concentration of most drugs is closer to the total plasma concentration than
the free plasma concentration due to efficient uptake by the liver, (2) protein
binding to microsomal proteins may serve to ‘‘cancel out’’ protein binding in
plasma, that is, the free concentration in microsomal incubations is closer to
the free concentration in plasma so the effect protein binding is already
accounted for in the in vitroparameters. Methods to account for protein
binding or lessen its effect are (1) to determine free concentration in
microsomal incubations and then calculatein vitroparameters based on free
drug (this would then be compared to free plasma clearance values), or (2) to
use as little protein in microsomal incubations as possible to lessen the effect of
binding. The scale-up from in vitroparameters to predict in vivohuman
clearance of new chemical entities is still a difficult task to do prospectively,
especially in cases where the CYP kinetic data does not follow classical
Michaelis–Menten behavior (Houston and Kenworthy, 2000). The uncertainty
of this calculation can be somewhat mitigated if the same scale-up methods
provide accurate results when applied to preclinical animal data.
This method has most often been applied in conjunction to rates of CYP-
mediated oxidative metabolism in microsomal systems but can also be applied
to conjugation (Lin and Wong, 2002; Miners et al., 2004, 2006; Soars et al.,
2002) or FMO-catalyzed reactions (Fisher et al., 2002) or data derived from
hepatocytes (Lau et al., 2002) or otherin vitrosystems (Yamamoto et al., 2005).
The scaled clearance values can be compared to the values determinedin vivo
for a single compound or a set of analogs to give some idea of the predictive
capacity of thein vitrosystems.
The use of microsomes along with UDPGA as cofactor assay to measure
UGT enzyme activity has been hampered historically by the fact that this
enzymatic activity in microsomes is often in a ‘‘latent’’ form and requires
activation by physical or detergent-induced disruption of the membrane
matrices. Recently, a generic method involving the addition of the pore-
forming peptide alamethicin to overcome the latency exhibited by this enzyme
system has been described (Fisher et al., 2000). The inclusion of alamethicin
seems to provide a more consistent method of assessing UGT enzyme activity.
8.2.3 In vivoMethods to Study Metabolism
The methods outlined above may not lead to a satisfactory determination of
mechanism of incomplete bioavailability and additional methods may be
necessary in some cases to fully characterize the factors responsible. Bile duct
cannulated animals provide a powerful model to examine incomplete bioavail-
ability issues, and the rat provides the most flexibility because terminal studies can
routinely be done. For compounds that are thought to have dissolution limitations
or instability in the GI tract, the GI tract can be removed at the end of the
experiment and the contents assayed for drug and metabolites. The amount of
parent in bile and urine can be quantitated by LC/MS/MS and this method can
also be used for any metabolites where authentic standards have been prepared.
242 DRUG METABOLISM RESEARCH