1 Several selective 5-HT reuptake inhibitors (SSRIs) are inhibitors of the genetically polymorphic drug metabolizing enzyme, CYP2D6. We studied the interaction of venlafaxine, a new SSRI, with CYP2D6 in human liver microsomes. 2 Venlafaxine was a less potent inhibitor of this enzyme activity in vitro than other SSRIs tested. The average apparent K i values determined using CY P2D6-dependent dextromethorphan 0-demethylation were: 33, 52 and 22 PM for rac-venlafaxine, R( +)-venlafaxine and S (-)-venlafaxine, respectively, us 0.065 to 1.8 p~ for paroxetine, fluoxetine, norfluoxetine, fluvoxamine and sertraline. 3 Microsomes from human livers ( n = 3 ) and from yeast transformed with an expression plasmid containing human CYP2D6 cDNA catalyzed the 0-demethylation of venlafaxine, which is the major metabolic pathway in uiuo. Intrinsic metabolic clearance values ( Vmax/Km) indicated that S( -)-venlafaxine was cleared preferentially via this pathway. 4 In microsomes from CYP2D6-deficient livers (n = 2), Vmax/Km of O-demethylation of venlafaxine was one to two orders of magnitude lower and was similar to the rate of N-demethylation. 5 Studies with chemical probes which preferentially inhibit P450 isoforms suggested that CYP3A3/4 is involved in venlafaxine N-demethylation. 6 These in vitro findings predict phenotypic differences in the kinetics of venlafaxine in vivo, although the clinical importance of this is unclear as 0-demethylvenlafaxine is pharmacologically similar to the parent drug. The findings also predict relatively limited pharmacokinetic interaction between venlafaxine and other CYP2D6 substrates.
Chenodeoxycholic acid (CDCA) is a liver-formed detergent and plays an important role in the control of cholesterol homeostasis. During cholestasis, toxic bile acids (BA) accumulate in hepatocytes causing damage and consequent impairment of their function. Glucuronidation, a conjugation reaction catalyzed by UDP-glucuronosyltransferase (UGT) enzymes, is considered an important metabolic pathway for hepatic BA. This study identifies the human UGT1A3 enzyme as the major enzyme responsible for the hepatic formation of the acyl CDCA-24glucuronide (CDCA-24G). Kinetic analyses revealed that human liver and UGT1A3 catalyze the formation of CDCA-24G with similar K m values of 10.6 to 18.6 mol/L, respectively. In addition, electrophoretic mobility shift assays and transient transfection experiments revealed that glucuronidation reduces the ability of CDCA to act as an activator of the nuclear farnesoid X-receptor (FXR). Finally, we observed that treatment of human hepatocytes with fibrates increases the expression and activity of UGT1A3, whereas CDCA has no effect. In conclusion, UGT1A3 is the main UGT enzyme for the hepatic formation of CDCA-24G and glucuronidation inhibits the ability of CDCA to act as an FXR activator. In vitro data also suggest that fibrates may favor the formation of bile acid glucuronides in cholestatic patients.
In this study, we established useful and reliable methods for the direct detection of the variants of CYP3A5 gene by polymerase chain reaction (PCR) and DdeI restriction analysis. The frequency of CYP3A5 related SNPs in 200 healthy Japanese male subjects was determined. The homozygous wild-type (*1/*1) frequency was 7.0% (14/200), the heterozygous (*1/*3) frequency was 32.5% (65/200) and the homozygous mutant-type (*3/*3) frequency was 60.5% (121/200). The *6 allele was not detected in any of the Japanese individuals. This result suggests that an estimated 40% of the Japanese express relatively high levels of metabolically active CYP3A5 protein. The proposed detection assays are useful for screening the CYP3A5 related SNPs in pharmacogenetic research.
The hydrolysis of cocaine and its N-demethylated product, norcocaine, by esterases was examined in liver and serum. Both liver and serum enzymatically formed ecgonine methyl ester from cocaine. The liver enzyme had a much lower affinity for cocaine than that of serum, indicating that a different form of esterase was present in liver. The liver enzyme had a similar affinity for both norcocaine and cocaine. Likewise, the serum enzyme showed similar affinities for both substrates. The Vmax estimates, however, were consistently higher for norcocaine than cocaine in both liver and serum. Benzoyl ecgonine, a major metabolite of cocaine formed by hydrolysis, was not produced enzymatically in either serum or liver; the rate of spontaneous formation at physiological pH suggests that this metabolite may arise nonenzymatically in the body.
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