Saady Kohanim

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.

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. [Mol Cancer Ther 2006;5(12):2991–3000]

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 (188Re).Methods.Sodium alginate (SA) and calcium chloride were used to create a hydrogel for delivery of CDDP and 188Re. In vitro studies were performed to evaluate cytotoxic effects of 188Re-hydrogel and sustained-release ability of the CDDP-hydrogel. Tumor-bearing rats were injected with 188Re-hydrogel (0.5–1 mCi/rat), 188Re-perrhenate (0.5–1 mCi/rat, intratumoral, I.T.), CDDP-hydrogel (3 mg/kg), and 188Re-hydrogel loaded with CDDP (3 mg/kg body weight, 0.5–1 mCi/rat), respectively, and groups receiving 188Re 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 188Re-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 188Re-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.

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 99mTc. The present study was aimed to develop 99mTc-cyclam-2-nitroimidazole (99mTc-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 99mTc-N4-NIM and 99mTc-N4 was obtained in 13762 rat mammary tumor cells and mesothelioma cells in 6-well plates. Tissue distribution of 99mTc-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 99mTc-N4-NIM was >96% by HPLC. Cell uptake of 99mTc-N4-NIM was higher than 99mTc-N4 in both cell lines. Biodistribution of 99mTc-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 99mTc-N4-NIM in animal models. Efficient synthesis of N4-NIM was achieved. 99mTc-N4-NIM is a novel hypoxic probe and may be useful in evaluating cancer therapy.

Synthesis of 99mTc-EC-AMT as an imaging probe for amino acid transporter systems in breast cancer

ObjectiveThis study was to develop a 99mTc-labeled α-methyl tyrosine (AMT) using L,L-ethylenedicysteine (EC) as a chelator and to evaluate its potential in breast tumor imaging in rodents. MethodsEC-AMT was synthesized by reacting EC and 3-bromopropyl AMT (N-BOC, ethyl ester) in ethanol/potassium carbonate solution. EC-AMT was labeled with 99mTc in the presence of tin (II) chloride. Rhenium-EC-AMT (Re-EC-AMT) was synthesized as a reference standard for 99mTc-EC-AMT. To assess the cellular uptake kinetics of 99mTc-EC-AMT, 13 762 rat breast cancer cells were incubated with 99mTc-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 99mTc-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 18F-2-fluoro-2-deoxy-glucose. Blocking uptake study using unlabeled AMT was conducted to investigate the transport mechanism of 99mTc-EC-AMT in vivo. ResultsStructures 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 99mTc-EC-AMT in 13 762 cells was increased as compared with that of 99mTc-EC and could be inhibited by L-tyrosine. Biodistribution in normal rats showed high in-vivo stability of 99mTc-EC-AMT. Planar scintigraphy at 30 and 90 min showed that 99mTc-EC-AMT could clearly visualize tumors. 99mTc-EC-AMT uptake could be significantly blocked by unlabeled AMT in vivo. ConclusionThe results indicate that 99mTc-EC-AMT, a new amino acid transporter-based radiotracer, is suitable for breast tumor imaging.

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 γ-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. γ-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 μCi/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. [Mol Cancer Ther 2009;8(3):703–10]

Abstract 306: Development of EC-DG as a molecular theranostic personalized medicine

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Purpose: D-glucosamine has been reported to inhibit proliferation of cancer cells in culture and in vivo. We have then synthesized Tc-99m- ethylenedicysteine-glucosamine (EC-DG). We found Tc-99m-EC-DG was involved in cell proliferation in lung, breast and head and neck cultures and could assess breast cancer treatment outcome in vivo by planar scintigraphy. Tc-99m-EC-DG is a safe imaging agent in lung cancer patients. This study was amied to (1) assess a novel response to unlabeled rhenium-EC-DG (Re-EC-DG) involving the translation regulation of hypoxia inducible factor (HIF)-1alpha expression in lymphoma cells, and, (2) evaluate feasibility of using EC-DG for theranostic approaches in cancers. Methods: For theranostic assessment studies, we synthesized cold Re-EC-DG. Re-EC-DG was synthesized via a two-step synthesis. The first step was to synthesize Re-EC by reacting rheniumoxo trichloride with EC. The second step was to react Re-EC with D-glucosamine tetraacetate, followed by de-acetylation. Twelve types of DLBCL cells were incubated with Re-EC-DG at various concentrations (0-10 mM) and TUNEL assays were used to determine cell apoptosis. To ascertain the mechanism of the anticancer properties for Re-EC-DG, DLBCL-LY10 cells were treated with Re-EC-DG (0-5 mM) for 48 hrs. Immunoblotting were then performed on nuclear extracts with 50 µg. For radiotheranostic assessment studies, 13762 breast tumor-bearing rats were imaged with In-111-EC-DG and tumor/muscle ratios were determined at 0.5-24 hrs. Radiation absorbed dose was estimated for the use of Y-90-EC-DG. Results: There was a dose response relationship of Re-EC-DG inhibition in DLBCL cells. Extensive apoptosis was observed at 24 hrs in lymphoma cell cultures. Re-EC-DG showed significant tumorcidal activity compared to normal B-lymphocyte activity at doses >0.17 µmol. Re-EC-DG caused a decreased expression of HIF-1alpha under normoxic conditions in DLBCL-LY10 cells. Tumor-to-muscle ratios for In-111-EC-DG were 5.43±0.45 to 7.80±0.05 whereas In-111-EC had 3.24±0.32 to 4.64±0.16 at 0.5-24 hrs. Radiation exposure of In-111-EC-DG to whole body, blood-forming organs, gonads, and effective dose equivalent for a single dose at 5 mCi was below the limits of 3 rad annually and 5 rad total. The absorbed dose in all other organs was below the limits of 5 rad annually and 15 rad total. Conclusion: EC-DG is a useful molecular theranostic compound. In-111-EC-DG has favorable dosimetry, providing a potential use of Y-90-EC-DG to treat cancers. Re-EC-DG inhibits HIF-1alpha expression and is an attractive anti-proliferation compound. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 306. doi:10.1158/1538-7445.AM2011-306