Evaluation in rats and primates of [11C]-mecamylamine, a potential nicotinic acetylcholine receptor radioligand for positron emission tomography

Mecamylamine is a well-described non specific antagonist of nicotinic acetylcholine receptors (nAChRs), used in therapy and in psychopharmacological studies. [(11)C]-Mecamylamine was prepared and evaluated as a putative radioligand for positron emission tomography to study nicotinic acetylcholine receptors. The radiosynthesis consisted in the [(11)C]-methylation of the desmethyl precursor within 40 min with 30-40% radiochemical yield decay corrected. Biodistribution studies in rats showed that radioligand crossed the blood-brain barrier (0.39% ID at 30 min) and only unmetabolized tracer was recovered from brain at 45 min. Ex vivo autoradiography studies in rats did not indicate preferential uptake, and pre-treatment mecamylamine or with chlorisondamine, an nicotinic receptor inhibitor, did not demonstrate a significant specific binding. To investigate possible specie differences and effects of anesthesia, in vivo positron emission tomography (PET) studies were carried out on anaesthetized baboons and conscious macaques. The regional brain distribution of [(11)C]-mecamylamine in the two species of primates exhibited similar kinetics as did the rat with steady state reached about 45-50 min after radiotracer administration. Uptake values were two-fold higher in brain of conscious macaque than in anaesthetized baboon (thalamus: 0.258% ID/(kg mL) in conscious macaques and 0.129% ID/(kg mL) in baboons). PET images showed a radioactivity distribution which was quite homogeneous throughout the brain but with somewhat higher uptake in grey matter than in white. Brain distribution was unaltered by saturation or displacement studies. Possible explanation for the failure to establish specific binding in vivo could be long-lived structural modifications of the ionotropic channel by the unlabeled ligand administered before the tracer. In conclusion, [(11)C]-mecamylamine did not satisfy the requirements for a PET tracer of nicotinic acetylcholine receptors.     

Characterisation of [¹¹C]PR04.MZ in Papio anubis baboon: a selective high-affinity radioligand for quantitative imaging of the dopamine transporter

N-(4-fluorobut-2-yn-1-yl)-2β-carbomethoxy-3β-(4'-tolyl)nortropane (PR04.MZ, 1) is a PET radioligand for the non-invasive exploration of the function of the cerebral dopamine transporter (DAT). A reliable automated process for routine production of the carbon-11 labelled analogue [(11)C]PR04.MZ ([(11)C]-1) has been developed using GMP compliant equipment. An adult female Papio anubis baboon was studied using a test-retest protocol with [(11)C]-1 in order to assess test-retest reliability, metabolism and CNS distribution profile of the tracer in non-human primates. Blood sampling was performed throughout the studies for determination of the free fraction in plasma (f(P)), plasma input functions and metabolic degradation of the radiotracer [(11)C]-1. Time-activity curves were derived for the putamen, the caudate nucleus, the ventral striatum, the midbrain and the cerebellum. Distribution volumes (V(T)) and non-displaceable binding potentials (BP(ND)) for various brain regions and the blood were obtained from kinetic modelling. [(11)C]-1 shows promising results as a selective marker of the presynaptic dopamine transporter. With the reliable visualisation of the extra-striatal dopaminergic neurons and no indication on labelled metabolites, the tracer provides excellent potential for translation into man.     

Synthesis of 11C-labelled (R)-OHDMI and CFMME and their evaluation as candidate radioligands for imaging central norepinephrine transporters with PET

(R)-1-(10,11-Dihydro-dibenzo[b,f]azepin-5-yl)-3-methylamino-propan-2-ol ((R)-OHDMI) and (S,S)-1-cyclopentyl-2-(5-fluoro-2-methoxy-phenyl)-1-morpholin-2-yl-ethanol (CFMME) were synthesized and found to be potent inhibitors of norepinephrine reuptake. Each was labelled efficiently in its methyl group with carbon-11 (t(1/2)=20.4 min) as a prospective radioligand for imaging brain norepinephrine transporters (NET) with positron emission tomography (PET). The uptake and distribution of radioactivity in brain following intravenous injection of each radioligand into cynomolgus monkey was examined in vivo with PET. After injection of (R)-[(11)C]OHDMI, the maximal whole brain uptake of radioactivity was very low (1.1% of injected dose; I.D.). For occipital cortex, thalamus, lower brainstem, mesencephalon and cerebellum, radioactivity ratios to striatum at 93 min after radioligand injection were 1.35, 1.35, 1.2, 1.2 and 1.0, respectively. After injection of [(11)C]CFMME, radioactivity readily entered brain (3.5% I.D.). Ratios of radioactivity to cerebellum at 93 min for thalamus, occipital cortex, region of locus coeruleus, mesencephalon and striatum were 1.35, 1.3, 1.3, 1.2 and 1.2, respectively. Radioactive metabolites in plasma were measured by radio-HPLC. (R)-[(11)C]OHDMI represented 75% of plasma radioactivity at 4 min after injection and 6% at 30 min. After injection of [(11)C]CFMME, 84% of the radioactivity in plasma represented parent at 4 min and 20% at 30 min. Since the two new hydroxylated radioligands provide only modest regional differentiation in brain uptake and form potentially troublesome lipophilic radioactive metabolites, they are concluded to be inferior to existing radioligands, such as (S,S)-[(11)C]MeNER, (S,S)-[(18)F]FMeNER-D(2) and (S,S)-[(18)F]FRB-D(4), for the study of brain NETs with PET in vivo.     

N-[18F]fluoroethyl-4-piperidyl acetate ([18F]FEtP4A): A PET tracer for imaging brain acetylcholinesterase in vivo

N-[(18)F]Fluoroethyl-4-piperidyl acetate ([(18)F]FEtP4A) was synthesized and evaluated as a PET tracer for imaging brain acetylcholinesterase (AchE) in vivo. [(18)F]FEtP4A was previously prepared by reacting 4-piperidyl acetate (P4A) with 2-[(18)F]fluoroethyl bromide ([(18)F]FEtBr) at 130 degrees C for 30 min in 37% radiochemical yield using an automated synthetic system. In this work, [(18)F]FEtP4A was synthesized by reacting P4A with 2-[(18)F]fluoroethyl iodide ([(18)F]FEtI) or 2-[(18)F]fluoroethyl triflate ([(18)F]FEtOTf in improved radiochemical yields, compared with [(18)F]FEtBr under the corresponding condition. Ex vivo autoradiogram of rat brain and PET summation image of monkey brain after iv injection of [(18)F]FEtP4A displayed a high radioactivity in the striatum, a region with the highest AchE activity in the brain. Moreover, the distribution pattern of (18)F radioactivity was consistent with that of AchE in the brain: striatum>frontal cortex>cerebellum. In the rat and monkey plasma, two radioactive metabolites were detected. However, their presence might not preclude the imaging studies for AchE in the brain, because they were too hydrophilic to pass the blood-brain barrier and to enter the brain. In the rat brain, only [(18)F]fluoroethyl-4-piperidinol ([(18)F]FEtP4OH) was detected at 30 min postinjection. The hydrolytic [(18)F]FEtP4OH displayed a slow washout and a long retention in the monkey brain until the PET experiment (120 min). Although [(18)F]FEtP4A is a potential PET tracer for imaging AchE in vivo, its lower hydrolytic rate and lower specificity for AchE than those of [(11)C]MP4A may limit its usefulness for the quantitative measurement for AchE in the primate brain.     

Synthesis and evaluation in vitro and in vivo of a 11C-labeled cyclooxygenase-2 (COX-2) inhibitor

The radiosynthesis and radiopharmacological evaluation of 1-[(11)C]methoxy-4-(2-(4-(methanesulfonyl)phenyl)cyclopent-1-enyl)-benzene [(11)C]5 as novel PET radiotracer for imaging of COX-2 expression is described. The radiotracer was prepared via O-methylation reaction with [(11)C]methyl iodide in 19% decay-corrected radiochemical yield at a specific activity of 20-25GBq/mumol at the end-of-synthesis within 35 min. The radiotracer [(11)C]5 was evaluated in vitro using various pro-inflammatory and tumor cell lines showing high functional expression of COX-2 at baseline or after induction. In vivo biodistribution of compound [(11)C]5 was characterized in male Wistar rats. Compound [(11)C]5 was rapidly metabolized in rat plasma, and more pronounced, in mouse plasma. In vivo kinetics and tumor uptake were demonstrated by dynamic small animal PET studies in a mouse tumor xenograft model. Tumor uptake of radioactivity was clearly visible overtime. However, radioactivity uptake in the tumor could not be blocked by the pre-injection of nonradioactive compound 5. Therefore, it can be concluded that radioactivity uptake in the tumor was not COX-2 mediated.     

Radiosynthesis and in vivo evaluation of 1-[18F]fluoroelacridar as a positron emission tomography tracer for P-glycoprotein and breast cancer resistance protein

Aim of this study was to label the potent dual P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) inhibitor elacridar (1) with (18)F to provide a positron emission tomography (PET) radiotracer to visualize Pgp and BCRP. A series of new 1- and 2-halogen- and nitro-substituted derivatives of 1 (4a-e) was synthesized as precursor molecules and reference compounds for radiolabelling and shown to display comparable in vitro potency to 1 in increasing rhodamine 123 accumulation in a cell line overexpressing human Pgp (MDCKII-MDR1). 1-[(18)F]fluoroelacridar ([(18)F]4b) was synthesized in a decay-corrected radiochemical yield of 1.7±0.9% by a 1-step no-carrier added nucleophilic aromatic (18)F-substitution of 1-nitro precursor 4c. Small-animal PET imaging of [(18)F]4b was performed in na?ve rats, before and after administration of unlabelled 1 (5 mg/kg, n=3), as well as in wild-type and Mdr1a/b((-/-))Bcrp1((-/-)) mice (n=3). In PET experiments in rats, administration of unlabelled 1 increased brain activity uptake by a factor of 9.5 (p=0.0002, 2-tailed Student's t-test), whereas blood activity levels remained unchanged. In Mdr1a/b((-/-))Bcrp1((-/-)) mice, the mean brain-to-blood ratio of activity at 60 min after tracer injection was 7.6 times higher as compared to wild-type animals (p=0.0002). HPLC analysis of rat brain tissue extracts collected at 40 min after injection of [(18)F]4b revealed that 93±7% of total radioactivity in brain was in the form of unchanged [(18)F]4b. In conclusion, the in vivo behavior of [(18)F]4b was found to be similar to previously described [(11)C]1 suggesting transport of [(18)F]4b by Pgp and/or BCRP at the rodent BBB. However, low radiochemical yields and a significant degree of in vivo defluorination will limit the utility of [(18)F]4b as a PET tracer.     

Radiosynthesis and in vivo evaluation of [11C]MP-10 as a PET probe for imaging PDE10A in rodent and non-human primate brain

2-((4-(1-[(11)C]Methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl)-quinoline (MP-10), a specific PDE10A inhibitor (IC(50)=0.18 nM with 100-fold selectivity over other PDEs), was radiosynthesized by alkylation of the desmethyl precursor with [(11)C]CH(3)I, ～45% yield, >92% radiochemical purity, >370 GBq/μmol specific activity at end of bombardment (EOB). Evaluation in Sprague-Dawley rats revealed that [(11)C]MP-10 had highest brain accumulation in the PDE10A enriched-striatum, the 30 min striatum: cerebellum ratio reached 6.55. MicroPET studies of [(11)C]MP-10 in monkeys displayed selective uptake in striatum. However, a radiolabeled metabolite capable of penetrating the blood-brain-barrier may limit the clinical utility of [(11)C]MP-10 as a PDE10A PET tracer.     

Synthesis and biological evaluation of novel carbon-11-labelled analogues of citalopram as potential radioligands for the serotonin transporter

Three serotonin reuptake inhibitors where the 5-cyano group in citalopram [1-(3-dimethylamino-propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile (1)] was replaced with a methyl, acetyl and piperidinyl carbonyl group, respectively, were synthesized. In a Stille reaction applying [(11)C]methyl iodide the labelled compound [5-methyl-(11)C][3-[1-(4-fluorophenyl)-5-methyl-1,3-dihydroisobenzofuran-1-yl]-propyl]-dimethylamine ([(11)C]-2) was synthesized in 60-90% radiochemical yield. [5-carbonyl-(11)C][1-[1-(3-dimethylaminopropyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-yl]-1-piperidin-1-yl-methanone] ([(11)C]-3) was synthesized in 62% radiochemical yield in a palladium mediated cross-coupling reaction utilizing [(11)C]carbon monoxide. The specific activity of [(11)C]-2 was highly dependent on whether the corresponding trimethyltin or tributyltin precursor was applied. In ex vivo rodent studies compound [(11)C]-2 exhibited a good blood-brain barrier (BBB) penetration whereas [(11)C]-3 did not. The brain distribution of [(11)C]-2 was investigated in a non-human primate using PET. There was a rapid uptake of radioactivity into the brain. Accumulation of the radiotracer was in agreement with the known distribution of serotonin transporters. The maximal thalamus to cerebellum ratio of 1.3 was reached after 85 min and the specific binding was partly blocked after pre-treatment with citalopram. Thus, [(11)C]-2 does not exhibit appropriate properties as radioligand for visualization of the serotonin transporter in vivo.     

A novel PET marker for in vivo quantification of myelination

C-11-labeled N-methyl-4,4'-diaminostilbene ([(11)C]MeDAS) was synthesized and evaluated as a novel radiotracer for in vivo microPET imaging of myelination. [(11)C]MeDAS exhibits optimal lipophilicity for brain uptake with a logP(oct) value of 2.25. Both in vitro and ex vivo staining exhibited MeDAS accumulation in myelinated regions such as corpus callosum and striatum. The corpus callosum region visualized by MeDAS is much larger in the hypermyelinated Plp-Akt-DD mouse brain than in the wild-type mouse brain, a pattern that was also consistently observed in Black-Gold or MBP antibody staining. Ex vivo autoradiography demonstrated that [(11)C]MeDAS readily entered the mouse brain and selectively labeled myelinated regions with high specificity. Biodistribution studies showed abundant initial brain uptake of [(11)C]MeDAS with 2.56% injected dose/whole brain at 5 min post injection and prolonged retention in the brain with 1.37% injected dose/whole brain at 60 min post injection. An in vivo pharmacokinetic profile of [(11)C]MeDAS was quantitatively analyzed through a microPET study in an Plp-Akt-DD hypermyelinated mouse model. MicroPET studies showed that [(11)C]MeDAS exhibited a pharmacokinetic profile that readily correlates the radioactivity concentration to the level of myelination in the brain. These studies suggest that MeDAS is a sensitive myelin probe that provides a direct means to detect myelin changes in the brain. Thus, it can be used as a myelin-imaging marker to monitor myelin pathology in vivo.     

Evaluation of 18F-labeled acetylcholinesterase substrates as PET radiotracers

Four 18F-labeled acetylcholinesterase (AChE) substrates, (S)-N-[18F]fluoroethyl-2-piperidinemethyl acetate (1), (R)-N-[18F]fluoroethyl-3-pyrrolidinyl acetate (2), N-[18F]fluoroethyl-4-piperidinyl acetate (3), and (R)-N-[18F]fluoroethyl-3-piperidinyl acetate (4), were evaluated for in vivo blood and brain metabolism in mice, brain pharmacokinetics in rats monkeys (M. nemistrina) using PET imaging. All 18F-labeled compounds were compared to N-[11C]methyl-4-piperidinyl propionate (PMP). Compound 1 was completely metabolized within 1 min in mouse blood and brain. This compound had relatively fast regional brain pharmacokinetics and poor discrimination between brain regions with different AChE concentration. Compound 4 showed relatively slower blood metabolism and slower pharmacokinetics than compound 1 but again poor discrimination between brain regions. Both compounds 1 and 4 showed different kinetic profiles than PMP in PET studies. Compound 3 had the slowest blood metabolism and slower pharmacokinetics than PMP. Compound 2 showed highly encouraging characteristics with an in vivo metabolism rate, primate brain uptake, and regional brain pharmacokinetics similar to [11C]PMP. The apparent hydrolysis rate constant k3 in primate cortex was very close to that of [11C]PMP. This compound has potential to be a good PET radiotracer for measuring brain AChE activity. The longer lifetime of 18F would permit longer imaging times and allows preparation of radiotracer batches for multiple patients and delivery of the tracer to other facilities, making the technique more widely available to clinical investigators.     

Development of new carbon-11 labelled radiotracers for imaging GABAA- and GABAB-benzodiazepine receptors

Two quinolines identified as positive allosteric modulators of γ-aminobutyric acid (GABA)(A) receptors containing the α(2) subunit, 9-amino-2-cyclobutyl-5-(6-methoxy-2-methylpyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one (4) and 9-amino-2-cyclobutyl-5-(2-methoxypyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one (5), were radiolabelled at the methoxy position with carbon-11 (half-life=20.4 min). These quinolines represent a new class of potential radiotracers for imaging the benzodiazepine site of GABA(A) receptors with positron emission tomography (PET). Both radiotracers were reliably isolated following reaction of their respective pyridinone/pyridinol tautomeric precursors with [(11)C]CH(3)I in clinically useful, formulated quantities (2.9% and 2.7% uncorrected radiochemical yield, respectively, relative to [(11)C]CO(2)) with high specific activities (>70 GBq μ mol(-1); >2 Ci μ mol(-1)) and high radiochemical purities (>95%). The radiosyntheses reported herein represent rare examples of selectively isolating radiolabelled compounds bearing [(11)C]2-methoxypyridine moieties. Although both radiotracers demonstrated promising imaging characteristics based on preliminary ex vivo biodistribution studies in conscious rodents, higher brain uptake was observed with [(11)C]5 and therefore this radiotracer was further evaluated. Carbon-11 labelled 5 readily penetrated the brain (>1 standard uptake value in cortical regions at 15 min post-injection of the radiotracer), had an appropriate regional brain distribution for GABA(A) receptors that appeared to be reversible, and did not show any appreciable radiometabolites in rat brain homogenates up to 15 min post-injection. Preadministration of flumazenil (1, 10 mg kg(-1)) or 5 (5 mg kg(-1)) effectively blocked >50% of [(11)C]5 binding to the GABA(A) receptor-rich regions, thereby suggesting that this radiotracer is worthy of further evaluation for imaging GABA(A) receptors. Additionally (R,S)-N-(1-(3-chloro-4-methoxyphenyl)ethyl)-3,3-diphenylpropan-1-amine, 6, an allosteric modulator of GABA(B) receptors, was efficiently labelled in one step using [(11)C]methyl iodide. Ex vivo biodistribution studies in conscious rats showed low brain uptake, therefore, efforts are underway to discover alternative radiotracers to image GABA(B). In conclusion, [(11)C]5 is worthy of further evaluation in higher species for imaging GABA(A) receptors in the central nervous system.     

11C-Fallypride: radiosynthesis and preliminary evaluation of a novel dopamine D2/D3 receptor PET radiotracer in non-human primate brain

Fallypride [benzamide, 5-(3-fluoropropyl)-2,3-dimethoxy-N-[(2S)-1-(2-propenyl)-2-pyrrolidinyl]methyl]-, CAS RN 166173-78-0] is a selective dopamine D2/D3 receptor antagonist. Carbon-11 labeled fallypride may serve as a radiotracer for use in biomedical imaging technique positron emission tomography (PET). The precursor, 5-(3-fluoropropyl)-2-hydroxy-3-methoxy-N-[(2S)-1-(2-propenyl)-2-pyrrolidinyl]methyl]benzamide was synthesized from 2-hydroxy-3-methoxy-5-(2-propenyl)benzoic acid, methyl ester in seven steps with approximately 10% overall chemical yield. Using this precursor (11)C-fallypride was produced by radiolabeling with (11)C-methyl iodide in 25-40% radiochemical yields with specific activities of 200-1000 Ci/mmol. PET imaging studies in nonhuman primates with (11)C-fallypride showed radiotracer localization in dopaminergic brain regions such as caudate, putamen, thalamus and cortex. This regional localization of (11)C-fallypride is similar to that observed previously for (18)F-fallypride. The results suggest (11)C-fallypride is a useful PET radiotracer for imaging dopamine D2/D3 receptors.     

Synthesis and radiopharmacological characterization of 2beta-carbo-2'-[18F]fluoroethoxy-3beta-(4-bromo-phenyl)tropane ([18F]MCL-322) as a PET radiotracer for imaging the dopamine transporter (DAT)

The fluoroalkyl-containing tropane derivative 2beta-carbo-2'-fluoroethoxy-3beta-(4-bromo-phenyl)tropane (MCL-322) is a highly potent and moderately selective ligand for the dopamine transporter (DAT). The compound was labeled with the short-lived positron emitter (18)F in a single step by nucleophilic displacement of the corresponding tosylate precursor MCL-323 with no-carrier-added [(18)F]fluoride. The positron emission tomography (PET) radiotracer 2beta-carbo-2'-[(18)F]fluoroethoxy-3beta-(4-bromo-phenyl)tropane [(18)F]MCL-322 was obtained in decay-corrected radiochemical yields of 30-40% at a specific radioactivity of 1.6-2.4Ci/mumol (60-90GBq/mumol) at the end-of-synthesis (EOS). Small animal PET, ex vivo and in vivo biodistribution experiments in rats demonstrated a high uptake in the striatum (3.2% ID/g) 5min after injection, which increased to 4.2% ID/g after 60min. The uptake in the cerebellum was 1.8% ID/g and 0.6% ID/g after 5min and 60min post-injection, respectively. Specific binding to DAT of [(18)F]MCL-322 was confirmed by blocking experiments using the high affinity DAT ligand GBR 12909. The radiopharmacological characterization was completed with metabolite and autoradiographic studies confirming the selective uptake of [(18)F]MCL-322 in the striatum. It is concluded that the simple single-step radiosynthesis of [(18)F]MCL-322 and the promising radiopharmacological data make [(18)F]MCL-322 an attractive candidate for the further development of a PET radiotracer potentially suitable for clinical DAT imaging in the human brain.     

Synthesis, radiosynthesis and in vivo evaluation of 5-[3-(4-benzylpiperidin-1-yl)prop-1-ynyl]-1,3-dihydrobenzoimidazol-2-[(11)C]one, as a potent NR(1A)/2B subtype selective NMDA PET radiotracer

Recently, a new series of potent and highly subtype-selective 1-(heteroarylalkynyl)-4-benzylpiperidine antagonists of the NMDA receptors has been described by Pfizer Laboratories. In this series, 5-[3-(4-benzylpiperidin-1-yl)prop-1-ynyl]-1,3-dihydrobenzoimidazol-2-one (1) was identified as a selective antagonist for the NR1(A)/2B subtype, displaying IC(50) values for inhibition of the NMDA responses of 5.3 nM for this subtype (compared to NR1(A)/2A: 35 microM and NR1(A)/2C>100 microM) and was active in rat at a relatively low dosage (10mg/kg po). Derivative 1 has been synthesized in four chemical steps in good overall yield and labelled with carbon-11 at its benzoimidazolone ring using [(11)C]phosgene. The pharmacological profile of [(11)C]-1 was evaluated in vivo in rats with biodistribution studies and brain radioactivity monitored with intracerebral radiosensitive beta-microprobes. The brain uptake of [(11)C]-1 was extremely low (0.07% I.D./mL on average at 30 min) and rather uniform across the different brain structures. This in vivo brain regional distribution of [(11)C]-1 did not match with autoradiographic or binding data obtained with other NR2B subtype-selective NMDA ligands. Competition studies with ifenprodil (20 mg/kg, ip, 30 min before the radiotracer injection) failed to demonstrate specific binding of the radiotracer in the brain. In view of these results, and especially considering the low brain penetration of the radiotracer, [(11)C]-1 does not have the required properties for imaging NMDA receptors using positron emission tomography.     

Radiosynthesis of [11C]BBAC and [11C]BBPC as potential PET tracers for orexin2 receptors

Radiosynthesis of [N-methyl-(11)C](S)-N-([1,1'-biphenyl]-2-yl)-1-(2-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)acetyl)pyrrolidine-2-carboxamide ([(11)C]BBAC or [(11)C]3) and [N-methyl-(11)C] (S)-N-([1,1'-biphenyl]-2-yl)-1-(3-(1-methyl-1H-benzo[d]imidazol-2-yl)propanoyl)pyrrolidine-2-carboxamide ([(11)C]BBPC or [(11)C]-4), two potential PET tracers for orexin2 receptors are described. Syntheses of non-radioactive standards 3, 4 and corresponding desmethyl precursors 1, 2 were achieved from common intermediate (S)-2-([1,1'-biphenyl]-2-yl)-1-(pyrrolidin-2-yl)ethanone. Methylation using [(11)C]CH(3)OTf in the presence of base in acetone afforded [(11)C]3 and [(11)C]4 in 30±5% yield (EOS) with >99 % radiochemical purities with a specific activity ranged from 2.5±0.5 Ci/μmol (EOB). The logP of [(11)C]3 and [(11)C]4 were determined as 3.4 and 2.8, respectively. The total synthesis time was 30 min from EOB. However, PET scans performed in a rhesus monkey did not show tracer retention or appropriate brain uptake. Hence [(11)C]3 and [(11)C]4 cannot be used as PET tracers for imaging orexin2 receptors.     

Synthesis and evaluation of 4-(2-fluoro-4-[11C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide for PET imaging of the metabotropic glutamate receptor 2 in the rat brain

Metabotropic glutamate receptor 2 (mGluR2) has been suggested as a therapeutic target for treating schizophrenia-like symptoms arising from increased glutamate transmission in the human forebrain. However, no reliable positron emission tomography (PET) radiotracer allowing for in vivo visualization of mGluR2 in the human brain is currently available. In this study, we synthesized 4-(2-fluoro-4-[¹¹C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide ([¹¹C]1) and evaluated its potential as a PET tracer for imaging mGluR2 in the rodent brain. Compound 1, a negative allosteric modulator (NAM) of mGluR2, showed high in vitro binding affinity (IC₅₀: 26?nM) for mGluR2 overexpressed in human cells. [¹¹C]1 was synthesized by O-[¹¹C]methylation of the phenol precursor 2 with [¹¹C]methyl iodide. After the reaction, HPLC purification and formulation, [¹¹C]1 of 7.4?±?2.8?GBq (n?=?8) was obtained from [¹¹C]carbon dioxide of 22.5?±?4.8?GBq (n?=?8) with >99% radiochemical purity and 70?±?32?GBq/μmol (n?=?8) molar activity at the end of synthesis. In vitro autoradiography for rat brains showed that [¹¹C]1 binding was heterogeneously distributed in the cerebral cortex, striatum, hippocampus, and cerebellum. This pattern is consistent with the regional distribution pattern of mGluR2 in the rodent brain. The radioactivity was significantly reduced by self- or MNI-137 (a mGluR2 NAM) blocking. Small-animal PET studies indicated a low in vivo specific binding of [¹¹C]1 in the rat brain. The brain uptake was increased in a P-glycoprotein and breast cancer resistant protein double knockout mouse, when compared to a wild-type mouse. While [¹¹C]1 presented limited potential as an in vivo PET tracer for mGluR2, we suggested that it can be used as a lead compound for developing new radiotracers with improved in vivo brain properties.     

Synthesis, radiofluorination and pharmacological evaluation of a fluoromethyl spirocyclic PET tracer for central σ1 receptors and comparison with fluoroalkyl homologs

The spirocyclic σ(1) receptor ligand 1 (1'-benzyl-3-(fluoromethyl)-3H-spiro[[2]benzofuran-1,4'-piperidine]) was prepared in four steps starting from methoxy derivative 5. Due to its high σ(1) affinity (K(i)=0.74nM) and selectivity against several other relevant targets, 1 was investigated as (18)F-labeled PET tracer and its biological properties were compared with those of homologous fluoroalkyl derivatives 2-4. The fluoromethyl derivative 1 was faster metabolized in vitro than homologs 2-4. In contrast to the radiosynthesis of [(18)F]2-4, the nucleophilic substitution of the tosylate 15 using the K[(18)F]F-K(222)-carbonate complex required heating to 150°C in DMSO to achieve high labeling efficiencies. Whereas radiometabolites of [(18)F]2-4 were not detected in vivo in the brain of mice, two radiometabolites of [(18)F]1 were found. Analysis of ex vivo autoradiography images provided rather low target-to-nontarget ratio for [(18)F]1 compared with [(18)F]2-4. [(18)F]1 showed a fast uptake in the brain, which decreased continuously over time. The brain-to-plasma ratio of the radiotracer [(18)F]1 was only exceeded by the fluoroethyl tracer [(18)F]2.     

Comparative evaluation of positron emission tomography radiotracers for imaging the norepinephrine transporter: (S,S) and (R,R) enantiomers of reboxetine analogs ([11C]methylreboxetine, 3-Cl-[11C]methylreboxetine and [18F]fluororeboxetine), (R)-[11C]nisoxetine, [11C]oxaprotiline and [11C]lortalamine

We have synthesized and evaluated several new ligands for imaging the norepinephrine transporter (NET) system in baboons with positron emission tomography (PET). Ligands possessing high brain penetration, high affinity and selectivity, appropriate lipophilicity (log P = 1.0-3.5), high plasma free fraction and reasonable stability in plasma were selected for further studies. Based on our characterization studies in baboons, including 11C-labeled (R)-nisoxetine (Nis), oxaprotiline (Oxap), lortalamine (Lort) and new analogs of methylreboxetine (MRB), in conjunction with our earlier evaluation of 11C and 18F derivatives of reboxetine, MRB and their individual (R,R) and (S,S) enantiomers, we have identified the superiority of (S,S)-[11C]MRB and the suitability of MRB analogs [(S,S)-[11C]MRB > (S,S)-[11C]3-Cl-MRB > (S,S)-[18F]fluororeboxetine] as potential NET ligands for PET. In contrast, Nis, Oxap and Lort displayed high uptake in striatum (higher than in thalamus). The use of these ligands is further limited by high non-specific binding and relatively low specific signal, as is characteristic of many earlier NET ligands. Thus, to our knowledge (S,S)-[11C]MRB remains by far the most promising NET ligand for PET studies.     

Synthesis and preliminary evaluation of (S)-[11C]-exaprolol, a novel beta-adrenoceptor ligand for PET

Positron-emitting beta-adrenoceptor ligands for the CNS could allow determination of changes in beta-adrenoceptor availability after treatment of patients with norepinephrine reuptake inhibitors or tricyclic antidepressants, and differential diagnosis between multiple sclerosis and other brain disorders in an early stage of the disease. No ligands suitable for this purpose are available for human use. In order to prepare a tracer for human studies, we labeled the biologically active enantiomer of the beta-blocker exaprolol with (11)C. Exaprolol has the appropriate lipophilicity (log P + 1.6) for entry of the CNS and is claimed to be a very potent beta-adrenoceptor antagonist. (S)-Desisopropyl-exaprolol was synthesized by reaction of 2-hexylphenol with (S)-glycidyl-nosylate followed by ring opening using ammonia gas. The desisopropyl precursor was reacted with (11)C-acetone in methanol to produce (S)-[(11)C]-exaprolol. Radiochemical purification was performed with RP-HPLC and was followed by Sep-Pak formulation. The labeled product was i.v. injected into male Wistar rats. Brain images were acquired using a microPET Focus 220 and the biodistribution of (11)C was assessed. The radiochemical yield of (S)-[(11)C]-exaprolol was 7% with a total synthesis time of 30 min. Specific activities were >10 GBq/micromol. Brain uptake of the tracer reached a maximum after 15 min. Standardized uptake values were moderate (0.5-0.9) but sufficient for imaging. However, beta-blockade (propranolol, 2.5mg/kg body weight) did not lower tracer uptake in any CNS region and washout from the brain was not accelerated when propranolol was administered 40 min after injection of (S)-[(11)C]-exaprolol. Tracer binding in lung, spleen and erythrocytes was lowered after beta-blockade, but the myocardial uptake of radioactivity was not affected. These data indicate that (S)-[(11)C]-exaprolol is not a suitable beta-adrenoceptor ligand for PET, probably because the in vivo affinity of exaprolol to beta-adrenoceptors is in the nM rather than the sub-nM range. The observed inhibition of tracer uptake in lung, spleen and erythrocytes seems due to an interaction of propranolol with amine transporters rather than beta-adrenoceptors.     

Synthesis and in vivo evaluation of [O-methyl-11C] N-[3,5-dichloro-2-(methoxy)phenyl]-4-(methoxy)-3-(1-piperazinyl)benzenesulfonamide as an imaging probe for 5-HT6 receptors

The serotonin receptor 6 (5-HT(6)) is implicated in the pathophysiology of cognitive diseases, schizophrenia, anxiety and obesity and in vivo studies of this receptor would be of value for studying the pathophysiology of these disorders. Therefore, N-[3,5-dichloro-2-(methoxy)phenyl]-4-(methoxy)-3-(1-piperazinyl)benzenesulfonamide (SB399885), a selective and high affinity (pK(i)=9.11) 5-HT(6) antagonist, has been radiolabeled with carbon-11 by O-methylation of the corresponding desmethyl analogue with [(11)C]MeOTf in order to determine the suitability of [(11)C]SB399885 to quantify 5-HT(6)R in living brain using PET. Desmethyl-SB399885 was prepared, starting from 1-(2-methoxyphenyl) piperazine hydrochloride, in excellent yield. The yield obtained for radiolabeling of [(11)C]SB399885 was 30±5% (EOS) and the total synthesis time was 30min at EOB. PET studies with [(11)C]SB399885 in baboon showed fast uptake followed by rapid clearance in the brain. Highest uptake of radioactivity of [(11)C]SB399885 in baboon brain were found in temporal cortex, parahippocampal gyrus, pareital cortex, amygdala, and hippocampus. Poor brain entry and inconsistent brain uptake of [(11)C]SB399885 compared to known 5-HT(6)R distribution limits its usefulness for the in vivo quantification of 5-HT(6)R with PET.