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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 Endothelial Cells
Gary N.Y. Chan, Ph. D. Candidate
Supervisor: Dr. Reina Bendayan
Department of Pharmaceutical Sciences
Leslie Dan Faculty of Pharmacy
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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)
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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/
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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
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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)
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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)
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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
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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
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1
In Vitro Luciferase Reporter
Gene Assay
PXR &
CAR
Screen for ligands of hPXR and hCAR
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Add ARVs
Luciferase Activity
In Vitro Luciferase Reporter Gene Assay Activation of hPXR & hCAR
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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
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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
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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
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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
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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
* *
*
* * * * * *
* *
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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
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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
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N = 5 per group, P
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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
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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
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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.
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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
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EXTRA INFORMATION
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Results: Luciferase Reporter Gene Assay Activation of Human PXR
Positive
Controls
N = 3, 10 µM
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Results: Luciferase Reporter Gene Assay Activation of Human CAR
Positive
Control
N = 3, 10 µM
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PROTEASE INHIBITORS STRUCTURE
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NNRTI
Abacavir
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ANTIRETROVIRAL TREATMENT OF ADULT HIV INFECTION: 2012 RECOMMENDATIONS OF THE INTERNATIONAL ANTIVIRAL SOCIETY-USA PANEL. (JAMA 2012)