(3) In some cases, bis-adducts may be formed in trapping studies. It is
recommended to extract MS spectra of these potential adducts in
chromatography at a molecular weight of two molecules of the trapping
agents plus the molecular weight of the test molecule in MS spectrum
analysis.
(4) Because of the natural tendency of oxidation of free thiols, dGSH
should be stored at 80 C prior to use. Aliquots of dGSH in water are
stable at 80 C for at least 1 year, but not very stable at 20 C.
(5) Use of dithiothreitol in MeOH reduces the oxidized dGSH to obtain
cleaner chromatograms. Dithiothreitol should be omitted when the
potential adduct of interest has a disulfide bond.14.2 PROTOCOLS FORIN VITROANDIN VIVOCOVALENT
PROTEIN BINDING STUDIES
14.2.1 Introduction
Bioactivation of xenobiotics leads to the formation of reactive intermediates,
which may covalently bind to cellular proteins. General approaches to
quantitatively assess the bioactivation potential of compounds includein vitro
covalent protein binding studies in liver microsomal preparations, and
hepatocytes from human and preclinical species, andin vivostudies in selected
preclinical species. In cases wherein the compounds of interest are mainly
metabolized by Phase I metabolism,in vitrobinding studies in liver microsomal
preparations should provide relatively relevant assessment for covalent protein
binding potentialsin vivo. On the contrary, if compounds are mainly cleared
through Phase II metabolism or mixed Phase I/II metabolism,in vitrobinding
studies in hepatocytes would be relatively more valuable.In vivocovalent
protein binding studies have been often performed in rats for practical reasons.
At Merck, a high concentration of 10mM and a dose of 20 mg/kg of
compounds are commonly used forin vitroandin vivocovalent protein binding
studies, respectively (Evans et al., 2004). This reflects the desire to balance
maximizing analytical sensitivity with standardizing protocols. Investigators
may use lower doses depending on the intended clinical use of testing
compounds.
Covalent protein binding studies require the use of radiolabeled compounds.
(^3) H-tracers are often used because they are relatively easy to synthesize and are
of high specific radioactivity. However, possibilities exist that the^3 H-label may
be lost if oxygenation of the test compound occurs directly on the carbons
where the^3 H-label is attached. As a consequence, observed binding values
could be underestimated.^14 C- tracers, on the contrary, are less likely to lose the
(^14) C- label, and therefore are preferred. However, possibilities of losing a
radiolabel should be considered for any given radiolabeled tracer. Therefore,
evaluation of the loss of radiolabels needs to be performed before covalent
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