Drug Metabolism in Drug Design and Development Basic Concepts and Practice

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Bioactivation, or metabolic activation, is a process where xenobiotics are
metabolized to proximate and ultimate reactive intermediates (Kalgutkar et al.,
2002, 2005). In general, these reactive intermediates are electrophiles and ready
to react with nucleophilic macromolecules, including protein and DNA. From
chemistry point of view, electrophiles include polarized double bonds,
epoxides, carbonium, and iminium ions (Coles, 1984–1985). The nucleophilic
portions of macromolecules in biological systems include the thiol groups of
cysteinyl residues in proteins and glutathione, amino groups in proteins, DNA
and RNA, and phosphate oxygen atoms in DNA and RNA. The chemical
hardness and softness of the electrophiles and nucleophiles are mainly
dependent on their polarizability. The polarized double bonds and epoxides
are considered as soft electrophiles, and iminium ions hard electrophiles. On
the contrary, the thiol group is the soft nucleophile in biological systems. The
amino and the phosphate groups in DNA and RNA are considered as hard
nucleophiles. Reactions occur more readily between electrophiles and
nucleophiles of similar hardness (Coles, 1984–1985).
Bioactivation can also lead to the formation of free radicals and reactive
oxygen (ROS)/nitrogen/chlorine species, and these species may cause oxidative
stress that is deemed as one of the factors in the development of drug-induced
toxicity (Comporti, 1989; Aust et al., 1993; Guengerich, 2001). Exposure to
ROS has been implicated in diseases such as cancer, Alzheimer’s, and
atherosclerosis (Butterfield, 1997; Harrison et al., 2003; Klaunig and
Kamendulis, 2004). Classic examples of free radical formation/oxidative stress
are cytochrome P450-mediated semiquinone radical formation from acetami-
nophen and reductive dehalogenation of CCl 4 and halothane, DT-diaphorase-
mediated semiquinone radical formation from menadione, and peroxidase-
mediated radical formation from phenylbutazone and butylated hydroxyto-
luene (Parkinson, 2001 and references therein). At present time, it is not a
common practice to screen new chemical entities (NCEs) for free radical/ROS
formation in the pharmaceutical industry. However, there have been excellent
reviews in the recent years on the methodologies of detection and measurement
of free radical/ROS (Degli Esposti, 2002; Halliwell and Whiteman, 2004). In
this section, measurement of intracellular GSH/GSSG levels, one of the
surrogate biomarkers of oxidative stress, will be discussed.
Common approaches to evaluate bioactivation potential include screening
for glutathione (GSH, Fig. 14.1), N-acetylcysteine (NAc, Fig. 14.1), and
cyanide adducts of unlabeled compounds in incubations with liver microsomal
preparations or recombinant cytochrome P450 enzymes in the presence of
corresponding trapping agents and evaluating covalent binding of radiolabeled
compounds to proteins of liver microsomal preparations or hepatocytes.
Evaluation of covalent binding of radiolabeled compounds gives a quantitative
measure of the extent of bioactivation, hence provides valuable guidance in
compound selection for further development. However, covalent protein
binding studies are resource intensive and do not provide mechanistic insight of
the bioactivation pathways. Trapping studies using trapping agents such as


448 PROTOCOLS FOR ASSESSMENT

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