Rat liver microsomes

NDD-713.HCl and -825.HCl were incubated (0.02, 0.1, or 0.5 μM at 37°C, 120 min) with human (XenoTech, Kansas City, KS, USA) or rat liver microsomes (BD Biosciences, Brea, CA, USA) at 0.4 mg/ml protein concentration. Metabolic reaction was initiated by addition of NADPH-regenerating system or NADPH-regenerating system with uridine diphosphate glucuronic acid. Samples were quenched at time points by acetonitrile addition. Control samples, which contained neither NADPH, nor uridine diphosphate glucuronic acid, were monitored for degradation. Compound concentrations were determined by ultraperformance LC-MS (UPLC-MS) relative to calibration standards that were prepared in quenched liver microsomes and fitted to exponential decay functions to determine first-order rate constants (k) for substrate depletion. These were used to calculate in vitro intrinsic clearance (CL_int) values: CL_int = k/microsomal protein content (0.4 mg protein/ml). Metabolic stability assays that used rat liver cytosol (BD Biosciences), human cryopreserved hepatocytes, or rat hepatocytes were carried out similarly. Average viable cell concentrations were determined by Trypan Blue exclusion methods (in the absence of test compounds). Studies were carried out by CDCO.

Paeoniflorin (purity >98%) and hyperoside (purity >98%) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). β-nicotinamide adenine dinucleotide phosphate (NADP⁺) and Lucifer yellow were provided by Sigma (St. Louis, MO, USA). Rat liver microsomes were purchased from BD (Woburn, MA, USA). Acetonitrile and methanol were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Ultrapure water was prepared with a Milli-Q water purification system (Millipore, Billerica, MA, USA). All other chemicals were of analytical grade or better.

Anastrozole (purity >98%), omeprazole (purity >98%) and psoralen (purity >98%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). β-Nicotinamide adenine dinucleotide phosphate (NADP) and lucifer yellow were provided by Sigma (St. Louis, MO, USA). Rat liver microsomes were purchased from BD (Franklin Lakes, NJ, USA). Dulbecco’s modified Eagle’s medium (DMEM) and non-essential amino acid (NEAA) solution were purchased from Thermo Scientific Corp. (Logan, UT, USA). Fetal bovine serum (FBS) was obtained from GIBCO BRL (Grand Island, NY, US). Penicillin G (10,000 U/mL) and streptomycin (10 mg/mL) were purchased from Amresco (Solon, OH, USA). Hanks’ balanced salt solution (HBSS) was purchased from GIBCO (Grand Island, NY, USA). Acetonitrile and methanol were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Ultrapure water was prepared with a Milli-Q water purification system (Millipore, Billerica, MA, USA). All other chemicals were of analytical grade or better.