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Miyamoto R.,Astellas Research Technologies Co. | Nozawa T.,Astellas Pharma Inc. | Kimura M.,Astellas Research Technologies Co. | Shiozuka K.,Astellas Research Technologies Co. | Tabata K.,Astellas Pharma Inc.
Assay and Drug Development Technologies | Year: 2015

Transport assays using P-gp-expressing cell lines are commonly used to identify P-gp substrates and inhibitors in drug discovery. The P-gp cell-based assay is performed manually in 12- or 24-well plates and requires improvement for high-throughput screening. In this study, we established an efficient semiautomated 96-well transport assay using LLC-PK1 cells transfected with human P-gp. The protocol was optimized with a microplate washer for exchanging media and buffer to enhance throughput. P-gp substrates and inhibitors, and the paracellular marker Dextran Texas Red® were used to validate the 96-well transport assay. Cell monolayer integrity after washing by a microplate washer was confirmed by measuring paracellular permeability of Dextran Texas Red. Permeability and net flux ratio of the P-gp substrates and the inhibitory potency of the P-gp inhibitors were comparable in 24- and 96-well plates. The regression value of net flux ratio of P-gp substrates was high between the two formats (r2=0.99). The optimized 96-well transport assay using the microplate washer was found to be an efficient high-throughput screening tool that provided the same quality data as the 24-well plate for the identification of P-gp substrates and inhibitors in drug discovery. © Copyright 2015, Mary Ann Liebert, Inc.

Ohtsu Y.,Astellas Pharma Inc. | Takanuki F.,Astellas Research Technologies Co. | Fukunaga Y.,Astellas Pharma Inc. | Noguchi K.,Astellas Pharma Inc.
Biomedical Chromatography | Year: 2015

The potent phosphodiesterase 4 inhibitor ASP3258 contains a carboxylic acid moiety and a naphthyridine ring and is a novel therapeutic agent for asthma and chronic obstructive pulmonary disease. To support the drug development of ASP3258, we developed and validated a simple method for its determination in rat plasma. Following the addition of the analog AS1406604-00 as an internal standard, plasma samples were processed using C18-bonded solid-phase extraction cartridges under acidic conditions and injected into a high-performance liquid chromatography system with fluorescence detection. Chromatographic separation was achieved on a Shiseido Capcell Pak C18 UG120 column (3.0×150mm, 5μm) with a mobile phase consisting of acetonitrile-0.5% acetic acid (50:50, v/v). HPLC eluent was monitored with a fluorescence detector set at a wavelength of 315nm for excitation and 365nm for emission. The calibration curve was linear over a range of 2.5-250ng/mL. Validation data demonstrated that the method is selective, sensitive and accurate. In addition, the present method was successfully applied to rat plasma samples from a pharmacokinetic study. © 2014 John Wiley & Sons, Ltd.

Minematsu T.,Astellas Pharma Inc. | Sonoda T.,Astellas Research Technologies Co. | Hashimoto T.,Astellas Pharma Inc. | Iwai M.,Astellas Pharma Inc. | And 5 more authors.
Biopharmaceutics and Drug Disposition | Year: 2012

YM155 monobromide is a novel small-molecule survivin suppressant. The pharmacokinetics, distribution and excretion of YM155/[ 14C]YM155 were investigated using males and pregnant or lactating female rats after a single intravenous bolus administration. For the 0.1, 0.3 and 1 mg/kg YM155 doses given to male rats, increases in area under the plasma concentration-time curves were approximately proportional to the increase in the dose level. After administering [ 14C]YM155, radioactivity concentrations in the kidney and liver were highest among the tissues in both male and pregnant rats: e.g. 14.8- and 5.24-fold, respectively, and higher than in plasma at 0.1 h after dosing to male rats. The YM155 concentrations in the brain were lowest: 25-fold lower than in plasma. The transfer of radioactivity into fetuses was low (about 2-fold lower than in plasma). In lactating rats, the radioactivity was transferred into milk at a level 8- to 21-fold higher than for plasma. Radioactivity was primarily excreted in feces (64.0%) and urine (35.2%). The fecal excretion was considered to have occurred mainly by biliary excretion and partly by secretion across the gastrointestinal membrane from the blood to the lumen. Copyright © 2012 John Wiley & Sons, Ltd.

Ohsumi K.,Astellas Pharma Inc. | Masaki T.,Astellas Research Technologies Co. | Takase S.,Astellas Pharma Inc. | Watanabe M.,Astellas Pharma Inc. | Fujie A.,Astellas Pharma Inc.
Journal of Antibiotics | Year: 2014

A novel antifungal agent, AS2077715, was isolated from the fermentation broth of a fungal strain (339855) identified as a new Capnodium species based on morphological characteristics and large-subunit ribosomal DNA sequencing. AS2077715 was isolated as a white powder via solvent extraction, HP-20 and ODS-B column chromatography and crystallization, and was determined to have the molecular formula C 25 H 41 NO 7. AS2077715 has a structure related to that of funiculosin, an inhibitor of mitochondrial cytochrome bc 1 complex (complex III), and showed antifungal activity against Trichophyton species.

Tanaka-Amino K.,Astellas Pharma Inc. | Matsumoto K.,Astellas Pharma Inc. | Matsumoto K.,Astellas Research Technologies Co. | Hatakeyama Y.,Astellas Pharma Inc. | And 2 more authors.
Acta Diabetologica | Year: 2010

ASP4000 ((2S)-1-{[(1R,3S,4S,6R)-6-hydroxy-2-azabicyclo[2.2.1]hept-3-yl] carbonyl}-2-pyrrolidinecarbonitrile hydrochloride) is a novel, potent and selective dipeptidyl peptidase 4 (DPP IV, EC inhibitor (Keiko Tanaka-Amino et al. in Eur J pharmacol 59:444-449, 2008). The aim of the present study was to characterize the kinetic profile of and identify the long duration effect of the antihyperglycemic activity of ASP4000. ASP4000 was found to inhibit human recombinant DPP4 activity with a K i of 1.05 nM, a k on value of 22.3 × 105 M-1 s -1, and a k off of 2.35 × 10-3 M -1 s-1, with higher affinity than that of vildagliptin. The kinetic studies indicate that both the formation and dissociation of ASP4000/DPP4 complex were faster than those of vildagliptin, and that ASP4000 slow-bindingly inhibits DPP4 with a different mode of inhibition than vildagliptin. In addition, ASP4000 augmented the insulin response and ameliorated the glucose excursion during the oral glucose tolerance test in Zucker fatty rats at 4 h post dosing. ASP4000 is expected to be a promising, long duration DPP4 inhibitor for type 2 diabetes. © 2009 Springer-Verlag.

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