9.3.1 Use of Radiolabeled Compound in ADME Studies
During the process of drug development there are numerous studies conducted
with radiolabeled material (C-14 or tritium), because these studies provide
quantitative information about parent and metabolites (Marathe et al., 2004). To
provide reliable information, it is critical that the position of the C-14 or tritium
label on the molecule be such that there is no loss of the label either due to
exchange or due to a small fragment that gets incorporated or through
expiration. The position of the label on the molecule is based on prior knowledge
about the metabolism of the compound conducted during lead optimization. In
cases where metabolism of a compound leads to splitting of the molecule into
large fragments, it is useful to label both sides of the molecule. This strategy has
been successfully used in ADME studies with omapatrilat and gemopatrilat.
Amide hydrolysis, resulting in the formation of (S)-2-thio-3-phenylpropionic
acid, was shown to be one of the major metabolic pathways for both of the
structurally similar ACE and NEP inhibitors. Therefore, the radioactivity studies
with a single [^14 C] label would not elucidate the metabolic fate of both sides of the
molecule. Based on this consideration, ADME studies were conducted with a
mixture of [^14 C]-labeled derivatives: one [^14 C] label at the exocyclic carbonyl
carbon, and the other [^14 C] label on the amine side of the molecule that is
generated after hydrolysis. This approach helped to track metabolites that arose
from both sides of the molecule (Iyer et al., 2001; Wait et al., 2006).
In addition to quantitation of parent and metabolites, radiolabeled studies
also aid in the detailed structural characterization of metabolites since
radioactivity can be used as a selective marker for isolation and identification
purposes. LC/MS/MS analysis can then be performed on the metabolite of
interest and the structure of the metabolite assigned based on mass spec
fragmentation patterns. One of the challenges faced during the analysis of a
complex matrix like bile or urine, where there is interference from endogenous
compounds, is to identify the base peak for MS/MS analysis. One approach to
circumvent this issue is to administer a stable-labeled drug along with
radiolabeled drug in a fixed proportion during the ADME studies. The use of
stable-labeled compound in toxicology studies with small molecules and
chemicals is well reported in the literature (Fennell et al., 1991; Fennell and
Summer, 1994; Sumner et al., 1992). This strategy was successfully used in
ADME studies with omapatrilat. The stable-labeled^13 C 2 omapatrilat was
included in the dose to help in the identification of the metabolites by LC/MS.
The metabolites that retained the C-13 labels showed a characteristic^12 C^13 C
isotope cluster in the full mass spectrum. LC/MS/MS analysis of the molecular
ions (m/zand^13 C 2 m/z) further confirmed the identity of the metabolites. The
observation that the daughter ions either retained or lost the^13 C 2 -label helped
establish the fragmentation pattern for the metabolites (Iyer et al., 2003).
Recently, mass defect filtering techniques were developed and effectively
applied to identify molecular ions of drug metabolites in complex biological
matrices such as plasma, urine, bile, and feces (Zhang et al., 2003).
IN VIVOADME STUDIES 267