Heterotropic activation of the midazolam hydroxylase activity of CYP3A by a positive allosteric modulator of mGlu5: In vitro to in vivo translation and potential impact on clinically relevant drug-drug interactions

Article abstract of DOI:10.1124/dmd.113.052662

Allosteric modulation of G protein-coupled receptors has gained considerable attention in the drug discovery arena because it opens avenues to achieve greater selectivity over orthosteric ligands. We recently identified a series of positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGlu₅) for the treatment of schizophrenia that exhibited robust heterotropic activation of CYP3A4 enzymatic activity. The prototypical compound from this series, 5-(4-fluorobenzyl)-2-((3-fluorophenoxy)methyl)-4,5,6, 7-tetrahydropyrazolo[1,5-a]pyrazine (VU0448187), was found to activate CYP3A4 to >100% of its baseline intrinsic midazolam (MDZ) hydroxylase activity in vitro; activation was CYP3A substrate specific and mGlu₅ PAM dependent. Additional studies revealed the concentration-dependence of CYP3A activation by VU0448187 in multispecies hepatic and intestinal microsomes and hepatocytes, as well as a diminished effect observed in the presence of ketoconazole. Kinetic analyses of the effect of VU0448187 on MDZ metabolism in recombinant P450 or human liver microsomes resulted in a significant increase in V_max (minimal change in K_m) and required the presence of cytochrome b₅. The atypical kinetics translated in vivo, as rats receiving an intraperitoneal administration of VU0448187 prior to MDZ treatment demonstrated a significant increase in circulating 1- and 4-hydroxy- midazolam (1-OH-MDZ, 4-OH-MDZ) levels compared with rats administered MDZ alone. The discovery of a potent substrate-selective activator of rodent CYP3A with an in vitro to in vivo translation serves to illuminate the impact of increasing intrinsic enzymatic activity of hepatic and extrahepatic CYP3A in rodents, and presents the basis to build models capable of framing the clinical relevance of substrate-dependent heterotropic activation. Copyright

Full text of DOI:10.1124/dmd.113.052662

Supplemental material to this article can be found at:
http://dmd.aspetjournals.org/content/suppl/2013/09/03/dmd.113.052662.DC1.html
1521-009X/41/12/2066–2075$25.00
DRUG METABOLISM AND DISPOSITION
http://dx.doi.org/10.1124/dmd.113.052662
Drug Metab Dispos 41:2066–2075, December 2013
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
Special Section on Prediction of Human Pharmacokinetic
Parameters from In Vitro Systems
Heterotropic Activation of the Midazolam Hydroxylase Activity of
CYP3A by a Positive Allosteric Modulator of mGlu5: In Vitro to In Vivo
Translation and Potential Impact on Clinically Relevant
Drug-Drug Interactionss
Anna L. Blobaum, Thomas M. Bridges, Frank W. Byers, Mark L. Turlington, Margrith E. Mattmann,
Ryan D. Morrison, Claire Mackie, Hilde Lavreysen, José M. Bartolomé, Gregor J. MacDonald,
Thomas Steckler, Carrie K. Jones, Colleen M. Niswender, P. Jeffrey Conn, Craig W. Lindsley,
Shaun R. Stauffer, and J. Scott Daniels
Drug Metabolism and Pharmacokinetics Laboratory (A.L.B., T.M.B., F.W.B., R.D.M., J.S.D.), Medicinal Chemistry Laboratory
(M.L.T., M.E.M., C.W.L., S.R.S.), and Molecular Pharmacology Laboratory (C.K.J., C.M.N., P.J.C.), Vanderbilt Center for
Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, Tennessee; CREATe ADME/Tox, (C.M.),
and Neuroscience (H.L., G.J.M., T.S.), Janssen Research and Development, Beerse, Belgium; and Jarama 75, Toledo,
Spain (J.M.B.)
Received May 2, 2013; accepted August 30, 2013
ABSTRACT
Allosteric modulation of G protein-coupled receptors has gained in the presence of ketoconazole. Kinetic analyses of the effect of
considerable attention in the drug discovery arena because it opens VU0448187 on MDZ metabolism in recombinant P450 or human liver
avenues to achieve greater selectivity over orthosteric ligands. We microsomes resulted in a significant increase in Vmax (minimal change
recently identified a series of positive allosteric modulators (PAMs) of in Km) and required the presence of cytochrome b5. The atypical
metabotropic glutamate receptor 5 (mGlu5) for the treatment of kinetics translated in vivo, as rats receiving an intraperitoneal ad-
schizophrenia that exhibited robust heterotropic activation of CYP3A4 ministration of VU0448187 prior to MDZ treatment demonstrated
enzymatic activity. The prototypical compound from this series, 5-(4- a significant increase in circulating 1- and 4-hydroxy- midazolam
fluorobenzyl)-2-((3-fluorophenoxy)methyl)-4,5,6,7-tetrahydropyrazolo[1,
(1-OH-MDZ, 4-OH-MDZ) levels compared with rats administered
5-a]pyrazine (VU0448187), was found to activate CYP3A4 to >100% MDZ alone. The discovery of a potent substrate-selective activator of
of its baseline intrinsic midazolam (MDZ) hydroxylase activity in vitro; rodent CYP3A with an in vitro to in vivo translation serves to illuminate
activation was CYP3A substrate specific and mGlu5 PAM dependent. the impact of increasing intrinsic enzymatic activity of hepatic and
Additional studies revealed the concentration-dependence of CYP3A extrahepatic CYP3A in rodents, and presents the basis to build mod-
activation by VU0448187 in multispecies hepatic and intestinal mi- els capable of framing the clinical relevance of substrate-dependent
crosomes and hepatocytes, as well as a diminished effect observed heterotropic activation.
Introduction
The neurotransmitter, L-glutamate, modulates cell excitability and
synaptic transmission via neuromodulatory glutamate receptors, called
This research was supported in part by the National Institutes of Health
National Institute of Mental Health [Grants R01-MH062646 and R01-MH89870]
metabotropic glutamate (mGlu) receptors, in the mammalian central ner-
and the National Institutes of Health National Institute of Neurological Disorders
vous system. The mechanisms by which mGlu receptors are activated, the
and Stroke [Grant R01-NS031373]; and an industry-sponsored contract from
proteins with which they interact, and the downstream second-messenger
Johnson & Johnson.
pathways involved in the signaling of these receptors have been exten-
sively investigated. Both orthosteric and allosteric small molecule ligands
dx.doi.org/10.1124/dmd.113.052662.
s This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: 1-OH-MDZ, 1-hydroxy midazolam; 4-OH-MDZ, 4-hydroxy midazolam; 9EP, 9-ethynylphenanthrene; CPHP, 4-(4-chlorophenyl)-
4-hydroxy piperidine; DDI, drug-drug interaction; HAL, haloperidol; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry;
MDZ, midazolam; mGlu5, metabotropic glutamate receptor subtype 5; P450, cytochrome P450; PAM, positive allosteric modulator; RHAL, reduced
haloperidol; VU0448187, 5-(4-fluorobenzyl)-2-((3-fluorophenoxy)methyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine; VU0464797, 2-(phenoxymethyl)-N-
(pyridin-3-ylmethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-4-amine).
2066

Heterotropic Activation of CYP3A by a Novel mGlu5 PAM
2067
can modulate the activity of the mGlu receptors; however, greater preliminary data suggesting that VU0448187 can activate CYP3A4-
selectivity, and often potency, can be achieved with positive allosteric mediated metabolism of haloperidol in human liver microsomes, suggests
modulators (PAMs) or negative allosteric modulators of each mGlu a potential to elicit a clinically relevant DDI that is a result of an acute
receptor. Due to the widespread distribution of mGlu receptors in the increase in the enzymatic activity of a drug-metabolizing enzyme.
central nervous system, these receptors represent attractive targets for
therapeutic intervention into a wide variety of neurologic and psy-
chiatric disorders such as Alzheimer’s disease, anxiety, depression,
Materials and Methods
Parkinson’s disease, and schizophrenia (Niswender and Conn, 2010).
We and others have developed selective PAMs for mGlu5 that have
potential utility in the treatment of schizophrenia and other disorders
that involve impaired cognitive function (Moghaddam, 2004; Conn
et al., 2009), as evidenced by the effects these compounds produce in
animal models of psychosis and cognition (Kinney et al., 2005; Darrah
et al., 2008; Liu et al., 2008).
Similar to other disease states, patients receiving treatment for schizo-
phrenia are commonly subjected to polypharmacy approaches intended
to alleviate the variety of symptoms of their disease. Many of these drugs,
Chemicals and Enzyme Sources. NADPH, MDZ, 1-OH-MDZ, 4-OH-
MDZ, HAL, reduced haloperidol (RHAL), 4-(4-chlorophenyl)-4-hydroxy
piperidine (CPHP), testosterone, 6b-OH-testosterone, progesterone, 6b-OH-
progesterone, phenacetin, acetaminophen, diclofenac, 4-OH-diclofenac, dextro-
methorphan, dextrorphan, ketoconazole, and miconazole were all purchased from
Sigma-Aldrich (St. Louis, MO). VU0448187 and VU0464797 (2-(phenoxymethyl)-
N-(pyridin-3-ylmethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-4-amine) (Fig. 1A)
were synthesized internally at the Vanderbilt Center for Neuroscience Drug
Discovery (Supplemental Schemes 1 and 2) with purity and structural charac-
terization confirmed by nuclear magnetic resonance. Human liver microsomes
(150-donor pool, mixed sex), human intestinal microsomes (20-donor pool, mixed
including haloperidol (HAL), are metabolized either exclusively or to sex), male murine microsomes, and rat liver microsomes were all purchased from
BD Biosciences (Woburn, MA). Human (20-donor pool, mixed sex), male
Sprague-Dawley rat, and male CD-1 mouse hepatocytes were purchased from
Celsis In Vitro Technologies (Baltimore, MD). The cDNA-expressed CYP3A4
and CYP3A5 enzymes with reductase and with or without cytochrome b5
(EasyCyps) were purchased from XenoTech, LLC (Kansas City, MO).
Inhibition or Activation Assays. A four-in-one (cocktail) assay for deter-
mining IC50 values against human CYP1A2, CYP2C9, CYP2D6, and CYP3A4 was
originally developed based on previous reports (Youdim et al., 2008) and used for
high-throughput screening of potential P450 inhibitors. Human liver microsomes
(final concentration of 0.1 mg/ml) and a substrate mixture containing the P450 probe
a great extent by CYP3A4 (Fang et al., 2001; Kalgutkar et al., 2003;
Avent et al., 2006). CYP3A4 is predominantly expressed in the liver, and
to a lesser extent in the intestine, and is involved in the metabolism
of a wide variety of drugs, xenobiotics, and steroids (Thummel and
Wilkinson, 1998; Guengerich, 2006). Significant clinical drug-drug
interactions (DDIs) have been observed in humans due to the inhibition
of CYP3A4 by other concomitantly administered drugs (Lin and Lu,
1998) or naturally occurring components of herbal remedies or fruits
(i.e., the grapefruit juice effect). CYP3A4 is also subject to induction by
a wide variety of ligands (Lin and Lu, 1998), resulting in a significant substrates phenacetin (10 mM), diclofenac (5 mM), dextromethorphan (5 mM), and
MDZ (2 mM) were added to a potassium phosphate-buffered solution (0.1 M, pH
7.4) and warmed to 37°C. The reaction mixture was divided evenly into a 96-well
plate and various dilutions of each inhibitor/compound of interest (in duplicate) were
increase in the levels of protein that are expressed in tissues. Generally,
the inhibition or induction of CYP3A4 has received the most attention
with regard to clinically relevant DDIs (Hutzler et al., 2005; Hafner et al.,
2010). However, the intrinsic enzymatic activity of the cytochrome
P450 (P450) family, including CYP3A4, has also been reported to be
significantly affected in vitro (and, in only a few examples, in vivo)
through homotropic or heterotropic activation of the enzyme (Hutzler
and Tracy, 2002; Obach, 2012). A wide variety of compounds and
known drugs have been shown to be activators of CYP3A4 enzymatic
activity, including 7,8-benzoflavone (Shou et al., 1994), quinidine
(Ngui et al., 2000), and steroids (Henshall et al., 2008). The activation of
CYP3A4 metabolic activity in vivo may represent an acute DDI scenario
that could increase the clearance of a parent drug and/or increase levels of
circulating, and perhaps toxic or active, metabolites.
Herein, we report the discovery of a novel mGlu5 PAM, 5-(4-
fluorobenzyl)-2-((3-fluorophenoxy)methyl)-4,5,6,7-tetrahydropyrazolo[1,
5-a]pyrazine (VU0448187), that significantly activated the midazolam
(MDZ) hydroxylase activity of CYP3A4 in multispecies hepatic and
intestinal microsomes and hepatocytes. Interestingly, this effect ap-
pears to be substrate dependent, as the CYP3A4-mediated hydroxyla-
tion of testosterone and progesterone was unaffected by VU0448187. In
addition, the effects of these mGlu5 PAMs on MDZ hydroxylase
activity by CYP3A4 appear to be structure dependent, as an mGlu5
PAM from a second distinct structural series was shown to be an
inhibitor of CYP3A4-mediated metabolism of MDZ to its 1-hydroxy
metabolite (1-OH-MDZ). Furthermore, ketoconazole, a potent and
selective inhibitor of CYP3A4, can block the ability of VU0448187 to
activate CYP3A MDZ hydroxylase activity in vitro. Kinetic studies of
MDZ metabolism with human liver microsomes and recombinant
CYP3A4 and CYP3A5 demonstrated that VU0448187 increases Vmax
(with minimal changes in Km) and requires the presence of cytochrome
b5. Importantly, we were able to show that the activation of CYP3A MDZ
metabolism to either 1-OH-MDZ or 4-OH-MDZ by VU0448187 could
Fig. 1. (A) Chemical structures of VU0448187 and VU0464797: two structurally
distinct, but active PAMs of human metabotropic glutamate receptor 5 (hmGlu5).
be translated to an in vivo rodent model. This observation, along with (B) The metabolism of MDZ by CYP3A4 in vitro.

2068
Blobaum et al.
then added to this reaction mixture such that the final concentration of each was serially collected over EDTA at 0.0333, 0.117, 0.25, 0.5, 1, 3, 7, and 24 hours
compound ranged from 100 nM to 30 mM. This mixture was allowed to preincubate postdose and plasma was isolated via centrifugation (4000 rpm, 4°C) for storage at
for 15 minutes while shaking at 37°C. Buffer or NADPH (1 mM) was added and the
reaction mixture was incubated for an additional 8 minutes at 37°C prior to
quenching with two volumes of ice-cold acetonitrile containing 50 nM carba-
mazepine (internal standard). The plates were centrifuged at 4000 rpm (4°C)
for 10 minutes and the supernatant was removed and diluted with water (1:4, v/v)
280°C until LC-MS/MS analysis. The sample extraction of plasma was performed
by protein precipitation employing three volumes of ice-cold acetonitrile containing
50 nM carbamazepine (internal standard). The samples were centrifuged (4000
rpm, 4°C) and the supernatants were transferred and diluted 1:1 (v/v) for LC-MS/
MS analysis. The final pharmacokinetic parameters for MDZ (clearance, CLp; half-
in preparation for liquid chromatography coupled to tandem mass spectrometry life, t1/2; volume of distribution predicted at steady state, Vss) were calculated by
(LC-MS/MS) analysis. The IC50 values for each compound were obtained for the noncompartmental analysis employing WinNonLin software (Phoenix, version 6.2;
individual P450 enzymes by quantitating the inhibition of metabolite formation Pharsight Inc., Mountain View, CA). Alternatively, VU0448187 was prepared in
for each probe substrate against a 12-point standard curve specific for each
10% Tween 80 (aq) for intraperitoneal administration (i.p.) of a 10 mg/kg dose to
metabolite: acetaminophen (CYP1A2), 4-OH-diclofenac (CYP2C9), dextro- dual-cannulated Sprague-Dawley male rats (n = 2) 1 hour prior to a 10 mg/kg i.p.
rphan (CYP2D6), and 1-OH-MDZ (CYP3A4). A 0 mM compound condition (or
control) was set to 100% enzymatic activity and the effect of increasing test
compound concentrations on enzymatic activity could then be calculated from
the percentage of control activity. Curves were fitted using XLfit 5.2.2 (four-
dose of MDZ. Blood was collected at various times postdose and handled as
described above for LC-MS/MS analysis. VU0448187, MDZ, 1-OH-MDZ, and
4-OH-MDZ levels were simultaneously monitored from each experiment and the
levels of each analyte were quantified against a 10-point standard curve.
parameter logistic model, Eq. 201) to determine the concentration that produces Concentrations were reported as nanograms per milliliter.
half-maximal inhibition (IC50). Miconazole was included as a positive control
for broad spectrum P450 inhibition (pan-P450 inhibitor). Experiments were
performed in duplicate unless noted otherwise.
LC-MS/MS Analysis. In vitro and in vivo samples were analyzed via
electrospray ionization LC-MS/MS on an AB Sciex API-4000 (Foster City, CA)
triple-quadrupole instrument that was coupled with Shimadzu LC-10AD pumps
Discrete assays for monitoring the activation or inhibition of CYP3A4 enzymatic (Columbia, MD) and a Leap Technologies CTC PAL autosampler (Carrboro,
activity in human, rat, or mouse liver and intestinal microsomes or hepatocytes were NC). Analytes were separated by gradient elution using a thermostated (40°C)
performed similarly to that described above, except that only one substrate was
present in any given assay and levels of substrate-specific metabolites were ex-
C18 column (Fortis 3.0 ꢀ 50 mm, 3 m; Fortis Technologies Ltd, Cheshire, UK).
Mobile phase A was 0.1% formic acid (aq) and mobile phase B was 0.1% formic
clusively monitored. Final concentrations of the substrates MDZ (2 mM), tes- acid in acetonitrile. The gradient started at 10% B with a 0.2-minute hold and was
tosterone (25 mM), progesterone (50 mM), and HAL (50 mM) were chosen to be then linearly increased to 90% over 1.3 minutes, followed by a return to 10% B in
reflective of reported Km values for CYP3A4 and the protein concentration, time of 0.1 minutes and subsequent re-equilibration (0.9 minutes). The total run time was
incubation with NADPH (when necessary), and reconstitution ratios for LC-MS/MS 2.5 minutes and the high-performance liquid chromatography flow rate was
analysis were optimized for each substrate and metabolite combination. Levels of 0.5 ml/min. The source temperature was set at 500°C and mass spectral analyses
each metabolite (1-OH or 4-OH-MDZ, 6b-OH-testosterone, 6b-OH-progesterone, were performed using multiple reaction monitoring, with transitions specific for
and CPHP) were quantitated against a 12-point standard curve and data were rep- each compound utilizing a Turbo-Ionspray source in positive ionization mode
resented as the percentage of control activity remaining. Experiments in human liver
microsomes involving ketoconazole (1 mM) were performed by preincubating
ketoconazole for 5 minutes prior to the addition of VU0488187 for 15 minutes and
then NADPH (1 mM) for an additional 8 minutes. Hepatocytes were thawed per the
vendor’s instructions, and incubations were conducted with 0.5 ꢀ 106 cells/ml.
Kinetic Analysis of CYP3A4 Activation. Studies to monitor the effect of
VU0448187 (10 mM) on the Km and Vmax for MDZ hydroxylation to 1-OH-MDZ
were performed in potassium phosphate-buffered solution (0.1 M, pH 7.4) with 5 mM
MgCl2. MDZ was dissolved in methanol and diluted over a range of concentrations.
Final protein concentrations were 0.5 mg/ml (human liver microsomes) and 5 pmol/ml
for CYP3A4 and CYP3A5 (EasyCYPs coexpressed with reductase and with or
without cytochrome b5) and the assay was optimized for linearity or per the vendor’s
instructions. A premixture composed of human liver microsomes or recombinant
CYP3A4 or CYP3A5 in assay buffer was distributed to tubes on ice that contained
either varied concentrations of MDZ alone or MDZ with VU0448187. Every 15
seconds, a tube was placed in the 37°C water bath so that all tubes were preincubated
for 5 minutes prior to the addition of NADPH (1 mM final) for an additional
5 minutes. Reactions were quenched with ice-cold acetonitrile containing 50 nM
carbamazepine (internal standard). The plates were centrifuged at 4000 rpm (4°C)
for 10 minutes and the supernatant was removed and diluted with water (1:1, v/v)
in preparation for LC-MS/MS analysis. 1-OH-MDZ levels were quantitated
against a 12-point standard curve and the data were fit to either Michaelis-Menten
(Eq. 1) or substrate-inhibition (Eq. 2) equations and represented as nanomoles per
minute per milligram of protein (human liver microsomes) or picomoles per
minute per picomole of P450 (CYP3A4 or CYP3A5). Because MDZ metabolism
undergoes substrate inhibition at higher concentrations (Martínez et al., 2000;
Khan et al., 2002), it was decided to report all data using Eq. 2 for completeness.
Experiments were n = 2 or more.
(5.0 kV spray voltage). All data were analyzed using AB Sciex Analyst 1.5.1
software.
Results
Assessment of CYP3A-Mediated MDZ Hydroxylase Activity in
Hepatic and Intestinal Microsomes and Hepatocytes. A mixed-age
and mixed-sex hepatic microsomal pool of 150 human donors was used
to monitor the effect of increasing concentrations of VU0448187 and
VU0464797 (Fig. 1A) on the ability of four major drug-metabolizing
P450 enzymes (CYP1A2, CYP2C9, CYP2D6, and CYP3A4) to me-
tabolize their respective probe substrates. Using a cocktail (four-in-one)
approach in human liver microsomes, we observed that VU0448187
displayed little to no effect on the enzymatic activities of CYP1A2 or
CYP2D6 (IC50 . 30 mM) and that the activity of CYP2C9 was
minimally affected (IC50 = 20 mM; inhibition of 4-OH-diclofenac
formation) (Fig. 2A). Interestingly, we observed an increase in the
CYP3A4-mediated metabolism of the probe substrate MDZ to its 1-OH-
MDZ metabolite in the presence of VU0448187. The CYP3A4 activation
discovered was concentration dependent and the maximal effect occurred
at 10 mM VU0448187 (Fig. 2A). To delineate what appeared to be
a CYP3A4-specific effect by VU0448187, we moved to a discrete as-
sessment of MDZ metabolism in human liver microsomes. Importantly, a
comparison of the data in Fig. 2, A and B, demonstrates that VU0448187
acts directly on the intrinsic activity of CYP3A4 to increase MDZ
metabolism. In our initial metabolic assessment of a series of mGlu5
PAMs, we discovered a structure-activity relationship of the substrate-
specific activation of CYP3A4 in human liver microsomes. As shown in
Fig. 2C, a structurally distinct mGlu5 PAM (VU0464797) does not
activate MDZ hydroxylase activity but, in fact, inhibits the CYP3A4-
mediated conversion of MDZ to 1-OH-MDZ in human liver microsomes.
Therefore, we investigated the ability of VU0448187 to activate
CYP3A metabolism of MDZ in multispecies and varied tissues (Fig. 3).
Y ¼ Vmax ꢀ X=ðKm þ XÞ
ð1Þ
ð2Þ
Y ¼ Vmax ꢀ X=ðKm þ X ꢀ ð1 þ X=KiÞÞ
In Vivo Pharmacokinetic Studies with MDZ. MDZ is metabolized by
CYP3A in vitro to 1-OH-MDZ and 4-OH-MDZ (Fig. 1B) (Kronbach et al., 1989).
MDZ was prepared as a solution in 10% ethanol/50% PEG-400/40% saline and
dosed intravenously (i.v.) at 1 mg/kg to dual-cannulated male Sprague-Dawley rats
(n = 2) or male CD-1 mice (n = 2) (Harlan Laboratories, Indianapolis, IN). Blood Interestingly, we found that increasing concentrations of VU0448187

Heterotropic Activation of CYP3A by a Novel mGlu5 PAM
2069
model for examining the effect of VU0448187 on CYP3A activity in
vivo.
As VU0464797 was found to be an inhibitor of CYP3A4 in human
liver microsomes (Fig. 2), we examined the effect of increasing con-
centrations of this mGlu5 PAM in human and rat hepatocytes. Data
provided in Fig. 4 demonstrate that VU0464797 dose-dependently
inhibits the conversion of MDZ to 1-OH-MDZ in hepatic microsomes
and hepatocytes from multiple species. Validating VU0464797 as a
structurally distinct mGlu5 PAM (negative control) that has a different
reaction phenotype to VU0448187 demonstrates that not all mGlu5
PAMs exhibit this CYP3A4 activation phenomenon and that any
potential acute effects caused by the activation of the intrinsic enzymatic
activity are likely drug and/or compound specific.
The Effects of a Specific CYP3A4 Inhibitor on VU0448187-
Mediated Enhancement of Enzymatic Activity and Observations
with Alternative Probe Substrates. We investigated the ability of
ketoconazole, a potent and selective inhibitor of CYP3A4, to inhibit the
activity of this enzyme in human liver microsomes in the presence of
increasing concentrations of VU0448187. Data in Fig. 5A show that
ketoconazole (at 1 mM) can significantly block the VU0448187-
mediated activation of MDZ hydroxylation, indicating that CYP3A4
and/or CYP3A5 are the targets of this heterotropic activator in human
liver microsomes and not other P450 enzymes. Although we cannot
confirm that the effects of the activator are mediated through CYP3A4
or CYP3A5 alone, our data are consistent with historical accounts that
the effects of a heterotropic activator (e.g., VU0448187) are likely
mediated through the enzyme and not simply through a direct in-
teraction of the activator with other partner proteins (e.g., reductase)
(Hutzler and Tracy, 2002; Keubler et al., 2012) In addition, the block-
ade of VU0488187-mediated CYP3A4 activation with MDZ as the probe
substrate can be titrated with varying concentrations of ketoconazole
(data not shown).
Understanding that CYP3A4 may catalyze the oxidation of multiple
substrates at topologically distinct sites within the protein, we investigated
the ability of VU0448187 to activate the metabolism of testosterone
and progesterone, two substrates known to bind to distinct sites within
CYP3A4. The results displayed in Fig. 5 demonstrate that the en-
hancement of CYP3A4 enzymatic activity in human liver microsomes
by VU0448187 is substrate-dependent, because the testosterone (Fig.
5B) and progesterone (Fig. 5C) hydroxylase activities of CYP3A4
(conversion to 6b-OH metabolites) were not activated by increasing
concentrations of VU0448187. In fact, we observed a slight dose-
Fig. 2. The effect of increasing concentrations of VU0448187 or VU0464797 on
the metabolic activity of CYP1A2, CYP2C9, CYP2D6, and CYP3A4 in human liver
microsomes from a 150-donor pool using a cocktail approach to assay for enzymatic
activity or inhibition (A) or a discrete assay in human liver microsomes monitoring
the conversion of MDZ to 1-OH-MDZ for CYP3A4 activity (B and C).
could dose-dependently activate the metabolism of MDZ to its dependent inhibition of testosterone and progesterone metabolism by
1-hydroxy (1-OH) metabolite in both mixed donor pools of human VU0448187 in human liver microsomes. These data underscore the
intestinal microsomes (Fig. 3B) and human hepatocytes (Fig. 3C). In importance of testing multiple potential perpetrators and victims when
fact, the activation of CYP3A activity in hepatocytes by VU0448187 examining atypical enzyme kinetics in vitro and further confirm
was significantly greater than that originally observed in human liver previous literature reports supporting the notion that CYP3A allostery
microsomes (Fig. 3A) with activation continuing out to 30 mM is substrate dependent (Wang et al., 2000).
(approximately 400% activity of control) and increased levels of 1-OH-
The Kinetics of CYP3A4 Activation of MDZ 1-Hydroxylase
MDZ were measured at the lowest concentration of VU0448187 tested Activity in cDNA-Expressed CYP3A4 and CYP3A5 and Human
(100 nM). The CYP3A activation effect observed with VU0448187 in Liver Microsomes. Previous studies examining the effect of efavirenz
hepatocytes versus that in human liver microsomes may be a direct on the metabolic activation of CYP3A4 MDZ metabolism found that the
result of a more complete physiologic test system (all proteins, co- Km of MDZ was relatively unaffected by the presence of efavirenz, but
factors at physiologically relevant expression and concentrations, that the Vmax significantly increased (Keubler et al., 2012). Therefore, we
respectively). The concentration-dependent effects of VU0448187 on investigated the impact of VU0448187 on the binding affinity (Km) and
CYP3A activity were also observed in rat hepatocytes (Fig. 3D) and catalytic efficiency (Vmax) of MDZ 1-hydroxylation in recombinant
hepatic microsomes (data not shown) and in murine liver microsomes CYP3A4 and CYP3A5 (with and without cytochrome b5), as well as in
(Fig. 3E) and hepatocytes (data not shown). These in vitro data pro- human liver microsomes. Not surprisingly, the kinetics of MDZ me-
vided confidence that our observations were not species specific, and tabolism (1-OH-MDZ formation) was linear at concentrations up to, but
that VU0448187 was membrane permeable and targeted CYP3A activity not exceeding, 10 mM; as expected, we observed substrate-inhibition
in hepatocytes from multiple species and distinct tissues. Together, these kinetics at higher concentrations (100 mM) (Martínez et al., 2000;
results provided support for the selection of an appropriate preclinical Lin et al., 2001; Khan et al., 2002). Consistent with previous accounts of

2070
Blobaum et al.
Fig. 3. The activation of MDZ 1-hydroxylase activity in pools of human liver microsomes (A), human intestinal microsomes (B), human hepatocytes (C), rat hepatocytes
(D), and murine liver microsomes (E) by increasing concentrations of VU0448187. Human donor pools were mixed sex/mixed age; rat and murine pools were male Sprague-
Dawley and male CD-1, respectively.
efavirenz-mediated activation, the presence of VU0448187 (10 mM) Although it is unclear at this time what precise role cytochrome b5
activated the maximal product formation rate (Vmax) by 1.2- to 1.4-fold in plays in the activation of CYP3A4-mediated MDZ metabolism by
CYP3A4 and CYP3A5 and 2.8-fold in human liver microsomes, with VU0448187 (or efavirenz), it is possible that cytochrome b5
little to no change in Km for cDNA-expressed CYP3A4 (Fig. 6 and induces a conformational change in CYP3A4 or CYP3A5 (Yamazaki
Table 1), CYP3A5 (data not shown) and in human liver microsomes (Fig. et al., 1996) or increases the coupling between the P450 protein and
6C and Table 1). The increase in Vmax associated with VU0448187 in the reductase (Lee et al., 1997; Locuson et al., 2007), such that the
recombinant P450 assay was dependent on the presence of cytochrome metabolism is enhanced by the presence of VU0448187.
b5 (Fig. 6, A and B), and the overall intrinsic enzymatic activity of either
MDZ Pharmacokinetics and In Vivo Models of VU0448187
CYP3A4 or CYP3A5 was higher with cytochrome b5. Importantly, P450 Activation of CYP3A in Rodents. Although MDZ hepatic clearance
enzymes have been reported to display compound-dependent variability in the rat is dependent upon the specific rat strain used for a given
in metabolic activity (or inhibition profiles) when reactions are fortified study, it is also well known that MDZ is generally a high-clearance
with cytochrome b5 (Jushchyshyn et al., 2005). In fact, efavirenz compound (55–80 ml/min per kilogram) in rat (Kotegawa et al., 2002)
activation of CYP3A4-mediated MDZ metabolism was also shown to be with the predominant metabolic pathways being CYP3A-mediated
dependent on the presence of cytochrome b5 (Keubler et al., 2012). conversion to 1-OH-MDZ and 4-OH-MDZ (at near equal amounts). In

Heterotropic Activation of CYP3A by a Novel mGlu5 PAM
2071
Fig. 5. (A) The effect of a specific CYP3A4 inhibitor, ketoconazole (1 mM), on the
activation of MDZ 1-hydroxylase activity in human liver microsomes by
VU0448187. (B and C) The effect of increasing concentrations of VU0448187 on
the hydroxylase activity of alternative CYP3A4 substrates testosterone (25 mM) and
progesterone (50 mM).
Fig. 4. The inhibition of MDZ 1-hydroxylase activity in pools of human liver
microsomes (A), human hepatocytes (B), and rat hepatocytes (C) by increasing
concentrations of VU0464797. Human donor pools were mixed sex/mixed age; rat
pools were male Sprague-Dawley.
(25 mM) displayed no inhibition of the glucuronidation of 1-OH-MDZ in
vitro in rat liver microsomes or rat hepatocytes (data not shown). In
a follow-up experiment in which VU0448187 (10 mg/kg i.p.) pretreated
rats received a single dose of MDZ (10 mg/kg i.p.), we also observed
a time-dependence in the increased formation of circulating levels of
1-OH and 4-OH-MDZ (Fig. 7B). Specifically, the time-concentration data
displayed in Fig. 7B indicated that compared with the rats receiving MDZ
alone, 1-OH-MDZ metabolite concentrations were maximal in the plasma
shortly after rats received an i.p. administration of MDZ (apparent Tmax of
7 minutes). Importantly, the concentrations of 1-OH-MDZ returned to
plasma levels that were observed in vehicle-treated rats within 2 hours, an
observation that is consistent with the notion that CYP3A activation of
MDZ hydroxylase activity is a transient effect dependent on VU0448187
exposure.
a single time point experiment in which rats (n = 2) were pretreated
with vehicle or VU0448187 (10 mg/kg i.p., 1 hour) followed by MDZ
administration (10 mg/kg i.p.), we observed a significant increase in
concentrations (in nanograms per milliliter) of both 1-OH-MDZ (Fig. 7A)
and 4-OH-MDZ (data not shown) at 15 minutes after MDZ treatment.
The 1-OH-MDZ metabolite was observed in plasma at approximately
5 ng/ml in rats treated with MDZ alone versus concentrations approaching
125 ng/ml in rats that were pretreated with VU0448187. The increase in
the metabolism of MDZ (approximately a 25-fold increase in 1-OH-MDZ
production) exceeded the fold increase that was predicted from the results
generated in vitro in rat liver microsomes or rat hepatocytes. Importantly,
the increase in 1-OH-MDZ that we observed in rodents was not an effect
stemming from the inhibition of the glucuronidation of this metabolite by
the mGlu5 PAM, because LC/MS analysis indicated that VU0448187

2072
Blobaum et al.
Fig. 7. Increases in 1-OH-MDZ levels in Sprague-Dawley rats after pre-treatment
with VU0448187. Single time point measurement (A) or time course (B) of 1-OH-
MDZ levels in male Sprague-Dawley rats (n=2) that were administered a 10 mg/kg i.p.
dose of MDZ after a 1-hour pretreatment with either vehicle or a 10 mg/kg i.p. dose of
VU0448187.
Fig. 6. The kinetics of CYP3A4 activation of 1-OH-MDZ activity by VU0448187
in recombinant systems with (B) or without (A) cytochrome b5 or human liver
microsomes (C). Km and Vmax parameters were determined by fitting the data to an
equation for substrate inhibition. The metabolite formation rate is expressed as
picomoles per minute per picomole of P450 for the recombinant P450 or as
nanomoles per minute per milligram of P450 for human liver microsomes.
Clinical Relevance of Heteroactivation of CYP3A4 Activity in
Human Liver Microsomes. The neuroleptic agent HAL is metabolized
nearly exclusively by CYP3A4 to multiple metabolites, including RHAL,
CPHP, HP+ (4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-pyr-
idinium), and RHP+ (4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-hydroxy-
butyl]-pyridinium) (Fang et al., 2001; Kalgutkar et al., 2003; Avent
et al., 2006). Importantly, HAL was also found to be back-oxidized
from RHAL in vitro. To determine the impact of VU0448187-mediated
CYP3A4 activation on the metabolism of HAL, we monitored the
conversion of HAL to its primary oxidized metabolite, CPHP, in human
liver microsomes. Because our data utilizing MDZ as the CYP3A4
probe substrate (victim) indicated that the resulting enzymatic acti-
vation was perpetrator specific, we conducted the experiments with
HAL in human liver microsomes in the presence or absence of
VU0448187 (CYP3A4 activator), VU0464797 (CYP3A4 inhibitor),
and miconazole (pan-P450 inhibitor). We observed that both
VU0464797 (10 mM) and miconazole (1 mM) significantly inhibited
the metabolism of HAL to CPHP (Fig. 8), whereas VU0448187 (10 mM)
enhanced CPHP product formation at an approximate 20% increase over
the control experiment. Although the extent of activation of CYP3A4
activity was not as significant as what was observed for MDZ hydrox-
ylation, the observed activation of a clinically relevant CYP3A4 substrate
is noteworthy. Likewise, compounds that inhibited MDZ hydroxylation in
human liver microsomes also showed inhibitory profiles with HAL. These
data suggest a substrate dependence to heteroactivation and imply that
drugs displaying heteroactivation of enzymatic activity in vitro may
mediate this effect in vivo.
TABLE 1
Kinetic effects of VU0448187 heteroactivation of CYP3A4 MDZ activity
Assay Condition
Vmax
CYP3A4
CYP3A4 + VU0448187
CYP3A4/b5
CYP3A4/b5 + VU0448187
Human liver microsomes
Human liver microsomes + VU0448187
5.30 6 0.26
5.31 6 0.14
7.35 6 0.43
10.1 6 0.21
1.16 6 0.08
3.29 6 0.16
Changes in Vmax for the conversion of MDZ to 1-OH-MDZ in the absence and presence of
VU0448187 (10 mM) for cDNA-expressed CYP3A4 and reductase with and without cytochrome
b5 and in human liver microsomes. The metabolite formation rate is expressed as picomoles per
minute per picomole of P450 for the recombinant P450 or as nanomoles per minute per milligram
of P450 for human liver microsomes.

Heterotropic Activation of CYP3A by a Novel mGlu5 PAM
2073
the F’ helix, b1–b2 loop region, and b4 loop; these and other protein
coordinates were previously described as a potential allosteric site in-
volved in the heterotropic activation of substrate-specific P450 enzymatic
activity (Harlow and Halpert, 1998; Davydov et al., 2012; Shah et al.,
2012). In the example of 9EP and the mechanism-based inactivation of
CYP2B4, the high-affinity site functions as an allosteric site to enhance
the efficiency of activation of the acetylenic group of 9EP and subsequent
covalent modification of the active site Thr302 residue. Considering the
well known consequences of P450 inhibition or mechanism-based in-
activation in vivo (adverse drug interactions and toxicity) and the
magnitude of the effects reported here, it is now prudent that the potential
for allosteric effects of a drug molecule either on activation and/or
inhibition of a P450 must also be considered during a preclinical DDI
assessment. The methods and studies described here served as an
example of an approach to define these types of interactions in vitro and
in animal models prior to clinical advancement.
A recent perspective has served to reintroduce the importance of the
kinetic phenomena of heterotropic activation (and atypical kinetics in
general) and its impact to clinical pharmacokinetics of coadministered
P450-metabolized drugs (Obach, 2012). Moreover, the perspective
highlighted the importance of establishing new approaches toward
the prediction of DDIs associated with atypical kinetics; particularly
noteworthy was the notion of identifying preclinical models capable of
mirroring the clinical setting. Importantly, there have been relatively
limited examples of translating observed in vitro heteroactivation to in
vivo animal models or humans. Tang et al. (1999) found an in vitro to
in vivo translation for the stimulation of diclofenac metabolism by
quinidine in monkeys; however, follow-up studies by Hutzler et al.
(2001a,b) on the observed robust in vitro activation of CYP2C9-mediated
flurbiprofen metabolism by dapsone showed only minimal effects in
vivo. More importantly, Yang et al., (2012) demonstrated evidence for
CYP3A allostery in healthy human volunteers by examining the in-
teraction between fluconazole and MDZ. In a clinical study, Bayer et al.
(2009) found that efavirenz rapidly increased MDZ metabolism, sug-
gesting an acute activation of CYP3A-mediated MDZ metabolism that
did not occur in a time frame suitable for induction of the enzyme via
mRNA transcription. Subsequent in vitro analyses confirmed that the
heteroactivation of CYP3A MDZ hydroxylase activity in human liver
microsomes and in recombinant CYP3A4 and CYP3A5 (Keubler et al.,
2012) was a Vmax-driven effect linked to the presence of cytochrome b5.
CYP3A4 is expressed in a variety of tissues, but is predominantly
localized to the liver and intestine. Recent focus has also been placed on
the metabolism and inhibition of CYP3A5, which is often expressed in
the same tissues as CYP3A4, but may or may not have overlapping
substrate specificities and/or show subtype-specific inhibition in vitro
(Gorski et al., 1994). In addition, rat and murine CYP3A catalytic activity
(or inhibition thereof) does not always translate to human CYP3A4 and
CYP3A5 activity (Komura and Iwaki, 2008). For example, there have
been reports (Kronbach et al., 1989; Perloff et al., 2000) indicating that
murine metabolism of MDZ correlates more closely to human metab-
olism (MDZ to 1-OH-MDZ) than rat (MDZ to 1-OH-MDZ and 4-OH-
MDZ in equal quantities). More recently, studies in humanized CYP3A4
transgenic mice using triazolam have shown stimulation of intestinal and
hepatic CYP3A activity (van Waterschoot et al., 2009). In addition,
Yamazaki et al. (2013) investigated the in vivo drug interaction between
MDZ and thalidomide in mice with humanized livers and found a
significant increase in the area under the curve for the production of
1-OH-MDZ in mice that were coadministered MDZ (intravenously) and
thalidomide (orally). In an attempt to find alternative rodent models to
examine the activation of CYP3A activity in vivo by VU0448187, we
examined the hepatic clearance of MDZ in male CD-1 mice (n = 2;
1 mg/kg i.v.). The hepatic clearance of MDZ was moderate at 50 ml/min
Fig. 8. The activation or inhibition of CYP3A4 HAL metabolism in human liver
microsomes. VU0448187 (10 mM), VU0464797 (10 mM), or miconazole (1 mM)
were incubated with HAL (50 mM) and NADPH for 40 minutes and the conversion
of HAL to its metabolite, CPHP, was monitored.
Discussion
Although the metabolism of a particular substrate by a P450 enzyme is
best described employing Michaelis-Menten kinetics, there are many
instances of substrate-enzyme interactions that follow more atypical
kinetic profiles, such as homotropic or heterotropic activation, substrate
inhibition, partial inhibition, or biphasic metabolism (Korzekwa et al.,
1998; Hutzler and Tracy, 2002; Atkins, 2005; Egnell et al., 2005). One of
the earliest demonstrations of the ability of a P450 activator (i.e.,
perpetrator) to enhance the metabolism of a concomitantly administered
compound (i.e., victim) was the CYP3A4-mediated hydroxylation of
benzo[a]pyrene by a-naphthoflavone reported by Shou et al. (1994).
Although multiple P450 enzymes experience heterotropic activation,
the majority of examples of this phenomenon to date have involved
CYP3A4. Due to the plethora of liganded/nonliganded x-ray crystal
structures available with this enzyme (Williams et al., 2004; Yano et al.,
2004), as well as a predominance of kinetics, mutagenesis, and various
spectral titration studies or mathematical models (Domanski et al., 1998,
2000; Harlow and Halpert, 1998; Shou et al., 1999; Hosea et al., 2000;
Kenworthy et al., 2001; Khan et al., 2002), multiple hypotheses exist to
explain the ability of a perpetrator to enhance the metabolism of a victim
substrate. The perpetrator may either bind in a separate or overlapping
binding site in the enzyme active site or it may bind to a distinct allosteric
site of the enzyme to influence the metabolism of the victim substrate;
however, it is generally accepted that the victim drug must bind close to
the center heme prosthetic and in close proximity to the activated-oxygen
species. Although evidence exists to support both theories, it is well
accepted that the mechanism of heteroactivation may be dependent upon
the specific victim-perpetrator combination and the enzyme itself (due to
the relative size of the active site).
The contribution of allosteric binding is not limited to atypical enzyme
kinetics, such as heterotropic activation, but, in fact, has been linked to
the mechanism-based inactivation of CYP2B4 by 9-ethynylphenanthrene
(9EP) (Zhang et al., 2013). Zhang and colleagues found that quench-
ing of 9EP fluorescence by unmodified or 9EP-modified CYP2B4 re-
vealed two binding sites with distinct affinities, with the high-affinity site
located on the protein periphery. The suggested location of this high-
affinity site is at the entrance of a substrate access channel surrounded by

2074
Blobaum et al.
Domanski TL, Liu J, Harlow GR, and Halpert JR (1998) Analysis of four residues within
substrate recognition site 4 of human cytochrome P450 3A4: role in steroid hydroxylase
activity and alpha-naphthoflavone stimulation. Arch Biochem Biophys 350:223–232.
Egnell AC, Houston JB, and Boyer CS (2005) Predictive models of CYP3A4 Heteroactivation: in
vitro-in vivo scaling and pharmacophore modeling. J Pharmacol Exp Ther 312:926–937.
Fang J, McKay G, Song J, Remillrd A, Li X, and Midha K (2001) In vitro characterization of the
metabolism of haloperidol using recombinant cytochrome p450 enzymes and human liver
microsomes. Drug Metab Dispos 29:1638–1643.
per kilogram (hepatic blood flow ꢁ90 ml/min per kilogram) and
1-OH-MDZ was readily detected in mice at this dose employing LC-
MS/MS analysis (data not shown). Due to the similarities in the
metabolism of MDZ in humans and mice, these preliminary data will
serve to direct future studies in which potential changes in MDZ
clearance and exposure as well as circulating metabolite exposures can
be monitored in the presence of VU0448187.
Gorski JC, Hall SD, Jones DR, VandenBranden M, and Wrighton SA (1994) Regioselective
biotransformation of midazolam by members of the human cytochrome P450 3A (CYP3A)
subfamily. Biochem Pharmacol 47:1643–1653.
Historical data surrounding the homotropic and heterotropic activation
of CYP3A4 enzymatic activity in vitro indicate that these observations
are likely to be compound (or perpetrator) specific, as well as substrate
(or victim) specific (Shou et al., 1994; Ngui et al., 2000; Wang et al.,
2000; Kenworthy et al., 2001). CYP3A4 and CYP3A5 metabolize over
half of all known drugs on the market today (Thummel and Wilkinson,
1998). The likelihood of patients receiving drugs and herbal remedies
that are metabolized by this same enzymatic pathway is high (antibiotics,
antidepressants, birth control, etc); in particular, the chance to precipitate
a clinically relevant DDI in patients is significantly increased with disease
states (e.g., neurologic disorders, tissue malignancies, and AIDS) that
require concomitant administration of multiple medications, the metab-
olism of which may be catalyzed at topological sites within the CYP3A
protein that are distinct from the MDZ binding site. Understanding the
potential off-target activities of a drug or its metabolite(s) is crucial for
drug development and clinical success (Hutzler et al., 2005). The dis-
covery and development of mGlu5 PAMs represents a novel approach in
the treatment of schizophrenia. With the understanding that many patients
are subjected to polypharmacy, in addition to the deleterious issues of
patient compliance and drug overdose, the potential for DDIs (target- or
off-mediated) is increased in this particular patient group. Although it is
uncertain at this time whether the heterotropic activation of P450 enzymes
is clinically relevant in terms of DDI potential, it is important to un-
derstand the effects of this phenomenon on increasing the clearance of
parent drug and/or increasing circulating metabolite levels that may have
pharmacological or toxicological significance. We have shown here that
an in vitro observation of potent activation of CYP3A in multispecies
liver and intestinal fractions by a novel mGlu5 PAM can be translated to
rodents in vivo, subsequently enabling the modeling of atypical clinical
pharmacokinetics with relevant therapeutic agents.
Guengerich FP (2006) Cytochrome P450s and other enzymes in drug metabolism and toxicity.
AAPS J 8:E101–E111.
Hafner V, Jäger M, Matthée AK, Ding R, Burhenne J, Haefeli WE, and Mikus G (2010) Effect of
simultaneous induction and inhibition of CYP3A by St John’s Wort and ritonavir on CYP3A
activity. Clin Pharmacol Ther 87:191–196.
Harlow GR and Halpert JR (1998) Analysis of human cytochrome P450 3A4 cooperativity:
construction and characterization of a site-directed mutant that displays hyperbolic steroid
hydroxylation kinetics. Proc Natl Acad Sci USA 95:6636–6641.
Henshall J, Galetin A, Harrison A, and Houston JB (2008) Comparative analysis of CYP3A
heteroactivation by steroid hormones and flavonoids in different in vitro systems and potential
in vivo implications. Drug Metab Dispos 36:1332–1340.
Hosea NA, Miller GP, and Guengerich FP (2000) Elucidation of distinct ligand binding sites for
cytochrome P450 3A4. Biochemistry 39:5929–5939.
Hutzler JM, Frye RF, Korzekwa KR, Branch RA, Huang SM, and Tracy TS (2001a) Minimal in
vivo activation of CYP2C9-mediated flurbiprofen metabolism by dapsone. Eur J Pharm Sci
14:47–52.
Hutzler JM, Hauer MJ, and Tracy TS (2001b) Dapsone activation of CYP2C9-mediated me-
tabolism: evidence for activation of multiple substrates and a two-site model. Drug Metab
Dispos 29:1029–1034.
Hutzler JM and Tracy TS (2002) Atypical kinetic profiles in drug metabolism reactions. Drug
Metab Dispos 30:355–362.
Hutzler M, Messing DM, and Wienkers LC (2005) Predicting drug-drug interactions in drug
discovery: where are we now and where are we going? Curr Opin Drug Discov Devel 8:
51–58.
Jushchyshyn MI, Hutzler JM, Schrag ML, and Wienkers LC (2005) Catalytic turnover of pyrene
by CYP3A4: evidence that cytochrome b5 directly induces positive cooperativity. Arch Bio-
chem Biophys 438:21–28.
Kalgutkar AS, Taylor TJ, Venkatakrishnan K, and Isin EM (2003) Assessment of the con-
tributions of CYP3A4 and CYP3A5 in the metabolism of the antipsychotic agent haloperidol to
its potentially neurotoxic pyridinium metabolite and effect of antidepressants on the bio-
activation pathway. Drug Metab Dispos 31:243–249.
Kenworthy KE, Clarke SE, Andrews J, and Houston JB (2001) Multisite kinetic models for
CYP3A4: simultaneous activation and inhibition of diazepam and testosterone metabolism.
Drug Metab Dispos 29:1644–1651.
Keubler A, Weiss J, Haefeli WE, Mikus G, and Burhenne J (2012) Drug interaction of efavirenz
and midazolam: efavirenz activates the CYP3A-mediated midazolam 19-hydroxylation in vitro.
Drug Metab Dispos 40:1178–1182.
Khan KK, He YQ, Domanski TL, and Halpert JR (2002) Midazolam oxidation by cytochrome
P450 3A4 and active-site mutants: an evaluation of multiple binding sites and of the metabolic
pathway that leads to enzyme inactivation. Mol Pharmacol 61:495–506.
Kinney GG, O’Brien JA, Lemaire W, Burno M, Bickel DJ, Clements MK, Chen TB, Wisnoski
DD, Lindsley CW, and Tiller PR, et al. (2005) A novel selective positive allosteric modulator
of metabotropic glutamate receptor subtype 5 has in vivo activity and antipsychotic-like effects
in rat behavioral models. J Pharmacol Exp Ther 313:199–206.
Komura H and Iwaki M (2008) Species differences in in vitro and in vivo small intestinal
metabolism of CYP3A substrates. J Pharm Sci 97:1775–1800.
Authorship Contributions
Korzekwa KR, Krishnamachary N, Shou M, Ogai A, Parise RA, Rettie AE, Gonzalez FJ,
and Tracy TS (1998) Evaluation of atypical cytochrome P450 kinetics with two-substrate
models: evidence that multiple substrates can simultaneously bind to cytochrome P450 active
sites. Biochemistry 37:4137–4147.
Participated in research design: Blobaum, Bridges, Byers, Mackie,
Lavreysen, Steckler, Daniels.
Conducted experiments: Blobaum, Byers.
Kotegawa T, Laurijssens BE, Von Moltke LL, Cotreau MM, Perloff MD, Venkatakrishnan K,
Warrington JS, Granda BW, Harmatz JS, and Greenblatt DJ (2002) In vitro, pharmacokinetic,
and pharmacodynamic interactions of ketoconazole and midazolam in the rat. J Pharmacol Exp
Ther 302:1228–1237.
Kronbach T, Mathys D, Umeno M, Gonzalez FJ, and Meyer UA (1989) Oxidation of midazolam
and triazolam by human liver cytochrome P450IIIA4. Mol Pharmacol 36:89–96.
Lee CA, Manyike PT, Thummel KE, Nelson SD, and Slattery JT (1997) Mechanism of cyto-
chrome P450 activation by caffeine and 7,8-benzoflavone in rat liver microsomes. Drug Metab
Dispos 25:1150–1156.
Lin JH and Lu AY (1998) Inhibition and induction of cytochrome P450 and the clinical impli-
cations. Clin Pharmacokinet 35:361–390.
Lin Y, Lu P, Tang C, Mei Q, Sandig G, Rodrigues AD, Rushmore TH, and Shou M (2001)
Substrate inhibition kinetics for cytochrome P450-catalyzed reactions. Drug Metab Dispos 29:
368–374.
Liu F, Grauer S, Kelley C, Navarra R, Graf R, Zhang G, Atkinson PJ, Popiolek M, Wantuch C,
and Khawaja X, et al. (2008) ADX47273 [S-(4-fluoro-phenyl)-3-[3-(4-fluoro-phenyl)-[1,2,4]-
oxadiazol-5-yl]-piperidin-1-yl-methanone]: a novel metabotropic glutamate receptor 5-selective
positive allosteric modulator with preclinical antipsychotic-like and procognitive activities. J
Pharmacol Exp Ther 327:827–839.
Locuson CW, Wienkers LC, Jones JP, and Tracy TS (2007) CYP2C9 protein interactions with
cytochrome b(5): effects on the coupling of catalysis. Drug Metab Dispos 35:1174–1181.
Martínez C, Gervasini G, Agúndez JA, Carrillo JA, Ramos SI, García-Gamito FJ, Gallardo L,
and Benítez J (2000) Modulation of midazolam 1-hydroxylation activity in vitro by neuro-
transmitters and precursors. Eur J Clin Pharmacol 56:145–151.
Contributed new reagents or analytic tools: Turlington, Mattmann, Morrison,
Bartolomé, MacDonald, Stauffer.
Performed data analysis: Blobaum, Morrison, Mackie.
Wrote or contributed to the writing of the manuscript. Blobaum, Bridges,
Morrison, Bartolomé, Jones, Niswender, Conn, Lindsley, Stauffer, Daniels.
References
Atkins WM (2005) Non-Michaelis-Menten kinetics in cytochrome P450-catalyzed reactions.
Annu Rev Pharmacol Toxicol 45:291–310.
Avent KM, DeVoss JJ, and Gillam EM (2006) Cytochrome P450-mediated metabolism of
haloperidol and reduced haloperidol to pyridinium metabolites. Chem Res Toxicol 19:914–
920.
Bayer J, Heinrich T, Burhenne J, Haefeli WE, and Mikus G (2009) Potential induction of
CYP3A4 after a single dose of efaverinz using midazolam pharmacokinetics as a marker
(Abstract 59). Basic Clin Pharmacol Toxicol 104:515.
Conn PJ, Lindsley CW, and Jones CK (2009) Activation of metabotropic glutamate receptors as
a novel approach for the treatment of schizophrenia. Trends Pharmacol Sci 30:25–31.
Darrah JM, Stefani MR, and Moghaddam B (2008) Interaction of N-methyl-D-aspartate and
group 5 metabotropic glutamate receptors on behavioral flexibility using a novel operant set-
shift paradigm. Behav Pharmacol 19:225–234.
Davydov DR, Rumfeldt JA, Sineva EV, Fernando H, Davydova NY, and Halpert JR (2012)
Peripheral ligand-binding site in cytochrome P450 3A4 located with fluorescence resonance
energy transfer (FRET). J Biol Chem 287:6797–6809.
Moghaddam B (2004) Targeting metabotropic glutamate receptors for treatment of the cognitive
symptoms of schizophrenia. Psychopharmacology (Berl) 174:39–44.
Domanski TL, He YA, Harlow GR, and Halpert JR (2000) Dual role of human cytochrome P450
3A4 residue Phe-304 in substrate specificity and cooperativity. J Pharmacol Exp Ther 293:
585–591.
Ngui JS, Tang W, Stearns RA, Shou M, Miller RR, Zhang Y, Lin JH, and Baillie TA (2000)
Cytochrome P450 3A4-mediated interaction of diclofenac and quinidine. Drug Metab Dispos
28:1043–1050.

Heterotropic Activation of CYP3A by a Novel mGlu5 PAM
2075
Niswender CM and Conn PJ (2010) Metabotropic glutamate receptors: physiology, pharmacol-
ogy, and disease. Annu Rev Pharmacol Toxicol 50:295–322.
Yamazaki H, Johnson WW, Ueng YF, Shimada T, and Guengerich FP (1996) Lack of
electron transfer from cytochrome b5 in stimulation of catalytic activities of cytochrome
P450 3A4. Characterization of a reconstituted cytochrome P450 3A4/NADPH-cytochrome
P450 reductase system and studies with apo-cytochrome b5. J Biol Chem 271:27438–
27444.
Yamazaki H, Suemizu H, Murayama N, Utoh M, Shibata N, Nakamura M, and Guengerich FP
(2013) In vivo drug interactions of the teratogen thalidomide with midazolam: heterotropic
cooperativity of human cytochrome P450 in humanized TK-NOG mice. Chem Res Toxicol 26:
486–489.
Yang J, Atkins WM, Isoherranen N, Paine MF, and Thummel KE (2012) Evidence of CYP3A
allosterism in vivo: analysis of interaction between fluconazole and midazolam. Clin Phar-
macol Ther 91:442–449.
Yano JK, Wester MR, Schoch GA, Griffin KJ, Stout CD, and Johnson EF (2004) The structure of
human microsomal cytochrome P450 3A4 determined by X-ray crystallography to 2.05-A
resolution. J Biol Chem 279:38091–38094.
Youdim KA, Lyons R, Payne L, Jones BC, and Saunders K (2008) An automated, high-
throughput, 384 well Cytochrome P450 cocktail IC50 assay using a rapid resolution LC-MS/
MS end-point. J Pharm Biomed Anal 48:92–99.
Zhang H, Gay SC, Shah M, Foroozesh M, Liu J, Osawa Y, Zhang Q, Stout CD, Halpert JR,
and Hollenberg PF (2013) Potent mechanism-based inactivation of cytochrome P450 2B4 by 9-
ethynylphenanthrene: implications for allosteric modulation of cytochrome P450 catalysis.
Biochemistry 52:355–364.
Obach RS (2012) Heterotropic effects on drug-metabolizing enzyme activities: in vitro curiosity
emerges as a clinically meaningful phenomenon (perhaps?). Clin Pharmacol Ther 91:385–387.
Perloff MD, von Moltke LL, Court MH, Kotegawa T, Shader RI, and Greenblatt DJ (2000)
Midazolam and triazolam biotransformation in mouse and human liver microsomes: relative
contribution of CYP3A and CYP2C isoforms. J Pharmacol Exp Ther 292:618–628.
Shah MB, Wilderman PR, Pascual J, Zhang Q, Stout CD, and Halpert JR (2012) Conformational
adaptation of human cytochrome P450 2B6 and rabbit cytochrome P450 2B4 revealed upon
binding multiple amlodipine molecules. Biochemistry 51:7225–7238.
Shou M, Grogan J, Mancewicz JA, Krausz KW, Gonzalez FJ, Gelboin HV, and Korzekwa KR
(1994) Activation of CYP3A4: evidence for the simultaneous binding of two substrates in
a cytochrome P450 active site. Biochemistry 33:6450–6455.
Shou M, Mei Q, Ettore MW, Jr, Dai R, Baillie TA, and Rushmore TH (1999) Sigmoidal kinetic
model for two co-operative substrate-binding sites in a cytochrome P450 3A4 active site: an
example of the metabolism of diazepam and its derivatives. Biochem J 340:845–853.
Tang W, Stearns RA, Kwei GY, Iliff SA, Miller RR, Egan MA, Yu NX, Dean DC, Kumar S,
and Shou M, et al. (1999) Interaction of diclofenac and quinidine in monkeys: stimulation of
diclofenac metabolism. J Pharmacol Exp Ther 291:1068–1074.
Thummel KE and Wilkinson GR (1998) In vitro and in vivo drug interactions involving human
CYP3A. Annu Rev Pharmacol Toxicol 38:389–430.
van Waterschoot RA, Rooswinkel RW, Sparidans RW, van Herwaarden AE, Beijnen JH,
and Schinkel AH (2009) Inhibition and stimulation of intestinal and hepatic CYP3A activity:
studies in humanized CYP3A4 transgenic mice using triazolam. Drug Metab Dispos 37:
2305–2313.
Wang RW, Newton DJ, Liu N, Atkins WM, and Lu AY (2000) Human cytochrome P-450 3A4: in
vitro drug-drug interaction patterns are substrate-dependent. Drug Metab Dispos 28:360–366.
Williams PA, Cosme J, Vinkovic DM, Ward A, Angove HC, Day PJ, Vonrhein C, Tickle IJ,
and Jhoti H (2004) Crystal structures of human cytochrome P450 3A4 bound to metyrapone
and progesterone. Science 305:683–686.
Address correspondence to: Dr. J. Scott Daniels, Drug Metabolism and
Pharmacokinetics Laboratory, Vanderbilt Center for Neuroscience Drug Discov-
ery, Vanderbilt University Medical Center, Nashville, TN 37232. E-mail: scott.
daniels@vanderbilt.edu