Development of simulated intestinal fluids containing nutrients as transport media in the Caco-2 cell culture model: assessment of cell viability, monolayer integrity and transport of a poorly aqueous soluble drug and a substrate of efflux mechanisms.
The purpose of this study was to identify simulated intestinal fluids (SIFs) containing nutrients compatible with the Caco-2 cell culture model and to examine the impact of the identified medium on the transport of a poorly aqueous soluble model compound, estradiol, and a substrate of efflux mechanisms, etoposide. Monolayer integrity was evaluated by transepithelial electrical resistance and cellular viability by release of lactate dehydrogenase to the apical compartment and cellular protein content. It was shown that the viability of Caco-2 cells was enhanced by use of the CO(2) independent nutritional medium, Leibovitz's L-15 compared to Hanks' balanced salt solution. SIF containing 5mM sodium taurocholate and 1.25 mM phosphatidylcholine or lysophosphatidylcholine in Leibovitz's L-15 induced less release of lactate dehydrogenase than the traditional transport medium, HBSS. Addition of lipolysis products, 0.5mM oleic acid and 0.25 mM monoolein, did only cause increase in lactate dehydrogenase in 3 of 12 comparisons. The presence of SIFs in the apical compartment was shown to decrease flux of estradiol due to incorporation of estradiol in micelles and hence a decreased fraction of free estradiol. Further, a concentration dependent increase in the apparent permeability of etoposide was observed from apical to basolateral compartment, which indicated that components in the SIFs affects efflux mechanisms.
Lind
Marianne L
ML
Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
Jacobsen
Jette
J
Holm
René
R
Müllertz
Anette
A
D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D004355:Drug Stability; D004958:Estradiol / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D005047:Etoposide / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D005502:Food; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007419:Intestinal Secretions / Q000737:chemistry / Q000502:physiology*; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008823:Micelles; D009994:Osmolar Concentration; D010539:Permeability; D010713:Phosphatidylcholines / Q000494:pharmacology; D012995:Solubility; D013501:Surface-Active Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013656:Taurocholic Acid / Q000494:pharmacology; D014867:Water / Q000737:chemistry
D003470:Culture Media; D008244:Lysophosphatidylcholines; D008565:Membrane Proteins; D008823:Micelles; D010713:Phosphatidylcholines; D013501:Surface-Active Agents; D014867:Water; D004958:Estradiol; D013656:Taurocholic Acid; D005047:Etoposide; D007770:L-Lactate Dehydrogenase