In Vitro Application of Hydrolase Reaction Phenotyping in Early Discovery Research

Abstract

<b>Abstract ID 52879</b> <b>Poster Board 171</b> In recent years, the biotransformation profile of novel chemical entities (NCE) has evolved away from predominant cytochrome P450 (P450)-mediated metabolism and shunted toward other pathways that can contribute to small molecule drug clearance. Hydrolases are an important class of drug metabolizing enzymes, that catalyze the hydrolysis of NCE containing ester and amide functional groups. Preliminary metabolite identification in&nbsp;vitro (mouse, rat, dog, human hepatocytes) and in&nbsp;vivo (mouse, rat, dog plasma) showed that our internal NCE, denoted here as compound A1, was highly susceptible to amide hydrolysis forming the metabolite M1. To deduce the potential enzyme(s) contributing to the formation of M1, compound A1 was incubated with mouse and human plasma and whole blood, but exhibited metabolic stability suggesting the hydrolysis product was formed in tissues and not in systemic circulation. In the current study, we conducted hydrolase reaction phenotyping in tissue fractionations to investigate the relative contribution to A1 hydrolysis by carboxylesterase 1 (CES1), CES2, serine proteases, arylacetamide deacetylase (AADAC) and aldehyde oxidase (AO), all of which have been reported to catalyze amide hydrolysis in liver. Also, a workflow for hydrolase phenotyping is proposed. Our data showed that the formation of M1 was time-dependent in the incubation of 10 μM A1 with mouse and human liver cytosol and microsomes, respectively, and the formation was significantly higher in mouse than in human liver subcellular fractions. The time-dependent formation of M1 was also observed in human intestinal S9, brain S9, and recombinant human CES1 and CES2 supersomes. Chemical inhibition studies were then performed in human liver S9 and intestinal S9 using troglitazone, a CES1 selective inhibitor, and telmisartan, a CES2 selective inhibitor, which both reduced M1 formation by &gt;90%. In contrast to these findings, phenylmethylsulfonyl fluoride (PMSF), a reported pan inhibitor of human CES and serine proteases, did not inhibit M1 formation in either human liver S9 or intestinal S9, suggesting that some unknown hydrolase(s), the activity of which is inhibited by telmisartan and troglitazone but not by PMSF, contribute to A1 hydrolysis. Additionally, hydralazine, a selective AO inhibitor, did not cause M1 formation. Because it has been reported in literature that AADAC activity is not inhibited by telmisartan, our telmisartan inhibition data suggested that AADAC does not play a significant role in A1 hydrolysis. Taken together, serine proteases, AADAC and AO are not involved in A1 compound metabolism while CES1, CES2 and other unknown hydrolases contribute to the amide hydrolysis of A1.