Oral Insulin Delivery Strategies Using Absorption Promoters, Absorption Enhancers, and Protease Inhibitors

April 2, 2006
Pharmaceutical Technology, Pharmaceutical Technology-04-02-2006, Volume 30, Issue 4

Absorption promoters and enhancers and enzyme inhibitors, either alone or in combination, can play an important role in improving the bioavailability of oral insulin.

The early years of oral insulin research inspired pharmaceutical polymer and biomedical researchers to explore the possibility of improving the oral bioavailability of insulin and other peptide and protein drugs. A historical retrospective of oral insulin delivery methods shows that the oral absorption of both native and modified insulin in animals significantly increases with the incorporation of absorption enhancers and promoters and enzyme inhibitors, alone and in combination. Many authors have suggested that various pharmaceutical excipients in different chemical forms and concentrations could help enhance the absorption of insulin in an oral dosage form. These excipients have improved insulin absorption in laboratory animal experiments. Studies were performed to observe if any of these agents have a toxic effect on the cellular structure of the intestinal epithelium.

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In vivo studies using various types of laboratory animal models have suggested that orally administered insulin is available for absorption when protected from enzymatic degradation. The experiments also were performed in vitro to observe the effect of these agents in insulin-containing formulations. Oil and water bases were prepared by using absorption enhancers and promoters. Evaluation studies on various types of enzyme inhibitors, selected based on their availability and suitability in the insulin containing formulations, also were reviewed.

Some of these studies are described in this article, with an emphasis on the delivery of oral insulin formulations containing either promoters and enhancers alone or in combination with enzyme inhibitors. This article also reviews the current state of use of absorption promoters and enhancers and enzyme inhibitors and assesses the progress and limitations of studies and reports. The article concludes with an examination of clinical outcomes of these agents characterized by increased permeability and no toxic effect.

Oral insulin delivery strategies

Enzyme-inhibiting agents. Enzyme inhibiting agents are becoming increasingly popular as a delivery strategy for oral insulin. Insulin, like many other protein and peptide drugs, may be degraded in the gastrointestinal tract (GIT) by digestive enzymes such as pepsin, proteases, peptidases, and other proteolytic enzymes. The proper selection of protease inhibitors depends on both the peptide and protein drug to be delivered and on the type of proteases and peptidases used to protect the drug in the GIT.

Several studies support using protease inhibitors as a protective cover to prevent the degradation of insulin by digestive enzymes, which are mostly located in the upper part of the intestine (see Table I). Investigators should conduct more in vitro experiments to evaluate the potential for cytotoxicity or other gastrointestinal side effects. Most studies cited in this article show increased permeability with little or no toxic effect.

Table I: Select penetration and permeation enhancers and enzyme inhibitors used in various studies.

In the 1980s, Ziv and his colleagues studied bile acid (sodium cholate) as a protease inhibitor, along with aprotinin (a trypsin inhibitor), in an insulin-containing solution. This solution was injected into rats' ileums. The results suggest that the combination of both inhibitors could improve insulin absorption in the intestinal lumen of rats (1). Interestingly, acarbose, an intestinal alpha glucosidase inhibitor, also showed some positive effects in diabetic animals (2).

A potent and specific inhibitor, 4-(4-isopropylpiper-adinocarnonyl) phenyl 1, 2, 3, 4-tetrahydro-1-naphthoate methanesulphonate (FK-448) of chymotrypsin improved intestinal absorption of insulin and resulted in a reduction in blood glucose in rats and dogs (3). Fujii's study, which used soybean trypsin inhibitors in the ileum, confirmed the earlier findings of Ziv et al. and Kidron et al. (1, 4). There is no evidence that soybean trypsin inhibitor enhanced insulin absorption in the ascending colon, however. Further attempts to increase the hypoglycemic levels in diabetic animals were made by introducing enzyme inhibitors and protease inhibitors (aprotinin or the Bowman–Birk inhibitor) to prevent the degradation of insulin in the intestine by pancreatic enzymes (5, 6).

In one study, the use of a trypsin inhibitor (protease inhibitor) caused greater hypoglycemic effect in a gelatin microspheres formulation containing insulin (7). The study suggested that aprotinin or bacitracin (protease inhibitors) caused a significant and prolonged plasma glucose reduction in diabetic rats (8).

Furthermore, another study suggested that the coadministration of aprotinin and soybean trypsin inhibitor (protease inhibitors) had little effect on the absorption of insulin when compared with aprotinin alone. The same study also suggested the use of a combination of camostat mesilate and bacitracin (protease inhibitors). This combinational strategy was more effective in reducing plasma glucose levels than any other combination previously used by the same authors (9).

A study by Damage et al. reported that poly (alkyl cyanoacrylate) insulin nanoscapsules dispersed in an oily medium containing poloxmer 188 and deoxycholic acid (surfactants) reduces fasted gylcemia in diabetic rats (10).

Qi and Ping studied the oral coadministration of insulin enteric microspheres with sodium N-(8-[2-hydroxybenzoyl] amino) caprylate (SNAC). The study confirmed that microsphere preparation facilitates insulin absorption in rats because of SNAC's enzyme inhibiting properties, which increase insulin permeability (11). In this study, insulin was solubilized in the form of anhydrous reverse micelles. Ethylene–diaminetetraacetic acid (EDTA) was administered before the insulin oil solution was given to rats. A decrease in glucose levels, which primarily resulted from EDTA's enzyme inhibiting properties, was observed (12).

A success with the coadministration of trypsin and chymotrypsin (protease inhibitors) that did not alter any physiological digestive process also was reported (13).

Morishita et al. used microspheres for insulin delivery in rats (14). Their study showed that L-microspheres carrying insulin and aprotinin enhanced insulin absorption. Four years later, Hosny investigated the use of sodium cholate as an effective enzyme inhibitor in different doses and found that smaller doses of sodium cholate were more effective in reducing plasma glucose levels than higher doses (15).

The issue of insulin stability in the presence of enzyme inhibitors should be addressed. If the incorporated enzyme inhibitors are not absorbed, then nutrition, or the digestion of protein, may be disturbed (16). If the enzyme inhibitors are absorbed, it may lead to systemic intoxication (17).

A novel strategy was developed in which the enzyme inhibitors (Bowman–Birk inhibitor and elastinal) were used as covalently attached inhibitors (Bowman–Birk inhibitor–carboxymethylcellulose and carboxymethylcellulose–elastinal). These were used as effective inhibitors in an oral insulin formulation and were focused only on drug delivery systems. They apparently did not cause any damage to intestinal cellular components. The result was a record decrease in blood glucose levels for more than 72 hours (18).

Tozaki and his colleagues used azopolymer-coated pellets containing 12.5 international units of insulin and camostat mesilate (a protease inhibitor) to produce a decrease in plasma glucose levels in rats (19). Other scientists studied the in vitro oral insulin delivery approach, using chicken ovomucoid and duck ovomucoid as enzyme inhibitors, to evaluate the preparation technique and dissolution stability of the polyethylacrylate-based microparticulate delivery system of insulin (20).

The glycoprotein ovomucoid from duck egg white was used later to immobilize the peptide drug (probably insulin) in a polymeric hydrogel formulation. Glycoprotein ovomucoid prevents the degradation of peptide drugs (insulin) by the proteolytic enzymes in the GIT (21). Finally, a double-liposomal formulation containing insulin and aprotinin effectively reduced plasma glucose levels in normal rats (22).

The results from in situ closed small and large intestinal loops in rats suggest that protease inhibitors could increase insulin absorption more effectively in the large intestine than in the small intestine. The protease inhibitors that improved insulin absorption in the intestine were, in order of increasing improvement, leupeptin, sodium glycocholate, bacitracin, bestatin, and cystatin (23).

Side effects such as systemic intoxication and disturbed digestion of food proteins are regularly associated with the use of insulin with enzyme inhibitors. These side effects have prompted caution in the use of enzyme inhibitors in oral insulin formulations. The abovementioned side effects can be reversed, however, if oral insulin formulations are designed to contain an inhibitor that releases insulin and an inhibiting agent at the same time and limit its action to a certain area within the intestine.

Absorption enhancers and promoters. An absorption enhancer or promoter is a chemical or natural product that enhances or promotes the absorption of oral insulin in the small and large intestines. It may act upon the intestines by altering the structural integrity of the mucosal and epithelial membranes or by simply improving insulin diffusion across the intestinal membrane. Bile salts, detergents, and fatty acids in formulations containing insulin in various capacities and concentrations often enhance or promote insulin absorption. Table I lists some common enhancers and promoters. One of these agents may greatly increase intestinal mucosal permeability in in vitro and in situ animal studies. The increase, however, does not ensure that the particular agent will also effectively improve the oral bioavailability of a drug (e.g., insulin).

As early as the 1970s, nonionic surfactant cetomacrogels were successfully used to enhance insulin's membrane permeability in rats. The formulation was injected directly into diabetic rats' jejunums (24). In an early attempt to achieve success with enhancers, a combination of BRIJ (fatty acid–polyoxyalkylene fatty ether) and stearic acid was used to improve the bioavailability of an insulin formulation (25). Later in the mid-1980s, studies by Touitou and Rubinstein further supported using polymeric-coated gelatin capsules containing sodium laurate in an oily medium to enhance insulin absorption (26). The scientists published their results after applying for a US patent in 1985 based on similar findings. Their study arguably represents the earliest success by any group using absorption enhancers and promoters.

Another study showed that the nonionic BRIJ surfactant–hydroxypropyl cellulose system was not effective in reducing plasma glucose levels in rabbits when compared with the salicylate–cellulose system. Two absorption promoters (BRIJ 35 and sodium salicylate) and two carriers (cellulose and hydroxypropyl cellulose) were used in the study (27).

In a separate study, sodium 5-methoxysalicylic acid was found to reduce plasma glucose levels in rats (28).

Bile salts have been used to influence the absorption of insulin through palmitic acid emulsion formulations. Nishikata et al. reported that the addition of different bile salts could enhance the reduction of plasma glucose levels, depending upon the type of bile salt used in the insulin–palmitic acid emulsion formulation (29).

In another study, soft gelatin capsules containing insulin and sodium salicylate as absorption enhancers were coated with a mixture of various Eudragit grades for use in rats. The administration of capsules significantly increased the hypoglycemic effect in rats (30). An attempt was made to modulate the epithelial structures by introducing zonula occludens toxin (ZOT) as an enhancer in an oral insulin formulation. ZOT produced a multifold increase in intestinal permeability and reduced plasma glucose levels in diabetic rats (31).

Cyclodextrin, which has the unique properties of solubilization and reducing enzymatic degradation, was used to increase the bioavailability of insulin in an alginate–chitson microsphere formulation (32).

The author of a review article about the use of absorption-enhancing agents elaborated on the positive outcome of using such agents with insulin for oral administration. The article discussed significant morphological changes in the cellular components of the gastrointestinal epithelium. The author also stressed that the study's limited success resulted from the use of a particular agent and its effect on the cell's morphology (33).

Studies of several low-molecular-weight absorption enhancers, including various bile salts, sodium salicylate, and ZOT, produced positive results. Although these agents sometimes damage intestinal tissue, one study confirmed that the damaging effect of bile acids does not occur consistently (34). Furthermore, studies to ensure the safe use of bile acids and other acids as absorption enhancers in different types of insulin formulations have not yet been completed.

A study conducted in Japan involving in situ intestinal experiments on rats indicates that a water-in-oil-in-water emulsion containing docosahexaenoic acid may improve insulin absorption without causing any serious damage to the intestinal epithelial cells (35).

Few studies discussed in this review article have raised questions about the toxicity and long-term clinical application of permeation enhancers in oral insulin formulations. Surface-acting agents act as permeation enhancers by causing exfoliation of the intestinal epithelium and may compromise its barrier functions (36).

Eley and Triumalashetty evaluated the suitability of several alkyglycosides as permeability enhancers. The study proved that most alkylglycosides are unsuitable because they cause varying degrees of cell membrane component solubilization. The cell membranes were badly disrupted and unable to recover fully all cell components above the concentration range of 0.01–0.1% used in the study (37).

One study conducted in 1996 tested sodium glycocholate (GC-Na), sodium taurocholate (TC-Na), sodium deoxycholate, EDTA, sodium salicylate, sodium caprate (Cap-Na), N-lauryl-beta-D-maltopyranoside (LM), diethyl maleate, and mixed micelles as absorption enhancers at a concentration of 20 mM to determine whether they caused intestinal damage in rats. Only GC-Na, TC-Na, and LM, which have low levels of toxicity at that concentration, promoted the absorption of phenol red and were effective absorption enhancers (38).

Intestinal in situ experiments also were conducted in rats using Labrasol (Gattefosse, NJ), a novel emulsifier and oral enhancing agent, and insulin. The results showed an increase in insulin absorption resulting from the enhancing effect of Labrasol (39).

A study published by Hosny et al. proved that sodium salicylate is a successful absorption promoter that reduced plasma glucose levels in diabetic dogs (40).

A chemical agent used in an oral insulin formulation to enhance or promote intestinal permeability should be nontoxic. Although such an agent may greatly increase intestinal mucosal permeability in in vitro and in situ animal studies, the increase does not ensure improved the oral bioavailability of a drug. Moreover, although chemical agents that enhance or promote intestinal permeability should be nontoxic, they may damage intestinal mucosa or the epithelium.

One of the main drawbacks of using permeation enhancers is that they attack the lipid structure's surface. An essential criterion is knowledge of the mechanism of absorption enhancement. The results described previously indicate the importance of optimizing the amount of absorption-enhancing agents in oral insulin formulation development in animal models. So far, not much data from studies of absorption enhancers and promoters in humans exist.

Combinational strategy. A group of researchers developed a strategy to use both an absorption promoter (sodium lauryl sulfate) and an enzyme inhibitor (aprotinin) separately in their formulation and reported a reduction in blood glucose levels in beagle dogs (41).

In the fall of 1993, leading pharmaceutical scientists participating in a symposium shared vital information about the gastrointestinal parameters that influence oral drugs. They reviewed various drug delivery approaches and concluded that certain chemical substances possess drug-absorption-enhancing properties. During the conference, the scientists suggested that most enhancers are amphiphilic compounds that include bile salts (e.g., deoxycholate) and nonionic (e.g., Triton X 100) and anionic detergents (e.g., sodium dodecyl sulfate). Several surfactants, lecithins, and medium-chain glycerides were also mentioned in the review study. Some of the work presented at the symposium was the continuation of the research studies mentioned earlier in this article. The data presented included the use of sodium cholate as an effective promoter for the absorption of insulin and showed a reduction in blood glucose levels after the insulin was orally delivered with sodium cholate, aprotinin, and a trypsin inhibitor. Several other absorption-enhancing agents that effectively promoted the absorption of insulin in the GIT were cited at this meeting (42).

Ziv and his colleagues demonstrated that insulin can be effective if given orally in conjunction with absorption promoters and enhancers. The group suggested a combination of sodium cholate and soybean trypsin inhibitor as an effective promoter of insulin absorption in diabetic dogs (43).

Sodium oleate and n-dodecyl-beta-D-maltopyranoside as absorption promoters and enhancers and sodium glycocholate and other protease inhibitors (aprotinin, bacitracin, and soybean trypsin inhibitor) improved the intestinal absorption of insulin in a colon-specific drug delivery system (44).

Another study by Hosny et al. showed that the successful incorporation of sodium salicylate in the presence of sodium cholate produced a significant reduction in plasma glucose levels in diabetic rabbits (45).

A study including several large peptide and protein drugs suggested that the inclusion of pharmaceutical additives such as absorption enhancers and protease inhibitors may improve the oral gastrointestinal absorption of insulin in rats (46). In 2002, researchers reported employing several absorption enhancers to protect orally delivered insulin-loaded polyethylcyanoacrylate nanospheres in diabetic rats. Almost all enhancers were found effective when used in insulin-nanosphere formulations. The same study also found that the addition of a protease inhibitor (aprotinin) significantly reduced plasma glucose levels in rats (47).

The latter studies suggest that the use of two different kinds of agents in oral insulin formulations has helped reduce toxic effects that were common when only one agent was used. More toxicological evaluation studies are needed, however, before combinational strategies can be tested in humans. Although a limited number of research studies has been performed on laboratory animals, the extent of absorption promotion of insulin by coadministration of absorption enhancers with various enzyme-inhibiting agents appears to have improved.

Conclusion

Absorption promoters and enhancers and enzyme inhibitors, either alone or in combination, can play an important role in improving the bioavailability of oral insulin in the GIT. In addition, the substances are effective in oral, nasal, buccal, and pulmonary drugs. Despite these agents' limitations, one should not overlook the successes achieved in various studies. Researchers should focus on minimizing the intestinal toxicity associated with these agents. The intestinal tissue samples taken from the animals used in the absorption studies should be examined using various histological techniques (e.g., microscopic examination) to assess the magnitude of tissue damage caused by the oral insulin formulation. If substantial damage to intestinal tissues is observed, the study should be stopped and reviewed for possible progression. The research studies discussed in this article mostly were performed on small animals (e.g., rats and rabbits) and reported success in enhancing the bioavailability of oral insulin. It is not yet clear how these techniques could produce similar outcomes in humans. It might take years before an oral insulin dosage form using any of these strategies becomes a reality.

Naushad M. Khan Ghilzai, PhD, is an associate professor in the Department of Pharmaceutical Sciences at LECOM School of Pharmacy, 1858 W. Grandview Blvd., Erie, PA 16509, tel. 814.860.5134, fax 814.860.5123, nghilzai@lecom.edu

References

1. Ziv et al., "Absorption of Protein via the Intestinal Walls: A Quantitative Model," Biochem. Pharmacol.39 (7), 1035–1039 (1987).

2. M.J. Katavoich and M.J. Meldrum, "Effect of Insulin and Acarbose Alone and in Combination with the Female Streptozotocin-Induced Diabetic Rat," J. Pharm. Sci. 82 (12), 1209–1213 (1993).

3. S. Fujii et al., "Promoting Effect of the New Chymotrypsin Inhibitor FK-448 on the Intestinal Absorption of Insulin in Rats and Dogs," J. Pharm. Pharmacol. 37 (8), 545–549 (1985).

4. Kidron et al., "The Absorption of Insulin from Various Regions of the Rat Intestine," Life Sci. 31 (25), 2837–2841 (1982).

5. Morisita et al., "Novel Oral Microspheres of Insulin with Protease Inhibitor Protecting from Enzymatic Degradation," Int. J. Pharm. 78 (1–3), 1–7 (1992).

6. Morisita et al., "Hypoglycemic Effect of Novel Oral Microspheres of Insulin with Protease Inhibitor in Normal and Diabetic Rats," Int. J. Pharm. 78 (1–3), 9–16 (1992).

7. R. Narayani, "Oral Delivery of Insulin Making Needles Needless," Trends Biomater. Artif. Organs. 15 (1), 12–16 (2001).

8. Kimura et al., "Oral Administration of Insulin as Poly (vinyl alcohol)-Gel Spheres in Diabetic Rats," Biol. Pharm. Bull. 19 (6), 897–900 (1996).

9. Yamamoto et al., "Effects of Various Protease Inhibitors on the Intestinal Absorption and Degradation of Insulin in Rats," Pharm. Res. 11 (10), 1496–1500 (1994).

10. C. Damage et al., "Poly (alkyl cyanoacrylate) Nanospheres for Oral Administration of Insulin," J. Pharm. Sci. 86 (12), 1403–1409 (1997).

11. R. Qi and Q.N. Ping, "Gastrointestinal Absorption Enhancement of Insulin by Administration of Enteric Microspheres and SNAC to Rats," J. Microencapsul. 21 (1), 37–45 (2004).

12. Chun-Lei Li and Ying-Jie Deng, "Oil-Based Formulations for Oral Delivery of Insulin," J. Pharm. Pharmacol. 56 (9), 1101–1107 (2004).

13. Lee et al., "Oral Route of Peptide and Protein Drug Delivery," in Peptide and Protein Drug Delivery, V.H. Lee, Ed. (Dekker, New York, NY, 1991), pp. 691–738.

14. Morishita et al., "Enteral Insulin Delivery by Microspheres in Three Different Formulations Using Eudragit L-100 and S-100," Int. J. Pharm. 91, 29–37 (1993).

15. E.A. Hosny, N.M. Khan Ghilzai, and M.M. Elmazar, "Promotion of Oral Insulin Absorption in Diabetic Rabbits Using pH-Dependent Coated Capsules Containing Sodium Cholate," Pharm. Acta Helv. 72 (4), 203–207 (1997).

16. M. Otsuki et al., "Effect of Synthetic Protease Inhibitor Camostate on Pancreatic Exocrine Function in Rats," Pancreas 2 (2), 164–169 (1987).

17. G. McCaffrey and J.C. Jamieson, "Evidence for the Role of a Cathepsin D-Like Activity in the Release of Gal Beta 1-4GlcNAcAlpha 2-6 Sialytransferase from Rat and Mouse Liver in Whole-Cell Systems," Comp. Biochem. Physiol. B. 104 (1), 91–94 (1993).

18. M.K. Marschuetz, P. Caliceti, and A. Bernkop-Schnuerch, "Design and In Vitro Evaluation of an Oral Delivery System for Insulin," Pharm. Res. 17 (12), 1468–1474 (2000).

19. H. Tozaki et al., "Enhanced Absorption of Insulin and (Asu(1,7)eel-calcitonin Using Novel Azopolymer-Coated Pellets for Colon-Specific Drug Delivery," J. Pharm. Sci. 90 (1), 89–97 (2001).

20. Agarwal et al., "Polyethyacrylate-Based Microparticulate of Insulin for Oral Delivery: Preparation and In Vitro Dissolution Stability in the Presence of Enzyme Inhibitors," Int. J Pharm. 225 (1–2), 31–39 (2001).

21. N.A. Plate et al., "Mucoadhesive Polymers with Immobilized Proteinase Inhibitors for Oral Administration of Protein Drugs," Biomaterials 23 (7), 1673–1677 (2002).

22. K. Katayama et al., "Double Liposomes: Hypoglycemic Effects of Liposomal Insulin on Normal Rats," Drug Dev. Ind. Pharm. 29 (7), 725–731 (2003).

23. H. Liu et al., "Potential Utility of Various Protease Inhibitors for Improving the Intestinal Absorption of Insulin in Rats," J. Pharm. Pharmacol. 55 (11), 1523–1529 (2003).

24. Touitou et al., "Effective Intestinal Absorption of Insulin in Diabetic Rats Using New Formulation Approach," J. Pharm. Pharmacol. 32 (2), 108–110 (1980).

25. M.S. Mesiha and H.I. El-Bitar, "Hypoglycemic Effect of Oral Insulin Preparation Containing Brij 35, 52, 58, or 92 and Stearic Acid," J. Pharm. Pharmacol. 33 (11) 733–734 (1981).

26. E. Touitou and A. Rubinstein, "Targeted Enteral Delivery of Insulin to Rats," Int. J. Pharm. 30 (2–3), 95–99 (1986).

27. Mesiha and Sidhom, "Increased Oral Absorption Enhancement of Insulin by Medium Viscosity Hydroxypropyl Cellulose," Int. J. Pharm. 114 (2), 137–140 (1995).

28. Nishikata et al., "Enhanced Intestinal Absorption of Insulin in Rats in the Presence of Sodium 5-Methoxysalicylate," Diabetes 30 (12), 1065–1067 (1981).

29. M.S. Mesiha, "Abstract 142," APhA Annual Meeting 1995, p. 158.

30. E.A. Hosny, N.M. Khan Ghilzai, and A.H. Al Dawalie, "Effective Intestinal Absorption of Insulin in Diabetic Rats Using Enteric Coated Capsules Containing Sodium Salicylate," Drug Dev. Ind. Res. 21 (13), 1583 (1995).

31. A. Fasano and S. Uzzau, "Modulation of Intestinal Tight Junctions by Zonula Occulens Toxin Permits Enteral Administration of Insulin and Other Macromolecules in an Animal Model," J. Clin. Invest. 99 (6), 1158–1164 (1997).

32. K.J. Dileep et al., "Modulation of Insulin Release from Chitosan–Alginate Microsphere," Trends Biomater. Artif. Organs 12 (2), 42–46 (1998).

33. J.A. Fix, "Strategies for Delivery of Peptides Utilizing Absorption Enhancing Agents," J. Pharm. Sci. 85 (12), 1282–1285 (1996).

34. A. Bernkop-Schnuerch et al., "The Use of Auxillary Agents to Improve the Mucosal Uptake of Peptides," Med. Chem. Reviews-Online. 1 (1), 1–10 (2004). http://www.bentham.org/mcro/mcro1-1.htm, accessed Jan. 2005

35. A. Suzuki et al., "Enhanced Colonic and Rectal Absorption of Insulin Using a Multiemulsion-Containing Eicosapentaenoic Acid and Docosahexaenoic Acid," J. Pharm. Sci. 87 (10), 1196–1202 (1998).

36. J. Hochman and P. Artursson, "Mechanisms of Absorption Enhancement and Tight Junction Regulation," J. Controlled Release 29 (3), 253–267 (1994).

37. J.G. Eley and P. Triumalashetty, "In Vitro Enhancement of Alkylglycosides as Permeability Enhancers," AAPS Pharmsci. Technol. 2 (3), article 19, 1–7 (2001).

38. T. Uchiyama et al., "Effectiveness and Toxicity Screening of Various Absorption Enhancers in the Large Intestine: Intestinal Absorption of Phenol Red and Protein and Phospholipid Release from the Intestinal Membrane," Biol. Pharm. Bull. 19 (12), 1618–1621 (1996).

39. S. Eaimtrakara et al., "Absorption-Enhancing Effect of Labrasol on the Intestinal Absorption of Insulin in Rats," J. Drug Target. 10 (3), 255–260 (2002).

40. Hosny et al., "Effect of Bioadhesive Polymers, Sodium Salicylate, Polyoxyethylene-9-Lauryl Ether, and Method of Preparation on the Relative Hypoglycemic Produced by Insulin Enteric-Coated Capsules in Diabetic Beagle Dogs," Drug Dev. Ind. Pharm. 28 (5), 563–570 (2002).

41. M.E.K. Kraeling and W.A. Ritschel, "Development of a Colonic Release Capsule Dosage Form and the Absorption of Insulin," Meth. Find. Exp. Clin. Pharmacol. 14 (3), 199–209 (1992).

42. Dressman et al., "Gastrointestinal Parameters that Influence Oral Medications," J. Pharm. Sci. 82 (9), 857–872 (1993).

43. Ziv et al., "Oral Administration of Insulin in Solid Form to Nondiabetic Dogs," J. Pharm. Sci. 83 (6), 792–794 (1994).

44. Tozaki et al., "Chitosan Capsules for Colon-Specific Drug Delivery: Improvement of Insulin Absorption from the Rat Colon," J. Pharm. Sci. 86 (9), 1016–1021 (1997).

45. E.A. Hosny et al., "Hypoglycemic Effect of Oral Insulin in Diabetic Rabbits Using pH–Dependent Coated Capsules Containing Sodium Salicylate without and with Sodium Cholate," Drug Dev. Ind. Pharm. 24 (3), 307–311 (1998).

46. A. Yamamoto, "Improvement of Transmucosal Absorption of Biologically Active Peptide Drugs," Yakugaku Zasshi 121 (12), 929–948 (2001).

47. M.A. Radwan and H.Y. Aboul-Eneim, "The Effect of Oral Absorption Enhancers on the In Vivo Performance of Insulin-Loaded Poly (ethylcyanoacrylate) Nanospheres in Diabetic Rats," J. Microencapsul. 19 (2), 225–235 (2002).