Targeting of drugs and vaccines to the gut

Pharmac.Ther.Vol.62, pp. 9%124, 1994

Pergamon 0163-7258(94)E0010-Y

Copyright© 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0163-7258/94$26.00

Specialist Subject Editor: C. J. HAWKEY

TARGETING OF DRUGS A N D VACCINES TO THE GUT I. R. WILDING,*~ S. S. DAVIS*t and D. T. O ' H A G A N t *Pharmaceutical Profiles Limited, 2 Faraday Building, Highfields Science Park, University Boulevard, Nottingham NG7 2QP, U.K, f Department of Pharmaceutical Sciences, Nottingham University, University Park, Nottingham, NG7 2RD, U.K. Abstract--Targeted delivery to the gastrointestinal tract requires a multi-disciplinary approach to research involving contributions from polymer and material scientists, gastroenterologists, pharmaceutical scientists and technologists. Intestinal delivery is important not only for drugs that act locally, but also for those with systemic activity. In particular, there is considerable interest in the oral delivery of peptides and it is felt that the colon may provide an advantageous absorption site for such molecules. The different targeting mechanisms available to the pharmaceutical scientist to provide site-specific delivery in the gastrointestinal tract will be critically assessed. Delivery systems and targeting agents, which are being developed for the delivery of drugs, may also be exploited for the delivery of vaccines, since many of the delivery problems are common to both areas. Recent developments in the design of oral antigen formulations will be discussed in this review. Keywords--Targeted delivery, drug delivery systems, peptides, oral vaccines, ~-scintigraphy.

CONTENTS I. General Introduction 2. Targeting Mechanisms 2.1. Pharmaceutical 2. l.l. Enteric coating 2.1.2. Timed-release systems 2.2. Chemical 2.2.1. Prodrugs for colonic delivery 2.3. Biological 2.3.1. Redox-sensitive polymers 2.3.2. Microbially degraded polymers 2.3.3. Bioadhesives 2.3.4. Passive targeting 3. Drug-targeting Sites 3. I. Introduction 3.2. Local action 3.2.1. Digestive enzyme supplements 3.2.2. Topical action in the treatment of inflammatory bowel disease 3.3. Systemic absorption 3.3.1. Prevention of gastric irritation 3.3.2. Oral delivery of peptides

98 99 99 99 101 103 103 104 104 105 106 107 107 107 108 108 108 109 109 109

JiCorresponding author.

Abbreviations--5-ASA, 5-aminosalicylic acid; CAP, cellulose acetate phthalate; CMIS, common mucosal immune system; CT, cholera toxin; CTB, cholera toxin B subunit; GALT, gut-associated lymphoid tissue; IgA, immunoglobulin A; IgG, immunoglobulin G; ISCOMS, immunostimulating complexes; LTB, heat-labile enterotoxin subunit-B; NSAID, non-steroidal anti-inflammatory drugs; PLG, poly (lactide-co-glycolide); VBI2, Vitamin B12; WC, whole cells. 97

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I.R. WILDINGet al. 4. Targeting of Oral Vaccines 4.1. Introduction 4.2. Non-living antigen-deliverysystems 4.2.1. Controlled release microparticles as oral vaccines 4.2.2. Lectin-like molecules as oral vaccines 4.2.3. Liposomes as oral vaccines 4.2.4. Immunostimulating complexes as oral vaccines 4.3. Live vectors for vaccine development 4.3.1. Salmonella-based oral vaccines 4.3.2. Alternative live vectors 5. Conclusions References

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1. GENERAL INTRODUCTION The oral route of drug delivery is still preferred by the vast majority of patients. In the past, it has been common for pharmaceutical preparations to disintegrate rapidly in the upper gastrointestinal tract, where drug absorption is typically the most efficient. For a number of drugs, this approach is generally adequate, however, as our understanding of drug action, or misaction, has improved so as the requirement for novel techniques for drug delivery. In some situations, it would be highly beneficial to target a drug to a particular site within the gastrointestinal tract, either to maximize a therapeutic response or to reduce side-effects caused by drug delivery to an inopportune region of the gut. The rational design of a drug delivery system for targeting delivery to the gut needs to take into account four essential and interrelated elements (Fig. 1): the drug, the disease, the destination and last but not least, the appropriate delivery system (Davis and Ilium, 1986). Targeted delivery to the gastrointestinal tract, therefore, requires a multi-disciplinary approach to research involving contributions from polymer and material scientists, gastroenterologists, pharmaceutical scientists and technologists. A review of the literature suggests that many attempts to target to the different regions of the gut have failed because of a lack of coordinated interaction between the different disciplines and little or no understanding of the relevant aspects of gastrointestinal pathophysiology. The objective of this review is to bring together published research on these interrelated disciplines, in order to allow the reader to critically appraise the key issues associated with targeted drug delivery to the gastrointestinal tract. We will review the different targeting mechanisms that are now available to the pharmaceutical scientist, to include so-called 'bioadhesives', which are designed to retain the preparation in the stomach or the upper small intestine, as well as the possibility of site-specific delivery to the large bowel, using colon-selective polymers. The potential drug targeting sites will be explored to include both drugs that act locally and those with systemic activity. Particular reference will be made to the oral delivery of peptides, which is the equivalent of the 'Holy Grail' in pharmaceutical research.

SYSTEM FIG. 1. The four basic elements of drug delivery. Delivery systems and targeting agents that are being developed for the delivery of drugs may also be exploited for the delivery of vaccines, since many of the delivery problems are common to both areas. Traditionally, vaccine research has mainly been concerned with the induction of systemic immunity through the use of parenteral vaccines. However, the majority of infectious

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agents first infect the host through mucosal sites, including the gut and parenteral vaccines do not normally induce mucosal immunity. Consequently, there has been a resurgence of interest in the delivery of vaccines by the oral route, with the objective of inducing both mucosal and systemic immunity. Unfortunately, poor absorption, antigen degradation, interaction with non-specific host factors and pre-existing immunity have all contrived to negatively influence the outcome following oral immunization. Nevertheless, in recent years novel antigen-delivery systems have emerged, including live antigen carriers, such as Salmonellae and non-living delivery systems, such as microparticles and liposomes. Recent research has indicated that these novel delivery systems have considerable potential for the development of new and improved oral vaccines. These recent developments in the design of oral antigen-delivery systems will be discussed in this review. 2. TARGETING MECHANISMS 2.1. PHARMACEUTICAL

2.1.1. Enteric Coating

Enteric coatings have traditionally been utilized for drug substances that cause gastric irritation, produce nausea if released in the stomach or are destroyed by acid or gastric enzymes (Healey, 1989). The principle by which enteric-coating polymers act is that their solubility is highly pH dependent, the polymers being insoluble in gastric acid, but dissolving in intestinal fluid. To ensure total gastric resistance, a coating should be impermeable until, at least, pH 5. With some polymers that dissolve at relatively high pH values (e.g. pH 8), concern arises as to whether the coating will dissolve promptly at the target site so as to provide adequate opportunity for drug absorption in all cases. The first reported use of enteric coating is credited to Unna in 1884, who introduced a medication based on keratin-coated pills. The range of materials used to produce enteric coatings has increased greatly since then, with polymers of natural or synthetic origin being the most popular and effective. These long-chain molecules characteristically display acidic or acidic ester groups, conferring the pH sensitivity necessary for enteric coating activity. There is not, in fact, a precise pH threshold above which a material is soluble, rather a range of about one pH unit over which a polymer coating varies from being virtually impermeable to being quite readily soluble and fast to rupture (Healey, 1989). Information on the threshold pH for the most widely used enteric-coating polymers is provided in Table 1. The enteric coating of orally administered dosage forms is an effective method of modifying and obtaining control of drug delivery to the small intestine (Murray and Tucker, 1990). The external TABLE 1. Commonly Used Enteric-coating Mater&& Enteric polymer Threshold pH CAT 4.81 HPMCP 4.5-4.8 PVAP 5.0 HPMCP 50 5.2 HPMCP 55 5.4 Eudragit L30D 5.6 Aquateric 5.8 CAP 6.0 Eudragit L 6.0 Eudragit S 6.8 Shellac 7.22 CAT, cellulose acetate trimellitate; HPMCP, hydroxypropylmethylcellulose phthalate; PVAP, polyvinyl acetate phthalate. JManufacturer's data. 2Increases on storage. Reprinted from Healey (1989), with permission of the author and the copyright holder, Ellis Horwood, a division of Simon and Schuster International Group, Chichester.

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appearances of enteric-coated and conventional dosage forms are very similar, but the in-vivo behaviour of the preparations is very different (Hardy et al., 1987a, b; Wilding et al., 1992b). The combined use of gamma scintigraphy with pharmacokinetic assessment (pharmacoscintigraphy) is routinely used to provide in-vivo information about the gastrointestinal behaviour of oral dosage forms and subsequent drug absorption (Wilding et al., 1991). This type of study is particularly useful for enteric-coated preparations, where it is important to verify that the formulation complies fully with the product rationale (Skelly et al., 1990). For example, the most frequently reported side-effects of non-steroidal anti-inflammatory drugs (NSAIDs), such as naproxen, are gastrointestinal discomfort, nausea and gastric bleeding (Haslock, 1989). The primary cause is considered to be the direct effect of the NSAID on the gastric mucosa (Pemberton and Strand, 1979). Enteric-coated NSAID tablets, designed to prevent direct contact between the drug and the mucosal tissue of the stomach, have been shown to reduce the incidence of gastrointestinal side-effects (Trondstad et al., 1985). In a recent pharmacoscintigraphic evaluation of an enteric-coated naproxen tablet formulation (Naprosyn ® EC), no loss of preparation integrity was observed in the stomach following administration under both fasted and fed conditions (Wilding et al., 1992b). Residence time in the small intestine, prior to tablet disintegration, was independent of food intake and a good correlation was observed between onset of subsequent tablet disintegration and commencement of drug absorption. The pH gradient in the human gastrointestinal tract has also been exploited to deliver drugs to the colon (Ashford et al., 1993a; Dew et al., 1982; Davies et al., 1990; Gwinup et al., 1991; Healey, 1990; Iamartino et al., 1989). It was originally believed that in humans, the pH of the gastrointestinal tract increased in progressing from the stomach through to the colon. However, Evans et al. (1988), using radiotelemetry, have reported that the pH gradient does not follow this pattern in normal healthy subjects. The mean pH in the proximal small intestine and terminal ileum was 6.6 +__0.5 and 7.5 +__0.4, respectively. There was a sharp fall in pH to a mean of 6.4 +_0.4, as the radiotelemetry capsule passed into the caecum, presumably due to the presence of short-chain fatty acids produced from the bacterial fermentation of dietary fibre. The pH then rose progressively from the right to the left colon, with a final value of 7.0 + 0.7. In addition, recent studies have shown that the pH of the luminal contents of the proximal colon of patients with active ulcerative colitis can be as low as 4.7 + 0.7 (Raimundo et al., 1992). Gruber et al. (1987) concluded that the change in the luminal pH may not be used reliably and routinely as a mechanism to deliver drug specifically in the colon. Nevertheless, a number of colonic delivery systems have been developed that are designed to exploit such a pH-triggered release. These include several commercially available 5-aminosalicylic acid (5-ASA) delayed-release systems, e.g. Asacol ~ and Claversal~'~ (Dew et al., 1982; Thomas et al., 1985; Hardy et al., 1987a, b). Both Asacol~'-~ ' and Claversal :'~ utilize copolymers of methacrylic acid and methyl methacrylate, available commercially as the EudragifR~range of polymers, to deliver drug to the colon. Asacol :" consists of 400 mg of 5-ASA, film coated with Eudragit ~t S, which has a threshold pH of 7. Radiological studies have shown that release of 5-ASA from Asacol~'~ generally occurs in the distal ileum and the proximal colon (Dew et al., 1983). Data obtained from studies in ileostomy patients suggest that Asacol ~:' delivers nearly 90% of the administered drug to the colon (Riley et al., 1988). The Claversal~";preparation is composed of 250 mg of 5-ASA coated with Eudragit"~ L, which has a threshold pH of 6. To compensate for this lower triggering pH, a thicker film coat has been applied. Scintigraphic and radiological studies have both demonstrated that drug release typically occurs in the terminal ileum or the ascending colon (Batham, 1991; Hardy et al., 1987a, b). The coating of capsules with cellulose acetate phthalate (CAP) has been used to deliver the topically effective corticosteroid, beclomethasone dipropionate, to the colon for the treatment of distal idiopathic ulcerative colitis (Levine, D. S. et al., 1987). The threshold pH of the polymer is 6 and drug delivery from CAP-coated preparations has been shown to take place in the distal small intestine and terminal ileum (Healey, 1990). Despite the potential limitations and difficulties associated with the use of pH-triggered release for colonic delivery, this strategy has proven useful in that it is capable of delivering drug to the colon in the majority of subjects. Therefore, whilst simple enteric coating is not as technically elegant as other approaches, its simplicity has a significant appeal to the pharmaceutical industry (Friend, 1992). The alternative colon-targeting mechanisms are shown schematically in Fig. 2.

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Prodrugs Activedrug is cleavedfrom the carrier molecule via the action of microbiallyderivedenzymes or the redox potential of the colon.

Microbially Degraded Polymers Degradation and fermentation of polysaccharides to component ~ monomers in the presence of ~

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Timed-Release Systems Transittime throughthe smallintestineis relatively constant. Delivery systems, which release drug at a pre-determined

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2.1.2. Timed-release Systems

Residence time of pharmaceutical preparation in the stomach is highly variable, ranging from minutes to hours, depending upon the size of the preparation and whether the stomach is in the fed or fasted state (Wilding et al., 1992b). On the other hand, transit time through the small intestine is relatively consistent, in the order of 3-4 hr (Davis et aL, 1986). Timed release, based on a constant small-intestinal transit time, has been used to design a delayed release osmotic dosage for delivery to the colon; 'Oros CT ~' (Theeuwes et al., 1990, 1991). The Oros CT :~; osmotic therapeutic system can be a single osmotic unit or can comprise as many as 5--6 small 4-mm tablets contained within a hard gelatin capsule. The individual units are enteric coated to prevent release in the stomach and the release process is triggered by the change in pH of the intestinal fluid upon gastric emptying. Following triggering, a delay period has been built into the system to coincide with the normal small intestinal residence time, i.e. 3 hr. Oral administration of 'colon-targeted' osmotic systems containing insulin and permeation enhancers to healthy volunteers has been shown to produce a significant decrease in blood glucose concentrations, indicating absorption of biologically active insulin from the gastrointestinal tract.* A miniature osmotic pump, the Osmet ~, has been developed, which will pass through the stomach and small intestine and then deliver its contents over 8 hr in the colon (Chacko et al., 1990). The device has been evaluated in prototype form, using gamma scintigraphy and the mean in-vivo start-up time was 5.3 + 0.2 hr. The authors report on unpublished studies in which they have used the system to deliver 15N-glycine to the caecum in order to follow the incorporation of ~SN into bacterial protein and also 13C-cholic acid in studies of caecal bile acid metabolism (Chacko et al., 1990). However, the primary value of this device is likely to be in research rather than therapy. Other groups have developed systems that have a predominantly research application and *Fox, D. A. (1991) Colon-targeted osmotic systems for oral delivery of peptides and proteins. Abstracts of a meeting on the oral delivery of proteins, peptides and other biopharmaceutical agents, Wakefield, Massachusetts.

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Pulsincap Dosage Form Waterinsoluble body ~ ' ~ , , d ~ ' " Drugformulation

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Swollenejectedplug Releaseddrugin small intestineor colon FIG. 3. Timed-release delivery: the PulsincapTM approach. Reprinted with permission from Scherer DDS, Clydebank. these include a computer-controlled site-specific delivery device (D'Andrea et al., 1991) and a remote control system, which, to date, has only been evaluated in the dog model.* Kobe et al. (1989) described an oral sustained-release device in which, when placed in the gastrointestinal tract, fluid penetrated through an outer membrane, resulting in swelling of the inner core. Drug is released rapidly when the outer membrane explodes due to internal pressures. By controlling the ingress of fluid into the device and the amount of swelling, the time before release can be predetermined. An oral drug delivery system, which releases drug at a predetermined time, or place, within the gastrointestinal tract, recently has been described (McNeill et al., 1990; Rashid, 1990). This dosage form, called Pulsincap ~, consists of an impermeable capsule body containing the drug, fitted with a hydrogel plug (Bakhshaee et al., 1992). In aqueous media, the plug hydrates, swells and after a time period, defined by the plug dimensions, is ejected from the device, thereby allowing release of the capsule contents (Fig. 3). Scintigraphic evaluation of Pulsincap ~ has enabled the in-vivo release properties of the device to be defined (Bakhshaee et al., 1992; Wilding et al., 1992a). Colonic absorption of captopril has been evaluated by the oral administration of a Pulsincap C~device, with an in-vitro pulse time of 5 hr. In six out of the eight subjects, the device reached the colon before the drug was released. The in-vivo pulse times ranged from 246 to 389 min with a mean time of 327 min, which is in good agreement with that recorded in vitro and suggests that the device performs as predicted. Measurable blood levels of free captopril were found in three subjects. Variable instability of the drug in the distal intestine was suggested as a possible reason for the lack of absorption of the drug in all subjects (Wilding et al., 1992a). The study demonstrated the utility of the pulsatile system, combined with scintigraphy, for investigating drug absorption in different regions of the gastrointestinal tract. Greater targeting specificity appears to have been achieved by the enteric coating of the device, which prevents hydration of the plug whilst in the stomach (Bakhshaee et al., 1992). Timed release has also been obtained from an oral dosage form, which comprises a tablet core coated with a mixture of hydrophobic material and surfactant (Pozzi et al., 1994). Drug release from the core of the Time-Clock ~" system occurs after a predetermined lag time, which depends mainly on the thickness of the hydrophobic layer and is independent of gastrointestinal pH. The preparation of the dosage form is carried out by using conventional industrial procedures *Casper, R., Parr, A., McCartney, M. and Jochem, W. (1990) Remotely controlled site specific gastrointestinal drug delivery. Proceedings of the 5th Annual Convention of the American Association of Pharmaceutical Scientists, Las Vegas, Nevada, p. 68.

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and standard industrial equipment. A series of scintigraphic studies has been carried out to characterize the in-vivo behaviour of the Time-Clock ® system (Pozzi et al., 1994). The results showed that in-vivo performance of the device was reproducible and demonstrated that the methodology for in-vitro testing was a good predictor of in-vivo release. When the primary objective is delivery to the colon, the timed-release tablet has been enteric coated to prevent the problem of drug release in an inopportune region of the gastrointestinal tract, caused by extended gastric residence. The timed-release coat would only then start to dissolve after the tablet had left the stomach. There are a number of other systems now available to give timed-release drug delivery, which can be preprogrammed to give the desired lag phase. These include a multi-particulate pellet system, which has been shown to be capable of targeting drug delivery to either the lower part of the small bowel or the colon (Klokkers-Bethke and Fisher, 1991) and a timed-release erosion-based system, which has been suggested for a number of applications, such as oral delivery of morphine and theophylline (Bar-Shalom and Kindt-Larsen, 1989). The rationale behind all these timed-release devices is valid, provided that small intestinal transit time remains relatively constant. However, the specificity of the systems for colonic targeting could be significantly affected by changes in the motility of the gastrointestinal tract (Kellow et al., 1986).

2.2. CHEMICAL 2.2.1. Prodrugs for Colonic Delivery Prodrugs consist of an active drug conjugated or bonded to a carrier molecule. The prodrug sulphasalazine has long been used in the management of inflammatory disorders of the colon. After passage through the small intestine, the active species, 5-ASA and the carrier moiety, sulphapyridine, are released from the prodrug after cleavage of the azo-linkage (Peppercorn and Goldman, 1972). The need for carrier moieties less toxic than sulphapyridine has led to the development of a number of alternative azo-prodrugs, of which olsalazine (Dipentum ®) is the only marketed product. Olsalazine is a dimer of 5-ASA, linked via an azo-bond and on reaching the large intestine, it is cleaved, delivering drug to the target site (Lauristen et al., 1984; Ryde et al., 1991). Clinical trials have shown that olsalazine is clinically effective, although diarrhoea has been reported in about 15% of patients. This side-effect appears to be caused by a combination of transit changes and a stimulation of secretion in the small intestine (Rao et al., 1987; Pamukco et al., 1988). Balsalazide consists of 5-ASA azo-linked to 4-aminobenzoyl-fl-alanine. The carrier moiety is designed to be less toxic than sulphapyridine, whilst minimizing absorption of the prodrug from the upper gastrointestinal tract. The carrier is only minimally absorbed following cleavage in the colon (Chan et al., 1983). Clinical trial data suggest that balsalazide is useful in maintaining remission in patients with ulcerative colitis and the side-effect profile was improved, compared with that with sulphasalazine-maintenance therapy (Mclntyre et aL, 1988). More recently, a number of prodrugs have been developed that are capable of targeting steroidal anti-inflammatory drugs to the large bowel. The colonic bacteria produce a wide array of glycosidases capable of hydrolysing simple glycosides and polysaccharides. The ability of the microflora to hydrolyse glycosides, therefore, has formed the basis for the design of steroid glycoside prodrugs intended for colon-positioned delivery (Friend and Chang, 1985; Friend and Tozer, 1992; Haeberlin et al., 1993; Kimura et al., 1991; Suzuki et al., 1991; Tozer et al., 1991). The successful delivery of dexamethasone from its glucoside prodrug has been demonstrated in several rodent species. The results support the hypothesis that efficacy can be maintained with lower doses, when drug is administered in this manner, thereby reducing the potential for side-effects (Friend and Tozer, 1992). No data have been obtained in humans, as yet, in order to substantiate this hypothesis. Prodrugs consisting of dextran-esters have also been reported as being effective in targeting to the colon for drugs containing a carboxylic acid functional group, such as naproxen (Harboe et aL, 1989).

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2.3.1. Redox-sensitive Polymers The discovery that sulphasalazine exerts its action in the large bowel by cleavage of the azo-bond to generate the active moiety, 5-ASA, has provided a focus for the development of novel polymers based on an analogous targeting mechanism. Under anaerobic conditions, the azo-reduction process represents a non-enzymatic reduction by enzymatically generated flavins (Brown, 1976). Redox mediators, such as benzyl viologen and flavin mononucleotide, act as electron shuttles between the intracellular enzyme and extracellular substrate (Grim and Kopecek, 1991). Reduction of azo bonds, therefore, is believed to be a general reaction caused by the redox potential of the colonic environment, rather than a specific reaction mediated by only a limited number of intestinal bacteria (Scheline, 1973). Redox potential is an expression of total metabolic and bacterial activity in the colon and is believed to be insensitive to dietary changes. Recent studies have demonstrated that the mean redox potential (mV + SD) is - 67 +__90 in the proximal small bowel, - 196 + 97 in the distal small bowel and - 415 + 72 in the right colon.* Therefore, the microflora-induced changes in the redox potential can be used as a highly specific mechanism for targeting to the large bowel. Coating of peptide preparations with polymers crosslinked with azo-aromatic groups is reported to protect the drugs from digestion in the stomach and small intestine. Drug is then released in the large intestine by the ability of the indigenous microflora to effect reduction of the azo-bonds (Hastewell et al., 1991; Saffran et al., 1986; Saffran and Neckers, 1987). An azo-polymer has been reported to deliver vasopressin and insulin to the systemic circulation after oral administration in rats (Saffran et al., 1986). Further studies have shown that release of insulin was delayed significantly when azo-polymer-coated capsules were administered to pancreatectomized dogs (Saffran et al., 1991). However, the biological response to released peptide was highly variable, so that the data are difficult to interpret and there was no direct evidence of cleavage in the colon. Recent studies have suggested that the balance between the hydrophobic and hydrophilic components is important in optimizing the azo-polymers for colon-selective delivery (Van den Mooter et al., 1992). A high proportion of hydrophilic components are required to ensure that the azo-groupings are accessible for bacterial-mediated reduction. However, the hydrophobic components provide the resistance to the conditions in the upper gastrointestinal tract. Radiological studies in dogs have been carried out to investigate the in-vivo behaviour of novel polyurethane systems containing azo-linkages (Kimura et al., 1990, 1992). The results suggested that the coated pharmaceutical compositions could deliver drug to the colon; however, it is important to note that no data were published to support the assertion that disintegration was a direct consequence of breakdown of the azo-bonds, rather than time-dependent failure of the delivery device. It is interesting that not only azo-linkages are susceptible to reduction in the colonic environment. Disulphide bonds can also be cleaved by the redox potential in the large bowel. Disulphide linkages in drug-cysteine conjugates have been shown to be cleaved in the colon to produce glutathione-linked thiols (Larsen et al., 1983). Non-crosslinked redox-sensitive polymers, containing an azo and/or a disulphide linkage in the backbone, have been developed recently (Schacht and Wilding, 1991). The density of the reductive-sensitive linkage can be varied widely so as to optimize the degradation properties of the polymer. An azo-crosslinked, pH-sensitive hydrogel system has been described (Bronsted and Kopecek, 1990). As the gel transits the gastrointestinal tract, the degree of swelling increases due to an increase in pH. Upon arrival in the colon, the gel has reached a degree of swelling that makes the crosslinks accessible to the reduction mediators, which cleave the crosslinks and release the drug. Preliminary studies in rats have indicated that enzymatic degradation of the hydrogel crosslinks was achieved but only over a period of several days (Bronsted and Kopecek, 1992). If *Stirrup, V., Evans, D. F., Ledingham, S., Thomas, M., Pye, G. and Hardcastle, J. D. (1990) Redox potential measurement in the gastrointestinal tract in man. Proceedings of the World Congress of Gastroenterology, Digestive Endoscopy and Colo-proctology, Sydney, Australia.

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this were the case in humans, then the applicability of the hydrogels for targeting to the colon may be poor since the preparation will have been excreted before significant release of the drug has occurred. To overcome this problem, new synthetic water-soluble polymeric systems have been developed, which couple the degradation properties of the azo-linkages with the concept of extending colonic residence time by the inclusion of polymeric carriers containing carbohydrate moieties, which are complementary to lectin-like structures in the colon (Grim and Kopecek, 1991; Kopecek et al., 1992). No in-vivo data have been reported on the modified system yet. 2.3.2. Microbially Degraded Polymers It is now widely recognized that dietary fibre is extensively degraded during transit through the colon (Cummings et al., 1976; Gear et aL, 1981). The main consequences of bacterial fermentation of such polysaccharides are faecal bulking, increased transit of the colonic contents, increased nitrogen utilization in the gut and the formation of short-chain fatty acids, which form a useful energy supply (Cummings and Macfarlane, 1991). Extensive work has been carried out on the metabolism of plant polysaccharides by the microflora of the large bowel and, in particular, the fermentation of non-starch polysaccharide (Macfarlane and Cummings, 1991). Enzymes responsible for degradation include fl-galactosidase, fl-glucosidase, fl-xylosidase and fl-fucosidase, which are secreted by the various bacterial species (Finegold et aL, 1983). Consequently, polysaccharides possess promise as agents for the selective delivery to the colon and as a result, a number of groups are currently exploring the potential of natural and modified polysaccharides for targeting to the large bowel. It has been found that certain types of amylose coatings are particularly suitable for colonspecific delivery. For a number of years, it was believed that starch was almost entirely digested by intestinal enzymes, such as ~-amylase; however, recent studies have suggested that a proportion is not degraded until it reaches the colon (Archer and Ring, 1989). This resistant starch takes the form of glassy amylose and in combination with other film-forming compounds, has shown promise in vitro as a delivery system for targeting to the large bowel (Allwood et aL, 1991). Starch crosslinking is widely used to provide the textural characteristics necessary in the food industry. The basic idea is to toughen starch granules by treatment with crosslinking agents, such as calcium chloride. Ionically crosslinked corn starch for enzymatically controlled oral drug delivery has been described recently (Kost and Shefer, 1990). The release rate of large molecules, such as bovine serum albumin, from bioerodible matrices was dependent on degradation of the matrix, whilst the release of small molecules was mainly by diffusion out of the matrix. Chrondroitin sulphate is a soluble mucopolysaccharide (Wastenson, 1971), which is utilized as a substrate by the microflora of the large intestine, in particular Bacteroides thetaiotaomicron and B. ovatus (Salyers, 1979). However, naturally occurring chrondroitin is readily water soluble, which prevents its use as a protective polymer for colon-specific drug delivery. In order to overcome this problem, various examples of crosslinked chrondroitin have been produced and their potential for colonic drug delivery has been assessed in the rat caecal model (Rubinstein et al., 1992a,b). The results suggest that, as expected, the degree of crosslinkage has a direct influence on drug release from tablets manufactured from these modified polysaccharides. Tablet cores have been compression coated with pectin and the in-vivo scintigraphic results suggest that these prototype systems may possess promise for colon-selective delivery. However, it would appear that in subjects with rapid small intestinal transit, the pectin coat may not have time to hydrate, which can limit access of the bacterial enzymes to the susceptible bonds (Ashford et al., 1993b). Matrices have also been prepared from calcium pectinate (Rubinstein et al., 1993) and studies in the rat caecal model suggest that these systems could serve as a colon-specific drug delivery system. Glycosidic polysaccharides have been assessed for their potential in colonic targeting (Lancaster and Wheatley, 1989). The susceptibility of cast films was evaluated in the rat caecal model and the preliminary results were encouraging from a degradation perspective. However, the study demonstrated several of the inherent disadvantages associated with the potential use of polysaccharides for site-specific delivery to the colon. Films cast directly from the polysaccharides were very brittle, which highlights the importance of formulating the product to maximize the film-forming characteristics of the polymers. Natural galactomannans, such as guar gum and locust bean gum,

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have been formulated with an insoluble carrier polymer, such as Eudragit ~ RL, to improve their film-forming properties (Lehmann and Dreher, 1991). However, the high aqueous water solubility of the glycosidic polysaccharides could still preclude their use for colon targeting. Therefore, although polysaccharides are known to possess promise as agents for selective delivery to the colon, their film-forming and solubility properties are less than ideal. It may be possible to modify the properties of polysaccharides in a controlled manner so as to improve their formulation characteristics, whilst retaining substrate recognition (Bauer and Wohlschlegel, 1992). The range of subsequent polymers that can be produced is wide and depends on the nature of the modifications made, i.e. backbone or side chain. An artificial polysaccharide based on food grade materials could be produced, which has both optimal film-forming properties coupled with a rapid rate of enzymic lysis. This would have the advantage over the redox-sensitive polymers, in that, at least, the starting materials would carry "generally regarded as safe" (GRAS) status. 2.3.3. Bioadhesives The ability to provide adhesion of a drug (or more particularly, a delivery system) to the wall of the gastrointestinal tract has a number of attractions. A formulation coated with a bioadhesive material, such as a polymer, could lead to a longer residence time in a particular organ site, thereby providing an improved effect in terms of local action or systemic absorption. Consequently, in recent years, there has been considerable debate in the pharmaceutical literature as to whether such a concept of bioadhesion can be successfully applied to targeting within the gastrointestinal tract. Unfortunately, in many of the instances, poor attention has been given to the relevant aspects of the anatomy and physiology of the human gut. For example, in some reports, the supposed attachment site for the bioadhesive system has not been made clear. Sometimes mucoadhesion seems to be inferred (i.e. the delivery system attaches to the mucus layer lining the gastrointestinal tract), whereas in other cases, the direct attachment of the delivery system to the epithelial surface is implied. In the former case, it is important to realize that the mucus layer within the gastrointestinal tract turns over continuously (Lehr et al., 1991). In addition, mucus can be found not only on the surface of the lumen but also within the lumen. This is particularly important in the stomach, where so-called "soluble mucus" is present in large quantities. Therefore, it is difficult to understand how certain mucoadhesive systems can identify the designated attachment site (Veillard, 1989). The subject of bioadhesion has been reviewed in detail by others (Gurny and Junginger, 1989; Lenaerts and Gurny, 1990), and, therefore, will be considered here briefly. The biomaterials proposed have included a range of polymers to include polycarbophils, polyurethanes and polyethylene oxide-polypropylene oxide co-polymers. While such systems may show some adhesive properties in vitro, few studies have demonstrated an ability to adhere in vivo and, as yet, no studies have demonstrated a preferential advantage in humans (Khosla and Davis, 1987; Harris et al., 1990). Some of the in-vitro models appear to have very little relevance to known physiology and, therefore, can be questioned as to their relevance. In animal experiments where bioadhesion has been demonstrated (Longer et al., 1985), the effect seems to be one associated more with the bulk of the polymer administered rather than a bioadhesive effect per se. As a consequence of these problems, many of the earlier workers in the field have now turned their attention to more appropriate sites for bioadhesion, such as the nasal cavity and the vagina. It is unlikely that a bioadhesion mechanism based on non-specific interactions will have a demonstrable benefit in the human gastrointestinal tract. Other groups have taken an alternative stance and have attempted to exploit natural mechanisms of bioadhesion based upon receptor-mediated events, i.e. lectins (Woodley and Naisbett, 1989). It is well established that certain plant lectins interact specifically with sugar groups resident in mucus or on the glycocalyx. The binding ability of various lectins to the gastrointestinal tract has been well described by Pusztai and others (Pusztai, 1989a; Kilpatrick et al., 1985). Materials such as tomato lectin have been proposed as bioadhesive systems and have been evaluated in in-vitro and in-vivo models (Woodley and Naisbett, 1989). In order for a lectin to have utility in a drug-targeting situation, the lectin would need to be non-toxic, available in large quantities at an acceptable price and demonstrate suitable specificity. Pusztai (1989b) has reviewed the possibilities and problems and has also discussed how lectins linked to drugs may be used to provide not only

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attachment to the epithelial surface but also their subsequent absorption through processes of endocytosis. Caston et al. (1990) have exploited a similar strategy of bioadhesion to that found with plant lectins, but have chosen to use the bioadhesive properties of micro-organisms that are known to colonize the gastrointestinal tract. Bacteria, such as Escherichia coli, are able to adhere in the small intestines through a bioadhesive process involving protein-like structures growing on the surface of the organism, known as fimbriae. These interact specifically with sugar groups in mucus or on the cell surface. For example, the fimbriae from type 1 E. coli interact with mannose receptors. Species of E. coli that overexpress fimbrial material can be grown in culture and the fimbriae can be harvested and purified. These fimbriae can then be attached to the surfaces of drug-loaded microspheres and used to provide a bioadhesive system within the gastrointestinal tract. Work so far conducted at Nottingham has demonstrated that this concept works well in vitro and in vivo. For example, recent studies in the non-anaesthetized rat have shown that the bioavailability of the marker molecule hydrochlorothiazide can be greatly enhanced if the drug is incorporated into a biodegradable microsphere coated with fimbriae. The bioadhesive region of the fimbrial structure has been identified. Fimbriae from E. coli are polyvalent and achieve their bioadhesive effect through a specific region designated Fim-H (Krogfelt and Klemm, 1988). Through techniques of genetic engineering, it is now possible to grow bacteria expressing only Fim-H and for this material to be exported into the culture media. Thus, Fim-H may offer important opportunities as a bioadhesive material in its own right or in combination with other materials, such as drugs or as a conjugate with synthetic polymers. 2.3.4. Passive Targeting Accumulated experimental evidence suggests that small microparticles, i.e. > 10/zm, may be taken up into the intestinal Peyer's patches following oral administration (O'Hagan, 1990). A specialized cell type, the M-cell, has been identified in the epithelium of the Peyer's patches, which is thought to be responsible for such particle uptake (Owen and Ermak, 1990). Following uptake, particles have been identified in the mesenteric lymph, lymph nodes, liver and spleen of experimental animals (O'Hagan, 1990). Therapeutic agents, such as drugs or antigens, may be entrapped in microparticles and administered orally in attempts to exploit this targeted uptake mechanism in the intestine. Attempts to use microparticles and nanoparticles for oral delivery purposes will be discussed in subsequent sections.

3. DRUG-TARGETING SITES 3.1. INTRODUCTION The targeting of drugs following any route of administration can be conducted at different levels of sophistication. Targeting to a particular organ, for example, the stomach, the different regions of the small intestine or the colon, could be termed "first-order" targeting. The more selective delivery to a particular cell type within an organ (for example, targeting of a vaccine to the M-cells residing in the Peyer's patch region of the small intestines) represents a more sophisticated targeting requirement and can be termed "second-order" targeting. A third level of targeting can also be identified where the target would be a structure within a particular cell type residing in a particular organ. At the present time, most examples of targeting to the gastrointestinal tract involve aspects of first-order targeting. There are a number of instances where targeting to the gastrointestinal tract may be beneficial. In some situations, it will be an advantage to deliver the drug to a specific region, either because this site is preferred for good absorption of the drug for local drug action or that delivery to other sites could lead to adverse reactions and side-effects. The enteric coating of a dosage form, such as the tablet, is a simple example where the delivery system is targeted to the proximal small intestines in order to avoid damage to the drug by the hostile environment within the stomach or, alternatively, to prevent mucosal damage caused by the drug. A well-known example is the enteric

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coating of NSAIDs and the enteric coating of penicillin derivatives. While it is clear that many drugs can be degraded under acid conditions, the practical problems of drug degradation in the stomach should not be overemphasized. Work conducted by Farraj and others (1988) has shown clearly that for certain unstable basic drugs it is advantageous for them to be delivered into the stomach, where they are well dissolved, in order to have good absorption subsequently from the small intestine and, thus, demonstrate high bioavailability. Notwithstanding the fact that the basic drug (Progabide) itself was unstable in acid conditions, (t05 = 20 min at a pH of 1.5, 37°C), the rapid gastric emptying of the drug solution prevented large degradative losses in the stomach. In comparison, if the formulation was enteric coated, the drug avoided degradation in the stomach, but was then faced with an extremely slow dissolution process within the more alkaline conditions of the intestines. Consequently, a strategy of enteric coating of the drug to prevent loss due to degradation in the stomach did not improve bioavailability, but actually led to a substantial loss. These competitive factors could be well represented by a computer model. Thus, only drugs that are extremely unstable in acid conditions may represent candidates for enteric coating where the procedure is designed to protect the drug molecule from degradation. 3.2. LOCALAc'rloN 3.2.1. Digestive Enzyme Supplements In certain disease conditions, such as cystic fibrosis and following pancreatectomy or chronic pancreatitis, enzyme supplements (pancreatin) are given to replace endogenous enzymes (Graham, 1977; Smalley, 1986; Linehan et al., 1986). These supplements assist in the digestion of fat, starch and protein. Pancreatin is a preparation of mammalian pancreas, containing enzymes having protease, lipase and amylase activity, which are inactivated by gastric acid, and, therefore, enteric-coated products have been developed to deliver high enzyme concentrations to the duodenum (Graham, 1979). Ideally, the product should be in particulate form and the small particles should mix with and empty together with the gastric contents (Meyer et al., 1988). However, studies in healthy subjects using gamma scintigraphy have suggested that some commercially marketed microparticulate systems of pancreatin will empty too slowly to be effective in the digestion of food (Meyer et al., 1988). 3.2.2. Topical Action in the Treatment o f Inflammatory Bowel Disease As mentioned in Section 2.2.1, the drug sulphasalazine, has been used for the treatment of ulcerative colitis for many years. The material itself is inactive, being a prodrug and it also displays low absorption from the gastrointestinal tract. However, in the colon, it is cleaved into sulphapyridine and 5-ASA by the reducing conditions present in the colon. Sulphapyridine appears to act solely as a carrier, but is responsible for some of the side-effects associated with the drug (Swift et al., 1992). Unfortunately, the clinical usefulness of sulphasalazine has been hampered by a high rate of adverse reactions (Sninsky et al., 1991). Sulphapyridine-free azo-bond analogs of sulphasalazine, such as olsalazine and balsalazide, have been prepared (Laursen et al., 1990; Mclntyre et al., 1988). It is believed that the low systemic load of 5-ASA provided by the new prodrugs reduces the potential risk of nephrotoxicity during long-term treatment. 5-ASA itself can be used for the treatment of ulcerative colitis; however, to prevent unwanted systemic absorption from the small intestine (that reduces the efficacy of the drug), coated delayed-release systems are available, which have been designed to target the drug to the colonic regions. These coated systems are given the generic name, Mesalazine (Crotty and Jewell, 1992). They rely upon pH-sensitive enteric coatings or a semi-permeable film of ethyl cellulose to provide a pH-sensitive-controlled release effect (Dew et al., 1983; Rasmussen et al., 1982). Details of the development of one of the pH-sensitive coating products and its evaluation in vivo has been reported (Hardy et al., 1987a,b; Healey, 1990). Hayllar and Bjarnason (1991) have noted that changing the delivery system for 5-ASA from an azo-bond cleavage process to mechanical or pH-dependent release has two important implications. Firstly, it can reduce the effectiveness of colonic 5-ASA delivery and secondly, it changes the

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pharmacokinetics of the drug and, in turn, its metabolic profile. The products coated with a semipermeable ethylcellulose layer allows gradual release of the drug during passage through the small intestine, while the pH-sensitive polymer coating used with products can allow rapid release of 5-ASA. Free 5-ASA is acetylated in the small bowel and colonic epithelium. This process can be overloaded by rapid release in the small bowel to permit 5-ASA itself to be absorbed. Interestingly, the unacetylated form of the drug is nephrotoxic. 3.3. SYSTEMICABSORPTION 3.3.1. Prevention of Gastric Irritation The enteric coating of certain drugs is undertaken in an effort to reduce the effect of the drug on the gastric mucosa. Enteric forms of NSAIDs are now available; however, it is probable that NSAIDs may affect other regions of the gastrointestinal tract to include the small intestine and the colon (Jenkins et al., 1991). NSAIDs have been reported to exacerbate ulcerative colitis. The laxative agent, bisacodyl and the anticonvulsant, sodium valproate, are enteric coated to avoid gastric irritation. 3.3.2. Oral Delivery of Peptides Polypeptide and protein drugs, such as insulin, growth hormone, interferon, granulocyte-colony stimulating factor, calcitonin, etc., now have an important place in therapy. Clearly, it would be a great advantage if these products of biotechnology and synthetic peptide chemistry could be administered by mouth rather than by injection or via transmucosal routes, such as the nose and the lung. Not surprisingly, the absorption of significant quantities of complex and labile polypeptide from the intestines will be fraught with difficulties. Polypeptides can be denatured or degraded by the harsh environmental conditions present within the gastrointestinal tract. These conditions include the acidic pH of the stomach and the presence of endogenous enzymes. Even if the molecule could survive such assault, it would be unlikely to pass across the mucosa to a large extent, simply because of size and hydrophilicity considerations. At first sight, one would expect the bioavailability of an orally administered polypeptide to be almost nil. However, it is now clear that small quantities of low molecular weight polypeptides and even smaller quantities of proteins can survive intact to reach the systemic circulation (Bloch et al., 1988; Gonella and Walker, 1987). In most cases, the measured bioavailabilities are about 1% and can even be a fraction of a percentage (Vilhardt and Lundin 1986). Such low absorption can be increased by the chemical modification of the polypeptide to render it less unstable and, perhaps, to increase its lipophilicity (Muranishi et al., 1992). This strategy has been successful in some instances, particularly for renin inhibitors (Hanson et al., 1989). However, with other molecules, particularly where they are dependent on tertiary structure for their biological effect, any change in chemical nature can often reduce biological activity or even render the material inactive. Some polypeptides are surprisingly well absorbed from the gastrointestinal tract. Cyclosporin-A is a well-quoted example, where bioavailabilities of 50% or more can be achieved through the use of the correct vehicle system (Drewe et al., 1992). Unfortunately, cyclosporin-A is not a good role model for most therapeutic peptides and proteins, since it is of a cyclical structure and has a high lipid solubility. A detailed understanding of the problems facing the absorption of peptides and proteins from the gastrointestinal tract can be gained using data for well-studied molecules, such as insulin. Such an analysis indicates that a maximum bioavailability of about 1% can be expected, unless the transport of the molecule across the intestinal barrier can be increased using some form of drug delivery or targeting strategy (Davis, 1990, 1992). Unfortunately, in this area, the fundamental difficulties of delivering peptides and proteins intact across the gastrointestinal tract seem to have been ignored and as a result, many wild claims have been advanced. For example, some groups have claimed it possible to deliver more than 50% of complex proteins, such as interferon-or and erythropoietin, across the human gastrointestinal tract. As one would expect, such claims have not been substantiated by well-conducted clinical investigations, and, indeed, it has been suggested that the race for commercial success has led to reckless science and even scientific malpractice (Morgan, 1991). Some of the novel delivery systems being proposed as an answer to the problem of peptide JPT 62/b2

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and protein delivery from the gastrointestinal tract are based on totally unscientific principles. For example, the belief that large quantities of particulate material can be taken intact across the human gastrointestinal tract and that this presents opportunities for the delivery of therapeutic agents, such as insulin, heparin and even growth factors, is without foundation (O'Hagan, 1990). The use of lipid vehicles to enhance the absorption of peptides and proteins from the gastrointestinal tract is another popular but misguided concept. Certainly, a polypeptide molecule can be afforded some degree of protection by incorporating it in a colloidal carrier, such as a liposome or emulsion system. In addition, the presence of formulation factors within the delivery system, such as surfactants, could lead to modification of membrane permeability and, hence, a greater uptake of the peptide agent. However, the suggestion that emulsion particles and lipid droplets can move intact across the gastrointestinal tract in large quantities and carry the therapeutic peptide on their surface for subsequent presentation to the systemic circulation is contrary to known mechanisms of fat digestion and chylomicron formation (Davis, 1992). Often where claims of high bioavailability have been published, the measured effect has been one based upon a pharmacodynamic response rather than the determination of a plasma concentration of the delivered drug. Obviously, if one uses a high enough dose via the oral route, one can match the pharmacological or therapeutic effect of a parenterally administered formulation. The few well-conducted studies that have been conducted in humans on the oral absorption of polypeptide molecules would suggest that 1% bioavailability is a realistic estimate (Bonuccelli et al., 1988; Davis, 1992). A careful analysis of the problem of peptide and protein absorption from the gastrointestinal tract, together with computer modelling, would suggest that the small intestine, though having a large surface area, is perhaps not the preferred site for uptake. Not only does the small intestine contain endogenous enzymes, but the transit time from stomach to the colon is surprisingly short--about 3 hr in normal individuals (Davis et al., 1986). Thus, even if the drug was relatively stable in the intestines, there could be little time for its absorption. Consequently, the colon is being examined as the preferential site for the absorption of polypeptide drugs. Although the colon has a much smaller surface area, it does have an extremely long transit time and the level of endogenous enzymes is low due to processes of rapid inactivation. The colon does, of course, contain micro-organisms, which have the ability to degrade proteins. However, by appropriate formulation strategies, it should be possible to create local environments and to thereby minimize protein degradation, as well as to enhance their uptake. Preliminary work conducted at Nottingham and elsewhere (Gwinup et al., 1991; Hastewell et al., 1992; Atchison et al., 1989; Wilding et al., 1992b) would indicate that the colon, indeed, does seem to be the best site within the gastrointestinal tract for peptide and protein delivery. In evaluating candidate delivery systems, it is often essential to undertake studies in animal models. Unfortunately, there is no animal model that is truly representative of the human gastrointestinal tract, particularly with regard to the colonic region. The pig is perhaps the closest species to humans for the evaluation of drug absorption and formulation strategies. If the colon does represent the best or only site for uptake of therapeutic polypeptides, then the systems described above that have the propensity to target to the colon are an essential requirement.

4. TARGETING OF ORAL VACCINES 4.1. INTRODUCTION The majority of the gut-associated lymphoid tissue (GALT) is organized into aggregates of lymphoid follicles called the Peyer's patches. The major physiological role of the Peyer's patches is the induction of secretory immune responses to ingested antigens. Peyer's patches have been identified in most species studied, but their number and distribution is dependent upon the microbial and antigenic load encountered during the evolution of a particular species. The Peyer's patches are commonly located at sites in the gastrointestinal tract where the intestinal contents are often resident for extended periods, e.g. near the ileocaecal sphincter and in the rectum (Owen and Ermak, 1990). In humans, the largest Peyer's patches are found in the terminal ileum, just above the ileocaecal valve (Cornes, 1965). The Peyer's patches are covered with a specialized lymphoepithelium, which is adapted to allow antigen sampling from the lumen and the uptake of

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micro-organisms. Antigens are then delivered into the underlying dome structures of the patches, which consist of lymphoid follicles, with an abundance of lymphoid cells, including B- and T-lymphocytes, macrophages and dendritic cells. Antigens are transported into the Peyer's patches by specialized epithelial cells, called M-cells. M-cells have been shown to be responsible for the uptake of a variety of materials, including macromolecules, bacteria, viruses and protozoa (Owen and Ermak, 1990). There are two important issues concerning the uptake and transport of antigens by M-cells: (i) antigens will probably escape degradation and (ii) the antigen will be released into an environment rich in immunocompetent cells. Thus, uptake by M-cells allows delivery of intact antigenic material into the immune-inductive environment of the Peyer's patches and restricts access of antigens to alternative areas of the intestine, where suppressor T-cells predominate (Bland and Warren, 1986). Consequently, antigens that are taken up into the Peyer's patches through M-cells are likely to induce potent immune responses, while antigens that are taken up by ordinary enterocytes are more likely to induce systemic tolerance. The available experimental and clinical evidence strongly suggests that secretory immunity is induced and regulated by mechanisms distinct and separate from those involved in the regulation of systemic immunity (Mestecky, 1987). This evidence includes the observation that secretory IgA is predominantly locally synthesized and is transported across the epithelium into the mucosal secretions, following binding to a specific receptor produced by the epithelial cells, the secretory component. Furthermore, it has been shown that intravenously administered IgA appears in intestinal and salivary secretions only at low concentrations (Delacroix et al., 1982; Kubagawa et al., 1987). Moreover, although patients with IgA multiple myeloma show very high levels of IgA in blood, only trace amounts are found in their saliva (Tomasi et al., 1965). Immune responses at the mucosal sites are linked through the existence of a common mucosal immune system (CMIS) (Fig. 4). The existence of the CMIS makes it possible to induce secretory immune responses at all the mucosal sites following oral administration of antigens. The CMIS is linked by migrating antigen-stimulated IgA precursor cells, which are induced in the Peyer's patches. The antigen-stimulated IgA-committed lymphoblasts migrate via the mesenteric lymphatics and the thoracic duct into the systemic circulation. Subsequently, they specifically localize in mucosal tissues through interaction with specific cellular receptors on high endothelial venules (Stoolman, 1989). Within the mucosal tissues, which are the effector sites of mucosal immunity, e.g. the lamina propria of the gut and the respiratory tract, the lymphoblasts mature into plasma cells and secrete IgA (Mestecky and McGhee, 1987). The IgA is then transported through the epithelial cells and into the external secretions after interaction with the secretory component. The most convincing evidence for the existence of the CMIS comes from studies that have demonstrated the induction of secretory antibody responses at distant mucosal sites, e.g. tears, saliva and genital secretions, following oral immunization (Bergmann and Waldman, 1988). The CMIS and the mechanisms of regulation of mucosal immune responses has been reviewed recently in detail by McGhee et al. (1992).

4.2. NON-LIVINGANTIGEN-DELIVERYSYSTEMS 4.2.1. Controlled Release Microparticles as Oral Vaccines In preliminary studies, it was demonstrated that the incorporation of an antigen into microparticles (O'Hagan et al., 1989a), or its adsorption to the surface of biodegradable microparticles (O'Hagan et al., 1989b), resulted in the induction of enhanced serum and secretory antibody responses, following oral administration. Subsequently, it was demonstrated that oral delivery of an antigen entrapped in poly (lactide-co-glycolide) (PLG) microparticles also resulted in the induction of enhanced immunity (Eldridge et al., 1990). The PLG polymers and related analogs have already been used for several alternative biomedical applications, and, therefore, they are the primary candidates for use in the preparation of controlled-release vaccines. PLG polymers have been used both as resorbable sutures (Reul, 1977) and as bone plates for internal fixation (Christel et al., 1982). In addition, PLG polymers have also been used for the preparation of a number of

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FIG. 4. Uptake of antigen by the M cells on the Peyer's patches of the GALT stimulates IgA-committed B cells and CD4 ÷ T helper cells, which migrate via the mesenteric lymph nodes (MLN) and the thoracic duct (TD) into the blood. The cells then "home" to mucosal sites and IgA is produced by plasma cells, which have developed from the migrating B cells. Polymeric IgA (PIgA) is formed from monomeric IgA (mlgA) through the J chain, and is transported across the epithelial cells after interaction with the secretory component (SC). Reprinted from O'Hagan (1992), with permission of the copyright holder, ADIS Press Ltd, Manchester. controlled-release drug-delivery systems (Maulding, 1987). Long experience with these polymers has shown that they are completely biodegradable in a physiological environment and degrade to toxicologically acceptable products, which are eliminated from the body (Wise et al., 1979). The biocompatibility of microparticles prepared from PLG polymers has been demonstrated by Visscher et al. (1987). A soluble protein antigen entrapped in PLG microparticles showed enhanced immunogenicity, following parenteral immunization (O'Hagan et al., 1991, 1993a). In addition, it was demonstrated that an antigen entrapped in PLG microparticles (3/~m) induced significantly enhanced secretory IgA and systemic IgG antibody responses following oral administration (Fig. 5). Optimally, the salivary IgA-antibody response to antigen in microparticles was 50 times greater than the response to soluble antigen (Challacombe et al., 1992). Oral immunization with antigen in microparticles has also been shown to result in the induction of a specific cytotoxic T-cell response in the spleen cells of immunized mice (O'Hagan et al., 1993b). Edelman et al. (1993) also described the use of PLG microparticles with an entrapped antigen for oral immunization. Following a single immunization, a vigorous serum IgG antibody response was induced to enterotoxigenic E. coli colonization factor-1 entrapped in microparticles. However, only one of three rabbits in the study showed a secretory IgA response. The microparticles used in this study ranged in size from < 10 to 200 #m (mean size 27 /~m). Therefore, the majority of the microparticles were too large for uptake into the Peyer's patches (O'Hagan, 1990). Consequently, the main purpose of microencapsulation, in this study, was to protect the entrapped antigen against degradation in the gut. Thus, microparticles, which are too large for uptake into the Peyer's patches, may also be exploited for the development of oral vaccines. A study by McQueen et al. (1993) showed that colonization factor-1 entrapped in microparticles was able to induce protective immunity following oral immunization in rabbits.

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If microparticles are to achieve widespread use in humans as oral vaccines, then the current formulations will need to be considerably improved. Ideally, the dose should be administered as a capsule or as a tablet, which would help to protect the incorporated antigens from degradation. Bodmeier et al. (1989) described one approach for the production of an acceptable final dosage form for oral administration of a microparticle formulation. Preformed microparticles were entrapped in beads prepared by ionotropic gelation of the polysaccharides, chitosan and sodium alginate. The beads could be coated with CAP to produce an entericcoated dosage form, although the polysaccharides themselves are capable of providing pH-dependent release profiles. Klipstein et al. (1983) were the first to describe the use of enteric-coated microparticles as oral vaccines. Heat-labile enterotoxin from E. coli (heat-labile enterotoxin subunit-B (LTB)) was encapsulated in pellets (3 mm) prepared from starch and cellulose, with hydroxypropylmethylcellulose phthalate as an enteric-coating polymer. After oral administration to rats, the microparticles induced serum and intestinal antibody responses that were comparable to those induced by oral immunization after ablation of gastric secretions with cimetidine. Maharaj et al. (1984) described a method for the preparation of microparticles (1-3 mm) with entrapped virus using CAP as a polymer coating. The microspheres were stable for 6 hr in simulated gastric fluid, but disintegrated rapidly in simulated intestinal fluid. A similar approach to oral vaccine development was described by Lin et al. (1991a, b), CAP microspheres (0.5-2 mm) with entrapped bacteria showed enhanced vaccine stability on storage and in the presence of low pH. Enteric-coatings have also been applied to a number of vaccines, which were subsequently used in clinical trials, including vaccines against Haemophilus influenzae (Yeung et al., 1987), typhoid fever

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(Levine, M.M. et al., 1987), tuberculosis (Ishihara et al., 1986) and hepatitis B virus (Lubeck et al., 1989). Hence, enteric-coating polymers may be utilized to improve the efficacy of orally administered vaccines.

4.2.2. Lectin-like Molecules as Oral Vaccines The ability of a range of different proteins to induce immune responses following oral administration was assessed by De Aizpura and Russell-Jones (1988). They found that proteins with 'lectin' or 'lectin-like' binding activity to the intestinal mucosa were capable of inducing immune responses, while proteins without binding activity were not. In a subsequent study, these observations were exploited through the conjugation of peptide and protein antigens to Vitamin B12 (VB12) (Russell-Jones and De Aizpura, 1988). VB12 is transported across the intestine by receptor-mediated endocytosis after binding to the intrinsic factor in the stomach. The intrinsic factor is endocytosed following interaction with an intestinal receptor, through lectin-like binding activity. The orally administered antigen-VB12 conjugates induced serum antibody responses in mice, while the unconjugated antigens did not. It remains to be seen if VB12 can be further developed for oral delivery purposes, but the carrier capacity of the complex would appear to be very limited. An extensive range of lectins from a variety of sources, including many from plants, may have potential as components of oral vaccines. However, a cause for concern is the reported toxicity of many lectins following oral administration (Pusztai, 1989a). Furthermore, the conjugation of antigens to lectins would probably be required, to prevent possible immune responses to unrelated antigens and the effects on lectin binding would need to be determined. Additional molecules with lectin-like binding activity to the intestinal epithelium, which may have potential as components of oral vaccines, include LTB and cholera toxin B subunit (CTB). Two new oral cholera vaccines have been evaluated in large-scale field trials in cholera endemic areas. The vaccines consisted of either killed whole cells (WC) alone, or WC in association with purified CTB. CTB is non-toxic and binds to specific receptors on the intestinal epithelium. In the trials, the WC-CTB vaccine showed an advantage over the WC vaccine of greater efficacy for the initial 4--6 months (85% protection against disease with WC-CTB, compared with 58% with WC alone). Thereafter, the level of protection was similar for the two vaccines, at about 60% for 3 years (Holmgren et al., 1992; Holmgren and Svennerholm, 1990). It has been shown that whole cholera toxin (CT) is a potent adjuvant for unrelated antigens (Lycke and Holmgren, 1986) and induces long-term immunological memory in the gut following oral immunization (Vajdy and Lycke, 1992). Consequently, there has been considerable interest in the possible use of CT or CTB as adjuvants in a wide range of oral vaccines. However, since CT and CTB themselves are very potent immunogens, pre-existing immunity is likely to limit their efficacy on repeated administration. The mucosal adjuvant activity of CT is thought to be due to a synergistic effect, involving both the CTB and the adenylate cyclase activity of the A-subunit (Wilson et al., 1990). However, more recently, the adjuvant effect of CT has been more closely linked to ADP-ribosyltransferase activity (Lycke et al., 1992). It has been shown that purified CTB alone is unable to act as an oral adjuvant (Lycke and Holmgren, 1986; Lycke et al., 1992). Hence, many of the reports describing the adjuvant effect of CTB alone may actually be due to the presence of small amounts of the whole CT. If CTB or CT were to be used as oral adjuvants, it is likely that they would need to be specifically conjugated to the vaccine antigens, since immune responses to unrelated 'bystander' antigens in the gut may otherwise occur. The adjuvant effect of CT has been shown to be associated with an increased permeability of the intestine for luminal antigens (Lycke et al., 1991). The effect of chemical conjugation to the antigens on the binding characteristics and efficacy of CT or CTB would need to be determined and it might not be easy to couple to antigens, while retaining adjuvant activity. The mechanism of the adjuvant activity of CT remains poorly understood and in view of its undoubted toxicity, its acceptability as a general vaccine component appears limited.

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4.2.3. Liposomes as Oral Vaccines Although some success has been achieved in experimental studies involving oral administration of antigens entrapped in liposomes (Michalek et al., 1989), disappointing results have also been reported by others (Clarke and Stokes, 1992a, b). Nevertheless, a liposomal vaccine has been shown to induce a salivary IgA response in a small number of human volunteers, following oral immunization (Childers et al., 1991). Furthermore, although it has been reported that liposomes are unstable in the gut and are not taken up by epithelial cells (Chiang and Weiner, 1987), the uptake of liposomes into Peyer's patches has been reported (Childers et al., 1990). 4.2.4. Immunostimulating Complexes as Oral Vaccines An alternative novel approach to vaccine development, which has aroused considerable interest in recent years, involves the use of immunostimulating complexes or ISCOMS (Morein et al., 1984). ISCOMS are a complex formed between the saponin adjuvant Quil A, cholesterol and amphipathic antigens and they have been used for parenteral immunization in many studies since 1984. Recently, ISCOMS have also been shown to be capable of inducing antibody responses and cell-mediated immunity, following oral administration (Mowat et al., 1991). A previous report had indicated that saponins alone were effective oral adjuvants for associated antigens (Chavali and Campbell, 1987). Nevertheless, the presentation of antigen and saponins in ISCOMS allowed a reduction in the dose of saponin and may prevent induction of adverse immune responses to 'bystander' antigens. However, there are no data to indicate whether the ISCOMS are actually stable in the gut and whether only a limited range of antigens may be effectively incorporated. 4.3. LIVE VECTORS FOR VACCINE DEVELOPMENT 4.3.1. Salmonella-based Oral Vaccines In recent years, considerable efforts have been directed towards the development of an effective oral vaccine against the causative agent of typhoid fever, Salmonella typhi. Strains of Salmonellae have been shown to colonize the GALT and persist in the host before inducing a disseminated infection of the reticuloendothelial system. In the 1970s, an avirulent mutant strain of S. typhi (Ty21 a) was developed using chemical mutagenesis. Subsequent field trials in humans demonstrated that the vaccine was safe and immunogenic, although protection against typhoid was only moderate and unduly sensitive to vaccine formulation and dose regimen (Levine, M. M. et al., 1987). Although this vaccine has been licensed in many countries as a live oral typhoid vaccine, reports have suggested that it may not be effective in previously unexposed individuals, e.g. travellers to an endemic area (Schwartz et al., 1990). Furthermore, the molecular basis of its attenuation is not understood, although it is thought to harbour mutations in galE. It is now possible to construct strains of a well-defined nature and it may be inappropriate to use Ty21a when the basis of attenuation is poorly understood. Indeed, when a S. typhi strain with a site-directed mutation in galE was tested in humans, it retained virulence (Hone et al., 1988). Second generation typhoid vaccines have now been developed by recombinant DNA techniques and Salmonella strains have been developed harbouring individual defined mutations (Chatfield et al., 1989). Salmonella strains with gene mutations in enzymes of the pre-chorismate metabolic pathway, such as aroA, aroC and aroD, which have an auxotrophic requirement for several aromatic compounds, have been shown to be effective oral vaccines in mice (Hoiseth and Stocker, 1981), sheep (Mukkur et al., 1987) and calves (Jones et al., 1991). In humans, the genetically modified vaccines have been shown to be safe and immunogenic (Hone et al., 1988, 1991; Chatfield et al., 1992). In anticipation of the construction of safe and immunogenic oral Salmonella vaccines, the possibility of using such organisms as carriers of heterologous antigens has been discussed (Cfirdenas and Clements, 1992). For example, live attenuated S. typhimurium (aroA mutant), expressing fragment C of tetanus toxin, has been used to achieve protective immunity against a lethal toxin challenge, following oral immunization (Fairweather et al., 1990). More recent developments have been concerned with the stable expression of heterologous proteins in vivo from

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genes that are chromosomally integrated (Chatfield et al., 1992). As well as the potential instability of the foreign gene, there are a number of additional factors that may restrict the use of Salmonella-based carrier systems. These include the level of antigen expression required, the site of expression of the antigen within the bacterial cell, the possible effects of the foreign gene on the carrier organism and the effect of pre-existing immunity. Perhaps the biggest potential problem for Salmonella-based vaccines is that pre-existing immunity to the carrier organism may limit replication in the host, resulting in reduced efficacy on repeated administration. Hence, the vaccine may be capable of being used successfully only once. Similar problems are also likely to occur with alternative live carriers, especially if they are used in endemic areas or in hosts who have been previously immunized against the carrier organism. These problems may seriously limit the use of live recombinant carriers in many areas and in many populations. A second l:otential problem concerns the unresolved issue of safety, particularly in immunocompromised individuals. However, aro mutants of Salmonella do not show enhanced pathogenicity in immunosuppressed mice (Izhar et al., 1990). 4.3.2. Alternative Live Vectors Alternative live vectors currently being investigated as possible oral vaccines include poxviruses, adenoviruses, polioviruses and mycobacterium BCG. Considerable success has been achieved in studies involving immunization of animals with recombinant vaccines. For example, a vaccinia-rabies recombinant virus has been successfully used to orally immunize foxes against rabies by distribution of vaccine-baited food in their feeding area (Brochier et al., 1990). In addition, vaccinia virus recombinants expressing human immunodeficiency virus antigens have been used to immunize human subjects in phase I clinical trials at several centers both in the United States and elsewhere (Picard et al., 1992). An alternative approach involves the use of adenoviruses as carrier systems for heterologous antigens. Adenovirus vaccine has been orally administered to several million U.S. soldiers and has been shown to offer excellent protection against acute respiratory disease. Recombinant adenoviruses, based on the vaccine strains, have been constructed and in studies in chimpanzees, have been shown to be capable of inducing immune responses to the expressed antigen (hepatitis B surface antigen), following oral immunization. Recently, this recombinant adenohepatitis B oral vaccine was assessed for safety and immunogenicity in a phase 1 study in human volunteers (Tacket et al., 1992). Another live attenuated organism under consideration for development as a possible recombinant vaccine for humans is poliovirus. The Sabin type 1 vaccine strain of poliovirus, which is probably the safest and most successful attenuated human vaccine, has been adapted to express antigens from alternative pathogens (Burke et al., 1988). However, the ability of this poliovirus chimaera to induce antibodies following oral administration remains to be assessed and the effect of pre-existing immunity needs to be determined. Mycobacterium BCG also offers promise as a recombinant oral vaccine (Stover et al., 1991). Genetic engineering has allowed the development of a new attenuated oral vaccine against cholera, which has produced very encouraging results in clinical trials (Suharyono et al., 1992). The attractions and possible limitations, of the different novel antigen delivery systems for oral immunization are highlighted in Table 2.

5. CONCLUSIONS The gastrointestinal tract, for many years, has been the most popular route for drug delivery and it is likely to remain so in the future, despite certain known disadvantages of oral dosage. For example, many drugs show little, if any, inherent specificity for selective delivery. This could limit their therapeutic effectiveness and possibly lead to significant side-effects. As a consequence, the current trend in oral therapy is to explore the possibilities for targeted drug delivery to a particular site within the gastrointestinal tract and, thereby, to maximize drug performance. Over recent years, there has been a significant increase in the number of targeting mechanisms available to the pharmaceutical scientist to provide site-specific delivery in the gastrointestinal tract. However, although these prototype drug delivery systems have been tested in vitro using various

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TABLE2. Advantages and Disadvantages of Alternative Antigen Delivery Systems of Oral Immunization Delivery systems

Advantages

Colonization of Peyer's patches with genetically engineered organisms (e.g. Salmonella, mycobacteria, adeno- and poliovirus)

Potential for potent stimulation of immune response, may include several antigens

Disadvantages Antibodies to carrier may preclude the use of the same organisms for booster immunization, safety concerns?

CTB subunit

Very potent adjuvant

Probably requires whole toxin for adjuvant effect, chemical coupling may be required, immunity to carrier may restrict use

Microparticles

Promotes uptake by Peyer's patches, protects antigen, controlled release, can include adjuvants and targeting agents

Unproven efficacy in humans, uptake of particles requires further study. Possible denaturation of antigens during microencapsulation

Liposomes

May include adjuvants and targeting agents, protects antigen from degradation

Stability problems, solubilization in the gut (bile salts, lipases, etc.)

Lectins

Wide range of materials available for assessment, with different specificities

Non-specific enhancement of immune responses to gut contents possible, may require chemical coupling, toxicity?

techniques designed to study drug release, it is also critical that such concepts are evaluated in the clinical target, i.e. humans. For example, despite a large number of in-vitro studies, there is no published evidence that suggests that bioadhesive systems can actually alter intestinal transit in humans nor is there any evidence that polysaccharides or redox-sensitive polymers actually guarantee selective delivery to the colon. Animal models clearly have some utility in this respect, but, presently, there is a growing tendency for new delivery systems to be tested, whenever possible, in human subjects. In particular, it has become common practice for the in-vivo performance of novel drug delivery systems to be evaluated in healthy volunteers or patients using scintigraphic techniques (Davis et al., 1992) to ascertain, for example, whether the designed system is performing correctly in providing optimal deposition at the preferred site of absorption or action, or whether it is releasing the drug according to a prescribed pattern or the intended rationale. Gamma scintigraphy has allowed basic but important questions in drug delivery to be addressed, such as: where is the dosage form?, what is it doing?, and is the system targeting/releasing in accordance with its intended mechanism or rationale? Currently, there is a great deal of effort being aimed at achieving effective delivery of novel therapeutic drugs, such as peptides, by the oral route. Opportunities have been identified that could lead to more convenient delivery systems for this class of drug. It is likely that a polypeptide, given unprotected into the gastrointestinal environment, will be degraded significantly. However, it is well known that small quantities of dietary proteins can be absorbed, even though these may have little or no physiological effects. It is felt that the colon may provide an advantageous absorption site for peptides. As a consequence, there has been considerable interest, not only in the development of colonic delivery systems, but also in the establishment of strategies designed to maximize peptide absorption from the colon. Traditionally, vaccine research has been concerned with producing systemic immunity by parenteral immunization. However, the gradual acceptance of the importance of IgA in protecting mucosal surfaces against infection from numerous pathogenic organisms has led to an increased interest in oral immunization. The recent advances in recombinant DNA technology and the development of antigen delivery systems, have given rise to significant optimism that several new and improved oral vaccines may be available by the next millennium.

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Whether live or non-living carrier systems are the most attractive option in the long term is an unresolved question and both may find specific applications against different pathogens. While the immune responses to live vaccines are generally more potent and of longer duration, non-living controlled-release delivery systems may also offer the possibility of long-lasting immune responses. The main drawback to using live vaccines, in the past, has been their propensity to cause severe infections in immunocompromised individuals. However, it is possible, with modern genetic techniques, that stable, non-reverting deletions may be introduced to specific genes in order to render the carrier completely safe. Nevertheless, with the use of live carriers, the immune system is subjected not only to the antigen of interest, but also to a vast array of additional antigens that constitute the structure of the carrier organism. This may not be to the advantage of the individual. Alternatively, a specific antigen may be selectively delivered in a non-immunogenic carrier vehicle into the sites of induction of the immune response in the intestine. This is certainly a 'cleaner' and, possibly, more appealing approach, which may have advantages over alternatives.

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