Metabolism of loratadine and further characterization of its in vitro metabolites.

The present study demonstrated that in addition to CYP3A4 and CYP2D6, the metabolism of loratadine is also catalyzed by CYP1A1, CYP2C19, and to a lesser extent by CYP1A2, CYP2B6, CYP2C8, CYP2C9 and CYP3A5. The biotransformation of loratadine was associated with the formation of desloratadine (DL) and further hydroxylation of both DL and the parent drug (loratadine). Based on the inhibition and correlation studies contribution of CYP2C19 in the formation of the major circulating metabolite DL seems to be minor. Reported clinical results suggest that the steady state mean (%CV) plasma Cmax and AUC(24hr) of loratadine were 4.73 ng/ml (119%) and 24.1 ng.hr/ml (157%), respectively, after dosing with 10 mg loratadine tablets for 10 days. High inter-subject variability in loratadine steady-state data is probably due to the phenotypical characteristics of CYP2D6, CYP2C19, and CYP3A4. The relative abundance of CYP3A4 in the human liver exceeds that of CYP2C19 and CYP2D6 and therefore the contribution of CYP3A4 in the metabolism of loratadine should be major (approximately 70%).

Identification of human liver cytochrome P450 enzymes responsible for the metabolism of lonafarnib (Sarasar).

Lonafarnib (Sarasar), a farnesyl transferase inhibitor, is currently under development for the treatment of solid tumors. Incubation of lonafarnib with human liver microsomes resulted in the formation of four oxidative metabolites (M1, M2, M3, and M4). Minor to trace levels of these metabolites were detected in humans after multiple-dose administration of lonafarnib. Liquid chromatography-mass spectrometry analyses exhibited a mass to charge ratio (m/z) for the (M+H)(+) ion of M1, M2, M3, and M4 at 653, 635, 669, and 653 Th, respectively. These metabolites, respectively, resulted from changes of +O, -2H, +2O, and +O relative to lonafarnib. Recombinant human CYP3A4 and CYP3A5 exhibited catalytic activity with respect to the formation of M1, M2, and M3, whereas CYP2C8 exhibited catalytic activity with respect to the formation of M4. There was a high correlation between the formation of M1, determined in 10 human liver microsomal samples, and 6beta-hydroxylation of testosterone catalyzed by CYP3A4/5 (r = 0.93). The IC(50) values of ketoconazole for inhibition of M1 and M2 were 0.61 and 0.92 microM, respectively. The formation of M4 by human liver microsomes was inhibited 72% by 50 microM quercetin, suggesting that the formation of M4 was mediated via CYP2C8. A CYP3A4/5-specific inhibitory monoclonal antibody inhibited the formation of M1, M2, and M3 by 85, 75, and 100%, respectively. In conclusion, the formation of metabolites M1, M2, and M3 from lonafarnib was mediated via CYP3A4 and CYP3A5.

Identification of human UDP-glucuronosyltransferase enzyme(s) responsible for the glucuronidation of 3-hydroxydesloratadine.

Desloratadine is a non-sedating antihistamine recently approved for the treatment of seasonal allergic rhinitis. The major metabolite of desloratadine in human plasma and urine is the glucuronide conjugate of 3-hydroxydesloratadine. 3-Hydroxydesloratadine-glucuronide is also the major in vitro metabolite of 3-hydroxydesloratadine formed by incubation of 3-hydroxydesloratadine with human liver microsomes supplemented with uridine 5'-diphosphate-glucuronic acid (UDPGA). The metabolite structure was confirmed by LC-MS and LC-MS/MS. Out of ten recombinant human UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A3, UGT1A8 and UGT2B15 exhibited catalytic activity with respect to the formation of 3-hydroxydesloratadine-glucuronide. Inhibition studies with known inhibitors of UGT (diclofenac, flunitrazepam and bilirubin) confirmed the involvement of UGT1A1, UGT1A3 and UGT2B15 in the formation of 3-hydroxydesloratadine-glucuronide. The results from this study demonstrated that the in vitro formation of 3-hydroxydesloratadine-glucuronide from 3-hydroxydesloratadine was mediated via UGT1A1, UGT1A3 and UGT2B15 in human liver.

Identification of human UDP-glucuronosyltransferase enzyme(s) responsible for the glucuronidation of ezetimibe (Zetia).

Ezetimibe [1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone] (Zetia; Schering-Plough, Kenilworth, NJ) is the first in a new class of cholesterol-lowering agents known as cholesterol absorption inhibitors. The objective of this study was to identify the isoform(s) of human liver and intestinal UDP-glucuronosyltransferase (UGT) enzymes responsible for the glucuronidation of ezetimibe. The main circulating metabolite of this drug in human plasma is SCH 60663, the phenolic glucuronide conjugate of ezetimibe. SCH 60663 [m/z = 584 Thompsons (Th)] is also the major in vitro metabolite formed by human liver microsomes supplemented with UDP glucuronic acid (UDPGA). In contrast to the liver, human jejunum microsomes supplemented with UDPGA converted ezetimibe to two glucuronides with the same mass (m/z = 584 Th) by liquid chromatography-mass spectrometry. One corresponds to the phenolic glucuronide (1-O-[4-trans-2S,3R)-1-(4-fluorophenyl)-4-oxo-3-[3(S)-hydroxy-3-(4-fluorophenyl)propyl]-2-azetidinyl]phenyl-beta-D-glucopyranuronic acid; SCH 60663) and the other was identified as the benzylic glucuronide of ezetimibe (1-O-[1(S)-(4-fluorophenyl)-3-[1-(4-fluorophenyl)-2(S)-(4-hydroxyphenyl)-4-oxo-3(R)-azetidinyl]propyl]-beta-D-glucopyranuronic acid; SCH 488128). Recombinant human UGT1A1, UGT1A3, and UGT2B15 all exhibited catalytic activity with respect to the formation of the phenolic glucuronide. However, UGT2B7 exclusively formed SCH 488128, a trace metabolite detected in dog and human plasma samples after oral administration of ezetimibe. In conclusion, the formation of SCH 60663 is mediated via UGT1A1, UGT1A3, and UGT2B15, and the formation SCH 488128 is mediated via UGT2B7.

Identification of human UDP-glucuronosyltransferase enzyme(s) responsible for the glucuronidation of posaconazole (Noxafil).

Posaconazole (Noxafil, SCH 56592), an orally available broad-spectrum triazole antifungal, is currently in phase III clinical studies for treating serious opportunistic fungal infections. The major in vitro metabolite of posaconazole formed by human liver microsomes supplemented with uridine 5'-diphosphate-glucuronic acid was a glucuronide of posaconazole (m/z877). Screening of 10 cDNA-expressed recombinant human UDP-glucuronosyltransferase (UGT) enzymes showed that only UGT1A4 exhibited catalytic activity with respect to the formation of the glucuronide of posaconazole. The formation of glucuronide by human liver microsomes and UGT1A4 was inhibited by bilirubin, a known inhibitor of UGT1A4. There was a high correlation (r =0.90) between the rate of formation of glucuronide, determined in 10 human liver microsomal samples, and trifluoperazine glucuronidation catalyzed by UGT1A4. These results confirmed that the formation of major posaconazole-glucuronide produced from human liver microsomes was mediated via UGT1A4.

Rapid determination of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytes.

Cytochrome P450 (CYP) substrates that yield fluorescent metabolites were used for rapid screening of drug metabolism activities of 13 recombinant human cytochromes P450, human liver microsomes and human hepatocytes. Reproducible results were obtained using a fluorescent plate reader (CytoFluor) more expediently than those generated using conventional HPLC methods. Typically, results for 96 samples were obtained with the plate reader in less than 10 min as opposed to 15-35 min/sample required by conventional HPLC. The fluorescent substrates used to measure CYP activities were as follows: 3-cyano-7-ethoxycoumarin (CEC) for CYP1A1, CYP1A2, CYP2C9 and CYP2C19; 7-ethoxyresorufin (7-ER) for CYP1A1, CYP1A2 and CYP1B1; 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) for CYP2D6; dibenzylfluorescein (DBF) for CYP3A4, CYP3A5 and CYP2C8; 7-methoxy-4-trifluoromethylcoumarin (7-MFC) for CYP2E1, CYP2B6 and CYP2C18; and coumarin for CYP2A6. The chemical inhibition and correlation data indicated that the following substrates can be used as specific functional probes for individual cytochrome P450 present in human liver microsomes: coumarin for CYP2A6 (r=0.82), AMMC for CYP2D6 (r=0.83) and DBF for CYP3A4 (r=0.92). The fluorescent plate reader was found to be useful for the rapid assessment of CYP activities (positive control) in both intact cells and subcellular fractions.