Preclinical Evaluation of [155/161Tb]Tb-Crown-TATE-A Novel SPECT Imaging Theranostic Agent Targeting Neuroendocrine Tumours.

Terbium radioisotopes (149Tb, 152Tb, 155Tb, 161Tb) offer a unique class of radionuclides which encompass all four medicinally relevant nuclear decay modalities (α, ß+, γ, ß-/e-), and show high potential for the development of element-matched theranostic radiopharmaceuticals. The goal of this study was to design, synthesise, and evaluate the suitability of crown-TATE as a new peptide-conjugate for radiolabelling of [155Tb]Tb3+ and [161Tb]Tb3+, and to assess the imaging and pharmacokinetic properties of each radiotracer in tumour-bearing mice. [155Tb]Tb-crown-TATE and [161Tb]Tb-crown-TATE were prepared efficiently under mild conditions, and exhibited excellent stability in human serum (>99.5% RCP over 7 days). Longitudinal SPECT/CT images were acquired for 155Tb- and 161Tb- labelled crown-TATE in male NRG mice bearing AR42J tumours. The radiotracers, [155Tb]Tb-crown-TATE and [161Tb]Tb-crown-TATE, showed high tumour targeting (32.6 and 30.0 %ID/g, respectively) and minimal retention in non-target organs at 2.5 h post-administration. Biodistribution studies confirmed the SPECT/CT results, showing high tumour uptake (38.7 ± 8.0 %ID/g and 38.5 ± 3.5 %ID/g, respectively) and favourable tumour-to-background ratios. Blocking studies further confirmed SSTR2-specific tumour accumulation. Overall, these findings suggest that crown-TATE has great potential for element-matched molecular imaging and radionuclide therapy using 155Tb and 161Tb.

In vivoquantitative SPECT imaging of actinium-226: feasibility and proof-of-concept.

Objective.225Ac radiopharmaceuticals have tremendous potential for targeted alpha therapy, however,225Ac (t1/2= 9.9 d) lacks direct gamma emissions forin vivoimaging.226Ac (t1/2= 29.4 h) is a promising element-equivalent matched diagnostic radionuclide for preclinical evaluation of225Ac radiopharmaceuticals.226Ac has two gamma emissions (158 keV and 230 keV) suitable for SPECT imaging. This work is the first feasibility study forin vivoquantitative226Ac SPECT imaging and validation of activity estimation.Approach.226Ac was produced at TRIUMF (Vancouver, Canada) with its Isotope Separator and Accelerator (ISAC) facility. [226Ac]Ac3+was radiolabelled with the bioconjugate crown-TATE developed for therapeutic targeting of neuroendocrine tumours. Mice with AR42J tumour xenografts were injected with either 2 MBq of [226Ac]Ac-crown-TATE or 4 MBq of free [226Ac]Ac3+activity and were scanned at 1, 2.5, 5, and 24 h post injection in a preclinical microSPECT/CT. Quantitative SPECT images were reconstructed from the 158 keV and 230 keV photopeaks with attenuation, background, and scatter corrections. Image-based226Ac activity measurements were assessed from volumes of interest within tumours and organs of interest. Imaging data was compared withex vivobiodistribution measured via gamma counter.Main results. We present, to the best of our knowledge, the first everin vivoquantitative SPECT images of226Ac activity distributions. Time-activity curves derived from SPECT images quantify thein vivobiodistribution of [226Ac]Ac-crown-TATE and free [226Ac]Ac3+activity. Image-based activity measurements in the tumours and organs of interest corresponded well withex vivobiodistribution measurements.Significance. Here in, we established the feasibility ofin vivo226Ac quantitative SPECT imaging for accurate measurement of actinium biodistribution in a preclinical model. This imaging method could facilitate more efficient development of novel actinium labelled compounds by providing accurate quantitativein vivopharmacokinetic information essential for estimating toxicities, dosimetry, and therapeutic potency.

Preclinical evaluation of [225Ac]Ac-crown-TATE - An alpha-emitting radiopharmaceutical for neuroendocrine tumors.

BACKGROUND: Targeted alpha therapy (TAT) of somatostatin receptor-2 (SSTR2) positive neuroendocrine tumors (NETs) involving Ac-225 ([225Ac]Ac-DOTA-TATE) has previously demonstrated improved therapeutic efficacy over conventional beta particle-emitting peptide receptor radionuclide therapy agents. DOTA-TATE requires harsh radiolabeling conditions for chelation of [225Ac]Ac3+, which can limit the achievable molar activities and thus therapeutic efficacy of such TAT treatments. Macropa-TATE was recently highlighted as a potential alternative to DOTA-TATE, owing to the mild radiolabeling conditions and high affinity toward [225Ac]Ac3+; however, elevated liver and kidney uptake were noted as a major limitation and a suitable imaging radionuclide is yet to be reported, which will be required for patient dosimetry studies and assessment of therapeutic benefit. Previously, [155Tb]Tb-crown-TATE has shown highly effective imaging of NETs in preclinical SPECT/CT studies, with high tumor uptake and low non-target accumulation; these favourable properties and the versatile coordination behavior of the crown chelator may therefore show promise for combination with Ac-225 for TAT. METHODS: Crown-TATE was labeled with Ac-225, and radiochemical yield was analyzed as the function of crown-TATE concentration. LogD7.4 was measured as the indication of hydrophilicity. Free [225Ac]Ac3+ release from [225Ac]Ac-crown-TATE in human serum was studied. Biodistribution studies of [225Ac]Ac-crown-TATE in mice bearing AR42J tumors was evaluated at 1, 4, 24, 48, and 120 h, and the absorbed dose to major organs calculated. Therapy-monitoring studies with AR42J tumor bearing mice were undertaken using 30 kBq and 55 kBq doses of [225Ac]Ac-crown-TATE and compared to controls treated with PBS or crown-TATE. RESULTS: [225Ac]Ac-crown-TATE was successfully prepared with high molar activity (640 kBq/nmol), and characterized as a moderately hydrophilic radioligand (LogD7.4 = -1.355 ± 0.135). No release of bound Ac-225 was observed over 9 days in human serum. Biodistribution studies of [225Ac]Ac-crown-TATE showed good initial tumor uptake (11.1 ± 1.7% IA/g at 4 h) which was sustained up to 120 h p.i. (6.92 ± 2.03% IA/g). Dosimetry calculations showed the highest absorbed dose was delivered to the tumors. Therapy monitoring studies demonstrated significant (log-rank test, P < 0.005) improved survival in both treatment groups compared to controls. CONCLUSIONS: This preclinical study demonstrated the therapeutic efficacy of [225Ac]Ac-crown-TATE for treatment of NETs, and highlights the potential of using crown chelator for stable chelation of Ac-225 under mild conditions.

Preclinical evaluation of MC1R targeting theranostic pair [155Tb]Tb-crown-αMSH and [161Tb]Tb-crown-αMSH.

BACKGROUND: Targeted radionuclide therapy is established as a highly effective strategy for the treatment of metastatic tumors; however, the co-development of suitable imaging companions to therapy remains significant challenge. Theranostic isotopes of terbium (149Tb, 152Tb, 155Tb, 161Tb) have the potential to provide chemically identical radionuclidic pairs, which collectively encompass all modes of nuclear decay relevant to nuclear medicine. Herein, we report the first radiochemistry and preclinical studies involving 155Tb- and 161Tb-labeled crown-αMSH, a small peptide-based bioconjugate suitable for targeting melanoma. METHODS: 155Tb was produced via proton induced spallation of Ta targets using the isotope separation and acceleration facility at TRIUMF with isotope separation on-line (ISAC/ISOL). The radiolabeling characteristics of crown-αMSH with 155Tb and/or 161Tb were evaluated by concentration-dependence radiolabeling studies, and radio-HPLC stability studies. LogD7.4 measurements were obtained for [161Tb]Tb-crown-αMSH. Competitive binding assays were undertaken to determine the inhibition constant for [natTb]Tb-crown-αMSH in B16-F10 cells. Pre-clinical biodistribution and SPECT/CT imaging studies of 155Tb and 161Tb labeled crown-αMSH were undertaken in male C57Bl/6 J mice bearing B16-F10 melanoma tumors to evaluate tumor specific uptake and imaging potential for each radionuclide. RESULTS: Quantitative radiolabeling of crown-αMSH with [155Tb]Tb3+ and [161Tb]Tb3+ was demonstrated under mild conditions (RT, 10 min) and low chelator concentrations; achieving high molar activities (23-29 MBq/nmol). Radio-HPLC studies showed [161Tb]Tb-crown-αMSH maintains excellent radiochemical purity in human serum, while gradual metabolic degradation is observed in mouse serum. Competitive binding assays showed the high affinity of [natTb]Tb-crown-αMSH toward MC1R. Two different methods for preparation of the [155Tb]Tb-crown-αMSH radiotracer were investigated and the impacts on the biodistribution profile in tumor bearing mice is compared. Preclinical in vivo studies of 155Tb- and 161Tb- labeled crown-αMSH were performed in parallel, in mice bearing B16-F10 tumors; where the biodistribution results showed similar tumor specific uptake (6.06-7.44 %IA/g at 2 h pi) and very low uptake in nontarget organs. These results were further corroborated through a series of single-photon emission computed tomography (SPECT) studies, with [155Tb]Tb-crown-αMSH and [161Tb]Tb-crown-αMSH showing comparable uptake profiles and excellent image contrast. CONCLUSIONS: Collectively, our studies highlight the promising characteristics of [155Tb]Tb-crown-αMSH and [161Tb]Tb-crown-αMSH as theranostic pair for nuclear imaging (155Tb) and radionuclide therapy (161Tb).

Quantitative SPECT imaging of 155Tb and 161Tb for preclinical theranostic radiopharmaceutical development.

BACKGROUND: Element-equivalent matched theranostic pairs facilitate quantitative in vivo imaging to establish pharmacokinetics and dosimetry estimates in the development of preclinical radiopharmaceuticals. Terbium radionuclides have significant potential as matched theranostic pairs for multipurpose applications in nuclear medicine. In particular, 155Tb (t1/2 = 5.32 d) and 161Tb (t1/2 = 6.89 d) have been proposed as a theranostic pair for their respective applications in single photon emission computed tomography (SPECT) imaging and targeted beta therapy. Our study assessed the performance of preclinical quantitative SPECT imaging with 155Tb and 161Tb. A hot rod resolution phantom with rod diameters ranging between 0.85 and 1.70 mm was filled with either 155Tb (21.8 ± 1.7 MBq/mL) or 161Tb (23.6 ± 1.9 MBq/mL) and scanned with the VECTor preclinical SPECT/CT scanner. Image performance was evaluated with two collimators: a high energy ultra high resolution (HEUHR) collimator and an extra ultra high sensitivity (UHS) collimator. SPECT images were reconstructed from photopeaks at 43.0 keV, 86.6 keV, and 105.3 keV for 155Tb and 48.9 keV and 74.6 keV for 161Tb. Quantitative SPECT images of the resolution phantoms were analyzed to report inter-rod contrast, recovery coefficients, and contrast-to-noise metrics. RESULTS: Quantitative SPECT images of the resolution phantom established that the HEUHR collimator resolved all rods for 155Tb and 161Tb, and the UHS collimator resolved rods ≥ 1.10 mm for 161Tb and ≥ 1.30 mm for 155Tb. The HEUHR collimator maintained better quantitative accuracy than the UHS collimator with recovery coefficients up to 92%. Contrast-to-noise metrics were also superior with the HEUHR collimator. CONCLUSIONS: Both 155Tb and 161Tb demonstrated potential for applications in preclinical quantitative SPECT imaging. The high-resolution collimator achieves < 0.85 mm resolution and maintains quantitative accuracy in small volumes which is advantageous for assessing sub organ activity distributions in small animals. This imaging method can provide critical quantitative information for assessing and optimizing preclinical Tb-radiopharmaceuticals.

A GATE simulation study for dosimetry in cancer cell and micrometastasis from the 225Ac decay chain.

BACKGROUND: Radiopharmaceutical therapy (RPT) with alpha-emitting radionuclides has shown great promise in treating metastatic cancers. The successive emission of four alpha particles in the 225Ac decay chain leads to highly targeted and effective cancer cell death. Quantifying cellular dosimetry for 225Ac RPT is essential for predicting cell survival and therapeutic success. However, the leading assumption that all 225Ac progeny remain localized at their target sites likely overestimates the absorbed dose to cancer cells. To address limitations in existing semi-analytic approaches, this work evaluates S-values for 225Ac's progeny radionuclides with GATE Monte Carlo simulations. METHODS: The cellular geometries considered were an individual cell (10 µm diameter with a nucleus of 8 µm diameter) and a cluster of cells (micrometastasis) with radionuclides localized in four subcellular regions: cell membrane, cytoplasm, nucleus, or whole cell. The absorbed dose to the cell nucleus was scored, and self- and cross-dose S-values were derived. We also evaluated the total absorbed dose with various degrees of radiopharmaceutical internalization and retention of the progeny radionuclides 221Fr (t1/2 = 4.80 m) and 213Bi (t1/2 = 45.6 m). RESULTS: For the cumulative 225Ac decay chain, our self- and cross-dose nuclear S-values were both in good agreement with S-values published by MIRDcell, with per cent differences ranging from - 2.7 to - 8.7% for the various radionuclide source locations. Source location had greater effects on self-dose S-values than the intercellular cross-dose S-values. Cumulative 225Ac decay chain self-dose S-values increased from 0.167 to 0.364 GyBq-1 s-1 with radionuclide internalization from the cell surface into the cell. When progeny migration from the target site was modelled, the cumulative self-dose S-values to the cell nucleus decreased by up to 71% and 21% for 221Fr and 213Bi retention, respectively. CONCLUSIONS: Our GATE Monte Carlo simulations resulted in cellular S-values in agreement with existing MIRD S-values for the alpha-emitting radionuclides in the 225Ac decay chain. To obtain accurate absorbed dose estimates in 225Ac studies, accurate understanding of daughter migration is critical for optimized injected activities. Future work will investigate other novel preclinical alpha-emitting radionuclides to evaluate therapeutic potency and explore realistic cellular geometries corresponding to targeted cancer cell lines.

Rearmed Bifunctional Chelating Ligand for 225Ac/155Tb Precision-Guided Theranostic Radiopharmaceuticals─H4noneunpaX.

Superior bifunctional chelating ligands, which can sequester both α-emitting radionuclides (225Ac, 213Bi) and their diagnostic companions (155Tb, 111In), remain a formidable challenge to translating targeted alpha therapy, with complementary diagnostic imaging, to the clinic. H4noneupaX, a chelating ligand with an unusual diametrically opposed arrangement of pendant donor groups, has been developed to this end. H4noneunpaX preferentially complexes Ln3+ and An3+ ions, forming thermodynamically stable (pLa = 17.8, pLu = 21.3) and kinetically inert complexes─single isomeric species by nuclear magnetic resonance and density functional theory. Metal binding versatility demonstrated in radiolabeling [111In]In3+, [155Tb]Tb3+, [177Lu]Lu3+, and [225Ac]Ac3+ achieved high molar activities under mild conditions. Efficient, scalable synthesis enabled in vivo evaluation of bifunctional H4noneunpaX conjugated to two octreotate peptides targeting neuroendocrine tumors. Single photon emission computed tomography/CT and biodistribution studies of 155Tb-radiotracers in AR42J tumor-bearing mice showed excellent image contrast, good tumor uptake, and high in vivo stability. H4noneunpaX shows significant potential for theranostic applications involving 225Ac/155Tb or 177Lu/155Tb.

SPECT imaging of226Ac as a theranostic isotope for225Ac radiopharmaceutical development.

Objective. The development of alpha-emitting radiopharmaceuticals using225Ac (t½ = 9.92 d) benefits from the quantitative determination of its biodistribution and is not always easy to directly measure. An element-equivalent matched-pair would allow for more accurate biodistribution and dosimetry estimates.226Ac (t½ = 29.4 h) is a candidate isotope forin vivoimaging of preclinical225Ac radiopharmaceuticals, given its 158 keV and 230 keV gamma emissions making it suitable for quantitative SPECT imaging. This work aimed to conduct a performance assessment for226Ac imaging and presents the first-ever226Ac SPECT images.Approach. To establish imaging performance with regards to contrast and noise, image quality phantoms were scanned using a microSPECT/CT system. To assess the resolution, a hot rod phantom with cylindrical rods with diameters between 0.85 and 1.70 mm was additionally imaged. Two collimators were evaluated: a high-energy ultra-high resolution (HEUHR) collimator and an extra ultra-high sensitivity (UHS) collimator. Images were reconstructed from two distinct photopeaks at 158 keV and 230 keV.Main results. The HEUHR SPECT image measurements of high activity concentration regions were consistent with values determined independently via gamma spectroscopy, within 9% error. The lower energy 158 keV photopeak images demonstrated slightly better contrast recovery. In the resolution phantom, the UHS collimator only resolved rods ≥1.30 mm and ≥1.50 mm for the 158 keV and 230 keV photopeaks, respectively, while the HEUHR collimator clearly resolved all rods, with resolution <0.85 mm.Significance. Overall, the feasibility of preclinical imaging with226Ac was demonstrated with quantitative SPECT imaging achieved for both its 158 keV and 230 keV photopeaks. The HEUHR collimator is recommended for imaging226Ac activity distributions in small animals due to its resolution <0.85 mm. Future work will explore the feasibility of using226Ac both as an element-equivalent isotope for225Ac radiopharmaceuticals, or as a standalone therapeutic isotope.