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New Horizons in Predictive Drug Metabolism and Pharmacokinetics / Edition 1

New Horizons in Predictive Drug Metabolism and Pharmacokinetics / Edition 1


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Drug metabolism, pharmacokinetics and toxicokinetics as determinants of drug attrition and the safety of xenobiotics are critically important. This book presents a comprehensive treatise on the current issues and challenges facing drug metabolism and pharmacokinetics. Readers will find a thorough exploration of their predictive role in impacting drug discovery and development and in improving the success rate and safety assessment of pharmaceuticals and industrial or occupational chemicals. Chapters not only focus on the current state of art, with distinct examples, but on future needs and approaches likely to improve our prediction of potential human risk.
Discussions of critical properties that are determinants of a compound’s metabolic and pharmacokinetic fate follow introductory chapters. The Drug Discovery process increasingly incorporates pharmacokinetics and drug metabolism screening and focus has shifted towards in silico, computational and systems biology approaches. Core chapters reflect this and the recent interest and need to assess the role of transporters, along with drug metabolizing enzymes, as potential determinants of pharmacokinetic behaviour, toxicity and drug-drug interactions. Lastly, chapters cover the issues and factors involved in translating pharmacokinetics from in silico to in vivo and from animal models to man, and postulate future directions and opportunities.
Leading experts from academia, industry and regulatory bodies across the globe contribute their knowledge to this book, which scientists involved in many aspects of the drug discovery process, as well as regulators and postgraduate students, will find a useful resource.

Product Details

ISBN-13: 9781849738286
Publisher: RSC
Publication date: 12/15/2015
Series: Drug Discovery Series , #49
Pages: 422
Product dimensions: 6.20(w) x 9.30(h) x 1.20(d)

About the Author

Alan G.E. Wilson is an expert in the area of drug metabolism, pharmacokinetics and toxicology. He is a Board Certified Toxicologist with over thirty years of experience working in governmental and industrial companies, conducting ADME, PK, Toxicology and risk assessments. He has over 120 peer reviewed publications, including edited books and book chapters and is a frequently invited speaker at International Symposia. He is currently Vice President of a Department of DMPK, Toxicology and Pathology.

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New Horizons in Predictive Drug Metabolism and Pharmacokinetics

By Alan G. E. Wilson

The Royal Society of Chemistry

Copyright © 2016 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-84973-828-6


How Physicochemical Properties of Drugs Affect Their Metabolism and Clearance

1.1 Introduction

The physicochemical properties of compounds have been used for more than a century to predict or estimate pharmacokinetic processes. The most well known property is lipophilicity, often defined as the partition coefficient between octanol and water. This property is related to passive diffusion across cell membranes, solubility, interaction with receptors, metabolism and toxicity. To activate proteins, e.g. receptors and enzymes, the compound needs to bind to a binding pocket. Besides lipophilicity, physicochemical properties of importance for binding include molecular size, hydrogen bond acceptors/donors and charge. This chapter discusses the physicochemical properties of importance for drug metabolism. The primary organ for drug metabolism is the liver and to reach the liver the compound must cross cellular barriers. Absorption from the gastrointestinal tract (GIT) is therefore of critical importance for orally administered drugs, before distribution into and out of the liver can occur. We introduce the GIT in this chapter, and all of these processes are discussed in detail in other chapters. Thereafter we describe the enzymes responsible for drug metabolism in different tissues; the biology of these enzymes is further discussed in later chapters. Finally, the role of the enzymes and that of transporters in drug clearance is presented together with an analysis of the structural features of molecules of importance for binding to enzymes and transporters.

1.2 Intestinal Absorption, Liver Disposition and Enzyme Expression

Only free molecules can pass through cell barriers and, hence, only the unbound fraction of drugs can pass over the intestinal epithelium. Solubility governs the concentration reached in the intestinal fluid and is therefore a major driving force for the absorption. Hydrophilic and small molecules may be absorbed by diffusing through the paracellular route. This transport route has limited capacity as its total surface area is much smaller than that of the transcellular (membrane related) pathway. Furthermore, the tight junction reduces the pore size. In the small intestine compounds greater than 4 Å have limited permeability through this pathway whereas those greater than 15 Å are excluded from permeation. To cross the cell membrane compounds have several options. The two most common pathways are passive diffusion through the lipoidal membrane or active transport mediated by transport proteins. The impact of these pathways is heavily debated. Kell and coworkers have challenged the theory that the majority of drugs use lipoidal passive diffusion to pass through cells. Their hypothesis is that most of the transport across cells involves active processes and transport proteins. This debate has spurred research to determine to what extent the two pathways are involved in drug distribution.

Once the compound has traversed the luminal membrane, it may either diffuse through the cytosol and cross the serosal membrane, or interact with enzymes, intracellular organelles (lysosomes, endoplasmic reticulum) or the cell nuclei. It has been proposed that there is a substrate overlap between cytochrome P450 3A4 (CYP3A4) and the efflux protein P-glycoprotein [P-gp; also known as multidrug resistance protein 1 (MDR1)]. Hence, these two different pathways may have synergistic effects in the clearance and detoxification of certain compounds.

When the compound has crossed the intestinal epithelium it reaches the portal vein from where the systemic circulation transports it to the main metabolic organ in the body — the liver. The compound can then reach the cytosol by either passive or active transport mechanisms across the basolateral membrane facing the bloodstream. The capacity of the liver as a detoxification organ is remarkable. Even compounds with high protein binding can be extracted to a large extent by the liver. This can be exemplified by atorvastatin, the cholesterol-lowering compound marketed as LIPITOR®. A high fraction of atorvastatin is absorbed from the intestine but is also highly bound (98%) to proteins in the blood. Therefore, only 2% is available in an unbound free form that can permeate the cell membrane. in spite of this, the absolute bioavailability after oral administration is only 14%. This low number is a result of the cooperation between active influx transporters [mainly organic anion-transporting polypeptides (OATP) 1B1 and 1B3] in the basolateral membrane and CYP3A4 in the cytosol. In addition to these processes, atorvastatin is thereafter cleared from the hepatocytes through canalicular efflux by P-gp. The drug transporters and metabolic enzymes in the gut and liver that are crucial for the first-pass effect are shown in Figure 1.1. The metabolic capacity of the gut and liver are discussed in more detail below.

1.3 Metabolic Capacity of the Intestine and Liver

The intestine is the most important extrahepatic site of drug metabolism and its involvement in the first-pass metabolism of orally administered drugs makes it a major determinant of drug bioavailability. The most abundant CYP in the small intestine is CYP3A, constituting 50–82% of the intestinal CYP content. However, compared with the liver, the total mass of CYP3A in the small intestine corresponds to only about 1% of the hepatic CYP3A levels. Other CYPs expressed in the small intestine, as determined using immunoblotting or liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based protein quantification, are CYP2C9, CYP2C19, CYP2D6 and CYP2J2 (table 1.1). Their expression levels vary in the different regions of the intestine. CYP3A, CYP2C and CYP2D6 show highest expression in the proximal intestinal region and decreasing levels in the distal regions. For CYP2J2, the expression is constant throughout the GIT.

More CYPs are expressed in human liver and at higher expression levels than in the intestine. In 1994 Shimada et al. determined the expression levels of the major drug metabolizing CYPs in human liver using P450-spectra of total CYP content and SDS-PAGE and immunoblotting in 60 people (30 Caucasians and 30 Japanese). Although the expression levels displayed both interindividual and interethnic variations, the average expressions levels in comparison with total CYP content were: CYP3A (28.8%) > CYP2C (18.2%) > CYP1A2 (12.7%) > CYP2E1 (6.6%) > CYP2A6 (4.0%) > CYP2D6 (1.5%) > CYP2B6 (0.2%). Since then the methodological development of more sophisticated methods, e.g. different types of LC-MS/MS-based proteomics, has allowed quantification of CYPs. Although the quality of the LC-MS/MS analyses may vary due to the level of method validation etc., the results are consistent with those obtained by Shimada et al. that identified the CYP3A and CYP2C families as the most abundant hepatic CYPs (table 1.2). All tissues have enzymatic activity to some extent; however, the gut and the liver are the two most important metabolic tissues. An overview of the metabolic profile of different tissues is provided in table 1.3. It should be noted that not only the type of enzymes differs between tissues; the expression levels of these enzymes differ as well.

Although the abundance of CYPs is of major interest it does not provide the full picture of the importance of the specific CYP enzymes for drug metabolism. One striking example showing discrepancy between expression levels and importance is the CYP2D6 enzyme. This enzyme is only expressed in low levels in human liver (1.5–2% of the total CYP content). However, it is one of the major drug-metabolizing enzymes and metabolizes up to 25% of clinically used drugs. Another example is CYP1A2. It constitutes approximately 18% of the human hepatic CYP content, but its relative importance in drug metabolism is only 3–9% (Table 1.4).

1.4 Pharmacogenomics

In addition to interindividual variation in expression levels, many drug-metabolizing enzymes, and especially some of the CYPs, are highly polymorphic. Approximately 40% of CYP-dependent phase I metabolism is performed by polymorphic CYPs, including CYP2D6, CYP2C9, CYP2C19 and CYP2B6. For CYP2D6 more than 100 different alleles and suballeles have been identified. These include alleles where the entire CYP2D6 gene is deleted, alleles with duplicated or multiduplicated CYP2D6 genes, and alleles containing single-nucleotide polymorphisms (SNPs). Such gene variants may of course have a major impact on the pharmacokinetics and may result in adverse effects of drugs that are CYP2D6 substrates (cf.). A classic example of this is the prodrug codeine, which is activated by CYP2D6 into the active drug morphine. For people who are poor CYP2D6 metabolizers, i.e. their CYP2D6 genes are deleted or contain mutations leading to non-functional enzymes, codeine does not give the desired analgesic effect. On the contrary, for individuals with alleles with duplicated or multiduplicated CYP2D6 genes, i.e. ultra-rapid metabolizers, codeine is activated rapidly, which can lead to codeine toxicity and central nervous system depression. There are also a few cases of infant mortality where ultra-rapid metabolizer mothers treated with codeine transferred fatally high morphine concentrations to their breastfed infants.

For other CYPs, e.g. CYP2C9, CYP2C19 and CYP2B6, many variant alleles and suballeles have been described, some of which have significant clinical impact. The most well known clinical CYP2C examples are the CYP2C9 polymorphisms involved in warfarin metabolism and CYP2C19 polymorphisms associated with clopidogrel activation. Both of these were highlighted in 2011 as important pharmacogenomics biomarkers. The warfarin (COUMADIN®) and the clopidogrel (PLAVIX®) Food and drug administration (FDA) drug labels have been updated to contain recommendations for initial doses based on e.g. CYP2C9 genotype and a warning about diminished effectiveness in CYP2C19 poor metabolizers, respectively. A complete and updated overview of CYP and CYP oxidoreductase (POR) polymorphisms can be found on the home page of The Human Cytochrome P450 (CYP) Allele Nomenclature Committee (

1.5 Molecular Features of Importance for Transporter Interactions: Substrates [Versus Inhibitors

Many substrates and inhibitors have been identified for transporters that are of importance for drug distribution into and out of cells. For a selection of these, see table 1.5. While a substrate of the transporter can also be an inhibitor of the transport protein and block transport of other compounds, compounds that have been identified as inhibitors may not be transported. The latter is related to the inactivation of the transport protein by binding to sites other than the one crucial for mediating transport. Interaction with the transport-mediating site allows the drug compound (or its metabolite) to traverse the lipophilic membrane. Hence, the molecular requirements of the different transporters have been studied to better understand what physicochemical properties of a compound will result in them being actively transported by a particular transport protein. While metabolism is a chemical reaction that turns a substrate into a product that is chemically different, the substrates of transport proteins remain the same; no chemical reaction occurs. However, the terminology of transporters and experimental procedures to study transport have been inspired by those in the metabolism field. So, for example, the Michaelis–Menten equation is often used to describe the efficiency of transporters to flux compounds across the membrane.

The structural requirements for transport by influx and efflux transport proteins have been heavily studied. The majority of studies have been directed towards investigation of transport protein inhibition. The reason for this is mainly methodological issues associated with substrate assays. While analyses of molecular features of substrates require determination of the intracellular concentration of a large number of compounds, inhibition assays rely on screening a large number of compounds for their inhibition of the transport of one substrate. Hence, analytical demands for the latter are reduced and a higher throughput mode is possible. The most important transport proteins for clearance are discussed below.

1.5.1 Efflux Proteins P-gp Substrate Recognition Pattern

One of the most studied transport proteins is the efflux protein P-gp since it is important for drug distribution to several tissues, including the gut and liver. Drug–drug interactions (DDIs) have also been identified that are mediated by P-gp. Among the most well known are those that occur between digoxin and the P-gp inhibitors amiodarone, cyclosporin A, quinine and verapamil. Seelig and coworkers were pioneers in the study of the recognition pattern of P-gp (cf.). Based on studies of ~100 compounds, they suggested that a special spatial separation of electron donor groups is required for compounds to be transported by P-gp. Their work was followed by a number of structure–activity relationship (SAR) studies in which P-gp substrates are predicted on the basis of chemical information calculated from the molecular structure. The SAR models are typically classification models used to distinguish compounds that are substrates from those that are not transported by the P-gp. One classification model used the sum of atomic electrotopological states (MolES), a descriptor of molecular bulkiness, to predict substrates. Compounds with a MolES >110 are regarded as substrates for P-gp whereas a MolES <49 indicates non-substrates. For compounds with a value between 49 and 110 other descriptors are needed to identify whether they would be substrates. A similar study using a classification approach established the rule of four. This rule states the following: compounds with (n + O) ≥ = 8, molecular weight > 400 and acid pKa > 4 are likely to be P-gp substrates. Compounds with (n + O) = 4, molecular weight < 400 and base pKa< 8 are likely to be non-substrates. Both of these SAR studies identified that P-gp transports larger molecules. Furthermore, it seems that compounds with many hydrogen bonds, and to some extent negative charges, are transportable by P-gp. The non-substrates have fewer hydrogen bond acceptors and are neutral, or at least not highly positively charged. The importance of n and O demonstrated by this study confirms the work by Seelig and colleagues. Finally, P-gp substrates are amphipathic and lipophilic. It has been suggested that the substrate binding pocket sits inside the cellular membrane and needs to be accessed by distribution into the lipid bilayer. Based on this, the lipophilic and amphiphilic nature of the substrates is to be expected. Inhibition of P-gp, BCRP, MRPs, BSEP: Specificity and Overlap

While it is important to understand molecular features that result in substances being substrates to efflux proteins, it is also of interest to look at which molecular features lead to inhibition of transport. Inhibition may result in severe DDIs. Inhibitors may be competitive (they bind to the same binding site as the substrate) or non-competitive (they bind to another site on the transport protein and thereby block the transport). Therefore, a substrate may inhibit the transport of another substrate, and an inhibitor is not necessarily transported by the protein. Artursson and colleagues have explored large compound series to identity inhibitors of the transport proteins most important for drug disposition. They identified specific molecular requirements of the different transporters and the extent to which the molecular requirements for inhibition of these transporters overlap. For example, the ABC transporters P-gp, breast cancer resistance protein (BCRP), multidrug resistance-associated protein 2 (MRP2) and bile salt export pump (BSEP), all of which are expressed in the canalicular membrane of the hepatocyte, have a significant overlap of inhibitors, i.e. the same compound may block several of these transporters at the same time. The impact on drug clearance, for instance from hepatocytes to bile, may therefore be greatly affected. Such inhibition may also result in reduced enterohepatic recycling of endogenous substances such as bile acids and bilirubin, which can result in, among others, fatal cholestasis. In a study of 122 compounds, all tested for their inhibition of P-gp, BCRP and MRP2, molecular features of specific inhibitors (interacting with only one of the transporters) and of those that interacted with all three transporters were identified. The inhibitors of P-gp were lipophilic, non-polar and had higher structure connectivity. BCRP inhibitors were also more lipophilic than non-inhibitors and the number of aromatic rings correlated positively with inhibition. Inhibitors of MRP2 had similar properties; lipophilicity and unsaturated bonds (double bonds) positively correlated with inhibition, as did shape. Thus, inhibitors of P-gp, BCRP and MRP2 are all lipophilic and aromatic, but to different degrees. The specific inhibitors of P-gp are less aromatic than those of MRP2 and BCRP, and the BCRP inhibitors generally have more aromatic nitrogens than the P-gp inhibitors. P-gp inhibitors are the most lipophilic (log DpH7.4 of 2.3) followed by BCRP (log DpH7.4 of 1.9) and MRP2 (log DpH7.4 of 1.2). By contrast, multi-specific inhibitors, i.e. compounds that inhibit all three proteins, are 100- to 1000-fold more lipophilic (log DpH7.4 of 4.5).


Excerpted from New Horizons in Predictive Drug Metabolism and Pharmacokinetics by Alan G. E. Wilson. Copyright © 2016 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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Table of Contents

How physicochemical properties of drugs affect their metabolism and clearance; Role of solubility, permeability and adsorption in drug discovery in development; Models for nonspecific binding and partitioning; Cytochrome P450 mediated drug metabolism; Non-cytochrome P450 enzymes and glucuronidation; Metabolite profiling; Application of humanised and other transgenic models to predict human responses to drugs; Stem cells and drug metabolism; Chemically reactive versus stable drug metabolites: Role in adverse drug reactions; Integrating metabolism and toxicity properties; Metabolomics-based approaches to determine drug metabolite profiles; Drug-drug ineteractions: regulatory and theoretical considerations, and an industrial perspective; Drug-drug interactions: Computational approaches; Induction of hepatic cytochrome P450 enzymes: Importance in drug development and toxicity; Current status and implications of transporters: QSAR analysis method to evaluate drug-drug interactions of human bile salt export pump (ABCB11/BSEP) and prediction of intrahepatic cholestasis risk; Formulation of optimizing bioavailability; Systems pharmacology modelling; Pharmacokinetic-pharmacodynamic modelling in drug development with special reference to oncology;

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