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

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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.

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Abbreviations

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

D aq :

aqueous diffusion coefficient

D m :

membrane diffusion coefficient

DOPC:

dioleoyl phosphatidylcholine

DOPC-PAMPA:

dioleoyl phosphatidylcholine PAMPA

DS-PAMPA:

double-sink PAMPA

f n :

neutral fraction of the solute

h :

membrane thickness

HDM-PAMPA:

hexadecane PAMPA

IAM:

immobilised artificial membrane

J(t):

time-dependent solute flux

K d :

distribution coefficient

K OCT :

octanol/water distribution coefficient

MDCK:

Madin-Darby canine kidney epithelial cell line

P :

(not specified) permeability coefficient

P 0 :

intrinsic permeability coefficient

PAMPA:

parallel artificial membrane permeation assay

P e :

effective (measured) permeability coefficient

P m :

membrane permeability coeffcient

PTFE:

polytetrafluoroethylene

P u :

unstirred water layer permeability coefficient

PVDF:

polyvinylidene fluoride

R :

fractional membrane retention

t :

time

UWL:

unstirred water layer

V :

volume

δ UWL :

unstirred water layer thickness

ν :

kinematic viscosity

τ LAG :

lag-time

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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.

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Authors and Affiliations

  1. School of Chemistry, University of Manchester, Oxford Road, Manchester, M13 9PL, UK

    Matěj Velický, Dan F. Bradley & Robert A. W. Dryfe

  2. AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK

    Kin Y. Tam

Authors
  1. Matěj Velický
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  2. Dan F. Bradley
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  3. Kin Y. Tam
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  4. Robert A. W. Dryfe
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Corresponding authors

Correspondence to Kin Y. Tam or Robert A. W. Dryfe.

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Velický, M., Bradley, D.F., Tam, K.Y. et al. In Situ Artificial Membrane Permeation Assay under Hydrodynamic Control: Permeability-pH Profiles of Warfarin and Verapamil. Pharm Res 27, 1644–1658 (2010). https://doi.org/10.1007/s11095-010-0150-6

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