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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

ABSTRACT

Purpose

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.

Methods

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.

Results

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.

Conclusions

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 WORDS

hydrodynamic control in situ permeation PAMPA permeability unstirred water layer 

ABBREVIATIONS AND SYMBOLS

A

membrane area

α

hydrodynamic exponent

BM-PAMPA

bio-mimetic PAMPA

c(t)

time-dependent solute concentration

Caco-2

colorectal adenocarcinoma cell epithelial line

CHES

2-(Cyclohexylamino)ethanesulfonic acid

Daq

aqueous diffusion coefficient

Dm

membrane diffusion coefficient

DOPC

dioleoyl phosphatidylcholine

DOPC-PAMPA

dioleoyl phosphatidylcholine PAMPA

DS-PAMPA

double-sink PAMPA

fn

neutral fraction of the solute

h

membrane thickness

HDM-PAMPA

hexadecane PAMPA

IAM

immobilised artificial membrane

J(t)

time-dependent solute flux

Kd

distribution coefficient

KOCT

octanol/water distribution coefficient

MDCK

Madin-Darby canine kidney epithelial cell line

P

(not specified) permeability coefficient

P0

intrinsic permeability coefficient

PAMPA

parallel artificial membrane permeation assay

Pe

effective (measured) permeability coefficient

Pm

membrane permeability coeffcient

PTFE

polytetrafluoroethylene

Pu

unstirred water layer permeability coefficient

PVDF

polyvinylidene fluoride

R

fractional membrane retention

t

time

UWL

unstirred water layer

V

volume

δUWL

unstirred water layer thickness

ν

kinematic viscosity

τLAG

lag-time

Notes

ACKNOWLEDGEMENTS

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)

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

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

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