Phospho-ibuprofen (MDC-917) is a novel agent against colon cancer: Efficacy, metabolism, and pharmacokinetics in mouse models

Article abstract of DOI:10.1124/jpet.111.180224

We have developed a novel chemical modification of conventional nonsteroidal anti-inflammatory drugs to reduce their toxicity and enhance their efficacy. Phospho-ibuprofen [(PI) 2-(4-isobutyl-phenyl)-propionic acid-4-(diethoxy-phosphoryloxy)-butyl ester (MDC-917)], a novel derivative of ibuprofen, strongly inhibited the growth of human colon cancer cells in vitro and SW480 human colon cancer xenografts in nude mice. PI was metabolized minimally by cultured cells, but extensively by liver microsomes and mice, undergoing regioselective oxidation to produce 1-OH-PI and carboxyl-PI, which can be hydrolyzed to 1-OH-ibuprofen and carboxyl-ibuprofen, respectively. PI also can be hydrolyzed to release ibuprofen, which can generate 2-OH-ibuprofen, carboxyl-ibuprofen, and ibuprofen glucuronide. After a single oral administration (400 mg/kg) of PI, ibuprofen and ibuprofen glucuronide are the main plasma metabolites of PI; they have, respectively, C_max of 530 and 215 μM, T_max of 1 and 2 h, elimination t_1/2 of 7.7 and 5.3 h, and area under the concentration-time curve (0-24 h) of 1816 and 832 μM X h. Intact PI was detected in several tissues but not in plasma; at a higher PI dose (1200 mg/kg), PI plasma levels were 12.4 μM. PI generated the same metabolites in mouse plasma as conventional ibuprofen, but with much lower levels, perhaps accounting for the enhanced safety of PI. The antitumor effect of PI was significantly associated with plasma ibuprofen levels (p = 0.016) but not with xenograft ibuprofen levels (p = 0.08), suggesting a complex anticancer effect. These results provide a pharmacological basis to explain, at least in part, the anticancer efficacy and safety of this promising compound and indicate that PI merits further evaluation as an anticancer agent.

Full text of DOI:10.1124/jpet.111.180224

0022-3565/11/3373-876–886
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
U.S. Government work not protected by U.S. copyright
JPET 337:876–886, 2011
Vol. 337, No. 3
180224/3691334
Printed in U.S.A.
Phospho-Ibuprofen (MDC-917) Is a Novel Agent against Colon
Cancer: Efficacy, Metabolism, and Pharmacokinetics in Mouse
Models
Gang Xie, Yu Sun, Ting Nie, Gerardo G. Mackenzie, Liqun Huang, Levy Kopelovich,
Despina Komninou, and Basil Rigas
Division of Cancer Prevention, Department of Medicine, Stony Brook University, Stony Brook, New York (G.X., Y.S., T.N.,
G.G.M., L.H., B.R.); Division of Cancer Prevention, National Cancer Institute, Bethesda, Maryland (L.K.); and Medicon
Pharmaceuticals, Inc., Stony Brook, New York (D.K.)
Received January 31, 2011; accepted March 8, 2011
ABSTRACT
We have developed a novel chemical modification of conven- olites of PI; they have, respectively, Cmax of 530 and 215 ␮M,
tional nonsteroidal anti-inflammatory drugs to reduce their tox- Tmax of 1 and 2 h, elimination t1/2 of 7.7 and 5.3 h, and area
icity and enhance their efficacy. Phospho-ibuprofen [(PI) 2-(4- under the concentration-time curve (0–24 h) of 1816 and 832
isobutyl-phenyl)-propionic acid-4-(diethoxy-phosphoryloxy)-butyl ␮M ϫ h. Intact PI was detected in several tissues but not in
ester (MDC-917)], a novel derivative of ibuprofen, strongly in- plasma; at a higher PI dose (1200 mg/kg), PI plasma levels were
hibited the growth of human colon cancer cells in vitro and 12.4 ␮M. PI generated the same metabolites in mouse plasma
SW480 human colon cancer xenografts in nude mice. PI was as conventional ibuprofen, but with much lower levels, perhaps
metabolized minimally by cultured cells, but extensively by liver accounting for the enhanced safety of PI. The antitumor effect
microsomes and mice, undergoing regioselective oxidation to of PI was significantly associated with plasma ibuprofen levels
produce 1-OH-PI and carboxyl-PI, which can be hydrolyzed to (p ϭ 0.016) but not with xenograft ibuprofen levels (p ϭ 0.08),
1-OH-ibuprofen and carboxyl-ibuprofen, respectively. PI also suggesting a complex anticancer effect. These results provide
can be hydrolyzed to release ibuprofen, which can generate a pharmacological basis to explain, at least in part, the anti-
2-OH-ibuprofen, carboxyl-ibuprofen, and ibuprofen glucuro- cancer efficacy and safety of this promising compound and
nide. After a single oral administration (400 mg/kg) of PI, ibu- indicate that PI merits further evaluation as an anticancer agent.
profen and ibuprofen glucuronide are the main plasma metab-
cancer through cyclooxygenase (COX)-dependent and COX-
independent mechanisms, and their COX-independent tar-
gets include nuclear factor-␬B, peroxisome proliferator-acti-
vated receptor, lipoxygenase, and apoptosis signaling (Ulrich
Introduction
Inflammation is a critical component of tumor progression,
and nonsteroidal anti-inflammatory drugs (NSAIDs) are ef-
ficacious in inhibiting early neoplastic progression and ma-
et al., 2006).
lignant conversion (Coussens and Werb, 2002). For example,
Conventional NSAIDs are associated with significant tox-
the NSAID ibuprofen is effective in reducing the risk of
icity, which makes their long-term use for cancer prevention
various human cancers, including those of the colon (Meier et
problematic (Piazza et al., 2009). For example, significant
al., 2002), breast (Harris et al., 1996), lung (Marnett, 1995),
side effects are seen with ibuprofen, including those of the
and prostate (Harris et al., 2005). Indeed, NSAIDs inhibit
central nervous system (McElwee et al., 1990), gastrointes-
tinal tract (Lanza, 1984), liver (Riley and Smith, 1998), and
respiratory tract (Antonicelli and Tagliabracci, 1995). On the
other hand, little has been accomplished over the same pe-
riod of time for the treatment of advanced colon cancer, with
its mortality remaining essentially unchanged for at least
three decades (American Chemical Society, 2008). These con-
This work was supported by the National Institutes of Health National
Cancer Institute [Grants STTR-CA156920A, R01-CA153662 and Contract
1N01-CN43302WA22].
B.R. has an equity position in Medicon Pharmaceuticals, Inc.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.111.180224.
ABBREVIATIONS: NSAID, nonsteroidal anti-inflammatory drug; COX, cyclooxygenase; PI, phospho-ibuprofen; RLM, rat liver microsomes; HLM,
human liver microsomes; PK, pharmacokinetics; PD, pharmacodynamics; HPLC, high-performance liquid chromatography; UGT, UDP-glucu-
ronosyltransferase; MS, mass spectrometry; MS/MS, tandem MS; AUC, area under the concentration-time curve; PBS, phosphate-buffered
saline.
876

Efficacy, Metabolism, and PK of Phospho-Ibuprofen
877
siderations provide a compelling need to develop new effec- colon cancer cell lines were purchased from American Type Culture
Collection (Manassas, VA). Rat and human liver microsomes and
NADPH-regenerating solution were purchased from BD Biosciences
(San Jose, CA).
tive agents for the control of cancer.
We developed a novel chemical modification of conven-
tional NSAIDs to reduce their toxicity and enhance their
efficacy (Mackenzie et al., 2010). One such modified NSAID
is phospho-ibuprofen (PI), a derivative of ibuprofen, consist-
ing of ibuprofen covalently attached to the diethylphosphate
group via a spacer moiety (Fig. 1, inset). PI has strong anti-
inflammatory properties both in vitro and in animal models
of arthritis, and it was shown to have far better gastrointes-
tinal safety than conventional ibuprofen (Huang et al., 2011).
PI inhibited cancer cell growth 16 to 23 times more potently
than its parent drug, ibuprofen. PI also inhibited the growth
of human colon cancer xenografts in nude mice, while man-
ifesting minimal or no animal toxicity.
Cell Culture. Human colon cancer cells were grown as monolay-
ers as recommended by the American Type Culture Collection. Cell
growth was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium assay (Promega, Madison, WI) following the
manufacturer’s instructions. To study PI metabolism in cultured
cells, HCT-15 human colon cancer cells were seeded in 6-cm plates at
a density of 105 cells/cm2 in 4 ml of culture media and allowed to
attach for 24 h. The cells were treated with 100 ␮M PI for 6 h and
subsequently washed three times with cold PBS and lysed by soni-
cation. Acetonitrile was added to extract PI and its metabolites from
cells, and the extracts were submitted for HPLC and LC-MS/MS
analysis.
HPLC Analysis. The HPLC system consisted of a Waters Alli-
ance 2695 Separations Module equipped with a Waters 2998 photo-
diode array detector (220 nm) (Waters, Milford, MA) and a Thermo
BDS Hypersil C18 column (150 ϫ 4.6 mm, particle size 3 ␮m)
(Thermo Fisher Scientific, Waltham, MA). The mobile phase followed
a gradient between buffer A [formic acid, acetonitrile, H2O (95:4.9:
0.1 v/v/v)] and buffer B (acetonitrile).
Given the promise of PI, we studied its metabolism, phar-
macokinetics, and pharmacodynamics in mice. Here, our re-
sults establish the metabolic pathways of PI in vitro and in
vivo and provide an insight into the interplay between its
metabolic transformations and pharmacological effects.
LC-MS/MS Analysis. The LC-MS/MS system consisted of a
Thermo TSQ Quantum Access triple quadrupole mass spectrom-
eter (Thermo Fisher Scientific) interfaced by an electrospray ion-
ization probe with an Ultimate 3000 HPLC system (Dionex Cor-
poration, Sunnyvale, CA). Chromatographic separations were
achieved on a Luna C18 column (150 ϫ 2 mm) (Phenomenex Inc.,
Torrance, CA), and the mobile phase consisted of a gradient from
10 to 95% acetonitrile.
Materials and Methods
Reagents. PI was provided by Medicon, Inc (Stony Brook, NY).
Ibuprofen and acetonitrile of HPLC grade and porcine liver esterase
were purchased from Sigma-Aldrich (St. Louis, MO). 1-OH-ibupro-
fen, 2-OH-ibuprofen, and ibuprofen glucuronide were purchased
from U.S. Biological (Swampscott, MA). RPMI medium 1640 and
antibiotics were obtained from Mediatech (Herndon, VA). Human
Metabolism of PI by Liver Microsomes. PI (120 ␮M) was
preincubated at 37°C for 5 min with NADPH-regenerating solution
(1.3 mM NADP, 3.3 mM D-glucose 6-phosphate, 3.3 mM MgCl2, and
0.4 U/ml glucose-6-phosphate dehydrogenase) in 0.1 M potassium
phosphate buffer, pH 7.4. Reaction was initiated by the addition of
liver microsomes (protein concentration 0.5 mg/ml), and samples
were maintained at 37°C for a given time. At the end of each incu-
bation, 0.3-ml aliquots were mixed with 0.6 ml of CH3CN containing
2 M 1% phosphoric acid, vortexed, and then centrifuged for 10 min at
5000g. The supernatants were subjected to HPLC analysis.
Hydrolysis of Phospho-NSAIDs by Liver Esterases. To re-
lease their NSAID moieties, phospho-NSAIDs (PI, 1-OH-PI, or car-
boxyl-PI) were treated with porcine liver esterase (10 mg/ml) at 37°C
for 30 min in 0.1 M potassium phosphate buffer, pH 7.4,. After this,
0.3-ml aliquots were mixed with 0.6 ml of CH3CN, vortexed, and then
centrifuged for 10 min at 5000g. The supernatants were subjected to
HPLC analysis.
In Vivo Inhibition of Colon Cancer Growth in the Xenograft
Mouse Model. The animal study was approved by the Institutional
Animal Care and Use Committee at Stony Brook University. SW480
human colon cancer cells (2 ϫ 106 cells suspended in 100 ␮l of PBS)
were injected subcutaneously in the left and right flanks of 5- to
6-week-old female NCr nude mice (Taconic Farms, Germantown,
NY). After the average tumor volume reached ϳ100 mm3, four
groups of animals (n ϭ 9/group) started receiving the following
treatments intraperitoneally once daily five times a week (5 days
continuously with a 2-day break) for 32 days: group 1 received
vehicle; group 2 received 125 mg/kg per day ibuprofen; group 3
received 225 mg/kg per day PI; and group 4 received 400 mg/kg per
day PI. The volume of each drug or vehicle dose was ϳ100 ␮l. Tumor
volume was measured every 3 or 4 days as specified and was calcu-
lated by measuring with a caliper its length (L) and width (W) and
using the formula L ϫ W ϫ (L ϩ W/2) ϫ 0.56. At the endpoint,
animals were euthanized following a standard protocol.
Fig. 1. Effect of phospho-ibuprofen on colon cancer cell growth in vitro
and in vivo. Top, IC50 values for colon cancer cells treated with PI or
ibuprofen for 24 h. Bottom, PI inhibits the growth of SW480 human colon
cancer xenografts in nude mice. The study was performed as described
under Materials and Methods. The doses of ibuprofen (125 mg/kg) and PI
(225 mg/kg) are equimolar. The structure of PI is also shown; the shaded
part of the molecule corresponds to conventional ibuprofen. Values are
mean Ϯ S.E.M. ء, p Յ 0.05 compared with the corresponding control
value.
Pharmacokinetic Studies in Mice. PI or ibuprofen dissolved in
corn oil was administered to female BALB/c mice as a single equimo-
lar dose by gastric gavage. Mice were sacrificed at various time

878
Xie et al.
points after drug administration, and the blood was collected and LC-MS/MS analysis. The mass spectrum of carboxyl-PI
showed a [M-H]ϩ ion at m/z 443.0, which was further frag-
immediately centrifuged. The resulting plasma was deproteinized by
immediately mixing it with 2-fold volume of acetonitrile. PI and its
metabolites were analyzed by HPLC as described above.
PK parameters in plasma were determined by noncompartmental
mented at m/z 153, 197, and 219 (Fig. 2B). The mass spec-
trum of 1-OH-PI showed a [MϩNa]ϩ ion at m/z 453.0, which
was further fragmented at m/z 176.8. (Fig. 2C). To determine
PK data analysis using PK Solutions 2.0 software (Summit Research
the position of the -OH group, 1-OH-PI was treated with
Services, Montrose, CO). The PK parameters determined include
porcine liver esterase as described under Materials and
maximal plasma concentration (Cmax), time to reach maximal
Methods, and it was completely hydrolyzed to release 1-OH-
ibuprofen, indicating that the -OH group was at the 1-posi-
tion of the isobutyl group. Thus PI undergoes hydrolysis and
plasma concentration (Tmax), half-life (t1/2), and the area under the
concentration-time curve (AUC).
Tissue Distribution in Mice. One hour after PI administration,
various tissues of the sacrificed mice were collected, washed three oxidation in RLM, and its oxidation regioselectively occurs at
times with 50% dimethyl sulfoxide, homogenized, and sonicated in
PBS. PI and its metabolites were extracted by adding a 2-fold volume
of acetonitrile. After centrifugation for 10 min at 5000g, the super-
natants were subjected to HPLC analysis.
Statistical Analyses. Results, obtained from at least three
independent experiments, were expressed as mean Ϯ S.E.M. and
analyzed by one-factor analysis of variance followed by the Tukey
test for multiple comparisons. p Ͻ 0.05 was considered statisti-
cally significant.
the 1- and 3-positions of the isobutyl group (Fig. 2D). Like
1-OH-PI, PI and carboxyl-PI were hydrolyzed by the same
esterase to release ibuprofen and carboxyl-ibuprofen, respec-
tively, indicating that the carboxyester bond of the phospho-
NSAIDs is susceptible to hydrolysis by liver esterase. These
results also support the identification of carboxyl-PI and
1-OH-PI.
As shown in Fig. 3A, the level of PI in the reaction mixture
continuously decreased during the entire period of observa-
tion, while, at the same time, the level of its hydrolysis
product, ibuprofen, continuously increased. The levels of
Results
PI Inhibits the Growth of SW480 Colon Cancer Cells 1-OH-PI and carboxyl-PI increased in the first 30 min but
In Vitro and In Vivo. To evaluate the effect of PI on cell slightly dropped afterward because they can also be hydro-
growth, we determined the 24-h IC50 values of PI and ibu- lyzed by RLM. The level of 1-OH-PI was much lower and
profen in HT-29, SW480, and HCT-15 human colon cancer decreased earlier than that of carboxyl-PI, suggesting that PI
cell lines (Fig. 1). The potency of PI was dramatically en- was oxidized preferentially at the 3-position over the 1-posi-
hanced (approximately 16–23 times) compared with conven- tion of its isobutyl group.
tional ibuprofen.
In addition, three minor metabolites (1-OH-ibuprofen,
We next evaluated the chemotherapeutic potential of PI 2-OH-ibuprofen, and carboxyl-ibuprofen) were detected. They are
and compared it with ibuprofen in a subcutaneous model of derived from the oxidation of ibuprofen and the hydrolysis
SW480 cell xenografts in nude mice. PI was administered of 1-OH-PI and carboxyl-PI, and their levels are generally
intraperitoneally at two doses, 225 and 400 mg/kg, the for- Ͻ10% of the major metabolites. These three minor metab-
mer being equimolar to the dose of ibuprofen (125 mg/kg). As olites shared similar kinetic behaviors in that their levels
shown in Fig. 1, both PI and ibuprofen inhibited tumor all were negligible in the first 10 min, after which they
growth throughout the observation period with the efficacy continued to increase until the end of the observation
being 400 mg/kg PI Ͼ 225 mg/kg PI Ͼ 125 mg/kg ibuprofen. period.
At sacrifice, the tumor volume of each study group was as
Drug-metabolizing enzymes differ between humans and
follows: control ϭ 530 Ϯ 197 (meanϮ S.E.M. for this and animals with respect to isoform composition, expression, and
subsequent values), ibuprofen ϭ 297 Ϯ 117 (not significantly catalytic activities (Martignoni et al., 2006). In addition, test-
different from control), 225 mg/kg PI ϭ 196 Ϯ 29 (p ϭ 0.05 ing drug metabolism by human liver microsomes (HLM) is
compared with control), and 400 mg/kg PI ϭ 171 Ϯ 42 mm3 required for the prediction of human pharmacokinetics of the
(p Ͻ 0.05 compared with control). This indicates a reduction drug (Liu et al., 2010). Therefore, we next explored the me-
in tumor volume of 44, 63, and 68% by ibuprofen, 225 mg/kg tabolism of PI by HLM. As was the case with RLM, three
PI, and 400 mg/kg PI, respectively. Whereas both PI doses major metabolites (ibuprofen, 1-OH-PI, and carboxyl-PI) as
were more effective than ibuprofen, the differences between well as the three minor metabolites (1-OH-ibuprofen, 2-OH-
either one of them and the effect of ibuprofen did not reach ibuprofen, and carboxyl-ibuprofen) were also detected in the
statistical significance. It is noteworthy that these treat- reaction mixture of PI and HLM; this finding suggested that
ments produced no apparent adverse effects in mice during the two species share the same metabolic pathways in the
the 32 days of administration.
metabolism of PI by liver microsomes.
The Metabolism of PI by Liver Microsomes. Given the
The level of PI decreased rapidly and was cleared by 82%
efficacy of PI, we explored its metabolism by rat liver micro- within the first 30 min of its reaction with HLM (Fig. 3B).
somes (RLM), an enriched source of major metabolic enzymes Consistently, the levels of 1-OH-PI and carboxyl-PI increased
(Bajrami et al., 2009). These enzymes, including oxidative rapidly during the initial reaction, suggesting that in the
enzymes, hydrolases, and transferases, function to make xe- beginning the formation of these two metabolites was much
nobiotic compounds more polar and more water-soluble, thus faster than their hydrolysis because of the initial high con-
facilitating their elimination from the body.
centration of PI. With the PI level decreasing rapidly, the
Three major metabolites of PI (ibuprofen, carboxyl-PI, and formation rate of 1-OH-PI and carboxyl-PI decreased, and
1-OH-PI) were readily detected in the reaction mixture of PI their consumption became dominant, resulting in the steady
and RLM (Fig. 2A). Ibuprofen was identified by comparing its decrease of their levels during the later phase of the reaction.
HPLC retention time and UV spectrum with that of authen- As was the case with RLM, the level of 1-OH-PI was much
tic compound; the other two metabolites were identified by lower and decreased earlier than that of carboxyl-PI.

Efficacy, Metabolism, and PK of Phospho-Ibuprofen
879
Fig. 2. Identification of the metabolites of
PI by RLM. A, HPLC chromatogram of an
extract of RLM incubated with PI for 30
min; PI and two of its metabolites are
identified. PI and its metabolites were
extracted and fractionated by HPLC as
described under Materials and Methods.
B, MS and MS/MS spectrum of hydroxyl-
PI fraction collected from HPLC. C, MS
and MS/MS spectrum of carboxyl-PI frac-
tion collected from HPLC. D, metabolic
transformations of PI by liver microsomes.
The level of ibuprofen increased initially and reached a mained primarily intact 6 h after treatment (Fig. 4, inset).
plateau at 30 min, suggesting equilibrium between its for- Consistent with this result, LC-MS analysis showed that
mation from PI and oxidation to other metabolites. Similar to the dominant ion of the highest intensity was at m/z 415,
the case with RLM, all of the minor metabolites (1-OH- which further generated fragment ions at m/z 155, 161,
ibuprofen, 2-OH-ibuprofen, and carboxyl-ibuprofen) contin- and 261 (Fig. 4). This spectrum was identical to that of
ued to increase during the entire period of observation.
authentic PI, confirming that the majority of PI taken up
Even though PI followed the same metabolic pathway in by the cells stays intact. This result also confirmed the
RLM and HLM, the kinetics of metabolite formation in the chemical structure of PI.
two species was distinct. PI seems to be more stable in RLM
Multiple minor metabolites of PI were detected and iden-
than in HLM. The t1/2 of PI in RLM was 4.5 times longer than tified in cultured cells using LC-MS/MS analysis. The mass
that in HLM (28.5 versus 6.4 min). PI was hydrolyzed more spectrum of the hydroxy-PI showed a [MϩH]ϩ ion at m/z 431,
rapidly in HLM than in RLM, because the rate constant of which was further fragmented at m/z 155, 177, and 277 (Fig. 5A).
the formation of ibuprofen was 3.8 times higher in HLM that Butanediol phosphate, the carboxyester hydrolysis product of
in RLM (0.088 versus 0.023 minϪ1). Thus, PI was metabo- PI, gave an [MϩH]ϩ ion at m/z 227 in the MS spectrum,
lized more extensively and rapidly in HLM than in RLM. which further generated fragment ions at 99, 127, and 155
Because of the slow clearance of PI in RLM, 1-OH-PI and (Fig. 5B). Desethyl-PI, the phosphate ester hydrolysis prod-
carboxyl-PI were formed more rapidly in RLM than in HLM, uct of PI, showed an [MϩH]ϩ ion at m/z 387, which was
and the observed highest concentrations of 1-OH-PI and car- further fragmented to 161 and 261 (Fig. 5C). Thus PI under-
boxyl-PI in RLM were 2.9 and 2.0 times higher than those in goes hydrolysis and oxidation transformations in cultured
HLM, respectively.
cells (Fig. 5D), which are generally similar to its metabolism
The Metabolism of PI by Cultured Cancer Cells. by liver microsomes.
Given its potency in inhibiting cancer cell growth, we also
We assessed the pharmacological activity of the various
explored the metabolism of PI by cultured HCT-15 human metabolites of PI in cultured cells. As shown in Table 1, each
colon cancer cells. Cell lysates were prepared as described of the three structural components of PI (plain ibuprofen,
under Materials and Methods and analyzed using HPLC and spacer, and diethyl phosphate) tested individually for its cell
MS. HPLC analysis using UV detection showed that PI re- growth inhibitory effect was essentially inactive compared

880
Xie et al.
Fig. 3. Kinetics of PI and its metabolites by
rat and human liver microsomes. PI was
incubated with rat (A) or human (B) liver
microsomes at 37°C for up to 90 min. PI
and its metabolites were extracted at the
designated time points and assayed as de-
scribed under Materials and Methods.
with PI. The same applied to three additional metabolites of
When PI was administered at a dose of 400 mg/kg, intact
PI (2-OH-ibuprofen, carboxyl-ibuprofen, and ibuprofen gluc- PI was detected in mouse plasma by the MS detector (data
uronide), all of which had IC50 values in the millimolar not shown), but was undetectable by the UV detector, sug-
range. These results suggested that the limited metabolism gesting high carboxyesterase activity in mouse plasma, as
of PI by cells may be important for its growth inhibitory has been shown previously (Li et al., 2005). However, PI at a
effect.
dose of 1200 mg/kg generated a significant plasma level of
Pharmacokinetics of PI in the Mouse. We studied the intact PI (12.4 ␮M). These results indicated that PI can
PK properties of PI after its single oral administration to indeed survive in the blood.
mice. The two major metabolites in plasma were ibuprofen
For purposes of comparison, we performed side-by-side PK
and ibuprofen glucuronide. As shown in Fig. 6A, the plasma studies of conventional ibuprofen and PI administered at
levels of ibuprofen increased rapidly and reached the maxi- equimolar doses. As shown in Fig. 6B, ibuprofen was metab-
mum concentration of 530 ␮M 1 h after oral gavage, suggest- olized to produce ibuprofen glucuronide, 1-OH-ibuprofen,
ing that PI was rapidly absorbed, metabolized, and distrib- 2-OH-ibuprofen, and carboxyl-ibuprofen in mouse plasma;
uted to the blood. The level of ibuprofen decreased this metabolic profile was the same as that after PI admin-
substantially 4 h after drug administration and became neg- istration. The plasma levels of ibuprofen and its metabolites
ligible at 8 h. The plasma levels of ibuprofen glucuronide increased rapidly and reached their peak values within ϳ0.5
followed a similar pattern, but the peak value was much h after ibuprofen administration (Fig. 6B), indicating that
smaller.
ibuprofen was rapidly absorbed, metabolized, and distrib-
As was the case with PI metabolism by liver microsomes, uted to the blood. Compared with the plasma levels of 2-OH-
the three minor metabolites (1-OH-ibuprofen, 2-OH-ibupro- ibuprofen and carboxyl-ibuprofen, those of 1-OH-ibuprofen
fen, and carboxyl-ibuprofen) were detected in mouse plasma were negligible (Fig. 6B).
after PI administration. These three metabolites shared sim-
Compared with PI, ibuprofen generated higher levels of
ilar kinetic behavior because they are derived from ibupro- metabolites with the AUC0–24h of “total metabolites” (the
fen. The levels of these metabolites reached their peak values sum of the five metabolites) being 2.2-fold higher (Table 2);
at 2 h after PI administration, but decreased dramatically this probably reflects the higher hydrophobicity of PI and
afterward. The Tmax of each of these metabolites was 1 h hence lower solubility in plasma. The Tmax of each metabolite
later than that of ibuprofen (Table 2), which is reasonable after ibuprofen administration was much shorter than those
because they are derived from ibuprofen.
after PI administration (Table 2). This finding suggests that

Efficacy, Metabolism, and PK of Phospho-Ibuprofen
881
Fig. 4. PI metabolism by cultured colon
cancer cells. PI and its metabolites were
extracted from the PI-treated HCT-15 cell
lysates and analyzed using HPLC and
LC/MS/MS as described under Materials
and Methods. Top, MS spectrum of PI
obtained from LC separation. Inset, HPLC
chromatogram of extract from HCT-15 cells
treated with PI; only PI was detected by
UV detector. Bottom, MS/MS spectrum of
fragment ions of the PI.
the absorption and distribution of PI are slower than those of cacy study. The major metabolite detected was ibuprofen,
ibuprofen, also probably caused by its higher hydrophobicity. and there was a statistically significant association between
Biodistribution of PI in the Mouse Model. We also the tumor volume and ibuprofen level in plasma (p ϭ 0.02),
measured the levels of PI and its metabolites in liver, heart, underscoring the relative importance of the metabolites of PI
kidney, and gastrointestinal tissues of mice after its oral in cancer control (Fig. 7). However, the association between
administration (Table 3). Significant levels of intact PI were the anticancer efficacy and tumor levels of ibuprofen was
detected in all of the tissues examined, confirming that PI statistically significant only for the trend (p ϭ 0.08), suggest-
can partially survive in mouse plasma and therefore be dis- ing that metabolite-independent mechanisms are also in-
tributed to other tissues. High levels of intact PI were de- volved in inhibiting cancer growth.
tected in gastrointestinal tissues, whereas its liver concen-
tration was the lowest, probably because of the high
metabolic capacity of this organ.
Discussion
As expected, the five metabolites detected in mouse plasma
Our work demonstrated the preclinical anticancer efficacy of
(ibuprofen, ibuprofen glucuronide, 1-OH-ibuprofen, 2-OH- PI, a novel derivative of ibuprofen, established its metabolism
ibuprofen, and carboxyl-ibuprofen) were also detected in in vitro and in vivo, defined its PK in mice, and explored its PD
these tissues. High levels of ibuprofen glucuronide were de- in tumor xenografts. Collectively, these findings provide strong
tected in the liver and kidney, suggesting that most of the support for the anticancer potential of this agent and set the
glucuronidation of ibuprofen occurs in these organs (Rudy et stage for its further pharmacological evaluation.
al., 1991). Similar to PI metabolism by liver microsomes, the
level of 1-OH-PI in liver tissue was much lower than that of potently than ibuprofen and also inhibited the growth of xeno-
carboxyl-PI. grafts of one of these cell lines in nude mice. These effects are
PI inhibited the growth of colon cancer cell lines much more
Blood and Xenograft Levels of PI Metabolites in quantitatively significant and, considering the efficacy of PI on
Mice: Correlations with Tumor Size. To gain an insight pancreatic and breast cancer from our unpublished data, con-
into the interplay between PI’s metabolic transformations stituted a firm justification for studying its metabolism and
and pharmacological effects, we measured the levels of PI PK/PD. Compared with ibuprofen, the two PI doses inhibited
metabolites in mouse blood and tumor tissue from the effi- tumor growth approximately 1.5 times more effectively. The

882
Xie et al.
Fig. 5. Identification of the metabolites from PI-treated cancer cells. The metabolites of PI were extracted from the PI-treated HCT-15 cells and
analyzed using LC/MS/MS as described under Materials and Methods. A, MS/MS spectrum of fragment ions of hydroxyl-PI. B, MS/MS spectrum of
fragment ions of butanediol phosphate. C, MS/MS spectrum of fragment ions of desethyl-PI. D, proposed metabolic transformations of PI by liver
microsomes.
TABLE 1
nine products. Three distinct pathways of PI metabolism are
readily recognizable: 1) oxidation at the 1-position of the
isobutyl group to form 1-OH-PI, which can be hydrolyzed to
form 1-OH-ibuprofen, 2) oxidation at the 3-position of the
isobutyl group to form carboxyl-PI, probably through 3-OH-
PI, and 3) carboxyl-ester hydrolysis to form ibuprofen, which,
in turn, can follow three metabolic routes: 1) conjugation
with glucuronic acid to form ibuprofen glucuronide, 2) oxida-
tion to form 2-OH-ibuprofen, or 3) oxidation at the 3-position
of the isobutyl group to form carboxyl-ibuprofen, probably
through 3-OH-ibuprofen. In general, hydrolysis, oxidation,
and glucuronidation of drugs represent mechanisms used by
an organism to inactivate and/or eliminate them (Ulrich et
al., 2006). Given that all PI metabolites are much weaker
than PI at inhibiting cancer cell growth and their aqueous
solubility is enhanced (evidenced by their retention times on
reverse-phase HPLC), it seems that PI follows the general
fate of compounds that are inactivated and eliminated by the
IC50 values of phospho-ibuprofen and its components/metabolites in
cultured HT-29 cancer cells
Compound
IC50
␮M
Phospho-ibuprofen
Ibuprofen
1,4-Butanediol (spacer)
Diethyl phosphate
2-OH-ibuprofen
Carboxyl-ibuprofen
Ibuprofen glucuronide
67
1516
Ͼ10,000
10,229
Ͼ4500
Ͼ4500
Ͼ4500
differences in efficacy between the two were statistically not
significant; such lack of significance is probably caused by a lack
of power. Nonetheless, we deem these differences as clinically
relevant, especially when the greater safety of PI is factored in
(current data and Huang et al., 2011).
As summarized in the metabolic diagram of Fig. 8, PI
undergoes extensive biotransformations, leading to at least mice.

Efficacy, Metabolism, and PK of Phospho-Ibuprofen
883
Fig. 6. Comparison of pharmacokinetics
of PI and ibuprofen in mice. Equimolar
doses (1 mmol/kg) of PI (A) and ibuprofen
(B) dissolved in corn oil were adminis-
tered to mice as a single dose by gastric
gavage. Plasma levels of PI metabolites
at the indicated time points were deter-
mined as described under Materials and
Methods.
TABLE 2
Pharmacokinetic parameters of phospho-ibuprofen (400 mg/kg) and ibuprofen (200 mg/kg) in mice
The doses of PI and ibuprofen are equimolar. Experiments were done independently three times; values were generally within 15%.
Measurement
Drug Administered
Cmax
Tmax
Elimination t1/2
AUC0–24h
Sum of AUC0–24h
␮M ϫ h
␮M
h
Phospho-ibuprofen
Ibuprofen
530
28
1.0
2.0
2.0
2.0
2.0
7.7
N.D.
N.D.
N.D.
5.3
1816
74
193
48
832
2963
1-OH-ibuprofen
2-OH-ibuprofen
Carboxyl-ibuprofen
Ibuprofen glucuronide
Ibuprofen
77
16
215
Ibuprofen
932
5
83
81
274
0.5
0.5
2.0
0.5
0.5
12.8
N.D.
N.D.
10.0
6.3
4146
11
572
311
1398
6437
1-OH-ibuprofen
2-OH-ibuprofen
Carboxyl-ibuprofen
Ibuprofen glucuronide
N.D., not determined.
TABLE 3
PI metabolite levels in mouse tissues 1 h after oral administration of 400 mg/kg PI
Experiments were done independently three times; values were generally within 15%.
PI
1-OH-PI
Carboxyl-PI
Ibuprofen
1-OH-Ibuprofen
2-OH-Ibuprofen
Carboxyl-Ibuprofen
Ibuprofen Glucuronide
pmol/mg protein
Liver
82
101
121
421
521
414
N.D.
22
1013
N.D.
N.D.
N.D.
36
739
2409
1946
462
19
34
33
96
220
N.D.
N.D.
N.D.
60
13
N.D.
104
N.D.
N.D.
N.D.
10
408
55
322
N.D.
50
64
121
Heart
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Kidney
Stomach
Small intestine
Colon
75
N.D.
N.D.
N.D.
24
913
20
431
530
Blood*
N.D.
N.D., not detected.
* Metabolite levels in the blood were expressed as ␮M in plasma.
The metabolic fate of PI is largely context-dependent. Cul- inhibiting cell growth; all others are Ͼ67-fold less potent
tured colon cancer cells preserve PI essentially intact, with than PI. Liver microsomes generate a different profile of
only a minor fraction (Ͻ10%) metabolized to mostly biologi- metabolites; at 90 min the four most abundant metabolites
cally inactive metabolites. Of these, the most active metabo- are (in descending order) ibuprofen, carboxyl-PI, PI, and
lite of PI is ibuprofen, which is 22-fold less potent than PI in 1-OH-PI. Human and rat liver microsomes generate the

884
Xie et al.
site of CYP450 and is preferentially oxidized at its C-H bond
close to the active oxygen of the heme moiety of CYP450
(Seifert et al., 2006). By analogy, we propose that the phos-
phate moiety of PI may affect its binding to CYP450, result-
ing in geometric preference of position 1 over position 2 for its
oxidation by CYP450.
Glucuronidation reactions are an important part of the
detoxification process (Zheng et al., 2002). The only glucuro-
nide derivative that we detected was ibuprofen glucuronide.
Such a transformation is well known for conventional ibu-
profen, whose carboxylic acid group is conjugated with the
hydroxyl group of UDP-glucuronic acid; this reaction is cat-
alyzed by UDP-glucuronosyltransferases (UGTs), especially
UGT2B7 and UGT1A9 (Kepp et al., 1997; Kuehl et al., 2005).
Glucuronidation of PI is unlikely to occur because of the lack
of a free carboxylic acid group. It is noteworthy that ibupro-
fen glucuronide can undergo spontaneous chemical reactions,
resulting in acyl migration and irreversible binding to endog-
enous proteins, which can have toxicological consequences
(Johnson et al., 2007).
HLM and RLM metabolize PI in much the same manner,
generating an identical array of metabolites. The principal
differences between them concern the rate and extent of each
metabolic step, with HLM metabolizing PI faster and gener-
ating more ibuprofen and less carboxyl-PI than RLM. The
faster metabolism of PI by HLM (evidenced by the much
faster formation of ibuprofen by HLM) is probably caused by
differences in esterase activity between HLM and RLM. Dif-
ferential metabolic kinetics in RLM and HLM has been re-
ported for other drugs. For example, p-nitrophenylacetate
was hydrolyzed more rapidly by HLM than RLM, whereas
the opposite was observed for isocarboxazid (Hosokawa et al.,
1990; Imai et al., 2003). Taken together, these findings indi-
cate significant differences in esterase activity between ani-
mals and humans (Kremers et al., 1981).
Fig. 7. Correlation of PI metabolite levels with tumor volume. Mice with
SW480 colon cancer xenografts were sacrificed 1 h after the last dosing,
and ibuprofen levels in mouse blood and tumors were determined as
described under Materials and Methods. A significant correlation (p ϭ
0.016) between plasma levels of ibuprofen and tumor volume (top) and a
trend between tumor levels of ibuprofen and tumor volume (bottom) were
observed.
We determined the basic PK parameters of PI in mice and
also assessed its biodistribution. PI is rapidly absorbed, me-
same metabolic profile, although the ratio between the four tabolized, and distributed to the blood and other tissues. PI,
most abundant metabolites is different between the two. administered at the dose of 400 mg/kg, was not detected in
Finally, a markedly different metabolite profile is obtained in plasma, whereas the Cmax of its main metabolites was in the
mice. AUC quantification shows that ibuprofen and ibupro- high micromolar range. All PI metabolites reached Tmax
fen glucuronide make up 90% of the total plasma metabo- within 2 h, and their t1/2 was relatively short (Ͻ8 h), indicat-
lites, while PI is conspicuous by its absence.
ing an efficient elimination process; the high degree of glucu-
Both liver and mice microsomes oxidized PI at the 1- and ronidation probably accounts to a large extent for this rapid
3-positions of its isobutyl group to form 1-OH-PI and 3-OH- elimination. Even though PI was not detectable in plasma,
PI, respectively, with the latter further oxidized to carboxyl- remarkably, it was found in all other tissues that we evalu-
PI. In contrast, ibuprofen, generated as a metabolite of PI, ated, albeit at low concentrations; its liver concentration was
was oxidized at the 2- and 3-positions of its isobutyl group, the lowest, probably reflecting the high metabolic capacity of
generating 2-OH-ibuprofen and 3-OH-ibuprofen, the latter this organ. The simplest interpretation of this apparent par-
leading to carboxyl-ibuprofen. The metabolism of ibuprofen adox is that PI partially and briefly escapes blood esterases
generated from PI is identical to that of conventional ibupro- and that once taken up by other tissues its degradation
fen (Hamman et al., 1997; Chang et al., 2008). The differen- becomes much slower. This is supported by the finding that
tial regioselectivity of the oxidation of ibuprofen and PI is PI at a 3-fold higher dose generated significant plasma levels
reflected in our finding that PI generated 15.6 times more of PI (12.4 ␮M). These results indicated that PI can indeed
1-OH-ibuprofen in murine plasma than ibuprofen (2.5 versus survive in the blood.
0.16%).
As a corollary, it is conceivable that protection of PI from
The CYP450 superfamily, and particularly CYP2C9, plays plasma esterases would lead to higher levels of intact PI in
a major role in the regioselective oxidation of ibuprofen target tissues and higher anticancer efficacy, because PI is
(Hamman et al., 1997; Chang et al., 2008). The molecular much more potent against cancer cells than any of its metab-
basis of regioselectivity of CYP2C9 has been examined in the olites, including ibuprofen. Nonspecific carboxylic ester hy-
case of the anticoagulant warfarin using molecular dynamics drolases, the suspected culprit in the hydrolysis of PI in
simulations (Seifert et al., 2006). Warfarin binds to the active plasma, are grouped into carboxyesterase, arylesterase, and

Efficacy, Metabolism, and PK of Phospho-Ibuprofen
885
Fig. 8. PI metabolism in vitro and in vivo. PI can be metabolized through three distinct pathways: 1) oxidation to form 1-OH-PI, 2) oxidation to form carboxyl-PI,
or 3) hydrolysis to form ibuprofen, which, in turn, can be metabolized through: 1) glucuronidation to form ibuprofen glucuronide, 2) oxidation to form 2-OH-
ibuprofen, or 3) oxidation to form carboxyl-ibuprofen.
acetylesterase, based on their substrate specificity (Xie et al., between the two (89.4 versus 86.1%); and 3) of the quantita-
2008); carboxyesterase can efficiently catalyze the hydrolysis tively minor metabolites, the most striking difference con-
of carboxyester drugs to release free acids (Imai et al., 2003). cerned 1-OH-ibuprofen and to a lesser extent carboxyl-ibu-
It is of great interest that in human plasma carboxyesterase profen; PI generated 15.6-fold more 1-OH-ibuprofen and
activity is either undetectable or very low compared with 3-fold less carboxyl-ibuprofen than ibuprofen. As discussed
mice and rats (Li et al., 2005). Thus, it is reasonable to above, the difference in 1-OH-ibuprofen was expected. Per-
predict that in humans, who have very low plasma car- haps the mechanistically most interesting observation was
boxyesterase activity, the plasma and tissue levels of PI will that PI, even though equimolar to ibuprofen, generated lower
be high. If this proves to be the case, PI should have a plasma AUC0–24h of ibuprofen (2.3-fold) and fewer total me-
stronger anticancer efficacy in humans. In agreement with tabolites (2.2-fold). Characteristically, the Cmax value of ibu-
this notion, irinotecan was reported to undergo much less profen derived from PI is approximately half that of ibupro-
hydrolysis by carboxyesterase in human plasma than in fen derived from conventional ibuprofen administered at the
mouse plasma (Mathijssen et al., 2002).
equimolar dose. The explanation of these differences is not
The side-by-side comparison of PI to conventional ibupro- readily apparent. However, it is conceivable that these dif-
fen in which these compounds were administered at equimo- ferences account, at least partially, for the greater safety of
lar doses, corresponding to the lower dose of PI was of inter- PI compared with ibuprofen.
est: 1) both compounds generated the same metabolites in
Pharmacodynamically, there was a statistically significant
plasma; 2) the relative abundance of the two major metabo- association between plasma levels of ibuprofen and the anti-
lites of PI, ibuprofen and ibuprofen glucuronide, was similar tumor effect of PI, determined as the volume of the xenograft.

886
Xie et al.
involvement of a pulmonary first-pass effect via carboxylesterase in the disposition
of a propranolol ester derivative after intravenous administration. J Pharmacol
Exp Ther 307:1234–1242.
Johnson CH, Wilson ID, Harding JR, Stachulski AV, Iddon L, Nicholson JK, and
Lindon JC (2007) NMR spectroscopic studies on the in vitro acyl glucuronide
migration kinetics of Ibuprofen ((ϩ/Ϫ)-(R,S)-2-(4-isobutylphenyl) propanoic acid),
its metabolites, and analogues. Anal Chem 79:8720–8727.
Kepp DR, Sidelmann UG, and Hansen SH (1997) Isolation and characterization of
major phase I and II metabolites of ibuprofen. Pharm Res 14:676–680.
Kremers P, Beaune P, Cresteil T, de Graeve J, Columelli S, Leroux JP, and Gielen
JE (1981) Cytochrome P-450 monooxygenase activities in human and rat liver
microsomes. Eur J Biochem 118:599–606.
Kuehl GE, Lampe JW, Potter JD, and Bigler J (2005) Glucuronidation of nonsteroi-
dal anti-inflammatory drugs: identifying the enzymes responsible in human liver
microsomes. Drug Metab Dispos 33:1027–1035.
Lanza FL (1984) Endoscopic studies of gastric and duodenal injury after the use of
ibuprofen, aspirin, and other nonsteroidal anti-inflammatory agents. Am J Med
77:19–24.
Li B, Sedlacek M, Manoharan I, Boopathy R, Duysen EG, Masson P, and Lockridge
O (2005) Butyrylcholinesterase, paraoxonase, and albumin esterase, but not car-
boxylesterase, are present in human plasma. Biochem Pharmacol 70:1673–1684.
Liu D, Zheng X, Tang Y, Zi J, Nan Y, Wang S, Xiao C, Zhu J, and Chen C (2010)
Metabolism of tanshinol borneol ester in rat and human liver microsomes. Drug
Metab Dispos 38:1464–1470.
Mackenzie GG, Sun Y, Huang L, Xie G, Ouyang N, Gupta RC, Johnson F, Komninou
D, Kopelovich L, and Rigas B (2010) Phospho-sulindac (OXT-328), a novel sulindac
derivative, is safe and effective in colon cancer prevention in mice. Gastroenterol-
ogy 139:1320–1332.
Marnett LJ (1995) Aspirin and related nonsteroidal anti-inflammatory drugs as
chemopreventive agents against colon cancer. Prev Med 24:103–106.
Martignoni M, Groothuis GM, and de Kanter R (2006) Species differences between
mouse, rat, dog, monkey and human CYP-mediated drug metabolism, inhibition
and induction. Expert Opin Drug Metab Toxicol 2:875–894.
The association between ibuprofen levels in the xenografts
and the antitumor effect of PI, however, did not reach statis-
tical significance (p ϭ 0.08). Whether this is caused by our
small sample size or an effect by additional PI metabolites
and/or effects triggered by intact PI has yet to be determined.
In conclusion, our work established the metabolic pathway
of PI in vitro and in vivo, defined its PK and biodistribution
in mice, and explored its PD in tumor xenografts. These
results provide a pharmacological basis to explain the anti-
cancer efficacy and safety of this promising compound, sug-
gest areas of potential improvements in terms of drug formu-
lation and administration, and set the stage for its further
evaluation.
Acknowledgments
We thank R. Rieger and T. Koller (Stony Brook University) for the
expert LC-MS/MS analysis of our samples.
Authorship Contributions
Participated in research design: Xie, Sun, and Rigas.
Conducted experiments: Xie, Sun, Nie, Mackenzie, and Huang.
Contributed new reagents or analytic tools: Kopelovich and Komninou.
Performed data analysis: Xie, Sun, Nie, Mackenzie, Huang, and
Rigas.
Mathijssen RH, Verweij J, de Bruijn P, Loos WJ, and Sparreboom A (2002) Effects
of St. John’s wort on irinotecan metabolism. J Natl Cancer Inst 94:1247–1249.
McElwee NE, Veltri JC, Bradford DC, and Rollins DE (1990) A prospective, popu-
lation-based study of acute ibuprofen overdose: complications are rare and routine
serum levels not warranted. Ann Emerg Med 19:657–662.
Meier CR, Schmitz S, and Jick H (2002) Association between acetaminophen or
nonsteroidal antiinflammatory drugs and risk of developing ovarian, breast, or
colon cancer. Pharmacotherapy 22:303–309.
Wrote or contributed to the writing of the manuscript: Xie, Kopelovich,
Komninou, and Rigas.
References
American Cancer Society (2008) Cancer Facts and Figures 2008. American Cancer
Society Atlanta, GA.
Piazza GA, Keeton AB, Tinsley HN, Gary BD, Whitt JD, Mathew B, Thaiparambil J,
Coward L, Gorman G, Li Y, et al. (2009) A novel sulindac derivative that does not
inhibit cyclooxygenases but potently inhibits colon tumor cell growth and induces
apoptosis with antitumor activity. Cancer Prev Res (Phila) 2:572–580.
Riley TR 3rd and Smith JP (1998) Ibuprofen-induced hepatotoxicity in patients with
chronic hepatitis C: a case series. Am J Gastroenterol 93:1563–1565.
Rudy AC, Knight PM, Brater DC, and Hall SD (1991) Stereoselective metabolism of
ibuprofen in humans: administration of R-, S- and racemic ibuprofen. J Pharmacol
Exp Ther 259:1133–1139.
Antonicelli L and Tagliabracci A (1995) Asthma death induced by ibuprofen. Mon-
aldi Arch Chest Dis 50:276–278.
Bajrami B, Zhao L, Schenkman JB, and Rusling JF (2009) Rapid LC-MS drug
metabolite profiling using microsomal enzyme bioreactors in a parallel processing
format. Anal Chem 81:9921–9929.
Chang SY, Li W, Traeger SC, Wang B, Cui D, Zhang H, Wen B, and Rodrigues AD
(2008) Confirmation that cytochrome P450 2C8 (CYP2C8) plays a minor role in
(S)-(ϩ)- and (R)-(Ϫ)-ibuprofen hydroxylation in vitro. Drug Metab Dispos 36:2513–
2522.
Seifert A, Tatzel S, Schmid RD, and Pleiss J (2006) Multiple molecular dynamics
simulations of human p450 monooxygenase CYP2C9: the molecular basis of sub-
strate binding and regioselectivity toward warfarin. Proteins 64:147–155.
Ulrich CM, Bigler J, and Potter JD (2006) Non-steroidal anti-inflammatory drugs for
cancer prevention: promise, perils and pharmacogenetics. Nat Rev Cancer 6:130–
140.
Xie G, Liu M, Zhu H, and Lei B (2008) Esterase SeE of Streptococcus equi ssp. equi
is a novel nonspecific carboxylic ester hydrolase. FEMS Microbiol Lett 289:181–
186.
Coussens LM and Werb Z (2002) Inflammation and cancer. Nature 420:860–867.
Hamman MA, Thompson GA, and Hall SD (1997) Regioselective and stereoselective
metabolism of ibuprofen by human cytochrome P450 2C. Biochem Pharmacol
54:33–41.
Harris RE, Beebe-Donk J, Doss H, and Burr Doss D (2005) Aspirin, ibuprofen, and
other non-steroidal anti-inflammatory drugs in cancer prevention: a critical review
of non-selective COX-2 blockade (review). Oncol Rep 13:559–583.
Harris RE, Namboodiri KK, and Farrar WB (1996) Nonsteroidal antiinflammatory
drugs and breast cancer. Epidemiology 7:203–205.
Zheng Z, Fang JL, and Lazarus P (2002) Glucuronidation: an important mechanism
for detoxification of benzo[a]pyrene metabolites in aerodigestive tract tissues.
Drug Metab Dispos 30:397–403.
Hosokawa M, Maki T, and Satoh T (1990) Characterization of molecular species of
liver microsomal carboxylesterases of several animal species and humans. Arch
Biochem Biophys 277:219–227.
Huang L, Mackenzie G, Ouyang N, Sun Y, Xie G, Johnson F, Komninou D, and Rigas
B (2011) The novel phospho-non-steroidal anti-inflammatory drugs, OXT-328,
MDC-22 and MDC-917, inhibit adjuvant-induced arthritis in rats. Br J Pharmacol
162:1521–1533.
Address correspondence to: Dr. Basil Rigas, Division of Cancer Prevention,
Stony Brook University, HSC, T17-080, Stony Brook, NY 11794-8173. E-mail:
basil.rigas@stonybrook.edu
Imai T, Yoshigae Y, Hosokawa M, Chiba K, and Otagiri M (2003) Evidence for the