P-Glycoprotein, CYP3A, and Plasma Carboxylesterase Determine Brain and Blood Disposition of the Mtor Inhibitor Everolimus (Afinitor) in Mice
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Published OnlineFirst April 11, 2014; DOI: 10.1158/1078-0432.CCR-13-1759 Clinical Cancer Cancer Therapy: Preclinical Research P-Glycoprotein, CYP3A, and Plasma Carboxylesterase Determine Brain and Blood Disposition of the mTOR Inhibitor Everolimus (Afinitor) in Mice Seng Chuan Tang1, Rolf W. Sparidans3, Ka Lei Cheung4, Tatsuki Fukami5, Selvi Durmus1, Els Wagenaar1, Tsuyoshi Yokoi5, Bart J.M. van Vlijmen4, Jos H. Beijnen2,3, and Alfred H. Schinkel1 Abstract Purpose: To clarify the role of ABCB1, ABCG2, and CYP3A in blood and brain exposure of everolimus using knockout mouse models. À À À À À À À À 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 À À À À À À 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 À À and recombinant human CES2, respectively. After correcting for carboxylesterase binding, Cyp3a / , but not À À À À À À Abcb1a/1b / , Abcg2 / ,orAbcb1a/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. Upre- gulated 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. Clin Cancer Res; 1–13. Ó2014 AACR. Introduction (PI3K)–protein kinase B signaling pathway (1, 2), which The mTOR is a serine–threonine protein kinase and controls cell growth, proliferation, survival, and metabo- downstream effector of the phosphoinositide 3-kinase lism (3, 4). Deregulation of the PI3K–AKT–mTOR signaling pathway occurs in many types of cancers (5–7). The mac- rocyclic lactone everolimus (Afinitor, Zortress/Certican, SDZ RAD or RAD001; Supplementary Fig. S1A), a derivative Authors' Affiliations: 1Division of Molecular Oncology, the Netherlands Cancer Institute; 2Department of Pharmacy and Pharmacology, Slotervaart of rapamycin (sirolimus), is an orally active inhibitor of Hospital, Amsterdam; 3Division of Pharmacoepidemiology and Clinical mTOR used in cancer therapy and as an immunosuppres- Pharmacology, Department of Pharmaceutical Sciences, Faculty of Sci- sant to prevent transplanted organ rejection. ence, Utrecht University, Utrecht; 4Department of Thrombosis and Hemo- stasis, Einthoven Laboratory for Experimental Vascular Medicine, Leiden Everolimus is used either alone or in combination for University Medical Center, Leiden, the Netherlands; and 5Drug Metabolism treating multiple cancers, including advanced renal cell and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa Univer- sity, Kakuma-machi, Kanazawa, Japan carcinoma (8), subependymal giant cell astrocytoma (9), advanced pancreatic neuroendocrine tumors (10), and Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). advanced hormone receptor–positive, HER-2–negative breast cancer (11). Clinical trials to assess its efficacy in Corresponding Author: Alfred H. Schinkel, Division of Molecular Oncol- ogy, the Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amster- gastric cancer, hepatocellular carcinoma, and lymphoma are dam, the Netherlands. Phone: 312-0512-2046; Fax: 312-0669-1383; ongoing, and it appears beneficial in refractory graft-versus- E-mail: [email protected] host disease after bone marrow transplantation. Given the doi: 10.1158/1078-0432.CCR-13-1759 sensitivity of human glioma cell lines to everolimus (12, 13), Ó2014 American Association for Cancer Research. and the alterations in the PI3K–AKT–mTOR pathway in www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst April 11, 2014; DOI: 10.1158/1078-0432.CCR-13-1759 Tang et al. treatment, or serious (life-threatening) side effects of this Translational Relevance potentially highly toxic drug. We aimed to clarify the in vivo Everolimus is currently used to treat patients with roles of ABCB1, ABCG2, and CYP3A in oral availability and breast cancer, who have a high risk of developing brain brain accumulation of everolimus using knockout mouse metastases. We show here that brain accumulation of models, to investigate a potential improvement of the everolimus is markedly restricted by ABCB1 in mice, therapeutic efficacy of everolimus, especially for brain providing a rationale for combining everolimus with tumors positioned behind an intact BBB. ABCB1 inhibitors to improve its therapeutic efficacy against primary and metastatic brain tumors. CYP3A Materials and Methods also strongly restricted the oral availability of everoli- mus, underscoring drug–drug interaction risks via Part of the Materials and Methods is presented in the CYP3A. Unexpectedly, several carboxylesterase (Ces) Supplementary Materials and Methods section. enzymes were upregulated in Abcb1a/1b and Abcg2 knockout mice, causing a strong increase in everolimus Blood pharmacokinetics and tissue disposition of blood levels, apparently by tight binding of everolimus everolimus in mice to plasma Ces1c. Numerous pharmacologic and phar- Everolimus was dissolved in ethanol:tween-80 (1:1, v/v) macokinetic studies of various drugs using these knock- to 2 mg/mL and further diluted with saline to yield solu- out strains in academia and pharmaceutical companies tions of 0.3 mg/mL and 0.4 mg/mL for oral and intravenous alike could be confounded by this Ces upregulation. administration, respectively. To minimize variation in Importantly, our results indicate that everolimus is a absorption on oral administration, mice were fasted for 3 human CES1 and CES2 inhibitor, which might be rel- hours before everolimus was administered (6.7 mL/kg) by evant in modulating the efficacy of (pro-)drugs hydro- gavage into the stomach, using a blunt-ended needle. Three lyzed especially by CES2. hours later, mice were anesthetized with isoflurane and blood was collected by cardiac puncture. Blood samples were collected in tubes containing Na2EDTA as an antico- agulant. Immediately thereafter, mice were sacrificed by >80% of glioblastoma (ref. 14; Cancer Genome Atlas cervical dislocation and livers and brains were rapidly removed. Brains and livers were homogenized with 1 mL Research Network, 2008), it might also benefit the treatment and 5 mL of 4% bovine serum albumin, respectively, and of these primary brain tumors. À The ATP-binding cassette (ABC) drug efflux transporters stored at 20 C until analysis. For intravenous adminis- P-glycoprotein (P-gp; ABCB1) and breast cancer resistance tration, mice were injected with a single bolus of everolimus protein (BCRP; ABCG2) are highly expressed in the intes- (5 mL/kg) via the tail vein. One hour later, mice were tinal epithelium and in the blood–brain barrier (BBB), as anesthetized with isoflurane and blood was collected by well as in many tumors. They can thus confer multidrug cardiac puncture. Immediately thereafter, mice were sacri- resistance and limit the oral absorption and brain pene- ficed by cervical dislocation, and livers and brains were tration of many clinically used anticancer drugs (15–19), processed as described above. which may well limit their therapeutic efficacy, especially against brain metastases. It is therefore important to know Blood cell distribution and tissue disposition of whether everolimus interacts with these transporters. In intravenous everolimus in mice vitro, everolimus is transported by ABCB1 (20) and it To obtain complete plasma pharmacokinetics and tis- inhibits ABCB1 and ABCG2 (21). However, the plasma sue concentration curves of everolimus, the experiment À/À AUC0–24h of everolimus in Abcb1a/1b mice was only 1.3- was initially terminated at 5, 30, and 60 minutes and later fold higher than in wild-type mice upon oral administra- extended to 2, 4, and 8 hours. Everolimus was adminis- tion of 0.25 mg/kg everolimus (22), suggesting little influ- tered intravenously to mice, and at the aforementioned ence of Abcb1 on oral availability. Nonetheless, everoli- time-points, mice were anesthetized and blood was col- mus coadministration could increase the brain accumula- lected by cardiac puncture. Immediately thereafter, mice tion of vandetanib, presumably by inhibiting Abcb1 and were sacrificed by cervical dislocation and livers and Abcg2 activity in wild-type mice (21). Although these brains were processed