p-glycoprotein, cyp3a, and plasma …...tinal epithelium and in the blood–brain barrier (bbb), as...

Cancer Therapy: Preclinical

P-Glycoprotein, CYP3A, and Plasma CarboxylesteraseDetermine Brain and Blood Disposition of themTOR InhibitorEverolimus (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

AbstractPurpose: 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 notAbcb1a/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. 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; 20(12); 3133–45. �2014 AACR.

IntroductionThe mTOR is a serine–threonine protein kinase and

downstream effector of the phosphoinositide 3-kinase

(PI3K)–protein kinase B signaling pathway (1, 2), whichcontrols cell growth, proliferation, survival, and metabo-lism (3, 4). Deregulation of the PI3K–AKT–mTOR signalingpathway occurs in many types of cancers (5–7). The mac-rocyclic lactone everolimus (Afinitor, Zortress/Certican,SDZRADor RAD001; Supplementary Fig. S1A), a derivativeof rapamycin (sirolimus), is an orally active inhibitor ofmTOR used in cancer therapy and as an immunosuppres-sant to prevent transplanted organ rejection.

Everolimus is used either alone or in combination fortreating multiple cancers, including advanced renal cellcarcinoma (8), subependymal giant cell astrocytoma (9),advanced pancreatic neuroendocrine tumors (10), andadvanced hormone receptor–positive, HER-2–negativebreast cancer (11). Clinical trials to assess its efficacy ingastric cancer, hepatocellular carcinoma, and lymphomaareongoing, and it appears beneficial in refractory graft-versus-host disease after bone marrow transplantation. Given thesensitivityof humangliomacell lines to everolimus (12,13),and the alterations in the PI3K–AKT–mTOR pathway in

Authors' Affiliations: 1Division of Molecular Oncology, the NetherlandsCancer Institute; 2Department of Pharmacy andPharmacology, SlotervaartHospital, Amsterdam; 3Division of Pharmacoepidemiology and ClinicalPharmacology, Department of Pharmaceutical Sciences, Faculty of Sci-ence, Utrecht University, Utrecht; 4Department of Thrombosis and Hemo-stasis, Einthoven Laboratory for Experimental Vascular Medicine, LeidenUniversity Medical Center, Leiden, the Netherlands; and 5DrugMetabolismand Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa Univer-sity, Kakuma-machi, Kanazawa, Japan

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Alfred H. Schinkel, Division of Molecular Oncol-ogy, the Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amster-dam, the Netherlands. Phone: 312-0512-2046; Fax: 312-0669-1383;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-1759

�2014 American Association for Cancer Research.

ClinicalCancer

Research

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>80% of glioblastoma (ref. 14; Cancer Genome AtlasResearchNetwork, 2008), itmight alsobenefit the treatmentof these primary brain tumors.

The ATP-binding cassette (ABC) drug efflux transportersP-glycoprotein (P-gp; ABCB1) and breast cancer resistanceprotein (BCRP; ABCG2) are highly expressed in the intes-tinal epithelium and in the blood–brain barrier (BBB), aswell as in many tumors. They can thus confer multidrugresistance and limit the oral absorption and brain pene-tration of many clinically used anticancer drugs (15–19),which may well limit their therapeutic efficacy, especiallyagainst brain metastases. It is therefore important to knowwhether everolimus interacts with these transporters. Invitro, everolimus is transported by ABCB1 (20) and itinhibits ABCB1 and ABCG2 (21). However, the plasmaAUC0–24h of everolimus in Abcb1a/1b�/�mice was only 1.3-fold higher than in wild-type mice upon oral administra-tion of 0.25 mg/kg everolimus (22), suggesting little influ-ence of Abcb1 on oral availability. Nonetheless, everoli-mus coadministration could increase the brain accumula-tion of vandetanib, presumably by inhibiting Abcb1 andAbcg2 activity in wild-type mice (21). Although thesestudies suggest an interaction of everolimus with ABCB1and ABCG2 in vitro and in vivo, the roles of Abcb1 andpossibly Abcg2 in brain accumulation of everolimusremain unknown.

In vitro studies supported by clinical data established thateverolimus ismetabolized by cytochromeP4503A (CYP3A;ref. 23). This is a concern for drug–drug interactions, ascoadministered drugs or food components may drasticallyalter CYP3A activity, and therefore the systemic levels oforally administered everolimus, resulting in either under-

treatment, or serious (life-threatening) side effects of thispotentially highly toxic drug. We aimed to clarify the in vivoroles of ABCB1, ABCG2, and CYP3A in oral availability andbrain accumulation of everolimus using knockout mousemodels, to investigate a potential improvement of thetherapeutic efficacy of everolimus, especially for braintumors positioned behind an intact BBB.

Materials and MethodsPart of the Materials and Methods is presented in the

Supplementary Materials and Methods section.

Blood pharmacokinetics and tissue disposition ofeverolimus in mice

Everolimus was dissolved in ethanol:tween-80 (1:1, v/v)to 2 mg/mL and further diluted with saline to yield solu-tions of 0.3mg/mL and 0.4mg/mL for oral and intravenousadministration, respectively. To minimize variation inabsorption on oral administration, mice were fasted for 3hours before everolimus was administered (6.7 mL/kg) bygavage into the stomach, using a blunt-ended needle. Threehours later, mice were anesthetized with isoflurane andblood was collected by cardiac puncture. Blood sampleswere collected in tubes containing Na2EDTA as an antico-agulant. Immediately thereafter, mice were sacrificed bycervical dislocation and livers and brains were rapidlyremoved. Brains and livers were homogenized with 1 mLand 5 mL of 4% bovine serum albumin, respectively, andstored at �20�C until analysis. For intravenous adminis-tration,micewere injectedwith a single bolus of everolimus(5 mL/kg) via the tail vein. One hour later, mice wereanesthetized with isoflurane and blood was collected bycardiac puncture. Immediately thereafter, mice were sacri-ficed by cervical dislocation, and livers and brains wereprocessed as described above.

Blood cell distribution and tissue disposition ofintravenous everolimus in mice

To obtain complete plasma pharmacokinetics and tis-sue concentration curves of everolimus, the experimentwas initially terminated at 5, 30, and 60 minutes and laterextended to 2, 4, and 8 hours. Everolimus was adminis-tered intravenously to mice, and at the aforementionedtime-points, mice were anesthetized and blood was col-lected by cardiac puncture. Immediately thereafter, micewere sacrificed by cervical dislocation and livers andbrains were processed as described above. Fifty micro-liters of blood samples was transferred to new Eppendorftubes and stored at �20�C for further analysis. Theremaining blood samples were immediately centrifugedat 2,100 � g for 6 minutes at 4�C, and plasma and bloodcell fractions were then collected and stored at �20�C forfurther analysis. The measured everolimus concentrationsin plasma and blood cell fractions were adjusted tocorrespond to the volume ratio between plasma and redblood cells in total blood composition, which is 0.63 and0.37, respectively.

Translational RelevanceEverolimus is currently used to treat patients with

breast cancer, who have a high risk of developing brainmetastases. We show here that brain accumulation ofeverolimus is markedly restricted by ABCB1 in mice,providing a rationale for combining everolimus withABCB1 inhibitors to improve its therapeutic efficacyagainst primary and metastatic brain tumors. CYP3Aalso strongly restricted the oral availability of everoli-mus, underscoring drug–drug interaction risks viaCYP3A. Unexpectedly, several carboxylesterase (Ces)enzymes were upregulated in Abcb1a/1b and Abcg2knockout mice, causing a strong increase in everolimusblood levels, apparently by tight binding of everolimusto plasma Ces1c. Numerous pharmacologic and phar-macokinetic studies of various drugs using these knock-out strains in academia and pharmaceutical companiesalike could be confounded by this Ces upregulation.Importantly, our results indicate that everolimus is ahuman CES1 and CES2 inhibitor, which might be rel-evant in modulating the efficacy of (pro-)drugs hydro-lyzed especially by CES2.

Tang et al.

Clin Cancer Res; 20(12) June 15, 2014 Clinical Cancer Research3134




Stability of everolimus in mouse plasma in vitroBlood was freshly collected by cardiac puncture in anes-

thetized wild-type, Abcb1a/1b�/�, Abcg2�/�, and Abcb1a/1b;Abcg2�/�mice, followed by centrifugation at 2,100� g for 6minutes at 4�C for the separation of plasma from bloodcells. The test was initiated by mixing 20 mL of everolimussolution with 980 mL of plasma pooled from mice of thesame genotype to achieve final concentrations of 250,1,000, or 4,000 ng/mL. The mixture was incubated at 37�Cfor 8 hours with gentle shaking. Samples (50 mL) werecollected at different time points until 8 hours, and werestored frozen at �20�C until analysis.

Stability of everolimus in knockout plasma dilutedwith increasing amounts of wild-type plasmaBlood was freshly collected as described above. Pooled

Abcb1a/1b�/�, Abcg2�/�, and Abcb1a/1b;Abcg2�/� plasmawas diluted with increasing amounts of wild-type plasma,at dilution factors between 2- and 125-fold. The reactionwas initiated by mixing 15 mL of everolimus with 735 mL ofknockout plasma with or without increasing amounts ofwild-type plasma. The final everolimus concentration was4,000 ng/mL and the mixture was incubated at 37�C for 8hours with gentle shaking. Samples (50 mL) were collectedat different time points until 8 hours and stored frozen at�20�C until analysis.

Statistical analysisData are presented as mean � SD. One-way ANOVA

was used to determine the significance between groups,after which post hoc tests with Bonferroni correction wereperformed for comparison between individual groups.Differences were considered statistically significant whenP < 0.05.

ResultsEverolimus pharmacokinetics and tissue dispositionin vivoTo assess the impact of Abcb1 and Abcg2 on oral bio-

availability and tissue disposition of everolimus, we admin-istered everolimus (2 mg/kg) orally or intravenously towild-type, Abcb1a/1b�/�, Abcg2�/�, and Abcb1a/1b;Abcg2�/�

mice, and measured blood and tissue concentrations byliquid chromatography/tandem mass spectrometry (LC/MS-MS). Three hours after oral administration, 7 of 10wild-typemice had low blood levels of everolimus, whereas3 had approximately 50-fold higher levels (Fig. 1A). Nowild-type mice had intermediate blood everolimus levels,implying the existence of two clearly distinct groups. Also inlater experiments a variable, but usually minor fraction ofwild-type mice displayed much higher everolimus bloodlevels. We therefore separately present data for the "low"and "high" everolimus wild-type mice. No "high" ever-olimus wild-type mice were present in the parallel intrave-nous experiment, assessed 1 hour after administration (Fig.1B). Everolimus blood levels in all the knockout strainswere approximately 80-fold higher upon oral administra-tion, and approximately 16-fold higher upon intravenous

administration than those obtained in the "low" wild-typemice.

In spite of the large differences in blood everolimus levels,the liver concentrations in wild-type (low and high) andknockout strains were quite similar, regardless of the routeof administration (Fig. 1C andD). This suggested that somefactor(s) affecting the blood–tissue distribution behavior ofeverolimus had drastically changed in the knockout strains,and likely also in the "high" wild-type mice, relative to the"low" wild-type mice.

To correct for possibly altered blood–tissue distributionbehavior of everolimus, we plotted both the direct brainconcentrations (Fig. 1E and F), and the brain-to-liver con-centration ratios in the different strains (Fig. 1G and H),rather than the brain-to-blood concentration ratios. Weassumed that altered free everolimus concentrations inplasma of knockout strains would similarly affect drugdistribution to liver and brain.We thus used liver as a probefor the level of free everolimus in plasma. The brain-to-liverconcentration ratios suggested that Abcb1a/1b�/� andAbcb1a/1b;Abcg2�/�mice had 10- to 14-fold increased brainaccumulation of everolimus relative to wild-type mice(both "low" and "high", P < 0.001), whereas Abcg2�/�micehad approximately 3-fold increased brain accumulation(P < 0.05) upon oral administration (Fig. 1G). Despite the50-fold higher blood concentration of everolimus in the"high" versus "low" wild-type mice, brain concentrationsand brain-to-liver ratios between these groups were notsignificantly different (Fig. 1E and G). Upon intravenousadministration, brain-to-liver ratios were approximately 8-fold increased in both Abcb1-deficient strains (P < 0.001),and not altered in the Abcg2

�/�strain (Fig. 1H). Collectively,

the data suggest that Abcb1 strongly restricts the brainaccumulation of everolimus, whereas Abcg2 has little, ifany, impact on brain accumulation of everolimus.

Blood cell distribution and tissue disposition ofeverolimus in vivo

The discrepancy between blood concentration and liver(plus brain) accumulation data of everolimus between thestrains suggested the existence of strong everolimus reten-tion factors in the blood of the knockout strains, andpresumably also the "high" wild-type mice. As everolimusin blood can sometimes distribute very extensively to redblood cells (e.g.,�80% in humans; ref. 24), we assessed thein vivoplasma to blood cell distribution of everolimus in thedifferent strains, as well as the liver and brain accumulation,at 5, 30, and 60minutes after intravenous administration at2 mg/kg. We again observed "low" and "high" wild-typemice in the 5- and 60-minute (but not the 30 minutes)groups (Fig. 2A and B), and while there was obvious (andsignificant) everolimus clearance in blood and plasma ofthe "low"wild-typemice between 5 and60minutes (3- to 4-fold decrease), this was not seen in the other strains. Bloodlevels of everolimus were greatly and similarly increased inall the knockout strains. Importantly, there was only littledistribution of everolimus to the blood cells relative towhole blood and plasma, ranging from about 6% in the

Everolimus Binds to and Inhibits Carboxylesterase

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Figure 1. Blood levels and tissuedisposition of everolimus. Bloodconcentration (ng/mL; A and B),liver accumulation (ng/g; C and D),brain concentration (ng/g; E and F),and brain-to-liver concentrationratio (G and H) of everolimus inmale wild-type, Abcb1a/1b�/�,Abcg2�/�, and Abcb1a/1b;Abcg2�/�mice 3 hours after oral(left) or 1 hour after intravenous(right) administration of 2 mg/kgeverolimus. All data are presentedas mean� SD (n¼ 3–7; �, P < 0.05;��, P < 0.001 when compared withwild-typemice with low everolimusblood levels; †,P<0.05; ††,P<0.01;†††, P < 0.001 when compared withwild-type mice with higheverolimus blood levels). One-percent of doses for liver are 452ng/g and 562 ng/g for oral andintravenous administration,respectively.

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"low" wild-type mice to well below 2% in all the knockoutstrains (Fig. 2C and D). Altered retention in blood cellscould therefore not explain the marked alterations in totalblood levels of everolimus. To better understand the tissueconcentrationduring the plasma clearance phase, especially

in the "high" wild-type and knockout strains, the experi-ment was extended to 2, 4, and 8 hours. Liver and brainaccumulation in this experiment (Fig. 2E–H) reflected thepatterns observed in Fig. 1, with low plasma clearance in allstrains except for the "low" wild-type mice, similar levels of

Figure 2. Blood, blood cell, andplasma distribution and tissuedisposition of everolimus in vivo.Blood concentrations (ng/mL; A),plasma concentrations (ng/mL; B),blood cells concentrations (ng/mL;C), blood cells-to-blood ratio (D),plasma concentration–time curve(ng/mL; E), liver concentration-timecurve (ng/g; F), brainconcentration–time curve (ng/g; G)and brain-to-liver ratio (H) ofeverolimus in male wild-type,Abcb1a/1b�/�, Abcg2�/�, andAbcb1a/1b;Abcg2�/�mice at 5, 30,or 60 minutes (panels A–D) as wellas 2, 4, and 8 hours (panels E-H)after intravenous administration of2 mg/kg everolimus. All data arepresented as mean � SD (n ¼ 1–5;�, P < 0.05; ��, P < 0.01; ��, P <0.001 when compared with wild-type mice with low everolimusblood levels; ††, P < 0.01; †††, P <0.001 when compared with wild-type mice with high everolimusblood levels). #, by chance no"high" wild-typemice were presentin the 30-minute group.






everolimus in liver, and highly increased brain concentra-tions and brain-to-liver ratios in Abcb1a/1b�/� and Abcb1a/1b;Abcg2�/�mice. Note that in all strains a substantialfraction (�30%of thedose) of everolimushad accumulatedin the liver within 5 minutes, which was then graduallycleared at similar rates (Fig. 2F).

The higher blood cells-to-blood ratios in the "low" wild-type mice versus all the knockout strains and the "high"wild-type mice (Fig. 2D) suggested higher retention ofeverolimus in the plasma of the latter strains. This mightalso explain why there was little clearance of everolimusfrom plasma in the knockout strains after intravenousadministration (Fig. 2A, B, and E). Note that at 4,000 ng/mL,a substantial fraction (�10%)of the administered everolimusdose was retained in the plasma.

Stability of everolimus in plasma of wild-type andknockout mice in vitro

Attempts to assess in vitro whether knockout and wild-type plasmahaddifferent levels of free and (protein-)boundamounts of everolimus failed because of the rapid disap-pearance of everolimus from wild-type plasma (data notshown). Indeed, a possible cause of the very differentplasma levels of everolimus might be greater stability ofeverolimus in knockout plasma relative to the ("low") wild-type plasma.We therefore incubated various concentrationsof everolimus (roughly covering the concentration rangeseen in Fig. 2B) in plasma of the different strains in vitro at37�C, and measured presence of everolimus over time.Everolimus itself was quite stable in saline at all concentra-tions (data not shown). Interestingly, while there was verylimited loss of everolimus at 250 ng/mL in plasma of allstrains, at 4,000 ng/mL there was marked loss in the wild-type plasma but not in the knockout plasmas. At 1,000ng/mL an intermediate pattern was observed (Fig. 3A–C).

The stability of everolimus in wild-type plasma at lowconcentrations and its relative instability at high concentra-tions suggested that a stabilizing plasma protein fully pro-tected low amounts of everolimus. Upon saturation of thisprotein, more free everolimus existed, that was degraded inwild-type plasma by an as yet unidentified plasma enzyme.The results of Fig. 3A–C imply a much higher level of thestabilizing protein in the knockout plasmas than in thewild-type plasma, whereas the level of the everolimus-degrading enzyme might be similar between the strains.Alternatively, wild-type plasma might have much higherlevels of an everolimus-degrading plasma enzyme, whereasall the strains had similar (low) levels of everolimus-stabi-lizing protein. To distinguish between these two hypothe-ses, we repeated the everolimus stability experiment at4,000 ng/mL with mixtures at various ratios of wild-typeand knockout plasmas. In case of much higher concentra-tions of an everolimus-degrading enzyme in wild-typeplasma, a 1 to 1 (i.e., 2-fold) dilution of wild-type plasmawith knockout plasma should result in a 2-fold lowerdegradation (loss) rate of everolimus. However, the resultsof Fig. 3D–F show that the everolimus loss rate was muchmore than 2-fold decreased in the 2-fold dilution mixtures

compared with undiluted wild-type plasma. For example,interpolation of the data in Fig. 3D indicated an initialeverolimus loss rate of 2,500 ng/mL/h in wild-type plasma,and 100 ng/mL/h in Abcb1a/1b�/� plasma. The predictedeverolimus degradation rate for the 1:1 dilution in case of ahigher concentration of everolimus-degrading enzyme inthe wild-type plasma would have been: 1/2� (100þ 2,500)¼ 1,300 ng/mL/h. This is clearly far higher than the inter-polated measured rate of 350 ng/mL/h in the 1:1 dilutionsamples (Fig. 3D). We can thus reject the hypothesis ofhigher everolimus-degrading enzyme in wild-type plasma.As for the alternative hypothesis, depending on the amountof excess of the everolimus-protecting protein (over ever-olimus) in knockout plasma, one can easily envisage that a5- to 10-fold reduction in the rate of everolimus degradationensues upon 1:1 dilution of wild-type plasma with knock-out plasma. We indeed observed a 7-fold reduced degra-dation rate, from 2,500 ng/mL/h to 350 ng/mL/h (Fig. 3D).Qualitatively similar data were obtained with the two otherknockout strains (Fig. 3E and F). Only when knockoutplasmas were between 5- and 25-fold diluted with wild-type plasma did the everolimus loss rates approach thoseseen in undiluted wild-type plasma. These results are there-fore more compatible with upregulation of an everolimus-stabilizing protein in the knockout plasmas, than withdownregulation of an everolimus-degrading protein in theknockout plasmas relative to wild-type plasma.

Indirect information on the presumed everolimus-degrading activity in plasma came from the detection inthe in vitro plasma incubations of (Fig. 3D–F) a prominenteverolimus metabolite, metabolite A (Fig. 3G–I). On thebasis of LC/MS-MS detection properties, metabolite A hadthe same mass over charge ratio as everolimus, but anapparently opened ring structure. Because its product spec-trum lacked the m/z 686.4 and m/z 518.3 peaks (25, 26),metabolite A was very likely the dehydrated ring-openedderivative of everolimus (Supplementary Fig. S1B). Theabsolute amount of metabolite A could not be determinedwithout reference material, but assuming an LC/MS-MSsignal strength similar to that of everolimus, a substantialfraction (�50%) of everolimuswas converted tometaboliteA in undiluted wild-type plasma (Fig. 3D–F and Fig. 3G–I).As with the disappearance rate of everolimus, the formationrate of metabolite A was much more than 2-fold decreasedin the 2-fold diluted wild-type plasmas (Fig. 3G–I). In fact,the metabolite A formation rate was already about 2-foldreduced in amixture of 80%wild-type and20%Abcb1a/1b�/�

plasma (5-fold dilution, Fig. 3G). This again suggests upre-gulation of an everolimus-stabilizing protein in the knock-out plasmas. Still,metabolite Amaybe furthermetabolized,thus complicating interpretation of its appearance profile.

Increased levels of an everolimus-stabilizing (and pre-sumably everolimus-binding) protein in the knockout plas-maswould likely also cause increased plasma retention, andreduced levels of free everolimus relative to total bloodconcentrations of everolimus. This would be compatiblewith the greatly reduced liver-to-blood ratios (as can bederived from Fig. 1), and reduced blood cells-to-blood

Tang et al.





ratios (Fig. 2A–D) in the knockout strains compared with("low") wild-type mice. Collectively, our data suggest thateverolimus in plasma of knockout strains is protected fromdegradation by an everolimus-binding protein.

Liver expression of Ces1 genes is highly upregulated inAbcb1a/1b�/�, Abcg2�/�, and Abcb1a/1b;Abcg2�/�miceWhile trying to identify the nature of the everolimus-

stabilizingplasmaprotein,wediscovered in an independentstudy that a range of carboxylesterase enzymes was highlyupregulated in, among others, Abcb1a/1b�/� mice (27). Assome carboxylesterases synthesized in the liver can be abun-dant inmouse plasma, perhaps one ormore of these plasmacarboxylesterases could tightly bind everolimus. One could

even speculate that there could be recognition (but nothydrolysis) of the lactone ring-internal carboxylester bondof everolimus (see Supplementary Fig. S1A), leading to atight but nonprocessive protein–substrate complex. Wetherefore tested RNA levels of the main liver-expressedmouse Ces genes, that is, Ces1b-Ces1g and Ces2a, in wild-type, Abcb1a/1b�/�, Abcg2�/�, and Abcb1a/1b;Abcg2�/�miceusing real-time reverse transcription-PCR (RT-PCR). Inter-estingly, Ces1b was about 8- to 10-fold upregulated, andCes1c was about 70-fold upregulated in all the knockoutstrains (Supplementary Fig. S2). The basal expression ofCes1d was virtually undetectable in male wild-type liver,leading to nominally approximately 40,000-fold upregula-tion in all the knockout strains (Supplementary Fig. S2),

Figure 3. Stability of everolimus in plasma of wild-type and knockout mice in vitro. A–C, concentration-time curves of everolimus in male wild-type,Abcb1a/1b�/�, Abcg2�/�, and Abcb1a/1b;Abcg2�/�plasma after incubation of 250 ng/mL (A), 1,000 ng/mL (B) or 4,000 ng/mL (C) spiked everolimus. D–I,stability of everolimus in knockout mouse plasmas diluted with increasing amounts of wild-type plasma in vitro. Concentration–time curves of everolimus(ng/mL, D–F) and metabolite A (response relative to internal standard, G–I) after incubation of 4,000 ng/mL everolimus in Abcb1a/1b�/� (D and G), Abcg2�/�

(E and H), and Abcb1a/1b;Abcg2�/�(F and I) pooled plasma diluted with increasing amounts of pooled wild-type plasma. Values below lower limit ofquantifications were replaced with 100 ng/mL and 0.1 response relative to internal standard for everolimus and metabolite A, respectively. Each data pointrepresents a single determination.






but the observedDCt values of these strains were in the sameorder as for theotherupregulatedCes1genes, suggesting thatthe final expression levels were not extremely high (Supple-mentary Table S1). Ces1e was 3- to 6-fold upregulated,whereas Ces1f, Ces1g, and Ces2a were not upregulated, andperhaps sometimes even downregulated (SupplementaryFig. S2). Strikingly, reminiscent of the everolimus pharma-cokinetic data, there was one "high" Ces1 wild-type mouse,which consistently displayed clearly increased expressionlevels ofCes1b,Ces1c,Ces1d, andCes1e relative to the2 "low"Ces1wild-type mice, albeit not completely up to the level ofthe knockout strains (Supplementary Fig. S2).

Of the upregulated Ces1 proteins, only Ces1b and Ces1clack the ER retention signal that prevents protein secretionfrom liver into plasma, and they are thus likely to occur inplasma. As Ces1b is hardly expressed in mouse liver (28,29), the substantially expressed carboxylesterase Ces1c isthe most likely candidate everolimus-binding protein inplasma. A range of other esterases, including Ces2e, Ces3a,Aadac, and Pon1, 2, and 3, were not upregulated in Abcb1a/1b�/�mice (27), and thus unlikely to be involved in ever-olimus protection in the knockout strains studied here.

The carboxylesterase inhibitor BNPP reversesstabilization of everolimus in mouse plasma

To provide more direct evidence that plasma carboxyles-terase was responsible for protecting everolimus from deg-radation in knockout plasma, we tested whether the stabi-lization of everolimus could be reversed using the carbox-ylesterase inhibitor BNPP. This organophosphate irrevers-ibly inhibits carboxylesterases through the generation of astable phosphate ester covalently attached to the catalyticserine residue in the enzyme active site (30). If everolimusnormally binds to the substrate binding site of carboxyles-terase, one would expect it to bind no longer if the carbox-ylesterase has bound BNPP. We therefore preincubatedBNPP (1 mmol/L) for 15 minutes in vitro with freshlycollected wild-type, Abcb1a/1b�/�, Abcg2�/�, and Abcb1a/1b;Abcg2�/�plasma, and measured disappearance of subse-quently spiked everolimus (4,000 ng/mL) over time. In theabsence of BNPP, everolimuswas rapidly decreased in 3of 5("low") wild-type plasmas, whereas everolimus concentra-tions remained similar over time in2of 5 ("high")wild-typeplasmas and all knockout plasmas (Fig. 4A). After preincu-bation with BNPP, however, all knockout plasmas and the"high" wild-type plasmas displayed similar rapid degrada-tion profiles as seen in the "low" wild-type plasma withoutorwith BNPPpreincubation (Fig. 4B). These results indicatethat upregulated carboxylesterases in the knockout and 2"high" wild-type plasmas are responsible for stabilizingeverolimus. Moreover, the similarity in everolimus degra-dation rates between all the strains in the presence of BNPPindicates that there were no pronounced differences in thepotential plasma everolimus-degrading activity between thestrains. Ces expression analysis of livers of the individualmice tested confirmed that the "high" wild-type mice hadmarked upregulation of Ces1b-e relative to the "low" wild-type mice, and this upregulation approached the levels

seen in the knockout strains (Fig. 4C–F and SupplementaryTable S2).

Everolimus inhibits the conversion of irinotecan toSN-38 by carboxylesterase 1c in knockout plasma

If everolimus binds tightly to the active site of plasmacarboxylesterase, it might also inhibit the hydrolytic activitytowards carboxylesterase substrates. The anticancer prodrugirinotecan is hydrolyzed to its active derivative SN-38 pri-marily by plasma Ces1c in mice (31). We therefore testedthe conversion of spiked irinotecan (5 mmol/L) to SN-38 inindividual wild-type and knockout plasmas in a 30-minutein vitro incubation and the effect of preincubation of theseplasmaswith everolimus (100mmol/L).Without inhibitors,we observed very little conversion of irinotecan to SN-38 inall wild-type plasmas, versus almost complete conversion inall knockout plasmas (Supplementary Fig. S3). The hydro-lase activity towards irinotecan in knockout plasmas was atmost weakly inhibited by the everolimus vehicle (0.25%ethanol and 0.25%polysorbate 80), whereas it was stronglyinhibited by both everolimus and the positive controlinhibitor BNPP (Supplementary Fig. S3). These resultsindicate that there is highly increased hydrolysis of irino-tecan in knockout plasmas, most likely due to the highlyupregulated Ces1c, and that preincubation with everolimuscould effectively inhibit this hydrolase activity. This stronglysupports that everolimus binds to plasma carboxylesterase1c, most likely to its active site.

In vivo knockdown confirms Ces1c involvement ineverolimus pharmacokinetics

To specifically test whether Ces1c was responsible for thealtered pharmacokinetic behavior of everolimus, we per-formed an in vivo Ces1c knockdown experiment in theAbcb1a/1b�/�mice, which had the most consistent upregu-lation of Ces1c and altered everolimus pharmacokinetics.We compared everolimus pharmacokinetics in Abcb1a/1b�/�

mice treated with either a specific Ces1c siRNA or anegative control siRNA. Pilot experiments showed thatthe selected siRNA was efficient in knocking down Ces1cin cultured primary hepatocytes of FVB mice, whereas thenegative control siRNA had no effect (data not shown).Subsequent in vivo siRNA experiments demonstrateda very extensive knockdown of hepatic Ces1c RNA 3 daysafter intravenous administration of the Ces1c siRNA, rel-ative to the negative control siRNA, as judged by real-timeRT-PCR (DCt 0.77 � 0.33 vs. �4.54 � 0.21, a 40.2-foldlinear decrease; P < 0.001; Supplementary Table S3).Pharmacokinetic analysis performed at this day 3 of orallyadministered everolimus showed that in the Ces1c siRNA-treated Abcb1a/1b�/�mice plasma levels were extensively,albeit not completely, reversed to those seen in (low)wild-type mice, whereas in the negative control siRNA-treated mice everolimus plasma levels were roughly thesame as seen previously in untreated Abcb1a/1b�/�mice(Fig. 5A and Supplementary Table S4). Liver and brainconcentrations were only modestly affected by thesechanges in the Ces1c siRNA-treated mice: the slightly lower

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liver concentration may reflect faster overall everolimuselimination, and the somewhat higher brain concentra-tion may reflect higher peak free plasma concentrations ofeverolimus (Fig. 5B and C). Nonetheless, these data fullyconfirm that Ces1c was the main factor responsible for theanomalous everolimus plasma pharmacokinetics seen inthe knockout strain.

Everolimus inhibits hydrolysis by recombinant humanCES1 and CES2

Although there are no straightforward orthologs betweenthe mouse Ces1 and Ces2 family members and the humanCES1 and CES2 enzymes, and substrate and inhibitorspecificity can differ between these species, we tested theinhibitory effect of everolimus on the p-nitrophenyl acetate

Figure 4. Stability of everolimus inmouse plasma after preincubationwith the irreversible CES inhibitorBNPP in vitro. Concentration ofeverolimus (% of control) afterincubation of 4,000 ng/mLeverolimus spiked into wild-type,Abcb1a/1b�/�, Abcg2�/�, andAbcb1a/1b;Abcg2�/�plasmaeither without (A), or with 1 mMBNPP pretreatment (B). All data arepresented as mean� SD (n¼ 2–3).Expression levels of Ces1b (C),Ces1c (D), Ces1d (E), or Ces1e (F)mRNA in livers of male wild-type,Abcb1a/1b�/�, Abcg2�/�, andAbcb1a/1b;Abcg2�/�mice used inthe stability experiment, asdetermined by real-time RT-PCR.Data are normalized to GAPDHexpression. Values representmeanfold change � SD, compared withwild-type mice with low Cesexpression (n ¼ 2–5; �, P < 0.05;��, P < 0.01; ��, P < 0.001 whencompared with wild-type mice withlow plasma everolimus levels).






hydrolase activity of recombinant humanCES1 andCES2.Asubstrate concentration of 100 mmol/L was used, similar tothe Km values of recombinant CES1 and CES2 (32). Ever-olimus inhibited both enzymes, albeit with relatively highIC50 values of 157.2 mmol/L and 19.4 mmol/L for CES1 andCES2, respectively (Fig. 6). These results indicate that ever-olimus is a better inhibitor of human CES2 than CES1in vitro.

Cyp3a, but not Abcb1, limits the oral availability ofeverolimus in mice

Notwithstanding the carboxylesterase upregulation andeverolimus binding in the knockout mouse strains, weaimed to assess the impact of Abcb1 and CYP3A on theoral availability of everolimus in mice. Everolimus wasorally administered at 2 mg/kg to wild-type and Abcb1a/1b, Cyp3a and combination Abcb1a/1b/Cyp3a knockoutstrains, and whole blood everolimus concentrations wereassessed (Supplementary Fig. S4). We again observed a"low" (n ¼ 5) and "high" (n ¼ 3) wild-type group.Importantly, the Abcb1a/1b knockout did not result ina significant increase in oral area under the curve (AUC)relative to the "high" wild-type group (SupplementaryFig. S4 and Supplementary Table S5). Considering thehigh upregulation of plasma Ces in both mouse groups,this indicates that Abcb1 had little impact on the oralavailability of everolimus at this dosage. Cyp3a�/�mice,however, which have a similar level of hepatic Ces1upregulation as Abcb1a/1b�/�mice, had a 7.8-fold highereverolimus blood AUC than the "high" wild-type group,indicating that Cyp3amarkedly restricts the oral availabilityof everolimus (Supplementary Fig. S4 and SupplementaryTable S5). Additional knockout of Abcb1a/1b in Abcb1a/1b;Cyp3a�/�mice did not result in a further increase in bloodAUC, consistent with the absence of a marked effect ofAbcb1a/1b on everolimus oral availability.

DiscussionWe demonstrated that Abcb1a/1b markedly reduces the

brain accumulation, but not the oral availability of ever-olimus, whereas Abcg2 does not affect either. Cyp3a,

Figure 5. Effect of Ces1cknockdown on plasmapharmacokinetics and tissuedisposition of everolimus in vivo.Plasma concentration–time curve(ng/mL; A), liver concentration(ng/g; B), brain concentration(ng/g; C) and brain-to-liverratio (D) of everolimus in negativecontrol siRNA (siNEG)- or siCes1c-treated male Abcb1a/1b�/�mice at3 hours after oral administration of2 mg/kg everolimus. All data arepresented as mean � SD(n ¼ 5–7; �, �� and �� indicateP < 0.05, P < 0.01 and P < 0.001when compared with siNEG-injected male Abcb1a/1b�/�

mice, respectively).

Figure 6. Inhibitory effect of everolimus on hydrolase activities byrecombinant human CES1 and CES2 in vitro. The activities weredetermined at 100 mmol/L p-nitrophenyl acetate substrateconcentrations. Each data point represents the mean of triplicatedeterminations. The control activities by recombinant CES1 andCES2 were 542 and 300 nmol/min/mg, respectively.

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however, strongly reduced the oral availability of everoli-mus. Most remarkably, upregulation of plasma Ces1c inknockout and "high" wild-type mice had a pronouncedeffect on the plasma pharmacokinetics of everolimus,which could be reversed by in vivo siRNA-mediated Ces1cknockdown. Apparently, everolimus binds tightly to plas-maCes1c,which largely prevents degradationof everolimusby another plasma protein and strongly reduces overallblood clearance. This is schematically illustrated in Supple-mentary Fig. S5. Everolimus also inhibited human CES1and especially CES2.Although we knew that plasma Ces enzymes are upregu-

lated in some knockout strains, affecting drugs hydrolyzedby these enzymes (27), we had not anticipated that strongCes binding of otherwise unhydrolyzed drugs might pro-foundly affect their blood pharmacokinetics. Becausemanyother drugs may be bound but not hydrolyzed by thesemultispecific enzymes, this confounder should be consid-ered in studies with these knockout strains. Note that not allknockout strains for detoxifying proteins display hepaticCes1 upregulation, whereas similar levels of Ces1b-e upre-gulation were observed in Abcb1a/1b, Abcg2, and Cyp3aknockout strains and combinations thereof (27 and thepresent study), Abcc2 and Abcc3 knockout strains did notshow altered everolimus blood pharmacokinetics (data notshown), and are thus unlikely to have upregulated Ces1c.The mechanism behind the upregulation of the Ces1b-e

genes is currently unknown. The similarity in upregulationprofiles between the different mouse strains suggests ashared induction mechanism between these genes. Becausea semisynthetic diet does not affect Ces1 upregulation (asjudged by everolimus blood pharmacokinetics) in the var-ious transporter knockout strains (Supplementary Fig. S6),it is unlikely that altered exposure to some dietary xenobi-otic is directly responsible. Perhaps some endogenousinducers are responsible, or possibly signaling pathwaysactivated by xenotoxins derived from the intestinal micro-flora, but their nature remains speculative.That some wild-type mice display very similar, albeit

slightly lower, upregulation of the same group ofCes1 genesasmany knockout strains do is also intriguing (genotypes of"high"wild-typemicewere double-checked). Therewere noobvious external clues to which wild-type mice displayed a"high" or "low" everolimus or Ces1 phenotype, and itvaried also among siblings from one litter. We currentlydo not understand themechanistic cause of incidental Ces1upregulation in wild-type mice. We observed no interme-diate Ces1c expression levels in wild-type mice, suggestingeither that Ces1 upregulation is a fixed situation in indi-vidual wild-typemice, or that a switch from "low" to "high"Ces1 expression (or vice versa) occurs abruptly.Regardless, the endogenous variation in Ces1 expression

inwild-typemicewill complicate pharmacokinetic analysesfor any drug that is hydrolyzed or bound by the upregulatedCes enzymes. For instance, oral everolimus pharmacoki-netics in nude female BALB/c mice (24) show everolimusblood levels that are compatible with the levels in our wild-type FVB "high" mice, but not the "low" mice. The tested

strain apparently had constitutively "high" plasma Ces1levels. This could also explain the extremely high plasmaprotein binding (99.9%) of everolimus reported for thismouse strain, as opposed to 92% in rats and 75% inhumans. To test whether the "nude" mutation might beresponsible for this presumed Ces1 upregulation, we testedFVB nude "wild-type" and Abcb1a/1b�/� and Abcb1a/1b;Abcg2�/�mice, but found similar low wild-type and highknockout Ces1 expression in liver as in the normal FVBbackground (data not shown). In addition, the level ofirinotecan hydrolysis observed in plasma of wild-typeB6D2mice byMorton and colleagues (31) suggests a "high"wild-type plasma Ces1c level. As carboxylesterases are nor-mally not substantially present in plasma of humans, and inview of the poorly predictable variation in plasma carbox-ylesterases in various mouse strains, it may be preferable toperform studies with drugs that may be hydrolyzed orbound by plasma carboxylesterases in Ces1c knockoutstrains, or in other species that lack plasma carboxyles-terases (33), thus avoiding this potential confounder.

The prolonged retention of everolimus in blood andplasma of mice with high Ces1 plasma levels suggests amuch reduced blood-to-tissue distribution of everolimus.However, our data indicate that, apart from the fraction ofeverolimus that is tightly bound to plasma Ces (�5% of thedose after oral and �10% of the dose after intravenousadministration in Ces upregulated mice; values derivedfrom Fig. 1A and B), there is also a "free" fraction of thedrug in blood. The concentration of this free fraction doesnot seem to differ much between all the mouse strains,judging from the similar levels of liver accumulationbetween the knockout strains and the "low" and "high"wild-typemice (Fig. 1). Thus, although a significant fractionof everolimus is rapidly and tightly bound to plasma Ces1,the remainder (90%–95% of the dose) seems to be nor-mally available for distribution. The overall impact ofplasma binding of everolimus on tissue distribution of thedrug is therefore limited, at least during the first few hours,and at the dosage tested.

The identity of the plasma enzyme that converts ever-olimus to metabolite A is unknown. Human liver micro-somes in both the absence and presence of NADPH converteverolimus to a lactone ring-opened product that is subse-quently dehydrated to its seco acid (34), which resemblesmetabolite A. The responsible enzyme is thus not a Cyto-chrome P450. A plasma-localized mouse analogue of thisenzyme might be responsible for the metabolism of freeeverolimus in mouse plasma. Its activity towards everoli-mus when Ces1c was blocked by BNPP did not differbetween all the wild-type and knockout mouse strains (Fig.4B). Of note, also in human plasma ring-opened everoli-mus metabolites predominate (FDA application 21-560s000).

Everolimus showed higher inhibitory effect towardshuman CES2 than towards human CES1. This differencecould be due to size-limited access of the bulky everolimus,as the active site of CES1 is smaller than that of CES2 (35).The inhibitory effects of everolimus were also different






between humanCES1 andmouse Ces1c, possibly reflectinga similar size-access difference, although little is knownabout Ces1c structure. Species differences in inhibitor sen-sitivity between human and rat liver Ces have been dem-onstrated previously (36).

The profound plasma carboxylesterase binding of ever-olimus observed inmice is unlikely to play a role in humansas, unlike mouse Ces1c, human CES1 or CES2 are notnormally substantially present in plasma (37). Also theinhibitory activity of everolimus towards human CES1 andCES2 is not very high, suggesting that itmaybind less tightlyto these proteins than tomouse Ces1c. However, inhibitionof the hepatic CES1 and especially CES2, which is primarilyfound in the intestine, by everolimus might play a role indrug–drug interactions with coadministered drugs. CES1and CES2 hydrolyze many drugs and prodrugs (30). Uponoral everolimus administration, local concentrations ofeverolimus might be high especially in the intestine, andpossibly surpass the Ki of approximately 20 mmol/L towardCES2. CES2 is for instance thought to be a main enzymeresponsible for the conversion of irinotecan to SN-38 inhumans (38), and for hydrolysis of a prodrug of gemcita-bine (39). Coadministration of everolimuswith ester (pro-)drugs affected by carboxylesterases, including the 5-FUanticancer prodrug capecitabine (40), should thus beassessed very carefully.

The limited brain accumulation of everolimus due to theactivity of ABCB1 may restrict the therapeutic efficacy ofeverolimus towards brain tumor parts or (micro-)metasta-ses that are effectively situated behind a functional blood–brain barrier. Very likely this limited accumulation could beimproved by coadministration of an effective ABCB1 inhib-itor such as elacridar (16). ABCB1 itself did not affect theoral availability of everolimus, when taking plasma Cesupregulation into account. Given the complications ofplasma carboxylesterase upregulation, we think that thesmall (1.3-fold) reported effect of ABCB1 on low-doseeverolimus oral availability assessed with Abcb1a/1b�/�mice(22) should be interpreted with caution.

Notwithstanding the plasma Ces1 upregulation, mouseCyp3a can considerably reduce the oral availability of ever-olimus (Supplementary Fig. S4 and Supplementary TableS5). This is consistentwith the demonstratedmetabolismof

everolimus by recombinant human CYP3A4, CYP3A5, andCYP2C8 in vitro, with CYP3A4 being the major enzymeinvolved (41). Moreover, drug–drug interaction studieswith various CYP3A inhibiting drugs further support thatCYP3A is amajor factor in the in vivo clearance of everolimus(FDA application 21-560s000). Accordingly, great cautionis indicated in the clinical coapplication of everolimus withdrugs that affect CYP3A activity.

Disclosure of Potential Conflicts of InterestThe research group of A.H. Schinkel benefits from the commercial

availability of knockout strains used in this study. No potential conflicts ofinterest were disclosed by the other authors.

DisclaimerThe content is solely the responsibility of the authors and does not

necessarily represent the official views of the funding agencies.

Authors' ContributionsConception and design: S.C. Tang, B.J.M. van Vlijmen, A.H. SchinkelDevelopment of methodology: R.W. Sparidans, B.J.M. van VlijmenAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): R.W. Sparidans, T. Fukami, S. Durmus,J.H. BeijnenAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): S.C. Tang, S.Durmus, B.J.M. van Vlijmen,A.H. SchinkelWriting, review, and/or revision of the manuscript: S.C. Tang,R.W. Sparidans, T. Fukami, J.H. Beijnen, A.H. SchinkelAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S.C. Tang, R.W. Sparidans,K.L. Cheung, E. Wagenaar, T. YokoiStudy supervision: A.H. Schinkel

AcknowledgmentsThe authors thank Anita van Esch, Dilek Iusuf, Gloria Mena Lozano, Fan

Lin, and Olaf van Tellingen for their assistance with bioanalytical experi-ments. The authors also thank Dilek Iusuf for critical reading of the article.

Grant SupportThis work was financially supported by an academic staff training scheme

fellowship from the Malaysian Ministry of Science, Technology and Inno-vation (to S.C. Tang) and in part by Dutch Cancer Society grant 2007–3764.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 26, 2013; revisedMarch 25, 2014; acceptedMarch 27, 2014;published OnlineFirst April 11, 2014.

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2014;20:3133-3145. Published OnlineFirst April 11, 2014.Clin Cancer Res Seng Chuan Tang, Rolf W. Sparidans, Ka Lei Cheung, et al. (Afinitor) in MiceBrain and Blood Disposition of the mTOR Inhibitor Everolimus P-Glycoprotein, CYP3A, and Plasma Carboxylesterase Determine

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