(C) Means to enhance penetration

Advanced Drug Deliver), Reviews, 8 (1992) 39 92

39

© 1992 Elsevier Science Publishers B.V. All rights reserved. / 0169-409X/92/$03.50 ADR 00106

(C) Means to Enhance Penetration (2) Intestinal permeability enhancement for proteins, peptides and other polar drugs: mechanisms and potential toxicity E. Scott Swenson and William J. Curatolo Pharmaceutical Research and Development Department, Central Research Division, Pfizer Inc., Groton, CT, USA (Received January 28, 1991) (Accepted May 6, 1991)

Key words: Bile salt; Non-ionic detergent; Oral delivery; Bioavailability; Peptide delivery; Protein delivery; Surfactant

Contents Summary .........................................................................................................

41

I. Introduction ............................................................................................

41

II. The components and structure of cell membranes ............................................

42

Abbreviations: AUC, area under the drug plasma concentration vs. time curve; CDOC, chenodeoxycholate; 6-CF, 6-carboxyfluorescein; CMC, critical micellar concentration; DDAVP, 1-deamino-8-Darginine vasopressin; DGAVP, desglycinamide arginine vasopressin; DOC, deoxycholate; DOSS, dioctyl sodium sulfosuccinate; DPPC, dipalmitoylphosphatidylcholine; EDTA, ethylenediaminetetraacetic acid; GalCer, galactosylceramide; GC, glycocholate; GCDOC, glycochenodeo×ycholate; GDOC, glycodeo×ycholate; GI, gastrointestinal: HCO-60, polyetho×ylated hydrogenated castor oil; HLB, hydrophile lipophile balance; LC, lithocholate; lyso-PC, l-acylglycerophosphocholine; MCG, medium-chain glyceride; MGK, a commercially available mixture of glycerol, octanoic acid and glycerylmono-, di- and trioctanoate; NSAID, non-steroidal anti-inflammatory drug; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PGE2, prostaglandin E2; POE, polyoxyethylene; rHGH, recombinant human growth hormone; SDS, sodium dodecyl sulfate; SPM, sphingomyelin: TC, taurocholate; TCDOC, taurochenodeoxycholate; TDHF, taurodihydrofusidate; TDOC, taurodeoxycholate; Tin, acyl-chain order~tisorder transition temperature; UDOC, ursodeoxycholate. Correspondence: W.J. Curatolo, Pharmaceutical Research and Development Department, Central Research Division, Pfizer Inc., Building 156, Eastern Point Road, Groton, CT 06340, USA. Fax: (1) (203) 441 3972.

40

E.S. S W E N S O N A N D W.J. C U R A T O L O Ill. The microstructure of the intestinal epithelial barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

45

IV. General effects of m e m b r a n e perturbants on cell m e m b r a n e structure and cell viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

47

V. Experimental considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

48

VI. Bile salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. General properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Interactions with model m e m b r a n e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. A b s o r p t i o n enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (a) Effect of bile salt structure and dosing regimen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (b) Mechanisms of absorption enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (c) Effect of bile salts on gastric emptying and water flux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (d) Reversibility o f bile salt-induced enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (e) Bile salt/lipid mixed micelles: effect in c o m p a r i s o n to bile salts alone .......... 4. Irritation and toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

49 49 53 54 54 56 57 58 58 60

VII. Non-ionic surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. General properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Interactions with model m e m b r a n e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. A b s o r p t i o n enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Irritation and toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

62 62 63 65 66

VIII. Anionic surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. General properties and interactions with model m e m b r a n e s . . . . . . . . . . . . . . . . . . . . . . . . . . 2. A b s o r p t i o n enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Irritation and toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

68 68 68 69

IX. Lysolecithin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. General properties interactions with model m e m b r a n e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. A b s o r p t i o n enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Irritation and toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

70 70 71 71

X. Acylcarnitines, acylcholines, and acyl amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. General properties and interactions with model and biological m e m b r a n e s ....... 2. A b s o r p t i o n enhancement and toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

72 72 72

XI. Medium chain glycerides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. A b s o r p t i o n enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Irritation and toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

73 73 74

XII. Salicylates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Interactions with model m e m b r a n e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2, A b s o r p t i o n enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Irritation and toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

74 74 75 76

XIII. Ethylenediaminetetraacetic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

77

XIV. Particulate carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

78

XV. Other absorption enhancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

79

XVI. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

79

INTESTINAL PERMEABILITY ENHANCEMENT

41

Acknowledgements.............................................................................................

81

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

81

Summary This review examines reported methods for the enhancement of the oral absorption of polar drugs, including polar peptides and proteins. The microstructure of the intestinal brush border is described, and the interactions of various absorption enhancers with this microstructure are examined. For each absorption enhancer class, this review examines the physical properties of the enhancer, its interactions with model membranes, its absorption enhancing effects, and its potential toxicity. The enhancers reviewed are the bile salts, anionic detergents, non-ionic detergents, medium chain glycerides, salicylates, acyl amino acids, acylcarnitines, lysolecithin, ethylenediaminetetraacetic acid and particulate carriers. I. Introduction The effective delivery of a therapeutic agent via the oral route requires that the agent have sufficient solubility, sufficient stability in the stomach and intestinal lumen, and the ability to pass through the intestinal wall. Each of these hurdles is a significant one, and the relative importance of each will depend upon the physical and chemical characteristics of the agent to be delivered. In these general respects, peptides and proteins do not differ significantly from other drugs. Solubility is generally not an overwhelming problem for peptides and proteins. However, exceptions do exist, such as the synthetic hydrophobic tripeptide renin inhibitors [1]. Sensitivity to lumenal proteases such as trypsin, chymotrypsin and carboxypeptidase, and to brush border aminopeptidases is a significant problem for any peptide or protein which contains specific enzyme-labile bonds. Intestinal wall permeability is a major problem for small polar peptides (as for any polar drug) and for larger peptides and proteins. Intestinal wall permeability is generally not problematic for simple underivatized dipeptides (which are actively transported) and for small peptides (up to approximately ten amino acids) which are nonpolar in nature. The problems associated with the absorption and metabolism of peptide drugs have been reviewed by Humphrey and Ringrose [2], and by Lee and Yamamoto [3,4]. An enormous literature exists on enhancement of the absorption of a wide variety of drugs which have solubility, stability or permeability deficits (or a combination of these deficits). In reviewing this literature, it is sometimes difficult to determine the relative degree to which a drug's incomplete absorption is due to solubility or to permeability difficulties. Ideally, absorption and absorption enhancement studies should provide the aqueous solubility and the partition coefficient for all drugs evaluated (partition

42

E.S. SWENSON AND W.J. C U R A T O L O

coefficients excepted for proteins). Unfortunately, most published studies do not provide this minimal information. In preparing this review, we have included only work which utilizes drugs which are clearly highly polar or possess high molecular weight (or both). Our goal has been to provide insight into the opportunities and difficulties involved in the oral delivery of large polar molecules, i.e., proteins. This review focuses on the problem of poor intestinal wall permeability. The microstructure of the intestinal wall is examined in detail, as is the interaction of membrane permeability enhancers with the intestinal wall. Excellent exhaustive reviews of the permeation enhancer literature have recently appeared [5,6]. The current review emphasizes enhancer mechanisms and potential toxicity. II. The components and structure of cell membranes

The major structural motif of the cell membrane is the lipid bilayer. The lipids of cell membranes are quite heterogeneous, and are primarily comprised of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol, sphingomyelin (SPM), the simple glycolipids glucosylceramide and galactosylceramide (GalCer), and cholesterol (Fig. 1). The composition of the acyl chains of the phospholipids and glycolipids exhibits extreme heterogeneity, with respect to both chain length and degree of unsaturation. An understanding of the physical properties of cell membranes is aided by consideration of the behavior of pure lipids. When a polar phospholipid such as PC is suspended in water, lipid bilayers are formed which are separated by layers of water. Although natural cell membranes are composed of only a single lipid bilayer, the multilamellar pure lipid dispersion exhibits physical properties which can be extrapolated to the behavior of the natural membrane. Aqueous PC dispersions can exist in a number of polymorphic states, most commonly in the 'gel' (rigid chain) and 'liquid crystalline' (melted or fluid chain) states (Fig. 2). At a characteristic temperature (Tm), an order-disorder transition occurs between these two states. On heating, this transition consists of melting of the acyl chains, without loss of the overall stacked bilayer structure [7]. The temperature Tm at which this transition occurs is directly proportional to the acyl chain length of PC and is inversely proportional to the degree of acyl chain unsaturation. The PE's exhibit higher Tm's, probably because the smaller ethanolamine headgroup permits tighter interlipid packing, resulting in a more stable 'gel' state. It is highly unusual for a natural membrane to contain phospholipids with two identical acyl chains. More commonly, mammalian phospholipids possess one saturated and one unsaturated chain, e.g., 1-palmitoyl-2-oleyl-PC. This common natural phospholipid exhibits its Tm at - 3 ° C [8]; thus at body temperature (37°C), this lipid provides fluidity to the non-polar interior of the plasma membrane. Glycolipids are generally ubiquitous but minor components of cell

INTESTINAL PERMEABILITY ENHANCEMENT

(a)

43

O II

R1~C--O--CH= R2--C--O--CH

I

oII

+

H=CmO--~O--CH=CH=N(CH3)~

(b)

O II

RI--C--O--CH2 R=--C--O~CH H=C~O~_~O--CH2CH=NH3

(C)

CH3(CH=)I=~CH = CH~CHOH R~C--NH~CH

I

H2COH (d)

CH~(CH=h~,--CH= CH--CHOH R--C--NH--CH

I

°II

+

H=C--O--I~--O--CH=CH=N(CH3)3 O_ (e)

CH3(CH=)I=--CH = CH--CHOH

I

H=C--O / O H

HO H OH Fig. 1. Chemical structures of some polar membrane lipids: (a) phosphatidylcholine, (b) phosphatidylethanolamine, (c) ceramide, (d) sphingomyelin, (e) glucosyl ceramide. R, Rh and R2 are generally saturated or unsaturated linear hydrocarbons of chain length 13-23.

44

E.S. SWENSON A N D W.J. C U R A T O L O H20

H20

ggg; H20

H20

"GEL"

"LIQUID CRYSTALLINE"

Fig, 2. Order~disorder transition in lamellar aqueous lipid dispersions. T,n is the acyl chain order-disorder transition temperature, and AH is the enthalpy of this transition.

membranes. The physical behavior of pure glycolipid bilayers reveals significant differences from the more c o m m o n phospholipids. GalCer bilayers exhibit the order~lisorder transition at a high temperature which is relatively independent of acyl chain composition [9-12]. For example, GalCer's with a 16:0, 18:0 or a 24:0 acyl chain all have Tm at 83°C [9]. The high acyl chain order-disorder transition temperature for glycolipids is likely due to interlipid hydrogen bonding via the hydroxyls of the glycosyl headgroup. Two highly specialized mammalian membranes are known to have high glycolipid contents: neural myelin and the apical (lumenal) membrane of the intestinal epithelial cell. In both cases, a likely function for glycolipids is to provide membrane stabilization, since the membrane glycolipids are generally below their acyl chain Tm at body temperature, and thus possess relatively rigid acyl chains. The function of neural myelin as an electrical insulator demands membrane stabilization. In the case of the intestinal brush-border membrane, stabilization is necessary because this membrane faces the harsh environment of the intestinal lumen, which is osmotically variable and contains the detergent bile salts. Any discussion of membrane lipids would be incomplete without attention to cholesterol, which is a c o m m o n constituent of essentially all mammalian plasma membranes. This sterol intercalates between phospholipid molecules with the non-polar fused sterol ring embedded in the hydrocarbon region of the

INTESTINAL PERMEABILITY ENHANCEMENT

45

bilayer, and the polar hydroxyl end located at the approximate level of the glycerol backbone of the phospholipids. Because cholesterol is rigid, it has the effect of making a fluid 'liquid crystalline' bilayer less fluid. Cholesterol has the opposite effect on a rigid-chain 'gel'-state bilayer. Cholesterol disrupts intermolecular interactions between the rigid-chain phospholipids, and thus fluidizes the 'gel'-state bilayer. Thus cholesterol decreases the cooperativity of lipid phase transitions (i.e., broadens their temperature range), and is said to 'buffer' such transitions [13]. Because natural membranes consist of a wide variety of lipids (and proteins), their order-disorder transitions are not sharp, but are spread over a broad temperature range. Furthermore, the order-disorder transition of natural membranes generally occurs at a temperature lower than body temperature. The cell membrane contains a variety of enzymes and protein 'pumps' which are involved in the active transport of amino acids, sugars and other compounds into and out of the cell. These membrane proteins are embedded in the cell membrane, and their proper functioning requires that the membrane interior be in the fluid 'liquid crystalline' state [14-17]. Thus cells will control the chain length and degree of unsaturation of their membrane lipids to assure that the membrane is above its order~tisorder transition at body temperature. This phenomenon has been coined 'homeoviscous adaptation' by Sinensky [18]. A more detailed discussion may be found in two reviews [19,20]. Lipid model membranes are relatively impermeable to inorganic ions at temperatures above and below the order-disorder transition temperature. However, at Tm a high permeability is observed, suggesting that polar transmembrane pathways exist when both gel and liquid crystalline phases coexist [21,22]. It is possible that these polar pathways result from bilayer defects at the phase boundary between contiguous gel and liquid crystalline regions of the bilayer membrane. Partitioning of non-polar organic molecules into the lipid bilayer also exhibits a maximum at the order-disorder transition temperature [23]. However, in this case significant partitioning is also observed at temperatures above and below Tin. Cell membranes must maintain impermeability to small inorganic ions (e.g., Na +), since many of the plasma membrane proteins which transport nutrients into the cell are 'powered' (directly or indirectly) by transmembrane ion gradients. To maintain these gradients, the cell must maintain its plasma membrane lipid composition in a range which assures that no lipid phase transition occurs at the environmental temperature (body temperature for an animal). This is accomplished by control of the length and degree of unsaturation of lipid acyl chains (which set Tin), and by the use of cholesterol to decrease (buffer) transition cooperativity. IlL The mierostrueture of the intestinal epithelial barrier

The intestinal epithelium consists of a monolayer of cells which are attached via tight junctions (Fig. 3). The tight junction, which forms a band around the

46

E.S. SWENSON

AND

W.J. CURATOLO

perimeter of the cell, separates the cell membrane into an apical surface (facing the intestinal lumen) and a basolateral surface (reviewed in Refs. 81,98). The compositions of the apical and basolateral membranes are different, both with respect to proteins and certain lipids, particularly glycolipids (reviewed in Ref. 177). This compositional compartmentalization permits vectorial active transport of nutrients from the lumen to the subepithelial extracellular space and into the portal blood and lymph. Although the structure of the tight junction is not known in detail, it is clear that it serves as a diffusional barrier which prevents mixing of the components of the contiguous apical and basolateral membranes [99]. Furthermore, the tight junction connects adjacent

Intestinallumen Microvillus Glycocalyx I '':t~

~

,

~

, :

I !

'tH

Apical surface" : Tight junction

~

'

•

.

:

.:!

•

..

•

•

.

.

:::'::'2"" .'i ~-

Basol surface Basalateral"~ l ~

.- :

"SI:jI''"""

~ •

.

,;.. ..

'.

ij:i!:::.

"i"::."." " [i

..~.; . . .

"

= '/

lamina Fig. 3.

Schematic representation of intestinal epithelial cells. Reproduced from

Ref. 216.

INTESTINAL PERMEABILITY ENHANCEMENT

47

epithelial cells, and controls passage of fluid intercellularly from the lumen to the subepithe!ial extracellular space. The apical membrane, which faces the intestinal lumen, is morphologically and compositionally unusual. This membrane is highly invaginated, forming microvilli which serve to increase the surface area available for absorption (Fig. 3). The apical membrane is rich in glycolipids [24,25], which would serve to rigidify this membrane at body temperature due to the high Tm of these lipids. In addition, the high glycolipid content may be involved in the genesis and/or maintenance of the highly curved microvillar surface [104]. The apical membrane is also rich in cholesterol, which in this case probably serves to fluidize the rigid-chain glycolipids (which are below Tm at body temperature), in order to provide the minimum membrane fluidity required to permit effective functioning of transmembrane protein pumps for sugars and amino acids. Scanning calorimetry and fluorescence polarization studies have demonstrated that the microvillus membrane is less fluid than most membranes, and that the membrane exhibits a broad order-disorder transition which ends (on heating) just at body temperature [26-28]. Thus, the composition of the brush border membrane is finely balanced to permit proper functioning of transmembrane pumps while assuring structural stability against the onslaughts of the osmotically variable and detergent-laden intestinal lumen. The apical microvillus membrane provides the major physical barrier to drug absorption from the small intestine. Most oral absorption enhancement approaches are aimed at causing disruption of this membrane or the intercellular tight junction. IV. General effects of membrane perturbants on cell membrane structure and cell viability

In the sections to follow, the behavior of a variety of chemical drug permeability enhancers will be examined. Most of these permeability enhancers are detergents, and it is their detergent properties which are the source of their permeability-enhancing capability. Before discussing specific permeability enhancers in detail, it is appropriate to touch upon the effects of such compounds on cells in general. Within this more general context, the efficacy and toxicity of these compounds can be better understood.The terminology of detergents and surfactants is confusing, and deserves some clarification. Amphiphilic (amphipathic) compounds are those which possess both polar and non-polar groups, usually at opposite ends of a generally elongated molecule or on opposite sides of a generally planar molecule. Most amphiphilic compounds are surfactants, i.e., they exhibit significant surface activity. Small [136] has classified amphiphiles into insoluble non-swelling amphiphiles, insoluble swelling amphiphiles, and soluble amphiphiles. The common phospholipids which form biological membranes are insoluble swelling amphiphiles; these aggregate in bilayer sheets of effectively infinite dimensions. Detergents such as bile salts, sodium dodecyl sulfate and Triton X-100 are soluble amphiphiles.

48

E.S. SWENSON A N D W.J. C U R A T O L O

These are generally more polar than insoluble swelling membrane lipids, and aggregate in small water-soluble micellar structures. The detergents have monomer water solubilities which can be in the millimolar range, while membrane phospholipids have monomer solubilities which are many orders of magnitude lower. The physical properties of detergents have been nicely reviewed by Helenius and Simons [76]. In the current review, we will follow the general habit of the permeability enhancement literature, which is to use the terms 'surfactant' and 'detergent' interchangeably to indicate micelle-forming soluble amphiphiles. Surfactants which increase cellular permeability generally do so by disturbing the cell's plasma membrane. Surfactant monomers are capable of partitioning into the plasma membrane, where they can form polar defects in the lipid bilayer. At high surfactant concentrations in the plasma membrane, surfactant-surfactant contacts occur, and the membrane can be dissolved into surfactant-membrane mixed micelles. Surfactants can also extract proteins from the plasma membrane of the cell. The interactions of surfactants with model membranes and with biological membranes have been extensively reviewed by Helenius and Simons [76] and by Lichtenberg and colleagues [105,149]. A generally assumed property of surfactant membrane permeability enhancers is non-specificity. Thus, in general, concentrations of a surfactant sufficient to increase the permeability of a cell membrane to a polar organic molecule, i.e. a drug, are also likely to be sufficient to cause increased flux of inorganic ions into and out of the cell. Many cellular functions, e.g. active transport, are 'driven' by transmembrane ion gradients. Treatments which drastically dissipate these ion gradients are toxic, and may be lethal to the cell. Furthermore, when a cell membrane has been permeabilized to the degree that macromolecules can pass, the cell is generally assumed to have undergone lysis [76], which is irreversible. Thus any physical treatment which increases cellular permeability will have cytotoxic effects. The important issues are: (1) the degree of cytotoxicity, e.g. temporarily deranged metabolism vs. total lysis; and (2) the ability of the tissue to overcome the loss of individual cells. V. Experimental considerations Permeability enhancement and toxicity have been studied for a wide variety of surfactants and other compounds, in a wide variety of experimental systems. These experimental systems include model membranes, everted intestinal sacs, intestinal epithelia in Ussing chambers, a variety of intestinal perfusion configurations, and intact animals. It is worth pointing out that the composition of the contents of the intestinal lumen is complex and variable, and that all experimental systems other than intact animals are model systems. In vivo, the physical properties of an enhancer or enhancer system may be significantly modified by interaction with endogenous bile salts. Furthermore, glyceride enhancers will be subject to degradation by lumenal lipases. Thus, for

INTESTINAL PERMEABILITY E N H A N C E M E N T

49

example, observations of surfactant-induced epithelial damage in perfusion experiments are not necessarily predictive of damage occurring in an intact animal or human. However, such observations should be useful in comparing the relative potential damage caused by a series of perfused compounds. A similar statement can be made about assessment of the relative permeability enhancement capability of a series of enhancers tested in the same experimental system. The best studies are those in which some chemical or physical property of an enhancer has been systematically varied (e.g., polyoxyethylene (POE) chainlength in a non-ionic surfactant) and has been correlated with permeability enhancement. It is common practice in intestinal perfusion experiments to measure the decrement in drug concentration as the perfusate passes through an intestinal loop or segment. In the case of permeability enhancement experiments, artifacts are possible, since permeability enhancers can also affect flux of water into and out of the intestinal lumen, thus affecting lumenal drug concentration in a manner which has nothing to do with the permeability of the intestinal wall to the drug. Even so-called 'non-absorbable' volume markers may be absorbed to a significant extent in the presence of highly effective permeability enhancers, potentially leading to an erroneous estimation of water flux into the lumen. In this regard, it is instructive to review the extensive published literature on the effects of bile salts on intestinal water flux (see for example Refs. 65,67,80). Everted intestinal sac experiments are further complicated by the fact that the measured transmucosal drug flux involves passage across both the mucosa and the muscular wall of the small intestine, while, in vivo, drug must only cross the mucosal epithelium and enter the portal or lymphatic circulation. In general the best permeability enhancement studies are those in which portal, lymphatic or systemic drug plasma levels are measured in the presence and absence of enhancers. VI. Bile salts

An enormous literature exists on the structure and function of bile salts, due to their importance in fat digestion and their involvement in the pathogenesis of cholesterol gallstone disease. For a more extensive treatment of this literature, the reader is referred to excellent reviews on the physical properties of bile salts [29-32], and their involvement in digestion [32-34] and cholesterol gallstone disease [32,35-38]. I/'1.1. General properties Bile salts are synthesized in the liver from cholesterol and enter the duodenal lumen in the form of mixed micelles with lecithin and cholesterol. In the intestinal lumen, bile salts serve as detergents which aid in the solubilization of dietary fats. This detergent activity results from the amphiphilic nature of these compounds, which possess a polar and a non-polar face (Fig. 4). In the gastrointestinal tract, bile salts are generally found in mixed micelles

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E.S. S W E N S O N A N D W.J. C U R A T O L O

with lecithin, cholesterol, monoglycerides, and fatty acids. Small et al. [39] have proposed a discoidal model for these mixed micelles, which is illustrated in Fig. 5G. In this model, a small discoidal lecithin/cholesterol bilayer is stabilized on its hydrophobic edges by bile salt molecules, which are oriented with their hydroxyls pointed outward into the aqueous environment. The radius of the mixed micelle is of the order of 2-8 nm, depending on lipid composition [40,41]. Various other micellar models have been proposed for the bile salt/lecithin/ cholesterol micelle, including the 'mixed disc' model of Mazer and colleagues [40] in which bile salt dimers are present in the center of Small's discoidal micelle [40-42], the spherical model of Muller [41] for bile salt-rich micelles [41,43], the 'capped rod' model of Nichols and Ozarowski [44] and the sphere/ rod model of Hjelm et al. [236]. The common bile salts of man are the glycine- and taurine-conjugates of the 22 18

HO p`

24 23

1 @ 23

~

COOH

OH

3

3 I

Inrn

I

Fig. 4. The structure of the trihydroxy bile acid cholic acid. The 3-~-, 7-~-, and 12-c~-OH groups are located on the same face of this roughly planar molecule. As a result, cholic acid has a polar and a non-polar face. From Carey and Small [46].

INTESTINAL PERMEABILITY ENHANCEMENT

E

F

ooo.o.

51

G

oooO ~

:oo ~o~oo

~,~

Fig. 5. Structuresproposed by Small for mixtures of PC, cholcsterol,and bile salts.A: PC: bilayerphase containing a small amount of ¢holestero|;B: PC bilayerphase containing a large amount of cholesterol;C: PC bilayerphase containing a small amount of bile salt;D: PC bilayerphase containing a large amount of bile salt;E: bile salt/PC micelle; F: bile salt/PC micelle containing a small amount of cholesterol;G: bile salt/PC micelle containing a large amount of cholesterol.From Small [52].

dihydro×y-bile salts deo×ycholate (DO(:::) and chenodeo×ycholate (CDOC), and of the trihydro×y-bile salt cholate. The structures of sodium cholate, taurocholate (TC), and glycocholate (OC) are presented in Fig. 6. Some physical properties of selected bile salts are presented in Table [. The unconjugated bile salts are sometimes referred to as 'free' bile salts. In the bile salt literature, the terms 'bile salt' and 'bile acid' are frequently used interchangeably, and generally do not indicate the state of protonation of the molecule. The mean bile salt composition found in human bile has been reported to be

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E.S. S W E N S O N A N D W.J. C U R A T O L O

COOeN ~a (a) HO .... v

v--%OH

°-~ (bl

(C)

cONHC(H22)S~ON3a

HO

~

cONHCH2COO°Na~

Fig. 6. Structures of (a) sodium cholate, (b) sodium taurocholate and (c) sodium glycocholate.

30 mol% GC, 30 mol% glycochenodeoxycholate (GCDOC), 15 mol% glycodeoxycholate (GDOC), 10 mol% TC, 10 mol% taurochenodeoxycholate (TCDOC), and 5 mol% taurodeoxycholate (TDOC) [45]. The major effects of conjugation with glycine and taurine are a lowering of the pKa, increased solubility, and an increased CMC [46]. From the point of view of bile salt physiological function, conjugation with glycine and taurine results in compounds which are resistant to enzymatic degradation by carboxypeptidase in the intestinal lumen, and do not undergo extensive passive absorption [47]. From the point of view of the use of bile salts as absorption enhancers, it is useful to note that the unconjugated bile salts will precipitate as the free acid at pH up to approximately 6.5, while the glycine conjugates are soluble at pH's down to approximately pH 4.5, and the taurine conjugates are soluble at all physiological pH's [29]. An interesting structural analogue of the bile salts is sodium fusidate, which exhibits micellar behavior which is similar to that of the bile salts [48]. An analogue of this fungal metabolite, sodium taurodihydrofusidate (TDHF), has been reported to be an effective nasal and intestinal absorption enhancer [49,50,73,74].

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TABLE I PHYSICAL PROPERTIES O F BILE SALTS Data were compiled from reports by Roda and Fini [235], Roda et al. [217,218] and Small [30]. ' Bile salt

Lithocholate Deoxycholate (DOC) Glyco-DOC Tauro-DOC Chenodeoxycholate (CDOC) Glyco-CDOC Tauro-CDOC Ursodeoxycholate (UDOC) Glyco-UDOC Tauro-UDOC Cholate Glycocholate Taurocholate Ursocholate Glycoursocholate Tauroursocholate

OH's

3c~ 3c~,12~ 3c~,12~ 3c~,12~ 3c~,7~ 3~,7~ 3c~,7~ 3:~,7/~ 37,7/3 3~,7/~ 3c~,7c~,12c~ 3~,7c~, 12c~ 3~,7~,12c~ 37,7~,12c~ 3~,7/~,12c~ 3~,7/L 12~

Aqueous solubility a (/~M) 0.05 28 6 27 7 9 3 235, 273 32 1670 150 -

CMC b (mM) water

0.15 M Na

10 6 6 9 6 7 19 12 8 13 12 10 60 35 52

3 2 2.4 4 2 3 7 4 2.2 11 10 6 39 30 40

pK~c

5.03 5.02, 5.3 4.69 1.93 5.07, 5.88 4.23 5.08 5.08, 4.98 3.95 1.85 5.06 -

~From Refs. 218,235. hFrom Refs. 217,218. CFrom Refs. 30,235.

VL2. Interactions with model membranes The interactions of bile salts with other membrane components have been extensively studied, and the quaternary cholate/lecithin/cholesterol/water phase diagram has been reported by Small and colleagues [51]. From the point of view of absorption enhancement, these interactions are nicely illustrated by Fig. 5, which is taken from an early review by Small [52]. In this model, at low bile salt contents the bile salt is intercalated between lecithin molecules, most likely as dimers or tetramers which minimize contact of the polar hydroxylcontaining face with the non-polar lecithin acyl chains (Fig. 5C). At higher bile salt contents, extensive bile salt/bile salt contacts occur in the bilayer plane, ultimately resulting in fragmentation of the bilayer and formation of mixed micelles (Fig. 5D-F). Numerous model membrane studies have been carried out which indicate that bile salts at low concentration (below the CMC) can elicit an increased bilayer permeability. At higher concentrations (usually above the CMC), bile salts cause bilayer disruption and micellar dissolution. For example, O'Connor et al. [53] observed that release of the entrapped marker 6-carboxyfluorescein (6-CF) from egg PC liposomes was enhanced by cholic acid and ursodeoxycholic acid at concentrations lower than the CMC, with 50% 6-CF release occurring at around the CMC. In a similar study, Walde et al. [54] incorporated TC (CMC ~ 3.2 mM) into egg-PC small unilamellar vesicles and multilamellar vesicles. Solubilization of the egg PC suspensions required a TC concentration

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above approximately 3 mM. At TC concentrations greater than 0.8 mM, a slow leakage of 6-CF from 6-CF-loaded egg-PC small unilamellar vesicles was noted. At TC concentrations greater than the CMC, 6-CF was released immediately, concurrent with a decrease in vesicle turbidity, indicating dissolution of the small unilamellar vesicles. Schubert et al. [55] studied the effects of a number of bile salts on the leakage of inulin (Mr 5000) or raffinose (Mr 505) from egg PC unilamellar vesicles of 700 nm and 1700 nm diameter. In all cases, a slightly higher bile salt concentration was required to release inulin than to release raffinose, suggesting an effect of solute size on permeability. In all cases, solute release occurred at lower bile salt concentrations than did PC dissolution. The monohydroxy bile salt lithocholate (LC) was more effective than the dihydroxy bile salts DOC or CDOC, which in turn were more effective than the trihydroxy bile salt cholate. Thus the most effective bilayer permeation enhancers were the more hydrophobic bile salts. Ursodeoxycholate (UDOC), which possesses a 7-/~-OH rather than the 7-~-OH of CDOC, was a much less effective permeability enhancer than CDOC. This is consistent with UDOC's greater hydrophilicity (relative to CDOC) [60], which would be expected to have a negative influence on UDOC's ability to interact with bilayers and membranes. These and other similar studies indicate that bile salts can permeabilize model membranes at bile salt concentrations lower than that required to dissolve the model membrane into a mixed micellar solution. Small's model for lecithin/bile salt interactions (Fig. 5) provides a conceptual context in which permeability enhancement by bile salts can be visualized. Reversed micelle aggregates of bile salt dimers, tetramers, etc. in the bilayer plane may serve as polar defects which permit transbilayer movement of polar solutes (Fig. 5D). Gordon and colleagues have proposed a reversed micelle model of this type for nasal absorption enhancement by bile salts [56].

VI.3. Absorption enhancement VI.3(a). Effect of bile salt structure and dosing regimen. The ability of bile salts to increase transmucosal solute flux has been studied by a variety of methods, allowing some general conclusions to be drawn. Conjugation reduces the overall hydrophobicity of the bile salt but conjugated bile salts generally retain absorption enhancing potency [56,58,59]. Thus, the hydrophobic steroid nucleus is the primary determinant of the effect of bile salts on mucosal permeability [56]. In contrast to trihydroxy bile salts, dihydroxy bile salts are more hydrophobic [60], more rapidly absorbed [61], more effective as absorption enhancers [56,58,59,61-63], and more damaging morphologically [61,64-66]. One exception to this general observation is ursodeoxycholate (UDOC), which is frequently less effective as an absorption enhancer than DOC and its conjugates, and is minimally irritating [61,62]. In order to more clearly elucidate the relationship between bile salt structure

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TABLE II PEAK BLOOD LEVELS OF SODIUM AMPICILLIN IN RATS AFTER RECTAL ADMINISTRATION IN THE PRESENCE OF BILE SALTS (12.5 mM) From Murakami et al. [62]. Enhancer

Peak Na ampicillin level (gg/ml)

Control

1.1

Trihydroxy bile salts Cholate Taurocholate Glycocholate

1.8 1.1 1.6

Dihydroxy bile salts Deoxycholate Taurodeoxycholate Chenodeoxycholate Glycochenodeoxycholate Ursodeoxycholate Triketo bile salt Dehydrocholate

23.9 17.1 28.3 10.8 5.0 1.0

and enhanced intestinal absorption of drugs, Chadwick et al. [61], studied nine unconjugated bile salts varying in number (2 or 3) and position (3/7, 3/12, 7/12, a n d / / c o n f o r m a t i o n s ) o f - OH and keto groups, in the perfused rat colon. The trihydroxy bile salt sodium cholate was ineffective as an absorption enhancer for polyethyleneglycol. The dihydroxy bile salts were effective only when the 2 - OH groups were present in the configuration 3-e, 7-e (CDOC), 3e, 12-c~(DOC), or 7-~, 12-e (see Fig. 4 and Table I for bile salt structures). The dihydroxy bile salt UDOC (3-~, 7-//was ineffective, due to the presence of the //-OH in the hydrophobic plane of the bile salt. Gullikson et al. [67] similarly found that dihydroxy bile salts, but not trihydroxy bile salts, enhanced the absorption of inulin, dextran and albumin in perfused rat jejunum. A series of free and conjugated bile salts was studied by Murakami et al. [62], who demonstrated that regardless of the state of conjugation, dihydroxy bile salts (DOC, TDOC, GDOC, CDOC and, to a lesser extent, UDOC) enhanced absorption of ampicillin from an in situ rectal loop in rats, while trihydroxy bile salts were ineffective (Table II). The absorption-promoting efficacy of the bile salts correlated with both the hydrophobicity and the hemolytic activity toward sheep erythrocytes [62]. Ziv and colleagues [59] demonstrated that both cholate and DOC promoted rectal insulin absorption from an aqueous solution, as measured by blood glucose lowering in normal and diabetic rats, but the dihydroxy DOC was more effective than the trihydroxy cholate [59]. The dependence of absorption enhancement upon bile salt dosing regimen has been studied by a variety of in vivo and in vitro methods. Feldman and Gibaldi [68] detected a small but significant increase in the rate of salicylate transfer into rat everted small intestine in the presence of submicellar concentrations of TDOC. This effect became pronounced above the CMC,

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and was not immediately reversible. Fasano et al. [71] found that submicellar concentrations (0.5 mM) of CDOC and UDOC were effective in enhancing lactulose permeation through rabbit jejunum and ileum in vitro (Ussing chambers). Feldman et al. [69] showed that simultaneous oral dosing of phenol red with micellar concentrations of DOC significantly increased the urinary excretion of phenol red in rats. Guarini and Ferrari [70] compared simultaneous oral dosing of DOC and heparin in rats to pretreatment with DOC by oral gavage followed by oral heparin administration 0.5-24 h later. DOC increased heparin absorption, as measured by plasma clearing activity, for all pretreatment intervals from 0 to 24 h vs. oral heparin alone, with maximum plasma clearing activity observed with a 1 h interval between dosing of DOC and heparin [70]. The fusidic acid derivative TDHF possesses structural properties similar to the bile salts and has been reported to be an effective absorption enhancer for both oral and nasal drug delivery [50,73,74]. The effect of TDHF on rectal absorption of cefoxitin and the vasopressin analog desglycinamide arginine vasopressin (DGAVP) was studied by Van Hoogdalem et al. [74] in rats using bolus or infusion delivery techniques. Significant improvement of rectal absorption of both drugs was obtained with TDHF concentrations as low as 0.5%, while a 4% w/v solution of TDHF yielded complete absorption of cefoxitin. Infusion gave greater bioavailability of both drugs for any given TDHF concentration than did bolus dosing, perhaps due to the reduced spreading of the solution on the rectal surface. Lundin et al. [73] demonstrated that a 15 mM solution of TDHF increased the permeation of a nonapeptide vasopressin analog DDAVP, as well as bovine serum albumin, through everted rat small intestine. The same investigators reported that oral gavage of 2.1 mmol/kg TDHF significantly increased the absorption of DDAVP in postclosure (30 day old) rat pups, but bovine serum albumin absorption was not increased [73].

VI.3(b). Mechanisms of absorption enhancement. The perturbation of membranes may occur as a result of attack on one or more membrane components. Bile salts have a high capacity for solubilization of phospholipid [76], suggesting that extraction of phospholipid may disrupt the brush-border membrane. Feldman et al. [77] showed that increased permeability of everted rat intestine to phenol red in the presence of micellar concentrations of TDOC correlated with the release of both phospholipid and protein. Unconjugated bile salts may also solubilize protein [76], potentially leading to alterations in both membrane and tight junction integrity. The enhanced permeation of inulin, dextran and albumin observed by Gullikson et al. [67] in the presence of the dihydroxy bile salt DOC (5 mM) correlated with several effects consistent with a membrane-damaging mechanism. Release of DNA and sucrase from the perfused intestine was increased, morphologic damage to the villus surface was observed, and fluid accumulation occurred in everted jejunal sacs. Furthermore, absorption-

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promoting bile salts were more damaging to human erythrocytes in vitro. The enhanced absorption of macromolecules and the evidence of membrane damage were not observed in the presence of the trihydroxy bile salt TC (10 mM) [67]. Similarly, Chadwick et al. [61] found that dihydroxy bile salts were effective in promoting polyethylene glycol absorption in the perfused rabbit colon, but caused net fluid secretion and loss of DNA, and caused histologic damage visible by light microscopic examination. The absorption-promoting ability of several compounds has been ascribed to their Ca 2 ÷-binding ability. Cassidy and Tidball [78] suggested that chelation of Ca 2÷ may alter the structure of the cell's tight junction, thus permitting intercellular penetration through the intestinal wall. Recently, Fasano et al. [71] observed alterations in tight junctions and increased permeation of lactulose following perfusion of CDOC or UDOC at low concentrations (below the CMC) in rabbit small intestine in vitro. However, at higher concentrations, the bile salt-induced absorption promotion appeared to occur through frank disruption of the brush-border membrane. The premise that bile salt-induced absorption enhancement occurs as a result of a harmful effect on intestinal epithelium is supported by the work of Erickson et al. [72], who perfused rat jejunum in vivo using CDOC. These investigators correlated CDOC-induced absorption of mannitol with progressive morphologic damage manifested in denudation of villus tips. In rats pretreated with PGE2, CDOC-induced absorption enhancement and morphologic damage were decreased. Pretreatment with indomethacin, which inhibits the production of endogenous PGE2 by cyclooxygenase, significantly potentiated the damaging effect of CDOC, but morphological damage was repaired within 2 h in rats treated with CDOC with or without indomethacin pretreatment.

I/I.3(c). Effect of bile salts on gastric emptying and water flux. In considering bile salts as pharmaceutical formulation components, one must consider other p h a r m a c o l o g i c c o n s e q u e n c e s in the GI tract besides m e m b r a n e permeabilization. Orally delivered bile salts can inhibit gastric emptying and intestinal transit time in a dose-dependent manner. Unconjugated bile salts are more effective in this respect than conjugated bile salts [79]. Furthermore, some bile salts exert an effect on water flux in the intestinal lumen. In man, DOC > CDOC >> cholate in terms of inducing water influx in the lumen [80], corresponding to the rank order of hydrophobicity [60]. Teem and Phillips [65] observed that CDOC, DOC and DOC conjugates (but not CDOC conjugates) induced fluid accumulation in the lumen of perfused hamster jejunum. A similar relationship was observed by Gullikson et al. [67] using everted rat jejunum, in which dihydroxy bile salts, but not trihydroxy or triketo-bile salts, inhibited water transport into the everted sac. These investigators also observed that DOC induced net fluid secretion into the lumen during perfusion of hamster small intestine in vivo. Thus, the bile salts most likely to induce fluid accumulation in the lumen are those identified as effective absorption

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E.S. SWENSON AND W.J. C U R A T O L O

enhancers. Though the mechanism of fluid accumulation is incompletely understood, a possible laxative effect of bile salts may restrict their practical use as absorption enhancers.

VI.3(d). Reversibility of bile salt-induced enhancement. Regardless of the mechanism of bile-salt-induced absorption e n h a n c e m e n t , a transient perturbation of the brush border might be acceptable if the cells recover quickly enough. Several studies have addressed the issue of pretreatment of mucosal tissues with absorption enhancer, and have shown permeability enhancement to be reversible within a short period (i.e., minutes or hours). Fasano et al. [71] demonstrated that the increased permeability of rabbit jejunum and ileum to lactulose following 0.5 m M CDOC pretreatment was reversible within 40 min of removal of the bile salt. Feldman et al. [69] demonstrated that oral dosing of phenol red with 150/~mol of DOC induced no long-term effect (3-day experiment) on urinary phenol red excretion in rats. Feldman and Gibaldi [82] had previously reported that the effect of the dihydroxy bile salt T D O C on the permeability of everted rat intestine to sodium salicylate was not reversible. The reversal of rectal permeability enhancement for the model drugs sulfanilic acid and creatinine was investigated by Nakanishi et al. [83]. Rectally infused sulfanilic acid was used as a marker of rectal-to-blood permeation, and intravenously dosed creatinine was used as a marker of blood-to-rectal clearance. 5 mM DOC rectal perfusion enhanced the permeability of both drugs and caused morphologic damage. However, the permeabilizing effect of DOC declined rapidly as the bile salt was removed from the rectum. The epithelial cells recovered their normal appearance within 2 h, but goblet cells remained altered for more than 24 h. VI.3(e). Bile salt/lipid mixed micelles- effect in comparison to bile salt alone. Under normal physiologic conditions, bile salts are not found in pure form, but in mixed micelles with other biliary lipids and with normal lipid digestion products, e.g., monoglycerides and fatty acids. It has been observed that mixed micelles containing dihydroxy bile salts are less toxic and less efficacious as absorption promoters than the dihydroxy bile salts alone. Mixed micelles containing trihydroxy bile salts tend to be more damaging and more effective enhancers than the trihydroxy bile salt alone. Feldman and Gibaldi [84] reported increased permeability of everted rat jejunum to salicylate from mixed micelles containing (TDOC + egg PC) or (TDOC + oleic acid + monoolein) versus buffer control, but TDOC-based mixed micelles were less potent enhancers of salicylate absorption than TDOC alone. Visually obvious evidence of toxicity was reduced by incorporation of lecithin or monoglyceride/fatty acid into T D O C micelles. In a rat jejunal perfusion model, Lamabadusuriya et al. [85] found that the toxic effects of DOC (inhibition of water absorption and glucose transport, inactivation of N a + / K + ATPase) were reduced by the addition of TC plus oleic acid,

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monoolein or caprylic acid. Absorption of DOC itself was decreased from these mixed micellar solutions (DOC + TC + oleic acid). When DOC/TC micelles were perfused, DOC absorption was not decreased, while toxic effects were decreased relative to DOC alone. These observations underscore the mechanistic complexity involved in enhancement by mixed micellar solutions, in which physical properties (e.g., CMC, surface activity) may be complicated non-linear functions of composition. The incorporation of fatty acids or glycerides may increase the absorption enhancement induced by trihydroxy bile salts, which by themselves are generally weak enhancers. Muranishi et al. [86] found that incorporation of monoolein into TC or GC micelles greatly enhanced absorption of heparin as compared to bile salt alone. These authors proposed a mechanism which involves the bile salt micelle as a carrier for monoolein, which can then partition into the brush-border membrane and disorder the hydrophobic environment. Taniguchi et al. [87] found that the absorption of heparin from a closed rat large intestinal loop was greatly enhanced by the mixed micelles TC/ monoolein, TC/oleic acid, HCO-60/oleic acid, and polysorbate 80/oleic acid, while bile salt or non-ionic surfactant alone did not enhance heparin absorption. The rapid absorption of monoolein or oleic acid from a TC mixed miceilar solution was associated with higher plasma heparin levels, while the relatively slow absorption of monoolein from HCO-60 micelles corresponded to low plasma heparin activity, suggesting that the partitioning of the fatty acid or monoglyceride into the lipid bilayer is potentially involved in the mechanism of enhancement. However, the complexity of the mixed micellar systems prevents absolute mechanistic interpretation. Tokunaga et al. [88] demonstrated that GC or TC mixed micelles with monoolein (40 mM each) in water (but to a much lesser extent from PO4 buffer) increased absorption of heparin from closed small intestinal loops in rats. These mixed micelles caused exsorption of intravenously dosed sulfanilic acid into the intestinal lumen, and the degree of concurrent protein release during the mixed micellar perfusion was elevated, suggesting a direct harmful effect of the mixed micelle on the membrane. However, pretreatment of the intestinal loop with mixed micelles did not affect heparin absorption, indicating that the mixed micelle must be present with the drug in the lumen for effective absorption enhancement to occur. The fatty acid or glyceride incorporated into a mixed micelle can affect the intestine-permeabilizing efficacy. For example, Muranushi et al. [89] observed increased absorption of streptomycin from the rat large intestine in the presence of mixed micelles of TC plus monoolein or fatty acid (lauric, oleic, linoleic, or linolenic). TC mixed micelles with long-chain saturated fatty acids (16:0, 18:0), or TC alone, were not effective. In general, lower melting fatty acids added to TC micelles gave enhanced streptomycin absorption. TC micelles with less surface active lipids (e.g., methyl oleate, oleyl alcohol, triolein and diolein) were ineffective.

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Ishizawa et al. [214] similarly demonstrated that mixed micelles composed of 1% w/v TC plus 1% w/v unsaturated fatty acid (C18:1, C18:2, C18:3) enhanced both colonic and jejunal absorption of fosfomycin in rats, while TC alone was ineffective.

VI.4. Irritation and toxicity Bile salts are endogenous residents of the gastrointestinal lumen and, as such, are generally thought to be non-toxic. However, as described in detail above, bile salts are capable of permeabilizing and dissolving membranes. Thus it is not truly surprising that these endogenous surfactants can exhibit some local toxicity. Lysis and dissolution of erythrocytes or erythrocyte membranes ('ghosts') have frequently been used as a model of membrane damage by surfactants. Coleman and Holdsworth demonstrated that the ability of three bile salts to solubilize e r y t h r o c y t e ghosts r a n k e d in the o r d e r d e o x y c h o l a t e >cholate>dehydrocholate [90]. This is consistent with the greater hydrophobicity and surface activity of the dihydroxy compound deoxycholate than the trihydroxy compound cholate. Dehydrocholate, which does not form micelles, did not solubilize erythrocyte membranes. A similar ranking of di- and trihydroxy bile salts was observed with respect to dissolution of hepatocyte membranes [91]. Coleman and colleagues [92] also demonstrated that erythrocyte membranes which possess a higher sphingomyelin content are more resistant to lysis by cholate. These authors proposed that the high sphingomyelin content of bile canalicular membranes may contribute to the resistance of these membranes to bile salt-mediated dissolution in vivo. This proposal is consistent with the relatively high order-disorder transition temperature of natural sphingomyelin, which suggests a general stabilizing role for sphingomyelin in membranes [93,94]. More recently, Graham and Northfield [95] reported that the extent of dissolution of bile c a n a l i c u l a r m e m b r a n e s r a n k e d in the o r d e r CDC > cholate > dehydrocholate, again consistent with the relative hydrophobicity and surface activity of these bile salts. Comparison of the glycine and taurine conjugates of both CDC and cholate revealed no consistent difference between these two conjugates with respect to capacity for membrane solubilization. A large body of work has demonstrated that bile salts can damage the mucosa of the small intestine. In the 1960's, Dawson et al. [96] and Donaldson [97] demonstrated in vitro histological damage of rat small intestinal mucosa by deoxycholate (but not by taurodeoxycholate). Low-Beer et al. [64] in 1970 demonstrated bile acid-induced histological damage in perfused guinea pig and hamster intestine. Mild to severe shedding of cells was observed which was more severe at higher bile salt concentration. The dihydroxy bile salt DOC was more damaging than the trihydroxy compound cholate, consistent with the greater hydrophobicity and surface activity of DOC. In the study of Low-Beer et al., the dihydroxy bile salt CDOC was similar to DOC with respect to small

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intestinal damage. Teem and Phillips [65] observed similar results in a perfusion study in hamsters. Perfusion with 4 mM DOC resulted in epithelial loss at villus tips. "Similar, but less striking" damage was observed after a perfusion of 4 mM CDOC. In this model, 8 mM cholate caused no significant histological damage. Furthermore, no significant damage resulted from perfusion (at 4 mM or 8 mM) of a variety of conjugated dihydroxy bile salts (GDOC, TDOC, GCDOC, TCDOC). Ammon and colleagues observed that sulfation of DOC (3-sulfodeoxycholate) resulted in reduced membrane damage, compared with DOC [100]. 3-Sulfo-DOC was significantly less potent (0.1 × ) than DOC in its ability to dissolve liposomes, and the reduced detergent activity of 3-sulfo-DOC is presumably the cause of its reduced toxicity [100]. Work from a variety of laboratories has indicated that treatment with DOC or CDOC results in histological damage to the colonic mucosa [61,101-103]. Interestingly, Craven et al. observed that rat colonic epithelium was capable of rapid repair of DOC-induced damage [102]. These authors suggested that, after a 4 h DOC infusion, non-dividing crypt epithelial cells may migrate upwards to replace damaged epithelial cells. The bile salt analogue T D H F has also been shown by van Hoogdalem et al. [106] to damage the rectal mucosa. However, bile salt-induced colonic damage has not been universally observed. For example, Mekhjian and Phillips [107] observed that perfusion of the canine colon with 10 mM DOC resulted in no detectable histological damage. While acute studies of surfactant-induced damage may be useful for identifying problems, chronic dosing studies are absolutely necessary for assessment of the safety of bile salts as absorption-enhancing adjuvants in drug formulations. In 1964, Fry and Staffeldt reported a morphological study of mice which were fed DOC as 2% of diet for 39 days [108]. Mice killed after the second day of dosing exhibited hyperemia of the small intestine, enlarged gallbladders and some histological damage. However, mice killed on the 39th day of dosing exhibited histologically normal small intestines. Gracey and colleagues [109] fed DOC at 125 mg/kg to rats for 3 days, and observed no significant alterations of the intestinal epithelium, using light microscopic examination. However, electron microscopy revealed numerous abnormalities, including reduction or absence of microvilli, shortened microvilli, mitochondrial swelling and disruption of the rough endoplasmic reticulum. Recovery from this damage was observed to occur gradually over a 4-day period, with only slight improvement at 24 h after cessation of dosing with bile salt. For now, the safety of bile salts as dosing adjuvants remains an open question. However, the indigenous nature of the bile salts would certainly suggest that one or more of these compounds might be used to advantage without any serious untoward effects. Furthermore, both CDOC and UDOC are currently dosed orally for treatment of gallstone disease, with typical doses in the 1 g/day range [110]. The toxicity and clinical use of CDOC have been reviewed by Schoenfield et al. [111], Iser and Sali [112], and Bell [113]. Briefly, CDOC toxicity exhibits wide interspecies variation, with toxicity apparently related to dehydroxylation of CDOC to the hepatotoxic lithocholic acid. Those

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species (including man) which are relatively resistant to the hepatotoxic effects of CDOC have the capacity to sulfate lithocholic acid, facilitating its fecal excretion. Clinically, the major side effect is diarrhea, which occurs in 30-50% of patients, and which sometimes reverts within 2-3 weeks or can be controlled by lowering the CDOC dose. The toxicology and clinical use of UDOC have been reviewed by Bachrach and Hofmann [114,115]. UDOC is dehydroxylated in most species to form the hepatotoxic lithocholic acid which, in man, is sulfated and excreted. Diarrhea is a rare side effect in UDOC-treated patients. Reports of a tumor-promotor role for bile salts are difficult to place in the perspective of long-term safety. Chronic therapeutic dosing with CDOC or UDOC has not resulted in any discernible increase in the incidence of carcinoma [111,112,114]. VII. Non-ionic surfactants

VII. 1. General properties Non-ionic surfactants possess a polar 'headgroup', generally polyoxyethylene (POE), and a non-polar hydrocarbon chain. Most commercial non-ionic detergents are polydisperse with respect to the length of the polar POE chain. The structures of some examples are presented in Fig. 7. Non-ionic surfactants are frequently characterized with a parameter called the hydrophile-lipophile balance (HLB) [116,117], and this scale has been widely used to classify oil-in-

(b)

~

(C)

~

(d)

~

O-1 c H 2 C H 20 )nH

~

C

'

~

~

-

-

O

O

-

O II

-

(

C H 2 C H 20)nH

O(CH2CH20)y H ! ( C H 2 C H 20 )xC H 2C"7"~C H O (C H 2 C H 20 )v)t H O I ~CHO(CH2CH20)z H

Fig. 7. Structures of some non-ionic surfactants, a: polyoxyethylene ethers (e.g., Brij series); b: p-toctylphenoxypolyoxyethylenes (e.g., Triton X-100); c: nonylphenoxypolyoxyethylenes (e.g., Igepal CO series); d: polyoxyethylene sorbitan esters (e.g., Tween series).

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TABLE III EFFECT OF E N H A N C E R ALKYL CHAIN LENGTH ON GASTRIC ABSORPTION OF PARAQUAT, FOR POLYOXYETHYLENE ETHERS WITH CONSTANT POE CHAIN = 10 From Walters et al. [125]. Surfactant name

Alkyl chain

HLB

Paraquat permeability constant ( x 103 cm.h i)

Control Brij 36T Brij 56 Brij 76 Brij 96

12 16 18 18:1

15.2 12.9 12.4 12.4

9 146 52 42 83

TABLE IV E F F E C T O F E N H A N C E R P O L Y O X Y E T H Y L E N E C H A I N LENGTH ON G A S T R I C ABSORPTION OF P A R A Q U A T , FOR P O L Y O X Y E T H Y L E N E ETHERS WITH CONSTANT A L K Y L CHAIN = 16 From Walters et al. [125]. Surfactant name Control Brij 52 Texaphor A6 Brij 56 Texaphor A14 Brij 58 Texaphor A60

Polyoxyethylene chain

HLB

2 6 10 14 20 60

5.4 10.5 a 12.9 14.4 15.7 18.3

~HLB was recalculated from the formula: HLB = 20 . 1

Paraq,uat permeability constant ( x 10~ cm.h i) 9 8 10 52 39 36 28 Mr hydrophobicgroup total Mr

water and water-in-oil emulsifiers. Characteristic HLB values are presented in Tables III and IV for a series of non-ionic surfactants. The shapes of the micelles formed by non-ionic surfactants are generally not known in detail. Robson and Dennis [118] have proposed two possible structures for micelles formed by Triton X-100: an oblate ellipsoid and a nonclassical spherical micelle in which there is no distinct boundary between the non-polar and polar portions of the micelle (Fig. 8). VII.2. Interactions with model membranes The interactions of model phospholipid membranes with the non-ionic detergent Triton X-100 have been extensively studied, and should serve as a prototype for similar systems. Triton X-100 is p-t-octyl phenol polyoxyethylene-9.5 (Fig. 7b). An early report of the permeabilization of bilayers by Triton X-100 was that of Weissmann et al. in 1965 [119]. These authors demonstrated that egg PC/cholesterol liposomes could trap glucose, and that addition of Triton X-100 resulted in glucose release. Ruiz et al. similarly demonstrated that Triton X-100 releases 50% of the 6-

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E.S. SWENSON AND W.J. C U R A T O L O

T 2.7am

_k A

[1 5.2nm q I

4.1nm

B Fig. 8. Two proposed structures for the Triton X-100 micelle. From Robson and Dennis [118].

carboxyfluorescein dye trapped in egg PC multilamellar liposomes at a phospholipid/surfactant ratio of 2.9 [120]. Under the same experimental conditions, a phospholipid/surfactant ratio of 0.6 was necessary to achieve 50% dissolution of the egg PC. Thus increased membrane permeability caused by a surfactant does not require frank dissolution of the membrane. Along similar lines, Bangham and Lea [121] demonstrated that the electrical conductance (i.e., ionic permeability) of egg-PC/egg-PE 'black lipid films' doubled at a Triton X-100 concentration of 0.05 mM, which is less than the Triton CMC of 0.24 mM. Inoue and Kitagawa [122] demonstrated that Triton X-100 elicited release of trapped glucose from egg PC multilamellar and unilamellar liposomes. Significant permeability changes were observed at Triton concentrations which were lower than the CMC. Incorporation of 50% cholesterol rendered egg PC liposomes slightly less sensitive to Triton-induced leakage. Anzai et al. [123] used electron spin resonance spectroscopy to demonstrate that the acyl chain order in egg-PC liposomes decreased with increasing Triton X-100 content at low Triton/PC ratios (below 0.4). This suggests that the Triton, which possesses a single 'fluid' hydrocarbon chain, partitions into the

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more ordered diacyl PC bilayer, thus fluidizing it. At Triton/PC ratios above 0.4 the turbidity decreased, indicating the destruction of PC bilayers and the formation of mixed micelles composed of Triton and PC. In a general way, the model of Small [52] for PC/bile salt interactions (Fig. 5) can also serve as a prototype for the interactions of PC with non-ionic surfactants. As the concentration of non-ionic surfactant in the PC bilayer increases, surfactant-surfactant interactions become favorable, resulting in formation of a transbilayer polar defect (or channel). At a higher threshold surfactant concentration in the bilayer, the bilayer becomes unstable and surfactant/PC mixed micelles form. Robson and Dennis [205] have proposed a structure for the PC/Triton X-100 micelle in which PC molecules are intercalated into micelles of the type shown in Fig. 8.

VII.3. Absorption enhancement The older literature concerning absorption enhancement by non-ionic surfactants is often complicated by the failure to distinguish between model drugs which have some degree of lipid solubility, and therefore could be solubilized in the hydrophobic environment of a micelle, and very polar model drugs, which are not solubilized in micelles. In the case of a non-polar model drug, the membrane-permeabilizing effect of a surfactant might be masked by the reduction of free drug solute due to its solubilization in micelles. A wide variety of non-ionic surfactants have been shown to increase drug absorption in vivo and in vitro. Excellent reviews of these reports are available [5,127]. We have restricted our review to the few studies which have systematically investigated the effect of non-ionic surfactant structure on the permeation of polar solutes. It has been generally observed that surfactants which are too hydrophobic to be water soluble are poor enhancers, while surfactants which are very hydrophilic cannot partition into the hydrophobic environment of the lipid bilayer. For non-ionic surfactants, the hydrophilic-lipophilic balance (HLB) alone is not a reliable predictor of absorption enhancing capability. The size and shape of both the alkyl chain and the polar group (e.g., POE) influence absorption-enhancing ability. Certain polyoxyethylene ethers were found by Ichikawa et al. [124] to be effective enhancers of rectal insulin absorption when incorporated into suppositories containing 0.5% surfactant in corn oil. Of all the surfactants tested, C12/POE-9 was the most effective enhancer of insulin absorption, as indicated by blood glucose lowering. The surfactants studied varied in the length of both alkyl chain (hydrophobic) and POE chain (hydrophilic). For a fixed C12 alkyl chain with varying POE chain, C12/POE-6 was also effective, while C12/POE-3, C12/POE-25 or C12/POE-40 were ineffective. C12/POE-3 was presumably too water-insoluble, while C 12/POE-25 and C 12/POE-40 were too hydrophilic. For a fixed POE chain of 9 with varying alkyl chain, C12/ POE-9 was most effective, followed by C16/POE-9, followed by C18/POE-9 = C10/POE-9. C8/POE-9 was ineffective.

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E.S. SWENSON AND W.J. C U R A T O L O

Although we are primarily concerned with intestinal permeability, a study of gastric mucosal transport of a polar solute in the presence of non-ionic surfactants provides useful comparative information. Walters et al. [125] studied the effect of surfactant structure on the transport kinetics of paraquat dichloride, a passively absorbed polar model drug which does not interact with micelles, across rabbit gastric mucosa in vitro. By varying both the alkyl chain length and the POE chain length, they demonstrated that there is no simple relationship between HLB and absorption promotion. Of all the surfactants tested, the most effective enhancer was C12/POE-10. Table III presents the effect of alkyl chain length, for a constant POE chain length of POE-10. Paraquat transfer occurred in the rank order C12/POE-10 > C18:I/POE-10 > C 16/POE-10 -- C 18/POE-10. Table IV presents the effect of POE chain length, for a constant alkyl chain length of C16. Of the CI6/POE ethers tested, maximal paraquat transfer was observed with C16/POE-10. Florence [127] noted that one unique aspect of the C12 chain is its intermediate solubility between oil and water. A medium length alkyl chain surfactant may penetrate the lipid bilayer easily, and because of its aqueous solubility has a greater monomer concentration and higher CMC than a longer alkyl chain surfactant. By increasing the length of the alkyl chain, one would expect to improve membrane penetration, but the monomer concentration would be reduced, perhaps offsetting the improvement in penetration [127,203]. It is interesting to note that among the anionic fatty acid soaps, sodium laurate (C12) was more active than soaps with shorter or longer chain lengths with respect to disruption of membrane integrity in cultured mammalian cells [204]. While the C12 alkyl chain appears especially well suited for membrane permeabilization, Guarini and Ferrari [128] found the C16 and C18 ethers more effective than C12 ethers in studies on the absorption of orally dosed heparin in rats. In their experiments, the C16, C18 and C18:1 ethers with POE 10 or 20 were effective, and C12/POE-10 was effective, but CI2/POE-9 was not. These results are at variance with the studies described above, in which C12/POE-9 was found to be generally very effective. VII.4. Irritation and toxicity Yonezawa [132] studied the effects of the non-ionic surfactants Tween-80 (polysorbate 80, POE-20-sorbitan-monooleate), Pluronic F68 (POE/polyoxypropylene copolymer; HLB 24), and Brij 35 (POE-23-1auroyl-ether) upon the morphology of rabbit intestine, by injecting 10% solutions into blind intestinal loops. The polar surfactants Tween-80 and Pluronic F68 caused significant mucus secretion by goblet cells. However, the more non-polar Brij 35 caused moderate desquamation of the epithelial surface, while Tween-80 and Pluronic F68 had almost no effect. Bryan and colleagues [133] reported that 1% and 10% solutions of the polar surfactant Myrj 52 (POE-40-~tearate) caused no histological damage to the rat small intestine. These studies suggest that local toxicity decreases with increasing polarity. The absorption promoting efficacy of C12/POE ethers for nasal insulin

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TABLE V SUSCEPTIBILITY OF BLUEGILL FISH TO VARIOUS ALKYLPHENOXY-POLYOXYETHYLENE A N D POLYOXYETHYLENE-ETHER NON-IONIC S U R F A C T A N T S From Macek and Krzeminski [134]. Surfactant

Number of POE units

Alkylphenoxy-POE's Triton X-45 Triton X-100 Triton X-305 Surfonic N-40 Igepal CO-520 Surfonic N-95 Igepal CO-630 Igepal CO-880

4-5 10 30 4 5 9 9 30

POE-ethers Neodol 25-3 Neodol 25-9 Tergitol 15-S-9 Alfonic 1012-60 Surfonic TD-90

3 9 9 6 9

Alkyl chain length 8 8 8 9 9 9 9 9 12 15 12 15 11-15 10-12 13

LCso a (mg/I) 3.5 (3.1-4.0) b 16.2 (13.3 19.8) 1080 (663-1470) 1.5 (1.3 1.8) 2.8 (2.4-3.2) 7.8 (6.2-9.9) 8.9 (5.9 13.6) > 1000 1.8 2.1 4.7 6.4 7.8

(1.2 2.4) (1.6,2.9) (3.7 5.9) (4.2-9.6) (6.~9.9)

aSurfactant concentration which results in a 50% response (death) within 24 h. b95% confidence interval.

delivery in rats was reported by Hirai et al. [126] to be correlated with the release of protein from the perfused nasal cavity and with the degree of erythrocyte hemolysis in vitro. Of the C12/POE ethers tested, blood glucose lowering, protein release and erythrocyte hemolysis were all maximal for C12/ POE-9. Macek and Krzeminski [134] studied the toxicity of a variety of non-ionic detergents in bluegill sunfish, measuring the surfactant concentration which resulted in death of 50% of fish during a specified time interval (LCs0). Table V demonstrates that for alkylphenoxy-POE's toxicity decreased (LCs0 increased) with increasing POE chainlength, at constant alkyl chainlength. The polar surfactants Igepal CO-880 and Triton X-305, which possess 30 POE units, are essentially non-toxic. The most toxic alkylphenoxy-POE surfactant studied was the most non-polar: surfonic N-40, with four PEO units and an alkyl chain nine carbons long. A similar relationship between PEO chainlength and toxicity was observed for the POE-ether non-ionic detergents (Table V). Macek and Krzeminski [134] reported no information on the mechanism of toxicity. Smyth and Calandra [135] have reviewed toxicology studies carried out on 22 commercial alkylphenoxy-POE non-ionic detergents. Oral dosing was carried out in rats and dogs for 90 days and for 2 years. In 90 day rat and dog studies, a no-effect level was generally observed at approximately 0.04 g.kg-l-day - 1 for nonylphenoxypolyoxyethylenes with POE chain lengths up to approximately 20 units. Those with longer POE chain lengths were generally less toxic, with the exception of compounds with 20 POE units (see below). NonylphenoxyPOE-9 showed no significant effects in 2-year studies at 0.14 g.kg- 1 .day- 1 and

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E.S. SWENSON AND W.J. C U R A T O L O

0.03 g.kg-~.day - ~ in rats and dogs, respectively. Two year no effect levels for nonylphenoxy-POE-4 were 0.2 g.kg-~.day-1 and 0.04 g.kg-1.day-~ for rats and dogs, respectively. The 2-year no-effect level for octylphenoxy-POE-40 was 0.7 g . k g - l . d a y -1 in rats, again demonstrating the reduced toxicity of more polar non-ionic surfactants. The major toxic effect observed in these studies was non-progressive myocardial toxicity caused only by nonylphenoxy-POE's with a POE chain length of 20 units. The mechanism for this structural specificity is unknown. The data of Smyth and Calandra indicate that a no effect level for nonylphenoxy-POE-9 lies at approximately 30 mg.kg-~.day -1 in the most sensitive species (dog). A 10-fold safety margin would place a safe human dose in the range of 3 m g . k g - l - d a y -1, which corresponds to approximately 200 m g . d a y - 1 for a 70 kg man. These calculations are meant only to be illustrative, in an effort to provide an 'order-of-magnitude' estimation of surfactant doses which might be toxicologically acceptable. The structure-toxicity relationship generally mirrors the structure-absorption enhancement relationship described above. Both absorption enhancement and toxicity decrease as the POE chain-length increases above approximately 20 POE units, i.e., as the surfactant becomes polar. VIII. Anionic surfactants

VIII.1. General properties and interactions with model membranes Aside from the bile salts, the two most pharmaceutically common anionic detergents are sodium dodecyl sulfate (SDS) and dioctyl sodium sulfosuccinate (DOSS). The CMC's of these detergents are 0.32 mM and 0.36 raM, respectively [67]. A laser light scattering study of SDS by Mazer et al. [137] indicated that SDS micellar size and shape depend on the SDS concentration, the temperature and the salt concentration. Depending on conditions, the SDS micelle shape can range from a minimally sized sphere (2.5 nm radius) to a prolate ellipsoid with a semiminor axis of 2.5 nm and a semimajor axis of 67.5 nm [137]. The interactions of SDS with model membranes are similar to those described for bile salts and non-ionic surfactants. For example, Bangham and Lea [121] demonstrated that SDS increases the permeability of 'black lipid membranes' at concentrations well below the SDS CMC. At concentrations above the CMC, SDS dissolves model membranes. VIII.2. Absorption enhancement The well-known ability of sodium dodecyl sulfate (SDS) to disrupt lipid membranes and denature proteins suggests its possible effectiveness as an absorption enhancer. Ichikawa et al. [124] found that several anionic surfactants, including SDS, significantly enhanced rectal insulin absorption in rats when incorporated into a corn oil-based suppository at a 3% surfactant concentration.

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Engel and Riggi demonstrated in 1969 that the absorption of heparin (100 mg) was significantly increased by incorporation of SDS or dioctyl sodium sulfosuccinate (DOSS) (25 mg/kg) in an enteric coated capsule dosage form in dogs [207]. These investigators also found simple dose-response relationships for both heparin and surfactant (SDS or DOSS) on the plasma heparin level, as measured by the lipemia clearing activity, in rats given a duodenal bolus dose. A 1% w/v SDS or potassium laurate solution was reported by Hirai et~al. [138] to be highly effective in enhancing nasal insulin absorption in rats. SDS has also been shown to increase the permeation of a variety of polar and nonpolar solutes across canine oral [139] and gastric [140] mucosa. However, SDS is almost never seriously considered for the purpose of permeation enhancement because it is generally believed to be too toxic. The therapeutic use of DOSS and ricinoleic acid as laxatives suggests that their use as absorption enhancers might be acceptable. Khalafallah and colleagues [141] conducted a study in which human subjects were given 20 mg phenol red concurrently with 250 or 500 mg DOSS. Urinary excretion of phenol red occurred earlier and to a greater extent with DOSS than in controls. Six days of pretreatment with DOSS at 200 rag/day did not affect the urinary excretion of phenol red given on the 7th day; thus the absorption enhancing effect was transient. Like the bile salts, anionic surfactants may alter water and ion flux in the lumen. In their study of the effect of ricinoleic acid on colonic permeability and water/ion flux, Bright-Asare and Binder [142] found that the clearance of intravenously dosed inulin from the blood into the lumen of rat colon perfused with 1-2 mM ricinoleic acid was significantly increased, and was accompanied by net secretion of water, Na + and CI- into the lumen. It should be pointed out, however, that in their study the ricinoleic acid was solubilized by the trihydroxy bile salt TC. While TC alone did not influence water or ion flux, it is not known whether the mixed character of the TC/ricinoleate micelle accounts for some of the observed effects. Gullikson et al. [67] similarly observed that SDS, DOSS and ricinoleic acid inhibited water absorption in the everted rat jejunum. Saunders et al. [143] also demonstrated that DOSS was a potent inhibitor of water absorption in human jejunum and in rat jejunum, ileum and colon. Histological and biochemical markers of intestinal damage indicated that these anionic surfactants damaged the mucosal surface [67,143]. As with the bile salts, the ability of anionic surfactants to inhibit water absorption in the lumen reflected the membrane damaging effects of the surfactants. VIII.3. Irritation and toxicity Sugimura studied the effects of 5% SDS on the morphology of the rabbit small intestine, and on the capacity of the intestine to transport the polar solute glucose [144-146]. SDS caused focal desquamation of the villus epithelium and a variety of epithelial cell abnormalities, including mitochondrial degeneration, dilatation of the lumen of the endoplasmic reticulum, and decreased height or

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E.S. SWENSON A N D W.J. C U R A T O L O

disappearance of microvilli. In the presence of 5% SDS, glucose transport (assessed by portal blood assay) was non-saturable and thus passive in nature, while glucose transport was saturable (and thus carrier mediated) in control experiments. Nadai and colleagues [147] carried out a histological study of rat small intestine which was perfused for 1 h with a recirculating solution of buffered 1% SDS. Degeneration of epithelial cells and villus erosion were observed, in addition to destruction of blood vessels and hemorrhage. Similarly, Kakemi et al. [148] observed erosion of the rat rectal mucosa after a 1 h perfusion with 0.4 mM (0.01% w/v) SDS. Saunders and colleagues [143] observed that perfusion of rat colon with 0.52.0 mM DOSS resulted in histological changes, including flattening of epithelial cells and disappearance of their brush borders. No histological damage was observed at 0.15 mM DOSS, which is below the CMC. Gullikson and colleagues [67] demonstrated that SDS and DOSS were more potent in an erythrocyte lysis assay than were dihydroxy or trihydroxy bile salts. The surfactant concentrations which caused 80% hemolysis were 0.036 mM and 0.051 mM for SDS and DOSS, respectively, while the CMC's for these detergents were much higher: 0.32 mM and 0.36 mM, respectively. However, intestinal wall effects, such as inhibition of water transport, required that SDS and DOSS be above their CMC's. These studies suggest that SDS and DOSS can cause significant local toxicity when contacted with the intestinal wall. However, DOSS is apparently relatively non-toxic in humans, since it is marketed as a stool softener (Docusate sodium) which can be administered at a daily dose as high as 200 mg.

IX. Lysoleeithin IX. 1. General properties and interactions with model membranes Lysolecithin is an analogue of lecithin in which the acyl chain has been removed from the 2-position of the glycerol backbone. The diacyl compound lecithin is roughly cylindrical in shape, and forms bilayer phases. Lysolecithin, on the other hand, is conical in shape, and thus favors more curved structures, e.g., micellar and cylindrical (hexagonal) phases. In water, lysolecithin forms micellar solutions [150]. The CMC of lysolecithin (C12-1yso-PC) is approximately 0.25 mM [151]. The physical properties of lysolecithin have been extensively reviewed by Weltzien [15l]. Kitagawa and colleagues demonstrated that leakage of glucose from egg PC liposomes increases with increasing lysolecithin (lyso-PC) content, and that incorporation of cholesterol reverses the permeabilizing effect of lyso-PC [152]. Light scattering measurements indicated that egg PC liposomes do not dissolve at the lyso-PC contents which result in permeabilization [152]. Small's PC/lysoPC/water phase diagram [153] indicates that lyso-PC/PC interactions fit the general model described above for bile salts and non-ionic surfactants: (1)

INTESTINAL PERMEABILITY ENHANCEMENT

71

incorporation of lyso-PC into the bilayer occurs until the lyso-PC content is large enough to favor lyso-PC/lyso-PC contacts and formation of polar defects or channels; and (2) at high lyso-PC contents, the bilayer is disrupted and mixed micelles form. IX.2. Absorption enhancement Lyso-PC has been explored for use as an absorption promoter for both oral and nasal drug delivery. While both palmitoyl and stearoyl lyso-PC have been shown by Illum et al. [154] to be comparable to C12/POE-9 ether in terms of blood glucose lowering after nasal insulin co-administration, questions remain regarding the irritation of nasal mucosa. Tagesson et al. [155] demonstrated that, at a lyso-PC concentration of 20 raM, the enhanced ileal permeation of M r 3000-70000 dextrans and bovine serum albumin was accompanied by morphologic damage, described in terms of loss of microvilli, swelling of mitochondria, and cytoplasmic vesiculation. The tight junctions appeared intact between viable cells. In comparing the effect of lyso-PC to 1% Triton X100, Triton induced a greater degree of absorption promotion and greater morphologic damage. Talbot et al. [156] reported that relatively low concentrations of lyso-PC (0.05 0.2 %) increased horseradish peroxidase penetration in guinea pig proximal jejunum without causing morphologic damage. HRP vesicles were localized in the enterocytes and in the extracellular space, but did not penetrate the macula adherens or the tight junction. Using tied loops of rat distal ileum, Bolin et al. [157] demonstrated that 10 mg of lyso-PC did not influence the absorption of ethylene glycol (Mr 62), but did enhance absorption of PEG (Mr 634-1206) and Mr 3000 dextran. This observed enhancement was accompanied by desquamation at villus tips. Thus it appears that the absorption-promoting efficacy of lyso-PC is due to its detergent activity upon the mucosal membrane. Whether a practical degree of absorption enhancement can be achieved with a sub-lytic concentration of lyso-PC remains to be proven. IX.3. Irritation and toxicity The hemolysis of erythrocytes by lysolecithin is well known, and has been extensively reviewed by Weltzien [151]. Talbot and colleagues reported that perfusion of guinea pig small intestine with 0.05-0.2% (w/v) (1~l mM) C16:0lyso-PC resulted in no detectable histological damage [156]. Tagesson et al. [155] observed that in situ infusion of tied rat intestinal loops with 20 mM egg lyso-PC resulted in mucosal damage, including desquamation, loss of microvilli and mitochondrial swelling. Like the bile salts, lyso-PC is a common resident of the intestinal lumen, where it is a lumenal digestive product of dietary phospholipids. However, during digestion, lyso-PC is always present in emulsions or mixed micelles with other lipids, and is present in only small quantities.

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X. Acylcarnitines, acylcholines and acyl amino acids X.1. General properties and interactions with model and biological membranes Acylcarnitincs are natural compounds which are involved in the transfer of fatty acids across mitochondrial membranes. These zwittcrionic detergents have been shown by Yalkowsky and Zografi [180] to exhibit CMC's (in 0.2 M KC1) of 9.0 raM, 0.9 m M and 0.08 mM for dccyl-, lauryl- and myristoylcarnitine, at neutral pH. Studies at lower pH indicate that palmitoylcarnitine exhibits a CMC which is approximately one order of magnitude lower than that of myristoylcarnitine. Haeyert and colleagues [181] have demonstrated that acylcarnitines destabilize dimyristoyl-PC and dipalmitoyl-PC liposomes, as do other detergents. Furthermore, acylcarnitines have been shown to lyse erythrocytes [182,183]. Accumulation of acylcarnitines has been proposed to be one of the important pathological effects of cardiac ischemia. Palmitoylcarnitine is believed to disrupt cardiac plasma membranes and sarcoplasmic reticulum via its detergent action, thus affecting the activitics of mcmbrane-bound enzymes such as Na,K-ATPase [57,75].Acyl cholines are detergents which have been shown to lyse erythrocytes [183]. X.2. Absorption enhancement and toxicity Fix and colleagues [184] have demonstrated that acylcarnitines enhance the absorption of cefoxitin and a cyclic hexapcptide analog of somatostatin, when dosed rectally or duodenally in the rat. Palmitoylcarnitine was generally the most effective absorption enhancer, but lauroyl-, myristoyl- and stearoylcarnitine were also effective. These acylcarnitines were above their CMC's in these experiments, which might suggest that they enhance absorption via detergent activity. However, short-chain (C2 and C6) acylcarnitines enhanced the absorption of the somatostatin analog, and it is unlikely that these shortchain acyl carnitines were micellar at the concentrations used. Fix et al. [184] reported that 1% palmitoylcarnitine caused minor rectal tissue alterations, i.e., minor alterations in the glycocalyx, variation in microvillar length and increased vacuole density. Under similar conditions, Fix et al. observed similar minor effects of 1% SDS. It should be noted that studies described in subsection VIII.3 above indicated that SDS was quite damaging to the mucosa of the small intestine and rectum. Very recently, Fix and colleagues [176] have reported that palmitoylcarnitine increases tight junction permeability, both in vivo (rats) and in Caco-2 cultured cell monolayers. Alexander and Fix [185] reported that medium- and long-chain choline esters enhanced the rectal absorption of cefoxitin and other polar drugs. Lauroylcholine was generally more effective than palmitoylcholine. No mechanistic or toxicological information was reported. Fix and Pogany [186] reported that lysine esters enhanced the rectal and duodenal absorption of cefoxitin and other drugs. The optimal ester chain length appeared to be drug-dependent. The enhancing activity of hcxadecylly-

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sinate was reversible in 60-120 min. No mechanistic or toxicological information was reported. Wu and colleagues studied the effects of N-acyl amino acids on the absorption of ampicillin in rat rectum [187]. Absorption enhancement efficacy increased with acyl chain length. Enhancement was greater for N-acylphenylglycine and N-acylphenylalanine than for N-acylglycine and N-acylalanine. These authors favored calcium ion sequestration as a potential mechanism for the observed enhancement, with possible subsequent alterations of the tight junction. Morphological data were not reported. The potential for detergent activity of these compounds was not discussed, although the amphipathic structures of the N-acyl amino acids suggest that such activity may exist.

XI. Medium-chain glyeerides XI.1. Absorption enhancement Several investigators have reported enhanced intestinal absorption of hydrophilic drugs in the presence of medium-chain glycerides (MCGs), which are mixtures of mono-, di- and triglycerides containing medium-chain-length fatty acids (C8, C10 or C12; caprylic, capric and lauric acids, respectively). Some MCG preparations may also contain free fatty acids and glycerol, but these are usually present in low proportion. Higaki et al. [194] demonstrated a 5-fold increase in the disappearance of phenol red from the small intestine during a recirculating perfusion in the presence of MCG emulsion. In a recent study, the same group showed that the kinetics of phenol red appearance in the plasma after small intestinal perfusion with MCG emulsion ( 0 . 5 4 % w/v) paralleled the kinetics of absorption of M C G itself, and was dependent on the M C G concentration [19J]. Furthermore, the elimination kinetics of phenol red from the plasma were delayed by the absorbed MCG, thus increasing the AUC (area under the curve) of phenol red [195]. Sekine et al. [200] observed that the rectal absorption of cefmetazole sodium in rabbits was increased from 0% to 65% bioavailability in the presence of MGK, a commercially available mixture (from Nikko Chem., Tokyo) containing approximately 56% monocaprylate, 29% dicaprylate, 3% tricaprylate, 3% caprylic acid and 8% glycerol. Yoshitomi and colleagues [197] demonstrated that trilaurate or monolaurate improved cefoxitin absorption in the duodenum to a greater extent than in the rectum, except when lipase was added. Since pancreatic lipase is present in the small intestine, but not in the rectum, lipase-generated fatty acids or monoglycerides may be responsible for the permeability increase [197]. The plasma AUC of duodenally administered cefoxitin was studied as a function of acyl chain length for triglycerides, fatty acids and their salts, and triglycerides preincubated with bile in the presence or absence of lipase. For each lipid class tested, the plasma AUC was maximized at an acyl chain length of 12, with fatty acids and fatty acid salts giving the greatest AUC. Triglycerides preincubated with lipase to generate fatty acids and monoglycerides were

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more effective than those which were not preincubated with lipase [197]. The MCG component primarily responsible for the enhancement of rectal cefmetazole absorption was shown by Sekine et al. [196] to be glycerylmonocaprylate. The mono-, di- and tricaprylate fractions yielded bioavailabilities of 37, 7 and 4%, respectively vs. 0% from an aqueous buffer. The relative cefmetazole absorption promoting efficacy of medium chain monoglycerides was monocaprylate > monocaprate > monolaurate [196]. Van Hoogdalem et al. [199] similarly demonstrated that the enhancement of rectally delivered cefazolin by M G K was almost entirely attributable to monocaprylate. Cefazolin absorption was not promoted by rectal infusion in a vehicle containing medium chain fatty acids, diglycerides, triglycerides, or glycerol. The same group also demonstrated the importance of rectal delivery rate on the enhancement of cefazolin absorption by MGK. A continuous rectal infusion yielded complete absorption of cefazolin, while only about half of the cefazolin was absorbed from a bolus dose of M G K [198], suggesting to the authors that minimal spreading of the vehicle on the mucosal surface may increase the local concentration of MGK. XI.2. Irritation and toxicity Van Hoogdalem et al. [106] reported that infusion of highly concentrated (93%, w/w) MGK, a commercially available mixture of medium chain glycerides, resulted in generalized detachment of rat rectal epithelial cells. The local toxicological effects of lower concentrations of MCG emulsions cannot be predicted from this observation. Medium chain triglycerides are commonly used in a variety of dietary supplements, including baby formulas. In the intestinal lumen, these medium chain triglycerides are degraded by lipase to form various medium chain monoglycerides (and fatty acids), which are subsequently absorbed. Thus MCG's are likely to be minimally toxic or nontoxic when present at low concentrations in the small intestine.

XII. Salicylates XII.1. Interactions with model membranes Casal et al. used infrared spectroscopy to investigate the interactions of dipalmitoylphosphatidylcholine (DPPC) with phenol, salicylic acid, and aspirin [160]. At pH 7.4, these three compounds (at 1:1 m/m with DPPC) caused downward shifts in the DPPC acyl chain order-disorder transition temperature (Tin) of approximately 9, 17 and 8°C, respectively. In the presence of these compounds, band-splitting of the acyl chain a(CH2) mode was maintained at low temperatures, and the cooperativity of the acyl chain transition was not significantly decreased relative to DPPC alone. These observations indicate that the three aromatic compounds are not embedded in the hydrocarbon chain region of DPPC (as surfactants are), and probably exert their effects on Tm indirectly through interactions at the polar surface of the bilayer. Kajii et al. [161] demonstrated that sodium salicylate had no effect on the release of 6-

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carboxyfluorescein from neutral or anionic phospholipid/cholesterol vesicles. These studies demonstrate that salicylates do not interact with membranes in a fashion similar to surfactants.

XII.2. Absorption enhancement Sodium salicylate and several of its derivatives have been investigated as non-surfactant absorption enhancers. The use of sodium salicylate as an enhancer of rectal insulin absorption from suppositories in normal humans was investigated by Nishihata et al. [162]. A significant increase in plasma insulin and decrease in plasma glucose were observed in volunteers receiving suppositories containing 300 mg sodium salicylate and 1.5 or 2 U/kg insulin in a Witepsol H-15 base. The same investigators demonstrated efficacious rectal insulin delivery in normal dogs from a suppository formulation containing sodium salicylate in a triglyceride base [163]. The incorporation of soy lecithin further increased the duration of this effect due to a decrease in the release rate of sodium salicylate from the suppository [163]. Moore et al. [164] demonstrated that the incorporation of sodium salicylate into cocoa butter-based suppositories significantly improved the rectal bioavailability of recombinant h u m a n growth h o r m o n e (rHGH, Mr 22 500) in rats. Further experiments indicated that sodium salicylate and a mineral oil vehicle synergistically improved the bioavailability of rHGH from ligated rat ileum and colon, but not from stomach or jejunum [164]. Saffran and colleagues [165] demonstrated that 0.025% 5-methoxy salicylate enhanced the rectal absorption of the peptides arginine vasopressin and 1deamino-8-D-arginine vasopressin. The efficacy of sodium-5-methoxysalicylate as an enhancer of duodenally delivered insulin was shown by Nishihata et al. [166] to improve when the spreading of solution upon the mucosal surface was reduced by the addition of gelatin to increase the viscosity. The mechanism of salicylate-enhanced absorption from rat rectum was studied by Fix et al. [167], who demonstrated that rectal gentamicin sulfate absorption was promoted by high sodium ionic strength formulations, but not from high potassium ionic strength formulations. The addition of sodium salicylate to the high sodium ionic strength formulation further enhanced rectal gentamicin sulfate absorption. Subsequently, this group demonstrated that active Na ÷ transport is likely involved in the salicylate promotion mechanism [168]. Pretreatment of rectal mucosa with ouabain, which inhibits active Na ÷ transport, significantly reduced salicylate-mediated (sodium salicylate, 5bromo salicylate) absorption enhancement. High ionic strength NaCI increased the effect of salicylate on drug absorption, while high ionic strength choline chloride, which is not actively transported, had no effect [168]. Nakanishi et al. [169] demonstrated that permeabilization of rat rectal membrane to sulfanilic acid and creatinine correlated with the accumulation of acetyl salicylic acid and other non-steroidal anti-inflammatory drugs (NSAID), such as phenylbutazone, diclofenac and indomethacin in the rectal tissue. The effect of NSAID was associated with only slight histologic alterations and

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minimal protein release, and was rapidly reversible upon switching from a NSAID-containing perfusate to a control solution. These investigators proposed that the mechanism underlying the absorption promotion of these model drugs by NSAID involved interaction of NSAID with both membrane protein and lipid [170]. The pretreatment of rectal mucosa with papain, which hydrolyzes surface proteins without disrupting the membrane, or HgCI2, which irreversibly modifies membrane protein sulfhydryls, reduced NSAID accumulation and permeability enhancement. Further, in vitro salicylate treatment of egg lecithin/cholesterol liposomes or rectal lipid liposomes increased the rate of release of trapped sulfanilic acid, supporting the possible interaction of salicylate with membrane lipids [170]. Nishihata et al. [171] reported that the effect of sodium salicylate on rectal theophylline absorption was rapidly reversible following washout, while SDSinduced absorption promotion persisted after the surfactant was removed, suggesting a difference in the promotion mechanism between surfactants and salicylates. However, salicylate and its derivatives may also enhance the absorption of macromolecules by grossly disrupting the brush border membrane. In a study by Peters et al. [172], sodium-5-methoxysalicylate at doses of 60 mg or more was shown to markedly increase the portal vein appearance of somatostatin (hexapeptide analog), insulin and horseradish peroxidase in a vascularly perfused rat small intestine model. Visible histologic damage was observed within 10 min, with progressive deterioration to extensive damage in 1 h. Caldwell et al. [174] and Nishihata et al. [173] have suggested that absorption enhancement by 5-methoxysalicylate in the rectum can be attributed to increased lymphatic drug uptake, even for polar drugs such as cefoxitin, phenol red, insulin and theophylline. Lymphatic uptake may further increase bioavailability by bypassing hepatic 'first pass' metabolism. At present, the mechanism of the absorption enhancing effect of salicylates remains an open question. XII.3. Irritation and toxicity Sithigorngul et al. [175] reported that exposure of the rat rectal epithelium to 2% sodium salicylate resulted in minimal damage at neutral pH, the most significant effect being an alteration of the cell surface glycocalyx. Salicylateinduced rectal epithelial cell permeability to the dye Trypan blue was shown to be reversible after a 15 min washout. Van Hoogdalem and colleagues [106] observed 'moderate' effects of 6% (w/v) sodium salicylate on rat rectal mucosa, with some epithelial detachment observed 2 h post-dose, which appeared to persist for 24 h. This mucosal damage was judged to be comparable to that caused by indomethacin suppositories and by the suppository bases Witepsol H15 and PEG 1540/6000 [106]. On the other hand, Peters and colleagues [172] observed extensive mucosal stripping of the rat small intestine when sodium 5-methoxysalicylate was perfused. The effects of aspirin on the mucosa of the canine stomach were

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studied by Meyer et al. [178]. Exposure to a low concentration (20 mM) of aspirin resulted in focal damage of approximately 10% of the epithelial surface, as assessed by light microscopy. Electron microscopy revealed alteration of 67% of the cells viewed, with the major alterations involving abnormalities in the tight junctions. The reversibility of these lesions was not evaluated. The salicylates are known to cause mild gastric bleeding and, in some cases frank ulceration, as do many non-steroidal anti-inflammatory drugs. These effects probably have both a topical and a systemic component [179]. However, as is well-known, salicylates have an extensive history of relatively safe dosing in humans.

XIII. Ethylenediaminetetraacetic acid The salts of ethylenediaminetetraacetate (EDTA) were recognized as effective enhancers of gastrointestinal absorption in 1961 by Windsor and Cronheim [208]. They found concurrent oral delivery of sodium EDTA with heparin significantly increased heparin in the blood of rats and dogs, as estimated from clotting time and lipemia clearing activity. Cassidy and Tidball in 1967 [78] established the foundation for the now widely held belief that EDTA enhances intestinal permeation of drugs by altering the fine structure of cellular tight junctions via a divalent cation chelation mechanism. In their work, phenol red absorption was greatly increased, while tissue levels of Ca 2+ and Mg 2+ were depleted, by sodium EDTA. Replacement of C a 2 + or Mg 2+ in the perfusion solutions restored the normally low permeability of phenol red and normalized tissue levels of these cations. Histologic evidence of widening and swelling at the tight junctions supported the conclusion that paracellular transport of phenol red had occurred. Based on the effect of an applied transmural potential difference on sulphanilic acid permeation through rat jejunum in vitro, Yamashita et al. [210] concluded that the permeation-enhancing effect of 10 mM EDTA occurred exclusively through paracellular pathways. Shiga et al. [211], using an in situ recirculating rectal perfusion in the rat, reported that a 1% EDTA solution increased the clearance of antipyrine from the lumen. EDTA also enhanced flux of water both into and out of the lumen. Pretreatment of the rectum with ouabain reduced both the water flux and antipyrine absorption, suggesting the involvement of active transport mechanisms. In a subsequent experiment, this group found that EDTA induced net water secretion into the lumen [212]. Similarly, Fix et al. [168] found that ouabain pretreatment reduced the observed increase in rectal permeability to gentamicin sulfate with 20 mM EDTA in the intact rat. Nishihata et al. [213] demonstrated improved rectal cefoxitin absorption in rats in the presence of EDTA, with further enhancement induced by addition of NaC1. EDTA induced release of protein from rat everted rectal sacs, perhaps implicating membrane disruption in the enhancement mechanism. An earlier study from the same laboratory [171] showed that only at high concentrations

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(30%) was EDTA effective in enhancing rectal theophylline absorption in rats, and that these EDTA concentrations were accompanied by the appearance of blood in the perfusate. The route of administration influences the efficacy of absorption promotion by EDTA. Ishizawa et al. [214] found significant enhancement of fosfomycin absorption from rat jejunum, but not colon, in the presence of 1% disodium EDTA. Aungst and Rogers [215] also demonstrated site-specific effects of Na2 EDTA on insulin absorption. In their study, EDTA effectively promoted insulin absorption from the rectum, but not from the nasal or buccal cavities. XIV. Particulate carriers

An interesting and unusual approach involves the delivery of drugs in small particles ('nanoparticles'), generally of size up to 10/~m, which are believed to cross the intestinal wall intact. In 1961, Sanders and Ashworth [222] observed that 220 nm polystyrene particles were absorbed intact in the rat intestine. In 1974, Volkheimer [223] observed absorption ('persorption') of intact starch particles (5-110 #m) in animals and humans. LeFevre et al. [224] similarly observed absorption of 2 #m polyvinyltoluene latex beads in mice fed latex suspensions in drinking fluid. This particulate uptake appeared to involve Peyer's patches, and was quantitatively small. Jani et al. [225,226] more recently demonstrated that 50 nm and 500 nm polystyrene particles are taken up by intestinal Peyer's patches and enter the lymphatic circulation. Delivery of insulin by this approach has been reported by Damge et al. [227], who demonstrated glucose-lowering in diabetic rats orally dosed with 220 nm insulin-loaded polyalkylcyanoacrylate 'nanocapsules'. Eldridge and colleagues [228] have recently demonstrated that orally dosed microspheres composed of the hydrophobic polymers polystyrene, poly(methyl methacrylate), and polyhydroxybutyrate were absorbed by mouse Peyer's patches to a greater extent than those composed of the more hydrophilic materials poly(D,L-lactide), poly(L-lactide), and poly(D,L-lactide-co-glycolide). Microspheres composed of the cellulosic polymers ethyl cellulose, cellulose acetate hydrogen phthalate, or cellulose triacetate were not absorbed. These authors reported that Peyer's patches absorbed microspheres of diameter less than 10/~m and that other portions of the GI mucosa did not absorb particles, even those of submicrometer size. The microsphere content of Peyer's patches was observed to decrease with time after oral dosing, with microspheres still present at 35 days after oral dosing. As the microsphere content of the Peyer's patches decreased, microspheres appeared in the mesenteric lymph nodes (peaking at day 7) and the spleen (peaking at day 14). Eldridge et al. also orally dosed mice with biodegradable poly(o,L-lactide-co-glycolide) microspheres which contained a staphylococcal enterotoxin B vaccine. This treatment resulted in induction of a disseminated mucosal IgA anti-toxin response. While the efficiency of particulate uptake appears to be low, it may be sufficient for delivery of vaccines. Reproducible and efficient delivery of

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therapeutic agents remains to be demonstrated. The toxicity of this approach is an issue which remains to be investigated. XV. Other absorption enhancers

A variety of additional chemical agents have exhibited the capability to enhance the oral or rectal absorption of polar drugs. These agents have widely varying structures and act by a variety of mechanisms. For example, azone [219], phenothiazines [220,231-234], and oleic acid vesicles ('ufasomes') [221] probably enhance permeability via their surfactant properties. The mechanism of enhancers such as diethoxymethylene malonate and diethyl maleate [159] is less clear. These and additional enhancer types have been reviewed by Van Hoogdalem et al. [5] and by Muranishi [6]. XVI. Discussion

Most approaches for enhancement of the permeability of the intestinal wall require alteration of the tight junction or the apical membrane of the intestinal epithelial cell. While some enhancement treatments may affect the tight junction (e.g., EDTA, low concentrations of bile salts), the majority of effective approaches act directly on the apical membrane. Rigorous mechanistic interpretation of absorption enhancement experiments is confounded both by mucosal damage and by the complex composition of the lumenal contents. For example, intestinal perfusion experiments with surfactants, emulsions or mixed micelles do not take into account the fact that, in vivo, the physical properties of the enhancer mix will likely be drastically altered by the presence of endogenous bile salt/lecithin/cholesterol micelles and in some cases by lumenal lipase. Future experimental work should be aimed at understanding the complexity of absorption enhancement in vivo. In most in vivo studies involving surfactants, enhanced polar drug absorption has been correlated with histological damage to the intestinal wall. It is possible that permeation enhancement and histological damage are independent sequelae of the partitioning of surfactants into the apical membrane of the brush-border cell. Alternatively, the enhanced permeation of polar solutes may occur directly through damaged regions. While this remains an open question, permeation enhancement and damage appear to be almost inseparable events. Accepting the association of permeability enhancement with mucosal damage, the important issue becomes "How serious is this damage?" Two points are important here. First, bile salt/monoglyceride and bile salt/fatty acid mixed micelle 'enhancers' have been shown to damage the intestinal epithelium. This result is interesting, because during the course of normal digestion bile salt/monoglyceride/fatty acid mixed micelles are present at relatively high concentrations in the intestinal lumen. Thus the damage caused by mixed micellar 'enhancers' may be a common physiological phenomenon. Second,

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repair of surfactant-induced epithelial damage may occur quickly. For example, a recent report by Moore and Madara [158] indicates that repair of focal epithelial denudation caused by Triton X- 100 in an Ussing chamber experiment may occur very rapidly, i.e., within approximately 1 h. This repair appears to involve two mechanisms: (1) migration of undamaged epithelial cells to cover the denuded area and (2) villus shortening, resulting in reduction of the size of the injured area. In a similar experiment, Fasano et al. [71] demonstrated that CDOC-enhanced mucosal permeability to lactulose was reversible within approximately 40 min. In addition, Erickson [189] demonstrated that mucosal restitution occurs within 1 h of the cessation of CDOC perfusion. Similarly, Nakanishi et al. [190] observed that histological damage to rat rectal epithelial cells by 5 mM DOC, 5 mM SDS, 25 mM EDTA, or 50% PEG-400 was reversed after a 2 h perfusion without enhancer. Recovery from damage to rectal goblet cells did not occur as quickly, with 40-95% recovery observed at 24 h post-dose [190]. These considerations lead to guarded optimism about the prospects for using absorption-enhancing excipients to enable the oral dosing of polar peptides and proteins. The availability of the bile salts chenodeoxycholate and ursodeoxycholate as marketed therapies for gallstone disease provides further encouragement. In the end, the question of enhancer toxicity can only be answered with proper toxicology studies designed to address scientific and regulatory concerns. However, three practical concerns must somehow be addressed. First, enhancer-induced epithelial damage, however transitory, could be exacerbated by drug interactions in patients who are undergoing multiple therapies. For example, coincident dosing with non-steroidal antiinflammatory drugs could be problematic because some drugs of this class elicit gastrointestinal side effects. Second, side effects could result from hyperabsorption of a coincidently absorbed drug which normally has a low bioavailability. This potential problem was pointed out in 1965 by Tucker [191], when the anionic surfactant DOSS was under consideration as a material for tablet coating. Two subsequent letters by Naess [192] and Godfrey [193] warned that formulations of oxyphenasatin which contained DOSS might unintentionally give a high (and potentially toxic) oxyphenasatin exposure, due to the surfactant properties of DOSS. It is not clear whether this issue was ever settled. One potential approach to assessing the significance of this problem would be to follow adverse drug reactions carefully in patients treated with chenodeoxycholate for gallstone therapy. Third and finally, immunological effects resulting from unintended enhanced absorption of environmental antigens (e.g., undigested dietary proteins) could be problematic. Choice of an enhancer and an enhancer dosing regimen is a complicated issue which has many correlates in the more common problem of selecting a drug candidate for development. The following considerations are important. (1) In vitro potency. A drug candidate must exhibit significant in vitro potency against a specific target. Likewise, an enhancer must have the capacity to affect the permeability of the tight junction or the apical epithelial membrane.

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(2) Pharmacokinetics. A drug candidate must reach a significant concentration in its therapeutic compartment and must maintain this concentration for a sufficient time to assure therapeutic efficacy. Similarly, an enhancer must reach a significant concentration in its therapeutic compartment, which is the small intestinal, colonic or rectal lumen. For some surfactant enhancers, this threshold concentration may be the CMC. Furthermore, this efficacious concentration must be maintained in the lumen for a period of time sufficient to assure that a therapeutic drug dose is absorbed. This constraint is affected by the size of the drug dose, the GI residence time and the time dependence of the enhancer concentration (i.e., the kinetics of disappearance of the enhancer due to spreading and the absorption of the enhancer itself). If the enhancer is quickly absorbed, then its concentration may fall below its threshold of efficacy (e.g., its CMC) quickly. (3) Safety. As with a drug candidate, an enhancer candidate must cause minimal toxicity. The enhancer must have an acceptable 'therapeutic index'. These considerations lead to the conclusion that the enhancer of choice for a polar drug may not necessarily be the enhancer which is most potent in vitro or in situ. The enhancer of choice will be one which enables the reproducible delivery of a therapeutic drug dose (which may be small for proteins) in the GI residence time available, with minimal toxicity. The oral delivery of proteins is obviously a complex problem which involves both stability and permeability issues. The successful development of oral protein dosage forms will require attention to both issues, and dosage forms have been recently described which contain both absorption enhancers and protease inhibitors [188,209,229,230]. In our opinion, sufficient evidence exists to support guarded optimism that the permeability problem can be solved, if the toxicological issues can be appropriately addressed.

Acknowledgements We thank Drs. J. Fix and J.H. Rytting for helpful discussions about salicylates. We are grateful to Dr. T. Hagen for a critical reading of the manuscript, and for his encouragement and support.

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