Imaging of I2-imidazoline receptors by small-animal PET using 2-(3-fluoro-[4-11C]tolyl)-4,5-dihydro-1H-imidazole ([11C]FTIMD)

IntroductionImidazoline receptors (IRs) have been established as distinct receptors, and have been categorized into at least two subtypes (I₁R and I₂R). I₂Rs are associated with depression, Alzheimer's disease, Huntington's disease and Parkinson's disease. A few positron emission tomography (PET) probes for I₂Rs have been synthesized, but a selective PET probe has not been evaluated for the imaging of I₂Rs by PET. We labeled a selective I₂R ligand 2-(3-fluoro-4-tolyl)-4,5-dihydro-1H-imidazole (FTIMD) with ¹¹C and performed the first imaging of I₂Rs by PET using 2-(3-fluoro-[4-¹¹C]tolyl)-4,5-dihydro-1H-imidazole ([¹¹C]FTIMD).Methods[¹¹C]FTIMD was prepared by a palladium-promoted cross-coupling reaction of the tributylstannyl precursor and [¹¹C]methyl iodide in the presence of tris(dibenzylideneacetone)dipalladium(0) and tri(o-tol)phosphine. Biodistribution was investigated in rats by tissue dissection. [¹¹C]FTIMD metabolites were measured in brain tissues and plasma. Dynamic PET scans were acquired in rats, and the kinetic parameters estimated.Results[¹¹C]FTIMD was successfully synthesized with a suitable radioactivity for the injection. Co-injection with 0.1 mg/kg of cold FTIMD and BU224 induced a significant reduction in the brain-to-blood ratio 15 and 30 min after the injection. In metabolite analysis, unchanged [¹¹C]FTIMD in the brain was high (98%) 30 min after the injection. In PET studies, high radioactivity levels were observed in regions with a high density of I₂R. The radioactivity levels and V_T values in the brain regions were prominently reduced by 1.0 mg/kg of BU224 pretreatment as compared with control.Conclusion[¹¹C]FTIMD showed specific binding to I₂Rs in rat brains with a high density of I₂R.     

Synthesis and evaluation of PET probes for the imaging of I2 imidazoline receptors in peripheral tissues

IntroductionTo explore the possible use of positron emission tomography (PET) probes for imaging of I₂-imidazoline receptors (I₂Rs) in peripheral tissues, we labeled two new I₂R ligands, 2-[2-(o-tolyl)vinyl]-4,5-dihydro-1H-imidazole (K_i for I₂Rs, 3.7 nM) and 2-[2-(o-tolyl)ethyl]-4,5-dihydro-1H-imidazole (K_i for I₂Rs, 1.7 nM) with ¹¹C ([¹¹C]metrazoline and [¹¹C]TEIMD), respectively, and evaluated these ligands and the recently developed I₂R ligand 2-[3-fluoro-[4-¹¹C]tolyl]-4,5-dihydro-1H-imidazole ([¹¹C]FTIMD) by in vivo studies.Methods[¹¹C]Metrazoline and [¹¹C]TEIMD were prepared by a palladium-promoted cross-coupling reaction of the tributylstannyl precursor and [¹¹C]methyl iodide. Their biodistribution in mice was investigated by tissue dissection. In addition, PET scans and metabolite analysis were performed.Results[¹¹C]Metrazoline and [¹¹C]TEIMD were successfully synthesized with a suitable radioactivity for injection. In the liver and pancreas expressing I₂Rs, coinjection with the high-affinity I₂R ligand, BU224, induced a reduction in the radioactivity level at 30 min after injection of [¹¹C]metrazoline and [¹¹C]FTIMD. However, the radioactivity level after injection of [¹¹C]TEIMD was unchanged. In the PET study, coinjection with BU224 induced a decrease in the radioactivity level in the liver and pancreas after more than 15 min of injection of [¹¹C]metrazoline and [¹¹C]FTIMD as compared with the results obtained for controls. In metabolite analysis, coinjection with BU224 induced a significant reduction in the percentage of unchanged [¹¹C]metrazoline at 30 min after injection as compared with that in the control, although no significant difference was observed in the percentage of unchanged [¹¹C]FTIMD.Conclusion[¹¹C]Metrazoline may be a more useful PET probe than [¹¹C]FTIMD for imaging of I₂Rs in peripheral tissues.     

Radiosynthesis and ex vivo evaluation of (R)-(−)-2-chloro-N-[1-11C-propyl]n-propylnorapomorphine

IntroductionSeveral dopamine D₂ agonist radioligands have been used with positron emission tomography (PET), including [¹¹C-]-(?)-MNPA, [¹¹C-]-(?)-NPA and [¹¹C]-(+)-PHNO. These radioligands are considered particularly powerful for detection of endogenous dopamine release, but they either provide PET brain images with limited contrast or have affinity for both D₂ and D₃ receptors. We here present the carbon-11 radiolabeling and ex vivo evaluation of 2-Cl-(?)-NPA, a novel PET-tracer candidate with high in vitro D₂/D₃ selectivity.Methods2-Cl-[¹¹C]-(?)-NPA and [¹¹C]-(?)-NPA were synthesized by a two step N-acylation-reduction process using [¹¹C]-propionyl chloride. Awake rats were injected with either tracer, via the tail vein. The rats were decapitated at various times, the brains were removed and quickly dissected, and plasma metabolites were measured. Radioligand specificity, and P-glycoprotein involvement in brain uptake, was also assessed.Results2-Cl-[¹¹C]-(?)-NPA and [¹¹C]-(?)-NPA were produced in high specific activity and purity. 2-Cl-[¹¹C]-(?)-NPA accumulated slower in the striatum than [¹¹C]-(?)-NPA, reaching maximum concentrations after 30 min. The maximal striatal uptake of 2-Cl-[¹¹C]-(?)-NPA (standard uptake value 0.72±0.24) was approximately half that of [¹¹C]-(?)-NPA (standard uptake value 1.37±0.18). Nonspecific uptake was similar for the two compounds. 2-Cl-[¹¹C]-(?)-NPA was metabolized quickly, leaving only 17% of the parent compound in the plasma after 30 min. The specific binding of 2-Cl-[¹¹C]-(?)-NPA was completely blocked and inhibition of P-glycoprotein did not alter the brain uptake.ConclusionEx vivo experiments showed, despite a favorable D₂/D₃ selectivity, that 2-Cl-[¹¹C]-(?)-NPA is inferior to [¹¹C]-(?)-NPA as a PET tracer in rat, because of slower brain uptake and lower specific to nonspecific binding ratio.     

Preclinical evaluation of [11C]NE40, a type 2 cannabinoid receptor PET tracer

IntroductionUp-regulation of the type 2 cannabinoid receptor (CB₂R) has been reported in (neuro)inflammatory diseases. In this study, we report the preclinical evaluation of [¹¹C]NE40 as positron emission tomography (PET) radioligand for visualization of the CB₂R.MethodsThe selectivity of NE40 for CB₂R and its toxicity and mutagenicity were determined. [¹¹C]NE40 was evaluated by biodistribution and autoradiography studies in normal rats and a microPET study in normal mice, rats and a rhesus monkey. Specific in vivo binding of [¹¹C]NE40 to human CB₂R (hCB₂R) was studied in a rat model with hCB₂R overexpression.Results[¹¹C]NE40 shows specific CB₂R binding in the spleen and blood of normal rats and high brain uptake in rhesus monkey. [¹¹C]NE40 showed specific and reversible binding to hCB₂R in vivo in a rat model with local hCB₂R overexpression.Conclusions[¹¹C]NE40 shows favorable characteristics as radioligand for in vivo visualization of the CB₂R and is a promising candidate for hCB₂R PET imaging.     

Measurement of dopamine D2 receptors in living human brain using [11C]raclopride with ultra-high specific radioactivity

IntroductionHigh specific radioactivity is preferable in the measurement of neuroreceptor bindings with positron emission tomography (PET) because receptor occupancy by mixed cold ligand hampers the accurate estimation of receptor binding. Recently, we succeeded in synthesizing [¹¹C]raclopride, a dopamine D₂ receptor ligand, with ultra-high specific radioactivity, i.e., several thousand GBq/μmol. In the present study, we compared the [¹¹C]raclopride bindings to dopamine D₂ receptors between radioligands with ultra-high specific radioactivity and ordinary high specific radioactivity in healthy human subjects.MethodsTwo PET studies using [¹¹C]raclopride with ultra-high specific radioactivity (4302–7222 GBq/μmol) or ordinary high specific radioactivity (133-280 GBq/μmol) were performed on different days in 14 healthy men. Binding potential (BP) was calculated by the simplified reference tissue method, peak equilibrium method, and area-under-the-curve method for each region-of-interest using time-activity data in the cerebellum as a reference brain region.ResultsBP values for radioligands with ultra-high specific radioactivity and ordinary high specific radioactivity calculated by the simplified reference tissue method were 4.06±0.29 and 4.10±0.25 in the putamen, 0.44±0.07 and 0.47±0.07 in the thalamus and 0.37±0.06 and 0.38±0.06 in the temporal cortex, respectively (mean±S.D.). No significant difference in BP was observed between ultra-high specific radioactivity and ordinary high specific radioactivity in any of the brain regions.ConclusionBP values of [¹¹C]raclopride with ultra-high specific radioactivity did not differ from those with ordinary high specific radioactivity in the measured brain regions, including striatal and extrastriatal regions.     

Synthesis and preclinical evaluation of [11C]D617, a metabolite of (R)-[11C]verapamil

Objectives(R)-[¹¹C]verapamil is widely used as a positron emission tomography (PET) tracer to evaluate P-glycoprotein (P-gp) functionality at the blood–brain barrier in man. A disadvantage of (R)-[¹¹C]verapamil is the fact that its main metabolite, [¹¹C]D617, also enters the brain. For quantitative analysis of (R)-[¹¹C]verapamil data, it has been assumed that the cerebral kinetics of (R)-[¹¹C]verapamil and [¹¹C]D617 are the same. The aim of the present study was to investigate whether the cerebral kinetics of (R)-[¹¹C]verapamil and [¹¹C]D617 are indeed similar and, if so, whether [¹¹C]D617 itself could serve as an alternative PET tracer for P-gp.Methods[¹¹C]D617 was synthesized and its ex vivo biodistribution was investigated in male rats at four time points following intravenous administration of [¹¹C]D617 (50 MBq) without (n=4) or with (n=4) pretreatment with the P-gp inhibitor tariquidar (15 mg·kg^?1, intraperitoneally). Brain distribution was further assessed using consecutive PET scans (n=8) before and after pretreatment with tariquidar (15 mg·kg^?1, intravenously), as well as metabolite analysis (n=4).ResultsThe precursor for the radiosynthesis of [¹¹C]D617, 5-amino-2-(3,4-dimethoxy-phenyl)-2-isopropyl-pentanitrile (desmethyl D617), was synthesized in 41% overall yield. [¹¹C]D617 was synthesized in 58%–77% decay-corrected yield with a radiochemical purity of ≥99%. The homogeneously distributed cerebral volume of distribution (V_T) of [¹¹C]D617 was 1.1, and this increased 2.4-fold after tariquidar pretreatment.ConclusionV_T of [¹¹C]D617 was comparable to that of (R)-[¹¹C]verapamil, but its increase after tariquidar pretreatment was substantially lower. Hence, (R)-[¹¹C]verapamil and [¹¹C]D617 do not show similar brain kinetics after inhibition of P-gp with tariquidar.     

Radiosynthesis and evaluation of [11C]YM-202074 as a PET ligand for imaging the metabotropic glutamate receptor type 1

IntroductionDeveloping positron emission tomography (PET) ligands for imaging metabotropic glutamate receptor type 1 (mGluR1) is important for studying its role in the central nervous system. N-cyclohexyl-6-{[N-(2-methoxyethyl)-N-methylamino]methyl}-N-methylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM-202074) exhibited high binding affinity for mGluR1 (K_i=4.8 nM), and selectivity over other mGluRs in vitro. The purpose of this study was to label YM-202074 with carbon-11 and to evaluate in vitro and in vivo characteristics of [¹¹C]YM-202074 as a PET ligand for mGluR1 in rodents.Methods[¹¹C]YM-202074 was synthesized by N-[¹¹C]methylation of its desmethyl precursor with [¹¹C]methyl iodide. The in vitro and in vivo brain regional distributions were determined in rats using autoradiography and PET, respectively.Results[¹¹C]YM-202074 (262–630 MBq, n=5) was obtained with radiochemical purity of >98% and specific activity of 27–52 GBq/μmol at the end of synthesis, starting from [¹¹C]CO₂ of 19.3–21.5 GBq. In vitro autoradiographic results showed that the high specific binding of [¹¹C]YM-202074 for mGluR1 was presented in the cerebellum, thalamus and hippocampus, which are known as mGluR1-rich regions. In ex vivo autoradiography and PET studies, the radioligand was specifically distributed in the cerebellum, although the uptake was low. Furthermore, the regional distribution was fairly uniform in the whole brain by pretreatment with JNJ16259685 (a mGluR1 antagonist). However, radiometabolite(s) was detected in the brain.ConclusionsFrom these results, especially considering the low brain uptake and the influx of radiometabolite(s) into brain, [¹¹C]YM-202074 may not be a useful PET ligand for in vivo imaging of mGluR1 in the brain.     

Evaluation of [11C]-DAA1106 for imaging and quantification of neuroinflammation in a rat model of herpes encephalitis

IntroductionMany neurological and psychiatric disorders are associated with neuroinflammation. Positron emission tomography (PET) with [¹¹C]-PK11195 can be used to study neuroinflammation in these disorders. However, [¹¹C]-PK11195 may not be sensitive enough to visualize mild neuroinflammation. As a potentially more sensitive PET tracer for neuroinflammation, [¹¹C]-N-(2,5-dimethoxybenzyl)-N-(4-fluoro-2-phenoxyphenyl)-acetamide (DAA1106) was evaluated in a rat model of herpes encephalitis.MethodsMale Wistar rats were intranasally inoculated with HSV-1 (HSE) or phosphate-buffered saline (control). At Day 6 or Day 7 after inoculation, small-animal [¹¹C]-DAA1106 PET scans were acquired, followed by ex vivo biodistribution. Arterial blood sampling was performed for quantification of uptake.ResultsIn HSE rats, a significantly higher ex vivo, but not in vivo, uptake of [¹¹C]-DAA1106 was found in almost all examined brain areas (24–71%, P<.05), when compared to control rats. Pretreatment with unlabeled PK11195 effectively reduced [¹¹C]-DAA1106 uptake in HSE rats (54–84%; P<.001). The plasma and brain time–activity curves showed rapid uptake of [¹¹C]-DAA1106 into tissue. The data showed a good fit to the Logan analysis but could not be fitted to a two-tissue compartment model.Conclusions[¹¹C]-DAA1106 showed a high and specific ex vivo uptake in the encephalitic rat brain. However, neuroinflammation could not be demonstrated in vivo by [¹¹C]-DAA1106 PET. Quantification of the uptake of [¹¹C]-DAA1106 using plasma sampling is not optimal, due to rapid tissue uptake, slow tissue clearance and low plasma activity.     

Test-retest reproducibility of dopamine D2/3 receptor binding in human brain measured by PET with [11C]MNPA and [11C]raclopride

Purpose

Dopamine D_2/3 receptors (D_2/3Rs) have two affinity states for endogenous dopamine, referred to as high-affinity state (D_2/3 ^HIGH), which has a high affinity for endogenous dopamine, and low-affinity state (D_2/3 ^LOW). The density of D_2/3 ^HIGH can be measured with (R)-2-¹¹CH₃O-N-n-propylnorapomorphine ([¹¹C]MNPA), while total density of D_2/3 ^HIGH and D_2/3 ^LOW (D_2/3Rs) can be measured with [¹¹C]raclopride using positron emission tomography (PET). Thus, the ratio of the binding potential (BP) of [¹¹C]MNPA to that of [¹¹C]raclopride ([¹¹C]MNPA/[¹¹C]raclopride) may reflect the proportion of the density of D_2/3 ^HIGH to that of D_2/3Rs. In the caudate and putamen, [¹¹C]MNPA/[¹¹C]raclopride reflects the proportion of the density of D₂ ^HIGH to that of D₂Rs. To evaluate the reliability of the PET paradigm with [¹¹C]MNPA and [¹¹C]raclopride, we investigated the test-retest reproducibility of non-displaceable BP (BP _ND) measured with [¹¹C]MNPA and of [¹¹C]MNPA/[¹¹C]raclopride in healthy humans.

Methods

Eleven healthy male volunteers underwent two sets of PET studies on separate days that each included [¹¹C]MNPA and [¹¹C]raclopride scans. BP _ND values in the caudate and putamen were calculated. Test-retest reproducibility of BP _ND of [¹¹C]MNPA and [¹¹C]MNPA/[¹¹C]raclopride was assessed by intra-subject variability (absolute variability) and test-retest reliability (intraclass correlation coefficient: ICC).

Results

The absolute variability of [¹¹C]MNPA BP _ND was 5.30?±?3.96 % and 12.3?±?7.95 % and the ICC values of [¹¹C]MNPA BP _ND were 0.72 and 0.82 in the caudate and putamen, respectively. The absolute variability of [¹¹C]MNPA/[¹¹C]raclopride was 6.11?±?3.68 % and 11.60?±?5.70 % and the ICC values of [¹¹C]MNPA/[¹¹C]raclopride were 0.79 and 0.80 in the caudate and putamen, respectively.

Conclusion

In the present preliminary study, the test-retest reproducibility of BP _ND of [¹¹C]MNPA and of [¹¹C]MNPA/[¹¹C]raclopride was reliable in the caudate and putamen.     

Imaging of peripheral-type benzodiazepine receptor in tumor: in vitro binding and in vivo biodistribution of N-benzyl-N-[C]methyl-2-(7-methyl-8-oxo-2-phenyl-7,8-dihydro-9H-purin-9-yl)acetamide

IntroductionThe aim of this study was to evaluate N-benzyl-N-[¹¹C]methyl-2-(7-methyl-8-oxo-2-phenyl-7,8-dihydro-9H-purin-9-yl)acetamide ([¹¹C]DAC) as a novel peripheral-type benzodiazepine receptor (PBR) ligand for tumor imaging.Methods[¹¹C]DAC was synthesized by the reaction of a desmethyl precursor with [¹¹C]CH₃I. In vitro uptake of [¹¹C]DAC was examined in PBR-expressing C6 glioma and intact murine fibrosarcoma (NFSa) cells. In vivo distribution of [¹¹C]DAC was determined using NFSa-bearing mice and small-animal positron emission tomography (PET).Results[¹¹C]DAC showed specific binding to PBR in C6 glioma cells, a standard cell line with high PBR expression. Specific binding of [¹¹C]DAC was also confirmed in NFSa cells, a target tumor cell line in this study. Results of PET experiments using NFSa-bearing mice, showed that [¹¹C]DAC was taken up specifically into the tumor, and pretreatment with PK11195 abolished the uptake.Conclusions[¹¹C]DAC was taken up into PBR-expressing NFSa. [¹¹C]DAC is a promising PET ligand that can be used for imaging PBR in tumor-bearing mice.     

Binding potential of (E)-[11C]ABP688 to metabotropic glutamate receptor subtype 5 is decreased by the inclusion of its 11C-labelled Z-isomer

Introduction[¹¹C]ABP688 is a promising positron emission tomography (PET) ligand for imaging of metabotropic glutamate receptor subtype 5 (mGlu5 receptor). Of the two geometric isomers of ABP688, (E)-ABP688 has a greater affinity towards mGlu5 receptors than (Z)-ABP688. Therefore, a high ratio of E-isomer is required when using [¹¹C]ABP688 as a PET probe for imaging and quantification of mGlu5 receptors. The aim of this study was to evaluate the effect (Z)-[¹¹C]ABP688 on the synthesis of [¹¹C]ABP688 to be used for binding (E)-[¹¹C]ABP688 in the brain.MethodsWe synthesized and separated (E)- and (Z)-[¹¹C]ABP688 by purification using an improved preparative high-performance liquid chromatography (HPLC) method equipped with a COSMOSIL Cholester column. We performed an in vitro binding assay in rat brain homogenates and PET studies of the rat brains using (E)- and (Z)-[¹¹C]ABP688.Results(E)- and (Z)-[¹¹C]ABP688 were successfully obtained with suitable radioactivity for application. In the in vitro assay, the K_d value of (E)-[¹¹C]ABP688 (5.7 nmol/L) was higher than that of (Z)-[¹¹C]ABP688 (140 nmol/L). In the PET study of the rat brain, high radioactivity after injection of (E)-[¹¹C]ABP688 was observed in regions rich in mGlu5 receptors such as the striatum and hippocampus. In contrast, after injection of (Z)-[¹¹C]ABP688, radioactivity did not accumulate in the brain. Furthermore, BP_ND in the striatum and hippocampus was highly correlated (R² = 0.99) with the percentage of (E)-[¹¹C]ABP688 of the total radioactivity of (E)- and (Z)-[¹¹C]ABP688 in the injection.ConclusionWe demonstrated that including (Z)-[¹¹C]ABP688 in the [¹¹C]ABP688 injection can decrease BP_ND in regions rich in mGlu5 receptors. Routine production of (E)-[¹¹C]ABP688 will be helpful for imaging and quantification of mGlu5 receptors in clinical studies.     

Rat Imaging and In Vivo Stability Studies using [11C]-Dimethyl-Diphenyl Ammonium,a Candidate Agent for PET-Myocardial Perfusion Imaging

BackgroundPET myocardial perfusion imaging (MPI) holds several advantages over SPECT for diagnosing coronary artery disease. The short half-lives of prevailing PET-MPI agents hamper wider clinical application of PET in nuclear cardiology; prompting the development of novel PET-MPI agents. We have previously reported on the potential of radiolabeled ammonium salts, and particularly on that of [¹¹C]dimethyl-diphenyl-ammonium ([¹¹C]DMDPA), for cardiac PET imaging. This study was designed to improve the radiosynthesis and increase the yield of [¹¹C]DMDPA, characterize more meticulously the kinetics of radioactivity distribution after its injection via micro-PET/CT studies, and further explore its potential for PET-MPI.MethodsThe radiosynthetic procedure of [¹¹C]DMDPA was improved with respect to the previously reported one. The kinetics of radioactivity distribution following injection of [¹¹C]DMDPA were investigated in juvenile and young adult male SD rats using microPET/CT, and compared to those of [¹³N]NH₃. Furthermore, the metabolic fate of [¹¹C]DMDPA in vivo was examined after its injection into rats.ResultsFollowing a radiosynthesis time of 25–27 min, 11.9 ± 1.1 GBq of [¹¹C]DMDPA was obtained, with a 43.7% ± 4.3% radiochemical yield (n = 7). Time activity curves calculated after administration of [¹¹C]DMDPA indicated rapid, high and sustained radioactivity uptake in hearts of both juvenile and young adult rats, having a two-fold higher cardiac radioactivity uptake compared to [¹³N]NH₃. Accordingly, at all time points after injection to both juvenile and young adult rats, image quality of the left ventricle was higher with [¹¹C]DMDPA compared to [¹³N]NH₃. In vivo stability studies of [¹¹C]DMDPA indicate that no radioactive metabolites could be detected in plasma, liver and urine samples of rats up to 20 min after injection, suggesting that [¹¹C]DMDPA is metabolically stable in vivo.ConclusionsThis study further illustrates that [¹¹C]DMDPA holds, at least in part, essential qualities required from a PET-MPI probe. Owing to the improved radiosynthetic procedure reported herein, [¹¹C]DMDPA can be produced in sufficient amounts for clinical use.     

Synthesis and preclinical evaluation of [11C-carbonyl]PF-04457845 for neuroimaging of fatty acid amide hydrolase

IntroductionFatty acid amide hydrolase (FAAH) has a significant role in regulating endocannabinoid signaling in the central nervous system. As such, FAAH inhibitors are being actively sought for pain, addiction, and other indications. This has led to the recent pursuit of positron emission tomography (PET) radiotracers targeting FAAH. We report herein the preparation and preclinical evaluation of [¹¹C-carbonyl]PF-04457845, an isotopologue of the potent irreversible FAAH inhibitor.MethodsPF-04457845 was radiolabeled at the carbonyl position via automated [¹¹C]CO₂-fixation. Ex vivo brain biodistribution of [¹¹C-carbonyl]PF-04457845 was carried out in conscious rats. Specificity was determined by pre-administration of PF-04457845 or URB597 prior to [¹¹C-carbonyl]PF-04457845. In a separate experiment, rats injected with the title radiotracer had whole brains excised, homogenized and extracted to examine irreversible binding to brain parenchyma.ResultsThe title compound was prepared in 5 ± 1% (n = 4) isolated radiochemical yield based on starting [¹¹C]CO₂ (decay uncorrected) within 25 min from end-of-bombardment in > 98% radiochemical purity and a specific activity of 73.5 ± 8.2 GBq/μmol at end-of-synthesis. Uptake of [¹¹C-carbonyl]PF-04457845 into the rat brain was high (range of 1.2–4.4 SUV), heterogeneous, and in accordance with reported FAAH distribution. Saturable binding was demonstrated by a dose-dependent reduction in brain radioactivity uptake following pre-treatment with PF-04457845. Pre-treatment with the prototypical FAAH inhibitor, URB597, reduced the brain radiotracer uptake in all regions by 71–81%, demonstrating specificity for FAAH. The binding of [¹¹C-carbonyl]PF-04457845 to FAAH at 40 min post injection was irreversible as 98% of the radioactivity in the brain could not be extracted.Conclusions[¹¹C-carbonyl]PF-04457845 was rapidly synthesized via an automated radiosynthesis. Ex vivo biodistribution studies in conscious rodents demonstrate that [11C PF-04457845 is a promising candidate radiotracer for imaging FAAH in the brain with PET. These results coupled with the known pharmacology and toxicology of PF-04457845 should facilitate clinical translation of this radiotracer.     

Radiosynthesis and evaluation of new α1-adrenoceptor antagonists as PET radioligands for brain imaging

IntroductionEvaluation of the α₁-adrenoceptors in relation to brain pathophysiology and drug treatment has been hindered by lack of α₁-adrenoceptor specific radioligands with sufficient brain exposure. Our aim was to develop an α₁-adrenoceptor specific PET radioligand for brain imaging.MethodsTwo sertindole analogues 1-(4-fluorophenyl)-5-(1-methyl-1H-1,2,4-triazol-3-yl)-3-(1-[¹¹C]methylpiperidin-4-yl)-1H-indole [¹¹C]3 and 1-(4-fluorophenyl)-3-(1-[¹¹C]methylpiperidin-4-yl)-5-(pyrimidin-5-yl)-1H-indole ([¹¹C]Lu AA27122) [¹¹C]4 were synthesized and evaluated as α₁-adrenoceptor PET radioligands in cynomolgus monkey. Compounds 3 and 4 were selected due to their promising in vitro preclinical profile; high affinity and selectivity for the α₁-adrenoceptor, favourable blood brain barrier permeability rates in Caco-2 monolayers and promising brain tissue/plasma ratio, assessed by equilibrium dialysis of free fraction in plasma and brain homogenate.ResultsCompounds [¹¹C]3 and [¹¹C]4 were synthesized from their desmethyl piperidine precursors with high specific radioactivity (> 370 GBq/μmol) using [¹¹C]methyl iodide. The 1,2,4-triazole analogue [¹¹C]3 exhibited poor brain uptake, but the corresponding pyrimidyl analogue [¹¹C]4 exhibited high brain exposure and binding in α₁-adrenoceptor rich brain regions. However, the binding could not be inhibited by pretreatment with prazosin (0.1 mg/kg and 0.3 mg/kg). The results were extended by autoradiography of [¹¹C]4 binding in human brain sections and competition with antagonists from different structural families, revealing that only a minor portion of the observed binding of [¹¹C]4 in brain was α₁-adrenoceptor specific.ConclusionThough [¹¹C]3 and [¹¹C]4 proved not suitable as PET radioligands, the study provided further understanding of structural features influencing brain exposure of the chemical class of compounds related to the antipsychotic drug sertindole. It provided valuable insight in the delicacy of blood brain barrier penetration for structurally related compounds and underlines the importance for improved protocols for evaluation of brain penetration of future PET ligands.     

Systemic catechol-O-methyl transferase inhibition enables the D1 agonist radiotracer R-[11C]SKF 82957

IntroductionR-[¹¹C]-SKF 82957 is a high-affinity and potent dopamine D₁ receptor agonist radioligand, which gives rise to a brain-penetrant lipophilic metabolite. In this study, we demonstrate that systemic administration of catechol-O-methyl transferase (COMT) inhibitors blocks this metabolic pathway, facilitating the use of R-[¹¹C]-SKF 82957 to image the high-affinity state of the dopamine D₁ receptor with PET.MethodsR-[¹¹C]SKF 82957 was administered to untreated and COMT inhibitor-treated conscious rats, and the radioactive metabolites present in the brain and plasma were quantified by HPLC. Under optimal conditions, cerebral uptake and dopamine D₁ binding of R-[¹¹C]SKF 82957 were measured ex vivo. In addition, pharmacological challenges with the receptor antagonist SCH 23390, amphetamine, the dopamine reuptake inhibitor RTI-32 and the dopamine hydroxylase inhibitor α-methyl-p-tyrosine were performed to study the specificity and sensitivity of R-[¹¹C]-SKF 82957 dopamine D₁ binding in COMT-inhibited animals.ResultsTreatment with the COMT inhibitor tolcapone was associated with a dose-dependent (EC₉₀ 5.3±4.3 mg/kg) reduction in the lipophilic metabolite. Tolcapone treatment (20 mg/kg) also resulted in a significant increase in the striatum/cerebellum ratio of R-[¹¹C]SKF 82957, from 15 (controls) to 24. Treatment with the dopamine D₁ antagonist SCH 23390 reduced the striatal binding to the levels of the cerebellum, demonstrating a high specificity and selectivity of R-[¹¹C]SKF 82957 binding.ConclusionsPre-treatment with the COMT inhibitor tolcapone inhibits formation of an interfering metabolite of R-[¹¹C]SKF 82957. Under such conditions, R-[¹¹C]SKF 82957 demonstrates high potential as the first agonist radiotracer for imaging the dopamine D₁ receptor by PET.     

Biodistribution and metabolism of the anti-influenza drug [11C]oseltamivir and its active metabolite [11C]Ro 64-0802 in mice

IntroductionOseltamivir phosphate (Tamiflu) is an orally active anti-influenza drug, which is hydrolyzed by esterase to its carboxylate metabolite Ro 64-0802 with potent activity to inhibit the influenza virus. The abnormal behavior and death associated with the use of oseltamivir have developed into a major problem in Japan where Tamiflu is often prescribed for seasonal influenza. It is critical to determine the amount of oseltamivir and Ro 64-0802 in the human brain and to elucidate the relationship between their amounts and neuropsychiatric side effects. The aim of this study was to evaluate [¹¹C]oseltamivir and [¹¹C]Ro 64-0802 in mice as promising positron emission tomography (PET) ligands for measuring their amounts in living brains.MethodsWhole-body biodistribution of [¹¹C]oseltamivir and [¹¹C]Ro 64-0802 was determined in mice using the dissection method and micro-PET. In vitro and in vivo metabolite assay was performed in the plasma and brain of mice.ResultsBetween 1 and 60 min after injection of [¹¹C]oseltamivir and [¹¹C]Ro 64-0802, 0.20–0.06% and 0.39–0.03% ID/g were detected in the mouse brains, respectively (dissection method). Radioactivity concentrations in the living brains between 0 and 90 min after injection were measured at standardized uptake values of 0.25–0.05 for [¹¹C]oseltamivir and 0.38–0.02 for [¹¹C]Ro 64-0802 (micro-PET). In vivo metabolite assay demonstrated the presence of [¹¹C]oseltamivir and [¹¹C]Ro 64-0802 in the brains after [¹¹C]oseltamivir injection.ConclusionThis study determined the distribution and metabolism of [¹¹C]oseltamivir and [¹¹C]Ro 64-0802 in mice. PET could be used to measure their amounts in the living brain and to elucidate the relationship between the amounts in the brain and the side effects of Tamiflu in the central nervous system.     

Assessment of cerebral P-glycoprotein expression and function with PET by combined [11C]inhibitor and [11C]substrate scans in rats

IntroductionThe adenosine triphosphate-binding cassette (ABC) transporter P-glycoprotein (Pgp) protects the brain from accumulation of lipophilic compounds by active efflux transport across the blood–brain barrier. Changes in Pgp function/expression may occur in neurological disorders, such as epilepsy, Alzheimer’s or Parkinson’s disease. In this work we investigated the suitability of the radiolabeled Pgp inhibitors [¹¹C]elacridar and [¹¹C]tariquidar to visualize Pgp density in rat brain with PET.MethodsRats underwent a first PET scan with [¹¹C]elacridar (n = 5) or [¹¹C]tariquidar (n = 6) followed by a second scan with the Pgp substrate (R)-[¹¹C]verapamil after administration of unlabeled tariquidar at a dose which half-maximally inhibits cerebral Pgp (3 mg/kg). Compartmental modeling using an arterial input function and Logan graphical analysis were used to estimate rate constants and volumes of distribution (V_T) of radiotracers in different brain regions.ResultsBrain PET signals of [¹¹C]elacridar and [¹¹C]tariquidar were very low (~ 0.5 standardized uptake value, SUV). There was a significant negative correlation between V_T and K₁ (i.e. influx rate constant from plasma into brain) values of [¹¹C]elacridar or [¹¹C]tariquidar and V_T and K₁ values of (R)-[¹¹C]verapamil in different brain regions which was consistent with binding of [¹¹C]inhibitors to Pgp and efflux of (R)-[¹¹C]verapamil by Pgp.ConclusionThe small Pgp binding signals obtained with [¹¹C]elacridar and [¹¹C]tariquidar limit the applicability of these tracers to measure cerebral Pgp density. PET tracers with higher (i.e. subnanomolar) binding affinities will be needed to visualize the low density of Pgp in brain.     

Synthesis, radiolabeling, in vitro and in vivo evaluation of [F]-FPECMO as a positron emission tomography radioligand for imaging the metabotropic glutamate receptor subtype 5

Introduction[¹⁸F]-(E)-3-((6-Fluoropyridin-2-yl)ethynyl)cyclohex-2-enone O-methyl oxime ([¹⁸F]-FPECMO) is a novel derivative of [¹¹C]-ABP688. [¹⁸F]-FPECMO was characterized as a PET imaging agent for the metabotropic glutamate receptor subtype 5 (mGluR5).Methods[¹⁸F]-FPECMO was synthesized in a one-step reaction sequence by reacting [¹⁸F]-KF-K₂₂₂ complex with (E)-3-((6-bromopyridin-2-yl)ethynyl)cyclohex-2-enone O-methyl oxime in dry DMSO. The in vitro affinity of FPECMO was determined by displacement assays using rat whole brain homogenates (without cerebellum) and the mGluR5-specific radioligand [³H]-M-MPEP. Further in vitro characterization involved metabolite studies, lipophilicity determination and autoradiographical analyses of brain slices. In vivo evaluation was performed by postmortem biodistribution studies and PET experiments using Sprague-Dawley rats.ResultsThe radiochemical yield after semipreparative HPLC was 35±7% and specific activity was >240 GBq/μmol. [¹⁸F]-FPECMO exhibited optimal lipophilicity (logD=2.1) and high metabolic stability in vitro. Displacement studies revealed a K_i value of 3.6±0.7 nM for FPECMO. Biodistribution studies and ex vivo autoradiography showed highest radioactivity accumulation in mGluR5-rich brain regions such as the striatum and hippocampus. Co-injection of [¹⁸F]-FPECMO and ABP688 (1 mg/kg body weight), an mGluR5 antagonist, showed 40% specific binding in the striatum, hippocampus and cortex, regions known to contain high densities of the mGluR5. PET imaging, however, did not allow the visualization of mGluR5-rich brain regions in the rat brain due to a fast washout of [¹⁸F]-FPECMO from mGluR5-expressing tissues and rapid defluorination.Conclusions[¹⁸F]-FPECMO showed significant potential for the detection of mGluR5 in vitro; however, its in vivo characteristics are not optimal for a clear-cut visualization of the mGluR5 in rats.     

[11C]NS8880, a promising PET radiotracer targeting the norepinephrine transporter

IntroductionPositron emission tomography (PET) imaging of the norepinephrine transporter (NET) is still hindered by the availability of useful PET imaging probes. The present study describes the radiosynthesis and pre-clinical evaluation of a new compound, exo-3-(6-methoxypyridin-2-yloxy)-8-H-8-azabicyclo[3.2.1]octane (NS8880), targeting NET. NS8880 has an in vitro binding profile comparable to desipramine and is structurally not related to reboxetine.MethodsLabeling of NS8880 with [¹¹C] was achieved by a non-conventional technique: substitution of pyridinyl fluorine with [¹¹C]methanolate in a Boc-protected precursor. The isolated [¹¹C]NS8880 was evaluated pre-clinically both in a pig model (PET scanning) and in a rat model (μPET scanning) and compared to (S,S)-[¹¹C]-O-methylreboxetine ([¹¹C]MeNER).ResultsThe radiolabeling technique yielded [¹¹C]NS8880 in low (<10%) but still useful yields with high purity. The PET in vivo evaluation in pig and rat revealed a rapid brain uptake of [¹¹C]NS8880 and fast obtaining of equilibrium. Highest binding was observed in thalamic and hypothalamic regions. Pretreatment with desipramine efficiently reduced binding of [¹¹C]NS8880.ConclusionBased on the pre-clinical results obtained so far [¹¹C]NS8880 displays promising properties for PET imaging of NET.     

A radiometabolite study of the serotonin transporter PET radioligand [11C]MADAM

Introduction¹¹C]MADAM is a radioligand suitable for PET studies of the serotonin transporter (SERT). Metabolite analysis in human and non-human plasma samples using HPLC separation has shown that [¹¹C]MADAM was rapidly metabolized. A possible metabolic pathway is the S-oxidation which could lead to SOMADAM and SO₂MADAM.In vitro evaluation of these two potential metabolites has shown that SOMADAM exhibited a good affinity for SERT and a good selectivity for SERT over NET and DAT.MethodsComparative PET imaging studies in non-human primate brain with [¹¹C]MADAM and [¹¹C]SOMADAM were carried out, and plasma samples were analyzed using reverse phase HPLC. We have explored the metabolism of [¹¹C]MADAM in rat brain with a view to understand its possible interference for brain imaging with PET.ResultsPET imaging studies in non-human primate brain using [¹¹C]SOMADAM indicated that this tracer does not bind with high amounts to brain regions known to be rich in SERT. The fraction of [¹¹C]SOMADAM in non-human primate plasma was approximately 5% at 4 min and 1% at 15 min after [¹¹C]MADAM injection. HPLC analysis of brain sample after [¹¹C]MADAM injection to rats demonstrated that [¹¹C]SOMADAM was not detected in the brain.Conclusions¹¹C]SOMADAM is not superior over [¹¹C]MADAM as a SERT PET radioligand. Nevertheless, [¹¹C]SOMADAM has been identified as a minor labeled metabolite of [¹¹C]MADAM measured in monkey plasma. [¹¹C]SOMADAM was not detected in rat brain.