Abstract
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 . The present study was aimed to develop -cyclam-2-nitroimidazole (-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 -N4-NIM and -N4 was obtained in 13762 rat mammary tumor cells and mesothelioma cells in 6-well plates. Tissue distribution of -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 -N4-NIM was >96% by HPLC. Cell uptake of -N4-NIM was higher than -N4 in both cell lines. Biodistribution of -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 -N4-NIM in animal models. Efficient synthesis of N4-NIM was achieved. -N4-NIM is a novel hypoxic probe and may be useful in evaluating cancer therapy.
1. Introduction
Recent studies demonstrated that the hypoxic environment induces more malignant neoplastic phenotypes [1]. Disruption of oxygen delivery to tumors could diminish apoptotic potential and increase the chemotherapy/radiation resistance, while an improvement in oxygen delivery to tumors increases tumor sensitivity to radiation and chemotherapy [2–4]. Due to its significant prognostic and therapeutic implication, efforts have been made to invent efficient noninvasive methods to assess the presence and extent of tumor hypoxia because information on the hypoxic fraction in tumors could aid to reveal the mechanisms of aggressive behavior. The success of the endeavor to noninvasively detect the hypoxic fraction of tumor by nuclear molecular imaging such as single-photon emission computed tomography (SPECT) allows physicians to select patients for additional or alternative treatment regimens that may circumvent or overcome the ominous impact of tumor hypoxia and improve disease control [5].
The nitro group of nitroimidazole is enzymatically reduced by ribonucleoside reductase within viable hypoxic cells. This mechanism is well understood through numerous in vitro and in vivo studies from the past three decades [6]. In aerobic cells the reduced nitroimidazole is immediately reoxidized and washed out rapidly. On the contrary, in cells with low oxygen concentration the reoxidation is slowed, which allows further reductive reactions to take place. This leads to the formation of reactive products that could covalently bind to cell components and thus diffuse more slowly out of the tissue in an oxygen-dependent manner [7]. 18F-Fluoromisonidazole (18F-FMISO) and 18F-fluoroerythronitroimidazole (18F-FETNIM), 2-nitroimidazole analogues, have been used with PET to evaluate tumor hypoxia, but the chemistries are dependent on a cyclotron to produce 18F in addition to slow serum clearances [8–10].
In an attempt to identify efficient and clinically user-friendly chelator-based hypoxia tracers, our team has developed a new class of 99mTc hypoxia SPECT tracers based on the nitroimidazole backbone. The nitrogen, oxygen, and sulfur combination has shown to be stable chelators for radiometals [11–18]. The chelator used in this paper is 1,4,8,11-tetraazabicyclohexadecane (N4) which has shown to be a stable chelator for 99mTc [11, 12]. In addition, N4-chemistry could be easily modified and selectively reacted with halogenated homing compounds. In this paper, the potential use of 99mTc-N4-NIM as tumor hypoxic imaging agent was evaluated. The advantage of 99mTc-N4-nitroimidazole over 18F-FMISO and 18F-FETNIM is its easier chemistry and its water solubility that may lead to the rapid clearance of unbounded tracers after intravenous injection.
2. Materials and Methods
2.1. Chemicals and Analysis
All chemicals of analytical grade and solvents of high performance liquid chromatography (HPLC) grade were obtained from Sigma-Aldrich (St. Louis, MO, USA). Nuclear magnetic resonance (NMR) was performed on a Bruker 300-MHz spectrometer (Bruker BioSpin Corporation, Billerica, MA, USA), and mass spectra were performed on a Waters Q-TOF Ultima mass spectrometer (Waters, Milford, MA, USA) at the chemistry core facility at The University of Texas MD Anderson Cancer Center (Houston, TX, USA). Sodium pertechnetate (Na 99mTcO4) was obtained from a 99Mo/99mTc generator (Covidien, Mansfield, MA, USA).
2.2. Sodium Salt of 2-Nitroimidazole (Compound 1)
The synthetic scheme of N4-NIM is shown in Figure 1. One molar equivalent of NaOH 1 M (0.3864 g, 9.66 mmol, 9.66 mL) was added to 2-nitroimidazole (1.09 g, 9.66 mmol) and warmed 30 minutes at 50°C to dissolve it. If the compound was not dissolved in the solution then NaOH was added drop by drop until the solid was dissolved completely and we continue heating for 15 minutes more. Water was removed on rotary evaporator. Crude compound was dissolved in minimum quantity of water, filtered, and lyophilized. Yield: 5.31 g, (90%).
2.3. Bromopropane Nitroimidazole (Compound 2)
In two-neck flask one molar equivalent of Sodium salt of 2-nitroimidazole (2.00 g, 14.7 mmol) in solid form was added in 40 mL of acetonitrile (anhydrous) to dissolve it, then 18-crown-6 (3.88 g, 14.7 mmol) was added to this mixture. In nitrogen atmosphere 12.5 molar equivalent of 1,3-dibromopropane (18.65 mL, 183.76 mmol, 37.09 g) was added to the reaction mixture. The reaction mixture was then refluxed in a nitrogen environment at 50°C overnight. The reaction was filtered to purify the precipitate of NaBr using the 0.22 μM filter paper. Soon after the solvent was evaporated, the crude compound was purified by column chromatography with a CHCl3 : CH3OH mixture (v : v, 9 : 1). Yield: 10.45 g (90%).
2.4. N1, N4-Dioxylyl-1,4,8,11-Tetraazacyclotetradecane (Compound 3)
1,4,8,11-Tetraazacyclotetradecance (N4) (2.997 g, 14.974 mmol) was dissolved in 50 mL of ethanol (anhydrous) ethanol, and after heating at 50°C, diethyl oxalate (2.188 g, 14.974 mmol, 2.03 mL) was added. The reaction mixture was refluxed for 18 hours at 75°C. The solvent was rotary evaporated and the crude product was dissolved in minimum quantity of ethanol and the turbid solution was filtered with 0.22 μM filter. Crude compound was dissolved in minimum amount of anhydrous ethanol and kept rotating on rotary evaporator for 40 minutes at 40–50°C. The compound was crystallized by adding 200 mL acetone (anhydrous) leaving the solution overnight in the refrigerator to yield white crystals of N1, N4-dioxylyl-1,4,8,11-tetraazacyclotetradecane. Yield: 8.64 g (52.04%) Ms (m/z) 255.19 [M] +.
2.5. 1-[3-(2-Nitroimidazole-1-yl)propyl]-(N1,N4-Dioxylyl-1,4,8,11-Tetraazacyclotetradecane) (Compound 4)
N1,N4-dioxylyl-1,4,8,11-tetraazacyclotetradecane (0.5746 g, 2.259 mmol) was placed into the two-neck flask and dissolved in 25 mL of DMF (anhydrous). While heating the solution at 50°C, bromonitroimidazole (0.5329 g, 2.259 mmol) dissolved in 2.0 mL of DMF (anhydrous) was added to the reaction mixture. After adding K2CO3 (anhydrous) (0.7805 g; 5.6475 mmol) in 2.5 molar equivalent solid to the reaction mixture, it was refluxed at 70°C overnight. Crude compound was filtered using 0.22 μM filter paper and checked by TLC in chloroform: methanol in an 8 : 2 solvent system. Solvent was then evaporated and crude compound was purified by column chromatography in (8 : 2) of chloroform and methanol. Yield: 6.96 g, (75.86%). Ms (m/z) 408.30 [M] +.
2.6. 1-[3-(2-Nitroimidazole-1-yl)propyl]-(1,4,8,11-Tetraazacyclotetradecane) (Compound 5, N4-NIM)
To a 1 equivalent solution of (N4OxaNIM) N1,N4-dioxylyl-1,4,8,11-tetraazacyclotetradecane (0.100 g; 0.246 mmol) in 1 mL of water, 10 equivalents of 10 N NaOH (0.246 mL) were added, stirred, and refluxed over night at 75°C. The solvent was evaporated under vacuum, giving off white solid. The crude compound was dissolved in minimum quantity of water and filtered, then purified by sephadex G25 column. The fractions were lyophilized and collected by checking 1H NMR as off white solid. Yield: 0.045 g (52.32%).
2.7. Radiolabeling of N4-NIM with 99mTc
Radiosynthesis of 99mTc-N4-NIM was achieved by adding a required amount of 99mTc-pertechnetate into a kit containing the lyophilized residue of N4-NIM (1 mg) and SnCl2 (100 μg). Final pH of the preparation was 5.5–7.4. Radiochemical purity was determined by TLC and HPLC. Radio-TLC (Waterman No. 1) was obtained by eluting 99mTc-N4-NIM with acetone and saline, respectively. Radio-HPLC (Waters) was obtained by eluting 99mTc-N4-NIM on a C-18 reverse phase column (4.5 × 250 mm, 5 μm diameter, Zorbax Extend C 18, Agilant Technologies, Santa Clara, CA, USA) with 8 : 2 acetonitrile: water solution (v : v) using a flow rate of 0.5 mL/minute at UV 210 and 254 nm. The concentration of 99mTc-N4-NIM was 1.0 mg/1.96 mCi/500 μL H2O. The concentration of unlabeled N4-NIM was 1.7 mg/200 μL H2O.
2.8. Cellular Uptake of 99mTc-N4-NIM
The 13762 mammary cancer cells and mesothelioma cells (IL-45) were plated to 6-well tissue culture plates that contained 2 × 105 cells/well and incubated with 99mTc-N4-NIM (0.05 mg/well, 8 μCi/well) and 99mTc-N4 (0.025 mg/well, 8 μCi/well) for 0–4 hours. After incubation, cells were washed with ice-cold phosphate-buffered solution twice and detached using a treatment of 0.5 mL of trypsin for 5 minutes. Cells were then collected and the radioactivity of the cells was measured in triplicate with a gamma counter. Radioactivity was expressed as mean ± standard deviation percent of cellular uptake (%Uptake).
2.9. Biodistribution and Radiation Dosimetry Estimates of 99mTc-N4-NIM
The animal experiments were carried out in compliance with the relevant national laws relating to the conduct of animal experimentation. The animal protocol was approved by The University of Texas M. D. Anderson Cancer Center Animal Care and Use Committee (IACUC). Female Fischer 344 rats (150 ± 25 g) (Harlan Sprague-Dawley, Indianapolis, IN) were inoculated subcutaneously with 0.1 mL of mammary tumor cells from the 13762 tumor cell line suspension (106 cells/rat, a tumor cell line specific to Fischer rats) into the hind legs using 25-gauge needles. Studies were performed 14 to 17 days after implantation when tumors reached approximately 1 cm diameter. The rats were divided into 3 groups and each rat was injected intravenously with 25 ± 0.5 μCi of 99mTc-N4-NIM and 99mTc-N4. Each group was examined at 1 of 3 time points (0.5, 2, or 4 hours after injection). At each time point, the rats were killed and the selected tissues were excised, weighed, and measured for radioactivity by gamma counter. For each sample, radioactivity was expressed as mean percentage of the injected dose per gram of tissue wet weight (%ID/g). Counts from a 1/10 diluted sample of the original injection were used as a reference.
Tumor/nontarget tissue count density ratios were calculated from the corresponding %ID/g values. Olinda software (version 1.1; Vanderbilt University, Nashville, TN, USA) was used to determine dosimetry, based upon preclinical source organ residence time estimates as followed: rat organ distribution data was processed using in-house software to determine residence times () based on AUC. The data was then converted to human residence time estimates using the correction factor for each organ, and subsequent values were entered into Olinda software to generate human dose estimates.
2.10. Polarographic pO2 Measurements of Mammary Tumors
To confirm tumor hypoxia, intratumoral pO2 measurements were performed using the Eppendorf computerized histographic system. Twenty to twenty-five pO2 measurements along each of two to three linear tracks were performed at 0.4 mm intervals on each tumor (40–75 measurements total). Tumor pO2 measurements were made on three tumor-bearing rats. Using an on-line computer system, the pO2 measurements of each track were expressed as absolute values relative to the location of the measuring point along the track and as the relative frequencies within a pO2 histogram between 0 and 100 mmHg with a class width of 2.5 mm.
2.11. Planar Scintigraphic Imaging in Tumor-Bearing Models
Two animal models were created. For Rabbit model, the VX-2 tumor mass was inoculated (im) to the thigh region of male New Zealand white rabbits (2 kg). For rat model, cells from the mammary tumor cell line 13762, suspended in phosphate-buffered solution (105 cells/0.1 mL solution per rat), were injected subcutaneously into the right calf muscle of female Fischer 344 rats. Planar scintigraphic imaging of 99mTc-N4-NIM was performed 12–14 days after inoculation when tumors reached approximately 1 cm in diameter. The animals were anesthetized and injected intravenously with 99mTc-N4-NIM (0.3 mg/rat, 300 μCi/rat; 1.5 mCi/rabbit), and images were acquired at 30, 120, and 240 minutes after administration of tracers. Planar scintigraphic images were obtained using M-CAM (Siemens Medical Solutions, Hoffman Estates, IL, USA) equipped with a low-energy high-resolution collimator. Computer-outlined regions of interest (ROIs in counts per pixel) between tumor and muscle tissue were used to calculate tumor-to-muscle (T/M) ratios. Percent of injected dose (%ID) in the tumor was also calculated from the reference standard, which was 1/10 of the original injection activity of 99mTc-N4-NIM.
3. Results
3.1. Chemistry and Radiochemistry
The synthetic scheme is shown in Figure 1. The total synthesis yield of precursor N4-NIM via our 5-step procedure was 14%. The structure and purity of N4-NIM were confirmed by 1H- and 13C-NMR, mass spectra, and HPLC. The N4-NIM 1H-NMR results (D2O δ, ppm) were as follows: 8.25 (s, H, imidazole ring), 3.74 (s, H, imidazole ring), 3.17–3.25 (t, 2H,CH2 of propyl group), 3.05–2.91 (t, 2H, CH2 of propyl group), 2.26–2.76 (m, 2H, CH2 of propyl group), 2.28–2.48 (m, 14H, CH2 of N4 ring), and 1.38–1.68 (m, 6H, CH2 of N4 ring). 13C-NMR results (D2O δ, ppm) were as follows: 180.27, 173.13, 164.13, 61.48, 54.13, 51.73, 50.20, 49.45, 48.29, 46.61, 24.64, 23.76, and 22.64. Ms (m/z) 377.24 [M]+. In radio-TLC analysis, 99mTc-N4-NIM did not migrate either in saline or acetone solvent. HPLC analysis showed that the retention time for N4-NIM was 3.506 min and 3.690 min at UV-254 and 210 nm, respectively. N4-NIM was labeled with 99mTc successfully with high radiochemical purity (>96%) (Figure 2). HPLC analysis showed the retention time for 99mTc-N4-NIM was 3.663 min (UV-254 nm), 3.650 min (UV-210 nm), and 4.200 min (NaI detector). Because 99mTc-N4-NIM is a kit product and labeled without any further purification, its radiochemical yield was assumed to be identical to its radiochemical purity.
3.2. In Vitro Cellular Uptake Assays
The cellular uptake kinetics of 99mTc-N4-NIM and 99mTc-N4 in rat mammary tumor cells and rat mesothelioma cells are shown in Figure 3. The uptake for 99mTc-N4-NIM increased dramatically up to 240 minutes, but this was not the case for the 99mTc-N4, suggesting that 99mTc-N4-NIM can enter tumor cells specifically and accumulate rapidly.
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3.3. Biodistribution and Radiation Dosimetry Estimates of 99mTc-N4-NIM
Tissue distribution of 99mTc-N4 and 99mTc-N4-NIM is shown in Tables 1 and 2. Biodistribution studies showed that tumor/blood and tumor/muscle count density ratios at 0.5–4 hr gradually increased for 99mTc-N4-NIM (Table 2). No significant uptake in thyroid and stomach suggests in vivo stability of 99mTc-N4-NIM. The optimal tumor imaging time for 99mTc-N4-NIM is at 2 hr post-administration of 99mTc-N4-NIM. Although tumor/blood and tumor/muscle count density ratios at 0.5–4 hr gradually increased for 99mTc-N4, yet there was almost no tumor uptake (Table 1). Based upon preclinical studies, dosimetry of 99mTc-N4-NIM was estimated from MIRDose. It is safe to use 99mTc-N4-NIM in human because the whole body, liver, and effective dose equivalent for the proposed single dose at 20 mCi of 99mTc-N4-NIM were less than the limits for 3 rem annual and 5 rem total dose equivalent, and other organs of single dose at 5 rem annual and total dose equivalent at 15 rem if the subject did not have any other radiation exposure (Table 3).
3.4. Polarographic Oxygen Microelectrode pO2 Measurements
Intratumoral pO2 measurements of mammary tumors indicated the tumor oxygen tension ranged 4.6 ± 1.4 mmHg as compared to normal muscle of 35 ± 10 mmHg. The data indicated that the tumors were hypoxic.
3.5. Planar Scintigraphic Imaging in Tumor-Bearing Models
The selected planar scintigraphic images of tumor-bearing rats and rabbits after 99mTc-N4-NIM injection are shown in Figures 4 and 5. Tumors could be clearly detected by 99mTc-N4-NIM. The T/M ratios at 60–120 minutes were 4.1–4.2 in tumor-bearing rats and 2.52–2.98 in tumor-bearing rabbits, respectively.
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4. Discussion
The development of new tumor hypoxia agents is clinically desirable for detecting primary and metastatic lesions as well as predicting radioresponsiveness and time to recurrence [10, 19]. None of the contemporary imaging modalities accurately measures hypoxia since the diagnosis of tumor hypoxia requires a pathologic examination. It is often difficult to predict the outcome of a therapy for hypoxic tumor without knowing at least the baseline of hypoxia in each tumor treated. Although the Eppendorf polarographic oxygen microelectrode can measure the oxygen tension in a tumor, this technique is invasive and needs a skillful operator. Additionally, this technique can only be used on accessible tumors (e.g., head and neck, cervical) and multiple readings are needed. Therefore, an accurate and easy method of measuring tumor hypoxia will be useful for patient selection. However, tumor-to-normal tissue uptake ratios vary and depend upon the radiopharmaceuticals used. Therefore, it would be rational to correlate tumor-to-normal tissue uptake ratios with the gold standard Eppendorf electrode measures of hypoxia when new radiopharmaceuticals are introduced to clinical practice.
In biodistribution, 99mTc-N4-NIM in tumor tissue was decreased as same as 99mTc-N4 (Tables 1 and 2). This decreased uptake might be due to slower uptake 99mTc-N4 and 99mTc-N4-NIM as a function of increased renal excretion. However, the tumor uptake as well as tumor/blood and tumor/muscle ratios in 99mTc-N4-NIM were higher than that in 99mTc-N4 group.
Hypoxia-Inducible Factor (HIF)-1α/β heterodimer is a master transcription factor for several genes involved in angiogenesis, glycolysis, pH balance, and metastasis. These HIF-1 target genes help tumors to overcome forthcoming metabolic obstacles as they grow. Under normoxic condition, the HIF-1α subunit is hydroxylated by its specific prolyl-4 hydroxylase 2, given the acronym PHD2, thus stabilizing it under normoxic conditions [20]. In vitro cellular uptake assay, the uptake of 99mTc-N4-NIM in tumor cells increases up to 240 min. However, this assay was performed under normoxic condition. The increased uptake of 99mTc-N4-NIM in tumor cells under normoxic condition might be due to stabilized HIF-1α. NaTcO4 was reduced to +5Tc [O] and bound to the three nitrogens of cyclam. The charge of 99mTc-N4-NIM is neutral. Cell uptake of 99mTc-N4-NIM was via passive diffusion.
In our animal model, tumor oxygen tension was determined to be 3.2 to 6.0 mmHg, whereas normal muscle tissue had 30 to 40 mmHg. Although another factor such as anemia may have influenced the level of tumor hypoxia, there was no attempt in identifying this factor. In biodistribution, 99mTc-N4-NIM in tumor tissue was decreased as same as 99mTc-N4 (Tables 1 and 2). This decreased uptake might be due to slower uptake of 99mTc-N4 and 99mTc-N4-NIM as a function of increased renal excretion. However, the tumor uptake as well as tumor/blood and tumor/muscle ratios in 99mTc-N4-NIM was higher than those in 99mTc-N4 group.
Due to better imaging characteristics and lower cost, attempts are made to replace the 123I-, 131I-, 67Ga-, and 111In-labeled compounds with corresponding 99mTc-labeled compounds when possible. Our radiochemistry data indicated N4-NIM could be labeled with 99mTc very easily and efficiently at room temperature with high radiochemical purity. In vivo tissue distribution studies showed that radiation dosimetry of blood-forming organs was within radiation dose limits. Our planar imaging studies indicate that 99mTc-N4-NIM is feasible to assess tumor hypoxia.
In summary, N4-NIM kits could be labeled with 99mTc 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 99mTc-N4-NIM for tumor hypoxia imaging.
Acknowledgments
This work was supported in part by a Sponsored Research Grant (MDA LS2005-00012803PL) established between MDACC and Cell>Point L.L.C. and the J. S. Dunn Foundation. The chemistry analysis and animal research are supported by M. D. Anderson Cancer Center (CORE) Grant NIH CA-16672.