Here we describe a high capacity and high-throughput, automated, 384-well CYP inhibition assay using well-known HLM-based MS probes. We provide consistently robust IC(50) values at the lead optimization stage of the drug discovery process. Our method uses the Agilent Technologies/Velocity11 BioCel 1200 system, timesaving techniques for sample analysis, and streamlined data processing steps. For each experiment, we generate IC(50) values for up to 344 compounds and positive controls for five major CYP isoforms (probe substrate): CYP1A2 (phenacetin), CYP2C9 ((S)-warfarin), CYP2C19 ((S)-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4/5 (testosterone and midazolam). Each compound is incubated separately at four concentrations with each CYP probe substrate under the optimized incubation condition. Each incubation is quenched with acetonitrile containing the deuterated internal standard of the respective metabolite for each probe substrate. To minimize the number of samples to be analyzed by LC-MS/MS and reduce the amount of valuable MS runtime, we utilize timesaving techniques of cassette analysis (pooling the incubation samples at the end of each CYP probe incubation into one) and column switching (reducing the amount of MS runtime). Here we also report on the comparison of IC(50) results for five major CYP isoforms using our method compared to values reported in the literature.

In the early stages of drug discovery, the formation of reactive metabolites is often assessed by co-incubating the drug in liver microsomes with a trapping agent in the presence of NADPH. Our group assessed the capability of commonly used trapping agents to reversibly inhibit major cytochrome P450 (CYP) isoforms. Glutathione and cyanide did not inhibit the enzymes at concentrations up to 10 mM; however methoxylamine did show inhibition, with IC(50) values of 0.53 mM for CYP1A2, 4.12 mM for CYP2C9, 2.04 mM for CYP2C19, 9.72 mM for CYP2D6, and 1.26 and >10 mM for CYP3A4/5 (for testosterone and midazolam, respectively, as substrates).

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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.

Early in the drug discovery process, the identification of cytochrome P450 (CYP) time-dependent inhibition (TDI) is an important step for compound optimization. Here we describe a high-throughput, automated method for the evaluation of TDI utilizing human liver microsomes and conventional CYP-specific mass spectrometer-based probes in a 384-well format. One of the key differences from other published TDI assays is the use of a shift in area the under curve of the percent activity remaining versus inhibitor concentration plot (AUC shift) rather than the traditional fold-shift in IC50, to determine the magnitude of TDI. An AUC shift of < 15% suggests negative TDI and > 15% suggests potential TDI. This AUC shift was used to achieve quantitative data reporting, even in the case of weak inhibitors for which IC50 values cannot be quantified. An Agilent Technologies BioCel 1200 System was programmed such that the TDI liability of up to 77 test compounds, incubated at four test concentrations, with and without NADPH in the pre-incubation, can be analyzed in a single run. The detailed automated methodology, assay validation, data reporting and the novel TDI AUC shift approach to describe magnitude of TDI are presented.