challenges in delivery of therapeutic genomics and proteomics || oral delivery of proteins and...

Challenges in Delivery of Therapeutic Genomics and Proteomics. DOI:© 2010 Elsevier Inc. All rights reserved.

10.1016/B978-0-12-384964-9.00010-42011

Oral Delivery of Proteins and Peptides: Concepts and ApplicationsGaurang Patel, Ambikanandan MisraPharmacy Department, TIFAC – Centre of Relevance and Excellence in New Drug Delivery Systems, The Maharaja Sayajirao University of Baroda, PO Box 51, Kalabhavan, Vadodara 390 001, Gujarat, India

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10.1 Introduction

Recombinant deoxyribonucleic acid (DNA) techniques have eased the commercial scale production of proteins and peptides and thus the number of protein drugs has been increased exponentially [1]. Pharmacological classes such as enzymes (e.g., tis-sue plasminogen activator), enzyme inhibitors (i.e., peptide inhibitor of angiotensin I), hormones (e.g., leutinizing hormone-releasing hormone, LHRH), immunomodulators (e.g., interferons, vaccines), and antimicrobial agents (e.g., phylloxin) include proteins or peptides as a drug substance. Although the therapeutic potential and specificity of proteins are well recognized, their use in therapies is still limited due to their low bio-availability via noninvasive routes [2]. Currently, proteins and peptides are adminis-tered only through the parenteral route. Significant efforts have been made to explore other noninvasive routes, including oral, buccal, nasal, pulmonary, vaginal, rectal, ocu-lar, and transdermal, because of the limitations of the parenteral route, that is, frequent dosing due to the short half-life of protein and peptide in blood, pain on administration, poor patient compliance, and sterility requirement [3–5].

Delivery of peptides, such as LHRH and calcitonin through the nasal route, has already been studied and has showed the advantage of high permeability of proteins and peptides across nasal mucosa. Chronic or prolonged use of the dosage form in the nasal cavity is associated with disadvantages such as potential irritation and tox-icity to ciliary cells. Variable mucus secretion among patients affects the residence time and absorption through mucosa [6]. Delivery of proteins and peptides across the vaginal and the rectal mucosa has poor patient compliance [7]. Due to disadvantages of other mucosa, much attention is paid to mucosa of the oral cavity, such as buccal, gingival, and sublingual regions, and small and large intestinal mucosa for systemic delivery of proteins and peptides [8].

Mucosa of the oral cavity has several positive features, including excellent acces-sibility, high patient acceptance and compliance, and significant robustness of the mucosa. Without the assistance of trained personnel, the patient can self-administer the

Challenges in Delivery of Therapeutic Genomics and Proteomics482

dosage form without any pain or discomfort. These features increase patient compli-ance and acceptance of the dosage form [9,10]. Due to routine exposure to a variety of foreign substances, oral mucosa becomes robust and thus less prone to irreversible irritation or damage by the drug, the dosage form, or additives such as absorption pro-moters. However, salivary production and composition may contribute to chemical modification of certain drugs [11,12]. Protein and peptide absorption increases several fold due to the presence of a great number of capillary blood vessels, lymphatic plexus, and Peyer’s patches.

Oral bioavailability below 1% is often recorded for proteins and peptides because of limiting factors such as inactivation due to stomach acid or to digestive protease enzymes, the larger size and hydrophilic nature of protein, the tendency to undergo denaturation and aggregation, and first-pass metabolism [13–15]. To overcome such problems, various approaches are useful, that is, site-specific drug delivery [16,17], chemical modification [18,19], use of some vehicles and adjuvants [20], bioadhesive drug delivery [21,22], use of penetration enhancers, protease inhibitors [23], nano and microparticulate carriers [24], and use of multifunctional polymeric excipients [25]. New tools that raise the membrane permeability of macromolecules have been incorporated into delivery systems; this is essential to attain the high oral bioavail-ability required for acceptance in clinical applications. Modification of the physi-cochemical properties of macromolecules [26], the addition of novel function to macromolecules, and the use of modified and specialized delivery carriers [27,28] improves the oral bioavailability from 1% to at least 30–50%. More importantly, it is essential that these approaches maintain the biological activity of the proteins.

The purpose of the chapter is to focus attention on factors affecting protein and peptide absorption, barriers to protein absorption, penetration pathways, various approaches to improve their oral delivery, methods to study oral absorption, and vari-ous strategies in oral immunization.

10.2 Anatomy and Physiology of Oral Mucosa

10.2.1 Oral Cavity

The oral cavity is composed of three tissue layers: the epithelium, the basement membrane, and connective tissues.

10.2.1.1 Epithelium

The epithelium is composed of epithelial cells originated from a layer of cuboi-dal-shaped basal cells. Approximately 40–50 layers of stratified squamous epithe-lial cells make up the epithelium. These layers were made because the basal cells undergo continuous mitosis and move to the surface. As the cells migrate to the sur-face through the intermediate layers, they differentiate and become larger, flattened, and surrounded by an external lipid matrix (membrane-coating granules). This exter-nal lipid matrix determines the drug permeability of the tissue. Although gingiva

Oral Delivery of Proteins and Peptides: Concepts and Applications 483

(gum) and the hard palate are keratinized, areas such as buccal, sublingual, and the soft palate are nonkeratinized. Buccal epithelium thickness varies with location and typically ranges from 500 m to 800 to 800 m in humans, dogs, and rabbits. The estimated cell turnover time is 5–6 days [8,15]. Intercellular gap junctions are also present in the buccal epithelium.

The mucosa of the oral cavity is divided into three functional zones. First, the mucus-secreting regions (consisting of the soft palate, the floor of the mouth, the undersurface of the tongue, and the labial and buccal mucosa) have a normally non-keratinized epithelium. These regions represent the major absorption sites in the oral cavity. Second, the hard palate and the gingiva are the regions of masticatory mucosa and have a keratinized epidermis. Third, specialized zones are the borders of the lips and the dorsal surface of the tongue with its highly selective keratinization.

10.2.1.2 Basement Membrane

The basement membrane forms a boundary between the basal layer of epithelium and the connective tissues of the lamina propria and the submucosa. The basement membrane is generally composed of extracellular materials. This membrane is fur-ther divided into the lamina lucida, lamina densa, and a sublayer of fibrous material. This layer generally provides adherence between epithelium and underlying connec-tive tissues, mechanical support for epithelium and act as a barrier to the passage of cells and some large molecules.

10.2.1.3 Connective Tissues

Connective tissues are further divided into the lamina propria and submucosa. Connective tissue, in the form of a continuous sheet, composed of blood capillaries and nerve fibers serving the oral mucosa, is known as lamina propria. Blood carried to the oral mucosa is principally by way of the lingual, facial, and retromandibular veins. These veins open into the internal jugular vein and thus avoid first-pass metabolism. The buccal mucosae from monkeys, apes, dogs, pigs, and rabbits possess physiology very similar to that of human buccal mucosa [8].

10.2.2 Gastrointestinal Tract Other Than Oral Cavity

After administration of peptides and proteins to the gastrointestinal (GI) tract, their absorption and other biochemical transformation largely depends on the variety of GI tract parameters such as morphology, geometry, function of different components of the cells, ultrastructure, biochemical processes, transport mechanisms, and hydro-dynamics in the intestinal lumen.

Passage from the mouth to the anus (oropharynx, esophagus, stomach, small intes-tine, and large intestine) and associated organs (salivary glands, liver, gallbladder, and pancreas) compose a digestive system. Among all parts, the small intestine is the pri-mary site of absorption for most drugs. The small intestine is further divided into three parts: the duodenum, jejunum, and ileum. The large intestine, which is also called the


colon, is divided into the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, rectum, and anus. The salivary glands, liver, gallbladder, and pancreas deliver digestive secretions and help in digestion and absorption of drugs.

10.2.2.1 Stomach

Surface of the stomach is divided into three well-defined tissue layers: the muscle, the submucosal and mucosal layers. Because of the smaller size, lack of villi, thick mucosal layer, short residence time, and smaller absorption surface area, absorption from the stomach is minimal. The major function of the gastric mucosal epithelium is to secrete hydrochloric acid, pepsin, intrinsic factor, and bicarbonate [29,30]. Acidic pH affects the folded structure of protein and causes protein denaturation. These denatured proteins are more likely to be attacked by pepsin because of increased exposure of the peptide bonds to pepsin, which breaks down the proteins into peptides. However, the peptide bond breakage in the stomach is incomplete because pepsin can only break peptide bonds between specific amino acid sequences in pep-tides and proteins.

10.2.2.2 Small Intestine

The small intestine is a tubelike structure approximately 200 in. long with a radius of approximately 0.75 in., which is subdivided into three anatomical regions: the duode-num (the first 10 in.), the jejunum (the second 80 in.), and the ileum (the last 110 in.) [29]. Ingested food and macromolecules are enzymatically hydrolyzed to smaller molecules in the small intestine. The wall of the intestinal membrane has four dif-ferent layers: the mucosa, submucosa, muscularis, and serosa. These tissue layers are depicted in Fig. 10.1. The innermost, or luminal, surface is the mucosal layer,

Surface epithelium

Lamina propria

Crypt of lieberkuhn

Lymphoid

Muscularis mucosae

Submucosa

Circular muscle ofmuscularis externa

Longitudinal muscle ofmuscularis externa

Figure 10.1 Generalhistology of the GI tract.


which is mainly responsible for the digestive and absorptive functions of the small intestine. The surface area of mucosal layer is increased by several more folds than predicted for a simple cylinder due to the presence of circular folds, fingerlike pro-jections called villi, and depressions called crypts. The mucosal surface area of the small intestine is approximately 250 m2, whereas the simple cylinder has the surface area of 0.5 m2. The height of each villi is 0.5–1.0 mm and is equal to the depth of the crypts. Each villus and crypt is lined by epithelial cells that are covered with many closely packed microvilli projecting into the intestinal lumen.

The mucosal layer is further divided into three layers: an absorptive layer (com-posed of a continuous single sheet of columnar epithelium), the lamina propria (which is heterogeneous in composition and cell type), and the muscularis mucosa (which sep-arates the mucosa and submucosa).

Each villi and crypt is covered by a layer of epithelial cells having thickness of one cell. A variety of cells are found and identified in this epithelial layer of the small intestine. These include enterocytes, which have the function of digestion and absorption; goblet cells, which act to secrete mucus; endocrine cells secreting hor-mones; and M cells, which are involved in the absorption of food and antigens.

Enterocytes of the epithelial layer, more commonly known as absorptive cells, are more commonly present on the tips of villi and mainly involved in the absorptive function of the layer. In humans, these cells are 20–30 m in height and 8–10 m in width. Large numbers of microvilli are present on the surface of the enterocytes. This structure resembles a brush, and thus the microvillus border of the intestinal epithelial absorptive cells is known as the brush border and has approximately 1.0 m height and 0.1–0.2 m width. The cell membrane of these microvilli has a trilaminar structure (70–90 Å thick) composed of proteins, neutral lipids, phospholipids, and glycolipids. The plasma membrane of microvilli has an unusually high protein–lipid ratio, owing to the presence of specialized proteins possessing enzymatic, receptor, and transport properties.

The glycocalyx is an integral and dynamic part of the plasma membrane. This gly-cocalyx is a uniform layer of filamentous glycoproteins [31]. The carbohydrate part of the glycocalyx has sialic acid residues that impart negative charge to the glycoca-lyx layer at physiological pH. The microvillus and its glycocalyx are responsible for the digestive and absorptive functions of the enterocytes.

A variety of enzymes (peptidases) involved in the hydrolysis of peptides and pro-teins are present in this microvillus plasma membrane. Cotransport proteins, involved in the transport of sodium and amino acids, are found in the microvillus plasma mem-brane. Certain substances are taken up by specific receptor proteins present on the microvilli of enterocytes in different regions of the small intestine. For example, the receptors for vitamin B12 complex are present in the microvilli of ileal enterocytes but are not found on jejunal cells. This is why vitamin B12 is absorbed exclusively from the ileum. Vitamin B12 has been explored as a delivery system for peptide and proteins by covalently conjugating vitamin B12 to peptides (LHRH) or proteins (bovine serum albumin, BSA) [32].

The absorptive cell membrane rests on the lamina propria, which consists pri-marily of connective tissue and supports the epithelium lining. Lymphoid cells and


associated structures of the lamina propria do not play an important role immunolog-ically. Small lymphoid nodules are present in the upper small intestine, whereas large organized aggregates of lymphoid tissue (Peyer’s patches) are present in the ileum.

The basolateral membrane differs from the apical membrane. The protein-to-lipid ratio is very low in the basolateral membrane; thus it is thinner and more permeable than the apical membrane. These membranes differ in their enzymatic composition. Tight junction, responsible for paracellular transport, is present between the cells (Fig. 10.2).

Aqueous Boundary LayerBesides providing cellular barriers, the aqueous boundary layer presents an impor-tant hydrodynamic barrier that a drug must traverse before reaching the surface of the mucosal membrane [33]. This barrier layer, often referred to as the dormant, unstirred, or aqueous diffusion cover, is located at the intestinal lumen and membrane interface that a drug molecule has to pass before it passes through the membrane. The thickness of the aqueous boundary layer varies from the tips of the villi to the crypts. It is thinner at the tips and thicker and less moved in the crypts. This aque-ous boundary layer offers great resistance to the hydrophobic molecules and is the rate-limiting step. But with hydrophilic drugs, including peptides, diffusion across the aqueous boundary layer is much faster because the layer has less resistance to hydrophilic molecules [34].

Figure 10.2 Intestinal epithelial absorptive cells.


Luminal and Membrane Metabolism of Peptides and ProteinsWhen peptides and proteins are administered through the oral route, they tend to metabolize at three major sites before reaching the blood: in the lumen, on the sur-face of the membrane, and within the cell context. In the intestinal lumen, various enzymes, such as trypsin, chymotrypsin, carboxypeptidase, and elastase, are secreted by the pancreas, which metabolizes peptides and proteins [33]. On the surface of the membrane, the metabolism of peptides and proteins is caused by the presence of various aminopeptidases located on the brush border. Prolidase, dipeptidase, tripep-tidase, and cytoplasmic peptidases are present within the cell and are responsible for intracellular metabolism of administered peptides and proteins [29].

10.2.2.3 Large Intestine (Colon)

The large intestine is a tubelike structure having approximately 60 in. length and 2 in. diameter. It is further subdivided into the cecum, which is the beginning, or opening, of the colon; the ascending colon; the transverse colon; the descending colon, and the sigmoid colon, which continues into the rectum. As in the small intestine, the wall of the large intestine is also divided into four layers: the serosa, the muscularis externa, the submucosa, and the mucosa. These tissue layers are depicted in Fig. 10.1. In the large intestine, villi, microvilli, and crypts are not present, and hence it offers much less surface area for the absorption of administered peptides and proteins. The cells are much less dense than those in the small intestine. A large amount of various bac-teria are present in the large intestine, whereas there is much less of a bacterial load in the small intestine and stomach. These bacteria are involved in the digestion of residual food into caloric substance for absorption. Colon targeting is achieved by delivering peptide and protein drugs coated with polymers that are only degraded by azoreductases released by colonic bacteria [13]. Because of low enzymatic activity in the colon, colon targeting has gained much attention for the delivery of peptide and protein drugs. Table 10.1 describes anatomical and physiological properties of each absorption area of the GI tract [35].

10.3 Transport Mechanisms in the GI Tract

There are four distinct mechanisms for molecules to cross the membrane: via para-cellular, transcellular, carrier-mediated, and receptor-mediated transport (Fig. 10.3).

10.3.1 Paracellular Transport

Drug molecules pass through aqueous pores created by epithelial tight junctions. This is the most likely route for polar, hydrophilic drugs because they exhibit poor membrane partitioning. Peptides are presumed to permeate through the aqueous pathways, in other words, the paracellular and aqueous pore paths. In the human small intestine, the average size of these water-filled pores is approximately 7–9 Å for the jejunum, 3–4 Å for the ileum, and 8–9 Å for the colon [36,37]. The extent of

Table 10.1 Anatomical and Physiological Characteristics of the Human GI Tract

Region Length (m) Absorbing Surface Area (m2)

Absorption Pathway

Transit (or Residence) Time of Solids (h)

pH Enzymes and Others Microorganism (Counts/g Content)Fasted Fed

Stomach 0.2 0.1 P, C, A 1–3 1.5–3 2–5 Pepsin, lipases, rennin, HCl, cathepsin

102

Small intestine 7 12 3–5Duodenum 0.3 0.1 P, C, A, F, I, E 5.5 5 Bile acids, trypsin,

a-chymotrypsin, and other peptidases, amylase, maltase, proteases, lipases, nucleases

102

Jejunam 3 60 P, C, A, F, I, E 6.1 No change Erepsin, amylase, maltase, lactase, sucrase, peptidases, lipases

105

Ileum 4 60 P, C, A, F, I, E 7–8 No change Enteropeptidase (enterokinase) and other peptidases, lipases, nucleases, nucleotidases

107

Large intestine 1.5 0.3 4–16 No change 1011

Cecum 0.06–0.07 0.05 P, C, A, E 5.7Colon 1.35 0.25 P, C, E 8.0 Reductases, esterases,

glycosidases, amidases, sulfatase

1011

Ref. [35], Table 4. (Reprinted with permission from Elsevier)

P: passive diffusion C: convective transport or aqueous channel transport A: active transport

F: facilitated transport I: ion-pair transport E: entero- or pinocytosis


paracellular transport is limited, as tight junctions comprise only about 0.01% of the total absorptive surface area of the intestine (villi) [38].

10.3.2 Transcellular Absorption

Drugs pass through the intestinal epithelial cells (enterocytes) and require partitioning across both the apical and the basolateral membranes. The transcellular permeability of a peptide is a complex function of various physicochemical properties, including size, lipophilicity, hydrogen bond potential, charge, and conformation [39]. This route is lim-ited to the transport of relatively low-molecular-weight lipophilic drugs. Furthermore, studies in humans have demonstrated that absorption by the transcellular route decreases significantly in the colon (small intestine ascending colon transverse colon), whereas no such gradient exists for the paracellular route [40].

10.3.3 Carrier-Mediated Transport

Drugs interact with a specific transporter or carrier, in which the drug is transferred across the cell membrane or entire cell and then released from the basal surface of

Figure 10.3 Mechanisms for the transport of drugs


the enterocyte into the circulation [41]. The process is saturable and utilized by small hydrophilic molecules [42].

10.3.4 Receptor-Mediated Transport

In receptor-mediated transport, drugs act either as a ligand for surface-attached recep-tors or as a receptor for surface-attached ligands [43]. This process involves cell invagi-nation, which leads to formation of a vesicle. This process, in general, is known as endocytosis and comprises phagocytosis, pinocytosis, receptor-mediated endocytosis (clathrin mediated), and potocytosis (nonclathrin mediated) [44]. Small peptides such as di- and tri-peptides, as well as monosaccharides and amino acids, are thought to be transported by carrier-mediated systems [8,45,46]. After a drug is absorbed in the GI tract, it gains access to the systemic circulation via two separate and functionally distinct absorption pathways: portal blood and the intestinal lymphatics. The physico-chemical and metabolic features of the drug and the characteristics of the formulation largely control the relative proportion of drug absorbed via these two pathways. Portal blood represents the major pathway for the majority of orally administered drugs as it has higher capacity to transport both water-soluble and poorly water-soluble com-pounds. During this process, hydrophilic molecules are carried to the liver via the hepatic portal vein, and then by the hepatic artery gain access to the systemic circula-tion, for subsequent delivery to their sites of action. On the other hand, highly lipophilic drugs (log P 5) that cross the same epithelial barrier are transported to the intestinal lymphatics, which directly deliver them to the vena cava, thereby bypassing the hepatic first-pass metabolism [47].

10.4 Barriers to Protein Absorption

Protein and peptide delivery via oral route is a keen area of research. The main draw-backs that should be considered during development of such a delivery system are barriers to protein absorptions. Such barriers include the mucus barrier, extracellular barriers, enzymatic barriers, and cellular barriers [48]. These barriers restrict the bio-availability of the orally given peptides to less than 1%. The ray of hope here is that there are some exceptions in which some proteins show higher bioavailability alone or in the presence of certain adjuvants.

10.4.1 Mucus Barrier

The glycocalyx, which is atop the epithelial cells, is a fuzzy and filamentous coat that is weakly acidic and consists of sulfated mucopolysaccharides. Goblet cells secrete mucus, which lines the top of the glycocalyx [49]. The mucus consists of mucin glycoproteins, enzymes, electrolytes, water, and so forth [50]. The cohesive and adhesive nature of the mucus layer is due to the presence of mucin glycoprotein [51,52]. Mucus and glycocalyx layers are the first and foremost barriers to peptides


and proteins, which must first diffuse through these layers to reach the cellular mem-brane. Due to viscosity and the interactive nature of these layers, they offer a certain level of resistance to the protein drug diffusion. After diffusing through the mucus and glycocalyx, the protein drug reaches the epithelial surface [53].

10.4.2 Extracellular and Enzymatic Barriers

The GI tract has a wide variety of proteolytic enzymes, commonly considered as extra-cellular barriers, which are involved in the degradation of peptides and proteins, mak-ing the oral delivery of proteins and peptides a thorny issue. Various parts of the GI tract such as the lumen, brush border, and the cytosol of the enterocytes are key loca-tions where peptides and proteins are degraded by such proteases [54] (Table 10.2).

In the GI tract, from the stomach (the first part) to the colon (the last part), families of proteinases are ready to degrade the ingested peptides and proteins. The stomach has the added drawback of acidic pH, from 2.0 to 3.0, as compared to other sites that have better degradation of peptides and proteins. Such acidic environment favors the activation of proteinases, mainly pepsins, and thereby the degradation of proteins and peptides.

Not only acidic pH but also rapid pH changes are responsible for degradation of ingested proteins and peptides. When proteins migrate from the stomach to the duo-denum, pH is rapidly changed from 2.0 to about 8.0. This wide pH range covers the isoelectric points of many peptides and proteins and precipitates them. These precipi-tated proteins do not rapidly redissolve upon pH change [55,56].

Table 10.2 A List of Various Proteases along with Their Sites of Action

Types Enzymes Major Site of Action

Gastric proteases Pepsins (aspartic proteases) Broad activity, hydrolyze many peptide bond peptides

Intestinal pancreatic proteases

Trypsin (endopeptidase) Peptide bonds of basic amino acids/peptides

a-chymotrypsin (endopeptidase) Peptide bonds of hydrophobic amino acids/peptides

Elastase (endopeptidase) Peptide bonds of smaller and nonaromatic amino acids/peptides

Carboxypeptidases (exopeptidase)

A: C-terminal amino acid B: C-terminal basic amino acid

Brush border proteases Aminopeptidase A Aminopeptidase N Aminooligopeptidase Dipeptidylaminopeptidase IV Carboxypeptidase

Aminopeptidases are N-terminopeptidases, degrading mostly 3–10 amino acid residue-dipeptides and amino acids

Cytosolic proteases Di- and tripeptidase 2–3 aminopeptiddamino acids

Ref. [65]. (Reprinted with permission from Taylor & Francis)


In the duodenum, apart from pH, enzymatic degradations make conditions peril-ous for ingested peptides and proteins. Two classes of pancreatic proteases, consisting of endopeptidases and exopeptidases, are present in the duodenum. Endopeptidases include trypsin, chymotrypsin, and elastase; and exopeptidases include carboxy-peptidase A [57]. Proteins and peptides are degraded into smaller peptides, and this sequence is continuous in the brush border, the cytosol of the enterocyte, and even in the lysosomes and other cell organelles. Amiopeptidases are from the exopeptidase family and act on the N-terminal of peptides [58]. Leucine aminopeptidase and ami-nopeptidases N, A, and B are generally present in the intestine. Apart from the areas these peptidases reach, there are areas in the jejunum and ileum where amiopepti-dases activity is about 20–30% of the aminopeptidases activity in other neighboring areas. Such areas are known as Peyer’s patches and are a potential targeting site for the delivery of proteins and peptides [59–64].

A wide variety of peptidases is present in brush border membranes of different sites. Such peptidases are present in different concentrations, and their target sites differ as well. An understanding of the distribution of peptidases in the GI tract and of their tar-get site provides an opportunity for targeted oral delivery of peptides and proteins [65].

10.4.3 Cellular Barriers

Transcellular and paracellular are the major pathways involved in the transfer of pep-tides and proteins across the epithelial barriers. In the transcellular pathway, peptides and proteins move across the epithelia through intracellular transfer from the lumen to the blood stream. In this case, proteins and peptides either transfer through a spe-cific uptake mechanism or follow simple partitioning from the aqueous lumen con-tent to the lipid membrane, and from there to the aqueous blood stream. Lipophilic molecules are generally transported through partition, whereas hydrophilic molecules require a specific transport mechanism.

The paracellular pathway involves the transfer of peptides and proteins through the space present between the adjacent cells. This space has radius 8 Å, so only smaller peptides can pass through the space. The only hindrance in the paracellular pathway is the tight intracellular junction of the villus cells [66]. The paracellular pathway avoids degradation of peptides and proteins by proteases present in the cells. Peptides and pro-teins have a log P value of less than zero, suggesting that they follow the paracellular pathway. Use of a penetration enhancer significantly improves the transport of peptides through paracellular pathways. Penetration enhancers such as zonula occludens toxin (a protein from vibrio cholera), Pz peptide, and chitosan reversibly open tight junctions between intestinal cells [67,68]. Once tight junctions have been opened, transport is enhanced not only for drugs but also for potentially toxic or unwanted molecules pres-ent in the GI tract [69,70].

Nonpolar compounds with a molecular weight (MW) of 3–4 kDa are absorbed orally to a significant extent because of their desired partition coefficient. Although the tight junctions of villus cells have been reported to be generally impermeable to molecules with radii of more than 11–15 Å, peptides have some conformational flex-ibility, and even larger molecules can perhaps permeate the tight junctions [71].


The absorbed drug molecule enters into either the blood or the lymphatic circula-tion. Because the blood flow rate is 1000–2000 times higher than that of lymphatic flow in humans, lymphatic absorption is not as important [72]. The advantage of lymphatic absorption, however, is that a drug can bypass the liver and enter directly into the circulation.

10.5 Factors Affecting Peptides and Proteins Absorption

10.5.1 Molecular Weight and Size of Molecule

Diffusion of the drug across the epithelial layer largely depends on MW and molecular size of the drug. Very large molecules have lower diffusion than small molecules (75–100 Da), which cross the mucosa barrier rapidly [73]. Drug diffusion decreases markedly with increase in molecular size. There are several reports showing the effects of MW on mucosal absorption of various hydrophilic compounds [74–76]. It was found that the apparent permeability coefficient of fluorescein isothiocyanate dextrans (FITCD), a neu-tral polysaccharide, decreased as MW increased [75–77]. Generally, absorption decreased exponentially with MWs above 300 Da [78]. MW varies largely among therapeutically used peptides and proteins, ranging from less than 600 to greater than 100,000 Da so that a direct comparison is not possible [79]. In vivo permeation studies on humans dem-onstrated that peptides such as protirelin (MW: 362 Da) and oxytocin (MW: 1007 Da) crossed the human buccal mucosa barrier, whereas buserelin (MW: 1239 Da) and calcito-nin (MW: 3500 Da) did not [80]. Outcomes of such studies led Merkle et al. [81] to pro-pose that the transfer of peptides with MWs above 500–1000 Da through mucosa would require use of an absorption enhancer. Peptides and proteins having a wide variety of MWs showed different absorption patterns [82–87] (Table 10.3).

10.5.2 Three-Dimensional Structure and Immunogenicity

Peptide drugs may have primary, secondary, and tertiary structures, and in solution they may exist in several different conformations depending upon their size [88–90]. Change in conformation influences membrane permeability, and this phenomenon can be utilized during formulation, but the potential problem is the preservation of the pharmacologically active conformation [91]. Stereoselectivity exists in the pro-cess of permeation, so care must be taken to preserve stereospecificity during for-mulation [45,92]. Peptides are also recognized as often being immunogenic, and the use of inert polymers for peptide delivery, for example, polyethylene glycol (PEG), dextran, polyvinylpyrrolidone (PVP), and albumin, has been shown to increase resis-tance to proteolysis and simultaneously decrease peptide immunogenicity [90,93].

10.5.3 Charge Distribution

Proteins and peptides exist as zwitterions at their isoelectric point (pI) and have a negative effect on membrane permeability [45]. The electrostatic charge on the


peptide chain may be even more important than the value of the partition coeffi-cient in the prediction of oral permeability. The change in pH of medium results in a change in charge density and degree of ionization, and thus permeability of the peptides [94]. At physiological pH or at a pH above the isoelectric point (pI), the epithelial proteins are negatively charged and are selective to positively charged sol-utes, and vice versa [95]. At the isoelectric point, the membrane is nondiscriminat-ing to either ion [96]. This phenomenon has a significant effect on the absorption of proteins and peptides and helps the formulator to design the composition. Due to the negative charge of insulin, it was found to be excluded from the aqueous paracellular pathway, whereas positively charged peptides, such as thyrotropin-releasing hormone (TRH), were taken up predominantly via this pathway [95].

10.5.4 Solubility, Lipophilicity, and the Partition Coefficient

Because of their amphoteric nature, the pH-dependent solubility profile is not uni-form for all peptides. At the isoelectric point, proteins exist in a zwitterion state and have minimum water solubility. Solubility is also dependent upon pH, metallic ions, ionic strength, and temperature. Proteins and peptides are usually water soluble and have a much smaller oil–water partition coefficient. The solubility of proteins can be reduced by blocking the C-terminal through cyclization, amide formation, or esterifi-cation [78,97–101] (Table 10.4).

These modifications have been utilized to increase lipophilicity of proteins and thus their absorption by the passive diffusion pathway. Although the octanol–water partition

Table 10.3 Oral Absorption of Peptide and Proteins in Relation to Molecular Weight

Peptides and Proteins MW Amount of Absorption (%)

References

Dietary di- and tripeptides 200–300 5–50 82Dietary tetrapeptides 400 5 83Thyrotropin-releasing hormone (TRH) analogs

400 5 83

Enkephalins 600 2 83Cyclic somatostatin 806 5 83Bradykinin 1060 2 83Vasopressin 1200 2 83Cyclosporine 1203 50 84Leuprolide 1208 5 85Beta-endorphin 3500 2 83Calcitonin 3500 2 83Corticotropin (ACTH) 4700 2 83Insulin 5700 0.5 85Growth hormone 22,600 2 83Horseradish peroxidase (HRP) 40,000 3 86Bovine serum albumin (BSA) 50,000 4.5 87


coefficient is a simple parameter that may predict mucosal permeability, its correlation with absorption of peptides is not always observed, as the oral bioavailability of pep-tides models varies parabolically with their lipophilicity [78,102] (Table 10.4).

Absorption of drugs through oral route follows the pH-partition hypothesis, which implies a passive diffusion mechanism [103,104], where the absorption rate is directly proportional to the concentration of drug molecules in the unionized form. Drug absorption decreases with increase in extent of ionization.

10.5.5 Aggregation

Aggregation self-association and hydrogen bonding affects the intrinsic properties of peptides. Insulin has a tendency toward aggregation that is accelerated in the pres-ence of ionic ingredients and phenolic preservatives. Complexation of insulin with zinc prevents aggregation and makes it more stable [105–107]. Nonionic surfactants, such as Pluronic F68, are used for stabilization of insulin [108].

Some peptides have a tendency to form a hydrogen bond with water molecules. This leads to the addition of a hydroxyl group and thus a decrease in partition coeffi-cient and the ability to penetrate a lipidic membrane, therefore resulting in a decrease in permeability [78,79,107,109–113]. However, hydrogen bonding sometimes leads to increased permeability. Hydrogen bonding among protein molecules results in the formation of a cyclic structure, which decreases hydrogen bonding with water, increases lipophilicity, and thus increases permeability [114–116].

10.6 Approaches to Improve Oral Protein and Peptide Delivery

10.6.1 Targeted Delivery of Peptides and Proteins

Different regions of the GI tract have different specificity for different peptides and pro-teins; thus their absorption dose is not the same throughout the GI tract. It is concluded

Table 10.4 Lipophilicity of Some Peptides

Peptide Partition Coefficient (n-Octanol/Buffer, pH 7.4)

Insulin 0.0215Luteinizing hormone–releasing hormone 0.0451Thyrotropin-releasing hormone 0.0376Glucagon 0.0633Substance P 0.275Met-enkephalin 0.0305Leu-enkephalin 1.12

Ref. [80]. (Reprinted with permission from Elsevier)


from various studies that cyclosporine is more favorably absorbed from the duode-num, and tetragastrin, from the upper GI tract and rectum. Other studies showed that the junction of the duodenum and the ileocecal is the most ideal site of absorption for desmopressin, and the upper GI tract is the ideal site for octreotide [117,118]. Due to less enzymatic activity and higher paracellular permeability, distal intestinal segments are the preferred zone over the proximal small intestine for absorption of oxytocin and vasopressin [119]. Experiments showed that absolute bioavailability for insulin when administered to the distal region of the rat intestine (0.133%) was higher than that absorbed from a proximal region (0.059%) of the intestine [120]. As described earlier, proteolytic enzymes are also present in the cytosol and brush border of the GI tract. Proteolytic ruin the ingested peptides and proteins within the cytosol remains the same throughout the GI tract, except in the brush border or for luminal fluid. The harshest circumstances for the proteins and peptide inside the GI tract exist in the stomach. Very low pH and high protease (mainly pepsin) enzyme activity make the condition inauspi-cious for proteins. The problem of peptide and protein degradation in the stomach is overcome by the use of an enteric coating technique, in which the release of peptides in such an unkind environment is prevented. Surface area is reduced when moving from the proximal to the distal region of the intestine, because of the reduced presence of small villi in the distal part.

10.6.1.1 Colon Targeting

Delivery of peptide to the colon offers the advantage of low enzymatic activity but suffers from drawbacks such as variable pH and interference of fecal matter. It was concluded from earlier studies that calcitonin degrades more in the small intestine than in the colon [121]. Peptides and proteins, when administered to the upper half of the colon, will be taken up by hepatic portal veins and delivered to the liver, thus reducing degradation. Polypeptides reach to the lymphatic circulation when absorbed from the lower colon and hence, hepatic first pass metabolism is avoided. Thickness and nature of the composition may be varied to get the drug to release in the lower colon, bypassing the hepatic vein. The pH-dependent properties of Eudragit have been utilized for improving delivery of insulin to lower colon [122]. Delivery of insulin-like growth factor I (IGF-I) to rat and minipig colonic mucosae under in vitro conditions has been investigated [123]. A novel time-based drug release system for colon-specific delivery has also been investigated, where the release of peptide starts after a predetermined lag time. This lag time is equal to the transit time of the deliv-ery system (from time of ingestion to time it takes to reach the colon). The lag time is independent of physiological conditions such as pH, digestive state, and the level of digestive enzymes.

The colon has a high population of anaerobic bacteria species. These bacteria have the capacity to reduce azo bonds, and this capacity can be utilized to develop a formulation where a peptide or protein is coated with polymers crosslinked with azo–aromatic groups to protect the peptide in the stomach and intestine. When the formulation reaches the colon, the azo bond is reduced by such flora and releases the peptide, which is absorbed through the mucosa [13,124].


10.6.1.2 Payer’s Patch Targeting

The gut-associated lymphoid tissue (i.e., Peyer’s patches) has been considered the primary site of particle uptake due to the presence of follicle-associated epithelium. Transcytosis of particles is facilitated by the M (microfold or membranous) cells pres-ent in this epithelium. Although there is some controversy in the literature on the extent of particle uptake by Peyer’s patches, there is evidence that particle translocation can occur in them. However, the complete mechanism of uptake and endocytic transport by M cells is unclear, although it is generally accepted that the entry of micro- and nanoparticles into M cells occurs via this type of transport. Various physicochemical parameters, such as particle size, zeta potential, hydrophobicity, and coating with adhe-sion factors of particulate systems, determines the extent of their absorption by M cells [125]. It has been proposed that M cells function to sample and transport luminal par-ticles, macromolecules, and antigens into lymphoid follicles for immunologic surveil-lance and initiation of appropriate immunologic responses. Because of this, M cells are considered the target sites of oral protein absorption. In order to gain efficiency in the delivery of peptides and proteins to the Peyer’s patch by particulate carriers, their uptake by the Peyer’s patch must be enhanced [65]. For this purpose, there is a require-ment to identify M cell-specific surface antigens and receptors that may permit the production of specific antibodies and ligands for M cell selective targeting. Another option is to develop particles having optimal surface properties for effective delivery to M cells [125].

10.6.2 Chemical Alteration in the Structure

Chemical modification of peptides, to fabricate their prodrugs and analogs, is a use-ful tool for increasing the lipophilicity of peptides and thus increasing the passage of peptides through biological membranes. But this type of modification is only useful for small peptides with less than 10 amino acid residues [126]. These types of modi-fications increase not only the lipophilicity but also the resistance power of peptides against proteolytic enzymes present at various sites of the GI tract. A prior condition for these types of modifications is that peptides retain their pharmacological actions, and such modifications are performed only after confirming the pharmacological activity of the modified peptide [127].

10.6.2.1 N-Terminal Acylation

The N-terminal of peptides is conjugated with lipophilic molecules by acylation or alkylation, to increase the lipophilic nature of the peptides [53,60,127]. Primarily, fatty acids are conjugated to peptides to increase their lipophilicity and their trans-port across the membrane. Lysozyme [128], insulin [129,130], thyroxin-releasing hormone [131–134], calcitonin [135], tetragastrin [136–139], and some enkephalin analogs [140] have been studied for acyl modification and have showed encouraging results in terms of improved absorption of these peptides and retention of pharmaco-logical activities. Bovine-insulin when acylated with fatty acids having an increasing


amount of carbon showed improved permeability due to increased lipophilicity with increased chain length and inhibition of insulin self-union [130,141]. Research has also been done on the modification of peptides, for example, TRH, LHRH, neuro-tensin, pepsin, gastrin, fibrinopeptides, and collagen (peptides containing the pyro-glutamyl group). These studies conclude that lipophilic alteration of these peptides resulted in a significant increase in their absorption, by overcoming poor perme-ability and enzymatic insecurity [142]. Buccal absorption of TRH tripeptide from the buccal patch has been enhanced when chemically modified to its lauryl deriva-tive [143,144]. However, an increase in the lipophilic nature of the peptide does not ensure increased permeability across the mucosa. This can be explained by the devel-oping myristoylated TRH tripeptide, which, having a high lipophilic nature, is unable to cross the mucosa and is retained in the epithelium [145].

10.6.2.2 Prodrug Approach

A prodrug itself has no pharmacological activity but elicits activity only after conver-sion to the parent drug. Before reaching the systemic circulation, prodrugs must retain their structure to avoid enzymatic degradation and to increase absorption. After reach-ing the systemic circulation, they must be transformed to their dynamic form to have a pharmacological effect [146]. This approach can be understood with the example of lauryl-TRH, which is a prodrug of TRH and is gradually converted to TRH in the plasma [132,133], as is demonstrated in the study conducted by Tanaka et al.

10.6.2.3 Methylation of Peptides and Proteins

Peptides are often methylated to improve lipophilicity and to reduce their potential to form hydrogen bonds. Methylation changes the conformation of peptides and makes them permeable across the cell membrane. Conradi et al. studied methylation of AcPhe3NH2 peptide and its effect on penetration and concluded that methylation of AcPhe3NH2 peptide at four different places significantly increases its penetration through the CACO-2 cell membrane [110]. The modified peptides must be demethyl-ated enzymatically in the blood after absorption, to liberate the active peptide so that its pharmacological action can take place [142,147].

10.6.2.4 Polymeric Conjugates

This approach involving covalent conjugation of peptides to polymers may be useful to increase peptide stability and plasma half-life. It has also showed its usefulness for decreasing the immunogenicity of peptides, because it involves partially or totally covering the immunogenic sites of the peptides with polymers [65]. Prerequisites for these types of modifications are that these polymers must be water soluble, biocom-patible, and nonimmunogenic. Lipophilicity is also increased by coupling the peptide with lipophilic polymers [148]. Research has already been done on the conjugation of peptides with polystyrene-co-maleic acid/anhydride, PEG, poly(styrene maleic acid), copolymer (SM), albumin, and dextrans [149], and the usefulness of these approaches has been demonstrated.


10.6.3 Vehicles to Improve Absorption

Transportation of peptides across the membrane is improved by the use of different solvent compositions. Peptides are given in soluble or in dispersed form, and these vehicles increase solubility of peptides in the membrane. This effect is due to a change in concentration gradients and a change in partition of peptide between vehicle and mucosa. Due to the presence of such solvents, partitioning of peptides in lipoidal mucosa is increased. Examples of such solvent system include the use of 10% lauric acid in propylene glycol to improve insulin absorption [150] and ethanol at different concentrations (5% and 30%) to improve absorption of various peptides [145,151]. Two or more absorption enhancers can be utilized to get a synergistic effect [152].

10.6.4 Bioadhesive Formulations

The most successful approach for mucosal delivery of peptides has been a bioadhe-sive formulation. Bioadhesive systems enhance peptide absorption by increasing con-tact time with the mucosa. To avoid rapid removal of the drug delivery system from the GI tract, mucoadhesive delivery systems are gaining much more attention and are being widely investigated for sustained oral delivery of a variety of molecules [153–156]. Mucoadhesive systems adhere to the GI tract mucosa and release the drug for a longer period of time; this will increase the overall time period of drug absorption. Researchers have also been successful in developing delivery systems where bioad-hesion occurs at a particular site of the GI tract. Bioadhesion of such delivery system depends on mucous turnover in the particular region of the GI tract. Low turnover of mucous in the colon and cecum makes these regions best suited for adhesive systems [157]. Bioadhesive polymers, such as polycarbophil, have also been shown to stabi-lize peptides by inhibiting proteolytic activity [158–160]. Carbopol 934P, 971P, and 974P strongly inhibited proteolysis of insulin, calcitonin, and IGF-I [65].

The properties and characteristics of materials used to develop such bioadhesive drug delivery systems are depicted in Table 10.5 [160].

Various bioadhesive polymers have been extensively studied for oral delivery of a wide variety of peptide drugs. Polycarbophil and chitosan derivatives have been reported to improve the permeation of the peptide drug 9-desglycinamide, 8-arginine vasopressin (DGAVP) across the mucosa [161]. Advantageous properties such as good bioadhesion, permeation enhancement, and protease inhibition is imparted in commonly used polymers by chemical modifications, for example, ethylene diamine tetra acetate (EDTA)-conjugated chitosan.

10.6.4.1 Bioadhesive Tablets

To avoid the disadvantages associated with conventional tablets, adhesive tablets have been developed that allow drinking and speaking without major discomfort. Two techniques are utilized to develop bioadhesive tablets: monolithic and multilay-ered formulation [162].

In monolithic tablets, the core is either prepared by direct compression or by wet granulation followed by coating the core with water impermeable materials on all the

Table 10.5 Properties and Characteristics of Some Bioadhesive Polymers

Bioadhesive Materials Properties Characteristics

Polycarbophil (polyacrylic acid crosslinked with divinyl glycol)

l MW 2.2 105 l 2000–22,500 cps (1% aqueous solution) l 15–35 ml/g in acidic media (pH 1–3) 100 ml/g in neutral and basic media l viscous colloid in cold water l Insoluble in water, but swells to varying degrees

in common organic solvents, strong mineral acids, and bases

l Synthesized by lightly crosslinking of 0.5–1% w/w divinyl glycol l Swellable depending on pH and ionic strength l Swelling increases as pH increases. l At pH 1–3, absorbs 15–35 ml of water per gram but absorbs 100 ml per gram at neutral and alkaline pH l Entangles the polymer with mucus on the surface of the tissue l Hydrogen bonding between the nonionized

carboxylic acid and mucinCarbopol/carbomer (carboxy polymethylene) empirical formula: (C3H4O2)x (C3H5–Sucrose)y

l Pharmaceutical grades: 934 P, 940 P, 971 P, and 974 P l MW 1 106–4 106 l 29,400–39,400 cps at 25°C with 0.5% neutralized aqueous solution l 5 g/cm3 in bulk, 1.4 g/cm3 tapped l pH 2.5–3.0 l water, alcohol, glycerin l White, fluffy, acidic, hygroscopic powder with a

slight characteristic odor

l Synthesized by a crosslinker of allyl sucrose or allyl pentaerythritol l Excellent thickening, emulsifying, suspending, gelling agent l Common component in bioadhesive dosage forms l Gel loses viscosity on exposure to sunlight. l Unaffected by temperature variations, hydrolysis, and oxidation, and resistant to bacterial growth l Contributes no off-taste and may mask the undesirable taste of the formulation l Incompatible with phenols, cationic polymers, high

concentrations of electrolytes, and resorcinolSodium carboxymethyl cellulose—SCMC (cellulose carboxymethyl ether sodium salt) empirical formula: [C6H7O2(OH)3x (OCH2–COONa)x]n

l An anionic polymer made by swelling cellulose with NaOH and then reacting it with monochloroacetic acid l Grades H, M, and L l MW 9 104–7 105

l Emulsifying, gelling, binding agent l Sterilization in dry and solution form, irradiation of solution loses the viscosity l Stable on storagel Incompatible with strongly acidic solutions

l 1200 cps with 1.0% solutionl 0.75 g/cm3 in bulkl pH 6.5–8.5l waterl White to faint yellow, odorless, hygroscopic

powder or granular material having faint paper-like taste

l In general, shows very good stability with monovalent salts; with divalent salts, good to marginal; with trivalent and heavy metal salts, poor, resulting in gelation or precipitation

l In CMC solutions offers good tolerance of water-miscible solvents, good viscosity stability over the pH 4–10 range, compatibility with most water-soluble nonionic gums, and synergism with HEC (hydroxy ethyl cellulose) and HPC (hydroxy propyl cellulose)

l Most CMC solutions are thixotropic; some are strictly pseudoplastic

l A reversible decrease in viscosity at elevated temperatures shown in all solutions; CMC solutions lack yield value

l Solutions susceptible to shear, heat, bacterial, enzyme, and UV degradation

l Good bioadhesive strengthl Cell immobilization via a combination of ionotropic

gelation and polyelectrolyte complex formation (e.g., with chitosan) in drug delivery systems and dialysis membranes

Hydroxypropyl cellulose partially substituted polyhydroxy propylether of cellulose HPC (cellulose 2-hydroxypropyl ether) empirical formula: (C15H28O8)n

l Grades: Klucel EF, LF, JF, GF, MF, and HFl MW 6 104–1 106

l 4–6500 cps with 2.0% aqueous solutionl pH 5.0–8.0l 0.5 g/cm3 in bulkl Soluble in water below 38°C, ethanol, propylene

glycol, dioxane, methanol, isopropyl alcohol, dimethyl sulfoxide, dimethyl formamide, etc.

l Insoluble in hot waterl White to slightly yellowish, odorless powder

l pH best between 6.0 and 8.0l Solutions of HPC susceptible to shear, heat, bacterial,

enzymatic and bacterial degradationl Inert and shows no evidence of skin irritation or

sensitizationl Compatible with most water-soluble gums and resinsl Synergistic with CMC and sodium alginatel Not metabolized in the bodyl May not tolerate high concentrations of dissolved

materials and tend to be salting out

(Continued)

Table 10.5 (Continued)


l Also incompatible with the substituted phenolic derivatives, such as methyl and propyl parahydroxy benzoate

l Granulating and film-coating agent for tabletl Thickening agent, emulsion stabilizer, suspending

agent in oral and topical solution or suspensionHydroxypropylmethyl cellulose—HPMC (cellulose 2-hydroxypropylmethyl ether) empirical formula: C8H15O6–(C10H18O6)n–C8H15O5

l Methocel E5, E15, E50, E4M, F50, F4M, K100, K4M, K15M, K100M.

l MW 8.6 104

l E15–15 cps, E4M–400 cps and K4M–4000 cps (2% aqueous solution.)

l Cold water, mixtures of methylene chloride and isopropylalcohol

l Insoluble in alcohol, chloroform, and etherl Odorless, tasteless, white or creamy white fibrous

or granular powder

l Mixed alkyl hydroxyalkyl cellulosic etherl Suspending, viscosity-increasing and film forming

agentl Tablet binder and adhesive ointment ingredientl E grades generally suitable as film formers, while K

grades are used as thickenersl Stable when dryl Solutions stable at pH 3.0 to 11.0l Incompatible to extreme pH conditions and oxidizing

materialsHydroxyethyl cellulose nonionic polymer made by swelling cellulose with NaOH and treating with ethylene oxide

l Available in grades ranging from 2 to 800,000 `cps at 2%

l Light tan or cream-to-white powder, odorless and tasteless; may contain suitable anticaking agents

l 0.6 g/mll pH 6–8.5l in hot or cold water and gives a clear, colorless

solution

l Solutions are pseudoplastic and show a reversible decrease in viscosity at elevated temperatures

l HEC solutions lacking yield valuel Solutions show only a fair tolerance with water-

miscible solvents (10–30% of solution weight).l Compatible with most water-soluble gums and resinsl Synergistic with CMC (carboxy methyl cellulose) and

sodium alginatel Susceptible to bacterial and enzymatic degradationl Polyvalent inorganic salts will salt out HEC at lower

concentrations than monovalent saltsl Shows good viscosity stability over the pH 2–12 range.l Used as a suspending or viscosity builderl Binder, film former

Xanthan gum anionic polysaccharide derived from the fermentation of plant bacteria Xanthamonas campestris

l Soluble in hot or cold water and gives visually hazy, neutral pH solutions

l Dissolves in hot glycerinl Solutions typically in the 1500–2500 cps range at

1%; pseudoplastic and especially shear thinning. In the presence of small amounts of salt, solution shows good viscosity stability at elevated temperatures

l Possesses excellent yield value in solutions

l More tolerant of electrolytes, acids, and bases than most other organic gums

l Can, nevertheless, be gelled or precipitated with certain polyvalent metal cations under specific circumstances

l Very good viscosity stability over the pH 2–12 range and good tolerance of water-miscible solvents shown in solutions

l More compatible with most nonionic and anionic gums, featuring useful synergism with galactomannans

l More resistant to shear, heat, bacterial, enzyme, and UV degradation than most gums

Guar gum (galactomannan polysaccharide) empirical formula: (C6H12O6)n consists chiefly of a high-molecular-weight hydrocolloid polysaccharide composed of galactan and mannan units combined through glycosidic linkages

l Obtained from the ground endosperms of the seeds of Cyamopsis tetragonolobus (family Leguminosae).

l MW approx. 220,000l 2000–22,500 cps (1% aqueous solution)l Forms viscous colloidal solution when hydrated in

cold water; optimum rate of hydration between pH 7.5 and 9.0.

l Stable in solution over a pH range of 1.0–10.5l Viscosity degraded by prolonged heatingl Bacteriological stability is improved by the addition of

a mixture of 0.15% methyl paraben or 0.1% benzoic acid.

l The FDA recognizes guar gum as a substance added directly to human food and has been affirmed as generally recognized as safe.

l Incompatible with acetone, tannins, strong acids, and the alkalis. Borate ions, if present in the dispersing water, will prevent hydration of guar.

l Used as a thickener for lotions and creams, as a tablet binder, and as an emulsion stabilizer.

Hydroxypropyl Guar nonionic derivative of guar, prepared by reacting guar gum with propylene oxide

l in hot and cold waterl Gives high viscosity, pseudoplastic solutions that

show reversible decrease in viscosity at elevated temperatures

l Lacks yield value

l Compatible with a high concentration of most saltsl Shows good tolerance of water-miscible solventsl Better compatibility with minerals than guar guml Good viscosity stability in the pH range of 2–13.l More resistance to bacterial and enzymatic degradation

(Continued)



Chitosan a linear polysaccharide composed of randomly distributed -(1-4)-linked d-glucosamine (deacetylated unit) and N-acetyl-d-glucosamine (acetylated unit)

l Prepared from chitin of crabs and lobsters by N-deacetylation with alkali

l dilutes acids to produce a linear polyelectrolyte with a high positive charge density and forms salts with inorganic and organic acids such as glutamic acid, hydrochloric acid, lactic acid, and acetic acid.

l The amino group in chitosan has a pKa value of 6.5; thus, chitosan is positively charged and soluble in acidic to neutral solutions with a charge density dependent on pH and the %DA-value.

l Mucoadhesive agent due to either secondary chemical bonds, such as hydrogen bonds, or ionic interactions between the positively charged amino groups of chitosan and the negatively charged sialic acid residues of mucus glycoproteins or mucins

l Possesses cell-binding activity due to polymer cationic polyelectrolyte structure and to the negative charge of the cell surface

l Biocompatible and biodegradablel Excellent gel-forming and film-forming abilityl Widely used in controlled delivery systems such as

gels, membranes, and microspheresl Chitosan enhances the transport of polar drugs across

epithelial surfaces. Purified qualities of chitosans are available for biomedical applications. Chitosan and its derivatives such as trimethylchitosan (where the amino group has been trimethylated) have been used in nonviral gene delivery. Trimethylchitosan, or quaternised chitosan, has been shown to transfect breast cancer cells. As the degree of trimethylation increases, the cytotoxicity of the derivative increases. At approximately 50% trimethylation, the derivative is the most efficient at gene delivery. Oligomeric derivatives (3–6 kDa) are relatively nontoxic and have good gene delivery properties.

Carrageenan an anionic polysaccharide, extracted from the red seaweed Chondrus crispus

l Available in sodium, potassium, magnesium, calcium, and mixed cation forms

l Three structural types: iota, kappa, and lambda, differing in solubility and rheology

l All solutions are pseudoplastic with some degree of yield value. Certain ca-Iota solutions are thixotropic. Lambda is non-gelling, kappa produces brittle gels; Iota produces elastic gels. All solutions

l Sodium form of all three types is soluble in both cold and hot water.

l Other cation forms of kappa and iota are soluble only in hot water.

l All forms of lambda are soluble in cold water.

show a reversible decrease in viscosity at elevated temperatures. Iota and lambda carrageenan have excellent electrolyte tolerance; kappa’s being somewhat less. Electrolytes will however decrease solution viscosity. The best solution stability occurs in the pH 6–10 range. It is compatible with most nonionic and anionic water-soluble thickeners. It is strongly synergistic with locust bean gum and strongly interactive with proteins. Solutions are susceptible to shear and heat degradation.l Excellent thermoreversible propertiesl Used also for microencapsulation

Sodium alginate consists chiefly of the alginic acid, a polyuronic acid composed of -d-mannuronic acid residues empirical formula: (C6H7O6Na)n anionic polysaccharide extracted principally from the giant kelp Macrocystis Pyrifera as alginic acid and neutralized to sodium salt

l Purified carbohydrate product extracted from brown seaweed by the use of dilute alkali

l Occurs as a white or buff powder, which is odorless and tasteless

l pH 7.2l 20–400 cps (1% aqueous solution)l Water, forming a viscous, colloidal solutionl Insoluble in other organic solvents and acids where

the pH of the resulting solution falls below 3.0

l Safe and nonallergenicl Incompatible with acridine derivatives, crystal violet,

phenyl mercuric nitrate and acetate, calcium salts, alcohol in concentrations greater than 5%, and heavy metals

l Stabilizer in emulsion, suspending agent, tablet disintegrating agent, tablet binder

l Also used as a hemostatic agent in surgical dressingsl Excellent gel formation propertiesl Biocompatiblel Microstructure and viscosity are dependent on the

chemical composition.l Used as immobilization matrices for cells and enzymes,

controlled release of bioactive substances, and injectable microcapsules for treating neurodegenerative and hormone deficiency diseases

l Lacks yield value

(Continued)



l Solutions show fair to good tolerance of water-miscible solvents (10–30% of volatile solvents; 40–70% of glycols).

l Compatible with most water-soluble thickeners and resins

l Solutions more resistant to bacterial and enzymatic degradation than many other organic thickeners

Poly (hydroxy butyrate), Poly (e-caprolactone) and copolymers

l Biodegradablel Properties can be changed by chemical

modification, copolymerization, and blending.

l Used as a matrix for drug delivery systems, cell microencapsulation

Poly (ortho esters) l Surface eroding polymers l Application in sustained drug delivery and ophthalmology

Poly (cyano acrylates) l Biodegradable depending on the length of the alkyl chain

l Used as surgical adhesives and gluesl Potentially used in drug delivery

Polyphosphazenes l Can be tailored with versatile side chain functionality

l Can be made into films and hydrogelsl Applications in drug delivery

Poly (vinyl alcohol) l Biocompatible l Gels and blended membranes are used in drug delivery and cell immobilization.

Poly (ethylene oxide) l Highly biocompatible l Its derivatives and copolymers are used in various biomedical applications.

Poly (hydroxytheyl methacrylate) l Biocompatible l Hydrogels have been used as soft contact lenses, for drug delivery, as skin coatings, and for immunoisolation membranes.

Poly (ethylene oxide-b-propylene oxide)

l Surfactants with amphiphilic properties l Used in protein delivery and skin treatments

Ref. [160]. (Reprinted with permission from Elsevier)


faces except the face which is in contact with the mucosa. Water-impermeable materials include Teflon, ethyl cellulose, cellophane, hydrogenated castor oil, and so on. Such a system begins unidirectional drug flow toward the mucosa and avoids drug loss [163].

Multilayered tablets are prepared by compressing the tablet layer by layer. They are generally composed of an adhesive layer that faces the mucosa, a rate-controlling layer, and a drug layer, with the drug layer sandwiched between the adhesive layer and rate-controlling layer. The drug is also incorporated into the adhesive layer to give drug release direct to the mucosa. This approach is useful to deliver drugs either systemically or locally [164].

10.6.4.2 Bioadhesive Patches

Mucoadhesive intestinal patches have also been investigated for oral delivery of conventional drug molecules [165]. Similar to methods employed with bioadhesive tablets, monolithic, and multilayered techniques are utilized to develop bioadhesive patch formulation. Methods to manufacture such patches include solvent casting and direct milling, where direct milling may or may not utilize the solvent. The backing membrane is applied to the monolithic or multilayer sheet to get unidirectional drug release and to avoid deformation and disintegration after application. In solvent cast-ing, the bioadhesive solution is casted on the backing membrane, which is mounted on stainless steel. Here the drug can either be incorporated into the adhesive layer or sandwiched between the adhesive layer and the backing membrane. Casting is fol-lowed by drying and cutting the patches. On the other hand, direct milling involves uniform mixing of the drug and an adhesive polymer, followed by compressing the mixture to get desired thickness and cut to appropriate size [165–167].

10.6.4.3 Bioadhesive Gels

Adhesive gels may also be tried to deliver drugs via the mucous membranes of the GI tract. Adhesive gels prolong the residence time of the delivery system to the site and thus improve drug absorption. Site-specific gelling polymers are utilized to get the drug release at the desired location or at the absorption window of the drug [168–170]. Such a site-specific approach can be utilized to protect the peptide drug from enzymes and an acidic environment.

10.6.5 Penetration Enhancers

Absorption of proteins and peptides is improved by the use of different penetra-tion enhancers. Penetration enhancers affect the mechanism by which the drug is absorbed through the mucosa. Transcellular or paracellular pathways of drug absorp-tion are significantly affected by the use of penetration enhancers in the formulation. Penetration enhancers may enhance the absorption of drugs preferentially in some specific region of the GI tract [171]. By changing the lipid orientation and arrange-ment within the cell wall, surfactants and fatty acids ease the transport of peptides and proteins through the membrane. Penetration enhancers may act by increasing the thermodynamic activity of peptide drugs. This may be affected by the vehicle


composition, which will influence solubility and micellization and also by ion-pair formation between the enhancer and the peptides or proteins Table 10.6.

Chelating agents, such as EDTA, form complexes with calcium and magnesium present around tight junction and destruct the integrity of tight junctions, thus easing permeation of peptides across the membrane [172].

Sodium lauryl sulfate (SLS) is an ionic surfactant, which disorganizes the entire membrane architecture, affecting both protein and lipid structures. Expansion of intercellular spaces and insertion of SLS molecules into the lipid structure has also been observed [173]. SLS proved to be efficient in promoting an extensive enhance-ment of the absorption of human calcitonin (hCT) [174] and insulin [175].

Table 10.6 Types of Absorption Enhancers with Examples

Type of Penetration Enhancer Examples

Water –Surfactants Sodium lauryl sulfate

Polyoxyethylene ethersTween

Fatty acids and derivatives Oleic acidCaprylic acidAcylcarnitinesCapric acidLinoleic acidMono- and diglycerides

Sulfoxides Dimethyl sulfoxideDiethyl sulfoxide

Pyrrolidones N-methyl-2-pyrrolidone2-pyrrolidone

Alcohols, fatty alcohols, and glycols EthanolPropylene Glycol

Metal ion chelators EDTACitric acidSalicylates

Bile salts Sodium glycocholateSodium taurocholateSodium deoxycholateSodium taurodihydrofusidateSodium glycodihydrofusidate

Essential oils, terpenes, and terpenoids Cineole, eucalyptolMenthol, carvoneLimonene, nerolidol

Others UreaAzonePhospholipidsCeramide analogsSolvents at high concentration


Sodium dodecyl sulfate is incorporated to enhance absorption of peptide drug vaso-pressin across the CACO-2 cell monolayer. This is due to loosening of the tight junction, thus easing paracellular transport [176]. Bile salts make up another important class of nat-ural or semisynthetic surfactants. Some reports suggest bile salts also increase absorption of proteins and peptides across the membrane. Sodium deoxycholate and sodium cholate dissociates insulin from its complexes and makes it available for absorption [177].

Mixed micellar systems, which are naturally formed during the absorption of lipids from the GI tract, have also shown enhanced absorption of proteins and peptides [178]. Absorption of hCT across the rat colon was increased ninefold when given as a mixed micellar form. Mixed micelles enhance the absorption of peptides and proteins over the MW range of 4000 to 40,000 without acute damage to the membrane [179].

Cyclodextrin and its derivatives have been studied to check its effect on absorp-tion of peptides and proteins. Cyclodextrins improves absorption of insulin from the lower jejunum to the upper ileum segment. Citric acid and castor oil derivatives are used to increase pharmacological activity of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats by affecting its absorption [180,181].

Due to lack of specificity and destructive nature, penetration enhancers may have long-term toxicities, and thus their use to increase absorption of proteins and pep-tides can only be accepted after extensive chronic studies. They are prone to destruct the membrane that is the barrier to external toxic chemicals and microorganisms. Thus their use can only be established after solving their safety issues [178].

10.6.6 Protease Inhibitors

Proteins are degraded by various proteases present in the GI tract and thus reduce its absorption when given via oral route. Degradation of proteins by proteases is a major drawback of the oral route for protein delivery. The GI tract presents a very unfavorable environment for the peptides and proteins, due to the presence of plentiful proteolytic enzymes at high concentration in the lumen and throughout the intestinal wall [182]. Therefore, an enteric coating alone is not sufficient to protect peptides and proteins. This problem is overcome by the use of protease inhibitors in a formulation that avoids proteolytic degradation. Protease inhibitors may be classified as follows:

1. Polypeptide protease inhibitors (e.g., aprotinin)2. Peptides and their derivatives (e.g., bacitracin, chymostatin, and amastatin)3. Amino acids and their derivatives (e.g., -aminoboronic acid derivatives)4. Miscellaneous (e.g., p-aminobenzamidine and camostat mesilate) [183].

It was concluded in various studies that aminopeptidase inhibitor (puromycin) was able to increase the absorption of metkephamid (MKA), a stable analog of met-enkephalin, across the rat intestine. Endopeptidase inhibitor (thiorphan) was ineffec-tive in the prevention of MKA metabolism because the enzyme participate in MKA metabolism during absorption is aminopeptidase [184]. Hydrolysis of the pentapeptide, leucine (Leu)-enkephalin (YGGFL) at a high pH was reported to be reduced by amastatin [185]. The endopeptidase inhibitors such as tripeptides YGG and GGF were found to be effective at lower pH (below 5.0).


Some dual-acting excipients are also used to obtain a synergistic effect on the total amount of drug transported. An example is bile salts, which act as penetra-tion enhancers and also as protease inhibitors, thus augmenting oral absorption. The mechanism behind its use as a protease inhibitor is inhibition of the membrane and cytosolic proteases [186]. Studies supported that a significant hypoglycemic effect was obtained following large intestinal administration of insulin with 20 mM sodium glycocholate, camostat mesilate, and bacitracin [187].

Researchers have also proposed the use of biodegradable and mucoadhesive poly-mers to encapsulate proteins in a particulate system and then attach the protease inhibi-tors covalently onto the surface of the particles. When such a system is given orally, particles adhere to the membrane and release the protein for its absorption through mucosa. Here, protease inhibitors create a microenvironment that is free from enzy-matic activity and thus act locally without affecting the activity of digestive enzymes. In another study, hydrogel beads have been developed containing enzyme inhibitor (ovomucoid or soyabean trypsin) covalently coupled on the polymeric material [188].

10.6.7 Specialized Drug Delivery Systems

10.6.7.1 Liposomes

Nowadays, liposomes have gained much more attention for their entrapment of pep-tides to improve stability and for their effective delivery. They entrap both lipophilic and hydrophilic drugs and thus are useful for a wide variety of bioactives. The main disadvantages of liposomal delivery of peptides include limited stability of liposomes and degradation of proteins during the formulation of proteins due to the use of organic solvents [189]. Efforts are continuously made to stabilize liposomes by formulating polymerized liposomes, which are stable even in the brutal environment present in the stomach and intestine. Liposomes are a useful tool for delivering peptides to the sur-face of mucosa, which in turn are useful in localizing a greater amount of the drug in the membrane. Researchers studied another approach where protease inhibitors are also being incorporated in the formulation to improve the performance of less effective liposomal peptide delivery systems; for example, aprotinin is incorporated into Factor VIII-loaded liposomes, made up of lecithin and phosphatidic acid [65].

Liposomes are also taken up by Peyer’s patches and thus increase the uptake of any entrapped drug. Negatively charged liposomes containing at least 25 mol% phos-phatidylserine have been reported to be taken up readily by the rat Peyer’s patches following intraluminal administration [190].

10.6.7.2 Microemulsions

Microemulsions may be used as the carrier for protein and peptide delivery to the GI tract. With all types of microemulsions, water–oil–water type microemulsion offers unique advantages, including these:

l Degradation of proteins is effectively reduced due to the use of relatively mild conditions during the formulation process of microemulsions.


l Use of protein-compatible ingredients such as surfactants, water, and oils. These excipients are used in pharmaceutical formulations and generally recognized as safe. Their toxicities and metabolic profiles are well-known.

l Microemulsions are stable for relatively long periods. Microemulsions are recognized as a thermodynamically stable system.

l Microemulsion-based formulations are prepared in liquid forms, which are preferable for patients with difficulties in swallowing solids.

Along with the stability of proteins, the stability of the microemulsion is also important for effective delivery to the GI tract [191–193]. The composition of the microemulsion greatly affects the stability of the microemulsion in the GI tract. Penetration enhancer and protease inhibitors may also be incorporated in microemul-sion composition to improve the delivery of peptides to the GI tract [191].

10.6.7.3 Micro- and Nanoparticles

Various materials including gel-forming polymers, pH-dependent polymers, and site-specific biodegradable polymers may be used to develop beads, microspheres, and nanoparticles for effective oral delivery of proteins and peptides. Among them, gel-forming, polymer-based formulations are extensively studied for their protein encap-sulation and release of the bioactive. pH-dependent polymers are also used to protect the peptides from the acidic environment present in the stomach by tailoring the release from the micro- and nanoparticles. These polymers prevent the release of peptides in the acidic environment and hence increase the amount of peptide available for absorp-tion. Biodegradable microparticles protect the peptide in the stomach and then gradually release them in intestine. Hydrogel-forming polymers are modified to get the material, which is only degraded by colonic microflora. Such a system releases the peptides in the colon where the pH is nearly neutral and proteolytic enzyme activity is very low.

The stability of some proteins and peptides after oral administration is also increased by encapsulating them in albumin [194]. In situ microparticle formation occurs when proteinoids come in contact with the acidic environment after oral administration. There are reports suggesting effective delivery of calcitonin encap-sulated in proteinoid microspheres to rats and monkeys [195]. Poly(d,l-lactide-co-glycolide) nanoparticles fail to deliver the drug after oral administration because they significantly accumulate the drug in the liver and have a short GI transit time [196].

Peyer’s patches are mainly responsible for the absorption of nanoparticles, espe-cially in the ileum after oral administration, whereas in the jejunum nanoparticles may be absorbed by the paracellular pathway. Nanoparticles may pass through the intercellular spaces formed by the desquamation of well-differentiated absorptive cells at the tip of the villi [197].

It has been concluded from the study that insulin-loaded polyalkylcyanoacrylate nanocapsules when administered to diabetic rats show significant reduction in the glycemia up to 50–60%. Hypoglycemic effect starts at day 2 and the effect persists even after 20 days [198]. The results are very superior to plain insulin delivery [199].

Sustained drug release has also been achieved from polyisobutylcyanoacrylate nanoparticles loaded with insulin and calcitonin. Initial absorption was less due to


slower drug release but relatively higher plasma concentration was seen at the later time points [200]. Polymethacrylic acid-grafted-poly(ethylene glycol) have a capac-ity to open tight junctions between epithelial cells in CACO-2 cell monolayers and have hydrogeling properties; thus its nanoparticles have also been investigated for oral protein delivery [201].

10.6.8 Other Formulation Approaches

Protein endocytosis in enterocyte is effectively increased by noncovalent linking of peptides to phospholipids. Insulin, calcitonin, porcine somatotropin, erythropoietin, and -interferon have been effectively complexed with phospholipids to develop effective oral formulations [202]. Enteric-coated microtablets containing insulin along with cholate and trypsin inhibitor protect insulin from stomach pH. Protease inhibitors protect drugs from proteolytic enzymes, and cholate increases the absorp-tion of protein along with fatty acids [203].

10.6.8.1 Cochleates

In a lipid-based system, cochleates have been demonstrated as a promising peptide delivery system via oral route. They are mainly composed of phosphatidylserine, cholesterol, and calcium. The phospholoid bilayer acquires a spiral shape, whereas in liposomes they are in the form of concentric bilayers. Cochleates do not contain an internal aqueous core. Cochleates have shown several advantages such as enhanced stability, nontoxic nature, and ease of lyophilization as well as effective delivery of peptides to mucosa [204]. Though there are several advantages, very little work has been done on cochleates, and thus only a small amount of information is available about this delivery system.

10.6.8.2 Polymersomes

Polymersomes are composed of hydrophilic–hydrophobic block copolymer, arranged in a bilayer vesicular system having a central aqueous core. They have a hydrophilic inner core and lipophilic bilayer, hence can be used for both hydrophilic and lipo-philic drugs. They differ from nanoparticles in that they contain a hydrophilic core rather than a lipophilic core, as in the case of nanoparticles [205]. Although they have a bilayer structure, they offer more stability than liposomes due to the presence of a thick and rigid bilayer. They contain a hydrophilic core that provides a protein-affable environment. The PEG chain is generally present as a hydrophilic block in most of the research carried out so far. Because proteins have the tendency to move away from the PEG chain, their entrapment in such a vesicular system is much less than 5% [206].

10.6.8.3 Polymeric Micelles

Polymeric micelles are also composed of hydrophilic–hydrophobic block copolymers. Today, they are gaining attention to deliver a wide variety of bioactives [207]. They


contain a hydrophobic core and encapsulate hydrophobic drugs. Polyion complex micelles entrap biomacromolecules such as enzymes and DNA, and may attain increased stability against various environmental factors [208]. Encouraging results have been obtained by researchers in the oral delivery of polymeric micelles, because they cross the intestinal barrier after oral administration [209]. Thus oral delivery of macromolecules, that is, peptides and proteins, is also possible by means of polymeric micelles.

10.7 Technique for Oral Absorption Studies

Newly synthesized peptides and proteins and their delivery systems may be evaluated for both in vitro and in vivo release and mucosal permeation by designing appropri-ate experiments.

10.7.1 In Vitro Studies

10.7.1.1 Diffusion Cells

Various types of diffusion cells are used to evaluate the transepithelial transport and metabolism of several compounds. The Frans diffusion cell and the Ussing chamber are the most widely used diffusion apparatuses for these purposes. Here intestinal segment is used as a semipermeable membrane that separates the donor and acceptor compartment [210]. Reduced leakage, better mixing, increased working tissue sur-face area, and easier cleaning are added features of modified diffusion cells. Certain modifications have also been made to maintain constant temperature of the donor and acceptor compartment throughout. Today there are novel systems which mea-sures electrophysiologic changes in the cell, such as transepithelial electrical resis-tance (TEER), which is a useful index of tissue integrity and viability [211].

10.7.1.2 Intestinal Segments

In vitro studies or ex vivo perfusion studies are performed using an isolated segment of intestine. Segments from various sites are isolated and perfused according to the need. An everted gut sac apparatus has been used to study site-specific absorption of insulin from various intestinal regions of the rat. Everted gut sac apparatus is named so because here the intestinal segments are everted on a thin stainless steel rod and ligated on the other end with a silk thread. The sac created by tying one end of seg-ment is filled with a known volume of modified Kreb’s ringer phosphate bicarbonate buffer and placed inside a test tube containing the test solution at 37oC, which was continuously bubbled with 95% O2/5% CO2 to provide the physiological condition. A definite amount of load was also applied to the everted sac to prevent peristaltic muscular contractions. At time intervals, samples are withdrawn from both serosal and mucosal compartments and analyzed for the amount of drug absorbed.

Even though physiological conditions are maintained, the isolated segment is only viable for 20 min and a maximum of up to 3 h in some cases. The short viable time is


the main drawback of these in vitro techniques. It has been suggested that active and paracellular solute transport is not compromised during these in vitro experiments [212]. Along with their drawbacks, these techniques have several advantages over in vivo and other in vitro methods. Advantages over in vivo methods include bypass-ing drug dissolution and stomach-emptying steps, avoiding degradation of peptides and proteins in the stomach, and allowing control over drug input and choice of the intestinal region to be perfused. In these techniques, lymphatic and blood vessels remain intact for solute uptake and have relatively extended tissue viability as com-pared to other in vitro techniques.

10.7.1.3 Brush Border Membrane Vesicles

Brush border membrane vesicles are isolated from human intestinal epithelial cells. These vesicles are further purified and used to evaluate drugs and their dosage form for intestinal transport studies [213,214]. The isolation and purification pro-cedure of brush border membrane vesicles was described by Schmitz et al. [215]. However, this process was complex in nature and was further simplified by Kessler et al. [216]. During preparation, brush border fragments should be separated from microsomal fragments (endoplasmic reticulum) using calcium ions (Ca2). Brush border fragments are not readily separable from microsomal fragments (endoplasmic reticulum) unless Ca2 ions are used [217]. Endoplasmic reticulum and the mito-chondria are converted in the larger particulate form after aggregation with the help of Ca2. Particles thus formed are easily separated by slow speed centrifugation at about 2000 g. Supernatant containing brush border fragment is centrifuged at 20,000 g to get a pellet of brush border fragment [215].

10.7.1.4 Cell Line Studies

Cell line studies have gained plenty of attention from researchers because of their extended viability. Intestinal cells are grown in the form of monolayers and used to study intestinal drug transport [218–222]. These monolayers have extended viability when kept in nutrient- and oxygen-rich culture media and hence are preferred over other in vitro methods. Generally, intestinal cells are difficult to culture because of the unavailability of a well-differentiated human intestinal cell line, but the CACO-2 cell line, obtained from the human colon, is isolated, differentiated, and grown in culture media. When CACO-2 cells are cultured in a medium, they undergo entero-cytic differentiation and develop occluding junctional complexes between adjacent cells within 3 days, and develop morphological characteristics of the small intestine, such as microvilli, desmosomes, occluding junctions, and cell polarity after about 15 days. Due to extended viability, cells grown on microporus membranes can be directly used for drug diffusion studies for at least 20 days. Generally, polycarbon-ate and nitrocellulose membranes are used because of their translucent nature, which allows microscopic examination, and also because they are permeable to both hydro-philic and hydrophobic solutes, whatever their MW [223].

The transport of proteins and peptides across intestinal membrane is studied using CACO-2 monolayers. More realistic results are obtained from these studies because


CACO-2 cell monolayers have major drug-metabolizing enzymes as well as an array of transporters found in the intestinal cells [224] (Fig. 10.4).

In the CACO-2 cell experiment, the apparent permeability coefficient (Papp) is determined by Eq. (10.1):

PAC

M

tappd

d

d

1

0,

⋅ (10.1)

where Cd,0 is the initial concentration of drug in the donor side and M is the mass of the drug in the receiver side at the time t. The apparent permeability coefficient is further defined in Eq. (10.2):

1 1 1 1

P P P Papp aq mono F

(10.2)

where PF is the permeability coefficient of the filter support, Paq is the perme-ability coefficient of the aqueous boundary layer, and Pmono is the permeability coef-ficient of the CACO-2 cell monolayer.

Figure 10.4 Transportation across CACO-2 cell monolayer.


10.7.2 In Vivo Studies

In vivo animal studies are generally carried out to check the dissolution profile of dos-age form, rate and extent of drug absorption, and stability of the drug and dosage form. These studies reduce the cost of development by reducing the number of failed clinical trials. Various animal models are used for such studies, and selection of animal model depends on drug, dosage form, indication, and type of study. But generally these pre-clinical in vivo studies are performed in dogs. Before doing such studies, metabolic and physiological differences between selected animal model and humans must be well characterized for such studies to be useful. Gastrointestinal motility affects the rate and extent of drug absorption, and thus drug absorption in the anesthetized animal is dif-ferent from the conscious animal. A study characterizing the upper GI pH, volumetric flow rate, and the activity of chymotrypsin in mongrel fistulated dogs as a function of fasted, GI motility phase has been reported [225]. The anatomical site where a peptide is released from its formulation prior to its absorption is determined by radioiodination of peptide in conjunction with -scintigraphy. The regional perfusion technique is gen-erally used when the in vivo approach is applied in humans [226]. A multichannel per-fusion tube is introduced orally, which has two inflatable balloons 10 cm apart from each other. Thus a segment of 10 cm length can be perfused without contamination of luminal contents from proximal and distal segments [227].

10.8 Strategic Use of Oral Route for Immunization

Generally, vaccines are administered through the parenteral route. But the site of entry of most bacteria and viruses is mucosa. So better protection can be offered when a vac-cine is given through the mucosa, where bacteria enter the body. Currently, only few oral vaccines are available, so this field of oral immunization should be exploited to a large extent to develop more oral vaccine for better patient compliance. Oral immuni-zation has another advantage, that of self-immunization, where the patient alone can administer a vaccine, without a healthcare professional; this is the case in countries where healthcare professionals are present in much fewer numbers [228,229].

The presence of M cells in Peyer’s patches makes oral immunization more advan-tageous. Antigens of the given vaccine are captured by M cells and transported to the lymphatic system. After gaining entry to the lymphatic system, antigens spread to different sites and produce immune response at multiple sites. Generally, the intes-tinal segment is considered to be a immunologically nonresponsive environment because it produces a poor response to food and environmental antigens. Thus, it is very difficult for an orally given antigen to produce an immune response in such a nonresponsive environment. The intestinal segment also acquires tolerance to fre-quently given antigen.

Relatively safe bacteria or viruses, erythrocytes, liposomes, and micro- or nanopar-ticles are used to target the antigen to inductive sites [228]. For this purpose, the size of such carriers should be in a range of 5–10 m to stimulate a mucosal response. Potent oral adjuvants, such as cholera toxin, have been tried for the induction


of mucosal immunoglobulin (Ig) A antibody responses to protein antigens [230]. A comparative evaluation of influenza virus antigen entrapped in proteinoid micro-spheres and free antigen was done and found that the antigen entrapped in proteinoid microspheres was able to induce a significant IgG response as early as 2 weeks after the administration of the dose, whereas free antigen showed no detectable antibody response [231]. Polylactic-co-glycolic acid (PLGA) microspheres are a striking car-rier for oral delivery of antigens because of their higher lipophilicity and protection of the entrapped antigen from proteolytic enzymes [197–232].

10.9 Conclusion

Therapeutic peptides and proteins can now be successfully delivered through oral route. We have a better understanding of the para- and transcellular routes of absorp-tion and proteolytic enzyme activity that may potentially degrade therapeutic peptides as well as the simultaneous degradation of compounds during the mucosal transport process. Oral delivery of therapeutic peptides and proteins is only successfully attained if the peptides and proteins bypass the various penetration or enzymatic barriers at each stage. The goal is to increase typical bioavailability from less than 1% to at least 10% to 20%. Methods to increase drug flux without associated toxicity (e.g., by the use of permeation enhancers) seeks to minimize proteolytic degradation and chemical modi-fication. Innovative approaches with regard to mucoadhesive dosage forms and tar-geted as well as controlled drug delivery will be employed to improve oral peptide and protein delivery. Several methods are employed to study oral absorption using these approaches. Promising results have begun to appear, and oral delivery of insulin is cur-rently in clinical trials.

Acknowledgment

The authors acknowledge the financial assistance from the Indian Council of Medical Research and TIFAC CORE in Novel Drug Delivery Systems (NDDS), Department of Science and Technology, Government of India, New Delhi, for providing research facilities to the team.

References

[1] Wengenmayer F. Synthesis of peptide hormones using recombinant DNA techniques. Angew Chem Int Ed Engl 2003;22:842–58.

[2] Antosova Z, Mackova M, Kral V, Macek T. Therapeutic application of peptides and pro-teins: parenteral forever? Trends Biotechnol 2009;27:628–35.

[3] Moeller EH, Jorgensen L. Alternative routes of administration for systemic delivery of protein pharmaceuticals. Drug Discovery Today: Technol 2008;5:e89–94.


[4] Zempsky WT. Alternative routes of drug administration—advantages and disadvantages (subject review). Pediatrics 1998;100:143–52.

[5] Van de Weert M, Jorgensen L, Horn Moeller E, Frokjaer S. Factors of importance for a[5] Van de Weert M, Jorgensen L, Horn Moeller E, Frokjaer S. Factors of importance for a successful delivery system for proteins. Expert Opin Drug Deliv 2005;2:1029–37.

[6] Arora P, Sharma S, Garg S. Permeability issues in nasal drug delivery. Drug Discov[6] Arora P, Sharma S, Garg S. Permeability issues in nasal drug delivery. Drug Discov Today 2002;7:967–75.

[7] Rohan LC, Sassi AB. Vaginal drug delivery systems for HIV prevention. AAPS J[7] Rohan LC, Sassi AB. Vaginal drug delivery systems for HIV prevention. AAPS J 2009;11:78–87.

[8] Harris D, Robinson JR. Drug delivery via the mucous membranes of the oral cavity.[8] Harris D, Robinson JR. Drug delivery via the mucous membranes of the oral cavity. J Pharm Sci 1992;81:1–10.

[9] Gupta H, Bhandari D, Sharma A. Recent trends in oral drug delivery: a review. Recent[9] Gupta H, Bhandari D, Sharma A. Recent trends in oral drug delivery: a review. Recent Pat Drug Deliv Formul 2009;3:162–73.

[10] Sastry SV, Nyshadham JR, Fix JA. Recent technological advances in oral drug delivery—a review. Pharm Sci Technol Today 2000;3:138–45.

[11] Madhav NV, Shakya AK, Shakya P, Singh K. Orotransmucosal drug delivery systems: a review. J Control Release 2009;140:2–11.

[12] Chiappin S, Antonelli G, Gatti R, De Palo EF. Saliva specimen: a new laboratory tool for diagnostic and basic investigation. Clin Chim Acta 2007;383:30–40.

[13] Saffran M, Kumar GS, Savariar C, Burnham JC, Williams F, Neckers DC. A new approach to the oral administration of insulin and other peptide drugs. Science 1986;233:1081–4.

[14] Fix JA. Oral controlled release technology for peptides: status and future prospects. Pharm Res 1996;13:1760–4.

[15] Vincent HLLee, Satish DK, George MG, Werner R. Oral route of protein and pep-tide drug delivery. In: Lee Vincent HL, editor. Protein and peptide drug delivery: oral approaches. New York, NY: Marcel Dekker; 1991. p. 691–738.

[16] Sinha V, Singh A, Kumar RV, Singh S, Kumria R, Bhinge J. Oral colon-specific drug delivery of protein and peptide drugs. Crit Rev Ther Drug Carrier Syst 2007;24:63–92.

[17] Pettit DK, Gombotz WR. The development of site-specific drug-delivery systems for protein and peptide biopharmaceuticals. Trends Biotechnol 1998;16:343–9.

[18] Shen WC. Oral peptide and protein delivery: unfulfilled promises? Drug Discov Today 2003;8:607–8.

[19] Goldberg M, Gomez-Orellana I. Challenges for the oral delivery of macromolecules. Nat Rev Drug Discov 2003;2:289–95.

[20] Lavelle EC, O’Hagan DT. Delivery systems and adjuvants for oral vaccines. Expert Opin Drug Deliv 2006;3:747–62.

[21] Bernkop-Schnürch A, Dundalek K. Novel bioadhesive drug delivery system protecting (poly) peptides from gastric enzymatic degradation. Int J Pharm 1996;138:75–83.

[22] Lehr CM. Bioadhesion technologies for the delivery of peptide and protein drugs to the gastrointestinal tract. Crit Rev Ther Drug Carrier Syst 1994;11:119–60.

[23] Semalty A, Semalty M, Singh R, Saraf SK, Saraf S. Properties and formulation of oral drug delivery systems of protein and peptides. Indian J Pharm Sci 2007;69:741–7.

[24] Allémann E, Leroux J, Gurny R. Polymeric nano- and microparticles for the oral delivery of peptides and peptidomimetics. Adv Drug Deliv Rev 1998;34:171–89.

[25] Vigl C. Multifunctional polymeric excipients in oral macromolecular drug delivery. In: Bernkop-Schnurch A, editor. Oral delivery of macromolecular drugs. New York, NY: Springer; 2009. p. 137–52.

[26] Tang P, Wang N, Steven MD. Oral delivery of macromolecular drugs. Polymeric Drug Delivery I. ACS Symp Ser 2006;923:68–83.


[27] Tan ML, Choong PF, Dass CR. Recent developments in liposomes, microparticles and nanoparticles for protein and peptide drug delivery. Peptides 2010;31:184–93.

[28] Sakuma S, Suzuki N, Kikuchi H, Hiwatari K, Arikawa K, Kishida A, et al. Oral peptide delivery using nanoparticles composed of novel graft copolymers having hydrophobic backbone and hydrophilic branches. Int J Pharm 1997;149:93–106.

[29] Kutchai HC. The gastrointestinal system. In: Berne RM, Levy MN, Koeppen BM, Stanton BA, editors. Physiology. 4th ed. St. Louis, MO: Mosby-Year Book; 1998. p. 587–674.

[30] Fox SI. The Digestive system. In: Fox SI, editor. Human physiology. 9th ed. Columbus, OH: McGraw Hill; 2003:558–95.

[31] Egberts HJ, Koninkx JF, Dijk J, Mouwen JM. Biological and pathobiological aspects of the glycocalyx of the small intestinal epithelium. a review. Vet Q 1984;6:186–99.

[32] Russell-Jones GJ, Aizpurua HJ. Vitamin B12: a novel carrier for orally presented anti-gens. Proc Int Symp Control Release Bioact Mater 1988;15:142–3.

[33] Park, JY. Biophysical model approach to the mechanistic studies of intestinal absorption of drugs. Ph.D. Thesis, The University of Michigan, Ann Arbor, Michigan; 1977.

[34] Sallee YL, Wilson FA, Dietschy JM. Determination of unidirectional uptake rates for lip-ids across the intestinal brush border. J Lipid Res 1972;13:184–92.

[35] Rouge N, Buri P, Doelker E. Drug absorption sites in the gastrointestinal tract and dosage forms for site specific delivery. Int J Pharm 1996;136:117–39.

[36] Madara JL. Functional morphology of epithelium of the small intestine Section 6. In: Field M, Raymond AF, editors. Intestinal absorption and secretion—handbook of physi-ology. Bethesda, MD: American Physiological Society; 1991. p. 83–120.

[37] Tomita M, Shiga M, Hayashi M, Awazu S. Enhancement of colonic drug absorption by the paracellular permeation route. Pharm Res 1988;5:341–6.

[38] Pappenheimer JR. Physiological regulation of transepithelial impedance in the intestinal mucosa of rats and hamsters. J Membr Biol 1987;100:137–48.

[39] Burton PS, Conradi RA, Hilgers AR. B) Mechanisms of peptide and protein absorption: (2) Transcellular mechanism of peptide and protein absorption: passive aspects. Adv Drug Deliv Rev 1991;7:365–86.

[40] Hebden JM, Wilson CG, Spiller RC, Gilchrist PJ, Blackshaw E, Frier ME, et al. Regional differences in quinine absorption from the undisturbed human colon assessed using a timed release delivery system. Pharm Res 1999;16:1087–92.

[41] Russell-Jones GJ. Carrier-mediated transport, oral drug delivery. In: Mathiowitz E, editor. Encyclopedia of controlled drug delivery, vol. 1. New York, NY: John Wiley & Sons; 1999. p. 173–84.

[42] Barthe L, Woodley J, Houin G. Gastrointestinal absorption of drugs: methods and stud-ies. Fundamental Clin Pharmacol 1999;13:154–68.

[43] Russell-Jones GJ. The potential use of receptor-mediated endocytosis for oral drug deliv-ery. Adv Drug Deliv Rev 1996;20:83–97.

[44] Swaan PW. Recent advances in intestinal macromolecular drug delivery via receptor-mediated transport pathways. Pharm Res 1998;15:826–34.

[45] Ho NFH, Day JS, Barsuhn CL, Burton PS, Raub TJ. Biophysical model approaches to mechanistic transepithelial studies of peptides. J Control Release 1990;11:3–24.

[46] Rathbone MJ, Hadgraft J. Absorption of drugs from the human oral cavity. Int J Pharm 1991;74:9–24.

[47] Charman WN, Porter CJH. Lipophilic prodrugs designed for intestinal lymphatic trans-port. Adv Drug Deliv Rev 1996;19:149–69.

[48] Hamman JH, Enslin GM, Kotzé AF. Oral delivery of peptide drugs: barriers and develop-ments. BioDrugs: Clin Immunotherap, Biopharm Gene Ther 2005;19:165–77.


[49] Macadam A. The effect of gastro-intestinal mucus on drug absorption. Adv Drug Deliv Rev 1993;11:201–20.

[50] Schachter H, Williams D. Biosynthesis of mucus glycoproteins. In: Chantler ENJ, Elden B, Elstein M, editors. Advances in experimental medicine and biology, vol. 144. New York, NY: Plenum Press; 1982. p. 3–28.

[51] Allen A, Garner A. Mucus and bicarbonate secretion in the stomach and their possible role in mucosal protection. Gut 1980;21:249–62.

[52] Kompella UB, Vincent HLLee. Delivery systems for penetration enhancement of peptide and protein drugs: design considerations. Adv Drug Deliv Rev 1992;46:115–62.

[53] Vincent HLLee, Yamamoto A. Penetration and enzymatic barriers to peptide and protein absorption. Adv Drug Deliv Rev 1989;4:171–207.

[54] Langguth P, Bohner V, Heizmann J, Merkle HP, Wolffram S, Amidon GL, et al. The challenge of proteolytic enzymes in intestinal peptide delivery. J Control Release 1997;46:39–57.

[55] Ganong WF. Regulation of gastrointestinal function. In: Ganong WF, editor. Review of medi-cal physiology. 12th ed. Los Altos, CA: Lange Medical Publications; 1983. p. 394–420.

[56] Mrsny RJ. Challenges for the oral delivery of proteins and peptidesPeptides: theoretical and practical approaches to their delivery. Greenwood, SC: Capsugel Symposia Series; 1991. p. 45–52.

[57] Woodley J. Enzymatic barriers. In: Bernkop-Schnurch A, editor. Oral delivery of macro-molecular drugs. New York, NY: Springer Science and Business Media; 2009. p. 1–19.

[58] Vazeux G, Iturrioz X, Corvol P, Llorens-Cortes C. A glutamate residue contributes to the exopeptidase specificity in aminopeptidase A. Biochem J 1998;334:407–13.

[59] Bai JP, Amidon GL. Structural specificity of mucosal-cell transport and metabolism of peptide drugs: implication for oral peptide drug delivery. Pharm Res 1992;9:969–78.

[60] Vincent HL Lee. Enzymatic barriers to peptide and protein absorption. Crit Rev Ther Drug Carrier Syst 1988;5:68–97.

[61] Lee HLV, Dodda-Kashi S, Grass GM, Rubas W. Oral route of peptide and protein drug delivery. In: Lee HLV, editor. Peptide and protein drug delivery. New York, NY: Marcel Dekker; 1991. p. 691–738.

[62] Wearley LL. Recent progress in protein and peptide delivery by noninvasive routes. Crit Rev Ther Drug Carrier Syst 1991;8:331–94.

[63] Sasaki I, Fujita T, Murakami M, Yamamoto A, Nakamura E, Imasaki H, et al. Intestinal absorption of azetirelin, a new thyrotropin-releasing hormone (TRH) analogue. I. Possible factors for the low oral bioavailability in rats. Biol Pharm Bull 1994;17:1256–61.

[64] Hayakawa E, Lee HLV. Aminopeptidase activity in the jejunal and ileal Peyer’s patches of the albino rabbit. Pharm Res 1992;9:535–40.

[65] Wang W. Oral protein drug delivery. J Drug Target 1996;4:195–232. [66] Mrsny RJ, Cromwell M, Villagran J, Rubas W. Oral delivery of peptides to the small

intestineCurrent status on targeted drug delivery to the gastrointestinal tract. Greenwood, SC: Capsugel Symposia Series; 1993. p. 45–56.

[67] Mahato RI, Narang AS, Thoma L, Miller DD. Emerging trends in oral delivery of peptide and protein drugs. Crit Rev Ther Drug Carrier Syst 2003;20:153–214.

[68] Fasano A. Novel approaches for oral delivery of macromolecules. J Pharm Sci 1998; 87:1351–6.

[69] Ward PD, Tippin TK, Thakker DR. Enhancing paracellular permeability by modulating epithelial tight junctions. Pharm Sci Technol Today 2000;3:346–58.

[70] Salamat-Miller N, Johnston TP. Current strategies used to enhance the paracellu-lar transport of therapeutic polypeptides across the intestinal epithelium. Int J Pharm 2005;294:201–16.


[71] Pauletti GM, Gangwar S, Knipp GT, Nerurkar MM, Okumu FW, Tamura K, et al. Structural requirements for intestinal absorption of peptide drugs. J Control Release 1996;41:3–17.

[72] Kararli TT. Gastrointestinal absorption of drugs. Crit Rev Ther Drug Carrier Syst 1989;6:39–86.

[73] Siegel IA. Permeability of the rat oral mucosa to organic solutes measured in vivo. Arch Oral Biol 1984;29:13–16.

[74] McMartin C, Hutchinson LE, Hyde R, Peters GE. Analysis of structural requirements for the absorption of drugs and macromolecules from the nasal cavity. J Pharm Sci 1987;76:535–40.

[75] Maitani Y, Machida Y, Nagai. T. Influence of molecular weight and charge on nasal absorption of dextran and DEAE-dextran in rabbits. Int J Pharm 1989;49:23–7.

[76] Donovan MD, Flynn GL, Amidon GL. Absorption of polyethylene glycols 600 through 2000: the molecular weight dependence of gastrointestinal and nasal absorption. Pharm Res 1990;7:863–8.

[77] Tavakoli-Saberi MR, Audus KL. Cultured buccal epithelium: an in vitro model derived from the hamster pouch for studying drug transport and metabolism. Pharm Res 1989;6:160–6.

[78] Siegel IA, Izutsu KT, Watson E. Mechanisms of nonelectrolyte penetration across dog and rabbit oral mucosa in vitro. Arch Oral Biol 1981;26:357–61.

[79] Sandow J, Ditzinger G, Rachs S, Merkle HP. Cyclopeptides as a novel class of transmu-cosal absorption enhancers. Proc Int Symp Control Release Bioact Mater 1990;17:11–12.

[80] Veuillez F, Kalia YN, Jacques Y, Deshusses J, Buri P. Factors and strategies for improv-ing buccal absorption of peptides. Eur J Pharm Biopharm 2001;51:93–109.

[81] Merkle HP, Wolany G. Buccal delivery for peptide drugs. J Control Release 1992;21:155–64.

[82] Nightingale CH, Greene DS, Quintiliani R. Pharmacokinetics and clinical use of cepha-losporin antibiotics. J Pharm Sci 1975;64:1899–927.

[83] McNally EJ, Park JY. Peptides and proteins: oral absorption. In: Swarbrick J, editor. Encyclopedia of pharmaceutical technology. 3rd ed., vol. 1. New York, NY: Informa Healthcare USA; 2007. p. 2713–30.

[84] Okada H, Yamazaki I, Ogawa Y, Hirai S, Yashiki T, Mima H. Vaginal absorption of a potent luteinizing hormone-releasing hormone analog (leuprolide) in rats I: absorption by various routes and absorption enhancement. J Pharm Sci 1982;71:1367–71.

[85] Crane CW, Luntz GR. Absorption of insulin from the human small intestine. Diabetes 1968;17:625–7.

[86] Heyman M, Ducroc R, Desjeux JF, Morgat JL. Horseradish peroxidase trans-port across adult rabbit jejunum in vitro. Am J Physiol Gastrointest Liver Physiol 1982;242:G558–64.

[87] Kimm MH, Curtis GH, Hardin JA, Gall DG. Transport of bovine serum albumin across rat jejunum: role of enteric nervous system. Am J Physiol Gastrointest Liver Physiol 1994;266:G186–93.

[88] Green PG, Hinz RS, Kim A, Szoka Jr FC, Guy RH. Iontophoretic delivery of a series of tripeptides across the skin in vitro. Pharm Res 1991;8:1121–7.

[89] Delie F, Letourneux Y, Nisato D, Puisieux F, Couvreur P. Oral administration of peptides: study of a glycerolipidic prodrug. Int J Pharm 1995;115:45–52.

[90] Burnham NL. Polymers for delivering peptides and proteins. Am J Hosp Pharm 1994;51:210–8.

[91] Palm K, Luthman K, Ungell AL, Strandlund G, Artursson P. Correlation of drug absorp-tion with molecular surface properties. J Pharm Sci 1996;85:32–9.


[92] Merkle HP, Wolany G. Intraoral peptide absorption. In: Audus KL, Raub TJ, edi-tors. Biological barriers to protein delivery. New York/London: Plenum Press; 1993. p. 131–60.

[93] Reddy IK, Madan PL. Controlled/sustained release: proteins and peptides. Pharmatimes 1992;58:132–40.

[94] Liaw J, Rojanasakul Y, Robinson JR. The effect of drug charge type and charge density on corneal transport. Int J Pharm 1992;88:111–24.

[95] Rojanasakul Y, Wang LY, Bhat M, Glover DD, Malanga CJ, Ma JK. The transport bar-rier of epithelia: a comparative study on membrane permeability and charge selectivity in the rabbit. Pharm Res 1992;9:1029–34.

[96] Gandhi RB, Robinson JR. Permselective characteristics of rabbit buccal mucosa. Pharm Res 1991;8:1199–202.

[97] Corbo DC, Huang YC, Chien YW. Nasal delivery of progestational steroid in ovariecto-mized rabbits. II. Effect of penetrant hydrophilicity. Int J Pharm 1989;50:253–60.

[98] Huang CH, Kimura R, Bawarshi-Nassar R. Mechanism of nasal absorption of drugs II: absorption of l-tyrosine and the effect of structural modification on its absorption. J Pharm Sci 1985;74:1298–301.

[99] Hirai S, Yashiki T, Matsuzawa T, Mima H. Absorption of drugs from the nasal mucosa of rat. Int J Pharm 1981;7:317–25.

[100] Hansen LB, Christrup LL, Bundgaard H. Enhanced delivery of ketobemidone through porcine buccal mucosa in vitro via more lipophilic ester prodrugs. Int J Pharm 1992; 88:237–42.

[101] Hansen LB, Jorgensen A, Rasmussen SN, Christrup LL, Bundgaard H. Buccal absorption of ketobemidone and various ester prodrugs in the rat. Int J Pharm 1992;88:243–50.

[102] Corbo DC, Liu JC, Chien YW. Drug absorption through mucosal membranes: effect of mucosal route and penetrant hydrophilicity. Pharm Res 1989;6:848–52.

[103] Beckett AH, Moffat AC. Correlation of partition coefficients in n-heptane aqueous sys-tems with buccal absorption data for a series of amines and acids. J Pharm Pharmacol 1969;21:144S–150S.

[104] Beckett AH, Moffat AC. Kinetics of buccal absorption of some carboxylic acids and the correlation of the rate constants and n-heptane: aqueous phase partition coefficients. J Pharm Pharmacol 1970;22:15–19.

[105] Toniolo C, Bonora GM, Stavropoulos G, Cordopatis P, Theodoropoulos D. Self-asso-ciation and solubility of peptides: solvent-titration study of N-protected C-terminal sequences of substance P. Biopolymers 1986;25:281–9.

[106] Touitou E. Enhancement of intestinal peptide absorption. J Control Release 1992;21:139–44.

[107] Banga AK, Chien YW. Systemic delivery of therapeutic peptides and proteins. Int J Pharm 1988;48:15–50.

[108] Chien YW, Banga AK. Potential developments in systemic delivery of insulin. Drug Dev Ind Pharm 1989;15:1601–34.

[109] Conradi RA, Hilgers AR, Ho NFH, Burton PS. The influence of peptide structure on transport across CACO-2 cells. Pharm Res 1991;8:1453–60.

[110] Conradi RA, Hilgers AR, Ho NFH, Burton PS. The influence of peptide structure on transport across CACO-2 cells-II. Peptide bond modification which results in improved permeability. Pharm Res 1992;9:435–9.

[111] Burton PS, Conradi RA, Hilgers AR, Ho NFH, Maggiora LL. The relationship between peptide structure and transport across epithelial cell monolayers. J Control Release 1992;19:87–98.


[112] Davis SS. Advanced delivery systems for peptides and proteins-pharmaceutical. In: Davis SS, Tomlinson E, editors. Delivery systems for peptide drugs. New York/London: NATO Advanced Science Institutes Series; 1986. p. 1–21.

[113] Chikhale EG, Ng KY, Burton PS, Borchardt RT. Hydrogen bonding potential as a deter-minant of the in vitro and in situ blood–brain barrier permeability of peptides. Pharm Res 1994;11:412–19.

[114] Siegel IA. Permeability of the oral mucosa. In: Meyer J, Squier CA, Gerson SJ, editors. The structure and function of oral mucosa. Oxford, England: Oxford University Press; 1984. p. 95–108.

[115] Chikhale EG, Burton PS, Borchardt RT. The effect of verapamil on the transport of pep-tides across the blood–brain barrier in rats: kinetic evidence for an apically polarized efflux mechanism. J Pharmacol Exp Ther 1994;273:298–303.

[116] Diamond JM, Wright EM. Molecular forces governing non-electrolyte permeation through cell membranes. Proc R Soc B 1969;172:273–316.

[117] Lee HLV. Oral route of peptide and protein drug delivery. BioPharm 1992;5:39–50. [118] Pantzar N, Lundin S, Westrom BR. Different properties of the paracellular pathway

account for the regional small intestinal permeability to the peptide desmopressin. J Pharm Sci 1995;84:1245–8.

[119] Lundin S, Pantzar N, Broeders A, Ohlin M, Westrom BR. Differences in transport rate of oxytocin and vasopressin analogues across proximal and distal isolated segments of the small intestine of the rat. Pharm Res 1991;8:1274–80.

[120] Schilling RJ, Mitra AK. Pharmacodynamics of insulin following intravenous and enteral administrations of porcine-zinc insulin to rats. Pharm Res 1992;9:1003–9.

[121] Lu RH, Kopeckova P, Kopecek J. Degradation and aggregation of human calcitonin in vitro. Pharm Res 1999;16:359–67.

[122] Touitou E, Rubinstein A. Targeted enteral delivery of insulin to rats. Int J Pharm 1986;30:95–9.

[123] Quadros E, Landzert NM, LeRoy S, Gasparani F, Worosila G. Colonic absorption of insulin-like growth factor I in vitro. Pharm Res 1994;11:226–30.

[124] Ashford M, Fell JT. Targeting drugs to the colon: delivery systems for oral administra-tion. J Drug Target 1994;2:241–57.

[125] Shakweh M, Ponchel G, Fattal E. Particle uptake by Peyer’s patches: a pathway for drug and vaccine delivery. Expert Opin Drug Deliv 2004;1:141–63.

[126] Zhou XH. Overcoming enzymatic and absorption barriers to non-parenterally adminis-tered protein and peptide drugs. J Control Release 1994;29:239–52.

[127] Hughes RA, Toth I, Ward P, Ireland SJ, Gibbons WA. Lipidic peptides. III: lipidic amino acid and oligomer conjugates of morphine. J Pharm Sci 1991;80:1103–5.

[128] Taniguchi T, Tomoda Y, Tanaka Y, Murakami M, Yamamoto A, Muranishi S. Synthesis of acyloyl lysozyme and improvement of its lymphatic transport following small intestinal administration in rats. Proc Int Symp Control Release Bioact Mater 1992;19:104–5.

[129] Asada H, Douen T, Waki M, Adachi S, Fujita T, Yamamoto A, et al. Absorption charac-teristics of chemically modified-insulin derivatives with various fatty acids in the small and large intestine. J Pharm Sci 1995;84:682–7.

[130] Asada H, Douen T, Murakami M, Yamamoto A, Muranishi S. Improvement of intestinal absorption of insulin by chemical modification with fatty acids. Proc Int Symp Control Release Bioact Mater 1992;19:212–3.

[131] Yamada K, Murakami M, Yamamoto A, Takada K, Muranishi S. Improvement of intes-tinal absorption of thyrotropin-releasing hormone by chemical modification with lauric acid. J Pharm Pharmacol 1991;44:717–21.


[132] Muranishi S, Murakami M, Hashidzume M, Yamada K, Tajima S, Kiso Y. Trials of lipid modification of peptide hormones for intestinal delivery. J Control Release 1992;19:179–88.

[133] Tanaka K, Fujita T, Yamamoto Y, Murakami M, Yamamoto A, Muranishi S. Enhancement of intestinal transport of thyrotropin-releasing hormone via a carrier-mediated transport sys-tem by chemical modification with lauric acid. Biochim Biophys Acta 1996;1283:119–26.

[134] Muranishi S, Sakai A, Yamada K, Murakami M, Takada K, Kiso Y. Lipophilic peptides: synthesis of lauroyl thyrotropin-releasing hormone and its biological activity. Pharm Res 1991;8:649–52.

[135] Fujita T, Morikawa K, Iemura O, Yamamoto A, Muranishi S. Improvement of intestinal absorption of human calcitonin by chemical modification with fatty acids: synergistic effects of acylation and absorption enhancers. Int J Pharm 1996;134:47–57.

[136] Tenma T, Yodoya E, Tashima S, Fujita T, Murakami M, Yamamoto A, et al. Development of new lipophilic derivatives of tetragastrin: physicochemical character-istics and intestinal absorption of acyl-tetragastrin derivatives in rats. Pharm Res 1993; 10:1488–92.

[137] Yodoya E, Uemura K, Tashima S, Fujita T, Murakami M, Yamamoto A, et al. Enhancement permeability of tetragastrin across the intestinal membrane and its reduced degradation by Acylation with various fatty acids in rats. J Pharmacol Exp Ther 1994;271:1509–13.

[138] Setoh K, Murakami M, Araki N, Fujita T, Yamamoto A, Muranishi S. Improvement of transdermal delivery of tetragastrin by lipophilic modification with fatty acids. J Pharm Pharmacol 1995;47:808–11.

[139] Fujita T, Kawahara I, Quan Y, Hattori K, Takenaka K, Muranishi S, et al. Permeability characteristics of tetragastrins across intestinal membranes using the CACO-2 mono-layer system: comparison between acylation and application of protease inhibitors. Pharm Res 1998;15:1387–92.

[140] Murakami M, Kotani A, Uchiyama T, Fujita T, Yamamoto A, Muranishi S. Enteric pep-tide delivery by chemical modification with fatty acids: epithelial permeability enhance-ment of Dadle. Proc Int Symp Control Release Bioact Mater 1997;24:349–50.

[141] Stoughton RB, McClure WO. Azone, a new non toxic enhancer of cutaneous penetra-tion. Drug Dev Ind Pharm 1983;9:725–44.

[142] Bundgaard H, Moss J. Prodrugs of peptides (part 4): bioreversible derivatization of the pyroglutamyl group by N-acylation and N-aminomethylation, effect protection against pyroglutamyl aminopeptidase. J Pharm Sci 1989;78:122–6.

[143] Veillard MM, Longer MA, Robinson JR. Buccal controlled delivery of peptides. Proc Int Symp Control Release Bioact Mater 1987;14:22.

[144] Veillard MM, Longer MA, Martens TW, Robinson JR. Preliminary studies of oral mucosal delivery of peptide drugs. J Control Release 1987;6:123–31.

[145] Veuillez F, Ganem-Quintanar A, Deshusses J, Falson-Rieg F, Buri P. Comparison of the ex-vivo oral mucosal permeation of tryptophan-leucine (Trp-Leu) and its myristoyl derivative. Int J Pharm 1998;170:85–91.

[146] Kahns AH, Bundgaard H. Prodrugs of peptides. 13. Stabilization of peptide amides against a-chymotrypsin by the prodrug approach. Pharm Res 1991;8:1533–8.

[147] Buur A, Bundgaard H. Prodrugs of peptides. III. 5-oxazolidinones as bioreversible derivatives for the x-amido carboxy moiety in peptides. Int J Pharm 1988;46:159–67.

[148] Shen W, Wan J, Ekrami H. C) Means to enhance penetration: (3) Enhancement of poly-peptide and protein absorption by macromolecular carriers via endocytosis end trans-cytosis. Adv Drug Deliv Rev 1992;8:93–113.


[149] Sezaki H, Takakura Y, Hashida M. Chemical modification and disposition of pro-teins and peptides: biopharmaceutical aspects in topics in pharmaceutical sciences. In: Crommelin DJA, editor. Topics in pharmaceutical sciences. Stuttgart, Germany: Medpharm Scientific; 1991. p. 47–56.

[150] Aungst BJ, Rogers NJ. Comparison of the effects of various transmucosal absorption promoters on buccal insulin delivery. Int J Pharm 1989;53:227–35.

[151] Steward A, Bayley DL, Howes C. The effect of enhancers on the buccal absorption of hybrid (BDBB) alpha-interferon. Int J Pharm 1994;104:145–9.

[152] Mollgaard B. Synergistic effects in percutaneous enhancement. In: Walters KA, Hadgraft J, editors. Pharmaceutical skin penetration enhancement. New York, NY: Marcel Dekker; 1993. p. 220–42.

[153] Duchene D, Touchard F, Peppas NA. Pharmaceutical and medical aspects of bioadhe-sive systems for drug administration. Drug Dev Ind Pharm 1988;14:283–318.

[154] Park K, Robinson JR. Bioadhesive polymers as platforms for oral-controlled drug deliv-ery: method to study bioadhesion. Int J Pharm 1984;19:107–27.

[155] Park K, Cooper SL, Robinson JR. Bioadhesive hydrogels. In: Peppas NA, editor. Hydrogels in medicine and pharmacy—properties and applications, vol. 3. Boca Raton, FL: CRC Press; 1987. p. 151–74.

[156] Pxlate NA, Valuev IL, Sytov GA, Valuev LI. Mucoadhesive polymers with immo-bilized proteinase inhibitors for oral administration of protein drugs. Biomaterials 2002;23:1673–77.

[157] Rubinstein A, Tirosh B. Mucus gel thickness and turnover in the gastrointestinal tract of the rat: response to cholinergic stimulus and implication for mucoadhesion. Pharm Res 1994;11:794–9.

[158] Li C, Bhatt PP, Johnston TP. Transmucosal delivery of oxytocin to rabbits using a mucoadhesive buccal patch. Pharm Dev Technol 1997;2:265–74.

[159] Lehr CM, Bouwstra JA, Kok W, De Boer AG, Tukker JJ, Verhoef JC, et al. Effects of the mucoadhesive polymer carbophil on the intestinal absorption of a peptide in the rat. J Pharm Pharmacol 1992;44:402–7.

[160] Sudhakar Y, Kuotsu K, Bandyopadhyay AK. Buccal bioadhesive drug delivery—a promising option for orally less efficient drugs. J Control Release 2006;114:15–40.

[161] LueBen HL, Lehr CM, Rentel CO, Noach ABJ, Deboer AG, Verhoef JC, et al. Bioadhesive polymers for the peroral delivery of peptide drugs. J Control Release 1994;29:329–38.

[162] Ponchel G. Formulation of oral mucosal drug delivery systems for the systemic delivery of bioactive materials. Adv Drug Deliv Rev 1994;13:75–87.

[163] Qiu Y, Zhang G. Development of modified-release solid oral dosage forms. In: Qiu Y, Chen Y, Zhang GGZ, Liu L, Porter WR, editors. Developing solid oral dosage forms. San Diego, CA: Elsevier Academic Press; 2009. p. 501–17.

[164] Abdul S, Poddar SS. A flexible technology for modified release of drugs: multi-layered tablets. J Control Release 2004;97:393–405.

[165] Shen Z, Mitragotri S. Intestinal patches for oral drug delivery. Pharm Res 2002;19:391–5.

[166] Harris D, Robinson JR. Bioadhesive polymers in peptide drug delivery. Biomaterials 1990;11:652–8.

[167] Bruschi ML, Freitas O. Oral Bioadhesive drug delivery systems. Drug Dev Ind Pharm 2005;31:293–310.

[168] Ishida M, Nambu N, Nagai T. Highly viscous gel ointment containing carbopol for application to the oral mucosa. Chem Pharm Bull 1983;31:4561–4.


[169] Bernkop-Schnürch A, Pasta M. Intestinal peptide and protein delivery: novel bioadhesive drug-carrier matrix shielding from enzymatic attack. J Pharm Sci 1998;87: 430–4.

[170] Bremecker KD, Stremple H, Klein G. Novel concept for a mucosal adhesive ointment. J Pharm Sci 1984;73:548–52.

[171] Gomez-Orellana I, Paton DR. Advances in the oral delivery of proteins. Expert Opin Ther Pat 1998;8:223–34.

[172] Hayashi M. Strategies to improve intestinal membrane transport of poorly absorbed drugs and peptidesCurrent status on targeted drug delivery to the gastrointestinal tract. Greenwood, SC: Capsugel Symposia Series; 1993. p. 153–60.

[173] Ganem-Quintanar A, Kalia YN, Falson-Rieg F, Buri P. Mechanism of oral permeation enhancer. Int J Pharm 1997;156:127–42.

[174] Nakada Y, Awata N, Nakamichi C, Sugimoto I. The effects of additives on the oral mucosal absorption of human calcitonin in rats. J Pharmacobio-Dyn 1988;11:395–401.

[175] Oh CK, Ritschel WA. Absorption characteristics of insulin through the buccal mucosa. Methods Find Exp Clin Pharmacol 1990;12:275–9.

[176] Anderberg EK, Artursson P. Epithelial transport of drugs in cell culture. 8. Effects of sodium dodecyl sulfate on cell membrane and tight junction permeability in human intestinal epithelial (CACO-2) cells. J Pharm Sci 1993;82:392–8.

[177] Ziv E, Kidron M, Berry EM, Bar-On H. Bile salts promote the absorption of insulin from the rat colon. Life Sci 1981;29:803–9.

[178] Hochman J, Artursson P. Mechanisms of absorption enhancement and tight junction regulation. J Control Release 1994;29:253–67.

[179] Hastewell J, Lynch S, Fox R, Williamson I, Skelton-Stroud P, Mackay M. Enhancement of human calcitonin absorption across the rat colon in vivo. Int J Pharm 1994;101:115–20.

[180] Takada K, Nakahigashi Y, Tanaka T, Kishimoto S, Ushirogawa Y, Takaya T. Pharmacological activity of tablets containing recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats. Int J Pharm 1994;101:89–96.

[181] Takaya T, Niwa K, Takada K. Pharmacological effect of recombinant human colony-stimulating factor (rhG-CSF) after administration into rat large intestine. Int J Pharm 1994;110:47–53.

[182] Woodley JF. Enzymatic barriers for GI peptide and protein delivery. Crit Rev Ther Drug Carrier Syst 1994;11:61–95.

[183] Bernkop-Schnurch A. The use of inhibitory agents to overcome the enzymatic bar-rier to perorally administered therapeutic peptides and proteins. J Control Release 1998;52:1–16.

[184] Taki Y, Yamashita S, Sakane T, Nadai T, Sezaki H, Langguth P, et al. Gastrointestinal absorption of metkephamid—quantitative evaluation of degradation and permeation. Proc Int Symp Control Release Bioact Mater 1994;21:814–5.

[185] Friedman DI, Amidon GL. Oral absorption of peptides: influence of pH and inhibitors on the intestinal hydrolysis of Leu-enkephalin and analogues. Pharm Res 1991;8:93–96.

[186] Bai JPF. Effects of bile salts on brush-border and cytosolic proteolytic activities of intestinal enterocytes. Int J Pharm 1994;111:147–52.

[187] Yamamoto A, Taniguchi T, Rikyuu K, Tsuji T, Fujita T, Murakami M, et al. Effects of various protease inhibitors on the intestinal absorption and degradation of insulin in rats. Pharm Res 1994;11:1496–1500.

[188] Gomez-Orellana I, Paton DR. Advances in the oral delivery of proteins. Expert Opin Ther Pat 1999;9:247–53.


[189] Rogers JA, Anderson KE. The potential of liposomes in oral drug delivery. Crit Rev Ther Drug Carrier Syst 1998;15:421–80.

[190] Tomizawa H, Aramaki Y, Fujii Y, Hara T, Suzuki N, Yachi K, et al. Uptake of phos-phatidylserine liposomes by rat Peyer’s patches following intraluminal administration. Pharm Res 1993;10:549–52.

[191] New, RRC, & Kirby, CJ. Hydrophobic preparations containing medium chain mono-glycerides. Cortecs LTD. WO9800169; 1998.

[192] Baudys M, Mix D, Kim SW. Stabilization and oral delivery of calcitonin. University of Utah Research Foundation. Salt Lake City, UT. WO9800155; 1998.

[193] Oral insulin delivery. Shire Laboratories, Inc. Gaithersburg, MD. US5824638; 1998.[194] Mora M, Pato J. Preparation of egg albumin microparticles for oral application.

J Control Release 1993;25:107–13. [195] Sarubbi D, Variano B, Haas S, Maher J, Falzarano L, Burnett B, et al. Oral delivery of

calcitonin in rats and primates using proteinoid microspheres. Proc Int Symp Control Release Bioact Mater 1994;21:288–9.

[196] Le Ray AM, Vert M, Gautier JC, Benoit JP. Fate of [C-14] poly(dl-lactide-co-gly-colide) nanoparticles after intravenous and oral administration to mice. Int J Pharm 1994;106:201–11.

[197] Couvreur P, Puisieux F. Nanoparticles and microparticles for the delivery of polypep-tides and proteins. Adv Drug Deliv Rev 1993;10:141–62.

[198] Michel C, Aprahamian M, Defontaine L, Couvreur P, Damge C. The effect of site of administration in the gastrointestinal tract on the absorption of insulin from nanocap-sules in diabetic rats. J Pharm Pharmacol 1991;43:1–5.

[199] Damge C, Michel C, Aprahamian M, Couvreur P. New approach for oral administra-tion of insulin with polyalkylcyanoacrylate nanocapsules as drug carrier. Diabetes 1988;37:246–51.

[200] Lowe PJ, Temple CS. Calcitonin and insulin in isobutylcyanoacrylate nanocap-sules: protection against proteases and effect on intestinal absorption in rats. J Pharm Pharmacol 1994;46:547–52.

[201] Torres-Lugo M, Garcia M, Record R, Peppas NA. Physicochemical behavior and cyto-toxic effects of p(methacrylic acid-g-ethylene glycol) nanospheres for oral delivery of proteins. J Control Release 2002;80:197–205.

[202] Story MJ. Oral delivery of peptides has become a realityPeptides: theoretical and practical approaches to their delivery. Greenwood, SC: Capsugel Symposia Series; 1991. p. 55–67.

[203] Ziv E, Kidron M, Raz I, Krausz M, Blatt Y, Rotman A, et al. Oral administration of insulin in solid form to nondiabetic and diabetic dogs. J Pharm Sci 1994;83:792–4.

[204] Gould-Fogerite S, Kheiri MT, Zhang F, Wang Z, Scolpino AJ, Feketeova E, et al. Targeting immune response induction with cochleate and liposome-based vaccines. Adv Drug Deliv Rev 1998;32:273–87.

[205] Yang S, Yuan W, Jin T. Formulating protein therapeutics into particulate forms. Expert Opin Drug Deliv 2009;6:1–11.

[206] Arifin D, Palmer A. Polymersome encapsulated hemoglobin: a novel type of oxygen carrier. Biomacromolecules 2005;6:2172–81.

[207] Aliabadi HM, Lavasanifar A. Polymeric micelles for drug delivery. Expert Opin Drug Deliv 2006;3:139–62.

[208] Yuan X, Harada A, Yamasaki Y, Kataoka K. Stabilization of lysozyme-incorporated polyion complex micelles by the omega-end derivatization of poly(ethylene glycol)-poly(alpha,betaaspartic acid) block copolymers with hydrophobic groups. Langmuir 2005;21:2668–74.


[209] Mathot F, Van Beijsterveldt L, Préat V, Brewster M, Ariën A. Intestinal uptake and bio-distribution of novel polymeric micelles after oral administration. J Control Release 2006;111:47–55.

[210] Smith P, Mirabelli C, Fondacaro J, Ryan F, Dent J. Intestinal 5-fluorouracil absorp-tion: use of Ussing chambers to assess transport and metabolism. Pharm Res 1988;5:598–603.

[211] Sutton SC, Forbes AE, Cargill R, Hochman JH, LeCluyse EL. Simultaneous in vitro measurement of intestinal tissue permeability and transepithelial electrical resistance (TEER) using Sweetana-grass diffusion cells. Pharm Res 1992;9:316–9.

[212] Lu HH, Thomas JD, Tukker JJ, Fleisher D. Intestinal water and solute absorption stud-ies: comparison of in situ perfusion with chronic isolated loops in rats. Pharm Res 1992;9:894–900.

[213] Thwaites DT, Simmons NL, Hirst BH. Thyrotropin-releasing hormone (TRH) uptake in intestinal brush-border membrane vesicles: comparison with proton-coupled dipeptide and Na-coupled glucose transport. Pharm Res 1993;10:667–73.

[214] Thwaites DT, Hirst BH, Simmons NL. Passive transepithelial absorption of thyrotropin-releasing hormone (TRH) via a paracellular route in cultured intestinal and renal epithe-lial cell lines. Pharm Res 1993;10:674–81.

[215] Schmitz J, Preiser H, Maestracci D, Ghosh BK, Cerda JJ, Crane RK. Purification of the human intestinal brush border membrane. Biochim Biophys Acta 1973;323:98–112.

[216] Kessler M, Acuto O, Storelli C, Murer H, Muller M, Semenza G. A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes and their use in investigating some properties of d-glucose and cho-line transport systems. Biochim Biophys Acta 1978;506:136–54.

[217] Osiecka I, Porter PA, Borchardt RT, Fix JA, Gardner CR. In vitro drug absorption mod-els. I. Brush border membrane vesicles, isolated mucosal cells and everted intestinal rings: characterization and salicylate accumulation. Pharm Res 1985;2:284–92.

[218] Artursson P. Epithelial transport of drugs in cell culture. I: a model for studying the passive diffusion of drugs over intestinal absorptive (CACO-2) cells. J Pharm Sci 1990;79:476–82.

[219] Hidalgo IJ, Hillgren KM, Grass GM, Borchardt RT. Characterization of the unstirred water layer in CACO-2 cell monolayers using a novel diffusion apparatus. Pharm Res 1991;8:222–7.

[220] Cogburn JN, Donovan MG, Schasteen CS. A model of human small intestinal absorp-tive cells. 1. Transport barrier. Pharm Res 1991;8:210–6.

[221] Buur A, Mork N. Metabolism of testosterone during in vitro transport across CACO-2 cell monolayers: evidence for b-hydroxysteroid dehydrogenase activity in differentiated CACO-2 Cells. Pharm Res 1992;9:1290–4.

[222] Laboisse CL, Jarry A, Bouhanna C, Merlin D, Vallette G. Intestinal cell culture models. Eur J Pharm Sci 1994;2:36–8.

[223] Borchardt RT. Rational delivery strategies to circumvent physical and metabolic barri-ers to the oral absorption of peptides. In: Peptides: theoretical and practical approaches to their delivery. Greenwood, SC: Capsugel Symposia Series; 1991. p. 11–19.

[224] Burton PS, Conradi RA, Hilgers AR, Ho NFH. Evidence for a polarized efflux system for peptides in the apical membrane of CACO-2 cells. Biochem Biophys Res Commun 1993;190:760–6.

[225] Sinko PJ. Intestinal absorption of peptides and peptide analogues: implications of fast-ing pancreatic serine protease levels and pH on the extent of oral absorption in dogs and humans. Pharm Res 1992;9:320–5.


[226] Schedl HP, Clifton JA. Cortisol absorption in man. Gastroenterology 1963;44:134–45. [227] Lennernas H, Ahrenstedt O, Hallgren R, Knutson L, Ryde M, Paalzow LK. Regional

jejunal perfusion, a new in vivo approach to study oral drug absorption in man. Pharm Res 1992;9:1243–51.

[228] Manganaro M, Ogra PL, Ernst PB. Oral immunization—turning fantasy into reality. Int Arch Allergy Immunol 1994;103:223–33.

[229] O’Hagan DT. Oral delivery of vaccines. Formulation and clinical pharmacokinetic con-siderations. Clin Pharmacokinet 1992;22:1–10.

[230] Snider DP, Marshall JS, Perdue MH, Liang H. Production of IgE antibody and allergic sensitization of intestinal and peripheral tissues after oral immunization with protein Ag and cholera toxin. J Immunol 1994;153:647–57.

[231] Santiago N, Milstein S, Rivera T, Garcia E, Zaidi T, Hong H, et al. Oral immunization of rats with proteinoid microspheres encapsulating influenza virus antigens. Pharm Res 1993;10:1243–7.

[232] Mestecky J, Moldoveanu Z, Novak M, Huang WQ, Gilley RM, Staas JK, et al. Biodegradable microspheres for the delivery of oral vaccines. J Control Release 1994;28:131–41.

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