) Functional expression and localization of P-glycoprotein in the central nervous system: relevance to the pathogenesis and treatment of neurological disorders. Pharm Res. 21(8):1313-30.

313 12. Kusuhara H, Sugiyama Y (2001b) Efflux transport systems for drugs at the blood-brain barrier and blood-cerebrospinal fluid barrier (Part 2). Drug Discov Today 6(4):206-212. 13. Butt AM, Jones HC, Abbott NJ (1990) Electrical resistance across the blood-brain barrier in anaesthetized rats: a developmental study. J Physiol. 429:47-62. 14. Cucullo L, Couraud P, Weksler B, Romero IA, Hossain M, Rapp E, Janigro D (2008) Immortalized human brain endothelial cells and flow-based vascular modeling: A marriage of convenience for rational neurovascular studies. J Cereb Blood Flow Metab. 28(2):312-328. 15. Weksler BB, Subileau EA, Perrière N, Charneau P, Holloway K, Leveque M, Tricoire- Leignel H, Nicotra A, Bourdoulous S, Turowski P, Male DK, Roux F, Greenwood J, Romero IA, Couraud PO (2005) Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J. 19(13): 1872-1874. 16. Roux F, Durieu-Trautmann O, Chaverot N, Claire M, Mailly P, Bourre J, Strosberg AD, Couraud P (1994) Regulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells. J of Cell Physiol. 159:101-113. 17. Walz W (2000) Controversy surrounding the existence of discrete functional classes of astrocytes in adult gray matter. Glia 31:95-103. 18. Ronaldson PT, Bendayan R (2008) HIV-1 viral envelope glycoprotein gp120 produces oxidative stress and regulates the functional expression of multidrug resistance protein-1 (Mrp1) in glial cells. J Neurochem. 106(3):1298-313. 19. Ronaldson PT, Bendayan R (2006) HIV-1 viral envelope glycoprotein gp120 triggers an inflammatory response in cultured rat astrocytes and regulates the functional expression of P-glycoprotein. Mol Pharmacol. 70(3): 1087-98. 20. Ronaldson PT, Bendayan M, Gingras D, Piquette-Miller M, R Bendayan (2004a) Cellular localization and functional expression of P-glycoprotein in rat astrocyte cultures. J Neurochem. 89(3):788-800. 21. Ronaldson PT, Lee G, Dallas S, Bendayan R(2004b) Involvement of P-glycoprotein in the transport of saquinavir and indinavir in rat brain microvessel endothelial and microglia cell lines. Pharm Res. 21(5): 811-8. 22. Colgan OC, Collins NT, Ferguson G, Murphy RP, Birney YA, Cahill PA, Cummins PM (2008) Influence of basolateral condition on the regulation of brain microvascular endothelial tight junction properties and barrier function. Brain Res. 1193(C): 84-92. 23. Perrière N, Yousif S, Cazaubon S, Chaverot N, Bourasset F, Cisternino S, Declèves X, Hori S, Terasaki T, Deli M, Scherrmann J, Temsamani J, Roux F, Couraud P (2007) A functional in vitro model of rat blood-brain barrier for molecular analysis of efflux transporters. Brain Res. 1150(1): 1-13. 24. Richert ND, Aldwin L, Nitecki D, Gottesman MM, Pastan I (1988) Stability and covalent modification of P-glycoprotein in multidrug-resistant KB cells. Biochemistry 27:7607- 7613.

314 25. Kiernan JA (2000) Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: What they are and what they do. Microscopy Today 00-1:8-12.

26. Lee G, Babakhanian K, Ramaswamy M, Prat A, Wosik K, Bendayan R(2007) Expression of the ATP-binding Cassette Membrane Transporter, ABCG2, in Human and Rodent Brain Microvessel Endothelial and Glial Cell Culture Systems. Pharm Res. 24(7):1262 – 1274. 27. Babakhanian K, Bendayan M, Bendayan R (2007) Localization of P-glycoprotein at the nuclear envelope of rat brain cells. Biochem Biophys Res Commun. 361(2):301-6. 28. Ashraf T, Ronaldson PT and Bendayan R (2009). Regulation of P-glycoprotein expression by the viral envelope protein gp120 and pro-inflammatory cytokines in human glial cells. AAPS Workshop on Drug Transporters in ADME: from the bench to the bedside. Baltimore, MD, USA. 29. Babakhanian K, Bendayan R (2007) Master Thesis. University of Toronto.

315 Appendix E. Chan G.N.Y., Hoque Md. T. and Bendayan R. 2013. The Role of Nuclear Receptors in Drug Transporters Regulation in the Brain. Trends in pharmacological sciences. 34(7):361-72.

This manuscript is reproduced in this thesis with permission from Elsevier.

Author Contribution: Chan G.N.Y. (primary author; full manuscript and responses to reviewer

comments); Hoque Md.T. (Editorial review of several drafts and responses to reviewer

comments) Bendayan R (Editorial review of several drafts and responses to reviewer comments)

316 Abstract

ATP-binding cassette membrane-associated drug efflux transporters and solute carrier

influx transporters, expressed at the blood-brain barrier, blood-cerebrospinal fluid barrier and in brain parenchyma are important determinants of drug disposition in the central nervous system.

Targeting the regulatory pathways that govern the expression of these transporters could provide novel approaches to selectively alter drug permeability in the brain. Nuclear receptors are ligand-

activated transcription factors which regulate the gene expression of several metabolic enzymes

and drug efflux/influx transporters. Although efforts have primarily been focused on

investigating these regulatory pathways in peripheral organs (i.e., liver and intestine), recent

findings demonstrate their significance in the brain. This review addresses the role of nuclear

receptors in the regulation of drug transporters functional expression in the brain. An in-depth

understanding of these pathways could guide the development of novel pharmacotherapy with

either enhanced efficacy in the central nervous system or minimal associated neurotoxicity.

Nuclear Receptors and Drug Transporters in the Brain

Pharmacological efficacy of central nervous system (CNS)-targeted drugs is often impaired by limited drug entry into the brain (Eyal et al. 2009). It has long been recognized that

drug permeability into the CNS can be restricted by the presence of brain barriers [i.e., the blood-

brain barrier (BBB)], which is primarily formed by brain microvessel endothelial cells, and the

blood-cerebrospinal fluid barrier (BCSFB), which comprises the epithelial cells of the choroid

plexus. Both barriers present a dynamic interface which regulate systemic blood-CNS exchanges

and play an instrumental role in the permeability of nutrients and several xenobiotics, including

drugs, into and out of the brain(Abbott et al. 2010). For effective CNS pharmacotherapy, drugs need to permeate through these barriers and reach their cellular targets in brain parenchyma

317 (Eyal et al. 2009). In the CNS, the functional expression of several drug efflux and influx

transporters, belonging to the ATP-binding cassette (ABC) or solute carrier (SLC) superfamilies,

is known to alter drug permeability across brain barriers and/or drug distribution in different

cellular compartments of brain parenchyma (i.e., neurons and astrocytes) (Eyal et al. 2009). In peripheral organs, it is well established that these transporters can be highly regulated by several nuclear receptors which become activated upon binding to a wide range of xenobiotics, environmental toxins and endogenous signalling molecules (Urquhart et al. 2007, Germain et al.

2006c). Recent in vitro and in vivo evidence suggest that nuclear receptor-mediated pathways

are also implicated in regulation of drug transporter functional expression in the central nervous

system. This knowledge can assist in the identification of nuclear receptor targets or pathways

whose modification can result in clinically significant alterations in drug concentrations in the

brain. For example, a selective down-regulation of drug efflux transporters mediated by nuclear

receptor inhibitors at the BBB could enhance permeability of CNS drugs into the brain, ensuring

that therapeutic concentrations are reached at the site of action. For drugs acting at peripheral

tissue sites, a selective induction of the same drug efflux transporters mediated by nuclear

receptor activators at the BBB could prevent the permeability of CNS drugs and avoid drug-

associated neurotoxicity. Through implementation of pharmacotherapeutic regimens that can

alter nuclear receptor activity in the brain, one may identify novel opportunities for selective

cerebral modulation of the functional expression of drug transporters. For example, a selective

down-regulation of drug efflux transporters mediated by nuclear receptor inhibitors at the BBB

could enhance permeability of CNS drugs into the brain, ensuring that therapeutic concentrations

are reached at the site of action. For drugs acting at peripheral tissue sites, a selective induction

of the same drug efflux transporters mediated by nuclear receptor activators at the BBB could

318 prevent the permeability of CNS drugs and avoid drug-associated neurotoxicity. Therefore, a better understanding of drug transporters regulatory pathways (i.e., nuclear receptors) is essential for the implementation of CNS pharmacotherapy with enhanced drug efficacy and/or minimal drug-associated neurotoxicity. The objective of this review is to discuss recent findings on the regulation of drug transporters functional expression by nuclear receptor pathways in the brain.

ABC and SLC Membrane Transporters in the Brain

Small, lipophilic and uncharged molecules can typically cross the lipid bilayer of biological cell membranes by passive diffusion, whereas large, hydrophilic and charged molecules have more restricted permeability. Furthermore, the pharmacokinetics of many CNS drugs and the degree of trafficking across the brain barriers can be significantly regulated by

ABC and/or SLC drug transporters expressed at the brain barriers and in the cellular compartments of brain parenchyma (Eyal et al. 2009). Within the superfamily of ABC transporters, several members belonging to the ABCB [e.g., ABCB1/P-glycoprotein (P-gp)],

ABCC (also known as Multidrug Resistance-Associated Proteins, MRPs; e.g., ABCC1/MRP1,

ABCC2/MRP2, ABCC4/MRP4) and ABCG [e.g., ABCG2/Breast Cancer Resistance Protein

(BCRP)] subfamilies constitute the major transport systems restricting drug permeability across the blood-CNS barriers ultimately affecting drug elimination and distribution in the brain.

Substrate specificity, function and localization of these ABC transporters have been comprehensively reviewed by several groups including ours and will not be discussed here (Lee et al. 2001, Dallas et al. 2006, Ronaldson et al. 2008, Miller 2010, Ashraf et al. 2012, Bendayan et al. 2002, Bendayan et al. 2006). Similarly, several members of the SLC family also mediate transport of a wide variety of pharmacotherapeutic agents at the brain barriers and brain parenchyma. Among these transporters, the organic anion transporting polypeptides (OATPs),

319 organic anion transporters (OATs), organic cation transporters (OCTs), novel organic cation transporters (OCTNs), concentrative nucleoside transporters (CNTs), equilibrative nucleoside transporters (ENTs) and peptides transporters (PEPTs) have been identified to play an important role in CNS drug clearance and distribution (Kusuhara & Sugiyama 2004, Kusuhara & Sugiyama

2005, Pastor-Anglada et al. 2008, Ashraf et al. 2012, Roth et al. 2012). A summary of the localization of the different ABC and SLC transporters at the BBB and BCSFB is depicted in

Figure E-1. Depending on the function and localization of these transporters, they can serve as a protective system against xenobiotics brain exposure and/or constitute a significant obstacle to

CNS pharmacotherapy by limiting drug entry (i.e., ABC transporters) or they can facilitate drug brain permeability (i.e., SLC transporters).

320 A

Figure E-1. Localization of selected ABC and SLC transport proteins in A) brain microvessel endothelial cells at the BBB and B) choroid plexus epithelial cells at the BCFB. Arrows indicate the direction of substrate transport.

321 Nuclear Receptors in the Brain

Nuclear receptors are members of a superfamily of DNA-binding transcriptional factors that regulate gene transcription in response to signaling small molecules and xenobiotics (Benoit

et al. 2006). They play a role in every aspect of developmental processes, cellular differentiation,

metabolic homeostasis and protective mechanisms against endogenous and exogenous chemicals

(Benoit et al. 2006). Although the size of nuclear receptors varies considerably, they all consist

of an assembly of functional domains (Benoit et al. 2006). The DNA-binding domain, the most

highly conserved region among nuclear receptors, is important for receptor dimerization [i.e.,

with Retinoid X Receptor α (RXRα)] and binding to the DNA response element. The ligand-

binding domains (LBD) allow recognition of small-molecule ligands, which is considered the

most important structural feature in drug discovery. Other domains in some of the nuclear

receptors [i.e., activation function 1 (AF-1)] can be important for the activation of nuclear

receptors through phosphorylation or interactions with co-activators and co-repressors (Germain

et al. 2006c). Mechanistically, pathways by which nuclear receptors regulate gene expression are

remarkably complex and the reader is encouraged to review the literature for further in-depth

information (Benoit et al. 2006, di Masi et al. 2009, Helsen et al. 2012, Brélivet et al. 2012,

Germain et al. 2006c). A detailed diagram of the overall nuclear receptor transcription regulation

is depicted in Figure E-2.

322

Figure E-2. Transcriptional regulation of drug transporters by nuclear receptors. Activation of cytosolic-residing nuclear receptors begins in the cytoplasm. (1) In the absence of ligand binding, the receptor may be associated with and retained by a large multi-protein complex of chaperones, such as the Heat Shock Protein 90 (Hsp90) (Squires et al. 2004, Kawamoto et al. 1999, Heinlein & Chang 2001, Kriechbaumer et al. 2012). (2) Upon ligand binding, the nuclear receptor dissociates from the cytoplasmic chaperones complex and translocates into the nucleus (Li & Wang 2010, Heinlein & Chang 2001). (3A) Once in the nucleus, the ligand-bound receptor may bind to response elements of its target gene promoter as a monomer, homodimer or RXR-heterodimer (Whitfield et al. 1999, Wang & LeCluyse 2003, Khorasanizadeh & Rastinejad 2001). Recruitment of multiple co-activator complexes is responsible for chromatin remodelling and histone acetyltransferase activities which allow the recruitment of RNA polymerase II and other transcriptional machineries (Rosenfeld et al. 2006, George et al. 2011). (3B) Some ligand-dissociated nuclear receptors may predominantly reside or be constitutively active in the nucleus (George et al. 2011). Inverse agonists can alter their basal activity and antagonists can oppose the action of agonists in a ligand-dependent manner (Hilser & Thompson 2011). Ligand-independent pathways, (i.e., phosphorylation of the nuclear receptor), may also regulate activity of the receptor or other receptor-associated signal transduction pathways (Hilser & Thompson 2011, Ward & Weigel 2009). (4) Sequentially, the nuclear receptor transcriptional complex stimulates gene transcription (mRNA production) of the

323 target genes (e.g., drug transporter) (Rosenfeld et al. 2006). Conversely, co-repressors can bind to the receptor or transcriptional complex to inhibit formation of transcriptionally active protein complex(Germain et al. 2006c, Benoit et al. 2006). (5) Once mature mRNA is produced, translation of the amino acid chain occurs across the endoplasmic reticulum and proper protein folding is achieved in the golgi apparatus. (6) The folded functional transporter is then cycled between the endosomal compartments and plasma membrane(Germain et al. 2006c, Li & Wang 2010). Taken together, these functional domains and interactions with other accessory proteins, in both the nucleus and cytoplasm, allow nuclear receptors to translate many different endogenous and exogenous chemical stimuli into cellular responses.

Forty-eight known nuclear receptors of the human genome have been classified into six evolutionary groups (1-6) according to nuclear receptor amino acid alignment and phylogenetic

tree construction (Germain et al. 2006c). Conventionally, these nuclear receptors are often

divided into three classes based on their ligand-binding specificities: i) steroid and endocrine

receptors; ii) true orphan receptors and iii) adopted orphan receptors (Germain et al. 2006c, Li &

Wang 2010). The steroid and endocrine receptors, such as glucocorticoid receptor (GR; NR3C1)

and estrogen receptors (ERs; NR3A1 & NR3A2) are well documented mediators of cellular

activities associated with their steroid hormones (Germain et al. 2006c). By contrast, orphan

receptors are those identified through molecular sequencing without prior knowledge of their

endogenous ligands or functions. Within this category, true orphan receptors are those whose

endogenous ligands have yet to be found or may not exist (Germain et al. 2006c). Adopted

orphan nuclear receptors, such as pregnane X receptor (PXR; NR1I2) and constitutive

androstane receptor (CAR; NR1I3), are receptors that were cloned without prior knowledge of

their endogenous ligand profiles or function but have recently been found to be high capacity and

low affinity xenobiotic sensors (Benoit et al. 2006, Li & Wang 2010) (Table E-1). At present,

several adopted orphan nuclear receptors, including PXR, CAR, vitamin D receptor (VDR;

NR1I1) and peroxisome proliferator-activated receptors (PPARs; NR1C1, NR1C2 & NR1C3) as

well as a ligand-activated transcriptional factor Aryl hydrocarbon receptor (AhR) have been

324 identified as key regulators of gene transcription of phase I [i.e., Cytochrome P450 enzymes

(CYP450)] and phase II drug metabolizing enzymes (i.e., Glutathione S-Transferases,

Sulfotransferases and UDP-Glucuronosyltransferases) and efflux/influx drug transporters from the ABC and SLC superfamilies. Both natural and synthetic compounds, including many current therapeutic agents, can alter receptor activity. Ligand-mediated activation of these receptors has been demonstrated to modulate expression of these enzymes and transporters, which can sequentially affect detoxicification processes, as well as drugs pharmacokinetic properties

(Urquhart et al. 2007, Li & Wang 2010). Current evidence suggests that some of these receptors

are also present in the brain in addition to their expression in the peripheral system, hence ligand-

receptor interactions could regulate functional expression of drug transporters which ultimately

could alter drug permeability into/out of the brain. In the next sections, we summarize recent

data on nuclear receptor pathways that have been demonstrated to play a role in the regulation of

drug transporters in the brain (Table E-1).

PXR

Belonging to the subfamily 1I of nuclear receptors, PXR (NR1I2) can regulate various

physiological processes, including steroid hormone balance, bile acid clearance and homeostasis

of cholesterol, glucose and lipids (Moreau et al. 2008). Human PXR was first cloned and identified in 1998 as a regulator of CYP450 3A4 gene expression (Kliewer et al. 1998,

Bertilsson et al. 1998). PXR exhibits marked species differences in amino acid sequences of the

LBD, which often leads to significant differences in ligand selectivity between species (Chang &

Waxman 2006). The growing list of human PXR ligands includes a remarkable diverse array of

chemicals with molecular weight ranging from 250 kDa to more than 800 kDa, such as

prescription drugs (e.g., rifampin, paclitaxel, taxol and protease inhibitors), pesticides (e.g.,

325 chlordane and transnonachlor), environmental toxins (e.g., phenols), natural herbal compounds

(e.g., colupulone and hyperforin), endogenous steroids (e.g., progesterone, 5α-pregnane-3,20- dione) and bile acids (i.e., cholic acid) (Chang & Waxman 2006, di Masi et al. 2009, Dussault et al. 2001) (Table E-1). More importantly, interactions between PXR and its ligands are known to play a central role in regulating the expression of phase I and II enzymes and drug transporters that are known to affect tissue distribution and clearance of many xenobiotics (Li & Wang 2010,

Urquhart et al. 2007). In hepatic and intestinal tissues, drug transporters known to be regulated by PXR include members of the ABC transporter superfamily (i.e., P-gp, BCRP, MRP2/Mrp2 and Mrp3)(Geick et al. 2001, Teng et al. 2003, Albermann et al. 2005) and SLC transporter superfamily [i.e., organic anion transporting polypeptide (OATP1A2) and Oatp2 or Oatp1a4]

(Guo et al. 2002, Meyer Zu Schwabedissen et al. 2008). At the BBB, P-gp and Mrp2 are the two drug transporters that have been shown to be regulated by PXR. Bauer et al. were the first to demonstrate that P-gp can be regulated by PXR in brain capillaries isolated from rats (Bauer et al. 2004) and a PXR-humanized mouse model (Bauer et al. 2006). Furthermore, the same group reported very interesting data showing that the anti-nociceptive effect of methadone (an opiate analgesic and P-gp substrate) was reduced in mice exhibiting P-gp induction in brain capillaries.

(Bauer et al. 2006). In vitro induction of P-gp mediated by PXR was later confirmed by several groups, including ours, using primary cultures of brain microvessel endothelial cells or an immortalized brain microvessel endothelial cell culture system derived from porcine (Ott et al.

2009), bovine (Perloff et al. 2007), rodent (Lombardo et al. 2008, Narang et al. 2008) and human

(Chan et al. 2011). Recently, applying quantitative cerebral microdialysis, our group also demonstrated that in vivo upregulation of P-gp mediated by dexamethasone, a steroid derivative and PXR ligand, at the mouse BBB could reduce brain extracellular concentrations of a P-gp

326 substrate (i.e., quinidine). These data further suggest that drug-drug interactions as a result of P- gp induction mediated by PXR at the BBB are possible. In addition to P-gp, a few studies have suggested that PXR can regulate MRP2/Mrp2 expression in a rodent brain microvessel endothelial cell line culture (Narang et al. 2008, Lombardo et al. 2008) and isolated rat brain capillaries (Bauer et al. 2008). Furthermore, a PXR response element in the promoter region of the OATP1A2 gene has been identified (Meyer Zu Schwabedissen et al. 2008). However, it remains to be demonstrated that OATP1A2 expressed at the luminal membrane of brain capillaries is also regulated by PXR. Currently, human PXR protein expression has been clearly detected in the hCMEC/D3 cell line, primary cultures of human brain microvessel endothelial cells (Chan et al. 2011) and human fetal brain tissue (Chan et al. 2010). Furthermore, human

PXR transcript expression was detected in specific regions of the human brain such as thalamus, pons and medulla (Nishimura et al. 2004, Miki et al. 2005). As well, both the transcript and protein expression of rodent and porcine PXR have also been reported in studies using isolated brain capillaries or in vitro brain microvessel endothelial cell line systems (Bauer et al. 2006,

Bauer et al. 2004, Ott et al. 2009, Narang et al. 2008). In contrast, a few studies failed to demonstrate human PXR transcript and protein expression in isolated human brain microvessels and brain cortical tissues, this could possibly be due to the limitation of the sensitivity of the analytic methods applied (Shawahna et al. 2011, Dauchy et al. 2008). At present, the function of human PXR at the BBB and cellular compartments of the brain parenchyma in the clinic remains unclear.

327 CAR

Functionally similar to PXR, CAR (NR1I3) has also been termed a “xenobiotic sensor” and can recognize many structural diverse compounds, such as androstane metabolites (i.e., androstenol and androstanol), bile acids (i.e., cholic acid, 6-ketolithocholic acid) and

pharmacological agents (i.e., clotrimazole and meclizine) (di Masi et al. 2009) (Table E-1). It is

well known that CAR activity overlaps with PXR in the regulation of CYP2B and CYP3A genes

(Urquhart et al. 2007). Recently, it has also been demonstrated that the overlap in CAR and PXR

target genes extends well beyond the CYP family and includes phase II metabolic enzymes (e.g.,

Sulfotransferase and UDP-Glucuronosyl Transferase families) and bile acid/drug transporters

(e.g., OATP1B1 and MRP2) (Tolson & Wang 2010, Huang et al. 2003). Other drug transporters

shown to be regulated by CAR include P-gp (Burk et al. 2005), BCRP (Jigorel et al. 2006),

MRP2 (Kast et al. 2002, Jigorel et al. 2006), Mrp3 (Xiong et al. 2002, Staudinger et al. 2003),

MRP4/Mrp4 (Assem et al. 2004), OCT1, OATP2B1, OATP1B3 (Jigorel et al. 2006) and

Oatp1a4 (Staudinger et al. 2003). In the brain, Wang et al. have recently shown CAR expression

in isolated rat and mouse brain capillaries and its activation was able to induce functional

expression of P-gp, Bcrp and Mrp2 in both ex vivo models of the BBB and in vivo (Wang et al.

2010). Our group has recently provided in vitro evidence for human CAR protein expression in

the hCMEC/D3 cell line, primary cultures of human brain microvessel endothelial cells (Chan et

al. 2011) and in human fetal brain tissue (Chan et al. 2010). In addition, we demonstrated that

human CAR could regulate P-gp mRNA and protein expression in the hCMEC/D3 cells (Chan et

al. 2011). Lemmen et al. also demonstrated the in vitro regulation of P-gp and Bcrp by CAR in

primary cultures of porcine brain capillary endothelial cells (Lemmen et al. 2013). Other groups

have reported a low level of CAR mRNA in human brain tissue samples (Nishimura et al.

328 2004), caudate nucleus (Lamba et al. 2004) and brain glioma cells (Malaplate-Armand et al.

2005). In human brain microvessels, although CAR mRNA expression was reported (Dauchy et

al. 2008), protein expression was not detected in these samples (Shawahna et al. 2011). Overall,

the function of human CAR in the clinic remains unclear.

VDR

VDR (NR1I1) belongs to the same subfamily as PXR and CAR. This receptor binds to its

ligand, 1α,25-dihydroxyvitamin D3 (VitD3) and regulates calcium and phosphate balance(Tirona

& Kim 2005). VDR is expressed ubiquitously, including BBB (Eyles et al. 2005, Durk et al.

2012) and several cellular compartments of the brain parenchyma (i.e., neurons and glial cells)

(Chow et al. 2011, Cui et al. 2013). The receptor has been shown to take part in a variety of physiological functions, including immunity, tumour suppression and regulation of CYP3A enzymes in response to bile acid activation (i.e., lithocholic acid) (Tirona & Kim 2005).

Recently, increasing evidence on VDR-mediated regulation of drug transporters in the periphery has emerged. For example, several studies have shown that VitD3 can induce P-gp expression in human intestinal (Aiba et al. 2005, Fan et al. 2009, Tachibana et al. 2009) and rat kidney cell lines (Chow et al. 2010, Chow et al. 2011). A VDR response element has also been identified in the MDR1 (P-gp) gene promoter, confirming the VDR regulatory effect on P-gp expression

(Saeki et al. 2008). In addition to P-gp, protein expression of MRP2/Mrp2, Mrp3, MRP4/Mrp4,

Oat1, Oat3 and PepT1 has been reported to be induced by VitD3 in a cell line of heterogeneous

human epithelial colorectal adenocarcinoma cells (Caco-2) (Fan et al. 2009) and rodents intestinal tissues (McCarthy et al. 2005, Chow et al. 2009, Chow et al. 2010, Maeng et al. 2011).

Interestingly, renal protein expression of PepT1 and Oat1 were decreased following treatment of

VitD3 in rats, suggesting tissue specificity of VDR regulation (Chow et al. 2009, Chow et al.

329 2010). In the brain, recent in vitro and in vivo studies using VitD3 have demonstrated VDR- mediated regulation of P-gp transcript and protein expression in whole brain membrane fraction from mice, brain capillaries from rats and in vitro culture systems of human and rat brain

microvessel endothelial cells (hCMEC/D3 and REB4) (Chow et al. 2011, Durk et al. 2012). In addition, the study performed by Chow et al. showed that P-gp induction in membrane fractions from whole brain isolated from VitD3–treated mice was associated with enhanced brain

clearance of a P-gp substrate digoxin (Chow et al. 2011). To date, the clinical role of human

VDR in the regulation of drug transporters at the BBB and cellular compartments of the brain

parenchyma has yet to be determined.

PPARs

PPARs (PPARα, NR1C1; PPARβ/δ, NR1C2 and PPARγ, NR1C3) belong to the same

family of nuclear receptors as the PXR, CAR and VDR, and are known to regulate several

biological processes, such as lipid metabolism, development, apoptosis, neoplastic

transformation and inflammation (Wang 2010). The three isotypes identified to date, PPARα,

PPARβ/δ and PPARγ, differ from each other in terms of ligand profiles, tissue distribution and

physiological functions (Wang 2010). PPARα and PPARγ are the major isotypes which have

been demonstrated to regulate the expression of drug transporters in rodent models and human

derived cell culture systems. PPARα agonists have been shown to induce in vivo hepatic P-gp

protein expression in mice (Kok et al. 2003, Moffit et al. 2006, Cui et al. 2009); however, Hirai

et al failed to reproduce these results (Hirai et al. 2007). Therefore, P-gp regulation by PPARα

remains unclear, although such discrepancy may be due to differences in mice strains used in

these studies. Furthermore, a PPARα response element has been identified in the promoter region

of PXR, it is also possible that activation of PPARα can have an indirect effect on P-gp

330 expression through a PXR pathway (Aouabdi et al. 2006). To the best of our knowledge, no

studies have investigated such mechanisms in the brain. In vivo studies in rodents implicated

PPARα in the regulation of Bcrp, Mrp1, Mrp3, Mrp4, Oatp1a1 Octn2 and Octn3 drug

transporters in hepatic and intestinal tissues (Kok et al. 2003, Maher et al. 2005, Moffit et al.

2006, Wen et al. 2010, Hirai et al. 2007). In human monocyte-derived dendritic cells, PPARγ

has been reported to regulate BCRP mRNA and protein expression (Szatmari et al. 2006).

Moreover, three PPARγ response elements have been identified, for direct binding of PPARγ-

RXRα, in the enhancer region upstream of ABCG2 (BCRP) gene (Szatmari et al. 2006).

Although the two PPAR isotypes (α and γ) were reported to be expressed in the brain,

particularly in the microvessel endothelial cells of the BBB, macrophages, glial cells and

neurons, only a few in vitro studies have demonstrated the regulation of drug transporters by

PPARα in the CNS (Heneka & Landreth 2007). In primary cultures of rat astrocytes, Octn3

protein expression and function were upregulated in the presence of PPARα ligands (e.g.,

clofibrate) (Januszewicz et al. 2009). Our group recently reported that in hCMEC/D3 cells,

PPARα is directly involved in the regulation of BCRP at the transcript and protein level as well

as function (Hoque et al. 2012).These results suggest the likelihood of a PPAR-mediated regulation of drug transporters at the BBB.

ERs

Estrogen receptors (ERα, NR3A1; ERβ, NR3A2) belong to the steroid hormone receptor

superfamily. Two subtypes, ERα and ERβ have been identified and are involved in processes of

reproduction, development, homeostasis, immune function, osteroporosis, cardiovascular

diseases and cancers (Deroo & Korach 2006). ERs located in the nucleus can act in the classical

manner by binding to estrogen or selective ligands (i.e., tamoxifen, raloxifene, fulvestrant,

331 genistein (Renoir et al. 2013)), ultimately altering target genes transcription. ERs located at the plasma membrane can mediate a rapid and nongenomic change in cellular processes that affect activity, localization and degradation rate of several signalling molecules, such as kinases (Deroo

& Korach 2006). Recently, several efflux and influx drug transporters, such as BCRP (Ee et al.

2004, Imai et al. 2005, Wang et al. 2006, Zhang et al. 2007, Wang et al. 2008, Yasuda et al.

2009, Tanaka et al. 2004), Mrp3 (Ruiz et al. 2006), Oatp1a4 (Cheng et al. 2006) and several organic anion transporters (i.e., Oat1, Oat 2, Oat3)(Buist et al. 2003) have been found to be tightly regulated by ERs and estrogens (i.e., 17β-estradiol) in rat liver or kidney. However, with the exception of BCRP, their regulation by ERs in the brain remains to be investigated. At the

BBB, Hartz et al. showed that ERα and ERβ are involved in a rapid (minutes) downregulation of

BCRP transport function by post-translational pathways, while BCRP expression remained unchanged in brain capillaries isolated from rats and ERα and ERβ single knockout mouse models (Hartz et al. 2010b). Using the same model, this group reported that estrogen acts through ERβ and signals via the phosphatase and tensin homolog (PTEN) / phosphoinositide 3- kinase (PI3K) / Akt / glycogen synthase kinase 3 (GSK3) cascade to stimulate degradation of

BCRP leading to a decrease in BCRP expression and transport activity (Hartz et al. 2010a). By using a similar model, Mahringer and Fricker were able to demonstrate a downregulation of

BCRP mRNA expression in rat brain capillaries after 6 h treatment with 17β estradiol

(Mahringer & Fricker 2010). Together, these data provide evidence that BCRP activity at the

BBB can be reduced for a short time by ER-mediated signalling pathways. These authors further suggested that ER pathways can provide potential venues to rapidly increase brain permeability of pharmacological agents known to serve as BCRP substrates. In the brain, ERs have been identified to play a role in neurodegenerative diseases because of their neuroprotective and

332 antioxidant effects and their ability to attenuate BBB disruption following ischemic stroke (Chi

et al. 2006, Cipolla et al. 2009). As well, the functional expression of both ERα and ERβ

subtypes has been identified in the brain microvasculature of rodents (Stirone et al. 2003, Krause

et al. 2006, Gonzales et al. 2007). These findings suggest that ERs could serve as potential

targets to alter endothelial function in the brain.

Glucocorticoid receptor (GR, NR3C1) regulates genes governing development,

metabolism and immune responses and specifically responds to steroidal and non-steroidal GR

modulators (i.e., budesonide and mometasone) (De Bosscher et al. 2010, Tirona & Kim 2005).

The presence of a functional glucocorticoid responsive element in the promoter region of drug

transporters in humans has yet to be identified, however, it is believed that GR activity can affect

transporter’s expression through its regulation on other nuclear receptors. For example, the

expression of PXR and CAR has been reported to be glucocorticoid dependent and a GRE has

been found within the promoter region of human PXR and CAR genes (Pascussi et al. 2000,

Pascussi et al. 2003). These observations suggest that GR may alter PXR and CAR expression

and affect indirectly the gene expression of numerous drug transporters. Indeed, it was reported

that exposure to GR antagonists (i.e., RU486, ketoconazole and miconazole) to human

hepatocytes can downregulate both PXR and CAR expression (Duret et al. 2006). Furthermore, the use of RU486 could reverse induction of P-gp mediated by dexamethasone, ligand of human

PXR and GR, in human retinal pigment epithelial cells (Zhang et al. 2012). Data from a recent publication utilizing primary cultures of rat brain microvessel endothelial cells also support this hypothesis at the BBB (Narang et al. 2008). In this study, ligand-mediated activation of GR was able to induce rat PXR protein expression, while the addition of a GR antagonist RU486 reversed

333 the induction. Together these results provide evidence for the involvement of GR-PXR and GR-

CAR pathways in the regulation of drug transporters in the periphery as well as at the BBB where GR, PXR and CAR are active. Furthermore, GR expression has been reported in cellular compartments of the brain (i.e., neurons and glial cells (Reul & De Kloet 1985, Pujols et al.

2002, Narang et al. 2008)), however the regulation on PXR and CAR expression by GR at these location has not been fully addressed.

Other hormone-activated and ligand-activated receptors

Liver X receptors (LXRα; NR1H3 and LXRβ; NR1H2) are generally activated by endogenous ligands such as, oxysterols, fatty acids and bile acids (i.e., taurine) (Hoang et al.

2012, Xiao et al. 2010). These receptors have been shown to regulate several lipid transporters belonging to the ABC transporter family, i.e., ABCA1, ABCG1, ABCG4, ABCG5 and ABCG8 and drug transporters i.e., ABCB1 (P-gp), ABCC2 (MRP2) and ABCC5 (MRP5) and ABCG2

(BCRP) (Repa et al. 2002, Kaneko et al. 2003, Langmann et al. 2006, Chisaki et al. 2009).

LXRs expression has been found in brain capillaries (Elali & Hermann 2011), neurons(Cao et al.

2007), pericytes (Saint-Pol et al. 2012), microglia (Terwel et al. 2011, Gilardi et al. 2009) and astrocytes (Liang et al. 2004, Gilardi et al. 2009), however the regulation of drug transporters expression by LXRs at these sites has not been fully investigated.

Interestingly, the in vivo effect of LXR ligand (T0901317) on P-gp and MRP1 protein expression in brain microvessels has been examined in mice with or without surgical-induced ischemia (Elali & Hermann 2011). P-gp induction in brain microvessels was observed only in mice without induced ischemia, whereas MRP1 induction was only found in ischemic mice

(Elali & Hermann 2011). Although the study was unable to delineate the direct transcriptional role of LXR on the two transporters, it demonstrated that LXR could attenuate activation of

334 several kinase pathways, such as JNK1/2 and caspase-3, which ultimately may affect P-gp and

MRP1 expression. Hepatocyte Nuclear Factor 4α (HNF4α; NR2A1) (Hwang-Verslues & Sladek

2010) can regulate hepatic transporters, including Oatp1a1, 1a4, 2b1, Oat2, Oct1, Octn2, Mdr1a,

MRP1, Mrp3 and Mrp4 (Lu et al. 2010, Tirona & Kim 2005, Niehof & Borlak 2009).

Interestingly, HNF4α is involved in the regulation of drug metabolizing processes mediated by

PXR and CAR (Tirona et al. 2003, Hwang-Verslues & Sladek 2010). Furthermore, in silico analysis suggests that HNF4α can play a role in the overall regulation of drug metabolizing enzymes and drug transporters by modulating PXR and PPARα expression (Torra et al. 2002,

Kamiya et al. 2003). Although both the transcript and protein expression of HNF4α have been detected in human and rat choroid plexus, the functional significance of HNF4α on transporter expression at the BBB, the BCSFB and brain parenchyma requires further investigation.

Farnesoid X Receptor (FXR; NR1H4) (Tirona & Kim 2005), through its binding with

cholic acids or the plant steroid, guggulsterone (Porez et al. 2012) can regulate several bile acids

transporters [e.g., organic solute transporter (OSTs) (Landrier et al. 2006) and bile salt excretory

pump (BSEP) (Ananthanarayanan et al. 2001, Yu et al. 2002)], however it does not seem to

participate in drug transporters regulation, with the exception of MRP2 (Kast et al. 2002) and

OATP1B3 in the liver (Jung et al. 2002). Currently, there is only ex vivo evidence suggesting the involvement of FXR in Mrp2 regulation in isolated rat brain capillaries, whereas its involvement at the BCSFB and other cellular compartments of the brain parenchyma is currently not known

(Bauer et al. 2008).

Three isoforms of the RAR receptor (α; NR1B1, β; NR1B2, γ; NR1B3) function as heterodimers with the three RXR isoforms to regulate cell growth and survival (Germain et al.

2006a). RARα has been associated with the in vitro regulation of hepatic and intestinal P-gp and

335 BCRP expression (Stromskaya et al. 1998, Hessel & Lampen 2010). In the brain, an increase in

P-gp function was also observed in the immortalized rat brain endothelial cell line (RBE4)

following treatment with retinoid acid (El Hafny et al. 1997). Overall, the in vivo role of RARs

in the regulation of drug transporter is poorly understood.

Retinoid X Receptors (RXRs: RXRα; NR2B1, RXRβ; NR2B2, RXRγ; NR2B3] are an

important component in the retinoid signalling pathway due to their ability to form heterodimers

with isoforms of retinoic acid receptor (RARs; NR1Bs) and specifically respond to retinoids,

such as retinoic acids and tretinoin (Mandrekar-Colucci & Landreth 2011, Germain et al. 2006b).

Differences in their expression pattern in humans have been reported, but all three isoforms of

RXR can be found in different regions of the brain (Krezel et al. 1999). They have an important

role in neuronal development and adult neuronal function (Maden 2007, Arfaoui et al. 2013).

One interesting aspect of RXRs is their ability to form functional RXR homodimers and also

serve as a common heterodimerizing partner with several nuclear receptors, including VDR,

PPARs, LXRs, FXR, PXR and CAR (Germain et al. 2006b). Heterodimerization with RXRs is an essential step which facilitates the activation and binding of these nuclear receptors to the promoter regions of their target genes (Mangelsdorf et al. 1991, Mader et al. 1993, Germain et al. 2006b). Activation of the RXR heterodimer can be regulated by agonists of RXR or agonists of the partnering receptor. Interestingly, the presence of both the agonist for RXR and agonist for its heterodimerizing partner (i.e., PPARs, LXRs, FXR, PXR and CAR) can produce a synergic activation (Westin et al. 1998). As mentioned, these partnering receptors have been demonstrated to regulate expression of several drug transporters in the brain. Therefore, RXRs, in particular RXRα, is considered to be involved directly and indirectly in these regulations.

Recently, the activation of RXRs by its selective agonist, methoprene, has been shown to

336 increase expression of cholesterol and phospholipid transporters, Abca1 and Abcg1, in rat

astrocytes, possibly through the activity of its heterodimerizing partners, LXRs or PPARs (Chen

et al. 2011). However, the contribution of RXR activation to the overall regulation of drug

transporters (i.e., P-g, MRPs and BCRP) mediated by its heterodimerizing partners in the brain

has not been fully investigated.

Endocrine receptors, such as thyroid hormone receptors (TRα; NR1A1, TRβ; NR1A2),

mineralocorticoid receptor (MR; NR3C2), progesterone receptor (PR; NR3C3) and the androgen

receptor (AR; NR3C4) are generally considered to play a lesser role in the regulation of

metabolic enzymes and drug transporters compared to other xenobiotic nuclear receptors. Their

involvement in the regulation of drug transporters in the brain is currently unclear.

The transcriptional factor aryl hydrocarbon receptor (AhR) which belongs to the basic

Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) family of developmental regulators can be

activated by environmental pollutants such as dioxins and a number of natural plant constituents

such as flavonoids, curcumin and indoles (Gu et al. 2000, Ashida et al. 2008). It has long been recognized to play a role in the regulation of phase I and II biotransformation enzymes, such as

CYP 1A1, CYP 1B1 and UGTs in the field of toxicology (Nguyen & Bradfield 2008, Bock &

Köhle 2006). Recently, it has also been identified to regulate the expression of P-gp (Jigorel et al. 2006), BCRP/Bcrp (Jigorel et al. 2006, Han & Sugiyama 2006), Mrp2 (Jigorel et al. 2006,

Han & Sugiyama 2006), Mrp3 (Maher et al. 2005), Mrp5 (Maher et al. 2005), OCT1 (Jigorel et al. 2006) and OATP2B1 (Jigorel et al. 2006). In the brain, AhR expression has been demonstrated in mouse neuroblastoma cells (Akahoshi et al. 2006), astrocytes, microvessel endothelial cells (Filbrandt et al. 2004), whole brain homogenate and brain capillaries of rodents and humans (Dauchy et al. 2008, Wang et al. 2011b), different regions of rat brain tissues

337 (Petersen et al. 2000) and the hCMEC/D3 human brain microvessel endothelial cell line (Dauchy

et al. 2009). Evidence on the functional regulation of CYPs expression by AhR at the BBB is

primarily generated from in vitro and in vivo studies (Wang et al. 2011b, Dauchy et al. 2009).

Wang et al also demonstrated in vivo P-gp, Mrp2 and Bcrp protein induction in isolated rat brain microvessels following treatment with a potent AhR ligand, 2,3,7,8-Tetrachlorodibenzo-p-dioxin

(TCDD), suggesting the in vivo involvement of AhR in the regulation of drug efflux transporters at the BBB in rodents (Wang et al. 2011b, Wang et al. 2013). In addition, in vivo upregulation of

P-gp functional expression mediated by TCDD at the rat BBB can be reversed by PKC-β1 activation, demonstrating AhR inhibition can also be achieved by upstream signaling pathways in addition to the conventional inhibitor approach (Wang et al. 2011a). At present, it remains unclear whether AhR is involved in the regulation of other drug transporters at the human BBB and in the brain parenchyma, despite the presence of AhR pathway in neurodevelopment and neuronal apoptosis processes (Nayyar et al. 2002, Kajta et al. 2009).

Conclusion

ABC and SLC drug transporters at the brain barriers and various cellular compartments of the brain parenchyma can alter CNS drug distribution and impair drug efficacy. However, these transporters also serve as important mechanisms which provide neuroprotection against harmful chemicals. Targeting the regulatory pathways that govern the expression of these transporters could provide novel strategies to selectively modulate drug permeability into the brain. Among these pathways, nuclear receptors present a useful molecular target to alter transporters expression in the brain through their ligand interactions with drugs and constituents of diets and/or herbal products. Therefore, a clear understanding of the mechanisms involved in these ligand interactions and the regulatory pathways between nuclear receptors and drug

338 transporters in the brain could better predict drug transporter functional expression following nuclear receptor activation or inhibition. At present, compelling data suggest that nuclear receptors play an important role in the regulation of drug transporters expression at the BBB, yet little is known about these regulatory pathways at the BCSFB and various cellular compartments of brain parenchyma (i.e., astrocytes, microglia and neurons). As well, these regulatory pathways have not been fully evaluated in humans. Hence, studies examining the expression of nuclear receptors in human brain tissues and primary cultures of human brain microvessel endothelial cells are needed. Also, the activity of many nuclear receptors is not limited to the brain.

Although modulation of nuclear receptors pathways may improve drug distribution in the brain, adverse effects in peripheral organs could occur in tissues where nuclear receptors are highly expressed (i.e., hepatic tissues, intestine, kidney). For this purpose, utilization of tissue-specific ligands of nuclear receptors presents a very attractive approach, however, the development of these agents poses a great challenge. In addition, target genes for most nuclear receptors are not only limited to drug transporters, but also include drug metabolizing enzymes (i.e., CYP450 enzymes). Indeed, the activities of these enzymes could be significantly different following their induction and could further alter drug distribution in the brain. Moreover, the overall contribution from each nuclear receptor in the regulation of drug transporters can be very complex and renders the goal to selectively modulate the activity of a single nuclear receptor challenging. Therefore, a full understanding of drug transporter regulatory pathway involving nuclear receptors in the brain is needed in the future. These studies are essential in the development of novel CNS pharmacotherapy with enhanced drug efficacy and/or minimal drug- associated neurotoxicity.

339

Table E-1: Regulation of Drug Transporters Mediated by Nuclear Receptors and Aryl Hydrocarbon Receptor. Targeted Drug Subtypes and Targeted Drug Transporters Transporters in Brain Examples of Abbreviation Name in Peripheral Tissues and Microvessel Endothelial Therapeutic and Dietary (Unified Organs Cells or Otherwise Ligands Nomenclature) Indicated RXRα-Heterodimerizing Partners P-gp(Bauer et al. 2004, P-gp(Geick et al. 2001, Rifampin, st. John’s Bauer et al. 2006, Perloff Albermann et al. 2005), Wort, statins, anti-HIV et al. 2007, Bauer et al. MRP2/Mrp2(Albermann et al. agents, ketoconazole, 2008, Lombardo et al. Pregnane X 2005), MRP3/Mrp3(Teng et al. herbal compounds and PXR (NR1I2) 2008, Narang et al. 2008, Receptor 2003, Albermann et al. 2005), other xenobiotics(Chang Ott et al. 2009, Chan et OATP1A2(Meyer Zu & Waxman 2006, di Masi al. 2011) and Schwabedissen et al. 2008) and et al. 2009, Dussault et al. MRP2/Mrp2(Bauer et al. Oatp1a4 (Guo et al. 2002) 2001) 2008) P-gp(Burk et al. 2005), MRP2(Jigorel et al. 2006, Huang et al. 2003, Kast et al. 2002), BCRP(Jigorel et al. 2006), MRP2(Xiong et al. P-gp(Wang et al. 2010, Phenobarbital, phenytoin, 2002, Staudinger et al. 2003), Chan et al. 2011, meclizine, clotrimazole, Constitutive Mrp3(Xiong et al. 2002, Lemmen et al. 2013), artemisinin, herbal Androstane CAR (NR1I3) Staudinger et al. 2003), Mrp2(Wang et al. 2010, compounds and other Receptor MRP4/Mrp4(Assem et al. Lemmen et al. 2013) and xenobiotics(Chang & 2004), OCT1(Jigorel et al. Bcrp(Wang et al. 2010, Waxman 2006) 2006), OATP1B1(Huang et al. Lemmen et al. 2013) 2003), OATP1B3(Jigorel et al. 2006) OATP2B1(Jigorel et al. 2006), and Oatp1a4(Staudinger et al. 2003) P-gp(Aiba et al. 2005, Saeki et al. 2008, Fan et al. 2009, Tachibana et al. 2009, Chow et al. 2010, Chow et al. 2011, Chow et al. 2009), MRP2/Mrp2(Fan et al. 2009, Chow et al. 2010, Maeng et al. P-gp(Durk et al. 2012), P- 2011), MRP3/Mrp3(McCarthy Vitamin D gp (Whole Brain Calcitriol (vitamin VDR (NR1I1) et al. 2005, Chow et al. 2009, Receptor Homogenate)(Chow et al. D)(Tirona & Kim 2005) Fan et al. 2009, Chow et al. 2011) 2010, Maeng et al. 2011), MRP4/Mrp4(Chow et al. 2010, Fan et al. 2009), PEPT1/Pept1(Fan et al. 2009, Maeng et al. 2011), Oat1(Chow et al. 2010) and Oat3(Chow et al. 2010). Mdr1a(Kok et al. 2003, Moffit et al. 2006, Cui et al. 2009), Mrp1(Hirai et al. 2007, Maher Peroxisome BCRP(Hoque et al. et al. 2005), Mrp3(Moffit et al. Fenofibrate and clofibrate Proliferators- 2012), Octn3 PPARα (NR1C1) 2006, Maher et al. 2005), (Mandrekar-Colucci & activated (Astrocytes)(Januszewicz Mrp4(Moffit et al. 2006), Landreth 2011) Receptors et al. 2009) BCRP/Bcrp(Moffit et al. 2006, Hirai et al. 2007), Oatp1a1(Kok et al. 2003), Octn2(Hirai et al.

340 2007, Wen et al. 2010) and Octn3(Januszewicz et al. 2009)

Thiazolidenediones (i.e., pioglitazone and PPARγ (NR1C3) BCRP(Szatmari et al. 2006) Not Determined rosiglitazone)(Mandrekar- Colucci & Landreth 2011) P-gp(Langmann et al. 2006), MRP2/Mrp2(Langmann et al. Liver X LXRα (NR1H2) P-gp and Mrp1(Elali & Taurine(Hoang et al. 2006, Chisaki et al. 2009), Receptors LXRβ (NR1H3) Hermann 2011) 2012) MRP5(Langmann et al. 2006), BCRP (Langmann et al. 2006) Oatp1a1, 2b1, Oat2,Oct1, Hepatocyte Oatp1a4, Octn2, Mdr1a, Mrp1, None reported(Hwang- Nuclear Factor HNF4α (NR2A1) Mrp3 and Mrp4 (Lu et al. 2010) Not Determined Verslues & Sladek 2010) 4α and MRP1(Niehof & Borlak 2009) Farnesoid X MRP2(Kast et al. 2002) and Guggulsterone(Porez et FXR (NR1H4) Mrp2(Bauer et al. 2008) Receptor OATP1B3(Jung et al. 2002) al. 2012) Retinoids (i.e., vitamin A RARα (NR1B1) P-gp(Stromskaya et al. 1998) Retinoic Acid P-gp(El Hafny et al. and tretinoin)(Mandrekar- RARβ (NR1B2) and BCRP(Hessel & Lampen Receptors 1997) Colucci & Landreth RARγ (NR1B3) 2010) 2011) Rexinoids (i.e., Potentially many genes bexarotene) and retinoids regulated by its Retinoid X (i.e., vitamin A, RXRα (NR2B1) heterodimerizing Receptor α tretinoin)(Mandrekar- partners(Westin et al. 1998, Colucci & Landreth Germain et al. 2006b) 2011) Endocrine Receptors Mrp3(Ruiz et al. 2006), BCRP/Bcrp(Ee et al. 2004, Tanaka et al. 2004, Imai et al. Bcrp(Hartz et al. 2010a, Tamoxifen, raloxifene, 2005, Wang et al. 2006, Cheng Estrogen ERα (NR3A1) Hartz et al. 2010b, fulvestrant, et al. 2006, Zhang et al. 2007, Receptors ERβ (NR3A2) Mahringer & Fricker genistein(Renoir et al. Wang et al. 2008, Yasuda et al. 2010) 2013) 2009) and Organic Anion Transporters (i.e., Oat1, Oat 2, Oat3)(Buist et al. 2003) Steroidal and non- P-gp, Bcrp * indirect effect steroidal selective GR through PXR and Glucocorticoid P-gp, Bcrp(Narang et al. modulators (i.e., GR (NR3C1) CAR(Pascussi et al. 2000, Receptor 2008) budesonide, Pascussi et al. 2003, Narang et mometasone)(De al. 2008, Zhang et al. 2012) Bosscher et al. 2010) P-gp(Jigorel et al. 2006), BCRP/Bcrp(Jigorel et al. 2006, Han & Sugiyama 2006), Glucobrassicin, Aryl Mrp2(Jigorel et al. 2006, Han & P-gp, Bcrp and flavonoids, curcumin, Hydrocarbon AhR Sugiyama 2006), Mrp3(Maher Mrp2(Wang et al. 2011b) indigoids(Ashida et al. Receptor et al. 2005), Mrp5(Maher et al. 2008) 2005), OCT1(Jigorel et al. 2006) and OATP2B1(Jigorel et al. 2006)

Conflict of Interest

None.

341 References

Abbott, N. J., Patabendige, A. A., Dolman, D. E., Yusof, S. R. and Begley, D. J. (2010) Structure and function of the blood-brain barrier. Neurobiology of Disease, 37(1), 13-25. Aiba, T., Susa, M., Fukumori, S. and Hashimoto, Y. (2005) The effects of culture conditions on CYP3A4 and MDR1 mRNA induction by 1alpha,25-dihydroxyvitamin D(3) in human intestinal cell lines, Caco-2 and LS180. Drug metabolism and pharmacokinetics., 20(4), 268-274. Akahoshi, E., Yoshimura, S. and Ishihara-Sugano, M. (2006) Over-expression of AhR (aryl hydrocarbon receptor) induces neural differentiation of Neuro2a cells: neurotoxicology study. Environmental Health, 5(0), 1-24. Albermann, N., Schmitz-Winnenthal, F. H., Z'Graggen, K., Volk, C., Hoffmann, M. M., Haefeli, W. E. and Weiss, J. (2005) Expression of the drug transporters MDR1/ABCB1, MRP1/ABCC1, MRP2/ABCC2, BCRP/ABCG2, and PXR in peripheral blood mononuclear cells and their relationship with the expression in intestine and liver. Biochemical Pharmacology, 70(6), 949-958. Ananthanarayanan, M., Balasubramanian, N., Makishima, M., Mangelsdorf, D. J. and Suchy, F. J. (2001) Human Bile Salt Export Pump Promoter Is Transactivated by the Farnesoid X Receptor/Bile Acid Receptor. Journal of Biological Chemistry, 276(31), 28857-28865. Aouabdi, S., Gibson, G. and Plant, N. (2006) Transcriptional regulation of the PXR gene: Identification and characterization of a functional peroxisome proliferator-activated receptor α binding site within the proximal promoter of PXR. Drug Metabolism and Disposition, 34(1), 138-144. Arfaoui, A., Lobo, M. V., Boulbaroud, S., Ouichou, A., Mesfioui, A. and Arenas, M. I. (2013) Expression of retinoic acid receptors and retinoid X receptors in normal and vitamin A deficient adult rat brain. Annals of Anatomy, 195(2), 111-121. Ashida, H., Nishiumi, S. and Fukuda, I. (2008) An update on the dietary ligands of the AhR. Expert Opinion on Drug Metabolism and Toxicology, 4(11), 1429-1447. Ashraf, T., Kis, O., Banerjee, N. and Bendayan, R. (2012) Drug Transporters at Brain Barriers: Expression and Regulation by Neurological Disorders. Advances in Experimental Medicine and Biology, 763(0): 20-69. Assem, M., Schuetz, E. G., Leggas, M., Sun, D., Yasuda, K., Reid, G., Zelcer, N., Adachi, M., Strom, S., Evans, R. M., Moore, D. D., Borst, P. and Schuetz, J. D. (2004) Interactions between hepatic Mrp4 and Sult2a as revealed by the constitutive androstane receptor and Mrp4 knockout mice. Journal of Biological Chemistry, 279(21), 22250-22257. Bauer, B., Hartz, A. M. S., Fricker, G. and Miller, D. S. (2004) Pregnane X receptor upregulation of P-glycoprotein expression and transport function at the blood-brain barrier. Molecular Pharmacology, 66(3), 413-419. Bauer, B., Hartz, A. M. S., Lucking, J. R., Yang, X., Pollack, G. M. and Miller, D. S. (2008) Coordinated nuclear receptor regulation of the efflux transporter, Mrp2, and the phase-II metabolizing enzyme, GSTπ, at the blood-brain barrier. Journal of Cerebral Blood Flow and Metabolism, 28(6), 1222-1234. Bauer, B., Yang, X., Hartz, A. M. S., Olson, E. R., Zhao, R., Kalvass, J. C., Pollack, G. M. and Miller, D. S. (2006) In vivo activation of human pregnane X receptor tightens the blood- brain barrier to methadone through p-glycoprotein up-regulation. Molecular Pharmacology, 70(4), 1212-1219.

342 Bendayan, R., Lee, G. and Bendayan, M. (2002) Functional expression and localization of P- glycoprotein at the blood brain barrier. Microscopy Research and Technique, 57(5), 365- 380. Bendayan, R., Ronaldson, P. T., Gingras, D. and Bendayan, M. (2006) In situ localization of P- glycoprotein (ABCB1) in human and rat brain. Journal of Histochemistry and Cytochemistry, 54(10), 1159-1167. Benoit, G., Cooney, A., Giguere, V., Ingraham, H., Lazar, M., Muscat, G., Perlmann, T., Renaud, J. P., Schwabe, J., Sladek, F., Tsai, M. J. and Laudet, V. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacological Reviews, 58(4), 798-836. Bertilsson, G., Heidrich, J., Svensson, K., Asman, M., Jendeberg, L., Sydow-Bäckman, M., Ohlsson, R., Postlind, H., Blomquist, P. and Berkenstam, A. (1998) Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proceedings of the National Academy of Sciences of the United States of America, 95(21), 12208-12213. Bock, K. W. and Köhle, C. (2006) Ah receptor: Dioxin-mediated toxic responses as hints to deregulated physiologic functions. Biochemical Pharmacology, 72(4), 393-404. Brélivet, Y., Rochel, N. and Moras, D. (2012) Structural analysis of nuclear receptors: From isolated domains to integral proteins. Molecular and Cellular Endocrinology, 348(2), 466-473. Buist, S. C. N., Cherrington, N. J. and Klaassen, C. D. (2003) Endocrine regulation of rat organic anion transporters. Drug Metabolism and Disposition, 31(5), 559-564. Burk, O., Arnold, K. A., Geick, A., Tegude, H. and Eichelbaum, M. (2005) A role for constitutive androstane receptor in the regulation of human intestinal MDR1 expression. Biological Chemistry, 386(6), 503-513. Cao, G., Bales, K. R., DeMattos, R. B. and Paul, S. M. (2007) Liver X receptor-mediated gene regulation and cholesterol homeostasis in brain: relevance to Alzheimer's disease therapeutics. Current Alzheimer Research, 4(2), 179-184. Chan, G. N. Y., Hoque, M. T. and Bendayan, R. (2010) Regulation of P-glycoprotein (P-gp) by Orphan Nuclear Receptors in Human Brain. Gordon Research Conference. Barriers of the CNS: Blood/Brain Interfaces in Health and Disease. New London, NH, USA. Chan, G. N. Y., Hoque, M. T., Cummins, C. L. and Bendayan, R. (2011) Regulation of P- glycoprotein by orphan nuclear receptors in human brain microvessel endothelial cells. Journal of Neurochemistry, 118(2), 163-175. Chang, T. K. H. and Waxman, D. J. (2006) Synthetic drugs and natural products as modulators of constitutive androstane receptor (CAR) and pregnane X receptor (PXR). Drug Metabolism Reviews, 38(1-2), 51-73. Chen, J., Costa, L. G. and Guizzetti, M. (2011) Retinoic acid isomers up-regulate ATP binding cassette A1 and G1 and cholesterol efflux in rat astrocytes: Implications for their therapeutic and teratogenic effects. Journal of Pharmacology and Experimental Therapeutics, 338(3), 870-878. Cheng, X., Maher, J., Lu, H. and Klaassen, C. D. (2006) Endocrine regulation of gender- divergent mouse organic anion-transporting polypeptide (Oatp) expression. Molecular Pharmacology, 70(4), 1291-1297.

343 Chi, O. Z., Hunter, C., Liu, X. and Weiss, H. R. (2006) Effects of 17β-estradiol on blood-brain barrier disruption during focal cerebral ischemia in younger and older rats. Hormone and Metabolic Research, 38(6), 377-381. Chisaki, I., Kobayashi, M., Itagaki, S., Hirano, T. and Iseki, K. (2009) Liver X receptor regulates expression of MRP2 but not that of MDR1 and BCRP in the liver. Biochimica et Biophysica Acta - Biomembranes, 1788(11), 2396-2403. Chow, E. C. Y., Durk, M. R., Cummins, C. L. and Pang, K. S. (2011) 1α,25-dihydroxyvitamin D3 up-regulates P-glycoprotein via the vitamin D receptor and not farnesoid X receptor in both fxr(-/-) and fxr(+/+) mice and increased renal and brain efflux of digoxin in mice in vivo. Journal of Pharmacology and Experimental Therapeutics, 337(3), 846-859. Chow, E. C. Y., Maeng, H. J., Liu, S., Khan, A. A., Groothuis, G. M. M. and Pang, K. S. (2009) 1α,25-dihydroxyvitamin D3 triggered vitamin D receptor and farnesoid X receptor-like effects in rat intestine and liver in vivo. Biopharmaceutics and Drug Disposition, 30(8), 457-475. Chow, E. C. Y., Sun, H., Khan, A. A., Groothuis, G. M. M. and Pang, K. S. (2010) Effects of 1α,25-dihydroxyvitamin D3 on transporters and enzymes of the rat intestine and kidney in vivo. Biopharmaceutics and Drug Disposition, 31(1), 91-108. Cipolla, M. J., Godfrey, J. A. and Wiegman, M. J. (2009) The effect of ovariectomy and estrogen on penetrating brain arterioles and blood-brain barrier permeability. Microcirculation, 16(8), 685-693. Cui, X., Pelekanos, M., Liu, P. Y., Burne, T. H., McGrath, J. J. and Eyles, D. W. (2013) The vitamin D receptor in dopamine neurons; its presence in human substantia nigra and its ontogenesis in rat midbrain. Neuroscience, 236(1), 77-87. Cui, Y. J., Cheng, X., Weaver, Y. M. and Klaassen, C. D. (2009) Tissue distribution, gender- divergent expression, ontogeny, and chemical induction of multidrug resistance transporter genes (Mdr1a, Mdr1b, Mdr2) in mice. Drug Metabolism and Disposition, 37(1), 203-210. Dallas, S., Miller, D. S. and Bendayan, R. (2006) Multidrug resistance-associated proteins: Expression and function in the central nervous system. Pharmacological Reviews, 58(2), 140-161. Dauchy, S., Dutheil, F., Weaver, R. J., Chassoux, F., Daumas-Duport, C., Couraud, P. O., Scherrmann, J. M., De Waziers, I. and Decleves, X. (2008) ABC transporters, cytochromes P450 and their main transcription factors: Expression at the human blood- brain barrier. Journal of Neurochemistry, 107(6), 1518-1528. Dauchy, S., Miller, F., Couraud, P. O., Weaver, R. J., Weksler, B., Romero, I. A., Scherrmann, J. M., De Waziers, I. and Declèves, X. (2009) Expression and transcriptional regulation of ABC transporters and cytochromes P450 in hCMEC/D3 human cerebral microvascular endothelial cells. Biochemical Pharmacology, 77(5), 897-909. De Bosscher, K., Haegeman, G. and Elewaut, D. (2010) Targeting inflammation using selective glucocorticoid receptor modulators. Current Opinion in Pharmacology, 10(4), 497-504. Deroo, B. J. and Korach, K. S. (2006) Estrogen receptors and human disease. Journal of Clinical Investigation, 116(3), 561-570. di Masi, A., Marinis, E. D., Ascenzi, P. and Marino, M. (2009) Nuclear receptors CAR and PXR: Molecular, functional, and biomedical aspects. Molecular Aspects of Medicine, 30(5), 297-343.

344 Duret, C., Daujat-Chavanieu, M., Pascussi, J. M., Pichard-Garcia, L., Balaguer, P., Fabre, J. M., Vilarem, M. J., Maurel, P. and Gerbal-Chaloin, S. (2006) Ketoconazole and miconazole are antagonists of the human glucocorticoid receptor: Consequences on the expression and function of the constitutive androstane receptor and the pregnane X receptor. Molecular Pharmacology, 70(1), 329-339. Durk, M. R., Chan, G. N. Y., Campos, C. R., Peart, J. C., Chow, E. C. Y., Lee, E., Cannon, R. E., Bendayan, R., Miller, D. S. and Pang, K. S. (2012) 1α,25-Dihydroxyvitamin D3-liganded vitamin D receptor increases expression and transport activity of P-glycoprotein in isolated rat brain capillaries and human and rat brain microvessel endothelial cells. Journal of Neurochemistry, 123(6), 944-953. Dussault, I., Lin, M., Hollister, K., Wang, E. H., Synold, T. W. and Forman, B. M. (2001) Peptide mimetic HIV protease inhibitors are ligands for the orphan receptor SXR. Journal of Biological Chemistry, 276(36), 33309-33312. Ee, P. L. R., Kamalakaran, S., Tonetti, D., He, X., Ross, D. D. and Beck, W. T. (2004) Identification of a Novel Estrogen Response Element in the Breast Cancer Resistance Protein (ABCG2) Gene. Cancer Research, 64(4), 1247-1251. El Hafny, B., Chappey, O., Piciotti, M., Debray, M., Boval, B. and Roux, F. (1997) Modulation of P-glycoprotein activity by glial factors and retinoic acid in an immortalized rat brain microvessel endothelial cell line. Neuroscience Letters, 236(2), 107-111. Elali, A. and Hermann, D. M. (2011) Liver X receptor activation enhances blood-brain barrier integrity in the ischemic brain and increases the abundance of ATP-binding cassette transporters ABCB1 and ABCC1 on brain capillary cells. Brain Pathology, 22(2), 175- 187. Eyal, S., Hsiao, P. and Unadkat, J. D. (2009) Drug interactions at the blood-brain barrier: Fact or fantasy? Pharmacology and Therapeutics, 123(1), 80-104. Eyles, D. W., Smith, S., Kinobe, R., Hewison, M. and McGrath, J. J. (2005) Distribution of the Vitamin D receptor and 1α-hydroxylase in human brain. Journal of Chemical Neuroanatomy, 29(1), 21-30. Fan, J., Liu, S., Du, Y., Morrison, J., Shipman, R. and Pang, K. S. (2009) Up-regulation of transporters and enzymes by the vitamin D receptor ligands, 1α,25-dihydroxyvitamin D3 and vitamin D analogs, in the caco-2 cell monolayer. Journal of Pharmacology and Experimental Therapeutics, 330(2), 389-402. Filbrandt, C. R., Wu, Z., Zlokovic, B., Opanashuk, L. and Gasiewicz, T. A. (2004) Presence and functional activity of the aryl hydrocarbon receptor in isolated murine cerebral vascular endothelial cells and astrocytes. NeuroToxicology, 25(4 SPEC. ISS.), 605-616. Geick, A., Eichelbaum, M. and Burk, O. (2001) Nuclear Receptor Response Elements Mediate Induction of Intestinal MDR1 by Rifampin. Journal of Biological Chemistry, 276(18), 14581-14587. George, C. L., Lightman, S. L. and Biddie, S. C. (2011) Transcription factor interactions in genomic nuclear receptor function. Epigenomics, 3(4), 471-485. Germain, P., Chambon, P., Eichele, G., Evans, R. M., Lazar, M. A., Leid, M., De Lera, A. R., Lotan, R., Mangelsdorf, D. J. and Gronemeyer, H. (2006a) International union of pharmacology. LX. Retinoic acid receptors. Pharmacological Reviews, 58(4), 712-725. Germain, P., Chambon, P., Eichele, G., Evans, R. M., Lazar, M. A., Leid, M., De Lera, A. R., Lotan, R., Mangelsdorf, D. J. and Gronemeyer, H. (2006b) International union of pharmacology. LXIII. Retinoid X receptors. Pharmacological Reviews, 58(4), 760-772.

345 Germain, P., Staels, B., Dacquet, C., Spedding, M. and Laudet, V. (2006c) Overview of nomenclature of nuclear receptors. Pharmacological Reviews, 58(4), 685-704. Gilardi, F., Viviani, B., Galmozzi, A., Boraso, M., Bartesaghi, S., Torri, A., Caruso, D., Crestani, M., Marinovich, M. and de Fabiani, E. (2009) Expression of sterol 27-hydroxylase in glial cells and its regulation by liver X receptor signaling. Neuroscience, 164(2), 530-540. Gonzales, R. J., Ansar, S., Duckles, S. P. and Krause, D. N. (2007) Androgenic/estrogenic balance in the male rat cerebral circulation: Metabolic enzymes and sex steroid receptors. Journal of Cerebral Blood Flow and Metabolism, 27(11), 1841-1852. Gu, Y. Z., Hogenesch, J. B. and Bradfield, C. A. (2000) The PAS superfamily: sensors of environmental and developmental signals. Annual Review of Pharmacology and Toxicology, 40(519-561. Guo, G. L., Staudinger, J., Ogura, K. and Klaassen, C. D. (2002) Induction of rat organic anion transporting polypeptide 2 by pregnenolone-16α-carbonitrile Is via interaction with pregnane X receptor. Molecular Pharmacology, 61(4), 832-839. Han, Y. and Sugiyama, Y. (2006) Expression and regulation of breast cancer resistance protein and multidrug resistance associated protein 2 in BALB/c mice. Biological and Pharmaceutical Bulletin, 29(5), 1032-1035. Hartz, A. M. S., Madole, E. K., Miller, D. S. and Bauer, B. (2010a) Estrogen receptor β signaling through phosphatase and tensin homolog/phosphoinositide 3-kinase/Akt/glycogen synthase kinase 3 down-regulates blood-brain barrier breast cancer resistance protein. Journal of Pharmacology and Experimental Therapeutics, 334(2), 467-476. Hartz, A. M. S., Mahringer, A., Miller, D. S. and Bauer, B. (2010b) 17-Β-Estradiol: A powerful modulator of blood-brain barrier BCRP activity. Journal of Cerebral Blood Flow and Metabolism, 30(10), 1742-1755. Heinlein, C. A. and Chang, C. (2001) Role of chaperones in nuclear translocation and transactivation of steroid receptors. Endocrine, 14(2), 143-149. Helsen, C., Kerkhofs, S., Clinckemalie, L., Spans, L., Laurent, M., Boonen, S., Vanderschueren, D. and Claessens, F. (2012) Structural basis for nuclear hormone receptor DNA binding. Molecular and Cellular Endocrinology, 348(2), 411-417. Heneka, M. T. and Landreth, G. E. (2007) PPARs in the brain. Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, 1771(8), 1031-1045. Hessel, S. and Lampen, A. (2010) All-trans retinoic acid enhances the transport of phase II metabolites of benzo[a]pyrene by inducing the Breast Cancer Resistance Protein expression in Caco-2 cells. Toxicology Letters, 197(2), 151-155. Hilser, V. J. and Thompson, E. B. (2011) Structural dynamics, intrinsic disorder, and allostery in nuclear receptors as transcription factors. Journal of Biological Chemistry, 286(46), 39675-39682. Hirai, T., Fukui, Y. and Motojima, K. (2007) PPARα agonists positively and negatively regulate the expression of several nutrient/drug transporters in mouse small intestine. Biological and Pharmaceutical Bulletin, 30(11), 2185-2190. Hoang, M. H., Jia, Y., Jun, H. J., Lee, J. H., Hwang, K. Y., Choi, D. W., Um, S. J., Lee, B. Y., You, S. G. and Lee, S. J. (2012) Taurine is a liver X receptor-α ligand and activates transcription of key genes in the reverse cholesterol transport without inducing hepatic lipogenesis. Molecular Nutrition and Food Research, 56(6), 900-911.

346 Hoque, M. T., Robillard, K. R. and Bendayan, R. (2012) Regulation of Breast Cancer Resistant Protein (BCRP) by Peroxisome Proliferator-Activated Receptor Alpha (PPARalpha) in Human Brain Microvessel Endothelial Cells. Molecular Pharmacology, 81(4), 598-609. Huang, W., Zhang, J., Chua, S. S., Qatanani, M., Han, Y., Granata, R. and Moore, D. D. (2003) Induction of bilirubin clearance by the constitutive androstane receptor (CAR). Proceedings of the National Academy of Sciences of the United States of America, 100(7), 4156-4161. Hwang-Verslues, W. W. and Sladek, F. M. (2010) HNF4α-role in drug metabolism and potential drug target? Current Opinion in Pharmacology, 10(6), 698-705. Imai, Y., Ishikawa, E., Asada, S. and Sugimoto, Y. (2005) Estrogen-mediated post transcriptional down-regulation of breast cancer resistance protein/ABCG2. Cancer Research, 65(2), 596-604. Januszewicz, E., Pajak, B., Gajkowska, B., Samluk, Ł., Djavadian, R. L., Hinton, B. T. and Nałecz, K. A. (2009) Organic cation/carnitine transporter OCTN3 is present in astrocytes and is up-regulated by peroxisome proliferators-activator receptor agonist. International Journal of Biochemistry and Cell Biology, 41(12), 2599-2609. Jigorel, E., Le Vee, M., Boursier-Neyret, C., Parmentier, Y. and Fardel, O. (2006) Differential regulation of sinusoidal and canalicular hepatic drug transporter expression by xenobiotics activating drug-sensing receptors in primary human hepatocytes. Drug Metabolism and Disposition, 34(10), 1756-1763. Jung, D., Podvinec, M., Meyer, U. A., Mangelsdorf, D. J., Fried, M., Meier, P. J. and KullakUblick, G. A. (2002) Human organic anion transporting polypeptide 8 promoter is transactivated by the farnesoid X receptor/bile acid receptor. Gastroenterology, 122(7), 1954-1966. Kajta, M., Wójtowicz, A. K., Maćkowiak, M. and Lasoń, W. (2009) Aryl hydrocarbon receptor- mediated apoptosis of neuronal cells: A possible interaction with estrogen receptor signaling. Neuroscience, 158(2), 811-822. Kamiya, A., Inoue, Y. and Gonzalez, F. J. (2003) Role of the hepatocyte nuclear factor 4α in control of the pregnane X receptor during fetal liver development. Hepatology, 37(6), 1375-1384. Kaneko, E., Matsuda, M., Yamada, Y., Tachibana, Y., Shimomura, I. and Makishima, M. (2003) Induction of intestinal ATP-binding cassette transporters by a phytosterol-derived liver X receptor agonist. Journal of Biological Chemistry, 278(38), 36091-36098. Kast, H. R., Goodwin, B., Tarr, P. T., Jones, S. A., Anisfeld, A. M., Stoltz, C. M., Tontonoz, P., Kliewer, S., Willson, T. M. and Edwards, P. A. (2002) Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor. Journal of Biological Chemistry, 277(4), 2908-2915. Kawamoto, T., Sueyoshi, T., Zelko, I., Moore, R., Washburn, K. and Negishi, M. (1999) Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene. Molecular and Cellular Biology, 19(9), 6318-6322. Khorasanizadeh, S. and Rastinejad, F. (2001) Nuclear-receptor interactions on DNA-response elements. Trends in Biochemical Sciences, 26(6), 384-390. Kliewer, S. A., Moore, J. T., Wade, L., Staudinger, J. L., Watson, M. A., Jones, S. A., McKee, D. D., Oliver, B. B., Willson, T. M., Zetterstro?m, R. H., Perlmann, T. and Lehmann, J.

347 M. (1998) An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell, 92(1), 73-82. Kok, T., Bloks, V. W., Wolters, H., Havinga, R., Jansen, P. L. M., Staels, B. and Kuipers, F. (2003) Peroxisome proliferator-activated receptor α (PPARα)-mediated regulation of multidrug resistance 2 (Mdr2) expression and function in mice. Biochemical Journal, 369(3), 539-547. Krause, D. N., Duckles, S. P. and Pelligrino, D. A. (2006) Influence of sex steroid hormones on cerebrovascular function. Journal of Applied Physiology, 101(4), 1252-1261. Krezel, W., Kastner, P. and Chambon, P. (1999) Differential expression of retinoid receptors in the adult mouse central nervous system. Neuroscience, 89(4), 1291-1300. Kriechbaumer, V., von Löffelholz, O. and Abell, B. M. (2012) Chaperone receptors: Guiding proteins to intracellular compartments. Protoplasma, 249(1), 21-30. Kusuhara, H. and Sugiyama, Y. (2004) Efflux transport systems for organic anions and cations at the blood-CSF barrier. Advanced Drug Delivery Reviews, 56(12), 1741-1763. Kusuhara, H. and Sugiyama, Y. (2005) Active efflux across the blood-brain barrier: Role of the solute carrier family. NeuroRx, 2(1), 73-85. Lamba, J. K., Lamba, V., Yasuda, K., Lin, Y. S., Assem, M., Thompson, E., Strom, S. and Schuetz, E. G. (2004) Expression of constitutive androstane receptor splice variants in human tissues and their functional consequences. Journal of Pharmacology and Experimental Therapeutics, 311(2), 811-821. Landrier, J. F., Eloranta, J. J., Vavricka, S. R. and Kullak-Ublick, G. A. (2006) The nuclear receptor for bile acids, FXR, transactivates human organic solute transporter-alpha and - beta genes. American Journal of Physiology-Gastrointestinal and Liver Physiology, 290(3), G476-485. Langmann, T., Mauerer, R. and Schmitz, G. (2006) Human ATP-binding cassette Transporter TaqMan Low-Density Array: Analysis of macrophage differentiation and foam cell formation. Clinical Chemistry, 52(2), 310-313. Lee, G., Dallas, S., Hong, M. and Bendayan, R. (2001) Drug transporters in the central nervous system: Brain barriers and brain parenchyma considerations. Pharmacological Reviews, 53(4), 569-596. Lemmen, J., Tozakidis, I. E. P., Bele, P. and Galla, H. J. (2013) Constitutive androstane receptor upregulates Abcb1 and Abcg2 at the blood-brain barrier after CITCO activation. Brain Research, 21(1501), 68-80. Li, H. and Wang, H. (2010) Activation of xenobiotic receptors: Driving into the nucleus. Expert Opinion on Drug Metabolism and Toxicology, 6(4), 409-426. Liang, Y., Lin, S., Beyer, T. P., Zhang, Y., Wu, X., Bales, K. R., DeMattos, R. B., May, P. C., Li, S. D., Jiang, X. C., Eacho, P. I., Cao, G. and Paul, S. M. (2004) A liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein E expression, secretion and cholesterol homeostasis in astrocytes. Journal of Neurochemistry, 88(3), 623-634. Lombardo, L., Pellitteri, R., Balazy, M. and Cardile, V. (2008) Induction of nuclear receptors and drug resistance in the brain microvascular endothelial cells treated with antiepileptic drugs. Current Neurovascular Research, 5(2), 82-92. Lu, H., Gonzalez, F. J. and Klaassen, C. (2010) Alterations in hepatic mRNA expression of phase II enzymes and xenobiotic transporters after targeted disruption of hepatocyte nuclear factor 4 alpha. Toxicological Sciences, 118(2), 380-390.

348 Maden, M. (2007) Retinoic acid in the development, regeneration and maintenance of the nervous system. Nature Reviews Neuroscience, 8(10), 755-765. Mader, S., Chen, J. Y., Chen, Z., White, J., Chambon, P. and Gronemeyer, H. (1993) The patterns of binding of RAR, RXR and TR homo- And heterodimers to direct repeats are dictated by the binding specificities of the DNA binding domains. EMBO Journal, 12(13), 5029-5041. Maeng, H. J., Durk, M. R., Chow, E. C. Y., Ghoneim, R. and Pang, K. S. (2011) 1α,25- dihydroxyvitamin D3on intestinal transporter function: Studies with the rat everted intestinal sac. Biopharmaceutics and Drug Disposition, 32(2), 112-125. Maher, J. M., Cheng, X., Slitt, A. L., Dieter, M. Z. and Klaassen, C. D. (2005) Induction of the multidrug resistance-associated protein family of transporters by chemical activators of receptor-mediated pathways in mouse liver. Drug Metabolism and Disposition, 33(7), 956-962. Mahringer, A. and Fricker, G. (2010) BCRP at the blood-brain barrier: Genomic regulation by 17β-estradiol. Molecular Pharmaceutics, 7(5), 1835-1847. Malaplate-Armand, C., Ferrari, L., Masson, C., Visvikis-Siest, S., Lambert, H. and Batt, A. M. (2005) Down-regulation of astroglial CYP2C, glucocorticoid receptor and constitutive androstane receptor genes in response to cocaine in human U373 MG astrocytoma cells. Toxicology Letters, 159(3), 203-211. Mandrekar-Colucci, S. and Landreth, G. E. (2011) Nuclear receptors as therapeutic targets for Alzheimer's disease. Expert Opinion on Therapeutic Targets, 15(9), 1085-1097. Mangelsdorf, D. J., Umesono, K., Kliewer, S. A., Borgmeyer, U., Ong, E. S. and Evans, R. M. (1991) A direct repeat in the cellular retinol-binding protein type II gene confers differential regulation by RXR and RAR. Cell, 66(3), 555-561. McCarthy, T. C., Li, X. and Sinal, C. J. (2005) Vitamin D receptor-dependent regulation of colon multidrug resistance-associated protein 3 gene expression by bile acids. Journal of Biological Chemistry, 280(24), 23232-23242. Meyer Zu Schwabedissen, H. E., Tirona, R. G., Yip, C. S., Ho, R. H. and Kim, R. B. (2008) Interplay between the nuclear receptor pregnane X receptor and the uptake transporter organic anion transporter polypeptide 1A2 selectively enhances estrogen effects in breast cancer. Cancer Research, 68(22), 9338-9347. Miki, Y., Suzuki, T., Tazawa, C., Blumberg, B. and Sasano, H. (2005) Steroid and xenobiotic receptor (SXR), cytochrome P450 3A4 and multidrug resistance gene 1 in human adult and fetal tissues. Molecular and Cellular Endocrinology, 231(1-2), 75-85. Miller, D. S. (2010) Regulation of P-glycoprotein and other ABC drug transporters at the blood- brain barrier. Trends in Pharmacological Sciences, 31(6), 246-254. Moffit, J. S., Aleksunes, L. M., Maher, J. M., Scheffer, G. L., Klaassen, C. D. and Manautou, J. E. (2006) Induction of hepatic transporters multidrug resistance-associated proteins (Mrp) 3 and 4 by clofibrate is regulated by peroxisome proliferator-activated receptor α. Journal of Pharmacology and Experimental Therapeutics, 317(2), 537-545. Moreau, A., Vilarem, M. J., Maurel, P. and Pascussi, J. M. (2008) Xenoreceptors CAR and PXR activation and consequences on lipid metabolism, glucose homeostasis, and inflammatory response. Molecular Pharmaceutics, 5(1), 35-41. Narang, V. S., Fraga, C., Kumar, N., Shen, J., Throm, S., Stewart, C. F. and Waters, C. M. (2008) Dexamethasone increases expression and activity of multidrug resistance

349 transporters at the rat blood-brain barrier. American Journal of Physiology - Cell Physiology, 295(2), C440-C450. Nayyar, T., Zawia, N. H. and Hood, D. B. (2002) Transplacental effects of 2,3,7,8- tetrachlorodibenzo-p-dioxin on the temporal modulation of Sp1 DNA binding in the developing cerebral cortex and cerebellum. Experimental and Toxicologic Pathology, 53(6), 461-468. Nguyen, L. P. and Bradfield, C. A. (2008) The search for endogenous activators of the aryl hydrocarbon receptor. Chemical Research in Toxicology, 21(1), 102-116. Niehof, M. and Borlak, J. (2009) Expression of HNF4alpha in the human and rat choroid plexus - Implications for drug transport across the blood-cerebrospinal-fluid (CSF) barrier. BMC Molecular Biology, 10(68), 1-14. Nishimura, M., Naito, S. and Yokoi, T. (2004) Tissue-specific mRNA expression profiles of human nuclear receptor subfamilies. Drug metabolism and pharmacokinetics, 19(2), 135- 149. Ott, M., Fricker, G. and Bauer, B. (2009) Pregnane X receptor (PXR) regulates P-glycoprotein at the blood-brain barrier: Functional similarities between pig and human PXR. Journal of Pharmacology and Experimental Therapeutics, 329(1), 141-149. Pascussi, J. M., Busson-Le Coniat, M., Maurel, P. and Vilarem, M. J. (2003) Transcriptional analysis of the orphan nuclear receptor constitutive androstane receptor (NR1I3) gene promoter: Identification of a distal glucocorticoid response element. Molecular Endocrinology, 17(1), 42-55. Pascussi, J. M., Drocourt, L., Fabre, J. M., Maurel, P. and Vilarem, M. J. (2000) Dexamethasone induces pregnane X receptor and retinoid X receptor-α expression in human hepatocytes: Synergistic increase of CYP3A4 induction by pregnane X receptor activators. Molecular Pharmacology, 58(2), 361-372. Pastor-Anglada, M., Cano-Soldado, P., Errasti-Murugarren, E. and Casado, F. J. (2008) SLC28 genes and concentrative nucleoside transporter (CNT) proteins. Xenobiotica, 38(7-8), 972-994. Perloff, M. D., VonMoltke, L. L., Fahey, J. M. and Greenblatt, D. J. (2007) Induction of P- glycoprotein expression and activity by ritonavir in bovine brain microvessel endothelial cells. Journal of Pharmacy and Pharmacology, 59(7), 947-953. Petersen, S. L., Curran, M. A., Marconi, S. A., Carpenter, C. D., Lubbers, L. S. and McAbee, M. D. (2000) Distribution of mRNAs encoding the arylhydrocarbon receptor, arylhydrocarbon receptor nuclear translocator, and arylhydrocarbon receptor nuclear translocator-2 in the rat brain and brainstem. Journal of Comparative Neurology, 427(3), 428-439. Porez, G., Prawitt, J., Gross, B. and Staels, B. (2012) Bile acid receptors as targets for the treatment of dyslipidemia and cardiovascular disease. Journal of Lipid Research, 53(9), 1723-1737. Pujols, L., Mullol, J., Roca-Ferrer, J., Torrego, A., Xaubet, A., Cidlowski, J. A. and Picado, C. (2002) Expression of glucocorticoid receptor α- and β-isoforms in human cells and tissues. American Journal of Physiology - Cell Physiology, 283(4 52-4), C1324-C1331. Renoir, J. M., Marsaud, V. and Lazennec, G. (2013) Estrogen receptor signaling as a target for novel breast cancer therapeutics. Biochemical Pharmacology, 85(4), 449-465. Repa, J. J., Berge, K. E., Pomajzl, C., Richardson, J. A., Hobbs, H. and Mangelsdorf, D. J. (2002) Regulation of ATP-binding cassette sterol transporters ABCG5 and ABCG8 by

350 the liver X receptors alpha and beta. Journal of Biological Chemistry, 277(21), 18793- 18800. Reul, J. M. H. M. and De Kloet, E. R. (1985) Two receptor systems for corticosterone in rat brain: Microdistribution and differential occupation. Endocrinology, 117(6), 2505-2511. Ronaldson, P. T., Persidsky, Y. and Bendayan, R. (2008) Regulation of ABC membrane transporters in glial cells: relevance to the pharmacotherapy of brain HIV-1 infection. GLIA, 56(16), 1711-1735. Rosenfeld, M. G., Lunyak, V. V. and Glass, C. K. (2006) Sensors and signals: A coactivator/corepressor/epigenetic code for integrating signal-dependent programs of transcriptional response. Genes and Development, 20(11), 1405-1428. Roth, M., Obaidat, A. and Hagenbuch, B. (2012) OATPs, OATs and OCTs: The organic anion and cation transporters of the SLCO and SLC22A gene superfamilies. British Journal of Pharmacology, 165(5), 1260-1287. Ruiz, M. L., Villanueva, S. S. M., Luquita, M. G., Vore, M., Mottino, A. D. and Catania, V. A. (2006) Ethynylestradiol increases expression and activity of rat liver MRP3. Drug Metabolism and Disposition, 34(6), 1030-1034. Saeki, M., Kurose, K., Tohkin, M. and Hasegawa, R. (2008) Identification of the functional vitamin D response elements in the human MDR1 gene. Biochemical Pharmacology, 76(4), 531-542. Saint-Pol, J., Vandenhaute, E., Boucau, M. C., Candela, P., Dehouck, L., Cecchelli, R., Dehouck, M. P., Fenart, L. and Gosselet, F. (2012) Brain pericytes abca1 expression mediates cholesterol efflux but not cellular amyloid-β peptide accumulation. Journal of Alzheimer's Disease, 30(3), 489-503. Shawahna, R., Uchida, Y., Declèves, X., Ohtsuki, S., Yousif, S., Dauchy, S., Jacob, A., Chassoux, F., Daumas-Duport, C., Couraud, P. O., Terasaki, T. and Scherrmann, J. M. (2011) Transcriptomic and quantitative proteomic analysis of transporters and drug metabolizing enzymes in freshly isolated human brain microvessels. Molecular Pharmaceutics, 8(4), 1332-1341. Squires, E. J., Sueyoshi, T. and Negishi, M. (2004) Cytoplasmic localization of pregnane X receptor and ligand-dependent nuclear translocation in mouse liver. Journal of Biological Chemistry, 279(47), 49307-49314. Staudinger, J. L., Madan, A., Carol, K. M. and Parkinson, A. (2003) Regulation of drug transporter gene expression by nuclear receptors. Drug Metabolism and Disposition, 31(5), 523-527. Stirone, C., Duckles, S. P. and Krause, D. N. (2003) Multiple forms of estrogen receptor-α in cerebral blood vessels: Regulation by estrogen. American Journal of Physiology - Endocrinology and Metabolism, 284(1 47-1), E184-E192. Stromskaya, T. P., Rybalkina, E. Y., Shtil, A. A., Zabotina, T. N., Filippova, N. A. and Stavrovskaya, A. A. (1998) Influence of exogenous RAR alpha gene on MDR1 expression and P-glycoprotein function in human and rodent cell lines. British Journal of Cancer, 77(11), 1718-1725. Szatmari, I., Vámosi, G., Brazda, P., Balint, B. L., Benko, S., Széles, L., Jeney, V., Özvegy- Laczka, C., Szántó, A., Barta, E., Balla, J., Sarkadi, B. and Nagy, L. (2006) Peroxisome proliferator-activated receptor γ-regulated ABCG2 expression confers cytoprotection to human dendritic cells. Journal of Biological Chemistry, 281(33), 23812-23823.

351 Tachibana, S., Yoshinari, K., Chikada, T., Toriyabe, T., Nagata, K. and Yamazoe, Y. (2009) Involvement of vitamin D receptor in the intestinal induction of human ABCB1. Drug Metabolism and Disposition, 37(8), 1604-1610. Tanaka, Y., Slitt, A. L., Leazer, T. M., Maher, J. M. and Klaassen, C. D. (2004) Tissue distribution and hormonal regulation of the breast cancer resistance protein (Bcrp/Abcg2) in rats and mice. Biochemical and Biophysical Research Communications, 326(1), 181- 187. Teng, S., Jekerle, V. and Piquette-Miller, M. (2003) Induction of ABCC3 (MRP3) by pregnane X receptor activators. Drug Metabolism and Disposition, 31(11), 1296-1299. Terwel, D., Steffensen, K. R., Verghese, P. B., Kummer, M. P., Gustafsson, J. A., Holtzman, D. M. and Heneka, M. T. (2011) Critical role of astroglial apolipoprotein E and liver X receptor-alpha expression for microglial Abeta phagocytosis. Journal of Neuroscience, 31(19), 7049-7059. Tirona, R. G. and Kim, R. B. (2005) Nuclear receptors and drug disposition gene regulation. Journal of Pharmaceutical Sciences, 94(6), 1169-1186. Tirona, R. G., Lee, W., Leake, B. F., Lan, L. B., Brimer Cline, C., Lamba, V., Parviz, F., Duncan, S. A., Inoue, Y., Gonzalez, F. J., Schuetz, E. G. and Kim, R. B. (2003) The orphan nuclear receptor HNF4α determines PXR- and CAR-mediated xenobiotic induction of CYP3A4. Nature Medicine, 9(2), 220-224. Tolson, A. H. and Wang, H. (2010) Regulation of drug-metabolizing enzymes by xenobiotic receptors: PXR and CAR. Advanced Drug Delivery Reviews, 62(13), 1238-1249. Torra, I. P., Jamshidi, Y., Flavell, D. M., Fruchart, J. C. and Staels, B. (2002) Characterization of the human PPARα promoter: Identification of a functional nuclear receptor response element. Molecular Endocrinology, 16(5), 1013-1028. Urquhart, B. L., Tirona, R. G. and Kim, R. B. (2007) Nuclear receptors and the regulation of drug-metabolizing enzymes and drug transporters: Implications for interindividual variability in response to drugs. Journal of Clinical Pharmacology, 47(5), 566-578. Wang, H. and LeCluyse, E. L. (2003) Role of Orphan Nuclear Receptors in the Regulation of Drug-Metabolising Enzymes. Clinical Pharmacokinetics, 42(15), 1331-1357. Wang, H., Unadkat, J. D. and Mao, Q. (2008) Hormonal regulation of BCRP expression in human placental BeWo cells. Pharmaceutical Research, 25(2), 444-452. Wang, H., Zhou, L., Gupta, A., Vethanayagam, R. R., Zhang, Y., Unadkat, J. D. and Mao, Q. (2006) Regulation of BCRP/ABCG2 expression by progesterone and 17β-estradiol in human placental BeWo cells. American Journal of Physiology - Endocrinology and Metabolism, 290(5), E798-E807. Wang, X., Cabrera, R. M., Li, Y., Miller, D. S. and Finnell, R. H. (2013) Functional regulation of P-glycoprotein at the blood-brain barrier in proton-coupled folate transporter (PCFT) mutant mice. FASEB Journal, 27(3), 1167-1175. Wang, X., Hawkins, B. T. and Miller, D. S. (2011a) Activating PKC-Β1 at the blood-brain barrier reverses induction of P-glycoprotein activity by dioxin and restores drug delivery to the CNS. Journal of Cerebral Blood Flow and Metabolism, 31(6), 1371-1375. Wang, X., Hawkins, B. T. and Miller, D. S. (2011b) Aryl hydrocarbon receptor-mediated upregulation of ATP-driven xenobiotic efflux transporters at the blood-brain barrier. FASEB Journal, 25(2), 644-652.

352 Wang, X., Sykes, D. B. and Miller, D. S. (2010) Constitutive androstane receptor-mediated upregulation of ATP-driven xenobiotic efflux transporters at the blood-brain barrier. molecular Pharmacology, 78(3), 376-383. Wang, Y. X. (2010) PPARs: Diverse regulators in energy metabolism and metabolic diseases. Cell Research, 20(2), 124-137. Ward, R. D. and Weigel, N. L. (2009) Steroid receptor phosphorylation: Assigning function to site-specific phosphorylation. BioFactors, 35(6), 528-536. Wen, G., Ringseis, R. and Eder, K. (2010) Mouse OCTN2 is directly regulated by peroxisome proliferator-activated receptor α (PPARα) via a PPRE located in the first intron. Biochemical Pharmacology, 79(5), 768-776. Westin, S., Kurokawa, R., Nolte, R. T., Wisely, G. B., McInerney, E. M., Rose, D. W., Milburn, M. V., Rosenfeld, M. G. and Glass, C. K. (1998) Interactions controlling the assembly of nuclear-receptor heterodimers and co-activators. Nature, 395(6698), 199-202. Whitfield, G. K., Jurutka, P. W., Haussler, C. A. and Haussler, M. R. (1999) Steroid hormone receptors: Evolution, ligands, and molecular basis of biologic function. Journal of Cellular Biochemistry, 76(SUPPL. 32/33), 110-122. Xiao, L., Xie, X. and Zhai, Y. (2010) Functional crosstalk of CAR-LXR and ROR-LXR in drug metabolism and lipid metabolism. Advanced Drug Delivery Reviews, 62(13), 1316-1321. Xiong, H., Yoshinari, K., Brouwer, K. L. R. and Negishi, M. (2002) Role of constitutive androstane receptor in the in vivo induction of Mrp3 and CYP2B1/2 by phenobarbital. Drug Metabolism and Disposition, 30(8), 918-923. Yasuda, S., Kobayashi, M., Itagaki, S., Hirano, T. and Iseki, K. (2009) Response of the ABCG2 promoter in T47D cells and BeWo cells to sex hormone treatment. Molecular Biology Reports, 36(7), 1889-1896. Yu, J., Lo, J. L., Huang, L., Zhao, A., Metzger, E., Adams, A., Meinke, P. T., Wright, S. D. and Cui, J. (2002) Lithocholic acid decreases expression of bile salt export pump through farnesoid X receptor antagonist activity. Journal of Biological Chemistry, 277(35), 31441-31447. Zhang, Y., Lu, M., Sun, X., Li, C., Kuang, X. and Ruan, X. (2012) Expression and activity of p- glycoprotein elevated by dexamethasone in cultured retinal pigment epithelium involve glucocorticoid receptor and pregnane X receptor. investigative Ophthalmology and Visual Science, 53(7), 3508-3515. Zhang, Y., Zhou, G., Wang, H., Zhang, X., Wei, F., Cai, Y. and Yin, D. (2007) Transcriptional upregulation of breast cancer resistance protein by 17β-estradiol in ERα-positive MCF-7 breast cancer cells. Oncology, 71(5-6), 446-455.

353