- Email: [email protected]
Oral delivery of macromolecules using intestinal patches: applications for insulin delivery
Journal of Controlled Release 98 (2004) 37 – 45 www.elsevier.com/locate/jconrel
Oral delivery of macromolecules using intestinal patches: applications for insulin delivery Kathryn Whitehead, Zancong Shen, Samir Mitragotri * Department of Chemical Engineering, University of California, Engineering II building, Santa Barbara, CA 93106, USA Received 4 March 2004; accepted 17 April 2004 Available online 8 June 2004
Abstract Oral drug delivery, though attractive compared to injections, cannot be utilized for the administration of peptides and proteins due to poor epithelial permeability and proteolytic degradation within the gastrointestinal tract. A novel method is described that utilizes mucoadhesive intestinal patches to deliver therapeutic doses of insulin into systemic circulation. Intestinal patches localize insulin near the mucosa and protect it from proteolytic degradation. In vitro experiments confirmed the secure adhesion of patches to the intestine and the release of insulin from the patches. In vivo experiments performed via jejunal administration showed that intestinal insulin patches with doses in the range of 1 – 10 U/kg induced dose-dependent hypoglycemia in normal rats with a maximum drop in blood glucose levels of 75% observed at a dose of 10 U/kg. These studies demonstrate that reduction in blood glucose levels comparable to that induced by subcutaneous injections can be achieved via enteral insulin absorption with doses only 2 – 10-fold higher than subcutaneous doses. D 2004 Elsevier B.V. All rights reserved. Keywords: Oral; Insulin; Hypoglycemia; Patch; Jejunum
1. Introduction Oral delivery is a convenient and patient-friendly route of drug administration compared to injections. However, the oral route cannot be utilized for macromolecules due to their low oral bioavailability [1]. This limitation is particularly aggravated for proteins and peptides because of their susceptibility to enzymatic degradation in the gastrointestinal tract and low permeability across the intestinal epithelium [2]. * Corresponding author. Tel.: +1-805-893-7532; fax: +1-805893-4731. E-mail address: [email protected] (S. Mitragotri). 0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2004.04.013
Several strategies have been proposed to boost oral insulin bioavailability, including permeation enhancers [3– 5], enzyme inhibitors [6], encapsulation technologies such as microspheres and nanoparticles [7,8], hydrogels [9], microemulsions [10,11] and liposomes [12,13]. Permeation enhancers, such as bile salts and fatty acids, increase the permeability of the epithelial cells of the gastrointestinal tract, thereby increasing oral bioavailability [14,15]. The use of protease inhibitors such as aprotinin and soybean trypsin inhibitor also aids protein absorption by reducing protein degradation in the intestinal tract [16]. Interestingly, some poly(acrylates) such as polycarbophil and chitosan derivatives have been shown to act as
38
K. Whitehead et al. / Journal of Controlled Release 98 (2004) 37–45
both permeation enhancers and enzyme inhibitors [17]. Poly(acrylates) are able to enhance paracellular epithelial transport by loosening the tight junctions of the mucosal epithelium [18] and inhibit calcium-dependent enzymes such as trypsin by competitively binding to local calcium ions [19]. Other delivery strategies have been primarily focused on utilization of microencapsulation technologies [20,21]. Micro/nanospheres can protect proteins from enzymatic degradation in the intestine, while nanospheres or nanocapsules can further facilitate protein transport across the epithelium by way of the Peyer’s patches that line the intestine [14]. Additionally, modified insulin, which results in higher enteral absorption, has been successfully delivered in human volunteers [2,22]. Despite significant research in this area, oral delivery of proteins still poses a significant scientific challenge. Many of these methods have been developed in the context of insulin delivery and they have shown improved insulin bioavailabilities over the basic liquid formulations. However, relatively high insulin doses are required to observe a physiologically significant reduction in blood glucose levels. Specifically, the doses required to reduce the blood glucose by 50% by some of the most promising oral delivery techniques have been reported in the range of 75– 100 U/kg [3]. These doses are quite high compared to a typical dose of f 1 U/kg required to induce the same degree of hypoglycemia via subcutaneous injections [3]. Therefore, many of the strategies surrounding oral insulin delivery have been cost prohibitive, as they require the administration of large quantities of insulin. Low insulin bioavailability has been one of the main hurdles in bringing oral insulin delivery into clinical practice. Herein, the use of intestinal patches for oral delivery of macromolecules is described and their capabilities are demonstrated using insulin, a very challenging model drug. Although the use of patchlike devices has been described in the literature for oral drug delivery [23 – 25], a design of intestinal patches that allows delivery of therapeutic doses of insulin has yet to be described. Herein, the fabrication and proof-of-concept evaluation of intestinal patches is described.
2. Experimental methods 2.1. Formulation of patches Patches were fabricated using a mixture of Carbopol 934 (BF Goodrich Cleveland, OH), pectin (Sigma, St. Louis, MO) and sodium carboxylmethylcellulose (SCMC, Aldrich, Milwaukee, WI) with a Carbopol/ pectin/SCMC weight ratio of 1:1:2. Bovine insulin (MW = 5733, 28.3 U/mg, Sigma) was added to this mixture such that the final insulin concentration in each patch was 0.25 –2.5 U/mg, which corresponds to a dry weight percentage of 0.275 –2.75% (w/w). A total of 100 mg of the mixture was compressed under a pressure of 0.5 – 4 tons using a hydraulic press (Carver Wabash, IN). This produced a 400 Am thick disk with a typical diameter of 13 mm. A hole punch was used to cut this disk into smaller disks with radii of 1– 4 mm. These disks were placed on a support and coated on all sides but one using a solution of 5% w/v ethylcellulose (EC, Sigma) in acetone. Acetone was evaporated at room temperature. This procedure produced an EC layer of about 50 Am. 2.2. In vitro perfusion experiments In vitro perfusion experiments were performed to observe dissolution of a capsule (loaded with patches) in the intestine and patch adhesion. In these experiments, a freshly harvested segment of porcine intestine was placed horizontally on a bench top and was connected to a tubing so that the lumen could be perfused with phosphate buffered saline (PBS, pH 7.4, 0.01 M) at a volumetric flow rate of 1 ml/min. A longitudinal incision was made in the intestine to observe the patches. A gelatin capsule containing three patches was placed in the intestine and was periodically imaged over a time frame of 30 min. 2.3. Unidirectional release of drug from patches Release of insulin from patches was measured in vitro into PBS. To distinguish drug release from the mucoadhesive side and the backing side of the patch, the patches were placed in a custom-designed diffusion cell. The cell was comprised of two chambers placed side by side with an opening provided between the chambers of about 3.14 mm2. A patch (4 mm in
K. Whitehead et al. / Journal of Controlled Release 98 (2004) 37–45
diameter) was placed between the two chambers and each chamber was filled with 6 ml PBS. Vacuum grease was used to seal the joints to avoid leakage of PBS. The amount of insulin released from either side of the patch into the solution was quantified by HPLC using a Lichrosorb RP18 column (MethaChem). The mobile phase consisted of acetonitrile: PBS (0.05 M Na2SO4, 0.05 M NaH2PO4, pH 3) at a ratio of 29:71. The flow rate was 1 ml/min and the absorption was measured at 280 nm. 2.4. Adhesion force measurement Experiments were performed to determine the adhesion force between the patch and the intestine. The adhesive force depended on the patch characteristics, intestinal fluid content, amount of time spent in the intestine and the method of measurement. Freshly harvested small intestine (Yorkshire pigs) was used in these studies. The intestine was rinsed with 100 ml PBS and then cut into 5 cm long loops. One end of the loop was tied off and different volumes of PBS (0.5, 1.0, 2.0, 3.0, 4.0 ml) were added to the lumen. Eight to ten intestinal patches (4 mm in diameter and 400 Am thick) were randomly inserted in the intestine sections. The other end of the section was also tied off. The whole intestinal section was placed on a rocker (Boekel Scientific, Feasterville, PA) and was shaken for 1 h. The force of adhesion between the patch and the intestine was measured using methods described by Bernkop-Schnu¨rch et al. [26]. Specifically, the intestine was longitudinally dissected and the mucosal surface (where the patches were still attached) was mounted onto a microbalance using clamps to secure it. A small piece of a plastic cylinder (2 cm in length and 1 mm in diameter) was super glued to the backing side of one of the patches on the mucosa. The other end of the cylinder was attached to a string and passed over a pulley. The cylinder was gradually pulled until the patch detached from the mucosa. The detachment force (force of adhesion) at which the adhesive bond between the patch and the mucosa failed was recorded. To assess whether the adhesion force of the patch was time-dependent, patches were inserted in the intestinal section filled with a fixed volume (1.0 ml) of PBS and incubated for 0.5, 1.0, 2.0, 3.0 or 4.0 h. At the end of the incubation period, patch detachment force was determined using methods discussed above.
39
2.5. Insulin delivery in non-diabetic rats All animal experiments were conducted under aseptic conditions using institutionally approved protocols. Male Sprague – Dawley rats, weighing 400 – 600 g and fasted for 12 – 16 h, were anesthetized using gas anesthesia (1.25 –4% isofluorane in oxygen). Due to the large size of the capsule used to contain the patches, direct oral administration of capsules was not performed. Instead, the efficacy of intestinal patches was assessed by a jejunal administration method, which has been effectively used for determining absorption efficiency in animals [27,28]. Briefly, rat intestine was exposed through a midline abdominal incision (2 cm). A small longitudinal incision (0.5 cm) was made about 5 cm from the proximal end of the small intestine. Three to six patches (1 mm in radius) containing insulin (0.25 – 1.2 U/patch, total dose of 1, 5 or 10 U/kg per animal) were inserted through the opening into the lumen. Alternatively, no. 9 capsules containing the patches (loaded with the same insulin doses mentioned above) as well as 10 mg SGC were delivered to the intestinal lumen in the same fashion. The patches or the capsules were inserted about 5 cm into the intestine to avoid direct contact of the patch or the capsule with the incision site. A total of 0.5 ml saline was added using a syringe following the patch or capsule insertion. The incisions were then sealed with surgical tissue glue (NEXABANDR, Veterinary Products Laboratories, Phoenix, AZ). Blood samples (0.2 ml) were collected from the tail vein or jugular vein every 1 h, up to 5 h, after the delivery of patches. Blood glucose levels were measured using a glucometer (ExciteR XL, Bayer, Elkhart, IN). Negative control experiments were performed by delivering 0.5 ml insulin solution (10 U/kg with and without 10 mg SGC), 0.5 ml PBS into which insulin patches were dissolved with and without 10 mg SGC (10 U/ kg), and blank patches (no insulin) with 10 mg SGC. All negative control experiments were performed under procedures identical to those used for insulin patch experiments. All solutions were added using a syringe. An additional negative control was performed in the form of no treatment. Positive controls were performed by subcutaneously injecting a pH 7 insulin solution (1 and 5 U/kg). In some experiments, blood samples (0.3 – 0.4 ml) were collected into heparinized tubes every hour for 5 h. Plasma insulin concentra-
40
K. Whitehead et al. / Journal of Controlled Release 98 (2004) 37–45
tions were measured using a bovine insulin ELISA kit (DRG Diagnostics, Marburg, Germany). 2.6. Tissue histology Histological studies were performed to evaluate the effect of patches on the intestine. In these experiments, capsules containing patches were administered and the animals were euthanized after 1 or 5 h. The intestine exposed to patches was excised, fixed in formalin, sectioned and stained using hematoxylin and eosin.
3. Results Intestinal patches utilized in this study were about 1 mm in radius and f 500 Am in thickness and consisted of a mucoadhesive matrix in which insulin was dispersed. The patches were coated on all sides but one with an impermeable polymeric layer. The patches were placed in a capsule (Fig. 1a). The capsule released the patches in the intestine (shown schematically in Fig. 1b), where they adhered to the intestinal mucosal layer (Fig. 1c and d) and delivered insulin across the epithelium. The adhesion of the patches localized insulin near the mucosa, thereby offering an increased concentration gradient for its transport. The protective layer minimized insulin loss into the intestinal lumen and promoted unidirectional diffusion of insulin towards the mucosa. It also minimized enzyme penetration into the patch, rendering further protection for polypeptide drugs like insulin. The efficacy of intestinal patches was evaluated by observing patch adhesion, insulin release, and insulininduced hypoglycemia in rats. A significant adhesion force (1.0 – 2.7 N/cm2) was observed between the patch and the mucosa over a period of 4 h, although there was no directed force to bring the patch in contact with the mucosa. Adhesion force decreased with an increase in fluid volume in the intestine (Fig. 2a). However, sufficient adhesion (>1 N/cm2) was observed even when 20% of the intestine was filled with fluids (an estimated value of fluid content in vivo). An adhesion force of 1 N/cm2 is quite significant and orders of magnitude greater than the detachment force arising from the weight of the patch (37 AN/cm2). Long-term contact of the patches with the
Fig. 1. (a) Images of capsules containing patches and a close up of patches themselves. The backing membrane is stained with sulforhodamine B to aid visualization. (b) A schematic representation of the mode of insulin delivery using intestinal patches. The patches are enclosed in an enteric-coated capsule and released in the intestine. The patches adhere to the intestinal mucosa and deliver insulin across the epithelium. (c) Adhesion of patches on porcine intestine in vitro. (d) A close-up of a patch adhering on rat intestine in vitro. The patch is in the early stages of swelling.
mucosa was confirmed by in vitro experiments, which demonstrated that about 90% of the patches adhered to the lumenal wall by their mucoadhesive side. No patches attached to the mucosa by their backing layer. When delivered in a capsule, the patches exited the capsule upon dissolution and adhered to the mucosa within 10 min (Fig. 2b). In vitro studies showed that insulin was released from patches over a period of f 4 h (Fig. 3), during which the patches dissolved. More than 99% of insulin was released from the mucoadhesive side of the patch, thus confirming the unidirectionality of the release. Upon adhesion to the intestinal mucosa, patches co-administered with sodium glycocholate (SGC, 10 mg/animal) successfully delivered insulin across the epithelium and induced dose-dependent hypoglycemia (Fig. 4a). Insulin patch doses of 5 and 10 U/kg decreased glucose levels by about 60% and 75%, respectively (open circles and closed circles).
K. Whitehead et al. / Journal of Controlled Release 98 (2004) 37–45
41
Fig. 2. (a) Adhesion force of the patches on intestinal mucosa. (b) Time-lapse photography of patch release from the capsule in the pig intestine. The capsule is about 1.5 cm long. Patches correspond to white circles in the figure. The total time elapsed between the first and the last image in this figure is 10 min.
No significant drop in glucose level was observed for a dose of 1 U/kg (closed triangles). Delivery of patches without insulin (blank patches) induced no hypoglycemia (closed squares). Additional controls including no treatment and delivery of 10 U/kg insulin solution with 10 mg SGC (open triangles and open
Fig. 3. Insulin release from the patches measured in vitro. (n = 3). No significant release was observed from the backing side. Error bars correspond to standard deviations.
circles, respectively, in Fig. 4b) also showed no hypoglycemia. At the end of the in vivo experiment, the intestine was excised to locate the patches. Five hours after their insertion into the intestine, only a few patches that had not yet dissolved could be found in the intestine. In some experiments, the animals were euthanized after 1 –2 h to locate the patches. Nearly all patches were found adhering to the mucosa and had undergone significant swelling. Patches that adhered did not move far, if at all, from the original location at which they were placed. Patches that did not adhere were fully dissolved after 1 –2 h. To compare hypoglycemia induced by 10 U/kg insulin patches (Fig. 4b, closed circles) with that induced by subcutaneous injections, rats were subcutaneously injected with 1 and 5 U/kg solutions of insulin (Fig. 4b, open squares and closed squares, respectively). Hypoglycemia induced by 10 U/kg intestinal patches fell between those induced by 1 and 5 U/kg subcutaneous injections, a result suggesting high insulin uptake from patches compared to that typically associated with oral insulin delivery via jejunal administration [29,30]. Intestinal insulin patches demonstrated that enteral insulin absorption can result in a reduction in blood glucose levels comparable to that induced by subcutaneous injections with doses only 2– 10-fold higher than subcutaneous doses.
42
K. Whitehead et al. / Journal of Controlled Release 98 (2004) 37–45
Fig. 4. (a) Blood glucose levels after intestinal administration of patches at three doses (10 U/kg—closed circles, 5 U/kg—open circles, 1 U/kg—closed triangles) and blank patches serving as a negative control (closed squares). A total of 10 mg of SGC was co-administered in all experiments. (n = 3 – 4). Error bars correspond to standard deviations. (b) Comparison of hypoglycemia induced by 10 U/kg patches administered with 10 mg SGC (closed circles) with subcutaneous injection (1 U/kg—open squares, 5 U/kg—closed squares). Negative controls were performed by inducing hypoglycemia by insulin solution (10 U/kg) co-administered with 10 mg SGC (open circles) and through no treatment (open triangles). (n = 3 – 4). Error bars correspond to standard deviations.
Although the addition of SGC enhanced insulin absorption, it was not fundamentally essential to the use of patches (Fig. 5a). Significant hypoglycemia (>50%) was induced by intestinal patches without SGC (10 U/kg, closed circles). Data on two additional controls, delivery of 10 U/kg insulin solution without
SGC (closed triangles) and a solution prepared by dissolution of patches in PBS (open squares) are also shown. None of these controls exhibited significant hypoglycemia. Patches also induced hypoglycemia when delivered from a capsule. SGC was included in the capsule
Fig. 5. (a) Hypoglycemia induced by patches (10 U/kg) without SGC (closed circles). Hypoglycemia induced by insulin solution (10 U/kg) without SGC (closed triangles) and patches dissolved in PBS (10 U/kg without SGC) (open squares) is also shown. (n = 3). Error bars correspond to standard deviations. (b) Hypoglycemia induced after administration of capsules containing insulin patches (10 U/kg) with SGC (open triangles). For comparison, hypoglycemia induced from direct administration of patches (10 U/kg) with SGC is shown by the closed circles. (n = 3). Error bars correspond to standard deviations.
K. Whitehead et al. / Journal of Controlled Release 98 (2004) 37–45
Fig. 6. Plasma insulin concentration after insulin patch administration (10 U/kg, closed circles). The open squares show plasma insulin concentrations after a subcutaneous injection of 1 U/kg. (n = 3 – 4). Error bars correspond to standard deviations.
for these experiments (Fig. 5b). The purpose of these experiments was to assess whether the capsules can dissolve and release the patches in a timely manner. Furthermore, since migration of enteric-coated capsules into the intestine has been extensively studied [31], the ability of the patches to exit from the capsule and adhere to the mucosa offers the next critical step in the validation of intestinal patches. The induction of hypoglycemia was slower in these experiments (open triangles) compared to that induced by directly delivered patches (closed circles). However, the minimum in blood glucose levels was identical to that obtained
43
by direct patch administration, thus confirming that the patches are able to exit the capsule, adhere to the mucosa and deliver insulin across the intestine. Plasma insulin concentrations were also measured after direct patch administration (Fig. 6). Plasma insulin levels achieved after patch delivery of 10 U/ kg (closed circles) were comparable to those achieved by a subcutaneous insulin injection of 1 U/kg (open squares, positive control). Histological studies showed no evidence of significant changes in the structure of the intestine exposed to patches compared to controls [Fig. 7a (control), b (1-h contact) and c (5-h contact)]. No significant presence of inflammatory cells was observed in samples. The villus structure was relatively normal. There was no evidence of necrosis or specific inflammation. No disruption of the epithelium was observed. Cellularity of the tissue was not significantly changed due to patch adhesion. Although further studies are necessary to arrive at a firm conclusion on the safety of intestinal patches, the histological studies did not indicate any significant safety issues associated with patches.
4. Discussion Oral delivery offers an attractive alternative to injections for the administration of proteins and peptides. It is a particularly attractive alternative for insulin administration due to: (i) reestablishment of the physiological ratio of portal to peripheral blood insulin concentration, thereby providing a more complete regulation of glucose metabolism in the liver [2]; (ii) proper long-term activation of the insulin-depen-
Fig. 7. (a) Histological section of a control rat intestine. (b) Histological section of rat intestine after contact with a patch for 1 h. (c) Histological section of rat intestine after contact with a patch for 5 h. All samples have been stained with hematoxylin and eosin.
44
K. Whitehead et al. / Journal of Controlled Release 98 (2004) 37–45
dent metabolic pathways of the liver that reduce medical complications associated with diabetes [2]; and (iii) improved patient compliance. Intestinal patches offer an attractive methodology for insulin delivery. Although patches are routinely used for drug delivery across the skin and buccal membrane [32,33], very little attention has been paid to their use in oral delivery [23,24] and none for insulin delivery. Intestinal patches offered significant hypoglycemia (>50%) at doses in the range of 5 –10 U/kg (Fig. 4a). The high efficacy of patches in insulin delivery is attributed to increased local concentration, reduced proteolytic protection and increased permeability. Although the insulin dose delivered to the animals is moderate (10 U/kg), the concentration of insulin in the patch is quite high ( f 400 U/cm3). This concentration is about 20-fold higher than that in controls. It is believed to be the primary reason for high insulin bioavailability. The benefit of the patch over a simple insulin solution is further enhanced since simple solutions get diluted over the gastrointestinal contents, thereby further reducing the concentration. At the same time, dilution also exposes insulin to proteolytic degradation. The important role of patch design is also evident from the lack of hypoglycemia observed after the delivery of a solution of dissolved patches (Fig. 5a), which shows that the efficacy of the patches originates from the engineering design and not the chemical composition of the patch. The data (Figs. 4a and 5a) also demonstrate that chemical enhancers work synergistically with the patches but are not necessarily an integral part of the intestinal patch technology. The co-administration of the SGC with the patches offers significant design flexibility. Safety studies indicated that patches did not induce adverse effects on the intestine. The mucosa under the intestine appeared normal. The ingredients used in the patch fabrication (Carbopol 934, SCMC and pectin) have been used previously in oral formulations and these substances (or their derivatives) are included in the list of inactive ingredients in FDA approved products [34]. SGC has also been used in a number of investigative oral products. Studies have reported that at the doses used in this study, SGC offers low toxicity as assessed by release of lactate dehydrogenase [4]. Several possibilities exist for clinical applications of intestinal patches, including delivery of basal
insulin doses over long periods of time, delivery of bolus doses over short periods of time and potentially a combination approach that delivers basal as well as boluses by using different types of patches. The kinetics of insulin absorption from patches depends on the adhesion time and patch dissolution time, both of which depend on the composition of the patch. By optimizing the patch composition, insulin absorption kinetics can be controlled over a wide range.
5. Conclusions Intestinal patches offer an effective mode for insulin delivery. The studies described here demonstrate that intestinal patches induce hypoglycemia comparable to that induced by subcutaneous injections with doses only 2 –10-fold higher than subcutaneous doses. With further research, intestinal patches could not only offer a novel methodology for the oral delivery of insulin, but for various other macromolecules, including growth hormones, heparin and vaccines.
Acknowledgements This work was supported by the American Diabetes Association. The authors acknowledge assistance from Elizabeth Chambers in animal experiments and Dr. L.D. McGill for assistance with histological studies. References [1] A. Bernkop-Schnurch, G. Walker, Multifunctional matrices for oral peptide delivery, Crit. Rev. Ther. Drug Carr. Syst. 18 (2001) 459 – 501. [2] J.G. Still, Development of oral insulin, Diabetes/Metab. Res. Rev. (2002 Jan. – Feb.) S29 – S37. [3] A. Fasano, S. Uzzau, Modulation of Intestinal tight junctions by zonula occludens toxin permits enteral administration of insulin and other macromolecules in an animal model, J. Clin. Invest. 99 (1997) 1158 – 1164. [4] T. Uchiyama, T. Sugiyama, Y. Quan, A. Kotani, N. Okada, T. Fujita, S. Muranishi, A. Yamamoto, Enhanced permeability of insulin across the rat intestinal membrane by various absorption enhancers: their intestinal mucosal toxicity and absorptionenhancing mechanism of n-lauryl-beta-D-maltopyranoside, J. Pharm. Pharmacol. 51 (1999) 1241 – 1250.
K. Whitehead et al. / Journal of Controlled Release 98 (2004) 37–45 [5] A. Yamamoto, T. Okagawa, A. Kotani, T. Uchiyama, T. Shimura, S. Tabata, S. Kondo, S. Muranishi, Effects of different absorption enhancers on the permeation of ebiratide, an ACTH analogue, across intestinal membranes, J. Pharm. Pharmacol. 49 (1997) 1057 – 1061. [6] A. Yamamoto, T. Taniguchi, K. Rikyuu, T. Tsuji, T. Fujita, M. Murakami, S. Muranishi, Effects of various protease inhibitors on the intestinal absorption and degradation of insulin in rats, Pharm. Res. 11 (1994) 1496 – 1500. [7] E. Mathiowitz, J.S. Jacob, Y.S. Jong, G.P. Carino, D.E. Chickering, P. Chaturvedi, C.A. Santos, K. Vijayaraghavan, S. Montgomery, M. Bassett, C. Morrell, Biologically erodable microspheres as potential oral drug delivery systems, Nature 386 (1997) 410 – 414. [8] X. Ma, G. Pan, Z. Lu, J. Hu, J. Bei, J. Jia, S. Wang, Preliminary study of oral polylactide microcapsulated insulin in vitro and in vivo, Diabetes Obes. Metab. 2 (2000) 243 – 250. [9] A. Lowman, M. Morishita, M. Kajita, T. Nagai, N. Peppas, Oral delivery of insulin using pH-responsive complexation gels, J. Pharm. Sci. 88 (1999) 933 – 937. [10] T. Ogiso, N. Funahashi, Y. Tsukioka, M. Iwaki, T. Tanino, Oral delivery of synthetic eel calcitonin, elcatonin in rats, Biol. Pharm. Bull. 24 (2001) 656 – 661. [11] S.T. Dogru, S. Calis, F. Oner, Oral multiple w/o/w emulsion formulation of a peptide salmon calcitonin: in vitro – in vivo evaluation, J. Clin. Pharm. Ther. 25 (2000) 435 – 443. [12] K. Iwanaga, S. Ono, K. Narioka, M. Kakemi, K. Morimoto, S. Yamashita, Y. Namba, N. Oku, Application of surfacecoated liposomes for oral delivery of peptide: effects of coating the liposome’s surface on the GI transit of insulin, J. Pharm. Sci. 88 (1999) 248 – 252. [13] M. Kisel, L. Kulik, I. Tsybovsky, A. Vlasov, M. Vorob’yov, E. Kholodova, Z. Zabarovskaya, Liposomes with phosphatidylethanol as a carrier for oral delivery of insulin: studies in the rat, Int. J. Pharm. 216 (2001) 105 – 114. [14] G.P. Carino, E. Mathiowitz, Oral insulin delivery, Adv. Drug Deliv. Rev. 35 (1999) 249 – 257. [15] B. Aungst, N. Rogers, Comparison of the effects of various transmucosal absorption promoters on buccal insulin delivery, Int. J. Pharm. 53 (1989) 227 – 235. [16] M. Morishita, I. Morishita, K. Takayama, Y. Machida, T. Nagai, Site-dependent effect of aprotinin, sodium caprate, Na2EDTA and sodium glycocholate on intestinal absorption of insulin, Biol. Pharm. Bull. 16 (1993) 68 – 72. [17] F. Madsen, N.A. Peppas, Complexation graft copolymer networks: swelling properties, calcium binding and proteolytic enzyme inhibition, Biomaterials 20 (1999) 1701 – 1708. [18] M. Thanou, J.C. Verhoef, H.E. Junginger, Oral drug absorption enhancement by chitosan and its derivatives, Adv. Drug Deliv. Rev. 52 (2001) 117 – 126. [19] H.L. Lueßen, C.-O. Rentel, A.F. Kotze, C.-M. Lehr, A.G. de Bohr, J.C. Verhoef, H.E. Junginger, Mucoadhesive polymers in peroral peptide drug delivery: IV. Polycarbophil and chito-
[20]
[21] [22]
[23] [24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
45
san are potent enhancers of peptide transport across intestinal mucosae in vitro, J. Control. Release 45 (1997) 15 – 23. H. Chung, J. Kim, J.Y. Um, I.C. Kwon, S.Y. Jeong, Selfassembled ‘‘nanocubicle’’ as a carrier for peroral insulin delivery, Diabetologia 45 (2002) 448 – 451. G. Carino, J. Jacob, E. Mathiowitz, Nanosphere based oral insulin delivery, J. Control. Release 65 (2000) 261 – 269. S.J. Milestein, H. Leipold, D. Sarubbi, A. Leone-Bay, G.W. Mlynek, J.R. Robinson, M. Kasimova, E. Freire, Partially unfolded proteins efficiently penetrate cell membranes— implications for oral drug delivery, J. Control. Release 53 (1998) 259 – 267. Z. Shen, S. Mitragotri, Intestinal patches for oral drug delivery, Pharm. Res. 19 (2002) 391 – 395. S. Eiamtrakarn, Y. Itoh, J. Kishimoto, Y. Yoshikawa, N. Shibata, M. Murakami, K. Takada, Gastrointestinal mucoadhesive patch system (GI-MAPS) for oral administration of G-CSF, a model protein, Biomaterials 23 (2002) 145 – 152. S.L. Tao, M.W. Lubeley, T.A. Desai, Bioadhesive poly(methyl methacrylate) microdevices for controlled drug delivery, J. Control Release 88 (2003) 215 – 228. A. Bernkop-Schnu¨rch, I. Apprich, Synthesis and evaluation of a modified mucoadhesive polymer protecting from alpha-chymotrypsinic degradation, Int. J. Pharm. 146 (1997) 247 – 254. S. Eiamtrakam, Y. Itoh, J. Kishimoto, Y. Yoshikawa, N. Shibata, M. Murakami, K. Takada, Gastrointestinal mucoadhesive patch system (GI-MAPS) for oral administration of G-CSF, a model protein, Biomaterials 23 (2002) 145 – 152. M. Thanou, J.C. Verhoef, J.H. Verheijden, H.E. Juginger, Intestinal absorption of octreotide using trimethyl chitosam chloride: studies in pigs, Pharm. Res. 18 (2001) 823 – 828. C. Michel, M. Aprahamian, L. Defontaine, P. Couvreur, C. Damge, The effect of site of administration in the gastrointestinal tract on the absorption of insulin from nanocapsules in diabetic rats, J. Pharm. Pharmacol. 43 (1991) 1 – 5. E.A. Hosny, N.M. Ghilzai, A.H. Al-Dhawalie, Effective intestinal absorption of insulin in diabetic rats using enteric coated capsules containing sodium aslicylate, Drug Dev. Ind. Pharm. 21 (1995) 1583 – 1589. E.T. Cole, R.A. Scott, A.L. Connor, I.R. Wilding, H.U. Petereit, C. Schminke, T. Beckert, D. Cade, Enteric coated HPMC capsules designed to achieve intestinal targetring, Int. J. Pharm. 231 (2002) 83 – 95. J.D. Bos, M.M. Meinardi, The 500 Dalton rule for the skin penetration of chemical compounds and drugs, Exp. Dermatol. 9 (2000) 165 – 169. C. Li, P.P. Bhatt, T.P. Johnston, Evaluation of a mucoadhesive buccal patch for delivery of peptides: in vitro screening of bioadhesion, Drug Dev. Ind. Pharm. 24 (1998) 919 – 926. F.D.A., Inactive ingredient search for approved drug products, http://www.accessdata.fda.gov/scripts/cder/iig/index.cfm (2003).
Oral delivery of macromolecules using intestinal patches: applications for insulin delivery Kathryn Whitehead, Zancong Shen, Samir Mitragotri * Department of Chemical Engineering, University of California, Engineering II building, Santa Barbara, CA 93106, USA Received 4 March 2004; accepted 17 April 2004 Available online 8 June 2004
Abstract Oral drug delivery, though attractive compared to injections, cannot be utilized for the administration of peptides and proteins due to poor epithelial permeability and proteolytic degradation within the gastrointestinal tract. A novel method is described that utilizes mucoadhesive intestinal patches to deliver therapeutic doses of insulin into systemic circulation. Intestinal patches localize insulin near the mucosa and protect it from proteolytic degradation. In vitro experiments confirmed the secure adhesion of patches to the intestine and the release of insulin from the patches. In vivo experiments performed via jejunal administration showed that intestinal insulin patches with doses in the range of 1 – 10 U/kg induced dose-dependent hypoglycemia in normal rats with a maximum drop in blood glucose levels of 75% observed at a dose of 10 U/kg. These studies demonstrate that reduction in blood glucose levels comparable to that induced by subcutaneous injections can be achieved via enteral insulin absorption with doses only 2 – 10-fold higher than subcutaneous doses. D 2004 Elsevier B.V. All rights reserved. Keywords: Oral; Insulin; Hypoglycemia; Patch; Jejunum
1. Introduction Oral delivery is a convenient and patient-friendly route of drug administration compared to injections. However, the oral route cannot be utilized for macromolecules due to their low oral bioavailability [1]. This limitation is particularly aggravated for proteins and peptides because of their susceptibility to enzymatic degradation in the gastrointestinal tract and low permeability across the intestinal epithelium [2]. * Corresponding author. Tel.: +1-805-893-7532; fax: +1-805893-4731. E-mail address: [email protected] (S. Mitragotri). 0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2004.04.013
Several strategies have been proposed to boost oral insulin bioavailability, including permeation enhancers [3– 5], enzyme inhibitors [6], encapsulation technologies such as microspheres and nanoparticles [7,8], hydrogels [9], microemulsions [10,11] and liposomes [12,13]. Permeation enhancers, such as bile salts and fatty acids, increase the permeability of the epithelial cells of the gastrointestinal tract, thereby increasing oral bioavailability [14,15]. The use of protease inhibitors such as aprotinin and soybean trypsin inhibitor also aids protein absorption by reducing protein degradation in the intestinal tract [16]. Interestingly, some poly(acrylates) such as polycarbophil and chitosan derivatives have been shown to act as
38
K. Whitehead et al. / Journal of Controlled Release 98 (2004) 37–45
both permeation enhancers and enzyme inhibitors [17]. Poly(acrylates) are able to enhance paracellular epithelial transport by loosening the tight junctions of the mucosal epithelium [18] and inhibit calcium-dependent enzymes such as trypsin by competitively binding to local calcium ions [19]. Other delivery strategies have been primarily focused on utilization of microencapsulation technologies [20,21]. Micro/nanospheres can protect proteins from enzymatic degradation in the intestine, while nanospheres or nanocapsules can further facilitate protein transport across the epithelium by way of the Peyer’s patches that line the intestine [14]. Additionally, modified insulin, which results in higher enteral absorption, has been successfully delivered in human volunteers [2,22]. Despite significant research in this area, oral delivery of proteins still poses a significant scientific challenge. Many of these methods have been developed in the context of insulin delivery and they have shown improved insulin bioavailabilities over the basic liquid formulations. However, relatively high insulin doses are required to observe a physiologically significant reduction in blood glucose levels. Specifically, the doses required to reduce the blood glucose by 50% by some of the most promising oral delivery techniques have been reported in the range of 75– 100 U/kg [3]. These doses are quite high compared to a typical dose of f 1 U/kg required to induce the same degree of hypoglycemia via subcutaneous injections [3]. Therefore, many of the strategies surrounding oral insulin delivery have been cost prohibitive, as they require the administration of large quantities of insulin. Low insulin bioavailability has been one of the main hurdles in bringing oral insulin delivery into clinical practice. Herein, the use of intestinal patches for oral delivery of macromolecules is described and their capabilities are demonstrated using insulin, a very challenging model drug. Although the use of patchlike devices has been described in the literature for oral drug delivery [23 – 25], a design of intestinal patches that allows delivery of therapeutic doses of insulin has yet to be described. Herein, the fabrication and proof-of-concept evaluation of intestinal patches is described.
2. Experimental methods 2.1. Formulation of patches Patches were fabricated using a mixture of Carbopol 934 (BF Goodrich Cleveland, OH), pectin (Sigma, St. Louis, MO) and sodium carboxylmethylcellulose (SCMC, Aldrich, Milwaukee, WI) with a Carbopol/ pectin/SCMC weight ratio of 1:1:2. Bovine insulin (MW = 5733, 28.3 U/mg, Sigma) was added to this mixture such that the final insulin concentration in each patch was 0.25 –2.5 U/mg, which corresponds to a dry weight percentage of 0.275 –2.75% (w/w). A total of 100 mg of the mixture was compressed under a pressure of 0.5 – 4 tons using a hydraulic press (Carver Wabash, IN). This produced a 400 Am thick disk with a typical diameter of 13 mm. A hole punch was used to cut this disk into smaller disks with radii of 1– 4 mm. These disks were placed on a support and coated on all sides but one using a solution of 5% w/v ethylcellulose (EC, Sigma) in acetone. Acetone was evaporated at room temperature. This procedure produced an EC layer of about 50 Am. 2.2. In vitro perfusion experiments In vitro perfusion experiments were performed to observe dissolution of a capsule (loaded with patches) in the intestine and patch adhesion. In these experiments, a freshly harvested segment of porcine intestine was placed horizontally on a bench top and was connected to a tubing so that the lumen could be perfused with phosphate buffered saline (PBS, pH 7.4, 0.01 M) at a volumetric flow rate of 1 ml/min. A longitudinal incision was made in the intestine to observe the patches. A gelatin capsule containing three patches was placed in the intestine and was periodically imaged over a time frame of 30 min. 2.3. Unidirectional release of drug from patches Release of insulin from patches was measured in vitro into PBS. To distinguish drug release from the mucoadhesive side and the backing side of the patch, the patches were placed in a custom-designed diffusion cell. The cell was comprised of two chambers placed side by side with an opening provided between the chambers of about 3.14 mm2. A patch (4 mm in
K. Whitehead et al. / Journal of Controlled Release 98 (2004) 37–45
diameter) was placed between the two chambers and each chamber was filled with 6 ml PBS. Vacuum grease was used to seal the joints to avoid leakage of PBS. The amount of insulin released from either side of the patch into the solution was quantified by HPLC using a Lichrosorb RP18 column (MethaChem). The mobile phase consisted of acetonitrile: PBS (0.05 M Na2SO4, 0.05 M NaH2PO4, pH 3) at a ratio of 29:71. The flow rate was 1 ml/min and the absorption was measured at 280 nm. 2.4. Adhesion force measurement Experiments were performed to determine the adhesion force between the patch and the intestine. The adhesive force depended on the patch characteristics, intestinal fluid content, amount of time spent in the intestine and the method of measurement. Freshly harvested small intestine (Yorkshire pigs) was used in these studies. The intestine was rinsed with 100 ml PBS and then cut into 5 cm long loops. One end of the loop was tied off and different volumes of PBS (0.5, 1.0, 2.0, 3.0, 4.0 ml) were added to the lumen. Eight to ten intestinal patches (4 mm in diameter and 400 Am thick) were randomly inserted in the intestine sections. The other end of the section was also tied off. The whole intestinal section was placed on a rocker (Boekel Scientific, Feasterville, PA) and was shaken for 1 h. The force of adhesion between the patch and the intestine was measured using methods described by Bernkop-Schnu¨rch et al. [26]. Specifically, the intestine was longitudinally dissected and the mucosal surface (where the patches were still attached) was mounted onto a microbalance using clamps to secure it. A small piece of a plastic cylinder (2 cm in length and 1 mm in diameter) was super glued to the backing side of one of the patches on the mucosa. The other end of the cylinder was attached to a string and passed over a pulley. The cylinder was gradually pulled until the patch detached from the mucosa. The detachment force (force of adhesion) at which the adhesive bond between the patch and the mucosa failed was recorded. To assess whether the adhesion force of the patch was time-dependent, patches were inserted in the intestinal section filled with a fixed volume (1.0 ml) of PBS and incubated for 0.5, 1.0, 2.0, 3.0 or 4.0 h. At the end of the incubation period, patch detachment force was determined using methods discussed above.
39
2.5. Insulin delivery in non-diabetic rats All animal experiments were conducted under aseptic conditions using institutionally approved protocols. Male Sprague – Dawley rats, weighing 400 – 600 g and fasted for 12 – 16 h, were anesthetized using gas anesthesia (1.25 –4% isofluorane in oxygen). Due to the large size of the capsule used to contain the patches, direct oral administration of capsules was not performed. Instead, the efficacy of intestinal patches was assessed by a jejunal administration method, which has been effectively used for determining absorption efficiency in animals [27,28]. Briefly, rat intestine was exposed through a midline abdominal incision (2 cm). A small longitudinal incision (0.5 cm) was made about 5 cm from the proximal end of the small intestine. Three to six patches (1 mm in radius) containing insulin (0.25 – 1.2 U/patch, total dose of 1, 5 or 10 U/kg per animal) were inserted through the opening into the lumen. Alternatively, no. 9 capsules containing the patches (loaded with the same insulin doses mentioned above) as well as 10 mg SGC were delivered to the intestinal lumen in the same fashion. The patches or the capsules were inserted about 5 cm into the intestine to avoid direct contact of the patch or the capsule with the incision site. A total of 0.5 ml saline was added using a syringe following the patch or capsule insertion. The incisions were then sealed with surgical tissue glue (NEXABANDR, Veterinary Products Laboratories, Phoenix, AZ). Blood samples (0.2 ml) were collected from the tail vein or jugular vein every 1 h, up to 5 h, after the delivery of patches. Blood glucose levels were measured using a glucometer (ExciteR XL, Bayer, Elkhart, IN). Negative control experiments were performed by delivering 0.5 ml insulin solution (10 U/kg with and without 10 mg SGC), 0.5 ml PBS into which insulin patches were dissolved with and without 10 mg SGC (10 U/ kg), and blank patches (no insulin) with 10 mg SGC. All negative control experiments were performed under procedures identical to those used for insulin patch experiments. All solutions were added using a syringe. An additional negative control was performed in the form of no treatment. Positive controls were performed by subcutaneously injecting a pH 7 insulin solution (1 and 5 U/kg). In some experiments, blood samples (0.3 – 0.4 ml) were collected into heparinized tubes every hour for 5 h. Plasma insulin concentra-
40
K. Whitehead et al. / Journal of Controlled Release 98 (2004) 37–45
tions were measured using a bovine insulin ELISA kit (DRG Diagnostics, Marburg, Germany). 2.6. Tissue histology Histological studies were performed to evaluate the effect of patches on the intestine. In these experiments, capsules containing patches were administered and the animals were euthanized after 1 or 5 h. The intestine exposed to patches was excised, fixed in formalin, sectioned and stained using hematoxylin and eosin.
3. Results Intestinal patches utilized in this study were about 1 mm in radius and f 500 Am in thickness and consisted of a mucoadhesive matrix in which insulin was dispersed. The patches were coated on all sides but one with an impermeable polymeric layer. The patches were placed in a capsule (Fig. 1a). The capsule released the patches in the intestine (shown schematically in Fig. 1b), where they adhered to the intestinal mucosal layer (Fig. 1c and d) and delivered insulin across the epithelium. The adhesion of the patches localized insulin near the mucosa, thereby offering an increased concentration gradient for its transport. The protective layer minimized insulin loss into the intestinal lumen and promoted unidirectional diffusion of insulin towards the mucosa. It also minimized enzyme penetration into the patch, rendering further protection for polypeptide drugs like insulin. The efficacy of intestinal patches was evaluated by observing patch adhesion, insulin release, and insulininduced hypoglycemia in rats. A significant adhesion force (1.0 – 2.7 N/cm2) was observed between the patch and the mucosa over a period of 4 h, although there was no directed force to bring the patch in contact with the mucosa. Adhesion force decreased with an increase in fluid volume in the intestine (Fig. 2a). However, sufficient adhesion (>1 N/cm2) was observed even when 20% of the intestine was filled with fluids (an estimated value of fluid content in vivo). An adhesion force of 1 N/cm2 is quite significant and orders of magnitude greater than the detachment force arising from the weight of the patch (37 AN/cm2). Long-term contact of the patches with the
Fig. 1. (a) Images of capsules containing patches and a close up of patches themselves. The backing membrane is stained with sulforhodamine B to aid visualization. (b) A schematic representation of the mode of insulin delivery using intestinal patches. The patches are enclosed in an enteric-coated capsule and released in the intestine. The patches adhere to the intestinal mucosa and deliver insulin across the epithelium. (c) Adhesion of patches on porcine intestine in vitro. (d) A close-up of a patch adhering on rat intestine in vitro. The patch is in the early stages of swelling.
mucosa was confirmed by in vitro experiments, which demonstrated that about 90% of the patches adhered to the lumenal wall by their mucoadhesive side. No patches attached to the mucosa by their backing layer. When delivered in a capsule, the patches exited the capsule upon dissolution and adhered to the mucosa within 10 min (Fig. 2b). In vitro studies showed that insulin was released from patches over a period of f 4 h (Fig. 3), during which the patches dissolved. More than 99% of insulin was released from the mucoadhesive side of the patch, thus confirming the unidirectionality of the release. Upon adhesion to the intestinal mucosa, patches co-administered with sodium glycocholate (SGC, 10 mg/animal) successfully delivered insulin across the epithelium and induced dose-dependent hypoglycemia (Fig. 4a). Insulin patch doses of 5 and 10 U/kg decreased glucose levels by about 60% and 75%, respectively (open circles and closed circles).
K. Whitehead et al. / Journal of Controlled Release 98 (2004) 37–45
41
Fig. 2. (a) Adhesion force of the patches on intestinal mucosa. (b) Time-lapse photography of patch release from the capsule in the pig intestine. The capsule is about 1.5 cm long. Patches correspond to white circles in the figure. The total time elapsed between the first and the last image in this figure is 10 min.
No significant drop in glucose level was observed for a dose of 1 U/kg (closed triangles). Delivery of patches without insulin (blank patches) induced no hypoglycemia (closed squares). Additional controls including no treatment and delivery of 10 U/kg insulin solution with 10 mg SGC (open triangles and open
Fig. 3. Insulin release from the patches measured in vitro. (n = 3). No significant release was observed from the backing side. Error bars correspond to standard deviations.
circles, respectively, in Fig. 4b) also showed no hypoglycemia. At the end of the in vivo experiment, the intestine was excised to locate the patches. Five hours after their insertion into the intestine, only a few patches that had not yet dissolved could be found in the intestine. In some experiments, the animals were euthanized after 1 –2 h to locate the patches. Nearly all patches were found adhering to the mucosa and had undergone significant swelling. Patches that adhered did not move far, if at all, from the original location at which they were placed. Patches that did not adhere were fully dissolved after 1 –2 h. To compare hypoglycemia induced by 10 U/kg insulin patches (Fig. 4b, closed circles) with that induced by subcutaneous injections, rats were subcutaneously injected with 1 and 5 U/kg solutions of insulin (Fig. 4b, open squares and closed squares, respectively). Hypoglycemia induced by 10 U/kg intestinal patches fell between those induced by 1 and 5 U/kg subcutaneous injections, a result suggesting high insulin uptake from patches compared to that typically associated with oral insulin delivery via jejunal administration [29,30]. Intestinal insulin patches demonstrated that enteral insulin absorption can result in a reduction in blood glucose levels comparable to that induced by subcutaneous injections with doses only 2– 10-fold higher than subcutaneous doses.
42
K. Whitehead et al. / Journal of Controlled Release 98 (2004) 37–45
Fig. 4. (a) Blood glucose levels after intestinal administration of patches at three doses (10 U/kg—closed circles, 5 U/kg—open circles, 1 U/kg—closed triangles) and blank patches serving as a negative control (closed squares). A total of 10 mg of SGC was co-administered in all experiments. (n = 3 – 4). Error bars correspond to standard deviations. (b) Comparison of hypoglycemia induced by 10 U/kg patches administered with 10 mg SGC (closed circles) with subcutaneous injection (1 U/kg—open squares, 5 U/kg—closed squares). Negative controls were performed by inducing hypoglycemia by insulin solution (10 U/kg) co-administered with 10 mg SGC (open circles) and through no treatment (open triangles). (n = 3 – 4). Error bars correspond to standard deviations.
Although the addition of SGC enhanced insulin absorption, it was not fundamentally essential to the use of patches (Fig. 5a). Significant hypoglycemia (>50%) was induced by intestinal patches without SGC (10 U/kg, closed circles). Data on two additional controls, delivery of 10 U/kg insulin solution without
SGC (closed triangles) and a solution prepared by dissolution of patches in PBS (open squares) are also shown. None of these controls exhibited significant hypoglycemia. Patches also induced hypoglycemia when delivered from a capsule. SGC was included in the capsule
Fig. 5. (a) Hypoglycemia induced by patches (10 U/kg) without SGC (closed circles). Hypoglycemia induced by insulin solution (10 U/kg) without SGC (closed triangles) and patches dissolved in PBS (10 U/kg without SGC) (open squares) is also shown. (n = 3). Error bars correspond to standard deviations. (b) Hypoglycemia induced after administration of capsules containing insulin patches (10 U/kg) with SGC (open triangles). For comparison, hypoglycemia induced from direct administration of patches (10 U/kg) with SGC is shown by the closed circles. (n = 3). Error bars correspond to standard deviations.
K. Whitehead et al. / Journal of Controlled Release 98 (2004) 37–45
Fig. 6. Plasma insulin concentration after insulin patch administration (10 U/kg, closed circles). The open squares show plasma insulin concentrations after a subcutaneous injection of 1 U/kg. (n = 3 – 4). Error bars correspond to standard deviations.
for these experiments (Fig. 5b). The purpose of these experiments was to assess whether the capsules can dissolve and release the patches in a timely manner. Furthermore, since migration of enteric-coated capsules into the intestine has been extensively studied [31], the ability of the patches to exit from the capsule and adhere to the mucosa offers the next critical step in the validation of intestinal patches. The induction of hypoglycemia was slower in these experiments (open triangles) compared to that induced by directly delivered patches (closed circles). However, the minimum in blood glucose levels was identical to that obtained
43
by direct patch administration, thus confirming that the patches are able to exit the capsule, adhere to the mucosa and deliver insulin across the intestine. Plasma insulin concentrations were also measured after direct patch administration (Fig. 6). Plasma insulin levels achieved after patch delivery of 10 U/ kg (closed circles) were comparable to those achieved by a subcutaneous insulin injection of 1 U/kg (open squares, positive control). Histological studies showed no evidence of significant changes in the structure of the intestine exposed to patches compared to controls [Fig. 7a (control), b (1-h contact) and c (5-h contact)]. No significant presence of inflammatory cells was observed in samples. The villus structure was relatively normal. There was no evidence of necrosis or specific inflammation. No disruption of the epithelium was observed. Cellularity of the tissue was not significantly changed due to patch adhesion. Although further studies are necessary to arrive at a firm conclusion on the safety of intestinal patches, the histological studies did not indicate any significant safety issues associated with patches.
4. Discussion Oral delivery offers an attractive alternative to injections for the administration of proteins and peptides. It is a particularly attractive alternative for insulin administration due to: (i) reestablishment of the physiological ratio of portal to peripheral blood insulin concentration, thereby providing a more complete regulation of glucose metabolism in the liver [2]; (ii) proper long-term activation of the insulin-depen-
Fig. 7. (a) Histological section of a control rat intestine. (b) Histological section of rat intestine after contact with a patch for 1 h. (c) Histological section of rat intestine after contact with a patch for 5 h. All samples have been stained with hematoxylin and eosin.
44
K. Whitehead et al. / Journal of Controlled Release 98 (2004) 37–45
dent metabolic pathways of the liver that reduce medical complications associated with diabetes [2]; and (iii) improved patient compliance. Intestinal patches offer an attractive methodology for insulin delivery. Although patches are routinely used for drug delivery across the skin and buccal membrane [32,33], very little attention has been paid to their use in oral delivery [23,24] and none for insulin delivery. Intestinal patches offered significant hypoglycemia (>50%) at doses in the range of 5 –10 U/kg (Fig. 4a). The high efficacy of patches in insulin delivery is attributed to increased local concentration, reduced proteolytic protection and increased permeability. Although the insulin dose delivered to the animals is moderate (10 U/kg), the concentration of insulin in the patch is quite high ( f 400 U/cm3). This concentration is about 20-fold higher than that in controls. It is believed to be the primary reason for high insulin bioavailability. The benefit of the patch over a simple insulin solution is further enhanced since simple solutions get diluted over the gastrointestinal contents, thereby further reducing the concentration. At the same time, dilution also exposes insulin to proteolytic degradation. The important role of patch design is also evident from the lack of hypoglycemia observed after the delivery of a solution of dissolved patches (Fig. 5a), which shows that the efficacy of the patches originates from the engineering design and not the chemical composition of the patch. The data (Figs. 4a and 5a) also demonstrate that chemical enhancers work synergistically with the patches but are not necessarily an integral part of the intestinal patch technology. The co-administration of the SGC with the patches offers significant design flexibility. Safety studies indicated that patches did not induce adverse effects on the intestine. The mucosa under the intestine appeared normal. The ingredients used in the patch fabrication (Carbopol 934, SCMC and pectin) have been used previously in oral formulations and these substances (or their derivatives) are included in the list of inactive ingredients in FDA approved products [34]. SGC has also been used in a number of investigative oral products. Studies have reported that at the doses used in this study, SGC offers low toxicity as assessed by release of lactate dehydrogenase [4]. Several possibilities exist for clinical applications of intestinal patches, including delivery of basal
insulin doses over long periods of time, delivery of bolus doses over short periods of time and potentially a combination approach that delivers basal as well as boluses by using different types of patches. The kinetics of insulin absorption from patches depends on the adhesion time and patch dissolution time, both of which depend on the composition of the patch. By optimizing the patch composition, insulin absorption kinetics can be controlled over a wide range.
5. Conclusions Intestinal patches offer an effective mode for insulin delivery. The studies described here demonstrate that intestinal patches induce hypoglycemia comparable to that induced by subcutaneous injections with doses only 2 –10-fold higher than subcutaneous doses. With further research, intestinal patches could not only offer a novel methodology for the oral delivery of insulin, but for various other macromolecules, including growth hormones, heparin and vaccines.
Acknowledgements This work was supported by the American Diabetes Association. The authors acknowledge assistance from Elizabeth Chambers in animal experiments and Dr. L.D. McGill for assistance with histological studies. References [1] A. Bernkop-Schnurch, G. Walker, Multifunctional matrices for oral peptide delivery, Crit. Rev. Ther. Drug Carr. Syst. 18 (2001) 459 – 501. [2] J.G. Still, Development of oral insulin, Diabetes/Metab. Res. Rev. (2002 Jan. – Feb.) S29 – S37. [3] A. Fasano, S. Uzzau, Modulation of Intestinal tight junctions by zonula occludens toxin permits enteral administration of insulin and other macromolecules in an animal model, J. Clin. Invest. 99 (1997) 1158 – 1164. [4] T. Uchiyama, T. Sugiyama, Y. Quan, A. Kotani, N. Okada, T. Fujita, S. Muranishi, A. Yamamoto, Enhanced permeability of insulin across the rat intestinal membrane by various absorption enhancers: their intestinal mucosal toxicity and absorptionenhancing mechanism of n-lauryl-beta-D-maltopyranoside, J. Pharm. Pharmacol. 51 (1999) 1241 – 1250.
K. Whitehead et al. / Journal of Controlled Release 98 (2004) 37–45 [5] A. Yamamoto, T. Okagawa, A. Kotani, T. Uchiyama, T. Shimura, S. Tabata, S. Kondo, S. Muranishi, Effects of different absorption enhancers on the permeation of ebiratide, an ACTH analogue, across intestinal membranes, J. Pharm. Pharmacol. 49 (1997) 1057 – 1061. [6] A. Yamamoto, T. Taniguchi, K. Rikyuu, T. Tsuji, T. Fujita, M. Murakami, S. Muranishi, Effects of various protease inhibitors on the intestinal absorption and degradation of insulin in rats, Pharm. Res. 11 (1994) 1496 – 1500. [7] E. Mathiowitz, J.S. Jacob, Y.S. Jong, G.P. Carino, D.E. Chickering, P. Chaturvedi, C.A. Santos, K. Vijayaraghavan, S. Montgomery, M. Bassett, C. Morrell, Biologically erodable microspheres as potential oral drug delivery systems, Nature 386 (1997) 410 – 414. [8] X. Ma, G. Pan, Z. Lu, J. Hu, J. Bei, J. Jia, S. Wang, Preliminary study of oral polylactide microcapsulated insulin in vitro and in vivo, Diabetes Obes. Metab. 2 (2000) 243 – 250. [9] A. Lowman, M. Morishita, M. Kajita, T. Nagai, N. Peppas, Oral delivery of insulin using pH-responsive complexation gels, J. Pharm. Sci. 88 (1999) 933 – 937. [10] T. Ogiso, N. Funahashi, Y. Tsukioka, M. Iwaki, T. Tanino, Oral delivery of synthetic eel calcitonin, elcatonin in rats, Biol. Pharm. Bull. 24 (2001) 656 – 661. [11] S.T. Dogru, S. Calis, F. Oner, Oral multiple w/o/w emulsion formulation of a peptide salmon calcitonin: in vitro – in vivo evaluation, J. Clin. Pharm. Ther. 25 (2000) 435 – 443. [12] K. Iwanaga, S. Ono, K. Narioka, M. Kakemi, K. Morimoto, S. Yamashita, Y. Namba, N. Oku, Application of surfacecoated liposomes for oral delivery of peptide: effects of coating the liposome’s surface on the GI transit of insulin, J. Pharm. Sci. 88 (1999) 248 – 252. [13] M. Kisel, L. Kulik, I. Tsybovsky, A. Vlasov, M. Vorob’yov, E. Kholodova, Z. Zabarovskaya, Liposomes with phosphatidylethanol as a carrier for oral delivery of insulin: studies in the rat, Int. J. Pharm. 216 (2001) 105 – 114. [14] G.P. Carino, E. Mathiowitz, Oral insulin delivery, Adv. Drug Deliv. Rev. 35 (1999) 249 – 257. [15] B. Aungst, N. Rogers, Comparison of the effects of various transmucosal absorption promoters on buccal insulin delivery, Int. J. Pharm. 53 (1989) 227 – 235. [16] M. Morishita, I. Morishita, K. Takayama, Y. Machida, T. Nagai, Site-dependent effect of aprotinin, sodium caprate, Na2EDTA and sodium glycocholate on intestinal absorption of insulin, Biol. Pharm. Bull. 16 (1993) 68 – 72. [17] F. Madsen, N.A. Peppas, Complexation graft copolymer networks: swelling properties, calcium binding and proteolytic enzyme inhibition, Biomaterials 20 (1999) 1701 – 1708. [18] M. Thanou, J.C. Verhoef, H.E. Junginger, Oral drug absorption enhancement by chitosan and its derivatives, Adv. Drug Deliv. Rev. 52 (2001) 117 – 126. [19] H.L. Lueßen, C.-O. Rentel, A.F. Kotze, C.-M. Lehr, A.G. de Bohr, J.C. Verhoef, H.E. Junginger, Mucoadhesive polymers in peroral peptide drug delivery: IV. Polycarbophil and chito-
[20]
[21] [22]
[23] [24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
45
san are potent enhancers of peptide transport across intestinal mucosae in vitro, J. Control. Release 45 (1997) 15 – 23. H. Chung, J. Kim, J.Y. Um, I.C. Kwon, S.Y. Jeong, Selfassembled ‘‘nanocubicle’’ as a carrier for peroral insulin delivery, Diabetologia 45 (2002) 448 – 451. G. Carino, J. Jacob, E. Mathiowitz, Nanosphere based oral insulin delivery, J. Control. Release 65 (2000) 261 – 269. S.J. Milestein, H. Leipold, D. Sarubbi, A. Leone-Bay, G.W. Mlynek, J.R. Robinson, M. Kasimova, E. Freire, Partially unfolded proteins efficiently penetrate cell membranes— implications for oral drug delivery, J. Control. Release 53 (1998) 259 – 267. Z. Shen, S. Mitragotri, Intestinal patches for oral drug delivery, Pharm. Res. 19 (2002) 391 – 395. S. Eiamtrakarn, Y. Itoh, J. Kishimoto, Y. Yoshikawa, N. Shibata, M. Murakami, K. Takada, Gastrointestinal mucoadhesive patch system (GI-MAPS) for oral administration of G-CSF, a model protein, Biomaterials 23 (2002) 145 – 152. S.L. Tao, M.W. Lubeley, T.A. Desai, Bioadhesive poly(methyl methacrylate) microdevices for controlled drug delivery, J. Control Release 88 (2003) 215 – 228. A. Bernkop-Schnu¨rch, I. Apprich, Synthesis and evaluation of a modified mucoadhesive polymer protecting from alpha-chymotrypsinic degradation, Int. J. Pharm. 146 (1997) 247 – 254. S. Eiamtrakam, Y. Itoh, J. Kishimoto, Y. Yoshikawa, N. Shibata, M. Murakami, K. Takada, Gastrointestinal mucoadhesive patch system (GI-MAPS) for oral administration of G-CSF, a model protein, Biomaterials 23 (2002) 145 – 152. M. Thanou, J.C. Verhoef, J.H. Verheijden, H.E. Juginger, Intestinal absorption of octreotide using trimethyl chitosam chloride: studies in pigs, Pharm. Res. 18 (2001) 823 – 828. C. Michel, M. Aprahamian, L. Defontaine, P. Couvreur, C. Damge, The effect of site of administration in the gastrointestinal tract on the absorption of insulin from nanocapsules in diabetic rats, J. Pharm. Pharmacol. 43 (1991) 1 – 5. E.A. Hosny, N.M. Ghilzai, A.H. Al-Dhawalie, Effective intestinal absorption of insulin in diabetic rats using enteric coated capsules containing sodium aslicylate, Drug Dev. Ind. Pharm. 21 (1995) 1583 – 1589. E.T. Cole, R.A. Scott, A.L. Connor, I.R. Wilding, H.U. Petereit, C. Schminke, T. Beckert, D. Cade, Enteric coated HPMC capsules designed to achieve intestinal targetring, Int. J. Pharm. 231 (2002) 83 – 95. J.D. Bos, M.M. Meinardi, The 500 Dalton rule for the skin penetration of chemical compounds and drugs, Exp. Dermatol. 9 (2000) 165 – 169. C. Li, P.P. Bhatt, T.P. Johnston, Evaluation of a mucoadhesive buccal patch for delivery of peptides: in vitro screening of bioadhesion, Drug Dev. Ind. Pharm. 24 (1998) 919 – 926. F.D.A., Inactive ingredient search for approved drug products, http://www.accessdata.fda.gov/scripts/cder/iig/index.cfm (2003).