endocannabinoid signaling at the g - protein 
 coupled receptors cb1 and cb2 is activated by 
 the lipid messengers n - arachidonoylethanolamine ( anadamide ) 
 and 2-arachidonoylglycerol 
 ( 2-ag ) , which are metabolized by fatty acid amide hydrolase ( faah ) 
 and monoacylglycerol lipase ( magl ) , respectively . anandamide 
 and 2-ag are thought to be produced on demand in postsynaptic neurons , 
 since both chemical messengers have low aqueous solubility that may 
 preclude vesicular storage . retrograde 
 signaling to cb1 on the presynaptic neurons then initiates 
 a cascade that regulates calcium and potassium ion channels . reuptake 
 of endocannabinoids is still a matter of some debate , though it is 
 believed to be preceded by amide hydrolysis , hence the tone of endocannabinoid 
 signaling in the central nervous system is regulated by faah and magl . distortion in the cannabinoid system has been linked to a wide variety 
 of pathologies including eating disorders , neurodegenerative disorders , 
 anxiety , depression , pain , and inflammation . faah in particular shows promise as a therapeutic target since selective 
 inhibitors of this enzyme have not been shown to contribute to hypomotility , 
 hyper - reflexia , and cb1 receptor downregulation , nor to 
 be candidates for substance abuse , in contrast to magl inhibitors . several faah inhibitors including urb597 and pf-04457845 ( figure 1 ) have advanced to phase i and ii clinical trials for treatment of 
 pain , cannabis dependence , and schizophrenia . structures of faah inhibitors 
 urb597 and pf-04457845 and pet imaging 
 agents [ c]curb and [ f]dopp . accurate quantification of faah activity in the 
 living human brain 
 under normal and diseased states would assist in understanding the 
 role that the endocannabinoid system plays in these conditions , and 
 could provide a valuable tool for drug development . positron emission 
 tomography ( pet ) is a noninvasive molecular imaging technique that 
 uses positron - emitting radionuclides to prepare functional probes 
 with high sensitivity and is administered at a dose that leads to 
 minimal perturbation of the underlying biological state . several pet 
 radiotracers for faah have been reported , including anadamide analogues , carbon-11 labeled pf-04457845 and a fluorine-18 labeled derivative , [ c]mk-3168 , a c - methyl labeled urb597 analogue , and related [ c - carbonyl]-o - arylcarbamates prepared by co2-fixation in the presence of an organic base . among pet radiotracers for faah , only two have 
 been reported in human studies , namely [ c]mk-3168 and [ c - carbonyl]-6-hydroxy-[1,1-biphenyl]-3-yl cyclohexylcarbamate ( [ c]curb ) . this 
 scaffold has been fruitful for development of selective faah cns probes 
 due to the mechanism of inhibition by covalent modification of the 
 ser241-ser217-lys142 active site , 
 which is unusual for serine hydrolases . quantification of faah activity with [ c]curb 
 was first 
 performed by ex vivo biodistribution studies in rats . high brain uptake ( 1.62.4 suv , standardized 
 uptake value ) was followed by low washout , which is expected from 
 an irreversible tracer . radiotracer uptake was irreversible , consistent 
 with known faah distribution , and could 
 be blocked by pretreatment with urb597 and other faah inhibitors . human imaging studies showed rapid and high brain uptake of [ c]curb with regional peak and plateau concentrations greatest 
 in the putamen and thalamus compared to other regions , matching human postmortem data . a two - tissue compartment model 
 with irreversible trapping was found to be optimal , and the composite 
 parameter k3 provided a sensitive 
 measure of faah activity . while [ c]curb appears 
 to be a very promising radiotracer 
 for faah , it is limited by the half - life of carbon-11 ( c , t1/2 = 20.4 min ) , which necessitates 
 an on - site cyclotron for radiotracer production and poses many practical 
 challenges for its widespread use . in light of the potential demonstrated 
 by [ c]curb , a fluorine-18 ( f , t1/2 = 109.7 min ) labeled o - arylcarbamate , 
 [ f]3-(4,5-dihydrooxazol-2-yl)phenyl ( 5-fluoropentyl)carbamate 
 ( [ f]dopp ) , has been prepared in a three - step radiosynthesis 
 and also evaluated in rats . similar to 
 [ c]curb , [ f]dopp showed irreversible and 
 specific binding , with regional distribution consistent with faah 
 distribution . however , the stability of [ f]dopp in rat 
 plasma was low , as less than 5% of radioactivity was associated with 
 the parent compound 5 min after radiotracer administration . though 
 the observed metabolism is likely associated with rodent carboxylesterases 
 that are not present in human plasma , rapid consumption 
 of the parent radiotracer could have a profound effect on modeling 
 outcome parameters of its distribution . in order to advance [ f]dopp for human studies , we sought to evaluate the kinetics 
 and metabolism of this radiotracer in higher species and with plasma 
 input function correction . we are also interested in determining the 
 sensitivity of [ f]dopp binding by pretreatment with faah 
 inhibitors in a dose dependent manner , prior to evaluation of this 
 radiotracer in human subjects . in the present study , we conducted 
 pet imaging of [ f]dopp in nonhuman primates to assess 
 brain uptake , washout , regional distribution , and kinetic modeling 
 of this tracer following pretreatment studies . precursors for the synthesis of [ f]dopp were prepared 
 by literature procedures with minor modifications . chemical yields and characterization data closely matched previously 
 published values . a ge pettrace 16.5 
 mev cyclotron was 
 used for [ f]fluoride production by the o(p , n)f nuclear reaction to irradiate o - enriched water . synthesis of [ f]dopp was performed manually using minor 
 modifications to the previously reported automated procedure . specifically , [ f]fluoride drying 
 was conducted as follows : a solution consisting of 14.4 mg of 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane 
 ( crypt-222 ) , and 3 mg of k2co3 in 1 ml of 10% 
 ( v / v ) water in acetonitrile was used to elute [ f]fluoride 
 from the ion - exchange resin and into a conical glass vial . the vented 
 vial was heated to 90 c under continuous nitrogen gas flow . 
 when no liquid was visible in the vial , it was removed from heat , 
 anhydrous acetonitrile ( 1 ml ) was added , and the heating was resumed 
 until dryness . the 
 remainder of the radiosynthesis was conducted as previously described , with the exceptions of reaction temperatures 
 for [ f]fluoride incorporation and amine deprotection , 
 which were increased to 100 c from the previously reported 80 
 c . urb597 was purchased from cayman chemicals 
 and formulated in saline with 5% ethanol and 5% tween 80 . all solutions 
 were filtered through a 0.22 m filter into a sterile empty 
 vial and were tested for sterility and pyrogen content . an aliquot 
 of the pretreatment solution for injection was withdrawn and analyzed 
 by hplc to determine urb597 concentration . all animal studies were performed in 
 accordance with the national institutes of health guide for the care 
 and use of laboratory animals and were approved by the massachusetts 
 general hospital institutional animal care and use committee . three adult baboons ( papio anubis , 2m/1f , 34 
 years old , 10.115.6 kg ) , deprived of food for 12 h prior to 
 imaging experiments , were included in the study . one baseline and 
 two pretreatment experiments were conducted for a total of three imaging 
 sessions . intramuscular ketamine ( 10 mg / kg ) was administered for animal 
 preparation and intubation . baboons were catheterized antecubitally 
 for hydration and radiotracer injection , and a radial arterial line 
 was placed for plasma and metabolite analysis . for maintenance of 
 anesthesia throughout the imaging study , baboons were provided 12% 
 isoflurane ( forane ) in a mixture of medical oxygen . vital signs including end 
 tidal co2 , spo2 , heart rate , respiration rate , 
 and blood pressure were monitored and maintained within normal ranges 
 during the studies . pet and mri images were acquired on a 3 t 
 siemens tim - trio with 
 a brainpet insert ( siemens , erlangen , germany ) . a custom pet / mri compatible 
 8-channel array coil for nonhuman primate brain imaging was used to 
 improve image signal and quality . dynamic pet image acquisition was 
 initiated concurrently with the administration of the radiotracer 
 ( 4.99  0.20 mci , 3 scans ) in a homogeneous solution of 10% ethanol 
 and 90% isotonic saline , and continued for a duration of 120 min . 
 a high - resolution anatomical scan using multiecho mprage sequence 
 ( tr = 2530 ms , te1/te2/te3/te4 = 1.64/3.5/5.36/7.22 ms , ti = 1200 
 ms , flip angle = 7 , and 1 mm isotropic ) was acquired for anatomic 
 coregistration . for pretreatment studies , baboons were administered 
 urb597 ( 200 or 300 g / kg , iv ) 10 min prior to radiotracer administration . blood was sampled from 
 the radial arterial line nominally every 10 s for the first 3 min 
 ( 1 ml each ) after radiotracer administration , followed by 
 sampling at 5 , 10 , 20 , 30 , 45 , 60 , and 90 min time points ( 3 
 ml each ) for plasma and metabolite analysis . arterial samples were 
 centrifuged to obtain plasma , which was then removed ( 200 l 
 for samples collected during the first 3 min ; 600 l for all 
 later samples ) and placed in an automated gamma counter using a 350600 
 kev window , and cross - calibrated with the pet scanner . beginning with 
 the arterial sample acquired at 5 min after radiotracer administration , 
 an aliquot ( 600 l ) of plasma was added to acetonitrile ( 600 
 l ) and centrifuged for 13 min to obtain protein - free 
 plasma ( pfp ) . an aliquot ( 600 l ) of pfp was diluted with deionized 
 water ( 3 ml ) and loaded onto a customized automated robot , fitted 
 with phenomenex strata - x 500 mg spe cartridges that were primed with 
 ethanol ( 2 ml ) and deionized water ( 20 ml ) . solutions of pfp were 
 loaded on spe cartridges and then extracted sequentially with 4 ml 
 of 100% ( twice ) , 90% , 85% , 80% , 75% , and 60% water , followed by 100% 
 acetonitrile . each eluent sample 
 was counted in an automated gamma counter to determine the presence 
 of radiolabeled metabolites . an aliquot 
 of [ f]dopp formulated in 10% ethanolic saline ( 10 l ) 
 was added 
 to a sample of baboon plasma ( 0.8 ml ) . the mixture was gently mixed 
 by repeated inversion and incubated for 10 min at room temperature . 
 following incubation , a small sample ( 20 l ) was removed to 
 determine the total radioactivity in the plasma sample ( at ; at = abound + aunbound ) . an additional 
 0.2 ml of the plasma sample was placed in the upper compartment of 
 a centrifree ultrafiltration device ( amicon , inc . the upper part of the device and the 
 membrane were discarded , and an aliquot ( 20 l ) from the collection 
 cup was removed to determine the fraction of radioactivity that passed 
 through the membrane ( aunbound ) . plasma 
 protein binding is reported as the free fraction of radioactivity 
 ( aunbound / at ) . pet data were registered 
 to the black baboon brain atlas using 
 jip tools optimized for nonhuman primate data processing ( www.nitrc.org/projects/jip ) . image registration was carried out on high - resolution t1-weighted anatomical mri images using a 12 
 degree of freedom linear algorithm and a nonlinear algorithm to the 
 atlas brain . twelve volumes 
 of interest ( vois ) were defined according to the black baboon brain 
 atlas . time  activity curves ( tacs ) 
 were extracted from the amygdala ( amyg ) , dorsal lateral prefrontal 
 cortex ( dlpfc ) , hippocampus ( hip ) , primary motor cortex ( m1 ) , nucleus 
 accumbens ( nac ) , posterior cingulate cortex ( pcc ) , putamen ( put ) , 
 orbitofrontal cortex ( ofc ) , supplementary motor area ( sma ) , thalamus 
 ( tha ) , caudate ( cau ) , and cerebellum ( cb ) vois for analysis . compartmental 
 models ( one - tissue ( 1tcm ) , two - tissue ( 2tcm ) , and three - tissue ( 3tcm ) 
 compartmental models ) with and without irreversible binding were carried 
 out in pmod using the metabolite - corrected arterial blood as input 
 function . a composite parameter k3 , where  equals k1/k2 , was determined for all vois from all scans and plotted 
 in figure 4 . a two - way anova was applied to 
 compare regional k3 values between 
 baseline and the two pretreatment scans using graphpad prism ( prism6 , 
 graphpad software inc . , la jolla , ca , usa ) . because a 2tcm with 
 irreversible binding ( 2tcmi ) provides the best 
 fits in regional analysis , voxelwise maps were then obtained using 
 2tcmi from the dynamic pet data using pmod ( pxmod module ) . in order 
 to implement a 2tcmi fitting on noisy voxelwise tacs , we implemented 
 a published method to reduce the number of parameters being estimated . specifically , instead of fitting the model for 
 voxelwise k2 , a k1/k2 ratio representing the distribution 
 volume of free plus nonspecific binding of a high binding region ( i.e. , 
 basal ganglia ) was first estimated . [ f]dopp was 
 prepared by a multistep radiosynthesis that involved radiofluorination 
 of a protected alkyl amine , deprotection , and coupling with a diaryl 
 carbonate under buffered conditions to form the desired fluorine-18 
 labeled carbamate . both the protected amine precursor and the diaryl 
 carbonate precursor were prepared using literature procedures with minor modifications . the synthesis of [ f]dopp 
 was adapted from the previously reported method , which made use of an automated radiosynthesis module for 
 synthesis and purification . in addition to a modified [ f]fluoride - drying procedure , in the manual synthesis both the radiofluorination 
 and deprotection steps were conducted at slightly higher temperatures 
 ( 100 c compared to 80 c ) . specific activities in excess 
 of 5.4 cimol were obtained for the 
 imaging studies . after radiotracer 
 injection , rapid brain uptake was observed in baboon brain at baseline 
 by in vivo pet imaging ( figure 2a and figure s1 in the supporting information ) . an initial whole - brain activity peak was observed at 45 s , and 
 uptake reached a maximum of 2.1 suv beginning at 55 min , with little 
 washout thereafter ( figure 2b ) . activity curves are 
 greatest for the putamen and anterior cingulate cortex , reaching 2.8 
 suv in each , and lowest in the temporal and occipital cortices , where 
 peak activity was only 2.2 and 2.3 suv , respectively . the nominal 
 rank order was maintained both in the initial uptake peak and during 
 the plateau phase . ( a ) summed baseline pet / mr image 090 min post 
 tracer injection . image data is normalized to injected dose and animal weight ( kbq / ml / mci / kg ) . ( b ) whole brain ( wb ) baboon time  activity curves for [ f]dopp at baseline and after pretreatment with urb597 . activity 
 curves after pretreatment . for summed pet / mr images after pretreatment , 
 see the supporting information . urb597 is a carbamate - based inhibitor that , within 
 the brain , is 
 selective for faah . pretreatment of baboons 
 with 200 or 300 g / kg urb597 iv showed only modest reduction 
 in whole brain radioactivity uptake at peak ( 1.8 suv ) and plateau 
 ( 1.9 suv ) levels ( figure 2b and figure s2 in 
 the supporting information ) . the modest 
 effect on radiotracer uptake likely reflects the opposing influences 
 of increased delivery of [ f]dopp due to peripheral blocking 
 ( figure 3 , vide infra ) and 
 blocking of faah binding sites in the central nervous system ( cns ) . there is scant literature on higher doses of urb597 administered intravenously 
 to nonhuman primates , and given the complete 
 blocking observed in rat , experimental 
 higher dosing regimens were not considered to be justified in the 
 present study . the limited whole brain blocking of faah observed in 
 nonhuman primates at this level of pretreatment with urb597 also provides 
 an excellent opportunity to evaluate the sensitivity of [ f]dopp and kinetic modeling approaches to quantification of faah 
 with high sensitivity . selectivity of [ f]dopp for faah 
 has been previously demonstrated in rats using relatively high doses 
 of urb597 ( 2 mg / kg ) , or a more potent urea - based faah inhibitor , pf-04457845 . parent fraction data from arterial plasma at baseline 
 and after 
 pretreatment at two different doses of urb597 ( 200 or 300 g / kg 
 iv ) . the rate of metabolism of [ f]dopp is rapid at baseline 
 and significantly decreased by pretreatment with the faah inhibitor 
 urb597 . this compares favorably with [ c]curb in humans , where free fraction in plasma was estimated 
 at 0.9% . unmetabolized [ f]dopp in arterial plasma peaked at 11.9 suv , 40 s after radiotracer 
 administration , and declined to 2.0 suv after just 3 min and to 0.05 
 suv at 70 min postinjection ( see figure s3 , supporting 
 information ) . less than 5% of radioactivity in plasma was 
 associated with the parent compound at 50 min postadministration ( figure 3 ) . following pretreatment with urb597 ( 300 g / kg , 
 iv ) , [ f]dopp in arterial plasma peaked at 13.1 suv at 
 29 s and declined to 2.0 suv at 3.7 min and to 0.05 suv at 71 min . the observed slight increase in plasma concentration at early time 
 points after pretreatment is consistent with moderate blocking of 
 faah in the brain as well as peripheral metabolic enzymes . indeed , the stability of [ f]dopp in vivo was significantly augmented after pretreatment with urb597 . by way 
 of comparison with baseline , at 50 min postadministration , metabolism of the parent compound did not reach 95% until 
 approximately 113 min postinjection . this confirms our previous hypothesis 
 that the rapid peripheral metabolism of [ f]dopp observed 
 in rodents would not translate to higher species . to determine the 
 optimal kinetic parameters 
 for faah quantification using [ f]dopp , a number of different 
 tissue - compartment models ( tcms ) were evaluated with the data from 
 a baseline scan and two pretreatment scans ( urb597 , 200 , and 300 g / kg 
 iv , 10 min prior to radiotracer administration ) . consistent 
 with a previous human study , the 2tcmi 
 was found to give a good fit for the data in all scans . the 3tcmi 
 did not outperform the 2tcmi as judged by the percent errors of the 
 fits and resulted in comparable outcome measurements . the composite 
 parameter k3 was used to quantify 
 faah activity in regional vois . the regional k3 values at baseline ranged from 0.09 to 0.16 ml / cm / min ( figure 4 ) . by way of comparison , the average k3 value for [ c]curb in selected human brain regions 
 was 0.14 ml / cm / min . the trend 
 and rank order of regional k3 values 
 also correspond well with previously reported human imaging analysis 
 with [ c]curb . regional k3 values showed significant reduction of faah binding when 
 comparing both pretreatment scans to baseline condition ( one - way anova 
 with tukey correction for multiple comparisons , p < 0.0001 ) , while the two pretreatment doses did not cause different 
 levels of blockade ( p > 0.05 ) . however , trends 
 of 
 dose - dependent blocking are evident for most brain structures evaluated . 
 putamen and amygdala showed the highest k3 at baseline , while cortical regions such as occipital ( v1 ) 
 and motor ( m1 ) cortices had the lowest values . across brain regions , k3 values were reduced by 42.2  
 4.5% by pretreatment with 200 g / kg urb597 , and by 45.1  
 6.0% at the 300 g / kg level . regional brain k3 values for 
 baseline and at both pretreatment doses with urb597 . amyg , amygdala ; 
 cau , caudate ; cb , cerebellum ; dlpfc , dorsal lateral prefrontal cortex ; 
 hip , hippocampus ; m1 , primary motor cortex ; nac , nucleus accumbens ; 
 ofc , orbitofrontal cortex ; pcc , posterior cingulate cortex ; put , putamen ; 
 sma , supplementary motor area ; tha , thalamus ; v1 , primary visual cortex . as a 3tcmi would significantly 
 increase the complexity and computational 
 burden of generating voxelwise maps , the 2tcmi was used to generate 
 voxelwise maps . figure 5 shows the voxelwise k3 maps overlaid on their high - resolution 
 anatomical mri under baseline conditions and with 300 g / kg 
 pretreatment . in accordance to regional analysis , basal ganglia ( in 
 particular the putamen ) and nonhuman 
 primate brain k3 maps 
 overlaid on their anatomical mri ( a ) at baseline and ( b ) after pretreatment 
 with 300 g / kg urb597 . among the challenges of developing irreversible covalent 
 enzyme 
 inhibitors for pet are achieving selectivity for the enzyme target 
 and a rate of enzyme inhibition that is not limited by flow . pretreatment 
 studies with faah inhibitors prior to administration of either [ c]curb or [ f]dopp have shown that both of these radiotracers exhibit 
 high selectivity for faah . human studies with [ c]curb 
 have also proven that faah activity as quantified by this radiotracer 
 is not dependent on cerebral blood flow . although the potential effects of cerebral blood flow were not directly 
 measured in the present study , it is possible to use the values of 
 microparameters derived from kinetic modeling to assess whether tracer 
 distribution is flow - limited . specifically , the ratio of k3/k2 has been proposed as 
 a useful metric for this purpose , and values within the range of 0.10.3 
 are predictive of an optimal balance of high tracer uptake without 
 flow - limited distribution . since a high k3 is indicative of rapid specific binding , a 
 value above this range is suggested to indicate flow dependence , while 
 a value below this range will likely translate to poor uptake and 
 low signal . over all of the rois analyzed , the mean k3/k2 value was 0.18 , which 
 is within the optimal range . since [ f]dopp is an 
 irreversible inhibitor of faah , 
 pet measurements with this compound provide a measure of enzyme activity , as opposed to enzyme availability , which could be measured with reversible inhibitors in the same 
 manner as a ligand  receptor interaction . the effects of anesthesia are known to play a role in pet 
 imaging of g protein coupled receptors and enzyme targets in the cns . the effects of isoflurane anesthesia used in 
 the present study , as necessary for imaging in nonhuman primates , 
 present a potential confounding factor for translation of [ f]dopp for quantification of faah activity in conscious humans . in summary , we report that [ f]dopp 
 rapidly crosses 
 the nonhuman primate blood brain barrier and shows low washout 
 in regions consistent with faah distribution and the distribution 
 of [ c]curb in human brain imaging studies . we further 
 report on the evaluation of kinetic modeling parameters and found 
 that k3 displays sensitivity to 
 pretreatment with a faah inhibitor , urb597 . as predicted , [ f]dopp is rapidly metabolized in vivo and at a reduced 
 rate after pretreatment with urb597 . in combination with the previously 
 reported data in rats , these data are 
 suggestive that [ f]dopp exhibits high specific binding 
 and that distribution is not flow dependent , attributes that will 
 ultimately be characterized in human subjects . the radiosynthesis 
 of [ f]dopp has been validated for human use , and preparations 
 for clinical research studies are underway .
fatty 
 acid amide hydrolase ( faah ) regulates endocannabinoid signaling . 
 [ 11c]curb , an irreversibly binding faah inhibitor , has 
 been developed for clinical research imaging with pet . 
 however , no 
 fluorine-18 labeled radiotracer for faah has yet advanced to human 
 studies . 
 [ 18f]dopp ( [ 18f]3-(4,5-dihydrooxazol-2-yl)phenyl 
 ( 5-fluoropentyl)carbamate ) has been identified as a promising 18f - labeled analogue based on rodent studies . 
 the goal of this 
 work is to evaluate [ 18f]dopp in nonhuman primates to support 
 its clinical translation . 
 high specific activity [ 18f]dopp 
 ( 56 cimol1 ) was administered 
 intravenously ( iv ) to three baboons ( 2m/1f , 34 years old ) . 
 the distribution and pharmacokinetics were quantified following a 
 2 h dynamic imaging session using a simultaneous pet / mr scanner . 
 pretreatment 
 with the faah - selective inhibitor , urb597 , was carried out at 200 
 or 300 g / kg iv , 10 min prior to [ 18f]dopp administration . 
 rapid arterial blood sampling for the first 3 min was followed by 
 interval sampling with metabolite analysis to provide a parent radiotracer 
 plasma input function that indicated 95% baseline metabolism 
 at 60 min and a reduced rate of metabolism after pretreatment with 
 urb597 . 
 regional distribution data were analyzed with 1- , 2- , and 
 3-tissue compartment models ( tcms ) , with and without irreversible 
 trapping since [ 18f]dopp covalently links to the active 
 site of faah . 
 consistent with previous findings for [ 11c]curb , the 2tcm with irreversible binding was found to provide the 
 best fit for modeling the data in all regions . 
 the composite parameter k3 was therefore used to evaluate whole 
 brain ( wb ) and regional binding of [ 18f]dopp . 
 pretreatment 
 studies showed inhibition of k3 across all brain regions ( wb baseline : 0.112 ml / cm3/min ; 
 300 g / kg urb597 : 0.058 ml / cm3/min ) , suggesting that 
 [ 18f]dopp binding is specific for faah , consistent with 
 previous rodent data .