Document Type

Dissertation

Date of Degree

Summer 2009

Degree Name

PhD (Doctor of Philosophy)

Degree In

Pharmacy

First Advisor

Maureen D. Donovan

Abstract

A human-derived, mammary epithelial cell culture model would allow drug transport in the mammary epithelium to be studied in greater detail while minimizing risks to mothers and nursing infants. MCF10A and primary human mammary epithelial cells (HMECs) were investigated for their utility as human, cell-based model systems for drug transport studies. Polarized monolayers are essential for transcellular flux studies of drug transporter function, and their formation was measured by transepithelial electrical resistance, immunofluorescence microscopy and vectoral flux studies. Both cell types failed to form adequately polarized monolayers despite various modifications to the cells or culture conditions.

Transporter-mediated drug uptake and efflux in MCF10A cells was measured using flow cytometry, a technique which enables the measurement of intracellular drug concentrations. The fluorescent drug, mitoxantrone, was used to assess active efflux transport by the ABC transporters ABCG2 (BCRP) and ABCB1 (MDR1). After accounting for the inter-day variability with a linear mixed effects model, inhibitor effects on intracellular drug concentrations were evident. Specific inhibition of MDR1 using verapamil increased mitoxantrone accumulation as expected; however, BCRP-specific inhibition with fumitremorgin C decreased accumulation. Flow cytometry studies on mitoxantrone uptake suggested that it is a substrate for an unidentified active uptake transporter.

PEPT1 and PEPT2 transporter functionality in MCF10A cells was evaluated using a fluorescently labeled dipeptide (A-K-AMCA). A-K-AMCA uptake showed an active component which was inhibited by a general metabolic inhibitor, the dipeptide Gly-Gln, and the peptidomimetic cefadroxil, indicating the involvement of a peptide transporter in A-K-AMCA uptake.

Drug transporter expression levels in MCF10A cells and HMECs were measured using RT-PCR. Transporter expression levels, which were similar in the MCF10A cells and the HMECs, were compared with expression levels in lactating and non-lactating mammary epithelial cells. Low expression of BCRP, MDR1 and PEPT1 was seen in MCF10A cells, yet the effects of these transporters could still be observed in functional flow cytometry transport assays. Flow cytometry studies MCF10A cells may useful as a mammary drug transport model for transporters which have similar expression levels to lactating mammary epithelial cells.

Pages

xix, 247 pages

Bibliography

Includes bibliographical references (pages 239-247).

Copyright

Copyright 2009 Joanne Elizabeth Reiland

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