Preclinical Evaluation of Novel Positron Emission Tomography (PET) Probes for Imaging Leucine-Rich Repeat Kinase 2 (LRRK2)

Parkinson’s disease (PD) is one of the most highly debilitating neurodegenerative disorders, which affects millions of people worldwide, and leucine-rich repeat kinase 2 (LRRK2) mutations have been involved in the pathogenesis of PD. Developing a potent LRRK2 positron emission tomography (PET) tracer would allow for in vivo visualization of LRRK2 distribution and expression in PD patients. In this work, we present the facile synthesis of two potent and selective LRRK2 radioligands [11C]3 ([11C]PF-06447475) and [18F]4 ([18F]PF-06455943). Both radioligands exhibited favorable brain uptake and specific bindings in rodent autoradiography and PET imaging studies. More importantly, [18F]4 demonstrated significantly higher brain uptake in the transgenic LRRK2-G2019S mutant and lipopolysaccharide (LPS)-injected mouse models. This work may serve as a roadmap for the future design of potent LRRK2 PET tracers.


INTRODUCTION
Parkinson's disease (PD) is a highly debilitating neurodegenerative disease that affects approximately 10% of people over 60 years old.Currently, there is no available diseasemodifying therapy.Although remarkable investment has been devoted to many clinical trials to advance potential diseasemodifying therapeutic intervention, the underlying disease mechanisms of PD still remain unclear, leading to significant clinical attrition. 1,2−5 Indeed, LRRK2 mutations, which are autosomal dominant, represent the most common known cause of PD worldwide.Approximately 10% of inherited PD patients and 2% of sporadic cases carry LRRK2 mutations (e.g., G2019S, I2020T, R1441C), suggesting LRRK2 mutation likely as a common etiology underlying both inherited and sporadic PD. 6−9 Notwithstanding growing evidence supporting the deep linkage of LRRK2 dysfunction with both inherited and non-inherited PD, it is still challenging to elucidate the physiological and/or pathological role of LRRK2 due to its complexity.−13 As part of our continuing efforts in LRRK2 inhibitor development, we are interested in identifying an appropriate LRRK2-selective PET agent. 14Indeed, an LRRK2selective PET agent could enable the investigation of target engagement for a specific LRRK2 inhibitor, thus advancing its clinical characterization and translation.Moreover, in light of numerous pieces of evidence indicating increased LRRK2 enzyme activity in LRRK2 mutation-related PD brains, we are eager to investigate whether the LRRK2 distribution and expression in the brain of PD patients could be visualized by PET imaging, which would in turn give rise to a more in-depth understanding of PD pathogenesis.To date, several LRRK2 inhibitors have been labeled with carbon-11 or fluorine-18, which included GNE-1023 15 and its analogues 16 as well as HG-10-102-01, 17 but limited biological data were disclosed (Figure 1A).As such, the development and validation of a blood−brain barrier (BBB)-penetrable LRRK2 PET tracer with excellent binding specificity are intensively motivated by the therapeutic potential of LRRK2 inhibitors.Toward this end, we were enlightened by PF-06447475 (3), an LRRK2 inhibitor lead initially disclosed by Pfizer. 14−22 Both 3 and 4 have been comprehensively validated in pharmacology screening, revealing favorable pharmacological and pharmacokinetic characteristics such as excellent potency and target selectivity toward LRRK2, reasonable clearance rate, clean safety profile, high passive permeability, and low P-glycoprotein (P-gp) efflux (Table 1). 14,18Moreover, we successfully radiolabeled 4 with fluorine-18 and performed a preliminary PET imaging study of [ 18 F]4 ([ 18 F]PF-06455943) in nonhuman primates (NHPs), which exhibited high brain uptake and binding specificity. 18During the course of preparing the current manuscript, based on the scaffold of 3, Li et al. 23 also developed two 18 F-labeled LRRK2 PET ligands [ 18 F]1 and [ 18 F]2, whereas Schaffer et al. 24 disclosed [ 18 F]FMN3PA and [ 18 F]FMN3PU, despite lack of comprehensive pharmacological and pharmacokinetic information (Figure 1A).Therefore, we focus on developing an appropriate PET radioligand with comprehensively validated pharmacological and pharmacokinetic properties to enable preclinical and clinical characterization of LRRK2 inhibitors.In this work, we present herein a novel synthesis of radioligands [ 11       rodents by autoradiography and PET imaging studies (Figure 1B).More importantly, in transgenic LRRK2-G2019S mutant and lipopolysaccharide (LPS)-injected mouse models, [ 18 F]4 exhibited significantly higher brain uptake compared to that of control mice.This work may serve as a roadmap for the future design of potent LRRK2 PET tracers.

Molecular Docking Study.
To probe the possible molecular interactions of compounds 3 and 4 with LRRK2, a preliminary molecular docking study was carried out.Considering the similarity (73%) of the ATP-binding site residues between LRRK2 and mammalian STE20-like protein kinase 3 (MST3), an MST3-inhibitor complex was constructed as a surrogate for the LRRK2-inhibitor complex by Autodock Vina with PDB ID 4W8E as the building template.As shown in Figure 2, compounds 3 and 4 both fell into the binding pocket of MST3.Significant hydrophobic and minor polar, glycine, and charged interactions were observed between compounds 3 and 4 with the binding pocket.Notably, a hydrogen bond between the cyano N atom in compounds 3 and 4 with the Leu102 residue of the binding domain may exist, which highlighted the significance of the cyano group for the high binding potencies of these two compounds.cyanide, whereas the radioligand [ 18 F]4 was prepared via a S N Ar nucleophilic substitution reaction of the corresponding nitro precursor 6 with [ 18 F]fluoride.Specifically, by heating the mixture of the bromide precursor 5, [ 11 C]cyanide, NH 4 HCO 3 , and CuI in N,N-dimethylformamide (DMF) at 180 °C for 5 min, the copper-mediated coupling reaction readily proceeded to produce [ 11 C]3 in 4.6% decay-corrected radiochemical yield (RCY).On the other hand, [ 18 F]4 was achieved in 18% RCY (nondecay-corrected) by reacting precursor 6 with [ 18 F]fluoride and K 2 CO 3 /K222 in dimethyl sulfoxide (DMSO) at 150 °C for 15 min.Notably, for both radioligands [ 11 C]3 and [ 18 F]4, excellent radiochemical purity and molar activity were obtained.

In Vitro Autoradiography.
To investigate the specific binding of [ 11 C]3 and [ 18 F]4 toward LRRK2, in vitro autoradiography was carried out in rat brain sections (Figures 4 and 5).As shown in Figure 4, in baseline studies, [ 11 C]3 revealed a heterogeneity of radioactivity levels in various rat brain regions.The highest uptake was seen in the hippocampus, followed by the striatum, cerebral cortex, thalamus, and cerebellum, and the lowest uptake was observed in the pons and midbrain.The radioactive distribution profile of [ 11 C]3 was in line with the LRRK2 expression pattern in rodents. 7,25,26Under self-blocking conditions (10 μM), radioactivity accumulations in all brain regions were remarkably reduced by 64−87%.Of particular note, brain regions featuring relatively high levels of LRRK2 exhibited much higher reduction of radioactive uptake, such as hippocampus (87%), striatum (85%), cerebral cortex (87%), thalamus (83%), and cerebellum (83%).By contrast, brain regions with low LRRK2 expression exhibited relatively lower reduced uptake of [ 11 C]3 (e.g., the pons, 64%).Similar to autoradiographic studies of [ 11 C]3, [ 18 F]4 provided a heterogeneous distribution (hippocampus > cerebral cortex > striatum > thalamus > cerebellum > midbrain) under baseline conditions as well as remarkably decreased uptake (34−65%) when compound 3 was used in the pretreatment studies (Figure 5), which was in line with the results from NHP autoradiographic studies. 18To further demonstrate the binding specificity of [ 18 F]4, a structurally diverse blocking reagent GNE-0877 was used in autoradiographic studies.As shown in Figure 5, the radioactivity levels in LRRK2-rich brain regions (hippocampus, cerebral cortex, striatum, and thalamus) were significantly reduced, whereas no obvious blocking results were observed in LRRK2-deficient brain regions (cerebellum and midbrain).These results indicated excellent in vitro specific binding of both [ 11 C]3 and [ 18 F]4 toward LRRK2.
2.4.Preliminary Rat PET Imaging.The promising pharmacological, pharmacokinetic, and in vitro autoradiographic data encouraged us to carry out preliminary dynamic PET imaging studies of [ 11 C]3 and [ 18 F]4.−28 Given the superiority of fluorine-18 such as clean positron emission, low positron range, and relatively long half-life compared with carbon-11, [ 18 F]4 was subjected to further investigation (Figure 6).A blocking scan was carried out with intravenous administration of compound 3 (1 mg/kg) prior to tracer injection.For both baseline and blocking studies, blood samples were extracted from the artery.Both whole-blood and plasma radioactivity concentrations were evaluated, and radiometabolites were measured in plasma samples by radio high-performance liquid chromatography (HPLC) to generate metabolite-corrected input function (Figure S2).Compartmental analyses with one-and two-tissue compartment models (1TCM and 2TCM) were carried out on regional time−activity curves.A 2TCM with reversible binding exhibited better fits to all brain regions with a stable volume of distribution (V T ).In the baseline study, V T of various brain regions ranged from 2.2 to 2.9 mL/cm 3 , confirming the high binding of the radiotracer.Pretreatment of compound 3 resulted in a significant decrease of uptake in various brain regions, which suggested encouraging binding specificity of [ 18 F]4.Furthermore, reasonable in vivo metabolic stability of [ 18 F]4 was also demonstrated with 38% and 23% parent fractions in the plasma of rats at 30 and 60 min post tracer administration, respectively (Figure S2).S2, initial high radioactive signals were seen in several peripheral organs such as the spleen, heart, lungs, pancreas, stomach, small intestine, kidneys, and liver (>4%ID/g, injected dose per gram of tissue), followed by rapid clearance in almost all of these organs except the stomach and small intestine.The slow radioactivity clearance in the small intestine, together with high radioactive signals in the small intestine and liver at 60 min post tracer injection, suggested the hepatobiliary and urinary elimination pathway of [ 18 F]4.Additionally, no remarkable de-radiofluorination was seen during the current study.To investigate the in vivo stability of [ 18 F]4 in mouse brains, we conducted a radiometabolic analysis in mouse brain homogenate at 30 min post tracer administration.The metabolism in the mouse brain was found to be reasonable with the parent fraction of 69% (Figure S3).

PET Imaging of [ 18 F]4 in Disease Models.
To further showcase the translational feasibility of [ 18 F]4, we carried out dynamic PET scans in model mice with the transgenic LRRK2-G2019S mutant and the corresponding wild-type (WT) mice.It is worth mentioning that G2019S is a dominant LRRK2 mutation, which replaces the glycine at amino acid 2019 with serine.Studies have demonstrated that G2019S leads to an increased LRRK2 kinase activity. 29urthermore, G2019S has also proved as related to not only familial and sporadic PD but also impairment of adult neurogenesis in mice. 30,31As shown in Figure 8, [ 18 F]4 rapidly accumulated in both LRRK2-G2019S and WT mouse brains within 3 min, followed by rapid elimination.[ 18 F]4 revealed statistically significant higher brain uptake in G2019S mice compared with that of WT mice.Quantitative analysis indicated that there was around 22% increase (p ≤ 0.001) of radioactivity accumulation based on the area under curve (AUC) in G2019S mice.Moreover, we measured ex vivo LRRK2 expression levels in both G2019S and WT mouse brains by Western blot, which demonstrated a 2.26-fold increase of LRRK2 enzymes in G2019S mouse brains.These results suggested that the increased uptake of [ 18 F]4 was consistent with the increased LRRK2 enzyme expression in LRRK2-G2019S mouse models, although in vivo PET results are not as profound as those obtained from in vitro Western blot analysis.Considering the significant involvement of LRRK2-G2019S mutation in PD, [ 18 F]4 may represent a promising PET ligand for studying LRRK2 changes in PD.−34 As a proof of concept, we utilized a neuroinflammatory mouse model by intracranial injection of LPS and conducted preliminary PET imaging studies with [ 18 F]4.As shown in Figure 9, the LPSinjected mice revealed much higher brain uptake of [ 18 F]4 compared to the sham group injected with phosphate-buffered saline (PBS), with 28% increase of radioactivity based on the AUCs, which is consistent with the Western blot results.This preliminary result built a foundation for the feasibility of probing LRRK2 changes in neuroinflammation rodent models.

CONCLUSIONS
Previously, PF-06447475 (3) and PF-06455943 (4) were identified as two highly potent LRRK2 inhibitors.Both compounds feature favorable pharmacological and pharmacokinetic characteristics, such as excellent binding affinity and target selectivity toward LRRK2, reasonable clearance rate, clean safety profile, high passive permeability, low P-gp efflux, and favorable unbound free fraction in the brain and plasma.As a step forward for our continuing interest in LRRK2 PET ligand development, we present herein our successful synthesis of two LRRK2 PET ligands via a copper-mediated cyanation reaction for [    uptake in the G2019S mutant and LPS-injected mice compared with that of control mice.Taken together, [ 18 F]4 may represent a novel promising PET tracer for studying LRRK2 changes during PD progression, which thereafter warrants more comprehensive preclinical and clinical validations.

EXPERIMENTAL SECTION
−38 All of the chemicals used in the synthesis of LRRK2 inhibitors and the corresponding precursor were directly acquired from commercial vendors without any purification.Silica gel was used for the purification of synthetic compounds by column chromatography, and 0.25 mm silica gel plates were used as indicators for TLC.All heating reactions were heated by a metal sand bath (WATTCAS, LAB-500).To obtain the NMR spectra of synthetic compounds, a 300 MHz Bruker spectrometer was used."ppm" was used to indicate the chemical shifts (δ), and "hertz" was the unit of coupling constants.The abbreviations of multiplicities for peaks in the HNMR and FNMR spectra were described as follows: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiple), and br (broad signal).For the measurement of mass spectrometry, an Agilent 6430 Triple Quad LC/MS was adopted with ESI as the ionization approach.No promiscuity was observed in the assay of PAINS (Pan Assay Interference Compounds) for compounds 3 and 4 with two in silico filters (http://zinc15.docking.org/patterns/home and http://www.swissadme.ch/index.php). 39High purity (≥95%) was also determined for compounds 3 and 4 by reversephase HPLC (Agilent 5 μm, Eclipse plus C18 column (4.6 mm ID × 100 mm)).Unless otherwise stated, molar activity was determined at the end of the synthesis.All animal studies were carried out following the ethical rules of our institutional policy.CD-1 mice (female, 22−24 g, 7 weeks), Sprague−Dawley (SD) rats (male, 210−230 g, 7−9 weeks), LRRK2-G2019S mutation knock-in mice, and wild-type mice (female, 25−28 g, 6−7 months) were fed ad libitum with food and water under a 12 h light/12 h dark cycle condition.The ammonium [ 11 C]cyanide solution obtained was transferred to a 1.5 mL reaction vial containing the precursor 5 (2.0 mg), CuI (1.2 mg), and anhydrous DMF (300 μL), and the reaction mixture was agitated at 180 °C for 5 min.
After cooling to room temperature, the reaction mixture was then diluted with the HPLC mobile phase (3.5 mL), followed by the injection into an HPLC column.HPLC purification was performed on a COSMOSIL Cholester column (10 mm × 250 mm, 5 μm) using a mobile phase of CH 3 CN/0.1 M NH 4 OAc (60/40) at a flow rate of 4.5 mL/min.The reaction time of [ 11 C]3 was 5.1 min.The radioactive fraction corresponding to the desired product was collected in a sterile flask, evaporated to dryness in vacuo, and reformulated in a saline solution (3 mL) containing 100 μL of 25% ascorbic acid in sterile water and 100 μL of 20% Tween 80 in ethanol.The synthesis time was 70 min from the end of bombardment.Radiochemical and chemical purity were measured by analytical HPLC COSMOSIL Cholester column (4.6 mm × 250 mm, 5 μm) using a mobile phase of CH 3 CN/0.1 M NH 4 OAc (60/40) at a flow rate of 1.0 mL/min.The identity of [ 11 C]3 was confirmed by the coinjection with unlabeled 3. The radiochemical yield was 4.6% nondecay-corrected based on [ 11 C]CO 2 with >99% radiochemical purity, and the molar activity was 2.5 Ci/μmol.

Radiosynthesis of [
18 F]4.The general procedure was described previously. 18The cyclotron-produced [ 18 F]HF (approximately 500 mCi) was separated from H 2 18 O using a Sep-Pak Accell Plus QMA Plus Light cartridge (Waters; Milford, Ma).The produced [ 18 F]HF was eluted from the cartridge with a solution of K 2 CO 3 (3 mg) and K222 (15 mg) in water (300 μL) and CH 3 CN (700 μL), and transferred to a reaction vessel in the hot cell as [ 18 F]KF.After drying the [ 18 F]KF solution at 150 °C for 30 min to remove water and CH 3 CN, a solution of nitro precursor 6 (1.5 mg) in anhydrous DMSO (700 μL) was then added.The vessel was heated at 150 °C for 15 min and then diluted with an HPLC mobile phase (3.5 mL), followed by injection into an HPLC column.HPLC purification was performed on an X-Select Prep C18 column (10 mm × 250 mm, 5 μm) using a mobile phase of CH 3 CN/0.1 M ammonium formate (AMF) (30/70) at a flow rate of 5.0 mL/min.The retention time of [ 18 F]4 was 16.9 min.The radioactive fraction corresponding to the desired product was collected in a sterile flask, diluted with 30 mL of water, and trapped on a Sep-Pak light HLB cartridge.After washing with 10 mL of water to remove the CH 3 CN residue, the product was washed out from the cartridge with 1 mL of ethanol and formulated with 10 mL of phosphate-buffered saline (PBS).The synthesis time was 70 min from the end of bombardment.Radiochemical and chemical purity were measured by analytical HPLC Gemini NX-C18 column (3 mm × 150 mm, 5 μm) using a mobile phase of CH 3 CN/ 0.1 M AMF (30/70) at a flow rate of 0.8 mL/min.The identity of [ 18 F]4 was confirmed by the coinjection with unlabeled 4. The radiochemical yield was 18% nondecay-corrected based on [ 18 F]F − with >99% radiochemical purity, and the molar activity was greater than 1.0 Ci/μmol.4.3.In Vitro Autoradiography.The general procedure for autoradiography studies was described previously with minor revision in this work. 36,40Brain sections from rats were preincubated with Tris-HCl buffer (50 mM), MgCl 2 (1.2 mM) and CaCl 2 (2 mM) solution for 20 min at ambient temperature, followed by incubation with [ 11 C]3 and [ 18 F]4 (0.48 nM).For blocking studies, PF-06447475 (10 μM), a known LRRK2 inhibitor, was added to the incubation solution in advance to determine the specificity of radioligand binding.After incubation, brain sections were rinsed with ice-cold buffer 3 times for 2 min and dipped in cold distilled water for 10 s.The brain sections were dried with cold air and then placed on imaging plates (BAS-MS2025, GE Healthcare, NJ) for optimized contact periods.Autoradiograms were obtained and regions of interest (ROIs) were carefully drawn with the reference of nakedeye observation.Radioactivity was measured using an Amersham Typhoon 5 analyzer system and expressed as photostimulated luminescence values per unit area (PSL/mm 2 ) or normalized to % of radioactivity vs control.
4.4.PET Imaging in Rats.The general procedure for PET studies was described previously 35,41 with minor modification in this work.Briefly, PET scans were carried out with an Inveon PET scanner (Siemens Medical Solutions, Knoxville, TN).Sprague−Dawley rats were kept under anesthesia using 1−2% (v/v) isoflurane during the scan.The radiotracer (ca.0.5 mCi/150 μL) was injected into the tail vein via a preinstalled catheter.A dynamic scan in the threedimensional (3D) list mode was acquired for 60 min.For pretreatment studies, a solution of PF06447475 (3 mg/kg) in 300 μL saline containing 10% ethanol and 5% Tween 80 was injected via the pre-embedded tail vein catheter at 30 min prior to tracer injection.As we previously reported, 40−42 the PET dynamic images were reconstructed using ASIPro VW software (Analysis Tools and System Setup/Diagnostics Tool, Siemens Medical Solutions).Volumes of interest, including the hippocampus, cortex, cerebellum, striatum, and thalamus, were placed using ASIPro software.The radioactivity was decay-corrected and expressed as the standard uptake value: SUV = (radioactivity per mL tissue/injected radioactivity) × body weight.

Ex Vivo Whole-Body Biodistribution of [ 18 F]4 in Mice.
The general procedure for ex vivo biodistribution studies was described previously 35,41 with minor modification in this work.Briefly, a solution of [ 18 F]4 (50 μCi/100 μL) was injected into CD-1 mice via tail vein.These mice (each time point n = 4) were sacrificed at 5, 15, 30, and 60 min post tracer injection.Major organs, including whole brain, heart, liver, lung, spleen, kidneys, small intestine (including contents), muscle, and blood samples, were quickly harvested and weighted.The radioactivity present in these tissues was measured using a Cobra Model 5002/5003 γ counter, and all radioactivity measurements were automatically decay-corrected based on the half-life of fluorine-18.The results are expressed as the percentage of injected dose per gram of wet tissue (%ID/g).
4.6.Animals and Treatments.The CD-1 mice were anesthetized and secured in a stereotaxic frame.The skull was exposed and stereotaxic coordinates (−2.6 mm dorsal/ventral, −1.5 mm lateral, and −0.2 mm anterior/posterior from bregma) according to the procedure by Haley and McCormick. 43The i.c.v.injections of 10 μg (in 2 μL of saline) of LPS and saline (control group) injections were administered using a microsyringe.The behavior of each mouse was characterized and recorded in the form of scores, including appearance, activity, level of consciousness, eyes, respiration rate, and respiration quality.Scores were determined by summing up the individual scores (Figure S10).4.7.Western Blot Analysis.Mouse brain tissues were homogenized using RIPA lysis buffer (Thermo Scientific, GA) supplemented with protease inhibitor (Thermo Scientific, GA).The homogenate was then centrifuged at 12,000g for 20 min at 4 °C.Equal amounts of protein from different experimental groups were subsequently separated via sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) and transferred onto a nitrocellulose membrane.Afterward, the membranes were blocked for 1 h using a 5% skim milk solution and incubated with LRRK2 primary antibody (ab133474, 1:1000 dilution, Abcam, MA) or β actin antibody (ab115777, 1:1000 dilution, Abcam, MA) overnight at 4 °C.Then, the membranes were incubated in a horseradish peroxidaseconjugated secondary antibody (A16096, 1:2000 dilution Thermo Scientific, GA) for 1 h at room temperature.The signals were detected using an enhanced chemiluminescence kit (1705061, Bio-Rad) with a ChemiDoc imaging system (Bio-Rad, MA), and the results were analyzed using image lab software.
4.8.PET Imaging in Mouse Models.The general procedure for PET studies was described previously 35,41 with minor modification in this work.Briefly, PET scans were carried out by a Genisys 4 PET scanner (Sofie Biosciences, Culver, CA).Mice were kept under anesthesia using 1−2% (v/v) isoflurane during the scan.The radiotracer (ca.45 μCi/100 μL) was injected into the tail vein via a preinstalled catheter.A dynamic scan in 3D list mode was acquired for 60 min.The PET dynamic images were reconstructed using G4 software (Analysis Tools and System Setup/Diagnostics Tool, Sofie Biosciences).The radioactivity was decay-corrected and expressed as the standardized uptake value: SUV = (radioactivity per mL tissue/ injected radioactivity) × body weight.

Data Availability Statement
The article contains the complete data used to support the findings of this study.
Experimental procedures; compound characterization data; computation details, and 1 H, 19 C]3 ([ 11 C]PF-06447475) via a facile copper-mediated cyanation reaction and [ 18 F]4 via a nucleophilic S N Ar displacement reaction.Although the radiosynthesis of [ 18 F]4 and its preliminary evaluation in nonhuman primates (NHPs) have

Figure 2 .
Figure 2. Molecular docking structures of compounds 3 (A) and 4 (B) onto MST3.The top insets at each panel exhibit the docking pose of each compound into the binding pocket.The bottom insets at each panel exhibit the significant hydrophobic and minor polar (light blue), glycine (white), and positively (dark blue) and negatively (red) charged interactions between each compound with the binding pocket.The PDB ID of the protein structure is4W8E.

2 . 5 .
Ex Vivo Biodistribution.In the present study, [ 18 F]4 was used for further evaluation due to its superior RCY and high molar activity, longer half-life, and improved brain distribution profile compared with that of [ 11 C]3.With the established good BBB penetration ability of [ 18 F]4, we then performed ex vivo biodistribution studies to obtain more indepth information on the whole-body distribution and clearance of [ 18 F]4.Mice were sorted into four groups based on their survival intervals (5, 15, 30, and 60 min) after intravenous administration of [ 18 F]4.As shown in Figure 7 and Table

4 . 1 .
Radiosynthesis of [ 11 C]3.[ 11 C]HCN was yielded from cyclotron-produced [ 11 C]CO 2 by the 14 N(p, α) 11 C nuclear reaction.In brief, [ 11 C]CO 2 was first converted to [ 11 C]CH 4 with H 2 on Ni at 400 °C and then to [ 11 C]HCN with NH 3 on Pt at 900 °C.He was used as a carrier gas.The [ 11 C]HCN was trapped in a solution of NH 4 HCO 3 in 1.7 mL of water (0.32 g/mL).We measured the amount of [ 11 C]CN that is captured by attaching the vent line of the reaction vial to a charcoal trap.Passing the [ 11 C]HCN gas mixture through NH 4 HCO 3 aqueous solution leads to an approximately 150 mCi of [ 11 C]HCN captured in solution at 10 min post bombardment.

Table 1 .
15,par23,24of the Pharmacological and Pharmacokinetic Properties of Representative LRRK2 Inhibitors15,18,23,24 a Biochemical LRRK2 assays (n ≥ 2).b Whole cellular LRRK2 assay (n ≥ 2); MDR1 efflux ratio.c Human liver microsomal clearance.d Transformed human liver epithelial (THLE) cell viability assay.e MDR1 efflux ratio.f Passive permeability as a rate in 1 × 10 −6 cm/s.g c log P and tPSA were calculated by ChemBioDraw Ultra 14.0 (CambridgeSoft Corporation, PerkinElmer).h Saturation assays.been described in our previous report, in this study, we aim to highlight the evaluation of [ 11 C]3 and [ 18 F]4 in rodent-based disease models.As a consequence, both radioligands demonstrated favorable brain uptake and specific bindings in National Institutes for Quantum and Radiological Science and Technology, Chiba 263-8555, Japan; orcid.org/0000-0002-3001-9605; Email: zhang.ming-rong@qst.go.jpSteven H. Liang − Department of Radiology and Imaging Sciences, Emory University, Atlanta, Georgia 30322, United States; Division of Nuclear Medicine and Molecular Imaging, Massachusetts General Hospital & Department of Radiology, Harvard Medical School, Boston, Massachusetts 02114, United States; orcid.org/0000-0003-1413-6315;Email: steven.liang@emory.eduTomoteru Yamasaki − Department of Radiopharmaceuticals Development, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Chiba 263-8555, Japan Hideki Ishii − Department of Radiopharmaceuticals Development, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Chiba 263-8555, Japan Yiding Zhang − Department of Radiopharmaceuticals Development, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Chiba 263-8555, Japan Tomomi Kokufuta − Department of Radiopharmaceuticals Development, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Chiba 263-8555, Japan Corresponding AuthorsMing-Rong Zhang − Department of Radiopharmaceuticals Development, National Institute of Radiological Sciences,