Drug Metabolism in Drug Design and Development Basic Concepts and Practice

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The cytotoxicity assay is an indirect measurement of the accumulation of
cytotoxic compounds in cells and is a frequently used surrogate assay to
determine whether a compound is a substrate or an inhibitor of a given
transporter. Transporter substrates can be identified by comparing the IC 50
(the concentration that inhibits the cell growth by 50%) in wildtype (naı ̈ve) and
transporter expressing (drug resistant) cells, while inhibitors can be identified
by their ability to potentiate (for efflux transporters) or attenuate (for uptake
transporters) the cytotoxicity of a substrate in transporter expressing or drug
resistant cells. The activity of the reversal agent is generally expressed as a fold
reversion or MDR ratio. The MDR ratio is the ratio of the IC 50 of a cytotoxic
drug alone to the IC 50 of a cytotoxic drug in the presence of a transporter
modulator.
Freshly isolated or cryopreserved hepatocyte suspension can be used for
hepatobiliary uptake transporter but not efflux transporter studies (Hirano
et al., 2004; Hoffmaster et al., 2004a; Shitara et al., 2003) because efflux
transporters are internalized after isolation (Hoffmaster et al., 2004a). The
uptake of compounds into suspended hepatocytes was determined by a
centrifugation filtration method (Hirano et al., 2004). Sandwich-cultured
hepatocytes have become a valuable tool to evaluate both uptake and efflux
hepatobiliary transporters since sandwich-cultured hepatocytes maintain the
hepatocyte architecture, including tight junctions, canalicular biliary network,
and the functional transporters. Because depletion of Ca2+can open tight
junctions, accumulation of compounds in hepatocytes, and bile ducts, or in
hepatocytes is determined in Ca2+and Ca2+free buffer, respectively (Liu
et al., 1999). Biliary elimination is then calculated as the difference of these two
measurements. Sandwich cultured hepatocytes can be used to evaluate
hepatobiliary transporter-mediated DDI and liver toxicities (Kemp et al.,
2005). Other potential advantages of the hepatocyte model include the
evaluation of the induction of transporters by xenobiotics and understanding
interspecies difference in hepatobiliary transporters. Although cultured
hepatocytes can provide information on an overall outcome of transporter-
related DDI and hepatotoxicity, its utility is limited by the lack of the details of
the individual interactions between a xenobiotic and an uptake or efflux
transporter. This limitation could be overcome by using a specific transporter
substrate or inhibitor or by using specific transporter expressedin vitrosystems.
The results obtained from one donor hepatocytes may not be reflective of the
whole human population because of the possible polymorphisms of
transporters.


6.6.1.3 Oocytes and Yeast Both yeast and Xenopus laevis oocyte over-
expressing transporters can be used to evaluate transporter functions. Yeast,
which grows rapidly, can be used for the production of large quantities of
transporter proteins and can be used for high throughput bioassay screening.
Genetic manipulations in yeast are easy and cheap. Oocytes can be directly
microinjected with mRNA into cytoplasm or cDNA into nucleus and they have


180 DRUG TRANSPORTERS IN DRUG DISPOSITION, DRUG INTERACTIONS

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