Lipid excipients Peceol and Gelucire 44/14 decrease P-glycoprotein mediated efflux of rhodamine 123 partially due to modifying P-glycoprotein protein expression within Caco-2 cells.
ABSTRACT The objective of this study was to determine the influence of two lipid excipients, Peceol(c) and Gelucire(c) 44/14 on P-glycoprotein (Pgp) activity and protein expression in human colon adenocarcinoma cells (Caco-2). Lipid excipients are increasingly used as drug delivery systems for hydrophobic drugs to increase their bioavailability by overcoming the barrier of low absorption. This study will probe a novel mechanism by which lipid excipients reduce Pgp-mediated efflux and thereby increase bioavailability of orally administered therapeutics.
Non-cytotoxic concentrations of Peceol(c) and Gelucire(c) 44/14 were determined for 24-hour treatments of Caco-2 cells using integrity of the cell membranes and mitochondrial respiration as markers. Pgp activity after treatment with non-cytotoxic concentrations of Peceol(c) and Gelucire(c) 44/14 was measured with a fluorescent Pgp substrate, rhodamine 123 (Rh123). The activity of Pgp was ascertained by measuring accumulation and the directional flux of Rh123 using the Transwell(c) semi-permeable cell culture support system. To assess the effect of Peceol(c) and Gelucire(c) 44/14 on Pgp protein expression, Western blotting with a specific Pgp antibody was performed. RESULTS. The two assays for cytotoxicity were in agreement and showed that concentrations of less than 0.5% (v/v) Peceol(c) and less than 0.02% (w/v) Gelucire(c) 44/14 were not toxic to Caco-2 cells. Rh123 accumulation was increased up to 3-fold in cells treated with sub-toxic concentrations of the excipients. The flux of Rh123 across the cell monolayer was unaffected by treatment in the absorptive (apical to basolateral) direction but the efflux transport was reduced after treatment with Peceol(c), Gelucire(c) 44/14 or the positive control , 100microM verapamil. Some of the reduction in Pgp efflux activity can be explained by the reduction in protein expression after treatment with the lipid excipients; treatment with 0.25% (v/v) and 0.5% (v/v) Peceol(c) reduced Pgp protein levels to 62.4% and 68.4% of the control respectively while Gelucire(c) 44/14 treatments of 0.01% (w/v) and 0.02% (w/v) reduced Pgp to 64.5% and 51.8% respectively.
In this study we utilized established methodologies to assess the inhibitory effect of the excipients on the Pgp-mediated efflux of the probe, Rh123 and tested the hypothesis that long-term treatment of Caco-2 cells with the lipid excipients, Peceol(c) and Gelucire(c) 44/14, decreased Pgp protein expression. The results suggest a new mechanism which may contribute to the improved bioavailability seen for drugs formulated with lipid-based excipients.
Article: Enhancing drug absorption using lipids: a case study presenting the development and pharmacological evaluation of a novel lipid-based oral amphotericin B formulation for the treatment of systemic fungal infections.[show abstract] [hide abstract]
ABSTRACT: The development of a safe and efficacious drug involves a balance between bioavailability, toxicity and disposition within the body. If the drug is hydrophobic or acid labile, oral administration may lead to poor systemic exposure, necessitating a parenteral treatment regime. Amphotericin B (AmpB) is one example of a well established, highly efficacious drug that has a 50 year history of intravenous therapy. AmpB formulated as a micellar dispersion (Fungizone; FZ) for IV use, remains one of the most effective agents in the treatment of systemic fungal infections, yet no oral formulations are currently commercially available. Recently, our laboratory has developed new oral lipid-based AmpB formulations with enhanced gastrointestinal (GI) tract absorption and antifungal activity with minimum renal toxicity. This review article will discuss these findings and present data to support two potential mechanisms for the enhanced GI tract absorption of AmpB when formulated in this oral lipid-based delivery system, namely an increase in lymphatic drug transport and a decrease in pre-systemic transporter-mediated drug efflux.Advanced Drug Delivery Reviews 04/2008; 60(6):692-701. · 11.50 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Intestinal lymphatic transport has been shown to be an absorptive pathway following oral administration of lipids and an increasing number of lipophilic drugs, which once absorbed, diffuse across the intestinal enterocyte and while in transit associate with secretable enterocyte lipoproteins. The chylomicron-associated drug is then secreted from the enterocyte into the lymphatic circulation, rather than the portal circulation, thus avoiding the metabolically-active liver, but still ultimately returning to the systemic circulation. Because of this parallel and potentially alternative absorptive pathway, first-pass metabolism can be reduced while increasing lymphatic drug exposure, which opens the potential for novel therapeutic modalities and allows the implementation of lipid-based drug delivery systems. This review discusses the physiological features of the lymphatics, enterocyte uptake and metabolism, links between drug transport and lipid digestion/re-acylation, experimental model (in vivo, in vitro, and in silico) of lymphatic transport, and the design of lipid- or prodrug-based drug delivery systems for enhancing lymphatic drug transport.Advanced drug delivery reviews 06/2011; 63(10-11):923-42. · 11.96 Impact Factor
Article: Effects of monoglycerides on rhodamine 123 accumulation, estradiol 17 beta-D-glucuronide bidirectional transport and MRP2 protein expression within Caco-2 cells.[show abstract] [hide abstract]
ABSTRACT: Oral drug development had been hindered by the bioavailability issue despite vast market popularity. Lipid excipients had shown to enhance bioavailability of a number of reformulated hydrophobic oral drugs, yet the underlying mechanisms of action by lipids are still unclear. One proposed mechanism is that lipid excipients could facilitate drug uptake by altering the activities of apical membrane intestinal efflux transporters. Thus, this study aimed to investigate the effects of 1-monopalmitin, 1-monoolein and 1-monostearin on the efflux activity and protein expression of multidrug resistance-associated protein 2 (MRP2) in vitro. The 24-hour non-cytotoxic ranges of these monoglycerides were first determined using MTS and LDH assays in Caco-2 cells. Then, both accumulation and bidirectional transport studies were conducted using 10 microM rhodamine 123 (Rh123) and 10 nM estradiol 17 beta-D-glucuronide (E(2)17betaG), respectively, to assess the functional activities of MRP2. 50 microM MK-571, a specific MRP1 and MRP2 inhibitor, was used as the positive control in both studies. Western blotting was followed to determine the effect of these monoglycerides on MRP2 protein expression. Caco-2 cells were viable when treated with 1-monopalmitin, 1-monostearin and 1-monoolein at concentrations equal or less than 1000 microM, 1000 microM and 500 microM, respectively. Cells treated with 1-monoplamitin, 1-monostearin, 1-monoolein and MK571 resulted in significant increases in Rh123 accumulation and decreases in E(2)17BetaG efflux ratio compared to the control (medium treated only). MRP2 protein expressions in 1-monopalmitin and 1-monoolein treated cells were decreased by 19% and 35% compared to the control; however, there was no change of MRP2 protein expression in 1- monostearin treated cells. These findings suggested that 1-monoolein, 1-monostearin and 1-monopalmitin could attenuate the activity of MRP2 and possibly other efflux transporters in Caco-2 cells. The reduction of efflux activity of MRP2 by 1-monoolein treatment could be partially accounted by the non-specific down-regulation of MRP2 protein expression.Journal of pharmacy & pharmaceutical sciences: a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques 02/2008; 11(3):45-62. · 1.65 Impact Factor