. The capture column was eluted with 1 acetonitrile (2 mL/ min) for four min then backflushed (60 acetonitrile/40 water + 0.1 N ammonium formate, pH = 4, two mL/min) onto a Phenomenex Luna C18 10m, 250 x four.six mm column. Both column effluents have been monitored by way of a flow detector (Bioscan Flow-Count) operated in coincidence mode. To monitor for very lipophilic metabolites, the HPLC eluent was switched to one hundred ethanol immediately after the parent radiotracer eluted. All radioactivity data have been corrected for physical decay and integrated. 2.six Irreversible binding of [11C]PF-04457845 to FAAH within the rat brain Following tail-vein injection of [11C]PF-04457845 groups of three conscious male SpragueDawley rats have been sacrificed and the entire brain was surgically removed in the skull, washed in saline, and kept on ice. To measure specific binding, rats in a single group were pretreated with URB597 (2 mg/kg in saline with five Tween80? ip) 1 h before radiotracer injection. Brains have been then homogenized (Polytron, setting 7) in five mL of cold 80 acetonitrile/20 aqueous hydrochloric acid (0.01 ) and centrifuged (17000 rpm, 10 min). Following careful decantation on the supernatants, the pellets had been resuspended in extraction solvent (five mL) and centrifuged again. Immediately after repeating the extraction process once extra, an aliquot from the combined supernatants from each rat was removed, weighed and counted for radioactivity. Pellets have been also counted for radioactivity.three. Results3.1 Blocking [11C]CURB with PF-04457845 We synthesized the identified FAAH inhibitor PF-04457845 as previously reported by Johnson et al [16]. To verify its ability to cross the blood-brain barrier and block FAAH, conscious male Sprague-Dawley rats have been pretreated with PF-04457845 (ip) at two different doses (0.1 or 1.0 mg/kg) then injected with [11C]CURB by means of the tail-vein and sacrificed 40 min post injection. Depending upon the area, uptake of radioactivity in rat brain regions decreased 53 ?83 for both ip doses of PF-04457845 (Fig. 1, p 0.05).Nucl Med Biol. Author manuscript; available in PMC 2014 August 01.Hicks et al.Page3.two Radiochemistry To radiolabel PF-04457845, we employed a [11C]CO2 fixation system made use of previously to prepare [11C]carbamates [35?7], [11C]ureas [37, 38] and [11C]oxazolidinones [39]. All experiments have been carried out by bubbling [11C]CO2 into a conical vial containing a fixating base (BEMP) and 2-(3-piperidin-4-ylidenemethyl-phenoxy)-5-trifluoromethyl-pyridine hydrochloride (PPP) in acetonitrile. Following HPLC purification and formulation, [11C]PF-04457845 was prepared in 4.five ?1.three radiochemical yield, based on beginning [11C]CO2 (uncorrected for decay) along with a radiochemical purity of 98.Benzofuran-4-carboxylic acid site 4 ?1.Formula of 3,5-Dibromo-1H-pyrazole-4-carbonitrile three using a total synthesis time of 25 ?two min (n = 4, Scheme 1).PMID:23398362 The reaction was carried out employing an automated synthesis module which needed no heating/cooling or manual manipulations, as previously described [20, 37?9]. Clinically helpful amounts (two.63 ?0.58 GBq) of [11C]PF-04457845, with a certain activity of 73.5 ?eight.two GBq/mol at end of synthesis, were obtained as a final formulated answer, appropriate for animal studies. 3.3 Lipophilicity as measured by Log P7.4 The partition coefficient, involving 1-octanol and 0.02 M phosphate buffer at pH 7.4, of [11C]PF-04457845 was measured by way of a shake-flask strategy [33] to become 3.48 ?0.08 (n = 16). 3.four Regional and temporal distribution of [11C]PF-04457845 in rat brain Following tail-vein injections of [11C]PF-04457845 into conscious rats, brain uptake was.