bind cellular macromolecules. Although there are many literature values for
covalent binding of compounds associated with toxicity, there are relatively
few reports of compounds that are clinically safe and thus the lower limit of
covalent binding values has not been well established.
Trapping experiments are most often done with unlabeled drug and
glutathione with detection by mass spectrometry. Because unlabeled drug is
used, these experiments can be done earlier in the discovery cycle than the
covalent binding experiments. The detection of glutathione adducts is aided by
the characteristic fragmentation pattern of glutathione. The use of this
fragmentation has recently been adapted into a relatively high throughput assay
based on the neutral loss of the lysine fragment of glutathione (Chen et al., 2001).
Other analytical strategies have been developed to provide qualitative and/or
quantitative determination of reactive metabolite formation at the screening
stage (Argoti et al., 2005; Dieckhaus et al., 2005; Gan et al., 2005; Mutlib et al.,
2005; Yan et al., 2005). These assays provide only qualitative information or
utilize a nonphysiological trapping agent and are thus somewhat limited in
utility. The approach most often employed to circumvent this problem is through
the use of radiolabeled trapping agents, typically tritiated glutathione. The
information generated from this approach can be utilized in a number of ways:
(1) after determination of the formation of adduct and quantitation of adduct
level, the adduct structure can be characterized. The adduct structure can then
lead to medicinal chemistry approaches with the goal of limiting the amount of
adduct formed. (2) as a trigger to perform more advanced covalent binding
studies. (3) as a trigger to do advanced toxicology studies in animals or in
hepatocytes. The link between the absolute amount of glutathione adduct
formed upon reaction of a compound with liver microsomes in the presence of
glutathione and prediction of toxicological endpoints has not been made and is
likely to remain elusive. For this reason, the information gained in trapping
experiments is usually used to modify structure to minimize the problem or to
trigger more definitive studies.
8.5.2 Reactive Metabolite StudiesIn vivo
Questions regarding the formation of metabolites that covalently bind
proteins can be addressedin vivowith experiments similar in nature to the
experiments described in thein vitrosection. Animal studies are often focused
on the liver proteins as a target for drug binding, while human studies are
usually focused on blood components due to obvious ethical limitations.
Baillie and colleagues have proposed the use of extent of covalent modification
as a measure of risk of adverse events for new chemical entities. In this
proposal, a level of greater than 50 pmol compound bound/mg microsomal
protein has been put forth as a level of binding that would provide a safety
margin (approximately 20-fold) over compounds that have shown liver
toxicities in the clinic and are thought to produce toxicity through formation
of reactive intermediate (Evans et al., 2004).
248 DRUG METABOLISM RESEARCH