A new 68Ga-labeled ornithine for tumor ornithine metabolism PET imaging

doi:10.21203/rs.3.rs-1947291/v1

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A new 68Ga-labeled ornithine for tumor ornithine metabolism PET imaging

https://doi.org/10.21203/rs.3.rs-1947291/v1

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published 21 Feb, 2023

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Ornithine metabolism plays a vital role in tumorigenesis. For cancer cells, ornithine is mainly used as a substrate for ornithine decarboxylase (ODC) to produce amounts of polyamines. The ODC as a key enzyme of polyamine metabolism has become an important target for cancer diagnosis and treatment. To non-invasively detect the levels of ODC expression in malignant tumors, we have synthesized a novel ⁶⁸Ga-labeled ornithine analog (⁶⁸Ga-NOTA-Orn). The synthesis time of ⁶⁸Ga-NOTA-Orn was about 30 min with a radiochemical yield of 45–50% (uncorrected), and the radiochemical purity was > 98%. ⁶⁸Ga-NOTA-Orn was stable in saline and rat serum. Cellular uptake and competitive inhibition assays using DU145 and AR42J cells demonstrated that the transport pathway of ⁶⁸Ga-NOTA-Orn was similar to that of L-ornithine, and it could interact with the ODC after transporting into the cell. Biodistribution and micro-positron emission tomography (Micro-PET) imaging studies showed that ⁶⁸Ga-NOTA-Orn exhibited rapid tumor uptake and was rapidly excreted through the urinary system. All above results suggested that ⁶⁸Ga-NOTA-Orn is a novel amino acid metabolic imaging agent with great potential of tumor diagnosis.

ornithine

polyamines

PET

molecular probes

Ornithine is a ubiquitous nonessential amino acid whose role in growth was partially elucidated early on. Studies have shown that ornithine utilization is higher in cancer patients compared to normal subjects (Milo M. Webber 1980; Muhling et al. 2004), with the excessive synthesis of polyamines from ornithine by different rate-limiting enzymes, which plays a vital role in regulating cancer progression (Vargas-Ramirez et al. 2016; Zhang et al. 2020; Sivashanmugam et al. 2017). Polyamines are naturally occurring polycationic alkylamines and are ubiquitously present in minute concentrations in all mammalian cells and are involved in many fundamental processes of cell growth and transformation (Igarashi and Kashiwagi 2019; Michael 2016; Agostinelli 2020). Numerous studies have shown that polyamines not only regulate cell proliferation but are also closely associated with tumorigenesis and metastasis. Polyamine and polyamine synthase levels are significantly elevated in tumor cells compared to normal cells (Asai et al. 2018; Allan Lipton 1975). Considering the crucial role of polyamines in tumor development, targeting key enzymes and transporters of polyamine metabolic pathways for diagnosis and treatment has become a meaningful strategy for cancer management (Chandra et al. 2021; Novita Sari et al. 2021; Nowotarski et al. 2013).

Ornithine decarboxylase (ODC) is the first rate-limiting enzyme of the polyamine synthesis pathway, and its close relationship with tumor development is well known (Wallace et al. 2003). Under normal conditions, the activity of ODC is strictly regulated by the cell cycle, and it has a very rapid response to growth stimulators (Nasizadeh et al. 2008). ODC has also been widely studied in tumor diagnosis and treatment (Xu et al. 2011; Samoylenko capital O et al. 2021) and is considered an important indicator of tumor staging and efficacy. The detection of ODC expression has significant clinical importance for the early diagnosis of tumors, the assessment of benign and malignant grades, and the determination of the presence of infiltration and metastasis (Y. Elitsur 1992; Samal et al. 2013; Lozier et al. 2015).

In order to achieve noninvasive detection of the levels of tumor ODC expression and tumor diagnosis, a series of radiolabeled ornithine derivatives based on ornithine metabolism have been reported, including ¹⁴C-ornithine, N⁵-¹⁸F-fluoroacetylornithine, ^99mTc-ornithine, N⁵-¹⁸F-fluoropropanylornithine (Kiichi Ishiwata 1987; Turkman et al. 2011; Wang et al. 2021). Some have been biologically evaluated and have proven to be promising nuclear molecular probes for tumor diagnosis. In recent years, the radionuclide ⁶⁸ Ga eluted as ⁶⁸GaCl₃ from ⁶⁸Ge/⁶⁸Ga generators has been widely used in clinical tumor PET imaging, such as ⁶⁸Ga-PSMA-11, and has good sensitivity and spatial resolution and high temporal resolution. Therefore, we radiolabeled an ornithine derivative modified by a difunctional chelator NOTA-Bn-SCN (NOTA-Bn-SCN-Orn) with ⁶⁸Ga and investigated the tumor diagnostic value of the probe (⁶⁸Ga-NOTA-Orn) through a series of in vivo and in vitro experiments.

Chemicals and equipment

All chemicals (reagent grade) and solvents were purchased from commercial suppliers without further purification. Generator ⁶⁸Ge/⁶⁸Ga: product of ITG Medical Isotopes Gmbh. The analysis and purification of radiopharmaceuticals were performed on an HPLC instrument obtained from Tianjin Lab Alliance Company (Tianjin, China) equipped with a B-FC-3600 high-energy radioactivity detector (Bioscan, USA) and a 201 UV detector (Tianjin Lab Alliance, China). The semi-preparative C18 reversed phase column (5 µm, 250 × 10 mm) and analytical C18 reversed-phase column (5 µm, 250 × 4.6 mm) were products of YMC (Japan) and GRACE/Hydro, respectively. The micro-PET/CT scanner (IRIS, Inviscan, France) was used for animal PET imaging.

Synthesis of NOTA-Bn-SCN-Orn

NOTA-Bn-SCN-Orn was synthesized with the following procedure. Wang resin (0.5 g) was activated with 20 ml N, N-dimethylformamide (DMF) at room temperature for 40 min. Boc-Orn(Fmoc)-OH (1 mmol), 4-dimethylaminopyridine (DMAP, 3 mmol) and 1,3-diisopropyl carbodiimide (DIC, 3 mmol) were added to the activated resin and allowed to react at room temperature under N₂ atmosphere for 3h. (b) After filtering and washing with DMF (6 ml×5), pyridine (3 ml) and acetic anhydride (3 ml) were added to Boc-Orn(Fmoc)-resin and stirred at room temperature for 30 min. (c) The resin was then filtered and washed with DMF (6 ml×5) again, 30% piperidine in DMF solution (10 ml) was added and allowed to react for 20 min to remove the Fmoc-protected ornithine moiety. (d) After filtering and washing with DMF (6 ml×5) again, the difunctional chelator NOTA-Bn-SCN (1.5 mmol) and DIEA (5 mmol) were added to the resin in DMF (30 ml) and reacted at room temperature for 40 min. The resin was then filtered and rewashed with DMF (6 ml×5). (e) Finally, the solution (95% trifluoroacetic acid (TFA), 2% triisopropylsilane (TIS), 2% 1, 2-Ethanedithiol (EDT) and 1% H₂O was used to remove side chain protection to obtain a NOTA-Bn-SCN-Orn crude product. (f) After purification by semi-HPLC, the final precursor NOTA-Bn-SCN-Orn was obtained and dispensed in a refrigerator at -20 °C.

HPLC purity identification and MS analysis

The purity of NOTA-Bn-SCN-Orn was determined by HPLC, and a reverse phase analytical C18 column (5 μm, 250 × 4.6 mm) was used. UV absorption spectra were detected at 220 nm, and the flow rate was 1 mL/min with the mobile phase starting with 12 % solvent A: acetonitrile (0.1% TFA) and 88% solvent B: H₂O (0.1% TFA) and then changed to 53% solvent A and 47% solvent B at 25 min. The molecular weight of NOTA-Bn-SCN-Orn was analyzed by mass spectrometry.

Radiolabeling with gallium-68

⁶⁸Ga was eluted as ⁶⁸GaCl₃ from a ⁶⁸Ge/⁶⁸Ga generator with HCl (5 ml, 0.04983 mol/l) for five fractions (1 ml). To the fraction that contains the most radioactivity (approximately 130 MBq), sodium acetate buffer (0.25 M, pH=4, 380 µl) and NOTA-Bn-SCN-Orn (50 µg) were added. The mixture was heated to 100 ° C for 15 min, followed by a dilution with H₂O (5 ml) and loaded onto a Sep-Pek C18 light cartridge (Waters). The C18 cartridge was rewashed with H₂O (5 ml), and ⁶⁸Ga-NOTA-Orn was eluted with 50 % ethanol (1 ml). The radiochemical purity (RCP) is determined by analytic HPLC. RCP was also determined by TLC using 85% acetonitrile as the eluent. The ⁶⁸Ga-NOTA-Orn eluent was diluted to ethanol <5% using saline and used for further studies.

Stability studies

For in vitro stability, ⁶⁸Ga-NOTA-Orn (7 MBq)was incubated in 0.5 ml of fetal bovine serum and saline at 37 ° C. After 30 min, 60 min and 120 min, respectively, the RCP was determined by radio-HPLC. For in vivo stability, blood and urine were collected 60 min after injection of ⁶⁸Ga-NOTA-Orn (7 MBq, 0.2 ml) in SD rats (100-110 g, male, clean) provided by the Shanxi Medical University Experimental Animal Center. The sample was precipitated, filtered, and analyzed by radio-HPLC.

Cell culture

The human prostate cancer cell line (DU145) and the rat pancreatic exocrine tumor cell line (AR42J) were purchased from the Shanghai cell bank (China). These two tumor cell lines were ODC positive and were used for cell uptake and competitive inhibition assays. The DU145 medium consisted of minimum essential medium (MEM), 10% fetal bovine serum (FBS), and 1% penicillin/ streptomycin (PS). The AR42J medium consisted of Ham's F-12K medium, 20% FBS and 1% P/S. Both cell lines were cultured in an incubator (37 °C, 5% CO₂/95% air).

In vitro cell uptake

For the cell uptake experiment, cells (at a density of 1×10⁵ cells/well) were inoculated in 24-well plates for 24 h and then the medium was discarded and washed with PBS (phosphate buffered saline) twice. Cells in each well were incubated with ⁶⁸Ga-NOTA-Orn (0.2 ml, 0.74 MBq) at 37 °C for 2, 5, 10, 15, 30 and 60 min, and each group contained four wells. After incubation, cells were washed twice with ice cold PBS and lysed with 1M NaOH (0.5 ml). Cell lysate was collected and detected using a gamma counter. The data obtained were normalized to the percentage of radioactivity ⁶⁸Ga-NOTA-Orn (0.2 ml, 0.74 MBq).

To investigate the transport mechanisms in cell uptake and the biological function of ⁶⁸Ga-NOTA-Orn, the inhibitors used in the competitive experiment were DFMO and L-ornithine. Cells in 24-well plates were divided into the control group and three inhibition groups, and each group contained four wells. For the control group, the cell wells were treated with 0.2 ml of ⁶⁸Ga-NOTA-Orn (0.74 MBq) and 0.2 ml of PBS. For the inhibited group, cell wells were treated with 0.2 ml of ⁶⁸Ga-NOTA-Orn (0.74 MBq) and 0.2 ml of inhibitor solution. Following incubation for 20 min, the cells were washed three times with ice-cold PBS and treated according to the uptake assay. Cell lysate was collected and counted using a gamma counter and the data normalized to the percentage of radioactivity in the control group.

Biodistribution

Twenty-one ICR (Institute of Cancer Research) mice (25-30 g, male, clean) were provided by the Shanxi Medical University Experimental Animal Center. To evaluate the biodistribution of ⁶⁸Ga-NOTA-Orn, ICR mice (n=3 per group) were injected with ⁶⁸Ga-NOTA-Orn (0.2ml, 2 MBq) intravenously into the tail vein and sacrificed by cervical dislocation at 2, 5, 10, 30, 60, 90 or 120 min after injection. Blood and main organs were harvested and weighed. The radioactivity was measured with a gamma counter, and the results were reported as decay-corrected percentage injected activity per gram (%ID/g).

Animal Models

All animal experimental procedures were performed following the animal care and use guidelines approved by the ethics committee of Shanxi Medical University. BALB/c nude male mice (approximately 4–6 weeks old, with a body weight of 18–20 g) were purchased from SPF Biotechnology Co., Ltd (Beijing). Then 0.15 ml of tumor cell suspension (concentration 1×10⁷ cells/ml) was subcutaneously injected into the right shoulder of each mouse to form a tumor xenograft. Tumor-bearing mice were selected as tumor models for micro-PET/CT imaging when tumor volume reached 600–800 mm³.

Micro-PET/CT imaging

PET/CT images are acquired with a Micro-PET/CT scanner and acquisition time was ten min with the energy window set to 250–750 keV and a time resolution of 1 s. For CT imaging, the radiographic tube was set at 80 kV with a tube current of 1 mA and an exposure time of 40 s. During image acquisition, mice were immobilized with isoflurane anesthesia of 3% for induction and 2% for acquisition, respectively. Mice were fasted for 12 h but without water before imaging and injected through the tail vein with 5 MBq ⁶⁸Ga-NOTA-Orn, and PET/CT images were obtained 10, 30, 60, 90, and 120 min after injection. All images were analyzed with PMOD software (PMOD Technologies LLC, Zurich, Switzerland) to draw the outline of the tumor, muscle, liver and other regions of interest (VOI) to determine the percentage ID / g per gram of tissue and to quantify their SUV_max and the radioactivity uptake ratio of tumor to tissues such as liver and muscle.

Immunohistochemical (IHC)

Tumor tissues were fixed in 10% formalin solution, paraffin embedded, and sectioned. Antigen repair buffer (pH=6.0) was used to thermally repair the antigen in an autoclave. 3% H₂O₂ was incubated at room temperature for 25 min to block endogenous peroxidase. 3% BSA (Bovine serum albumin) was blocked at room temperature for 30 min to block nonspecific background reactions. The primary antibody is then added and incubated overnight at 4 °C in a wet box. Secondary antibody is added dropwise and incubated at room temperature for 50 min. Hematoxylin is used to stain the nuclei, dehydrate, and seal the cells for microscopic observation.

Statistical analysis

All quantitative data are expressed as mean ± SEM (standard error of the mean). Statistical analyzes were performed using SPSS. Two independent samples were analyzed by the t-test, and p values <0.05 were considered statistically significant.

Synthesis of NOTA-Bn-SCN-Orn

The HPLC purity and mass spectrometric analysis are shown in Fig.1. The purity of the synthesized NOTA-Bn-SCN-Orn was more than 95% (Fig.1a), and the molecular weight [M+H⁺] of NOTA-Bn-SCN-Orn was 583.50 (Fig.1b).

Radiosynthesis of ⁶⁸Ga-NOTA-Orn

⁶⁸Ga-NOTA-Orn was synthesized by a one-step chelated reaction (Fig. 2). The whole radiosynthesis was performed within 30 min with a radiochemical yield of about 45-50% (uncorrected). The radiochemical purity of ⁶⁸Ga-NOTA-Orn was more than 98% determined by radio-HPLC (Figs. 3a and 3b). The TLC plot is shown in Figure 3c, and the Rf values of ⁶⁸Ga-NOTA-Orn were 0.45~0.6, and the Rf value of ⁶⁸Ga-⁶⁸GaCl₃ was less than 0.2.

Stability Assays

No significant degradation of ⁶⁸Ga-NOTA-Orn was observed after incubation for up to 2 h in fetal bovine serum and saline at 37 ° C. Representative radio-HPLC were shown in Fig.4a-4f. ⁶⁸Ga-NOTA-Orn also showed excellent stability in SD rat serum and urine. The results (Figs.4g and 4h) showed that the RCP of the probes remained over 95%.

In vitro cell uptake

The cellular uptake properties of ⁶⁸Ga-NOTA-Orn were performed using DU145 and AR42J cell lines. Uptake levels of ⁶⁸Ga-NOTA-Orn in DU145 and AR42J cells are shown in Figs.5a and 5b. The overall trend of cell uptake of 68Ga-NOTA-Orn for these two tumor cell lines was similar. There was rapid uptake of ⁶⁸Ga-NOTA-Orn in the first 10 min and showed a maximum cellular uptake value of 10.6±0.47% for DU145 cells and 18.5±0.7% for AR42J cells, respectively, then as a function of time, showed a tendency to decrease uptake, which stabilized at 60 min.

To clarify the transport mechanism of ⁶⁸Ga-NOTA-Orn, we performed a competitive inhibition assay, and the results are shown in Figs.5c and 5d. The addition of L-ornithine reduced the uptake of the probe by AR42J by 21.1% (p=0.014); DU145 by 25.4% (p=0.002). After adding DFMO, AR42J reduced the uptake of ⁶⁸Ga-NOTA-Orn by 52.4% (p=0.005); DU145 decreased by 39.2% (p=0.021). The synergistic effect of L-ornithine and DFMO, probe uptake was significantly decreased in both cells. The difference between this synergistic effect and the separate effect of L-ornithine was statistically significant, while it was not statistically significant compared to the DFMO group.

Biodistribution

The biodistribution of ⁶⁸Ga-NOTA-Orn was performed in ICR mice, and the mice were sacrificed at 2, 5, 10, 30, 60, 90, or 120 min after injection of ⁶⁸Ga-NOTA-Orn. The calculated %ID/g of the major organs and tissues are shown in Fig.6. After injection of the probe, the radioactivity was rapidly and widely distributed.⁶⁸Ga-NOTA-Orn 2 to 90 min of blood radioactivity clearance was approximately 76%. In agreement with PET / CT scans, the probe showed low accumulation in bones and brain. At the first 5 min after tail vein injection, radioactivity rapidly accumulated in the kidneys (9.97 ± 0.84 %ID/g) but decreased to 2.67 ± 1.41%ID/g at 30 min. The predominant renal uptake of ⁶⁸Ga-NOTA-Orn indicated dominant renal-urinary clearance of the probe. In particular, the maximum uptake of ⁶⁸Ga-NOTA-Orn in the brain was 0.27 ± 0.15% ID/g, representing a small background.

Micro-PET/CT imaging

Static PET/CT scans were performed on DU145 and AR42J tumor-bearing nude mice10, 30, 60, 90, and 120 min after injection of ⁶⁸Ga-NOTA-Orn. Representative decay-corrected coronal images are shown in Fig.7a and 7b. After injection, DU145 and AR42J showed specific uptake of ⁶⁸Ga-NOTA-Orn over time, as evidenced by a significant radioactive concentration in the tumor on PET/CT images. In addition to uptake in the tumor, ⁶⁸Ga-NOTA-Orn was also visible in the kidney and bladder, indicating a dominant renal-urinary clearance pathway. In addition to tumors and kidneys, probe uptake was low in other organs or tissues, especially the brain. With time extension, the radioactivity is quickly excreted via the renal system. All these results are consistent with biodistribution studies.

In particular, while both tumors showed varying degrees of concentration reduction in radioactive uptake over time, the SUV_max of the AR42J tumor was relatively higher than that of the DU145 tumor. And we collected information of PET/CT images of DU145 and AR42J tumor-bearing nude mice from 10 to 120 min after postinjection of ⁶⁸Ga-NOTA-Orn. We plotted the SUV_max values of tumors and tumor-muscle (Tu/Mu) curves over time as shown in Figs.7c and 7d. For DU145 tumor-bearing mice, the highest Tu/Mu ratio of 2.23 ± 0.27 was obtained with an SUV_max of 0.87 ID/g for the tumor 30 min after injection. For AR42J tumor-bearing mice, the highest Tu/Mu ratio of 8.82±0.17 was obtained after 90 min of injection, and the SUV_max of the tumor was 0.67 ID/g.

Immunohistochemical (IHC)

The expression of ODC in tumor xenografts and some other tissues is shown in Fig. 8. The positive marker was defined by the brownish yellow or brownish color of the cell membrane and cytoplasm. Therefore, significantly high expression of ODC was observed in both DU145 and AR42J tumor xenografts (Fig.8a and 8b), while it was barely expressed in the heart, liver, spleen, lung, kidney and muscle (from Fig.8c to 8h). Meanwhile, there were much more positive markers in AR42J tumor xenografts, demonstrating a significantly higher level of ODC expression than that in DU145 tumor xenografts.

Amino acids are essential nutrients for cell growth and proliferation. After cellular malignancy, the demand for amino acids increases and the transport of amino acids is enhanced accordingly. Therefore, some natural amino acids and their derivatives are transported by the tumor cell transport system after radiolabeling and accumulate in tumor tissue. This is the primary mechanism by which radiolabeled amino acid imaging agents are used for tumor imaging. In recent years, PET tumor imaging with amino acid imaging agents has made significant progress, especially in areas where the application of ¹⁸F-FDG imaging is limited, such as brain tumor imaging and differential diagnosis of inflammation and tumor (Chen and Chen 2011).

Ornithine is a ubiquitous amino acid and a critical substrate for the synthesis of polyamines. It also plays a vital role in the regulation of metabolic processes that lead to diseases such as cancer. Studies have shown that ornithine is mainly involved in tumor metabolic processes as a substrate of ODC, a key enzyme of the polyamine synthesis pathway. ODC has become a target for cancer diagnosis and treatment, and accurate detection of ODC expression in diseased tissues has an important role in improving early detection and precise treatment of cancer. Therefore, positron radionuclide labeled ornithine as a PET imaging agent is of great significance for achieving noninvasive detection of ODC and diagnosis of tumors. As one of the widely used PET isotopes, the most critical characteristic of ⁶⁸Ga is its cyclotron-independent availability through the ⁶⁸Ge/⁶⁸Ga generator system (Velikyan 2015; Zhernosekov et al. 2007). However, the chemical properties of ⁶⁸Ga are not suitable for directly labeling amino acids. NOTA is a well-known chelator that exhibits a strong affinity for metal ions and can be used for the radiolabel of metal radionuclides easily and efficiently with high stability. Therefore, in this study, we used the difunctional chelator NOTA-Bn-SCN to modify ornithine to prepare an ornithine derivative NOTA-Bn-SCN-Orn. Then, NOTA-Bn-SCN-Orn as the precursor was radiolabeled with ⁶⁸Ga for the preparation of ⁶⁸Ga-NOTA-Orn without HPLC purification, a characteristic that facilitates clinical translation.

DU145 and AR42J as ODC positive tumor cells (Arisan et al. 2014) both showed a significantly increased uptake of ⁶⁸Ga-NOTA-Orn, and the cellular uptake of ⁶⁸Ga-NOTA-Orn tends to increase rapidly and decrease rapidly, which may be related to the short half-life of ODC. To determine the transport properties of the probe by tumor cells and its subsequent intracellular metabolism, in vitro cell competitive inhibition assays were performed and the results showed that the transport mechanism of ⁶⁸Ga-NOTA-Orn was similar to that of L-ornithine. DFMO is an irreversible inhibitor of ODC. Significant inhibitions of uptake of DMFO or the synergistic effect of L-ornithine and DFMO demonstrated that DFMO plays a major inhibitory role in the uptake of ⁶⁸Ga-NOTA-Orn by tumor cells. So, inhibition assays partly demonstrated that ⁶⁸Ga-NOTA-Orn could bind to ODC after being transported to cells. In addition, cellular uptake studies of other radionuclide-labeled ornithine derivatives such as DFMO, also show similar results (Wang et al. 2021). The study of subcellular distribution or cell uptake with ODC-negative tumor cells would be investigated in further research to confirm the exact metabolic mechanism of ⁶⁸Ga-NOTA-Orn.

In a biodistribution study, ⁶⁸Ga-NOTA-Orn showed rapid blood clearance and low accumulations in most normal tissues, such as the brain, lung and liver. And low bone uptake indicated high stability in vivo of ⁶⁸Ga-NOTA-Orn, as free Ga³⁺ is known to accumulate in bone by binding to transferrin (Bernstein 1998). The biodistribution profile of ⁶⁸Ga-NOTA-Orn was similar to that of other amino acid radiotracers (Kiichi Ishiwata YA 1987). And significant accumulations of ⁶⁸Ga-NOTA-Orn were observed in tumors from both DU145 and AR42J tumor-bearing mice in micro-PET/CT images, and high tumor to the muscle ratios (Tu/Mu) were obtained due to the low uptake of other organs or tissues. Furthermore, the SUV_max value of the AR42J tumor for ⁶⁸Ga-NOTA-Orn after 60 min after injection was significantly higher than that of the DU145 tumor, which was consistent with the IHC results. Furthermore, the relationship between ODC tumor expression in vivo by micro-PET imaging and its effects on ODC resistance will be investigated in further study.

Currently, multiple clinical studies targeting ODC are still in progress. DFMO as the ODC inhibitor has been proven to be beneficial for cancer treatment, and new inhibitors for ODC are also being developed. Although its performance in clinical trials for DFMO is still unsatisfactory, it has good clinical application prospects for combination therapy from DFMO and other therapeutic drugs. Therefore, the accurate detection of ODC expression in tumor tissues is of great importance for tumor treatment. Our experiments demonstrated that PET imaging of ODC in tumors with ⁶⁸Ga labeled ornithine derivative provided a noninvasive detection of ODC expression, which could be applied to evaluate the effect of ODC inhibition for tumor therapy.

In this study, we successfully synthesized a novel ⁶⁸Ga-labeled ornithine derivative ⁶⁸Ga-NOTA-Orn with good stability. In vitro and in vivo results demonstrated that ⁶⁸Ga-NOTA-Orn could be uptaken by ODC positive tumor cells with the same transport system as L-ornithine, and metabolized by ODC. Animal PET imaging showed high tumor uptake and low nonspecific uptake in normal organs, and excreted via the urinary system. Therefore, ⁶⁸Ga-NOTA-Orn is a promising amino acid probe for PET imaging of ODC positive tumor ornithine metabolism.

PET positron emission tomography

CT computed tomography

ODC ornithine decarboxylase

T/NT target-to-nontarget

ICR Institute of Cancer Research

SD Sprague-Dawley

HPLC high performance liquid chromatography

TLC thin-layer chromatography

RCP radiochemical purity

MEM minimum essential medium

FBS fetal bovine serum

PBS phosphate buffered saline

P/S penicillin / streptomycin

DFMO difluoromethylornithine

%ID/g percent of injected dose per gram

ROI Region of interest

SUV Standardized uptake value

BSA bovine serum albumin

Authors’ contributions

Corresponding authors: HW, ZW and SL. KQ and DS: methodology, software, writing: original draft. YZ and XH: data curation, formal analysis, PW and JG: visualization. HL and ZW: validation, formal analysis. GL and SL: resources. HW: writing, review and editing. SL: writing, review, supervision.

Funding This work was supported by the National Natural Science Foundation (Nos. 81471695, 81971655, 82027804, 82001873). Supported by Program for the Outstanding Innovative Teams of Higher Learning Institutions of Shanxi (2016, OIT), Applied Basic Research Program of Shanxi Province (No. 201801D211004). The research was also supported by the Shanxi “1331 Project” Key Innovative Research Team, the Construction Fund for Shanxi Key Subjects and the Four “Batches” Innovation Project of Invigorating Medical through Science and Technology of Shanxi Province.

Conflict of interest No conflicts of interest, financial or otherwise, are declared by the author(s).

Ethical approval: All animal procedures were performed in according to the protocol approved by the Animal Care and Use Committee of Shanxi Medical University.

Financial support and potential conflict of interest: none.

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A new 68Ga-labeled ornithine for tumor ornithine metabolism PET imaging

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