Evaluation of polydentate picolinic acid chelating ligands and an α-melanocyte-stimulating hormone derivative for targeted alpha therapy using ISOL-produced ²²⁵Ac

Abstract

Background

Actinium-225 (²²⁵Ac, t_1/2 = 9.9 d) is a promising candidate radionuclide for use in targeted alpha therapy (TAT), though the currently limited global supply has hindered the development of a suitable Ac-chelating ligand and ²²⁵Ac-radiopharmaceuticals towards the clinic. We at TRIUMF have leveraged our Isotope Separation On-Line (ISOL) facility to produce ²²⁵Ac and use the resulting radioactivity to screen a number of potential ²²⁵Ac-radiopharmaceutical compounds.

Results

MBq quantities of ²²⁵Ac and parent radium-225 (²²⁵Ra, t_1/2 = 14.8 d) were produced and separated using solid phase extraction DGA resin, resulting in a radiochemically pure ²²⁵Ac product in > 98% yield and in an amenable form for radiolabeling of ligands and bioconjugates. Of the many polydentate picolinic acid (“pa”) containing ligands evaluated (H₄octapa [N₄O₄], H₄CHXoctapa [N₄O₄], p-NO₂-Bn-H₄neunpa [N₅O₄], and H₆phospa [N₄O₄]), all out-performed the current gold standard, DOTA for ²²⁵Ac radiolabeling ability at ambient temperature. Moreover, a melanocortin 1 receptor-targeting peptide conjugate, DOTA-modified cyclized α-melanocyte-stimulating hormone (DOTA-CycMSH), was radiolabeled with ²²⁵Ac and proof-of-principle biodistribution studies using B16F10 tumour-bearing mice were conducted. At 2 h post-injection, tumour-to-blood ratios of 20.4 ± 3.4 and 4.8 ± 2.4 were obtained for the non-blocking (molar activity [M.A.] > 200 kBq/nmol) and blocking (M.A. = 1.6 kBq/nmol) experiment, respectively.

Conclusion

TRIUMF’s ISOL facility is able to provide ²²⁵Ac suitable for preclinical screening of radiopharmaceutical compounds; [²²⁵Ac(octapa)]⁻, [²²⁵Ac(CHXoctapa)]⁻, and [²²⁵Ac(DOTA-CycMSH)] may be good candidates for further targeted alpha therapy studies.

Background

Due to recent clinical results demonstrating the exceptional ability of ²²⁵Ac-radiopharmaceuticals for the treatment of late stage castration resistant prostate cancer (Sathekge et al. 2018; Kratochwil et al. 2016; Kratochwil et al. 2018), considerable efforts within the field of nuclear medicine have been directed towards development of new radiopharmaceuticals for targeted alpha therapy (TAT) containing ²²⁵Ac or other suitable alpha-emitting radionuclides (Poty et al. 2018a; Poty et al. 2018b; Seidl 2014; Kim and Brechbiel 2012; 11th International Symposium on Targeted Alpha Therapy (TAT11), 2019; Elgqvist et al. 2014; Morgenstern et al. 2018; Baidoo et al. 2013). Despite these promising preliminary findings, the progression of ²²⁵Ac drugs towards the clinic has been obstructed by the limited radionuclide supply and the limited investigation of radiochemical protocols for chelating ²²⁵Ac^III under mild conditions. Currently, the total global supply of ²²⁵Ac is approximately 63 GBq (1.7 Ci) per year, slowing clinical trials and resulting in high costs that make ²²⁵Ac inaccessible to many researchers. The reader is referred to the following reviews for a more detailed overview of ²²⁵Ac supply (Robertson et al. 2018; Engle 2018).

Moreover, the absence of a stable surrogate isotope makes the study of actinium chemistry particularly challenging, and only very recently have insights into the fundamental chemical properties and coordination preferences of the actinide appeared in the literature (Ferrier et al. 2017; Ferrier et al. 2016). The tetraaza macrocycle DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, N₄O₄), is currently the “gold standard” in ²²⁵Ac chelation, despite the chelating ligand’s sub-optimal properties for encapsulating this large actinide. DOTA forms kinetically inert complexes with ²²⁵Ac, albeit at a cost of slow radiolabeling kinetics which requires heating to elevated temperatures and extended reaction times (Ramogida and Orvig 2013; Price and Orvig 2014). The requirement to heat the current gold standard DOTA, above physiological temperatures for extended periods to facilitate incorporation of the ²²⁵Ac^III ion is adequate yet not ideal for small molecule or peptide bioconjugates that can withstand such temperatures but results in denaturing of sensitive biomolecules such as antibodies (Maguire et al. 2014; McDevitt et al. 2002). Consequently, two-step radiolabeling methods are often required for antibody radiolabeling. For example, the DOTA-isothiocyanate (DOTA-NCS) bifunctional chelate can be radiolabeled with ²²⁵Ac at elevated temperatures first, followed by conjugation to the antibody at mild temperature, unfortunately the hydrolysis of the isothiocyanate in the first step results in low radiochemical yields (8–11%) (McDevitt et al. 2002). Recently, Poty et al. have demonstrated that a DOTA-tetrazine bifunctional chelate can be radiolabeled in a two-step method to a transcyclooctene-modified antibody by exploiting the facile inverse electron-demand Diels-Alder reaction yielding bioconjugates with molar activities that are satisfying for TAT (Poty et al. 2018c). Nonetheless, a chelating ligand with the ability to quantitatively incorporate the ²²⁵Ac^III ion under conditions commensurate for radiopharmaceutical use (such as sub-micromolar ligand concentrations, mild ambient reaction temperatures, and fast reaction times), that also forms thermodynamically stable and kinetically inert complexes would be of high interest and utility in the field of ²²⁵Ac TAT. Furthermore, access to myriad chelate options will allow greater flexibility during future discovery work of ²²⁵Ac radiopharmaceuticals, since the radiometal-complex can be used to influence the pharmacokinetics of the overall construct, particularly for small molecule or peptide-based radiopharmaceuticals. In an effort to produce a viable alternative, an octadentate bispidine, H₂bispa² (Comba et al. 2017), and an 18-membered macrocycle, macropa (Thiele et al. 2017), have been reported that show improved properties for ²²⁵Ac radiolabeling, and to date are the only chelators that permit rapid, ambient temperature chelation (Deal et al. 1999; Chappell et al. 2000).

The aims, herein, were to (1) determine the feasibility of producing ²²⁵Ac via Isotope Separation On-Line (ISOL) at TRIUMF and (2) test the suitability of ISOL-derived ²²⁵Ac (in terms of its (radio) chemical properties and amount of radioisotope produced) to drive radiolabeling studies and preclinical radiopharmaceutical development. Within the second aim, we sought to answer two questions: (2a) can the ISOL-derived ²²⁵Ac be used to evaluate chelating ligands for Ac coordination; and (2b) is the ²²⁵Ac-ISOL product produced in sufficient amounts to drive preclinical radiopharmaceutical development? In terms of radiopharmaceutical development, a DOTA-bioconjugate was chosen since DOTA is currently the industry standard in ²²⁵Ac TAT.

ISOL facilities have been used to produce medical radionuclides that are otherwise challenging to obtain (Dilling et al. 2014; dos Santos Augusto et al. 2014; Crawford et al. 2017a; Crawford et al. 2017b; Crawford et al. 2018). Herein we report for the first time the ISOL-production of ²²⁵Ac, at ISAC, TRIUMF’s ISOL facility. The ²²⁵Ac isolated from these experiments was used in radiolabeling studies with a class of picolinic acid (“pa”) ligands developed in our laboratories (Price et al. 2014; Ramogida et al. 2015; Price et al. 2012; Spreckelmeyer et al. 2017) (Fig. 1) and compared to DOTA as well as macropa and H₂bispa² (Comba et al. 2017; Thiele et al. 2017). The “pa” chelating ligand family has demonstrated versatility for coordinating a number of imaging (e.g., gallium-67/68, indium-111, copper-64) and therapeutic isotopes (e.g., copper-64, lutetium-177, yttrium-90), making them extremely amenable for incorporation into a “theranostic” radiopharmaceutical. The octadentate and nonadentate “pa” ligands H₆phospa (N₄O₄) (Price et al. 2014), p-NO₂-Bn-H₄neunpa (N₅O₄) (Spreckelmeyer et al. 2017), H₄octapa (N₄O₄) (Price et al. 2012; Price et al. 2013), and H₄CHXoctapa (N₄O₄) (Ramogida et al. 2015) used in these radiolabeling experiments, were chosen because they were designed to incorporate larger metal ions such as actinium. Ac^III has a documented ionic radius of 112 ppm (coordination number, CN 6) (Shannon 1976) and is likely suited to large polydentate chelators of high denticities. In addition, two DOTA-peptide conjugates, DOTA-D-Phe¹-Tyr³-octreotide (DOTA-TOC) (Graf et al. 2014; Eppard et al. 2017) and DOTA-Cyclized α-melanocyte-stimulating hormone (DOTA-CycMSH, Fig. 1) (Zhang et al. 2017) was radiolabeled with our ISAC derived ²²⁵Ac. Finally, a pilot in vivo study with the ²²⁵Ac-DOTA-CycMSH conjugate on tumour-bearing mice was performed to establish the feasibility of using ISOL-derived ²²⁵Ac to drive preclinical radiopharmaceutical development. Malignant melanoma is the most lethal form of skin cancer, accounting for 75% of deaths from this type of cancer. Receptor-targeted radiopharmaceuticals for melanoma imaging and therapy are promising strategies for diagnosis and treatment. In particular, the melanocortin-1 receptor (MC1R) is highly expressed in 83% of malignant melanoma cell lines, and has low expression in normal tissues making it an attractive target for radionuclide therapy and imaging (Zhang et al. 2017; Raposinho et al. 2010). Analogues of the tridecapeptide α-melanocyte-stimulating hormone (αMSH) have been studied extensively for MC1R-targeted imaging and less so for therapy. The most stable and successful analogues to date are based on lactam-bridged cyclized α-MSH (Ac-Nle4-cyclo [Asp⁵-His-D- Phe⁷-Arg-Trp-Lys¹⁰]-NH₂, or Nle-CycMSH_hex). Most recently our lab has developed a DOTA-CycMSH conjugate incorporating a cationic piperidine linker for positron emission tomography (PET) imaging with the positron-emitter gallium-68 (⁶⁸Ga, t_1/2 = 68 min) that exhibited promising ability to localize and internalize in melanoma cells, referred to herein as DOTA-CycMSH or CCZ01048 (Fig. 1). The [⁶⁸Ga]Ga-CCZ01048 tracer exhibited high binding affinity to MC1R with sub-nanomolar K_i values, rapid internalization into B16F10 melanoma cells, high in vivo stability in blood plasma, and exceptional PET image contrast in vivo with tumour-to-blood, tumour-to-muscle, tumour-to-bone and tumour-to-kidney ratios of 96.4 ± 13.9, 210.9 ± 20.9, 39.6 ± 11.9 and 4.0 ± 0.9, respectively, at 2 h post-injection (Zhang et al. 2017). Given this ⁶⁸Ga-tracer’s receptor binding affinity, rapid internalization, and low background organ uptake, we hypothesized that CCZ01048 would be a promising candidate for targeted alpha therapy with ²²⁵Ac, and biodistribution studies were therefore conducted to evaluate the ability of CCZ01048 to deliver ²²⁵Ac to B16F10 tumours in vivo.

Fig. 1

Ligand structures discussed in this work: macrocyclic commercial standard DOTA, previously reported ligands macropa and H₂bispa², acyclic picolinic acid “pa” ligands p-NO₂-Bn-H₄neunpa, H₆phospa, H₄CHXoctapa, and H₄octapa, and DOTA-CycMSH bioconjugate (CCZ01048)

Materials and methods

All solvents and reagents were purchased from commercial suppliers (Fisher Scientific, Sigma Aldrich) and used as received. Ultrapure HCl (TraceSELECT®) and NaOH (99.99% trace metal grade) was purchased from Sigma Aldrich; nitric acid (TraceMetal™ Grade) was purchased from Fisher Scientific. Branched DGA resin (N,N,N′,N′-tetrakis-2-ethylhexyldiglycolamide, 50–100 μm particle size) was purchase from Eichrom Technologies (Lisle, IL, USA). Picolinate-based ligands H₄octapa, H₄CHXoctapa, H₆phospa, and p-NO₂-Bn-H₄neunpa were prepared as previously described (Price et al. 2014; Ramogida et al. 2015; Price et al. 2012), as were H₂bispa² and macropa (Comba et al. 2017; Thiele et al. 2017). DOTA and human serum were purchased from Sigma Aldrich (St. Louis, MO). Pharmaceutical grade DOTATOC was purchased from ABX, DOTA-CycMSH was prepared as previously described (Zhang et al. 2017). Deionized water (> 18 MΩ cm) was prepared on site using a Millipore Direct-Q® 3UV water purification system.

All radioactivity measurements were performed using gamma ray spectroscopy on an N-type co-axial high purity germanium (HPGe) gamma spectrometer (Canberra Industries) calibrated with a 20 mL ¹⁵²Eu and ¹³³Ba source. After thorough mixing, aliquots (5–100 μL) from a sample were removed and diluted to the 20 mL standard volume for measurement. Samples were counted at a distance at least 15 cm from the detector for at least 30 min or until peak areas were below 5%; dead time was kept below 5%. Spectra were analyzed using the Genie 2000 software package (version X, Canberra Industries), using 40 keV, 218 keV, and 440 keV gamma lines for ²²⁵Ra, ²²¹Fr, and ²¹³Bi, respectively. ²²⁵Ac was quantified either directly via its 188 keV emission, or indirectly via ²²¹Fr after waiting at least 30 min to ensure equilibrium between ²²⁵Ac and ²²¹Fr.

The High Performance Liquid Chromatography (HPLC) system used for analysis of ²²⁵Ac-bioconjugates consisted of an Agilent 1260 Infinity II Quaternary Pump, Agilent 1260 autosampler, Raytest Gabi Star NaI (Tl) radiation detector, Agilent 1260 variable wavelength detector, and Agilent 1260 analytical scale fraction collector. Phenomenex Luna 5 μm C18 100 Å liquid chromatography analytical (250 × 4.6 mm) and semi-preparative (250 × 10 mm) columns were used for the quality assurance analysis and purification of ²²⁵Ac-bioconjugates, respectively.

²²⁵Ac production and purification

Ion beams of ²²⁵Ra and ²²⁵Ac were created using the isotope separation on-line (ISOL) technique in operation at TRIUMF’s ISAC facility (Dilling et al. 2014). Detailed descriptions of the collection of radioactivity from these beams is described elsewhere (Robertson et al. 2018; Crawford et al. 2017a; Crawford et al. 2017b; Crawford et al. 2018; Kunz et al. 2018). Briefly, a uranium carbide target is bombarded with 9.8 μA of 480 MeV protons, which cause spallation, fission and fragmentation of the ²³⁸U target atoms. Simultaneous heating of the target under vacuum results in the diffusion and effusion of volatile reaction products out of the target into the transfer tube/ionizer region. The reaction products, among them ²²⁵Ra and ²²⁵Ac, are ionized via surface or resonant laser ionization and extracted as a secondary particle beam with an energy of 20–40 keV, which is subsequently mass separated and delivered to experiments – in our case an aluminum implantation target for isotope collection. The resolving power of the ISAC mass separator is such that the delivered A = 225 beam contains isotopes of Fr, Ra, and Ac. Fr, and to some degree Ac and Ra, ionize in the ISAC surface ion source; however, higher ionization efficiencies can be obtained by means of element selective resonant laser ionization. Efficient laser ionization schemes for Ac and Ra can now be applied at ISAC simultaneously for co-implantation of Ac and Ra. After implantation, the aluminum target containing the implanted radioactivity was removed from the beamline and the ²²⁵Ra and ²²⁵Ac retrieved from the aluminum material by etching the surface of the aluminum with 0.1 M HCl (~ 0.5 mL) as described previously (Kunz et al. 2018).

The purification of ²²⁵Ac from ²²⁵Ra and other non-radioactive contaminants was adapted from previously reported methods for isolation of ²²⁵Ac from solid thorium targets (Zielinska et al. 2007; Apostolidis et al. 2005; Radchenko et al. 2015; Horwitz et al. 2005). Briefly, the recovered ²²⁵Ra/²²⁵Ac solution (0.1 M HCl, ~ 0.5 mL) was diluted to 4 M HNO₃ (5 mL), and passed through a DGA column (35–40 mg of DGA branched resin in a 6 mm diameter reservoir, pre-conditioned with H₂O (2 mL), 0.5 M HNO₃ (2 mL), and 4 M HNO₃ (2 mL)) (Horwitz et al. 2005). Under these conditions, Ac^III binds to the DGA while Ra passes through. The loaded DGA column was then washed with 4 M HNO₃ (4 mL). After air drying the column, the ²²⁵Ac was eluted from the resin using 0.05 M HNO₃ (0.5 mL). This separation scheme is illustrated in Fig. 2. The effluent (load fraction) containing ²²⁵Ra^II and the bulk of the non-radioactive Al^III impurities was collected and retained to decay, allowing for additional grow-in of ²²⁵Ac from ²²⁵Ra. Approximately every 1–3 weeks, the purification procedure was repeated to isolate a new batch of ²²⁵Ac.

Fig. 2

²²⁵Ra/²²⁵Ac radiochemical separation method used for purification of ISOL-produced ²²⁵Ac. The solution resulting from etching ²²⁵Ra and ²²⁵Ac from the aluminum target was diluted with nitric acid (to 4 M HNO₃) and loaded onto a preconditioned DGA resin (Horwitz et al. 2005). After washing the resin (with 4 M HNO₃) to remove residual ²²⁵Ra^II, the ²²⁵Ac^III was eluted from the resin 0.5 mL of 0.05 M HNO₃

The chemical purity of the ²²⁵Ac product was assessed via inductively coupled plasma optical emission spectrometry (ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS) to quantify the presence of any stable cations that may compete with ²²⁵Ac in radiolabeling reactions. Samples of both the initial solution containing ²²⁵Ra and ²²⁵Ac (before separation via DGA) and the final purified ²²⁵Ac solution were analyzed for elemental composition using an Agilent 7700× inductively-coupled plasma mass-spectrometer (ICP-MS). All samples were prepared by drying on a hotplate, treated with concentrated HNO₃ and then again dried to convert all salts into the nitrate form. Samples were then diluted with a 1% HNO₃, 0.05% HF solution containing 10 ppb Indium (internal standard). All acids used were sub-boiled to produce solutions containing a very low level of trace elements. Standard solutions were prepared from both mixed and single-element standards purchased from Inorganic Ventures (Christiansburg, VA, USA). Elements assayed included: Be, Al, Ca, Sc, Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Sr, Y, Zr, Nb, Mo, Sn, W, and Pb.

²²⁵Ac radiolabeling studies and complex stability assays

The pro-ligands H₄octapa, H₄CHXoctapa, H₆phospa, p-NO₂-Bn-H₄neunpa and standard DOTA were made up as stock solutions (10 mg/mL, ~ 10^− 2 M) in deionized water. A serial dilution was used to prepare ligand solutions at 10^− 3, 10^− 4, 10^− 5, and 10^− 6 M in water. A 10 μL aliquot of each ligand solution (or water, as a radiolabeling negative control) was diluted with ammonium acetate buffer (170 μL; 0.15 M; pH 5, 6, or 7). ²²⁵Ac(NO₃)₃ (5–30 kBq, 10–20 μL) was added and mixed gently to begin the radiolabeling reaction at ambient temperature (or 80 °C in the case of DOTA). Reaction pH (5.5 or 7) and temperatures (ambient or 40 °C) were applied and chosen because they are favourable conditions amenable for radiolabeling antibodies. An exhaustive list of radiolabeling reactions tested – repeated in triplicate – can be found in Additional file 1: Section S1.

With the exceptions of [²²⁵Ac(phospa)]³⁻ and [²²⁵Ac(neunpa)]⁻ complexes, for each stock ligand concentration and reaction pH, the reaction progress and radiochemical yield at varying time points (15–120 min) was analyzed by spotting a portion (1–5 μL) of the reaction mixture onto the bottom of an instant thin layer chromatography (iTLC) plate or aluminum backed silica TLC plate. After waiting at least 6 h to ensure equilibrium between ²²⁵Ac and its decay chain, plates were counted on a BioScan System 200 imaging scanner equipped with a BioScan Autochanger 1000.

Varying TLC/iTLC plate systems were selected for different ligands based from results of positive and negative radiolabeling controls:

• Method A, for [²²⁵Ac(octapa)]⁻ and [²²⁵Ac(CHXoctapa)]⁻: iTLC-SG plates (Agilent, 2 cm × 10 cm, baseline at 1.5 cm), developed using 10 mM NaOH/9% NaCl solution. Under these conditions free ²²⁵Ac^III remains at the baseline (R_f = 0), while the ²²⁵Ac-ligand complex migrates upwards with the solvent (R_f > 0); or

• Method B, for [²²⁵Ac(bispa²)]⁺ and [²²⁵Ac(macropa)]⁺: aluminum-backed ultrapure silica gel 60 with 250 μm thickness TLC plates (Fisher, 2 cm × 10 cm, baseline at 1.5 cm), developed using 0.4 M sodium citrate (pH 4) with 10% methanol. Under these conditions free ²²⁵Ac^III migrates with the solvent front (R_f = 1), while ²²⁵Ac-ligand complex remains at the baseline (R_f = 0); (Comba et al. 2017; Thiele et al. 2017) or

• Method C, for [²²⁵Ac(DOTA)]⁻: iTLC-SA plates (Agilent, 2 × 10 cm, baseline at 1.5 cm) and developed using 50 mM EDTA buffer, pH 4. Under these conditions free ²²⁵Ac^III migrates with the solvent front (R_f = 1), while ²²⁵Ac-ligand complex remains at the baseline (R_f = 0); or

• Method D, for human serum stability (below): aluminum-backed ultrapure silica gel 60 with 250 um thickness TLC plates (Fisher, 2 cm × 10 cm, baseline at 1.5 cm), developed in 50 mM EDTA, pH 5. Under these conditions, chelate-bound ²²⁵Ac remains at the baseline (R_f = 0), and any ²²⁵Ac^III that has de-complexed from the chelate will travel with the solvent front (R_f = 1). Method D was validated by incubating “free” ²²⁵Ac^III with human serum under analogous conditions to ensure all the activity eluted with the solvent front (R_f ~ 1).

Radiolabeling yields of [²²⁵Ac(phospa)]³⁻ and [²²⁵Ac(p-NO₂-Bn-neunpa)]⁻ were determined as follows: Briefly, to a 1 mL resin filled with Chelex (100–150 μg, sodium form, 50–100 mesh, equilibrated with NH₄OAc [0.15 M, pH 5.5]), the entire radiolabeling reaction mixture was loaded (Full). The resin was eluted with NH₄OAc (0.15 M, pH 5.5, 2 mL) (Eluted) to collect the ²²⁵Ac-complex while “free” unbound ²²⁵Ac^III remained on the resin. The eluate was diluted to 20 mL with H₂O in a scintillation vial and activity quantified via HPGe gamma spectroscopy. The radiolabeling yield was calculated by comparing the fraction of ²²⁵Ac collected in the eluate (Elute) with the amount in the initial radiolabeling reaction (Full); i.e. % RCY = (Elute)/(Full)*100.

To investigate the stabilities of the ²²⁵Ac-complexes, a competition experiment was performed in the presence of excess human serum and the displacement of radioactivity from the chelator to serum proteins was tracked over the course of seven days. Endogenous metal-binding proteins in serum such as apo-transferrin and metallothionein can compete for and displace chelator-bound metal ions in vivo, preventing successful delivery of the radionuclide to the desired target. The ²²⁵Ac-complexes were prepared using the radiolabeling protocols described above and added to an equal volume (170–180 μL) of human serum (stored at − 5 °C and thawed at ambient temperature). At varying time points (0.5 h to 7 days), small aliquots (1–5 μL) were spotted on iTLC plates using Methods B or D, described above. Representative TLC radio-chromatograms of a serum competition assay with ligand and control are presented in Additional file 1: Figures S2 and S3.

The stability of a radiometal-chelate complex is also often assessed by its ability to withstand transchelation in the presence of excess stable isotopes of the radiometal. Though this is an impossible experiment to perform with Ac since all Ac isotopes are unstable, lanthanum(III) has a similar ionic radius (103–116 ppm, CN = 6–9) and charge to actinium(III) and is often considered a chemical surrogate to Ac^III. Using non-radioactive metal ions such as La^III as a competitor for ligand complexation is a straightforward method to probe the kinetic off-rate of a pre-formed Ac^III complex, since many ligands that bind Ac^III may also efficiently complex La^III. Herein, ²²⁵Ac-complexes were prepared using the radiolabeling protocols described above and combined with La(NO₃)₃ (0.1 M, 50 M equivalents compared to the ligand) and allowed to react at ambient temperature. At varying time points (1 h – 7 d), aliquots of the competition reaction mixture (1–5 μL) were spotted on aluminum backed TLC plates and developed using the TLC methods described above.

Bioconjugate labeling studies and pilot in vivo studies with ²²⁵Ac-DOTA-CycMSH

Preliminary radiolabeling studies with DOTATOC using ISOL-derived ²²⁵Ac was performed. To prepare ²²⁵Ac-labeled DOTATOC, ²²⁵Ac^III (37 kBq or 1.5 MBq) was added to a vial containing precursor (25 μg, in 25 μL deionized water) and ammonium acetate buffer (1 M, pH 7) to give a final ligand concentration between 19 and 9.8 × 10^− 5 M and heated at 85 °C for 40 min. Reaction progress and RCYs were determined using iTLC method B (see ²²⁵Ac radiolabeling studies and complex stability assays section).

To further demonstrate the utility of ISAC-ISOL derived ²²⁵Ac and to explore the potential of novel ²²⁵Ac-radiopharmaceuticals, preliminary radiolabeling and in vivo studies were conducted with a previously reported lactam bridge-cyclized α-melanocyte-stimulating hormone peptide conjugated to the DOTA chelator (DOTA-CycMSH or CCZ01048) (Fig. 1).

To prepare ²²⁵Ac-labeled CCZ01048, the entire ²²⁵Ac fraction from a ²²⁵Ra/²²⁵Ac separation (see ²²⁵Ac production and purification section) was evaporated to dryness and reconstituted with 25 μL of 0.05 M HNO₃. This ²²⁵Ac solution was combined with CCZ01048 (20–80 μg, 25 μL in deionized water) and an ammonium acetate buffer (1 M, pH 7, 35 μL). The reaction mixture was subsequently heated to 85 °C for 30 min, and reaction progress and RCYs determined using iTLC Method B (see ²²⁵Ac radiolabeling studies and complex stability assays section). More details regarding the selection of conditions for labeling CCZ01048 with ISAC-derived ²²⁵Ac can be found in Additional file 1: Section S2.

Following the radiolabeling reaction for the non-blocked in vivo experiments, HPLC purification was performed to remove unbound ²²⁵Ac^III and to separate radiolabeled product from precursor to ensure high molar activity of the final product for preclinical biodistribution studies. The reaction mixture was separated by HPLC using a semi-preparative column eluted with 22% acetonitrile containing 0.1% TFA for ²²⁵Ac-CCZ01048 at a flow rate of 4.5 mL/min. The retention time of the ²²⁵Ac-labeled peptide was 9.5 min, compared to 10.0 min for the unlabeled peptide. The collected radiolabeled peptide was diluted with deionized water and retained on a C18 SepPak cartridge (Waters, preconditioned with 5 mL ethanol and 10 mL deionized water) to remove acetonitrile and TFA. The purified peptide was eluted with ethanol and diluted with 0.9% NaCl saline. HPLC purification was not performed for the blocking studies, but instead the radiolabeling reaction was diluted with ammonium formate (0.05 M, 10 mL), loaded onto a pre-conditioned C18 SepPak to remove unlabelled ²²⁵Ac^III, and the purified peptide was eluted with ethanol and diluted with 0.9% NaCl. Quality control of both reactions was conducted by iTLC-SG developed in citric acid (0.05 M, pH 5); under these conditions unbound ²²²⁵Ac^III travels with the solvent front (R_f = 1) while ²²⁵Ac-bound to peptide remains at the baseline (R_f = 0). Both ²²⁵Ac tracers had radiochemical purity > 98%. To determine the final molar activities of the purified tracer prior to in vivo injection, an aliquot of known volume (5–10 μL) was counted on the HPGe gamma spectrometer, and ²²⁵Ac activity per unit volume was calculated. In parallel, a known volume of final diluted tracer was analyzed using analytical HPLC eluted with 22% acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min, and the amount of peptide was quantified by integrating the peaks in the UV-Vis spectra (at 220 nm) and comparing to a standard curve of the [La(CCZ01048)] complex. The retention times of the unlabeled peptide and La-complex were 11.4 and 10.8 min, respectively.

The B16F10 melanoma cell line (Mus musculus) used in the tumour model was purchased from ATTC (CRL-6475), and handled as previously described (Zhang et al. 2017). The cell line was confirmed pathogen-free by the IMPACT 1 mouse profile test (IDEXX BioResearch). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, StemCell Technologies) supplemented by 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in a humidified incubator containing 5% CO₂. Cells grown to approximately 90% confluence were washed with sterile 1× PBS (pH 7.4), followed by trypsinization.

Biodistribution studies were performed using male C57BL/6 J mice. For tumour implantation, mice were anesthetized using isoflurane on a precision vaporizer (5% for induction, and 2% for maintenance) in 2.0 L/min of oxygen, and 1 × 10⁶ B16F10 cells were implanted subcutaneously on the right back at the level of the forelimbs. Tumours were allowed to grow and reach approximately 9 mm in diameter and used for biodistribution studies. Mice were anesthetized, and injected with 12–20 kBq of ²²⁵Ac-CCZ01048 prepared at either high molar activity (> 200 kBq/nmol for non-blocking, n = 4) or low molar activity (1.2 kBq/nmol for blocking studies, n = 4).

After injection, the mice were allowed to roam freely in their cages and sacrificed 2 h post-injection by CO₂ asphyxiation under isoflurane anesthesia. Cardiac puncture was promptly performed to recover blood, and the organs of interest were harvested, rinsed with 1× PBS, and blotted dry. Each organ was weighed, and the radioactivity of the collected tissue was measured using a calibrated gamma counter 931(Packard Cobra II Auto-gamma counter, Perkin Elmer, Waltham, MA, USA) using three energy windows: 60–120 keV (window A), 180–260 keV (window B), and 400–480 keV (window C). Counting was performed both immediately following sacrifice and after 7 days to ensure equilibrium of the ²²⁵Ac decay chain (Note: secular equilibrium of ²²⁵Ac with its daughters is reached at about 24 h; samples in this study were counted after 7 days due to availability of the gamma counter). Counts were decay corrected from the time of injection and total organ weights were used for the calculation of injected dose per gram of tissue (%ID/g). No differences were noted between the data; therefore, the biodistributions are reported using the data acquired using window A.

Results

²²⁵Ac production and purification

Mass A = 225, singly-charged (+ 1) ion beams produced at TRIUMF’s ISAC facility contained ²²⁵Ra and ²²⁵Ac intensities between 4.0 × 10⁶–1.6 × 10⁸ and 3.8 × 10⁶–1.3 × 10⁸ ions/s, respectively, depending on the state of the extraction electrode and the availability of the laser ionisation source. ²²⁵Fr (t_1/2 = 4.0 min) was also present but decayed rapidly to ²²⁵Ra. For durations between 13.3–80.7 h, the 40 keV ions beams were implanted into an aluminum disk to a depth of 15–25 nm as determined by SRIM (Ziegler et al. 2010). Etching of the radioactivity from the aluminum target post-implantation with 500 μL of 0.1 M HCl resulted in 0.2–7.5 MBq of ²²⁵Ra and 0.16–18.0 MBq of ²²⁵Ac. A summary of the production runs is shown in Table 1.

Table 1 Summary of ²²⁵Ra and ²²⁵Ac production runs at ISAC, TRIUMF’s ISOL facility. ^aEE = extraction electrode; ^bLIS = laser ionization source

One-step purification of ²²⁵Ac from ²²⁵Ra on the DGA column reliably yielded a pure ²²⁵Ac product, free from parent isotope ²²⁵Ra, with yields > 90% as determined by gamma ray spectroscopy. A second elution with an additional 0.5 mL of 0.05 M HNO₃ is typically able to recover the remaining ²²⁵Ac. The overall purification yield of ²²⁵Ac from the target solution was > 98%. Example results from a single separation are shown in Fig. 3. The load fraction containing ²²⁵Ra was retained and used as an ²²⁵Ac generator, providing a supply of ²²⁵Ac for nearly 3 months.

Fig. 3

Representative elution profile for ²²⁵Ra/²²⁵Ac separation on DGA branched resin in nitric acid media

To quantify the amount of stable metal impurities present in the ²²⁵Ac product that could compete with ²²⁵Ac during radiolabeling reactions, the chemical purity of the ²²⁵Ac was assessed via ICP-OES and ICP-MS. Modest values of aluminum with 7709 ± 1281 and 10175 ± 872 ppb (3.9 ± 0.6 and 5.1 ± 0.4 μg) were present in the first and second eluates, respectively, compared to 166,000 ppb (830 μg) present in the load fraction (Table 2). The ~ 200-fold decrease of Al in the eluate 1 compared to the initial load solution suggests that the DGA resin does a satisfactory job of removing excess Al^III from the ²²⁵Ac^III product with a separation factor of ~ 10²; however, an additional washing step may further decrease the amount of metal impurity, which may in turn positively impact radiolabeling yields. Calcium, iron, copper, nickel and zinc were found in the ²²⁵Ac product (elute 1) with amounts of 1392 ± 208, 203 ± 20, 47 ± 12, 19 ± 6, and 137 ± 21 ppb, respectively. If one assumes the entire solution of eluate 1 (0.5 mL) were to contain 10 MBq of ²²⁵Ac, the non-radioactive impurities would account for 6970 ± 1158 atoms of aluminum, 847 ± 127 atoms of calcium, 88 ± 9 atoms of iron, 51 ± 8 atoms of zinc, 8 ± 2 atoms of nickel, and 18 ± 5 atoms of copper per atom of actinium. Of concern are the high Mⁿ⁺:Ac^III ratios of Al^III, Fe^III, and Ca^II in the ²²⁵Ac eluate, since the gold standard in actinium chelation, DOTA, also has a strong affinity for these metals (Clarke and Martell 1991). Additional ICP-MS results for other elements (in ppb and μg) can be found in Additional file 1: Section S3.

Table 2 Trace metal content in ppb (μg/L) determined by ICP-MS (n = 2)

²²⁵Ac radiolabeling studies

The phosphinic acid ligand (phospa)⁶⁻ was able to efficiently complex ²²⁵Ac^III (radiochemical yield, RCY 95%) in one hour at ambient temperature, pH 5.5, and 10^− 3 M ligand concentrations; with decreasing ligand concentrations (10^− 4, 10^− 5, 10^− 6 M), RCYs dropped sequentially from 82, 14, to 0%, respectively. At even 10^− 3 M ligand concentration, nonadentate ligand (p-NO₂-Bn-neunpa)⁴⁻ displayed only a moderate radiochemical yield (56%) after 1 h at ambient temperature. Heating increased the RCY to 87% under the same ligand concentration and time (Additional file 1: Table S1).

The octadentate ligand (octapa)⁴⁻ efficiently complexed (RCY > 95%) ²²⁵Ac^III at 10^− 4 and 10^− 5 M ligand concentrations after one hour at ambient temperature, while the RCY dropped to 7% at a ligand concentration of 10^− 6 M. After only 30 min, radiolabeling was determined to be incomplete even at the 10^− 4 M ligand concentration (RCY = 77%), and thus 60-min timepoints were used to test the remaining labeling reactions for (octapa)⁴⁻. The chiral derivative, (CHXoctapa)⁴⁻ was able to efficiently complex ²²⁵Ac^III at ambient temperature in one hour at 10^− 4, 10^− 5 and 10^− 6 M, exhibiting RCYs of 91, 94 and 94%, respectively; at ligand concentration of 10^− 7 M, RCY decreased to 4%. Radiochemical yields > 90% were also obtained at 10^{–4 to − 6} M ligand after 30 min.

DOTA was inefficient at complexing ²²⁵Ac^III at ambient temperature, and required heating to 85 °C to induce ²²⁵Ac^III labeling (Additional file 1: Table S1).

Previously published ²²⁵Ac radiolabeling results with ligands (macropa)²⁻ and (bispa²)²⁻, (Comba et al. 2017; Thiele et al. 2017), which were performed with the same source of ²²⁵Ac are also plotted in Fig. 4 for comparison. As previously reported, octadentate bispidine, (bispa²)²⁻, was able to efficiently complex ²²⁵Ac after one hour at ambient temperature with ligand concentrations of 10^− 4 and 10^− 5 M (RCYs of 98 and 94%, respectively). At ligand concentrations of 10^− 6, 10^− 7, and 10^− 8 M, ²²⁵Ac RCYs decreased sequentially to 64, 15, and 2%, respectively. Unlike the gold standard DOTA and the rest of the tested pro-ligands (H₄octapa, H₄CHXoctapa, p-NO₂-Bn-H₄neunpa, H₆phospa), the 18-membered macrocycle, macropa, can quantitatively complex ²²⁵Ac (RCYs > 95%) at ligand concentrations as low as 10^− 7 M, in 5 min at ambient temperature (Thiele et al. 2017).

Fig. 4

Radiochemical yields (RCY, %) for ²²⁵Ac^III radiolabeling reactions of DOTA (RT, 2 h, pH 5.5; 85 °C, 30 min, pH 7, H₄octapa (RT, 1 h, pH 5.5), H₃CHXoctapa (RT, 1 h, pH 5.5), H₄neunpa (RT, 1 h, pH 5.5), H₆phospa (RT, 1 h, pH 5.5), H₂bispa² (RT, 1 h, pH 5.5) and macropa (RT, 5 min, pH 7) for comparison, at ambient temperature (RT) and ligand concentrations 10^− 3 to 10^− 8 M

The stability of ²²⁵Ac complexes in human serum for [²²⁵Ac(octapa)]⁻, [²²⁵Ac(CHXoctapa)]⁻, [²²⁵Ac(phospa)]³⁻ [²²⁵Ac(DOTA)]⁻, [²²⁵Ac(bispa²)]⁺, and [²²⁵Ac(macropa)]⁺ is shown in Table 3. The inability of p-NO₂-Bn-H₄neunpa to quantitatively complex ²²⁵Ac^III under any tested conditions precluded us from testing the stability of the resulting [²²⁵Ac(p-NO₂-Bn-neunpa)]⁻ complex. The [²²⁵Ac(octapa)]⁻ and [²²⁵Ac(CHXoctapa)]⁻ complexes exhibited favourable stabilities in human serum, remaining 92.9 ± 1.0 and 95.6 ± 1.6% intact after 7 days, respectively, while [²²⁵Ac(phospa)]³⁻ was moderately stable in serum, remaining 77.1 ± 6.4% intact after 7 days. The commercial standard [²²⁵Ac(DOTA)]⁻ remained sufficiently intact after 7 days (84.7 ± 8.1%). Both the previously reported [²²⁵Ac(bispa²)]⁺ and [²²⁵Ac(macropa)]⁺ complexes, which remained 88.9 ± 2.8 and 90% intact after 7 days in serum, respectively, show promising complex stabilities (Thiele et al. 2017), but slightly lower than the [²²⁵Ac(octapa)]⁻ and [²²⁵Ac(CHXoctapa)]⁻ complexes.

Table 3 Stability of ²²⁵Ac-labelled chelate complexes in human serum at ambient Temperatures (n = 3 unless otherwise noted)

The results of the La^III exchange competition assay are summarized in Table 4 and compared to previously reported values for [²²⁵Ac(macropa)]⁺ and [²²⁵Ac(bispa²)]⁺ (Comba et al. 2017; Thiele et al. 2017). The ²²⁵Ac^III was readily displaced from the [²²⁵Ac(phospa)]³⁻ complex upon addition of excess La^III. After 1 h, none of the ²²⁵Ac^III remained complexed to (phospa)⁶⁻, suggesting that (phospa)⁶⁻ does not form kinetically inert complexes with Ac^III and readily forms complexes with La^III. Significant decomplexation of the [²²⁵Ac(octapa)]⁻ complex was observed over a short period of time, with 78.9 ± 2.9, 42.7 ± 0.3, 4.7 ± 1.5, 6.1 ± 2.6% of the complex remaining intact after 1 h, 5 h, 1 d, and 2 d, respectively. The [²²⁵Ac(CHXoctapa)]⁻ complex displayed slightly delayed decomplexation kinetics compared to the (octapa)⁴⁻ complex, with 81.8 ± 4.3, 45.7 ± 3.1, 26.5 ± 8.9, 18.1 ± 0.4, 5.0 ± 0.4, 3.9 ± 0.6% intact ²²⁵Ac-complex remaining after 5 h, 1, 2, 3, 6, and 7 d, respectively. The cyclohexyl backbone of H₄CHXoctapa was designed to pre-organize the donor atoms of the linear chelating ligand, in hopes to form more kinetically inert complexes compared to the achiral analogue H₄octapa (Ramogida et al. 2015). The results of the La^III exchange study suggest that the introduction of the cyclohexyl diamine backbone indeed delay the kinetics of exchange of (CHXoctapa)⁴⁻ and its metal complexes compared to (octapa)⁴⁻ complexes, though neither [²²⁵Ac(octapa)]⁻ or [²²⁵Ac(CHXoctapa)]⁻ were able to sufficiently withstand transchelation at later time points. In contrast, [²²⁵Ac(macropa)]⁻, [²²⁵Ac(DOTA)]⁻, and [²²⁵Ac(bispa²)]⁻ remained 91, 77.1 ± 3.6, and 71.1 ± 2.7% intact after 7 days. In summary, taking into consideration the results from the La^III exchange competition assay, a trend in the robustness of the resulting ²²⁵Ac-complexes can be drawn as follows: (phospa)⁶⁻ < (octapa)⁴⁻ < (CHXoctapa)⁴⁻ < (bispa²)²⁻ ~ (DOTA)⁴⁻ < (macropa)²⁻.

Table 4 Stability of ²²⁵Ac-Labelled Chelate Complexes in 50-fold excess La^III at ambient temperature

Bioconjugate labeling studies and pilot in vivo studies with ²²⁵Ac-DOTA-CycMSH

The DOTA-bioconjugates, DOTATOC were radiolabeled with ISOL-derived ²²⁵Ac with moderate to poor yields. After heating at 85 °C for 40 min, [²²⁵Ac]Ac-DOTATOC radiolabeling yields of 91 ± 5% (n = 3, 37 kBq ²²⁵Ac used per reaction) or 43 ± 14% (n = 3, 1.5 MBq ²²⁵Ac used per reaction) were obtained, heating for longer periods of time did not increase radiolabeling yield.

[²²⁵Ac]Ac-CCZ01048 radiotracers were prepared with molar activities of > 200 kBq/nmol or 1.6 kBq/nmol, for non-blocked and blocked in vivo biodistribution experiments, respectively. Results from CCZ01048 radiolabeling studies with varying concentrations of ²²⁵Ac and precursor are shown in Additional file 1: Section S2. In general, high ²²⁵Ac radiolabeling yields (> 95%) of CCZ01048 were obtained when 60 μg (39 nmol) or more precursor was added, and radiolabeling yields dropped dramatically (57%) when 50 μg (33 nmol) of precursor was used.

Biodistribution results of the non-blocked experiment revealed tumour uptake at 2 h post injection of 5.23 ± 1.78%ID/g, with tumour-to-blood, tumour-to-bone, and tumour-to-kidney ratios of 20.42 ± 3.38, 23.16 ± 10.32, and 1.09 ± 0.11%ID/g, respectively (see Table 5). Uptake in other organs is shown in Fig. 5. To confirm the tumour uptake was receptor mediated, a blocking experiment was conducted by injecting low molar activity ²²⁵Ac-tracer (1.6 kBq/nmol) in tumour-bearing mice. As expected tumour uptake at 2 h post-injection decreased to 1.15 ± 0.21%ID/g, while kidney uptake increased to 8.85 ± 1.19% ID/g (compared to 4.83 ± 1.58%ID/g from non-blocked experiment). These results agree with the previously reported ⁶⁸Ga-labeled tracer (Zhang et al. 2017).

Table 5 Summary of in vivo biodistribution studies of [²²⁵Ac]Ac-CCZ01048 at 2 h post-injection. Statistical analysis between non-blocked and blocked experiments was performed using the Student’s t-test (* p < 0.05; ** p < 0.005; n = 4 for each animal experiment)

Fig. 5

Blocked and non-blocked biodistribution results (both n = 4) of [²²⁵Ac]Ac-CCZ01048 at 2 h post-injection

Discussion

²²⁵Ac obtained from TRIUMF’s ISOL facility was purified using a simple method with a high yield (> 98%), high radionuclidic purity (> 98%) and chemical purity suitable for preclinical screening of potential targeted alpha therapy compounds (Comba et al. 2017; Thiele et al. 2017). The entire purification process, from target dissolution to radiochemical purification is typically completed in under 2 h and produces less than 10 mL of radioactive liquid waste. In comparison, the current supply of ²²⁵Ac which is separated from a ²²⁹Th stock requires at least four separate column purifications to remove the multi-gram quantities of Th from Ra/Ac, evaporation procedures, and produces several litres of radioactive liquid waste with anywhere between 85 and 95% Ac recovery yields and > 99% radionuclidic purity (Robertson et al. 2018). For a detailed discussion of the current production and purification methods of ²²⁵Ac the reader is referred to the following review article (Robertson et al. 2018). Since the mass separator is of high resolution and efficiency, ISOL derived ²²⁵Ac can provide a radiochemically pure product without the need to chemically separate from multi-gram Th quantities or other target materials, allowing facile and rapid ²²⁵Ac purification using minimal solvent volumes. The main current disadvantage of the ISOL method for ²²⁵Ra/²²⁵Ac production is the limited yield that can be produced; the highest amount of ²²⁵Ac and ²²⁵Ra was 18.0 MBq and 8.6 MBq, respectively, from any one implantation (not including additional ²²⁵Ac activity isolated from the ²²⁵Ra generator).

Potential improvements to this ISOL ²²⁵Ac source are possible and include: using higher proton beam currents (up to 100 μA) to increase ²²⁵Ra and ²²⁵Ac production inside the uranium target; increasing ISAC beam availability for ²²⁵Ac production to allow for longer implantations; and utilization of alternative implantation target materials (other than aluminum) to increase ²²⁵Ac product chemical purity. For example, future work to directly implant ion beams into metal-free media (e.g., ultra-pure water, buffers, dilute acids) is underway and would significantly decrease nonradioactive metal-impurities in the ²²⁵Ac fraction. In principal, ISOL-medical isotope production can provide radioisotopes of the highest radiochemical purity, as it relies on extraction and high-resolution mass separation of the ion beams. Using the current ISAC facilities, ISOL could theoretically produce up to 370 MBq of ²²⁵Ac per month (given the maximum measured ISAC beam intensities of 1.3 × 10⁸ ions/s and 1.6 × 10⁸ ions/s for ²²⁵Ac and ²²⁵Ra, respectively, and three 10-day implantations) (Robertson et al. 2018). Increasing the beam current from 10 to 100 μA would further increase yields 10x and replacing uranium carbide targets with thorium targets should increase yields by 8.39x. Given these potential improvements, maximum annual ²²⁵Ac production per ISOL facility (191 GBq/year) could surpass the production via ²²⁹Th decay (63 GBq/year) (Robertson et al. 2018). While ²²⁵Ac produced via ISOL has the advantage of low cost for grant-funded academic researchers, compared to other ²²⁵Ac suppliers, with the current facility, there are disadvantages related to the reliability, consistency, and frequency of production runs (see Table 1). In our experience, this has made it challenging to plan more involved in vivo studies. As new sources of ²²⁵Ac begin to emerge (11th International Symposium on Targeted Alpha Therapy (TAT11), 2019; Robertson et al. 2017), medical radionuclide production at TRIUMF’s ISAC facility will transition towards less accessible radionuclides in order to harness the main advantage of ISOL facilities for medical isotope production – the flexibility to provide quick access to a diverse range of high-purity medical radionuclides without needing to establish radionuclide-specific production infrastructure or processes (Robertson et al. 2018; dos Santos Augusto et al. 2014; Kunz et al. 2018). The limitations of ISAC-produced ²²⁵Ac for preclinical research are highlighted by challenges associated with the ²²⁵Ac-DOTA-CycMSH study: the relatively low levels of ²²⁵Ac isolated by the ISOL method required the entire isolated ²²⁵Ac fraction for one radiolabeling reaction to isolate enough purified radiotracer for one set of preclinical in vivo studies (see Additional file 1: Section S2). The larger amount of precursor needed to provide high RCY consequently resulted in a lower molar activity product, and though HPLC purification can be used to remove excess unlabeled peptide and increase molar activity, this also results in a loss of product. Preliminary ²²⁵Ac radiolabeling of DOTATOC (with 25 μg) was also accomplished, using low (37 kBq) or high (1.5 MBq) ²²⁵Ac radioactivities, and a similar trend in radiolabeling yield was observed. Moderately high radiolabeling yields (91 ± 5%, n = 3, with calculated molar activities between 1.8–2.0 kBq/nmol) were obtained when low activities were used, while yields decreased significantly (43 ± 14%, n = 3, with calculated molar activities between 25 and 50 kBq/nmol) when 1.5 MBq of ²²⁵Ac was used in the labeling reaction. Presumably, the added non-radioactive impurities introduced by the increased amount of ²²⁵Ac added to the radiolabeling solution in the high radioactivity labeling reactions hampers radiolabeling yields. In comparison, previously published reports of DOTATOC radiolabeling yields using (commercially available) ²²⁹Th-derived ²²⁵Ac were 90 ± 4% (Miederer et al. 2008) which yielded molar activities between 57 and 171 kBq/nmol.

The moderate to poor ²²⁵Ac radiolabeling yields of DOTATOC and DOTA-CycMSH with decreasing ligand concentration further highlights the need to develop chelating ligands that have high affinity for ²²⁵Ac^III. Of the ligands reported in the ²²⁵ Ac radiolabeling studies section, the high radiochemical yields and favourable stabilities in serum of the two new ²²⁵Ac-complexes studied here, [²²⁵Ac(octapa)]⁻ and [²²⁵Ac(CHXoctapa)]⁻, suggests that these ligands may be good candidates for incorporation into a bioconjugate for ²²⁵Ac targeted alpha therapy. Though in vitro human serum stability assays are a widely used and accepted assay to predict the in vivo robustness of radiometal-chelate complexes (Ramogida and Orvig 2013; Price and Orvig 2014), in vivo biodistribution studies of [²²⁵Ac(octapa)]⁻ and [²²⁵Ac(CHXoctapa)]⁻ compared to [²²⁵Ac(DOTA)]⁻ or [²²⁵Ac(macropa)]⁺ would validate the in vitro results and assess the clearance and uptake profiles of the radiometal complexes and indeed are planned for future studies as we secure other higher sources of ²²⁵Ac. The results of the La^III exchange studies should also be interpreted with caution; this assay is helpful to assess the kinetic inertness or the kinetic off rates of the pre-formed ²²⁵Ac-complexes, but the use of a “surrogate element” (i.e. La^III) introduces potential bias as it is not known if the chelators will have the same affinity for Ac^III as they do La^III. For example, the [²²⁵Ac(CHXoctapa)]⁻ complex outperforms [²²⁵Ac(macropa)]⁺ in the 7 day serum stability challenge (95.6 ± 1.6 and 90%, respectively), yet [²²⁵Ac(CHXoctapa)]⁻ dissociates (3.9 ± 0.6% intact after 7 days) in the presence of excess La^III; this may be due to (CHXoctapa)⁴⁻ affinity for La^III and less so than the inertness of [²²⁵Ac(CHXoctapa)]⁻. Additional competitive radiolabeling experiments in which excess of non-radioactive ions such as Na^I, Ca^II, Mg^II, Fe^III followed by ²²⁵Ac^III and chelator would further corroborate the selectivity of a chelator for Ac^III (Deri et al. 2014).

Based on this preliminary data, macropa remains the most efficient ligand for ²²⁵Ac radiolabeling, though all the “pa” ligands tested represent an advantage compared to DOTA as they can complex ²²⁵Ac to at least some degree at ambient temperature.

The preliminary biodistribution studies of [²²⁵Ac]Ac-CCZ01048 revealed elevated tumour uptake of 5.23 ± 1.78%ID/g at 2 h p.i. While blocking studies confirmed that the uptake was receptor mediated, the excess amount of unlabelled precursor delayed the clearance of [²²⁵Ac]Ac-CCZ01048, and thus higher background levels in the kidneys and liver were observed, which was expected and in line with the results of the [⁶⁸Ga]Ga-CCZ01048 PET imaging study (Zhang et al. 2017). The previously reported [⁶⁸Ga]Ga-CCZ01048 tracer exhibited a higher tumour uptake of 21.9 ± 4.6%ID/g at 2 h p.i. (Zhang et al. 2017); this large discrepancy in tumour uptake may be explained by the differences in molar activity of the final injected tracers. The molar activity of the ²²⁵Ac-bioconjugate (200 kBq/nmol) is equivalent to one in every ~ 2440 peptide molecules labelled with ²²⁵Ac, while the reported molar activity of the injected ⁶⁸Ga-tracer (236.8 ± 66.6 MBq/nmol) (Zhang et al. 2017) is equivalent to one in every ~ 433 peptide molecules labelled with ⁶⁸Ga; a 5.6x difference. The lower radioactivity levels obtainable with ISOL derived ²²⁵Ac, coupled with the moderate radiolabeling yields precluded the isolation of an ²²⁵Ac-labelled conjugate with higher molar activity. Nonetheless, these preliminary studies show ISOL-derived ²²⁵Ac can be used to drive preclinical radiopharmaceutical development. Additionally, in vivo studies show proof-of-concept that CCZ01048 may be a promising candidate for further therapy studies with ²²⁵Ac targeted alpha therapy, which can be enabled by ²²⁵Ac obtained via other ²²⁵Ac production routes.

Conclusions

²²⁵Ra/²²⁵Ac ion beams produced at TRIUMF’s ISOL facility, were successfully used to produce ²²⁵Ac quantities sufficient for preclinical radiopharmaceutical screening (up to 18.0 MBq). The radiochemical separation of ²²⁵Ac from ²²⁵Ra was achieved on a solid phase extraction resin in high yields (> 98%) and as a radionuclidically pure product. The purified ²²⁵Ac was in a form amenable for radiolabeling and was used to screen a library of acyclic picolinic polydentate chelators for their ability to bind ²²⁵Ac^III, and perform preliminary radiolabeling of DOTA-bioconjguates. Finally, a DOTA-CycMSH conjugate was radiolabeled with ISOL-derived ²²⁵Ac, and proof-of-principle biodistribution studies were conducted in mice bearing B16F10 tumours. Receptor-mediated localization of the ²²⁵Ac-DOTA-CycMSH tracer was confirmed by a blocking experiment, suggesting that this peptide would be a promising candidate for further preclinical testing in vivo for targeted alpha therapy with ²²⁵Ac. In conclusion, together these studies show that ISOL-derived ²²⁵Ac can successfully be used to screen new chelating ligands for Ac^III coordination and be used to drive preclinical radiopharmaceutical development.