Transport of a GABA_A Receptor Modulator and Its Derivatives from Valeriana officinalis L. s. l. Across an in Vitro Cell Culture Model of the Blood-Brain Barrier

Abstract

The roots and rhizome of Valeriana officinalis L. s. l. are therapeutically used for their sedative and sleep-enhancing effects. Some of the active compounds found in commonly used extracts are the sesquiterpenic acids, especially valerenic acid, which was recently identified as a GABA_A receptor modulator. To interact with this receptor in the brain, substances such as valerenic acid and its derivatives acetoxyvalerenic acid and hydroxyvalerenic acid have to cross the blood-brain barrier (BBB). The aim of our study was to obtain BBB permeability data of these compounds for the first time and to elucidate possible transport pathways across our BBB in vitro model. Transport of valerenic acid, acetoxyvalerenic acid and hydroxyvalerenic acid was compared with the permeability of the GABA_A modulator diazepam, which is known to penetrate into the central nervous system transcellularly by passive diffusion. Experiments were carried out with an established Transwell in vitro model based on the human cell line ECV304. Results indicated clearly that all three acids permeated significantly slower than diazepam. The ranking was confirmed in group studies as well as in single-substance studies after normalization to diazepam. Valerenic acid (1.06 ± 0.29 μm/min, factor 0.03 related to diazepam) was the slowest to permeate in the group study, followed by hydroxyvalerenic acid (2.72 ± 0.63 μm/min, factor 0.07 related to diazepam) and acetoxyvalerenic acid (3.54 ± 0.58 μm/min, factor 0.09 related to diazepam). To elucidate the contribution of the paracellular transport, studies were performed at different tightness status of the cell layers reflected by different transendothelial electrical resistance (TEER) values. Results showed an exponential correlation between transport and TEER for all three acids, whereas diazepam permeated TEER independently. In summary, it is hypothesized that the investigated compounds from Valeriana officinalis L. s. l. can probably only pass through the BBB by a still unknown transport system and not transcellularly by passive diffusion.

Abbreviations

ACM: astrocyte-conditioned medium

BBB: blood-brain barrier

BMEC: brain microvascular endothelial cells

DMP: 2,2-dimethoxypropane

ESEM: environmental scanning electron microscopy

GABA: γ-aminobutyric acid

SEM: scanning electron microscopy

TEER: transendothelial electrical resistance

TEM: transmission electron microscopy

Key words

Valeriana officinalis L. s. l. - Valerianaceae - blood-brain barrier - valerenic acid - ECV304 - GABA_A receptor

References

• 1 Houghton P J. The scientific basis for the reputed activity of Valerian.  J Pharm Pharmacol. 1999;  51 505-12
• 2 ESCOP monographs, 2nd edition. Stuttgart; Thieme 2003: 539-46
• 3 Donath F, Quispe S, Diefenbach K, Maurer A, Fietze I, Roots I. Critical evaluation of the effect of valerian extract on sleep structure and sleep quality.  Pharmacopsychiatry. 2000;  33 47-53
• 4 Khom S, Baburin I, Timin E, Hohaus A, Trauner G, Kopp B. et al . Valerenic acid potentiates and inhibits GABA_A receptors: molecular mechanism and subunit specificity.  Neuropharmacology. 2007;  53 178-87
• 5 Trauner G, Khom S, Baburin I, Benedek B, Hering S, Kopp B. Modulation of GABA_A receptors by valerian extracts is related to the content of valerenic acid.  Planta Med. 2008;  74 19-24
• 6 Anderson G D, Elmer G W, Kantor E D, Templeton I E, Vitiello M V. Pharmacokinetics of valerenic acid after administration of valerian in healthy subjects.  Phytother Res. 2005;  19 801-3
• 7 Joo F. Endothelial cells of the brain and other organ systems: some similarities and differences.  Prog Neurobiol. 1996;  48 255-73
• 8 Takahashi K, Sawasaki Y, Hata J J, Mukai K, Goto T. Spontaneous transformation and immortalization of human endothelial cells.  In Vitro Cell Dev Biol. 1990;  25 265-74
• 9 Tan K H, Dobbie M S, Felix R A, Barrand M A, Hurst R D. A comparison of the induction of immortalized endothelial cell impermeability by astrocytes.  Neuroreport. 2001;  12 1329-34
• 10 Scism J L, Laska D A, Horn J W, Gimple J L, Pratt S E, Shepard R L. et al . Evaluation of an in vitro coculture model for the blood-brain barrier: comparison of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources.  In Vitro Cell Dev Biol Anim. 1999;  35 580-92
• 11 Youdim K A, Dobbie M S, Kuhnle G, Proteggente A R, Abbott N J, Rice-Evans C. Interaction between flavonoids and the blood-brain barrier: in vitro studies.  J Neurochem. 2003;  85 180-92
• 12 Janzer R C, Raff M C. Astrocytes induce blood-brain barrier properties in endothelial cells.  Nature. 1987;  325 253-7
• 13 Kuchler-Bopp S, Delaunoy J P, Artault J C, Zaepfel M, Dietrich J B. Astrocytes induce several blood-brain barrier properties in non-neural endothelial cell.  Neuroreport. 1999;  10 1347-53
• 14 Garberg P, Ball M, Borg N, Cecchelli R, Fenart L, Hurst R D. et al . In vitro models for the blood-brain barrier.  Toxicol In Vitro. 2005;  19 299-334
• 15 Neuhaus W, Plattner V E, Wirth M, Germann B, Lachmann B, Gabor F. et al .Validation of in vitro cell culture models of the blood-brain barrier: Tightness characterization of two promising cell lines. J Pharm Sci, in press
• 16 Benda P, Lightbody J, Sato G, Levine L, Sweet W. Differential rat glial cell strain in tissue culture.  Science. 1968;  161 370-1
• 17 Neuhaus W, Bogner E, Wirth M, Trzeciak J, Lachmann B, Gabor F. et al . A novel tool to characterize paracellular transport: the APTS-dextran Ladder.  Pharm Res. 2006;  23 1491-501
• 18 Baguneid M, Murray D, Salacinsky H J, Fuller B, Hamilton G, Walker M. et al . Shear-stress preconditioning and tissue-engineering-based paradigms for generating arterial substitutes.  Biotechnol Appl Biochem. 2004;  39 151-7
• 19 Collins S P, Pope R K, Scheetz R W, Ray R I, Wagner P A, Little B J. Advantages of environmental scanning electron microscopy in studies of microorganisms.  Microsc Res Tech. 1993;  25 398-405
• 20 Dolman D EM, Anderson P, Rollison C, Abbott N J. Characterisation of a new in vitro model of the blood-brain barrier (BBB).  J Physiol. 1998;  505 56-7
• 21 Easton A S, Abbott N J. Bradykinin increases permeability by calcium and 5-lipoxygenase in ECV304/C6 cell culture model of the blood-brain barrier.  Brain Res. 2002;  953 157-69
• 22 Arendt R M, Greenblatt D J, Liebisch D C, Luu M D, Paul S M. Determinants of benzodiazepine brain uptake: lipophilicity versus binding affinity.  Psychopharmacology. 1987;  93 72-6
• 23 Dubey R K, McAllister C B, Inoue M, Wilkinson G R. Plasma binding and transport of diazepam across the blood-brain barrier. No evidence for in vivo enhanced dissociation.  J Clin Invest. 1989;  84 1155-9
• 24 Gaillard P J, de Boer A G. Relationship between permeability status of the blood-brain barrier and in vitro permeability coefficient of a drug.  Eur J Pharm Sci. 2000;  12 95-102