Radiosynthesis and Evaluation of [11C]HC070, a Promising PET Tracer for ImagingTransient Receptor Potential Canonical 5 (TRPC5)

151 Introduction: Transient Receptor Potential Canonical 5 (TRPC5) belongs to the seven mammalian TRPC members (TRPC1-7) which are Ca2+-permeable nonselective cationic channels. The brain widely expressed channel has been implicated in a wide range of physiological and pathological mechanisms. These findings have driven the clinical trials of several TRPC5 inhibitors for CNS disorders and kidney disease while no conclusion has been reported yet. We have previously reported the first TRPC5 PET tracer [11C]HC608,its relative low maximum brain standard uptake value (SUV) of 0.9 at 5 min, preventing it to be a suitable PET radiotracer for assessing the TRPC5 in vivo. HC070, a compound structural close to HC608 with different chemo-physical properties is reported as one of the most potent (IC50 = 9.3 nM) and selective TRPC5 inhibitors. To identify a novel PET radiotracer for imaging TRPC5, herein, we reported our efforts on radiosynthesizing [11C]HC070 and initial in vitro and in vivo comparison studies of two radiotracers [11C]HC070 and[11C]HC608. Our data suggested [11C]HC070 is more promising than [11C]HC608 to be a PET radiotracer for imaging TRPC5 in vivo. Methods: : The radiosynthesis of [11C]HC070 was achieved by N-[11C]methylation of its precursor reacting with [11C]CH3I in DMF using K2CO3 as the base, followed by semi-preparative reverse-phase HPLC purification. The biodistribution study of [11C]HC070 was performed in male Sprague Dawley (SD) rats at three-time points (5, 30, and 60 min) after intravenous injection of the radiotracer. The uptake of radioactivity was calculated as percentage injected dose per gram (%ID/gram). In vitro autoradiography (ARG) was performed with rat brain slides incubated with [11C]HC070 (0.56 MBq/slide). MicroPET study was conducted in male macaques (9-10 kg) after administration of ∼0.35 GBq of radiotracer via the venous catheter. For each study, a 2 hours dynamic scan was performed for data collection using a Focus 220 microPET scanner. Radioactive metabolites analysis for NHP plasma samples at 5, 15, 30, and 60 min post-injection of radiotracer was performed by HPLC. Each radioactive peak was collected, counted in a gamma counter and then calculated. Results: The radiotracer [11C]HC070 was successfully afforded in 45 ± 5% (n = 15) radiochemical yield (RCY) with high molar activity (> 37 GBq/μmol) and high radiochemical purity (> 99%) at the end of synthesis (EOS). The brain uptake (%ID/gram) was 0.47 at 5 min, 0.18 at 30 min, and 0.09 at 60 min. At 5 min, high uptake (>1% ID/g) was observed in tissues including liver, kidney, pancreas and small intestine, followed up by washout rapidly. Strong ARG signal shows up on rat brain sections, especially on the colliculus, striatum, cortex, and thalamus regions. The ARG signal is significantly blocked in the presence of TRPC5 antagonist HC608 (1 μM). The NHP SUV for [11C]HC070 reached a maximum (~2.2) at 5 min after tracer injection. The uptake (SUV) ratio of [11C]HC070 at 4 min versus 120 min reached 3.7-fold. For radiometabolite analysis, the percentage of the parent compound [11C]HC070 was 85 %, 70 %, 50 %, and 36% at 5, 15, 30, and 60 min post-injection respectively. While two hydrophilic radio-metabolites with a retention time of ~3 and ~7 min were observed. Conclusions: Our preliminary ex-vivo biodistribution and in vitro autoradiography results suggested that [11C]HC070 owes good brain uptake and it specifically binds towards TRPC5. The MicroPET data suggest that [11C]HC070 has much higher initial brain uptake than [11C]HC608 and possesses fast washout kinetics in the nonhuman primates. This tracer also exhibits relative metabolic stability in vivo. Together, [11C]HC070 has great potential to be a PET tracer for imaging TRPC5 in vivo. Further evaluation and optimization to support the TRPC5 tracer translation for clinical use are underway in our laboratory.Research support: USA NIH/NINDS #NS103988, NS075527, and NS103957.