# LB 49

ABC transporters in the blood-brain barrier limit the brain penetration of the PARP inhibitor ABT-888
Lin Fan, Mark C. de Gooijer, Jan H. Beumer, Susan M. Christner, Jos H. Beijnen and Olaf van Tellingen.
Netherlands Cancer Institute, Amsterdam, The Netherlands/ University of Pittsburgh Cancer Institute, Pittsburgh, USA .

BACKGROUND OBJECTIVES
•ABT-888 is a potent poly(ADP-ribose) polymerase (PARP) inhibitor and The aim of this study was to establish the impact of P-glycoprotein (ABCB1/Abcb1a/b) and Bcrp1 (ABCG2/Abcg2) in
is currently in phase 2 clinical trials. PARP inhibitors enhance the the blood-brain barrier on brain penetration and the intracranial antitumor efficacy of ABT-888.
activity of DNA damaging therapies, due to the critical function of PARP-
1 and PARP-2 in base excision repair. The combination of PARP
inhibitors with chemo-radiation therapies for the treatment of glioma
patients is receiving considerable interest because this combination has Plasma concentration of ABT888
RESULTS
shown promise in several preclinical models. 500

400
In vitro studies
•High-grade glioma patients have a very poor prognosis, in particular
300 • LLC-PK1 cells translocate ABT-888 into the apical direction.
due to the inability to achieve complete surgical resection. Importantly,

ng/ml
200
This is probably due to endogenous (porcine) P-gp and can
the residual tumor cells that have infiltrated surrounding normal brain be inhibited by 5µM of the P-gp inhibitor zosuquidar.
100
tissue are using pre-existing brain vasculature and are thus protected by
0 •LLC-Mdr1a cells translocate ABT-888 even better and this
the blood-brain barrier (BBB). The ABC transporters ABCB1/Abcb1a/b; FVB
Wildtype Mdr1
Abcb1a/b Bcrp1
Abcg2 Bcrp1;Mdr1
Abcg2;Abcb1
transport can also be inhibited by 5 µM of zosuquidar (=P-gp
MDR1/mdr1a/b P-glycoprotein; P-gp) and ABCG2/Abcg2 (BCRP/Bcrp1; inhibitor).
Breast cancer resistance protein) are important factors limiting the brain Brain concentration of ABT888
•MDCK-Bcrp1 cells also efficiently translocate ABT-888 to the
entry of many agents. Consequently, the ability of potential
2500

apical direction. This transport can be inhibited by 5 µM of

chemotherapeutics to cross the BBB, including the role of ABC-
2000

elacridar (=P-gp and BCRP inhibitor).

transporters in the brain penetration, needs to be established.
1500

•There was no background translocation in the MDCK-parent

ng/g
1000

cell line
500

METHODS 0
Wildtype
FVB Abcb1a/b
Mdr1 Abcg2
Bcrp1 Abcg2;Abcb1
Bcrp1;Mdr1
In vivo studies
Drug solutions: ABT-888 was obtained from Selleck Chemicals.
•The plasma concentration of ABT-888 was not different
Brain-plasma ratio of ABT888
between the 4 genotypes, indicating that the clearance is
Transwell assays: In vitro equilibrium transwell studies were performed 10

with LLC-PK1 (parent) and LLC-Mdr1a cells and MDCK-parent vs likely not affected by the absence of Abcb1a/b and Abcg2.
8

MDCK-Bcrp1. ABT-888 0.5 µM in Optimem was added to both the •The brain-to-plasma ratio was significantly (p=0.006) higher
6

apical and basolateral sides and samples were taken at 0.5, 1, 2 and 4 in Abcb1a/b knockout mice versus wildtype controls.
h. Samples in Optimem were diluted 5-fold in 0.1% formic acid and 4

•The brain concentration in Abcg2 knockout mice was not

injected. ABT-888 was analyzed by HPLC on a Zorbax SB C18 column 2

higher, probably because Abcb1a/b alone is already sufficient.

(75 x 4.6 mm) and a linear gradient from 2.5% to 30% (v/v) acetonitrile 0

in 0.1% formic acid in water (8:92, v/v) and UV detection at 295 nm. Wildtype
FVB Abcb1a/b
Mdr1 Abcg2
Bcrp1 Abcg2;Abcb1
Bcrp1;Mdr1
•The brain concentration in Abcg2;Abcb1a/b mice, however,
was significantly higher (p=0.022) than in Abcb1a/b knockouts
Pharmacokinetic studies: The i.v. studies were performed in FVB wild- demonstrating that Abcg2 is also a limiting factor
type, Mdr1a/b, Bcrp1 and Bcrp1;Mdr1a/b knockout mice. Animals
received ABT-888 by i.v. bolus injection in the tail vein. Blood was taken CONCLUSION
by cardiac puncture at 1 h after drug administration. Plasma separated
by centrifugation. Brains were dissected on ice and homogenized in 4% The brain penetration of ABT-888 is limited primarily by P-glycoprotein (ABCB1) but also by BCRP (ABCG2). The
(w/v) BSA in water. Samples were stored at -20ºC until analysis.
Brain-to-plasma ratio was 7.5-fold higher when both ABC transporters were absent. Further in vivo experiments to
Analysis of ABT-888 in biological specimens was performed using a
validated LC-MS method (Parise et al., J. Chromatogr. B 2008; establish the impact of these transporters on the efficacy of ABT-888 are warranted.
872:141-147

Statistical analyses: ANOVA with Bonferroni post hoc test for multiple
comparison was used.