enhancing paracellular permeability by modulating epithelial tight junctions

1461-5347/00/$ – see front matter ©2000 Elsevier Science Ltd. All rights reserved. PII: S1461-5347(00)00302-3

▼ Epithelial cellular sheets act as barriers to sep-arate the body from the external environment aswell as to maintain distinct compartments withinthe body.This is achieved by the presence of thecell membrane and membrane-associated trans-porters, intracellular enzymes and junctionalcomplexes. Even the movement of ions across the epithelium is restricted, giving rise to electricalpotential gradients across the epithelia.

Molecules cross the intestinal epithelium intothe blood by three main pathways (Fig. 1): (1)passive diffusion across the cell membranes(transcellular pathway); (2) passive diffusion be-tween adjacent cells (paracellular pathway); and(3) carrier-mediated transport (carrier-mediatedtranscellular pathway). Lipophilic molecules easilycross the cell membrane by transcellular dif-fusion. By contrast, hydrophilic molecules thatare not recognized by a carrier cannot partitioninto the hydrophobic membrane and thus trav-erse the epithelial barrier via the paracellularpathway. The transport of hydrophilic moleculesvia the paracellular pathway is, however, severelyrestricted by the presence of the tight junctions1.

This barrier function of the tight junction isdynamic and appears to be modulated by cellularprocesses that regulate the movement of hydro-philic molecules across the epithelium. For example, many hydrophilic nutrients, such as glucose, have been hypothesized to cross the epi-thelium at least in part through the paracellularpathway2. The intestinal epithelial barrier there-fore serves a dual role: to keep potentially harmfulexternal agents out of the body and to allow beneficial nutrients, ions and water into the body.

The therapeutic problemMany useful therapeutic agents are hydrophilicand thus cannot be delivered via the oral route,which is the most favored route of drug delivery.For example, the hydrophilic broad-spectrumantibiotic cefoxitin has an oral bioavailability of,5% in animals owing to poor intestinal perme-ability3 and is currently only marketed as an intravenous formulation. Enalaprilat, an angio-tensin-converting enzyme inhibitor, is alsopoorly absorbed and marketed as an intravenousformulation. Enalaprilat was chemically modifiedto produce a more lipophilic prodrug, enalapril,which is well absorbed4 and converted to the active enalaprilat, thereby achieving therapeuticconcentrations of the active hydrophilic agentafter oral administration.

The enalapril modification illustrates one wayto increase the absorption of hydrophilic drugsacross the intestinal epithelium: convert theminto a more lipophilic prodrug, thus increasingtranscellular flux of the drug. Another approachinvolves redesigning the drug so that it is a sub-strate for a carrier (or linked to a substrate for acarrier). Some of the cephalosporin antibioticsrepresent such a class of drugs. Several of theirstructural features make them a substrate for the dipeptide transporter PepT1 (Ref. 5), and the carrier-mediated transport of these drugs

Enhancing paracellular permeability bymodulating epithelial tight junctionsPeter D. Ward, Tim K. Tippin and Dhiren R. Thakker

Peter D. WardDepartment of Pharmacology

School of MedicineUniversity of North Carolina at

Chapel HillChapel Hill

NC 27599, USATim K. Tippin and Dhiren R.

Thakker*Division of Drug Delivery and

DispositionSchool of Pharmacy

University of North Carolina atChapel HillChapel Hill

NC 27599, USATim K. Tippin

Division of Bioanalysis andDrug MetabolismGlaxo Wellcome

Research Triangle ParkNC 27709, USA

*tel: 11 919 962 0092fax: 11 919 966 0197

e-mail:[email protected]

reviews research focus

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PSTT Vol. 3, No. 10 October 2000

The intestinal epithelium is a major barrier to the absorption of

hydrophilic drugs. The presence of intercellular junctional complexes,

particularly the tight junctions (zona occludens), renders the epithelium

impervious to hydrophilic drugs, which cannot diffuse across the cells

through the lipid bilayer of the cell membranes. There have been

significant advances in understanding the structure and cellular

regulation of tight junctions over the past decade. This article reviews

current knowledge regarding the physiological regulation of tight

junctions and paracellular permeability, and recent progress towards

the rational design of agents that can effectively and safely increase

paracellular permeability via modulation of tight junctions.

contributes to an increase in their flux across the intestinal epi-thelium. One member of this class, cephadroxil, is reported tohave nearly 100% oral bioavailability6.

The controlled and reversible opening of the tight junctionrepresents a third way to increase the absorption of hydrophilicdrugs across the intestinal epithelium.This approach is attrac-tive because it could be applied to many different hydrophilicdrugs. In addition, it would avoid degradation of the activeagent by intracellular enzymes, another barrier for drug absorp-tion, especially peptide- and protein-related therapeutic agents.In recent years, the identification of compounds that selectivelyopen the tight junction [paracellular permeability enhancers(PPEs)] has been actively pursued in the field of pharmaceutics.Co-administering these compounds with hydrophilic drugsimproves the bioavailability of these drugs in vivo.

However, PPEs also cause toxicity in vivo, suggesting that theimproved bioavailability is the result of nonspecific mecha-nisms. For example, palmitoyl carnitine was found to increasethe intestinal bioavailability of cefoxitin from ,5% to as muchas 70% in rats3; however, this effect was associated with re-versible mucosal damage7. In the past, a lack of knowledgeabout the physiological mechanism(s) that regulates the struc-ture and function of the tight junctions as well as paracellularpermeability has prevented the development of safe and effectivePPEs that can be used pharmaceutically.

Exciting research in the past decade from many groups hasunfolded many different proteins and biochemical signalingpathways that play an important role in the physiological regu-lation of the cellular functions of the epithelial tight junctions8.Furthermore, the development of in vitro models of the intesti-nal epithelium, such as Caco-2 cell monolayers9,10, have pro-vided excellent tools to identify and design safe, effective PPEsthat work by specific cellular mechanisms.

Studies with these models have shown that some PPEs,within a limited concentration range, could selectively increaseparacellular permeability or disrupt tight junction functionwithout compromising the integrity of the cell monolayers.For example, non-toxic concentrations of palmitoyl carnitinecan increase paracellular permeability across Caco-2 mono-layers11. This article will review current knowledge regardingthe physiological regulation of tight junctions and paracellularpermeability, and recent progress towards the rational designof agents that can effectively and safely increase paracellularpermeability by modulating tight junctions.

The Caco-2 cell modelCaco-2 cell monolayers are the most widely used in vitro cellculture model for intestinal epithelium9,10.This model has alsobeen used extensively to investigate the efficacy and mode ofaction of the PPEs12,13. Caco-2 cells originate from human

colon adenocarcinoma cells and, when cultured for threeweeks, differentiate into polarized cells with distinct mucosal(apical) and serosal (basolateral) cell membrane domains.Although they originate from colon cells, Caco-2 cells havemany of the properties of small intestinal absorptive cells,including microvilli, intercellular junctions and many of theenzymes, nutrient transporters and efflux transporters that arepresent in the small intestinal absorptive cells. A recent reviewprovides an excellent description of the morphological, bio-chemical and drug permeability properties of the Caco-2 cellmodel14.

For drug transport studies, Caco-2 cells can be grown onporous filters in multi-well plastic plates or on filters that areplaced in side-by-side Ussing-type diffusion chambers. In bothsystems, the transport of solutes can be measured in the absorptive direction (mucosal to serosal) or in the secretorydirection (serosal to mucosal). The solutions in the apical orbasolateral compartment can be changed independently with-out significantly affecting the exposure of the cells in the othercompartment. For example, the absence of Ca21 in the apicalchamber is sometimes desired to improve the solubility of apharmaceutical agent15, but the absence of Ca21 in the basolat-eral chamber results in a dramatic decrease in the tightness ofthe intercellular junctional complex16.

The tightness of the intercellular junctional complex can becharacterized in a leaky to moderately leaky epithelium bymeasuring the transepithelial electrical resistance (TEER) thatexists across the cell monolayers because of the restriction ofmovement of ions imposed by the tight junctions. It was

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PSTT Vol. 3, No. 10 October 2000 reviews research focus

Figure 1. Transport across intestinal epithelium. Molecules cross theintestinal epithelium into the blood primarily by three pathways:passive diffusion across the cell membranes (transcellular pathway);passive diffusion between adjacent cells (paracellular pathway); andcarrier-mediated transport (carrier-mediated pathway).

Pharmaceutical Science & Technology Today

Paracellular

Tight junction

Apical Transcellular

Carrier-mediated

Passivediffusion

Basolateral

shown conclusively in the 1970s that the permeability of ionsthrough a simple epithelium (gall bladder from Necturus) wasprimarily paracellular, through the tight junctions and the lateral space between the cells, rather than across the cell mem-brane itself17. A leaky epithelium has a low transepithelial elec-trical resistance (TEER) of 10–50 V cm2 (high permeability),whereas a tight epithelium has a high TEER (low permeability)of .1000 V cm2. TEER values for a variety of epithelial tissues and cell lines that serve as in vitro models for the epithelial tissues are listed in Table 1.

Clearly, a wide range of TEER values has been reported, re-flecting very significant differences in the ‘leakiness’ of differ-ent epithelial tissues. An important question that needs to beanswered is whether or not these differences in TEER reflect adifference in tight-junction structure or regulation. In vivo, theintestinal epithelium is composed of intercellular junctionalcomplexes that vary in tightness along the length of the

gastrointestinal tract – from leaky epithelium in the jejunum,with its primary function of rapidly absorbing nutrients, to atighter epithelium in the colon, with its primary function ofabsorbing water and ions but at the same time preventing theentry of bacterial toxins.

Evaluating PPEs with Caco-2 cell monolayersIn the presence of PPEs, changes to the tight junction are moni-tored by measuring the TEER. If the tight junction opens in thepresence of a PPE, this reduces the TEER by increasing the ionflow through the tight junctions. By taking this together withan increase in the flux of a hydrophilic molecule, such as man-nitol, the efficacy of a PPE can be characterized. As the paracel-lular permeability of solutes depends on their molecular radii,paracellular markers of different molecular sizes can be used toassess the efficacy of PPEs in opening the restricted paracellularspace for the enhanced transport of hydrophilic molecules.

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PSTT Vol. 3, No. 10 October 2000reviews research focus

Table 1. Transepithelial electrical resistance of various epithelial tissues and cell lines

Adapted from Refs 17,18,89,90.

aNecturus is a large American salamander.

Abbreviation: TEER, transepithelial electrical resistance.

Classification Epithelial tissue TEER (V cm2)

Very leaky Proximal renal tubule 6–7(rat, rabbit, dog)

Leaky Gallbladder 20–30(rabbit, goose, trout)

Gallbladder 110(fish)

Proximal convoluted renal tubule 70–80(aNecturus)

Small intestine, duodenum 100(rat)

Small intestine, jejunum 30–70(rat)

Small intestine, ileum 90–100(rat, rabbit)

Choroid plexus 80(frog)

IEC-18 55(rat ileal cell line)

2/4/A1 25(rat fetal intestinal cell line)

LLC-PK1 170(pig, kidney cell line)

Classification Epithelial tissue TEER (V cm2)

Intermediate to tight Gallbladder 300(Necturus)

Colon 290–500(rabbit, turtle)

Caco-2 230–1000(human, colon cell line)

MDCK II(dog, kidney cell line) 150–350

Stomach 500(frog)

Very tight Gastric mucosa 1700–2200(Necturus)

MDCK I 2000–10000(dog, kidney cell line)

Urinary bladder 800–4500(frog, toad)

Urinary bladder 5000–10000(rabbit)

Skin 2000–8700(frog)

Like any simplified in vitro model, Caco-2 cells do not pos-sess all of the properties of the intestinal epithelium in vivo.Understanding these differences, especially with regard totight junctions, is critical to extrapolating the results obtainedwith PPEs in Caco-2 monolayers to in vivo systems. Caco-2monolayers resemble colonic epithelia in their relative tight-ness. Other in vitro models, such as IEC-18 cells or 2/4/A1cells, better approximate the tightness of the jejunum18. Theheterogeneity of cell types in the small intestine contribute tothe ‘leakier’ intercellular junctions in vivo. Although Caco-2 cellmonolayers are homogeneous, composed of fully differenti-ated absorptive cells, the intestinal epithelium contains celltypes that differ in their maturity (increasing from crypt to villus) and function.

It has been shown that the tight junctions between absorptiveand mucus-secreting cell types are leakier than those betweencells of the same type19.The secretions and endocytic nature ofthese other cell types might also influence the permeability ofmolecules in vivo. For example, goblet cells, which secrete a pro-tective layer of mucus on top of the epithelium, are not presentin Caco-2 cell monolayers.The presence of mucus is a barrier tothe passive diffusion of lipophilic compounds like testosterone20

and appears to be the reason for the reduced in vivo potency ofcertain surfactant enhancers21.The lack of mucus in the Caco-2cell model could lead to an overestimate of the PPE efficacy rela-tive to the in vivo efficacy of these agents. Caco-2 monolayers alsolack M cells, which are highly endocytic and present antigens toimmune cells. Some investigators have created mixed mono-layers of Caco-2 and goblet cells22 or added lymphocytes toCaco-2 monolayers, which effectively converts the homogenousCaco-2 cell monolayers to ones containing mixed populationsof enterocytic cells and M cells23, in order to approximate moreclosely the environment of the intestinal tract. Other con-founding factors also affect our ability to assess the in vivo effi-cacy of the PPEs based on in vitro studies with Caco-2 cellmonolayers (or other cell culture models). For example, theconcentrations of the PPE and co-administered hydrophilicdrug are continually changing in vivo because they are dilutedby intestinal fluids. In addition, the PPE and therapeutic agentare exposed in the gastrointestinal tract to environments withdifferent pH, mucosal enzymes and bacterial microflora.

This highly dynamic setting in vivo is clearly more complexthan the controlled, relatively static setting of the in vitrocell monolayer system. Thus, an effective PPE in Caco-2 cell monolayers might, under some circumstances, not be an effective PPE in vivo. However, the ease of manipulation and measurement of PPE modulation in Caco-2 cell mono-layers is an invaluable tool for understanding the structure–activity relationships of many PPEs and their mechanism of action.

The tight junctionA century ago, the term ‘tight junction’ was given to the epi-thelial intercellular junctional complex because it appeared as afused region between cells. Based on research over the past40 years, the concept of the tight junction as an impermeablebarrier, analogous to cement, has undergone dramatic change.Although the rapidly evolving understanding about the archi-tecture and function of the tight junction is far from complete,it is clear that the tight junction is a dynamic and complexmultiprotein structure (Fig. 2) that is selectively permeable tocertain hydrophilic molecules (ions, nutrients and drugs).

In recent years, the morphology and regulation of the tightjunction have been studied in great detail.The current analogyfor the tight junction is a gate, because the tight junction allows the passage of small hydrophilic compounds but acts asa barrier to larger hydrophilic ones. The tight junction is also referred to as a fence because it forms an intramembrane dif-fusion barrier that restricts the intermixing of apical and baso-lateral membrane components24,25.This ‘fence’ function of thetight junction maintains the polarity of enterocytes and is notdirectly related to its ‘gate’ function26.Thus, the function of thetight junction is complex, and this complexity is also reflectedin its multiprotein architecture, which spans extracellular,transmembrane and intracellular domains of the cells.

The tight junction is composed of a group of transmem-brane and cytosolic proteins that interact not only with eachother but also with the membrane and the cytoskeleton8

(Fig. 2). Occludin was first identified in the tight junctions ofchicken liver27 and various mammalian species28,29. This pro-tein is an integral membrane protein that is predicted to con-tain four transmembrane domains. Occludin plays a dual role:to provide structural integrity to the tight junction and to reg-ulate the barrier function of the tight junction.The N terminusand extracellular domains of occludin provide the structuralintegrity necessary for the barrier function of the tight junc-tion. Thus, a construct of occludin that lacked the N terminusand extracellular domains was found to impair the barrierfunction of the tight junctions30. The presence of occludin is,however, not necessary for the formation of the tight junction,because disruption of the occludin gene in embryonic stemcells did not prevent the formation of functional strands31.TheC-terminal cytoplasmic tail of occludin confers a regulatoryrole by binding to the tight-junction-associated proteins thatinteract with the cytoskeleton32 and by directly binding to thecytoskeleton33. Interactions of the tight junction with the cyto-skeleton and the phosphorylation status of occludin might playan important role in the regulation of tight junction function.

Recently, a new class of proteins, claudins, have been suggested to have an important role in the structure and func-tion of tight junctions. Claudins, like occludin, contain four

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putative transmembrane domains. To date, 15 claudin-like proteins have been identified34–37. Expression of claudins 1 and2 in fibroblasts (which lack constitutive tight junctions) inducedtight-junction strand formation38, suggesting that claudins arethe major structural components of tight junction strands(Fig. 2).

Three cytosolic proteins, referred to as tight junction-associated proteins (TJAPs), localize occludin in the tight junction fibrils and couple the tight junction to the scaffold ofthe cytoskeleton (Fig. 2). ZO-1 was the first TJAP to be identi-fied39. Its N-terminal half interacts with the C-terminal tail ofoccludin40,41, and its C-terminal half interacts with F-actin ofthe cytoskeleton41. ZO-2, a 160 kDa protein42, has also beenfound to interact with the C-terminal half of occludin42 andthe N-terminal half of ZO-1 (Ref. 41).The expression of ZO-2is restricted exclusively to the tight junctions in epithelial cells,whereas ZO-1 is also found at some types of adherens junc-tions43. ZO-3, a 130 kDa protein, interacts with ZO-1 and occludin, but not with ZO-2 (Ref. 44). Recently, independent

ZO-1–ZO-2 and ZO-1–ZO-3 complexeshave been found in vivo33 (Fig. 2) andtherefore the TJAPs might not form a single trimeric grouping (e.g. a ZO-1–ZO-2–ZO-3 complex).

Physiological regulationThe physiological regulation of thetightness of tight junctions of variousepithelial tissues is consistent with theirphysiological role. For example, thetightness of the intestinal epithelial tightjunction is decreased after a meal, pre-sumably in order to allow hydrophilicnutrients like glucose and amino acids topass through the paracellular spaces. Ithas been proposed that this convective,paracellular transport, termed ‘solventdrag’, represents a significant proportionof the total nutrient absorption relativeto the carrier-mediated pathways45, butthis proposal remains controversial46.Studies in support of this hypothesishave shown that activation of thesodium-dependent glucose transporter 1corresponds with an increase in para-cellular permeability across Caco-2monolayers that are transfected with thistransporter47.

The concept of physiological regu-lation of the tight junctions is further

substantiated by reports that several endogenous agents can in-crease paracellular permeability. For example, hormones andneurotransmitters such as vasopressin, angiotensin II and epi-nephrine have been found to increase paracellular permeabilityin hepatocytes48. Cytokines, such as tumor necrosis factor a,have also been found to increase epithelial permeability bymodulating the tight junctions49. Although some of these factors have a response time that is too long to be consideredas useful PPEs, it is clear that the tight junctions are modulatedby endogenous factors.

Cellular and molecular regulationOccludin, ZO-1, ZO-2 and ZO-3 might serve not only to linkthe tight junction from the membrane to the cytoskeleton butalso to regulate tight-junction function. Many classic secondmessengers and protein kinases of signaling pathways influ-ence both the assembly and the barrier properties of tightjunctions. These include tyrosine kinases, Ca21 and protein kinase C (PKC)8,50–52. The final effect of modulating many of

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Figure 2. Schematic representation of interactions among tight junction proteins. The tight junctionis composed of a group of transmembrane and cytosolic proteins that interact not only with eachother but also with the membrane and cytoskeleton. The C-terminal cytoplasmic tail of occludinconfers a regulatory role by binding to tight junction-associated proteins (TJAP) that interact withthe cytoskeleton. Occludin might also bind to the cytoskeleton directly. The N-terminus of ZO-1interacts with the C-terminal tail of occludin, whereas the C-terminus of ZO-1 interacts with F-actinof the cytoskeleton. ZO-2 interacts with the C-terminus of occludin and the N-terminus of ZO-1. ZO-3 interacts with ZO-1 and occludin but not with ZO-2. Claudins serve a structural role in thetight junction. The photograph (from Ref. 91) is an electron micrograph of the intercellularjunctional complex between two Caco-2 cells. The tight junction is circled. Abbreviations: D, desmosome; LS, lateral space; mv, microvilli; ZA, zonula adherens (adherensjunction); ZO, zonula occludens (tight junction).


ZO-2

C-terminus ofoccludin

Actin filamentsof cytoskeleton

Occludin

Cell membrane

Actin filamentsof cytoskeleton

Paracellularspace

Cell 1 Cell 2

ClaudinClaudinmV

ZOZAD

LS

ZO-3 ZO-1

N

C

ZO-1

N

C

Occludin

these signaling pathways is the phos-phorylation of the tight junction pro-teins or the displacement (i.e. contractionor relaxation) of the perijunctionalactin–myosin ring (Fig. 3).

Phosphorylation of the tight junctioncomponents is an important step in theregulation of tight junction function.Theregulatory proteins of the tight junction(ZO-1, ZO-2, ZO-3 and occludin) arephosphoproteins53–55. The phosphoryl-ation of occludin correlates with itslocalization and function at the tightjunction56. Thus, the phosphorylation ofoccludin might be involved in tightjunction formation54. Disruption of thetight junction integrity by ATP depletioninduced a decrease in phosphorylationof all the tight junction regulatory pro-teins, followed by an increase inphosphorylation during ATP repletion55.Furthermore, the tyrosine kinase in-hibitor, genistein, markedly inhibited theformation of the tight junctions duringATP repletion55. In Madin–Darby caninekidney (MDCK) cells, increased tyrosinephosphorylation of ZO-1 and ZO-2 cor-relates with a decreased TEER induced bytreatment with a tyrosine phosphatase inhibitor57. Furthermore, displacementof ZO-1 accompanies the increased para-cellular permeability induced by thistreatment50.

Different phosphorylation states of the tight junction pro-teins might explain the differences in permeability between in vitro models (Table 1). Two strains of MDCK cells that differmarkedly (by a factor of 30) in TEER have similar numbers oftight junction strands and content of ZO-1 (Ref. 58).The levelof phosphorylation of ZO-1 in the low resistance strain is,however, approximately twice that of the high resistance strain59.Interestingly, one domain of ZO-1 binds to an uncharacterizedserine protein kinase60.

The same signaling pathways that induce the phosphoryl-ation of the tight junction proteins might also modulate theactin cytoskeleton8. The actin cytoskeleton associates with theplasma membrane, specifically through a network of actin fila-ments underneath the tight junction and through a ring ofactin filaments at the level of the adherens junction61. In guineapig ileum, disruption of the actin cytoskeleton by drugs such as cytochalasin D increased sodium and mannitol flux62,

suggesting the importance of the integrity of the cytoskeletonto the function of junctional complexes.

Disruption of the actin cytoskeleton is not the only mecha-nism that increases paracellular permeability through modu-lation of actin structure. The ring of actin filaments, which is at the level of the adherens junction, contains myosin II.Phosphorylation of myosin light chain (MLC) induces theactin–myosin ring to contract. Contraction of this actin–myosinring has been associated with a loosening of the tight junction.For example, increased paracellular permeability induced byvasopressin has been correlated with an increase in intracellularCa21 and in MLC phosphorylation63.

Modulation of the tight junction by PPEsGeneralA variety of exogenous compounds have been identified thatincrease paracellular permeability (Table 2), including Ca21

351


Figure 3. The role of the phospholipase-C (PLC)-dependent signaling pathway in modulating thestructure and function of the tight junction. The PLC-dependent signaling pathway is initiated by thecleavage of phosphatidyl-inositol (4,5)-bisphosphate (PIP2) into two products: inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 is released into the cytosol and induces Ca21 releasefrom the endoplasmic reticulum92,93. Increased intracellular levels of DAG and Ca21 or DAG aloneactivate the conventional or novel protein kinase C (PKC) isoenzymes, respectively94–96. Furthermore,increased intracellular Ca21 concentrations activate calmodulin-dependent kinase. PKC andcalmodulin-dependent kinase phosphorylate and alter the myosin light chain kinase activity63,68.Myosin light chain kinase phosphorylates the myosin light chain and induces a contraction in theperijunctional actin–myosin ring63,68. This ring is connected to the cell membrane61 and itsdisplacement has been postulated to alter tight junction function63,68.


Phospholipase C

IP3

DAGPIP2Protein

kinase C

Ca2+

Calmodulin-dependent kinase

Myosin lightchain kinase

Perijunctionalactin-myosin ring

ZO-1ZO-1

ZO-3 TightjunctionEndoplasmic

reticulum

IP3-sensitiveCa2+ channel

Cell 2

Occludin/claudins

Apical membrane

Cell 1Paracellular

space

ZO-2

ZO-1 ZO-1

ZO-2

Myosin II

ZO-3

chelators, surfactants and cationic polymers.The concentrationsat which PPEs increase the permeability of selective paracellularmarkers in Caco-2 cells cover several orders of magnitude(Table 2). Many different mechanisms of action have been proposed and investigated to some extent for PPEs; however,the mechanism of action of most PPEs is not fully understood.

Most PPEs have little separation between their cytotoxicityand ability to increase paracellular permeability (efficacy).Thecytotoxicity of PPEs is determined by several methods, includ-ing inhibition of mitochondrial dehydrogenase activity assay[oxidation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-zolium bromide (MTT) results in a blue formazan product],

352


Table 2. Paracellular permeability enhancers in Caco-2 cell monolayers

Class Specific example Effective concentration Tentative mechanism Refs

Ca21 1 mM increased FITC-Dextran4 Extracellular chelation of Ca21, 75chelators flux 6-fold disrupt cell-to-cell contact

(cadherin) and associated intracellular events

Ethylene diamine tetraacetate sodium

Bile salts 20 mM; (CMC=5.6); Membrane perturbation, alteration 21increased mannitol flux 5-fold of intracellular events: incr Ca21,

actin disbandment

Sodium taurocholate

Anionic 0.4 mM; (CMC=4.1 mM); Membrane perturbation, incr Ca21, 72surfactants increased mannitol flux 4-fold ATP depletion

Sodium dodecyl sulfate

Medium-chain 10 mM; (CMC=13 mM); Alteration of several intracellular 67,97,98fatty acids increased mannitol flux 8-fold events: incr Ca21, dec ATP, PLC

mediationSodium caprate

Fatty-acid 0.2 mM; (CMC=0.075 mM); Membrane perturbation, ATP 11,99esters increased mannitol flux 10-fold depletion; no effect on Ca21 or

actin

Palmitoyl carnitine

Phosphate 0.75 mM; (CMC=1 mM); Modulation of tight junctions 12esters via PLC inhibition increased mannitol

flux 10-fold

Dodecylphosphocholine (DPC)

Cationic 0.3 mM (170 kDa, degree of Mucus adhesion; increased apical 77,78polymers acetylation 35%); increased membrane permeability; actin

mannitol flux 8-fold at pH 5.5 depolymerization; change in ZO-1 localization

Chitosan

Abbreviations: incr, increase; dec, decrease; CMC, critical micelle concentration; FITC, fluorescein isothiocynate.

OO

OO

OO

NN O2Na1

2

Na1

Na1

2O2Na1

S O

ON OHOH

OHO

O

2

Na1

O

O S

O2Na1

O

O

O2Na1

N

OO

O

O

21

OON P

O

O2

1

*HO

OO

O

On

N

NH31

HO

increased membrane permeability (increased dye penetrationinto the cell) to propidium iodide and trypan blue, increasedcytosolic enzyme release (e.g. lactate dehydrogenase) and his-tological examination (increased cell denudation or membranedamage). For example, it has been shown that the separationbetween the cytotoxicity and efficacy is slight (a difference ofless than a factor of two) for PPEs such as dodecylphospho-choline, palmitoyl carnitine, sodium dodecyl sulfate (SDS),1-lauroyl-sn-glycero-3-phosophocholine and sodium 1-myris-toyl-sn-glycero-3-phosphate12. Structural modifications ofthese agents have yielded some increase in the separation between the cytotoxicity and efficacy of PPEs but significantimprovement in this separation has not been achieved. For example, the separation between the cytotoxicity and efficacyof these compounds was only increased to a factor of three bystructural modification of synthetic phospholipids13.

Although the separation between the cytotoxicity and efficacyof PPEs is small, the ability of many PPEs to increase paracellularpermeability is not a direct result of their toxic actions. For exam-ple, palmitoyl carnitine, which shows little separation betweencytotoxicity and efficacy64, appears to act via specific disruptionof the tight junctions. At a concentration of 0.2 mM, it increasesparacellular permeability across Caco-2 monolayers within thefirst minute11.This effect is reversible and does not involve lysis ofthe apical membrane11. Instead, palmitoyl carnitine appears todisrupt tight junction integrity, as indicated by the accumulationof fluorescent dextrans and the electron dense marker lanthanumnitrate in paracellular spaces11. Furthermore, transmission elec-tron microscopy and freeze fracture electron microscopy indicatethat this treatment produces significant structural modificationsto the tight junctions11. However, at slightly higher concen-trations (0.4 mM), it is toxic64. This example illustrates that,although the separation between the cytotoxicity and efficacy ofpalmitoyl carnitine is small, this compound can increase paracel-lular permeability by a reversible and controlled modulation ofthe tight junction within a narrow concentration range.

Mechanisms of action of PPEsIn this section, we will address possible mechanisms behind theability of PPEs to increase paracellular permeability safely.We will concentrate on the ability of PPEs to modulate cellularsignaling pathways. PPEs can be used as tools to investigate signaling pathways that regulate tight junction structure andfunction. Elucidating these pathways will provide targets for thecontrolled and reversible modulation of the tight junctions, andthus a new generation of safer and more potent PPEs.

Phospholipase-C-dependent pathwayThe activation of the phospholipase-C (PLC)-dependent sig-

naling pathway has been implicated in the assembly, regulation

and barrier properties of the tight junction65. Recently, defini-tive evidence has been forthcoming for the involvement of PLCin the regulation of tight junctions (P.D. Ward et al., un published). This pathway involves an inositol-triphosphate-dependent Ca21 release from the endoplasmic reticulum anddiacylglycerol plus Ca21-dependent activation of PKC (Fig. 3).Increased intracellular Ca21 levels, through the activation ofcalmodulin-dependent kinases, activate MLC kinase (MLCK)(Fig. 3). MLCK phosphorylates MLC and thereby induces con-traction of the perijunctional actin–myosin ring, which hasbeen reported to loosen the tight junction63. The increase inparacellular permeability caused by ethanol66 and mediumchain fatty acids, such as capric and lauric acid67, is attributedto this mechanism. Ethanol caused the disassembly and dis-placement of the perijunctional actin–myosin ring and stimu-lated MLCK66. An inhibitor of MLCK (ML-7) attenuated boththe ethanol-mediated increase in paracellular permeability andMLCK activity66. Similarly, chelation of cytosolic Ca21 and in-hibition of MLCK attenuated the ability of capric and lauricacid to increase paracellular permeability67.

The effects on paracellular permeability of diacylglycerol,the other hydrolytic product of PLC, and subsequent Ca21-dependent activation of PKC are less clear. It has been reportedthat activation of PKC attenuates the increase in paracellularpermeability induced by lauric acid67. Furthermore, activationof PKC leads to inhibition of MLCK and decreased phosphoryl-ation of the MLC68. Turner et al.68 proposed that this reducedphosphorylation of MLC might cause a relaxation of the peri-junctional actin–myosin ring, leading to decreased tight junc-tion permeability. These results contradict other publicationson the involvement of PKC in tight junction regulation. For ex-ample, treating MDCK cells with PKC activators induced a rapidincrease in paracellular permeability69,70.

Additional controversies on the role of the PLC-dependentpathway in tight junction regulation exist. For example, it hasbeen reported that increasing the intracellular Ca21 concen-trations by treatment of the cells with ionomycin, a calciumionophore, does not affect the paracellular permeability as in-dicated by unchanged mannitol flux70. Recent data supportthese results, showing that changes in intracellular Ca21

concentrations do not lead to changes in paracellular perme-ability (P.D.Ward et al., unpublished).

Few studies have directly measured the effects of PPEs on theactivities of specific enzymes in the PLC-dependent pathway(PKC and PLC) by cellular and biochemical assays. Most stud-ies only infer the effect of PPEs on these enzymes. This inference is usually determined from the ability of otherknown enzyme modulators to attenuate the PPE-induced in-crease in paracellular permeability. Such results might lead toconclusions that are open to many interpretations. Therefore,

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additional research is needed to elucidate the role of the PLC-dependent pathway on tight junction regulation.

ATP depletionATP depletion is an effective way to increase paracellular per-meability. For example, inhibitors of glycolysis and oxidativephosphorylation induced a rapid drop in the TEER that corre-lated well with declining ATP levels55. Such inhibitors also in-duced a twofold increase in flux of the paracellular markermannitol across LLC-PK1 monolayers71. Although ATP de-pletion is an effective method to increase paracellular perme-ability, depleting the cells of their necessary energy resourceproduces cytotoxicity. For example, ATP depletion results inmembrane damage and lipid abnormalities in energy-depletedLLC-PK1 monolayers. These injuries were, however, reversiblein the case of treatments that decreased ATP levels down to 5%of control for less than 2 h (Ref. 71).

The ability of anionic surfactants such as SDS to increaseparacellular permeability might be due to the transient deple-tion of cellular ATP. Changes in cellular morphology inducedin LLC-PK1 cells after reversible ATP depletion were similar tothose after SDS treatment, including shortening of microvilliand induction of apical membrane wounds72. These effectswere reversible and absorption enhancement was not dimin-ished after repair of the apical cell membrane72.

The use of ATP depletion as a way to increase paracellularpermeability is probably not appropriate because of the cyto-toxicity produced by this method. ATP depletion could, how-ever, be a useful research tool for the discovery of signal trans-duction pathways that regulate paracellular permeability. Asdiscussed earlier,ATP depletion alters the degree of phosphoryl-ation of the regulatory proteins of the tight junction55, andthereby inhibits the ability of kinases to phosphorylate theseproteins (probably by depleting their substrate, ATP).

Tyrosine kinase–phosphatase pathwayThe tyrosine kinase–phosphatase cascade might be a good targetfor the development of novel PPEs. Inhibition of tyrosine phos-phatases by nonselective phosphatase inhibitors (sodium vana-date and H2O2) decreased the TEER in MDCK cells and displacedZO-1 from the tight junctions50. Similarly, phenylarsine oxide,a selective tyrosine phosphatase inhibitor, decreased TEER inMDCK cells and also increased the tyrosine phosphorylation ofZO-1 and ZO-2 (Ref. 57).Although the toxicity of these tyrosinephosphatases has not been fully explored, the potential target forthe development of PPEs is evident.

Depletion of extracellular calcium: disruption of cell–cell adhesionInteractions between components of adherens junctions on ad-jacent epithelial cells maintain cell–cell adhesion and thus the

paracellular space. The mechanism behind the ability of Ca21

chelators such as ethylene diamine tetraacetic acid (EDTA) toincrease paracellular permeability might be the disruption ofadherens junctions. Ca21 chelators might disrupt cell–cell adhesion by depleting the extracellular Ca21 required for the interaction of components of adherens junctions. Therefore,chelation of Ca21 might induce tight junction separation bythe physical disruption of cell–cell adhesion.

The chelation of extracellular Ca21 might also activate intra-cellular protein kinases that induce the disruption of junctionalintegrity.The increase in paracellular permeability and disrup-tion of the junctional proteins induced by a low extracellularCa21 concentration is reversed by the inhibition of protein kinases73. Ca21 chelators might function by the activation oftyrosine kinases and the subsequent phosphotyrosine-regulatedincreases in paracellular permeability effected by the E-cadherin–catenin complex that composes the adherens junc-tion74.Therefore, chelation of Ca21 might not just be physicallyabolishing cell–cell adhesion; instead, it might be specificallyactivating a signal transduction cascade that regulates junctional integrity.

If titrated carefully, Ca21 chelators can increase paracellularpermeability without causing gross cytotoxicity. At concen-trations that increased the permeation of fluorescein isothio-cyanate (FITC)–dextran 4000 by 6.2 times over the control(1–5 mM), EDTA did not significantly decrease mitochondrialdehydrogenase activity75. Clearly, chelators cannot be adminis-tered to lower Ca21 levels enough to increase the paracellularpermeability sufficiently in vivo76 without disrupting a varietyof physiological junctions. Although not useful clinically, Ca21

chelators are useful tools for research into the regulation ofparacellular permeability.

Polymeric enhancersChitosans (Table 2) have been investigated for their absorption-enhancing properties in Caco-2 cells77–79. Depending on thesize and degree of acetylation, these cationic polymers canachieve an adequate separation between efficacy and cyto-toxicity. Confocal laser scanning microscopy confirmed that N-trimethyl chitosan chloride opens the tight junctions of intestinal epithelial cells to allow increased transport of hydrophilic compounds through the paracellular pathway. Nodeleterious effects to the cells could be demonstrated by thetrypan blue exclusion technique79. Many chitosans are, how-ever, insoluble at neutral pH.At pH 6.2, all the chitosans causeda pronounced reduction in TEER across Caco-2 cells; however,the increase in paracellular permeability was minimal atpH 7.4 (Refs 80,81).

Chitosans appear to be useful PPEs in vitro but recent studiesin animals have shown less dramatic increases in the paracellular

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permeability of hydrophilic markers82. It was suggested that thepresence of the anionic mucus layer in the intestine was pre-venting chitosan interaction with the epithelial surface, therebylowering efficacy.This was confirmed by studies in the mucus-producing cell line HT-29-H.Thus, chitosans, like other PPEs,will require high local concentrations at the epithelial cell sur-face, which might be achieved with particulate dosage forms82.

Finally, a new category of PPEs has recently been discovered.These are proteins that have been postulated to interact selec-tively with the extracellular domains of the tight junction pro-teins83,84. They modulate the tight junctions by either cleavingthe extracellular tight junction strands [e.g. Der p1 (Ref. 83)]or simply binding to the tight junction strands [e.g. the C-ter-minal fragment of Clostridium perfringens enterotoxin84 (C-terminalCPE)]. In MDCK cell monolayers, Der p1 increased mannitolpermeability tenfold and delocalized ZO-1 from the tight junc-tion region after a 2.5 h incubation period.This decrease in thebarrier function of the tight junction was reversible83.

For the C-terminal CPE, a twofold increase in the permeabil-ity of FITC–dextran was induced after incubation in the baso-lateral side of MDCK monolayers. It was suggested that this increase in permeability was caused by binding to claudins 3and 4 (but not 1 or 2) and consequent disruption of thehomophilic interaction of the tight junction strands from adja-cent cells84. It is surprising that such high molecular weightpeptides and proteins would affect the paracellular permeabilityvia direct interactions with the extracellular components of thetight junction because it is unlikely that such molecules wouldpermeate through the paracellular space.

Implications of enhanced paracellular permeability on systemic toxicityClearly, the tight junction barrier function is physiologicallyimportant, in that it maintains electrolyte gradients and mem-brane polarity and keeps macromolecules out of the body85.Indeed, certain intestinal inflammatory diseases, such as in-flammatory bowel syndrome (IBS), are characterized byheightened paracellular permeability86. Although the etiologyof IBS is still speculative, one hypothesis is that IBS patientshave an abnormally leaky colonic epithelium that allows lumi-nal bacterial fragments to penetrate into the subepithelialspaces, resulting in an inflammatory response from macro-phages or monocytes that reside in the lamina propria86. In addition, non-steroidal anti-inflammatory drug enteropathy ishypothesized to be caused by increased paracellular perme-ability to luminal toxins, resulting in infiltration of neutrophilsand subsequent inflammation87.

It has recently been shown that tight junctions in lung epithelia are dissolved by proteases found in dust mite fecal pel-lets83. It was suggested that the resulting increase in paracellular

permeability initiates a sensitization that results in an allergicresponse to dust mites.Thus, the increased intestinal paracellu-lar permeability caused by PPEs, especially in the colon (whichhas a higher population of bacteria), might cause local intesti-nal inflammation similar to that observed in intestinal inflam-matory disease. However, it is premature to assume that PPEswill cause systemic toxicity regardless of their selectivity, po-tency and reversibility.The key to finding a successful PPE willbe to show that the controlled, transient and reversible openingof intestinal tight junctions does not result in increased exposureto intestinal bacteria or their byproducts.

ConclusionsThe search for safe, effective agents to improve the intestinal ab-sorption of hydrophilic drugs is ongoing. During the past fewyears, scientists have come closer to a viable approach for the im-proved oral administration of peptides, proteins and other hydro-philic compounds. Many of the advances have been achievedthrough studies of in vitro models of intestinal epithelium, such asCaco-2 monolayers. These studies have produced a wealth ofknowledge about the regulation and modulation of the epithelialtight junctions, the major impediment to the absorption ofhydrophilic drugs.These advances have been complemented bythe elucidation of the efficacy, toxicity and mechanism of actionof several PPEs with the help of cell monolayers.

For the most part, current PPEs do not have sufficient sep-aration between efficacy and cellular toxicity. Because of thisnarrow therapeutic window, the requirement for high doses ofthe PPEs for efficacy and the complex and dynamic environ-ment of the in vivo system, current agents (e.g. sodiumcaprate88) are not viable candidates for pharmaceutical use.Future development of safer, more potent and pharmaceuti-cally useful PPEs should exploit advances in the emergingknowledge about tight junction regulation. Clearly, the con-cerns regarding systemic toxicity associated with enhancedparacellular permeability caused by PPEs continue to persist.However, such concerns should not prevent continued effortsto search for agents that can modulate the tight junctions andenhance paracellular permeability in a controlled and transientfashion via modulation of specific cellular processes implicatedin the regulation of the structure and function of tight junctions.

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Announcement...Platform Computing Corporation (San Jose, CA, USA) have announced their combined marketing initiative with LIONBioscience (Heidelberg, Germany) at The Institute for Genomic Research’s (TIGR) 12th International GenomeSequencing and Analysis Conference (Miami Beach, FL, USA). This joint initiative is to help accelerate internationalresearch on the human genome by integrating Platform’s LSF technology for DRM (distributed resource management)into LION Bioscience’s impressive data integration platform, thus enabling scientists to easily manipulate human genomestatistical data.

‘Now that sequencing for the human genome is nearly complete, there will be a manifold increase in the amount ofsequence analysis that has to happen,’ said Phil Weaver, President and Chief Operating Officer of Platform ComputingCorporation. ‘In the race against time to discover the next new medicine and the next new gene, Platform Computing and LION Bioscience are strengthening their commitment to the Human Genome Project by combining complementarytechnologies that help researchers solve real-world medical issues.’

enhancing paracellular permeability by modulating epithelial tight junctions

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