Download pharmacokinetics, tissue distribution, metabolism, and excretion of

0090-9556/00/2805-0514–521$03.00/0
DRUG METABOLISM AND DISPOSITION
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
DMD 28:514–521, 2000 /1797/817858
Vol. 28, No. 5
Printed in U.S.A.
PHARMACOKINETICS, TISSUE DISTRIBUTION, METABOLISM, AND EXCRETION OF
CELECOXIB IN RATS
SUSAN K. PAULSON, JI Y. ZHANG, ALAN P. BREAU, JEREMY D. HRIBAR, NORMAN W. K. LIU, SUSAN M. JESSEN,
YVETTE M. LAWAL, J. NITA COGBURN, CHRISTOPHER J. GRESK, CHARLES S. MARKOS, TIMOTHY J. MAZIASZ,
GRANT L. SCHOENHARD,1 AND EARL G. BURTON
Departments of Clinical Pharmacokinetics and Bioavailability (S.K.P.), Metabolism and Safety Evaluation (J.Y.Z., A.P.B., C.J.G., C.S.M., E.G.B.),
Physical Methodology (J.D.H., N.W.K.L.), Regulatory Affairs (S.M.J., Y.M.L.), and COX-2 Technology (T.J.M.), G.D. Searle & Co., Skokie, Illinois;
and Nutrition and Consumer Products, Monsanto Company, St. Louis, Missouri (J.N.C.)
(Received September 27, 1999; accepted January 10, 2000)
This paper is available online at http://www.dmd.org
The pharmacokinetics, tissue distribution, metabolism, and excretion of celecoxib, 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1Hpyrazol-1-yl] benzenesulfonamide, a cyclooxygenase-2 inhibitor,
were investigated in rats. Celecoxib was metabolized extensively
after i.v. administration of [14C]celecoxib, and elimination of unchanged compound was minor (less than 2%) in male and female
rats. The only metabolism of celecoxib observed in rats was via a
single oxidative pathway. The methyl group of celecoxib is first
oxidized to a hydroxymethyl metabolite, followed by additional
oxidation of the hydroxymethyl group to a carboxylic acid metabolite. Glucuronide conjugates of both the hydroxymethyl and carboxylic acid metabolites are formed. Total mean percent recovery
of the radioactive dose was about 100% for both the male rat (9.6%
in urine; 91.7% in feces) and the female rat (10.6% in urine; 91.3%
in feces). After oral administration of [14C]celecoxib at doses of 20,
80, and 400 mg/kg, the majority of the radioactivity was excreted in
the feces (88–94%) with the remainder of the dose excreted in the
urine (7–10%). Both unchanged drug and the carboxylic acid metabolite of celecoxib were the major radioactive components excreted with the amount of celecoxib excreted in the feces increasing with dose. When administered orally, celecoxib was well
distributed to the tissues examined with the highest concentrations of radioactivity found in the gastrointestinal tract. Maximal
concentration of radioactivity was reached in most all tissues
between 1 and 3 h postdose with the half-life paralleling that of
plasma, with the exception of the gastrointestinal tract tissues.
Prostaglandins (PGs)2 are local mediators of cellular activity that
produce biological responses that include the ability to induce pain,
fever, and symptoms associated with inflammation (Davies et al.,
1984; Needleman et al., 1986; Robinson, 1987). The first step in the
synthesis of PGs is the conversion of arachidonic acid to PGH2, which
is catalyzed by the enzyme cyclooxygenase (COX). Since the early
1970s, the mechanism of action of nonsteroidal anti-inflammatory
drugs has been attributed to the blockade of the production of PGs by
inhibition of COX (Smith and Willis, 1971; Vane, 1971). Two isoforms of COX are known to exist, COX-1 and COX-2, which differ
in their regulation and tissue distribution (Merlie et al., 1988; Fu et al.,
1990; Masferrer et al., 1990; Kujubu et al., 1991; Xie and Chipman,
1991; DeWitt and Smith, 1988). The gene for COX-1 is constitutively
expressed and believed to be responsible for maintaining physiolog-
ical processes in tissues such as the platelet and gastrointestinal (GI)
tract. COX-2 is an inducible enzyme and the expression of the COX-2
gene has been shown to increase in certain inflammatory states. These
data led to the hypothesis that specific COX-2 inhibitors would have
the anti-inflammatory efficacy of traditional nonsteroidal anti-inflammatory drugs but not adverse effects in the GI tract and platelet
(Masferrer et al., 1994; Isakson et al., 1998).
Celecoxib is an inhibitor of COX-2 that has analgesic and antiinflammatory effects in patients with rheumatoid arthritis and no
effect on COX-1 activity at therapeutic plasma concentrations (Penning et al., 1997; Isakson et al., 1998). Celecoxib was recently
approved in the United States in 1998 for relief of the signs and
symptoms of osteoarthritis and rheumatoid arthritis in adults.
Celecoxib is extensively metabolized in humans via a single oxidative pathway (Paulson et al., 2000). The metabolic pathway of
celecoxib in humans involved oxidation of the methyl group of
celecoxib to form a methylhydroxy metabolite, and then to its carboxylic acid. A glucuronide conjugate of the carboxylic acid metabolite is also produced in humans. The objective of the present study
was to characterize the metabolic disposition of celecoxib in rats.
1
Current address: Genentech Inc., South San Francisco, CA.
Abbreviations used are: PG, prostaglandins; COX, cyclooxygenase; LSC,
liquid scintillation counting; MS, mass spectrometry; SPE, solid-phase extraction;
CID, collision-induced dissociation; AUC, area under the plasma concentrationtime curve; Vd, volume of distribution; Cmax, maximum plasma concentration;
NSAIDs, nonsteroidal anti-inflammatory drugs; GI, gastrointestinal; PEG, polyethylene glycol.
2
Materials and Methods
Chemicals. Celecoxib and radiolabeled celecoxib, 4-[5-(4-methylphenyl)3-(trifluoromethyl)-1H-pyrazol-1-yl-5-14 C] were synthesized at Searle
(Skokie, IL) (Fig. 1). The specific activity of the [14C]celecoxib was approximately 44.2 ␮Ci/mg. The methylhydroxy and carboxylic acid metabolites of
514
Send reprint requests to: Susan K. Paulson, Ph.D., G.D. Searle, 4901 Searle
Pkwy., Skokie, IL 60077. E-mail: [email protected]
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 17, 2017
ABSTRACT:
DISPOSITION OF [14C]CELECOXIB IN RATS
515
FIG. 1. Proposed metabolic pathway for celecoxib in rats.
celecoxib (Fig. 1) were synthesized at Searle, and the structures were confirmed by mass spectrometry (MS) and NMR. All other chemicals and reagents
were analytical grade and commercially available.
Radiolabeled celecoxib was synthesized using Friedel-Crafts acylation of
toluene with [1-14C]acetyl chloride to produce 4⬘-methyl[2-14C]acetophenone.
The labeled intermediate was condensed sequentially with trifluoroethylacetate
catalyzed by sodium methoxide followed by 4-sulfonamidophenylhydrazine
catalyzed by dilute hydrochloric acid to provide [14C]celecoxib in an overall
yield of 37% after chromatography and crystallization.
The hydroxymethyl metabolite of celecoxib (M3) was synthesized from
celecoxib by photocatalyzed bromination of the methyl group using Nbromosuccinimide followed by hydrolysis of the bromomethyl products. Sodium borohydride reduction of the hydrolysis mixture converted the mixture of
hydroxymethyl and aldehyde products to the desired compound. Oxidation of
the hydroxymethyl metabolite with Jone’s chromic acid in acetone afforded the
carboxylic acid metabolite of celecoxib (M2). All other chemicals and reagents
were analytical grade and commercially available.
Animal Studies. i.v. pharmacokinetics. Twelve male and twelve female
Sprague-Dawley rats were administered i.v. a single dose of celecoxib at 1
mg/kg in a solution of polyethylene glycol (PEG) 400/saline (2:1, v/v). Blood
(approximately 1.0 ml) was collected from the jugular vein into chilled tubes
containing sodium heparin at 5, 15, and 30 min, and 1, 2, 4, 8, and 24 h after
administration of the dose. Each animal was bled twice. Three blood samples
were collected for each time period. All animals were sacrificed by exsanguination 48 h after dosing. Plasma was prepared by centrifugation of blood. The
plasma was stored at ⫺20°C until analysis for celecoxib concentrations.
Metabolism and excretion studies. Three male and three female SpragueDawley rats were administered i.v. a single dose of [14C]celecoxib at 1 mg/kg
(44.2 ␮Ci/kg). The [14C]celecoxib was administered as a solution in PEG
400/saline (2:1, v/v). After dosing, the animals were housed in individual glass
metabolism cages designed for the separation and collection of urine and feces.
Urine and feces were collected at ⫺18 to 0 h predose and at 24-h intervals for
the five consecutive days after dose administration. Urine was collected into
containers surrounded by dry ice. Feces were frozen immediately on dry ice at
the end of each collection interval. Urine and feces were stored frozen at
approximately ⫺20°C until analyzed for radioactivity and metabolic profile.
Male and female Sprague-Dawley rats (n ⫽ 3/sex/dose) were administered
by oral gavage a single dose of [14C]celecoxib at doses of 20, 80, and 400
mg/kg. At each dose, the animals were administered a radioactive dose of 87
␮Ci/kg. The [14C]celecoxib was administered in a suspension in 0.5% methylcellulose, 0.1% polysorbate 80. After dosing, the animals were housed in
individual glass metabolism cages designed for the separation and collection of
urine and feces. Urine and feces were collected at ⫺18 to 0 h predose and at
24-h intervals for the seven consecutive days after dose administration. Urine
was collected into containers surrounded by dry ice. Feces were frozen
immediately on dry ice at the end of each collection interval. Urine and feces
were stored frozen at approximately ⫺20°C until analyzed for radioactivity
and metabolic profile.
Bile duct-cannulated rat study. Cannulas were surgically inserted into the
bile duct of male Sprague-Dawley rats (n ⫽ 6). A 100 ␮Ci/kg dose of
[14C]celecoxib was administered by oral gavage in a solution of PEG 400/
saline (2:1, v/v) to the rats. Three rats were given radiolabeled celecoxib at a
dose of 5 mg/kg, and the other three rats were administered a 20 mg/kg dose.
Bile was collected from the rats for up to 8 h for the rats given 5 mg/kg and
6.5 h for the rats administered the 20 mg/kg dose. Bile samples were stored at
approximately ⫺20°C until they were analyzed for total radioactivity, celecoxib, and metabolites.
Tissue distribution study. Thirty male Long-Evans rats were administered a
single oral dose of [14C]celecoxib at a dose of 2 mg/kg (18.4 ␮Ci/animal).
Long-Evans rats were used to evaluate the distribution of drug-related radioactivity to both pigmented and nonpigmented skin. The [14C]celecoxib was
administered as a solution in PEG 400/water (2:1, v/v). Three animals each
were sacrificed at 0.5, 1, 3, 8, 24, 48, 72, 96, 144, or 168 h after dose
administration. One additional rat was sacrificed predose to provide control
tissues for analysis. The following tissues and organs were collected at the time
of sacrifice for determination of radioactivity: adrenal glands, aorta, blood,
bone (femur, no marrow), bone marrow (femur), brain, cecum, eye lens, eye
(remainder), fat (brown), fat (s.c.), heart, kidneys, lacrimal glands, large
intestine, liver, lungs, lymph nodes, mesenteric, muscle (skeletal), pancreas,
pituitary, plasma, prostate, red blood cells, skin (nonpigmented), skin (pigmented), small intestine, spinal cord, spleen, stomach, testes, thymus, thyroid,
urinary bladder, and vena cava. Whole samples of aorta, bone marrow, eye
lens, pituitary, thyroid and vena cava were analyzed. Adrenal glands, cecum,
eye, lacrimal glands, lymph nodes, prostate, spinal cord, and urinary bladder
were split into two aliquots and analyzed. Fat samples were minced and
allowed to extract into scintillation cocktail before analysis. Brain, kidney,
large intestine, liver, lungs, muscle, small intestine, stomach, and testes were
homogenized with a probe homogenizer and duplicate aliquots (about 0.2 g)
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 17, 2017
FIG. 2. Plasma concentrations of celecoxib after i.v. administration of 1 mg/kg
celecoxib to male and female rats.
516
PAULSON ET AL.
TABLE 1
Percentage (mean ⫾ S.D.) of radioactivity excreted in urine and feces of rats administered i.v. [14C]celecoxib at 1 mg/kg
Sex
Male
Female
a
Collection
Period
[14C] Excreted
in Urine
[14C] Excreted
in Feces
Othera
Total [14C]
Excreted
h
%
%
%
0–24
24–48
48–72
72–96
96–120
0–120
0–24
24–48
48–72
72–96
96–120
0–120
8.76 ⫾ 2.34
0.74 ⫾ 0.12
0.08 ⫾ 0.04
0.03 ⫾ 0.01
0.02 ⫾ 0.00
9.63 ⫾ 2.48
6.14 ⫾ 2.00
2.92 ⫾ 0.79
0.99 ⫾ 0.32
0.42 ⫾ 0.21
0.14 ⫾ 0.09
10.6 ⫾ 3.3
71.8 ⫾ 7.5
17.4 ⫾ 4.8
2.24 ⫾ 1.68
0.27 ⫾ 0.15
0.05 ⫾ 0.01
91.7 ⫾ 2.0
26.4 ⫾ 6.9
39.2 ⫾ 6.5
19.7 ⫾ 5.3
4.62 ⫾ 1.02
1.36 ⫾ 0.67
91.3 ⫾ 4.0
80.6 ⫾ 7.0
18.1 ⫾ 4.8
2.32 ⫾ 1.71
0.30 ⫾ 0.16
0.06 ⫾ 0.01
101 ⫾ 1
32.5 ⫾ 7.9
42.2 ⫾ 5.8
20.7 ⫾ 5.3
5.04 ⫾ 1.23
1.50 ⫾ 0.76
102 ⫾ 1
0.14
0.43
Percentage of the dose recovered in cage wipes and washes.
The extracts were combined and evaporated in a heated water bath under a
stream of nitrogen. The residues were reconstituted in 3.0 ml of methanol and
centrifuged at approximately 2000g for 10 min at 4°C. Aliquots of the
reconstituted residues were evaporated in a heated water bath under a stream
of nitrogen, and reconstituted in 15% acetonitrile containing 0.025 M sodium
acetate, pH 4.5, and directly injected onto the HPLC system. Extraction
efficiency were greater than 95%.
HPLC analyses of urine and fecal samples was performed using a Beckman
System Gold autosampler, UV detector, and pump (Beckman Instruments,
Fullerton, CA) equipped with a Novapak C18 column (3.9 ⫻ 150 mm, 5 ␮M;
Waters Associates, Milford, MA), and a Novapak C18 guard column (Waters
Associates). A linear gradient was used from 25% acetonitrile in 0.01 M
sodium phosphate buffer, pH 7.4 (mobile phase A) to 70% acetonitrile in 0.01
M sodium phosphate buffer, pH 7.4 (mobile phase B) over 15 min followed by
a linear gradient from mobile phase B to mobile phase A over 1 min and
equilibration for 4 min at mobile phase A. The flow rate was 1 ml/min. Eluates
from the HPLC column after injection of urine samples and fecal extracts were
mixed with Ready Flow III (Beckman Instruments) at a ratio of 1:3 (v/v) and
analyzed for radioactivity using a Beckman model 171 radioactivity detector
(Beckman Instruments).
Extraction and HPLC Profiling of Bile. Bile was extracted using a C-18
Bond Elut SPE column (size 3 cc; Varian, Harbor City, CA), which was
preconditioned with 6 ml of acetonitrile and 6 ml of methanol. The SPE
columns were rinsed with 6 ml of water and left hydrated before application of
the sample. After application of the sample, the column was washed with 6 ml
of water. The radioactive compounds retained on the column were eluted with
6 ml of acetonitrile followed by 3 ml of methanol. The extracts were evaporated to dryness in a heated water bath under a gentle stream of nitrogen. The
residues were reconstituted in 20% acetonitrile in 0.025 M ammonium acetate,
pH 4.5, before being injected on the HPLC for determination of metabolic
profile.
HPLC analysis of bile extracts was performed a 1050 series autosampler
and pump (Hewlett-Packard, Wilmington, DE) equipped with a Novapak C18
column (3.9 ⫻ 150 mm, 4 ␮M; Waters Instruments, Marlborough, MA). A
linear gradient system was run from acetonitrile/0.025 M ammonium acetate,
pH 4.5 (20:80) to acetonitrile/0.025 M ammonium acetate, pH 4.5 (60:40) over
a 20-min period. Fractions were collected at 5 to 6.5, 6.5 to 7.5, 7.5 to 8.5, and
8.5 to 11.2 min, respectively. The fractions were evaporated to dryness under
a stream of nitrogen in preparation for LC/MS.
MS. LC/MS analysis was carried out using a Perkin-Elmer ISS 200 LC
autosampler (Perkin-Elmer, Norwalk, CT), a 1050 HPLC pump (HewlettPackard, Naperville, IL), and a API-III Plus triple quadrupole mass spectrometer (PE Sciex, Thornhill, Ontario, Canada). The separation was performed on
an Inertsil ODS-3 HPLC column (2 ⫻ 150 mm, 5 ␮M; MetaChem Technologies Inc., Torrance, CA). An isocratic system consisting of 0.1% triethylamine in acetonitrile and water (30:70, v/v) was used. The flow rate of mobile
phase was 100 ␮l/min. The eluent from the HPLC was analyzed by negative
ion ionspray MS. The ionspray interface was set at a voltage of ⫺3700 V and
the nebulizer gas (nitrogen) was set at 50 psi. The nitrogen curtain gas was
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 17, 2017
were analyzed. Bone, heart, pancreas, spleen, and thymus were cut into small
pieces, and the whole sample was analyzed in duplicate. Skin (pigmented and
nonpigmented) were cut into small pieces and the samples were digested in 1
N NaOH at 40°C overnight. The digested skin samples were homogenized with
probe homogenizer, and duplicate aliquots (about 0.2 g) were analyzed.
Organ and Tissue Radioactivity Determinations. All organ and tissue
sample combustions were performed using a Packard model 306 or 307
oxidizer (Packard Instrument Company, Downers Grove, IL). Combusted
samples were analyzed for radioactivity by liquid scintillation counting (LSC)
using a Model 1900TR or 1500 liquid scintillation counter (Packard Instrument
Company) using the external standardization and an instrument-stored quench
curve to determine counting efficiency. Samples were counted for at least 5
min or until 100,000 counts accumulated. Radioanalytical procedures were
validated by analyzing aliquots of control tissues fortified with known amounts
of radioactivity. The mean recovery of radioactivity was 98.2%.
Tissue levels of radioactivity were expressed as microgram equivalents per
gram of tissue and were calculated by dividing the dpm per gram of tissue by
the specific activity of the [14C]celecoxib administered. Pharmacokinetic parameters [time to maximal plasma concentration (Cmax), tmax, ␤, t1/2, area
under the plasma concentration-time curve (AUC)0-⬁] for mean radioactivity
levels in tissues were calculated. The tissue radioactivity concentration-time
curve was plotted and the elimination rate constant (␤) was determined from
the slope of the regression line of the terminal phase. The half-life was
calculated as ln 2/␤. AUC0-t was calculated by trapezoidal summation, and the
AUCt-⬁ was calculated by dividing the concentration at time t by the elimination rate constant. AUC0-⬁ was the sum of AUC0-t and AUCt-⬁.
Urine and Feces Radioactivity Determinations. Urine samples were analyzed for radioactivity by LSC after samples were mixed with Ultima-Gold
scintillation cocktail (Packard Instruments).
Approximately 2 to 3 times the fecal sample weight of ethanol was added.
The resulting mixture was weighed and then homogenized using a probe
homogenizer. Duplicate weighed aliquots (approximately 0.5 g) were combusted in a Packard Oxidizer. The resulting 14CO2 was trapped using CarboSorb (Packard Instruments). Perma-Flour E (Packard Instruments) was added
and samples were analyzed for radioactivity by LSC. The percentage of dose
excreted in urine or feces was calculated as dpm in urine or feces divided by
total dpm dosed ⫻ 100.
Quantitative Metabolic Profiling of Urine and Feces. To determine
metabolic profiles, urine samples were subjected to the same solid-phase
extraction (SPE) procedure as described below for bile and the same HPLC
procedure as was described for feces below.
To determine the distribution of radioactivity in feces, aliquots (approximately 0.2 g) of fecal homogenates were pooled on a percentage of weight
basis and extracted with 15 ml of methanol by end-over-end rotation for 1.5 h
at room temperature. The fecal extracts were centrifuged at approximately
2000g for 10 min at 4°C using a Sorvall RT6000D centrifuge (DuPont,
Wilmington, DE). The supernatant volume was measured, aliquots were taken,
and the radioactivity was determined by LSC. The pellet was resuspended in
15 ml of methanol, vortexed briefly, and then extracted as described above.
DISPOSITION OF [14C]CELECOXIB IN RATS
517
TABLE 2
Percentage of the radioactivity excreted as celecoxib and metabolites in urine and feces of rats administered i.v. [14C]celecoxib at 1 mg/kg
Percentage of Radiolabeled Dose Excreted as
Sex
Collection
Interval
Matrix
Celecoxib
Methylhydroxy
metabolite M5
Carboxylic acid
metabolite M4
0.04
0.57
0.02
1.77
0.04
3.19
0.05
5.18
8.64
83.9
5.97
74.1
h
Male
Female
0–24
0–48
0–24
0–72
Urine
Feces
Urine
Feces
FIG. 3. Representative radiochromatogram of a extracted rat urine sample.
Results
Pharmacokinetics. There was a gender difference in the pharmacokinetics of celecoxib. Celecoxib was eliminated from plasma approximately four times faster in the male (t1/2 ⫽ 3.73 h) than in the
female (t1/2 ⫽ 14.0 h) animal (Fig. 2). The clearances of celecoxib in
the male and female were 7.76 and 1.99 ml/min/kg, respectively. The
Vd of celecoxib in male (2.51 liters/kg) and female (2.42 liters/kg)
animals were similar. The AUC0-⬁ was higher in female (8.38 ␮g/
ml 䡠 h) compared with male (2.15 ␮g/ml 䡠 h) rats.
Metabolism and Excretion Study. i.v. route. The primary route of
excretion of the administered [14C]celecoxib was biliary excretion
and/or intestinal secretion, because 91.7% of the dose for male and
91.3% of the dose for female was recovered in the feces (Table 1).
The percentage of the radioactive dose excreted in urine was 9.63%
for male rat and 10.6% for female rat. However, there was a sexrelated difference in the rate of excretion of the radioactive dose. In
male rats, about 80.6% of the dose was excreted within the first 24 h
compared with 32.5% for their female counterparts during the same
time period. Excretion of the administered radioactivity appeared to
be complete for both sexes by 120 h after dose administration.
[14C]Celecoxib was extensively metabolized after i.v. administration with less then 2% of the dose recovered in urine or feces as parent
drug (Table 2). Representative radiochromatograms of urine and fecal
extracts are in Figs. 3 and 4, respectively. The majority of the urinary
FIG. 4. Representative radiochromatogram of an extracted rat fecal sample.
and fecal radioactivity consisted of the carboxylic acid metabolite of
celecoxib (M2), which accounted for 92.5% of the dose for the male
rat, and 80.0% of the dose for female rat. The remainder of the
radioactive dose was excreted as the hydroxymethyl metabolite of
celecoxib (M3), which accounted for 3.2% of the dose in male rats
and 5.2% of the dose in female rats.
Oral. The cumulative excretion of drug-related radioactivity after
single oral dose administration is given in Table 3. After single oral
doses of [14C]celecoxib ranging from 20 to 400 mg/kg to male rats,
approximately 7% of the dose was recovered in the urine, 88 to 94%
of the dose was recovered in feces, for a total recovery of 95 to 101%.
After single oral doses of [14C]celecoxib ranging from 20 to 400
mg/kg to female rats, approximately 9 to 10% of the dose was
recovered in the urine, approximately 90% of the dose was recovered
in feces, for a total recovery of about 99%.
The radiolabeled components in urine and feces collected after oral
administration of [14C]celecoxib were analyzed for celecoxib and its
metabolites by HPLC (Table 4). The radioactivity excreted in urine
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 17, 2017
adjusted to a constant flow rate of 1.8 liters/min. For collision-induced dissociation (CID) experiments, the collision energy was ⫺30 electron volts and
collision gas thickness was set at 250 ⫻ 1013 molecules/cm2.
Assay for Plasma Celecoxib. Rat plasma (0.3 ml) containing an internal
standard, was treated with 100 ␮l 1.0 N phosphoric acid. The plasma sample
was extracted with a cation exchange/hydrophobic mixed mode SPE column
(Jones Chromatography, Lakewood, CO) preconditioned with 2⫻ 1 ml acetonitrile followed by 2⫻ 1 ml water. The sample was eluted from the SPE
column with 1.0 ml of 0.6% ammonium hydroxide in methanol. The extract
was evaporated under nitrogen, and the sample was dissolved into 200 ␮l
HPLC mobile phase. An aliquot of the sample extract was injected onto a
Novapak TM C18 HPLC column (3.9 ⫻ 150 mm, 4 ␮M; Waters Associates)
using a 3.2 ⫻ 15 mm, 7 ␮M RP-18 New Guard Cartridge (Brownlee Labs,
Inc., Santa Clara, CA). The mobile phase, acetonitrile/0.01 M sodium phosphate buffer (pH 9) (50:50, v/v), was run at 1.0 ml/min. The analyte was
quantified by peak height comparison to the internal standard using a FL2000
fluorescence detector (Thermo Separation Product, Schaumburg, IL) with
excitation at 240 nm and emission at 380 nm. The analyte was compared
against a standard curve of rat plasma fortified at concentrations of 0.01 to 10
␮g celecoxib/ml, prepared as described above.
Pharmacokinetic Calculations. The celecoxib plasma concentration-time
curves after i.v. administration were analyzed by a one compartment model
using NONLIN for final parameter estimates (Metzler et al., 1974). The curve
was described by the equation (Ct ⫽ Ae ⫺ kelt) where Ct is the plasma
concentration at time t, A is the coefficient, and kel is the first order rate
constant for the elimination phase. The volume of distribution (Vd) was
calculated from Vd ⫽ Dose/k 䡠 AUC0-⬁. Clearance (Cl) was calculated from
Cl ⫽ Dose/AUC0-⬁. The half-life (t1/2) was calculated as ln2/kel.
518
PAULSON ET AL.
TABLE 3
Percentage (mean ⫾ S.D.) of radioactivity excreted in urine and feces of rats administered [14C]celecoxib orally at 20, 80, and 400 mg/kg
Sex
Male
Female
a
Dose
Collection
Period
[14C] Excreted in
Urine
[14C] Excreted
in Feces
mg/kg
h
%
%
20
80
400
20
80
400
0–168
0–168
0–168
0–168
0–168
0–168
7.48 ⫾ 1.94
7.00 ⫾ 1.98
7.63 ⫾ 1.19
8.86 ⫾ 1.84
10.3 ⫾ 1.97
9.42 ⫾ 2.61
88.0 ⫾ 3.8
94.3 ⫾ 4.54
90.3 ⫾ 1.06
91.2 ⫾ 2.11
88.3 ⫾ 2.07
88.5 ⫾ 2.93
Othera
Total [14C]
Excreted
0.29
0.18
0.32
0.15
0.43
0.90
95.8 ⫾ 3.58
101 ⫾ 2.43
98.2 ⫾ 0.38
99.9 ⫾ 2.69
98.8 ⫾ 1.37
98.9 ⫾ 0.90
%
Percentage of the dose recovered in cage wipes and washes.
TABLE 4
Percentage of the radioactivity excreted as celecoxib and metabolites in urine and feces of rats administered [14C]celecoxib orally at 20, 80, and 400 mg/kg
% of the Radiolabeled Dose Excreted as
Sex
Dose
Collection
Interval
h
20
Male
80
Male
400
0–24
0–72
0–24
0–72
0–24
0–72
0–24
0–72
0–24
0–72
0–24
0–72
Female
20
Female
80
Female
400
Urine
Feces
Urine
Feces
Urine
Feces
Urine
Feces
Urine
Feces
Urine
Feces
Celecoxib
Methylhydroxy
metabolite
(M3)
Carboxylic acid
metabolite
(M2)
ND
31.9
ND
47.5
ND
65.6
ND
26.8
ND
50.0
ND
77.0
ND
1.31
ND
1.05
ND
0.33
ND
0.73
ND
1.09
ND
ND
4.5
48.7
2.4
31.6
1.1
12.2
5.2
43.0
2.2
26.3
0.8
9.6
ND, value is below the limit of detection of the assay.
was primarily the carboxylic acid metabolite of celecoxib (M2). No
unchanged drug was excreted in the urine. The excretion of celecoxib
and its metabolites was dose-dependent. The amount of celecoxib
excreted in the feces increased with dose, and the amount of the
carboxylic acid metabolite of celecoxib excreted in the feces decreased with increasing dose.
Tissue Distribution Study. After oral administration of 2 mg/kg
[14C]celecoxib to male Long-Evans rats, radioactivity was shown to be
distributed to all 35 tissues examined (Table 5, Fig. 5). Maximum levels
of radioactivity were seen in the majority of tissues by 1 h, with the
exception of large intestine and cecum, where peak radioactivity was
observed at 8 h. The GI tract tissue exhibited the highest exposures. The
mean Cmax values for radioactivity in stomach, small intestine, large
intestine, and cecum were 21.0, 3.97, 4.17, and 10.7 ␮g equivalents/g,
respectively. Other tissues with high exposure were liver, red blood cell,
adrenal glands, lacrimal glands, and bone marrow. The concentrations of
radioactivity in pigmented and nonpigmented skin were similar and
decreased at similar rates, indicating no irreversible or extensive binding
of celecoxib and/or it metabolites to melanin. The tissues with the lowest
exposure were eye lens (0.025 ␮g equivalent/g) and bone (0.620 ␮g
equivalent/g). By 96 h post dose, concentrations of radioactivity in most
tissues were below the limit of detection.
Identification of Biliary Metabolites. The HPLC chromatogram
of bile extract contained four peaks containing radioactivity eluting at
5 to 6.5, 6.5 to 7.5, 7.5 to 8.5, and 8.5 to 11.2 min that were further
analyzed by LC/MS (Fig. 6). A summary of the mass spectral data are
given in Table 6.
The peak eluting from 8.5 to 11.2 min (M2) had the same HPLC
retention time as that of the carboxyl acid metabolite of celecoxib. The
deprotonated molecular ion (M⫺H)⫺ of M2 at m/z 410 in its negative
ion mass spectrum, 30 Da higher than the parent compound celecoxib,
suggested that M2 was a carboxylic acid metabolite of celecoxib. The
CID spectrum of m/z 410 generated a series of product ions at m/z 366,
302, 282, 262, 233, 179, and 159, which were similar to those of an
authentic standard of the carboxylic acid metabolite of celecoxib. The
sequential losses of 44 (CO2), 64 (SO2), 20 (HF), and 20 (HF) daltons
from m/z 410 generated the product ions at m/z 366, 302, 282, and
262, respectively. The product ion at m/z 233 was generated by a loss
of a CF3 from m/z 302. Based on these data, M2 was identified as a
carboxylic acid metabolite of celecoxib.
The LC/MS chromatogram of the fraction eluting from 5 to 6.5 min
(M1) contained two peaks. The negative ion ionspray mass spectra of the
two peaks showed the same deprotonated molecular ion (M⫺H)⫺ at m/z
586, which was 176 Da higher than that of M2, suggesting that they are
glucuronide conjugates of the carboxylic acid metabolite of celecoxib.
The CID spectrum of m/z 586 generated a series of product ions at m/z
410, 366, 302, 282, and 113. The product ions at m/z 410, 366, 302, and
282 were similar to those of M2, indicating it was related to M2. The
sequential losses of 176 (dehydroglucuronic acid), 44 (CO2) daltons from
m/z 586 in its CID spectrum suggested that it was a glucuronide conjugate
of M2 with the conjugation occurring at the carboxyl acid group. Therefore, based on the MS data, the peak radioactivity eluting at 5 to 6.5 min
contained two positional isomers of the glucuronide conjugate of the
carboxylic acid metabolite of celecoxib (M1) with the conjugation occurring at the carboxylic acid group.
The LC/MS chromatogram of the 6.5- to 7.5-min fraction contained
two peaks. The negative ion ionspray mass spectra of the two peaks
showed deprotonated ions (M⫺H)⫺ at m/z 572 and 586, which were
consistent with the glucuronide conjugate of the hydroxymethyl metabolite of celecoxib (M5) and the glucuronide conjugate of the
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 17, 2017
mg/kg
Male
Matrix
DISPOSITION OF [14C]CELECOXIB IN RATS
519
TABLE 5
Pharmacokinetic parameters of radioactivity in male rats after a single oral dose of 2 mg/kg [14C]celecoxib
[14C] at 96 h
tmax
␮g Eq./g
␮g Eq./g
h
Adrenal glands
Aorta
Blood
Bone (femur, no marrow)
Bone marrow (femur)
Brain
Cecum
Eye lens
Eye (remainder)
Fat (brown)
Fat (subcutaneous)
Heart
Kidneys
Lacrimal glands
Large intestine
Liver
Lungs
Lymph nodes (mesenteric)
Muscle (skeletal)
Pancreas
Pituitary
Plasma
Prostate
Red Blood Cells
Skin (nonpigmented)
Skin (pigmented)
Small intestine
Spinal cord
Spleen
Stomacha
3.31
1.17
4.18
0.62
2.99
1.03
10.7
0.025
0.665
2.32
2.58
2.23
2.39
3.24
4.17
6.28
2.23
1.51
0.775
2.42
1.70
0.966
1.24
5.70
0.684
0.661
3.97
1.03
2.06
21.0
ND
ND
ND
0.002
ND
ND
0.004
0.005
ND
ND
ND
0.001
ND
ND
ND
0.001
0.003
0.005
ND
ND
ND
⬍0.0005
ND
ND
ND
0.002
ND
0.002
ND
ND
1
1
1
1
1
1
8
3
1
1
1
1
1
3
8
1
1
3
1
1
1
1
1
1
1
3
0.5
3
1
0.5
0.744
0.918
1.34
0.751
1.07
ND
ND
ND
ND
ND
Testes
Thymus
Thyroid
Urinary bladder
Vena cava
3
3
1
3
3
␤ h⫺1
0.172
0.151
0.165
0.098
0.161
0.180
0.007
0.024
0.159
0.155
0.003
0.180
0.171
0.143
0.024
0.011
0.174
0.173
0.201
0.137
0.113
0.197
0.140
0.012
0.133
0.136
0.011
0.173
0.155
0.161
(0.002)
0.178
0.187
0.176
0.196
0.166
t1/2
AUC0–⬁
h
␮g Eq./gh/g
4.0
4.6
4.2
7.0
4.3
3.8
95.9
28.8
4.4
4.5
234
3.9
4.1
4.8
28.9
65.7
4.0
4.0
3.5
5.1
6.1
3.5
5.0
58.0
5.2
5.1
66.0
4.0
4.5
4.28
(433)
3.9
3.7
3.9
3.5
4.2
29.7
83.48
41.9
7.57
34.1
9.97
184
1.21
6.61
21.8
24.4
2039
22.4
35.1
72.4
50.1
20.3
16.2
7.25
23.2
16.1
7.27
10.0
60.6
7.45
7.27
43.2
12.0
20.4
54.1
(54.8)
8.17
9.91
13.9
7.07
15.7
ND, value is below the limit of detection of the assay.
a
Radioactivity levels were below background in all animals by 72 h post dose. One animal had radioactivity levels that were just above background at 144 h post dose. Value in parentheses
represents analysis conducted including the data from the 144-h sample in that one animal.
carboxylic acid metabolite of celecoxib (M1), respectively. The CID
spectrum of m/z 572 gave a series of product ions at 510, 396, 380,
316, 302, and 113. The sequential losses of 62 (CH2OHCH2OH), 176
(dehydroglucuronic acid), and 16 (O) daltons from m/z 572 to yield
the product ions at m/z 510, 396, and 380 in its CID spectrum
suggested that it was a glucuronide conjugate of the hydroxymethyl
metabolite of celecoxib (M5) with the conjugation occurring at the
hydroxymethyl group. The product ions at m/z 316 and 302 were
generated from the losses of 80 (SO2 ⫹ O) and 94 (OCH2 ⫹ SO2)
daltons from m/z 396. The CID spectrum of m/z 586 was not obtained.
However, because it had the same molecular weight as that of M1, it
is likely to be a positional isomer of M1, which was formed via an
acyl migration mechanism. Based on these data, the 6.5- to 7.5-min
fraction was confirmed as a mixture of the glucuronide conjugates of
the hydroxymethyl and the carboxylic acid metabolites of celecoxib.
The 8.5- to 11.2-min fraction had the same deprotonated molecular
ion (M⫺H)⫺ at m/z 586. The CID spectra of m/z 586 were very
similar to that of M1. These results demonstrated that the 8.5- to
11.2-min fraction contained a positional isomer of M1, a glucuronide
conjugate of the carboxylic acid metabolite of celecoxib with the
glucuronide attached at the carboxylic acid group.
Discussion
There is a gender difference in the clearance of celecoxib in rats with
elimination of parent drug from plasma occurring faster in male com-
pared with female animals. As a result of the gender difference in
celecoxib pharmacokinetics, female rats achieve a higher exposure to the
drug than the males when administered the same dose. Sex differences in
pharmacokinetics of xenobiotics that undergo metabolism are not unusual
for the rat and have been, in part, attributed to sex-specific expression of
rat CYP2C and CYP3A genes (Waxman et al., 1985). There were no
gender differences in celecoxib pharmacokinetics in healthy subjects or in
patients as determined either by classical or population pharmacokinetic
methods (Dr. Aziz Karim, personnel communication).
After i.v. or oral administration, total recovery of the radioactive dose
of celecoxib was about 100% for both male and female rats. The majority
of the i.v. administered dose (approximately 90%) was recovered in the
feces for both sexes, indicating that the primary route of excretion was
biliary. Approximately 10% of the i.v. dose represented renal elimination
for both sexes. The excretion of the radioactivity after i.v. administration
was also faster in male than in female animals. Approximately 81% of the
dose was excreted in the first 24 h for the male rats, whereas only about
33% was excreted in the same time period for the female animals. In male
rats, 98% of the dose was excreted by 48 h after dosing, whereas
excretion of the same percentage took 96 h for female rats. After oral
administration, the majority of the dose was excreted primarily in the
feces (about 90%) with the remaining percentage was excreted in the
urine. The pattern of excretion of radioactivity after oral administration
was the same over the 40-fold dose range tested.
Both male and female rats extensively metabolized celecoxib after
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 17, 2017
Cmax
Matrix
520
PAULSON ET AL.
TABLE 6
Ionspray mass spectral data of celecoxib metabolites in rats
HPLRC
Elution Time
Metabolite
[M⫺H]⫺
5–6.5
M1
586
6.5–7.5
M1
M5
586
572
7.5–8.5
M1
M1
586
586
8.5–11.2
M2
410
min
FIG. 5. Tissue to plasma ratio of radioactivity at 3 h after oral administration of
2 mg/kg [14C]celecoxib to male rats.
an i.v. dose, with less than 2% of the radioactive dose excreted as
unchanged drug by either sex. after After oral administration, the
majority of the radioactivity is excreted as celecoxib and its carboxylic acid metabolite in the feces, with the amount of celecoxib
excreted increasing with dose. The celecoxib excreted in the feces at
the higher oral doses likely represents unabsorbed drug.
The metabolic pathway for celecoxib involved oxidation of the aromatic methyl group to form a hydroxymethyl metabolite, which underwent additional oxidation to a carboxylic acid metabolite (Fig. 1). Glucuronide conjugates of both the hydroxymethyl and the carboxylic acid
metabolites of celecoxib were minor metabolites found only in bile. Four
glucuronide conjugates of the carboxylic acid metabolite were produced.
One of the biliary acyl glucuronide metabolites was a 1-O-glucuronide.
The position of the glucuronide on the other acyl glucuronides was not
established. It is possible that the other three glucuronide conjugates of
the carboxylic acid metabolite are positional isomers formed as the result
of acyl migration (Spahn-Langguth and Benet, 1992). No glucuronide
CID Mass Spectral Data
m/z (%)
410 (25), M-H-C6H8O6;
366 (70), M-H-C6H8O6-CO2;
302 (22), M-H-C6H8O6-CO2-SO2;
282 (5), M-H-C6H8O6-CO2-SO2-HF;
113 (100), C6H7O6-CH2OHCH2OH.
Not acquired
510 (50), M-H-CH2OHCH2OH;
396 (100), M-H-C6H8O6;
380 (74), M-H-C6H8O6-O;
316 (20), M-H-C6H8O6-O-SO2;
302 (20), M-H-C6H8O6-O-CH2-SO2;
113 (100), C6H7O6-CH2OHCH2OH.
Not acquired
410 (28), M-H-C6H8O6;
366 (60), M-H-C6H8O6-CO2;
302 (25), M-H-C6H8O6-CO2-SO2;
282 (5), M-H-C6H8O6-CO2-SO2-HF;
113 (100), C6H7O6-CH2OHCH2OH;
366 (25), M-H-CO2;
302 (80), M-H-CO2-SO2;
282 (65), M-H-CO2-SO2-HF;
262 (100), M-H-CO2-SO2-2HF;
233 (65), M-H-CO2-SO2-CF3;
179 (48), M-H-CO2-SO2-C6H4CHCH2-HF;
159 (29), M-H-CO2-SO2-C6H4CHCH2-2HF.
conjugates were present in feces, suggesting that hydrolysis of the acyl
and ether glucuronide metabolites occurred in the GI tract. The carboxylic acid was the major metabolite of celecoxib excreted in urine and
feces of rats, representing 80% of the dose excreted by female animals
and 92% of the dose excreted by male animals. A minor portion of the
dose was excreted as the hydroxymethyl celecoxib in the feces of male
(5%) and female (3%) rats.
Celecoxib is also extensively metabolized in humans. The metabolism
of celecoxib in humans is similar to rat occurring primarily through a
single metabolic pathway with the formation of the methylhydroxy (M3)
and carboxylic acid (M2) metabolites (Paulson et al., 2000). M2 is also
the major metabolite of celecoxib excreted in humans. The carboxylic
acid glucuronide (M1) is only a minor metabolite in humans, representing
less than 2% of an oral dose excreted. Unlike the rat, the carboxylic acid
glucuronide (M1) is found in human urine and was present only as the
1-O-glucuronide (Paulson et al., 2000).
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 17, 2017
FIG. 6. Representative radiochromatogram of a bile extract.
DISPOSITION OF [14C]CELECOXIB IN RATS
Celecoxib is well distributed to tissues as indicated by most tissue
to plasma ratios greater than one for most of the tissues examined in
the tissue distribution study. The distribution to tissues is rapid with
the maximal concentration occurring in most tissues at about 1 h. The
highest concentrations of radioactivity were found in the GI tract.
Total radioactivity concentrations were below background in most
tissues by 96 h postdose, indicating that there was no retention of drug
and/or metabolites in the animals.
In conclusions, celecoxib is readily distributed to tissues. Celecoxib
is eliminated by a single metabolic pathway, i.e., hydroxylation of the
aromatic methyl group of celecoxib and additional oxidation of the
hydroxymethyl metabolite to a carboxylic acid metabolite. The major
route of elimination of celecoxib is metabolism followed by excretion
of the metabolites in feces (90%), with the remainder of the metabolites excreted by the kidney.
References
Davies P, Bailey PJ, Goldenberg MM and Ford-Hutchinson AW (1984) The role of arachidonic
acid oxygenation products in pain and inflammation. Annu Rev Immunol 2:335–357.
DeWitt DL and Smith WL (1988) Primary structure of prostaglandin G/H synthase from sheep
vesicular gland determined by complimentary DNA sequence. Proc Natl Acad Sci USA
85:1412–1416.
Fu J-Y, Masferrer JL, Seibert K, Raz A and Needleman P (1990) The induction and suppression
of prostaglandin H2 synthase (cyclooxygenase) in human monocytes. J Biol Chem 265:16737–
16740.
Isakson P, Zweifel B, Masferrer J, Koboldt C, Seibert K, Hubbard R, Geis S and Needleman P
(1998) Specific COX-2 inhibitors: From bench to bedside, in Selective COX-2 Inhibitors:
Pharmacology, Clinical Effects and Therapeutic Potential (Vane J and Botting J eds) pp.
127–133, Kluwer Academic Publishers and William Harvey Press, London, UK.
Kujubu DA, Fletcher BS, Varnum BC, Lim RW and Herschman HR (1991) TIS10, a phorbol
ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin
synthase/cyclooxygenase homologue. J Biol Chem 266:12866 –12872.
Masferrer JL, Zweifel BS, Seibert K and Needleman P (1990) Selective regulation of cellular
cyclooxygenase by dexamethasone and endotoxin in mice. J Clin Invest 86:1375–1379.
Masferrer JL, Zweifel BS, Manning PT, Hauser DS, Leahy KM, Smith WG, Isakson PC and
Seibert K (1994) Selective inhibition of inducible cyclooxygenase 2 in vivo is antiinflammatory and nonulcerogenic. Proc Natl Acad Sci USA 91:3228 –3232.
Merlie JP, Fagan D, Mudd J and Needleman P (1988) Isolation and characterization of sheep
seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase). J Biol Chem 263:
3550 –3553.
Metzler CM, Elfring GK and McEween AJ (1974) A package of computer programs for
pharmacokinetic modeling. Biometrics 30:562–563.
Needleman P, Turk J, Jakschik BA, Morrison AR and Lefkowith JB (1986) Arachidonic acid
metabolism. Annu Rev Biochem 55:69 –102.
Paulson SK, Hribar JD, Liu NWK, Hajdu E, Bible R, Piergies A and Karim A (2000) Metabolism
and excretion of [14C] celecoxib in healthy male volunteers. Drug Metab Dispos 28:308 –314.
Penning TD, Talley JJ, Bertenshaw SR, Carter JD, Collins PW, Docter S, Graneto MJ, Lee
LF, Malecha JW, Miyashiro JM, Rogers RS, Rogier DJ, Yu SS, Anderson GD, Burton EG,
Cogburn JN, Gregory SA, Koboldt CM, Perkins WE, Seibert K, Veenhuizen AW, Zhang
YY and Isakson PC (1997) Synthesis and biological evaluation of the 1,5-diarylpyrazole
class of cyclooxygenase-2 inhibitors: Identification of 4-[5-(4-methylphenyl)-3(trifluoromethyl)-1H-pyrazol-1-yl] bezenesulfonamide (SC-58635, celecoxib). J Med
Chem 40:1347–1365.
Robinson DR (1987) Lipid mediators of inflammation in rheumatic disease, in Clinics of North
America (Zvaifler N ed) pp 385– 405, WB Saunders Company, Philadelphia, PA.
Smith JB and Willis AL (1971) Aspirin selectivity inhibitors prostaglandins production in human
platelets. Nat New Biol 231:235–237.
Spahn-Langguth H and Benet L (1992) Acyl glucuronides revisited: Is the glucuronidation
process a toxification as well as a detoxification mechanism? Drug Metab Rev 241:5– 48.
Vane JR (1971) Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like
drugs. Nat New Biol 231:232–235.
Waxman DJ, Dannan GA and Guengerich FP (1985) Regulation of rat hepatic cytochrome P450:
Age-dependent expression, hormonal imprinting, and xenobiotic inducibility of sex-specific
isoenzymes. Biochemistry 24:4409 – 4417.
Xie W and Chipman JG (1991) Expression of a mitogen-responsive gene encoding prostaglandin
synthase is regulated by mRNA splicing. Proc Natl Acad Sci USA 88:2692–2696.
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 17, 2017
Acknowledgments. Bioanalytical assays for celecoxib were performed by CEDRA Corporation (Austin, TX). Radioactivity measurements were conducted by Covance Labs (Madison, WI). Radiolabeled
compounds were synthesized by Scott Harring Ph.D., at the Radiochemistry Department at G.D. Searle.
521