Multiple P450 substrates in a single run: rapid and comprehensive in vitro interaction assay

Abstract

The dramatically increased number of new chemical entities (NCE) used in drug discovery has raised a demand for efficient and rapid drug metabolism screening techniques. The aim of this study was to develop a global in vitro metabolic interaction screening test utilising the N-in-1 approach. A cocktail consisting of 10 CYP-selective probes with known kinetic, metabolic and interaction properties in vivo was incubated in a pool of human liver microsomes, and metabolites of melatonin (CYP1A2), coumarin (CYP2A6), bupropion (CYP2B6), amodiaquine (CYP2C8), tolbutamide (CYP2C9), omeprazole (CYP2C19 and CYP3A4), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1), midazolam (CYP3A4) and testosterone (CYP3A4) were analysed simultaneously using LC/TOF–MS. Performance of the method was assessed with cDNA expressed P450s and diagnostic CYP-specific inhibitors. The results were in good accordance with literature and our previous studies. The cocktail developed is suitable for fast and reliable in vitro screening of the interaction potential and characteristics of NCEs.

Introduction

The clinical usefulness of a new drug is determined largely by its ADME properties, of which metabolism is an important determinant affecting, e.g. bioavailability, detoxication, drug–drug interactions and interindividual pharmacokinetic and pharmacodynamic variability. A majority of crucial steps within drug metabolism are in connection with cytochrome P450 (CYP) enzymes. In the drug discovery phase, the knowledge of a drug candidate's metabolic properties helps eliminate unfavourable molecules and highlight the potentially safer ones. In the clinical phase, a lack of knowledge can lead to morbidity or mortality, therapeutic failure and toxicity from unanticipated overdose or metabolic reactions.

The dramatically increased number of new chemical entities (NCE) in the field of drug discovery has raised a demand for efficient and rapid drug metabolism screening techniques.

So-called cocktail or N-in-1-assays have been used in recent years for in vivo phenotyping (Frye et al., 1997, Scott et al., 1999, Streetman et al., 2000, Christensen et al., 2003). Recently, as part of high-throughput screening techniques, some tests for the simultaneous analysis of CYP enzymes in vitro have also been developed (Bu et al., 2001, Dierks et al., 2001, Testino and Patonay, 2003). These assays have some limitations as not all major drug-metabolizing CYP isoforms can be detected (e.g. CYP2B6), selection of unselective probes, and in some cases, the probe substrate concentrations may not be in the appropriate range as determined by enzyme kinetics.

The aim of this work was to develop a rapid and comprehensive in vitro assay utilising the N-in-1 approach, which would give a “first glimpse” of the interaction properties of any compound, and which would serve as background information for further decisions about the fate of a compound in terms of development and usability. Substrates covering all major CYP enzymes were selected on the basis of selectivity and on reported metabolic, kinetic and drug–drug interaction properties in vivo. Analysis of the metabolites and disappearance of the parent were conducted simultaneously using LC/TOF–MS, and the final combination of substrates was the most suitable compromise for this analytical tool. The method was further assessed with diagnostic CYP-selective inhibitors and recombinant enzymes. The ultimate intention was to develop a cost-effective screening method for ranking hit/lead candidates for selection purposes or to characterize interaction potential of candidate drugs. This test should point out which CYP enzymes are potentially inhibited by a compound and to give a tentative IC₅₀ value for all relevant drug-metabolising CYP enzymes. If something risky is found, it should be verified with additional experiments employing standard enzymatic approaches.