Many promising drug candidates metabolized by aldehyde oxidase (AOX) fail during clinical trial owing to underestimation of their clearance. AOX is species-specific, which makes traditional allometric studies a poor choice for estimating human clearance. Other studies have suggested using half-life calculated by measuring substrate depletion to measure clearance. In this study, we proposed using numerical fitting to enzymatic pathways other than Michaelis-Menten (MM) to avoid missing the initial high turnover rate of product formation. Here, product formation over a 240-minute time course of six AOX substrates-, and zoniporide-have been provided to illustrate enzyme deactivation over time to help better understand why MM kinetics sometimes leads to underestimation of rate constants. Based on the data provided in this article, the total velocity for substrates becomes slower than the initial velocity by 3.1-, 6.5-, 2.9-, 32.2-, 2.7-, and 0.2-fold, respectively, in human expressed purified enzyme, whereas the K m remains constant. Also, our studies on the role of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, show that ROS did not significantly alter the change in enzyme activity over time. Providing a new electron acceptor, 5-nitroquinoline, did, however, alter the change in rate over time for mumerous compounds. The data also illustrate the difficulties in using substrate disappearance to estimate intrinsic clearance.

No abstract

No abstract

No abstract

Although aldehyde oxidase (AO) is an important hepatic drug-metabolizing enzyme, it remains understudied and is consequently often overlooked in preclinical studies, an oversight that has resulted in the failure of multiple clinical trials. AO’s preclusion to investigation stems from the following: (1) difficulties synthesizing metabolic standards due to the chemospecificity and regiospecificity of the enzyme and (2) significant inherent variability across existing in vitro systems including liver cytosol, S9 fractions, and primary hepatocytes, which lack specificity and generate discordant expression and activity profiles. Here, we describe a practical bacterial biotransformation system, ecoAO, addressing both issues simultaneously. ecoAO is a cell paste of MoCo-producing Escherichia coli strain TP1017 expressing human AO. It exhibits specific activity toward known substrates, zoniporide, 4-trans-(N,N-dimethylamino)cinnamaldehyde, O6-benzylguanine, and zaleplon; it also has utility as a biocatalyst, yielding milligram quantities of synthetically challenging metabolite standards such as 2-oxo-zoniporide. Moreover, ecoAO enables routine determination of kcat and V/K, which are essential parameters for accurate in vivo clearance predictions. Furthermore, ecoAO has potential as a preclinical in vitro screening tool for AO activity, as demonstrated by its metabolism of 3-aminoquinoline, a previously uncharacterized substrate. ecoAO promises to provide easy access to metabolites with the potential to improve pharmacokinetic clearance predictions and guide drug development.

We investigate the mechanism of time-dependent inhibition (TDI) of human cytochrome P450 2D6 (CYP2D6) by 3,4-methylenedioxymethamphetamine (MDMA, ecstasy), one of the most widespread recreational drugs of abuse. In an effort to unravel the kinetic mechanism of the formation of metabolic inhibitory complex (MIC) of CYP2D6 with MDMA-derived carbene we carried out a series of spectrophotometric studies paralleled with registration of the kinetics of time-dependent inhibition (TDI) in CYP2D6-incorporated proteoliposomes. The high amplitude of spectral signal in this system allowed us to characterize the spectral properties of the formed MIC in details and obtain an accurate spectral signature of MIC formation. This information was then used in the studies with CYP2D6-containing microsomes of insect cells (CYP2D6 Supersomes™). Our results demonstrate that in both systems the formation of the ferrous carbene-derived MIC is relatively slow, reversible and is not associated with the accumulation of the ferric carbene intermediate, as takes place in the case of CYP3A4 and podophylotoxin. Furthermore, the limited amplitude of MIC formation suggests that only a fraction (∼50%) of spectrally detectable CYP2D6 in both proteoliposomes and Supersomes participates in the formation of MIC and is therefore involved in the MDMA metabolism. This observation reveals yet another example of a cytochrome P450 that exhibits persistent functional heterogeneity of its population in microsomal membranes. Our study provides a solid methodological background for further mechanistic studies of MIC formation in human liver microsomes and demonstrates that the potency and physiological relevance of MDMA-dependent TDI of CYP2D6 may be overestimated.

Cytochrome P450 (CYP) enzyme kinetics often do not conform to Michaelis-Menten assumptions, and time-dependent inactivation (TDI) of CYPs displays complexities such as multiple substrate binding, partial inactivation, quasi-irreversible inactivation, and sequential metabolism. Additionally, in vitro experimental issues such as lipid partitioning, enzyme concentrations, and inactivator depletion can further complicate the parameterization of in vitro TDI. The traditional replot method used to analyze in vitro TDI datasets is unable to handle complexities in CYP kinetics, and numerical approaches using ordinary differential equations of the kinetic schemes offer several advantages. Improvement in the parameterization of CYP in vitro kinetics has the potential to improve prediction of clinical drug-drug interactions (DDIs). This manuscript discusses various complexities in TDI kinetics of CYPs, and numerical approaches to model these complexities. The extrapolation of CYP in vitro TDI parameters to predict in vivo DDIs with static and dynamic modeling is discussed, along with a discussion on current gaps in knowledge and future directions to improve the prediction of DDI with in vitro data for CYP catalyzed drug metabolism.

No abstract