Demonstration of intracellular pH-weighting PET imaging using a new-type PET probe responsible for monoacylglycerol lipase activity in the brain

Objectives: The brain acidosis is caused by intracellular hyper-accumulation of acidic sources (H+, lactate, and carbonic acid) by switching the cellular energy metabolism from aerobic to anaerobic by the hypoxia. Such intra cellular acidosis give curucial injury to central nurves system of the brain. Therefore, monitoring intracellular pH would be very important to diagnosis neuronal condition. Recently, covalent inhibitors for monoacylglycerol lipase (MAGL), an enzyme intracellur lacated on neuron and astrocyte in the brain and regulates endocannabinoid system, were identified by Bulter et al [1]. Among these inhibitors, 1,1,1,3,3,3-hexafluoropropan-2-yl 3-(1-phenyl-1H-pyrazol-3-yl)azetidine-1-carboxylate (1) showed reversible inhibitory effect to MAGL. The purpose of this study is to establish quantification method of hydrolysis rate of compound 1 mediated by MAGL and to demonstrate pH-weighted PET imaging in the brain of ischemic rat.Methods: To estimate interaction between compound 1 and MAGL, docking simulations were conducted comparing to similar chemical structural irreversible-type inhibitor (2). In addition, to evaluate influents of pH shifts, molecular dynamics (MD) simulations of compound 1 were also performed under the neutral (pH 7) or acidic (pH 6) conditions. Radiosynthesis of [11C]1 and [11C]2 was described in another presentation in this meeting (Mori W, et a1.). To confirm MAGL-hydrolysis of [11C]1, in vitro assessments using rat brain homogenates were conducted. PET imaging with [11C]1 was carried out using middle cerebral artery occlusion (MCAO) rat as an acute hypoxia model and hydrolysis rate (KH) of [11C]1 with MAGL was estimated by monoexponential fitting on time-activity curves of ipsilateral region.Results: MD simulations predicted that azetidine carbamate moiety of 1 was easily hydrolyzed by MAGL due to close distance from water molecule, compared to 2 containing piperidine carbamate. Moreover, the acylated azetidine in 1 has been shown to react differently than piperidine ring size amide in 2 due to decreased planarity in the amide moiety itself, conveyed by the ring strain associated with the azetidine itself. Additionally, it was simulated that the hydrolysis rate of 1 would be slower under the acidic condition because of changing interaction of 1 against water molecule. In vitro assessments showed that generation rate of 11CO2, the final product derived from hydrolysis of [11C]1, would become slower depending on pH shifts. In PET study with [11C]1 using MCAO rat, KH value in ipsilateral region was significantly slower than that in contralateral region.Conclusions: We successfully established the method for quantifying hydrolysis rate of MAGL using new-type PET probe and demonstrated pH-weighted imaging in vivo

Synthesis of [11C]carbonyl-labeled cyclohexyl (5-(2-acetamidobenzo[d]thiazol-6-yl)-2-methylpyridin-3-yl)carbamate ([11C-carbonyl]PK68) as a potential PET tracer for receptor-interacting protein 1 kinase

Background: Receptor-interacting protein 1 kinase (RIPK1) is a key enzyme in the regulation of cellular necroptosis. Recently, cyclohexyl (5-(2-acetamidobenzo[d]thiazol-6-yl)-2-methylpyridin-3-yl)carbamate (PK68, 5) has been developed as a potent inhibitorof RIPK1. Herein, we synthesized [11C]carbonyl-labeled PK68 ([11C-carbonyl]PK68, [11C]PK68) as a potential PET tracer for imaging RIPK1 and evaluated its brain uptake in vivo.Results: We synthesized [11C]PK68 by reacting amine precursor 14 with [11C]acetyl chloride. At the end of synthesis, we obtained [11C]PK68 of 1200–1790 MBq with a radiochemical yield of 9.1 ± 5.9 % (n = 10, decay-corrected to the end of irradiation) and radiochemical purity of >99%, and a molar activity of 37–99 GBq/μmol starting from 18–33 GBq of [11C]CO2. The fully automated synthesis took 30 min from the end of irradiation. In a small-animal PET study, [11C]PK68 was rapidly distributed in the liver and kidneys of healthy mice after injection, and subsequently cleared from their bodies via hepatobiliary excretion and the intestinal reuptake pathway. Although there was no obvious specific binding of RIPK1 in the PET study, [11C]PK68 demonstrated relatively high stability in vivo and provided useful structural information further candidate development.Conclusions: In the present study, we successfully radiosynthesized [11C]PK68 as a potential PET tracer and evaluated its brain uptake. We are planning to optimize the chemical structure of [11C]PK68 and conduct further PET studies on it using pathological models

Radiolabeling and evaluation of cyclohexyl (5-(2-acetamidobenzo[d]thiazol-6-yl)-2-methylpyridin-3-yl) carbamate (PK68), a potent inhibitor for receptor interacting protein 1 kinase (RIP1)

Objectives: The receptor interacting protein 1 kinase (RIP1) is well-known as a key enzyme to regulate neuronal cell death, indicating relationship with several central nervous system disorders. Recently, Hou J et al has identified potent inhibitors for RIP1 [1]. Among, cyclohexyl (5-(2-acetamidobenzo[d]thiazol-6-yl)-2-methylpyridin-3-yl)carbamate (PK68) showed the highest inhibitory efficacy (mIC50 = 13 nM) for RIP1. The aim of this study is to radiolabel PK68 with 11C and to evaluate its distribution in healthy mice.Methods: [11C]PK68 was synthesized by the reaction of an amine precursor with [11C]acetyl chloride ([11C]AcCl) that was prepared by the reaction of methyl magnesium bromide with [11C]CO2, followed by chlorination with oxallyl chloride and distillation (Scheme 1), using an automated synthesis system developed in house [2]．Dynamic PET scans were conducted for 60 min (1 min × 4 frames, 2 min × 8 frames, and 5 min × 8 frames) using healthy mice. For the blocking study, the mouse was intravenously administrated with unlabeled PK68 (1 mg/kg) before staring a PET scan. Metabolite analysis of [11C]PK68 was performed using the plasma and liver samples by radio-HPLC system. Biodistribution of [11C]PK68 in mice was evaluated by measuring radioactivity in tissues of mice sacrificed at several time points (1, 5, 15, 30, and 60 min) after the injection.Results: At the end of synthesis, 670 ± 68 MBq (n = 6) of [11C]PK68 was obtained with >99% radiochemical purity and > 37 GBq/μmol of molar activity, starting from about 24 GBq of [11C]CO2. The fully-automated synthesis time was 30 min from the end of irradition. This radioactive product showed > 95% radiochemical purity within 60 min after the formulation. PET imaging with [11C]PK68 showed high uptakes in the liver and kidney of mouse in early time after the injection. Although the uptake of radioactivity in the liver was slightly decreased by treatment with unlabeled PK68, there was no significant difference between control and blocking subjects. Metabolite analysis exhibited relative high stablilty of [11C]PK68 in the plasma and liver in vivo. In ex vivo biodistribution study, it was suggested that [11C]PK68 was metabolised in the liver and removed from body via the hepatobiliary excretion and intestinal reuptake pathway. Conclusions: In this study, we successfully radiosynthesized [11C]PK68 and evaluated its dynamics and stability in vivo

Upregulation of striatal metabotropic glutamate receptor subtype 1 (mGluR1) in rats with excessive glutamate release induced by N-acetylcysteine

Aim of this study is to investigate the changes in expression of metabotropic glutamate (Glu) receptor subtype 1 (mGluR1), a key molecule involved in neuroexcitetoxicity, during excessive Glu release in the brain by PET imaging. An animal model of excessive Glu release in the brain was produced by intraperitoneally implanting an Alzet osmotic pump containing N-acetylcysteine (NAC), an activator of the cysteine/Glu antiporter, into the abdomen of rats. Basal Glu concentration in the brain was measured by microdialysis, which showed that basal Glu concentration in NAC-treated rats (0.31 µM) was higher than that in saline-treated rats (0.17 µM) at day 7 after the implantation of the osmotic pump. Similarly, PET studies with [11C]ITDM, a useful radioligand for mGluR1 imaging exhibited that the striatal binding potential (BPND) of [11C]ITDM for mGluR1 in PET assessments was increased in NAC-treated animals at day 7 after implantation (2.30) compared with before implantation (1.92). The dynamic changes in striatal BPND during the experimental period were highly correlated with basal Glu concentration. In conclusion, density of mGluR1 is rapidly upregulated by increases in basal Glu concentration, suggesting that mGluR1 might to be a potential biomarker of abnormal conditions in the brain

Automated radiosynthesis of the 18F-labeled BF2-chelated tetraaryl-azadipyrromethenes photosensitizer using isotopic exchange

Objectives: A family of the BF2-chelated tetraaryl-azadipyrromethenes (ADPMs) was developed as a nonporphyrin photosensitizer (PS) for photodynamic therapy (PDT) [1]. Among ADPMs, ADPM06 displayed excellent photochemical and photophysical properties [1]. In addition, PDT using ADPM06 elicited impressive complete response rates in various tumor models when a short drug-light interval was applied [2]. Molecular imaging is a promising PDT planning and monitoring tool, and the PS biodistribution is a relevant issue for PDT planning that radiolabeled PSs may address efficiently. To evaluate efficiency for PDT using ADPM06 and also side effects of ADPM, we synthesized [18F]ADPM06 using an automated 18F-labeling synthesizer, and evaluated its in vivo properties.Methods: [18F]ADPM06 was synthesized using an automated 18F-labeling synthesizer by Lewis acid-assisted isotopic 18F-19F exchange [3]. The [18F]F– was extracted from a Sep-Pak Accell Plus QMA Carbonate Plus Light cartridge with a mixture of tetrabutylanmmonium bicarbonate aqueous solution and acetonitrile. The solution was concentrated by evaporation at 100 °C for 10 min under nitrogen gas flow. After the reaction vessel was cooled, the mixture of ADPM06 in acetonitrile and tin(IV) chloride (SnCl4) solution was added to the reaction vessel, and then was agitated using magnetic stirrer at room temperature for 10 min. After the reaction, the mixture was diluted with water for injection, and transferred to the injector for semi-preparative radio-HPLC. The HPLC fractions were collected in a flask, to which Tween 80 in ethanol was added prior to radiosynthesis. The solution was subsequently evaporated to dryness and the residue was dissolved in physiological saline. The product was analyzed by HPLC with radioactivity and UV-VIS detection. The in vivo biodistribution study was performed using mice.Results:To radio-synthesize efficiently [18F]ADPM06, we performed semi-automated radiosynthesis. By increasing the concentration of SnCl4 from 100 to 400 μmol, the radiochemical conversion (RCC) of [18F]ADPM06 from [18F]TBAF was increased until 60%. Also, the RCC using 0.8 μmol of ADPM06 was 1.2-fold higher than that using 0.4 μmol of ADPM06. In the radiosynthesis using an automated 18F-labeling synthesizer, we successfully synthesized [18F]ADPM06 for in vivo applications. The radiochemical yield (RCY) from [18F]F- was 13 ± 2.7 % (n = 5; 0.4 μmol of ADPM06, 200 μmol of SnCl4) at the end of irradiation. The radiosynthesis time was within 60 min, and radiochemical purity remained >95% after maintaining it for 1 hour after the end of synthesis. In the biodistribution study within 120 min after the injection, radioactivity levels in heart, lung, liver, pancreas, spleen, kidney, small intestine, muscle, and brain gradually decreased after initial uptake.Conclusions: We enabled to synthesize [18F]ADPM06 using an automated 18F-labeling synthesizer, and to evaluate biodistribution of [18F]ADPM06 in mice