1mol/L sodium hydroxide

solution were mixed, followed by the addition of sodium chloride. All other materials were of analytical reagent grade, and deionized double-distilled water was used. 2.2. Spectroscopic Studies Fluorescence and circular dichroism (CD) spectra were measured at 25°C using a HITACHI fluorescence spectrophotometer F-2500 (Tokyo, Japan) and a JASCO J-720 polarimeter (Tokyo, Japan), respectively. 2.3. Solubility Studies Excess amounts of insulin glargine were shaken in phosphate buffer (pH 9.5, I = 0.2) in the absence Inhibitors,research,lifescience,medical and presence of the selected Abiraterone P450 (e.g. CYP17) anionic β-CyDs at 25°C. After equilibrium was attained, the solutions were filtered with Millex GV filter 0.22 μm, and the insulin Inhibitors,research,lifescience,medical glargine dissolved was determined by high-performance liquid chromatography (HPLC) with Agilent 1100 series (Tokyo, Japan) under the following conditions: Merck Superspher 100RP-18 column (4 μm, 3mm × 250mm, Tokyo, Japan), a mobile phase of phosphate buffer (pH 2.5) and acetonitrile and a gradient flow, increasing the ratio of the acetonitrile Inhibitors,research,lifescience,medical (25–40%) over 30min, a flow rate of 0.55mL/min, a detection of UV at 214nm. 2.4. Ultrafiltration Studies Ultrafiltration studies

were performed using stirred ultrafiltration cells model 8010 (Millipore, Tokyo, Japan) applied with YM30 ultrafiltration discs (MWCO = 30,000) in phosphate buffer (pH 9.5, I = 0.2) in the absence and presence Inhibitors,research,lifescience,medical of the selected anionic β-CyDs at 25°C under nitrogen current. Insulin glargine levels in filtrates were determined by HPLC as http://www.selleckchem.com/products/epz-5676.html described above. 2.5. Particle Size Determination Particle sizes of insulin glargine (0.1mM) with or without the selected anionic β-CyDs (10mM) in phosphate buffer (pH 9.5, I = 0.2) were Inhibitors,research,lifescience,medical measured by Zetasizer Nano (Malvern Instruments, Worcestershire, UK). 2.6. Dissolution Study of Insulin Glargine Insulin glargine (0.1mM) dissolved in phosphate buffer (pH 9.5,

I = 0.2) in the absence and presence of the selected anionic β-CyDs (10mM) was precipitated by a pH shift to 7.4. After centrifugation (2,500rpm, 10min), the AV-951 supernatant was discarded, and then phosphate buffer (pH 7.4, I = 0.2) was newly added to the precipitate at 25°C. At appropriate intervals, an aliquot of the dissolution medium was withdrawn, centrifuged at 2,500rpm for 10min, and analyzed for the insulin glargine by HPLC as described above. 2.7. Stability of Insulin Glargine against Tryptic Cleavage Insulin glargine (0.1mM) in phosphate buffer (pH 9.5, I = 0.2) was incubated with recombinant trypsin (0.02mg/mL) in the absence and presence of the selected anionic β-CyDs at 37°C. At appropriate intervals, 5 μL of sample solution was withdrawn and determined intact insulin glargine level by HPLC.