Characterization of an Etoposide-Glutathione Conjugate Derived from Metabolic Activation by Human Cytochrome P450

Abstract

Etoposide (VP-16), a DNA topoisomerase II poison widely used as an antineoplastic agent is also known to cause leukemia. One of its major metabolic pathways involves O-demethylation to etoposide catechol (etoposide-OH) by cytochrome P450 3A4 (CYP3A4). The catechol metabolite can undergo sequential one- and two-electron oxidations to form etoposide semi-quinone (etoposide-SQ) and etoposide quinone (etoposide-Q), respectively, which have both been implicated as cytotoxic metabolites. However, etoposide-Q is known to react with glutathione (GSH), which can protect DNA from oxidative damage by this reactive metabolite. In this study, etoposide-Q was reacted with GSH and the two etoposide-GSH conjugates were characterized. The major conjugate was etoposide-OH-6'-SG and the minor product was etoposide-OH-2'-SG. Etoposide-OH-6'-SG, which arose from Michael addition of GSH to etoposide-Q, was characterized by mass spectrometry and 2-D NMR. It was identified as the sole product from in vitro metabolism experiments using recombinant human CYP3A4 or liver microsomes incubated with etoposide in the presence of GSH. Etoposide-OH-6'-SG was also detected from incubations of etoposide-OH and GSH alone. Therefore, the presence of etoposide-OH, which can be formed from etoposide metabolism by CYP3A4, is essential for formation of the GSH conjugate. The oxidation of etoposide-OH to a quinone intermediate is likely the precursor in the formation of etoposide-OH-6'-SG.

A Phase I Trial of the HIV Protease Inhibitor Nelfinavir with Concurrent Chemoradiotherapy for Unresectable Stage IIIA/IIIB Non-small Cell Lung Cancer: A Report of Toxicities and Clinical Response

“…There is conflicting data whether this interaction would result in potential antagonism or enhancement of etoposide by nelfinavir as the CYP3A4 metabolites of etoposide have cytotoxic effect. (25) At present, although data are limited, there is no evidence of enhancement of grade 4 toxicities in trials of HIV patients receiving etoposide while on nelfinavir. (26, 27) Therefore the standard platform of platinum-etoposide was employed in this phase I trial.…”

Myeloperoxidase-Dependent Oxidation of Etoposide in Human Myeloid Progenitor CD34+Cells

“…Oxidation of etoposide results in the formation of several metabolites (Fan et al, 2006;Zheng et al, 2006). Patients receiving etoposide accumulate appreciable levels of etoposide catechol catalyzed by the action of CYP3A4 and CYP3A5 Zhuo et al, 2004).…”

“…Etoposide catechol can be converted to semiquinone radicals by one-electron oxidation catalyzed by MPO. MPO may also catalyze (in cells and in vitro) the formation of a two-electron oxidation product of etoposide, etoposide ortho-quinone that forms conjugates with GSH (Mans et al, 1992;Fan et al, 2006;Zheng et al, 2006) and may be responsible in part for the cyto-and genotoxic effects of etoposide because of its ability to stabilize topoisomerase II/DNA covalent complexes as studied in vitro (Lovett et al, 2001). …”

Mass spectrometry in the pharmacokinetic studies of anticancer natural products

“…In addition to DNA topoisomerase II‐mediated chromosomal breakage reported above, cytotoxicity of etoposide also involves a secondary mechanism related to the etoposide catechol, one of etoposide major metabolite obtained through the O‐demethylation by cytochrome P450 3A4 (Zheng et al, ). Indeed, this catechol metabolite can undergo sequential one‐ and two‐electron oxidations to form etoposide semi‐quinone and etoposide quinone, respectively, which have been recognized as cytotoxic metabolites.…”

“…Following redox cycling results in the generation of reactive oxygen species, such as hydrogen peroxide and superoxide, which undergo Fenton chemistry form hydroxil radicals. These hydroxil radicals are able to either damage DNA directly or induce the formation of lipid hydroperoxides, which break down to DNA‐reactive aldehydic bifunctional electrophiles (Zheng et al, ).…”