Utility of the Three-dimensional Cultured Human Skin Model as a Tool to Evaluate Skin Permeation of Drugs

Tetsuya Watanabe¹, Tetsuya Hasegawa¹, Hidekazu Takahashi², Takuya Ishibashi², Kozo Takayama³ and Kenji Sugibayashi¹
1 Josai University, 2 Toyobo Co., Ltd., 3 Hoshi University
Correspondence: Kenji Sugibayashi, Ph.D.
Faculty of Pharmaceutical Sciences, Josai University
1-1 Keyakidai, Sakado, Saitama 350-0295, Japan. Tel: 0492-71-7943, Fax: 0492-71-7943, E-mail: sugib@josai.ac.jp

Original paper :AATEX 8(1):1-14
Abstract
Three-dimensional cultured human skin model (cultured human skin) was evaluated for prediction of skin permeability of drugs. The permeation data of different drugs from their aqueous solution in the cultured human skin were compared with those through excised human and rat skin. Although the cumulative amount of each drug that permeated through the cultured human skin was about 10-fold higher than those through the excised human and hairless rat skin, a good correlation was found between the cultured human skin and excised skin permeabilities. In addition, time profiles for skin permeation of each drug were very analogical for both the skin membranes. This was probably due to similar partition parameters of the drugs from aqueous solution to both skin preparations, although the diffusion parameters in skin barrier were markedly different among the membranes. The rate-limiting layer for cultured human skin permeation was evaluated using isosorbide-5-mononitrate (ISMN) and isosorbide-dinitrate (ISDN). The resistances of stratum corneum in the cultured human skin and excised hairless rat skin were 74.8 and 95.4 % of the total resistance for ISMN permeation, and 66.1 and 94.7 % for ISDN permeation. Those of the total epidermis in the cultured human skin were 99.4 and 93.5 % for ISMN and ISDN permeation, respectively. It is clear from these results that the total epidermis is the primary barrier in the cultured human skin, whereas the stratum corneum alone is the barrier in excised hairless rat skin. These results suggested that one has to use this cultured human skin model under consideration of different barrier properties of the model from those of excised hairless rat and human skin when predicting skin permeation of drugs.

Keywords: three-dimensional cultured human skin model, skin permeation, high through-put screening, alternative skin

Effect of Penetration Enhancers on the Permeation of Drugs across a Three-Dimensional Cultured Human Skin Model: Comparison with the Effects Using Excised Hairless Rat Skin

Tetsuya Watanabe¹, Tetsuya Hasegawa¹, Hidekazu Takahashi², Takuya Ishibashi² and Kenji Sugibayashi¹
1 Josai University, 2 Toyobo Co., Ltd.
Correspondence: Kenji Sugibayashi, Ph.D.
Faculty of Pharmaceutical Sciences, Josai University
1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
Tel: 0492-71-7943, Fax: 0492-71-7943, E-mail: sugib@josai.ac.jp

Original paper :AATEX 8(1):15-22
Abstract
Effect of penetration-enhancers on the skin permeation of drugs was evaluated using a three-dimensional cultured human skin model, Living Skin Equivalent-high (LSE-high), which consists of stratum corneum, viable epidermis and dermis. Seven kinds of potential enhancers (diisopropyl adipiate, isopropyl myristate, lauryl alcohol, d-limomen, l-menthol, N-methyl-2-pyrrolidone and oleic acid) were used, and isosorbide-5-mononitrate (ISMN) and isosorbide dinitrate (ISDN) were selected as a model hydrophilic and a lipophilic drug, respectively. The membrane permeation experiments were carried out using the LSE-high as well as excised hairless rat skin as a control. Every enhancer at a concentration of 3 % showed a penetration-enhancing effect on the drug permeation across the LSE-high as it did across the hairless rat skin. Interestingly, the rank order of the enhancement effect in LSE-high was similar to that in the hairless rat skin, although the effect across the LSE-high was lower than that across the hairless rat skin. In addition, the enhancing effect against the ISMN permeation was more marked than that against the ISDN permeation for both types of skin. Concentration dependence of enhancers for their promoting effect was then evaluated using l-menthol, that showed the highest effect among the enhancer systems used in this study. As results, 3 and 10 % l-menthol exhibited the highest effect on the skin permeation of ISMN and ISDN, respectively, for both skins. These findings suggest that LSE-high may be utilized as an alternative membrane when testing the promoting effect of absorption enhancers on the skin permeation of drugs.
Keywords: three-dimensional cultured human skin model, alternative membrane, penetration enhancer, skin permeation, isosorbide-5-mononitrate, isosorbide dinitrate

The Utility of Whole Embryo Culture for Assessment on Embryotoxicity of Estrogenic Compounds in Rats

Makiko Kuwagata¹, Chiaki Watanabe¹, Makio Takeda², Hiroaki Aoyama², Toshiaki Watanabe³ and Hiroshi Ono¹
1 Food and Drug Safety Center, 2 Institute of Environmental Toxicology, 3 Yamagata University School of Medicine
Correspondence: Makiko Kuwagata, D.V.M.
Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano, Kanagawa, 257-8523, Japan
Tel: 0463-82-4751, Fax: 0463-82-9627, E-mail: kuwagata.m@fdsc.or.jp

Original paper :AATEX 8(1):23-30
Abstract
We evaluated the utility of whole embryo culture for the screening technique of the potency of endocrine active chemicals in rat embryos. Rat embryos on embryonic day 11 (ED11) were exposed to two estrogenic chemicals, estradiol benzoate (EB) and diethylstilbestrol (DES), for 42 hr in culture. The treated embryos showed some morphological defects in the cardiovascular system, such as edema around umbilical vessels (EB- and DES-treated groups), enlargement of the heart (EB) and hemorrhage of the head (DES) at high doses. To determine the mediation of estrogen receptors to these morphological defects, we also studied the expression of estrogen receptor a (ER-a) in embryos both by immunohistochemistry and by Western blotting. Immunohistochemistry revealed positive grains stained with anti-ER- a antibody in both the cytoplasm and nucleus of heart and yolk sac in EDs 12 and 13 rat embryos. The staining pattern of anti-ER-a antibody in embryos was different from the positive control, the uterus of adult rats. In addition, the expression of ER-a protein in these embryos was not detected by Western blotting. These results suggest that the embryotoxicity in this study is not mediated by ER-a. Further modifications of whole embryo culture as a screening technique are needed to detect the embryotoxicity of endocrine active compounds.

Keywords: estrogenic compounds, estrogen receptor-a, immunohistochemistry, Western blotting, whole rat-embryo culture

In Vitro Embryotoxicity Testing of Polymeric Substances for Dental Use by Differentiation of Embryonic Stem Cells

Koichi Imai¹, Horst Spielmann², Gabi Scholz², Ingeborg pohl² and Masaaki Nakamura¹
1 Osaka Dental University, 2 ZEBET (National German Center for the Documentation and Evaluation of Alternatives to Testing in Animals) at BgVV (National Center for Health Protection of Consumers and Veterinary Medicine )
Correspondence: Koichi Imai, Ph.D.
Department of Biomaterials, Osaka Dental University
8-1, Kuzuhahanazono-cho, Hirakara-city, Osaka573-1121, Japan
Tel: 072-864-3056, Fax: 072-864-3156, E-mail:imai@cc.osaka-dent.ac.jp

Original paper :AATEX 8(1):31-39
Abstract
2,2-bis [4-(2-hydroxy-3-methacryloy-loxypropoxy) phenyl] propane (Bis-GMA), di(methacryloyloxyethyl) trimethyl hexamethylene-diurethane (UDMA), 2,2-bis (4-methacryloyloxypoly ethoxy phenyl) propane (Bis-MPEPP) and triethylen glycoldimethacrylate (TEGDMA) were used as main component monomers for the tooth restorative composites resin or preventive filling sealant. These monomers absorbed could possibly be metabolized in the living body. The effects of four dental monomers on the capability of embryonic stem (ES) cells of the mouse cell-line D3 to differentiate into contracting myocard was examined using an embryonic stem cell test (EST), together with the cytotoxic effect on differentiated mouse 3T3 fibroblasts and on differentiated ES cells. The half-inhibition concentration for differentiation (ID50) on ES cells was 10.5ug/mL for Bis-GMA, 9.8ug/mL for UDMA, 7.9ug/mL for Bis-MPEPP was and 15.4ug/mL for TEGDMA. The IC50 ES for Bis-GMA was 23.6ug/mL and declined in the order of Bis-MPEPP, UDMA and TEGDMA. On the contrary, the IC503T3 for Bis-GMA was 19.8ug/mL and declined in the order of UDMA, TEGDMA and Bis-MPEPP in 3T3 cells. The embryotoxic potential was obtained from a biostatistically based prediction model of EST, which was based upon the three endpoints, ID50, IC50ES and IC503T3. It is considered that the four dental monomers are classified as “weak embryotoxic”. The in vitro test using ES cells holds promise to be used to examine in vitro embryotoxicity of dental biomaterials.

Keywords: embryonic stem cell, EST, in vitro embryotoxicity test, dental, monomer