AM 630

All experiments were conducted double-blinded with animals randomly assigned into groups. Cisplatin and paclitaxel were used to produce chemotherapy-induced neuropathy. Cisplatin (3 mg/kg i.p.) or saline vehicle was injected four times once weekly
[69 (link)] in a volume of 10 ml/kg. Cisplatin/saline-treated animals were assessed for mechanical paw withdrawal thresholds and cold withdrawal frequencies every four days. Paclitaxel (2 mg/kg i.p.) or cremophor EL: ethanol: saline (1: 1: 4) vehicle was administered to rats four times every two days
[70 (link)] in a volume of 1 ml/kg. Animals with paclitaxel/cremophor treatment were assessed for paw withdrawal thresholds to mechanical stimulation every two days and paw withdrawal frequencies to cold stimulation every four days. On the days animals received cisplatin/saline or paclitaxel/cremophor treatments, behavioral testing was performed prior to pharmacological manipulations.
Effects of pharmacological manipulations on mechanical and cold allodynia were assessed on day 28 in animals receiving cisplatin/saline treatments or day 20 in paclitaxel/cremophor-treated animals. On the test days, animals received either vehicle (DMSO), AM1710 either alone or in combination with the CB₂ antagonist AM630 or the CB₁ antagonist AM251, or the CXCR4 antagonist AMD3100. Withdrawal thresholds to mechanical stimulation and withdrawal frequencies to cold stimulation were measured before drug administration (−60 min) and at 30, 90, 150 min post drug administration in cisplatin/saline-treated animals, or at 30 min and 3 h post drug in paclitaxel/cremophor-treated animals. A subset of cisplatin- and paclitaxel-treated animals was additionally tested at 6 h and 24 h post drug administration.
In Experiments 1 and 2, antinociceptive effects of AM1710 in chemotherapy-induced neuropathy evoked by cisplatin or paclitaxel treatments were studied. Effects of AM1710 (0.1, 1 or 5 mg/kg i.p.)
[19 (link)] or vehicle were assessed in animals receiving cisplatin or paclitaxel treatment. The high dose of AM1710 was also administered to animals that received saline or cremophor-vehicle in lieu of cisplatin or paclitaxel, respectively. To further evaluate the duration of action of the compound, a subset of paclitaxel-treated animals receiving AM1710 (5 mg/kg i.p.) or DMSO vehicle were tested from 30 min to 24 h post injection. Pharmacological specificity of anti-allodynic effects of AM1710 was assessed in both models by co-administering AM1710 (5 mg/kg i.p.) with the CB₂ antagonist AM630 (3 mg/kg i.p.)
[71 (link)] or CB₁ antagonist AM251 (3 mg/kg i.p.)
[72 (link)]. Separate groups received AM630 (3 mg/kg i.p.) or AM251 (3 mg/kg i.p.) alone. In Experiments 3 and 4, the CXCR4 antagonist AMD3100 (10 mg/kg i.p.)
[73 (link)] was administered to animals to examine the impact of blockade of CXCR4 signaling on established neuropathy produced by cisplatin or paclitaxel treatment. AMD3100 (10 mg/kg i.p.) was administered to paclitaxel-treated animals either in absence or presence of AM1710 (5 mg/kg i.p.) to evaluate whether blockade of CXCR4 signaling would enhance CB₂ agonist efficacy.

To determine the impact of miR-142-3p on WFS1, we overexpressed FAM-labeled locked nucleic conjugated miR-142-3p mimics (Qiagen Inc) in primary human HCN2 neuronal cells (ATCC, USA) as WFS1 protein expression was strong and exclusively expressed in BG neurons. HCN2 cells were cultured in CnT Prime medium (CnT-PR, CELLnTEC Advanced Cell Systems AG, Bern, Switzerland) in 8 well chamber slides (Thermo-Fisher Scientific Waltham, MA USA) at 37 °C in a humidified atmosphere with 5% CO₂. At 50–60% confluency, cells were transfected with 30 nM of FAM-LNA- miR-142-3p or FAM-LNA-negative control mimic using the Lipojet transfection reagent (Signagen, DE). Cells were fixed with 2% paraformaldehyde at 96 h post-transfection and immunostained with WFS1 and later with DAPI for nuclear localization.
To determine the impact of THC and the endocannabinoid mechanisms regulating WFS1 expression, miR-142-3p transfected cells (cultured as described in the previous section) were either treated with 3 µM THC or preincubated with the 10 µM cannabinoid receptor 1 inverse agonist AM251 (Tocris Bioscience, Minneapolis, MN) or the cannabinoid receptor 2 antagonist AM630 (Tocris Bioscience, Minneapolis, MN) for 1 h followed by 3 µM THC treatment and incubation for 18 h. At the end of the incubation period, cells were fixed with 2% paraformaldehyde in PBS for WFS1 immunofluorescence staining.

After 24-h exposure to JWH-133 and AM630, cell culture supernatants were collected to measure iron (III). The assay was performed by using the Iron Assay Kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. Briefly, standards and macrophages supernatant were pipetted into the wells and were incubated with an acidic buffer to allow iron release. Then, an iron probe at 25 °C for 60 min was added, protected from light. Released iron reacted with the chromogen resulting in a colorimetric (593 nm) product, proportional to the iron amount. The optical density was measured at a wavelength of 593 nm by using the DAS Italy plate reader (DAS Italy, Palombara Sabina—Italia.). Iron (II) and Total Iron (II + III) contents of the test samples (nmol/μL) were determined against a standard concentration curve. Iron (III) content can be calculated as: Iron (III) = Total Iron (II + III) − Iron (II).

JWH-133 and AM630 powder were purchased from Tocris (Avonmouth, UK) and were dissolved in PBS containing DMSO. DMSO final concentration on cultures was 0.01%. DMD-associated macrophages were treated with JWH-133 [100 nM] and AM630 [10 µM] for 24 h. Non-treated cultured macrophages were maintained in incubation media during the relative treatment time with or without vehicle (DMSO 0.01%).