Jyuhn-Huarng Juang

In vivo evaluation of safety and efficacy of self-assembled nanoparticles for oral insulin delivery

A variety of approaches have been studied in the past to overcome the problems encountered with the oral delivery of insulin, but with little success. In this study, self-assembled nanoparticles (NPs) with a pH-sensitive characteristic were prepared by mixing the anionic poly-gamma-glutamic acid solution with the cationic chitosan solution in the presence of MgSO(4) and sodium tripolyphosphate. The in vitro results found that the transport of insulin across Caco-2 cell monolayers by NPs appeared to be pH-dependent; with increasing pH, the amount of insulin transported decreased significantly. An in vivo toxicity study was performed to establish the safety of the prepared NPs after oral administration. Additionally, the impact of orally administered NPs on the pharmacodynamics (PD) and pharmacokinetics (PK) of insulin was evaluated in a diabetic rat model. The in vivo results indicated that the prepared NPs could effectively adhere on the mucosal surface and their constituted components were able to infiltrate into the mucosal cell membrane. The toxicity study indicated that the NPs were well tolerated even at a dose 18 times higher than that used in the PD/PK study. Oral administration of insulin-loaded NPs demonstrated a significant hypoglycemic action for at least 10h in diabetic rats and the corresponding relative bioavailability of insulin was found to be 15.1+/-0.9%. These findings suggest that the NPs prepared in the study are a promising vehicle for oral delivery of insulin.

Enteric-coated capsules filled with freeze-dried chitosan/poly(γ-glutamic acid) nanoparticles for oral insulin delivery

A pH-sensitive nanoparticle (NP) system composed of chitosan and poly(gamma-glutamic acid) was prepared for the oral delivery of insulin. The biodistribution study in a rat model showed that some of the orally administered NPs were retained in the stomach for a long duration, which might lead to the disintegration of NPs and degradation of insulin. To overcome these problems, we freeze-dried NPs and filled them in an enteric-coated capsule. The small angle X-ray scattering (SAXS) profiles indicated that the freeze-drying process did not significantly disrupt the internal structure of NPs; additionally, their pH-sensitivity was preserved and the insulin release was pH-dependent. The results obtained in the native PAGE analysis indicated that the released insulin molecules were neither fragmented nor aggregated. Upon oral administration, the enteric-coated capsule remained intact in the acidic environment of the stomach, but dissolved rapidly in the proximal segment of the small intestine. Consequently, all the NPs loaded in the capsule were brought into the small intestine, thus enhancing the intestinal absorption of insulin and providing a prolonged reduction in blood glucose levels. The relative bioavailability of insulin was found to be approximately 20%. These results suggest that the formulation developed in the study might be employed as a potential approach for the oral delivery of insulin.

Biodistribution, pharmacodynamics and pharmacokinetics of insulin analogues in a rat model: Oral delivery using pH-Responsive nanoparticles vs. subcutaneous injection

In this study, we report the biodistribution of aspart-insulin, a rapid-acting insulin analogue, following oral or subcutaneous (SC) administration to rats using the single-photon emission computed tomography (SPECT)/computed tomography (CT). Oral delivery of aspart-insulin was achieved using a pH-responsive nanoparticle (NP) system composed of chitosan (CS) and poly(gamma-glutamic acid). The results obtained in the SPECT/CT study indicate that the orally administered aspart-insulin was absorbed into the systemic circulation, while the drug carrier (CS) was mainly retained in the gastrointestinal tract.Via the SC route, the peak aspart-insulin concentration in the peripheral tissue/plasma was observed at 20 min after injection. Within 3 h, half of the initial dose (ID) of aspart-insulin was degraded and excreted into the urinary bladder. In contrast, via oral delivery, there was constantly circulating aspart-insulin in the peripheral tissue/plasma during the course of the study, while 20% of the ID of aspart-insulin was metabolized and excreted into the urinary bladder. In the pharmacodynamic (PD) and pharmacokinetic (PK) evaluation in a diabetic rat model, the orally administered aspart-insulin loaded NPs produced a slower hypoglycemic response for a prolonged period of time, whereas the SC injection of aspart-insulin produced a more pronounced hypoglycemic effect for a relatively shorter duration. Finally, comparison of the PD/PK profiles of the orally administered aspart-insulin with those of the SC injection of NPH-insulin, an intermediate-acting insulin preparation, suggests the suitability of our NP system to be used as a non-invasive alternative for the basal insulin therapy.

Beneficial effects of hyperbaric oxygen therapy on islet transplantation.

We envisage that hyperbaric oxygen (HBO) would ameliorate islet anoxia, preventing early graft failure. Thus, treatment of HBO to diabetic recipients should improve the outcome of islet transplantation. We tested this hypothesis by syngeneically transplanting insufficient number of islets (150 islets) into streptozotocin-diabetic C57BL/6 mice, each followed by HBO (2.4 ATA, 100% O2) therapy for 1.5 h from day 0 to 28, once daily (group A) or twice daily (group B), or from day 5 to 28, once daily (group C) or twice daily (group D), 6 days/week. Recipients without HBO treatment served as controls. At day 28 after transplantation, groups B, C, and D gained weight and had lower blood glucose compared with their baseline values. In addition, groups B and D had higher insulin content of the graft than the controls. To determine the optimal timing of HBO therapy, groups B and D were compared with recipients treated with HBO twice daily, 6 days/week, from day –14 to 0 (group E) and from day –14 to 28 (group F). At day 28 after transplantation, groups B, D, E, and F had significantly lower blood glucose than their individual baseline values and higher insulin content of the graft than controls. But only group F had more β-cell mass of the graft than controls. These findings lend credence to the expectation that peritransplant application of adequate frequency of HBO to diabetic recipients would enhance the performance and growth of the islet graft, resulting in an improvement of the outcome of the transplantation.

Binding characteristics of 9-fluoropropyl-(+)-dihydrotetrabenzazine (AV-133) to the vesicular monoamine transporter type 2 in rats ☆

UNLABELLEDnThe vesicular monoamine transporter type 2 (VMAT2) is highly expressed in pancreatic beta-cells and thus has been proposed to be a potential target for measuring beta-cell mass (BCM) by molecular imaging. C-11- and F-18-labeled tetrabenazine derivatives targeting VMAT2 have shown some promising results as potential biomarkers for BCM. In the present study, we examined the binding characteristics of 9-fluoropropyl-(+)-dihydrotetrabenzazine ([(18)F]AV-133), a potential PET tracer for BCM imaging, in rat pancreas and rat brain.nnnMETHODSnPancreatic exocrine cells and pancreatic islet cells were isolated and purified from Sprague-Dawley rats. Membrane homogenates, prepared from both pancreatic exocrine and islet cells as well as from brain striatum and hypothalamus regions, were used for in vitro binding studies. In vitro and ex vivo autoradiography studies with [(18)F]AV-133 were performed on rat brain and rat pancreas sections. Immunohistochemistry studies were performed to confirm the distribution of VMAT2 on islet beta-cells.nnnRESULTSnExcellent binding affinities of [(18)F]AV-133 were observed in rat striatum and hypothalamus homogenates with K(d) values of 0.19 and 0.25 nM, respectively. In contrast to single-site binding observed in rat striatum homogenates, rat islet cell homogenates showed two saturable binding sites (site A: K(d)=6.76 nM, B(max)=60 fmol/mg protein; site B: K(d)=241 nM, B(max)=1500 fmol/mg protein). Rat exocrine pancreas homogenates showed only a single low-affinity binding site (K(d)=209 nM), which was similar to site B in islet cells. In vitro autoradiography of [(18)F]AV-133 using frozen sections of rat pancreas showed specific labeling of islets, as evidenced by co-localization with anti-insulin antibody. Ex vivo VMAT2 pancreatic autoradiography in the rat, however, was not successful, in contrast to the excellent ex vivo autoradiography of VMAT2 binding sites in the brain. In vivo/ex vivo islet labeling may be complicated by the presence of the low-affinity/high-capacity site B binding in rat pancreas.nnnCONCLUSIONSn[(18)F]AV-133 is an excellent imaging agent for mapping VMAT2 sites in rat brain and specifically binds rat islet cells in vitro and postmortem. Additional optimization may be required to achieve ex vivo islet beta-cell labeling in rats.

Assessment of the function and effect of diabetes education programs in Taiwan

A multi-center prospective study was conducted to assess the function and impact of diabetic education programs on diabetic control. A total of 208 subjects with type 2 diabetes were recruited. Diabetes self-care, assessed by questionnaire, was evaluated before, and 4 months after attending a diabetes education course. A total of 121 subjects who received advanced diabetes education courses were designated as the experimental group. A second group of 87 cases receiving a basic course served as controls. In addition to basic knowledge, the advanced education programs included dietary control, blood glucose monitoring, management of hypoglycemia, medication compliance, foot care and exercise. Diabetes self-care techniques were significantly improved in the experimental group. The overall score for diabetes self-care techniques improved in both groups at the 4th month over baseline values. The change was significant with the controls (P < 0.001). Multiple regression analysis confirmed the intensity of diabetic education was the only significant variable correlated with the decrease of fasting blood glucose and systolic blood pressure. In conclusion, integrated and intensive diabetes education program in diabetes education centers provides an effective method for improving diabetes self-care techniques and metabolic outcome.

Augmentation of diabetic wound healing and enhancement of collagen content using nanofibrous glucophage-loaded collagen/PLGA scaffold membranes

This work developed nanofibrous drug-loaded collagen/poly-D-L-lactide-glycolide (PLGA) scaffold membranes that provided the sustained release of glucophage for the wounds associated with diabetes. PLGA, glucophage, and collagen were firstly dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol and were spun into nanofibrous membranes by electrospinning. High-performance liquid chromatography assay was used to characterize the in vivo and in vitro release rates of the pharmaceuticals from the membranes. High concentrations of glucophage were released for over three weeks from the nanofibrous membranes. The nanofibrous glucophage-loaded collagen/PLGA membranes were more hydrophilic than collagen/PLGA membranes and exhibited a greater water-containing capacity. The glucophage-loaded collagen/PLGA membranes markedly promoted the healing of diabetic wounds. Moreover, the collagen content of diabetic rats using drug-eluting membranes was higher than that of the control rats, because of the down-regulation of matrix metalloproteinase 9. The experimental results herein suggest that the nanofibrous glucophage-loaded collagen/PLGA membranes had effect for increasing collagen content in treating diabetic wounds and very effective promoters of the healing of such wounds in the early stages.

Three-dimensional optical method for integrated visualization of mouse islet microstructure and vascular network with subcellular-level resolution

Microscopic visualization of islets of Langerhans under normal and diabetic conditions is essential for understanding the pathophysiology of the disease. The intrinsic opacity of pancreata, however, limits optical accessibility for high-resolution light microscopy of islets in situ. Because the standard microtome-based, 2-D tissue analysis confines visualization of the islet architecture at a specific cut plane, 3-D representation of image data is preferable for islet assessment. We applied optical clearing to minimize the random light scattering in the mouse pancreatic tissue. The optical-cleared pancreas allowed penetrative, 3-D microscopic imaging of the islet microstructure and vasculature. Specifically, the islet vasculature was revealed by vessel painting-lipophilic dye labeling of blood vessels-for confocal microscopy. The voxel-based confocal micrographs were digitally processed with projection algorithms for 3-D visualization. Unlike the microtome-based tissue imaging, this optical method for penetrative imaging of mouse islets yielded clear, continuous optical sections for an integrated visualization of the islet microstructure and vasculature with subcellular-level resolution. We thus provide a useful imaging approach to change our conventional planar view of the islet structure into a 3-D panorama for better understanding of the islet physiology.

Magnetic Resonance Imaging of Transplanted Mouse Islets Labeled With Chitosan-Coated Superparamagnetic Iron Oxide Nanoparticles

Although only 10% of islet recipients maintain insulin independence, 80% of them are C-peptide positive at 5 years after transplantation. To better understand the fate of transplanted islets, a magnetic resonance imaging (MRI) technique has been used to detect Feridex-labeled islet grafts in rodents. In this study, we used a novel MRI contrast agent, chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles, to monitor mouse islet grafts. Male inbred C57BL/6 mice were used as donors and recipients of islet transplantation. The islet cytotoxicity was evaluated by fluorescein diacetate and propidium iodide staining for RAW cells incubated with CSPIO. After being incubated overnight with and without CSPIO (10 mg/mL), 300 islets were transplanted under the left kidney capsule of each mouse. After transplantation, 3.0-Tesla MRI of the recipients was performed biweekly until 19 weeks. At the end of study, the islet graft was removed for insulin and Prussian blue staining. The cell death rates in RAW cells did not increase with increasing CSPIO concentrations or incubation time. The grafts of CSPIO-labeled islets were visualized on MRI scans as distinct hypointense spots homogeneously located at the upper pole of left kidney. Their MRI signal was 30%-50% that of control islets and was maintained throughout the follow-up period. At 18 weeks, the histology of CSPIO-labeled islet graft revealed the insulin- and iron-stained areas to be almost identical. Our results indicate that isolated mouse islets labeled with CSPIO nanoparticles can be effectively and safely imaged by using MRI as long as 18 weeks after transplantation.

Enhancement of Diabetic Wound Repair Using Biodegradable Nanofibrous Metformin-Eluting Membranes: in Vitro and in Vivo

This work developed biodegradable nanofibrous drug-eluting membranes that provided sustained release of metformin for repairing wounds associated with diabetes. To prepare the biodegradable membranes, poly-d-l-lactide-glycolide (PLGA) and metformin were first dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and were spun into nanofibrous membranes by electrospinning. An elution method and an HPLC assay were utilized to characterize the in vivo and in vitro release rates of the pharmaceuticals from the membranes. The biodegradable nanofibrous membranes released high concentrations of metformin for more than three weeks. Moreover, nanofibrous metformin-eluting PLGA membranes were more hydrophilic and had a greater water-containing capacity than virgin PLGA fibers. The membranes also improved wound healing and re-epithelialization in diabetic rats relative to the control. The experimental results in this work suggest that nanofibrous metformin-eluting membranes were functionally active in the treatment of diabetic wounds and very effective as accelerators in the early stage of healing of such wounds.