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Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the clinical treatment of pain, inflammation and
fever[1,2]. These drugs (eg indomethacin, diclofenac and meloxicam) are thought to act by inhibiting cyclooxygenase (COX), an
enzyme that limits the biosynthesis rate of prostaglandins from arachidonic
acid[3_5]. Two forms of cyclooxygenase,
designated COX-I and COX-II,
exist[6,7]. COX-I is the major form, and is located in healthy tissues, catalyzes the formation of
prostaglandins under normal physiological conditions, and plays a role in the maintenance of the gastrointestinal mucosa as
well as platelet function[8]. In comparison, COX-II is an inducible enzyme that is predominantly expressed in association with
inflammation[9]. It is believed that NSAID-induced gastrointestinal damage results from the inhibition of COX-I, whereas the
therapeutic benefit results from the inhibition of COX-II expressed at the site of
inflammation[10]. However, the division
between the biological functions of COX-I and COX-II is not clear-cut. Moreover, treatment with COX-II selective inhibitors
could theoretically lead to problems with thrombosis, and salt and water balance. Recently, much attention has been focused
on the increased risk of cardiovascular events associated with COX-II selective NSAIDs (such as celecoxib and rofecoxib) as
compared with nonselective
NSAIDs[11_13]. Therefore, NSAIDs that preferentially inhibit COX-II with moderate selectivity
seem more promising. Imrecoxib,
[4-(4-methane-sulfonyl-phenyl)-1-propyl-3-p-tolyl-1,5-dihydro-pyrrol-2 -one] (Figure 1), is
a novel and moderately selective COX-II
inhibitor[14_16]. The drug inhibits COX-I and COX-II with
IC50 values of 115±28 nmol/L and 18±4 nmol/L, respec-tively. Imrecoxib exerts its anti-inflammatory effect by inhibiting COX-II mRNA
expression[16]. In fact, imrecoxib is currently undergoing clinical trials in China for the treatment of acute and chronic inflammatory disease. We
previously found that imrecoxib is extensively metabolized in rats, with less than 2% of the dose excreted unchanged in urine
and feces (unpublished data). We characterized 7 metabolites of imrecoxib in rats: the 4¡¯-hydroxymethyl (M4), 4¡¯-carboxylic
acid (M2), 4¡¯-hydroxymethyl-5-hydroxy (M3), and 4¡¯-hydroxymethyl-5-carbonyl (M5) metabolites, and glucuronide
conjugates of M2, M3, and M4. The major route of metabolism appears to be 4¡¯-methyl hydroxylation, with further oxidation of the
corresponding carboxylic acid (Figure 1). The purpose of the present study was to study the in vitro metabolism of imrecoxib
in rat liver microsomes and to identify the cytochrome P450 (CYP) forms involved in its metabolism.
Materials and methods
Chemicals Imrecoxib, the 4¡¯-hydroxymethyl (M4) and 4¡¯-carboxylic acid (M2) metabolites of imrecoxib, and BAP 910 (an
analogue of imrecoxib, used as internal standard) were supplied by the Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College (Beijing, China). 4¡¯-Hydroxymethyl-5-hydroxyl (M3) and
4¡¯-hydroxy-methyl-5-carbonyl (M5) metabolites of imrecoxib, used as reference substances, were isolated from rat urine in our laboratory
and identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy.
a-Naphthoflavone, b-naphthoflavone, methylpyrazole, quinine, codeine, and morphine were all purchased from Sigma-Aldrich (Deisenhofen,
Germany). Cimetidine was obtained from Kaili Pharmaceutical Co (Jiangsu, China), ketoconazole from Dragon Pharmachemical
Co (Zhejiang, China), dexamethasone from Tianjin Pharmaceutical Group Co (Tianjin, China), isoniazid from Jiangbei
Pharmaceutical Co (Zhejiang, China), nifedipine from Zhongnuo Pharmaceutical Co (Shijiazhuang, China), diphenhydramine from
Beijing Taiyang Pharmaceutical Co (Beijing, China), and gliclazide from Tianjin Zhongxin Pharmaceutical Co (Tianjin, China).
Dehydronifedipine was synthesized in the Department of Pharmaceutical Chemistry, Shenyang Pharmaceutical University
(Shenyang, China). b-Nicotinamine adenine dinucleotide phosphate (reduced form, NADPH) was obtained from Xinjingke
Biotechnology Co (Beijing, China). DL-Dithio-threitol (DTT, ultrapure grade) and tris (hydroxymethyl)aminomethane (Tris,
ultrapure grade) were obtained from Ameresco (Solon, Ohio, USA). Methanol and acetonitrile were of high performance
liquid chromatography grade (Yuwang Co, Shandong, China). All other chemicals were of analytical grade.
Animal preparation Male Wistar rats (7*8 weeks old) weighing 200 to 250 g were supplied by the Laboratory Animal
Center of Shenyang Pharmaceutical University (grade II, certificate
No 042). Animals were fed a standard diet
ad libitum and kept in a 12 h light/dark cycle. Groups of rats
(n=6 per group) were treated daily (intraperitoneally) for 5 d with
b-naphthoflavone (50 mg/kg, in corn oil, CYP 1A), isoniazid (100 mg/kg, in saline, CYP 2E), or dexamethasone (50 mg/kg, in corn oil, CYP 3A).
Control animals (n=6 per group, 2 groups) were treated with an equivalent volume of corn oil or saline. The rats were fasted
for 18 h before being killed, and were killed 24 h after the last injection. All experimental procedures were performed in
accordance with the guidelines of the Experimental Animal Care and Use Committee of Shenyang Pharmaceutical University
(Shenyang, China).
Preparation of rat liver microsomes Pooled liver microsomes from 6 rat livers in each group were prepared as previously
described[17]. The microsomal protein
concentrations were determined by using the method of Lowry
et al[18]. Total cytochrome P450 contents were measured according to the method of Omura and
Sato[19].
Incubation of imrecoxib with rat liver
microsomes To determine the formation rate of the 4¡¯-hydroxymethyl metabolite
(M4), the basic incubation medium contained 0.1 mol/L Tris-HCl buffer (pH 7.4), 1.0 mmol/L NADPH, 10
mmol/L KCl, 10 mmol/L MgCl2, 1.0 g/L microsomal protein and 5_600 µmol/L imrecoxib in a final volume of 200 µL. The mixture
was incubated at 37 °C for various times (0, 5, 15, 30, or 60 min). The reactions were initiated by the addition of NADPH after
5 min preincubation and were terminated by the addition of 50 µL cold methanol. Then 20 µL internal standard (BAP 910, 20
µmol/L in methanol) and 200 µL of 20 µmol/L
NH4H2PO4 buffer (pH 3) were added to the reaction mixtures. The samples were
extracted with 2 µL ethyl acetate and the supernatant was evaporated under a stream of nitrogen at 40 °C. The residue was
dissolved in 100 µL of the mobile phase for
LC/MSn (liquid chromatography-ion trap mass spectrometry) assay. Controls
were prepared in the same manner, except for the presence of NADPH. Blank samples were assayed without substrate to
exclude analytical interference by the matrix.
Metabolite identification To identify the
in vitro phase I metabolites of imrecoxib formed from rat liver microsomes, 200
µmol/L of imrecoxib (saturated concentration) was used as substrate. The final volume was 1 mL and the mixtures were
incubated at 37 °C for 60 min. The reactions were initiated by addition of NADPH and were terminated with 1 mL of 20
µmol/L cold
NH4H2PO4 buffer (pH 3). Then the mixtures were applied to preconditioned 2.5 mL
C18 cartridges (Tianjin Fuji Co, China). The columns were washed with 2 mL water and the metabolites were eluted with 1 mL methanol. The eluting solvents
were evaporated and the residue was dissolved in 100 µL of the mobile phase for
LC/MSn analysis.
Enzyme kinetics Linear conditions for the formation of M4 were established with respect to protein content and
incubation time. The rate of formation was linear over 60 min incubation and 0.5 to 1.5 g/L of microsomal protein. The
Michaelis-Menten kinetics of imrecoxib 4¡¯-methyl hydroxylation by rat liver microsomes was determined by using 14
substrate concentrations in the range of 5 to 600 µmol/L at 37 °C for 15 min. The rate of imrecoxib metabolism was analyzed by
using the MULTI program, using a nonlinear least-squares
method[20]. Data were also analyzed by linear transformation
(Eadie-Hofstee plot) to confirm a single
Km model. The following Michaelis-Menten equation was used to analyze the
relation between velocity and substrate concentra-tion:
V=Vm·S/(Km+S),
where V, S, Km, and
Vm are the velocity of metabolite formation, the substrate concentration, the apparent Michaelis-Menten
constant, and the maximum velocity of metabolism, respectively.
Inhibition study of imrecoxib metabolism
Inhibitory effects on the 4¡¯-methyl hydroxylation of imrecoxib in rat liver
microsomes prepared from induced rats and control rats were determined at substrate concentrations of 50 µmol/L imrecoxib
(~Km) and at 3 inhibitor concentrations in the range of 2_50 µmol/L. Incubations were performed at 37 °C for 15 min. The
selective P450 inhibitors a-naphthoflavone (CYP 1A), quinine (CYP 2D), methylpyrazole (CYP 2E), cimetidine (CYP 2C), and
ketoconazole (CYP 3A) were used. Because all the inhibitors were dissolved in methanol, an equivalent volume of methanol
(without inhibitors) was included in the control incubations to correct for any effects of the solvent on microsomal activity.
The concentration of methanol was 0.5%.
LC/MSn analysis A Finnigan LCQ liquid chromatography-ion trap mass spectrometer (San Jose, USA) was used to
identify the in vitro metabolites of imrecoxib formed by rat liver microsomes. The instrument was operated in positive
electrospray ionization mode. The source voltage was held at 4.5 kV. The capillary voltage was fixed at 13 V, and its
temperature was set at 200 °C. Nitrogen was used as the sheath gas (0.75 L/min) and auxiliary gas (0.15 L/min). The
MS2 spectra were produced by collision-induced dissociation (CID) of the selected precursor ions with helium in the mass
analyzer, and the relative collision energies were individually optimized for each compound. Liquid chromatography was
performed with a Shimadzu LC-10 AD solvent delivery system (Kyoto, Japan). The samples were separated on a Diamonsil
C18 column (200 mm×4.6 mm ID; 5 µm, Dikma Technologies, Beijing, China). A mobile phase consisting of
methanol-ammonium acetate 10 mmol/L (60:40, v/v) was used at a flow rate of 0.5 mL/min. The injection volume was 20 µL. All data were
analyzed by using Xcalibur software (version 1.2, Thermo Finnigan MAT).
The formation rate of M4 was determined by using the same
LC/MSn system, except that a mobile phase composed of
acetonitrile-water-formic acid (75:25:0.5, v/v/v) was used and quantification was performed using selected reaction
monitoring (SRM) of the transitions m/z 386
® m/z 356, 278 for M4 and m/z 374 ®m/z 278 for BAP 910 (IS). Calibration standards were
prepared by spiking 20 µL of appropriate standard solution of M4 into 200 µL of blank medium. The linear regressions of the
peak area ratios versus concentrations were fitted over the concentration range of 0.10_50.0 µmol/L. The method was
validated by determining quality control (QC) samples at 3 concentration levels on 3 consecutive days. The precisions were
expressed as relative standard deviation (RSD) and the accuracy as relative error (RE%). The intra- and inter-day precision
values were less than 8%. The accuracy for M4 was 7.5%, -5.3% and -4.8% at 0.1 µmol/L, 4.0 µmol/L and 50.0 µmol/L levels,
respectively.
Codeine O-demethylase activity assay
Codeine O-demethylation was carried out as a probe assay for CYP
2D[21,22]. The substrate concentration of codeine was 20
µmol/L. Incubations were performed at 37 °C for 20 min. After the reactions were finished, 40 µL internal standard
(diphenhydramine, 20 µmol/L in methanol) and 200 µL of 0.1 mol/L
Na2CO3 were added to the mixtures. The samples were
extracted with 3 mL diethyl ether and the supernatant was evaporated under a stream of nitrogen at 40 °C. The residue was
dissolved in 100 µL of the mobile phase. An aliquot of 20 µL of the solution was analyzed by using the same
LC/MSn system as described for
LC/MSn analysis except that the source voltage was held at 4.25 kV and its temperature was
set at 180 °C; the SRM of the transitions of
m/z 286 ®
m/z 201, 229 for morphine and m/z 256
® m/z 167 for diphenhydramine (IS) were used for quantification; and the mobile phase
consisted of methanol-water-formic acid (60:40:0.5,
v/v/v).
Nifedipine dehydrogenase activity assay
Nifedipine dehydrogenation was used to probe the activity of CYP
3A[23]. Nifedipine (80 µmol/L) was incubated with microsomal protein at 37 °C for 10 min in a final volume of 200 µL. After the
reactions were finished, 20 µL internal standard (gliclazide, 400 µmol/L in methanol) and 100 µL of 0.1 mol/L NaOH were added
to the mixtures, respectively. The samples were extracted with 2 mL
n-hexane-dichloromethane-iso-propyl alcohol (20:10:1,
v/v/v). The dehydronifedipine formed was analyzed using the same
LC/MSn system as described earlier except that the SRM
of the transitions of m/z 345 ® m/z 284 for dehydronifedipine and
m/z 324 ® m/z 127, 168 for gliclazide (IS) were used for
quantification; and the mobile phase consisted of acetonitrile-water-formic acid (85:15:0.5, v/v/v).
Data analysis All data are the means of 3 individual incubations. The significance of differences between means were
evaluated by using ANOVA followed by the one-tailed Student¡¯s
t-test.
Results
Metabolism of imrecoxib in rat liver microsomes
Compared with the controls, 3 metabolites were found in rat liver
microsomal incubates, in addition to the substrate imrecoxib (Figure 2). The structures of the metabolites were identified by
investigation of their chromatographic behavior, and electrospray ionization MS and
MS2 spectra relative to reference substances. The retention times and main characteristic ions in mass spectra of imrecoxib and its metabolites are summarized
in Table 1.
The compound eluting at 30.2 min had the same pseudomolecular ion
([M+H]+), full scan MS2 spectrum, and
chromatographic behavior as imrecoxib, therefore, it was identified as unchanged imrecoxib. By using the same method, the
metabolites eluting at 8.0 min, 10.5 min, and 29.3 min were identified as the 4¡¯-hydroxymethyl-5-hydroxyl metabolite (M3), the
4¡¯-hydroxymethyl metabolite (M4), and the 4¡¯-hydroxymethyl-5-carbonyl (M5) metabolite, respectively. No metabolites were
detected in the absence of NADPH, indicating that metabolite formation is enzymatic and NADPH-dependent.
Enzyme kinetics Overall, the formation of M4 conformed to saturable kinetics and a representative Michaelis-Menten
plot is shown in Figure 3. The Eadie-Hofstee plot (Figure 3, inset) for the formation of M4 from imrecoxib was indicative of
monophasic behavior. Accordingly, a simple Michaelis-Menten kinetic analysis was used to estimate
Km and Vm (Table 2).
The Vm of M4 in dexamethasone-induced micro-somes increased significantly, to 7.5-fold higher than that in control microsomes.
The same parameter in isoniazid-induced microsomes was 2-fold higher than that in control microsomes from rats treated with
saline. The results suggest that CYP 3A and CYP 2E enzymes play important roles in the 4¡¯-methyl hydroxylation of imrecoxib
in rat liver microsomes.
Effects of inducers on the formation of
M4 Imrecoxib (50 µmol/L or 200 µmol/L) was incubated at 37 °C for 15 min with
induced and control rat liver microsomes (1.0 g/L) in the presence of NADPH. Among typical P450 inducers administered
intraperitoneally, dexamethasone caused the most induction of M4 formation, followed by isoniazid (Table 3). The formation
rates of M4 in dexamethasone-induced microsomes were 3.4-fold and 6.1-fold higher than those in control microsomes from
rats treated with corn oil, respec-tively, when substrate concentrations of 50 µmol/L and 200 µmol/L were used, respectively.
The corresponding values in isoniazid-induced microsomes were 1.2-fold and 1.4-fold higher than those in control
microsomes from rats treated with saline, respectively. However,
b-naphthoflavone had no significant effect on the metabolism
of imrecoxib. The results were in accordance with those regarding the kinetics of imrecoxib 4¡¯-methyl hydroxylation (Table 2).
Effects of inhibitors on the formation of
M4 The effects of inhibitors on the formation of M4 in rat liver microsomes are
shown in Figures 4_6. The inhibitory activities are expressed as a ratio in comparison with the control activity without
inhibitors. Ketoconazole, a selective inhibitor of CYP 3A, was shown to effectively decrease the formation rate of M4. When
2 µmol/L of ketoconazole was used, the hydroxylation activity was reduced to approximately 70% of the control activity in
microsomes from control rats treated with saline or corn oil (Figure 4), whereas much lower enzyme activity (28% of the
control activity) was observed in microsomes from dexamethasone-induced rats at the same ketoconazole concentration
(Figure 5). In addition, a significant inhibition was observed in the presence of quinine (CYP 2D-selective) in control
microsomes, but the other chemical inhibitors, a-naphthoflavone, cimetidine, and methylpyra-zole, did not produce any
significant effects on the 4¡¯-methyl hydroxylation of imrecoxib (Figure 4). The effects of inhibitors on the formation of M4 by
liver microsomes from
isoniazid-induced rats and from b-naphthoflavone-induced rats were similar to those observed in control microsomes (Figure
6).
Discussion
Three phase I metabolites of imrecoxib were observed in rat liver microsomal incubates using the
LC/MSn method with a saturated substrate concentration (200 µmol/L imrecoxib), a longer incubation time (60 min), and a larger final volume (1 mL).
The identities of the metabolites were confirmed by chromatographic and mass spectra comparison with reference substances.
These metabolites were identified as the 4¡¯-hydroxymethyl-5-hydroxyl metabolite (M3), the 4¡¯-hydroxymethyl metabolite
(M4), and the 4¡¯-hydroxymethyl-5-carbonyl (M5) metabolite. The formation of these metabolites was enzymatic and
NADPH-dependent. However, when 50 µmol/L imrecoxib was incubated at 37 °C for 60 min in a final volume of 200 µL, imrecoxib
metabolites other than M4 were not detected. Under these conditions, approximately 40% of imrecoxib was converted to M4
and the remainder was unchanged imrecoxib. It seems that 4¡¯-methyl hydroxylation is the major metabolic pathway in
NADPH-fortified rat liver microsomes. Although further oxidation of the 4¡¯-hydroxymethyl metabolite to form the
4¡¯-carboxylic acid metabolite (M2) was the predominant pathway in vivo, no carboxylic acid metabolite was found in the present study.
This suggests that the generation of M2 in
vitro may require the presence of cytosolic enzymes as in the metabolism of
celecoxib[24]. This hypothesis is under investigation.
To identify the CYP isozymes involved in the 4¡¯-methyl hydroxylation of imrecoxib, the effects of specific inducers and
inhibitors of CYP on this reaction were examined. M4 was produced to the greatest extent by microsomes from
dexamethasone-induced rats (Table 2 and 3). Dexamethasone is considered to be a specific inducer of CYP
3A[25,26]. However, ketoconazole, a well-known inhibitor of CYP
3A[22,26], strongly inhibited the reaction in a concentration-dependent manner (Figure 4_6).
These results indicate that CYP 3A is the principal enzyme involved in the 4¡¯-methyl hydroxylation of imrecoxib in rat liver
microsomes.
In addition, 10 mmol/L of quinine (an inhibitor specific for CYP
2D[22,27]) significantly inhibited the rate of formation of M4
by approximately 20% compared with control activities in all types of microsomes except the microsomes obtained from
dexamethasone-pretreated rats (Figure 4_6). When the concentration of quinine was increased to 50
µmol/L, the formation rate was decreased to a much lower level. With respect to the results mentioned above, participation
of CYP 2D in the 4¡¯-methyl hydroxylation of imrecoxib is also thought to be involved in control rats, and in
b-naphthoflavone-induced and in isoniazid-induced rats.
a-Naphthoflavone, which is often used as inhibitor of CYP
1A[22,26,28], did not decrease the rate of hydroxylation of
imrecoxib. This result is in agreement with those of the induction experiments.
b-naphthoflavone, an inducer of CYP 1A, did not significantly increase the formation of M4 (Tables 2 and 3). Cimetidine was used to confirm the participation of CYP
2C[27,28] in imrecoxib biotransformation; however, the formation of M4 was not inhibited by cimetidine in microsomes either from
control rats or from induced rats. Thus, substantial participation of CYP 2C was unlikely in this reaction. Methylpyrazole, a
selective inhibitor of CYP 2E[26], had no significant inhibitory effect on the 4¡¯-methyl hydroxylation of imrecoxib. However, the
reaction was elevated significantly in microsomes from isoniazid-pretreated rats compared with those from control rats
treated with saline (Table 3). Isoniazid is an inducer of CYP
2E[25]. There appears to be a discrepancy between these two
different phenomena. The results of inhibition studies clearly show that CYP 3A and 2D catalyze the 4¡¯-methyl hydroxylation
of imrecoxib, so the effects of isoniazid on CYP 3A and 2D were investigated in the present study. Codeine
O-demethylation and nifedipine dehydrogenation were carried out as probe assays for CYP 2D and 3A, respectively, and their activities were
determined as described in the materials and methods section. The activities of codeine
O-demethylase in control microsomes from rats treated with saline and in isoniazid-induced microsomes were 45.8±5.9
pmol·min-1·mg-1 protein and
59.4±7.5 pmol·
min-1·mg-1 protein, respectively. The activities of nifedipine dehydrogenase in control microsomes from rats treated with
saline and in isoniazid-induced microsomes were 759.8±35.4
pmol·min-1·mg-1 protein and 722.6±20.8
pmol·min-1·mg-1 protein, respectively. There was no significant difference in the activities of CYP 3A between saline-treated control microsomes and
isoniazid-induced microsomes, but the activity of CYP 2D in isoniazid-induced microsomes was significantly higher than that
in saline-treated control microsomes. The results suggest that the faster formation rate of M4 observed in isoniazid-induced
microsomes was caused by the higher activity of CYP 2D in isoniazid-induced microsomes. So the involvement of CYP 2E in
the 4¡¯-methyl hydroxylation of imrecoxib may be excluded.
Interestingly, despite CYP 3A and 2D being shown to catalyze the 4¡¯-methyl hydroxylation of imrecoxib, the data obtained
from rat liver microsomes were described by a single
Km model (Figure 3). This finding indicates that CYP 3A and 2D are likely
to be characterized by similar apparent
Km values.
In conclusion, imrecoxib was metabolized to 3 metabolites by rat liver microsomes: 4¡¯-hydroxymethyl imrecoxib (M4),
4¡¯-hydroxymethyl-5-hydoxyl imrecoxib (M3), and 4¡¯-hydroxymethyl-5-carbonyl imrecoxib (M5). This biotransformation study
of imrecoxib in rat liver microsomes indicates that 4¡¯-methyl hydroxylation represents the major metabolic pathway of imrecoxib.
The reaction was mainly catalyzed by CYP 3A. CYP2D also played a role in control rats based on the results of inhibition
studies. Other CYP (such as CYP 1A, 2C, and 2E) seem to not participate in the metabolic pathway.
Acknowledgements
We would like to thank Dr Xue-qing LI (Drug Metabolism and Pharmacokinetics and Bioanalytical Chemistry, AstraZeneca
Research and Development, Mölndal, Sweden) and Prof Shu-qiu ZHANG (Department of Pharmaceutics, Shanxi Medical
University, Shanxi, China) for their advice and assistance.
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