Terfenadine

2,5-dihydroxybenzoic acid (DHB), 9-Aminiacridin hydrochloride (9-AA) trifluoroacetic acid (TFA), paraplast histology grade paraffin, terfenadine, dextromethorphan hydrobromide and diphenhydramine hydrochloride, nitrotetrazolium blue chloride, sodium chloride, 2 mM sodiumhydroxide solution, lactic acid, Nicotinamide adenine dinucleotide hydrate, polypep and Gly-Gly were purchased from Merck (Darmstadt, Germany). Methanol, water, iso-pentane, ethanol, xylene, isopropanol and acetonitrile (ACN) were obtained from Fisher Scientific (Waltham, MA, USA). Losartan-potassium salt was obtained from Cambridge Bioscience (Cambridge, UK). All solvents used were of analytical grade or higher.

Terfenadine and nifedipine (Sigma, Steinheim, Germany) were prepared as 50 mM stock solutions in dimethylsulphoxide (DMSO) and stored at −20 °C. Prior to application, stock solutions were diluted in ERM (medaka) or E3 (zebrafish) to the desired final concentrations; light-sensitive nifedipine was prepared freshly for each experiment. DMSO (vehicle control) and drug incubation experiments were performed with single mounted embryos. For each plate a baseline was recorded in ERM/E3 at 28 °C (see automated microscopy) followed by exchange of the medium with test substances. Prior to the first loop of imaging during drug incubation, embryos were allowed to equilibrate in the incubation chamber of the microscope for min. 15 min to max. 30 min after drug transfer to the plate (equals first point in time).

HDC (>98.0%) and H. helix extract were obtained from the Lab. of Pharmacognosy, College of Pharmacy, Yonsei University (Incheon, Korea). The extract was prepared by extracting the pulverized ivy leaves with 30% ethanol for 1 h using sonication. A voucher specimen of H. helix extract (HY-2016-01-05) was deposited at the Herbarium of the College of Pharmacy, Hanyang University, Ansan, Korea. H. helix extract contains 8.2% of HDC. The content of HDC was determined by liquid chromatography–tandem mass spectrometry (LC-MS/MS) [10 (link)] and the representative chromatogram is shown in Figure 4. Pooled human liver microsomes and recombinant CYP2C8, CYP2C19, and CYP2D6 isozymes were purchased from BD Gentest (Woburn, MA, USA). Glucose-6-phosphate, β-NADP+, Glucose-6-phosphate dehydrogenase, coumarin, phenacetin, diclofenac, midazolam, mephenytoin, dextromethorphan, ketoconazole, and terfenadine were purchased from Sigma Chemical Co. (Saint Louis, MO, USA). All other solvents used were of HPLC grade and were obtained from J. T. Baker (Phillipsburg, NJ, USA). Distilled water was prepared using a Milli-Q purification system (Millipore, Billerica, MA, USA). All standard solutions and mobile phases were passed through a 0.22 µm membrane filter before use.

Human liver microsomes (0.25 mg/mL), 0.1 M phosphate-buffered solution (pH 7.4), substrate drug cocktail of 5 drug-metabolizing enzymes (Phenacetin (50 µM), Diclofenac (10 µM), S-mephenytoin (100 µM), Dextromethorphan (5 µM), and Midazolam (2.5 µM)), and Ce6 were added at a concentration of 0 and 10 µM and were pre-incubated at 37 °C for 5 min. NADPH generation system solution was added and incubated at 37 °C for 15 min. Then, an acetonitrile solution containing an internal standard (Terfenadine, Sigma Aldrich, T9652, St. Louis, MO, USA) was added to terminate the reaction, and it was centrifuged (14,000 rpm, 4 °C) for 5 min. The supernatant was injected into an LC–MS/MS system to simultaneously analyze the metabolites of the substrate drug, thereby evaluating the drug metabolizing enzyme inhibition ability of Ce6.

Apogossypol and Leflunomide from ApexBio (Boston, MA, USA), A-1331852, A-1210477, ABT-199, Z-VAD.FMK and CCCP from Selleck (Houston, TX, USA), Teriflunomide, norhydroguaiaretic acid (NDGA), Ivermectin, Terfenadine, Suloctidil, orotate and uridine from Sigma Aldrich (St Louis, MO, USA), MitoTracker Deep Red FM from Thermo Fisher (Loughborough, UK) were used. Antibodies against BAP31, RTN4, BiP, PDI, CHOP, DHODH and tubulin from Abcam (Cambridge, UK), CLIMP-63 and BAX (6A7) from Enzo Life Sciences (Exeter, UK), TIM22 and KNT-1 from Sigma, HSP60, Cytochrome c, BAX, OPA1 and DRP-1 from BD Biosciences (San Jose, CA, USA), phospho-DRP-1 (S616), phospho-DRP-1 (S637), MFN1 and MFN2 from Cell Signaling Technologies (Danvers, MA, USA), BAK (AB-1) from Calbiochem (Watford, UK), MFF, MID49 and MID51 from ProteinTech (Manchester, UK) and GAPDH from Santa Cruz Biotechnologies (Santa Cruz, CA, USA) were used. For RNA interference, cells were transfected with 10 nM of siRNAs against DHODH (SI00363384 and SI00363391) purchased from Qiagen Ltd. (Manchester, UK), using Interferin (Polyplus Transfection Inc, NY), according to the manufacturer’s protocol and processed 72 h after transfection.

Serum samples that were stored at −80 °C were used for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. For sample preparation, 100 μL of the sera, 800 μL of 70% methanol, and 10 μL of the internal standard were briefly vortexed and then left on ice for 10 min. The sample mixture was then obtained by centrifugation at 10,000 rpm for 5 min at 4 °C, and the supernatant was lyophilized overnight at −84 °C. Then, 100 μL of 10% methanol was added to the freeze-dried supernatant, vortexed, and centrifuged at 10,000 rpm for 5 min at 4 °C. The supernatant (90 μL) was stored at 4 °C until further analysis. For the internal standard, 10 μg/mL of reserpine, acetaminophen, sulfadimethoxine, and terfenadine (Sigma-Aldrich, Oakville, ON, Canada) solutions were prepared in 70% acetonitrile in autoclaved water. Then, the same volumes of each solution were mixed well and stored at 4 °C until sample preparation.