Multiple forms of cytochrome P450 are involved in the metabolism of ondansetron in humans.

Ondansetron is cleared primarily by metabolism in humans, with hydroxylation of the indole moiety in the 7- and 8-positions being the major identified phase I pathways. In vitro studies using lymphoblastoid cell lines expressing single human cytochrome P450 forms and hepatic microsomes were undertaken to investigate the forms involved in the metabolism of ondansetron in humans. The cell lines that expressed CYP1A1, CYP1A2, and CYP2D6 were shown to be capable of metabolizing [14C]ondansetron. Studies with human hepatic microsomes and the specific inhibitors furafylene, quinidine, and ketoconazole confirmed the role of CYP1A2 and CYP2D6 and also demonstrated the involvement of the CYP3A subfamily. The data in this study collectively indicate that multiple cytochrome P450 forms, including CYP1A1, CYP1A2, CYP2D6, and the CYP3A subfamily, are probably involved in the clearance of ondansetron in humans, with no single form of cytochrome P450 dominating the overall metabolism of ondansetron. The role played by CYP2D6 in the metabolism of [14C]ondansetron by human hepatic microsomes in vitro was shown to be minor. This finding is consistent with the lack of bimodality in the clinical pharmacokinetics of ondansetron. It is therefore concluded that ondansetron is metabolized by multiple forms of cytochrome P450, and this limits the likelihood of a clinically relevant interaction with ondansetron by a modulator of a single form of cytochrome P450.

High-performance liquid chromatographic purification, optimization of the assay, and properties of ribonucleoside diphosphate reductase from rabbit bone marrow.

Partial purification of ribonucleoside diphosphate reductase from rabbit bone marrow was achieved by size exclusion HPLC of the crude homogenate. This step, requiring < 15 min, led to 9- to 13-fold purification of the reductase and removal of 64% of the contaminating kinase/phosphatase activities, which in the crude extract degrade > 95% of substrate CDP when reductase is assayed. A systematic study was conducted to evaluate the influence of contaminating kinase/phosphatase activities on CDP concentration during the reductase-catalyzed reaction with either ATP or its kinase-inhibiting analog, 5'-adenylylimidodiphosphate (AMP-PNP), as the allosteric effector. Our studies demonstrated that in the presence of ATP, CDP levels fell instantly to < 24% but thereafter remained fairly constant due to recycling via CTP. In contrast, in the presence of AMP-PNP, CDP levels decreased continuously. The Km values of the reductase for CDP determined in the presence of ATP were significantly higher than those in the presence of AMP-PNP. Furthermore, we also found that the concentration of the ultimate electron donor dithiothreitol (DTT) required for optimum activity of the reductase varies significantly with the level of purity of the reductase preparation. Interestingly, DTT is an inhibitor of the reductase above the optimum concentration. This purification method and the optimized assay together with the understanding of the fate of CDP in partially purified preparations should find application in studies with reductases from other eukaryotic sources.

Synthesis and characterization of the oxygen and desthio analogues of glutathione as dead-end inhibitors of glutathione S-transferase.

The oxygen analogue, gamma-L-Glu-L-SerGly (GOH) and desthio analogue, gamma-L-Glu-L-AlaGly (GH) have been synthesized by a simple three step procedure involving active ester coupling of N-t-BOC-alpha-(4-nitrophenyl)-L-glutamate to L-SerGly and L-AlaGly, respectively. The two peptides are excellent dead-end inhibitors of isozymes 3-3 and 4-4 of rat liver glutathione S-transferase. At low fixed concentrations of 1-chloro-2,4-dinitrobenzene (CDNB) GOH and GH are linear competitive inhibitors of isozyme 3-3 vs glutathione with KI values of 13.0 and 116 microM, respectively. Both peptides are non-competitive (mixed-type) inhibitors vs CDNB when glutathione is the fixed substrate. Similar results are obtained with both peptides and isozyme 4-4. The results rule out ordered or ping-pong kinetic mechanisms where the electrophile adds first.

Investigation of the kinetic and stereochemical recognition of arene and azaarene oxides by isozymes A2 and C2 of glutathione S-transferase.

The stereoselectivity of the closely related isozymes A2 and C2 of rat liver glutathione S-transferase toward several arene and azaarene oxides is examined. Isozyme C2 is stereospecific, catalyzing attack of glutathione at the oxirane carbon of R absolute configuration for a series of K-region arene oxides including phenanthrene 9,10-oxide, 1. Substitution of nitrogen in the biphenyl system of 1 causes a loss in stereospecificity. Isozyme A2 exhibits a low degree of stereoselectivity toward both arene and azaarene oxides. Kinetic studies of the two isozymes show that although isozyme C2 turns over 1 faster than does isozyme A2 the opposite is true when 4,5- diazaphenanthrene 9,10-oxide is the substrate. The kinetic and stereochemical behavior of the homodimeric isozymes A2 and C2 can be used to predict the stereoselectivity of the heterodimeric isozyme AC perhaps suggesting that catalysis is insensitive to different subunit-subunit interactions in the three isozymes.