Determination of in vivo Bmax and Kd for 11C-GR103545, an agonist PET tracer for κ-opioid receptors: a study in nonhuman primates.

UNLABELLED: 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, (11)C-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 (11)C-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 Kd(ND) = Kd/fND, the Kd value with respect to the cerebellar concentration. Individual data were first analyzed separately and then pooled together. When Kd(ND) was allowed to vary among ROIs, results were variable; therefore, Kd(ND) 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 Kd(ND). Estimated Bmax ranged from 0.3 to 6.1 nM across ROIs and animals. The Kd estimate of 0.048 nM, obtained by correcting Kd(ND) 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 (11)C-GR103545 permitted the successful estimation of Bmax and Kd(ND) 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.

Equivalence of arterial and venous blood for [11C]CO2-metabolite analysis following intravenous administration of 1-[11C]acetate and 1-[11C]palmitate.

PURPOSE: Sampling of arterial blood for metabolite correction is often required to define a true radiotracer input function in quantitative modeling of PET data. However, arterial puncture for blood sampling is often undesirable. To establish whether venous blood could substitute for arterial blood in metabolite analysis for quantitative PET studies with 1-[(11)C]acetate and 1-[(11)C]palmitate, we compared the results of [(11)C]CO2-metabolite analyses performed on simultaneously collected arterial and venous blood samples. METHODS: Paired arterial and venous blood samples were drawn from anesthetized pigs at 1, 3, 6, 8, 10, 15, 20, 25 and 30min after i.v. administration of 1-[(11)C]acetate and 1-[(11)C]palmitate. Blood radioactivity present as [(11)C]CO2 was determined employing a validated 10-min gas-purge method. Briefly, total blood (11)C radioactivity was counted in base-treated [(11)C]-blood samples, and non-[(11)C]CO2 radioactivity was counted after the [(11)C]-blood was acidified using 6N HCl and bubbled with air for 10min to quantitatively remove [(11)C]CO2. RESULTS: An excellent correlation was found between concurrent arterial and venous [(11)C]CO2 levels. For the [(11)C]acetate study, the regression equation derived to estimate the venous [(11)C]CO2 from the arterial values was: y=0.994x+0.004 (r(2)=0.97), and for the [(11)C]palmitate: y=0.964x-0.001 (r(2)=0.9). Over the 1-30min period, the fraction of total blood (11)C present as [(11)C]CO2 rose from 4% to 64% for acetate, and 0% to 24% for palmitate. The rate of [(11)C]CO2 appearance in venous blood appears similar for the pig model and humans following i.v. [(11)C]-acetate administration. CONCLUSION: Venous blood [(11)C]CO2 values appear suitable as substitutes for arterial blood samples in [(11)C]CO2 metabolite analysis after administration of [(11)C]acetate or [(11)C]palmitate ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Quantitative PET studies employing 1-[(11)C]acetate and 1-[(11)C]palmitate can employ venous blood samples for metabolite correction of an image-derived tracer arterial input function, thereby avoiding the risks of direct arterial blood sampling.

A new facile synthetic route to [11C]GSK189254, a selective PET radioligand for imaging of CNS histamine H3 receptor.

GSK189254 and its corresponding precursor GSK185071B were synthesized from 3-methoxyphenylacetic acid with 6-chloropyridine-3-carbolic acid or 6-chloronicotinamide in 8 and 7 steps with either 6% or 7% and either 14% or 16% yield, respectively. [(11)C]GSK189254 was prepared from GSK185071B with [(11)C]CH(3)OTf through N-[(11)C]methylation and isolated by HPLC combined with solid-phase extraction (SPE) in 50-60% radiochemical yield based on [(11)C]CO(2) and decay corrected to end of bombardment (EOB), with 370-740 GBq/µmol specific activity at EOB.

An automated SPE-based high-yield synthesis of [11C]acetate and [11C]palmitate: no liquid-liquid extraction, solvent evaporation or distillation required.

INTRODUCTION: An automated method is described for the rapid and high-yield synthesis of two of the most commonly used radioactive fatty acids: [(11)C]acetate and [(11)C]palmitate. METHODS: Reaction of [(11)C]CO(2) with the respective Grignard reagents in diethyl ether solution proceeded for 2 min at 40°C. Quenching of the reaction and liberation of nonreacted [(11)C]CO(2) occurred upon addition of a fourfold molar excess of aqueous 0.1 M HCl (acetate) or nonaqueous HCl/Et(2)O (palmitate). Labeled products were then purified by adsorption to an Alumina-N Sep-Pak Plus cartridge and eluted with either aqueous NaH(2)PO(4) solution (acetate) or 100% ethanol (palmitate). RESULTS: High-performance liquid chromatography analysis confirmed that the radiochemical purity of each product was >98%, and decay-corrected radiochemical yields averaged 33% for [(11)C]palmitate and 40% for [(11)C]acetate. CONCLUSION: The method requires no liquid-liquid extraction, solvent evaporation or distillation capabilities and can be readily adapted to existing radiosynthesis modules.

Behavioral depression and positron emission tomography-determined serotonin 1A receptor binding potential in cynomolgus monkeys.

CONTEXT: Current animal models of depression are inadequate to further our understanding of depression. New models that allow for analysis of cognitive function and sex differences are needed. OBJECTIVE: To characterize serotonin 1A (5-HT(1A)) receptor binding potential (BP) and its relationship with specific characteristics of behavioral depression in cynomolgus monkeys. DESIGN: A 23-month case-control study. SETTING: Small social groups in the laboratory. Subjects Seventeen adult female cynomolgus monkeys. MAIN OUTCOME MEASURES: Serotonin 1A receptor BP was examined by positron emission tomography using the radioligand 4,2"-(methoxyphenyl)-1-[2"-(N-2"-pyridinyl)-p-fluorobenzamido]ethylpiperazine in the raphe, amygdala, hippocampus, and anterior cingulate cortex in monkeys characterized by behavioral observation as depressed or not depressed. Aggression, submission, affiliation, pathologic behaviors, and activity levels were determined by behavioral observation. Heart rate and hypothalamic-pituitary-adrenal function were also determined. RESULTS: Throughout the brain areas examined, there was a reduction in 5-HT(1A) BP in depressed monkeys. The 5-HT(1A) BP in the amygdala and hippocampus was associated with aggression and submission. Friendly interaction, grooming, and locomotion were associated with 5-HT(1A) BP in the left cingulate cortex, whereas attention directed toward the environment was associated with 5-HT(1A) BP in the right cingulate cortex. The 5-HT(1A) receptor BP was inversely associated with heart rate in the raphe, left cingulate, and right amygdala. CONCLUSIONS: This is the fourth in a series of studies that suggest that depressive behavior in adult female cynomolgus monkeys is similar to that observed in humans. It has been observed in 2 large groups of monkeys randomly selected from feral populations, suggesting that the capacity for depression is inherent in the species. This animal model holds promise to further our understanding of the basic mechanisms of affective behavior, the neuropathophysiologic characteristics of depression and the cognitive dysfunction that accompanies them, genetic and environmental factors that may affect depression risk, and the role of reproductive function in the excess depression risk in women.

Distribution of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) uracil in mice bearing colorectal cancer xenografts: rationale for therapeutic use and as a positron emission tomography probe for thymidylate synthase.

PURPOSE: In colorectal, breast, and head and neck cancers, response to 5-fluorouracil is associated with low expression of thymidylate synthase. In contrast, tumors with high expression of thymidylate synthase may be more sensitive to prodrugs such as 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) uracil (FAU) that are activated by thymidylate synthase. These studies were designed to evaluate FAU as a potential therapeutic and diagnostic probe. EXPERIMENTAL DESIGN: [18F]-FAU and [3H]-FAU were synthesized with >97% radiochemical purity. [3H]-FAU or [18F]-FAU was administered intravenously to severe combined immunodeficient mice bearing either HT29 (low thymidylate synthase) or LS174T (high thymidylate synthase) human colon cancer xenografts. Four hours after [3H]-FAU dosing, tissue distribution of total radioactivity and incorporation of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) 5-methyluracil (FMAU), derived from thymidylate synthase activation of FAU, into tumor DNA was measured. Positron emission tomography (PET) images were obtained for 90 minutes after injection of [18F]-FAU. Thymidylate synthase activity was determined in vitro in tumors from untreated mice by [3H] release from [3H]dUMP. Each cell line was incubated in vitro with [3H]-FAU or [3H]-FMAU in the absence or presence of 5-fluoro-2'-deoxyuridine (FdUrd) and then was analyzed for incorporation of radiolabel into DNA. RESULTS: Thymidylate synthase enzymatic activity in LS174T xenografts was approximately 3.5-fold higher than in HT29 xenografts, and incorporation of radioactivity derived from [3H]-FAU into LS174T DNA was approximately 2-fold higher than into HT29 DNA. At 240 minutes, radioactivity derived from [3H]-FAU was approximately 2-fold higher in tumors than in skeletal muscle. At times up to 90 minutes, PET imaging detected only small differences in uptake of [18F]-FAU between the tumor types. Fluorine-18 in skeletal muscle was higher than in tumor for the first 90 minutes and plateaued earlier, whereas [18F] in tumor continued to increase during the 90-minute imaging period. For both cell lines in vitro, FdUrd decreased the rate of incorporation of [3H]-FAU into DNA, whereas the incorporation of [3H]-FMAU was increased. CONCLUSIONS: These results for FAU incorporation into DNA in vitro and in vivo further support clinical evaluation of FAU as a therapeutic agent in tumors with high concentrations of thymidylate synthase that are less likely to respond to 5-fluorouracil treatment. The high circulating concentrations of thymidine reported in mice may limit their utility in evaluating FAU as a PET probe.