Enhanced brain accumulation of pazopanib by modulating P-gp and Bcrp1 mediated efflux with canertinib or erlotinib

Abstract

Primary objective of this investigation was to delineate the differential impact of efflux transporters P-glycoprotein (P-gp/Abcb1) and breast cancer resistance protein (Bcrp1/Abcg2) on brain disposition and plasma pharmacokinetics of pazopanib. In addition, this research investigated whether inhibition of these efflux transporters with clinically relevant efflux modulators canertinib or erlotinib could be a viable strategy for improving pazopanib brain delivery. In vitro assays with MDCKII cell monolayers suggested that pazopanib is a high affinity substrate for Bcrp1 and a moderate substrate for P-gp. Co-incubation with specific transport inhibitors restored cell accumulation and completely abolished the directionality of pazopanib flux. Brain and plasma pharmacokinetic studies were conducted in FVB wild type mice in the absence and presence of specific transport inhibitors. Drug levels in plasma and brain were determined using a validated high performance liquid chromatography method using vandetanib as an internal standard. In vivo studies indicated that specific inhibition of either P-gp (by zosuquidar or LY335979) or Bcrp1 (by Ko143) alone did not significantly alter pazopanib brain accumulation. However, dual P-gp/Bcrp1 inhibition by elacridar (GF120918), significantly enhanced pazopanib brain penetration by ∼5-fold without altering its plasma concentrations. Thus, even though Bcrp1 showed higher affinity towards pazopanib in vitro, in vivo at the mouse BBB both P-gp and Bcrp1 act in concert to limit brain accumulation of pazopanib. Furthermore, erlotinib and canertinib as clinically relevant efflux modulators efficiently abrogated directionality in pazopanib efflux in vitro and their co-administration resulted in 2–2.5-fold increase in pazopanib brain accumulation in vivo. Further pre-clinical and clinical investigations are warranted as erlotinib or canertinib may have a synergistic pharmacological effect in addition to their primary role of pazopanib efflux modulation as a combination regimen for the treatment of recurrent brain tumors.

Introduction

Recurrent brain tumors still remain one of the most lethal forms of solid tumors with poor prognosis. Antiangiogenic therapy is at the forefront of current clinical practice for management of highly vascularized brain tumors. The role of epidermal growth factor receptor (EGFR), platelet derived growth factor receptor (PDGFR) and vascular endothelial growth factor receptors (VEGFR) in angiogenesis and cell proliferation is very well established (Hermanson et al., 1992, Plate et al., 1992). Pazopanib (GW786034, Votrient^®) is an orally active, second generation tyrosine kinase inhibitor (TKI) that targets VEGFR-1, -2, and -3, PDGFR-α, PDGFR-β, and c-Kit. It has been recently approved by the United States Food and Drug Administration (FDA) for the treatment of metastatic renal cell carcinoma (Ward and Stadler, 2012). Recently in a Phase II study, pazopanib was evaluated for its efficacy in recurrent glioblastoma (Iwamoto et al., 2010). Although pazopanib showed in situ biological activity, but as monotherapy was unable to prolong the progression free survival (PFS) in patients.

It is now well established that ATP binding cassette (ABC) efflux transporters restrict the entry of chemotherapeutic agents across BBB (Miller, 2010). Apical localization and overlapping substrate specificity of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) regulate central nervous system penetration and toxicity. Breast cancer resistance protein in addition to P-gp has shown significantly higher affinity toward small molecule TKIs (Agarwal et al., 2011, Breedveld et al., 2005); overexpression of P-gp and BCRP at tumor cell surface further compromises brain penetration of anti-tumor agents (Gottesman et al., 2002). No reports yet exist relating to the failure of pazopanib in glioblastoma treatment. We hypothesize that active efflux at the BBB could be partially responsible for sub-therapeutic concentrations of pazopanib attained in tumor tissue.

A viable strategy to improve brain penetration of compounds that are substrates of efflux proteins is to inhibit the efflux activity at the BBB (Breedveld et al., 2006). Such inhibitors may significantly enhance the brain distribution of substrate drug molecules to therapeutically relevant concentrations in the target tumor tissue located behind an intact BBB. Inhibition of P-gp at BBB using selective transport inhibitors has yielded encouraging results in preclinical studies. Kemper et al. (2004) reported enhanced brain penetration (∼5.6-fold) of paclitaxel in mice upon co-administration of a specific P-gp inhibitor zosuquidar trihydrochloride (LY335979). Similar results were reported by Hubensack et al. (2008). More recently, co-operative role of efflux proteins in limiting the brain penetration of several antitumor agents has been shown to be reversed by simultaneous chemical inhibition of P-gp and Bcrp1 using elacridar at mouse BBB (Agarwal et al., 2010, Tang et al., 2012, Chen et al., 2009, Yamasaki et al., 2011). Although much of the data regarding improved brain penetration upon efflux modulation are available from pre-clinical studies, first clinical study was reported by Sasongko et al. (2005) wherein, enhanced brain penetration of verapamil; a probe P-gp substrate, was demonstrated using PET imaging upon co-administration of a potent and specific P-gp inhibitor; cyclosporine A. In another report, Wagner et al. (2009) published similar findings upon co-administration of tariquidar as a P-gp inhibitor using PET imaging.

Thus far, many attempts have been made to overcome multidrug resistance in oncology leading to development of numerous transport inhibitors, with limited clinical success (Tamaki et al., 2011). Although potent, specific efflux modulators may cause systemic toxicity at doses required to modulate efflux activity (Katragadda et al., 2005). Hence, a dual advantage could be achieved if the co-administered molecule has a synergistic pharmacological effect in addition to its primary role of efflux modulation. Erlotinib and canertinib are currently under investigation for the treatment of recurrent brain tumors (Raizer et al., 2010, Slichenmyer et al., 2001). Both these compounds act by inhibiting the tyrosine kinase domain of the EGFR; known to play a pivotal role in tumor cell proliferation. Furthermore, it has been reported that EGFR is overexpressed in 60% of brain tumors (Smith et al., 2001). In addition both erlotinib and canertinib are know to reverse MDR1 and BCRP mediated resistance to paclitaxel and mitoxantrone or topotecan, respectively in cancer cells (Erlichman et al., 2001, Shi et al., 2007). Hence, we selected canertinib and erlotinib as potential candidates for modulating P-gp and BCRP mediated efflux of pazopanib.

The primary aim of this investigation was to demonstrate the interaction of pazopanib with efflux proteins P-gp and BCRP at the BBB and to elucidate their role in pazopanib brain disposition. Furthermore, from the clinical viewpoint, we wanted to investigate if co-administration of erlotinib and canertinib, could prove to be a viable strategy for modulating P-gp and BCRP mediated active efflux of pazopanib at the BBB.