Cancer Therapy: Preclinical P-Glycoprotein, CYP3A, and Plasma Carboxylesterase Determine Brain and Blood Disposition of themTOR 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 thehydrolysis of irinotecan and p-nitrophenyl acetatebymouseplasma carboxylesterase and recombinant humanCES2, respectively. After correcting for carboxylesterase binding,Cyp3a / , but not Abcb1a/1b / , Abcg2 / , or Abcb1a/1b;Abcg2 / mice, displayed highly (>5-fold) increased oral availability