IN VITRO–IN VIVO SCALE-UP OF DRUG TRANSPORT ACTIVITIES - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 566

proteins. Hagenbuch et al. have investigated the effect of coinjection of trans-
porter (Ntcp or Oatp1a1)-specific antisense oligonucleotide on the uptake of bro-
mosulfophthalein (BSP) and TCA in Xenopus oocytes injected with total rat liver
mRNA. 23 They succeeded in obtaining a significant reduction in the expression level
of target transporter specifically and concluded that the Na + -dependent and Na + -
independent uptakes of TCA were due almost entirely to Ntcp and Oatp1a1, re-
spectively, whereas only half of the BSP uptake could be explained by Oatp1a1.
Nakai et al. adopted the same approach to estimate the contribution of OATP1B1
to the hepatic uptake of pravastatin and E 2 17 G in humans. 24 They demon-

strated that oocytes microinjected with human liver poly(A) mRNA exhibited Na + -
independent uptake of pravastatin and E 2 17 G and that a simultaneous injection

of OATP1B1 antisense oligonucleotides abolished this uptake completely, suggest-
ing that OATP1B1 is a major transporter involved in their uptake. In this approach
we should note the assumption that the relative expression level of each uptake
transporter in Xenopus oocytes is the same as that in rat liver. Currently, we can-
not identify the transport activity mediated by a specific transporter only by us-
ing specific inhibitors. Takagi et al. have constructed 2 - O ,4 - C -ethylene-bridged
nucleic acid residues that can be incorporated into antisense oligonucleotides tar-
geted to rat Oatp1a1, 1a4, and 1a5, and they checked the selective inhibition of
Oatp subtypes. 25 Recently, some groups have succeeded in the construction of small
interference RNAs (siRNAs), which can efficiently reduce the expression level of
specific transporters such as MDR1 and MRP2. 26 , 27 However, it is fairly difficult to
apply these gene-silencing techniques to primary cultured hepatocytes because long-
term culture dramatically reduces the expression level of several transporters, 8 − 10
although generally it takes a few days to knock down the protein by depletion
of mRNA expression, and optimization of the culture conditions is required for
this analysis.
Regarding the evaluation of efflux transport in liver, one of the most popular
experimental systems involves the use of canalicular membrane vesicles (CMVs).
It is difficult to evaluate the transport activity of efflux transporters in cell systems
because substrates cannot easily reach the intracellular compartment, so this system
is often used for the rapid determination of adenosine triphosphate (ATP)-dependent
efflux transport of substrates across the bile canalicular membrane. Aoki et al. have
compared the in vitro transport clearance of nine substrates in rat CMVs defined as the
initial velocity for the ATP-dependent uptake divided by the substrate concentration
of the incubation medium with in vivo biliary excretion clearance, defined as the
biliary excretion rate normalized by the unbound concentration in rat liver at steady
state. They found a significant correlation between in vitro and in vivo clearance,
suggesting that in vivo biliary excretion clearance can be predicted from in vitro
transport studies using CMVs. 28
To evaluate the biliary excretion clearance in humans, several methods have been
created. 29 Niinuma et al. have shown that the transport activities of several substrates
were observed in human CMVs prepared from six independent batches of human
liver blocks, and interestingly, the initial uptake clearance of glucuronides was not
so different between rat and human CMVs, 30 whereas that of other compounds in
human CMVs was significantly lower than that in rat CMVs. This result suggests