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Pharmacokinetic and metabolite identification studies were conducted to understand the clearance pathways of EPZ011652 [(2-aminoethyl)(methyl)({3-[4-(propan-2-yloxy)phenyl]-1H-pyrazol-4-yl} methyl)amine], a potent protein arginine N-methyltransferase inhibitor. Metabolic clearance was the major pathway of EPZ011652 elimination in rats with structural elucidation of metabolites via liquid chromatography -mass spectrometry (LC-MS n ) accurate mass measurement revealing the formation of a novel aliphatic N-acetylated metabolite (M1) located on the terminal nitrogen of the ethylenediamine side chain. EPZ015564, a synthetic standard of the N-acetyl product, was prepared and was also generated by human and rat, but not dog hepatocytes. In rat hepatocytes, on incubation with EPZ011652, the concentration of EPZ015564 initially increased before decreasing with incubation time, suggesting that the metabolite is itself a substrate for other metabolizing enzymes, in agreement with the identification of metabolites M2, M3, and M4 in rat bile, all N-acetylated metabolites, undergoing sequential phase I (demethylation, oxidation) or phase II (sulfation) reactions. Reaction phenotyping with recombinant human N-acetyltransferase (NAT) isoforms revealed that both NAT1 and NAT2 are capable of acetylating EPZ011652, although with different catalytic efficiencies. Kinetic profiles of EPZ015564 formation followed classic Michaelis-Menten behavior with apparent K m values of >1000 mM for NAT1 and 165 6 14.1 mM for NAT2. The in vitro intrinsic clearance for EPZ011652 by NAT2 (110 mL/min/mg) was 500-fold greater than by NAT1. In summary, we report the unusual N-acetylation of an aliphatic amine and discuss the implications for drug discovery and clinical development.

An increased understanding of the cellular pathways involved in toxicity responses, coupled with a simultaneous advance in technology, has allowed for a shift in the way that the safety assessment of novel chemicals is performed. The development of assays that offer a high-throughput and low-cost option in comparison to more traditional approaches has been a focus of recent years.Early identifi cation of compounds which have the potential to cause Drug Induced Liver Injury (DILI) remains a major challenge for the pharmaceutical industry. Improvements in the mechanistic understanding of the cellular processes involved in this complex response have allowed for models to be generated and more reliable predictions to be made.High-Content Screening (HCS) describes an approach whereby multiple end points can be monitored in a single assay. The focus of this chapter is to introduce HCS with relation to using automated fl uorescence microscopy in order to assess phenotypic changes within cells and, more specifi cally, how this can be incorporated into the drug discovery process.We discuss the advantages that HCS can offer, whilst highlighting important factors to take into account when considering establishing the approach within a laboratory. Four papers whereby HCS has been used to highlight the potential of compounds to cause DILI are reviewed and compared. In addition, an option for including HCS as part of a wider workfl ow to identify environmental toxicants is introduced.

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