Chapter 1. Introduction
Xenobiotics are chemical compounds that do not belong to the normal composition of the human body. These compounds enter the body via the diet, air and medication. The principal route of elimination of xenobiotics from the body is biotransformation. They are eliminated by microsomal phase I and microsomal and cytosolic phase II drug-metabolising enzymes. These enzymes add functional groups to make lipophilic molecules more hydrophilic and hence easier to eliminate. The oxidative reactions are mainly catalysed by cytochrome P450 (CYP or P450) enzymes (phase I metabolism) and, after that, by conjugating enzymes (phase II metabolism), such as UDP-glucuronosyl transferases and N-acetyl transferases (Wrighton & Stevens 1992; Wrighton et al. 1993b). On the other hand, some drugs (called “prodrugs”) need to be metabolically activated before they are pharmacologically active. This activation usually occurs via CYP or hydrolytic enzymes.
The CYP superfamily of microsomal hemoproteins catalyses the monooxygenation of a large number of endogenous and exogenous compounds. They play a key role in the metabolism of a wide variety of xenobiotics, such as drugs, pesticides and (pre)carcinogens (Pelkonen & Breimer 1994; Pelkonen et al. 1998). The CYP superfamily is divided into families and subfamilies on the basis of their nucleotide sequence homology. Members of the subfamilies exhibit quite strict specificity in metabolising xenobiotics with a wide variety of substrates as a whole family. Some CYPs play a role in both the formation and the elimination of endogenous compounds, while some other CYPs, especially those belonging to the families 1-3, seem to be there principally for xenobiotic metabolism purposes (Nelson et al. 1996).
The early knowledge about the metabolism of a new chemical entity (NCE) and its affinity to certain drug-metabolising enzymes helps in the drug development process by providing important information for the selection of a lead compound from among a number of substances pharmacologically equally effective in their therapeutic response (Obach et al. 1997). In vitro metabolic studies give information about the metabolic stability and possible interactions between compounds that have affinity for the same drug-metabolising enzymes (Pelkonen et al. 1998; Wrighton et al. 1993b). In vitro metabolic studies and affinity screenings have a role in determining the reasonable dose ranges in in vivo studies of test species and human volunteers (Obach et al. 1997).
When several drugs are used simultaneously or in sequence, there is always a risk of metabolic interactions in case these compounds are metabolised by the same CYP enzyme or one compound affects the metabolism of the other compound (Boobis 1995). Before an NCE becomes a drug candidate and is subjected to clinical trials, it has to be tested for safety and the possibility of drug-drug interactions. These preclinical experiments must be reliable and give relevant information about the studied properties of an NCE. Safety and interactions are, even today, examined to a considerable extent by animal tests. Extrapolation of the results from animal studies to the human situation is usually difficult and contains many sources of errors. The most important reasons for this are the species-specific differences in drug-metabolising enzymes, both qualitative (different metabolic pathways) and quantitative (different intrinsic clearances), between the human being and the test species. The current tendency is to increasingly make in vitro preclinical trials, which makes it possible to use human-derived cell organelle fractions, primary cultured cells or cDNA-expressed proteins as a source of drug-metabolising enzymes. This would enable more reliable results with fewer animal trials in the early preclinical phase.
The effects of an NCE on drug-metabolising CYP enzymes can be tested by using CYP isoform-specific model substrates and reactions. The effects of the studied compound on metabolite formation in the selected model system (human liver microsomes, hepatocytes, etc.) are evaluated by incubating the substrate and the studied compound with the enzymes and by observing the metabolite formation in incubations with the studied compound and by comparing it to the formation in incubations without the tested compound (Boobis 1995; Pelkonen et al. 1998; Wrighton et al. 1993b).
Testing the effects of an NCE on CYP-specific model activities and the effects of CYP-specific reference inhibitors on the metabolism of an NCE in human liver microsomes in vitro gives information about the affinity of an NCE for CYP enzymes and permits in vivo predictions about the behaviour of the NCE in man (metabolic pathways, intrinsic clearance, etc.), which helps to design in vivo studies for revealing possible interactions (Yuan et al. 1999). The determination of IC50 (the concentration causing 50% inhibition compared to the control activity) and Ki values (the affinity of the compound for the enzyme at the initial velocity conditions) for the studied compound produces information about the inhibitory effect of an NCE on CYP isoforms, and enzyme kinetic studies can be made to evaluate the possibilities of drug-drug interactions (Yuan et al. 1999).
Many different models for the prediction of drug metabolism and drug-drug interactions in vitro have been introduced recently. One of the best characterised models is the use of the microsomal fraction derived from the human liver tissue samples. The collection of human liver tissue has become easier globally, although there are some practical and especially ethical questions associated with their use. However, studies using human liver preparations produce a “golden standard” for other studies, given naturally their inherent limitations, and they should only be exploited to the extent feasible (Boobis 1995; Guillouzo et al. 1995; Pelkonen et al. 1998; Yuan et al. 1999).
Fig. 1 presents an approximate time course of drug development. As one can see, preclinical studies start at the very beginning of a lead compound selection and continue up to the time of the first phase I clinical studies. Metabolic stability assays employing different test species and human liver make it possible to select species that best represent the human in the metabolic fate of an NCE. These results can be utilised in, for example, selecting test species for toxicological tests.
The affinities to CYP enzymes and the enzymes that participate in the biotransformation of an NCE are valuable information for the selection of lead compounds and for the planning of early clinical studies. On the basis of in vitro studies, a tentative prediction of the clearance and interaction potential of an NCE can be made, and the first clinical studies can be based on these results.
