S1304 Intestinal Cell Culture Model System to Test the Effect of Natural Products on Drug Transport: Effect of Naringin on the Transport of Digoxin

Abstract

INTRODUCTION: Certain foods can modify the luminal plasma membrane pumps of intestinal absorptive cells (enterocytes) and significantly change drug absorption. This project aims to create a cell culture model and a series of drug immunoassays to assess the ability of specific natural products to change enterocyte pump activity. Current work focuses upon naringin, a flavonoid that occurs naturally in citrus fruits, especially grapefruit. Both naringin and slow-release naringin microparticles will be assessed for their ability to inhibit the ABC transporter. Inhibition of this pump increases drug absorption in vivo. METHODS: Intestinal enteroids and a cell line derived from a human colonic adenocarcinoma (T84) were obtained from collaborators. A survey of drugs whose absorption was stimulated by ABC pump inhibition was cross-referenced to commercially available antibodies, and digoxin was chosen as the model drug. A digoxin-bovine serum albumin (BSA) conjugate was prepared by NaIO4 oxidation of the glucose moiety in digoxin. The newly generated aldehydes were then reacted with the lysine residues on the BSA to create a Schiff base, which was then reduced to stabilize the covalent bond. Our in-house synthesized conjugate was then compared to commercially available digoxin-BSA conjugate as a capture reagent in a competitive ELISA for digoxin. RESULTS: The competitive ELISA using in-house made digoxin-BSA conjugate was ∼ 10-fold more sensitive than the assay formatted with the commercial conjugate. The limit of detection of the final ELISA was 0.4 ng/mL and the IC50 was 5.0 ng/mL. The assay was not affected by the addition of up 25% of culture media or ethanol (used as a solvent for digoxin). CONCLUSION: An assay to detect the levels of digoxin in culture media has been optimized. The next steps will be to grow intestinal cell monolayers on transwell plates and measure any increase in digoxin reaching the basolateral side in the presence of naringin. This could ultimately lead to a novel model system to test food-drug interactions that affect intestinal transporter pump activity.