Yinhan Zhang

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.

Development of tyrosine-based radiotracer 99mTc-N4-Tyrosine for breast cancer imaging.

The purpose of this study was to develop an efficient way to synthesize 99mTc-O-[3-(1,4,8,11-tetraazabicyclohexadecane)-propyl]-tyrosine (99mTc-N4-Tyrosine), a novel amino acid-based radiotracer, and evaluate its potential in breast cancer gamma imaging. Precursor N4-Tyrosine was synthesized using a 5-step procedure, and its total synthesis yield was 38%. It was successfully labeled with 99mTc with high radiochemical purity (>95%). Cellular uptake of 99mTc-N4-Tyrosine was much higher than that of 99mTc-N4 and the clinical gold standard 18F-2-deoxy-2-fluoro-glucose (18F-FDG) in rat breast tumor cells in vitro. Tissue uptake and dosimetry estimation in normal rats revealed that 99mTc-N4-Tyrosine could be safely administered to humans. Evaluation in breast tumor-bearing rats showed that although 99mTc-N4-Tyrosine appeared to be inferior to 18F-FDG in distinguishing breast tumor tissue from chemical-induced inflammatory tissue, it had high tumor-to-muscle uptake ratios and could detect breast tumors clearly by planar scintigraphic imaging. 99mTc-N4-Tyrosine could thus be a useful radiotracer for use in breast tumor diagnostic imaging.

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 99mTc-O-[3-(1,4,8,11-tetraazabicyclohexadecane)-propyl]-α-methyl tyrosine (99mTc-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 99mTc-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 99mTc-N4-AMT was significantly higher than that of 99mTc-N4. Planar imaging revealed that 99mTc-N4-AMT rendered greater tumor/muscle ratios than 99mTc-N4. Conclusions. N4-AMT could be synthesized with a considerably high yield. Our in vitro and in vivo data suggest that 99mTc-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.

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.

Automated Synthesis of 18F-Fluoropropoxytryptophan for Amino Acid Transporter System Imaging

Objective. This study was to develop a cGMP grade of [18F]fluoropropoxytryptophan (18F-FTP) to assess tryptophan transporters using an automated synthesizer. Methods. Tosylpropoxytryptophan (Ts-TP) was reacted with K18F/kryptofix complex. After column purification, solvent evaporation, and hydrolysis, the identity and purity of the product were validated by radio-TLC (1M-ammonium acetate : methanol = 4 : 1) and HPLC (C-18 column, methanol : water = 7 : 3) analyses. In vitro cellular uptake of 18F-FTP and 18F-FDG was performed in human prostate cancer cells. PET imaging studies were performed with 18F-FTP and 18F-FDG in prostate and small cell lung tumor-bearing mice (3.7 MBq/mouse, iv). Results. Radio-TLC and HPLC analyses of 18F-FTP showed that the Rf and Rt values were 0.9 and 9 min, respectively. Radiochemical purity was >99%. The radiochemical yield was 37.7% (EOS 90 min, decay corrected). Cellular uptake of 18F-FTP and 18F-FDG showed enhanced uptake as a function of incubation time. PET imaging studies showed that 18F-FTP had less tumor uptake than 18F-FDG in prostate cancer model. However, 18F-FTP had more uptake than 18F-FDG in small cell lung cancer model. Conclusion. 18F-FTP could be synthesized with high radiochemical yield. Assessment of upregulated transporters activity by 18F-FTP may provide potential applications in differential diagnosis and prediction of early treatment response.

Synthesis and biological evaluation of O-[3-18F-fluoropropyl]-α-methyl tyrosine in mesothelioma-bearing rodents.

Radiolabeled tyrosine analogs enter cancer cells via upregulated amino acid transporter system and have been shown to be superior to 18F-fluoro-2-deoxy-D-glucose (18F-FDG) in differential diagnosis in cancers. In this study, we synthesized O-[3-19F-fluoropropyl]-α-methyl tyrosine (19F-FPAMT) and used manual and automated methods to synthesize O-[3-18F-fluoropropyl]-α-methyl tyrosine (18F-FPAMT) in three steps: nucleophilic substitution, deprotection of butoxycarbonyl, and deesterification. Manual and automated synthesis methods produced 18F-FPAMT with a radiochemical purity >96%. The decay-corrected yield of 18F-FPAMT by manual synthesis was 34% at end-of-synthesis (88 min). The decay-corrected yield of 18F-FPAMT by automated synthesis was 15% at end-of-synthesis (110 min). 18F-FDG and 18F-FPAMT were used for in vitro and in vivo studies to evaluate the feasibility of 18F-FPAMT for imaging rat mesothelioma (IL-45). In vitro studies comparing 18F-FPAMT with 18F-FDG revealed that 18F-FDG had higher uptake than that of 18F-FPAMT, and the uptake ratio of 18F-FPAMT reached the plateau after being incubated for 60 min. Biodistribution studies revealed that the accumulation of 18F-FPAMT in the heart, lungs, thyroid, spleen, and brain was significantly lower than that of 18F-FDG. There was poor bone uptake in 18F-FPAMT for up to 3 hrs suggesting its in vivo stability. The imaging studies showed good visualization of tumors with 18F-FPAMT. Together, these results suggest that 18F-FPAMT can be successfully synthesized and has great potential in mesothelioma imaging.

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