accurately reflect the true metabolic profile of the compound and may reflect
experimental or analytical issues (i.e., the radiolabel located on a metabolically
unstable position, specific metabolites not extracting or being retained on the
HPLC column). This calculation can only be conducted for radiolabeled
components that are present on the radiochromatogram and it requires well-
defined chromatographic separation of the metabolic peaks of interest.
Radiolabeled components that are only observed utilizing mass spectroscopy
would be considered trace amounts.
15.7.2 In vivoQuantitative ADME Studies
The extent of formation of a particular metabolite and its impact on overall
drug elimination is ultimately evaluated in a radiolabeledin vivoabsorption,
distribution, metabolism, and excretion (ADME) study. Human radiolabeled
ADME studies are often conducted to determine overall elimination pathways
of a drug of interest. If a polymorphic enzyme primarily metabolizes the
compound of interest, the subjects may be genotyped for this enzyme and a
poor metabolizer cohort added to the study. Essentially the concentration time
profiles of a drug and its metabolites as well as the final routes of elimination of
an administered dose of drug are determined in a radiolabeled ADME study.
The matrices that are commonly collected in human radiolabeled ADME
studies include blood, plasma, urine, and feces with additional matrices
collected for metabolite characterization if needed and available (i.e., bile). The
total amount of the radioactive dose eliminated is determined for each
excretory matrix (i.e., urine and feces). The excretory matrices are then profiled
utilizing HPLC-radiometric detection and the observed radiometric peaks are
then characterized utilizing LC/MS/MS and NMR (if regiochemical identifica-
tion is required).
The % dose (or fraction metabolized) for each metabolite is calculated by
summing the % dose calculated for each excretion pathway (urine or feces) of
the metabolite and any subsequent secondary metabolites. The % dose for
each excretion pathway is calculated by multiplying the percent abundance of a
characterized metabolite on a radiometric chromatogram by the total
radioactivity present in the evaluated sample (once extraction and column
efficiencies have been taken into account). More detailed analysis would be
required to calculate the fraction metabolized if secondary metabolites were
formed from more than one primary metabolite (Bu et al., 2005, 2006).
The systemic exposure of circulating metabolite is calculated by estimating
the area under the curve for a mean radioactivity versus time plot (Fig. 15.7).
The fraction of the systemic exposure contributed by a particular metabolite is
calculated by dividing the AUC estimated for a particular metabolite by the
AUC of the total radioactivity. Alternatively, the metabolite concentration
time profile and metabolite concentrations can be constructed
using nonradiometric bioanalytical techniques if metabolite standards are
available.
RADIOLABELED REACTION PHENOTYPING 501