Introduction

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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 physiological 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 anti-inflammatory 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.

	Materials and Methods

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Chemicals. Celecoxib and radiolabeled celecoxib, 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl-5-¹⁴C] were synthesized at Searle (Skokie, IL) (Fig. 1). The specific activity of the [¹⁴C]celecoxib was approximately 44.2 µCi/mg. The methylhydroxy and carboxylic acid metabolites of 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.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Proposed metabolic pathway for celecoxib in rats.

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 Sprague-Dawley rats were administered i.v. a single dose of [¹⁴C]celecoxib at 1 mg/kg (44.2 µCi/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.

Bile duct-cannulated rat study. Cannulas were surgically inserted into the bile duct of male Sprague-Dawley rats (n = 6). A 100 µCi/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 µCi/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) 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%.

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Urine and Feces Radioactivity Determinations. Urine samples were analyzed for radioactivity by LSC after samples were mixed with Ultima-Gold scintillation cocktail (Packard Instruments).

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.

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.

MS. LC/MS analysis was carried out using a Perkin-Elmer ISS 200 LC autosampler (Perkin-Elmer, Norwalk, CT), a 1050 HPLC pump (Hewlett-Packard, 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 µM; 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 µl/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 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 µl 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 µl HPLC mobile phase. An aliquot of the sample extract was injected onto a Novapak TM C18 HPLC column (3.9 × 150 mm, 4 µM; Waters Associates) using a 3.2 × 15 mm, 7 µM 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 µg 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 (C_t = Ae  k_elt) where C_t is the plasma concentration at time t, A is the coefficient, and k_el is the first order rate constant for the elimination phase. The volume of distribution (V_d) was calculated from V_d = Dose/k · AUC_0-. Clearance (Cl) was calculated from Cl = Dose/AUC_0-. The half-life (t_1/2) was calculated as ln2/k_el.

	Results

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Pharmacokinetics. There was a gender difference in the pharmacokinetics of celecoxib. Celecoxib was eliminated from plasma approximately four times faster in the male (t_1/2 = 3.73 h) than in the female (t_1/2 = 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 V_d of celecoxib in male (2.51 liters/kg) and female (2.42 liters/kg) animals were similar. The AUC_0- was higher in female (8.38 µg/ml · h) compared with male (2.15 µg/ml · h) rats.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Plasma concentrations of celecoxib after i.v. administration of 1 mg/kg celecoxib to male and female 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 sex-related 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.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Representative radiochromatogram of a extracted rat urine sample.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Representative radiochromatogram of an extracted rat fecal sample.

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%.

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 C_max values for radioactivity in stomach, small intestine, large intestine, and cecum were 21.0, 3.97, 4.17, and 10.7 µg 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 µg equivalent/g) and bone (0.620 µg equivalent/g). By 96 h post dose, concentrations of radioactivity in most tissues were below the limit of detection.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Tissue to plasma ratio of radioactivity at 3 h after oral administration of 2 mg/kg [14C]celecoxib to male rats.

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.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Representative radiochromatogram of a bile extract.

	Discussion

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There is a gender difference in the clearance of celecoxib in rats with elimination of parent drug from plasma occurring faster in male compared 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 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 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).

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.