Dose-Dependent Targeted Suppression of P‑glycoprotein Expression and Function in Caco‑2 Cells

The efflux transporter P-glycoprotein (Pgp), encoded by the <i>ABCB1</i> gene, decreases the bioavailability of a wide range of orally administered drugs. Drug permeability studies using the in vitro Caco-2 cell model commonly rely on small molecule modulators to estimate the contribution of Pgp to drug efflux. The use of such modulators may be limited by their interactions with other membrane transporters. RNA interference, a tool allowing for the specific degradation of a target gene’s mRNA, has emerged as a technique to study gene expression and function. This manuscript describes the use of chemically modified small interfering RNA (siRNA) for a dose-dependent suppression of <i>ABCB1</i> in Caco-2 cells and the subsequent drug permeability assay. We transfected Caco-2 cells while in suspension with chemically modified synthetic siRNA–lipid complexes and then seeded the cells on polycarbonate semipermeable supports. Once the monolayer of Caco-2 cells formed tight junctions and expressed brush border enzymes, we determined the dose-dependent suppression of the <i>ABCB1</i> gene using RT-qPCR. We measured the duration of silencing at the optimal siRNA dose by Western blot for Pgp protein. The utility of this in vitro model was determined by performing bidirectional transport studies using a well-established substrate for Pgp, rhodamine 123. A single 4 h transfection of the Caco-2 cells with ≥100 nM siRNA reduced the expression of <i>ABCB1</i> mRNA by >85% at day five in culture. The time-course study revealed that the single transfection reduces Pgp protein levels for 9 days in culture. This magnitude of silencing was sufficient to reduce the efflux of rhodamine 123 as measured by the apparent permeability coefficient and intracellular accumulation. In this study, we demonstrate the dose-dependent, targeted degradation of Pgp in Caco-2 cells as a new model for assessing drug efflux from enterocytes. The dose-dependent nature of the Pgp silencing in this study offers significant improvements over other approaches to creating a Caco-2 model with suppressed <i>ABCB1</i> expression. We envision that this technique, in conjunction with better small molecule inhibitors, will provide a useful tool for future drug permeability studies

Evaluation of the Contribution of the ATP Binding Cassette Transporter, P‑glycoprotein, to <i>in Vivo</i> Cholesterol Homeostasis

P-glycoprotein (Pgp, encoded by <i>ABCB1</i>, commonly known as MDR1), an ATP-dependent transporter with a broad range of hydrophobic drug substrates, has been associated with the <i>in vitro</i> intracellular transport of cholesterol; however, these findings have not been confirmed <i>in vivo</i>. In this manuscript we tested the contributions of Pgp to <i>in vivo</i> cholesterol homeostasis by comparing the cholesterol phenotype of wild type mice with mice lacking both murine isoforms of Pgp (<i>Abcb1a</i>^–/–/<i>1b</i>^–/–) by measuring cholesterol absorption, circulating cholesterol, and lipoprotein cholesterol profiles. The mice were fed diets containing normal or high levels of dietary fat (25% vs 45% kcal from fat) and cholesterol (0.02% vs 0.20% w/w) for 8 weeks to challenge their capacity to maintain homeostasis. There were no significant differences in cholesterol absorption, circulating cholesterol levels, and lipoprotein profiles between Pgp knockout and wild type mice fed matching diets. Compensatory shifts were observed in the activation of two key transcription factors involved in maintaining cholesterol balance, the Liver X Receptor and SREBP-2, which may have maintained the wild type phenotype in the knockout mice. Deletion of Pgp affected the molar composition of gallbladder bile, when the mice were fed diets containing high levels of dietary fat, cholesterol, or both. The mole fraction of bile salts was reduced in the gallbladder bile of Pgp knockout mice, while the mole fraction of cholesterol was increased. In this paper, we provide evidence that Pgp knockout mice maintain cholesterol homeostasis, even when challenged with high cholesterol diets. We suggest that the specific shifts in cholesterol regulatory networks identified in the jejunum and liver of the knockout mice may have compensated for the lack of Pgp. Our finding that Pgp knockout mice were unable to maintain gallbladder bile composition when challenged with high dietary fat and/or cholesterol compliments recent reports that Pgp may be a secondary bile salt export pump

Decomposition rate of AmpB in lipid formulations.

<p>Data indicate mean rate of loss of AmB ± SD (n = 4). Results derived from the data in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000913#pntd-0000913-g001" target="_blank">Fig. 1</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000913#pntd-0000913-g002" target="_blank">2</a>.</p

Stability of AmB in lipid suspensions at 30°C over 60 days.

<p>Symbols: solid diamonds: iCo-010; solid squares: iCo-011; crosses: iCo-012; open circles: iCo-013. Data represent mean ± SD (n = 4).</p

Stability of AmB in lipid formulations in fasted-state simulated intestinal fluid (FaSSIF) at 37°C.

<p>Symbols: solid diamonds: iCo-010; solid squares: iCo-011; crosses: iCo-012; open circles: iCo-013.Data represent mean ± SD (n = 3).</p