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Chondrial Ca2+ Overload. Eur J Ph 誌 上 発 表 （ 原 著 論 文 ） 185 誌 上 発 表 （原著論文） Summaries of Papers Published in Other Journals (Original Papers) Namekata, I.*, Shimada, H.*, Kawanishi, T., Tanaka, H.* were in good agreement with those previously reported in and Shigenobu, K*: Reduction by SEA0400 of myo- voltage clamped cardiomyocytes. cardial ischemia-induced cytoplasmic and mito- Keywords: Na+-Ca2+ exchange, cardiomyocyte, fl uorescence chondrial Ca2+ overload. microscopy （ ） *1 Eur J Pharmacol ., 543, 108-115 2006 東邦大学薬学部 + The cardioprotective effects of SEA0400, a novel Na - *2 大妻大学家政学部 Ca2+ exchanger inhibitor, were examined in isolated guinea pig myocardial tissue and ventricular myocytes. In a coro- Tanaka, H.*1, Shimada, H.*1, Namekata, I.*1, Kawanishi, T., nary-perfused right ventricular tissue preparation, SEA0400 Iida-Tanaka, N.*2, Shigenobu, K.*1: Involvement of the had no cardiosuppressive effect during normoxia and exper- Na+/Ca2+ exchanger in ouabain-induced inotropy imental ischemia, but enhanced the recovery of contractile and arrhythmogenesis in guinea-pig myocardium force during reperfusion. SEA0400 had no effect on tissue as revealed by SEA0400. ATP content during normoxia, but attenuated its decrease J Pharmacol Sci. , 103, 241-246（ 2007） during ischemia. Treatment of ventricular myocytes with Involvement of the Na+/Ca2+ exchanger in ouabain- an ischemia mimetic solution（ high K+, glucose free, pH induced inotropy and arrhythmogenesis was examined with 6.0, gassed with N2） resulted in the depolarization of the a specific inhibitor, SEA0400. In right ventricular papil- mitochondrial membrane potential and an increase in cyto- lary muscle isolated from guinea-pig ventricle, 1 microM plasmic and mitochondrial Ca2+ concentration, which had a SEA0400, which specifi cally inhibits the Na+/Ca2+ exchang- similar time course. SEA0400 significantly delayed these er by 80%, reduced the ouabain（ 1 μM） -induced posi- changes. These results suggest that SEA0400 maintains mi- tive inotropy by 40%, but had no effect on the inotropy tochondrial function and tissue ATP content during ischemia induced by 100μM isobutyl methylxantine. SEA0400 sig- through the inhibition of cytoplasmic and mitochondrial nifi cantly inhibited the contracture induced by low Na+ so- Ca2+ overload. lution. In HEK293 cells expressing the Na+/Ca2+ exchanger, Keywords: Na+-Ca2+ exchange, Calcium, cardiomyocyte 1 microM ouabain induced an increase in intracellular Ca2+, *東邦大学薬学部 which was inhibited by SEA0400. The arrhythmic contrac- tions induced by 3 μM ouabain were signifi cantly reduced Namekata, I.*1, Kawanishi, T., Iida-Tanaka, N.*2, Tanaka, by SEA0400. These results provide pharmacological evi- H.*1 and Shigenobu, K.*1: Quantitative fluorescence dence that the Na+/Ca2+ exchanger is involved in ouabain- measurement of cardiac Na+/Ca2+ exchanger in- induced inotropy and arrhythmogenesis. hibition by kinetic analysis in stably transfected Keywords: Na+/Ca2+ exchange, cardiomyocyte, fl uorescence HEK293 cells. microscopy J Pharmacol Sci ., 101, 356-360（ 2006） *1 東邦大学薬学部 We developed a method to quantitatively evaluate the *2 大妻大学家政学部 potency of Na+/Ca2+ exchanger（ NCX） inhibitors with fl uo- rescence microscopy in NCX1-transfected HEK 293 cells. Izutsu, K., Yomota, C., and Aoyagi, N.: Inhibition of The reverse mode and forward mode NCX activities were mannitol crystallization in frozen solutions by so- measured as the ascending slope of the early phase increase dium phosphates and citrates. in cytoplasmic Ca2+ concentration after change to low Na+ Chem. Pharm. Bull., 55, 565-570（ 2007） extracellular solution and the descending rate（ inverse of Effects of co-solutes on the physical property of mannitol the exponential time constant） on return to normal solu- and sorbitol in frozen solutions and freeze-dried solids were tion, respectively. Both modes of NCX were inhibited by studied as a model of controlling component crystallinity SEA0400（ 2-[4-[（2,5-difl uorophenyl）methoxy]phenoxy]- in pharmaceutical formulations. A frozen mannitol solution 5-ethoxyaniline） and KB-R7943（ 2-[2-[4-（4-nitrobenzy- （500 mM） showed a eutectic crystallization exotherm at loxy）phenyl]ethyl]isothiourea methanesulfonate）, and the -22.8 degrees C, whereas sorbitol remained amorphous in concentration-inhibition relationships for both inhibitors the freeze-concentrated fraction in the thermal scan. Various 186 国 立 衛 研 報 第 125 号（2007） inorganic salts reduced the eutectic mannitol crystalliza- lowing derivatization with 2-nitrophenylhydrazine tion peak. Trisodium and tripotassium phosphates or cit- by high-performance liquid chromatography-UV rates prevented the mannitol crystallization at much lower detection-electrospray ionization mass spectrom- concentrations than other salts. They also raised transition etry. temperatures of the frozen mannitol and sorbitol solutions J.Chromatogr . A, 1142, 231-235（2007） （T（g）’: glass transition temperature of maximally freeze- Currently, biotin is typically determined in Japan using a concentrated amorphous phase）. Crystallization of some microbiological method. Such microbiological assays are sen- salts（ e.g., NaCl） induced crystallization of mannitol at sitive, but they are not always highly specific and are also above certain salt concentration ratios. Thermal and near- rather tedious and time-consuming. In the present study, RP- infrared analyses of cooled-melt amorphous sorbitol solids HPLC and LC-MS methods for the determination of biotin indicated increased intermolecular hydrogen-bonding in the have been developed by coupling the carboxyl group with presence of trisodium phosphate. The sodium phosphates 2-nitrophenylhydrazine hydrochloride. 2-Nitrophenylhydrazine and citrates should prevent crystallization of mannitol in is used for the derivatization of carboxylic acids, and these frozen solutions and freeze-dried solids by the intense derivatives are known to be applicable to LC-MS detection. hydrogen-bonding and reduced molecular mobility in the Good recovery rates of over 80% were obtained for the ad- amorphous phase. dition of 0.20~0.41μ mol of biotin per formulation. The Keywords: amorphous, crystallization, formulation, freeze- detection limit in HPLC at 400 nm was 5 ng per injection, drying, thermal analysis with good linearity being obtained over the concentration range 0.01-1.5 μ g per injection. Further, derivatives were Yonezawa, Y. *1, Izutsu, K., Tokunaga, H. *1, Maeda, determined by LC-MS with electrospray ionization, where H. *1, Arakawa, T. *2 and Tokunaga, M. *1: Dimeric the spectra indicated the molecular-related ions [M+H]+. The structure of nucleoside diphosphate kinase from detection limit was 0.2 ng per injection in SIMS analysis, moderately halophilic bacterium: contrast to te- and linearity was observed in the range of 5~50 ng per in- trameric Pseudomonas counterpart. jection. The proposed method could be used to specifically FEMS Microbiol Lett., 268, 52-58（ 2007） determine the presence of biotin for relatively clean samples Light scattering and chemical cross-linking analyses with almost pharmacologic amounts of biotin. of nucleoside diphosphate kinase（ NDK） from moderate Keywords: biotin, 2-nitrophenylhydrazine derivative, LC- halophile, Halomonas sp. 593（ HaNDK）, unambiguously MS, electrospray ionization demonstrated that this enzyme formed a dimeric structure, in contrast to the Pseudomonas NDK（ PaNDK）, a non- 四方田千佳子，保立仁美，伊豆津健一，青柳伸男：皮 halophilic counterpart, and other NDKs from Gram-negative 膚適用製剤の溶出試験に関する研究． bacteria, which all formed a tetrameric structure. Com- 医薬品研究, 38, 235-241（2007） parison of HaNDK and PaNDK showed that the HaNDK 皮膚適用製剤は，皮膚への作用を目的とした局所製剤 was less thermally stable than the PaNDK: the optimum の他，経口投与でバイオアベイラビリティーが低い医薬 temperature of PaNDK enzyme activity was 20 degrees C 品等の注射剤に変わる剤形としても広く検討されてい higher than that of HaNDK. However, the HaNDK readily る．現在，欧州薬局方（EP）や米国薬局方（USP）には， refolded and reassembled back to the active dimeric struc- 皮膚適用製剤の品質評価法として溶出試験のベッセル内 ture, upon heat denaturation at 0.2 M NaCl, as soon as the に製剤を固定する装置を設置する方法，パドル部分を改 temperature was lowered. On the contrary, the thermally 変した溶出試験法等が数種収載されている．しかし，日 more stable PaNDK was irreversibly denatured at its melt- 本薬局方（日局）では，皮膚適用製剤の品質評価法を収 ing temperature. 載しておらず，日局への適切な取り込みを目指すことと Keywords: protein structure, stability, halophilic, nucleoside した．それぞれの収載試験法を試みると共に，新たなメ diphosphate kinase ンブランフィルターを熱溶着する手法を開発し，有用な *1 Faculty of Agriculture, Kagoshima University 試験法であることを示した． *2 Alliance Protein Laboratories 簡便な試験法として，貼付剤の他，軟膏剤等への広い 応用が期待される． Yomota,C., Ohnishi,Y.: Determination of biotin fol- Keywords: 貼付剤，放出試験，溶出試験器，メンブラン 誌 上 発 表 （ 原 著 論 文 ） 187 フィルター brary. Our results clearly demonstrate the importance of the improved protocol for the library preparation of antibodies Kawamura, M.*1, Shibata, H., Kamada, H.*2, Oka- and the resulting isolation of antibodies for clinical and re- moto, T.*1, Mukai, Y.*1, Sugita, T.*1, Abe, Y.*1, Imai, S.*1, search applications. Nomura, T.*1, Nagano, K.*1, Mayumi, T.*3, Nakagawa, Keywords: non-immune antibody library, phage display S.*1, Tsutsumi, Y.*2, Tsunoda, S.*2 : A novel method system, single-chain Fv, high-throughput screening, vascular for constructing of gene fragment library to endothelial growth factor receptor 2 searching epitopes. *1 大阪大学大学院薬学研究科 Biochem. Biophys . Res. Commun ., 346 ,198-201（2006） *2 医薬基盤研 Identification of the epitope sequence or the functional domain of proteins is a laborious process but a neces- Mukai, Y.*1, Sugita, T.*1, Yamato, T.*1, Yamanada, N.*1 sary one for biochemical and immunological
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