for the calculation of the RAF value(s) particularly for enzymes that
demonstrate substrate dependent kinetic relationships (i.e., CYP3A4). Other
potential confounding factors using the RAF approach are nonspecific protein
binding and potential differences between expressed enzyme systems. Since
higher protein concentrations are usually used with microsomal incubations,
compared to expressed enzyme incubations, nonspecific protein binding may
be different for the same substrate across the two matrices, especially for
lipophilic amines. Also in consideration of P450 scaling, expressed systems
often contain excess cytochrome b5, over expressed P450 reductase, or a
different lipid environment when compared to microsomes. Therefore, an
RAF value calculated for one expression system or lot of expressed enzyme
may not be relevant for a different expression system or lot of expressed
enzyme.
Another quantitative approach, scales the expressed enzyme activity using
the relative abundance of the enzymes of interest. The recombinant or purified
enzyme reaction rate is scaled utilizing the relative abundance of the particular
enzyme in microsomes, hepatocytes, or liver (Proctor et al., 2004; Rodrigues,
1999). A modified approach that has been utilized for P450s incorporates a
scaling factor (Inter System Extrapolation factor: ISEF) to account for the
potential differences in intrinsic activity per unit enzyme between expressed
enzyme and human liver microsomes (Proctor et al., 2004). These approaches
based on enzyme abundance require accurate immunohistochemical data
regarding the enzyme of interest. A comparison of the relative abundance
approach, RAF based on Vmax, and RAF based on CLinthas also been
conducted utilizing the substrate depletion approach in early discovery; where
the most accurate prediction method was determined to be RAF based on
CLint(Emoto et al., 2006). Currently, scaling by relative abundance for
metabolic enzymes has only been conducted for P450s.
15.7 Radiolabeled Reaction Phenotyping
Radiolabeled reaction phenotyping studies are usually conducted in the later
stages of drug development due to the timing of the availability of the
radiolabeled compound. However the timing of radiolabeledin vitrostudies
(and preclinicalin vivostudies) can be adjusted due to the development needs
of a NCE. Tritium is often used instead of^14 Cin early drug discovery studies
depending on the particular isotopic challenges in the radiosynthesis of a
compound. Isotopic stability (loss of the tritium) is a particular problem with
tritium labeled compounds and should be evaluated prior to the conduct of
reaction phenotyping studies. As a compound moves through the drug
discovery–drug development continuum, the evaluation of its metabolic
processes and the subsequent evaluations (if risk is projected) of interindividual
variability (due to polymorphic enzymes) and drug–drug interactions become
more definitive.
RADIOLABELED REACTION PHENOTYPING 499