Development of (99m)Tc-N4-NIM for molecular imaging of tumor hypoxia.

The nitro group of 2-nitroimidazole (NIM) enters the tumor cells and is bioreductively activated and fixed in the hypoxia cells. 1,4,8,11-tetraazacyclotetradecane (N4) has shown to be a stable chelator for (99m)Tc. The present study was aimed to develop (99m)Tc-cyclam-2-nitroimidazole ((99m)Tc-N4-NIM) for tumor hypoxia imaging. N4-NIM precursor was synthesized by reacting N4-oxalate and 1,3-dibromopropane-NIM, yielded 14% (total synthesis). Cell uptake of (99m)Tc-N4-NIM and (99m)Tc-N4 was obtained in 13762 rat mammary tumor cells and mesothelioma cells in 6-well plates. Tissue distribution of (99m)Tc-N4-NIM was evaluated in breast-tumor-bearing rats at 0.5-4 hrs. Tumor oxygen tension was measured using an oxygen probe. Planar imaging was performed in the tumor-bearing rat and rabbit models. Radiochemical purity of (99m)Tc-N4-NIM was >96% by HPLC. Cell uptake of (99m)Tc-N4-NIM was higher than (99m)Tc-N4 in both cell lines. Biodistribution of (99m)Tc-N4-NIM showed increased tumor-to-blood and tumor-to-muscle count density ratios as a function of time. Oxygen tension in tumor tissue was 6-10 mmHg compared to 40-50 mmHg in normal muscle tissue. Planar imaging studies confirmed that the tumors could be visualized clearly with (99m)Tc-N4-NIM in animal models. Efficient synthesis of N4-NIM was achieved. (99m)Tc-N4-NIM is a novel hypoxic probe and may be useful in evaluating cancer therapy.

Molecular imaging of Bcr-Abl phosphokinase in a xenograft model.

The purpose of this study was to determine whether the Bcr-Abl tyrosine kinase can be assessed by gamma-imaging using an 111In-labeled anti-phosphotyrosine (APT) antibody, and if the response to treatment with imatinib could be detected using this imaging technique. APT antibody was labeled with 111In using ethylenedicysteine (EC) as a chelator. To determine if 111In-EC-APT could assess a nonreceptor tyrosine kinase, xenografts of the human chronic myelogenous leukemia cell line K562 were used. gamma-Scintigraphy of the tumor-bearing mice, before and after imatinib treatment, was obtained 1, 24, and 48 h after they were given 111In-EC-APT (100 microCi/mouse i.v.). 111In-EC-APT is preferentially taken up by Bcr-Abl-bearing tumor cells when compared with 111In-EC-BSA or 111In-EC-IgG1 controls and comparable with the level of uptake of 111In-EC-Bcr-Abl. Imatinib treatment resulted in decreased expression of phospho-Bcr-Abl by Western blot analysis, which correlated with early (4 days after starting imatinib) kinase down-regulation as assessed by imaging using 111In-EC-APT. The optimal time to imaging was 24 and 48 h after injection of 111In-EC-APT. Although tumor regression was insignificant on day 4 after starting imatinib treatment, it was marked by day 14. 111In-EC-APT can assess intracellular phosphokinase activity, and down-regulation of phosphokinase activity predates tumor regression. This technique may therefore be useful in the clinic to detect the presence of phosphokinase activity and for early prediction of response.

Imaging and dosimetry of 99mTc EC annexin V: preliminary clinical study targeting apoptosis in breast tumors.

BACKGROUND: Early detection of cellular events is important to predict the outcome of the patients. This study was aimed to use (99m)Tc EC-annexin V to image tumor cells undergoing apoptosis. METHODS: In 10 patients with breast cancer, scintigraphic images and dosimetric estimates were obtained after administering (99m)Tc EC-annexin V. RESULTS: Nine of the 10 cases showed detectable (99m)Tc EC-annexin V uptake in tumor. Higher values of T/N ratios are associated with patient after treatment. CONCLUSIONS: Apoptosis can be quantified using (99m)Tc EC-annexin V.

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.

Novel in vivo imaging shows up-regulation of death receptors by paclitaxel and correlates with enhanced antitumor effects of receptor agonist antibodies.

Susceptibility to apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is mediated through cognate death receptor signaling. We hypothesized that auto-amplification of this apparatus would enhance antitumor effects in vivo and could be optimized using the results obtained from novel imaging techniques. We therefore imaged mice bearing human colorectal cancer (Colo205) tumor xenografts with HGS-ETR1 and HGS-ETR2 agonist antibodies to TRAIL receptor-1 (TRAIL-R1) and TRAIL-R2, respectively, after radiolabeling the antibodies. Paclitaxel significantly increased in vivo expression of TRAIL-R1 and TRAIL-R2 in a time-dependent manner. The imaging results were confirmed by immunoblots for steady-state protein levels (>20-fold increase in TRAIL-R1 and TRAIL-R2 levels in tumor xenografts by 48 h after paclitaxel administration). TRAIL-R1 and TRAIL-R2 mRNA expression did not change, suggesting that these effects were posttranscriptional. Sequential treatment with paclitaxel followed by HGS-ETR1 or HGS-ETR2 after 48 h resulted in markedly enhanced antitumor activity against Colo205 mouse xenografts. Our experiments suggest that sequential taxane treatment followed by TRAIL-R agonist antibodies could be applied in the clinic, and that novel imaging techniques using radiolabeled receptor antibodies may be exploitable to optimize sequence timing and patient selection.

Radiolabeled L-lysine for tumor imaging.

RATIONALE AND OBJECTIVES: The aims of this study were to label the versatile amino acid l-lysine with (99m)Tc using 2,3-dimercapto-succinic acid (DMSA) as a chelator, and to assess its tumor imaging feasibility under in vivo and in vitro conditions, and finally to determine the subcellular biodistribution of this radiopharmaceutical. MATERIALS AND METHODS: DMSA-l-lysine was chemically synthesized and labeled with sodium pertechnetate. Nuclear magnetic resonance (NMR) and mass spectral analysis of DMSA-l-lysine were conducted. Radiochemical purity was determined by thin-layer chromatography (TLC) and paper chromatography. Cellular uptake, competition and subcellular localization studies were performed in rat breast cancer cells (13762). In vivo studies of planar imaging and biodistribution studies were performed on female Fischer 344 rats. Medical Internal Radiation Dose (MIRD) dosimetry estimates were calculated. RESULTS: Radiochemical purity (determined by radio-TLC and high-performance liquid chromatography) of these compounds was >95%. (99m)Tc-DMSA-l-lysine showed good uptake in in vitro cell culture assays and uptake was reduced in competition studies. (99m)Tc-DMSA-l-lysine accumulates in the nucleus as much as in the cytoplasm and it was also shown that accumulation of the (99m)Tc-DMSA-l-lysine in the nucleus increases as a function of a time. There was an increase in tumor-to-blood and tumor-to-muscle count density ratios. Tumor/background ratios were 5.75 at 1 hour and 6.87 at 2 hours. In vivo tissue distribution studies revealed that radiation dosimetry of blood-forming organs were within radiation dose limits. CONCLUSION: DMSA-l-lysine kits can be labeled with (99m)Tc easily and efficiently, with high radiochemical purity and cost-effectiveness. In vitro cellular uptake and scintigraphic imaging studies demonstrated the pharmacokinetic distribution and feasibility of using (99m)Tc-DMSA-l-lysine for tumor imaging.

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.

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.

Synthesis of (99m)Tc-EC-AMT as an imaging probe for amino acid transporter systems in breast cancer.

OBJECTIVE: This study was to develop a (99m)Tc-labeled alpha-methyl tyrosine (AMT) using L,L-ethylenedicysteine (EC) as a chelator and to evaluate its potential in breast tumor imaging in rodents. METHODS: EC-AMT was synthesized by reacting EC and 3-bromopropyl AMT (N-BOC, ethyl ester) in ethanol/potassium carbonate solution. EC-AMT was labeled with (99m)Tc in the presence of tin (II) chloride. Rhenium-EC-AMT (Re-EC-AMT) was synthesized as a reference standard for (99m)Tc-EC-AMT. To assess the cellular uptake kinetics of (99m)Tc-EC-AMT, 13 762 rat breast cancer cells were incubated with (99m)Tc-EC-AMT for 0-2 h. To investigate the transport mechanism, the same cell line was used to conduct the competitive inhibition study using L-tyrosine. Tissue distribution of (99m)Tc-EC-AMT was determined in normal rats at 0.5-4 h. Planar imaging of breast tumor-bearing rats was performed at 30 and 90 min. The data were compared with those of (18)F-2-fluoro-2-deoxy-glucose. Blocking uptake study using unlabeled AMT was conducted to investigate the transport mechanism of (99m)Tc-EC-AMT in vivo. RESULTS: Structures of EC-AMT and Re-EC-AMT were confirmed by nuclear magnetic resonance, high performance liquid chromatography and mass spectra. In-vitro cellular uptake of (99m)Tc-EC-AMT in 13,762 cells was increased as compared with that of (99m)Tc-EC and could be inhibited by L-tyrosine. Biodistribution in normal rats showed high in-vivo stability of (99m)Tc-EC-AMT. Planar scintigraphy at 30 and 90 min showed that (99m)Tc-EC-AMT could clearly visualize tumors. (99m)Tc-EC-AMT uptake could be significantly blocked by unlabeled AMT in vivo. CONCLUSION: The results indicate that (99m)Tc-EC-AMT, a new amino acid transporter-based radiotracer, is suitable for breast tumor imaging.

Molecular imaging of gefitinib activity in an epidermal growth factor receptor (EGFR)-bearing xenograft model.

Finding noninvasive methods to discern which patients' tumors bear a specific target molecule, and are presumably more likely to respond, remains a critical challenge. An anti-phospho-tyrosine antibody was labeled with indium ((111)In) using ethylenedicysteine (EC) as a chelator ((111)In-EC-P-Tyr). We hypothesized that tumor phosphokinase activity would be discernible by imaging with (111)In-EC-P-Tyr. A xenograft of A431 cells, a human epithelial carcinoma cell line overexpressing epidermal growth factor receptor (EGFR), was employed. Biodistribution studies confirmed increased tumor/muscle ratios of (111)In-EC-P-Tyr in the A431 model. Imaging demonstrated that a marked decrease in tumor uptake of (111)In-EC-P-Tyr occurred after 3 d of gefitinib therapy in A431 cells (gefitinib-sensitive), but not in H441 cells (gefitinib-resistant). Our results indicate that (111)In-EC-P-Tyr can detect tumor phospho-tyrosine kinase activity in animal models. This type of agent merits investigation in the clinic to determine if it can predict patient responses to kinase inhibitors based on phosphokinase imaging.

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.

Regional radiochemotherapy using in situ hydrogel.

PURPOSE: To evaluate the feasibility of regional radiochemotherapy of mammary tumors using in situ hydrogel loaded with cisplatin (CDDP) and rhenium-188 ((188)Re). METHODS: Sodium alginate (SA) and calcium chloride were used to create a hydrogel for delivery of CDDP and (188)Re. In vitro studies were performed to evaluate cytotoxic effects of (188)Re-hydrogel and sustained-release ability of the CDDP-hydrogel. Tumor-bearing rats were injected with (188)Re-hydrogel (0.5-1 mCi/rat), (188)Re-perrhenate (0.5-1 mCi/rat, intratumoral, I.T.), CDDP-hydrogel (3 mg/kg), and (188)Re-hydrogel loaded with CDDP (3 mg/kg body weight, 0.5-1 mCi/rat), respectively, and groups receiving (188)Re were imaged at 24 and 48 h postinjection. Tumor volume, body weight, imaging, and kidney function were assessed as required for each group. RESULTS: Successful formation of the hydrogel was demonstrated by cytotoxic effects of (188)Re-hydrogel and slow release of CDDP-hydrogel in vitro. Tumor volume measurements showed significant delay in tumor growth in treated vs. control groups with minimal variation in normal kidney function for the CDDP-hydrogel group. Scintigraphic images indicated localization of (188)Re-hydrogel in the tumor site up to 48 h postinjection. CONCLUSIONS: Our data demonstrate the feasibility of using hydrogel for delivery of chemotherapeutics and radiation locally. This technique may have applications involving other contrast modalities as well as treatment in cases where tumors are inoperable.

(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.