Characterization of insulin protection properties of complexation hydrogels in gastric and intestinal enzyme fluids

Journal of Controlled Release 112 (2006) 343 – 349 www.elsevier.com/locate/jconrel

Characterization of insulin protection properties of complexation hydrogels in gastric and intestinal enzyme fluids Tetsuo Yamagata a , Mariko Morishita a,⁎, Nikhil J. Kavimandan b , Koji Nakamura a,1 , Yu Fukuoka a , Kozo Takayama a , Nicholas A. Peppas b b

a Department of Pharmaceutics, Hoshi University, Ebara 2-4-41, Shinagawa, Tokyo, 142-8501, Japan Departments of Chemical and Biomedical Engineering, and Division of Pharmaceutics, The University of Texas at Austin, Austin, TX 78712-0231, USA

Received 5 September 2005; accepted 10 March 2006 Available online 2 May 2006

Abstract The objective of this study was to elucidate the mechanisms contributing to oral bioavailability of insulin by poly(methacrylic acid grafted with poly(ethylene glycol)) (P(MAA-g-EG)) hydrogels using the gastric and intestinal fluids from rats. P(MAA-g-EG) hydrogels successfully protected the incorporated insulin from enzymatic degradation by forming interpolymer complexes in the gastric fluid. The hydrogels also showed the insulin protection ability by itself. In the intestinal fluid, P(MAA-g-EG) hydrogels significantly decreased the insulin degradation rate and calcium ion levels, while protein levels was not changed. Insulin protecting effects were dependent on the fraction of the carboxylic group in the polymer networks. Moreover, the insulin degradation inhibitory effect was significantly correlated with Ca2+ deprivation ability of P(MAA-g-EG) hydrogels in the intestinal fluid, implying that the Ca2+ deprivation ability plays an important role in the inhibition of the intestinal enzyme activities. Insulin-loaded P(MAA-g-EG) (ILPs) hydrogels showed a rapid and almost complete insulin release even in the presence of intestinal proteases. These results suggested that the insulin protection ability of the hydrogels contributed to improve oral insulin absorption and that P (MAA-g-EG) hydrogels can be an excellent carrier for protecting insulin during their transit through the GI tract. © 2006 Elsevier B.V. All rights reserved. Keywords: Insulin; Complexation hydrogel; Enzyme inhibition; Ca2+ deprivation

1. Introduction In recent years, numerous researchers have attempted to fabricate oral delivery system for therapeutic peptides and proteins, because the oral delivery route is the most acceptable route of administration, being more natural and less invasive. One of the most desirable characteristics of oral delivery of peptides and proteins is the ability to protect these drugs from proteolytic attack during their transit through the gastrointestinal (GI) tract. In the absence of any mechanisms to prevent protein digestion in the GI tract, the bioavailability of orally administered therapeutic proteins is generally quite low [1,2]. Therefore, in order to overcome the enzymatic barriers, various ⁎ Corresponding author. Tel./fax: +81 3 5498 5782. E-mail address: [email protected] (M. Morishita). 1 Present affiliation: R&D Center, Terumo Corporation, Inokuchi 1500, Nakai-machi, Ashigarakami-gun, Kanagawa 259-0151, Japan. 0168-3659/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2006.03.005

approaches were developed such as the utilization of enzyme inhibitors [3,4] and functional polymers [5,6]. Novel complexation hydrogels of poly(methacrylic acid grafted with poly(ethylene glycol)) (P(MAA-g-EG)), which have exhibit pH-dependent swelling behavior due to the formation/dissociation of interpolymer complexes, have been thought to be potential carriers for insulin via the oral route [7– 9]. The interpolymeric hydrogen-bonding complexes between etheric groups of the graft PEG chains and the acid protons of the PMAA network at low pH result in low mesh size of the network. The low mesh size severely limits the diffusion of drug into the gastric environment, and the drug is protected within the network structure. On exposure to the near-neutral pH environment of the intestine, the network swells to a high degree and results in rapid release of the entrapped drug. In previous studies, the oral administration of insulin-loaded P(MAA-g-EG) hydrogels demonstrated significant insulin

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absorption and blood glucose reduction in healthy and diabetic rats [10–12]. This implies that P(MAA-g-EG) hydrogels have a direct mucosal absorption enhancing effect on insulin. In fact, Madsen and Peppas [13] showed that these complexation hydrogels could bind Ca2+ at the pH of the small intestine and inhibited the activity of the Ca2+-dependent intestinal enzymes. However, the precise mechanisms behind the improvement in oral insulin bioavailability have not been fully elucidated. Understanding the mechanisms by which the hydrogel carriers can prevent insulin from getting degraded in the GI tract can help in formulating strategies to improve the oral bioavailability of insulin using complexation hydrogels. Therefore, the aim of this study was to evaluate the ability of P(MAA-g-EG) hydrogels to protect the incorporated insulin from proteolytic degradation in the gastric and intestinal enzyme fluids. 2. Materials and methods 2.1. Materials Methacrylic acid (MAA), dimethoxy propyl acetophenone (DMPA) and lactate dehydrogenase reagent were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Poly(ethylene glycol) (PEG) monomethacrylate (PEGMA, with PEG of molecular weight 1000, namely 23 repeating units of EG) and tetraethylene glycol dimethacrylate (TEGDMA) were obtained from Polysciences Inc. (Warrington, PA, USA). Carbopol 934P was gifts from B.F. Goodrich, Cleveland, OH, USA. Recombinant human insulin (26 units/mg) was purchased from Wako Pure Chemical Industries Co., Ltd (Osaka, Japan). All other chemicals were of analytical grade and commercially available. 2.2. Hydrogel synthesis P(MAA-g-EG) hydrogels were prepared by a free-radical solution UVpolymerization of MAA and PEGMA with PEG of molecular weight 1000 as described elsewhere [14]. 1 wt.% of TEGDMA and 2 wt.% of DMPA were used as a crosslinking agent and a photoinitiator, respectively. The P(MAA-g-EG) hydrogels were prepared with initial monomer feed ratios of 1:1, 4:1 and 1:0 MAA/EG repeating units and abbreviated as P (MAA-g-EG)(1:1), P(MAA-g-EG)(4:1) and P(MAA-g-EG) (1:0), respectively. All the hydrogels were crushed and sieved to < 53 μm sizes.

(ILP) microparticles were dried under vacuum and stored at 4 °C until further use. The degree of loading was determined using HPLC as described previously [14]. All the ILPs were loaded so as to contain 60 μg of amount of insulin in 1 mg ILP. 2.4. Preparation of the gastric and intestinal fluids This research complied with the regulations of the Committee on Ethics in the Care and Use of Laboratory Animals of Hoshi University. Male Wistar rats (200–300 g) were purchased from Sankyo Lab Service Co., Ltd. (Tokyo, Japan). Animals were housed in rooms controlled between 23 ± 1 °C and 55 ± 5% relative humidity and allowed free access to water and food during acclimatization. The rats were fasted for 24 h and anesthetized by i.p. injection of 50 mg/kg sodium pentobarbital. The rats were secured to the operating table in a supine position and midline abdominal incision was performed. The stomach was excised from the rats, and ligated at both ends. A sonde needle was carefully inserted into the stomach from the pylorus. Five milliliters of HCl–KCl buffer (pH 1.2) was injected into the stomach and then the gastric contents were collected as the gastric fluid [15]. The total protein concentration of the purified gastric fluid was determined (Protein Quantification Kit, Dojindo Molecular Technologies, Inc. Gaithersburg, MD, USA). To obtain the intestinal fluid, a sonde needle was inserted into the upper portion of the small intestine and the intestine was cannulated on the lower side (length = 20 cm) to remove the intestinal fluid. The small intestinal contents were washed out with 20 ml of PBS. The intestinal fluid has high lipid contents which may interfere with the HPLC analysis of insulin. Hence this efflux was treated with two volumes of methylene chloride to remove the lipids [16]. The purification was repeated by 5 times. In the case of the gastric fluid, the lipid content is very low, so this step could be omitted. The total protein concentration of the purified intestinal fluid was determined using the Protein Quantification Kit. To compare the results from different set of insulin degradation studies, the concentration of the gastric or intestinal enzyme fluid was adjusted to yield 50% degradation of the initial insulin level in 30 min. For protein incorporation study and calcium deprivation study, the intestinal fluid was diluted with PBS to yield a protein concentration of 400–500 μg/ml. 2.5. Insulin degradation study in the gastric fluid

2.3. Preparation of insulin loaded P(MAA-g-EG) microparticles (ILP) Insulin loading was performed by equilibrium partitioning in PBS as described previously [14]. Briefly, 140 mg of P(MAAg-EG) microparticles (MAA/EG = 1:1, 4:1, 1:0) were added to 20 ml of insulin solution containing of specific amounts of human insulin in phosphate-buffered saline (PBS, pH 7.4). The mixture was stirred for 2 h. The particles were collapsed by addition of 20 ml of 0.1 M HCl. The loading mixture was then filtered through 1 μm pore size filter (47 mm: Advantec Toyo Co., Ltd, Tokyo, Japan). The insulin loaded P(MAA-g-EG)

Two milliliters of the gastric fluid was incubated at 37 °C for 15 min. Ten milligrams of ILP (MAA/EG = 1:1, 4:1, 1:0) was added to the enzyme solution and the mixture was incubated at 37 °C for 1 h. Following incubation, the solution was centrifuged at 3000 rpm for 5 min and the supernatant was removed to separate the particles. Forty milliliters of PBS was then added to the particles and the total amount of insulin released from the particles in 3 h was measured by HPLC. For the control experiments, the amount of insulin equal to the amount incorporated in ILPs was exposed to 2 ml of gastric fluid at 37 °C for 1 h.

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In addition, 1.9 ml of the gastric fluid was incubated at 37 °C for 15 min. Ten milligrams of P(MAA-g-EG) microparticles (MAA/EG = 1:1, 4:1, 1:0) were added to the enzyme solution and immediately after the addition of microparticles, 0.1 ml of insulin solution (4 mg/ml) was added to this solution. Samples of 50 μl were withdrawn at predetermined time intervals after addition of the insulin solution. The enzyme activity in the samples was completely inhibited by addition of 50 μl of icecold acetonitrile solution (acetonitrile: 0.1% TFA = 31:69, v/v). The samples were then analyzed by HPLC to determine the amount of insulin remaining in the solution at different time intervals. Insulin remaining as a percentage of the initial insulin present in the solution was plotted against time and compared with the control (without polymer). The degradation rate constant was determined from the slope of the semi log plot of percentage insulin remaining plotted against time.

After adding 50 μl of ice-cold acetonitrile solution to terminate the reaction, the samples were then analyzed by HPLC. The fractional release of insulin from ILPs, defined as the ratio of the amount released at any time (Mt) to the total amount released after 60 min (M∞) was calculated. Additionally, the intestinal fluid added Ca2+ (Ca2+ concentration: 0.5 mM) was prepared by diluting intestinal fluid with PBS containing Ca2+. Twenty milliliters of the intestinal fluid added Ca2+ was incubated at 37 °C for 15 min. Ten milligrams of ILPs (MAA/EG = 1:1) were added to the enzyme solution and incubated at 37 °C. At predetermined times up until 60 min, samples of 50 μl were withdrawn from the incubation mixture. After adding 50 μl of ice-cold acetonitrile solution to terminate the reaction, the samples were then analyzed by HPLC.

2.6. Insulin degradation study in the intestinal fluid

Each value was expressed as the mean ± S.D. For group comparisons, the one-way layout ANOVA with duplication was applied. Significant differences in the mean values were evaluated by the Student's unpaired t-test. A p value of less than 0.05 was considered significant.

2.7. Protein incorporation study in the intestinal fluid Two milliliters of the intestinal fluid was incubated at 37 °C for 15 min. Ten milligrams of P(MAA-g-EG) microparticles (MAA/EG = 1:1, 4:1, 1:0) or Carbopol 934P were added to the enzyme solution and incubated at 37 °C for 1 h. The concentration of protein in the fluid was determined by the Protein Quantification Kit. 2.8. Ca2+ deprivation study in the intestinal fluid Two milliliters of the intestinal fluid was incubated at 37 °C for 15 min. Ten milligrams of P(MAA-g-EG) microparticles (MAA/EG = 1:1, 4:1, 1:0) or Carbopol 934P were added to the intestinal fluid and incubated at 37 °C for 1 h. The concentration of calcium in the fluid was determined using Calcium test kit (Wako Pure Chemical Industries Co., Ltd, Osaka, Japan).

3. Results and discussion 3.1. Insulin protection by complexation hydrogels in the gastric fluid In order to examine the protection ability of the complexation hydrogels in the harsh environment of the stomach, the native insulin and ILP were treated with the gastric fluid. After the treatment in the gastric fluid, the amount of insulin remaining inside the particles was determined. The results are shown in Fig. 1 for hydrogels with different molar ratios of MAA and EG repeating units. The unprotected insulin in the control experiment was significantly degraded in the gastric fluid and only 20% of the insulin remained after 1 h. In contrast, more than 80% of the insulin remained undegraded in all the three ILPs. It is evident that all the three hydrogel compositions were effective in Insulin remaining (% of initial concentration)

Two milliliters of the intestinal fluid was incubated at 37 °C for 15 min. Ten milligrams of P(MAA-g-EG) microparticles (MAA/EG = 1:1, 4:1, 1:0) or Carbopol 934P were added to the enzyme solution and incubated at 37 °C for 1 h. Following incubation, the particles were separated by centrifugation at 3000 rpm for 5 min and supernatant was collected. The pH of the fluid was adjusted to 7.4, and insulin solution was added, which contained 2 mg/ml as the final insulin concentration in the solution. At predetermined times up to 30 min samples of 50 μl were withdrawn from the incubation mixture. The samples were analyzed after adding 50 μl of ice-cold acetonitrile solution to terminate the reaction. The samples were then analyzed by HPLC.

2.10. Statistical analysis

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Twenty milliliters of the intestinal fluid was incubated at 37 °C for 15 min. Ten milligrams of ILPs (MAA/EG = 1:1, 4:1, 1:0) were added to the enzyme solution and samples of 50 μl were withdrawn at predetermined time intervals up to 60 min.

Fig. 1. Insulin remaining in ILPs after exposure to gastric fluid at 37 °C for 1 h. Control indicates only insulin was exposed to gastric fluid. Each value represents the mean ± S.D. Statistically significant difference: p < 0.01, ⁎⁎ against control.

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Fig. 2. Degradation profiles of insulin as a function of time in the gastric fluid at 37 °C for 1 h. Keys: insulin solution (control) (□), insulin solution with P (MAA-g-EG) (1:1) (♦), (4:1) (▴) and (1:0) (●). Each value represents the mean ± S.D.

(a) Degradation rate constant (x10-2/min)

protecting the entrapped insulin from enzymatic degradation. Morishita et al. [14] reported that in pH 1.2 media, only 6% of the insulin was released from the P(MAA-g-EG) (1:1) as the hydrogel was in a collapsed state, suggesting that the ability of the P(MAA-g EG) hydrogel to protect the entrapped insulin can be mainly attributed to the ineffective diffusion of insulin out of the hydrogels at low pH. On the other hand, it has been reported that both of the P(MAA-g-EG) (4:1) and (1:0) released almost 30–40% of the insulin in 1 h at pH 1.2 media [14]. However, in the present studies, even the hydrogels with 4:1 or 1:0 molar ratio of MAA/EG repeating units effectively prevented the degradation of entrapped insulin. Although the experimental conditions in the present study, such as the release volume, were different from that of Morishita's study [14], these results imply that other factors may play a critical role in the insulin protection ability. To further characterize the protection ability of the hydrogels, we investigated whether P(MAA-g-EG) microparticles could directly inhibit the gastric enzymes, thus preventing insulin degradation. Insulin was incubated in the presence or absence of P(MAA-g-EG) microparticles at 37 °C. The results are shown in Fig. 2. The degradation rate constants determined from each slope treated with P(MAA-g-EG) (1:1), (4:1) and (1:0) were 1.42 ± 0.17 × 10− 2/min, 0.62 ± 0.03 × 10− 2/min and 0.32 ± 0.06 × 10− 2/min, respectively. As expected, insulin was rapidly degraded in the absence of microparticles, and the insulin degradation rate constant was 1.45 ± 0.05 × 10− 2/min. Interestingly, even in the presence of P(MAA-g-EG) (1:1), the degradation rate constant did not change significantly and less than 20% of insulin was present at 60 min. On the other hand, in the presence of P(MAA-g-EG) (4:1) and (1:0), the insulin remaining amount at 1 h remarkably increased and the degradation rate constants were significantly decreased. Additionally, the insulin loss was not observed in the presence of any P(MAA-g-EG) microparticles at 4 °C (data not shown), suggesting that the decrease of insulin amount was caused by the only enzymatic degradation and these hydrogels could directly inhibit enzymatic activity in the gastric fluid. Fig. 3(a) shows the effect of amount of P(MAA-g-EG)(1:1) microparticles on the insulin degradation rate in the gastric

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0

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Fig. 3. Effects of P(MAA-g-EG) (1:1) (a) and (1:0) (b) on insulin degradation rate in the gastric fluid. Each value represents the mean ± S.D.

fluid. The degradation rate constant was almost constant even at high amount of the particles. Therefore, the difference in the inhibitory characteristics of P(MAA-g-EG)(1:1) and P(MAA-gEG)(4:1) or (1:0) seemed to be due to the difference in the amount of free MAA units in the polymer network. At low pH, most of the MAA units in P(MAA-g-EG)(1:1) are expected to be completely complexed with the EG units, implying that P (MAA-g-EG)(1:1), if any, possesses few free MAA units. On the other hand, P(MAA-g-EG)(4:1) and (1:0) contain significant number of free MAA units. The inhibitory behavior may thus be linked to the presence of free MAA units in the network. This hypothesis was further supported by the results presented in Fig. 3(b). The increase in the amount of P(MAA-g-EG)(1:0) resulted in a decrease in the insulin degradation rate constant. However, at higher concentrations, no further decrease in the insulin degradation rate constants was observed with increasing concentration of P(MAA-g-EG)(1:0) in the solution. The precise mechanism of this inhibition remains unclear but the observed inhibitory effect might be due to some interactions between the MAA units and/or the pendant EG units and the active sites on the pepsin. Complexation between the MAA units and the pendant EG units reduces the interaction, resulted in decreasing the inhibitory effects of the polymer microparticles to pepsinic degradation. It is worth noting that the inability of P(MAA-g-EG)(1:1) to inhibit the gastric enzyme is a desirable feature because this means that the hydrogel carriers

3.2. Enzyme inhibition by complexation hydrogels in the intestinal fluid It is well known that there are several luminally secreted proteases, such as trypsin, chymotrypsin and elastase, are present in the small intestine [17]. Insulin is very sensitive to these proteases and insulin degradation by proteases occurs in both intestinal fluid and the mucus/glycocalyx layers [18]. Therefore, it is essential for oral insulin delivery system to protect insulin from enzymatic degradation. To investigate that whether P(MAA-g-EG) hydrogels could inhibit the insulin degradation in the intestinal fluid, insulin degradation study in the intestinal fluid was conducted. In order to evaluate its own effect of P(MAA-g-EG) hydrogels, and therefore, to avoid interaction between P(MAA-g-EG) hydrogels and insulin, the intestinal fluid was preincubated in the presence or absence of P(MAA-g-EG) microparticles in the intestinal fluid at 37 °C for 1 h. After adjusting pH of the fluid to 7.4, insulin was incubated with the fluid at 37 °C. The results are shown in Fig. 4. The insulin degradation rate constants determined from the each slope treated with P(MAA-g-EG) (1:1), (4:1), (1:0) and Carbopol 934P were 0.76 ± 0.05 × 10− 2/ min, 0.44 ± 0.06 × 10− 2/min, 0.36 ± 0.03 × 10− 2/min and 0.02 ± 0.01 × 10− 2/min, respectively. The results indicated that in the intestinal fluid containing no hydrogels, insulin was rapidly degraded and less than 50% of insulin was detected at 30 min, and the insulin degradation rate constant was 1.12 ± 0.03 × 10− 2/min. On the other hand, in the intestinal fluid pretreated with P(MAA-g-EG) hydrogels or Carbopol 934P, the amount of insulin remaining was significantly increased. It is noteworthy that the enzyme inhibitory action of these hydrogels in the intestinal fluid was dependent on the density of the ionized carboxylic groups in the hydrogel structure. Carbopol 934P was previously reported to inhibit the protease activities strongly [13,19]. The enzyme inhibition activities of P

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Time (min) Fig. 4. Degradation profiles of insulin as a function of time in the intestinal fluid pretreated with P(MAA-g-EG) (1:1), (4:1), (1:0) or Carbopol 934P at 37 °C for 1 h. Keys: Control (○), P(MAA-g-EG) (1:1) (■), (4:1) (▴), (1:0) (●) and Carbopol 934P (⋄). Control indicates only insulin was exposed to the intestinal fluid. Each value represents the mean ± S.D.

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Molar ratio of MAA and EG repeating units Fig. 5. Changes in protein (closed bars) and Ca2+ (open bars) levels in the intestinal fluid following pretreatment with P(MAA-g-EG) (1:1), (4:1), (1:0) or Carbopol 934P at 37 °C for 1 h. Each value represents the mean ± S.D.

(MAA-g-EG) hydrogels were lower compared to that of Carbopol 934P. 3.3. Ca2+ deprivation and protein incorpration by complexation hydrogels in the intestinal fluid It is thought that the main reason for the observed enzyme inhibitory effect of hydrogels is the Ca2+ deprivation of the enzyme structures. For instance, trypsin is a serine protease in the intestinal fluid, which has a binding site for Ca2+ [20]. Ca2+ plays an important role in maintaining the theramodynamic stability of this enzyme. Therefore, if the Ca2+ was removed from the enzyme structure, the activity is inhibited [19]. For another possibility, some direct interactions between the hydrogels and enzymes such as the incorporation of the enzymes into hydrogels may explain this inhibitory effect. In order to investigate the exact mechanism of this inhibitory effect, protein incorporation and Ca2+ deprivation studies in the intestinal fluid were performed. The intestinal fluid was incubated in the presence or absence of P(MAA-g-EG) microparticles in the intestinal fluid at 37 °C for 1 h. The changes of the protein or the Ca2+ levels were then measured. The results are shown in Fig. 5. No changes in the intestinal protein levels were observed in the presence of hydrogels, suggesting that the direct interaction between the hydrogels and enzymes was negligible. On the other hand, in the intestinal fluid treated with all the three hydrogels and Carbopol 934P, the significant reduction of Ca2+ levels was observed. The Ca2+ deprivation activity of these hydrogels was dependent on the amount of the carboxylic groups in the hydrogel structure. Madsen and Peppas reported that the affinity of the hydrogels towards Ca2+ was dependent on the density of the ionized carboxylic groups [13]. The amount of Ca2+ sequestered from the solution in the present study was lower but comparable to Madsen's data. Fig. 6 shows the relationship between degradation rate and Ca2+ level reduction in the solution. A linear and significant relationship (r = 0.997) was observed, suggesting that the higher the affinity toward Ca2+, the stronger the enzyme inhibition. Thus the Ca2+ deprivation by the hydrogels plays an important role in the inhibition of the intestinal enzymes.

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Degradation rate constant (x10-2/min)

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Fig. 6. Relationship between the insulin degradation rate constants and Ca2+ reduction in the intestinal fluid pretreated with P(MAA-g-EG) (1:1), (4:1), (1:0) or Carbopol 934P at 37 °C for 1 h. Each value represents the mean ± S.D.

insulin

Fig. 8. Effects of Ca2+ addition on insulin degradation rate of ILP (MAA/ EG = 1:1) in the intestinal fluid. ILP or only insulin was added to the intestinal fluid. Each value represents the mean ± S.D. Statistically significant difference: p < 0.01, ⁎⁎, p < 0.05, ⁎ against ‘ILP (MAA/EG = 1:1) in the intestinal fluid’.

3.4. Insulin release from ILPs in the intestinal fluid P(MAA-g-EG) (1:1), (4:1) and (1:0) microparticles typically showed different drug loading efficiencies [14]. These differences influence the insulin release rate (data not shown). Hence, the loading efficiencies of the microparticles were adjusted by changing the initial insulin concentration of the loading solution such that all types of microparticles released the same amount of insulin under the experimental conditions. The fractional release of insulin, defined here as the ratio of the amount released at any time (Mt) in the intestinal fluid to the total amount released in PBS after 1 h (M∞), is shown in Fig. 7. All types of the particles rapidly reached the swollen state and a very rapid release of considerable amount of insulin was observed. The insulin release from ILPs reached equilibrium when approximately 70% of initially entrapped insulin was released. The released insulin was then gradually degraded in the intestinal fluid. In the preliminary experiment using the same intestinal fluid, it was found that insulin was far rapidly degraded in the intestinal fluid in the absence of microparticles (data not shown). The observation implies that in the harsh environment of the intestinal fluid, a condition of ‘high local insulin concentration’ could be maintained for a short time due to the very rapid release of insulin. Previously, Morishita et al. reported that insulin released from ILPs was rapidly absorbed from the ileal

(Mt/M") x100%

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membrane in rats [11]. Therefore, it is believed that maintaining the condition of ‘high local insulin concentration’ in the small intestine for even a short time resulted in an increase the oral insulin bioavailability. All the three types of hydrogels were able to protect insulin from proteolytic attack in the intestinal environment. To clarify whether the enzyme inhibition activity of hydrogels was due to the Ca 2+ deprivation ability, we compared the insulin degradation rate in the intestinal fluid and the intestinal fluid added Ca2+. In this study, ILP (MAA/EG = 1:1) was used because this was the formulation employed for in vivo trials of the previous study [11,12]. These results are shown in Fig. 8. Adding Ca2+ in the intestinal fluid markedly increased the insulin degradation rate constant, suggesting that extra added Ca2+ decreased the enzyme inhibition activity of P(MAA-gEG) hydrogels. The Ca2+ binding sites of P(MAA-g-EG) hydrogels were saturated by excess amount of Ca2+, therefore, it might not deplete the Ca2+ in the structure of the enzyme. It is noteworthy that the insulin degradation rate constant in the intestinal fluid added Ca2+ was almost the same as that of insulin in the intestinal fluid, suggesting that Ca2+ addition completely abolished the enzyme inhibition effects of P(MAAg-EG) hydrogels. These results imply that the insulin released from ILPs in the small intestine may be protected by the enzyme inhibition effects of P(MAA-g-EG) hydrogels due to mainly Ca2+ deprivation from enzyme structure.

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Fig. 7. Insulin release profiles from ILP (MAA/EG = 1:1) (a), (MAA/EG = 4:1) (b) and (MAA/EG = 4:1) (c) in the intestinal fluid (▴) and PBS (■) at 37 °C. Each value represents the mean ± S.D.

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4. Conclusions In conclusions, this work indicated that the complexation hydrogels could completely protect insulin which was entrapped in the hydrogels in the gastric fluid. In the small intestine, the hydrogels inhibit protease activity due to the Ca2+ deprivation from the structure of enzymes, thus, this effect may significantly contribute to the prevention of insulin degradation. These results suggested that P(MAA-g-EG) hydrogels can be an excellent carrier for protecting insulin during their transit through the GI tract. Acknowledgements This work was sponsored by grants from the National Institutes of Health of the USA (R01-EB000246) and from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References [1] P. Langguth, V. Bohner, J. Heizmann, H.P. Merkle, S. Wolffram, G.L. Amidon, S. Yamashita, The challenge of proteolytic enzymes in intestinal peptide delivery, J. Control. Release 46 (1997) 39–57. [2] J.F. Woodley, Enzymatic barriers for GI peptide and protein delivery, Crit. Rev. Ther. Drug Carr. Syst. 11 (1994) 61–95. [3] V. Agarwal, s. Nazzal, I.K. Reddy, M.A. Khan, Transport studies of insulin across rat jejunum in the presence of chicken and duck ovomucoids, J. Pharm. Pharmacol. 53 (2001) 1131–1138. [4] A. Yamamoto, T. Taniguchi, K. Rikyuu, T. Tsuji, T. Fujita, M. Murakami, S. Muranishi, Effect of various protease inhibitors on the intestinal absorption and degradation of insulin in rats, Pharm. Res. 11 (1994) 1496–1500. [5] A.F. Kotze, H.L. Lueβen, B.J. de Leeuw, B.G. de Boer, J.C. Verhoef, H.E. Junginger, N-trimethyl chitosan chloride as a potential absorption enhancer across mucosal surfaces: in vitro evaluation in intestinal epithelial cells (Caco-2), Pharm. Res. 14 (1997) 1197–1202. [6] A. Bernkop-Schnurch, A.H. Krauland, V.M. Leitner, T. Palmberger, Thiomers: potential excipients for non-invasive peptide delivery systems, Eur. J. Pharm. Biopharm. 58 (2004) 253–263. [7] N.A. Peppas, P. Bures, W. Leobandung, H. Ichikawa, Hydrogels in pharmaceutical formulations, Eur. J. Pharm. Biopharm. 50 (2000) 27–46.

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