CR4056

Kinase experiment:

Binding assays on monoamine oxidases in rat cerebral cortical membranes are conducted. In summary, for the MAO-A binding assay, tritiated N-(2-aminoethyl)-5-(m-fluorophenyl)-4-thiazole carboxamide HCl ([3H]Ro 41-1049, 10 nM) is incubated in the absence or presence of CR4056 for 60 minutes at 37°C and non specific binding is determined in the presence of 1 μM clorgyline. For the MAO-B binding assay, incubation of [3H]Ro 19-6327(tritiated lazabemide) (15 nM) is carried out for 90 minutes at 22°C and non specific binding is determined in the presence of 10 μM (R)-deprenyl [1].

Cell experiment:

To rule out any interference of CR4056 with bortezomib (BTZ)-induced cytotoxicity, non-small cell lung cancer and MM cell lines are simultaneously treated for 72 hours with BTZ and CR4056. Cells are exposed to the IC50 of BTZ, estimated in the cytotoxicity study, with or without three concentrations of CR4056 (3, 10, and 30 μM). The incubations with CR4056 alone (3, 10, and 30 μM) and with the highest dose of vehicle (DMSO) are also performed. Growth inhibition is assessed by MTT assay. In these experiments, the CR4056 is tested in vitro in a range of concentrations spanning from pharmacological to toxicological levels[2].

Animal experiment:

Rats: Rats are fasted overnight before drug administration. A first measurement of pain threshold is undertaken before capsaicin injection. Capsaicin is administered at time t=0 by the intraplantar route in the right hind paw (10 μL of a 1 mg/mL Tween 80/saline solution). Sixty minutes later, animals are dosed by the oral route with CR4056 (3 to 30 mg/kg) or its vehicle in a volume of 5 mL/kg. A sham control group is always present for comparison. In mechanistic studies, antagonists are administered 30 minutes after capsaicin and 15 minutes before CR4056 administration[1].

References:

[1]. Ferrari F, et al. Analgesic efficacy of CR4056, a novel imidazoline-2 receptor ligand, in rat models of inflammatory and neuropathic pain. J Pain Res. 2011;4:111-25.
[2]. Meregalli C, et al. CR4056, a new analgesic I2 ligand, is highly effective against bortezomib-induced painful neuropathy in rats. J Pain Res. 2012;5:151-67.