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DRUG METABOLISM AND DISPOSITION
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
DMD 28:1244–1254, 2000
Vol. 28, No. 10
51/856965
Printed in U.S.A.
THE ABSORPTION, DISTRIBUTION, METABOLISM AND EXCRETION OF ROFECOXIB, A
POTENT AND SELECTIVE CYCLOOXYGENASE-2 INHIBITOR, IN RATS AND DOGS
RITA A. HALPIN, LESLIE A. GEER, KANYIN E. ZHANG,1 TINA M. MARKS, DENNIS C. DEAN, ALLEN N. JONES,
DAVID MELILLO, GEORGE DOSS, AND KAMLESH P. VYAS
Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania and Rahway, New Jersey
(Received March 16, 2000; accepted July 20, 2000)
This paper is available online at http://www.dmd.org
ABSTRACT:
Absorption, distribution, metabolism, and excretion studies were
conducted in rats and dogs with rofecoxib (VIOXX, MK-0966), a
potent and highly selective inhibitor of cyclooxygenase-2 (COX-2).
In rats, the nonexponential decay during the terminal phase (4- to
10-h time interval) of rofecoxib plasma concentration versus time
curves after i.v. or oral administration of [14C]rofecoxib precluded
accurate determinations of half-life, AUC0–ⴥ (area under the
plasma concentration versus time curve extrapolated to infinity),
and hence, bioavailability. After i.v. administration of [14C]rofecoxib
to dogs, plasma clearance, volume of distribution at steady state,
and elimination half-life values of rofecoxib were 3.6 ml/min/kg, 1.0
l/kg, and 2.6 h, respectively. Oral absorption (5 mg/kg) was rapid in
both species with Cmax occurring by 0.5 h (rats) and 1.5 h (dogs).
Bioavailability in dogs was 26%. Systemic exposure increased with
increasing dosage in rats and dogs after i.v. (1, 2, and 4 mg/kg), or
oral (2, 5, and 10 mg/kg) administration, except in rats where no
additional increase was observed between the 5 and 10 mg/kg
doses. Radioactivity distributed rapidly to tissues, with the highest
concentrations of the i.v. dose observed in most tissues by 5
min and by 30 min in liver, skin, fat, prostate, and bladder. Excretion occurred primarily by the biliary route in rats and dogs, except
after i.v. administration of [14C]rofecoxib to dogs, where excretion
was divided between biliary and renal routes. Metabolism of rofecoxib was extensive. 5-Hydroxyrofecoxib-O-␤-D-glucuronide was
the major metabolite excreted by rats in urine and bile. 5-Hydroxyrofecoxib, rofecoxib-3ⴕ,4ⴕ-dihydrodiol, and 4ⴕ-hydroxyrofecoxib
sulfate were less abundant, whereas cis- and trans-3,4-dihydrorofecoxib were minor. Major metabolites in dog were 5-hydroxyrofecoxib-O-␤-D-glucuronide (urine), trans-3,4-dihydro-rofecoxib
(urine), and 5-hydroxyrofecoxib (bile).
Aspirin has been marketed since the latter part of the nineteenth
century as an anti-inflammatory agent, but its mechanism of action
remained obscure until 1971 when Sir John Vane proposed that
aspirin served as an inhibitor of prostaglandin (PG)2 synthesis (Vane,
1971). This inhibition occurred by an irreversible acetylation of cyclooxygenase (COX), the key enzyme involved in PG biosynthesis
(Roth et al., 1975).
COX [E.C. 1.14.99], also known as prostaglandin endoperoxide
synthase (PGHS), is a bifunctional, membrane-bound hemeprotein
that catalyzes the addition of two molecules of molecular oxygen to
arachidonic acid to form PGG2, followed by reduction of the endoperoxide moiety to give PGH2 (Smith and Marnett, 1991; Smith and
DeWitt, 1996). The enzyme exists as two isoforms: a constitutive
form, designated as COX-1, and an inducible form, referred to as
COX-2 (Fu et al., 1990; Masferrer et al., 1990, 1992). The constitutive
COX-1 is expressed in most tissues, generating PGs that function to
protect the gastric mucosal lining, regulate blood flow to the kidney,
and support platelet aggregation. In contrast, COX-2 is constitutively
expressed in limited healthy tissues, e.g., kidney. However, when
induced, the PGs produced by this isoform are associated with the
pain and swelling of inflammation. Expression of COX-2 is stimulated by growth factors, cytokines, phorbol esters, and mitogens, but
is inhibited by steroidal anti-inflammatories, e.g., glucocorticoids,
which have little or no effect on COX-1 levels (reviewed by Vane and
Botting, 1996; Donnelly and Hawkey, 1997; Jouzeau et al., 1997).
Currently marketed NSAIDs generally are nonselective COX-1/
COX-2 inhibitors whose therapeutic utility is due to inhibition of COX-2,
but whose side effect profile (i.e., gastrointestinal irritation and/or bleeding) results from inhibition of COX-1. Clearly, the development of highly
selective inhibitors of COX-2 are an attractive target, because such agents
retain the anti-inflammatory, analgesic, and antipyretic properties of current
NSAIDs, while reducing the risk of gastrointestinal side effects (Vane and
Botting, 1996; Donnelly and Hawkey, 1997; Jouzeau et al., 1997).
Rofecoxib
(3-phenyl-4-[4-(methysulfonyl)phenyl]-2-(5H)-furanone, VIOXX,3 MK-0966; Fig. 1), a potent and highly selective
inhibitor of COX-2 (Prasit et al., 1999), has been approved by the
Food and Drug Administration for the treatment of osteoarthritis and
pain. The high selectivity of rofecoxib as an inhibitor of COX-2 has
been examined in several in vitro preparations and in vivo rodent
models (Chan et al., 1999). In clinical trials, rofecoxib showed effec-
1
Current address: Agouron Pharmaceuticals, Inc., 4215 Sorrento Valley Blvd.,
San Diego CA, 92121.
2
Abbreviations used are: PG, prostaglandin; COX, cyclooxygenase; PGHS,
prostaglandin endoperoxide synthase; TFA, trifluoroacetic acid; DMSO, dimethyl
sulfoxide.
Send reprint requests to: Kamlesh P. Vyas, Ph.D., Merck Research Laboratories, Merck & Co., Inc., WP75A-203, West Point, PA 19486. E-mail: kamlesh
[email protected]
3
1244
VIOXX is a registered trademark of Merck & Co., Inc.
ADME OF ROFECOXIB IN RAT AND DOG
FIG. 1. Chemical structures of rofecoxib and internal standard. The asterisk
indicates the location of the 14C radiolabel.
tive analgesia in a dental pain model (Ehrich et al., 1999) and effective
antipyretic activity in monkeys and humans (Schwartz et al., 1999).
The objectives of the present investigation were to determine the
absorption, distribution, metabolism, and excretion (ADME) of rofecoxib in rats and dogs, the two species used in the toxicological
evaluation of this compound.
Materials and Methods
Chemicals and Dosing Solutions. Chemicals. [Furanone-4-14C]rofecoxib
was prepared as outlined in Fig. 2. All chemicals for the synthesis were
obtained from Aldrich Chemical Co. (Milwaukee, WI), except that [14C]sodium acetate was obtained from ChemSyn Laboratories (Lenexa, KS). The
label was introduced by Friedel-Crafts acylation of thioanisole (1) using
[14C]acetyl chloride (prepared from [14C]sodium acetate) to give acetophenone
2. Oxidation of 2 was achieved using monoperoxyphthalic acid magnesium salt
to afford sulfone 3, which was brominated via formation of the enol triflate.
The resulting bromosulfone 4 was esterified using phenylacetic acid to yield
ketoester 5. Cyclization was effected by treatment with diisopropylethylamine.
The crude product was purified by preparative HPLC and crystallized from
ethyl acetate. The overall radiochemical yield for the synthesis was 30% from
[14C]sodium acetate. Structural identification of the tracer was confirmed by
chromatographic coelution with unlabeled standard. After purification, chemical and radiochemical purity were at least 99% (as determined by HPLC
analysis), and the specific activity of the stock material was 12.62 mCi/mmol
(40.14 ␮Ci/mg). Unlabeled rofecoxib was synthesized by the Process Research
group (Merck and Company, Inc., Rahway, NJ), whereas rofecoxib metabolites and the internal standard (the 4⬘-methylphenyl analog of rofecoxib, Fig. 1)
were synthesized by Medicinal Chemistry (Merck Frosst Canada, Kirkland,
Quebec). Due to the sensitivity of rofecoxib to natural light, the parent
compound, the internal standard, and all biological samples were handled
under yellow lights.
Other chemicals. ␤-Glucuronidase (Helix pomatia, type H-5) was obtained
from Sigma Chemical Co. (St. Louis, MO), trifluoroacetic acid (TFA) was
purchased from Aldrich Chemical Co., and dimethyl sulfoxide (DMSO),
ammonium hydroxide (NH4OH), sodium acetate, acetonitrile (CH3CN), and
methanol (CH3OH) were obtained from Fisher (Pittsburgh, PA).
Dosing solutions. [14C]Rofecoxib or unlabeled rofecoxib was administered
to rats and dogs for absorption, metabolism, excretion, and pharmacokinetic
studies, and to rats for the tissue distribution study.
[14C]Rofecoxib was used without dilution of the specific activity for i.v.
administration to rats. The weighed material was dissolved directly in DMSO
before dosing. The specific activity of [14C]rofecoxib was diluted with unlabeled rofecoxib for i.v. administration to dogs and oral administration to rats
and dogs. For these solutions, the radiolabeled and unlabeled compounds were
combined and dissolved in organic solvent (either toluene or methylene chlo-
1245
ride). After evaporation of the solvent to dryness (N2, 44°C), the residue was either
dissolved in DMSO for i.v. dosing or suspended in 0.5% methylcellulose (w/v) for
oral dosing. The concentrations were 4 mg/ml (rats) or 10 mg/ml (dogs) for the i.v.
solutions or 1 mg/ml for the oral dosing suspensions. The specific activities ranged
from 0.56 to 40.1 ␮Ci/mg, and each animal received approximately 20 to 25 ␮Ci.
For those studies requiring only unlabeled rofecoxib, the i.v. solutions were
prepared in DMSO, and the oral solutions were prepared as 0.5% methylcellulose
suspensions at the required concentrations.
Animal Studies. Male Sprague-Dawley rats weighing approximately 225 to
350 g were obtained from Taconic Farms (Germantown, NY). Male beagle dogs
weighing approximately 8 to 10 kg were obtained from Marshall Farms (North
Rose, NY). All procedures were approved by the Merck Research Laboratories
Institutional Animal Care and Use Committee. Rats and dogs were housed in
temperature-controlled rooms with a 12-h light/dark cycle. Animals were surgically prepared with a cannula in the jugular vein and/or bile duct for sample
collection. For all studies described below, except the tissue distribution study in
rats and the food effect study in dogs, the animals were deprived of food 14 to 18 h
before dosing but were allowed free access to water. Food was provided to rats and
dogs 6 to 8 h after dosing. Blood samples were collected in heparinized syringes,
and plasma subsequently was obtained by centrifugation. With the exception of
blood samples, which were stored at 4°C, all biological samples were stored at
⫺20°C until analyzed. Finally, all biological samples were centrifuged before
analysis to remove particulate matter.
Disposition and metabolism of [14C]rofecoxib in rats and dogs. [14C]Rofecoxib was administered i.v. to rats (n ⫽ 4) by tail vein injection (0.5 ml/kg)
at a dose of 2 mg/kg. The oral dose (5 mg/kg) was administered to a second
group of animals (n ⫽ 4) by oral gavage (5 ml/kg). In dogs (n ⫽ 4), the i.v.
dose (2 mg/kg) of [14C]rofecoxib was administered as a 10-min infusion (0.2
ml/kg) via either the cephalic or saphenous veins. After a 3-week wash-out
period, the same dogs received an oral dose (5 mg/kg) of [14C]rofecoxib by
gavage. In both studies, blood was collected at time points of 0, 0.08 (i.v. only),
0.17 (i.v. only), 0.25, 0.5, 1, 2, 4, 6, 8, 10, 24, 48, 72, and 96 h. Urine and feces
were collected at time intervals of 0 to 8, 8 to 24, 24 to 48, 48 to 72, and 72
to 96 h. The urine samples from 0 to 8 and 8 to 24 h were collected over dry
ice, and the remaining samples were collected at room temperature.
Biliary excretion of [14C]rofecoxib in rats and dogs. [14C]Rofecoxib was
administered to bile duct-cannulated rats (n ⫽ 3– 4 per group) and dogs (n ⫽ 1 per
group) as i.v. (2 mg/kg) or oral (5 mg/kg) doses. Bile samples were collected on
dry ice at hourly intervals for 6 h (rat) or 8 h (dog) and then until 24 h.
Dose dependence of rofecoxib in rats and dogs. Rats (n ⫽ 4) and dogs (n ⫽
4) received i.v. doses of unlabeled rofecoxib of 1, 2, and 4 mg/kg, and oral
doses of 2, 5, and 10 mg/kg. Blood samples were collected to 48 h.
Effect of food on absorption of rofecoxib in dogs. The effect of food on the
absorption kinetics of rofecoxib was studied in dogs (n ⫽ 4) at an oral dose of
5 mg/kg. The fasted study was carried out as described above. After a 1-week
wash-out period, dogs were fed 1 h before and 10 h after a second oral dose.
Blood was collected at specific time points to 48 h.
Tissue distribution of [14C]rofecoxib in rats. The tissue distribution study
was performed by Covance Laboratories Inc. (Madison, WI). [14C]Rofecoxib
was administered as an i.v. dose (2 mg/kg) to rats (n ⫽ 12) by tail vein
injection. Groups of three animals were sacrificed by exsanguination under
halothane anesthesia at 5 min, and at 0.5, 2, and 24 h postdose. Blood (4 –10
ml) was collected via cardiac puncture at the time of sacrifice. Specified tissues
were excised, rinsed with water, blotted dry, weighed, and placed on ice until
transferred to a freezer.
Metabolism of rofecoxib in rats after administration of a high oral dose.
Rats (n ⫽ 3 per group) received either unlabeled or radiolabeled rofecoxib
(100 mg/kg, 5 ml/kg, 0.5% methylcellulose) by oral gavage. Urine was
collected over dry ice for 24 h.
Radioactivity Measurements. Total radioactivity in plasma, bile, and urine
was determined directly by adding aliquots of sample (0.025–1.0 ml) to
polyethylene vials containing scintillation cocktail (15 ml, Ready Safe, Beckman Instruments, Fullerton, CA). Fecal samples were homogenized in 4 to 5
volumes of water using a homogenizer (Omni International, Waterbury, CT).
Aliquots of the homogenate (⬃1 g) were weighed into tared combustion cups,
and, after drying overnight, the samples were combusted in a Packard Tricarb
sample oxidizer (model B306; Packard Instruments, Downers Grove, IL). The
resultant carbon dioxide was trapped in a Carbosorb/Permafluor mixture
1246
HALPIN ET AL.
FIG. 2. Radiochemical synthesis of [14C]rofecoxib.
The asterisks denote the location of the
14
C radiolabel.
(Packard Instruments). Radioactivity in all samples was determined with a
Beckman LS5000CE liquid scintillation spectrometer. In the tissue distribution
study, all tissue samples, with the exception of skin samples, were homogenized and subjected to combustion (model 306 or 307 sample oxidizer;
Packard Instruments) before liquid scintillation counting as described above
for the fecal samples. Skin samples were digested in 1 N sodium hydroxide at
40°C, homogenized, and counted. Plasma from this study was analyzed directly by liquid scintillation counting (model 1900TR; Packard Instruments).
Quench correction was automatic and employed the H-number method.
Rofecoxib Assay. Concentrations of rofecoxib in rat and dog plasma from
all the kinetics studies were determined by HPLC with fluorescence detection
of a product resulting from postcolumn photochemical activation (Woolf et al.,
1999). Briefly, an aliquot of plasma (0.15 ml) was added to 0.85 ml of buffer
(0.1 M sodium acetate, pH 5), followed by the addition of internal standard (25
␮l of 150 ng/ml solution in CH3CN). Liquid-liquid extraction was carried out
with a 50:50 mixture of methylene chloride and hexane (4 ml). After centrifugation (3000g, 5 min), the tubes were placed in an acetone/dry ice bath to
freeze the aqueous layer. The organic phase was isolated and evaporated to
dryness under a flow of nitrogen and mild heat (44°C, Reacti-Therm heating
module). The residue was reconstituted in HPLC mobile phase (35% aqueous
CH3CN) immediately before analysis.
HPLC analysis was carried out on a Hewlett-Packard 1090 instrument
equipped with a photochemical reactor (Aura Industries, Staten Island, NY)
and fluorescence detector (Perkin-Elmer LC 240, Norwalk, CT). The mobile
phase was delivered isocratically at a flow rate of 1 ml/min. Samples (100 ␮l)
were injected onto an Upchurch guard column (Uptight C-135B with Perisorb
C18 packing; Thompson Instruments, Clearbrook, VA) and chromatographed
on a Base Deactivated Silica Hypersil analytical column (4.6 ⫻ 100 mm, 5
␮m; Keystone, Bellefonte, PA) at ambient temperature. Fluorescence detection
was achieved by excitation at ␭ ⫽ 250 nm and detection at ␭ ⫽ 375 nm. The
run time was 15 min. The assay demonstrated good linearity and reproducibility over the plasma concentration range of 2.5 to 250 ng/ml ([14C]rofecoxib
studies) or 1.0 to 250 ng/ml (unlabeled rofecoxib studies). The assay accuracy,
defined as the mean percentage of recovery, was ⱖ95%. The intra- and
interday precision, expressed as the coefficient of variance (%CV), was ⱕ10%.
Radiochromatographic Analysis of Urine and Bile for Metabolite Profiles. Urine and bile from rats and dogs were analyzed by HPLC with both UV
and radiochemical detection. To aliquots of bile or urine (10 ␮l to 1 ml), four
volumes of CH3CN were added, and after centrifugation and evaporation of
solvent, the residues were reconstituted in mobile phase (10 –200 ␮l) for
injection directly onto the HPLC column. In the study where rats received a
high oral dose (100 mg/kg) of [14C]rofecoxib, the urine sample (1 ml) was
adjusted to pH 2 with TFA (20 ␮l).
The chromatographic system included a Hewlett-Packard 1050 instrument
equipped with a Zorbax RX C8 analytical column (4.6 ⫻ 250 mm, 5 ␮m)
heated to 40°C. The mobile phase, consisting of 0.1% aqueous TFA (TFAaq)/
NH4OH (pH 3, solvent A) and CH3CN (solvent B), was delivered at a flow rate
of 1 ml/min, starting at 15% solvent B and increasing to 60% solvent B as a
linear gradient (1%/min) for a total run time of 45 min. The effluent was
monitored by a photodiode array UV detector, set at 280 as well as 220 nm (i.v.
samples) or 225 nm (p.o. samples), and by an in-line radiochemical detector
using a 3 ml/min flow rate for the scintillation cocktail. For profiles of rat urine
from the 100 mg/kg oral dose of [14C]rofecoxib, the gradient began at 20%
solvent B and increased to 40% solvent B (0.5%/min) for a total run time of
40 min. Percentage of recovery of radioactivity from rat samples ranged from
39 to 100% (average ⬃70%) and from dog samples were 60 and 43% after i.v.
and oral dosing, respectively.
␤-Glucuronidase Treatment of Urine and Bile. ␤-Glucuronidase (2 mg/
ml) was dissolved in 0.2 M sodium acetate (pH 5). Aliquots of urine (1 ml) and
bile (20 –100 ␮l diluted to 1 ml with acetate buffer), obtained after i.v. or p.o.
administration of [14C]rofecoxib to rats and dogs, were incubated at 37°C with
a final ␤-glucuronidase concentration of 1 mg/ml (600 U/ml). Concomitantly,
samples were incubated in acetate buffer in the absence of enzyme to serve as
controls. The reaction was terminated after ⬃16 h by the addition of four
volumes of CH3CN. After centrifugation, the supernatants were evaporated to
dryness under nitrogen. The residues were reconstituted in 15% CH3CN in
0.1% TFAaq/NH4OH (pH 3) and injected for radiochromatography as described above.
Isolation and Characterization of Rofecoxib Metabolites. Metabolite
isolation and purification by HPLC. Metabolites of rofecoxib were isolated for
identification from urine that was collected as part of low dose (2 mg/kg, i.v.)
radiolabeled studies in rats and dogs and a high dose (100 mg/kg, p.o.)
unlabeled study in rats. During initial HPLC analysis to determine the retention
times of drug-related material in these samples, metabolites from the radiolabeled studies were monitored by a combination of UV and radiochemical
detection, whereas metabolites from the high dose unlabeled study were
followed by UV and fluorescence detection. For isolation and purification of
metabolites from biological samples, fluorescence detection was not employed.
From the radiolabeled i.v. study in rats, urine from three animals was pooled
and subjected to solid phase extraction before chromatographic separation and
isolation of metabolites. The solid phase extraction cartridges (C18, Baker)
were preconditioned by sequential additions of CH3OH (1 ml) and 0.1%
TFAaq/NH4OH (pH 3, 2 ml). Pooled urine (1 ml, containing ⬃281,000 dpm)
was transferred to the cartridge. The cartridge was washed with 0.1% TFAaq/
NH4OH (pH 3, 1 ml), the analytes were eluted with CH3OH (2 ml), and ⱖ85%
of total radioactivity was recovered. The solvent was evaporated to dryness
under nitrogen, and the residue was reconstituted in 10% CH3CN in 0.1%
TFAaq/NH4OH (pH 3, 1 ml) before HPLC analysis. Urine from other studies
were only filtered and/or centrifuged to remove particulate material before
HPLC analysis.
Rat urine (1 ml) was injected onto a semipreparative C18 column (9.4 ⫻ 250
mm). The mobile phase consisted of 0.1% TFAaq/NH4OH (pH 3, solvent A)
and CH3CN (solvent B) and was delivered at 3 ml/min. A gradient elution
began with 10% solvent B and increased to 60% solvent B at a rate of 2%/min,
followed by a hold (8-min) at 60% solvent B, to give a total run time of 33 min.
Analysis of dog urine was carried out as described above, except that a C8
analytical column was used, solvent A was 0.1% TFAaq (pH 1.8), and the
ADME OF ROFECOXIB IN RAT AND DOG
gradient involved a rate of increase in solvent B of 0.5%/min. From the
unlabeled, high dose study in rats, final HPLC purification of metabolites
utilized deionized water for solvent A, and the gradient began with 10%
solvent B and increased to 40% solvent B at 1%/min. The peaks of interest
were collected, the solvent was evaporated, and the residues were submitted
for 1H NMR analysis.
1
H NMR characterization of metabolites. All NMR spectra were obtained
on a Varian Unity instrument (500 MHz; Varian, Palo Alto, CA). Samples
were dissolved in deuterated methanol (CD3OD), DMSO (DMSO-d6), or
acetonitrile (CD3CN), and chemical shifts were reported in ppm (␦) downfield
from tetramethylsilane. Residual protonated solvent signals were used as an
internal reference (CD3OD, 3.3; DMSO-d6, 2.49; CD3CN, 1.93 ppm). Coupling constants were given in Hz.
Protein Binding. The binding of rofecoxib to plasma proteins was determined by incubating the compound with pooled plasma from rat and dog in a
concentration range of 0.05 to 5.0 ␮g/ml. [14C]Rofecoxib, dissolved in DMSO
(1% v/v), was added to plasma samples (adjusted to pH 7.4 with 1 M sodium
phosphate buffer, pH 4.0, and equilibrated to 37°C) and incubated for 15 to 30
min. The samples were transferred to Centrifree ultrafiltration units (Amicon/
Millipore, Bedford, MA), and, after centrifugation of the sample (2000g,
37°C), the ultrafiltrates were removed and analyzed by liquid scintillation
counting. The free fraction of drug was determined by dividing the amount
(dpm) of drug in the ultrafiltrate by the amount in the original plasma sample.
The percentage of protein bound values were not corrected for nonspecific
binding (ⱕ5%).
Blood-to-Plasma Ratio. The partitioning of rofecoxib into erythrocytes
was determined by incubating radiolabeled drug with fresh, heparinized whole
blood from rat and dog. [14C]Rofecoxib, dissolved in DMSO (1% v/v), was
added to blood (1 ml) to give a final concentration of 1 ␮g/ml, and the samples
were incubated at 37°C for 15 min. Plasma was separated by centrifugation,
and aliquots were taken for scintillation counting.
Pharmacokinetic Analysis. The plasma concentration versus time curves
from both i.v. and oral studies in rats displayed nonexponential decay behavior
during the terminal phase (4- to 10-h interval), which precluded calculation of
the elimination half-life (t1/2), and hence, area under the plasma concentration
versus time curve extrapolated to infinity (AUC0 –⬁), plasma clearance (Clp),
volume of distribution at steady state (Vdss), and bioavailability (F). Due to
interanimal variability observed in the terminal phase of the plasma concentration data, the partial AUC from time zero to the last quantifiable time point,
Cmax, and Tmax values were estimated from mean plasma concentration data.
The plasma concentration at time zero (C0) after the i.v. dose in rats was
obtained by back extrapolation of the mean plasma concentration versus time
curve. The pharmacokinetic parameters in dogs were determined using modelindependent methods. The terminal elimination rate constant (␤) for each
animal was determined from the slope of the unweighted regression line fitted
to the terminal phase of the log-linear plasma concentration versus time data
using the method of least-squares. The half-life was obtained by dividing ln2
by ␤. The plasma clearance (Clp) was calculated as
Cl⫽
Dose
AUC0 –⬁
(1)
The steady-state volume of distribution was estimated by the following
equation:
Vdss ⫽
Dosei.v. ⫻ AUMC0 –⬁
(AUC0 –⬁)2
(2)
where AUMC0 –⬁ is the total area under the first moment of the drug concentration versus time curve from time zero to infinity. Bioavailability was
estimated from the dose-normalized ratios of [AUC]0 –⬁, p.o. to [AUC]0 –⬁, i.v..
Results
Absorption and Disposition of [14C]Rofecoxib. Mean plasma
concentration versus time curves of rofecoxib and total radioactivity
after a single i.v. (2 mg/kg, Fig. 3A) or oral (5 mg/kg, Fig. 3B) dose
of [14C]rofecoxib to rats exhibited a nonexponential decline over the
time interval of 4 to 10 h, precluding a reliable estimate of the
1247
FIG. 3. Mean concentrations of total radioactivity and rofecoxib in rat plasma
after administration of [14C]rofecoxib at 2 mg/kg, i.v. (A) and 5 mg/kg, p.o. (B).
elimination rate constant, and hence, half-life, AUC0 –⬁, plasma clearance, volume of distribution, and bioavailability. The time to peak
plasma concentration (205 ng/ml) after oral dosing was 30 min.
Comparison of partial AUC values of unchanged rofecoxib and total
radioactivity after i.v. or oral dosing showed that parent drug represented 62 or 29%, respectively, of circulating drug-related material.
The corresponding AUC values for rofecoxib and total radioactivity
after each dose were 1357 and 2195 ng 䡠 h/ml (0 –10 h, i.v.) and 687
and 2368 ng 䡠 h/ml (0 –24 h, p.o.).
The plasma concentration of rofecoxib and total radioactivity in
dogs (Fig. 4), unlike rats, declined exponentially after i.v. (2 mg/kg)
and oral (5 mg/kg) administration. After a short distribution phase
after i.v. dosing (Fig. 4A), the decline in concentrations of parent drug
and total radioactivity was essentially monoexponential with an elimination half-life of 2.6 h. Total plasma clearance and steady-state
volume of distribution values were 33.6 ml/min/kg and 0.8 l/kg,
respectively. The Cmax (1114 ng/ml) after the oral dose occurred at
1.5 h, and half-life and bioavailability were 3.3 h and 26.1%. Plasma
concentrations of unchanged rofecoxib after i.v. or oral dosing comprised 66 and 43%, respectively, of total radioactive drug equivalents
in the systemic circulation of dogs. The AUC0 –⬁ values for rofecoxib
and total radioactivity were 9,225 and 13,116 ng 䡠 h/ml (i.v.) and
5,997 and 13,708 ng 䡠 h/ml (p.o.).
1248
HALPIN ET AL.
TABLE 1
Excretion of radioactivity in urine, bile, and feces from rats and dogs after
administration of [14C]rofecoxib at 2 mg/kg (i.v.) or 5 mg/kg (p.o.)
Percentage of Dose Excreteda
Time
(h)
Urine
Feces
2 mg/kg, i.v.
5 mg/kg, p.o.
19.2 (1.7)
23.1 (1.9)
61.3 (4.3)
74.5 (2.3)
20.1 (1.1)
25.6 (2.0)
51.9 (13.6)
72.3 (7.0)
47.8 (4.6)
49.2 (5.2)
27.7 (2.9)
34.8 (2.6)
11.4 (1.2)
17.5 (1.4)
70.5 (3.7)
76.0 (3.1)
0–96
97.6 (2.6)
97.9 (6.9)
84.0 (7.0)
93.5 (2.7)
Bileb
0–24
59.4 (30.6)
68.7 (9.9)
28.2
26.5
b
Oral administration of rofecoxib to dogs after a meal resulted in
slight increases of the mean AUC0 –⬁, Cmax, and Tmax values compared with fasted controls. The mean AUC0 –⬁ and Cmax values for
dogs that were fed 1 h before the oral dose were 34 and 13%,
respectively, higher than values observed in fasted animals. It was
noted that, in individual dogs, the effect of food led to variability of
the Tmax, which ranged from no effect to a 2- to 4-fold increase over
the value in fasted dogs (1.8 h). Overall, the presence of food before
oral dosing had a modest impact on the absorption kinetics of rofecoxib in dogs.
Excretion of Radioactivity after Administration of [14C]Rofecoxib. Recovery of total radioactivity in the excreta of rats after i.v. (2
mg/kg) and oral (5 mg/kg) administration of [14C]rofecoxib was
⬎97% over the 96-h collection period (Table 1). Urinary excretion
accounted for 23.1% of the i.v. dose, whereas 74.5% was recovered in
the feces. The corresponding values after the oral dose were 25.6 and
72.3%. Based on comparative recoveries of radioactivity in urine after
oral and i.v. dosing, absorption of rofecoxib in rats was quantitative.
That biliary excretion was the major route of elimination was supported further by a study in rats in which, over a 24-h interval, 59%
of an i.v. dose of [14C]rofecoxib (2 mg/kg) was excreted into bile,
whereas 69% was recovered after an oral dose (5 mg/kg).
In dogs, after an i.v. dose of [14C]rofecoxib (2 mg/kg), urinary
Dog
5 mg/kg, p.o.
Total
a
FIG. 4. Mean concentrations of total radioactivity and rofecoxib in dog plasma
after administration of [14C]rofecoxib at 2 mg/kg, i.v. (A) and 5 mg/kg, p.o. (B).
0–24
0–96
0–24
0–96
Rat
2 mg/kg, i.v.
Data are expressed as mean (S.D.), n ⫽ 4.
For the rat study, n ⫽ 3, and for the dog study, n ⫽ 1, per dose.
excretion accounted for 49% of the dose, whereas 35% was recovered
in feces (Table 1). The corresponding values after an oral dose of
[14C]rofecoxib (5 mg/kg) were 17.5 and 76%. Total recovery of
radioactivity over the 96-h sampling period was 84% (i.v.) and 94%
(p.o.). Comparison of the oral and i.v. urinary recoveries indicated that
rofecoxib was less well absorbed in dog (36%) than in rat. A biliary
excretion study in dogs (n ⫽ 1 per dose) showed that 28% of an i.v.
dose of [14C]rofecoxib (2 mg/kg) was excreted into bile, while 27% of
the dose was recovered after an oral dose (5 mg/kg) over the 24-h
collection period.
The Effect of Dose on Rofecoxib Kinetics. The mean pharmacokinetic parameters for rats and dogs that received unlabeled doses of
rofecoxib at 1, 2, and 4 mg/kg (i.v.) and 2, 5, and 10 mg/kg (p.o.) are
presented in Table 2. As had been noted with the 2 mg/kg, i.v. dose
of [14C]rofecoxib in rats, an estimate of t1/2 for the 1 mg/kg, i.v. dose
in this dose dependence study could not be determined due to the
nonexponential decline over the 2- to 10-h interval. However, for the
2- and 4-mg/kg doses, an estimate of t1/2 was 6.6 and 6.8 h, respectively. In both rats and dogs the increase in AUC values suggested that
the pharmacokinetics were nearly linear with increasing i.v. doses
over the range of 1 to 4 mg/kg. After administration of the oral doses
to rats, the increase in AUC values was greater than proportional
between the low and middle doses, with no significant difference in
AUC or Cmax values between the middle and high oral doses. The
AUC and Cmax values in dogs increased with all three oral doses, but
the increases were less than proportional.
Tissue Distribution of [14C]Rofecoxib in the Rat. Radioactivity
was widely distributed to most tissues of rats after i.v. administration
of [14C]rofecoxib at 2 mg/kg (Table 3). The maximum concentration
of radioactivity in plasma was 2.25 ␮g equivalents/g at 5 min postdose, and the level declined to 0.051 ␮g equivalents/g at the 24-h
sampling time. Although concentrations of radioactivity were highest
in most tissues at 5 min postdose, maximum levels in the urinary
bladder, prostate, skin, and fat occurred at 30 min. Concentrations in
the liver were sustained at ⬃7.5 ␮g equivalents/g (⬃17% of the dose)
from 5 to 30 min postdose and declined to 0.4 ␮g equivalents/g (⬍1%
of the dose) at 24 h. In general, tissue concentrations declined with
time to 24 h, with the amount remaining in the body confined
primarily to the gastrointestinal tract and liver.
Plasma Protein Binding and Blood-to-Plasma Ratio. [14C]Rofecoxib was highly bound to plasma proteins from rat and dog and
showed species dependence. At [14C]rofecoxib concentrations ranging from 0.05 to 5.0 ␮g/ml, the bound value percentages were 93 and
82%, respectively. [14C]Rofecoxib partitioned into erythrocytes at a
blood-to-plasma ratio of 0.76, indicating that blood clearance would
be higher than plasma clearance.
1249
ADME OF ROFECOXIB IN RAT AND DOG
TABLE 2
The effect of intravenous and oral dose levels on the pharmacokinetics of rofecoxib in rats (n ⫽ 4) and dogs (n ⫽ 4)
i.v.
Species
p.o.
Dose
(mg/kg)
AUC0–⬁
(ng 䡠 h/ml)
Clp
(ml/min/kg)
Vdss
(l/kg)
t1/2
(h)
Dose
(mg/kg)
AUC0⬁
(ng 䡠 h/ml)
t1/2
(h)
Cmax
(ng/ml)
Tmax
(h)
Rata
1
2
4
409
1156
2271
—b
—
—
—
—
—
—
6.6
6.8
2
5
10
179
3001
2963
6.2
4.3
5.0
58.3
373
400
1.0
1.8
2.3
Dogc
1
2
4
4458
9255
26174
3.8
3.6
2.6
0.7
1.0
0.7
2.2
2.5
3.2
2
5
10
5195
7490
11213
4.0
3.4
3.8
a
b
c
798
1096
1506
1.8
1.6
1.3
The AUC value for rats is a partial value from 0 to 10 h (i.v.) or 0 to 24 h (p.o.).
—, values could not be determined.
For the 5 mg/kg oral dosing study in dogs, n ⫽ 8.
TABLE 3
Tissue distribution of total radioactivity in male rats (n ⫽ 3 per time point) following i.v. administration of [14C]rofecoxib at 2 mg/kg
5 min
Tissue
Adrenal glands
Bladder (urinary)
Blood
Bone marrow (both femurs)
Brain
Eyes
Fat (reproductive)
Heart
Kidneys
Large intestine
Large intestinal contents and wash
Liver
Lungs
Lymph nodes (cervical)
Lymph nodes (mesenteric)
Muscle (thigh)
Pancreas
Pituitary gland
Plasma
Prostate
Skin (dorsal, shaven)
Small intestine
Small intestinal contents and wash
Spleen
Stomach
Stomach contents and wash
Testes
Thymus
Thyroid
30 min
2h
24 h
␮g Eq/g
% of Dose
␮g Eq/g
% of Dose
␮g Eq/g
% of Dose
␮g Eq/g
% of Dose
6.93
0.887
1.43
1.50
1.93
0.608
0.720
2.69
3.46
1.11
0.114
7.38
2.61
1.63
1.57
1.42
3.17
3.92
2.25
1.86
0.775
2.48
0.710
1.38
1.92
0.078
0.940
2.14
3.44
0.06
0.02
0.11a
0.01a
0.62
0.03
0.17a
0.44
1.34
0.52
0.50
17.1
0.78
0.04
0.06
0.57a
0.71
0.01
2.69
1.39
0.939
1.00
0.667
0.396
1.81
1.37
2.90
0.833
0.149
7.47
1.37
0.948
1.15
1.05
1.70
1.39
1.61
2.10
1.10
3.12
10.2
0.865
1.46
0.325
0.917
0.874
1.33
0.02
0.02
3.11b
0.17b
0.20
0.02
6.05b
0.21
1.04
0.39
0.93
15.8
0.33
0.03
0.05
22.7b
0.34
⬍0.005
0.690
0.393
0.269
0.198
0.141
0.126
0.483
0.306
0.867
0.502
0.597
2.25
0.338
0.219
0.296
0.240
0.396
0.401
0.443
0.349
0.267
6.00
21.7
0.194
0.300
0.158
0.222
0.181
0.403
0.01
0.01
0.92b
0.03b
0.04
0.01
1.67b
0.05
0.33
0.24
3.13
5.29
0.09
0.01
0.01
5.35b
0.07
⬍0.005
0.073
0.056
0.042
0.021
0.010
0.014
0.026
0.035
0.134
0.762
1.51
0.397
0.039
0.020
0.032
0.016
0.045
ND
0.051
0.052
0.035
0.586
1.63
0.022
0.083
0.119
0.019
0.016
0.069
⬍0.005
⬍0.005
0.14b
⬍0.005b
⬍0.005
⬍0.005
0.09b
0.01
0.05
0.43
16.3
0.90
0.01
⬍0.005
⬍0.005
0.36b
0.01
ND
0.06
0.35a
2.79
2.13
0.15
0.50
0.14
0.55
0.17
0.01
0.08
9.39b
2.60
25.9
0.09
0.35
1.41
0.51
0.08
⬍0.005
0.01
2.35b
5.22
62.9
0.02
0.07
0.52
0.12
0.02
⬍0.005
⬍0.005
0.31b
0.63
8.43
⬍0.005
0.02
0.70
0.01
⬍0.005
⬍0.005
ND, not detectable.
a
Residual carcass was analyzed at the 5-min time point and contained 59.2% of the dose; therefore, the percentages of dose for blood, bone marrow, fat, muscle, and skin were not extrapolated
based on total body weight as in the other time points.
b
Percentage of dose at 30 min, 2 h, and 24 h is based on the percentages of total body weight represented by the specified tissue as follows: blood [7.00% (Ringler and Dabich, 1979)], bone
marrow (0.35%), fat (7.08%), muscle (45.5%), and skin (18.0%) (Caster et al., 1956).
Identification of Rofecoxib Metabolites in Urine and Bile of
Rats and Dogs. Metabolites of rofecoxib were isolated from urine of
rat and dog and from rat bile after i.v. or oral administration of
rofecoxib. After further purification, the metabolites were characterized by the combined application of HPLC, UV, and NMR. The 1H
NMR data for the identified metabolites are presented in Table 4. At
the time of these studies, liquid chromatography tandem mass spectrometry analyses in the positive ionization mode showed that rofecoxib ionized poorly under these conditions, limiting the use of this
analytical technique to support the identification of rofecoxib metabolites. Reference standards of some of the metabolites were prepared
by chemical synthesis, which allowed further confirmation of the
pathway of rofecoxib metabolism in laboratory animals (Fig. 5).
Metabolism of [14C]Rofecoxib (2 mg/kg, i.v. and 5 mg/kg, p.o.).
Rats. Rofecoxib was extensively metabolized in rats as indicated by
the absence of unchanged drug in radiochromatograms of urine and
bile (Fig. 6). The profiles of urine (Fig. 6A) and bile (Fig. 6B) from
rats after i.v. (2 mg/kg) or oral (5 mg/kg) administration were qualitatively and quantitatively similar, showing two radioactive components. The metabolites were identified by NMR spectroscopy as
5-hydroxyrofecoxib-O-␤-D-glucuronide (major) and unconjugated
5-hydroxyrofecoxib (minor). The most notable change in the NMR
spectra of the two metabolites as compared with a reference spectrum
of synthetic rofecoxib occurred at the C-5 protons (Hd) of the furanone ring (Table 4). A significant downfield shift from 5.34 ppm to
6.59 or 6.87 ppm and the loss of one proton supported the conclusion
that hydroxylation had occurred at this position. The five additional
signals appearing in the spectrum of the major urinary and biliary
1250
HALPIN ET AL.
TABLE 4
1
H NMR data for rofecoxib and metabolites isolated from rat and dog urine (500 MHz, CD3OD)a
Protons
Rofecoxib
5-Hydroxyrofecoxib
5-Hydroxyrofecoxib-O-␤-D-glucuronide
a
b
c
c⬘
c⬙
c⬘⬙
d
d⬘
e
f
g
h
i
j
7.93 (AA“XX”, 2H, J ⫽ 8.6)
7.62 (AA“XX”, 2H, J ⫽ 8.6)
7.39 (m, 5H)
—b
—
—
5.34 (s, 2H)
—
3.12 (s, 3H)
—
—
—
—
—
7.92 (AA“XX”, 2H, J ⫽ 8.6)
7.67 (AA“XX”, 2H, J ⫽ 8.6)
7.39 (m, 5H)
—
—
—
6.59 (s, 1H)
—
3.12 (s, 3H)
—
—
—
—
—
7.88 (AA“XX”, 2H, J ⫽ 8.6)
7.73 (AA“XX”, 2H, J ⫽ 8.6)
7.41 (m, 5H)
—
—
—
6.87 (s, 1H)
—
3.12 (s, 3H)
4.92 (d, 1H, J ⫽ 7.9)
3.98 (d, 1H, J ⫽ 9.7)
3.54, 3.45, 3.22 (dd, 1H each)
—
—
Protons
a
b
c
c⬘
c⬙
c⬘⬙
d
d⬘
e
f
g
h
i
j
trans-3,4-Dihydro-rofecoxib
7.90 (AA“XX”, 2H, J ⫽ 8.4)
7.60 (AA“XX”, 2H, J ⫽ 8.4)
7.22 (m, 2H, ortho)
7.31 (m, 2H, meta)
7.27 (m, 1H, para)
—
4.76 (dd, 1H, J ⫽ 8.4, 8.7)
4.41 (dd, 1H, J ⫽ 10.6, 8.7)
3.08 (s, 3H)
—
—
—
4.26 (d, 1H, J ⫽ 12.4)
4.12 (ddd, 1H, J ⫽ 12.4, 10.6, 8.4)
Rofecoxib-trans-3⬘,4⬘-dihydrodiol
7.96 (AA“XX”, 2H, J ⫽ 8.5)
7.66 (AA“XX”, 2H, J ⫽ 8.5)
6.20 (t, 1H, J ⫽ 2.0)
5.85 (dd, 1H, J ⫽ 9.9, 2.4)
5.54 (d, 1H, J ⫽ 9.9, 2.0)
4.39 (m, 2H, J3⬘4 ⫽ 11.3)
5.17, 5.06 (AB, 2H, J ⫽ 17.3)
—
3.08 (s, 3H)
—
—
—
—
—
4⬘-Hydroxyrofecoxib Sulfate
7.96 (AA“XX”, 2H, J ⫽ 8.5)
7.64 (AA“XX”, 2H, J ⫽ 8.5)
7.26 (AA“XX”, 2H, J ⫽ 8.8)
7.20 (AA“XX”, 2H, J ⫽ 8.8)
—
—
5.36 (s, 2H)
—
3.25 (s, 3H)
—
—
—
—
—
a
Splitting patterns: s ⫽ singlet; d ⫽ doublet; t ⫽ triplet; dd ⫽ doublet of doublets; ddd ⫽ doublet of doublet of doublets; m ⫽ multiplet; AA“XX” ⫽ multiplet characteristic of a para-substituted
benzene ring. Coupling constants ( J ) are expressed in Hz.
b
—, signals were not detected.
metabolite were characteristic of protons corresponding to a glucuronosyl moiety with a ␤ configuration. The structure of the glucuronide conjugate was confirmed by treatment of urine and bile with
␤-glucuronidase and subsequent HPLC analyses, which showed the
disappearance of the glucuronide conjugate peak and an increase in
the area of the 5-hydroxyrofecoxib peak. The UV and NMR spectra,
as well as HPLC retention time of the 5-hydroxy metabolite were
identical with those of its synthetic reference standard. The retention
time of the glucuronide conjugate was variable, ranging from 11 to 17
min in these studies (Figs. 6 and 7, where the same chromatographic
conditions were used). These shifts were attributed to batch-to-batch
changes in the HPLC column stationary phase, although biological
matrix effects could not be ruled out.
Dogs. Radiochromatographic analysis of urine (0 –24 h) from dogs
(Fig. 7) that received either an i.v. (2 mg/kg) or oral (5 mg/kg) dose
of [14C]rofecoxib showed a more complex metabolite profile than that
observed in rats. Similar to rats, rofecoxib was extensively metabolized and unchanged parent drug was not detected in urine (Fig. 7A)
or bile (Fig. 7B). 5-Hydroxyrofecoxib was barely detected in urine
(Fig. 7A) but was the most abundant component in bile (Fig. 7B). The
ADME OF ROFECOXIB IN RAT AND DOG
1251
FIG. 5. Metabolism of rofecoxib in rat and dog.
Asterisks indicate metabolites observed in rats only. Absolute stereochemistry was not determined for the cis- and trans-3,4-dihydro-rofecoxib metabolites.
presence of 5-hydroxyrofecoxib-O-␤-D-glucuronide in dog urine was
supported by comparison of its UV spectrum with that of the metabolite identified in rat urine. Isolation and purification of metabolites
from the 8- to 24-h urine sample led to the identification of only one
metabolite by NMR spectroscopy, namely trans-3,4-dihydro-rofecoxib (Fig. 7A). The 1H NMR spectrum showed loss of the signal for
the pair of protons at C-5 of rofecoxib (Hd, 5.34 ppm) and the
appearance of four novel signals at 4.76 (Hd), 4.41 (Hd), 4.26 (Hi), and
4.12 (Hj) consistent with a CH2-CH-CH spin system. The splitting
patterns and coupling constants (12.6 Hz) of the two new proton
signals led to identification of this metabolite as 3,4-dihydro-rofecoxib with protons Hi and Hj in a trans-orientation (Table 4). The
characteristic drug-related UV absorbance band at 280 to 300 nm was
absent in the UV spectrum of the trans-dihydro derivative due to loss
of the extended conjugation of the ␲-electrons. Only a very weak
absorbance band with some fine structure at 260 to 270 nm could be
observed. The UV and NMR spectra and HPLC retention time of this
metabolite were closely similar to those of the synthetic standard. The
cluster of metabolites that eluted between 15 and 18 min were not
characterized due to poor stability of these components during isolation. However, work is ongoing to resolve, isolate, and characterize
these metabolites.
Metabolism of Rofecoxib in Rats (100 mg/kg, p.o.). Two additional metabolites of rofecoxib were isolated from rat urine (Fig. 8A)
after oral administration of unlabeled rofecoxib at 100 mg/kg, one of
the doses used in toxicological studies. Fluorescence detection of
products from postcolumn photoirradiation of the HPLC effluent,
which provided a highly sensitive analytical method for the determination of rofecoxib concentrations in plasma samples (Woolf et al.,
1999), was exploited to detect urinary metabolites in studies where
unlabeled drug was administered. Fluorescence detection and UV
absorption (photodiode array spectra) both were utilized to identify
drug-related material. Those components that gave only UV peaks
were considered to be endogenous material. Unlike the three metabolites described above, in which metabolism perturbed the proton
signals of the furanone ring, the NMR spectral data for these two
components indicated that the unsubstituted phenyl ring was the site
of metabolism (Table 4). The metabolites were identified as rofecoxib-3⬘,4⬘-trans-dihydrodiol and 4⬘-hydroxyrofecoxib sulfate (retention times of 17 and 19 min, respectively, Fig. 8A). In the NMR
spectrum of the dihydrodiol derivative, the phenyl ring resonances of
the rofecoxib Hc multiplet were conspicuously absent, and three novel
signals at 6.20, 5.85, and 5.54 ppm, consistent with a conjugated diene
system, appeared. In addition, two CH-(OH) signals were evident at
4.39 ppm. The large vicinal coupling (11.3 Hz) between these two
methine protons indicated a trans configuration, supporting the identification of this metabolite as rofecoxib-3⬘,4⬘-trans-dihydrodiol.
The NMR spectrum of the second metabolite indicated that metabolism occurred at the para position of the unsubstituted phenyl ring of
rofecoxib. The AA“XX” multiplet signal of the aromatic protons
(characteristic of para substitution), in addition to the absence of a
1252
HALPIN ET AL.
FIG. 6. HPLC radiochromatograms of urine (0 –24 h, A) and bile (1–2 h, B)
from rat after oral administration of [14C]rofecoxib at 5 mg/kg.
signal for the phenolic OH proton (8 –12 ppm), supported the identification of this metabolite as 4⬘-hydroxyrofecoxib sulfate.
When [14C]rofecoxib was administered orally to rats at the same
high dose (100 mg/kg), the radiochromatogram of urine (0 –24 h)
showed the presence of two major and several minor radioactive
peaks (Fig. 8B). The HPLC gradient conditions were modified from
those used for the profiles of rat and dog urine and bile (Figs. 6 and
7), resulting in a slight shift of retention times for the known metabolites in this profile. Two of the four late-eluting peaks were characterized as 5-hydroxyrofecoxib (retention time ⫽ 25 min) and unchanged rofecoxib (retention time ⫽ 32 min) by comparing their
respective HPLC retention times and UV spectra with those of authentic standards. UV Spectra of the two small peaks that eluted
between 5-hydroxyrofecoxib and parent drug were identical with one
another and displayed the same weak absorbance band at 260 to 270
nm that was observed with authentic trans-3,4-dihydro-rofecoxib. The
retention times of these two minor radiochemical peaks at 26 and 29
min corresponded to those of reference cis- and trans-3,4-dihydrorofecoxib, respectively, suggesting that both isomers were formed in
vivo. A more rigorous characterization of the cis-isomer will be
described elsewhere.4
Discussion
The pharmacokinetics and metabolism of rofecoxib displayed distinct species differences in rats and dogs. After oral administration,
the drug was well absorbed in rats, but less so in dogs. In rats, the
4
R. Zamboni, unpublished data, Merck Frosst Canada, Kirkland, Quebec.
FIG. 7. HPLC radiochromatograms of urine (0 –24 h, A) and bile (2–3 h, B)
from dog after oral administration of [14C]rofecoxib at 5 mg/kg.
nonexponential decay during the terminal phase that was observed in
plasma concentration versus time curves after administration of radiolabeled rofecoxib (i.v. or p.o.) or unlabeled rofecoxib (1 mg/kg,
i.v.) did not permit determination of the elimination rate constant, and
consequently, calculation of such pharmacokinetic parameters as the
terminal half-life, or Clp, Vdss, AUC0 –⬁, and bioavailability. The
appearance of secondary plasma concentration peaks at approximately
8 h after either i.v. or oral administration at the doses examined
suggested the involvement of enterohepatic recirculation. That these
secondary peaks appeared after i.v. as well as oral routes of administration ruled out the possibility of localized sites of absorption. In
addition, the absence of rofecoxib in bile after an i.v. dose excluded
the possibility of re-absorption of unchanged drug in the lower gastrointestinal tract. The glucuronide conjugate identified in rat bile
(Fig. 6B) was that of 5-hydroxyrofecoxib, not of unchanged drug,
indicating that re-entry of rofecoxib into the systemic circulation
involved more than hydrolysis of a glucuronide conjugate with subsequent re-absorption of parent drug. A preliminary study in which
5-hydroxyrofecoxib was administered orally (5 mg/kg) to a rat provided evidence that the metabolite underwent reversible metabolism
to rofecoxib. The extensive studies undertaken to examine this phenomenon and its role in the mechanism by which these secondary
peaks occurred in rats are the subject of a separate report.5
The plasma concentration versus time curves of rofecoxib in dogs
did not display the secondary peaks observed in rats. In fact, the decay
curves were essentially monoexponential after a short distribution
5
T. A. Baillie and coworkers, manuscript in preparation, Merck Research
Laboratories, West Point, PA.
ADME OF ROFECOXIB IN RAT AND DOG
FIG. 8. HPLC chromatograms of rat urine (0–24 h) after an oral dose (100 mg/kg)
of unlabeled rofecoxib employing fluorescence detection (A) and after an oral dose
(100 mg/kg) of [14C]rofecoxib employing radiochemical detection (B).
Peaks labeled with an X were determined to be unrelated to drug.
phase. Rofecoxib was a low clearance drug in dogs, with a Vdss
slightly greater than that of total body water, and a bioavailability of
26%. The presence of food before an oral dose of rofecoxib resulted
in a modest increase in AUC0 –⬁ and Cmax values.
Rofecoxib appeared to display linear kinetics over the i.v. dose range
of 1 to 4 mg/kg in rats and dogs (Table 2). However, after increasing oral
doses, increases of AUC0 –⬁ and Cmax values in dogs were less than
proportional, whereas, in rat, after a greater than proportional increase in
partial AUC and Cmax values between the low and middle doses, no
further increase occurred above the middle dose (Table 2).
The structure of rofecoxib is that of a constrained cis-stilbene,
which, when exposed to bright light, undergoes a photon-induced
rearrangement to a highly fluorescent phenanthrene-like product,
6-(methylsulfonyl)phenanthro[9,10-c]furan-1(3H)-one, (Woolf et al.,
1999). This reaction was the basis for the development of a highly
sensitive, accurate, and reproducible HPLC assay with fluorescence
detection for the determination of rofecoxib concentrations in rat and
dog plasma. Furthermore, fluorescence detection in combination with
UV detection facilitated isolation of drug-related metabolites from
biological samples of rats that had received an oral dose (100 mg/kg)
of unlabeled drug.
Metabolism of rofecoxib was extensive and species-dependent in
rats and dogs. No unchanged drug was observed in urine or bile of
either species after administration of [14C]rofecoxib by i.v. (2 mg/kg)
or oral (5 mg/kg) routes (Figs. 6 and 7). In rats, the major route of
1253
metabolism involved hydroxylation at the C-5 position of the furanone ring followed by the formation of its O-␤-glucuronide conjugate,
the major radioactive component in rat urine and bile (Fig. 6).
Metabolism of rofecoxib was more complex in dogs than in rats,
with several polar metabolites observed in urine (Fig. 7A). 5-Hydroxyrofecoxib, its O-␤-D-glucuronide, and trans-3,4-dihydro-rofecoxib were identified in dog urine, whereas 5-hydroxyrofecoxib was
the most abundant metabolite in dog bile (Fig. 7B). Attempts to isolate
and characterize several components in dog urine proved futile due to
their instability either on the HPLC column or during workup after
isolation. However, work is ongoing to isolate and characterize these
metabolites.
Two additional metabolites of rofecoxib were identified in rat urine
with the aid of fluorescence and radiochemical detection (Fig. 8).
Rofecoxib-3⬘,4⬘-dihydrodiol and 4⬘-hydroxyrofecoxib sulfate were
products of epoxidation at the 3⬘,4⬘-position on the unsubstituted
phenyl ring of rofecoxib. The relative abundance of these metabolites
could not be determined, because synthetic standards were not available. The corresponding oral high dose study with [14C]rofecoxib
revealed the presence of two novel minor metabolites in rat urine,
namely, cis- and trans-3,4-dihydro-rofecoxib. These metabolites, resulting from reduction of the furanone ring double bond, were not
fluorescent due to loss of extended ␲-electron conjugation.
Drugs that contain a substituted 2(5H)-furanone ring similar to that
of rofecoxib are limited. Digoxin, a cardiac glycoside used in the
treatment of congestive heart failure, is the most noted. In addition to
the sequential metabolic removal of its three 2,6-dideoxyglucoside
groups, digoxin undergoes reduction of the furanone ring double bond
to yield inactive 20,22-dihydrodigoxin (Brown et al., 1962; Watson et
al., 1973; Shomo et al., 1988). This highly stereoselective reduction
leads to the creation of a chiral center at the steroidal C-20 of digoxin.
Only the R-enantiomer has been identified in human urine (Reuning et
al., 1985). Unlike the monosubstituted furanone ring of digoxin,
reduction of the disubstituted furanone ring of rofecoxib yields a pair
of diastereomers, wherein the substituted and unsubstituted aromatic
rings are cis and trans to each other. The absolute stereochemistry of
the two reduced metabolites observed in this study, cis- and trans3,4-dihydro-rofecoxib, has not been determined.
Losigamone, in early development as an anticonvulsant drug, possesses a 4,5-disubstituted 2(5H)-furanone ring and is a racemic mixture with two chiral centers (Torchin et al., 1996). The in vitro
metabolism studies with (⫾)-losigamone and its (⫹)- and (⫺)-enantiomers in human liver microsomal preparations or with recombinant
P450 enzymes yielded five metabolites, two of which were characterized
as phenolic derivatives by mass spectrometry (Torchin et al., 1996).
Although one of the unidentified UV-absorbing metabolites was thought
to be nonphenolic due to its lack of electrochemical response, no evidence was presented in this report to support the possibility that losigamone had undergone reduction of the double bond.
In conclusion, rofecoxib displayed notable species differences in
pharmacokinetic and metabolic behavior in laboratory animals. The
role of reversible metabolism and subsequent enterohepatic recirculation in the disposition of rofecoxib in rats has been examined and
will be described in a subsequent report.
Acknowledgments. We thank M. Cramer, S. White, J. Brunner, K.
Michel, and C. Henry for excellent technical support with the animal
studies. We are grateful to Drs. R. Tillyer and D. Tschaen (Process
Research Group, MRL) for the preparation of unlabeled rofecoxib and
Dr. E. L. Grimm, Y. Leblanc, J. Y. Gauthier, P. Roy, M. Therien, and
S. Leger (Medicinal Chemistry, Merck Frosst Canada) for providing
synthetic rofecoxib metabolites and internal standard (the 4⬘-methyl-
1254
HALPIN ET AL.
phenyl analog of rofecoxib). We also thank Dr. K. M. Schultz for
assistance in preparing this manuscript.
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