Blood-Brain Barrier Transporters in Ischemic Stroke:Focus on Organic Anion Transporting Polypeptides (Oatps)
Patrick T. Ronaldson, Ph.D.Associate Professor
Department of PharmacologyUniversity of Arizona College of Medicine
Invited Presentation:
Solvo Biotechnology Meet the Experts Transporter Conference 2019
September 4, 2019
Disclosures
• Research Funding– NINDS/NIH R01-NS084941 (PT Ronaldson, PI)
– American Heart Association 19TPA34910113 (PT Ronaldson, PI)
– Arizona Biomedical Research Commission ADHS16-162406 (PT Ronaldson, PI)
The BBB – A Vast Microvascular Network
A. Zlokovic and Apuzzo, 1998; B. Rodriguez-Baeza et al., 2003; C. Hartz et al., 2006
400 miles of
capillaries
in the human brain
One capillary per
neuron and
average distance
~8-20 µm
> 100 billion
capillaries in the
human brain
comprising ~215
ft2 surface area
Challenge: Neuroprotective Drugs for Stroke - Many Failures
• Preclinical success in neuroprotective drug development has not resulted in translation of new therapeutics to the clinic.– As noted by Jun Chen’s group (University of Pittsburgh), 95% of published
neuroprotective studies between 1990 and 2018 describing positive results in animal models - none have progressed to phase III trial success. (Shi et al., 2018. J Cerebral Blood Flow Metabolism 38,12. 2073-2091).
• Failures may be attributed, in part, to the fact that most preclinical stroke studies do not evaluate biological mechanisms that can deliver these drugs successfully to ischemic brain tissue.– Targeting uptake transporters such as Organic Anion Transporting Polypeptides
(Oatps) may address this problem !
Why study Oatps in Stroke?• Statins have been shown to improve functional outcomes in stroke patients.
– Amarenco et al. 2006. New Engl J Med. 355: 549-559.
– Castilla-Guerra et al. 2006. Stroke. 37: 1153.
– Montaner et al. 2008. Eur J Neurol. 15: 82-90.
– Salat et al. 2009. Expert Rev Cardiovasc Ther. 7: 1219-1230.
– Huisa et al. 2010. Vasc Health Risk Manag. 6: 229-236.
– Montecucco et al. 2012. Curr Pharm Biotechnol. 13: 68-76.
– Montaner et al. 2016. Stroke. 47: 2870-2873.
– Lee et al. 2017. J Am Heart Assoc. 6.
– Zhang et al. 2017. Int J Neurosci. 127: 92-97.
• Statins are transport substrates for Oatps– Work from our group has shown, for the first time, that Oatp1a4 enables these drugs to
permeate the BBB and accumulate in the CNS (Thompson et al. 2014. J Cereb Blood Flow Metab. 34: 699-707; Abdullahi et al. 2018. Molecular Pharmacology. 94: 1321-1333).
These papers
provide CLINICAL
evidence that statins
are EFFECTIVE in
providing
neuroprotection to
stroke patients.
Modified from: Ronaldson & Davis. 2015. Brain Res. 1623: 39-52.
1 2 1 2
0
10
20
30
40
50
Dru
g U
pta
ke
(pm
ol /
mg
bra
in t
issu
e)
ATV
PRV**
**
** p < 0.01
CTL E3S
(100 uM)
CTL E3S
(100 uM)
Oatp-Mediated Brain
Uptake of Statins
Adapted from: Abdullahi et al. 2018. Mol
Pharmacol. 94: 1321-1333. E3S=OATP
inhibitor.
Targeting Oatp1a4 for CNS Drug Delivery
Endothelial Cell
Oatp1a4
Drug Delivery byTransporter (Oatp1a4) Targeting
Control
TGF-β Signaling
Increased Drug Transport/Uptake into the Brain
Oatp1a4
Blood
Brain
ALK5 ALK1
tMCAO Model of
Focal Cerebral Ischemia
Hypothesis: Oatp1a4 expression and activity at the BBB is
an absolute requirement for statins to exert neuroprotective
effects in the brain following ischemic stroke.
Oatp1a4 Localization: Brain Microvascular Endothelium
Tomato Lectin Oatp1a4 Merge
Control
Microvessel
tMCAO
(Ipsilateral CTX)
tMCAO
(Contralateral CTX)
Dr. Jeff Lochhead
tMCAO:
90 min MCAO
22.5 hrs reperfusion
Oatp1a4 Localization: Pericytes
Control
Microvessel
tMCAO
(Ipsilateral CTX)
tMCAO
(Contralateral CTX)
tMCAO:
90 min MCAO
22.5 hrs reperfusion
PDGFR-β Oatp1a4 Merge
Statins require Functional Expression of Oatp1a4 at the
BBB to exert Neuroprotective Effects in Stroke
n = 8 animals per group
Reperfusion (22.5 h)MCAO (1.5 h)
ATV (20 mg/kg, i.v.) given 2 h following reperfusion(+/- 3.2 mg/kg FEX, i.v.)
A
B C
D
MCAO/RMCAO/R
+ ATV
MCAO/R+ ATV/FEX
Male Sprague-Dawley Rats Female Sprague-Dawley Rats
MCAO/RMCAO/R
+ ATV
MCAO/R+ ATV/FEX
MCAO/R MCAO/R+ ATV
MCAO/R+ ATV/FEX
0
10
20
30
40
50
60
Infa
rct
Vo
lum
e
(% H
em
isp
here
) Males
Females
** ff
**
ff
** p < 0.01
(vs. Male MCAO/R)
ff p < 0.01
(vs. Female MCAO/R)
**
MCAO/R MCAO/R+ ATV
MCAO/R+ ATV/FEX
0.00
0.05
0.10
0.15
0.20
0.25
0.30
Ed
em
a R
ati
o
Males
Females
**
ff
** p < 0.01
(vs. Male MCAO/R)
ff p < 0.01
(vs. Female MCAO/R)
**
From: Lochhead et al. 2019. Stroke. Submitted
Statins require Functional Expression of Oatp1a4 at the
BBB to exert Neuroprotective Effects in Stroke
Functional Neuroscore
(4 point scale)
Sensorimotor Performance
(Grip Tape Test)
n = 16 animals per group (males); n = 8 animals per group (females)
A
B
Sham MCAO/R MCAO/R+ ATV
MCAO/R+ ATV/FEX
0
1
2
3
Neu
rolo
gica
l Def
icit
Sco
re
**
**
**
** p < 0.01
Sham MCAO/R MCAO/R+ ATV
MCAO/R+ ATV/FEX
0
20
40
60
80
100
120
140
Tim
e (s
econ
ds)
**
****
** p < 0.01
Sham MCAO/R MCAO/R+ ATV
MCAO/R+ ATV/FEX
0
1
2
3
Neu
rolo
gica
l Def
icit
Sco
re
**
**
**** p < 0.01
C
D
Sham MCAO/R MCAO/R+ ATV
MCAO/R+ ATV/FEX
0
20
40
60
80
100
120
140
Tim
e (s
econ
ds)
**
**
**** p < 0.01
Males
Females
TGF-β Signaling Pathway
Adapted from: Abdullahi et al. 2017. AAPS J. 19: 931-937
(+)Endoglin
(CD105)Betaglycan
(+)
TGF-βBMP-9
Regulation of Oatp1a4 Expression by Transforming Growth Factor-β Signaling
From: Abdullahi et al. 2017. J Cereb Blood Flow Metab. 37: 2340-2345.
Abdullahi et al. 2018. Mol Pharmacol. 94: 1321-1333.
Abdullahi et al. 2019. Drug Metab Dispos. Submitted.
0 5 10 15 200
20
40
60
80
100
120
140
Time (minutes)
Ato
rvasta
tin
Up
take
(pm
ol / m
g b
rain
tis
su
e)
Control
BMP-9 (1 ug/kg)
LDN (10 mg/kg) + BMP-9 (1 ug/kg)
**
*** p < 0.05
** p < 0.01
*
**
A
Contr
ol
BMP-
9 (1
ug/k
g)
LDN (1
0 m
g/kg)
+ BMP-
9 (1
ug/k
g)
0
500
1000
1500
2000
AU
C (
pm
ol *
min
/mg
bra
in t
issu
e)
Control
BMP-9 (1 ug/kg)
LDN (10 mg/kg)
+ BMP-9 (1 ug/kg)
** **** p < 0.01
B
Dr. Wazir Abdullahi
0 5 10 15 200
20
40
60
80
100
120
140
Time (minutes)
Ato
rvasta
tin
Up
take
(pm
ol /
mg
bra
in t
issu
e)
Control
BMP-9 (1 ug/kg)
LDN (10 mg/kg) + BMP-9 (1 ug/kg)
**
*** p < 0.05
** p < 0.01
*
**
A
Contr
ol
BMP-9
(1 u
g/kg)
LDN (1
0 mg/k
g)
+ BMP-9
(1 u
g/kg)
0
500
1000
1500
2000
AU
C (
pm
ol *
min
/mg
bra
in t
issu
e)
Control
BMP-9 (1 ug/kg)
LDN (10 mg/kg)
+ BMP-9 (1 ug/kg)
** **** p < 0.01
B
Regulation of Oatp1a4 Expression by Transforming Growth Factor-β Signaling
ChIP Analysis
Smad Binding
Element:
GC-rich
sequences
flanking CAGA
boxes
From: Abdullahi et al. 2018. Mol Pharmacol. 94: 1321-1333.
Functional Expression of OATP1A2 in HUVECs
“Emphasizes Translational Potential of our Oatp1a4 studies”
(HUVEC cells kindly provided by Dr. Gregory Bix at the University of Kentucky)
ATV ATV + E3S
0
100
200
300
400
500
Ato
rvasta
tin
Up
take
(pm
ol/m
g p
rote
in)
Control
BMP-9 (0.01 uM)
SB431542 (10 uM)
****
**
****
** p < 0.01
OATP1A2
β-actin
HeLa
HUVEC
+0.01uMBMP-9
HUVEC
+10uMSB431542HUVEC
80kDa
43kDa
A
B
ATV ATV + E3S
0
100
200
300
400
500
Ato
rvasta
tin
Up
take
(pm
ol/m
g p
rote
in)
Control
BMP-9 (0.01 uM)
SB431542 (10 uM)
****
**
****
** p < 0.01
OATP1A2
β-actin
HeLa
HUVEC
+0.01uMBMP-9
HUVEC
+10uMSB431542HUVEC
80kDa
43kDa
A
BHe
La
HUVE
C
HUVE
C
+ 0.
01 u
M BMP
-9
HUVE
C
+ 10
uM
SB43
1542
0.0
0.5
1.0
1.5
2.0
Rela
tive O
AT
P1A
2 E
xp
ressio
n
** **
** p < 0.01
ATV ATV + E3S
0
100
200
300
400
500
Ato
rvasta
tin
Up
take
(pm
ol/m
g p
rote
in)
Control
BMP-9 (0.01 uM)**
**
**
** p < 0.01
B
From: Ronaldson et al. 2019. J Pharm Sci. Submitted
Conclusions • Our data shows, for the first time, that an uptake transporter (i.e.,
Oatp1a4) at the BBB is a CRITICAL DETERMINANT of atorvastatin neuroprotection in ischemic stroke.– Data are clinically relevant because they demonstrate that an endogenous
BBB transporter is required for statins to be effective in stroke treatment.
• Results from this study emphasize the need to consider transport mechanisms in the development of neuroprotective treatment strategies for stroke.
• We have also identified a molecular pathway (i.e., TGF-β/ALK1 signaling) that can be targeted to control Oatp-mediated delivery of statins to the brain– Opportunity to improve neuroprotective therapy with statins for stroke.
Summary
Endothelial Cell
Oatp1a4
Drug Delivery byTransporter (Oatp1a4) Targeting
Control
TGF-β Signaling
Increased Drug Transport/Uptake into the Brain
Oatp1a4
Blood
Brain
ALK5 ALK1
Statin Delivery to Ischemic
Brain by Targeting Oatp1a4
Accumulation of Statins in
Ischemic Brain Tissue
Reduced Infarction Volume/Edema &
Improved Neurocognitive Performance
ISCHEMIC
STROKE
AcknowledgementsArizona Blood-Brain Barrier Research Group
- Dr. Thomas P. Davis, Ph.D. (Professor & Collaborator)
- Dr. Tally Largent-Milnes, Ph.D. (Assistant Professor)
- Dr. Jeffrey Lochhead, Ph.D. (Res. Asst. Professor)
- Dr. Hrvoje Brzica, Ph.D. (Postdoctoral Fellow)
- Dr. Wazir Abdullahi, Ph.D. (Ph.D. Student)
- Qianying He, B.S. (Ph.D. Student)
- Erica Williams, B.S. (Ph.D. Student)
- Junzhi Yang, B.S. (Ph.D. Student)
- Tianhong Fu, B.S. (M.S. Student, Perfusion Sciences)
- Nicholas Hirsch, B.S. (M.S. Student, Perfusion Sciences)
- Raul Nava, B.S. (M.S. Student)
- Ayman Sami, B.S. (M.S. Student)
- Robert Betterton (Undergrad Res. Associate)
- Bianca Reilly (Undergrad Res. Associate)
- Samantha Serna (Undergrad Res. Associate)
- Joshua Stanton (Undergrad Res. Associate)
Zlokovic: Neurosurgery, Volume 43(4).October 1998.877-878
Questions?