IN VITRO–IN VIVO SCALE-UP OF DRUG TRANSPORT ACTIVITIES - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 563

transporters other than Oatp1a1 are involved in their uptake. Now, other hepatic uptake
transporters, such as Oatp1a4, Oatp1b2, and Oat2, have also been characterized, and
they can accept a variety of anions. 16 − 18 Therefore, E 2 17 G can no longer be used as

a reference compound for Oatp1a1, but Crespi and Penman's concept is applicable
to estimation of the relative contribution.
Hirano et al. have applied this concept to human hepatocytes to estimate the rel-
ative contribution of OATP1B1 and OATP1B3 to the hepatic uptake of E 2 17

G
and pitavastatin, a novel 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) re-
ductase inhibitor, in cryopreserved human hepatocytes (Figure 19.2 a ). 7 As reference
compounds, they used estrone 3-sulfate (E-sul) for OATP1B1 and cholecystokinin
octapeptide (CCK-8) for OATP1B3. As with the previous method, they calculated the
ratio of the uptake clearance of the reference compounds in human hepatocytes to that
in the expression systems and defined R act for OATP1B1 and 1B3. By multiplying
the R act value by the uptake clearance of test compounds (CL test ), it is possible to
estimate the uptake clearance of the test compounds mediated by a specific trans-
porter in human liver. Assuming that the hepatic uptake clearance (CL hep ) could be
explained by OATP1B1- and OATP1B3-mediated transport, the following equation
applies:

CL hep = R act , OATP1B1 CL test , OATP1B1 + R act , OATP1B3 CL test , OATP1B3 (12)

They demonstrated that both pitavastatin and E 2 17 G were taken up mainly by

OATP1B1 in three independent batches of human hepatocytes and that the uptake
clearance observed in human hepatocytes was almost identical to the sum of the es-
timated clearance mediated by OATP1B1 and 1B3. They also confirmed their results
by two different approaches. 7 , 19 One involves direct estimation of the ratio of the
expression level of OATP1B1, 1B3, and 2B1 in human hepatocytes to that in expres-
sion systems by comparing the band density of Western blot analysis and estimating
their contributions using that ratio instead of the R act value shown above (Figure
19.2 b ). 7 , 19 The other approach is to estimate the inhibitable portion of the uptake
of test compounds in human hepatocytes in the presence of a specific inhibitor for
each transporter (Figure 19.2 c ). 19 E-sul can be used as a specific inhibitor as well
as a specific substrate for OATP1B1 (Figure 19.3 a ), whereas CCK-8 inhibited both
OATP1B1 and OATP1B3 to the same extent, indicating that CCK-8 could not be used
as a specific inhibitor, although it is a specific substrate for OATP1B3. The uptake of
pitavastatin and E 2 17

G was completely inhibited by excess concentration of E-sul
(Figure 19.3 b ). Therefore, the importance of OATP1B3 in their hepatic uptake was
supported by these different approaches.
Shimizu et al. examined the uptake of fexofenadine in OATP1B1-, 1B3-, and
2B1-expressing cells, and significant uptake could be observed only via OATP1B3,
although the uptake in OATP1B1- and 2B1-expressing cells was slightly higher com-
pared with that in control cells. 20 They estimated the contribution of OATP1B3 to
the uptake of fexofenadine based on the method using reference compounds and
concluded that OATP1B3 rather than OATP1B1 is mainly involved in fexofenadine
transport because its observed clearance in OATP1B1-expressing cells was much