Automated synthesis, preclinical toxicity, and radiation dosimetry of [F-18]MC225 for clinical use:a tracer for measuring P-glycoprotein function at the blood-brain barrier

Introduction: [18F]MC225 is a selective substrate for P-glycoprotein (P-gp) that has good metabolic stability and shows higher baseline uptake compared with other P-gp substrates such as (R)-[11C]Verapamil. Prior to clinical translation, it is necessary to perform process validation of the radiosynthesis, assessment of preclinical toxicity, and radiation dosimetry. Methods: The production of [18F]MC225 was automated on a CFN-MPS200 multipurpose synthesizer. The acute toxicity of MC225 was evaluated at a dose of 2.5 mg/kg bodyweight, which is more than 10,000-fold the postulated maximum clinical dose of [18F]MC225. The acute toxicity of [18F]MC225 injection at a 200-fold dose, to administer a postulated dose of 185 MBq of [18F]MC225, was also evaluated after the decay-out of 18F. The mutagenicity of MC225 was studied by a reverse mutation test using Salmonella typhimurium and Escherichia coli (Ames test). In vivo biodistribution and dosimetry studies of [18F]MC225 were carried out in normal mice. Human dosimetry was estimated using OLINDA software. Results: The mean decay-corrected yields of [18F]MC225 at end of synthesis were 13%, with > 99% radiochemical purity, > 1000 GBq/!mol molar activity, and ! 1.5 !g/185 MBq of total chemical contents. All process validation batches complied with the product specifications and the process was confirmed to be appropriate for the production of [18F]MC225. No acute toxicity of MC225 or [18F]MC225 injection was found. No mutagenic activity was observed for MC225. The biodistribution study demonstrated both hepatobiliary and renal excretion of radioactivity. The most critical organ was the pancreas, with (63.8 !Gy/MBq) or without urination (63.9 !Gy/MBq) at 360 min after injection. The estimated effective dose (!Sv/MBq) with and without urination at 360 min after injection was calculated as 15.7 and 16.9, respectively. Conclusion: [18F]MC225 shows acceptable pharmacological safety at the dose required for adequate PET imaging. The potential risk associated with [18F]MC225 PET imaging is well within acceptable dose limits

Test-retest reproducibility of cerebral adenosine A(2A) receptor quantification using [C-11]preladenant

Objective To evaluate the reproducibility of cerebral adenosine A(2A) receptor (A(2A)R) quantification using [C-11]preladenant ([C-11]PLN) and PET in a test-retest study. Methods Eight healthy male volunteers were enrolled. Dynamic 90 min PET scans were performed twice at the same time of the day to avoid the effect of diurnal variation. Subjects refrained from caffeine from 12 h prior to scanning, and serum caffeine was measured before radioligand injection. Arterial blood was sampled repeatedly during scanning and the fraction of the parent compound in plasma was determined. Total distribution volume (V-T) was estimated using 1- and 2-tissue compartment models (1-TCM and 2-TCM, respectively) and Logan graphical analysis (Logan plot) (t* = 30 min). Plasma-free fraction (f(P)) of [C-11]PLN was measured and used for correction of V-T values. Distribution volume ratio (DVR) was calculated from V-T of target and reference regions and obtained by noninvasive Logan graphical reference tissue model (LGAR) (t* = 30 min). Absolute test-retest variability (aTRV), and intra-class correlation coefficient (ICC) of V-T and DVR were calculated as indexes of repeatability. Correlation between DVR and serum concentration of caffeine (a nonselective A(2A)R blocker) was analyzed by Pearson's correlation analysis. Results Regional time-activity curves were well described by 2-TCM models. Estimation of V-T by 2-TCM produced some erroneous values; therefore, the more robust Logan plot was selected as the appropriate model. Global mean aTRV was 20% for V-T and 14% for V-T/f(P) (ICC, 0.72 for V-T and 0.87 for V-T/f(P)). Global mean aTRV of DVR was 13% for Logan plot and 10% for LGAR (ICC, 0.70 for Logan plot and 0.81 for LGAR). DVR estimates using LGAR and Logan plot were in good agreement (r(2) = 0.96). Coefficients of variation for V-T, V-T/f(P), DVR (Logan plot), and DVR (LGAR) were 47%, 47%, 27%, and 18%, respectively. Despite low serum caffeine levels, significant concentration-dependent effects on [C-11]PLN binding to target regions were observed (p < 0.01). Conclusions In this study, moderate test-retest reproducibility and large inter-subject differences were observed with [C-11]PLN PET, possibly attributable to competition by baseline amount of caffeine. Analysis of plasma caffeine concentration is recommended during [C-11]PLN PET studies

First clinical assessment of [ 18 F]MC225, a novel fluorine-18 labelled PET tracer for measuring functional P-glycoprotein at the blood-brain barrier

Objective: 5-(1-(2-[18F]fluoroethoxy))-[3-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-propyl]-5,6,7,8-tetrahydronaphthalen ([18F]MC225) is a selective substrate for P-glycoprotein (P-gp), possessing suitable properties for measuring overexpression of P-gp in the brain. This is the first-in-human study to examine safety, radiation dosimetry and P-gp function at the blood-brain barrier (BBB) of [18F]MC225 in healthy subjects. Methods: [18F]MC225 biodistribution and dosimetry were determined in 3 healthy male subjects, using serial 2 h and intermittent 4 and 6 h whole-body PET scans acquired after [18F]MC225 injection. Dynamic [18F]MC225 brain PET (90 min) was obtained in 5 healthy male subjects. Arterial blood was sampled at various time intervals during scanning and the fraction of unchanged [18F]MC225 in plasma was determined. T1-weighted MRI was performed for anatomical coregistration. Total distribution volume (VT) was estimated using 1- and 2-tissue-compartment models (1-TCM and 2-TCM, respectively). VT was also estimated using the Logan graphical method (Logan plot) (t* = 20 min). Surrogate parameters without blood sampling (area-under the curve [AUC] of regional time-activity curves [TACs] and negative slope of calculated TACs) were compared with the VT values. Results: No serious adverse events occurred throughout the study period. Although biodistribution implied hepatobiliary excretion, secretion of radioactivity from liver to small intestine through the gallbladder was very slow. Total renal excreted radioactivity recovered during 6 h after injection was 0.9). AUCs of TACs were positively correlated with VT (2-TCM) values (r2: AUC0-60 min = 0.61, AUC0-30 min = 0.62, AUC30-60 min = 0.59, p < 0.0001). Negative slope of SUV TACs was negatively correlated with VT (2-TCM) values (r2 = 0.53, p < 0.0001). Conclusions: This initial evaluation indicated that [18F]MC225 is a suitable and safe PET tracer for measuring P-gp function at the BBB. Keywords: Blood–Brain barrier; Dosimetry; First-in-human; P-glycoprotein; Positron emission tomography

Pharmacokinetic Modeling of [ 18 F]MC225 for Quantification of the P-Glycoprotein Function at the Blood-Brain Barrier in Non-Human Primates with PET

[18F]MC225 has been developed as a weak substrate of P-glycoprotein (P-gp) aimed to measure changes in the P-gp function at the blood–brain barrier with positron emission tomography. This study evaluates [18F]MC225 kinetics in non-human primates and investigates the effect of both scan duration and P-gp inhibition. Three rhesus monkeys underwent two 91-min dynamic scans with blood sampling at baseline and after P-gp inhibition (8 mg/kg tariquidar). Data were analyzed using the 1-tissue compartment model (1-TCM) and 2-tissue compartment model (2-TCM) fits using metabolite-corrected plasma as the input function and for various scan durations (10, 20, 30, 60, and 91 min). The preferred model was chosen according to the Akaike information criterion and the standard errors (%) of the estimated parameters. For the 91-min scan duration, the influx constant K1 increased by 40.7% and the volume of distribution (VT) by 30.4% after P-gp inhibition, while the efflux constant k2 did not change significantly. Similar changes were found for all evaluated scan durations. K1 did not depend on scan duration (10 min—K1 = 0.2191 vs 91 min—K1 = 0.2258), while VT and k2 did. A scan duration of 10 min seems sufficient to properly evaluate the P-gp function using K1 obtained with 1-TCM. For the 91-min scan, VT and K1 can be estimated with a 2-TCM, and both parameters can be used to assess P-gp function

Head-to-head comparison of (R)-[C-11]verapamil and [F-18]MC225 in non-human primates, tracers for measuring P-glycoprotein function

PURPOSE: P-glycoprotein (P-gp) function is altered in several brain disorders; thus, it is of interest to monitor the P-gp function in vivo using PET. (R)-[11C]verapamil is considered the gold standard tracer to measure the P-gp function; however, it presents some drawbacks that limit its use. New P-gp tracers have been developed with improved properties, such as [18F]MC225. This study compares the characteristics of (R)-[11C]verapamil and [18F]MC225 in the same subjects.METHODS: Three non-human primates underwent 4 PET scans: 2 with (R)-[11C]verapamil and 2 with [18F]MC225, at baseline and after P-gp inhibition. The 30-min PET data were analyzed using 1-Tissue Compartment Model (1-TCM) and metabolite-corrected plasma as input function. Tracer kinetic parameters at baseline and after inhibition were compared. Regional differences and simplified methods to quantify the P-gp function were also assessed.RESULTS: At baseline, [18F]MC225 VT values were higher, and k2 values were lower than those of (R)-[11C]verapamil, whereas K1 values were not significantly different. After inhibition, VT values of the 2 tracers were similar; however, (R)-[11C]verapamil K1 and k2 values were higher than those of [18F]MC225. Significant regional differences between tracers were found at baseline, which disappeared after inhibition. The positive slope of the SUV-TAC was positively correlated to the K1 and VT of both tracers.CONCLUSION: [18F]MC225 and (R)-[11C]verapamil show comparable sensitivity to measure the P-gp function in non-human primates. Moreover, this study highlights the 30-min VT as the best parameter to measure decreases in the P-gp function with both tracers. [18F]MC225 may become the first radiofluorinated tracer able to measure decreases and increases in the P-gp function due to its higher baseline VT.</p

Advances in the Development of PET Ligands Targeting Histone Deacetylases for the Assessment of Neurodegenerative Diseases

Epigenetic alterations of gene expression have emerged as a key factor in several neurodegenerative diseases. In particular, inhibitors targeting histone deacetylases (HDACs), which are enzymes responsible for deacetylation of histones and other proteins, show therapeutic effects in animal neurodegenerative disease models. However, the details of the interaction between changes in HDAC levels in the brain and disease progression remain unknown. In this review, we focus on recent advances in development of radioligands for HDAC imaging in the brain with positron emission tomography (PET). We summarize the results of radiosynthesis and biological evaluation of the HDAC ligands to identify their successful results and challenges. Since 2006, several small molecules that are radiolabeled with a radioisotope such as carbon-11 or fluorine-18 have been developed and evaluated using various assays including in vitro HDAC binding assays and PET imaging in rodents and non-human primates. Although most compounds do not readily cross the blood-brain barrier, adamantane-conjugated radioligands tend to show good brain uptake. Until now, only one HDAC radioligand has been tested clinically in a brain PET study. Further PET imaging studies to clarify age-related and disease-related changes in HDACs in disease models and humans will increase our understanding of the roles of HDACs in neurodegenerative diseases

18F-Fluorination of 2-methyl-6-nitrobenzenesulfonate ester and its application to the synthesis of a 18F-labeled amino acid.

In the development of an efficient 18F-labeling method for the synthesis of PET tracers, it is essential not only to improve the efficiency of [18F]fluorine incorporation into a carrier but also to minimize non-radioactive side products from the precursor. Highly reactive sulfonate esters are promising precursors for 18F-nucleophilic fluorination under mild conditions. However, the expected labeling efficiency from the precursor is often hampered by its competitive degradation due to coexisting bases during 18F-fluorination. In this report, we designed a 2-methyl-6-nitrobenzenesulfonate (2-MeNs) ester as a precursor for 18F-fluorination, in which the methyl group suppresses hydrolysis of the sulfonate ester via steric hindrance. An increase in labeling efficiency in the 18F-labeling of a neopentyl labeling group was observed for the neopentyl 2-MeNs ester compared with that of the 2-nitrobenzenesulfonate ester. Ultimately, we achieved the automated synthesis of an 18F-labeled amino acid using a neopentyl labeling group by using a 2-MeNs ester as a precursor

Comparison of the binding characteristics of [(18)F]THK-523 and other amyloid imaging tracers to Alzheimer\u27s disease pathology.

PurposeExtensive deposition of senile plaques and neurofibrillary tangles in the brain is a pathological hallmark of Alzheimer disease (AD). Although several PET imaging agents have been developed for in vivo detection of senile plaques, no PET probe is currently available for selective detection of neurofibrillary tangles in the living human brain. Recently, [18F]THK-523 was developed as a potential in vivo imaging probe for tau pathology. The purpose of this study was to compare the binding properties of [18F]THK-523 and other amyloid imaging agents, including PiB, BF-227 and FDDNP, to synthetic protein fibrils and human brain tissue.\nMethodsIn vitro radioligand binding assays were conducted using synthetic amyloid beta42 and K18 deltaK280-tau fibrils. Nonspecific binding was determined by the addition of unlabelled compounds at a concentration of 2 micro M. To examine radioligand binding to neuropathological lesions, in vitro autoradiography was conducted using sections of AD brain.\nResults[18F]THK-523 showed higher affinity for tau fibrils than for Abeta fibrils, whereas the other probes showed a higher affinity for Abeta fibrils. The autoradiographic analysis indicated that [18F]THK-523 accumulated in the regions containing a high density of tau protein deposits. Conversely, PiB and BF-227 accumulated in the regions containing a high density of Abeta plaques.\nConclusionThese findings suggest that the unique binding profile of [18F]THK-523 can be used to identify tau deposits in AD brain

Characteristics of Tau and Its Ligands in PET Imaging

Tau deposition is one of the neuropathological hallmarks in Alzheimer’s disease as well as in other neurodegenerative disorders called tauopathies. Recent efforts to develop selective tau radiopharmaceuticals have allowed the visualization of tau deposits in vivo. In vivo tau imaging allows the assessment of the regional distribution of tau deposits in a single human subject over time for determining the pathophysiology of tau accumulation in aging and neurodegenerative conditions as well as for application in drug discovery of anti-dementia drugs as surrogate markers. However, tau deposits show complicated characteristics because of different isoform composition, histopathology, and ultrastructure in various neurodegenerative conditions. In addition, since tau radiopharmaceuticals possess different chemotype classes, they may show different binding characteristics with heterogeneous tau deposits. In this review, we describe the characteristics of tau deposits and their ligands that have β-sheet binding properties, and the status of tau imaging in clinical studies

Synthesis of functionalized (-)-Epigallocatechin gallates by amidomethylation with amidomethyl 2-alkynylbenzoates and its application to the synthesis of 18F-labled labled EGCG

In this study, we report a gold-catalyzed electrophilic aromatic substitution of protected (-)-epigallocatechin gallates (EGCG) with amidomethyl 2-alkynylbenzoates. Treatment of the amidomethyl 2-alkynylbenzoates with (Ph3P)AuOTf provided N-acyl imines under neutral conditions. The N-acyl imines underwent electrophilic aromatic substitution of EGCG at the electron-rich 8 and 6 positions of the A ring. This method facilitated direct modification of a protected EGCG to produce EGCG derivatives containing a (2,2-dimethyl-1,3-dioxan-5-yl)methyl sulfonyloxy group on the A ring, serving as synthetic precursors for 18F-labeled EGCGs. Importantly, the introduction of the radiohalogen labeling unit at the A ring of EGCG did not diminish their cytotoxicity against U266 cells compared to EGCG. Finally, we successfully synthesized a 18F-labeled EGCG via 18F-fluorination of the precursors followed by acidic deprotection