SPECT Imaging Agent Research Articles

Abstract I18: Retooling a blood-based biomarker into a theranostic agent

Abstract Over the past 30 years, our ability to noninvasively diagnose, localize, and treat many forms of cancer has grown significantly. However, the biomolecular basis of many malignancies—most notably pancreatic cancer—remains convoluted, and technical advances have not translated to significantly improved survival rates. This is reflected in 1- and 5-year survival rates of 27% and 6%, respectively, for pancreatic cancer diagnoses. Given the tremendous current focus on the creation of new therapeutics for treating pancreatic cancer, the development of companion technologies that can safely, accurately, and unambiguously aid in the early diagnosis, staging, and treatment monitoring of pancreatic cancer is a vitally important—yet unmet—clinical need. The remarkable specificity and selectivity of antibodies for cancer biomarkers have made immunoglobulins some of the most flexible and adaptable tools in modern medicine. For therapeutic purposes, a wide range of nonlabeled antibodies has now entered the clinic. Antibody-based PET and SPECT imaging agents are not far behind. For example, an array of 89Zr-labeled radioimmunoconjugates has shown significant promise in both preclinical and clinical studies. CA19-9 levels in the serum are routinely used for diagnosis, risk stratification, and follow-up of PDAC. While CA19-9 is a shed antigen entering the circulation, the concentration is much higher in the tumor tissue, making it a potential target for in vivo imaging with radiolabeled antibodies. Our studies show that a tumor antigen secreted to the circulation can be used for sensitive detection of primary tumors and metastatic disease by immuno-PET. For example, in proof-of-concept preclinical studies, we were able to visualize pancreatic cancer xenograft and orthotopic models of early- and late-stage disease with excellent tumoral uptake and target-to-non-target ratios using the 89Zr-labeled human monoclonal antibody [89Zr]Zr-DFO-HuMab-5B1 (MVT-2163, NCT02687230). This 5B1 radioimmunoconjugate scaffold has also been deployed with therapeutic beta- and alpha-emitting radionuclides, creating an effective radiotherapeutic paradigm for the imaging and therapy of PDAC. In conclusion, our study shows that a tumor antigen secreted by PDAC to the circulation can be used for sensitive detection of primary tumors and metastatic disease by immuno-PET. This significantly broadens the number of molecular targets that can be used for PET imaging and provides new opportunities for noninvasive characterization of tumors in pancreatic cancer patients. Citation Format: Jason S. Lewis. Retooling a blood-based biomarker into a theranostic agent [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr I18.

Modular Synthesis of DOTA-Metal-Based PSMA-Targeted Imaging Agents for MRI and PET of Prostate Cancer.

A practical, convergent synthesis of prostate-specific membrane antigen (PSMA) targeted imaging agents for MRI, PET, and SPECT of prostate cancer has been developed. In this approach, metals chelated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were placed on the side chains of lysine early in the synthesis to form imaging modules. These are coupled to targeting modules, in this case consisting of the PSMA-binding urea DCL, bonded to an activated linker. The modular approach to targeted molecular imaging agents (TMIAs) offers distinct advantages. By chelating the MRI contrast metal Gd early, it doubles as a protecting group for DOTA. Standard coupling and deprotection steps may be utilized to assemble the modules into peptides, and the need for tri-tert-butyl protection of DOTA requiring removal by strong acid is averted. This enables mild conjugation of the imaging module to a wide variety of targeting agents in the final step. It was further discovered that two labile metals, La3+ or Ce3+ , can be used as placeholders in DOTA during the synthesis, then transmetalated in mild acid by Cu2+ , Ga3+ , In3+ , and Y3+ , metals used in PET/SPECT. This enables the efficient synthesis of nonradioactive analogues of targeted molecular imaging agents that may be transported or stored until needed. A simple and mild two-step transmetalation, involving de-metalation in dilute acid, followed by rapid chelation of the radioactive metal, may be conveniently performed later at the clinic to provide the TMIAs for PET or SPECT.

Synthesis, characterisation and bioconjugation of [109Cd]CdSe/ZnS core/shell quantum dots as “proof of principle” for the potential development of an anti-cancer theranostic

This work describes the synthesis and analysis of radioactive multifunctional nanoparticle probes based on CdSe/ZnS semiconductor quantum dots (QDs) for potential in vitro and in vivo anti-cancer theranostics. The synthetic design involved the radiosynthesis of luminescent CdSe/ZnS QDs using the SPECT imaging agent Cd-109 as the cadmium elemental source. These radioactive nanoparticle materials were then rendered water dispersed and bioconjugated to the tumor-targeting ligand Rituximab.

PP#178 - Development of FR-targeting agent for SPECT imaging of atherosclerosis

PP#178 - Development of FR-targeting agent for SPECT imaging of atherosclerosis

PP#173 - Improving excretory pathways of technetium-99m SPECT imaging agents based on bioorthogonal Diels-Alder click chemistry

PP#173 - Improving excretory pathways of technetium-99m SPECT imaging agents based on bioorthogonal Diels-Alder click chemistry

Pre-labelling versus direct labelling of anthrax proteins for imaging of matrix metalloproteinases activity using DOTA-GA

IntroductionIncreased activity of matrix metalloproteases (MMPs) is associated with reduced survival in several cancer subtypes. Aiming to produce an MMP tumour cell-selective cytotoxin, we genetically modified both components of the AB–type lethal toxin from Bacillus anthracis. Component A, Protective Antigen (PA-WT), was re-engineered to form an oligomeric pore in cell membranes only when cleaved by MMPs (PA-L1). The pore-translocation domain (LFn – N-terminal, 30 kDa) of the Lethal Factor (LF), component B, was fused to the catalytic domain of Pseudomonas exotoxin-A to increase its cytotoxic effect when delivered to cancerous cells. Here, we develop radiolabelled forms of LFn for MMP activity imaging by SPECT using the LFn/PA-L1 system. MethodsDOTA-GA-maleimide was conjugated to LFn to allow radiolabelling with 111In via two different routes: (1) LFn was conjugated with maleimide-DOTA-GA under mild conditions, and then radiolabelled in acidic conditions at 95°C, or (2) 111In was coordinated to maleimide-DOTA-GA first and then conjugated via maleimide chemistry to LFn. Circular Dichroism Spectroscopy of LFn was performed to evaluate changes in its secondary structure. Cell uptake assays using the differently labelled forms of [111In]In-DOTA-GA-LFn in the presence or not of PA-WT or PA-L1 were performed. ResultsLFn was successfully radiolabelled by either strategy. Comparison of the secondary structure content of LFn exposed to 37°C or 95°C, showed a loss of alpha helix content at higher temperatures. Cell uptake of both forms of [111In]In-DOTA-GA-LFn, labelled directly or indirectly, was significantly higher in MMP-positive cells, in the presence of PA-L1, compared to controls. Notably, despite being exposed to high temperatures, uptake of directly labelled [111In]In-DOTA-GA-LFndir was higher than indirectly labelled [111In]In-DOTA-GA-LFnindir. Conclusions111In-radiolabelling of LFn results in a functional molecule that targets MMP-activity in cells when combined with PA-L1. [111In]In-LFn/PA-L1 is a promising MMP activity imaging agent for SPECT imaging.

New 99m Tc(CO)3 -radiolabeled arylpiperazine pharmacophore as potent 5HT1A serotonin receptor radiotracer: Docking studies, chemical synthesis, radiolabeling, and biological evaluation.

In spite of previous efforts, there is lack of a radiotracer for imaging the 5HT1A receptor density in human brain, which is involved in several neurological brain disorders. The aim of this study was to prepare a new derivative of 1-(2-methoxyphenyl)piperazine (MPP) as a main chemical structure of 5HT1A receptor antagonist with 3-carbon linker and radiolabeled by [99m Tc][Tc(CO)3 (H2 O)3 ]+ precursor. Docking studies before chemical synthesis showed similar fashion of interaction for both WAY100635 (potent 5HT1A receptor antagonist) and new designed ligand, despite of addition of 99m Tc(CO)3 group in the structure of new ligand. MPP-(CH2 )3 -N3 was synthesized via three efficient and reliable chemical synthesis steps (more than 80% yield) then radiolabeled by addition of 2-ethynylpyridine and [99m Tc][Tc(CO)3 (H2 O)3 ]+ precursor in one pot procedure (more than 95% radiochemical efficiency) through click chemistry method. After incubation, radiotracer was found stable in vitro up to 2hours. Binding assays showed about 33% specific binding of radiotracer to the 5HT1A receptors. Brain biodistribution studies indicated (0.26±0.05)% ID/g hippocampus uptake at 30minutes post injection, which its specificity was verified through blocking studies. These results suggested that new designed radioligand might serve as a potent SPECT imaging agent to estimate status of 5HT1A receptors.

Trimodal Nanoparticle Contrast Agent for CT, MRI and SPECT Imaging: Synthesis and Characterization of Radiolabeled Core/Shell Iron Oxide@Gold Nanoparticles

Recently, nanoparticles have emerged as promising contrast agents for various imaging applications. In this paper, we present the synthesis and characterization of a novel hybrid nano-structure, co...

Dual-isotope imaging allows in vivo immunohistochemistry using radiolabelled antibodies in tumours

While radiolabelled antibodies have found great utility as PET and SPECT imaging agents in oncological investigations, a notable shortcoming of these agents is their propensity to accumulate non-specifically within tumour tissue. The degree of this non-specific contribution to overall tumour uptake is highly variable and can ultimately lead to false conclusions. Therefore, in an effort to obtain a reliable measure of inter-individual differences in non-specific tumour uptake of radiolabelled antibodies, we demonstrate that the use of dual-isotope imaging overcomes this issue, enables true quantification of epitope expression levels, and allows non-invasive in vivo immunohistochemistry. The approach involves co-administration of (i) an antigen-targeting antibody labelled with zirconium-89 (89Zr), and (ii) an isotype-matched non-specific control IgG antibody labelled with indium-111 (111In). As an example, the anti-HER2 antibody trastuzumab was radiolabelled with 89Zr, and co-administered intravenously together with its 111In-labelled non-specific counterpart to mice bearing human breast cancer xenografts with differing HER2 expression levels (MDA-MB-468 [HER2-negative], MDA-MB-231 [low-HER2], MDA-MB-231/H2N [medium-HER2], and SKBR3 [high-HER2]). Simultaneous PET/SPECT imaging using a MILabs Vector4 small animal scanner revealed stark differences in the intratumoural distribution of [89Zr]Zr-trastuzumab and [111In]In-IgG, highlighting regions of HER2-mediated uptake and non-specific uptake, respectively. Normalisation of the tumour uptake values and tumour-to-blood ratios obtained with [89Zr]Zr-trastuzumab against those obtained with [111In]In-IgG yielded values which were most strongly correlated (R = 0.94; P = 0.02) with HER2 expression levels for each breast cancer type determined by Western blot and in vitro saturation binding assays, but not non-normalised uptake values. Normalised intratumoural distribution of [89Zr]Zr-trastuzumab correlated well with intratumoural heterogeneity HER2 expression.

Multi-atlas cardiac PET segmentation

PurposeWe propose a multi-atlas based segmentation method for cardiac PET and SPECT images to deal with the high variability of tracer uptake characteristics in myocardium. In addition, we verify its performance by comparing it to the manual segmentation and single-atlas based approach, using dynamic myocardial PET. MethodsTwelve left coronary artery ligated SD rats underwent ([18F]fluoropentyl) triphenylphosphonium salt PET/CT scans. Atlas-based segmentation is based on the spatial normalized template with pre-defined region-of-interest (ROI) for each anatomical or functional structure. To generate multiple left ventricular (LV) atlases, each LV image was segmented manually and divided into angular segments. The segmentation methods performances were compared in regional count information using leave-one-out cross-validation. Additionally, the polar-maps of kinetic parameters were estimated. ResultsIn all images, the highest r2 template yielded the lowest root-mean-square error (RMSE) between the source image and the best-matching templates ranged between 0.91–0.97 and 0.06–0.11, respectively. The single-atlas and multi-atlas based ROIs yielded remarkably different perfusion distributions: only the multi-atlas based segmentation showed equivalent high correlation results (r2 = 0.92) with the manual segmentation compared with the single-atlas based (r2 = 0.88). The high perfusion value underestimation was remarkable in single-atlas based segmentation. ConclusionsThe main advantage of the proposed multi-atlas based cardiac segmentation method is that it does not require any prior information on the tracer distribution to be incorporated into the image segmentation algorithms. Therefore, the same procedure suggested here is applicable to any other cardiac PET or SPECT imaging agents without modification.

A study of 99mTc/Re-tricarbonyl complexes of 4-amino-1,8-naphthalimides.

Three 4-amino-1,8-naphthalimide analogues were synthesized, consisting of the tridentate chelators di-2-picolylamine, (pyridin-2-ylmethyl)glycinate, and iminodiacetate conjugated to the naphthalimide scaffold. Coordination with fac-99mTc/Re(CO)3 resulted in metal complexes with overall charges of -1, 0, or +1. Upon coordination of Re(i), the initial naphthalimide-based fluorescence is largely maintained for both negative and neutral complexes compared to their free ligand forms, while an increase in fluorescence quantum yield was observed for the positively charged complex. OVCAR-8 ovarian cancer cells were stained with each of the complexes, demonstrating that the positive complex is more lipophilic and cell membrane permeable than the neutral and negative complexes. Each of the three technetium-99m labelled naphthalimide complexes were successfully produced from fac-[99mTc(CO)3(H2O)3]+ in 15 minutes at 70 °C and isolated in radiochemical yields ranging from 60-95% with radiochemical purities greater than 95%. These fluorescent metal complexes of various charges can be used to tune pharmacokinetic and cellular uptake properties of 99mTc/Re-naphthalimide-bioconjugates, while still maintaining desirable fluorescence properties.

Preparation and clinical translation of 99mTc-PSMA-11 for SPECT imaging of prostate cancer.

This study explores the feasibility of radiolabeling the HBED-CC-PSMA (PSMA-11) ligand with Tc-99m for SPECT imaging of prostate cancer patients. 68Ga-HBED-CC-PSMA (PSMA-11) is used clinically for PET/CT imaging of prostate cancer (PCa) patients. However, a PET/CT facility may not be affordable and/or accessible to remotely located health centers. Thus, economic considerations require development of a SPECT-based tracer to provide low cost effective health care to the entire global population. Hence, radiochemical parameters were varied and optimized to obtain the maximum radiochemical yield of 99mTc-PSMA-11. 99mTc-PSMA-11 could be prepared in 60 ± 5% radiochemical yield and >98% radiochemical purity with a specific activity of 15 ± 5 GBq μmol-1. The radiotracer exhibited high stability in vitro in human serum after 24 h. A cell uptake of 15.2 ± 1.2% was observed for 99mTc-PSMA-11 in PSMA-positive prostate carcinoma LNCaP cells. Rapid clearance from blood, liver, intestine, lungs and other major organs was observed during normal biodistribution studies. The radiotracer, 99mTc-PSMA-11, exhibited physiological distribution in salivary and lacrimal glands similar to that of 68Ga-PSMA-11 in mice and successfully identified primary tumors as well as metastatic lesions in human patients. This study thus highlights successful radiolabeling of HBED-CC-PSMA with Tc-99m and the potential of 99mTc-PSMA-11 as a SPECT imaging agent for PCa.

Evaluation of 111In-DOTA-5D3, a Surrogate SPECT Imaging Agent for Radioimmunotherapy of Prostate-Specific Membrane Antigen.

5D3 is a new high-affinity murine monoclonal antibody specific for prostate-specific membrane antigen (PSMA). PSMA is a target for the imaging and therapy of prostate cancer. 111In-labeled antibodies have been used as surrogates for 177Lu/90Y-labeled therapeutics. We characterized 111In-DOTA-5D3 by SPECT/CT imaging, tissue biodistribution studies, and dosimetry. Methods: Radiolabeling, stability, cell uptake, and internalization of 111In-DOTA-5D3 were performed by established techniques. Biodistribution and SPECT imaging were done on male nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice bearing human PSMA(+) PC3 PIP and PSMA(-) PC3 flu prostate cancer xenografts on the upper right and left flanks, respectively, at 2, 24, 48, 72, and 192 h after injection. Biodistribution was also evaluated in tumor-free, healthy male CD-1 mice. Blocking studies were performed by coinjection of a 10-fold and 50-fold excess of 5D3 followed by biodistribution at 24 h to determine PSMA binding specificity. The absorbed radiation doses were calculated on the basis of murine biodistribution data, which were translated to a human adult man using organ weights as implemented in OLINDA/EXM. Results:111In-DOTA-5D3 was synthesized with specific activity of approximately 2.24 ± 0.74 MBq/μg (60.54 ± 20 μCi/μg). Distribution of 111In-DOTA-5D3 in PSMA(+) PC3 PIP tumor peaked at 24 h after injection and remained high until 72 h. Uptake in normal tissues, including the blood, spleen, liver, heart, and lungs, was highest at 2 h after injection. Coinjection of 111In-DOTA-5D3 with a 10- and 50-fold excess of nonradiolabeled antibody significantly reduced PSMA(+) PC3 PIP tumor and salivary gland uptake at 24 h but did not reduce uptake in kidneys and lacrimal glands. Significant clearance of 111In-DOTA-5D3 from all organs occurred at 192 h. The highest radiation dose was received by the liver (0.5 mGy/MBq), followed by the spleen and kidneys. Absorbed radiation doses to the salivary and lacrimal glands and bone marrow were low. Conclusion:111In-DOTA-5D3 is a new radiolabeled antibody for imaging and a surrogate for therapy of malignant tissues expressing PSMA.

Design, physico-chemical and pre-clinical evaluation of a homo-bivalent99mTc-(BTZ)2DTPA radioligand for targeting dimeric 5-HT1A/5-HT7receptors

A99mTc-labelled bis-benzothiazolone-DTPA radio-complex as a SPECT neuroimaging agent.

Development of a New FR-Targeting Agent 99mTc-HYNFA with Improved Imaging Contrast and Comparison of Multimerization and/or PEGylation Strategies for Radio-Folate Modification.

This study aims to develop a new folate receptor (FR)-targeting agent for SPECT imaging with improved contrast and evaluate the modification strategies of multimerization and/or PEGylation in the development of new radio-folates. A series of novel folate derivatives have been synthesized and radiolabeled with 99mTc using tricine and TPPTS as coligands. To better investigate their pharmacokinetics, cell uptake, biodistribution, and microSPECT/CT imaging were evaluated. Four radioligands displayed high KB cell uptake after incubation for 2 and 4 h. Presaturated with excess folic acid (FA) resulted in a significant blocking effect in KB cells, indicating specificity of these radioligands toward FR. Biodistribution and microSPECT imaging studies in KB tumor-bearing mice showed that the folate conjugate 99mTc-HYNFA with poly(ethylene glycol) (PEG) and triazole linkage displayed the highest tumor uptake (16.30 ± 2.01 %ID/g at 2 h p.i. and 14.9 ± 0.62 %ID/g at 4 h p.i. in mice biodistribution) and best imaging contrast, indicating promising application prospect. More interestingly, the in vivo performance of this monomeric 99mTc-HYNFA was much better than that of FA multimers and non-PEGylated monomers, suggesting that multimerization may not be a feasible method for the design of radio-folates. PEG linkage rather than FA multimerization should be taken into consideration in the development of folate-based radiopharmaceuticals in the future.

Synthesis and preliminary biological evaluation of a 99m Tc-chlorambucil derivative as a potential tumor imaging agent.

Technetium-99m-based radiopharmaceuticals have been used widely as diagnostic agents in the nuclear medicine. Chlorambucil (CLB) as one typical alkylating drug exhibits excellent inhibition effects against many human malignancies. To develop and explore a novel potential imaging agent for early diagnosis of tumors, tricarbonyl technetium-99m and rhenium complexes on the basis of the tridentate ligand dipicolylamine (DPA) bound to the chlorambucil pharmacophore were designed and synthesized: 99m Tc-DPA-CLB (3) and Re-DPA-CLB (4). The high performance liquid chromatography analyses showed that the retention time of 3 and 4 was 13.5 and 13.6minutes, respectively. Radiolabeling efficiency of the 99m Tc-DPA-CLB tracer was 97%, and the radiochemical purity was larger than 95% after 6hours stored in phosphate buffered saline or human serum as observed by thin layer chromatography and high performance liquid chromatography. Biodistribution studies in a mouse model of breast cancer showed 99m Tc-DPA-CLB exhibited a favorable tumor affinity. The radiotracer cleared quickly in the first hour via hepatobiliary and renal routes of excretion, resulted in a very low background at 4hours post injection (p.i.). It had moderate uptake ratios of tumor to blood and tumor to muscle. These results suggested 99m Tc-DPA-CLB might be a promising SPECT imaging agent for tumor diagnosis.

Development of Radiolabeled Ligands Targeting the Glutamate Binding Site of the N-Methyl-d-aspartate Receptor as Potential Imaging Agents for Brain.

Abnormal activity of various N-methyl-d-aspartate receptor (NMDAR) subtypes has been implicated in a wide variety of neurological disorders such as Alzheimer's disease, schizophrenia, and epilepsy. Imaging agents for PET and SPECT that target NMDARs in a subtype-selective fashion may enable better characterization of those disorders and enhance drug development. On the basis of a pyrazoline derivative that demonstrated neuroprotective effects in vivo, we synthesized a series of para-substituted analogues and measured their affinities to various NMDAR subtypes. Compounds 4a-c and 4e showed greater, nanomolar affinity for the GluN1/2A subtype versus GluN1/2B. Dicarbomethoxy (pro-drug) analogues of [124/125I]4d and [11C]4e (i.e., [124/125I]11d and [11C]11e) were generated and tested for NMDAR binding specificity in ex vivo autoradiography and brain biodistribution studies. Although NMDAR-specific binding could be demonstrated for [125I]11d and [11C]11e through autoradiography and biodistribution studies, imaging of neither [124I]11d nor [11C]11e could demonstrate brain penetration sufficient for detection by PET.

Synthesis and Evaluation of Tricarbonyl (99m)Tc-Labeled 2-(4-Chloro)phenyl-imidazo[1,2-a]pyridine Analogs as Novel SPECT Imaging Radiotracer for TSPO-Rich Cancer.

The 18-kDa translocator protein (TSPO) levels are associated with brain, breast, and prostate cancer progression and have emerged as viable targets for cancer therapy and imaging. In order to develop highly selective and active ligands with a high affinity for TSPO, imidazopyridine-based TSPO ligand (CB256, 3) was prepared as the precursor. 99mTc- and Re-CB256 (1 and 2, respectively) were synthesized in high radiochemical yield (74.5% ± 6.4%, decay-corrected, n = 5) and chemical yield (65.6%) by the incorporation of the [99mTc(CO)3(H2O)3]+ and (NEt4)2[Re(CO)3Br3] followed by HPLC separation. Radio-ligand 1 was shown to be stable (>99%) when incubated in human serum for 4 h at 37 °C with a relatively low lipophilicity (logD = 2.15 ± 0.02). The rhenium-185 and -187 complex 2 exhibited a moderate affinity (Ki = 159.3 ± 8.7 nM) for TSPO, whereas its cytotoxicity evaluated on TSPO-rich tumor cell lines was lower than that observed for the precursor. In vitro uptake studies of 1 in C6 and U87-MG cells for 60 min was found to be 9.84% ± 0.17% and 7.87% ± 0.23% ID, respectively. Our results indicated that 99mTc-CB256 can be considered as a potential new TSPO-rich cancer SPECT imaging agent and provides the foundation for further in vivo evaluation.

Preparation of an estrogen receptor SPECT imaging agent 99Tcm-GAP-EDL and its preliminary study in breast tumor-bearing nude mice

Objective To prepare a kind of 99Tcm-labeled estrogen receptor (ER) SPECT imaging agent, and evaluate its binding characteristics with ER by in vitro and in vivo studies. Methods EDL was prepared from estrone and then reacted with GAP to synthesize GAP-EDL. Then, GAP-EDL was labeled with 99Tcm to obtain 99Tcm-GAP-EDL. Cell uptake and blocking assays were performed in vitro on MCF-7 and MDA-MB-231 cells. The biodistribution study of 99Tcm-GAP-EDL was performed on normal BALB/c mice at 30, 60, 120, 180 and 240 min post injection. Nude mice bearing either MCF-7 or MDA-MB-231 derived tumors were injected with 99Tcm-GAP-EDL, and subjected to γ imaging at 1, 2, 4 h post injection. The mice injected with excess unlabeled GAP-EDL or estradiol were used as the blocking control. Two-sample t test was used for data analysis. Results The radiolabeling yield of 99Tcm-GAP-EDL was (98.8±0.5)% and the radiochemical purity after 24 h was over 90%. The cell uptake rate of MCF-7 cells at 240 min was (4.84±0.21)%, which was significantly higher than that of MDA-MB-231 cells ((2.11±0.21)%; t=15.96, P<0.05). 99Tcm-GAP-EDL uptake rate of MCF-7 cells in blocking groups ((2.31±0.28)% and (2.05±0.35)%) decreased significantly compared to that of non-blocking group(t=11.52, 11.16, both P<0.05). Biodistribution studies showed that 99Tcm-GAP-EDL was mainly metabolized by the liver and kidneys, and exhibited quick blood clearance. Gamma imaging showed high uptake in MCF-7 tumors 1 h post injection and the uptake reached the highest at 4 h, while there was little 99Tcm-GAP-EDL uptake in MDA-MB-231 tumors and blocked MCF-7 tumors. Conclusions 99Tcm-GAP-EDL may be prepared under mild conditions with high labeling purity and stability. The in vitro and in vivo characteristics of 99Tcm-GAP-EDL suggest that it may be a promising probe for ER positive tumor imaging. Key words: Receptors, estrogen; GAP-EDL; Isotope labeling; Technetium; Radionuclide imaging; Breast neoplasms; Mice

Synthesis and Preclinical Evaluation of Folate-NOTA-Al(18)F for PET Imaging of Folate-Receptor-Positive Tumors.

Folate-receptor-targeted PET radiotracers can potentially serve as versatile imaging agents for the diagnosis, staging, and prediction of response to therapy of patients with folate-receptor (FR)-expressing cancers. Because current FR-targeted PET reagents can be compromised by complex labeling procedures, low specific activities, poor radiochemical yields, or unwanted accumulation in FR negative tissues, we have undertaken to design an improved folate-PET agent that might be more amenable for clinical development. For this purpose, we have synthesized a folate-NOTA-Al(18)F radiotracer and examined its properties both in vitro and in vivo. Radiochemical synthesis of folate-NOTA-Al(18)F was achieved by incubating (18)F(-) with AlCl3 for 2 min followed by heating in the presence of folate-NOTA for 15 min at 100 °C. Binding of folate-NOTA-Al(18)F to FR was quantitated in homogenates of KB and Cal51 tumor xenografts in the presence and absence of excess folic acid as a competitor. In vivo imaging was performed on nu/nu mice bearing either FR+ve (KB cell) or FR-ve (A549 cell) tumor xenografts, and specific accumulation of the radiotracer in tumor and other tissues was assessed by high-resolution micro-PET and ex vivo biodistribution in the presence and absence of excess folic acid. Image quality of folate-NOTA-Al(18)F was compared with that of (99m)Tc-EC20, a clinically established folate-targeted SPECT imaging agent. Total radiochemical synthesis and purification of folate-NOTA-Al(18)F was completed within 37 min, yielding a specific activity of 68.82 ± 18.5 GBq/μmol, radiochemical yield of 18.6 ± 4.5%, and radiochemical purity of 98.3 ± 2.9%. Analysis of FR binding revealed a Kd of ∼1.0 nM, and micro-PET imaging together with ex vivo biodistribution analyses demonstrated high FR-mediated uptake in an FR+ tumor and the kidneys. Folate-NOTA-Al(18)F constitutes an easily prepared FR-targeted PET imaging agent with improved radiopharmaceutical properties and high specificity for folate receptor expressing tumors. Given its improved properties over (99m)Tc-EC20 (i.e., higher resolution, shorter image acquisition time, etc.), we conclude that folate-NOTA-Al(18)F constitutes a viable alternative to (99m)Tc-EC20 for use in identification, diagnosis, and staging of patients with FR-expressing cancers.