Mol Imaging Biol (2014) DOI: 10.1007/s11307-014-0789-1 * World Molecular Imaging Society, 2014
RESEARCH ARTICLE
Synthesis and Preclinical Evaluation of [18F]FCHC for Neuroimaging of Fatty Acid Amide Hydrolase Timothy M. Shoup,1 Ali A. Bonab,1 Alan A. Wilson,2 Neil Vasdev1 1
Division of Nuclear Medicine and Molecular Imaging, Center for Advanced Medical Imaging Sciences, Massachusetts General Hospital, and Department of Radiology, Harvard Medical School, Boston, MA, USA 2 Research Imaging Centre, Centre for Addiction and Mental Health & Department of Psychiatry, University of Toronto, Toronto, ON, M5T 1R8, Canada
Abstract Purpose: Fatty acid amide hydrolase (FAAH), a catabolic enzyme which regulates lipid transmitters in the endocannabinoid system, is an avidly sought therapeutic and positron emission tomography (PET) imaging target for studies involving addiction and neurological disorders. We report the synthesis of a new fluorine-18-labeled FAAH inhibitor, trans-3-(4, 5-dihydrooxazol-2-yl)phenyl-4[18F]fluorocyclohexylcarbamate ([18F]FCHC), and its evaluation in rat brain. Procedures: The synthesis of [18F]FCHC was conducted via a 3-step, 1-pot reaction, resulting in uncorrected radiochemical yields between 10 and 20 % (n=5) relative to [18F]fluoride, with specific activities of 95 Ci/μmol at the end of the synthesis. The radiosynthesis was seamlessly automated using a commercial radiofluorination apparatus. Ex vivo biodistribution and preliminary PET imaging studies were carried out in male Sprague-Dawley rats. Results: Rat brain biodistribution at 2 min post-injection showed a standard uptake value of 4.6± 0.1 in the cortex, which increased to 7.8±0.1 at 40 min. Pretreatment with the selective FAAH inhibitor URB597 reduced uptake of radioactivity in all brain regions by 990 %, with 98 % blockade in the FAAH-rich cortex. PET imaging was consistent with biodistribution studies. Conclusions: [18F]FCHC appears to be a highly sensitive 18F-labeled radiotracer for imaging FAAH in the central nervous system, and these results warrant further imaging in nonhuman primates. Key words, [18F]FCHC, Fatty acid amide hydrolase, FAAH, Positron emission tomography, PET, Rodents
Introduction
F
atty acid amide hydrolase (FAAH) is a dimeric intra-
membrane enzyme responsible for the catabolism of bioactive fatty acid amides such as, N-acylethanolamines, the endocannabinoids anandamide (AEA), and 2arachidonoyl glycerol (2-AG), as well as the sleep-
Correspondence to: Neil Vasdev; e-mail: [email protected]
inducing lipid oleamide [1, 2]. A member of the serine hydrolase family of enzymes, FAAH utilizes a unique SerSer-Lys catalytic triad to effect hydrolysis of fatty acid amides to free fatty acids and ethanolamine [3–5]. FAAH is mainly present in the pancreas, brain, kidney, and skeletal muscle in humans, whereas in rats, it is detected in the liver, small intestine, brain, kidney, and spleen. In the central nervous system (CNS), this enzyme is located in the cerebral cortex, cerebellar cortex, hippocampus, and olfactory bulb, with expression restricted to principal neurons and hypertrophic astrocytes [6]. Within the neurons, FAAH is post-
T.M. Shoup et al.: Evaluation of [18F]FCHC for imaging FAAH
synaptic to axon fibers expressing CB1 and CB2 cannabinoid G protein-coupled receptors and is the major contributor to the clearance and inactivation of AEA and 2-AG after endocannabinoid reuptake [7]. Due to its ability to regulate anandamide levels, FAAH is a recognized target for the development of therapeutically useful drugs for a range of conditions including pain [8–12], loss of appetite, immunosuppression, peripheral vascular disease, neuropsychiatric disorders, and inflammation in the human brain and elsewhere [13–19]. Additionally, there is evidence for a link between defects in the endocannabinoid system and substance abuse [20] indicating that selective inhibitors could aid in treating drug addiction. Consequently, FAAH inhibitors including URB597 [21] and PF-04457845 [22] have advanced to phases I and II clinical trials for treatment of pain, cannabis dependence, and schizophrenia. In vivo molecular imaging of the endocannabinoid pathway has been pursued using positron emission tomography (PET); various radiotracers for the CB1 receptor have been developed and translated to human PET studies [23– 27]. For FAAH, a number of PET radiotracers [28] have been reported [29–35], but only [11C]CURB [33, 36] and [11C]MK-3168 [31] have been translated for FAAH imaging in humans. While these radiotracers show much promise, they are labeled with the short-lived radionuclide carbon-11 (t1/2 =20.4 min), and thus, their use is confined to sites that have an on-site cyclotron for the production of carbon-11. Fluorine-18 (t1/2 =109.7 min) is the most commonly used radionuclide for PET and not only allows for longer imaging protocols to be carried out but can be also shipped for use at remote locations, thereby enabling multicenter trials. Herein, we describe the synthesis of a new fluorine-18-labeled FAAH inhibitor, trans-3-(4,5-dihydrooxazol-2-yl)phenyl-4[18F]fluorocyclohexylcarbamate ([18F]FCHC). The potential of [18F]FCHC as a FAAH imaging agent was evaluated by ex vivo biodistribution and PET imaging studies in rats. Baseline and blocking results were compared to [11C]CURB and our earlier F-18-labeled n-pentyl carbamate analog, [18F]DOPP [35].
Materials and Methods Chemistry and Radiochemistry All chemicals and solvents were ACS grade and used as received. Melting points were determined using a Thomas Hoover model apparatus and are uncorrected. 1H NMR was recorded in CDCl3 or DMSO-d6 on a Bruker 300 MHz spectrometer, and resonances are relative to TMS. All water used was distilled and deionized. Column chromatography purifications were performed using silica gel 60 (63–200 um, Selecto). Electrospray ionization mass spectrometry was conducted with an Advion Expression MS. A GE PETtrace 17 MeV cyclotron was used for radionuclide production. Purifications and analyses of radioactive mixtures were performed by a high-performance liquid chromatography (HPLC) with an in-line UV (254 nm) detector in series with a radioactivity
detector (purifications). Radiochemical incorporations were determined using a Bioscan AR-2000 Radio-TLC and Imaging Scanner. Radiochemical yields were determined with a dose calibrator (Capintec, CRC-712M), and unless stated otherwise, all radioactivity measurements were normalized for radioactive decay.
trans-4-Fluorocyclohexanamine hydrochloride (2) A solution of diethylaminosulfur trifluoride (0.8 g, 4.8 mmol) in dichloromethane (8 ml) was added dropwise to a suspension of benzyl cis-4-hydroxycyclohexyl carbamate [37] (1) (1.0 g, 4 mmol) in dichloromethane (40 ml) at −78 °C. After stirring at −78 °C for 30 min, the clear reaction mixture was allowed to warm to 25 °C and stirred for 1 h. The mixture was poured into a saturated NaHCO3 aqueous solution and extracted with dichloromethane, and the organic layer was dried (Na2SO4) and evaporated in vacuo. The residue was purified by column chromatography on silica gel eluting with n-hexanes/ethyl acetate (95:5) to give benzyl trans-4fluorocyclohexyl carbamate (140 mg, 14 %) as a solid; melting point (mp) 74–76 °C. 1H NMR (CDCl3): δ (ppm), 1.26–1.27 (m, 2H, cyclohexyl), 1.30–1.32 (m, 2H, cyclohexyl), 1.55–1.59 (m, 4H, cyclohexyl), 3.55–3.56 (m, 1H), 4.40–4.62 (m, JHF =45Hz, 1H), 4.62–4.68 (m, 1H), 5.10 (s, 2H), 7.30–7.39 (m, 5H); 19F NMR (CDCl3) δ (ppm), −187.8. A suspension of benzyl trans-4-fluorocyclohexyl carbamate (130 mg) and 5 % palladium hydroxide 50 % wet (50 mg) in methanol (5 ml) was stirred at an ambient temperature for 2 h under 1 atm H2 atmosphere. After filtration through Celite, the reaction mixture was evaporated in vacuo to give trans-4-fluorocyclohexanamine (17 mg, 40 % yield). 1H NMR (CDCl3): δ (ppm), 1.23–1.69 (m, 6H), 1.79–1.97 (m, 2H), 2.64–2.89 (m, 1H), 4.47–4.89 (d, m, 1H, JHF =45 Hz). The amine was converted to the hydrochloride salt by adding HCl in ether to an ether solution of the corresponding amine followed by vacuum filtration of the precipitate; m/z calcd for [M+H]+ C6H13FN 118.10, found 118.14.
trans-3-(4,5-Dihydrooxazol-2-yl)phenyl(4-fluorocyclohexyl) carbamate (4) 3-(4,5-Dihydrooxazol-2-yl)phenyl-(4-nitrophenyl) carbonate (3) was prepared by literature procedures [35] with minor modifications for the preparation of 3-(4,5-dihydrooxazol-2-yl)phenol [38]. A solution of 2 (100 mg, 0.65 mmol) in acetonitrile/water (4:1, 5 ml) was added to a stirred mixture of 3 (212 mg, 0.65 mmol) and potassium bicarbonate (200 mg, 2.0 mmol) in acetonitrile (5 ml). The mixture was stirred at room temperature for 3 h, and then dichloromethane (10 ml) and saturated aqueous sodium bicarbonate (50 ml) were added. The aqueous layer was extracted with dichloromethane; the combined organic layers were filtered, and the solvent was evaporated. Chromatography (acetonitrile/dichloromethane, 10:90) on silica gel gave 4 as a white solid (76 mg, 38 % yield); mp 132–137 °C. 1H NMR (CDCl3): δ (ppm), 1.60–1.73 (m, 4H), 2.04–2.14 (m, 4H), 3.58–3.68 (m, 1H), 4.07 (t, J=9.52 Hz, 2H), 4.42 (t, J=9.52 Hz, 2H, OCH2), 4.61, 4.49 (d, m, 1H, JHF = 48 Hz), 44.96 (br, 1H, NH), 7.23 (m, 1H), 7.40 (t, J=8.00 Hz, 1H), 7.77 (s, 1H), 7.80 (d, J=7.80 Hz, 1H). 19F NMR (CDCl3): δ (ppm), −180; m/z calcd for [M+H]+ C16H19FN2O3 306.14, found 307.1.
T.M. Shoup et al.: Evaluation of [18F]FCHC for imaging FAAH
cis-tert-Butyl-N-[4(methanesulfonyloxy)cyclohexyl] carbamate (5) Methanesulfonyl chloride (9.6 g, 83.81 mmol) was added dropwise to a solution of cis-tert-butyl-N-(4-hydroxycyclohexyl) carbamate [39] (1.5 g, 6.96 mmol, 1.0 equiv.) and triethylamine (15.5 g, 153.18 mmol, 2.0 equiv.) in dichloromethane (150 ml) cooled to 0 °C. The resulting solution was stirred for 30 min at 0 °C and then for 1 h at room temperature. Water (100 ml) was added, and phases were separated and extracted with dichloromethane (2×20 ml). The combined organic layer was washed with saturated sodium bicarbonate solution, and brine, dried with sodium sulfate, and concentrated under vacuum. Chromatography (silica gel, methanol/ methylene chloride 5:95) gave 1.6 g (76 %) of cis-methanesulfonic ester as an off-white solid; mp 119–121 °C; 1H NMR (300 mHz, CDCl3): δ (ppm), 1.45 (s, 9H), 1.60 (m, 2H), 1.78 (m, 4H), 2.05 (m, 2H), 3.02 (s, 3H), 3.53 (br s, 1H), 4.47 (br s, 1H), 4.89 (m, 1H).
[18F]-trans-3-(4,5-dihydrooxazol-2-yl)phenyl-4fluorocyclohexylcarbamate ([18F]FCHC) [18F]fluoride was produced by the 18O(p, n)18F nuclear reaction using 995 % enriched [18O]H2O. The [18F]fluoride was trapped on a Chromafix ion exchange 30-PS-HCO3 resin and eluted into a sealed 5-ml glass reaction V-vial (Reacti-Vial™) with a solution consisting of 14.4 mg of 4,7,13,16,21,24-hexaoxa-1;10-diazabicyclo[8.8.8]hexacosane (Kryptofix 222), and 3 mg of K2CO3 in 1 ml of 4 % (v/v) water in acetonitrile. The solution was dried at 120 °C using a nitrogen flow. The contents were further azeotroped dry by the addition of 1 ml of acetonitrile followed by evaporation at 120 °C of the solvent using a nitrogen stream. This process was repeated three times. A solution of cis-tert-butyl-N-[4-(methanesulfonyloxy)cyclohexyl] carbamate (5) (5 mg, 14 mmol) in acetonitrile (0.5 ml) was added to the vial and heated at 120 °C for 10 min. The vial was cooled to 80 °C for addition of 2 % aq. H2SO4 (0.5 ml). Further heating at 120 °C was continued for 10 min. After cooling the vial to 80 °C, the solution was neutralized with a phosphate buffer (0.5 ml, stock solution prepared by dissolving K2HPO4 (34.84 g) and KOH (2.81 g) in water (250 ml)). A solution of 3 (7 mg, 21.3 mmol) in 70 % aq. acetonitrile (1 ml) was added with heating at 80 °C continued for an additional 7 min. The mixture was cooled to G45 °C, quenched with water (2 ml), and purified by HPLC using a Phenomenex Luna C18 5 μm, 250×10 mm column using acetonitrile/0.1 N ammonium formate (gradient, 0–2 min 0/ 100, 2–5 min 10/90, 5–10 15/85, 10–25 min 35/65) as the eluent at 7 ml/min. The radioactive fraction eluting between 17 and 19 min was collected, diluted with sterile water (25 ml), and immobilized on a C18 Sep-Pak cartridge. After washing the cartridge with water (5 ml), [18F]FCHC was eluted with ethanol (1 ml) and diluted with a citrate buffer (10 ml, pH 6) prepared from trisodium citrate dehydrate (0. 588 g) and disodium hydrogen citrate 1.5-hydrate (0. 263 g) in water (100 ml). The buffered solution containing [18F]FCHC was passed through a sterile filter (Millex-GV, 0.22 μm, 33 mm, Millipore) into an empty vial. The identity of the radiotracer was established by co-injection of the radioactive product with an authentic nonradioactive standard. Uncorrected radiochemical yields relative to starting [18F]fluoride were 10–20 %
with a radiosynthesis time of 90 min and a radiochemical purity of 998 %; specific activity 95 Ci/μmol at EOS. For rodent dissection studies, the radiotracer was synthesized by a fully automated procedure (n=3) using a Synthra RNplus module with essentially the same outcomes as described above.
Biodistribution Studies of [18F]FCHC in Rat Brain All animal experiments were carried out under humane conditions, with approval from the Animal Care Committee at the Centre for Addiction and Mental Health, in accordance with the guidelines set forth by the Canadian Council on Animal Care, and consistent with those of Partners Healthcare. Rats (male, Sprague-Dawley, 300– 350 g) were kept on a reversed 12-h light/12-h dark cycle and allowed food and water ad libitum. The distribution of radioactivity was determined in brain tissues of conscious male Sprague-Dawley rats (n=5/time point) after tailvein injection of 135–190 mCi (5–7 MBq) of high-specific-activity [18F]FCHC (95 Ci/μmol) in 0.3 ml. The activity per unit volume was obtained from standards. The animals were sacrificed by decapitation at 2 and 40 min post-injection; bloods were collected, and the whole brain surgically removed. Excised brain tissues were blotted, weighed, and assayed using an automated gamma counter, and the raw counts were decay corrected. Pretreatment of rats with URB597 (Cayman Chemicals) at 2 mg/kg in saline containing 5 % DMSO and 5 % Tween 80, IP, or vehicle alone, was performed 30 min prior to radiotracer injection. All results are expressed as standard uptake values (SUVs; mean±standard deviation) defined as percent injected dose/gram of tissue divided by the rat weight in kilogram. Results are evaluated by Student’s t test, and statistical comparisons were considered significant when pG0.05.
Metabolite Studies As per the biodistribution studies (vide supra), rats (n=3–4) were administered [18F]FCHC, and following decapitation, the trunk blood was collected into a heparinized tube and was centrifuged to separate the plasma. Using the method described by Hilton [40] with minor modifications, the plasma was treated with 10 % by volume of acetic acid then used directly for column-switching HPLC analysis.
PET Imaging Data was acquired using a Siemens Focus 220 μPET in line with a CereTom CT scanner acquired for 60 min in list mode and histogrammed into 22 frames (eight 15-s frames, three 1-min frames, and eleven 5-min frames); then, sonograms were reconstructed using Fourier rebinning and a 2D filter back projection with a zoom of 6 in 256×256 matrix. Whole brain ROIs were constructed, and time activity curves were generated using ASIPro software from Siemens. The field of view is 20 cm transaxial by 7.6 cm axial with a sensitivity of 4 %. The rats (n = 2) were fasted for 24 h prior to imaging, anesthetized with isoflurane-N2-O2, and positioned with a customfabricated head holder; list-mode acquisition started with a tail-vein
T.M. Shoup et al.: Evaluation of [18F]FCHC for imaging FAAH
Scheme 1 Synthesis of FCHC: a DAST −78 °C to 25 °C, 14 %; b H2/Pd(OH)2; HCl/ethanol, 40 %; c 3, K2CO3 25 °C, 38 % injection of ~2 mCi (~74 MBq) of [18F]FCHC for 60 min. The subsequent imaging study was similarly conducted in a rat pretreated with URB597 (2 mg/kg, IP) 30 min prior to radiotracer injection. URB597 was formulated in saline with 5 % DMSO and 5 % Tween 80 at 30 min prior to injection in a total volume of 1 ml/kg.
Results Chemistry and Radiochemistry FCHC was synthesized in four steps starting from cis-4aminocyclohexanol hydrochloride (Scheme 1). Fluorination of 1 with diethylaminosulfur trifluoride (DAST) at −78 °C to 25 °C gave benzyl trans-4-fluorocyclohexyl carbamate in 14 % yield, and the removal of the CBz group by catalytic hydrogenation gave trans-4-fluorocyclohexanamine (40 % yield) which was converted to the hydrochloride salt, 2. Coupling of the fluorocyclohexylamine hydrochloride with the p-nitrophenylcarbonate of 3-(4,5-dihydrooxazol-2yl)phenol (3) provided 4 in 38 % yield. The N-tert-butoxycarbonyl (t-Boc)-protected, O-mesylate derivative of cis-4-aminocyclohexanol, precursor for radiolabeling (5; Scheme 2), was synthesized from cis-4-aminocyclohexanol hydrochloride using di-tert-butyl dicarbonate and triethylamine followed by treatment of the resultant carbonate (90 % yield) with methanesulfonyl chloride affording t-Boc-protected mesylate (76 % yield). [18F]FCHC was manually synthesized in a one-pot, threestep reaction starting from cis-4-(t-Boc-amino)cyclohexyl methanesulfonate (5) and K18F/Kryptofix as shown in Scheme 2. The displacement of the mesylate group by [18F]fluoride in acetonitrile at 120 °C proceeded to give the
t-Boc-protected 4-[18F]fluorocyclohexylamine with incorporation yields of 70–80 % (radio-TLC). The removal of the tBoc group was effected by addition of 2 % sulfuric acid with 85–95 % efficiency to yield [18F]4. After neutralizing the acid with addition of phosphate buffer to pH=7, [18F]fluorocyclohexylamine was coupled with carbonate 3 at 80 °C for 7 min to generate [18F]FCHC in 10–20 % radiochemical conversion. Reversed-phase HPLC purification, followed by formulation via solid phase extraction and sterile filtration, gave [18F]FCHC suitable for animal studies in an isolated uncorrected radiochemical yield (from [18F]fluoride) of 10– 20 % (n=5). The synthesis was completed within 90 min from the end of bombardment with radiochemical purity 998 % and specific activities of 95 Ci/μmol at the end of synthesis. The radiosynthesis was subsequently automated using a Synthera RN plus module with equivalent results.
Biodistribution, Metabolism and PET Imaging of [18F]FCHC in Rat Brain High-specific-activity [18F]FCHC (G1 nmol/kg) was injected into the tail-vein of conscious rats. The brain uptake of radioactivity was rapid and high as measured by ex vivo dissection followed by counting of radioactivity in specific brain regions (Fig. 1). The radioactivity in the FAAH-rich cortex [41] was high at 2 min post-injection, showing 4.6± 0.1 SUV, which increased to 7.8±0.1 SUV at 40 min postinjection. The hypothalamus had radioactivity levels of 2.2± 0.1 and 2.4±0.1 SUVs at 2 and 40 min post-injection, respectively, and is consistent with our expectations for this comparatively FAAH-low region. To demonstrate that radiotracer uptake was mediated by FAAH binding, groups of rats were pretreated with the established FAAH inhibitor
Scheme 2 One-pot, three-step radiosynthesis of [18F]FCHC: a K18F/K222, CH3CN, 120 °C, 7 min; b 2 % H2SO4, 120 °C, 7 min; c K2HPO4 buffer; 3, 80 °C, 7 min, 10–20 % radiochemical yield
T.M. Shoup et al.: Evaluation of [18F]FCHC for imaging FAAH
Fig. 3 Summed transaxial brain slices from PET/CT images acquired over 60 min in a rat using a Siemens Focus 220 scanner after administration of approximately 2 mCi of [18F]FCHC, depicting: a a control rat revealing enhanced uptake in cortical and related regions of high FAAH expression and b a pretreated rat which received URB597 (2 mg/kg, IP) 30 min before radiotracer injection showing a significantly decreased radiotracer uptake in the CNS. Fig. 1 Biodistribution of [18F]FCHC in rat brain at 2 and 40 min post-injection (n=4/group, mean±SD). The blocked groups received URB597 (2 mg/kg, IP) 30 min before radiotracer injection. *pG0.05.
URB597 (2 mg/kg, IP) at 30 min prior to radiotracer administration [42–45]. Pretreatment studies showed reduced uptake of radioactivity in all brain regions studied. A blockage of 98 % was seen in the cortex, 92 % was seen in the hypothalamus, and 96 % reduction in uptake in the whole brain was observed compared with controls. Ex vivo regional brain uptake of [18F]FCHC and regional FAAH enzyme activity in rat brain compared with other FAAH radiotracers is shown in Fig. 2. Reversed-phase radio-HPLC analysis of rat plasma 2 min post-radiotracer injection showed that metabolism had occurred. Fortunately, there was no sign of any lipophilic
Fig. 2 Correlation between ex vivo regional brain uptake of FAAH radiotracers and regional FAAH enzyme activity in rat brain. Data taken from [18F]FCHC uptake, [11C]CURB, and [18F]DOPP uptake; [35] and Thomas et al., (FAAH enzyme activity).[40] Error bars represent standard deviation from the mean.
metabolites present. The radioactivity associated with hydrophilic metabolites showed parent was 75 % present at 2 min and 16 % intact at 40 min post-injection. For pretreated rats, 13 % of the parent compound remained at 40 min post-injection. PET imaging demonstrated that the tracer had high brain penetration (Fig. 3) and showed rapid uptake (93 SUV) for the first 4 min which slowly increased to 95 SUV at 60 min with a distribution which correlated with reported regional FAAH enzyme activity. A tracer uptake was clearly seen in the cerebral cortex, cerebellar cortex, hippocampus, and olfactory bulb. Pretreatment with the potent and selective FAAH inhibitor URB597 showed 995 % blockade in all regions.
Discussion The chemical structure for the new probe [18F]FCHC was chosen on the basis of previous preclinical studies with 11Clabeled FAAH inhibitors which showed that N-alkyl-3-(4,5dihydrooxazol-2-yl)phenyl carbamates, as compared to analogous substituted biphenyl carbamates, possess superior properties as FAAH imaging radiotracers, including higher brain uptake and specific binding [34]. Concurrent with our recent development of an 18F-labeled FAAH inhibitor based on this scaffold, [18F]DOPP [35], we are developing a series of 18F-labeled radiotracers for imaging of this enzyme. Of the many N-alkyl substituents, the cyclohexyl moiety was selected based on the FAAH binding affinity of the nonfluorocyclohexyl analog, N-cyclohexyl-3-(4,5-dihydrooxazol-2-yl)phenyl carbamate (IC50 of 1.2 nM), which was reported as having a 10-fold increase in potency over that of the cyclopentyl analog (13 nM) and a 28-fold increase over that of the N-n-propyl analog (33 nM) [46]. In our previous work, N-hexyl and N-cyclohexylcarbamates were also potent inhibitors of FAAH with effective inhibitory concentrations in low nanomolar region with various O-aryl groups including dihydrooxazole [34]. In addition, both selective FAAH inhibitors URB694
T.M. Shoup et al.: Evaluation of [18F]FCHC for imaging FAAH
([11C]CURB) [33] used in clinical research studies [36] and URB597 for clinical trials in humans contain a N-cyclohexyl moiety; therefore, an 18F-fluorocyclohexyl derivative was pursued in the present work. While one-step radiolabeling is desirable, direct introduction of fluorine-18 to generate [18F]FCHC using a sulfonate ester as a precursor proved fruitless. Under basic (KryptofixK2CO3) or mildly basic (TBA-bicarbonate) conditions required for F-18 nucleophilic substitution reactions, such aryl carbamates undergo a facile and rapid elimination reaction which precluded introduction of [18F]fluoride directly [35]. However, careful selection of reaction conditions that efficiently generated [18F]2 allowed [18F]FCHC to be prepared in an efficient, one-pot, three-step reaction (Scheme 2). This reaction can be readily automated using a commercial radiosynthesis module. The suitability of [18F]FCHC as a radiotracer for imaging FAAH by PET was demonstrated by rat brain biodistribution and PET imaging studies. These studies showed that brain penetration of [18F]FCHC was markedly high and mediated by FAAH based on pretreatment with the established and selective FAAH inhibitor URB597 and blocked 98 % of the uptake in the cortex, a FAAH-rich region (Fig. 1). In addition, regional brain uptake of radioactivity at 40 min post-administration of [18F]FCHC correlated with the reported regional FAAH enzyme activity in rat brain. Brain regions of high FAAH activity (cortex) and a region of low FAAH activity (hypothalamus) were differentiated. A similar correlation was found with [11C]CURB [33] and [18F]DOPP [35] in the same assay, albeit [18F]FCHC showed substantially greater brain uptake in these regions (Fig. 2). As compared to our other FAAH radiotracers based on this scaffold and evaluated in the same manner [34], [18F]FCHC demonstrated faster blood clearance, higher brain uptake, and lower nonspecific binding at 2 and 40 min post-injection. [18F]FCHC exhibited a significantly higher control/blocked ratio in the cortex than [11C]CURB and [18F]DOPP and reflects higher specific binding. A comparative correlation of [18F]FCHC and other FAAH radiotracers uptake (SUV) versus regional FAAH enzyme activity (0.82 to 1.90 nmol/min/mg) in rat brain is presented in Fig. 2. The uptake of [18F]FCHC in the regions with lowest FAAH activity (0.82 nmol/min/mg) is nearly twice as that of [11C]CURB and [18F]DOPP whereas in the region with highest FAAH activity (1.90 nmol/min/mg), the uptake of the tracer is 3.3 and 1.9 times higher than that of these radiotracers, respectively. These results indicate that [18F]FCHC is a highly sensitive radiotracer for imaging FAAH in the CNS. Dynamic PET imaging with [18F]FCHC in rats was also carried out for a qualitative comparison, and the results were consistent with the ex vivo biodistribution studies. Radioactivity was seen in the cerebral cortex, cerebellar cortex, hippocampus, and olfactory bulb, the areas of typical FAAH activity. PET-CT neuroimaging with [18F]FCHC after pretreatment with the FAAH inhibitor URB597 showed
995 % tracer binding inhibition (Fig. 3). This work demonstrates that [18F]FCHC is worthy of further evaluation in higher species, and nonhuman primate imaging with this radiotracer, as well as the cis-isomer, are planned.
Conclusions We have identified and characterized a novel F-18-labeled radiotracer for PET imaging of FAAH. As demonstrated by ex vivo biodistribution studies, in conjunction with PET imaging, [18F]FCHC demonstrated a combination of high brain uptake and high specific binding in rats. These properties suggest that this promising 18F-labeled FAAH radiotracer is suitable for neuroimaging and worthy of further evaluation in higher species. Acknowledgments. This work was supported by the NIH Grant No. 1R21MH094424 to AAW. We would like to thank Dr. Jun Parkes for her assistance with the radiochemistry and animal dissection experiments. Conflict of Interest. The authors declare no conflicts of interest.
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31. Li W, Sanabria-Bohorquez S, Aniket J et al (2011) The discovery and characterization of [C-11]MK-3168, a novel PET tracer for imaging fatty acid amide hydrolase (FAAH). J Labelled Compd Radiopharm 54:S38 32. Skaddan MB, Zhang L, Johnson DS et al (2012) The synthesis and in vivo evaluation of [18F]PF-9811: a novel PET ligand for imaging brain fatty acid amide hydrolase (FAAH). Nucl Med Biol 39:1058– 1067 33. Wilson AA, Garcia A, Parkes J et al (2011) [11C]CURB: evaluation of a novel radiotracer for imaging fatty acid amide hydrolase by positron emission tomography. Nucl Med Biol 38:247–253 34. Wilson AA, Hicks JW, Sadovski O et al (2013) Radiosynthesis and evaluation of [(11)C-carbonyl]-labeled carbamates as fatty acid amide hydrolase radiotracers for positron emission tomography. J Med Chem 56:201–209 35. Sadovski O, Hicks JW, Parkes J et al (2013) Development and characterization of a promising fluorine-18 labelled radiopharmaceutical for in vivo imaging of fatty acid amide hydrolase. Bioorg Med Chem 21:4351–4357 36. Rusjan PM, Wilson AA, Mizrahi R et al (2013) Mapping human brain fatty acid amide hydrolase activity with PET. J Cereb Blood Flow Metab 33:407–414 37. Buettner A, Cottin T, Xu J et al (2010) Serotonin derivatives as a new class of non-ATP-competitive receptor tyrosine kinase inhibitors. Bioorganic & Medicinal Chemistry 18:3387–3402 38. Luston J, Kronek J, Bohme F (2006) Synthesis and polymerization reactions of cyclic imino ethers. Ring-opening homopolyaddition of AB-type hydroxyphenyl-substituted 2-oxazolines. J Polym Sci A Polym Chem 44:343–355 39. Vennerstrom JL, Dong Y, Charman SA et al (2009) Preparation of dispiro 1,2,4-trioxolanes for use as prodrugs treating malaria, schistosomiasis, and cancer. PCT Int Appl 2009058859 40. Hilton J, Yokoi F, Dannals RF et al (2000) Column-switching HPLC for the analysis of plasma in PET imaging studies. Nucl Med Biol 27:627–630 41. Thomas EA, Cravatt BF, Danielson PE et al (1997) Fatty acid amide hydrolase, the degradative enzyme for anandamide and oleamide, has selective distribution in neurons within the rat central nervous system. J Neurosci Res 50:1047–1052 42. Piomelli D, Tarzia G, Duranti A et al (2006) Pharmacological profile of the selective FAAH inhibitor KDS-4103 (URB59). CNS Drug Rev 12:21–38 43. Ahn K, Smith SE, Liimatta MB et al (2011) Mechanistic and pharmacological characterization of PF-04457845: a highly potent and selective fatty acid amide hydrolase inhibitor that reduces inflammatory and noninflammatory pain. J Pharmacol Exp Ther 338:114–124 44. Ahn K, Johnson D, Fitzgerald L et al (2007) Novel mechanistic class of fatty acid amide hydrolase inhibitors with remarkable selectivity. Biochem 46:13019–13030 45. Zhang D, Saraf A, Kolasa T et al (2007) Fatty acid amide hydrolase inhibitors display broad selectivity and inhibit multiple carboxylesterases as off-targets. Neuropharmacol 52:1095–1105 46. Myllymäki MJ, Käsnänen H, Kataja AO et al (2009) Chiral 3-(4,5dihydrooxazol-2-yl)phenyl alkylcarbamates as novel FAAH inhibitors: insight into FAAH enantioselectivity by molecular docking and interaction fields. Eur J Med Chem 44:4179–4191
RESEARCH ARTICLE
Synthesis and Preclinical Evaluation of [18F]FCHC for Neuroimaging of Fatty Acid Amide Hydrolase Timothy M. Shoup,1 Ali A. Bonab,1 Alan A. Wilson,2 Neil Vasdev1 1
Division of Nuclear Medicine and Molecular Imaging, Center for Advanced Medical Imaging Sciences, Massachusetts General Hospital, and Department of Radiology, Harvard Medical School, Boston, MA, USA 2 Research Imaging Centre, Centre for Addiction and Mental Health & Department of Psychiatry, University of Toronto, Toronto, ON, M5T 1R8, Canada
Abstract Purpose: Fatty acid amide hydrolase (FAAH), a catabolic enzyme which regulates lipid transmitters in the endocannabinoid system, is an avidly sought therapeutic and positron emission tomography (PET) imaging target for studies involving addiction and neurological disorders. We report the synthesis of a new fluorine-18-labeled FAAH inhibitor, trans-3-(4, 5-dihydrooxazol-2-yl)phenyl-4[18F]fluorocyclohexylcarbamate ([18F]FCHC), and its evaluation in rat brain. Procedures: The synthesis of [18F]FCHC was conducted via a 3-step, 1-pot reaction, resulting in uncorrected radiochemical yields between 10 and 20 % (n=5) relative to [18F]fluoride, with specific activities of 95 Ci/μmol at the end of the synthesis. The radiosynthesis was seamlessly automated using a commercial radiofluorination apparatus. Ex vivo biodistribution and preliminary PET imaging studies were carried out in male Sprague-Dawley rats. Results: Rat brain biodistribution at 2 min post-injection showed a standard uptake value of 4.6± 0.1 in the cortex, which increased to 7.8±0.1 at 40 min. Pretreatment with the selective FAAH inhibitor URB597 reduced uptake of radioactivity in all brain regions by 990 %, with 98 % blockade in the FAAH-rich cortex. PET imaging was consistent with biodistribution studies. Conclusions: [18F]FCHC appears to be a highly sensitive 18F-labeled radiotracer for imaging FAAH in the central nervous system, and these results warrant further imaging in nonhuman primates. Key words, [18F]FCHC, Fatty acid amide hydrolase, FAAH, Positron emission tomography, PET, Rodents
Introduction
F
atty acid amide hydrolase (FAAH) is a dimeric intra-
membrane enzyme responsible for the catabolism of bioactive fatty acid amides such as, N-acylethanolamines, the endocannabinoids anandamide (AEA), and 2arachidonoyl glycerol (2-AG), as well as the sleep-
Correspondence to: Neil Vasdev; e-mail: [email protected]
inducing lipid oleamide [1, 2]. A member of the serine hydrolase family of enzymes, FAAH utilizes a unique SerSer-Lys catalytic triad to effect hydrolysis of fatty acid amides to free fatty acids and ethanolamine [3–5]. FAAH is mainly present in the pancreas, brain, kidney, and skeletal muscle in humans, whereas in rats, it is detected in the liver, small intestine, brain, kidney, and spleen. In the central nervous system (CNS), this enzyme is located in the cerebral cortex, cerebellar cortex, hippocampus, and olfactory bulb, with expression restricted to principal neurons and hypertrophic astrocytes [6]. Within the neurons, FAAH is post-
T.M. Shoup et al.: Evaluation of [18F]FCHC for imaging FAAH
synaptic to axon fibers expressing CB1 and CB2 cannabinoid G protein-coupled receptors and is the major contributor to the clearance and inactivation of AEA and 2-AG after endocannabinoid reuptake [7]. Due to its ability to regulate anandamide levels, FAAH is a recognized target for the development of therapeutically useful drugs for a range of conditions including pain [8–12], loss of appetite, immunosuppression, peripheral vascular disease, neuropsychiatric disorders, and inflammation in the human brain and elsewhere [13–19]. Additionally, there is evidence for a link between defects in the endocannabinoid system and substance abuse [20] indicating that selective inhibitors could aid in treating drug addiction. Consequently, FAAH inhibitors including URB597 [21] and PF-04457845 [22] have advanced to phases I and II clinical trials for treatment of pain, cannabis dependence, and schizophrenia. In vivo molecular imaging of the endocannabinoid pathway has been pursued using positron emission tomography (PET); various radiotracers for the CB1 receptor have been developed and translated to human PET studies [23– 27]. For FAAH, a number of PET radiotracers [28] have been reported [29–35], but only [11C]CURB [33, 36] and [11C]MK-3168 [31] have been translated for FAAH imaging in humans. While these radiotracers show much promise, they are labeled with the short-lived radionuclide carbon-11 (t1/2 =20.4 min), and thus, their use is confined to sites that have an on-site cyclotron for the production of carbon-11. Fluorine-18 (t1/2 =109.7 min) is the most commonly used radionuclide for PET and not only allows for longer imaging protocols to be carried out but can be also shipped for use at remote locations, thereby enabling multicenter trials. Herein, we describe the synthesis of a new fluorine-18-labeled FAAH inhibitor, trans-3-(4,5-dihydrooxazol-2-yl)phenyl-4[18F]fluorocyclohexylcarbamate ([18F]FCHC). The potential of [18F]FCHC as a FAAH imaging agent was evaluated by ex vivo biodistribution and PET imaging studies in rats. Baseline and blocking results were compared to [11C]CURB and our earlier F-18-labeled n-pentyl carbamate analog, [18F]DOPP [35].
Materials and Methods Chemistry and Radiochemistry All chemicals and solvents were ACS grade and used as received. Melting points were determined using a Thomas Hoover model apparatus and are uncorrected. 1H NMR was recorded in CDCl3 or DMSO-d6 on a Bruker 300 MHz spectrometer, and resonances are relative to TMS. All water used was distilled and deionized. Column chromatography purifications were performed using silica gel 60 (63–200 um, Selecto). Electrospray ionization mass spectrometry was conducted with an Advion Expression MS. A GE PETtrace 17 MeV cyclotron was used for radionuclide production. Purifications and analyses of radioactive mixtures were performed by a high-performance liquid chromatography (HPLC) with an in-line UV (254 nm) detector in series with a radioactivity
detector (purifications). Radiochemical incorporations were determined using a Bioscan AR-2000 Radio-TLC and Imaging Scanner. Radiochemical yields were determined with a dose calibrator (Capintec, CRC-712M), and unless stated otherwise, all radioactivity measurements were normalized for radioactive decay.
trans-4-Fluorocyclohexanamine hydrochloride (2) A solution of diethylaminosulfur trifluoride (0.8 g, 4.8 mmol) in dichloromethane (8 ml) was added dropwise to a suspension of benzyl cis-4-hydroxycyclohexyl carbamate [37] (1) (1.0 g, 4 mmol) in dichloromethane (40 ml) at −78 °C. After stirring at −78 °C for 30 min, the clear reaction mixture was allowed to warm to 25 °C and stirred for 1 h. The mixture was poured into a saturated NaHCO3 aqueous solution and extracted with dichloromethane, and the organic layer was dried (Na2SO4) and evaporated in vacuo. The residue was purified by column chromatography on silica gel eluting with n-hexanes/ethyl acetate (95:5) to give benzyl trans-4fluorocyclohexyl carbamate (140 mg, 14 %) as a solid; melting point (mp) 74–76 °C. 1H NMR (CDCl3): δ (ppm), 1.26–1.27 (m, 2H, cyclohexyl), 1.30–1.32 (m, 2H, cyclohexyl), 1.55–1.59 (m, 4H, cyclohexyl), 3.55–3.56 (m, 1H), 4.40–4.62 (m, JHF =45Hz, 1H), 4.62–4.68 (m, 1H), 5.10 (s, 2H), 7.30–7.39 (m, 5H); 19F NMR (CDCl3) δ (ppm), −187.8. A suspension of benzyl trans-4-fluorocyclohexyl carbamate (130 mg) and 5 % palladium hydroxide 50 % wet (50 mg) in methanol (5 ml) was stirred at an ambient temperature for 2 h under 1 atm H2 atmosphere. After filtration through Celite, the reaction mixture was evaporated in vacuo to give trans-4-fluorocyclohexanamine (17 mg, 40 % yield). 1H NMR (CDCl3): δ (ppm), 1.23–1.69 (m, 6H), 1.79–1.97 (m, 2H), 2.64–2.89 (m, 1H), 4.47–4.89 (d, m, 1H, JHF =45 Hz). The amine was converted to the hydrochloride salt by adding HCl in ether to an ether solution of the corresponding amine followed by vacuum filtration of the precipitate; m/z calcd for [M+H]+ C6H13FN 118.10, found 118.14.
trans-3-(4,5-Dihydrooxazol-2-yl)phenyl(4-fluorocyclohexyl) carbamate (4) 3-(4,5-Dihydrooxazol-2-yl)phenyl-(4-nitrophenyl) carbonate (3) was prepared by literature procedures [35] with minor modifications for the preparation of 3-(4,5-dihydrooxazol-2-yl)phenol [38]. A solution of 2 (100 mg, 0.65 mmol) in acetonitrile/water (4:1, 5 ml) was added to a stirred mixture of 3 (212 mg, 0.65 mmol) and potassium bicarbonate (200 mg, 2.0 mmol) in acetonitrile (5 ml). The mixture was stirred at room temperature for 3 h, and then dichloromethane (10 ml) and saturated aqueous sodium bicarbonate (50 ml) were added. The aqueous layer was extracted with dichloromethane; the combined organic layers were filtered, and the solvent was evaporated. Chromatography (acetonitrile/dichloromethane, 10:90) on silica gel gave 4 as a white solid (76 mg, 38 % yield); mp 132–137 °C. 1H NMR (CDCl3): δ (ppm), 1.60–1.73 (m, 4H), 2.04–2.14 (m, 4H), 3.58–3.68 (m, 1H), 4.07 (t, J=9.52 Hz, 2H), 4.42 (t, J=9.52 Hz, 2H, OCH2), 4.61, 4.49 (d, m, 1H, JHF = 48 Hz), 44.96 (br, 1H, NH), 7.23 (m, 1H), 7.40 (t, J=8.00 Hz, 1H), 7.77 (s, 1H), 7.80 (d, J=7.80 Hz, 1H). 19F NMR (CDCl3): δ (ppm), −180; m/z calcd for [M+H]+ C16H19FN2O3 306.14, found 307.1.
T.M. Shoup et al.: Evaluation of [18F]FCHC for imaging FAAH
cis-tert-Butyl-N-[4(methanesulfonyloxy)cyclohexyl] carbamate (5) Methanesulfonyl chloride (9.6 g, 83.81 mmol) was added dropwise to a solution of cis-tert-butyl-N-(4-hydroxycyclohexyl) carbamate [39] (1.5 g, 6.96 mmol, 1.0 equiv.) and triethylamine (15.5 g, 153.18 mmol, 2.0 equiv.) in dichloromethane (150 ml) cooled to 0 °C. The resulting solution was stirred for 30 min at 0 °C and then for 1 h at room temperature. Water (100 ml) was added, and phases were separated and extracted with dichloromethane (2×20 ml). The combined organic layer was washed with saturated sodium bicarbonate solution, and brine, dried with sodium sulfate, and concentrated under vacuum. Chromatography (silica gel, methanol/ methylene chloride 5:95) gave 1.6 g (76 %) of cis-methanesulfonic ester as an off-white solid; mp 119–121 °C; 1H NMR (300 mHz, CDCl3): δ (ppm), 1.45 (s, 9H), 1.60 (m, 2H), 1.78 (m, 4H), 2.05 (m, 2H), 3.02 (s, 3H), 3.53 (br s, 1H), 4.47 (br s, 1H), 4.89 (m, 1H).
[18F]-trans-3-(4,5-dihydrooxazol-2-yl)phenyl-4fluorocyclohexylcarbamate ([18F]FCHC) [18F]fluoride was produced by the 18O(p, n)18F nuclear reaction using 995 % enriched [18O]H2O. The [18F]fluoride was trapped on a Chromafix ion exchange 30-PS-HCO3 resin and eluted into a sealed 5-ml glass reaction V-vial (Reacti-Vial™) with a solution consisting of 14.4 mg of 4,7,13,16,21,24-hexaoxa-1;10-diazabicyclo[8.8.8]hexacosane (Kryptofix 222), and 3 mg of K2CO3 in 1 ml of 4 % (v/v) water in acetonitrile. The solution was dried at 120 °C using a nitrogen flow. The contents were further azeotroped dry by the addition of 1 ml of acetonitrile followed by evaporation at 120 °C of the solvent using a nitrogen stream. This process was repeated three times. A solution of cis-tert-butyl-N-[4-(methanesulfonyloxy)cyclohexyl] carbamate (5) (5 mg, 14 mmol) in acetonitrile (0.5 ml) was added to the vial and heated at 120 °C for 10 min. The vial was cooled to 80 °C for addition of 2 % aq. H2SO4 (0.5 ml). Further heating at 120 °C was continued for 10 min. After cooling the vial to 80 °C, the solution was neutralized with a phosphate buffer (0.5 ml, stock solution prepared by dissolving K2HPO4 (34.84 g) and KOH (2.81 g) in water (250 ml)). A solution of 3 (7 mg, 21.3 mmol) in 70 % aq. acetonitrile (1 ml) was added with heating at 80 °C continued for an additional 7 min. The mixture was cooled to G45 °C, quenched with water (2 ml), and purified by HPLC using a Phenomenex Luna C18 5 μm, 250×10 mm column using acetonitrile/0.1 N ammonium formate (gradient, 0–2 min 0/ 100, 2–5 min 10/90, 5–10 15/85, 10–25 min 35/65) as the eluent at 7 ml/min. The radioactive fraction eluting between 17 and 19 min was collected, diluted with sterile water (25 ml), and immobilized on a C18 Sep-Pak cartridge. After washing the cartridge with water (5 ml), [18F]FCHC was eluted with ethanol (1 ml) and diluted with a citrate buffer (10 ml, pH 6) prepared from trisodium citrate dehydrate (0. 588 g) and disodium hydrogen citrate 1.5-hydrate (0. 263 g) in water (100 ml). The buffered solution containing [18F]FCHC was passed through a sterile filter (Millex-GV, 0.22 μm, 33 mm, Millipore) into an empty vial. The identity of the radiotracer was established by co-injection of the radioactive product with an authentic nonradioactive standard. Uncorrected radiochemical yields relative to starting [18F]fluoride were 10–20 %
with a radiosynthesis time of 90 min and a radiochemical purity of 998 %; specific activity 95 Ci/μmol at EOS. For rodent dissection studies, the radiotracer was synthesized by a fully automated procedure (n=3) using a Synthra RNplus module with essentially the same outcomes as described above.
Biodistribution Studies of [18F]FCHC in Rat Brain All animal experiments were carried out under humane conditions, with approval from the Animal Care Committee at the Centre for Addiction and Mental Health, in accordance with the guidelines set forth by the Canadian Council on Animal Care, and consistent with those of Partners Healthcare. Rats (male, Sprague-Dawley, 300– 350 g) were kept on a reversed 12-h light/12-h dark cycle and allowed food and water ad libitum. The distribution of radioactivity was determined in brain tissues of conscious male Sprague-Dawley rats (n=5/time point) after tailvein injection of 135–190 mCi (5–7 MBq) of high-specific-activity [18F]FCHC (95 Ci/μmol) in 0.3 ml. The activity per unit volume was obtained from standards. The animals were sacrificed by decapitation at 2 and 40 min post-injection; bloods were collected, and the whole brain surgically removed. Excised brain tissues were blotted, weighed, and assayed using an automated gamma counter, and the raw counts were decay corrected. Pretreatment of rats with URB597 (Cayman Chemicals) at 2 mg/kg in saline containing 5 % DMSO and 5 % Tween 80, IP, or vehicle alone, was performed 30 min prior to radiotracer injection. All results are expressed as standard uptake values (SUVs; mean±standard deviation) defined as percent injected dose/gram of tissue divided by the rat weight in kilogram. Results are evaluated by Student’s t test, and statistical comparisons were considered significant when pG0.05.
Metabolite Studies As per the biodistribution studies (vide supra), rats (n=3–4) were administered [18F]FCHC, and following decapitation, the trunk blood was collected into a heparinized tube and was centrifuged to separate the plasma. Using the method described by Hilton [40] with minor modifications, the plasma was treated with 10 % by volume of acetic acid then used directly for column-switching HPLC analysis.
PET Imaging Data was acquired using a Siemens Focus 220 μPET in line with a CereTom CT scanner acquired for 60 min in list mode and histogrammed into 22 frames (eight 15-s frames, three 1-min frames, and eleven 5-min frames); then, sonograms were reconstructed using Fourier rebinning and a 2D filter back projection with a zoom of 6 in 256×256 matrix. Whole brain ROIs were constructed, and time activity curves were generated using ASIPro software from Siemens. The field of view is 20 cm transaxial by 7.6 cm axial with a sensitivity of 4 %. The rats (n = 2) were fasted for 24 h prior to imaging, anesthetized with isoflurane-N2-O2, and positioned with a customfabricated head holder; list-mode acquisition started with a tail-vein
T.M. Shoup et al.: Evaluation of [18F]FCHC for imaging FAAH
Scheme 1 Synthesis of FCHC: a DAST −78 °C to 25 °C, 14 %; b H2/Pd(OH)2; HCl/ethanol, 40 %; c 3, K2CO3 25 °C, 38 % injection of ~2 mCi (~74 MBq) of [18F]FCHC for 60 min. The subsequent imaging study was similarly conducted in a rat pretreated with URB597 (2 mg/kg, IP) 30 min prior to radiotracer injection. URB597 was formulated in saline with 5 % DMSO and 5 % Tween 80 at 30 min prior to injection in a total volume of 1 ml/kg.
Results Chemistry and Radiochemistry FCHC was synthesized in four steps starting from cis-4aminocyclohexanol hydrochloride (Scheme 1). Fluorination of 1 with diethylaminosulfur trifluoride (DAST) at −78 °C to 25 °C gave benzyl trans-4-fluorocyclohexyl carbamate in 14 % yield, and the removal of the CBz group by catalytic hydrogenation gave trans-4-fluorocyclohexanamine (40 % yield) which was converted to the hydrochloride salt, 2. Coupling of the fluorocyclohexylamine hydrochloride with the p-nitrophenylcarbonate of 3-(4,5-dihydrooxazol-2yl)phenol (3) provided 4 in 38 % yield. The N-tert-butoxycarbonyl (t-Boc)-protected, O-mesylate derivative of cis-4-aminocyclohexanol, precursor for radiolabeling (5; Scheme 2), was synthesized from cis-4-aminocyclohexanol hydrochloride using di-tert-butyl dicarbonate and triethylamine followed by treatment of the resultant carbonate (90 % yield) with methanesulfonyl chloride affording t-Boc-protected mesylate (76 % yield). [18F]FCHC was manually synthesized in a one-pot, threestep reaction starting from cis-4-(t-Boc-amino)cyclohexyl methanesulfonate (5) and K18F/Kryptofix as shown in Scheme 2. The displacement of the mesylate group by [18F]fluoride in acetonitrile at 120 °C proceeded to give the
t-Boc-protected 4-[18F]fluorocyclohexylamine with incorporation yields of 70–80 % (radio-TLC). The removal of the tBoc group was effected by addition of 2 % sulfuric acid with 85–95 % efficiency to yield [18F]4. After neutralizing the acid with addition of phosphate buffer to pH=7, [18F]fluorocyclohexylamine was coupled with carbonate 3 at 80 °C for 7 min to generate [18F]FCHC in 10–20 % radiochemical conversion. Reversed-phase HPLC purification, followed by formulation via solid phase extraction and sterile filtration, gave [18F]FCHC suitable for animal studies in an isolated uncorrected radiochemical yield (from [18F]fluoride) of 10– 20 % (n=5). The synthesis was completed within 90 min from the end of bombardment with radiochemical purity 998 % and specific activities of 95 Ci/μmol at the end of synthesis. The radiosynthesis was subsequently automated using a Synthera RN plus module with equivalent results.
Biodistribution, Metabolism and PET Imaging of [18F]FCHC in Rat Brain High-specific-activity [18F]FCHC (G1 nmol/kg) was injected into the tail-vein of conscious rats. The brain uptake of radioactivity was rapid and high as measured by ex vivo dissection followed by counting of radioactivity in specific brain regions (Fig. 1). The radioactivity in the FAAH-rich cortex [41] was high at 2 min post-injection, showing 4.6± 0.1 SUV, which increased to 7.8±0.1 SUV at 40 min postinjection. The hypothalamus had radioactivity levels of 2.2± 0.1 and 2.4±0.1 SUVs at 2 and 40 min post-injection, respectively, and is consistent with our expectations for this comparatively FAAH-low region. To demonstrate that radiotracer uptake was mediated by FAAH binding, groups of rats were pretreated with the established FAAH inhibitor
Scheme 2 One-pot, three-step radiosynthesis of [18F]FCHC: a K18F/K222, CH3CN, 120 °C, 7 min; b 2 % H2SO4, 120 °C, 7 min; c K2HPO4 buffer; 3, 80 °C, 7 min, 10–20 % radiochemical yield
T.M. Shoup et al.: Evaluation of [18F]FCHC for imaging FAAH
Fig. 3 Summed transaxial brain slices from PET/CT images acquired over 60 min in a rat using a Siemens Focus 220 scanner after administration of approximately 2 mCi of [18F]FCHC, depicting: a a control rat revealing enhanced uptake in cortical and related regions of high FAAH expression and b a pretreated rat which received URB597 (2 mg/kg, IP) 30 min before radiotracer injection showing a significantly decreased radiotracer uptake in the CNS. Fig. 1 Biodistribution of [18F]FCHC in rat brain at 2 and 40 min post-injection (n=4/group, mean±SD). The blocked groups received URB597 (2 mg/kg, IP) 30 min before radiotracer injection. *pG0.05.
URB597 (2 mg/kg, IP) at 30 min prior to radiotracer administration [42–45]. Pretreatment studies showed reduced uptake of radioactivity in all brain regions studied. A blockage of 98 % was seen in the cortex, 92 % was seen in the hypothalamus, and 96 % reduction in uptake in the whole brain was observed compared with controls. Ex vivo regional brain uptake of [18F]FCHC and regional FAAH enzyme activity in rat brain compared with other FAAH radiotracers is shown in Fig. 2. Reversed-phase radio-HPLC analysis of rat plasma 2 min post-radiotracer injection showed that metabolism had occurred. Fortunately, there was no sign of any lipophilic
Fig. 2 Correlation between ex vivo regional brain uptake of FAAH radiotracers and regional FAAH enzyme activity in rat brain. Data taken from [18F]FCHC uptake, [11C]CURB, and [18F]DOPP uptake; [35] and Thomas et al., (FAAH enzyme activity).[40] Error bars represent standard deviation from the mean.
metabolites present. The radioactivity associated with hydrophilic metabolites showed parent was 75 % present at 2 min and 16 % intact at 40 min post-injection. For pretreated rats, 13 % of the parent compound remained at 40 min post-injection. PET imaging demonstrated that the tracer had high brain penetration (Fig. 3) and showed rapid uptake (93 SUV) for the first 4 min which slowly increased to 95 SUV at 60 min with a distribution which correlated with reported regional FAAH enzyme activity. A tracer uptake was clearly seen in the cerebral cortex, cerebellar cortex, hippocampus, and olfactory bulb. Pretreatment with the potent and selective FAAH inhibitor URB597 showed 995 % blockade in all regions.
Discussion The chemical structure for the new probe [18F]FCHC was chosen on the basis of previous preclinical studies with 11Clabeled FAAH inhibitors which showed that N-alkyl-3-(4,5dihydrooxazol-2-yl)phenyl carbamates, as compared to analogous substituted biphenyl carbamates, possess superior properties as FAAH imaging radiotracers, including higher brain uptake and specific binding [34]. Concurrent with our recent development of an 18F-labeled FAAH inhibitor based on this scaffold, [18F]DOPP [35], we are developing a series of 18F-labeled radiotracers for imaging of this enzyme. Of the many N-alkyl substituents, the cyclohexyl moiety was selected based on the FAAH binding affinity of the nonfluorocyclohexyl analog, N-cyclohexyl-3-(4,5-dihydrooxazol-2-yl)phenyl carbamate (IC50 of 1.2 nM), which was reported as having a 10-fold increase in potency over that of the cyclopentyl analog (13 nM) and a 28-fold increase over that of the N-n-propyl analog (33 nM) [46]. In our previous work, N-hexyl and N-cyclohexylcarbamates were also potent inhibitors of FAAH with effective inhibitory concentrations in low nanomolar region with various O-aryl groups including dihydrooxazole [34]. In addition, both selective FAAH inhibitors URB694
T.M. Shoup et al.: Evaluation of [18F]FCHC for imaging FAAH
([11C]CURB) [33] used in clinical research studies [36] and URB597 for clinical trials in humans contain a N-cyclohexyl moiety; therefore, an 18F-fluorocyclohexyl derivative was pursued in the present work. While one-step radiolabeling is desirable, direct introduction of fluorine-18 to generate [18F]FCHC using a sulfonate ester as a precursor proved fruitless. Under basic (KryptofixK2CO3) or mildly basic (TBA-bicarbonate) conditions required for F-18 nucleophilic substitution reactions, such aryl carbamates undergo a facile and rapid elimination reaction which precluded introduction of [18F]fluoride directly [35]. However, careful selection of reaction conditions that efficiently generated [18F]2 allowed [18F]FCHC to be prepared in an efficient, one-pot, three-step reaction (Scheme 2). This reaction can be readily automated using a commercial radiosynthesis module. The suitability of [18F]FCHC as a radiotracer for imaging FAAH by PET was demonstrated by rat brain biodistribution and PET imaging studies. These studies showed that brain penetration of [18F]FCHC was markedly high and mediated by FAAH based on pretreatment with the established and selective FAAH inhibitor URB597 and blocked 98 % of the uptake in the cortex, a FAAH-rich region (Fig. 1). In addition, regional brain uptake of radioactivity at 40 min post-administration of [18F]FCHC correlated with the reported regional FAAH enzyme activity in rat brain. Brain regions of high FAAH activity (cortex) and a region of low FAAH activity (hypothalamus) were differentiated. A similar correlation was found with [11C]CURB [33] and [18F]DOPP [35] in the same assay, albeit [18F]FCHC showed substantially greater brain uptake in these regions (Fig. 2). As compared to our other FAAH radiotracers based on this scaffold and evaluated in the same manner [34], [18F]FCHC demonstrated faster blood clearance, higher brain uptake, and lower nonspecific binding at 2 and 40 min post-injection. [18F]FCHC exhibited a significantly higher control/blocked ratio in the cortex than [11C]CURB and [18F]DOPP and reflects higher specific binding. A comparative correlation of [18F]FCHC and other FAAH radiotracers uptake (SUV) versus regional FAAH enzyme activity (0.82 to 1.90 nmol/min/mg) in rat brain is presented in Fig. 2. The uptake of [18F]FCHC in the regions with lowest FAAH activity (0.82 nmol/min/mg) is nearly twice as that of [11C]CURB and [18F]DOPP whereas in the region with highest FAAH activity (1.90 nmol/min/mg), the uptake of the tracer is 3.3 and 1.9 times higher than that of these radiotracers, respectively. These results indicate that [18F]FCHC is a highly sensitive radiotracer for imaging FAAH in the CNS. Dynamic PET imaging with [18F]FCHC in rats was also carried out for a qualitative comparison, and the results were consistent with the ex vivo biodistribution studies. Radioactivity was seen in the cerebral cortex, cerebellar cortex, hippocampus, and olfactory bulb, the areas of typical FAAH activity. PET-CT neuroimaging with [18F]FCHC after pretreatment with the FAAH inhibitor URB597 showed
995 % tracer binding inhibition (Fig. 3). This work demonstrates that [18F]FCHC is worthy of further evaluation in higher species, and nonhuman primate imaging with this radiotracer, as well as the cis-isomer, are planned.
Conclusions We have identified and characterized a novel F-18-labeled radiotracer for PET imaging of FAAH. As demonstrated by ex vivo biodistribution studies, in conjunction with PET imaging, [18F]FCHC demonstrated a combination of high brain uptake and high specific binding in rats. These properties suggest that this promising 18F-labeled FAAH radiotracer is suitable for neuroimaging and worthy of further evaluation in higher species. Acknowledgments. This work was supported by the NIH Grant No. 1R21MH094424 to AAW. We would like to thank Dr. Jun Parkes for her assistance with the radiochemistry and animal dissection experiments. Conflict of Interest. The authors declare no conflicts of interest.
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