Ciclopirox olamine

Peripheral blood mononuclear cells [PBMC] were isolated from buffy coats obtained from healthy blood bank donors, using Ficoll density gradient centrifugation. PBMC of two individual donors were cultured in a 1:1 ratio for 48 h. Subsequently, anti-TNF [infliximab] or control IgG [Sigma Aldrich, Zwijndrecht, The Netherlands] was added, to a final concentration of 10 µg/ml. Where appropriate, additional compounds were added at the concentrations indicated. Albendazole [#A4673], mebendazole [#46404], docetaxel [#01885], paclitaxel [#T7402], budesonide [#PHR1178], fenbendazole [#35032], pyrythione zinc [#H6377], ciclopirox olamine [#C0415], pyrimethamine [#46706], quinacrine [#Q3251], mycophenolic acid [#M3536], adapalene [#A7486], amitryptiline [#A8404], duloxetine [#SML0474], triamterene [#T4143], and colchicine [#C9754] were all obtained from Sigma Aldrich. AMPK activator A769662 was obtained from SelleckChem [#S2697, Munich, Germany] and Compound C from Calbiochem [#171260–1, San Diego, CA]. Cultures were incubated for another 4–5 days and analysed by flow cytometry.
For analysis of effects on isolated macrophages, mixed lymphocyte reactions (MLR) were cultured for 48 h. CD14-expressing cells were isolated using magnetic cell sorting [CD14-microbeads, Miltenyi, Bergisch Gladbach, Germany] and cultured for an additional 72 h in the presence of anti-TNF and/or Albendazole.

SH-SY5Y and NTERA-2cl.D1 (NT2/D1) cells were obtained from ATCC and cultured in DME (D6046)/F12 1:1 (Sigma-Aldrich, St. Louis, MO, USA) and DME (D0547)/F12 (Sigma-Aldrich) supplemented with 0.21% NaHCO₃, respectively. The murine hippocampal cell line HT22 was a kind gift from D. Schubert, Salk Institute, La Jolla, CA, USA and cultured in DME (D0547)/F12 1:1 supplemented with 0.21% NaHCO3. Media for all cells were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). All cells were cultured in a humidified 5% CO₂ atmosphere at 37 °C. Hypoxia culture experiments were performed in an O₂-regulatable incubator (Binder GmbH, Tuttlingen, Germany) or in a H135 HEPA hypoxystation (Don Whitley Scientific Limited, West Yorkshire, UK) in a humidified 5% CO₂ and 1% O₂ atmosphere. Ciclopirox olamine and deferoxamine were obtained from Sigma-Aldrich. Cells were plated in 24-well plates 1 day before transient transfection with plasmid constructs for 24–48 h using Lipofectamine 3000 (Invitrogen) or DNAfectinTM Plus (ABM Inc., Vancouver, Canada). The pRL-TK plasmid (Promega) was used as a transfection control. Luciferase activities were measured using the Dual Luciferase Reporter Assay System (Promega) as described [15 (link)]. Results are representative of two or more experiments, each performed in quadruplicate, and are shown as means + SD.

Cobalt chloride hexahydrate was a product of Sigma Chemical Co. and was used as a 100 mM stock solution in water. Ciclopirox olamine was also purchased from Sigma and was used as a 250 mM stock in methanol. Dimethyloxalylglycine was used as a 100 mM stock solution in water and was obtained from EMD Millipore. All culture medium and associated reagents were a product of Corning Cellgro (Mediatech, Inc). The fetal bovine serum used in the culture medium was a product of HyClone (Thermo Fisher Corp.).

Compounds were prepared in 100% DMSO, at the following concentrations: benznidazole (supplied by EpiChem Pty Ltd., Perth, Australia): 34.7 mM, nifurtimox (Sigma Aldrich, St. Louis, MO, USA): 34.7 mM, ciclopirox olamine: 40 mM, camptothecin 20 mM, clotrimazole 17.5 mM (all purchased from Sigma Aldrich, USA). Ethyl oxo{[2-(4-phenyl-1-piperazinyl)-2-(3-pyridinyl)ethyl]amino} acetate: 20 mM (E155-0206; Chemdiv, San Diego, CA, USA). MMV688958 and MMV688796 were sourced from Enamine (Kyiv, Ukraine) and prepared at 20 mM in 100% DMSO. Compounds were tested in assays in 14 log dilutions to determine IC₅₀ values, ranging from 73 µM to 3.7 × 10⁻³ µM for camptothecin, E155-0206, MMV688958 and MMV688796; 64.1 µM to 2.6 µM for clotrimazole; 146 µM to 2.6 µM for ciclopirox olamine and 127 µM to 6.4 × 10⁻³ µM for nifurtimox and benznidazole, to determine IC₅₀ values.