A gold-complex initiated functionalization of biologically active polyphenols applied to a 18F-labeled chemical probe.

(-)-Epigallocatechin gallate (EGCG), a key component of green tea, exerts therapeutic anticancer and antiallergic properties through its binding to the 67 kDa laminin receptor. The functionalization of EGCG is a promising strategy for creating new drug candidates and chemical probes. In our study, we developed a method for effectively modifying the A ring of EGCG through an electrophilic aromatic substitution with amidomethyl 2-alkynylbenzoates initiated with a gold complex. The 2-alkynylbenzoates treated with (Ph3P)AuOTf under neutral conditions yielded N-acylimines. A further electrophilic aromatic substitution resulted in a mixture of EGCG substituted with acylaminomethyl groups at the 6 and 8 positions with a significant amount noted at the 6 position. We then explored the synthesis of 18F-labeled EGCG with a neopentyl labeling group, an effective labeling group for radiohalogens of not only fluorine-18 but also of astatine-211. To achieve this, we prepared precursors that possessed acid-sensitive protecting groups and base-unstable leaving groups using our established method. Substitution of EGCG with a neopentyl labeling group at either the C6 or C8 position did not affect its anticancer efficacy in U266 cells. Finally, we investigated the preparation of 18F-labeled EGCG. The 18F-fluorination of a mixture of 6- and 8-substituted precursors yielded the corresponding 18F-labeled compounds in 4.5% and 3.0% radiochemical yields (RCYs), respectively. Under acidic conditions, the 18F-labeled 8-substituted compound produced 18F-labeled EGCG in 37% RCY, which heralds the potential of our functionalization approach.

Test-retest reproducibility of cerebral adenosine A2A receptor quantification using [11C]preladenant.

OBJECTIVE: To evaluate the reproducibility of cerebral adenosine A2A receptor (A2AR) quantification using [11C]preladenant ([11C]PLN) and PET in a test-retest study. METHODS: Eight healthy male volunteers were enrolled. Dynamic 90 min PET scans were performed twice at the same time of the day to avoid the effect of diurnal variation. Subjects refrained from caffeine from 12 h prior to scanning, and serum caffeine was measured before radioligand injection. Arterial blood was sampled repeatedly during scanning and the fraction of the parent compound in plasma was determined. Total distribution volume (VT) was estimated using 1- and 2-tissue compartment models (1-TCM and 2-TCM, respectively) and Logan graphical analysis (Logan plot) (t* = 30 min). Plasma-free fraction (fP) of [11C]PLN was measured and used for correction of VT values. Distribution volume ratio (DVR) was calculated from VT of target and reference regions and obtained by noninvasive Logan graphical reference tissue model (LGAR) (t* = 30 min). Absolute test-retest variability (aTRV), and intra-class correlation coefficient (ICC) of VT and DVR were calculated as indexes of repeatability. Correlation between DVR and serum concentration of caffeine (a nonselective A2AR blocker) was analyzed by Pearson's correlation analysis. RESULTS: Regional time-activity curves were well described by 2-TCM models. Estimation of VT by 2-TCM produced some erroneous values; therefore, the more robust Logan plot was selected as the appropriate model. Global mean aTRV was 20% for VT and 14% for VT/fP (ICC, 0.72 for VT and 0.87 for VT/fP). Global mean aTRV of DVR was 13% for Logan plot and 10% for LGAR (ICC, 0.70 for Logan plot and 0.81 for LGAR). DVR estimates using LGAR and Logan plot were in good agreement (r2 = 0.96). Coefficients of variation for VT, VT/fP, DVR (Logan plot), and DVR (LGAR) were 47%, 47%, 27%, and 18%, respectively. Despite low serum caffeine levels, significant concentration-dependent effects on [11C]PLN binding to target regions were observed (p < 0.01). CONCLUSIONS: In this study, moderate test-retest reproducibility and large inter-subject differences were observed with [11C]PLN PET, possibly attributable to competition by baseline amount of caffeine. Analysis of plasma caffeine concentration is recommended during [11C]PLN PET studies. TRIAL REGISTRATION: UMIN000030040.

Reversal of spatial memory impairment by phosphodiesterase 3 inhibitor cilostazol is associated with reduced neuroinflammation and increased cerebral glucose uptake in aged male mice.

The nucleotide second messenger 3', 5'-cyclic adenosine monophosphate (cAMP) and 3', 5'-cyclic guanosine monophosphate (cGMP) mediate fundamental functions of the brain, including learning and memory. Phosphodiesterase 3 (PDE3) can hydrolyze both cAMP and cGMP and appears to be involved in the regulation of their contents in cells. We previously demonstrated that long-term administration of cilostazol, a PDE3 inhibitor, maintained good memory performance in aging mice. Here, we report on studies aimed at determining whether cilostazol also reverses already-impaired memory in aged male mice. One month of oral 1.5% cilostazol administration in 22-month-old mice reversed age-related declines in hippocampus-dependent memory tasks, including the object recognition and the Morris water maze. Furthermore, cilostazol reduced neuroinflammation, as evidenced by immunohistochemical staining, and increased glucose uptake in the brain, as evidence by positron emission tomography (PET) with 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG). These results suggest that already-expressed memory impairment in aged male mice that depend on cyclic nucleotide signaling can be reversed by inhibition of PDE3. The reversal of age-related memory impairments may occur in the central nervous system, either through cilostazol-enhanced recall or strengthening of weak memories that otherwise may be resistant to recall.

Automated synthesis, preclinical toxicity, and radiation dosimetry of [18F]MC225 for clinical use: a tracer for measuring P-glycoprotein function at the blood-brain barrier.

INTRODUCTION: [18F]MC225 is a selective substrate for P-glycoprotein (P-gp) that has good metabolic stability and shows higher baseline uptake compared with other P-gp substrates such as (R)-[11C]Verapamil. Prior to clinical translation, it is necessary to perform process validation of the radiosynthesis, assessment of preclinical toxicity, and radiation dosimetry. METHODS: The production of [18F]MC225 was automated on a CFN-MPS200 multipurpose synthesizer. The acute toxicity of MC225 was evaluated at a dose of 2.5 mg/kg bodyweight, which is more than 10,000-fold the postulated maximum clinical dose of [18F]MC225. The acute toxicity of [18F]MC225 injection at a 200-fold dose, to administer a postulated dose of 185 MBq of [18F]MC225, was also evaluated after the decay-out of 18F. The mutagenicity of MC225 was studied by a reverse mutation test using Salmonella typhimurium and Escherichia coli (Ames test). In vivo biodistribution and dosimetry studies of [18F]MC225 were carried out in normal mice. Human dosimetry was estimated using OLINDA software. RESULTS: The mean decay-corrected yields of [18F]MC225 at end of synthesis were 13%, with > 99% radiochemical purity, > 1000 GBq/µmol molar activity, and ≤ 1.5 µg/185 MBq of total chemical contents. All process validation batches complied with the product specifications and the process was confirmed to be appropriate for the production of [18F]MC225. No acute toxicity of MC225 or [18F]MC225 injection was found. No mutagenic activity was observed for MC225. The biodistribution study demonstrated both hepatobiliary and renal excretion of radioactivity. The most critical organ was the pancreas, with (63.8 µGy/MBq) or without urination (63.9 µGy/MBq) at 360 min after injection. The estimated effective dose (µSv/MBq) with and without urination at 360 min after injection was calculated as 15.7 and 16.9, respectively. CONCLUSION: [18F]MC225 shows acceptable pharmacological safety at the dose required for adequate PET imaging. The potential risk associated with [18F]MC225 PET imaging is well within acceptable dose limits.

Radiosynthesis and preliminary evaluation of an 18 F-labeled tubastatin A analog for PET imaging of histone deacetylase 6.

Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family because of its characteristics, namely, its cytoplasmic localization and ubiquitin binding. HDAC6 has been implicated in cancer metastasis and neurodegeneration. In the present study, we performed radiosynthesis and biological evaluation of a fluorine-18-labeled ligand [18 F]3, which is an analog of the HDAC6-selective inhibitor tubastatin A, for positron emission tomography (PET) imaging. [18 F]3 was synthesized by a two-step reaction composed of 18 F-fluorination and formation of a hydroxamic acid group. IC50 values of 3 against HDAC1 and HDAC6 activities were 996 nM and 33.1 nM, respectively. A biodistribution study in mice demonstrated low brain uptake of [18 F]3. Furthermore, bone radioactivity was stable at around 2% ID/g after injection, suggesting high tolerance to defluorination. Regarding metabolic stability, 70% of the compound was observed as the unchanged form at 30 minutes post injection in mouse plasma. A small animal PET study in mice showed that pretreatment with cyclosporine A had no effect on initial brain uptake of [18 F]3, suggesting low brain uptake of [18 F]3 was not caused by the P-glycoprotein-mediated efflux. While PET imaging using [18 F]3 has a limitation with respect to neurodegenerative diseases, further studies evaluating its utility for certain cancers are worth evaluating.

Searching for diagnostic properties of novel fluorine-18-labeled D-allose.

OBJECTIVE: Two fluorine-18-labeled analogues, 3-deoxy-3-[18F]fluoro-D-allose (3-[18F]FDA) and 6-deoxy-6-[18F]fluoro-D-allose (6-[18F]FDA), were synthesized and their potentials of diagnostic property were characterized. METHODS: In vitro rat red blood cell (RBC) transport and phosphorylation by yeast hexokinase were evaluated in comparison with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG). The rate of protein binding in pooled human serum was measured by an ultrafiltration method. In vivo metabolite analysis in mice was also performed. Biodistribution, urine excretion, and in vivo renal kinetics in mice were compared with 2-deoxy-2-[18F]fluorosorbitol ([18F]FDS). RESULTS: Rat RBC uptake of 3- and 6-[18F]FDA (7.8 ± 2.5%ID and 10.2 ± 4.8%ID, respectively) was significantly lower than that of [18F]FDG (44.7 ± 8.7%ID). RBC uptake of 3-[18F]FDA was inhibited by D-glucose (30%) and cytochalasin B (40%), indicating the involvement of GLUT1-dependent transport. In contrast, 6-[18F]FDA transport was not inhibited by D-glucose and cytochalasin B. 3- and 6-[18F]FDA were not phosphorylated by yeast hexokinase under the conditions that result in 60% conversion of [18F]FDG into [18F]FDG-6-phosphate within 30 min. Serum protein binding of 3- and 6-[18F]FDA was negligible. Metabolic transformation of both tracers was not detected in plasma and urine at 30 min after injection. The highest tissue uptake of both tracers was observed in kidneys. Heart and brain uptake of both tracers was below blood levels throughout the biodistribution studies (until 120 min after injection). No significant uptake in the bone was observed, indicating the absence of de-fluorination in mice. In vivo PET imaging visualized rapid excretion of the administered 3- and 6-[18F]FDA from the kidneys, with minimal tracer accumulation in other organs. The urine excretion rate of 3-[18F]FDA was much lower than that of 6-[18F]FDA and [18F]FDS. CONCLUSIONS: 3- and 6-[18F]FDA might be unsatisfactory for tumor imaging. In contrast, these tracers demonstrated high levels of kidney uptake and excretion, low serum protein binding, and high metabolic stability as preferable properties for renal imaging. Notably, the urine excretion rate and kidney uptake kinetics of 6-[18F]FDA were comparable with those of the potential renal imaging agent [18F]FDS. Further validation studies in animal models are required to confirm the feasibility of 6-[18F]FDA as a functional renal imaging agent.

Monoamine Oxidase B Binding of 18F-THK5351 to Visualize Glioblastoma and Associated Gliosis: An Autopsy-Confirmed Case.

An 86-year-old woman with cognitive impairment and left hemiparesis underwent F-THK5351 PET 4 months before her death. In addition to "normal" off-target binding in the basal ganglia, abnormal accumulation was observed along the pyramidal tract and around the right basal ganglia as ring-shaped uptake that overlapped a gadolinium-enhanced lesion. Postmortem pathological examination revealed that she had glioblastoma multiforme with associated gliosis, in which monoamine oxidase B (MAO-B) activity is increased. In vitro autoradiography of the corresponding lesion demonstrated specific binding of F-THK5351, which was blocked by an MAO-B-selective ligand. Thus, F-THK5351 PET may reflect glioblastomas and associated gliosis by binding to MAO-B.

Characterization of the binding of tau imaging ligands to melanin-containing cells: putative off-target-binding site.

OBJECTIVE: Amyloid-ß plaques and neurofibrillary tangles composed of tau protein are the neuropathological hallmarks of Alzheimer's disease. In recent years, marked progress has been made in Alzheimer's disease research using tau ligands for positron emission tomography (PET). However, the issue of off-target binding, that is, the binding of ligands to regions without tau pathology, remains unresolved. Tissues with melanin-containing cells (MCCs) have been suggested as binding targets for tau ligands. In the present study, we characterized the MCC-binding properties of representative tau PET ligands. METHODS: Autoradiographic studies of [18F]AV-1451 and [18F]THK5351 were conducted using postmortem human midbrain sections. Saturation-binding assays of [18F]AV-1451 and [18F]THK5351 were performed with B16F10 melanoma cells. The blocking effects of 25 compounds against [18F]THK5351 binding to B16F10 cells were used to investigate the relationship between chemical structure and MCC binding. RESULTS: Autoradiography demonstrated specific binding of the radioligands in the substantia nigra. [18F]AV-1451 and [18F]THK5351 exhibited saturable binding to melanoma cells ([18F]AV-1451: Kd = 669 ± 196 nM, Bmax = 622 ± 269 pmol/mg protein; [18F]THK5351: Kd = 441 ± 126 nM, Bmax = 559 ± 75.5 pmol/mg protein). In blocking studies with melanoma cells, compounds bearing multiple aromatic rings and an aminopyridine group, including tau ligands such as AV-1451, PBB3, and a lead compound of MK-6240, exhibited the inhibition of [18F]THK5351 binding comparable to self-blocking by THK5351 (> 70% at 10 µM). CONCLUSIONS: These studies suggest that the binding properties of [18F]AV-1451 and [18F]THK5351 are sufficient to expect highlighting of tissues with a high density of MCCs. The findings of the present study should aid the development of neuroimaging ligands that do not bind to MCC.