the fragment ion or neutral fragment used in the PIS or CNLS experiments, the
metabolite will not be detected. This is typically not a major concern since as a
compound advances toward late stage drug discovery, a more detailed
metabolite characterization with radiolabeled drugs will likely detect any
missed metabolite.
During late stage drug discovery, it will be important to characterize major
metabolites to establish whether there are significant metabolic differences
between species and to identify potential pharmacologically active, reactive, or
toxic metabolites (Nassar and Talaat, 2004). At this time, the tritium (^3 H)-
labeled parent drug is often available; therefore, radiometric detectors can be
coupled in-line with mass spectrometers to facilitate metabolite identification.
The radiochromatograms assist in locating the metabolites in the chromato-
gram, as well as to provide a semiquantitative estimate for each metabolite.
Structural characterization of all major metabolites (>10% of the total
chromatographic radioactivity) is attempted (Cox, 2005). Using the parent
drug and its corresponding fragmentation pattern as a reference, the site(s) of
biotransformation can often be narrowed down to a certain region of the
molecule based on a corresponding shift inm/zvalue of a characteristic product
ion after comparing the product ion (MS/MS or MSn) spectra of a metabolite
with that from the parent drug.
Identification of reactive intermediates/metabolites is critical in drug
discovery research (Evans et al., 2004; Ma and Subramanian, 2006).
Reactive intermediates are short-lived; therefore chemical trapping agents
(e.g., glutathione, methoxylamine, cyanide) are often usedin vitroto form
stable adducts with reactive intermediates that can be characterized by LC/MS/
MS and LC/NMR (Baillie, 2006; Evans and Baillie, 2005). These experiments
provide valuable indirect information on the identity of the reactive
intermediates, thereby defining a potential bioactivation mechanism and hence
a rationale on which to base a synthetic intervention strategy to minimize the
formation of reactive intermediates. Detecting and characterizing reactive
metabolites will be discussed in more details in the later section of this chapter
(Section 11.8).
11.3.2 Metabolite Identification in Preclinical and Clinical Development
In the early stages of preclinical development, metabolite profiling is performed
usingin vitrosystems from animal and human, mainly to identify potential
species-dependent metabolism early in the development process and to support
the selection of the animal species employed in safety assessment studies. As
the compound moves further into development,in vivoanimal ADME studies
are performed. The compound is usually dosed into a rodent and nonrodent
species along with an efficacy model. Metabolism data from the animal studies
are then used in species selection for safety assessment to insure that all
expected human metabolic transformations will be represented in the animal
models used in the safety study.
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