Permeabilization of the Tight Junction: A Perspective for Tissue-Specific Drug Targeting

Epithelial barrier properties are determined by three components, namely transcellular permeability, the paracellular pathway, and locally limited peaks of permeability caused by apoptoses. Whereas transcellular permeability is strongly dependent on polarity or specific substrate recognition by membrane receptors and transport proteins, a promising target for specific permeabilization is the tight junction. Within this structure, tetraspan tight junction proteins have been shown to determine paracellular barrier properties, including occludin, tricellulin and the family of claudins. Especially members of the claudin family have been shown to be key determinants for epithelial barrier function in many different epithelia, including intestine, skin, and brain capillary endothelium. This review focuses on the strategy of specific tight junction perturbation in order to develop novel stategies for drug targeting. Tight junction proteins: molecular correlate of epithelial barrier function A number of tetraspan proteins have been identified within tight junction (TJ) strands, which are regarded as significant contributors to barrier function. Three types of proteins been identified by the group of Shoichiro Tsukita, namely occludin [1], the family of claudins [2], and most recently, tricellulin [3]. Whereas the function of occludin due to unchanged intestinal barrier properties in occludin-deficient mice is still discussed, numerous studies have demonstrated a primary role of claudins for the charge- and size selective barrier against ions and larger molecules in many organs and tissues, including intestine [4], kidney [5], blood brain barrier [6] and epidermis [7]. Within different organs and tissues, specific predominant expression of single members of the claudin family is responsible for barrier properties in accordance with local requirements for epithelial functions (Fig. 9.1 ). Many members of the claudin family functionally contribute to a tightening of the paracellular pathway, but some some members of the claudin family have been shown to form paracellular channels, or contribute to distinct transport functions of TJs. In intestine, for example, claudin-1, -3, -4, -5, and -8 belong to the tightening group, claudin-2 mediates paracellular permeability as a channel for small cations and – as has been shown recently [8] – also for water, and claudin-7, -12, and -15 have ambiguous functions. For specific permeabilization of the paracellular pathway, tightening claudins are regarded as a promising target, as a perturbation may lead to a temporary opening of the paracellular pathway. Fig. 9.1 Localization of claudins in intestine, blood brain barrier and nephron segments. Whereas leaky epithelia as e.g. small intestine and proximal tubule show a strong expression of channel-forming claudin-2 in combination with tightening TJ proteins, tight epithelia as colon and collecting duct show a more consistent expression of tightening claudins. The tight junction as a target for drug development A variety of substances have been considered for the formulation of medication in context with a specific and reversible opening of the epithelial barrier. These substances include Ca 2+ chelators, fatty acids, and other organic bioactive compounds. Some molecules have been shown to interact with TJ proteins directly, or with different cellular signalling cascades leading to a temporarily opening of TJs (examples listed in Table 9.1 ). Table 9.1 A selection of paracellular absorption enhancers: compounds and mechanisms. Compound Mechanism Reference decanoylcarnitine via Ca 2+ [11] EGTA/EDTA via Ca 2+ [14] sodium caprate via Ca 2+ [11] CPE claudin binding [9] ZOt via PKC [19] Mechanisms of action versus the epithelial barrier vary: Some compounds interact with single TJ proteins as e.g. demonstrated for Clostridium perfringens enterotoxin (CPE), binding to claudin-3, -4, -6, -7, -8, and -14 but not claudin-5 and -10 [9]. Further studies revealed a direct interaction of the C-terminus of Clostridium perfringens enterotoxin, leading to a degradation of epithelial cells and therefore to an opening of the barrier dependent on the expression of single claudins [10]. Another major mechanism of barrier perturbation is an action via protein kinases, leading to perijunctional actomyosin ring contraction which results in a fast and reversible opening of TJs, as shown for both EGTA and sodium caprate [11]. Ca 2+ chelators Study of absorption enhancers was initiated in 1961 when ethylenediaminetetraacetic acid (EDTA) was shown to increase absorption of heparin in rats and dogs [12]. Since then, the molecular structure and regulation of the target, namely the TJ, has been characterized in detail. Today, the effect of Ca 2+ chelators on barrier properties is attributed to a condensation of the perijunctional actomyosin ring, leading to an internalization of TJ proteins which are connected with the cytoskeleton via PDZ-binding protein ZO-1 and additional scaffolding proteins of the TJ complex [13]. Moreover, this specific effect can be used to determine an opening of the paracellular route in in vitro studies, as demonstrated for two path impedance spectroscopy [14]. Fatty acids Sodium caprate is a fatty acid licensed as a compound of an ampicillin suppository, and has been analyzed concerning functional effects on barrier properties [15], demonstrating a fast and reversible decrease of TJ proteins in intestinal cells. In human keratinocytes, claudin-1 and occludin reversibly dispersed into fragmented patterns on the cell–cell contact region and translocated as granular structures in the cytoplasm [16]. Therefore, sodium caprate is regarded as a potential promoter of drug delivery in different epithelia. One major challenge is the development of oral formulations, which comprises the co-release of absorption enhancer and substrate in sufficient concentrations at the small intestinal epithelium in order to rapidly and reversibly increase permeability. Whether the observed mechanism can be transferred to human duodenum in vivo, however, remains to be elucidated. At least, one advantage of the compound is its safety profile as an intact epithelial barrier is essential for physiological homeostasis and defense against extrinsic antigens [17]. However, sodium caprate compares well with other dietary constituents and endogenous secretions, and is significantly better than other agents, as e.g. aspirin or alcohol [18]. Perspective A variety of substances are currently discussed in context with a specific and reversible opening of the paracellular barrier. These substances include Ca 2+ chelators, fatty acids, and other organic bioactive compounds. Therefore, further studies are necessary to evaluate whether absorption enhancers provide a valuable tool for intestinal drug targeting, to show whether cell culture results can be transferred to human epithelium, and last but not least to rule out that an application represents an avoidable risk for the patient. References [1] Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S. Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol 1993; 123: 1777–1788 [2] Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 1998; 141: 1539–1550 [3] Ikenouchi J, Furuse M, Furuse K et al. Tricellulin constitutes a novel barrier at tricellular contacts of epithelial cells. J Cell Biol 2005; 171: 939–945 [4] Rahner C, Mitic LL, Anderson JM. Heterogeneity in expression and subcellular localization of claudins 2, 3, 4, and 5 in the rat liver, pancreas, and gut. Gastroenterology 2001; 120: 411–422 [5] Kiuchi-Saishin Y, Gotoh S, Furuse M, Takasuga A, Tano Y, Tsukita S. Differential expression patterns of claudins, tight junction membrane proteins, in mouse nephron segments. J Am Soc Nephrol 2002; 13: 875–886 [6] Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H, Hashimoto N, Furuse M, Tsukita S. Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice. J Cell Biol 2003; 161: 653–660 [7] Kirschner N, Houdek P, Fromm M, Moll I, Brandner JM. Tight junctions form a barrier in human epidermis. Eur J Cell Biol 2010; in press [8] Rosenthal R, Milatz S, Krug SM, Oelrich B, Schulzke JD, Amasheh S, Günzel D, Fromm M. Claudin-2, a component of the tight junction, forms a paracellular water channel. J Cell Sci 2010; 123: 1913–1921 [9] Fujita K, Katahira J, Horiguchi Y, Sonoda N, Furuse M, Tsukita S. Clostridium perfringens enterotoxin binds to the second extracellular loop of claudin-3, a tight junction integral membrane protein. FEBS Lett 2000; 476: 258–261 [10] Kondoh M, Takahashi A, Fujii M, Yagi K, Watanabe Y. A novel strategy for a drug delivery system using a claudin modulator. Biol Pharm Bull 2006; 29: 1783–1789 [11] Tomita M, Hayashi M, Awazu S. Absorption-enhancing mechanism of EDTA, caprate, and decanoylcarnitine in Caco-2 cells. J Pharm Sci 1996; 85: 608–611 [12] Windsor E, Cronheim GE. Gastro-intestinal absorption of heparin and synthetic heparinoids. Nature 1961; 190: 263–264 [13] Rothen-Rutishauser B, Riesen FK, Braun A, Günthert M, Wunderli-Allenspach H. Dynamics of tight and adherens junctions under EGTA treatment. J Membr Biol 2002; 188: 151–162 [14] Krug SM, Fromm M, Günzel D. Two-path impedance spectroscopy for measuring paracellular and transcellular epithelial resistance. Biophys J 2009; 97(8): 2202–2211 [15] Lindmark T, Kimura Y, Artursson P. Absorption enhancement through intracellular regulation of tight junction permeability by medium chain fatty acids in Caco-2 cells. J Pharmacol Exp Ther 1998; 284: 362–369 [16] Kurasawa M, Kuroda S, Kida N, Murata M, Oba A, Yamamoto T, Sasaki H. Regulation of tight junction permeability by sodium caprate in human keratinocytes and reconstructed epidermis. Biochem Biophys Res Commun 2009; 381: 171–175 [17] Bjarnason I. Intestinal permeability. Gut 1994; 35: S18–S22 [18] Wolfe MM, Lichtenstein DR, Singh G. Gastrointestinal toxicity of nonsteroidal antiinflammatory drugs. N Engl J Med 1999; 340: 1888–1899 [19] Fasano A, Uzzau S, Fiore C, Mararetten K. The enterotoxic effect of Zonula Occludens toxin on rabbit small intestine involves the paracellular pathway. Gastroentrology 1997; 112: 839–846 Correspondence Priv.-Doz. Dr. Salah Amasheh salah.amasheh@charite.de