Nuclear Receptors Mediated Induction of P-glycoprotein by Antiretroviral Drugs in Human Brain Microvessel Endothelial Cells

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Under The Microscope: The Impact of ARTs Nuclear Receptors Mediated Induction of P-glycoprotein by Antiretroviral Drugs in Human Brain Microvessel

1 Under The Microscope: The Impact of ARTs Nuclear Receptors Mediated Induction of P-glycoprotein by Antiretroviral Drugs in Human Brain Microvessel Endothelial Cells Gary N.Y. Chan, Ph. D. Candidate Supervisor: Dr. Reina Bendayan Department of Pharmaceutical Sciences Leslie Dan Faculty of Pharmacy

Blood-brain Barrier (BBB): Neurovascular Unit At the site of brain

the blood and brain Essential for the health and function of the

Laboratory, Texas A&M University) (Cardoso et al.

2 Blood-brain Barrier (BBB): Neurovascular Unit At the site of brain microvasculature Dynamic physical and biochemical barrier between the blood and brain Essential for the health and function of the CNS Whole brain vasculature visualization (Brain Network Laboratory, Texas A&M University) (Cardoso et al. 2010) Electron microscopy of a blood microvasculature from rat brain tissue (Bendayan et al. 2002)

3 Brain Permeability of Antiretroviral Drugs (ARVs) Protease Inhibitors (PIs) Nucleoside/ nucleotide Reverse-Transcriptase inhibitors (NRTIs) Non-nucleotide Reverse- Transcriptase inhibitors (NNRTIs) CSF Concentration (ng/ml) Effective Drug Concentration to inhibit 50% of wildtype HIV-1 replication (IC50) (ng/ml) Atazanavir ND Darunavir Emtricitabine Tenofovir Efavirenz Nevirapine Integrase Inhibitor Raltegravir Entry Inhibitor Maraviroc Adapted from McGee et al & Ene et al & assessed on 2012 Oct

4 Consequences from Limited Brain Permeability of Antiretroviral Drugs I. Increase CNS viral load II. Development of viral sanctuary in the brain III. HIV-associated Neurocognitive Disorders (HAND) a. major cause of morbidity and mortality b. Additional disease burden on people living with HIV Cognitive Motor Behavioral Impaired memory Difficulty to focus Decrease psychomotor speed Impaired coordination Impaired motor disturbances Ocular dysmetria Depression Seizures Apathy Vivithanaporn et al. 2010, Persidsky & Poluektova 2006

Most HIV protease inhibitors Abacavir Zidovudine Nelfinavir Amprenavir Maraviroc Raltegravir (Kis

5 A TP B inding C assette Membrane-associated Drug Transporter P-glycoprotein (P-gp) at the BBB Systemic Circulation Endothelial Cells of Brain Microvasculature Brain Parenchyma P-gp substrates Most HIV protease inhibitors Abacavir Zidovudine Nelfinavir Amprenavir Maraviroc Raltegravir (Kis et al. 2010) P-gp P-gp P-gp P-gp Tight Junctions and Adhesion Molecules Brain Microvessel Endothelial Cells

6 Regulation of P-gp by Orphan Nuclear Receptors P-gp Drugs Pregnane X Receptor (PXR) Constitutive Androstane Receptor (CAR) Adapted from Stanley et al. 2006

Relevance of human PXR and CAR During cart Ritonavir and efavirenz can activate hpxr Chronic exposure to antiretroviral drugs can lead to the induction of CYP3A4 and P-gp expression in peripheral

7 Relevance of human PXR and CAR During cart Ritonavir and efavirenz can activate hpxr Chronic exposure to antiretroviral drugs can lead to the induction of CYP3A4 and P-gp expression in peripheral tissues Ritonavir: booster agent for other HIV PIs because of its inhibitory role on CYP3A4 in the liver CYP3A4 activities in the brain is low, drug transport processes becomes a major factor P-gp induction at the human BBB during chronic exposure to antiretroviral drugs

8 Hypotheses I. Several ARVs currently used in the clinic can activate xenobiotic receptors such as human PXR (hpxr) and CAR (hcar) II. III. ARVs identified as ligands of hpxr and/or hcar can induce the functional expression of membrane-associated drug efflux transporters, such as P-glycoprotein (P-gp), at the human BBB P-gp induction at the BBB can further restrict brain permeability of P-gp substrates

9 In Vitro Luciferase Reporter Gene Assay PXR & CAR Screen for ligands of hpxr and hcar 1

10 In Vitro Luciferase Reporter Gene Assay Activation of hpxr & hcar Luciferase Activity Add ARVs

11 In Vitro Luciferase Reporter Gene Assay Activation of hpxr & hcar Protease Inhibitors (PIs) Nucleoside/nucleotide Reverse-Transcriptase inhibitors (NRTIs) Non-nucleotide Reverse- Transcriptase inhibitors (NNRTIs) Integrase Inhibitor Entry Inhibitor Antiretroviral Drugs Atazanavir Amprenavir Darunavir Lopinavir Ritonavir Abacavir Emtricitabine Lamivudine Tenofovir Zidovudine Efavirenz Nevirapine Raltegravir Maraviroc Human PXR Human CAR

12 In Vitro Luciferase Reporter Gene Assay Screen for ligands of hpxr and hcar 1 PXR & CAR In Vitro Human Brain Microvessel Endothelial Cells 2 Induction of P-gp by ARVs at human BBB

13 In Vitro Model of the Human BBB: Immortalized Human Brain Microvessel Endothelial Cell Culture System (hcmec/d3) Tight Junction proteins & Brain Microvessel Endothelial Cell Markers HCMEC/D3 Under Light Contrast Microscopy (Weksler et al. 2005) Nuclear Receptors Major Drug Efflux Transporters P-gp (Zastre et al. 2009) (Chan et al. 2011)

14 P-gp Protein Expression (% Control) Positive Control Positive Control Positive Control In Vitro Induction of P-gp by ARVs in hcmec/d3 PXR Ligands CAR Ligands PXR + CAR * * * * * * * * * * * N = 3, * P < 0.05

15 P-gp Substrate (R6G) Accumulation (% Control) In Vitro Induction of P-gp Function by ARVs in hcmec/d * * * * * * * * * * * N = 3, * P < 0.05

16 In Vitro Luciferase Reporter Gene Assay Screen for ligands of hpxr and hcar 1 PXR & CAR In Vitro Human Brain Microvessel Endothelial Cells 2 Induction of P-gp by ARVs at human BBB 3 In Vivo Mouse Brain Microdialysis P-gp induction can further restrict brain entry of P-gp substrate

17 In Vivo: Mouse Brain Microdialysis Direct and continuous sampling of brain ECF of quinidine (Pgp substrate) following P-gp Induction at the mouse BBB C Brain ECF > C Dialysate Diffusion Brain Parenchyma Brain ECF (Muller M. 2002)

18 Decrease Brain ECF/Plasma Ratio of Quinidine (P-gp Substrate) Following P-gp Induction at the Mouse BBB (DEX = dexamethasone) P-gp Expression in Mouse Brain Capillary Control P-gp Induced (Vehicle) (DEX) N = 5 per group, P <0.05

19 Summary In vitro - Luciferase Reporter Gene Assays hpxr: lopinavir, amprenavir, darunavir, atazanavir, ritonavir & efavirenz hcar : efavirenz, abacavir & nevirapine In vitro - Human Brain Microvessel Endothelial Cells (hcmec/d3) ARVs at relevant clinical plasma concentrations can upregulate P-gp protein expression and function In vivo - Brain Microdialysis in Mouse Induction of P-gp at the BBB can reduce brain entry of P-gp substrates, i.e., quinidine

20 Significance P-gp P-gp P-gp P-gp Human PXR & CAR Human BBB Systemic exposure to ARVs could potentially lead to an increase in P-gp expression at the human BBB possibly through the activity of PXR and CAR. P-gp induction by these agents could further contribute to limit drug entry into the brain

21 Can be useful for the design of novel HIV pharmacotherapy with limited P-gp induction and ultimately improve permeability of antiretroviral drugs into the brain.

22 Dr. Reina Bendayan (Career Scientist, Ontario HIV Treatment Network, MHO) Members of the Bendayan Lab Leslie Dan Faculty of Pharmacy, U of T Dr. Carolyn L. Cummins Rucha Patel NoAb Biodiscoveries Inc. Dr. Ines de Lannoy Victor Saldivia Dr. Yingbo Yang Dr. Henrianna Pang Acknowledgement

23 EXTRA INFORMATION

25 Results: Luciferase Reporter Gene Assay Activation of Human PXR Positive Controls N = 3, 10 µm

26 Results: Luciferase Reporter Gene Assay Activation of Human CAR Positive Control N = 3, 10 µm

28 PROTEASE INHIBITORS STRUCTURE

29 NNRTI Abacavir

30 ANTIRETROVIRAL TREATMENT OF ADULT HIV INFECTION: 2012 RECOMMENDATIONS OF THE INTERNATIONAL ANTIVIRAL SOCIETY- USA PANEL. (JAMA 2012)

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

Drug Transport in the Brain: Models of BBB and Brain Parenchyma Reina Bendayan, Pharm.D. Professor and Career Scientist, MHO Department of Pharmaceutical Sciences Leslie Dan Faculty of Pharmacy University