Nuclear Medicine and Biology - Articles in Press

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

Zhude Tu, Jinbin Xu, Lynne A. Jones, Shihong Li, Robert H. Mach — 2010-03-08

Abstract: Papaverine, 1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline, a specific inhibitor of phosphodiesterase (PDE) 10A with IC50 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 [11C]papaverine and the in vitro and in vivo evaluation of [11C]papaverine as a potential positron emission tomography (PET) radiotracer for imaging PDE10A in the central nervous system (CNS). The radiosynthesis of papaverine with 11C was achieved by O-methylation of the corresponding des-methyl precursor with [11C]methyl iodide. [11C]papaverine was obtained with ∼70% radiochemical yield and a specific activity >10 Ci/μmol. In vitro autoradiography studies of rat and monkey brain sections revealed selective binding of [11C]papaverine to PDE10A enriched regions: the striatum of rat brain and the caudate and putamen of rhesus monkey brain. The biodistribution of [11C]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 [11C]papaverine from brain. Our initial evaluation suggests that despite papaverine's utility for in vitro studies and as a pharmaceutical tool, [11C]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.

[18F]FE@SUPPY and [18F]FE@SUPPY:2 — metabolic considerations - Corrected Proof

2010-03-08

Abstract: Introduction: Recently, [18F]FE@SUPPY and [18F]FE@SUPPY:2 were introduced as the first positron emission tomography (PET) tracers for the adenosine A3 receptor. Thus, aim of the present study was the metabolic characterization of the two adenosine A3 receptor PET tracers.Methods: In vitro carboxylesterase (CES) experiments were conducted using incubation mixtures containing different concentrations of the two substrates, porcine CES and phosphate-buffered saline. Enzymatic reactions were stopped by adding acetonitrile/methanol (10:1) after various time points and analyzed by a high-performance liquid chromatography (HPLC) standard protocol. In vivo experiments were conducted in male wild-type rats; tracers were injected through a tail vein. Rats were sacrificed after various time points (n=3), and blood and brain samples were collected. Sample cleanup was performed by an HPLC standard protocol.Results: The rate of enzymatic hydrolysis by CES demonstrated Michaelis–Menten constants in a micromolar range (FE@SUPPY, 20.15 μM, and FE@SUPPY:2, 13.11 μM) and limiting velocities of 0.035 and 0.015 μM/min for FE@SUPPY and FE@SUPPY:2, respectively. Degree of metabolism in blood showed the following: 15 min pi 47.7% of [18F]FE@SUPPY was intact compared to 33.1% of [18F]FE@SUPPY:2; 30 min pi 30.3% intact [18F]FE@SUPPY was found compared to 15.6% [18F]FE@SUPPY:2. In brain, [18F]FE@SUPPY:2 formed an early hydrophilic metabolite, whereas metabolism of [18F]FE@SUPPY was not observed before 30 min piConclusion: Knowing that metabolism in rats is several times faster than in human, we conclude that [18F]FE@SUPPY should be stable for the typical time span of a clinical investigation. As a consequence, from a metabolic point of view, one would tend to decide in favor of [18F]FE@SUPPY.

Improved synthesis of 2′-deoxy-2′-[18F]-fluoro-1-β-d-arabinofuranosyl-5-iodouracil ([18F]-FIAU) - Corrected Proof

Harry Anderson, NagaVaraKishore Pillarsetty, Melchor Cantorias, Jason S. Lewis — 2010-03-08

Abstract: An improved synthesis of 2′-[18F]-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodouracil ([18F]-FIAU) has been developed. The method utilizes trimethylsilyl trifluoromethanesulfonate (TMSOTf) catalyzed coupling of 2-deoxy-2-[18F]-fluoro-1,3,5-tri-O-benzoyl-d-arabinofuranose with 2,4-bis(trimethylsilyloxy)-5-iodouracil to yield the protected dibenzoyl-[18F]-FIAU. Dibenzoyl-[18F]-FIAU was deprotected with sodium methoxide to yield a mixture of α- and β-anomers in a ratio of 1:1, which were purified by HPLC. The procedure described in this article eliminates the need for HBr activation of the sugar prior to coupling with silylated iodouracil and is suitable for automation. The total reaction time was about 110 min, starting from [18F]-fluoride. The average isolated yield of the required β-anomer was 10±6% (decay corrected) with average specific activity of 125 mCi/μmol.

The effects of chemical and radioactive properties of Tl-201 on human erythrocyte glucose 6-phosphate dehydrogenase activity - Corrected Proof

Ali Sahin, Murat Senturk, Mehmet Ciftci, Erhan Varoglu, Omer Irfan Kufrevioglu — 2010-02-11

Abstract: Aim: The inhibitory effects of thallium-201 (201Tl) solution on human erythrocyte glucose 6-phosphate dehydrogenase (G6PD) activity were investigated.Methods: For this purpose, erythrocyte G6PD was initially purified 835-fold at a yield of 41.7% using 2′,5′-Adenosine diphosphate sepharose 4B affinity gel chromatography. The purification was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed a single band for the final enzyme preparation. The in vitro and in vivo effects of the 201Tl solution including Tl+, Fe+3 and Cu+2 metals and the in vitro effects of the radiation effect of the 201Tl solution and non-radioactive Tl+, Fe+3 and Cu+2 metals on human erythrocyte G6PD enzyme were studied. Enzyme activity was determined with the Beutler method at 340 nm using a spectrophotometer. All purification procedures were carried out at +4°C.Results: 201Tl solution and radiation exposure had inhibitory effects on the enzyme activity. IC50 value of 201Tl solution was 36.86 μl ([Tl+]: 0.0036 μM, [Cu+2]: 0.0116 μM, [Fe+3]: 0.0132 μM), of human erythrocytes G6PD. Seven human patients were also used for in vivo studies of 201Tl solution. Furthermore, non-radioactive Tl+, Fe+3 and Cu+2 were found not to have influenced the enzyme in vitro.Conclusion: Human erythrocyte G6PD activity was inhibited by exposure for up to 10 minutes to 0.057 mCi/kg 201Tl solution. It was detected in in vitro and in vivo studies that the human erythrocyte G6PD enzyme is inhibited due to the radiation effect of 201Tl solution.

Site-specifically 89Zr-labeled monoclonal antibodies for ImmunoPET - Corrected Proof

2010-02-11

Abstract: Three thiol reactive reagents were developed for the chemoselective conjugation of desferrioxamine (Df) to a monoclonal antibody via engineered cysteine residues (thio-trastuzumab). The in vitro stability and in vivo imaging properties of site-specifically radiolabeled 89Zr-Df-thio-trastuzumab conjugates were investigated.Methods: The amino group of desferrioxamine B was acylated by bromoacetyl bromide, N-hydroxysuccinimidyl iodoacetate, or N-hydroxysuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate to obtain thiol reactive reagents bromoacetyl-desferrioxamine (Df-Bac), iodoacetyl-desferrioxamine (Df-Iac) and maleimidocyclohexyl-desferrioxamine (Df-Chx-Mal), respectively. Df-Bac and Df-Iac alkylated the free thiol groups of thio-trastuzumab by nucleophilic substitution forming Df-Ac-thio-trastuzumab, while the maleimide reagent Df-Chx-Mal reacted via Michael addition to provide Df-Chx-Mal-thio-trastuzumab. The conjugates were radiolabeled with 89Zr and evaluated for serum stability, and their positron emission tomography (PET) imaging properties were investigated in a BT474M1 (HER2-positive) breast tumor mouse model.Results: The chemoselective reagents were obtained in 14% (Df-Bac), 53% (Df-Iac) and 45% (Df-Chx-Mal) yields. Site-specific conjugation of Df-Chx-Mal to thio-trastuzumab was complete within 1 h at pH 7.5, while Df-Iac and Df-Bac respectively required 2 and 5 h at pH 9. Each Df modified thio-trastuzumab was chelated with 89Zr in yields exceeding 75%. 89Zr-Df-Ac-thio-trastuzumab and 89Zr-Df-Chx-Mal-thio-trastuzumab were stable in mouse serum and exhibited comparable PET imaging capabilities in a BT474M1 (HER2-positive) breast cancer model reaching 20–25 %ID/g of tumor uptake and a tumor to blood ratio of 6.1–7.1.Conclusions: The new reagents demonstrated good reactivity with engineered thiol groups of trastuzumab and very good chelation properties with 89Zr. The site-specifically 89Zr-labeled thio-antibodies were stable in serum and showed PET imaging properties comparable to lysine conjugates.

Biodistribution and stability studies of [18F]Fluoroethylrhodamine B, a potential PET myocardial perfusion agent - Corrected Proof

2010-02-11

Abstract: Introduction: Fluorine-18-labeled rhodamine B was developed as a potential positron emission tomography (PET) tracer for the evaluation of myocardial perfusion, but preliminary studies in mice showed no accumulation in the heart suggesting that it was rapidly hydrolyzed in vivo in mice. A study was therefore undertaken to further evaluate this hypothesis.Methods: [18F]Fluoroethylrhodamine B was equilibrated for 2 h at 37°C in human, rat and mouse serum and in phosphate-buffered saline. Samples were removed periodically and assayed by high-performance liquid chromatography. Based on the results of the stability study, microPET imaging and a biodistribution study were carried out in rats.Results: In vitro stability studies demonstrated that [18F]fluoroethylrhodamine B much more stable in rat and human sera than in mouse serum. After 2 h, the compound was >80% intact in rat serum but <30% intact in mouse serum. The microPET imaging and biodistribution studies in rats confirmed this result showing high and persistent tracer accumulation in the myocardium compared with the absence of uptake by the myocardium in mice thereby validating our original hypothesis that 18F-labeled rhodamines should accumulate in the heart.Conclusions: [18F]Fluoroethylrhodamine B is more stable in rat and human sera than it is in mouse serum. This improved stability is demonstrated by the high uptake of the tracer in the rat heart in comparison to the absence of visible uptake in the mouse heart. These observations suggest that 18F-labeled rhodamines are promising candidates for more extensive evaluation as PET tracers for the evaluation of myocardial perfusion.

Synthesis and evaluation of a technetium-99m labeled cytotoxic bombesin peptide conjugate for targeting bombesin receptor-expressing tumors - Corrected Proof

Subhani M. Okarvi, Ibrahim Al Jammaz — 2010-02-11

Abstract: Conjugation of the cytotoxic drugs to receptor-binding peptides is an attractive approach for the targeted delivery of cytotoxic peptide conjugates to tumor cells. In an attempt to develop an efficient peptide-based radiopharmaceutical for targeting bombesin (BN) receptor-expressing tumors (i.e., breast and prostate), we have prepared by solid-phase peptide synthesis, a novel BN analog derived from the universal sequence of BN and conjugated to a widely characterized antineoplastic agent, methotrexate (MTX). MTX-BN, after radiolabeling with 99mTc via stannous-tartrate exchange, showed a good stability against cysteine and histidine transchelation as well as a high in vitro metabolic stability in human plasma. In vitro cell-binding and internalization on MDA-MB-231, MCF-7, T47-D breast cancer and PC-3 prostate cancer cell lines demonstrated high affinity and specificity of 99mTc-MTX-BN towards both human breast and prostate cancer cells (binding affinities in nanomolar range). In addition, the radioconjugate displayed a significant internalization (values ranged between 19–35%) into the tumor cells. In vivo biodistribution and clearance kinetics in Balb/c mice are characterized by an efficient clearance from the blood and excretion mainly through the renal-urinary pathway with some elimination via the hepatobiliary system. In vivo tumor uptake in nude mice bearing MDA-MB-231 cells was 2.70±0.44% ID/g at 1 h, whereas in nude mice with human epidermoid KB cells the accumulation in the tumor was found to be 1.48±0.31% ID/g at 1 h post injection. The tumor uptake was always higher than in the blood and muscle, with good tumor retention and good tumor-to-blood and tumor-to-muscle ratios. The accumulation/retention in the major organs (i.e., lungs, stomach, liver, intestines, etc.) was low to moderate (<6% ID/g) in both healthy and tumor-bearing mice. However, the uptake/retention in the kidneys was rather high (up to 11.05±1.80% ID/g), which is of a concern, particularly for radionuclide therapy. This initial study towards the development of a novel cytotoxic BN conjugate suggest that the combination of favorable in vitro and in vivo properties may render 99mTc-MTX-BN a potential candidate for the targeted imaging and eventually for radionuclide therapy (when labeled with an appropriate radionuclide) of BN receptor-positive tumors and deserves further evaluation.

The feasibility of using a baculovirus vector to deliver the sodium-iodide symporter gene as a reporter - Corrected Proof

Xiang Zhou, Biao Li, Jun Wang, Hongyan Yin, Yifan Zhang — 2010-02-11

Abstract: Purpose: To evaluate the efficiency of baculovirus vectors in transducing FTC-133 cells and to examine the feasibility of using baculovirus vectors for the delivery of the sodium-iodide symporter (NIS) gene as a reporter through co-transduction to monitor the expression of the target gene.Method: Two recombinant baculoviruses were constructed to express NIS and green fluorescent protein (GFP) respectively. FTC-133, 8050C, SW1116, A549 cells, were infected with Bac-GFP. The infection efficiency of Bac-GFP and the intensity of fluorescence, in either the presence or absence of sodium butyrate, were monitored by flow cytometry. The iodine uptake by FTC-133 cells infected with Bac-NIS was measured using a γ counter. FTC-133 cells were infected with a mixture of equal amounts of Bac-NIS and Bac-GFP at different setting of multiplicity of infection (MOI). The changes of GFP fluorescence intensity and iodine uptake were monitored 24 h after infection in the coinfected cells.Results: We have successfully constructed recombinant baculoviruses carrying NIS and GFP under the control of the cytomegalovirus IE-1 promoter. We found that transduced efficiency of baculovirus in 8505C, SW1116, A549 cells are low in absence of sodium butyrate. Yet Bac-GFP infects FTC-133 cells at a high efficiency, 77.67%, 85.57% and 93.23% with MOI of 100, 200 and 400, respectively. The fluorescence intensity of the Bac-GFP infected tumor cells correlated positively with the MOI of the virus. Sodium butyrate induction increased both the infection efficiency and the fluorescence intensity, but increase of infection efficiency was insignificant in FTC-133 cells. Reporter gene (GFP) expression in FTC-133 is stable within 7 days after infection. The radioactivity incorporated by the tumor cells infected with Bac-NIS correlated positively with the MOI of Bac-NIS as well. In tumor cells co-infected with Bac-NIS and Bac-GFP, the amount of radioactivity incorporated significantly correlated with the GFP fluorescence intensity (r=0.922).Conclusion: Baculovirus vectors are powerful vehicles for studying FTC-133 tumor cells in gene delivery. It is feasible to use a baculovirus vector to deliver NIS as a reporter gene to monitor the expression of target genes. This is therefore an effective approach for the detection of target gene expression in gene therapy.

Autoradiography study and SPECT imaging of reporter gene HSV1-tk expression in heart - Corrected Proof

2010-02-11

Abstract: Aim: To demonstrate the feasibility and optimal conditions of imaging herpes simplex virus 1-thymidine kinase (HSV1-tk) gene transferred into hearts with 131I-2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodouracil (131I-FIAU) using autoradiography (ARG) and single photon emission computed tomography (SPECT) in animal models.Methods: HSV1-tk inserted into adenovirus vector (Ad5-tk) and adenovirus (Ad5-null) was prepared. Rats or rabbits were divided into a study group receiving intramyocardial injection of Ad5-tk, and a control group receiving Ad-null injection. In the study group of rats, two sets of experiments, time-course study and dose-dependence study, were performed. In time-course experiments, rats were injected with 131I-FIAU on Days 1, 2, 3, 5 and 7, after transfection of 1×108 pfu Ad5-tk, to study the feasibility and suitable time course for reporter gene imaging. In dose-dependence study, various titers of Ad5-tk (5×108, 1×108, 5×107 and 1×107 pfu) were used to determine the threshold and optimal viral titer needed for detection of gene expression. The gamma counts of hearts were measured. The rat myocardium was analyzed by ARG and reverse transcriptase-polymerase chain reaction (RT-PCR). SPECT whole-body planar imaging and cardiac tomographic imaging were performed in the rabbit models.Results: From the ARG images, rats injected with Ad5-tk showed significant 131I-FIAU activity in the anterolateral wall compared with background signals seen in the control Ad5-null rats. In time-course study, the highest radioactivity in the focal myocardium could be seen on Day 1, and then progressively declined with time. In dose-dependence study, the level of 131I-FIAU accumulation in the transfected myocardium declined with the decrease of Ad viral titers. From the ARG analysis and gamma counting, the threshold viral titer was 5×107 pfu, and the optimal Ad titer was 1×108 pfu. The ARG images in region of interest-derived semi-quantitative study correlated well with ex vivo gamma counting and mRNA levels from RT-PCR analysis. The gamma counting and RT-PCR also correlated well with each other in both sets of experiments. Both SPECT planar and tomographic images showed clear uptake of 131I-FIAU in the anterolateral wall where Ad5-tk was injected.Conclusion: The study confirmed the feasibility of cardiac SPECT reporter gene imaging with HSV1-tk as a reporter gene and 131I-FIAU as a reporter probe. The optimal Ad5-tk titer for imaging was 1×108 pfu and the optimal imaging time was 1–2 days after gene transfer. Thus, the imaging of HSV1-tk transgene expression in the heart is feasible and may be used for the noninvasive SPECT imaging of gene therapy in cardiac diseases.

Efficient automated one-step synthesis of 2-[18F]fluoroethylcholine for clinical imaging: optimized reaction conditions and improved quality controls of different synthetic approaches - Corrected Proof

2010-02-11

Abstract: [18F]-labelled choline analogues, such as 2-[18F]fluoroethylcholine (18FECH), have suggested to be a new class of choline derivatives highly useful for the imaging of prostate and brain tumours. In fact, tumour cells with enhanced proliferation rate usually exhibit an improved choline uptake due to the increased membrane phospholipids biosynthesis. The aim of this study was the development of a high yielding synthesis of 18FECH. The possibility of shortening the synthesis time by reacting all the reagents in a convenient and rapid one-step reaction was specially considered.Methods: 18FECH was synthesized by reacting [18F]fluoride with 1,2-bis(tosyloxy)ethane and N,N-dimethylaminoethanol. The synthesis was carried out using both a one- and a two-step reaction in order to compare the two procedures. The effects on the radiochemical yield and purity by using different [18F]fluoride phase transfer catalysts, reagents amounts and purification methods were assessed. Quality controls on the final products were performed by means of radio-thin-layer chromatography, gas chromatography and high-performance liquid chromatography equipped with conductimetric, ultraviolet and radiometric detectors.Results: In the optimized experimental conditions, 18FECH was synthesized with a radiochemical yield of 43±3% and 48±1% (not corrected for decay) when the two-step or the one-step approach were used, respectively. The radiochemical purity was higher than 99% regardless of the different synthetic pathways or purification methods adopted. The main chemical impurity was due to N,N-dimethylmorpholinium. The identity of this impurity in 18FECH preparations was not previously reported.Conclusion: An improved two-step and an innovative one-step reaction for synthesizing 18FECH in a high yield were reported. The adaptation of a multistep synthesis to a single step process, opens further possibilities for simpler and more reliable automations.

Copper-64-diacetyl-bis (N4-methylthiosemicarbazone) accumulates in rich regions of CD133+ highly tumorigenic cells in mouse colon carcinoma - Corrected Proof

2010-02-11

Abstract: Introduction: 64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM) is a potential imaging agent of hypoxic tumor for use with PET. Recent literature demonstrated that cancer cells expressing CD133, which is a frequently used marker for so-called cancer stem cells or cancer stem cell-like cells (collectively referred to here as CSCs), contribute to tumor's therapeutic resistance and metastasis ability. Culturing under hypoxia is also reported to enlarge the proportion of CD133+ cells, which would indicate survival advantage of CD133+ cells under hypoxia. Here, we investigated the relationships between 64Cu-ATSM accumulation and existence of CD133+ cells using mouse colon carcinoma (colon-26) tumor.Methods: Intratumor distribution of 64Cu-ATSM and 18F-fluorodeoxyglucose (18FDG) was compared with immunohistochemical staining for CD133 with a colon-26 model. In vitro characterization of CD133+ colon-26 cells was also performed.Results: In colon-26 tumors, 64Cu-ATSM localized preferentially in regions with a high density of CD133+ cells. The percentage of CD133+ cells was 11-fold higher in 64Cu-ATSM high-uptake regions compared with 18FDG high- (but 64Cu-ATSM low-) uptake regions. CD133+ colon-26 cells showed characteristics previously linked with CSCs in other cancer cell lines, such as high colony-forming ability, high tumor-initiating ability and enrichment under hypoxic cultivation. The proportion of CD133+ cells was enlarged by culturing under glucose starvation as well as hypoxia, and 64Cu-ATSM uptake was increased under such conditions.Conclusions: Our findings showed that, in colon-26 tumors, 64Cu-ATSM accumulates in rich regions of CD133+ cells with characteristics of CSCs. Therefore 64Cu-ATSM could be a potential imaging agent for rich regions of CD133+ cells, associated with CSCs, within tumors.

In vivo and in vitro characterization of [18F]-FE-(+)-DTBZ as a tracer for beta-cell mass - Corrected Proof

2010-01-18

Abstract: Introduction: The positron emission tomography (PET) tracer 9-[18F]fluoroethyl-(+)-dihydrotetrabenazine ([18F]-FE-(+)-DTBZ) is a potential candidate for quantifying beta-cell mass in vivo. The purpose was to investigate in vitro and in vivo utility of this tracer for the assessment of beta-cell mass.Methods: Three pigs were intravenously administered [18F]-FE-(+)-DTBZ and examined by PET/computed tomography. Binding parameters were estimated by kinetic modeling. In vitro kD and Bmax were determined by saturation binding studies of endocrine and exocrine human tissue homogenates. In vitro pancreatic uptake was determined by tissue autoradiography with pancreases from patients with types 1 (T1DM) and 2 diabetes mellitus (T2DM) and healthy controls.Results: [18F]-FE-(+)-DTBZ had a kD of 3.5±1.0 nM, a Bmax of 382±108 fmol/mg protein and a specificity of 89±1.8% in islet homogenates. The total exocrine uptake was lower and 65% was nondisplaceable. No uptake difference was observed in pancreatic tissue slices from patients with T1DM, T2DM or healthy controls. The in vivo porcine pancreatic uptake reached a peak of standardized uptake value (SUV) of 2.8 with a low distribution volume ratio in all animals. Moderate to high tracer uptake was identified in the bile system and in bone.Conclusions: [18F]-FE-(+)-DTBZ binds to vesicular monoamine transporter 2 (VMAT2) with high specificity in pure islet tissue in vitro. However, there is high nondisplaceable binding to exocrine tissue. In addition, in vivo tracer metabolism and dehalogenation result in severe underestimation of porcine pancreatic VMAT2 expression and BCM. The results do not support [18F]-FE-(+)-DTBZ as a suitable tracer for in vivo beta-cell imaging.

Some thoughts on the mechanism of cellular trapping of Cu(II)-ATSM - Corrected Proof

Jason L.J. Dearling, Alan B. Packard — 2010-01-15

Abstract: Cu(II)-ATSM continues to be investigated, both in the laboratory and in the clinic, as a tumor hypoxia imaging agent. However, meaningful interpretation of these images requires a more complete understanding of the mechanism by which the tracer is trapped within the cell. Cu(II)-ATSM is a simple molecule and its biochemical interaction with cells is similarly simple, mainly based upon redox chemistry. Here we suggest that the trapping mechanism is biphasic. The first phase is a reduction/oxidation cycle involving thiols and molecular oxygen. This can be followed by interaction with proteins in the mitochondria leading to more permanent retention of the tracer. The uptake mechanism is complicated by this second step because of the changes in the cell resulting from hypoxia, such as an increase in nicotinamide adenine dinucleotide (NADH) redox state and differences in cellular biochemistry and cell proteomes. These changes may lead to differences in the extent of trapping and retention of the 64Cu in different cell types. For example, copper uptake might be increased in cells with lower pH due to the lower stability of metal bis(thiosemicarbazones) under acidic conditions. Reaction rates with cellular reductants also vary with pH, which differs between cellular organelles. For Cu(II)-ATSM to reach its full potential, more complete characterization of the mechanism of cellular trapping in different cell types is required.

Preparation and biological evaluation of cyclopentadienyl-based 99mTc-complexes [(Cp-R)99mTc(CO)3] mimicking benzamides for malignant melanoma targeting - Corrected Proof

2010-01-15

Abstract: The biological evaluation of half-sandwich 99mTc-complexes that surrogate iodobenzamide with a high affinity for melanin tumor tissue is described. We have synthesized via retro Diels–Alder reaction two models of 99mTc complexes which possess the piano stool [Cp99mTc(CO)3] motif instead of a phenyl ring as in the original iodobenzamide 123I-N-(N-benzylpiperidin-4-yl)-2-iodobenzamide (2-IBP) and N-(2-diethylaminoethyl)-4-iodobenzamide (BZA). Diels–Alder products – (HCp-CONHR)2 (, R=2-diethylaminoethyl; , R=benzylpiperidin-4-yl) were prepared and reacted with fac-[99mTc(H2O)3(CO)3)]+ 1 in water to produce the corresponding 99mTc complexes [()99mTc(CO)3)] and [()99mTc(CO)3)] . The structures of the 99mTc complexes on the no-carrier-added level have been confirmed by chromatographic comparison with the corresponding rhenium complexes and , macroscopically characterized by IR, NMR, ESI-MS and X-ray crystallography for [triclinic, P-1, a=7.3518(1) Å, b=8.0309(2) Å, c=17.5536(3) Å, α=99.1260(5)°, β=90.4215(14)°, γ=117.0187(11)°]. The radioconjugate showed good in vitro stability. In murine melanoma B16F1 cells, significant cellular uptake (43.9% of the total applied activity) was attained after 4 h at 37°C with about 50% of the cell-associated radioactivity being internalized in the cells (22% of the applied activity). Furthermore, in melanoma-bearing C57BL6 mice, tumor uptake values of 3.39±0.50 %ID g−1 and 3.21±0.26 %ID g−1 at 1 and 4 h postinjection, respectively, were observed indicating a good retention of in the tumor.

Synthesis and evaluation of new imaging agent for central nicotinic acetylcholine receptor α7 subtype - Corrected Proof

2010-01-15

Abstract: Introduction: The nicotinic acetylcholine receptor (nAChR) α7 subtype (α7 nAChR) is one of the major nAChR subtypes in the brain. We synthesized C-11 labeled α7 nAChR ligands, (R)-2-[11C]methylamino-benzoic acid 1-aza-bicyclo[2.2.2]oct-3-yl ester ([11C](R)-MeQAA) and its isomer (S)-[11C]MeQAA, for in vivo investigation with positron emission tomography (PET). Then, the potential of (R)- and (S)-[11C]MeQAA for in vivo imaging of α7 nAChR in the brain was evaluated in mice and monkeys.Methods: The binding affinity for α7 nAChR was measured using rat brain. Biodistribution and in vivo receptor blocking studies were undertaken in mice. Dynamic PET scans were performed in conscious monkeys.Results: The affinity for α7 nAChR was 41 and 182 nM for (R)- and (S)-MeQAA, respectively. The initial uptake in the mouse brain was high ([11C](R)-MeQAA: 7.68 and [11C](S)-MeQAA: 6.65 %dose/g at 5 min). The clearance of [11C](R)-MeQAA was slow in the hippocampus (α7 nAChR-rich region) but was rapid in the cerebellum (α7 nAChR-poor region). On the other hand, the clearance was fast for [11C](S)-MeQAA in all regions. The brain uptake of [11C](R)-MeQAA was decreased by methyllycaconitine (α7 nAChR antagonist) treatment. In monkeys, α7 nAChRs were highly distributed in the thalamus and cortex but poorly distributed in the cerebellum. The high accumulation was observed in the cortex and thalamus for [11C](R)-MeQAA, while the uptake was rather homogeneous for [11C](S)-MeQAA.Conclusions: [11C](R)-MeQAA was successfully synthesized and showed high uptake to the brain. However, since the in vivo selectivity for α7 nAChR was not enough, further PET kinetic analysis or structure optimization is needed for specific visualization of brain α7 nAChRs in vivo.

In vivo binding of [68Ga]-DOTATOC to somatostatin receptors in neuroendocrine tumours — impact of peptide mass - Corrected Proof

2010-01-15

Abstract: Objectives: The aim of this pilot study was to explore the impact of peptide mass on binding of [68Ga]-DOTATOC to neuroendocrine tumour somatostatin receptors in vivo using a tracer of variable specific radioactivity (SRA) and to show the logistic feasibility of sequential PET scans in the same patient.Material and Methods: Nine patients with gastroenteropancreatic neuroendocrine tumours were included. Six of them underwent three sequential PET-CT examinations with intravenous injections of [68Ga]-DOTATOC proceeded by 0, 50 and 250 or 500 μg of octreotide, administered 10 min before the tracer. Three patients were examined by dynamic and static PET/CT for pharmacokinetic and dosimetric calculations. The [68Ga]-DOTATOC synthesis included preconcentration and purification of the generator eluate and microwave heating in a semi-automated in-house procedure.Results: [68Ga]-DOTATOC synthesis and quality control were accomplished within 30 min and radiochemical purity was >95%. The tracer accumulation in the tumours varied and depended on the total amount of the administered peptide. In five of six patients, the highest tumour-to-normal tissue ratio was found when 50 μg of octreotide was preadministered. One patient showed a continuously increasing tumour uptake. Dosimetrically, a large variation in organ doses was found (kidney: 0.086–0.168 mSv/MBq; liver: 0.026–0.096 mSv/MBq; spleen: 0.046–0.226 mSv/MBq). The effective dose (0.015, 0.0067 and 0.0042 mSv/MBq) was correlated to the total amount of decays.Discussion: Three sequential PET-CT examinations using 68Ga-based tracer was carried out in 1 day. The use of high SRA [68Ga]-DOTATOC and unlabelled octreotide indicates an optimal mass leading to better image contrast. [68Ga]-DOTATOC-PET-CT employing variable SRA may be utilised for accurate quantification of tumour uptake with subsequent dosimetry for personalized therapy management.

Combination therapy in A549 cells - Corrected Proof

2010-01-15

Abstract: Background and aim: We investigated the anti-tumor effect induced by the combination of the radiotherapeutic agent 131I-RC-160 and the prodrug 5-FC in human non-small cell lung cancer (NSCLC) A549 cells that were co-expressing the human somatostatin receptor 2 gene (hSSTR2) and E. coli cytosine deaminase gene (CD).Methods: We cloned both hSSTR2 and CD into a bicistronic mammalian expression plasmid and stably transfected it into A549 cells (pCIS-A549 cells). After antibiotic selection, SSTR expression in stable clones was determined by reverse transcription and polymerase chain reaction (RT-PCR), Western blot, flow cytometry and immunofluorescence analyses. To assess the in vivo targeting efficiency of the “engineered” A549 cells, the cells were subcutaneously injected into nude mice and the biodistribution of 99mTc-RC-160 was assessed at different time points. The tumor inhibitory effects of 131I-RC-160 and/or 5-FC were evaluated by measurement of tumor growth and immunohistochemical analysis.Results: Multiple analyses demonstrated the successful expression of hSSTR2 in A549 cells. In vivo radioimaging revealed specific targeting of RC-160 to the tumors derived from pCIS-A549 cells when compared to those from control A549 cells. The tumor inhibitory rate of pCIS-A549 tumors in the 131I-RC-160 plus 5-FC-treated group was significantly higher than that in the single agent-treated group, control group and control tumors.Conclusion: Co-expression of the hSSTR2 and CD genes in tumor cells can selectively sensitize these cells to the infra-additive effects of radioisotope-labeled RC-160 and 5-FC in vivo. This approach offers a potential therapeutic strategy for the treatment of lung cancer.

In vivo tracking of 111In-labeled bone marrow mesenchymal stem cells in acute brain trauma model - Corrected Proof

2010-01-15

Abstract: Introduction: This study was to evaluate the in vivo distribution of intravenously transplanted bone marrow-derived mesenchymal stem cells (BMSCs) in an acute brain trauma model by 111In-tropolone labeling.Methods: Rat BMSCs were labeled with 37 MBq 111In-tropolone. Their labeling efficiency and in vitro retention rate were measured. The viability and proliferation of labeled BMSCs were evaluated for 14 days after labeling. The biodistribution of 111In-labeled BMSCs in trauma models was compared with those of sham-operated rats and normal rats on gamma camera images. The migration of 111In-BMSCs to the traumatic brain was evaluated using confocal microscope.Results: The labeling efficiency of 111In-BMSCs was 66±5%, and their retention rate was 85.3% at 1 h after labeling. There was no difference in the number of viable cells between 111In-BMSCs and controls at 48 h after labeling. However, the proliferation of 111In-BMSCs was inhibited after the third day of labeling, and it did not reach confluency. On gamma camera images, most of the 111In-BMSCs uptake was observed in the liver and spleen at the second day of injection. The brain uptake of 111In-BMSCs was detected prominently in trauma models (1.4%) than in sham-operated (0.5%) or normal rats (0.3%). Radiolabeled BMSCs were observed at the traumatic brain on the confocal microscope as they have a homing capacity, although its proliferation capacity was suppressed.Conclusion: Although growth inhibition by 111In-labeling need to be evaluated further prior to use in humans, 111In-labeled BMSCs are useful for the tracking of intravenously transplanted mesenchymal stem cells in brain disease models.

18F-FDG PET/CT-related metabolic parameters and their value in early prediction of chemotherapy response in a VX2 tumor model - Corrected Proof

2010-01-15

Abstract: Objective: This study acquired fluorine-18-deoxyglucose (FDG) kinetic parameters prior and subsequent to cisplatin chemotherapy so as to define the optimal parameters for early prediction of chemotherapy response.Methods: A total of 12 non-tumor-bearing rabbits were used to obtain noninvasive input function, five VX2 tumor-bearing rabbits were used for validation and 32 tumor-bearing rabbits underwent 4 mg kg−1 cisplatin chemotherapy. Dynamic FDG PET/CT was performed at pretherapy, Day 0, Day 1, Day 7 and Day 14 after cisplatin administration. With the application of a three-compartment model, influx index (Ki), k1, k2, k3 and k4 were noninvasively obtained.Results: Sensitive (SG) and insensitive groups (ISG) were defined based on their volume on Day 7. k1, Ki, SUVmean, and SUVmax showed significant decreases in SG vs. ISG at Day 0 (P .05). Soon after cisplatin administration, GLUT-1 expression was greatly decreased in SG vs. ISG.Conclusions: The parameters of SUVmax, SUVmean, Ki and k1 were valuable for the early prediction of chemotherapy response. k1 had a wider observation window compared to SUVmean, SUVmax and Ki, and k1 also reflected the changes in GLUT-1 expression.