P-Glycoprotein, CYP3A, and Plasma Carboxylesterase Determine Brain and Blood Disposition of the mTOR Inhibitor Everolimus (Afinitor) in Mice

Purpose: To clarify the role of ABCB1, ABCG2, and CYP3A in blood and brain exposure of everolimus using knockout mouse models. Experimental Design: We used wild-type, Abcb1a/1b^-/-, Abcg2^-/-, Abcb1a/1b;Abcg2 ^-/-, and Cyp3a^-/- mice to study everolimus oral bioavailability and brain accumulation. Results: Following everolimus administration, brain concentrations and brain-to-liver ratios were substantially increased in Abcb1a/1b^-/- and Abcb1a/1b;Abcg2 ^-/-, but not Abcg2^-/- mice. The fraction of everolimus located in the plasma compartment was highly increased in all knockout strains. In vitro, everolimus was rapidly degraded in wild-type but not knockout plasma. Carboxylesterase 1c ( Ces1c), a plasma carboxylesterase gene, was highly upregulated (∼80-fold) in the liver of knockout mice relative to wild-type mice, and plasma Ces1c likely protected everolimus from degradation by binding and stabilizing it. This binding was prevented by preincubation with the carboxylesterase inhibitor BNPP. In vivo knockdown experiments confirmed the involvement of Ces1c in everolimus stabilization. Everolimus also markedly inhibited the hydrolysis of irinotecan and p-nitrophenyl acetate by mouse plasma carboxylesterase and recombinant human CES2, respectively. After correcting for carboxylesterase binding, Cyp3a^-/-, but not Abcb1a/1b^-/-, Abcg2^-/-, or Abcb1a/1b;Abcg2^-/- mice, displayed highly (>5-fold) increased oral availability of everolimus. Conclusions: Brain accumulation of everolimus was restricted by Abcb1, but not Abcg2, suggesting the use of coadministered ABCB1 inhibitors to improve brain tumor treatment. Cyp3a, but not Abcb1a/1b, restricted everolimus oral availability, underscoring drug-drug interaction risks via CYP3A. Upregulated Ces1c likely mediated the tight binding and stabilization of everolimus, causing higher plasma retention in knockout strains. This Ces upregulation might confound other pharmacologic studies. © 2014 AACR.