Date of Award

5-12-2012

Degree Type

Thesis

Degree Name

Honors Thesis

Department

Pharmacy

Abstract

Background: The overexpression of an efflux protein, p-glycoprotein (P-gp), is a leading cause of multi drug resistance (MDR). This research project is based on designing emulsions containing optimal doses of cyclosporine (CyA) and Pluronic® P-85 (P85), two agents found to be effective in inhibiting P-gp. Study

Objective: To investigate whether CyA exposure affects the quantity or functionality of P-gp in vitro.

Methods: To study the functionality of P-gp, MDR cells were incubated with CyA solutions containing the P-gp substrate and fluorescent dye Rhodamine 123 (RI23). P-gp inhibition was measured by fluorescence spectrophotometry, which indicates the accumulation of R123 inside the cells. This was repeated with the surfactant P85, a component of emulsions, in solution with R123. Optimal doses were selected and used to prepare O/W sub-micron emulsions loaded with CyA, R123, and P85. The accumulation of R123 in the MDR cells when incubated with emulsions was then compared to the accumulation seen with the CyA or P85 solutions. To study the quantity of P-gp as the cell line ages, Western blotting was performed on aging cell passages.

Results: Out of the concentration range 1u.M - 25 uM for CyA and 100-1 000 ug/ml, of P85, 15 uM CyA and 200-500 ug/ml. P85 led to the greatest R123 uptake. The R123 accumulation with CyA solutions was fivefold greater than with CyA emulsions. Other studies suggest that surfactants such as P85 encapsulate CyA or R123 by micelle formation making them unavailable to interact with the cell. Western blotting showed that the quantity of P-gp decreases as the cell line ages. However, earlier passages had greater R 123 accumulation than later passages when exposed to CyA solutions which remains unexplained.

Conclusions: Although both CyA and P85 led to greater P-gp inhibition at higher concentrations, their combined effect in emulsions was not synergistic; this may be explained by the micellization of the substrates by P85. The effect of aging on P-gp functionality was inconclusive.

Download

Share

COinS