been published to date fall into four categories: (1) expert systems designed to
identify probable sites of metabolism based on comparison to database
information (Erhardt, 2003; Hawkins, 1999), (2) programs to predict the site of
metabolism based on molecular orbital theory (Jones et al., 2002), (3) programs
to predict the rate of metabolism based on physical chemical attributes of the
molecule and QSAR development (Andrews et al., 2000; Balakin et al., 2005;
de Groot and Ekins, 2002; Ekins and Obach, 2000; Ekins et al., 2005; Lewis,
2003, 2004; Long and Walker, 2003; Taskinen et al., 2003; Yoshida and
Topliss, 2000) and (4) the prediction of site of metabolism based on knowledge
of the CYP active site (Lewis, 2005; Lewis et al., 2006).
8.3 Metabolite Profiling
Although advances in LC/MS/MS technology have made the determination of
metabolic profiles without radiolabeled material more reliable, methods for
obtaining quantitative, complete profiles using cold material are still lacking.
The use of radiolabeled material for the rigorous determination of metabolic
profiles remains the ‘‘gold standard’’ at all stages of drug discovery and
development. However, as mentioned above, new HPLC technology and
techniques have made qualitative metabolite profiling ofin vitroandin vivo
samples much more robust. New highly sensitive triple quadrapole and ion-
trap mass spectrometers along with techniques such as neutral loss and product
ion scanning allow routine detection of metabolites even from complex
matrices. Future advances in technology, such as accurate mass–mass
spectrometers coupled with mass filters and nanospray technologies will likely
lead to significant improvements in both qualitative and quantitative
metabolite profiling data and will continue to make metabolite profiling at
the early stage more definitive.
The majority ofin vitrometabolite profiling experiments are designed to
evaluate interspecies differences in profiles and are generally done to provide
background context for toxicology studies. These studies generally involve
HPLC separation of metabolites followed by quantitation by flow or standard
scintillation counting. There have been recent advances in technology that have
made the coupling of mass spectrometers in-line with radioactive flow detectors
possible so that metabolites can be identified in the same run that they are
being quantified (Egnash and Ramanathan, 2002; Nassar et al., 2003, 2004).
Also, new scintillation counters and technologies are available that allow much
greater sensitivity and thus reduce the amount of radioactivity necessary to
detect low level metabolites (Nassar et al., 2003, 2004).
A major use of radiolabeled drugs in thein vivosituation is for metabolic
profiling. These studies can be utilized to investigate interspecies comparisons
of metabolic profiles in plasma urine and feces (or bile) in conjunction with
toxicology or carcinogenicity studies or for performing definitive metabolite
profiling in human ADME studies.
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