In Situ Artificial Membrane Permeation Assay under Hydrodynamic Control: Permeability-pH Profiles of Warfarin and Verapamil
- 439 Downloads
To investigate the permeation of two ionisable drug molecules, warfarin and verapamil, across artificial membranes. For the first time since the introduction of the parallel artificial membrane permeation assay (PAMPA) in 1998, in situ permeation-time profiles of drug molecules are studied.
The method employs a rotating-diffusion cell where the donor and acceptor compartments are separated by a lipid-impregnated artificial membrane. The permeation of the solute is investigated under well-defined hydrodynamic conditions with control over the unstirred water layer. The flux of the permeating molecule is analysed in situ using UV spectrophotometry.
In situ permeation-time profiles are obtained under hydrodynamic control and used to determine permeability coefficients. An advanced analytical transport model is derived to account for the membrane retention, two-way flux and pH gradient between the two compartments. Moreover, a numerical permeation model was developed to rationalise the time-dependent permeation profiles. The membrane permeability, intrinsic permeability and unstirred water permeability coefficients of two drug molecules are obtained from two independent methods, hydrodynamic extrapolation and pH profiling, and the results are compared.
Both warfarin and verapamil exhibit high permeability values, which is consistent with the high fraction absorbed in human. Our results demonstrate that a considerable lag-time, varying with the solute lipophilicity and stirring rate, exists in membrane permeation and leads to incorrect compound ranking if it is not treated properly. Comparison of the permeability data as a function of pH and stirring rate suggests that some transport of the ionized molecules occurs, most likely via ion-pairing.
KEY WORDShydrodynamic control in situ permeation PAMPA permeability unstirred water layer
ABBREVIATIONS AND SYMBOLS
time-dependent solute concentration
colorectal adenocarcinoma cell epithelial line
aqueous diffusion coefficient
membrane diffusion coefficient
dioleoyl phosphatidylcholine PAMPA
neutral fraction of the solute
immobilised artificial membrane
time-dependent solute flux
octanol/water distribution coefficient
Madin-Darby canine kidney epithelial cell line
(not specified) permeability coefficient
intrinsic permeability coefficient
parallel artificial membrane permeation assay
effective (measured) permeability coefficient
membrane permeability coeffcient
unstirred water layer permeability coefficient
fractional membrane retention
unstirred water layer
unstirred water layer thickness
We thank our industrial collaborator, AstraZeneca, and EPSRC for funding and Dr. J. Matthew Wood (AstraZeneca, Alderley Park) for consultation and training in the industrial PAMPA method.
- 8.Avdeef A. Absorption and Drug Development: Solubility, Permeability, and Charge State, Wiley-Interscience, 2003.Google Scholar
- 9.Avdeef A. High-throughput measurement of permeability profiles, Drug Bioavailability, Wiley-VCH Weinheim, 2003.Google Scholar
- 11.Kansy M, Fischer H, Kratzat K, Senner F, Wagner B, and Parilla I. High-Throughput Artificial Membrane Permeability Studies in Early Lead Discovery and Development, Pharmacokinetic Optimization in Drug Research. Helvetic Chim Acta. 2001.Google Scholar
- 35.Levich VG. Physicochemical hydrodynamics. London: Englewood Cliffs; 1962.Google Scholar
- 41.Atkins P, de Paula J. Physical chemistry for the life sciences. Oxford University Press, 2006.Google Scholar
- 46.Dollery CT. Therapeutic drugs. Churchill Livingstone, 1999.Google Scholar