Ketoconazole ke

The compounds used as the references—caffeine (CFN), doxorubicin (DX), ketoconazole (KE), nonyl-4-hydroxyquinoline-N-oxide (NQNO), norfloxacin (NFX), sulfaphenazole (SE), quinidine (QD) and warfarin (WFN)—were obtained from Sigma-Aldrich (St. Louis, MO, USA). The references for Pgp activity and pharmacokinetic studies—verapamil (VL) and Na₃VO₄—were provided with the luminescent Pgp-Glo^TM Assay System (Promega, Madison, WI, USA).

The metabolic interactions of H3R antagonist 4 were determined by the luminescent P450-Glo™ 3A4 and 2D6 assays purchased from Promega^®. All enzymatic reactions were performed according to the manufacturer protocols and as previously described [26 (link)]. The references ketoconazole (KE, CYP3A4 inhibitor) and quinidine (QD, CYP2D6 inhibitor) were obtained from Sigma-Aldrich. The luminescence was measured with a microplate reader EnSpire (PerkinElmer). The final concentration of H3R antagonist 4 was in the range of 0.1–25 µM, whereas reference inhibitory effects ranged from 0.01 µM to 10 µM. The IC₅₀ values’ calculations were performed by GraphPad Prism™ software (version 5.01, San Diego, CA, USA).

The influence on CYP3A4 activity by 10f was analyzed with luminescent CYP3A4 P450-Glo (Madison, WI, USA). The compounds were tested in triplicate in a final concentration 10 μM. The luminescent signal was measured with the EnSpire PerkinElmer (Waltham, MA, USA) microplate reader. Reference inhibitor ketoconazole (KE) was obtained from Sigma-Aldrich (St. Louis, MO, USA).