A Handbook of Bioanalysis and Drug Metabolism

Gary Evans

• Publisher: CRC Press
• Year: 2021
• ISBN:
  9781134477364
• Edition: 1
• Topic: Medicine
• Subtopic: Biochemistry in Medicine

Recent years have seen a greater industrial emphasis in undergraduate and postgraduate courses in the pharmaceutical and chemical sciences. However, textbooks have been slow to adapt, leaving the field without a text/reference that is both instructional and practical in the industrial setting – until now.

A Handbook of Bioanalysis and Drug Metabolism is a stimulating new text that examines the techniques, methodology, and theory of bioanalysis, pharmacokinetics, and metabolism from the perspective of scientists with extensive professional experience in drug discovery and development. These three areas of research help drug developers to optimize the active component within potential drugs thereby increasing their effectiveness, and to provide safety and efficacy information required by regulators when granting a drug license. Professionals with extensive experience in drug discovery and development as well as specialized knowledge of the individual topics contributed to each chapter to create a current and well-credentialed text. It covers topics such as high performance liquid chromatography, protein binding, pharmacokinetics and drug–drug interactions. The unique industrial perspective helps to reinforce theory and develop valuable analytical and interpreting skills.

This text is an invaluable guide to students in courses such as pharmaceutical science, pharmacology, chemistry, physiology and toxicology, as well as professionals in the biotechnology industry.

CHAPTER 1Introduction

DOI: 10.1201/9780203642535-1

Gary Evans

1.1 Bioanalysis, pharmacokinetics and drug metabolism (BPDM)

The prime function of the Research and Development subsidiaries of pharmaceutical companies is to discover and develop new medicines. Achieving these objectives is not easy, only a small percentage of the chemical compounds synthesised become medicines, most compounds prove unsuitable for reasons of efficacy, potency or toxicity.

Both the discovery and development phases are time-consuming processes which take between five and ten years to complete. They involve scientists from many different disciplines, working together, to identify disease targets and, to discover suitable chemical entities which have the appropriate biological, chemical and pharmacological/toxicological properties to be quality medicines.

When the potential drug is selected for development, extensive safety and clinical studies are conducted to provide sufficient data for a regulatory submission for registration of a new medicine.

One group of scientists whose contribution is particularly important in both drug discovery and drug development are those working in the discipline which may be described as bioanalysis, pharmacokinetics and drug metabolism (BPDM). Whilst other names have been used to describe this discipline, it has broad range of activities which seek the same objectives – to understand what happens to the drug after it is administered and to determine what implications a knowledge of the fate of the dosed drug has for either improving the drug or for dosage regimens and safety.

This discipline consists of three main areas which are closely related. Bioanalysis is a term generally used to describe the quantitative measurement of a compound (drug) in biological fluids primarily blood, plasma, serum, urine or tissue extracts.

Pharmacokinetics is the technique used to analyse these data and to define a number of parameters which describe the absorption, distribution, clearance (including metabolism) and excretion (often referred by the acronym ADME). The ability to monitor the presence of the drug in the body and to measure its removal is critical in understanding the safety, dosage and efficacy of any medicine.

Drug metabolism is the study of the metabolism of a drug. It can be used to discover the nature and route of metabolism of the drug and the information permits predictions to be made concerning the potential for interactions with co-administered drugs through knowledge of the enzymes involved in the metabolism of drugs. Increasingly predictions can be made about the metabolism of a putative drug using the knowledge base from existing drugs. These three areas have developed together because of the use of common techniques and knowledge. The scientists conducting these functions may be geographically separated or, increasingly, scientists may specialise in one of the functional areas. However, the functions are closely inter-related and may be considered a single scientific discipline. In this book this discipline will be referred to as BPDM.

1.2 The role of BPDM in drug discovery and drug development

The roles and techniques of BPDM used in supporting drug discovery and development are similar but the information is required for different purposes, hence the priorities and approaches differ. Information is used for decision-making in both phases; however, the nature of the decisions affects the quality and quantity of information required.

For discovery, the priority is to examine a large number of compounds and determine which pharmacologically active compounds are most suitable for drug development. In practice when a compound is obtained which has the required biological activity, a number of analogues or chemically similar compounds will be synthesised and tested to optimise the preferred characteristics of the compound (a process is known as lead optimisation). Figure 1.1 shows an illustration of a possible scenario in discovering drugs which are active in vitro and improving these by modification of the chemical structure optimised for in vivo activity.

FIGURE 1.1 Process diagram representing some of the BPDM steps in drug discovery.

In drug development, a single compound is progressed and information relating to the safety of the drug and the dosage required for efficacy in man is obtained. Figure 1.2 shows the studies which are conducted on a drug under development – the exact experimental design and priorities will depend on the particular drug under development.

FIGURE 1.2 Process diagram representing some of the BPDM steps in drug development.

There is a significant overlap in the techniques and methodology used in BPDM for drug discovery and drug development and often the difference is in the experimental design. Consequently, the chapters in this book have been organised on the basis of functional topics and where there are different approaches for discovery and development these are discussed in each chapter.

Physiochemical properties of compounds are also an important consideration in drug design as they will effect absorption and clearance. They will also be of concern in the development of an analytical method or determining a suitable drug formulation. These aspects are discussed in detail in Chapter 2. A sensitive and specific bioanalytical method is developed to allow the monitoring of drug levels in plasma (systemic circulating levels) and urine (excreted levels) in clinical studies. The assay is also used to monitor the levels of exposure in pre-clinical safety studies. Whilst the analytical methodology used in discovery and development will require different levels of sensitivity and validation the basic aspects remain the same. A bioanalytical method consists of two main components:

1. Sample preparation – extraction of the drug from the biological fluid usually including a concentration step to enhance sensitivity of the method; and
2. Detection of the compound – usually following chromatographic separation from other components present in the biological extract. The detector of choice is a mass spectrometer.

These issues are discussed in Chapter 3 (Sample preparation), Chapter 4 (Chromatographic separation: HPLC) and Chapter 5 (Quantitative mass spectrometry). Whilst traditionally other chromatographic techniques have been used, the method of choice is now high-performance liquid chromatography (HPLC) which is used almost universally in both discovery and development methods. The use of tandem mass spectrometry has reduced the need for extensive chromatographic separation because of the enhanced specificity and selectivity of this methodology. It is especially valuable in lead optimisation for studying the pharmacokinetics of multiple compounds administered simultaneously. In addition to monitoring the drug there is an increasing need (with the advent of biochemically active compounds) to monitor surrogate and biomarkers. These are endogenous compounds whose profile reflects the pharmacological action of the drug (biomarker) or disease (surrogate). Immunoassay is the chosen technique for most endogenous compounds and surrogate markers. The use of immunoassay forms the subject of Chapter 6. Plasma levels of the drug are normally monitored to permit the calculation of pharmacokinetic parameters. Whilst preliminary pharmacokinetic data is obtained in drug discovery in pre-clinical species, the definitive kinetics is obtained in drug development by conducting single dose experiments in pre-clinical species and in humans. The importance and definition of the pharmacokinetic parameters are discussed in detail in Chapter 7. These data are essential in defining the dosage regimen in man and ensuring that the therapeutic benefit is maximised.

Plasma samples are also taken from the pre-clinical species used in safety testing. The kinetics derived from these data are often referred to as ‘toxicokinetics’ because they represented exposure after repeated administration of high levels of drug and the data may indicate drug accumulation, inhibition or induction of clearance mechanisms. In addition the calculation of total drug exposure in the safety studies is critical to calculating the margin of safety in clinical studies, and by scaling data from different species predictions can be made of the parameters in humans. All of these issues are discussed in Chapter 9.

The need to monitor both drug and biomarker levels is important for both discovery and development work. The priority for discovery is to ensure suitable pharmacokinetic properties of the chosen drug and to establish the relationship between systemic levels of drug (pharmacokinetics, PK) and the pharmacodynamics (PD) of the drug. This PK–PD relationship is discussed more fully in Chapter 8.

Pharmacological and toxicological effects are normally only produced by the free drug in the body. Most drugs are, to a greater or lesser degree, bound to proteins, notably serum albumen. The techniques and importance of measuring protein binding are discussed in Chapter 10, illustrated with a case study on a highly protein-bound drug.

Many of the studies conducted in the development phase involve the use of radiolabelled drug which is not available at the earlier discovery phase. This allows the absorption, distribution, excretion and metabolism of the drug-related material to be investigated in the pre-clinical species, and where appropriate, in man. These studies are essential in determining the elimination from the animal of all drug-related material. The initial experiments conducted are known as ‘excretion balance studies’ because following administration of radiolabelled drug, urine, faeces and exhaled air are collected over a 7-day period to measure the percentage of the dose eliminated. The excreta is used to examine the form of radioactivity and identify the metabolites. In addition, whole body autoradiography is used to follow the distribution of radioactivity in the organs and the time period of elimination. This data is used quantitatively to determine the dose of radiolabelled drug which can be administered to human volunteers; however, these studies are becoming less common as stable isotope alternatives are developed. All of these issues are discussed in Chapters 11 and 12.

The major routes of metabolism and the enzyme systems involved are well documented although it is an area under continuing development, particularly the Phase 2 enzyme systems. Phase 1 metabolism (Chapter 13) primarily consists of oxidation and hydrolysis of the parent molecule whilst conjugation is the main feature of Phase 2 metabolism (Chapter 14). In both instances the result is to render the molecule more polar and thus suitable for elimination from the animal. There are many in vitro techniques used to investigate the metabolism of compounds. The pros and cons of different models are discussed in Chapter 15. These in vitro techniques allied to tools provided by molecular biological techniques (Chapter 18) permit the scientist to identify the enzymes involved in the metabolism of a drug. By considering the metabolism of co-administered drugs, predictions can be made about the potential for drug–drug interactions and reduce the need to conduct expensive clinical studies (Chapter 16).

It is important to identify the metabolites and to show that the metabolites which were present in the pre-clinical species used in toxicity testing are the same as those observed in humans. Traditionally metabolite identification involved painstaking extraction of radiolabelled drug from biological material and the use of spectrometric methods for identification. In recent years developments in nuclear magnetic resonance or NMR spectroscopy linked to chromatographic systems and mass spectrometry have revolutionised the ability to identify metabolites without extensive extraction, and from much smaller quantities of material. Examples of the use of these techniques are presented in Chapter 17.

Whilst the pharmacologist or biochemist can develop a screening method for determining which compounds show biological activity against a particular target, these data are of limited value without the knowledge to determine whether the compound can be developed into a commercially viable medicine. Indeed many homologous compounds may show similar biological activities in screens but may behave significantly differently when administered in vivo.

The bioanalytical methods used in discovery are designed to be more generic and suitable to monitor a range of analogous compounds. The assay does not require the high sensitivity which is required in drug development because the concentrations of compound used in the pharmacological screening models and initial in vivo testing are higher than will be encountered in the human studies. In addition the pharmacokinetic experiments are not designed to obtain the definitive data but to obtain comparative data between a series of analogous compounds permitting these to be ranked in order of, for example, plasma half-life. Indeed only a small number of sampling times need to be taken to derive this information and many compou...

Table of contents

• Cover Page
• Half-Title Page
• Title Page
• Copyright Page
• Table of Contents
• Editor’s preface
• Preface
• Grieves Harnby: In memoriam
• Chapter 1 Introduction
• Chapter 2 The importance of the physicochemical properties of drugs to drug metabolism
• Chapter 3 Sample preparation
• Chapter 4 High-performance liquid chromatography in pharmaceutical bioanalysis
• Chapter 5 Mass spectrometry and quantitative bioanalysis
• Chapter 6 Immunoassay in pharmacokinetic and pharmacodynamic bioanalysis
• Chapter 7 Pre-clinical pharmacokinetics
• Chapter 8 Pharmacokinetic/pharmacodynamic modelling in pre-clinical drug discovery
• Chapter 9 Toxicokinetics
• Chapter 10 Protein binding in plasma: a case history of a highly protein-bound drug
• Chapter 11 Isotope drug studies in man
• Chapter 12 Whole body autoradiography
• Chapter 13 Phase I metabolism
• Chapter 14 Phase II enzymes
• Chapter 15 In vitro techniques for investigating drug metabolism
• Chapter 16 Drug–drug interactions: an in vitro approach
• Chapter 17 Identification of drug metabolites in biological fluids using qualitative spectroscopic and chromatographic techniques
• Chapter 18 Molecular biology
• Chapter 19 The role of drug metabolism and pharmacokinetics in drug discovery: past, present and future
• Index