⁴⁷Sc as useful β^–-emitter for the radiotheragnostic paradigm: a comparative study of feasible production routes

Abstract

Background

Radiotheragnostics makes use of the same molecular targeting vectors, labeled either with a diagnostic or therapeutic radionuclide, ideally of the same chemical element. The matched pair of scandium radionuclides, ⁴⁴Sc and ⁴⁷Sc, satisfies the desired physical aspects for PET imaging and radionuclide therapy, respectively. While the production and application of ⁴⁴Sc was extensively studied, ⁴⁷Sc is still in its infancy. The aim of the present study was, therefore, to investigate and compare two different methods of ⁴⁷Sc production, based on the neutron irradiation of enriched ⁴⁶Ca and ⁴⁷Ti targets, respectively.

Methods

⁴⁷Sc was produced by thermal neutron irradiation of enriched ⁴⁶Ca targets via the ⁴⁶Ca(n,γ)⁴⁷Ca → ⁴⁷Sc nuclear reaction and by fast neutron irradiation of ⁴⁷Ti targets via the ⁴⁷Ti(n,p)⁴⁷Sc nuclear reaction, respectively. The product was compared with regard to yield and radionuclidic purity. The chemical separation of ⁴⁷Sc was optimized in order to obtain a product of sufficient quality determined by labeling experiments using DOTANOC. Finally, preclinical SPECT/CT experiments were performed in tumor-bearing mice and compared with the PET image of the ⁴⁴Sc labeled counterpart.

Results

Up to 2 GBq ⁴⁷Sc was produced by thermal neutron irradiation of enriched ⁴⁶Ca targets. The optimized chemical isolation of ⁴⁷Sc from the target material allowed formulation of up to 1.5 GBq ⁴⁷Sc with high radionuclidic purity (>99.99%) in a small volume (~700 μL) useful for labeling purposes. Three consecutive separations were possible by isolating the in-grown ⁴⁷Sc from the ^46/47Ca-containing fraction. ⁴⁷Sc produced by fast neutron irradiated ⁴⁷Ti targets resulted in a reduced radionuclidic purity (99.95–88.5%). The chemical purity of the separated ⁴⁷Sc was determined by radiolabeling experiments using DOTANOC achievable at specific activities of 10 MBq/nmol. In vivo the ⁴⁷Sc-DOTANOC performed equal to ⁴⁴Sc-DOTANOC as determined by nuclear imaging.

Conclusion

The production of ⁴⁷Sc via the ⁴⁶Ca(n,γ)⁴⁷Ca nuclear reaction demonstrated significant advantages over the ⁴⁷Ti production route, as it provided higher quantities of a radionuclidically pure product. The subsequent decay of ⁴⁷Ca enabled the repeated separation of the ⁴⁷Sc daughter nuclide from the ⁴⁷Ca parent nuclide. Based on the results obtained from this work, ⁴⁷Sc shows potential to be produced in suitable quality for clinical application.

Background

Over the past few years, the concept of personalized medicine, where patient treatment is performed according to an individually tailored treatment regime, has gained much recognition (Kraeber-Bodere and Barbet 2014). In nuclear medicine, this approach is realized by exploiting diagnostic techniques, such as non-invasive imaging by means of Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT), together with individualized radiotherapeutic treatment (Velikyan 2012). The resulting combination became known as the theragnostic approach and comprises the use of the same molecular targeting vectors, labeled either with a diagnostic or therapeutic radionuclide (Baum and Kulkarni 2012). Ideally, the employed radionuclides represent a matched pair, where both are radioisotopes of the same chemical element. Only a limited number of matching radionuclides entail suitable decay characteristics for radiotheragnostic application (Rösch and Baum 2011); of those the radionuclides of scandium, ⁴⁴Sc/⁴³Sc and ⁴⁷Sc, are interesting candidates. Based on the physical and chemical characteristics, the β⁻-emitter ⁴⁷Sc is particularly interesting for radionuclide therapy, while the decay characteristics of ⁴⁴Sc and ⁴³Sc are well-suited for diagnostic PET imaging (Table 1) (Rösch 2012; Müller et al. 2014a, 2014b; Walczak et al. 2015).

Table 1 Nuclear data of theragnostic radionuclides for therapy and PET imaging

This matched pair would present an attractive alternative to ⁶⁸Ga and ¹⁷⁷Lu, which are currently used in clinics for PET imaging and therapy, respectively (Oh et al. 2011). Ga(III) and Lu(III) can be coordinated by 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-complexes (Majkowska-Pilip and Bilewicz 2011), however, they do not share the same coordination chemistry. Lu(III) is coordinated by all carboxyl groups of the octadentate DOTA (Viola-Villegas and Doyle 2009; Parus et al. 2015), while Ga(III) has a preference for the coordination number six, leaving two uncoordinated carboxyl groups in the Ga-DOTA-complex (Viola-Villegas and Doyle 2009; Majkowska-Pilip and Bilewicz 2011). As a result, these structural differences may have an influence on the radioconjugate’s chemical properties and, consequently, on the in vivo kinetics and receptor binding affinity (Reubi et al. 2000; Majkowska-Pilip and Bilewicz 2011). By using chemically identical radionuclides such as ⁴⁴Sc/⁴⁷Sc–known to form stable complexes with DOTA–this limitation could be addressed.

The physical half-life of ⁴⁴Sc of 3.97 h (recently re-determined as 4.04 h (Garcia-Torano et al. 2016), is almost 4-fold longer than that of ⁶⁸Ga (T_1/2 = 68 min) and, hence, allows its use with biomolecules with slower kinetics. Due to the possibility of shipping ⁴⁴Sc-radiopharmaceuticals over long distances, it can also facilitate logistics as it would allow centralized production and distribution to remote hospitals (Chakravarty et al. 2014; van der Meulen et al. 2015). The increased availability of ⁴⁴Sc has initiated a number of preclinical in vitro and in vivo studies with DOTA-conjugated biomolecules (Müller et al. 2013a, 2013b; Hernandez et al. 2014) and, recently, labeling of NODAGA (1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid)-functionalized peptides and DTPA (N-[(R)-2-amino-3-(para-isothiocyanato-phenyl)propyl]trans-(S,S)-cyclohexane-1,2-diamine N,N,N’,N”N”-pentaacetic acid)-functionalized antibodies was also demonstrated (Chakravarty et al. 2014; Domnanich et al. 2016).

The emission of low-energy β⁻-particles from ⁴⁷Sc (Eβ⁻ _av = 162 keV, Table 1) is particularly interesting for targeted radionuclide therapy of small tumors and cancer metastases, similar to the clinically-established ¹⁷⁷Lu (Eβ⁻ _av = 134 keV, T_1/2 = 6.65 d, Table 1). Moreover, the shorter half-life of ⁴⁷Sc (T_1/2 = 3.35 d) would encourage its use, in conjunction with small molecules, with relatively fast pharmacokinetic profiles. In analogy to ¹⁷⁷Lu, the decay of ⁴⁷Sc is characterized by the co-emission of γ-rays with an ideal energy (Eγ = 159 keV, Table 1) for SPECT imaging (Müller et al. 2014a).

The availability of high ⁴⁷Sc activity with adequate purity becomes a crucial issue for the realization of more detailed preclinical investigations and future clinical applications. So far, successful production of ⁴⁷Sc was described by two different neutron induced reactions: ⁴⁷Ti(n,p)⁴⁷Sc and ⁴⁶Ca(n,γ)⁴⁷Ca → ⁴⁷Sc (Fig. 1) (Bartoś et al. 2012; Müller et al. 2014a). To produce ⁴⁷Sc from ⁴⁷Ti, fast neutrons (E_n > 1 MeV) are required, while the ⁴⁶Ca(n,γ)⁴⁷Ca reaction is induced by thermal neutrons (E_n = 0.025 eV) (Bartoś et al. 2012). Proton irradiation of enriched ⁴⁸Ti targets made ⁴⁷Sc available via the ⁴⁸Ti(p,2p)⁴⁷Sc nuclear reaction, however, too much of the long-lived ⁴⁶Sc was co-produced (Srivastava 2012). An alternative ⁴⁷Sc production route considers photonuclear reactions on enriched ⁴⁸Ti and ⁴⁸Ca targets, respectively (Yagi and Kondo 1977; Mamtimin et al. 2015; Rane et al. 2015; Starovoitova et al. 2015). So far only the former route was studied in detail with enriched targets (Yagi and Kondo 1977), while for the latter only natural target material was used for initial benchmark experiments.

Fig. 1

Nuclear reactions for production of ⁴⁷Sc from ⁴⁶Ca via ⁴⁶Ca(n,γ)⁴⁷Ca → ⁴⁷Sc (a) and from ⁴⁷Ti via ⁴⁷Ti(n,p)⁴⁷Sc (b)

The aim of the present study was to optimize the parameters of the previously-reported production process for ⁴⁷Sc from enriched ⁴⁶Ca targets (Müller et al. 2014a) in order to reproducibly obtain increased ⁴⁷Sc yields in a formulation allowing direct preclinical application after radiolabeling and to compare it with the method using ⁴⁷Ti as target material. ⁴⁷Sc labeling experiments with DOTANOC were performed as part of the quality control. In the second part of the study the stability of ⁴⁷Sc-labeled DOTANOC was investigated and SPECT/CT imaging studies were performed in tumor-bearing mice to compare the performance of ⁴⁷Sc-DOTANOC with the previously obtained PET images of the ⁴⁴Sc-labeled counterpart. Moreover, comparison of SPECT images obtained with mice injected with ¹⁷⁷Lu-DOTANOC were also performed.

Methods

Chemicals

Enriched ⁴⁶CaCO₃ (83.09% ⁴⁰Ca, 1.19% ⁴²Ca, 0.36% ⁴³Ca, 8.55% ⁴⁴Ca, 5.00 ± 0.50% ⁴⁶Ca, 1.81% ⁴⁸Ca, Trace Sciences International, USA) was used as target material for thermal neutron irradiation. Enriched ⁴⁷TiO₂ (0.41% ⁴⁶Ti, 95.7 ± 0.3% ⁴⁷Ti, 3.61% ⁴⁸Ti, 0.15% ⁴⁹Ti, 0.13% ⁵⁰Ti, Isoflex, USA) was reduced to ⁴⁷Ti metal and used as target material for fast neutron irradiation. Prior to irradiation, a precursory scan for trace metals by ICP-OES (Perkin Elmer Optima 3000) was performed.

The chemical separation of Sc from Ca was performed on a N,N,N’,N’-tetra-n-octyldiglycolamide, non-branched resin (DGA, particle size 50–100 μm, TrisKem International, France). SCX cation exchange cartridges (100 mg Bond Elut SCX, particle size 40 μm, Agilent Technologies Inc., USA) or DGA extraction chromatographic resin were used for the preconcentration of Sc. Chemical separations were performed with MilliQ water, hydrochloric acid (HCl, 30% Suprapur, Merck KGaA, Germany) and sodium chloride (NaCl, Trace Select, ≥99.999%, Fluka Analytical, Germany). For the ⁴⁷TiO₂ reduction process, calcium hydride (CaH₂, metals basis, Mg <1%, Alfa Aesar, Germany), argon (Ar, 99.9999%, Linde, Germany) and acetic acid (CH₃COOH, 100% Suprapur, Merck KGaA, Germany) were used. Nitric acid (HNO₃, 65% Suprapur, Merck KGaA, Germany) was required for the preparation of the ⁴⁶Ca targets. DOTANOC acetate was obtained from ABX GmbH, advanced biochemical compounds, Germany.

Production of ⁴⁷Sc from enriched ⁴⁷Ti

The reduction of ⁴⁷TiO₂ was performed at Helmholtz Center for Heavy Ion Research (GSI) in Darmstadt as described elsewhere (Lommel et al. 2014). Briefly, the enriched ⁴⁷TiO₂ was combined with 40% surplus of calcium hydride and the reduction process was performed under constant argon flow at 900 °C for 1 h. Dilute acetic acid was used for the isolation of the reduced ⁴⁷Ti metal from the co-produced calcium oxide.

To prepare the targets, 0.6–19.9 mg reduced ⁴⁷Ti powder was placed in a quartz glass ampoule (Suprasil, Heraeus, Germany) and sealed. The targets were irradiated with neutrons at the spallation-induced neutron source, SINQ, at Paul Scherrer Institut (PSI) at a fast neutron flux (>1 MeV) of 3.3–3.5 × 10¹¹ n cm⁻² s⁻¹ for 1.5–18.9 days and in the BR2 reactor at SCK.CEN, Mol, Belgium in a reflector channel at a fast neutron flux (>1 MeV) of 5.7 × 10¹³ n cm⁻² s⁻¹ for 7 days. ⁴⁷Sc was formed via the ⁴⁷Ti(n,p)⁴⁷Sc nuclear reaction with fast neutrons.

Production of ⁴⁷Sc from enriched ⁴⁶Ca

To prepare the targets, 65–91 mg enriched ⁴⁶CaCO₃ powder was dissolved in concentrated nitric acid and evaporated to complete dryness at 60–70 °C. The ⁴⁶Ca(NO₃)₂ residue was taken up in dilute nitric acid (~1 M HNO₃) and an aliquot of the aqueous solution (0.14–0.35 mg ⁴⁶Ca) transferred into a quartz glass ampoule, evaporated to dryness and sealed.

⁴⁷Sc was produced by the irradiation of the described ⁴⁶Ca targets with thermal neutrons at the high flux reactor of Institut Laue-Langevin (ILL) in Grenoble, France at a thermal neutron flux of 1.0–1.4 × 10¹⁵ n cm⁻² s⁻¹, for 4 to 11 days, and at the BR2 reactor at SCK.CEN, Mol, Belgium at a thermal neutron flux of 3.2 × 10¹⁴ n cm⁻² s⁻¹ for 7 days, respectively. ⁴⁷Sc was generated by the decay of the formed ⁴⁷Ca (T_1/2 = 4.54 d) occurring during the irradiation, but also after removal of the ampoule from the reactor.

Separation of ⁴⁷Sc from ⁴⁶Ca and ⁴⁷Ca

The irradiated ⁴⁶Ca ampoules were delivered to PSI several days post-irradiation (2.6–12.4 d) and the ⁴⁷Sc separation was performed immediately, similarly to previously reported (Müller et al. 2014a). Each ampoule was transferred into a hot cell and the glass surface was cleaned twice with ~20 mL 1.0 M HCl and rinsed twice with ~20 mL MilliQ water. The crushing of the quartz glass ampoule was performed within a plastic target tube in a separate hot cell. Subsequently, the target tube containing the crushed ampoule was attached to the separation panel with the aid of manipulators. The design of the separation panel, including the adaptation of its operation inside the hot cell, was a crucial part of the method development (Fig. 2). The ⁴⁶Ca(NO₃)₂ (~10–25 mg) from the ampoule was dissolved in 4 mL 3.0 M HCl and transferred from the target tube to the reaction vessel. A system of syringes, peristaltic pumps and three-way valves (see schematic of the panel in Fig. 2) was used to transfer the reagents from outside into the hot cell. To ensure complete dissolution of the target material, the solution was pumped from the target tube to the reaction vial and back several times. The solution was loaded on a pre-conditioned DGA column (1 mL cartridge filled with 50–70 mg of DGA resin). A second rinse cycle of the crushed glass ampoule with 2.5 mL 3.0 M HCl ensured collection of final traces of the ⁴⁷Sc activity, which were subsequently sorbed onto the DGA resin column. Radioactivity detection probes were attached in the vicinity of the target tube and the DGA column to follow the transfer of the ⁴⁷Sc radioactivity. Further application of 2 mL 3.0 M HCl removed the stable ⁴⁶Ca and radioactive ⁴⁷Ca from the DGA resin. The entire Ca-containing effluent was collected in a separate vessel and kept for consecutive separation of further in-growing ⁴⁷Sc from the decaying ⁴⁷Ca. The sorbed ⁴⁷Sc was eluted from the resin column with 4 mL 0.1 M HCl and sorbed on a second column containing SCX cation exchange resin (Method A). Alternately, the ⁴⁷Sc-containing eluate was collected, acidified to yield a 3.0 M HCl solution and sorbed on a second, smaller DGA resin column (1 mL cartridge filled with 20–25 mg DGA resin) at a slow flow rate of ~0.3 mL/min (Method B), as described by Domnanich et al. (Domnanich et al. 2016). The elution of ⁴⁷Sc from the second column was performed with 700 μL 4.8 M NaCl/0.1 M HCl (for Method A) and with 1.7 mL 0.05 M HCl (for Method B) via a separate valve. In order to collect ⁴⁷Sc in a small volume, the 0.05 M HCl (Method B) was fractionized into three Eppendorf vials; the first contained ~700 μL and the other two ~500 μL each. Fractionized collection was not necessary for Method A, as the highest proportion of the ⁴⁷Sc radioactivity was trapped in a low quantity of eluate.

Fig. 2

Schematic diagram of the ⁴⁷Sc production panel. The components drawn in green are used only to perform separations according to Method A, the parts required for Method B are shown in red, while black indicates the apparatus components used for both methods

The renewed generation of ⁴⁷Sc, by the decay of radioactive ⁴⁷Ca in the Ca-containing fraction (⁴⁷Ca and stable ⁴⁶Ca), enabled subsequent separations after a minimum in-growth time of 3 days.

Radionuclidic purity

To identify the nuclide inventory of the samples, γ-ray spectrometry with an N-type high-purity germanium (HPGe) coaxial detector (EURISYS MESURES, France) and the Ortec InterWinner 7.1 software were employed. The aliquot of ⁴⁷Sc eluate was in the range of 3–15 MBq, while the entire neutron irradiated glass ampoules containing the ⁴⁷Ti were used for the measurements. The counting time was determined by ensuring the measurement error was <4%. To determine small activities of long-lived radionuclidic impurities, γ-spectrometry measurements of the same samples were performed with an extensive counting time several days post-irradiation.

Radiolabeling for quality control of the produced ⁴⁷Sc

After quantitative determination of the ⁴⁷Sc activity in the eluate with a dose calibrator (ISOMED 2010, Nuclear-Medizintechnik Dresden, GmbH, Germany), the required activity for radiolabeling in 0.05 M HCl was withdrawn from the product vial and 0.5 M sodium acetate solution (pH 8) was added to the ⁴⁷Sc eluate to obtain a pH value of ~4.5. DOTANOC (0.7 mM solution in MilliQ water) was added to the ⁴⁷Sc solution (~50 MBq) to obtain a specific activity of 10–25 MBq/nmol (2–5 nmol DOTANOC). The reaction mixture was incubated at 95 °C for 15 min. The preparation of ¹⁷⁷Lu-DOTANOC (25 MBq/nmol) was carried out under standard labeling conditions (pH 4.5, 95 °C) using no carrier-added ¹⁷⁷Lu (purchased from Isotope Technologies Garching GmbH, Germany) (Müller et al. 2013a).

High-performance liquid chromatography (HPLC, Merck Hitachi, LaChrom) with a C-18 reversed-phase column (Xterra^TM MS, C18, 5 μm, 150 × 4.6 mm; Waters) was used for determination of the radiolabeled fraction of DOTANOC. The detection was performed with a UV (LaChrom L-7400) and radiodetector (Berthold, HPLC Radioactivity Monitor, LB 506B). The mobile phase consisted of MilliQ water containing 0.1% trifluoracetic acid (A) and acetonitrile (B) with a gradient of 95% A and 5% B to 20% A and 80% B, over a period of 15 min, at a flow rate of 1.0 mL/min.

In vitro stability of ⁴⁷Sc- and ¹⁷⁷Lu-labeled DOTANOC

The in vitro stability of ⁴⁷Sc- and ¹⁷⁷Lu-labeled DOTANOC (radiochemical purities >95%) was investigated in phosphate buffered saline (PBS, pH 7.4). An activity of 50 MBq of ⁴⁷Sc- or ¹⁷⁷Lu-DOTANOC was diluted with PBS (pH 7.4) to a total volume of 500 μL and incubated at room temperature for 3 days. Once every 24 h an aliquot was withdrawn to determine the integrity of the labeled compound by means of HPLC.

SPECT/CT imaging with ⁴⁷Sc- and ¹⁷⁷Lu-DOTANOC

In vivo experiments were approved by the local veterinarian department and conducted in accordance with the Swiss law of animal protection. Female, athymic nude mice (CD-1 nude) at the age of 5–6 weeks were obtained from Charles River Laboratories, Sulzfeld, Germany. AR42J cells (rat exocrine pancreatic tumor cells, European Collection of Cell Cultures ECACC, Salisbury, U.K.) were suspended in PBS (5 × 10⁶ cells in 100 μL) and subcutaneously inoculated on each shoulder. SPECT/CT experiments were performed about 2 weeks after tumor cell inoculation, when the tumor reached a size of about 400 mm³.

Imaging studies were performed using a small-animal SPECT camera (NanoSPECT/CT™, Mediso Medical Imaging Systems, Budapest, Hungary) as previously reported (Müller et al. 2014b). The energy peaks for the camera were set at 159.4 keV (± 10%) for the scans with ⁴⁷Sc and 56.1 keV (± 10%), 112.9 keV (± 10%) and 208.4 keV (± 10%) for the scans with ¹⁷⁷Lu. SPECT/CT scans were followed by CT scans. The images were acquired using Nucline Software (version 1.02, Bioscan Inc., Poway, California, US). The reconstruction was performed iteratively with HiSPECT software (version 1.4.3049, Scivis GmbH, Göttingen, Germany). SPECT and CT data were automatically co-registered and the fused datasets were analyzed with the VivoQuant post-processing software (version 2.50, inviCRO Imaging Services and Software, Boston, USA).

The mice were injected intravenously with ⁴⁷Sc-DOTANOC (12 MBq, 1.2 nmol, 100 μL) and ¹⁷⁷Lu-DOTANOC (40 MBq, 1.2 nmol, 100 μL), respectively. The in vivo SPECT/CT scans of 35 min duration were acquired 3 h after injection of ⁴⁷Sc-DOTANOC. During the scans, the mice were anesthetized by inhalation of a mixture of isoflurane and oxygen. Post-mortem scans of 1.3–3.5 h were performed 6 h after injection of ⁴⁷Sc- and ¹⁷⁷Lu-DOTANOC. The SPECT acquisitions were performed in such a manner to obtain the same total number of counts for each scan.

Results

Production of ⁴⁷Sc from ⁴⁷Ti via the (n,p) reaction

The irradiation of enriched ⁴⁷Ti targets at both SINQ and the BR2 reactor resulted in the formation of 0.07–4.9 MBq ⁴⁷Sc at the end of irradiation (EOI). The respective ⁴⁷Sc saturation yields were determined to be between 1.8 and 10.0 MBq ⁴⁷Sc/mg ⁴⁷Ti 10⁻¹³ n cm⁻² s⁻¹ (summarized in Table 2) by taking the irradiation time, mass of enriched ⁴⁷Ti and fast neutron flux into consideration. γ-spectrometry measurements of the neutron-irradiated ⁴⁷Ti ampoules revealed that, other than ⁴⁷Sc, the long-lived radionuclidic impurity ⁴⁶Sc was formed. The amount of generated ⁴⁶Sc was influenced by the irradiation period and the neutron energy (Bokhari et al. 2010; Zerkin 2016) and ranged from 3.8 to 11.5% ⁴⁶Sc/for the irradiations at SINQ (Additional file 1: Figure S3). Considerably less ⁴⁶Sc (0.05%) was produced by the irradiation at the BR2 reactor, however. In view of the high percentage of co-produced ⁴⁶Sc and the relatively low ⁴⁷Sc production yield at both facilities, the production of sufficiently high ⁴⁷Sc activities for radiopharmaceutical applications was not considered feasible and, thus, chemical isolation of ⁴⁷Sc from neutron irradiated ⁴⁷Ti targets was not performed.

Table 2 Activity and yield of ⁴⁷Sc at the end of irradiation (EOI) with fast neutrons (>1 MeV) at SINQ (irradiations PSI 1, PSI 2 and PSI 3) and at the BR2 reactor (irradiation SCK.CEN)

Production of ⁴⁷Sc from ⁴⁶Ca via the (n,γ) reaction

The irradiation of ⁴⁶Ca targets with thermal neutrons resulted in the formation of ⁴⁷Ca, which decayed to ⁴⁷Sc and yielded 210–2140 MBq ⁴⁷Sc at the time the separation was performed. The ⁴⁷Sc saturation yield was determined by taking the mass of ⁴⁶Ca, the irradiation time (t_irr), the decay time after EOI (t_wait) and the thermal neutron flux (Φ_th) into account and was within the range of 85–98 MBq ⁴⁷Sc/mg ⁴⁶Ca 10⁻¹³ n cm⁻² s⁻¹, which is comparable with the calculated ⁴⁷Sc saturation yield of 92 MBq ⁴⁷Sc/mg ⁴⁶Ca 10⁻¹³ n cm⁻² s⁻¹ (summarized in Table 3). ⁴⁷Sc is formed during the irradiation but also, however, for some time after the end of irradiation by the decay of ⁴⁷Ca. This implies that both the irradiation time (t_irr = 7.0–11.0 d) and the elapsed post-irradiation time until the start of the separation (t_wait = 3.0–12.5 d) are part of the yield-determining factors. The highest ⁴⁷Sc activities under the applied irradiation conditions become accessible at an optimal post irradiation waiting time (t_opt) and are represented as relative activity a(⁴⁷Sc)_opt in Table 3. The measured relative ⁴⁷Sc activities (a(⁴⁷Sc)_meas) are lower than the optimal relative ⁴⁷Sc activities (a(⁴⁷Sc)_opt), as the separations were performed several days after the optimal waiting time. The variable f(⁴⁷Sc) describes the ratio of the ⁴⁷Sc activity at t_opt and the ⁴⁷Ca activity at EOI. It can be considered as a measure for the maximal obtainable ⁴⁷Sc activity, since both activities are referred to the time point of their maximum. The formulae used for the calculation of A(⁴⁷Sc)_calc, t_opt, a(⁴⁷Sc)_opt and f(⁴⁷Sc) are given in Additional file 1: Figure S1 a-d.

Table 3 Activity of ⁴⁷Sc at the time of separation (A(⁴⁷Sc)), comparison of the calculated and measured ⁴⁷Sc saturation yield (A(⁴⁷Sc)_calc and A(⁴⁷Sc)_meas) and of the optimal and measured relative ⁴⁷Sc activity (a(⁴⁷Sc)_opt and a(⁴⁷Sc)_meas) after irradiation with thermal neutrons at ILL (irradiations ILL 1–5) and BR2 (irradiation SCK.CEN)

The measured relative ⁴⁷Sc activities (a(⁴⁷Sc)_meas) of the irradiations ILL 1, ILL 5 and SCK.CEN are represented by single data points in Fig. 3. After an optimal post irradiation waiting time (t_opt) of 2.8 days, the relative ⁴⁷Sc activity (a(⁴⁷Sc)_opt) reaches the maximum with 0.43. The separations ILL 1 and ILL 5 were performed after waiting times of 6.7 and 6.8 days, resulting in lower relative ⁴⁷Sc activities of 0.34 and 0.37, respectively. The waiting time after the irradiation at SCK.CEN (3.0 days) is close to t_opt, thus the obtained relative ⁴⁷Sc activity of 0.44 is comparable with the optimum value.

Fig. 3

The relative ⁴⁷Sc activities (a(⁴⁷Sc)_meas) of the irradiations ILL 1, ILL 5 and SCK.CEN accessible (and measured) at the time of separation (single data points). The activation functions of ⁴⁷Sc and ⁴⁷Ca at an irradiation period of 7.0 days (solid blue and red line) and their subsequent decay functions after the EOI (dashed blue and red line) are calculated for the respective irradiations

Separation of ⁴⁷Sc from ⁴⁶Ca and ⁴⁷Ca

After the ampoule was crushed, it was moved to the hot cell containing the production panel (Fig. 2), where the target material was dissolved by repeated application of 6.5 mL 3.0 M HCl. The ⁴⁷Ca and ⁴⁷Sc activity was transferred from the crushed glass ampoule to the DGA column, leaving only 1.1 ± 0.5% ⁴⁷Ca and 3.3 ± 0.4% ⁴⁷Sc attached to the glass. Direct application of 2 mL 3.0 M HCl quantitatively removed the Ca (⁴⁷Ca and stable Ca isotopes) from the resin. The collected Ca fraction contained 99.8 ± 0.2% of the total ⁴⁷Ca activity. The ⁴⁷Sc activity was eluted from the DGA column with 4 mL 0.1 M HCl and only 1.5 ± 0.6% of the ⁴⁷Sc activity remained on the column. Using Method A, the solution was concentrated on the SCX cation exchange resin (used as the second column) and eluted with 700 μL 4.8 M NaCl/0.1 M HCl solution (pH 0–0.5), collecting 94.8 ± 2.1% of the total ⁴⁷Sc activity. When using Method B, the molarity of the ⁴⁷Sc eluate was increased from 0.1 to 3.0 M HCl and the resulting solution adsorbed on a second smaller DGA column and eluted with 1.7 mL 0.05 M HCl. Fractionized collection revealed that about ~90% of the eluted ⁴⁷Sc activity was obtained in the first 700 μL (pH ~0).

With the installation of the chemical separation system in a hot cell, yields of up to 1.9 GBq ⁴⁷Sc could be isolated from the irradiated ⁴⁶Ca target. The renewed generation of ⁴⁷Sc from the β⁻-decay of ⁴⁷Ca (T_1/2 = 4.54 d) in the Ca-containing fraction, reached the maximum ⁴⁷Sc activity after an in-growth period of 5.6 days (Fig. 4) and, thus, enabled repeated separations. As a result of experimental conditions, separations were performed after an in-growth time of 3–7 days. The separation process was successfully repeated 2–4 times, until the eluted ⁴⁷Sc activity was ~100 MBq.

Fig. 4

The radioactive decay of the parent nuclide ⁴⁷Ca (T_1/2 = 4.54 d) to the daughter nuclide ⁴⁷Sc (T_1/2 = 3.35 d) reaches the maximum of ⁴⁷Sc activity after 135 h (5.6 d). The grey-shaded area indicates the time frame wherein the next separation was performed

Radionuclidic purity of ⁴⁷Sc produced from ⁴⁶Ca via (n,γ) reaction

The γ-ray spectrum of the neutron-irradiated ⁴⁶Ca target material (Additional file 1: Figure S2a) showed, exclusively, the γ-lines of ⁴⁷Sc (159 keV) and the parent nuclide ⁴⁷Ca (489, 808 and 1297 keV). After chemical separation and concentration of ⁴⁷Sc on SCX resin (Method A), the radionuclidic purity of the final ⁴⁷Sc eluate was 99.6 ± 0.7%. When the second DGA column (Method B) was used, the radionuclidic purity increased to 99.99 ± 0.03% (Additional file 1: Figure S2b). The long-lived radionuclidic impurity ⁴⁶Sc was only present in the eluate obtained from the first separation at a maximum of 0.005% and could be only detected by performing long-term γ-spectrometry measurements several days post separation. The isolated ⁴⁷Sc which was generated by the decay of ⁴⁷Ca in the Ca-containing fraction did not contain any ⁴⁶Sc, due to its entire removal within the first separation.

Radiolabeling and stability of ⁴⁷Sc labeled DOTANOC

Radiolabeling of ⁴⁷Sc was reproducible at a specific activity of 10 MBq ⁴⁷Sc per nmol DOTANOC, with >96% radiochemical purity. Depending on the activity concentration of the ⁴⁷Sc solution, it was also possible to label at higher specific activity of up to 25 MBq/nmol.

The stability of ⁴⁷Sc-labeled DOTANOC in PBS (pH 7.4) was investigated over a period of 3 days and compared to the stability of the ¹⁷⁷Lu-labeled analogue (Fig. 5). Directly after the radiosynthesis of ⁴⁷Sc with DOTANOC, the amount of intact radiolabeled product was 96.6–99.0% with less than 2% of radiolysis products visible as pre-peaks on the HPLC chromatogram. After 1 day at room temperature, the amount of intact radiolabeled ⁴⁷Sc-DOTANOC decreased to 81.3%, while 18.3% were subjected to radiolysis. Over the whole investigation period of 3 days, the percentage of radiolysis products increased to 44.4%, however, the amount of free ⁴⁷Sc was always below 2.1%. The stability of ⁴⁷Sc-labeled DOTANOC was found to be comparable with the clinically-used analogue ¹⁷⁷Lu-DOTANOC. After 3 days, the amount of intact ⁴⁷Sc-DOTANOC (54.1%) was similar to the amount of intact ¹⁷⁷Lu-DOTANOC (43.2%).

Fig. 5

Stability of ⁴⁷Sc-DOTANOC (a) and the comparison of the stability of ⁴⁷Sc- and ¹⁷⁷Lu-DOTANOC (b) in PBS (pH 7.4) investigated at room temperature over a 3-day period after radiolabeling. The retention times of free ⁴⁷Sc and ¹⁷⁷Lu are 2.2 ± 0.1 min and for ⁴⁷Sc- and ¹⁷⁷Lu-DOTANOC 9.5 ± 0.1 min and 9.3 ± 0.2 min, respectively

Imaging with ⁴⁷Sc-DOTANOC in comparison to ⁴⁴Sc-DOTANOC and ¹⁷⁷Lu-DOTANOC

SPECT/CT experiments performed with AR42J tumor-bearing mice allowed excellent visualization of the accumulated ⁴⁷Sc-DOTANOC in tumor xenografts, which express the somatostatin receptor (Fig. 6a). Activity accumulation was also observed in the kidneys, which was due to renal excretion of the radiopeptide. The SPECT/CT image obtained with ⁴⁷Sc-DOTANOC showed an equal activity distribution profile as was previously demonstrated by a PET/CT scan of an AR42J tumor-bearing mouse 3 h after injection of ⁴⁴Sc-DOTANOC (Fig. 6b) (Domnanich et al. 2016).

Fig. 6

In vivo SPECT/CT scan of a tumor-bearing mouse 3 h after injection of ⁴⁷Sc-DOTANOC (~12 MBq, ~1.2 nmol) (a). In vivo PET/CT scan of a tumor-bearing mouse 3 h after injection. ⁴⁴Sc-DOTANOC (~10 MBq, ~1 nmol), image reproduced from Domnanich et al. 2016 (Domnanich et al. 2016) (b). The scan durations were 35 min and 20 min, respectively (Tu = AR42J tumor xenograft, Ki = kidney, Bl = urinary bladder)

To compare the image quality of ⁴⁷Sc with the clinically-employed ¹⁷⁷Lu, mice with AR42J tumors were injected with either ⁴⁷Sc- or ¹⁷⁷Lu-labeled DOTANOC and scanned 6 h after injection (Fig. 7). Both images visualized the uptake of the radiopeptides in tumor xenografts located on each shoulder of the mouse and in the kidneys. The distribution pattern was equal for both radiopeptides, as expected, based on similar coordination of ⁴⁷Sc and ¹⁷⁷Lu when using DOTA.

Fig. 7

Post-mortem SPECT/CT scans of tumor-bearing mice 6 h after injection of the corresponding radiopeptide. Mouse injected with ⁴⁷Sc-DOTANOC (~12 MBq, ~1.2 nmol) (a) and mouse injected with ¹⁷⁷Lu-DOTANOC (~40 MBq, ~1.2 nmol) (b). The scan durations were 3.5 h and 1.3 h, respectively. (Tu = AR42J tumor xenograft, Ki = kidney)

Discussion

Recently, the use of ⁴⁷Sc for therapeutic purposes, as part of the ⁴⁴Sc/⁴⁷Sc theragnostic radionuclide pair, has attracted considerable attention in the field of nuclear medicine (Rösch and Baum 2011). In the present study we report, to our knowledge, the first reproducible production of MBq to GBq activities of ⁴⁷Sc by the irradiation of enriched ⁴⁶Ca target material with thermal neutrons. Concurrently, an alternative production route was investigated, using ⁴⁷Ti as target material and, by irradiation with fast neutrons, ⁴⁷Sc was formed via the (n,p) nuclear reaction. Irradiation of enriched ⁴⁷Ti at the spallation source SINQ (PSI, Switzerland) and the BR2 reactor (SCK.CEN, Mol, Belgium) resulted in the formation of 1.8–10.0 MBq ⁴⁷Sc/mg ⁴⁷Ti 10⁻¹³ n cm⁻² s⁻¹. The production of ⁴⁷Sc by neutron irradiation of enriched ⁴⁷Ti target material was performed previously by Mausner and Kolsky in the fission neutron spectrum of the HFIR reactor in Oak Ridge National Laboratory (ORNL) and Brookhaven National Laboratory (BNL) (Kolsky et al. 1998, Mausner, Kolsky et al. 1998) and by Bartoś et al. at the Maria reactor in Świerk, Poland (Bartoś et al. 2012). The reported activity of produced ⁴⁷Sc was within the range of 1.4–4.2 MBq ⁴⁷Sc/mg ⁴⁷Ti 10⁻¹³ n cm⁻² s⁻¹, which is comparable with the ⁴⁷Sc radioactivity obtained in our experiment at BR2. The irradiations at SINQ generated higher ⁴⁷Sc activities, due to the larger proportion of fast neutrons (> 1 MeV) at the spallation source (Lehmann 2016) than at the reactor (Chrysanthopoulou et al. 2014). The production of 85–98 MBq ⁴⁷Sc/mg ⁴⁶Ca 10⁻¹³ n cm⁻² s⁻¹ was feasible via the ⁴⁶Ca(n,γ)⁴⁷Ca → ⁴⁷Sc route with thermal neutrons from nuclear reactors at ILL and BR2, however. The nearly ten-fold higher accessible ⁴⁷Sc activity from ⁴⁶Ca irradiations can be attributed to the higher nuclear cross section of the ⁴⁶Ca(n,γ)⁴⁷Ca reaction (σ = 0.7 barn) (Magill et al. 2015) in comparison to the ⁴⁷Ti(n,p)⁴⁷Sc nuclear reaction (Additional file 1: Figure S4), as well as to an increased flux of thermal neutrons in comparison to fast neutrons. Lower measured relative ⁴⁷Sc activities than the optimal activities were obtained for the irradiations ILL 1–ILL 5, because the post-irradiation waiting period was too long and the built up ⁴⁷Sc already started to decay. Shorter waiting periods were prevented due to logistical issues. The waiting period after the irradiation at SCK.CEN, however, was close to the optimum time period, thus the measured relative ⁴⁷Sc activity obtained from the irradiation at SCK.CEN is in good correlation with the optimal value.

γ-spectra of the irradiated ⁴⁶Ca indicated the presence of ⁴⁷Ca and ⁴⁷Sc, however, due to high dead time a precise determination of both activities before separation was not possible. After chemical separation a product of high radionuclidic purity, containing only 0.005% ⁴⁶Sc, was obtained (Additional file 1: Figure S2). The acquired radionuclide inventory of ⁴⁷Ti targets, neutron irradiated at the spallation source SINQ, indicated a higher percentage of ⁴⁶Sc (Additional file 1: Figure S3), which increased proportionally with irradiation time (3.8–11.5% ⁴⁶Sc for 1.5–18.9 days irradiation), whereas a significantly smaller amount of ⁴⁶Sc (0.05%) was produced after 7 days of irradiation at the BR2 reactor (SCK.CEN). Trace activities of ¹²²Sb and ¹²⁴Sb were identified in the γ-ray spectrum of the irradiated ⁴⁷Ti ampoule at SINQ (Additional file 1: Figure S3), conceivably produced by neutron activation reactions on natural Sb impurities (Additional file 1: Table S5). The presence of ²²Na and ⁷Be can be attributed to spallation reactions with the capsulation material. The scan for trace metals revealed impurities of Ca, Sr, Sb and Zr in the reduced ⁴⁷Ti metal (Additional file 1: Table S5), which were probably introduced by the reduction process. Neutron activation reactions were only observed with Sb, but not with any of the other determined impurities, however.

The formation of ⁴⁶Sc from ⁴⁷Ti via the ⁴⁷Ti(n,n + p)⁴⁶Sc nuclear reaction is known to be only induced by very fast neutrons above the threshold of 10.7 MeV (Additional file 1: Figure S4) (Zerkin 2016). The considerably decreased ⁴⁶Sc impurity of the sample irradiated at the BR2 reactor can, therefore, be attributed to the lower proportion of very energetic neutrons in the fission spectrum of the BR2 reactor (Chrysanthopoulou et al. 2014), compared to the spallation neutron spectrum in the SINQ (Lehmann 2016). The percentage of ⁴⁶Sc obtained from the ⁴⁷Ti irradiation at the BR2 reactor was in agreement with those from previous experiments at ORNL, BNL and the Maria reactor, which was reported to be 0.06–0.64% ⁴⁶Sc (Kolsky et al. 1998; Mausner et al. 1998; Bartoś et al. 2012). With respect to radiopharmaceutical applications, the ten-fold higher ⁴⁷Sc production from ⁴⁶Ca targets, together with the absence of long-lived radionuclidic impurities, intensified our research towards the further development of the more attractive ⁴⁶Ca route.

In order to meet the requirements for radiopharmaceutical applications, the obtained ⁴⁷Sc eluate needed to be of high chemical purity and concentrated into a small volume of moderately acidic eluate to facilitate efficient radiolabeling and subsequent in vivo application. Initially, SCX cation exchange resin (Method A) was used and 94.8% ± 2.1% of the total ⁴⁷Sc activity was recovered in only 700 μL eluate (4.8 M NaCl/0.1 M HCl). The use of this resin is already established for the concentration of the ⁶⁸Ga eluate from the ⁶⁸Ge generator (Mueller et al. 2013); however, direct preclinical in vivo application is not feasible due to the high osmolarity of the obtained eluate. In a modification of the separation procedure, a second, smaller DGA column was used (Method B), allowing the elution of ~90% using 700 μL 0.05 M HCl. This enabled labeling and preclinical application as previously shown with ⁴⁴Sc (Domnanich et al. 2016).

The labeling of DOTANOC with ⁴⁷Sc was performed to verify a consistent chemical purity of the obtained eluate. Our results demonstrated reproducible radiosynthesis of ⁴⁷Sc-DOTANOC at specific activities of 10 MBq/nmol, whereas radiolabeling at 25 MBq/nmol proved possible. The obtained results indicated good quality of the produced ⁴⁷Sc achieving radiolabeling yields, in agreement with the previously-performed ⁴⁷Sc-radiolabeling of a DOTA-folate conjugate (Müller et al. 2014a).

In PBS ⁴⁷Sc remained stably coordinated by the DOTA-chelator over 3 days (<6% release), a result which was comparable to the ¹⁷⁷Lu-labeled peptide. ⁴⁷Sc- and ¹⁷⁷Lu-DOTANOC were, however, affected by radiolytic decomposition, which decreased the amount of intact product over time. It is likely the radiolytic stability could be enhanced by the addition of radical scavengers, such as ascorbic or gentisic acids, which were previously successfully employed for the stabilization of ⁹⁰Y- and ¹⁷⁷Lu-labeled DOTA-peptides (Liu and Edwards 2001; Liu et al. 2003).

In a proof-of-concept study, ⁴⁷Sc-DOTANOC was utilized for SPECT/CT imaging of AR42J tumor-bearing mice. The equal distribution profile of ⁴⁷Sc-DOTANOC and ⁴⁴Sc-DOTANOC, previously demonstrated using PET/CT, demonstrated the successful realization of the “matched pair” principle using scandium radionuclides. Moreover, the in vivo distribution of ⁴⁷Sc-DOTANOC was comparable to ¹⁷⁷Lu-DOTANOC. Due to the higher percentage of emitted γ-radiation in the case of ⁴⁷Sc, it is expected that less activity of ⁴⁷Sc-labeled compounds would be necessary for clinical SPECT as compared to the activity necessary for ¹⁷⁷Lu-labeled counterparts.

Conclusions

The reproducible production of activities of up to 2 GBq ⁴⁷Sc at high radionuclidic purity via the ⁴⁶Ca(n,γ)⁴⁷Ca nuclear reaction in the thermal neutron flux of a reactor was demonstrated. The subsequent decay of ⁴⁷Ca to ⁴⁷Sc creates a “pseudo-generator” system, which enables the repeated separation of the ⁴⁷Sc daughter nuclide from the ⁴⁷Ca parent nuclide. Together with the high radionuclidic purity and the superior yield of the isolated ⁴⁷Sc activity, the ⁴⁶Ca production route bears significant advantages over the ⁴⁷Ti production route with fast neutrons. Even though the high price of enriched ⁴⁶Ca represents a drawback, implementation of a suitable recovery method will limit the expenses. Based on the results obtained from this proof-of-concept study, ⁴⁷Sc has the potential to be produced in a suitable quality for clinical applications, however, the quantity of radioactivity still needs to be expanded to meet the requirements for radionuclide therapy.