Drug Metabolism in Drug Design and Development Basic Concepts and Practice

11.8 DETECTION AND CHARACTERIZATION OF REACTIVE

METABOLITES AND INTERMEDIATES

Bioactivation is a long standing issue for drug development because of
potential risks associated with drug–protein adduct and organ toxicity
(Williams et al., 2002). There has been an increasing emphasis on screening
drug candidates for their tendency to generate reactive metabolites and
characterizing the nature of the reactive metabolites. Potential bioactivation
mechanism(s) can then be defined and rationalized, which may provide a
synthetic intervention strategy at an early stage to modify or screen out
problematic compounds.

11.8.1 Trapping Reactive Metabolites

Most reactive metabolites are electrophiles that can react with nucleophiles
(such as proteins). In vitro chemical trapping approaches are generally
employed to examine the bioactivation potential of drug candidates. These
experiments are often conducted in liver microsomes with NADPH and
appropriate nucleophilic trapping agents, such as thiols (glutathione, its ethyl
ester derivative, cysteine, or N-acetylcysteine), amines (semicarbazide and
methoxylamine), or cyanide anion (Evans et al., 2004; Kalgutkar and Soglia,
2005). Glutathione (GSH) contains a free sulfhydryl group, a soft nucleophile
capable of reacting with a broad range of reactive electrophiles, including
Michael acceptors, epoxides, arene oxides, nitrenium ions, and alkyl halides.
The use of the corresponding ethyl ester analogue of GSH has been shown to
increase the MS sensitivity of the detection of reactive metabolites (Soglia et
al., 2004). Semicarbazide and methoxylamine are hard nucleophiles, which will
preferentially react with hard electrophiles such as aldehydes (Chauret et al.,
1995; Zhang et al., 1996). The cyanide anion is a hard nucleophile that can be
used to effectively trap iminium species (Argoti et al., 2005).

N

Br Cl

Br

N

O

N

H 2 N O H 2 N H 2 N HN

2

N

Br Cl

Br

N

O

N

O

HO

N

Br Cl

Br

N

O

N

O

MeO

N

Br Cl

Br

N

O

N

O

Lonafarnib Metabolite A Metabolite B Metabolite C

SCHEME 11.7 Chemical structures of Lonafarnib, metabolite A, B, and C.

DETECTION AND CHARACTERIZATION OF REACTIVE METABOLITES 353