interindividual variability in exposure via significant drug–drug interactions
or polymorphic expression coupled with the advanced development of tools
such as specific chemical or antibody inhibitors of the major P450s,in vitro–
in vivoscaling techniques, and recombinant enzyme systems (Crespi et al.,
1993; Lee et al., 1995; Remmel and Burchell, 1993) have all made reaction
phenotyping a staple approach across the pharmaceutical industry. In
addition, regulatory expectations now exist whereby new chemical entities
(NCEs) are to be sufficiently characterized during development such that
potential risks to a patient population can be weigheden route to final
dosing and labeling (US Department of Health and Human Services et al.,
1997, 2001, 2006).
Optimal application of the various reaction phenotyping techniques is
dependent upon the scientific issue at hand, required robustness of data to
enable decision making, and whether data are being used in compound
selection and design versus the full characterization of a single compound.
During the earliest stages of the novel drug discovery process, it is generally
not known which specific enzymes are responsible for the metabolism of a
newly synthesized compound although the role that biotransformation
contributes to the total clearance of a compound may be estimated. The
extent to which this metabolism is governed by P450s, non-P450s or
conjugative enzymes, may be delineated by manipulatingin vitrosystems to
assess the impact of (1) eliminating cofactors (e.g., NADPH for P450s,
UDPGA for glucuronidation) on metabolic turnover, (2) coincubating with
P450 pan-inhibitors (e.g., aminobenzotriazole, sulconazole, SKF-525A), or
(3) preboiling preparations to destroy enzymatic activity to establish the
contribution of chemical instability. Such evaluations may be incorporated
into a general high throughput screening strategy or performed on small
subsets of prototypical compounds representative of the template of interest
to learn if observed attributes are associated with the structural core or offer
the potential to be designed out in a facile manner. Drug discovery strategies
often call for the desired development candidate to be eliminated by multiple
pathways that may include several P450 enzymes, conjugation or direct
elimination into bile or urine. Typically, P450s do play a role in the metabolic
processing of small organic molecules so it is often desirable to determine the
specific P450 enzyme contribution for mature leads generated during
discovery to enable the advancement of lower risk candidates into the clinic.
However, such a profile is sometimes not achievable within a chemical
series and an overall risk assessment of the clinical prospects will need to
be made.
Metabolic reaction phenotyping usually involves the use of enzyme specific
chemical inhibitors in fully competent microsomal systems or recombinant
enzyme systems individually expressing single enzymes. Once a drug candidate
has entered the clinic, efforts will be made to perform a more definitive reaction
phenotyping characterization as well as the consideration of clinical probe drug
evaluations if patient risk is perceived.
478 REACTION PHENOTYPING