In vitro and in vivo evaluation of [18F]-FDEGPECO as a PET tracer for imaging the metabotropic glutamate receptor subtype 5 (mGluR5)

(E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-2-(2-(18)F-fluoroethoxy)ethyl oxime, ([(18)F]-FDEGPECO), a novel high affinity radioligand for the metabotropic glutamate receptor subtype 5 (mGluR5) was assessed for its potential as a PET imaging agent. In vitro autoradiography on rat brain slices resulted in a heterogeneous and displaceable binding to mGluR5-rich brain regions. [(18)F]-FDEGPECO showed high stability in rat plasma and brain homogenate as well as in human plasma and microsomes. Good blood-brain barrier passage was predicted from an in vitro transport assay with P-glycoprotein-transfected hMDR1-MDCK cells. In vivo PET imaging on rats revealed specific uptake of radioactivity in the mGluR5-rich brain regions such as hippocampus, striatum and cortex while the cerebellum, a region with low mGluR5-expression, showed negligible uptake. Blockade experiments by co-injection of [(18)F]-FDEGPECO and M-MPEP (6mg/kg), an antagonist for mGluR5, reduced the level of radioactivity in mGluR5-regions to that of the cerebellum, pointing to an effective blockade of specifically bound [(18)F]-FDEGPECO. Postmortem biodistribution studies at 15min p.i. confirmed the distribution pattern observed in PET. HPLC analysis of rat brain extracts indicated that 98.5% and 91% of the total radioactivity were parent compound at 5min and 17min p.i., respectively. Taken together, the high affinity and the high in vivo specificity of [(18)F]-FDEGPECO for mGluR5 in the rat brain as well as the lack of in vivo defluorination make this new [(18)F]-labeled ABP688 derivative a suitable ligand for the preclinical PET imaging of mGluR5. These favorable characteristics warrant further evaluation in humans.     

Positron Emission Tomography Studies on Binding of Central Nervous System Drugs and P-Glycoprotein Function in the Rodent Brain

The permeability of the blood–brain barrier (BBB) is one of the factors determining the bioavailability of drugs in the brain. The BBB only allows passage of lipophilic drugs by passive diffusion. However, some lipophilic drugs hardly enter the brain. The transmembrane protein P-glycoprotein (P-gp) is one of the carrier systems that is responsible for transportation of drugs out of the brain. P-Glycoprotein affects the pharmacokinetics of many drugs and can be inhibited by administration of modulators or competitive substrates. Identification and classification of central nervous system (CNS) drugs as P-gp substrates or inhibitors are of crucial importance in drug development. Positron emission tomography (PET) studies can play an important role in the screening process as a follow-up of high-throughput in vitro assays. Several rodent studies have shown the potential value of PET to measure the effect of P-gp on the pharmacokinetics and brain uptake of radiolabeled compounds. P-Glycoprotein-mediated effects were observed for two 5-HT_1a receptor ligands, [¹⁸F]MPPF vs. [carbonyl-¹¹C]WAY100635. Under control conditions, the specific brain uptake of [¹⁸F]MPPF is five- to eightfold lower than that of [¹¹C]WAY100635. After cyclosporin A (CsA) modulation, [¹⁸F]MPPF uptake in the rat brain increased five- to tenfold. Cerebral uptake of [carbonyl-¹¹C]WAY100635 was also increased by modulation, but in general the increase was lower than that observed for [¹⁸F]MPPF (two- to threefold). Brain uptake of the -adrenergic receptor ligands [¹¹C]carazolol and [¹⁸F]fluorocarazolol was increased in P-gp knockout mice and CsA-treated rats. Both the specific and nonspecific binding of [¹⁸F]fluorocarazolol were doubled by CsA. Cerebral uptake of [¹¹C]carazolol in rats was much lower than that of [¹⁸F]fluorocarazolol and no specific binding was measured. After CsA modulation, the uptake of [¹¹C]carazolol increased five- to sixfold, but this uptake was not receptor-mediated. Quantitative PET studies in rodents on P-gp functionality demonstrated a dose-dependent increase of radioligands after administration of CsA. Studies with [¹¹C]verapamil and [¹¹C]carvedilol showed that complete modulation was achieved at 50 mg/kg CsA. The distribution volume of [¹¹C]carvedilol increased from 0.25 in the control study to 1.0 after full modulation with CsA. By quantitative PET measurement of P-gp function, the dose of modulators required to increase the concentration of CNS drugs may be determined, which may result in improved drug therapy.     

In vivo evaluation of P-glycoprotein function at the blood-brain barrier in nonhuman primates using [11C]verapamil

P-glycoprotein (P-gp) is a major efflux transporter contributing to the efflux of a range of xenobiotic compounds at the blood-brain barrier (BBB). In the present study, we evaluated the P-gp function at the BBB using positron emission tomography (PET) in nonhuman primates. Serial brain PET scans were obtained in three rhesus monkeys after intravenous administration of [(11)C]verapamil under control and P-gp inhibition conditions ([PSC833 ([3'-keto-Me-Bmt(1)]-[Val(2)]-cyclosporin) 20 mg/kg/2 h]). The parent [(11)C]verapamil and its metabolites in plasma were determined by HPLC with a positron detector. The initial brain uptake clearance calculated from the integration plot was used for the quantitative analysis. After intravenous administration, [(11)C]verapamil was taken up rapidly into the brain (time to reach the peak, 0.58 min). The blood level of [(11)C]verapamil decreased rapidly, and it underwent metabolism with time. The inhibition of P-gp by PSC833 increased the brain uptake of [(11)C]verapamil 4.61-fold (0.141 versus 0.651 ml/g brain/min, p < 0.05). These results suggest that PET measurement with [(11)C]verapamil can be used for the evaluation of P-gp function at the BBB in the living brain.     

Comparison of in vitro and in vivo models of drug transcytosis through the blood-brain barrier

Drug and solute transport through in vitro and in vivo models of the blood-brain barrier (BBB) were compared to provide a measure of how well the in vitro model predicted BBB permeability found in vivo. The in vitro model employed bovine brain capillary endothelial cells in either primary tissue culture or as a continuous line grown on Transwells and placed in side-by-side diffusion chambers. The in vivo model of BBB transport utilized an internal carotid artery perfusion/capillary depletion method in anesthetized rats. BBB permeability in vivo and in vitro was measured for 15 radiolabeled drugs and for L-[3H]dopa, D-[14C]glucose and [3H]albumin. [3H]- or [14C]sucrose was used in vivo as a blood volume reference. Lipid solubility of each drug was measured based on the 1-octanol/Ringer's partition coefficient. The morphology of the endothelial cell in primary tissue culture was spindle-shaped and the morphology of the endothelial cell in continuous culture was cuboid-shaped. The cuboidal morphology demonstrated a 2-fold greater resistance to solute transport and was used for the majority of the in vitro studies. Drug and solute permeability coefficients (Pe) ranged from 3.9 X 10(-3) to 2.5 X 10(-1) cm/min in vitro and from 1.0 X 10(-5) to 2.1 X 10(-2) cm/min in vivo. The In of the permeability.surface area product in vitro correlated with the In partition coefficient (r = 0.62, P less than .0125) and the In permeability.surface area product in vivo correlated with the In partition coefficient (r = 0.84, P less than .0005).(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of a physiologically based pharmacokinetic model to study the time to reach brain equilibrium: an experimental analysis of the role of blood-brain barrier permeability, plasma protein binding, and brain tissue binding

This study was designed 1) to examine the effects of blood-brain barrier (BBB) permeability [quantified as permeability-surface area product (PS)], unbound fraction in plasma (f(u,plasma)), and brain tissue (f(u,brain)) on the time to reach equilibrium between brain and plasma and 2) to investigate the drug discovery strategies to design and select compounds that can rapidly penetrate the BBB and distribute to the site of action. The pharmacokinetics of seven model compounds: caffeine, CP-141938 [methoxy-3-[(2-phenyl-piperadinyl-3-amino)-methyl]-phenyl-N-methyl-methane-sulfonamide], fluoxetine, NFPS [N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine], propranolol, theobromine, and theophylline in rat brain and plasma after subcutaneous administration were studied. The in vivo log PS and log f(u,brain) calculated using a physiologically based pharmacokinetic model correlates with in situ log PS (R(2) = 0.83) and in vitro log f(u,brain) (R(2) = 0.69), where the in situ PS and in vitro f(u,brain) was determined using in situ brain perfusion and equilibrium dialysis using brain homogenate, respectively. The time to achieve brain equilibrium can be quantitated with a proposed parameter, intrinsic brain equilibrium half-life [t(1/2eq,in) = V(b)ln2/(PS . f(u,brain))], where V(b) is the physiological volume of brain. The in vivo log t(1/2eq,in) does not correlate with in situ log PS (R(2) < 0.01) but correlates inversely with log(PS . f(u,brain)) (R(2) = 0.85). The present study demonstrates that rapid brain equilibration requires a combination of high BBB permeability and low brain tissue binding. A high BBB permeability alone cannot guarantee a rapid equilibration. The strategy to select compounds with rapid brain equilibration in drug discovery should identify compounds with high BBB permeability and low nonspecific binding in brain tissue.     

Gender-dependent increases with healthy aging of the human cerebral cannabinoid-type 1 receptor binding using [(18)F]MK-9470 PET

The endocannabinoid system (ECS) is implicated as a regulator of homeostasis of several cerebral functions and is a novel target for drug treatment of neuropyschiatric disorders. So far, the cerebral cannabinoid-type 1 receptor (CB1R) has only been studied using in vitro, animal model, electrophysiological and post-mortem data. We have used positron emission tomography (PET) using a high-affinity, subtype-selective radioligand, [(18)F]MK-9470, to assess the in vivo cerebral CB1R distribution and its variation with healthy aging and gender. Fifty healthy volunteers (25 M/25 F, 18-69 years) underwent [(18)F]MK-9470 PET. Parametric [(18)F]MK-9470 binding maps were constructed, corrected for partial volume effects and analyzed using statistical parametric mapping in a combined categorical (gender) and covariate (age) design. We found that [(18)F]MK-9470 binding to CB1R increased with aging but only in women (p(FWE)<0.05, corrected for multiple comparisons); this was most pronounced in the basal ganglia, lateral temporal cortex and limbic system, especially in the hippocampus. Men showed higher [(18)F]MK-9470 binding then women (p<0.001, uncorrected), in clusters of the limbic system and cortico-striato-thalamic-cortical circuit. Region-dependent and gender-related upregulation of [(18)F]MK-9470 binding with aging is in line with ex vivo findings in rodent studies and may be associated with a changing homeostatic capacity or compensation mechanisms in the ECS that is modulated by sex hormones. In vivo PET of the CB1R will likely improve our understanding of the ECS in several neurological and psychiatric disorders.     

Radiodefluorination of 3-fluoro-5-(2-(2-[18F](fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile ([18F]SP203), a radioligand for imaging brain metabotropic glutamate subtype-5 receptors with positron emission tomography, occurs by glutathionylation in rat brain

Metabotropic glutamate subtype-5 receptors (mGluR5) are implicated in several neuropsychiatric disorders. Positron emission tomography (PET) with a suitable radioligand may enable monitoring of regional brain mGluR5 density before and during treatments. We have developed a new radioligand, 3-fluoro-5-(2-(2-[(18)F](fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile ([(18)F]SP203), for imaging brain mGluR5 in monkey and human. In monkey, radioactivity was observed in bone, showing release of [(18)F]-fluoride ion from [(18)F]SP203. This defluorination was not inhibited by disulfiram, a potent inhibitor of CYP2E1. PET confirmed bone uptake of radioactivity and therefore defluorination of [(18)F]SP203 in rats. To understand the biochemical basis for defluorination, we administered [(18)F]SP203 plus SP203 in rats for ex vivo analysis of metabolites. Radio-high-performance liquid chromatography detected [(18)F]fluoride ion as a major radiometabolite in both brain extract and urine. Incubation of [(18)F]SP203 with brain homogenate also generated this radiometabolite, whereas no metabolism was detected in whole blood in vitro. Liquid chromatography-mass spectrometry analysis of the brain extract detected m/z 548 and 404 ions, assignable to the [M + H](+) of S-glutathione (SP203Glu) and N-acetyl-S-l-cysteine (SP203Nac) conjugates of SP203, respectively. In urine, only the [M + H](+) of SP203Nac was detected. Mass spectrometry/mass spectrometry and multi-stage mass spectrometry analyses of each metabolite yielded product ions consistent with its proposed structure, including the former fluoromethyl group as the site of conjugation. Metabolite structures were confirmed by similar analyses of SP203Glu and SP203Nac, prepared by glutathione S-transferase reaction and chemical synthesis, respectively. Thus, glutathionylation at the 2-fluoromethyl group is responsible for the radiodefluorination of [(18)F]SP203 in rat. This study provides the first demonstration of glutathione-promoted radiodefluorination of a PET radioligand.     

Synthesis, characterization, and monkey positron emission tomography (PET) studies of [18F]Y1-973, a PET tracer for the neuropeptide Y Y1 receptor

Neuropeptide Y receptor subtype 1 (NPY Y1) has been implicated in appetite regulation, and antagonists of NPY Y1 are being explored as potential therapeutics for obesity. An NPY Y1 PET tracer is useful for determining the level of target engagement by NPY Y1 antagonists in preclinical and clinical studies. Here we report the synthesis and evaluation of [(18)F]Y1-973, a novel PET tracer for NPY Y1. [(18)F]Y1-973 was radiolabeled by reaction of a primary chloride with [(18)F]KF/K2.2.2 followed by deprotection with HCl. [(18)F]Y1-973 was produced with high radiochemical purity (>98%) and high specific activity (>1000 Ci/mmol). PET studies in rhesus monkey brain showed that the distribution of [(18)F]Y1-973 was consistent with the known NPY Y1 distribution; uptake was highest in the striatum and cortical regions and lowest in the pons, cerebellum nuclei, and brain stem. Blockade of [(18)F]Y1-973 uptake with NPY Y1 antagonist Y1-718 revealed a specific signal that was dose-dependently reduced in all regions of grey matter to a similarly low level of tracer uptake, indicative of an NPY Y1 specific signal. In vitro autoradiographic studies with [(18)F]Y1-973 in rhesus monkey and human brain tissue slices revealed an uptake distribution consistent with the in vivo PET studies. Highest binding density was observed in the dentate gyrus, caudate-putamen, and cortical regions; moderate binding density in the hypothalamus and thalamus; and lowest binding density in the globus pallidus and cerebellum. In vitro saturation binding studies in rhesus monkey and human caudate-putamen homogenates confirmed a similarly high B(max)/K(d) ratio for [(18)F]Y1-973, suggesting the tracer may provide a specific signal in human brain of similar magnitude to that observed in rhesus monkey. [(18)F]Y1-973 is a suitable PET tracer for imaging NPY Y1 in rhesus monkey with potential for translation to human PET studies.     

Correlation between serotonin synthesis and 5-HT1A receptor binding in the living human brain: a combined alpha-[11C]MT and [18F]MPPF positron emission tomography study

Serotonin (5-HT) is one of the major neurotransmitters and has been implicated in a wide variety of cerebral functions. Several lines of evidence indicate that 5HT(1A) receptors exert a negative feedback in the synthesis and release of serotonin. While most of what is known about serotonin comes from studies in animals, much less empirical evidence exists about the serotonergic system in the living human brain. This study aims to assess the correlation between serotonin synthesis and 5-HT(1A) receptor binding using positron emission tomography (PET) in humans. Six healthy male volunteers underwent 2 PET scans in the same day: one measuring alpha-[(11)C]MT K [ml/g/min] trapping constant (a measure of serotonin synthesis) and one measuring 5-HT(1A) receptor binding potential BP(ND) with [(18)F]MPPF. Volumes of interest (VOIs) selected a priori included: anterior cingulate cortex (ACC), anterior insula, hippocampus, amygdala, thalamus, hypothalamus and midbrain raphe nuclei. Correlation analyses were conducted voxel-by-voxel and with manually traced VOIs. A significant negative correlation between serotonin synthesis and 5-HT(1A) binding potential was found bilaterally in hippocampus and anterior insula and in the left ACC. The combination of [(18)F]MPPF and alpha-[(11)C]MT PET offers a means to investigate key determinants of 5-HT neurotransmission under physiological and psychopathological conditions in the human brain in vivo.     

In vivo imaging of adenosine A1 receptors in the human brain with [18F]CPFPX and positron emission tomography

The important roles played by the A(1) adenosine receptor (A(1)AR) in brain physiology and pathology make this receptor a target for in vivo imaging. Here we describe the distribution of A(1)ARs in the living human brain with PET, made possible for the first time by the highly potent and selective A(1)AR antagonist 8-cyclopentyl-3-(3-[(18)F]fluoropropyl)-1-propylxanthine ([(18)F]CPFPX). In vivo data demonstrate a rapid cerebral uptake, peaking at 2.9 +/- 0.6% injected dose/liter at 3.3 +/- 1.3 min, followed by a gradual washout. Consistent with the results of autoradiography, high receptor densities occurred in the putamen and the mediodorsal thalamus. Neocortical regions showed regional differences in [(18)F]CPFPX binding, with high accumulation in temporal > occipital > parietal > frontal lobes and a lower level of binding in the sensorimotor cortex. Ligand accumulation was low in cerebellum, midbrain, and brain stem. Metabolism of [(18)F]CPFPX is rapid outside the central nervous system, but the metabolites do not penetrate the blood-brain barrier. In conclusion, in vivo application of [(18)F]CPFPX, a highly potent and selective PET ligand, for the first time allows the imaging of A(1)ARs in the living human brain.     

Effects of fluvoxamine treatment on the in vivo binding of [F-18]FESP in drug naive depressed patients: a PET study

This study investigates the effect of chronic treatment with Fluvoxamine, a potent and specific serotonin reuptake sites inhibitor (SSRI), on 5HT(2) serotonin and D(2) dopamine receptors in the brain of drug naive unipolar depressed patients. Drug effect was evaluated in different cortical areas and in the basal ganglia by positron emission tomography (PET) and fluoro-ethyl-spiperone ([(18)F]FESP), an high affinity 5HT(2) serotonin and D(2) dopamine receptors antagonist. Patients underwent a PET study at recruitment and after clinical response to Fluvoxamine treatment. Nine of the 15 patients recruited completed the study. Fluvoxamine treatment significantly improved clinical symptoms and modified [(18)F]FESP binding in the frontal and occipital cortex of all of the nine patients who completed the study; in these regions a mean 31% increase in the in vivo [(18)F]FESP binding was found (P < 0.01). On the contrary, no significant changes in the in vivo [(18)F]FESP binding were found in the basal ganglia where [(18)F]FESP binds mainly to D(2) dopamine receptors. Chronic treatment with Fluvoxamine significantly increases the in vivo binding of [(18)F]FESP in the frontal and occipital cortex of drug naive unipolar depressed patients. The increase of the in vivo binding of [(18)F]FESP may reflect a modification in 5HT(2) binding capacity secondary to changes in cortical serotonin activity.     

Quantitation of cell number by a positron emission tomography reporter gene strategy.

PURPOSE: An important potential of positron emission tomography (PET) is the capacity for quantitation of cell signals in an anatomic regions of interest. However, little is known about the constraints and parameters for using PET signal detection to establish cell numbers in regions of interest. In this study, we determined the correlation of PET signal to cell number, and characterized the cellular limit of detection for PET imaging. PROCEDURES: Cells expressing the herpes simplex virus type I thymidine kinase PET reporter gene (HSV1-sr39TK) were detected following accumulation of [(18)F]FHBG (9-[4-[(18)F]-fluoro-3-(hydroxymethyl) butyl]guanine) by microPET scanning and quantitation. RESULTS: When cells were cultured with [(18)F]FHBG in vitro, and then transferred to a model vascularized site, 73% retention was observed one hour post-transfer. Using this information, and the measured attenuation of PET signal in whole mouse scans, we assessed the per-cell uptake of [(18)F]FHBG in the vascularized site following standard parenteral administration of the substrate. We observed a cell number-dependent signal, with a limit of detection calculated as 10(6) cells in a region of interest of 0.1 mL volume. Quantitatively similar parameters were observed with stably tranfected N2a glioma cells and retrovirally transduced primary T lymphocytes. CONCLUSION: These methods and findings provide a strategy for quantitation of cellularity using PET imaging that has implications for both experimental models and clinical diagnosis.     

Basic Evaluation of FES-hERL PET Tracer-Reporter Gene System for <Emphasis Type="Italic">In Vivo</Emphasis> Monitoring of Adenoviral-Mediated Gene Therapy

PURPOSE: The purpose of the study is to evaluate the feasibility of human estrogen receptor alpha ligand binding domain (hERL) as a reporter gene in combination with positron emission tomography (PET) probe, 16alpha-[(18)F]fluoro-17beta-estradiol (FES), in an adenovirus gene delivery system. METHODS: An adenoviral vector (test), carrying hERL gene and a model angiogenesis therapeutic gene (human thymidine phosphorylase, hTP) was constructed along with a control vector. In vitro radioligand binding and expression studies were performed on various cell lines. The control and test viruses were injected into contralateral adductor muscles of a rat followed by FES-PET imaging and immunohistochemical staining of resected muscle samples. RESULTS: A high FES uptake accompanied by hERL and hTP expression was obtained both in vitro and in vivo by the test adenovirus infection. CONCLUSION: Considering the versatile tissue permeability of the probe, highly efficient gene expression, and safeness for human use, we expect our reporter gene system to have favorable characteristics for clinical application.     

The pyridinyl-6 position of WAY-100635 as a site for radiofluorination--effect on 5-HT1A receptor radioligand behavior in vivo.

PURPOSE: We aimed to evaluate radiofluorination at the pyridinyl-6 position of the selective 5-HT(1A) receptor antagonist, WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazinyl)ethyl))-N-(2-pyridinyl)cyclohexanecarboxamide)], on 5-HT(1A) receptor radioligand behavior in vivo. PROCEDURES: The pyridinyl-6 [(18)F]fluoro derivative of WAY-100635 ([(18)F]6FPWAY) was obtained by direct nucleophilic substitution with [(18)F]fluoride ion in a bromo precursor. After intravenous injection of [(18)F]6FPWAY into Cynomolgus monkey, the uptake of radioactivity into brain regions was assessed with positron emission tomography (PET) and blood samples analyzed by high performance liquid chromatography (HPLC) for parent radioligand and radioactive metabolites. The experiment was repeated after pretreatment of the monkey with a dose of WAY-100635 that blocks brain 5-HT(1A) receptors. RESULTS: After intravenous injection of [(18)F]6FPWAY into Cynomolgus monkey, the uptake of radioactivity into whole brain reached 4.33% of injected dose at 7.5 min. Uptake was highest in 5-HT(1A) receptor-rich regions. Pretreatment with WAY-100635 reduced uptake in these regions to near the levels in receptor-devoid cerebellum. [(18)F]6FPWAY was rapidly metabolized in vivo, as evidenced by the rapid appearance of radioactive metabolites in plasma. CONCLUSION: [(18)F]6FPWAY is selective and moderately useful for imaging brain 5-HT(1A) receptors in vivo. The pyridinyl-6 position is resistant to defluorination and may be an attractive site for the (18)F-labeling of 6FPWAY analogs that resist hydrolysis.     

A database of [(11)C]WAY-100635 binding to 5-HT(1A) receptors in normal male volunteers: normative data and relationship to methodological, demographic, physiological, and behavioral variables

PET studies of [(11)C]WAY-100635 binding are proving to be a useful tool to evaluate 5-HT(1A) receptor function in vivo in humans. We describe the pattern of [(11)C]WAY-100635 binding in 61 healthy male brains and examine its variability. For all PET scans, binding potential (BP) values for [(11)C]WAY-100635 in different regions were calculated using a simplified reference tissue model, with the cerebellum as reference region. Specifically we describe (1) region of interest and SPM databases of PET [(11)C]WAY-100635 binding, including test-retest variability; (2) the sensitivity of [(11)C]WAY-100635 binding to manipulations of endogenous 5-HT; and (3) correlations between [(11)C]WAY-100635 binding and radiochemical, demographic, physiological, and behavioral variables. The regional distribution of [(11)C]WAY-100635 binding in healthy human brain was similar to that reported in vitro. The test-retest variability was approximately 12% (range 9-16%) and was similar for all methods of regional sampling. The binding of [(11)C]WAY-100635 was insensitive to changes in brain 5-HT induced by tryptophan infusion and depletion. Although BP values varied greatly across subjects (range 2.9-6.8), there were no significant correlations of regional and global BP with common radiochemical, demographic, physiological, and personality variables. Specifically, in contrast with two recent small studies, we found no decline of [(11)C]WAY-100635 binding with age in our large cohort over the age range of 24 to 53 years. Assessment of 5-HT(1A) receptors in vivo using PET and [(11)C]WAY-100635 gives reliable measures of 5-HT(1A) binding. The large between-subject variability observed could not be explained by common methodological, physiological, or behavioral factors and hence the biological basis of this variability remains to be clarified.     

Pharmacological characterization of (E)-N-(4-fluorobut-2-enyl)-2beta-carbomethoxy-3beta-(4'-tolyl)nortropane (LBT-999) as a highly promising fluorinated ligand for the dopamine transporter

In the aim to develop an efficient fluorinated probe for positron emission tomography (PET) exploration of the dopamine transporter (DAT), we studied several in vitro and in vivo characteristics of the phenyltropane derivative (E)-N-(4-fluorobut-2-enyl)-2beta-carbomethoxy-3beta-(4'-tolyl)nortropane (LBT-999). In vitro on rat striatal membrane, [(3)H]LBT-999 bound to a single site with a K(d) of 9 nM, B(max) of 17 pmol/mg protein, and a very high selectivity for the DAT [IC(50) for 1-{2-[bis-(4-fluorophenyl)-methoxy]ethyl}-4-(3-phenylpropyl)piperazine (GBR 12909) and (E)-N-(3-iodoprop-2-enyl)-2beta-carbomethoxy-3beta-(4'-methylphenyl)nortropane (PE2I): 2.4 and 18 nM, respectively; IC(50) for paroxetine, citalopram, N,N-dimethyl-2-(2-amino-4-methylphenyl thio)benzylamine, nisoxetine, and desipramine >1 muM]. In vitro on post-mortem human brain sections, LBT-999 bound with high intensity to the caudate-putamen, weakly to the thalamus, and not in the neocortex and cerebellum. This binding was totally abolished in the presence of PE2I. Ex vivo cerebral biodistribution of [(11)C]LBT-999 in rats showed striatum/cerebellum radioactivity ratios of 18 and 25 at 30 and 60 min postinjection, respectively. This accumulation was strongly prevented by preinjection of GBR 12909, whereas paroxetine and nisoxetine had no effect. An in vivo kinetic PET study in three baboons showed a fast and very high uptake in the striatum, with a plateau at 30 min postinjection and a maximal putamen/cerebellum ratio of 30. Taken together, these findings demonstrate that LBT-999 is a highly promising agent for in vivo exploration of the DAT. This probe is currently labeled with (18)F for further characterizations.     

Comparison of [<Superscript>11</Superscript>C]Choline ([<Superscript>11</Superscript>C]CHO) and [<Superscript>18</Superscript>F]Bombesin (BAY 86-4367) as Imaging Probes for Prostate Cancer in a PC-3 Prostate Cancer Xenograft Model

Purpose

Carbon-11- and fluorine-18-labeled choline derivatives are commonly used in prostate cancer imaging in the clinical setting for staging and re-staging of prostate cancer. Due to a limited detection rate of established positron emission tomography (PET) tracers, there is a clinical need for innovative tumor-specific PET compounds addressing new imaging targets. The aim of this study was to compare the properties of [¹⁸F]Bombesin (BAY 86-4367) as an innovative biomarker for prostate cancer imaging targeting the gastrin-releasing peptide receptor and [¹¹C]Choline ([¹¹C]CHO) in a human prostate tumor mouse xenograft model by small animal PET/X-ray computed tomography (CT).

Procedures

We carried out a dual-tracer small animal PET/CT study comparing [¹⁸F]Bombesin and [¹¹C]CHO. The androgen-independent human prostate tumor cell line PC-3 was implanted subcutaneously in the flanks of nu/nu NMRI mice (n?=?10) (PET/CT measurements of two [¹¹C]Choline mice could not be analyzed due to technical reasons). [¹⁸F]Bombesin and [¹¹C]CHO PET/CT imaging was performed about 3–4 weeks after the implantation of PC-3 cells on two separate days. After the intravenous tail vein injection of 14 MBq [¹⁸F]Bombesin and 37 MBq [¹¹C]CHO, respectively, a dynamic study over 60 min was acquired in list mode using an Inveon animal PET/CT scanner (Siemens Medical Solutions). The sequence of [¹⁸F]Bombesin and [¹¹C]CHO was randomized. Image analysis was performed using summed images as well as dynamic data. To calculate static and dynamic tumor-to-muscle (T/M), tumor-to-blood (T/B), liver-to-blood (L/B), and kidney-to-blood (K/B) ratios, 4?×?4?×?4 mm³ volumes of interest (VOIs) of tumor, muscle (thigh), liver, kidney, and blood derived from transversal slices were used.

Results

The mean T/M ratio of [¹⁸F]Bombesin and [¹¹C]CHO was 6.54?±?2.49 and 1.35?±?0.30, respectively. The mean T/B ratio was 1.83?±?0.79 for [¹⁸F]Bombesin and 0.55?±?0.10 for [¹¹C]CHO. The T/M ratio as well as the T/B ratio for [¹⁸F]Bombesin were significantly higher compared to those for [¹¹C]CHO (p?<?0.001, respectively). Kidney and liver uptake was statistically significantly lower for [¹⁸F]Bombesin (K/B 3.41?±?0.81, L/B 1.99?±?0.38) compared to [¹¹C]CHO [K/B 7.91?±?1.85 (p?<?0.001), L/B 6.27?±?1.99 (p?<?0.001)]. The magnitudes of the time course of T/M and T/B ratios (T/M and T/B_dyn ratios) were statistically significantly different (showing a higher uptake of [¹⁸F]Bombesin compared to [¹¹C]CHO); additionally, also the change of the T/M and T/B ratios over time was significantly different between both tracers in the dynamic analysis (p?<?0.001, respectively). Furthermore, there was a statistically significantly different change of the K/B and L/B ratios over time between the two tracers in the dynamic analysis (p?=?0.026 and p?<?0.001, respectively).

Conclusions

[¹⁸F]Bombesin (BAY 86-4367) visually and semi-quantitatively outperforms [¹¹C]CHO in the PC-3 prostate cancer xenograft model. [¹⁸F]Bombesin tumor uptake was significantly higher compared to [¹¹C]CHO. [¹⁸F]Bombesin showed better imaging properties compared to the clinically utilized [¹¹C]CHO due to a higher tumor uptake as well as a lower liver and kidney uptake.

     

Quantitative assessment of P-glycoprotein function in the rat blood-brain barrier by distribution volume of [11C]verapamil measured with PET

The blood-brain barrier (BBB) is a functional barrier that hampers the delivery of various drugs to the brain by its physicoanatomical properties and by the presence of ATP-driven drug efflux pumps, such as P-glycoprotein (P-gp). The aims of this study were (1) to study whether the distribution volume (DV) is useful for quantification of (labeled) P-gp substrate kinetics over the BBB and (2) to study how brain DV is affected by P-gp modulation. We measured the kinetics of the P-gp substrate [11C]verapamil (0.1 mg/kg) in rat brains using positron emission tomography (PET) and arterial blood sampling. Cyclosporin A (CsA) at 0, 10, 15, 25, 35, and 50 mg/kg of body weight was used as a P-gp modulator. The [11C]verapamil kinetics were very well described by DV, computed by noncompartmental Logan analysis. Logan analysis resulted in excellent fits of dynamic PET data, revealing the reversible behavior of [11C]verapamil and its associated DV. The DV in unmodulated rats was 0.65 ml/ml +/- 0.23 (mean +/- SD). After modulation with 10, 15, 25, 35, and 50 mg/kg of CsA, DV values increased to 0.82 +/- 0.06, 1.04 +/- 0.20, 2.85 +/- 0.51, 2.91 +/- 0.64, and 3.77 +/- 1.23, respectively. The [11C]Verapamil kinetics were saturable at modulation levels above 25 mg/kg of CsA. The data fitted well by a four-parameter Hill plot (R2 = 0.79). In conclusion, the DV of [11C]verapamil is a valid and potent tool to measure the kinetics of (labeled) P-gp substrates in vivo at the BBB. The brain DV of [11C]verapamil increases dose dependently by P-gp modulation. Quantitative insight into in vivo P-gp modulation may be a promising step toward assessment of P-gp substrate delivery to human brains.     

PET imaging of cholinergic deficits in rats using [18F]fluoroethoxybenzovesamicol ([18F]FEOBV)

[(18)F]fluoroethoxybenzovesamicol ([(18)F]FEOBV) is one of the most promising radioligands for imaging the vesicular ACh transporter (VAChT) with positron emission tomography (PET). We report here that this method can detect subtle cholinergic terminals losses such as those associated with aging, or those following a partial lesion of the nucleus basalis magnocellularis (NBM). Twenty-one adult rats were evenly distributed in three groups including 1) aged rats (18 months); 2) young rats (3 months); and 3) rats with unilateral lesion of the NBM, following a local stereotaxic infusion of 192 IgG-saporin. In both normal and lesioned rats, our results revealed the highest [(18)F]FEOBV binding to be in the striatum, followed by similar values in both frontal cortex and thalamus, while lower values were observed in both hippocampus and temporo-parietal cortex. This binding distribution is consistent with the known anatomy of brain cholinergic systems. In the lesioned rats, [(18)F]FEOBV binding was found to be reduced mostly in the ventral frontal cortex on the side of the lesion, but some reductions were also observed in the homologous region of the contralateral hemisphere. Aging was found to be associated with a [(18)F]FEOBV binding reduction limited to the hippocampus of both hemispheres. [(18)F]FEOBV appears to be a very promising marker for the in vivo quantification of the brain VAChT; PET imaging of this agent allows in vivo detection of both physiological and pathological reductions of cholinergic terminals density.     

Limited distribution of new quinolone antibacterial agents into brain caused by multiple efflux transporters at the blood-brain barrier

Transport of new quinolone antibacterial agents (quinolones) at the blood-brain barrier (BBB) was studied in vitro by using immortalized rat brain capillary endothelial cells RBEC1, and in vivo by using the brain perfusion method in rats and multidrug-resistant mdr1a/1b gene-deficient mice. The permeability coefficient of grepafloxacin measured by brain perfusion was increased by an excess of unlabeled grepafloxacin, suggesting a participation of a saturable BBB efflux system. Uptake coefficients of [(14)C]grepafloxacin, [(14)C]sparfloxacin, and [(14)C]levofloxacin by RBEC1 cells at the steady state were increased in the presence of the unlabeled quinolones. The steady-state uptake of [(14)C]grepafloxacin was increased in the presence of various quinolones. Brain distributions of [(14)C]grepafloxacin and [(14)C]sparfloxacin evaluated in terms of the brain-to-plasma free concentration ratio in mdr1a/1b gene-deficient mice were significantly higher than those in wild-type mice, demonstrating an involvement of P-glycoprotein as the efflux transporter. Anionic compounds, including 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and genistein, increased the steady-state uptake of [(14)C]grepafloxacin by RBEC1 cells. Because [(14)C]grepafloxacin was transported by multidrug resistance-associated protein (MRP), in MRP1-overexpressing cells and because RBEC1 and primary cultured brain capillary endothelial cells expressed MRP1, this protein may be an additional efflux transporter for quinolones. Furthermore, the permeability coefficient of [(14)C]grepafloxacin across the BBB was increased by DIDS or in the absence of bicarbonate ions in the brain perfusion method. DIDS or bicarbonate ion did not affect MRP1 function. Accordingly, the brain distribution of quinolones is restricted by the action of multiple efflux transporters, including P-glycoprotein, MRP1, and an unknown anion exchange transporter.