Characterization of T-5 N-oxide formation as the first highly selective measure of CYP3A5 activity

Article abstract of DOI:10.1124/dmd.113.054726

Almost half of prescription medications are metabolized by cytochrome P450 3A4 and 3A5. CYP3A4 and 3A5 have significant substrate overlap, and there is currently no way to selectively monitor the activity of these two enzymes, which has led to the erroneous habit of attributing the cumulative activity to CYP3A4. While CYP3A4 expression is ubiquitous, CYP3A5 expression is polymorphic, with large individual differences in CYP3A5 expression level. The CYP3A5 genotype has been shown to alter the pharmacokinetics of drugs in clinical trials. We report the first tool compound capable of determining CYP3A5 activity in biologic samples containing both enzymes. Oxidation of T-5 by CYP3A5 yields an N-oxide metabolite that is over 100-fold selective over CYP3A4. Formation of T-5 N-oxide highly correlates with the CYP3A5 genotype and CYP3A5 expression levels in human liver microsomes and human hepatocytes. Copyright

Full text of DOI:10.1124/dmd.113.054726

Supplemental Material can be found at:
http://dmd.aspetjournals.org/content/suppl/2013/12/11/dmd.113.054726.DC1.html
1521-009X/42/3/334–342$25.00
http://dx.doi.org/10.1124/dmd.113.054726
DRUG METABOLISM AND DISPOSITION
Drug Metab Dispos 42:334–342, March 2014
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
Characterization of T-5 N-Oxide Formation as the First Highly
Selective Measure of CYP3A5 Activity
Xiaohai Li, Valer Jeso, Scott Heyward, Gregory S. Walker, Raman Sharma, Glenn C. Micalizio,
and Michael D. Camerons
Department of Molecular Therapeutics, Scripps Research Institute, Jupiter, Florida (M.D.C., X.L.); Celsis In Vitro Technologies,
Baltimore Maryland (S.H.); Department of Chemistry, Dartmouth College, Hanover, New Hampshire (V.J., G.C.M.),
Pharmacokinetics, Dynamics, and Metabolism Department, Pfizer Global Research and Development, Groton, Connecticut
(G.S.W., R.S.)
Received September 3, 2013; accepted December 6, 2013
ABSTRACT
Almost half of prescription medications are metabolized by cyto-
chrome P450 3A4 and 3A5. CYP3A4 and 3A5 have significant sub-
strate overlap, and there is currently no way to selectively monitor
genotype has been shown to alter the pharmacokinetics of drugs in
clinical trials. We report the first tool compound capable of deter-
mining CYP3A5 activity in biologic samples containing both enzymes.
the activity of these two enzymes, which has led to the erroneous Oxidation of T-5 by CYP3A5 yields an N-oxide metabolite that is over
habit of attributing the cumulative activity to CYP3A4. While CYP3A4
expression is ubiquitous, CYP3A5 expression is polymorphic, with
large individual differences in CYP3A5 expression level. The CYP3A5
100-fold selective over CYP3A4. Formation of T-5 N-oxide highly
correlates with the CYP3A5 genotype and CYP3A5 expression levels
in human liver microsomes and human hepatocytes.
Introduction
and CYP3A5 can be accounted for and the importance of genetic
polymorphisms can be predicted.
Selective cytochrome P450 substrates are used in pharmaceutic
research to determine the likelihood that a candidate compound would
be involved in pharmacokinetic drug-drug interactions. Quality selective
substrates exist for all the major drug-metabolizing P450s except
CYP3A4 and CYP3A5 (Yuan et al., 2002). This is ironic because these
enzymes are involved in the metabolism of more drugs than any other
P450 enzyme (Wilkinson, 1996; Thummel and Wilkinson, 1998; Bu,
2006; Niwa et al., 2008). The frequently used CYP3A substrates
include testosterone, midazolam, nifedipine, and erythromycin. Because
those substrates are metabolized by both CYP3A enzymes, they do not
allow researchers to determine whether a tested compound preferen-
tially inhibits CYP3A4 or CYP3A5.
There exists considerable overlap in substrate specificity between
CYP3A4 and CYP3A5, (Liu et al., 2007) resulting in the inability to
differentiate their activity or inhibition in biologically relevant samples.
The substrate promiscuity of has made the identification of a specific
substrate for CYP3A5 a challenge (Yuan et al., 2002). A selective
readout of CYP3A5 activity will refine current models for pharmaco-
kinetic drug-drug interactions where the catalytic efficiency of CYP3A4
Four members of the CYP3A family have been identified in humans:
CYP3A4, CYP3A5, CYP3A7, and CYP3A43 (Daly, 2006). CYP3A7
is predominantly a fetal form and decreases rapidly after birth, and
CYP3A43 is expressed at very low levels (Daly, 2006). Neither is
considered to play a significant drug metabolism role in children or
adults. CYP3A4 is highly expressed in the liver and intestine. In
contrast, CYP3A5 is polymorphically expressed. CYP3A5*1 leads to
active, full-length CYP3A5, with individuals homozygous or heterozy-
gous for the *1 allele reported to have similar levels of CYP3A5
expression (Hustert et al., 2001; Kuehl et al., 2001). The frequency of
having at least one CYP3A5*1 allele (expressing active enzyme) has
varied with reports, but averages 10%–30% in the Caucasian population
and rises to 50%–70% in African Americans (Daly, 2006). The
CYP3A5*3 allele (22893A→G) in intron 3 leads to a frame shift in the
majority of the CYP3A5 mRNA, yielding inactive protein and loss of
CYP3A5 expression. Individuals lacking at least one CYP3A5*1 allele
have minimal correctly spliced mRNA coding for CYP3A5 and only
trace levels of functional CYP3A5 protein (Hustert et al., 2001; Kuehl
et al., 2001; Lamba et al., 2002; Roy et al., 2005). For patients with at
least one CYP3A5*1 allele, hepatic CYP3A5 expression is approxi-
mately 40% of the total CYP3A protein (Perrett et al., 2007). Thus,
substantial amounts of hepatic CYP3A5 protein are available to
metabolize CYP3A substrates in CYP3A5*1 individuals.
This work was supported by a research grant from the National Institutes of
Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant
R21 DK091630] (to M.D.C.).
A number of chemicals have been shown to have moderate (5- to
10-fold) IC50 and/or intrinsic clearance differences between CYP3A4
dx.doi.org/10.1124/dmd.113.054726.
s This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: ACN, acetonitrile; FMO, flavin-containing monooxygenase; HLM, human liver microsomes; HPLC, high-performance liquid
chromatography; LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry; MS/MS, tandem mass spectrometry; SR-9186,
1-(4-(3H-imidazo[4,5-b]pyridin-7-yl)phenyl)-3-(49-cyano-[1,19-biphenyl]-4-yl)urea; T-1032, methyl 2-(4-aminophenyl)-1-oxo-7-(pyridin-2-ylmethoxy)-4-
(3,4,5-trimethoxyphenyl)-1,2-dihydroisoquinoline-3-carboxylate; vincristine M1 [2b, 3b,4b,5a,12R,19a)-4-(acetyloxy)-6,7-didehydro-1-formyl-3-
hydroxy-16-methoxy-15-[(5S,7S)-1,2,3,4,5,6,7,8-octahydro-7-(methoxycarbonyl)-5-(2-oxobutyl)azonino[5,4-b]indol-7-yl]aspidospermidine-3-carboxylic
acid methyl ester.
334

Selective CYP3A5 Marker Reaction
335
and 3A7) and three recombinant human flavin-containing monooxygenases
(FMO1, FMO3, and FMO5) were purchased from BD Biosciences (Woburn,
MA). Recombinant P450 enzymes were microsomal preparations coexpressing
P450, NADPH-oxidoreductase, and cytochrome b5. Recombinant P450 enzymes
were supplied from the vendor at a concentration of 1 pmol enzyme per
microliter.
and CYP3A5 (Ekins et al., 2003). Individuals taking specific drug
combinations with differing CYP3A4/5 intrinsic clearance or in-
hibitory potential are likely to have altered susceptibility to drug-drug
interactions depending on their CYP3A5 genotype.
The clinical significance of CYP3A5 polymorphisms has been demon-
strated in pharmacokinetic and drug-drug interaction studies. The immu-
nosuppressant tacrolimus requires trough concentration to be maintained
between 5 and 15 ng/ml. Patients who express active CYP3A5 (*1/*1
or *1/*3) require approximately twice the dose compared with *3/*3
patients to maintain an appropriate tacrolimus concentration (Zhao
et al., 2005; Barry and Levine, 2010). The CYP3A5 genotype was also
shown to be involved in drug-drug interactions caused by fluconazole,
which inhibits CYP3A4 2 to 5 times more potently than CYP3A5.
Concomitant dosing of fluconazole increased the area under the curve
of intravenously administered midazolam, resulting in larger increases
in midazolam exposure in patients lacking the *1 allele, 124 versus
198 nM*h, P = 0.02 (Isoherranen et al., 2008).
CYP3A5 *3/*3 single-donor microsomes can be used to essentially
isolate CYP3A4 activity, but there is not a similar CYP3A4 loss-of-
function mutation allowing for the isolation of CYP3A5 activity. A
selective CYP3A5 substrate will facilitate more accurate predictions
when using human microsomes than those that could be achieved with
recombinant P450. Human liver microsomes (HLM) have full-length
P450 enzyme without modification of the membrane spanning region
and have physiologic ratios of P450s, NADPH-oxidoreductase, and
cytochrome b5. Studies using CYP3A4 coexpressed with NADPH-
oxidoreductase in bacteria (Escherichia coli), yeast (Saccharomyces
cerevisiae), and human B-lymphoblastoid cells demonstrated 9-fold
differences in the maximal rates of midazolam and diazepam
metabolism between the different expression systems, leading to high
variability in the predicted intrinsic clearance (Andrews et al., 2002).
Batch-to-batch variability is also a concern, where published IC50
values using recombinant 3A4 showed up to 29-fold variation
(Stresser et al., 2000; Wang et al., 2000; Andrews et al., 2002).
We describe the CYP3A5 selective catalysis of an N-oxide metabolite of
T-5, T-1032 [methyl 2-(4-aminophenyl)-1-oxo-7-(pyridin-2-ylmethoxy)-4-
(3,4,5-trimethoxyphenyl)-1,2-dihydroisoquinoline-3-carboxylate]. T-1032
was first synthesized by Tanabe Seiyaku Co. (Osaka, Japan). Before the
clinical trials were discontinued, T-1032 had advanced to phase II as
a selective cyclic GMP phosphodiesterase-5 inhibitor, intended to compete
with Viagra (sildenafil citrate; Pfizer, New York, NY). We refer to the
compound as T-5 to reflect the role of Tenabe and the properties of the
molecule as a selective inhibitor of phosphodiesterase-5 and a selective
marker substrate for evaluation of CYP3A5 activity.
CYP3A5 genotyped individual donor hepatic microsomes were purchased
from both Xenotech and BD Biosciences. Donor lots were as follows (Xenotech
lots start with the HH prefix, and donor sex is indicated with an M or F):
CYP3A5 *1/*1 donors were HH739(F), HH47(F), HH867(M), HH785(M),
HH860(F), 07100271(F), 0710272(F), and 0810554(F); CYP3A5 *1/*3 donors
were HH757(M), HH868(M), HH54(F), 0710239(F), 0710231(M), and
0710232(F); CYP3A5 *3/*3 donors were HH61(M), HH792(F), HH189(F),
HH837(F), 0710233(F), 0710253(M), and 0710234(M). Cryopreserved human
hepatocytes were obtained from Celsis In Vitro Technologies (Baltimore, MD).
The donor lot information is as follows: CYP3A5*1/*1 donors were RQM(F),
LUH(F), and ZQM(M); CYP3A5*1/*3 donors were OJE(M), NRJ(F), and
VLS(F); CYP3A5*3/*3 donors were KQN(F), JCM(F), and XPD(F).
Synthesis of T-5 and T-5 N-Oxide. The synthesis of T-5 was modified from
a previously published method (Ukita et al., 2001). A detailed procedure noting
which procedures were changed along with the NMR characterization data for
intermediates are included as Supplemental Material and Supplemental Figures
14–17.
Incubations with cDNA-Expressed P450 and Human FMO Enzymes.
Triplicate 200-ml incubations were set up containing 10 pmol/ml recombinant
P450 (0.2 mg protein/ml for FMO incubations), 5 mM T-5, 5 mM MgCl2, and
100 mM phosphate buffer, pH 7.4. In accord with the manufacturer recom-
mendations, TRIS buffer was used for CYP2A6, 2C9, and 3A7 incubations.
The reactions were shaken at 37°C for 3 minutes to equilibrate temperature
before the addition of 1 mM NADPH to start the reaction. After 15 minutes,
incubations were stopped with an equal volume of ice-cold acetonitrile (ACN)
(containing 0.1 mM dextrorphan as an internal standard), chilled, and cen-
trifuged. The solution was filtered and submitted for LC-MS/MS analysis.
To determine the Michaelis-Menten parameters, a range of T-5 concentrations
(0.1–50 mM) were tested, and T-5 N-oxide was quantitated using a standard
curve (5, 10, 25, 50, 100, 250, 500, 2500 nM). The LC-MS/MS data were
processed using analyst 1.6 software. The standard curve was fit by quadratic
regression with correlation coefficients between 0.9997–1.0000 and calculated
concentrations for each standard were within 20% of their nominal concentration.
The in vitro clearance was calculated for individual enzymes by determining the
T-5 half-life with each recombinant enzyme and then fitting the data to the
equation CLin vitro = (Incubation volume in ml/pmol enzyme) * ln2/Half-life.
HLM Incubations. Hepatic microsomal incubations using pooled HLM
(150 donors, mixed gender) and individual-donor 3A5-genotyped HLM were
conducted at 37°C in 100 mM potassium phosphate buffer, pH 7.4, in a final
volume of 250 ml. Each reaction consisted of 0.1 mg protein/ml and contained
5 mM T-5 unless otherwise indicated. All reactions were initiated by the
addition of 1 mM NADPH and stopped after 15 minutes by the addition of an
equal volume of ACN.
For inhibition studies, the inhibitors included: 10 mM furafylline (CYP1A2),
30 mM phenyl-piperidinyl propane (CYP2B6), 25 mM ticlopidine (CYP2B6/2C19),
where the first three inhibitors are time-dependent inhibitors and were
preincubated for 30 minutes before initiating the reaction by the addition of
T-5; 10mM tranylcypromine (CYP2A6/2B6), 20 mM sulfaphenazole (CYP2C9),
1 mM quinidine (CYP2D6), 1 mM montelukast (CYP2C8), 1 mM SR-9186
(CYP3A4), and 1 mM ketoconazole and ritonavir for total CYP3A. Incubation
reactions were conducted in 96-well plates on a shaking incubator maintained at
37°C. At the end of the assay, the reactions were stopped by the addition of an
equal volume of cold ACN (containing 0.1 mM internal standard), centrifuged,
passed through a Millipore MultiScreen Solvinert 0.45 mm low-binding
polytetrafluoroethylene (PTFE) hydrophilic filter plate (Millipore, Billerica, MA),
and analyzed by LC-MS/MS. All reactions were performed in triplicate.
Vincristine M1 [2b,3b,4b,5a,12R,19a)-4-(acetyloxy)-6,7-didehydro-1-formyl-
3-hydroxy-16-methoxy-15-[(5S,7S)-1,2,3,4,5,6,7,8-octahydro-7-(methoxycarbonyl)-
5-(2-oxobutyl)azonino[5,4-b]indol-7-yl]aspidospermidine-3-carboxylic acid
Materials and Methods
Chemicals. Unless otherwise indicated, chemicals were purchased from
Sigma-Aldrich (St. Louis, MO). T-5 (T-1032) was originally purchased from
Sigma Chemical as part of a high-throughput screening library (LOPAC-1080).
T-5 was discontinued by Sigma-Aldrich and an alternative commercial vendor
could not be found. Additional T-5 and T-5 N-oxide was obtained through
contract synthesis from WuXi PharmaTech (Shanghai, People’s Republic of
China) and was later synthesized at Scripps Florida (Scripps Research Institute,
Jupiter, FL). T-5 (#A622635) and T-5 N-oxide (#A622640) can now be
obtained from Toronto Research Chemical (Toronto, Ontario, Canada). SR-9186
[1-(4-(3H-imidazo[4,5-b]pyridin-7-yl)phenyl)-3-(49-cyano-[1,19-biphenyl]-4-yl)
urea] (Li et al., 2012; Song et al., 2012) was synthesized at Scripps Florida. All
solvents used for liquid chromatography coupled with tandem mass spectrom-
etry (LC-MS/MS) were chromatographic grade. Pooled HLM was purchased
from Xenotech (Lenexa, KS).
Recombinant CYP3A4 and CYP3A5 were obtained from PanVera Corpora- methyl ester] formation in genotyped individual donor microsomes was
tion (Madison, WI). In later phenotyping studies, 11 recombinant human performed using previously published methods (Dennison et al., 2006; Li et al.,
cytochromes P450 (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, 3A5, 2012).

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LI et al.
Fig. 1. Chromatograms from T-5 incubations. Incubations contained 5 mM T-5 and 10 pmol/ml recombinant P450 and were initiated with 1 mM NADPH. After 15 minutes,
the incubation was stopped with an equal volume of ACN containing 0.1 mM dextrorphan as an internal standard. The multiple reaction monitoring (MRM) chromatograms
obtained from incubations containing CYP3A4 and CYP3A5 are presented with traces representing T-5 (gray), along with +16 AMU (red) and 291 AMU (blue) metabolites.
Cryopreserved Human Hepatocyte Incubations. Cryopreserved human
hepatocytes were obtained from Celsis In Vitro Technologies. Nine individual
donors comprising three donors each of CYP3A5 *1/*1, *1/*3, and *3/*3
genotype were evaluated. Hepatocytes were thawed according to Celsis’s
protocol and resuspended in Krebs-Henseleit buffer to 1,500,000 cells/ml. Then
50 ml of cells were incubated with T-5 (0.021–45 mM) or midazolam (0.18–400 mM)
in a final reaction volume of 100 ml, containing 0.5% dimethylsulfoxide.
Incubations were stopped after 30 minutes by the addition of an equal volume
of methanol. The concentration of 19-OH midazolam and T-5 N-oxide were
determined by LC-MS/MS, and the data were expressed as the pmol of metab-
olite generated per minute per million live cells.
Relative Quantification of CYP3A5 Content in 3A5-Genotyped
Individual-Donor HLM. To correlate T-5 N-oxide activity with CYP3A5
levels, the relative content of CYP3A5 in individual-donor HLM was determined
using a previously published LC-MS/MS method (Williamson et al., 2011). A
refined protocol can be downloaded from the AB Sciex Web site (AB Sciex,
2011).
Hepatic microsomes (95 ml at a concentration of 10 mg microsomal protein/
ml) were denatured for 5 minutes at 37°C by the addition of 5 ml of 20%
N-octyl glucoside. The sample was reduced by adding 10 ml of 50 mM tris(2-
carboxyethyl) phosphine and incubated at 50°C for 30 minutes before the
addition of 10 ml of 0.2 M iodoacetamide. The mixture was digested overnight
at 37°C by adding 20 mg of trypsin. In the morning, 100 ml of ice cold ACN
containing 0.5% formic acid was added to the trypsin digested solution. The
sample was filtered and subjected to LC-MS/MS analysis.
Chromatographic separations were performed on a Shimadzu Prominence
Series 20 High-Performance Liquid Chromatography (HPLC) system (Shimadzu
Scientific Instruments Inc., Columbia, MD) fit with a C18 column (Jupiter
5 mm C18 300A, 150 Â 2.0 mm i.d.; Phenomenex Inc., Torrance, CA) with
a flow rate of 0.7 ml/min. The mobile phase consisted of 0.1% formic acid
water (mobile phase A) and 0.1% formic acid in ACN (mobile phase B).
Tryptic peptides were eluted using a series of linear gradients: 5% B 0–0.1
minutes, increased to 30% B at 7.0 minutes, 90% B at 10–13 minutes, and back
to 5% B from 13.1–18.0 minutes.
An AB Sciex 6500 QTRAP (AB Sciex, Framingham, MA) was used to
quantitate the peptides. Initially, all nine multiple reaction monitoring transitions
(MRM) listed in Williamson et al. (2011) were followed, representing three
transitions for each of three unique CYP3A5 peptides. However, only five
transitions had acceptable chromatograms for all samples. Peptides used for
quantitation were DTINFLSK(244-251) where the doubly charged parent ion
(m/z = 469.5) was fragmented to singly charged fragments, 608.3 and 721.4 AMU.
Similarly, the doubly charged ion of the peptide SLGPVGFMK(107-115) was
detected using a parent ion of 468.3 and a fragment ion of 735.5. The triple
charged ion of GSMVVIPTYALHHDPK(391-406) was detected using a parent
ion of 589.1 and fragment ions of 696.0 and 745.5. In the same analytic run,
CYP3A4 levels were determined using the following transitions: parent ion
Fig. 2. Formation of T-5 N-oxide in recombinantly expressed enzymes. Twelve
P450 and three FMO enzymes were compared. Incubations contained 5 mM T-5 and
10 pmol/ml recombinant enzyme and were initiated with 1 mM NADPH. To
minimize the potential for thermal inactivation, FMO incubations were initiated by
the addition of FMO. (A) After 15 minutes, the incubation was stopped with an
equal volume of ACN, and T-5 N-oxide formation was evaluated by LC-MS/MS.
(B) T-5 depletion was monitored in parallel incubations using similar conditions,
and the data are expressed in terms of in vitro clearance.

Selective CYP3A5 Marker Reaction
337
T-5 and T-5 N-oxide corresponded to fragmentation at the ether bond yielding
a fragment of 476.2 AMU. Due to in-source fragmentation, both compounds had
significant “crosstalk” with the 477.2→445.2 transition used to monitor the
O-dealkylated metabolite of T-5.
NMR of Metabolites. Metabolites generated by recombinant CYP3A5 were
HPLC purified. Peaks were resolved using an ACE Excel 2, C18-PFP, 150 Â
4.6 mm analytical column with a 20:1 in-line diverter located between the
column and the mass spectrometer. This allowed for simultaneous analysis and
sample collection. Samples were concentrated by vacuum centrifugation,
resuspended in 0.05 ml of dimethyl sulfoxide-D6 (Cambridge Isotope
Laboratories, Andover, MA), and placed in 1.7 mm diameter NMR tubes.
The NMR spectra were recorded on a Bruker Avance 600 MHz system
(Bruker, Karlsruhe, Germany) controlled by TopSpin v2.0 (Bruker), equipped
with a 1.7-mm triple (TCI) cryoprobe. One-dimensional spectra were recorded
using a sweep width of 12,000 Hz and a total recycle time of 7.2 seconds. The
resulting time-averaged free induction decays were transformed using an
exponential line broadening of 1.0 Hz to enhance signal to noise. All spectra
were referenced using residual dimethyl sulfoxide-d6 (1H d = 2.5 ppm and 13C
d = 39.5 relative to TMS, d = 0.00). Phasing, baseline correction, and
integration were all performed manually. If needed, the BIAS- and SLOPE-
functions for the integral calculation were adjusted manually.
Other supporting NMR experiments were recorded using the standard pulse
sequences provided by Bruker, including homonuclear correlation spectroscopy,
total correlation spectroscopy, multiplicity edited heteronuclear single-quantum
correlation spectroscopy, and heteronuclear multiple-bond correlation spectros-
copy data. Two-dimensional experiments were typically acquired using a 1K Â
128 data matrix with 16 dummy scans. The data were zero-filled to a size of 1K Â
1K. Unless otherwise noted, for 2D experiments a relaxation delay of 1.5 seconds
was used between transients. Postacquisition data analysis was performed with
Mnova V8.1.1 software (Mestrelab Research, Santiago de Compostela, Spain).
Data Analysis. Data were curve fitted to the Michaelis-Menten equation
using nonlinear regression analysis in GraphPad Prism (GraphPad Software
Inc., San Diego, CA). Correlation analysis was performed using the Pearson
method with the two-tailed P value determination, which assumes the data are
from Gaussian populations. Calculation of P values testing the statistical
significance of the CYP3A5 genotype on T-5 N-oxide formation were done
with a one-tailed t test assuming unequal variance. A one-tailed test was chosen
instead of two-tailed because the result of having the CYP3A5*1 allele would
be increased CYP3A5 expression, and the Gaussian distribution would
primarily present with a positive tail for the increased CYP3A5 expression.
Fig. 3. Inhibition of T-5 N-oxide formation in human liver microsomes. Incubations
contained 5 mM T-5 and 0.2 mg/ml HLM protein and were initiated with the
addition of 1 mM NADPH. After 15 minutes, the incubation was stopped with an
equal volume of ACN (with 0.1 mM dextrorphan as internal standard), and the T-5
N-oxide formation was evaluated by LC-MS/MS. Selective inhibitors included
furafylline (CYP1A2), tranylcypromine (CYP2A6/2B6), 2-phenyl-2-(1-piperidinyl)
propane (PPP) (CYP2B6), ticlopidine (CYP2B6/2C19), montelukast (CYP2C8),
sulfaphenazole (CYP2C9), quinidine (CYP2D6), SR-9186 (CYP3A4), ketoconazole
(CYP3A4 and CYP3A5), and ritonavir (CYP3A4 and CYP3A5).
439.7 and fragment ions of 532.3, 549.3, and 650.4; parent ion 564.3 and
fragment ions of 689.4, 745.9, and 789.5; parent ion 798.4 and fragment ions of
819.4, 932.5, and 1003.5. The peak area of the individual-donor microsomes
were normalized against the 150-donor pool and expressed as a percentage. A
representative chromatogram showing the detected peptide signals for different
genotypes is available in Supplemental Figures 11–13.
Detection of T-5 and Its Metabolites. A 5.5-minute chromatographic
separation was achieved using an ACE C18-PFP column (50 Â 3.0 mm;
Advanced Chromatography Technologies Ltd., Aberdeen, Scotland, United
Kingdom) with an aqueous mobile phase containing 0.1% formic acid (solvent-
A) and ACN with 0.1% formic acid (solvent-B) run at a flow rate of 0.4 ml/
min. Linear gradients were used as follows: 0–0.2 minutes = 5% B; 2.0–3.0
minutes = 95% B; and 3.1–5.5 = 5% B.
Results
Metabolite characterization was performed by LC-MS/MS using an API4000
Q-trap (Applied Biosystems, Foster City, CA) or an AB Sciex 6500 QTRAP
mass spectrometer. T-5, T-5 N-oxide, and O-dealkylated T-5 were detected using
the mass transitions of m/z 568.2→476.2, m/z 584.2→476.2, and m/z
477.2→445.2, respectively. The optimized declustering potential and collision
energy were 110 and 37 eV, respectively. The most intense fragment ion for both
Preliminary incubations of T-5 with recombinant CYP3A4 and
CYP3A5 resulted in the formation of metabolites corresponding to
a mass increase of +16 AMU, consistent with the addition of an oxygen,
or a loss of 291 AMU, corresponding to O- or N-dealkylation of the
methylpyridine or amino phenyl, respectively. Statistically significant
differences between CYP3A4 and CYP3A5 were observed for the
major +16 AMU metabolite (Fig. 1), where the peak area of the
CYP3A5 specific (+O) metabolite was almost 100-fold greater than that
from CYP3A4 incubations. CYP3A5 was also more efficient at
generating the 291 AMU metabolite, but the ratio was smaller than for
the +16 AMU metabolite. A second 291 AMU peak was observed in
the mass spectrometer, but this was caused by in-source fragmentation
of T-5 as mentioned in the Materials and Methods section. The tandem
mass spectrometry (MS/MS) spectra for each peak is included as
Supplemental Figure 10.
A larger panel of recombinant P450s and FMOs were examined
after T-5 depletion and metabolite formation. As shown in Fig. 2A,
formation of the major +16 AMU metabolite eluting at 2.48 minutes
was found to be highly CYP3A5 selective. The concentration of T-5 in
Fig. 2 was 5 mM, but similar selectivity was observed when 40 mM
T-5 was used (data not shown). T-5 depletion was similarly evaluated
using recombinant human P450 enzymes and FMOs (Fig. 2B). The in
vitro clearance of T-5 was at least 3 times greater than other enzymes.
Fig. 4. T-5 N-oxide formation in genotyped HLM. Incubations contained 5 mM T-5,
0.1 mg/ml CYP3A5-genotyped individual-donor hepatic microsomes and 1 mM
NADPH. T-5 N-oxide quantitation was based on peak area and normalized to the
average for the *1/*1 donors. Error bars are standard deviation, and P values are
one-tailed t test assuming unequal variance.

338
LI et al.
Fig. 5. Activity correlation. The relative rates of
vincristine M1 and T-5 N-oxide formation were
compared. Incubations contained 0.1 mg/ml hepatic
microsomes and 1 mM NADPH with either 5 mM T-5
or 20 mM vincristine. Activity was normalized against
a 150-donor HLM pool, represented by a black circle
that is partially obscured by the *3/*3 donor in the
upper right-hand side of the boxed-in area. The dashed
lines of the regression are the 90% prediction bands.
The Pearson r value of the correlation was 0.982, P ,
0.0001.
The MS/MS fragmentation pattern of the selective +16 AMU metabolite
was ambiguous as the incorporated oxygen could be assigned to either the
amino phenyl or the methylpyridine ring (structure shown in Fig. 10). Each
would result in a fragment of C6H7NO, 109.0528 AMU. The metabolite
was tentatively assigned as an N-oxide based on incorporation of deuterium
(data not shown). Deuterium incorporation was tested by diluting an
incubated sample into D2O and changing the water fraction of the HPLC
mobile phase to deuterated water. The observed mass of both T-5 and its
selective +16 AMU (+oxygen) metabolite both increased 2 AMU due to
exchange of two amino hydrogen with deuterium. If the oxygen had been
incorporated through hydroxylation of T-5 on the methylpyridine or amino
phenyl rings, this would have resulted in an additional 1 AMU increase in
the observed molecular mass because the acidic phenolic (or pyridine-ol)
hydrogen would have exchanged with deuterium. Incorporation of the
oxygen through formation of an N-oxide would not change the number of
exchangeable hydrogen.
Further evidence of the formation of an N-oxide metabolite comes
from the addition of TiCl3. The proposed N-oxide metabolite was
reduced upon the addition of TiCl3 (data not shown) to reform T-5.
TiCl3 is known to efficiently reduce nitrogen-oxide back to its amine
form, but does not reduce hydroxylated phenyl or pyridine (Seaton
et al., 1984; Kulanthaivel et al., 2004).
The selective N-oxide metabolite was formed in incubations con-
taining T-5 and human liver microsomes (Fig. 3). Selective inhibitors of
several P450 enzymes, including SR-9186, a selective inhibitor of
CYP3A4 (Song et al., 2012), did not decrease formation of the N-oxide
metabolite. Addition of 1 mM ketoconazole inhibited the reaction by
69%, which is consistent with the published potency of ketoconazole
against CYP3A5 (ketoconazole IC50 = 20–50 nM for CYP3A4 and
150-300 nM for CYP3A5) (Soars et al., 2006). Ritonavir inhibited by
89%, consistent with ritonavir being a potent inhibitor of both CYP3A
enzymes. The ritonavir incubation was repeated to include a 20-minute
preincubation in the presence of NADPH, which resulted in increased
inhibition due to the time-dependent properties of ritonavir’s CYP3A
inhibition (data not shown).
T-5 was used to characterize individually genotyped HLM. At least
six individual donors with CYP3A5 *1/*1, *1/*3, or *3/*3 genotype
were tested (Fig. 4). Donors who had a least one copy of the *1 allele
had statistically significantly higher rates of T-5 N-oxide formation.
Minimal T-5 N-oxide formation was observed in the *3/*3 donors. Of
interest, a commercial 150-donor mixed gender pool of HLM behaved
the most similar to the *3/*3 genotyped donors.
Fig. 6. Correlation of T-5 N-oxide formation versus CYP3A5 expression level. The
relative rate of T-5 N-oxide formation was determined and normalized (expressed as
a percentage of the level in pooled HLM). Incubations contained 0.1 mg/ml hepatic
microsomes, 5 mM T-5 and 1 mM NADPH. CYP3A4 and 3A5 content was
estimated by digesting 1 mg microsomal protein with 20 mg trypsin in an overnight
incubation at 37°C. P450 specific peptides were evaluated by LC-MS/MS. All error
bars reflect standard deviation of triplicate analysis and the dashed lines of the
regression are the 90% prediction band. The Pearson r value of the correlation for
CYP3A5 (A) was 0.9736, P , 0.0001, and CYP3A4 (B) was 0.2791, and the
correlation was not statistically significant.

Selective CYP3A5 Marker Reaction
339
N-oxide formation in individually genotyped HLM was compared injections were combined, lyophilized, and analyzed by NMR using
with vincristine M1 formation (Fig. 5). Vincristine M1 is 5-fold to 10- a 1.7-mm cryoprobe. The two metabolites were referred to as M1 and
fold selective for CYP3A5 over CYP3A4 (Dennison et al., 2006, M2 until their structures were determined. The complete 1D and 2D data
1
2007), which makes it useful for correlation studies; however, M1 sets as well as complete H and selected 13C assignments for T-5, T-5
formation is not sufficiently selective for CYP3A5 inhibition studies N-oxide (M1), and desmethyl pyridine T-5 (M2) are contained in
1
because the levels of CYP3A4 greatly exceed CYP3A5 in commercial Supplemental Figures 1–9. The H spectrum T-5-M1 retained all of the
batches of pooled hepatic microsomes, resulting in similar levels of resonances observed in T-5 with only minor changes in the chemical
M1 formed by both 3A4 and 3A5 in an unbiased pool with a large shifts to those resonances of the pyridine ring (Fig. 7B). This strongly
number of donors. Additionally, M1 is unstable and degrades if left on supports the proposed N-oxide structure of T5-M1. In the 1H spectrum of
the autosampler or under basic conditions.
T-5-M2, the resonances of the methyl pyridine hydrogens are absent, and
To further evaluate the correlation of T-5 N-oxide formation and the there is an additional singlet with a chemical shift of 10.21 ppm (Fig. 7C).
CYP3A5 genotype, individual-donor microsomes were digested with These data are consistent with the T-5 metabolites depicted in Fig. 8.
trypsin, and unique CYP3A5 peptides were monitored (Fig. 6A). The
In collaboration with the Micalizio group, the published synthesis of
activity and peptide levels were both normalized to the Xenotech 150- T-5 was significantly improved, and a simple detour was established
donor pooled liver microsomes. One of the donors in Fig. 5 had been to generate the N-oxide. A detailed synthesis along with spectral
depleted and could not be reordered, so a new lot of genotyped properties for all intermediates is included in the Supplemental
individual-donor microsomes was substituted. Similar to the activity Material. The authentic standards were used to confirm the metabolites
assays, peptide quantification indicated that the 150-donor pool most by NMR and LC-MS/MS.
closely reflected the *3/*3 donors. A similar analysis with CYP3A4
showed no correlation (Fig. 6B).
The kinetics of T-5 N-oxide formation were repeated when a T-5
N-oxide standard was available. This allowed for absolute rather than
T-5 (T-1032) was discontinued by Sigma-Aldrich before comple- relative quantitation. The activity versus concentration curve for
tion of the characterization. Additional compound could be obtained CYP3A4 and CYP3A5 are shown in Fig. 9. The CYP3A4 curve is lost
only through contracting for custom synthesis. The contracted synthesis, in the baseline, so an insert is provided where the y-axis is 250-times
which was based on methods from the original patent, worked very zoomed. Determined KM and Vmax along with calculated Vmax/KM
poorly. After pooling the final product from three attempts, 13 mg was values are provided in Table 1.
obtained (ꢀ10% of the target quantity). However, this was sufficient to
To ensure that T-5 can be used in cellular environments, hepatocyte
set up parallel incubations of T-5 in recombinant CYP3A5. A 20:1 incubations were conducted, and the rate of T-5 N-oxide formation
splitter was inserted between the HPLC and the mass spectrometer to was determined. Similar to microsomal incubations, the level of T-5
allow the majority of the volume to be collected while still getting real- N-oxide formation was minimal in *3/*3 donors when compared with
time MS/MS verification of the eluted peaks. The fractions from multiple individuals with at least one *1 allele. Comparison incubations were
Fig. 7. Aromatic portion of the 1H spectrum of (A) T-5, (B) T-5 N-oxide, and (C) dealkylated T-5. Additional annotated NMR spectra are included in Supplemental Figures 1–9.

340
LI et al.
Fig. 8. Major metabolites generated by CYP3A4
and CYP3A5.
conducted using midazolam to ensure that the lower level of T-5 N-oxide to predict special populations such as patients with hepatic or renal
formation in the *3/*3 donors was due to lower levels of CYP3A5 and dysfunction or those with specific P450 polymorphisms. Predictions are
not due to low levels of total CYP3A protein. Calculated Km values for needed to evaluate the potential of drugs to be both the victim and
the hepatocyte incubations were 6.2 6 1.4 mM for the *1/*1 donors, 6.6 6 perpetrator in drug-drug interactions. All of these models are predicated
2.4 mM for the *1/*3 donors, and 3.1 mM for the *3/*3 donors. The on the ability to obtain high-quality preclinical data.
calculated KM in hepatocyte incubations were similar to what was
found with pooled HLM: 5.1 mM (Table 1).(Fig.10).
In the case of CYP3A4 and CYP3A5, these two enzymes have long
been acknowledged to have different catalytic efficiencies for different
compounds, but the lack of quality substrates and inhibitors has given
rise to the longstanding practice of lumping the two together and
pretending that they reflect a single enzyme.
Discussion
Using recombinantly expressed enzymes, several substrates have
been shown to have higher catalytic efficiency with either CYP3A4 or
CYP3A5 (Ekins et al., 2003), but the tools were not available for the
evaluation of these activities in microsomal or hepatocyte incubations.
With the introduction of two selective CYP3A4 inhibitors in 2012, the
suicide inhibitor Cyp3cide (Walsky et al., 2012) and competitive
inhibitor SR-9186 (Li and Cameron, 2012; Song et al., 2012), along
with the substrate detailed in our study, the tools to evaluate differences
in CYP3A4/5 and the importance of the CYP3A5 genotype have
significantly improved.
The ability to predict human pharmacokinetics based on in vitro
experimentation and preclinical animal studies has long been
a challenge. Before 1990, one of the major causes of clinical failure
was poor pharmacokinetics, particularly oral bioavailability, which to
approximately 40% of clinical failures are attributed. By the year
2000, this number was below 10% (Kola and Landis, 2004), and today
figures of 1%–3% are quoted. This improvement reflects a major
advancement in the field of drug metabolism and pharmacokinetics.
As the field continues to progress, we are no longer limited to
pharmacokinetics predictions for the average patient; now, we can start
Fig. 9. Kinetics of T-5 N-oxide formation. Multiple
concentrations of T-5 (0.1–40 mM) were incubated with
10 pmol/ml recombinant CYP3A4 or CYP3A5 in the
presence of 1 mM NADPH for 15 minutes. Incubations
were stopped by the addition of an equal volume of ACN.
Results were fit to the Michaelis-Menten equation. When
plotted on the same scale, CYP3A4 results cannot be
discerned from the x-axis and are presented as an insert to
aid in evaluation of the data. Calculated kinetic constants
are provided in Table 1.

Selective CYP3A5 Marker Reaction
341
TABLE 1
digestion of the microsomes, there appears to be a low level of
background T-5 N-oxide formation. This may be a cumulative
contribution from all other P450 enzymes, FMO, and unknown
enzymes toward the generation of T-5 N-oxide or may be due to low
levels of a hydroxyl metabolite that coelutes with the N-oxide and is
observed with the same mass transitions. In most cases, this
background level is likely to be of little consequence, but it becomes
more prominent as CYP3A5 levels decrease. It should be pointed out
that all samples regardless of genotype did contain CYP3A5.
Chromatographic peaks for CYP3A5 specific peptides were detectable
for all of the *3/*3 donors. Control samples using digested mouse and
rat microsomes did not contain peaks corresponding to the CYP3A5
peptide (data not shown).
T-5 N-oxide kinetic constants
Kinetic constants were determined using 10 pmol/ml recombinant P450 or 0.1 mg/ml HLM
and are reported as pmol/min/pmol P450 except with pooled HLM where the units are pmol/min/
mg microsomal protein.
Enzyme
Km
Vmax
Vmax/Km
mM
pmol/min/pmol
rCYP2C8
rCYP2D6
rCYP3A4
rCYP3A5
HLM
1.6
31
6.4
2.6
5.1
.22
.30
.06
13.9
19.6a
0.14
0.01
0.01
5.4
aUnits are pmol/min/mg microsomal protein.
T-5 was in clinical trials as a selective phosphodiesterase type
We found the 150-donor mixed-gender pool of HLM from 5 inhibitor (T-1032). Unfortunately, the data are not publicly
Xenotech to have minimal CYP3A5 protein levels, and the CYP3A5 available to determine whether there was an associated change in
activity was similar to *3/*3 donors. This likely reflects a low T-1032 pharmacokinetics depending on the patient’s CYP3A5
percentage of *1/*1 donors used to generate the pool. If the genotype. Based on the in vitro experimentation presented in our
commercial pools from other vendors have a similar profile, this study, it would be expected that patients with at least one CYP3A5
may have significant consequences as microsomal experiments *1 allele may have significantly higher concentrations of the
would be unlikely to identify the metabolic contribution of CYP3A5 N-oxide and/or desmethylpyridine metabolites, and it is possible
even for compounds with significant preference for CYP3A5 over that overall T-5 clearance would be altered. How this might affect
CYP3A4.
the pharmacodynamics or safety of T-1032 in CYP3A5*1 patients
The enzymatic conversion of T-5 to T-5 N-oxide highly correlated to would be of interest. Because the *1 allele is much more prevalent
CYP3A5 levels and represents a CYP3A5 selective marker reaction. in African Americans than in Caucasian or Asian populations, it is
We hesitate to term T-5 as a CYP3A5 selective substrate because other possible that there could be a racial component to the drug efficacy
enzymes can metabolize T-5 to generate alternate metabolites. The in or safety.
vitro clearance of T-5 by CYP3A5 was three times greater than other
The idea of personalized medicine has primarily focused on pairing
enzymes tested, implying that CYP3A5 may be a major metabolic patients with the best drug for optimal efficacy. However, personal-
pathway for CYP3A5*1 individuals. For CYP3A5*3/*3 individuals, ized medicine should not be reserved to efficacy. As the contribution
CYP2C8, 2C9, and 3A4 would be expected to play significant roles in of genetics and disease state become understood, improvement in
T-5 metabolism.
patient safety should be achievable, with optimal drug pairings or dose
Increased T-5 N-oxide formation was statistically significant for adjustments determined in a proactive rather than reactionary setting.
microsomes from patients with a CYP3A5 *1/*1 genotype over those To achieve this goal, having the appropriate in vitro tools to advance
with *1/*3. This difference was not observed with hepatocytes. It is the science is paramount.
unclear if the difference is due to the low number of tested microsome
We believe T-5 represents an exciting new discovery in the ability
and hepatocyte donors, or if there are additional undetermined factors to elucidate the influence of the CYP3A5 genotype on currently
to consider. In all cases there was a large and statistically significant marketed drugs and those being developed in the future. Cases where
difference between those donors with at least one *1 allele and those the CYP3A5 genotype appears to have a high potential to influence
without.
exposure or metabolite profile can be studied with intelligently
Based on the intercept of the regression line when T-5 N-oxide designed clinical trials, similar to those currently used for patients with
formation was correlated to CYP3A5 content, determined by tryptic high and low CYP2D6 activity.
Fig. 10. Kinetic comparison in genotyped human hepatocytes. T-5 N-oxide formation was evaluated in human hepatocytes from nine individual donors, comprising three
donors each of the CYP3A5 *1/*1, *1/*3, and *3/*3 genotype. Incubations were 100 ml volume containing 75,000 cells and were stopped after 30 minutes by the addition of
an equal volume of methanol. Parallel incubations using the same hepatocyte lots were set up after midazolam hydroxylation to show that the hepatocytes were metabolically
viable and that the low level of T-5 N-oxide formation in CYP3A5 *3/*3 donor hepatocytes was not due to low total CYP3A expression.

342
LI et al.
Li X, Song X, Kamenecka TM, and Cameron MD (2012) Discovery of a highly selective
CYP3A4 inhibitor suitable for reaction phenotyping studies and differentiation of CYP3A4 and
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Liu YT, Hao HP, Liu CX, Wang GJ, and Xie HG (2007) Drugs as CYP3A probes, inducers, and
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Niwa T, Murayama N, Emoto C, and Yamazaki H (2008) Comparison of kinetic parameters for
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Authorship Contributions
Participated in research design: Cameron, Li, Jeso.
Conducted experiments: Li, Jeso, Heyward, Walker, Sharma.
Contributed new reagents or analytic tools: Jeso, Micalizio.
Performed data analysis: Cameron, Li, Jeso, Walker.
Wrote or contributed to the writing of the manuscript: Cameron, Li, Jeso.
Perrett HF, Barter ZE, Jones BC, Yamazaki H, Tucker GT, and Rostami-Hodjegan A (2007)
Disparity in holoprotein/apoprotein ratios of different standards used for immunoquantification
of hepatic cytochrome P450 enzymes. Drug Metab Dispos 35:1733–1736.
Roy JN, Lajoie J, Zijenah LS, Barama A, Poirier C, Ward BJ, and Roger M (2005) CYP3A5
genetic polymorphisms in different ethnic populations. Drug Metab Dispos 33:884–887.
Seaton QF, Lawley CW, and Akers HA (1984) The reduction of aliphatic and aromatic N-oxides
to the corresponding amines with titanium(III) chloride. Anal Biochem 138:238–241.
Soars MG, Grime K, and Riley RJ (2006) Comparative analysis of substrate and inhibitor
interactions with CYP3A4 and CYP3A5. Xenobiotica 36:287–299.
Song X, Li X, Ruiz CH, Yin Y, Feng Y, Kamenecka TM, and Cameron MD (2012) Imidazo-
pyridines as selective CYP3A4 inhibitors. Bioorg Med Chem Lett 22:1611–1614.
Stresser DM, Blanchard AP, Turner SD, Erve JC, Dandeneau AA, Miller VP, and Crespi CL
(2000) Substrate-dependent modulation of CYP3A4 catalytic activity: analysis of 27 test
compounds with four fluorometric substrates. Drug Metab Dispos 28:1440–1448.
Thummel KE and Wilkinson GR (1998) In vitro and in vivo drug interactions involving human
CYP3A. Annu Rev Pharmacol Toxicol 38:389–430.
Ukita T, Nakamura Y, Kubo A, Yamamoto Y, Moritani Y, Saruta K, Higashijima T, Kotera J,
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inhibitors: synthesis and biological activities of a series of 4-aryl-1-isoquinolinone derivatives.
J Med Chem 44:2204–2218.
Walsky RL, Obach RS, Hyland R, Kang P, Zhou S, West M, Geoghegan KF, Helal CJ, Walker
GS, and Goosen TC, et al. (2012) Selective mechanism-based inactivation of CYP3A4 by
CYP3cide (PF-04981517) and its utility as an in vitro tool for delineating the relative roles of
CYP3A4 versus CYP3A5 in the metabolism of drugs. Drug Metab Dispos 40:1686–1697.
Wang RW, Newton DJ, Liu N, Atkins WM, and Lu AY (2000) Human cytochrome P-450 3A4:
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Address correspondence to: Michael D. Cameron, Scripps Florida, Department
of Molecular Therapeutics, 130 Scripps Way, Jupiter, FL 33458. E-mail:
cameron@scripps.edu