Louis, MO) and were used without further purification. 2.2. Solubility, Solid-State Properties, and Formulation Evaluation of the Free Base The solubility of Compound 1 was assessed by stirring a small amount of crystalline free base in scintillation vials that contained 5mL of various pH buffers and FASSIF (fasted state simulated intestinal fluid). Samples were checked periodically to ensure that they were saturated with excess solid. At the end of 48hrs, a final pH reading was taken for each sample and a representative amount of the slurry was aliquoted into centrifuge tubes. These were centrifuged at 14,000rpm for a period of two hours. Supernatants were transferred into

individual HPLC vials, and the concentration was determined by HPLC Inhibitors,research,lifescience,medical (DAD). The remaining solid form was analyzed by PXRD. Formulations with mTOR inhibitor aqueous media were prepared by suspending bulk drug in a vehicle containing 0.5% Methylcellulose and 0.1% Tween 80 in distilled water. Formulation concentrations were adjusted to dose with a fixed dosing volume for all doses Inhibitors,research,lifescience,medical (total dose 20mL/Kg/day). Particle size distribution of each formulation was determined on a Beckman Coulter LS 230 particle Inhibitors,research,lifescience,medical size analyzer. 2.2.1. In Vivo Methodology For in vivo work, male Sprague-Dawley (SD) rats were purchased from Charles River Laboratories (Wilmington, MA). This animal study

was approved by the St. Louis Pfizer Institutional Animal Care and Use Committee. The animal care and use program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. All oral doses other than standard

Inhibitors,research,lifescience,medical Pharmacokinetic studies were performed under “fed” condition to better estimate the multiday toxicology study. The oral dose volume was based on 20mL/Kg/day of body weight for all studies. All doses were based on mg/Kg of body weight. Rats were catheterized in the jugular vein and carotid artery for iv dosing and sampling, respectively. At each time Inhibitors,research,lifescience,medical point, 150uL of blood was withdrawn from each animal, and replaced by saline. Blood sampling was carried out using a Culex Automated Blood Sampling System (West Lafayette, IN) and collected in microtainer plasma separator tubes with lithium heparin using heparinized capillary tubes. Plasma samples were obtained by centrifugation at 8000rpm for 10 oxyclozanide minutes, and 20μL of the plasma sample was extracted with 180μL of acetonitrile containing 0.25μM of the internal standard (prepared in house). The precipitated samples were centrifuged, and supernatant was transferred to a 96-well plate. Analytical standards were prepared by spiking known amount of standards into control plasma and followed the above extraction procedure. Tandem dosing (three times) was performed at 50, 100, and 200mg/kg and dose intervals were 1, 1.5, and 2.5hrs. Plasma samples were analyzed by LC/MS/MS. A Shimadzu LC (LC 20 AD) multiple solvent pump system was used for the gradient elution.