Covalent Binding of Radioactivity from [c]rofecoxib, but Not [c]celecoxib or [c]cs-706, to the Arterial Elastin of Rats

Rofecoxib is a cyclooxygenase-2 (COX-2) inhibitor that has been withdrawn from the market because of an increased risk of cardiovascular (CV) events. With a special focus on the arteries, the distribution profiles of radioactivity in rats orally administered [C]rofecoxib were investigated in comparison with two other COX-2 inhibitors, [C]celecoxib and [C]CS-706 (2-(4-ethoxyphenyl)-4-methyl 1-(4-sulfamoylphenyl)-1H-pyrrole), a novel selective COX-2 inhibitor. Whole-body autoradioluminography and quantitative determination of the tissue concentrations showed that considerable radioactivity is retained by and accumulated in the thoracic aorta of rats after oral administration of [C]rofecoxib, but not [C]celecoxib or [C]CS-706. Acid, organic solvent, and proteolytic enzyme treatments of aorta retaining high levels of radioactivity from [C]rofecoxib demonstrated that most of the radioactivity is covalently bound to elastin. In agreement with this result, the radioactivity was found to be highly localized on the elastic fibers in the aorta by microautoradiography. The retention of radioactivity on the elastic fibers was also observed in the aortic arch and the coronary artery. These findings indicate that [C]rofecoxib and/or its metabolite(s) are covalently bound to elastin in the arteries. These data are consistent with the suggestion of modified arterial elasticity leading to an increased risk of CV events after long-term treatment with rofecoxib. Rofecoxib (VIOXX) is a potent and highly selective cyclooxygenase-2 (COX-2) inhibitor that has been widely used as a nonsteroidal anti-inflammatory drug (NSAID). However, this drug was withdrawn from the worldwide market because of an increased risk of cardiovascular (CV) events found in the Adenomatous Polyp Prevention on VIOXX (APPROVe) trial, which was conducted for the prevention of the recurrence of colorectal polyps and included 2600 patients with no history of CV disease before enrollment (Merck, 2004). The study had originally been intended to last for 3 years but was halted in midcourse because of the increased CV risks among patients in the group that was taking 25 mg of rofecoxib, in which the incidence of risk was more than twice that in the group taking the placebo. In the last few years, it has been reported that other selective COX-2 inhibitors (e.g., etoricoxib, parecoxib/valdecoxib, and celecoxib) and nonselective NSAIDs (e.g., naproxen) may also have a potential for increased CV risk (Bombardier et al., 2000; Ott et al., 2003; FDA, 2004; FDA Advisory Committee.com, 2004; NIH, 2004; Aldington et al., 2005; Krotz et al., 2005; Nussmeier et al., 2005). However, rofecoxib differs in the following ways: 1) a significantly greater frequency and higher odds of CV events (Mamdani et al., 2004; Solomon et al., 2004a; Graham et al., 2005; Kimmel et al., 2005); 2) a shorter period and a lower dose (even at the clinical dose) leading to the incidence of CV events; and 3) an earlier onset and a greater hypertensive effect correlating closely with CV risk (Brinker et al., 2004; Solomon et al., 2004b; Wolfe et al., 2004; Fredy et al., 2005). Therefore, it is suggested that rofecoxib could have distinctive mechanisms or more potent toxic activity leading to CV risks, in comparison with other selective COX-2 inhibitors or nonselective NSAIDs. Consequently, it is vital to investigate rofecoxib by comparing it with other selective COX-2 inhibitors to clarify its relevance to increased CV events. Several hypotheses have been proposed to explain the mechanism for the increased risk of CV events with rofecoxib. McAdam et al. (1999) suggested the endothelial prostacyclin (PGI2)/thromboxane A2 (TxA2) imbalance theory, whereby the CV complications caused by selective COX-2 inhibitors might be partially due to an imbalance of the concentration ratio of two prostanoids with major CV actions: PGI2, a vasodilator and inhibitor of platelet aggregation, and TxA2, a vasoconstrictor and promotor of platelet aggregation. That is, selective COX-2 inhibitors diminish the production of PGI2 in the endothelium, but not TxA2 in the platelets, so that the relative concentration of TxA2 increases around the affected area, which might increase the risks. Walter et al. (2004) proposed the pro-oxidant effect theory, whereby rofecoxib, which is a sulfone-type COX-2 inhibitor, but not celecoxib (sulfonamide-type COX-2 inhibitor) or nonselective NSAIDs, promotes oxidative damage to low-density lipoprotein and phospholipids in vitro, and this action might lead to atherogenesis in vivo. In addition, Pope et al. (1993) and Johnson et al. (1994) Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.106.009860. ABBREVIATIONS: COX-2, cyclooxygenase-2; CV, cardiovascular; NSAID, nonsteroidal anti-inflammatory drug; PGI2, prostacyclin; TxA2, thromboxane A2; CS-706, 2-(4-ethoxyphenyl)-4-methyl 1-(4-sulfamoylphenyl)-1H-pyrrole; TCA, trichloroacetic acid; Kp, the ratio of concentration in tissue to that in plasma (tissue/plasma). 0090-9556/06/3408-1417–1422$20.00 DRUG METABOLISM AND DISPOSITION Vol. 34, No. 8 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 9860/3125747 DMD 34:1417–1422, 2006 Printed in U.S.A. 1417 at A PE T Jornals on A ril 9, 2017 dm d.aspurnals.org D ow nladed from proposed the blood pressure elevation theory, in which NSAIDs and selective COX-2 inhibitors disrupt the production of prostaglandins, which play an important homeostatic role in the kidney. The resulting sodium and water retention can contribute to blood pressure elevation. The higher risk potential of rofecoxib on the CV events can be supported only by the Walter et al. (2004) hypothesis. However, it may not be enough to distinguish rofecoxib from other COX-2 inhibitors and NSAIDs in terms of the risks. CS-706 is a novel sulfonamide-type selective COX-2 inhibitor, having the strongest anti-inflammatory activity in animal models in its class (Sankyo Co., Ltd., data on file; S. Ushiyama, T. NakajimaYamada, Y. Murakami, S. Kumakura, S. Inoue, K. Suzuki, A. Nakao, A. Kawara, and T. Kimura, Sankyo Co., Ltd., in preparation). This drug is currently in phase 2 studies in the United States, and its efficacy and safety have been demonstrated (Sankyo Co., Ltd., data on file; J. B. Moberly, P. J. Desjardins, D. P. Bandy, A. Link, J. Xu, and K. E. Truitt, Daiichi Sankyo Pharma Development, in preparation). In the present study, we investigated the distribution profiles of radioactivity after oral administration of [C]rofecoxib to rats, especially in its retention by and accumulation in the thoracic aorta, by comparing it with [C]celecoxib and [C]CS-706 as references (Fig. 1). Aorta samples were collected after administration of [C]rofecoxib and treated with acid, organic solvent, and proteolytic enzymes to demonstrate that [C]rofecoxib and/or its metabolite(s) are covalently bound to a protein of the aorta. Microautoradiographic observation of the radioactivity localized in the aorta was also conducted. Materials and Methods Chemicals and Reagents. [C]Rofecoxib (59 mCi/mmol for microautoradiography and 17 mCi/mmol for the other studies), [C]celecoxib (13 mCi/mmol), and [C]CS-706 (14 mCi/mmol) were synthesized at GE Healthcare Bio-Sciences (Little Chalfont, Buckinghamshire, UK; Fig. 1). The radiochemical purity of these compounds was more than 98% by radiodetectionhigh-performance liquid chromatography analysis. Nonlabeled rofecoxib, celecoxib, and CS-706 were synthesized at Sankyo Co., Ltd. (Tokyo, Japan). All other reagents and solvents used were commercially available and were of extra pure, guaranteed, or high-performance liquid chromatography grade. Dosing of Animals. Male Sprague-Dawley rats (7 weeks old, Charles River Japan, Inc., Yokohama, Japan) were used after one or more weeks of acclimatization. Rats were housed in a temperature-controlled room with a 12-h light/dark cycle. Their body weights ranged from 160 g to 260 g at the start of administration. For the single oral administration, rats were fasted overnight before dosing and through 8 h postdose. For the repeated administrations, rats were fasted only on the first day. Water was available ad libitum throughout the study. [C]Rofecoxib was orally administered at 2 ml/kg as a solution in polyethylene glycol 400. [C]Celecoxib and [C]CS-706 were orally administered at 2 ml/kg as a solution in a mixture of N,N-dimethylacetamide, Tween 80, and distilled water (5/20/75, v/v/v). The dosing solutions were prepared shortly before dosing at the same specific radioactivity within the same study (range 68–370 Ci/kg body weight). Whole-Body Autoradioluminography. Rats (one animal at each time point) were euthanized by deep anesthesia with diethyl ether at 0.5, 2, 6, 24, and 48 h after single oral administration (2 mg/kg) of [C]rofecoxib, [C]celecoxib, or [C]CS-706, at 48 h after 3-day repeated administrations of [C]rofecoxib (2 mg/kg) and at 48 h and 10 days after 7-day repeated administrations of [C]rofecoxib (2 mg/kg). The rats were frozen in n-hexane/ dry ice, embedded in a gel of carboxymethyl cellulose (ca. 5% w/v), and frozen again in n-hexane/dry ice. The frozen blocks were sliced with Cryomacrocut (CM3600; Leica Microsystems Nussloch GmbH, Nussloch, Germany) at approximately 25°C to prepare whole-body sections of 50m thickness. The whole-body sections obtained were freeze-dried at approximately 25°C for 48 h. After freeze-drying, the sections were covered with a protective film (4 m, DIAFOIL membrane; Mitsubishi Polyester Film Corp., Tokyo, Japan) and placed in contact with imaging plates (BAS-MS2040; Fuji Photo Film Co., Ltd., Tokyo, Japan) for approximately 24 h. Finally, the imaging plates were subjected to image analysis using a Bio Imaging Analyzer (BAS-2500; Fuji Photo Film Co., Ltd.) to obtain whole-body autoradioluminograms (resolution 50 m, gradation 256, sensitivity 10,000, latitude 4). Quantitative Tissue Distribution. After single oral administration of [C]rofecoxib, [C]celecoxib, or [C]CS-706 to the rats (2 mg/kg, n 3), blood samples were taken from the abdominal aorta under diethyl ether anesthesia at 0.5, 2, 6, 24, and 48 h postdose. Subsequently, the liver, kidney, lung, aorta, cartilage (auricular), and skin (back) were collected from each carcass. After repeated administrations of [C]rofecoxib to rats (2 mg/kg, n 3) for 3 days or 7 days, the blood and the above described tissues were collected at 48 h postdose. Blood samples were centrifuged (4°C, 1680g, 10 min) to obtain plasma samples. Acid, Organic Solvent, and Proteolytic Enzyme Treatments of Thoracic Aortas. At 24 h after single oral administration of [C]rofecoxib (2 mg/kg, n 10), rats were euthanized by exsanguination under diethyl ether anesthesia and the aorta samples were collected, pooled, and weighed. The samples were homogenized in isotonic saline (5% w/v) using a motor-driven homogenizer, and 0.2 ml of the homogenate was removed for determination of the radioactivity. To the remaining homogenate, 30 ml of 0.9 M trichloroacetic acid (TCA) was added to precipitate the proteins. Then, the mixture was centrifuged at 2000g at room temperature for 10 min. The supernatant was discarded, and the precipitate was washed by resuspension and centrifugation successively with 0.6 M TCA, 80% (v/v) methanol, and 100% methanol. This process was