QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.107.016121v1 35/10/1846    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Oitate, M. Articles by Ikeda, T. Search for Related Content PubMed PubMed Citation Articles by Oitate, M. Articles by Ikeda, T.

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

Drug Metabolism & Pharmacokinetics Research Laboratories (M.O., T.M., S.M., T.I.), and Global Project Management Department (T.H.), Daiichi Sankyo Co., Ltd., Tokyo, Japan

(Received April 12, 2007; accepted July 6, 2007)

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
In rats, it has been reported that rofecoxib, a cyclooxygenase-2 (COX-2) inhibitor, reacts with the aldehyde group of allysine in elastin to give a condensation covalent adduct, thereby preventing the formation of cross-linkages in the elastin and causing degradation of the elastic fibers in aortas in vivo. Acid, organic solvent, and proteolytic enzyme treatments of human aortic homogenate after incubation with [¹⁴C]rofecoxib demonstrated that most of the radioactivity is covalently bound to elastin. The in vitro covalent binding was inhibited in the presence of ß-aminopropionitrile, D-penicillamine, and hydralazine, which suggested that the aldehyde group of allysine in human elastin was relevant to the covalent binding. The in vitro covalent binding of [¹⁴C]rofecoxib was significantly decreased by the addition of only nonradiolabeled rofecoxib but not the other COX-2 inhibitors, celecoxib, valdecoxib, etoricoxib, and CS-706 [2-(4-ethoxyphenyl)-4-methyl 1-(4-sulfamoylphenyl)-1H-pyrrole], a novel selective COX-2 inhibitor. All the above COX-2 inhibitors except for rofecoxib had no reactivity with the aldehyde group of benzaldehyde used as a model compound of allysine aldehyde under a physiological pH condition. On the other hand, no retention of the radioactivity of [¹⁴C]rofecoxib was observed in human aortic endothelial cells in vitro, suggesting that rofecoxib is not retained in aortic endothelial cells in vivo. These results suggest that rofecoxib, but not other COX-2 inhibitors, is capable of covalently binding to the aldehyde group of allysine in human elastin. This might be one of the main causes of cardiovascular events by rofecoxib in clinical situations.

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). This was also strongly supported by an in vitro reaction using benzaldehyde, a model compound of allysine, in which rofecoxib reacted with the aldehyde group of benzaldehyde in a kind of condensation reaction under a physiological pH condition. Similar to collagen, elastin is a key extracellular matrix protein which provides CV tissues, e.g., arteries and heart valves, with tensile strength and elasticity (Vrhovski and Weiss, 1998). The elastic property is due to the existence of covalent cross-linkages, such as desmosine and isodesmosine, and allysine is the most important precursor of physiologically essential cross-linkage formation in elastin and collagen and is formed from the lysine residue by lysyl oxidase (LOX) (Fig. 1A). 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).

View larger version (23K): [in this window] [in a new window]   FIG. 1. Schematic diagram illustrating the structure and route of the formation of elastin cross-linkages under normal conditions (A) and a proposed reaction mechanism for the covalent binding of rofecoxib with elastin (B).

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Chemicals and Reagents. [¹⁴C]Rofecoxib (17 mCi/mmol) and [¹⁴C]celecoxib (13 mCi/mmol) were synthesized at GE Healthcare Biosciences (Little Chalfont, Buckinghamshire, UK; radiochemical purities; >98%), and nonradiolabeled rofecoxib, celecoxib, valdecoxib, etoricoxib, and CS-706 were synthesized at Daiichi Sankyo Co., Ltd. (Tokyo, Japan) (Fig. 2). ß-Aminopropionitrile (BAPN), D-penicillamine, hydralazine, and benzaldehyde were purchased from Sigma-Aldrich Inc. (St. Louis, MO). All other reagents and solvents used were commercially available and were of extra pure, guaranteed or high-performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS) grade.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Chemical structures of rofecoxib, celecoxib, valdecoxib, etoricoxib, and CS-706. Asterisks indicate the positions of the radiolabels.

Procurement of Human Aorta Sample. A human abdominal aorta (Caucasian, female, 69 years old) was obtained from the Human Animal Bridging Research Organization in Chiba, Japan. The sample was legally procured from the National Disease Researcher Interchange in Philadelphia, PA, with permission to use the aorta for research purposes only, based on the international partnership between the National Disease Researcher Interchange and Human Animal Bridging Research Organization.

Cell Culture. Human aortic endothelial cells (HAOEC) were obtained from Cell Applications, Inc. (San Diego, CA). The cells were cultured in endothelial cell medium (HAOEC Total Kit; Cell Applications, Inc.) according to the supplier's instructions. For the uptake and efflux experiments, HAOEC were seeded at 9.0 x 10⁴ cells/cm² in 24-well dishes (type I collagen-coated plate; Asahi Techno Glass, Tokyo, Japan) and used at 90% confluence.

Acid, Organic Solvent, and Proteolytic Enzyme Treatments of Human Aorta. After the removal of the adhering tissues, the human aortic sample was weighed and homogenized [5% (w/v)] in isotonic saline using a motor-driven homogenizer. The homogenate in final volume of 10 ml was incubated with [¹⁴C]rofecoxib (100 µM) at 37°C for 2 h (n = 1). After the incubation, to the homogenates 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 until only background radioactivity was measured in each wash. According to a previous report (Oitate et al., 2006), the resulting precipitate was enzymatically treated successively with collagenase (25,000 units, from Clostridium histolyticum; Wako Pure Chemical Industries, Ltd., Osaka, Japan) in 0.1 M Tris-HCl buffer, pH 8.0, at 37°C for 16 h, elastase (4000 units, from porcine pancreas; Wako Pure Chemical Industries, Ltd.) in 0.1 M Tris-HCl buffer, pH 8.5, at 37°C for 16 h, and pronase (10,000 units, from Streptomyces griseus; Merck, Darmstadt, Germany) in 0.1 M sodium phosphate buffer, pH 7.5, at 37°C for 16 h, and then the radioactivity recovered in each proteolytic fraction was measured.

In Vitro Covalent Binding of [¹⁴C]Rofecoxib to Human Aorta Homogenate and Effect of Protein Modifiers. 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 [¹⁴C]rofecoxib (100 µM) at 37°C for 2 h (n = 3). As a control, the homogenate was pretreated with buffer alone. For evaluating the nonspecific binding, the same treatments were performed at 4°C. After the incubation, 3-fold volume of 0.9 M TCA was added to the incubation mixture to precipitate the proteins. Then, the mixture was centrifuged at 4°C for 10 min. The supernatant was removed for radiodetection HPLC analysis, and the precipitate was washed by resuspension and centrifugation successively with 0.6 M TCA, 80% (v/v) methanol, and 100% methanol until only background radioactivity was measured in the centrifuged supernatants of each washing step. The resulting precipitates were air-dried and subjected to radioactivity measurement. 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).

In Vitro Covalent Binding of [¹⁴C]Rofecoxib to Rat and Human Aorta Homogenates and Effect of Several COX-2 Inhibitors. After the removal of the adhering tissues, the rat and human aortic samples were weighed and homogenized [5% (w/v)] in isotonic saline using a motor-driven homogenizer. The homogenates were incubated with [¹⁴C]rofecoxib (50 µM) in the presence or absence of 0.5 mM of nonradiolabeled COX-2 inhibitors rofecoxib, celecoxib, valdecoxib, etoricoxib, or CS-706 at 37°C for 2 h (n = 3). In a separate study, the homogenate was incubated with 25 µM [¹⁴C]rofecoxib (only for humans), 100 µM [¹⁴C]rofecoxib, or 50 µM [¹⁴C]celecoxib at 37°C for 2 h (n = 3). For evaluating the nonspecific binding, the same treatments were performed at 4°C for 2 h (n = 3). After the incubation, the samples were washed and treated as described above, and the amount of net covalent binding was calculated. The LOX activity in the samples was also measured as described above.

Condensation Reaction of COX-2 Inhibitors with Benzaldehyde As a Model Reaction. 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 µM 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 -80°C until analysis using LC/MS.

Uptake and Efflux Experiments with HAOEC. Immediately before the uptake experiment, the culture medium was removed and the cells were washed three times with 1 ml of Hanks' balanced salt solution (HBSS; pH 7.4, 37°C; Invitrogen Corp., Carlsbad, CA). Uptake was initiated by adding 1 ml of endothelial cell medium containing 10 µM [¹⁴C]rofecoxib or [¹⁴C]celecoxib (final concentration of dimethyl sulfoxide, 0.1%), and the cells were incubated at 37°C up to 360 min. After the incubation, the cells were washed three times with 1 ml of ice-cold HBSS to stop the uptake and to remove any extracellular ¹⁴C-compounds. For the quantification of the compounds taken up, the cells were dissolved in 0.5 ml of HBSS containing Triton X-100 (final 0.1%) by sonication.

For the efflux experiment, each ¹⁴C-compound (10 µM) was incubated with the cells for 120 min at 37°C, and then the cells were washed in the same manner as described above. After that, the cells were incubated at 37°C with 1 ml of endothelial cell medium containing 20 µM of each nonradiolabeled compound to start the efflux. At the designated time, the cells were washed and dissolved as described above.

Both the uptake and efflux experiments were done in triplicate. The cellular protein content was measured using a DC Protein Assay Kit (Bio-Rad Laboratories, Inc., Hercules, CA) in duplicate.

Radiodetection HPLC Analyses. After the incubation of [¹⁴C]rofecoxib with aortic homogenates, the supernatants were analyzed by radiodetection HPLC as previously reported (Oitate et al., 2007). The chromatographic system included a LC-10A system (Shimadzu Corp., Kyoto, Japan) equipped with an Xterra MS C18 column (4.6 x 150 mm, 5 µm; Waters Corp., Milford, MA) heated to 40°C. The mobile phase, consisting of distilled water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B), was delivered at a flow rate of 1 ml/min, starting at 20% solvent B in solvent A and increasing the proportion of solvent B to 60% linearly for 20 min, and holding for 10 min at 60% solvent B. The effluent was monitored using a SPD-10A UV detector (280 nm; Shimadzu Corp.) and a Radiomatic 525TR radiochemical detector (PerkinElmer Life and Analytical Sciences, Boston, MA) with a 3 ml/min flow rate for the scintillation cocktail ULTIMA-FLO (PerkinElmer Life and Analytical Sciences).

View larger version (10K): [in this window] [in a new window]   FIG. 3. Effect of protein modifiers on the covalent binding of [14C]rofecoxib to human aorta homogenate (mean ± S.D., n = 3). [14C]Rofecoxib (100 µM) was incubated with human aortic homogenate [5% (w/v)] with or without the pretreatment of each of the protein modifiers (10 mM) and the amount of covalent binding was measured as described under Materials and Methods. ***, p < 0.001, significantly different from the control by ANOVA followed by Dunnett's test.

Radioactivity Measurements. Samples were solubilized with a tissue solubilizer NCS-II (1-2 ml; GE Healthcare Biosciences) under constant shaking at approximately 55°C. After solubilization, these samples were mixed with 10 ml of scintillator HIONIC-FLUOR (PerkinElmer Life and Analytical Sciences) and were subjected to radioactivity measurement by a liquid scintillation counter (model 2300TR; PerkinElmer Life and Analytical Sciences). The radioactivity value of each proteolytic fraction of the aorta was converted to a percentage of the total covalent-binding radioactivity, which is not extractable by washing with TCA and methanol. The radioactivity in the aorta was calculated as an equivalent (Eq) value of ¹⁴C-compound and expressed as a concentration per gram of aorta. For the cell experiments, the intracellular concentrations were expressed as the amount per milligram of protein of cells (nmol/mg protein).

Data Analyses. Statistical analyses were performed by analysis of variance (ANOVA) followed by Dunnett's test using SAS System Release version 9.1 (SAS Institute Inc., Cary, NC). Differences were considered to be significant when p < 0.05.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Acid, Organic Solvent, and Proteolytic Enzyme Treatments of Human Aorta. After incubation with [¹⁴C]rofecoxib, the human abdominal aortic homogenate was treated by successive extraction and washing with 0.6 M TCA, 80% methanol, and 100% methanol, and then the resultant precipitated fraction was enzymatically treated successively with collagenase, elastase, and pronase (n = 1). As shown in Table 1, the largest amount of the radioactivity from [¹⁴C]rofecoxib was recovered in the elastolytic fraction (51.9% of the total covalent binding). The second largest amount of radioactivity was recovered in the collagenolytic fraction (40.6% of the total). The radioactivity recovered in the fraction after pronase treatment was 4.4%, and the radioactivity in the residue after the treatments with the proteolytic enzymes was 3.0%.

Effect of Protein Modifiers on in Vitro Covalent Binding of [¹⁴C]Rofecoxib to Human Aorta Homogenate. Homogenate of human aorta, which had been pretreated with 10 mM of protein modifiers, was incubated with [¹⁴C]rofecoxib (100 µM) under a physiological pH condition (pH 7.4), and the amount of covalent binding to the proteins was measured (Fig. 3). The radioactivity from [¹⁴C]rofecoxib bound covalently to the aorta without any protein modifiers was 1.10 ± 0.06 µg Eq/g (control). Pretreatment of the aortic homogenate with the protein modifiers BAPN, D-penicillamine, or hydralazine decreased the amount of covalent binding to 56, 52, and 31% of the control, respectively. Radiodetection HPLC analysis of the reaction mixture supernatant after the incubation demonstrated that the major component is the unchanged rofecoxib (>95%, data not shown). In a separate experiment, BAPN and hydralazine significantly decreased the LOX activity to 4.1 and 0% of the control, respectively, whereas rofecoxib did not decrease it at all. The LOX activity in the presence of D-penicillamine could not be measured because the fluorescence in the assay mixture was too strong, perhaps because of the production of some unknown compound(s) with fluorescence.

Effect of Several COX-2 Inhibitors on in Vitro Covalent Binding of [¹⁴C]Rofecoxib to Rat and Human Aorta Homogenates. Homogenate of rat aorta was incubated with [¹⁴C]rofecoxib (50 µM) under a physiological pH condition (pH 7.4) in the presence or absence of nonradiolabeled COX-2 inhibitors (0.5 mM), and the amount of the radioactivity that bound covalently to the proteins was measured (Table 2). The covalently bound aortic radioactivity from [¹⁴C]rofecoxib in the control was 1.66 ± 0.07 µg Eq/g. The amount was significantly decreased in the presence only of nonradiolabeled rofecoxib to 53% of the control, but not of the other COX-2 inhibitors at all. On the other hand, the binding amount of 50 µM[¹⁴C]celecoxib (0.19 ± 0.17 µg Eq/g) was significantly lower than that of [¹⁴C]rofecoxib (control).

Human aortic homogenate was treated in the same way as described above, and the effect of nonlabeled COX-2 inhibitors (0.5 mM) on the covalent binding of [¹⁴C]rofecoxib (50 µM) was investigated (Table 2). In the control, the covalent binding of [¹⁴C]rofecoxib was 0.46 ± 0.02 µg Eq/g. The addition of nonradiolabeled rofecoxib to the aortic homogenate significantly decreased it to 22% of the control, whereas the other COX-2 inhibitors had no such effect at all. The binding of 50 µM[¹⁴C]celecoxib (0.09 ± 0.09 µg Eq/g) was significantly lower than that of [¹⁴C]rofecoxib (control). The binding of 25 and 100 µM [¹⁴C]rofecoxib was 0.24 ± 0.01 and 0.88 ± 0.05 µg Eq/g, respectively.

Radiodetection HPLC analysis of the reaction mixtures of rat and human samples demonstrated that the major component in the reaction mixtures was unchanged rofecoxib (>95%, data not shown). All the COX-2 inhibitors tested had no significant inhibitory effect on the LOX activity in rat and human aortas.

View larger version (26K): [in this window] [in a new window]   FIG. 4. Reconstructed ion chromatograms of m/z 488 (A), 421 (B), 465 (C), 463 (D), and 421 (E), corresponding to the estimated protonated molecules of the adducts formed from each COX-2 inhibitor with benzaldehyde. Each COX-2 inhibitor (100 µM) was incubated with benzaldehyde (1 mM) in potassium phosphate buffer (pH 7.4) at 37°C for 24 h. Positive ion mode results are representatively shown. Nominal mass: celecoxib (381), valdecoxib (314), etoricoxib (358), CS-706 (356), rofecoxib (314), and benzaldehyde (106). Peaks shown as asterisks were confirmed not to be peaks derived from the adducts with benzaldehyde.

View larger version (13K): [in this window] [in a new window]   FIG. 5. Uptake and efflux of [14C]rofecoxib (circle) and [14C]celecoxib (triangle) in HAOEC (n = 3, mean ± S.D.). For the uptake experiment (closed symbols), each compound (10 µM) was incubated with HAOEC at 37°C up to 360 min. For the efflux experiment (open symbols), each compound (10 µM) was incubated with HAOEC for 120 min, and then the cells were washed and incubated at 37°C with 20 µM of each nonlabeled compound.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
After incubation with [¹⁴C]rofecoxib, human abdominal aortic homogenate was washed and extracted using TCA/methanol, and the residue was successively enzymatically treated with collagenase, elastase, and pronase and then the radioactivity recovered in each fraction was measured. As shown in Table 1, the largest amount of the radioactivity from [¹⁴C]rofecoxib that was covalently bound to the aorta was recovered in the elastolytic fraction, suggesting that rofecoxib is covalently bound to elastin in the human aorta. This result was consistent with that obtained in rat aorta (Oitate et al., 2006).

To investigate the reactivity of rofecoxib with the allysine aldehyde in human elastin compared with that in rat elastin (Oitate et al., 2007), in vitro binding studies using human aortic homogenate were conducted in the presence of some protein modifiers (Fig. 3). Pretreatment of BAPN, a specific inhibitor of LOX (Tang et al., 1983), significantly decreased the covalent binding of [¹⁴C]rofecoxib, probably via the prevention of allysine production from lysine. The covalent binding of [¹⁴C]rofecoxib was also significantly decreased by pretreatment with D-penicillamine and hydralazine, which have been reported to react with protein aldehydes in connective tissues and to form thiazolidine or hydrazone analog (hydralazine can also inhibit LOX activity) (Pinnell et al., 1968; Deshmukh and Nimni, 1969; Gallop and Paz, 1975; Numata et al., 1981; Howard-Lock et al., 1986). These results strongly suggested that the aldehydic functional group in human elastin, i.e., the aldehyde group of allysine, is relevant to the covalent binding with rofecoxib, as it is in rat elastin (Oitate et al., 2007). In addition, the covalent binding of [¹⁴C]rofecoxib to human aorta was concentration-proportional in the range between 25 to 100 µM (Table 2), suggesting that the binding capacity is fairly high.

In the enzymatic fractionation of the human aorta (Table 1), the rank order of recovery of radioactivity was similar to that in rat aortas after oral administration of [¹⁴C]rofecoxib in vivo (Oitate et al., 2006). However, it differed in one way: in the rat aorta almost all of the radioactivity (>90%) was recovered in the elastolytic fraction with a small percentage in the collagenolytic fraction, whereas in the human aorta approximately 41% of the total binding radioactivity was recovered in the collagenolytic fraction as well as in the elastolytic fraction. This difference might come from dissimilarities in the parts of the aortas used (human, abdominal; rat, thoracic). It has been reported that the content of elastin in the aortic wall decreases progressively, whereas in contrast, the content of collagen increases, since the aorta is traversed longitudinally from the arch to abdominal (Grant, 1967). In collagen, allysine is also formed from the lysine residue by LOX and contributes importantly to the elasticity of CV tissues, as well as elastin (Eyre et al., 1984). Pinnell and Martin (1968) have reported that, in the case of elastin, 5 to 16 lysyl residues per 10³ amino acids are converted to allysine residues, compared with one lysyl residue per 10³ amino acids in collagen. From the above results in the human abdominal aorta, it was suggested that rofecoxib is capable of covalently binding to the allysine aldehyde in elastin and partially binding to that in collagen.

After the incubation of [¹⁴C]rofecoxib with rat aortic homogenate in vitro in the presence or absence of nonradiolabeled rofecoxib, celecoxib, valdecoxib, etoricoxib, and CS-706, the amount of covalent binding was measured (Table 2). The binding of [¹⁴C]celecoxib was significantly lower than that of [¹⁴C]rofecoxib, supporting our previous result; that is, there was no retention of radioactivity in the aorta after oral administration of [¹⁴C]celecoxib to rats in vivo, whereas the radioactivity from [¹⁴C]rofecoxib was retained by and accumulated in the rat aorta (Oitate et al., 2006). The covalent binding of [¹⁴C]rofecoxib to rat aortic homogenate in vitro was significantly decreased by the addition of only rofecoxib, whereas the other COX-2 inhibitors had no such effect. As well as the above results in rats, only the addition of nonradiolabeled rofecoxib to human aortic homogenate could significantly reduce the covalent binding of [¹⁴C]rofecoxib, and the binding of [¹⁴C]celecoxib was much less than that of [¹⁴C]rofecoxib (Table 2). In the cases of both rats and humans, rofecoxib did not affect the LOX activity at all. Therefore, the decrease of [¹⁴C]rofecoxib binding by nonradiolabeled rofecoxib was thought to be due to a competitive inhibition of allysine aldehyde. This suggested that other COX-2 inhibitors except for rofecoxib have no reactivity with the allysine aldehyde in human elastin, the same as in rats and collagen.

To estimate the reactivity of COX-2 inhibitors with the allysine aldehyde in human elastin and collagen with certainty, they were incubated with benzaldehyde, a model compound of allysine, under a physiological pH condition, as previously reported (Oitate et al., 2007). In the case of rofecoxib (as a positive control), the two peaks P_A and P_B were detected as reported previously (Fig. 4). By LC/MS and LC-NMR analyses, they had been identified as the covalent adducts of rofecoxib with benzaldehyde and diastereomers (Oitate et al., 2007). On the other hand, in the cases of other COX-2 inhibitors, no reaction products that were considered to be adducts with benzaldehyde were detected at all by LC/MS analyses operated in both the positive and negative ion modes. The C5 position in rofecoxib was considered to be sufficiently nucleophilic to react with aldehyde (Oitate et al., 2007). The other COX-2 inhibitors tested structurally do not contain a carbon atom carrying hydrogen or allylic to the carbonyl group (Fig. 2), and therefore it is reasonable to conclude that these compounds did not react with benzaldehyde.

In clinical situations, the C_max of rofecoxib at a general therapeutic dose (25 mg) was reported to be 1 µM (Davies et al., 2003). On the other hand, the in vitro binding of [¹⁴C]rofecoxib to the human aorta was linear, at least in the concentration range of 25 to 100 µM (Table 2), and the binding capacity was high, as mentioned above. Considering that rofecoxib binds to allysine in a covalent manner, rofecoxib has often been chronically dosed in clinical situations, and because both elastin and collagen have very slow turnover rates (Maroudas et al., 1992; Petersen et al., 2002), rofecoxib would be increasingly accumulated in the human aorta depending on the dose frequency. From the above results, it is presumed that in clinical situations rofecoxib, but not other COX-2 inhibitors, might prevent the cross-linking process of elastin and collagen, leading to the degradation of elastin and collagen, the dysfunction of arteries, and finally CV events.

It has been generally accepted that CV events 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 vaso-constrictor and promoter 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 CV risks (McAdam et al., 1999). Both [¹⁴C]rofecoxib and [¹⁴C]celecoxib were taken up by HAOEC, and the intracellular concentration of [¹⁴C]celecoxib at 120 min was about 3-fold higher than that of [¹⁴C]rofecoxib (Fig. 5). On the other hand, the efflux of both compounds was very rapid without any retention of radioactivity in the cells, suggesting that both compounds might not be retained by and accumulated in human aortic endothelial cells in vivo. When the concentrations in the plasma are high immediately after the administrations, it is thought that the concentrations in the endothelial cells and platelets are also high and that all selective COX-2 inhibitors, including rofecoxib and celecoxib, would theoretically cause a PGI₂/TxA₂ imbalance. However, it is quite difficult to postulate that rofecoxib has an especially strong effect in causing an imbalance compared with other compounds.

In conclusion, it was suggested that rofecoxib, but not the other COX-2 inhibitors, can covalently bind to the allysine aldehyde in human elastin as well as that in rats (Fig. 1B) and that it can bind partially in human collagen. This would lead to the prevention of the normal cross-linking processes and dysfunction of the arteries. This might be one of the main causes of CV events by rofecoxib in clinical situations. In this view, other COX-2 inhibitors could be less toxic in terms of CV risks than rofecoxib.

	Acknowledgments

 
We would like to thank Philip Snider for editing the manuscript.

	Footnotes

 
doi:10.1124/dmd.107.016121.

ABBREVIATIONS: COX-2, cyclooxygenase-2; CV, cardiovascular; PGI₂, prostacyclin; TxA₂, thromboxane A₂; LOX, lysyl oxidase; CS-706, 2-(4-ethoxyphenyl)-4-methyl 1-(4-sulfamoylphenyl)-1H-pyrrole; BAPN, ß-aminopropionitrile; HPLC, high-performance liquid chromatography; LC/MS, liquid chromatography/mass spectrometry; TCA, trichloroacetic acid; HBSS, Hanks' balanced salt solution; HAOEC, human aortic endothelial cell(s); ANOVA, analysis of variance.

Address correspondence to: Masataka Oitate, Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan. E-mail: oitate.masataka.i3{at}daiichisankyo.co.jp

	References

Top Abstract Materials and Methods Results Discussion References

 


Aldington S, Shirtcliffe P, Weatherall M, and Beasley R (2005) Systematic review and meta-analysis of the risk of major cardiovascular events with etoricoxib therapy. N Z Med J 118: U1684.[Medline]

Andrews EJ, White WJ, and Bullock LP (1975) Spontaneous aortic aneurysms in blotchy mice. Am J Pathol 78: 199-210.[Abstract]

Davies NM, Teng XW, and Skjodt NM (2003) Pharmacokinetics of rofecoxib: a specific cyclo-oxygenase-2 inhibitor. Clin Pharmacokinet 42: 545-556.[CrossRef][Medline]

Deshmukh K and Nimni ME (1969) A defect in the intramolecular and intermolecular cross-linking of collagen caused by penicillamine. II. Functional groups involved in the interaction process. J Biol Chem 244: 1787-1795.[Abstract/Free Full Text]

Eyre DR, Paz MA, and Gallop PM (1984) Cross-linking in collagen and elastin. Annu Rev Biochem 53: 717-748.[CrossRef][Medline]

Fredy J, Diggins Jr DA, and Morrill GB (2005) Blood pressure in Native Americans switched from celecoxib to rofecoxib. Ann Pharmacother 39: 797-802.[Abstract/Free Full Text]

Gallop PM and Paz MA (1975) Posttranslational protein modifications, with special attention to collagen and elastin. Physiol Rev 55: 418-487.[Abstract/Free Full Text]

Graham DJ, Campen D, Hui R, Spence M, Cheetham C, Levy G, Shoor S, and Ray WA (2005) Risk of acute myocardial infarction and sudden cardiac death in patients treated with cyclooxygenase 2 selective and non-selective non-steroidal anti-inflammatory drugs: nested case-control study. Lancet 365: 475-481.[Medline]

Grant RA (1967) Content and distribution of aortic collagen, elastin and carbohydrate in different species. J Atheroscler Res 7: 463-472.[Medline]

Hashimoto K, McEvoy B, and Belcher R (1981) Ultrastructure of penicillamine-induced skin lesions. J Am Acad Dermatol 4: 300-315.[Medline]

Herd K and Orbison JL (1966) Lathyrism in the fetal rat. Evidence for impairment of elastin formation. Arch Pathol 81: 60-66.[Medline]

Howard-Lock HE, Lock CJ, Mewa A, and Kean WF (1986) D-Penicillamine: chemistry and clinical use in rheumatic disease. Semin Arthritis Rheum 15: 261-281.[CrossRef][Medline]

Junker P, Helin G, Jensen BA, Oxlund H, and Lorenzen I (1982) D-Penicillamine-induced angiopathy in rats. Changes in aortic collagen, glycosaminoglycans, DNA and RNA in rats treated with D-penicillamine. Atherosclerosis 45: 17-31.[CrossRef][Medline]

Light N, Meyrick ThomasRH, Stephens A, Kirby JD, Fryer PR, and Avery NC (1986) Collagen and elastin changes in D-penicillamine-induced pseudoxanthoma elasticum-like skin. Br J Dermatol 114: 381-388.[CrossRef][Medline]

Maroudas A, Palla G, and Gilav E (1992) Racemization of aspartic acid in human articular cartilage. Connect Tissue Res 28: 161-169.[Medline]

McAdam BF, Catella-Lawson F, Mardini IA, Kapoor S, Lawson JA, and FitzGerald GA (1999) Systemic biosynthesis of prostacyclin by cyclooxygenase (COX)-2: the human pharmacology of a selective inhibitor of COX-2. Proc Natl Acad Sci U S A 96: 272-277.[Abstract/Free Full Text]

Numata Y, Takei T, and Hayakawa T (1981) Hydralazine as an inhibitor of lysyl oxidase activity. Biochem Pharmacol 30: 3125-3126.[CrossRef][Medline]

Nussmeier NA, Whelton AA, Brown MT, Langford RM, Hoeft A, Parlow JL, Boyce SW, and Verburg KM (2005) Complications of the COX-2 inhibitors parecoxib and valdecoxib after cardiac surgery. N Engl J Med 352: 1081-1091.[Abstract/Free Full Text]

Oitate M, Hirota T, Koyama K, Inoue S, Kawai K, and Ikeda T (2006) Covalent binding of radioactivity from [¹⁴C]rofecoxib, but not [¹⁴C]celecoxib or [¹⁴C]CS-706, to the arterial elastin of rats. Drug Metab Dispos 34: 1417-1422.[Abstract/Free Full Text]

Oitate M, Hirota T, Takahashi M, Murai T, Miura S, Senoo A, Hosokawa T, Oonishi T, and Ikeda T (2007) Mechanism for covalent binding of rofecoxib to elastin of rat aorta. J Pharmacol Exp Ther 320: 1195-1203.[Abstract/Free Full Text]

Pinnell SR and Martin GR (1968) The cross-linking of collagen and elastin: enzymatic conversion of lysine in peptide linkage to alpha-aminoadipic-delta-semialdehyde (allysine) by an extract from bone. Proc Natl Acad Sci U S A 61: 708-716.[Free Full Text]

Pinnell SR, Martin GR, and Miller EJ (1968) Desmosine biosynthesis: nature of inhibition by D-penicillamine. Science 161: 475-476.[Abstract/Free Full Text]

Tang SS, Trackman PC, and Kagan HM (1983) Reaction of aortic lysyl oxidase with ß-aminopropionitrile. J Biol Chem 258: 4331-4338.[Abstract/Free Full Text]

Vrhovski B and Weiss AS (1998) Biochemistry of tropoelastin. Eur J Biochem 258: 1-18.[Medline]

Yoshikawa H, Wang S, Seo H, Kurotaki T, Ueki H, and Yoshikawa T (2001) Ultrastructure of aortic elastic fibers in copper-deficient Sika deer (Cervus nippon Temminck). J Vet Med Sci 63: 163-165.[CrossRef][Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.107.016121v1 35/10/1846    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Oitate, M. Articles by Ikeda, T. Search for Related Content PubMed PubMed Citation Articles by Oitate, M. Articles by Ikeda, T.