Kazuhiko Yanamoto

11C-AC-5216: A Novel PET Ligand for Peripheral Benzodiazepine Receptors in the Primate Brain

Developing a PET ligand for imaging of the peripheral benzodiazepine receptor (PBR; Translocator Protein [18 kDa] TSPO) is of great importance for studying its role in glial cells in the injured brain and in neurodegenerative disorders, such as Alzheimers disease. The aim of this study was to synthesize and evaluate N-benzyl-N-ethyl-2-(7-11C-methyl-8-oxo-2-phenyl-7,8-dihydro-9H-purin-9-yl)acetamide (11C-AC-5216) as a PET ligand for imaging PBR in the primate brain. Methods: AC-5216 and its desmethyl precursor (compound 1) were synthesized starting from commercially available compounds. The radiosynthesis of 11C-AC-5216 was performed through the reaction of compound 1 with 11C-CH3I in the presence of NaH. The in vivo brain regional distribution was determined in mice (dissection) and a monkey (PET). Results: 11C-AC-5216 (800–1,230 MBq; n = 25) was obtained with a radiochemical purity of 98% and a specific activity of 85–130 GBq/μmol at the end of synthesis. After injection of 11C-AC-5216 into mice, a high accumulation of radioactivity was found in the lungs, heart, adrenal glands, and other PBR-rich organs. In the mouse brain, high radioactivity was observed in the olfactory bulb and cerebellum. Radioactivity in these regions was inhibited by nonradioactive AC-5216 or PK11195 but was not decreased by central benzodiazepine receptor–selective flumazenil and Ro15-4513. A PET study of the monkey brain determined that 11C-AC-5216 had a relatively high uptake in the occipital cortex, a rich PBR-dense area in the primate brain. Pretreatment with nonradioactive AC-5216 and PK11195 reduced the radioactivity of 11C-AC-5216 in the occipital cortex significantly, suggesting its high specific binding with PBR in the brain. Metabolite analysis demonstrated that 11C-AC-5216 was stable in vivo in the mouse brain, although it was metabolized in the plasma of mice and the monkey. Conclusion: 11C-AC-5216 is a promising PET ligand for imaging PBR in rodent and primate brains.

Evaluation of Limiting Brain Penetration Related to P-glycoprotein and Breast Cancer Resistance Protein Using [11C]GF120918 by PET in Mice

PurposeGF120918 has a high inhibitory effect on P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). We developed [11C]GF120918 as a positron emission tomography (PET) probe to assess if dual modulation of P-gp and BCRP is useful to evaluate brain penetration.ProceduresPET studies using [11C]GF120918 were conducted on P-gp and/or Bcrp knockout mice as well as wild-type mice.ResultsIn PET studies, the AUCbrain[0–60 min] and K1 value in P-gp/Bcrp knockout mice were nine- and 26-fold higher than that in wild-type mice, respectively. These results suggest that brain penetration of [11C]GF120918 is related to modulation of P-gp and BCRP and is limited by two transporters working together.ConclusionsPET using [11C]GF120918 may be useful for evaluating the function of P-gp and BCRP. PET using P-gp/Bcrp knockout mice may be an effective method to understand the overall contributions the functions of P-gp and BCRP.

Synthesis and evaluation of 6-[1-(2-[18F]fluoro-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline for positron emission tomography imaging of the metabotropic glutamate receptor type 1 in brain

The purpose of this study was to synthesize 6-[1-(2-[(18)F]fluoro-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline ([(18)F]FPTQ, [(18)F]7a) and to evaluate its potential as a positron emission tomography ligand for imaging metabotropic glutamate receptor type 1 (mGluR1) in the rat brain. Compound [(18)F]7a was synthesized by [(18)F]fluorination of 6-[1-(2-bromo-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline (7b) with potassium [(18)F]fluoride. At the end of synthesis, 1280-1830MBq (n=8) of [(18)F]7a was obtained with >98% radiochemical purity and 118-237GBq/μmol specific activity using 3300-4000MBq of [(18)F]F(-). In vitro autoradiography showed that [(18)F]7a had high specific binding with mGluR1 in the rat brain. Biodistribution study using a dissection method and small-animal PET showed that [(18)F]7a had high uptake in the rat brain. The uptake of radioactivity in the cerebellum was reduced by unlabeled 7a and mGluR1-selective ligand JNJ-16259685 (2), indicating that [(18)F]7a had in vivo specific binding with mGluR1. Because of a low amount of radiolabeled metabolite present in the brain, [(18)F]7a may have a limiting potential for the in vivo imaging of mGluR1 by PET.

18F-FEAC and 18F-FEDAC: PET of the Monkey Brain and Imaging of Translocator Protein (18 kDa) in the Infarcted Rat Brain

We evaluated two 18F-labeled PET ligands, N-benzyl-N-ethyl-2-[7,8-dihydro-7-(2-18F-fluoroethyl)-8-oxo-2-phenyl-9H-purin-9-yl]acetamide (18F-FEAC) and N-benzyl-N-methyl-2-[7,8-dihydro-7-(2-18F-fluoroethyl)-8-oxo-2-phenyl-9H-purin-9-yl]acetamide (18F-FEDAC), by investigating their kinetics in the monkey brain and by performing in vitro and in vivo imaging of translocator protein (18 kDa) (TSPO) in the infarcted rat brain. Methods: Dissection was used to determine the distribution of 18F-FEAC and 18F-FEDAC in mice, whereas PET was used for a monkey. With each 18F-ligand, in vitro autoradiography and small-animal PET were performed on infarcted rat brains. Results: 18F-FEAC and 18F-FEDAC had a high uptake of radioactivity in the heart, lung, and other TSPO-rich organs of mice. In vitro autoradiography showed that the binding of each 18F-ligand significantly increased on the ipsilateral side of rat brains, compared with the contralateral side. In a small-animal PET study, PET summation images showed the contrast of radioactivity between ipsilateral and contralateral sides. Pretreatment with TSPO ligands N-benzyl-N-ethyl-2-(7-methyl-8-oxo-2-phenyl-7,8-dihydro-9H-purin-9-yl)acetamide (AC-5216) or (R)-N-methyl-N-(1-methylpropyl)-1-(2-chlorophenyl)isoquinoline-3-carboxamide (PK11195) diminished the difference in uptake between the 2 sides. The PET study showed that each 18F-ligand had uptake and distribution patterns in the monkey brain similar to those of 11C-AC-5216. After injection into the monkey during PET, the uptake of each 18F-ligand in the brain decreased over time whereas 11C-AC-5216 did not. In the brain homogenate of mice, the percentage of the fraction corresponding to intact 18F-FEAC and 18F-FEDAC was 68% and 75% at 30 min after injection. In monkey plasma, each 18F-ligand was scarcely metabolized until the end of the PET scan. Conclusion: 18F-FEAC and 18F-FEDAC produced in vitro and in vivo signals allowing visualization of the increase in TSPO expression in the infarcted rat brain. The kinetics of both 18F-ligands in the monkey brain and tolerance for in vivo metabolism suggested their usefulness for imaging studies of TSPO in primates.

Visualization of early infarction in rat brain after ischemia using a translocator protein (18 kDa) PET ligand [11C]DAC with ultra-high specific activity.

The aim of this study was to visualize early infarction in the rat brain after ischemia using a translocator protein (TSPO) (18 kDa) PET ligand [(11)C]DAC with ultra-high specific activity (SA) of 3670-4450 GBq/μmol. An infarction model of rat brain was prepared by ischemic surgery and evaluated 2 days after ischemia using small-animal PET and in vitro autoradiography. Early infarction with a small increase of TSPO expression in the brain was visualized using PET with high SA [(11)C]DAC (average 4060 GBq/μmol), but was not distinguished clearly with usually reported SA [(11)C]DAC (37 GBq/μmol). Infarction in the rat brain 4 days after ischemia was visualized using high and usually reported SAs [(11)C]DAC. Displacement experiments with unlabeled TSPO-selective AC-5216 or PK11195 diminished the difference in radioactivity between ipsilateral and contralateral sides, confirming that the increased uptake on the infracted brain was specific to TSPO. In vitro autoradiography with high SA [(11)C]DAC showed that the TSPO expression increased on early infarction in the rat brain. High SA [(11)C]DAC is a useful and sensitive biomarker for the visualization of early infarction and the characterization of TSPO expression which was slightly elevated in the infarcted brain using PET.

Radiosynthesis and evaluation of [11C]YM-202074 as a PET ligand for imaging the metabotropic glutamate receptor type 1

INTRODUCTION Developing positron emission tomography (PET) ligands for imaging metabotropic glutamate receptor type 1 (mGluR1) is important for studying its role in the central nervous system. N-cyclohexyl-6-{[N-(2-methoxyethyl)-N-methylamino]methyl}-N-methylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM-202074) exhibited high binding affinity for mGluR1 (K(i)=4.8 nM), and selectivity over other mGluRs in vitro. The purpose of this study was to label YM-202074 with carbon-11 and to evaluate in vitro and in vivo characteristics of [(11)C]YM-202074 as a PET ligand for mGluR1 in rodents. METHODS [(11)C]YM-202074 was synthesized by N-[(11)C]methylation of its desmethyl precursor with [(11)C]methyl iodide. The in vitro and in vivo brain regional distributions were determined in rats using autoradiography and PET, respectively. RESULTS [(11)C]YM-202074 (262-630 MBq, n=5) was obtained with radiochemical purity of >98% and specific activity of 27-52 GBq/mumol at the end of synthesis, starting from [(11)C]CO(2) of 19.3-21.5 GBq. In vitro autoradiographic results showed that the high specific binding of [(11)C]YM-202074 for mGluR1 was presented in the cerebellum, thalamus and hippocampus, which are known as mGluR1-rich regions. In ex vivo autoradiography and PET studies, the radioligand was specifically distributed in the cerebellum, although the uptake was low. Furthermore, the regional distribution was fairly uniform in the whole brain by pretreatment with JNJ16259685 (a mGluR1 antagonist). However, radiometabolite(s) was detected in the brain. CONCLUSIONS From these results, especially considering the low brain uptake and the influx of radiometabolite(s) into brain, [(11)C]YM-202074 may not be a useful PET ligand for in vivo imaging of mGluR1 in the brain.

Evaluation of Chemotherapy Response in VX2 Rabbit Lung Cancer with 18F-Labeled C2A Domain of Synaptotagmin I

The C2A domain of synaptotagmin I can target apoptotic cells by binding to exposed anionic phospholipids. The goal of this study was to synthesize and develop 18F-labeled C2A-glutathione-S-transferase (GST) as a molecular imaging probe for the detection of apoptosis and to assess the response of paclitaxel chemotherapy in VX2 rabbit lung cancer. Methods: 18F-C2A-GST was prepared by labeling C2A-GST with N-succinimidyl 4-18F-fluorobenzoate (18F-SFB). 18F-C2A-GST was confirmed by high-performance liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The binding of 18F-C2A-GST toward apoptosis was validated in vitro using camptothecin-induced Jurkat cells. Biodistribution of 18F-C2A-GST was determined in mice by a dissection method and small-animal PET. Single-dose paclitaxel was used to induce apoptosis in rabbits bearing VX2 tumors (n = 6), and 2 VX2 rabbits without treatment served as control. 18F-C2A-GST PET was performed before and at 72 h after therapy, and 18F-FDG PET/CT was also performed before treatment. To confirm the presence of apoptosis, tumor tissue was analyzed and activated caspase-3 was measured. Results: 18F-C2A-GST was obtained with more than 95% radiochemical purity and was stable for 4 h after formulation. 18F-C2A-GST bound apoptotic cells specifically. Biodistribution in mice showed that 18F-C2A-GST mainly excreted from the kidneys and rapidly cleared from blood and nonspecific organs. High focal uptake of 18F-C2A-GST in the tumor area was determined after therapy, whereas no significant uptake before therapy was found in the tumor with 18F-FDG–avid foci. The maximum standardized uptake value after therapy was 0.47 ± 0.28, significantly higher than that in the control (0.009 ± 0.001; P < 0.001). The apoptotic index was 79.81% ± 8.73% in the therapy group, significantly higher than that in the control (5.03% ± 0.81%; P < 0.001). Activated caspase-3 after paclitaxel treatment increased to 69.55% ± 16.27% and was significantly higher than that in the control (12.26% ± 5.39%; P < 0.001). Conclusion: 18F-C2A-GST was easily synthesized by conjugation with 18F-SFB and manifested a favorable biodistribution. Our results demonstrated the feasibility of 18F-C2A-GST for the early detection of apoptosis after chemotherapy in a VX2 lung cancer model that could imitate the human lung cancer initiation, development, and progress.

Evaluation of N‐benzyl‐N‐[11C]methyl‐2‐ (7‐methyl‐8‐oxo‐2‐phenyl‐7,8‐dihydro‐9H‐purin‐9‐yl)acetamide ([11C]DAC) as a novel translocator protein (18 kDa) radioligand in kainic acid‐lesioned rat

The aim of this study was to evaluate N‐benzyl‐N‐[11C]methyl‐2‐(7‐methyl‐8‐oxo‐2‐phenyl‐7,8‐dihydro‐9H‐purin‐9‐yl)acetamide ([11C]DAC) as a new translocator protein (18 kDa) [TSPO, formerly known as the peripheral‐type benzodiazepine receptor (PBR)] positron emission tomography (PET) ligand in normal mice and unilateral kainic acid (KA)‐lesioned rats. DAC is a derivative of AC‐5216, which is a potent and selective PET ligand for the clinical investigation of TSPO. The binding affinity and selectivity of DAC for TSPO were similar to those of AC‐5216, and DAC was less lipophilic than AC‐5216. The distribution pattern of [11C]DAC was in agreement with TSPO distribution in rodents. No radioactive metabolite of [11C]DAC was found in the mouse brain, although it was metabolized rapidly in mouse plasma. Using small‐animal PET, we examined the in vivo binding of [11C]DAC for TSPO in KA‐lesioned rats. [11C]DAC and [11C]AC‐5216 exhibited similar brain uptake in the lesioned and nonlesioned striatum, respectively. The binding of [11C]DAC to TSPO was increased significantly in the lesioned striatum, and [11C]DAC showed good contrast between the lesioned and nonlesioned striatum (the maximum ratio was about threefold). In displacement experiments, the uptake of [11C]DAC in the lesioned striatum was eventually blocked using an excess of either unlabeled DAC or PK11195 injected. [11C]DAC had high in vivo specific binding to TSPO in the injured rat brain. Therefore, [11C]DAC is a useful PET ligand for TSPO imaging, and its specific binding to TSPO is suitable as a new biomarker for brain injury. Synapse 63:961–971, 2009.

Synthesis and in vivo evaluation of 18F-fluoroethyl GF120918 and XR9576 as positron emission tomography probes for assessing the function of drug efflux transporters

The purpose of this study was to synthesize two new positron emission tomography (PET) probes, N-(4-(2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl)phenyl)-9,10-dihydro-5-[¹⁸F]fluoroethoxy-9-oxo-4-acridine carboxamide ([¹⁸F]3) and quinoline-3-carboxylic acid [2-(4-{2-[7-(2-[¹⁸F]fluoroethoxy)-6-methoxy-3,4-dihydro-1H-isoquinolin-2-yl]ethyl}phenylcarbamoyl)-4,5-dimethoxyphenyl]amide ([¹⁸F]4), and to evaluate the potential of these PET probes for assessing the function of two major drug efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). [¹⁸F]3 and [¹⁸F]4 were synthesized by ¹⁸F-alkylation of each O-desmethyl precursor with [¹⁸F]2-fluoroethyl bromide for injection as PET probes. In vitro accumulation assay showed that treatment with P-gp/BCRP inhibitors (1 and 2) enhanced the intracellular accumulation capacity of P-gp- and BCRP-overexpressing MES-SA/Dx5 cells. In PET studies, the uptake (AUC(brain[0-)₆₀ (min])) of [¹⁸F]3 and [¹⁸F]4 in wild-type mice co-injected with 1 were approximately sevenfold higher than that in wild-type mice, and the uptake of [¹⁸F]3 and [¹⁸F]4 in P-gp/Bcrp knockout mice were eight- to ninefold higher than that in wild-type mice. The increased uptake of [¹⁸F]3 and [¹⁸F]4 was similar to that of parent compounds ([¹¹C]1 and [¹¹C]2) previously described, indicating that radioactivity levels in the brain after injection of [¹⁸F]3 and [¹⁸F]4 are related to the function of drug efflux transporters. Also, these results suggest that the structural difference between parent compounds ([¹¹C]1 and [¹¹C]2) and fluoroethyl analogs ([¹⁸F]3 and [¹⁸F]4) do not obviously affect the potency against drug efflux transporters. In metabolite analysis of mice, the unchanged form in the brain and plasma at 60 min after co-injection of [¹⁸F]4 plus 1 were higher (95% for brain; 81% for plasma) than that after co-injection of [¹⁸F]3 plus 1. [¹⁸F]4 is a promising PET probe to assess the function of drug efflux transporters.

Imaging of I2-imidazoline receptors by small-animal PET using 2-(3-fluoro-[4-11C]tolyl)-4,5-dihydro-1H-imidazole ([11C]FTIMD)

INTRODUCTION Imidazoline receptors (IRs) have been established as distinct receptors, and have been categorized into at least two subtypes (I(1)R and I(2)R). I(2)Rs are associated with depression, Alzheimers disease, Huntingtons disease and Parkinsons disease. A few positron emission tomography (PET) probes for I(2)Rs have been synthesized, but a selective PET probe has not been evaluated for the imaging of I(2)Rs by PET. We labeled a selective I(2)R ligand 2-(3-fluoro-4-tolyl)-4,5-dihydro-1H-imidazole (FTIMD) with (11)C and performed the first imaging of I(2)Rs by PET using 2-(3-fluoro-[4-(11)C]tolyl)-4,5-dihydro-1H-imidazole ([(11)C]FTIMD). METHODS [(11)C]FTIMD was prepared by a palladium-promoted cross-coupling reaction of the tributylstannyl precursor and [(11)C]methyl iodide in the presence of tris(dibenzylideneacetone)dipalladium(0) and tri(o-tol)phosphine. Biodistribution was investigated in rats by tissue dissection. [(11)C]FTIMD metabolites were measured in brain tissues and plasma. Dynamic PET scans were acquired in rats, and the kinetic parameters estimated. RESULTS [(11)C]FTIMD was successfully synthesized with a suitable radioactivity for the injection. Co-injection with 0.1 mg/kg of cold FTIMD and BU224 induced a significant reduction in the brain-to-blood ratio 15 and 30 min after the injection. In metabolite analysis, unchanged [(11)C]FTIMD in the brain was high (98%) 30 min after the injection. In PET studies, high radioactivity levels were observed in regions with a high density of I(2)R. The radioactivity levels and V(T) values in the brain regions were prominently reduced by 1.0 mg/kg of BU224 pretreatment as compared with control. CONCLUSION [(11)C]FTIMD showed specific binding to I(2)Rs in rat brains with a high density of I(2)R.