P317 Creation and assessment of adenovirus-mediated drug transporter model expression system for the prediction of pharmacokinetic profiles in humans

Daisuke Sugiyama , Medicine, The University of Western Ontario, London, ON, Canada
Wendy A. Teft , Medicine, The University of Western Ontario, London, ON, Canada
Mike J. Knauer , Medicine, The University of Western Ontario, London, ON, Canada
Marianne K. DeGorter , Division of Clinical Pharmacology, Department of Medicine, The University of Western Ontario, London, ON, Canada
Rommel G. Tirona , Medicine, The University of Western Ontario, London, ON, Canada
Richard B. Kim , Medicine, The University of Western Ontario, London, ON, Canada

Introduction. Predicting the in vivo role and effects of functional genetic polymorphisms to observed PK profile of drugs in humans is important for optimal drug therapy. Currently, IVIVE (in vitro to in vivo extraporation) algorithms are widely utilized for the prediction of drugs which are metabolized by CYP enzymes, but not transporters. However, for many drugs, the rate-limiting step in their elimination is transporters, particularly those that mediate uptake into tissues or target organs. In addition, the extent of drug transporter expression in human tissues or organs, particularly in a quantitative fashion, has not been clearly delineated. Therefore, our goal is to create an in vitro transporter expression system capable of expressing multiple drug transporters simultaneously so that the cell-based system better reflects human organs such as liver. Towards this goal, we have cloned a number of hepatic bile acid and drug transporters into an adenovirus-based expression construct and assess their expression as well as transport activity in a number of cell lines. 

Methods. Adenovirus constructs containing drug transporters such as NTCP (Na/taurocholate cotransporting polypeptide), members of the human OATPs (organic anion transporting polypeptides) were constructed, and their transport activities as well as cell viabilities were assessed.

Results. After incubation with adenoviruses containing NTCP, OATP1B1, 1B3 and 2B1, their substrate transport activities were determined. For OATP1B1 and 1B3 in HeLa cells transport activity at 48 hr incubation with adenovirus was higher than at 72 hr, and cell viability after 48 hr was also better that at 72 hr for the same Multiplicity of Infection (MOI). Although transport activity for NTCP and OATP2B1 could be readily detected in HeLa, LLC-PK1, MDCKII and Caco-2 cells, overall activity was greatest in HeLa cells and lowest in Caco-2 cells. In terms of cell viability, Hela, LLC-PK1 and Caco-2 were able to tolerate an MOI of 1000, but for MDCKII cells an MOI of 300. Interestingly, MDCKII cells exhibited higher transport activity compared to LLC-PK1 cells when expressing NTCP, but LLC-PK1 was better when expressing OATP2B1.  

Conclusions. Our findings show that although a number of cell lines can be transduced to express hepatic transporters using the adenoviral expression system, the effects of adenovirus-mediated transporter expression differed significantly among the tested cell lines. Thus an optimal condition has to be identified based on the cell line as well as the transporter of interest. In our hands, it would appear that HeLa cells may be better for optimal expression of uptake transporters and the LLC-PK1 cells better for expressing efflux transporters. Overall, our newly created in vitro system appears to have the desired properties to serve as a physiologically relevant model for predicting in vivo PK.