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 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)

Electron microscopy of a blood microvasculature from rat brain tissue (Bendayan et al. 2002)

(Cardoso et al. 2010)

Brain Permeability of Antiretroviral Drugs (ARVs)

CSF Concentration

(ng/mL)

Effective Drug

Concentration to

inhibit 50% of wild-

type HIV-1 replication

(IC50) (ng/mL)

Protease Inhibitors (PIs) Atazanavir ND - 40 3.7 – 7.5

Darunavir 15.9 – 212.0 2.5 – 5.5

Nucleoside/ nucleotide

Reverse-Transcriptase inhibitors

(NRTIs)

Emtricitabine 3.0 – 9.8 0.1 – 0.2

Tenofovir 2.0 – 8 0.1 – 30

Non-nucleotide Reverse-

Transcriptase inhibitors

(NNRTIs)

Efavirenz 0.019 - 0.29 8.0 – 52

Nevirapine 4.9 - 40.9 23 – 140

Integrase Inhibitor Raltegravir 2.0 - 126 4.1 – 15

Entry Inhibitor Maraviroc 1.83 – 12.2 50 – 640

Adapted from McGee et al. 2006 & Ene et al. 2011 & www.hivclinic.ca assessed on 2012 Oct

http://www.hivclinic.ca/

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

Impaired memory

Difficulty to focus

Decrease psychomotor speed

Motor

Impaired coordination

Impaired motor disturbances

Ocular dysmetria

Behavioral

Depression

Seizures

Apathy

Vivithanaporn et al. 2010, Persidsky & Poluektova 2006

ATPBindingCassette Membrane-associated Drug Transporter P-glycoprotein (P-gp) at the BBB

Systemic Circulation Brain

Parenchyma

Tight Junctions and Adhesion Molecules

Endothelial Cells of Brain Microvasculature

Brain Microvessel Endothelial Cells

P-gp

P-gp

P-gp

P-gp P-gp substrates Most HIV

protease inhibitors

Abacavir Zidovudine Nelfinavir Amprenavir Maraviroc Raltegravir (Kis et al. 2010)

Regulation of P-gp by Orphan Nuclear Receptors

Adapted from Stanley et al. 2006

Drugs

P-gp Pregnane X Receptor (PXR) Constitutive Androstane Receptor (CAR)

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

Hypotheses

I. Several ARVs currently used in the clinic can activate xenobiotic receptors such as human PXR (hPXR) and CAR (hCAR)

II. 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

III. P-gp induction at the BBB can further restrict brain permeability of P-gp substrates

1

In Vitro Luciferase Reporter

Gene Assay

PXR &

CAR

Screen for ligands of hPXR and hCAR

Add ARVs

Luciferase Activity

In Vitro Luciferase Reporter Gene Assay Activation of hPXR & hCAR

In Vitro Luciferase Reporter Gene Assay Activation of hPXR & hCAR

Antiretroviral Drugs Human

PXR

Human

CAR

Protease Inhibitors (PIs)

Atazanavir

Amprenavir

Darunavir

Lopinavir

Ritonavir

Nucleoside/nucleotide

Reverse-Transcriptase

inhibitors (NRTIs)

Abacavir

Emtricitabine

Lamivudine

Tenofovir

Zidovudine

Non-nucleotide Reverse-

Transcriptase inhibitors

(NNRTIs)

Efavirenz

Nevirapine

Integrase Inhibitor Raltegravir

Entry Inhibitor Maraviroc

1

In Vitro Luciferase Reporter

Gene Assay

2

In Vitro Human Brain Microvessel

Endothelial Cells

PXR &

CAR

Screen for ligands of hPXR and hCAR

Induction of P-gp by ARVs at human

BBB

In Vitro Model of the Human BBB: Immortalized Human Brain Microvessel Endothelial Cell Culture System (hCMEC/D3)

Major Drug Efflux Transporters

(Weksler et al. 2005) Nuclear Receptors

(Chan et al. 2011) (Zastre et al. 2009)

P-gp

Tight Junction proteins & Brain Microvessel Endothelial Cell Markers

HCMEC/D3 Under Light Contrast Microscopy

0.0

50.0

100.0

150.0

200.0

250.0

300.0

350.0P

-gp

Pro

tein

Exp

ress

ion

(%

Co

ntr

ol)

In Vitro Induction of P-gp by ARVs in hCMEC/D3

N = 3, * P < 0.05

*

*

* * *

*

* *

* *

*

PXR Ligands CAR Ligands PXR + CAR

Po

siti

ve C

on

tro

l

Po

siti

ve C

on

tro

l

Po

siti

ve C

on

tro

l

In Vitro Induction of P-gp Function by ARVs in hCMEC/D3

0

20

40

60

80

100

120

P-g

p S

ub

stra

te (

R6

G)

Acc

um

ula

tio

n (

%

Co

ntr

ol)

N = 3, * P < 0.05

* *

*

* * * * * *

* *

1

In Vitro Luciferase Reporter

Gene Assay

2

In Vitro Human Brain Microvessel

Endothelial Cells

PXR &

CAR

Screen for ligands of hPXR and hCAR

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

In Vivo: Mouse Brain Microdialysis

Brain ECF Brain

Parenchyma

Direct and continuous sampling of brain ECF of quinidine (P-gp substrate) following P-gp Induction at the mouse BBB

(Muller M. 2002)

C Brain ECF > C Dialysate

Diffusion

N = 5 per group, P

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 up-regulate 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

Significance

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

P-gp

P-gp

P-gp

P-gp

Human BBB

Human PXR &

CAR

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.

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

EXTRA INFORMATION

Results: Luciferase Reporter Gene Assay Activation of Human PXR

Positive

Controls

N = 3, 10 µM

Results: Luciferase Reporter Gene Assay Activation of Human CAR

Positive

Control

N = 3, 10 µM

PROTEASE INHIBITORS STRUCTURE

NNRTI

Abacavir

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