(b) mechanisms of peptide and protein absorption: (1) paracellular intestinal transport: modulation...

Advanced Drug Delivery Reviews, 7 (1991) 339 364 (Q 1991 Elsevier Science Publishers B.V. All rights reserved. / 0169-409X/91/$03.50 ADONIS 0169409X91004023

ADR 00103

339

I

(B) Mechanisms of Peptide and Protein Absorption

(l) Paracellular intestinal transport: modulation of absorption

Hugh N. Nellans Abbott Laboratories, Department of Pharmacology, G1 Pharm. Sec., Abbott Park, IL, USA

(Received December 12, 1990) (Accepted May 6, 1991)

Key words: Intestinal absorption; Intestinal secretion; Cellular; Paracellular; Permeability; Partition coefficient; Molecular size; Tight junction; Convective flow; Solvent drag

Contents

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 I. Intestinal paracellular pathway: functional properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 2. Intestinal paracellular pathway: physiological control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342

II.Passive modulation of intestinal paracellular transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 1. Descriptive equations for transcellular and paracellular fluxes . . . . . . . . . . . . . . . . . . . . . . . 344 2. Relative transport contributions of transcellular and paracellular pathways ...... 347 3. Convective flow and paracellular flux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 4. Net intestinal transport as a difference in unidirectional fluxes . . . . . . . . . . . . . . . . . . . . . . 348 5. Rectified convective water flow: physiological barrier to absorption . . . . . . . . . . . . . . . 349

|II. Active control of paracellular absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 352 1. Mechanism of change in intestinal permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 2. Other epithelia and in vitro/in vivo comparisons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354 3. Permeability changes and in vivo absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354

Abbreviations: PEG, poly(ethylene glycol); MDCK, Madine Darby canine kidney.

Correspondence: H.N. Nellans, Abbott Laboratories, Department of Pharmacology (46R), Pharmaceu- tical Products Division, G1 Pharm. Sec., Building AP9, One Abbott Park Road, Abbott Park, IL 60064- 3500, USA. Fax: (1) (708) 938-5286.

340 H.N. N E L L A N S

IV. The parace l lu lar pa thway in disease states . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

V. Paracel lu lar probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358

VI. Exp lo ra to ry studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

VII. Conc lus ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360

Summary

The significance of the paracellular pathway in both the absorption and secretion of therapeutic compounds by the intestine has received little attention to date. A growing body of literature firmly documents the importance of this aqueous pathway as a major contributor to the transepithelial flow of water and solutes in a wide spectrum of epithelia, including both the small and large intestine. The relative contributions of the paracellular and cellular pathways to intestinal absorption are discussed with respect to both diffusion and convective flow of water. Emphasis is placed on the interaction of convective flow with net intestinal absorption. The discussion of convective flow is extended to the intriguing observations that the permeability of the paracellular tight junction may be under active regulation by glucose and amino acids through cytoskeletal connections to the perijunctional actomyosin complex. The review cites studies from other epithelia, suggesting the universal character of the importance of the transport between, as well as through, cells and attempts to establish physiological criteria for assessing the contribution of paracellular transport to the intestinal absorption of therapeutic compounds, including peptides and peptidomimetics.

1. Introduction

Historically, solute transport across biological 'membranes' has been dominated by the concept of diffusion through the lipid barrier of cell plasma membranes. The classic studies of Collander [1], relating membrane permeability to the partition coefficient of an array of non-electrolytes, have served several generations of students and investigators as the appropriate paradigm for considering most biological transport phenomena. However, distinct deficiencies arise within this framework when considering epithelia; it is the objective of this review to point to alternative pathways for transport, particularly those dominated by high water permeation. The specific target will be the pathway between, rather than through, mucosal epithelial cells of the intestinal tract. Despite the fact that a significant body of data identifies the extracellular or paracellular pathway of the intestine as a dominant route for water and electrolyte transport [2-5], many current models of drug absorption

PARACELLULA R INTESTINAL TRANSPORT: MODULATION OF ABSORPTION 341

[7,8] have disregarded the potential role of this pathway in the intestinal absorption of therapeutic drugs. The appreciation of this route for drug delivery across the intestine is only beginning to be explored [9,10]. The intent of this review is to discuss the physiological role of the paracellular pathway in intestinal water homeostasis and to explore the implications of control mechanisms for paracellular permeation which may impact oral drug delivery.

H.1. Intestinal paracellular pathway." functional properties For nearly 20 years, it has been recognized that the small intestine of

common laboratory mammals, such as rat and rabbit [3,6], moves as much as 85% of the transepithelial electrolyte flow via the aqueous environment of the extracellular or paracellular pathway between epithelial cells. This is to say that seven times more ion traffic traverses the spaces and intercellular junctions between intestinal epithelial cells than the route through the cells. In fact, for some electrolytes, such as calcium, the paracellular pathway may be the primary route of intestinal absorption under conditions of adequate intake [4]. Several reviews have focused on the role of the epithelial tight junction of intact tissue or of cells maintained in cultured monolayers. Cereijido and coworkers [11-13] have been particularly active in the characterization of MDCK (Madine-Darby canine kidney) cells. These pivotal studies have established a strong correlation between the electrical conductance of the monolayer and the integrity of the tight junctions. Cation selectivity has been established and the requisite role of extracellular calcium for maintaining tight junctional structure has been identified. Virtually all of these kidney epithelial characteristics are shared by the intestine, although the small intestinal epithelium has not been satisfactorily maintained as an isolated monolayer. Cho and coworkers [14,15] have recently begun more intensive work on the MDCK monolayer to characterize size and charge selectivity of the paracellular pathway. Lord and Di Bona [16] have also demonstrated that the septate junctions of invertebrate epithelia serve essentially identical functions to those of vertebrate tight junctions. Powell [6] has provided a comprehensive review of the intestinal and gastric barriers to passive diffusion. In that review he has characterized the role of the tight junction in distinguishing gastrointestinal tissues which transport large quantities of solutes against small gradients (small intestine) from those transporting smaller quantities against large gradients (stomach and colon). It is primarily the small intestine, capable of large mass transfer against relatively small gradients of concentration and pressure, which will be the focus of this review. The paracellular pathway provides the route for such large volume transport and manipulations of the driving forces and selectivity of this pathway may provide a feasible mode for enhancing oral delivery of small peptides and peptidomimetics.

The limiting dimensions of the paracellular pathway under fasting conditions is of the order of 10-15 ,~ ec~uivalent pore diameter in rat and rabbit [3] and may be slightly smaller (4-8 A), as reported by Fordtran and coworkers [17] in man. These dimensions are not fixed, however, and factors such as osmotic and

342 H.N. NELLANS

hydrostatic pressure differences, as well as recently described cellular control mechanisms [5,32], may modulate the limiting dimensions of the paracellular pathway. It has been known for some time that under conditions of water absorption, the dog jejunum increases the equivalent pore dimensions of the convective (paracellular) pathway for water and solutes by at least threefold, from 10 to 30 A diameter [18-20]. Such increases in equivalent pore size are attributable to expansion of the lateral intercellular space and may include changes in dimension of the perijunctional complexes binding adjacent cells to one another.

Differences in response of the paracellular pathway to changes in osmotic and hydrostatic gradients are quite striking and reveal an intriguing degree of asymmetry. From the early work of Lifson and his coworkers [18-20], it has become clear that mucosal hypertonicity collapses the lateral space between cells and markedly decreases water absorption, while a reverse tonicity gradient expands the space and increases water absorption. Further, the effect of hydrostatic pressure also alters dimensions of the paracellular pathway in a rectified fashion. Mucosal pressure as high as 22 cm H20 failed to cause any detectable increase in water absorption, while pressures as small as 2-6 cm, applied from the serosal side, totally abolished net water absorption. Further, increases in serosal pressure to 10 cm H20 led to frank water secretion with corresponding increases in paracellular equivalent dimensions allowing inulin, ferritin and Evans blue to access the lumenal solution.

H.2. Intestinal paracellular pathway: physiological control These observations suggest that both hydrostatic and osmotic forces are able

to alter the dimensions of the intercellular space and perhaps the tight junctional complexes. Extrapolating such observations to homeostatic control of water and solute transport reaffirms that hypertonicity in the serosal space beneath intestinal epithelial cells is capable of inducing water absorption, whereas serosal interstitial hydrostatic pressure and lumenal (mucosal) hypertonicity cause water secretion into the intestine. These results are entirely consistent with the physical factors known to control water flow in other epithelial such as the renal proximal tubule [21-23]. The similarity between intestine and tubule suggests that common mechanisms may, in fact, modulate whole body water homeostasis at the two sights. For example, the role of angiotensin II in increasing proximal tubular water reabsorption is well established [24]. It is striking that similar observations have been made in the small intestine where the renin-angiotensin system has been shown to stimulate water absorption [25,26]. Although the mechanism has not been established with certainty, an effect of angiotensin II to decrease intestinal interstitial pressure could result in increased water absorption. Such an effect would lead to water conservation similar to that in the kidney.

Other provocative examples of pressure effects on intestinal absorption have been observed with in vitro preparations of small intestine. Nellans and Kimberg [27,28] and Munck and Rasmussen [29,30] have observed that rat

PARACELLULA R INTESTINAL TRANSPORT: M O D U L A T I O N OF ABSORPTION 343

small intestine, in the absence of intact blood supply, displays a remarkable asymmetry in hydrophilic solute transport under conditions which normally stimulate net water absorption. Solutes, such as mannitol, poly(ethylene glycol) (PEG) and thiourea, have been shown to display net secretory fluxes in the presence of glucose-stimulated ion and water uptake by the tissue. This net secretion occurs in the absence of a concentration-dependent difference in driving force for the solutes. The explanation for this anomaly lies in the generation of hydrostatic pressure within the subepithelial space of these isolated intestinal preparations. As water uptake into tissue spaces is stimulated by glucose through active ion absorption, pressure builds in the lateral space between cells. In the absence of sufficient capillary and/or lymphatic outlets for this water, pressure continues rising to a point at which water flows back to the mucosal compartment, presumably through the junctional complexes of the paracellular pathway. Such water flow contributes to a significant increase in solvent drag of solutes in the serosal to mucosal direction leading to net mannitol, PEG and thiourea fluxes (see Fig. 2). These studies highlight not only the influence of hydrostatic pressure on net intestinal solute flux, but also raise the cautionary note that in vitro transport studies must be continually compared to in vivo counterparts to ascertain the relevance of observations from isolated tissues to those in the intact animal. It is highly probable that tissue hydrostatic pressure in the intact animal plays a significant role in water and water-driven solute absorption (or secretion) through the paraceilular pathway. The influence of physiological factors controlling such tissue pressure, such as prostaglandins [41] and the renin-angiotensin system [25,26], remains to be delineated and will be discussed in greater detail in subsequent sections. However, such pharmacological interactions must be considered when evaluating anomalies in intestinal absorption for compounds which may influence intestinal tissue water flow through effects on intestinal blood pressure.

Intestinal hydrostatic pressure beneath the mucosal epithelium may also be modulated by influences of intestinal smooth muscle. It is suggested that increased intestinal motility may contribute to either transient or sustained intestinal tissue pressure. Such a pressure increase, if of sufficient magnitude, may be capable of either inhibiting absorption through the paracellular pathway and/or causing secretion of absorbed solutes residing in the subepithelial space. Either or both effects would reduce the net intestinal absorption of lumenal solutes as well as the systemic bioavailability. Limited observations from this laboratory suggest that erythromycin derivatives, acting as agonists for smooth muscle contraction, may hamper their own intestinal absorption through such a pharmacological mechanism. To date the role of smooth muscle motility in intestinal absorption has been limited to the consideration of mixing and transit time. The introduction of an influence of motility on paracellular transport of water and contained solutes opens a new arena for interpretation of atypical pharmacokinetics of orally administered compounds. These passive pressure effects, together with the active control of

344 H.N. N E L L A N S

paracellular pathway permeability, provide the focus for the following discussions.

II. Passive modulation of intestinal paraeellular transport

H.1. Descriptive equations for transcellular and paracellularfluxes A comprehensive view of intestinal transport in the current era demands

inclusion of both cellular and extracellular (paracellular) routes of translocation. As an initial attempt to consider the relative contributions of both pathways to absorption and secretion, the following set of descriptive flux equations is advanced to account for mass transfer without consideration of the small effects of epithelial potential on fluxes of solutes with low charge to mass ratios. The fundamental assumption for developing a formalism for intestinal transport is that both absorption (mucosal to serosal flux) and secretion (serosal to mucosal flux) may occur through the parallel pathways of the columnar epithelial cells and the junctions and associated lateral spaces between the cells, as depicted in Fig. 1. In this model, the resistance to transcellular flux is considered to include both the apical and basolateral plasma membranes, as well as the aqueous/membranous route through the cellular cytosol.

Intestinal Epithelial Cel l

Lumen

J c e l l

JparaceII

Subepithelial __~ ~ S p a c e r _

. . . . Paracellular Pathway

Fig. 1. Parallel pathways for intestinal absorption and secretion. The cellular and paracellular pathways for solute and water flux (J) across the intestinal epithelium are depicted using two adjacent cells with the

tight junctional complex illustrated by broken lines.

PARACELLULAR INTESTINAL TRANSPORT: MODULATION OF ABSORPTION 345

The primary resistance to paracellular flux is considered to reside in the tight junctional complexes [3,6,32], binding the epithelial cells together near their apical (lumenal) domains. Changes in the geometry of the space between epithelial cells (lateral intercellular space, LIS) may also contribute impedance to flow. Changes in LIS dimensions observed with osmotic and/or hydrostatic pressure gradients are believed to reflect capacitative changes as opposed to resistive changes. If the driving forces for solute flux across the intestinal epithelium are initially limited to diffusion and convection, the following equations describe the contributions of cellular and paracellular flux:

Jcell = Dceu" Kcell . C . Ace H (1) ~cell

Jparacell = ( Dparacell + Dparacell Jvl " C DH20 (2)

(2)The diffusion coefficients (D, cm2/h) for a solute through the transcellular (cell) or paracellular (paracell) pathways are incorporated in a diffusional permeability term of the form (DK/z), where K represents the partition coefficient and the length of the relevant diffusional pathway. In the case of the transcellular flux, K is a lumped partition coefficient representing both plasma membranes and possible aqueous/membranous partitioning in the cytosol. For the paracellular pathway, the partition coefficient is considered to be unity. For initial approximation, the transport path lengths for the two pathways are considered equivalent. At this point, both the transcellular and paracellular pathways are described with unidirectional diffusional flux equations, consisting of a permeability term (cm/h), multiplied by the diffusional driving force (concentration in compartment of origin, C, mol/cm 3) and the total transporting area (A, cm 2) of the relevant pathway. For all uncharged compounds and most charged compounds with relatively low charge to mass ratios, the above equations would be sufficient to describe mass transfer or flux (tool/h) in the unidirectional dimension. Jv represents the volume or convective water flux (#l.cm-2.h - l) and will be considered below.

As discussed previously, however, an additional driving force must be incorporated in the intestinal model to provide for the influence of convective water flux across the paracellular pathway. Although some bulk water transport (convection) may occur through the cells, it is considered insignificant relative to the mass transfer between the cells. The significance of convective water flow across the paracellular pathway lies in the capacity of such flow to carry dissolved solutes in the convective stream. Specifically, convective water absorption or secretion may induce solvent drag of solutes from the lumenal or subepithelial compartments, respectively. This solvent drag phenomenon is completely ignored in transport treatments which do not

346 H.N. NELLANS

recognize the paracellular pathway as a significant parallel route for intestinal mass transfer.

The form of the solvent drag or convective component of paracellular flux is dependent on concentration (C) and area (A), as previously described for the diffusional fluxes, but the sieving characteristics of the paracellular pathway must also be incorporated in this component of Eqn. 2. For the sake of parallel arguments in Eqns. 1 and 2, the conventional use of the solute reflection coefficient [5] has been replaced by the ratio of the solute diffusion coefficient through the paracellular pathway with respect to the diffusion coefficient of water through the same pathway [33]. The significance of this dimensionless ratio can be appreciated by use of two limiting examples. The first case is characterized by the absence of a detectable diffusion coefficient for a solute of interest. A resulting value of zero for the diffusion coefficient ratio is associated with complete sieving of the solute from the convective water stream with no convection-dependent mass transport. Because the diffusional permeability t e r m (oParacell/XParacell)of Eqn. 2 is also dependent on D paracell, no solute would be expected to traverse the paracellular pathway by either mechanism. The diffusion coefficient for solutes in the aqueous pathway may, however, approach or in unusual circumstances exceed, the diffusion coefficient for water. As a second example, let the solute diffusion coefficient be identical to that of water. In this instance, the solute entrained in the convective water stream across the intestine would enter the receiving compartment at the concentration in the originating compartment. This is to say that bulk water transport would move such a solute without any sieving, filtering or reflection.

It is not difficult to imagine that most solutes of interest as therapeutic agents would possess paracellular diffusion coefficients between the extremes of zero and that of water. Consequently, most convectively driven solutes would be filtered to varying degrees by the paracellular tight junction and may even accumulate at higher concentration than in the bulk medium. The implication of such sieving or filtering as a result of convective flow, is that for a sufficiently soluble compound, the diffusional driving force through the cellular pathway may be enhanced. It should be added that the concentration on the opposite side of the paracellular pathway could be simultaneously decreased through wash-out. The effect of this sieving would be to enhance transcellular diffusional net flux independent of mass transfer via the convective stream of water flow.

The physical dimensions of the paracellular pathway have been discussed previously and will no doubt be subject to continuing investigations. For the sake of establishing common ground for discussion, the equilavent pore diameter for this pathway appears to lie between 10 and 30-50 A. These dimensions suggest that molecules slightly in excess of 3-5 kDa may traverse this pathway. Such evidence exists for both animal [18-20] and human [17] investigations and recent studies have implicated a regulatory mechanism for changing the dimensions of this pathway [5]. Discussion of the active modulation of paracellular permeability will be found in a subsequent section.


11.2. Relative transport contributions of transcellular and paracellular pathways If the dimensions of the paracellular pathway are large enough to

accommodate compounds with molecular radius approaching 15 A (~ 3.5 kDa), what is the significance of this pathway with respect to absorption and secretion relative to the cellular pathway? One approach to this question is to divide the expression for the paracellular flux (Eqn. 2) by the expression for cellular flux (Eqn. 1).

J~aracei, Dparacell ] . ce l l mparace,l {/Jr Y.paracel, + 1) • l Jcell Dccll ~paracell "'4ccll "k, OH20 / R (3)

After simple rearrangement and simplification, the ratio of paracellular-to- cellular flux can be analyzed with respect to ratios of several contributing parameters. The ratio of diffusion coefficients between paracellular and cellular pathways Dparacell/D call is likely to be greater than 1 in most cases, since free solution viscosity is significantly less than that of the cellular plasma membranes. For the sake of simplicity, we will assume the ratio of pathway lengths (XCeU/gparacen) is approximately 1, although it is conceivable that the cellular diffusion route may be significantly longer than the paracellular route.

The contribution of relative cross-sectional areas (Aparacen/Acen) for transport represents a significant difference between the two pathways. If one assumes that the width of the tight junctional complex is 0.1 #m, then the area of the paracellular pathway would be approximately 8% of that of a single cell of 5 #m diameter. With inclusions of area amplification of epithelial cells due to microvilli and limited diffusional area through the tight junctions, a more reasonable ratio for the two pathways would be closer to 0. 1%. That is to say that the paracellular pathway represents only 0.1% of the available intestinal epithelial transport cross-section. Pappenheimer and Reiss [5] have calculated this cross-sectional ratio to be < 0.1% from functional transport studies in the rat.

The cellular partition coefficient (K) represents another significant factor which can dramatically alter the balance of solute flux between cell and tight junction. If it is assumed that the majority of compounds of therapeutic interest possess partition coefficients which lie between 10 -2 and 10 +4, then the product of the area ratio 0.1 and the reciprocal partition coefficient ranges from 1 to 10 -6. This is to say that for hydrophilic compounds (K,-- 10-2), the area and partition coefficient product would predict that roughly equal mass transfer would occur across the two parallel pathways. This has, in fact, been confirmed for water transport in the rat [5]. For lipophilic compounds (K,-~ 104), no more than 1 in 106 of the transported molecules would be expected to traverse the paracellular pathway. The contributions of the area and cell partition terms weigh heavily in favoring the transcellular pathway for transport of many compounds considered for oral delivery. At best, to this point of the discussion, the paracellular pathway may contribute an equivalent fraction of transported mass when compared to the transcellular pathway.

348 H.N. NELLANS

11.3. Convective flow and paracellular flux The contributions of convective water flux, (Jv/DH2o) + 1, normalized to unit

path length, to intestinal transport are capable of significantly enhancing paracellular mass transfer. In the absence of convective water flux, the dimensionless ratio between Jv and DH~o is zero and the area ratio and cellular partition coefficient dominate balance between paracellular and cellular flux. At non-zero values of Jv, the contribution of the paracellular pathway as a transport route increases. The value of the free diffusion coefficient for water at 35°C is approximately 3.55 × 10 -5 cm2/s [34]. For a value of unity for Jv/DH~o, the volume flow of water required is approximately 2 / d - 1. c m - 2. h - r. This is a very feasible value for intestinal water transport under physiological conditions where values for absorption can frequently approach 20 #1 1.cm-2. h-1 Consequently, paracellular convective flow of 20 kt l - l -cm-2, h-1 could increase the ratio of Jv/DH2o to 10 and dramatically enhance the relative contribution of paracellular flux. The discussion of increasing absorptive paracellular water flux as a mode of enhancing paracellular solute flux will be discussed in the section dealing with active regulation of paracellular dimensions.

H.4. Net intestinal transport as a difference in unidirectional fluxes The discussion of intestinal transport via diffusive and convective pathways,

as represented by Eqns. 1 and 2, has focused explicitly on unidirectional transfer of both solutes and water. The unidirectional fluxes are often difficult to measure empirically, particularly in vivo where the variables driving the flux from subepithelial space to lumen are not well controlled and are often entirely unknown. As a result, the net flux or difference of the two unidirectional fluxes is frequently measured simply as the rate of appearance of solute in portal blood or often as the disappearance of lumenal solute. In these measurements, little or no consideration is given to the magnitude of the flux from tissue to lumen. The significance of this back flux is that the net flux becomes the difference between the forward or absorptive, flux and the back or secretory flux. The importance of this observation is that net flux can be changed appreciably by changes in either the secretory or absorptive unidirectional flux. Eqn. 4 combines the terms of Eqns. 1 and 2 to yield an expression for net flux which provides one novel insight with respect to the influence of convective water flow.

( . Acell • Dcell " K + Ap aracell . Op aracell x Jnet (Cm Cs) \ /

o

Zcell Zparacell

m s s i n

+ mparacell" Dparacell ' (Jv • C m - Jv • Cs) (4) OH20

Note that the first two bracketed terms simply describe the diffusive net flux in terms of permeabilities (DK/x), areas (A) and the concentration difference


(C, - C,) between the mucosal and serosal bathing media. Owing to the fact that diffusional fluxes are most frequently governed by entropic forces, the simple difference in concentration becomes the driving force for the net flux. Implicit in this characterization is the fact that diffusional fluxes in opposite directions through the same pathway are essentially independent of one another. This is to say that no significant energy transfer occurs between solute or water molecules diffusing in opposite directions across the intestine. The random dissipation of the concentration gradient determines the net flux. The practical implication of this principle is that for solutes which are transport- limited by their epithelial permeation, the concentration in the serosal or subepithelial space (C,) is frequently quite small compared to the musosal concentration (C,). Consequently, the mucosal concentration is the primary driving force for net diffusive flux. Under conditions, such as inadequate serosal vascular perfusion [35] or where epithelial permeation is not rate limiting, C, may increase to such an extent that net diffusive absorption is substantially reduced. These observations on diffusive flux are well-accepted principles of mass transfer; the inclusion of a component in Eqn. 4 based on volume or convective flow represents the novel contribution to the net flux expression.

The contribution of convective water flow to solute transport occurs as a result of solvent drag of dissolved solute molecules. If we assume for the moment that paracellular cross-sectional area (Aparacelt) and solute sieving

(Drarace~~) are invariant, then the controlling factor for convective transport lies in the difference of the products of unidirectional volume flow and source compartment concentration (FC, - JVSm C,). Convective water flow, contrary to diffusive movement, does transfer significant energy to oppositely directed streams. The result of this phenomenon is that the net convective flux of solute is essentially a unidirectional flux as contrasted to the net diffusional flux which is the difference between what may be large unidirectional fluxes. The significance of this net unidirectional flow for solvent drag (convective) flux is that the intestine displays distinct rectification properties with respect to hydrostatic pressure differences. Hydrostatic pressure differences directed from lumen to serosa induce a contraction of the lateral intercellular space and increased resistance to volume flow [18-201. However, when hydrostatic pressure is applied from the serosal surface, the cellular spaces expand, resistance to flow drops and flow increases markedly toward the lumen [18- 20,931. This rectified behavior of the intestinal epithelium to pressure-driven convective flow has significant implications for net intestinal solute absorption.

11.5. RectiJied convective water flow: physiological barrier to absorption Rectification of epithelial resistance to convective flow, although not well

understood on mechanistic grounds, does have appeal on the teleological level. Namely, pressure from exterior to the organism (lumen) does not efficiently drive water into the organism, while pressure originating internally is readily

350 H.N. NELLANS

dissipated through water flow to the outside. The efficiency of such a scheme in the control of internal water homeostasis is both simple and elegant.

If lumenal pressure is of relatively little significance in the control of intestinal water flow, what are the factors modulating water flow from tissue space to lumen and what effect do such flows have on net solute absorption from the lumen? The principle driving force for water flow from subepithelial tissue space to intestinal lumen is hydrostatic pressure. Very small osmotic pressure gradients within the lateral intercellular space are responsible for water absorption, but hydrostatic pressure is the dominant force in controlling water secretion. Interstitial pressure is controlled by capillary hydrostatic pressure leading to lymphatic drainage and intestinal secretion. Although the magnitude of intestinal interstitial pressure is extremely difficult to measure accurately [36,37], Levens and Suvannapura [25,26] have shown that in cat small intestine, vasoactive angiotensin II (AII) is a significant factor controlling absorption of water. The mechanism for the AII effect has not yet been elucidated, but a striking parallel surfaces with respect to effects of AII in the kidney proximal tubule where this peptide exerts sensitive control over water balance through constriction of efferent glomerular capillary sphincters. Such a constriction decreases hydrostatic pressure and increases osmotic pressure in downstream capillaries resulting in increased water reabsorption from the proximal tubule lumen [24]. Similar hemodynamic changes may be occurring in the small intestine such that AII stimulates water absorption and AII antagonism may lead to decreased absorption or frank secretion. One interesting observation in this regard is the continuing difficulty in the pharmaceutical industry associated with the oral delivery of putative renin inhibitors. Is it possible that these highly potent compounds may lead to local reductions in AII levels in the intestinal vasculature which in turn blunts further absorption due to an increase in interstitial hydrostatic pressure? Such a hydrostatic pressure increase could be envisioned to carry absorbed solute in the lateral interstitial spaces back to the intestinal lumen in a cycle of futile turnover with little net absorption (Fig. 2). Testing of such a hypothesis requires sensitive measurements of hydrostatic pressure changes as well as identification of the sites of AII vasoconstriction.

An additional source of control of interstitial hydrostatic pressure in the intestine emanates from intestinal smooth muscle. One can envision, that hydrostatic pressure within the intestinal tissue mass could be elevated during waves of either spontaneous contractions in the fed state or during migrating myoelectric complexes in the fasted state. The rise in interstitial pressure could initiate either a decrease in net water absorption or potentially, water secretion. Recently, a striking correlation between duodenal smooth muscle activity and duodenal water transport was reported in humans [38]. These studies demonstrated that as smooth muscle activity increases, the rate of water absorption declines and finally reverses to net secretion. Similar observations have been made in response to toxigenic bacteria, such as Vibrio cholerae and Escherichia coli [39,40]. In the latter cases, cellular and paracellular pathways of


secretion are stimulated making the identification of the paracellular secretory component more difficult.

Another example of a causal link between motility and absorption lies in unpublished observations from this laboratory with derivatives of erythromycin. These compounds are documented to possess a broad range of motility- inducing properties in the dog. When oral bioavailability was assessed, a striking inverse correlation was observed between motility induction potency and systemic bioavailability. While far from definitive, such a correlation suggests that similar to the human studies above [38], intestinal motility may change interstitial hydrostatic pressure such that absorption of lumenal solutes is either slowed or in some cases halted. The mechanism for such an effect would be by recycling diffusionally absorbed solutes from the lateral intercellular space back to the lumen before such solutes can gain access to lymphatics or mucosal capillaries.

The above findings suggest that net flux of solute transport by the intestine may possibly be influenced by paracellular convective water transport. The absorption of solutes by entirely predictable diffusional routes may be thwarted by physiologically based mechanisms, as illustrated in Fig. 2. These effects stem from influences of the absorbed solutes on water flow from intestinal tissue to lumen. The common mechanism for these effects is, conceivably, an increase in

In te s t ina l Epithelial

Cell

Lumen _ ~ ~ L y m p h a t i c s

~ Capil laries

Fig. 2. Reduction of absorption by paracellular water secretion. The influence of secretory water flow between adjacent epithelial cells is depicted to include a reduction in net absorption through a solvent drag effect on solutes (X)in the lateral intercellular space. Recycling of absorbed solutes and wash-out of diffusional fluxes diminishes absorption. Hydrostatic pressure in this space regulates intestinal secretion as

well as lymphatic and capillary absorption.

352 H.N. NELLANS

the tissue or interstitial hydrostatic pressure. When coupled to the propensity of the small intestine to lower resistance and thereby increase flow when subjected to pressure from the serosal side, this effect may manifest itself as a dramatic rise in paracellular solvent drag. This convective effect can wash out solutes deposited in the tissue space as a result of absorptive diffusion through the cells. Consequently, independent of partitioning properties, intestinally absorbed solutes can be restricted from access to systemic compartments by local recycling from epithelial tissue space to intestinal lumen.

III. Active control of paracellular absorption

III.1. Mechanism of change in intestinal permeability Secretory water flow and associated mechanisms giving rise to increases in

pressure in the subepithelial space are associated with passive responses of the paracellular pathway, resulting in increased water and solute flow. In addition to such passive reactions to physical forces, the paracellular pathway, particularly the tight junction, may have the capacity to alter actively its resistance to solute and water transport. The active components of these structurally based changes are the actin microfilaments of the tight junctional complex. From the extensive structural studies of Madara, as well as the functional studies of Madara and Pappenheimer and their coworkers, a significant body of literature has emerged, suggesting that paracellular pathway permeability may be increased several-fold in response to nutrients, such as glucose and amino acids. Discrimination among solutes absorbed by such changes is both intriguing and perplexing, since it is not currently known how selective absorption can be controlled by such a process. The following discussion will identify, however, both the structural basis for such active, cellularly controlled changes in paracellular permeability as well as functional transport results correlated with such changes.

Investigations from Madara's laboratory [32,46] have suggested that the cytoskeleton may control the physical geometry of the tight junction through contraction of actin microfilaments. Other laboratories have advanced similar hypotheses [47,48]. Observations that protein kinase C [49], calcium [50,52] and c-AMP [53] may also control permeation of solutes through the paracellular pathway of cultured epithelial cells have all added to the evidence in favor of a modulated barrier function for the tight junction. Madara and coworkers [46] have also been able to demonstrate that perturbations of the tight junctions of cultured human colonic epithelial cells by cytochalasin D lead to decreases in electrical resistance. These changes in electrical conductance are paralleled by increased fluxes of sodium and the specific paracellular probe, mannitol. Studies by Meza and associates [54] in kidney epithelial cells have also demonstrated a specific effect of the cytoskeletal perturbing activity of cytochalasins on tight junctions. Similar findings have also been reported for intact intestinal epithelia [46,55] by Madara and collaborators. Further, Stevenson et al. [56,57] and D'Angelo-Siliciano and Goodenough [61] have


recently isolated a protein (ZO-1) from intestinal epithelium, which may represent the junctional complex linking cytoskeleton with tight junction. Madara's group has also been able to demonstrate, in guinea pig small intestine, that osmotic loads may alter tight junction structure [59]; but this observation could well be linked to physical factors such as osmotic pressure rather than a specific effect on a cellular control system through the cytoskeleton. It is of interest to speculate, however, that, since the effects of hydrostatic and osmotic pressure exhibit distinct properties of rectification [18- 20,60], depending on the direction of the gradient, these 'passive' effects may also be under the control of the cellular cytoskeletal system. It remains to be determined if cellular signal transduction in addition to simple mechanical stress is requisite for induction of rectified responses in both water and solute flux.

Marcial and Madara [62] have suggested that the cells at the tips of intestinal villi may be more vulnerable to changes in junctional structure than cells of the crypts. These studies, which employed osmotic gradients to induce changes in horseradish peroxidase (HRP) translocation, also revealed that the junctional strands, believed to contribute mechanical integrity to the tight junctional complex [63], decreased their crosslinking. Further, the number of strands did not decrease and the most apical strand never displayed discontinuities despite significant increases in HRP translocation. Schulzke and coworkers [64] have reported that the density and depths of tight junctional strands decreases from crypt to villus in rat jejunum and that in experimentally induced blind loops, the density and depth of strands increased at all sites, presumably to maintain solute gradients. Mora-Golindo [65], on the other hand, reports that strand density and depth increase with cell age and migration up the villus in guinea pig cecum. Madara [66] in a subsequent investigation explored the possibility that the villus tip may be the most likely site for significant paracellular flux as the result of shedding of mature cells. His morphological studies revealed that while, indeed, villar tip cells did exfoliate, the resultant void was filled simultaneously. He concluded that voids in the continuous villar tip epithelium were so transient that such a pathway could not account for the magnitude of paracellular flux observed with intact tissue. As a consequence of the above studies, it is undetermined whether a spontaneous tip-to-crypt villar gradient of tight junctional leakiness may exist. The villar tips may be more susceptible to decreases in tight junctional integrity, but the villus tips have also been implicated as the primary sites of small intestinal water absorption. Absorption efficiency would be significantly limited by increased leakiness. The secretory crypt cells may benefit more from such increased junctional leakiness owing to their function as a source of net salt and water movement into the lumen [67]. Additional inquiry is required before meaningful conclusions can be drawn with respect to variations in paracellular conductance as a function of a tip-to- crypt villar gradient.

The charge selectivity of the paracellular pathway appears to favor cations [2,51]. Pappenheimer and Reiss [5] have been able to demonstrate in rat small

354 H.N. NELLANS

intestine that with a charge to mass ratio high enough, an anionic solute may be completely impermeant through the paracellular pathway. These investigators reported that the ferrocyanide ion, with four negative charges, behaved as an ideal osmotic solute; they could not detect any significant intestinal transport despite a relatively small molecular mass (175 Da). Gustke et al. [58] have investigated cation selectivity for human ileum and jejunum and report that up to 55% of total conductance is attributable to the cation selective paracellular pathway of jejunum.

111.2. Other epithelia and in vitro/in vivo comparisons Additional studies are necessary to elucidate the signal transduction system

between extracellular control factors and cellular mediated changes in paracellular permeability. The cytoskeleton, through a link to the tight junction, most probably serves as the mechanism of action to explain changes in the geometry and, consequently, the permeability of the intestinal paracellular pathway. Many other epithelia have been characterized with respect to plasticity of tight junctional conductance and permeability, including proximal kidney tubule [21-23], cortical collecting duct [70], gall bladder [95], cornea [99] and pancreas [101]. Caution must be exercised, however, in extrapolating conclusions drawn from studies on isolated tissues or cultured cell monolayers to the in vivo state. It is quite likely that cellular responses may remain intact following isolation, but the balance of forces determining net water and solute fluxes may be dramatically altered by the in vitro conditions. For example, tight junctional permeability may increase in the presence of luminal glucose, as suggested by Madara and Pappenheimer [32] and Atisook et al. [71], but if interstitial hydrostatic pressure is decreased due to experimentally induced decreases in capillary blood pressure, effects on absorption may be greatly overestimated. Consequently, while the specific elements of paracellular permeability may be studied in detail with isolated or cultured systems, the final arbiter of physiological significance is the intact animal where nuance of driving force, as well as the possibility of additional control signals, determine the significance of changes observed in isolation. It is conceivable, in the example above, that with increased interstitial hydrostatic pressure in vivo, higher permeability may be offset by secretory water flow such that little or no net increase in absorptive flux is observed. Several attempts from this laboratory (unpublished results) to stimulate paracellular solute flux in intact rats using lumenal glucose have not met with success. Other investigators [68] have published similar experiences, suggesting that the full complement of flows, forces and resistances for the paracellular pathway have not yet been accounted for in the in vitro or in situ studies reported to date.

III.3. Permeability changes and in vivo absorption If for the moment we assume that the cytoskeleton is responsible for changes

in tight junctional geometry and permeability, what is the magnitude of such changes and what size solutes are likely to cross this barrier? Pappenheimer and


Reiss [5] have reported an intriguing series of studies on paracellular transport using anesthetized rats. In these studies, the dimensions of small intestinal paracellular tight junctions were calculated at 100 A diameter, passing molecules the size of inulin (5.5 kDa) and PEG (4 kDa) with only slight restriction. The absorption of creatinine, PEG and inulin were all demonstrated to be linear functions of water absorption in the small intestine, suggesting transport by convective flow (solvent drag), as illustrated in Fig. 3. Further, lumenal glucose (23 mM) doubled net water absorption and the increase in the flux of the three hydrophilic solutes doubled as well. Since glucose absorption was also observed to be a linear function of water absorption, Pappenheimer and Reiss postulated that active sodium transport, stimulated by the addition of glucose, generates the requisite driving force for water absorption through the paracellular pathway, accounting for 50% of total water absorption. Independent investigations by Madara and Pappenheimer [32] demonstrate that glucose and amino acids, indeed, lead to not only increases in water absorption but also an increase in the permeability of the tight junction. These structural studies were carried out, not in rat, but in isolated segments of hamster small intestine or in anesthetized hamster in situ. Functional electrical impedance measurements [69] as well as tight junctional morphology studies

Intestinal Epi the l i a l

Cell

Lumen Lymphatics

I-I20

Capillaries

Fig. 3. Enhanced absorption via paracellular diffusion and convection. The cross-section of the tight junction (broken lines) has been postulated to increase in the presence of glucose and amino acids as discussed in the text. Such an increase is suggested to be associated with an increase in intestinal absorption via both diffusion and convection (solvent drag) when lymphatic and capillary drainage allow

adequate removal of water and solutes.

356 H.N. NELLANS

[32] support the hypothesis that glucose, alanine and leucine may be responsible for initiating contraction of the junctional actomyosin leading to expanded geometry of the occluding junctions and, consequently, increased permeability. The increased electrical impedance observed with glucose and amino acids was both oxygen dependent and reversible, lending additional credence to the physiological significance of these changes. Further, Pappenheimer [69] reports that his survey of the literature indicates glucose-dependent changes in electrical impedance of rat small intestine are seldom observed in isolated sheets and may be limited to tissues that are oxygenated comparably to in vivo conditions.

The critical dependence of the permeability (impedance) change on adequate oxidative metabolism also suggests that significant differences may exist between anesthetized and unanesthetized rat models. Indeed, Pappenheimer and Reiss [5] report that rats given access to 20% glucose in water excrete approximately 1.5-fold more exogenously administered oral creatinine than controls consuming glucose-free water. While this observation is qualitatively supportive of the glucose-induced permeability effect seen in anesthetized rats, the rates of glucose-stimulated absorption of creatinine in the anesthetized animals were nearly threefold greater than in glucose-free controls. Certainly, the design of the unanesthetized animal study may not optimize the absorption promoting potential of glucose. However, the magnitude of the results was appreciably lower than the prediction of the anesthetized model, suggesting that the uncompromised animal may not respond as dramatically to glucose stimulation as the anesthetized animal.

One possible explanation for such results may lie in lower secretory convective flow in anesthetized rats resulting from lower mucosal capillary perfusion pressure. Lower secretory water flow may cause less recycling of absorbed solute and, consequently, greater net absorption. Decreased net creatinine absorption in t h e unanesthetized rat could, consequently, be explained as a result of paracellular recycling. It will be necessary to critically evaluate intact animal models to ascertain whether the absorption promoting effects of glucose and amino acids observed in vitro or in situ can be translated to comparable enhancement in uncompromised animals.

Passive permeability characteristics of intestinal epithelium are often measured in vitro with flat sections of the intestinal mucosa stripped of underlying musculature. Such studies, as recently exemplified by investigations of Gross and Sweetana [81], provide consistent permeability data allowing rank ordering of compounds. Where permeation through the paracellular pathway is a significant fraction of total flux, however, the in vitro model may bear little or no relation to the in vivo state. The influence of additional in vivo factors, such as solvent drag, active modulation of tight junctional permeation and the more traditional role of capillary blood flow clearing solute from the subepithelial space [35], can all play significant roles in the dynamic, integrated permeation of solute from lumen to blood. These dynamic factors may, in fact, lead to both significant decreases or increases in integrated permeation such that in vitro

P A R A C E L L U L A R INTESTINAL TRANSPORT: M O D U L A T I O N OF ABSORPTION 357

studies must always be compared to the observed in vivo results to confirm utility of a selected in vitro technique.

Recently, Fleisher and coworkers [68] have attempted to capitalize on the absorption enhancing potential of glucose, employing phenytoin as the solute targeted for solvent drag-promoted absorption. In these studies, given significant lumen to blood concentration gradients, only marginal improve- ment in absorption was noted in glucose-treated rats. These results suggest that other driving forces may be influencing net intestinal absorption in addition to the elegantly characterized component of absorptive solvent drag, described by Pappenheimer, Madara and their coworkers.

IV. The paracellular pathway in disease states

Changes in permeability of the intestine have been attributed to specific alterations in the paracellular pathway associated with a variety of pathological conditions. Hollander et al. [44,73,74] and Dawson et al. [75] have suggested that both the small and large intestine demonstrate increases in paracellular pathway permeability in Crohn's and possibly celiac disease. With the infection of Clostridium difficile, bacterial toxin A is responsible for a massive loss of mucosal integrity due to disruption of the tight junctional complexes as demonstrated in both the human colonic T-84 epithelial cell line, Hecht et al. [76] and in mice, Heyman et al. [77]. Further, inflammation and leukocyte transmigration across T-84 monolayers have also been associated with reversible decreases in electrical resistance and increased fluxes of paracellular probes consistent with increased tight-junctional permeability, as demonstrated by Madara et al. [78]. Madara and Trier [79] note, as well, a significant disruption of tight junctional complexes of both villus tip and crypt cells in celiac spruce. These observations of decreases in intestinal mucosal restriction of lumenal molecules from subepithelial tissues suggest that breaching the epithelial barrier could be a significant factor in the etiology of these conditions. It is not unlikely that the ability to break the barrier between internal and external (lumenal) environments is a key factor in the progress of these disease states. At the current time, virtually nothing is known about methods to prevent such disease-induced functional degradation of the intestinal epithelial barrier. Model systems exist for in vitro investigation, but a satisfactory cell culture system for small intestine has not been developed to date. The well-known Caco-2 and T-84 colonic cell lines have certainly provided initial insight to transport pathways of the colon and perhaps the small intestine. However, significantly lower paracellular permeability of the colon will require development of cultured small intestinal cell lines before tissue specific data are available from these in vitro models.

Another intriguing example of paracellular plasticity has been described in the studies of McRoberts and coworkers [72]. These investigators have recently demonstrated a unique effect of insulin in the T84, human colonic epithelial cell line. Insulin is reported to be responsible for a significant reversible increase in

358 H,N. NELLANS

tight-junctional permeability generated over a period of 3-4 days. The physiological implications of such a long time-course for an insulin effect in the intestine are not readily apparent, although an argument could be made for increased efficiency of glucose absorption under conditions of restricted dietary intake. The implications of hormonal control of paracellular permeability add an additional dimension to the dynamic control of this route of absorption. Such hormonal regulation is a possibility which calls for additional study of the active cellular mechanism for permeability modulation.

In an effort to increase the paracellular route of drug delivery, the tight junctional complex is frequently perturbed in animal studies with agents that chelate calcium ion, such as EDTA, EGTA [14,15] and citrate. While these agents may be affecting an increase in junctional permeation by inhibiting perijunctional actomyosin [31], the effects are frequently irreversible. This irreversible degradation of tight junctional integrity may lead to indiscriminate penetration of the epithelium by lumenal solutes, including enterotoxins or inflammatory peptides as in the cases above. Reversible modes of increasing junctional permeability, as may be the case for sugars and amino acids are possible; however, calcium chelation [31] and some bile salts [80,82-84], alcohol [85] and other irreversible procedures are not likely to be viable alternatives for clinical utility in drug delivery.

V. Paraceilular probes

The identification of an appropriate probe molecule(s) for the paracellular pathway is of great utility in assessing the site of action of agents which may modulate intestinal permeability. Recently, Hollander and his coworkers [42] have provided compelling evidence that poly(ethylene glycol), PEG-900, may serve such a function. PEG-900 represents a spectrum of polymers with molecular weights from 200 to 1100 and hydrophilic properties favoring paracellular translocation. It is a more desirable candidate as a paracellular probe for peptidic drugs than more conventional hydrophilic molecules, such as mannitol, owing to greater molecular size. Bioavailability of PEG-898 monomer has been reported [102] to be in the 5-10% range in man. This result suggests that peptides in this size range, with appropriate formulation and water flow conditions, might be expected to approach bioavailabilities as high as 20-30%.

Hollander and his collaborators, looking for a probe to assess changes in human intestinal permeability [43,44,73], have investigated the characteristics of PEG transport in the rat intestine. With an octanol/water partition coefficient of 7.9 • 10 4, PEG-900 was shown to be nearly completely excluded from entry across isolated brush-border membranes of rabbit intestinal epithelial cells. The aqueous environment of the paracellular pathway is, consequently, the most probable route for the observed intestinal translocation of the compound. The Hollander group found that PEG-900 behaved as if the only pathway for translocation was identical to that available for bulk water

PARACELLULA R INTESTINAL TRANSPORT: M O D U L A T I O N OF ABSORPTION 359

flow. They reported reduced absorption with increased lumenal osmolarity and increased lumenal hydrostatic pressure, both conditions which reduce net water absorption. Stimulation of water absorption increased PEG absorption. These investigators conclude that PEG-900, by virtue of both its size and low partition coefficient, is a viable probe for paracellular permeability and would be of utility in both clinical and animal studies for monitoring changes in paracellular permeability. Results from our own laboratory, as well as several others [86-92], have confirmed the utility of PEG as a paracellular probe and the synthesis of tritium-labeled polymers of known molecular weight [45] has been helpful in determining the size discrimination of the paracellular pathway both in vitro and in vivo.

VI. Exploratory studies

The argument that secretory convective water flux may decrease absorption of lumenal solutes may be extended, with editorial license, to techniques which may stimulate solute absorption via the paracellular route. From the observations of Levens [25,26], it appears that the intestine via the renin- angiotensin system participates, in parallel with the kidney, in the homeostatic control of water balance. The implication for such a control mechanism is that factors favoring water conservation (increased absorption and/or decreased secretion) may be used to test the hypothesis that convective water flow is a dominant factor in controlling both the direction and magnitude of paracellular drug transport. Consequently, factors such as sodium loading and/or volume depletion may result in increased intestinal solute absorption through the actions of angiotensin II and aldosterone. Further, the osmotic activity of the extracellular fluid may determine the efficacy of stimulation of intestinal fluid (and solute) absorption by glucose and amino acids. In this case, if net water reabsorption is not required by the organism, water recycling from lateral space to lumen may occur as a result of the renin-angiotensin control of mucosal capillary uptake. As another example, aldosterone is known to have water conserving effects on the colon [100], suggesting that colonic drug delivery might also be modulated by convective water flux. Because of the myriad interactions of sodium loading, volume depletion and osmotic activity with aldosterone and angiotensin II in control of whole animal water homeostasis, many combinations of factors can be explored in regulating intestinal drug absorption. Such exploratory studies may well identify a drug delivery niche for exploiting the aqueous intestinal paracellular pathway.

VII. Conclusion

Intestinal absorption through the aqueous environment of the paracellular pathway represents an unexploited route for the absorption of molecules whose weights may approach 5 kDa. With equivalent pore diameters as large as 30 to 100 A, this pathway provides a portal of significant specific cross-section for

360 H.N. NELLANS

absorption of high-potency therapeutic compounds, such as peptides and peptidomimetics. While the total area of the paracellular pathway relative to the cellular pathway may approach 0.1%, the influence of convective water flow provides a compensating factor in balancing the relative transport contribution of the two routes. The importance of hydrostatic pressure in the subepithelial space can dramatically alter net absorption, stimulating and in some cases inhibiting, delivery to the central systemic compartment. The importance of including the physiological effects of orally delivered drugs on hydrostatic pressure has been discussed in the context of divergences between in vitro and in vivo observations. Whether the paracellular junctional resistance will prove to be a portal for oral delivery, which can be actively controlled in a reversible fashion, remains to be established. The possibilities are beginning to be explored and dogma that intestinal drug permeation is dominated exclusively by high partition coefficients and absence of charge must be challenged. The more integrated approach must include an aqueous route of delivery between cells for potent compounds with significantly higher hydrophilic character. The paracellular pathway is capable of both increasing and decreasing intestinal absorption; the challenge of the future is to harness its plasticity to selectively and reversibly enhance oral bioavailability of highly potent peptides.

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(b) mechanisms of peptide and protein absorption: (1) paracellular intestinal transport: modulation...

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