Amitriptyline

KB-R7943 is an isothiourea derivative, quaternary ammonium-containing molecule, with a molecular weight 372, logP 3,83, logD from 1,41 to 1,52 (for pH from 1,7 to 8,0), HLB 16,31 pKa 10 (data from Chemaxone) (Fig. 1).Fig. 1

KB-R7943 [(2-{4-[(4-nitrophenyl)methoxy]phenyl}ethyl)sulfanyl]methanimidamide.

Fig. 1KB-R7943 was from Tocris Bioscience (Bristol, UK). STZ, citrate buffer, and amitriptyline were from Merck KGaA (Darmstadt, Germany), and OraPlus from Perrigo (Dublin, Ireland). The formalin test used a freshly prepared 0.5% formaldehyde solution in saline. amitriptyline (purity not less than 98%) was obtained from Merck (USA), аcetonitrile (ACN, HPLC grade) manufactured by ITW Group (USA), hexane was obtained from Cryochrom (Russia), formic acid produced by Sigma-Aldrich (USA), purified water was prepared in the laboratory with a Milli-Q system from Millipore (USA), ethylacetate (purity not less than 99,8%) produced by Vekton (Russia), reserpine (99%, HPLC grade) manufactured by “Sigma-Aldrich” (USA) was used as an internal standard.

LPS, fluoxetine (FXT), and amitriptyline (AMT) were purchased from Sigma-Aldrich. Tin protoporphyrin IX (SnPP; HO-1 inhibitor) and cobalt protoporphyrin IX (CoPP; HO-1 inducer) were purchased from Santa Cruz Biotechnology (San Diego, CA). Murine BV-2 microglial cells were obtained from Dr. SW Chae (Korea Institute of Oriental Medicine) and cultured in Dulbecco's Modified Eagle's medium (DMEM; Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Carlsbad, MA, USA) and 100 μg/mL penicillin-streptomycin (Gibco) at 37°C in a humidified atmosphere containing 5% CO₂.

Beta-cyclodextrin (BCD), terephthaloyl chloride (TPC), Trimesoyl chloride (TMC), polysulfone, triethylamine (TEA) and N,N′-bis(3-aminopropyl)ethylenediamine (BAPEDA) were purchased from Sigma Aldrich, St. Louis, MO, USA. For the filtration test, different salts (MgCl₂, CaCl₂, MgSO_4, Na₂SO₄, NaCl) and pharmaceutically active compounds (Caffeine, Sulfamethoxazole, Amitriptyline, Loperamide) were also bought from Sigma.
The membranes were characterized using an ATR-Fourier-transform infrared spectroscopy (Thermo, Waltham, MA, USA, Smart iTR NICOLET iS10), a scanning electron microscope (JEOL JSM6610LV, Tokyo, Japan), an atomic force microscope (Agilent 550, Amsterdam, The Netherlands) and water contact angle (KRUSS DSA25). The feed and permeate solution were tested using a conductivity meter (Ultrameter II, Hanna, Woonsocket, RI, USA) for salts and a JASCO V-750 UV-Vis spectophotometer for pharmaceutically active compounds. The membranes were tested for their performance using the Sterlitech CF042 Membrane test system, United States of America.

All treatments, except amitriptyline, were performed on diluted washed RBCs. To activate PIEZO1, RBCs were incubated with 0.5 µM Yoda1 (Biotechne, Minneapolis, MD, USA) for 20 s at 22 °C. To inhibit voltage-dependent Ca²⁺ channels, RBCs were incubated with 1 µM ω-agatoxin (Alomone Labs, Jerusalem, Israel) for 20 min at 22 °C. To modulate the intracellular Ca²⁺, RBCs were preincubated either in a Ca²⁺-free medium containing 1 mM EGTA (Sigma-Aldrich) for 10 min at 22 °C or with 20–40 µM BAPTA-AM (Abcam) for 60–90 min at 37 °C. For cholesterol depletion, RBCs were preincubated with 0.9 mM methyl-β-cyclodextrin (mβCD; Sigma-Aldrich) for 30 min at 37 °C. To decrease oxidative stress, RBCs were preincubated with 1 mM ascorbic acid for 1 h at 37 °C. Lysophosphatidylserine (lysoPS; Sigma) was inserted into membranes at 3 µM in 0.01% (w/v) defatted bovine serum albumin (BSA; Sigma)-containing medium for 5 or 40 min at 37 °C and was maintained during the whole experiment. For plasma acid sphingomyelinase (aSMase) inhibition, whole blood was incubated with 5 µM amitriptyline (Sigma-Aldrich, Saint-Louis, MO, USA) for 1 h at 37 °C, then diluted and washed as above.