"Off-Label Use" of the Siderophore Enterobactin Enables Targeted Imaging of Cancer with Radioactive Ti(IV).

The development of inert, biocompatible chelation methods is required to harness the emerging positron emitting radionuclide 45Ti for radiopharmaceutical applications. Herein, we evaluate the Ti(IV)-coordination chemistry of four catechol-based, hexacoordinate chelators using synthetic, structural, computational, and radiochemical approaches. The siderophore enterobactin (Ent) and its synthetic mimic TREN-CAM readily form mononuclear Ti(IV) species in aqueous solution at neutral pH. Radiolabeling studies reveal that Ent and TREN-CAM form mononuclear complexes with the short-lived, positron-emitting radionuclide 45Ti(IV), and do not transchelate to plasma proteins in vitro and exhibit rapid renal clearance in naïve mice. These features guide efforts to target the 45Ti isotope to prostate cancer tissue through the design, synthesis, and evaluation of Ent-DUPA, a small molecule conjugate composed of a prostate specific membrane antigen (PSMA) targeting peptide and a monofunctionalized Ent scaffold. The [45Ti][Ti(Ent-DUPA)]2- complex forms readily at room temperature. In a tumor xenograft model in mice, selective tumor tissue accumulation (8±5 %, n=5), and low off-target uptake in other organs is observed. Overall, this work demonstrates targeted imaging with 45Ti(IV), provides a foundation for advancing the application of 45Ti in nuclear medicine, and reveals that Ent can be repurposed as a 45Ti-complexing cargo for targeted nuclear imaging applications.

Exploration of commercial cyclen-based chelators for mercury-197 m/g incorporation into theranostic radiopharmaceuticals.

A comprehensive investigation of the Hg2+ coordination chemistry and 197m/gHg radiolabeling capabilities of cyclen-based commercial chelators, namely, DOTA and DOTAM (aka TCMC), along with their bifunctional counterparts, p-SCN-Bn-DOTA and p-SCN-Bn-TCMC, was conducted to assess the suitability of these frameworks as bifunctional chelators for the 197m/gHg2+ theranostic pair. Radiolabeling studies revealed that TCMC and DOTA exhibited low radiochemical yields (0%-6%), even when subjected to harsh conditions (80°C) and high ligand concentrations (10-4 M). In contrast, p-SCN-Bn-TCMC and p-SCN-Bn-DOTA demonstrated significantly higher 197m/gHg radiochemical yields (100% ± 0.0% and 70.9% ± 1.1%, respectively) under the same conditions. The [197 m/gHg]Hg-p-SCN-Bn-TCMC complex was kinetically inert when challenged against human serum and glutathione. To understand the differences in labeling between the commercial chelators and their bifunctional counterparts, non-radioactive natHg2+ complexes were assessed using NMR spectroscopy and DFT calculations. The NMR spectra of Hg-TCMC and Hg-p-SCN-Bn-TCMC suggested binding of the Hg2+ ion through the cyclen backbone framework. DFT studies indicated that binding of the Hg2+ ion within the backbone forms a thermodynamically stable product. However, competition can form between isothiocyanate binding and binding through the macrocycle, which was experimentally observed. The isothiocyanate bound coordination product was dominant at the radiochemical scale as, in comparison, the macrocycle bound product was seen at the NMR scale, agreeing with the DFT result. Furthermore, a bioconjugate of TCMC (TCMC-PSMA) targeting prostate-specific membrane antigen was synthesized and radiolabeled, resulting in an apparent molar activity of 0.089 MBq/nmol. However, the complex demonstrated significant degradation over 24 h when exposed to human serum and glutathione. Subsequently, cell binding assays were conducted, revealing a Ki value ranging from 19.0 to 19.6 nM. This research provides crucial insight into the effectiveness of current commercial chelators in the context of 197m/gHg2+ radiolabeling. It underscores the necessity for the development of specific and customized chelators to these unique "soft" radiometals to advance 197m/gHg2+ radiopharmaceuticals.

Evaluation of a novel hexadentate 1,2-hydroxypyridinone-based acyclic chelate, HOPO-O6-C4, for 43Sc/47Sc, 68Ga, and 45Ti radiopharmaceuticals.

INTRODUCTION: Chelators play a crucial role in the development of metal-based radiopharmaceuticals, and with the continued interest in 68Ga and increasing availability of new radiometals such as 43Sc/47Sc and 45Ti, there is a growing demand for tailored chelators that can form stable complexes with these metals. This work reports the synthesis and characterization of a hexadentate tris-1,2-hydroxypyridonone chelator HOPO-O6-C4 and its in vitro and in vivo evaluation with the above mentioned radiometals. METHODS: To investigate the affinity of HOPO-O6-C4, macroscopic studies were performed with Sc3+, and Ga3+ followed by DFT structural optimization of the Sc3+, Ga3+ and Ti4+ complexes. Further tracer studies with 43Sc (and 47Sc), 45Ti, and 68Ga were performed to determine the potential for positron emission tomography (PET) imaging with these complexes. In vitro stability studies followed by in vivo imaging and biodistribution studies were performed to understand the kinetic stability of the resultant radiometal-complexes of HOPO-O6-C4. RESULTS: Promising radiolabeling results with HOPO-O6-C4 were obtained with 43Sc, 47Sc, 45Ti, and 68Ga radionuclides; rapid radiolabeling was observed at 37 °C and pH 7 in under 30-min. Apparent molar activity measurements were performed for radiolabeling of HOPO-O6-C4 with 43Sc (4.9 ± 0.26 GBq/µmol), 47Sc (1.58 ± 0.01 GBq/µmol), 45Ti (11.5 ± 1.6 GBq/µmol) and 68Ga (5.74 ± 0.7 GBq/µmol), respectively. Preclinical in vivo imaging studies resulted in promising results with [68Ga]Ga-HOPO-O6-C4 indicating a rapid clearance through hepatic excretion route and no decomplexation whereas [43Sc]Sc-HOPO-O6-C4, [47Sc]Sc-HOPO-O6-C4 and [45Ti]Ti-HOPO-O6-C4 showed modest and significant evidence of decomplexation, respectively. CONCLUSIONS: The tris-1,2-HOPO chelator HOPO-O6-C4 is a promising scaffold for elaboration into a 68Ga- based radiopharmaceutical.

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.

Rollover Cyclometalation vs Nitrogen Coordination in Tetrapyridyl Anticancer Gold(III) Complexes: Effect on Protein Interaction and Toxicity.

In this work, a pair of gold(III) complexes derived from the analogous tetrapyridyl ligands H2biqbpy1 and H2biqbpy2 was prepared: the rollover, bis-cyclometalated [Au(biqbpy1)Cl ([1]Cl) and its isomer [Au(biqbpy2)Cl ([2]Cl). In [1]+, two pyridyl rings coordinate to the metal via a Au-C bond (C∧N∧N∧C coordination) and the two noncoordinated amine bridges of the ligand remain protonated, while in [2]+ all four pyridyl rings of the ligand coordinate to the metal via a Au-N bond (N∧N∧N∧N coordination), but both amine bridges are deprotonated. As a result, both complexes are monocationic, which allowed comparison of the sole effect of cyclometalation on the chemistry, protein interaction, and anticancer properties of the gold(III) compounds. Due to their identical monocationic charge and similar molecular shape, both complexes [1]Cl and [2]Cl displaced reference radioligand [3H]dofetilide equally well from cell membranes expressing the Kv11.1 (hERG) potassium channel, and more so than the tetrapyridyl ligands H2biqbpy1 and H2biqbpy2. By contrast, cyclometalation rendered [1]Cl coordinatively stable in the presence of biological thiols, while [2]Cl was reduced by a millimolar concentration of glutathione into metastable Au(I) species releasing the free ligand H2biqbpy2 and TrxR-inhibiting Au+ ions. The redox stability of [1]Cl dramatically decreased its thioredoxin reductase (TrxR) inhibition properties, compared to [2]Cl. On the other hand, unlike [2]Cl, [1]Cl aggregated into nanoparticles in FCS-containing medium, which resulted in much more efficient gold cellular uptake. [1]Cl had much more selective anticancer properties than [2]Cl and cisplatin, as it was almost 10 times more cytotoxic to human cancer cells (A549, A431, A375, and MCF7) than to noncancerous cells (MRC5). Mechanistic studies highlight the strikingly different mode of action of the two compounds: while for [1]Cl high gold cellular uptake, nuclear DNA damage, and interaction with hERG may contribute to cell killing, for [2]Cl extracellular reduction released TrxR-inhibiting Au+ ions that were taken up in minute amounts in the cytosol, and a toxic tetrapyridyl ligand also capable of binding to hERG. These results demonstrate that bis-cyclometalation is an appealing method to improve the redox stability of Au(III) compounds and to develop gold-based cytotoxic compounds that do not rely on TrxR inhibition to kill cancer cells.