Comparative in vitro metabolism of phospho-tyrosol-indomethacin by mice, rats and humans

Article abstract of DOI:10.1016/j.bcp.2013.01.031

Phospho-tyrosol-indomethacin (PTI; MPI 621), a novel anti-cancer agent, is more potent and safer than conventional indomethacin. Here, we show that PTI was extensively metabolized in vitro and in vivo. PTI was rapidly hydrolyzed by carboxylesterases to generate indomethacin as its major metabolite in the liver microsomes and rats. PTI additionally undergoes cytochromes P450 (CYP)-mediated hydroxylation at its tyrosol moiety and O-demethylation at its indomethacin moiety. Of the five major human CYPs, CYP3A4 and CYP2D6 catalyze the hydroxylation and O-demethylation reactions of PTI, respectively; whereas CYP1A2, 2C9 and 2C19 are inactive towards PTI. In contrast to PTI, indomethacin is primarily O-demethylated by CYP2C9, which prefers acidic substrates. The hydrolyzed and O-demethylated metabolites of PTI are further glucuronidated and sulfated, facilitating drug elimination and detoxification. We observed substantial inter-species differences in the metabolic rates of PTI. Among the liver microsomes from various species, PTI was the most rapidly hydrolyzed, hydroxylated and O-demethylated in mouse, human and rat liver microsomes, respectively. These results reflect the differential expression patterns of carboxylesterase and CYP isoforms among these species. Of the human microsomes from various tissues, PTI underwent more rapid carboxylesterase- and CYP-catalyzed reactions in liver and intestine microsomes than in kidney and lung microsomes. Together, our results establish the metabolic pathways of PTI, reveal significant inter-species differences in its metabolism, and provide insights into the underlying biochemical mechanisms.

Full text of DOI:10.1016/j.bcp.2013.01.031

Biochemical Pharmacology 85 (2013) 1195–1202
Contents lists available at SciVerse ScienceDirect
Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm
Comparative in vitro metabolism of phospho-tyrosol-indomethacin by
mice, rats and humans
a
a
Gang Xie a, , Dingying Zhou , Ka-Wing Cheng , Chi C. Wong a,b, Basil Rigas a,b
*
a Division of Cancer Prevention, Department of Medicine, Stony Brook University, HSC, T17-080, Stony Brook, NY 11794, USA
b Medicon Pharmaceuticals, Inc., Stony Brook, NY 11790, USA
A R T I C L E I N F O
A B S T R A C T
Article history:
Phospho-tyrosol-indomethacin (PTI; MPI 621), a novel anti-cancer agent, is more potent and safer than
conventional indomethacin. Here, we show that PTI was extensively metabolized in vitro and in vivo. PTI
was rapidly hydrolyzed by carboxylesterases to generate indomethacin as its major metabolite in the
liver microsomes and rats. PTI additionally undergoes cytochromes P450 (CYP)-mediated hydroxylation
at its tyrosol moiety and O-demethylation at its indomethacin moiety. Of the five major human CYPs,
CYP3A4 and CYP2D6 catalyze the hydroxylation and O-demethylation reactions of PTI, respectively;
whereas CYP1A2, 2C9 and 2C19 are inactive towards PTI. In contrast to PTI, indomethacin is primarily O-
demethylated by CYP2C9, which prefers acidic substrates. The hydrolyzed and O-demethylated
metabolites of PTI are further glucuronidated and sulfated, facilitating drug elimination and
detoxification. We observed substantial inter-species differences in the metabolic rates of PTI. Among
the liver microsomes from various species, PTI was the most rapidly hydrolyzed, hydroxylated and O-
demethylated in mouse, human and rat liver microsomes, respectively. These results reflect the
differential expression patterns of carboxylesterase and CYP isoforms among these species. Of the human
microsomes from various tissues, PTI underwent more rapid carboxylesterase- and CYP-catalyzed
reactions in liver and intestine microsomes than in kidney and lung microsomes. Together, our results
establish the metabolic pathways of PTI, reveal significant inter-species differences in its metabolism,
and provide insights into the underlying biochemical mechanisms.
Received 17 December 2012
Accepted 30 January 2013
Available online 8 February 2013
Keywords:
Phospho-tyrosol-indomethacin
Cytochrome P450
Liver microsomes
Glucuronidation
ß 2013 Elsevier Inc. All rights reserved.
synthesis of prostaglandins. In order to reduce its toxicity and
improve its anticancer efficacy, we chemically modified IND at its –
1. Introduction
COOH group. Phospho-tysosol-IND (PTI) consists of IND with a
diethylphosphate group covalently linked to it via a tyrosol spacer
(Fig. 1A inset). Our preclinical study has demonstrated that PTI
strongly inhibits colon and lung cancer in mice, and showed
markedly reduced gastrointestinal toxicity compared to conven-
tional IND [11].
Given the important role of drug metabolism in defining drug
efficacy and toxicity, we systematically examined the metabolism of
PTI in vitro using mouse, rat and human microsomes from various
tissues, as well as in vivo in rats. Our findings reveal extensive
biotransformations of PTI and significant inter-species differences in
its metabolism, and provide insights into the biochemical mecha-
nisms underlying its metabolic transformations.
Inflammatory responses play critical roles in tumorigenesis, and
nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cancer pro-
gression and its malignant conversion [1]. In 1976, indomethacin
(IND), a well-known NSAID, was reported to inhibit growth of
experimental tumors in mouse [2]. Following that, IND has been
reported to be effective against various cancers, including those of
colon [3], breast [4], lung [5] and skin [6]. IND inhibits tumor
development at different stages including tumor initiation and
promotion [7], invasion [8] and metastasis [9]. Mechanistically, it
inhibits cancer cell proliferation and induces cancer cell apoptosis [10].
Despite the promising anti-cancer properties of IND, it has
significant dose-limiting toxicity, particularly gastrointestinal
toxicity due to its strong ability to inhibit cyclooxygenase-catalyzed
2. Materials and methods
Abbreviations: CES, carboxylesterase; CYP, cytochrome P450; DFP, diisopropyl
fluorophosphates; HLMs, human liver microsomes; IND, indomethacin; PTI,
phospho-tyrosol-indomethacin; UGT, UDP-glucuronosyltransferases.
* Corresponding author. Tel.: +1 631 444 9538; fax: +1 631 444 9553.
E-mail addresses: basil.rigas@stonybrook.edu, gangxie2002@yahoo.com
(G. Xie).
2.1. Reagents
PTI was synthesized as reported [11]. IND, demethyl-IND,
debenzoyl-IND, demethyl-debenzoyl-IND, IND glucuronide,
0006-2952/$ – see front matter ß 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bcp.2013.01.031

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G. Xie et al. / Biochemical Pharmacology 85 (2013) 1195–1202
Fig. 1. Kinetics of PTI metabolism by mouse, rat and human liver microsomes. (A) Time courses of the levels of PTI and IND in PTI-treated liver microsomes. PTI (75
mM) was
incubated with mouse, rat and human liver microsomes (protein concentration 0.5 mg/ml) at 37 8C for up to 3 h. PTI and its metabolites were extracted at the designated time
points and assayed using HPLC. Inset, the structure of PTI. (B) Time course of metabolite formation in PTI-treated liver microsomes. (C) Comparison of metabolite formation in
PTI-treated HLMs in the presence (+) versus absence (ꢁ) of NADPH, 20 min after the initiation of the reaction.
diisopropyl fluorophosphates (DFP), ketoconazole and quinidine
were purchased from Toronto Research Chemicals (Toronto,
Canada). 30-Phosphoadenosine-50-phosphosulfate lithium salt,
trifluoroacetic acid, and CH3CN of HPLC grade were purchased
from Sigma–Aldrich, St. Louis, MO. Mouse, rat and human liver
microsomes, human liver cytosol, recombinant human CYPs
(CYP1A2, 2C9, 2C19, 2D6 and 3A4), UGT2B7, NADPH regenerating
solution, and UGT reaction solution were purchased from BD
Biosciences, San Jose, CA. Human intestine, kidney and lung
microsomes were purchased from XenoTech LLC (Lenexa, KS).
CH3CN at a flow rate of 1 ml/min at 30 8C. We applied gradient
elution from 0% to 100% CH3CN for 15 min, and it was
maintained at 100% CH3CN for 5 min.
2.3. LC–MS/MS analysis
The LC–MS/MS system consisted of Thermo TSQ Quantum
Access (Thermo-Fisher) triple quadrupole mass spectrometer
interfaced by an electrospray ionization probe with an Ultimate
3000 HPLC system (Dionex Corporation, Sunnyvale, CA). Chro-
matographic separations were achieved using a Luna C18 column
(150 mm ꢀ 2 mm), and the mobile phase consisted of a gradient
from 10% to 95% CH3CN.
2.2. HPLC-UV analysis
The HPLC system consisted of a Waters Alliance 2695
Separations Module equipped with a Waters 2998 photodiode
array detector (260 nm) and a Thermo Hypersil BDS C18
2.4. The metabolism of PTI by mouse, rat and human liver microsomes
and human intestine, kidney and lung microsomes
column (150 mm ꢀ 4.6 mm, particle size 3
phase consisted of gradient between aqueous phase
(Trifluoroacetic acid, CH3CN, H2O (0.1:4.9:95, v/v/v)) and
mm). The mobile
a
PTI was preincubated at 37 8C for 5 min with NADPH-regenerat-
ing solution (1.3 mM NADP, 3.3 mM D-glucose 6-phosphate, 3.3 mM

G. Xie et al. / Biochemical Pharmacology 85 (2013) 1195–1202
1197
Table 1
MgCl2, and 0.4 U/ml glucose-6-phosphate dehydrogenase) in
0.1 M potassium phosphate buffer (pH 7.4). The reaction was
initiated by the addition of mouse, rat and human liver
microsomes (protein concentration 0.5 mg/ml) or human intes-
tine, kidney, and lung microsomes (protein concentration
0.25 mg/ml) and samples were maintained at 37 8C for various
time periods. At each of the designated time-points, 0.1-ml
aliquots were mixed with 0.2 ml of CH3CN, vortexed, and then
centrifuged for 10 min at 13,000 ꢀ g. The supernatants were
subjected to HPLC analyses. The HPLC peaks corresponding to
each metabolite of PTI were collected and subjected to mass
spectrometry analysis.
The stability (t1/2 and CLint) of PTI in mouse, rat and human microsomes from
various tissues.
t1/2 (min)
CLint (ml/min/g)
Mouse liver microsomes
Rat liver microsomes
8.0
37.6
13.9
13.2
77.3
348.0
172.4
36.9
100.0
210.0
35.9
8.0
Human liver microsomes
Human intestine microsomes
Human kidney microsomes
Human lung microsomes
equation (GraphPad Prism 5.0; GraphPad Software Inc., San
Diego, CA).
2.5. Stability of PTI in liver and intestinal microsomes
2.7. Glucuronidation of PTI by human liver microsomes
The half-life (t1/2
) of PTI was determined by non-linear
regression analysis using one-phase decay model (GraphPad
Prism, version 5). Intrinsic clearance (CLint) of PTI was calculated
using the formula CLint = (0.693/t1/2) ꢀ (V/P), where V is the
incubation volume, and P is the mass of microsomal proteins in
the incubation mixture.
PTI was preincubated at 37 8C for 5 min with UGT reaction
solution (UDP glucuronic acid 2 mM, alamethicin 25
mg/ml and
MgCl2 8 mM) in 50 mM Tris–HCl buffer (pH 7.5). The reaction was
initiated by the addition of human liver microsomes (protein
concentration 0.5 mg/ml) and samples were maintained at 37 8C
for various time periods. At the end of each of the incubations, 0.1-
ml aliquots were mixed with 0.2 ml of CH3CN, vortexed, and then
centrifuged for 10 min at 13,000 ꢀ g. The supernatants were
subjected to HPLC analyses.
2.6. <!——!>Enzymatic kinetics of the metabolism of PTI by human CYP
isoforms
Human recombinant CYPs were pre-incubated with diiso-
propyl fluorophosphates (DFP) (final 200
to abrogate their esterase activities, and were subsequently
treated with PTI ranging from 2 to 200 M and an NADPH-
regenerating solution in 0.1 M potassium phosphate buffer (pH
7.4) for 1 h. The resultant reaction mixtures (100 l) were mixed
with 200 l CH3CN, vortexed, and then centrifuged for 10 min at
13,000 ꢀ g. The supernatants were subjected to HPLC analysis.
The kinetic parameters Km and Vmax were calculated using a
nonlinear curve fitting program based on the Michaelis–Menten
m
M) at 37 8C for 15 min
2.8. Preparation of demethyl-IND sulfate
m
Demethyl-IND (100
cytosol (1 mg protein/ml) and 30-phosphoadenosine-50-phospho-
sulfate lithium salt (100 M) in 100 mM Tris–HCl buffer (pH 7.4) at
mM) was incubated with human liver
m
m
m
37 8C for 7 h. The resulting reaction mixture was deproteinized by
adding acetonitrile prior to HPLC fractionation. The structure of the
HPLC peak corresponding to demethyl-IND sulfate was confirmed
by LC–MS/MS analysis.
Fig. 2. Identification of the microsomal metabolites of PTI using LC–MS/MS analysis. MS (top) and MS2 (bottom) ion spectra were shown. (A) Top, the protonated demethyl-PTI
was observed at m/z 600.2. Bottom, the ion above was fragmented to generate ions at m/z 121.1 and 297.9. (B) Top, the protonated debenzoyl-PTI was observed at m/z 476.5.
Bottom, the ion above was fragmented to generate ions at m/z 120.9 and 173.7.(C) Top, the protonated hydroxy-PTI was observed at m/z 630.5. Bottom, the ion above was
fragmented to generate ions at m/z 138.4 and 311.4.

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G. Xie et al. / Biochemical Pharmacology 85 (2013) 1195–1202
2.9. The metabolism of PTI in rats
hydrolysis at its carboxylester bond, hydroxylation at its tyrosol
moiety, O-demethylation and N-debenzoylation reactions at its
IND moiety in liver microsomes, with its hydrolysis being the
major reaction. Demethyl-PTI and hydroxy-PTI shared similar
kinetic features in liver microsomes (Fig. 1B). Their levels
increased rapidly reaching their peak values ꢃ20 min after
initiation of reaction, then decreased dramatically due to
esterase-mediated hydrolysis. Of the microsomes from the three
species, human liver microsomes (HLMs) generated by far the
highest hydroxy-PTI level, while rat liver microsomes generated
the highest demethyl-PTI level.
All animal care and experimental procedures were approved by
the Institutional Animal Care and Use Committee at Stony Brook
University. An equimolar single dose of PTI (10 mg/kg) or
conventional IND (5 mg/kg) in corn oil was administered orally
to rats, which were then sacrificed 3 h post-dosing. Rat blood was
collected and immediately centrifuged. The resulting plasma was
deproteinized by immediate mixing it with a 2-fold volume of
CH3CN, and then subjected to HPLC analyses.
2.10. Statistical analysis
To determine whether these metabolic reactions are NADPH-
dependent, we also examined the metabolism of PTI by HLMs in
the absence of NADPH. Hydroxy-PTI, demethyl-PTI and demethyl-
IND were not detected in the absence of NADPH, whereas
debenzoyl-IND was detected (Fig. 1C). Thus, the hydroxylation
and O-demethylation reactions of PTI and IND are NADPH-
dependent, while the N-debenzoylation reaction is not.
Results were analyzed using the Student’s t-test; p ꢂ 0.05 was
considered statistically significant.
3. Results
3.1. Phase I metabolism of PTI by mouse, rat and human liver
microsomes
3.2. Phase I metabolism of PTI by human intestine, kidney and lung
microsomes
We first explored the metabolism of PTI by mouse, rat and
human liver microsomes. PTI was rapidly hydrolyzed at its
carboxylester bond to yield IND by the liver microsomes
(Fig. 1A). Twenty minutes after initiation of the reactions, 22%,
52%, and 80% of PTI was hydrolyzed to yield IND by rat, human and
mouse liver microsomes, respectively. Esterase-mediated hydro-
lysis of PTI essentially accounted for its microsomal instability.
Consequently, the half-life (t1/2) of PTI in liver microsomes
decreased in the order: rat > human > mouse (Table 1).
PTI was efficacious against human colon and lung cancer in
preclinical models [11]. Therefore, we examined the metabolism of
PTI in human intestine, lung and kidney microsomes. The
dominant metabolic reaction of PTI in these microsomes is its
hydrolysis to yield IND; the hydrolysis rate decreased in the order:
intestine > kidney > lung (Fig. 3). As a result, intrinsic clearance
(CLint) of PTI in human intestine microsomes is much higher than
that in human kidney and lung microsomes (Table 1). Minimal
levels of demethyl-PTI and hydroxy-PTI were also detected in PTI-
treated human intestine microsomes, but not in human kidney and
lung microsomes (Fig. 3).
In addition to IND, multiple minor metabolites of PTI were
identified by LC–MS/MS analysis, including demethyl-PTI,
debenzoyl-PTI and hydroxy-PTI (Fig. 2). Thus, PTI undergoes
Intesꢀne
Kidney
Lung
80
60
40
20
0
80
60
40
20
0
0
1
2
3
4
0
1
2
3
4
0.4
0.2
0.0
0.2
0.1
0.0
0
1
2
3
4
0
1
2
3
4
Time, h
Fig. 3. Kinetics of PTI metabolism by human intestine, kidney, and lung microsomes. Time courses of the levels of PTI and its metabolites in PTI-treated human microsomes.
PTI (80 M) was incubated with human intestine, kidney and lung microsomes (protein concentration 0.25 mg/ml) at 37 8C for up to 4 h. PTI and its metabolites were
extracted at the designated time points and assayed as described in Section 2.
m

G. Xie et al. / Biochemical Pharmacology 85 (2013) 1195–1202
1199
3.3. The metabolism of PTI by human CYPs
3.4. Glucuronidation reaction in human liver and intestinal
microsomes
Cytochrome P450s (CYPs) are major drug-metabolizing
enzymes that account for ꢃ75% of the metabolism of clinically
used drugs and other xenobiotics [12]. We evaluated the
metabolism of PTI by five major human CYPs. The recombinant
CYPs contained significant esterase activity, resulting in substan-
tial hydrolysis of PTI. Therefore, we pre-treated CYPs with
diisopropyl fluorophosphate (DFP) to inhibit the esterase activity,
and then examined the kinetics of PTI metabolite formation by
CYPs. As shown in Table 2, CYP3A4 exclusively catalyzed the
hydroxylation PTI, while CYP2D6 predominantly catalyzed its O-
demethylation. In contrast, CYP1A2, 2C9 and 2C19 were not
appreciably active towards PTI.
As NSAIDs can be glucuronidated at their free –COOH group, we
examined the metabolism of PTI in the presence of UDP-glucuronic
acid in HLMs. PTI was rapidly hydrolyzed to yield IND, which was
then significantly glucuronidated (Fig. 4A). The level of IND
glucuronide increased steadily throughout the entire period of
observation. Human intestine microsomes generated similar
results (data not shown). However, PTI itself is not glucuronidated,
as evidenced by a lack of glucuronidation in the presence of DFP
(Fig. 4B). Likewise, UDP-glucuronosyltransferase (UGT 2B7) was
capable of catalyzing the glucuronidation of IND, but not that of
PTI. Thus, the free –COOH group of IND is required for its acyl
glucuronidation.
We next evaluated the effects of ketoconazole (CYP3A4
inhibitor) and quinidine (CYP2D6 inhibitor) on the metabolism
of PTI by HLMs. We observed that ketoconazole (10
CYP3A4 activity by 98.0%, and that quinidine (20
m
M) inhibited
3.5. The metabolism of PTI in rats
m
M) inhibited
CYP2D6 activity by 98.5%. We then evaluated the effects of these
inhibitors on PTI metabolism by DFP-treated HLMs. Ketoconazole
An equimolar single dose of PTI or IND was administered orally
to rats, which were sacrificed 3 h post-dosing. In rat plasma, we
detected six metabolites: IND, demethyl-IND, debenzoyl-IND,
demethyl-IND glucuronide, demethyl-IND sulfate and IND glucu-
ronide (Fig. 5). Demethyl-IND glucuronide and demethyl-IND
sulfate were identified by LC-MS/MS analysis (Fig. 6). We also
prepared authentic demethyl-IND glucuronide and demethyl-IND
sulfate by treating demethyl-IND with liver microsomes and
cytosol, respectively, and their structures were confirmed using
LC–MS/MS analysis. The plasma level of IND was significantly
higher than that of any other metabolites (P < 0.01) following IND
or PTI administration (Fig. 5). PTI and IND generated similar
metabolite level in rat plasma. Intact PTI, however, was not
detected in the rat plasma.
(10
m
M) inhibited the hydroxylation of PTI in HLMs by 97.4%.
M) inhibited the O-demethylation of PTI in HLMs
Quinidine (20
m
by 61.5%. These results indicate that CYP3A4 and CYP2D6 play
major roles in the hydroxylation and O-demethylation of PTI by
HLMs, respectively.
4. Discussion
This work establishes the metabolic pathways of PTI in vitro and
in vivo. As illustrated in Fig. 7, PTI undergoes the following metabolic
reactions: (1) hydrolysis at its carboxylester bond to yield IND; (2) O-
demethylation to form demethyl-PTI; (3) hydroxylation at its tyrosol
moiety to form hydroxy-PTI; (4) N-debenzoylation to form deben-
zoyl-PTI; and (5) phase II glucuronidation and sulfation of IND and
demethyl-IND to form conjugated metabolites.
40
PTI treatment
Indomethacin treatment
20
0
Fig. 4. Glucuronidation of PTI by human liver microsomes. (A) Time course of the
levels of PTI and its metabolites in PTI-treated HLMs in the presence of UDP-
glucuronic acid. (B) Comparison of glucuronide formation from PTI versus IND in
HLMs treated with the esterase inhibitor DFP. HLMs were treated with PTI (dotted
line) or IND (solid line) in the presence of DFP for 2 h, and the metabolite levels were
determined by HPLC. The respective chromatograms are shown. The off-scale peak
values of IND and PTI were 0.41 and 0.25 units, respectively.
Fig. 5. Drug levels in the plasma of PTI- or IND-treated rats. Equimolar amounts of
PTI (10 mg/kg) or IND (5 mg/kg) in corn oil were administered orally to rats, and
their blood was obtained 3 h later. Plasma levels of the metabolites were
determined using HPLC, as described in Section 2.

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G. Xie et al. / Biochemical Pharmacology 85 (2013) 1195–1202
Table 2
Kinetic parameters for the hydroxylation and O-demethylation reactions of PTI by human recombinant CYPs.
Hydroxylation of PTI O-demethylation of PTI
Km
(
mM)
Vmax (pmol/min/pmol CYP)
Vmax/Km
(
m
l/min/pmol CYP)
Km
(
m
M)
Vmax (pmol/min/pmol CYP)
Vmax/Km (ml/min/pmol CYP)
CYP1A2
CYP2C9
CYP2C19
CYP2D6
CYP3A4
ND
ND
ND
ND
2.5
ND
ND
ND
4.0
70.0
1.5
17.5
0.03
27.5
11.0
44.5
ND, not detectable.
The rapid hydrolysis of PTI to IND is catalyzed by carbox-
ylesterase (CES) [13]. In mammals, there are two major isoforms of
CES, CES1 (liver) and CES2 (intestine). CES1 mainly catalyzes the
hydrolysis of esters with a large acyl group, while CES2 prefers
substrates with bulky alcohol group. Liver is a major site for CES-
mediated drug elimination. While the expression of CES1 in liver is
similar in mouse, rat and human, the liver expression of CES2
decreases in the order: mouse > human > rat [14]. As both CES1
and CES2 can efficiently hydrolyze PTI [13], it is reasonable to
observe that the hydrolysis rates of PTI in liver microsomes from
various species decreased in the order: mouse > human > rat. The
human intestine microsomes are also highly efficient in hydrolyz-
ing PTI, likely a result of high expression of CES2 in human intestine
[15]. In comparison, the expressions of CES1 and CES2 are
considerably lower in human kidney and lung [15], thus
accounting for the higher stability of PTI in these microsomes.
CYPs also play a major role in the phase I metabolism of PTI by
catalyzing its hydroxylation and O-demethylation. Of the five
major human CYPs, CYP3A4 uniquely catalyzes the hydroxylation
of PTI at its tyrosol moiety, presumably because the active site of
CYP3A4 is large and flexible. Indeed, CYP3A4 has extremely broad
substrate specificity and is responsible for the metabolism of ꢃ50%
of drugs in current use [16]. CYP3A4 is abundant (40–60%) in
human liver, but absent in mouse or rat [17,18]. This fact explains
the far more rapid hydroxylation of PTI in HLMs than in rodent
microsomes.
On the other hand, O-demethylation of PTI is primarily
catalyzed by CYP2D6. CYP2D6 substrate binding is uniquely
governed by an ion-pair interaction between a positively charged
nitrogen atom and a negatively charged Asp301 residue [19]. Thus,
the nitrogen atom in the IND moiety of PTI may play an important
role in its interaction with CYP2D6 and the subsequent
Fig. 6. Identification of the conjugated metabolites derived from demethyl-IND. MS (top) and MS2 (bottom) ion spectra were shown. (A) Top, the deprotonated demethyl-IND
glucuronide was observed at m/z 518.40. Bottom, the ion above was fragmented at its glucuronide bond to generate an ion at m/z 297.89. (B) top, the deprotonated demethyl-
IND sulfate was observed at m/z 422.36. Bottom, the ion above was fragmented at its sulfate bond to generate an ion at m/z 298.11 (Fig. 6B).

G. Xie et al. / Biochemical Pharmacology 85 (2013) 1195–1202
1201
Fig. 7. Overall metabolic pathways of PTI in vitro and in vivo. PTI undergoes hydrolysis, hydroxylation, O-demethylation and N-debenzoylation reactions to generate IND,
hydroxy-PTI, demethyl-PTI and debenzoyl-PTI, respectively. The resulting IND can be transformed to demethyl-IND and debenzoyl-IND, which, in turn, can be
glucuronidated. Demethyl-IND can also be sulfated. SULTs, sulfotransferases.
O-desmethylation of PTI. In contrast to its hydroxylation reaction,
PTI is more rapidly O-demethylated in rat liver microsomes than in
HLMs. This observation reflects the low abundance (2%) of CYP2D6
in human liver [20]. Our observation that the hydroxylation and O-
demethylation of PTI are catalyzed by distinct CYP isoforms not
only suggests that different molecular mechanisms are involved in
these two reactions, but also demonstrates the unique CYP-
catalyzed reaction specificity within a given substrate.
glucuronides (IND glucuronide and demethyl-IND glucuronide)
and demethyl-IND sulfate in the plasma of IND- and PTI-treated
rats. To our knowledge, this is the first report of the sulfated
metabolite derived from IND. These phase II conjugation reactions
substantially enhance drug hydrophilicity, which generally facil-
itates drug elimination and detoxification. However, both glucur-
onidation [24] and sulfation reactions [25] have also been
implicated in substrate activation and reactivity.
In contrast to PTI, IND (parent compound of PTI) is primarily O-
demethylated by CYP2C9, which prefers acidic substrates like IND
[21]. The interaction of Arg108 of CYP2C9 with the acidic ligands is
critical for its substrate binding [22]. CYP2C9 is abundant in the
human but absent in mouse or rat [18], which explains our
observation that the IND was O-demethylated far more rapidly in
HLMs than in rodent microsomes (Fig. 1B). The differential
metabolism of PTI and IND by distinct CYP isoforms is primarily
due to their strikingly different physicochemical properties, such
as acidity and lipophilicity. As is the case with PTI and IND, CYP3A4
primarily catalyzed the hydroxylation of phospho-ibuprofen,
whereas CYP2C9 exclusively hydroxylated ibuprofen [23]. These
observations account, at least in part, for the characteristic
metabolic profiles and pharmacokinetic properties of phospho-
NSAIDs [23].
N-debenzoylation is a minor metabolic reaction for PTI and IND,
involving the hydrolysis of an amide bond to form debenzoyl-PTI
and debenzoyl-IND, respectively. This reaction is presumably
catalyzed by an amidase/esterase, and is therefore NADPH-
independent. In contrast, CYP-catalyzed O-demethylation of PTI
and IND is NADPH-dependent. Similar to our observations, CYP-
catalyzed O-deethylation of a phosphoric acid ester compound is
NADPH-dependent, while esterase-catalyzed hydrolysis of its
amide bond is NADPH-independent [26].
The metabolic fate of PTI has important implications for its
efficacy. CYP3A4-mediated hydroxylation of PTI at its tyrosol
moiety may contribute to its potent biological activity. Unlike
tyrosol, hydroxy-tyrosol is
a polyphenol that possess anti-
inflammatory and anti-cancer activities [27]. Hydroxy-tyrosol
has been shown to induce cancer cell apoptosis, inhibit cell
proliferation and prevent cancer metastasis. Its molecular targets
include ERK1/2, cyclin D1, 5- and 12-lipooxygenase [28,29]. Given
the significant biological activities of hydroxy-tyrosol, hydroxy-PTI
may play an important role in cancer control.
CES- and CYP-catalyzed hydrolysis and O-demethylation of PTI
introduce polar –COOH and –OH functional groups, which are
recognized by phase II conjugation enzymes for acyl glucuronida-
tion and sulfation reactions, respectively. We detected two acyl

1202
G. Xie et al. / Biochemical Pharmacology 85 (2013) 1195–1202
transition in A549 human lung cancer cells: a novel cyclooxygenase-inhibi-
On the other hand, CES-catalyzed hydrolysis of PTI may be
tion-independent effect. Biochem Pharmacol 2011;82:1781–91.
[10] Shiff SJ, Koutsos MI, Qiao L, Rigas B. Nonsteroidal antiinflammatory drugs
inhibit the proliferation of colon adenocarcinoma cells: effects on cell cycle
and apoptosis. Exp Cell Res 1996;222:179–88.
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anticancer effect of phospho-tyrosol-indomethacin (MPI 621), a novel phos-
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involved in metabolic activation of prodrugs. Molecules 2008;13:412–31.
[15] Nishimura M, Naito S. Tissue-specific mRNA expression profiles of human
phase I metabolizing enzymes except for cytochrome P450 and phase II
metabolizing enzymes. Drug Metab Pharmacokinet 2006;21:357–74.
[16] Kapelyukh Y, Paine MJ, Marechal JD, Sutcliffe MJ, Wolf CR, Roberts GC. Multiple
substrate binding by cytochrome P450 3A4: estimation of the number of
bound substrate molecules. Drug Metab Dispos 2008;36:2136–44.
[17] Davis MP, Homsi J. The importance of cytochrome P450 monooxygenase
CYP2D6 in palliative medicine. Support Care in Cancer 2001;9:442–51.
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detrimental to its bioactivity in vivo. We previously demonstrated
that the intact PTI is more efficacious against cancer than
conventional IND [13]. Given the strong CES activity in the
intestine and liver, it is not surprising that intact PTI cannot be
found following its oral administration in rats. It is thus
conceivable that the bioactivity of PTI can be further improved
if co-administered with CES inhibitors or formulated in nanocar-
riers that protect PTI from hydrolysis [30].
In summary, PTI undergoes extensive metabolic transforma-
tions in vitro and in vivo, generating numerous metabolites. Our
results reveal significant differences in its metabolism between
humans and laboratory rodents, which would help to predict
clinical outcomes based on preclinical animal data.
Statement of conflicts of interest
The authors have nothing to disclose except for BR, who has an
equity position in Medicon, Pharmaceuticals, Inc.
Acknowledgements
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This work was supported by the National Institute of Health
Grant R01CA139454. We thank R. Rieger and T. Koller, Stony Brook
University, for their expert LC–MS/MS analysis and the shared
instrumentation grant, NIH/NCRR 1 S10 RR023680-1. We also
thank Carol Ann Amella for critically reading the manuscript.
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