Human Induced Pluripotent Stem Cell-Based Skin for Assessing Transdermal Drug Permeability and Irritancy.

To establish a system for assessing drug permeation and irritation of the skin, the permeation of benzoic acid and isosorbide dinitrate, which are listed in the Pharmacopoeia, and the chemical irritation were evaluated using skin generated from human induced pluripotent stem cells (iPSCs). Multilayer structures and cellular markers (keratin 14 and 10, which are in basal and suprabasal epidermal layers) were clearly detected in our iPSC-based skin. Transepidermal water loss (TEWL) decreased after iPSC-derived keratinocytes were cultured on collagen gels from human primary fibroblasts. These results indicate that the barrier function was partly increased by formation of the living epidermis. The cumulative amount of benzoic acid and isosorbide dinitrate across human iPSC-based skin gradually increased after an initial lag time. Moreover, the irritancy of various chemicals (non-irritants: ultrapure water, allyl phenoxy-acetate, isopropanol, and hexyl salicylate and irritants: 5% sodium dodecyl sulfate (SDS), heptanal, potassium hydroxide (5% aq.) and cyclamen aldehyde) to iPSC-based skin was almost met the irritation criteria of the Organisation for Economic Co-operation and Development (OECD) guideline. The results of our iPSC-based skin evaluation provide useful basic information for developing an assessment system to predict the permeation and safety of new transdermal drugs in human skin.

Utility of Three-Dimensional Skin From Human-Induced Pluripotent Stem Cells as a Tool to Evaluate Transdermal Drug Permeation.

Transdermal drug delivery is an attractive route for administration of drugs, and it offers several advantages such as painless administration. To accurately predict the rate of human skin permeation for new transdermal drug formulations, we developed a novel assessment system using induced pluripotent stem cells (iPSCs). Skin was generated from iPSC-derived keratinocytes and fibroblasts. In the histological and immunohistochemical examination, cellular markers (keratin 14 and keratin 10) for the epidermal basal and suprabasal layers were clearly detected within the multilayer structures produced in the human iPSC-based three-dimensional skin model. The results from our permeation study indicate that an initial lag time exists during permeation of 5(6)-carboxyfluorescein and fluorescein isothiocyanate dextran 4000. Furthermore, the permeation for these model drugs in human iPSC-based skin was inversely proportional to the molecular weight of the drugs. These results of the present iPSC-based skin are useful basic information as a first step for developing a new assessment system to predict the efficacy of drug permeation in human skin by using iPSC-based skin.

Development of a Phosphoric Acid-Mediated Hyaluronic Acid Gel Sheet for Efficient Transdermal Delivery of Alendronate for Anti-Osteoporotic Therapy.

For efficient transdermal delivery of alendronate (ALN) for anti-osteoporotic therapy, we developed a hyaluronic acid (HA) gel sheet that was prepared simply by enhancing HA noncovalent interactions using phosphoric acid and polyhydric alcohol (propanediol and glycerin). HA solution viscosity increased after addition of phosphoric acid, and the HA gel sheet formed after heated drying. The HA gel sheet could be converted to high viscosity state by addition of water. These results indicate that phosphoric acid enhances the noncovalent interactions of HA molecules. The HA gel sheet elicited no skin irritation over 7 days after a 24-h application. The permeation of ALN across rat and human skin was 109 and 7.17 µg/cm2, respectively, up to 24 h after application of the ALN-loaded HA gel sheet, which is sufficient for clinical treatment of osteoporosis. The bioavailability of ALN in rats was ~20% after application of the ALN-loaded HA gel sheet, and plasma calcium levels were effectively reduced 3 days after sheet application. Furthermore, in a rat osteoporosis model, the reduction in tibial bone density was suppressed by treatment with the ALN-loaded HA gel sheet. These results indicate that our phosphoric acid-mediated HA gel sheet is a promising transdermal formulation for efficient ALN delivery.

Self-Dissolving Microneedle Arrays for Transdermal Absorption Enhancement of Human Parathyroid Hormone (1-34).

Human parathyroid hormone (1-34) (PTH) has been widely used as the subcutaneous injection formulation for the treatment of osteoporosis. In the present study, we developed an efficient transdermal delivery system of PTH by using dissolving microneedle arrays (MNs) composed of hyaluronic acid (HA) for the treatment of osteoporosis. PTH-loaded MNs, with needle length 800 µm, were fabricated via a micro-molding method. The stability of PTH in MNs was found to be 6-fold higher than that of PTH solution when stored at room temperature (15⁻20 °C) for one month. Micron-scale pores were clearly visible in rat skin following application of PTH-loaded MNs. PTH-loaded MNs were completely dissolved by 60 min following application to rat skin. The bioavailability (BA) of PTH relative to subcutaneous injection was 100 ± 4% following application of PTH-loaded MNs in rats. In addition, PTH-loaded MNs were found to effectively suppress decreases in bone density in a rat model of osteoporosis. Furthermore, no skin irritation was observed at the site of application in rats. These findings indicate that our dissolving MNs have a potential use in formulations for the transdermal delivery of PTH and for the treatment of osteoporosis.

Development of PEGylated aspartic acid-modified liposome as a bone-targeting carrier for the delivery of paclitaxel and treatment of bone metastasis.

To prevent bone metastasis, we developed polyethylene glycol (PEG)-conjugated aspartic acid (Asp)-modified liposomes (PEG-Asp-Lipo) as a bone-targeting carrier of paclitaxel (PTX) by using Asp-modified 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-Asp). The affinity of Asp-modified liposomes to hydroxyapatite increased as the concentration of DPPE-Asp increased. The bone accumulation of [3H]-labeled PEG(2)-Asp(33)-Lipo was approximately 24.6% 360 min after intravenous injection in mice, in contrast to 5.4% and 6.7% of [3H]-labeled normal Lipo and PEG(2)-Lipo, respectively. Similarly, [14C]-labeled PTX encapsulated into PEG(2)-Asp(33)-Lipo predominantly accumulated in the bone. Furthermore, using an in situ imaging experiment, we observed that near-infrared fluorescence-labeled PEG(2)-Asp(33)-Lipo selectively accumulated in the bone near the joint after intravenous injection in mice. We also found that FITC-labeled PEG(2)-Asp(33)-Lipo predominantly accumulated on eroded and quiescent bone surfaces. In a bone metastatic tumor mouse model, in which B16-BL6/Luc cells were injected into the left ventricle of female C57BL/6 mice, metastatic bone tumor growth was significantly inhibited by an intravenous injection of PEG(2)-Asp(33)-liposomal PTX. In contrast, PEGylated liposomal PTX hardly affected the growth of metastatic bone tumors. These findings indicate that PEG(2)-Asp(33)-Lipo is a promising bone-targeting carrier for the delivery of PTX and treatment of bone metastasis.

Changes in aquatic toxicity of potassium dichromate as a function of water quality parameters.

Potassium dichromate (K2Cr2O7) is used as a general reference toxicant in aquatic toxicity testing, but relatively little is known regarding the effects of water quality parameters on K2Cr2O7 toxicity to Daphnia magna. The acute toxicity of K2Cr2O7 to D. magna was comparatively examined in one very hard (M4 medium for D. magna assay), four hard, one moderately hard and one soft dilution water samples. The 48-h EC50 (50% effective concentration) of K2Cr2O7 to D. magna was reproducible (coefficient of variation [CV]: 13%) in tests using the same dilution water sample, but reproducibility was poor (CV: 62%) in tests using seven different dilution water samples. The observed 48-h EC50 value increased with increasing water hardness (28-250 mg CaCO3/L) and Na+ concentration (4.3-19.7 mg Na/L). The effect of Ca2+ and Mg2+ on K2Cr2O7 toxicity was equivalent in terms of molar concentration. The 48-h EC50 for K2Cr2O7 was determined according to OECD TG 202 by six contract laboratories using M4 medium and were shown to be reproducible (CV: 15%), indicating that the toxicity level can be determined with high accuracy if holding and dilution water samples are standardized. Multiple regression analysis revealed that the 48-h EC50 was strongly correlated (r2 = 0.927) with the Ca2+, Mg2+, Na+ concentration, and alkalinity of the dilution water samples. Detailed monitoring of water quality characteristics thus facilitates intra- and inter-laboratory comparisons of toxicity data and enables predictions of changes in the susceptibility of test animals.