Zirconium tetraazamacrocycle complexes display extraordinary stability and provide a new strategy for zirconium-89-based radiopharmaceutical development

89 Zr–Tetraazamacrocycle complexes display extraordinary stability

Development of Targeted Alpha Particle Therapy for Solid Tumors

Abstract: Targeted alpha-particle therapy (TAT) aims to selectively deliver radionuclides emitting α-particles (cytotoxic payload) to tumors by chelation to monoclonal antibodies, peptides or small molecules that recognize tumor-associated antigens or cell-surface receptors. Because of the high linear energy transfer (LET) and short range of alpha (α) particles in tissue, cancer cells can be significantly damaged while causing minimal toxicity to surrounding healthy cells. Recent clinical studies have demonstrated the remarkable efficacy of TAT in the treatment of metastatic, castration-resistant prostate cancer. In this comprehensive review, we discuss the current consensus regarding the properties of the α-particle-emitting radionuclides that are potentially relevant for use in the clinic; the TAT-mediated mechanisms responsible for cell death; the different classes of targeting moieties and radiometal chelators available for TAT development; current approaches to calculating radiation dosimetry for TATs; and lead optimization via medicinal chemistry to improve the TAT radiopharmaceutical properties. We have also summarized the use of TATs in pre-clinical and clinical studies to dat

Evaluation of a 3-hydroxypyridin-2-one (2,3-HOPO) Based Macrocyclic Chelator for 89 Zr 4+ and Its Use for ImmunoPET Imaging of HER2 Positive Model of Ovarian Carcinoma in Mice

Abstract A novel octadentate 3-hydroxypyridin-2-one (2,3-HOPO) based di-macrocyclic ligand was evaluated for chelation of 89 Zr; subsequently, it was used as a bi-functional chelator for preparation of 89 Zr-labeled antibodies. Quantitative chelation of 89 Zr 4+ with the octadentate ligand forming 89 ZrL complex was achieved under mild conditions within 15 minutes. The 89 Zr-complex was stable in vitro in presence of DTPA, but a slow degradation was observed in serum. In vivo, the hydrophilic 89 Zr-complex showed prevalently renal excretion; and an elevated bone uptake of radioactivity suggested a partial release of 89 Zr 4+ from the complex. The 2,3-HOPO based ligand was conjugated to the monoclonal antibodies, HER2-specific trastuzumab and an isotypic anti-gD antibody, using a p-phenylene bis-isothiocyanate linker to yield products with an average loading of less than 2 chelates per antibody. Conjugated antibodies were labeled with 89 Zr under mild conditions providing the PET tracers in 60-69% yield. Despite the limited stability in mouse serum; the PET tracers performed very well in vivo. The PET imaging in mouse model of HER2 positive ovarian carcinoma showed tumor uptake of 89 Zr-trastuzumab (29.2 ± 12.9 %ID/g) indistinguishable (p = 0.488) from the uptake of positive control 89 Zr-DFO-trastuzumab (26.1 ± 3.3 %ID/g). In conclusion, the newly developed 3-hydroxypyridin-2-one based di-macrocyclic chelator provides a viable alternative to DFO-based heterobifunctional ligands for preparation of 89 Zr-labeled monoclonal antibodies for immunoPET studies

Recent Advances in Zirconium-89 Chelator Development

The interest in zirconium-89 (89Zr) as a positron-emitting radionuclide has grown considerably over the last decade due to its standardized production, long half-life of 78.2 h, favorable decay characteristics for positron emission tomography (PET) imaging and its successful use in a variety of clinical and preclinical applications. However, to be utilized effectively in PET applications it must be stably bound to a targeting ligand, and the most successfully used 89Zr chelator is desferrioxamine B (DFO), which is commercially available as the iron chelator Desferal®. Despite the prevalence of DFO in 89Zr-immuno-PET applications, the development of new ligands for this radiometal is an active area of research. This review focuses on recent advances in zirconium-89 chelation chemistry and will highlight the rapidly expanding ligand classes that are under investigation as DFO alternatives

Evaluation of macrocyclic hydroxyisophthalamide ligands as chelators for zirconium-89.

The development of bifunctional chelators (BFCs) for zirconium-89 immuno-PET applications is an area of active research. Herein we report the synthesis and evaluation of octadentate hydroxyisophthalamide ligands (1 and 2) as zirconium-89 chelators. While both radiometal complexes could be prepared quantitatively and with excellent specific activity, preparation of 89Zr-1 required elevated temperature and an increased reaction time. 89Zr-1 was more stable than 89Zr-2 when challenged in vitro by excess DTPA or serum proteins and in vivo during acute biodistribution studies. Differences in radiometal complex stability arise from structural changes between the two ligand systems, and suggest further ligand optimization is necessary to enhance 89Zr chelation

Imaging of Fibroblast Activation Protein Alpha Expression in a Preclinical Mouse Model of Glioma Using Positron Emission Tomography

Glioblastoma multiforme (GBM) is the most aggressive glioma of the primary central nervous system. Due to the lack of effective treatment options, the prognosis for patients remains bleak. Fibroblast activation protein alpha (FAP), a 170 kDa type II transmembrane serine protease was observed to be expressed on glioma cells and within the glioma tumor microenvironment. To understand the utility of targeting FAP in this tumor type, the immuno-PET radiopharmaceutical [89Zr]Zr-Df-Bz-F19 mAb was prepared and Lindmo analysis was used for its in vitro evaluation using the U87MG cell line, which expresses FAP endogenously. Lindmo analysis revealed an association constant (Ka) of 10−8 M−1 and an immunoreactivity of 52%. Biodistribution studies in U87MG tumor-bearing mice revealed increasing radiotracer retention in tumors over time, leading to average tumor-to-muscle ratios of 3.1, 7.3, 7.2, and 8.3 at 2, 24, 48 and 72 h, respectively. Small animal PET corroborated the biodistribution studies; tumor-to-muscle ratios at 2, 24, 48, and 72 h were 2.0, 5.0, 6.1 and 7.8, respectively. Autoradiography demonstrated accumulated activity throughout the interior of FAP+ tumors, while sequential tumor sections stained positively for FAP expression. Conversely, FAP− tissues retained minimal radioactivity and were negative for FAP expression by immunohistochemistry. These results demonstrate FAP as a promising biomarker that may be exploited to diagnose and potentially treat GBM and other neuroepithelial cancers

Imaging of Fibroblast Activation Protein Alpha Expression in a Preclinical Mouse Model of Glioma Using Positron Emission Tomography

Glioblastoma multiforme (GBM) is the most aggressive glioma of the primary central nervous system. Due to the lack of effective treatment options, the prognosis for patients remains bleak. Fibroblast activation protein alpha (FAP), a 170 kDa type II transmembrane serine protease was observed to be expressed on glioma cells and within the glioma tumor microenvironment. To understand the utility of targeting FAP in this tumor type, the immuno-PET radiopharmaceutical [89Zr]Zr-Df-Bz-F19 mAb was prepared and Lindmo analysis was used for its in vitro evaluation using the U87MG cell line, which expresses FAP endogenously. Lindmo analysis revealed an association constant (Ka) of 10−8 M−1 and an immunoreactivity of 52%. Biodistribution studies in U87MG tumor-bearing mice revealed increasing radiotracer retention in tumors over time, leading to average tumor-to-muscle ratios of 3.1, 7.3, 7.2, and 8.3 at 2, 24, 48 and 72 h, respectively. Small animal PET corroborated the biodistribution studies; tumor-to-muscle ratios at 2, 24, 48, and 72 h were 2.0, 5.0, 6.1 and 7.8, respectively. Autoradiography demonstrated accumulated activity throughout the interior of FAP+ tumors, while sequential tumor sections stained positively for FAP expression. Conversely, FAP− tissues retained minimal radioactivity and were negative for FAP expression by immunohistochemistry. These results demonstrate FAP as a promising biomarker that may be exploited to diagnose and potentially treat GBM and other neuroepithelial cancers.</jats:p

Abstract 517: An MC1R targeted 225Ac radiopharmaceutical agent for treatment of uveal melanoma

Abstract Prognosis in metastatic uveal melanoma is poor with median survival being less than one year. There is no approved therapy for metastatic disease. Hence, new targeted therapies are needed. The melanocortin 1 receptor (MC1R) is expressed in 94% of uveal melanomas but is not expressed in normal tissues of concern for toxicity. We have developed a MC1R specific ligand (MC1RL), conjugated imaging contrast agents to it, demonstrated high selectivity for MC1R expressing tumors in mice, and demonstrated rapid systemic clearance, without retention in tissues of concern for toxicity. The aim of this study is to use MC1RL as a targeting scaffold for development of a radiopharmaceutical by conjugation of 225Ac chelate. 225Ac is a therapeutic alpha emitting radionuclide. A targeted approach to deliver 225Ac to uveal melanoma without causing toxicity in normal tissues would provide clinicians with a novel and powerful therapeutic option for treatment of metastatic disease. Here, we conjugated 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA) to the MC1R specific scaffold (DOTA-MC1RL) and chelated the nonradioactive surrogate 139La (substitute for 225Ac) and demonstrated high binding affinity to MC1R (0.2 nM Ki). We anticipate that the radioactive conjugates will have comparable affinities. We also synthesized 225Ac-DOTA-MC1RL and showed a radiochemical yield greater than 95% and high radiochemical purity (≥99.8%) as determined by radio-TLC, radio-HPLC, and gamma-counter quantification. Moreover, 225Ac -DOTA-MC1RL showed excellent in vitro stability, i.e. 90% after 10 days in human serum at 37ºC. A maximum tolerated dosage (MTD) study was performed by administration of 0, 9, 18, 28, 37, 56, 74 and 148 kBq of 225Ac-DOTA-MC1RL. Animals were followed for 120 days and there were no signs of altered behavior among the groups. The group that received the highest dosage had a slightly but significantly lower increase in body weight over the course of the study, suggesting that 225Ac-DOTA-MC1RL is tolerated extremely well. Blood work and organ pathology did not show any significant deleterious effects even at the highest dose. In vitro cytotoxicity assays showed significant cell death in uveal and cutaneous cell lines in an MC1R dependent manner. Biodistribution studies showed tumor selectivity and a combination of renal and hepatic clearance with minimal retention in other normal tissues. In vivo efficacy studies in SCID mice bearing MC1R expressing human A375 subcutaneous xenograft tumors showed complete loss of tumor within one week of intravenous administration of 225Ac-DOTA-MC1RL relative to controls that had continued tumor growth. In conclusion, we have demonstrated radiosynthesis of 225Ac-DOTA-MC1RLwith high yield, purity and stability. In vitro studies demonstrated MC1R dependent cytotoxicity in uveal melanoma cells. In vivo studies demonstrated favorable biodistribution and significant antitumor efficacy. Citation Format: Narges K. Tafreshi, Michael L. Doligalski, Darpan N. Pandya, Hyun Joo Kil, Mikalai Budzevich, Thaddeus J. Wadas, Mark L. McLaughlin, David Morse. An MC1R targeted 225Ac radiopharmaceutical agent for treatment of uveal melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 517.</jats:p