Development and Evaluation of an Electrochemical Method for Studying Reactive Phase-I Metabolites:  Correlation to in Vitro Drug Metabolism

Abstract

A reactive metabolite may react covalently with proteins or DNA to form adducts that ultimately may lead to a toxic response. Reactive metabolites can be formed via, for example, cytochrome P450-mediated phase 1 reactions, and in this study, we report the development and evaluation of an electrochemical method for generating reactive metabolites. Paracetamol was used as a test compound to develop the method. The stability of the electrochemically generated N-acetyl-p-benzoquinoneimine (NAPQI) from paracetamol was investigated at 37 degrees C at pH 5.0, 7.4, and 9.0. The highest stability of NAPQI was observed at pH 7.4. The reaction rate between NAPQI and glutathione (GSH) was studied with cyclic voltammetry. NAPQI reacted quantitatively with GSH within 130 ms. The reactivity of NAPQI toward other nucleophiles was investigated, and for the reaction with N-acetyltyrosine, a time-dependent formation of a conjugate with N-acetyltyrosine was observed from 0 to 4 min. The applicability of the method was evaluated with compounds that were able to form quinone imines (amodiaquine), quinones (3-tert-butyl-4-hydroxyanisole and p-cresol), imine methides (3-methylindole; trimethoprim), quinone methides (3,5-di-tert-butyl-4-hydroxytoluene), and nitrenium ions (clozapine). The compounds were oxidized in an analytical electrochemical cell, and the formed reactive metabolites were trapped with GSH. The samples were then analyzed by LC-MS and LC-MS/MS. For comparison, all compounds were incubated with GSH in rat and human liver microsomes, and the formation of GSH conjugates was compared with that observed by electrochemical oxidation. Furthermore, the electrochemical method was used to synthesize a GSH conjugate of clozapine, which made it possible to obtain structural information by NMR. In summary, a high degree of similarity was observed between the conjugates identified from electrochemical oxidation and GSH conjugates identified from incubation with liver microsomes. In conclusion, we have developed a method that is useful for studies on reactive metabolites and furthermore can be scaled up for the synthesis of GSH conjugates for NMR.

Role of Residue 87 in the Activity and Regioselectivity of Clozapine Metabolism by Drug-Metabolizing CYP102A1 M11H: Application for Structural Characterization of Clozapine GSH Conjugates

“…The spectrum shown in Fig. 5A is in full agreement with the 1 H NMR spectra of C-6 glutathionyl clozapine, which was identified previously as the major GSH conjugate formed by peroxidases and electrochemical oxidation of CLZ (Fischer et al, 1991;Liu and Uetrecht, 1995;Madsen et al, 2007). Two doublets at 6.96 and 7.23 ppm correspond to the protons at positions 9 and 7, respectively, with a small coupling constant of 2.5 Hz due to the proton in the meta position.…”

“…Fischer et al (1991) previously assigned a doublet at 6.96 ppm to H 7 and the signal at 7.23 ppm to H 9 . However, Madsen et al (2007) assigned a doublet at 6.96 ppm to H 9 and the signal at 7.23 ppm to H 7 (Madsen et al, 2007). Which signal corresponds to which proton could not be determined unequivocally only based on chemical shift and coupling pattern (Liu and Uetrecht, 1995).…”

“…1) (Maggs et al, 1995;Dragovic et al, 2010). Unequivocal structure determination by 1 H NMR has been published for only two of the GSH conjugates of CLZ (Fischer et al, 1991;Liu and Uetrecht, 1995;Madsen et al, 2007). The major GSH conjugate (CG-1; Fig.…”

On-line electrochemistry/liquid chromatography/mass spectrometry for the simulation of pesticide metabolism

“…Using this set-up, valuable information on the polarity of the oxidation products and on the formation of different isomers can be obtained, as was shown in our group for the antipsychotic agent clozapine [15] and for paracetamol [16]. Particularly for the investigation of reactive metabolites, on-line EC/LC/MS proved to offer some interesting aspects concerning the preparation [17] and the direct detection of short-lived species that often cannot be seen in biological systems [18]. Detailed information on the state-of-the-art in the field of EC/MS and EC/LC/MS has been published in recent reviews [19 -21].…”

Antimalarial benzoheterocyclic 4-aminoquinolines: Structure–activity relationship, in vivo evaluation, mechanistic and bioactivation studies

“…25 Other researchers have employed electrochemistry to mimic phase I oxidation of paracetamol, clozapine, trimethoprim and diclofenac. 26,27 The coupling of EC with MS means that MS/MS can be used to provide important structural information about the reactive metabolites formed. …”