ABC transporters in the blood-brain barrier limit the brain penetration of the PARP inhibitor ABT-888Lin 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•ABT-888 is a potent poly(ADP-ribose) polymerase (PARP) inhibitor and is currently in phase 2 clinical trials. PARP inhibitors enhance the 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 shown promise in several preclinical models.•High-grade glioma patients have a very poor prognosis, in particular due to the inability to achieve complete surgical resection. Importantly, the residual tumor cells that have infiltrated surrounding normal brain tissue are using pre-existing brain vasculature and are thus protected by the blood-brain barrier (BBB). The ABC transporters ABCB1/Abcb1a/b; MDR1/mdr1a/b P-glycoprotein; P-gp) and ABCG2/Abcg2 (BCRP/Bcrp1; Breast cancer resistance protein) are important factors limiting the brain entry of many agents. Consequently, the ability of potential chemotherapeutics to cross the BBB, including the role of ABC-transporters in the brain penetration, needs to be established.

METHODSDrug solutions: ABT-888 was obtained from Selleck Chemicals.

Transwell assays: In vitro equilibrium transwell studies were performed with LLC-PK1 (parent) and LLC-Mdr1a cells and MDCK-parent vsMDCK-Bcrp1. ABT-888 0.5 µM in Optimem was added to both the apical and basolateral sides and samples were taken at 0.5, 1, 2 and 4 h. Samples in Optimem were diluted 5-fold in 0.1% formic acid and injected. ABT-888 was analyzed by HPLC on a Zorbax SB C18 column (75 x 4.6 mm) and a linear gradient from 2.5% to 30% (v/v) acetonitrilein 0.1% formic acid in water (8:92, v/v) and UV detection at 295 nm.

Pharmacokinetic studies: The i.v. studies were performed in FVB wild-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 by cardiac puncture at 1 h after drug administration. Plasma separated by centrifugation. Brains were dissected on ice and homogenized in 4% (w/v) BSA in water. Samples were stored at -20ºC until analysis.Analysis of ABT-888 in biological specimens was performed using a validated LC-MS method (Parise et al., J. Chromatogr. B 2008; 872:141-147

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

CONCLUSION

The brain penetration of ABT-888 is limited primarily by P-glycoprotein (ABCB1) but also by BCRP (ABCG2). The Brain-to-plasma ratio was 7.5-fold higher when both ABC transporters were absent. Further in vivo experiments to establish the impact of these transporters on the efficacy of ABT-888 are warranted.

OBJECTIVESThe aim of this study was to establish the impact of P-glycoprotein (ABCB1/Abcb1a/b) and Bcrp1 (ABCG2/Abcg2) in the blood-brain barrier on brain penetration and the intracranial antitumor efficacy of ABT-888.

RESULTS

In vitro studies• LLC-PK1 cells translocate ABT-888 into the apical direction. This is probably due to endogenous (porcine) P-gp and can be inhibited by 5µM of the P-gp inhibitor zosuquidar.•LLC-Mdr1a cells translocate ABT-888 even better and this transport can also be inhibited by 5 µM of zosuquidar (=P-gpinhibitor).•MDCK-Bcrp1 cells also efficiently translocate ABT-888 to the apical direction. This transport can be inhibited by 5 µM of elacridar (=P-gp and BCRP inhibitor).•There was no background translocation in the MDCK-parent cell line

In vivo studies•The plasma concentration of ABT-888 was not different between the 4 genotypes, indicating that the clearance is likely not affected by the absence of Abcb1a/b and Abcg2.•The brain-to-plasma ratio was significantly (p=0.006) higher in Abcb1a/b knockout mice versus wildtype controls.•The brain concentration in Abcg2 knockout mice was not higher, probably because Abcb1a/b alone is already sufficient.•The brain concentration in Abcg2;Abcb1a/b mice, however, was significantly higher (p=0.022) than in Abcb1a/b knockouts demonstrating that Abcg2 is also a limiting factor

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