Involvement of the CYP1A Subfamily in Stereoselective Metabolism of Carvedilol in .BETA.-Naphthoflavone-Treated Caco-2 Cells

Abstract

Carvedilol is a b-adrenoceptor antagonist, and has been clinically used to treat chronic heart failure as well as hypertension, angina pectoris, and cardiac arrhythmias.1,2) Orally administered carvedilol undergoes stereoselective first-pass metabolism, and the blood concentration of R-enantiomer with low b-blocking activity is approximately 2-fold higher than that of S-enantiomer with high b-blocking activity. [3][4][5] Carvedilol is metabolized extensively via aliphatic side-chain oxidation, aromatic ring oxidation, and conjugation pathways.6) Oldham and Clarke reported that oxidative activity of carvedilol is observed in cytochrome P450 (CYP) 2D6, 1A2, 3A4, and 2C9.7) In addition, Ohno et al. reported that UDPglucuronosyltransferase (UGT) 2B7, 1A1, and 2B4 are capable of catalyzing the glucuronidation of carvedilol.8) However, it has been unknown whether the intestine also plays a role in the first-pass presystemic metabolism of carvedilol, although several CYP and UGT isoforms are expressed in human intestinal epithelial cells. 9,10) We previously investigated the metabolism of carvedilol in human intestinal epithelial Caco-2 cells.9) The metabolism of R-carvedilol was not significant in Caco-2 cells cultured on plastic dishes, whereas S-carvedilol was significantly metabolized in the cells. The metabolism of S-carvedilol was further increased by the treatment of Caco-2 cells with 50 m M b-naphthoflavone (b-NF) for 3 d. In addition, the mRNA expression level of the UGT1A subfamily in b-NF-treated Caco-2 cells was higher than that in non-treated cells, whereas the expression level of UGT2B4 and UGT2B7 mRNA in b-NF-treated Caco-2 cells were lower than that in non-treated cells.9) On the other hand, Takekuma et al. have recently evaluated the glucuronidation of racemic carvedilol by recombinant UGT1A1 and UGT2B7, and reported that UGT1A1 and UGT2B7 was mainly involved in the glucuronidation of R-and S-carvedilol, respectively. 11) These findings suggested that UGT was not involved in the stereoselective metabolism of carvedilol in b-NF-treated Caco-2 cells.The aim of the present study was to identify the CYP enzyme responsible for the stereoselective metabolism of carvedilol in b-NF-treated Caco-2 cells. We evaluated the effect of b-NF-treatment on the expression of CYP mRNA in Caco-2 cells, and then investigated the effect of CYP-specific inhibitors on the metabolism of carvedilol in b-NF-treated Caco-2 cells cultured on plastic dishes. Moreover, we examined whether the oxidation of carvedilol in microsomes of b-NF-treated Caco-2 cells was faster than glucuronidation of the drug. That is, the oxidation of carvedilol in microsomes was evaluated in the presence of NADPH, whereas glucuronidation was evaluated in the presence of UDP-glucuronic acid (UDPGA). 10) MATERIALS AND METHODS MaterialsCarvedilol was kindly supplied by Daiichi Pharmaceutical (Tokyo, Japan). 1a,25-Dihydroxyvitamin D 3 (VD 3 ), b-NF, and ketoconazole were purchased from Wako Pure Chemicals (Osaka, Japan). Quinidine hydrochloride, furafylline, ...

Inhibitory and Stimulative Effects of Amiodarone on Metabolism of Carvedilol in Human Liver Microsomes

“…According to previous reports, the concentration of CYP-specific inhibitors was determined as follows: 25 mM furafylline (inhibitor of CYP1A2), 10 mM sulfaphenazole (inhibitor of CYP2C9), 1 mM quinidine (inhibitor of CYP2D6), and 1 mM ketoconazole (inhibitor of CYP3A4). 9,10) The concentrations of amiodarone (50 mM) and desethylamiodarone (25 mM) were determined according to the report of Ohyama et al 13) In the pooled HLM (#18888), the oxidation activity for Rcarvedilol was significantly decreased by quinidine (to 49.7% of control), whereas that for S-carvedilol was decreased by furafylline (to 70.6% of control). The results reconfirmed that CYP2D6 and CYP1A2 in the pooled HLM contributed mainly to the oxidation of R-and S-carvedilol, respectively.…”

“…[13][14][15] Assay of Carvedilol Enantiomers The amount of carvedilol in the samples was measured using chiral high performance liquid chromatography (HPLC) as described by Saito et al with minor modifications. [7][8][9][10]16) Briefly, carvedilol was extracted from the samples (0.1 ml) with 5 ml diethylether after alkalization in 3 ml of 0.1 M Britton-Robinson buffer (pH 8.5). The organic phase was transferred and evaporated dry in a water bath at 45°C.…”

“…6) In addition, our previous findings indicated that R-carvedilol is metabolized mainly by CYP2D6 and partly by CYP1A2, 2C9, and 3A4, and that S-carvedilol is metabolized mainly by CYP1A2 and partly by CYP2C9, 2D6, and 3A4. [7][8][9][10] On the other hand, Ohno et al found that uridine 5Ј-diphosphate (UDP)-glucuronosyltransferase (UGT) 2B7, 2B4, and 1A1 are capable of catalyzing the glucuronidation of carvedilol using microsomes from insect cells expressing human UGT. 11) In addition, they have demonstrated that glucuronidation of R-carvedilol is mediated by UGT1A1 and 2B4, and that glucuronidation of S-carvedilol is mediated by UGT2B7 and 2B4.…”

Stereoselective Glucuronidation of Carvedilol in Human Liver and Intestinal Microsomes

“…The enzymes of humans involved in carvedilol metabolism have been identified to be cytochrome P450 (CYP) for oxidative metabolism, and UDP-glucuronosyltransferase (UGT) for conjugative metabolism [13,14]. In addition, CYP2D6 and CYP1A2 have been suggested to be predominant enzymes for oxidative metabolism of r-and s-carvedilol, respectively [10,[15][16][17]. With respect to the glucuronidation of carvedilol, it has been reported that of r-diastereomeric formation is mediated by UGT1A1 and UGT2B4, and that s-diastereomeric formation is mediated by UGT2B4 and UGT2B7 [16,18].…”

<i>In Vitro</i> Enhancement of Carvedilol Glucuronidation by Amiodarone-Mediated Altered Protein Binding in Incubation Mixture of Human Liver Microsomes with Bovine Serum Albumin

“…5) We previously demonstrated that R-carvedilol is metabolized mainly by CYP 2D6 and partly by CYP1A2, 2C9, and 3A4, and that S-carvedilol is metabolized mainly by CYP1A2 and partly by CYP2C9, 2D6, and 3A4. [6][7][8][9] On the other hand, Ohno et al found that uridine 5′-diphosphate (UDP)-glucuronosyltransferase (UGT) 2B7, 2B4, and 1A1 are capable of catalyzing the glucuronidation of carvedilol using microsomes from insect cells expressing human UGT. 10) They also reported that glucuronidation of R-carvedilol is mediated by UGT1A1 and 2B4, and glucuronidation of S-carvedilol is mediated by UGT2B7 and 2B4.…”