activation status of the pregnane x receptor (pxr ......2015/09/11 · humanized for cyp3a4/3a7,...

Vemurafenib Pharmacokinetics in Humanized Mice

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Title:

Activation status of the Pregnane X Receptor (PXR) influences Vemurafenib availability in humanized mouse models

Authors:

A. Kenneth MacLeod, Lesley A. McLaughlin, Colin J. Henderson and C. Roland Wolf*.

Affiliations:

Medical Research Institute, University of Dundee, Dundee DD1 9SY, United Kingdom.

Running title:


Keywords:

Vemurafenib, melanoma, humanized mice, pharmacology, pregnane X receptor

Financial support:

This work was supported by Cancer Research UK programme grant C4639/A12330.

on June 6, 2021. © 2015 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 11, 2015; DOI: 10.1158/0008-5472.CAN-15-1454

http://cancerres.aacrjournals.org/


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* Corresponding author:

C. Roland Wolf,

Medical Research Institute,

University of Dundee,

Dundee DD1 9SY,

United Kingdom.

Tel +441382383134

Email: [email protected]

Conflict of interest statement:

The authors disclose no potential conflicts of interest.





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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, we examined how

vemurafenib disposition, particularly through cytochrome P450-mediated oxidation pathways, could

potentially influence these outcomes using a panel of knockout and transgenic humanized mouse

models. We identified CYP3A4 as the major enzyme involved in the metabolism of vemurafenib in in

vitro assays with human liver microsomes. However, mice expressing human CYP3A4 did not process

vemurafenib to a greater extent than CYP3A4 null animals, suggesting that other pregnane X

receptor (PXR)-regulated pathways may contribute more significantly to vemurafenib metabolism in

vivo. Activation of PXR, but not of the closely related constitutive androstane receptor (CAR),

profoundly reduced circulating levels of vemurafenib in humanized mice. This effect was

independent of CYP3A4 and was negated by co-treatment with the drug efflux transporter inhibitor,

elacridar. Finally, vemurafenib strongly induced PXR activity in vitro, but only weakly induced PXR in

vivo. Taken together, our findings demonstrate that vemurafenib is unlikely to exhibit a clinically

significant interaction with CYP3A4, but that modulation of bioavailability through PXR-mediated

regulation of drug transporters (for example by other drugs) has the potential to markedly influence

systemic exposure and thereby therapeutic outcomes. Activation status of the Pregnane X Receptor

(PXR) influences Vemurafenib availability in humanized mouse models.





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Introduction

The BRAF gene is mutated in approximately 50% of all malignant melanomas (1). In three quarters

of these cases, mutation results in a valine to glutamic acid substitution at codon 600 (V600E),

conferring an enhancement of kinase activity that promotes cellular proliferation and tumorigenesis

(1-4). Vemurafenib (RG7204, PLX4032) was the first therapeutic agent clinically approved for the

treatment of BRAFV600E-positive malignant melanoma. A highly specific inhibitor of the mutant

kinase, vemurafenib elicits an objective tumour response in 48% of all patients with BRAFV600E-

positive metastatic melanoma when given as a single agent, with increases in both overall and

progression-free survival when compared to conventional chemotherapy with dacarbazine (5-7).

Vemurafenib is both a revolutionary treatment for melanoma and, along with the companion

diagnostic assay (8), a widely-cited example of the potential for therapeutic stratification based on

mutational analysis. However, as with many recently developed clinically-approved kinase inhibitors,

drug resistance is a major problem. The magnitude of initial response to vemurafenib is highly

variable (5-7). While approximately half of patients meet the RECIST (Response Evaluation in Criteria

in Solid Tumours) threshold for an objective response after 14 weeks of therapy, the remainder

occupy a spectrum from non-objective response to disease progression, suggestive of varying

degrees of innate resistance (7). Moreover, the median duration of response is only 6.7 months due

to the acquisition of resistance (6). A large number of potential mechanisms of both innate and

acquired resistance have been identified, most of which invoke the plasticity of signalling networks

within the tumour to circumvent blockade to BRAFV600E function (9, 10). The majority of these studies

have been conducted in cultured cell lines and tumour biopsy samples. There has been

comparatively little effort to investigate whether the high inter-individual variability in drug

exposure at steady state, with coefficients of variation of both Cmax and AUC of 30 – 40%, is a

determinant of therapeutic outcome and/or resistance, despite the trends towards an exposure-

response relationship in both overall and progression-free survival identified by population





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pharmacokinetic (PK) analyses(11-13), is a determinant of therapeutic outcome and/or resistance,

with the notable exception of a recent study in which low circulating vemurafenib concentrations

were associated with disease progression (14).

During pre-clinical development, vemurafenib was found to be a substrate and possible inducer of

CYP3A4, which was the major enzyme involved in its metabolism in vitro (11, 12, 15). Moreover,

CYP3A4-generated monohydroxy-vemurafenib (OH-vemurafenib) was detected in a mass balance

analysis in patients at steady-state (11, 12, 15). In this latter study, OH-vemurafenib levels were low

compared to the parental compound but, as the absolute bioavailability of vemurafenib is unknown,

it is impossible to draw any conclusions as to the relative contribution of metabolism in general, and

CYP3A4 in particular, to elimination of the drug. Therefore if only a small percentage of vemurafenib

reaches the systemic circulation, metabolism may yet constitute a major route of elimination and an

important determinant of drug exposure. In addition to these metabolic considerations, post-

approval studies using knockout mouse lines have identified Phase III drug efflux transporters as

critical determinants of vemurafenib bioavailability (16, 17).

CYP3A4 shows high inter- and intra-individual variation in expression, with differences in excess of

100-fold (18). The primary mode of CYP3A4 regulation is transcriptional, with activation of two

nuclear hormone receptors in particular, PXR and CAR, by exogenous ligands, endogenous ligands, or

upstream signalling pathways known to increase its levels (19). In the present study, we have

assessed the role of these metabolic factors in mediating vemurafenib disposition in both in vitro

assays and in humanized mouse models in vivo. We have compared the relative contributions of

metabolic and efflux pathways to vemurafenib PK, and have investigated the potential of

vemurafenib to influence its own disposition.





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Materials and Methods

Chemicals and reagents

Crystalline vemurafenib and PLX4720 were purchased from Selleck chemicals (Houston, TX, USA).

Microprecipitated bulk powder (MBP) vemurafenib was obtained through the Genentech Research

Projects and Reagents Program (Genentech, San Francisco, CA, USA). Klucel LF Pharm was obtained

from Ashland Inc. (Tadworth, UK). Elacridar hydrochloride was purchased from Sequoia Research

Products (Pangbourne, UK). NADPH was purchased from Melford laboratories (Ipswich, UK).

Protease inhibitors (cOmplete ULTRA, EDTA-free) were obtained from Roche (Burgess Hill, UK). All

other chemicals were purchased from Sigma-Aldrich (Poole, UK).

Animal lines and husbandry

The generation and characterisation of Cyp3aKO/Cyp3a13+/+, huCYP3A4/3A7,

huPXR/huCAR/huCYP3A4/3A7 and huPXR/huCAR mice has been described previously (20, 21).

Briefly, the Cyp3aKO/Cyp3a13+/+ line bears a deletion of seven of the eight murine Cyp3a genes and

exhibits low and uninducible oxidative metabolism of the CYP3A4 probe substrates triazolam,

dibenzylfluorescein and midazolam (20). The huCYP3A4/3A7 line carries a large genomic insertion of

human CYP3A4 and CYP3A7 in place of the Cyp3a locus (20). In this line, CYP3A4 is maintained at a

low basal level but can be upregulated following ligand-activated nuclear translocation of the

transcription factor, Pxr, most effectively with a synthetic glucocorticoid, 5-pregnenolone-3β-ol-20-

one-16α-carbonitrile (PCN) (20). The huPXR/huCAR/huCYP3A4/3A7 line has been similarly

humanized for CYP3A4/3A7, with additional humanisations for Pxr/PXR and Car/CAR (20). In this

line, CYP3A4 can be induced by rifampicin (RIF), a ligand activator of human PXR (20). The

huPXR/huCAR line carries the human transcription factors but retains the murine Cyp3a cluster (21).

All animals were maintained under standard animal house conditions, with free access to food (RM1

diet, Special Diet Services, Essex, UK) and water, and a 12-h light/12-h dark cycle. All animal work





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was carried in accordance with the Animal Scientific Procedures Act (1986) and after local ethical

review.

Subcellular fractionation

Livers 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 potassium chloride,

10mM potassium phosphate, pH 7.4) and homogenised by rotor-stator. Debris was pelleted by

centrifugation (11 000 x g at 4°C for 15 minutes) and the supernatant withdrawn for

ultracentrifugation (100 000 x g at 4°C for 60 minutes). After ultracentrifugation, the supernatant

(cytosolic fraction) was retained and the pellet (microsomal fraction) was resuspended in KCl buffer

containing 0.25 M sucrose. Protein content of microsomal and cytosolic fractions was quantified by

Bradford assay (Bio-Rad, Hemel Hempstead UK).

In vitro studies

All in vitro analyses were carried out with crystalline vemurafenib in 50 mM HEPES pH 7.4 containing

30 mM MgCl2. Incubations were initiated ay the addition of NADPH to a final concentration of 1mM

and terminated by the addition of 1x volume of ice cold acetonitrile. Microsomal stability assays

were performed in triplicate with 1 µM compound and 0.2 mg/mL pooled human liver microsomes

(HLM, 150 donors, Life Technologies, Paisley, UK) with or without NADPH for a period of up to 50

min. HLM and mouse liver microsome (MLM, pooled from 3 wild-type mice) incubations for

formation of the OH-vemurafenib metabolite contained 0.4 mg/ml microsomal protein and 60 µM

vemurafenib. Incubations with HLM from individual donors for Spearman’s rank correlation were

performed with 50 µM vemurafenib and 0.1 mg/mL protein for 6 min. Initial incubations with

recombinant P450s were carried out with 60 µM compound and 400 pmol/mL enzyme for 15 min.

Assays to determine the apparent kinetic parameters of OH-vemurafenib formation in HLM and

MLM were performed in triplicate under conditions of linearity for time and protein: 10 min at 0.2





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mg/mL for HLM, 15 min at 0.4 mg/mL for MLM, 6 min at 160 pmol/mL for recombinant CYP3A4, and

9 min at 160 pmol/mL for CYP2C9. For inhibition studies, pooled HLM were incubated at 0.2 mg/mL

with 60 µM vemurafenib for 10 min in the presence of 1 µM ketoconazole, 2 µM sulfaphenazole, or

both together. Inhibition of 7-benzyloxy quinolone (BQ) turnover by vemurafenib was measured

using a BQ concentration of 30 µM and 0.2 mg/ml HLM with increasing concentrations of

vemurafenib, and the fluorescent product measured using a Fluoroskan Ascent FL fluorimeter

(Thermo Fisher, UK)

PXR-transactivation assay

Cells stably transfected with the PXR gene and a reporter construct with luciferase under the control

of PXR-responsive elements of the CYP3A4 promoter (DPX2 cells, Puracyp Inc., Carlsbad, CA, USA)

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 µM) in triplicate for 48 hours and assayed for luminescence and fluorescence as per

the manufacturer’s instructions. The top two concentrations of vemurafenib were highly toxic so

data from these points were excluded from the analysis. PXR activation (fold change) was calculated

by dividing luminescence by fluorescence and normalising to vehicle control.

In vivo studies

All animal work was carried out on 8- to 12-week-old male mice (except for Figure 7, where female

mice were used). PCN was suspended in corn oil at 1 mg/mL for administration by intraperitoneal

(i.p.) injection at 10 mg/kg (10 µL per g body weight). RIF was administered in the same way at 60

mg/kg. Phenobarbital (PB) was dissolved in PBS for i.p. administration at 80 mg/kg. For

pharmacokinetic analyses, crystalline vemurafenib was first dissolved in DMSO then further diluted

in a mixture of Polysorbate 80, ethanol and water (20:13:67) for immediate administration by oral

gavage at a final dose of 50 mg/kg, as described by Durmus et. al. (16). The same method was used

for administration of elacridar at 100 mg/kg (16). For sample collection, 10 µL of whole blood was





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withdrawn from the tail vein at the indicated time points. Samples were immediately added to a

tube containing heparin solution (10 µL, 15 IU/mL) and stored at -20°C until processing. For

induction studies, MBP vemurafenib was suspended in water containing 2% Klucel LF Pharm which

had been adjusted to pH 4.0 by the addition of hydrochloric acid, and administered twice daily (eight

hours apart) by oral gavage, as described by Yang et. al. (22).

Sample processing for LC-MS/MS

100 µL of water containing 100 µg/mL internal standard (PLX4720) was added to in vitro and in vivo

samples, followed by 500 µL of diethyl ether (Fisher Scientific, Loughborough, UK). This mixture was

incubated at room temperature for 30 minutes with gentle mixing. Samples were centrifuged for 10

minutes at 16 000 x g and the organic solvent phase withdrawn to a fresh tube for drying under a

vacuum (20 minutes at 30°C). The dried extract was re-dissolved in 100 µL of acetonitrile (Sigma) at

37°C with agitation for 5 minutes and samples were added to 96-well plates for LC-MS/MS.

LC-MS/MS

Analysis of in vitro incubation and in vivo blood PK samples was carried out on a Waters Acquity

UPLC and Micromass Quattro Premier mass spectrometer (both Micromass, Machester, UK). LC

separation was performed on a Kinetex 1.7µ C19 100A; 50 x 2.1 mm column (Phenomenex,

Macclesfield, UK) at a temperature of 45°C with an injection volume of 5 µL and flow rate of 0.6

mL/min. Mobile phases were water containing 0.1% (v/v) formic acid (A) and acetonitrile containing

0.1% (v/v) formic acid (B). Isocratic elution was carried out at 35%/65% A/B for 1 minute. Multiple

reaction monitoring data in ESI-positive mode were acquired for vemurafenib (490.10 > 255.04, cone

voltage (CV) = 65 V, collision energy (CE) = 40 kV), OH-vemurafenib (505.99 > 255.24, CV = 65 V, CE =

40 kV) and PLX4720 (414.20 >306.97, CV = 60 V, CE = 37 kV). Acquired data were analysed in

Quanlynx relative to analyte standard curves spanning the range of concentrations under study.





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Data analysis

In vitro data exhibited non-Michaelis–Menten kinetics and therefore was fitted with the Hill

equation using GraFit version 5 (Erithacus Software, Horley, UK). Standard deviations given are from

the fit of the calculated curve. Pharmacokinetic parameters of in vivo data were calculated with a

simple non-compartmental model using WinNonLin software, v4.1 (Pharsight, Munich, Germany)

and are shown with standard deviations. P values were calculated using an unpaired t-test.

Western blotting

Microsomal and cytosolic samples were adjusted to 1 mg/mL in LDS sample buffer (Life

Technologies) for electrophoresis through 10% acrylamide gels for 1 hr at 150 V, followed by transfer

onto nitrocellulose membranes. Primary antibodies used for immunoblotting included anti-CYP3A4

(458234, BD Biosciences, Oxford, UK), Cyp1a, Cyp2b, Cyp2c, Cyp3a/CYP3A and Por (23, 24). Anti-

GRP78 (ab21685, Abcam, Cambridge, UK) and anti-GAPDH (G9545, Sigma) were used as loading

controls for microsomal and cytosolic fractions, respectively. In blots for Cyp3a/CYP3A and CYP3A4,

pooled HLM material was the same as used in microsomal incubations, while low, medium and high

expression individuals were taken from the panel of 15 characterised donors based on testosterone

6β-hydroxylation activity.

mRNA analysis

Total RNA was isolated from snap-frozen small intestinal samples using TRIzol (Life Technologies).

The purity of this RNA was increased by processing with the RNeasy Mini Kit (Qiagen, Crawley, UK)

followed by treatment with RG1 DNase (Promega, Southampton, UK). Conversion to cDNA was

carried out using the ImProm-II Reverse Transcription System with random primers (Promega). The

relative levels of mRNA species were determined by TaqMan RT-PCR (Life Technologies) using the

following assays: Abcb1a (Mm00440761_m1), Abcb1b (Mm00440736_m1), Abcg2





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(Mm00496364_m1) and CYP3A4 (Hs00604506_m1). Fold changes were calculated by the

comparative Ct method with 18S rRNA as an endogenous control (4319413E, Life Technologies).





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Results

Vemurafenib is metabolised by CYP3A4 in vitro

Metabolic stability of vemurafenib was assessed in pooled HLM (Figure 1A). At 1 µM, the compound

was highly stable in both the presence and absence of NADPH, however formation of OH-

vemurafenib was detectable in the presence of NADPH (Figure 1B). Apparent kinetic parameters of

OH-vemurafenib formation by HLM and MLM were found to be sigmoidal, therefore were fitted

using the Hill equation. The Vmax for HLM was approximately 10-fold higher than that observed in

MLM (4879 ± 108 vs. 467 ± 54 peak area/min/mg, respectively) while the S50 was similar (23.2 ±0.8

vs. 23.8 ± 4.9 µM, respectively, Figure 1C). Hill coefficients are detailed in Supplementary Table S1,

alongside parameters calculated using the Michaelis-Menten model. In a panel of HLM from 15

donors which had been characterised by the vendor with regard to their activity with probe

substrates for individual CYP isoforms, formation of OH-vemurafenib correlated most strongly with

that of 6β-hydroxy-testosterone, the marker for CYP3A4 (Supplementary Table S2 and Figure 1D).

There was also a strong correlation with CYP2A6 activity (Supplementary Table S2), but this was

deemed artefactual as CYP3A4 and CYP2A6 probe activities within the HLM panel correlate (data not

shown), an observation which has previously been made in a separate batch of samples (23). Indeed,

in incubations with recombinant CYP containing high concentrations of both protein and substrate,

OH-vemurafenib was formed by CYP3A4, CYP2C9 and, to a lesser extent, by CYP2C8 and CYP1A1, but

not by CYP2A6 (Figure 1E). The in vitro apparent kinetics of OH-vemurafenib formation by CYP3A4

and CYP2C9 were, similarly to HLM and MLM, sigmoidal in nature. CYP3A4 had a 2-fold higher

affinity for vemurafenib (S50: 10.0 ± 0.9 vs. 20.4 ± 1.7 µM) and a 3.4-fold higher Vmax (11693 ± 471 vs.

3481 ± 216 peak area/min/nmol) than CYP2C9. As with the hepatic microsomal preparations, Hill

coefficients and Michaelis-Menten analyses for recombinant P450s are detailed in Supplementary

Table S1. In pooled HLM, co-incubation with the CYP3A4 inhibitor, ketoconazole, significantly

inhibited OH-vemurafenib formation (Figure 1G). The CYP2C9 inhibitor, sulfaphenazole, effected a





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partial decrease in OH-vemurafenib formation. As expected, co-incubation with both inhibitors

completely ablated OH-vemurafenib formation. Also in pooled HLM, vemurafenib inhibited the

activity of the CYP3A4 probe drug substrate BQ with an IC50 of 14.9 µM (Figure 1H).

Single-dose vemurafenib is not extensively metabolised by CYP3A4 in vivo

In order to study the contribution of CYP3A4 to metabolism of vemurafenib in vivo,

Cyp3aKO/Cyp3a13+/+ and huCYP3A4/3A7 mice were administered either PCN or vehicle (CO) alone

for three days prior to administration of vemurafenib, as described in Materials and Methods. 24

hours after the third dose of PCN, CYP3A4 is expressed in the liver of huCYP3A4/3A7 at a level similar

to that observed in pooled HLM (data not shown). As stated above, Cyp3aKO/Cyp3a13+/+ mice are

essentially null for Cyp3a/CYP3A activity, both basal and in response to activation of Pxr. Therefore

any difference in the pharmacokinetics of vemurafenib between huCYP3A4/3A7 PCN and

Cyp3aKO/Cyp3a13+/+ PCN is attributable to the activity of CYP3A4. Although not statistically

significant, there was a decrease in both Cmax and AUCall, and an increase in CL, in the huCYP3A4/3A7

PCN group, relative to the Cyp3aKO/Cyp3a13+/+ PCN group (Figure 2A, Supplementary Table S3).

More pronounced, however, were these same changes in the PCN groups of both genotypes,

relative to their respective vehicle control groups. The fact that PCN treatment enhanced clearance

equally well in Cyp3a null and huCYP3A4/3A7 animals suggests that PXR-regulated pathways other

than CYP3A4 predominate in the clearance of vemurafenib in vivo. Immediately after the final blood

sample collection, livers were harvested and Western blotting carried out to confirm that CYP3A4

had been effectively induced by PCN in the huCYP3A4/3A7 mice (Figure 2B). Due to this harvest

occurring 48 hours after the last dose of PCN, CYP3A4 expression was lower than in pooled HLM due

to turnover of the enzyme.

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

CAR





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As PCN pre-treatment decreased the systemic concentration of vemurafenib independently of

CYP3A4 (Figure 2A, Supplementary Table S3), we further investigated the role of nuclear hormone

receptor activity in mediating disposition to vemurafenib. HuPXR/huCAR/huCYP3A4/3A7 mice were

pre-treated with RIF, a potent activator of human PXR and thus inducer of CYP3A4, leading to a

marked reduction in the AUCall of vemurafenib from 432 ± 169 to 121 ± 18 hr*µg/mL (p = 0.0106,

Figure 3A, Supplementary Table S4). Western blotting confirmed induction of CYP3A4 in the liver

and, due to the high initial magnitude of this induction, CYP3A4 levels remained several fold higher

than pooled HLM (and equivalent to HLM expressing high CYP3A4) despite 48 hours having elapsed

after administration of the final dose of RIF (Figure 3B). In addition, we observed a 4.7-fold increase

in mRNA for Abcb1a, but no change for Abcb1b or Abcg2, in the small intestine of

huPXR/huCAR/huCYP3A4/3A7 mice treated with RIF (Figure 3C). Activation of PXR in the

huPXR/huCAR mouse line led to a similar decrease in AUCall of vemurafenib from 349 ± 24 to 75 ± 33

hr*µg/mL (p = 0.0003, Figure 3A, Supplementary Table S4).

In order to determine whether CAR might play a role in modulating vemurafenib disposition,

huPXR/huCAR/huCYP3A4/3A7 mice were dosed for three days with PB, a CAR activator (25), prior to

vemurafenib administration. This had no effect on any of the kinetic parameters assessed (Figure 4A,

Supplementary Table S5). Western blotting of liver samples demonstrated induction of the well-

characterised Car target protein, Cyp2b10, confirming the effective activation of CAR by PB (Figure

4B).

Activation of PXR reduces vemurafenib exposure through induction of efflux transporters

Activation of PXR by RIF in huPXR/huCAR/huCYP3A4/3A7 mice decreased the AUCall of vemurafenib

(Figures 3A, 4A and 5A, Supplementary Tables S3, S4 and S5). This decrease was almost completely

ablated by pre-treatment of mice, 90 minutes before administration of vemurafenib, with elacridar,

an effective inhibitor of Abcb1 and Abcg2, but not CYP3A4, at the dose used (100 mg/kg p.o., Figure





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5A) (16, 26, 27). Relative to mice dose with RIF alone, AUCall of vemurafenib increased from 90 ± 13

to 330 ± 85 hr*µg/mL (p = 0.0085) in animals dosed with RIF then elacridar, while Cmax increased

from 11.1 ± 1.8 to 21.3 ± 2.2 µg/mL (p = 0.0032, Supplementary Table S6). Moreover,

huPXR/huCAR/huCYP3A4/3A7 mice which had been pre-dosed with vehicle (CO) alone were subject

to a similar magnitude of increase in exposure to vemurafenib following elacridar pre-treatment,

with AUCall increasing from 391 ± 112 to 684 ± 85 hr*µg/mL (p = 0.0224) and Cmax increasing from

29.1 ± 4.3 to 37.8 ± 3.5 µg/mL (p = 0.0507, Figure 5A). Again, effective activation of PXR by RIF was

confirmed by Western blot of CYP3A4 in liver samples (Figure 5B).

Vemurafenib is a strong inducer of PXR in vitro

We 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 in the

range of 0.01 – 10 µg/mL (approximately 20 nM – 20 µM) at physiological pH (12). An EC50 of 8.13

µM and an EMAX of 8.54-fold (Figure 6) was determined. The positive control, rifampicin, gave an EC50

of 1.01 µM and an EMAX of 14.10-fold (data not shown).

Vemurafenib is a weak inducer of PXR in vivo

We administered the high bioavailability MBP form of vemurafenib at a dose of 100 mg/kg b.i.d. for

four days to huPXR/huCAR/huCYP3A4/3A7 mice, as described in Materials and Methods. The PK

parameters of a single 100 mg/kg dose (Supplementary Table S7) were in good agreement with

those calculated in a previous study (22). Western blotting of liver proteins demonstrated weak

induction of CYP3A4 and Cyp2b, and moderate induction of Cyp1a (Figure 7A). Analysis of small

intestinal mRNA found no change in expression in CYP3A4, Abcb1a or Abcb1b (Figure 7B).





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Discussion

Vemurafenib is a revolutionary treatment for advanced melanoma and, along with the companion

diagnostic test for BRAF mutation (8), is a successful example of cancer therapy stratification based

on molecular-level information. As with other oncogenic kinases, however, clinical response to

inhibition of BRAFV600 is highly variable and the acquisition of resistance in patients initially

responsive to therapy is usually rapid (5-7). A wide variety of mechanisms of innate and acquired

resistance have been identified, most of which are a function of the redundancy of the pro-

proliferative signalling cascade being targeted (9, 10, 28). Data generated during the pre-clinical

development of vemurafenib suggested several points of interaction with proteins involved in

xenobiotic metabolism. These have the capacity to alter PK parameters and hence the therapeutic

effectiveness of this agent (11, 12).

In agreement with the pre-clinical data (11, 12), we found that CYP3A4 was the major CYP isoform

involved in vemurafenib metabolism in vitro, but that the rate of metabolism was low. We also

found that vemurafenib has the capacity to inhibit CYP3A4, with an IC50 for BQ activity of 14.9 µM. In

addition, and in conflict with the manufacturer’s findings (11, 12), we identified a minor role of

CYP2C9 in generating OH-vemurafenib, evidenced by a small but statistically significant decrease in

OH-vemurafenib in pooled HLM co-incubated with the 2C9-specific inhibitor, sulfaphenazole.

In a previous study, mass balance analysis in seven patients at steady state found that, in serum

samples withdrawn up to 48 hours after administration of a radiolabelled dose of vemurafenib, 95%

of recovered 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 ~4% over this time period. It took 18 days

for 95% of the radioactive dose to be recovered; 94% in faeces, 1% in urine (15). Metabolic profiling

was carried out on the 68.5% of the total dose which was recovered within the first 96 hours. Over

the 0 – 48 hr period, ≥ 94% of the radioactivity recovered was in the parental form. Over the 48 – 96





17

hr period, however, this value decreased to 55.5 ± 20.1%. OH-vemurafenib accounted for 13.7% of

the radioactivity recovered from the 48 – 96 hr samples (3.4% of the total dose). Conjugated

(glucosylated and glucuronidated) forms of vemurafenib were the only other metabolites definitively

identified in these samples (11, 12, 15). Although the percentage of vemurafenib excreted in the

form of metabolites was low relative to the parent compound, it was acknowledged that a large

proportion of the parent compound eliminated during the first 48 hr might constitute drug which

had not been absorbed, and a significant proportion of the vemurafenib excreted during 48 - 96 h

post-dose might have been generated by hepatobiliary recirculation. Indeed, it was also reported

that metabolites predominated over the parent compound in a bile sample from one of the patients

receiving vemurafenib (15). As the absolute bioavailibility of vemurafenib is unknown, and hence the

contribution of metabolism to its elimination cannot be calculated, Phase IV clinical trials into the

effect of an inhibitor and an inducer of CYP3A4 into the pharmacokinetics of vemurafenib have been

requested by both the FDA and EMA. At present, an inhibitor (ketoconazole) study (NCT01765556)

has been withdrawn and an inducer (rifampicin) study (NCT01765543) is recruiting

(clinicaltrials.gov). Despite the occurrence of CYP-dependent hydroxylation of vemurafenib in vitro,

we found that the in vivo effect of CYP3A4 in modulating the systemic levels of a single dose of the

compound in humanized mice was small. Far more apparent was a decrease in AUCall following

activation of Pxr, which was independent of CYP3A4 as it occurred in both the Cyp3aKO/Cyp3a13+/+

and huCYP3A4/3A7 lines. This decrease was even more pronounced following activation of PXR in

the huPXR/huCAR and huPXR/huCAR/huCYP3A4/3A7 mouse lines with RIF. The likely explanation for

the differing magnitudes of this decrease is that, at the doses used, PCN-mediated activation of mPxr

(and thereby induction of the downstream effector proteins) is less effective than RIF-mediated

activation of hPXR (20).

Pre-incubation of vemurafenib-resistant clones of the BRAFV600E-positive A375 melanoma cell line

with verapamil, an ABCB1 inhibitor, partially increased their sensitivity to the drug (29). Studies with





18

the MDCK-ABCB1 cell line demonstrated that vemurafenib was both an ABCB1 substrate and

inhibitor (12), and a post-authorisation report mentions evidence of an interaction with ABCG2 (30).

These observations have been confirmed independently, and studies in mice nulled for these

transporters either separately or in combination have found that they modulate both intestinal

bioavailability and permeability at the blood-brain barrier (16, 17, 31). This synergistic interaction of

Abcb1 and Abcg2 is a phenomenon which has been shown to modulate the bioavailability of other

therapeutics (32). ABCB1 is a transcriptional target of PXR in human cells (33, 34), and this

interaction 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 mRNA levels in the

small intestine of huPXR/huCAR/huCYP3A4/3A7 mice demonstrate that, of these, only the first was

induced following RIF treatment. Activation of CAR by PB induced Cyp2b10 to the same extent as

activation of PXR by RIF, while also weakly inducing CYP3A4, but this pre-treatment had no effect on

the PK parameters of vemurafenib. Taken together, these observations suggest that the Pxr/PXR-

dependent modulation of vemurafenib PK is likely to be due to the upregulation of Abcb1a, and

hence a decrease in the bioavailability of vemurafenib, rather than to induction of drug metabolism

enzymes. Although PXR regulates the transcription of other drug metabolising enzymes in

humanized mice, such as UDP-glucuronsyltransferases (UGT) of the 1a subfamily (38), our

contention that the observed effects are due to the modulation of transport are supported by the

finding that administration of the Abcb1/Abcg2 inhibitor, elacridar, 90 minutes prior to

administration of vemurafenib completely ablated the effect of RIF pre-treatment in

huPXR/huCAR/huCYP3A4/3A7 mice. Furthermore, elacridar pre-treatment of uninduced (i.e. not

pre-treated with RIF) huPXR/huCAR/huCYP3A4/3A7 mice increased AUCall of vemurafenib by a

degree comparable to pre-treatment of induced mice, suggesting that inhibition of efflux

transporters in the basal state can profoundly increase vemurafenib bioavailability. It should be

noted that, although elacridar is often cited as a specific inhibitor of ABCB1/Abcb1 and

ABCG2/Abcg2, this is not the case. At the dose used in this study, the predicted plasma





19

concentration of elacridar is an order of magnitude below the IC50 for CYP3A4 and other P450s (26,

27). But this compound is known to inhibit SLCO1B1 (39) and is highly likely to inhibit other

transporters (40). Therefore, although we cannot conclusively state that PXR-directed modulation of

Abcb1a level in the small intestine is the pivotal determinant of AUCall of vemurafenib, this remains

the most likely explanation. In support of this, studies in the Abcb1a/b and Bcrp knockout mouse

lines suggested a predominant role for the former transporter (16). Increased drug efflux mediated

by ABC transporter proteins in the cancer cell is a mechanism of multidrug resistance which has

been understood for nearly 40 years (41). As a class of therapeutics, tyrosine kinase inhibitors, such

as vemurafenib, are no less vulnerable to this process than conventional chemotherapeutics (42).

The demonstration that drug transporters can markedly affect in vivo vemurafenib pharmacokinetics

implies that changes in their expression in the tumour itself will also be a factor in drug response.

Surprisingly, to date, this possibility has not been investigated.

During pre-clinical development it was posited that decreases in the steady state blood plasma

concentrations of vemurafenib in dogs on a twice-daily dosing regimen between 29 and 92 days

might be due to the induction of metabolism (11). Furthermore, in patients at steady state,

vemurafenib decreased AUC0-last of the CYP3A4-specific probe, midazolam, by 39%, increased AUC0-

last of the CYP1A2 probe, caffeine, by 160%, and increased AUC0-last of the CYP2D6 probe,

dextromethorphan, by 47% (11, 12). This defined vemurafenib as an inducer of CYP3A4, a moderate

inhibitor of CYP1A2 and a weak inhibitor of CYP2D6. In agreement with these findings, we observed

strong induction of CYP3A4-promoter-driven luciferase activity in a cell-line based reporter assay for

PXR with crystalline vemurafenib. During the Phase I clinical trial, vemurafenib was reformulated

from a crystalline to an MBP form to improve bioavailability (5). With this formulation, AUC0-24 is

proportional to doses of between 240 and 960 mg bid (2, 5, 43). In the present study, we

administered MBP vemurafenib to huPXR/huCAR/huCYP3A4/3A7 mice to assess the drug-drug

interaction potential of this compound in this mouse model in vivo. Moreover, the AUCall of 1142 ±





20

242 hr*µM observed at this dose is approximately four times the AUC0-24 of 300 hr*µM cited as

being necessary to effect regression of COLO205 xenografts (44) and, with twice daily dosing, was

considered to be sufficiently representative of the steady state level of exposure in humans (AUC0-24

= 1741 ± 639 hr*µM, (5)). In addition, the Cmax of this single dose, at 121.7 ± 19.7 µM, is above the

mean value of 86 ± 32 µM reported in patients at steady state (5). With these parameters, in silico

analysis predicted that steady state concentrations in mice would be achieved within approximately

32 hours (i.e. with the fourth dose) on a schedule of two doses per day, 8 hours apart (data not

shown). Under these conditions we observed induction of CYP3A4 in the liver of

huPXR/huCAR/huCYP3A4/3A7 mice administered a high dose of the MBP formulation twice daily for

four days, although the level of this induction was slight and highly variable. Nevertheless, our in

vitro and in vivo data demonstrate that vemurafenib has the capacity to induce CYP3A4. In addition,

we also observed induction of Cyp1a and Cyp2b proteins in huPXR/huCAR/huCYP3A4/3A7 mice.

These effects may, in part, be due to activation of PXR, but are likely also due to the activation of

one or more additional transcription factors, such as the aryl hydrocarbon receptor (45). In addition

to possible overall effects on drug PK, the capacity of vemurafenib to activate transcription factors

involved in drug disposition raises the possibility that it may induce its own metabolism and efflux in

tumour cells. This could potentially be a significant factor in the acquisition of drug resistance.

Similar effects might also be invoked by co-medications which activate these transcription factors.

In conclusion, we have found that vemurafenib interacts with the P450 system in vitro, but that PXR-

directed regulation of efflux transporters and the consequent modulation of bioavailability is likely

to be a more important factor in defining disposition in vivo. In addition, we have demonstrated that

vemurafenib has the capacity to activate PXR, and potentially other transcription factors, at

clinically-relevant concentrations. Taken together, our observations support the case for therapeutic

monitoring of vemurafenib levels in patients over the course of therapy to evaluate whether low





21

systemic drug concentrations coincide with innate or acquired resistance, and to determine whether

modulators of PXR/CYP3A4/ABCB1 activity influence the outcome of vemurafenib therapy.

Acknowledgements

We would like to thank Julia Carr, Catherine Meakin and Susanne van Schelven for assistance with

animal work, and Cheryl Wood, Mairi Lennie and Rumen Kostov for additional technical support. We

thank Dr. Yury Kapelyukh for critical reading of the manuscript. We would also like to acknowledge

Taconic Biosciences for the supply of the mouse lines used in this study.

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Figure Legends

Figure 1. Vemurafenib is metabolised by CYP3A4 and CYP2C9 in vitro.

(A) Microsomal stability of vemurafenib with HLM in the absence (open circles) and presence (filled

circles) of NADPH. (B) Formation of OH-vemurafenib from vemurafenib when incubated with HLM in

the absence (open circles) and presence (filled circles) of NADPH. (C) Vemurafenib kinetics with HLM

(open circles) and MLM (filled circles) fitted with the Hill equation. (D) Correlation of conversion of

6β-OH-testosterone formation in characterised liver microsomes from 15 individuals with formation

of OH-vemurafenib from vemurafenib. (E) Generation of OH-vemurafenib by recombinant CYP. (F)

Vemurafenib kinetics with recombinant CYP2C9 (filled circles) and CYP3A4 (open circles) fitted with

the Hill equation. (G) Inhibition of OH-vemurafenib formation in pooled HLM by ketoconazole and

sulfaphenazole. (H) Inhibition of BQ activity in pooled HLM by vemurafenib (IC50 = 14.9 µM). In all

cases data comprise mean ± SD of triplicate incubations.

Figure 2. CYP3A4 has a modest effect on the pharmacokinetic profile of a single dose of

vemurafenib in vivo.

(A) Pharmacokinetic profiles of vemurafenib administered to huCYP3A4/3A7 PCN (filled circles, n=5),

Cyp3aKO/Cyp3a13+/+ PCN (filled squares, n=4), huCYP3A4/3A7 Vehicle (open circles, n=5), and

Cyp3aKO/Cyp3a13+/+ Vehicle (open squares, n=4) mice. Mean data ± SEM are shown. (B) Western

blot of hepatic microsomal fractions from Cyp3aKO/Cyp3a13+/+ and huCYP3A4/3A7 mice dosed with

either PCN or CO vehicle alone, alongside pooled HLM.

Figure 3. Activation of PXR reduces exposure to vemurafenib independently of CYP3A4 activity.

(A) Pharmacokinetic profiles of vemurafenib in huPXR/huCAR/huCYP3A4/3A7 Vehicle (open circles,

n=4), huPXR/huCAR/huCYP3A4/3A7 RIF (closed circles, n=4), huPXR/huCAR Vehicle (open squares,

n=3) and huPXR/huCAR RIF (closed squares, n=3) mice. Mean data ± SEM are shown. (B) Western





25

blot of hepatic microsomal fractions from huPXR/huCAR/huCYP3A4/3A7 mice dosed with either RIF

or CO vehicle alone, alongside HLM preparations; pooled and low, medium and high CYP3A4 activity.

(C) TaqMan RT-PCR analysis of Abcb1a, Abcb1b and Abcg2 mRNA levels in whole small intestine of

huPXR/huCAR/huCYP3A4/3A7 mice dosed with vehicle or RIF.

Figure 4. Activation of CAR does not alter vemurafenib pharmacokinetics.

(A) Pharmacokinetic profiles of vemurafenib in huPXR/huCAR/huCYP3A4/3A7 Vehicle (open circles,

n=3), PB (closed circles, n=3) and RIF (closed squares, n=3). Mean data ± SEM are shown. (B)

Western blots of hepatic microsomal fractions from huPXR/huCAR/huCYP3A4/3A7 mice dosed with

either vehicle (PBS), PB, or RIF alongside HLM preparations; pooled and low, medium and high

CYP3A4 activity.

Figure 5. PXR regulates vemurafenib bioavailability through the modulation of efflux transporters.

(A) Pharmacokinetic profiles of vemurafenib in huPXR/huCAR/huCYP3A4/3A7 mice treated with

vehicle/vehicle (open circles, n=3), vehicle/elacridar (filled circles, dashed line, n=3), RIF/vehicle

(open squares, n=3) and RIF/elacridar (filled squares, dashed line, n=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 either DMSO/Tween 80/ethanol/water (vehicle) or

elacridar 90 mins prior to vemurafenib, alongside HLM preparations; pooled and low, medium and

high CYP3A4 activity.

Figure 6. Vemurafenib activates PXR in vitro.

DPX2 cells were used to calculate the EC50 (8.13 µM) and EMAX (8.54-fold) for vemurafenib mediated

trans-activation of PXR

Figure 7. Vemurafenib activates PXR and possibly other transcription factors in vivo.





26

(A) Western blots of hepatic microsomal fractions from huPXR/huCAR/huCYP3A4/3A7 mice dosed

with either vehicle (4% Klucel LF, pH 4.0, n = 4) or MBP vemurafenib (n = 4) twice daily (8 hours

apart) for four days. (B) TaqMan RT-PCR of CYP3A4, Abcb1a and Abcb1b in duodenum (Duo),

jejunum (Jej) and ileum (Ile) of huPXR/huCAR/huCYP3A4/3A7 mice dosed with either vehicle or

vemurafenib. Data shown are mean of triplicate analyses ± SD.




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Figure 1




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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 (PXR) influences

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activation status of the pregnane x receptor (pxr ......2015/09/11 · humanized for cyp3a4/3a7,...

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