Anamorelin

Anamorelin was provided by Toronto Research Chemical, Inc. (North York, ON, Canada). Pooled HLM, c-DNA-expressing CYP3A4, and three human flavin-containing monooxygenases (FMO) isoforms (FMO1, FMO3, and FMO5) were obtained from BD Gentest Corp. (Woburn, MA, USA). Glucose 6-phosphate (G6P), β-NADP⁺, glucose 6-phosphate dehydrogenase (G6Pd), and ammonium formate were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All chemicals were analytical grade and were used as received. HPLC-grade acetonitrile (ACN) and distilled water (DW) were purchased from J.T. Baker (Phillipsburg, NJ, USA). Formic acid was purchased from Junsei Chemical Co. (Chou-ku, Japan).

We procured pure standards of anamorelin and diclofenac (experimental control) from Toronto Research Chemicals (Toronto, ON, Canada) and Sigma-Aldrich (Milan, Italy), respectively. Stock solutions of the standards were prepared at 1 mg/mL in methanol and stored at −20 °C. LC-MS grade acetonitrile, water, methanol, and formic acid were obtained from Carlo Erba (Cornaredo, Italy). Cryopreserved human hepatocytes (pooled from ten donors), thawing medium, and trypan blue (0.4%) were obtained from Lonza (Basel, Switzerland). Williams’ medium E (WME), l-glutamine, and HEPES buffer (2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid), were also procured from Sigma-Aldrich. HEPES (20 mmol/L) and l-glutamine (2 mmol/L) were used in preparing Supplemented Williams’ Medium E (SWM) and stored at 4 °C until incubation.