Hepatocytes—the choice to investigate drug metabolism and toxicity in man: In vitro variability as a reflection of in vivo
Introduction
Drugs and other xenobiotics usually have a low solubility in aqueous systems and require biotransformation to metabolites that are more hydrophilic and more readily eliminated. Typically, drug metabolism occurs in two phases: Phase I and Phase II. Phase I of biotransformation is the oxidative pathway in which the compound undergoes oxidation to a more polar substance. Two groups of enzymes, cytochrome P-450 (P450)-depending monooxygenases, a large group of membrane-associated heme proteins and flavin monooxygenases, are major role players in the oxidative metabolism of a wide range of structurally diverse xenobiotics and endogenous compounds. This process is followed by the Phase II reactions in which metabolites are further conjugated by hepatocytes with endogenous molecules by glucuronidation, sulfation, methylation, acetylation and mercapture formation, rendering derivatives that are much more soluble, thus facilitating their elimination [1], [2], [3]. Biotransformation reactions generally follow a detoxification process rendering metabolites inactive. Nevertheless, many drug intermediary products generated during metabolism are highly reactive and toxic, causing hepatotoxicity [4], [5], [6].
The human P450 enzymatic system consists of a great number of different enzymes that demonstrate interindividual variation in activity, which are susceptible to induction and inhibition by a number of compounds. This results in several drug interactions and an increased risk of drug-induced liver injury. The liver is the best-equipped body organ to deal with toxins to prevent or minimize damage caused by reactive intermediates. Although many enzymatic and non-enzymatic pathways of bioinactivation are present in the liver, reactive intermediates may escape the detoxification process and initiate radical-chain reactions. The relationship between bioactivation and the occurrence of hepatic injury is not simple. Such reactive species may either directly or indirectly inflict a toxic injury on the cell by acting as a hapten and initiating an immune-mediated reaction [7], [8].
Drug-induced liver injury is the most frequent cause of post-market withdrawal of an approved drug. Most drug-induced hepatic injuries that occur in humans are unpredictable and poorly understood [5]. A major goal for the pharmaceutical industry is to market safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug–drug interactions. Drug metabolism is the major source of pharmacokinetic variability in human beings. At the root of this variability are the phenotypic as well genotypic differences in the expression of the enzymes involved in drug metabolism. In addition, qualitative and quantitative interspecies differences in the regulation, expression and functional activity of key ADMET (absorption, distribution, metabolism, excretion and toxicity) processes, particularly P450-mediated metabolism, confound the extrapolation from animals to man. Collectively, deficiencies in ADMET properties and drug-drug interactions are the major causes of attrition during drug development [9], [10]. In vitro assays developed for the evaluation of drug-like properties can accelerate the drug development process. However, there is still an increasing need to develop robust, enhanced-throughput in vitro assays which accurately extrapolate metabolic parameters to humans.
Section snippets
Key issues to be addressed at early stages of drug development
From a commercial perspective, it is desirable that poorly performing compounds are removed early in the discovery phase rather than during the more costly drug development phases. Consequently, over the past decade, in vitro-based strategies in lead optimization screening in conjunction with ADMET screening studies have been incorporated earlier in the drug discovery phase [11], [12]. Studies in the late 1990s indicated that poor pharmacokinetics, metabolism, drug–drug interactions and
P450 variability in human liver
Progressive advances in the knowledge of metabolic routes and enzymes responsible for drug biotransformation have contributed to understanding the great metabolic variations existing in human beings. Phenotypic and genotypic differences in the expression of the enzymes involved in drug metabolism are the main causes of this variability. Analyzing the activity levels of major P450s responsible for drug metabolism in microsomes from a human liver bank revealed the existence of considerable
P450 expression in human hepatocytes
Human hepatocytes in culture show active levels of major P450s involved in drug metabolism [105], [106]. Similarly to that observed in human liver microsomes, high preparation-to-preparation differences in P450 activity levels are found in cultured human hepatocytes from different donors [70], [107], [108], [109] (Fig. 7). This variability is observed in individual P450 enzymes at both activity and mRNA levels. A consequence of this variability, markedly higher than that found in other species,
Drug metabolism by cultured human hepatocytes. How far are we from in vivo?
Human hepatocytes are recognized to be the closest model to the human liver [125]. Once isolated, cells are placed in chemically defined culture conditions where they express typical hepatic biochemical functions, among which is the ability to metabolize drugs [19], [70], [125]. This model is presently considered to be a very useful tool for anticipating drug metabolism and drug hepatotoxicity in man [12], [24], [70], [109]. However, as shown above, the fact that cells are kept in an artificial
Acknowledgements
The authors thank the financial support of the ALIVE Foundation, the Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III of Spain (03/0339), and the European Commission (LSHB-CT-2004-504761 and LSHB-CT-2004 512051).
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