Hidemasa Katsumi

The development and characteristics of novel microneedle arrays fabricated from hyaluronic acid, and their application in the transdermal delivery of insulin

The aim of the present study was to develop novel insulin-loaded microneedle arrays (MNs) fabricated from hyaluronic acid (HA), and characterize their applicability in the transdermal delivery of insulin. The shape of MNs was observed via scanning electron microscopy. The characteristics of these novel insulin-loaded MNs, including hygroscopy, stability, drug release profiles, and dissolution properties, were evaluated from a clinical application point-of-view. Transepidermal water loss (TEWL) was measured to investigate the piercing properties of MNs, and the recovery of the skin barrier after the removal of MNs to confirm their safety. Additionally, the transdermal absorption of insulin from MNs was examined via an in vivo absorption study in diabetic rats. The length of MNs was 800 μm with a base diameter of 160 μm and a tip diameter of 40 μm. MNs were found to maintain their skin piercing abilities for at least 1h, even at a relative humidity of 75%. After storing insulin-loaded MNs for a month at -40, 4, 20, and 40 °C, more than 90% of insulin remained in MNs at all temperatures, indicating that insulin is highly stable in MNs at these storage conditions. It was also found that insulin is rapidly released from MNs via an in vitro release study. These findings were consistent with the complete dissolution of MNs within 1h of application to rat skin in vivo. Therefore, the novel HA MNs possess self-dissolving properties after their dermal application, and insulin appears to be rapidly released from these MNs. A significant increase in TEWL was observed after the application of MNs. However, this parameter recovered back to baseline within 24h after the removal of MNs. These findings indicate that the transdermal transport pathway of insulin, which was created by the MNs, disappeared within 24h, and that the skin damage induced by the MNs was reversible. Furthermore, a dose-dependent hypoglycemic effect and transdermal delivery of insulin were observed after a dermal treatment with insulin-loaded MNs in vivo. A continuous hypoglycemic effect was observed after 0.25 IU of insulin was administered to skin via MNs. Additionally, lower peak plasma insulin levels, but higher plasma insulin concentrations after 2 h, were achieved with 0.25 IU of insulin administered via MNs as compared to the subcutaneous administration of insulin of the same dose. Pharmacodynamic and pharmacokinetic parameters indicated that insulin administered via MNs was almost completely absorbed from the skin into the systemic circulation, and that the hypoglycemic effect of insulin-loaded MNs was almost similar to that of the subcutaneous injection of insulin. These findings indicate that the novel insulin-loaded MNs fabricated from HA are a very useful alternative method of delivering insulin via the skin into the systemic circulation without inducing serious skin damage. Therefore, HA MNs may be an effective and safe method of transdermal insulin delivery in the clinic.

Transdermal delivery of relatively high molecular weight drugs using novel self-dissolving microneedle arrays fabricated from hyaluronic acid and their characteristics and safety after application to the skin

The purpose of this study was to develop novel dissolving microneedle arrays fabricated from hyaluronic acid (HA) as a material and to improve the transdermal permeability of relatively high molecular weight drugs. In this study, fluorescein isothiocyanate-labeled dextran with an average molecular weight of 4kDa (FD4) was used as a model drug with a relatively high molecular weight. The microneedle arrays significantly increased transepidermal water loss (TEWL) and reduced transcutaneous electrical resistance (TER), indicating that they could puncture the skin and create drug permeation pathways successfully. Both TEWL and TER almost recovered to baseline levels in the microneedle array group, and relatively small pathways created by the microneedles rapidly recovered as compared with those created by a tape stripping treatment. These findings confirmed that the microneedle arrays were quite safe. Furthermore, we found that the transdermal permeability of FD4 using the microneedle arrays was much higher than that of the FD4 solution. Furthermore, we found that the microneedle arrays were much more effective for increasing the amount of FD4 accumulated in the skin. These findings indicated that using novel microneedle arrays fabricated from HA is a very useful and effective strategy to improve the transdermal delivery of drugs, especially relatively high molecular weight drugs without seriously damaging the skin.

Improvement of intestinal absorption of insulin and water-soluble macromolecular compounds by chitosan oligomers in rats

The effects of five chitosan oligomers on the intestinal absorption of fluorescein isothiocyanate-labeled dextrans (FDs) and insulin were studied by an in situ loop method. The absorption of FD4 from the jejunum was effectively improved in the presence of 0.5% (w/v) chitosan hexamer and dimer. However, chitosan hexamer did not improve the colonic absorption of FD4, although we found a moderate increase in the colonic absorption of FD4 in the presence of chitosan pentamer and dimer. The absorption enhancing effect of chitosan hexamer decreased as the molecular weights of FDs increased. In addition, we found a remarkable increase in plasma insulin levels and a significant hypoglycemic effect after jejunal administration of insulin with chitosan hexamer. In the toxicity studies of chitosan hexamer, we found no significant increase in the release of total protein and activity of lactate dehydrogenase (LDH) from the intestinal epithelium in the presence of chitosan hexamer (0.5%, w/v), indicating that this compound was a safe absorption enhancer for improving the intestinal absorption of poorly absorbable drugs. Finally, the transepithelial electrical resistance (TEER) and the permeability of FD4 in rat jejunal membranes with or without chitosan hexamer (0.5%, w/v) were examined by an in vitro diffusion chamber method. We observed a moderate decrease in the TEER values of rat jejunal membranes and a corresponding increase in the permeability of FD4 in the presence of chitosan hexamer (0.5%, w/v). These findings suggested that chitosan hexamer might loosen the tight junction of the intestinal epithelium, thereby improving the intestinal permeability of hydrophilic macromolecular compounds via a paracellular pathway.

Polyamidoamine dendrimers as novel potential absorption enhancers for improving the small intestinal absorption of poorly absorbable drugs in rats

Effects of polyamidoamine (PAMAM) dendrimers on the intestinal absorption of poorly absorbable drugs were examined by an in situ closed loop method in rats. 5(6)-Carboxyfluorescein (CF), fluorescein isothiocyanate-dextrans (FDs) with various molecular weights, calcitonin and insulin were used as model drugs of poorly absorbable drugs. The absorption of CF, FD4 and calcitonin from the rat small intestine was significantly enhanced in the presence of PAMAM dendrimers. The absorption-enhancing effects of PAMAM dendrimers for improving the small intestinal absorption of CF were concentration and generation dependent and a maximal absorption-enhancing effect was observed in the presence of 0.5% (w/v) G2 PAMAM dendrimer. However, G2 PAMAM dendrimer had almost no absorption-enhancing effect on the small intestinal absorption of macromolecular drugs including FD10 and insulin. Overall, the absorption-enhancing effects of G2 PAMAM dendrimer in the small intestine decreased as the molecular weights of drug increased. However, G2 PAMAM dendrimer did not enhance the intestinal absorption of these drugs with different molecular weights in the large intestine. Furthermore, we evaluated the intestinal membrane damage with or without G2 PAMAM dendrimer. G2 PAMAM dendrimer (0.5% (w/v)) significantly increased the activities of lactate dehydrogenase (LDH) and the amounts of protein released from the intestinal membranes, but the activities and amounts of these toxic markers were less than those in the presence of 3% Triton X-100 used as a positive control. Moreover, G2 PAMAM dendrimer at concentrations of 0.05% (w/v) and 0.1% (w/v) did not increase the activities and amounts of these toxic markers. These findings suggested that PAMAM dendrimers at lower concentrations might be potential and safe absorption enhancers for improving absorption of poorly absorbable drugs from the small intestine.

Improvement of intestinal absorption of water-soluble macromolecules by various polyamines: Intestinal mucosal toxicity and absorption-enhancing mechanism of spermine

The absorption-enhancing effects of three different polyamines, spermine (SPM), spermidine (SPD) and putrescine (PUT) on the intestinal absorption of water-soluble macromolecules were examined in rats. Fluorescein isothiocyanate-labeled dextrans (FDs) with different average molecular weights were chosen as models of water-soluble macromolecules and intestinal absorption of FDs with or without these polyamines was examined by an in situ closed loop method. The intestinal absorption of fluorescein isothiocyanate-labeled dextran with an average molecular weight of 4400 (FD4) was relatively low in the absence of these polyamines. However, its absorption was improved in the presence of 5-10mM SPM and 10mM SPD in the jejunum and 10mM SPM in the colon, while 10mM PUT had almost no absorption-enhancing effect on the intestinal absorption of FD4. Overall, the enhancing effects of these polyamines were greater in the jejunal membranes than in the colonic membranes. The absorption-enhancing effect of SPM decreased as the molecular weights of FDs increased. The intestinal membrane toxicity of 10mM SPM was evaluated by measuring the amount of protein and activity of lactate dehydrogenase (LDH) released from the intestinal epithelial cells. We also observed the morphological changes of intestinal mucosa in the presence or absence of SPM. The results indicated that the amount of protein and LDH was not changed in the presence of 10mM SPM, although we observed a significant increase in these biological markers in the presence of 3% Triton X-100, as a positive control. Furthermore, we found no significant change in the intestinal membrane with 10mM SPM by the morphological observation. These findings suggested that 10mM SPM did not cause any significant membrane damage to the intestinal epithelium. To investigate the absorption-enhancing mechanism of SPM, the transepithelial electrical resistance (TEER) of the rat jejunal membranes was studied by using a diffusion chamber method. SPM decreased the TEER values in a concentration dependent manner and 10mM SPM had almost the same effect to decrease the TEER value compared with 10mM EDTA as a positive control. These findings suggest that SPM may loosen the tight junction of the epithelium, thereby increasing the intestinal absorption of drugs via a paracellular route. In summary, polyamines, especially SPM would be one of the suitable absorption enhancers with high effectiveness and low intestinal membrane toxicity.

Polyamidoamine dendrimers can improve the pulmonary absorption of insulin and calcitonin in rats

The absorption-enhancing effects of polyamidoamine (PAMAM) dendrimers with various generations (G0-G3) and concentrations [0.1%-1.0% (w/v)] on the pulmonary absorption of peptide and protein drugs were studied in rats. Insulin and calcitonin were chosen as models of peptide and protein drugs, and their pulmonary absorption with or without PAMAM dendrimers was examined by in vivo pulmonary absorption studies. PAMAM dendrimers significantly increased the pulmonary absorption of insulin and calcitonin in rats, and their absorption-enhancing effects were generation dependent. The rank order of absorption enhancement effect of these PAMAM dendrimers was G3 > G2 > G1 > G0. For the same generation, the absorption-enhancing effects of PAMAM dendrimers were shown to be concentration dependent. The toxicity of these PAMAM dendrimers in the lung tissues was evaluated by measuring the release of protein and the activities of lactate dehydrogenase (LDH) in bronchoalveolar lavage fluid (BALF). The PAMAM dendrimers with various generations and concentrations did not significantly increase the release of protein and the activities of LDH in BALF, indicating that these dendrimers did not cause any membrane damage to the lung tissues. The zeta potentials of insulin and calcitonin solutions changed to positive by the addition of PAMAM dendrimers, and the degree of positive charge as determined by the zeta potentials was linearly correlated with the absorption-enhancing effects of the PAMAM dendrimers. This positive charge of the PAMAM dendrimers might be related to their absorption-enhancing mechanisms for improving the pulmonary absorption of insulin and calcitonin in rats. In conclusion, the PAMAM dendrimers are suitable absorption enhancers to improve the pulmonary absorption of insulin and calcitonin without any membrane damage to the respiratory tissues.

Development of nitric oxide donors for the treatment of cardiovascular diseases.

Nitric oxide (NO) is a molecule that dynamically modulates the physiological functions of the cardiovascular system, which include relaxation of vascular smooth muscle, inhibition of platelet aggregation, and regulation of immune responses. Because a reduced NO level has been implicated in the onset and progression of various disease states, NO is expected to provide therapeutic benefits in the treatment of cardiovascular diseases, such as essential hypertension, stroke, coronary artery disease, atherosclerosis, platelet aggregation after percutaneous transluminal coronary angioplasty, and ischemia/reperfusion injury. To date, pharmacologically active compounds that can release NO within the body, such as organic nitrates and sodium nitroprusside, have been used as therapeutic agents, but their efficacy is significantly limited by their rapid NO release, poor distribution to the target site, toxicity, and induction of tolerance. Therefore, new NO donors with better pharmacological and pharmacokinetic properties would be highly desirable. In this review, recent challenges in the development of new NO donors and NO delivery systems are summarized. Then, future developments of novel NO donors are also discussed in order to optimize NO delivery in the treatment of cardiovascular diseases.

Development of a Novel Self-Dissolving Microneedle Array of Alendronate, a Nitrogen-Containing Bisphosphonate: Evaluation of Transdermal Absorption, Safety, and Pharmacological Effects After Application in Rats

Alendronate is a nitrogen-containing bisphosphonate that is widely used for the treatment of osteoporosis. In this study, we developed a novel self-dissolving micron-size needle array (microneedle array) containing alendronate, which was fabricated by micromodeling technologies using hyaluronic acid as a basic material. Micron-scale pores in the skin were seen after the application of the alendronate-loaded microneedle array, verifying establishment of transdermal pathways for alendronate. The absorption of alendronate after the application of alendronate-loaded microneedle array was almost equivalent to that after subcutaneous administration, and the bioavailability of alendronate was approximately 90% in rats. Furthermore, delivery of alendronate via this strategy effectively suppressed the decrease in the width of the growth plate in a rat model of osteoporosis. Although mild cutaneous irritation was observed after the application of the alendronate-loaded microneedle array, it resolved by day 15. These findings indicate that this alendronate-loaded microneedle array is a promising transdermal formulation for the treatment of osteoporosis.

Development of polyethylene glycol-conjugated alendronate, a novel nitrogen-containing bisphosphonate derivative: Evaluation of absorption, safety, and effects after intrapulmonary administration in rats

Bisphosphonates are widely used for the treatment of bone diseases, including hypercalcemia and osteoporosis. However, the bioavailability (BA) of orally administered bisphosphonates is low, at approximately 0.9%-1.8%. In addition, the oral administration of bisphosphonates is associated with mucosal damage, including gastritis, gastric ulcer, and erosive esophagitis. Here, to develop a new delivery system for bisphosphonates that improve their BA and safety, we developed polyethylene glycol (PEG)-conjugated alendronate, a novel nitrogen-containing bisphosphonate derivative. We evaluated the absorption and safety of PEG-alendronate in rats following intrapulmonary administration. The BA of PEG-alendronate after intrapulmonary administration was approximately 44 ± 10% in rats, which was similar to that of alendronate (54 ± 3.9%). Alendronate significantly increased total protein concentration and lactate dehydrogenase activity in bronchoalveolar lavage fluid, suggesting that pulmonary epithelium was locally damaged by intrapulmonary administration of alendronate. In marked contrast, PEG-alendronate did not significantly increase the markers following intrapulmonary administration. In an osteoporosis model in rats, intrapulmonary administration of PEG-alendronate effectively inhibited decreases in the width of the growth plate to a level similar to that achieved by intrapulmonary administration of alendronate. These results indicate that pulmonary delivery of PEG-alendronate is a promising approach for the treatment of bone diseases.

Synthesis of Tat tagged and folate modified N-succinyl-chitosan self-assembly nanoparticles as a novel gene vector.

The purpose of this research was to prepare a novel type of Tat tagged and folate modified N-succinyl-chitosan (Tat-Suc-FA) self-assembly nanoparticles, to provide a new vector for tumor gene therapy. In this study, Tat-Suc-FA polymers was synthesized and characterized using (1)H NMR and FT-IR. The copolymer had a mean diameter of 65 ± 22.6 nm, a zeta potential of 40 ± 0.2 mV. The cytotoxicity assay showed that Tat-Suc-FA polymers were less toxic than chitosan in the tested concentration range (from 2 to 500 μg/ml). Tat-Suc-FA/DNA complexes at various weight ratios were formulated and characterized. Particle sizes of Tat-Suc-FA/DNA complexes were between 54 and 106 nm as determined by dynamic light scattering. Accordingly, Transmission electron microscope photo of Tat-Suc-FA/DNA complexes exhibited a spherical and compact morphology. Zeta potentials of these complexes changed as the weight ratio varied (from 3 to 44 mV). Agarose gel electrophoresis assay showed that Tat-Suc-FA could efficiently condense the DNA, when the weight ratio was above 1.5/1. Together, these results suggest that the low toxic Tat-Suc-FA cationic polymers could be considered for use as a novel type of gene delivery vectors.