Alternatively, bile and urine metabolites can be estimated with HPLC–UV
chromatography using the extinction coefficient of the parent. These measure-
ments will begin to define the total absorption of the compound and how that
relates to the systemic bioavailabilty. The availability of radiolabeled compound at
this point makes the bile duct cannulated rat experiment especially powerful.
A secondin vivomodel system that is very useful in sorting through problem
of low oral bioavailability is portal vein cannulated animals. There are two
ways this experiment can be conducted to determine hepatic extraction: (1)
measure systemic plasma concentration after oral, portal vein and systemic
administration and (2) measure portal vein and hepatic vein concentrations
after an oral dose. Both methods yield information on hepatic extraction and
the percentage of dose reaching the portal circulation (the product of the
fraction absorbed and the fraction metabolized by the gut wall).
Both the bile duct cannulated model and the portal vein cannulated model
can be combined with a number of methods of modulating absorption or
metabolism to ask specific mechanistic questions regarding stability,
permeability, and metabolism. The following are several methods that can be
used to modulate metabolismin vivo. The most commonly used method to
modulate oxidative metabolismin vivois to coadminister either ketoconazole
or 1-aminobenzotriazole (Balani et al., 2002) to inhibit CYP enzymes. Both of
these compounds will inhibit intestinal and liver CYP enzymes after oral
administration. Care must be exercised when using ketoconazole for this
purpose because the compound also has effects on transporters and this effect
may make interpretation of results ambiguous. Alternatively, a CYP inducer
can be coadministered to determine the effect on the clearance of the
compound of interest. This can be accomplished with one of many known
inducers of CYP enzymes. For inhibition of esterase enzyme activity,
bis-[p-nitrophenyl]phosphate (BNPP) can be coadministered because this
compound does not inhibit cholinesterase activity (Buch et al., 1969).
Methods for inhibiting conjugation enzymesin vivoare not as well defined,
since in most cases good inhibitors of the enzymes have not been identified. It is
possible to deplete cofactor stores by coadministering large amounts of
substrate to ask mechanistic questions, although this method is most easily
applied to sulfotransferase (SULT) enzymes (Kim et al., 1995). Many
conjugation enzymes are subject to induction, which may be an avenue
available for modulating enzyme activitiesin vivo.
8.2.4 Screening Strategies
The approach often taken in candidate optimization is to try to isolate
bioavailability problems for a member of a chemotype of interest with a
combination ofin vitroandin vivoexperiments and then to try to devise rapid
techniques to screen for the liability (Fig. 8.1). This approach relies on a good
deal of up-front work to fully understand the bioavailability limitations and
periodic checking of the property to ensure that the screen is providing reliable
METABOLIC CLEARANCE 243