Naloxone

The following drugs and chemicals were used: δ-CNTX-Pn1a was purified by a combination of preparative reverse phase HPLC (RP-HPLC), ion exchange HPLC and analytical reverse phase HPLC as previously described [52 (link)]. µ-Conotoxin MVIIA was purchased from Latoxan (Valence, France). Carrageenan (Sigma, St Louis, MO, USA), Prostaglandin E₂ (Enzo Life Sciences, Farmingdale, NY, USA), AM251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; Tocris, Pittsburg, PA, USA), AM630 (6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl(4-ethoxyphenyl) methanone Tocris, Pittsburgh, PA, USA), Naloxone (Sigma, St. Louis, MO, USA), Clocinnamox (Tocris, Pittsburgh, PA, USA), Naltrindole (Tocris, Pittsburgh, PA, USA), Nor-BNI (Nor-Binaltorphimine dihydrochloride; Sigma, St. Louis, MO, USA) were dissolved as follows: PGE₂ (2% ethanol in saline); AM251 and AM630 (12% DMSO in saline); Carrageenan, δ-CNTX-Pn1a, µ-Conotoxin MVIIA (MVIIA), Naloxone, Clocinnamox, Naltrindole and Nor-BNI (saline).

The rats were randomly divided into 4 groups (n=7 in each group), while receiving one of following 1-day treatments: 1) The control group received 4 subcutaneous (sc) normal saline injections at 2-h intervals; 2) the METH group was administered with the subcutaneous injection of METH (Catalog ID M8750; Sigma-Aldrich Co., St. Louis, MO) dissolved in normal saline at 4 events (4×6 mg/kg at 2-h intervals) [7 ]; 3) the Nal group received the intraperitoneal (ip) injection of naloxone (Sigma-Aldrich Co., St. Louis, MO) dissolved in normal saline at 4 events (4×1 mg/kg at 2-h intervals) [13 ]; and 4) the Nal-METH group were injected with METH (6 mg/kg, sc) and naloxone (1 mg/kg, ip) at 2-h intervals. In the last group, the animals were administered with naloxone 30 min prior to each METH injection [12 , 18 ]. The animals were evaluated in the Morris Water Maze (MWM) task for spatial learning and memory one week later.

Diclofenac sodium and tramadol were gifted by Alliance Pharmaceuticals and Aries Pharmaceuticals, Peshawar, KPK, Pakistan. Silica, TLC plates, and bicuculline were purchased from Sigma-Aldrich, Germany. Methanol, chloroform, hexane, ethyl acetate, butanol, naloxone, acetic acid and formalin were bought from Merck (Darmstadt, Germany).

SR-14968 and SR-17018 were synthesized according to the previously published procedure (Supplementary Material, File S1) [11 (link)]. Morphine (Pharma Cosmetic, Warszawa, Poland) was used as a control as well as to study the effects of SR compounds on morphine antinociception, tolerance and dependence. All drugs were prepared in a vehicle (1% DMSO, 10% Kolliphor EL (Sigma Aldrich, St. Louis, MO, USA), 89% dH2O). Drug solutions were freshly prepared from powder and vortexed before each series of injections. In the experiments in which morphine was used, it was dissolved in the same vehicle as SR agonists. For precipitated withdrawal measurement, mice were injected with a non-selective opioid antagonist, naloxone (Merck, Warszawa, Poland). Drugs were administered intraperitoneally (i.p.), only in the experiment assessing the impact of SR compounds on morphine dependence; morphine was administered subcutaneously (s.c.). SR-14698 was administered to mice at doses of 0.3, 1 or 3 mg/kg; SR-17018 was administered at doses of 8, 24, or 48 mg/kg; and morphine was administered at doses of 5 or 10 mg/kg, depending on the experimental schedule.