Biotransformation of the Antiretroviral Drug Etravirine: Metabolite
Identification, Reaction Phenotyping, and Characterization of
Autoinduction of Cytochrome P450-Dependent Metabolism
Lindsay J. Yanakakis and Namandje ´ N. Bumpus
Department of Biology, Johns Hopkins University, Baltimore, Maryland (L.J.Y.); and Departments of Medicine and
Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland (N.N.B.)
Received December 20, 2011; accepted January 23, 2012
Etravirine (ETR) is a second-generation non-nucleoside reverse
transcriptase inhibitor prescribed for the treatment of HIV-1. By
using human liver microsomes (HLMs), cDNA-expressed cyto-
chromes P450 (P450s), and UDP-glucuronosyltransferases (UGTs),
the routes of ETR metabolism were defined. Incubations with
cDNA-expressed P450 isozymes and chemical inhibition studies
using HLMs indicated that CYP2C19 is primarily responsible for the
formation of both the major monohydroxylated and dihydroxylated
metabolites of ETR. Tandem mass spectrometry suggested that
these metabolites were produced via monomethylhydroxylation
and dimethylhydroxylation of the dimethylbenzonitrile moiety. For-
mation of these monohydroxy and dihydroxy metabolites was de-
creased by 75 and 100%, respectively, in assays performed using
HLMs that were genotyped as homozygous for the loss-of-function
CYP2C19*2 allele compared with formation by HLMs genotyped as
CYP2C19*1/*1. Two monohydroxylated metabolites of lower abun-
dance were formed by CYP3A4, and interestingly, although
CYP2C9 showed no activity toward the parent compound, this
enzyme appeared to act in concert with CYP3A4 to form two minor
dihydroxylated products of ETR. UGT1A3 and UGT1A8 were dem-
onstrated to glucuronidate a CYP3A4-dependent monohydroxy-
lated product. In addition, treatment of primary human hepato-
cytes with ETR resulted in 3.2-, 5.2-, 11.8-, and 17.9-fold increases
in CYP3A4 mRNA levels 6, 12, 24, and 72 h after treatment. The
presence of the pregnane X receptor antagonist sulforaphane
blocked the ETR-mediated increase in CYP3A4 mRNA expression.
Taken together, these data suggest that ETR and ETR metabolites
are substrates of CYP2C19, CYP3A4, CYP2C9, UGT1A3, and
UGT1A8 and that ETR is a PXR-dependent modulator of CYP3A4
Reverse transcription of single-stranded human immunodeficiency
virus (HIV) RNA into double-stranded DNA is a crucial, complex
step in the replication of the virus and requires the use of two HIV
reverse transcriptase active sites (Go ¨tte et al., 1999). Because HIV
reverse transcriptase is essential for HIV replication, the enzyme is a
major target for antiretroviral drug development (Parniak and Sluis-
Cremer, 2000). HIV-1 and HIV-2 are the two known species of the
virus, with HIV-1 being the more virulent and thus the cause of the
majority of HIV infections globally. To date, there are two classes of
reverse transcriptase inhibitors that are routinely used in the clinic to
treat HIV-1 infection: the nucleoside/nucleotide reverse transcriptase
inhibitors and the non-nucleoside reverse transcriptase inhibitors
(NNRTIs). NNRTI-based treatment approaches are effective in sup-
pressing HIV RNA levels, causing a concomitant increase in immune
function in a large majority of patients (Staszewski et al., 1999;
Gallant et al., 2004; Squires et al., 2004; Gulick et al., 2006; Riddler
et al., 2008). To maximally suppress viral loads and decrease inci-
dence of drug resistance, NNRTIs are prescribed in combination with
other antiretrovirals (i.e., protease inhibitors, integrase inhibitors, fu-
sion inhibitors, and CCR5 antagonists) as part of highly active anti-
dimethylbenzonitrile (etravirine; ETR) is a second-generation NNRTI that,
like other NNRTIs, functions by binding noncompetitively to HIV reverse
the virus. As a diarylpyrimidine, ETR has a high capacity for isomerization,
which allows it to effectively bind to and inhibit common mutated forms of
the viral enzyme (Andries et al., 2004; Vingerhoets et al., 2005; Gupta et al.,
This work was supported by the PhRMA Foundation (Research Starter Grant in
Pharmacology/Toxicology; to N.N.B.). The AB SCIEX QTRAP 5500 mass spec-
trometer was purchased using a grant from the National Institutes of Health
National Center for Research Resources [Grant 1S10-RR27733]; the Waters
Acquity ultraperformance liquid chromatograph interfaced with the AB SCIEX
QTRAP 5500 mass spectrometer was purchased with funds provided by the
Pendleton Foundation Trust.
Article, publicationdate,and citation
ABBREVIATIONS: HIV, human immunodeficiency virus; NNRTI, non-nucleoside reverse transcriptase inhibitor; ETR, etravirine; P450, cytochrome
P450; UGT, UDP-glucuronosyltransferase; HLM, human liver microsomes; PXR, pregnane X receptor; PPP, 2-phenyl-2-(1-piperidinyl)propane;
SFN, sulforaphane; RIF, rifampin; UDPGA, UDP-glucuronic acid; UPLC-MS, ultraperformance liquid chromatography-mass spectrometry; DMSO,
dimethyl sulfoxide; TIS, TurboIonSpray; MS/MS, tandem mass spectrometry; PCR, polymerase chain reaction; qPCR, quantitative polymerase
chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
DRUG METABOLISM AND DISPOSITION
Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
DMD 40:803–814, 2012
Vol. 40, No. 4
2011; Picchio et al., 2011). Therefore, ETR has a higher genetic barrier to
viral resistance and as such is often prescribed for treatment-experienced
patients who have developed mutations that confer resistance to first-gener-
ation NNRTIs (Lazzarin et al., 2007; Madruga et al., 2007; Nadler et al.,
2007). Because ETR is always taken concurrently with other antiretrovirals
that are substrates, inhibitors, and inducers of drug-metabolizing enzymes
(Kakuda et al., 2010; Calcagno et al., 2011), gaining a comprehensive
important to minimize drug-drug interactions and adverse events. For in-
ing dolutegravir (by 70%) (Song et al., 2011) and maraviroc (by 53%)
(Kakuda et al., 2011). CYP3A4 has been shown to play a central role in the
N-dealkylation of maraviroc, which is the major route of maraviroc metab-
olism, raising the possibility that ETR decreases exposure to maraviroc in
vivo via modulation of CYP3A4 expression and/or activity (Hyland et al.,
2008). The ETR product label (Tibotec, Inc., 2011) and a review article
(Scho ¨ller-Gyu ¨re et al., 2009) state that cytochromes P450 (P450s) 3A4,
ETR is an inducer of CYP3A4 expression; however, primary data that
comprehensively describe the metabolites formed, the contribution of these
P450s to ETR metabolism, and the mechanism(s) by which ETR increases
CYP3A4 levels have yet to be published. In addition, although glucuronida-
tion of oxidative metabolites of ETR by UDP-glucuronosyltransferases
(UGTs) has been noted, the enzymes involved in the formation of these
metabolites have yet to be reported (Scho ¨ller-Gyu ¨re et al., 2009).
With this in mind, the primary objectives of the present study were
to gain a comprehensive understanding of the routes of ETR metab-
olism as well as to characterize the ability of ETR to modulate the
expression of hepatic CYP3A4. By using HLMs, cDNA-expressed
P450s, cDNA-expressed UGTs, and primary human hepatocytes, the
products of ETR metabolism and the relative contributions of the
enzymes involved were defined. CYP2C19 was found to be primarily
responsible for the formation of both the major monooxygenated and
dioxygenated metabolites of ETR. Of interest, the monohydroxy
product was markedly reduced and the dihydroxy metabolite was
undetectable in assays performed using HLMs genotyped as homozy-
gous for the loss-of-function CYP2C19*2 allele. Thus, ETR may have
novel utility as a probe substrate for phenotyping CYP2C19 activity.
In addition, it is demonstrated using primary human hepatocytes that
the mRNA levels of CYP3A4 are modulated by ETR in a pregnane X
receptor (PXR)-dependent manner. Taken together, these studies pro-
vide a mechanistic foundation for understanding and potentially pre-
dicting drug-drug interactions involving ETR.
Materials and Methods
Materials. Etravirine was supplied by the National Institutes of Health
AIDS Research and Reference Reagent Program (Germantown, MD). Keto-
conazole, furafylline, quinidine, sulfaphenazole, and (?)-N-3-benzylnirvanol
were purchased from Sigma-Aldrich (St. Louis, MO), and 2-phenyl-2-(1-
piperidinyl)propane (PPP) was obtained from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). Sulforaphane (SFN) was purchased from Toronto Research
Chemicals Inc. (North York, ON, Canada). Rifampin (RIF) was obtained from
sanofi-aventis (Bridgewater, NJ).
Metabolism Assays. For metabolite identification experiments, ETR (20
?M) was incubated with HLMs (50 donor pool; BD Biosciences, San Jose,
CA), Supersomes expressing individual human cytochromes P450 (BD Bio-
sciences; CYP1A2, -2B6, -2C8, -2C9, -2C19, -2D6, -3A4, and -3A5) or both
P450 Supersomes and Supersomes expressing individual UGTs (BD Biosci-
ences; UGT1A1, -1A3, -1A4, -1A6, -1A7, -1A8, -1A9, -1A10, -2B4, -2B7,
-2B15, and -2B17). The final concentrations of microsomes, P450s, and UGTs
were 2 mg/ml, 20 pmol/ml, and 0.2 mg/ml, respectively. After a 5-min
equilibration period at 37°C in 100 mM potassium phosphate buffer, pH 7.4,
the reaction was initiated by addition of an NADPH-regenerating system (BD
Biosciences) and allowed to proceed for 30 min at 37°C. For the UGT
experiments, reactions also contained 2 mM UDP-glucuronic acid (UDPGA)
and UGT reaction mix (Tris buffer, pH 7.5, alamethicin, and MgCl2; BD
Biosciences), and reaction mixtures were incubated for a total of 60 min at
37°C because 30-min incubations did not result in the formation of glucuroni-
dated metabolites that could be detected above the level of background. The
total reaction volume was 500 ?l. For the enzyme kinetics experiments, the
incubations were performed under initial rate conditions, using CYP3A4 (5
pmol) and CYP2C19 (0.5 pmol). These reactions were allowed to proceed for
20 min at 37°C using concentrations of ETR ranging from 0 to 160 ?M for the
CYP3A4 incubation and from 0 to 40 ?M for the CYP2C19 experiments in a
total reaction volume of 100 ?l. All reactions were terminated by the addition
of acetonitrile followed by centrifugation at 3000g for 10 min at 4°C. The
resulting supernatant was removed and evaporated to dryness under nitrogen
gas stream. The residue was reconstituted in 200 ?l of methanol, and 5 ?l was
injected onto an ultraperformance liquid chromatography-mass spectrometry
(UPLC-MS) system for analysis.
P450 inhibition assays were conducted using 0.5 mg/ml HLMs in the
presence of an NADPH-regenerating system, 2 mM UDPGA and UGT reac-
tion mix, and 20 ?M ETR, with or without the following P450 inhibitors: 10
?M (?)-N-3-benzylnirvanol (CYP2C19 inhibitor), 20 ?M furafylline
(CYP1A2 inhibitor), 1 ?M ketoconazole (CYP3A4 inhibitor), 30 ?M PPP
(CYP2B6 inhibitor), 1 ?M quinidine (CYP2D6 inhibitor), or 20 ?M sulfa-
phenazole (CYP2C9 inhibitor). The concentrations of inhibitors and HLMs
used in these assays were selected on the basis of those reported previously
(Suzuki et al., 2002; Ward et al., 2003; Walsky and Obach, 2007). All
inhibitors were dissolved in DMSO except ketoconazole, which was dissolved
in methanol, and control reactions were conducted using solvents alone. The
total reaction volume was 250 ?l in 100 mM potassium phosphate buffer, pH
7.4. HLMs, NADPH, UDPGA, UGT reaction mix, and inhibitors were incu-
bated for 5 min at 37°C followed by the addition of ETR. The reactions were
allowed to proceed for 30 min before termination by the addition of acetoni-
trile. Inhibition assays using cDNA-expressed CYP2C19 contained 0.5 pmol
of protein, and reaction components and conditions were the same as those
used for inhibition assays using HLMs.
For coincubations with P450s 3A4 and 2C9, 5 pmol of each P450 were
incubated together with 20 ?M ETR in the presence of NADPH for 30 min at
37°C. For sequential incubations, 5 pmol of either CYP3A4 or CYP2C9 was
incubated with 20 ?M ETR with or without NADPH for 60 min at 37°C. These
reactions were then stopped using acetonitrile, centrifuged to remove protein,
and dried down under nitrogen gas stream. DMSO (5 ?l) was added to
reconstitute ETR and its metabolites and 1 ?l was then added to a mixture
containing 5 pmol CYP3A4 or CYP2C9 and NADPH. This second reaction
was allowed to proceed for 30 min at 37°C, at which point the reactions were
terminated by the addition of acetonitrile and later analyzed using UPLC-MS
for the presence of M5 and M6. Final reaction volumes were 100 ?l in 100 mM
potassium phosphate buffer, pH 7.4.
UPLC-MS. An UPLC-MS assay method was developed for the quantifi-
cation and identification of ETR metabolites, using an Acquity UPLC system
(Waters, Milford, MA) coupled to an AB SCIEX QTRAP 5500 mass spec-
trometer. The samples were resolved using a XTerra MS C18column (2.5 ?m,
21 ? 50 mm; Waters) at a flow rate of 0.5 ml/min. A gradient was generated
that consisted of mobile phases A (water, 0.1% formic acid) and B (acetoni-
trile, 0.1% formic acid) that was held at 30% B from 0 to 0.4 min, 30% B to
85% B from 0.4 to 4.5 min, 85% B to 100% B from 4.5 to 4.6 min, and 100%
B to 15% B from 4.6 to 4.8 min and maintained at 15% B until 5.5 min.
Standard curves were generated using ETR because ETR metabolite standards
were not commercially available. The electrospray ionization interface was set
to positive ion mode and the following instrument parameters: TIS tempera-
ture, 600°C; TIS voltage, 4500 V; curtain gas, nitrogen, 35; nebulizing gas, 40;
TIS gas, 40; collision energy, 51; declustering potential, 171 V; entrance
potential, 10 V; and collision cell exit potential, 26 V. Dwell times were 75 ms
and unit mass resolution was used. Metabolite identification was performed in
product ion (MS/MS) mode, and multiple reaction monitoring was used for
quantification and initial detection. The following transitions (Q1 3 Q3) were
monitored: m/z 435.3 3 304.1 (ETR), m/z 451.3 3 304.1 (monohydroxy ETR;
M1 and M2), m/z 451.3 3 353.1 (monohydroxy ETR; M3), m/z 467.3 3 369
YANAKAKIS AND BUMPUS
pharmacokinetics of ETR in vivo. Indeed, several polymorphisms
exist for the CYP2C19 allele, including the CYP2C19*3 allele, which
also encodes a nonfunctional protein product (Ozawa et al., 2004).
Of the three dihydroxy metabolites that were identified during our
study, only M4 was initially detected after incubation of ETR with the
cDNA-expressed P450s, whereas M5 and M6 were not. The fragmen-
tation patterns of M5 and M6 were similar although these metabolites
were readily separated chromatographically. A plausible explanation
for this result is that one of these metabolites is produced via oxygen
insertion at the position in the benzonitrile ring that is meta to the
hydroxylated methyl group, whereas the other is formed via oxygen-
ation of the benzonitrile ring at the position that is para to the
hydroxylated methyl group. To identify the P450s involved in the
catalysis of M5 and M6, studies were performed using a number of
P450 inhibitors, namely, (?)-N-3-benzylnirvanol (CYP2C19 inhibi-
tor), furafylline (CYP1A2 inhibitor), ketoconazole (CYP3A4 inhibi-
tor), sulfaphenazole (CYP2C9 inhibitor), PPP (CYP2B6 inhibitor),
and quinidine (CYP2D6 inhibitor). Of interest, ketoconazole abro-
gated the formation of M5 and M6, suggesting that CYP3A4 plays a
role in formation of these dihydroxy metabolites. Furthermore, the
presence of the CYP2C9 inhibitor sulfaphenazole also decreased the
formation of M5 and M6, indicating that these two P450s may act in
concert to metabolize ETR to M5 and M6. This was demonstrated
definitively via coincubation of CYP2C9 and CYP3A4 in the pres-
ence of ETR. Of interest, although tandem mass spectra indicated that
M5 and M6 involve methylhydroxylation of ETR, CYP2C19 was not
shown to be involved in the production of these metabolites even
though this P450 was primarily responsible for the formation of the
monomethylhydroxylated and dimethylhydroxylated metabolites M3
and M4. Further studies will be performed to probe whether oxygen-
ation of the benzonitrile ring of ETR affects the ability of this
molecule to bind to CYP2C19, thereby preventing the participation of
this enzyme in the formation of M5 and M6.
In summary, the present study provides a comprehensive analysis
of the in vitro biotransformation of ETR as well as the autoinduction
of ETR metabolism via up-regulation of CYP3A4 mRNA. Because
ETR is only prescribed as part of combination therapy to treat HIV, a
mechanistic understanding of the routes of ETR metabolism and
pathways that may be modulated by ETR is essential for predicting
potential drug-drug interactions. Finally, because our data indicate
that the dimethylhydroxy metabolite of ETR is formed exclusively by
CYP2C19, this metabolite may be a useful tool for probing CYP2C19
Participated in research design: Yanakakis and Bumpus.
Conducted experiments: Yanakakis.
Performed data analysis: Yanakakis and Bumpus.
Wrote or contributed to the writing of the manuscript: Yanakakis and
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Address correspondence to: Dr. Namandje ´ N. Bumpus, Department of Med-
icine, Johns Hopkins University School of Medicine, 600 N. Wolfe St., Osler 527,
Baltimore, MD 21287. E-mail: email@example.com
YANAKAKIS AND BUMPUS