*Article* **Production of GMP-Compliant Clinical Amounts of Copper-61 Radiopharmaceuticals from Liquid Targets**

**Alexandra I. Fonseca <sup>1</sup> , Vítor H. Alves 1,2 , Sérgio J. C. do Carmo 1,3 , Magda Silva <sup>1</sup> , Ivanna Hrynchak <sup>1</sup> , Francisco Alves 3,4 , Amílcar Falcão 1,5 and Antero J. Abrunhosa 1,3,\***


4

**Abstract:** PET imaging has gained significant momentum in the last few years, especially in the area of oncology, with an increasing focus on metal radioisotopes owing to their versatile chemistry and favourable physical properties. Copper-61 (t1/2 = 3.33 h, 61% β + , Emax = 1.216 MeV) provides unique advantages versus the current clinical standard (i.e., gallium-68) even though, until now, no clinical amounts of <sup>61</sup>Cu-based radiopharmaceuticals, other than thiosemicarbazone-based molecules, have been produced. This study aimed to establish a routine production, using a standard medical cyclotron, for a series of widely used somatostatin analogues, currently labelled with gallium-68, that could benefit from the improved characteristics of copper-61. We describe two possible routes to produce the radiopharmaceutical precursor, either from natural zinc or enriched zinc-64 liquid targets and further synthesis of [61Cu]Cu-DOTA-NOC, [61Cu]Cu-DOTA-TOC and [61Cu]Cu-DOTA-TATE with a fully automated GMP-compliant process. The production from enriched targets leads to twice the amount of activity (3.28 ± 0.41 GBq vs. 1.84 ± 0.24 GBq at EOB) and higher radionuclidic purity (99.97% vs. 98.49% at EOB). Our results demonstrate, for the first time, that clinical doses of <sup>61</sup>Cu-based radiopharmaceuticals can easily be obtained in centres with a typical biomedical cyclotron optimised to produce <sup>18</sup>F-based radiopharmaceuticals.

**Keywords:** radiometals; copper-61; liquid targets; post-processing; [61Cu]Cu-DOTA-NOC; [61Cu]Cu-DOTA-TOC; [61Cu]Cu-DOTA-TATE

#### **1. Introduction**

The use of advanced imaging technologies, especially nuclear medicine (i.e., PET and SPECT), can enhance diagnosis, staging, treatment planning and evaluation of treatment response in cancer care. Over the last two decades, an emerging quantity of small biomolecules (e.g., peptides, antibodies, antibodies fragments or nanoparticles) have been labelled with beta- or alpha-emitting metal radionuclides (e.g., gallium-68, copper-64, luthetium-177, actinium-225 and astatine-211) for imaging and therapeutic applications [1–5]. The wide variety of physical decay properties and half-lives, the simple and fast one-step radiolabelling chemistry [6,7]—easily adaptable to any type of vector for any target delivery—and the easy translation of metal-based radiopharmaceuticals into a theranostic approach [8] have primarily contributed to their interest and, currently, are major contributors to its success.

<sup>68</sup>Ge/68Ga generators play a substantial role in this growing phenomenon by allowing worldwide access to gallium-68 (t1/2 = 68 min, 89% β + , Emax = 1.899 MeV)—even in small

**Citation:** Fonseca, A.I.; Alves, V.H.; do Carmo, S.J.C.; Silva, M.; Hrynchak, I.; Alves, F.; Falcão, A.; Abrunhosa, A.J. Production of GMP-Compliant Clinical Amounts of Copper-61 Radiopharmaceuticals from Liquid Targets. *Pharmaceuticals* **2022**, *15*, 723. https://doi.org/ 10.3390/ph15060723

Academic Editor: Giorgio Treglia

Received: 22 April 2022 Accepted: 4 June 2022 Published: 7 June 2022

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hospital radiopharmacies (not requiring an onsite cyclotron)—which simplifies the translation of <sup>68</sup>Ga-conjugated peptides from the bench to routine clinical use [9]. Notwithstanding this, because of the worldwide shortage of gallium-68 generators, they are gradually losing out to more cost-effective production methods (i.e., accelerator-produced radiometals by the irradiation of natural or enriched targets). These methods are able to produce higher amounts of activity, without waiting time between productions (unlike the typical 3–4 h interval between elutions of <sup>68</sup>Ge/68Ga generators), aiming at fulfilling the ever-increasing clinical needs of gallium-68 [10,11]. The recently approved monograph of gallium-68 chloride solution produced from zinc-68 irradiation (Eur. Ph. 3109) [12] is a clear sign of the need for accelerator-produced methods in radiochemistry centres worldwide, which have access to this technology. Furthermore, since <sup>68</sup>Ge/68Ga generators are no longer a discriminatory advantage for using gallium-68 over other metal radionuclides on a routine basis, new promising radionuclides with better physicochemical properties are arising (e.g., scandium-43, scandium-44, copper-61, copper-64 and zirconium-89) with significant advantages over gallium-68: (1) easier distribution to centres that do not have onsite cyclotrons, (2) lower maximum positron emission energies that meet the requirements for a new generation of tomographs with higher resolution and (3) nuclides having a close therapeutic match, which is determinant for personalised medicine as we enter the theranostic era [13,14].

Copper-61 (t1/2 = 3.33 h, 61% β + , Emax = 1.216 MeV) [15] is a positron-emitting radionuclide presenting decay characteristics comparable to gallium-68 but with the advantage of presenting lower maximum positron energy (Emax = 1.216 MeV vs. Emax = 1.899 MeV) and a substantially more practical half-life (3.33 h vs. 68 min). In the past few years, several groups have attempted to find the best production and purification methods for cyclotron-produced copper-61. Liquid and solid target irradiations have both been explored. In 2012, the production of copper-61 from natural cobalt solid targets, following the natCo(α,xn)61Cu nuclear reaction, resulted in high-purity copper-61 [16]. Later, Asad et al. [17,18] and Thieme et al. [19] detailed the production of copper-61 from natural zinc and zinc-64, also in solid targets. In 2017, our group described the production of copper-61 from the irradiation of liquid targets at low proton energies [20] and later its automated purification [21]. More recently, the possibility of producing copper-61 from solid natural nickel targets, following natNi(d,x)61Cu nuclear reaction, was also outlined, along with its fully automated purification process [22]. Despite increasing efforts being made in the development of the above-mentioned methods towards being capable of producing high-purity copper-61, to date, only a handful of molecules have been labelled with this radioisotope—mostly thiosemicarbazone-based molecules (i.e., ATSM, PTSM, APTS and TATS) [23–27], which are well known for presenting high affinity for copper.

Considering the above, the aim of the current work is to demonstrate that the production of chelator-based copper-61 labelled radiopharmaceuticals can easily be performed and can be made Good Manufacturing Practise (GMP)-compliant for routine clinical use. For that purpose, we present the production, synthesis and quality control of [61Cu]Cu-DOTA-TATE, [61Cu]Cu-DOTA-TOC and [61Cu]Cu-DOTA-NOC, the copper-61 equivalents of the somatostatin (SST) analogues extensively used with gallium-68, in current clinical practice [28].

Initial work on targeting and staging neuroendocrine tumours (NETs) through the labelling of SST analogues begun with [123I]I-Tyr3-octreotide [29]. The first Food and Drug Administration (FDA)-approved radiopharmaceutical was Octreoscan®, in 1994 ([111In]In-DTPA-Octreotide) [30,31]. Today, <sup>68</sup>Ga-labelled radiopharmaceuticals such as [ <sup>68</sup>Ga]Ga-DOTA-NOC, [68Ga]Ga-DOTA-TOC and [68Ga]Ga-DOTA-TATE are in current clinical practice to diagnose, with PET, solid tumours which over-express SST receptors (SSTRs) [32,33]. The ready-to-label "cold kit" with DOTA-TATE was approved by the FDA in 2016 (NetspotTM), and the equivalent with DOTA-TOC (Somakit-TOC) was approved by the EMA in 2017 [34].

Most previous works regarding the labelling of SST analogues with copper focused on copper-64 (t1/2 = 12.7 h, 18% β + , Emax = 0.653 MeV). A first-in-human study with [ <sup>64</sup>Cu]Cu-DOTA-TATE revealed several advantages, i.e., higher lesion detection, better image quality and lower radiation doses, when compared with [111In]In-DTPA-octreotide when used for SPECT imaging [35]. More recent clinical studies, particularly head-to-head comparisons of [64Cu]Cu-DOTA-TATE with both [111In]In-DOTA-TATE [36] and [68Ga]Ga-DOTA-TOC [37] revealed an overall better performance of the <sup>64</sup>Cu-conjugated in terms of sensitivity, resolution and rate of lesion detection. Additionally, other first-in-human studies with [64Cu]Cu-DOTA-TOC also showed high lesion detection rate, safety of use and high effectiveness for predicting treatment planning [38]. Even more recently, Loft et.al. confirmed the extended imaging window of [64Cu]Cu-DOTA-TATE from 1 h to 3 h [39], without a decrease in performance. There is still work in progress aiming at clarifying the dosimetric parameters and predicting both the overall survival (OS) and progression-free survival (PFS) ability of these radiopharmaceuticals labelled with copper-64 [40–42]. These results lead us to conclude that the substitution of gallium for copper on these SST analogues has most likely a positive impact on their performance as PET radiopharmaceuticals. Moreover, the favourable physical properties of copper-61 when compared with copper-64 (shorter half-life, higher β + -emission) makes it an ideal nuclide for this purpose.

In this context, the simple, cost-effective production and separation methods herein described could pave the way for the widespread clinical use of copper-61 radiopharmaceuticals, providing an even better alternative to the scarce and expensive-to-obtain gallium-68.

#### **2. Results and Discussion**
