Abstract
In some cases, the formation of reactive species from the metabolism of xenobiotics has been linked to toxicity and therefore it is imperative to detect potential bioactivation for candidate drugs during drug discovery. Reactive species can covalently bind to trapping agents in in vitro incubations of compound with human liver microsomes (HLM) fortified with β-nicotinamide adenine dinucleotide phosphate (NADPH), resulting in a stable conjugate of trapping agent and reactive species, thereby facilitating analytical detection and providing evidence of short-lived reactive metabolites. Since reactive metabolites are typically generated by cytochrome P450 (CYP) oxidation, it is important to ensure high concentrations of trapping agents are not inhibiting the activities of CYP isoforms. Here we assessed the inhibitory properties of fourteen trapping agents against the major human CYP isoforms (CYP1A2, 2C9, 2C19, 2D6 and 3A). Based on our findings, eleven trapping agents displayed inhibition, three of which had IC50 values less than 1 mM (2-mercaptoethanol, N-methylmaleimide and N-ethylmaleimide (NEM)). Three trapping agents (dimedone, N-acetyl-lysine and arsenite) did not inhibit CYP isoforms at concentrations tested. To illustrate effects of CYP inhibition by trapping agents on reactive intermediate trapping, an example drug (ticlopidine) and trapping agent (NEM) were chosen for further studies. For the same amount of ticlopidine (1 μM), increasing concentrations of the trapping agent NEM (0.007–40 mM) resulted in a bell-shaped response curve of NEM-trapped ticlopidine S-oxide (TSO-NEM), due to CYP inhibition by NEM. Thus, trapping studies should be designed to include several concentrations of trapping agent to ensure optimal trapping of reactive metabolites.
Highlights
The relationship between drug bioactivation in vivo and subsequent toxicity is not straightforward; there have been reported instances of drug toxicity directly resulting from reactive metabolite formation [1,2]
Since hepatic cytochrome P450 (CYP) enzymes are mainly responsible for known bioactivation reactions [8] these assays are typically performed with human liver microsomes (HLM) fortified with reduced β-nicotinamide adenine dinucleotide phosphate (NADPH) [9]
CYP1A2 metabolism was activated by N-methylpiperidine at concentrations up to 12.5 mM, followed by inhibition up to 100 mM, resulting in an approximate IC50 of 50 mM
Summary
The relationship between drug bioactivation in vivo and subsequent toxicity is not straightforward; there have been reported instances of drug toxicity directly resulting from reactive metabolite formation [1,2]. Detection of sulfenic acid is especially challenging, as it is a highly elusive and unstable intermediate with a very short half-life (less than 1 min) [12] It is quite reactive, and can undergo a multitude of reactions resulting in a wide variety of chemical species. Sulfenic acid can undergo further oxidations to more stable metabolites (such as sulfinic and sulfonic acids), react with another sulfenic acid (disproportionation reaction) to form a free thiol and a sulfinic acid, react with nucleophiles (like thiols) resulting in mixed disulfides [13], react with cyanide (resulting in thiocyanate), react with thiocyanate (resulting in dithiocyanate) [14], or even react with nucleophilic moieties of proteins, commonly with cysteine residues, to form covalent adducts [11] Due to this highly reactive behavior of sulfenic acid intermediates, detection is especially challenging. Chhememicicaallsstrtruuccttuurreess ooff ((AA)) nnuucclleeoopphhiilleess,;((BB))rreedduucicninggagaegnetnst,s(;C()CM) Michicahëlaëalccaecpcteoprtsoarns dand didenieonpohpihleilsesanadnd(D(D) )aararaddicicaallssccaavveennggeerr
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