Pharmaceutical Research

, Volume 27, Issue 8, pp 1644–1658 | Cite as

In Situ Artificial Membrane Permeation Assay under Hydrodynamic Control: Permeability-pH Profiles of Warfarin and Verapamil

  • Matěj Velický
  • Dan F. Bradley
  • Kin Y. Tam
  • Robert A. W. Dryfe
Research Paper



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.


hydrodynamic control in situ permeation PAMPA permeability unstirred water layer 



membrane area


hydrodynamic exponent


bio-mimetic PAMPA


time-dependent solute concentration


colorectal adenocarcinoma cell epithelial line


2-(Cyclohexylamino)ethanesulfonic acid


aqueous diffusion coefficient


membrane diffusion coefficient


dioleoyl phosphatidylcholine


dioleoyl phosphatidylcholine PAMPA


double-sink PAMPA


neutral fraction of the solute


membrane thickness


hexadecane PAMPA


immobilised artificial membrane


time-dependent solute flux


distribution coefficient


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


polyvinylidene fluoride


fractional membrane retention




unstirred water layer




unstirred water layer thickness


kinematic viscosity





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.

Supplementary material

11095_2010_150_MOESM1_ESM.doc (1.8 mb)
Appendix (DOC 1812 kb)


  1. 1.
    Fade V. Link between drug absorption solubility and permeability measurements in Caco-2 cells. J Pharm Sci. 1998;87:1604–7.CrossRefGoogle Scholar
  2. 2.
    Artursson P, Karlsson J. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (CACO-2) cells. Biochem Biophys Res Commun. 1991;17:880–5.CrossRefGoogle Scholar
  3. 3.
    Irvine JD, Takahashi L, Lockhart K, Cheong J, Tolan JW, Selick HE et al. MDCK (Madin-Darby canine kidney) cells: a tool for membrane permeability screening. J Pharm Sci. 1999;88:28–33.CrossRefPubMedGoogle Scholar
  4. 4.
    Galinis-Luciani D, Nguyen L, Yazdanian M. Is PAMPA a useful tool for discovery? J Pharm Sci. 2007;96:2886–92.CrossRefPubMedGoogle Scholar
  5. 5.
    Avdeef A, Bendels S, Di L, Faller B, Kansy M, Sugano K et al. PAMPA - Critical factors for better predictions of absorption. J Pharm Sci. 2007;96:2893–909.CrossRefPubMedGoogle Scholar
  6. 6.
    Avdeef A. The rise of PAMPA. Expert Opin Drug Metab Toxicol. 2005;1:325–42.CrossRefPubMedGoogle Scholar
  7. 7.
    Mälkia A, Murtomäki L, Urtti A, Kontturi K. Drug permeation in biomembranes: In vitro and in silico prediction and influence of physicochemical properties. Eur J Pharm Sci. 2004;23:13–47.CrossRefPubMedGoogle Scholar
  8. 8.
    Avdeef A. Absorption and Drug Development: Solubility, Permeability, and Charge State, Wiley-Interscience, 2003.Google Scholar
  9. 9.
    Avdeef A. High-throughput measurement of permeability profiles, Drug Bioavailability, Wiley-VCH Weinheim, 2003.Google Scholar
  10. 10.
    Kansy M, Senner F, Gubernator K. Physicochemical high throughput screening: parallel artificial membrane permeation assay in the description of passive absorption processes. J Med Chem. 1998;41:1007–10.CrossRefPubMedGoogle Scholar
  11. 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
  12. 12.
    Avdeef A, Strafford M, Block E, Balogh MP, Chambliss W, Khan I. Drug absorption in vitro model: Filter-immobilized artificial membranes: 2. Studies of the permeability properties of lactones in Piper methysticum Forst. Eur J Pharm Sci. 2001;14:271–80.CrossRefPubMedGoogle Scholar
  13. 13.
    Bermejo M, Avdeef A, Ruiz A, Nalda R, Ruell JA, Tsinman O et al. PAMPA - a drug absorption in vitro model 7. Comparing rat in situ, Caco-2, and PAMPA permeability of fluoroquinolones. Eur J Pharm Sci. 2004;21:429–41.CrossRefPubMedGoogle Scholar
  14. 14.
    Avdeef A, Nielsen PE, Tsinman O. PAMPA - A drug absorption in vitro model: 11. Matching the in vivo unstirred water layer thickness by individual-well stirring in microtitre plates. Eur J Pharm Sci. 2004;22:365–74.PubMedGoogle Scholar
  15. 15.
    Sugano K, Hamada H, Machida M, Ushio H, Saitoh K, Terada K. Optimized conditions of bio-mimetic artificial membrane permeation assay. Int J Pharm. 2001;228:181–8.CrossRefPubMedGoogle Scholar
  16. 16.
    Sugano K, Nabuchi Y, Machida M, Aso Y. Prediction of human intestinal permeability using artificial membrane permeability. Int J Pharm. 2003;257:245–51.CrossRefPubMedGoogle Scholar
  17. 17.
    Sugano K, Takata N, Machida M, Saitoh K, Terada K. Prediction of passive intestinal absorption using bio-mimetic artificial membrane permeation assay and the paracellular pathway model. Int J Pharm. 2002;241:241–51.CrossRefPubMedGoogle Scholar
  18. 18.
    Wohnsland F, Faller B. High-throughput permeability pH profile and high-throughput alkane/water log P with artificial membranes. J Med Chem. 2001;44:923–30.CrossRefPubMedGoogle Scholar
  19. 19.
    Faller B, Grimm HP, Loeuillet-Ritzler F, Arnold S, Briand X. High-throughput lipophilicity measurement with immobilized artificial membranes. J Med Chem. 2005;48:2571–6.CrossRefPubMedGoogle Scholar
  20. 20.
    Chen X, Murawski A, Patel K, Crespi CL, Balimane PV. A novel design of artificial membrane for improving the PAMPA model. Pharm Res. 2008;25:1511–20.CrossRefPubMedGoogle Scholar
  21. 21.
    Flaten GE, Skar M, Luthman K, Brandl M. Drug permeability across a phospholipid vesicle based barrier: 3. Characterization of drug-membrane interactions and the effect of agitation on the barrier integrity and on the permeability. Eur J Pharm Sci. 2007;30:324–32.CrossRefPubMedGoogle Scholar
  22. 22.
    Flaten GE, Dhanikula AB, Luthman K, Brandl M. Drug permeability across a phospholipid vesicle based barrier: a novel approach for studying passive diffusion. Eur J Pharm Sci. 2006;27:80–90.CrossRefPubMedGoogle Scholar
  23. 23.
    Flaten GE, Bunjes H, Luthman K, Brandl M. Drug permeability across a phospholipid vesicle-based barrier. 2. Characterization of barrier structure, storage stability and stability towards pH changes. Eur J Pharm Sci. 2006;28:336–43.CrossRefPubMedGoogle Scholar
  24. 24.
    Di L, Kerns EH, Fan K, McConnell OJ, Carter GT. High throughput artificial membrane permeability assay for blood-brain barrier. Eur J Med Chem. 2003;38:223–32.CrossRefPubMedGoogle Scholar
  25. 25.
    Przybylo M, Olzynska A, Han S, Ozyhar A, Langner M. A fluorescence method for determining transport of charged compounds across lipid bilayer. Biophys Chem. 2007;129:120–5.CrossRefPubMedGoogle Scholar
  26. 26.
    Gjelstad A, Rasmussen KE, Pedersen-Bjergaard S. Electrokinetic migration across artificial liquid membranes. Tuning the membrane chemistry to different types of drug substances. J Chrom. 2006;1124:29–34.CrossRefGoogle Scholar
  27. 27.
    Balon K, Riebesehl BU, Müller BW. Drug liposome partitioning as a tool for the prediction of human passive intestinal absorption. Pharm Res. 1999;16:882–8.CrossRefPubMedGoogle Scholar
  28. 28.
    Seo PR, Teksin ZS, Kao JPY, Polli JE. Lipid composition effect on permeability across PAMPA. Eur J Pharm Sci. 2006;29:259–68.CrossRefPubMedGoogle Scholar
  29. 29.
    Avdeef A, Artursson P, Neuhoff S, Lazorova L, Gråsjö J, Tavelin S. Caco-2 permeability of weakly basic drugs predicted with the Double-Sink PAMPA pKa flux method. Eur J Pharm Sci. 2005;24:333–49.CrossRefPubMedGoogle Scholar
  30. 30.
    Adson A, Burton PS, Raub TJ, Barsuhn CL, Audus KL, Ho NFH. Passive diffusion of weak organic electrolytes across Caco-2 cell monolayers: Uncoupling the contributions of hydrodynamic, transcellular, and paracellular barriers. J Pharm Sci. 1995;84:1197–204.CrossRefPubMedGoogle Scholar
  31. 31.
    Karlsson J, Artursson P. A method for the determination of cellular permeability coefficients and aqueous boundary layer thickness in monolayers of intestinal epithelial (Caco-2) cells grown in permeable filter chambers. Int J Pharm. 1991;71:55–64.CrossRefGoogle Scholar
  32. 32.
    Lennernäs H. Human intestinal permeability. J Pharm Sci. 1998;87:403–10.CrossRefPubMedGoogle Scholar
  33. 33.
    Molloy BJ, Tam KY, Wood JM, Dryfe RAW. A hydrodynamic approach to the measurement of the permeability of small molecules across artificial membranes. Analyst. 2008;133:655–9.CrossRefPubMedGoogle Scholar
  34. 34.
    Mayer PT, Anderson BD. Transport across 1, 9-decadiene precisely mimics the chemical selectivity of the barrier domain in egg lecithin bilayers. J Pharm Sci. 2002;91:640–6.CrossRefPubMedGoogle Scholar
  35. 35.
    Levich VG. Physicochemical hydrodynamics. London: Englewood Cliffs; 1962.Google Scholar
  36. 36.
    Albery WJ, Burke JF, Leffler EB, Hadgraft J. Interfacial transfer studied with a rotating diffusion cell. J Chem Soc Faraday Trans I. 1976;72:1618–26.CrossRefGoogle Scholar
  37. 37.
    Guyand RH, Honda DH. Solute transport resistance at the octanol - water interface. Int J Pharm. 1984;19:129–37.CrossRefGoogle Scholar
  38. 38.
    Leahy DE, Wait AR. Solute transport resistance at water-oil interfaces. J Pharm Sci. 1986;75:1157–61.CrossRefPubMedGoogle Scholar
  39. 39.
    Amidon GE, Higuchi WI, Ho NFH. Theoretical and experimental studies of transport of micelle-solubilized solutes. J Pharm Sci. 1982;71:77–84.CrossRefPubMedGoogle Scholar
  40. 40.
    Shore PA, Brodie BB, Hogben CAM. The gastric secretion of drugs - a pH partition hypothesis. J Pharmacol Exp Ther. 1957;119:361–9.PubMedGoogle Scholar
  41. 41.
    Atkins P, de Paula J. Physical chemistry for the life sciences. Oxford University Press, 2006.Google Scholar
  42. 42.
    Charcosset C, Bernengo JC. Comparison of microporous membrane morphologies using confocal scanning laser microscopy. J Membr Sci. 2000;168:53–62.CrossRefGoogle Scholar
  43. 43.
    Sarveiya V, Templeton JF, Benson HAE. Ion-pairs of ibuprofen: Increased membrane diffusion. J Pharm Pharmacol. 2004;56:717–24.CrossRefPubMedGoogle Scholar
  44. 44.
    Takacs-Novak K, Szasz G. Ion-pair partition of quaternary ammonium drugs: the influence of counter ions of different lipophilicity, size, and flexibility. Pharm Res. 1999;16:1633–8.CrossRefPubMedGoogle Scholar
  45. 45.
    Neubert R. Ion pair transport across membranes. Pharm Res. 1989;6:743–7.CrossRefPubMedGoogle Scholar
  46. 46.
    Dollery CT. Therapeutic drugs. Churchill Livingstone, 1999.Google Scholar
  47. 47.
    Obach RS, Lombardo F, Waters NJ. Trend analysis of a database of intravenous pharmacokinetic parameters in human for 670 drug compounds. Drug Metab Dispos. 2008;36:1385–405.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.School of ChemistryUniversity of ManchesterManchesterUK
  2. 2.AstraZeneca, MeresideCheshireUK

Personalised recommendations