comparative metabolite profiling. On the contrary, if the metabolism rate of a
test drug in hepatocytes is relatively low, resulting in difficulty in metabolite
detection, or if metabolite profiles between human liver microsomes and
hepaotcytes are qualitatively similar, the liver microsome model is a good
choice for the profiling ofin vitrometabolites across species.
For the purpose of metabolite profiling, in vitro models have several
advantages overin vivoADME studies (1)In vitrometabolism studies need
much less resources. (2) Due to lower amounts of endogenous components
present inin vitrosystems than those in plasma, urine, and feces, identification
ofin vitrometabolites by LC/MS and quantitative analysis by LC/UV can be
readily accomplished without using radiolabeled compounds. (3) In vitro
metabolism models allow for determining the enzymes involved in a
bioactivation reaction. (4) In vitro metabolism experiments can provide
meaningful assessment of reactive metabolite formation by measuring
covalent protein binding or using trapping agents. However, in vitro
metabolite profiles are not consistent with those in plasma in many cases.
Additionally,in vitrometabolism models often underestimated the secondary
metabolites or metabolites formed from non-CYP-mediated biotransformation
reactions.
7.3.3 ADME Studies
In vivometabolite profiling data are routinely obtained from ADME studies
using radiolabeled material (Marathe, 2004; Dalvie, 2000). An ADME study
consists of two parts: (1) mass balance measurement by analyzing the total
radioactivity in excreta, such as urine and feces, and/or bile, and (2) quantitative
and qualitative metabolite profiling of biological matrices, including plasma,
urine, bile, and/or feces (see Chapters 9 and 18). Radiochromatograms in
plasma reveal the relative abundance (percentage of the total radioactivity) of
circulating radiolabeled components. Consequentially, concentrations of
individual radioactive components in the plasma, which are utilized for
calculating exposure to metabolites, can be derived from the relative abundance
of radioactive peaks and the concentration of total radioactivity in the plasma.
The total amount of a metabolite as a percent of dose can be calculated based
on the total radioactivity excreted into urine and feces (or bile), and the relative
abundance of this metabolite in urine and feces (or bile). Results from ADME
studies, together with pharmacological and/or toxicological activity data of the
metabolite, are essential for the determination of whether a specific metabolite
needs to be monitored in toxicological and clinical studies.
7.3.4 Analytical Methods for Metabolite Profiling
Metabolite profiling involves detection, quantitative analysis, and structural
elucidation of drug metabolites present in a biological matrix using HPLC
coupled with a UV detector, radioactivity detector, and/or mass spectrometer.
METABOLISM STUDIES RELEVANT TO METABOLITE SAFETY ASSESSMENT 213