Research Articles
Comparison of blood–brain barrier permeability assays: in situ brain perfusion, MDR1-MDCKII and PAMPA-BBB

https://doi.org/10.1002/jps.21580Get rights and content

Abstract

Permeability data from MDR1-MDCKII and PAMPA-BBB assays were compared to data from in situ brain perfusion to evaluate the accuracy of in vitro assays in predicting in vivo blood–brain barrier (BBB) permeability. PAMPA-BBB significantly correlated to in situ brain perfusion, however, MDR1-MDCKII had no correlation with in situ brain perfusion. PAMPA-BBB also significantly correlated with MDR1-MDCKII. The differential correlation of PAMPA-BBB and MDR1-MDCKII to in situ brain perfusion appears to be mainly due to the difference in membrane characteristics rather than binding to brain tissue. The MDR1-MDCKII cell membrane has lower ratios of: phospholipid to cholesterol, unsaturated to saturated acyl chains, and phosphatidyl-choline (PC) to sphingomyelin (SM) than brain endothelial cells, making it a poor passive permeability model for BBB. The BBB is more hydrophobic, rigid, and less fluidic than MDR1-MDCKII cell membrane. PAMPA-BBB more closely matches the BBB membrane in these characteristics and is a more accurate passive diffusion permeability model for BBB than MDR1-MDCKII. PAMPA-BBB is high throughput, low cost and has good prediction of in vivo BBB permeability, and therefore, it is a valuable tool in drug discovery to screen compounds for the rate of brain penetration. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:1980–1991, 2009

Section snippets

Abbreviations:

    ACS

    American Chemical Society

    AUC

    area under the curve

    BBB

    blood–brain barrier

    BBMEC

    bovine brain microvessel endothelial cells

    BMEC

    brain microvessel endothelial cells

    B/P

    brain AUC (or concentration)/plasma AUC (or concentration)

    Caco-2

    human colon carcinoma cell line

    Cmax

    maximum concentration

    CNS

    central nerve system

    MDCK

    Madin-Darby canine kidney cells

    MDR

    multi-drug resistance

    PAMPA

    parallel artificial membrane permeability assay

    P

    BBB permeability for in situ brain perfusion (×10−3 cm/s)

    Papp

    apparent permeability

INTRODUCTION

Determination of brain penetration is of great importance not only for CNS drug candidates, but also for non-CNS therapeutic areas where brain penetration can cause unwanted side effects.1., 2., 3. BBB research is very challenging and active, and new findings continue to be discovered. For example, inclusion of the term “BBB” in the ACS publications has increased 100% from 2000 to 2005.3 Concerted international strategies among academia, government, and industry have been developed to address

Materials

Thirty-seven commercial drugs were obtained from Sigma (St. Louis, MO), ChemPacific (Baltimore, MD), Toronto Research Chemicals (North York, ON, Canada), Wyeth Research (Princeton, NJ). The porcine polar brain lipid (PBL) (catalog no. 141101) was from Avanti Polar Lipids, Inc. (Alabaster, AL). Universal buffer was obtained from pION Inc. (Woburn, MA). DMSO was reagent grade from Aldrich. Dodecane was from EM Science (Gibbstown, NJ). The acceptor plate was a 96-well filter plate (Multiscreen™,

RESULTS AND DISCUSSION

All the compounds listed in Ref.39 were used in the study, except for those that had weak UV absorbance, were impure or were not available at the time of the experiment. The test compounds were CNS drugs with a wide range of physico-chemical properties. This is the first large set of data published on the rate of brain penetration on marketed drugs.39 Permeability values of the 37 compounds were compared using data from three different assays: physicochemically based PAMPA-BBB, cell-based

CONCLUSIONS

The rate of brain penetration is an essential property of CNS drug candidates. In situ brain perfusion is a gold standard method for measuring BBB permeability, however, high test compound concentration in serum-free perfusion fluid can saturate Pgp efflux transport and make passive diffusion the dominant pathway. The cell-based MDR1-MDCKII assay has been widely used in the pharmaceutical industry to predict Pgp efflux transport and it is highly effective. Even though MDR1-MDCKII is an

Acknowledgements

The authors would like to thank Dr. Magid Abou-Gharbia for his support, encouragement and leadership, thank people in Pharmaceutical Profiling for their contribution and Konstantin L. Tsinman at pION Inc. for useful discussions.

REFERENCES (67)

  • T. Terasaki et al.

    New approaches to in vitro models of blood-brain barrier drug transport

    Drug Discov Today

    (2003)
  • L. Di et al.

    Profiling drug-like properties in discovery research

    Curr Opin Chem Biol

    (2003)
  • J.D. Irvine et al.

    MDCK (Madin-Darby canine kidney) cells: A tool for membrane permeability screening

    J Pharm Sci

    (1999)
  • Q. Wang et al.

    Evaluation of the MDR-MDCK cell line as a permeability screen for the blood-brain barrier

    Int J Pharm

    (2005)
  • S. Carrara et al.

    Evaluation of in vitro brain penetration: Optimized PAMPA and MDCKII-MDR1 assay comparison

    Int J Pharm

    (2007)
  • K. Sugano et al.

    Prediction of human intestinal permeability using artificial membrane permeability

    Int J Pharm

    (2003)
  • E.H. Kerns et al.

    Combined application of parallel artificial membrane permeability assay and Caco-2 permeability assays in drug discovery

    J Pharm Sci

    (2004)
  • C. Zhu et al.

    A comparative study of artificial membrane permeability assay for high throughput profiling of drug absorption potential

    Eur J Med Chem

    (2002)
  • J.A. Ruell et al.

    PAMPA-a drug absorption in vitro model 5. Unstirred water layer in iso-pH mapping assays and pKaflux-optimized design (pOD-PAMPA)

    Eur J Pharm Sci

    (2003)
  • K.A. Youdim et al.

    Flavonoid permeability across an in situ model of the blood-brain barrier

    Free Rad Biol Med

    (2004)
  • J.-.L. Delaunay et al.

    Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100

    Biochim Biophys Acta (BBA)—Biomembranes

    (2008)
  • M. Shinitzky et al.

    Dynamics of the hydrocarbon layer in liposomes of lecithin and sphingomyelin containing dicetylphosphate

    J Biol Chem

    (1974)
  • J.M. Bourre et al.

    Possible role of the choroid plexus in the supply of brain tissue with polyunsaturated fatty acids

    Neurosci Lett

    (1997)
  • K.L. Audus et al.

    Evidence for 21-aminosteroid association with the hydrophobic domains of brain microvessel endothelial cells

    Free Rad Biol Med

    (1991)
  • C. Benistant et al.

    Fatty acid composition of brain capillary endothelial cells: Effect of the coculture with astrocytes

    J Lipid Res

    (1995)
  • A.A. Spector et al.

    Membrane lipid composition and cellular function

    J Lipid Res

    (1985)
  • V. Pata et al.

    Effect of membrane characteristics on phase separation and domain formation in cholesterol-lipid mixtures

    Biophys J

    (2005)
  • A. Avdeef et al.

    Parallel artificial membrane permeability assay (PAMPA)-critical factors for better predictions of absorption

    J Pharm Sci

    (2007)
  • L. Di et al.

    Strategies to assess blood–brain barrier penetration

    Expert Opinion on Drug Discovery

    (2008)
  • E.H. Kerns et al.

    Drug-like properties: Concepts, structure design and methods: From ADME to toxicity optimization

    (2008)
  • S.A. Hitchcock et al.

    Structure-brain exposure relationships

    J Med Chem

    (2006)
  • X. Liu et al.

    Strategies to optimize brain penetration in drug discovery

    Curr Opin Drug Discov Dev

    (2005)
  • M. Hammarlund-Udenaes et al.

    On the rate and extent of drug delivery to the brain

    Pharm Res

    (2008)
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