Synthesis and evaluation of 11C-labeled naphthalene derivative as a novel non-peptidergic probe for the β-secretase (BACE1) imaging in Alzheimer's disease brain

IntroductionAs a first trial for in vivo imaging of β-secretase (BACE1) in Alzheimer's disease brain, we applied a novel non-peptidergic small molecule which has high affinity to the enzyme, naphthalene-1-carboxylic acid (3′-chloro-4′-fluoro-4-piperazin-1-yl-biphenyl-3-yl)amide (NCFB) into positron emission tomography (PET) probe. In the current study, N-¹¹C-methylated compound of NCFB, [¹¹C]Me-NCFB was synthesized and evaluated for the visualization of BACE1 in brain.MethodsBACE1 inhibitory constant was measured by FRET assay. [¹¹C]Me-NCFB was synthesized from NCFB with [¹¹C]methyl triflate. To evaluate properties of [¹¹C]Me-NCFB, log P value, stability in mouse plasma and brain uptake index were measured. The biodistribution in 6-week-old ddY mice was also studied.ResultsBACE1 inhibitory constant showed an affinity of Me-NCFB to the enzyme (IC₅₀ = 2.3 ± 0.80 μM). [¹¹C]Me-NCFB was synthesized in a 3.0% ± 0.55% decay-corrected radiochemical yield. [¹¹C]Me-NCFB showed high lipophilicity, high stability in mouse plasma and blood–brain barrier (BBB) permeability. Injected to 6-week-old ddY mice, [¹¹C]Me-NCFB penetrated BBB and was retained in the brain (0.79% ± 0.22% ID/g at 2 min and 0.75% ± 0.08% ID/g at 60 min after injection, respectively), moreover, rapid blood clearance was observed.Conclusion[¹¹C]Me-NCFB could have a potential as a PET probe for the imaging of BACE1 in the brain.     

Synthesis,metabolite analysis,and in vivo evaluation of [11C]irinotecan as a novel positron emission tomography (PET) probe

IntroductionIrinotecan is a semisynthetic derivative of camptothecin that exerts potent antitumor activity by inhibiting topoisomerase I. Despite much research into the complex pharmacokinetic profile and pharmacodynamic effects of irinotecan, unpredictable and severe side effects are still commonly observed. In this study, we synthesized [¹¹C]irinotecan as a positron emission tomography (PET) probe, performed the metabolite analysis, and evaluated the biodistribution and kinetics of [¹¹C]irinotecan using small animal PET.Methods[¹¹C]Irinotecan was synthesized by two routes using [¹¹C]phosgene and [¹¹C]carbon dioxide fixation. Metabolites in the plasma of mice following injection of [¹¹C] irinotecan were investigated using a combination of column-switching high-performance liquid chromatography (HPLC) and on-line solid-phase extraction (SPE). Whole-body PET studies were conducted in wild-type mice and P-glycoprotein and breast cancer resistance protein (Pgp/Bcrp) knockout mice.Results[¹¹C]Irinotecan was successfully synthesized by the two abovementioned routes. Decay-corrected radiochemical yields based on [¹¹C]carbon dioxide using [¹¹C]phosgene and [¹¹C]carbon dioxide fixation were 8.8 ± 2.0% (n = 8) and 16.9 ± 2.9 % (n = 5), respectively. Metabolite analysis of the plasma of mice following injection of [¹¹C]irinotecan was successfully performed using the column-switching HPLC and on-line SPE combination resulting in greater than 87 % recovery of radioactivity from HPLC. In the PET study in mice, the radioactivity levels in the brain, liver, and small intestine were slightly increased by inhibition of the Pgp/Bcrp function for more than 30 min after [¹¹C]irinotecan injection. This result demonstrated that in vivo behavior of [¹¹C] irinotecan and radioactive metabolites are influenced by the Pgp/Bcrp function.ConclusionPET studies using [¹¹C]irinotecan combined with metabolite analysis may be a useful tool for evaluating irinotecan pharmacokinetics and toxicity.     

Longitudinal observation of [11C]4DST uptake in turpentine-induced inflammatory tissue

IntroductionLongitudinal changes of 4′-[methyl-¹¹C]thiothymidine ([¹¹C]4DST) uptake were evaluated in turpentine-induced inflammation.MethodsTurpentine (0.1 ml) was injected intramuscularly into the right hind leg of male Wistar rats. Longitudinal [¹¹C]4DST uptake was evaluated by the tissue dissection method at 1, 2, 4, 7, and 14 days after turpentine injection (n = 5). The tumor selectivity index was calculated using the previously published biodistribution data in C6 glioma-bearing rats. Dynamic PET scan was performed on day 4 when maximum [¹¹C]4DST uptake was observed during the longitudinal study. Histopathological analysis and Ki-67 immunostaining were also performed.ResultsThe uptake of [¹¹C]4DST in inflammatory tissue was significantly increased on days 2–4 after turpentine injection, and then decreased. On day 14, tracer uptake returned to the day 1 level. The maximum SUV of inflamed muscle was 0.6 and was 3 times higher than that of the contralateral healthy muscle on days 2–4 after turpentine injection. However, tumor selectivity index remains very high (> 10) because of the low inflammation uptake. A dynamic PET scan showed that the radioactivity in inflammatory tissues peaked at 5 min after [¹¹C]4DST injection, and then washed out until 20 min. At intervals > 20 min, radioactivity levels were constant and double that of healthy muscle. The changes in Ki-67 index were paralleled with those of [¹¹C]4DST uptake, indicating cell proliferation-dependent uptake of [¹¹C]4DST in inflammatory tissues.ConclusionIn our animal model, low but significant levels of [¹¹C]4DST uptake were observed in subacute inflammation.     

Biodistribution and radiation dosimetry in humans of [11C]FLB 457, a positron emission tomography ligand for the extrastriatal dopamine D2 receptor

Purpose[¹¹C]FLB 457, a radioligand with very high affinity and selectivity for dopamine D_2/3 receptors, is used to measure receptor binding in extrastriatal regions showing low density of the receptors. The purpose of this study was to estimate the whole-body biodistribution of radioactivity and the radiation absorbed doses to organs after intravenous injection of [¹¹C]FLB 457 in healthy human subjects.MethodsWhole-body images were acquired for 2 h after an injection of [¹¹C]FLB 457 in six healthy humans. Radiation absorbed doses were estimated by the MIRD scheme implemented in OLINDA/EXM 1.1 software.ResultsOrgans with the longest residence time were the liver, lungs, and brain. The organs with the highest radiation doses were the kidneys, liver, and pancreas. The effective dose delivered by [¹¹C]FLB 457 is 5.9 μSv/MBq, similar to those of other ¹¹C-labeled tracers.ConclusionsThis effective dose would allow multiple scans in the same individual based on prevailing maximum recommended-dose guidelines in the USA and Europe.     

Radiosynthesis,biodistribution and imaging of [11C]YM155, a novel survivin suppressant,in a human prostate tumor-xenograft mouse model

IntroductionSepantronium bromide (YM155) is an antitumor drug in development and is a first-in-class chemical entity, which is a survivin suppressant. We developed a radiosynthesis of [¹¹C]YM155 to non-invasively evaluate its tissue and tumor distribution in mice bearing human prostate tumor xenografts.MethodsMethods utilizing [¹¹C]acetyl chloride and [¹¹C]methyl triflate, both accessible with automated radiosynthesis boxes, were evaluated. The O-methylation of ethanolamine-alkolate with [¹¹C]methyl triflate proved to be the key development toward a rapid and efficient process. The whole-body distribution of [¹¹C]YM155 in PC-3 xenografted mice was examined using a planar positron imaging system (PPIS).ResultsSufficient quantities of radiopharmaceutical grade [¹¹C]YM155 were produced for our PET imaging and distribution studies. The decay corrected (EOB) radiochemical yield was 16–22%, within a synthesis time of 47 min. The radiochemical purity was higher than 99%, and the specific activity was 29–60 GBq/μmol (EOS). High uptake levels of radioactivity (%ID/g, mean ± SE) were observed in tumor (0.0613 ± 0.0056), kidneys (0.0513 ± 0.0092), liver (0.0368 ± 0.0043) and cecum (0.0623 ± 0.0070). The highest tumor uptake was observed at an early time point (from 10 min after) following injection. Tumor-to-blood and tumor-to-muscle uptake ratios of [¹¹C]YM155, at 40 min after injection, were 26.5 (± 2.9) and 25.6 (± 3.6), respectively.ConclusionA rapid method for producing a radiopharmaceutical grade [¹¹C]YM155 was developed. An in vivo distribution study using PPIS showed high uptake of [¹¹C]YM155 in tumor tissue. Our methodology may facilitate the evaluation and prediction of response to YM155, when given as an anti-cancer agent.     

Synthesis and evaluation of new radioligands [11C]A-833834 and [11C]A-752274 for positron-emission tomography of α7-nicotinic acetylcholine receptors

Introductionα7-nicotinic acetylcholine receptor (α7-nAChR) is one of the major neuronal nAChR subtypes. α7-nAChR is involved in variety of neuronal processes and disorders including schizophrenia and Alzheimer's disease. A number of α7-nAChR PET radioligands have been developed, but a quality radiotracer remains to be discovered.MethodsHigh binding affinity α7-nAChR ligands A-833834 and A-752274 were radiolabeled with ¹¹C. Baseline and blockade biodistribution studies in the mouse brain of [¹¹C]A-833834 (5-(6-(5-[¹¹C]methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyridazin-3-yl)-1H-indole) and [¹¹C]A-752274 (2-(6-[¹¹C]methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)-7-(6-methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)-9H-fluoren-9-one) were performed. [¹¹C]A-752274 was evaluated in a baseline baboon PET study.Results[¹¹C]A-833834 and [¹¹C]A-752274 were synthesized by radiomethylation of corresponding des-methyl precursors. The radioligands were prepared with radiochemical yield of 12%–32%, high specific radioactivity (330–403 GBq/μmol) and radiochemical purity > 95%. Dissection studies with [¹¹C]A-833834 demonstrated low specific α7-nAChR binding in the mouse brain. [¹¹C]A-752274 specifically (~ 50%) labeled α7-nAChR in the mouse thalamus. However, [¹¹CA-752274 exhibited low brain uptake in baboon (%SUV < 100).ConclusionTwo novel α7-nAChR ligands radioligands were synthesized and studied in animals. Specific binding of [¹¹C]A-833834 in the mouse brain is low due to the insufficient binding affinity of the radioligand. The very high binding affinity [¹¹C]A-752274 exhibited good specific binding in the α7-nAChR-rich mouse brain regions. The low uptake of [¹¹C]A-752274 in the baboon brain is due to its high hydrophilicity, rapid metabolism or other properties. Future development of α7-nAChR PET radioligands will be based on compounds with high binding affinities and good blood–brain barrier permeability.     

Novel fluorine-18 PET radiotracers based on flumazenil for GABAA imaging in the brain

IntroductionTwo 7-fluoroimidazobenzodiazepines (AH114726 and GEH120348), analogs of flumazenil, were labeled with fluorine-18 and evaluated as alternative radioligands for in vivo imaging of the GABA_A/benzodiazepine receptor by comparing them to [¹¹C]flumazenil in rhesus monkey.MethodsRadiotracers were prepared from the corresponding nitro-precursors in an automated synthesis module, and primate imaging studies were conducted on a Concorde MicroPET P4 scanner. The brain was imaged for 60 (12 × 5 min frames) or 90 min (18 × 5 min frames), and data was reconstructed using the 3D MAP algorithm. Specificity of [¹⁸F]AH114726 and [¹⁸F]GEH120348 was confirmed by displacement studies using unlabeled flumazenil.Results[¹⁸F]GEH120348 and [¹⁸F]AH114726 were obtained in 13–24% yields (end of synthesis) with high chemical (> 95%) and radiochemical (> 99%) purities, and high specific activities (2061 ± 985 Ci/mmol). The in vivo pharmacokinetics of [¹⁸F]AH114726 and [¹⁸F]GEH120348 were determined in a non-human primate and directly compared with [¹¹C]flumazenil. Both fluorine-18 radioligands showed time-dependent regional brain distributions that correlated with the distribution of [¹¹C]flumazenil and the known concentrations of GABA_A/benzodiazepine receptors in the monkey brain. [¹⁸F]AH114726 exhibited maximal brain uptake and tissue time-radioactivity curves that were most similar to [¹¹C]flumazenil. In contrast, [¹⁸F]GEH120348 showed higher initial brain uptake but very different pharmacokinetics with continued accumulation of radioactivity into the cortical regions of high GABA/benzodiazepine receptor concentrations and very little clearance from the regions of low receptor densities. Rapid washout of both radiotracers occurred upon treatment with unlabeled flumazenil.ConclusionThe ease of the radiochemical synthesis, together with in vivo brain pharmacokinetics most similar to [¹¹C]flumazenil, support that [¹⁸F]AH114726 is a suitable option for imaging the GABA_A receptor.     

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.     

Equivalence of arterial and venous blood for [11C]CO2-metabolite analysis following intravenous administration of 1-[11C]acetate and 1-[11C]palmitate

PurposeSampling of arterial blood for metabolite correction is often required to define a true radiotracer input function in quantitative modeling of PET data. However, arterial puncture for blood sampling is often undesirable. To establish whether venous blood could substitute for arterial blood in metabolite analysis for quantitative PET studies with 1-[¹¹C]acetate and 1-[¹¹C]palmitate, we compared the results of [¹¹C]CO₂-metabolite analyses performed on simultaneously collected arterial and venous blood samples.MethodsPaired arterial and venous blood samples were drawn from anesthetized pigs at 1, 3, 6, 8, 10, 15, 20, 25 and 30 min after i.v. administration of 1-[¹¹C]acetate and 1-[¹¹C]palmitate. Blood radioactivity present as [¹¹C]CO₂ was determined employing a validated 10-min gas-purge method. Briefly, total blood ¹¹C radioactivity was counted in base-treated [¹¹C]-blood samples, and non-[¹¹C]CO₂ radioactivity was counted after the [¹¹C]-blood was acidified using 6 N HCl and bubbled with air for 10 min to quantitatively remove [¹¹C]CO₂.ResultsAn excellent correlation was found between concurrent arterial and venous [¹¹C]CO₂ levels. For the [¹¹C]acetate study, the regression equation derived to estimate the venous [¹¹C]CO₂ from the arterial values was: y = 0.994x + 0.004 (r² = 0.97), and for the [¹¹C]palmitate: y = 0.964x ? 0.001 (r² = 0.9). Over the 1–30 min period, the fraction of total blood ¹¹C present as [¹¹C]CO₂ rose from 4% to 64% for acetate, and 0% to 24% for palmitate. The rate of [¹¹C]CO₂ appearance in venous blood appears similar for the pig model and humans following i.v. [¹¹C]-acetate administration.ConclusionVenous blood [¹¹C]CO₂ values appear suitable as substitutes for arterial blood samples in [¹¹C]CO₂ metabolite analysis after administration of [¹¹C]acetate or [¹¹C]palmitateAdvances in Knowledge and Implications for Patient CareQuantitative PET studies employing 1-[¹¹C]acetate and 1-[¹¹C]palmitate can employ venous blood samples for metabolite correction of an image-derived tracer arterial input function, thereby avoiding the risks of direct arterial blood sampling.     

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.     

Enhanced radiosyntheses of [11C]raclopride and [11C]DASB using ethanolic loop chemistry

IntroductionTo improve the synthesis and quality control of carbon-11 labeled radiopharmaceuticals, we report the fully automated loop syntheses of [¹¹C]raclopride and [¹¹C]DASB using ethanol as the only organic solvent for synthesis module cleaning, carbon-11 methylation, HPLC purification, and reformulation.MethodsEthanolic loop chemistry is fully automated using a GE TRACERLab FX_C-Pro synthesis module, and is readily adaptable to any other carbon-11 synthesis apparatus. Precursors (1 mg) were dissolved in ethanol (100 μL) and loaded into the HPLC loop. [¹¹C]MeOTf was passed through the HPLC loop and then the labeled products were purified by semi-preparative HPLC and reformulated into ethanolic saline.ResultsBoth [¹¹C]raclopride (3.7% RCY; > 95% RCP; SA = 20831 Ci/mmol; n = 64) and [¹¹C]DASB, both with (3.0% RCY; > 95% RCP; SA = 15152 Ci/mmol; n = 9) and without (3.0% RCY; > 95% RCP; SA = 10931 Ci/mmol; n = 3) sodium ascorbate, have been successfully prepared using the described methodology. Doses are suitable for human use and the described methods are now employed for routine clinical production of both radiopharmaceuticals at the University of Michigan.ConclusionsEthanolic loop chemistry is a powerful technique for preparing [¹¹C]raclopride and [¹¹C]DASB, and we are in the process of adapting it for other carbon-11 radiopharmaceuticals prepared in our laboratories ([¹¹C]PMP, [¹¹C]PBR28 etc.).     

Radiolabeling of the cannabinoid receptor agonist AZD1940 with carbon-11 and PET microdosing in non-human primate

IntroductionN-(2-tert-butyl-1-((4,4-difluorocyclohexyl)methyl)-1 H-benzo[d]imidazol-5-yl)ethanesulfonamide (AZD1940) is a candidate drug for treatment of neuropathic pain. As part of the preclinical evaluation of AZD1940, a microdosing study with positron emission tomography (PET) was conducted to assess brain exposure.MethodsAZD1940 was radiolabeled with carbon-11 in the benzimidazole moiety. The radioactive precursor, lithium [¹¹C]pivalate was obtained via ¹¹C-carboxylation of tert-butyl lithium. The target compound, [¹¹C]AZD1940, was in turn obtained by the microwave assisted reaction between lithium [¹¹C]pivalate and the o-phenylene diamine analog of AZD1940 (N-(3-amino-4-((4,4-difluorocyclohexyl)methylamino)phenyl)ethanesulfonamide) in neat phosphorous oxychloride. A brain PET measurement was performed in cynomolgus monkey.ResultsThe overall radiochemical yield of final formulated radiochemically pure (> 99%) [¹¹C]AZD1940 was 0.4% (uncorrected for decay) and the specific radioactivity was 13 GBq/μmol at time of administration (58 min after end of bombardment). After intravenous injection to cynomolgus monkey, the maximum concentration of radioactivity detected in the brain region of interest was 0.7% of the total injected radioactivity. The regional distribution of radioactivity within brain was homogenous.ConclusionsAZD1940 was radiolabelled with carbon-11 and its brain exposure, assessed using PET, was relatively low in comparison to peripheral organ exposure.     

Synthesis and preclinical evaluation of the radiolabeled P-glycoprotein inhibitor [11C]MC113

ObjectivesWith the aim to develop a PET tracer to visualize P-glycoprotein (Pgp) expression levels in different organs, the Pgp inhibitor MC113 was labeled with ¹¹C and evaluated using small-animal PET.Methods[¹¹C]MC113 was synthesized by reaction of O-desmethyl MC113 with [¹¹C]methyl triflate. Small-animal PET was performed with [¹¹C]MC113 in FVB wild-type and Mdr1a/b^(-/-) mice (n = 3 per group) and in a mouse model of high (EMT6Ar1.0) and low (EMT6) Pgp expressing tumor grafts (n = 5). In the tumor model, PET scans were performed before and after administration of the reference Pgp inhibitor tariquidar (15 mg/kg).ResultsBrain uptake of [¹¹C]MC113, expressed as area under the time-activity curve from time 0 to 60 min (AUC_0-60), was moderately but not significantly increased in Mdr1a/b^(-/-) compared with wild-type mice (mean ± SD AUC_0-60, Mdr1a/b^(-/-): 88 ± 7 min, wild-type: 62 ± 6 min, P = 0.100, Mann Whitney test). In the tumor model, AUC_0-60 values were not significantly different between EMT6Ar1.0 and EMT6 tumors. Neither in brain nor in tumors was activity concentration significantly changed in response to tariquidar administration. Half-maximum effect concentrations (IC₅₀) for inhibition of Pgp-mediated rhodamine 123 efflux from CCRFvcr1000 cells were 375 ± 60 nM for MC113 versus 8.5 ± 2.5 nM for tariquidar.Conclusion[¹¹C]MC113 showed higher brain uptake in mice than previously described Pgp PET tracers, suggesting that [¹¹C]MC113 was only to a low extent effluxed by Pgp. However, [¹¹C]MC113 was found unsuitable to visualize Pgp expression levels presumably due to insufficiently high Pgp binding affinity of MC113 in relation to Pgp densities in brain and tumors.     

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.     

[11 C]Rhodamine-123: Synthesis and biodistribution in rodents

IntroductionRhodamine-123 is a known substrate for the efflux transporter, P-glycoprotein (P-gp). We wished to assess whether rhodamine-123 might serve as a useful substrate for developing probes for imaging efflux transporters in vivo with positron emission tomography (PET). For this purpose, we aimed to label rhodamine-123 with carbon-11 (t_1/2 = 20.4 min) and to study its biodistribution in rodents.Methods[¹¹ C]Rhodamine-123 was prepared by treating rhodamine-110 (desmethyl-rhodamine-123) with [¹¹ C]methyl iodide. The biodistribution of this radiotracer was studied with PET in wild-type mice and rats, in efflux transporter knockout mice, in wild-type rats pretreated with DCPQ (an inhibitor of P-gp) or with cimetidine (an inhibitor of organic cation transporters; OCT), and in P-gp knockout mice pretreated with cimetidine. Unchanged radiotracer in forebrain, plasma and peripheral tissues was also measured ex vivo at 30 min after radiotracer administration to wild-type and efflux transporter knockout rodents.Results[¹¹ C]Rhodamine-123 was obtained in 4.4% decay-corrected radiochemical yield from cyclotron-produced [¹¹ C]carbon dioxide. After intravenous administration of [¹¹ C]rhodamine-123 to wild-type rodents, PET and ex vivo measurements showed radioactivity uptake was very low in brain, but relatively high in some other organs such as heart, and especially liver and kidney. Inhibition of P-gp increased uptake in brain, heart, kidney and liver, but only by up to twofold. Secretion of radioactivity from kidney was markedly reduced by OCT knockout or pretreatment with cimetidine.Conclusions[¹¹ C]Rhodamine-123 was unpromising as a PET probe for P-gp function and appears to be a strong substrate of OCT in kidney. Cimetidine appears effective for blocking OCT in kidney in vivo.     

Preclinical and the first clinical studies on [11C]ITMM for mapping metabotropic glutamate receptor subtype 1 by positron emission tomography

IntroductionPreclinical studies and first positron emission tomography (PET) imaging studies were performed using N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-4-[¹¹C]methoxy-N-methylbenzamide ([¹¹C]ITMM) to map metabotropic glutamate receptor type 1 (mGluR1) in the human brain.Methods[¹¹C]ITMM was synthesized by O-methylation of the desmethyl precursor with [¹¹C]methyl triflate in the presence of NaOH at room temperature. In vitro selectivity and brain distributions of [¹¹C]ITMM in mice were characterized. Radiation absorbed-dose by [¹¹C]ITMM in humans was calculated from mouse distribution data. Acute toxicity of ITMM at 4.72 mg/kg body weight (> 74,000-fold clinical equivalent dose of [¹¹C]ITMM) was evaluated. Mutagenicity of ITMM was studied by the Ames test. Clinical PET imaging of mGluR1 with [¹¹C] ITMM was performed in a healthy volunteer.ResultsITMM had low activity for a 28-standard receptor binding profile. Regional brain uptake of [¹¹C]ITMM in mice was heterogeneous and consistent with known mGluR1 distributions. The radiation absorbed-dose by [¹¹C]ITMM in humans was sufficiently low for clinical use, and no acute toxicity or mutagenicity of ITMM occurred. A 90-min dynamic PET scan with [¹¹C]ITMM in a healthy volunteer showed a gradual increase of radioactivity in the cerebellum. Total distribution volume of [¹¹C]ITMM was highest in the cerebellum, followed by thalamus, cerebral cortex, and striatum; regional differences in brain radioactivity corresponded to the mGluR1 distribution in the brain. Peripherally, [¹¹C]ITMM was stable in humans: 60% of the plasma radioactivity remained in the unchanged form for 60 min.Conclusions[¹¹C] ITMM is a suitable radioligand for imaging mGluR1 in the human brain providing acceptable dosimetry and pharmacological safety at the dose required for PET.     

Synthesis and biological evaluation of [11C]SB366791: A new PET-radioligand for in vivo imaging of the TRPV1 receptor

IntroductionThe transient receptor potential vanilloid subfamily member 1 (TRPV1) receptor, a non-selective cation channel, is known for its key role in pain nociception and neurogenic inflammation. TRPV1 expression has been demonstrated in diverse tissues and an essential role for TRPV1 in various disorders has been suggested. A TRPV1-specific PET-radioligand can serve as a useful tool for further in vivo research in animals and directly in humans. In this study, we report the synthesis and biological evaluation of a carbon-11 labelled analogue of N-(3-methoxyphenyl)-4-chlorocinnamide (SB366791) which was reported as a specific high-affinity antagonist for TRPV1.MethodsThe new tracer was evaluated with respect to log D and biodistribution in control, pretreated and TRPV1^?/? mice. The percentage of radiometabolites of [¹¹C]SB366791 was determined in mouse plasma and brain.Results[¹¹C] SB366791 was obtained in good yield (69% ± 11%; isolated amounts 3034–5032 MBq) and high specific activity (390 ± 215 GBq/μmol). The tracer was efficiently cleared from blood and all major organs via hepatobiliary and renal pathways. Initial brain uptake was high (1.6% ID) and wash-out from brain was rapid. The retention of [¹¹C] SB366791 in the trigeminal nerve of control mice was prominent. The in vitro binding affinity of SB366791 was determined to be 280 ± 56 nM and 780 ± 140 nM for human and rat TRPV1, respectively.Conclusions[¹¹C] SB366791 has favourable biodistribution characteristics in mice. However the obtained low binding affinity for TRPV1 may not be sufficient to use the current compound as PET tracer.     

Palladium mediated 11C-cyanation and characterization in the non-human primate brain of the novel mGluR5 radioligand [11C]AZD9272

IntroductionThe aims of the present positron emission tomography (PET) study were to set up a system for ¹¹C-cyanation labeling of the selective mGluR5-antagonist [¹¹C]AZD9272 and to perform the first in vivo characterization of [¹¹C]AZD9272 binding in cynomolgus monkeys.Methods[¹¹C]AZD9272 was labeled using palladium mediated ¹¹C-cyanation. Altogether seven PET measurements were performed in three cynomolgus monkeys including baseline and co-injection experiments with unlabelled AZD9272 (0.04 and 0.4 mg/kg). Radiometabolites in plasma were measured using HPLC.Results[¹¹C]AZD9272 was prepared in over 50% incorporation yield from hydrogen [¹¹C]cyanide in a total synthesis time of 45–50 min. The radiochemical purity of the radioligand in its final formulation was high (> 99%) and the mean specific radioactivity was 47 GBq/ μmol (1278 Ci/mmol, n = 7) calculated at end of bombardment (EOB). In the baseline measurements 10% of the total injected radioactivity was present in monkey brain at five minutes after i.v. injection. The radioactivity concentration was high in the caudate, cingulate gyrus and thalamus whereas it was moderate in the temporal cortex and lower for the cerebellum. After co-injection with cold AZD9272 the binding of [¹¹C]AZD9272 was reduced in a dose-dependent fashion. Analysis of radiometabolites showed relatively slow metabolism and resulted only in hydrophilic radiometabolites.ConclusionA fast and efficient method was developed to label AZD9272 with ¹¹C. PET-examination in Cynomolgus monkeys showed that [¹¹C]AZD9272 entered the brain to a high extent, that binding was saturable and that the regional radioactivity pattern was in accordance with the known distribution of mGluR5. The results support further examination of [¹¹C]AZD9272 binding in human subjects.     

11C-Labeling of a potent hydroxyethylamine BACE-1 inhibitor and evaluation in vitro and in vivo

IntroductionThe enzyme β-secretase 1 (BACE-1) is associated with the catalytic cleavage of amyloid precursor protein (APP) which leads to the production of amyloid-β, an amyloidogenic peptide that forms insoluble fibrils and is linked to neurodegeneration and Alzheimer's disease (AD). A PET-radioligand for the quantification of BACE-1 would be useful for the understanding of AD. In this report, we describe the synthesis and carbon-11 radiolabeling of a potent hydroxyethylamine BACE-1 enzyme inhibitor (BSI-IV) and its evaluation in vitro and in vivo.Methods¹¹[C]-N¹-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-1-phenylbutan-2-yl)-5-(N-methylmethyl-sulfonamido)-N³-((R)-1-phenylethyl)isophthalamide, a β-secretase inhibitor, denoted here as [¹¹C]BSI-IV was synthesized through a palladium-mediated aminocarbonylation with an aryl halide precursor (I or Br) and [¹¹C]CO. The effect of different palladium/ligand-complexes on radiochemical yield in the carbonylative reaction was investigated. The binding of the labeled compound to BACE-1 enzyme was studied in vitro by frozen section autoradiography from brains of healthy rats. Dynamic small animal PET-CT studies and ex vivo biodistribution were performed in male rats.ResultsThe halide precursors were synthesized in six steps starting from methyl-3-nitrobenzoate with an overall yield of 21–26%. [¹¹C]BSI-IV was obtained in 29 ± 12% decay corrected radiochemical yield (n = 12) with a specific activity of 790 ± 155 GBq/μmol at the end of synthesis with a radiochemical purity of > 99%. The preclinical studies showed that [¹¹C]BSI-IV has a rapid metabolism in rat with excretion to the small intestines.Conclusion¹¹[C]BSI-IV was obtained in sufficient amount and purity to enable preclinical investigation. The preclinical studies showed low specific binding in vitro and fast clearance in vivo and a low uptake in the brain. These findings suggests that [¹¹C]BSI-IV has limited use as a PET-ligand for the study of BACE-1 or AD.     

Radiosynthesis and ex vivo evaluation of [11C-carbonyl]carbamate- and urea-based monoacylglycerol lipase inhibitors

IntroductionMonoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) are the two primary enzymes that regulate the tone of endocannabinoid signaling. Although new PET radiotracers have been discovered for imaging FAAH in vivo, no such radiotracer exists for imaging MAGL. Here we report the radiosynthesis of five candidate MAGL radiotracers and their ex vivo evaluations in mice and rats.MethodsCandidate carbamate and urea MAGL inhibitors were radiolabeled at the carbonyl position by [¹¹C]CO₂ fixation. Radiotracers were administered (tail-vein injection) to rodents and brain uptake of radioactivity measured at early and late time points ex vivo. Specificity of uptake was explored by pretreatment with unlabeled inhibitors (2 mg/kg, ip) 30 min prior to radiotracer administration.ResultsAll five candidate MAGL radiotracers were prepared in high specific activity (> 65 GBq/μmol) and radiochemical purity (> 98%). Moderate brain uptake (0.2–0.8 SUV) was observed for each candidate while pretreatment did not reduce uptake for four of the five tested. For two candidates ([¹¹C]12 and [¹¹C]14), high retention of radioactivity was observed in the blood (ca. 10 and 4 SUV at 40 min) which was blocked by pretreatment with unlabeled inhibitors. The most promising candidate, [¹¹C]18, demonstrated moderate brain uptake (ca. 0.8 SUV) which showed circa 50% blockade by pretreatment with unlabeled 18.ConclusionOne putative and four reported potent and selective MAGL inhibitors have been radiolabeled via [¹¹C]CO₂ fixation as radiotracers for this enzyme. Despite the promising in vitro pharmacological profile, none of the five candidate radiotracers exhibited in vivo behavior suitable for PET neuroimaging.