Fumio Kamiyama

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.

Transcutaneous immunization using a dissolving microneedle array protects against tetanus, diphtheria, malaria, and influenza.

Transcutaneous immunization (TCI) is an attractive alternative vaccination route compared to the commonly used injection systems. We previously developed a dissolving microneedle array for use as a TCI device, and reported that TCI with the dissolving microneedle array induced an immune response against model antigens. In the present study, we investigated the vaccination efficacy against tetanus and diphtheria, malaria, and influenza using this vaccination system. Our TCI system induced substantial increases in toxoid-specific IgG levels and toxin-neutralizing antibody titer and induced the production of anti-SE36 IgG, which could bind to malaria parasite. On influenza HA vaccination, robust antibody production was elicited in mice that provided complete protection against a subsequent influenza virus challenge. These findings demonstrate that TCI using a dissolving microneedle array can elicit large immune responses against infectious diseases. Based on these results, we are now preparing translational research for human clinical trials.

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.

A transcutaneous vaccination system using a hydrogel patch for viral and bacterial infection

Abstract One of the most important anthropic missions is preventing the global spread of infectious diseases. Vaccination is the only available preventive treatment for infectious diseases, but the availability of vaccines in developing countries is not adequate. We report a simple, easy-to-use, noninvasive hydrogel patch transcutaneous vaccination system. Antigen (Ag)-specific IgG production was induced by applying an Ag-immersed patch to non-pretreated mouse auricle or hairless rat back skin. Immunofluorescence histochemical analysis revealed that Langerhans cells resident in the epidermal layer captured the antigenic proteins delivered by the hydrogel patch, which promoted the penetration of antigenic proteins through the stratum corneum, and that Ag-capturing Langerhans cells migrated into draining lymph nodes. Humoral immunity elicited by our transcutaneous vaccination system demonstrated neutralizing activity in both adenoviral infection and passive-challenge tetanus toxin experiments. The use of this hydrogel patch transcutaneous vaccination system will facilitate the global distribution of effective and convenient vaccines.

A low-invasive and effective transcutaneous immunization system using a novel dissolving microneedle array for soluble and particulate antigens

Transcutaneous immunization (TCI) is a promising needle-free, easy-to-use, and low-invasive vaccination method. The hydrogel patch-based TCI system induced immune responses against soluble antigens (Ags) like toxoids, but could not induce immune responses against particulate Ags. Here, as an effective TCI system against every form of Ag, we developed a dissolving microneedle array of three lengths (200, 300, or 800 μm) made of hyaluronate as a novel TCI device. Unlike conventional microneedles, the microneedles of our dissolving microneedle arrays dissolved in the skin after insertion. Each dissolving microneedle array effectively delivered both soluble and particulate Ags under the stratum corneum. TCI using these dissolving microneedle arrays induced effective immune responses in rats regardless of the Ag form that were comparable to conventional vaccination using subcutaneous immunization. In addition, application of these dissolving microneedle arrays caused only slight skin irritation. These findings suggest that our TCI system can simply, safely, and effectively improve protective immune responses for every vaccine Ag.

Development and Clinical Study of a Self-Dissolving Microneedle Patch for Transcutaneous Immunization Device

ABSTRACTPurposeWe previously reported the safety and efficacy in animal experiments of transcutaneous immunization (TCI) using a self-dissolving microneedle patch (MicroHyala; MH) made of hyaluronic acid and collagen. However, this MH was an unsuitable TCI device for the human skin, as collagen is suspected to induce inflammation. In this study, we developed an improved collagen-free MH (new-MH) and conducted clinical study to evaluate the fundamental properties and safety in human.MethodsMicroneedle dissolution, skin irritation, and antigen-specific antibody production about new-MH were measured in mice and/or rats. On the basis of the results, the clinical study was conducted in healthy volunteers to evaluate local and systemic adverse events caused by new-MH application.ResultsWe confirmed that the microneedles of new-MH, as well as those on our old-MH that contained collagen, could easily pierce stratum corneum without severe skin irritation, and that TCI using new-MH efficiently increased antibody titer with comparable to TCI using old-MH. Application of new-MH caused no severe adverse reactions in 20 healthy volunteers enrolled in a clinical study.ConclusionsThese results verified that new-MH is a safe TCI device in human, and greatly encouraged us to advance PI/PII clinical studies of antigen-loaded new-MH.

Transcutaneous vaccination using a hydrogel patch induces effective immune responses to tetanus and diphtheria toxoid in hairless rat.

Transcutaneous immunization (TCI) targeting the Langerhans cells (LCs) of the epidermal layer is a promising needle-free, easy-to-use, and non-invasive vaccination method. We developed a hydrogel patch formulation to promote the penetration of antigenic proteins into the stratum corneum. Here, we investigated the characteristics of the immune responses induced by this vaccination method and the vaccine efficacy of TCI using a hydrogel patch containing tetanus and diphtheria toxoids. Our TCI system induced toxoid-specific IgG production in an antigen dose-, patch area-, and application period-dependent manner. Moreover, IgG subclass analysis indicated that our TCI predominantly elicited a Th2-type immune response rather than a Th1-type immune response. Importantly, our TCI system induced antigen-specific immune memory based on the booster effect and showed potent efficacy, comparable to that of subcutaneous immunization in toxin-challenge experiments. On the basis of these results, we are now performing translational research to apply TCI for tetanus and diphtheria.

Performance and characteristics evaluation of a sodium hyaluronate-based microneedle patch for a transcutaneous drug delivery system

The MicroHyala(®) microneedle (MN) patch was developed to provide a simple, safe, and effective drug delivery system. In this study, we examined the performance and characteristics of our fabricated MN patch to identify potential quality issues with future commercial application. Mechanical failure force analysis identified that the strength of the MN patch was affected by environmental humidity, because higher moisture levels weakened the strength of the MN. Incorporation of all-trans retinoic acid (ATRA) or ovalbumin (OVA) into the MN patch decreased the mechanical failure force by almost 50% of the strength of placebo (without drug) patches. ATRA-loaded MN patches displayed good stability after storage at 4 °C, with more than 90% and 85% of the drug remaining in the patch after 8 and 24 weeks of storage, respectively. Tetanus toxoid- and diphtheria toxoid-loaded MN patches stored for 12 months induced robust antigen-specific immune responses similar to the responses by freshly prepared MN patches. Fluorescence imaging findings suggested that prolonged antigen deposition was induced by MN-mediated fluorescein isothiocyanate-labeled (FITC)-OVA vaccination. Overall, although the strength of MN requires improvement, our developed MN patch appears to be an effective pharmaceutical product providing a simple, safe, and relatively painless approach.

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 a novel transdermal patch of alendronate, a nitrogen‐containing bisphosphonate, for the treatment of osteoporosis

Bisphosphonates are widely used for the treatment and prevention of bone diseases, including Paget disease, hypercalcemia of malignancy, and postmenopausal osteoporosis. In this study, we developed a novel transdermal patch of alendronate, a nitrogen‐containing bisphosphonate, for the treatment of bone diseases. The maximum permeation fluxes of alendronate through rat and human skin after application of this patch were 1.9 and 0.3 µg/cm2 per hour, respectively. The bioavailability (BA) of alendronate in rats was approximately 8.3% after the application of alendronate patch and approximately 1.7% after oral administration. These results indicated that the transdermal permeation of alendronate using this patch system was sufficient for the treatment of bone diseases. The plasma calcium level was effectively reduced after application of the alendronate patch in 1α‐hydroxyvitamin D3–induced hypercalcemia model rats. The alendronate patch also effectively suppressed the decrease in bone mass in model rats with osteoporosis. Modest alendronate‐induced erythema of rat skin was observed after application of the alendronate patch. Incorporation of butylhydroxytoluene in the alendronate patch almost completely suppressed this alendronate‐induced skin damage while maintaining the transdermal permeation and pharmacologic effects of alendronate. These findings indicate that our novel transdermal delivery system for alendronate is a promising approach to improve compliance and quality of life of patients in the treatment of bone diseases.