Hepatocytes—the choice to investigate drug metabolism and toxicity in man: In vitro variability as a reflection of in vivo

Abstract

The pharmaceutical industry is committed to marketing safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug–drug interactions. Drug metabolism is a major determinant of drug clearance and interindividual pharmacokinetic differences, and an indirect determinant of the clinical efficacy and toxicity of drugs. 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 as well genotypic differences in the expression of the enzymes involved in drug metabolism are the main causes of this variability. However, only a minor part of phenotypic variability in man is attributable to gene polymorphisms, thus making the definition of a normal liver complex. At present, the use of human in vitro hepatic models at early preclinical stages means that the process of selecting drug candidates is becoming much more rational. Cultured human hepatocytes are considered to be the closest model to human liver. However, the fact that hepatocytes are located in a microenvironment that differs from that of the cell in the liver raises the question: to what extent does drug metabolism variability observed in vitro actually reflect that of the liver in vivo? By comparing the metabolism of a model compound both in vitro and in vivo in the same individual, a good correlation between the in vitro and in vivo relative abundance of oxidized metabolites and the hydrolysis of the compound was observed. Thus, it is reasonable to consider that the variability observed in human hepatocytes reflects the existing phenotypic heterogeneity of the P450 expression in human liver.

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.