We report here the preclinical profile of etoricoxib (MK-0663) [5-chloro-2-(6-methylpyridin-3-yl)-3-(4-methylsulfonylphenyl) pyridine], a novel orally active agent that selectively inhibits cyclooxygenase-2 (COX-2), that has been developed for high selectivity in vitro using whole blood assays and sensitive COX-1 enzyme assays at low substrate concentration. Etoricoxib selectively inhibited COX-2 in human whole blood assays in vitro, with an IC50 value of 1.1 ± 0.1 μM for COX-2 (LPS-induced prostaglandin E2synthesis), compared with an IC50 value of 116 ± 8 μM for COX-1 (serum thromboxane B2 generation after clotting of the blood). Using the ratio of IC50 values (COX-1/COX-2), the selectivity ratio for the inhibition of COX-2 by etoricoxib in the human whole blood assay was 106, compared with values of 35, 30, 7.6, 7.3, 2.4, and 2.0 for rofecoxib, valdecoxib, celecoxib, nimesulide, etodolac, and meloxicam, respectively. Etoricoxib did not inhibit platelet or human recombinant COX-1 under most assay conditions (IC50 > 100 μM). In a highly sensitive assay for COX-1 with U937 microsomes where the arachidonic acid concentration was lowered to 0.1 μM, IC50 values of 12, 2, 0.25, and 0.05 μM were obtained for etoricoxib, rofecoxib, valdecoxib, and celecoxib, respectively. These differences in potency were in agreement with the dissociation constants (K i) for binding to COX-1 as estimated from an assay based on the ability of the compounds to delay the time-dependent inhibition by indomethacin. Etoricoxib was a potent inhibitor in models of carrageenan-induced paw edema (ID50 = 0.64 mg/kg), carrageenan-induced paw hyperalgesia (ID50 = 0.34 mg/kg), LPS-induced pyresis (ID50 = 0.88 mg/kg), and adjuvant-induced arthritis (ID50 = 0.6 mg/kg/day) in rats, without effects on gastrointestinal permeability up to a dose of 200 mg/kg/day for 10 days. In squirrel monkeys, etoricoxib reversed LPS-induced pyresis by 81% within 2 h of administration at a dose of 3 mg/kg and showed no effect in a fecal 51Cr excretion model of gastropathy at 100 mg/kg/day for 5 days, in contrast to lower doses of diclofenac or naproxen. In summary, etoricoxib represents a novel agent that selectively inhibits COX-2 with 106-fold selectivity in human whole blood assays in vitro and with the lowest potency of inhibition of COX-1 compared with other reported selective agents.
Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to prostaglandin H2, which serves as the common precursor for the synthesis of prostaglandins, prostacyclins, and thromboxanes. Cyclooxygenases exist as two isoforms, which exhibit similar catalytic properties but differ in terms of regulation of expression (Jouzeau et al., 1997; Garavito and DeWitt, 1999; Hawkey, 1999). COX-1 is the major isoform expressed in healthy tissue and generates prostanoids for different functions such as gastric cytoprotection, maintenance of renal homeostasis, and platelet aggregation. The second isoform, COX-2, is also present at a basal level in certain tissues but its expression is up-regulated in response to inflammatory or mitogenic stimuli. The discovery of this second isoform of cyclooxygenase has provided the rationale for development of novel anti-inflammatory agents with improved gastrointestinal tolerability compared with nonselective NSAIDs, which inhibit both COX-1 and COX-2 (Jouzeau et al., 1997;Garavito and DeWitt, 1999; Hawkey, 1999). The efficacy of agents that selectively inhibit COX-2 in the treatment of the symptoms of osteoarthritis and the relief of acute pain, and their lower incidence of gastrointestinal-related adverse events compared with nonselective NSAIDs, have been demonstrated in several clinical studies (Ehrich et al., 1999a,b; Langman et al., 1999; Simon et al., 1999).
The difficulty of evaluating the selectivity of COX-2 inhibitors based on in vitro enzyme and cell-based assays is well recognized and has been extensively discussed (Jouzeau et al., 1997; Pairet and van Ryn, 1998; Brooks et al., 1999). Whole blood assays, based on the production of LPS-induced PGE2 for COX-2 and on the synthesis of TXB2 following clotting of the blood for COX-1, are considered to provide the most meaningful index of selectivity (Patrignani et al., 1994; Brideau et al., 1996; Brooks et al., 1999). In these assays, the selectivity of cyclooxygenase inhibition is measured in a physiological milieu taking into account the binding of the drugs to plasma proteins. These procedures have been adapted for ex vivo assays and have shown that rofecoxib and celecoxib have no significant effects on COX-1 activity at recommended therapeutic doses, providing the basis for specific COX-2 inhibition in humans (Brooks et al., 1999). Celecoxib has been reported to inhibit COX-1-mediated TXB2 production when administered in excess of the therapeutic doses (McAdam et al., 1999).
Although selective COX-2 inhibitors do not affect COX-1 in most cell and enzyme assay systems, they can inhibit COX-1 under certain in vitro conditions. The inhibitory effects on COX-1 activity are strongly dependent on the concentration of free arachidonic acid that can compete with inhibitor binding at the active site. Assays at low substrate concentrations thus offer particularly sensitive prescreening assays to detect inhibitory effects on COX-1 (Riendeau et al., 1997a). Furthermore, compounds with low potencies in such assays will also show a reduced potential for the inhibition of COX-1 in tissues where the availability of arachidonic acid might be limiting. Although there is no evidence from clinical studies that agents that selectively inhibit COX-2 do affect COX-1, the possibility that COX-1 inhibition could occur in certain tissues, such as the kidney or in others under certain pathological conditions, cannot be totally excluded.
In the present study, we describe the properties of etoricoxib (MK-0663, L-791,456), a novel dipyridinyl inhibitor (Fig.1) that has been developed for high in vitro selectivity of COX-2 inhibition using a combination of whole blood and sensitive COX-1 inhibition assays. The preclinical pharmacological profile of etoricoxib is reported, together with comparative data on the selectivity of other agents that have been reported to selectively inhibit COX-2.
Assays with Purified Recombinant Human COX-1 and COX-2.
Recombinant human COX-1 and COX-2 were expressed in baculovirus-Sf9 cells and enzymes were purified as previously described (Percival et al., 1994; Cromlish and Kennedy, 1996). Enzymatic activity of the purified COX-2 was measured using a chromogenic assay based on the oxidation ofN,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) during the reduction of PGG2 to PGH2 (Copeland et al., 1994) with minor modifications (Chan et al., 1999). This assay includes a 15-min preincubation of the enzyme with inhibitor before the initiation of the reaction with 100 μM arachidonic acid and was performed in the presence or absence of the detergent Genapol X-100 (2 mM). Assays for the assessment of inhibition of purified COX-1 at 0.1 μM arachidonic acid substrate concentration, the determination of the stoichiometry of the COX-2-inhibitor complex, the rate constant of dissociation of the enzyme-inhibitor complex by recovery of enzymatic activity, and the recovery of intact inhibitor from that complex were all performed as previously described (Riendeau et al., 1997b). HPLC analysis of etoricoxib was performed on a Novapak C18 column (3.9 × 150 mm) (Waters, Milford, MA), using acetonitrile/water/acetic acid (50:50:0.1) as solvent, a flow rate of 2 ml/min, and monitoring at 275 nm (retention time = 6.1 min).
The ability of inhibitors to function as substrates for the peroxidase reaction of COX-2 was examined using the following assay. Purified (desalted) COX-2 holoenzyme (0.8 μg) in buffer (200 μl, 100 mM Tris-HCl, pH 8.0) was treated for 1 min with either 100 μM etoricoxib, 100 μM indomethacin, 100 μM epinephrine, or vehicle (DMSO) control. The reaction was initiated with 13-hydroperoxy-9,11-octadecadienoic acid (final concentration 5 μM) and after 1 min the reaction was quenched with 800 μl of aqueous acetonitrile (25%). The reaction products were separated by injection of 100 μl onto a C18 Novapak column, which was developed with 60:40:0.1 acetonitrile/water/acetic acid at 2 ml/min with detection at 235 and 280 nm. 13-Hydroperoxy-9,11-octadecadienoic acid eluted at 2.9 min and the reduction product 13-hydroxy-9,11-octadecadienoic acid eluted at 2.6 min.
Mechanism of Inhibition of COX-2.
The time-dependent inhibition of COX-2 was analyzed using the model developed for the inhibition of ovine cyclooxygenase (Rome and Lands, 1975). In this model (Scheme 1), an initial reversible binding of enzyme and inhibitor (characterized by the dissociation constant K i), is followed by a conversion to a tight enzyme-inhibitor complex (characterized by a first order rate constant, k on). The rate of reversal of this process (k off) is considered to be negligible. This model predicts that the observed rate constant (k obs) for the exponential loss of cyclooxygenase activity during the preincubation of enzyme and inhibitor is given by the following equation: The remaining enzyme activity after a range of preincubation periods with purified COX-2 was determined for a series of inhibitor concentrations (1–300 μM), and was computer fitted to a first order equation to give a series of first order rate constants (k obs) for the onset of inhibition. The values of K i andk on were obtained by computer fitting the values of k obs at each inhibitor concentration to the above equation. Thek off value was estimated from the recovery of activity following dialysis of the COX-2-etoricoxib complex. The overall dissociation constant (K i*) for the purified COX-2-etoricoxib complex (EI*) was calculated using the kinetic constants determined above for the equilibrium defined in Scheme 1.
Measurement of Dissociation Constants for Human COX-1.
Human COX-1 was solubilized as previously described (Percival et al., 1994) except that phenylmethylsulfonyl fluoride and homovanillic acid were replaced by 50 μg/ml Pefabloc (Roche Molecular Biochemicals, Laval, Quebec, Canada) and 1 mM phenol, respectively, in the homogenization buffer. The enzyme was solubilized in homogenization buffer containing 1.5% decyl maltoside and was partially purified using a UNO Q1 anion exchange column (Bio-Rad, Richmond, CA) using a gradient of 0 to 400 mM KCl. The fractions were assayed using the TMPD assay and the active fractions were pooled and concentrated using a centriprep-30 (Amicon, Beverly, MA).
The K i andk on values for the time-dependent inhibition of COX-1 by indomethacin was determined as previously reported (Riendeau et al., 1997b) with the following modifications. The concentration of hematin in the reaction buffer was decreased to 0.5 μM and 0.5 mM TMPD was included. TheK i values for the time-independent, rapidly reversible inhibition of COX-1 by celecoxib, valdecoxib, rofecoxib, and etoricoxib were determined by measuring the observed first order loss of enzyme activity in the presence of indomethacin together with each of the aforementioned inhibitors. Briefly, 20 μg of COX-1 was preincubated in the presence of 2 μM indomethacin with or without the four inhibitors. Because of the difference in potency of the various inhibitors, preliminary experiments were performed to determine the concentration of each inhibitor that would cause a significant delay in the time-dependent inhibition caused by indomethacin. The concentrations used were 2 μM celecoxib, 3 μM valdecoxib, 30 μM rofecoxib, and 200 μM etoricoxib. An additional experiment was also performed to obtain comparative data at a single fixed inhibitor concentration (10 μM). At set times, aliquots (180 μl) were removed and assayed for remaining COX-1 activity by the addition of 100 μM arachidonic acid and TMPD. The observed first order rate constants (k obs) for the time-dependent loss of activity for COX-1 were obtained by fitting the data to the equation y = a +b · exp(−k obs t) using Kaleidagraph software. The K i values of the four inhibitors were obtained using the following equationk obs =k on/[1 + (K i/[I])(1 + ([I′]/K i′))] where [I], k on, andK i are the concentration and kinetic constants, respectively, for the inhibition of COX-1 by indomethacin, and [I′] and K i′ are the concentration and dissociation constant, respectively, of the rapidly reversible inhibitor tested (Kulmacz and Lands, 1985).
Whole Cell Assays with Transfected Chinese Hamster Ovary (CHO) Cells Expressing COX-1 and COX-2.
Stably transfected CHO cells expressing human COX-1 and COX-2 were cultured and assayed for the production of PGE2 following stimulation by arachidonic acid as previously described (Kargman et al., 1996). The cells were preincubated in Hanks' balanced salts solution containing 15 mM HEPES, pH 7.4, with the test drug or DMSO vehicle for 15 min at 37°C before a 15 min challenge with arachidonic acid (10 μM arachidonic acid for COX-2 and of 0.5 μM arachidonic acid for COX-1). Compounds were typically tested at 8 concentrations in duplicate using 3-fold serial dilutions in DMSO. Cyclooxygenase activity in the absence of test compounds is determined as the difference in PGE2 levels of cells challenged with arachidonic acid versus the PGE2 levels in cells mock-challenged with ethanol vehicle.
Assay of Microsomal COX-1 at Low Arachidonic Acid Concentration.
The assay of human COX-1 at low arachidonic acid concentration was performed using a microsomal preparation from U937 cells (Riendeau et al., 1997a). This assay system has been found to be more sensitive to inhibition than other COX-1 enzyme or cell assays and has been used to compare the relative potency of nonselective NSAIDs and agents that inhibit COX-2. Inhibitors were preincubated with the microsomal preparation for 15 min at room temperature in 0.1 M Tris-HCl, pH 7.4, 10 mM EDTA, 0.5 mM phenol, 1 mM reduced glutathione and 1 μM hematin. Arachidonic acid was added to a final concentration of 0.1 μM and the samples were further incubated for 40 min before quantitation of PGE2 by radioimmunoassay. Cyclooxygenase activity is defined as the difference in PGE2 levels of microsomes incubated with arachidonic acid versus the PGE2 levels in microsomes incubated with ethanol vehicle.
TXB2 Production by Calcium Ionophore-Activated Human Platelets.
Human washed platelets in Hanks' balanced salts solution buffered with 15 mM HEPES, pH 7.4, were preincubated at a final concentration of 4 × 107 cells/ml in the absence or presence of etoricoxib (from a 125-fold concentrated solution in DMSO). After 10 min, platelets were stimulated with 2 μM calcium ionophore A23187 for TXB2 production (Riendeau et al., 1997b).
Human Whole Blood Assays for COX-2 and COX-1.
The assays were done using identical procedures as reported previously (Brideau et al., 1996). Fresh human blood was collected from volunteers that had no apparent inflammatory conditions and had not taken and NSAID for at least 7 days before blood collection. Briefly, for the COX-2 assay, fresh heparinized whole blood from male volunteers (500 μl) was incubated with LPS at 100 μg/ml and with 2 μl vehicle (DMSO) or a test compound for 24 h at 37°C. Inhibitors were typically tested at five different concentrations using 3-fold serial dilutions of the highest tested concentration. PGE2 levels in the plasma were measured after deproteination using derivatization to the methyl oxime and radioimmunoassay (Amersham, Oakville, Ontario, Canada). COX-2 activity is defined as the production of PGE2 in the vehicle-treated and LPS-treated blood over that of background levels in unstimulated blood at time zero. For the COX-1 assay, an aliquot of fresh blood from male or female volunteers collected into vacutainers containing no anticoagulants was mixed with either DMSO or a test compound and was allowed to clot for 1 h at 37°C. TXB2 levels in the serum were measured using an enzyme immunoassay (Cayman Chemicals, Ann Arbor, MI) after deproteination. IC50 values were derived using a four-parameter fit of the titration with the software program SigmaPlot.
In Vivo Assays.
All procedures used in the in vivo assays were approved by the Animal Care Committees or Institutional Animal Care and Use Committee at the Merck Frosst Centre for Therapeutic Research (Kirkland, Quebec, Canada); Merck, Sharp & Dohme Neuroscience Research Centre (Harlow, UK); Merck Research Laboratories (Rahway, NJ), and were performed according to guidelines established by the Canadian Council on Animal Care, the British Home Office, and the U.S. Department of Agriculture and National Institutes of Health, respectively. Etoricoxib was given orally as a suspension in 0.5% (adjuvant arthritis and hyperalgesia assay) or 1% (other assays) methocellulose in water. The suspensions were used at volumes of 10 ml/kg in rats and 1 ml/kg in squirrel monkeys.
Acute Models of Efficacy.
The efficacy of etoricoxib and other compounds was evaluated in rat models, including carrageenan-induced paw edema assay, carrageenan-induced paw hyperalgesia assay, and endotoxin-induced pyresis using previously described procedures (Chan et al., 1995). A nonhuman primate model of endotoxin-induced pyresis using telemetric body temperature measurement was also used (Chan et al., 1997). Test compounds were given 2 h after injection of endotoxin in conscious, unrestrained squirrel monkeys. The reversal of increases in body temperature was measured 2 h after administration of tested compounds.
Chronic Model of Efficacy.
The efficacy of etoricoxib in chronic inflammatory conditions was evaluated in adjuvant-induced arthritis in rats as previously described (Fletcher et al., 1998). Test compounds were given orally twice daily starting on day 0 and continued until euthanasia 21 days later (Chan et al., 1999). In this study, body weight, paw volume, thymus, and spleen weights were measured. In addition, radiographs were taken from adjuvant injected and noninjected hind paws on day 0 and day 21.
Models of Gastrointestinal Integrity.
The effect of etoricoxib on the integrity of the gastrointestinal mucosa was tested using a 51Cr-EDTA urinary excretion assay in rats. This assay has been used in rats (Davies et al., 1994) and in humans (Bjarnason et al., 1986) and was found to be more sensitive to detect effects of NSAIDs on gastrointestinal integrity compared with the 51Cr-fecal excretion assay used previously (Chan et al., 1995). 51Cr-EDTA is an inert compound that is not taken up by extravascular tissue following its absorption and that is excreted completely via the kidney. Thus, urinary excretion of 51Cr can be used as an index for gastrointestinal permeability. Male Sprague-Dawley rats (∼300–360 g) were dosed orally with a test compound either once or twice daily for 3 to 10 days. Immediately after the administration of the last dose, the rats were given orally 2 ml of water containing 10 μCi of 51Cr-EDTA. The animals were placed individually in metabolism cages with food and water ad libitum. Urine was collected for a 24-h period and the amount of radioactivity was determined. Urinary 51Cr excretion was calculated as a percentage of the total administered dose. In the 10-day chronic studies with etoricoxib, an interim urinary 51Cr excretion readout was obtained after 5 days of dosing. In addition, the effect of etoricoxib on gastrointestinal integrity was also examined in nonhuman primates in a model where 51Cr fecal excretion is measured following i.v. administration of51CrCl3 in saline (Chan et al., 1995).
Results are expressed as means ± S.E. Unless otherwise specified, differences between vehicle control and treatment groups were tested using one-way ANOVA followed by multiple comparison by the Dunnett's test. A p value of <0.05 was considered statistically significant. Dose-response curves for percentage of inhibition were fitted by a four-parameter logistic function using a nonlinear least-squares regression. IC50 or ID50 was derived by interpolation from the fitted four-parameter equation.
The following compounds were synthesized by the Medicinal Chemistry Department at Merck Frosst Centre for Therapeutic Research: etoricoxib (Friesen et al., 1998), rofecoxib (Prasit et al., 1999), celecoxib (Penning et al., 1997), meloxicam (Engelhardt et al., 1996), JTE-522 (Wakitani et al., 1998), valdecoxib (Talley et al., 2000), and 6-methoxy-2-naphthaleneacetic acid. Sources of other compounds were diclofenac, nimesulide, etodolac, ibuprofen, nabumetone, lipopolysaccharide (Sigma-Aldrich, Oakville, Ontario, Canada); indomethacin (Merck, Sharp & Dohme Canada, Kirkland, Quebec, Canada), and M. Butyricum (Difco, Detroit, MI).
Selective Inhibition of COX-2 by Etoricoxib in Whole Cell, Enzyme, and Whole Blood Assays.
The IC50 values for the inhibition of COX-1 and COX-2 by etoricoxib in various in vitro assays are summarized in Table 1, together with those of indomethacin as a reference for a nonselective inhibitor. Etoricoxib was a potent inhibitor of the production of PGE2 in CHO cells expressing human COX-2 (IC50 = 79 ± 12 nM) with a high selectivity compared with its effects on COX-1 (IC50 > 50 μM in CHO cells). In addition, etoricoxib did not affect the synthesis of TXB2 mediated by COX-1 in calcium ionophore-stimulated human platelets (IC50 > 100 μM). Etoricoxib inhibited the cyclooxygenase activity of purified human COX-2 (spectrophotometric assay), with IC50values of 5.0 ± 1.1 μM (n = 8) and 4.1 ± 0.6 μM (n = 8) for the assays performed either in the absence or presence of the detergent Genapol X-100, respectively.
Etoricoxib did not significantly affect the activity of purified COX-1 when assayed using a [14C]arachidonic acid HPLC assay in which substrate concentration was 0.1 μM (IC50 > 100 μM). An inhibition of COX-1 by etoricoxib could be detected in a more sensitive assay with U937 microsomes at 0.1 μM arachidonic acid, with a potency (IC50 = 12.1 ± 2.5 μM) about 600-fold lower than that of indomethacin (Table 1). In the human whole blood assay, etoricoxib inhibits COX-2 with an IC50 of 1.1 ± 0.1 μM, showing a 106-fold selectivity compared with the inhibition of COX-1 (IC50 = 116 ± 18 μM).
Potency and Selectivity of Inhibitors in Whole Blood Assays.
The results are summarized in Table 2. Among the compounds showing more than a 5-fold selectivity in whole blood assays, all showed IC50 values against COX-2 within a 2-fold range (0.5–1.1 μM). Using the ratio of COX-1 IC50/COX-2 IC50, selectivity ratios for the inhibition of COX-2 of 106, 35, 30, 7.6, and 7.3 were obtained for etoricoxib, rofecoxib, valdecoxib, celecoxib, and nimesulide, respectively. Lower selectivity ratios were observed for diclofenac, etodolac, and meloxicam (2- to 3-fold). JTE-522, nabumetone, and 6-methoxy-2-naphthaleneacetic acid (the active metabolite of nabumetone) were not potent inhibitors of either COX-2 or COX-1 in whole blood assays (Table 2).
Effects of Inhibitors on COX-1 Activity.
Under certain conditions, selective COX-2 inhibitors show weak but detectable effects on COX-1. The production of PGE2 by U937 microsomes incubated with a low concentration of arachidonic acid (0.1 μM) represents the most sensitive assay to detect inhibitory effects on COX-1 (Riendeau et al., 1997a). The data for this assay are summarized in Table 3 for etoricoxib and other inhibitors that have shown at least a 2-fold selectivity for the inhibition of COX-2 in the whole blood assay. On average, the inhibitors were found to be 10 to 100 times more potent in the COX-1 microsomal assay compared with the COX-1 whole blood assay. The U937 microsomal assay indicates significant differences in the ability of COX-2 selective agents to inhibit COX-1 under these assay conditions (Fig. 2) with IC50values of 12, 2, 0.25, and 0.05 μM for etoricoxib, rofecoxib, valdecoxib, and celecoxib, respectively. To further confirm the differences detected by the U937 COX-1 assay,K i values were estimated with human recombinant COX-1 using an assay based on the ability of the inhibitors to compete with the time-dependent inhibition of indomethacin. Figure3 shows representative data for the effects of inhibitors on the time-dependent inhibition by 2 μM indomethacin, using a fixed concentration of 10 μM of each inhibitor. Celecoxib and valdecoxib showed direct inhibitory effects at time zero and also decreased the inhibitory effects of indomethacin, whereas etoricoxib and rofecoxib had little or no effect. Using additional data at different inhibitor concentrations,K i values for COX-1 (Table4) of 167, 18, 1.6, and 0.3 μM for etoricoxib, rofecoxib, valdecoxib, and celecoxib, respectively, were obtained. This rank order of the K ivalues is completely consistent with the inhibitory data of the microsomal assay and further confirms the lowest ability of etoricoxib to interact with COX-1 among this group of COX-2 selective agents.
Mechanism of Inhibition of COX-2 by Etoricoxib.
The kinetic mechanism of inhibition of COX-1 and COX-2 by etoricoxib was investigated using purified recombinant human enzymes. The inhibition of COX-2 by etoricoxib was time-dependent and slowly reversible. Analysis of the time-dependent inhibition data gave a value of 67 ± 22 μM for the initial reversible binding constantK i, and a value of 0.015 ± 0.002 s−1 for the rate constant of isomerization to the tightly bound enzyme-inhibitor complexk on (n = 3; data not shown). The second order rate constant for the onset of inhibition (k on/K i) of COX-2 by etoricoxib was 2.2 ± 1.0 × 10−4 μM−1s−1, compared with a value of 3.6 ± 1.0 × 10−3 μM−1s−1 for rofecoxib.
The effect of the substrate arachidonic acid on the time-dependent inhibition of COX-2 by etoricoxib was investigated. In the presence of etoricoxib at a concentration of 30 μM, COX-2 was inhibited in a time-dependent manner with a t 1/2 of 1.9 min. The inclusion of arachidonic acid (5 and 30 μM) in the preincubation mixture reduced the rate of the onset of inhibition in a concentration-dependent manner, such that less than 10% inhibition was observed after 6 min in the presence of 30 μM arachidonic acid. The results are consistent with the reversible binding of etoricoxib to COX-2 being competitive with arachidonic acid and occurring at the cyclooxygenase site. Etoricoxib did not serve as a substrate for the peroxidase reaction catalyzed by COX-2, in contrast to epinephrine (data not shown).
The stoichiometry of the tightly bound enzyme-inhibitor complex (EI*, Scheme 1) was determined by a titration of purified COX-2 (6.8 μM) with 0 to 14 μM etoricoxib for 1 h before the measurement of the remaining cyclooxygenase activity. A breaking point in the titration was observed at 7.4 μM etoricoxib, therefore consistent with the formation of an EI* complex having 1:1 stoichiometry. Intact etoricoxib could be recovered from the COX-2-etoricoxib complex. Purified COX-2 was treated with 1 equivalent of etoricoxib and incubated for 60 min, resulting in 77% loss of activity. The enzyme-inhibitor complex was then denatured and any released inhibitor extracted from the protein by treatment with organic solvent and analyzed by HPLC. A single peak with the same retention time and UV spectrum as that of authentic etoricoxib was quantitatively recovered compared with an identically treated enzyme control. These results are therefore consistent with the noncovalent nature of the COX-2-etoricoxib complex.
The inhibitor dissociation rate constantk off (Scheme 1) was evaluated by recovery of enzyme activity following dialysis. The time course for the recovery of enzyme activity during dialysis of the preformed etoricoxib-COX-2 complex followed a first order process with a rate constant (k off) of 0.088 ± 0.015 h−1 (t 1/2 = 7.8 h). The overall dissociation constantK i* calculated from the kinetic constants gives an estimated value of 109 ± 63 nM.
Effect of Etoricoxib in Models of Inflammation, Hyperalgesia, and Pyresis.
As summarized in Table 5, etoricoxib was effective in models of carrageenan-induced paw edema, carrageenan-induced paw hyperalgesia, and endotoxin-induced pyresis in rats with a potency (0.3–0.9 mg/kg) comparable to that of rofecoxib and indomethacin. It should be noted that dose-dependent inhibition was observed with etoricoxib in these models at a dosing range of 0.1 to 30 mg/kg. In the rat hyperalgesia study, a complete reversal of hyperalgesia response was observed with etoricoxib at doses of 10 mg/kg or above (data not shown).
In adjuvant-induced arthritis in rats, etoricoxib significantly reduced the secondary paw volume and radiographic scores with a similar potency to that of rofecoxib. As shown in Fig. 4, there was a clear reduction in adjuvant-induced radiographic changes of bone and joint structures for the group treated with etoricoxib compared with the vehicle group. The protection by etoricoxib was similar to that achieved with indomethacin. Etoricoxib was also effective in reversing endotoxin-induced increases in body temperature in squirrel monkeys at a dose of 3 mg/kg, as did rofecoxib and the NSAID diclofenac at the same dose (Table 5).
Gastrointestinal Tolerability of Etoricoxib.
Etoricoxib was tested in a model of urinary excretion of 51Cr following oral administration of 51Cr-EDTA in rats to evaluate its effects on the integrity of the gastrointestinal mucosa. In this model, acute oral dosing of indomethacin (3 mg/kg) or diclofenac (3 mg/kg) resulted in a significant increase in urinary51Cr excretion in rats. The total urinary51Cr excretion in the vehicle-, indomethacin-, and diclofenac-treated groups was 1.3 ± 0.1% (n= 11), 2.6 ± 0.1% (n = 5, p < 0.001 versus control), and 6.2 ± 1.5% (n = 6,p < 0.005 versus control), respectively. In a 3 day multiple dosing study, oral administration of indomethacin or diclofenac b.i.d. caused a significant dose-dependent increase in urinary 51Cr excretion (historical controls, Fig.5). In contrast, multiple oral dosing of etoricoxib at a dose of 200 mg/kg/day (administered with b.i.d. dosing) for up to 10 days had no effect on urinary 51Cr excretion (Fig. 5).
In squirrel monkeys, oral administration of the dosing vehicle (1% methocellulose) for 4 to 5 days resulted in excretion of fecal51Cr of 1.0 ± 0.1% (n = 8) over a 24-h collection period. Chronic oral administration of etoricoxib at 100 mg/kg/day (administered with b.i.d. dosing) for 5 days had no significant effect on fecal 51Cr excretion (Fig. 6). The plasma concentration of etoricoxib was 134 μM 1 h after the last dose. In contrast, both diclofenac and naproxen caused a significant increase in fecal chromium excretion in this assay (historical controls, Fig.6).
Etoricoxib is a novel dipyridinyl agent that selectively inhibits COX-2 and that shows an efficacy similar to traditional NSAIDs in various rodent models of inflammation, pain, and fever and also in a primate model of pyresis. Etoricoxib is more than 100-fold selective for COX-2 versus COX-1 in various cell and whole blood assays. In addition, no detectable effects were noted against human prostacyclin synthase (IC50 > 100 μM; data not shown) or in a diverse array of receptor and enzyme assays performed by a contract laboratory (MDS Panlabs, Bothell, WA).
Various inhibitors that demonstrate selectivity for COX-2 have been shown to inhibit COX-2 and COX-1 by different mechanisms, apparently due to an Ile-Val difference at the active site (Gierse et al., 1996;Wong et al., 1997). The weak inhibition of COX-1 is of a simple competitive nature, whereas COX-2 shows an additional time-dependent component with formation of a tight enzyme-inhibitor complex (Ouellet and Percival, 1995; Gierse et al., 1999). Because of this difference, the selectivity observed in a particular assay is strongly dependent on the concentration of arachidonic acid used as substrate and on the preincubation time of the inhibitor with the enzyme before the initiation of the reaction. Thus, a large number of articles have reported different COX-1/COX-2 selectivity ratios for the same compounds using different test systems and assay conditions (Jouzeau et al., 1997; Pairet and van Ryn, 1998). Whole blood assays have the advantage of being performed under conditions where arachidonic acid is generated endogenously in the presence of the test compound. In this system, the potency of drugs is often significantly reduced, due to binding of the drug to plasma proteins and the presence of arachidonic acid at the time of COX-2 induction. This is in contrast to most other in vitro assays, which are typically performed with preincubation of the enzyme and drug in the absence of arachidonic acid, allowing formation of the tight inhibitor-COX-2 complex and resulting in a strong inhibition of COX-2 and high selectivity (Ouellet and Percival, 1995; Riendeau et al., 1997b; Pairet and van Ryn, 1998). For example, shifts in IC50 values of 12-, 20-, and 1,000-fold were observed for etoricoxib, rofecoxib, and celecoxib in the whole blood assay compared with the values for the COX-2-transfected CHO cells. Similarly, valdecoxib has been reported to show a 28-fold selectivity for COX-2 in whole blood assays, in close agreement with the present results, but much lower than the 28,000-fold selectivity observed in enzyme assays (Talley et al., 2000). Meloxicam, etodolac, and nimesulide showed a 2- to 7-fold selectivity in COX-2 whole blood assays, which is considerably lower than in other assays (Riendeau et al., 1997b; Cullen et al., 1998; Brooks et al., 1999; Tustin, 1999), but in agreement with values reported for similar whole blood assays (Glaser et al., 1995; Panara et al., 1999; Warner et al., 1999).
It is of interest to compare the level of COX-2 selectivity as measured with the in vitro whole blood assays to clinical data, where selectivity has been assessed with ex vivo readouts. Meloxicam, which showed a 2-fold selectivity for COX-2 inhibition in the in vitro whole blood assay (Table 2), caused significant inhibitory effects of COX-1-mediated platelet TXB2 production in volunteers ex vivo at clinically relevant doses, although to a lesser extent than diclofenac (Panara et al., 1999; Tegeder et al., 1999). Celecoxib and nimesulide showed about a 7-fold selectivity in the present whole blood assays, slightly higher than the ratios of 1.4 and 5.3, respectively, reported for these inhibitors by Warner et al. (1999), but lower than the 40-fold value reported for celecoxib byTalley et al. (2000). In humans, nimesulide had no effect on serum TXB2 levels (Cullen et al., 1998), but caused a 50% reduction of platelet TXB2 production in ex vivo assays after administration of a single dose of 100 mg (Panara et al., 1998). Modest, but significant effects on platelet TXB2 production have been reported for celecoxib at a dose of 800 mg (McAdam et al., 1999), without effect on agonist-induced platelet aggregation or bleeding time at a dose 600 mg b.i.d. (Leese et al., 2000). Rofecoxib showed a 35-fold selectivity for COX-2 in the current assay and a 77-fold selectivity in the assay described by Warner et al. (1999). The higher selectivity detected in the latter assay is due to a lower potency against COX-1, which might be related to the use of calcium ionophore to stimulate thromboxane production. For rofecoxib, no effect on platelet function or TXB2 production has been observed, even at supratherapeutic doses (up to a single dose of 1000 mg, 20- to 80-fold the recommended dose) (Ehrich et al., 1999a).
The clinical data summarized above indicate a clear trend toward the reduction of effects on COX-1-mediated platelet TXB2 synthesis as the selectivity of the drug increases in the whole blood assays. A selectivity ratio of 6- to 7-fold for COX-2 appears to be sufficient to spare agonist-induced platelet functions, but not so completely as to eliminate their effects on COX-1-mediated TXB2 production. In contrast, rofecoxib, with a 35-fold selectivity, does not affect platelet function or thromboxane production. Whole blood assays, however, may not reflect the situation in other tissues where a lower arachidonic acid availability may exacerbate COX-1 inhibition. The present assays for the determination of COX-1 K ivalues and IC50 values for the inhibition of microsomal COX-1 were designed to compare the potential of the various inhibitors to inhibit COX-1 at low arachidonic acid concentration. The two assays gave the same rank order of potency of the various inhibitors and showed that etoricoxib is significantly less potent against COX-1 than the other agents that selectively inhibit COX-2. Whether the higher selectivity of etoricoxib in vitro will confer therapeutic advantages in normal or pathological situations remains to be fully evaluated. Clinical studies in progress have shown that etoricoxib is well tolerated and efficacious in the treatment of the symptoms of osteoarthritis (Gottesdiener et al., 1999) and is selective for COX-2 in vivo even at single doses more than 8-fold the clinically effective dose in osteoarthritis (Dallob et al., 2000). In addition, its major metabolites have no significant effects on COX-1 activity (N. Chauret, J. A. Yergey, C. Brideau, R. W. Friesen, J. Mancini, D. Riendeau, J. Scheigetz, J. Silva, A. Styhler, L. A. Trimble, and D. A. Nicoll-Griffith, submitted). Further clinical studies with etoricoxib should contribute to fully explore the therapeutic potential and tolerability of selective inhibitors of COX-2 in inflammatory conditions, cancer, and neurological diseases.
We thank Dr. K. Gottesdiener for helpful comments on the manuscript; Dr. W. R. Widmer (Purdue University, West Lafayette, IN), A. Christen, C. Orevillo, and S. O'Brien for help in the adjuvant arthritis studies; and the members of Laboratory Animal Resources (Merck Frosst and Rahway, NJ) for assistance in the studies with the animal models.
- Received June 8, 2000.
- Accepted August 30, 2000.
Send reprint requests to: Dr. D. Riendeau, Merck Frosst Centre for Therapeutic Research, 16711 TransCanada Hgwy., Kirkland, Quebec, Canada H9H 3L1. E-mail:
- nonsteroidal anti-inflammatory drug
- high performance liquid chromatography
- dimethyl sulfoxide
- Chinese hamster ovary
- The American Society for Pharmacology and Experimental Therapeutics