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.


Introduction
Drug discovery is accelerating due to mapping of molecular targets and the rapid synthesis of high-throughput in vitro testing of compounds in their early stage of the drug development process. The development of radiolabeled biochemical compounds, understanding molecular pathways and imaging devices to detect the radioactivity by external imaging, has expanded the use of nuclear molecular imaging studies in drug development. Nuclear molecular imaging provides vascular angiogenesis, cellular translational, and transcriptional information. The important applications in molecular imaging in oncology are at the characterization of tumors (degree of malignancy), optimal dosing determination, differentiation (i.e., inflammation/infection versus recurrence, sensitive versus resistant, low versus high grade), and prediction of treatment response (i.e., select patient who may respond to therapy). Thus, molecular imaging helps to control and monitor dosage for increased safety and effectiveness. The focus of molecular imaging in oncology is to identify tumor-specific markers and apply these markers for evaluation of patient response to treatment. Nuclear molecular imaging could noninvasively assess diseases treatment endpoints which used to rely almost exclusively on biopsies and histopathological assays. 18 F-Fluoro-deoxyglucose ( 18 F-FDG), a gold standard for molecular imaging, utilizes glucose transporters and hexokinase phosphorylated processes for tumor imaging [1]. However, 18 F-FDG has several limitations that give rise to false positive/negative results [2]. 18 F-FDG has poor differentiation between tumor and inflammation/infection due to its high uptake in granulocytes and macrophages. Therefore, it is amenable to develop a radiotracer as an alternative for better differentiation in tumor imaging. Glucosamine enters into cells via hexosamine biosynthetic pathway and its regulatory products of glucosamine-6-phosphate mediate insulin activation, downstream signaling, glycosylation, and tumor growth. In the hexosamine pathway, upregulated glucose transporters induce overexpression of glutamine: fructose-6-phosphate amidotransferase (GFAT). GFAT uses the amide group of glutamine to convert fructose 6-phosphate to glucosamine 6-phosphate and forms hexosamine products [3]. Phosphorylated glucosamine interacts with uridine diphosphate (UDP) to form UDPN-acetylglucosamine (UDP-GlcNAc). The dynamic glycosylation of serine or threonine residues on nuclear and cytosolic proteins by O-linked protein N-acetylglucosamine (O-GlcNAc) transferase is abundant in all multicellular eukaryotes. Glycosylation is a part of posttranslational modification and appears to modify a large number of nucleocytoplasmic proteins. O-GlcNAc transferase (OGT) activity is exquisitely responsive to intracellular UDP-GlcNAc and UDP concentrations, which are in turn highly sensitive to glucose concentrations and other stimuli [4]. In cell nucleus, the ubiquitous transcription factor Sp1 is extensively modified by O-GlcNAc. Sp1 becomes hyperglycosylated in response to hyperglycemia or elevated glucosamine [4]. Because O-GlcNAc is involved in hexosamine pathway and nucleus activity, it becomes an attractive imaging agent for differential diagnosis in cancers. 68 Ga (89% positron, 68 min half-life) and 99m Tc (140 keV, 6 hrs half-life) are obtained from generators onsite and have significant commercial interest. 68 Ga and 99m Tc could provide serial images which are pivotal to clinical applications by positron emission tomography (PET) and single photon emission-computed tomography (SPECT). PET/SPECT/CT is better than PET and SPECT alone because multiple slices by CT and serial images by PET and SPECT provide a better delineation in tumor volumes. L,L-ethylenedicysteine (EC), a family of bisaminoethanethiol, is known to form stable metal complexes [5,6]. The strong metal complexing property of such EC systems is also applied for labeling of small molecules, proteins, and peptides after conjugation to EC derivatives. EC-technology platform has shown to coordinate radiometals and metals for image-guided target assessment, theranostic applications, and the selection of patient for treatment [7][8][9][10][11][12][13][14][15][16][17][18][19].
Mesothelioma is a cancer caused by exposure to asbestos. Asbestos fibers have been shown to alter the function and secretory properties of macrophages, ultimately creating conditions which favor the development of mesothelioma. Following asbestos phagocytosis, macrophages generate increased amounts of hydroxyl radicals which are thought to promote asbestos carcinogenicity. Furthermore, genetic alterations in asbestos-activated macrophages may result in the release of potent mesothelial cell mitogens such as plateletderived growth factor and transforming growth factor-β which, in turn, may induce the chronic stimulation and proliferation of mesothelial cells after injury by asbestos fibers. Although there have been some modest improvements in prognosis from newer chemotherapies and multimodality treatments, the prognosis for malignant mesothelioma remains poor. The standard approaches such as radiation, chemotherapy, and surgery have been investigated to treat patients with malignant pleural mesothelioma. Treatment of malignant mesothelioma at earlier stages has a better prognosis, but cures are exceedingly rare. Immunohistochemical analysis has shown that GlcNAc-specific binding sites are useful for distinguishing metastatic carcinoma from mesothelioma in human [20]. Subsequently, EC-glucosamine (ECG), a glucose analogue, is developed to trace the glucose transport system and glucosamine binding sites in mesothelioma. 99m Tc-ECG and 68 Ga-ECG may assess the staging, restaging, and response monitoring in mesothelioma for early and right medication of this disease.

2.2.2.
Step 2: Reduction Reaction. Sodium was added piece by piece to a solution of T-G-(Ac) 4 (4.08 g, 8.8 mmol) in liquid ammonia (170 g). The color of the solution was slowly changed to dark blue. After 30 minutes, a little of ammonium chloride was added. The liquid ammonia was removed by reduced pressure. The residual solid was triturated with methanol (100 mL). The solid was then filtered and washed with additional methanol (50 mL) to yield crude product 4.16 g. To obtain analytical pure ECG, the crude product (0.1 g) was dissolved in 1.0 mL of HCl (0.1 N) and purified with sephadex column by eluting with H 2 O. The aqueous fractions were combined and lyophilized to yield EC-G 0.029 g (46.7%). NMR, mass spectrometry, and HPLC were used to confirm the structure of ECG. 69 GaCl 3 (20 mg, 0.11 mmol) in 0.2 mL H 2 O was added to a solution of ECG (60 mg, 0.1 mmol) in 0.5 mL H 2 O. The pH value was adjusted to 4-5 with 0.1 N NaOH (50 μL). The solution was heated for 30 min at 60 • C. The product was purified by a sephadex column eluting with H 2 O to yield Ga-ECG. After lyophilization, Ga-ECG was obtained as white solid (52 mg, 78.1%). NMR, mass spectrometry, and HPLC were used to confirm the structure of 69 Ga-ECG.

Biodistribution of Radiotracers in Mesothelioma-Bearing
Rats. Female Fischer 344 rats (150 ± 25 g) (Harlan Sprague-Dawley, Indianapolis, IN) (n = 3 rats/time point) were inoculated with malignant pleural mesothelioma cells derived from the IL-45 cell line. Tumor cells (10 6 cells/rat) were injected (i.m.) into the hind legs. Studies were performed 14 to 17 days after inoculation when tumors were approximately 1 cm in diameter. In tissue distribution studies, each animal was injected (iv, 10 μCi/rat, 10 μg/rat) with 99m Tc-ECG, 68 Ga-ECG, and 18 F-FDG. Rats were sacrificed at 0.5-4 hrs. The selected tissues were excised, weighed, and counted for radioactivity by using a gamma counter (Packard Instruments, Downers Grove, IL). The biodistribution of tracer in each sample was calculated as percentage of the injected dose per gram of tissue wet weight (%ID/g).

Scintigraphic Imaging Studies.
Female Fischer 344 rats (150 ± 25 g) bearing malignant pleural mesothelioma (at hind legs) derived from the IL-45 cell line were used for imaging studies. Studies were performed 14 to 17 days after inoculation when tumors were approximately 1 cm in diameter. Scintigraphic images were obtained either from a micro-PET (Inveon) embedded in the gantries coordinate PET/CT data acquisition or from an M-gamma camera (Siemens Medical Systems, Inc., Hoffman Estates, IL) equipped with low-energy parallel-hole collimator. Each animal was administered with 99m Tc-ECG (300 μCi/rat, iv), 68 Ga-ECG, and 18 F-FDG (400 μCi/rat, iv), and the images were obtained at 0.5-4 hrs. To demonstrate 68 Ga-ECG could be used for image-guided therapy, the same mesothelioma-bearing rats (n = 3) at tumor volume 1.5 cm were treated with paclitaxel (20 mg/kg, iv, single injection). Prior to treatment and postpaclitaxel treatment on day 7, the tumor-bearing rats were imaged with 68 Ga-ECG. Computer-outlined regions of interest (ROI) (counts per pixel) were used to determine tumor-to-background count density ratios for 99m Tc-ECG. Computer outlined regions of interest (ROI) (counts per pixel) for tumor and muscle at the corresponding time interval were used to generate a dynamic plot for 68 Ga-ECG and 18 F-FDG. Dynamic plot was from 0 to 45 minutes. Paclitaxel was selected because it produced antiproliferative effects by inhibition of glucose transporters (Glut-1) in cell line studies [21]. Also, it has been reported that mesothelioma responds to paclitaxel treatment in the animal model [22].

In Vivo Biodistribution Studies.
Tumor and tissue uptake (%ID/g) of 68 Ga-ECG, 99m Tc-ECG and 18 F-FDG are shown in Tables 1, 2, and 3. The highest tumor uptake of 99m Tc-ECG is 0.47 at 30 min after injection, and declined to 0.08 at 240 min after injection. Tumor uptake (%ID/g), tumor/lung, tumor/blood, and tumor/muscle count density ratios for

Scintigraphic Imaging Studies.
Scintigraphic images of rats administered 68 Ga-ECG, 99m Tc-ECG, and 18 F-FDG showed that tumors could be clearly visualized at 0.5-4 hrs ( Figures 5-7). Dynamic plot of tumor uptake with 68 Ga-ECG and 18 F-FDG showed similar transporter pattern ( Figure 5). 68 Ga-ECG was able to monitor paclitaxel treatment response in the same mesothelioma-bearing rats ( Figure 6). Two rats receiving 99m Tc-ECG (middle and right) were randomly selected to compare to that of the rat receiving 99m Tc-EC (left) under the same imaging panel. Tumor in 99m Tc-ECG group showed much higher uptake than 99m Tc-EC (control) group at 1 and 2 hrs (Figure 7).

Discussion
Previous studies have shown that 99m Tc-ECG exhibits characteristics similar to 18 F-FDG in terms of the glucose membrane transport process and tumor uptake [11]. These glucose membrane transporters are involved in both the glycolytic pathway and the hexosamine biosynthetic pathway. Cell cycle analysis revealed that 99m Tc-ECG was able to transport across the nucleus membrane and involved in proliferation activity in all phases cell cycles [14]. Moreover, the thymidine incorporation assay studies showed similar uptake patterns for both unlabeled ECG and glucose, suggesting that both ECG and glucose were involved in the proliferation/growth activity of cells, whereas unlabeled FDG was not [14].  The lack of 18 F-FDG involvement in DNA proliferation is attributable to the presence of the fluorine atom at position 2 of the molecule, which prevents its metabolism and consequently its utilization in cell proliferation and growth [23]. As both molecules enter a cell and become phosphorylated by hexokinase, it can be diverted from the main glycolytic/glycogen pathways into accessory pathways. For example, under normal conditions, cells utilize 95% glucose which is transported via transporters 1 and 3 through the glycolytic pathway. 18 F-FDG uses transporters 1 and 3. 99m Tc-ECG uses transporters 2 and 4 which are the transporters for glucosamine and transport glucose by the hexosamine pathway under normal conditions [24]. In addition, under abnormal conditions, only 3 to 5% of glucose is being utilized by the glycolytic pathway. However, in the case where an abnormal condition exists, there is a dramatic shift from 95% and above [25]. It should be noted that when the cell senses stress or a disease state occurs, the cells become hyper and glucose deficient/depleted, which in turn deactivates the glycolytic pathway. The hexosamine biosynthesis pathway then becomes active and takes over. The proliferation rates for tumors are with 100-1000x more  than normal cells and thus a relatively greater concentration of 99m Tc-ECG than normal cells. 99m Tc-ECG can be utilized by cancer cells; thus the imaging studies are successful. Clinical phase 1 and 2 trials revealed that 99m Tc-ECG was safe and had favorable radiation dosimetry. 99m Tc-ECG was able to differentiate tumor and inflammation in lung cancer patients [26].
In the present study, we are able to place different radiometals in ECG. Both 99m Tc-ECG and 68 Ga-ECG were able to image mesothelioma in the animal model. 68 Ga-ECG was able to provide image-guided therapy assessment. From biodistribution (Tables 1-3), tumor/brain count density ratios of 99m Tc-ECG and 68 Ga-ECG were better than those of FDG. It may have advantage in brain tumor imaging than FDG. However, tumor/blood count density ratios of 99m Tc-ECG and 68 Ga-ECG were less than that of FDG. Additional experiments such as mechanistic studies and differential diagnosis would warrant their applications in oncology. In summary, efficient synthesis of ECG was achieved with high yield. 68 Ga-ECG and 99m Tc-ECG were prepared with high radiochemical purities. Biodistribution and planar imaging studies demonstrated the pharmacokinetic distribution and feasibility of using 68 Ga-ECG and 99m Tc-ECG to image mesothelioma. 68 Ga-ECG and 99m Tc-ECG showed an increased uptake in mesothelioma in the model tested, indicating that they are feasible to assess tumor volume. 68 Ga-ECG and 99m Tc-ECG may be useful for screening, diagnosing, staging, and assessing the efficacy of treatment in respect to all cancer types.