Thioridazine hydrochloride

For the in vitro experiments we used DCX (Sigma-Aldrich Corporation, Denmark) and TDZ (thioridazine hydrochloride, Sigma-Aldrich Corporation, Denmark) in its racemic form. For the in vivo experiments DCX capsules (Dicillin® 250 mg; Sandoz, Denmark) were purchased and used as commercially registered in Denmark for clinical use [24 ]. The purchase of TDZ tablets (Thioridazin-neuraxpharm® 25 mg; thioridazine hydrochloride, Neuraxpharm, Germany) was licenced by the Danish Health and Medicines Authority (reference no.: 2013041542).

Tag-lite labeling medium (LABMED), SNAP-Lumi4-Tb, and the PPHT ((±)-2-(n-phenethyl-n-propyl)amino-5-hydroxytetralin hydrochloride;1-Naphthalenol,5,6,7,8-tetrahydro-6-[(2-phenylethyl)propylamino]) derivative labeled with a red fluorescent probe (PPHT-red) was obtained from Cisbio Bioassays (Bagnols-sur-Cèze, France). Ninety-six-well polypropylene plates (Corning) were purchased from Fisher Scientific UK (Loughborough, UK) and 384-well optiplate plates were purchased from PerkinElmer (Beaconsfield, UK). GppNHp, risperidone, chlorpromazine hydrochloride, quetiapine hemifumarate, ziprasidone hydrochloride monohydrate, zotepine, sertindole, thioridazine hydrochloride, fluphenazine dihydrochloride, molindone hydrochloride, loxapine succinate, perphenazine, trifluoperazine dihydrochloride, spiperone, (−)-sulpiride, droperidol, and (+)-butaclamol used in competition assays were obtained from Sigma-Aldrich (Poole, UK). Olanzapine, nemonapride, remoxipride hydrochloride, flupenthixol dihydrochloride, paliperidone, amisulpride, melperone hydrochloride, clozapine, raclopride, domperidone, asenapine maleate and haloperidol hydrochloride used for competition assays were obtained from Tocris Bioscience (Avonmouth, Bristol).

Hep G2 cells (ATCC HB-8065) were seeded at a density of 2 × 10⁴ cells/well in a 96-well, cell culture-treated microplate. They were incubated for 24 h at 37 °C in 5% CO₂ to allow the cells to adhere. Cells were then exposed to a 2-fold serial dilution series of compound 2, in duplicate, giving a final concentration range of 0.2–100 µM. Thioridazine hydrochloride (Sigma Aldrich) was used as a positive control. The viability of the cells was determined, after a further 24 h incubation, using CellTiter-Glo (Promega) according to the manufacturer’s instructions. Inhibition curves were generated to give the concentration of compound inhibiting 50% of cell viability (IC₅₀) and percentage viability of the cells at the top tested concentration (100 µM).

Thioridazine hydrochloride, phosphate-buffered saline (PBS), 3,4-dihydroxy-l-phenylalanine (l-DOPA) and amphotericin B were purchased from Sigma-Aldrich Inc. (USA). Neomycin sulphate was obtained from Amara (Poland). Penicillin was acquired from Polfa Tarchomin (Poland). Growth medium M-254 and human melanocyte growth supplement-2 (HMGS-2) were obtained from Cascade Biologics (UK). Trypsin/EDTA was obtained from Cytogen (Poland). Cell Proliferation Reagent WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulphonate) was purchased from Roche GmbH (Germany). The remaining chemicals were produced by POCH S.A. (Poland).

MAC (ATCC 700898) stock cultures were stored in Middlebrook 7H9 broth and 10% glycerol at −80°C. Bacterial stock was thawed and incubated at 37°C in a water bath in Middlebrook 7H9 broth with 10% oleic acid-albumin-dextrose-catalase (OADC) under shaking conditions for 4 days to achieve exponential-phase growth for each experiment. Methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 29213) was cultured in Mueller-Hinton broth at 37°C overnight for the protein binding studies. Hollow-fiber cartridges were purchased from FiberCell (Frederick, MD). RPMI 1640 was purchased from Sigma (St. Louis, MO), and fetal bovine serum (FBS) was purchased from SAFC Biosciences (St. Louis, MO) and Sigma. FBS was heat inactivated and filtered prior to use. Thioridazine hydrochloride in powder form was obtained from Sigma. The drug was serially diluted using distilled water to the drug concentrations required for study. Throughout all experiments, thioridazine was shielded from direct light.

Dicloxacillin (Diclocil, Bristol-Myers Squibb) and Vancomycin (Vancomycin, Fresenius Kabi, Denmark) were purchased and used as the commercial product registered in Denmark for parenteral clinical use. Thioridazine (Thioridazine hydrochloride, Sigma-Aldrich Corporation, Denmark) was purchased and used in its racemic form.
Dosages were set on behalf of the given references in Table 1 and considerations on clinical applicability in humans. However, VAN was intentionally and according to the study by Docobo-Perez et al. [15 (link)] set at a high dose compared to the equivalent dose in humans. The rationale was to make sure that Vancomycin had been administered in adequate dosages in order to be a useful positive control, and to minimize the risk of a type II error when comparing other treatments to Vancomycin.
According to the equivalent daily dosages in mice (Table 1), the antimicrobial agents were dissolved in isotonic saline (Amgros I/S, Denmark) at concentrations fitted for an injection of 0.5 ml twice a day. Mice treated with the combination treatment (DCX +TDZ) had two injections at different sites to avoid the possibility of crystallization or altered absorption when the drugs were mixed. Hence, this group of mice had a total volume of 1 ml twice a day. TDZ was at all times before injection protected from sunlight due to its decomposing effect on the drug.

Human breast cancer MCF-7 cells were obtained from the ATCC and cultured in DMEM basal medium at 37 °C in 5% CO₂. The medium was supplemented with foetal bovine serum (10%), amphotericin B (2.5 μg/mL), and Penicillin-Streptomycin (50 U/mL). Chlorpromazine hydrochloride, fluphenazine dihydrochloride, thioridazine hydrochloride, anisomycin, H₂O₂, DMSO, HEPES buffer pH 7.5, NaCl, Triton X-100, bromophenol, MgCl₂, EDTA, Protease Inhibitor Cocktail, and PhosSTOP™ were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Gibco™ BenchStable™ DMEM, Gibco™ Fetal Bovine Serum (qualified, heat-inactivated, United States), amphotericin B (250 µg/mL), Penicillin-Streptomycin (5000 U/mL), and StemPro^® Accutase^® Cell Dissociation Reagent were obtained from Thermo Fischer Scientific (Waltham, MA, USA). Tris(hydroxymethyl)aminomethane (TRIS, Trometamol) Electran^® for electrophoresis and dithiothreitol were obtained from VWR (Randor, PA, USA). Glycine was obtained from Alfa Aesar (Haver Hill, Fullerton, CA, USA), while sodium dodecyl sulfate was obtained from Merck (Darmstadt, Germany). INR119 was synthesized as previously described [14 (link)].