Reaction Phenotyping: Advances in the Experimental Strategies Used to Characterize the Contribution of Drug-Metabolizing Enzymes

Abstract

During the process of drug discovery, the pharmaceutical industry is faced with numerous challenges. One challenge is the successful prediction of the major routes of human clearance of new medications. For compounds cleared by metabolism, accurate predictions help provide an early risk assessment of their potential to exhibit significant interpatient differences in pharmacokinetics via routes of metabolism catalyzed by functionally polymorphic enzymes and/or clinically significant metabolic drug-drug interactions. This review details the most recent and emerging in vitro strategies used by drug metabolism and pharmacokinetic scientists to better determine rates and routes of metabolic clearance and how to translate these parameters to estimate the amount these routes contribute to overall clearance, commonly referred to as fraction metabolized. The enzymes covered in this review include cytochrome P450s together with other enzymatic pathways whose involvement in metabolic clearance has become increasingly important as efforts to mitigate cytochrome P450 clearance are successful. Advances in the prediction of the fraction metabolized include newly developed methods to differentiate CYP3A4 from the polymorphic enzyme CYP3A5, scaling tools for UDP-glucuronosyltranferase, and estimation of fraction metabolized for substrates of aldehyde oxidase. IntroductionIn an era where combination drug therapy to treat several conditions simultaneously is common, drug companies emphasize the need for optimal absorption, distribution, metabolism, and excretion (ADME) properties, with the purpose of optimization of efficacy and minimization of the risk of adverse events. This includes a proper assignment and an extensive understanding of the routes of metabolism to aid in the prediction of human pharmacokinetics, and to help avoid a potential "object drug" scenario when coadministration is likely. The attributes of the coadministered drug and/or the patient has the potential to increase the probability of a drug-drug interaction (DDI), i.e., enzyme inhibitor, inducer, polymorphic genotype. Hence, the greater the percentage attributed to a single metabolic route, the greater the potential for a DDI and possible "black box warning" being issued as part of the drug package insert. Furthermore, the pharmacokinetic implications of a single polymorphic enzyme being responsible for a majority of the metabolism of a drug may have either an efficacy (extensive metabolizers) or toxicological (poor metabolizers) impact on exposure. To address these concerns, early drug discovery teams strive for balanced metabolism across multiple enzymes and clearance mechanisms (hepatic, renal, biliary). These discovery efforts center on in vitro reaction phenotyping to support enhanced chemical design.There is a general agreement among the major regulatory agencies that pharmaceutical companies should provide a characterization of the metabolic profile of a new chemical entity (NCE) and understand the enzymology of the major clearance mechanisms ...

Quantitative Characterization of Major Hepatic UDP-Glucuronosyltransferase Enzymes in Human Liver Microsomes: Comparison of Two Proteomic Methods and Correlation with Catalytic Activity

“…A possible ramification of such apparent relationships can be compromised reaction phenotyping of substrates of such enzymes if phenotyping is carried out using correlation of activity with enzyme content in an in vitro system, such as human liver microsomes. Although correlation of UGT-isoform selective activity with metabolic turnover of a new chemical entity is useful in identifying the UGT isoforms responsible for metabolism, confirmatory approaches utilizing isoform-selective chemical inhibitors, recombinantly expressed UGT enzyme analyses, and genotyped tissue fractions should also be employed (Zientek and Youdim, 2015).…”

“…In particular, reaction phenotyping for drug-metabolizing enzymes using correlation approaches is crucially dependent on robust analytical methods used to measure both activity and expression levels in individual samples (Zientek and Youdim, 2015). However, despite recent efforts aimed at developing assays to characterize the abundance and activity of drug-metabolizing enzymes, with considerable success especially in the case of cytochrome P450 enzymes (Walsky and Obach, 2004;Gröer et al, 2014), this level of understanding is still hindered by the lack of standard and consistent methods for quantifying uridine-59-diphospho-glucuronosyltransferase (UGT) expression and function (Guillemette et al, 2014).…”

Evaluation of a New Molecular Entity as a Victim of Metabolic Drug-Drug Interactions--an Industry Perspective

“…It is therefore recommended to thoroughly assess and optimize conditions for f m of non-P450 enzymes when using the strategies described for the non-P450s as good starting guidelines. Relative contributions of individual P450 enzymes to total human hepatic microsomal clearance can be assessed in vitro by some commonly used techniques (Rodrigues, 1999;Wienkers and Stevens, 2003;Zhang et al, 2007;Ogilvie et al, 2008;Korzekwa, 2014;Zientek and Youdim, 2015). 1) Recombinant P450 kinetics scaled to HLM: Commonly referred to as the RAF/ISEF method (Supplemental Materials), this involves determination of enzyme kinetic parameters for the metabolism of the NME in a panel of recombinant human (rh) P450 (with predetermined specific activity and normalized for protein content) and scaling the rhCYP intrinsic clearance (CL int or V max /K m ) to HLM CL int via a RAF/ISEF approach.…”

Low-Turnover Drug Molecules: A Current Challenge for Drug Metabolism Scientists

“…For example, most of the studies performed to evaluate different models for estimating intrinsic clearance have done so using predominantly cytochrome P450 substrates. While this family of drug-metabolizing enzymes remains the most important in drug metabolism, other metabolic enzymes such as UDPglucuronosyltransferases and aldehyde oxidase are becoming increasingly relevant (Hutzler et al, 2013;Oda et al, 2015;Zientek and Youdim, 2015). Thus, the long-term stability and activity of these and other non-P450 enzymes in the discussed monoculture and coculture models still deserve better characterization.…”