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The effect of two novel cholesterol-lowering agents, disodium ascorbyl phytostanol phosphate (DAPP) and nanostructured aluminosilicate (NSAS) on the expression and activity of P-glycoprotein within Caco-2 cells.

Sachs-Barrable K, Darlington JW, Wasan KM - Lipids Health Dis (2014)

Bottom Line: This down-regulation of P-glycoprotein also resulted in decreased activity of P-glycoprotein compared to untreated control.These findings have to be taken into consideration when DAPP is concurrently given with other drugs that are substrates for P-gp since drug-drug interactions harbour a safety issue and alter bioavailability profiles.NSAS does not have any P-gp altering properties and therefore might not affect drug-drug interactions.We conclude from this study that NSAS might make a safer drug candidate compared to DAPP for lowering LDL-cholesterol.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmaceutics and Biopharmaceutics, Faculty of Pharmaceutical Sciences, The University of British Columbia, 2405 Wesbrook Mall, Vancouver, V6T 1Z3, British Columbia, Canada. Kishor.Wasan@usask.ca.

ABSTRACT

Background: Many drugs are substrates for P-glycoprotein (P-gp) and interactions involving P-gp may be relevant to clinical practice. Co-administration with P-gp inhibitors or inducers changes the absorption profile as well as the risk for drug toxicity, therefore it is important to evaluate possible P-gp alterations. The purpose of this study was to investigate the effect of two novel cholesterol-lowering agents, disodium ascorbyl phytostanol phosphate (DAPP) and nanostructured aluminium silicate (NSAS), a protonated montmorillonite clay, on mdr-1 gene expression and its protein, P-glycoprotein (P-gp) within Caco-2 cells.

Methods: The effects of DAPP and NSAS on the regulation of mdr-1 gene, P-gp protein expression and activity within Caco-2 cells, were determined using cell viability and cytotoxicity tests, RT-PCR, Western Blot analysis and bi-directional transport studies.

Results: We observed a significant down-regulation of mdr-1 mRNA (e.g. 38.5 ± 17% decrease vs. control at 5 μM DAPP and 61.2 ± 25% versus control at 10 μM DAPP; n = 6, P* < 0.05) within Caco-2 cells. Western Blot analysis of P-gp expression showed that changes in mdr-1 gene expression lead to correlating changes in P-gp protein expression. This down-regulation of P-glycoprotein also resulted in decreased activity of P-glycoprotein compared to untreated control. In contrast, when Caco-2 cells were treated with NSAS, no changes in mdr-1 gene expression, P-gp protein expression nor P-gp activity were observed.

Conclusions: DAPP but not NSAS decreases P-gp mediated drug efflux through decreased mdr-1 gene expression and consequently decreased P-gp protein expression. These findings have to be taken into consideration when DAPP is concurrently given with other drugs that are substrates for P-gp since drug-drug interactions harbour a safety issue and alter bioavailability profiles.NSAS does not have any P-gp altering properties and therefore might not affect drug-drug interactions. We conclude from this study that NSAS might make a safer drug candidate compared to DAPP for lowering LDL-cholesterol.

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Related in: MedlinePlus

Cell viability and cytotoxicity after treatment with DAPP. Effect on Caco-2 cells. MTS-Assay (A) and LDH-Assay (B) after incubation with different concentrations of DAPP for 24 hours. Results are compared to untreated control cells (in A) and Triton X-100 (in B). Each bar represents the mean ± SD. *p < 0.05. N = 4.
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Fig4: Cell viability and cytotoxicity after treatment with DAPP. Effect on Caco-2 cells. MTS-Assay (A) and LDH-Assay (B) after incubation with different concentrations of DAPP for 24 hours. Results are compared to untreated control cells (in A) and Triton X-100 (in B). Each bar represents the mean ± SD. *p < 0.05. N = 4.

Mentions: Treatment with concentrations between 0 and 500 μg/ml NSAS and 0 and 250 μM DAPP showed no significant difference in cell viability compared to untreated control as measured by MTS assay (Figures 3A and 4A). Results from the LDH assay showed no significant cytoxicity for concentrations between 0 and 300 μg/mL NSAS and 0 and 100 μM DAPP compared to untreated control (Figures 3B and 4B). These nontoxic concentrations were subsequently used to treat cells for cholesterol uptake, transporter protein expression, Rh123 accumulation and transport studies across the Caco-2 cell monolayer.Figure 3


The effect of two novel cholesterol-lowering agents, disodium ascorbyl phytostanol phosphate (DAPP) and nanostructured aluminosilicate (NSAS) on the expression and activity of P-glycoprotein within Caco-2 cells.

Sachs-Barrable K, Darlington JW, Wasan KM - Lipids Health Dis (2014)

Cell viability and cytotoxicity after treatment with DAPP. Effect on Caco-2 cells. MTS-Assay (A) and LDH-Assay (B) after incubation with different concentrations of DAPP for 24 hours. Results are compared to untreated control cells (in A) and Triton X-100 (in B). Each bar represents the mean ± SD. *p < 0.05. N = 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4195884&req=5

Fig4: Cell viability and cytotoxicity after treatment with DAPP. Effect on Caco-2 cells. MTS-Assay (A) and LDH-Assay (B) after incubation with different concentrations of DAPP for 24 hours. Results are compared to untreated control cells (in A) and Triton X-100 (in B). Each bar represents the mean ± SD. *p < 0.05. N = 4.
Mentions: Treatment with concentrations between 0 and 500 μg/ml NSAS and 0 and 250 μM DAPP showed no significant difference in cell viability compared to untreated control as measured by MTS assay (Figures 3A and 4A). Results from the LDH assay showed no significant cytoxicity for concentrations between 0 and 300 μg/mL NSAS and 0 and 100 μM DAPP compared to untreated control (Figures 3B and 4B). These nontoxic concentrations were subsequently used to treat cells for cholesterol uptake, transporter protein expression, Rh123 accumulation and transport studies across the Caco-2 cell monolayer.Figure 3

Bottom Line: This down-regulation of P-glycoprotein also resulted in decreased activity of P-glycoprotein compared to untreated control.These findings have to be taken into consideration when DAPP is concurrently given with other drugs that are substrates for P-gp since drug-drug interactions harbour a safety issue and alter bioavailability profiles.NSAS does not have any P-gp altering properties and therefore might not affect drug-drug interactions.We conclude from this study that NSAS might make a safer drug candidate compared to DAPP for lowering LDL-cholesterol.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmaceutics and Biopharmaceutics, Faculty of Pharmaceutical Sciences, The University of British Columbia, 2405 Wesbrook Mall, Vancouver, V6T 1Z3, British Columbia, Canada. Kishor.Wasan@usask.ca.

ABSTRACT

Background: Many drugs are substrates for P-glycoprotein (P-gp) and interactions involving P-gp may be relevant to clinical practice. Co-administration with P-gp inhibitors or inducers changes the absorption profile as well as the risk for drug toxicity, therefore it is important to evaluate possible P-gp alterations. The purpose of this study was to investigate the effect of two novel cholesterol-lowering agents, disodium ascorbyl phytostanol phosphate (DAPP) and nanostructured aluminium silicate (NSAS), a protonated montmorillonite clay, on mdr-1 gene expression and its protein, P-glycoprotein (P-gp) within Caco-2 cells.

Methods: The effects of DAPP and NSAS on the regulation of mdr-1 gene, P-gp protein expression and activity within Caco-2 cells, were determined using cell viability and cytotoxicity tests, RT-PCR, Western Blot analysis and bi-directional transport studies.

Results: We observed a significant down-regulation of mdr-1 mRNA (e.g. 38.5 ± 17% decrease vs. control at 5 μM DAPP and 61.2 ± 25% versus control at 10 μM DAPP; n = 6, P* < 0.05) within Caco-2 cells. Western Blot analysis of P-gp expression showed that changes in mdr-1 gene expression lead to correlating changes in P-gp protein expression. This down-regulation of P-glycoprotein also resulted in decreased activity of P-glycoprotein compared to untreated control. In contrast, when Caco-2 cells were treated with NSAS, no changes in mdr-1 gene expression, P-gp protein expression nor P-gp activity were observed.

Conclusions: DAPP but not NSAS decreases P-gp mediated drug efflux through decreased mdr-1 gene expression and consequently decreased P-gp protein expression. These findings have to be taken into consideration when DAPP is concurrently given with other drugs that are substrates for P-gp since drug-drug interactions harbour a safety issue and alter bioavailability profiles.NSAS does not have any P-gp altering properties and therefore might not affect drug-drug interactions. We conclude from this study that NSAS might make a safer drug candidate compared to DAPP for lowering LDL-cholesterol.

Show MeSH
Related in: MedlinePlus