Improvement of Transdermal Delivery of Exendin-4 Using Novel Tip

Dec 9, 2015 - Shu Liu†, Dan Wu†, Ying-shu Quan†‡, Fumio Kamiyama‡, Kosuke Kusamori†, Hidemasa Katsumi†, Toshiyasu Sakane†, and Akira Y...
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Article pubs.acs.org/molecularpharmaceutics

Improvement of Transdermal Delivery of Exendin‑4 Using Novel Tip-Loaded Microneedle Arrays Fabricated from Hyaluronic Acid Shu Liu,† Dan Wu,† Ying-shu Quan,†,‡ Fumio Kamiyama,‡ Kosuke Kusamori,† Hidemasa Katsumi,*,† Toshiyasu Sakane,† and Akira Yamamoto† †

Department of Biopharmaceutics, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan CosMED Pharmaceutical Co. Ltd, Higashikujo Kawanishi-cho 32, Minami-ku, Kyoto 601-8014, Japan

‡

ABSTRACT: The purpose of this study was to evaluate the characteristics of exendin-4 tip-loaded microneedle arrays and to compare their acute efficacy with subcutaneous injections in type 2 diabetic GK/Slc rats. Fluorescein isothiocyanate labeled dextran with an average molecular weight of 4,000 (FD4) was selected as a model drug, and FD4 tip-loaded microneedle arrays were prepared in this study. In addition, intraperitoneal glucose tolerance tests after application of exendin-4 tip-loaded microneedle arrays were also compared with those after subcutaneous injection in type 2 diabetic GK/Slc rats. The release of FD4 from the tip-loaded microneedle arrays was very rapid, particularly in the initial 30 s, and most of the FD4 was released within 5 min. In addition, glucose tolerance was improved and the insulin secretion was enhanced after application of exendin-4 tip-loaded microneedle arrays, and these effects were comparable to those after subcutaneous injection of exendin-4. Similar plasma concentration profiles were seen after application of exendin-4 tip-loaded microneedle arrays, as was the case with subcutaneous injection in type 2 diabetic GK/Slc rats. These findings indicate that exendin-4 tip-loaded microneedle arrays can be used as an alternative to achieve sufficient delivery of exendin-4 for treatment of type 2 diabetes. To our knowledge, this is the first report of transdermal exendin-4 delivery using tip-loaded microneedle arrays. KEYWORDS: transdermal absorption, exendin-4, microneedle array, hyaluronic acid, transdermal drug delivery, percutaneous, absorption, skin, peptide delivery, protein delivery a sustained release formulation.13−15 Therefore, an alternative approach is required to improve patient convenience and therapeutic efficacy. On the other hand, traditional noninvasive transdermal patch systems are simple and comfortable to use, but the choice of therapeutics is limited to small molecules due to the existence of stratum corneum. To overcome this skin barrier, microneedle arrays have gained increasing attention as a novel minimally invasive method, and they are able to deliver a variety of molecules into the skin, including small drugs, macromolecules, nanoparticles, and fluid extracts.16−18 Microneedles have been demonstrated to effectively penetrate the stratum corneum into the epidermis and/or superficial dermis to administer compounds into the skin for local or systemic administration. Their sharp tips and target insertion depth reduce the risk of encountering the nerves that perceive pain.

1. INTRODUCTION Exendin-4 is the first glucagon-like peptide-1 (GLP-1) receptor agonist to be approved for therapeutic use in humans. This peptide has 39 amino acids originally isolated from the saliva of the gila monster. It shares approximately 53% sequence homology with the mammalian gut hormone, GLP-1.1 Due to changes in amino acid sequence, exendin-4 is resistant to degradation against the enzyme dipeptidyl peptidase-4 (DPP-4),2−4 and has a longer half-life than native GLP-1. As a GLP-1 receptor agonist, exendin-4 shows numerous antidiabetic actions, including glucose-dependent simulation of insulin secretion,5,6 suppression of glucagon secretion,6,7 reduction of gastric mobility and food intake,6−8 and improvement in pancreatic endocrine function.6,7 Twice-daily subcutaneous injection has been associated with improvements in glycemic control in type 2 diabetic subjects that are inadequately treated with existing antidiabetic agents.9,10 However, its therapeutic utility is limited because of the frequent injections required and the associated inconvenience to patients. To address this issue, numerous research efforts have focused on chemical and/or structural modification of exendin-4,11,12 as well as the development of © 2015 American Chemical Society

Received: Revised: Accepted: Published: 272

October 9, 2015 December 1, 2015 December 9, 2015 December 9, 2015 DOI: 10.1021/acs.molpharmaceut.5b00765 Mol. Pharmaceutics 2016, 13, 272−279

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Molecular Pharmaceutics

used in IPGTTs. Rats were purchased from Japan SLC, Inc. (Shizuoka, Japan). All animal experiments were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The protocols for animal experiments were approved by the Animal Experimentation Committee of the Kyoto Pharmaceutical University. 2.2. Fabrication of FD4 and Exendin-4 Tip-Loaded Microneedle Arrays. The microneedle arrays were fabricated by micromolding from hyaluronic acid as a base material.19,20 The FD4 or exendin-4 was coated onto the tip of microneedle arrays using a dip-coating process with formulations composed of FD4 or exendin-4 and hyaluronic acid. Briefly, 5% hyaluronic acid solution was mixed well to prepare the dipping solutions containing 0.1−2% FD4 or exendin-4 for 0.5−10 μg of FD4 or exendin-4/microneedle array. The tips of microneedle arrays were then dipped into 500 μL of dipping solution quickly within 2 s. Depth of dipping was well adjusted with the control of dipping solution level. After the dip-coating process, the coated microneedle arrays were allowed to dry in a desiccator for 24 h at room temperature and then examined using a microscope to evaluate coating uniformity. To visualize the coated part of microneedle arrays, brilliant blue was coated onto the tip of microneedle arrays in the same manner. The mechanical strength of the resulting microneedle arrays was measured with a stress−strain gauge (FS1K, Imada, Japan) by pressing microneedle arrays against a stainless steel plate. When the tops of microneedle arrays were bent more than 45°, the force value was read from the meter. The failure force was calculated with the force divided by the area of microneedle arrays. The failure force of the microneedle arrays was 17.5 N/cm2. 2.3. Determination of Drug Contents in Tip-Loaded Microneedle Arrays. In this study, we prepared FD4 tiploaded microneedle arrays with doses of 0.5, 1, 2, and 4 μg of FD4/microneedle array. For determination of FD4 contents in the tip-loaded microneedle arrays, needles on arrays were collected and dissolved in phosphate buffered saline (pH 7.4). Concentrations of FD4 were determined by fluorescence HPLC as described below. 2.4. In Vitro Release Study. Release of FD4 from the tiploaded microneedle arrays across Silescol membranes was investigated using in vitro modified Franz cells. The content of FD4 in tip-loaded microneedle arrays was 4 μg/microneedle array. After application of microneedle arrays, the membranes with tip-loaded microneedle arrays were mounted onto Franz cells. Cells were positioned upright, and receptor compartments were filled with 2.4 mL of phosphate buffered saline (pH 7.4) maintained at 32 °C throughout the test period. At predetermined intervals, 0.2 mL of supernatant was withdrawn and replaced with an equal volume of fresh release medium. FD4 was analyzed by fluorescence HPLC as described below. 2.5. Histological and Microscopic Analysis. FD4 tiploaded microneedle arrays (10 μg FD4/microneedle array) were inserted into human cadaver skin and left in place for 5 min. In this study, microneedle arrays were applied into the skin by an applicator. The applicator can apply microneedle arrays into the membrane or skin with a definite force (15 N/cm2). With this applicator, the energy applied on microneedle arrays is well controlled, and the force is enough for the insertion of microneedle arrays into the skin. Therefore, our microneedle arrays can be inserted to the skin with a consistent depth using the applicators. Upon removal of the microneedle arrays, skin samples were subsequently embedded in OCT compound, flash

Therefore, this novel approach was proposed as a hybrid to combines the advantages of conventional injection needles and transdermal patch while minimizing their disadvantages. Among numerous research works regarding microneedle arrays, soluble microneedle arrays with drugs encapsulated within the needle matrix appear to be an attractive drug delivery system, due to the advantages of direct drug delivery along with dissolution of the needles in skin. Moreover, the materials for such microneedle arrays are safe and nontoxic after their application to skin. Based on these observations, in this study, hyaluronic acid was selected as a basis material to produce microneedle arrays. As a major component of skin, hyaluronic acid is reasonably expected to overcome the safety issues when applied to silicon and metal microneedle arrays. Moreover, hyaluronic acid is inexpensive and suitable for mass production, in contrast to other published methods that require more complex multistep fabrication schemes. In addition, the absence of organic solvents and elevated temperatures are notable advantages for preserving peptide and biomolecule stability. Due to these advantages, we have already developed microneedle arrays containing insulin and alendronate fabricated from hyaluronic acid, and have achieved sufficient transdermal delivery of these drugs using these novel microneedle arrays.19,20 However, in previous reports, drug was localized in whole microneedle arrays when microneedle arrays containing drugs were prepared.21−24 It is difficult to accurately control the drug dose delivered to skin, and to effectively deliver drug into skin, because only part of the needle could be inserted into skin due to skin deformation during insertion in many previous studies.23,25,26 In order to further improve the efficacy of soluble microneedle arrays, drug tip-loaded microneedle arrays were recently designed and developed. Using these tip-loaded microneedle arrays, the drug was only localized in the needle tips, which can be inserted into skin despite skin deformation. This allows drug delivery to be controlled and minimizes drug loss. Moreover, drugs with higher concentration microneedle arrays can be more quickly delivered to the skin, when compared with drugloaded whole microneedle arrays. Therefore, in this study, we designed and developed drug tip-loaded microneedle arrays on the transdermal delivery of drugs, particularly exendin-4. In addition, we evaluated the characteristics of exendin-4 tip-loaded microneedle arrays and compared their acute efficacy with subcutaneous injections by intraperitoneal glucose tolerance tests (IPGTTs) in type 2 diabetic GK/Slc rats in vivo.

2. MATERIALS AND METHODS 2.1. Materials. Hyaluronic acid was purchased from Kikkoman Biochemifa Company (Tokyo, Japan). FD4 was purchased from Sigma-Aldrich (St Louis, MO). Tissue-TeK O.C.T. Compound was purchased from Sakura Finetek Japan Co., Ltd. (Tokyo, Japan). Glucose CII-Test kits and exendin-4 were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Rat Insulin ELISA kits were purchased from Shibayagi Co., Ltd. (Gunma, Japan). Exendin-4 EIA kits were purchased from Phoenix Pharmaceuticals, Inc. (Belmont, CA). All other chemicals and reagents were of analytical reagent grade. Male Wistar rats aged 8 weeks and weighing 220−240 g were used for dermatoscope observation of test skin sites. Male GK/Slc rats aged 7−8 weeks and weighing 190−200 g were 273

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Molecular Pharmaceutics frozen in dry ice acetone (−78 °C), and sectioned into 10 μm slices using a Cryomicrotome (Histostat Cryostat Microtome, Buffalo, NY). Diffusion of FD4 and the pathway created by microneedle arrays in skin sections were observed under an all-in-one fluorescence microscope (BZ-8000; Keyence, Kyoto, Japan). 2.6. In Vivo Dermatoscope Observation. Back skin of Wistar rats was shaved 24 h prior to the experiment. Just before application, healthy rats were selected and skins in normal sections were used for subsequent studies. Microneedle arrays were inserted into the skin and left in place for 5 min. The subcutaneous injection was conducted with a 26G needle as a control. After removal of the microneedle arrays and subcutaneous injection with a 26G needle, test sites were observed at 0, 24, and 72 h with a Dermatoscope (DermaShotScope; Fineopto, Kyoto, Japan). 2.7. Acute Efficacy in IPGTTs. The acute efficacy of exendin-4 tip-loaded microneedle arrays was evaluated by IPGTTs in type 2 diabetic GK/Slc rats27after application of microneedle arrays, as compared with subcutaneous injection. GK/Slc rats were fasted for 14 h and anesthetized by intraperitoneal injection of 35 mg/kg pentobarbital sodium, and their abdominal regions were carefully shaved. GK/Slc rats were randomly allocated into five groups and then treated as follows: (a) transdermal application of exendin-4 tip-loaded microneedle arrays containing 10 μg/kg dose of exendin-4 (b) transdermal application of exendin-4 tip-loaded microneedle arrays containing 50 μg/kg dose of exendin-4 (c) subcutaneous injection of 10 μg/kg dose of exendin-4 solution (d) subcutaneous injection of 50 μg/kg dose of exendin-4 solution (e) transdermal application of microneedle arrays without exendin-4 Thirty minutes after administration of exendin-4 in each group, glucose (dose; 2 g/kg) was then intraperitoneally administered to each rat. At predetermined times, blood samples were collected from the jugular vein and were centrifuged at 10,000 rpm for 5 min in order to separate plasma immediately. Plasma concentrations of glucose, endogenous insulin, and exendin-4 were determined as described below. 2.8. Determination of Drugs. FD4 was analyzed using a fluorescence HPLC system (Hitachi, Kyoto, Japan). Samples were eluted on a C18 column (5C18-AR- II, 4.6 × 150 mm; Nacalai Tesque, Kyoto, Japan) using a mobile phase consisting of 0.1% phosphoric acid and acetonitrile (80:20). A flow rate of 1 mL/min was maintained. The excitation wavelength was 485 nm, and the emission wavelength was 585 nm. Plasma glucose levels were determined using a glucose CIITest kit, and plasma glucose levels at 0 min were considered to be 100%. Using this value, the percentage change in plasma glucose levels at each time point after dosing was calculated. Plasma concentrations of endogenous insulin were measured using a rat insulin ELISA kit, and plasma concentrations of exendin-4 were determined using an exendin-4 EIA kit. 2.9. Statistical Analyses. All statistical analyses were performed using GraphPad Prism Software (GraphPad Software Inc., San Diego, CA). Descriptive statistics are presented as mean values ± SE. Student’s paired t-test was used to compare the significance between data points. Multiple data sets between

groups were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s test when appropriate. Multiple data sets within groups were analyzed by ANOVA, followed by Dunnett’s test when appropriate. For all comparisons, P < 0.05 was considered to be statistically significant.

3. RESULTS 3.1. Determination of Drug Contents in Tip-Loaded Microneedle Arrays. As shown in Figure 1, the drug was

Figure 1. Schematic representation of drug tip-loaded microneedle arrays. To visualize the coated part of microneedle arrays, brilliant blue was coated onto the tip of microneedle arrays. Scale bars represent 300 μm. White arrowheads indicate the coated part of microneedle arrays.

designed to be loaded into tips (blue part) of microneedle arrays with a length of about 200 μm. Each needle was approximately 800 μm in height, with a diameter of 160 μm at the base and 40 μm at the tip, and an interspacing of 600 μm between the rows of needles. There were approximately 140 needles in a circular area with a diameter of 10 mm. In this study, FD4 was loaded into the tips of microneedle arrays, and the calculated doses of FD4 in these microneedle arrays were 1, 2, and 4 μg/microneedle array. The experimentally measured contents of FD4 in the tip-loaded microneedle arrays were determined and are summarized in Table 1. We found that the Table 1. FD4 Content of FD4 Tip-Loaded Microneedle Arrays dose of FD4 (μg/patch)

contenta (% of dose)

1 2 4

106.58 ± 8.90 102.43 ± 4.70 99.12 ± 5.68

Results are presented as means ± SE of four experiments in each group.

a

experimentally measured contents of FD4 in these microneedle arrays were almost constant, with a very small standard error. We observed less than 10% standard error values in the case of microneedle arrays with higher FD4 contents. 3.2. In Vitro Release Study. The cumulative release of FD4 from tip-loaded microneedle arrays was determined via an in vitro release study (Figure 2). FD4 tip-loaded microneedle arrays were readily dissolved, and FD4 was rapidly released from the microneedle arrays, particularly in the initial 30 s after 274

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that FD4 tip-loaded microneedle arrays were rapidly dissolved in the skin after application and that FD4 was released from the soluble microneedle arrays and was delivered to the skin by diffusion. 3.4. In Vivo Dermatoscope Observation. Figure 4 shows the surface images of rat skin after insertion of microneedle arrays and subcutaneous injection with a 26G needle. In the microneedle array treated group, pores corresponding to microneedle arrays were observed on test skin sites after removal of microneedle arrays. These pores gradually reduced with time and had almost disappeared at 24 h after application. In contrast, pores created by subcutaneous injection with a 26G needle were clearly observed, even at 72 h after application, although they were smaller when compared to 24 h after application. 3.5. Acute Efficacy in IPGTTs. Figure 5 compares exendin-4 tip-loaded microneedle arrays and subcutaneous injection of exendin-4 on glucose tolerance in IPGTTs. As shown in Figure 5A, blood glucose levels in the control group rapidly increased and reached a maximum value of 235.47 ± 10.91% of initial levels at 30 min after intraperitoneal glucose challenge. Dose-dependent suppression of the increased blood glucose levels was observed after subcutaneous preadministration of 10 μg/kg and 50 μg/kg exendin-4 when compared with the control group, and blood glucose values at 30 min in subcutaneous injection of 10 μg/kg and 50 μg/kg exendin-4 groups after intraperitoneal glucose challenge were 188.63 ± 9.14% and 126.57 ± 17.73%, respectively. In the case of the exendin-4 tip-loaded microneedle array group, normalization effects on blood glucose were found to be similar to the effects in the subcutaneous injection groups, and blood glucose values at 30 min in 10 μg/kg and 50 μg/kg exendin-4 tip-loaded microneedle array groups after intraperitoneal glucose challenge were 176.17 ± 13.35% and 137.66 ± 2.91%, respectively. Moreover, as shown in Figure 5B, calculated glucose AUC0−120min values in both of the microneedle arrays and the subcutaneous injection groups significantly decreased when compared with the control group. In addition, there were no significant differences in the calculated glucose AUC0−120min values between microneedle array groups and subcutaneous injection groups after administration of exendin-4 at the same dose. Figure 6A shows plasma insulin levels in the control group, the exendin-4 tip-loaded microneedle array group, and the subcutaneous injection group in IPGTTs. In the control group, insulin secretion increased slightly after intraperitoneal glucose challenge with a Cmax value of 1.89 ± 0.32 ng/mL at 15 min. In contrast, insulin secretion was markedly increased by subcutaneous injection of 10 μg/kg and 50 μg/kg exendin-4. The Cmax value in the case of 10 μg/kg subcutaneous injection of exendin-4 was 3.10 ± 0.56 ng/mL, while 50 μg/kg subcutaneous injection of exendin-4 had a Cmax value of 4.83 ± 0.32 ng/mL. On the other hand, similar results were observed on insulin secretion in the exendin-4 tip-loaded microneedle array groups. Cmax values of 10 μg/kg and 50 μg/kg exendin-4 tip-loaded microneedle array groups were 3.13 ± 0.24 ng/mL and 4.45 ± 0.48 ng/mL, respectively. In addition, as shown in Figure 6B, calculated insulin AUC0−60 min values in both the exendin-4 tip-loaded microneedle array and subcutaneous injection groups significantly increased when compared with controls. A dose-dependent increase in insulin AUC0−60 min values was observed in microneedle arrays and subcutaneous groups, and there were no significant differences in insulin secretion between the exendin-4 tip-loaded microneedle arrays group and the subcutaneous injection group.

Figure 2. In vitro release of FD4 from FD4 tip-loaded microneedle arrays in phosphate buffered saline (pH 7.4) maintained at 32 °C. Results are expressed as means ± SE of four experiments.

the start of the experiment. In the present study, most FD4 was released within 5 min, suggesting that the release of FD4 was very rapid from its tip-loaded microneedle arrays. 3.3. Histological and Microscopic Analyses. Figure 3 shows the cross section of human skin untreated and treated

Figure 3. Fluorescence microscope images of untreated skin section (A) and skin section after insertion of FD4 tip-loaded microneedle arrays for 5 min (B). Scale bars represent 100 μm. White arrowheads indicate the puncture site of microneedle arrays.

with FD4 tip-loaded microneedle arrays. The cross section of human skin was intact and no pores were observed in the skin without microneedle array treatment, as indicated in Figure 3A. On the other hand, as shown in Figure 3B, we observed that pores were created after application of FD4-tip loaded microneedle arrays, and that these microneedle arrays could successfully deliver FD4 into the skin. We also found that FD4 was localized in the resulting microchannels, thus suggesting 275

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Figure 4. Dermatoscopy of rat skin before and at 0, 24, and 72 h after treatment with microneedle arrays (A) and subcutaneous injection with a 26G needle (B).

Figure 6. Acute effects of exendin-4 (S.C. and M.N.) on plasma insulin response to glucose (2 g/kg) in GK rats (A), and calculated insulin AUC0−60min (B). Intraperitoneal glucose tolerance tests (IPGTTs) were carried out at 30 min after administration of exendin-4 (S.C. and M.N.). Results are presented as means ± SE of at least five experiments in each group. (***) p < 0.001 and (*) p < 0.05, compared with controls.

Figure 5. Acute effects of administration of exendin-4 by subcutaneous injection (S.C.) and exendin-4 tip-loaded microneedle arrays (M.N.) on glucose tolerance in GK rats (A), and calculated glucose AUC0−120min (B). Intraperitoneal glucose tolerance tests (IPGTTs) were carried out 30 min after administration of exendin-4 (S.C. and M.N.). Results are presented as means ± SE of at least five experiments in each group. (***) p < 0.001 and (*) p < 0.05, compared with controls.

of exendin-4 groups, plasma concentrations of exendin-4 rapidly increased and peaked at Cmax values of 19.86 ± 9.93 ng/mL (10 μg/kg subcutaneous injection of exendin-4) and 66.70 ± 26.11 ng/mL (50 μg/kg subcutaneous injection of exendin-4), respectively. The values decreased to baseline at 120 min after glucose challenge. In the case of exendin-4 tip-loaded microneedle arrays, similar plasma concentration−time profiles of exendin-4 were observed as with subcutaneous injection.

Figure 7A shows plasma profiles of exendin-4 in GK rats after the application of exendin-4 tip-loaded microneedle arrays and subcutaneous injection in IPGTTs. In subcutaneous injection 276

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experimentally measured amount of FD4 loaded in microneedle arrays largely coincided with the calculated dose, although there was some standard deviation, particularly in the case of exendin-4 administration at low dose. Due to the tip-specific localization of drug in microneedle arrays, a rapid release profile of FD4 was observed from its tiploaded microneedle arrays by in vitro release study. Namely, an initial burst release of FD4 was seen from FD4 tip-loaded microneedle arrays within the first 30 s, and most FD4 was released from the microneedle arrays within 5 min (Figure 2). These rapid release profiles for FD4 are superior when compared with dissolving microneedle arrays. For instance, the time needed for contained drug release varies from 25 min in galactose microneedle arrays29 to 120 h in amylopectin microneedle arrays.22 Moreover, in our previous studies, insulin was released within 1 h from whole microneedle arrays fabricated from hyaluronic acid (same material).20 As compared with our previous microneedle arrays, it is clear that the drug release time can be shortened by drug tip-loading. In this study, we prepared drug tip-loaded microneedle arrays in order to incorporate drug in their tips, as these tip-loaded microneedle arrays can efficiently deliver various drugs to the skin and avoid the loss of drug loaded in microneedle arrays. To confirm this, we observed a cross section of human skin after insertion of FD4 tip-loaded microneedle arrays. Figure 3 clearly shows the pathways of needle penetration, as well as the release of FD4 from tip-loaded microneedle arrays. We found that the penetration of FD4 after application of tip-loaded microneedle arrays was more effective than that after the application of whole drug-loaded microneedle arrays. This is due to skin deformation during insertion of microneedle arrays, as previously reported by Kolli et al.23 and Martanto et al.25 Moreover, the length inserted into skin was shown to be much longer than the FD4-loaded tips. These findings indicate that FD4 was completely and efficiently delivered into the skin by 200 μm tip-loaded microneedle arrays. In the in vitro release study, FD4 was rapidly released within 5 min after application of FD4 tip-loaded microneedle arrays, which is well correlated with their rapid dissolution. FD4 released from its tip-loaded microneedle arrays can then be delivered to skin by diffusion. In previous reports, when microneedle arrays were dissolved in the skin after application, fluorescence tracer was released from the dissolving microneedle arrays and diffused along the pathway created by the microneedle arrays.22,30 In contrast, in this study, deposition of FD4 was only observed at the bottom of the pathway created by microneedle arrays corresponding to tip of the microneedle arrays. We also confirmed that the tips of microneedle arrays were completely dissolved within 5 min after insertion of FD4 tip-loaded microneedle arrays (data not shown). Thus, when compared with previous microneedle arrays, the drug tiploaded microneedle arrays are more suitable for drug delivery into the dermis, and they can achieve rapid drug absorption from the skin into systemic circulation. In order to estimate the disruption and recovery of skin by the tip-loaded microneedle arrays, we compared skin injury and damage after the application of tip-loaded microneedle arrays with that after subcutaneous injection with a 26G needle using a dermatoscope. Figure 4 shows uniform insertion into skin by microneedle arrays. Moreover, the number of the pores created by microneedle arrays was much greater than the one pore created by subcutaneous injection, but the pore size was much smaller. These small pores reduced with time and almost

Figure 7. Plasma concentration−time profiles of exendin-4 (S.C. and M.N.) in GK rats (A) and calculated exendin-4 AUC−30−120min (B). Results are presented as means ± SE of at least five experiments in each group. (***) p < 0.001 and (*) p < 0.05, compared with controls.

As illustrated in Figure 7B, the calculated AUC−30−120min values of exendin-4 were significantly higher in both the exendin-4 tiploaded microneedle array group and the subcutaneous injection of exendin-4 group than in that of the control group. Moreover, a dose-dependent increase in AUC−30−120min values of exendin-4 was seen in the exendin-4 tip-loaded microneedle array group. No significant differences were observed in AUC−30−120min values of exendin-4 between the exendin-4 tip-loaded microneedle array group and the subcutaneous injection of exendin-4 group after application of the same dose of exendin-4.

4. DISCUSSION In previous papers, microneedle arrays were only partially inserted into skin due to skin deflection and elasticity. Thus, controlled dosing administered by dissolving microneedle arrays plays a critical role in their eventual use in medicine. To overcome the difficulties of controlling and delivering a specified drug dose using microneedle arrays, we developed a novel drug tip-loaded microneedle array, as shown in Figure 1, in which the drug is only incorporated in the 200 μm tip region of the needles. Although Sullivan et al. have reported microneedles with model drug located only in part of needles,28 FD4 in our tip-loaded microneedle arrays is more concentrated to the top 1/4 of needles compared with their needles. Moreover, there is a clear borderline between the part with drug loaded and the part without drug loaded as shown in Figure 1. In the present study, FD4 was used in order to simply and easily evaluate loaded drug dose, reproducibility, and physicochemical properties of tip-loaded microneedle arrays. As shown in Table 1, the amount of FD4 loaded in the tip of needles was determined at three doses. We found that the 277

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dose-dependent pharmacokinetics of exendin-4 in patients with type 2 diabetes mellitus.39 This difference may be due to different sources of peptide and assay methods utilized in each study. The presence of interspecies and/or interstrain differences may also contribute to such differences. Our present studies demonstrated the acute phase effect of exendin-4 tip-loaded microneedle arrays fabricated from hyaluronic acid. Further studies are necessary to assess efficacy and tolerability of exendin-4 using microneedle arrays with a longer period of time.

disappeared at 24 h after application, while the pores created by subcutaneous injection remained visible even at 72 h after application. Based on these findings, we confirmed that microneedle arrays did not cause serious skin damage or irritation, and that they were safe after application. Moreover, even though microneedle arrays were degraded in the skin, they were very safe, as they were composed of hyaluronic acid, an endogeneous biodegradable substance. In order to compare the efficacy of exendin-4 tip-loaded microneedle arrays with traditional subcutaneous injection, we examined glucose tolerance, insulin secretion, and plasma concentrations of exendin-4 in IPGTTs in type 2 diabetic GK/Slc rats in vivo. The GK rat is a lean model of spontaneous type 2 diabetes. It is derived from inbreeding of Wistar rats using glucose intolerance as the selection index. From around 5 weeks of age, GK rats show hyperglycemia, mild insulin resistance, impaired glucose-induced insulin secretion, and decreased β cell mass. This model differs from most type 2 diabetic animal models by being lean, but it has a characteristic resemblance with the human syndrome because of the polygenic origin.31 Exendin-4 is known to have promising antidiabetic effects in GK rats, such as HbA1c decrease,32 and delays the onset of overt diabetes.33 Moreover, it has been reported that acute administration of exendin-4 ameliorates hyperglycemia in animals by stimulating insulin secretion or directly promoting glucose metabolism.27,34−36 In this study, therefore, we used GK rats to assess and compare the efficacy of exendin-4 in tip-loaded microneedle arrays with that after subcutaneous injection. The experimental conditions were designed according to the method of Arakawa et al.,27 and we conducted administration of exendin-4 30 min before glucose challenge for better observation of insulin secretion in response to glucose change. We found that dose-dependent normalization of blood glucose was present in both the exendin-4 tiploaded microneedle array group and the subcutaneous injection group during IPGTTs (Figure 5). No significant differences were observed between the two different administration methods of exendin-4 at the same dose. Moreover, stimulation of insulin secretion by exendin-4 appeared to be dependent on glucose levels in both groups (Figure 6). This behavior is similar to previous studies, in which acute administration of exendin-4 lowered the glycemic response to glucose challenge as a result of augmented insulin secretion.27 We also observed that glucose normalization effects lasted longer than stimulation of insulin secretion, which is consistent with a previous report by Arakawa et al.27 It is possible that the insulin response was blunted, even though glucose was at much higher basal levels after rapid release during the initial phase (0−10 min), as shown in the pancreas perfusion study in GK rats by Edholm et al.37 In addition, we found that the pharmacokinetic properties of exendin-4 tip-loaded microneedle arrays were closely correlated with subcutaneous injection of exendin-4 at the same dose. From these observations, we also confirmed that exendin-4 was rapidly released from the tip-loaded microneedle arrays. Taken together, these results suggest that the efficacy of exendin-4 tip-loaded microneedle arrays is almost equivalent to that after subcutaneous injection. With regard to pharmacokinetics of exendin-4 in both the microneedle array and subcutaneous injection groups, Cmax and AUC−30−120min values increased with dose, but not proportionally (Figure 7). This is consistent with a report by Ai et al., who demonstrated nonlinear serum pharmacokinetics of exendin-4 in monkeys,38 although Kotlerman et al. reported

5. CONCLUSION In this study, we examined the potential use of exendin-4 tiploaded microneedle arrays as a useful alternative to develop a novel administration method for exendin-4. We found that the tip-loaded microneedle arrays improved transdermal delivery of exendin-4 without any skin damage. Furthermore, the exendin4 tip-loaded microneedle arrays had comparable effects on glucose tolerance, insulin secretion, and plasma concentration profiles, as compared with subcutaneous injection, in type 2 diabetic GK/Slc rats. Although we have to develop a process that allows for extensive scale-up in a safe, sterile, and reproducible system to produce clinical-grade microneedle arrays, this novel tip-loaded microneedle array may be useful for the clinical application of exendin-4.

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AUTHOR INFORMATION

Corresponding Author

*Department of Biopharmaceutics, Kyoto Pharmaceutical University, Misasagi-Nakauchi-cho 5, Yamashina-ku, Kyoto 607-8414, Japan. Tel: +81-75-595-4662. Fax: +81-75-595-4674. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by JSPS KAKENHI Grant Number 22590155, and by a grant from Uehara Memorial Foundation.

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REFERENCES

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