Regioselective oxidation of phospho-NSAIDs by human cytochrome P450 and flavin monooxygenase isoforms: Implications for their pharmacokinetic properties and safety

Article abstract of DOI:10.1111/j.1476-5381.2012.01982.x

BACKGROUND AND PURPOSE Phospho-ibuprofen (MDC-917) and phospho-sulindac (OXT-328) are highly effective in cancer and arthritis treatment in preclinical models. Here, we investigated their metabolism by major human cytochrome P450s (CYPs) and flavin monooxygenases (FMOs). EXPERIMENTAL APPROACH The CYP/FMO-catalysed metabolism of phospho-ibuprofen and phospho-sulindac was studied by using in silico prediction modelling and a direct experimental approach. KEY RESULTS The CYP isoforms catalyse the oxidation of non-steroidal anti-inflammatory drugs (NSAIDs) and phospho-NSAIDs, with distinct activity and regioselectivity. CYP1A2, 2C19, 2D6 and 3A4 oxidize phospho-ibuprofen, but not ibuprofen; whereas CYP2C9 oxidizes ibuprofen, but not phospho-ibuprofen. All CYPs tested oxidize phospho-sulindac, but not sulindac. Among the five CYPs evaluated, CYP3A4 and 2D6 are the most active in the oxidation of phospho-ibuprofen and phospho-sulindac respectively. FMOs oxidized phospho-sulindac and sulindac, but not phospho-ibuprofen or ibuprofen. FMOs were more active towards phospho-sulindac than sulindac, indicating that phospho-sulindac is a preferred substrate of FMOs. The susceptibility of phospho-NSAIDs to CYP/FMO-mediated metabolism was also reflected in their rapid oxidation by human and mouse liver microsomes, which contain a full complement of CYPs and FMOs. Compared with conventional NSAIDs, the higher activity of CYPs towards phospho-ibuprofen and phospho-sulindac may be due to their greater lipophilicity, a key parameter for CYP binding. CONCLUSIONS AND IMPLICATIONS CYPs and FMOs play an important role in the metabolism of phospho-NSAIDs, resulting in differential pharmacokinetic profiles between phospho-NSAIDs and NSAIDs in vivo. The consequently more rapid detoxification of phospho-NSAIDs is likely to contribute to their greater safety.

Full text of DOI:10.1111/j.1476-5381.2012.01982.x

DOI:10.1111/j.1476-5381.2012.01982.x
www.brjpharmacol.org
British Journal of
Pharmacology
BJP
Correspondence
RESEARCH PAPERbph_1982 222..232
Dr Basil Rigas, Division of Cancer
Prevention, Stony Brook
University, HSC, T17-080; Stony
Brook, NY 11794-8173, USA.
E-mail:
Regioselective oxidation of
phospho-NSAIDs by human
cytochrome P450 and flavin
monooxygenase isoforms:
implications for their
pharmacokinetic properties
and safety
Gang Xie1, Chi C Wong1, Ka-Wing Cheng1, Liqun Huang1,
Panayiotis P Constantinides2 and Basil Rigas1
basil.rigas@stonybrook.edu
----------------------------------------------------------------
Keywords
phospho-NSAIDs; NSAIDs;
cytochrome P450; regioselective
oxidation; anticancer drugs;
flavin monooxygenase
----------------------------------------------------------------
Received
14 February 2012
Revised
20 March 2012
Accepted
3 April 2012
1Department of Medicine, Division of Cancer Prevention, Stony Brook University, Stony Brook,
NY, USA, and 2Medicon Pharmaceuticals, Inc, Stony Brook, NY, USA
BACKGROUND AND PURPOSE
Phospho-ibuprofen (MDC-917) and phospho-sulindac (OXT-328) are highly effective in cancer and arthritis treatment in
preclinical models. Here, we investigated their metabolism by major human cytochrome P450s (CYPs) and flavin
monooxygenases (FMOs).
EXPERIMENTAL APPROACH
The CYP/FMO-catalysed metabolism of phospho-ibuprofen and phospho-sulindac was studied by using in silico prediction
modelling and a direct experimental approach.
KEY RESULTS
The CYP isoforms catalyse the oxidation of non-steroidal anti-inflammatory drugs (NSAIDs) and phospho-NSAIDs, with distinct
activity and regioselectivity. CYP1A2, 2C19, 2D6 and 3A4 oxidize phospho-ibuprofen, but not ibuprofen; whereas CYP2C9
oxidizes ibuprofen, but not phospho-ibuprofen. All CYPs tested oxidize phospho-sulindac, but not sulindac. Among the five
CYPs evaluated, CYP3A4 and 2D6 are the most active in the oxidation of phospho-ibuprofen and phospho-sulindac
respectively. FMOs oxidized phospho-sulindac and sulindac, but not phospho-ibuprofen or ibuprofen. FMOs were more active
towards phospho-sulindac than sulindac, indicating that phospho-sulindac is a preferred substrate of FMOs. The susceptibility
of phospho-NSAIDs to CYP/FMO-mediated metabolism was also reflected in their rapid oxidation by human and mouse liver
microsomes, which contain a full complement of CYPs and FMOs. Compared with conventional NSAIDs, the higher activity of
CYPs towards phospho-ibuprofen and phospho-sulindac may be due to their greater lipophilicity, a key parameter for CYP
binding.
CONCLUSIONS AND IMPLICATIONS
CYPs and FMOs play an important role in the metabolism of phospho-NSAIDs, resulting in differential pharmacokinetic
profiles between phospho-NSAIDs and NSAIDs in vivo. The consequently more rapid detoxification of phospho-NSAIDs is likely
to contribute to their greater safety.
Abbreviations
BNPP, bis(4-nitro phenyl)-phosphate; CYP, cytochrome P450; FMO, flavin monooxygenases; HLM, human liver
microsomes; MLM, mouse liver microsomes; NSAIDs, non-steroidal anti-inflammatory drugs; ROS, reactive oxygen
species
© 2012 The Authors
British Journal of Pharmacology © 2012 The British Pharmacological Society
222 British Journal of Pharmacology (2012) 167 222–232

Oxidation of phospho-NSAIDs by CYPs and FMOs
BJP
preferentially oxidized to form phospho-sulindac sulfone,
which upon hydrolysis gives sulindac sulfone as the major
metabolite (Xie et al., 2012). These findings suggest that cyto-
chrome P450s (CYPs), the key enzymes involved in oxidative
transformation of xenobiotics, have different activity and
regioselectivity towards NSAIDs and phospho-NSAIDs.
CYPs, consisting of 43 subfamilies and 57 individual
enzymes in humans, are involved in endogenous cellular
functions, such as hormone biosynthesis (Martignoni et al.,
2006). However, CYPs are best recognized as the major drug-
metabolizing enzymes that account for ~75% of the metabo-
lism of clinically used drugs and other xenobiotics (Sweeney
and Bromilow, 2006). In addition, CYP1A2, 2C9, 2C19, 2D6
and 3A4 are the major human CYP isoforms accounting for
95% of CYP-mediated drug metabolism (Lamb et al., 2007).
Flavin monooxygenases (FMOs), comprising five isoforms
(FMO1 to FMO5), also play important roles in detoxifying
xenobiotics. While CYPs can oxidize non-nucleophilic sub-
strates at their C-H bond, FMOs oxidize substrates containing
a ‘soft nucleophile’ such as nitrogen and sulfur (Krueger
and Williams, 2005). Both CYPs and FMOs are primarily
expressed in the liver and, to a lesser extent, in the gastroin-
testinal tract. In general, CYP/FMO-mediated oxidation leads
to detoxification and increased water solubility of drugs,
which facilitates their excretion from the body.
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely
used as anti-inflammatory, analgesic and antipyretic agents
to treat a number of diseases (Suleyman et al., 2010). NSAIDs
have also been shown to be effective in reducing the inci-
dence of colon and other cancers (Gravitz, 2011). However,
chronic use of conventional NSAIDs induces significant gas-
trointestinal and renal toxicities (Vonkeman and van de Laar,
2010). To address this issue, we developed novel phospho-
derivatives of NSAIDs, such as phospho-ibuprofen (MDC-
917) and phospho-sulindac (OXT-328), which exhibit greater
efficacy and are safer than their respective parent NSAIDs in
preclinical models of cancer and arthritis (Huang et al., 2010;
2011; Mackenzie et al., 2010).
Prompted by these promising results, we studied the
metabolism and pharmacokinetics of phospho-ibuprofen
(Xie et al., 2011) and phospho-sulindac (Xie et al., 2012)
in mice. Interestingly, we observed that the phospho-
modification has a significant impact on the regioselectivity
and the extent of NSAID oxidation in vitro and in vivo.
Phospho-ibuprofen was oxidized at the 1- and 3-positions of
its isobutyl group to form 1-OH-phospho-ibuprofen and
3-OH-phospho-ibuprofen, respectively, the latter leading to
carboxy-phospho-ibuprofen; whereas, ibuprofen was oxi-
dized at 2- and 3-positions to give 2-OH-ibuprofen and
3-OH-ibuprofen, respectively, the latter leading to carboxy-
ibuprofen (Figure 1A) (Xie et al., 2011). On the other hand,
phospho-sulindac and sulindac can be oxidized at their sul-
foxide groups to generate phospho-sulindac sulfone and
sulindac sulfone respectively (Figure 1B). Phospho-sulindac
and sulindac have markedly different metabolite profiles in
mice: while sulindac sulfide (the reduced form of sulindac) is
the predominant metabolite of sulindac, phospho-sulindac is
Here, we used ADMET (absorption, distribution, metabo-
lism, excretion and toxicity) modelling to predict the CYP
isoforms involved in the oxidation of phospho-NSAIDs and
NSAIDs, and experimentally characterized the activity and
regioselectivity of oxidation by human recombinant CYPs/
FMOs and liver microsomes. We found that phospho-
ibuprofen and phospho-sulindac were preferentially oxidized
by CYPs/FMOs relative to ibuprofen and sulindac, respec-
tively, leading to their rapid oxidation by liver microsomes.
Figure 1
Oxidation pathway of phospho-NSAIDs and NSAIDs. The oxidation pathway of phospho-ibuprofen versus ibuprofen (A) and that of phospho-
sulindac versus sulindac (B) in vitro and in vivo are shown.
British Journal of Pharmacology (2012) 167 222–232 223

G Xie et al.
BJP
These findings largely explain the characteristic pharmacoki-
netic parameters of phospho-NSAIDs compared to conven-
tional NSAIDs, which may contribute to their improved
safety profile.
of acetonitrile, vortexed and then centrifuged for 10 min at
13 000 ¥ g. The supernatants were analysed by HPLC.
Oxidation of phospho-NSAIDs and NSAIDs
by BNPP-treated human and mouse
liver microsomes
Methods
Human or mouse liver microsomes were pre-incubated with
BNPP (final 250 mM) at 37°C for 15 min and were subse-
quently treated with phospho-NSAIDs or conventional
NSAIDs (150 mM for phospho-ibuprofen or ibuprofen; 100 mM
for phospho-sulindac or sulindac) at 37°C for up to 9 h with
NADPH-regenerating solution in 0.1 M potassium phosphate
buffer (pH 7.4) (final 0.5 mg protein mL-1 of liver microsomes)
in a total volume of 1 mL. The oxidized products were
extracted at various time points and assayed by HPLC.
In silico simulations of drug metabolism by
human CYPs
Predictions of the metabolism of phospho-NSAIDs by the
major human CYP isoforms (CYP1A2, 2C9, 2C19, 2D6 and
3A4) were performed using ADMET Predictor version 5.5
(Simulations Plus Inc., Lancaster, CA) based on Accelrys
metabolite database and Drugbank database, as well as pub-
lished datasets of drug metabolism and general review arti-
cles. The possibility of being a metabolic site was indicated by
a score ranging from 0 to 1, with higher scores indicating a
greater likelihood, and the highest scoring atom is high-
lighted with a red hashed circle (Figure 2).
Determination of reactive oxygen species (ROS)
ROS assay kit (Cell Biolabs, Inc. San Diego, CA) was used in
this study according to the protocol provided by the manu-
facturer. Briefly, equimolar phospho-ibuprofen or ibuprofen
at 150 mM was incubated with CYP3A4 (25 pmol·mL-1) or
BNPP-treated HLM (0.5 mg protein mL-1) for 1 h, and 50 mL
of sample was incubated with 150 mL of dichlorodihy-
drofluorescein solution for 30 min at room temperature in a
black 96-well plate, and the fluorescence of the mixtures was
HPLC-UV analysis
The HPLC system comprised a Waters Alliance 2695 Separa-
tions Module equipped with a Waters 2998 photodiode array
detector (220 and 328 nm) and a Thermo BDS Hypersil C18
column (150 ¥ 4.6 mm, particle size 3 mm). The mobile phase
consisted of a gradient between aqueous solvent A [trif-
luroacetic acid, acetonitrile, H2O (0.1:4.9:95 v/v/v)] and
organic solvent B (acetonitrile) at a flow rate of 1 mL·min-1 at
30°C. We applied gradient elution from 0% to 100% B from 0
to 15 min, and it was maintained at 100% B until 18 min.
determined on
530 nm.
a fluorometric plate reader at 480 nm/
Data analysis
Data are shown as means Ϯ SEM unless otherwise indicated.
Results were analysed using the Student’s t-test; P Յ 0.05 was
considered statistically significant.
Isolation and LC-MS/MS analysis of
HPLC peaks
Materials
Phospho-ibuprofen and phospho-sulindac were provided by
Medicon Pharmaceuticals, Inc (Stony Brook, NY). Ibuprofen,
3-OH-ibuprofen, bis(4-nitro phenyl)-phosphate (BNPP),
sulindac, porcine liver esterase and acetonitrile were pur-
chased from Sigma-Aldrich (St. Louis, MO). 2-OH-ibuprofen
was purchased from US Biological (Swampscott, MA). a-OH-
ibuprofen, ketoconazole and quinidine were purchased from
Toronto Research Chemicals (North York, ON, Canada).
cDNA-expressed human CYPs (CYP1A2, 2C9, 2C19, 2D6 and
3A4) and FMOs (FMO1, FMO3 and FMO5), human and
mouse liver microsomes, and NADPH regenerating solution
were purchased from BD Biosciences (San Jose, CA).
The HPLC peaks corresponding to 3-OH-phospho-ibuprofen
and a-OH-phospho-ibuprofen were collected, concentrated
under vacuum and subjected to LC-MS/MS analysis. The
LC-MS/MS system consisted of Thermo TSQ Quantum Access
(Thermo-Fisher, San Jose, CA, USA) triple quadrupole mass
spectrometer interfaced by an electrospray ionization probe
with an Ultimate 3000 HPLC system (Dionex Corporation,
Sunnyvale, CA). Chromatographic separations were achieved
on a Luna C18 column (150 ¥ 2 mm), and the mobile phase
consisted of a gradient from 10% to 95% acetonitrile.
Oxidation of phospho-NSAIDs and NSAIDs
by human CYP and FMO isoforms
Phospho-NSAIDs or conventional NSAIDs (150 mM for
phospho-ibuprofen or ibuprofen; 100 mM for phospho-
sulindac or sulindac) were pre-incubated at 37°C for 5 min
with an NADPH-regenerating solution (1.3 mM NADP,
3.3 mM D-glucose 6-phosphate, 3.3 mM MgCl2 and
0.4 U·mL-1 glucose-6-phosphate dehydrogenase) in 0.1 M
potassium phosphate buffer (pH 7.4). Reaction was initiated
by the addition of individual recombinant human CYP iso-
forms (25 pmol·mL-1) or human FMO isoforms (0.125 mg
protein mL-1) in a total volume of 1 mL, and samples were
maintained at 37°C for various time periods. At each desig-
nated time point, an aliquot was mixed with twofold volume
Results
In silico prediction of the oxidation of
phospho-NSAIDs and NSAIDs by CYPs
Predictions of the metabolism of phospho-NSAIDs by the
major human CYP isoforms (CYP1A2, 2C9, 2C19, 2D6 and
3A4) were obtained using the ADMET Predictor. It was pre-
dicted that phospho-NSAIDs and conventional NSAIDs
would be oxidized by distinct CYPs. Thus, 2C19 and 3A4
would oxidize phospho-ibuprofen; while 1A2, 2C9 and 2C19
oxidize ibuprofen (Figure 2). 3A4 would oxidize phospho-
224 British Journal of Pharmacology (2012) 167 222–232

Oxidation of phospho-NSAIDs by CYPs and FMOs
BJP
Figure 2
In silico prediction of drug metabolism by CYP isoforms using ADMET modelling. A. Phospho-ibuprofen (PI) is predicted to be oxidized by
CYP2C19 and 3A4. The scores ranging from 0 to 1 indicate the possibility of being a metabolic site, and the highest scoring atom is highlighted
with a red hashed circle. B. Ibuprofen is predicted to be oxidized by CYP1A2, 2C9 and 2C19.
Table 1
Lipophilicity (LogP), modelling prediction and experimental results on the metabolism of phospho-ibuprofen, phospho-sulindac and the parent
NSAIDs by human CYPs
Human CYP isoforms
Compound
LogP
Prediction
Experimental
Phospho-ibuprofen
Ibuprofen
5.4
3.8
3.9
2.3
2C19, 3A4
1A2, 2C9, 2C19
3A4
1A2, 2C19, 2D6, 3A4
2C9
Phospho-sulindac
Sulindac
1A2, 2C9, 2C19, 2D6, 3A4
None
1A2, 2C9
LogP (P is octanol–water partition coefficient) is determined using ChemDraw 7.0.
sulindac, while 1A2 and 2C9 would oxidize sulindac
Regioselective oxidation of phospho-ibuprofen
and ibuprofen by CYPs
(Table 1). Moreover, the isobutyl group of phospho-ibuprofen
or ibuprofen, as well as the sulfoxide group of phospho-
sulindac or sulindac were predicted to be the most likely sites
of oxidation by CYPs, which we have independently demon-
strated (Xie et al., 2011; 2012).
To evaluate the in silico predictions experimentally, we exam-
ined the metabolism of phospho-ibuprofen and ibuprofen by
the recombinant major human CYPs. As shown in Figure 3A,
British Journal of Pharmacology (2012) 167 222–232 225

G Xie et al.
BJP
Figure 3
Kinetics of the oxidation of phospho-ibuprofen (PI) or ibuprofen by CYPs. Equimolar phospho-ibuprofen (A) or ibuprofen (B) at 150 mM was
incubated with individual human CYP isoforms and NADPH-regenerating solution at 37°C for up to 20 h. The oxidized products were extracted
at the designated time points and assayed by HPLC. Values are mean Ϯ SEM.
CYP1A2, 2C19, 2D6 and 3A4 catalyzed the oxidation of
phospho-ibuprofen, with 3A4 being the most active. In con-
trast, CYP2C9 was inactive towards phospho-ibuprofen
(data not shown). Interestingly, the four CYPs oxidized
phospho-ibuprofen with differential regioselectivity. CYP1A2
oxidized phospho-ibuprofen primarily at the 1-position of
the isobutyl group to produce 1-OH-phospho-ibuprofen,
whereas 2C19 and 2D6 oxidized this substrate exclusively at
the 3-position to form 3-OH-phospho-ibuprofen. On the
other hand, 3A4 generated both 1-OH- and 3-OH-phospho-
ibuprofen at similar rates and uniquely oxidized phospho-
ibuprofen at the a-position of the ibuprofen moiety to give
a-OH-phospho-ibuprofen.
and plateaued afterwards (Figure 3A). In contrast, the other
three CYPs (1A2, 2C19 and 2D6) exhibited similar kinetic
patterns; the levels of their products gradually increased in
a
time-dependent manner during the entire period of
observation.
3-OH-phospho-ibuprofen and a-OH-phospho-ibuprofen
were identified by LC-MS/MS analysis. The mass spectrum of
the 3-OH-metabolite showed a [M + Na]+ ion at m/z 453.0,
which was fragmented to yield ions at m/z 153 and 317
(Figure 4A). Similarly, the mass spectrum of a-OH-phospho-
ibuprofen showed a [M + Na]+ ion at m/z 453.0, which was
fragmented to produce m/z at 177 and 249 (Figure 4B). The
position of the – OH group of the 3-OH- and the a-OH-
phospho-ibuprofen was determined by treating them with
esterases from porcine liver, which completely hydrolysed
CYP3A4 also displayed a unique kinetic behaviour: all the
three products reached their peak levels rapidly within ~2 h
226 British Journal of Pharmacology (2012) 167 222–232

Oxidation of phospho-NSAIDs by CYPs and FMOs
BJP
Figure 4
Identification of the metabolites of phospho-ibuprofen (PI) by CYPs. A. MS and MS/MS spectrum of 3-OH-PI fraction collected from HPLC. B. MS
and MS/MS spectrum of a-OH-PI fraction collected from HPLC.
these metabolites to release 3-OH-ibuprofen and a-OH-
ibuprofen, respectively.
phospho-sulindac sulfone being detected. The metabolism of
phospho-sulindac was not a result of non-enzymatic oxida-
tion, as no phospho-sulindac sulfone could be detected in a
negative control reaction without NADPH. All five CYPs
tested failed to appreciably oxidize sulindac (data not
shown). This indicated that phospho-sulindac, but not sulin-
dac, was a substrate for CYPs.
We next evaluated the oxidation of phospho-sulindac and
sulindac by major human FMO isoforms (FMO1, FMO3 and
FMO5). All the FMOs oxidized phospho-sulindac far more
rapidly and extensively than sulindac (Figure 6), indicating
that phospho-sulindac was a preferred substrate of FMOs. On
the other hand, FMOs were inactive towards phospho-
ibuprofen or ibuprofen (data not shown). Thus, phospho-
sulindac was oxidized by both CYPs and FMOs, while
phospho-ibuprofen was oxidized exclusively by CYPs.
We have also examined the oxidation of ibuprofen by
individual CYPs. CYP2C9 was highly active towards ibupro-
fen, generating 2-OH-ibuprofen and 3-OH-ibuprofen
(Figure 3B); this regioselectivity was distinct from that of
phospho-ibuprofen. In contrast to CYP2C9, none of the other
four CYPs appreciably oxidized ibuprofen (data not shown).
Thus, the selectivity of CYPs for phospho-ibuprofen and ibu-
profen are strikingly different: CYP2C9 exclusively oxidizes
ibuprofen, while the other four CYPs exclusively oxidize
phospho-ibuprofen. This finding unambiguously explains our
previous observation that phospho-ibuprofen and ibuprofen
were oxidized with differential regioselectivity (Figure 1A).
Oxidation of phospho-sulindac and sulindac
by CYPs and FMOs
Comparison of the oxidation of phospho-
ibuprofen and ibuprofen by liver microsomes
We next compared the oxidation of phospho-ibuprofen and
ibuprofen by human liver microsomes (HLM), which contain
a full complement of CYPs. Carboxylesterases are abundant
We also evaluated the oxidation of phospho-sulindac and
sulindac by human CYPs. CYP2D6 was by far the most active
in oxidizing phospho-sulindac to form phospho-sulindac
sulfone (Figure 5). In comparison, all the other four CYPs
modestly oxidized phospho-sulindac with quite low levels of
British Journal of Pharmacology (2012) 167 222–232 227

G Xie et al.
BJP
Figure 5
Kinetics of the oxidation of phospho-sulindac (PS) by CYPs. Phospho-
sulindac at 100 mM was incubated with individual human CYP iso-
forms and NADPH-regenerating solution at 37°C for up to 20 h. The
oxidized products were extracted at the designated time points and
analysed by HPLC. Values are mean Ϯ SEM.
in HLM, leading to significant hydrolysis of phospho-
ibuprofen (Xie et al., 2011). To inhibit the hydrolysis of
phospho-ibuprofen, we pre-treated HLM with BNPP, a non-
specific esterase inhibitor, and then evaluated the oxidation
of phospho-ibuprofen or ibuprofen by the BNPP-treated HLM
(Figure 7A). Half an hour after the initiation of the reaction,
2% of ibuprofen and 20% of phospho-ibuprofen were oxi-
dized, indicating that phospho-ibuprofen was more rapidly
and extensively oxidized by HLM than ibuprofen.
Our data in Figure 3A suggested that CYP3A4, the most
abundant isoform in human liver, played a major role in
the oxidation of phospho-ibuprofen. To test this, we
assessed the effect of ketoconazole (CYP3A4 inhibitor) and
quinidine (CYP2D6 inhibitor) on the oxidation of phospho-
ibuprofen. We observed that ketoconazole (0.5 mM) com-
pletely inhibited CYP3A4 activity and quinidine (10 mM)
inhibited CYP2D6 activity by 90%. We then evaluated the
effects of these inhibitors on the oxidation of phospho-
ibuprofen by BNPP-treated HLM. Ketoconazole (0.5 mM)
inhibited the oxidation of phospho-ibuprofen in HLM by
84%, whereas quinidine (10 mM) had no appreciable effect
on phospho-ibuprofen oxidation. This result indicated that
CYP3A4 plays a major role in the oxidation of phospho-
ibuprofen by HLM.
Figure 6
Kinetics of the oxidation of phospho-sulindac (PS) or sulindac by
FMOs. Equimolar phospho-sulindac or sulindac at 100 mM was incu-
bated with individual human FMOs (FMO1, FMO3 and FMO5) and
NADPH-regenerating solution at 37°C for up to 20 h. The oxidized
products were extracted at the designated time points and analysed
by HPLC. Results were expressed as % of input drug. Values are
mean Ϯ SEM.
We have recently determined the pharmacokinetic
parameters of phospho-ibuprofen and ibuprofen in mice, but
mice differ from humans in composition and expression of
CYP isoforms (Martignoni et al., 2006). Therefore, we also
compared the oxidation of phospho-ibuprofen and ibuprofen
by BNPP-treated mouse liver microsomes (MLM) (Figure 7A).
Phospho-ibuprofen was far more rapidly oxidized by the
MLM than ibuprofen; the oxidation of phospho-ibuprofen
was essentially complete after a 9 h incubation, while only
12% of ibuprofen was oxidized over the same time period
(Figure 7A).
228 British Journal of Pharmacology (2012) 167 222–232

Oxidation of phospho-NSAIDs by CYPs and FMOs
BJP
Figure 7
Kinetics of the oxidation of phospho-NSAIDs or NSAIDs by BNPP-treated human and mouse liver microsomes. Human or mouse liver microsomes
were pre-incubated with BNPP (final 250 mM) at 37°C for 15 min and subsequently treated with phospho-ibuprofen (PI) or ibuprofen (A) at
150 mM or phospho-sulindac (PS) or sulindac (B) at 100 mM at 37°C for up to 9 h. The oxidized products extracted at the designated time points
and assayed by HPLC. Results were expressed as % of input drug. Values are mean Ϯ SEM.
was reduced by 88% at pH 9.5 compared with pH 7.4, sug-
gesting that CYPs, rather than FMOs, predominantly oxidize
phospho-sulindac in HLM.
Comparison of the oxidation of
phospho-sulindac and sulindac by
liver microsomes
We next examined and compared the oxidation of phospho-
sulindac and sulindac by BNPP-treated liver microsomes,
Induction of ROS by phospho-ibuprofen
which contain both CYPs and FMOs. Phospho-sulindac was
oxidized more rapidly than sulindac by HLM and to a much
larger extent by MLM (Figure 7B). These data further substan-
tiate our findings that phospho-sulindac is more susceptible
to CYP/FMO-mediated oxidation than sulindac, leading to
enhanced clearance in vivo.
As both CYPs and FMOs can oxidize phospho-sulindac,
we determined which enzyme primarily oxidizes phospho-
sulindac in HLM. The activities of CYPs, but not of FMOs, are
abolished at alkaline pH (Cashman, 2005). We observed com-
plete inhibition of CYPs at pH 9.5, while the activities of
FMOs were essentially the same at pH 7.4 and pH 9.5 (data
not shown). We next compared the oxidation of phospho-
NSAIDs by HLM at pH 7.4 and pH 9.5. HLM was inactive
towards phospho-ibuprofen at pH 9.5, supporting the notion
that CYPs exclusively oxidize phospho-ibuprofen. On the
other hand, the activity of HLM towards phospho-sulindac
or ibuprofen
As CYP-mediated oxidation of various substrates can generate
ROS (Puntarulo and Cederbaum, 1998), we measured and
compared the ROS level generated by the CYP-mediated oxi-
dation of phospho-ibuprofen and ibuprofen. Equimolar
concentrations of phospho-ibuprofen or ibuprofen were
incubated with CYP3A4 for 1 h, and ROS were determined
using dichlorodihydrofluorescein, a general ROS probe. Ibu-
profen generated a negligible level of ROS during its incuba-
tion with CYP3A4, whereas phospho-ibuprofen markedly
increased ROS levels during CYP3A4-mediated oxidation,
with their difference being statistically significant (P < 0.01)
(Figure 8A). Likewise, ibuprofen generated a minimal level of
ROS in BNPP-treated HLM, whereas phospho-ibuprofen
greatly enhanced the ROS level, with their difference being
statistically significant (P < 0.01) (Figure 8B).
British Journal of Pharmacology (2012) 167 222–232 229

G Xie et al.
BJP
highly correlated with their binding affinity with CYPs (Lewis
et al., 2004), as the lower desolvation energy of lipophilic
substrates contributes to their high binding affinity to CYPs
(Jacobsen et al., 2000). Phospho-ibuprofen and phospho-
sulindac are more lipophilic than ibuprofen and sulindac,
respectively, as shown by their higher LogP values (Table 1).
Another critical factor to their binding by CYPs is the
altered pKa of phospho-NSAIDs. The major human CYPs,
1A2, 2C9, 2D6 and 3A4, all prefer neutral or basic substrates
(e.g. phospho-ibuprofen and phospho-sulindac). An excep-
tion is CYP2C9, which primarily oxidizes acidic or anionic
substrates such as ibuprofen, with its positively charged Arg108
residue contributing to high-affinity binding (Dickmann
et al., 2004; Locuson et al., 2004). Thus, the phospho-
modification of conventional NSAIDs at their – COOH group
enhances lipophilicity and masks their acid group, thus pro-
moting their binding by CYPs.
CYP3A4 displays three unique features in the oxidation
of phospho-ibuprofen. First, CYP3A4 displays
a broad
regioselectivity, uniquely generating all the three hydroxy-
phospho-ibuprofen metabolites (1-OH-, 3-OH- and a-OH-
phospho-ibuprofen); whereas other CYPs generate either the
1-OH- or 3-OH-metabolite. Second, CYP3A4 generated the
highest levels of each product during the entire period of
observation. Third, ketoconazole, the CYP3A4 inhibitor,
inhibited the oxidation of phospho-ibuprofen by 84%.
Together, these observations suggest that CYP3A4, the most
abundant isoform in human liver, is the major isoform con-
tributing to the oxidation of phospho-ibuprofen. Indeed,
CYP3A4 accounts for 45–60% of the metabolism of all drugs
currently used (Hustert et al., 2001). Substrate binding to
CYP3A4 induces large increase in the volume of its active site
(Ekroos and Sjogren, 2006), which could account for its pref-
erence for bulky substrates such as phospho-ibuprofen. In
addition, the structure of CYP3A4–ligand complex is remark-
ably flexible (Ekroos and Sjogren, 2006; Bonn et al., 2010),
which may lead to its broad regioselectivity in phospho-
ibuprofen oxidation.
Among the CYPs, CYP2D6 is the most active in mediating
the oxidation of phospho-sulindac. The CYP2D6 substrate
binding uniquely relies on an ion pair between the basic
moiety (e.g. amine group) of the substrates and the negatively
charged residues of CYP2D6 including Asp301 (Ellis et al.,
1995) and Glu216 (Guengerich et al., 2003). It is probable that
the partially positively charged sulfur and phosphorus atoms
of phospho-sulindac may interact with the Asp301 and Glu216
residues of CYP2D6, thereby facilitating its binding and
favourable orientation in the active site.
FMOs oxidized phospho-sulindac and sulindac, but not
phospho-ibuprofen or ibuprofen. This result was entirely
consistent with the notion that FMOs act on substrates con-
taining soft nucleophiles such as nitrogen and sulfur. FMOs
oxidized phospho-sulindac far more rapidly than sulindac.
Unlike the case of CYPs, substrate binding does not affect the
reaction rate of FMO-mediated oxidation (Krueger and Wil-
liams, 2005). The difference in the oxidation rate of phospho-
sulindac and sulindac by FMOs can be explained by two
reasons. First, compounds containing a negative charge (e.g.
sulindac) are generally poor substrates of FMOs (Krueger and
Williams, 2005). Second, phospho-sulindac is much larger
than sulindac (Figure 1B), and the rate of FMO-mediated
Figure 8
Induction of ROS by phospho-ibuprofen (PI) or ibuprofen. Equimolar
phospho-ibuprofen or ibuprofen at 150 mM was incubated with
CYP3A4 (A) or BNPP-treated HLM (B) for 1 h, and ROS were deter-
mined as described in Methods. Values are mean Ϯ SEM. *, P < 0.01.
Discussion and conclusions
Here, we report the unique activity and regioselectivity of the
oxidation of phospho-NSAIDs by human CYPs and FMOs. Our
data establish that these enzymes are highly active towards
phospho-NSAIDs compared with their parent NSAIDs, leading
to the rapid oxidation of phospho-NSAIDs by liver micro-
somes. The CYP/FMO-mediated clearance of phospho-NSAIDs
results in their unique pharmacokinetic profiles, which poten-
tially contribute to their improved safety.
Phospho-NSAIDs were preferentially oxidized by human
CYPs compared to conventional NSAIDs. Phospho-ibuprofen
was oxidized by CYP1A2, 2C9, 2D6 and 3A4; while phospho-
sulindac was a substrate for all five CYPs. On the contrary, the
CYPs tested did not appreciably catalyze the oxidation of
ibuprofen and sulindac except for CYP2C9, which specifically
oxidizes ibuprofen. With the use of the in silico modelling
program, we were able to identify the potential site(s) of
attack by CYPs. However, the in silico predictions tend to
underestimate the range of CYP isoforms that can act on
phospho-sulindac or phospho-ibuprofen. The disparity may
arise from the fact that in silico prediction is based upon an
existing substrate database, which may not be sufficiently
comprehensive to take into account the structural features of
the novel phospho-NSAIDs.
The interaction of CYPs with drugs depends on several
factors, such as molecular size, lipophilicity (LogP) and ioni-
zation constant (pKa) (Lewis, 2000). Substrate lipophilicity is
230 British Journal of Pharmacology (2012) 167 222–232

Oxidation of phospho-NSAIDs by CYPs and FMOs
BJP
Table 2
Pharmacokinetic parameters of phospho-ibuprofen, phospho-sulindac and their parent compounds
Drug administered
Dose (mmol kg-1
)
Cmax (mM)
Elimination t1/2 (h)
AUC0-24h (mM h)
Reference
Phospho-ibuprofen
Ibuprofen
1.0
530
932
234
261
7.7
12.8
3.5
1816
4146
707
Xie et al. (2011)
Xie et al. (2012)
1.0
Phospho-sulindac
Sulindac
0.28
0.28
5.4
1476
reactions increases with increasing size of the substrate
(Nagata et al., 1990).
FMOs (Krueger and Williams, 2005) generate significant ROS
levels while oxidizing various substrates. Indeed, phospho-
ibuprofen (in contrast to ibuprofen) generated significant
levels of ROS during its oxidation by CYP3A4 or HLM. It
is thus conceivable that ROS generation via CYP/FMO-
mediated oxidation of phospho-NSAIDs may potentially con-
tribute to excess oxidative stress, leading to apoptosis and
inhibition of tumour growth.
In conclusion, phospho-NSAIDs are preferred substrates
of CYPs/FMOs, leading to their rapid oxidation by liver
microsomes. These findings largely explain the characteristic
pharmacokinetic features of phospho-NSAIDs that may
account for their improved safety.
In agreement with the CYP activity profiles, liver micro-
somes oxidized phospho-NSAIDs more rapidly than their cor-
responding parent NSAIDs. However, we observed significant
differences in their metabolism between mouse and human
liver microsomes. While phospho-ibuprofen was oxidized at
similar rates in HLM and MLM, ibuprofen was oxidized much
more rapidly in HLM. This occurs probably because CYP2C9,
the isoform efficiently oxidizing ibuprofen, is abundant in
humans but absent in mice (Martignoni et al., 2006). While
sulindac was oxidized at similar rates in HLM and MLM,
phospho-sulindac was oxidized more rapidly in MLM. This is
not surprising since in humans only CYP2D6 can signifi-
cantly oxidize phospho-sulindac. Whether other CYP iso-
forms in mouse can efficiently oxidize phospho-sulindac
remains to be determined.
Acknowledgements
This work was supported by the National Institute of Health
Grant R01CA139454 and Department of Defense grant
W81XWH1010873. We thank R Rieger and T Koller, Stony
Brook University, for the expert LC-MS/MS analysis of our
samples and the shared instrumentation grant NIH/NCRR 1
S10 RR023680-1.
The rapid and extensive oxidation of phospho-NSAIDs by
liver microsomes has important implications for their dis-
tinct pharmacokinetic properties and safety profiles in animal
models. The rapid clearance of phospho-NSAIDs by CYPs/
FMOs leads to their lower peak concentration (Cmax), shorter
elimination half-life (t1/2) and lower AUC0–24 h compared with
those of their parent compounds (Table 2). Thus, phospho-
NSAIDs reduce the systemic exposure of drugs in blood,
which is a favourable factor in terms of safety. It is conceiv-
able that phospho-NSAIDs may also be metabolized and
eliminated rapidly in humans, leading to their better safety
profile. In the case of phospho-sulindac, preferential oxida-
tion to form sulindac sulfone as the major metabolite, rather
than reduction to form sulindac sulfide, further contributes
to its improved safety. Sulindac sulfone does not inhibit COX
activity and is therefore safer than sulindac sulfide (Glavin
and Sitar, 1986; Piazza et al., 2009). On the other hand, the
rapid clearance of phospho-NSAIDs in vivo may affect drug
efficacy, which could be addressed by optimizing the dosing
regimen. Importantly, we have demonstrated that phospho-
ibuprofen (Xie et al., 2011) and phospho-sulindac (Mackenzie
et al., 2010) are more efficacious in cancer treatment in mice
than ibuprofen and sulindac respectively.
Conflicts of interest
The authors have nothing to disclose except for BR, who has
an equity position in Medicon Pharmaceuticals, Inc.
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