Mechanism for Covalent Binding of Rofecoxib to Elastin of Rat Aorta

Abstract

We have previously reported that oral administration of [14 C]rofecoxib to rats resulted in the long retention of radioactivity by the aorta as a consequence of covalent binding to elastin. Treatment of rats with ␣-phenyl-␣-propylbenzeneacetic acid 2-[diethylamino]-ethyl ester hydrochloride (SKF-525A), a cytochrome P450 inhibitor, significantly decreased the systemic exposure of 5-hydroxyrofecoxib, one of the main metabolites of rofecoxib, whereas there was no statistically significant change in the retention of radioactivity from [14 C]rofecoxib in the aorta. On the other hand, the aortic retention of radioactivity closely correlated to the systemic exposure of unchanged rofecoxib in the dose range between 2 and 10 mg/kg. A covalent binding study of [14 C]rofecoxib in vitro using rat aorta homogenate in the presence of D-penicillamine, hydralazine, ␤-aminopropionitrile, and sodium borohydride suggested that the aldehyde group of allysine in elastin was relevant to the covalent binding. In a model reaction using benzaldehyde, rofecoxib but not 5-hydroxyrofecoxib reacted with the aldehyde group of benzaldehyde in a manner of condensation reaction under a physiological pH condition. A histopathological examination using an electron microscope demonstrated that multiple oral administration of rofecoxib to rats caused marked degradation of the elastic fiber system of the aorta. These results suggested that rofecoxib as such is reactive in vivo, undergoing a condensation reaction with allysine, thereby preventing the formation of cross-linkages in elastin, i.e., desmosine and isodesmosine, and causing the degradation of the elastic fibers.

Covalent Binding of Rofecoxib, but Not Other Cyclooxygenase-2 Inhibitors, to Allysine Aldehyde in Elastin of Human Aorta

“…The experiment to examine the reaction of COX-2 inhibitors with benzaldehyde was performed according to a previous report (Oitate et al, 2007). In brief, 100 M of each compound (control; buffer only) was incubated with 1 mM benzaldehyde in potassium phosphate buffer (pH 7.4) at 37°C for up to 24 h. The reaction mixtures were frozen to stop the reaction and stored at Ϫ80°C until analysis using LC/MS.…”

“…Homogenate samples of the human aorta were prepared as described above. According to a previous report (Oitate et al, 2007), the homogenates were each pretreated with 10 mM of protein modifiers BAPN, D-penicillamine, or hydralazine in potassium phosphate buffer (100 mM, pH 7.4) at 37°C for 0.5 h, and then incubated with [ 14 C]rofecoxib (100 M) at 37°C for 2 h (n ϭ 3). As a control, the homogenate was pretreated with buffer alone.…”

“…The net amount of covalent binding was calculated by subtracting the nonspecific binding from the total. The LOX activity in the samples was measured as previously reported (Oitate et al, 2007).…”

“…It has been reported that the prevention of these cross-linkages in elastin or collagen causes serious lesions in the connective tissues in animals and humans (Herd and Orbison, 1966;Andrews et al, 1975;Hashimoto et al, 1981;Junker et al, 1982;Light et al, 1986;Yoshikawa et al, 2001). In fact, multiple oral administration of rofecoxib to rats caused a marked degradation of the elastic fibers in the aorta in vivo, conceivably by covalently binding to allysine aldehyde and by the inhibition of the normal cross-linking process of elastin (Oitate et al, 2007). In the present study, to investigate whether the above observations seen in rats also occur in clinical situations, that is, whether rofecoxib can bind with allysine aldehyde in human elastin, we conducted in vitro binding studies using a human aorta sample.…”

“…In our previous study using rat aortas (Oitate et al, 2007), it was suggested that rofecoxib itself reacts with the aldehyde group of allysine (␣-aminoadipic-␦-semialdehyde) in elastin to give a condensation covalent adduct (Fig. 1B).…”