Copper-61 is an advantageous alternative to gallium-68 for PET imaging of somatostatin receptor-expressing tumors: a head-to-head comparative preclinical study

Background: Gallium-68 positron emission tomography (⁶⁸Ga-PET) with the two registered somatostatin analogs, [⁶⁸Ga]Ga-DOTA-Tyr³-octreotide ([⁶⁸Ga]Ga-DOTA-TOC) and [⁶⁸Ga]Ga-DOTA-Tyr³-octreotate ([⁶⁸Ga]Ga-DOTA-TATE), where DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, is routinely used for imaging of somatostatin receptor (SST)-expressing tumors. We investigated copper-61 (⁶¹Cu) as an alternative radiometal for PET imaging of SST-expressing tumors. Compared to gallium-68, copper-61 (t_1/2 = 3.33 h, E_β⁺_max = 1.22 MeV) can be produced on a large scale, enables late time point imaging, and has the therapeutic twin copper-67. Herein, DOTA-TOC and 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA)-TOC were labeled with copper-61 and compared with the clinically used [⁶⁸Ga]Ga-DOTA-TOC.

Methods: [⁶¹Cu]CuCl₂ was produced from an irradiated natural nickel target. DOTA-TOC and NODAGA-TOC were labeled with [⁶¹Cu]CuCl₂ in ammonium acetate buffer so to achieve a reaction pH of 5–6 and a temperature of 95°C for DOTA-TOC or room temperature for NODAGA-TOC. The radioligands were evaluated head-to-head in vitro using human embryonic kidney (HEK)-SST₂ cells (affinity, binding sites, cellular uptake, and efflux) and in vivo using HEK-SST₂ xenografts [PET/computed tomography (CT) imaging, biodistribution, and pharmacokinetics] and compared with [⁶⁸Ga]Ga-DOTA-TOC, which was prepared using a standard procedure. Dosimetry estimates were made for [⁶¹Cu]Cu-NODAGA-TOC.

Results: [⁶¹Cu]Cu-DOTA-TOC and [⁶¹Cu]Cu-NODAGA-TOC were prepared at an apparent molar activity of 25 MBq/nmol with radiochemical purities of ≥96% and ≥98%, respectively. In vitro, both presented a sub-nanomolar affinity for SST₂ (IC₅₀ = 0.23 and 0.34 nM, respectively). They were almost entirely internalized upon binding to SST₂-expressing cells and had similar efflux rates at 37°C. In vivo, [⁶¹Cu]Cu-DOTA-TOC and [⁶¹Cu]Cu-NODAGA-TOC showed the same accumulation in SST₂-expressing tumors. However, PET/CT images and biodistribution analyses clearly showed an unfavorable biodistribution for [⁶¹Cu]Cu-DOTA-TOC, characterized by accumulation in the liver and the abdomen. [⁶¹Cu]Cu-NODAGA-TOC displayed favorable biodistribution, comparable with [⁶⁸Ga]Ga-DOTA-TOC at 1 h post-injection (p.i.). Notwithstanding, [⁶¹Cu]Cu-NODAGA-TOC showed advantages at 4 h p.i., due to the tumor retention and improved tumor-to-non-tumor ratios. The effective dose (2.41 × 10⁻³ mSv/MBq) of [⁶¹Cu]Cu-NODAGA-TOC, but also the dose to the other organs and the kidneys (9.65 × 10⁻² mGy/MBq), suggested a favorable safety profile.

Conclusion: Somatostatin receptor ⁶¹Cu-PET imaging not only matches the performance of ⁶⁸Ga-PET at 1 h p.i. but has advantages in late-time imaging at 4 h p.i., as it provides improved tumor-to-non-tumor ratios. [⁶¹Cu]Cu-NODAGA-TOC is superior to [⁶¹Cu]Cu-DOTA-TOC in vivo. The use of the chelator NODAGA allows quantitative labeling with copper-61 at room temperature and enables the straightforward use of a kit formulation for simple manufacturing in medical centers.

1 Introduction

Positron emission tomography (PET) imaging of somatostatin receptors (SST) has a high clinical impact on the management of neuroendocrine neoplasms (1, 2). Gallium-68 (⁶⁸Ga)-labeled DOTA-Tyr³-octreotate (DOTA-TATE) (NETSPOT®) and DOTA-Tyr³-octreotide (DOTA-TOC) (SOMAKIT TOC®), where DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, are two radiolabeled somatostatin analogs approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for SST-PET imaging of neuroendocrine tumors (NETs). Kit-based formulation and an authorized ⁶⁸Ge/⁶⁸Ga-generator enable decentralized (on-site) preparation of these two radiotracers. This concept gained rapid adoption worldwide as it is easy to implement in clinical practice. However, there are certain limitations that are becoming increasingly apparent: (i) authorized generators are expensive and have a limited production capacity (the maximum number of patient doses per synthesis is 2–3, depending on the age of the generator), and (ii) access to gallium-68 is increasingly limited because another ⁶⁸Ga-labeled tracer ([⁶⁸Ga]Ga-PSMA-11, where PSMA = prostate specific membrane antigen) has also received FDA/EMA approval for PET imaging of prostate cancer and is in broad clinical use. Furthermore, the approved ⁶⁸Ga-PET tracers have lutetium-177 (¹⁷⁷Lu)-labeled therapeutic companions, i.e., [¹⁷⁷Lu]Lu-DOTA-TATE (Lutathera®) and [¹⁷⁷Lu]Lu-PSMA-617 (Pluvicto®). With SST- and PSMA-targeted radioligand therapy being important therapeutic options, the demand for ⁶⁸Ga-PET scans is expected to expand rapidly. This demand is expected to further expand when new tracers are approved, for example, ⁶⁸Ga-labeled fibroblast activation protein (FAP) inhibitors.

To address the clinical demand and upscaling of production, large-scale production of gallium-68 in cyclotrons has been explored but not implemented in clinical practice. Moreover, the short half-life of gallium-68 (t_1/2 = 1.13 h) prevents the shipment of the radiotracer from central producers to smaller centers that are located beyond a 2 h journey away. Fluorine-18 (t_1/2 = 1.83 h) addresses some of the hurdles of gallium-68. This comes, however, at the cost of facile chelator-based radiolabeling and the possibility of a therapeutic companion (theranostics); these are options that are possible with radiometals. Thus, somatostatin analogs labeled via aluminium-[¹⁸F]fluoride ([¹⁸F]AlF) were proposed, such as [¹⁸F]F-NOTA-octreotide (3), where NOTA = 1,4,7-triazacyclononane-1,4,7-triacetic acid, but these are limited to the chelator NOTA.

The cyclotron-produced β⁺ emitter copper-61 (⁶¹Cu, t_1/2 = 3.33 h, E_β⁺_max = 1.22 MeV, E_β⁺_mean = 500 keV, I_β⁺ = 61%) is a valuable alternative to gallium-68 (t_1/2 = 1.13 h, E_β⁺_max = 1.90 MeV, I_β⁺ = 89%). Copper-61 can be produced in medical cyclotrons at a large scale and distributed to locations further away than gallium-68. Its lower positron energy (1.22 vs. 1.90 MeV) and mean range (1.3 vs. 2.4 mm in water) provide superior spatial resolution and improved imaging quality (4), suggesting enhanced detection of small lesions. Furthermore, its longer half-life allows imaging at multiple time points. This facilitates dosimetry estimates and potentially improves the identification of lesions due to the lower background over time. Last, but not least, copper-61 has a therapeutic twin, copper-67 [t_1/2 = 2.58 days, E_β⁻_max = 577 keV, E_β⁻_mean = 141 keV, E_{γ max} = 185 keV (49%)]. Of note, even though copper-64 is more established for PET imaging, copper-61 offers valuable advantages that are addressed in the discussion part of the manuscript.

Despite the suitable physical characteristics of copper-61 for PET imaging, there are scarce reports on ⁶¹Cu-labeled tracers. The literature focuses mainly on production routes, separation methods, and optimization for high-purity copper-61 production. These have been the obstacles to overcome in the development of ⁶¹Cu-PET tracers. In 1999, McCarthy et al. utilized diacetyl-bis(N⁴-methylthiosemicarbazone (ATSM) and TETA-octreotide (where TETA = 1,4,8,11-tetraazacyclotetradecane-N,N’,N”,N”’-tetraacetic acid) to highlight the potential applications of copper-61 and copper-60 (5). Since then, only a very limited number of ⁶¹Cu-labeled tracers followed, primarily referring to ATSM (6, 7). Recently, ⁶¹Cu-labeled NAPamide for PET imaging of melanoma was reported (8, 9), while our group developed ⁶¹Cu-labeled PSMA and reported the first human ⁶¹Cu-PET (10). Today, large-scale production is feasible using either liquid targets via proton bombardment of natural zinc or enriched zinc-64 [^nat/64Zn(p,α)⁶¹Cu] or using solid targets via deuteron bombardment of natural nickel or enriched nickel-61 [^nat/60Ni(d,n)⁶¹Cu reaction] (11, 12). The optimization of separation and purification methods allows copper-61 production for direct radiolabeling and opens the way for the development of ⁶¹Cu-based PET tracers.

We explored the potential of copper-61 in combination with established somatostatin analogs given their proven clinical value in PET-SST imaging in neuroendocrine neoplasms. The GMP production of clinical doses of ⁶¹Cu-labeled DOTA-TATE and DOTA-TOC was recently reported by Fonseca et al., focusing on the radiochemical aspects (11). We report herein a comprehensive in vitro and in vivo evaluation of [⁶¹Cu]Cu-DOTA-TOC and its NODAGA (1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid) derivative ([⁶¹Cu]Cu-NODAGA-TOC). The two ⁶¹Cu-labeled tracers were compared head-to-head with [⁶⁸Ga]Ga-DOTA-TOC in terms of radiochemistry, affinity, and in vitro and in vivo performance.

2 Materials and methods

2.1 Reagents, instrumentation, and cell line

DOTA-TOC and NODAGA-TOC were purchased from piCHEM (Raaba-Grambach, Austria). All solvents and reagents were purchased and used as supplied by Sigma Aldrich (Switzerland) and VWR International (Switzerland) in high-performance liquid chromatography (HPLC) or analytical grades.

The human embryonic kidney (HEK) cell line expressing the T7-epitope-tagged human SST₂ receptor (HEK-SST₂) was provided by Prof. Stefan Schulz (Institute of Pharmacology and Toxicology, Jena University Hospital, Jena, Germany) and cultured at 37°C and 5% carbon dioxide (CO₂) in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin, 200 µmol/ml L-Glutamin, and 500 mg/ml G418. All the reagents were purchased from BioConcept (Allschwill, Switzerland) and Biochrom GmbH, Merck Millipore (Darmstadt Germany).

Quantitative γ-counting was carried out on a Cobra 5003 γ-system well counter from Packard Instruments (Meriden, CT, USA).

2.2 ⁶¹Cu-labeled tracers and reference tracer

Copper-61 was produced by irradiating natural nickel electroplated on silver coins at 40 µA for 120 min in a GE HealthCare medical cyclotron at the University Hospital Zurich, Switzerland. Target dissolution and purification were based on Svedjehed et al. (12). A [⁶¹Cu]CuCl₂ solution in 0.05 M HCl was produced at an activity concentration of approximately 1 GBq/ml for direct radiolabeling.

An aliquot of DOTA-TOC or NODAGA-TOC (3–6 nmol, 1 mg/ml in TraceSelect water) was diluted in 0.25–0.30 ml ammonium acetate (0.5 M, pH 8), followed by the addition of [⁶¹Cu]CuCl₂ (30–150 MBq). The reaction mixture was incubated for 15 min at 95°C (DOTA-TOC) or at room temperature (NODAGA-TOC). The pH of the reaction mixture was between 5 and 6. Quality control was performed on a Shimadzu 2020 HPLC system connected to a Berthold radio detector (Flow Star 513). A sample of the labeling solution (1–5 μl) was withdrawn, diluted in 50 μl ethylenediaminetetraacetic acid (EDTA, 0.1 M), and analyzed on a Proteo Jupiter C12 (4.6 mm × 250 mm, 4 µm particle size), using a gradient of 15%–65% solvent B in 15 min [A = H₂O (0.1% TFA), B = ACN (0.1% TFA)], at a flow rate of 1 ml/min. [⁶¹Cu]Cu-DOTA-TOC and [⁶¹Cu]Cu-NODAGA-TOC labeling solutions were stored at room temperature and analyzed up to 4 h after the end of synthesis by radio-HPLC.

The complexes of DOTA-TOC and NODAGA-TOC with natural copper (^natCu) were prepared by incubating each conjugate (1–1.5 mg) with a 1.5-fold excess of ^natCuCl₂ × 2 H₂O in the same buffer used for radiolabeling, at 95°C for 30 min (DOTA-TOC) or 15 min (NODAGA-TOC). Uncomplexed natural copper ions were eliminated by SepPak C-18 purification. The ^natCu-complexes were eluted with methanol followed by evaporation to dryness and lyophilization after dissolution in water. Analysis was performed via liquid chromatography-mass spectrometry (LC-MS) on an LCMS-2020 Shimadzu system equipped with a Waters X Bridge C18 column (4.6 mm × 150 mm, 5 µm particle size), using a gradient of 15%–65% solvent B for 15 min [A = H₂O (0.1% TFA), B = ACN (0.1% TFA)] at a flow rate of 2 ml/min.

[⁶⁸Ga]Ga-DOTA-TOC was synthesized in an automatic Modular-Lab Pharm Tracer module connected to a ⁶⁸Ge/⁶⁸Ga-generator IGG100 (Eckert & Ziegler), following the synthesis template and kit reagents of the manufacturer. ^natGa-DOTA-TOC was synthesized using ^natGa(NO₃)₃ × H₂O and purified and analyzed as described above for the natural copper complexes.

2.3 Determination of lipophilicity

The lipophilicity of [⁶¹Cu]Cu-DOTA-TOC and [⁶¹Cu]Cu-NODAGA-TOC was assessed by the determination of the distribution coefficient (D), expressed as log D (pH = 7.4), between an aqueous and an organic phase following the “shake-flask” method. Briefly, the radiotracer (10 µl, 1 µM) was added to 1 ml of a 1:1 pre-saturated mixture of 1-octanol and phosphate-buffered saline (PBS pH 7.4). The solution was vortexed for 30 min and then centrifuged at 3,000 rpm to achieve phase separation. Aliquots of 0.1 ml from each phase were collected and measured in a γ-counter. The distribution coefficient was calculated as the average of the logarithmic values (n = 3) of the ratio between the radioactivity in the organic and PBS phases. [⁶⁸Ga]Ga-DOTA-TOC was used as a reference.

2.4 Affinity studies

Competition binding and saturation binding experiments were conducted in HEK-SST₂ cell membranes, and the data were analyzed using GraphPad Prism 9 software. The preparation of the cell membranes and the experimental details are as previously described (13).

For the competition binding experiments, membrane suspensions were incubated in 96 wells (10 µg/well) for 1 h at 37°C with the radioligand ¹²⁵I-Tyr-SS-14 [Chelatec (Saint-Herblain, France), where SS-14 = somatostatin-14], at a concentration of 0.05 nM, and increasing concentrations of ^natCu-DOTA-TOC or ^natCu-NODAGA-TOC (ranging from 0.001 to 100 nM). Filtration on a Brandel 48-well Cell Harvester followed and the filtered membranes were measured in a γ-counter. ^natGa-DOTA-TOC and SS-14 were used as references. The half-maximal inhibitory concentration (IC₅₀) was determined using the “log(inhibitor) vs. response” equation.

The saturation binding experiments were performed to determine the dissociation constant (K_D) and the maximum number of binding sites (B_max). The cell membrane suspension was incubated with different concentrations (ranging from 0.075 to 10 nM) of ^61/natCu-DOTA-TOC and ^61/natCu-NODAGA-TOC for 1 h at 37°C, followed by membrane harvesting on a Brandel 48-well Cell Harvester, as described above.

In both experiments, non-specific binding was determined in the presence of a 1,000-fold excess of SS-14.

2.5 Cellular uptake and efflux

The cellular uptake and distribution of [⁶¹Cu]Cu-DOTA-TOC and [⁶¹Cu]Cu-NODAGA-TOC were evaluated on intact HEK-SST₂ cells seeded in 6-well plates. The cells were each incubated with the radiotracer (2.5 nM/well) for 0.5, 1, 2, and 4 h at 37°C, either alone or in the presence of 1,000-fold excess of SS-14 to distinguish between specific and non-specific uptake. At the defined time points, the medium was removed, and the cells were washed twice with ice-cold PBS. The cells were then treated twice for 5 min with ice-cold glycine solution (0.05 M, pH 2.8) to detach the cell membrane-bound fraction (acid released). Afterward, the cells containing the internalized fraction were detached using NaOH 1 M at 37°C and collected for measurement. [⁶⁸Ga]Ga-DOTA-TOC was evaluated as a reference.

The efflux was studied after 1 h incubation of the cells with the radiotracer at 37°C and the removal of the unbound fraction and the cell membrane-bound fraction, as described above (acid-wash). The cells were then incubated with fresh medium at 37°C either alone or with a competitor (1,000-fold excess of DOTA-TOC, 2.5 µM/well) for 10, 20, 30, 45, 60, 90, 120, 150, 180, and 240 min. At each time point, the efflux (or retention) was measured after the removal of the medium without or with the competitor and replacement with fresh pre-warmed (37°C) medium alone or with the competitor. At the end of the experiment, the cells were detached using 1 M NaOH at 37°C and collected to determine the remaining cell-associated radiotracer.

2.6 Animal studies

Animal experiments were conducted in accordance with Swiss animal welfare laws and regulations under license number 30515 granted by the Veterinary Office (Department of Health) of the Canton Basel-Stadt. Female athymic nude-Foxn1^nu/Foxn1⁺ mice (Envigo, The Netherlands), 4–6 weeks old, were inoculated subcutaneously with HEK-SST₂ cells (10⁷ cells/100 μl), freshly suspended in sterile PBS, in the shoulder. The tumors were allowed to grow for 2–3 weeks, reaching a volume of 100–200 mm³.

2.7 PET/CT imaging studies

[⁶¹Cu]Cu-DOTA-TOC and [⁶¹Cu]Cu-NODAGA-TOC (100 µl/200 pmol/4–5 MBq) were intravenously injected in the tail vein of the HEK-SST₂ xenografts. The mice were anesthetized with 1.5% isoflurane and dynamic PET scans were acquired from 0 to 1 h post-injection (p.i.). The mice were euthanized by CO₂ at 4 h p.i., their bladders were mechanically emptied, and static PET scans were acquired for 30 min. PET scans of [⁶⁸Ga]Ga-DOTA-TOC (100 µl/200 pmol/5 MBq) were acquired in a static mode 1 h p.i.

The PET images were acquired in list mode using a small-animal PET scanner (β-CUBE, Molecubes, Ghent, Belgium) with a spatial resolution of 0.85 mm and an axial field-of-view of 13 cm. All PET scans were decay corrected and reconstructed into a 192 × 192 × 384 matrix by an ordered subsets maximization expectation (OSEM) algorithm using 30 iterations, a voxel size of 400 µm × 400 µm × 400 µm, and15 min per frame. Computed tomography (CT) data was used to apply attenuation correction on the PET data. The CT was imaged supine, headfirst, using the nanoSPECT/CT™ scanner (Bioscan Inc.). Topograms and helical CT scans of the whole mouse were first acquired using the following parameters: x-ray tube current: 177 µA, x-ray tube voltage 45 kVp, 90 s, and 180 frames per rotation, pitch 1. The CT images were reconstructed using CTReco (version r1.146) with a standard filtered back projection algorithm (exact cone beam) and post-filtered (Ram-Lak, 100% frequency cut-off), resulting in a pixel size of 0.2 mm. Co-registered PET/CT images were visualized using maximum intensity projection (MIP) with VivoQuant software (version 4.0).

2.8 In vivo specificity

The specificity of the ⁶¹Cu-labeled tracers was assessed in HEK-SST₂ xenografts by administering DOTA-TOC (100 µl/200 nmol) 2–5 min pre-injection, followed by the injection of the radiotracer (100 µl/200 pmol/4 MBq). The mice were euthanized 1 h p.i. and static PET/CT scans were performed, as described above.

2.9 Biodistribution

Quantitative biodistribution studies were performed for [⁶¹Cu]Cu-DOTA-TOC and [⁶¹Cu]Cu-NODAGA-TOC (100 µl/200 pmol/2–4 MBq) at 1 and 4 h p.i. The mice were euthanized at the time point of investigation by CO₂ asphyxiation. Organs of interest and blood were collected, rinsed of excess blood, blotted dry, weighed, and counted in a γ-counter. The samples were counted against a suitably diluted aliquot of the injected solution as the standard. The results are expressed as a percentage of injected activity per gram of tissue (%IA/g) and represent the mean ± standard deviation (SD) of n = 4–9 mice/group. The biodistribution of [⁶⁸Ga]Ga-DOTA-TOC was assessed at 1 h p.i. for comparison.

2.10 Dosimetry

Additional biodistribution data were generated in healthy BALB/c mice using [⁶⁴Cu]Cu-NODAGA-TOC at 1, 4, 12, and 24 h p.i. and were combined with the data of [⁶¹Cu]Cu-NODAGA-TOC at 1 and 4 h p.i. [⁶⁴Cu]CuCl₂ was provided by the University Hospital Tübingen, Germany. The non-decay corrected biodistribution data for copper-61 (t_1/2 = 3.33 h) were used to generate time-activity curves for [⁶¹Cu]Cu-NODAGA-TOC. OLINDA/EXM 1.0 was used to integrate the fitted time-activity curves and to estimate the organ and effective doses using the whole-body adult female model. For all the calculations, the assumption was made that the mouse biodistribution, determined as the %IA/organ, was the same as the human biodistribution. The dosimetry estimates were performed as previously described (14).

2.11 Statistics

Statistical analysis was performed by unpaired t-test with Welch's correction using GraphPad Prism software (GraphPad Inc., version 9). P-values < 0.05 were considered significant. All data were evaluated as mean ± standard deviation.

3 Results

3.1 ⁶¹Cu-labeled tracers and reference (radio)tracers

[⁶¹Cu]Cu-DOTA-TOC and [⁶¹Cu]Cu-NODAGA-TOC were synthesized with radiochemical purities of ≥96% and ≥98%, respectively, at an apparent molar activity of 25 MBq/nmol, without the need for a post-labeling purification step. [⁶¹Cu]Cu-DOTA-TOC required an elevated temperature (95°C), while [⁶¹Cu]Cu-NODAGA-TOC was synthesized at room temperature. Both ⁶¹Cu-labeled tracers remained stable in their buffered solution for up to 4 h at room temperature. The analytical data of the radiotracers are summarized in Table 1. [⁶⁸Ga]Ga-DOTA-TOC was prepared at an apparent molar activity of 40 MBq/nmol and followed an established standard labeling procedure. Representative (radio)chromatograms of all radiotracers are provided in Supplementary Figure S1.

Table 1

Table 1. Radiochemical purity, retention time (t_R), and stability data of the ⁶¹Cu-labeled tracers.

The reference non-active complexes ^natCu-DOTA-TOC and ^natCu-NODAGA-TOC were prepared with a purity of >98% and characterized by reverse phase (RP)-HPLC and LC-MS. The analytical data are provided in Supplementary Figure S1 and Supplementary Table S1.

3.2 Determination of lipophilicity and in vitro characterization

The results of the log D determination and affinity measurements of [⁶¹Cu]Cu-DOTA-TOC and [⁶¹Cu]Cu-NODAGA-TOC, in comparison to [⁶⁸Ga]Ga-DOTA-TOC, are summarized in Table 2.

Table 2

Table 2. Lipophilicity and in vitro characteristics in HEK-SST₂ cell membranes after 1 h incubation at 37°C.

The labeling of DOTA-TOC with copper-61 vs. gallium-68 impacted lipophilicity (log D = −2.81 ± 0.29 vs. −3.18 ± 0.11, respectively). Among the three radiotracers, [⁶¹Cu]Cu-NODAGA-TOC was the most lipophilic (log D = −2.60 ± 0.24). Nevertheless, the log D values indicate that all three radiotracers are highly hydrophilic.

The two ^natCu-complexes, ^natCu-DOTA-TOC, and ^natCu-NODAGA-TOC, showed a very high affinity for SST₂ [IC₅₀ = 0.23 nM (95% CI: 0.21–0.26) and 0.34 nM (95% CI: 0.30–0.38), respectively] and were in the same low sub-nanomolar level as the reference, ^natGa-DOTA-TOC [IC₅₀ = 0.18 nM (95% CI: 0.16–0.20)] and the SS-14 [IC₅₀ = 0.11 nM (95% CI: 0.09–0.14)]. The saturation binding experiments showed no significant difference between ^61/natCu-DOTA-TOC and ^61/natCu-NODAGA-TOC by means of the maximum number of binding sites (B_max) recognized by the radiotracers and their dissociation constant (K_D). The results are summarized in Table 2. The dose–response curves from the competition binding experiments vs. ¹²⁵I-Tyr-SS-14 and the saturation binding curves are shown in Supplementary Figures S2 and S3, respectively.

3.3 Cellular uptake and efflux

[⁶¹Cu]Cu-DOTA-TOC showed a higher cellular uptake than [⁶¹Cu]Cu-NODAGA-TOC (78.3 ± 1.3% and 70.9 ± 3.5%, respectively, after 4 h at 37°C, p = 0.0007) and was comparable to [⁶⁸Ga]Ga-DOTA-TOC (79.0 ± 3.1%, after 4 h at 37°C, p = 0.59) (Figure 1A). The radiotracers were almost entirely internalized upon binding to SST₂, while only a negligible amount remained on the cell membrane (<2% of the total added activity at 4 h). The cellular uptake and distribution over time are reported in Supplementary Figure S4. Blocking experiments with SS-14 proved the SST₂-mediated uptake of both ⁶¹Cu-labeled tracers in vitro.

Figure 1

Figure 1. (A) Cellular uptake (surface bound + internalized) of the tested radiotracers in the HEK-SST₂ cells after 4 h at 37°C. The results represent the mean ± standard deviation of the specific (=total − non-specific) uptake from a minimum of two separate experiments, each in triplicate. Cell membrane-bound and internalized fractions are indicated. (B) Efflux rate in the absence or in the presence of 1,000-fold excess of a competitor (DOTA-TOC) at 37°C. The data were analyzed according to the one-phase exponential decay equation (GraphPad Software Inc., Prism 9).

[⁶¹Cu]Cu-DOTA-TOC and [⁶¹Cu]Cu-NODAGA-TOC were externalized using a one-phase exponential decay model (Figure 1B). The efflux reached 80% after 4 h at 37°C when DOTA-TOC (competitor) was added to the medium. In the absence of a competitor, the efflux reached a plateau of approximately 40% after 2 h, without a further increase at 4 h, suggesting a rebinding of the externalized fraction of the radiotracer that remained in the proximity of the receptors.

3.4 PET/CT imaging studies

Dynamic PET/CT images from between 0–1 h p.i. (Figure 2) revealed fast accumulation of [⁶¹Cu]Cu-DOTA-TOC and [⁶¹Cu]Cu-NODAGA-TOC in the tumor as early as 15 min p.i., and which further increased up to 1 h p.i. for both radiotracers. However, their total body distribution was distinctly different. [⁶¹Cu]Cu-DOTA-TOC accumulated rapidly in the liver, gallbladder, kidneys, and intestine (Figure 2A). In contrast, [⁶¹Cu]Cu-NODAGA-TOC accumulated primarily in the kidneys (Figure 2B). Over time, the uptake of [⁶¹Cu]Cu-DOTA-TOC in the abdomen remained persistent, while no improvement of the tumor-to-background contrast was observed. In contrast, the tumor-to-non-tumor ratios were improved for [⁶¹Cu]Cu-NODAGA-TOC, especially the tumor-to-kidney ratio (see Figures 2C,D, respectively). PET/CT imaging of [⁶¹Cu]Cu-NODAGA-TOC compared well with the reference, [⁶⁸Ga]Ga-DOTA-TOC, at 1 h p.i., while [⁶¹Cu]Cu-DOTA-TOC was clearly inferior (Figure 3).

Figure 2

Figure 2. MIPs of the dynamic PET/CT scans of HEK-SST₂ xenografts after an injection of 200 pmol/4-5mMBq [⁶¹Cu]Cu-DOTA-TOC (A) or [⁶¹Cu]Cu-NODAGA-TOC (B) from 0 to 1 h p.i., in 15 min frames. MIPs of static PET/CT scans at 4 h after an injection of 200 pmol/4-5 MBq [⁶¹Cu]Cu-DOTA-TOC (C) or [⁶¹Cu]Cu-NODAGA-TOC (D) into the HEK-SST₂ xenografts. T, tumor; K, kidneys; L, liver; Gb, gallbladder; Bl, bladder; SUV, standard uptake value.

Figure 3

Figure 3. MIPs of the PET/CT scans of [⁶¹Cu]Cu-DOTA-TOC and [⁶¹Cu]Cu-NODAGA-TOC (100 µl/200 pmol/4-5 MBq) at 1 h p.i., with and without a pre-injection administration of a blocking agent (DOTA-TOC, 200 µmol). A PET/CT scan of [⁶⁸Ga]Ga-DOTA-TOC (100 µl/200 pmol/5 MBq,) at 1 h p.i. is provided for visual comparison. The ⁶¹Cu-PET/CT scans were acquired in mice under anesthesia (full bladder), while the ⁶⁸Ga-PET/CT scan was acquired in an euthanized mouse (bladder has been emptied mechanically). The PET images scale in 0-4 standardized uptake values (SUVs) for the ⁶¹Cu-PET images and 0-2 SUVs for the ⁶⁸Ga-PET image.

High excess DOTA-TOC pre-injection resulted in a significant reduction of the accumulation of both ⁶¹Cu-labeled tracers in the tumors and SST₂-positive organs, confirming their receptor-mediated uptake in these tissues (Figure 3). Nevertheless, the abdominal uptake of [⁶¹Cu]Cu-DOTA-TOC, especially in the liver and intestine, was not suppressed, suggesting that this uptake was non-specific. The relatively high liver and intestine uptake of [⁶¹Cu]Cu-DOTA-TOC can be attributed, at least partially, to the release of copper-61 from the [⁶¹Cu]Cu-DOTA complex and the bio-reduction of Cu(II) to Cu(I) that can be incorporated into Cu-binding proteins (15).

3.5 Biodistribution studies

The biodistribution data are presented in Table 3. [⁶¹Cu]Cu-DOTA-TOC and [⁶¹Cu]Cu-NODAGA-TOC showed similar tumor uptake at 1 h p.i. (7.44 ± 2.33%IA/g and 8.88 ± 3.19%IA/g, respectively, p = 0.46) and at 4 h p.i. (6.85 ± 2.48%IA/g and 7.39 ± 1.36%IA/g, p = 0.67, respectively). Tumor uptake remained the same between 1 and 4 h p.i. for both the radiotracers (p = 0.72 for [⁶¹Cu]Cu-DOTA-TOC and p = 0.37 for [⁶¹Cu]Cu-NODAGA-TOC).

Table 3

Table 3. Biodistribution of [⁶¹Cu]Cu-DOTA-TOC, [⁶¹Cu]Cu-NODAGA-TOC, and [⁶⁸Ga]Ga-DOTA-TOC in the HEK-SST₂ xenografts.

Distinct differences were observed between the two radiotracers in their total body distribution, especially in the liver, abdomen, and kidneys. At the early time point of investigation (1 h p.i.), [⁶¹Cu]Cu-DOTA-TOC showed a significantly lower uptake in the kidneys compared with [⁶¹Cu]Cu-NODAGA-TOC (5.32 ± 0.58%IA/g vs. 12.5 ± 2.25%IA/g, p = 0.0013), but a significantly higher uptake in the liver (3.59 ± 0.49%IA/g vs. 0.29 ± 0.04%IA/g, p = 0.0009) and intestine (2.94 ± 0.15%IA/g vs. 0.92 ± 0.12%IA/g, p < 0.0001). At the late time point of investigation (4 h p.i.), these differences remained comparatively the same. Despite the washout from all organs but the tumors, [⁶¹Cu]Cu-DOTA-TOC retained a clearly higher background activity, especially in the abdomen, and ∼5-fold higher blood values compared with [⁶¹Cu]Cu-NODAGA-TOC. In contrast, [⁶¹Cu]Cu-NODAGA-TOC had a fast blood and body clearance, with persistent uptake mainly in the kidneys and tumor.

With the exception of the tumor-to-kidney ratio, all the other tumor-to-non-tumor ratios, including the tumor-to-blood ratio, were in favor of [⁶¹Cu]Cu-NODAGA-TOC. All ratios, including the tumor-to-kidneys ratio, were significantly improved from 1 to 4 h p.i., due to the background clearance and the persistent tumor uptake.

[⁶¹Cu]Cu-DOTA-TOC showed very similar accumulation in the SST₂-expressing tissues (e.g., tumor, stomach, or pancreas) as the reference radiotracer [⁶⁸Ga]Ga-DOTA-TOC (Table 3). However, as described above, it had significantly higher liver (3.59 ± 0.49%IA/g vs. 0.58 ± 0.07%IA/g, p = 0.001) and intestinal (2.94 ± 0.15%IA/g vs. 1.39 ± 0.28%IA/g, p = 0.0003) uptake. [⁶¹Cu]Cu-NODAGA-TOC had a similar uptake in the tumor (8.88 ± 3.19%IA/g vs. 6.64 ± 1.11%IA/g, p = 0.207) and a higher uptake in the kidneys (12.5 ± 2.25%IA/g vs. 8.37 ± 0.84%IA/g, p = 0.011) compared with [⁶⁸Ga]Ga-DOTA-TOC at 1 h p.i. [⁶¹Cu]Cu-NODAGA-TOC showed improved tumor-to-non-tumor ratios for all the considered organs, except the kidneys. This advantage can be attributed to copper-61 and/or to the conjugate NODAGA-TOC, thus it is potentially possible to also gain this advantage with [⁶⁸Ga]Ga-NODAGA-TOC. However, [⁶¹Cu]Cu-NODAGA-TOC demonstrated the best ratios at 4 h p.i., (a time point compatible with the physical half-life of copper-61, but not that of gallium-68) among the three radiotracers and the two time points of investigation.

3.6 Dosimetry

The pharmacokinetic data of [^61/64Cu]Cu-NODAGA-TOC from 1 up to 24 h p.i. are provided in Supplementary Table S2 and were used for the dosimetry estimates. Table 4 shows the estimated radiation dose of [⁶¹Cu]Cu-NODAGA-TOC for human females with an effective dose of 0.00241 mSv/MBq.

Table 4

Table 4. Total absorbed doses of [⁶¹Cu]Cu-NODAGA-TOC in different organs calculated by OLINDA/EXM version 1.0 with a standard adult female phantom.

4 Discussion

In the era of precision medicine, patient-tailored treatments rely on advanced imaging tools. A neuroendocrine tumor is an exemplary case where precision medicine is realistic via PET/CT imaging with radiolabeled somatostatin analogs. This study aimed to provide insights into the advantages of ⁶¹Cu-labeled somatostatin analogs in comparison with the established ⁶⁸Ga-labeled analogs for PET imaging of SST-expressing tumors. Furthermore, it aimed to compare copper-61 and copper-64 in view of the recently FDA-approved [⁶⁴Cu]Cu-DOTA-TATE (Detectnet®) and other ⁶⁴Cu-labeled somatostatin analogs.

In this study, copper-61 was produced in a medical cyclotron after deuteron irradiation of a natural nickel solid target. Target dissolution and purification were adapted and adjusted from Svedjehed et al. (12) and the details will be published elsewhere (manuscript in preparation). Copper-61 was produced with a radionuclidic purity exceeding 99.99% at 12 h post-synthesis and in a very high purity for direct radiolabeling. [⁶¹Cu]Cu-DOTA-TOC and [⁶¹Cu]Cu-NODAGA-TOC were synthesized with high radiochemical purities (≥96% and ≥98%, respectively), at an apparent molar activity of 25 MBq/nmol without the need for a post-labeling purification step. ⁶¹Cu-labeling of the SST analogs DOTA-TOC, DOTA-TATE, and DOTA-NOC with a labeling yield >97% has been reported in the literature when using copper-61 produced from an enriched zinc-64 liquid target (11). The use of an inexpensive natural nickel solid target to produce high-purity copper-61 reported in this study is economically more sustainable and, thus, more attractive.

In comparison with the reference, [⁶⁸Ga]Ga-DOTA-TOC, the two ⁶¹Cu-tracers were more lipophilic (p < 0.0001 for [⁶¹Cu]Cu-DOTA-TOC vs. [⁶⁸Ga]Ga-DOTA-TOC and p < 0.0001 for [⁶¹Cu]Cu-NODAGA-TOC vs. [⁶⁸Ga]Ga-DOTA-TOC). The in vitro assessment of the SST₂-expressing intact cells and membranes did not indicate significant differences between the two ⁶¹Cu-tracers and in comparison with [⁶⁸Ga]Ga-DOTA-TOC. However, significant differences were observed among the three radiotracers in vivo. More specifically, while the tumor uptake of TOC was not impacted by the radionuclide (copper-61 or gallium-68) or the chelator (DOTA or NODAGA), the distribution in the other organs was significantly impacted. In comparison with [⁶⁸Ga]Ga-DOTA-TOC, [⁶¹Cu]Cu-DOTA-TOC showed major differences, while [⁶¹Cu]Cu-NODAGA-TOC showed a similar biodistribution pattern at 1 h p.i. In contrast with the other two, [⁶¹Cu]Cu-DOTA-TOC showed unfavorable biodistribution, characterized by high and persistent accumulation in the liver and the abdomen, which are proven to be non-SST-mediated (see specificity studies). The results indicate that ⁶¹Cu-based PET tracers using DOTA as a chelator have an unfavorable biodistribution.

Though, when [⁶⁴Cu]Cu-DOTA-TATE was compared with [⁶⁸Ga]Ga-DOTA-TOC in the same NET patients, it showed the same sensitivity on a patient-by-patient basis and a higher lesion detection rate (16). However, this was not attributed to the radiotracer itself but mainly to the shorter β⁺ range of copper-64 compared with gallium-68. Currently, there are no conclusive data on whether [⁶⁴Cu]Cu-DOTA-TATE or [⁶⁸Ga]Ga-DOTA-TOC/-TATE is superior (17). Moreover, it remains unclear whether the liver uptake of [⁶⁴Cu]Cu-DOTA-TATE is a limitation in detecting lesions, especially in the liver, the primary organ of metastasis of NETs. The liver uptake of ⁶⁴Cu-tracers or other ^xCu-based radiopharmaceuticals, especially those based on DOTA and DOTA-derivatives, is mainly attributed to the biologically triggered reduction of Cu(II) to Cu(I) and the inability of DOTA to stabilize Cu(I), leading to accumulation in the liver and other off-target tissues (10, 18–20). In our recent study, in which the in vivo metabolic stability of [⁶¹Cu]Cu-NODAGA-PSMA and [⁶¹Cu]Cu-DOTAGA-PSMA was investigated, we demonstrated that a substantial amount of copper-61 was released from the DOTAGA-complex and accumulated in the liver and the abdomen, similar to [⁶¹Cu]CuCl₂ (10). These common biodistribution features of [⁶¹Cu]Cu-DOTAGA-PSMA and [⁶¹Cu]CuCl₂ were also observed in the biodistribution of [⁶¹Cu]Cu-DOTA-TOC, suggesting the release of copper-61. Among the alternative chelators developed for copper, the cage amine ligand MeCOSAR {5-[8-methyl-3,6,10,13,16,19-hexaaza-bicyclo(6.6.6)icosan-1-ylamino]-5-oxopentanoic acid}, called sarcophagine (Sar), was used successfully in combination with TATE. In comparison with [⁶⁸Ga]Ga-DOTA-TATE, [⁶⁴Cu]Cu-SAR-TATE had very similar outcomes in the PET images of NET patients at 1 h p.i., and clear advantages at 4 and 24 h p.i. compared with 1 h p.i. due to the increased lesion-to-liver ratios (21).

Our findings with [⁶¹Cu]Cu-NODAGA-TOC are in line with the clinical observations of [⁶⁴Cu]Cu-SAR-TATE, supporting the advantages of imaging at a later time point. [⁶¹Cu]Cu-NODAGA-TOC had a fast blood and background clearance, with persistent uptake mainly in the kidneys and tumor. With the exception of the tumor-to-kidney ratio, all the other tumor-to-non-tumor ratios and the tumor-to-blood ratio were in favor of [⁶¹Cu]Cu-NODAGA-TOC at 1 h p.i. and they were all, including the tumor-to-kidney ratio, significantly improved from 1 h to 4 h p.i. due to the background clearance and the persistent tumor uptake. Furthermore, the tumor uptake of the radiolabeled somatostatin analogs continued to increase up to approximately 12 h p.i (22). Even though an uptake time of 1 h p.i. is recommended for ⁶⁸Ga-PET scans (23), there are indications that late-time imaging improves the detection rate (21, 24, 25). This might be valuable for unclear or difficult cases, especially regarding liver lesions. The half-life of copper-61 is compatible with the pharmacokinetics of somatostatin analogs and with early- and late-time imaging, i.e., 1 and 3–4 h after injection. Furthermore, NODAGA is a chelator that forms a stable complex with Cu(II), in contrast with DOTA, thus liver uptake is not of concern. Furthermore, NODAGA has the advantage of fast kinetics and radiolabeling at room temperature within a few minutes. These characteristics are very attractive for kit formulation and routine daily clinical use.

In principle, copper-64 also allows for late-time imaging and alleviates central production and shipment issues when compared with the logistical challenges of gallium-68. However, there are two main reasons to consider copper-61 over copper-64. The first is the positron-branching fraction as it is only 18% for copper-64 vs. 61% for copper-61. This implies that copper-64 requires a much higher injected dose or longer scanning time than copper-61 to obtain the same photon count statistics, while the image reconstruction parameters might be challenging. The low positron-branching fraction in combination with the longer half-life of copper-64 (t_1/2 = 12.7 h) and its β⁻ component (39%) account for the higher level of radiation exposure with copper-64. The reported effective dose of [⁶⁴Cu]Cu-DOTA-TATE in humans ranges from 0.0315 to 0.0454 mSv/MBq (21, 26), while for [⁶⁸Ga]Ga-DOTA-TOC or [⁶⁸Ga]Ga-DOTA-TATE it is 0.021–0.0257 mSv/MBq (27, 28). This difference increases due to the higher injected activity demand in the case of copper-64. In our study, we used biodistribution data from mice and assumed that humans have the same pharmacokinetics. [⁶¹Cu]Cu-NODAGA-TOC had an effective dose of 0.00241 mSv/MBq and a total-body absorbed dose of 1.27 × 10⁻³ mGy/MBq, while the organ with the highest dose was the kidneys (9.65 × 10⁻² mGy/MBq). In comparison with the dosimetry data for [⁶⁸Ga]Ga-DOTA-TATE extrapolated from mice for humans (e.g., a total-body absorbed dose of 1.27 × 10⁻³ mGy/MBq and kidney dose of 9.01 × 10⁻² mGy/MBq) (29), the data confirmed that copper-61 is within the safe radiation limits for PET imaging.

The second argument for copper-61 vs. copper-64 is related to the production. Copper-61 can be produced through the deuteron bombardment of natural nickel. For increased production volumes, this process can also utilize isotopically enriched nickel-60 or nickel-61. The yield of copper-61 can vary from 3 to 100 GBq, depending on the enrichment of the starting nickel material and beam parameters. This variability enables the adjustment of production to meet specific patient demands. Generating copper-61 necessitates 1–3 h of cyclotron beam time and an additional 30 min for purification. Conversely, copper-64 necessitates 4–12 h of beam time, with lower yields ranging from 3 to 10 GBq. The extensive beam time makes copper-64 production less practical for busy hospitals or manufacturing organizations involved in the production of multiple radiopharmaceuticals. Furthermore, the use of highly enriched (>98%) nickel-64 is required for copper-64 production to achieve the necessary radionuclidic purity and specific activity. Given that the cost of the target material and beam time are significant contributors to the overall production cost, the scalability of copper-61 production from an economic standpoint is more attractive.

Overall, copper-61 is a PET radionuclide with balanced characteristics and logistics that is between the short-lived, high-energy gallium-68 and the long-lived, low-energy copper-64 (4). In the era of radio-theranostics, research on new or unexplored radionuclides—especially in view of new discoveries, high clinical demand, and shortage of different radionuclides—is more pressing than ever. Radiometals are major players in this era, especially those with theranostic twins. Copper-61/copper-67 are among these major players.

5 Conclusion

Somatostatin receptor ⁶¹Cu-PET imaging not only matches the performance of ⁶⁸Ga-PET at 1 h p.i. but has advantages in late-time imaging at 4 h p.i. This improves image contrast due to the increased tumor-to-non-tumor ratios, allows flexibility, and provides options for dosimetry estimates. [⁶¹Cu]Cu-NODAGA-TOC is superior to [⁶¹Cu]Cu-DOTA-TOC in vivo. The use of the chelator NODAGA allows quantitative labeling with copper-61 at room temperature and enables the straightforward use of kit formulation for simple manufacturing in medical centers. Copper-61 has advantages over copper-64 in terms of dosimetry and production logistics. Copper-61 and its therapeutic twin copper-67 comprise an ideal theranostic pair for Cu-based radiopharmaceuticals.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the corresponding author on reasonable request.

Ethics statement

The animal study was approved by the Veterinary Office (Department of Health) of the Canton Basel-Stadt, Switzerland. The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

TB: Data curation, Investigation, Methodology, Writing – original draft. RM: Data curation, Formal Analysis, Methodology, Validation, Visualization, Writing – original draft. LDP: Data curation, Investigation, Methodology, Writing – review & editing. RG: Data curation, Investigation, Methodology, Writing – review & editing. LMcD: Data curation, Investigation, Methodology, Software, Writing – review & editing. AJ: Methodology, Resources, Writing – review & editing. MB: Investigation, Methodology, Writing – review & editing. FDR: Data curation, Investigation, Methodology, Writing – review & editing. LJ-T: Conceptualization, Funding acquisition, Resources, Writing – review & editing, Validation. MF: Conceptualization, Funding acquisition, Resources, Supervision, Writing – review & editing, Formal Analysis, Validation, Writing – original draft.

Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study received support from Innosuisse, Project Nr. 37014.1 IP-LS, and Nuclidium AG (matching funding).

Acknowledgments

We acknowledge Molecubes (Gent, Belgium) for their support on copper-61 image reconstruction and quantification with the small-animal PET scanner.

Conflict of interest

MF acts as a scientific advisor of Nuclidium AG and is co-inventor on patent applications filed by Nuclidium AG and the University of Basel related to ⁶¹Cu-labeled tracers. FDR and LJ-T are employees of Nuclidium AG and co-inventors in a series of patent related to ⁶¹Cu-labeled tracers.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The authors declare that this study received funding from Nuclidium AG. The funder had the following involvement in the study: Production of copper-61 and involvement in the study design and project supervision.

Publisher's note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnume.2024.1481343/full#supplementary-material

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