title: A liposomal fluorescence assay to study permeation kinetics of drug-like weak bases across the lipid bilayer.
abstract: Lipid bilayer permeation is considered the major route for in vivo barrier passage of drugs. Despite this fact, no technique is currently available to measure the kinetics of permeation across a single lipid bilayer of structurally unrelated drug-like solutes. We developed a liposomal fluorescence assay capable to determine permeation kinetics of basic drug-like solutes across lipid bilayers. The assay is based on the hypothesis that permeation of a weak base along a concentration gradient results in net proton release at the cis-side and net proton capture at the trans-side of the bilayer. The resulting pH changes were monitored with pH-sensitive fluorophores: Test compounds were incubated with liposomes containing a pH-sensitive fluorophore at the bilayer surfaces or in the aqueous lumen and fluorescence changes were monitored with a stopped-flow apparatus in solution or by total internal reflection fluorescence microscopy with surface-captured liposomes on a microfluidic platform. Incubation with lipophilic basic drugs resulted in the expected fluorescence changes while incubation with compounds without basic functionality or high polarity did not affect fluorescence. Kinetics of fluorescence changes followed bi-exponential functions. Logarithmic permeation coefficients (logPermapp) determined in solution and by microfluidics technology showed a good correlation (r(2)=0.94, n=7) and logPermapp increased with increasing lipophilicity. Neither diffusion in the aqueous phase nor partitioning into the bilayer was rate-limiting. PEGylation of 2% of the liposomal lipids reduced Permapp by a factor ~300. In conclusion, the presented liposomal fluorescence assay is capable to determine permeation kinetics of weak basic drug-like solutes across lipid bilayers. The method is adaptable to microfluidics technology for high-throughput measurements and can potentially be modified to work for weak acid solutes.
authors.affiliation: Bioanalytics, Laboratory of Organic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland.
authors.forename: Klaus
authors.initials: K
authors.lastname: Eyer
authors.affiliation: Biopharmacy and Radiopharmaceutical Sciences, Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland.
authors.forename: Franziska
authors.initials: F
authors.lastname: Paech
authors.affiliation: Bioanalytics, Laboratory of Organic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Schuler
authors.affiliation: Bioanalytics, Laboratory of Organic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland.
authors.forename: Phillip
authors.initials: P
authors.lastname: Kuhn
authors.affiliation: Bioinorganic and Solution Chemistry, Laboratory of Inorganic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Kissner
authors.affiliation: Biopharmacy and Radiopharmaceutical Sciences, Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland.
authors.forename: Sara
authors.initials: S
authors.lastname: Belli
authors.affiliation: Bioanalytics, Laboratory of Organic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland. Electronic address: dittrich@org.chem.ethz.ch.
authors.forename: Petra S
authors.initials: PS
authors.lastname: Dittrich
authors.affiliation: Biopharmacy and Radiopharmaceutical Sciences, Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland. Electronic address: stefanie.kraemer@pharma.ethz.c.
authors.forename: Stefanie D
authors.initials: SD
authors.lastname: Krämer
mesh_terms: D000468:Alkalies / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D056656:Lab-On-A-Chip Devices; D008051:Lipid Bilayers / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism*; D046210:Microfluidic Analytical Techniques; D010539:Permeability; D004364:Pharmaceutical Preparations / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D000468:Alkalies; D008051:Lipid Bilayers; D008081:Liposomes; D004364:Pharmaceutical Preparations
keywords: Chlorpromazine (PubChem CID: 2726); Desipramine (PubChem CID: 2995); Domperidone (PubChem CID: 3151); Fluorescence; Lab-on-a-chip; Labetalol (PubChem CID: 3869); Lipid bilayer; Liposomes; Loperamide (PubChem CID: 3955); Microfluidics; Permeation; Permeation coefficient; Propranolol (PubChem CID: 4946); Verapamil (PubChem CID: 2520)