Abstract
To clarify the role of ABCB1, ABCG2, and CYP3A in blood and brain exposure of everolimus using knockout mouse models. We used wild-type, Abcb1a/1b(-/-), Abcg2(-/-), Abcb1a/1b;Abcg2(-/-), and Cyp3a(-/-) mice to study everolimus oral bioavailability and brain accumulation. 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. 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.
Highlights
We show here that brain accumulation of everolimus is markedly restricted by ABCB1 in mice, providing a rationale for combining everolimus with ABCB1 inhibitors to improve its therapeutic efficacy against primary and metastatic brain tumors
We aimed to clarify the in vivo roles of ABCB1, ABCG2, and CYP3A in oral availability and brain accumulation of everolimus using knockout mouse models, to investigate a potential improvement of the therapeutic efficacy of everolimus, especially for brain tumors positioned behind an intact blood–brain barrier (BBB)
Everolimus blood levels in all the knockout strains were approximately 80-fold higher upon oral administration, and approximately 16-fold higher upon intravenous administration than those obtained in the "low" wild-type mice
Summary
Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Deregulation of the PI3K–AKT–mTOR signaling pathway occurs in many types of cancers [5,6,7]. The macrocyclic lactone everolimus (Afinitor, Zortress/Certican, SDZ RAD or RAD001; Supplementary Fig. S1A), a derivative of rapamycin (sirolimus), is an orally active inhibitor of mTOR used in cancer therapy and as an immunosuppressant to prevent transplanted organ rejection. Clinical trials to assess its efficacy in gastric cancer, hepatocellular carcinoma, and lymphoma are ongoing, and it appears beneficial in refractory graft-versushost disease after bone marrow transplantation. Given the sensitivity of human glioma cell lines to everolimus [12, 13], and the alterations in the PI3K–AKT–mTOR pathway in www.aacrjournals.org
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