Synthesis and evaluation of amino acid-based radiotracer 99mTc-N4-AMT for breast cancer imaging.

PURPOSE: This study was to develop an efficient synthesis of (99m)Tc-O-[3-(1,4,8,11-tetraazabicyclohexadecane)-propyl]-α-methyl tyrosine ((99m)Tc-N4-AMT) and evaluate its potential in cancer imaging. METHODS: N4-AMT was synthesized by reacting N4-oxalate and 3-bromopropyl AMT (N-BOC, ethyl ester). In vitro cellular uptake kinetics of (99m)Tc-N4-AMT was assessed in rat mammary tumor cells. Tissue distribution of the radiotracer was determined in normal rats at 0.5-4 h, while planar imaging was performed in mammary tumor-bearing rats at 30-120 min. RESULTS: The total synthesis yield of N4-AMT was 14%. Cellular uptake of (99m)Tc-N4-AMT was significantly higher than that of (99m)Tc-N4. Planar imaging revealed that (99m)Tc-N4-AMT rendered greater tumor/muscle ratios than (99m)Tc-N4. CONCLUSIONS: N4-AMT could be synthesized with a considerably high yield. Our in vitro and in vivo data suggest that (99m)Tc-N4-AMT, a novel amino acid-based radiotracer, efficiently enters breast cancer cells, effectively distinguishes mammary tumors from normal tissues, and thus holds the promise for breast cancer imaging.

Functional imaging of estrogen receptors with radiolabeled-GAP-EDL in rabbit endometriosis model.

RATIONALE AND OBJECTIVES: Endometriosis is a common women's health problem. Animal models provide an invaluable tool to study the natural history of endometriosis. We previously have reported that (99m)Tc-labeled glutamate peptide-estradiol ((99m)Tc-GAP-EDL) is a useful agent for imaging functional estrogen receptor (ER) via an ER-mediated process. This study was to evaluate the feasibility of using radiolabeled GAP-EDL to image ER-positive (ER +) endometriosis in nonprimate animal models. MATERIALS AND METHODS: 3-Aminoethyl estradiol (EDL) was conjugated to glutamate peptide (GAP) to yield GAP-EDL. In vitro cellular uptake studies of (99m)Tc and (68)Ga-GAP-EDL inhibition with cold estrone were conducted in 13,762 rat mammary tumor cells. To create a rabbit model with endometriosis, part of uterine tissue was dissected and grafted in the peritoneal wall. Eight weeks after surgery, scintigraphic images were obtained after intravenous injection of (99m)Tc-GAP-EDL (1 mCi/rabbit, intravenous) at 0.5-2.0 hours, and (68)Ga-GAP-EDL at 45 minutes. We also performed (68)Ga-GAP-EDL blocking study in rabbit model by using tamoxifen. The rabbits were sacrificed and the grafts were excised for histologic examination. RESULTS: In vitro uptake study of (99m)Tc- and (68)Ga-GAP-EDL in 13,762 rat breast cancer cells showed gradually increasing uptake of both tracers. Accumulation of (68)Ga-GAP-EDL in 13,762 cells was inhibited with cold estrone in a dose-dependent manner. In the endometriosis model, the grafted uterine tissue could be visualized by (99m)Tc-GAP-EDL. Necropsy was performed at 2.5 hours after injection time. Four follicular endometrial lesions in eight implanted endometrial tissues were detected, and all lesions could be detected by (99m)Tc-GAP-EDL. Planar scintigraphy of uterus, ovary and implants of necropsy specimen revealed an increased uptake of (99m)Tc-GAP-EDL in comparison with surrounding abdominal wall tissue. Microscopic examinations support that (99m)Tc-GAP-EDL was accumulated in the microinvasive endometrial tissue. After blocking with tamoxifen, (68)Ga-GAP-EDL accumulation in the endometrial grafts could not be visualized, and endometrial tissue-to-normal tissue count ratios were statistically higher in a nonblocked image than that in the blocked image. CONCLUSIONS: Endometriosis uptake of radiolabeled GAP-EDL was via an estrogen receptor-mediated process. Radiolabeled-GAP-EDLs are useful agents for imaging endometriosis.

PET and planar imaging of tumor hypoxia with labeled metronidazole.

RATIONALE AND OBJECTIVES: This study was aimed to develop 99mTc- and 68Ga-labeled metronidazole (MN) using ethylenedicysteine (EC) as a chelator and evaluate their potential use to assess tumor hypoxia. MATERIALS AND METHODS: EC-MN was labeled with 99mTc in the presence of tin (II) chloride. Labeling EC-MN with 68Ga was achieved by adding 68GaCl3 (2 mCi with 3.4 microg cold GaCl3). In vitro cellular uptakes of 99mTc- and 68Ga-EC-MN were obtained in various types of tumor cells at 0.5-4 hours. Tissue distribution and PET imaging of 99mTc and 68Ga-EC-MN were evaluated in breast tumor-bearing rats at 0.5-4 hours. Tumor oxygen tension was measured using an oxygen probe. RESULTS: There were similar cellular uptakes (2-10%) between 99mTc- and 68Ga-EC-MN at 0.5-4 hours. In vivo biodistribution of 99mTc- and 68Ga-EC-MN in breast tumor-bearing rats showed increased tumor-to-blood and tumor-to-muscle count density ratios as a function of time. Positron emission tomography images confirmed that the tumors could be visualized clearly with 68Ga-EC-MN. Oxygen tension in tumor tissue was determined to be 6-10 mm Hg compared with 40-50 mm Hg in normal muscle tissue. CONCLUSIONS: The results indicated that it is feasible to use 99mTc- and 68Ga-EC-MN for assessment of tumor hypoxia. These agents may be useful in selecting and evaluating cancer therapy.

Assessment of therapeutic tumor response using 99mtc-ethylenedicysteine-glucosamine.

PURPOSE: The aim of this study was to evaluate 99mTc-ethylenedicysteine-glucosamine (EC-DG) for the assessment of tumor growth. METHOD: To evaluate whether 99mTc-EC-DG is involved in cell nuclei activity, in vitro thymidine incorporation, and cell-cycle assays of EC-DG were conducted using lung and breast cancer cells. Biodistribution of 99mTc-EC-DG in lung tumor-bearing mice (0.5-4 hours, 1 Ci/mouse, i. v.) was used to estimate the radiation-absorbed dose. Autoradiograms of 99mTc-EC-DG and 18F-FDG were compared in nude mice bearing uterine sarcoma. Rabbits inoculated with VX-2 cells were imaged with 99mTc-EC-DG and 99mTc-EC. For therapeutic assessment studies, scintigraphic imaging studies with 99mTc-EC-DG in mammary tumor-bearing rats were conducted at various days after treatment with paclitaxel and cisplatin. The imaging findings were correlated immunohistochemical assays (mRNA expression, apoptosis, and cell-cycle changes in tumor), and flow cytometry analysis was performed. RESULTS: In vitro cellular uptake assays indicated that cell nuclei activity could be assessed by 99mTc-EC-DG. Scintigraphy and autoradiograms in animal models demonstrated that the tumor could be clearly visualized by 99mTc-EC-DG. The efficacy of paclitaxel and cisplatin treatment in rodent models could be assessed using tumor/muscle ratios. Immunohistochemical staining indicated a reduced expression of bFGF and an increased apoptosis and cell-cycle changes after paclitaxel and cisplatin treatment. CONCLUSION: 99mTc-EC-DG is involved in cell nuclei activity and could assess the therapeutic tumor response.

Synthesis and evaluation of Tc-99m DTPA-glutathione as a non-invasive tumor imaging agent in a mouse colon cancer model.

PURPOSE: Glutathione (GSH) plays a critical role in detoxification reactions by reducing the levels of reactive oxygen species in cancer cells. This study aimed to develop technetium (Tc)-99m diethylenetriaminepentaacetic acid (DTPA)-GSH as a tumor imaging agent, and to evaluate the diagnostic performance of Tc-99m DTPA-GSH in terms of its ability to differentiate tumors from inflammatory lesions. METHODS: DTPA-GSH was synthesized by reaction of GSH with DTPA anhydride under anhydrous conditions in a nitrogen atmosphere. DTPA-GSH was then reacted with Tc-99m sodium pertechnetate in a tin (II) chloride (SnCl2) solution. Gamma camera imaging was performed after intravenous injection of Tc-99m DTPA-GSH into a mouse CT-26 colon cancer model, or a mouse model of inflammation induced by the intramuscular injection of Freund's complete adjuvant. RESULTS: DTPA-GSH was successfully prepared via a straightforward synthetic procedure and radiolabeled with Tc-99m at a high labeling efficiency (>95%). Tc-99m DTPA-GSH was strongly internalized by tumors in colon cancer model mice, with the tumor-to-normal muscle ratio of the complex reaching 4.3±0.9 at 4 h. By contrast, Tc-99m DTPA-GSH showed relatively weak uptake in inflammatory lesions (target-to-non-target ratio=2.0±0.3 at 4 h). A competition study showed that the uptake of Tc-99m DTPA-GSH into tumors was blocked by co-injection with high concentrations of free GSH. CONCLUSIONS: The results of this work indicate that Tc-99m DTPA-GSH is a good candidate for development as a non-invasive tumor imaging agent. Furthermore, Tc-99m DTPA-GSH effectively distinguished between cancerous tissue and inflammatory lesions.

Sulfonylurea receptor as a target for molecular imaging of pancreas beta cells with (99m)Tc-DTPA-glipizide.

OBJECTIVE: This study was aimed to assess pancreas beta cell activity using (99m)Tc-diethyleneaminepentaacetic acid-glipizide (DTPA-GLP), a sulfonylurea receptor agent. The effect of DTPA-GLP on the blood glucose level in rats was also evaluated. METHODS: DTPA dianhydride was conjugated with GLP in the presence of sodium amide, yielding 60%. Biodistribution and planar images were obtained at 30-120 min after injection of (99m)Tc-DTPA-GLP (1 mg/rat, 0.74 and 11.1 MBq per rat, respectively) in normal female Fischer 344 rats. The control group was given (99m)Tc-DTPA. To demonstrate pancreas beta cell uptake of (99m)Tc-DTPA-GLP via a receptor-mediated process, a group of rats was pretreated with streptozotocin (a beta cell toxin, 55 mg/kg, i.v.) and the images were acquired at immediately-65 min on day 5 post-treatment. The effect on the glucose levels after a single administration (ip) of DTPA-GLP was compared to glipizide (GLP) for up to 6 h. RESULTS: The structure of DTPA-GLP was confirmed by NMR, mass spectrometry and HPLC. Radiochemical purity assessed by ITLC was >96%. (99m)Tc-DTPA-GLP showed increased pancreas-to-muscle ratios, whereas (99m)Tc-DTPA showed decreased ratios at various time points. Pancreas could be visualized with (99m)Tc-DTPA-GLP in normal rat, however, (99m)Tc-DTPA has poor uptake suggesting the specificity of (99m)Tc-DTPA-GLP. Pancreas beta cell uptake could be blocked by pre-treatment with streptozotocin. DTPA-GLP showed an equal or better response in lowering the glucose levels compared to the existing GLP drug. CONCLUSIONS: It is feasible to use (99m)Tc-DTPA-GLP to assess pancreas beta cell receptor recognition. (99m)Tc-DTPA-GLP may be helpful in evaluating patients with diabetes, pancreatitis and pancreatic tumors.

Targeted functional imaging of estrogen receptors with 99mTc-GAP-EDL.

PURPOSE: To evaluate the feasibility of using (99m)Tc-glutamate peptide-estradiol in functional imaging of estrogen receptor-positive [ER(+)] diseases. METHODS: 3-Aminoethyl estradiol (EDL) was conjugated to glutamate peptide (GAP) to yield GAP-EDL. Cellular uptake studies of (99m)Tc-GAP-EDL were conducted in ER(+) cell lines (MCF-7, 13762 and T47D). To demonstrate whether GAP-EDL increases MAP kinase activation, Western blot analysis of GAP-EDL was performed in 13762 cells. Biodistribution was conducted in nine rats with 13762 breast tumors at 0.5-4 h. Each rat was administered (99m)Tc-GAP-EDL. Two animal models (rats and rabbits) were created to ascertain whether tumor uptake of (99m)Tc-GAP-EDL was via an ER-mediated process. In the tumor model, breast tumor-bearing rats were pretreated with diethylstilbestrol (DES) 1 h prior to receiving (99m)Tc-GAP-EDL. In the endometriosis model, part of the rabbit uterine tissue was dissected and grafted to the peritoneal wall. The rabbit was administered with (99m)Tc-GAP-EDL. RESULTS: There was a 10-40% reduction in uptake of (99m)Tc-GAP-EDL in cells treated with DES or tamoxifen compared with untreated cells. Western blot analysis showed an ERK1/2 phosphorylation process with GAP-EDL. Biodistribution studies showed that tumor uptake and tumor-to-muscle count density ratio in (99m)Tc-GAP-EDL groups were significantly higher than those in (99m)Tc-GAP groups at 4 h. Among (99m)Tc-GAP-EDL groups, region of interest analysis of images showed that tumor-to muscle ratios were decreased in blocking groups. In the endometriosis model, the grafted uterine tissue could be visualized by (99m)Tc-GAP-EDL. CONCLUSION: Cellular or tumor uptake of (99m)Tc-GAP-EDL occurs via an ER-mediated process. (99m)Tc-GAP-EDL is a useful agent for imaging functional ER(+) disease.

(99m)Tc-EC-guanine: synthesis, biodistribution, and tumor imaging in animals.

PURPOSE: DNA markers are useful in assessing cell proliferation. The purpose of this study was to synthesize (99m)Tc-ethylenedicysteine-guanine (EC-Guan) for evaluation of cell proliferation. METHODS: Tumor cells were incubated with (99m)Tc-EC-Guan for cell cycle analysis. Prostate tumor cells that were overexpressing the HSV thymidine kinase gene, or various tumor cells were incubated with (99m)Tc-EC-Guan at 0.5-2 h. Thymidine incorporation assays were performed in lung cancer cells incubated with EC-Guan at 0.1-1 mg/well. Tissue distribution, autoradiography, and planar scintigraphy of (99m)Tc-EC-Guan and (99m)Tc-EC (control) were determined in tumor-bearing rodents at 0.5-4 h. RESULTS: Cell culture assays indicated that EC-Guan was incorporated in DNA, and there was no significant uptake difference between HSVTK overexpressed and normal groups. Biodistribution and scintigraphic imaging studies of (99m)Tc-EC-Guan showed increased tumor/tissue count density ratios as a function of time. CONCLUSIONS: Our results indicate that (99m)Tc-EC-Guan may be useful as a tumor proliferation imaging agent.

Assessment of cyclooxygense-2 expression with 99mTc-labeled celebrex.

Cyclooxygenase-2 (COX-2) plays an important role in angiogenesis and cancer progression. Since many tumor cells exhibit COX-2 expression, functional imaging of COX-2 expression using celebrex (CBX, a COX-2 inhibitor) may provide not only a non-invasive, reproducible, quantifiable alternative to biopsies, but it also greatly complements pharmacokinetic studies by correlating clinical responses with biological effects. Moreover, molecular endpoints of anti-COX-2 therapy could also be assessed effectively. This study aimed at measuring uptake of Tc-EC-CBX in COX-2 expression in tumor-bearing animal models. In vitro Western blot analysis and cellular uptake assays were used to examine the feasibility of using Tc-EC-CBX to measure COX-2 activity. Tissue distribution studies of Tc-EC-CBX were evaluated in tumor-bearing rodents at 0.5-4 h. Dosimetric absorption was then estimated. Planar scintigraphy was performed in mice, rats and rabbits bearing tumors. In vitro cellular uptake indicated that cells with higher COX-2 expression (A549 and 13762) had higher uptake of Tc-EC-CBX than lower COX-2 expression (H226). In vivo biodistribution of Tc-EC-CBX in tumor-bearing rodents showed increased tumor:tissue ratios as a function of time. In vitro and biodistribution studies demonstrated the possibility of using Tc-EC-CBX to assess COX-2 expression. Planar images confirmed that the tumors could be visualized with Tc-EC-CBX from 0.5 to 4 h in tumor-bearing animal models. We conclude that Tc-EC-CBX may be useful to assess tumor COX-2 expression. This may be useful in the future for selecting patients for treatment with anti-COX-2 agents.

Imaging with 99mTc ECDG targeted at the multifunctional glucose transport system: feasibility study with rodents.

PURPOSE: To evaluate the feasibility of technetium 99m ((99m)Tc) ethylenedicysteine-deoxyglucose (ECDG) imaging in tumor-bearing rodents. MATERIALS AND METHODS: ECDG was synthesized by means of reacting ethylenedicysteine with glucosamine, with carbodiimide as the coupling agent. Hexokinase assays were performed at an ultraviolet wavelength of 340 nm. To determine whether blood glucose level could be altered, ECDG or glucosamine was injected into six rats. In a separate study, ECDG followed by insulin was administered to three rats. To determine biodistribution, lung tumor cells were intramuscularly injected into the hind legs of 18 nude mice. The animals were then injected with (99m)Tc ECDG or fluorine 18 ((18)F) fluorodeoxyglucose (FDG) (0.037-0.074 MBq per mouse). Radioactivity was measured in tissue excised from the animals. Scintigraphy was performed in three groups: in group 1 to demonstrate that different-sized tumors could be imaged after (99m)Tc ECDG administration, in group 2 to ascertain whether tumor uptake of (99m)Tc ECDG was perfusion related, and in group 3 to demonstrate that tumor uptake of (99m)Tc ECDG occurred by means of a glucose-mediated process. RESULTS: ECDG was positive for phosphorylation at hexokinase assay. Blood glucose level increased with ECDG injection and decreased with insulin administration. Tumor-to-brain tissue and tumor-to-muscle tissue ratios of (99m)Tc ECDG uptake were higher than those of (18)F FDG uptake. Scintigraphic results demonstrated the feasibility of (99m)Tc ECDG imaging. CONCLUSION: There are similarities between (99m)Tc ECDG uptake and (18)F FDG uptake in tumors, and study findings supported the potential use of (99m)Tc ECDG as a functional imaging agent.

Assessment of epidermal growth factor receptor with 99mTc-ethylenedicysteine-C225 monoclonal antibody.

Epidermal growth factor receptor (EGFR) plays an important role in cell division and cancer progression, as well as angiogenesis and metastasis. Since many tumor cells exhibit the EGFR on their surface, functional imaging of EGFR provides not only a non-invasive, reproducible, quantifiable alternative to biopsies, but it also greatly complements pharmacokinetic studies by correlating clinical responses with biological effects. Moreover, molecular endpoints of anti-EGFR therapy could be assessed effectively. C225 is a chimeric monoclonal antibody that targets the human extracellular EGFR and inhibits the growth of EGFR-expressing tumor cells. Also, it has been demonstrated that C225, in combination with chemotherapeutic drugs or radiotherapy, is effective in eradicating well-established tumors in nude mice. We have developed 99mTc-labeled C225 using ethylenedicysteine (EC) as a chelator. This study aimed at measuring uptake of 99mTc-EC-C225 in EGFR+ tumor-bearing animal models and preliminary feasibility of imaging patients with head and neck carcinomas. In vitro Western blot analysis and cytotoxicity assays were used to examine the integrity of EC-C225. Tissue distribution studies of 99mTc-EC-C225 were evaluated in tumor-bearing rodents at 0.5-4 h. In vivo biodistribution of 99mTc-EC-C225 in tumor-bearing rodents showed increased tumor-to-tissue ratios as a function of time. In vitro and biodistribution studies demonstrated the possibility of using 99mTc-EC-C225 to assess EGFR expression. SPECT images confirmed that the tumors could be visualized with 99mTc-EC-C225 from 0.5 to 4 h in tumor bearing rodents. We conclude that 99mTc-EC-C225 may be useful to assess tumor EGFR expression. This may be useful in the future for selecting patients for treatment with C225.