Use of Electrochemical Oxidation and Model Peptides To Study Nucleophilic Biological Targets of Reactive Metabolites: The Case of Rimonabant

Abstract

Electrochemical oxidation of drug molecules is a useful tool to generate several different types of metabolites. In the present study we developed a model system involving electrochemical oxidation followed by characterization of the oxidation products and their propensity to modify peptides. The CB1 antagonist rimonabant was chosen as the model drug. Rimonabant has previously been shown to give high covalent binding to proteins in human liver microsomes and hepatocytes and the iminium ion and/or the corresponding aminoaldehyde formed via P450 mediated α-carbon oxidation of rimonabant was proposed to be a likely contributor. This proposal was based on the observation that levels of covalent binding were significantly reduced when iminium species were trapped as cyanide adducts but also following addition of methoxylamine expected to trap aldehydes. Incubation of electrochemically oxidized rimonabant with peptides resulted in peptide adducts to the N-terminal amine with a mass increment of 64 Da. The adducts were shown to contain an addition of C5H4 originating from the aminopiperidine moiety of rimonabant. Formation of the peptide adducts required further oxidation of the iminium ion to short-lived intermediates, such as dihydropyridinium species. In addition, the metabolites and peptide adducts generated in human liver microsomes were compared with those generated by electrochemistry. Interestingly, the same peptide modification was found when rimonabant was coincubated with one of the model peptides in microsomes. This clearly indicated that reactive metabolite(s) of rimonabant identical to electrochemically generated species are also present in the microsomal incubations. In summary, electrochemical oxidation combined with peptide trapping of reactive metabolites identified a previously unobserved bioactivation pathway of rimonabant that was not captured by traditional trapping agents and that may contribute to the in vitro covalent binding.

Liabilities Associated with the Formation of “Hard” Electrophiles in Reactive Metabolite Trapping Screens

“…Interestingly, liver microsomal covalent binding was also reduced by approximately 30% upon coincubation with methoxylamine, but no corresponding aldoxime adducts expected from the reaction with the putative aminoaldehyde intermediate 84 were detected in HLM . Subsequent incubation of electrochemically oxidized [ 14 C]- 82 with tryptic BSA digest and model peptides such as leucine-enkephalinamide or angiotensin II resulted in the characterization of a peptide adduct (compound 90 ) derived from the reaction of the peptide N -terminal amino group with a electrophilic metabolite of 82 . The peptide adduct, which contained a C 5 H 4 fragment originating from the aminopiperidine motif in 82 , was also generated in rimonabant and leucine-enkephalinamide coincubations in HLM suggesting that reactive metabolite(s) of 82 identical to electrochemically generated species are also present in HLM incubations .…”

Paraquat–Melanin Redox-Cycling: Evidence from Electrochemical Reverse Engineering

“…Electrochemistry is emerging as an in vitro tool for investigations in redox biology since it permits highly sensitive measurements of oxidation and reduction reactions . For instance, electrochemical methods have been investigated to evaluate the antioxidant activities of food phenolics, − mimic oxidative drug metabolism (e.g., phase I P450 enzymes), − and correlate the biological activities of bioreductive antitumor drugs to their redox potentials . In addition, electrochemistry allows arbitrarily complex potential inputs to be imposed while the output currents are in a convenient format for analysis by advanced algorithms (e.g., signal processing).…”

Idiosyncratic Drug-Induced Liver Injury (IDILI): Potential Mechanisms and Predictive Assays

“…For reactive metabolite counts, GSH trapping can be used to quantify the level of oxidative metabolites before being run through HPLC and MS [144]. Additionally, adduct formation of proteins can also be measured using LC-MS, providing insight towards the mechanism of injury [117, 145]. …”