DRUG TRANSPORT IN THE LIVER - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 380

hepatobiliary drug transport and metabolism processes can be considered as the most
attractive feature of these in vivo models.
13.8. TOOLS FOR STUDYING HEPATOBILIARY DRUG TRANSPORT
The various tools employed to study transport proteins are often utilized in combina-
tion with the many model systems discussed previously. As the nature of science is
constantly evolving with new discoveries, so are the tools used to make these discov-
eries. Many of the tools currently in use have been borrowed from other fields, such
as molecular biology or engineering.
Molecular biology techniques often are employed to assess the expression, local-
ization and function of the hepatic transport proteins. For example, Northern blot,
Western blot, or real-time polymerase chain reaction (RT-PCR) are utilized to deter-
mine relative protein or mRNA levels. These data should be interpreted with caution
because quantification of bands on a Western blot using densitometry gives only the
relative amount of protein on a single membrane. The same is true for RT-PCR, where
quantification of the mRNA expression of transport proteins is not usually correlated
with protein level in terms of the relative amount. Studies often examine the regulation
of hepatic transport proteins by assessing the induction or repression of protein or
mRNA using Western blot or RT-PCR analysis. 68 , 121 , 122 Animal studies that employ
genetic knockout of a particular transport protein or spontaneous genetic mutations re-
sulting in a transport protein deficiency often use Western or Northern blot to confirm
that the transport protein is absent. 120 , 292 Theoretically, the function of a particular
transport protein could be assessed using probe substrates. However, specific probes
for most proteins have yet to be identified, making it difficult to determine the role of
any single transport protein in the disposition of a given substrate.
The broad overlap in substrate specificity of the hepatic transport proteins also
holds true for compounds used as inhibitors. A single compound is often not a spe-
cific inhibitor of one particular transport protein, which confounds data interpretation
in deciphering the role of a single transport protein in vivo. More recently, the use
of small interfering RNA (siRNA) to knock down the expression and function of
a transport protein in primary hepatocytes has been demonstrated. Tian et al. uti-
lized siRNA to modulate both Mrp2 and Mrp3 in primary sandwich-cultured rat
hepatocytes. 305 RNA interference (RNAi) is reversible, unlike the knockout rodent
models or naturally occurring genetic mutations, and this technology could be applied
to human-derived cells and/or in vivo. Knockdown of a target gene using RNAi is
more potent than with antisense technology. 125 RNAi is also more specific because
it targets the mRNA, unlike chemical inhibitors that interact with the transport pro-
teins, which have a high degree of homology and similar structures. More recently,
morpholino antisense oligos have been shown to have higher specificity and longer
stability than siRNA. Morpholino oligos, named after the morpholine rings in their
backbone, contain nucleic acid bases and a nonionic phosphorodiamidate intersubunit
linkage that either blocks translation initiation in the cytosol or modifies pre-mRNA
splicing in the nucleus. 126 , 127