Richard Mendez

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.

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.

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 99mTc-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 99mTc-EC–CBX to measure COX-2 activity. Tissue distribution studies of 99mTc-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 99mTc-EC–CBX than lower COX-2 expression (H226). In vivo biodistribution of 99mTc-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 99mTc-EC–CBX to assess COX-2 expression. Planar images confirmed that the tumors could be visualized with 99mTc-EC–CBX from 0.5 to 4 h in tumor-bearing animal models. We conclude that 99mTc-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.

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.

Targeting the hexosamine biosynthetic pathway and O-linked N-acetylglucosamine cycling for therapeutic and imaging capabilities in diffuse large B-cell lymphoma

The hexosamine biosynthetic pathway (HBP) requires two key nutrients glucose and glutamine for O-linked N-acetylglucosamine (O-GlcNAc) cycling, a post-translational protein modification that adds GlcNAc to nuclear and cytoplasmic proteins. Increased GlcNAc has been linked to regulatory factors involved in cancer cell growth and survival. However, the biological significance of GlcNAc in diffuse large B-cell lymphoma (DLBCL) is not well defined. This study is the first to show that both the substrate and the endpoint O-GlcNAc transferase (OGT) enzyme of the HBP were highly expressed in DLBCL cell lines and in patient tumors compared with normal B-lymphocytes. Notably, high OGT mRNA levels were associated with poor survival of DLBCL patients. Targeting OGT via small interference RNA in DLBCL cells inhibited activation of GlcNAc, nuclear factor kappa B (NF-κB), and nuclear factor of activated T-cells 1 (NFATc1), as well as cell growth. Depleting both glucose and glutamine in DLBCL cells or treating them with an HBP inhibitor (azaserine) diminished O-GlcNAc protein substrate, inhibited constitutive NF-κB and NFATc1 activation, and induced G0/G1 cell-cycle arrest and apoptosis. Replenishing glucose-and glutamine-deprived DLBCL cells with a synthetic glucose analog (ethylenedicysteine-N-acetylglucosamine [ECG]) reversed these phenotypes. Finally, we showed in both in vitro and in vivo murine models that DLBCL cells easily take up radiolabeled technetium-99m-ECG conjugate. These findings suggest that targeting the HBP has therapeutic relevance for DLBCL and underscores the imaging potential of the glucosamine analog ECG in DLBCL.

Molecular Imaging of Mesothelioma with 99mTc-ECG and 68Ga-ECG

We have developed ethylenedicysteine-glucosamine (ECG) as an alternative to 18F-fluoro-2-deoxy-D-glucose (18F-FDG) for cancer imaging. ECG localizes in the nuclear components of cells via the hexosamine biosynthetic pathway. This study was to evaluate the feasibility of imaging mesothelioma with 99mTc-ECG and 68Ga-ECG. ECG was synthesized from thiazolidine-4-carboxylic acid and 1,3,4,6-tetra-O-acetyl-2-amino-D-glucopyranose, followed by reduction in sodium and liquid ammonia to yield ECG (52%). ECG was chelated with 99mTc/tin (II) and 68Ga/69Ga chloride for in vitro and in vivo studies in mesothelioma. The highest tumor uptake of 99mTc-ECG is 0.47 at 30 min post injection, and declined to 0.08 at 240 min post injection. Tumor uptake (%ID/g), tumor/lung, tumor/blood, and tumor/muscle count density ratios for 99mTc-ECG (30–240 min) were 0.47 ± 0.06 to 0.08 ± 0.01; 0.71 ± 0.07 to 0.85 ± 0.04; 0.47 ± 0.03 to 0.51 ± 0.01, and 3.49 ± 0.24 to 5.06 ± 0.25; for 68Ga-ECG (15–60 min) were 0.70 ± 0.06 to 0.92 ± 0.08; 0.64 ± 0.05 to 1.15 ± 0.08; 0.42 ± 0.03 to 0.67 ± 0.07, and 3.84 ± 0.52 to 7.00 ± 1.42; for 18F-FDG (30–180 min) were 1.86 ± 0.22 to 1.38 ± 0.35; 3.18 ± 0.44 to 2.92 ± 0.34, 4.19 ± 0.44 to 19.41 ± 2.05 and 5.75 ± 2.55 to 3.33 ± 0.65, respectively. Tumor could be clearly visualized with 99mTc-ECG and 68Ga-ECG in mesothelioma-bearing rats. 99mTc-ECG and 68Ga-ECG showed increased uptake in mesothelioma, suggesting they may be useful in diagnosing mesothelioma and also monitoring therapeutic response.

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.

99mTc-N4amG: Synthesis Biodistribution and Imaging in Breast Tumor-bearing Rodents

(99m)Tc-N4-guanine ((99m)Tc-N4amG) was synthesized and evaluated in this study. Cellular uptake and cellular fraction studies were performed to evaluate the cell penetrating ability. Biodistribution and planar imaging were conducted in breast tumor-bearing rats. Up to 17%ID uptake was observed in cellular uptake study with 40% of (99m)Tc-N4amG was accumulated in the nucleus. Biodistribution and scintigraphic imaging studies showed increased tumor/muscle count density ratios as a function of time. Our results demonstrate the feasibility of using (99m)Tc-N4amG in tumor specific imaging.

Abstract 218: The hexosamine biosyntheic pathway and O-linked glycosylation for targeted therapy in diffuse large B-cell lymphoma

Mammalian cells fuel their growth and proliferation through the catabolism of two main substrates: glucose and glutamine. Both nutrients are required for the synthesis of glucosamine, the precursor substrate of the hexosamine biosynthetic pathway. The hexosamine signaling pathway terminating in O-linked N-acetyl glucosamine (O-GlcNAc) cycling has been implicated in cellular signaling cascades and regulation of transcription factors involved in cancer biology. Biological functions of the hexosamine biosynthetic signaling pathways need elucidation, to determine whether altered O-GlcNAc metabolism plays a significant role in hematologic tumors such as diffuse large B-cell lymphoma (DLBCL), and utilize this bifunctional pathway as a targeted therapeutic strategy in DLBCL. We have identified a key enzyme of the hexosamine biosynthetic pathways to be highly-expressed in DLBCL cell lines and patient tumor cells. In contrast to normal circulating and tonsillar B cells, DLBCL cells expressed high levels of the terminating enzyme O-GlcNAc transferase (OGT). OGT mRNA expression is highly expressed in DLBCL in comparaison to other cancers. We discovered that several key growth and survival transcription factors, such as NF-kB and NFAT, known to be highly-activated in DLBCL, are linked to the hexoasmine biosynthetic pathway. We demonstrated that both NF-kB (p65) and NFATc1 directly associated with OGT, and down-regulation of OGT by siRNA inhibits these transcription factors activation, suggesting that both NF-kB-p65 and NFATc1 require O-GlcNAc glycosylation by OGT for their activation. These results suggest that the hexosamine pathway is highly active and utilized in DLBCL, and that exploiting this bi-functional pathway(s) as a therapeutic approach is feasible. We have previously developed an imaging agent, 99mTc-ethylenedicysteine-glucosamine (99mTc-EC-G) because EC-G mimics phosphorylated N-acetylglucosamine. ECG treatment in DLBCL cells enhances p65 and NFATc1 nuclear translocation. For therapeutic strategies, we developed metallic unlabeled Platinum (Pt) derivatives-EC-G as potential therapeutic agents. Pre-clinical in vitro studies have shown that our two lead compounds, Pt-9 and Pt-DACH-EC-G effectively inhibit lymphoma cell growth and induce apoptosis. These lead compounds can also induce DNA damage in DLBCL cells, through the up-regulation of phosphorylated histone 2AX (pH2AX), leading to the disruption of p65 and NFATc1 binding to DNA. This data importantly demonstrates that the hexosamine biosynthetic pathway is linked to key growth and survival pathways involved in the pathophysiology of DLBCL. Targeting these pathways with novel platinum EC-G compounds as a theranostic approach should lead to new, more effective treatments and diagnosis for DLBCL, particularly for relapsed/refractory DLBCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 218. doi:1538-7445.AM2012-218

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