Effect of co-ligands on chemical and biological properties of 99mTc(III) complexes [99mTc(L)(CDO)(CDOH)2BMe] (L = Cl,F, SCN and N3; CDOH2 = cyclohexanedione dioxime)

Introduction^99mTc-Teboroxime ([^99mTcCl(CDO)(CDOH)₂BMe]) is a member of the BATO (boronic acid adducts of technetium dioximes) class of ^99mTc(III) complexes. This study sought to explore the impact of co-ligands on solution stability, heart uptake and myocardial retention of [^99mTc(L)(CDO)(CDOH)₂BMe] (^99mTc-Teboroxime: L = Cl; ^99mTc-Teboroxime(F): L = F; ^99mTc-Teboroxime(SCN): L = SCN; and ^99mTc-Teboroxime(N₃): L = N₃).MethodsRadiotracers ^99mTc-Teboroxime(L) (L = F, SCN and N₃) were prepared by reacting ^99mTc-Teboroxime with NaF, NaSCN and NaN₃, respectively. Biodistribution and imaging studies were carried out in Sprague–Dawley rats. Image quantification was performed to compare their heart retention and liver clearance kinetics.ResultsComplexes ^99mTc-Teboroxime(L) (L = F, SCN and N₃) were prepared in high yield with high radiochemical purity. All new radiotracers were stable for > 6 h in the kit matrix. In its HPLC chromatogram, ^99mTc-Teboroxime showed one peak at ~ 15.5 min, which was shorter than that of ^99mTc-Teboroxime(F) (~ 16.4 min). There were two peaks for ^99mTc-Teboroxime(SCN) at 16.5 and 18.3 min. ^99mTc-Teboroxime(N₃) appeared as a single peak at 18.4 min. Their heart retention and liver clearance curves were best fitted to the bi-exponential decay function. The half-times of fast/slow components were 1.6 ± 0.4/60.7 ± 8.9 min for ^99mTc-Teboroxime, 0.8 ± 0.2/101.7 ± 20.7 min for ^99mTc-Teboroxime(F), 1.2 ± 0.3/84.8 ± 16.6 min for ^99mTc-Teboroxime(SCN), and 2.9 ± 0.9/51.6 ± 5.0 min for ^99mTc-Teboroxime(N₃). The 2-min heart uptake followed the order of ^99mTc-Teboroxime (3.00 ± 0.37%ID/g) > ^99mTc-Teboroxime(N₃) (2.66 ± 0.01 %ID/g) ≈ ^99mTc-Sestamibi (2.55 ± 0.46 %ID/g) > ^99mTcN-MPO (2.38 ± 0.15 %ID/g). ^99mTc-Teboroxime remains the best in first-pass extraction. The best image acquisition window is 0–5 min for ^99mTc-Teboroximine and 0–15 min for ^99mTc-Teboroximine(N₃).ConclusionCo-ligands had significant impact on the heart uptake and myocardial retention of complexes [^99mTc(L)(CDO)(CDOH)₂BMe] (L = Cl, F, SCN and N₃). Future studies should be directed towards minimizing the liver uptake and radioactivity accumulation in the blood vessels while maintaining their high heart uptake.     

Comparison of 99mTc-TRODAT-1 SPECT and 18 F-AV-133 PET imaging in healthy controls and Parkinson’s disease patients

^99mTc-TRODAT-1 is the first clinical routine ^99mTc radiopharmaceutical to evaluate dopamine neurons loss in Parkinson's disease (PD). ¹⁸ F-AV-133 is a novel PET radiotracer targeting the vesicular monoamine transporter type 2 (VMAT2) to detect monoaminergic terminal reduction in PD patients. The aim of this study is to compare both images in the same health control (HC) and PD subjects.MethodsEighteen subjects (8 HC and 10 PD) were recruited for ^99mTc-TRODAT-1 SPECT, ¹⁸ F-AV-133 PET and MRI scans within two weeks. The SPECT images were performed at 4-h post-injection for 45 min, and the PET images were performed at 90 min post-injection for 10 min. Each PET and SPECT image was normalized into Montreal Neurological Institute template aided from individual MRI for comparison. For regional analysis, volume of interest (VOIs) of bilateral caudate nuclei, anterior, posterior putamen and occipital cortex (as reference region) were delineated from the normalized MRI. The specific uptake ratio (SUR) was calculated as (regional mean counts/reference mean counts − 1). The nonparametric Mann–Whitney U test was used to evaluate the power of differentiating control from PD subjects for both image modalities. The correlations of the SURs to the clinical parameters were examined. For voxelwise analysis, two-sample t-test for group comparison between HC and PD was computed in both image modalities.ResultsThe SURs of caudate nucleus and putamen correlated well between two image modalities (r = 0.81, p < 0.001), and showed significant different between HC and PD subjects. Of note, the ¹⁸ F-AV-133 SUR displayed a better correlation to PD clinical laterality index as compared to ^99mTc-TRODAT-1 (r = 0.73 vs. r = 0.33). Voxelwise analysis showed more lesions for PD subjects from ¹⁸ F-AV-133 image as compared to ^99mTc-TRODAT-1 especially at the substantia nigra region.Conclusion¹⁸ F-AV-133 PET demonstrated similar performance in differentiation PD from control, and a better correlation to clinical characteristics than that of ^99mTc-TRODAT-1 SPECT. ¹⁸ F-AV-133 PET also showed additional information in substantia nigra integrity in PD subjects by voxelwise analysis. Collectively, ¹⁸ F-AV-133 could be a promising and better tracer for clinical use to detect monoaminergic terminal reduction in PD patients.     

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.     

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.     

[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.     

Imaging of rat cerebral ischemia-reperfusion injury using99mTc-labeled duramycin

ObjectivesPrompt identification of necrosis and apoptosis in the infarct core and penumbra region is critical in acute stroke for delineating the underlying ischemic/reperfusion molecular pathologic events and defining therapeutic alternatives. The objective of this study was to investigate the capability of ^99mTc-labeled duramycin in detecting ischemia-reperfusion injury in rat brain after middle cerebral artery (MCA) occlusion.MethodsIschemic cerebral injury was induced in ten rats by vascular insertion of a nylon suture in the left MCA for 3 hr followed by 21–24 hr reperfusion. After i.v. injection of ^99mTc-duramycin (1.0-3.5 mCi), dynamic cerebral images were acquired for 1 hr in six rats using a small-animal SPECT imager. Four other rats were imaged at 2 hr post-injection. Ex vivo images were obtained by autoradiography after sacrifice. Histologic analyses were performed to assess cerebral infarction and apoptosis.ResultsSPECT images showed that ^99mTc-duramycin uptake in the left cerebral hemisphere was significantly higher than that in the right at 1 and 2 hr post-injection. The level of radioactive uptake in the ischemic brain varied based on ischemic severity. The average ratio of left cerebral hot-spot uptake to right hemisphere radioactivity, as determined by computerized ROI analysis, was 4.92 ± 0.79. Fractional washout at 1 hr was 38.2 ± 4.5% of peak activity for left cerebral hot-spot areas and 80.9 ± 2.0% for remote control areas (P < 0.001). Based on triphenyltetrazolium chloride staining and autoradiograph image data, the hotspot uptake may be associated primarily with the ischemic penumbra, in which high apoptotic activity was observed by cleaved caspase-3 immunocytochemical staining.Conclusions^99mTc-duramycin SPECT imaging may be useful for detecting and quantifying ongoing apoptotic neuronal cell loss induced by ischemia-reperfusion injury.     

Design and biological evaluation of 99mTc-N2S2-Tat(49–57)-c(RGDyK): A hybrid radiopharmaceutical for tumors expressing α(v)β(3) integrins

The α(ν)β(3) integrin is over-expressed in the tumor neovasculature and the tumor cells of glioblastomas. The HIV Tat-derived peptide has been used to deliver various cargos into cells. The aim of this research was to synthesize and assess the in vitro and in vivo uptake of ^99mTc-N₂S₂-Tat(49–57)-c(RGDyK) (^99mTc-Tat-RGD) in α(ν)β(3) integrin positive cancer cells and compare it to that of a conventional ^99mTc-RGD peptide (^99mTc-EDDA/HYNIC-E-[c(RGDfK)]₂). Methods: The c(RGDyK) peptide was conjugated to a maleimidopropionyl (MP) moiety through Lys, and the MP group was used as the branch position to form a thioether with the Cys¹² side chain of the Tat(49–57)-spacer-N₂S₂ peptide. ^99mTc-Tat-RGD was prepared, and stability studies were carried out by size exclusion HPLC analyses in human serum. The in vitro affinity for α(v)β(3) integrin was determined by a competitive binding assay. In vitro internalization was determined using glioblastoma C6 cells. Biodistribution studies were accomplished in athymic mice with C6 induced tumors that had blocked and unblocked receptors. Images were obtained using a micro-SPECT/CT. Results: ^99mTc-Tat-RGD was obtained with a radiochemical purity higher than 95%, as determined by radio-HPLC and ITLC-SG analyses. Protein binding was 15.7% for ^99mTc-Tat-RGD and 5.6% for ^99mTc-RGD. The IC₅₀ values were 6.7 nM (^99mTc-Tat-RGD) and 4.6 nM (^99mTc-RGD). Internalization in C6 cells was higher in ^99mTc-Tat-RGD (37.5%) than in ^99mTc-RGD (10%). Biodistribution studies and in vivo micro-SPECT/CT images in mice showed higher tumor uptake for ^99mTc-Tat-RGD (6.98% ± 1.34% ID/g at 3 h) than that of ^99mTc-RGD (3.72% ± 0.52% ID/g at 3 h) with specific recognition for α(v)β(3) integrins. Conclusions: Because of the significant cell internalization (Auger and internal conversion electrons) and specific recognition for α(v)β(3) integrins, the hybrid ^99mTc-N₂S₂-Tat(49–57)-c(RGDyK) radiopharmaceutical is potentially useful for the imaging and possible therapy of tumors expressing α(v)β(3) integrins.     

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.     

Inflammation imaging of atherosclerosis in Apo-E-deficient mice using a 99mTc-labeled dual-domain cytokine ligand

Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) play a critical role in initiating and accelerating atherosclerosis. This study evaluated the imaging properties of ^99mTc-TNFR2-Fc-IL-1RA (^99mTc-TFI), a dual-domain cytokine radioligand that targets TNF-α and IL-1β pathways, in assessing atherosclerosis development in apolipoprotein-E-deficient (ApoE^−/−) mice.MethodsThe feasibility and specificity of detecting atherosclerosis with ^99mTc-TFI SPECT imaging were investigated in ApoE^−/− and ApoE^+/+ mice. Fifty-four ApoE^−/− mice were fed either an atherogenic diet (AGD) or a normal diet (ND) beginning at 5 weeks of age. Eighteen Apo-E wild-type (ApoE^+/+) mice were fed an ND. Two groups of ApoE^−/− mice (n = 12 each group) on AGD and ND were imaged three times with ^99mTc-TFI and a high-resolution SPECT system at 20–25, 30–40, and 48–52 weeks to study the evolution of atherosclerotic plaque.ResultsFocal radioactive accumulations in the aortic arch region were observed in the ApoE^−/− mice (n = 12) on AGD but not in the ApoE^+/+ mice on ND (n = 10). Apo-E^−/− mice on ND (n = 11) exhibited lower radioactive uptake than ApoE^−/− mice on AGD (P < 0.05). Co-injection of an excess of cold ligand with ^99mTc-TFI resulted in significant reduction of ^99mTc-TFI uptake in the ApoE^−/− mice on AGD. Longitudinal studies showed that ^99mTc-TFI uptake in the aortas of ApoE^−/− mice progressively increased from 20 to 48 weeks. Real-time PCR assays demonstrated that atherosclerotic aortas expressed significantly higher IL-1β and TNF-α than the aortas from wild-type controls.ConclusionsAtherosclerotic plaques were detected by ^99mTc-TFI imaging in ApoE^−/− mice. ^99mTc-TFI is promising for specific detection of inflammatory response in atherosclerotic plaques.     

Synthesis and in vivo preclinical evaluation of an 18F labeled uPA inhibitor as a potential PET imaging agent

IntroductionThe urokinase plasminogen activator (uPA) system is a proteolytic cascade involved in tumor invasion and metastasis. uPA and its inhibitor PAI-1 are described as biomarkers for breast cancer with the highest level of evidence. The present study describes the synthesis and first in vivo application of an activity based uPA PET probe.MethodsBased on the design of a small irreversible and selective uPA inhibitor we developed an ¹⁸F-labeled activity based probe for uPA imaging. Human uPA expressing MDA-MB-231-luc2-GFP cells were inoculated in the mammary fat pads of nude mice and treated with the probe once tumors reached a volume of 150 mm³. Scans were performed at 0.25, 0.75, 1.5, 4 and 6 h post injection. To evaluate tumor uptake in vivo and ex vivo data were gathered. Biodistribution data of the organs and tissues of interest were collected at all time points. Due to a relatively low tumor uptake, probe stability was further evaluated.ResultsThe uPA targeting PET tracer was produced in high purity and with good specific radioactivity. In vivo PET data showed a maximum tumor uptake of 2,51 ± 0,32 %ID/g at 4 h p.i. A significant correlation between in vivo and ex vivo tumor uptake calculation was found (R = 0.75; p < 0.01). Due to a high blood signal at all time points, probe stability was further examined revealing high plasma protein binding and low plasma stability.ConclusionsIn vivo and ex vivo results clearly demonstrate that uPA expressing tumors can be detected with non-invasive PET imaging. Stability tests suggest that further optimization is needed to provide a better tumor-to-background contrast.     

64Cu-Labeled tetraiodothyroacetic acid-conjugated liposomes for PET imaging of tumor angiogenesis

IntroductionWe synthesized and evaluated ⁶⁴Cu-labeled tetraiodothyroacetic acid (tetrac)-conjugated liposomes for PET imaging of tumor angiogenesis, because tetrac inhibits angiogenesis via integrin α_Vβ₃.MethodsTetrac-PEG-DSPE and DOTA-PEG-DSPE were synthesized and formulated with other lipids into liposomes. The resulting tetrac/DOTA-liposomes were labeled with ⁶⁴Cu at 40 °C for 1 h and purified using a PD-10 column. ⁶⁴Cu-DOTA-liposomes were also prepared for comparison. Human aortic endothelial cell (HAEC) binding studies were performed by incubating the liposomes with the cells at 37 °C. MicroPET imaging followed by tissue distribution study was carried out using U87MG tumor-bearing mice injected with tetrac/⁶⁴Cu-DOTA-liposomes or ⁶⁴Cu-DOTA-liposomes.ResultsHAEC binding studies exhibited that tetrac/⁶⁴Cu-DOTA-liposomes were avidly taken up by the cells from 1.02 %ID at 1 h to 11.89 %ID at 24 h, while ⁶⁴Cu-DOTA-liposomes had low uptake from 0.47 %ID at 1 h to 1.57 %ID at 24 h. MicroPET imaging of mice injected with tetrac/⁶⁴Cu-DOTA-liposomes showed high radioactivity accumulation in the liver and spleen. ROI analysis of the tumor images revealed 1.93 ± 0.12 %ID/g at 1 h and 2.70 ± 0.36 %ID/g at 22 h. In contrast, tumor ROI analysis of ⁶⁴Cu-DOTA-liposomes revealed 0.54 ± 0.08 %ID/g at 1 h and 0.52 ± 0.09 %ID/g at 22 h. Tissue distribution studies confirmed that the tumor uptakes of tetrac/⁶⁴Cu-DOTA-liposomes and ⁶⁴Cu-DOTA-liposomes were 1.75 ± 0.03 %ID/g and 0.36 ± 0.01 %ID/g at 22 h, respectively.ConclusionThese results demonstrate that tetrac/⁶⁴Cu-DOTA-liposomes have significantly enhanced tumor uptake compared to ⁶⁴Cu-DOTA-liposomes due to tetrac conjugation. Further studies are warranted to reduce the liver and spleen uptake of tetrac/⁶⁴Cu-DOTA-liposomes.     

Synthesis,radiolabeling and bioevaluation of a novel arylpiperazine derivative containing triazole as a 5-HT1A receptor imaging agents

IntroductionIt has been recognized that serotonin plays a main role in various pathological conditions such as anxiety, depression, aggressiveness, schizophrenia, suicidal behavior, panic and autism. 1-(2-Methoxyphenyl) piperazine pharmacophore, a fragment of the true 5-HT_1A antagonist WAY100635, is found in numerous selective 5-HT_1A imaging agents. In this paper, we have reported the synthesis of a novel derivative of 1-(2-methoxyphenyl) piperazine that is labeled with ^99mTc (CO)₃ via click chemistry.MethodsThe bidentate alkyne, propargylglycine was reacted with phenyl piperazine triazole derivative in the presence of a catalytic amount of Cu (I) to form tridentate ligand. The ligand was radiolabeled with the precursor [^99mTc] [(H₂O)₃ (CO)₃]⁺ and characterized by HPLC. The bioevaluation of radio labeled ligand was carried out in rats.ResultsTriazole complex was labeled by ^99mTc-tricarbonyl and its radiochemical yield was more than > 95% which was determined by HPLC. In vivo stability studies in human serum albumin show a 93% ratio of complex after a 24 h period. The calculated partition coefficient (logP) was 0.34 ± 0.02. Receptor binding assays indicated about 70% specific binding of radioligand to 5-HT_1A receptors. Biodistribution studies have shown brain hippocampus uptake of 0.40 ± 0.08 %ID/g at 30 min post injection.ConclusionsResults indicate that this ^99mTc-tricabonyl-arylpiperazine derivative has specific binding to 5-HT_1A receptors and presented suitable characters for its use as a CNS imaging agent.     

Pharmacokinetic Alteration of 99mTc-MAG3 using Serum Protein Binding Displacement Method

IntroductionWhen a radiopharmaceutical is simultaneously administered with a medicine that has high affinity for the same plasma protein, the radiopharmaceutical is released at higher concentrations in blood, leading to enhanced transfer into target tissues. This is known as the serum protein binding displacement method. In this study, we investigated the pharmacokinetic alteration of technetium-99m-labeled mercaptoacetylglycylglycylglycine (^99mTc-MAG3) using the serum protein binding displacement method.MethodsRat and human serum protein binding rates of ^99mTc-MAG3 were measured by ultrafiltration with or without displacers of human serum albumin (HSA) binding sites I and II (200 μM and 400 μM loading). Male Wistar rats were injected with ^99mTc-MAG3 (740 kBq/0.3 mL saline) via the tail vein, and biodistribution was assessed at 2, 5, 10 and 15 min. Dynamic whole-body images were obtained for ^99mTc-MAG3 (11.1 MBq/0.3 mL saline)-injected rats, with or without HSA displacers.Results^99mTc-MAG3 strongly bound to HSA (87.37%±2.13%). Using HSA site I displacers, the free fraction of ^99mTc-MAG3 increased significantly (1.20 to 1.47 times) when compared with controls. For biodistribution and imaging, rapid blood clearance was observed with bucolome (BCL) loading, which is an HSA site I displacer. With BCL loading, peak times for rat renograms were respectively shifted from 240 s to 110 s, and from 170 s to 120 s.ConclusionsWe found that ^99mTc-MAG3 bound to the HSA binding site I. It was confirmed that pharmacokinetic distribution of ^99mTc-MAG3 is altered by presence of BCL, which leads to increases in the free fraction of ^99mTc-MAG3, and BCL produced rapid blood clearance and fast peak times on rat renograms. The serum protein binding displacement method using ^99mTc-MAG3 and BCL, a safe displacer for humans, may be applicable to clinical study and lead to better diagnostic images with shorter waiting times and lower radiation doses for patients.     

Imaging the myocardium at risk with 99mTc-lactadherin administered after reperfusion in a porcine model

IntroductionPhosphatidylserine is translocated from the inner to the outer leaflet of the plasma membrane in the early stages of apoptosis and necrosis and in reversibly injured cells. In rabbit hearts, ischemia followed by reperfusion results in exposure of phosphatidylserine on myocytes unaffected by apoptosis or necrosis. Lactadherin was recently introduced as a highly sensitive phosphatidylserine ligand. We hypothesized that ischemic myocardial cell damage can be identified by radio-labeled lactadherin and that the ischemic area at risk (AAR) can be visualized retrospectively after reperfusion.MethodsLeft anterior descending coronary artery in pigs was occluded for 20 minutes, 45 minutes or 45 minutes preceded by ischemic preconditioning. In all three groups, ^99mTc-lactadherin was injected intravenously 30 minutes after reperfusion. The AAR was demarcated by Evans blue and the infarct size by 2,3,5,-triphenyltetrazodium chloride staining.ResultsThe regional myocardial uptake of ^99mTc-lactadherin closely correlated with the AAR (r = .83, P = .001). The area of ^99mTc–lactadherin uptake was unaltered by a shorter duration of ischemia and ischemic preconditioning (P = .23) despite significantly different infarct development (P = .001).ConclusionThe results suggest that ^99mTc–lactadherin can be used as a sensitive marker for AAR imaging when injected 30 minutes after reperfusion following acute ischemia.     

Characterization of (99m)Tc-labeled cytokine ligands for inflammation imaging via TNF and IL-1 pathways

IntroductionTNFR2-Fc and IL-1ra-Fc are recombinant cytokine ligands that target TNF and IL-1. TNFR2-Fc-IL-1ra, a dual-domain agent that incorporates both ligands, allows bifunctional binding of IL-1 receptors and TNF. This study was designed to characterize ^99mTc-labeled forms of these ligands, ^99mTc-IL-1ra-Fc (IF), ^99mTc-TNFR2-Fc (TF), and ^99mTc-TNFR2-Fc-IL-1ra (TFI), for inflammation imaging.MethodsThe cytokine ligands were labeled with ^99mTc by a direct approach via 2-iminothiolane (2-IT) reduction at various 2-IT/protein molar ratios. In vivo inflammation targeting studies were carried out in a mouse ear edema model created by topical application of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the right ear of ICR mice.ResultsRadiolabeling yields increased with increasing amounts of 2-IT. When the 2-IT/protein ratio reached 1000, the radiolabeling yield was greater than 90% without significant colloid production. TPA-treated ears showed high radioligand uptake, which was clearly detected by SPECT and autoradiographic imaging. The activities (%ID/g) in the inflamed and control ears at 3 h after injection were 2.76 ± 0.20 vs. 0.69 ± 0.12 for IF, 5.86 ± 0.40 vs. 2.86 ± 0.61 for TF, and 7.61 ± 0.86 vs. 1.99 ± 0.31 for TFI (P < 0.05 vs. controls). TFI showed significantly higher uptake in the inflamed ears compared to TF and IF (P < 0.05). Blocking study results indicated specificity of radioligand binding with decreased radioactive uptake in the inflamed ears. Western blotting and ELISA analysis further confirmed a high expression of IL-1β and TNF-α in the inflamed ears.Conclusions^99mTc-labeled cytokine ligands are a promising approach for detecting and understanding the inflammatory process. TFI may be more useful than the single-domain ligands for noninvasive detection of inflammatory sites.     

The preparation and biological characterization of a new HL91-derivative for hypoxic imaging on stroke mice

Aim^99mTc-HL91 (Prognox, GE-Healthcare) was the first nonnitro-aryl-based radiotracer for evaluating hypoxic fraction in neoplasm, stroke and myocardium infarction regions. However, the high hydrophilicity of ^99mTc-HL91 might hamper its penetration into cells. In this study, we prepared a new ligand 4,4,11,11-tetramethyl- 5,10-diazatetradecane- 3,12-dionedioxime (HL91-ET) with higher lipophilicity but structurally similar compared with that of HL91. The chemical and biological characterizations of ^99mTc-HL91-ET as a scintigraphic probe for hypoxia were performed with a stroke-bearing mouse model.Materials and MethodsHL91-ET was synthesized and formulated with stannous chloride and buffer to afford kits. After mixing with ^99mTc-pertechnetate, ^99mTc-HL91-ET can be prepared in high yield and high radiochemical purity (both >96%). The partition coefficient of ^99mTc-HL91-ET was determined in n-octanol/PBS system. Cellular uptake assays under normoxic and hypoxic conditions were performed in an oxygen-controlled CO₂ incubator. Brain stroke in the mouse model was induced by the electrocautery of the middle cerebral artery. After intravenous injection of ^99mTc-HL91-ET into the Balb/c mouse suffering brain stroke, small-animal SPECT images were acquired at designated time points and autoradiography of the brain slides was conducted. Parallel studies of ^99mTc-HL91 were also conducted at the same conditions for comparison.ResultsThe higher partition coefficient of ^99mTc-HL91-ET (0.294±0.007) indicated higher lipophlicity compared with that of ^99mTc-HL91 (0.089±0.005). The ^99mTc-HL91-ET preparation was stable at ambient temperature for 24 h. Cellular uptake assay showed that ^99mTc-HL91-ET was less selectively retained in hypoxic cells than ^99mTc-HL91. The target-to-normal brain ratios derived from the autoradiograms of the brains of stroke mice were 1.31±0.02 and 17.47±0.10 (n=3), respectively, at 2 h post injection of ^99mTc-HL91-ET and ^99mTc-HL91.ConclusionsThis study revealed that ^99mTc-HL91-ET, though with higher lipophilicity than ^99mTc-HL91, did not suggest better specific accumulation in hypoxic cells or tissues than ^99mTc-HL91. The uptake mechanism of ^99mTc-HL91 was at least not solely by passive diffusion. Lipophilicity should not be the major consideration in designing HL91-derivatives for hypoxia imaging.     

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.     

Comparison between two labeled agents in mice using a coinjection-ratio approach in contrast to a conventional group approach

IntroductionThe differences between two agents often need to be accurately defined in vivo. Usually they are injected respectively into two groups of subjects. However, if the two agents do not interact with each other in vivo, a coinjection would serve the same purpose. We believe some individual differences in biodistribution may be circumvented through this approach by calculating organ level ratios.MethodsA model system of MORF/cMORF pretargeting (MORF/cMORF is a complementary pair of DNA analogues) was employed in connection with an on-going tumor therapeutic project. Human LS174T cells were implanted into the flank of severely immuno-compromised NOD-scid IL2rg^null mice. The tumor was confirmed to express TAG-72 antigens. At 16 days post tumor inoculation, mice received IV 60 μg of MORF-conjugated CC49 (an antiTAG-72 antibody), followed 2 days later by a low-mass-dose IV coinjection containing 2.5 μg of ⁹⁰Y-cMORF and 2.5 μg of ^99mTc-cMORF. At 3 h post radioactivity injection, the distribution of ^99mTc was imaged on a SPECT/CT camera and then organs were excised and counted for ⁹⁰Y and ^99mTc. Because the two labeled cMORFs do not react or interact with each other in vivo, the two groups of ⁹⁰Y and ^99mTc data enabled a conventional group comparison. In a new effort, ⁹⁰Y/^99mTc ratios were calculated. Student’s t-test and retrospective power analysis were performed for both approaches. In the new approach, the ratios were set at 1 as the null hypothesis.ResultsThe Student’s t-test in the conventional group approach indicated that the two labeled cMORFs distributed similarly, but significant differences were observed in salivary gland and large intestines. The coinjection-ratio approach certainly did not subvert the results of the conventional approach but revealed subtler differences. The P values were reduced, the powers were increased in most organs, and more significant differences were observed. The increased sensitivity was due to the reduced CV%s (SD/average*100%) of the ⁹⁰Y/^99mTc ratios. Therefore, some individual differences were circumvented and notably the ratio approach differentiated individual differences into ratio-correctable and ratio-uncorrectable.ConclusionsAlthough the conventional approach is reliable, the coinjection-ratio approach using organ level ratios is more sensitive and therefore is recommended whenever possible. In addition, it differentiates individual differences into “coinjection correctable” and “coinjection uncorrectable”.     

Comparison of two new angiogenesis PET tracers 68Ga-NODAGA-E[c(RGDyK)]2 and 64Cu-NODAGA-E[c(RGDyK)]2; in vivo imaging studies in human xenograft tumors

IntroductionThe aim of this study was to synthesize and perform a side-by-side comparison of two new tumor-angiogenesis PET tracers ⁶⁸Ga-NODAGA-E[c(RGDyK)]₂ and ⁶⁴Cu-NODAGA-E[c(RGDyK)]₂ in vivo using human xenograft tumors in mice. Human radiation burden was estimated to evaluate potential for future use as clinical PET tracers for imaging of neo-angiogenesis.MethodsA ⁶⁸Ge/⁶⁸Ga generator was used for the synthesis of ⁶⁸Ga-NODAGA-E[c(RGDyK)]₂. ⁶⁸Ga and ⁶⁴Cu labeled NODAGA-E[c(RGDyK)]₂ tracers were administrated in nude mice bearing either human glioblastoma (U87MG) or human neuroendocrine (H727) xenograft tumors. PET/CT scans at 3 time points were used for calculating the tracer uptake in tumors (%ID/g), integrin α_Vβ₃ target specificity was shown by blocking with cold NODAGA-E[c(RGDyK)]₂, and biodistribution in normal organs were also examined. From biodistribution data in mice human radiation-absorbed doses were estimated using OLINDA/EXM software.Results⁶⁸Ga-NODAGA-E[c(RGDyK)]₂ was synthesized with a radiochemical purity of 89%–99% and a specific activity (SA) of 16–153 MBq/nmol. ⁶⁴Cu-NODAGA-E[c(RGDyK)]₂ had a purity of 92%–99% and an SA of 64–78 MBq/nmol.Both tracers showed similar uptake in xenograft tumors 1 h after injection (U87MG: 2.23 vs. 2.31%ID/g; H727: 1.53 vs. 1.48%ID/g). Both RGD dimers showed similar tracer uptake in non-tumoral tissues and a human radiation burden of less than 10 mSv with an administered dose of 200 MBq was estimated.Conclusion⁶⁸Ga-NODAGA-E[c(RGDyK)]₂ and ⁶⁴Cu-NODAGA-E[c(RGDyK)]₂ can be easily synthesized and are both promising candidates for PET imaging of integrin α_Vβ₃ positive tumor cells. ⁶⁸Ga-NODAGA-E[c(RGDyK)]₂ showed slightly more stable tumor retention. With the advantage of in-house commercially ⁶⁸Ge/⁶⁸Ga generators, ⁶⁸Ga-NODAGA-E[c(RGDyK)]₂ may be the best choice for future clinical PET imaging in humans.     

Synthesis and radiopharmacological evaluation of a high-affinity and metabolically stabilized 18F-labeled bombesin analogue for molecular imaging of gastrin-releasing peptide receptor-expressing prostate cancer

IntroductionBombesin (BBN) and BBN analogues have attracted much attention as high-affinity ligands for selective targeting of the gastrin-releasing peptide (GRP) receptor. GRP receptors are overexpressed in a variety of human cancers including prostate cancer. Radiolabeled BBN derivatives are promising diagnostic probes for molecular imaging of GRP receptor-expressing prostate cancer. This study describes the synthesis and radiopharmacological evaluation of various metabolically stabilized fluorobenzoylated bombesin analogues (BBN-1, BBN-2, BBN-3).MethodsThree fluorobenzoylated BBN analogues containing an aminovaleric (BBN-1, BBN-2), or an aminooctanoic acid linker (BBN-3) were tested in a competitive binding assay against ¹²⁵I-[Tyr⁴]-BBN for their binding potency to the GRP receptor. Intracellular calcium release in human prostate cancer cells (PC3) was measured to determine agonistic or antagonistic profiles of fluorobenzoylated BBN derivatives. Bombesin derivative BBN-2 displayed the highest inhibitory potency toward GRP receptor (IC₅₀ = 8.7 ± 2.2 nM) and was subsequently selected for radiolabeling with fluorine-18 (¹⁸F) through acylation with N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). The radiopharmacological profile of ¹⁸F-labeled bombesin [¹⁸F]BBN-2 was evaluated in PC3 tumor-bearing NMRI nude mice involving metabolic stability studies, biodistribution experiments and dynamic small-animal PET studies.ResultsAll fluorobenzoylated BBN derivatives displayed high inhibitory potency toward the GRP receptor (IC₅₀ = 8.7–16.7 nM), and all compounds exhibited antagonistic profiles as determined in an intracellular calcium release assay. The ¹⁸F-labeled BBN analogue [¹⁸F]BBN-2 was obtained in 30% decay-corrected radiochemical yield with high radiochemical purity > 95% after semi-preparative HPLC purification. [¹⁸F]BBN-2 showed high metabolic stability in vivo with 65% of the radiolabeled peptide remaining intact after 60 min p.i. in mouse plasma. Biodistribution experiments and dynamic small-animal PET studies demonstrated high tumor uptake of [¹⁸F]BBN-2 in PC3 xenografts (2.75 ± 1.82 %ID/g after 5 min and 2.45 ± 1.25 %ID/g after 60 min p.i.). Specificity of radiotracer uptake in PC3 tumors was confirmed by blocking experiments.ConclusionThe present study demonstrates that ¹⁸F-labeled BBN analogue [¹⁸F]BBN-2 is a suitable PET radiotracer with favorable metabolic stability in vivo for molecular imaging of GRP receptor-positive prostate cancer.