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Dissertation
Modeling the transport and inhibition kinetics of the human multidrug resistance transporter P-glycoprotein in MDCK-II cells
Doctor of Philosophy (Ph.D.), Drexel University
2007
DOI:
https://doi.org/10.17918/etd-3474
Abstract
The human multidrug resistance transporter P-glycoprotein (P-gp) effluxes a wide range of substrates and can be affected by a wide range of inhibitors or modulators. Many studies have presented classifications for these binding interactions, within either the context of equilibrium binding or the Michaelis-Menten enzyme analysis of the ATPase activity of P-gp. Our approach is to study P-gp transport and its inhibition using a physiologically relevant confluent monolayer of hMDR1-MDCKII cells. We measure the elementary rate constants for P-gp efflux of radiolabeled substrates and study inhibition using pair wise combinations with a different unlabeled substrate acting as the inhibitor. Our current kinetic model for P-gp has only a single kinetically relevant efflux-connected binding site. We conclude that there are at least two kinetically distinct efflux pathways through P-gp and the binding sites connected to these pathways may not be exclusive forany drug. Binding of these substrates must be cooperative which can be either positive or negative based on the substrate-inhibitor pair. From the transport and inhibition of digoxin and loperamide, we found that other transporters are responsible for uptake of these drugs into the cells and for recycling them into and from the apical membrane once P-gp has pumped them out. Each drug has a particular IC-50 for inhibiting transport of another. Exhaustive computer simulations of drug transport in the presence of "virtual inhibitors" showed that the fitted IC50 values overestimate the intrinsic dissociation constant, KI,Aq and an equation showed that this overestimate is contributed by a convolution of cell and substrate parameters. The smaller the passive permeability of the substrate, the greater is the magnitude of the overestimate of the inhibitor's dissociation constant by its IC50. These conclusions are valid for any membrane transporter whose substrates and inhibitors must pass a permeability barrier to reach their binding site on the transporter.
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Details
- Title
- Modeling the transport and inhibition kinetics of the human multidrug resistance transporter P-glycoprotein in MDCK-II cells
- Creators
- Poulomi Acharya - DU
- Contributors
- Joseph Edward Bentz (Advisor) - Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- College of Arts and Sciences; Drexel University
- Other Identifier
- 3474; 991014632548204721