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VOL. 73 (1), 5-26, 2007 IN VITRO INVESTIGATION OF DRUG METABOLISM AND TOXICITY...

differences between animals and man exist, not only because of the
different P450 enzymes expressed in man and other species, but also
because of their relative abundance (18-20). Therefore, metabolite
profiles in different animal models (i.e. microsomes or hepatocytes
from several species) are analyzed and compared with the human
profile toward a more astute selection of animal species for
subsequent pharmacokinetic or toxicological studies. As metabolites
may be inert, pharmacologically active or reactive, drug metabolite
profile evaluation and metabolite identification are essential to
design new safer drug candidates with improved ADME capabilities.
Incubating the drug candidate with recombinant P450s individually
expressed in various host systems can generate large amounts of
metabolites for chemical structure identification (Figure 2).
Analytically speaking, liquid chromatography tandem mass
spectrometry is the most widely used tool to identify metabolites
and determine both structure and metabolite profiles (21).

PREDICTION OF IN VIVO PHARMACOKINETIC
PARAMETERS FROM IN VITRO DATA

Human liver-derived models are invaluable tools in elucidating
the pharmacokinetic parameters of a drug candidate and the
selection of lead compounds with favorable properties during the
drug discovery and development process (22, 23). Clearance of a
drug from the body depends on the intrinsic ability of the organs,
such as liver and kidney, to metabolize and excrete. Systemic
clearance of a drug that is eliminated by hepatic metabolism is a
function of the hepatic blood flow and the intrinsic clearance of the
liver (CLint, in vivo), which is defined as the ability of the liver to
remove xenobiotics from the blood in the absence of other
confounding factors. Disease conditions of the liver or administration
of drugs that are inducers or inhibitors of P450 can therefore
influence systemic clearance. CLint, in vitro is a measure of enzyme
activity towards a drug which is not influenced by other physiological
determinants of liver clearance, such as hepatic blood flow or drug
binding to blood proteins (24). As with all clearance terms, it has
units of volume rate and acts as a proportionality constant to
describe the relationship between the metabolism rate of a drug and

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