PF-592379

Kinase experiment:

The binding affinities of PF-592379 (PF-592,379) and 7-OH-DPAT are characterized at each the five dopamine receptor subtypes using the following radioligands: [3H]SCH23390 (D1 and D5, 70 Ci mmol, 1 nM), [3H]U-86170 (D2, 62 Ci/mmol, 2 nM), and [3H]spiperone (D3 and D4, 96 Ci/mmol, 0.2 nM). CHO cells expressing recombinant human D1, D2, D3, D4, and D5 receptors are rinsed with, and harvested in, ice-cold Ca2+/Mg2+-free phosphate-buffered saline prior to pelleting (500g, 5 min), resuspension in 25 mM Tris, 5 mM EDTA, and 5 mM EGTA, pH 7.5, and freezing the cells in liquid nitrogen. Upon thawing, the cells are homogenized and centrifuged at 1,000g to remove nuclei and unbroken cells, with the supernatant subsequently centrifuged at 47,000g. The membrane pellet is then washed with Tris, EGTA, EDTA, resuspended in 20 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl2, and 1 mM EDTA, and frozen in liquid nitrogen prior to storage of membrane aliquots at -70°C. Membranes are then thawed and diluted into 20 mM HEPES, pH 7.4, 150 mM NaCl, 10 mM MgCl2, or 1 mM EDTA, 10 mM MgSO4, with binding reactions carried out in a total volume of 0.9 ml for 1 h at room temperature, and stopped by vacuum filtration. Nonspecific binding is assessed with 3 µM SCH23390 (D1-like antagonist) or 3 µM haloperidol (D2-like antagonist). Competition binding experiments employ 11 concentrations of PF-592379 or 7-OH-DPAT run in duplicate. IC50 values are determined by fitting the data to a one-site model by nonlinear least-squares minimization, and Ki values are calculated with the Cheng-Prusoff equation[3].

Animal experiment:

Rats[1]Male Sprague-Dawley rats (~8-10 weeks and 250 g) are surgically prepared with an indwelling jugular vein cannula at least 2 days before administration of dose. Rats had free access to food (rat diet pellets) and water throughout the duration of the study. Four rats receive PF-592379 either by IV dosing via the caudal vein (n=2) or via oral dosing (n=2) (both routes 2 mg/kg, 1 mL/kg). Blood samples (175 μL) are collected into heparinised tubes before dosing and at time points over a 24 h period from the jugular vein cannula, which is flushed with heparinised saline (100 μL, 10 units/mL), and plasma is prepared by centrifugation and stored at -20°C until analysis[1].

References:

[1]. Attkins N, et al. Pharmacokinetics and elucidation of the rates and routes of N-glucuronidation of PF-592379, an oral dopamine 3 agonist in rat, dog, and human. Xenobiotica. 2010 Nov;40(11):730-42.
[2]. Wager TT, et al. Dopamine D3/D2 Receptor Antagonist PF-4363467 Attenuates Opioid Drug-Seeking Behavior without Concomitant D2 Side Effects. ACS Chem Neurosci. 2017 Jan 18;8(1):165-177.
[3]. Collins GT, et al. Lack of abuse potential in a highly selective dopamine D3 agonist, PF-592,379, in drug self-administration and drug discrimination in rats. Behav Pharmacol. 2012 Jun;23(3):280-91.