Midazolam

Uridine diphosphate glucuronic acid (UDPGA), diazepam, and alamethicin were obtained from Sigma-Aldrich (St. Louis, MO). Ezetimibe and Ezetimibe-D4 were purchased from Toronto Research Chemical (Toronto, ON, Canada). Morphine sulfate, serotonin, and midazolam were obtained from Cerilliant Corporation (Round Rock, TX). Formic acid, methanol, and acetonitrile (ACS grade) were purchased from Avantor Performance Materials, Inc. (Center Valley, PA). Acetic acid and ammonium formate (ACS grade) were obtained from Fisher Scientific (Grand Island, NY). The miRNA mimic library (miRIDIAN microRNA Mimic Library, v.19), the miRNA mimics for miR-21–3p, miR-103b, miR-141–3p, miR-200a-3p, and miR-376b-3p, the miRNA mimic negative controls (Negative controls 1 and 2), and the short interfering RNA directed against the UGT1A 3’-UTR [positive control (siUGT1A), 5’-CCU AGU CAU UUC CAA ACU UUU-3’] were purchased from GE Dharmacon (Lafayette, CO). The pMIR-REPORT plasmid was from Life Technologies (Grand Island, NY) and the Renilla luciferase vector pRL-CMV was purchased from Promega (Madison, WI). All synthesized DNA oligos used in this study were obtained from Integrated DNA Technologies, Inc. (Coralville, IA). Lipofectamine RNAiMAX was from Life Technologies and the transfection reagent DharmaFECT Duo was purchased from GE Dharmacon.

Midazolam, 1-hydroxyMidazolam, and ²H₄-Midazolam (internal standard) were purchased from Cerilliant (Round Rock, TX, USA), and 4-hydroxyMidazolam [Figure 1], reagent-grade formic acid (~98%), and β-glucuronidase from Helix promatia were purchased from Sigma-Aldrich (St. Louis, MO, USA). The different individual (either male or female) lots of drug-free (blank) human plasma prepared with citrate phosphate dextrose (CPD) as an anti-coagulant were obtained from the blood bank at The Children’s Hospital of Philadelphia. Six different lots of drug-free (blank) human plasma prepared with lithium heparin as an anti-coagulant were obtained from BioreclamationIVT (Baltimore MD). HPLC grade methanol and acetonitrile were purchased from Fisher-Scientific (Pittsburgh, PA, USA). Deionized water from Barnstead Nanopure™ water purifying system (Thermo Fisher Scientific, Marietta, OH, USA) was used for all experiments.

Cortisol was purchased from Sigma-Aldrich (St. Louis, MO). Midazolam and 1-OHMidazolam-d₅ were purchased from Cerilliant Corporation (Round Rock, TX). Acetic acid and acetonitrile were obtained from Fisher Scientific (Pittsburgh, PA). William’s Medium E, Dulbecco’s phosphate-buffered saline (DPBS), GltaMAX-I, insulin-transferrin-selenium, penicillin-streptomycin(10,000 U/ml), Trizol reagent, TaqMan® reverse transcription reagents, universal Polymerase Chain Reaction Master Mix, CYP3A4, CYP3A5, GR, PXR, Tyrosine aminotransferase(TAT), UDP-glucuronosyltransferase(UGT) 1A1, and human beta glucuronidase(GUSB) Taqman primer-probe sets and CYP3A4, CYP3A5, GR, and PXR siRNAs were purchased from Life Technologies (Carlsbad, CA).