Troleandomycin

A mixed gender 50-donor pool of human liver microsomes, and supersomes made from baculovirus-infected insect cells that express recombinant human CYP3A4 with P450 reductase and cytochrome b₅, were purchased from Corning (Corning, NY). NADPH was purchased from Merck (Darmstadt, Germany). Glucose-6-phosphate and azamulin were from Sigma (NSW, Australia), Glucose-6-phosphate dehydrogenase was from Roche (NSW, Australia) and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) was from Cerestar (Hammond, IN). Troleandomycin was from Enzo Life Sciences (Farmingdale, NY). All solvents used were of HPLC grade, and were purchased from Merck (Darmstadt, Germany). 20(OH)D3 was synthesised enzymatically by the action of recombinant bovine CYP11A1 on vitamin D3 and was purified by TLC and reverse-phase HPLC (2 (link),4 (link)). 20,25(OH)₂D3, 20,24R(OH)₂D3 and 20,24S(OH)₂D3 were produced by the action of recombinant rat CYP24A1 on 20(OH)D3 (21 (link),22 (link)). 20,26(OH)₂D3 was produced from the action of recombinant CYP27A1 on 20(OH)D3 (25 ). The structures of these metabolites including the stereochemistry have been determined previously by NMR (21 (link)–23 (link)). Concentrations of hydroxyvitamin D standards were determined from their absorbance at 263 nm using an extinction coefficient of 18,000 M⁻¹ cm⁻¹ (35 (link)).

All buffers, media, and supplements were purchased from Life Technologies (Carlsbad, CA, USA) except for BSA (Thermofisher, Waltham, MA, USA), ascorbic acid (Sigma, St. Louis, MO, USA), collagenase (Sigma, St. Louis, MO, USA), Percoll^™ (GE, Pittsburgh, PA, USA), FBS (Hyclone, Logan, UT, USA), Matrigel^™ (BD Corning, Tewksbury, MA, USA), BCA protein assay kit (Pierce Thermofisher, Waltham, MA, USA), midazolam (Cambridge Isotope Laboratories, Andover, MA, USA), 4OH-midazolam (Sigma, St. Louis, MO, USA), troleandomycin (Enzo Life Sciences, Farmingdale, NY, USA), dexamethasone (Cayman Chemical Company, Ann Arbor, MI, USA), and diltiazem (Sigma, St. Louis, MO, USA). Multi-well cell culture plates were purchased from BD Corning (Tewksbury, MA, USA).