Recent advances in measurement of metabolic clearance, metabolite profile and reaction phenotyping of low clearance compounds

ABSTRACT

Introduction

Low metabolic clearance is usually a highly desirable property of drug candidates in order to reduce dose and dosing frequency. However, measurement of low clearance can be challenging in drug discovery. A number of new tools have recently been developed to address the gaps in the measurement of intrinsic clearance, identification of metabolites, and reaction phenotyping of low clearance compounds.

Areas covered

The new methodologies of low clearance measurements are discussed, including the hepatocyte relay, HepatoPac®, HμREL®, and spheroid systems. In addition, metabolite formation rate determination and in vivo allometric scaling approaches are covered as alternative methods for low clearance measurements. With these new methods, measurement of ~ 20-fold lower limit of intrinsic clearance can be achieved. The advantages and limitations of each approach are highlighted.

Expert opinion

Although several novel methods have been developed in recent years to address the challenges of low clearance, these assays tend to be time and labor intensive and costly. Future innovations focusing on developing systems with high enzymatic activities, ultra-sensitive universal quantifiable detectors, and artificial intelligence will further enhance our ability to explore the low clearance space.

KEYWORDS:

• Low clearance
• hepatocyte relay
• long-term hepatocyte culture
• intrinsic clearance
• metabolite identification

Acknowledgement

The author greatly appreciates the help of Carolyn Leverett in editing the manuscript.

Article highlights

• Significant advances have recently been made to enable measurement of intrinsic clearance, identification of metabolites, and reaction phenotyping of low clearance compounds.

• Methods currently available for low clearance measurements include the hepatocyte relay method, HepatoPac®, HμREL®, spheroids, metabolite formation rate, and allometric scaling from preclinical species.

• For long-term cocultured systems, it is important to evaluate the contribution of stromal cells to compound metabolism, as they possess certain metabolic competency.

• Down-regulation of some of the drug metabolizing enzymes in the long-term culture systems needs to be considered when scaling from in vitro to in vivo clearance.

• Future innovations in the low clearance field call for novel approaches to increase speed, reduce complexity, and decrease the cost of the low clearance studies.

Abbreviations

ADME, absorption, distribution, metabolism, and excretion; AI, artificial intelligence; AKR, aldo-keto reductase; AO, aldehyde oxidase; BSA, bovine serum albumin; CES, carboxylesterases; CL_h, hepatic clearance; CL_int, intrinsic clearance; CL_int,u, unbound intrinsic clearance; CYP, cytochrome P450; DDI, drug-drug interaction; DMPK, drug metabolism and pharmacokinetics; f_m, fraction metabolized; FMO, flavin-containing monooxygenase; f_u, plasma fraction unbound; HEP, hepatocyte; HHEP, human hepatocytes; HLM, human liver microsomes; IVIVE, in vitro-in vivo extrapolation; K_m, Michaelis constant; LM, liver microsomes; MIST, metabolites in safety testing; mRNA. messenger ribonucleic acid; OAT, organic anion transporter; OATP, organic anion transporting polypeptide; OATP1B1, organic anion transporting polypeptide 1B1; PK, pharmacokinetics; PM, poor metabolizer; QSAR, quantitative structure–activity relationships; rCYP, recombinant cytochrome P450; SAR, structure–activity relationships; SCHH, sandwich-cultured human hepatocytes; SULT, sulfotransferase; UGT, uridine 5’-diphospho-glucuronosyltransferase; V_max, maximal reaction rate

Declaration of interest

L Di is an employee of Pfizer Inc. She has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This manuscript was not funded.