Activation Status of the Pregnane X Receptor ﬂuences ......transgenic humanized mouse models. We...

Therapeutics, Targets, and Chemical Biology

Activation Status of the Pregnane X ReceptorInfluences Vemurafenib Availability inHumanized Mouse ModelsA. Kenneth MacLeod, Lesley A. McLaughlin, Colin J. Henderson, and C. Roland Wolf

Abstract

Vemurafenib is a revolutionary treatment for melanoma,but the magnitude of therapeutic response is highly variable,and the rapid acquisition of resistance is frequent. Here, weexamine how vemurafenib disposition, particularly throughcytochrome P450-mediated oxidation pathways, could poten-tially influence these outcomes using a panel of knockout andtransgenic humanized mouse models. We identified CYP3A4as the major enzyme involved in the metabolism of vemur-afenib in in vitro assays with human liver microsomes. How-ever, mice expressing human CYP3A4 did not process vemur-afenib to a greater extent than CYP3A4-null animals, suggest-ing that other pregnane X receptor (PXR)–regulated pathwaysmay contribute more significantly to vemurafenib metabolism

in vivo. Activation of PXR, but not of the closely relatedconstitutive androstane receptor, profoundly reduced circulat-ing levels of vemurafenib in humanized mice. This effectwas independent of CYP3A4 and was negated by cotreatmentwith the drug efflux transporter inhibitor elacridar. Finally,vemurafenib strongly induced PXR activity in vitro, but onlyweakly induced PXR in vivo. Taken together, our findingsdemonstrate that vemurafenib is unlikely to exhibit a clinicallysignificant interaction with CYP3A4, but that modulation ofbioavailability through PXR-mediated regulation of drug trans-porters (e.g., by other drugs) has the potential to markedlyinfluence systemic exposure and thereby therapeutic outcomes.Cancer Res; 75(21); 4573–81. �2015 AACR.

IntroductionThe BRAF gene is mutated in approximately 50% of all malig-

nant melanomas (1). In three quarters of these cases, mutationresults in a valine to glutamic acid substitution at codon 600(V600E), conferring an enhancement of kinase activity that pro-motes cellular proliferation and tumorigenesis (1–4). Vemurafe-nib (RG7204, PLX4032) was the first therapeutic agent clinicallyapproved for the treatment of BRAFV600E-positive malignantmelanoma. A highly specific inhibitor of the mutant kinase,vemurafenib elicits an objective tumor response in 48% of allpatients with BRAFV600E-positive metastatic melanoma whengiven as a single agent, with increases in both overall and pro-gression-free survival when compared with conventional chemo-therapy with dacarbazine (5–7).

Vemurafenib is both a revolutionary treatment for melanomaand, along with the companion diagnostic assay (8), a widely citedexample of the potential for therapeutic stratification based onmutational analysis. However, as with many recently developedclinically approved kinase inhibitors, drug resistance is a major

problem. The magnitude of initial response to vemurafenib ishighly variable (5–7). Although approximately half of patientsmeet the RECIST threshold for an objective response after 14weeksof therapy, the remainder occupy a spectrum from nonobjectiveresponse to disease progression, suggestive of varying degrees ofinnate resistance (7).Moreover, themedianduration of response isonly 6.7 months due to the acquisition of resistance (6). A largenumber of potential mechanisms of both innate and acquiredresistance have been identified, most of which invoke the plasticityof signaling networks within the tumor to circumvent blockade toBRAFV600E function (9, 10). Themajority of these studies have beenconducted in cultured cell lines and tumor biopsy samples. Therehas been comparatively little effort to investigate whether the highinterindividual variability in drug exposure at steady state, withcoefficients of variation of bothCmax and AUC of 30% to 40%, is adeterminant of therapeutic outcome and/or resistance, despite thetrends toward an exposure–response relationship in both overalland progression-free survival identified by population pharmaco-kinetic (PK) analyses (11–13), is a determinant of therapeuticoutcome and/or resistance, with the notable exception of a recentstudy in which low circulating vemurafenib concentrations wereassociated with disease progression (14).

During preclinical development, vemurafenib was found to bea substrate and possible inducer of CYP3A4, which was the majorenzyme involved in itsmetabolism in vitro (11, 12, 15).Moreover,CYP3A4-generated monohydroxy-vemurafenib (OH-vemurafe-nib) was detected in a mass balance analysis in patients at steadystate (11, 12, 15). In this latter study,OH-vemurafenib levels werelow compared with the parental compound but, as the absolutebioavailability of vemurafenib is unknown, it is impossible todraw any conclusions as to the relative contribution of metabo-lism in general, and CYP3A4 in particular, to elimination of the

Division of Cancer, School of Medicine, Jacqui Wood Cancer Centre,University of Dundee, Ninewells Hospital, Dundee, DD1 9SY, UnitedKingdom.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: C. Roland Wolf, University of Dundee, Ninewells Hos-pital andMedical School, DundeeDD19SY, UK. Phone: 44-13-8238-3134; Fax: 44-13-8238-6419; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-1454

�2015 American Association for Cancer Research.

CancerResearch

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drug. Therefore, if only a small percentage of vemurafenib reachesthe systemic circulation, metabolism may yet constitute a majorroute of elimination and an important determinant of drugexposure. In addition to these metabolic considerations, post-approval studies using knockout mouse lines have identifiedphase III drug efflux transporters as critical determinants ofvemurafenib bioavailability (16, 17).

CYP3A4showshigh inter- and intraindividual variation inexpres-sion, with differences in excess of 100-fold (18). The primary modeof CYP3A4 regulation is transcriptional, with activation of twonuclear hormone receptors in particular, pregnane X receptor (PXR)and constitutive androstane receptor (CAR), by exogenous ligands,endogenous ligands, or upstream signaling pathways known toincrease its levels (19). In the present study, we have assessed theroleof thesemetabolic factors inmediatingvemurafenibdispositionin both in vitro assays and in humanized mouse models in vivo. Wehave compared the relative contributions of metabolic and effluxpathways with vemurafenib PK, and have investigated the potentialof vemurafenib to influence its own disposition.

Materials and MethodsChemicals and reagents

Crystalline vemurafenib and PLX4720 were purchased fromSelleck chemicals. Microprecipitated bulk powder (MBP) vemur-afenibwas obtained through theGenentech Research Projects andReagents Program (Genentech). Klucel LF Pharm was obtainedfrom Ashland Inc. Elacridar hydrochloride was purchased fromSequoia Research Products. NADPHwas purchased fromMelfordlaboratories (Ipswich, UK). Protease inhibitors (cOmpleteULTRA, EDTA-free) were obtained from Roche. All other chemi-cals were purchased from Sigma-Aldrich.

Animal lines and husbandryThe generation and characterization of Cyp3aKO/Cyp3a13þ/þ,

huCYP3A4/3A7, huPXR/huCAR/huCYP3A4/3A7, and huPXR/huCAR mice have been described previously (20, 21). Briefly,the Cyp3aKO/Cyp3a13þ/þ line bears a deletion of seven of theeight murine Cyp3a genes and exhibits low and uninducibleoxidative metabolism of the CYP3A4 probe substrates triazolam,dibenzylfluorescein, and midazolam (20). The huCYP3A4/3A7line carries a large genomic insertion of human CYP3A4 andCYP3A7 in place of the Cyp3a locus (20). In this line, CYP3A4 ismaintained at a low basal level, but can be upregulated followingligand-activated nuclear translocation of the transcription factor,Pxr, most effectively with a synthetic glucocorticoid, 5-pregnen-olone-3b-ol-20-one-16a-carbonitrile (PCN; ref. 20). The huPXR/huCAR/huCYP3A4/3A7 line has been similarly humanized forCYP3A4/3A7, with additional humanizations for Pxr/PXR andCar/CAR (20). In this line, CYP3A4 can be induced by rifampicin(RIF), a ligand activator of human PXR (20). The huPXR/huCARline carries thehuman transcription factors, but retains themurineCyp3a cluster (21). All animals were maintained under standardanimal house conditions, with free access to food (RM1 diet,SpecialDiet Services) andwater, and a12-hour light/12-hour darkcycle. All animal work was carried in accordance with the AnimalScientific Procedures Act (1986) and after local ethical review.

Subcellular fractionationLivers were excised and snap-frozen in liquid nitrogen for

storage at �80�C until processing. Briefly, samples were thawed

by the addition of 3 volumes of KCl buffer (1.15%w/v potassiumchloride, 10 mmol/L potassium phosphate, pH 7.4) and homog-enized by rotor-stator. Debris was pelleted by centrifugation(11,000 � g at 4�C for 15 minutes) and the supernatant with-drawn for ultracentrifugation (100,000 � g at 4�C for 60 min-utes). After ultracentrifugation, the supernatant (cytosolic frac-tion) was retained and the pellet (microsomal fraction) wasresuspended in KCl buffer containing 0.25mol/L sucrose. Proteincontent of microsomal and cytosolic fractions was quantified byBradford assay (Bio-Rad).

In vitro studiesAll in vitro analyses were carried out with crystalline vemur-

afenib in 50 mmol/L HEPES pH 7.4 containing 30 mmol/LMgCl2. Incubations were initiated ay the addition of NADPH toa final concentration of 1mmol/L and terminated by the additionof 1x volume of ice cold acetonitrile. Microsomal stability assayswere performed in triplicate with 1 mmol/L compound and 0.2mg/mL pooled human liver microsomes (HLM, 150 donors; LifeTechnologies) with or without NADPH for a period of up to 50minutes. HLM andmouse liver microsome (MLM, pooled from 3wild-type mice) incubations for formation of the OH-vemurafe-nib metabolite contained 0.4 mg/mLmicrosomal protein and 60mmol/L vemurafenib. Incubations with HLM from individualdonors for Spearman's rank correlation were performed with50 mmol/L vemurafenib and 0.1 mg/mL protein for 6 minutes.Initial incubations with recombinant P450s were carried out with60 mmol/L compound and 400 pmol/mL enzyme for 15minutes.Assays to determine the apparent kinetic parameters of OH-vemurafenib formation in HLM and MLM were performed intriplicate under conditions of linearity for time and protein: 10minutes at 0.2 mg/mL for HLM, 15 minutes at 0.4 mg/mL forMLM, 6minutes at 160 pmol/mL for recombinant CYP3A4, and 9minutes at 160 pmol/mL for CYP2C9. For inhibition studies,pooled HLM were incubated at 0.2 mg/mL with 60 mmol/Lvemurafenib for 10 minutes in the presence of 1 mmol/L ketoco-nazole, 2 mmol/L sulfaphenazole, or both together. Inhibition of7-benzyloxy quinolone (BQ) turnover by vemurafenib was mea-sured using a BQ concentration of 30 mmol/L and 0.2 mg/mLHLM with increasing concentrations of vemurafenib, and thefluorescent product measured using a Fluoroskan Ascent FLfluorimeter (Thermo Fisher Scientific).

PXR-transactivation assayCells stably transfected with the PXR gene and a reporter

construct with luciferase under the control of PXR-responsiveelements of the CYP3A4 promoter (DPX2 cells; Puracyp Inc.)were treated with a range of concentrations of vemurafenib (0.06,0.17, 0.51, 1.54, 4.62, 13.89, 41.67, and 125 mmol/L) in triplicatefor 48 hours and assayed for luminescence andfluorescence as perthe manufacturer's instructions. The top two concentrations ofvemurafenib were highly toxic so data from these points wereexcluded from the analysis. PXR activation (fold change) wascalculated by dividing luminescence by fluorescence and normal-izing to vehicle control.

In vivo studiesAll animal work was carried out on 8- to 12-week-old male

mice (except for those shown in Fig. 7, where female mice wereused). PCN was suspended in corn oil at 1 mg/mL for adminis-tration by i.p. injection at 10 mg/kg (10 mL/g body weight).

MacLeod et al.

Cancer Res; 75(21) November 1, 2015 Cancer Research4574




RIFwas administered in the sameway at 60mg/kg. Phenobarbital(PB) was dissolved in PBS for i.p. administration at 80mg/kg. ForPK analyses, crystalline vemurafenib was first dissolved in DMSOand then further diluted in a mixture of Polysorbate 80, ethanol,and water (20:13:67) for immediate administration by oralgavage at a final dose of 50 mg/kg, as described by Durmus andcolleagues (16). The samemethodwas used for administration ofelacridar at 100mg/kg (16). For sample collection, 10mL ofwholeblood was withdrawn from the tail vein at the indicated timepoints. Samples were immediately added to a tube containingheparin solution (10 mL and 15 IU/mL) and stored at�20�Cuntilprocessing. For induction studies, MBP vemurafenib was sus-pended in water containing 2%Klucel LF Pharm, which had beenadjusted to pH 4.0 by the addition of hydrochloric acid, andadministered twice daily (8 hours apart) by oral gavage, asdescribed by Yang and colleagues (22).

Sample processing for LC-MS/MSWater (100 mL) containing 100 mg/mL internal standard

(PLX4720) was added to in vitro and in vivo samples, followedby 500 mL of diethyl ether (Thermo Fisher Scientific). Thismixturewas incubated at room temperature for 30 minutes with gentlemixing. Samples were centrifuged for 10 minutes at 16,000 � gand the organic solvent phasewithdrawn to a fresh tube for dryingunder a vacuum (20 minutes at 30�C). The dried extract wasredissolved in 100 mL of acetonitrile (Sigma) at 37�C with agita-tion for 5 minutes and samples were added to 96-well plates forLC-MS/MS.

LC-MS/MSAnalysis of in vitro incubation and in vivo blood PK samples was

carried out on a Waters Acquity UPLC and Micromass QuattroPremier mass spectrometer (both Micromass). LC separation wasperformed on a Kinetex 1.7m C19 100A; 50 � 2.1 mm column(Phenomenex) at a temperature of 45�Cwith an injection volumeof 5 mL and flow rate of 0.6 mL/min. Mobile phases were watercontaining 0.1% (v/v) formic acid (A) and acetonitrile containing0.1% (v/v) formic acid (B). Isocratic elution was carried out at35%/65%A/B for 1minute. Multiple reactionmonitoring data inESI-positive mode were acquired for vemurafenib [490.10 >255.04, cone voltage (CV) ¼ 65 V, collision energy (CE) ¼ 40kV), OH-vemurafenib (505.99 > 255.24, CV¼ 65 V, CE¼ 40 kV]andPLX4720 (414.20 >306.97, CV¼ 60V, CE¼ 37 kV). Acquireddata were analyzed in Quanlynx relative to analyte standardcurves spanning the range of concentrations under study.

Data analysisIn vitro data exhibited non-Michaelis–Menten kinetics, and

therefore was fitted with the Hill equation using GraFit version5 (Erithacus Software). Standard deviations given are from the fitof the calculated curve. PK parameters of in vivo data werecalculated with a simple non-compartmental model using Win-NonLin software, v4.1 (Pharsight) and are shown with SDs.P values were calculated using an unpaired t test.

Western blottingMicrosomal and cytosolic sampleswere adjusted to 1mg/mL in

LDS sample buffer (Life Technologies) for electrophoresisthrough 10% acrylamide gels for 1 hour at 150 V, followed bytransfer onto nitrocellulose membranes. Primary antibodies used

for immunoblotting included anti-CYP3A4 (458234; BD Bios-ciences), Cyp1a, Cyp2b, Cyp2c, Cyp3a/CYP3A, and Por (23, 24).Anti-GRP78 (ab21685; Abcam) and anti-GAPDH (G9545;Sigma) were used as loading controls for microsomal and cyto-solic fractions, respectively. In blots for Cyp3a/CYP3A andCYP3A4, pooled HLM material was the same as used in micro-somal incubations, whereas individuals with low, medium, andhigh expression were taken from the panel of 15 characterizeddonors based on testosterone 6b-hydroxylation activity.

mRNA analysisTotal RNA was isolated from snap-frozen small intestinal sam-

ples using TRizol (Life Technologies). The purity of this RNA wasincreased by processing with the RNeasy Mini Kit (Qiagen) fol-lowed by treatment with RG1 DNase (Promega). Conversion tocDNA was carried out using the ImProm-II Reverse TranscriptionSystem with random primers (Promega). The relative levels ofmRNA species were determined by TaqMan RT-PCR (Life Tech-nologies) using the following assays:Abcb1a (Mm00440761_m1),Abcb1b (Mm00440736_m1), Abcg2 (Mm00496364_m1), andCYP3A4 (Hs00604506_m1). Fold changes were calculated by thecomparative Ct method with 18S rRNA as an endogenous control(4319413E; Life Technologies).

ResultsVemurafenib is metabolized by CYP3A4 in vitro

Metabolic stability of vemurafenibwas assessed inpooledHLM(Fig. 1A). At 1 mmol/L, the compound was highly stable in boththe presence and absence of NADPH; however, formation of OH-vemurafenib was detectable in the presence of NADPH (Fig. 1B).Apparent kinetic parameters of OH-vemurafenib formation byHLM and MLM were found to be sigmoidal, and therefore werefitted using the Hill equation. The Vmax for HLM was approxi-mately 10-fold higher than that observed inMLM(4879� 108 vs.467 � 54 peak area/min/mg, respectively) whereas the S50 wassimilar (23.2 � 0.8 vs. 23.8 � 4.9 mmol/L, respectively, Fig. 1C).Hill coefficients are detailed in Supplementary Table S1, alongsideparameters calculated using the Michaelis–Menten model. In apanel of HLM from 15 donors that had been characterized by thevendor with regard to their activity with probe substrates forindividual CYP isoforms, formation of OH-vemurafenib corre-lated most strongly with that of 6b-hydroxy-testosterone, themarker for CYP3A4 (Supplementary Table S2 and Fig. 1D). Therewas also a strong correlationwithCYP2A6activity (SupplementaryTable S2), but this was deemed artefactual asCYP3A4andCYP2A6probe activities within the HLM panel correlate (data not shown),anobservation thathaspreviouslybeenmade ina separatebatchofsamples (23). Indeed, in incubations with recombinant CYPcontaining high concentrations of both protein and substrate,OH-vemurafenib was formed by CYP3A4, CYP2C9 and, to a lesserextent, by CYP2C8 andCYP1A1, but not by CYP2A6 (Fig. 1E). Thein vitro apparent kinetics of OH-vemurafenib formation byCYP3A4 andCYP2C9were, similarly toHLMandMLM, sigmoidalin nature. CYP3A4 had a 2-fold higher affinity for vemurafenib(S50: 10.0� 0.9 vs. 20.4� 1.7 mmol/L) and a 3.4-fold higher Vmax(11693 � 471 vs. 3,481 � 216 peak area/min/nmol) thanCYP2C9. As with the hepaticmicrosomal preparations, Hill coeffi-cients and Michaelis–Menten analyses for recombinant P450s aredetailed in Supplementary Table S1. In pooledHLM, coincubationwith the CYP3A4 inhibitor, ketoconazole, significantly inhibited

Vemurafenib Pharmacokinetics in Humanized Mice

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OH-vemurafenib formation (Fig. 1G). The CYP2C9 inhibitor,sulfaphenazole, effected a partial decrease in OH-vemurafenibformation. As expected, coincubation with both inhibitorscompletely ablated OH-vemurafenib formation. Also in pooledHLM,vemurafenib inhibited theactivity of theCYP3A4probedrugsubstrate BQ with an IC50 value of 14.9 mmol/L (Fig. 1H).

Single-dose vemurafenib is not extensively metabolized byCYP3A4 in vivo

To study the contribution of CYP3A4 tometabolism of vemur-afenib in vivo, Cyp3aKO/Cyp3a13þ/þ and huCYP3A4/3A7 micewere administered either PCN or vehicle (CO) alone for 3 daysbefore administration of vemurafenib, as described in Materials

and Methods. Twenty-four hours after the third dose of PCN,CYP3A4 is expressed in the liver of huCYP3A4/3A7 at a levelsimilar to that observed in pooled HLM (data not shown). Asstated above, Cyp3aKO/Cyp3a13þ/þ mice are essentially null forCyp3a/CYP3A activity, both basal and in response to activation ofPxr. Therefore, any difference in the PK of vemurafenib betweenhuCYP3A4/3A7 PCN and Cyp3aKO/Cyp3a13þ/þ PCN is attrib-utable to the activity of CYP3A4. Although not statistically sig-nificant, there was a decrease in both Cmax and AUCall, and anincrease in CL, in the huCYP3A4/3A7 PCN group, relative to theCyp3aKO/Cyp3a13þ/þPCNgroup (Fig. 2A; Supplementary TableS3). More pronounced, however, were these same changes in thePCN groups of both genotypes, relative to their respective vehiclecontrol groups. The fact that PCN treatment enhanced clearanceequally well in Cyp3a null and huCYP3A4/3A7 animals suggeststhat PXR-regulated pathways other than CYP3A4 predominate inthe clearance of vemurafenib in vivo. Immediately after the finalblood sample collection, livers were harvested and Western blot-ting carried out to confirm that CYP3A4 had been effectivelyinduced by PCN in the huCYP3A4/3A7mice (Fig. 2B). Because ofthis harvest occurring 48 hours after the last dose of PCN, CYP3A4expression was lower than in pooled HLM due to turnover of theenzyme.

Exposure to vemurafenib is decreased following activation ofPxr/PXR, but unaffected by activation of CAR

As PCN pretreatment decreased the systemic concentration ofvemurafenib independently of CYP3A4 (Fig. 2A; SupplementaryTable S3), we further investigated the role of nuclear hormonereceptor activity inmediating disposition to vemurafenib.HuPXR/huCAR/huCYP3A4/3A7 mice were pretreated with RIF, a potentactivator of human PXR, and thus inducer of CYP3A4, leading to amarked reduction in the AUCall of vemurafenib from432� 169 to121� 18 h�mg/mL (P¼ 0.0106, Fig. 3A; Supplementary Table S4).

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Figure 1.Vemurafenib is metabolized by CYP3A4 and CYP2C9 in vitro. A, microsomalstability of vemurafenib with HLM in the absence (open circles) and presence(filled circles) of NADPH. B, formation of OH-vemurafenib from vemurafenibwhen incubated with HLM in the absence (open circles) and presence(filled circles) of NADPH. C, vemurafenib kinetics with HLM (open circles) andMLM (filled circles)fittedwith the Hill equation. D, correlation of conversion of6b-OH-testosterone formation in characterized liver microsomes from 15individuals with formation of OH-vemurafenib from vemurafenib. E,generation of OH-vemurafenib by recombinant CYP. F, vemurafenib kineticswith recombinant CYP2C9 (filled circles) and CYP3A4 (open circles) fittedwith the Hill equation. G, inhibition of OH-vemurafenib formation in pooledHLM by ketoconazole and sulfaphenazole. H, inhibition of BQ activity inpooled HLM by vemurafenib (IC50¼ 14.9 mmol/L). In all cases, data comprisemean � SD of triplicate incubations.

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MacLeod et al.





Western blotting confirmed induction of CYP3A4 in the liver and,due to the high initial magnitude of this induction, CYP3A4 levelsremained several fold higher than pooled HLM (and equivalent toHLM expressing high CYP3A4) despite 48 hours having elapsedafter administration of the final dose of RIF (Fig. 3B). In addition,weobserveda4.7-fold increase inmRNAforAbcb1a, but no changefor Abcb1b or Abcg2, in the small intestine of huPXR/huCAR/huCYP3A4/3A7mice treated with RIF (Fig. 3C). Activation of PXRin the huPXR/huCARmouse line led to a similar decrease inAUCallof vemurafenib from 349 � 24 to 75 � 33 h�mg/mL (P ¼ 0.0003,Fig. 3A; Supplementary Table S4).

To determine whether CAR might play a role in modulatingvemurafenib disposition, huPXR/huCAR/huCYP3A4/3A7 micewere dosed for 3 days with PB, a CAR activator (25), beforevemurafenib administration. This had no effect on any of thekinetic parameters assessed (Fig. 4A; Supplementary Table S5).Western blotting of liver samples demonstrated induction of thewell-characterized CAR target protein, Cyp2b10, confirming theeffective activation of CAR by PB (Fig. 4B).

Activation of PXR reduces vemurafenib exposure throughinduction of efflux transporters

Activation of PXR by RIF in huPXR/huCAR/huCYP3A4/3A7mice decreased the AUCall of vemurafenib (Figs. 3A, 4A,

and 5A; Supplementary Tables S3–S5). This decrease wasalmost completely ablated by pretreatment of mice, 90 minutesbefore administration of vemurafenib, with elacridar, an effec-tive inhibitor of Abcb1 and Abcg2, but not CYP3A4, at the doseused (100 mg/kg orally, Fig. 5A; refs. 16, 26, 27). Relative tomice dosed with RIF alone, AUCall of vemurafenib increasedfrom 90 � 13 to 330 � 85 h�mg/mL (P ¼ 0.0085) in animalsdosed with RIF followed by elacridar, whereas Cmax increasedfrom 11.1 � 1.8 to 21.3 � 2.2 mg/mL (P ¼ 0.0032; Supple-mentary Table S6). Moreover, huPXR/huCAR/huCYP3A4/3A7mice, which had been predosed with vehicle (CO) alone, weresubject to a similar magnitude of increase in exposure tovemurafenib following elacridar pretreatment, with AUCallincreasing from 391 � 112 to 684 � 85 hr�mg/mL (P ¼0.0224) and Cmax increasing from 29.1 � 4.3 to 37.8 � 3.5mg/mL (P ¼ 0.0507, Fig. 5A). Again, effective activation of PXRby RIF was confirmed by Western blot analysis of CYP3A4 inliver samples (Fig. 5B).

Vemurafenib is a strong inducer of PXR in vitroWe tested the capacity of crystalline vemurafenib to activate a

PXR-driven luciferase reporter construct in the DPX2 cell line(Puracyp Inc.). The reported aqueous solubility of this form is inthe range of 0.01 to 10 mg/mL (approximately 20 to 20 mmol/L) atphysiologic pH (12). An EC50 value of 8.13mmol/L and an Emax of8.54-fold (Fig. 6) was determined. The positive control, rifampi-cin, gave an EC50 value of 1.01 mmol/L and an Emax of 14.10-fold(data not shown).

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Figure 3.Activation of PXR reduces exposure to vemurafenib independently of CYP3A4activity. A, PK profiles of vemurafenib in huPXR/huCAR/huCYP3A4/3A7vehicle (open circles; n ¼ 4), huPXR/huCAR/huCYP3A4/3A7 RIF (closedcircles;n¼4), huPXR/huCARvehicle (opensquares;n¼ 3), andhuPXR/huCARRIF (closed squares; n¼ 3)mice. Mean data� SEMare shown. B,Western blotanalysis of hepatic microsomal fractions from huPXR/huCAR/huCYP3A4/3A7mice dosed with either RIF or CO vehicle alone, alongside HLM preparations;pooled and low, medium, and high CYP3A4 activity. C, TaqMan RT-PCRanalysis ofAbcb1a,Abcb1b, andAbcg2mRNA levels inwhole small intestine ofhuPXR/huCAR/huCYP3A4/3A7 mice dosed with vehicle or RIF.

A

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] (μg

/mL)

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Time (h)0 10 20 30

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70VehiclePBRIF

Figure 4.Activation of CAR does not alter vemurafenib PK. A, PK profiles ofvemurafenib in huPXR/huCAR/huCYP3A4/3A7 vehicle (open circles; n ¼ 3),PB (closed circles; n¼ 3), and RIF (closed squares; n ¼ 3). Mean data � SEMare shown. B, Western blots of hepatic microsomal fractions from huPXR/huCAR/huCYP3A4/3A7 mice dosed with either vehicle (PBS), PB, or RIFalongside HLM preparations; pooled and low, medium, and high CYP3A4activity.






Vemurafenib is a weak inducer of PXR in vivoWe administered the high bioavailability MBP form of

vemurafenib at a dose of 100 mg/kg twice daily for 4 days to

huPXR/huCAR/huCYP3A4/3A7mice, as described inMaterials andMethods. The PK parameters of a single 100-mg/kg dose (Supple-mentary Table S7) were in good agreement with those calculated ina previous study (22). Western blotting of liver proteins demon-strated weak induction of CYP3A4 and Cyp2b, and moderateinduction of Cyp1a (Fig. 7A). Analysis of small-intestinal mRNArevealed no change in expression in CYP3A4, Abcb1a, or Abcb1b(Fig. 7B).

DiscussionVemurafenib is a revolutionary treatment for advanced melano-

ma and, along with the companion diagnostic test for BRAFmuta-tion (8), is a successful exampleof cancer therapy stratificationbasedon molecular-level information. As with other oncogenic kinases,however, clinical response to inhibition of BRAFV600 is highlyvariable, and the acquisition of resistance in patients initiallyresponsive to therapy is usually rapid (5–7). A wide variety ofmechanisms of innate and acquired resistance have been identified,most of which are a function of the redundancy of the pro-prolif-erative signaling cascade being targeted (9, 10, 28). Data generatedduring the preclinical development of vemurafenib suggested sev-eral points of interaction with proteins involved in xenobioticmetabolism. These have the capacity to alter PK parameters, andhence the therapeutic effectiveness of this agent (11, 12).

In agreement with the preclinical data (11, 12), we found thatCYP3A4 was the major CYP isoform involved in vemurafenibmetabolism in vitro, but that the rate of metabolism was low. We

A

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RIFelacridar

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Time (h)0 10 20 30

0

10

20

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40 Vehicle, vehicleVehicle, elacridarRIF, vehicleRIF, elacridar

Figure 5.PXR regulates vemurafenib bioavailability through the modulation of effluxtransporters. A, PK profiles of vemurafenib in huPXR/huCAR/huCYP3A4/3A7mice treated with vehicle/vehicle (open circles; n ¼ 3), vehicle/elacridar(filled circles, dashed line; n ¼ 3), RIF/vehicle (open squares; n ¼ 3), andRIF/elacridar (filled squares, dashed line; n¼ 3). Mean data� SEM are shown.B, Western blots of hepatic microsomal fractions from huPXR/huCAR/huCYP3A4/3A7 mice dosed with either CO (vehicle) or RIF, then with eitherDMSO/Tween 80/ethanol/water (vehicle) or elacridar 90 minutes beforevemurafenib, alongside HLMpreparations; pooled and low,medium, and highCYP3A4 activity.

log [Vemurafenib] (μmol/L)

Fold

cha

nge

–2 –1 0 1 20

2

4

6

8

Figure 6.Vemurafenib activates PXR in vitro. DPX2 cells were used to calculate theEC50 value (8.13 mmol/L) and Emax (8.54-fold) for vemurafenib-mediatedtransactivation of PXR.

CYP3A4

Cyp1a

Cyp2b

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VemurafenibHLM

Pool Low Med HighA

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4Duo vehicleDuo vemurafenibJej vehicleJej vemurafenibIle vehicleIle vemurafenib

CYP3A4 Mdr1a Mdr1b

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cha

nge

Figure 7.Vemurafenib activates PXR and possibly other transcription factors in vivo.A, Western blots of hepatic microsomal fractions from huPXR/huCAR/huCYP3A4/3A7mice dosedwith either vehicle (4% Klucel LF, pH 4.0, n¼ 4) orMBP vemurafenib (n ¼ 4) twice daily at 100 mg/kg (8 hours apart) for 4 days.B, TaqManRT-PCRofCYP3A4,Abcb1aandAbcb1b induodenum(Duo), jejunum(Jej), and ileum (Ile) of huPXR/huCAR/huCYP3A4/3A7 mice dosed with eithervehicle or vemurafenib. Data shown are mean of triplicate analyses � SD.

MacLeod et al.





also found that vemurafenib has the capacity to inhibit CYP3A4,with an IC50 value for BQ activity of 14.9mmol/L. In addition, andin conflict with themanufacturer's findings (11, 12), we identifieda minor role of CYP2C9 in generating OH-vemurafenib,evidenced by a small but statistically significant decrease inOH-vemurafenib in pooled HLM coincubated with the 2C9-specific inhibitor, sulfaphenazole.

In a previous study,mass balance analysis in 7patients at steadystate found that, in serum sampleswithdrawn up to 48 hours afteradministration of a radiolabeled dose of vemurafenib, 95% ofrecovered radioactivity was in the parental form (11, 12, 15).There was an increase in (the CYP3A4-generated form of) OH-vemurafenib from 0.5% to approximately 4% over this timeperiod. It took 18 days for 95% of the radioactive dose to berecovered; 94% in feces, 1% in urine (15).Metabolic profilingwascarried out on the 68.5% of the total dose that was recoveredwithin the first 96 hours. Over the 0- to 48-hour period,�94% ofthe radioactivity recovered was in the parental form. Over the 48-to 96-hour period, however, this value decreased to 55.5% �20.1%.OH-vemurafenib accounted for 13.7%of the radioactivityrecovered from the 48- to 96-hour samples (3.4% of the totaldose). Conjugated (glucosylated and glucuronidated) forms ofvemurafenib were the only other metabolites definitively identi-fied in these samples (11, 12, 15). Although the percentage ofvemurafenib excreted in the form of metabolites was low relativeto the parent compound, it was acknowledged that a largeproportion of the parent compound eliminated during the first48 hoursmight constitute drug that had not been absorbed, and asignificant proportion of the vemurafenib excreted during the 48-to 96-hour post-dose period might have been generated byhepatobiliary recirculation. Indeed, it was also reported thatmetabolites predominated over the parent compound in a bilesample from one of the patients receiving vemurafenib (15). Asthe absolute bioavailibility of vemurafenib is unknown, andhence the contribution of metabolism to its elimination cannotbe calculated, phase IV clinical trials assessing the effect of aninhibitor and an inducer of CYP3A4 in the PK of vemurafenibhave been requested by both the FDA and EMA. At present, aninhibitor (ketoconazole) study (NCT01765556) has been with-drawn and an inducer (rifampicin) study (NCT01765543) isrecruiting (clinicaltrials.gov). Despite the occurrence of CYP-dependent hydroxylation of vemurafenib in vitro, we found thatthe in vivo effect of CYP3A4 in modulating the systemic levels of asingle dose of the compound in humanized mice was small. Farmore apparent was a decrease in AUCall following activation ofPxr, which was independent of CYP3A4 as it occurred in both theCyp3aKO/Cyp3a13þ/þ and huCYP3A4/3A7 lines. This decreasewas even more pronounced following activation of PXR in thehuPXR/huCAR and huPXR/huCAR/huCYP3A4/3A7 mouse lineswith RIF. The likely explanation for the differing magnitudes ofthis decrease is that, at the doses used, PCN-mediated activationofmPxr (and thereby induction of the downstream effector pro-teins) is less effective than RIF-mediated activation of hPXR (20).

Preincubation of vemurafenib-resistant clones of theBRAFV600E-positive A375 melanoma cell line with verapamil, anABCB1 inhibitor, partially increased their sensitivity to the drug(29). Studies with the MDCK-ABCB1 cell line demonstrated thatvemurafenib was both an ABCB1 substrate and inhibitor (12),and a post-authorization report mentions evidence of an inter-actionwithABCG2 (30). These observations havebeen confirmedindependently, and studies in mice nulled for these transporters

either separately or in combination have found that they mod-ulate both intestinal bioavailability and permeability at theblood–brain barrier (16, 17, 31). This synergistic interaction ofAbcb1 and Abcg2 is a phenomenon that has been shown tomodulate the bioavailability of other therapeutics (32). ABCB1is a transcriptional target of PXR in human cells (33, 34), and thisinteraction is thought to be conserved for Abcb1a in the mouse(35, 36). There is some evidence that Pxr also regulates Abcg2(37). Our measurements of Abcb1a, Abcb1b, and Abcg2 mRNAlevels in the small intestine of huPXR/huCAR/huCYP3A4/3A7mice demonstrate that, of these, only the first was inducedfollowing RIF treatment. Activation of CAR by PB inducedCyp2b10 to the same extent as activation of PXR by RIF, whilealsoweakly inducing CYP3A4, but this pretreatment had no effecton the PK parameters of vemurafenib. Taken together, theseobservations suggest that the Pxr/PXR-dependent modulation ofvemurafenib PK is likely to be due to the upregulation of Abcb1a,and hence a decrease in the bioavailability of vemurafenib, ratherthan to induction of drug metabolism enzymes. Although PXRregulates the transcription of other drug-metabolizing enzymes inhumanized mice, such as UDP-glucuronsyltransferases (UGT) ofthe 1a subfamily (38), our contention that the observed effects aredue to the modulation of transport are supported by the findingthat administration of the Abcb1/Abcg2 inhibitor, elacridar, 90minutes before administration of vemurafenib completely ablat-ed the effect of RIF pretreatment in huPXR/huCAR/huCYP3A4/3A7mice. Furthermore, elacridar pretreatment of uninduced (i.e.,not pretreated with RIF) huPXR/huCAR/huCYP3A4/3A7 miceincreased AUCall of vemurafenib by a degree comparable withpretreatment of inducedmice, suggesting that inhibition of effluxtransporters in the basal state can profoundly increase vemura-fenib bioavailability. It should be noted that, although elacridar isoften cited as a specific inhibitor of ABCB1/Abcb1 and ABCG2/Abcg2, this is not the case. At the dose used in this study, thepredicted plasma concentration of elacridar is an order of mag-nitude below the IC50 value for CYP3A4 andother P450s (26, 27).However, this compound is known to inhibit SLCO1B1 (39) andis highly likely to inhibit other transporters (40). Therefore,although we cannot conclusively state that PXR-directed modu-lation of Abcb1a level in the small intestine is the pivotal deter-minant of AUCall of vemurafenib, this remains the most likelyexplanation. In support of this, studies in the Abcb1a/b and Bcrpknockout mouse lines suggested a predominant role for theformer transporter (16). Increased drug efflux mediated by ABCtransporter proteins in the cancer cell is amechanismofmultidrugresistance that has been understood for nearly 40 years (41). As aclass of therapeutics, tyrosine kinase inhibitors, such as vemur-afenib, are no less vulnerable to this process than conventionalchemotherapeutics (42). The demonstration that drug transpor-ters can markedly affect in vivo vemurafenib PK implies thatchanges in their expression in the tumor itself will also be a factorin drug response. Surprisingly, to date, this possibility has notbeen investigated.

During preclinical development, it was posited that decreasesin the steady-state blood plasma concentrations of vemurafenibin dogs on a twice-daily dosing regimen between 29 and 92 daysmight be due to the induction of metabolism (11). Furthermore,in patients at steady state, vemurafenib decreased AUC0-last of theCYP3A4-specific probe, midazolam, by 39%, increased AUC0-lastof the CYP1A2 probe, caffeine, by 160%, and increased AUC0-lastof the CYP2D6 probe, dextromethorphan, by 47% (11, 12). This






defined vemurafenib as an inducer of CYP3A4, a moderateinhibitor of CYP1A2 and a weak inhibitor of CYP2D6. In agree-ment with these findings, we observed strong induction ofCYP3A4-promoter–driven luciferase activity in a cell line–basedreporter assay for PXR with crystalline vemurafenib. During thephase I clinical trial, vemurafenib was reformulated from acrystalline to an MBP form to improve bioavailability (5). Withthis formulation, AUC0–24 is proportional to doses of between240 and 960 mg twice daily (2, 5, 43). In the present study, weadministered MBP vemurafenib to huPXR/huCAR/huCYP3A4/3A7 mice to assess the drug–drug interaction potential of thiscompound in this mouse model in vivo. Moreover, the AUCall of1,142� 242 h�mmol/L observed at this dose is approximately fourtimes the AUC0–24 of 300 h�mmol/L cited as being necessary toeffect regression of COLO205 xenografts (44) and, with twice-daily dosing, was considered to be sufficiently representative ofthe steady state level of exposure in humans (AUC0–24¼ 1,741�639 h�mmol/L; ref. 5). In addition, the Cmax of this single dose, at121.7� 19.7 mmol/L, is above the mean value of 86� 32 mmol/Lreported in patients at steady state (5). With these parameters, insilico analysis predicted that steady-state concentrations in micewould be achieved within approximately 32 hours (i.e., with thefourth dose) on a schedule of two doses per day, 8 hours apart(data not shown). Under these conditions we observed inductionof CYP3A4 in the liver of huPXR/huCAR/huCYP3A4/3A7 miceadministered a high dose of theMBP formulation twice daily for 4days, although the level of this induction was slight and highlyvariable. Nevertheless, our in vitro and in vivo data demonstratethat vemurafenib has the capacity to induce CYP3A4. In addition,we observed induction of Cyp1a and Cyp2b proteins in huPXR/huCAR/huCYP3A4/3A7mice. These effectsmay, in part, be due toactivation of PXR, but are likely also due to the activation of one ormore additional transcription factors, such as the aryl hydrocar-bon receptor (45). In addition to possible overall effects on drugPK, the capacity of vemurafenib to activate transcription factorsinvolved in drug disposition raises the possibility that it mayinduce its own metabolism and efflux in tumor cells. This couldpotentially be a significant factor in the acquisition of drugresistance. Similar effectsmight also be invoked by comedicationsthat activate these transcription factors.

In conclusion, we have found that vemurafenib interacts withthe P450 system in vitro, but that PXR-directed regulation of efflux

transporters and the consequent modulation of bioavailabilityare likely to be a more important factor in defining disposition invivo. In addition, we have demonstrated that vemurafenib has thecapacity to activate PXR, and potentially other transcriptionfactors, at clinically relevant concentrations. Taken together, ourobservations support the case for therapeutic monitoring ofvemurafenib levels in patients over the course of therapy toevaluate whether low systemic drug concentrations coincide withinnate or acquired resistance, and to determine whether mod-ulators of PXR/CYP3A4/ABCB1 activity influence the outcome ofvemurafenib therapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: A.K. MacLeod, L.A. McLaughlin, C.J. Henderson,C.R. WolfDevelopment of methodology: A.K. MacLeod, L.A. McLaughlin, C.R. WolfAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.K. MacLeod, L.A. McLaughlinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.K. MacLeod, L.A. McLaughlin, C.R. WolfWriting, review, and/or revision of the manuscript: A.K. MacLeod,L.A. McLaughlin, C.J. Henderson, C.R. WolfAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L.A. McLaughlin, C.R. WolfStudy supervision: C.J. Henderson, C.R. Wolf

AcknowledgmentsThe authors thank Julia Carr, Catherine Meakin, and Susanne van Schelven

for assistance with animal work, and Cheryl Wood, Mairi Lennie, and RumenKostov for additional technical support. The authors thank Dr. Yury Kapelyukhfor critical reading of the article. The authors also acknowledge TaconicBiosciences for the supply of the mouse lines used in this study.

Grant SupportThis work was supported by Cancer Research UK program grant C4639/

A12330.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 28, 2015; revised July 20, 2015; accepted August 4, 2015;published OnlineFirst September 11, 2015.

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2015;75:4573-4581. Published OnlineFirst September 11, 2015.Cancer Res A. Kenneth MacLeod, Lesley A. McLaughlin, Colin J. Henderson, et al. Vemurafenib Availability in Humanized Mouse ModelsActivation Status of the Pregnane X Receptor Influences

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http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-15-1454http://cancerres.aacrjournals.org/content/suppl/2015/12/12/0008-5472.CAN-15-1454.DC1http://cancerres.aacrjournals.org/content/75/21/4573.full#ref-list-1http://cancerres.aacrjournals.org/content/75/21/4573.full#related-urlshttp://cancerres.aacrjournals.org/cgi/alertsmailto:[email protected]://cancerres.aacrjournals.org/content/75/21/4573http://cancerres.aacrjournals.org/

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