Amitryptiline

The nine drugs tested (amitryptiline, atenolol, carbamazepine, fleroxacin, loperamide, norfloxacin, pefloxacin, propranolol and zolpidem) and HPLC grade solvents (acetonitrile, methanol and water) were purchased from Sigma-Aldrich (Barcelona, Spain). MDCK cell line was purchased from ATCC (USA) and MDCK-MDR1 cells were provided by Dr. Gottessman, MM (Nathional Institutes of Health, Bethesda). Pig brain homogenate was kindly supplied by a local slaughterhouse and fresh unfertilized chicken eggs were bought in a local supermarket. Table 2 shows the molecular properties of the nine drugs mentioned above.
Dulbecco’s modified Eagle’s medium (DMEM) with high content of glucose, L-glutamine, HEPES, MEM non-essential aminoacid, penicillin−streptomycin, trypsin-EDTA, Hank’s balanced salt solution (HBSS) and fetal bovine serum (FBS) for the cell culture of MDCK and MDCK-MDR1 cell lines were purchased from Sigma-Aldrich.

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.

C57BL/6J wild-type mice (8 weeks) were injected intraperitoneally with amitryptiline (5 mg/kg, dissolved in saline, Sigma Aldrich,) once a day during five days. The fourth day, animals were overnight fasted, and one hour after the last administration of amitryptiline, animals were injected intraperitoneally with APAP (300 mg/kg) and sacrificed 6 hours later. Serum and liver sections were collected for further analyses. A similar protocol was used for 25-HC administration (30 mg/Kg). 25-HC was dissolved in (2-hydroxypropyl)-β-cyclodextrin (Sigma Aldrich) at 45% (w/v). One hour after the last 25-HC administration animals were injected intraperitoneally with APAP (300 mg/kg) and sacrificed 6 hours later. Serum and liver sections were collected for further analyses.