Sunitinib is an orally active, multitargeted tyrosine kinase inhibitor which has been used for the treatment of metastatic renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumors. We aimed to investigate the in vivo roles of the ATP-binding cassette drug efflux transporters ABCB1 and ABCG2 in plasma pharmacokinetics and brain accumulation of oral sunitinib, and the feasibility of improving sunitinib kinetics using oral coadministration of the dual ABCB1/ABCG2 inhibitor elacridar. We used in vitro transport assays and Abcb1a/1b 2/2 , Abcg2 2/2 and Abcb1a/1b/Abcg2 2/2 mice to study the roles of ABCB1 and ABCG2 in sunitinib disposition. In vitro, sunitinib was a good substrate of murine (mu)ABCG2 and a moderate substrate of human (hu)ABCB1 and huABCG2. In vivo, the systemic exposure of sunitinib after oral dosing (10 mg kg 21 ) was unchanged when muABCB1 and/or muABCG2 were absent. Brain accumulation of sunitinib was markedly (23-fold) increased in Abcb1a/b/Abcg2 2/2 mice, but only slightly (2.3-fold) in Abcb1a/b 2/2 mice, and not in Abcg2 2/2 mice. Importantly, a clinically realistic coadministration of oral elacridar and oral sunitinib to wild-type mice resulted in markedly increased sunitinib brain accumulation, equaling levels in Abcb1a/1b/Abcg2 2/2 mice. This indicates complete inhibition of the blood-brain barrier (BBB) transporters. High-dose intravenous sunitinib could saturate BBB muABCG2, but not muABCB1A, illustrating a dose-dependent relative impact of the BBB transporters. Brain accumulation of sunitinib is effectively restricted by both muABCB1 and muABCG2 activity. Complete inhibition of both transporters, leading to markedly increased brain accumulation of sunitinib, is feasible and safe with a clinically realistic oral elacridar/sunitinib coadministration.

P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) combination knockout mice display disproportionately increased brain penetration of shared substrates, including topotecan and several tyrosine kinase inhibitors, compared to mice deficient for only one transporter. To better study the interplay of both transporters also in vitro, we generated a transduced polarized MDCKII cell line stably coexpressing substantial levels of human ABCB1 and ABCG2 (MDCKII-ABCB1/ABCG2). Next, we measured concentration-dependent transepithelial transport of topotecan, sorafenib and sunitinib. By blocking either one or both of the transporters simultaneously, using specific inhibitors, we aimed to mimic the ABCB1-ABCG2 interplay at the blood-brain barrier in wild-type, single or combination knockout mice. ABCB1 and ABCG2 contributed to similar extents to topotecan transport, which was only partly saturable. For sorafenib transport, ABCG2 was the major determinant at low concentrations. However, saturation of ABCG2-mediated transport occurred at higher sorafenib concentrations, where ABCB1 was still fully active. Furthermore, sunitinib was transported equally by ABCB1 and ABCG2 at low concentrations, but ABCG2-mediated transport became saturated at lower concentrations than ABCB1-mediated transport. The relative impact of these transporters can thus be affected by the applied drug concentrations. A comparison of the in vitro observed (inverse) transport ratios and cellular accumulation of the drugs at low concentrations with in vivo brain penetration data from corresponding Abcb1a/1b⁻/⁻, Abcg2⁻/⁻ and Abcb1a/1b;Abcg2⁻/⁻ mouse strains revealed very similar qualitative patterns for each of the tested drugs. MDCKII-ABCB1/ABCG2 cells thus present a useful in vitro model to study the interplay of ABCB1 and ABCG2.

Crizotinib is an oral tyrosine kinase inhibitor approved for treating patients with non-small cell lung cancer (NSCLC) containing an anaplastic lymphoma kinase (ALK) rearrangement. We used knockout mice to study the roles of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) in plasma pharmacokinetics and brain accumulation of oral crizotinib, and the feasibility of improving crizotinib kinetics using coadministration of the dual ABCB1/ABCG2 inhibitor elacridar. In vitro, crizotinib was a good transport substrate of human ABCB1, but not of human ABCG2 or murine Abcg2. With low-dose oral crizotinib (5 mg/kg), Abcb1a/1b 2/2 and Abcb1a/1b;Abcg2 2/2 mice had an approximately twofold higher plasma AUC than wild-type mice, and a markedly (~40-fold) higher brain accumulation at 24 hr. Also at 4 hr, crizotinib brain concentrations were~25-fold, and brain-to-plasma ratios~14-fold higher in Abcb1a/1b 2/2 and Abcb1a/1b;Abcg2 2/2 mice than in wild-type mice. High-dose oral crizotinib (50 mg/kg) resulted in comparable plasma pharmacokinetics between wild-type and Abcb1a/1b 2/2 mice, suggesting saturation of intestinal Abcb1. Nonetheless, brain accumulation at 24 hr was still~70-fold higher in Abcb1a/1b 2/2 than in wild-type mice. Importantly, oral elacridar coadministration increased the plasma and brain concentrations and brain-toplasma ratios of crizotinib in wild-type mice, equaling the levels in Abcb1a/1b;Abcg2 2/2 mice. Our results indicate that crizotinib oral availability and brain accumulation were primarily restricted by Abcb1 at a non-saturating dose, and that coadministration of elacridar with crizotinib could substantially increase crizotinib oral availability and delivery to the brain. This principle might be used to enhance therapeutic efficacy of crizotinib against brain metastases in NSCLC patients.Lung cancer remains the leading cause of cancer deaths in western countries. 1 Patients with non-small cell lung cancer (NSCLC), accounting for approximately 80% of all lung cancer cases, 2 are often diagnosed at advanced stages of the disease. A subset of NSCLC was shown to have a small inversion within chromosome 2p, which results in the formation of a fusion gene comprising portions of the echinoderm microtubule associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene. 3 The EML4-ALK fusion results in constitutive activation of ALK kinase activity, which promotes cell proliferation, survival and cell cycling. 4 Since the identification of ALK rearrangements in 7% of NSCLC as a new and promising molecular target for treatment, 3 considerable effort has been focused on developing Key words: crizotinib, P-glycoprotein, ALK inhibitor, elacridar, brain accumulation Abbreviations: ABC: ATP-binding cassette; ABCB1: P-glycoprotein; ABCG2: breast cancer resistance protein; ALK: anaplastic lymphoma kinase; ANOVA: analysis of variance; AUC: area under plasma concentration-time curve; C brain : brain concentration; C max : maximum drug concentration in plasma; EML4: echinoderm mic...