Personalised medicine
Co-administration strategy to enhance brain accumulation of vandetanib by modulating P-glycoprotein (P-gp/Abcb1) and breast cancer resistance protein (Bcrp1/Abcg2) mediated efflux with m-TOR inhibitors

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Abstract

The objectives of this study were (i) to characterize the interaction of vandetanib with P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp1) in vitro and in vivo (ii) to study the modulation of P-gp and BCRP mediated efflux of vandetanib with specific transport inhibitors and m-TOR inhibitors, everolimus and temsirolimus. Cellular accumulation and bi-directional transport studies in MDCKII cell monolayers were conducted to delineate the role of efflux transporters on disposition of vandetanib. Brain distribution studies were conducted in male FVB wild-type mice with vandetanib administered intravenously either alone or in the presence of specific inhibitors and m-TOR inhibitors. In vitro studies suggested that vandetanib is a high affinity substrate of Bcrp1 but is not transported by P-gp. Interestingly, in vivo brain distribution studies in FVB wild type mice indicated that vandetanib penetration into the brain is restricted by both Bcrp1 and P-gp mediated active efflux at the blood brain barrier (BBB). Co-administration of elacridar, a dual P-gp/BCRP inhibitor increased the brain to plasma concentration ratio of vandetanib upto 5 fold. Of the two m-TOR pathway inhibitors examined; everolimus showed potent effect on modulating vandetanib brain penetration whereas no significant affect on vandetanib brain uptake was observed following temsirolimus co-administration. This finding could be clinically relevant as everolimus can provide synergistic pharmacological effect in addition to primary role of vandetanib efflux modulation at BBB for the treatment of brain tumors.

Introduction

ATP binding cassette (ABC) transporters P-glycoprotein (P-gp, ABCB1), multi-drug resistance associated protein (MRPs, ABCC) and breast cancer resistance protein (BCRP, ABCG2) are involved in translocation of a wide array of substrates across biological barriers. Apical localization of these membrane proteins on the gut, liver and kidney limits oral absorption and provides a mechanism for drug elimination (Leslie et al., 2005, Shugarts and Benet, 2009). The protective BBB also harbors these efflux proteins, which restrict the central nervous system (CNS) penetration of xenobiotics (Miller, 2010). Over expression of these efflux pumps in brain tumor tissues further compromises drug delivery (Gottesman et al., 2002, Loscher and Potschka, 2005). Many groups have previously reported the significance of ABC efflux pumps, especially P-gp and Bcrp1 in restricting the entry of numerous antitumor agents across BBB. Lapatinib, imatinib, gefitinib, erlotinib, and more recently axitinib and sunitnib have been reported to have restricted brain penetration due to P-gp and/or Bcrp1 mediated active efflux at the BBB (Polli et al., 2009, Agarwal et al., 2010a, Agarwal et al., 2010b, Breedveld et al., 2005, Kodaira et al., 2010, Poller et al., 2011, Tang et al., 2012).

Vandetanib (ZD6474), is an orally active inhibitor of vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR), and rearranged during transfection (RET) tyrosine kinases recently approved by the FDA for the treatment of metastatic medullary thyroid carcinoma (http://www.cancer.gov/cancertopics/druginfo/fda-vandetanib). In addition, phase III studies are currently ongoing for testing its efficacy against metastatic non small cell lung cancer (Heymach et al., 2008) (http://clinicaltrials.gov, NCT00418886, NCT00312377 and NCT00404924). Furthermore, potent antitumor effects have been suggested in preclinical orthotopic brain tumor models (Sandstrom et al., 2004, Sandstrom et al., 2008) and several Phase I/II trials of vandetanib in newly diagnosed brain tumors are also ongoing (Broniscer et al., 2010, Drappatz et al., 2010). Adequate delivery of a chemotherapeutic agent to the invasive glioma cells located behind an intact BBB is a characteristic feature for optimal treatment of brain tumors. Numerous anti-cancer agents are subjected to active efflux leading to significantly reduced brain accumulation (Gottesman et al., 2002). Since clinical efficacy of vandetanib is currently under investigation for brain tumor treatment, it is relevant to know the interaction of vandetanib with these efflux proteins with respect to its brain penetration.

Given the fact that most of the anti-tumor agents are subject to efflux, a viable strategy to improve brain penetration is to inhibit the efflux activity at the BBB. Such inhibitors could significantly enhance the brain distribution of the substrate to therapeutically relevant concentrations in the target tumor tissue located behind an intact BBB. However, specific efflux modulators may cause systemic toxicity at doses required to modulate efflux activity and may not possess any inherent pharmacological action (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. Apart from inhibiting VEGF mediated angiogenesis, there is emerging pre-clinical evidence suggesting pharmacological inactivation of m-TOR (mammalian target of rapamycin) pathway reduces neoplastic proliferation and brain tumor size (Galanis et al., 2005, Goudar et al., 2005). In human brain tumors, Akt/m-TOR pathway is activated in approximately 70% of the cases, in association with loss of PTEN and/or activation of EGFR and PDGFR tyrosine kinases (Hu et al., 2005). For this reason, everolimus and temsirolimus; two FDA approved m-TOR pathway inhibitors for the treatment of brain tumors, were chosen as clinically relevant efflux modulators for improving vandetanib brain delivery in this study.

Therefore one of the primary aim of this investigation was to determine whether vandetanib is a substrate for P-gp and/or BCRP and to delineate its effect on plasma and brain pharmacokinetics using appropriate cell culture and animal model. Further, we investigated if inhibition of the active efflux at the BBB could enhance the brain penetration of vandetanib using specific P-gp and BCRP inhibitors. In addition, from a clinical perspective, a combination therapy approach was evaluated to improve brain penetration of vandetanib upon co-administration of m-TOR inhibitors (everolimus and temsirolimus). We hypothesize, if m-TOR inhibitors are able to modulate P-gp and BCRP activity at the BBB, a dual advantage could be realized for inclusion of these anticancer agents to vandetanib treatment regimen of brain tumor therapy. In addition to blocking both VEGF and m-TOR pathways; modulation of P-gp and BCRP mediated active efflux at the BBB could result in increased brain accumulation of vandetanib.

Section snippets

Chemicals

Vandetanib, everolimus, temsirolimus and pazopanib were purchased from LC Laboratories (Woburn, MA). [3H]Digoxin and [3H]abacavir were purchased from PerkinElmer Life Sciences (Boston, MA) and Moravek Biochemicals (Brea, CA) respectively. Elacridar [GF120918, N-[4-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]-5-methoxy-9-oxo-10H-acridine-4-carboxamide]] was a generous gift from GlaxoSmithKline Ltd. (Research Triangle, NC). Ko143, (a fumitremorgin C analog) was procured from Enzo Life

Cellular uptake of vandetanib in MDCKII cells

Intracellular accumulation of vandetanib (100 nM) was studied in MDCK parent (WT), MDR1 and Bcrp1 overexpressing cells. Vandetanib accumulation was approximately 50% lower in Bcrp1 overexpressing cells in comparison to that of WT cells, suggesting Bcrp1 is involved in efflux of vandetanib (A Fig. 1). However, vandetanib accumulation in MDR1 overexpressing cells was not statistically different from WT cells, indicating P-gp might not be playing a role in its efflux (Fig. 1B). Pre-treatment with

Discussion

Vandetanib is a potent VEGFR and EGFR receptor tyrosine kinase inhibitor (TKI) currently being evaluated in clinic for its application in brain tumor therapy (NCT00441142, http://clinicaltrials.gov). Active efflux via P-gp and BCRP at the BBB can be a rate limiting step for adequate delivery of chemotherapeutics to the target brain tumor cells located behind an intact BBB. Previously, one report attempted to demonstrate the interaction of vandetanib with these efflux proteins (Azzariti et al.,

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