Absorption Mechanism of a Physical Complex of Monomeric Insulin

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Absorption mechanism of a physical complex of monomeric insulin and deoxycholyl-L-lysyl-methylester in the small intestine Foyez Mahmud, Ok-Cheol Jeon, Taslim A. Al-Hilal, Seho Kweon, Victor C. Yang, Dong Soo Lee, and Youngro Byun Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp500626a • Publication Date (Web): 19 Apr 2015 Downloaded from http://pubs.acs.org on May 2, 2015

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Molecular Pharmaceutics

Absorption mechanism of a physical complex of monomeric insulin and deoxycholyl-L-lysylmethylester in the small intestine Foyez Mahmud,† Ok-Cheol Jeon,† Taslim A. Al-Hilal,‡ Seho Kweon,† Victor C. Yang,†,∥ Dong Soo Lee,†,§ Youngro Byun,*,†, ‡ †

Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of

Convergence Science and Technology, Seoul National University, Seoul 151-742, South Korea ‡

Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University,

Seoul 151-742, South Korea ∥Department

of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann

Arbor, MI 48109-1065, USA §Department

of Nuclear Medicine, Seoul National University College of Medicine, Seoul 110-

744, South Korea Corresponding Author *Youngro Byun E-mail: [email protected], Tel: +82-2-880-7866, Fax: +82-2-872-7864

1 ACS Paragon Plus Environment


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ABSTRACT

Currently oral administration of insulin still remains the best option to avoid the burden of repeated subcutaneous injections and to improve its pharmacokinetics. The objective of the present investigation was to demonstrate the absorption mechanism of insulin in the physical complexation of deoxycholyl-L-lysyl-methylester (DCK) for oral delivery. The oral insulin/DCK complex was prepared by making a physical complex of insulin aspart with DCK through ionpair interaction in water. For the cellular uptake study, fluorescein labeled insulin or DCK were prepared according to a standard protocol and applied to Caco-2 or MDCK cell lines. For the PK/PD studies, we performed intrajejunal administration of different formulation of insulin/DCK complex to diabetic rats. The resulting insulin and DCK complex demonstrated greatly enhanced lipophilicity

as

well

as

increased

permeation

across

Caco-2

monolayers.

The

immunofluorescence study revealed the distribution of the complex in the cytoplasm of Caco-2 cells. Moreover, in the apical sodium bile acid transporter (ASBT) transfected MDCK, the insulin/DCK complex showed interaction with ASBT, and also demonstrated absorption through passive diffusion. We could not find that any evidence of endocytosis in relation to the uptake of insulin complex in vitro. In the rat intestine model, the highest absorption of insulin complex was observed in the jejunum at 1 h and then in the ileum at 2-4 h. In PK/PD study, the complex showed a similar PK profile to that of SC insulin. Overall, the study showed that the effect of DCK on enhancing the absorption of insulin resulted from transcellular processes as well as bile acid transporter activity.

KEYWORDS: insulin, bile acid, enhancer, physical complex, oral absorption, tight junction


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1. INTRODUCTION

Despite ongoing researches in oral insulin therapy, it still remains a major challenge to develop orally active insulin that is the most convenient and effective in treating diabetes patients. Among various routes, oral insulin is able to mediates the similar physiological advantages in diabetic patients; this is because the absorbed insulin in the intestine can directly reach the liver through the hepatic circulation and can thus effectively regulate the glucose level.1-2 In practice, the application of insulin in oral delivery is restricted due to extensive degradation by gastric proteases, acidic environment of stomach, and its high molecular weight that altogether limits its penetration.3-4 To facilitate insulin absorption through the oral route, intestinal permeation enhancers, such as fatty acids, steroidal detergents and surfactants are used to increase the drug transport by paracellular and transcellular pathways through different mechanisms; this includes the alteration of various factors such as the drug formulation, physiological variables, the reduction of mucous viscosity, and the opening of tight junctions. However, the great risk of permeation enhancers is that it could solubilize the lipid membrane thus potentially increasing the uptake of toxins and pathogens into the bloodstream.5-6 One of the approaches for oral delivery of macromolecular drugs is to exploit the bile acid carrier mediated transport in the GI tract. Bile acids are absorbed both by passive diffusion in the intestine and by active transport in the terminal ileum via the apical sodium dependent bile acid transporter (ASBT). ASBT (SLC10A2) is expressed on the apical membrane of enterocytes in the small intestine and it mediates the reabsorption of conjugated bile acids in the ileum.7-8 Thus essentially, bile acid derivatives would be an ideal option for oral drug delivery agents with poor bioavailability considering its favorable properties, such as amphiphilicity, high chemical



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stability and binding ability. As reported by Lee et al., deoxycholic acid (DOCA) conjugated heparin was successfully delivered through oral administration without any morphological changes of the mucous membrane in the intestines.9-10 Based on this observation, we have prepared a novel oral delivery carrier by using DOCA and a lysine called Nα-deoxycholyl-Llysyl-methylester (DCK) (Fig. 1), which was formed into a physical complex with insulin by simple ion-pair interaction. In our previous study, we observed that the insulin/DCK complex significantly increased the lipophilicity and apparent permeability of insulin, and also showed a higher resistance against digestive enzymes. We also reported that the insulin/DCK physical complex increased the stability of the complex thereby displaying optimal glucodynamic effects of insulin in diabetic rats. Moreover, DCK also significantly affected the pharmacokinetic and pharmacodynamic parameters of orally administered insulin in a dose dependent manner. In addition, the effectiveness of oral insulin/DCK complex was proven through reproducible results in large animals and confirmed normalized blood glucose levels in pancreatectomized diabetic canines. It is noteworthy to mention that the oral administration of DCK alone could not be attributed to glucose lowering effects or alter plasma insulin level.11-12 However, the focus of our previous study was the physiological properties and also the biological activities of orally administered insulin. Despite demonstrating the promise of insulin/DCK as a substrate for the bile acid transporter in the ileum, the previous study had failed to generate any supportive data that confirms uptake of oral insulin through the ASBT. Besides, the integrity of the insulin/DCK complex at the site of absorption still remains unexplored. Neither of our previously published articles demonstrated any evidence of the exact uptake mechanisms operated during insulin absorption. Furthermore, we did not investigate the main sites of absorption and duration of uptake in different intestinal tissues. Indeed, it holds an immense significance to explore the


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uptake mechanism of oral insulin, because bile acid derivatives often exhibit permeation enhancer-like effects, that is, the increased chance of opening of tight junctions and subsequent accumulation of toxic substances in enterocytes. To address these points, we designed this study to shed light on the uptake pathways involved in insulin’s oral absorption. Unlike human zinc insulin used in the previous studies, the insulin/DCK complex was prepared using rapid-acting insulin aspart, which was selected for its monomeric form. It is reported that monomeric rather than hexameric insulin could be absorbed at a higher magnitude into the small intestine.2 The aim of this study was to evaluate how the insulin/DCK complex overcomes various obstacles of the GI tract and why it is better transported through the intestine. For this purpose, we investigated various contributing pathways and absorption mechanistic models for prediction of oral delivery of the complex.5,

13

To clarify the insulin uptake

enhancement in related with its absorption mechanism, we carried out the transport and immunofluorescence studies using Caco-2 and ASBT transfected MDCK cell lines, respectively. We also investigated the relative uptake of the complex throughout the intestinal segments in vivo. Finally, the abilities of insulin/DCK complex in regulating the glucose level and also in maintaining effective insulin concentration in the plasma were evaluated in diabetic rats.

2. MATERIALS AND METHODS

2.1. Materials. Insulin aspart (NovoLog®, MW: 5826 Da) was obtained from Novo Nordisk (Denmark). Deoxycholic acid (DOCA), N-methylmorpholine, ethyl chloroformate, Nε-Boc-Llysine methylester hydrochloride [H-Lys(Boc)-OMe·HCl], chloroform, tetrahydrofuran (THF) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO). Ethyl



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acetate, methanol, dimethylformamide (DMF) and acetyl chloride were purchased from Merck (Darmstadt, Germany). Rhodamine B isothiocyanate (RITC) and Fluorescein (NHS-Fluorescein) were obtained from Sigma-Aldrich and Thermo scientific (Rockford, IL), respectively. All other chemicals and reagents used for this research were of an analytical grade. The physical complex of insulin aspart and DCK was formed by ion-pair interactions in distilled water. We also checked turbidity of the complex in water of different mole ratios such as 1:1, 1:3, 1:5, 1:7, 1:10, 1:15, and 1:20 between insulin and DCK. 2.2. Synthesis of oral absorption enhancer, DCK. The solution of deoxycholic acid (26 g) in anhydrous tetrahydrofuran (800 mL) was prepared, and both ethyl chloroformate (6.4 mL) and N-methylmorpholine (7.4 mL) were added to the solution in a dropwise manner, followed by reacting for 30 min in an ice bath that was further continued for 2 h at room temperature. HLys(Boc)-OMe.HCl (20 g) and N-methylmorpholine (7.4 mL) were added to the deoxycholic acid solution by refluxing for 2 h. The solution was cooled to room temperature, and the reaction was allowed to continue overnight under stirring. After filtration, the resulting solution was evaporated and the residues were purified with chloroform/methanol solution (20:1; v/v) by column chromatography. The crude product was dissolved in acetyl chloride (23.4 mL)/methanol (100 mL) solution and reacted for 12 h at room temperature. The solvent was evaporated under reduced pressure and the product was dissolved in water. Finally, the resulting solution was extracted with chloroform three times, and the aqueous layer collected and lyophilized to obtain the final product in the form of a white powder. 1H NMR (JEOL JNM-LA 300 WB FTNMR, Tokyo) and FT-IR (Nicolet 6700, Thermo Scientific) spectrum in KBr pellets were used to check the purity and chemical structure. 1H NMR (DMSO-d6); δ 0.57 (3H, s), 0.82 (3H, s), 0.90 (2H,


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d, J = 6.3 Hz), 1.11–1.83 (31H, m), 1.95–2.19 (1H, m), 2.71 (2H, dt, J = 6.0, 12.9 Hz), 3.29–3.44 (1H, m), 3.77 (1H, brs), 4.09–4.37 (11H, m), 7.94 (3H, brs). 2.3. Synthesis of F-DCK and RITC-insulin. The chemical conjugate of DCK and NHSFluorescein was synthesized by the formation of amide bond. For this purpose, we protected the primary amine group of DCK by creating a new amine group in the terminal side of lysine. The Lys(Boc)-DOCA was then synthesized as mentioned elsewhere in literature and used as a precursor for F-DCK.11 The mixture of Lys(Boc)-DOCA in methanol was added dropwise to ethylene diamine (EDA) solution and stirred for 3 h in an ice cool condition, followed by reacting for 72 h under a nitrogen atmosphere. After evaporation and precipitation, the product was lyophilized. Both ethylene amine Lys(Boc)-DOCA (Et-Lys(Boc)-DOCA) and n-hydroxy succinimide fluorescein (NHSF) were dissolved in DMF separately, and then NHSF was gradually added to the Et-Lys(Boc)-DOCA solution. After overnight reaction, the solvent was evaporated from the mixture and the resulting product was precipitated and washed with cold water until no fluorescence was detected in the supernatant. The synthesized amine group of Lys(Boc)-DOCA-NHSF was deprotected by acetyl chloride in methanol for 12 h. The solvent was evaporated from the mixture and the consequent dried product was dissolved in water to be extracted in chloroform. The aqueous layer was lyophilized and the purity of the obtained material was determined by MALDI-TOF. RITC labeled insulin was synthesized as described in the literature with minor modifications.14 Briefly, 20 mg of insulin aspart and a fluorescent dye (1:2, w/w) were dissolved in sodium carbonate buffer (0.1 M) and DMSO (200 µL), respectively. The RITC was then added slowly into the insulin solution while stirring at 4 °C. After incubation for 12 h in the dark, the coupling reaction was stopped by adding 10 mL NH4Cl (50 mM) and the mixture was stirred for 2 h at 4



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°C. The unconjugated RITC was separated by a fluorescent dye removal column (Thermo scientific). Finally, the resulting RITC-insulin conjugate was lyophilized and stored at 4 °C in the dark until further use. 2.4. Permeability of insulin/DCK complex in a Caco-2 cell model. To study the potential of oral absorption enhancement of DCK, the human colon cancer derived Caco-2 cells were used as the transport model as previously described.12 Caco-2 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and were grown in DMEM with 10% fetal calf serum, 1% non-essential amino acids (NEAA), and 1% antibiotic solution at 37 °C under 5% CO2 humidified atmosphere. The Caco-2 cells were seeded on 12-well polyester inserts (1.12 cm2, 0.4 µm pore size, Transwell, Corning Costar, NY) at a density of 5 x 105 cells/well. The cells were cultured for 21-28 days to form a differentiated confluent monolayer and fed with complete media on alternate days. An epithelial volt-ohm meter (ERS-2, Millicel) with electrode (STX 100M, World Precision Instruments, Sarasota, FL) was used to measure the transepithelial electrical resistance (TEER). Caco-2 cell monolayers were washed twice with cold Hanks Balanced Salt Solution (HBSS; Sigma-Aldrich) and preincubated for 30 min at 37 °C in a 5% CO2 incubator, with a transport media (pH 7.4) supplemented with HBSS, 10 mM HEPES (N-2hydroxyethyl piperazine-N′-2-ethanesulfonic acid; Biowhittaker), 25 mM D-glucose, and 10 mM sodium azide (Sigma-Aldrich) as a metabolic inhibitor. Insulin aspart (0.05 mg/mL) as the control and insulin/DCK complexes of four different feed mole ratios (1:5, 1:10, 1:15, and 1:20) were introduced in the apical part of the transwell, respectively. The inserts were transferred to a fresh medium at each time point (10, 20, 30, 40, 50, 60, 75, 90, 105 and 120 min) and the basolateral solutions were withdrawn for analysis. In order to confirm that the permeation of insulin or insulin/DCK complex was not occurring due to the damage of the Caco-2 cell


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monolayer, the TEER value was repetitively measured after treatment of samples. The amount of transported insulin was analyzed using the Iso-insulin ELISA kit (Mercodia, Uppsala). 2.5. Transport pathways study by using Caco-2 and MDCK cells. Caco-2 cells were grown to confluence in the transwell inserts and maintained under incubation conditions. The media was replaced with HBSS transport medium and the cells were equilibrated at least for 1 h before the experiments. The cells were treated with 200 µL RITC-insulin or RITC-insulin/DCK complex (mole ratio 1:10) at the concentration of 0.05 mg/mL for 60 min at 37 °C. The drug solutions were removed by washing three times with HBSS and the cells were fixed with 4% cold paraformaldehyde in PBS solution for 20 min at room temperature. The cells were washed again three times with washing buffer containing 0.1% bovine serum albumin (BSA) in PBS and permeabilized using 0.3% Triton X-100 and 10% goat serum in PBS for 60 min at room temperature. For actin filament visualization, the blocking solution was removed and the cells were counterstained with FITC labeled phalloidin (0.2 µg/mL, Sigma-Aldrich) for 20 min at room temperature. In cases of zonula occludens (ZO-1) and claudin-1 staining, the cells were incubated overnight with either Alexa Fluor 488 conjugated mouse monoclonal anti-ZO-1 antibody or rabbit anti-claudin-1 polyclonal antibody (Molecular Probes) at 4 °C. After washing, the claudin-1 treated cells were further incubated with Alexa Fluor 488 conjugated anti-rabbit secondary antibody for 1 h at room temperature. The nucleus was counterstained with Hoechst (Sigma-Aldrich, 10 µg/mL) for 5 min. The following filters were excised from the insert, mounted on a glass slide and subsequently covered with a cover slip using Vectashield antifade solution (Vector Laboratories, Burlingame, CA). The optical images were obtained using LSM Meta 710 inverted confocal laser scanning microscope (Carl Zeiss, Germany).



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2.6. Uptake of insulin/DCK complex through apical sodium bile acid transporter (ASBT). In order to demonstrate the bile acid transporter activity with the uptake of insulin/DCK complex, the stably transfected MDCK-ASBT cell line was used. MDCK cell line was obtained from ATCC and stably transfected with ASBT using Lipofectamine® 2000 according to methods described in literature.15 MDCK and MDCK-ASBT cells were seeded at the density of 4 x 104 cells and grown to confluence in 35 mm dishes with glass coverslip bottoms (SPL Life Sciences, South Korea). The cells were treated with the drug complex, fixed and permeabilized using similar procedures as described earlier. Then the blocking solution was removed and cells were treated with 200 µL of goat anti-SLC10A2 polyclonal antibody (Sigma-Aldrich) at 1:100 dilutions overnight at 4 °C. The cells were washed again for three times and incubated with Alexa Fluor 488 conjugated secondary antibody (Molecular Probes) at 1:200 dilutions for 1 h at room temperature. Followed by washing, the cells were incubated with Hoechst for DNA staining. Finally, the optical images were taken by confocal microscope. 2.7. Inhibition studies using flow cytometry. Inhibition of endocytosis: To assess the effect of various inhibitors on the uptake of insulin/DCK complex, 5 x 104 Caco-2 cells seeded in a 6-well cell culture plate were preincubated with chlorpromazine (10 µg/mL), wortmannin (50 nM), cytochalasin D (10 µg/mL), methyl-β-cyclodextrin (5 mM) and nystatin (25 µM) for 30 min at 37 °C.16-20 Following exposure to the insulin/DCK complex, the inhibitor solutions were replaced with freshly prepared media containing inhibitors with insulin/DCK complex (fluorescein-labeled; mole ratio 1:10) and further incubated for 1 h at 37 °C. Afterwards, the cells were washed with PBS, trypsinized, and analyzed by flow cytometry (Beckman Coulter, Fullerton, CA).


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Inhibition of bile acid transporter-mediated absorption: For the inhibition of bile acid transporter-mediated absorption, sodium taurocholate was used as a competitive inhibitor on account of its high affinity. Prior to the addition of complex, Caco-2 cells were pre-incubated with varied concentrations of sodium taurocholate (5, 10, 25, and 50 µM), within the tolerable range.21 Following the preincubation for 30 min at 37 °C, RITC-insulin/F-DCK complex (mole ratio 1:10) was added and the cells further incubated for 1 h. Subsequently, the cells were washed, trypsinized and analyzed by flow cytometry. The population of cells treated with the insulin/DCK complex was considered under gating in comparison to non-treated cells. Afterwards, we quantified the mean fluorescence intensity of each group using histogram statistics. In addition, the group containing the complex without inhibitors was used as control and considered as having 100% fluorescence intensities. Any morphological changes of Caco-2 cells was not observed during inhibition experiment using optical microscopy however we did not check for cell viability via methods such as the PI dye. 2.8. Animal studies. All animal studies were performed in accordance with the “Regulations for the Care of Animals” approved by the Institutional Animal Ethics Committee of Seoul National University Animal Care Facility (2008, No. 8852). Absorption mechanism study of insulin/DCK complex in rat intestine: Male Sprague-Dawley (SD) rats weighing from 230 to 250 g with free access to water were fasted for 12 h and were intrajejunally (IJ) administered with 80 IU of RITC labeled insulin aspart and fluorescein labeled DCK complex (mole ratio 1:10). The rats were sacrificed after a specific time period and different intestinal segments were removed and washed with the normal saline. The selected jejunum, ileum and colon segments were immediately frozen in cryoembedding media (OCT) for subsequent cryostat sectioning. Each 10 µm thick sections were applied onto microscope slides



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and fixed with freshly prepared 4% paraformaldehyde in PBS for 10 min at room temperature. Afterwards, the sections were stained with DAPI (Sigma-Aldrich) for visualizing the nuclei and imaging by CLSM (Carl Zeiss, Germany). In vivo evaluation of insulin/DCK complex bioactivity in diabetic rats: Diabetes was induced in male SD rats by intraperitoneal (IP) administration of streptozotocine at a dose of 60 mg/kg body weight. Pharmacokinetic studies were performed to investigate the efficacy of oral insulin/DCK complex in comparison with subcutaneously (SC) administered insulin. SD rats weighing from 230 to 250 g with free access to water were fasted for 12 h and were administered either insulin/DCK complex intrajejunally or insulin alone subcutaneously. The surgery was performed aseptically to the rat anesthetized with IP ketamine-xylazine solution and placed in dorsal recumbency.22 Midline laparotomy was made and the drug formulation was carefully administered next to the duodenojejunal junction. The abdominal muscle was sealed with absorbable suture and the skin incision was closed with wound clips. Blood samples were collected from the ocular orbital of predetermined time points. The collected blood was centrifuged at 4500 rpm for 15 min, after which plasma was withdrawn and stored at -70 °C until assay. Insulin concentration and the blood glucose levels were measured by Iso-Insulin ELISA kit (Mercodia Inc.) and CONTOUR®TS blood glucose meter (Bayer HealthCare LLC, NJ). 2.9. Statistical analysis. The quantitative data were stated as mean ± SEM, and the statistical analysis were performed by Student’s t test. The difference between the groups was considered significant when p < 0.05.

3. RESULTS


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3.1. Physical properties of insulin/DCK complex. The FT-IR spectrum of the synthesized DCK revealed that the vibrational band associated with carboxyl group of deoxycholic acid near 1700 cm-1 had disappeared and a new band of C=O stretch from ester conjugation had formed at the 1734 cm-1 position. Moreover, the N-H stretching peaks, which represented the primary amine group of DCK, were observed at 3412 and 3282 cm-1 in the IR spectra (Figure S1). In the case of fluorescein conjugated DCK (F-DCK), the MALDI-TOF spectrum showed peaks at m/z 921.39 [M], 922 [M+H], 923.40 [M+2H], 943.37 [M+Na] (Figure S2). Since there were no other peaks except for these spectrums, the fluorescein material well conjugated with DCK. Insulin/DCK complex was formed by electrostatic interaction due to the fact that the cationic amino group of DCK interacts with the anionic carboxyl group of insulin. The FT-IR analysis revealed that the stretching band representing the amine group of DCK disappeared after forming the complex, thus affirming the hypothesis (data not shown). It was found that the physical complex having a higher mole ratio of DCK to insulin had better water solubility than those of lower mole ratios. The partition coefficient of insulin/DCK was measured in the n-octanol/water system to evaluate its solubility in tissue. Because of its hydrophilic nature, only insulin exhibited a low partition coefficient (Ko/w= 0.04 ± 0.01), whereas the complex (1:10) showed highly increased lipophilicity (Ko/w= 1.82 ± 0.23). 3.2. Permeability of insulin/DCK complex in a Caco-2 cell model. The well-established Caco-2 monolayers with TEER higher than 600 Ω cm2 were used to mimic the intestinal epithelial cells. In order to assess the capability of insulin/DCK complex in enhancing insulin transport, Caco-2 monolayers were treated with 0.05 mg/mL concentration of insulin containing various mole ratios of DCK (1:5 to 1:20). As shown in Figure 2, Papp values of insulin and the insulin/DCK (1:10) complex were 4.44 x 10-8 and 1.99 x 10-7 cm/s, respectively; that is, insulin



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permeability had increased 4.49 fold due to physical complexation with DCK. Even though the ratio of DCK in the complex was increased up to 1:20, the Papp value did not increase further (2.08 x 10-7 cm/s). Thus, a linear response with respect to the mole ratio of complex in the Caco2 cell transport study was not observed. The results indicated that the complex system at higher than 1:10 mole ratio may be saturated at the uptake site, resulting in steadfast apparent permeability in the transport study. 3.3. Transport pathways in Caco-2 and MDCK cells. Confocal laser scanning microscope was used to explore the cellular uptake mechanism of insulin/DCK complex. To visualize actin filament as a paracellular marker for tight junctions, Caco-2 monolayers were stained with FITC labeled phalloidin, which could be observed as a continuous rings between adjacent cells.23 After 1 h of treatment, RITC-insulin/DCK complex was detected in the intracellular space of the monolayers, which indicated that the complex was well taken up into the cells (Figure 3). The confocal images, taken at different depths, also confirmed the internalization of insulin/DCK complex into the Caco-2 cells (data not shown). Moreover, the drug complex accumulated chiefly in the cytoplasm rather than the cell-cell contact regions. However, the merged images indicated very weak interactions of actin (green) with RITC-insulin/DCK complex (red), which suggests that the paracellular permeation of the insulin/DCK complex was negligible. In addition, the strong fluorescence and the smooth chicken-wire pattern of the actin staining confirmed that the paracellular pathway may not be actively involved in the drug uptake. From the confocal images, we could not observe any changes in the pattern of actin staining during drug treatment. Furthermore, the fluorescence intensity of actin protein was almost similar to that of non-treated cells and the drug complexes did not co-localized with actin proteins. To confirm the possibility of paracellular uptake, the tight junctions of Caco-2 cells were stained using ZO-1


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and claudin-1. Like actin staining, the complex was mostly localized in the cytoplasm, indicating the transcellular permeation of the drug. Moreover, the monolayers that were treated with the drug maintained intact morphology of tight junction encircling the apical domain of epithelial cells that accompanies the minimal paracellular transport.24-25 To elucidate the uptake mechanism in other representative intestinal cell lines, MDCK cell was chosen for additional studies. It was found that the insulin/DCK complex was well absorbed and accumulated mostly in the cytoplasm. In addition, we observed no ruffling of the membrane of treated cells, which suggests that the transcellular pathway is the most likely uptake mechanism of insulin/DCK complex. 3.4. Endocytosis in Caco-2 cells. In order to identify whether the enhancement of Caco-2 cells uptake of insulin was in connection with the endocytosis pathways, uptake experiments were performed in the presence of various inhibitors. The cellular uptake of RITC-insulin/DCK complex in the absence of endocytosis inhibitors was used as the control, and its intensity was considered as 100%. The incubation of Caco-2 cells with the complex in the presence of chlorpromazine, cytochalasin D and wortmannin lightly decreased the uptake of insulin. However, nystatin or methyl-β-cyclodextrin (MβCD) had almost no effect on the uptake of complex. This result concludes that there was no significant contribution of the endocytic pathway in the uptake of insulin/DCK complex into Caco-2 cells (Figure 4). 3.5. Uptake of insulin/DCK complex through apical sodium bile acid transporter. To evaluate the bile acid transporter mediated absorption of insulin/DCK complex, we used ASBT gene transfected MDCK cell line. ASBT is a sodium dependent bile acid transporter that is located in the ileum. As shown in Figure 5, since there was no expression of bile acid transporters in the non-transfected MDCK cell lines, this resulted in the absorption of the RITC-



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insulin/DCK complex through passive diffusion. On the contrary, ASBT protein (green) was readily expressed in the MDCK-ASBT cell line, and the merge images indicate that the complex (red) had interacted with the ASBT at the apical membrane. Based on these images, it was confirmed that the insulin/DCK complex was transported through the bile acid transporter in the presence of ASBT. In addition to ASBT involvement, high uptake of the complex into the cytoplasmic area in MDCK-ASBT cell line suggests that simultaneous transcellular uptake and bile acid transporter-mediated uptake were the key mechanisms for insulin absorption in the intestinal cells. Sodium taurocholate was used as competitive inhibitor to bile acid transporter since it has a high-binding affinity to ASBT. The taurocholate is regarded as a primary bile acid whereas deoxycholate is known as a secondary bile acid, formed by 7-dehydroxylation of cholate. Both taurocholate and deoxycholate are substrates for ASBT. 26-27 To confirm the role of ASBT in the enhancement of insulin/DCK complex uptake, cells were pretreated with excess amounts of sodium taurocholate ensuring blockage of ASBT. In this experiment, the permeated amount of insulin inversely correlated with the given sodium taurocholate concentration. In the presence of various concentrations of sodium taurocholate such as 5, 10, 25, and 50 µM, the relative amounts of insulin that permeated were 81.08 ± 3.38%, 74.85 ± 0.65%, 71.33 ± 1.71% and 69.19 ± 0.28%, respectively, compared to the control (Figure 6). 3.6. Absorption mechanism of insulin/DCK complex in a rat intestine. The location and pattern of absorption of rhodamine labeled insulin/DCK complex in the rat intestine was visualized by CLSM following the intrajejunal administration. The results demonstrated that RITC-insulin/DCK complex was absorbed into numerous intestinal segments, predominantly of the jejunum and ileum. Moreover, we observed that RITC-insulin/F-DCK was mainly absorbed


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in the jejunum within 1 h, and the intensity of RITC labeled insulin became more prominent after two to four hours in the ileum. We assumed that insulin would permeate by passive diffusion in the jejunum and be absorbed by bile acid transporters in ileum (Figure 7). Furthermore, the merged images from jejunum and ileum suggested that the insulin/DCK complex was not dissociated from each other during the absorption process in gut tissues. Surprisingly, any insulin/DCK complex in the colon was not observed throughout the study periods. 3.7. Plasma glucose concentration profiles in rat. In order to evaluate the plasma insulin profile and blood glucose level of intrajejunally administered insulin/DCK complex, we performed pharmacokinetic analysis in diabetic rats by its comparison it with subcutaneous administration.2 To minimize the possibility of drug degradation in the oral formulation, we incorporated camostat mesylate (6.2:2.5, w/w with insulin) as protease inhibitors.28 The plasma insulin concentration levels of intrajejunally delivered insulin aspart were negligible, whereas that of all oral doses ranging from 20 to 80 IU/kg of insulin/DCK complex increased more prominently. As shown in Figure 8a, the oral formulation of insulin/DCK complex demonstrated a slow absorption process, reaching Cmax at 2 h, whereas the Cmax of subcutaneous administration was at 1 h. Afterwards, the plasma insulin levels of the orally treated group decreased gradually within 6 h post-administration. To apprehend the efficacy of oral insulin, we evaluated the glucose lowering effects of various doses of the intrajejunally delivered insulin/DCK complex. As shown in Figure 8b, various doses of insulin/DCK complex produced strong hypoglycemic responses, whereas the IJ insulin alone failed to exhibit any pharmacological effect. Moreover, 80 IU/kg dose of the complex achieved better hypoglycemic effects with a maximum of 64% reduction of blood glucose at 1.5 h, compared with 74% reduction at 1 h post-administration for subcutaneous administration. The



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robust hypoglycemic effects of the oral drug persisted throughout 6 h study period following the intrajejunal administration.

4. DISCUSSION

Deoxycholic acid is an amphiphilic endogenous substance extensively used for the purpose of oral drug delivery for its absorption capability in the small intestine via bile acid transporter. To exploit the path of drug delivery via the bile acid transporter, deoxycholic acid derivatives had been proposed. Therefore, we developed a cationic chemical conjugate named DCK that consists of a hydrophobic core of deoxycholate and a positively charged amine group of lysine. The positively charged DCK can bind with the anionic amino acids of insulin aspart by charge-tocharge interaction. In the previous study, we found that our insulin/DCK complex showed a higher resistance against digestive enzymes (native insulin 62.3 min vs. insulin/DCK complex 111.4 min) and maintained a normal blood glucose level in a diabetic canine models, indicating a further need for identifying its uptake mechanism as a delivery agent for monomeric insulin aspart in the intestine.11-12 Herein, we focused on three key issues: i) favorable physicochemical properties towards oral therapy of insulin aspart physically complexed with DCK, ii) the absorption pathways of insulin/DCK complex including the bile acid-sensing mechanism, iii) in vivo application of insulin/DCK complex for diabetic animal models as as an oral insulin therapy. We found that the insulin/DCK complex of the feed mole ratio of 1:10 was highly soluble in water; however, its partition coefficient in octanol/water increased to aprroximately 45 times the value of insulin alone. In the preliminary study, lower mole ratios (1:1 to 1:7) of insulin/DCK


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complexes precipitated and formed larger particles in water. It followed that increasing the mole ratio reduced the chance of particle formation and led to complete solubilization of the complex at the 1:10 ratio. We assumed that larger particle size of the lower ratios could restrict insulin uptake in the octanol phase; however on the contrary, the soluble complex (1:10 ratio) had a higher surface area and thus increased contact with the oil phase. Therefore, increasing the complex ratio greatly enhanced the lipophilicity of insulin, a key factor required for increasing drug permeability across cell membranes, while maintaining the ability to be recognized by bile acid transporters in the intestinal membranes. In addition, complexes of greater lipophilicity enhanced insulin permeability across Caco-2 monolayers, as demonstrated in the in vitro epithelium model. To investigate the pathway of this uptake, immunofluorescence staining using cytoskeletal components such as F-actin and tight junction proteins, ZO-1 & claudin-1, were performed. Tight junctions are the most apical intercellular contacts that restrict paracellular permeation between individual cells and thus limit the passage of macromolecules, toxins, microbes, etc. It is worthwhile

to

mention

that

the

total

area

of

paracellular

space

in

the

intestinal mucosa is less than 1%. Among the membrane proteins, ZO-1 is the multi-domain scaffold that is tethered directly to the cortical actin cytoskeleton and also bound with claudin proteins, which are mainly responsible for maintaining the epithelial barrier against the flux of ions and molecules. Thus, ZO-1 is proposed to be involved in leak pathway, and claudins for the pore pathway.29 In order to permeate through the paracellular space, the complex must disrupt the tight junction barrier and also initiate its remodeling. The insulin/DCK complex could be successfully observed to be predominant in cytoplasmic area, in contrast to the fact that the tight junction proteins are located mainly in the intercellular space in both Caco-2 and MDCK cell



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lines. Furthermore, the images obtained from ZO-1 and claudin-1 staining revealed that the insulin fluorescence intensity was uniformly distributed in the intracellular space of Caco-2 cells. However, the membrane proteins stained in cell junctions indicate that tight junctions were unaffected during drug treatment. These results suggest that the insulin/DCK complex prefers the transcellular pathway by passive diffusion. In evaluating the involvement of endocytic processes in the transcellular pathway with regard to drug absorption, the effects of various endocytosis inhibitors were examined by FACS analysis. Chlorpromazine was chosen as an inhibitor of clathrin-mediated endocytosis, a classical endocytic pathway. The result indicated that the uptake of insulin/DCK complex did not depend on this pathway. Although the uptake was slightly lower than that of the control, due to the cationic amphiphilic nature of chlorpromazine it may have been incorporated into lipid bilayers, increasing lipid fluidity to affect the uptake of the insulin/DCK complex. As reported by JeanLouis et al., hydrophobic bile acids such as deoxycholic acids may integrate into the lipid bilayer and cause redistribution of cholesterol in the plasma membrane. This is because bile acids themselves are cholesterol derivatives that compete against membrane cholesterol.30 In our evaluations using both MβCD and nystatin as a cholesterol depleting agent and sequestering agent, respectively, any evidence of involvement of the lipid raft or caveolae-mediated endocytosis in the uptake of the insulin/DCK complex could not be found. Subsequently, similar results were also obtained in treatment of wortmannin (macropinocytosis inhibitors) and cytochalasin D. It has been reported that orally delivered macromolecules cannot be absorbed through membrane-based transporters in the intestinal tissues.31 However, in our recent study, the vesicular transport mechanism of ASBT for delivering bile acid-conjugated macromolecules was


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proposed.32 In this study, it was reported that oligomeric deoxycholic acid could enhance the uptake of low molecular weight heparin (LMWH) by binding with ASBT that enables the “receptor-like functional transformation” of ASBT; it was also noted that with the help of ileal bile acid-binding protein (IBABP), macromolecules are eventually transported to the basolateral side of the membrane. Therefore, we made an assumption that DCK could induce a similar uptake mechanism of insulin through interaction with ASBT. From the confocal images, we found that the complex co-localized with ASBT in the transfected cell line, confirming bile acid transporter mediated drug absorption. Also in despite of absence of ASBT, insulin uptake by means of simple passive diffusion occurred in non-transfected MDCK cell line. We then quantified the bile acid transporter activity in insulin uptake using sodium taurocholate as a competitive inhibitors. As a result, the relative uptake of insulin/DCK complex was diminished by up to 30% during the inhibition of the bile acid transporter. In order to demonstrate the uptake mechanism in vivo, we applied the complex in SD rats following the intrajejunal administration. It was found that the RITC-insulin/F-DCK was adequately internalized into the intestine villi (in both jejunum and ileum) instead of adsorption onto the surface of epithelial cells. It was also observed that the absorption of insulin was greatly enhanced for prolonged periods and also the strong fluorescence at the jejunum and the ileum showed perfect co-localization (yellow color) of insulin and DCK. These results imply that the insulin/DCK complex was absorbed into the enterocytes via the transcellular pathway along with the bile acid transporter mediated pathway, and to enter the systemic circulation. However, we have not investigated the stability of insulin complex in the gastrointestinal fluids, nor its possible toxic effects in the intestinal mucosa. To confirm the effectiveness of oral insulin therapy in vivo, we evaluated the plasma insulin concentration and blood glucose level in



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diabetic SD rats. In this study, intrajejunal administrations were performed for two reasons, firstly because ASBT is expressed specifically in the ileum, and secondly due to the involvement of the anion exchange mechanism in the uptake of bile acids in the jejunum. We assumed that the jejunum and ileum are the main active sites for the absorption of insulin/DCK complex. In addition, for the preclinical study, we specifically delivered the complex in an enteric coated formulations. In this study, the results demonstrated a striking correlation of plasma insulin levels and the glucose lowering effects of different doses of insulin/DCK complex. We found that 80 IU/kg insulin/DCK complex showed superior effects in the same time frame as when administered subcutaneously. The greatest glucose lowering effects of SC and IJ insulin were shown at 1 h and 1.5 h, respectively, and this was sustained throughout the study period. On the other hand, the lowest blood glucose level of fast acting SC insulin aspart has been shown to be 65 min in fasting healthy male volunteers, as reported previously.33 The results from human subjects are similar to our findings in terms of rapid decrease of blood glucose level. As expected, neither insulin nor glucose concentration in the blood was altered from the basal level following the IJ administration of insulin aspart (80 IU/kg) alone. It is quite evident that IJ administered insulin takes longer times than SC to achieve maximal effect due to the hepatic circulation, despite their similarity in both efficacy and duration of action. However, it is obvious that IJ administered insulin needs higher doses to achieve to the same level of blood glucose reduction than SC insulin. Future studies will explore the safety profiles as well as clinical benefits of the oral insulin complex to diabetic subjects in the clinic.

5. CONCLUSIONS


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In this study, we have examined the mechanism of insulin/DCK complex absorption both in vivo and in vitro model. The results showed that the effect of DCK in enhancing the absorption of insulin resulted mainly from transcellular processes and also from prominent bile acid transporter activity in the MDCK-ASBT cell line. In comparison with studies using the rat intestine, the insulin/DCK complex was highly absorbed through simple diffusion and the bile acid transporter mediated in the jejunum and ileum, respectively. Moreover, the PK profile of insulin/DCK complex delivered orally was comparable to that of insulin aspart administered subcutaneously. On the basis of these results, the insulin/DCK complex can be considered as a strong candidate for the oral delivery of insulin.

AUTHOR INFORMATION Corresponding Author *Youngro Byun E-mail: [email protected] Tel: +82-2-880-7866, Fax: +82-2-872-7864 Notes The authors declare no competing financial interest ACKNOWLEDGEMENTS This study was supported by grants from Basic Science Research Program (grant no. 20100029407), the Bio & Medical Technology Development Program (grant no. 2012028833) funded through the National Research Foundation of Korea (NRF) of the Ministry of Education, Science



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and Technology, and the Korea Health Technology R&D Project (grant no. HI12C1853) of the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Republic of Korea. Supporting Information The FT-IR of DCK and MALDI-TOF of F-DCK were listed in Figure S1 and Figure S2, respectively. This information is available free of charge via the Internet at http://pubs.acs.org/.

ABBREVIATIONS USED DCK, Nα-deoxycholyl-L-lysyl-methylester (DCK); DOCA, deoxycholic acid; RITC-Insulin, rhodamine B isothiocyanate labeled insulin aspart; F-DCK, N-hydroxysuccinimide fluorescein labeled DCK; ASBT, apical sodium dependent bile acid transporter; Papp, apparent permeability; MDCK, madin-darby canine kidney; SC, subcutaneous; PK, pharmacokinetics; Cmax, the peak plasma concentration; PD, pharmacodynamics; ZO-1, zonula occludens-1.

REFERENCES

1.

Cefalu, W. T. Concept, strategies, and feasibility of noninvasive insulin delivery. Diabetes Care 2004, 27, 239-246.

2.

Sonaje, K.; Lin, K. J.; Wey, S. P.; Lin, C. K.; Yeh, T. H.; Nguyen, H. N.; Hsu, C. W.; Yen, T. C.; Juang, J. H.; Sung, H. W. Biodistribution, pharmacodynamics and pharmacokinetics of insulin analogues in a rat model: Oral delivery using pH-responsive nanoparticles vs. subcutaneous injection. Biomaterials 2010, 31, 6849-6858. 24 ACS Paragon Plus Environment

Page 25 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.

Peppas, N. A.; Kavimandan, N. J. Nanoscale analysis of protein and peptide absorption: insulin absorption using complexation and pH-sensitive hydrogels as delivery vehicles. Eur. J. Pharm. Sci. 2006, 29, 183-197.

4.

Madara, J. L.; Trier, J.S. Functional morphology of the mucosa of the small intestine. In Physiology of the Gastrointestinal Tract, Johnson, L. R., Ed. Raven Press: New York, 1986; pp 1209-1250.

5.

Carino, G. P.; Mathiowitz, E. Oral insulin delivery. Adv. Drug Deliv. Rev. 1999, 35, 249257.

6.

Aungst, B. J. Intestinal permeation enhancers. J. Pharm. Sci. 2000, 89, 429-442.

7.

Dietschy, J. M. Mechanisms for the intestinal absorption of bile acids. J. Lipid Res. 1968, 9, 297-309.

8.

Alrefai, W. A.; Gill, R. K. Bile acid transporters: structure, function, regulation and pathophysiological implications. Pharm. Res. 2007, 24, 1803-1823.

9.

Lee, Y.; Nam, J. H.; Shin, H. C.; Byun, Y. Conjugation of low-molecular-weight heparin and deoxycholic acid for the development of a new oral anticoagulant agent. Circulation 2001, 104, 3116-3120.

10. Lee, Y.; Kim, S. H.; Byun, Y. Oral delivery of new heparin derivatives in rats. Pharm. Res. 2000, 17, 1259-1264. 11. Lee, S.; Lee, J.; Lee, D. Y.; Kim, S. K.; Lee, Y.; Byun, Y. A new drug carrier, Nαdeoxycholyl-L-lysyl-methylester, for enhancing insulin absorption in the intestine. Diabetologia 2005, 48, 405-411.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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12. Kim, S. K.; Lee, S.; Jin, S.; Moon, H. T.; Jeon, O. C.; Lee, D. Y.; Byun, Y. Diabetes correction in pancreatectomized canines by orally absorbable insulin-deoxycholate complex. Mol. Pharm. 2010, 7, 708-717. 13. Gamboa, J. M.; Leong, K. W. In vitro and in vivo models for the study of oral delivery of nanoparticles. Adv. Drug Deliv. Rev. 2013, 65, 800-810. 14. Clausen, A. E.; Bernkop-Schnurch, A. In vitro evaluation of the permeation-enhancing effect of thiolated polycarbophil. J. Pharm. Sci. 2000, 89, 1253-1261. 15. Balakrishnan, A.; Sussman, D. J.; Polli, J. E. Development of stably transfected monolayer overexpressing the human apical sodium-dependent bile acid transporter (hASBT). Pharm. Res. 2005, 22, 1269-1280. 16. von Gersdorff, K.; Sanders, N. N.; Vandenbroucke, R.; De Smedt, S. C.; Wagner, E.; Ogris, M. The internalization route resulting in successful gene expression depends on both cell line and polyethylenimine polyplex type. Mol. Ther. 2006, 14, 745-753. 17. Araki, N.; Johnson, M. T.; Swanson, J. A. A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages. J. Cell. Biol. 1996, 135, 1249-1260. 18. Perumal, O. P.; Inapagolla, R.; Kannan, S.; Kannan, R. M. The effect of surface functionality on cellular trafficking of dendrimers. Biomaterials 2008, 29, 3469-3476. 19. Vercauteren, D.; Vandenbroucke, R. E.; Jones, A. T.; Rejman, J.; Demeester, J.; De Smedt, S. C.; Sanders, N. N.; Braeckmans, K. The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls. Mol. Ther. 2010, 18, 561-569. 20. Matveev, S.; Li, X.; Everson, W.; Smart, E. J. The role of caveolae and caveolin in vesicledependent and vesicle-independent trafficking. Adv. Drug Deliv. Rev. 2001, 49, 237-250.


Page 27 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


21. Kim, S. K.; Lee, D. Y.; Lee, E.; Lee, Y. K.; Kim, C. Y.; Moon, H. T.; Byun, Y. Absorption study of deoxycholic acid-heparin conjugate as a new form of oral anti-coagulant. J. Control Release 2007, 120, 4-10. 22. Park, J. W.; Hwang, S. R.; Jeon, O. C.; Moon, H. T.; Byun, Y. Enhanced oral absorption of ibandronate via complex formation with bile acid derivative. Journal of pharmaceutical sciences 2013, 102, 341-346. 23. Jeon, O. C.; Hwang, S. R.; Al-Hilal, T. A.; Park, J. W.; Moon, H. T.; Lee, S.; Park, J. H.; Byun, Y. Oral delivery of ionic complex of ceftriaxone with bile acid derivative in nonhuman primates. Pharm. Res. 2013, 30, 959-967. 24. Salama, N. N.; Eddington, N. D.; Fasano, A. Tight junction modulation and its relationship to drug delivery. Adv. Drug Deliv. Rev. 2006, 58, 15-28. 25. Smith, J. M.; Dornish, M.; Wood, E. J. Involvement of protein kinase C in chitosan glutamate-mediated tight junction disruption. Biomaterials 2005, 26, 3269-3276. 26. Balakrishnan, A.; Polli, J. E. Apical sodium dependent bile acid transporter (ASBT, SLC10A2): a potential prodrug target. Mol. Pharm. 2006, 3, 223-230. 27. Tolle-Sander, S.; Lentz, K. A.; Maeda, D. Y.; Coop, A.; Polli, J. E. Increased acyclovir oral bioavailability via a bile acid conjugate. Mol. Pharm. 2004, 1, 40-48. 28. Del Curto, M. D.; Maroni, A.; Foppoli, A.; Zema, L.; Gazzaniga, A.; Sangalli, M. E. Preparation and evaluation of an oral delivery system for time-dependent colon release of insulin and selected protease inhibitor and absorption enhancer compounds. Journal of pharmaceutical sciences 2009, 98, 4661-4669.



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29. Fanning, A. S.; Van Itallie, C. M.; Anderson, J. M. Zonula occludens-1 and -2 regulate apical cell structure and the zonula adherens cytoskeleton in polarized epithelia. Molecular biology of the cell 2012, 23, 577-590. 30. Jean-Louis, S.; Akare, S.; Ali, M. A.; Mash, E. A., Jr.; Meuillet, E.; Martinez, J. D. Deoxycholic acid induces intracellular signaling through membrane perturbations. J. Biol. Chem. 2006, 281, 14948-14960. 31. Kramer, W.; Wess, G.; Neckermann, G.; Schubert, G.; Fink, J.; Girbig, F.; Gutjahr, U.; Kowalewski, S.; Baringhaus, K. H.; Boger, G.; et al. Intestinal absorption of peptides by coupling to bile acids. J. Biol. Chem. 1994, 269, 10621-10627. 32. Al-Hilal, T. A.; Chung, S. W.; Alam, F.; Park, J.; Lee, K. E.; Jeon, H.; Kim, K.; Kwon, I. C.; Kim, I. S.; Kim, S. Y.; Byun, Y. Functional transformations of bile acid transporters induced by high-affinity macromolecules. Sci. Rep. 2014, 4, 4163. 33. Kang, S.; Brange, J.; Burch, A.; Volund, A.; Owens, D. R. Absorption kinetics and action profiles of subcutaneously administered insulin analogues (AspB9GluB27, AspB10, AspB28) in healthy subjects. Diabetes Care 1991, 14, 1057-1065.


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Figure captions

Figure 1. Chemical structure of bile acid based enhancer DCK. Figure 2. Transport studies of insulin/DCK complex across Caco-2 monolayers at different ratios. The quantitative data represents as mean ± SEM (n = 3). *p < 0.05 versus control. Figure 3. Confocal images of Actin, ZO-1, and Claudin-1 proteins treated with RITCinsulin/DCK complex (red) in Caco-2 cells (a) and Actin staining in MDCK cells (b). The cells without any insulin treatment referred as control (Scale bars, 5 µm). Figure 4. Uptake comparison of insulin/DCK complex in the presence of various inhibitors in Caco-2 cell line by FACS analysis. The bar diagram represents the mean fluorescence intensities of RITC-insulin/DCK complex (Mean ± SEM, n = 3). Figure 5. Confocal images showing uptake of insulin/DCK complex through passive diffusion in MDCK cells and also ASBT mediated uptake in transfected MDCK-ASBT cells (Scale bars, 5 µm). Figure 6. Bile acid mediated uptake inhibition studies using sodium taurocholate by FACS analysis in Caco-2 cells. The quantitative data represents as mean ± SEM (n = 3). *p < 0.05 versus control. Figure 7. Confocal images of fluorescently labeled insulin (red) and DCK (green) in SD rat intestinal segment after intrajejunal administration of insulin/DCK complex in different time points (Scale bars, 50 µm).


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Figure 8. Plasma insulin profiles (a) and Blood glucose level (b) in diabetic rats following intrajejunal administration of insulin/DCK complex; Vehicle treatment (x), 80 IU/kg insulin/DCK (●), 40 IU/kg insulin/DCK (▲), 20 IU/kg insulin/DCK (♦), and subcutaneous administration of 1 IU/kg insulin (■).The quantitative data represents as mean ± SEM (n = 3-4).



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Graphical abstract 177x89mm (300 x 300 DPI)

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Figure 1. Chemical structure of bile acid based enhancer DCK. 44x30mm (600 x 600 DPI)



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Figure 2. Transport studies of insulin/DCK complex across Caco-2 monolayers at different ratios. The quantitative data represents as mean ± SEM (n = 3). *p < 0.05 versus control. 144x156mm (300 x 300 DPI)


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Figure 3. Confocal images of Actin, ZO-1, and Claudin-1 proteins treated with RITC-insulin/DCK complex (red) in Caco-2 cells (a) and Actin staining in MDCK cells (b). The cells without any insulin treatment referred as control (Scale bars, 5 µm). 177x162mm (300 x 300 DPI)



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Figure 4. Uptake comparison of insulin/DCK complex in the presence of various inhibitors in Caco-2 cell line by FACS analysis. The bar diagram represents the mean fluorescence intensities of RITC-insulin/DCK complex (Mean ± SEM, n = 3). 142x156mm (300 x 300 DPI)


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Figure 5. Confocal images showing uptake of insulin/DCK complex through passive diffusion in MDCK cells and also ASBT mediated uptake in transfected MDCK-ASBT cells (Scale bars, 5 µm). 177x99mm (300 x 300 DPI)



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Figure 6. Bile acid mediated uptake inhibition studies using sodium taurocholate by FACS analysis in Caco-2 cells. The quantitative data represents as mean ± SEM (n = 3). *p < 0.05 versus control. 139x96mm (300 x 300 DPI)


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Figure 7. Confocal images of fluorescently labeled insulin (red) and DCK (green) in SD rat intestinal segment after intrajejunal administration of insulin/DCK complex in different time points (Scale bars, 50 µm). 127x113mm (300 x 300 DPI)



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Figure 8. Plasma insulin profiles (a) and Blood glucose level (b) in diabetic rats following intrajejunal administration of insulin/DCK complex; Vehicle treatment (x), 80 IU/kg insulin/DCK (●), 40 IU/kg insulin/DCK (▲), 20 IU/kg insulin/DCK (♦), and subcutaneous administration of 1 IU/kg insulin (■).The quantitative data represents as mean ± SEM (n = 3-4). 264x481mm (300 x 300 DPI)


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Absorption Mechanism of a Physical Complex of Monomeric Insulin

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