Original article
Mechanistic insights from comparing intrinsic clearance values between human liver microsomes and hepatocytes to guide drug design

https://doi.org/10.1016/j.ejmech.2012.06.043Get rights and content

Abstract

Metabolic stability of drug candidates are often determined in both liver microsome and hepatocyte assays. Comparison of intrinsic clearance values between the two assays provides additional information to guide drug design. Intrinsic clearance values from human liver microsomes and hepatocytes were compared for a set of commercial drugs with known metabolic pathways and transporter characteristics. The results showed that for compounds that were predominately metabolized by CYP mediated mechanisms, the intrinsic clearance values from the two assays were comparable. For compounds with non-CYP pathways, such as UGT and AO, intrinsic clearance was faster in hepatocytes than in microsomes. Substrates of uptake or efflux transporters in this study did not have significant differences of intrinsic clearance between microsomes and hepatocytes, when uptake into the hepatocytes was not the rate-limiting step. When hepatic uptake was rate limiting, intrinsic clearance in microsomes was faster than that in hepatocytes, which was more prevalent for compounds with rapid metabolism. Low passive permeability can limit the exposure to drug molecules to the metabolizing enzymes in the hepatocytes in relationship to the rate of metabolism. The faster the rate of metabolism, the higher permeability is needed for molecule to enter the cells and not becoming rate-limiting. The findings are very useful for drug discovery programs to gain additional insights on mechanistic information to help drug design without added experiments. Follow-up studies can then be designed to address specific questions.

Highlights

► Mechanistic insights can be derived by comparing liver microsome and hepatocyte stability. ► Liver microsomes and hepatocytes give comparable intrinsic clearance for CYP mediated clearance. ► Liver microsomes give lower metabolic rate than hepatocytes for non-CYP mechanisms. ► Comparison of intrinsic clearance from liver microsomes and hepatocytes cannot easily identify hepatic uptake or efflux transporter substrates. ► Whether passive permeability will limit hepatocyte metabolism is dependent on the metabolic rate.

Introduction

Metabolic stability is one of the most important ADME properties of drug candidates [1]. It affects clearance, half-life and oral bioavailability. Early in vitro screening of metabolic stability has been a successful strategy in the pharmaceutical industry to identify and address metabolic liabilities and reduce attrition in the clinic [2], [3], [4]. High throughput metabolic stability assays have been widely implemented and applied in drug discovery to prioritize chemical series, guide structural modification, develop SAR, predict clearance, establish in vitroin vivo correlations (IVIVC) and estimate doses [5].

Both liver micosomes and hepatocytes of various species are most commonly used in vitro models for clearance prediction [6], [7], [8], [9], [10]. Human liver microsomal stability assay is often setup as a tier 1 ADME screen in the pharmaceutical industry to profile drug candidates [3]. The advantages of liver microsomes are their richness of metabolizing enzymes [especially Cytochrome P450 (CYP) enzymes], convenient for use and relatively low cost, which outweigh the limitation of incomplete metabolic pathways. Hepatocytes are also widely used in drug discovery and development since they possess a more complete complement of the drug metabolic enzymes and cofactors for the different clearance pathways [10], [11], [12], [13], [14]. Besides CYP, hepatocytes also contain several non-CYP enzymes of Phase I and Phase II, such as UGT, AO and MAO. In addition, hepatocytes preserve the cell membrane structure and most of the transporter functions [11], which more closely mimic the in vivo system than liver microsomes. The key drug uptake transporters in the human hepatocytes are OATP1B1, OATP1B3, OATP2B1, NTCP, OAT2 and OCT1; and the major drug efflux transporters are BCRP, MRP2, BSEP and P-gp [15]. Hepatocytes are particularly valuable in studying the interplay between metabolizing enzymes and transporters in drug disposition [16]. Both microsomes and hepatocytes are indispensable tools for clearance prediction in spite of the limitations of the in vitro models [8], [10]. Many drug research organizations use both assays to support drug discovery projects in order to answer specific questions and intrinsic clearance values are available for many drug discovery compounds in both assays.

Comparisons of liver microsomes and hepatocytes have been studied by several groups to address the causes of the differences between the two systems in predicting clearance, such as scaling factors, static and dynamic liver models, mismatch of donor liver activity, permeability/cofactor limited clearance and plasma protein binding [7], [17], [18], [19], [20]. These earlier investigations have laid out the foundation for this study to compare the intrinsic clearance values in both microsomes and hepatocytes to gain additional insights on metabolizing enzymes and transporters involved in the clearance of a drug, which might not be otherwise available by using a single assay alone. A set of commercial drugs with known metabolic pathways and transporter characteristics were used to determine if additional information can be extrapolated from comparison of the two assays and strategies can be developed to best apply the information.

Section snippets

Materials

Test compounds were obtained from Pfizer Global Material Management (Groton, CT) or purchased from Sigma–Aldrich (St. Louis, MO). Other reagents were obtained from Sigma–Aldrich (St. Louis, MO) unless specified. Polypropylene plates of 96- and 384-wells were from Axygen Scientific Inc. (Union City, CA). Tips of 96 blocks were obtained from Apricot Designs (Monrovia, CA). Tips of 384 blocks for both Sciclone® and Biomek® liquid handlers were obtained from Axygen, Inc. (Union City, California).

Results and discussion

The comparisons of apparent intrinsic clearance values in human liver microsomes and hepatocytes for about 34,000 Pfizer research compounds are shown in Fig. 1. Many compounds have apparent intrinsic clearance values within two fold between microsome and hepatocyte assays. However, some compounds have higher apparent intrinsic clearance in microsomes and others have higher values in hepatocytes. The compounds in Fig. 1 were also colored by MDCK-LE [21] passive permeability values as high (Papp

Conclusions

Comparisons between human liver microsome and hepatocyte apparent intrinsic clearance values provided additional mechanistic insights on metabolic pathways (Fig. 9), which was not available using a single metabolic stability assay alone. The two assays gave very similar apparent intrinsic clearance values for compounds that were mostly eliminated by CYP mediated pathways. For compounds that had non-CYP metabolic pathways, such as UGT and AO, hepatocytes gave higher rate of metabolism than

Acknowledgments

The authors would like to thank Hui Zhang, Carrie Funk, Jillian Bailey Van Hausen, JianHua Liu, Carrie Whitney-Pickett at ATG for providing the high throughput ADME data; Ted Liston and Larry Tremaine for their leadership and support.

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