15.7.1 QuantitativeIn vitroRadiolabeled Reaction Phenotyping Studies
Experimental methodologies utilizing radiolabeled compound are similar to
those where authentic standard is available (see Section 15.6.3). Radiolabeled
studies can be conducted using the following: expressed enzyme and/or human
liver microsomes for the evaluation ofKm–Vmaxchemical inhibition, and/or
correlation analysis using a characterized bank of human liver microsomes
(Tuvesson et al., 2005; Zhu et al., 2005; Zhang et al., 2007).
Often in the early stages of drug development the relative abundance of a
metabolite (in the absence of a synthesized standard or radiolabeled material)
is estimated utilizing multiple detection methods. These methods include
comparing either the ionization or the UV absorbance of a metabolite to the
parent drug utilizing mass spectroscopy or UV detection (within the limits
outlined above in Section 15.6.3.2), respectively. However, once radiolabeled
material becomes available the relative abundance of a metabolite in anin vitro
reaction phenotyping study can be determined by utilizing HPLC––radio-
metric detection via a comparison of the characterized radiometric peaks
(Fig. 15.6). This assumes however that the metabolite of interest contains either
the radiolabeled moiety or there is a clear and well-understood relationship
between a radiolabeled component and the metabolite of interest (i.e., single
cleavage product).
To estimate the total abundance of a particular metabolite in anin vitro
reaction phenotyping sample using a single^14 Clabel and HPLC-radiometric
detection, the amount of the metabolite in a given sample is estimated by
multiplying the percent abundance of the metabolite obtained from a
radiometric chromatogram of the sample (Fig. 15.6) to the molar amount of
the initial radiolabeled parent drug present in the sample (assuming overall
radioactive recovery is greater than 90%: taking into account incubation
recovery, sample extraction efficiency, and column recovery). If the overall
radioactive recovery is poor, then the metabolic profile obtained may not
FIGURE 15.6 Example radiochromatogram.500 REACTION PHENOTYPING