R to radiotracer injection. Brains were then homogenized (Polytron, setting 7) in 5 mL of cold 80 acetonitrile/20 aqueous hydrochloric acid (0.01 ) and centrifuged (17000 rpm, ten min). Following cautious decantation from the supernatants, the pellets had been resuspended in extraction solvent (5 mL) and centrifuged again. Soon after repeating the extraction process after a lot more, an aliquot in the combined supernatants from every rat was removed, weighed and counted for radioactivity. Pellets have been also counted for radioactivity.3. Results3.1 CCR9 Purity & Documentation 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 had been pretreated with PF-04457845 (ip) at two unique doses (0.1 or 1.0 mg/kg) then injected with [11C]CURB by way of the tail-vein and sacrificed 40 min post injection. Based upon the area, uptake of radioactivity in rat brain regions decreased 53 83 for each ip doses of PF-04457845 (Fig. 1, p 0.05).Nucl Med Biol. Author manuscript; out there in PMC 2014 August 01.Hicks et al.Page3.two Radiochemistry To radiolabel PF-04457845, we employed a [11C]CO2 fixation process utilized previously to prepare [11C]carbamates [357], [11C]ureas [37, 38] and [11C]oxazolidinones [39]. All experiments had 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 ready in four.five 1.3 radiochemical yield, according to beginning [11C]CO2 (uncorrected for decay) in addition to a radiochemical purity of 98.four 1.three with a total synthesis time of 25 2 min (n = four, Scheme 1). The reaction was carried out employing an automated synthesis module which necessary no heating/cooling or manual manipulations, as previously described [20, 379]. Clinically beneficial amounts (2.63 0.58 GBq) of [11C]PF-04457845, having a particular activity of 73.5 eight.two GBq/mol at end of synthesis, had been obtained as a final formulated resolution, suitable for animal research. 3.three Lipophilicity as measured by Log P7.4 The partition coefficient, between 1-octanol and 0.02 M phosphate buffer at pH 7.four, of [11C]PF-04457845 was measured ACAT Species through a shake-flask approach [33] to become three.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 higher with SUV ranging from 1.two to four.4, reaching a plateau 40 min post injection (Table 1). Radioactivity was considerably decrease in the plasma than the brain with cortex-to-plasma ratios growing from two:1 to 34:1 between two and 40 min post injection. A heterogeneous uptake of radioactivity was observed with highest levels in the cortex, intermediate amounts within the cerebellum and lowest uptake inside the hypothalamus. This distribution of radioactivity in a variety of brain regions is equivalent to [11C]CURB and in accordance using the identified expression of FAAH inside the rat brain (Fig. 2) [402]. three.5 Specificity of binding of [11C]PF-04457845 To demonstrate that binding of [11C]PF-04457845 is saturable, rats were pretreated (ip) with two doses of PF-04457845 (0.05 or 0.5 mg/kg; 0.11 or 1.1 mol/kg) 1h prior to injection with all the radiotracer (Fig. three). At both of the doses utilized, uptake of radioactivity was lowered by 67 85 , based on the r.