Metabolite Study and Structural Authentication for the First-in-Human Use Sphingosine-1-phosphate Receptor 1 Radiotracer.

The sphingosine-1-phosphate receptor 1 (S1PR1) radiotracer [11C]CS1P1 has shown promise in proof-of-concept PET imaging of neuroinflammation in multiple sclerosis (MS). Our HPLC radiometabolite analysis of human plasma samples collected during PET scans with [11C]CS1P1 detected a radiometabolite peak that is more lipophilic than [11C]CS1P1. Radiolabeled metabolites that cross the blood-brain barrier complicate quantitative modeling of neuroimaging tracers; thus, characterizing such radiometabolites is important. Here, we report our detailed investigation of the metabolite profile of [11C]CS1P1 in rats, nonhuman primates, and humans. CS1P1 is a fluorine-containing ligand that we labeled with C-11 or F-18 for preclinical studies; the brain uptake was similar for both radiotracers. The same lipophilic radiometabolite found in human studies also was observed in plasma samples of rats and NHPs for CS1P1 labeled with either C-11 or F-18. We characterized the metabolite in detail using rats after injection of the nonradioactive CS1P1. To authenticate the molecular structure of this radiometabolite, we injected rats with 8 mg/kg of CS1P1 to collect plasma for solvent extraction and HPLC injection, followed by LC/MS analysis of the same metabolite. The LC/MS data indicated in vivo mono-oxidation of CS1P1 produces the metabolite. Subsequently, we synthesized three different mono-oxidized derivatives of CS1P1 for further investigation. Comparing the retention times of the mono-oxidized derivatives with the metabolite observed in rats injected with CS1P1 identified the metabolite as N-oxide 1, also named TZ82121. The MS fragmentation pattern of N-oxide 1 also matched that of the major metabolite in rat plasma. To confirm that metabolite TZ82121 does not enter the brain, we radiosynthesized [18F]TZ82121 by the oxidation of [18F]FS1P1. Radio-HPLC analysis confirmed that [18F]TZ82121 matched the radiometabolite observed in rat plasma post injection of [18F]FS1P1. Furthermore, the acute biodistribution study in SD rats and PET brain imaging in a nonhuman primate showed that [18F]TZ82121 does not enter the rat or nonhuman primate brain. Consequently, we concluded that the major lipophilic radiometabolite N-oxide [11C]TZ82121, detected in human plasma post injection of [11C]CS1P1, does not enter the brain to confound quantitative PET data analysis. [11C]CS1P1 is a promising S1PR1 radiotracer for detecting S1PR1 expression in the CNS.

Phase 1 Evaluation of 11C-CS1P1 to Assess Safety and Dosimetry in Human Participants.

This study evaluated the safety, dosimetry, and characteristics of 3-((2-fluoro-4-(5-(2'-methyl-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-1,2,4-oxadiazol-3-yl)benzyl)(methyl-11C)amino)propanoic acid (11C-CS1P1), a radiotracer targeting sphingosine-1-phosphate receptor (S1PR) 1 (S1PR1). S1PR1 is of clinical interest because of its role in multiple sclerosis (and other conditions), with an expanding class of S1PR modulators approved for relapsing multiple sclerosis. 11C-CS1P1 binds S1PR1 with high specificity and has shown promise in animal models of inflammatory diseases. Methods: 11C-CS1P1 was injected into 5 male and 6 female healthy participants. Ten participants were imaged with PET using a multipass whole-body continuous-bed-motion acquisition, and one had dedicated head and neck PET and MRI. Participants were continuously monitored for safety events. Organ time-activity curve data were collected, integrated, and normalized to the injected activity. Organ radiation doses and effective dose were computed using the adult male and female models in OLINDA, version 2.2. SUV images were evaluated for qualitative biodistribution. Results: No adverse events were observed after the dose, including no bradycardia. The liver was the critical organ from dosimetry analysis (mean ± SD: female, 23.12 ± 5.19 µSv/MBq; male, 21.06 ± 1.63 µSv/MBq). The whole-body effective dose (as defined by International Commission on Radiological Protection publication 103) was 4.18 ± 0.30 µSv/MBq in women and 3.54 ± 0.14 µSv/MBq in men. Using a maximum delivered dose of 740 MBq (20 mCi), the effective dose for women would be 3.1 mSv (0.31 rem), with a liver dose of 17.1 mSv (1.7 rem); the effective dose for men would be 2.6 mSv (0.26 rem), with a liver dose of 15.6 mSv (1.56 rem). Brain uptake was seen predominantly in gray matter and correlated with regional S1PR1 RNA expression (r = 0.84). Conclusion: These results support the safety of 11C-CS1P1 for evaluation of inflammation in human clinical populations. Dosimetry permits repeated measures in the same participants. Brain uptake correlates well with known target topography.

Acute Rodent Tolerability, Toxicity, and Radiation Dosimetry Estimates of the S1P1-Specific Radioligand [11C]CS1P1.

PURPOSE: In preclinical studies with rodent models of inflammatory diseases, [11C]CS1P1 has been identified as a promising imaging agent targeting sphingosine-1-phosphate receptor 1 (S1P1) in the central nervous system and other tissues. In preparation for USA Food and Drug Administration (FDA) approval of [11C]CS1P1 for human use, an acute biodistribution study in mice and an acute tolerability and toxicity evaluation in rats were conducted. PROCEDURES: Acute organ biodistribution and excretion data was obtained using male and female Swiss Webster mice intravenously (IV) injected with 4.8-10 MBq of [11C]CS1P1. The organ residence times for each harvested organ were calculated using the animal biodistribution data, and were entered in the program OLINDA/EXM for C-11 to obtain human radiation dosimetry estimates. Acute tolerability and toxicity studies were conducted in male and female Sprague Dawley rats. Rats were administered an IV bolus of either the vehicle control or 0.3 mg/kg CS1P1. Blood samples were collected and a gross post-mortem examination was conducted at day 2 or day 15 post-injection. RESULTS: The extrapolated human radiation dose estimates revealed that the highest organ dose was received by the liver with 24.05 µGy/MBq in males and 32.70 µGy/MBq in females. The effective dose (ED) estimates of [11C]CS1P1 were calculated at 3.5 µSv/MBq in males and 5.9 µSv/MBq in females. The acute tolerability and toxicity study identified 0.3 mg/kg as a no observable adverse effect level (NOAEL) dose, which is a ~ 300-fold dose multiple of the human equivalent dose of the mass to be injected for positron emission tomography (PET) imaging studies in humans as a no-observable-effect limit. CONCLUSIONS: The toxicity study in rats suggested that injection dose of radiotracer [11C]CS1P1 with mass amount < 10 µg is safe for performing a human PET study. The dosimetry data supported an injection of 0.74 GBq (20 mCi) dose for human studies would be acceptable.

The role of beta-arrestin2 in shaping fMRI BOLD responses to dopaminergic stimulation.

RATIONALE: The dopamine D2 receptor (D2R) couples to inhibitory Gi/o proteins and is targeted by antipsychotic and antiparkinsonian drugs. Beta-arrestin2 binds to the intracellular regions of the agonist-occupied D2R to terminate G protein activation and promote internalization, but also to initiate downstream signaling cascades which have been implicated in psychosis. Functional magnetic resonance imaging (fMRI) has proven valuable for measuring dopamine receptor-mediated changes in neuronal activity, and might enable beta-arrestin2 function to be studied in vivo. OBJECTIVES: The present study examined fMRI blood oxygenation level dependent (BOLD) signal changes elicited by a dopamine agonist in wild-type (WT) and beta-arrestin2 knockout (KO) mice, to investigate whether genetic deletion of beta-arrestin2 prolongs or otherwise modifies D2R-dependent responses. METHODS: fMRI BOLD data were acquired on a 9.4 T system. During scans, animals received 0.2 mg/kg apomorphine, i.v. In a subset of experiments, animals were pretreated with 2 mg/kg of the D2R antagonist, eticlopride. RESULTS: Following apomorphine administration, BOLD signal decreases were observed in caudate/putamen of WT and KO animals. The time course of response decay in caudate/putamen was significantly slower in KO vs. WT animals. In cingulate cortex, an initial BOLD signal decrease was followed by a positive response component in WT but not in KO animals. Eticlopride pretreatment significantly reduced apomorphine-induced BOLD signal changes. CONCLUSIONS: The prolonged striatal response decay rates in KO animals might reflect impaired D2R desensitization, consistent with the known function of beta-arrestin2. Furthermore, the apomorphine-induced positive response component in cingulate cortex may depend on beta-arrestin2 signaling downstream of D2R.

Radiosyntheses and in vivo evaluation of carbon-11 PET tracers for PDE10A in the brain of rodent and nonhuman primate.

The radiosyntheses and in vivo evaluation of four carbon-11 labeled quinoline group-containing radioligands are reported here. Radiolabeling of [(11)C]1-4 was achieved by alkylation of their corresponding desmethyl precursors with [(11)C]CH3I. Preliminary biodistribution evaluation in Sprague-Dawley rats demonstrated that [(11)C]1 and [(11)C]2 had high striatal accumulation (at peak time) for [(11)C]1 and [(11)C]2 were 6.0-fold and 4.5-fold at 60 min, respectively. Following MP-10 pretreatment, striatal uptake in rats of [(11)C]1 and [(11)C]2 was reduced, suggesting that the tracers bind specifically to PDE10A. MicroPET studies of [(11)C]1 and [(11)C]2 in nonhuman primates (NHP) also showed good tracer retention in the striatum with rapid clearance from non-target brain regions. Striatal uptake (SUV) of [(11)C]1 reached 1.8 at 30 min with a 3.5-fold striatum:cerebellum ratio. In addition, HPLC analysis of solvent extracts from NHP plasma samples suggested that [(11)C]1 had a very favorable metabolic stability. Our preclinical investigations suggest that [(11)C]1 is a promising candidate for quantification of PDE10A in vivo using PET.

Synthesis, [¹8F] radiolabeling, and evaluation of poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors for in vivo imaging of PARP-1 using positron emission tomography.

Imaging of poly (ADP-ribose) polymerase-1 (PARP-1) expression in vivo is a potentially powerful tool for developing PARP-1 inhibitors for drug discovery and patient care. We have synthesized several derivatives of benzimidazole carboxamide as PARP-1 inhibitors, which can be (18)F-labeled easily for positron emission tomographic (PET) imaging. Of the compounds synthesized, 12 had the highest inhibition potency for PARP-1 (IC50=6.3 nM). [(18)F]12 was synthesized under conventional conditions in high specific activity with 40-50% decay-corrected yield. MicroPET studies using [(18)F]12 in MDA-MB-436 tumor-bearing mice demonstrated accumulation of [(18)F]12 in the tumor that was blocked by olaparib, suggesting that the uptake of [(18)F]12 in the tumor is specific to PARP-1 expression.

Quantitative receptor-based imaging of tumor proliferation with the sigma-2 ligand [(18)F]ISO-1.

The sigma-2 receptor is expressed in higher density in proliferating (P) tumor cells versus quiescent (Q) tumor cells, thus providing an attractive target for imaging the proliferative status (i.e., P:Q ratio) of solid tumors. Here we evaluate the utility of the sigma-2 receptor ligand 2-(2-[(18)F]fluoroethoxy)-N-(4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)butyl)-5-methyl-benzamide, [(18)F]ISO-1, in two different rodent models of breast cancer. In the first study, small animal Positron Emission Tomography (PET) imaging studies were conducted with [(18)F]ISO-1 and (18)FDG in xenografts of mouse mammary tumor 66 and tracer uptake was correlated with the in vivo P:Q ratio determined by flow cytometric measures of BrdU-labeled tumor cells. The second model utilized a chemically-induced (N-methyl-N-nitrosourea [MNU]) model of rat mammary carcinoma to correlate measures of [(18)F]ISO-1 and FDG uptake with MR-based volumetric measures of tumor growth. In addition, [(18)F]ISO-1 and FDG were used to assess the response of MNU-induced tumors to bexarotene and Vorozole therapy. In the mouse mammary 66 tumors, a strong linear correlation was observed between the [(18)F]ISO-1 tumor: background ratio and the proliferative status (P:Q ratio) of the tumor (R = 0.87). Similarly, measures of [(18)F]ISO-1 uptake in MNU-induced tumors significantly correlated (R = 0.68, P<0.003) with changes in tumor volume between consecutive MR imaging sessions. Our data suggest that PET studies of [(18)F]ISO-1 provide a measure of both the proliferative status and tumor growth rate, which would be valuable in designing an appropriate treatment strategy.

Development of (18)F-labeled PET probes for imaging cell proliferation.

In recent years, two different methods have been developed to image cell proliferation with the functional imaging technique, Positron emission Tomography (PET), proliferation rate and proliferative status. Proliferation rate is a measure of the tumor doubling time and uses radiolabeled analogs of the DNA precursor thymidine. This approach measures the activity of the enzyme thymidine kinase 1 (TK1) and provides a pulse label of the S phase fraction of a tumor. Proliferative status provides a measure of the ratio of proliferating (P) and quiescent (Q) cells in a tumor. This imaging approach for measuring proliferative status involves measuring the sigma-2 (σ(2)) receptor status of a tumor, the only protein which has been validated for making this measurement in vivo with PET. This article provides an overview of the biological information obtained from these different imaging strategies, and the development of radiotracers for imaging proliferation rate and proliferative status.

Positron emission tomography imaging of dopamine D2 receptors using a highly selective radiolabeled D2 receptor partial agonist.

A series of microPET imaging studies were conducted in anesthetized rhesus monkeys using the dopamine D2-selective partial agonist, [(11)C]SV-III-130. There was a high uptake in regions of brain known to express a high density of D2 receptors under baseline conditions. Rapid displacement in the caudate and putamen, but not in the cerebellum, was observed after injection of the dopamine D2/3 receptor nonselective ligand S(-)-eticlopride at a low dosage (0.025mg/kg/i.v.); no obvious displacement in the caudate, putamen and cerebellum was observed after the treatment with a dopamine D3 receptor selective ligand WC-34 (0.1mg/kg/i.v.). Pretreatment with lorazepam (1mg/kg, i.v. 30min) to reduce endogenous dopamine prior to tracer injection resulted in unchanged binding potential (BP) values, a measure of D2 receptor binding in vivo, in the caudate and putamen. d-Amphetamine challenge studies indicate that there is a significant displacement of [(11)C]SV-III-130 by d-Amphetamine-induced increases in synaptic dopamine levels.

Radiolabeled isatin binding to caspase-3 activation induced by anti-Fas antibody.

INTRODUCTION: Noninvasive imaging methods that can distinguish apoptosis from necrosis may be useful in furthering our understanding of diseases characterized by apoptotic dysregulation as well as aiding drug development targeting apoptotic pathways. We evaluated the ability of radiolabeled isatins to quantify caspase-3 activity induced by the activation of the extrinsic apoptotic pathway by the anti-Fas antibody in mice. METHODS: The behavior of three different radiolabeled isatins ([(18)F]WC-II-89, [(18)F]WC-IV-3 and [(11)C]WC-98) was characterized in mice with and without anti-Fas antibody treatment by microPET imaging and biodistribution studies. The activity of [(18)F]WC-II-89 was also compared with [(99m)Tc]mebrofenin. The effect of pan-caspase inhibition with quinolyl-valyl-O-methylaspartyl-[2,6-difluorophenoxy]-methyl ketone (Q-VD-OPh) on [(18)F]WC-II-89 uptake was studied. Caspase-3 activity was confirmed by a fluorometric enzyme assay. RESULTS: All three tracers behaved similarly in microPET and biodistribution studies. Increased retention of all tracers was observed in the livers of treated animals and several other organs, all of which demonstrated increased caspase-3 enzyme activity; however, impaired hepatobiliary excretion made attribution of these findings to caspase-3 activity difficult. The isatin [(18)F]WC-II-89 was retained at statistically significantly higher levels in the organs after anti-Fas antibody treatment while [(99m)Tc]mebrofenin activity cleared, suggesting specific binding to activated caspase-3, but the magnitude of increased binding was still relatively low. Caspase inhibition with Q-VD-OPh partially blocked [(18)F]WC-II-89 retention but completely blocked caspase-3 enzyme activity in the liver. CONCLUSIONS: The radiolabeled isatins appear to bind specifically to caspase-3 in vivo, but their sensitivity is limited. Further optimization is required for these tracers to be useful for clinical applications.

Characterization and evaluation of two novel fluorescent sigma-2 receptor ligands as proliferation probes.

We synthesized and characterized two novel fluorescent sigma-2 receptor selective ligands, SW120 and SW116, and evaluated these ligands as potential probes for imaging cell proliferation. Both ligands are highly selective for sigma-2 receptors versus sigma-1 receptors. SW120 and SW116 were internalized into MDA-MB-435 cells, and 50% of the maximum fluorescent intensity was reached in 11 and 24 minutes, respectively. In vitro studies showed that 50% of SW120 or SW116 washed out of cells in 1 hour. The internalization of SW120 was reduced ≈30% by phenylarsine oxide, an inhibitor of endocytosis, suggesting that sigma-2 ligands are internalized, in part, by an endocytotic pathway. Subcellular localization studies using confocal and two-photon microscopy showed that SW120 and SW116 partially colocalized with fluorescent markers of mitochondria, endoplasmic reticulum, lysosomes, and the plasma membrane, suggesting that sigma-2 receptors localized to the cytoplasmic organelles and plasma membrane. SW120 did not colocalize with the nuclear dye 4',6-diamidino-2-phenylindole. In vivo studies showed that the uptake of SW120 in solid tumors and peripheral blood mononuclear cells of mice positively correlated with the expression level of the cell proliferation marker Ki-67, suggesting that sigma-2 fluorescent probes may be used to image cell proliferation in mice.

Effect of cyclosporin A on the uptake of D3-selective PET radiotracers in rat brain.

INTRODUCTION: Four benzamide analogs having a high affinity and selectivity for D(3) versus D(2) receptors were radiolabeled with (11)C or (18)F for in vivo evaluation. METHODS: Precursors were synthesized, and the four D(3) selective benzamide analogs were radiolabeled. The tissue distribution and brain uptake of the four compounds were evaluated in control rats and rats pretreated with cyclosporin A, a modulator of P-glycoprotein and an inhibitor of other ABC efflux transporters that contribute to the blood brain barrier. Micro-positron emission tomographic (PET) imaging was carried out for [(11)C]6 in a control and a cyclosporin A pretreated rat. RESULTS: All four compounds showed low brain uptake in control rats at 5 and 30 min post-injection; despite recently reported rat behavioral studies conducted on analogs 6 (WC-10) and 7 (WC-44). Following administration of cyclosporin A, increased brain uptake was observed with all four PET radiotracers at both 5 and 30 min post-intravenous injection. An increase in brain uptake following modulation/inhibition of the ABC transporters was also observed in the microPET study. CONCLUSIONS: These data suggest that D3 selective conformationally-flexible benzamide analogs which contain a N-2-methoxyphenylpiperazine moiety are substrates for P-glycoprotein or other adenosine triphosphate (ATP)-binding cassette transporters expressed at the blood-brain barrier, and that PET radiotracers containing this pharmacophore may display low brain uptake in rodents due to the action of these efflux transporters.

Radiosynthesis and in vivo evaluation of [11C]MP-10 as a PET probe for imaging PDE10A in rodent and non-human primate brain.

2-((4-(1-[(11)C]Methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl)-quinoline (MP-10), a specific PDE10A inhibitor (IC(50)=0.18 nM with 100-fold selectivity over other PDEs), was radiosynthesized by alkylation of the desmethyl precursor with [(11)C]CH(3)I, ∼45% yield, >92% radiochemical purity, >370 GBq/µmol specific activity at end of bombardment (EOB). Evaluation in Sprague-Dawley rats revealed that [(11)C]MP-10 had highest brain accumulation in the PDE10A enriched-striatum, the 30 min striatum: cerebellum ratio reached 6.55. MicroPET studies of [(11)C]MP-10 in monkeys displayed selective uptake in striatum. However, a radiolabeled metabolite capable of penetrating the blood-brain-barrier may limit the clinical utility of [(11)C]MP-10 as a PDE10A PET tracer.

Endogenous dopamine (DA) competes with the binding of a radiolabeled D3 receptor partial agonist in vivo: a positron emission tomography study.

A series of microPET imaging studies were conducted in anesthetized rhesus monkeys using the dopamine D3-selective partial agonist, [¹8F]5. There was variable uptake in regions of brain known to express a high density of D3 receptors under baseline conditions. Pretreatment with lorazepam (1 mg/kg, i.v. 30 min) to reduce endogenous dopamine activity before tracer injection resulted in a dramatic increase in uptake in the caudate, putamen, and thalamus, and an increase in the binding potential (BP) values, a measure of D3 receptor binding in vivo. These data indicate that there is a high level of competition between [¹8F]5 and endogenous dopamine for D3 receptors in vivo.

Click synthesis and biologic evaluation of (R)- and (S)-2-amino-3-[1-(2-[18F]fluoroethyl)-1H-[1,2,3]triazol-4-yl]propanoic acid for brain tumor imaging with positron emission tomography.

The (R)- and (S)-enantiomers of 2-amino-3-[1-(2-[18F]fluoroethyl)-1H-[1,2,3]triazol-4-yl]propanoic acid (4) were synthesized and evaluated in the rat 9L gliosarcoma brain tumor model using cell uptake assays, biodistribution studies, and micro-positron emission tomography (microPET). The (R)- and (S)-enantiomers of [18F]4 were radiolabeled separately using the click reaction in 57% and 51% decay-corrected yields, respectively. (S)-[18F]4 was a substrate for cationic amino acid transport and, to a lesser extent, system L transport in vitro. In vivo biodistribution studies demonstrated that (S)-[18F]4 provided higher tumor uptake and higher tumor to brain ratios (15:1 at the 30- and 60-minute time points) compared to the (R)-enantiomer (7:1 at the 30- and 60-minute time points). MicroPET studies with (S)-[18F]4 confirmed that this tracer provides good target to background ratios for both subcutaneous and intracranial 9L gliosarcoma tumors. Based on these results, the 1H-[1,2,3]triazole-substituted amino acid (S)-[18F]4 has promising PET properties for brain tumors and represents a novel class of radiolabeled amino acids for tumor imaging.

Radiosynthesis and biological evaluation of a promising sigma(2)-receptor ligand radiolabeled with fluorine-18 or iodine-125 as a PET/SPECT probe for imaging breast cancer.

Sigma-2 receptors represent an endogenous marker for proliferation in solid tumors. The high affinity, high selectivity sigma(2) receptor ligand N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-fluoroethoxy)-5-iodo-3-methoxybenzamide (3) was separately radiolabeled with F-18 and I-125. The radiolabeling yield was 30% and 70% for [(18)F]3 and [(125)I]3, respectively. Studies of [(125)I]3 using murine 66 breast tumor membrane homogenates and evaluation of [(18)F]3 and [(125)I]3 in 66 tumor-bearing mice indicate that this ligand has potential as a PET or a SPECT probe for imaging sigma(2) receptors in breast cancer.

Carbon-11 labeled papaverine as a PET tracer for imaging PDE10A: radiosynthesis, in vitro and in vivo evaluation.

Papaverine, 1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline, a specific inhibitor of phosphodiesterase (PDE) 10A with IC(50) values of 36 nM for PDE10A, 1,300 nM for PDE3A and 320 nM for PDE4D, has served as a useful pharmaceutical tool to study the physiological role of PDE10A. Here, we report the radiosynthesis of [(11)C]papaverine and the in vitro and in vivo evaluation of [(11)C]papaverine as a potential positron emission tomography (PET) radiotracer for imaging PDE10A in the central nervous system (CNS). The radiosynthesis of papaverine with (11)C was achieved by O-methylation of the corresponding des-methyl precursor with [(11)C]methyl iodide. [(11)C]papaverine was obtained with approximately 70% radiochemical yield and a specific activity >10 Ci/mumol. In vitro autoradiography studies of rat and monkey brain sections revealed selective binding of [(11)C]papaverine to PDE10A enriched regions: the striatum of rat brain and the caudate and putamen of rhesus monkey brain. The biodistribution of [(11)C]papaverine in rats at 5 min demonstrated an initially higher accumulation in striatum than in other brain regions, however the washout was rapid. MicroPET imaging studies in rhesus macaques similarly displayed initial specific uptake in the striatum with very rapid clearance of [(11)C]papaverine from brain. Our initial evaluation suggests that despite papaverine's utility for in vitro studies and as a pharmaceutical tool, [(11)C]papaverine is not an ideal radioligand for clinical imaging of PDE10A in the CNS. Analogs of papaverine having a higher potency for inhibiting PDE10A and improved pharmacokinetic properties will be necessary for imaging this enzyme with PET.

[3H]4-(dimethylamino)-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl) butyl)benzamide: a selective radioligand for dopamine D(3) receptors. II. Quantitative analysis of dopamine D(3) and D(2) receptor density ratio in the caudate-putamen.

4-(Dimethylamino)-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)benzamide (WC-10), a N-phenyl piperazine analog, displays high affinity and moderate selectivity for dopamine D(3) receptors versus dopamine D(2) receptors (Chu et al. [2005] Bioorg Med Chem 13:77-87). In this study, WC-10 was radiolabeled with tritium (specific activity = 80 Ci/mmol), and quantitative autoradiography studies were conducted using rhesus monkey and Sprague-Dawley rat brain sections. K(d) values for the binding of [3H]WC-10 to D(3) receptors obtained from quantitative autoradiography with rhesus monkey and rat brain sections are in agreement with K(d) values obtained from cloned human and rat receptors (Xu et al. [2009] Synapse 63:717-728). The D(2) selective antagonist [3H]raclopride binds with 11-fold higher affinity to human HEK D(2L) (K(d) = 1.6 nM) than HEK D(3) (K(d) = 18 nM) receptors; [3H]raclopride binds to rat Sf9 rD(2L) receptors with a K(d) of 6.79 nM, a value that is 4-fold lower than binding to human HEK D(2L) receptors and 2.5-fold higher than binding to rat Sf9 rD(3) receptors. In vitro quantitative autoradiography studies with [3H]WC-10 and [3H]raclopride were conducted on adult rat and rhesus monkey brain sections. A mathematical model for calculating the absolute densities of dopamine D(2) and D(3) receptors based on the in vitro receptor binding data of [3H]WC-10 and [3H]raclopride was developed.

Evaluation of 5-ethynyl-2'-deoxyuridine staining as a sensitive and reliable method for studying cell proliferation in the adult nervous system.

Recently, a novel method for detection of DNA synthesis has been developed based on the incorporation of 5-ethynyl-2'-deoxyuridine (EdU), a thymidine analogue, into cellular DNA and the subsequent reaction of EdU with a fluorescent azide in a copper-catalyzed [3+2] cycloaddition ("Click" reaction). In the present study, we evaluated this method for studying cell proliferation in the adult central nervous system in comparison with the "gold standard" method of 5-bromo-2'-deoxyuridine (BrdU) staining using two behavioral paradigms, voluntary exercise and restraint stress. Our data demonstrate that the number of EdU-positive cells in the dentate gyrus of the hippocampus (DG) slightly increased in an EdU dose-dependent manner in both the control and voluntary exercise (running) mouse groups. The number of EdU-labeled cells was comparable to the number of BrdU-labeled cells in both the control and running mice. Furthermore, EdU and BrdU co-localized to the same cells within the DG. Voluntary exercise significantly increased the number of EdU- and BrdU-positive cells in the DG. In contrast, restraint stress significantly decreased the number of EdU-positive cells. The EdU-positive cells differentiated into mature neurons. EdU staining is compatible with immunohistochemical staining of other antigens. Moreover, our data demonstrated EdU staining can be combined with BrdU staining, providing a valuable tool of double labeling DNA synthesis, e.g., for tracking the two populations of neurons generated at different time points. In conclusion, our results suggest that EdU staining is a fast, sensitive and reproducible method to study cell proliferation in the central nervous system.

Comparison of radiolabeled isatin analogs for imaging apoptosis with positron emission tomography.

INTRODUCTION: Caspase-3 is one of the executioner caspases activated as a result of apoptosis. Radiolabeled isatins bind to caspase-3 with high affinity and are potential tracers for use with positron emission tomography to image apoptosis. We compared the ability of two novel radiolabeled isatins, [18F]WC-IV-3 and [11C]WC-98, to detect caspase-3 activation in a rat model of cycloheximide-induced liver injury. METHODS: Male Sprague-Dawley rats were treated with cycloheximide and then imaged with microPET 3 h later with [18F]WC-IV-3 and [11C]WC-98. Biodistribution studies were also performed simultaneously, with caspase-3 activation verified by fluorometric enzyme assay and Western blots. RESULTS: MicroPET imaging studies demonstrated similar behavior of both tracers but with a lower maximum peak with [11C]WC-98 than with [18F]WC-IV-3. Biodistribution studies demonstrated increased uptake of both tracers in the liver and spleen, but this was statistically significant only in the liver with both compounds. The level of [18F]WC-IV-3 uptake appeared to correlate roughly with rates of caspase-3 activation by the enzyme assay, but the magnitude of difference between treated and control groups was lower than that observed in previously published data with [18F]WC-II-89, another radiolabeled isatin analog. Activation was also confirmed in the liver and spleen but not in fat by Western blot. CONCLUSION: [18F]WC-IV-3 uptake appears to correlate with increased caspase-3 enzyme activity, but the dynamic range of uptake of these two tracers appears to be less than that seen with [18F]WC-II-89. Studies are ongoing to verify these results in other animal models of apoptosis.