3p-C-NETA: A versatile and effective chelator for development of Al18F-labeled and therapeutic radiopharmaceuticals.

Background: Radiolabeled somatostatin analogues (e.g. [68Ga]Ga-DOTATATE and [177Lu]Lu-DOTATATE) have been used to diagnose, monitor, and treat neuroendocrine tumour (NET) patients with great success. [18F]AlF-NOTA-octreotide, a promising 18F-labeled somatostatin analogue and potential alternative for 68Ga-DOTA-peptides, is under clinical evaluation. However, ideally, the same precursor (combination of chelator-linker-vector) can be used for production of both diagnostic and therapeutic radiopharmaceuticals with very similar (e.g. Al18F-method in combination with therapeutic radiometals 213Bi/177Lu) or identical (e.g. complementary Tb-radionuclides) pharmacokinetic properties, allowing for accurate personalised dosimetry estimation and radionuclide therapy of NET patients. In this study we evaluated 3p-C-NETA, as potential theranostic Al18F-chelator and present first results of radiosynthesis and preclinical evaluation of [18F]AlF-3p-C-NETA-TATE. Methods: 3p-C-NETA was synthesized and radiolabeled with diagnostic (68Ga, Al18F) or therapeutic (177Lu, 161Tb, 213Bi, 225Ac and 67Cu) radionuclides at different temperatures (25-95 °C). The in vitro stability of the corresponding radiocomplexes was determined in phosphate-buffered saline (PBS) and human serum. 3p-C-NETA-TATE was synthesized using standard solid/liquid-phase peptide synthesis. [18F]AlF-3p-C-NETA-TATE was synthesized in an automated AllinOne® synthesis module and the in vitro stability of [18F]AlF-3p-C-NETA-TATE was evaluated in formulation buffer, PBS and human serum. [18F]AlF-3p-C-NETA-TATE pharmacokinetics were evaluated using µPET/MRI in healthy rats, with [18F]AlF-NOTA-Octreotide as benchmark. Results: 3p-C-NETA quantitatively sequestered 177Lu, 213Bi and 67Cu at 25 °C while heating was required to bind Al18F, 68Ga, 161Tb and 225Ac efficiently. The [18F]AlF-, [177Lu]Lu- and [161Tb]Tb-3p-C-NETA-complex showed excellent in vitro stability in both PBS and human serum over the study period. In contrast, [67Cu]Cu- and [225Ac]Ac-, [68Ga]Ga-3p-C-NETA were stable in PBS, but not in human serum. [18F]AlF-3p-C-NETA-TATE was obtained in good radiochemical yield and radiochemical purity. [18F]AlF-3p-C-NETA-TATE displayed good in vitro stability for 4 h in all tested conditions. Finally, [18F]AlF-3p-C-NETA-TATE showed excellent pharmacokinetic properties comparable with the results obtained for [18F]AlF-NOTA-Octreotide. Conclusions: 3p-C-NETA is a versatile chelator that can be used for both diagnostic applications (Al18F) and targeted radionuclide therapy (213Bi, 177Lu, 161Tb). It has the potential to be the new theranostic chelator of choice for clinical applications in nuclear medicine.

Preclinical evaluation of [18 F]MA3: a CB2 receptor agonist radiotracer for PET.

BACKGROUND AND PURPOSE: Non-invasive in vivo imaging of cannabinoid CB2 receptors using PET is pursued to study neuroinflammation. The purpose of this study is to evaluate the in vivo binding specificity of [18 F]MA3, a CB2 receptor agonist, in a rat model with local overexpression of human (h) CB2 receptors. METHODS: [18 F]MA3 was produced with good radiochemical yield and radiochemical purity. The radiotracer was evaluated in rats with local overexpression of hCB2 receptors and in a healthy non-human primate using PET. KEY RESULTS: Ex vivo autoradiography demonstrated CB2 -specific binding of [18 F]MA3 in rat hCB2 receptor vector injected striatum. In a PET study, increased tracer binding in the hCB2 receptor vector-injected striatum compared to the contralateral control vector-injected striatum was observed. Binding in hCB2 receptor vector-injected striatum was blocked with a structurally non-related CB2 receptor inverse agonist, and a displacement study confirmed the reversibility of tracer binding. This study identified the utility of mutated inactive vector model for evaluation of CB2 receptor agonist PET tracers. [18 F]MA3 PET scans in the non-human primate showed good uptake and fast washout from brain, but no CB2 receptor-specific binding was observed. CONCLUSION AND IMPLICATIONS: Evaluation of [18 F]MA3 in a rat model with local overexpression of hCB2 receptors showed CB2 receptor-specific and reversible tracer binding. [18 F]MA3 showed good brain uptake and subsequent washout in a healthy non-human primate, but no specific binding was observed. Further clinical evaluation of [18 F]MA3 in patients with neuroinflammation is warranted. LINKED ARTICLES: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Cerebral dopaminergic and glutamatergic transmission relate to different subjective responses of acute alcohol intake: an in vivo multimodal imaging study.

Converging preclinical evidence links extrastriatal dopamine release and glutamatergic transmission via the metabotropic glutamate receptor 5 (mGluR5) to the rewarding properties of alcohol. To date, human evidence is lacking on how and where in the brain these processes occur. Mesocorticolimbic dopamine release upon intravenous alcohol administration and mGluR5 availability were measured in 11 moderate social drinkers by single-session [18 F]fallypride and [18 F]FPEB positron emission tomography, respectively. Additionally, baseline and postalcohol glutamate and glutamine levels in the anterior cingulate cortex (ACC) were measured by using proton-magnetic resonance spectroscopy. To investigate differences in reward domains linked to both neurotransmitters, regional imaging data were related to subjective alcohol responses. Alcohol induced significant [18 F]fallypride displacement in the prefrontal cortex (PFC), temporal and parietal cortices and thalamus (P < 0.05, corrected for multiple comparisons). Dopamine release in the ACC and orbitofrontal and ventromedial PFCs were correlated with subjective 'liking' and 'wanting' effects (P < 0.05). In contrast, baseline mGluR5 availability was positively correlated with the 'high' effect of alcohol in dorsolateral, ventrolateral and ventromedial PFCs and in the medial temporal lobe, thalamus and caudate nucleus (P < 0.05). Although neither proton-magnetic resonance spectroscopy glutamate nor glutamine levels were affected by alcohol, baseline ACC glutamate levels were negatively associated with the alcohol 'liking' effect (P < 0.003). These data reveal new mechanistic understanding and differential neurobiological underpinnings of the effects of acute alcohol consumption on human behavior. Specifically, prefrontal dopamine release may encode alcohol 'liking' and 'wanting' effects in specific areas underlying value processing and motivation, whereas mGluR5 availability in distinct prefrontal-temporal-subcortical regions is more related to the alcohol 'high' effect.

New transient receptor potential vanilloid subfamily member 1 positron emission tomography radioligands: synthesis, radiolabeling, and preclinical evaluation.

The transient receptor potential vanilloid subfamily member 1 (TRPV1) cation channel is known to be involved in pain nociception and neurogenic inflammation, and accumulating evidence suggests that it plays an important role in several central nervous system (CNS)-related disorders. TRPV1-specific positron emission tomography (PET) radioligands can serve as powerful tools in TRPV1-related (pre)clinical research and drug design. We have synthesized several potent TRPV1 antagonists and accompanying precursors for radiolabeling with carbon-11 or fluorine-18. The cinnamic acid derivative [(11)C]DVV24 and the aminoquinazoline [(18)F]DVV54 were successfully synthesized, and their biological behavior was studied. In addition, the in vivo behavior of a (123)I-labeled analogue of iodo-resiniferatoxin (I-RTX), a well-known TRPV1 antagonist, was evaluated. The binding affinities of DVV24 and DVV54 for human TRPV1 were 163 ± 28 and 171 ± 48 nM, respectively. [(11)C]DVV24, but not [(18)F]DVV54 or (123)I-RTX, showed retention in the trigeminal nerve, known to abundantly express TRPV1. Nevertheless, it appears that ligands with higher binding affinities will be required to allow in vivo imaging of TRPV1 via PET.

Preclinical evaluation of 18F-JNJ41510417 as a radioligand for PET imaging of phosphodiesterase-10A in the brain.

UNLABELLED: Phosphodiesterases are enzymes that inactivate the intracellular second messengers 3',5'-cyclic adenosine-monophosphate and/or cyclic guanosine-monophosphate. Of all 11 known phosphodiesterase families, phosphodiesterase-10A (PDE10A) has the most restricted distribution, with high expression in the striatum. PDE10A inhibitors are pursued as drugs for treatment of neuropsychiatric disorders. We have synthesized and evaluated (18)F-JNJ41510417 as a selective and high-affinity radioligand for in vivo brain imaging of PDE10A using PET. METHODS: The biodistribution of (18)F-JNJ41510417 was evaluated in rats. Rat plasma and perfused brain homogenates were analyzed by high-performance liquid chromatography to quantify radiometabolites. Dynamic small-animal PET was performed in rats and in wild-type and PDE10A knock-out mice and compared with ex vivo autoradiography. Blocking and displacement experiments were performed using the nonradioactive analog and other selective PDE10A inhibitors. RESULTS: Tissue distribution studies showed predominant hepatobiliary excretion, sufficient brain uptake (0.56 ± 0.00 percentage injected dose at 2 min after tracer injection), and continuous accumulation of the tracer in the striatum over time; rapid washout of nonspecific binding from other brain regions was observed. Polar radiometabolites were detected in plasma and brain tissue. Dynamic small-animal PET showed continuous tracer accumulation in the striatum, with rapid decline in the cortex and cerebellum. Pretreatment and chase experiments with PDE10A inhibitors showed that the tracer binding to PDE10A was specific and reversible. Imaging in PDE10A knock-out and wild-type mice further confirmed that binding in the striatum was specific for PDE10A. CONCLUSION: Experiments in rats and PDE10A knock-out mice indicate that (18)F-JNJ41510417 binds specifically and reversibly to PDE10A in the striatum, suggesting that this new fluorinated quinoline derivative is a promising candidate for in vivo imaging of PDE10A using PET.

An in vivo [18F]MK-9470 microPET study of type 1 cannabinoid receptor binding in Wistar rats after chronic administration of valproate and levetiracetam.

There is substantial evidence that the endocannabinoid system and in particular the type 1 cannabinoid receptor (CB1R) is involved in epilepsy. We evaluated the in vivo effect of chronic administration of the anti-epileptic drugs valproate (VPA) and levetiracetam (LEV) on rat brain CB1 receptors using the positron emission tomography (PET) tracer [(18)F]MK-9470. Six Wistar rats were treated with VPA (200mg/kg) or LEV (50mg/kg) IP daily for 2 weeks. Dynamic imaging after intravenous injection of 18 MBq [(18)F]MK-9470 was performed on a FOCUS 220 microPET at baseline and after chronic treatment. Six animals were used as controls and were injected with saline, using the same protocol. Parametric images based on standardized uptake values (SUV) were generated and were spatially normalized to Paxinos space. These CB1R images were analyzed using a predefined volume of interest (VOI)-based analysis. Differences in SUV values between chronic and baseline scans in each condition (saline, VPA and LEV treatment) were calculated in each VOI. Direct binding affinity of the drugs at CB1R was assessed by competitive binding assay in Chinese hamster ovarian cells expressing human CB1R. Chronic injections of saline did not produce significant changes in global [(18)F]MK-9470 binding (p=0.43), nor in tracer binding in individual VOIs. We found a significant increase in global cerebral [(18)F]MK-9470 binding after chronic VPA administration compared to sham treated animals (+32.5%, p<0.001), as well as in tracer binding in all individual VOIs. After chronic administration of LEV, there was no significant change in global cerebral CB1R binding (+6.9%, p=0.81), nor in tracer binding in individual VOIs. As VPA does not exhibit high affinity for CB1R (displacement of [(3)H]-SR141716A 1.3+/-14.0%), such upregulation is most likely caused by an indirect effect on the endocannabinoid system. This increase in CB1R tracer binding and possibly signaling may represent a supplementary and new mechanism of VPA, but not LEV, since activation of CB1Rs has been shown to decrease excitability and excitotoxicity on-demand.

Non-invasive detection and quantification of acute myocardial infarction in rabbits using mono-[123I]iodohypericin microSPECT.

AIMS: Mono-[(123)I]iodohypericin ([(123)I]MIH) has been reported to have high avidity for necrosis. In the present study, by using rabbit models of acute myocardial infarction, we explored the suitability of [(123)I]MIH micro single photon emission computed tomography (microSPECT) for non-invasive visualization of myocardial infarcts in comparison with [(13)N]ammonia micro positron emission tomography (microPET) imaging, postmortem histomorphometry, and [(123)I]MIH autoradiography. METHODS AND RESULTS: Fourteen rabbits were divided into four groups. The left circumflex coronary artery was permanently occluded in group A (n = 3), reperfused by releasing the ligature after 15 min in group B (n = 3) or 90 min in group C (n = 6), or not occluded in group D (n = 2). Animals received [(13)N]ammonia microPET perfusion imaging 18 h after infarct induction followed by microSPECT imaging at 2-3.5, 9-11, and 22-24 h post injection (p.i.) of [(123)I]MIH. The cardiac images were assembled into polar maps for assessment of tracer uptake. Animals were sacrificed and the excised heart was sliced for autoradiography, triphenyl tetrazolium chloride, and haematoxylin-eosin staining. Using [(123)I]MIH microSPECT, infarcts were well delineated at 9 h p.i. Mean microSPECT infarct size was 38.8 and 32.7% of left ventricular area for groups A and C, respectively, whereas group B showed low uptake of [(123)I]MIH. Highest mean infarct/viable tissue activity ratio of 61/1 was obtained by autoradiography in group C animals at 24 h p.i. CONCLUSION: The study indicates the suitability of [(123)I]MIH for in vivo visualization of myocardial infarcts.

Synthesis and evaluation of a (99m)Tc-MAMA-propyl-thymidine complex as a potential probe for in vivo visualization of tumor cell proliferation with SPECT.

INTRODUCTION: Cytosolic thymidine kinase (TK1) catalyzes phosphorylation of thymidine to its monophosphate. TK1 activity is closely related with DNA synthesis, and thymidine analogs derivatized with bulky carboranylalkyl groups at the N-3 position were reported to be good substrates for TK1. Accordingly, we have synthesized (99m)Tc-MAMA-propyl-thymidine and evaluated it as a potential tumor tracer. METHODS: The bis(S-trityl)-protected MAMA-propyl-thymidine precursor (3-N-[S-trityl-2-mercaptoethyl]-N-[N'-(S-trityl-2-mercaptoethyl)amidoacetyl]-aminopropyl-thymidine) was prepared in three steps, and its structure was confirmed with (1)H NMR and mass spectrometry. Deprotection of the thiols and labeling with (99m)Tc were done in a two-step, one-pot procedure, yielding (99m)Tc-MAMA-propyl-thymidine, which was analyzed with high-performance liquid chromatography, radio-LC-MS analysis (ESI+) and electrophoresis, and its log P was determined. The biodistribution in normal mice was evaluated, and its biodistribution in a radiation-induced fibrosarcoma (RIF) tumor mouse was compared with that of 3'-deoxy-3'-[(18)F] fluorothymidine [(18)F]FLT. RESULTS: (99m)Tc-MAMA-propyl-thymidine was obtained with a radiochemical yield of 70%. Electrophoresis indicated that the complex is uncharged, and its log P was 1.0. The molecular ion mass of the Tc complex was 589 Da, which is compatible with the hypothesized N(2)S(2)-oxotechnetium structure. Tissue distribution showed fast clearance from plasma primarily by the hepatobiliary pathway. Whole-body planar imaging after injection of (99m)Tc-MAMA-propyl-thymidine in an RIF tumor-bearing mouse showed high uptake in the liver and the intestines. No uptake was observed in the tumor, in contrast to the clear uptake observed for [(18)F] FLT visualized with muPET. CONCLUSIONS: Although it has been reported that TK1 accepts large substituents at the N-3 position of the thymine ring, the results of this study show that (99m)Tc-MAMA-propyl-thymidine cannot be used as a single photon emission computed tomography tumor tracer, probably because the (99m)Tc-MAMA ligand is too bulky to be tolerated by TK1.

Evaluation of (99m)Tc-labeled tropanes with alkyl substituents on the 3beta-phenyl ring as potential dopamine transporter tracers.

Technetium(V)-oxo-3beta-(4-chlorophenyl)-8-methyl-8-azabicyclo[3.2.1]oct-2-yl[N-(2-mercaptoethyl), N-(N'-(2-mercaptoethyl)-2-aminoethyl)]-aminomethyl ((99m)Tc-TRODAT-1) and three derivatives with one or two substituents on the 3beta-phenyl ring (4-methylphenyl, 4-ethylphenyl and 2,4-dimethylphenyl) were prepared and evaluated as potential imaging agents for the central nervous dopamine transporter (DAT). Labeling of the ligands with (99m)Tc yielded for each of them a mixture of two radiolabeled species, which were purified and isolated using reversed-phase high-performance liquid chromatography. Employing radio-LC-MS, we found both species to have the same molecular mass suggesting diastereoisomers. After intravenous injection in mice and rats, the compounds were stable in vivo and no important metabolites were found in plasma or urine. Replacement of the 4-chloro atom on the 3beta-phenyl ring by a methyl group causes no loss of affinity for the DAT system. However, substitution of an ethyl group for the 4-chloro atom or introduction of a second methyl group in the 2-position of the phenyl ring results in a serious reduction of the affinity for the DAT transporter. Ex vivo autoradiography on mice brain slices and biodistribution studies in rats showed specific uptake of (99m)Tc-TRODAT-1 and the 4-methylphenyl derivative in striatum and putamen. Although the 4-ethylphenyl and 2,4-dimethylphenyl derivatives show brain uptake in rats and mice, no specific uptake in striatum was found. In addition, differences in biological behavior between the different diastereomers were observed. In conclusion, small changes to (99m)Tc-TRODAT-1 at the phenyl ring in the 3beta position of the tropane moiety significantly change the biological behavior of the studied compounds.

First preclinical evaluation of mono-[123I]iodohypericin as a necrosis-avid tracer agent.

PURPOSE: We have labelled hypericin, a polyphenolic polycyclic quinone found in St. John's wort (Hypericum perforatum), with( 123)I and evaluated mono-[(123)I]iodohypericin (MIH) as a potential necrosis-avid diagnostic tracer agent. METHODS: MIH was prepared by an electrophilic radioiodination method. The new tracer agent was evaluated in animal models of liver infarction in the rat and heart infarction in the rabbit using single-photon emission computed tomography (SPECT), triphenyltetrazolium chloride (TTC) histochemical staining, serial sectional autoradiography and microscopy, and radioactivity counting techniques. RESULTS: Using in vivo SPECT imaging, hepatic and cardiac infarctions were persistently visualised as well-defined hot spots over 48 h. Preferential uptake of the tracer agent in necrotic tissue was confirmed by perfect match of images from post-mortem TTC staining, autoradiography (ARX) and histology. Radioactivity concentration in infarcted tissues was over 10 times (liver; 3.51% ID/g in necrotic tissue vs 0.38% ID/g in normal tissue at 60 h p.i.) and over 6 times (myocardium; 0.36% ID/g in necrotic tissue vs 0.054% ID/g in normal tissue; ratios up to 18 for selected parts on ARX images) higher than in normal tissues. CONCLUSION: The results suggest that hypericin derivatives may serve as powerful necrosis-avid diagnostic agents for assessment of tissue viability.

Biological evaluation of a technetium-99m-labeled integrated tropane-BAT and its piperidine congener as potential dopamine transporter imaging agents.

INTRODUCTION: Recently, we have reported modification of (99m)Tc-TRODAT-1 by integrating the N2S2 metal chelating unit and the tropane skeleton. Results of a preliminary biodistribution study in rats were promising with respect to brain uptake. The present report deals with the further biological characterization of the (99m)Tc-labelled integrated TRODAT derivatives ((99m)Tc-TropaBAT and (99m)Tc-norchloro-TropaBAT) and with the synthesis and biological evaluation of a novel (99m)Tc-labelled piperidine-based derivative ((99m)Tc-PipBAT). METHODS: Biodistribution of all radiolabelled complexes was studied in normal mice. A more detailed ex vivo intracerebral distribution study of the two (99m)Tc-TropaBAT complexes was additionally performed in normal rats. Autoradiography of brain sections of normal mice (with or without pretreatment with FP-beta-CIT or haloperidol) and rats was performed. Affinity for the dopamine transporter (DAT) was also assessed in vitro in the presence or absence of cocaine. RESULTS: Both (99m)Tc-TropaBAT complexes show a slightly higher brain uptake than (99m)Tc-TRODAT-1, but the striatum/cerebellum activity ratio is less favourable. Nevertheless, significant striatal uptake was detected after ex vivo autoradiography, but this uptake was also observed after pretreatment with FP-beta-CIT. Unexpectedly, no striatal uptake was detected after in vitro incubation of mouse brain sections with the tracer agents. For (99m)Tc-PipBAT, neither brain uptake nor in vitro striatal uptake was found. CONCLUSION: Both (99m)Tc-TropaBAT complexes exhibit similar diffusion into brain as (99m)Tc-TRODAT-1, and ex vivo autoradiography shows significant striatal uptake. However, the inferior striatum/cerebellum activity ratio, the striatal uptake in mice pretreated with FP-beta-CIT or haloperidol, and the lack of striatal uptake during in vitro incubation prove that the DAT is not targeted. Brain uptake disappears when the tropane skeleton is replaced by a piperidine ring, and also in this case no striatal uptake is found in vitro.

Synthesis and biological evaluation of a technetium-99m(I)-tricarbonyl-labelled phenyltropane derivative.

A new tropane derivative was synthesized by combining a tridentate ligand, N-(2-picolylamine)-N-acetic acid (2-PAA), and a phenyltropane derivative. It was labelled with a [(99m)Tc(CO)(3)](+) moiety, resulting in the formation of two stable and neutral lipophilic isomers. Their identity was confirmed using radio-LC-MS. In normal mice, no brain uptake was observed for any of the isomers and in vitro autoradiography using mouse brain sections showed no specific uptake in the striatal area.