JPET Fast Forward. Published on December 12, 2006 as DOI: 10.1124/jpet.106.114447 JPET FastThis Forward. article has not Published been copyedited on andDecember formatted. The 12, final 2006 version as mayDOI:10.1124/jpet.106.114447 differ from this version.
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Title Page
Mechanism for Covalent Binding of Rofecoxib to Elastin of Rat Aorta
Masataka Oitate, Takashi Hirota, Makoto Takahashi, Takahiro Murai, Shin-ichi Miura, Akira Senoo, Downloaded from
Tsunemichi Hosokawa, Tadahiro Oonishi, and Toshihiko Ikeda
jpet.aspetjournals.org
Drug Metabolism and Pharmacokinetics Research Laboratories (M.O., M.T., T.M., S.M, T.I.), at ASPET Journals on September 27, 2021
Core Technology Research Laboratories (T.Ho.), R&D Headquarters (T.O.),
and R&D Project Management Department (T.Hi.), Sankyo Co., Ltd., Tokyo, Japan.
Department of Nursing, Kiryu Junior College, Gunma, Japan (A.S.).
1
Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on December 12, 2006 as DOI: 10.1124/jpet.106.114447 This article has not been copyedited and formatted. The final version may differ from this version.
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Running Title Page
Running Title: Covalent binding mechanism of rofecoxib to aortic elastin
Corresponding Author:
Masataka Oitate Downloaded from
Drug Metabolism and Pharmacokinetics Research Laboratories, Sankyo Co., Ltd.
1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan jpet.aspetjournals.org
TEL: (81)3-3492-3131
FAX: (81)3-5436-8567 at ASPET Journals on September 27, 2021
E-mail: [email protected]
Text pages: 38
Figures: 7
Tables: 1
References: 38
Abstract: 223 words
Introduction: 713 words
Discussion: 1,028 words
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Abbreviations:
COX-2, cyclooxygenase-2; CV, cardiovascular; LOX, lysyl oxidase; allysine,
α-aminoadipic-δ-semialdehyde; BAPN, β-aminopropionitrile; NaBH4, sodium borohydride.
Downloaded from
Recommended section: Cardiovascular jpet.aspetjournals.org at ASPET Journals on September 27, 2021
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Abstract
We have previously reported that oral administration of [14C]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 SKF-525A, a cytochrome P450 inhibitor, significantly decreased the systemic exposure of
5-hydroxyrofecoxib, one of the main metabolites of rofecoxib, while there was no statistically Downloaded from significant change in the retention of radioactivity from [14C]rofecoxib in the aorta. On the other
hand, the aortic retention of radioactivity closely correlated to the systemic exposure of unchanged jpet.aspetjournals.org rofecoxib in the dose range between 2 and 10 mg/kg. Covalent binding study of [14C]rofecoxib in vitro using rat aorta homogenate in the presence of D-penicillamine, hydralazine, at ASPET Journals on September 27, 2021
β-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. 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.
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Introduction
Rofecoxib (3-phenyl-4-[4-(methylsulfonyl)phenyl]-2-(5H)-furanone; VIOXX) is a potent and highly selective cyclooxygenase-2 (COX-2) inhibitor and had been widely used as a non-steroidal anti-inflammatory drugs (NSAIDs). In 2004, however, it was withdrawn from the market based on
the results of some clinical studies indicating association of its treatment with increased risk of Downloaded from adverse cardiovascular (CV) events, such as heart attack and stroke (Merck, 2004).
In the last few years, it has been reported that other selective COX-2 inhibitors (e.g. etoricoxib, jpet.aspetjournals.org parecoxib and valdecoxib) and non-selective NSAIDs (e.g. naproxen) may also have a potential for increased CV risk (Aldington et al., 2005; Nussmeier et al., 2005; NIH, 2004). However, rofecoxib at ASPET Journals on September 27, 2021 differs in the following ways: 1) a significantly greater frequency and higher odds of CV events
(Mamdani et al., 2004; Graham et al., 2005; Kimmel et al., 2005; Solomon et al., 2004a); 2) a shorter period and a lower dose (even at a clinical dose) leading to the incidence of CV events (Solomon et al., 2006); 3) an earlier onset and a greater hypertensive effect correlating closely with CV risk
(Solomon et al., 2004b; Wolfe et al., 2004; Brinker et al., 2004; Fredy et al., 2005). Therefore, it is suggested that rofecoxib could have distinctive mechanisms or more toxic potential leading to CV risks, in comparison with other selective COX-2 inhibitors or non-selective NSAIDs.
Regarding its mechanism, several hypotheses have been proposed so far. However, most of them are common to all selective COX-2 inhibitors or non-selective NSAIDs, and not specific to
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rofecoxib, e.g. prostacyclin/thromboxane A2 imbalance in arteries (McAdam et al., 1999) and disruption of the production of prostaglandins, which play an important homeostatic role in the kidney (Pope et al., 1993; Johnson et al., 1994). On the other hand, Walter et al. (2004) proposed the pro-oxidant effect theory; whereby rofecoxib, which is a sulfone-type COX-2 inhibitor, promotes
oxidative damage to low-density lipoprotein and phospholipids in vitro, and this action might lead to Downloaded from atherogenesis in vivo. This theory might be able to account for the high incidence of CV events by
rofecoxib, but it has not yet been proven in vivo. Reddy and Corey (2005) reported that the lactone jpet.aspetjournals.org ring in rofecoxib is capable of undergoing spontaneous oxidation. Furthermore, they report that if one of the resulting metabolites, its maleic anhydride form, is generated transiently in vivo, even at ASPET Journals on September 27, 2021 though this has not been detected at all, it could react with nucleophilic groups in the biomolecules, especially amino acids. Even considering this possibility, however, it is difficult to explain why the radioactivity is retained only in a limited number of tissues, such as the aorta, ligament, and cartilage
(Oitate et al., 2006).
In our previous study, we demonstrated that the radioactivity from [14C]rofecoxib was covalently bound to the arterial elastin of rats (Oitate et al., 2006). Elastin, a natural elastomer, is a key extracellular matrix protein, which provides CV tissues, e.g. arteries and heart valves, with tensile strength and elasticity, and maintains the tissue architecture (Vrhovski and Weiss, 1998). This physical property of elastin is due to covalent cross-linkage structures, such as desmosine and
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isodesmosine (Figure 7A). These cross-linkages are preceded by selective lysine oxidation by the enzyme lysyl oxidase (LOX) (EC 1.4.3.13) to produce a reactive aldehyde,
α-aminoadipic-δ-semialdehyde (allysine), which can spontaneously react with neighboring aldehydes or ε-amino groups to form cross-linkages. It has been reported that an anti-rheumatic
amino thiol, D-penicillamine, reacts with the aldehyde group of allysine to form a thiazolidine-type Downloaded from complex (Deshmukh and Nimni, 1969; Pinnell et al., 1968; Howard-Lock et al., 1986). Chronic
administration of this compound has also been known to cause deep lesions in the connective tissues, jpet.aspetjournals.org such as angiopathy and pseudoxanthoma elasticum in rats and humans (Junker et al., 1982; Light et al., 1986; Hashimoto et al., 1981). From this information, we hypothesized that rofecoxib and/or at ASPET Journals on September 27, 2021 its metabolite(s) might also form a covalent adduct with allysine in elastin, and cause damage to the elastin.
In the present study, we investigated the mechanism for covalent binding of rofecoxib to elastin utilizing in vivo and in vitro approaches, especially focusing on the possibility that rofecoxib, but not
5-hydroxyrofecoxib, a metabolite of rofecoxib, can bind to the aldehyde group of allysine.
Furthermore, the histopathological changes in the aortic walls of rats were examined by transmission electron microscopy after multiple oral administration of rofecoxib.
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Methods
Chemicals and Reagents
[Furanone-4-14C]rofecoxib (17 mCi/mmol) was synthesized at GE Healthcare Bio-Sciences (Little
Chalfont, Buckinghamshire, UK). The radiochemical purity was more than 99% in analysis by Downloaded from radiodetection-high performance liquid chromatography (HPLC). Non-radiolabeled rofecoxib,
5-hydroxyrofecoxib and valdecoxib, which was used as an internal standard substance for analysis of jpet.aspetjournals.org rofecoxib and 5-hydroxyrofecoxib by liquid chromatography-tandem mass spectrometry
(LC-MS/MS), were synthesized at Sankyo Co., Ltd. (Tokyo, Japan). Polyethylene glycol 400 at ASPET Journals on September 27, 2021
(PEG 400) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
D-Penicillamine, hydralazine, β-aminopropionitrile (BAPN), sodium borohydride (NaBH4) and benzaldehyde were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Deuterated
acetonitrile (CD3CN, 99.8% D) and deuterium oxide (D2O, 99.9% D) were purchased from Isotec
Inc. (Miamisburg, OH, USA) and used to prepare the solvents for LC-nuclear magnetic resonance
(NMR) analysis. All other reagents and solvents used were commercially available and were of extra pure, guaranteed or HPLC or LC/MS grade.
Dosing of Animals and Sample Collection
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Male Sprague-Dawley rats (6 weeks of age) were obtained from Charles River Japan, Inc.
(Yokohama, Japan), and were used after one week of acclimatization. The rats were housed in a temperature-controlled room with a 12-h light/dark cycle. Their body weights ranged from 210 g to 230 g at the start of dosing. The rats were fasted overnight before the dosing. Water was
available ad libitum throughout the study. Downloaded from
[14C]Rofecoxib (for radioactivity measurement in the aorta) or non-radiolabeled rofecoxib (for
analysis of rofecoxib and 5-hydroxyrofecoxib in plasma) was dissolved in PEG 400, and orally jpet.aspetjournals.org administered to rats at a dose of 2, 5 or 10 mg/2 mL/kg (N = 3). SKF-525A (50 mg/kg, Funakoshi
Co., Ltd., Tokyo, Japan) in isotonic saline was injected intraperitoneally into the rats at 0.5 h before at ASPET Journals on September 27, 2021 the administration of [14C]rofecoxib (2 mg/kg) or non-radiolabeled rofecoxib (2 mg/kg). Blood samples were collected with heparinized syringes at 0.5, 1, 2, 4, 6, 8, 10 and 24 h after the dosing of non-radiolabeled rofecoxib under diethyl ether anesthesia, and plasma was subsequently obtained by centrifugation. At 24 h after administration of [14C]rofecoxib, the thoracic aortas were collected from the rats after being euthanized by exsanguination under diethyl ether anesthesia.
For the histopathological study, rofecoxib was orally administered repeatedly to rats once daily for
4 weeks except on weekends (5 times a week) at a dose of 10 mg/2 mL/kg (N = 6). As a control, the vehicle (PEG 400) was orally administered (2 mL/kg, N = 6). At 72 h after the final dosing, the rats were euthanized by exsanguination under diethyl ether anesthesia, and the thoracic aortas were
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collected.
Covalent binding of [14C]rofecoxib in vitro to rat aorta homogenate and effect of protein modifiers
The thoracic aorta samples were obtained from untreated rats (N = 10). After removal of the Downloaded from adhering tissues, the aortic samples were pooled, weighed (ca. 600 mg, wet weight), and
homogenized in isotonic saline (5% w/v) using a motor-driven homogenizer. According to jpet.aspetjournals.org previous reports (Ohta et al., 1998; Wilmarth and Froines, 1992; Tang et al., 1983), the homogenates were pretreated with protein modifiers, D-penicillamine (10 mM), hydralazine (10 mM), BAPN (10 at ASPET Journals on September 27, 2021
mM) or NaBH4 (20 mM) 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 non-specific binding, the same treatments were performed at 4°C for 2 h (N = 3). After the incubation, 0.9 M trichloroacetic acid (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. This process was repeated twice. The resulting precipitates were air-dried and subjected to radioactivity measurement. The net amount of covalent-binding was
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calculated by subtracting the non-specific binding from the total.
LOX activity in the presence of either of the protein modifiers or rofecoxb was measured using an
Amplex Red fluorescence assay (Molecular Probes, OR, USA) (Payne et al., 2005). The assay reaction mixture consisted of 55.6 mM sodium borate (pH 8.2), 1.33 M urea, 55.6 µM Amplex Red,
0.11 units/mL horseradish peroxidase, and 11.1 mM 1, 5-diaminopentane (cadaverine) substrate Downloaded from
(final concentration: 50 mM, 1.2 M, 50 µM, 0.1 units/mL and 10 mM, respectively). To 0.9 mL of
the mixture, 0.1 mL of each of the aortic homogenate samples was added and incubated at 37°C for jpet.aspetjournals.org
15 min in the presence of rofecoxib and either of the protein modifiers, of rofecoxib only or of potassium phosphate buffer only. The fluorescent product was excited at 535 nm and the emission at ASPET Journals on September 27, 2021 was read at 595 nm using a fluorescence spectrophotometer GENious Plus (TECAN, Salzburg,
Austria). For evaluating the non-specific fluorescence, the same treatments as above were performed without homogenate samples, and subtracted from the total fluorescence. LOX activity was expressed as the mean relative intensity (%) of the activity when only rofecoxib was added
(control, 100%). The experiments were carried out in triplicate.
Condensation reaction of rofecoxib with benzaldehyde as a model reaction
[14C]Rofecoxib or 5-hydroxyrofecoxib (100 µM) was incubated with benzaldehyde (1 mM) in potassium phosphate buffer (pH 7.4) at 37°C for up to 24 h. The incubation was started by the
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addition of [14C]rofecoxib or 5-hydroxyrofecoxib dissolved in acetonitrile to a solution of benzaldehyde in the buffer. As a control, the incubation was performed in the absence of benzaldehyde. For LC-NMR analysis, non-radiolabeled rofecoxib (10 mM) was incubated with benzaldehyde (100 mM) in 90% (v/v) acetonitrile (pH 8.1) at 37°C for 2 h. The reaction mixtures
were frozen in a deep freezer to stop the reaction, and stored at -80°C until analysis using Downloaded from radiodetection-HPLC, LC/MS or LC-NMR.
jpet.aspetjournals.org
Analyses by radiodetection-HPLC, LC-MS/MS, LC/MS, and LC-NMR
Radiodetection-HPLC analysis. HPLC was performed on a model LC-10A system (Shimadzu at ASPET Journals on September 27, 2021
Corp., Kyoto, Japan) equipped with an Xterra MS C18 column (4.6 × 150 mm, 5 µm, Waters Corp.,
MA, USA) maintained at 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 column effluent was monitored by a SPD-10A UV detector (Shimadzu Corp., 280 nm), and by a Radiomatic
525TR radiochemical detector (PerkinElmer Life and Analytical Sciences, Boston, USA) using a 3 mL/min flow rate for the scintillation cocktail ULTIMA-FLO (PerkinElmer Life and Analytical
Sciences). The total recovery of the chromatographed radioactivity was greater than 95%.
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LC-MS/MS for rofecoxib and 5-hydroxyrofecoxib analysis in plasma. The plasma samples with an internal standard (I.S., valdecoxib) were extracted using a solid-phase extraction plate (VersaPlate
96-well-C2, 100 mg, Varian, Inc., CA, USA) under an acidic condition. The analytes and I.S. were separated by a NANOSPACE SI-2 HPLC (Shiseido Co., Ltd., Tokyo, Japan) using an Inertsil CN-3
column (2.1 × 150 mm, 5 µm, GL Sciences, Inc., Tokyo, Japan) with a mobile phase of Downloaded from acetonitrile/water/ammonium acetate/formic acid (410/590/0.77/1, v/v/w/v). The eluent from the
column was analyzed by an API-5000 LC-MS/MS system (Applied Biosystems/MDS SCIEX, CA, jpet.aspetjournals.org
USA) using the electrospray ionization (ESI) interface in the negative ion mode. The transitions m/z 313 → 285 for rofecoxib, m/z 329 → 257 for 5-hydroxyrofecoxib and m/z 313 → 118 for at ASPET Journals on September 27, 2021 the I.S. were detected in the multiple reaction monitoring mode. The calibration curve for rofecoxib and 5-hydroxyrofecoxib were linear at the concentration range of 5 to 2,500 ng/mL, and tenfold dilution of the sample had no influence on the assay reliability.
LC/MS analysis. The reaction mixtures, obtained after incubation of [14C]rofecoxib or
5-hydroxyrofecoxib with benzaldehyde, were analyzed by a Waters Q-Tof Ultima mass spectrometer
(Waters Corp.) operated in the positive and negative ion ESI mode for structural analysis. For obtaining the product ion spectra, argon gas was used as the collision gas and collision energy was set at 25 eV. The LC system used was a Waters Alliance 2695 separation module coupled with a
2996 photodiode array detector, scanned from 200 to 340 nm for 1 second. Chromatographic
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separations were carried out on an XTerra MS C18 column (2.1 × 150 mm, 5 µm, Waters Corp.) maintained at 30°C in a column oven. Solvent A and solvent B were delivered at a flow rate of 0.2 mL/min. The mobile phase contained 10% solvent B in solvent A initially, and the proportion of solvent B was increased linearly to 70% in 30 min.
LC-NMR analysis. A 200 µL aliquot of the reaction mixture, obtained after incubation of Downloaded from non-radiolabeled rofecoxib with benzaldehyde, was evaporated and reconstituted in 40 µL of 35%
acetonitrile, and 20 µL of the reconstituted sample was subjected to LC-NMR analysis. On-flow jpet.aspetjournals.org
LC-NMR analysis was performed using a Varian Inova 500 MHz NMR spectrometer (Palo Alto, CA,
USA) equipped with a flow probe having an active volume of 60 µL. The LC system used was a at ASPET Journals on September 27, 2021
Varian ProStar model 230 solvent delivery module and a model 310 UV/VIS detector. An XTerra
MS C18 column (2.1 × 150 mm, 5 µm, Waters Corp.) was isocratically eluted at an ambient
temperature with CD3CN/D2O (35/65, v/v) at a flow rate of 0.2 mL/min. The NMR spectra were referenced to the signal of deuterated acetonitrile at 2.0 ppm. The residual solvent signals of deuterium oxide and deuterated acetonitrile were suppressed by water elimination through transverse gradients (WET) solvent suppression.
Radioactivity measurement
Isolated aorta samples were measured for the wet weight, and each sample was solubilized with a
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tissue solubilizer NCS-II (GE Healthcare Bio-Sciences, 2 mL) under constant shaking at ca. 55°C.
Precipitated aortic homogenate samples were solubilized with 1 mL of NCS-II. After solubilization, these samples were mixed with 10 mL of liquid 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 in the aorta Downloaded from was calculated as an equivalent (Eq) value of [14C]rofecoxib, and expressed as a concentration per g
aorta. jpet.aspetjournals.org
Histopathological analysis at ASPET Journals on September 27, 2021
Aorta samples from the rats treated with rofecoxib (N = 6) or the vehicle (N = 6) were fixed using
1/2 Karnovsky’s solution in 0.1 M phosphate buffer (pH 7.4) for 3 h at room temperature. After the fixation, samples were processed and embedded in epoxy resin (TAAB Laboratories equipment Ltd.,
Berkshire, England). Sections were stained with tannic acid to demonstrate the elastic fibers
(Cotta-Pereira et al., 1976), in addition to the uranyl acetate and lead citrate staining, and were observed with a transmission electron microscope (Type H7500, Hitachi Science system, Ibaraki,
Japan).
Data analyses
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The area under the plasma concentration-time curve (AUC) to the last quantifiable time point
(AUC0-t) was calculated using the computer program, WinNonlin Professional (Version 4.0.1,
Pharsight Corporation, CA, USA) with a non-compartment model.
Statistical analysis of the experimental data was performed using an unpaired t-test. Differences
were considered to be significant when p < 0.05. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021
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Results
Pharmacokinetics in rats
After a single oral administration of rofecoxib (2 mg/kg) to the rats with or without treatment of
SKF-525A (50 mg/kg), plasma concentrations of rofecoxib and 5-hydroxyrofecosib were measured, Downloaded from
and AUC0-t values were calculated (Table 1). In a separate experiment, the rats with or without
treatment of SKF-525A were dosed with [14C]rofecoxib (2 mg/kg), and the radioactive jpet.aspetjournals.org concentrations in the aorta were measured (Table 1). Treatment with SKF-525A significantly
decreased the AUC0-t of only 5-hydroxyrofecoxib to one third of that without the treatment, but did at ASPET Journals on September 27, 2021
not decrease the AUC0-t of rofecoxib. However, the treatment with SKF-525A caused no significant change in the concentration of radioactivity in the aorta.
In the experiment to examine the dose-response of pharmacokinetics, the rats were dosed orally
with rofecoxib at a dose of 2, 5 or 10 mg/kg, and the AUC0-t values of unchanged rofecoxib were determined. Other rats were dosed orally with [14C]rofecoxib at a dose of 2, 5 or 10 mg/kg, and the concentrations of radioactivity in the aorta were determined. As shown in Figure 1, there was a
2 very good correlation (r = 0.959) between the AUC0-t of unchanged rofecoxib and the radioactive concentration in the aorta.
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Covalent binding of [14C]rofecoxib in vitro to rat aorta homogenate and effect of protein modifiers
Homogenate of rat aorta, which had been pretreated with protein modifiers, D-penicillamine (10
14 mM), hydralazine (10 mM), BAPN (10 mM) or NaBH4 (20 mM), was incubated with [ C]rofecoxib
(100 µM) under a physiological pH condition (pH 7.4), and the amount of the radioactivity bound Downloaded from covalently to the proteins was measured (Figure 2). The radioactivity bound covalently to the aorta
after incubation with the aortic homogenate without pretreatment with the protein modifiers was jpet.aspetjournals.org
12.9 ± 1.3 µg Eq/g (control). On the other hand, pretreatment of the aortic homogenate with the protein modifiers decreased the covalently bound aortic radioactivity to 15 to 40 % of the control. at ASPET Journals on September 27, 2021
Radiodetection-HPLC analysis of the deproteinated supernatant fraction demonstrated that the major component is the unchanged rofecoxib (> 90%, data not shown). In a separate experiment,
hydralazine, BAPN and NaBH4 all significantly decreased the LOX activity to 0%, 19% and 63% 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 due to its own fluorescence and/or the production of some unknown compound(s) with fluorescence.
Condensation reaction of rofecoxib with benzaldehyde as a model reaction
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[14C]Rofecoxib (100 µM) was incubated with benzaldehyde (1 mM) in the phosphate buffer under a physiological pH condition (pH 7.4), and the reaction mixture was analyzed by radiodetection-HPLC. As shown in Figure 3, in addition to the unchanged rofecoxib (eluted at 13.4
min), peaks of two radioactive components designated as PA and PB were observed at 15.6 and 16.4 Downloaded from min, respectively. The peak areas of PA and PB were increased with time during the incubation.
At 24 h, the sum of the peak areas of PA and PB accounted for approximately 30% of the total jpet.aspetjournals.org radioactivity chromatographed. No PA or PB was observed in the mixture in the absence of benzaldehyde.
The chemical structures of PA and PB were analyzed by LC/MS (Figure 4). Both PA and PB at ASPET Journals on September 27, 2021 exhibited the protonated molecule ion [M+H]+ at m/z 421, demonstrating that the molecular weight
is greater than that of rofecoxib (314) by a mass of benzaldehyde (106). As shown in Figure 4, PA
and PB showed the same mass spectra, indicating that PA and PB are diastereomers. By analyzing
the product ion spectra and fragmentation schemes for PA and PB, both PA and PB were proposed to be covalent adducts of rofecoxib with benzaldehyde formed by a condensation reaction. On the other hand, no reaction product was detected by LC/MS after incubation of 5-hydroxyrofecoxib with benzaldehyde.
Since PA and PB interconverted each other after isolation by HPLC (data not shown), we
determined the NMR spectra of PA and PB using an on-flow LC-NMR technique. Integration of the
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1 H NMR resonances of both PA and PB (Figure 5) established fourteen aromatic protons (6.99 to 7.72
ppm for PA, 7.20 to 7.78 ppm for PB), a pair of weak doublets corresponding to Ha and Hb (6.09 and
5.12 ppm for PA, 5.88 and 4.86 ppm for PB), and a methyl proton (3.16 ppm for PA and PB), clearly
demonstrating that PA and PB are covalent adducts of rofecoxib with benzaldehyde.
Downloaded from
Histopathological analysis
The histopathological changes of the elastic fiber system of thoracic aorta after 4-week multiple jpet.aspetjournals.org oral administration of rofecoxib (10 mg/kg) to rats were evaluated by transmission electron microscopy (Figure 6, representative electron micrographs). In the control (A), the elastic lamellae at ASPET Journals on September 27, 2021 of all samples (6 of 6) appeared as continuous thick bands and were regularly parallel-arranged, among which many smooth muscle cells were found easily. On the other hand, in the rofecoxib-treated aorta (B), disruption and swelling of the elastic lamellae were noticed (6 of 6).
Between the irregularly-branching elastic lamellae, the smooth muscle cells were decreased in number and focally replaced by an abnormally large amount of collagenous fibers.
20 JPET Fast Forward. Published on December 12, 2006 as DOI: 10.1124/jpet.106.114447 This article has not been copyedited and formatted. The final version may differ from this version.
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Discussion
In our previous study (Oitate et al., 2006), we demonstrated that the radioactivity from
[14C]rofecoxib was covalently bound to the arterial elastin after oral administration to rats, but we could not identify the reactive components, i.e. whether rofecoxib or its metabolite was involved in
the binding. In rats after administration of rofecoxib, the main components in plasma were Downloaded from reported to be unchanged rofecoxib, 5-hydroxyrofecoxib that was partially produced from the parent
by some isoforms of cytochrome P450, and its glucuronide jpet.aspetjournals.org
(5-hydroxyrofecoxib-O-β-D-glucuronide) (Halpin et al., 2000, Slaughter et al., 2003). Among them, the glucuronide was thought to be chemically inactive, so we hypothesized that either at ASPET Journals on September 27, 2021 rofecoxib or 5-hydroxyrofecoxib or both were bound to the elastin. As shown in Table 1, the treatment of rats with SKF-525A, a non-specific inhibitor of cytochrome P450, significantly
decreased the systemic exposure (AUC0-t) of 5-hydroxyrofecoxib with no statistical change in that of rofecoxib, while no significant change in the concentration of radioactivity retained by the aorta was observed, indicating that rofecoxib itself is bound covalently to elastin. Supporting this idea, there was a very good correlation between the systemic exposure of unchanged rofecoxib and the aortic radioactive concentration (Figure 1).
The elastic property of elastin is due to the existence of covalent cross-linkages (Vrhovski and
Weiss, 1998). The first step in this cross-linking is the formation of a highly reactive aldehyde,
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allysine, through oxidation of lysyl ε-amino groups in elastin by LOX. Once formed, the aldehyde side chain of allysine is thought to react chemically with the aldehyde groups of other allysine molecules via an aldol condensation reaction or with ε-amino groups of an unoxidized lysine residues via a Schiff’s base-formation producing the tetra-functional cross-linkages demosine and
isodemosine (Figure 7A). It has been demonstrated that the prevention of this cross-linking in Downloaded from elastin causes serious lesions of connective tissues in animals and humans (Yoshikawa et al., 2001;
Junker et al., 1982; Light et al., 1986; Hashimoto et al., 1981; Herd and Orbison, 1966; Andrews et jpet.aspetjournals.org al., 1975).
As shown in Figure 2, the covalent binding of [14C]rofecoxib to aortic homogenate in vitro was at ASPET Journals on September 27, 2021 significantly decreased by pretreatment of the homogenate with D-penicillamine or hydralazine, which has been reported to react with protein aldehydes in connective tissues and to form thiazolidine or hydrazone analogue (hydralazine is also able to inhibit LOX activity) (Deshmukh and
Nimni, 1969; Pinnell et al., 1968; Howard-Lock et al., 1986; Gallop and Paz, 1975; Numata et al.,
1981). BAPN, a specific inhibitor of LOX (Tang et al., 1983), also significantly decreased the binding, probably via the prevention of allysine production from lysine, or by the reduction of a number of aldehyde groups of allysine by reacting chemically with aldehyde groups through Schiff’s
base formation. In addition, reductive pretreatment of aortic homogenate with NaBH4, significantly blocked the binding, quite conceivably due to all existing aldehyde groups of allysine
22 JPET Fast Forward. Published on December 12, 2006 as DOI: 10.1124/jpet.106.114447 This article has not been copyedited and formatted. The final version may differ from this version.
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being reduced to alcohol. Regarding hydralazine and NaBH4 having a LOX inhibitory activity, they might decrease the binding by the prevention of allysine production, the same as BAPN, in addition to the above mentioned mechanisms. From these results, it was strongly suggested that the aldehydic functional group in elastin, i.e. the aldehyde group of allysine, is relevant to the covalent
binding with rofecoxib. Downloaded from
These findings were also strongly supported by the other in vitro experiment using benzaldehyde
as an aldehyde model compound. In this experiment, rofecoxib, but not 5-hydroxyrofecoxib, jpet.aspetjournals.org formed covalent adducts with benzaldehyde under a physiological pH condition, presumably by a condensation reaction (Figures 3, 4 and 5). The C5 position in rofecoxib was considered to be at ASPET Journals on September 27, 2021 sufficiently nucleophilic to react with aldehyde under a physiological pH condition (Figure 4).
This is consistent with the fact that 5-hydroxyrofecoxib, in which the C5 position is not nucleophilic because of hydroxylation, did not react with benzaldehyde.
In order to make it clear whether the covalent binding of rofecoxib to the allysine aldehyde has any effects on the arterial function, histopathological changes in the aortic wall were evaluated ultrastructurally after 4-week multiple oral administration of rofecoxib to rats (Figure 6). In the rofecoxib-treated group, the elastic lamellae in the tunica media of the thoracic aorta were focally disrupted, resulting in a greater degree of branching than those in the control aorta. These observations are similar to the findings in animals and humans under conditions inhibiting
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cross-linking of elastin fibers (Yoshikawa et al., 2001; Light et al., 1986; Hashimoto et al., 1981;
Herd and Orbison, 1966; Andrews et al., 1975). In our previous report (Oitate et al., 2006), we demonstrated that the radioactivity from [14C]rofecoxib was localized on the elastic fibers of rat aortas by microautoradiography, and that the accumulation of radioactivity in the aortas increased in
a dose-frequency dependent manner. Histopathological changes in the rat aortas after a 2-week Downloaded from treatment of rofecoxib was investigated in addition to the 4-week treatment, and we found that the
degree of elastin degradation was also dose-frequency dependent (data not shown), suggesting that jpet.aspetjournals.org the degree of rofecoxib-binding correlated with the degree of elastin degradation. It is thought to be possible that a degradation of elastic fibers similar to that observed in the aortas also occurs in the at ASPET Journals on September 27, 2021 coronary arteries and the cerebral arteries, in which elastin also plays an important role in maintaining elasticity. Heart attacks and strokes, which were observed in the clinical cases of rofecoxib administration, might be evoked as a consequence of dysfunction of these arteries.
In conclusion, we propose the mechanism for the covalent binding of rofecoxib with rat elastin as schematically shown in Figure 7. Under normal conditions (A), lysine in elastin is converted to allysine by LOX, and the subsequent spontaneous condensation between allysine and lysine residues produces further cross-linkages, desmosine and isodesmosine. However, rofecoxib reacts with the aldehyde group of allysine to give a condensation covalent adduct, leading to the prevention of normal cross-linking (B), which was considered to cause the degradation of elastic fibers as a
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consequence (Figure 6). Since allysine is commonly present in the arteries in experimental animals and humans, it is presumed that in clinical situations the rofecoxib-induced degradation of elastic fibers in the arteries would lead to a dysfunction of the arteries and to further increased risk of CV events. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021
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Acknowledgments
The authors would like to thank Dr. Yusuke Kondo, Ms. Miyuki Yamada, and Dr. Takafumi
Kohama for their invaluable discussions, advice, and technical assistance with the histopathological analysis; Mr. Takanori Hayashi for his skilled technical assistance; and Mr. Philip Snider for editing
the manuscript. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021
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Footnotes
Reprint request to:
Masataka Oitate
Drug Metabolism and Pharmacokinetics Research Laboratories, Sankyo Co., Ltd. Downloaded from
1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
TEL: (81)3-3492-3131 jpet.aspetjournals.org
FAX: (81)3-5436-8567
E-mail: [email protected] at ASPET Journals on September 27, 2021
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Legends for Figures
Figure 1. Relationship between the AUC0-t of rofecoxib and the radioactive concentration in the aorta after single oral administration of rofecoxib or [14C]rofecoxib to rats at a dose of 2, 5 or 10
mg/kg (Mean ± SD, N = 3) Downloaded from
Figure 2. Effect of protein modifiers on the covalent binding of [14C]rofecoxib to rat aorta jpet.aspetjournals.org homogenate (Mean ± SD, N = 3). [14C]Rofecoxib (100 µM) was incubated with rat aortic homogenate (5% w/v) with or without pretreatment of each protein modifiers, and the amount of at ASPET Journals on September 27, 2021 covalent binding was measured as described under Methods.
Figure 3. Radiodetection-HPLC chromatograms of samples after incubation of [14C]rofecoxib
(100 µM) with benzaldehyde (1 mM) under a physiological pH condition (pH 7.4)
Figure 4. Product ion spectra and proposed fragmentation schemes of PA and PB
1 Figure 5. H NMR spectra of PA and PB obtained by on-flow LC-NMR analysis
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Figure 6. Representative elastic fibers of the aortic media after 4-week multiple administration of rofecoxib (10 mg/kg) to rats. Transmission electron microscopy with tannic acid stain, magnification ×2,300. A, control (vehicle); B, rofecoxib treatment.
Figure 7. Structure and route of formation of elastin cross-linkages under normal conditions (A) Downloaded from and a proposed reaction mechanism for covalent binding of rofecoxib with elastin (B) jpet.aspetjournals.org at ASPET Journals on September 27, 2021
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Table 1. Effect of treatment of rats with SKF-525A on pharmacokinetics of rofecoxib
Rats were treated with SKF-525A (50 mg/kg, i.p.) before the dosing of rofecoxib or [14C]rofecoxib (2 mg/kg). Data are shown as the mean ± SD (N = 3). An asterisk indicates significant difference from the control (non-treatment) (p < 0.05).
Downloaded from
AUC0-t (µg•h/mL) Concentration in aorta Group
Rofecoxib 5-Hydroxyrofecoxib (µg Eq/g) jpet.aspetjournals.org
Control 10.3 ± 2.79 0.934 ± 0.135 0.74 ± 0.17
SKF-525A 14.6 ± 6.38 0.342 ± 0.130 * 0.98 ± 0.40 at ASPET Journals on September 27, 2021
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