Synthesis and Preclinical Evaluation of Fluorinated 5-Azaindoles as CB2 PET Radioligands.

Several classes of cannabinoid receptor type 2 radioligands have been evaluated for imaging of neuroinflammation, with successful clinical translation yet to take place. Here we describe the synthesis of fluorinated 5-azaindoles and pharmacological characterization and in vivo evaluation of 18F-radiolabeled analogues. [18F]2 (hCB2 Ki = 96.5 nM) and [18F]9 (hCB2 Ki = 7.7 nM) were prepared using Cu-mediated 18F-fluorination with non-decay-corrected radiochemical yields of 15 ± 6% and 18 ± 2% over 85 and 80 min, respectively, with high radiochemical purities (>97%) and molar activities (140-416 GBq/µmol). In PET imaging studies in rats, both [18F]2 and [18F]9 demonstrated specific binding in CB2-rich spleen after pretreatment with CB2-specific GW405833. Moreover, [18F]9 exhibited higher brain uptake at later time points in a murine model of neuroinflammation compared with a healthy control group. The results suggest further evaluation of azaindole based CB2 radioligands is warranted in other neuroinflammation models.

Utilizing PET and MALDI Imaging for Discovery of a Targeted Probe for Brain Endocannabinoid α/ß-Hydrolase Domain 6 (ABHD6).

Multimodal imaging provides rich biological information, which can be exploited to study drug activity, disease associated phenotypes, and pharmacological responses. Here we show discovery and validation of a new probe targeting the endocannabinoid α/ß-hydrolase domain 6 (ABHD6) enzyme by utilizing positron emission tomography (PET) and matrix-assisted laser desorption/ionization (MALDI) imaging. [18F]JZP-MA-11 as the first PET ligand for in vivo imaging of the ABHD6 is reported and specific uptake in ABHD6-rich peripheral tissues and major brain regions was demonstrated using PET. A proof-of-concept study in nonhuman primate confirmed brain uptake. In vivo pharmacological response upon ABHD6 inhibition was observed by MALDI imaging. These synergistic imaging efforts used to identify biological information cannot be obtained by a single imaging modality and hold promise for improving the understanding of ABHD6-mediated endocannabinoid metabolism in peripheral and central nervous system disorders.

A binge high sucrose diet provokes systemic and cerebral inflammation in rats without inducing obesity.

While the dire cardiometabolic consequences of the hypercaloric modern 'Western' diet are well known, there is not much information on the health impact of a high sucrose diet not inducing weight gain. Here, we tested the hypothesis that rats reared with intermittent binge access to sucrose in addition to normal chow would develop an inflammatory response in brain. To test this hypothesis, we undertook serial PET/MRI scans with the TSPO ligand [18F]DPA714 in a group of (n=9) rats at baseline and again after voluntarily consuming 5% sucrose solution three days a week for three months. Compared to a control group fed with normal chow (n=9), the sucrose rats indeed showed widespread increases in the availability of cerebral binding sites for the microglial marker, despite normal weight gain compared to the control diet group. Subsequent immunofluorescence staining of the brains confirmed the PET findings, showing a widespread 20% increase in the abundance of IBA-1-positive microglia with characteristic 'semi-activated' morphology in the binge sucrose rats, which had 23% lower density of microglial endpoints and 25% lower mean process length compared to microglia in the control rats with ordinary feeding. GFAP immunofluorescence showed no difference in astroglial coverage in the sucrose rats, except for a slight reduction in hypothalamus. The binge sucrose diet-induced neuroinflammation was associated with a significant elevation of white blood cell counts. Taking these results together, we find that long-term intake of sucrose in a binge paradigm, similar in sucrose content to the contemporary Western diet, triggered a low-grade systemic and central inflammation in non-obese rats. The molecular mechanism of this phenomenon remains to be established.

GABAa receptor density alterations revealed in a mouse model of early moderate prenatal ethanol exposure using [18F]AH114726.

INTRODUCTION: Prenatal ethanol exposure (PEE) has been shown to alter the level and function of receptors in the brain, one of which is GABAa receptors (GABAaR), the major inhibitory ligand gated ion channels that mediate neuronal inhibition. High dose PEE in animals resulted in the upregulation of GABAaR, but the effects of low and moderate dose PEE at early gestation have not been investigated. This study aimed at examining GABAaR density in the adult mouse brain following PEE during a period equivalent to the first 3 to 4 weeks in human gestation. It was hypothesized that early moderate PEE would cause alterations in brain GABAaR levels in the adult offspring. METHODS: C57BL/6J mice were given 10% v/v ethanol during the first 8 gestational days. Male offspring were studied using in-vivo Positron Emission Tomography (PET)/Magnetic Resonance Imaging (MRI), biodistribution, in-vitro autoradiography using [18F]AH114726, a novel flumazenil analogue with a high affinity for the benzodiazepine-binding site, and validated using immunohistochemistry. RESULTS: In vivo PET and biodistribution did not detect alteration in brain tracer uptake. In vitro radiotracer studies detected significantly reduced GABAaR in the olfactory bulbs. Immunohistochemistry detected reduced GABAaR in the cerebral cortex, cerebellum and hippocampus, while Nissl staining showed that cell density was significantly higher in the striatum following PEE. CONCLUSION: Early moderate PEE may induce long-term alterations in the GABAaR system that persisted into adulthood.

Synthesis and evaluation of 6-[18F]fluoro-3-(pyridin-3-yl)-1H-indole as potential PET tracer for targeting tryptophane 2, 3-dioxygenase (TDO).

INTRODUCTION: The increase in expression of tryptophan 2, 3-dioxygenases (TDO) and indoleamine 2,3-dioxygenase (IDO) have been reported as potential tumor biomarkers. TDO and IDO are enzymes that catalyze the first and rate-limiting step of the kynurenine pathway. Positron emitting tomography (PET) tracers investigating the kynurenine pathway may allow for the detection of different disease pathologies in vivo including cancer. However, current PET tracers being developed for TDO and IDO have suffered from either multi-step low yielding syntheses or de-fluorination of the tracer in vivo. RESULTS: TDO inhibitors based on 6-fluoroindole with C3 substituents are a class of small molecules that have been shown to bind to TDO effectively, restore tryptophan concentration and decrease the production of immunosuppressive metabolites. The compound 6-fluoro-3-(pyridine-3-yl)-1H-indole has been reported to have high in vitro affinity for TDO. Herein we report the fully automated radiosynthesis of 6-[18F]fluoro-3-(pyridine-3-yl)-1H-indole [18F]4 using a copper-mediated nucleophilic 18F-fluorination resulting in a non-corrected yield of 5 to 6% of the tracer with a radiochemical purity of >99% after 4 h. Small animal dynamic PET/CT imaging of [18F]4 intravenously injected into normal C57BL/6 mice revealed rapid accumulation in heart and brain, reaching maximum occupancy in heart (10.9% ID/g) and brain (8.1% ID/g) at 1.75 min and 2.25 min, respectively. Furthermore, these in vivo studies revealed no de-fluorination of the tracer, as evidence by the absence of [18F]fluoride accumulation in bone. CONCLUSION: In vitro studies demonstrate that 4 has good affinity for hTDO and the radiolabeled analogue [18F]4 can be synthesized with suitable radiochemical yields. [18F]4 demonstrates good uptake in the brain and the radiolabeled compound shows no de-fluorination in vivo in C57BL/6 mice.

Investigation on the impact of three different quaternary methyl ammonium cartridges on the radiosynthetic yields of [18 F]fluoromethyl tosylate.

Our recent investigations for the radiosynthesis of [18 F]fluoromethyl tosylate have highlighted that choice of quaternary methyl ammonium (QMA) cartridge used during the radiosynthesis can significantly impact the radiochemical yields. Often the details of the QMA cartridge used in fluourine-18 syntheses are not fully described. However, our studies demonstrate that the type, the size, and nature (method by which it has been conditioned) of the QMA cartridge used during the radiosynthesis can make a significant impact in the labelling efficiency. This paper investigates the use of three QMA cartridges and demonstrates that radiochemical yield (decay corrected) of [18 F]fluoromethyl tosylate can increase from 46% to 60% by simply changing the QMA cartridge (and leaving all other reagents and labelling conditions exactly the same). These learnings may be applied to improve the radiochemical yields of a number of [18 F]-fluorinated tracers (and synthons), where the labelling step is base-sensitive to increase the radiochemical yield, thereby significantly benefiting the radiochemistry and nuclear medicine community. This paper also highlights the necessity of the radiochemistry community to ensure the details of QMA cartridges used in fluorine-18 chemistry are fully and accurately described, since this will improve the translation of radiochemical methods from one laboratory to another.

Synthesis and in vitro evaluation of fluorine-18 benzimidazole sulfones as CB2 PET-radioligands.

Cannabinoid type 2 receptor (CB2) is up-regulated on activated microglial cells and can potentially be used as a biomarker for PET-imaging of neuroinflammation. In this study the synthesis and pharmacological evaluation of novel fluorinated pyridyl and ethyl sulfone analogues of 2-(tert-butyl)-5-((2-fluoropyridin-4-yl)sulfonyl)-1-(2-methylpentyl)-1H-benzo[d]imidazole (rac-1a) are described. In general, the ligands showed low nanomolar potency (CB2 EC50 < 10 nM) and excellent selectivity over the CB1 subtype (>10 000×). Selected ligands 1d, 1e, 1g and 3l showing high CB2 binding affinity (Ki < 10 nM) were radiolabelled with fluorine-18 from chloropyridyl and alkyl tosylate precursors with good to high isolated radioactive yields (25-44%, non-decay corrected, at the end of synthesis). CB2-specific binding of the radioligand candidates [18F]-1d and [18F]-3l was assessed on rat spleen cryosections using in vitro autoradiography. The results warrant further in vivo evaluation of the tracer candidates as prospective CB2 PET-imaging agents.

Synthesis of 18 F-radiolabeled diphenyl gallium dithiosemicarbazone using a novel halogen exchange method and in vivo biodistribution.

18 F-radiolabeled diphenyl gallium thiosemicarbazone was prepared by [18 F] fluoride exchange of a nitrato anion under mild conditions. The diphenyl gallium thiosemicarbazone chloride is easily prepared in gram quantities and can be used at room temperature in the presence of oxygen. The corresponding nitrate complex is prepared using silver nitrate in methanol solvent and can be stored under nitrogen for weeks before radiolabeling. The biodistribution of this new tracer was studied in mice using positron emission tomography (PET).

N-(4-[18F]fluorobenzyl)cholylglycine, a novel tracer for PET of enterohepatic circulation of bile acids: Radiosynthesis and proof-of-concept studies in rats.

INTRODUCTION: Enterohepatic circulation (EHC) of conjugated bile acids is an important physiological process crucial for regulation of intracellular concentrations of bile acids and their function as detergents and signal carriers. Only few bile acid-derived imaging agents have been synthesized and hitherto none have been evaluated for studies of EHC. We hypothesized that N-(4-[18F]fluorobenzyl)cholylglycine ([18F]FBCGly), a novel fluorine-18 labeled derivative of endogenous cholylglycine, would be a suitable tracer for PET of the EHC of conjugated bile acids, and we report here a radiosynthesis of [18F]FBCGly and a proof-of-concept study by PET/MR in rats. METHODS: A radiosynthesis of [18F]FBCGly was developed based on reductive alkylation of glycine with 4-[18F]fluorobenzaldehyde followed by coupling to cholic acid. [18F]FBCGly was investigated in vivo by dynamic PET/MR in anesthetized rats; untreated or treated with cholyltaurine or rifampicin. Possible in vivo metabolites of [18F]FBCGly were investigated by analysis of blood and bile samples, and the stability of [18F]FBCGly towards enzymatic de-conjugation by Cholylglycine Hydrolase was tested in vitro. RESULTS: [18F]FBCGly was produced with a radiochemical purity of 96% ±â€¯1% and a non-decay corrected radiochemical yield of 1.0% ±â€¯0.3% (mean ±â€¯SD; n = 12). PET/MR studies showed that i.v.-administrated [18F]FBCGly underwent EHC within 40-60 min with a rapid transhepatic transport from blood to bile. In untreated rats, the radioactivity concentration of [18F]FBCGly was approximately 15 times higher in bile than in liver tissue. Cholyltaurine and rifampicin inhibited the biliary secretion of [18F]FBCGly. No fluorine-18 metabolites of [18F]FBCGly were observed. CONCLUSION: We have developed a radiosynthesis of a novel fluorine-18 labeled bile acid derivative, [18F]FBCGly, and shown by PET/MR that [18F]FBCGly undergoes continuous EHC in rats without metabolizing. This novel tracer may prove useful in PET studies on the effect of drugs or diseases on the EHC of conjugated bile acids.