Predicting the Safety of Ocular Iontophoresis Using Reconstructed Human Corneal Epithelial Cells.

We aimed to investigate eye damage caused by ocular iontophoresis (IP) based on an in vitro eye irritation test using a reconstructed human corneal cell. In this study, the LabCyte CORNEA-MODEL was selected as the reconstructed corneal cell. The test procedure was performed according to Test Guideline No.492 of the Organisation for Economic Co-operation and Development, which was partially revised for the IP. From the relationship between the cell viability of the cornea model and the electric field intensity [current density (mA/cm2) × application time (min)] of the IP, we predicted that the intensity values of 465 mA/cm2 × min and 930 mA/cm2 × min caused reversible eye irritation and irreversible eye damage, respectively. However, further studies are required to improve the accuracy and reproducibility of the prediction. This report provides essential knowledge on the clinical safety of ocular IP.

Potential of TAK-593 Ophthalmic Emulsion for the Treatment of Age-Related Macular Degeneration.

Intravitreal injection therapy of anti-vascular endothelial growth factor (VEGF) antibody or steroids is the mainstream for patients with age-related macular degeneration (AMD). However, since intravitreal injection is invasive administration, side effects such as endophthalmitis are major problems. In this study, we selected eye drops as a non-invasive treatment method, and aimed to develop eye drops that can deliver TAK-593 (VEGF receptor tyrosine kinase inhibitor) to the posterior segment of the eye. Since TAK-593 is a poorly water-soluble drug, the TAK-593 emulsion was formulated. The solubility of TAK-593 in various oils was measured, and the oil used for the emulsion was selected. Furthermore, viscosity enhancers were added to the emulsion in order to improve the drug delivery into the eye. As viscosity enhancer, xanthan gum was selected based on the properties and the viscosity of the emulsion. The delivery of TAK-593 to the posterior eye was increased by the formulation concentration and the addition of viscosity enhancers. In the laser-induced choroidal neovascularization model, TAK-593 emulsion eye drops showed the same angiogenesis-suppression efficacy as anti-VEGF antibody intravitreal injection. From these results, it was revealed that TAK-593 with an effective drug concentration can be delivered to the posterior eye by non-invasive eye drop administration.

Suitability of high-molecular-weight tissue-derived elastin polypeptides and their particles as cosmetic biomaterials.

We aimed to determine the coacervation properties of high-molecular-weight (HMW) tissue-derived elastin (TDE) and to examine the potential use of TDE particles as a cosmetic biomaterial. TDE solutions were filtered and divided into three fractions (1-3) according to the molecular weight of the elastin. The turbidity of fraction 2, which contained a large portion (58%) of HMW elastin polypeptides (>100 kDa), was measured under several pH values (3.0-11.0) and NaCl concentrations (0-1000 mM) to examine its coacervation ability. HMW TDE exhibited coacervation under the physiological conditions (temperature, pH, and NaCl concentration) of the skin surface. We performed inclusion and release experiments using three model chemicals with different molecular weights and measured the size and zeta potential of the fraction 3 particles to investigate the suitability of HMW elastin polypeptides. Fraction 3, which contained a larger portion (64%) of HMW elastin polypeptides, displayed a strong coacervation property at a phase transition temperature of 19.8 ± 0.1°C. The inclusion ratio of the model chemical Biebrich Scarlet (BS) with a molecular weight of <600 was approximately 92.1 ± 0.7%. The release profiles of BS from the particles linearly increased and reached a plateau after 15 days. Moreover, the average size of the particles with BS was 474.2 ± 24.6 nm. The low-molecular-weight (LMW) elastin peptides have moisturizing and whitening functions for the skin. We concluded that TDE, as a mixture of HMW polypeptides and LMW peptides, can potentially serve as a multifunctional and effective cosmetic biomaterial.

Fabrication of novel-shaped microneedles to overcome the disadvantages of solid microneedles for the transdermal delivery of insulin.

In this study, we fabricated two different microneedles (MNs) - semi-hollow and bird-bill - to overcome the limitations of solid and coated MNs, respectively. The two MN arrays were developed using a general injection molding process to obtain high-quality MNs with uniform shape. The semi-hollow and bird-bill MNs could penetrate the micropores of swine skin up to depths of 178.5 ± 27.6 µm and 232.1 ± 51.3 µm, respectively. When the semi-hollow MNs were used for the transdermal delivery of insulin in diabetic rats, it was observed that the blood glucose concentration (BGC) decreased remarkably within 30 min, and the desired effect of insulin was maintained for an additional 3 h after the removal of insulin from the skin surface. The bird-bill MN was able to load a coating gel at a maximum capacity of 3.20 ± 0.21 mg per MN array, and the BGC continued to decrease significantly after MN application for up to 2-6 h. In summary, we fabricated semi-hollow and bird-bill MN arrays using the injection molding method; these can be mass produced and are capable of effectively producing micro-holes in the stratum corneum. The two MN arrays could provide effective transdermal delivery of large-molecular-weight drugs such as insulin.

A phase I study of high dose camostat mesylate in healthy adults provides a rationale to repurpose the TMPRSS2 inhibitor for the treatment of COVID-19.

Camostat mesylate, an oral serine protease inhibitor, is used to treat chronic pancreatitis and reflux esophagitis. Recently, camostat mesylate and its active metabolite 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA) were reported to inhibit the infection of cells by severe acute respiratory syndrome coronavirus 2 by inhibiting type II transmembrane serine protease. We conducted a phase I study to investigate high-dose camostat mesylate as a treatment for coronavirus disease 2019. Camostat mesylate was orally administered to healthy adults at 600 mg 4 times daily under either of the following conditions: fasted state, after a meal, 30 min before a meal, or 1 h before a meal, and the pharmacokinetics and safety profiles were evaluated. In addition, the time of plasma GBPA concentration exceeding the effective concentration was estimated as the time above half-maximal effective concentration (EC50 ) by using pharmacokinetic/pharmacodynamic modeling and simulation. Camostat mesylate was safe and tolerated at all dosages. Compared with the fasted state, the exposure of GBPA after a meal and 30 min before a meal was significantly lower; however, no significant difference was observed at 1 h before a meal. The time above EC50 was 11.5 h when camostat mesylate 600 mg was administered 4 times daily in the fasted state or 1 h before a meal. Based on the results of this phase I study, we are currently conducting a phase III study.

Investigation of water-soluble elastin as a multifunctional cosmetic material: Moisturizing and whitening effects.

Elastin and collagen are extracellular matrix proteins that are widely distributed in the body. Although elastin essentially functions as a skin moisturizer, there have been few reports on its other fundamental chemical and biological functions. In this study, we investigated the moisturizing and whitening (tyrosinase inhibition) effects of elastin to examine its usefulness as a cosmetic material. Water-soluble hot alkali pig aorta (HAPA)-elastin was prepared from pig aorta using the hot alkali method. HAPA-elastin showed a widely distributed molecular weight and had a coacervation property that mediated reversible self-assembly of its molecules with increasing temperature. Amino acid analysis of HAPA-elastin showed a high content (81.5%) of hydrophobic amino acids such as Gly, Ala, Val, and Pro. Des (desmosine) and Ide (isodesmosine), which are characteristic amino acids of elastin, accounted for more than 0.4% of the total amino acid content. HAPA-elastin showed a moisture-retaining property. The water content of skin samples treated with and without HAPA-elastin was 77.2% ± 7.8% and 49.4% ± 10.1%, respectively. HAPA-elastin also inhibited tyrosinase activity by 11.3% ± 3.9%. The results obtained indicate that elastin has a useful function as a cosmetic material.

Effect of diffusive direction across the skin on the penetration profile of chemicals in vitro.

Skin has various types of transporters and is a biochemically active organ. These aspects of skin influence the distribution of chemicals in skin and their elimination from skin. The biochemical and histological variations of the skin must be taken into account when conducting transdermal penetration research. Here we used hairless mouse skin to investigate the percutaneous absorption of chemicals in vitro from the stratum corneum (SC) side to the viable skin (VS) side (forward direction) and from the VS side to the SC side (backward direction). We examined the effects of molecular weight, lipophilicity (Log Ko/w), electric charge, and the molecular structure of penetrants. The penetration flux of verapamil hydrochloride (VRP) for the backward direction was 3.2 times larger than that for the forward direction. The flux values of benzoic acid (BA) and para-hydroxybenzoic acid (pHBA) for the forward direction were 2.1 and 4.6 times larger than those for the backward direction, respectively. This directional difference was caused by the active transporter for VRP, the histological distribution of BA solubility, and the intermolecular hydrogen bonding between pHBA and skin tissue in the stripped skin. Across intact skin, in contrast, there was no difference in the skin penetration profile between the forward direction and backward directions.

Skin accumulation and penetration of a hydrophilic compound by a novel gemini surfactant, sodium dilauramidoglutamide lysine.

We investigated a novel peptide-based gemini amphiphilic compound, sodium dilauramidoglutamide lysine (DLGL), as a chemical enhancer for the skin penetration of l-ascorbic acid 2-glucoside (AAG). A three-dimensional cultured human skin product, TESTSKIN™ LSE-high (LSE-high), was used as a skin model. The penetration flux of AAG with DLGL and that obtained with sodium lauramidoglutamide (LG) as a conventional surfactant across LSE-high were increased by 12.56 and 69.29 times compared to the control, respectively. The ratio of AAG amount with DLGL in the skin (21.78% total dose) was significantly increased (p<0.05) compared to the control (7.23%) and to the AAG amount with LG (8.13%). The AAG amounts in receptor were 1.06% (control), 3.19% (+DLGL) and 21.00% (+LG). Thus, DLGL preserved AAG in skin, resulting in enhanced AAG penetration flux. However, LG might create the pathways through the skin. We conclude that DLGL is a gemini surfactant that accumulates a hydrophilic compound in skin and enhances the penetration flux. DLGL may therefore be a novel addition agent for skin local therapy.

Prediction of percutaneous absorption in human using three-dimensional human cultured epidermis LabCyte EPI-MODEL.

The objective of this study is to establish a relationship of the skin penetration parameters between the three-dimensional cultured human epidermis LabCyte EPI-MODEL (LabCyte) and hairless mouse (HLM) skin penetration in vitro and to predict the skin penetration and plasma concentration profile in human. The skin penetration experiments through LabCyte and HLM skin were investigated using 19 drugs that have a different molecular weight and lipophilicity. The penetration flux for LabCyte reached 30 times larger at maximum than that for HLM skin. The human data can be estimated from the in silico approach with the diffusion coefficient (D), the partition coefficient (K) and the skin surface concentration (C) of drugs by assuming the bi-layer skin model for both LabCyte and HLM skin. The human skin penetration of ß-estradiol, prednisolone, testosterone and ethynylestradiol was well agreed between the simulated profiles and in vitro experimental data. Plasma concentration profiles of ß-estradiol in human were also simulated and well agreed with the clinical data. The present alternative method may decrease human or animal skin experiment for in vitro skin penetration.

Mechanisms of synergistic skin penetration by sonophoresis and iontophoresis.

The mechanism of skin penetration enhancement by ultrasound under sonophoresis (US) or by an electrical field under iontophoresis (IP) was investigated using hairless mouse skin in vitro. The seven model chemicals with different molecular weights (122-1485) were dissolved in a hydrophilic gel. Donor gel with the chemicals was loaded on the skin surface and then the skin was treated with US (300 kHz, 5.2 W/cm(2), 5.4% duty-cycle) and IP (0.32+/-0.03 mA/cm(2)) individually or with US and IP in combination (US+IP). The penetration profiles of the chemicals with a molecular weight of less than 500 were influenced by the presence of an electric charge, the profiles of ionized chemicals for US+IP were the same as profiles for IP, while the penetration flux of a non-ionized chemical synergistically increased with US+IP compared with the individual flux of US and IP. The chemicals with molecular weight of more than 1000 showed synergistic effects with US+IP. The mathematical simulation assuming a bilayer skin model revealed that the synergistic effects were mainly influenced by electroosmosis in the stratum corneum (SC). Therefore the synergistic effects of US+IP was mainly caused by the SC diffusivity of chemicals increased by US and the electroosmotic water flow by IP application.

Bioequivalence of marketed transdermal delivery systems for tulobuterol.

Tulobuterol permeation through skin from various transdermal delivery systems has been compared for the bioequivalence among devices marketed. Both the permeation profiles across the hairless mouse skin and the release profiles from the devices were measured under well-controlled in vitro conditions. The release rate of the drug from the devices was appreciably higher than the penetration rate across the intact skin, indicating the skin-controlled delivery systems. However the deviation between the release rate and the permeation rate differs depending upon the system design. The brand patch showed the least difference between the release and permeation profiles among the brand and three generic devices examined. From the in vitro permeation profiles for both intact and stripped skins, the diffusion coefficient and the partition coefficient were evaluated on the basis of bi-layer skin model. The effect of the stratum corneum thickness was then simulated by SKIN-CAD. The simulated profile has suggested that the clinical performance for transdermal tulobuterol delivery is influenced not only by the thickness of the stratum corneum but by the device design as well. This is particularly the case for the stratum corneum, thinner than about 10 microm or damaged skin with the decreased barrier capacity. For the stratum corneum thicker than 20 microm, on the other hand, the clinical performance may not be significantly influenced by the device designs investigated in this study.

Skin penetration flux and lag-time of steroids across hydrated and dehydrated human skin in vitro.

To study the effect of hydration on skin absorption, we investigated penetration across human skin of twelve model chemicals having steroidal structure but different molecular weight and compared the steady-state penetration rate (J) and lag-time (t) across hydration intact skin (Jh and th) with that across dehydrated intact skin (Jd and td). Stratum corneum (SC) thickness of hydrated (52 microm) is 3.3 times that of dehydrated skin (16 microm). Transepidermal water loss (TEWL) of hydrated (7.6+/-2.1 g/m2/h) is twice that of dehydrated skin (3.4+/-1.6 g/m2/h, p<0.05) which are similar to in vivo values, suggesting the SC barrier function was recovered. The ratio of Jh/Jd ranged between 0.7 and 3.6 (average of 1.9). On the other hand, the ratio of th/td was almost constant (average of 0.8). Ratios of Jh/Jd and th/td were independent of MW and Ko/w. In percutaneous absorption experiments in vitro, skin was preserved in culture medium until use and SC might swell during that time. Therefore, we consider the possibility that J and t varied between hydrated and dehydrated skin. We confirmed the difference of J and t between hydrated and dehydrated skin in vitro and now need to define these results under in vivo condition.

Comparison of skin distribution of hydrolytic activity for bioconversion of beta-estradiol 17-acetate between man and several animals in vitro.

We have investigated the distribution of hydrolytic enzymes which metabolize beta-estradiol 17-acetate (EA) to beta-estradiol (E) in man and animal skins in vitro. The distribution of hydrolytic enzymes in human cadaver, hairless dog, rat and hairless mouse skin, was investigated by a skin-slicing technique. We performed histological studies with hematoxylin and eosin stain. The highest amount of metabolite (E) appeared in the layers of 80-120 microm from the skin surface, the basement layer in human skin, while the amount of metabolite was distributed evenly in the hairless dog skin from 0 to 180 microm. In the rat and hairless mouse skin, on the other hand, peak levels of metabolite were observed in the basement layer of dermis, the surrounding area of the cutaneous plexus. The total metabolic activities in the area of epidermis in human, hairless dog and hairless mouse skin were 2.59, 8.03 and 0.33 x 10(-4) microg/ml/microm/h, respectively. The values in whole skin layers in the hairless dog and hairless mouse skin were 3.35 and 1.85 x 10(-4)microg/ml/microm/h, respectively. EA transported across the human and hairless dog skin can be effectively metabolized before entering the capillary. Among animal models investigated, hairless dog skin might be the most facile model in simulating drug metabolism for human skin under the clinical (in vivo) conditions. Hairless mouse skin, on the other hand, was also an excellent model in excised human skin under in vitro conditions.