The Why and How of Absorption Distribution Metabolism Excretion and Toxicity Research

H Van de Waterbeemd, AstraZeneca, Macclesfield, UK B Testa, University Hospital Centre, Lausanne, Switzerland

© 2007 Elsevier Ltd. All Rights Reserved.

5.01.1 Evolving Paradigm in Drug R&D 1

5.01.2 The Increasing Role of Absorption, Distribution, Metabolism,

Excretion, and Toxicity (ADME-Tox) in Pharmaceutical R&D 3

5.01.2.1 Issues in Absorption, Distribution, Metabolism, Excretion, and Toxicity 3

5.01.2.1.1 Transporters 3

5.01.2.1.2 Metabolite identification 4

5.01.2.1.3 Drug-Drug interactions 4

5.01.2.1.4 Toxicology and safety prediction 4

5.01.2.2 Technological Issues 4

5.01.2.2.1 In vitro screening 4

5.01.2.2.2 In silico absorption, distribution, metabolism, and excretion 5

5.01.2.2.3 Simulations: physiologically-based pharmacokinetic (PBPK) and pharmacokinetic/pharmacodynamic (PK/PD) 5

5.01.3 Concept of the Volume 5 References 7

5.01.1 Evolving Paradigm in Drug R&D

Not so very long ago, pharmacokinetics, drug metabolism, and toxicology of selected clinical candidates were studied mainly during preclinical and clinical development. In those days the mission of medicinal chemistry was to discover and supply very potent compounds, with less interest being given to their behavior in the body. However, the R&D paradigm in the pharmaceutical industry has undergone dramatic changes since the 1970s and particularly since the mid-1990s. High-throughput biological assays were developed that have enabled large series of compounds to be screened. This was driven by the increasing size of proprietary depositories and the availability of new reagents and detection technologies.

Simultaneously, medicinal chemists have developed new synthetic strategies such as combinatorial chemistry and parallel synthesis. The number of compounds synthesized increased dramatically. In addition, specialized biotech companies as well as universities began offering compound collections and focused libraries. As a result, much attention is currently being paid to the design and/or purchase criteria of lead- and drug-like compounds.1-3 Increasingly, this includes considerations on ADME-related physicochemical properties as well as ADME properties themselves. The concept of property-based design,4 in addition to structure-based design where target structures are available, is now commonly used to address ADME issues as early as possible. Thus, the former traditional in vivo animal ADME evaluation could no longer cope with the demand and in vitro ADME screens became widely used. Despite rapid advances in the use of automation and robotics to increase the throughput of the in vitro ADME assays,5 screening all available compounds was not necessarily the most efficient and cost-effective strategy. It thus became reasonable and even essential to develop in silico tools to predict and simulate various physicochemical and ADME properties and to balance these in decision-making processes together with combined in vivo and in vitro approaches (in combo).

Rigorous analyses of the root causes of attrition during development revealed that lack of efficacy, toxicity, as well as inappropriate absorption, distribution, metabolism, and excretion (ADME) are among the major determinants of the failure of candidates.6'7 Lack of efficacy, in addition to insufficient response of the target, may of course be caused by poor absorption, inadequate distribution, and/or rapid metabolism, leading to drug concentrations at the target site that are too low.8 And since toxicity is also a major factor of attrition, the development of compounds is often halted even before a detailed human pharmacokinetics or efficacy study can be performed. Hence, the real impact of absorption, distribution, metabolism, excretion, and toxicity (ADMET) processes remains somewhat hidden in (incomplete)

attrition data. During the 1990s, it became good practice to collect ADME and toxicity data during the drug discovery stage in order to use them in decision making to select the best clinical candidates.9 Today, the drug discovery process has also become strongly dependent on departments providing data, guidance, and insight on issues of drug metabolism and pharmacokinetics (DMPK), toxicology, and safety.

Yet, the success of this move to early involvement of DMPK has been questioned. According to some practitioners the main contribution of a discovery (research) department of drug metabolism and pharmacokinetics has been to enable the design of pharmacokinetically adequate rather than optimal compounds and thus to make it possible to work on difficult targets.10 However, despite all preclinical efforts there will always remain an essential need for extensive clinical pharmacokinetics to lay the ground for safe prescription once the drug is on the market.10

This bird's-eye view of ADMET research can be summarized in two statements. First, and in a more industrial perspective, it is now entirely clear that ADMET profiling must be initiated as early as possible in the discovery process, using high-throughput and in silico methods characterized by the best possible balance between good relevance to clinical properties on the one hand, and high speed, efficiency, and capacity on the other. Second, and in a more fundamental perspective, it took decades for pharmacologists and biologists to realize that there is an unseverable relation between pharmacodynamic effects (what the drug does to the organism) and pharmacokinetic effects (what the organism does to the drug) (Figure 1).11-13 [A word of caution is necessary here about the meaning of the noun 'pharmacokinetics' and the adjective 'pharmacokinetic.' In its first meaning, the adjective refers to everything 'the organism does to the drug,' in other words to its disposition (absorption, distribution, metabolism, and excretion = ADME) in the body. This meaning is seen in the expression 'pharmacokinetic effects.' However, such effects can be investigated and described at the qualitative level (e.g., drug D yields metabolite M but not metabolite N), at the quantitative level (e.g., the oral availability of drug D in humans is in the range 70-90%), and at the kinetic (quantitative versus time) level (e.g., the halflife of drug D in humans is in the range 3-4h). In its second meaning, the adjective refers strictly to the kinetic levels (e.g., when one speaks of 'pharmacokinetic parameters'). In contrast, the noun 'pharmacokinetics' refers exclusively to the kinetic aspects of drug disposition, at least in the English language. Unfortunately, this unambiguous and restrictive meaning is not found in all languages; witness the French language where 'la pharmacocinetique' can mean either disposition or pharmacokinetics.] For well over a century, these two components of the interaction between drug and organism were investigated separately and in complete ignorance of any influence the other component might have. The importance now given to early ADME screening is a belated recognition of the interdependence of pharmacodynamic and pharmacokinetic effects. Indeed, the influence of pharmacokinetic effects on a drug's actions is common knowledge, be it in the duration and intensity of these actions, or even in their nature when active metabolites are produced. As for the changes in its disposition that result directly from a drug's pharmacodynamic effects, these may be due to modifications in blood flow, gastrointestinal transit time, or enzyme responses, to name a few.

Pharmacodynamic events (what the drug does to the biological system)

Basic Pharmacodynamic

Figure 1 The two basic modes of interaction between bioactive agents and biological systems, namely pharmacodynamic events (activity and toxicity) and pharmacokinetic events (ADME). (Reproduced with modifications from Testa, B.; Vistoli, G.; Pedretti, A. Chem. Biodiv. 2005, 2, 1411-1427 with the kind permission of the copyright owner, Verlag Helvetica Chimica Acta in Zurich.)

Pharmacokinetic events (what the biological system does to the drug)

Figure 1 The two basic modes of interaction between bioactive agents and biological systems, namely pharmacodynamic events (activity and toxicity) and pharmacokinetic events (ADME). (Reproduced with modifications from Testa, B.; Vistoli, G.; Pedretti, A. Chem. Biodiv. 2005, 2, 1411-1427 with the kind permission of the copyright owner, Verlag Helvetica Chimica Acta in Zurich.)

Oral form

Lead Distribution Excretion

Figure 2 A schematic description of the major processes of drug disposition showing absorption (passive and active), distribution (passive including binding, and active including efflux), metabolism ( = biotransformation), and excretion (passive and active including efflux). Elimination is not indicated explicitly, since metabolism is chemical elimination and excretion is physical elimination.

Figure 2 A schematic description of the major processes of drug disposition showing absorption (passive and active), distribution (passive including binding, and active including efflux), metabolism ( = biotransformation), and excretion (passive and active including efflux). Elimination is not indicated explicitly, since metabolism is chemical elimination and excretion is physical elimination.

5.01.2 The Increasing Role of Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADME-Tox) in Pharmaceutical R&D

ADME studies aim at obtaining an early estimate of human pharmacokinetic and metabolic profiles.14 However, drug behavior in the body is a highly complex process involving numerous components, as presented in very simplified form in Figure 2.15 This diversity and complexity is reflected in the ADME studies themselves, which include absorption, bioavailability, clearance and its mechanism, volume of distribution, plasma half-life, involvement of major metabolizing enzymes, nature and level of metabolites, dose estimates, dose intervals, potential for drug-drug interactions, etc.

Early toxicology and safety studies should weed out compounds before they enter lengthy and costly clinical trials. As a result of the recent withdrawal of a number of marketed drugs, more pressure is now being put on pharmaceutical companies by regulatory agencies such as the Food and Drug Administration (FDA) and the European Agency for the Evaluation of Medicinal Products (EMEA) with regard to safety evaluations including pharmacological and toxicological safety. Investigation of the potential to cause QT prolongation is now routine. However, the interpretation of data is not straightforward, since many marketed drugs can prolong the QT interval.16

The increased role of early and preclinical ADME and safety/tox studies has led to an important growth of the supporting departments and a considerable development of various technologies to address the key issues. A short overview of the hot issues is given below17; the important challenges in investigating the disposition of new chemical entities (NCEs) and candidates are highlighted first, followed by some technological issues.

5.01.2.1 Issues in Absorption, Distribution, Metabolism, Excretion, and Toxicity

5.01.2.1.1 Transporters

Transporter proteins constitute a significant fraction of membrane-bound proteins. They are typically expressed in all organs involved in the uptake, distribution, and elimination of drugs, including the gastrointestinal tract, the blood-brain barrier, the liver, and the kidneys. The sequence of many transporters is known (see 5.04 The Biology and Function of

Transporters; 5.32 In Silico Models for Interactions with Transporters). There is hope that in the near future the experimental 3D structures of the key transporters will be elucidated. Interaction of drugs with transporters can alter their behavior in membrane transport, which may result in, for example, active uptake, efflux, and rapid elimination. In other words, the pharmacokinetics of a drug may be influenced by transporters. Apart from a metabolic component (see Section 5.01.2.1.3), drug-drug and drug-nutrient interactions may involve transporters.18'19 However, while the basic knowledge on transporters is rapidly growing, their real clinical significance remains open to debate despite some convincing theories.20 A number of P-glycoprotein (P-gp) assays have been developed, as well as some double- and triple-transfected assays. More transporter assays will be available in the near future. The challenge will be to translate the flood of experimental data into relevant information for drug design projects.

5.01.2.1.2 Metabolite identification

With increasing resolution of mass spectrometry and nuclear magnetic resonance (NMR), it is now feasible to detect minute amounts of metabolites. Debate is ongoing as to how to define major versus minor metabolites. Some metabolites might be pharmacologically active and contribute to the overall pharmacokinetics (PK). Reactive metabolites might bind to proteins and cause idiosyncratic reactions.21 The challenge remains to detect these metabolites as early as possible. It has been suggested that time-dependent inhibition should become routine in vitro screening protocols.22 Good progress has been made in computational (in silico) metabolite prediction.23-25

5.01.2.1.3 Drug-Drug interactions

Regulatory authorities require information to be submitted on the potential for interactions to cause adverse effects. Thanks to the availability of in vitro systems this aspect is often considered during the early stages of discovery, including hit evaluation. Oxidative metabolism by cytochromes P450 (CYPs) is the major route of elimination of most drugs. Since CYPs are also able to metabolize multiple substrates, their inhibition is the major focus of drug-drug interaction (DDI) studies. Thus, CYP3A4 is not only the most abundant hepatic CYP, but is also present in the gut wall and is responsible for the metabolism of 50-60% of all drugs. Therefore, this enzyme is highly susceptible to both reversible and irreversible (mechanism-based) inhibition.26 Most CYP3A4 substrates or inhibitors are also P-gp substrates or inhibitors. It is believed that CYP3A4 and P-gp in the gastrointestinal tract work in concert to limit uptake of xenobiotics including drugs.27 Current inhibition studies are based on Ki or IC50 values,28 but more kinetic approaches would be of benefit.29 Great progress has been made in the reliable simulation of DDIs, even taking into account variability in the population.30 A further question is how metabolites contribute to DDIs; a better understanding of the allosteric kinetics of CYPs is also needed.31

Although clinically somewhat less important than enzyme inhibition, enzyme induction is also an inescapable issue and adequate protocols are being developed for its characterization. It remains to be agreed when to carry out such assays or screens during the discovery process.22

5.01.2.1.4 Toxicology and safety prediction

Drug safety is of great concern to patients, medical professionals, and regulatory bodies. As a result, early toxicity predictions and safety estimates are receiving ever-increasing attention in all drug discovery programs. Simple in vitro screening assays, e.g., for hERG and other cardiac ion channels, genetic toxicology, and cytotoxicity, are now routinely added to the growing battery of biology, ADME, and tox/safety screens. Despite encouraging progress,32 in silico predictive toxicology is still in its infancy.33 A promising tool is the integration of ADME-Tox, and pharmacology data to predict side effects, as in the BioPrint approach.34

5.01.2.2 Technological Issues

5.01.2.2.1 In vitro screening

Based on the experience gained with pharmacodynamic high-throughput screening, many in vitro ADME screens can now run in medium or high-throughput modes using automation, robotics, and miniaturization.35-37 Physicochemical properties are now recognized to play a key role in modulating DMPK properties,4,38-40 and their assessment and understanding are therefore receiving greater attention. Owing to the nature of many high-throughput physicochemistry and ADME assays, the typical analytical endpoint is often liquid chromatography-mass spectrometry (LC/MS). Cell-based assays such as the Caco-2 screen for permeability/absorption are quite expensive due to considerable reagent costs, particularly when run in screening mode with many compounds. The trend is to invest in either cheaper in vitro alternatives such as the PAMPA (parallel artificial membrane permeability assay) method or in silico approaches. A proper synergistic hybrid combination of in vitro and in silico methods,41 which has been called the 'in combo' approach,42 may be the most cost-effective approach to ADME screening in drug discovery.

5.01.2.2.2 In silico absorption, distribution, metabolism, and excretion

Prediction and simulation of various ADME properties is considerably cheaper than in vitro screening. Therefore, great efforts have been made to turn all available data into predictive computational models using quantitative structure-activity relationship (QSAR) methods and molecular modeling.42'43 In vitro data are now generated for many ADME and physicochemical endpoints and can be used to build more robust models. Model updating will need to be automated and fitted in the data generation cycle. There is a need for both local (project-specific) as well as global (general, encompassing a wide range of chemotypes) models. Unfortunately, there is still a paucity of human in vivo data, and thus models based upon these will need to be handled with care. Of course interindividual variability within the population is another key factor to take into account for human predictions. Predictions will be ranges rather than hard numbers. Many drug companies have web-based cheminformatics and ADME predictions deployed via their intranet giving the medicinal chemist easy access to 'web screening.'44 For the development of potential drugs, predictions may also contribute to high-throughput pharmaceutics and rational drug delivery.45

5.01.2.2.3 Simulations: physiologically-based pharmacokinetic (PBPK) and pharmacokinetic/ pharmacodynamic (PK/PD)

Physiologically-based pharmacokinetic (PBPK) models rely on principles well known in chemical engineering and describe the human or animal body as a series of pipes and tanks.46,47 Convenient software packages are now available and make PBPK modeling more accessible to drug discovery and development to predict various PK parameters and concentration-time profiles in the body. Population variability48 and specific groups such as children and the elderly can also be taken into account. The more experimental data that are available, the better the simulations, making these approaches of interest in drug discovery and clinical development. Some commercial programs have put considerable effort into simulating the absorption process, which is of interest in optimizing pharmaceutical formulations.

Pharmacokinetic/pharmacodynamic (PK/PD) modeling links dose-concentration relationships (PK) to concentration-effect relationships (PD). This approach helps to simulate the time course of drug effects depending on the dose regimen.49 Specialized software and a better understanding of the applications may help to speed up clinical development. As a whole the pharmaceutical industry lags behind engineering-based industries such as car and airplane manufacturers in using prediction, modeling, and simulation. Often, resources remain allocated to producing experimental data rather than developing the information technology (IT) tools to move toward in silico pharma; in other words, toward web screening rather than wet screening and to a 'will do' rather than a 'can do' attitude.

5.01.3 Concept of the Volume

ADME-Tox issues were far from the forefront in the first edition of Comprehensive Medicinal Chemistry, being implicit rather than explicit in a number of chapters on 'drug design.' Numerous and fast developments since the early 1990s now fully justify a complete volume being devoted to the strategies and technologies currently aimed at evaluating and assessing biotransformation, pharmacokinetics, toxicology, and safety of new chemical entities in the preclinical phases. A schematic diagram of drug discovery, development, and clinical assessment is shown in Figure 3a. Pharmacodynamics (i.e., activity) is obviously the first object of study, but the new paradigm of drug R&D now dictates that ADMET screening must be initiated rapidly. Activity (PD) and ADMET (PK) screening and evaluation thus run in parallel throughout the preclinical phases, and this is when medicinal chemists find themselves in close collaboration with pharmacologists, biologists, biochemists, bioanalysts, physicochemists, computer scientists, and other experts. Assessment of efficacy and tolerance, to merge into utility assessment, then become the objectives of clinical trials.

The present volume is organized mainly according to the methodologies and technologies available to researchers, with due consideration being given to the biochemical/biological background underlying them. However, such a concept taken per se offers a necessary but not sufficient rationale for a logical and mission-oriented organization of this volume. The second concept underlying the organization of the volume is the overwhelming fact that the ultimate 'target' of medicinal chemistry and drug discovery is the human patient, and that all methodologies, technologies, and tools used during the preclinical phases to screen, assess, and understand the biological behavior of hits, leads, and candidates must be relevant to this ultimate 'target.' In other words, each and every biological model, physicochemical method, and computer software package used in drug discovery and development must demonstrate a useful degree of predictivity to behavior in the human patient.

Activity □ □

ADMET ■■ Z

>Tolerance i

Utility qi

!

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