title: Mechanism of pluronic effect on P-glycoprotein efflux system in blood-brain barrier: contributions of energy depletion and membrane fluidization.
abstract: Pluronic block copolymer, P85, inhibits the P-glycoprotein (Pgp) drug efflux system and increases the permeability of a broad spectrum of drugs in the blood-brain barrier (BBB). This study examines the mechanisms by which P85 inhibits Pgp using bovine brain microvessel endothelial cells (BBMEC) as an in vitro model of the BBB. The hypothesis was that simultaneous alterations in intracellular ATP levels and membrane fluidization in BBMEC monolayers by P85 results in inhibition of the drug efflux system. The methods included the use of 1) standard Pgp substrate rhodamine 123 to assay the Pgp efflux system in BBMEC, 2) luciferin/luciferase assay for ATP intracellular levels, and 3) 1,6-diphenyl-1,3,5-hexatriene for membrane microviscosity. Using 3H-labeled P85 and fluorescein-labeled P85 for confocal microscopy, this study suggests that P85 accumulates in the cells and intracellular organelles such as the mitochondria where it can interfere with metabolic processes. Following exposure of BBMEC to P85, the ATP levels were depleted, and microviscosity of the cell membranes was decreased. Furthermore, P85 treatment decreased Pgp ATPase activity in membranes expressing human Pgp. A combination of experiments examining the kinetics, concentration dependence, and directionality of P85 effects on Pgp-mediated efflux in BBMEC monolayers suggests that both energy depletion (decreasing ATP pool available for Pgp) and membrane fluidization (inhibiting Pgp ATPase activity) are critical factors contributing to the activity of the block copolymer in the BBB.
authors.affiliation: Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68198-6025, USA.
authors.forename: E V
authors.initials: EV
authors.lastname: Batrakova
authors.forename: S
authors.initials: S
authors.lastname: Li
authors.forename: S V
authors.initials: SV
authors.lastname: Vinogradov
authors.forename: V Y
authors.initials: VY
authors.lastname: Alakhov
authors.forename: D W
authors.initials: DW
authors.lastname: Miller
authors.forename: A V
authors.initials: AV
authors.lastname: Kabanov
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D001812:Blood-Brain Barrier / Q000187:drug effects*; D001921:Brain / Q000187:drug effects / Q000201:enzymology; D002417:Cattle; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D004735:Energy Transfer / Q000187:drug effects*; D005454:Fluorescence Polarization; D066298:In Vitro Techniques; D007700:Kinetics; D008566:Membranes / Q000187:drug effects / Q000201:enzymology; D008856:Microscopy, Fluorescence; D011060:Poloxalene / Q000378:metabolism / Q000494:pharmacology*; D014783:Viscosity
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; C422648:pluronic block copolymer p85; D000255:Adenosine Triphosphate; D011060:Poloxalene; D000251:Adenosine Triphosphatases