IN VITRO–IN VIVO SCALE-UP OF DRUG TRANSPORT ACTIVITIES - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 561

activity of E 2 17 G and TCA, about 40% of the batches tested showed adequate trans-

port activity, while the other batches exhibited no detectable transport activity. Also,
the transport activity exhibited by each batch of hepatocytes did not always corre-
late with the metabolic activities described in the batch sheets. Therefore, before we
check the transport characteristics of several compounds using human hepatocytes,
we prescreen the uptake clearance of E 2 17 G and TCA in many batches and select in

advance at least three batches of hepatocytes with high transport activity. 7 Cultured
hepatocytes can also be used, due to their ease of handling; however, we must keep in
mind that several reports have indicated that long-term (

>

1 day) culture on collagen-
coated dishes results in a drastic reduction of the mRNA and protein levels of several
transporters and in the uptake activity of organic anions, such as pravastatin. 8 − 10
Theoretically, the hepatic uptake intrinsic clearance can be estimated simply by
scaling up the uptake clearance in hepatocytes to the in vivo level. By multiplying
the uptake clearance per cell by the number of cells per gram liver, it is possible
to extrapolate in vitro uptake data to obtain the in vivo intrinsic uptake clearance.
Miyauchi et al. have demonstrated that the uptake clearance of 15 drugs calculated
from isolated hepatocytes correlated well with the values estimated by the in situ
multiple indicator dilution (MID) method, although the in situ clearance appeared
to reach an upper limit possibly because the diffusion of compounds in the unstirred
water layer became the rate-determining process. 11 Kato et al. have also shown that the
uptake clearance of four types of endothelin antagonists obtained from integration
plot analysis after intravenous administration of the compounds to rats is almost
comparable with that calculated from the uptake clearance in isolated rat hepatocytes,
assuming the well-stirred model 12 (Figure 19.1). This evidence suggests that isolated
hepatocytes are a good model for predicting hepatic uptake clearance.
Regarding uptake transport in human liver, some SLC (solute carrier) transporters
are expressed, such as NTCP (Na + -taurocholate cotransporting polypeptide) and
OATP (organic anion transporting polypeptide) family transporters, as discussed in
other chapters. In particular, OATP1B1, OATP1B3, and OATP2B1 show very broad
substrate specificities, and these overlap with one another, so that one compound is
often recognized by multiple transporters. The transport properties of each transporter
can be evaluated by using gene expression systems (e.g., mammalian cells, Xenopus
oocytes). However, the transporters that accept one compound are not always im-
portant for overall hepatic uptake if the relative contribution is very minor compared
with that of other transporters. Therefore, it is essential to determine the quantita-
tive contribution to hepatic uptake of each transporter to show the importance of each
transporter under in vivo conditions. When the function and/or expression level of one
transporter is changed due to genetic polymorphisms, pathophysiological conditions
and transporter-mediated drug-drug interactions information about the contribution
is necessary to predict the change in the in vivo pharmacokinetics from in vitro data.
Several estimation methods have been developed for this purpose. Kouzuki et al.
have proposed a method using reference compounds to determine the contribution of
rat Oatp1a1 and Ntcp to the hepatic uptake of bile acids and organic anions. 13 , 14 This
concept was originally established in the field of metabolic enzymes by Crespi and
Penman, 15 who named it the relative activity factor (RAF) method . Using this method