Lynne A. Jones

Comparative studies of Cu-64-ATSM and C-11-acetate in an acute myocardial infarction model: ex vivo imaging of hypoxia in rats.

Copper labeled diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) is a promising agent for the imaging of hypoxic tissues. In the present study 64Cu(t1/2 = 12.8 h) labeled Cu-ATSM was used in combination with 11C (t1/2 = 20.3 min) labeled acetate as a regional perfusion marker to visualize hypoxic rat heart tissue in an acute left anterior descending (LAD) coronary artery occluded rat model using an ex vivo tissue slice imaging technique. 64Cu-ATSM was injected intravenously c.a. 10 min after occlusion and rats were sacrificed by cervical dislocation 10 min after injection. Carbon-11-acetate was injected 1 min before sacrifice to obtain a measure of blood flow. The heart was dissected, frozen, and cut into 1-mm thick slices with a gauged slicer, and 11C images were obtained with an electronic autoradiography instrument. After decay of 11C, 64Cu images were obtained in the same manner. In ischemic regions, where there was low 11C accumulation, 64Cu showed high accumulation when compared with normal regions. In rats with a large occlusion, the center of the ischemia did not show any accumulation of either 11C or 64Cu, indicating no blood supply. Cu-ATSM appears to be useful for the detection of hypoxia with contrast being observed at short times (10 min) postinjection.

Selective sigma-2 ligands preferentially bind to pancreatic adenocarcinomas: applications in diagnostic imaging and therapy

BackgroundResistance to modern adjuvant treatment is in part due to the failure of programmed cell death. Therefore the molecules that execute the apoptotic program are potential targets for the development of anti-cancer therapeutics. The sigma-2 receptor has been found to be over-expressed in some types of malignant tumors, and, recently, small molecule ligands to the sigma-2 receptor were found to induce cancer cell apoptosis.ResultsThe sigma-2 receptor was expressed at high levels in both human and murine pancreas cancer cell lines, with minimal or limited expression in normal tissues, including: brain, kidney, liver, lung, pancreas and spleen. Micro-PET imaging was used to demonstrate that the sigma-2 receptor was preferentially expressed in tumor as opposed to normal tissues in pancreas tumor allograft-bearing mice. Two structurally distinct sigma-2 receptor ligands, SV119 and WC26, were found to induce apoptosis to mice and human pancreatic cancer cells in vitro and in vivo. Sigma-2 receptor ligands induced apoptosis in a dose dependent fashion in all pancreatic cell lines tested. At the highest dose tested (10 μM), all sigma-2 receptor ligands induced 10–20% apoptosis in all pancreatic cancer cell lines tested (p < 0.05). In pancreas tumor allograft-bearing mice, a single bolus dose of WC26 caused approximately 50% apoptosis in the tumor compared to no appreciable apoptosis in tumor-bearing, vehicle-injected control animals (p < 0.0001). WC26 significantly slowed tumor growth after a 5 day treatment compared to vehicle-injected control animals (p < 0.0001) and blood chemistry panels suggested that there is minimal peripheral toxicity.ConclusionWe demonstrate a novel therapeutic strategy that induces a significant increase in pancreas cancer cell death. This strategy highlights a new potential target for the treatment of pancreas cancer, which has little in the way of effective treatments.

MicroPET assessment of androgenic control of glucose and acetate uptake in the rat prostate and a prostate cancer tumor model

PET has been used to monitor changes in tumor metabolism in breast cancer following hormonal therapy. This study was undertaken to determine whether PET imaging could evaluate early metabolic changes in prostate tumor following androgen ablation therapy. Studies were performed comparing two positron-emitting tracers, 18F-FDG and 11C-acetate, in Sprague-Dawley male rats to monitor metabolic changes in normal prostate tissue. Additional studies were performed in nude mice bearing the CWR22 androgen-dependent human prostate tumor to evaluate metabolic changes in prostate tumor. In rats, for the androgen ablation pretreatment, 1 mg diethylstilbestrol (DES) was injected subcutaneously 3 and 24 hours before tracer injection. For androgen pretreatment, 500 microg dihydrotestosterone (DHT) was injected intraperitoneally 2 and 6 hours before tracer injection. The rats were divided into three groups, Group A (no-DES, no-DHT, n = 18), Group B (DES, no-DHT, n = 18) and Group C (DES, DHT, n = 18). In each group, 10 animals received 18F-FDG, whereas the remaining eight animals were administered 11C-acetate. Rats were sacrificed at 120 min post-injection of 18F-FDG or 30 min post-injection of 11C-acetate. Pretreatment of the mouse model using DHT (200 microg of DHT in 0.1 mL of sunflower seed oil) or DES (200 microg of DES in 0.1 mL of sunflower seed oil) was conducted every 2 days for one week. Mice were imaged with both tracers in the microPET scanner (Concorde Microsystems Inc.). DES treatment caused a decrease in acetate and glucose metabolism in the rat prostate. Co-treatment with DHT maintained the glucose metabolism levels at baseline values. In the tumor bearing mice, similar effects were seen in 18F-FDG study, while there was no significant difference in 11C-acetate uptake. These results indicate that changes in serum testosterone levels influence 18F-FDG uptake in the prostate gland, which is closely tied to glucose metabolism, within 24 hours of treatment and in the prostate tumor within 1 week. These early metabolic changes could enable monitoring metabolic changes in prostate tumor following treatment by imaging using 18F-FDG PET. Further studies are needed to clarify the reason for the insensitivity of 11C-acetate for measuring metabolic change in prostate tumor.

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.

Synthesis and in Vitro and in Vivo Evaluation of 18F-Labeled Positron Emission Tomography (PET) Ligands for Imaging the Vesicular Acetylcholine Transporter

A new class of vesicular acetylcholine transporter inhibitor that incorporates a carbonyl group into the benzovesamicol structure was synthesized, and analogues were evaluated in vitro. (+/-)-trans-2-Hydroxy-3-(4-(4-[(18)F]fluorobenzoyl)piperidino)tetralin (9e) has K(i) values of 2.70 nM for VAChT, 191 nM for sigma(1), and 251 nM for sigma(2). The racemic precursor (9d) was resolved via chiral HPLC, and (+/-)-[(18)F]9e, (-)-[(18)F]9e, and (+)-[(18)F]9e were respectively radiolabeled via microwave irradiation of the appropriate precursors with [(18)F]/F(-) and Kryptofix/K(2)CO(3) in DMSO with radiochemical yields of approximately 50-60% and specific activities of >2000 mCi/micromol. (-)-[(18)F]9e uptake in rat brain was consistent with in vivo selectivity for the VAChT with an initial uptake of 0.911 %ID/g in rat striatum and a striatum/cerebellum ratio of 1.88 at 30 min postinjection (p.i.). MicroPET imaging of macaques demonstrated a 2.1 ratio of (-)-[(18)F]9e in putamen versus cerebellum at 2 h p.i. (-)-[(18)F]9e has potential to be a PET tracer for clinical imaging of the VAChT.

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 papaverines 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.

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.

New N-substituted 9-azabicyclo[3.3.1]nonan-3α-yl phenylcarbamate analogs as σ2 receptor ligands: synthesis, in vitro characterization, and evaluation as PET imaging and chemosensitization agents

A series of N-substituted 9-azabicyclo[3.3.1]nonan-3alpha-yl phenylcarbamate analogs were synthesized. Among them, WC-26 and WC-59 were identified as the most potent sigma(2) receptor ligands (K(i)=2.58 and 0.82 nM, respectively) with high selectivity against sigma(1) (K(i) of sigma(1)/sigma(2) ratio=557 and 2087, respectively). [(18)F]WC-59 was radiolabeled via a nucleophilic substitution of a mesylate precursor by [(18)F]fluoride, and in vitro direct binding studies of [(18)F]WC-59 were conducted using membrane preparations from murine EMT-6 solid breast tumors. The results indicate that [(18)F]WC-59 binds specifically to sigma(2) receptors in vitro (K(d)= approximately 2 nM). Biodistribution studies of [(18)F]WC-59 in EMT-6 tumor-bearing mice indicated that the tracer was a less suitable candidate for clinical imaging studies than existing F-18 labeled sigma(2) receptor ligands. The ability of WC-26 to enhance the cytotoxic effects of the chemotherapy drug, doxorubicin, was evaluated in cell culture using the mouse breast tumor EMT-6 and the human tumor MDA-MB435. WC-26 greatly increased the ability of doxorubicin to kill these two tumor cell lines in vitro. These results indicate that WC-26 is potentially a useful chemosensitizer for the treatment of cancer when combined with conventional chemotherapeutics.

[3H]4‐(dimethylamino)‐N‐(4‐(4‐(2‐methoxyphenyl)piperazin‐1‐yl) butyl)benzamide: A selective radioligand for dopamine D3 receptors. II. Quantitative analysis of dopamine D3 and D2 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 D3 receptors versus dopamine D2 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. Kd values for the binding of [3H]WC‐10 to D3 receptors obtained from quantitative autoradiography with rhesus monkey and rat brain sections are in agreement with Kd values obtained from cloned human and rat receptors (Xu et al. [ 2009 ] Synapse 63:717‐728). The D2 selective antagonist [3H]raclopride binds with 11‐fold higher affinity to human HEK D2L (Kd = 1.6 nM) than HEK D3 (Kd = 18 nM) receptors; [3H]raclopride binds to rat Sf9 rD2L receptors with a Kd of 6.79 nM, a value that is 4‐fold lower than binding to human HEK D2L receptors and 2.5‐fold higher than binding to rat Sf9 rD3 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 D2 and D3 receptors based on the in vitro receptor binding data of [3H]WC‐10 and [3H]raclopride was developed. Synapse 64:449–459, 2010.

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-[18F]fluoroethoxy)-N-(4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)butyl)-5-methyl-benzamide, [18F]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 [18F]ISO-1 and 18FDG 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 [18F]ISO-1 and FDG uptake with MR-based volumetric measures of tumor growth. In addition, [18F]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 [18F]ISO-1 tumor: background ratio and the proliferative status (P:Q ratio) of the tumor (R = 0.87). Similarly, measures of [18F]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 [18F]ISO-1 provide a measure of both the proliferative status and tumor growth rate, which would be valuable in designing an appropriate treatment strategy.