Assessment of DNA/RNA Deregulation in Cancer Using 99m Tc-Labeled Thiopurine

Abstract

Background:

DNA biomarkers are useful for the assessment of tumor cell proliferation. The authors aimed to synthesize a thiopurine-based ligand for evaluation of nuclear uptake and tumor localization.

Materials and Methods:

A 2-hydroxypropyl spacer was incorporated between a chelator (cyclam) and thiopurine ligand to produce SC-06-L1. In vitro cellular uptake and the cell/media ratios of [^99mTc]Tc-SC-06-L1 were assessed in breast (MCF-7, MDA-MB-231) and ovarian (TOV-112D, OVCAR3) cancer cells. The nuclear and cytosolic uptake ratio of [^99mTc]Tc-SC-06-L1 was determined in OVCAR-3 and MCF-7 cells. Cytotoxicity assays and flow cytometric analysis of cell cycle apoptosis were conducted in cancer cells treated with SC-06-L1. Imaging was conducted in tumor-bearing mice; fluorine-18-2′-fluorodeoxyglucose ([¹⁸F]FDG) was used as a control.

Results:

The radiochemical purity of [^99mTc]Tc-SC-06-L1 was >95%. [^99mTc]Tc-SC-06-L1 exhibited higher cell-to-media ratios than [¹⁸F]FDG in cancer cells. [^99mTc]Tc-SC-06-L1 had high uptake in the nuclear fractions in OVCAR-3 and MCF-7 cells, with nuclear/cytosolic ratios of 8 and 2, respectively. Cytotoxicity assays showed that SC-06-L1 was non-toxic compared with azathioprine in breast and ovarian cancer cells.

Conclusions:

[^99mTc]Tc-SC-06-L1 was stable and involved in nuclear activities. [^99mTc]Tc-SC-06-L1 showed non-toxic to cancer cells and exhibited fast kinetic uptake patterns for tumor imaging. [^99mTc]Tc-SC-06-L1 represents a promising biomarker for imaging purine pathway-directed systems.

Introduction

Diagnostic imaging modalities such as computed tomography (CT), magnetic resonance imaging (MRI), X-ray imaging, and ultrasound provide morphological and anatomical information, but do not provide data on cellular targets. Treatment endpoints rely almost exclusively on the analysis of biopsies by molecular and histopathological methods, which are invasive and subjected to sampling errors.^1
–3 Thus, existing assessments of the effectiveness of the responses of pathway-directed systems are insufficient. Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging radiopharmaceuticals can be used to measure target site activity and have made it possible to assess the efficacy of tumor therapy by measuring changes in proteasomal and proliferative activity.

Cyclotron-produced tracers are constrained by the poor availability of local cyclotrons; alternatively, radionuclide generator systems can be produced in well-controlled facilities, and radionuclides have a long history of successful clinical application. A generator uses a parent–daughter nuclide pair, wherein a relatively long-lived parent isotope decays to a short-lived daughter isotope that is used for imaging. The parent isotope, which is produced at a cyclotron facility, can be shipped to a clinical site where the daughter isotope is eluted on site for clinical use. The choice of molecules for clinical imaging should be determined not only by the biological behavior of radiopharmaceuticals but also by their ease of preparation. Thus, generator-produced isotopes are preferred for chelation–conjugate kit-based imaging.

^99mTc has been preferred to label radiopharmaceuticals due to its convenience from a ⁹⁹Mo/^99mTc generator, inexpensive free pertechnetate cost (U.S. $1.82 per mCi vs. $250 for ¹⁸F at 2023) (Noridian; https://med.noridianmedicare.com), easy instant kit-based manufacturing, favorable low energy (140 keV), and short physical half-life (6 h) for imaging purpose. In addition, [^99mTc]Tc^- (6 h half-life) and ⁶⁸Ga-based (68 min half-life) have significant commercial potential because the isotopes can be produced from the generators on-site and represent convenient alternatives to cyclotron-produced isotopes, such as [¹⁸F]F^- or [¹²⁴I]I^-. The uniqueness of the chelator–conjugate kit is that the instant kit can easily trap the radioisotope [^99mTc]Tc (SPECT) and [⁶⁸Ga]Ga- (PET) via a process known as “instant kit click chemistry” at high radiochemical purity and stability for imaging, or be used as a therapeutic agent by entrapping a therapeutic metal. Thus, chelation technology platforms represent a foundation for the development of theranostic molecules.

Achievement of noninvasive detection of proliferation activity in tumors would enable physicians to select personalized treatments or alternative regimens, which may lead to optimal response rates and reduce costs and side-effects by avoiding unnecessary treatment. The genes in DNA encode RNA, and RNA is transcribed into various proteins in cellular pathways. Proteomic activity dynamically changes in real time, and many proteins are overexpressed in various tumors. Sequential measurements of proteasome concentrations and DNA proliferative activity using specific molecular imaging agents would enable the assessment of tumor targets by whole-body imaging and potentially help to monitor the efficacy of therapy. In addition, molecular DNA agents could potentially be used to differentiate between inflammation or scar tissue and tumor recurrence.

Moreover, molecular DNA agents provide opportunities to predict the response to chemotherapy and radiation therapy, which may allow ineffective treatments to be discontinued earlier, which would be beneficial to patients. Incorporation of chelation-based PET/SPECT radiopharmaceuticals with radiometals and nonradioactive metals could transform these molecules from imaging tracers to biomarkers that enable therapeutic predictions. These developments are the foundation of molecular- and cellular pathway-directed system “imaging and therapy” (theranostics). The potential value of radiomic theranostic agents that target glucose transporters, nucleoside transporters, amino acid transporters, and lipid metabolism for more accurate staging and restaging of cancer, accelerated response prediction, improved efficacy of treatment, and a lower cost ratio are shown in Figure 1.

FIG. 1.

Potential value of radiomic theranostic agents in cancer: more accurate staging and restaging, accelerated response prediction, improved efficacy of treatment, and lower cost ratio.

Several efforts have been made to assess proliferative activity in tumors. Fluorine-18-2′-fluorodeoxyglucose ([¹⁸F]FDG) uptake has been reported to be an indicator of tumor proliferative activity.^4
–6 However, Higashi et al. showed that [¹⁸F]FDG uptake is strongly related to the number of viable cells.⁷ Another approach is to use radiolabeled amino acids as a marker of tumor cell proliferation.^8

–12 However, the structures of these agents are not based on purine or pyrimidine, which are the essential building blocks of DNA/RNA. Several radiolabeled pyrimidines and purines have been developed and used as probes for imaging herpes virus type 1 thymidine kinase (HSV1-tk) expression and other reporter genes.^{13

–26} However, the disadvantage of these imaging techniques is that HSV1-tk enzyme expression requires transduction of the HSV1-tk gene using an adenoviral type vector.

The level of HSV1-tk enzyme expression is likely to vary in different transduced cells and tissues; thus, applications of the HSV1-tk probe are limited. To improve the efficiency of gene therapy using HSV1-tk probes, researchers have synthesized and attempted to incorporate several pyrimidine and purine nucleosides/nucleotides into DNA/RNA.^{13,14,25

–28} For instance, the tracer 3′-deoxy-3′-¹⁸F-fluorothymidine ([¹⁸F]FLT) enters the salvage pathway of DNA synthesis and can be used to image cellular proliferation. Although [¹⁸F]FLT uptake correlates significantly with proliferative activity in certain types of cancer, tumor uptake is low.^25,26 Therefore, the applications of [¹⁸F]FLT for evaluation of primary or recurrent low-grade tumors or measurement of post-treatment outcomes are limited.

Others have reported the use of solid lipid magnetic nanoparticles and amino-modified silica-coated gadolinium-copper nanoparticles conjugated to AS1411 aptamer (Apt-ASGCuNCs) as delivery systems for MRI contrast concentrations in tumors by enhanced permeability and retention effect.^29,30 The biological behavior of ^99mTc-labeled systems showed that their targeting properties and their small size have improved biomedical imaging; however, ^99mTc was not labeled directly in the molecules. Thus, it is desirable to develop a radiopharmaceutical to measure proteasome and DNA activities that could enable therapeutic predictions beyond [¹⁸F]FDG and [¹⁸F]FLT. In addition, the choice of molecules should be determined not only by the biological behavior of the radiopharmaceuticals but also by their ease of preparation, as well as the logistics of imaging in real time with software to quantify changes in tumors to enable better treatment planning.

Purine structures are derivatives of adenine, thiopurine, or guanine and are crucial for providing cellular energy and in intracellular signaling. Purines can also be incorporated into more complex biomolecules that serve as cofactors, such as nicotinamide adenine dinucleotide and coenzymes, to promote cell survival and proliferation.³¹ The intracellular purine levels and expression of numerous enzymes are upregulated via biosynthetic pathways when tumor cell proliferation increases. Thiopurines are chemically more reactive than normal DNA purines. For instance, azathioprine, a thiopurine drug, has been used as an immunosuppressant to inhibit T cell and B cell proliferation for the treatment of hematologic malignancies, rheumatologic diseases, and inflammatory bowel disease and in solid organ transplantation.

Azathioprine is metabolized by reduction by glutathione and then enzymatically converted by hepatic xanthine oxidase (XO) to its active metabolite 6-mercaptopurine (6-MP). 6-MP is further metabolized by hypoxanthine-guanine phosphoribosyl transferase (HPRT) into 6-thioguanosine-5′-phosphate and 6-thioinosine monophosphate (6-thio-IMP), both of which inhibit nucleotide conversion and de novo purine synthesis. This leads to the inhibition of DNA, RNA, and protein synthesis. Ultimately, thiopurine can be incorporated into replicating DNA and can also block the de novo pathway of purine synthesis. Thiopurine methyltransferase, XO, and HPRT are thought to contribute to the relative specificity of 6-thio-IMP to lymphocytes.^32
–34

In this study, the authors report the synthesis of a chelator–thiopurine conjugate that mimics purine pathway-activated systems and can be used to map the RNA and DNA activity. The structural design of this analogue of thiopurine is shown in Figure 2A. A 2-hydroxypropyl or propyl spacer was incorporated between a chelator (cyclam, L) and thiopurine to produce SC-06-L-1.

FIG. 2.

(A) Structural design of an analogue of thiopurine: SC-06-L1 and (B) synthesis of [^99mTc]Tc-SC-06-L1.

The authors conducted biological evaluation studies in ovarian cancer (TOV-112D and OVCAR3) and breast cancer (MDA-MB-231 and MCF-7) cell lines. These cellular uptake and imaging findings may serve as a basis for evaluation of the differential responsiveness of tumors to chemo/radiotherapy or radionuclide therapy in individual patients. The authors ascertained the feasibility of imaging various tumors using the novel radiolabeled chelator–thiopurine conjugate. Chelator-based purine imaging agents may provide opportunities to characterize tumor aggressiveness and assess the efficacy and customize therapy for purine pathway-directed systems. In addition, this chelation–conjugate provides an opportunity for internal radionuclide therapy when coordinated with a therapeutic radionuclide.

Materials and Methods

Chemicals and analysis

Most chemicals were purchased from Sigma–Aldrich Chemical (Milwaukee, WI). Silica gel-coated thin-layer chromatography (TLC) plates were purchased from Whatman (Clifton, NJ). NMR data were collected using 300 or 500 MHz Varian Inova NMR spectrometers (Palo Alto, CA) equipped with a 5 mm PFG Triple ¹H-¹³C-¹⁵N probe, 5 mm PFG ¹H-¹⁹C-¹⁵N-³¹P switchable probe, or 4 mm ¹H-¹³C nanoprobe. Mass spectrometry was performed using a Bruker Solarix (Bremen, Germany). High-performance liquid chromatography (HPLC) data were collected using a Waters 2695 Separations Module (Milford, MA) equipped with a PC HILIC column (5 μm, 2.0 mm I.D. × 150 mm). ^99mTc-pertechnetate (Na^99mTcO₄) was obtained from a commercial ⁹⁹Mo/^99mTc generator (Taipei Veterans General Hospital, Taiwan).

Synthesis of compound SC-06-L1 (synthetic scheme shown in Fig. 2B)

Step 1: synthesis of the protected cyclam-bromohydrin conjugate (compound 2)

Diisopropylethylamine (7.34 mL, 42.16 mmol) was added dropwise to a solution of tri-BOC protected compound 1 (1.41 g, 2.81 mmol) and acetonitrile (MeCN, 20 mL) at room temperature (r.t.). The mixture was stirred at r.t. for 30 min, and then, 1,3-dibromo-2-propanol (2.87 mL, 28.11 mmol) was added dropwise. The reaction was stirred, refluxed for 18 h, and then cooled to r.t. The solvent was removed under reduced pressure. The crude product was purified by column chromatography (ethyl acetate: hexane, 1/1) to give compound 2 (869.8 mg, 1.36 mmol, 49%) as a white solid. ¹H-NMR (CDCl₃, 300 MHz): δ 3.64 (tt, 1H), 3.41–3.22 (m, 12H), 2.99 (t, 3H), 2.76–2.44 (m, 6H), 1.89 (tt, 2H), 1.69 (tt, 2H), 1.45 (s, 27H).

Step 2: synthesis of protected cyclam-hydroxypropyl-azathioprine conjugate (compound 3)

Cesium carbonate (Cs₂CO₃, 54.5 mg, 0.17 mmol) was added to a solution of azathioprine (23.2 mg, 0.08 mmol) and dimethylformamide (DMF, 5 mL) at r.t. The mixture was stirred at r.t. for 30 min and a solution of compound 2 (106.7 mg, 0.17 mmol) in DMF (3 mL) was added dropwise. The reaction was warmed, stirred at 100°C for 18 h, cooled to r.t., (ethyl acetate, 150 mL) was added, and the reaction was extracted three times with water (150 mL × 3). The organic layer was dried over magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography (5% ethyl acetate in methanol) and TLC (same eluant) analysis to give compound 3 (24.3 mg, 0.03 mmol, 35%) as a yellow solid. ¹H-NMR (CDCl₃, 300 MHz): δ 8.52 (s, 1H), 8.23 (s, 1H), 7.72 (s, 1H), 4.38 (dd, 1H), 3.91 (m, 1H), 3.73 (s, 3H), 3.37–3.16 (m, 13H), 2.54–2.36 (m, 6H), 1.76 (tt, 2H), 1.62 (tt, 2H), 1.40 (s, 18H), 1.37 (s, 9H).

Step 3: synthesis of cyclam-hydroxypropyl-thioprine conjugate (SC-06-L1)

Trifluoroacetic acid (3 mL) was added dropwise to a solution of compound 3 (96 mg, 0.12 mmol) dissolved in dichloromethane (CH₂Cl₂, 6 mL) in an ice-bath. The mixture was stirred at r.t. for 18 h. The solvent and reagent were removed under reduced pressure. The crude product was purified by reverse-phase column chromatography to give SC-06-L1 (80.5 mg, 76.7% yield) as a white solid containing 3TFA salt (C₂₈H₃₈F₉N₁₁O₉S; exact mass: 875.2431). The structure of SC-06-L1 was confirmed by both ¹H-NMR and ¹³C-NMR. ¹H-NMR (D₂O, 500 MHz): δ 8.56 (s, 1H), 8.48 (s, 1H), 8.08 (s, 1H), 4.46–4.44 (dd, 1H), 4.32 (m, 1H), 4.29–4.27 (dd, 1H), 3.78 (s, 3H), 3.39–2.46 (m, 18H), 2.06–1.84 (m, 2H), 1.91–1.64 (m, 2H); ¹³C-NMR (D₂O, 125 MHz): δ 163.07, 162.79, 156.04, 151.76, 149.70, 149.14, 147.23, 139.99, 130.69, 117.93, 115.17, 66.01, 54.07, 51.48, 49.99, 49.53, 48.58, 46.34, 46.31, 45.26, 43.92, 33.14, 24.61, 22.05. Compound SC-06-L-1 was also analyzed using mass spectrometry and HPLC.

Synthesis of [^99mTc]Tc-SC-06-L1

SC-06-L1 (5 mg) was dissolved in 0.3 mL of water (pH 5–6), SnCl₂ (0.1 mL, prepared from 10 mg SnCl₂ in 10 mL water) was added, and [^99mTc] sodium pertechnetate (NaTcO₄) (185 MBq in 0.1 mL) was added. The total volume was 1 mL after diluting with water. The radiolabeling experiment was repeatedly conducted at least five times. Radiochemical purity for [^99mTc]Tc-SC-06-L1 was determined by using an instant thin-layer chromatographic (ITLC) paper and eluting with saline or acetone.

Biological evaluation

Cell lines

Human ovarian cancer (TOV-112D and OVCAR3) and breast cancer (MDA-MB-231 and MCF-7) cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD). These cell lines were selected as they have varied hormonal status: OVCAR-3 cells are HER-2/estrogen receptor (ER)α and ERβ strong positive; TOV-112D cells are ERα-negative and ERβ moderate positive; MCF-7 and MDA-MB-231 cells are ER (+) and ER (−).^35
–37 OVCAR-3 cells were cultured in RPMI 1640 medium (Cat. No. 11875093; Gibco, Grand Island, NY) with 5% low-endotoxin fetal bovine serum (Cat. No. A5256701; Gibco). MCF-7 cells were cultured in MEM (Cat. No. A1048901; Gibco) with 5% low-endotoxin fetal bovine serum and supplemented with 0.1 mM non-essential amino acids and 1.0 mM sodium pyruvate. All cells were cultured in a humidified incubator with 5% CO₂ at 37°C.

In vitro cellular uptake studies

Six-well plates were used for cell uptake studies with breast cancer (MDA-MB-231, MCF-7) and ovarian cancer (OVCAR3, TOV-112D) cells. The cells were cultured in high-glucose Dulbecco's modified Eagle's medium supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO₂ at 37°C until 60%–70% confluent (∼50,000 cells per well). After washing the monolayers three times with fresh media, the cells were incubated with fresh cell culture media. For cell uptake studies, [^99mTc]Tc-SC-06-L1 was manufactured as follows: SC-06-L1 (5.3 mg, 0.01 mmol) was dissolved in 0.3 mL water (pH 5–6). SnCl₂ (0.1 mL, prepared from 10 mg SnCl₂ in 10 mL water) and NaTcO₄ (185 MBq in 0.1 mL) were added.

The total volume was 1 mL after diluting with water for molar activity at 0.01 mmol/185 MBq/1 mL. Each well contained 200 nmol (20 μL) SC-06-L1. [^99mTc]Tc-SC-06-L1 and [¹⁸F]FDG (3.7 MBq/20 μL/well) were then added to each well, and the cells were incubated for different intervals (0–2 h). Subsequently, the cells were washed twice with ice-cold phosphate-buffered saline (PBS), trypsinized with 0.5 mL of trypsin solution to detach the cells, and then, the cells were lysed in lysis buffer containing protease inhibitors (Roche Diagnostics, Mannheim, Germany).

The protein concentration of the cell lysates was quantified using the Bradford method as described by the manufacturer (Bio-Rad, Hercules, CA). The Bradford dye was diluted in distilled water (1:4) and filtered through filter paper (Whatman No. 1; Advantec Co. Ltd., Tokyo). Bovine serum albumin (concentrations of 1000, 500, 250, 125, 62.5, and 31.25 μg/mL) was used to build a standard curve. Protein samples were diluted in lysis buffer at 1:9. Diluted protein samples or standard were mixed with Bradford dye in 96-well plates. Thereafter, the absorbance values were recorded at 595 nm.

The radioactivity counts of the cells and culture medium were measured with a γ-counter (Packard, Meriden, CT) and expressed as counts per minute per gram of cells and counts per minute per gram of medium. Cell-to-medium radioactivity concentration ratios were calculated and plotted versus time to evaluate the radiotracer accumulation kinetics.

In vitro cellular distribution studies

The nuclear and cytosolic fractions of MCF-7 and OVCAR-3 cells were prepared as previously described.³⁸ To ascertain whether the cellular uptake of [^99mTc]Tc-SC-06-L1 was associated with cellular nuclear activity, MCF-7 and OVCAR-3 cells (10,000 cells per well) were incubated with [^99mTc]Tc-SC-06-L1 for 2 h (200 nmol/3.7 MBq/20 μL/well, n = 3), then rinsed with ice-cold PBS, and harvested into 1.5 mL microcentrifuge tubes on ice. The cytosolic and nuclear fractions were isolated by centrifugation at 3500 rpm for 2 min. After centrifugation, the pellets were resuspended in PBS containing 0.1% NP-40, centrifuged, and the pellets were resuspended in 1 × Laemmli sample buffer containing 50 M Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% β-mercaptoethanol, and 0.5% bromophenol blue; these samples was designed as the nuclear fraction.

The cell pellets and 0.1 mL of the radioactive supernatants were weighed, and the radioactivity was counted using a Packard 5500 γ-counter (Perkin-Elmer, Billerica, MA). The radioactivity counts for the cellular distribution and the ratio of [^99mTc]Tc-SC-06-L1 uptake between the nuclear and cytosolic fractions in OVCAR-3 and MCF-7 cells were determined.

In vitro cytotoxicity assays

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (CellTiter 96 Non-Radioactive Cell Proliferation Assay; Promega, Madison, WI) was used to quantify cell survival and assess the cytotoxicity of the compounds. The MTT assay is based on the cellular conversion of a tetrazolium salt into a formazan product that is easily detected using a 96-well plate reader.

MCF-7 and OVCAR-3 cells (1 × 10⁶ cells per well in 100 μL media) were seeded into 96-well plates, cultured for 24 h, incubated with PBS (vehicle control) or SC-01-L1 at the indicated concentrations for 24 h, and then incubated with WST-1 (Cat. No. ab155902; Abcam, Cambridge, United Kingdom) for 3 h. The absorbance values of the media were assessed using a UV/VIS Spectrophotometer at 450 nm (Beckman DU-800; Beckman Coulter, Brea, CA). The cell viability of MCF-7 and OVCAR-3 cells incubated with azathioprine was measured as a positive control.

In vitro and in vivo stability assays of [^99mTc]Tc-SC-06-L1

To ascertain the stability, [^99mTc]Tc-SC-06-L1 (3.7 MBq) was incubated in 100 μL PBS as well as in human serum and incubated at 37°C from 1 to 6 h. An aliquot of [^99mTc]Tc-SC-06-L1 (185 kBq) was spotted on ITLC paper and eluted with saline or acetone. In vivo stability of [^99mTc]Tc-SC-06-L1 was determined by uptake in thyroid and stomach during imaging studies. Computer-outlined count density technique was used to determine tracer uptake in the thyroid and stomach in rodents.

Cell cycle analysis using flow cytometry

To ascertain the effects of SC-06-L1 on the cell cycle distribution (i.e., distribution of cells in S, G, and M phases) of cancer cells, MCF-7 and OVCAR-3 cells (10,000 cells in each well) were incubated with SC-06-L1 (0.67 mM, n = 3) for 24 h and then harvested and fixed in 4% paraformaldehyde for 20 min, as previously described.^39,40 The cells were rinsed with PBS, centrifuged, resuspended in 70% cold ethanol for permeabilization, centrifuged, and the cell pellet was incubated with RNase (100 μg/mL), followed by PI staining solution (40 μg/mL) for 20 min.

The percentages of SC-06-L1 and PBS-treated MCF-7 and OVCAR-3 cells in the sub-G1, G0/G1, S, and G2/M phases were quantified by flow cytometry (Coulter^® Epics XL™, Miami, FL). A blue laser (488 nm) and 610 nm band pass filter were used to detect PI. The fluorescence intensity of singlet events was analyzed.

γ-imaging in mice bearing breast and ovarian tumors

All animal experiments were carried out on 8- to 9-week-old female nude mice and were approved by the National Yang-Ming University Institutional Animal Care and Use Committee (IACUC No: 1050910). The animals were housed with free access to water and maintained under controlled temperature (22°C ± 2°C) and humidity conditions (55%–65%) under a 12-h light/dark cycle. Athymic nude mice were inoculated subcutaneously with human breast cancer cells (MCF-7, MDA-MB-231) or ovarian cancer cells (OVCA-3 and TOV-112d) under both armpits of the front legs. When the tumor volumes reached 0.5 cm³, the mice were administered either [^99mTc]Tc-SC-06-L1 (0.1 mg/mouse) or [¹⁸F]FDG (20 ng/mouse, specific activity 1 Ci/mmol; control standard) at 4.4 MBq per mouse (0.1 mL, n = 3–6 per compound, intravenous). After injection of radiotracer, the mice were anesthetized by inhalation of 2% isoflurane and imaged using a nano-SPECT/CT camera or a handheld eZ-Scope (Anzai Medical Company, Japan).

The planar, SPECT, and CT fusion images were obtained using automatic fusion software (InterView Fusion; Mediso Medical Imaging Systems, Budapest, Hungary) and analyzed with PMOD 4.0 software (PMOD Technologies Ltd., Zurich, Switzerland). The PET images were obtained using a small animal 7T PETMR Inline (Bruker) for 10 min, with the energy window set to 350–650 keV. T1 and T2 MRI were obtained to determine the anatomical structure of the brain. The MRI sequences included 0.5 mm-thick coronal T2 Turbo RARE high-resolution images (repetition time = 3455 ms, time to echo = 36 ms, matrix = 256 × 256, average = 8, slice number = 30). The depth of anesthesia, pulse, and respiration of the animals were constantly monitored during the imaging procedure; in the rare event that an animal regained consciousness, the scanning was immediately stopped and the animal was removed (to be humanely euthanized and removed from the study).

After imaging acquisition, the PET images were reconstructed through three-dimensional ordered-subset expectation maximization. The regional radioactivity concentration (kBq/cc) of [¹⁸F]FDG was estimated from the mean pixel values within the volumes of interest (VOI) corresponding to the MRI results. Image data were decay-corrected to injection time. The radioactivity concentration (kBq/cc) of the VOI was converted to a standard uptake value, and the mean and standard error values of mean (SEM) radiotracer accumulation in various tissues were calculated. The PET/MR data were analyzed with PMOD 4.0 software (PMOD Technologies Ltd.). Counts per pixel in the tumor and muscle regions were acquired at various time points. Computer-outlined counts per pixel in the tumor (T) and muscle (M) regions were acquired at various time points from planar images. Data were expressed as percentage of injected dosage per wet tissue weight. T/M count density ratios and tumor uptake (% ID) were determined.

Statistical analysis

All data are expressed as the mean ± SEM and were analyzed using the Student's t-test. A p-value <0.05 was considered statistically significant and is marked with an asterisk in the figures.

Results

Chemistry

The intermediate used for the synthesis of SC-06-L1 is based on the reaction between 1,3-dibromo hydroxypropane and protects the nitrogen atoms in cyclam, which is the key step required to optimize SC-06-L1. Pure SC-06-L1 was synthesized by conjugation of thiopurine with brominated cyclam, followed by a de-protection step. The structural assignments in the ¹H- and ¹³C-NMR spectra are presented in Supplementary Figures S1 and S2.

SC-06-L1 was also analyzed by mass spectrometry (free base, C₂₂H₃₆N₁₁O₃S; exact mass 534.2718), and the purity was assessed by HPLC (Supplementary Figs. S3 and S4). In HPLC analysis (mobile phase: 50% water with 0.05% formic acid and 50% acetonitrile; column: AQ-C18; injection volume: 5 μL; wavelength: 220 nm), SC-06-L1 showed a retention time of 4.065 min and the chemical purity was >97%. Mass spectrometry of SC-06-L1 showed that the m/z was 534.3 with an exact mass of 534.2718. The radiochemical purity of [^99mTc]Tc-SC-06-L1 was >95% based on the ITLC cut and count method (eluant: saline and acetone, Rf = 0.2). The factor that considered for the optimization of radiochemical purity was the use of SnCl₂. In the preparation of [^99mTc]Tc-SC-06-L1, the authors used distilled water and also assured that there was no colloidal formation during manufacturing of [^99mTc]Tc-SC-06-L1. SnCl₂ tends to produce a large amount of ^99mTc-colloid if dissolved directly in water; thus, it is recommended to be dissolved in dilute hydrochloric acid.

Biological evaluation

In vitro cellular uptake studies

[^99mTc]Tc-SC-06-L1 had higher in vitro cell/media ratios (also known as the volume of distribution, Vd) in breast cancer and ovarian cancer cells than [¹⁸F]FDG in time-dependent patterns (Fig. 3). [^99mTc]Tc-SC-06-L1 exhibited a high Vd in MCF-7 and MB-231 breast cancer cells, whereas [¹⁸F]FDG had poor uptake in the same cells. [^99mTc]Tc-SC-06-L1 had a 10-fold higher Vd in breast cancer cells than in ovarian cancer cells. The Vd of [^99mTc]Tc-SC-06-L1 was significantly higher in MCF-7 cells than in MB-MB-231 cells, but there was no significant difference between OVCAR-3 and TOV-112d cells. These findings indicate that [^99mTc]Tc-SC-06-L1 has a fast kinetic uptake pattern in breast and ovarian cancer cells. As [^99mTc]Tc-SC-06-L1 showed high uptake in MCF-7 and OVCAR-3 cancer cells, these cell lines were selected for further cellular fraction and cytotoxicity assays and imaging studies.

FIG. 3.

In vitro cell/media ratios of breast and ovarian cancer cells. (A–D) [^99mTc]Tc-SC-06-L1 had higher uptake ratios than ¹⁸F-FDG in time-dependent patterns.

In vitro cellular distribution studies

Analysis of the cellular distribution of [^99mTc]Tc-SC-06-L1 revealed high uptake in the nuclear fraction. The ratios of uptake between the nuclear and cytosolic fractions in OVCAR-3 and MCF-7 cells were 8 and 2, respectively (Fig. 4A, B).

FIG. 4.

In vitro cellular distribution. The cellular distribution of [^99mTc]Tc-SC-06-L1 in the nuclear and cytosolic fractions (A), and the ratios of [^99mTc]Tc-SC-06-L1 uptake between the nuclear and cytosolic fractions in OVCAR-3 and MCF-7 cells (B). [^99mTc]Tc-SC-06-L1 showed high uptake in the nuclear fractions with the nuclear and cytosolic uptake ratios of 8 and 2 in OVCAR-3 and MCF-7 cells, respectively. *p < 0.05.

In vitro and in vivo stability assays of [^99mTc]Tc-SC-06-L1

In vitro stability assay of [^99mTc]Tc-SC-06-L1 using ITLC paper indicated that [^99mTc]Tc-SC-06-L1 remained at Rf = 0.2 (95%) of the time interval (1–6 h) tested in PBS and serum samples (Supplementary Fig. S5 represents samples tested in PBS). In vivo stability of [^99mTc]Tc-SC-06-L1 showed no thyroid and stomach uptake in rodents as determined by using computer-outlined count density technique. Both in vitro and in vivo assays indicated that [^99mTc]Tc-SC-06-L1 was a stable compound.

In vitro cytotoxicity assays

MTT cytotoxicity assays showed that SC-06-L1 at a concentration of 3 mM was not toxic to the breast or ovarian cancer cells (Fig. 5A–D); in contrast, the positive control thiopurine standard azathioprine significantly reduced the cell viability.

FIG. 5.

Cytotoxicity assays showed that SC-06-L1 was not toxic compared with azathioprine (control) in MCF-7 breast cancer cells (A) or OVCAR-3 ovarian cancer cells (B). Flow cytometry analysis indicated that treatment with SC-06-L1 (3 mM) for 24 h (n = 3) did not alter the cell cycle distribution of MCF-7 cells (C) or OVCAR-3 cells (D).

Cell apoptosis and flow cytometry analysis indicated that treatment with SC-06-L1 (3 mM) for 24 h (n = 3) did not alter the cell cycle distribution (S, Go, G1, G2) of MCF-7 or OVCAR-3 cells (Supplementary Figs. S6 and S7).

γ-imaging in mice bearing breast and ovarian tumors

Planar scintigraphy revealed high [^99mTc]Tc-SC-06-L1 uptake in athymic nude mice bearing human ovarian tumors (OVCAR-3 and TOV-2d cells), whereas [¹⁸F]FDG exhibited poor uptake (Fig. 6A–C). Compared with [¹⁸F]FDG, the tumor/muscle count density ratios of SC-06-L1 were significantly increased by 10- and 6-fold, respectively, in ovarian and breast tumors (MCF-7 and MB-231 cells) (Figs. 7A–C and 8D). Imaging studies revealed that [^99mTc]Tc-SC-06-L1 exhibited high tumor uptake and tumor-to-muscle ratios in nude mice bearing human ovarian and breast tumors. The optimal time of tumor uptake (% ID) was at 1–2 h in the tumor type selected (Tables 1 and 2). Thus, the imaging time was acquired at 1 h. Imaging studies of [^99mTc]Tc-SC-06-L1 demonstrated superior pharmacokinetic properties and high contrast visualization in the ovarian tumors (Fig. 8A–D). The imaging findings indicate that [^99mTc]Tc-SC-06-L1 is a sensitive biomarker for the assessment of DNA proliferation that could potentially be used for imaging various types of tumors.

FIG. 6.

[^99mTc]Tc-SC-06-L1 imaging at 1 h revealed high tumor-to-muscle ratios in athymic nude mice bearing human ovarian tumors (OVCAR-3 and TOV-112D) (A), whereas [¹⁸F]FDG had poor uptake in the same model (B). (C) Quantitative analysis of tracer uptake in tumor and muscle of OVCAR-3 and TOV-112D tumor-bearing mice, respectively. ***p < 0.005.

FIG. 7.

[^99mTc]Tc-SC-06-L1 exhibited higher tumor-to-muscle ratios than [¹⁸F]FDG in nude mice bearing MDA-MB231 and MCF-7 tumors (A). A handheld EZ-scope (Anzai Medical Japan) was used for imaging (B). (C) Quantitative analysis of tracer uptake in tumor and muscle of MDA-MB231 and MCF-7 tumor-bearing mice, respectively. **p < 0.01; ***p < 0.005.

FIG. 8.

[^99mTc]Tc-SC-06-L1 exhibited high tumor uptake and tumor-to-muscle ratios in nude mice bearing human ovarian (OVCAR-3 and TOV-112D) (A, B) and breast (MDA-MB-231 and MCF) tumors (C, D).

Table 1.

Tumor-to-Muscle Count Density Ratios (T/M) of [⁹⁹ ^mTc]Tc-SC-06-L1 in Mice Bearing OVCAR-3 and TOV-112D Tumors

Time point, h	OVCAR-3 tumor, % ID/g	TOV-112D tumor, % ID/g	Muscle, % ID/g	OVCAR-3 T/M	TOV-112D T/M
1	3.53 ± 1.57	2.72 ± 0.65	0.62 ± 0.17	5.54 ± 1.04	4.41 ± 0.14
2	2.80 ± 1.29	2.15 ± 0.39	0.51 ± 0.11	5.32 ± 1.74	4.26 ± 1.03
4	2.54 ± 1.23	1.85 ± 0.37	0.32 ± 0.08	7.80 ± 2.84	5.93 ± 0.92
6	2.12 ± 1.01	1.49 ± 0.42	0.26 ± 0.06	8.72 ± 5.41	6.21 ± 2.87

Biodistribution studies were conducted at the end of the imaging studies. Computer-outlined counts per pixel in the tumor (T) and muscle (M) regions were acquired at various time points from planar images. Tumor and tissue were excised. Data are expressed as the percentage of injected dosage per wet tissue weight. T/M count density ratios and tumor uptake (% ID) were determined.

Table 2.

Tumor-to-Muscle Count Density Ratios (T/M) by [⁹⁹ ^mTc]Tc-SC-06-L1 in Mice Bearing MDA-MB231 and MCF-7 Tumors

Time point, h	MDA-MB231 tumor, % ID/g	MCF-7 tumor, % ID/g	Muscle, % ID/g	MBA-MB-231 T/M	MCF-7 T/M
1	7.43 ± 3.07	8.17 ± 3.59	3.66 ± 1.14	1.95 ± 1.14	2.13 ± 0.23
2	8.74 ± 2.93	9.78 ± 0.94	3.39 ± 1.19	2.59 ± 0.04	3.17 ± 0.83
4	5.62 ± 1.12	5.94 ± 0.44	2.62 ± 0.41	2.13 ± 0.10	2.30 ± 0.19
6	6.73 ± 1.59	5.78 ± 1.16	2.26 ± 0.43	2.95 ± 0.15	2.56 ± 0.03

Discussion

At present, there is no standard nature [^99mTc]Tc (V) oxo chelation to validate the structure of [^99mTc]Tc (V) oxo chelation. Natural Re(V) oxo complex of the nitrogen-based chelation has been used to support the structural conformation for radioactive [^99mTc]Tc (V) oxo complex.^41,42 [^99mTc]Tc (V) oxo complexes in cyclam–vector conjugates have been reported by this group and others.^35,43
–45 The postulated structure of [^99mTc]Tc (V) oxo SC-06-L1 is presented in Figure 2. There are several advantages by using cyclam chemistry. The advantages include the following: (1) cyclam could react with covalent ¹⁸F-fluorine-PET precursors (-tosylate, -triflate, -mesylate) via displacement reactions and transform to cyclam conjugates. The end product could be water soluble for intravenous injection, and (2) cyclam conjugates could chelate various metallic substances (^99mTc, ⁶⁸Ga, ⁶¹Cu, ¹⁸⁸Re, ¹⁷⁷Lu, ⁹⁰Y, ¹¹¹In, ²²⁵Ac, etc.) that fulfill the theranostic concept.

TLC analysis in saline showed that Na^99mTcO₄ and ^99mTc-cyclam migrated, whereas ^99mTc-SC-06-L1 and ^99mTcO₂ colloid stayed at origin. Radioactive HPLC results showed that Na^99mTcO₄ and ^99mTc-cyclam were eluted, whereas ^99mTcO₂ colloid retained in the column (Supplementary Fig. S5).

There is unmet demand for theranostic radiopharmaceuticals that are paired at the molecular and cellular level to precisely diagnose, stage, and re-stage certain metabolic disorders and cancer. Various chelators have been reported to coordinate various metallic isotopes for the application of theranostic concepts, including ^99mTc (γ-emitter), ⁶⁸Ga, ⁶¹Cu, ⁸⁹Zr (β⁺-emitters), ¹⁷⁷Lu, ¹⁸⁸Re (β⁻/γ-emitters), ⁹⁰Y (a pure β⁻-emitter), ²²⁵Ac, and ²²³Ra (α-emitters). Thus, chelation–conjugate pairs are designed to measure changes in the target due to genomic and proteomic alterations via imaging. The application of chelation–conjugates within imaging platforms offers the potential to select patients who may respond to treatment and predict optimal treatment dosages for individual patients. Chelation–conjugate pairs also provide the opportunity for internal targeted radionuclide therapy by incorporating a therapeutic radionuclide as an alternative if/when chemotherapy/external radiation treatment failure.

The most advanced agents in clinical practices are ⁶⁸Ga/¹⁷⁷Lu/²²⁵Ac-prostate-specific membrane antigen (PSMA), and ⁶⁸Ga/¹⁷⁷Lu/²²⁵Ac-DOTA-octreotide for somatostatin receptor (SSTR) systems in neuroendocrine tumors (NETs); ⁶⁸Ga-DOTATATE PET/CT was used to assess for SSTR therapy with ¹⁷⁷Lu-DOTATATE and monitor treatment in NETs. ²²⁵Ac-DOTATATE was an alternative after failure of ¹⁷⁷Lu-DOTATATE treatment.^{46

–51 68}Ga-PSMA ligand binds to cells that express PSMA, including malignant prostate cancer cells, which usually overexpress PSMA. Subsequently, ⁶⁸Ga-PSMA ligand was used as a theranostic pair to monitor ¹⁷⁷Lu-PSMA and ²²⁵Ac-PSMA ligand treatment outcome.^52

–56 Theranostic paired radiopharmaceuticals will advance the understanding of pathway-directed systems and holds potential to create first-in-class or first-in-field chelation–conjugate pairs for theranostic avenues to modulate cancer and neuronal and immune dysregulation.

In this article, the authors have developed [^99mTc]Tc-SC-06-L1 for the assessment of RNA/DNA activities. The molecule also offers theranostic options based on chelation–conjugate platforms. From imaging perspective, [^99mTc]Tc-SC-06-L1 may help clinicians to identify the right dosage regimen to achieve the optimal dose response. In addition, [^99mTc]Tc-SC-06-L1 could help clinicians to identify the occurrence of drug resistance in late-stage cancer and help to reduce adverse events by switching to different drugs. From therapeutic perspective, SC-06-L1 may help clinicians to select patients with unresectable locally advanced or metastatic cancer for radionuclide therapy such as ¹⁷⁷Lu to improve the overall response rate and prolong progression-free survival of patients with cancer.

Currently, repeated high doses of chemotherapy are administered to eradicate solid tumors. The success of such high-dose therapy is often limited by the myelosuppressive and toxic effects of these drugs on bone marrow cells and by the intrinsic resistance of cancer cells to chemotherapy. Increased levels of multidrug resistance and resistance to chemotherapy are associated with proteasome and DNA proliferative activity.^40,57 Tumor proliferation rates are directly related to survival and prognosis. [¹⁸F]FDG, a gold standard for PET, is complementary to CT and MRI and enables the detection of unsuspected distant metastases. Although PET [¹⁸F]FDG imaging is concordant with the findings of CT and MRI for the diagnosis of various tumors, [¹⁸F]FDG has limited potential for personalized cancer treatment due to the lack of downstream-specific cellular pathways related to this tracer.

A significant amount (>95%) of [¹⁸F]FDG concentrates in the mitochondrial fraction, and this can result in an apparent false-positive lesions due to the difficulty of distinguishing between inflammation/infection and tumor recurrence.⁵⁸ Thus, [¹⁸F]FDG is a sensitive, but not a specific, biomarker for prediction of therapeutic responses. ¹⁸F-Fluorothymidine ([¹⁸F]FLT) was developed for imaging changes in genes, RNA, or DNA in primary and recurrent tumors; however, this tracer exhibits low uptake and also has complex chemistry.^25,26,59 Others have developed N4-guanine analogue derived from penciclovir.⁴³ The results were promising; however, the chemistry took multisteps and the chemistry yield was low at each step.

Thiopurine drugs, such as mercaptopurine, azathioprine, and thioguanine, are converted active metabolites to the endogenous purine-based guanine. The molecule consists of a purine attached to an endogenous phosphorylated ribose via purine de novo synthesis for its integration into the RNA and its deoxyribose derivative into DNA of leucocytes and leads to the inhibition of purine and cell death.⁶⁰ The authors hypothesize that the de novo synthesis of ribose and deoxyribose moieties in thiopurines are responsible for cytotoxicity. Placing a hydroxy aliphatic chain to the base of purine may mimic structural similarity of phosphorylated ribose purine in RNA and DNA activities. The chelator in SC-06-L1 would provide an opportunity for quantitative measurement of purine pathway-directed systems imaging.

To ascertain whether drugs are incorporated with RNA and DNA activities, the phenotype and genotype assays are commonly conducted. For instance, an Ames genomic assay is for single and double DNA strand breaks, point mutations, deletions, chromosomal aberrations, micronuclei formation, DNA repair, and cell cycle interactions.⁶¹ Thymidine incorporation and cell cycle analysis are considered as phenotype assays.⁶² These assays have different approaches, which are toward the direct measurement of DNA activities, but they are robust and costly. For proof of concept, the authors selected a simple and cost-effective centrifugation method to ascertain whether the cellular uptake of [^99mTc]Tc-SC-06-L1 was associated with cellular nuclear activity. The authors have isolated the cytosolic and nuclear fractions by centrifugation. For the cell uptake studies, a negative control ([^99mTc]Tc-cyclam) and a positive control ([¹⁸F]FDG) were selected to ascertain the sensitivity of [^99mTc]Tc-SC-06-L1 toward cancer cells.

The breast tumor cell line MCF-7 was ER strong positive, whereas MB-MB-231 was ER negative. The OVCAR-3 was HER-2/ERα and ERβ strong positive, whereas TOV-112D was ERα negative and ERβ moderate positive.³⁵ In vitro cell uptake of [^99mTc]Tc-SC-06-L1 showed higher cell/media ratios in MCF-7 cells than in MB-MB-231 cells, but there was no significant difference between OVCAR-3 and TOV-112d cells. MCF7 and OVCAR-3 had stable uptake patterns and were selected for further determination of ratios between the nuclear and cytosolic fractions. [^99mTc]Tc-SC-06-L1 showed significant higher Vd than [¹⁸F]FDG in breast cancer cells (Fig. 3). MCF7 had higher cell/media ratios than OVCAR-3 cells, and the authors found that the ratios of uptake between the nuclear and cytosolic fractions in OVCAR-3 and MCF-7 cells were 8 and 2, respectively (Fig. 4A, B).

In addition, there were no significant changes in the cell cycle distribution (S, Go, G1, G2) with dosage escalation for SC-06-L1 as shown in apoptosis and flow cytometry assays (Supplementary Figs. S7 and S8). In vitro cell uptake studies revealed that placing a hydroxy aliphatic chain between thiopurine and a chelator drastically reduced the cytotoxicity. Further studies would warrant to evaluate the amplification of DNA activities in these cancer cells. To assess whether cell uptake of [^99mTc]Tc-SC-06-L1 is via nucleoside transporters, further studies using nucleoside transporter inhibitors to block cell uptake of [^99mTc]Tc-SC-06-L1 is warranted. In addition, the measurement of partition coefficient of [^99mTc]Tc-SC-06-L1 in the future studies would support its penetration power in tumor tissue.

The findings of this study suggest that radiolabeled SC-06-L1 could be used to measure cellular nuclear activity and thus may allow precise measurement of tumor targets in whole-body images. [^99mTc]Tc-SC-06-L1 may be useful to monitor and predict responses to chemotherapy and radiation therapy. The authors used two different instruments to detect tumors: the tumors could be visualized by whole-body imaging and quantified by SPECT/CT (Fig. 6). Moreover, local/regional tumor activity could be quantified using a high-sensitivity handheld scope (Fig. 7). The whole-body images also showed a lot of nonspecific uptake in organs such as the liver, kidneys, and bones, which might be due to the lipophilic character of [^99mTc]Tc-SC-06-L1.

It would also need to evaluate whether the uptake in the bones or bone marrow by autoradiographic method. Nevertheless, the nonspecific uptake could influence the lesion uptake in the vicinity of these organs. Detailed tissue distribution studies for the determination of radiation dosimetry absorption in organs are planned. Overall, this study demonstrates the potential and feasibility of [^99mTc]Tc-SC-06-L1 for imaging tumors. Ultimately, chelator–purine probes may help clinicians to discontinue ineffective treatment earlier and beneficially improve the treatment outcomes of patients with cancer.

Perspectives

Molecular imaging agents enable comprehensive characterization of the effects of therapeutic interventions and can be used for patient selection, pharmacokinetic studies, dosage finding, and proof-of-concept studies. Molecular imaging agents based on chelation provide advantages in terms of high batch-to-batch reproducibility of radiochemical yield, purity, low production costs, and the availability of the agents for routine clinical practice. In addition to existing radionuclides, chelators could also be coordinated with other therapeutic radionuclides. Advances in theranostic (imaging and therapy) approaches, in parallel with development of instrumentation, could enhance the responses and outcomes to treatment.

In this report, the authors have demonstrated a technology platform that integrates a metal ion, chelator, and a homing agent to access DNA-related cell proliferation activity. Installation of protecting groups within chelators facilitates the purification of chelation–conjugates. The chelator-based thiopurine provides an efficient kit formulation for theranostic isotopes that do not require complex radiochemistry production; this strategy could be used to identify ineffective treatments at an early phase of treatment and enable patients to switch to more efficient treatments, which could improve patient outcomes overall.

Conclusions

Understanding the different types of proliferative activity in tumors could aid in the selection of individual patients for optimal therapeutic strategies. The radiolabeled chelator–thiopurine conjugate could be used to assess cellular proliferative activity on whole-body imaging, which could enable monitoring and prediction of the responses to chemotherapy, radiation therapy, and radionuclide therapy. Ultimately, chelator–thiopurine probes may help clinicians to discontinue ineffective treatment earlier, which could be beneficial to patients. In summary, [^99mTc]Tc-SC-06-L1 was developed with high radiochemical purity. [^99mTc]Tc-SC-06-L1 exhibited higher tumor uptake than [¹⁸F]FDG in breast and ovarian tumor models. The findings of this study on ^99mTc-SC-06-L1 provide an opportunity for the assessment of tumor proliferation activity to achieve optimal outcomes in cancer management.

Footnotes

Acknowledgments

The authors thank the Molecular Imaging Facility Small Animal 7T PET/MR and Brain Research Center at the National Yang Ming Chaio Tung University for technical support. The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan is also gratefully acknowledged.

Authors' Contributions

S.H.-H.Y. and D.J.Y. designed the content of the article. W.-C.C., M.-C.C., and Y.-C.L. designed the chemistry and radiochemistry. D.J.Y., S.H.-H.Y., and H.-J.T. executed the biological experiments and analyzed the data. C.-H.C., Y.-C.L., T.-H.Y., I.-J.C., P.-Y.W., and C.-P.L. conducted the biological evaluation. D.J.Y. and S.H.-H.Y. helped with final editing of the article. All authors contributed to and approved the final version of the article.

Disclosure Statement

There are no existing financial conflicts.

Funding Information

This work was funded through a sponsored research agreement established with SeeCure LLC (Taiwan). Funding for the imaging study was provided by the Ministry of Science and Technology, Taiwan (NSTC 110-2314-B-016-029, 111-2314-B-A49-041, NSTC 112-2314-B-016-060, and NSTC 112-2314-B-016-062), Medical Affairs Bureau of the Ministry of National Defense, Taiwan (Grant number: MAB-112-157), and Tri-Service General Hospital, National Defense Medical Center (Grant number: TSGH-D-112090 and TSGH-E-113240).

Supplementary Material

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Figure S4

Supplementary Figure S5

Supplementary Figure S6

Supplementary Figure S7

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Supplementary Material

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1.88 MB

Assessment of DNA/RNA Deregulation in Cancer Using 99m Tc-Labeled Thiopurine

Abstract

Background:

Materials and Methods:

Results:

Conclusions:

Introduction

Materials and Methods

Chemicals and analysis

Synthesis of compound SC-06-L1 (synthetic scheme shown in Fig. 2B)

Step 1: synthesis of the protected cyclam-bromohydrin conjugate (compound 2)

Step 2: synthesis of protected cyclam-hydroxypropyl-azathioprine conjugate (compound 3)

Step 3: synthesis of cyclam-hydroxypropyl-thioprine conjugate (SC-06-L1)

Synthesis of [99mTc]Tc-SC-06-L1

Biological evaluation

Cell lines

In vitro cellular uptake studies

In vitro cellular distribution studies

In vitro cytotoxicity assays

In vitro and in vivo stability assays of [99mTc]Tc-SC-06-L1

Cell cycle analysis using flow cytometry

γ-imaging in mice bearing breast and ovarian tumors

Statistical analysis

Results

Chemistry

Biological evaluation

In vitro cellular uptake studies

In vitro cellular distribution studies

In vitro and in vivo stability assays of [99mTc]Tc-SC-06-L1

In vitro cytotoxicity assays

γ-imaging in mice bearing breast and ovarian tumors

Discussion

Perspectives

Conclusions

Footnotes

Acknowledgments

Authors' Contributions

Disclosure Statement

Funding Information

Supplementary Material

References

Supplementary Material

Synthesis of [^99mTc]Tc-SC-06-L1

In vitro and in vivo stability assays of [^99mTc]Tc-SC-06-L1

In vitro and in vivo stability assays of [^99mTc]Tc-SC-06-L1