Preclinical evaluation of [11 C]L-235 as a radioligand for Positron Emission Tomography cathepsin K imaging in bone.

The cathepsin K (CatK) enzyme is abundantly expressed in osteoclasts, and CatK inhibitors have been developed for the treatment of osteoporosis. In our effort to support discovery and clinical evaluations of a CatK inhibitor, we sought to discover a radioligand to determine target engagement of the enzyme by therapeutic candidates using positron emission tomography (PET). L-235, a potent and selective CatK inhibitor, was labeled with carbon-11. PET imaging studies recording baseline distribution of [11 C]L-235, and chase and blocking studies using the selective CatK inhibitor MK-0674 were performed in juvenile and adult nonhuman primates (NHP) and ovariectomized rabbits. Retention of the PET tracer in regions expected to be osteoclast-rich compared with osteoclast-poor regions was examined. Increased retention of the radioligand was observed in osteoclast-rich regions of juvenile rabbits and NHP but not in the adult monkey or adult ovariectomized rabbit. Target engagement of CatK was observed in blocking studies with MK-0674, and the radioligand retention was shown to be sensitive to the level of MK-0674 exposure. [11 C]L-235 can assess target engagement of CatK in bone only in juvenile animals. [11 C]L-235 may be a useful tool for guiding the discovery of CatK inhibitors.

A simplified radiosynthesis of [18 F]MK-6240 for tau PET imaging.

[18 F]MK-6240 (6-(fluoro)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine) is a highly selective PET radiotracer for the in vivo imaging of neurofibrillary tangles (NFTs). [18 F]MK-6240 was synthesized in one step from its bis-Boc protected precursor N-[(tert-butoxy)carbonyl]-N-(6-nitro-3-[1H-pyrrolo[2,3-c]pyridin-1-yl]isoquinolin-5-yl) carbamate in DMSO using [18 F] fluoride with TEA HCO3 with step-wise heating up to 150°C, resulting in an isolated radiochemical yield of 9.8% ± 1.8% (n = 3) calculated from the end of bombardment (5.2% ± 1.0% calculated from the end of synthesis). This new synthetic approach eliminates the acidic deprotection of the bis-Boc 18 F-labeled intermediate, which reduces the number of operations necessary for the synthesis as well as losses, which occur during deprotection and neutralization of the crude product mixture prior to the HPLC purification. The synthesis was performed automatically with a single-use cassette on an IBA Synthera+ synthesis module. This synthesis method affords the radioligand with a reliable radiochemical yield, high radiochemical purity, and a high molar activity. [18 F]MK-6240 synthesized with this method has been regularly (n > 60) used in our ongoing human and animal PET imaging studies.

cGMP production of the radiopharmaceutical [18 F]MK-6240 for PET imaging of human neurofibrillary tangles.

Fluorine-18-labelled 6-(fluoro)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine ([18 F]MK-6240) is a novel potent and selective positron emission tomography (PET) radiopharmaceutical for detecting human neurofibrillary tangles, which are made up of aggregated tau protein. Herein, we report the fully automated 2-step radiosynthesis of [18 F]MK-6240 using a commercially available radiosynthesis module, GE Healthcare TRACERlab FXFN . Nucleophilic fluorination of the 5-diBoc-6-nitro precursor with potassium cryptand [18 F]fluoride (K[18 F]/K222 ) was performed by conventional heating, followed by acid deprotection and semipreparative high-performance liquid chromatography under isocratic conditions. The isolated product was diluted with formulation solution and sterile filtered under Current Good Manufacturing Practices, and quality control procedures were established to validate this radiopharmaceutical for human use. At the end of synthesis, 6.3 to 9.3 GBq (170-250 mCi) of [18 F]MK-6240 was formulated and ready for injection, in an uncorrected radiochemical yield of 7.5% ± 1.9% (relative to starting [18 F]fluoride) with a specific activity of 222 ± 67 GBq/µmol (6.0 ± 1.8 Ci/µmol) at the end of synthesis (90 minutes; n = 3). [18 F]MK-6240 was successfully validated for human PET studies meeting all Food and Drug Administration and United States Pharmacopeia requirements for a PET radiopharmaceutical. The present method can be easily adopted for use with other radiofluorination modules for widespread clinical research use.

Palladium-mediated oxidative carbonylation reactions for the synthesis of (11) C-radiolabelled ureas.

Palladium(II)-mediated oxidative carbonylation reactions have been used to synthesize (11) C-radiolabelled ureas via the coupling of amines with [(11) C]carbon monoxide, in a one-pot process. Following trapping of (11) CO in a solution of copper(I) tris(3,5-dimethylpyrazolyl)borate, homocoupling reactions of primary aliphatic amines proceed in the presence of Pd(PPh3 )2 Cl2 to give the corresponding N,N-disubstituted [(11) C]ureas. Secondary amines do not produce the corresponding N,N,N,N-tetrasubsituted [(11) C]ureas under these conditions. This difference in reactivity allows for the formation of unsymmetrical N,N',N'-trisubstituted [(11) C]ureas using a mixture of a primary amine and a reactive secondary amine. The potential use of this method in positron emission tomography (PET) was demonstrated by the synthesis of the M1 muscarinic acetylcholine receptor radiotracer, [(11) C-carbonyl]GSK1034702.

Monoamine transporter occupancy of a novel triple reuptake inhibitor in baboons and humans using positron emission tomography.

The selection of a therapeutically meaningful dose of a novel pharmaceutical is a crucial step in drug development. Positron emission tomography (PET) allows the in vivo estimation of the relationship between the plasma concentration of a drug and its target occupancy, optimizing dose selection and reducing the time and cost of early development. Triple reuptake inhibitors (TRIs), also referred to as serotonin-norepinephrine-dopamine reuptake inhibitors, enhance monoaminergic neurotransmission by blocking the action of the monoamine transporters, raising extracellular concentrations of those neurotransmitters. GSK1360707 [(1R,6S)-1-(3,4-dichlorophenyl)-6-(methoxymethyl)-4-azabicyclo[4.1.0]heptane] is a novel TRI that until recently was under development for the treatment of major depressive disorder; its development was put on hold for strategic reasons. We present the results of an in vivo assessment of the relationship between plasma exposure and transporter blockade (occupancy). Studies were performed in baboons (Papio anubis) to determine the relationship between plasma concentration and occupancy of brain serotonin reuptake transporter (SERT), dopamine reuptake transporter (DAT), and norepinephrine uptake transporter (NET) using the radioligands [(11)C]DASB [(N,N-dimethyl-2-(2-amino-4-cyanophenylthio) benzylamine], [(11)C]PE2I [N-(3-iodoprop-2E-enyl)-2ß-carbomethoxy-3ß-(4-methylphenyl)nortropane], and [(11)C]2-[(2-methoxyphenoxy)phenylmethyl]morpholine (also known as [(11)C]MRB) and in humans using [(11)C]DASB and [(11)C]PE2I. In P. anubis, plasma concentrations resulting in half-maximal occupancy at SERT, DAT, and NET were 15.16, 15.56, and 0.97 ng/ml, respectively. In humans, the corresponding values for SERT and DAT were 6.80 and 18.00 ng/ml. GSK1360707 dose-dependently blocked the signal of SERT-, DAT-, and NET-selective PET ligands, confirming its penetration across the blood-brain barrier and blockade of all three monoamine transporters in vivo.

Use of LC-MS for the quality control of radiopharmaceuticals: example of [(18)F]ML10.

[(18)F]ML10 is a promising novel low molecular weight positron emission tomography probe for apoptosis. As part of the quality control to support clinical studies for cancer therapy monitoring in the GSK Clinical Imaging Centre, a simple and sensitive liquid chromatography mass spectrometry method has been developed and validated for the quantification of total ML10 and impurity content in the final product. Chromatographic separation of ML10 and its radiolabelling precursor and impurities was achieved. Mass curves were constructed from a concentration range of ML10 and known impurities and were linear. Quantification was achieved by comparison of the area under the curve for ML10 content (m/z = 205) and the mass curve. The method was validated over a concentration range of 0.1-1 µg/ml.

Evaluation of 18F-flutemetamol amyloid PET image analysis parameters on the effect of verubecestat on brain amlyoid load in Alzheimer's disease.

PURPOSE: The BACE inhibitor verubecestat was previously found to reduce amyloid load as assessed by 18F-flutemetamol positron emission tomography (PET) composite cortical standard uptake value ratio (SUVr) in patients with mild-to-moderate Alzheimer's disease (AD) in a substudy of the EPOCH trial. Here, we report on additional analyses relevant to the EPOCH PET data, to help inform on the use of PET for assessing amlyloid load in AD clinical trials. PROCEDURES: The analyses addressed (1) identification of an optimal 18F-flutemetamol reference region, (2) determination of the threshold to characterize the magnitude of the longitudinal change, and (3) the impact of partial volume correction (PVC). Pons and subcortical white matter were evaluated as reference regions. The SUVr cutoffs and final reference region choice were determined using 162 18F-flutemetamol PET scans from the AIBL dataset. 18F-flutemetamol SUVrs were computed at baseline and at Week 78 in EPOCH participants who received verubecestat 12 mg (n = 14), 40 mg (n = 20), or placebo (n = 20). Drug effects on amyloid load were computed using either Meltzer (MZ), or symmetric geometric transfer matrix (SGTM) PVC and compared to uncorrected data. RESULTS: The optimal subcortical white matter and pons SUVr cutoffs were determined to be 0.69 and 0.62, respectively. The effect size to detect longitudinal change was higher for subcortical white matter (1.20) than pons (0.45). Hence, subcortical white matter was used as the reference region for the EPOCH PET substudy. In EPOCH, uncorrected baseline SUVr values correlated strongly with MZ PVC (r2 = 0.94) and SGTM PVC (r2 = 0.92) baseline SUVr values, and PVC did not provide improvement for evaluating treatment effects on amyloid load at Week 78. No change from baseline was observed in the placebo group at Week 78, whereas a 0.02 and a 0.04 decrease in SUVr were observed in the 12 mg and 40 mg arms, with the latter representing a 22% reduction in the amyloid load above the detection threshold. CONCLUSIONS: Treatment-related 18F-flutemetamol longitudinal changes in AD clinical trials can be quantified using a subcortical white matter reference region without PVC. CLINICAL TRIAL REGISTRATION: clinicaltrials.gov NCT01739348.

Further evaluation of quantum chemical methods for the prediction of non-specific binding of positron emission tomography tracers.

The non-specific binding of candidate positron emission tomography (PET) radiotracers causes resulting PET images to have poor contrast and is a key determinant for the success or failure of imaging drugs. Non-specific binding is thought to arise when radiotracers bind to cell membranes and moieties other than their intended target. Our previous preliminary work has proposed the use of the drug-lipid interaction energy descriptor to predict the level of non-specific binding in vivo using a limited set of ten well known PET radiotracers with kinetic modelling data taken from the literature. This work validates and extends the use of the drug-lipid interaction energy descriptor using a new set of twenty-two candidate PET radiotracers with non-specific binding data recently collected at the same imaging centre with consistent methodology. As with the previous set of radiotracers, a significant correlation is found between the quantum chemical drug-lipid interaction energy and in vivo non-specific binding experimental values. In an effort to speed up the calculation process, several semi-empirical quantum chemical methods were assessed for their ability to reproduce the ab initio results. However no single semi-empirical method was found to consistently reproduce the level of correlation achieved with ab initio quantum chemical methods.

PET/CT Imaging of 89Zr-N-sucDf-Pembrolizumab in Healthy Cynomolgus Monkeys.

PURPOSE: Programmed cell death-1 receptor (PD-1) and its ligand (PD-L1) are the targets for immunotherapy in many cancer types. Although PD-1 blockade has therapeutic effects, the efficacy differs between patients. Factors contributing to this variability are PD-L1 expression levels and immune cells present in tumors. However, it is not well understood how PD-1 expression in the tumor microenvironment impacts immunotherapy response. Thus, imaging of PD-1-expressing immune cells is of interest. This study aims to evaluate the biodistribution of Zirconium-89 (89Zr)-labeled pembrolizumab, a humanized IgG4 kappa monoclonal antibody targeting PD-1, in healthy cynomolgus monkeys as a translational model of tracking PD-1-positive immune cells. PROCEDURES: Pembrolizumab was conjugated with the tetrafluorophenol-N-succinyl desferal-Fe(III) ester (TFP-N-sucDf) and subsequently radiolabeled with 89Zr. Four cynomolgus monkeys with no previous exposure to humanized monoclonal antibodies received tracer only or tracer co-injected with pembrolizumab intravenously over 5 min. Thereafter, a static whole-body positron emission tomography (PET) scan was acquired with 10 min per bed position on days 0, 2, 5, and 7. Image-derived standardized uptake values (SUVmean) were quantified by region of interest (ROI) analysis. RESULTS: 89Zr-N-sucDf-pembrolizumab was synthesized with high radiochemical purity (> 99 %) and acceptable molar activity (> 7 MBq/nmol). In animals dosed with tracer only, 89Zr-N-sucDf-pembrolizumab distribution in lymphoid tissues such as mesenteric lymph nodes, spleen, and tonsils increased over time. Except for the liver, low radiotracer distribution was observed in all non-lymphoid tissue including the lung, muscle, brain, heart, and kidney. When a large excess of pembrolizumab was co-administered with a radiotracer, accumulation in the lymph nodes, spleen, and tonsils was reduced, suggestive of target-mediated accumulation. CONCLUSIONS: 89Zr-N-sucDf-pembrolizumab shows preferential uptake in the lymphoid tissues including the lymph nodes, spleen, and tonsils. 89Zr-N-sucDf-pembrolizumab may be useful in tracking the distribution of a subset of immune cells in non-human primates and humans. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02760225.

Test-retest characteristic of [18F]MK-6240 quantitative outcomes in cognitively normal adults and subjects with Alzheimer's disease.

[18F]MK-6240 is a selective, high-affinity PET radiotracer for imaging neurofibrillary tangles (NFT) in Alzheimer's disease (AD). Herein, we report test-retest (T-RT) reproducibility of [18F]MK-6240 in AD and healthy volunteers (HV). Twelve subjects with AD and three cognitively normal HV were enrolled in the study and dynamically scanned for 150 min with [18F]MK-6240 under a T-RT protocol. Two radioactivity doses were investigated: 165 ± 3 MBq (n = 6) and 300 ± 40 MBq (n = 9). Serial arterial blood samples were taken for each scan to obtain metabolite-corrected input functions. Following intravenous administration of [18F]MK-6240, the tracer rapidly partitioned into the brain and its heterogenous distribution pattern was consistent with known NFT pathology in AD. In contrast, uptake in HV was low and uniform across the brain parenchyma. Across all subjects, average T-RT variabilities in NFT-rich regions were ∼21%, ∼14% and ∼6% for various quantitative metrics: total distribution volume (VT), binding potential (BPND), and standardized uptake ratio (SUVR90-120), respectively. No significant differences in SUVR T-RT variability were observed between the high and low injected radioactivity groups (5.6% and 6.1%, respectively). This work suggests [18F]MK-6240 has adequate SUVR T-RT characteristics supporting the use of this outcome in future studies.

Preclinical Safety Evaluation and Human Dosimetry of [18F]MK-6240, a Novel PET Tracer for Imaging Neurofibrillary Tangles.

PURPOSE: [18F]MK-6240 is a selective, high-affinity positron emission tomography tracer for imaging neurofibrillary tangles, a key pathological signature that correlates with cognitive decline in Alzheimer disease. This report provides safety information from preclinical toxicology studies and first-in-human whole-body biodistribution and dosimetry studies of [18F]MK-6240 for its potential application in human brain imaging studies. PROCEDURES: MK-6240 was administered intravenously (IV) in a 7-day rat toxicity study at × 50, × 100, and × 1000 dose margins relative to projected highest clinical dose of 0.333 µg/kg. The IV formulation of MK-6240 for clinical use and the formulation used in the 7-day rat toxicity study was tested for hemolysis potential in human and Wistar rat whole blood. Sequential whole-body positron emission tomography scans were performed in three healthy young subjects after IV bolus injection of 180 ± 0.3 MBq [18F]MK-6240 to characterize organ biodistribution and estimate whole-body radiation exposure (effective dose). RESULTS: MK-6240 administered IV in a 7-day rat toxicity study did not show any test article-related changes. The no-observed-adverse-effect level in rats was ≥ 333 µg/kg/day which provides a margin 1000-fold over an anticipated maximum clinical dose of 0.333 µg/kg. Additionally, the MK-6240 formulation was not hemolytic in human or Wistar rat blood. [18F]MK-6240 activity was widely distributed to the brain and the rest of the body, with organ absorbed doses largest for the gall bladder (202 µGy/MBq). The average (±SD) effective dose was 29.4 ± 0.6 µSv/MBq, which is in the typical range for F-18 radiolabeled ligands. CONCLUSIONS: Microdoses of [18F]MK-6240 are safe for clinical positron emission tomography imaging studies. Single IV administration of 185 MBq (5 mCi) [18F]MK-6240 is anticipated to result in a total human effective dose of 5.4 mSv and thus allows multiple positron emission tomography scans of the same subject per year.

Dopamine D3 receptor antagonists: the quest for a potentially selective PET ligand. Part 3: Radiosynthesis and in vivo studies.

Compound 1 is a potent and selective antagonist of the dopamine D(3) receptor. With the aim of developing a carbon-11 labeled ligand for the dopamine D(3) receptor, 1 was selected as a potential PET probe. [(11)C]1 was obtained by palladium catalyzed cross coupling using [(11)C]cyanide and 4 with a specific activity of 55.5+/-25.9GBq/micromol (1.5+/-0.7Ci/micromol). [(11)C]1 was tested in porcine and non-human primate models to assess its potential as a radioligand for PET imaging of the dopamine D(3) receptor. We conclude that in both species and despite appropriate in vitro properties, [(11)C]1 does not show any specific signal for the dopamine D(3) receptor.

Brain Imaging of Alzheimer Dementia Patients and Elderly Controls with 18F-MK-6240, a PET Tracer Targeting Neurofibrillary Tangles.

18F-MK-6240 (18F-labeled 6-(fluoro)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine) is a highly selective, subnanomolar-affinity PET tracer for imaging neurofibrillary tangles (NFTs). Plasma kinetics, brain uptake, and preliminary quantitative analysis of 18F-MK-6240 in healthy elderly (HE) subjects, subjects with clinically probable Alzheimer disease (AD), and subjects with amnestic mild cognitive impairment were characterized in a study that is, to our knowledge, the first to be performed on humans. Methods: Dynamic PET scans of up to 150 min were performed on 4 cognitively normal HE subjects, 4 AD subjects, and 2 amnestic mild cognitive impairment subjects after a bolus injection of 152-169 MBq of 18F-MK-6240 to evaluate tracer kinetics and distribution in brain. Regional SUV ratio (SUVR) and distribution volume ratio were determined using the cerebellar cortex as a reference region. Total distribution volume was assessed by compartmental modeling using radiometabolite-corrected input function in a subgroup of 6 subjects. Results:18F-MK-6240 had rapid brain uptake with a peak SUV of 3-5, followed by a uniformly quick washout from all brain regions in HE subjects; slower clearance was observed in regions commonly associated with NFT deposition in AD subjects. In AD subjects, SUVR between 60 and 90 min after injection was high (approximately 2-4) in regions associated with NFT deposition, whereas in HE subjects, SUVR was approximately 1 across all brain regions, suggesting high tracer selectivity for binding NFTs in vivo. 18F-MK-6240 total distribution volume was approximately 2- to 3-fold higher in neocortical and medial temporal brain regions of AD subjects than in HE subjects and stabilized by 60 min in both groups. Distribution volume ratio estimated by the Logan reference tissue model or compartmental modeling correlated well (R2 > 0.9) to SUVR from 60 to 90 min for AD subjects. Conclusion:18F-MK-6240 exhibited favorable kinetics and high binding levels to brain regions with a plausible pattern for NFT deposition in AD subjects. In comparison, negligible tracer binding was observed in HE subjects. This pilot study suggests that simplified ratio methods such as SUVR can be used to quantify NFT binding. These results support further clinical development of 18F-MK-6240 for potential application in longitudinal studies.

In vivo quantification of neurofibrillary tangles with [18F]MK-6240.

BACKGROUND: Imaging agents capable of quantifying the brain's tau aggregates will allow a more precise staging of Alzheimer's disease (AD). The aim of the present study was to examine the in vitro properties as well as the in vivo kinetics, using gold standard methods, of the novel positron emission tomography (PET) tau imaging agent [18F]MK-6240. METHODS: In vitro properties of [18F]MK-6240 were estimated with autoradiography in postmortem brain tissues of 14 subjects (seven AD patients and seven age-matched controls). In vivo quantification of [18F]MK-6240 binding was performed in 16 subjects (four AD patients, three mild cognitive impairment patients, six healthy elderly individuals, and three healthy young individuals) who underwent 180-min dynamic scans; six subjects had arterial sampling for metabolite correction. Simplified approaches for [18F]MK-6240 quantification were validated using full kinetic modeling with metabolite-corrected arterial input function. All participants also underwent amyloid-PET and structural magnetic resonance imaging. RESULTS: In vitro [18F]MK-6240 uptake was higher in AD patients than in age-matched controls in brain regions expected to contain tangles such as the hippocampus, whereas no difference was found in the cerebellar gray matter. In vivo, [18F]MK-6240 displayed favorable kinetics with rapid brain delivery and washout. The cerebellar gray matter had low binding across individuals, showing potential for use as a reference region. A reversible two-tissue compartment model well described the time-activity curves across individuals and brain regions. Distribution volume ratios using the plasma input and standardized uptake value ratios (SUVRs) calculated after the binding approached equilibrium (90 min) were correlated and higher in mild cognitive impairment or AD dementia patients than in controls. Reliability analysis revealed robust SUVRs calculated from 90 to 110 min, while earlier time points provided inaccurate estimates. CONCLUSIONS: This evaluation shows an [18F]MK-6240 distribution in concordance with postmortem studies and that simplified quantitative approaches such as the SUVR offer valid estimates of neurofibrillary tangle load 90 min post injection. [18F]MK-6240 is a promising tau tracer with the potential to be applied in the disease diagnosis and assessment of therapeutic interventions.

Preclinical Characterization of 18F-MK-6240, a Promising PET Tracer for In Vivo Quantification of Human Neurofibrillary Tangles.

A PET tracer is desired to help guide the discovery and development of disease-modifying therapeutics for neurodegenerative diseases characterized by neurofibrillary tangles (NFTs), the predominant tau pathology in Alzheimer disease (AD). We describe the preclinical characterization of the NFT PET tracer 18F-MK-6240. METHODS: In vitro binding studies were conducted with 3H-MK-6240 in tissue slices and homogenates from cognitively normal and AD human brain donors to evaluate tracer affinity and selectivity for NFTs. Immunohistochemistry for phosphorylated tau was performed on human brain slices for comparison with 3H-MK-6240 binding patterns on adjacent brain slices. PET studies were performed with 18F-MK-6240 in monkeys to evaluate tracer kinetics and distribution in the brain. 18F-MK-6240 monkey PET studies were conducted after dosing with unlabeled MK-6240 to evaluate tracer binding selectivity in vivo. RESULTS: The 3H-MK-6240 binding pattern was consistent with the distribution of phosphorylated tau in human AD brain slices. 3H-MK-6240 bound with high affinity to human AD brain cortex homogenates containing abundant NFTs but bound poorly to amyloid plaque-rich, NFT-poor AD brain homogenates. 3H-MK-6240 showed no displaceable binding in the subcortical regions of human AD brain slices and in the hippocampus/entorhinal cortex of non-AD human brain homogenates. In monkey PET studies, 18F-MK-6240 displayed rapid and homogeneous distribution in the brain. The 18F-MK-6240 volume of distribution stabilized rapidly, indicating favorable tracer kinetics. No displaceable binding was observed in self-block studies in rhesus monkeys, which do not natively express NFTs. Moderate defluorination was observed as skull uptake. CONCLUSION: 18F-MK-6240 is a promising PET tracer for the in vivo quantification of NFTs in AD patients.

Discovery of 6-(Fluoro-(18)F)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine ([(18)F]-MK-6240): A Positron Emission Tomography (PET) Imaging Agent for Quantification of Neurofibrillary Tangles (NFTs).

Neurofibrillary tangles (NFTs) made up of aggregated tau protein have been identified as the pathologic hallmark of several neurodegenerative diseases including Alzheimer's disease. In vivo detection of NFTs using PET imaging represents a unique opportunity to develop a pharmacodynamic tool to accelerate the discovery of new disease modifying therapeutics targeting tau pathology. Herein, we present the discovery of 6-(fluoro-(18)F)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine, 6 ([(18)F]-MK-6240), as a novel PET tracer for detecting NFTs. 6 exhibits high specificity and selectivity for binding to NFTs, with suitable physicochemical properties and in vivo pharmacokinetics.

Quantification of the specific translocator protein signal of 18F-PBR111 in healthy humans: a genetic polymorphism effect on in vivo binding.

UNLABELLED: PET is used to image active inflammatory processes by targeting the translocator protein (TSPO). In vitro, second-generation TSPO radioligands, such as PBR111, have been shown to bind to human tissue samples with either high affinity (high-affinity binders, HABs), low affinity (low-affinity binders, LABs), or an intermediate, mixed affinity (mixed-affinity binders, MABs). We previously explained these differences in affinity in human tissue via the rs6971 polymorphism in the TSPO gene and predicted that the specific signal from PET ligands in vivo would vary accordingly. In silico modeling predicted that (18)F-PBR111 would have a moderate to high specific-to-nonspecific ratio in the normal human brain. To test these predictions, we present here the analysis and modeling of (18)F-PBR111 data in healthy humans. METHODS: Twenty-one subjects (9 HABs, 8 MABs, and 4 LABs), 28-62 y old, genotyped for the rs6971 polymorphism, underwent 120-min PET scans with arterial sampling after a bolus injection of (18)F-PBR111. Compartmental models and Logan graphical methods enabled estimation of the total volume of distribution (VT) in regions of interest (ROIs). To evaluate the specific signal, we developed 2 methods to estimate the nondisplaceable volume of distribution (V(ND)): the first assumed that the in vitro affinity ratio of (18)F-PBR111 in HABs relative to LABs (4-fold) is preserved in vivo; the second modeled the difference in the HAB and MAB signals in the context of an occupancy plot. RESULTS: A 2-tissue-compartment model described the data well, and a significant difference was found between the VT of HABs, MABs, and LABs across all ROIs examined (P < 0.05). We also found a significant correlation between VT and age for both HABs and MABs in most ROIs. The average V(ND) estimated by the 2 methods was 1.18 ± 0.35 (method I: V(ND) = 0.93, method II: V(ND) = 1.42), implying that the (18)F-PBR111 BPND was 2.78 ± 0.46 in HABs, 1.48 ± 0.28 in MABs, and 0.51 ± 0.17 in LABs and that the in vivo affinity ratio was similar to that measured in vitro. CONCLUSION: (18)F-PBR111 shows a high specific signal in the healthy human brain in vivo. A large component of the variability in the signal across subjects is explained by genetic variation and age, indicating that (18)F-PBR111 can be used for the quantitative assessment of TSPO expression.

An 18-kDa translocator protein (TSPO) polymorphism explains differences in binding affinity of the PET radioligand PBR28.

[(11)C]PBR28 binds the 18-kDa Translocator Protein (TSPO) and is used in positron emission tomography (PET) to detect microglial activation. However, quantitative interpretations of signal are confounded by large interindividual variability in binding affinity, which displays a trimodal distribution compatible with a codominant genetic trait. Here, we tested directly for an underlying genetic mechanism to explain this. Binding affinity of PBR28 was measured in platelets isolated from 41 human subjects and tested for association with polymorphisms in TSPO and genes encoding other proteins in the TSPO complex. Complete agreement was observed between the TSPO Ala147Thr genotype and PBR28 binding affinity phenotype (P value=3.1 × 10(-13)). The TSPO Ala147Thr polymorphism predicts PBR28 binding affinity in human platelets. As all second-generation TSPO PET radioligands tested hitherto display a trimodal distribution in binding affinity analogous to PBR28, testing for this polymorphism may allow quantitative interpretation of TSPO PET studies with these radioligands.

Mixed-affinity binding in humans with 18-kDa translocator protein ligands.

UNLABELLED: 11C-PBR28 PET can detect the 18-kDa translocator protein (TSPO) expressed within macrophages. However, quantitative evaluation of the signal in brain tissue from donors with multiple sclerosis (MS) shows that PBR28 binds the TSPO with high affinity (binding affinity [Ki], ∼4 nM), low affinity (Ki, ∼200 nM), or mixed affinity (2 sites with Ki, ∼4 nM and ∼300 nM). Our study tested whether similar binding behavior could be detected in brain tissue from donors with no history of neurologic disease, with TSPO-binding PET ligands other than 11C-PBR28, for TSPO present in peripheral blood, and with human brain PET data acquired in vivo with 11C-PBR28. METHODS: The affinity of TSPO ligands was measured in the human brain postmortem from donors with a history of MS (n=13), donors without any history of neurologic disease (n=20), and in platelets from healthy volunteers (n=13). Binding potential estimates from thirty-five 11C-PBR28 PET scans from an independent sample of healthy volunteers were analyzed using a gaussian mixture model. RESULTS: Three binding affinity patterns were found in brains from subjects without neurologic disease in similar proportions to those reported previously from studies of MS brains. TSPO ligands showed substantial differences in affinity between subjects classified as high-affinity binders (HABs) and low-affinity binders (LABs). Differences in affinity between HABs and LABs are approximately 50-fold with PBR28, approximately 17-fold with PBR06, and approximately 4-fold with DAA1106, DPA713, and PBR111. Where differences in affinity between HABs and LABs were low (∼4-fold), distinct affinities were not resolvable in binding curves for mixed-affinity binders (MABs), which appeared to express 1 class of sites with an affinity approximately equal to the mean of those for HABs and LABs. Mixed-affinity binding was detected in platelets from an independent sample (HAB, 69%; MAB, 31%), although LABs were not detected. Analysis of 11C-PBR28 PET data was not inconsistent with the existence of distinct subpopulations of HABs, MABs, and LABs. CONCLUSION: With the exception of 11C-PK11195, all TSPO PET ligands in current clinical application recognize HABs, LABs, and MABs in brain tissue in vitro. Knowledge of subjects' binding patterns will be required to accurately quantify TSPO expression in vivo using PET.