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Kinetic Design for Establishing Long-Term Stationary Cytosol Concentrations During Drug Transport across P-gp Expressing Confluent Cell Monolayers to Facilitate Measuring Cytosol Concentration, Fitting Drug Molar Partition Coefficients into the Cytosolic Monolayer of the Plasma Membrane, and Kinetically Identifying Drug Uptake Transporters
Book chapter

Kinetic Design for Establishing Long-Term Stationary Cytosol Concentrations During Drug Transport across P-gp Expressing Confluent Cell Monolayers to Facilitate Measuring Cytosol Concentration, Fitting Drug Molar Partition Coefficients into the Cytosolic Monolayer of the Plasma Membrane, and Kinetically Identifying Drug Uptake Transporters

Joe Bentz
Quantitative Analysis of Cellular Drug Transport, Disposition, and Delivery, pp 41-65
08 Jun 2021
DOI: https://doi.org/10.1007/978-1-0716-1250-7_2

Abstract

Drug–drug interactions (DDI) In vitro–in vivo extrapolation (IVIVE) Microvilli morphology P-glycoprotein Structural mass action kinetic model
The human multidrug resistance membrane transporter P-glycoprotein, P-gp, has been extensively studied due to its importance to human health and disease, e.g., as a major cause of drug–drug interactions in humans, DDI. P-gp is also an exemplary model of a membrane transporter found throughout the animal kingdom whose substrate specificity appears not to have evolved significantly beyond its apparent original function of effluxing positively charged amphiphiles out of cells, e.g., soaps. Since eukaryotic cell membranes are negatively charged, positively charged soaps are particularly disruptive of membrane integrity. Our structural mass action kinetic model has fitted the mass action kinetic parameters for many P-gp substrates based upon the time dependence of transported concentrations into the apical and basolateral aqueous chambers. However, because the cytosolic concentrations could not be accurately measured, molar partition coefficients for these drugs to the cytosolic monolayer have only been estimated using unilamellar liposomes with simple lipid compositions that roughly mimic the major lipid components of the relevant monolayers of the plasma membrane. In this chapter, we have developed a kinetic design for the confluent monolayer of P-gp expressing cells that predicts stationary cytosolic concentrations over time periods adequate to allow statistically robust measurements of the cytosolic concentrations. This kinetic design allows direct fitting of the P-gp substrate’s molar partition coefficient to the cytosolic monolayer, determination of whether P-gp expressing cell lines also express uptake transporters for P-gp substrates and will provide far more accurate and complete fittings of all the kinetic parameters that drive transcellular transport, there by yielding better mechanistically based predictions for in vitro–in vivo extrapolations, IVIVE.

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