Radiosynthesis of a ¹⁸F-labeled 2,3-diarylsubstituted indole via McMurry coupling for functional characterization of cyclooxygenase-2 (COX-2) in vitro and in vivo

The radiosynthesis of 3-(4-[(18)F]fluorophenyl)-2-(4-methylsulfonylphenyl)-1H-indole [(18)F]-3 as potential PET radiotracer for functional characterization of cyclooxygenase-2 (COX-2) in vitro and in vivo is described. [(18)F]-3 was prepared by McMurry cyclization of a (18)F-labeled intermediate with low valent titanium and zinc via a two-step procedure in a remote controlled synthesizer unit including HPLC purification and solid phase extraction. In this way [(18)F]-3 was synthesized in 80 min synthesis time in 10% total decay corrected yield from [(18)F]fluoride in radiochemical purity >98% and a specific activity of 74-91 GBq/μmol (EOS). [(18)F]-3 was evaluated in vitro using pro-inflammatory stimulated THP-1 and COX-2 expressing tumor cell lines (FaDu, A2058, HT-29), where the radiotracer uptake was shown to be consistent with up regulated COX-2 expression. The stability of [(18)F]-3 was determined by incubation in rat whole blood and plasma in vitro and by metabolite analysis of arterial blood samples in vivo, showing with 75% of original compound after 60 min an acceptable high metabolic stability. However, no substantial tumor accumulation of [(18)F]-3 could be observed by dynamic small animal PET studies on HT-29 tumor-bearing mice in vivo. This may be due to the only moderate COX-1/COX-2 selectivity of 3 as demonstrated by both cellular and enzymatic cyclooxygenase inhibition assay in vitro. Nevertheless, the new approach first using McMurry cyclization in (18)F-chemistry gives access to (18)F-labeled diarylsubstituted heterocycles that hold promise as radiolabeled COX-2 inhibitors.     

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.     

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.     

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.     

Evaluation of 2'-deoxy-2'-flouro-5-methyl-1-beta-D-arabinofuranosyluracil as a potential gene imaging agent for HSV-tk expression in vivo

2'-Deoxy-2'-flouro-5-methyl-1-beta-D-arabinofuranosyluracil (FMAU) has been evaluated in HT-29 cells as a potential positron emission tomography (PET) radiotracer for imaging HSV-tk gene expression in vivo. In vitro experiments demonstrate that the accumulation of [14C]-FMAU in HSV-tk-expressing cells is 2.4-fold (p < .02), 4.0-fold (p < .001), and 5.3-fold (p < .001) higher than the wild-type cells at 1, 3, and 5 hr, respectively. In vivo studies revealed that the tumor uptake in HSV-tk-expressing cells was 2.3-fold (p < .001), 3.0-fold (p < .001), and 5.5-fold (p < .001) higher than the control cells at 1, 2, and 5 hr, respectively. FMAU was found to be more sensitive compared to our earlier studies using 9-[(3-18F-fluoro-1-hydroxy-2-propoxy)methyl]-guanine ([18F]-FHPG) and 9-(4-[18F]-fluoro-3-hydroxy-methylbutyl)guanine ([18F]-FHBG) in the same cell lines, although, the specificity was less than FHBG. These results suggest that while FMAU labeled with PET isotopes may be useful for imaging HSV-tk-expressing tumors in vivo, multitracer studies across additional tumor models are necessary in order to identify an optimal PET radiotracer.     

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.     

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.     

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.     

Synthesis and evaluation of 18F- and 11C-labeled phenyl-galactopyranosides as potential probes for in vivo visualization of LacZ gene expression using positron emission tomography

3-Hydroxy-2-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside, a derivative of the chromogenic beta-galactosidase (beta-gal) substrate o-nitrophenyl beta-D-galactopyranoside (ONPG) was synthesized using a Koenigs-Knorr glycosylation reaction. It was alkylated with 2-[(18)F]fluoroethyl triflate or [(11)C]methyl triflate, followed by deacetylation of the sugar hydroxyl groups to obtain radiolabeled 3-(2'-[(18)F]fluoroethoxy)-2-nitrophenyl beta-D-galactopyranoside ([(18)F]-2c) and 3-[(11)C]methoxy-2-nitrophenyl beta- d-galactopyranoside ([(11)C]-3c), which were evaluated as potential reporter probes for in vivo visualization of LacZ gene expression with positron emission tomography (PET). In vitro, [(18)F]- 2c and [(11)C]-3c were good substrates of beta-gal and showed, respectively, a 7.5- and 2.5-fold higher uptake into beta-gal expressing cells (LacZ cells) compared to control cells. However, reversed-phase HPLC analysis of the LacZ cell lysate and supernatant showed that labeled 3-(2'-[(18)F]fluoroethoxy)-2-nitrophenol, the hydrolysis product formed by beta-gal-mediated cleavage of [(18)F]-2c, substantially leaked out of the cells, which would lead to loss of PET signal. In a microPET study of [(18)F]-2c in a mouse with a beta-gal expressing tumor, high retention was observed in liver and kidneys, but only negligible accumulation was seen in the tumor. As a general conclusion, it can be stated that the synthesized PET tracers [ (18)F]-2c and [(11)C]-3c are not suitable for use as LacZ reporter probes. Further structural modifications to improve the diffusion over the tumor cell membrane and to increase retention in beta-gal expressing cells may lead to more favorable in vivo imaging probes.     

Synthesis and in vivo evaluation of a new PET radioligand for studying sigma-2 receptors

The cyclohexyl piperazine 1 (1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydro-naphthalen-1-yl)-propyl]-piperazine) has been shown to be a potent and selective sigma-2 receptor ligand. In the present study, we prepared [(11)C]1 by O-alkylation of the phenolic precursor 2 with [(11)C]CH(3)I. [(11)C]1 was obtained in a 29% non-decay corrected yield and specific activity of 9299 mCi/micromol calculated at end-of-synthesis. The biodistribution of [(11)C]1 in mouse brain demonstrated rapid and homogenous concentration in all brain structures, which included the cortex, thalamus, cerebellum and striatum. Co-administration of unlabelled 1 (1 mg/kg) or the sigma-2 selective ligand SM-21 (1 mg/kg) failed to show any significant inhibition of [(11)C]1 uptake in the mouse brain. The evaluation of this radioligand in vivo in the mouse clearly indicates that it does not possess the required properties for studying sigma-2 receptors in the brain using PET.     

11C]Glycylsarcosine: synthesis and in vivo evaluation as a PET tracer of PepT2 transporter function in kidney of PepT2 null and wild-type mice

[11C]Glycylsarcosine (Gly-Sar) was synthesized as a potential radiotracer to investigate the localization and in vivo function of the peptide transporter PepT2 in mouse kidney. Its C-11 labeled diketopiperazine derivative, [11C]cyclo(Gly-Sar) [1-methylpiperazine-2,5-dione], was also evaluated as a potential tracer. [11C]Gly-Sar exhibited rapid initial uptake into kidneys with slow clearance from the medulla, consistent with uptake and retention of the radiotracer through the actions of PepT2. In contrast, the corresponding cyclized dipeptide [11C]cyclo(Gly-Sar) showed rapid clearance and accumulation only in the renal pelvis region. Involvement of PepT2 in reabsorption and delayed clearance of [11C]Gly-Sar was confirmed using the PepT2 knockout mouse, where rapid renal elimination of [11C]Gly-Sar and the absence of radioactivity in medulla were observed. This study demonstrates using in vivo imaging technique that PepT2 is primarily responsible for renal tubular active reabsorption of Gly-Sar, and provides a new tool for studying tubular peptide reabsorption and clearance.     

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.     

Synthesis and biodistribution of [11C]R107474, a new radiolabeled alpha2-adrenoceptor antagonist

R107474, 2-methyl-3-[2-(1,2,3,4-tetrahydrobenzo[4,5]furo[3,2-c]pyridin-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one, was investigated using in vitro and in vivo receptor assays and proved to be a potent and relatively selective alpha(2)-adrenoceptor antagonist. Performed assays in vitro were inhibition of binding to a large number of neurotransmitter receptor sites, drug receptor binding sites, ion channel binding sites, peptide receptor binding sites, and the monoamine transporters in membrane preparations of brain tissue or of cells expressing the cloned human receptors. The compound has subnanomolar affinity for halpha(2A)- and halpha(2C)-adrenoceptors (K(i) = 0.13 and 0.15 nM, respectively) and showed nanomolar affinity for the halpha(2B)-adrenoceptors and 5-hydroxytryptamine(7) (h5-HT(7)) receptors (K(i) = 1 and 5 nM, respectively). R107474 interacted weakly (K(i) values ranging between 81 and 920 nM) with dopamine-hD(2L), -hD(3) and -hD(4), h5-HT(1D)-, h5-HT(1F)-, h5-HT(2A)-, h5-HT(2C)-, and h5-HT(5A) receptors. The compound, tested up to 10 microM, interacted only at micromolar concentrations or not at all with any of the other receptor or transporter binding sites tested in this study. In vivo alpha(2A)- and alpha(2C)-adrenoceptor occupancy was measured by ex vivo autoradiography 1h after subcutaneous (sc) administration of R107474. It was found that R107474 occupies the alpha(2A)- and alpha(2C)-adrenoceptors with an ED(50) (95% confidence limits) of 0.014 mg/kg sc (0.009-0.019) and 0.026 mg/kg sc (0.022-0.030), respectively. Radiolabeled 2-methyl-3-[2-([1-(11)C]-1,2,3,4-tetrahydrobenzo[4,5]furo[3,2-c]pyridin-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one ([(11)C]R107474) was prepared and evaluated as a potential positron emission tomography (PET) ligand for studying central alpha(2)-adrenoceptors. [(11)C]R107474 was obtained via a Pictet-Spengler reaction with [(11)C]formaldehyde in 33 +/- 4% overall decay-corrected radiochemical yield. The total synthesis time was 55 min and the specific activity was 24-28 GBq/micromol. The biodistribution of [(11)C]R107474 in rats revealed that the uptake of [(11)C]R107474 after in vivo intravenous administration is very rapid; in most tissues (including the brain) it reaches maximum concentration at 5 min after tracer injection. In agreement with the known distribution of alpha(2)-adrenoceptors in the brain, highest uptake of radioactivity was observed in septum (3.54 +/- 0.52 ID/g, 5 min pi) and entorhinal cortex (1.57 +/- 0.10 ID/g, 5 min pi). Tissue/cerebellum concentration ratios for septum (5.38 +/- 0.45, 30 min pi) and entorhinal cortex (3.43+/-0.24, 30 min pi) increased with time due to rapid uptake followed by a slow washout. In vivo blocking experiments using the non-selective alpha(2)-adrenoceptor antagonist mirtazapine demonstrated specific inhibition of [(11)C]R107474 binding in selective brain areas. The receptor binding profile of mirtazapine is reported and the selectivity of inhibition of binding is discussed. These results suggest that [(11)C]R107474 deserves further investigation as a potential radioligand for studying alpha(2)-adrenoceptors using PET.     

Synthesis and evaluation of [¹¹C]Cimbi-806 as a potential PET ligand for 5-HT₇ receptor imaging

2-(2',6'-Dimethoxy-[1,1'-biphenyl]-3-yl)-N,N-dimethylethanamine has been identified as a potent ligand for the serotonin 7 (5-HT(7)) receptor. In this study, we describe the synthesis, radiolabeling and in vivo evaluation of [(11)C]2-(2',6'-dimethoxy-[1,1'-biphenyl]-3-yl)-N,N-dimethylethanamine ([(11)C]Cimbi-806) as a radioligand for imaging brain 5-HT(7) receptors with positron emission tomography (PET). Precursor and reference compound was synthesized and subsequent (11)C-labelling with [(11)C]methyltriflate produced [(11)C]Cimbi-806 in specific activities ranging from 50 to 300 GBq/μmol. Following intravenous injection, brain uptake and distribution of [(11)C]Cimbi-806 was assessed with PET in Danish Landrace pigs. The time-activity curves revealed high brain uptake in thalamic and striatal regions (SUV ～2.5) and kinetic modeling resulted in distribution volumes (V(T)) ranging from 6 mL/cm(3) in the cerebellum to 12 mL/cm(3) in the thalamus. Pretreatment with the 5-HT(7) receptor antagonist SB-269970 did not result in any significant changes in [(11)C]Cimbi-806 binding in any of the analyzed regions. Despite the high brain uptake and relevant distribution pattern, the absence of appropriate in vivo blocking with a 5-HT(7) receptor selective compounds renders the conclusion that [(11)C]Cimbi-806 is not an appropriate PET radioligand for imaging the 5-HT(7) receptor in vivo.     

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.     

Kinetics of four 11C-labelled enkephalin peptides in the brain, pituitary and plasma of rhesus monkeys

The kinetics of four 11C-labelled enkephalin peptides: Tyr-Gly-Gly-Phe-Met (Met-enkephalin), Tyr-D-Met-Gly-Phe-Pro-NH2 [D-Met2,Pro5)-enkephalinamide), Tyr-D-Ala-Gly-Phe-Met-NH2 (DALA) and Tyr-D-Ala-D-Ala-Phe-Met-NH2 (TAAFM) all labelled at the methyl group of methionine was studied in the Rhesus monkey. After intravenous administration, the regional kinetics in the head, lungs, liver and kidneys were followed by means of positron emission tomography (PET). The total radioactivity in blood and urine was measured and the composition of 11C-labelled peptide fragments in plasma in vivo and in vitro was analysed by liquid chromatography. With PET, an increased radioactivity was observed in the brain and pituitary over the 60-90 min investigation period after i.v. injection of the peptides. The highest radioactivities were noted for Met-enkephalin, followed by DALA and D-Met2, Pro5-enkephalinamide, while very low radioactivities were found for TAAFM. The uptake of Met-enkephalin- and DALA-derived radioactivity was of the same order as has previously been shown for morphine in the brain and considerably higher than that of D-Met2,Pro5-enkephalinamide and TAAFM, respectively. A large fraction of the brain radioactivity derived from Met-enkephalin and DALA probably emanated from [11C]methionine as indicated by plasma and urine analysis. Met-Enkephalin was rapidly eliminated from plasma in vitro with an half-life of less than two minutes, whereas DALA was stable suggesting clearance by other tissues than plasma. In conclusion, both Met-enkephalin and DALA, were rapidly hydrolyzed in vivo to [11C]methionine. [11C]Methionine was probably taken up in the brain, as the radioactivity increased with time in different brain regions as measured with PET.D-Met2,Pro5-Enkephalinamide and TAAFM were virtually stable in vivo and at least part of the radioactivity observed in the brain may have represented the intact peptide.     

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.     

Mechanistic Positron Emission Tomography Studies: Demonstration of a Deuterium Isotope Effect in the Monoamine Oxidase-Catalyzed Binding of [11C]l-Deprenyl in Living Baboon Brain

The application of positron emission tomography (PET) to the study of biochemical transformations in the living human and animal body requires the development of highly selective radiotracers whose concentrations in tissue provide a record of a discrete metabolic process. L-N-[11C-methyl]Deprenyl ([11C]L-deprenyl), a suicide inactivator of monoamine oxidase (MAO) type B, has been developed as a radiotracer for mapping MAO B in the living human and animal brain. In this investigation, [11C]L-deprenyl (1) and [11C]L-deprenyl-alpha, alpha-2H2 (2) have been compared in three different baboons by PET measurement of carbon-11 uptake and retention in the brain and the measurement of the amount of unchanged tracer in the arterial plasma over a 90-min time interval. For one baboon, N-[11C-methyl-2H3]L-deprenyl (3) was also studied. Kinetic parameters calculated using a three-compartment model revealed a deuterium isotope effect of 3.8 +/- 1.1. Comparison of the two tracers (1 and 2) in mouse brain demonstrated that deuterium substitution significantly reduced the amount of radioactivity bound to protein. HPLC and GLC analysis of the soluble radioactivity in mouse brain after injection of [11C]L-deprenyl showed the presence of [11C]methamphetamine as a major product along with unidentified labeled products. Sodium dodecyl sulfate-polyacrylamide electrophoresis with carbon-14-labeled L-deprenyl showed that a protein of molecular weight 58,000 was labeled. These results establish that MAO-catalyzed cleavage of the alpha carbon-hydrogen bond on the propargyl group is the rate limiting (or a major rate contributing) step in the retention of carbon-11 in brain and that the in vivo detection of labeled products in brain after the injection of [11C]L-deprenyl provides a record of MAO activity.     

Radiosynthesis of PET radiotracer as a prodrug for imaging group II metabotropic glutamate receptors in vivo

Group II metabotropic glutamate receptors (mGluRs) have been implicated in a variety of neurological and psychiatric disorders in recent studies. As a noninvasive medical imaging technique and a powerful tool in neurological research, positron emission tomography (PET) offers the possibility to visualize and study group II mGluRs in vivo under physiologic and pathologic conditions. We synthesized a PET tracer, (S,S,S)-2-(2-carboxycyclopropyl)-2-(3-[(11)C]methoxyphenethyl) glycine dimethyl ester ([(11)C]CMGDE), as a prodrug for group II mGluRs, and studied its preliminary biological properties in Sprague-Dawley rats to visualize group II mGluRs. The microPET studies demonstrated that [(11)C]CMGDE readily penetrated into the brain and the radiotracer generated from [(11)C]CMGDE had fast reversible binding in the group II mGluRs rich regions including striatum, hippocampus and different cortical areas. Blocking studies with LY341495 showed 20-30% decrease of binding of the radiotracer generated from [(11)C]CMGDE in all brain areas with the highest decrease in the striatum 31.5±3.2%. The results show [(11)C]CMGDE is the first PET tracer that is brain penetrating and can be used to image group II mGluRs in vivo.     

Radiosynthesis and biodistribution of cyclic RGD peptides conjugated with novel [18F]fluorinated aldehyde-containing prosthetic groups

Achieving high-yielding, robust, and reproducible chemistry is a prerequisite for the (18)F-labeling of peptides for quantitative receptor imaging using positron emission tomography (PET). In this study, we extend the toolbox of oxime chemistry to include the novel prosthetic groups [(18)F]-(2-{2-[2-(2-fluoroethoxy)ethoxy]ethoxy}ethoxy)acetaldehyde, [(18)F]5, and [(18)F]-4-(3-fluoropropoxy)benzaldehyde, [(18)F]9, in addition to the widely used 4-[(18)F]fluorobenzaldehyde, [(18)F]12. The three (18)F-aldehydes were conjugated to the same aminooxy-bearing RGD peptide and the effect of the prosthetic group on biodistribution and tumor uptake studied in mice. The peptide conjugate [(18)F]7 was found to possess superior in vivo pharmacokinetics with higher tumor to blood, tumor to liver, tumor to muscle, and tumor to lung ratios than either [(18)F]10 or [(18)F]13. The radioactivity from the [(18)F]7 conjugate excreted more extensively through the kidney route with 79%id passing through the urine and bladder at the 2 h time point compared to around 55%id for the more hydrophobic conjugates [(18)F]10 and [(18)F]13. The chemical nature of a prosthetic group can be employed to tailor the overall biodistribution profile of the radiotracer. In this example, the hydrophilic nature of the ethylene glycol containing prosthetic group [(18)F]5 clearly influences the overall excretion pattern for the RGD peptide conjugate.