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

PurposeTo 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.MethodTwo 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.ResultsWe 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).ConclusionBaculovirus 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.     

Therapeutic gene expression in transduced mesenchymal stem cells can be monitored using a reporter gene

AimWe constructed a recombinant adenovirus construct Ad5-sr39tk-IRES-VEGF₁₆₅ (Ad5-SIV) that contained a mutant herpes viral thymidine kinase reporter gene (HSV1-sr39tk) and the human vascular endothelial growth factor 165 (VEGF₁₆₅) gene for noninvasive imaging of gene expression. The recombinant adenovirus Ad5-SIV was transfected into rat bone marrow-derived mesenchymal stem cells (MSCs), and we measured the expression of HSV1-sr39tk and VEGF₁₆₅ to evaluate the feasibility of monitoring VEGF₁₆₅ expression using reporter gene expression.MethodsThe MSCs were infected with Ad5-SIV at various levels of infection (MOI), ranging from 0 to 100 infectious units per cell (IU/cell). The mRNA and protein expression levels of the reporter and therapeutic genes were determined using real-time RT-PCR, Western blot, ELISA and immunofluorescence. The HSV1-sr39tk expression in the MSCs was also detected in vitro using a cellular uptake study of the reporter probe ¹³¹I-FIAU. Gene expression was also evaluated in vivo by micro-Positron Emission Tomography/Computed Tomography (micro-PET/CT) imaging 1 day after injecting Ad5-SIV-tranfected MSCs into the left foreleg of the rat. The right foreleg was injected with non-transfected MSCs and served as an internal control.ResultsThe real-time RT-PCR results demonstrated a good correlation between the expression levels of HSV1-sr39tk mRNA and VEGF₁₆₅ mRNA (R² = 0.93, P < 0.05). The cellular uptake of ¹³¹I-FIAU increased with increasing viral titers (R² = 0.89; P < 0.05), and in the group that received an MOI of 100, a peak value of 30.15% ± 1.11% was found at 3 hours of incubation. The uptake rates increased rapidly between 30 and 150 minutes and reached a plateau after 150 minutes. The uptake rates of ¹³¹I-FIAU by the Ad5-SIV-infected cells were significantly higher than by the Ad5-EGFP-infected cells for all time points (t = 18.43-54.83, P < 0.05). Moreover, the rate of VEGF₁₆₅ protein secretion was highly correlated with the uptake rate of ¹³¹I-FIAU (R² = 0.84, P < 0.05). The radioactivity on the micro-PET/CT images was significantly higher in the left foreleg (which received the transfected MSCs) compared with the control foreleg.ConclusionsThese results suggest that radionuclide reporter gene imaging may be used to monitor gene expression in vivo.     

Heptamethine cyanine based 64Cu-PET probe PC-1001 for cancer imaging: Synthesis and in vivo evaluation

PurposeDevelopment of a heptamethine cyanine based tumor-targeting PET imaging probe for noninvasive detection and diagnosis of breast cancer.MethodsTumor-specific heptamethine–cyanine DOTA conjugate complexed with Cu-64 (PC-1001) was synthesized for breast cancer imaging. In vitro cellular uptake studies were performed in the breast cancer MCF-7 and noncancerous breast epithelial MCF-10A cell lines to establish tumor specificity. In vivo time-dependent fluorescence and PET imaging of breast tumor xenografts in mice were performed. Blood clearance, biodistribution, and tumor-specific uptake and plasma binding of PC-1001 were quantified. Tumor histology (H&E staining) and fluorescence imaging were examined.ResultsPC-1001 displayed similar fluorescence properties (ε = 82,880 cm^? 1 M^? 1, E_x/E_m = 750/820 nm) to the parental dye. Time-dependent cellular accumulation indicated significantly higher probe uptake (> 2-fold, 30 min) in MCF-7 than MCF-10A cells and the uptake was observed to be mediated by organic anion transport peptides (OATPs) system. In vivo studies revealed that PC-1001 has desirable accumulation profile in tumor tissues, with tumor versus muscle uptake of about 4.3 fold at 24 h and 5.8 fold at 48 h post probe injections. Blood half-life of PC-1001 was observed to be 4.3 ± 0.2 h. Microscopic fluorescence imaging of harvested tumor indicated that the uptake of PC-1001 was restricted to viable rather than necrotic tumor cells.ConclusionsA highly efficient tumor-targeting PET/fluorescence imaging probe PC-1001 is synthesized and validated in vitro in MCF-7 breast cancer cells and in vivo in mice breast cancer xenograft model.     

Comparison of Cerenkov Luminescence Imaging (CLI) and gamma camera imaging for visualization of let-7 expression in lung adenocarcinoma A549 Cells

Background and AimmiRNA is an important factor for tumorigenesis which could act as a potential molecular target for tumor diagnosis. The goal of this study was to explore a new method for visualizing the expression of let-7 in lung adenocarcinoma A549 cells by Cerenkov luminescence imaging (CLI) and gamma camera imaging.MethodsThe human sodium/iodine symporter (hNIS) and 3′-UTR sequence of the ras gene (RU) that complementarily binds to let-7 were cloned with hNIS serving as the reporter gene. The expression of hNIS regulated by let-7 in the fusion gene hNIS-RU was constructed; the let-7 primer (pri-let-7), which could specifically bind to RU and the mir-143 primer (pri-mir143) not binding with RU, was cloned. A549 cells were transfected with hNIS or hNIS-RU, and additional cells were cotransfected with hNIS-RU and different concentrations of pri-let-7 or pri-mir143. The cells were incubated with 740 kBq ¹³¹I-containing media for 1 h, 24 h after transfection. CLI, gamma camera imaging, and γ counting were subsequently conducted, and the correlation among CLI, gamma camera imaging, and γ counting was compared when cotransfected with pri-let-7.ResultsCLI, gamma camera imaging, and radioactive counting showed that hNIS-transfected A549 cells had significantly higher uptake of ¹³¹I compared to non-transfected cells. The uptake of ¹³¹I in hNIS-RU transfected A549 cells decreased to approximately 70% compared to hNIS-transfected cells, since hNIS-RU expression was suppressed by intracellular let-7. After cotransfection with hNIS-RU and various concentrations of pri-let-7, ¹³¹I uptake gradually decreased with increasing pri-let-7, while ¹³¹I uptake remained roughly unchanged in the presence of hNIS-RU cotransfected with different amounts of pri-mir143. CLI was highly correlated with gamma camera imaging (r² = 0.9893) and radioactivity counting (0.9779).ConclusionsBased upon miRNA-regulated reporter genes which mediate the uptake of the radionuclide, both CLI and gamma camera imaging can noninvasively detect miRNA expression in cells, which may provide a new way for the visualization of miRNA expression.     

High tumor uptake of 64Cu: Implications for molecular imaging of tumor characteristics with copper-based PET tracers

IntroductionThe use of copper-based positron emission tomography (PET) tracers in cancer studies is increasing. However, as copper has previously been found in high concentrations in human tumor tissue in vivo, instability of PET tracers could result in tumor accumulation of non-tracer-bound radioactive copper that may influence PET measurements. Here we determine the degree of ⁶⁴Cu uptake in five commonly used human cancer xenograft models in mice. Additionally, we compare copper accumulation in tumor tissue to gene expression of human copper transporter 1 (CTR1).MethodsSmall animal PET scans were performed on five different human cancer xenograft mice models 1 h and 22 h post injection (p.i.) of ⁶⁴CuCl₂. Regions of interest (ROIs) were drawn on tumor tissue and sections of various organs on all images. Quantitative real-time PCR (qPCR) gene expression measurements of CTR1 were performed on tumor samples obtained after the 22 h scan.ResultsA relatively high tumor uptake of ⁶⁴Cu was seen in four out of five tumor types and an increase in ⁶⁴Cu accumulation was seen in three out of five tumor types between 1 h and 22 h p.i. No relationship was found between tumor uptake of ⁶⁴Cu and gene expression of CTR1.ConclusionsThe relatively high, time- and tumor type dependent ⁶⁴Cu uptake demonstrated here in five different human cancer xenograft models in mice, emphasizes the importance of validating tracer uptake and indicates that high in vivo stability of copper-based PET tracers is of particular importance because non-tracer-bound copper can accumulate in tumor tissue to a level that could potentially lead to misinterpretation of PET data.     

Efficacy of lentiviral-mediated transfection of hTSHR in poorly differentiated thyroid carcinoma cell line

IntroductionDedifferentiated thyroid cancer is often incurable because it does not respond to radioiodine. This study aimed to investigate iodide uptake and the expressions of thyroid-specific molecules after the transfection of human thyrotropin receptor (hTSHR) gene in poorly differentiated follicular thyroid cancer cell line (FTC-133).MethodspGC-FU-hTSHR-GFP-lentivirus and pGC-FU-GFP-lentivirus were added into FTC-133 cells respectively. The parental cells were defined as the blank group. Cells transduced with pGC-FU-GFP and pGC-FU-hTSHR-GFP were defined as the control group and experimental group respectively. The efficiency of transfection was observed under a fluorescence microscope. ¹²⁵I uptake by FTC-133 was analyzed by measuring the radioactivity. Real time-PCR, western blotting and radioimmunoassay were applied to detect the expressions of mRNAs and proteins of Na⁺/I^? symporter (NIS), thyroid-stimulating hormone receptor (TSHR), thyroid peroxidase (TPO) and thyroglobulin (Tg) in FTC-133.ResultsThe green fluorescence was present in 80% of the transduced cells under fluorescence microscope. The iodine uptake of cells transduced with pGC-FU-TSHR-GFP was 3.3 times higher than that in the other two groups (P < 0.01). NIS, TSHR, TPO and Tg had been significantly up-regulated in the experimental group as compared to the control group (P < 0.01) and the blank group (P < 0.01).ConclusionThe hTSHR transfection in FTC-133 improved the expression of thyroid-specific molecules including TSHR, NIS, TPO and Tg and radioiodide uptake.     

Radiosynthesis and initial evaluation of 18F labeled nanocarrier composed of poly(L-lactic acid)-block-poly(sarcosine) amphiphilic polydepsipeptide

IntroductionWith the aim of developing radiotracers for in vivo positron emission tomography (PET) imaging of solid tumors based on the enhanced permeability and retention effect of nanocarriers, we have developed a polymer micelle named “Lactosome”, which is composed of the amphiphilic polydepsipeptide, poly(L-lactic acid)-block-poly(sarcosine). This paper describes and evaluates the initial evaluation of the ¹⁸F-labeled Lactosome as a novel contrast agent for the tumor PET imaging technique carried out.Methods¹⁸F-labeled Lactosomes were prepared by a film hydration method under sonication in water at 50 °C from a mixture of 4-[¹⁸F]fluoro-benzoyl poly-L-lactic acid (¹⁸F-BzPLLA₃₀) and the amphiphilic polydepsipeptide. For biodistribution studies, BALB/cA Jcl-nu/nu mice bearing HeLa cells in the femur region were used. We took both PET and near-infrared fluorescence (NIRF) images of tumor bearing mice after co-injection of ¹⁸F-labeled Lactosome and NIRF-labeled Lactosome.Results¹⁸F-labeled Lactosomes were prepared at good yields (222–420 MBq) and more than 99% of ¹⁸F-BzPLLA₃₀ was incorporated into ¹⁸F-labeled Lactosome. The radioactivity of ¹⁸F-labeled Lactosome was found to be stable and maintained at high level for up to 6 h after injection into the blood stream. Tumor uptake increased gradually after the injection. The uptake ratio of tumor/muscle was 2.7 at 6 h from the time of injection. Tumor PET imaging with ¹⁸F-labeled Lactosome was as capable as tumor NIRF imaging with NIRF-labeled Lactosome.ConclusionTumor PET imaging using Lactosome as a nanocarrier may be therefore a potential candidate for a facile and general solid tumor imaging technique.     

Kit-like 18F-labeling of RGD-19F-Arytrifluroborate in high yield and at extraordinarily high specific activity with preliminary in vivo tumor imaging

IntroductionPositron Emission Tomography (PET) is a rapidly expanding, cutting edge technology for preclinical evaluation, cancer diagnosis and staging, and patient management. A one-step aqueous ¹⁸F-labeling method, which can be applied to peptides to provide functional in vivo images, has been a long-standing challenge in PET imaging. Over the past few years, we have sought a rapid and mild radiolabeling method based on the aqueous radiosynthesis of in vivo stable aryltrifluoroborate (ArBF₃^?) conjugates. Recent access to production levels of ¹⁸F-Fluoride led to a fluorescent-¹⁸F-ArBF₃^? at unprecedentedly high specific activities of 15 Ci/μmol. However, extending this method to labeling peptides as imaging agents has not been explored.MethodsIn order to extend these results to a peptide of clinical interest in the context of production-level radiosynthesis, we applied this new technology for labeling RGD, measured its specific activity by standard curve analysis, and carried out a preliminary evaluation of its imaging properties.ResultsRGD was labeled in excellent radiochemical yields at exceptionally high specific activity (~ 14 Ci/μmol) (n = 3). Preliminary tumor-specific images corroborated by ex vivo biodistribution data with blocking controls show statistically significant albeit relatively low tumor uptake along with reasonably high tumor:blood ratios (n = 3).ConclusionsIsotope exchange on a clinically useful ¹⁸F-ArBF₃^? radiotracer leads to excellent radiochemical yields and exceptionally high specific activities while the anionic nature of the aryltrifluoroborate prosthetic results in very rapid clearance. Since rapid clearance of the radioactive tracer is generally desirable for tracer development, these results suggest new directions for varying linker arm composition to slightly retard clearance rather than enhancing it.Advances in Knowledge and Implications for patient CareThis work is the first to use production levels of ¹⁸F-activity to directly label RGD at specific activities that are an order of magnitude higher than most reports and thereby increases the distribution window for radiotracer production and delivery.     

Specific uptake of 99mTc-NC100692, an αvβ3-targeted imaging probe,in subcutaneous and orthotopic tumors

IntroductionThe α_vβ₃ integrin, which is expressed by angiogenic epithelium and some tumor cells, is an attractive target for the development of both imaging agents and therapeutics. While optimal implementation of α_vβ₃-targeted therapeutics will require a priori identification of the presence of the target, the clinical evaluation of these compounds has typically not included parallel studies with α_vβ₃-targeted diagnostics. This is at least partly due to the relatively limited availability of PET radiopharmaceuticals in comparison to those labeled with ^99mTc. In an effort to begin to address this limitation, we evaluated the tumor uptake of ^99mTc-NC100692, a cyclic RGD peptide that binds to α_vβ₃ with ~ 1-nM affinity, in an α_vβ₃-positive tumor model as well as its in vivo specificity.MethodsMicroSPECT imaging was used to assess the ability of cilengitide, a therapeutic with high affinity for α_vβ₃, to block and displace ^99mTc-NC100692 in an orthotopic U87 glioma tumor. The specificity of ^99mTc-NC100692 was quantitatively evaluated in mice bearing subcutaneous U87MG tumors, by comparison of the biodistribution of ^99mTc-NC100692 with that of the non-specific structural analogue ^99mTc-AH-111744 and by blocking uptake of ^99mTc-NC100692 with excess unlabeled NC100692.ResultsMicroSPECT imaging studies demonstrated that uptake of ^99mTc-NC100692 in the intracranial tumor model was both blocked and displaced by the α_vβ₃-targeted therapeutic cilengitide. Biodistribution studies provided quantitative confirmation of these imaging results. Tumor uptake of ^99mTc-NC100692 at 1 h post-injection was 2.8 ± 0.7% ID/g compared to 0.38 ± 0.1% ID/g for ^99mTc-AH-111744 (p < 0.001). Blocking ^99mTc-NC100692 uptake by pre-injecting the mice with excess unlabeled NC100692 reduced tumor uptake by approximately five-fold, to 0.68 ± 0.3% ID/g (p = 0.01).ConclusionThese results confirm that ^99mTc-NC100692 does, in fact, target the α_vβ₃ integrin and may, therefore, be useful in identifying patients prior to anti-α_vβ₃ therapy as well as monitoring the response of these patients to therapy.     

68Ga-NODAGA-VEGF121 for in vivo imaging of VEGF receptor expression

PurposeVascular endothelial growth factor (VEGF) is a crucial regulator of angiogenesis. In this study, we labeled VEGF₁₂₁ with ⁶⁸Ga using a hydrophilic chelating agent, NODAGA and evaluated the resulting ⁶⁸Ga-NODAGA-VEGF₁₂₁ for in vivo imaging of VEGF receptor (VEGFR) expression.MethodsNODAGA-VEGF₁₂₁ was prepared and its binding affinity for VEGFR2 was measured using ¹²⁵I-VEGF₁₂₁. ⁶⁸Ga-NODAGA-VEGF₁₂₁ was prepared by labeling NODAGA-VEGF₁₂₁ with ⁶⁸GaCl₃ followed by purification using a PD-10 column. Human aortic endothelial cell (HAEC) binding studies of ⁶⁸Ga-NODAGA-VEGF₁₂₁ were performed at 37 °C for 4 h. MicroPET imaging followed by biodistribution studies were performed in U87MG tumor-bearing mice injected with ⁶⁸Ga-NODAGA-VEGF₁₂₁. Immunofluorescence staining of the tumor tissues was performed to verify VEGFR2 expression.ResultsBinding affinity of NODAGA-VEGF₁₂₁ for VEGFR2 was found to be comparable to that of VEGF₁₂₁. ⁶⁸Ga-NODAGA-VEGF₁₂₁ was prepared in 47.8% yield with specific activity of 3.4 GBq/mg. ⁶⁸Ga-NODAGA-VEGF₁₂₁ was avidly taken up by HAECs with a time-dependent increase from 9.88 %ID at 1 h to 20.86 %ID at 4 h. MicroPET imaging of mice demonstrated high liver and spleen uptake with clear visualization of tumor at 1 h after injection. ROI analysis of tumors revealed 2.53 ± 0.11 %ID/g at 4 h after injection. In the blocking study, tumor uptake was inhibited by 29% at 4 h. Subsequent biodistribution studies demonstrated tumor uptake of 2.38 ± 0.15 %ID/g. Immunofluorescence staining of the tumor tissues displayed high level of VEGFR2 expression.ConclusionsThese results demonstrate that ⁶⁸Ga-NODAGA-VEGF₁₂₁ led to VEGFR-specific distribution in U87MG tumor-bearing mice. This study also suggests that altered physicochemical properties of VEGF₁₂₁ after radiolabeling may affect biodistribution of the radiolabeled VEGF₁₂₁.     

Biological evaluation of an ornithine-modified 99mTc-labeled RGD peptide as an angiogenesis imaging agent

IntroductionRadiolabeled RGD peptides that specifically target integrin α_νβ₃ have great potential in early tumor detection through noninvasive monitoring of tumor angiogenesis. Based on previous findings of our group on radiopeptides containing positively charged aminoacids, we developed a new cyclic cRGDfK derivative, c(RGDfK)-(Orn)₃-CGG. This new peptide availing the polar linker (Orn)₃ and the ^99mTc-chelating moiety CGG (Cys-Gly-Gly) is appropriately designed for ^99mTc-labeling, as well as consequent conjugation onto nanoparticles.MethodsA tumor imaging agent, c(RGDfK)-(Orn)₃-[CGG-^99mTc], is evaluated with regard to its radiochemical, radiobiological and imaging characteristics.ResultsThe complex c(RGDfK)-(Orn)₃-[CGG-^99mTc] was obtained in high radiochemical yield (> 98%) and was stable in vitro and ex vivo. It presented identical to the respective, fully analytically characterized ^185/187Re complex retention time in RP-HPLC. In contrary to other RGD derivatives, we showed that the new radiopeptide exhibits kidney uptake and urine excretion due to the ornithine linker. High tumor uptake (3.87 ± 0.48% ID/g at 60 min p.i.) was observed and was maintained relatively high even at 24 h p.i. (1.83 ± 0.05 % ID/g), thus providing well-defined scintigraphic imaging. Accumulation in other organs was negligible. Blocking experiments indicated target specificity for integrin receptors in U87MG glioblastoma cells.ConclusionDue to its relatively high tumor uptake, renal elimination and negligible abdominal localization, the new ^99mTc-RGD peptide is considered promising in the field of imaging α_νβ₃-positive tumors. However, the preparation of multifunctional SPECT/MRI contrast agents (RGD-conjugated nanoparticles) for dual modality imaging of integrin expressing tumors should be further investigated.     

Synthesis and evaluation of [11C]PyrATP-1, a novel radiotracer for PET imaging of glycogen synthase kinase-3β (GSK-3β)

IntroductionThe dysfunction of glycogen synthase kinase-3β (GSK-3β) has been implicated in a number of diseases, including Alzheimer’s disease. The ability to non-invasively quantify GSK-3β activity in vivo is therefore of critical importance, and this work is focused upon development of inhibitors of GSK-3β radiolabeled with carbon-11 to examine quantification of the enzyme using positron emission tomography (PET) imaging.Methods¹¹C PyrATP-1 was prepared from the corresponding desmethyl-piperazine precursor in an automated synthesis module. In vivo rodent and primate imaging studies were conducted on a Concorde MicroPET P4 scanner to evaluate imaging properties and in vitro autoradiography studies with rat brain samples were carried out to examine specific binding.Results2035 ± 518 MBq (55 ± 14 mCi) of [¹¹C]PyrATP-1 was obtained (1%–2% non-corrected radiochemical yield at end-of-synthesis based upon [¹¹C]CO₂) with high chemical (> 95%) and radiochemical (> 99%) purities, and good specific activities (143 ± 52 GBq/μmol (3874 ± 1424 Ci/mmol)), n = 5. In vivo microPET imaging studies revealed poor brain uptake in rodents and non-human primates. Pretreatment of rodents with cyclosporin A resulted in moderately increased brain uptake suggesting Pgp transporter involvement. Autoradiography demonstrated high levels of specific binding in areas of the rodent brain known to be rich in GSK-3β.Conclusion¹¹C PyrATP-1 is readily synthesized using standard carbon-11 radiochemistry. However the poor brain uptake in rodents and non-human primates indicates that the radiotracer is not suitable for the purposes of quantifying GSK-3β in neurological and psychiatric disorders.     

Evaluation of 18F-labeled BODIPY dye as potential PET agents for myocardial perfusion imaging

IntroductionDespite the great potential of positron emission tomography/computed tomography (PET/CT) in cardiovascular disease imaging, one of the major limitations is the availability of PET probes with desirable half-lives and reasonable cost. In this report, we hypothesized that lipophilic cationic BODIPY dye could be selectively accumulated in cardiac muscle, possibly for the development of novel PET myocardial perfusion imaging (MPI) probes.MethodsA ¹⁸F-labeled BODIPY dye ([¹⁸F]1) was synthesized efficiently through a fluoride exchange reaction catalyzed by the Lewis acid tin chloride (SnCl₄). The compound was first evaluated by a cellular uptake assay in vitro, followed by biodistribution and microPET imaging studies in vivo.Results[¹⁸F]1 was obtained in more than 90% labeling yield, with > 98% radiochemical purity. The HEK-293 cellular uptake assay showed that the preferential uptake of [¹⁸F]1 could be related to the cell membrane potential. The biodistribution data demonstrated high levels of [¹⁸F]1 accumulation in the heart. In the biodistribution study in mice, the radioactivity uptake in the heart, blood, liver and lung was 3.01 ± 0.44, 0.39 ± 0.09, 0.69 ± 0.07, 1.71 ± 0.27%ID/g, respectively, at 3 h post-injection (p.i.). The heart-to-lung and heart-to-liver ratios are 1.76 ± 0.14 and 4.37 ± 0.51 at 3 h p.i., respectively. Volume-of-interest analysis of the microPET images correlated well with the biodistribution studies in mice. The heart was clearly visualized in normal rats, with 0.72 ± 0.18, 0.69 ± 0.18, 0.67 ± 0.20 and 0.59 ± 0.17%ID/g uptake at 0.5, 1, 2 and 4 h p.i., respectively.Conclusions¹⁸F-labeled BODIPY dye showed good heart uptake and heart-to-blood and heart-to-lung contrast. A ¹⁸F-labeled BODIPY dyes may represent a new category of cationic PET agents for myocardial perfusion imaging.     

68Ga-DOTA-NGR as a novel molecular probe for APN-positive tumor imaging using MicroPET

Aminopeptidase N (APN) is selectively expressed on many tumors and the endothelium of tumor neovasculature, and may serve as a promising target for cancer diagnosis and therapy. Asparagine–glycine–arginine (NGR) peptides have been shown to bind specifically to the APN receptor and have served as vehicles for the delivery of various therapeutic drugs in previous studies. The purpose of this study was to synthesize and evaluate the efficacy of a ⁶⁸Ga-labeled NGR peptide as a new molecular probe that binds to APN.MethodsNGR peptide was conjugated with 1,4,7,10-tetraazacyclododecane-N,N’,N”,N”’-tetraacetic acid (DOTA) and labeled with ⁶⁸Ga at 95 °C for 10 min. In vitro uptake and binding analysis was performed with A549 and MDA-MB231 cells. Biodistribution of ⁶⁸Ga-DOTA-NGR was determined in normal mice by dissection method. ⁶⁸Ga-DOTA-NGR PET was performed in A549 and MDA-MB231 xenografts, and included dynamic and static imaging. APN expression in tumors and new vasculatures was analyzed by immunohistochemistry.ResultsThe radiochemical purity of ⁶⁸Ga-DOTA-NGR was 98.0% ± 1.4% with a specific activity of about 17.49 MBq/nmol. The uptake of ⁶⁸Ga-DOTA-NGR in A549 cells increased with longer incubation times, and could be blocked by cold DOTA-NGR, while no specific uptake was found in MDA-MB231 cells. In vivo biodistribution studies showed that ⁶⁸Ga-DOTA-NGR was mainly excreted from the kidney, and rapidly cleared from blood and nonspecific organs. MicroPET imaging showed that high focal accumulation had occurred in the tumor site at 1 h post-injection (pi) in A549 tumor xenografts. A significant reduction of tumor uptake was observed following coinjection with a blocking dose of DOTA-NGR, whereas only mild uptake was found in MDA-MB231 tumor xenografts. Tumor uptake, measured as the tumor/lung ratio, increased with time peaking at 12.58 ± 1.26 at 1.5 h pi. Immunohistochemical staining confirmed that APN was overexpressed on A549 cells and neovasculature.Conclusions⁶⁸Ga-DOTA-NGR was easily synthesized and showed favorable biodistribution and kinetics. ⁶⁸Ga-DOTA-NGR could also specifically bind to the APN receptor in vitro and in vivo, and might be a potential molecular probe for the noninvasive detection of APN-positive tumors and neovasculature.     

Synthesis and evaluation of 2-amino-5-(4-[18F]fluorophenyl)pent-4-ynoic acid ([18F]FPhPA): A novel 18F-labeled amino acid for oncologic PET imaging

Introduction¹⁸ F-labeled amino acids are important PET radiotracers for molecular imaging of cancer. This study describes synthesis and radiopharmacological evaluation of 2-amino-5-(4-[¹⁸ F]fluorophenyl)pent-4-ynoic acid ([¹⁸ F]FPhPA) as a novel amino acid radiotracer for oncologic imaging.Methods¹⁸ F]FPhPA was prepared using Pd-mediated Sonogashira cross-coupling reaction between 4-[¹⁸ F]fluoroiodobenzene ([¹⁸ F]FIB) and propargylglycine. The radiopharmacological profile of [¹⁸ F]FPhPA was evaluated in comparison with O-(2-[¹⁸ F]fluoroethyl)-L-tyrosine ([¹⁸ F]FET) using the murine breast cancer cell line EMT6 involving cellular uptake studies, radiotracer uptake competitive inhibition experiments and small animal PET imaging.Results¹⁸ F]FPhPA was prepared in 42 ± 10% decay-corrected radiochemical yield with high radiochemical purity >95% after semi-preparative HPLC purification. Cellular uptake of L-[¹⁸ F]FPhPA reached a maximum of 58 ± 14 % radioactivity/mg protein at 90 min. Lower uptake was observed for racemic and D-[¹⁸ F]FPhPA.Radiotracer uptake inhibition studies by synthetic and naturally occurring amino acids suggested that Na⁺-dependent system ASC, especially ASCT2, and Na⁺-independent system L are important amino acid transporters for [¹⁸ F]FPhPA uptake into EMT6 cells. Small animal PET studies demonstrated similar high tumor uptake of [¹⁸ F]FPhPA in EMT6 tumor-bearing mice compared to [¹⁸ F]FET reaching a maximum standardized uptake value (SUV) of 1.35 after 60 min p.i.. Muscle uptake of [¹⁸ F]FPhPA was higher (SUV_30min = 0.65) compared to [¹⁸ F]FET (SUV_30min = 0.40), whereas [¹⁸ F]FPhPA showed a more rapid uptake and clearance from the brain compared to [¹⁸ F]FET.ConclusionL-[¹⁸ F]FPhPA is the first ¹⁸ F-labeled amino acid prepared through Pd-mediated cross-coupling reaction.Advances in Knowledge and Implications for patient CareL-[¹⁸ F]FPhPA displayed promising properties as a novel amino acid radiotracer for molecular imaging of system ASC and system L amino acid transporters in cancer.     

Radiosynthesis and ex vivo evaluation of [11C-carbonyl]carbamate- and urea-based monoacylglycerol lipase inhibitors

IntroductionMonoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) are the two primary enzymes that regulate the tone of endocannabinoid signaling. Although new PET radiotracers have been discovered for imaging FAAH in vivo, no such radiotracer exists for imaging MAGL. Here we report the radiosynthesis of five candidate MAGL radiotracers and their ex vivo evaluations in mice and rats.MethodsCandidate carbamate and urea MAGL inhibitors were radiolabeled at the carbonyl position by [¹¹C]CO₂ fixation. Radiotracers were administered (tail-vein injection) to rodents and brain uptake of radioactivity measured at early and late time points ex vivo. Specificity of uptake was explored by pretreatment with unlabeled inhibitors (2 mg/kg, ip) 30 min prior to radiotracer administration.ResultsAll five candidate MAGL radiotracers were prepared in high specific activity (> 65 GBq/μmol) and radiochemical purity (> 98%). Moderate brain uptake (0.2–0.8 SUV) was observed for each candidate while pretreatment did not reduce uptake for four of the five tested. For two candidates ([¹¹C]12 and [¹¹C]14), high retention of radioactivity was observed in the blood (ca. 10 and 4 SUV at 40 min) which was blocked by pretreatment with unlabeled inhibitors. The most promising candidate, [¹¹C]18, demonstrated moderate brain uptake (ca. 0.8 SUV) which showed circa 50% blockade by pretreatment with unlabeled 18.ConclusionOne putative and four reported potent and selective MAGL inhibitors have been radiolabeled via [¹¹C]CO₂ fixation as radiotracers for this enzyme. Despite the promising in vitro pharmacological profile, none of the five candidate radiotracers exhibited in vivo behavior suitable for PET neuroimaging.     

[18F]Altanserin and small animal PET: Impact of multidrug efflux transporters on ligand brain uptake and subsequent quantification of 5-HT2A receptor densities in the rat brain

IntroductionThe selective 5-hydroxytryptamine type 2a receptor (5-HT_2AR) radiotracer [¹⁸F]altanserin is a promising ligand for in vivo brain imaging in rodents. However, [¹⁸F]altanserin is a substrate of P-glycoprotein (P-gp) in rats. Its applicability might therefore be constrained by both a differential expression of P-gp under pathological conditions, e.g. epilepsy, and its relatively low cerebral uptake. The aim of the present study was therefore twofold: (i) to investigate whether inhibition of multidrug transporters (MDT) is suitable to enhance the cerebral uptake of [¹⁸F]altanserin in vivo and (ii) to test different pharmacokinetic, particularly reference tissue-based models for exact quantification of 5-HT_2AR densities in the rat brain.MethodsEighteen Sprague-Dawley rats, either treated with the MDT inhibitor cyclosporine A (CsA, 50 mg/kg, n = 8) or vehicle (n = 10) underwent 180-min PET scans with arterial blood sampling. Kinetic analyses of tissue time–activity curves (TACs) were performed to validate invasive and non-invasive pharmacokinetic models.ResultsCsA application lead to a two- to threefold increase of [¹⁸F]altanserin uptake in different brain regions and showed a trend toward higher binding potentials (BP_ND) of the radioligand.ConclusionsMDT inhibition led to an increased cerebral uptake of [¹⁸F]altanserin but did not improve the reliability of BP_ND as a non-invasive estimate of 5-HT_2AR. This finding is most probable caused by the heterogeneous distribution of P-gp in the rat brain and its incomplete blockade in the reference region (cerebellum). Differential MDT expressions in experimental animal models or pathological conditions are therefore likely to influence the applicability of imaging protocols and have to be carefully evaluated.     

Synthesis and preclinical evaluation of the radiolabeled P-glycoprotein inhibitor [11C]MC113

ObjectivesWith the aim to develop a PET tracer to visualize P-glycoprotein (Pgp) expression levels in different organs, the Pgp inhibitor MC113 was labeled with ¹¹C and evaluated using small-animal PET.Methods[¹¹C]MC113 was synthesized by reaction of O-desmethyl MC113 with [¹¹C]methyl triflate. Small-animal PET was performed with [¹¹C]MC113 in FVB wild-type and Mdr1a/b^(-/-) mice (n = 3 per group) and in a mouse model of high (EMT6Ar1.0) and low (EMT6) Pgp expressing tumor grafts (n = 5). In the tumor model, PET scans were performed before and after administration of the reference Pgp inhibitor tariquidar (15 mg/kg).ResultsBrain uptake of [¹¹C]MC113, expressed as area under the time-activity curve from time 0 to 60 min (AUC_0-60), was moderately but not significantly increased in Mdr1a/b^(-/-) compared with wild-type mice (mean ± SD AUC_0-60, Mdr1a/b^(-/-): 88 ± 7 min, wild-type: 62 ± 6 min, P = 0.100, Mann Whitney test). In the tumor model, AUC_0-60 values were not significantly different between EMT6Ar1.0 and EMT6 tumors. Neither in brain nor in tumors was activity concentration significantly changed in response to tariquidar administration. Half-maximum effect concentrations (IC₅₀) for inhibition of Pgp-mediated rhodamine 123 efflux from CCRFvcr1000 cells were 375 ± 60 nM for MC113 versus 8.5 ± 2.5 nM for tariquidar.Conclusion[¹¹C]MC113 showed higher brain uptake in mice than previously described Pgp PET tracers, suggesting that [¹¹C]MC113 was only to a low extent effluxed by Pgp. However, [¹¹C]MC113 was found unsuitable to visualize Pgp expression levels presumably due to insufficiently high Pgp binding affinity of MC113 in relation to Pgp densities in brain and tumors.     

[99mTc]O2-AMD3100 as a SPECT tracer for CXCR4 receptor imaging

PurposeCXCR4 plays an important role in HIV infection, tumor progression, neurogenesis, and inflammation. In-vivo imaging of CXCR4 could provide more insight in the role of this receptor in health and disease. The aim of this study was to investigate [^99mTc]O₂-AMD3100 as a potential SPECT tracer for imaging of CXCR4.MethodAMD3100 was labelled with [^99mTc]pertechnetate. A cysteine challenge assay was performed to test the tracer stability. Heterologous and homologous receptor binding assay and internalization assay were performed in CXCR4 expressing Jurkat-T cells. Ex vivo biodistribution was studied in healthy mice at 30, 60, and 120 min after tracer injection. Tumor uptake of the tracer was determined by microSPECT imaging in nude mice xenografted with human PC-3 prostate tumor. Specificity of tracer uptake was determined by blocking studies using an excess of unlabelled AMD3100.ResultsAMD3100 was labelled with technetium-99 m with a radiochemical yield of > 98%. The tracer was stable in PBS and mouse plasma for at least 6 h at 37 °C. Heterologous and homologous binding assays with AMD3100 showed IC₅₀ values of 240 ± 10 μM, and 92 ± 5 μM for [¹²⁵I]SDF-1α and [^99mTc]O₂-AMD3100 respectively, with negligible receptor internalisation. The tracer showed high uptake in liver, lungs, spleen, thymus, intestine and bone. Blocking dose of AMD3100.8HCl (20 mg/kg) decreased the uptake in these organs (p < 0.05). [^99mTc]O₂-AMD3100 showed specific tumor accumulation in mice bearing PC-3 xenografts model. Time activity curves (TAC) in AMD3100 pre-treated animals tracer showed 1.7 times less tumor uptake as compared to control animals (p < 0.05).Conclusion[^99mTc]O₂-AMD3100 is readily labelled, is stable in plasma and displays a favourable binding affinity for the CXCR4 receptors. [^99mTc O₂-AMD3100 shows specific binding in organs with high CXCR4 expression and in CXCR4 positive tumors. These results justify further evaluation of this radiopharmaceutical as a potential biomarker for the non-invasive imaging of CXCR4 receptors.