Giampaolo Tomasi

Optimization of supervised cluster analysis for extracting reference tissue input curves in (R)-[11C]PK11195 brain PET studies

Performance of two supervised cluster analysis (SVCA) algorithms for extracting reference tissue curves was evaluated to improve quantification of dynamic (R)-[11C]PK11195 brain positron emission tomography (PET) studies. Reference tissues were extracted from images using both a manually defined cerebellum and SVCA algorithms based on either four (SVCA4) or six (SVCA6) kinetic classes. Data from controls, mild cognitive impairment patients, and patients with Alzheimers disease were analyzed using various kinetic models including plasma input, the simplified reference tissue model (RPM) and RPM with vascular correction (RPMV b ). In all subject groups, SVCA-based reference tissue curves showed lower blood volume fractions (V b ) and volume of distributions than those based on cerebellum time-activity curve. Probably resulting from the presence of specific signal from the vessel walls that contains in normal condition a significant concentration of the 18u2009kDa translocation protein. Best contrast between subject groups was seen using SVCA4-based reference tissues as the result of a lower number of kinetic classes and the prior removal of extracerebral tissues. In addition, incorporation of V b in RPM improved both parametric images and binding potential contrast between groups. Incorporation of V b within RPM, together with SVCA4, appears to be the method of choice for analyzing cerebral (R)-[11C]PK11195 neurodegeneration studies.

Synthesis and Evaluation of 11C-LY2795050 as a κ-Opioid Receptor Antagonist Radiotracer for PET Imaging

Kappa-opioid receptors (KOR) are believed to be involved in the pathophysiology of depression, anxiety disorders, drug abuse, and alcoholism. To date, only 1 tracer, the KOR agonist 11C-GR103545, has been reported to be able to image KOR in primates. The goal of the present study was to synthesize the selective KOR antagonist 11C-LY2795050 and evaluate its potential as a PET tracer to image KOR in vivo. Methods: The in vitro binding affinity of LY2795050 was measured in radioligand competition binding assays. Ex vivo experiments were conducted using microdosing of the unlabeled ligand in Sprague–Dawley rats and in wild-type and KOR knockout mice, to assess the ligand’s potential as a tracer candidate. Imaging experiments with 11C-LY2795050 in monkeys were performed on the Focus-220 scanner with arterial blood input function measurement. Binding parameters were determined with kinetic modeling analysis. Results: LY2795050 displays full antagonist activity and high binding affinity and selectivity for KOR. Microdosing studies in rodents and ex vivo analysis of tissue concentrations with liquid chromatography–tandem mass spectrometry identified LY2795050 as an appropriate tracer candidate able to provide specific binding signals in vivo. 11C-LY2795050 was prepared in an average yield of 12% and greater than 99% radiochemical purity. In rhesus monkeys, 11C-LY2795050 displayed a moderate rate of peripheral metabolism, with approximately 40% of parent compound remaining at 30 min after injection. In the brain, 11C-LY2795050 displayed fast uptake kinetics (regional activity peak times of <20 min) and an uptake pattern consistent with the distribution of KOR in primates. Pretreatment with naloxone (1 mg/kg, intravenously) resulted in a uniform distribution of radioactivity. Further, specific binding of 11C-LY2795050 was reduced by the selective KOR antagonist LY2456302 in a dose-dependent manner. Conclusion: 11C-LY2795050 displayed favorable pharmacokinetic properties and binding profiles in vivo and therefore is a suitable ligand for imaging the KOR in primates. This newly developed KOR antagonist tracer has since been advanced to PET imaging of KOR in humans and constitutes the first successful KOR antagonist radiotracer.

A spectral analysis approach for determination of regional rates of cerebral protein synthesis with the L-[1-11C]leucine PET method

A spectral analysis approach was used to estimate kinetic model parameters of the L-[1-11C]leucine positron emission tomography (PET) method and regional rates of cerebral protein synthesis (rCPS) in predefined regions of interest (ROIs). Unlike analyses based on the assumption that tissue ROIs are kinetically homogeneous, spectral analysis allows for heterogeneity within a region. To improve estimation performance, a new approach was developed—spectral analysis with iterative filter (SAIF). In simulation SAIF produced low bias, low variance estimates of the influx rate constant for leucine (K1), blood volume fraction (V b ), fraction of unlabeled leucine in the tissue precursor pool for protein synthesis derived from arterial plasma (λ), and rCPS. Simulation of normal count rate studies showed that SAIF applied to ROI time-activity curves (TACs) performed comparably to the basis function method (BFM) applied to voxel TACs when voxelwise estimates were averaged over all voxels in the ROI. At low count rates, however, SAIF performed better. In measured L-[1-11C]leucine PET data, there was good agreement between ROI-based SAIF estimates and average voxelwise BFM estimates of K1, V b , λ, and rCPS. We conclude that SAIF sufficiently addresses the problem of tissue heterogeneity in ROI data and provides a valid tool for estimation of rCPS, even in low count rate studies.

Voxel-Based Estimation of Kinetic Model Parameters of the l-[1-11C]Leucine PET Method for Determination of Regional Rates of Cerebral Protein Synthesis: Validation and Comparison with Region-of-Interest-Based Methods

We adapted and validated a basis function method (BFM) to estimate at the voxel level parameters of the kinetic model of the l-[1-11C]leucine positron emission tomography (PET) method and regional rates of cerebral protein synthesis (rCPS). In simulation at noise levels typical of voxel data, BFM yielded low-bias estimates of rCPS; in measured data, BFM and nonlinear least-squares parameter estimates were in good agreement. We also examined whether there are advantages to using voxel-level estimates averaged over regions of interest (ROIs) in place of estimates obtained by directly fitting ROI time-activity curves (TACs). In both simulated and measured data, fits of ROI TACs were poor, likely because of tissue heterogeneity not taken into account in the kinetic model. In simulation, rCPS determined from fitting ROI TACs was substantially overestimated and BFM-estimated rCPS averaged over all voxels in an ROI was slightly underestimated. In measured data, rCPS determined by regional averaging of voxel estimates was lower than rCPS determined from ROI TACs, consistent with simulation. In both simulated and measured data, intersubject variability of BFM-estimated rCPS averaged over all voxels in a ROI was low. We conclude that voxelwise estimation is preferable to fitting ROI TACs using a homogeneous tissue model.

Determination of In Vivo Bmax and Kd for 11C-GR103545, an Agonist PET Tracer for κ-Opioid Receptors: A Study in Nonhuman Primates

The κ-opioid receptors (KOR) are involved in mood disorders and addictive conditions. In vivo imaging studies of this receptor in humans have not been reported because of the lack of a selective ligand. We used a recently developed selective KOR agonist tracer, 11C-GR103545, and performed a study in rhesus monkeys to estimate the in vivo receptor concentration (Bmax) and dissociation equilibrium constant (Kd). Methods: Four rhesus monkeys underwent 12 scans with 11C-GR103545 on a PET scanner under baseline and self-blocking conditions. The injected mass was 0.042 ± 0.014 μg/kg for the baseline scans and ranged from 0.16 to 0.3 μg/kg for the self-blocking scans. The radiotracer was administered in a bolus-plus-infusion protocol, and cerebellum was used as the reference region in kinetic analysis. Binding potential (BPND) values were computed as [(CROI/CREF) − 1], where CROI and CREF are the mean of the radioactivity concentrations from 90 to 120 min after tracer administration in a given region of interest (ROI) and in the cerebellum. In 6 scans, arterial input functions and free fraction in plasma (fp) were measured. In addition, a 2-tissue-compartment model was used to compute the volume of distribution in the cerebellum (VT_REF), which was then used to estimate the free–to–nondisplaceable concentration ratio (fND) as fp/VT_REF. A Scatchard plot was used to estimate Bmax, and KdND = Kd/fND, the Kd value with respect to the cerebellar concentration. Individual data were first analyzed separately and then pooled together. When KdND was allowed to vary among ROIs, results were variable; therefore, KdND was constrained to be constant across ROIs, whereas Bmax was allowed to be ROI-dependent and animal-dependent. Results: A global estimate of 1.72 nM was obtained for KdND. Estimated Bmax ranged from 0.3 to 6.1 nM across ROIs and animals. The Kd estimate of 0.048 nM, obtained by correcting KdND by the factor fND, was in good agreement with the half maximal inhibitory concentration (IC50) of 0.018 nM determined from functional assays in rabbit vas deferens and inhibition constant (Ki) of 0.02 nM measured in radioligand competition assays using cloned human receptors. On the basis of these data, a suitable tracer dose of 0.02 μg/kg was selected for use in humans. Conclusion: The use of a bolus-plus-infusion protocol with the KOR agonist tracer 11C-GR103545 permitted the successful estimation of Bmax and KdND in vivo. On the basis of the estimated Kd value, a tracer dose of 1.4 μg (3.38 nmol) for an average body weight of 70 kg was chosen as the mass dose limit in human studies using this novel agonist radiotracer.

Evaluation of the agonist PET radioligand [11C]GR103545 to image kappa opioid receptor in humans: Kinetic model selection, test–retest reproducibility and receptor occupancy by the antagonist PF-04455242

INTRODUCTIONnKappa opioid receptors (KOR) are implicated in several brain disorders. In this report, a first-in-human positron emission tomography (PET) study was conducted with the potent and selective KOR agonist tracer, [(11)C]GR103545, to determine an appropriate kinetic model for analysis of PET imaging data and assess the test-retest reproducibility of model-derived binding parameters. The non-displaceable distribution volume (V(ND)) was estimated from a blocking study with naltrexone. In addition, KOR occupancy of PF-04455242, a selective KOR antagonist that is active in preclinical models of depression, was also investigated.nnnMETHODSnFor determination of a kinetic model and evaluation of test-retest reproducibility, 11 subjects were scanned twice with [(11)C]GR103545. Seven subjects were scanned before and 75 min after oral administration of naltrexone (150 mg). For the KOR occupancy study, six subjects were scanned at baseline and 1.5 h and 8 h after an oral dose of PF-04455242 (15 mg, n=1 and 30 mg, n=5). Metabolite-corrected arterial input functions were measured and all scans were 150 min in duration. Regional time-activity curves (TACs) were analyzed with 1- and 2-tissue compartment models (1TC and 2TC) and the multilinear analysis (MA1) method to derive regional volume of distribution (V(T)). Relative test-retest variability (TRV), absolute test-retest variability (aTRV) and intra-class coefficient (ICC) were calculated to assess test-retest reproducibility of regional VT. Occupancy plots were computed for blocking studies to estimate occupancy and V(ND). The half maximal inhibitory concentration (IC50) of PF-04455242 was determined from occupancies and drug concentrations in plasma. [(11)C]GR103545 in vivo K(D) was also estimated.nnnRESULTSnRegional TACs were well described by the 2TC model and MA1. However, 2TC VT was sometimes estimated with high standard error. Thus MA1 was the model of choice. Test-retest variability was ~15%, depending on the outcome measure. The blocking studies with naltrexone and PF-04455242 showed that V(T) was reduced in all regions; thus no suitable reference region is available for the radiotracer. V(ND) was estimated reliably from the occupancy plot of naltrexone blocking (V(ND)=3.4±0.9 mL/cm(3)). The IC50 of PF-04455242 was calculated as 55 ng/mL. [(11)C]GR103545 in vivo K(D) value was estimated as 0.069 nmol/L.nnnCONCLUSIONSn[(11)C]GR103545 PET can be used to image and quantify KOR in humans, although it has slow kinetics and variability of model-derived kinetic parameters is higher than desirable. This tracer should be suitable for use in receptor occupancy studies, particularly those that target high occupancy.

Kinetic Modeling of 11C-LY2795050, A Novel Antagonist Radiotracer for PET Imaging of the Kappa Opioid Receptor in Humans

11C-LY2795050 is a novel kappa opioid receptor (KOR) antagonist tracer for positron emission tomography (PET) imaging. The purpose of this first-in-human study was to determine the optimal kinetic model for analysis of 11C-LY2795050 imaging data. Sixteen subjects underwent baseline scans and blocking scans after oral naltrexone. Compartmental modeling and multilinear analysis-1 (MA1) were applied using the arterial input functions. Two-tissue compartment model and MA1 were found to be the best models to provide reliable measures of binding parameters. The rank order of 11C-LY2795050 distribution volume (VT) matched the known regional KOR densities in the human brain. Blocking scans with naltrexone indicated no ideal reference region for 11C-LY2795050. Three methods for calculation of the nondisplaceable distribution volume (VND) were assessed: (1) individual VND estimated from naltrexone occupancy plots, (2) mean VND across subjects, and (3) a fixed fraction of cerebellum VT. Approach (3) produced the lowest intersubject variability in the calculation of binding potentials (BPND, BPF, and BPP). Therefore, binding potentials of 11C-LY2795050 can be determined if the specific binding fraction in the cerebellum is presumed to be unchanged by diseases and experimental conditions. In conclusion, results from the present study show the suitability of 11C-LY2795050 to image and quantify KOR in humans.

Modeling analysis of the new kappa opioid receptor antagonist tracer [11C]LY2795050 in humans

Methods: Seven healthy volunteers (age=26±6) underwent 120-min scans with [C]LY2795050 on the HRRT scanner. The subjects received a bolus injection of 482±171 MBq, the specific activitywas 23±8 MBq/nmol, and the injectedmasswas 8.8±1.2 μg. Imageswere reconstructedwith a list-mode reconstruction algorithm which incorporates event-by-event motion correction. Regional TACs were created by registering an early summed image to eachsubjectsMRwhichwas then registered to theAAL template. TheseTACswere fitted to the2Tmodelwithmetabolite-corrected plasma input function to estimate VT. BPND values were computed as [VT(ROI)/VT(CER)]−1, with cerebellum as the reference region. ROI TACs were also fitted using SRTM and BPND results were compared with those derived from the 2T model. In addition, BPND values were computed employing only the first 90 min of scan data, and these estimates were compared to the 120-min values. Finally, parametric images of BPND were created with SRTM2, matching ROIs were applied to these images, and the resulting BPND values were compared to those obtained by regional TAC fitting.

Synthesis and evaluation of [C-11]LY2795050, an antagonist PET imaging tracer for the kappa opioid receptors

Introduction: The kappa opioid receptors (KOR) are believed to be involved in the pathophysiology of depression, anxiety disorders, drug abuse and alcoholism. To date only one tracer, the kappa opioid receptor agonist [C-11]GR103545, has been reported to be able to image KOR in nonhuman primates. The goal of the present study was to synthesize the selective KOR antagonist [C-11]LY2795050 and evaluate its potential as a PET tracer to image KOR in non-human primates.

Imaging the kappa opioid receptor in humans with [11C]LY2795050: Tracer evaluation and test–retest reproducibility study

Introduction: In our laboratories we have developed [C]LY2795050 as a novel, selective antagonist tracer for the kappa opioid receptor (KOR). Imaging studies in rhesus monkeys have demonstrated the tracers suitability to image KOR in vivo. Thus, a first-in-human study was conducted to evaluate the potential of this novel tracer for PET imaging of KOR in humans and to assess the test–retest variability of binding parameters derived from kinetic modeling of imaging data.