Targeted Alpha Therapy: All We Need to Know about 225Ac’s Physical Characteristics and Production as a Potential Theranostic Radionuclide

The high energy of α emitters, and the strong linear energy transfer that goes along with it, lead to very efficient cell killing through DNA damage. Moreover, the degree of oxygenation and the cell cycle state have no impact on these effects. Therefore, α radioisotopes can offer a treatment choice to individuals who are not responding to β− or gamma-radiation therapy or chemotherapy drugs. Only a few α-particle emitters are suitable for targeted alpha therapy (TAT) and clinical applications. The majority of available clinical research involves 225Ac and its daughter nuclide 213Bi. Additionally, the 225Ac disintegration cascade generates γ decays that can be used in single-photon emission computed tomography (SPECT) imaging, expanding the potential theranostic applications in nuclear medicine. Despite the growing interest in applying 225Ac, the restricted global accessibility of this radioisotope makes it difficult to conduct extensive clinical trials for many radiopharmaceutical candidates. To boost the availability of 225Ac, along with its clinical and potential theranostic applications, this review attempts to highlight the fundamental physical properties of this α-particle-emitting isotope, as well as its existing and possible production methods.


Introduction
At the end of the 1800s, Pierre and Marie Curie, along with Alexander Graham Bell in the early 1900s, conducted research linked to cancer-targeted α therapy (TAT), which represented one of the earliest non-surgical cancer treatments [1].Furthermore, α-particle emitters have significant curative effects, particularly in patients with limited therapeutic options and metastatic spread [2][3][4].They can target very small clusters of metastatic cancer cells.
There are many benefits of using these radioisotopes in cancer therapy over common methods.α particles can selectively destroy tumour cells while preserving adjacent normal tissues due to their narrow extent in human tissue, corresponding to less than 0.1 mm [5].Meanwhile, highly efficient cell destruction through DNA double-strand and DNA cluster damage is caused by the high energy of α emitters, in addition to the strong linear energy transfer (LET) (80 keV/µm) that goes along with it.These effects are mainly unaffected by the state of the cell cycle and oxygenation [6][7][8].Thus, α radioisotopes can provide a [5].Meanwhile, highly efficient cell destruction through DNA double-strand and DNA cluster damage is caused by the high energy of α emitters, in addition to the strong linear energy transfer (LET) (80 keV/µm) that goes along with it.These effects are mainly unaffected by the state of the cell cycle and oxygenation [6][7][8].Thus, α radioisotopes can provide a therapeutic option for patients who are resistant to therapy with β− or gamma radiation or chemotherapeutic medications [9][10][11].According to research estimations, tens of thousands of β− particles are needed to reach a single-cell killing rate of 99.99%, whereas only a few α decays are needed to accomplish a similar killing potential [4,12].
The high-LET radiation's biological efficacy is explained by its tendency to cause complex multiple clusters and double-strand or single-strand breaks in a target cells' DNA, rendering cellular repair mechanisms ineffective [4,13].Additionally, reactive oxygen species (ROS), which are produced when emitted particles interact with water, can react with biomolecules such as proteins, phospholipids, RNA, and DNA, leading to permanent cell deterioration [14].Moreover, during this type of therapy, the primary tumour and any additional cancerous lesions in the body that the radiation did not directly target may decrease as a result of "the abscopal effect" [14].It is thought that the immune system is a key player in this process, even though the precise biological mechanisms underlying the phenomenon are as yet unknown [4, 15,16] (Figure 1).Considering the clinical application of TAT, only a limited number of α-particle emitters are appropriate [17].The use of 225 Ac and its short-lived daughter nuclide 213 Bi represents the vast majority of available experience in clinical research [5].Furthermore, applying γ decays, which are produced during the radioactive 225 Ac cascade [5] in SPECT imaging, raises the possibility of theranostic nuclear medicine applications.
Although interest in using 225 Ac as an α-emitting radiolabel has been steadily increasing [18], substantial clinical investigations of many radiopharmaceutical candidates cannot be supported due to 225 Ac's limited worldwide accessibility [19].Notwithstanding the Considering the clinical application of TAT, only a limited number of α-particle emitters are appropriate [17].The use of 225 Ac and its short-lived daughter nuclide 213 Bi represents the vast majority of available experience in clinical research [5].Furthermore, applying γ decays, which are produced during the radioactive 225 Ac cascade [5] in SPECT imaging, raises the possibility of theranostic nuclear medicine applications.
Although interest in using 225 Ac as an α-emitting radiolabel has been steadily increasing [18], substantial clinical investigations of many radiopharmaceutical candidates cannot be supported due to 225 Ac's limited worldwide accessibility [19].Notwithstanding the significant financial investments made by numerous laboratories to establish production pathways, the widespread use of 225 Ac-labeled radiopharmaceuticals in human patients is still not achievable [19].This ongoing shortage in 225 Ac supply can be explained by the practical production techniques that need difficult logistical tasks, such as using controlled nuclear materials or highly irradiating radioactive accelerator targets [19].In order to increase the availability of 225 Ac and thus boosting the clinical use of α-particle-emitter therapeutics and potential theranostic applications, this review aims to outline the fundamental physical characteristics of 225 Ac in addition to its existing and potential production routes.

225 Ac: Physical Characteristics
Actinium is a radioactive component with atomic number 89 [20].Only two of its 32 isotopes, 228 Ac and 227 Ac, are naturally produced as a result of the disintegration of 232 Th and 235 U, respectively [20,21].With its long half-life of 21.7 years and predominant β− emissions decay, 227 Ac represents the most common actinium isotope.However, 228 Ac, which is also a β− emitter, is highly uncommon [20,21]. 225Ac is the initial element in the actinide family, and its radioactive parents are parts of the now-extinct "neptunium series" [19,21].This α-emitter isotope has a long half-life of 9.9 days [5,22].
significant financial investments made by numerous laboratories to establish production pathways, the widespread use of 225 Ac-labeled radiopharmaceuticals in human patients is still not achievable [19].This ongoing shortage in 225 Ac supply can be explained by the practical production techniques that need difficult logistical tasks, such as using controlled nuclear materials or highly irradiating radioactive accelerator targets [19].
In order to increase the availability of 225 Ac and thus boosting the clinical use of αparticle-emitter therapeutics and potential theranostic applications, this review aims to outline the fundamental physical characteristics of 225 Ac in addition to its existing and potential production routes.

225 Ac: Physical Characteristics
Actinium is a radioactive component with atomic number 89 [20].Only two of its 32 isotopes, 228 Ac and 227 Ac, are naturally produced as a result of the disintegration of 232 Th and 235 U, respectively [20,21].With its long half-life of 21.7 years and predominant β− emissions decay, 227 Ac represents the most common actinium isotope.However, 228 Ac, which is also a β− emitter, is highly uncommon [20,21]. 225Ac is the initial element in the actinide family, and its radioactive parents are parts of the now-extinct "neptunium series" [19,21].This α-emitter isotope has a long half-life of 9.9 days [5,22].

225 Ac and Its Potential Theranostic Use
225 Ac is considered a "nanogenerator", since one decay of this element produces a total of four α and three β particles, in addition to two γ emissions [24].Taking into account its α particle emissions, along with the fact that the non-tumour binding activity can be eliminated before most of its dose is deposited in organs, 225 Ac is considered an appealing choice for TAT [24,25].However, it is important to give attention to the notable

225 Ac and Its Potential Theranostic Use
225 Ac is considered a "nanogenerator", since one decay of this element produces a total of four α and three β particles, in addition to two γ emissions [24].Taking into account its α particle emissions, along with the fact that the non-tumour binding activity can be eliminated before most of its dose is deposited in organs, 225 Ac is considered an appealing choice for TAT [24,25].However, it is important to give attention to the notable 225 Ac cytotoxicity, including renal toxicity [26], due to its extended half-life and the various α particles produced throughout its decay chain [5].
A theranostic-based approach, characterised by the imaging-therapeutic duality, is the process of obtaining positron emission tomography (PET) and SPECT scans by exchanging the therapeutic α-emitting radionuclide with a positron or gamma diagnostic imaging radionuclide.Significant information on dosimetry and TAT reactions is obtained from these relevant nuclear medicine images.
Chemical characteristics, half-life, radioactive emission type and intensity, related dosimetry, ease and scalability of production, radionuclidic purity, economics, and radionuclide progeny considerations are the factors that determine "the ideal" imaging surrogates for targeted alpha therapy [27,28].
Therapeutic use of 225 Ac is often paired with imperfect PET imaging surrogates, such as 68 Ga, 89 Zr, or 111 In, despite significant differences in their half-lives or chelation chemistry [29].Studies are being conducted to address the limitations of imaging radionuclides by utilising lanthanum (La) as a potential alternative, especially 132 La (T 1/2 = 4.8 h, 42% β+) and 133 La (T 1/2 = 3.9 h, 7% β+) [30,31].However, the half-lives of these isotopes are much shorter than that of 225 Ac, limiting their applicability in PET imaging [29].In this regard, the production of 134 Ce (T 1/2 = 3.2 d) has recently been started by the U.S. Department of Energy (DOE) Isotope Program [32].The long 134 Ce T 1/2 and the similar chemical properties of 225 Ac and 134 Ce were considered potential benefits for monitoring in vivo pharmacokinetics.For PET imaging of the chelate and the antibody trastuzumab, 134 Ce has been demonstrated to bind with diethylenetriamine pentaacetate (DTPA) [32] and dodecane tetraacetic acid (DOTA) [33].On the other hand, greater molar ratios and higher temperatures are needed for isotope combinations with DOTA and DTPA [29].In contrast, N, N -bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6 (macropa) has shown great stability for nonradioactive cerium and better chelate characteristics for 225 Ac [34], indicating that it might be useful for the theranostic development of 134 Ce/ 225 Ac [35].
The potential use of γ disintegrations, obtained by the decay of the intermediate 221 Fr (218 keV, 11.6% emission probability) and 213 Bi (440 keV, 26.1% emission probability) [5], in SPECT in vivo imaging could lead the 225 Ac radioactive cascade to a possible theranostic prospective in nuclear medicine applications.Nonetheless, planar SPECT imaging would be challenging because of the effectiveness of 225 Ac, which results in modest administered doses (~50-200 kBq/kg [5]), along with low γ emissions [24,25].As a possible solution to this limitation, we can notice the suitable use of 213 Bi, which can be isolated from the 225 Ac decay cascades [24].Nevertheless, it is mandatory to consider the short half-life of 213 Bi (45.6 min), which poses difficulties for processing, radiolabelling, and radiopharmaceutical delivery [24].In addition, it is necessary to point out that these radiations make reaction monitoring complicated.Moreover, the secular equilibrium must be attained (for at least 6 h) before measuring a trustworthy radiochemical yield (RCY) [21].Actinium's chemistry lacks advancement because of its restricted availability; all Ac isotopes need specific management and facilities [20].

Radiochemistry
During the production of radionuclides, it is mandatory to take into consideration a set of important aspects, such as safety, the co-generation of a few long-lived radionuclidic impurities, and adjustability, to enable delivery through clinical sites [27].Once the target material has been irradiated, potent chemical purification methods are required to isolate the radioisotope [27,[36][37][38].Furthermore, the alpha particle may radiolytically damage the radiopharmaceutical itself, reducing in vivo targeting and producing more radioactive deposits in nontarget tissue.[27].
Since radiopharmaceuticals are considered typical pharmaceuticals, special manuals have been developed in the European Pharmacopoeia to deal with quality control issues [39].Additionally, optimised protocols for preparing 225 Ac agents in therapeutic doses have been established [40] (Table 1).¢ The labelling of the DOTA-conjugated peptide was carried out under good manufacturing practice within a shielded hot cell using a multifunctional automated radiosynthesis module (Trasis, AllinOne mini).¢ The radiolabelled products were characterised using thin-layer chromatography, high-pressure liquid chromatography, gamma counting, and high-energy resolution gamma spectroscopy.
225 Ac-DOTA-conjugated peptide >99%/>95% Dumond.et al., 2022 [42] ¢ PSMA-617 precursor was dissolved in 25 µL metal-free water (0.67 mg/mL) and combined with 500 µL 0.05M Tris buffer, pH 9. 225 Ac solution (~65 µCi in 15 µL) was added and the reaction was heated at 120 • C for 40-50 min.The resulting reaction was cooled and 0.6 mL gentisic acid solution (4 mg/mL in 0.2 M NH 4 OAc) was added.To formulate the dose for injection, sterile saline (8 mL) was added and the pH was adjusted by the addition of 100 µL 0.05 M Tris buffer (pH 9) to give a final pH of ~7.2.The final solution was filtered using a 0.22 µm GV sterile filter into a sterile dose vial.
¢ pH was determined using pH paper.
¢ RCP of 225 Ac-PSMA-617 was determined by ITLC.¢ 225 Ac was diluted into 0.1 M HCl.Stock solutions (10 mL) were proceeded in quartz-coated sterile vials.All purchased chemicals were prepared with Milli-Q water.Stock solutions prepared the day before labelling were 1 M HCl (from 37% HCl), 10 M NaOH, and 0.1 M TRIS-buffer pH 9. Two stock solutions were prepared on the day of labelling: First, 20% ascorbic acid was prepared; the ascorbic acid solution was transformed to ascorbate by the addition of 10 M NaOH to a pH 5.8.Secondly, PSMA-I&T (250 µg) was dissolved in 0.1 M TRIS buffer (pH 9) to a concentration of 600 µg/mL.Directly after labelling, 4 mg/mL diethylenetriaminepentaacetic acid (DTPA) was added to the labelling mixture.A solution for injection was prepared by the addition of ascorbate (50% v/v) and ethanol (6% v/v, 96%) into saline. 225Ac-PSMA-I&T >95%/>90%

225 Ac Radiopharmaceuticals and Clinical Applications
The delivery of the radiopharmaceutical via the circulatory system enables the targeting of both the main tumour and its metastases.Whether a radiopharmaceutical is intended for therapeutic or diagnostic purposes depends on the decay properties of the linked radioisotope.For the purpose of curing, controlling, or palliating symptoms, TAT aims to provide an adequate amount of ionising radiation to intended malignities areas [27].This means that any TAT agent must have a thorough understanding of its stability, pharmacokinetics, and dosimetry.
Investigations on 225 Ac have shown potential in treating neuroendocrine tumours, acute myeloid leukaemia, and metastatic prostate cancer, and more radiopharmaceuticals are being developed for other cancer types [46][47][48][49][50][51][52] (Table 2). 225Ac-lintuzumab Jurcic, 2018 [68] 225 Ac-lintuzumab Jurcic et al., 2016 [69] 225 Ac-lintuzumab Jurcic et al., 2011 [70] 225 Ac-lintuzumab The use of 225 Ac in clinical practice is limited by its low availability.Breaking through this barrier would allow 225 Ac therapy to spread widely.Automated synthesis and consistent patient doses are essential, regardless of the production route chosen for this α-isotope acquisition. 225Ac can be adapted for the commonly accessible DOTA-conjugated peptides for therapy [41], which are already capable of labelling 177 Lu or 90 Y. Marc Pretze et al. [71] studied the effectiveness and consistency of the radiosynthesis process for creating 225 Aclabelled DOTA-conjugated peptides.Additionally, the research aimed to establish whether this process could be adapted for clinical production purposes through an automated synthesis platform (cassette-based module-Modular-Lab EAZY, Eckert & Ziegler) [72].After comparing two purification methods, the researchers obtained 225 Ac-labelled peptides in an RCY of 80-90% for tumour therapy in patients [71].Thus, the whole process was meticulously validated in accordance with the regulations of the German Pharmaceuticals Act §13.2b, knowing that the estimated costs for the automated synthesis of 1 MBq 225 Ac is around EUR 300-390, taking into account that the peptides would cost EUR 600-1000, the cassettes would cost EUR 180-200, and the ML EAZY would cost EUR ~30,000 [71].

The Production Routes of 225 Ac
As already mentioned, 225 Ac is part of the 237 Np disintegration family that has vanished in nature.This radioactive element could be artificially reproduced [21].In addition to direct production paths, 225 Ac is conveniently reachable at numerous points along the decay chain, in particular via 233 U (T 1/2 =159200 y, 100% α), 229 Th (T 1/2 = 7340 y, 100% α), and 225 Ra (T 1/2 = 14.9 d, 100% β−) [19]. 225Ac possesses many fewer nucleons than other actinide nuclei that are more stable to be employed as production targets, such as 232 Th and 226 Ra [19].Thus, production methods should, with rare exceptions, rely on radioactive decay or greater energy bombardments.
The available production routes of 225 Ac and its parents are listed below (Figure 3) [14]: Pharmaceuticals 2023, 16, x FOR PEER REVIEW 7 of 20 Kratochwil et al., 2015 [66] 225 Ac-DOTATOC Acute myeloid leukaemia Rosenblat et al., 2022 [67] 225 Ac-lintuzumab Jurcic, 2018 [68] 225 Ac-lintuzumab Jurcic et al., 2016 [69] 225 Ac-lintuzumab Jurcic et al., 2011 [70] 225 Ac-lintuzumab The use of 225 Ac in clinical practice is limited by its low availability.Breaking through this barrier would allow 225 Ac therapy to spread widely.Automated synthesis and consistent patient doses are essential, regardless of the production route chosen for this αisotope acquisition. 225Ac can be adapted for the commonly accessible DOTA-conjugated peptides for therapy [41], which are already capable of labelling 177 Lu or 90 Y. Marc Pretze et al. [71] studied the effectiveness and consistency of the radiosynthesis process for creating 225 Ac-labelled DOTA-conjugated peptides.Additionally, the research aimed to establish whether this process could be adapted for clinical production purposes through an automated synthesis platform (cassette-based module-Modular-Lab EAZY, Eckert & Ziegler) [72].After comparing two purification methods, the researchers obtained 225 Aclabelled peptides in an RCY of 80-90% for tumour therapy in patients [71].Thus, the whole process was meticulously validated in accordance with the regulations of the German Pharmaceuticals Act §13.2b, knowing that the estimated costs for the automated synthesis of 1 MBq 225 Ac is around EUR 300-390, taking into account that the peptides would cost EUR 600-1000, the cassettes would cost EUR 180-200, and the ML EAZY would cost EUR ~30,000 [71].

The Production Routes of 225 Ac
As already mentioned, 225 Ac is part of the 237 Np disintegration family that has vanished in nature.This radioactive element could be artificially reproduced [21].In addition to direct production paths, 225 Ac is conveniently reachable at numerous points along the decay chain, in particular via 233 U (T1/2 =159200 y, 100% α), 229 Th (T1/2 = 7340 y, 100% α), and 225 Ra (T1/2 = 14.9 d, 100% β−) [19]. 225Ac possesses many fewer nucleons than other actinide nuclei that are more stable to be employed as production targets, such as 232 Th and 226 Ra [19].Thus, production methods should, with rare exceptions, rely on radioactive decay or greater energy bombardments.
The available production routes of 225 Ac and its parents are listed below (Figure 3) [14]:

Radiochemical Extraction from 229 Th
For more than two decades, the main source of 225 Ac has been the accumulation of 229 Th (T1/2 = 7340 y) from the disintegration of 233 U (T1/2 = 160,000 y) reserves.At this time,

Radiochemical Extraction from 229 Th
For more than two decades, the main source of 225 Ac has been the accumulation of 229 Th (T 1/2 = 7340 y) from the disintegration of 233 U (T 1/2 = 160,000 y) reserves.At this time, all clinical trials and a large number of pre-clinical studies involving 225 Ac and 213 Bi have so far used this type of generation route [5].
A large portion of 233 U was created between 1954 and 1970 by neutron irradiating 232 Th when it was being researched for use in nuclear weapons and reactors that were never completely implemented [73,74].A significant stockpile of 233 U was kept after the thorium fuel cycle was abandoned in favour of fast reactors powered by plutonium at the end of the 1970s [21].From supplies kept at the Oak Ridge National Laboratory (ORNL, Oak Ridge, TN, USA), 229 Th produced via 233 U disintegration was recovered between 1995 and 2005 [74].Currently, there are three principal sources for this 229 Th: at ORNL (5.55 GBq (150 mCi), or 704 mg) [74,75], at the Directorate for Nuclear Safety and Security of the Joint Research Centre (JRC) of the European Commission (JRC, Karlsruhe, Germany) (1.7 GBq (46 mCi), or 215 mg), formerly known as the Institute for Transuranium Elements (ITU) [74,76], and at the Leipunskii Institute for Physics and Power Engineering (IPPE, Obninsk, Russia) (5.55 GBq (150 mCi), or 704 mg) [74,77].Canadian Nuclear Laboratories (CNL) has more recently announced the isolation of an important 229 Th source [5].Very pure sources of 229 Th were also discovered, prepared, and used for pre-clinical research at the Belgian Nuclear Research Centre (SCK CEN) in Mol, Belgium [14].
By producing approximately 33 GBq (893,23 mCi) (ORNL) [78] and 13.1 GBq (350 mCi) (JRC) [74,76] of 225 Ac annually, the ORNL and JRC represent, up to now, the principal worldwide providers of 225 Ac and its parent 225 Ra (T 1/2 = 14.9 d).Anion exchange and extraction chromatography are combined to produce 225 Ac from 229 Th at JRC Karlsruhe, whereas anion [52] and cation exchange are used in the process at ORNL [78].Even though the IPPE source has the same amount of 229 Th as the ORNL source, the recorded values show that the IPPE source intermittently produces 225 Ac [74,77,79].According to Samsonov MD et al., IPPE 225 Ac production could reach 22 GBq per year [80].
Additionally, it has been noted that starting from 2019, a very considerable rise in the availability of 229 Th will be produced through the extraction of 229 Th from historical wastes kept by the US DOE [4, 52,78].According to estimations, there could be up to 45 g of total 229 Th available, which could result in a 40-fold boost in the supply of 225 Ac above current levels [78].
The 225 Ac developed at JRC Karlsruhe and ORNL is considered safe for human use and has been significantly utilised for patient treatment [5], although there have been no reports to date about the direct clinical application of 225 Ac made at IPPE [5].
Approximately 68 GBq of 225 Ac from 229 Th are generated per year on a global scale [5].Knowing that the 225 Ac-labelled ligands' given activities typically range from 4 to 50 MBq per therapeutic dosage [5], the amount of this isotope's supply is sufficient to treat several hundred patients annually and permits the performance of pre-clinical research.Although a major benefit of this production method is that the resulting 225 Ac is free of other actinium isotopes, the globally generated 229 Th is not enough to satisfy the extensive use and implementation in healthcare applications across the world [74,81].Therefore, the development of 225 Ac radiopharmaceuticals is hindered by the limited supply and high cost that make 225 Ac inaccessible to many researchers [74].In addition, the production of 233 U (T 1/2 = 160,000 y) is not viewed as a realistic solution for addressing expected short-term 225 Ac demand, because decades of steady growth are necessary to boost 229 Th (T 1/2 = 7340 y) supply [19,82,83].As a result, numerous other techniques for generating 225 Ac on a wide scale have been researched.
Exposing radium targets to high fluxes of thermal neutrons is considered an effective procedure to induce 229 Th production [19].This approach has been carefully investigated by ORNL researchers with access to the High Flux Isotope Reactor's (HFIR) > 1015 n cm −2 s thermal fluxes, noticing the production of 229   229 Th is the predominant generation pathway from 226 Ra targets and is driven by a combination of neutron capture probability and decay kinetics [19].The short half-lives of 227 Ra (T 1/2 = 42.2 min, 100% β−) and 228 Ac (T 1/2 = 6.15 h, 100% β−) represent the important restrictions for these possible 229 Th generation routes [19].The magnitude of the 226 Ra(n, γ) 229 Th cross section has the biggest impact on the amount of 229 Th that can be produced [19].Unfortunately, this predominant pathway passes through 228 Th.This Th isotope is a dosimetrically undesirable contaminant that can only be eliminated from 229 Th by mass isolation or burnup and lowers the yield of 229 Th that may be produced [19].The handling of the radium target and the generation of 228 Th (T 1/2 = 1.9 y) intermediate represent important challenges of this process [14,52,85].In addition, there is still a sizable gap between the theoretically predicted yields and the measured ones.In HFIR, ideal 5-cycle activations are expected to provide approximately 0.8 GBq (20 mCi g −1 ) of 229 Th for every gram of 226 Ra [19].
Whereas pure 227 Ac or 228 Ra targets are projected to generate somewhat more 229 Th, the current supply of these radionuclides is substantially less than that of 226 Ra [19].Although improving the cost effectiveness of centralised recovery and distribution from 229 Th stocks, the dedication of even relatively small quantities of 226 Ra to such irradiations will significantly help to ease the current 225 Ac shortages.Yet, the full scope of the predicted need cannot be promptly met using this production technique.Thus, other production methods will undoubtedly be pursued simultaneously.

The Spallation of 232 Th
This method is based on the spallation of 232 Th to produce 225 Ac.As a target material, 232 Th (4.1103 Bq/g, 110 nCi/g) is widely accessible, not excessively radioactive, and presents fewer radiation risks [74].Many countries are known to have stocks of tens of kilograms of thorium metal and hundreds of tonnes of thorium oxide or thorium nitrate, which are created every year as a byproduct of rare-earth mining and used to make more thorium metal in large amounts [74,86].
Waste recycling of the irradiated 232 Th target material might not be necessary because of its important accessibility [74].
The irradiation of 232 Th with highly energetic protons (0.6-2 GeV) accessible at large accelerators has been shown to produce considerable amounts of 225 Ac [5,87,88].Production yields of several GBq have been recorded for 10-day irradiations utilising highly energetic proton beams [5,89,90].From the irradiations of 5 g cm −2 targets throughout their roughly 8-month annual running durations, Los Alamos National Laboratory (LANL) can create between 40 and 80 GBq (1-2 Ci) every 10 days.This method is considered to be the most developed production procedure [78] and was validated at the Institute for Nuclear Research (INR), Russian Academy of Sciences (RAS) in Troitsk, Russia, and LANL in the US [78].Furthermore, the routine use of this technique was introduced by the US DOE Tri-Lab (ORNL, Brookhaven National Laboratory (BNL), LANL) [78].Once the targets are being handled and the completed product is delivered from ORNL, irradiations can be carried out at BNL (200 MeV at 165 mA) and LANL (100 MeV at 275 mA) [78,91].
The co-production of long-lived 227 Ac (T 1/2 = 21.8 y) is the process' primary constraint [27,78].A large amount of these radionuclidic impurities is simultaneously produced by the spallation of 232 Th and needs to be eliminated using the proper multi-step chemical separation methods [5,[92][93][94].The effects of the isotopic impurity on the therapeutic application of the produced 225 Ac need to be taken into account because 225 Ac and 227 Ac cannot be totally chemically separated (0.1-0.2% of the relative activity of 225 Ac) [21,88].Even with this limitation, the 225 Ac produced from high-energy accelerators may still be perfectly suitable for the manufacturing of 225 Ac/ 213 Bi generators, as all actinium daughters will be kept on the generator [14].According to preliminary research, the 227 Ac impurity will not significantly affect patient dosimetry [78].Recently, new purifying techniques that enable a reduction in the 227 Ac level and the recovery of 225 Ac with better purity, such as isotope separation (isotope separation on-line (ISOL) at Canada's particle accelerator centre (TRIUMF)) or a manufacturing method using 225 Ra produced after the proton irradiation of 232 Th, have been developed [4,21,[95][96][97].Nonetheless, there are still challenges to be resolved regarding long-lived 227 Ac licensing and accessibility in medical applications.In addition, due to the 21.8-year half-life, waste management is still a serious issue and will necessitate measures with possibly high related costs.

The Irradiations of 226 Ra
The Proton Irradiation of 226 Ra Compared with the 232 Th spallation reaction, the generation of 225 Ac from 226 Ra targets by proton irradiation in a cyclotron has several benefits.This method is based on the reaction 226 Ra(p,2n) 225 Ac.In medium-sized cyclotrons, at proton energies below 20 MeV (around 16 MeV), this procedure can be carried out with excellent results and at a reasonable cost [5,78,98].About 5 GBq 225 Ac, which is comparable to 500 patient doses of 10 MBq 225 Ac, should be produced after a 24 h exposure of 50 mg 226 Ra to the highest excitation function at 15-16 MeV with a current of 100 mA protons [78].It is noteworthy that research, both fundamental and applied, is believed to have relevance to medical cyclotrons that produce radioisotopes at energies between 15 and 25 MeV [14,99].
Since no other long-lived actinium isotopes, such as 227 Ac, are created during the chemical purification of the irradiation targets, 225 Ac with high isotopic purity is obtained.By choosing the right proton energies, it is possible to reduce the co-production of the short-lived 226 Ac (T 1/2 = 29 h) and 224 Ac (T 1/2 = 2.9 h) impurities produced by the reactions 226 Ra(p,n) 226 Ac and 226 Ra(p,3n) 224 Ac [5,78].Furthermore, during the time needed for target cooling and reprocessing, their activity will continue to decrease to low levels.Handling targets that contain milligram amounts of radioactive 226 Ra (T 1/2 = 1600 y) and controlling its highly radiotoxic gaseous decay product 222 Rn (T 1/2 = 3.8 d) [5,14,98,100] pose significant challenges in the production, processing, and control procedures [5,78].In addition, due to the limited availability of the target material, it is necessary to consider its recycling process [20].Currently, facilities in North and South America, Europe, and Asia are researching how to utilise this production strategy.For instance, work on the investigation and development of 225 Ac generation using 226 Ra (stored as radioactive waste) has started at the National Institutes for Quantum Science and Technology (QST), Chiba, Japan [100].These amounts of 226 Ra have previously been used as a sealed source for brachytherapy.Even in this resource-constrained country, some 226 Ra was accessible as a target for proton irradiation thanks to the national waste management program [100].
The Deuterons' Irradiation of 226 Ra An improved method for producing 225 Ac, which involves irradiating 226 Ra with deuterons through the reaction 226 Ra(d,3n) 225 Ac, has been proposed [101].Although experimental measurements of the reaction's cross sections are still in development, simulations indicate that the process will have a greater production yield than the 226 Ra(p,2n) 225 Ac reaction and a maximum cross section of 864 mb at 18.5 MeV [78].It is important to consider the prolonged cooling period by the 226 Ac decay, since deuteron irradiation might result in an increased co-production of 226 Ac (T 1/2 = 29 h) [78].Moreover, there are only a few accelerators that can produce deuteron beams with enough energy.

Ra
The photonuclear reaction 226 Ra(γ,n) 225 Ra, followed by the beta decay of 225 Ra to 225 Ac is a different method for producing 225 Ac by irradiating 226 Ra.It is noticed that the photon energy cutoff for the reaction is 6.4 MeV.However, experimentally established cross-section data are not yet available [78].Modelling data predict modest reaction yields and high-intensity electron beams from modern accelerators are required for commercially viable production.At JRC Karlsruhe, the process's fundamentals have been experimentally verified [78].A zircaloy capsule containing 1 mg of 226 Ra embedded in 800 mg of a BaCl 2 matrix underwent 3.5 h of 52 MeV betatron irradiation to generate 0.24 mCi of 225 Ac [78].At the INR in Dubna, Russia [102], as well as the Illawarra Cancer Centre (ICC) in Wollongong, Australia [103], the procedure's viability has also been effectively validated.At a maximum photon energy of 24 MeV, a radiation yield of 550 Bq/(mAh mg 226 Ra) was recorded [102].For a more precise estimate of production yields, it is extremely important to quantify the cross-section data in detail in this reaction.
The main challenges in this method are the recycling requirement of the 226 Ra target and some handling issues with the 222 Rn daughter [20].However, large-scale 225 Ac manufacturing using this procedure is already being implemented at several plants [104].It was reported that SCK CEN is capable of generating high-grade GMP-grade 225 Ac and also continually supplying it using a backup system [18,100].During the creation of GMP-grade 225 Ac, SCK CEN has been collaborating with the Institute of Radioelements Environment & Lifescience Technology (IRE Elit) and Global Morpho Pharma (GMP) (France) [100].Starting in 2019, SCK-CEN began irradiating their stock of several hundred grammes of 226 Ra.This Belgian research centre is also equipped with a BR2 reactor and an acceleratordriven subcritical reactor named Multi-purpose hYbrid Research Reactor for High-tech Application (MYRRHA) that are used in this approach [100].Additionally, utilising an IBA (Ion Beam Applications S.A., EURONEXT) Rhodotron, SCK CEN could produce GMPgrade 225 Ac at a weekly rate of 37 GBq (1000 mCi) by irradiating with 40 MeV electrons at 125 kW [100].The prospects should be kept an eye on, as SCK CEN and IBA established a research and development partnership agreement for the joint production of 225 Ac in 2021 [105] (Table 3).In the middle of the 1990s, the JRC was the first laboratory to offer 225 Ac/ 213 Bi to clinical partners [5].Ever since, the JRC has produced these radioisotopes on an annual basis for preclinical research and clinical testing carried out at JRC Karlsruhe or in partnership with a large network of healthcare partners.
In order to produce the short-lived 213 Bi (T 1/2 = 45.6 min) on-site, 225 Ac can either be utilised directly as a therapeutic nuclide [50,106] or set onto 225 Ac/ 213 Bi generators [78,83].All patient investigations with 213 Bi up to now have utilised 225 Ac/ 213 Bi generators.
There are numerous generator types available, including those based on ion exchange, extraction chromatography, and inorganic sorbents [106].The most widely used type is a single-column "direct" generator that was invented at the ITU and based on the strongly acidic cation-exchange sorbent AG MP-50 [106].
In this well-known approach, 213 Bi is obtained starting from 225 Ac, which is tightly bound to the sorbent and drowned in 0.05M HNO 3 solution [14,78,83].At roughly every 3 h [14,78], 213 Bi ( 213 BiI 4 -and 213 BiI 5 2-) is obtained for immediate use through elution with a mixture of 0.1 M HCl/0.1 M NaI [104] (Figure 4) [14].for preclinical research and clinical testing carried out at JRC Karlsruhe or in partnership with a large network of healthcare partners.
In order to produce the short-lived 213 Bi (T1/2 = 45.6 min) on-site, 225 Ac can either be utilised directly as a therapeutic nuclide [50,106] or set onto 225 Ac/ 213 Bi generators [78,83].All patient investigations with 213 Bi up to now have utilised 225 Ac/ 213 Bi generators.
There are numerous generator types available, including those based on ion exchange, extraction chromatography, and inorganic sorbents [106].The most widely used type is a single-column "direct" generator that was invented at the ITU and based on the strongly acidic cation-exchange sorbent AG MP-50 [106].
In this well-known approach, 213 Bi is obtained starting from 225 Ac, which is tightly bound to the sorbent and drowned in 0.05M HNO3 solution [14,78,83].At roughly every 3 h [14,78], 213 Bi ( 213 BiI4 -and 213 BiI5 2-) is obtained for immediate use through elution with a mixture of 0.1 M HCl/0.1 M NaI [104] (Figure 4) [14].The high-activity generator technology created at JRC Karlsruhe enables the generator to function reliably even when supplied with up to 4 GBq 225 Ac of activities [5,78].Although the penetration of 225 Ac is less than 0.2 ppm, the yields of 213 Bi elution may be more than 80% [107].The process of distributing 225 Ac activity uniformly over about two- The high-activity generator technology created at JRC Karlsruhe enables the generator to function reliably even when supplied with up to 4 GBq 225 Ac of activities [5,78].Although the penetration of 225 Ac is less than 0.2 ppm, the yields of 213 Bi elution may be more than 80% [107].The process of distributing 225 Ac activity uniformly over about two-thirds of the generator resin ensures stable performance over several weeks and minimises radiolytic degradation of the organic resin [5,78].
Injection-ready therapeutic dosages of 213 Bi-labeled peptides with activities of up to 2.3 GBq have been successfully prepared using the generator for clinical applications [78] including the locoregional therapy of brain tumours [5,13].Due to the relatively long parent half-life, which enables the transport of the generator to radiopharmacy facilities over vast distances, these generators may be employed clinically.

Conclusions
Taking into account its α-particle emissions, along with the ability to eliminate the nontumour binding activity before most of its dose is deposited in organs, 225 Ac is considered an appealing choice for TAT.Nevertheless, because of its long half-life and the different α particles created throughout its decay chain, it is crucial to pay attention to the considerable cytotoxicity of 225 Ac.Additionally, the γ disintegrations that result from the intermediate 221 Fr and 213 Bi disintegration may be used in SPECT clinical imaging.Thus, the radioactive cascade of 225 Ac could be used in nuclear medicine, especially in theranostic applications.However, the small 225 Ac doses given lead to low γ emissions, which makes planar SPECT imaging difficult.A potential alternative for this constraint is to make appropriate use of 213 Bi, which can be isolated from the decay cascades of 225 Ac.However, the brief half-life of 213 Bi must be taken into account since it presents challenges for radiopharmaceutical distribution, processing, and radiolabelling.
Apart from direct production pathways, 225 Ac can be easily accessed at many points in the decay chain, especially through 233 U, 229 Th, and 225 Ra.Compared with other actinide nuclei, including 232 Th and 226 Ra, which are more stable to use as production targets, 225 Ac has many fewer nucleons.As a result, production techniques must, for the most part, rely on radioactive decay or higher energy bombardments.
All the production techniques discussed in this paper are expensive and will all struggle to satisfy demand at the expected level if they are used separately.
It is necessary to readjust the facilities that are accessible throughout the world, to use suitable production methods that are adapted to the available infrastructure, and take into consideration the advantages and disadvantages of every used production modality.In addition, fruitful collaboration between the different centres and experienced scientific staff will pave the way for the widespread clinical use of actinium-based radiopharmaceuticals as a new standard of care.
The European medical isotope programme: Production of High-Purity Isotopes by Mass Separation Project (PRISMAP) represents an important initiative of this type of collaboration.Coordinated by the European Laboratory for Nuclear Research (CERN), the project partners come from thirteen nations: Austria, Belgium, Denmark, France, Germany, Italy, Latvia, Norway, Portugal, Poland, Sweden, Switzerland, and the United Kingdom.Nine significant EU, national, or regional infrastructures, four developing infrastructures, leader research institutes, medical facilities, the European Joint Research Centre, and one small and midsize enterprise (SME) are among the twenty-three partners that make up the PRISMAP Consortium.With the help of these considerable facilities, the programme goal is to create a sustainable source of high-purity-grade new radionuclides for medical use.It also aims to offer an accessible point of entry for all researchers working in this field, including those from SMEs, global pharmaceutical companies, nuclear centres, hospitals, and universities, by implementing standardised access procedures.
Several PRISMAP partners, including JRC Belgium, Narodowe Centrum Bada ń J ądrowych (NCBJ), Poland, Institut Max von Laue-Paul Langevin (ILL), France, and SCK CEN, Belgium, are additionally implicated in another promising project in the field of the sustainability of medical isotope production and its safe application in Europe, named the Strengthening the European Chain of sUpply for next-generation medical Radionu-clidEs (SECURE).The project focuses on encouraging advancements in the creation of irradiation targets and manufacturing processes for both new and existing isotopes used in nuclear medicine and diagnostics.A list of crucial alpha-emitting radioisotopes in nuclear medicine was created, and 225 Ac was selected at the top of this list.The research aims to overcome the primary challenges to ensure the future availability of these isotopes by: (1) creating a framework of guidelines and recommendations that enable investigating the full clinical potential of alpha and beta particle therapy and its safe application; (2) offering significant insights that serve as a model for resolving challenges with upscaling and continuous isotope production; (3) removing critical obstacles along the production of specific alpha-and beta-emitting isotopes that restrict a sustainable production.

Figure 1 .
Figure 1.Schematic representation of the biological effects following the use of α-particle emitter radiopharmaceutical for cancer therapy.SSD = Single-Strand Break, DSB = Double-Strand Break, ROS = Reactive Oxygen Species.

Figure 1 .
Figure 1.Schematic representation of the biological effects following the use of α-particle emitter radiopharmaceutical for cancer therapy.SSD = Single-Strand Break, DSB = Double-Strand Break, ROS = Reactive Oxygen Species.

Figure 2 .
Figure 2. The decay chain of 233 U to 225 Ac and 213 Bi.

Figure 2 .
Figure 2. The decay chain of 233 U to 225 Ac and 213 Bi.

¢ 46 . 6
MBq of the225 Ac source dissolved in 0.2 M HCl was loaded under vacuum in the initial vial for radiolabelling with the DOTA-conjugated precursor (200 µg) on day 5 postsource purification.The source was transferred to the one-pot radiolabelling reactor cassette, in which the reaction occurred in Tris buffer (1 M, pH 7.2) at 85 • C for 70 min in the presence of 20% v/v L-ascorbic acid at pH 6-8.The radiolabelled peptide was transferred in saline and passed through a 0.2 µm sterilizing filter, resulting in a final volume of 9.7 mL.

¢
225 Ac (9.25 MBq) was obtained from a thorium generator at Canadian Nuclear Laboratories and supplied as the dried [ 225 Ac]AcCl 3 salt.The [ 225 Ac]AcCl 3 was dissolved in 1 mL 1 M NH 4 OAc, pH 7.0, transferred by pipette to a 50 mL centrifuge tube, and diluted to 45 mL in 1 M NH 4 OAc.Stock solution (1 mL), containing approximately 205 kBq [ 225 Ac]Ac(OAc) 3 , was transferred by pipette to a plastic Eppendorf tube placed on a digital TermoMixer heating block.Then, 20 µL of the ligand stock solution (0.01-1 mg/mL of PSMA or DOTA or macropa) was added and the reaction was shaken at 300 rpm at either 25 • C or 95 • C. A 3 µL aliquot of the reaction mixture was withdrawn and deposited on the origin of a silica-gel-60-coated aluminium plate (Sigma Aldrich) after incubating the reaction for 1 min, 5 min, and 15 min.

Figure 3 .
Figure 3.The principal production routes for 225 Ac.

Figure 3 .
Figure 3.The principal production routes for 225 Ac.

Table 3 .¢
Advantages and disadvantages of the potential 225 Ac production methods.A large portion of 233 U was created by neutron irradiating 232 Th ¢ 229 Th and 233 U have long T 1/2 values ¢ A significant stockpile of 233 U was kept after the thorium fuel cycle was abandoned in favour of fast reactors powered by plutonium ¢ The globally generated 229 Th is not enough to satisfy the extensive use and implementation in healthcare applications across the world ¢ The CNL has more recently announced the isolation of an important 229 Th source ¢ The development of 225 Ac radiopharmaceuticals is hindered by the limited supply and high cost that make 225 Ac inaccessible to many researchers ¢ Very pure sources of 229 Th were discovered, prepared, and used for pre-clinical research at the SCK CEN ¢ The short half-lives of 227 Ra and 228 Ac represent important restrictions for the possible 226 Ra(n,γ) 227 Ra(β−) 227 Ac(n,γ) 228 Ac(β−) 228 Th(n,γ) 229 Th routes ¢ Starting from 2019, a considerable rise in the availability of 229 Th will be produced through the extraction of 229 Th from historical wastes kept by the US DOE ¢ The cross section of 226 Ra(n, γ) 229 Th greatly impacts 229 Th production but is hindered by undesirable contaminant 228 Th ¢ The resulting 225 Ac is free of other actinium isotopes ¢ There is still a sizable gap between the theoretically predicted yields and the measured ones ¢ Exposing radium targets to high fluxes of thermal neutrons is considered an effective procedure to induce 229 Th production ¢ Whereas pure 227 Ac or 228 Ra targets are projected to generate somewhat more 229 Th, the current supply of these radionuclides is substantially less than that of 226 Ra

Table 2 .
Clinical research based on 225 Ac.

Table 3 .
Cont.The recycling requirement of the 226 Ra target ¢ Issues with handling the 222 Rn daughter 6.3. 225Ac/ 213 Bi Radionuclide Generators