Intestinal Permeation Enhancers

MINI-REVIEW Intestinal Permeation Enhancers BRUCE J. AUNGST DuPont Pharmaceuticals Co., P. O. Box 80400, Wilmington, DE 19880-0400

Received 20 December 1999; accepted 4 January 2000

This review addresses the field of improving oral bioavailability through the use of excipients that increase intestinal membrane permeability. The critical issues to consider in evaluating these approaches are 1) the extent of bioavailability enhancement achieved, 2) the influence of formulation and physiological variables, 3) toxicity associated with permeation enhancement, and 4) the mechanism of permeation enhancement. The categories of permeation enhancers discussed are surfactants, fatty acids, medium chain glycerides, steroidal detergents, acyl carnitine and alkanoylcholines, N-acetylated ␣-amino acids and N-acetylated non-␣-amino acids, and chitosans and other mucoadhesive polyers. Some of these approaches have been developed to the stage of initial clinical trials. Several seem to have potential to improve oral bioavailabilities of poorly absorbed compounds without causing significant intestinal damage. In addition, the possible use of excipients that inhibit secretory transport is reviewed. © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci

ABSTRACT:

89:429–442, 2000

INTRODUCTION There is both great interest and a medical need for improving the oral bioavailabilities of various poorly bioavailable drugs. Maximizing oral bioavailability is therapeutically important because the extent of bioavailability directly influences plasma concentrations, as well as the therapeutic and toxic effects, resulting after oral drug administration. Poorly bioavailable drugs are inefficient because a major portion of a dose never reaches the plasma or exerts its pharmacologic effect. Moreover, a compilation of published results on structurally diverse drugs showed that the intersubject variability in bioavailability was inversely Correspondence to: B. J. Aungst (E-mail: bruce.j.aungst@ dupontpharma.com) Journal of Pharmaceutical Sciences, Vol. 89, 429–442 (2000) © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association

correlated with the extent of bioavailability.1 Therefore, low oral bioavailability leads to high variability and poor control of plasma concentrations and effects. Intersubject variability would be particularly of concern for a drug with a narrow safety margin or a steep dose vs effect profile. Incomplete oral bioavailability has various causes. These include poor dissolution or low aqueous solubility, degradation of the drug in gastric or intestinal fluids, poor intestinal membrane permeation, and presystemic intestinal or hepatic metabolism. This review addresses improving bioavailability of those compounds with poor membrane permeability and the use of excipients that increase intestinal permeability. What types of compounds have poor intestinal permeability? Some physicochemical properties that have been associated with poor membrane permeability are low octanol/aqueous partition-

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ing, the presence of strongly charged functional groups, high molecular weight, a substantial number of hydrogen-bonding functional groups, and high polar surface area. The compounds that may benefit most from intestinal absorption– enhancing formulations usually have one or more of these characteristics. Typically the drugs involved in absorption enhancement studies have been peptides, peptide analogs, or other polar, high molecular weight drugs, such as heparin. For some compounds, permeation through the intestinal epithelium is hindered by their active transport from the enterocyte back into the intestinal lumen. The secretory transporters involved may include P-glycoprotein (Pgp), the family of multidrug resistance–associated proteins (MRP), and possibly others. For substrates of these secretory transporters, inhibiting secretory transport can increase permeation in the absorptive direction. This can be accomplished with pharmacologically inactive excipients. Before permeation-enhancing excipients are evaluated for a particular drug of interest, it is important to understand the cause of its low oral bioavailability. Ideally, first-pass metabolism and solubility are not limiting oral bioavailability. Furthermore, the role of secretory transport should be assessed, because excipients that increase absorption by inhibiting secretory transport are different from those that increase absorption by other mechanisms. This review is not intended to be comprehensive. Rather, the focus is to address the most critical issues for the success of intestinal permeation–enhancing excipients and to enable readers to evaluate how far some of the more advanced technologies have come toward fulfilling these criteria. Previous reviews on this subject2–5 are recommended for more detailed information or for different perspectives.

CRITICAL ISSUES TO CONSIDER How should one evaluate the usefulness of any particular formulation or excipient for enhancing intestinal permeation? Certainly the most obvious indicator of success is the extent of bioavailability enhancement realized. Bioavailability is, of course, measured by performing in vivo studies. One important point to consider is that although an agent may greatly increase intestinal permeability in vitro or in situ, that does not ensure that the agent will significantly improve oral bioavailJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 4, APRIL 2000

ability. Generally, the effects of an absorptionenhancing excipient are related to its concentration at the site of drug absorption. The absorption of the drug could be intestinal site dependent, and the effects of the excipient are often also intestinal site dependent. The drug and the absorption promoter must be delivered to the absorption site simultaneously, and a sufficient concentration of the absorption promoter must be achieved and maintained there. This could be subject to the influence of both formulation and physiologic variables, such as dissolution rate, intestinal fluid pH, and intestinal motility, and may influence intersubject and intrasubject variability. As discussed previously, low bioavailability is undesirable because it is associated with large intersubject variability in plasma concentrations and effects. So another desired effect of an absorptionenhancing formulation would be a reduction of intersubject variability. Finally, an agent that increases intestinal permeability must do so without causing toxicity. The potential to cause cytotoxicity or gastrointestinal side effects must be evaluated. Toxicity is probably often closely related to the mechanism of intestinal permeation enhancement. Some mechanisms may inherently have lower potential to cause toxicity than other mechanisms. For example, a transient opening of tight junctions would seem less damaging than a disruption of cell membrane structure. Thus, it is important to develop an understanding of the mechanism of an absorption-enhancing formulation or excipient.

IN VITRO PERMEATION ENHANCEMENT, MECHANISMS, AND TOXICITY Often it is easiest to evaluate the effectiveness, mechanism, and potential for toxicity of absorption enhancers using in vitro models of the intestinal epithelium, typically Caco-2 cell monolayers or intestinal segments of animals. These in vitro studies can provide an initial indication of the extent of permeation enhancement possible for a drug of interest and whether this can be accomplished without damaging the membrane. More detailed in vitro studies often provide evidence of the mechanism of permeation enhancement. Many types of intestinal permeation enhancers have been evaluated in such in vitro studies. Table 1 presents an overview of representative in vitro studies describing the properties of some of the absorption enhancers discussed in this re-

Table 1. Overview of Representative Studies Describing in vitro Effects of Intestinal Permeation Enhancers

Enhancer

Membrane

Permeability Enhancing Effects

Concentration ↑ Permeability

Caco-2

↓ TEER, ↑ mannitol & PEG 4000 Papp

<0.58 mM

Dioctyl Na sulfosuccinate

Caco-2

↓ TEER, ↑ mannitol & PEG 4000 Papp

ⱕ0.48 mM

Na caprate

Caco-2

↑ fluorescein, FITC-dextran 4000, & rhodamine 123 Papp

0.2% (10 mM)

Na caprate MCG:PC (3:1)

Rat ileum Caco-2

1–10 mM ⱖ4 mM

Deoxycholate

Caco-2

↑ [51Cr]-EDTA Papp ↓ TEER, ↑ mannitol & low MW heparin Papp ↑ fluorescein, FITC-dextran 4000 & rhodamine 123 Papp

Glycocholate Taurodihydrofusidate

Caco-2 Caco-2

10 mM 2.8 mM

Saponin, DS-1

Caco-2

Palmitoylcarnitine

Caco-2

↑ mannitol Papp ↓ TEER, ↑ fluorescein & FITC-dextran 4000 Papp ↓ TEER, ↑ mannitol & decapeptide Papp ↓ TEER, ↑ mannitol & PEG 4000 Papp

Palmitoylcarnitine

Rat colon

↓ TEER, ↑ lucifer yellow Papp

Concentration vs. Toxicity

MTT IC50 ⳱ 0.36 mM, morphologic changes at 0.58 mM MTT IC50 ⳱ 0.36 mM, morphologic changes at 0.48 mM Paracellular, ↓ Trypan blue exclusion, ↑ transcellular protein release & DNA staining at 0.5% (26 mM) Paracellular Altered morphology at ⱖ8 mM

0.025–0.2%

Paracellular, ↓ Trypan blue exclusion, ↑ transcellular protein release & DNA staining at 0.1% (2.4 mM) MTT IC50 ⳱ 24 mM Paracellular ↓ Trypan blue exclusion at <8 mM Transcellular No morphologic effect at 0.1%

ⱖ0.4 mM

Paracellular

ⱕ1 mM

Paracellular

0.05% (1.2 mM)

↑ LDH at ⱖ0.2 mM, ↑ DNA staining & ↓ neutral red retention at ⱖ0.4 mM Minor histologic change at 1 mM, moderate at 5 mM

Reference 6

6

7, 8

9 10 7, 8

11 12 13 14

15

LDH ⳱ lactate dehydrogenase; MTT ⳱ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, a substrate of mitochondrial dehydrogenases; TEER ⳱ transepithelial electrical resistance.

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Na lauryl sulfate

Mechanism

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view. Both permeability enhancement and cytotoxicity depend on the time of exposure, so comparisons among studies performed using different protocols may not be valid. However, it is apparent that many, if not most, of the compounds examined as membrane permeation enhancers in vitro cause cytotoxicity or membrane damage. The issue then becomes whether the concentrations associated with membrane permeation enhancement are separated from concentrations causing membrane damage. A closer examination of the studies cited in Table 1 suggests that enhancers often have steep concentration vs effect profiles in vitro, with relatively small safety margins. Intact intestinal membranes often are found to be more resistant to the cytotoxic effects of permeation enhancers than are cell culture models. For example, dioctyl sodium sulfosuccinate (docusate) is used as a stool softener at suggested doses up to 200 mg, which if dissolved in a volume of gastrointestinal fluids of 250 mL would give a concentration of 1.8 mM. Although this concentration was cytotoxic to Caco-2 cells,6 docusate has few side effects in clinical practice. Some enhancers have been clearly cytotoxic in Caco-2 studies but caused relatively little damage when dosed to animals at doses effective for absorption enhancement. This difference could be because the intact tissue has mechanisms for recovery from trauma, which may not be present in cell cultures. Also, the intact intestine has a protective mucous layer, whereas cell culture monolayers do not. Despite the differences between in vitro and in vivo effects, in vitro studies with cell culture systems are useful for predicting the rank order of in vivo permeation-enhancing effects. Quan et al16 showed a good rank order correlation between Caco-2 and rat colonic permeation of FITC-dextran 4000 using five structurally diverse absorption enhancers. Enhancer concentrations producing similar Papp values were 1 mM for Caco-2 and 20 mM for rat colon.

FACTORS INFLUENCING IN VIVO PERFORMANCE Success in enhancing oral bioavailability in vivo requires the simultaneous delivery of the drug and effective concentrations of the absorption promoter to the absorption site. The targeted absorption site has to be a site where the absorption promoter has significant effects. This co-delivery JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 4, APRIL 2000

requirement can be challenging. It is quite common for in vivo studies to take advantage of dosing the drug and enhancer directly to a particular intestinal site. It has also often been the case that the bioavailability of a drug administered with an enhancer is profoundly lower after oral dosing than after administration directly into the intestine. An orally administered enhancer will be diluted by the fluids of the gastrointestinal tract and could spread over a large surface. The volume of fluid present in the gastrointestinal tract and gastric emptying and intestinal motility will affect the dilution and residence time at any particular site. In vivo studies administering enhancer formulations into intestinal ports of dogs showed that low dosing volumes and bolus input into the intestine provided optimal conditions for improving bioavailability. 17 Formulation approaches such as enteric coating have been used to deliver the drug and enhancer to the intestine more in a bolus. Enhancers may differ in their delivery requirements for optimal performance. For example, sodium decanoate was more effective in enhancing cefazolin absorption from the rat jejunum when administered at a concentration of 50 mM over 30 minutes than when administered at 100 mM over 15 minutes.18 In this case a sustained-release matrix was proposed for controlling the release of both drug and enhancer. A number of enhancers have been shown to have greater effects on absorption from the colon or ileum compared with upper small intestine. In these cases the enhancer and drug should be released in the lower small intestine or colon for optimal effect. Some enhancers, such as certain chitosans, have pH-dependent absorptionenhancing effects. In these cases maintaining the local gastrointestinal pH is important. As the various categories of intestinal permeation enhancers are discussed below, factors most likely to influence in vivo performance will be addressed. Surfactants Various nonionic, anionic, and cationic surfactants have been investigated as intestinal permeation enhancers. Readers are referred to the review by Swenson and Curatolo5 for in-depth information on this subject. This article only briefly addresses this category of compounds and their effects. For nonionic surfactants the size and structure of both the alkyl chain and the polar group influence absorption-enhancing activity. Generally, these seem to affect membranes by

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solubilizing membrane components. Sakai examined polyoxyethylene (POE) ethers, POE esters, and POE sorbitan esters and showed a good correlation between the enhancement of colonic absorption of p-aminobenzoic acid in rats and lactic dehydrogenase (LDH) release from the intestine.19 Within a series of nonylphenoxypolyoxyethylene surfactants, there was also a good correlation between enhancement of intestinal absorption of phenol red in rats and LDH release or phospholipid release.20 LDH and phospholipid release correlated with histologic observations of intestinal damage in rats, although the intestinal epithelial damage was rapidly reversible.21 More recently, dodecylmaltoside (DM) has been proposed as an absorption enhancer. DM (20 mM) increased the small intestinal and colonic absorption of phenol red in rats while not significantly elevating membrane protein and phospholipid release.22,23 However, in another study 20 mM DM increased in vitro permeability of an adrenocorticotropic hormone (ACTH) analog through rat jejunum and colon, but in this instance significant protein and phospholipid release was noted.24 DM increased the colonic absorption of azetirelin 8.7-fold in rats, and increased oral bioavailability in dogs from 15% to 44% when combined with citric acid in an entericcoated capsule formulation.25

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greater for rat colon than rat jejunum.24 Control colonic permeability was lower than control jejunal permeability, but in the presence of sodium caprate jejunal and colonic permeabilities were similar. Exposure of Caco-2 monolayers to 10 to 24 mM sodium caprate resulted in dilatations of the tight junctions.29 A mechanism by which sodium caprate increases paracellular permeation was proposed by Tomita et al30 and Lindmark et al31 and is outlined in Figure 1. This mechanism was proposed based on Caco-2 studies, but other results using isolated rat and human colon specimens were consistent with this mechanism.32 Sodium caprate may also affect transcellular permeation because it caused a release of phospholipids into rat colonic lumen in situ and affected permeability and fluorescence polarization of brush border membrane vesicles.27,28 The in vitro effects of sodium caprate on Caco-2 TEER and mannitol permeability acutely depend on caprate concentra-

Fatty Acids Although various fatty acids have been shown to have membrane permeation–enhancing activity, sodium caprate has been the most thoroughly characterized for use as an absorption-enhancing excipient. Sodium caprate (10-13 mM) increased the Caco-2 permeabilities of mannitol, PEGs, argvasopressin, and FITC-dextrans of 4000 and 20,000 molecular weight, and those compounds with molecular weight <1200 had Papp values in a range consistent with significant oral absorption in vivo.26 In rats the in situ absorption of cefmetazole from closed colonic loops was increased approximately 10-fold by sodium caprate, whereas the increases with sodium caprylate and sodium laurate were twofold and sevenfold, respectively.27 Under similar experimental conditions, the increase of in situ jejunal absorption of cefmetazole was only sixfold, suggesting that the colon was more sensitive than the jejunum to absorption enhancement with sodium caprate.28 Also, the extent of enhancement by sodium caprate of in vitro permeation of a peptide was

Figure 1. Proposed mechanism by which sodium caprate increases intestinal permeability. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 4, APRIL 2000

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tions.29 Concentrations greater than the upper limit of those used to increase permeability (e.g., >24 mM) were associated with cytotoxicity.8,29 However, sodium caprate is used as an enhancer in a rectal suppository formulation of ampicillin in some countries. This suppository was shown to induce reversible damage to the rectal mucosa of human subjects, which was suggested to be partly due to sodium caprate and partly due the other suppository components.33 In vivo absorption enhancement with sodium caprate depends on the formulation and how the drug and sodium caprate are released. The oral absorption of a polar, nonmetabolized peptide was enhanced in rats and dogs using capsules containing a semisolid matrix of sodium caprate, polyethylene glycol, and water, whereas other formulations were less effective.2,34 The extent of absorption enhancement reported ranged from < twofold to fourfold to fivefold, with maximum bioavailabilities in rats and dogs of approximately 7 and 19%, respectively. Intraduodenal bioavailability of calcein in rats was improved from 2% to 25– 37% with microemulsion formulations containing a mixture of caprylic acid or capric acid and their sodium salts, as well as medium chain glycerides.35 Longer chain fatty acids may also be effective as membrane-permeation enhancers, but because of their lower water solubilities, these are often combined with emulsifying agents. As an example, emulsions (w/o/w) containing oleic, linoleic, or linolenic acid were effective in increasing the in situ colonic absorption of insulin in rats.36

Medium-Chain Glycerides The term medium-chain glycerides (MCGs) generally refers to monoglycerides and diglycerides of caprylic and capric acid. These often are supplied as mixtures that may also contain small amounts of triglycerides as well as monoglycerides and diglycerides of shorter and longer chain fatty acids. Medium-chain triglycerides are used as pharmaceutical excipients and as nutritional agents, but these are much less active than monoglycerides and diglycerides as membrane-permeation enhancers. MCGs may be metabolized to free fatty acids in the intestinal lumen, but there have been no results suggesting that metabolism to fatty acids is required for absorption enhancement. Because MCGs are lipophilic and poorly water soluble, they have often been studied in combination with emulsifying or solubilizing agents, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 4, APRIL 2000

which probably affect their performance as absorption enhancers. A medium-chain glyceride/phosphatidylcholine (3:1) mixture (MCG/PC) reduced Caco-2 TEER and increased Papp of mannitol and a low molecular weight heparin approximately 10-fold at concentrations ⱖ4 mM.10 Concentrations of MCG/PC ⱖ8 mM caused irreversible increases in permeability and altered the morphology of the cell monolayer. Intraduodenal bioavailabilities of the poorly absorbed antibiotics cefamandole, cefotaxime, moxalactam, mezlocillin, penicillin G, ampicillin, cefoxitin, and carumonam were markedly improved when administered in an MCG vehicle in rats (0.5 mL/rat).37 In vitro permeability studies with mannitol and cephalexin indicated that the distal colon of rabbit was much more sensitive to the absorption-enhancing effect of MCG than was the ileum.38 And the in situ absorption of cefmetazole in dogs administered a MCG vehicle had the rank order ileum and middle jejunum > upper jejunum > duodenum.39 The in vivo safety of MCG has been assessed in a study in which an MCG vehicle was administered rectally to rats (0.25 mL/kg), rabbits (1.75 g), and dogs (1.75 g), and no remarkable morphologic changes to the rectal mucosa were observed.40 MCG has been delivered orally in several types of formulations. A self-emulsifying w/o microemulsion formulation containing 22% MCG was administered intraduodenally to rats (3.3 mL/kg); the bioavailability of calcein improved from 2.4% to 45%, and the bioavailability of a hydrophilic peptide improved from 0.5 to 27%.41 To aid delivery to the lower intestine, capsules containing cefmetazole in an MCG solution were enteric coated.39 Cefmetazole bioavailability in dogs was 65% with the enteric-coated MCG capsules, 21% with uncoated MCG capsules, and 6% with enteric-coated capsules without MCG. Enteric coating of capsules also improved bioavailability of ceftriaxone in monkeys when administered in MCG.42 Monoglycerides other than MCG, and other types of MCG formulations, have also been considered. Monohexanoin was more effective than MCG in increasing oral absorption of dDAVP in rats.43 A monoolein/sodium taurocholate combination (40 mM) increased the colonic absorption of calcitonin, horseradish peroxidase, and polyethylene glycol (PEG) 4000 in rats, while causing no morphologic damage to the colonic mucosa.44 The intracellular appearance of horseradish per-

INTESTINAL PERMEATION ENHANCERS

oxidase suggested enhancement of transcellular permeation. Steroidal Detergents Swenson and Curatolo5 previously provided a thorough summary of the properties of bile salts as absorption enhancers. Bile salts are produced in the liver and are usually found in the gastrointestinal tract in the form of mixed micelles with lecithin, monoglycerides, fatty acids, and cholesterol. Both conjugated and unconjugated bile salts have membrane permeation–enhancing effects. Bile salts have a high capacity for phospholipid solubilization, and a number of investigators have correlated membrane permeation–enhancing effects with the release of membrane phospholipids and protein. This effect of bile salts could be damaging to the intestinal mucosa. However, various studies have also shown that the absorption-enhancing effects of bile salts are reversible. The safety of bile salts as excipients has not yet been resolved. There is some history of use of chenodeoxycholate (CDCA) and ursodeoxycholate (UDCA) in oral products for treating gallstones, with doses of approximately 1 g/day. CDCA and UDCA were recently evaluated as absorption promoters for octreotide, a somatostatin analog.45 CDCA (1%) increased Caco-2 Papp of octreotide approximately threefold, whereas UDCA was not effective. In rats, 1% CDCA increased the absorption of intrajejunally administered octreotide from 0.26% to 20%, whereas UDCA gave 5% absorption. CDCA and UDCA were further evaluated in humans at 100-mg doses, and octreotide oral bioavailability was 1.26% with CDCA and 0.13% with UDCA; a control formulation without bile salt was not administered. So CDCA was consistently more effective than UDCA in enhancing octreotide absorption through Caco-2 and rat jejunum and after oral dosing to humans, although the extent of enhancement attained varied among those three experiments. The colon may be more sensitive than the small intestine to the absorption-enhancing effects of bile acids, as shown in the effects of glycocholate on insulin absorption in rats.46 Bile salts are normally present in the intestinal lumen in the form of mixed micelles. The in vitro effects of bile salts on epithelial membranes can be quite different when they are incorporated with other agents in mixed micelles. For example, the permeation-enhancing effects and cytotoxicity of taurocholate on Caco-2 monolayers was greatly

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reduced when incorporated with phospholipids or cholesterol in mixed micelles, but a mixed micelle composed of taurocholate and oleic acid had much more potent membrane effects than taurocholate alone.47 Other compounds with more unusual steroidal structures have also been examined as absorption enhancers. Saponins, which are found in plants, are glycosides having steroid or triterpenoid aglycones. A purified and semisynthesized saponin, DS-1, increased transcellular Caco-2 permeation of mannitol and a peptide drug, while having little adverse effect on cell viability.13 Glycyrrhizinate, which is found in licorice, contains two glucuronosyl moieties linked to a steroid. Glycyrrhizinate itself had little or no effect on Caco-2 permeability to reference compounds.7 However, glycyrrhizinate was metabolized to glycyrrhetinic acid by glucuronidase in colonic flora, and glycyrrhetinic acid increased Caco-2 permeability and enhanced colonic absorption of calcitonin in rats.48 A novel group of glycosylated bile acid analogs was prepared and some of these were shown to be more effective than taurocholate in increasing the intestinal absorption of gentamicin, vancomycin, and calcitonin in rats. 49 Furthermore, at least one of the compounds had much lower hemolytic potential than taurocholate, an indication of the membrane-damaging potential. This compound was also used in dogs to increase the absorption of gentamicin.50 The increase in bioavailability depended on the site of administration. Bioavailabilities in controlled and enhanced states, respectively, were 6 and 54% when administered to the ileum, 4 and 23% for jejunal dosing, and 2 and 10% when administered orally. Acylcarnitines and Alkanoylcholines Medium and long chain fatty acid esters of carnitine and choline have shown many useful properties as intestinal absorption enhancers. Palmitoyl-DL-carnitine chloride (PCC) significantly increased the rectal absorption of cefoxitin, gentamicin, cytarabine, and ␣-methyldopa, as well as duodenal and rectal absorption of a somatostatin analog, in rats.51 PCC increased cefoxitin absorption from ligated loops of rat jejunum (22-fold), ileum (16-fold), and colon (>32fold), but duodenal absorption was greater than from other sites and was not improved with PCC.52 Dosing into unligated intestinal segments resulted in lower absorption than with ligated JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 4, APRIL 2000

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segments, apparently because of the spreading and dilution of the enhancer. Intestinal absorption of cefoxitin in dogs dosed by means of an intestinal port increased when administered with 10 to 20 mg PCC.51 In dogs dosed orally, cefoxitin bioavailability increased from 2.4% to 29% with an enteric-coated tablet containing 600 mg PCC, but uncoated tablets containing PCC resulted in no improvement of absorption.53 Human colon and rat colon were shown to have similar sensitivities to permeation enhancement with PCC in vitro.32 Treatment of Caco-2 monolayers with PCC resulted in dilated paracellular spaces, and permeation of fluorescent compounds through the paracellular spaces was observed.54,55 By studying the influence of PCC on permeabilities of solutes of various size, it was shown that increasing concentrations of PCC in the 0.05 to 0.35 mM range resulted in a concentration-dependent increase in the effective pore radius of Caco-2 monolayers.56 The mechanism by which PCC affects tight junctions was calcium independent,54 and the mechanism of decanoyl carnitine was different from the mechanism of sodium caprate shown in Figure 1.30 Prolonged exposure of jejunum or colon to permeation-enhancing concentrations of PCC or lauroyl choline caused only slight alterations of the mucosal cell structure.15,51 However, in a study of PCC effects on Caco-2 monolayers, concentrations increasing mannitol or PEG 4000 Papp values also caused LDH release and increased uptake of propidium iodide and reduced neutral red retention, each indicative of reduced cell viability.14 The in vivo absorption-enhancing effects of acylcarnitines were also correlated with their capacity to decrease the lipid order of intestinal brush border membrane vesicles,57 suggesting that increased fluidity of the membrane structure could also be involved in the mechanism of absorption enhancement. The metabolism of PCC to fatty acids has not been addressed in the literature, so its role in absorption enhancement is not known. However, it is known that medium-chain alkanoylcholines are rapidly hydrolyzed by rat intestine.58

N-Acetylated ␣-Amino Acids and N-Acetylated Non-␣-Amino Acids The development of N-acetylated ␣-amino acids and N-acetylated non-␣-amino acids as absorption promoters, and most of the literature on the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 4, APRIL 2000

subject, originated from Emisphere Technologies. This technology began with work on proteinoid microspheres, which were formed from thermally condensed ␣-amino acid mixtures. Further studies characterized the effects of structurally defined N-acetylated amino acids and combinations thereof. Relationships between the structure of N-acetylated ␣-amino acids and enhancement of calcitonin absorption in rats and monkeys were described, with examples of effective enhancers being N-cyclohexanoylleucine and N-(phenylsulphonyl)leucine.59 The oral bioavailability of cromolyn in rats increased from 0.5% to 4.6% when co-administered with 200 mg/kg Ncyclohexanoylleucine, and in vitro rat intestinal permeability of cromolyn and lucifer yellow increased fivefold to sevenfold in the presence of 20 mg/mL of the enhancer.60 Absorption of recombinant human growth hormone (rhGH) in rats was most significantly improved using 4-[4-[(2hydroxybenzoyl)amino]phenyl]butyric acid, among a series of N-acetylated non-␣-amino acids tested.61 Colonic delivery of this agent required lower doses to produce plasma rhGH concentrations in the range seen after oral dosing, and there was no damage to the intestine on histologic examination after absorption-enhancing doses. More recent literature reports have focused on sodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC) as an absorption-promoting adjuvant. SNAC was identified from a series of structurally related compounds on the basis of its enhancement of colonic absorption of heparin in rats.62 Oral administration of SNAC at doses of 300 mg/ kg increased heparin absorption, measured by use of a pharmacologic endpoint.63 SNAC increased the rat ileal permeation of heparin in situ without altering mucosal histologic findings.64 In Caco-2 permeation studies, 25 mg/mL SNAC had no effect on mannitol Papp values, whereas 50 mg/ mL greatly reduced TEER, increased mannitol Papp values, and caused cytotoxicity.64 SNAC itself has a relatively high Caco-2 Papp value, indicating high absorption potential, and it was not metabolized by the Caco-2 monolayer. In another report, the Caco-2 Papp value of human growth hormone in the presence of 37.5 mg/mL SNAC was >10 × 10−6 cm/s, which is generally considered highly permeable, and LDH release was only slightly altered.65 For this category of enhancers, an as yet undefined interaction of the drug and the enhancer that enables transcellular permeation is thought to be involved in the mechanism of absorption enhancement. 65–67 The SNAC/

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heparin combination has been evaluated in phase I and phase II clinical trials. A decanoate analog of SNAC is claimed by Emisphere scientists to be more efficient than SNAC for oral heparin delivery.

Chitosans and Other Mucoadhesive Polymers Several mucoadhesive polymers have been used as absorption promoters, including the anionic polyacrylic acid derivatives, polycarbophil and carbomer (i.e., Carbopol 934P), and cationic chitosan. Chitosan is a polysaccharide derived from crustacean shells and is comprised of glucosamine and N-acetylglucosamine subunits. Chitosan has numerous food additive, food processing, and pharmaceutical uses. In vitro studies have shown that chitosan opens epithelial tight junctions in a concentration- and pH-dependent way. At acidic pH, 0.1% chitosan increased Caco-2 permeability to mannitol.68 Chitosan increased Caco-2 permeation of the peptide DGAVP at 0.4 and 1% concentrations at pH 5.6.69 At pH 7, however, a 1% concentration of chitosan had no effect on Caco-2 TEER, and a 1.5% concentration had no effect on mannitol permeation.70 Nevertheless, a vehicle containing 1.5% chitosan at pH 6.7 increased the bioavailability of intraduodenally administered buserelin in rats from 0.1 to 5.1%.71 As shown by Schipper et al,72 the degree of acetylation and molecular weight of chitosans influence their effects on Caco-2 permeability and cytotoxicity. Although some chitosans were ineffective or cytotoxic, one chitosan with an intermediate degree of acetylation and high molecular weight had good absorption-enhancing characteristics and low cytotoxicity. To overcome the solubility limitations of chitosan at neutral and basic pH, an Ntrimethyl quaternized chitosan derivative was prepared.73 At 1.5 to 2.5% concentrations, trimethyl chitosan reduced Caco-2 TEER and increased Papp of mannitol (32- to 60-fold), FITCdextran 4000 (167- to 373-fold), and buserelin (28to 73-fold), without damaging the cells.74 Moreover, trimethyl chitosan greatly increased mannitol Caco-2 Papp at pH 7.4, whereas other chitosans have no effect at neutral pH.74 The effects of chitosans on epithelial membranes are mediated through their positive charge, and when combined with negatively charged heparin, the effects of chitosan were inhibited.75 Other polycations, such as polylysine, have also been shown to increase epithelial paracellular permeability,76 al-

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though there have been far fewer studies of these agents as oral absorption promoters. The anionic polymers, polycarbophil and carbomer, also increased Caco-2 permeation of DDAVP, mannitol, and FITC dextran 4000 at acidic pH.69,70 Their effects are also by opening tight junctions, which may be partly due to their calcium-binding activity. These polymers also inhibit proteolytic enzymes and can increase the oral absorption of peptides and proteins through this mechanism. Secretory Transport Inhibitors The intestinal epithelial cells express transport systems that actively remove certain compounds from the cells, transporting them in the blood-tolumen direction. For these compounds, secretory transport can limit intestinal permeation in the absorptive direction. The secretory transport systems present in the intestinal epithelium include Pgp and several related MRPs, transporters first identified in multidrug-resistant tumor cells. The substrates of Pgp and MRP are structurally very diverse, but many are lipophilic. Substrates of secretory transport are not necessarily poorly absorbed. Verapamil, quinidine, and digoxin are examples of Pgp substrates that are well absorbed. For these compounds, absorptive permeation by passive diffusion is sufficient to overcome the negative contribution of secretory transport. Secretory transport would be expected to have a more significant role for compounds with low or moderate passive permeability, as for example, with the more hydrophilic compounds, peptides, and peptidomimetics that have been recently recognized as substrates.77 Inhibition of intestinal secretory transport can lead to an increase in net absorptive permeation and an increase in oral bioavailability. A 10-fold increase in the oral bioavailability of paclitaxel was observed in mice when co-administered the Pgp inhibitor SDZ PSC 833.78 A similar magnitude of enhanced bioavailability was found in human subjects administered paclitaxel with 15 mg/ kg cyclosporin, another Pgp inhibitor.79 Oral absorption of digoxin in mice was increased by co-administered SDZ PSC 833, and brain concentrations and brain/plasma ratios were also increased by the Pgp inhibitor, raising the possibility that systemic absorption of Pgp inhibitors could alter the pharmacologic and toxic effects of co-administered drugs.80 Rather than co-administer a pharmacologically JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 4, APRIL 2000

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active compound that also affects nonintestinal Pgp or MRP, the goal of increasing oral bioavailability would be better addressed by co-administration of a nonabsorbed, pharmacologically inactive excipient. Various pharmaceutical excipients have been investigated for their effects on Pgp activity in multidrug-resistant tumor cells, with the aim of overcoming resistance to antitumor agents. Polysorbate 80 and POE 35 castor oil (Cremophor EL威), which were known to be inhibitors of Pgp in MDR tumor cells, reduced the secretory transport of a peptidic Pgp substrate in Caco-2 monolayers and increased absorptive permeation of the compound.81 These surfactants did not appear to be absorbed through the Caco-2 monolayer. Poloxamers are polyoxyethylenepolyoxypropylene copolymer, nonionic surfactants, which are available as pharmaceutical excipients and which have also been shown to affect intestinal secretory transporters. The poloxamer Pluronic P85威 increased the absorptive permeation of fluorescein, doxorubicin, paclitaxel, etoposide, and azidodeoxythymidine through Caco-2 cells. 82 The relationship between poloxamer structure and inhibition of Pgp in multidrugresistant tumor cells was described, and inhibition increased with increasing hydrophobicity.83 Poloxamers have also been shown to inhibit secretory transport in an MRP-expressing cell model. 84 Although pharmaceutical excipients have been shown to inhibit intestinal secretory transport and increase absorptive permeation in vitro, there have not yet been in vivo studies demonstrating the usefulness of this mechanism for improving oral bioavailability.

OTHER TECHNOLOGIES IN DEVELOPMENT Cortecs International Ltd. has begun clinical trials with oral calcitonin (now discontinued) and oral insulin formulations that presumably enhance their oral bioavailabilities. Although these formulations have not been made public, results have been presented for an oral insulin formulation containing the permeation enhancers sodium ursodeoxycholate and MCGs.85 Unigene Laboratories is reported to be close to human clinical trials with an oral formulation of calcitonin. The prospective clinical formulation has not been disclosed, but Unigene scientists have published results of animal studies that use formulations containing taurodeoxycholate or lauroylcarnitine, with citric acid added to control the pH.17 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 4, APRIL 2000

CONCLUSIONS In vitro studies are useful for identification of absorption enhancers and defining their mechanisms. It is not clear whether cytotoxicity evaluations performed in vitro are predictive of in vivo toxicity. Several of the absorption enhancers discussed seem to have potential to improve the oral bioavailabilities of poorly absorbed compounds in animals, without causing significant intestinal damage. So far, there have been only scant reports of human bioavailability studies with permeation enhancers, although a few clinical studies have been performed on these potential new excipients. It is hoped that more of these results will become publicly available soon. There is also little information available on the long-term safety of compounds that alter intestinal permeability, and this subject needs to be addressed. The co-delivery of drug and enhancer most likely will require specific formulations to attain maximum bioavailability improvement. The inhibition of intestinal secretory transport with acceptable pharmaceutical excipients seems quite possible, although bioavailability studies supporting this approach are still required.

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