Abstract
Intracellular drug exposure is influenced by cell- and tissue-dependent expression of drug-transporting proteins and metabolizing enzymes. Here, we introduce the concept of intracellular bioavailability (Fic) as the fraction of extracellular drug available to bind intracellular targets, and we assess how Fic is affected by cellular drug disposition processes. We first investigated the impact of two essential drug transporters separately, one influx transporter (OATP1B1; SLCO1B1) and one efflux transporter (P-gp; ABCB1), in cells overexpressing these proteins. We showed that OATP1B1 increased Fic of its substrates, while P-gp decreased Fic. We then investigated the impact of the concerted action of multiple transporters and metabolizing enzymes in freshly-isolated human hepatocytes in culture configurations with different levels of expression and activity of these proteins. We observed that Fic was up to 35-fold lower in the configuration with high expression of drug-eliminating transporters and enzymes. We conclude that Fic provides a measurement of the net impact of all cellular drug disposition processes on intracellular bioavailable drug levels. Importantly, no prior knowledge of the involved drug distribution pathways is required, allowing for high-throughput determination of drug access to intracellular targets in highly defined cell systems (e.g., single-transporter transfectants) or in complex ones (including primary human cells).
Highlights
Unbound drug accumulation ratio (Kpuu), a term commonly used in pharmacokinetic studies of blood-to-tissue concentration ratios[22]
We studied the influence of an important uptake transporter (Fig. 1) by using HEK293 cells transfected with human organic anion-transporting polypeptide 1B1 (OATP1B1) or with an empty vector
This indicates a negligible contribution from OATP1B1 to the total binding to cellular proteins, which can be explained by previous observations that a non-saturable component, most likely the membrane lipids, is the major cellular binding site[25,32,33,34]
Summary
Unbound drug accumulation ratio (Kpuu), a term commonly used in pharmacokinetic studies of blood-to-tissue concentration ratios[22]. There are situations in which the Kpuu term is not applicable (e.g., when experiments are performed in the presence of serum proteins). We used well defined cell lines overexpressing the organic anion-transporting polypeptide 1B1 (OATP1B1; SLCO1B1*1a) and P-glycoprotein (P-gp; ABCB1), two of the transporters most commonly involved in drug disposition[1]. We studied the parallel impact of multiple transporters and enzymes in a more complex system, freshly isolated human hepatocytes. The hepatocytes were used in two configurations known to display different gene expression and activity of transporters and enzymes: directly after isolation and after 24 h of culture in monolayer format[29,30,31]. Our simple methodology provides a good estimate of compound available to bind targets in the cell interior, even in complex cell systems
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