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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

Bias in Estimation of Transporter Kinetic Parameters from Overexpression Systems: Interplay of Transporter Expression Level and Substrate Affinity

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland (A.B., N.H., P.G., P.W.S., J.E.P.); and Department of Pharmacy and Technology, University of Valencia, Valencia, Spain (M.B.)

The objective was to investigate the interplay between transporter expression levels and substrate affinity in controlling the influence of aqueous boundary layer (ABL) resistance on transporter kinetics in an over-expression system. Taurocholate flux was measured across human apical sodium-dependent bile acid transporter (hASBT)-Madin-Darby canine kidney monolayers on different occasions and kinetic parameters estimated with and without considering ABL. In error-free simulation/regression studies, flux values were generated across a range of J_max, K_t, and substrate concentrations. Similar evaluation was performed for transport inhibition studies. Additionally, simulation/regression studies were performed, incorporating 15% random error to estimate the probability of successfully estimating K_t. Across different occasions, experimental J_max and K_t estimates for taurocholate were strongly associated (p < 0.001; r² = 0.82) when ABL was not considered. Simulation/regression results indicate that not considering ABL caused this association, such that K_t estimates were highly positively biased at high hASBT expression. In reanalyzing taurocholate flux data using the ABL-present model, K_t was relatively constant across occasions (5 µM) and not associated with J_max (p = 0.24; r² = 0.13). Simulations suggest that J_max and K_t collectively determined ABL influence, which is most prominent under conditions of low monolayer resistance. Additionally, not considering ABL lead to negatively biased K_i estimates, especially at high J_max. Error-inclusive simulation/regression studies indicated that the probability of successfully estimating K_t depended on the contribution of ABL resistance to flux; when flux became increasingly ABL-limited, probability of success decreased. Results indicate that ABL resistance can bias K_t and K_i estimates from overexpression systems, where the extent of bias is determined by transporter expression level and substrate affinity.

Address correspondence to: Dr. James E. Polli, Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, 20 Penn St., HSF2 Rm 623, Baltimore, MD 21201. E-mail: jpolli{at}rx.umaryland.edu

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