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Identification of system independent and dependent parameters for reliable in vitro-in vivo extrapolation of P-glycoprotein using a mass action kinetic model
Dissertation   Open access

Identification of system independent and dependent parameters for reliable in vitro-in vivo extrapolation of P-glycoprotein using a mass action kinetic model

Zhou Meng
Doctor of Philosophy (Ph.D.), Drexel University
Nov 2015
DOI:
https://doi.org/10.17918/etd-7214
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Abstract

Biology P-glycoprotein
The purpose of this work is to investigate the best kinetic parameters for incorporation into mechanistic PBPK model for P-gp using a mass action kinetic model. We first showed that by extending the P-gp kinetics analysis from confluent MDCKII-hDMR1 cells monolayers to Caoc-2 cells monolayers, the elementary rate constants governing P-gp's binding and efflux (on-, off- and efflux rate constants: k1, kr and k2) remain constant between two cell lines, therefore independent of the in vitro system in which they are generated. In addition, we kinetically identified the involvement of a basolateral uptake transporters for digoxin and loperamide in Caco-2 cells, which were also seen in MDCKII-hMDR1 cells. This again demonstrates the value of our P-gp structural model as a diagnostic tool in detecting the presence of other transporters, which cannot be done unambiguously by Michaelis-Menten approach. Our results suggest the hypothesis that the elementary rate constants of P-gp obtained from in vitro would be reliable for incorporation into mechanistic PBPK models for transporters. We also investigated the P-gp expression-activity relationship and showed that in Caco-2 cells total P-gp is much greater than efflux active P-gp, while in MDCKII-hMDR1 cells the values are fairly similar. In addition, we found that the microvilli in Caco-2 cells are taller and more densely packed than those in MDCK-hMDR1 cells, which would lead to a substantial fraction of P-gp in Caco-2 cells not contributing to final release of drug into the apical chamber, but instead being involved in a futile cycle of drug efflux followed by reabsorption. The effect of microvilli morphology differences between in vitro and in vivo systems should be considered when scaling transporter activity for P-gp and possibly other efflux transporters. In summary, all our findings as shown in this thesis will allow for a future robust PBPK model building.

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