Blood-Brain Barrier Transporters in Ischemic Stroke: Focus on … · (HUVEC cells kindly provided by Dr. Gregory Bix at the University of Kentucky) AT V ATV + E3 S 0 100 200 300 400

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?