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).

Preclinical Evaluation of Gastrin-Releasing Peptide Receptor Antagonists Labeled with 161Tb and 177Lu: A Comparative Study.

To elucidate potential benefits of the Auger-electron-emitting radionuclide 161Tb, we compared the preclinical performance of the gastrin-releasing peptide receptor antagonists RM2 (DOTA-Pip5-d-Phe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Sta13-Leu14-NH2) and AMTG (α-Me-Trp8-RM2), each labeled with both 177Lu and 161Tb. Methods: 161Tb/177Lu labeling (90°C, 5 min) and cell-based experiments (PC-3 cells) were performed. In vivo stability (30 min after injection) and biodistribution studies (1-72 h after injection) were performed on PC-3 tumor-bearing CB17-SCID mice. Results: Gastrin-releasing peptide receptor affinity was high for all compounds (half-maximal inhibitory concentration [nM]: [161Tb]Tb-RM2, 2.46 ± 0.16; [161Tb]Tb-AMTG, 2.16 ± 0.09; [177Lu]Lu-RM2, 3.45 ± 0.18; [177Lu]Lu-AMTG, 3.04 ± 0.08), and 75%-84% of cell-associated activity was receptor-bound. In vivo, both AMTG analogs displayed distinctly higher stability (30 min after injection) and noticeably higher tumor retention than their RM2 counterparts. Conclusion: On the basis of preclinical results, [161Tb]Tb-/[177Lu]Lu-AMTG might reveal a higher therapeutic efficacy than [161Tb]Tb-/[177Lu]Lu-RM2, particularly [161Tb]Tb-AMTG because of additional Auger-electron emissions at the cell membrane level.

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.

Comparative Study of a Decadentate Acyclic Chelate, HOPO-O10, and Its Octadentate Analogue, HOPO-O8, for Radiopharmaceutical Applications.

Radiolanthanides and actinides are aptly suited for the diagnosis and treatment of cancer via nuclear medicine because they possess unique chemical and physical properties (e.g., radioactive decay emissions). These rare radiometals have recently shown the potential to selectively deliver a radiation payload to cancer cells. However, their clinical success is highly dependent on finding a suitable ligand for stable chelation and conjugation to a disease-targeting vector. Currently, the commercially available chelates exploited in the radiopharmaceutical design do not fulfill all of the requirements for nuclear medicine applications, and there is a need to further explore their chemistry to rationally design highly specific chelates. Herein, we describe the rational design and chemical development of a novel decadentate acyclic chelate containing five 1,2-hydroxypyridinones, 3,4,3,3-(LI-1,2-HOPO), referred to herein as HOPO-O10, based on the well-known octadentate ligand 3,4,3-(LI-1,2-HOPO), referred to herein as HOPO-O8, a highly efficient chelator for 89Zr[Zr4+]. Analysis by 1H NMR spectroscopy and mass spectrometry of the La3+ and Tb3+ complexes revealed that HOPO-O10 forms bimetallic complexes compared to HOPO-O8, which only forms monometallic species. The radiolabeling properties of both chelates were screened with [135La]La3+, [155/161Tb]Tb3+, [225Ac]Ac3+ and, [227Th]Th4+. Comparable high specific activity was observed for the [155/161Tb]Tb3+ complexes, outperforming the gold-standard 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, yet HOPO-O10 surpassed HOPO-O8 with higher [227Th]Th4+ affinity and improved complex stability in a human serum challenge assay. A comprehensive analysis of the decadentate and octadentate chelates was performed with density functional theory for the La3+, Ac3+, Eu3+, Tb3+, Lu3+, and Th4+ complexes. The computational simulations demonstrated the enhanced stability of Th4+-HOPO-O10 over Th4+-HOPO-O8. This investigation reveals the potential of HOPO-O10 for the stable chelation of large tetravalent radioactinides for nuclear medicine applications and provides insight for further chelate development.

A simple and automated method for 161Tb purification and ICP-MS analysis of 161Tb.

BACKGROUND: 161Tb is a radiolanthanide with the potential to replace 177Lu in targeted radionuclide therapy. 161Tb is produced via the neutron irradiation of [160Gd]Gd2O3 targets, and must be purified from 160Gd and the decay product 161Dy prior to use. Established purification methods require complex conditions or high-pressure ion chromatography (HPIC) which are inconvenient to introduce in a broad user community. This study aims to find a simpler small solid-phase extraction (SPE) column method for 161Tb purification that is more suitable for automation with commercially available systems like TRASIS. RESULTS: We first tested the distribution coefficients on TK211 and TK212 resins for the separation of Gd, Tb, and Dy, and subsequently developed a method to separate these metal ions, with an additional TK221 resin to concentrate the final product. A side-by-side comparison of the products purified using this new method with the HPIC method was undertaken, assessing the radionuclidic purity, chemical purity regarding Gd and Dy, and labeling efficiency with a standard chelate (DOTA) and a novel chelate (crown). The two methods have comparable radionuclidic purity and labeling efficiency. The small SPE column method reduced Gd content to nanogram level, although still higher than the HPIC method. An ICP-MS method to quantify 161Tb, 159Tb, 160Gd, and 161Dy was developed with the application of mass-shift by ammonia gas. Last, 161Tb produced from the small SPE column method was used to assess the biodistribution of [161Tb]Tb-crown-αMSH, and the results were comparable to the HPIC produced 161Tb. CONCLUSIONS: 161Tb was successfully purified by a semi-automated TRASIS system using a combination of TrisKem extraction resins. The resulting product performed well in radiolabelling and in vivo experiments. However, improvement can be made in the form of further reduction of 160Gd target material in the final product. An ICP-MS method to analyze the radioactive product was developed. Combined with gamma spectroscopy, this method allows the purity of 161Tb being assessed before the decay of the product, providing a useful tool for quality control.

Exploring the Potential of High-Molar-Activity Samarium-153 for Targeted Radionuclide Therapy with [153Sm]Sm-DOTA-TATE.

Samarium-153 is a promising theranostic radionuclide, but low molar activities (Am) resulting from its current production route render it unsuitable for targeted radionuclide therapy (TRNT). Recent efforts combining neutron activation of 152Sm in the SCK CEN BR2 reactor with mass separation at CERN/MEDICIS yielded high-Am 153Sm. In this proof-of-concept study, we further evaluated the potential of high-Am 153Sm for TRNT by radiolabeling to DOTA-TATE, a well-established carrier molecule binding the somatostatin receptor 2 (SSTR2) that is highly expressed in gastroenteropancreatic neuroendocrine tumors. DOTA-TATE was labeled with 153Sm and remained stable up to 7 days in relevant media. The binding specificity and high internalization rate were validated on SSTR2-expressing CA20948 cells. In vitro biological evaluation showed that [153Sm]Sm-DOTA-TATE was able to reduce CA20948 cell viability and clonogenic potential in an activity-dependent manner. Biodistribution studies in healthy and CA20948 xenografted mice revealed that [153Sm]Sm-DOTA-TATE was rapidly cleared and profound tumor uptake and retention was observed whilst these were limited in normal tissues. This proof-of-concept study showed the potential of mass-separated 153Sm for TRNT and could open doors towards wider applications of mass separation in medical isotope production.

Radiolabeling of Human Serum Albumin With Terbium-161 Using Mild Conditions and Evaluation of in vivo Stability.

Targeted radionuclide therapy (TRNT) is a promising approach for cancer therapy. Terbium has four medically interesting isotopes (149Tb, 152Tb, 155Tb and 161Tb) which span the entire radiopharmaceutical space (TRNT, PET and SPECT imaging). Since the same element is used, accessing the various diagnostic or therapeutic properties without changing radiochemical procedures and pharmacokinetic properties is advantageous. The use of (heat-sensitive) biomolecules as vector molecule with high affinity and selectivity for a certain molecular target is promising. However, mild radiolabeling conditions are required to prevent thermal degradation of the biomolecule. Herein, we report the evaluation of potential bifunctional chelators for Tb-labeling of heat-sensitive biomolecules using human serum albumin (HSA) to assess the in vivo stability of the constructs. p-SCN-Bn-CHX-A"-DTPA, p-SCN-Bn-DOTA, p-NCS-Bz-DOTA-GA and p-SCN-3p-C-NETA were conjugated to HSA via a lysine coupling method. All HSA-constructs were labeled with [161Tb]TbCl3 at 40°C with radiochemical yields higher than 98%. The radiolabeled constructs were stable in human serum up to 24 h at 37°C. 161Tb-HSA-constructs were injected in mice to evaluate their in vivo stability. Increasing bone accumulation as a function of time was observed for [161Tb]TbCl3 and [161Tb]Tb-DTPA-CHX-A"-Bn-HSA, while negligible bone uptake was observed with the DOTA, DOTA-GA and NETA variants over a 7-day period. The results indicate that the p-SCN-Bn-DOTA, p-NCS-Bz-DOTA-GA and p-SCN-3p-C-NETA are suitable bifunctional ligands for Tb-based radiopharmaceuticals, allowing for high yield radiolabeling in mild conditions.

Production of Sm-153 With Very High Specific Activity for Targeted Radionuclide Therapy.

Samarium-153 (153Sm) is a highly interesting radionuclide within the field of targeted radionuclide therapy because of its favorable decay characteristics. 153Sm has a half-life of 1.93 d and decays into a stable daughter nuclide (153Eu) whereupon ß- particles [E = 705 keV (30%), 635 keV (50%)] are emitted which are suitable for therapy. 153Sm also emits γ photons [103 keV (28%)] allowing for SPECT imaging, which is of value in theranostics. However, the full potential of 153Sm in nuclear medicine is currently not being exploited because of the radionuclide's limited specific activity due to its carrier added production route. In this work a new production method was developed to produce 153Sm with higher specific activity, allowing for its potential use in targeted radionuclide therapy. 153Sm was efficiently produced via neutron irradiation of a highly enriched 152Sm target (98.7% enriched, σth = 206 b) in the BR2 reactor at SCK CEN. Irradiated target materials were shipped to CERN-MEDICIS, where 153Sm was isolated from the 152Sm target via mass separation (MS) in combination with laser resonance enhanced ionization to drastically increase the specific activity. The specific activity obtained was 1.87 TBq/mg (≈ 265 times higher after the end of irradiation in BR2 + cooling). An overall mass separation efficiency of 4.5% was reached on average for all mass separations. Further radiochemical purification steps were developed at SCK CEN to recover the 153Sm from the MS target to yield a solution ready for radiolabeling. Each step of the radiochemical process was fully analyzed and characterized for further optimization resulting in a high efficiency (overall recovery: 84%). The obtained high specific activity (HSA) 153Sm was then used in radiolabeling experiments with different concentrations of 4-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA). Even at low concentrations of p-SCN-Bn-DOTA, radiolabeling of 0.5 MBq of HSA 153Sm was found to be efficient. In this proof-of-concept study, we demonstrated the potential to combine neutron irradiation with mass separation to supply high specific activity 153Sm. Using this process, 153SmCl3 suitable for radiolabeling, was produced with a very high specific activity allowing application of 153Sm in targeted radionuclide therapy. Further studies to incorporate 153Sm in radiopharmaceuticals for targeted radionuclide therapy are ongoing.

Stability of europium(ii) in aqueous nitrate solutions.

In the lanthanide series, Eu3+ is most easily reduced to its divalent state. Reduction of Eu3+ has been studied extensively in aqueous media that are insensitive to reducing conditions. Recently, it has been reported that reduction of Eu3+ is also feasible in aqueous nitrate solutions and that Eu2+ remained sufficiently stable in these media to conduct separation experiments. However, additional fundamental research on the reduction efficiency of Eu3+ and stability of Eu2+ in these media has not been reported yet. In this paper, cyclic voltammetry, magnetic susceptibility measurements, UV-vis absorption spectroscopy and X-ray absorption near edge structure (XANES) spectroscopy were used to gain more insights into the reduction of Eu3+ in aqueous nitrate media. Within the parameters used in this work, near-quantitative reduction of Eu3+ could be achieved within 120 min in highly concentrated nitrate salt solutions, using both chemical and electrochemical reduction techniques. Moreover, Eu2+ was remarkably stable in these solutions, showing just a small percentage of back-oxidation after 5 h in a sealed measurement cell.

Separation of samarium and europium by solvent extraction with an undiluted quaternary ammonium ionic liquid: towards high-purity medical samarium-153.

Long-lived europium-154 impurities are formed during the production of medical samarium-153 in a high-flux nuclear reactor. A method to separate these europium impurities from samarium was investigated using the hydrophobic quaternary ammonium ionic liquid Aliquat 336 nitrate. The separation method consists of the selective reduction of Eu3+ by zinc metal in an aqueous feed solution containing a high nitrate salt concentration. Subsequent extraction using undiluted Aliquat 336 nitrate leads to an efficient separation of both lanthanides in a relatively short time frame. Sm3+ was extracted to the neat ionic liquid phase much more efficiently than Eu2+. An initial approach using the addition of dicyclohexano-18-crown-6 to capture Eu2+ in the ionic liquid phase was less efficient.