Nuclear Medicine and Biology - Articles in Press

Radiation dosimetry of the translocator protein ligands [18F]PBR111 and [18F]PBR102 - Corrected Proof

2012-02-02

Abstract: Introduction: The translocator protein (TSPO) ligands [18F]PBR111 and [18F]PBR102 show promise for imaging neuroinflammation. Our aim was to estimate the radiation dose to humans from primate positron emission tomography (PET) studies using these ligands and compare the results with those obtained from studies in rodents.Methods: [18F]PBR111 and [18F]PBR102 PET–computed tomography studies were carried out in baboons. The cumulated activity in the selected source organs was obtained from the volume of interest time–activity curves drawn on coronal PET slices and adjusted for organ mass relative to humans. Radiation dose estimates were calculated in OLINDA/EXM Version 1.1 from baboon studies and compared with those calculated from Sprague–Dawley rat tissue concentration studies, also adjusted for relative organ mass.Results: In baboons, both ligands cleared rapidly from brain, lung, kidney and spleen and more slowly from liver and heart. For [18F]PBR111, the renal excretion fraction was 6.5% and 17% for hepatobiliary excretion; for [18F]PBR102, the renal excretion was 3.0% and 15% for hepatobiliary excretion. The estimated effective dose in humans from baboon data was 0.021 mSv/MBq for each ligand, whilst from rat data, the estimates were 0.029 for [18F]PBR111 and 0.041 mSv/MBq for [18F]PBR102.Conclusion: Biodistribution in a nonhuman primate model is better suited than the rat model for the calculation of dosimetry parameters when translating these ligands from preclinical to human clinical studies. Effective dose calculated from rat data was overestimated compared to nonhuman primate data. The effective dose coefficient for both these TSPO ligands determined from PET studies in baboons is similar to that for [18F]FDG.

A fast chemoenzymatic synthesis of [11C]-N5,N10-methylenetetrahydrofolate as a potential PET tracer for proliferating cells - Corrected Proof

Muhammad Saeed, Timothy J. Tewson, Colbin E. Erdahl, Amnon Kohen — 2012-02-02

Abstract: Introduction: Thymidylate synthase and folate receptors are well-developed targets of cancer therapy. Discovery of a simple and fast method for the conversion of 11CH3Ito[11C]-formaldehyde (11CH2O) encouraged us to label the co-factor of this enzyme. Preliminary studies conducted on cell lines have demonstrated a preferential uptake of [11-14C]-(R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (14CH2H4folate) by cancerous cell vs. normal cells from the same organ (Saeed M., Sheff D. and Kohen A. Novel positron emission tomography tracer distinguishes normal from cancerous cells. J Biol Chem 2011;286:33872–33878), pointing out 11CH2H4folate as a positron emission tomography (PET) tracer for cancer imaging. Herein we report the synthesis of 11CH2H4folate, which may serve as a potential PET tracer.Methods: In a remotely controlled module, methyl iodide (11CH3I) was bubbled into a reaction vial containing trimethylamine N-oxide in N,N-Dimethylformamide (DMF) and heated to 70°C for 2 min. Formaldehyde (11CH2O) formed after the completion of reaction was then mixed with a solution of freshly prepared tetrahydrofolate (H4folate) by using a fast chemoenzymatic approach to accomplish synthesis of 11CH2H4folate. Purification of the product was carried out by loading the crude reaction mixture on a SAX cartridge, washing with water to remove unbound impurities and finally eluting with a saline solution.Results: The synthesis and purification of 11CH2H4folate were completed within 5 min. High-performance liquid chromatography analysis of the product after SAX purification indicates that more than 90% of the radioactivity that was retained on the SAX cartridge was in 11CH2H4folate, with minor (<10%) radioactivity due to unreacted 11CH2O.Conclusion: We present a fast (∼5 min) synthesis and purification of 11CH2H4folate as a potential PET tracer. The final product is received in physiologically compatible buffer (100 mM sodium phosphate, pH 7.0 containing 500 mM NaCl) and ready for use in vivo.

Bombesin analogues for gastrin-releasing peptide receptor imaging - Corrected Proof

2012-01-20

Abstract: Objectives: The present study describes the design and development of a series of new bombesin (BBN) antagonist peptide ligands of the form [64Cu-(NO2A-X-D-Phe6-BBN(6-13)NHEt)], where Cu-64=a positron emitting radiometal; NO2A=1,4,7-triazacyclononane-1,4-diacetic acid; X=6-amino hexanoic acid, 8-amino octanoic acid or 9-Aminononanoic acid; and BBN(6-13)NHEt=Gln-Trp-Ala-Val-Gly-His-Leu-NHEt, an antagonist analogue of bombesin peptide for specific targeting of the gastrin-releasing peptide receptor (GRPR).Methods: [NO2A-X-D-Phe6-BBN(6-13)NHEt] conjugates were manually conjugated with NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), and the resulting conjugates were labeled with 64Cu to yield [64Cu-(NO2A-X-D-Phe6-BBN(6-13)NHEt)]. The metallated and nonmetallated conjugates were purified via reversed-phase high-performance liquid chromatography and characterized by electrospray ionization–mass spectrometry.Results: Competitive displacement binding assays displayed nanomolar binding affinities toward human GRPR for all of the newly formed peptide analogues. Biodistribution studies showed very high uptake and retention of tumor-associated radioactivity in PC-3 (a prostate tumor model known to express the GRPR) tumor-bearing rodent models. The radiolabeled conjugates also exhibited rapid urinary excretion and very high tumor to background ratios. Micro-positron emission tomography (PET) molecular imaging investigations showed clear visualization of tumors in female PC-3 tumor-bearing mice 15 h postinjection.Conclusion: The biodistribution and molecular imaging study suggests that these conjugates can be considered as potential PET tracer candidates for the diagnosis of GRPR-positive tumors in human patients.

Quantification of regional cerebral blood flow in rats using an arteriovenous shunt and micro-PET - Corrected Proof

2012-01-20

Abstract: Introduction: Measurement of regional cerebral blood flow (rCBF) in rodents can provide knowledge of pathophysiology of the cerebral circulation, but generally requires blood sampling for analysis during positron emission tomography (PET). We therefore tested the feasibility of using an arteriovenous (AV) shunt in rats for less invasive blood analysis.Methods: Six anesthetized rats received [15O]H2O and [15O]CO PET scans with their femoral artery and vein connected by an AV shunt, the activity within which was measured with a germanium ortho-oxysilicate scintillation detector. The [15O]H2O was intravenously injected either at a faster or slower injection rate, while animals were placed either with their head or heart centered in the gantry. The time–activity curve (TAC) from the AV shunt was compared with that from the cardiac ventricle in PET image. The rCBF values were calculated by a nonlinear least-square method using the dispersion-corrected AV-shunt TAC as an input.Results: The AV-shunt TAC had higher signal-to-noise ratio, but also had delay and dispersion compared with the image-derived TAC. The delay time between the AV-shunt TAC and image-based TAC ranged from 11 to 21 s, while the dispersion was estimated to be ∼5 s as a time constant of the dispersion model of exponential function, and both were properly corrected. In a steady-state condition of [15O]CO PET, the blood activity concentration by AV-shunt TAC was also comparable in height with the image-based TAC corrected for partial volume. Whole-brain CBF values measured by [15O]H2O were 0.37±0.04 (mean±S.D.) ml/g/min, partition coefficient was 0.73±0.04 ml/g, and the CBF varied in a linear relationship with partial pressure of carbon dioxide during each scan.Conclusions: The AV-shunt technique allows less invasive, quantitative and reproducible measurement of rCBF in [15O]H2O PET studies in rats than direct blood sampling and radioassay.

A historical perspective on the specific activity of radiopharmaceuticals: what have we learned in the 35 years of the ISRC? - Corrected Proof

Suzanne E. Lapi, Michael J. Welch — 2012-01-20

Abstract: Specific activity (SA), defined as the amount of radioactivity per unit mass of a compound, is arguably one of the most important parameters in radiopharmaceutical development, particularly in quality control of carbon-11- and fluorine-18-labeled compounds. This review article will outline the progression of improvements in SA over the last few decades. The International Symposium of Radiopharmaceutical Chemistry abstracts were an excellent source of materials for this review and will be referenced throughout.

Varenicline increases in vivo striatal dopamine D2/3 receptor binding: an ultra-high-resolution pinhole [123I]IBZM SPECT study in rats - Corrected Proof

2012-01-20

Abstract: Introduction: Ex vivo storage phosphor imaging rat studies reported increased brain dopamine D2/3 receptor (DRD2/3) availability following treatment with varenicline, a nicotinergic drug. However, ex vivo studies can only be performed using cross-sectional designs. Small-animal imaging offers the opportunity to perform serial assessments. We evaluated whether high-resolution pinhole single photon emission computed tomography (SPECT) imaging in rats was able to reproduce previous ex vivo findings.Methods: Rats were imaged for baseline striatal DRD2/3 availability using ultra-high-resolution pinhole SPECT (U-SPECT-II) and [123I]IBZM as a radiotracer, and randomized to varenicline (n=7; 2 mg/kg) or saline (n=7). Following 2 weeks of treatment, a second scan was acquired.Results: Significantly increased striatal DRD2/3 availability was found following varenicline treatment compared to saline (time⁎treatment effect): posttreatment difference in binding potential between groups corrected for initial baseline differences was 2.039 (P=.022), indicating a large effect size (d=1.48).Conclusions: Ultra-high-resolution pinhole SPECT can be used to assess varenicline-induced changes in DRD2/3 availability in small laboratory animals over time. Future small-animal studies should include imaging techniques to enable repeated within-subjects measurements and reduce the amount of animals.

In vitro and in vivo evaluation of a 64Cu-labeled NOTA-Bn-SCN-Aoc-bombesin analogue in gastrin-releasing peptide receptor expressing prostate cancer - Corrected Proof

2012-01-20

Abstract: Introduction: Bombesin (BN) is an amphibian peptide that binds to the gastrin-releasing peptide receptor (GRPR). It has been demonstrated that BN analogues can be radiolabeled for potential diagnosis and treatment of GRPR-expressing malignancies. Previous studies have conjugated various chelators to the eight C-terminal amino acids of BN [BN(7-14)] for radiolabeling with 64Cu. Recently, (1,4,7-triazacyclononane-1,4,7-triacetic acid) (NOTA) has been evaluated as the five-coordinate 64Cu complex, with results indicating GRPR-specific tumor uptake. This study aimed to conjugate S-2-(4-isothiocyanatobenzyl)-NOTA (p-SCN-Bn-NOTA) to BN(7-14) such that it could form a six-coordinate complex with 64Cu and to evaluate the resulting peptide.Methods: p-SCN-NOTA was conjugated to 8-aminooctanoic acid (Aoc)-BN(7-14) in solution to yield NOTA-Bn-SCN-Aoc-BN(7-14). The unlabeled peptide was evaluated in a cell binding assay using PC-3 prostate cancer cells and 125I-Tyr4-BN to determine the IC50 value. The peptide was radiolabeled with 64Cu and evaluated for internalization into PC-3 cells and for tumor uptake in mice bearing PC-3 xenografts using biodistribution and micro-positron emission tomography imaging studies.Results: The binding assay demonstrated that NOTA-Bn-SCN-Aoc-BN(7-14) bound with high affinity to GRPR with an IC50 of 1.4 nM. The radiolabeled peptide demonstrated time-dependent internalization into PC-3 cells. In vivo, the peptide demonstrated tumor-specific uptake and imaging that were comparable to those of previously reported 64Cu-labeled BN analogues.Conclusions: These studies demonstrate that 64Cu-NOTA-Bn-SCN-Aoc-BN(7-14) binds to GRPR-expressing cells and that it can be used for imaging of GRPR-expressing prostate cancer.

Evaluation of the relationship between [18F]FDG and P-glycoprotein expression: an experimental study - Corrected Proof

Chunjing Yu, Weixing Wan, Bin Zhang, Shengming Deng, Tzu-Chen Yen, Yiwei Wu — 2012-01-20

Abstract: Introduction: P-glycoprotein (P-gp) is a cell-membrane-associated protein that transports a variety of drug substrates. We sought to evaluate the relationship between 2-[18F]fluoro-2-deoxy-d-glucose ([18F]FDG) and P-gp expression using breast carcinoma Bcap37/multidrug resistant (MDR1) and Bcap37 in vitro and in vivo.Methods: The function of P-gp expressed in Bcap37/MDR1 cells was evaluated using verapamil (VER), a classical inhibitor of P-gp. The accumulation of 99mTc-methoxyisobutylisonitrile ([99mTc]MIBI) in vitro was measured. In vivo imaging of severe combined immune deficiency (SCID) mice implanted with Bcap37 and Bcap37/MDR1 cells was performed by scintigraphy and micro-positron emission tomography (PET).Results: The uptake of [99mTc]MIBI was 0.62%±0.05% in the Bcap37/MDR1 cells and 2.02%±0.28% in the Bcap37 cells. VER significantly increased the uptake of [99mTc]MIBI in the Bcap37/MDR1 cells (1.90%±0.09%) but not in the Bcap37 cells (2.15%±0.27%). In vivo, neither the Bcap37 nor Bcap37/MDR1 tumors grown in the SCID mice could be detected by [99mTc]MIBI scintigraphy. Both the Bcap37 and Bcap37/MDR1 tumors were visible by micro-PET. The mean standardized uptake value (SUV) was significantly higher in the Bcap37 tumors (1.00±0.06) than in the Bcap37/MDR1 (0.67±0.11) tumors. VER significantly increased the mean SUV in the Bcap37/MDR1 tumors (1.02±0.16) but not in the Bcap37 tumors (1.09±0.22).Conclusions: [18F]FDG combined with VER may be an effective noninvasive method of determining P-gp expression in tumors.

Evaluation of 68Ga-labeled tracers for PET imaging of myocardial perfusion in pigs - Corrected Proof

2012-01-20

Abstract: Purpose: We evaluated four potential gallium-68 (68Ga)-labeled tracers for positron emission tomography (PET) imaging of myocardial perfusion in comparison with oxygen-15-labeled water ([15O]water) in healthy pigs. Four hexadentate salicylaldimine ligands derived from bis(3-aminopropyl)ethylenediamine (BAPEN) that showed promise in previous rat experiments were selected for this study.Methods: Following an evaluation of myocardial blood flow with [15O]water PET, the pigs (total n=14) underwent a dynamic 90-min PET study with one of four 68Ga-labeled BAPEN derivatives (n=3–5 per tracer) either at rest or under adenosine stress. Serial arterial blood samples were collected during the imaging for the measurements of total radioactivity, radiometabolites, plasma protein binding and blood-to-plasma ratio for the 68Ga chelates. Time–activity curves of the left ventricular blood pool and myocardium were derived from PET images, and metabolite-corrected arterial input function was used for kinetic modeling. Also, ex vivo biodistribution of 68Ga radioactivity was analyzed.Results: All four 68Ga tracers showed undesirably slow myocardial accumulation over time, but their in vivo stability, clearance from blood and the kinetics of the myocardium uptake varied. [68Ga][Ga-(sal)2BAPDMEN]1+ showed the highest myocardial uptake in PET images and tissue samples (myocardium-to-blood ratio 7.63±1.89, myocardium-to-lung ratio 3.03±0.33 and myocardium-to-liver ratio 1.80±0.82). However, there was no correlation between the myocardial perfusion measured with [15O]water and the net uptake rates or K1 values of the 68Ga chelates.Conclusion: Our results revealed that myocardial accumulation of the 68Ga chelates proposed for myocardial perfusion imaging with PET was slow and not determined by myocardial perfusion in a large animal model. These findings suggest that the studied tracers are not suitable for clinical imaging of myocardial perfusion.

64Cu-NO2A-RGD-Glu-6-Ahx-BBN(7-14)NH2: a heterodimeric targeting vector for positron emission tomography imaging of prostate cancer - Corrected Proof

2012-01-09

Abstract: Introduction: The present study describes the design and development of a new heterodimeric RGD-bombesin (BBN) agonist peptide ligand for dual receptor targeting of the form 64Cu-NO2A-RGD-Glu-6-Ahx-BBN(7-14)NH2 in which Cu-64=a positron emitting radiometal; NO2A=1,4,7-triazacyclononane-1,4-diacetic acid; Glu=glutamic acid; 6-Ahx=6-aminohexanoic acid; RGD=the amino acid sequence [Arg-Gly-Asp], a nonregulatory peptide that has been used extensively to target αvβ3 receptors up-regulated on tumor cells and neovasculature; and BBN(7-14)NH2=Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2, an agonist analogue of bombesin peptide for specific targeting of the gastrin-releasing peptide receptor (GRPr).Methods: RGD-Glu-6-Ahx-BBN(7-14)NH2 was manually coupled with NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), and the resulting conjugate was labeled with 64Cu to yield 64Cu-NO2A-RGD-Glu-6-Ahx-BBN(7-14)NH2. Purification was achieved via reversed-phase high-performance liquid chromatography and characterization confirmed by electrospray ionization–mass spectrometry.Results: Competitive displacement binding assays displayed single-digit nanomolar IC50 values showing very high binding affinities toward the GRPr for the new heterodimeric peptide analogues. In vivo biodistribution studies showed high uptake and retention of tumor-associated radioactivity in PC-3 tumor-bearing rodent models with little accumulation and retention in nontarget tissues. The radiolabeled conjugate also exhibited rapid urinary excretion and high tumor-to-background ratios. Micro-positron emission tomography (microPET) molecular imaging investigations produced high-quality, high-contrast images in PC-3 tumor-bearing mice 15 h postinjection.Conclusions: Based on microPET imaging experiments that show high-quality, high-contrast images with virtually no residual gastrointestinal radioactivity, this new heterodimeric RGD-BBN conjugate can be considered as a promising PET tracer candidate for the diagnosis of GRPr-positive tumors in human patients.

An automated module for the separation and purification of cyclotron-produced 99mTcO4− - Corrected Proof

2012-01-09

Abstract: Introduction: The shortage of reactor-produced molybdenum-99 (99Mo, t½=66 h) has renewed interest in alternative production methods of its daughter isotope, technetium-99m (99mTc, t½=6.02 h). While adsorption chromatography serves as a mechanism for selective elution of sodium pertechnetate from technetium generators, this method of purification is not sufficient for many alternative production methods. Several ion-separation/solid phase extraction chromatography methods are known, yet none have been demonstrated on cyclotron-produced [99mTc]TcO4−. Herein we describe the design, manufacture and optimization of a remotely operated module for the purification of sodium pertechnetate from a bulk solution of molybdate.Methods: The automated purification module was designed to separate [99mTc]TcO4− using either Dowex 1x8 or an Aqueous Biphasic Extraction Chromatography (ABEC) resin. 100Mo composite targets were irradiated with 18.5 MeV protons for 10 μA·h using an ASCI TR19 cyclotron. Once purified, the radiopharmaceutical quality of 99mTcO4− isolated from each process (Dowex and/or ABEC) was established by assaying for molybdate breakthrough, alumina levels and, in the case of the Dowex approach, residual organics.Results: The separation processes are efficient (75% for Dowex, 90% for ABEC) and complete in less than 30 min. Overall, up to 2.1 GBq of 99mTc was produced using the 100Mo(p,2n)99mTc transformation, processed using the separation module and subjected to a detailed chemical and radionuclidic analysis. Due to its expense and limited availability, 100MoO42− was recovered in >90% yield using a precipitation/filtration/lyophilization approach.Conclusions: Na[99mTc]TcO4 was produced using a medical cyclotron, recovered using an automated purification module and found to exceed all established quality control parameters.

Preclinical studies of potential amyloid binding PET/SPECT ligands in Alzheimer's disease - Corrected Proof

Marie M. Svedberg, Obaidur Rahman, Håkan Hall — 2012-01-09

Abstract: Visualizing the neuropathological hallmarks amyloid plaques and neurofibrillary tangles of Alzheimer's disease in vivo using positron emission tomography (PET) or single photon emission computed tomography will be of great value in diagnosing the individual patient and will also help in our understanding of the disease. The successful introduction of [11C]PIB as a PET tracer for the amyloid plaques less than 10 years ago started an intensive research, and numerous new compounds for use in molecular imaging of the amyloid plaques have been developed. The candidates are based on dyes like thioflavin T, Congo red and chrysamine G, but also on other types such as benzoxazoles, curcumin and stilbenes. In the present review, we present methods of the radiochemistry and preclinical evaluation as well as the main properties of some of these compounds.

Synthesis, radiolabeling, biodistribution and fluorescent imaging of histidine-coupled hematoporphyrin - Corrected Proof

Yupeng Liu, Bo Shen, Fei Liu, Bin Zhang, Taiwei Chu, Jing Bai, Shanglian Bao — 2012-01-09

Abstract: Introduction: Hematoporphyrin (Hp) and hematoporphyrin derivatives (HpDs) have been widely used as photosensitizers in photodynamic therapy (PDT). Radiolabeling of HpDs is helpful for preclinical and clinical studies of PDT.Methods: The histidine-coupled hematoporphyrin (His-Hp) was synthesized and radiolabeled with [99mTc(CO)3(H2O)3]+. Biodistribution of the radioligand and fluorescent imaging of His-Hp in mice bearing S180 tumor were investigated.Results: [99mTc(CO)3]+-labeled His-Hp was electrically neutral, hydrophilic and stable. The biodistribution of the radioligand in S180 tumor-bearing mice was similar with that of nonlabeled HpD in the literature. The uptake of His-Hp in tumors and livers was confirmed by fluorescent imaging.Conclusions: The complex [99mTc(CO)3]+–His-Hp might be suitable for in vivo dose evaluation of HpD in PDT.

Synthesis and preclinical evaluation of [11C]D617, a metabolite of (R)-[11C]verapamil - Corrected Proof

2012-01-09

Abstract: Objectives: (R)-[11C]verapamil is widely used as a positron emission tomography (PET) tracer to evaluate P-glycoprotein (P-gp) functionality at the blood–brain barrier in man. A disadvantage of (R)-[11C]verapamil is the fact that its main metabolite, [11C]D617, also enters the brain. For quantitative analysis of (R)-[11C]verapamil data, it has been assumed that the cerebral kinetics of (R)-[11C]verapamil and [11C]D617 are the same. The aim of the present study was to investigate whether the cerebral kinetics of (R)-[11C]verapamil and [11C]D617 are indeed similar and, if so, whether [11C]D617 itself could serve as an alternative PET tracer for P-gp.Methods: [11C]D617 was synthesized and its ex vivo biodistribution was investigated in male rats at four time points following intravenous administration of [11C]D617 (50 MBq) without (n=4) or with (n=4) pretreatment with the P-gp inhibitor tariquidar (15 mg·kg−1, intraperitoneally). Brain distribution was further assessed using consecutive PET scans (n=8) before and after pretreatment with tariquidar (15 mg·kg−1, intravenously), as well as metabolite analysis (n=4).Results: The precursor for the radiosynthesis of [11C]D617, 5-amino-2-(3,4-dimethoxy-phenyl)-2-isopropyl-pentanitrile (desmethyl D617), was synthesized in 41% overall yield. [11C]D617 was synthesized in 58%–77% decay-corrected yield with a radiochemical purity of ≥99%. The homogeneously distributed cerebral volume of distribution (VT) of [11C]D617 was 1.1, and this increased 2.4-fold after tariquidar pretreatment.Conclusion: VT of [11C]D617 was comparable to that of (R)-[11C]verapamil, but its increase after tariquidar pretreatment was substantially lower. Hence, (R)-[11C]verapamil and [11C]D617 do not show similar brain kinetics after inhibition of P-gp with tariquidar.

Synthesis and preliminary evaluation of [18F]-labeled 2-oxoquinoline derivatives for PET imaging of cannabinoid CB2 receptor - Corrected Proof

2012-01-09

Abstract: Introduction: The cannabinoid receptor type 2 (CB2) is an important target for development of drugs and imaging agents for diseases, such as neuroinflammation, neurodegeneration and cancer. Recently, we reported synthesis and results of in vitro receptor binding of a focused library of fluorinated 2-oxoquinoline derivatives as CB2 receptor ligands. Some of the compounds demonstrated to be good CB2-specific ligands with Ki values in the nanomolar to subnanomolar concentrations; therefore, we pursued the development of their 18F-labeled analogues that should be useful for positron emission tomography (PET) imaging of CB2 receptor expression. Here, we report the radiosynthesis of two 18F-labeled 2-oxoquinoline derivatives and the preliminary in vitro and ex vivo evaluation of one compound as a CB2-specific radioligand.Methods: 4-[18F]fluorobenzyl amine [18F]-3 was prepared by radiofluorination of 4-cyano-N,N,N-trimethylanilinium triflate salt followed by reduction with LiAlH4 and then coupled with acid chlorides 11 and 12 to afford [18F]-13 and [18F]-14. In vitro CB2 receptor binding assay was performed using U87 cells transduced with CB2 and CB1 receptor. Ex vivo autoradiography was performed with [18F]-14 on spleen and on CB2- and CB1-expressing and wild-type U87 subcutaneous tumors grown in mice.Results: The radiochemical yields of [18F]-13 and [18F]-14 were 10%–15.0% with an average of 12% (n=10); radiochemical purity was >99% with specific activity 1200 mCi/μmol. The dissociation constant Kd for [18F]-14 was 3.4 nM. Ex vivo autoradiography showed accumulation of [18F]-14 in the CB2-expressing tumor.Conclusion: Two new [18F]-labeled CB2 ligands have been synthesized. Compound [18F]-14 appears to be a potential PET imaging agent for the assessment of CB2 receptor expression; however, poor solubility restrain its use in vivo.

Synthesis, radioiodination and in vitro and in vivo sigma receptor studies of N-1-allyl-N´-4-phenethylpiperazine analogs - Corrected Proof

2011-12-15

Abstract: Introduction: Sigma-1 (σ1) receptor radioligands are useful for basic pharmacology studies and for imaging studies in neurology, psychiatry and oncology. We derived a hybrid structure, N-1-allyl-N´-4-phenethylpiperazine, from known ligands TPCNE and SA4503 for use as a scaffold for development of radioiodinated σ1 receptor ligands.Methods: E-and Z-N-1-(3′-iodoallyl)-N´-4-(3″,4″-dimethoxyphenethyl)-piperazine (E-1 and Z-1), N-1-allyl-N´-4-(3′,4′-dimethoxyphenethyl)-piperazine (2) and E-N-1-(3′-iodoallyl)-N´-4-(3″-methoxy-4′´-hydroxyphenethyl)-piperazine (3) were synthesized. Affinities for σ1 and σ2 receptors were determined. [125I]E-1 and [125I]Z-1 were prepared and evaluated in vivo in mice. [125I]E-1 was further evaluated in σ1 receptor binding assays in vitro.Results: E-1 displayed moderately high apparent affinity (15 nM) for σ1 sites and 84-fold selectivity against σ2 sites. Z-1 showed similar σ1 affinity, but only 23-fold selectivity. In contrast, 2 exhibited poor binding to both subtypes, while 3 had good affinities but poor selectivity. E-1 profiled as a probable antagonist in the phenytoin shift assay. [125I]E-1 and [125I]Z-1 were prepared in good yields and with high specific radioactivities. Log D7.4 values (2.25 and 2.27) fall within the optimal range for in vivo studies. Both radioligands selectively labeled σ1 receptors in mouse brain and peripheral organs in vivo. [125I]E-1 showed a higher level of specific binding than [125I]Z-1 and displayed good metabolic stability. Further, [125I]E-1 selectively labeled σ1 receptors in mouse brain homogenates (Kd 3.79 nM; Bmax=599 fmol/mg protein).Conclusions: [125I]E-1 is a selective σ1 receptor radioligand that exhibits properties amenable to in vitro and in vivo studies, with possible extension to single photon emission computed tomography using iodine-123.

Measuring diffuse metabolic activity on FDG-PET/CT: new method for evaluating Langerhans cell histiocytosis activity in pulmonary parenchyma - Corrected Proof

2011-12-15

Abstract: Introduction: Pulmonary Langerhans cell histiocytosis (PLCH) is a rare cause of interstitial lung disease characterized by formation of nodules in the active phase of the disease that evolve into nonactive cystic lesions later on. To evaluate PLCH activity in patients, we developed a new method for measuring diffuse metabolic activity on fluorine-18-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) using a lung-to-liver activity ratio.Material and Methods: We retrospectively studied a series of 4 FDG-PET and 23 FDG-PET/CT scans from 7 patients with PLCH and analyzed a sample of 100 randomly chosen FDG-PET/CT studies free from any known lung or hepatic diseases. Maximum standardized uptake value (SUVmax) in a spherical volume (6–8 cm in diameter) in the right lung was put into relation with SUVmax in a spherical volume (9–10 cm in diameter) in the reference liver parenchyma to set up the SUVmaxPULMO/SUVmaxHEPAR index. The index values were compared to the disease course in each patient.Results: In patients with PLCH, a close correlation between the index value and the disease course was found in all seven subjects, where the increasing index values indicated disease activity, while decreasing index values were observed after therapy administration. In the group of 100 healthy control subjects, we found index values lower than 0.3 in 80% and lower than 0.4 in 96% [range: 0.14–0.43; 0.24±0.07 (100)].Conclusion: Measuring SUVmaxPULMO/SUVmaxHEPAR values and their time-trend monitoring represent simple, noninvasive screening tools allowing an early diagnosis and treatment response follow-up assessment in patients with PLCH.

11C-CHO PET in optimization of target volume delineation and treatment regimens in postoperative radiotherapy for brain gliomas - Corrected Proof

2011-12-15

Abstract: Objective: We explored the clinical values of 11C-choline (11C-CHO) PET in optimization of target volume delineation and treatment regimens in postoperative radiotherapy for brain gliomas.Methods: Sixteen patients with the pathological confirmation of the diagnosis of gliomas prior to receiving radiotherapy (postoperative) were included, and on whom both MRI and CHO PET scans were performed at the same position for comparison of residual tumors with the two techniques. 11C-CHO was used as the tracer in the PET scan. A plain T1-weighted, T2-weighted and contrast-enhanced T1-weighted imaging scans were performed in the MRI scan sequence. The gliomas' residual tumor volume was defined as the area with CHO-PET high-affinity uptake and metabolism (VCHO) and one with MRI T1-weighted imaging high signal intensity (VGd), and was determined by a group of experienced professionals and clinicians.Results: (1) In CHO-PET images, the tumor target volume, i.e., the highly metabolic area with a high concentration of isotopes (SUV 1.016–4.21) and the corresponding contralateral normal brain tissues (SUV0.1–0.62), was well contrasted, and the boundary between lesions and surrounding normal brain tissues was better defined compared with MRI and 18F-FDG PET images. (2) For patients with brain gliomas of WHO Grade II, the SUV was 1.016–2.5; for those with WHO Grades III and IV, SUVs were >26–4.2. (3) Both CHO PET and MRI were positive for 10 patients and negative for 2 patients. The residual tumor consistency between these two studies was 75%. Four of the 10 CHO-PET-positive patients were negative on MRI scans. The maximum distance between VGd and VCHO margins was 1.8 cm. (4) The gross tumor volumes (GTVs) and the ensuing treatment regimens were changed for 31.3% (5/16) of patients based on the CHO-PET high-affinity uptake and metabolism, in which the change rate was 80% (4/5), 14.3 % (1/7) and 0% (0/4) for patients with WHO Grade II III, and IV gliomas, respectively.Conclusion: Our data demonstrate that difference exists between CHO PET and MRI by which to judge and identify residual tumor for patients with brain gliomas. CHO PET is considered to be a supplementary diagnostic approach for MRI. Biological tumor target volume (BTV) displayed in the CHO PET images is useful in determining or delineating the radiotherapy target volume and making decisions in selecting treatment regimens. Tumor target volume may be defined more accurately and rationally when the CHO PET is combined with MRI.

In vitro and in vivo evaluation of selected 68Ga-siderophores for infection imaging - Corrected Proof

2011-12-15

Abstract: Introduction: Siderophores are low-molecular-mass iron chelators serving as iron transporters for almost all bacteria, fungi and some plants. Iron is an essential element for majority of organisms and plays an important role in virulence of pathogenic organisms. 68Ga is a positron emitter with complexing properties comparable to those of Fe(III) and readily available from a generator. Initial studies with 68Ga-triacetylfusarinine C (TAFC) showed excellent targeting properties in a rat infection model. We report here on the in vitro and in vivo evaluation of other siderophores radiolabelled with 68Ga as potential radiopharmaceuticals for infection imaging.Methods: 68Ga labelling was performed using acetate buffer. Stability, log P and protein binding values were determined. In vitro uptake was tested using iron-deficient and iron-sufficient Aspergillus fumigatus (A.f.) cultures. Biodistribution of 68Ga-siderophores was studied in Balb/c mice.Results: Significant differences among studied siderophores were observed in labelling efficiency, stability and protein binding. Uptake in A.f. cultures was highly dependent on iron load and type of the siderophore. In mice, 68Ga-TAFC and 68Ga-ferrioxamine E (FOXE) showed rapid renal excretion and low blood values even at a short period after injection; in contrast, 68Ga-ferricrocin and 68Ga-ferrichrome revealed high retention in blood and 68Ga-fusarinine C showed very high kidney retention.Conclusions: Some of the studied siderophores bind 68Ga with high affinity and stability, especially 68Ga-TAFC and 68Ga-FOXE. Low values of protein binding, high and specific uptake in A.f., and excellent in vivo biodistribution make them favourable agents for Aspergillus infection imaging.

Simple preparation and purification of ethanol-free solutions of 3′-deoxy-3′-[18F]fluorothymidine by means of disposable solid-phase extraction cartridges - Corrected Proof

2011-12-15

Abstract: Introduction: 3′-Deoxy-3′-[18F]fluorothymidine ([18F]FLT) shows great potential as a tracer for proliferative studies with PET. However, its regular application is often limited by low radiochemical yields and the use of a troublesome HPLC separation. Moreover, a high content of ethanol, at least one-fold higher than the European Pharmacopoeia and US Pharmacopoeia's established limit, is always present in the final product. The present study reports an optimization of the reaction conditions and a simple and straightforward purification step which affords a solution of [18F]FLT suitable for human use.Methods: Several conditions and materials were tested for both the nucleophilic substitution and purification step. The latter was achieved by means of a series of commercial solid-phase extraction cartridges. Very conveniently, the whole one-pot synthesis was carried out on commercial automated modules using basically the same setup employed for the synthesis of [18F]FDG.Results: Under routine conditions, radiochemical yields of 37% [decay-corrected to start of synthesis (SOS)] were achieved in ca. 39 min from SOS, with radiochemical purities >98% (usually >99%). The negligible radiolysis observed could be easily suppressed by adding 0.5% of EtOH. Typical unlabelled chemical impurities detected were thymidine (0.15 ppm), thymine (0.28 ppm) and stavudine (0.05 ppm).Conclusions: A reliable, simple and efficient preparation of [18F]FLT has been developed, able to afford an ethanol-free solution of the tracer with no need for any HPLC purification. Because of its similarity to the [18F]FDG synthesis, the method can be readily implemented on basically all the commercial modules developed for this common radiotracer.

An allogenic site-specific rat model of bone metastases for nuclear medicine and experimental oncology - Corrected Proof

2011-12-15

Abstract: Bone metastases are a major problem in several tumor entities affecting the therapeutic decision and the patient's prognosis. Single photon emission computed tomography (SPECT) and positron emission tomography (PET) are promising techniques for identifying bone tumors using gamma- or positron-emitting labeled radiotracers, but the same tracers if labeled with beta-emitters may also be used to apply therapeutic radionuclides for localized irradiation. For the tracer development specifically accumulating in osseous lesions, animal models of bone metastasis are needed. A technique was developed for tumor cell injection into the circulation of the hind limb of rats. For tumor implantation, the arteria epigastrica caudalis superficialis (a branch of the femoral artery) was cannulated, and 2×105 cells were injected. By using the allogenic Walker 256 mammary carcinoma cell line, isolated bone metastases were induced. For visualizing of the tumor growth, PET with 18F-fluoride was performed weekly on a μ-PET system. After 2–3 weeks, tumor invasion was confirmed by histology. Three weeks after tumor cell inoculation, PET images showed signs of bone metastases in 9 out of 11 animals. The tumors were located either in the proximal tibia/fibula or in the distal femur. At this time, the animals showed no restrictions in mobility. The tumors grew constantly over time. The final histological analysis showed tumors growing invasively into the bone matrix. With this model, new SPECT or PET tracers can be evaluated for their potency of accumulating in bone metastases in vivo and to determine which are therefore suitable for diagnosis and/or therapy.

68Ga-labeling and in vivo evaluation of a uPAR binding DOTA- and NODAGA-conjugated peptide for PET imaging of invasive cancers - Corrected Proof

Morten Persson, Jacob Madsen, Søren Østergaard, Michael Ploug, Andreas Kjaer — 2011-12-15

Abstract: Introduction: The urokinase-type plasminogen activator receptor (uPAR) is a well-established biomarker for tumor aggressiveness and metastatic potential. DOTA-AE105 and DOTA-AE105-NH2 labeled with 64Cu have previously been demonstrated to be able to noninvasively monitor uPAR expression using positron emission tomography (PET) in human cancer xenograft mice models. Here we introduce 68Ga-DOTA-AE105-NH2 and 68Ga-NODAGA-AE105-NH2 and evaluate their imaging properties using small-animal PET.Methods: Synthesis of DOTA-AE105-NH2 and NODAGA-AE105-NH2 was based on solid-phase peptide synthesis protocols using the Fmoc strategy. 68GaCl3 was eluted from a 68Ge/68Ga generator. The eluate was either concentrated on a cation-exchange column or fractionated and used directly for labeling. For in vitro characterization of both tracers, partition coefficient, buffer and plasma stability, uPAR binding affinity and cell uptake were determined. To characterize the in vivo properties, dynamic microPET imaging was carried out in nude mice bearing human glioma U87MG tumor xenograft.Results: In vitro experiments revealed uPAR binding affinities in the lower nM range for both conjugated peptides and identical to AE105. Labeling of DOTA-AE105-NH2 and NODAGA-AE105-NH2 with 68Ga was done at 95°C and room temperature, respectively. The highest radiochemical yield and purity were obtained using fractionated elution, whereas a negative effect of acetone on labeling efficiency for NODAGA-AE105-NH2 was observed. Good stability in phosphate-buffered saline and mouse plasma was observed. High cell uptake was found for both tracers in U87MG tumor cells. Dynamic microPET imaging demonstrated good tumor-to-background ratio for both tracers. Tumor uptake was 2.1% ID/g and 1.3% ID/g 30 min postinjection and 2.0% ID/g and 1.1% ID/g 60 min postinjection for 68Ga-NODAGA-AE105-NH2 and 68Ga-DOTA-AE105-NH2, respectively. A significantly higher tumor-to-muscle ratio (P<.05) was found for 68Ga-NODAGA-AE105-NH2 60 min postinjection.Conclusions: The use of 68Ga-DOTA-AE105-NH2 and 68Ga-NODAGA-AE105-NH2 as the first gallium-68 labeled uPAR radiotracers for noninvasive PET imaging is reported, which combine versatility with good imaging properties. These new tracers thus constitute an interesting alternative to the 64Cu-labeled version (64Cu-DOTA-AE105 and 64Cu-DOTA-AE105-NH2) for detecting uPAR expression in tumor tissue. In our hands, the fractionated elution approach was superior for labeling of peptides, and 68Ga-NODAGA-AE105-NH2 is the favored tracer as it provides the highest tumor-to-background ratio.

Initial evaluation in healthy humans of [18F]DPA-714, a potential PET biomarker for neuroinflammation - Corrected Proof

2011-12-15

Abstract: Introduction: The translocator protein 18 kDa (TSPO), although minimally expressed in healthy brain, is up-regulated in pathological conditions, coinciding with microglial activation. It is thereby a suitable in vivo biomarker of neuroinflammation for detection, evaluation and therapeutic monitoring of brain diseases. We aimed to estimate the radiation dosimetry of the positron emission tomography (PET) TSPO radioligand [18F]DPA-714, and we evaluated in healthy volunteers its whole-body uptake and cerebral kinetics.Methods: Biodistribution data from mice were used for the prediction of radiation dosimetry. In human studies, a 90-min dynamic PET scan was performed in seven healthy volunteers after injection of [18F]DPA-714 (245±45 MBq). Arterial and venous samples were collected from two subjects, and two additional subjects were submitted to whole-body acquisition. Regions of interest were defined over cerebral structures to obtain mean time–activity curves and to estimate the distribution volume ratios by Logan graphical analysis, and over peripheral organs to obtain standard uptake values.Results: The effective dose estimated from biodistribution in mice was 17.2 μSv/MBq. Modeling of regional brain and plasma data showed good in vivo stability of [18F]DPA-714 in humans, with only 20% of blood metabolites 20 min postinjection (p.i.). Maximum cerebral uptake was observed 5 min p.i., followed by two decreasing phases: a rapid washout (5–30 min) followed by a slower phase for the remainder of PET acquisition. Whole-body images demonstrate high activity in the gallbladder, heart, spleen and kidneys.Conclusions: This initial study in humans shows that [18F]DPA-714 is a promising PET radioligand with excellent in vivo stability and biodistribution, and acceptable effective dose estimation. Therefore, [18F]DPA-714 could provide a sensitive measure of neuroinflammatory changes in subsequent clinical investigations.

Preclinical evaluation of anti-HER2 Affibody molecules site-specifically labeled with 111In using a maleimido derivative of NODAGA - Corrected Proof

2011-12-15

Abstract: Introduction: Affibody molecules have demonstrated potential for radionuclide molecular imaging. The aim of this study was to synthesize and evaluate a maleimido derivative of the 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) for site-specific labeling of anti-HER2 Affibody molecule.Methods: The maleimidoethylmonoamide NODAGA (MMA-NODAGA) was synthesized and conjugated to ZHER2:2395 Affibody molecule having a C-terminal cysteine. Labeling efficiency, binding specificity to and cell internalization by HER2-expressing cells of [111In-MMA-NODAGA-Cys61]-ZHER2:2395 were studied. Biodistribution of [111In-MMA-NODAGA-Cys61]-ZHER2:2395 and [111In-MMA-DOTA-Cys61]-ZHER2:2395 was compared in mice.Results: The affinity of [MMA-NODAGA-Cys61]-ZHER2:2395 binding to HER2 was 67 pM. The 111In-labeling yield was 99.6%±0.5% after 30 min at 60°C. [111In-MMA-NODAGA-Cys61]-ZHER2:2395 bound specifically to HER2-expressing cells in vitro and in vivo. Tumor uptake of [111In-MMA-NODAGA-Cys61]-ZHER2:2395 in mice bearing DU-145 xenografts (4.7%±0.8% ID/g) was lower than uptake of [111In-MMA-DOTA-Cys61]-ZHER2:2395 (7.5%±1.6% ID/g). However, tumor-to-organ ratios were higher for [111In-MMA-NODAGA-Cys61]-ZHER2:2395 due to higher clearance rate from normal tissues.Conclusions: MMA-NODAGA is a promising chelator for site-specific labeling of targeting proteins containing unpaired cysteine. Appreciable influence of chelators on targeting properties of Affibody molecules was demonstrated.

Authors' reply to letter to the editor by Robert Freudenberg - Corrected Proof

2011-12-15

Thank you for your interest in our recent article . The direct cell labeling of bone marrow mesenchymal stem cells (BMSCs) using 99mTc-HMPAO was performed based on the assumption that 99mTc-HMAPO can enter BMSCs in the same way it does with leukocytes. 99mTc is taken up by BMSCs through HMPAO which is lipophilic and able to carry 99mTc into the cells. Within the cell, 99mTc-HMPAO is cleaved by the enzyme reaction and free 99mTc is retained in the cytoplasm. In fact, however, a certain fraction of uncleaved 99mTc-HMPAO comes out from the cells and, thus, the retention rate of 99mTc in BMSCs decreases over time. Based on our measurements, the retention rates in vitro were 72%, 45%, 31% and 16% at 1, 3, 6 and 24 h after radiolabeling, respectively. Therefore, the radiation to BMSCs in the current study would be much less than in case radioisotopes are tightly bound to cells.

Influence of cations on the complexation yield of DOTATATE with yttrium and lutetium: a perspective study for enhancing the 90Y and 177Lu labeling conditions - Corrected Proof

2011-12-15

Abstract: The DOTA macrocyclic ligand can form stable complexes with many cations besides yttrium and lutetium. For this reason, the presence of competing cationic metals in yttrium-90 and lutetium-177 chloride solutions can dramatically influence the radiolabeling yield. The aim of this study was to evaluate the coordination yield of yttrium- and lutetium-DOTATATE complexes when the reaction is performed in the presence of varying amounts of competing cationic impurities. In the first set of experiments, the preparation of the samples was performed by using natural yttrium and lutetium (20.4 nmol). The molar ratio between DOTATATE and these metals was 1 to 1. Metal competitors (Pb2+, Zn2+, Cu2+, Fe3+, Al3+, Ni2+, Co2+, Cr3+) were added separately to obtain samples with varying molar ratio with respect to yttrium or lutetium (0.1, 0.5, 1, 2 and 10). The final solutions were analyzed through ultra high-performance liquid chromatography with an UV detector. In the second set of experiments, an amount of 90Y or 177Lu chloride (6 MBq corresponding to 3.3 and 45 pmol, respectively) was added to the samples, and a radio-thin layer chromatography analysis was carried out. The coordination of Y3+ and Lu3+ was dramatically influenced by low levels of Zn2+, Cu2+ and Co2+. Pb2+ and Ni2+ were also shown to be strong competitors at higher concentrations. Fe3+ was expected to be a strong competitor, but the effect on the incorporation was only partly dependent on its concentration. Al3+ and Cr3+ did not compete with Y3+ and Lu3+ in the formation of DOTATATE complexes.

Synthesis of oncological [11C]radiopharmaceuticals for clinical PET - Corrected Proof

2011-12-15

Abstract: Positron emission tomography (PET) is a nuclear medicine modality which provides quantitative images of biological processes in vivo at the molecular level. Several PET radiopharmaceuticals labeled with short-lived isotopes such as 18F and 11C were developed in order to trace specific cellular and molecular pathways with the aim of enhancing clinical applications. Among these [11C]radiopharmaceuticals are N-[11C]methyl-choline ([11C]choline), l-(S-methyl-[11C])methionine ([11C]methionine) and 1-[11C]acetate ([11C]acetate), which have gained an important role in oncology where the application of 2-[18F]fluoro-2-deoxy-d-glucose ([18F]FDG) is suboptimal. Nevertheless, the production of these radiopharmaceuticals did not reach the same level of standardization as for [18F]FDG synthesis. This review describes the most recent developments in the synthesis of the above-mentioned [11C]radiopharmaceuticals aiming to increase the availability and hence the use of [11C]choline, [11C]methionine and [11C]acetate in clinical practice.

Evaluation of an automated double-synthesis module: efficiency and reliability of subsequent radiosyntheses of FHBG and FLT - Corrected Proof

2011-12-15

Abstract: We optimized the synthesis methods for 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT) and 9-(4-[18F]fluoro-3-[hydroxymethyl]butyl)guanine) ([18F]FHBG) and automated them on an Explora General Nucleophilic double-synthesis module. Furthermore, the synthesis efficiency and reliability and the formation of cross-contaminations of the products when preparing two consecutive batches were evaluated. Whereas the preinstalled FLT synthesis conditions required substantial modification in reaction and neutralization conditions to achieve radiochemical yields of up to 60% within 70±10 min including high-performance liquid chromatography purification, the synthesis of FHBG had to be implemented to the module to obtain competitive radiochemical yields of up to 40% in an overall synthesis time of 60±10 min. The radiochemical purities obtained were ≥99% and ≥96% for the synthesis of [18F]FLT and [18F]FHBG, respectively. No significant changes in yield or purity could be observed between both batch productions. We found that the yields and purities also did not change when performing FLT after FHBG syntheses and vice versa. Hence, we developed a synthesis setup that offers the opportunity to perform two subsequent syntheses of either [18F]FLT, [18F]FHBG or [18F]FLT after [18F]FHBG without decrease in radiochemical yields and purities. Also, no cross-contaminations were observed, which can be attributed to the use of separate product delivery tubes, purification columns and an automated intermediate cleaning program. These results open up the possibility of producing consecutively either two equal 18F-fluorinated tracers or two different ones in high yields on the same synthesis module.

Combined-modality radioimmunotherapy: synergistic effect of paclitaxel and additive effect of bevacizumab - Corrected Proof

2011-12-15

Abstract: Introduction: This study was undertaken to investigate the effect of paclitaxel and bevacizumab on the therapeutic efficacy of 90Y-labeled B3 monoclonal antibody, directed against Ley antigen, for the treatment of Ley-positive A431 tumors implanted subcutaneously in the right hind flank of nude mice.Methods: When the tumor size reached ∼200 mm3, the mice received a single dose of intravenous (iv) 90Y-labeled B3 (60 μCi/150 μg or 100 μCi/150 μg B3), intraperitoneal paclitaxel (40 mg/kg) or iv bevacizumab (5 mg/kg) for monotherapy. To investigate the effect of combined therapies on survival, the mice were treated with two or three agents in the following combinations: 90Y-B3 on day 0 and paclitaxel on day 1; bevacizumab on −1 day and 90Y-B3 on day 0; bevacizumab on −1 day and paclitaxel on day 1; bevacizumab, 90Y-B3 and paclitaxel each at 1-day intervals. The mice with no treatment were used as a control. The tumor volume at 1000 mm3 was used as a surrogate end point of survival.Results: Compared to control animals, paclitaxel delayed tumor growth with a significantly longer median survival time (P .10). Fluorescence microscopy analysis indicated that paclitaxel increased, whereas bevacizumab decreased, the accumulation and penetration of Alexa Fluor 647-B3 into tumor microenvironment compared to the control (P<.05).Conclusion: Our findings on the paclitaxel effect support a hypothesis that the increased tumor accumulation and penetration of 90Y-B3 as well as the high radiosensitization of tumor cells by paclitaxel may be the major factors responsible for the synergistic effect of the combined therapy involving 90Y-B3 with paclitaxel.

Preclinical evaluation of [11C]NE40, a type 2 cannabinoid receptor PET tracer - Corrected Proof

2011-12-12

Abstract: Introduction: Up-regulation of the type 2 cannabinoid receptor (CB2R) has been reported in (neuro)inflammatory diseases. In this study, we report the preclinical evaluation of [11C]NE40 as positron emission tomography (PET) radioligand for visualization of the CB2R.Methods: The selectivity of NE40 for CB2R and its toxicity and mutagenicity were determined. [11C]NE40 was evaluated by biodistribution and autoradiography studies in normal rats and a microPET study in normal mice, rats and a rhesus monkey. Specific in vivo binding of [11C]NE40 to human CB2R (hCB2R) was studied in a rat model with hCB2R overexpression.Results: [11C]NE40 shows specific CB2R binding in the spleen and blood of normal rats and high brain uptake in rhesus monkey. [11C]NE40 showed specific and reversible binding to hCB2R in vivo in a rat model with local hCB2R overexpression.Conclusions: [11C]NE40 shows favorable characteristics as radioligand for in vivo visualization of the CB2R and is a promising candidate for hCB2R PET imaging.

Difficulties in dopamine transporter radioligand PET analysis: the example of LBT-999 using [18F] and [11C] labelling: Part II: Metabolism studies - Corrected Proof

2011-12-12

Abstract: Introduction: LBT-999, (E)-N-(4-fluorobut-2-enyl)-2β-carbomethoxy-3β-(4'-tolyl)nortropane, has been developed for PET imaging of the dopamine transporter. [18F]LBT-999 PET studies in baboons showed a lower brain uptake than [11C]LBT-999 and a high bone uptake, suggesting the presence of interfering metabolites. Therefore, in vitro and in vivo metabolism of these radiotracers was investigated.Methods: Rat and human liver microsomal incubations, baboon plasma and rat brain extracts were analyzed by radio-HPLC and LC-MS-MS.Results: In vitro experiments demonstrated the formation by P450s of five polar metabolites. The main routes of LBT-999 metabolism proposed were N-dealkylation, tolyl-hydroxylation and dealkylation plus tolyl-hydroxylation. In vivo in baboons, [18F]LBT-999 was rapidly converted into a [18F]hydroxylated metabolite likely oxidized in plasma into a [18F]carboxylic acid and into unlabeled N-dealkyl-LBT-999. The latter was detected in baboon plasma and in rat brain by LC-MS-MS. The time course of unchanged [18F]LBT-999 decreased rapidly in plasma and was higher than that of [11C]LBT-999 due to the formation of unlabeled N-dealkyl-LBT-999. In rats, striatum-to-cerebellum ratios of [18F]LBT-999, [18F]hydroxylated and [18F]acidic metabolite were 20, 4.2 and 1.65, respectively, suggesting a possible accumulation of the hydroxylated compound in the striatum.Conclusion: P450s catalyzed the formation of dealkylated and hydroxylated metabolites of LBT-999. In baboons, an extensive metabolism of [18F]LBT-999, with formation of unlabeled N-dealkyl-LBT-999, [18F]fluorobutenaldehyde (or its oxidation product) and [18F]hydroxy-LBT-999 able to penetrate the brain, prevented an easy and accurate estimation of the input function of the radiotracer. CYP3A4 being the main P450 involved in the metabolism of LBT-999, a similar pathway may occur in humans and confound PET quantification.

Dosimetry of 99mTc-HMPAO in mesenchymal stem cells - Corrected Proof

Robert Freudenberg, Jörg Kotzerke — 2011-12-12

We read the article by Park et al. with great interest. The authors describe a method of using bone marrow mesenchymal stem cells (BMSCs) labeled with 99mTc-HMPAO to evaluate acute brain trauma in rats. Concomitantly to the evaluation of the in vivo distribution, the authors investigated the proliferation, apoptosis and necrosis of 99mTc-BMSCs. As described in the article, the authors divided 40,000 cells into dishes and observed the cell growth for 13 days. Apoptosis and necrosis were monitored after 3 h, on Day 3 and on Day 7 after incubation. Activities were between 4.6 and 18.5 Bq/cell.

Insights into the failure of the potential, neutral myocardial imaging agent TcN-NOET: physicochemical identification of by-products and degradation species - Corrected Proof

2011-12-02

Abstract: Introduction: The neutral complex [99mTc(N)(NOEt)2], often referred to as TcN-NOET [NOEt=N-ethoxy,N-ethyldithiocarbamate(1−)], was proposed several years ago as a myocardial imaging agent. Despite some favorable clinical properties evidenced during phase I and phase II studies, the overall results of the European and American phase III clinical studies have been judged insufficient for a successful approval process by the regulatory agencies.Methods: Non-carrier-added and carrier-added experiments using short-lived 99mTc and long-lived 99gTc have been utilized to prepare a series of bis-substituted [Tc(N)(DTC)2] complexes [DTC=dithiocarbamate(1−)]. They have been purified by means of chromatographic techniques (high-performance liquid chromatography and thin-layer chromatography) and identified via double detection (UV-vis and radiometry) by comparison with authenticated samples of 99gTc compounds prepared by conventional coordination chemistry procedures.Results: The molecular structure of the lipophilic, neutral complex cis-[Tc(N)(NOEt)2] has been assigned by comparison with similar nitrido-Tc(V) complexes already reported in the literature. Novel bis-substituted nitrido-Tc complexes containing hydrolyzed portions of coordinated NOEt, namely, N-ethyldithiocarbamate [NHEt(1−)] and N-hydroxy, N-ethyldithiocarbamate [NOHEt(1−)], have been prepared and characterized by means of multinuclear nuclear magnetic resonance spectroscopy and mass spectrometry.Conclusions: Despite the identification of these “hydrolyzed” species, it is still unclear whether the failure to reach the clinical goal of the perfusion tracer [99mTc(N)(NOEt)2] is related to the degradation processes evidenced in this study or is the result of the mediocre imaging properties of the tracer.

Fluorine-18 labeling of three novel d-peptides by conjugation with N-succinimidyl-4-[18F]fluorobenzoate and preliminary examination by postmortem whole-hemisphere human brain autoradiography - Corrected Proof

2011-12-02

Abstract: Introduction: β-Amyloid (Aβ) plaques and neurofibrillary tangles are the main characteristics of Alzheimer's disease (AD). Positron emission tomography (PET), a high-resolution, sensitive, and noninvasive imaging technique, has been widely utilized in visualizing the localization of plaques and tangles and thereby distinguishing between AD and healthy controls. A small 12-mer d-enantiomeric peptide (amino acid sequence=QSHYRHISPAQV), denoted as D1, has high binding affinity to Aβ in vitro in the sub-micromolar range, and consequently, its radiolabeled analogues have a potential as radioligands for visualizing amyloid plaques in vivo by PET.Aim: The aims of the present work were to develop three different potent D1 derivative peptides labeled with fluorine-18 and to examine them in the AD and control postmortem human brain by autoradiography (ARG).Methods: Three different D1 derivative peptides were radiolabeled with fluorine-18 ([18F]ACI-87, [18F]ACI-88, [18F]ACI-89) using the prosthetic group N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) and purified by high performance liquid chromatography (HPLC). Preliminary ARG measurements were performed in AD and control brains.Results: The three fluorine-18-labeled d-peptides were obtained in a total synthesis time of 140 min with radiochemical purity higher than 98%. The specific radioactivities of the three different D1 derivative peptides were between 9 and 113 GBq/μmol. ARG demonstrated a higher radioligand uptake in the cortical gray matter and the hippocampus in the AD brain as compared to age-matched control brain.Conclusions: Fluorine-18 labeling of the three novel D1 derivative peptides using [18F]SFB was successfully accomplished. Higher contrast between AD and control brain slices demonstrates their potential applicability for further use in vivo by PET.

Preclinical evaluation of a 68Ga-labeled biotin analogue for applications in islet transplantation - Corrected Proof

2011-12-02

Abstract: Introduction: Islet transplantation is a promising treatment for type 1 diabetes mellitus, but the fate of the cells after intraportal infusion is unclear. It is therefore imperative to develop novel techniques for noninvasive imaging and quantification of events following islet transplantation.Methods: Small islet-like microbeads, avidin-covered agarose resins (AARs), were used as a model system for islet transplantation. Capability for specific [68Ga]Ga-DOTA-(PEG)2-biotin uptake and retention for either AARs or human islets conjugated with avidin by means of a heparin scaffold was studied in vitro. Biodistribution of the novel positron emission tomography (PET) tracer [68Ga]Ga-DOTA-(PEG)2-biotin was evaluated in mice treated by intraportal transplantation of AARs by μPET/computed tomography and ex vivo organ distribution and compared with control mice.Results: AARs had high capability to bind [68Ga]Ga-DOTA-(PEG)2-biotin, close to 50% of administrated tracer/μl in vitro (>0.25 MBq/μl). Avidin-tagged human islets could bind on average 2.2% of administered tracer/μl. Specificity (>90%) and retention (>90% after 1 h) were high for both AARs and avidin-tagged islets. Hepatic tracer uptake and retention were increased in mice transplanted with AARs [standardized uptake value (SUV)=2.6] compared to the untreated group (SUV=1.4). In vivo uptake of tracer to AARs was blocked by preadministration of unlabeled biotin.Conclusions: Avidin-tagged islet-like objects can be tracked in hepatic volume after intraportal transplantation by using [68Ga]Ga-DOTA-(PEG)2-biotin and PET.

Evaluation of 111In-labeled macrocyclic chelator-amino acid derivatives for cancer imaging - Corrected Proof

2011-12-02

Abstract: Purpose: We evaluated new 111In-labeled amino acid derivatives, in which the amino acids are conjugated with1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A) or 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A).Methods: DOTA-aminoalanine (DOTA-A), DOTA-aminohomoalanine (DOTA-H), DOTA-lysine (DOTA-L), DO2A-alanine (DO2A-A), DO3A-alanine (DO3A-A) and DO3A-homoalanine (DO3A-H) were labeled with 111In. In vitro cell uptake assays were performed usingHep3B (a human hepatoma cell line), CT26 (a mouse colon cancer cell line) and U87MG (a human glioma cell line). In vitro cell uptake inhibition assays were performed using U87MG and 111In-DO3A-H. U87MG bearing xenografted mice were subject to biodistribution, SPECT imaging, autoradiography, and immunohistochemistry studies.Results: Of the amino acid derivatives and cell lines examined, U87MG and 111In-DO3A-H showed highest uptake in vitro. This uptake was blocked by 2-aminobicyclo-[2,2,1] heptane-2-carboxylic acid (BCH) and by tryptophan. 111In-DO3A-HSPECT imaging of U87MG bearing xenografted mice visualized tumors (mean tumor-to-muscle ratio 3.16±0.74). Autoradiography and immunohistochemistry revealed that 111In-DO3A-H uptake matched L-type amino acid transporter 1 expression.Conclusion: Tumor uptake was successfully imaged using 111In-DO3A-H in U87MG bearing xenografted mice. 111In-DO3A-H appears to be useful for imaging tumors expressing L-type amino acid transporter.

Study of [18F]FLT and [123I]IaraU for cellular imaging in HSV1 tk-transfected murine fibrosarcoma cells: evaluation of the tracer uptake using 5-fluoro, 5-iodo and 5-iodovinyl arabinosyl uridines as competitive probes - Corrected Proof

2011-11-30

Abstract: As one of the most intensively studied probes for imaging of the cellular proliferation, [18F]FLT was investigated whether the targeting specificity of thymidine kinase 1 (TK1) dependency could be enhanced through a synergistic effect mediated by herpes simplex type 1 virus (HSV1) tk gene in terms of the TK1 or TK2 expression. 5-[123I]Iodo arabinosyl uridine ([123I]IaraU) was prepared in a radiochemical yield of 8% and specific activity of 21 GBq/μmol, respectively. Inhibition of the cellular uptake of these two tracers was compared by using the arabinosyl uridine analogs such as 5-iodo, 5-fluoro and 5-(E)-iodovinyl arabinosyl uridine along with 2′-fluoro-5-iodo arabinosyl uridine (FIAU). Due to potential instability of the iodo group, accumulation index of 1.6 for [123I]IaraU by HSV1-TK vs. control cells could virtually be achieved at 1.5 h, but dropped to 0.2 compared to 2.0 for [18F]FLT at 5 h. The results from competitive inhibition by these nucleosides against the accumulation of [18F]FLT implied that FLT exerted a mixed TK1- and TK2-dependent inhibition with HSV1-tk gene transfection because of the shifting of thymidine kinase status. Taken together, the combination of [18F]FLT and HSV1-TK provides a synergistic imaging potency.

Combination of nitric oxide stimulation with high-dose 18F-FDG promotes apoptosis and enhances radiation therapy of endothelial cells - Corrected Proof

Jin-Young Paik, Jin-Won Park, Kyung-Ho Jung, Eun Jeong Lee, Kyung-Han Lee — 2011-11-14

Abstract: Introduction: High-dose 18F-FDG can provide targeted nuclear therapy of cancer. Endothelial cell injury is a key determinant of tumor response to radiotherapy. Here, we tested the hypothesis that activation of endothelial cell glycolytic metabolism with nitric oxide can enhance the therapeutic effect of high-dose 18F-FDG.Methods: Calf pulmonary artery endothelial (CPAE) cells were treated with graded doses of 18F-FDG. Glycolysis was stimulated by 24 h of exposure to the nitric oxide donor, sodium nitroprusside (SNP). Cell viability was assessed by MTT and clonogenic assays. Apoptosis was evaluated by ELISA of cytosolic DNA fragments and Western blots of cleaved caspase-3.Results: SNP stimulation (0.1 and 1 mM) augmented CPAE cell 18F-FDG uptake to 2.6- and 4.6-fold of controls without adverse effects. Treatment with 333 μCi/ml 18F-FDG alone reduced viable cell number to 35.4% of controls by Day 3. Combining 0.1 mM SNP stimulation significantly enhanced the killing effect, reducing cell numbers to 19.2% and 39.2% of controls by 333 and 167 μCi/ml of 18F-FDG, respectively. 18F-FDG also suppressed clonogenic survival to 80.8% and 43.2% of controls by 83 and 167 μCi/ml, which was again intensified by SNP to 59.7% and 21.1% of controls. The cytotoxic effect of 18F-FDG was attributed to induction of apoptosis as shown by increased cytosolic fragmented DNA and cleaved caspase-3 levels (26.4% and 30.7% increases by 167 μCi/ml). Combining SNP stimulation significantly increased both of these levels to 1.8-fold of control cells.Conclusion: High-dose 18F-FDG combined with nitric oxide-stimulated glycolysis is an effective method to inhibit endothelial cell survival and promote apoptosis. These results suggest a potential role of this strategy for targeted radiotherapy of angiogenic vasculature.