DRUG TRANSPORT IN THE LIVER - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 377

substrates and inhibitors of transport proteins. 290 , 291 , 95 - 97 Although cLPM and bLPM
preparation is labor intensive, it remains an attractive model system for determining
differences in the functional activity of transporters obtained from various hepatic
tissue sources without the need to isolate intact cells. For example, this approach has
been used successfully to compare functional expression of taurocholate transport
in the basolateral liver membrane of developing rats compared to adult rats. 98 This
model system is less suitable for assessing the role of a specific transport protein
in hepatic drug uptake or biliary excretion because multiple transport proteins are
expressed in cLPM and bLPM that may mediate transport of a single substrate.
Cloning and expression of (human) transport proteins in polarized mammalian
cell lines (predominantly the MDCK cell line) has evolved rapidly to one of the
most widely used techniques for generating efficient and flexible in vitro tools for
identification of substrates and inhibitors of hepatic transport proteins in humans. 242
Expression of at least two transport proteins (one for uptake and one for excre-
tion) has now become a standard approach used to mimic the in vivo expres-
sion of uptake and excretory transporters at the basolateral (sinusoidal) and apical
(canalicular) membrane domains. 242 , 99 Moreover, a correlation was found between in
vitro vectorial transport of substrates across Oatp1b2 (Oatp4)- and Mrp2-transfected
MDCKII monolayers and in vivo biliary clearance in rats. 100 Recently, Kopplow
et al. 101 reported the construction under well-defined conditions of quadruple-
transfected MDCKII cells allowing simultaneous screening for OATP1B1/1B3 and
2B1 substrates along with MRP2-mediated excretion at the apical membrane. The
use of polarized cell lines mimics the polarized phenotype of the hepatocyte, thus al-
lowing for study of the vectorial transport of nonmetabolized substrates. On the other
hand, relative expression levels of multiple cotransfected transport proteins remain
difficult to assess, resulting in a rather artificial model exhibiting differences in rate-
limiting steps in hepatobiliary drug disposition compared to in vivo. Nonpolarized cell
systems such as insect cells and oocytes also have been used for human transporter
expression. 102 For example, a characterization of OAT2, which is highly expressed in
human liver, was conducted recently with Xenopus laevis oocytes, resulting in novel
information regarding substrate specificity and transport mechanism. 11 Important ad-
vantages of the latter model are that they can be constructed and validated rapidly and
that drug transport by a specific transport protein can be characterized without inter-
ference from other (transport) processes. Indeed, Saito et al. recently demonstrated
that membrane vesicles prepared from BCRP-transfected insect cells provide a high-
speed assay to establish a structure-activity relationship for inhibitors. 103 However,
the main disadvantage of these model systems is that the relative contribution of the
particular transport protein to overall transport processes cannot be determined. 104
In addition, the actual functional activity of a given transport protein is likely to de-
pend on the cellular environment, which is clearly different in a transfected system
compared to the normal hepatocyte. 102
Freshly isolated hepatocytes can be considered the most comprehensive cell-based
model for the study of hepatic drug transport. Suspended hepatocytes remain a useful
and convenient system for studying drug uptake mechanisms. Unfortunately, hepa-
tocytes lose their cellular polarity rapidly upon isolation, 105 resulting in the absence