Stereoselective Metabolism of Carvedilol by the .BETA.-Naphthoflavone-Inducible Enzyme in Human Intestinal Epithelial 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][3][4] Carvedilol is highly lipophilic and is absorbed rapidly from the gastrointestinal tract after oral administration. Orally administered carvedilol undergoes stereoselective first-pass metabolism, and the blood concentration of R-enantiomer with very low b-blocking activity is approximately 2-fold higher than that of S-enantiomer with high b-blocking activity. 5,6) Both enantiomers are eliminated predominantly by hepatic metabolism, with renal excretion accounting for only 0.3% of the administered dose. 7) Carvedilol is metabolized extensively via aliphatic side-chain oxidation, aromatic ring oxidation, and conjugation pathways.8) Oldham and Clarke reported that oxidative activity for carvedilol is observed in cytochrome P450 (CYP) 2D6, 2C9, 3A4, and 1A2. 9) In addition, Ohno et al. reported that UDP-glucuronosyltransferase (UGT) 2B7, 2B4, and 1A1 are capable of catalyzing the glucuronidation of carvedilol. 10) However, it is still unclear which enzyme is responsible for the stereoselective presystemic clearance of carvedilol. Furthermore, although CYP3A4 and several UGTs are expressed in intestinal epithelial cells, it is also unknown whether the intestine as well as the liver is involved in the first-pass metabolism of carvedilol.The approach for investigating intestinal drug absorption and metabolism is to use human intestinal cell lines, such as the frequently used line Caco-2. This line spontaneously differentiates, after which it shows enterocyte-like morphology and forms polarized monolayers via tight junctions. It also expresses various transporters and efflux pumps such as P-glycoprotein.11,12) Therefore, it has been utilized to examine the mechanism responsible for the intestinal absorption of various compounds. 12,13) In addition, several UGTs, such as UGT1A1, 1A6, and 2B7, are expressed in Caco-2 cells, and the expression of some UGTs is induced by aromatic hydrocarbon receptor (AhR) ligands, such as b-naphthoflavone (b-NF).14,15) Therefore, glucuronidation of drugs in the intestine has been studied using Caco-2 cells. [16][17][18] In contrast, the Caco-2 cell line had not been employed to examine the intestinal metabolism of CYP3A4 substrates due to its lack of CYP3A4 expression. Recently, however, it has been reported that CYP3A4 in Caco-2 cells is induced by 1a,25-dihydroxyvitamin D 3 (VD 3 ). 11,19) The finding suggests that this cell line may also be utilized to investigate intestinal drug metabolism by CYP3A4. In the present study, we investigated whether carvedilol is metabolized stereoselectively in Caco-2 cells, and also evaluated the changes in the metabolic rate of carvedilol in b-NF-and VD 3 -treated Caco-2 cells. MATERIALS AND METHODS MaterialsCarvedilol was kindly supplied by Daiichi Pharmaceutical (Tokyo, Japan). b-NF was obtained from Nacalai Tesque (Kyoto, Japan). VD 3 and 3Ј-azido-3Ј-deoxythimidine (AZT...

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

“…Carvedilol was dissolved in a mixture of dimethylformamide and acetonitrile, and the final concentrations of dimethylformamide and acetonitrile in the culture medium were 0.05% and 0.02% (v/v), respectively. 9,10) To evaluate the metabolism of carvedilol, the cells were incubated for 24 h in an atmosphere of 5% CO 2 -95% air at 37°C. 9) We also evaluated the effect of quinidine (1 mM), furafylline (25 mM), ketoconazole (1 mM), and sulfaphenazole (10 mM) on the metabolism of carvedilol in b-NF-treated Caco-2 cells.…”

“…9,10) To evaluate the metabolism of carvedilol, the cells were incubated for 24 h in an atmosphere of 5% CO 2 -95% air at 37°C. 9) We also evaluated the effect of quinidine (1 mM), furafylline (25 mM), ketoconazole (1 mM), and sulfaphenazole (10 mM) on the metabolism of carvedilol in b-NF-treated Caco-2 cells. 7,9,16) The cells and culture medium were collected at 24 h after the addition of carvedilol, and stored at Ϫ30°C for the assay 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.…”

“…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.…”

“…9,10) We previously investigated the metabolism of carvedilol in human intestinal epithelial Caco-2 cells.…”