Masakatsu Kanazawa

Novel PET Probes 18F-BCPP-EF and 18F-BCPP-BF for Mitochondrial Complex I: A PET Study in Comparison with 18F-BMS-747158-02 in Rat Brain

We developed novel PET probes, 2-tert-butyl-4-chloro-5-{6-[2-(2-18F-fluoroethoxy)-ethoxy]-pyridin-3-ylmethoxy}-2H-pyridazin-3-one (18F-BCPP-EF) and 2-tert-butyl-4-chloro-5-[6-(4-18F-fluorobutoxy)-pyridin-3-ylmethoxy]-2H-pyridazin-3-one (18F-BCPP-BF), for quantitative imaging of mitochondrial complex I (MC-I) activity in the brain and preliminarily evaluated their properties in comparison with 18F-BMS-747158-02 (18F-BMS). Methods: The affinity of 18F-BCPP-EF, 18F-BCPP-BF, and 18F-BMS to MC-I was analyzed using in vitro binding assays with 3H-dihydrorotenone and bovine cardiomyocyte submitochondrial particles. 18F-BCPP-EF, 18F-BCPP-BF, or 18F-BMS was intravenously injected into rats, and the uptake (standardized uptake value) in each organ was determined by dissection method. The effects of rotenone, a specific MC-I inhibitor, on the uptake of each probe were assessed by whole-body PET imaging in rats. Ischemic brain model rats were imaged using 18F-BCPP-EF. Results: The rank order of affinity to MC-I was 18F-BCPP-BF > 18F-BMS > 18F-BCPP-EF. The uptake of 18F-BCPP-EF and 18F-BMS was high in the heart, intermediate in brain, and low in muscle and bone 60 min after the injection. 18F-BCPP-BF provided increasing bone uptake with time after the injection. The uptake of 18F-BCPP-EF and 18F-BMS into the brain and heart was significantly decreased by preadministration of rotenone; however, the reduction degree of 18F-BCPP-EF was more pronounced than that of 18F-BMS. Rotenone did not affect 18F-BCPP-BF uptake in either the brain or the heart. 18F-BCPP-EF imaged the cortical ischemic neuronal damage without any disturbance by microglial activation even on day 7 when 18F-FDG showed high uptake in the damaged area. Conclusion: The present study demonstrated that 18F-BCPP-EF could be a potential PET probe for quantitative imaging of MC-I activity and its ischemic damage in the living brain with PET.

Development of novel PET probes, [18F]BCPP-EF, [18F]BCPP-BF, and [11C]BCPP-EM for mitochondrial complex 1 imaging in the living brain†

We developed three novel positron-emission tomography (PET) probes, 2-tert-butyl-4-chloro-5-{6-[2-(2[(18)F]fluoroethoxy)-ethoxy]-pyridin-3-ylmethoxy}-2H-pyridazin-3-one ([(18)F]BCPP-EF), 2-tert-butyl-4-chloro-5-[6-(4-[(18) F]fluorobutoxy)-pyridin-3-ylmethoxy]-2H-pyridazin-3-one ([(18)F]BCPP-BF), and 2-tert-butyl-4-chloro-5-{6-[2-(2-[(11)C]methoxy-ethoxy)-ethoxy]-pyridin-3-ylmethoxy}-2H-pyridazin-3-one ([(11)C]BCPP-EM), for quantitative imaging of mitochondrial complex 1 (MC-1) activity in vivo. These three PET probes were successfully labeled by nucleophilic [(18)F]fluorination or by [(11)C]methylation of their corresponding precursor with sufficient radioactivity yield, good radiochemical purity, and sufficiently high specific radioactivity for PET measurement. The specificity of these probes for binding to MC-1 was assessed with rotenone, a specific MC-1 inhibitor, by a rat brain slice imaging method in vitro. Rat whole-body imaging by small-animal PET demonstrated that all probes showed high uptake levels in the brain as well as in the heart sufficient to image them clearly. The rank order of uptake levels in the brain and the heart just after injection was as follows: high in [(18)F]BCPP-BF, intermediate in [(11)C]BCPP-EM, and low in [(18) F]BCPP-EF. The kinetics of [(18)F]BCPP-EF and [(11)C]BCPP-EM provided a reversible binding pattern, whereas [(18)F]BCPP-BF showed nonreversible accumulation-type kinetics in the brain and heart. Metabolite analyses indicated that these three compounds were rapidly metabolized in the plasma but relatively stable in the rat brain up to 60 min post-injection. The present study demonstrated that [(18)F]BCPP-EF could be a useful PET probe for quantitative imaging of MC-1 activity in the living brain by PET.

PET imaging of ischemia-induced impairment of mitochondrial complex I function in monkey brain

To assess the capability of 18F-2-tert-butyl-4-chloro-5-{6-[2-(2-fluoroethoxy)-ethoxy]-pyridin-3-ylmethoxy}-2H-pyridazin-3-one (18F-BCPP-EF), a novel positron emission tomography (PET) probe for mitochondrial complex I (MC-I) activity, as a specific marker of ischemia-induced neuronal death without being disturbed by inflammation, translational research was conducted using an animal PET in ischemic brains of Cynomolgus monkeys (Macaca fascicularis). Focal ischemia was induced by the right middle cerebral artery occlusion for 3 hours, then PET scans were conducted at Day-7 with 15O-gases for regional cerebral blood flow (rCBF) and regional cerebral metabolism of oxygen (rCMRO2), and 18F-BCPP-EF for MC-I with arterial blood sampling. On Day-8, the additional PET scans conducted with 11C-flumazenil (11C-FMZ) for central-type benzodiazepine receptors, 11C-PBR28 for translocator protein, and 18F-fluoro-2-deoxy-D-glucose (18F-FDG) for regional cerebral metabolic rate of glucose (rCMRglc). The total distribution volume (VT) values of 18F-BCPP-EF showed the significant reduction in MC-I activity in the damaged area at Day-7. When correlated with rCBF and rCMRO2, the VT values of 18F-BCPP-EF provided better correlation with rCMRO2 than with rCBF. In the inflammatory regions (region of interest, ROIPBR) of the ischemic hemisphere detected with 11C-PBR28, higher 18F-FDG uptake and lower VT of 18F-BCPP-EF, 11C-FMZ, and rCMRO2 than those in normal contralateral hemisphere were observed. These results strongly suggested that 18F-BCPP-EF could discriminate the neuronal damaged areas with neuroinflammation, where 18F-FDG could not owing to its high uptake into the activated microglia.

Comparing amyloid-β deposition, neuroinflammation, glucose metabolism, and mitochondrial complex I activity in brain: a PET study in aged monkeys.

PurposeThe aim of the present study was to compare amyloid-β (Aβ) deposition, translocator protein (TSPO) activity, regional cerebral metabolic rate of glucose (rCMRglc), and mitochondrial complex I (MC-I) activity in the brain of aged monkeys.MethodsPET scans with 11C-PIB (Aβ), 18F-BCPP-EF (MC-I), 11C-DPA-713 (TSPO), and 18F-FDG (rCMRglc) were performed in aged monkeys (Macaca mulatta) in the conscious state and under isoflurane anaesthesia. 11C-PIB binding to Aβ and 11C-DPA-713 binding to TSPO were evaluated in terms of standard uptake values (SUV). The total volume of distribution (VT) of 18F-BCPP-EF and rCMRglc with 18F-FDG were calculated using arterial blood sampling.ResultsIsoflurane did not affect MC-I activity measured in terms of 18F-BCPP-EF uptake in living brain. There was a significant negative correlation between 18F-BCPP-EF binding (VT) and 11C-PIB uptake (SUVR), and there was a significant positive correlation between 11C-DPA-713 uptake (SUV) and 11C-PIB uptake. In contrast, there was no significant correlation between rCMRglc ratio and 11C-PIB uptake.Conclusion18F-BCPP-EF could be a potential PET probe for quantitative imaging of impaired MC-I activity that is correlated with Aβ deposition in the living brain.

PET Imaging of Mitochondrial Complex I with 18F-BCPP-EF in the Brains of MPTP-Treated Monkeys

18F-BCPP-EF was applied to assess mitochondrial complex I (MC-I) activity in the brains of Parkinson disease model monkeys prepared by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and also presynaptic dopamine parameters. Methods: 11C-β-CFT for the dopamine transporter; 11C-3,4-dihydroxy-phenyl-L-alanine (β-11C-L-DOPA), L-6-18F-fluorodopa (18F-FDOPA), or 6-11C-methyl-m-tyrosine (11C-6MemTyr) for dopamine synthesis; or 2-tert-butyl-4-chrolo-5-{6-[2-(2-18F-fluoroethoxy)-ethoxy]-pyridin-3-ylmethoxy}-2H-pyridazin-3-one (18F-BCPP-EF) for MC-I was intravenously injected into normal and MPTP monkeys in order to analyze their uptake in the striatum. Results: Significant reductions in presynaptic dopamine parameters and MC-I activity were detected in the striatum of MPTP monkeys. Correlations were observed between MC-I activity and dopamine transporter as well as between MC-I activity and dopamine synthesis in the striatum. The order of detectability of impaired MC-I activity was 11C-6MemTyr >> β-11C-L-DOPA > 18F-FDOPA. Conclusion: 18F-BCPP-EF has potential as a PET probe for the quantitative imaging of MC-I damage in the living brains of Parkinson disease model monkeys using PET.

Evaluation of 6-11C-Methyl-m-Tyrosine as a PET Probe for Presynaptic Dopaminergic Activity: A Comparison PET Study with β-11C-l-DOPA and 18F-FDOPA in Parkinson Disease Monkeys

We recently developed a novel PET probe, 6-11C-methyl-m-tyrosine (11C-6MemTyr), for quantitative imaging of presynaptic dopamine synthesis in the living brain. In the present study, 11C-6MemTyr was compared with β-11C-l-DOPA and 6-18F-fluoro-l-dopa (18F-FDOPA) in the brains of normal and Parkinson disease (PD) model monkeys (Macaca fascicularis). Methods: PD model monkeys were prepared by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, and 11C-β-CFT was applied to assess neuronal damage as dopamine transporter (DAT) availability. 11C-6MemTyr, β-11C-l-DOPA, or 18F-FDOPA was injected with and without carbidopa, a specific inhibitor of peripheral aromatic L-amino acid decarboxylase. In normal and PD monkeys, the dopamine synthesis rates calculated using PET probes were analyzed by the correlation plot with DAT availability in the striatum. Results: In normal monkeys, whole-brain uptake of β-11C-l-DOPA and 18F-FDOPA were significantly increased by carbidopa at the clinical dose of 5 mg/kg by mouth. In contrast, 11C-6MemTyr was not affected by carbidopa at this dose, and the metabolic constant value of 11C-6MemTyr in the striatum was significantly higher than those of the other 2 PET probes. Significant reduction of the presynaptic DAT availability in the striatum was detected in MPTP monkeys, and correlation analyses demonstrated that 11C-6MemTyr could detect dopaminergic damage in the striatum with much more sensitivity than the other PET probes. Conclusion: 11C-6MemTyr is a potential PET probe for quantitative imaging of presynaptic dopamine activity in the living brain with PET.

Effect of MPTP on Serotonergic Neuronal Systems and Mitochondrial Complex I Activity in the Living Brain: A PET Study on Conscious Rhesus Monkeys

The objective of the present PET study was to compare the effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on serotonergic neuronal systems and mitochondrial complex I (MC-I) activity with that of dopamine in conscious rhesus monkeys (Macaca mulatta). Methods: A Parkinson disease monkey model was prepared by repeated administration of MPTP. For the PET measurements, normal and MPTP-treated conscious monkeys received an intravenous injection of 11C-DASB for serotonin transporter, 18F-MPPF for serotonin 1A receptor, 11C-PE2I for dopamine transporter, 11C-6MemTyr for dopamine synthesis, 11C-raclopride for dopamine D2 receptor, or 18F-BCPP-EF for MC-I. Serotonin and dopamine parameters were calculated using time–activity curves in the cerebellum as the input function. The total distribution volume of 18F-BCPP-EF was assessed using Logan plot graphical analysis with metabolite-corrected plasma as the input function. Results: MPTP-induced diffuse reductions in MC-I activity were observed throughout the brain, except the cerebellum. Significant reductions in the presynaptic dopamine parameters—dopamine transporter and dopamine synthesis—were detected in the striatum and substantia nigra pars compacta of MPTP-treated monkeys, whereas no significant differences in postsynaptic dopamine D2 receptor binding were observed. Serotonin transporter binding was reduced by MPTP not only in striatal regions but also in extrastriatal regions. In contrast, serotonin 1A receptor binding was unaffected by MPTP anywhere in the brain. In the cortex, the reduction of serotonin transporter binding correlated with that of MC-I. Conclusion: The results obtained by multiparametric PET measurements in a Parkinson disease monkey model demonstrated that chronic MPTP treatment induced reductions not only in the dopaminergic system in the nigrostriatal pathway but also in serotonin transporter in the cortical and subcortical regions. These results suggest that the neurotoxicity of MPTP is not exclusive to the nigrostriatal pathway, as predicted from MC-I damage in the extrastriatal regions of the brain.

Comparing α7 nicotinic acetylcholine receptor binding, amyloid‐β deposition, and mitochondria complex‐I function in living brain: A PET study in aged monkeys

This study was aimed to assess the correlations among α7 nicotinic acetylcholine receptor (α7‐nAChR) binding, amyloid‐β (Aβ) deposition, and mitochondrial complex I (MC‐I) activity in the brain of aged monkeys (Macaca mulatta). Positron emission tomography (PET) measurements with [11C](R)‐MeQAA, [11C]PIB, and [18F]BCPP‐EF were conducted in monkeys in a conscious condition. [11C](R)‐MeQAA binding was analyzed by a simplified reference tissue model to calculate nondisplaceable binding potential (BPND), [11C]PIB uptake was calculated by standard uptake value ratio (SUVR), and [18F]BCPP‐EF binding was determined by Logan graphical analysis to calculate total distribution volume (VT) with arterial blood sampling. Higher brain uptake was determined in the thalamus, hippocampus, striatum, and cortical regions for [11C](R)‐MeQAA, while being lower in the cerebellum. Significant age‐related reduction of [11C](R)‐MeQAA binding to α7‐nAChR was determined only in the occipital cortex. The plot of Vt of [18F]BCPP‐EF against BPND of [11C](R)‐MeQAA indicated a significant negative correlation in the hippocampus and cortical regions in aged animals. Plotting of SUVR of [11C]PIB against BPND of [11C](R)‐MeQAA showed a positive correlation. The in vivo binding of [11C](R)‐MeQAA could reflect the upregulation of α7‐nAChR induced by neurodegenerative damage determined by Aβ deposition as well as impaired MC‐I activity in living brain. Synapse 69:475–483, 2015.

Synthesis of 6-[11C]methyl-m-tyrosine ([11C]6MemTyr) for dopamine synthesis imaging in living brain using PET

A novel PET probe, 6-[(11)C]methyl-m-tyrosine ([(11)C]6MemTyr), was developed for quantitative imaging of presynaptic dopamine (DA) synthesis in the living brain using positron emission tomography (PET). This probe was evaluated by comparison with conventional 6-[(18)F]fluoro-l-dopa ([(18)F]FDOPA). [(11)C]6MemTyr was labeled using rapid Pd(0)-mediated C-[(11)C]methylation with [(11)C]methyl iodide. The synthesis time was only 35min, and its radiochemical yield was 76%, with radiochemical purity of >99%. PET measurements indicated that [(11)C]6MemTyr could image presynaptic DA synthesis in the striatum of living monkey brain, providing much higher contrast between the striatum and the cerebellum than that with [(18)F]FDOPA.

Monitoring Mitochondrial Complex-I Activity Using Novel PET Probe 18F-BCPP-EF Allows Early Detection of Radiotherapy Effect in Murine Squamous Cell Carcinoma

Objectives Aerobic glycolysis, the main pathway of energy production in tumors (Warburg effect) allows detection of tumors by positron emission tomography (PET) using 18F-fluoro-2-deoxy-D-glucose (18F-FDG). Since ionizing radiation (IR) is reported to switch aerobic glycolysis to mitochondrial oxidative phosphorylation, radiotherapeutic efficacy was monitored by the activity of mitochondrial complex I (MC-I), using a new PET probe 18F-BCPP-EF, 18F-2-tert-butyl-4-chloro-5-{6-[2-(2-fluoro-ethoxy)-ethoxy] -pyridine-3-ylmethoxy}-2H-pyridazin-3-one, compared with 18F-FDG uptake and the apoptosis index. Methods Tumor uptake of 18F-BCPP-EF or 18F-FDG was examined in C3H/HeN mice inoculated with murine squamous cell carcinoma SCCVII at various time points after a single dose of x-ray irradiation at 0, 6, 15, or 30 Gy. Apoptosis incidence was determined by TUNEL staining in excised tumor tissue. Results Tumor growth suppression was dose-dependent; tumor grew 10-fold (0 Gy), 5-fold (6 Gy), 2-fold (15 Gy), and reduced to half in its volume (30 Gy) 14 days after treatment. 18F-BCPP-EF uptake was significantly increased as early as 3 days after 15 Gy or 30 Gy, when tumor size and apoptosis index showed no difference among radiation doses. In contrast, 18F-FDG uptake was initially increased dose-dependently, remained elevated up to 7 days, and eventually decreased 10 days after 30 Gy and also 14 days after 15 Gy when tumor size was already reduced. Apoptosis index was increased after irradiation but failed to correlate with tumor response. Conclusion Tumor uptake of 18F-BCPP-EF was increased dose-dependently early after effective doses of IR when 18F-FDG uptake as well as apoptosis incidence were not indicative of tumor response. The results suggest that 18F-BCPP-EF is a promising “positive” MC-I imaging PET probe for early detection of efficacy of tumor radiotherapy.