Istradefylline

IM-HCF cells were cultured in six-well plates at a density of 1.5 × 10⁶ cells/well; the day after, cells were stimulated with TGF-β at the concentration of 10 ng/mL for 24 h to induce a fibrotic phenotype. Cells were treated with two different A_2A receptor antagonists, Istradefylline (Tocris Bioscience Bristol, Bristol, UK) and ZM241385 (Tocris Bioscience Bristol, Bristol, UK), for 24 h, at concentrations of 10 µM and 1 µM, respectively, alone or following TGF-β challenge, as previously described [53 (link),54 (link)].

Clozapine-N-Oxide (Sigma) was diluted in sterile saline and administered at 1 mg/kg i.p. 30 min prior to behavioral testing. Istradefylline (Tocris # 5417) was dissolved in DMSO and diluted to 10% DMSO, 1% NP-40 in sterile saline immediately prior to i.p. administration at 3 mg/kg. Velneperit (MEdChem Express #342577-38-2) has very low solubility in water, thus it was dissolved in pure DMSO prior to injection and injected at 100 mg/kg in 0.03 ml using an insulin syringe. Bexarotene (Tocris # 5819) also has limited solubility in water, thus it was dissolved in pure DMSO prior to injection and injected i.p. at 50 mg/kg in a volume of 0.03 ml using an insulin syringe. Control animals received equal volumes of vehicle. These volumes of pure DMSO have been previously tested and validated to cause no adverse health effects in adult mice.

CHPG [(RS)-2-Chloro-5-hydroxyphenylglycine sodium salt], ZM241385 [4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol], DHPG [(RS)-3,5-Dihydroxyphenylglycine], and CGS21680 [4-[2-[[6-Amino-9-(N-ethyl-β-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride] (Tocris Biosciences) were dissolved in dimethyl sulfoxide (DMSO).
Istradefylline [8-[(1E)-2-(2-(3,4-Dimethoxyphenyl)ethenyl]-1,3-diethyl-3,7-dihydro-7-methyl-1H-purine-2,6-dione] (Tocris Biosciences, hereinafter referred to as KW6002) was orally administered solubilized in the vehicle prepared according to Orr et al.^{44 (link)}. The weight of the animals and the fluid intake were assessed three times a week, and the concentration of the solution was adjusted so that the drug intake was maintained at 4 mg/kg per day^{44 (link)}.