Cyclotron production of 43Sc for PET imaging

Background Recently, significant interest in 44Sc as a tracer for positron emission tomography (PET) imaging has been observed. Unfortunately, the co-emission by 44Sc of high-energy γ rays (Eγ = 1157, 1499 keV) causes a dangerous increase of the radiation dose to the patients and clinical staff. However, it is possible to produce another radionuclide of scandium—43Sc—having properties similar to 44Sc but is characterized by much lower energy of the concurrent gamma emissions. This work presents the production route of 43Sc by α irradiation of natural calcium, its separation and purification processes, and the labeling of [DOTA,Tyr3] octreotate (DOTATATE) bioconjugate. Methods Natural CaCO3 and enriched [40Ca]CaCO3 were irradiated with alpha particles for 1 h in an energy range of 14.8–30 MeV at a beam current of 0.5 or 0.25 μA. In order to find the optimum method for the separation of 43Sc from irradiated calcium targets, three processes previously developed for 44Sc were tested. Radiolabeling experiments were performed with DOTATATE radiobioconjugate, and the stability of the obtained 43Sc-DOTATATE was tested in human serum. Results Studies of natCaCO3 target irradiation by alpha particles show that the optimum alpha particle energies are in the range of 24–27 MeV, giving 102 MBq/μA/h of 43Sc radioactivity which creates the opportunity to produce several GBq of 43Sc. The separation experiments performed indicate that, as with 44Sc, due to the simplicity of the operations and because of the chemical purity of the 43Sc obtained, the best separation process is when UTEVA resin is used. The DOTATATE conjugate was labeled by the obtained 43Sc with a yield >98 % at elevated temperature. Conclusions Tens of GBq activities of 43Sc of high radionuclidic purity can be obtainable for clinical applications by irradiation of natural calcium with an alpha beam.

relevant positron emission tomography tracers for imaging such tumors and their metastases [3]. DOTATOC labeled with 68 Ga showed high binding affinity to the human somatostatin receptor subtype 2, improving tumor imaging capabilities and offering the possibility of low dose imaging followed by higher dose treatment. However, the halflife of 68 Ga (T 1/2 = 67.71 min) may limit the spectrum of clinical applications of 68 Galabeled radiopharmaceuticals. Furthermore, the relatively high cost of the generators and perhaps more importantly the requirement for postelution purification and concentration of 68 Ga solution to small volume make this isotope of limited utility in clinical applications [4]. Therefore, the use of radionuclides of extended physical half-life is currently being reconsidered.
An alternative could be cyclotron-produced 64 Cu (T 1/2 = 12.7 h) which has been applied in a large number of preclinical and clinical positron emission tomography (PET) studies [5]. The longer half-life offers the possibility to label bigger molecules like mAB fragments and to use 64 Cu radiopharmaceuticals in hospitals without cyclotrons and radiopharmaceutical units. However, 64 Cu exists in three oxidation states and forms unstable in vivo chelate complexes. In addition, 64 Cu presents a relatively low positron branching ratio (17.6 %) and high co-emission of β − particles (39 % branching ratio) which significantly contribute to an additional patient dose.
In 2010, the 44 Sc radionuclide was proposed by Roesch as a potential alternative to 68 Ga in clinical PET diagnosis [6,7]. 44 Sc decays by the emission of low-energy positrons E β+ , with a half-life of T 1/2 = 3.97 h, which is almost four times longer than that of 68 Ga. 44 Sc can be obtained from the 44 Ti/ 44 Sc generator [8] or produced in the 44 Ca(p,n) 44 Sc reaction on small-or medium-sized medical cyclotrons that currently supply 18 F to hospitals [9][10][11][12][13][14]. These properties make it highly attractive for clinical PET applications because they enable transportation of 44 Sc-labeled radiopharmaceuticals to hospitals that are located several hundred kilometers away from the radiopharmaceutical production site. Moreover, it was found that Sc 3+ likewise Y 3+ and Lu 3+ , forms in aqueous solutions of DOTA complexes with the same coordination sphere (CN = 8) and with similar stability constants [15], whereas the relatively small Ga 3+ forms octahedral complexes. As a result, the chemical properties of 44 Sc-labeled DOTA conjugates are almost the same as those of the 90 Y-and 177 Lu-labeled versions; therefore, we can presume that 44 Sc-DOTA bioconjugates will demonstrate similar properties in vivo (i.e., receptor affinity, kidney clearance) to the 177 Lu-and 90 Y-conjugates currently applied in therapy.
However, the co-emission of high-energy γ rays (E γ = 1157, 1499 keV) has to be taken into consideration with regard to the radiation dose to the patients and clinical staff. Also, co-production of longer lived 44m Sc (T 1/2 = 58.6 h) increases the radiation dose.
In our work, we propose to use another radionuclide of scandium, i.e., 43 Sc, which shows properties similar to 44 Sc, but with much lower energy concurrent gamma emissions (Table 1). 43 Sc can be produced either by the 43 Ca(p,n), or 42 Ca(d,n) reactions, but unfortunately, the cost of enriched calcium targets is prohibitive. A more promising method of 43 Sc production is alpha irradiation of a natural calcium target via the 40 Ca(α,p) and 40 Ca(α,n) channels. The possibility of 43 Sc production by this route has already been mentioned in four conference communications. The availability of cyclotrons with intense α beams is limited; however, with a near-to-4-h half-life and predicted production cross-section approaching 1 b [20], the potential exists for regional distribution following mass production at a single cyclotron unit. The aim of this study was to investigate the possibility of 43 Sc production at an accelerator, allowing its use for preclinical and clinical PET imaging.
Natural CaCO 3 of chemical purity >99.999 purchased from Sigma Aldrich and enriched [ 40 Ca]CaCO 3 (99.99 %) purchased from Isoflex (USA) were used as target materials. The isotopic composition of enriched 40 Ca was 99.99 % of 40 Ca and 0.01% of 44 Ca, while the amounts of other calcium isotopes were below 0.01 %.

Irradiation of nat CaCO 3 and [ 40 Ca]CaCO 3 targets
Irradiations of natural targets were performed using the Scanditronix MC 40 cyclotron at the European Commission's Joint Research Centre (Ispra, Italy). Irradiations of enriched [ 40 Ca]CaCO 3 targets were performed using the Warsaw Heavy Ion Cyclotron operating at the Heavy Ion Laboratory of the University of Warsaw. The Ispra cyclotron is capable of accelerating positive ions such as protons, deuterons, and alphas to variable energies. The Warsaw machine accelerates heavy ions from +He up to Ar with energies from 2 up to about 8 AMeV. For irradiation at the Ispra cyclotron, the target material was wrapped in an aluminum foil of a 25-μm thickness. The samples were irradiated in aluminum capsules with an inner diameter of 10 mm. Each target capsule was inserted in a holder that allowed direct water cooling from both the rear and the front sides. In the Warsaw cyclotron, targets in the form of pellets bundled in thin aluminum foils produced from CaCO 3 powder using a hydraulic press were irradiated with an internal α-particle beam. Al energy degraders were used when alpha particle energies lower than maximal were necessary. In order to optimize the yield of 43 Sc production by the 40 Ca(α,p) 43 Sc and nat Ca(α,n) 43-Ti→ 43 Sc nuclear reactions,~100-mg nat CaCO 3 samples (target thickness~375 μm) were irradiated for 28-34 min by an alpha beam of 13-25 MeV on the target with an alpha current of 0.5 pμA at the Scanditronix MC 40 cyclotron.
Enriched [ 40 Ca]CaCO 3 targets of~100 mg were irradiated for 30 min by an alpha beam of 20 MeV with an alpha current of 0.25 pμA (He + ) at the Warsaw Heavy Ion Cyclotron.

Measurement of radioactivity
The absolute radioactivity of 43 Sc and other obtained radionuclides was measured by γspectrometry using two high-purity germanium (HPGe) detectors. The detectors were energy and efficiency calibrated in different geometries using certified standard radioactive sources (ENEA Italy, DAMRI and CERCA France). The gamma-ray spectrum analysis software package Genie 2000 (CANBERA, USA) was used to collect the data. The γ-ray peak at 372.8 keV was chosen for 43 Sc detection, and the peaks at 1157.00, 271.24, and 159.38 keV were chosen to detect 44 Sc, 44m Sc, and 47 Sc, respectively. Three peaks at 983.52, 1037.52, 1312.10 keV were used to quantify yields of 48 Sc [21]. The uncertainty of all the determined activities was below 1 %.

Separation of 43 Sc from the target
In order to find the optimal method for the separation of 43 Sc from the irradiated calcium targets, three procedures were tested: In the first method, described by Valdovinos et al. [22], the irradiated nat CaCO 3 target was dissolved in 1 ml of 9 M HCl solution. The dissolved target solution was passed through a column containing 50 mg of UTEVA resin, and after adsorption of 43 Sc, the column was washed with 5 ml of 9 M HCl. The scandium radionuclides were eluted with a 400-μl portion of H 2 O.
The second method, reported in the paper by Mueller et al. [10], consists of dissolving the CaCO 3 targets in 3 M HCl and adsorption of scandium radionuclides in a column filled with 70 mg of DGA. The adsorbed 43 Sc was eluted from the DGA resin with HCl (0.1 M, 2-3 ml). Afterwards, the acidic 43 Sc solution was loaded on a second column filled with 100 mg of cation exchange resin DOWEX 50 (hydrogen form, 200-400 mesh). Finally, 43 Sc was eluted using 1 M ammonium acetate adjusted to pH = 4 using HCl solution.
The third method, developed by our group [9], The stability of the labeled DOTATATE in human serum was assessed by adding 20 μl of the radioconjugate solution to 500 μl of the human serum. The mixture was incubated at 37°C, and the stability was measured by taking aliquots of the human serum solutions at different times and measuring the liberated scandium radionuclide by ITLC analysis.

Results and discussion
Optimization of 43 Sc cyclotron production The first step towards developing a simple, fast, and inexpensive method of 43 Sc-DOTATATE production is the optimization of the cyclotron production parameters. For this purpose, we measured the radioactivity yield as a function of alpha particle energy on target. The results are presented in Table 2. The analysis of the results obtained shows that the optimum on-target α particle energies are in the range of 24-26.5 MeV, which is a little higher than that predicted by Levkovskij [23].
The 43 Sc activity obtained after irradiation of a~100 mg nat CaCO 3 target for 34 min with a 25-MeV alpha particle beam of a 0.5-μA beam current was about 29 MBq. The produced activity can be increased by extending the irradiation time and using a higher Table 2 Activity of 43 Sc as a function of alpha energy on target. The optimum proton beam energy is highlighted in gray beam current, as in the case of 44 Sc production by proton irradiation of a [ 44 Ca]CaCO 3 target [24]. Extrapolating to an irradiation time of 4 h at 20 μA, the end of bombardment (EOB) yields are expected to approach 5.7 GBq, enough for the preparation of more than 25 patient doses. Of course, the proposed 40-fold scale-up of the current will bring challenging problems with heat dissipation from the CaCO 3 target, which can be solved by the use of a metallic Ca target previously tested by Severin et al. [11] for high-current proton irradiation.
Production of 43 Sc is accompanied by small co-production of other scandium radionuclides such as 44 Sc, 44m Sc, 46 Sc, and 47 Sc ( Table 3). The scandium radioisotopes 44g Sc and 44m Sc were synthesized from 42 Ca present in the natural target (0.65 %) via the (α,pn) reaction. 46 Sc and 47 Sc were produced from the 2 % of 44 Ca and the 0.13 % of 43 Ca components of the natural calcium in (α,pn) and (α,p) reactions, respectively. Table 3

Separation of 43 Sc from the target
Three methods previously developed for 44 Sc production were tested for the separation of 43 Sc from the natural Ca target. One method was based on the application of chelating resin and was developed in our group [9], and in the other two methods, the extraction resins developed by Valdovinos et al. [13] and Mueller et al. [10] were used. These methods were compared with respect to 43 Sc recovery, the volume and composition of the 43 Sc fraction (Table 4), and the possibility of separation from metallic impurities which could negatively affect the effectiveness of 43 Sc bioconjugate labeling.
All separation procedures studied are fast and simple. In the case of Chelex 100 and UTEVA resins, the target dissolution and separation of 43 Sc were performed in 30 min and the two-step separation process DGA + Dowex 50 in 45 min. All methods render possible the effective separation of 43 Sc from calcium. The efficiency of the separation methods is consistent with the previously reported procedures for separation of 44 Sc from enriched and natural targets [9,10,13]. Ca(α,p) 5 Irradiation of a 100-mg target by a 0.5-μA alpha beam current of 25 MeV. The radioactivity of the produced 43 Sc is equal to 28.9 MBq a Energies of the γ-photon used for activity measurement of the radionuclide High chemical purity of the final 43 Sc fraction is important, since the presence of other metals may interact with the DOTA chelator. The most dangerous is Fe 3+ for which the log of the stability constant with the DOTA ligand is 29.4 [25] which is greater than that for Sc 3+ (log K = 27 [15]). Influence of other possible impurities like Zn 2+ and Co 2+ is negligible due to the much lower stability constants of their DOTA complexes (log K Zn = 19.3, log K Co = 19.3 [25]. Fe concentration, measured with the ICP-MS technique in the dissolved calcium target in HCl solutions, varied between 58 and 87 ppm. After the separation processes, the total level of Fe in the 43 Sc samples decreased to 10.50 ppm for separation with Chelex resin, 0.56 ppm for DGA + DOWEX 50 and <0.001 ppm for UTEVA. The amount of Ca 2+ in the 43 Sc fractions was less than 1 ppm.
Two additional important factors in the labeling processes are the volume of the 43 Sc fraction and the composition of the eluate. In the three methods tested here, the volumes of eluates containing more than 80 % of 43 Sc which was used for reprocessing varied between 0.4 and 0.65 ml. The best composition of the eluate was obtained using the tandem of DGA and Dowex 50 resins where the eluate containing ammonium acetate buffer can be used directly to label DOTA or DTPA bioconjugates. The acidic eluates from Chelex 100 and UTEVA resins need neutralization. From the experiments performed, it can be concluded that, as with 44 Sc [13], due to the simplicity of the operations, the best methods for isolation of 43 Sc from the target material are procedures in which Chelex 100 or UTEVA resins are used. In respect to the chemical purity of the obtained 43 Sc solutions, the best separation is obtained using UTEVA resin. Therefore, for further experiments related to the labeling of DOTATATE, we chose this process.

Radiolabeling and stability studies of DOTATATE conjugate
The DOTATATE was used as a model system for radiolabeling with the 43 Sc radionuclide. High efficiency of labeling the DOTATATE with 43 Sc was achieved as shown by the labeling yield exceeding 98 % for an amount of bioconjugate equal to or higher than 15 nmol ( Table 5). The high yield showed that highly pure 43 Sc was obtained after the separation process with UTEVA resin and is suitable for labeling biomolecules. When the reaction yield is not high enough, the labeled peptide can be easily purified using the Sep-Pak® C-18 column.  The labeled DOTATATE radioconjugate exhibited high stability in human serum at 37°C. After 14 h of incubation in the serum, more than 98 % of 43 Sc remained in the radioconjugate.
In the present study, we found that synthesis of 43 Sc-DOTATATE using cyclotron produced 43 Sc could be adequate for nuclear medicine applications. Therefore, we believe that our method could be suitable for labeling different bioconjugates, regardless of whether they are other somatostatin analogs or a useful diagnostic peptide such as bombesin, substance P, or an oxytocin analog.
The procedure for labeling with 43 Sc is as easy as that in the case of 68 Ga and 44 Sc which makes it possible to use commercially available kits. The 4 h half-life and obtainable GBq activities of 43 Sc make possible the production and transport of the labeled bioconjugates to satellite PET centers, in analogy to 18 F-FDG.

Conclusions
The production of 43 Sc in (α,n) and (α,p) nuclear reactions on a natural CaCO 3 target was successfully performed, and extrapolation of the results obtained creates the opportunity to produce activity levels of 43 Sc sufficient for medical applications. The 43 Sc radionuclide has several advantages in comparison to the 44 Sc recently proposed for PET imaging. Firstly, in contrast to 44 Sc, it does not emit high-energy gamma rays that should be taken into consideration with regard to the radiation dose delivered to the patients and clinical staff. Emission of high-energy gamma rays also generates radiolytic decomposition of biomolecules, which is thought to be mediated by the formation of free radicals. This becomes an important issue when high quantities of radioactivity are used for labeling, as is necessary for clinical applications [26]. Also, co-production of the longer lived 44m Sc (T 1/2 = 58.6 h) increases the radiation dose. Furthermore, in contrast to 44 Sc, the production of 43 Sc does not require an expensive highly enriched 44 CaCO 3 target, the price of which currently exceeds 14 USD/mg.
The proposed separation process of 43 Sc from the calcium target is simple, reliable, efficient, and fast. Therefore, it is possible to use the same commercially available modular entity as that commonly used for preparation of 68 Ga-radiopharmaceuticals. Unfortunately, availability of cyclotrons with high-current alpha beams is limited. Despite this, regional distribution following massive production at a single alpha facility is possible. We believe that the 43 Sc obtained could be used instead of 68 Ga in PET imaging and in planning peptide receptor radionuclide therapy with 177 Lu-and 90 Y-DOTA radiobioconjugates.

Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors. studies. FS prepared targets from natural calcium in JRC Ispra. AS designed and prepared targets from natural and enriched [ 40 Ca]CaCO 3 . AT and WZ performed the analysis of gamma spectra of irradiated enriched targets and performed the statistical analysis. AB presented the idea of this work and wrote the publication. All authors read and approved the final manuscript.