Challenges and Opportunities with Non-CYP Enzymes Aldehyde Oxidase, Carboxylesterase, and UDP-Glucuronosyltransferase: Focus on Reaction Phenotyping and Prediction of Human Clearance

Abstract

Over the years, significant progress has been made in reducing metabolic instability due to cytochrome P450-mediated oxidation. High throughput metabolic stability screening has enabled advancement of compounds with little to no oxidative metabolism. Furthermore, high lipophilicity and low aqueous solubility of presently pursued chemotypes reduces the probability of renal excretion. As such, these low microsomal turnover compounds are often substrates for non CYP-mediated metabolism. UGTs, esterases and aldehyde oxidase are major enzymes involved in catalyzing such metabolism. Hepatocytes provide an excellent tool to identify such pathways including elucidation of major metabolites. To predict human PK parameters for P450-mediated metabolism, in vitro–in vivo extrapolation using hepatic microsomes, hepatocytes, and intestinal microsomes has been actively investigated. However, such methods have not been sufficiently evaluated for non-P450 enzymes. In addition to the involvement of liver, extra-hepatic enzymes (intestine, kidney, lung) are also likely to contribute to these pathways. While there has been considerable progress in predicting metabolic pathways and clearance primarily mediated by the liver, progress in characterizing extra-hepatic metabolism and prediction of clearance has been slow. Well-characterized in vitro systems or in vivo animal models to assess drug-drug interaction potential and inter-subject variability due to polymorphism are not available. Here we focus on the utility of appropriate in vitro studies to characterize non CYP-mediated metabolism; understand the enzymes involved followed by pharmacokinetic studies in the appropriately characterized surrogate species. The review will highlight progress made in establishing in vitro-in vivo correlation; predicting human clearance and avoid costly clinical failures when non-CYP mediated metabolic pathways are predominant.

Metabolic Profiling of Human Long-Term Liver Models and Hepatic Clearance Predictions from In Vitro Data Using Nonlinear Mixed-Effects Modeling

“…Interestingly, the conventional scaling showed better performance for the mainly CYP-metabolized reference compounds in the long-term co-culture system HepatoPac as compared to the direct scaling approach. The better performance of direct scaling for the in vivo clearance prediction of Roche compounds might be due to the involvement of non-CYP enzymes and transporters, which gives rise to larger underprediction compared to CYP-metabolized reference compounds (42,43). These results demonstrated that the performance of the scaling approach was dependent on the applied in vitro liver model, the chemical space of the selected compound set, i.e., their physicochemical properties, such as plasma protein binding, and the involved clearance routes.…”

Rifampin modulation of xeno‐ and endobiotic conjugating enzyme mRNA expression and associated microRNAs in human hepatocytes

“…Successful mitigation of CYP450 metabolic liabilities during drug development has increased the importance of non‐CYP450 enzyme contributions to drug metabolism. However, evaluation of non‐CYP450 mediated xenobiotic metabolism continues to pose research, development, and regulatory challenges . Standardized in vitro models to assess conjugating enzyme contributions to xenobiotic clearance and approaches to predict clinical consequences are evolving.…”

“…Of the non‐CYP450 enzymes that contribute to xenobiotic metabolism, UGTs are the most important in terms of abundance in hepatic and extrahepatic tissues as well as in the wide range of xenobiotics including many drugs and endobiotics they metabolize. Drug–drug interactions mediated via UGT induction are inherently difficult to predict from in vitro data . LC‐MS/MS approaches to directly quantify UGT protein content within in vitro systems and human tissues have enhanced in vitro–‐in vivo extrapolation of UGT mediated metabolism.…”

Predicting mammalian metabolism and toxicity of pesticides in silico

“…Unfortunately, other analytical difficulties limit the amount of useful kinetic information available for UGTs and makes them difficult to model . In particular, the active sites of UGTs are internally oriented (facing the lumen) in liver microsomes .…”

“…Hence esters that are readily cleaved in plants, fungi or insects are also likely to be cleaved in the human gastrointestinal tract or liver. 28 There is rarely much point in trying to build a QSAR model for such a reaction. The better approach to pro-pesticides is to identify ethyl and other short-chain, unbranched esters 29 and 'cleave' them in silico before applying QSAR models.…”