title: 31P and1H NMR Studies of the Molecular Organization of Lipids in the Parallel Artificial Membrane Permeability Assay.
abstract: The parallel artificial membrane permeability assay (PAMPA) has emerged as a widely used primary in vitro screen for passive permeability of potential drug candidates. However, the molecular structure of the permeation barrier (consisting of a filter-supported dodecane-egg lecithin mixture) has never been characterized. Here, we investigated the long-range order of phospholipids in the PAMPA barrier by means of31P static solid-state NMR. Diffusion constants of PAMPA membrane components were derived from liquid state NMR and, in addition, drug distribution between the PAMPA lipid phase and buffer (log DPAMPAat pH 7.4) was systematically investigated. Increasing concentration of n-dodecane to the system egg lecithin-water (lamellar phase, Lα) induces formation of inverted hexagonal (Hii) and isotropic phases. At n-dodecane concentrations matching those used in PAMPA (9%, w/v) a purely "isotropic" phase was observed corresponding to lipid aggregates with a diameter in the range 4-7 nm. Drug distribution studies indicate that these reverse micelles facilitate the binding to, and in turn the permeation across, the PAMPA dodecane barrier, in particular for amphiphilic solutes. The proposed model for the molecular architecture and function of the PAMPA barrier provides a fundamental, hitherto missing framework to evaluate the scope but also limitations of PAMPA for the prediction of in vivo membrane permeability.
authors.affiliation: Roche Pharmaceutical Research and Early Development, Pharmaceutical Sciences, Innovation Center Basel, F. Hoffmann-La Roche Ltd. , Grenzacherstrasse 124, 4070 Basel, Switzerland.|Division of Biophysical Chemistry, Biozentrum, University of Basel , Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland.
authors.forename: Frauke
authors.identifier: 0000-0002-1146-3452
authors.initials: F
authors.lastname: Assmus
authors.affiliation: Roche Pharmaceutical Research and Early Development, Therapeutic Modalities, pCMC, Innovation Center Basel, F. Hoffmann-La Roche Ltd. , Grenzacherstrasse 124, 4070 Basel, Switzerland.
authors.forename: Alfred
authors.initials: A
authors.lastname: Ross
authors.affiliation: Roche Pharmaceutical Research and Early Development, Pharmaceutical Sciences, Innovation Center Basel, F. Hoffmann-La Roche Ltd. , Grenzacherstrasse 124, 4070 Basel, Switzerland.
authors.forename: Holger
authors.initials: H
authors.lastname: Fischer
authors.affiliation: Division of Biophysical Chemistry, Biozentrum, University of Basel , Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland.
authors.forename: Joachim
authors.initials: J
authors.lastname: Seelig
authors.affiliation: Division of Biophysical Chemistry, Biozentrum, University of Basel , Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland.
authors.forename: Anna
authors.initials: A
authors.lastname: Seelig
mesh_terms: D000473:Alkanes / Q000737:chemistry; D001681:Biological Assay / Q000379:methods; D004058:Diffusion; D054709:Lecithins / Q000737:chemistry; D008055:Lipids / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008567:Membranes, Artificial; D008823:Micelles; D010539:Permeability; D010743:Phospholipids / Q000737:chemistry
chemical_list: D000473:Alkanes; D054709:Lecithins; D008055:Lipids; D008567:Membranes, Artificial; D008823:Micelles; D010743:Phospholipids; C007548:n-dodecane
keywords: NMR; PAMPA; membrane; phase transition; structure