Development of zirconium-89 chelators for use in positron emission tomography imaging

Positron emission tomography (PET) is used for non-invasive high sensitivity molecular imaging to diagnose diseases, to follow biological processes or to monitor treatment response. Immuno-PET is an imaging technique used to improve the visualisation of a target site by combining the high sensitivity of PET with the specificity of monoclonal antibodies (mAbs). Due to the relatively slow pharmacokinetics of antibodies, the half-life of the radioisotope and the stability of radioisotope attachment are key factors in the development of immuno-PET imaging agents. Zirconium-89 is an excellent candidate to be the immuno-PET radionuclide of choice as it decays with a half-life of 78.41 hours.DFO (acyclic structure) is the most common chelator used to bind to [⁸⁹Zr]Zr(IV) for imaging applications. However, it has been reported that the [⁸⁹Zr]ZrDFO-mAb conjugate showed decomplexation and significant uptake of radioactivity in bones. The bone uptake of free [⁸⁹Zr]Zr(IV) results in increased radiation dose to the bone marrow and a decrease in target-to-background ratio. Therefore, the development of a more suitable chelator for [⁸⁹Zr]Zr(IV) is of high interest to biomedical researchers.The tetraazamacrocycle cyclen was functionalised with three different pendant arms; phosphonic acid, phosphinic acid and picolinic acid, to offer at least eight binding sites for Zr(IV) to form complexes with the metal ion either “in cavity” or “out of cavity”. A preliminary study of [⁸⁹Zr]Zr(IV) radiolabelling of compounds 1, 5 and 9 was performed in acetate buffer at pH 7, with [⁸⁹Zr]zirconium(IV) oxalate incubated at 95oC for 2 hours to obtain 5.0%, 6.1% and 8.4% labelling yields respectively. Labelling with [⁸⁹Zr]zirconium(IV) chloride was performed either in water or acetate buffer to form [⁸⁹Zr]Zr9 in a 49.3% labelling yield when using 0.5 MBq of [⁸⁹Zr]Zr(IV) in 0.2 M acetate buffer at pH 6 incubated at 95oC for 2 hours but achieving this yield required a higher concentration of the chelator. Extended picolinic acid arm chelators were synthesised with both cyclen and cyclam backbones. Compounds 16 and 20 were synthesised and labelled with [⁸⁹Zr]zirconium(IV) chloride. Unexpectedly [⁸⁹Zr]Zr9 gives higher radiolabelling yields than [⁸⁹Zr]Zr16 and [⁸⁹Zr]Zr20. It was possible that 16 and 20 formed lower stability complexes with Zr(IV) with a higher chelator to metal ratio than the anticipated 1:1 ratio, and so the chelators are not octadentate.Novel macrocycle-linked DFO chelators were designed as the linear chelator DFO rapidly forms a complex with Zr(IV) at ambient temperature and then the metal could transfer to the cyclic ring giving a higher stability thermodynamic product (the “in-cavity” complex). Compounds 21, 22 and 23 were synthesized and labelled with [⁸⁹Zr]Zr(IV). The labelled compounds were stable in 100-fold excess EDTA and fetal bovine serum but did not show any clear improvement over DFO

Development of Chelators for Enhancing Radiometal-based Radiopharmaceuticals

Zirconium-89 is one of the few radionuclides among positron emitters which have a relatively long physical half-life compatible with the biological half-life of most antibodies. Zirconium-89’s low positron energy, well studied and established production and purification protocols, cost effective and wide availability illustrates it as the best currently available radionuclide for immuno-positron emission tomography (immuno-PET). To employ zirconium-89 in monoclonal antibody-based radiotracers a bifunctional chelator is required to attach the radiometal securely to the antibody and assure site specific delivery of the radiometal. Desferrioxamine (DFO) has been used as the “gold standard” chelator for zirconium-89 labeled monoclonal antibodies preclinically and clinically. However, the hexadentate nature of DFO is insufficient to saturate the coordination sphere of [89Zr]Zr4+, thus in vivo sub-optimal stability of it has been reported in various studies. The released oxophilic and osteophilic zirconium (IV) ion accumulates in healthy tissue particularly in bone. Furthermore, DFO, apart from binding to diagnostic radiometal like zirconium-89, it is not known to stably bind to any therapeutic radiometals which means it has little theranostic potential. Thus, the development of optimized chelators for zirconium-89 is ongoing and demands for new bifunctional chelators which can stably bind the radiometal and conjugate to targeting vectors such as antibodies has grown considerably in the last decade. This thesis describes the design, synthesis and evaluation of novel high denticity (8-12) desferrioxamine based ligands/chelators for zirconium-89 and potentially other high valent therapeutic radiometals. Following the synthesis and characterization of the new chelators with standard chemical characterization methods, the radiolabeling properties and in vitro/in vivo stability of unconjugated “bare” chelators and antibody conjugates were evaluated by using various radiochemical assays and animal studies. Two families of chelators are the highlights of this thesis: the DFO2 family with 12 possible coordination sites, and an amino acid-based chelator family with 8-coordination sites. Each of the families consist of two chelators, for which their stabilities with zirconium-89 have been found to surpass the “gold standard” chelator DFO, and their higher denticities compared to DFO might offer the potential for utilizing other high valent and oxophilic radiometal ions such as thorium-227 or actinium-225 for theranostic applications

DESIGN AND SYNTHESIS TOWARDS BIFUNCTIONAL 89ZR AND 44SC CHELATORS FOR IMMUNO-POSITRON EMISSION TOMOGRAPHY

The design and synthesis of novel zirconium and scandium complexes, ZrIV(p-NO2-sal-DFO) and ScIII(p-NO2-Bn-NOTPi) respectively, and their potential for use in immuno-positron emission tomography (immuno-PET) are discussed. The design for a linear octadentate ligand for Zr was hypothesized and derived from deferoxamine B (DFO), a well-known hexadentate siderophore for Fe complexation. A p-NO2-salicyclic acid derivative was prepared to provide a bifunctional handle for antibody conjugation. Deferoxamine B chelates with hydroxamate moieties, which have proven useful for chelation of hard acids. While difficulties in purification led to no complexation studies being performed, this chelator was confirmed via 1H NMR , 13C NMR, and time-of-flight electron ionization mass spectrometry (TOF ESI-MS). A cyclic hexadentate ligand for Sc derived from 1,4,7-triazacyclononane (TACN) was chosen because it can be readily derived. The design and viability for this ligand has been worked on computationally using a model cyclic, octadentate chelator derived from 1,4,7,10-tetraazacyclododecane (cyclen). This work led to design and undertaking of a multistep synthesis to the novel complex, ScIII(p-NO2-Bn-NOTPi), which incorporates phosphinate pendant arms and a para-nitrobenzyl group for bioconjugation. While the synthesis of p-NO2-Bn-NOTPi has not been completed, intermediates have been analyzed via 1H NMR, 13C NMR

Alternative Chelator for 89Zr Radiopharmaceuticals: Radiolabeling and Evaluation of 3,4,3-(LI-1,2-HOPO)

Zirconium-89 is an effective radionuclide for antibody-based positron emission tomography (PET) imaging because its physical half-life (78.41 h) matches the biological half-life of IgG antibodies. Desferrioxamine (DFO) is currently the preferred chelator for 89Zr4+; however, accumulation of 89Zr in the bones of mice suggests that 89Zr4+ is released from DFO in vivo. An improved chelator for 89Zr4+ could eliminate the release of osteophilic 89Zr4+ and lead to a safer PET tracer with reduced background radiation dose. Herein, we present an octadentate chelator 3,4,3-(LI-1,2-HOPO) (or HOPO) as a potentially superior alternative to DFO. The HOPO ligand formed a 1:1 Zr-HOPO complex that was evaluated experimentally and theoretically. The stability of 89Zr-HOPO matched or surpassed that of 89Zr-DFO in every experiment. In healthy mice, 89Zr-HOPO cleared the body rapidly with no signs of demetalation. Ultimately, HOPO has the potential to replace DFO as the chelator of choice for 89Zr-based PET imaging agents

Heptadentate chelates for 89Zr-radiolabelling of monoclonal antibodies

Herein, we report the synthesis of three new bifunctional heptadentate metal ion binding chelates derived from desferrioxamine B (DFO) linked to a tripeptide unit that comprises of a glutamic acid and two glycine residues. The three DFO derivatives were also functionalised with a photoactivatable aryl azide unit for light-triggered labelling of proteins. The chelates were obtained in 3 synthetic steps in good overall yields by using solid phase peptide synthesis (SPPS). Density Functional Theory (DFT) calculations were used to estimate thermodynamic formation constants (log β) of the corresponding Zr4+ complexes. Quantitative zirconium-89 radiolabelling (>95%) was obtained in <5 min at room temperature, and the stability of the radioconjugates toward different competitors (human serum, EDTA and Fe3+) was assessed in vitro. One-pot 89Zr-photoradiosynthesis produced [89Zr]Zr-2-onartuzumab directly from the formulated, clinical-grade sample MetMAb™, without pre-purifying the monoclonal antibody (mAb) component, with an isolated decay-corrected radiochemical yield of 36.4 ± 2.4%. PET imaging and biodistribution studies were performed in female athymic nude mice bearing subcutaneous xenografts derived from the MKN-45 human gastric cancer cell line to assess the pharmacokinetic profile and tumour binding of [89Zr]Zr-2-onartuzumab. Specific tumour uptake of [89Zr]Zr-2-onartuzumab was confirmed by using competitive inhibition (blocking) studies and bone uptake was significantly reduced compared to the parent DFO analogue

A solid phase-assisted approach for the facile synthesis of a highly water-soluble zirconium-89 chelator for radiopharmaceutical development

International audienceNuclear medicine has seen impressive growth in recent years. An important development in this field occurred through the application of new radionuclides, e.g., 89Zr (t1/2 = 78.4 h, β+ 0.396 MeV), the physical properties of which allow the use of antibodies as biological vectors for specific cancer targeting in combination with high resolution imaging by positron emission tomography (PET). The most commonly used chelator for 89Zr-based PET imaging is the hexadentate desferrioxamine (DFO) chelator. However, due to the instability of this complex, there has been a strong push towards the development of octadentate chelators. We report an ether derivative, oxoDFO*, resembling the motif of DFO with four hydroxamic acid groups for the binding of the radiometal and four ether linkages to increase the water solubility. Very importantly, the synthesis of this chelator follows a solid phase-assisted approach allowing for the development of an attractive synthetic methodology and widening the scope for the access to DFO-like chelators in highly efficient synthetic sequences

Zirconium-89: Radiochemistry and Ligand Development toward Improved PET Applications

Antibodies are attractive targeting vectors for positron emission tomography (PET) based imaging agents due to their high affinity and selectivity to a variety of biological targets. Creating such imaging agents requires a radionuclide with suitable decay characteristics. This role has been filled by zirconium-89 which has a 78.41 h half-life that ideally matches the multi-day biological half-life of IgG antibodies. However, the use of a radiometal necessitates the use of a bifunctional chelator to bind the metal and conjugate to the antibody. For 89Zr this has been the purview of various derivatives of desferrioxamine B (DFO); however, the observed uptake of radioactivity in the bones of mice is evidence of in vivo release of 89Zr4+. A more robust chelator designed specifically around the chemistry of Zr4+ could eliminate the release of the bone-seeking 89Zr4+ cation in vivo and thus make for a safer, more effective PET tracer with reduced dose to non-target tissues. The investigation of alternative ligands will be described with an emphasis on the lead compound: a hydroxypyridinone based chelator -- 3,4,3-(LI-1,2-HOPO) or HOPO. The evaluation of the chelator itself will be detailed leading to the development of a bifunctional derivative -- p-SCN-Bn-HOPO -- that can be applied within the world of PET imaging

A Semi Rigid Novel Hydroxamate AMPED-Based Ligand for 89Zr PET Imaging

In this work, we designed, developed, characterized, and investigated a new chelator and its bifunctional derivative for 89Zr labeling and PET-imaging. In a preliminary study, we synthesized two hexadentate chelators named AAZTHAS and AAZTHAG, based on the seven-membered heterocycle AMPED (6-amino-6-methylperhydro-1,4-diazepine) with the aim to increase the rigidity of the 89Zr complex by using N-methyl-N-(hydroxy)succinamide or N-methyl-N-(hydroxy)glutaramide pendant arms attached to the cyclic structure. N-methylhydroxamate groups are the donor groups chosen to efficiently coordinate 89Zr. After in vitro stability tests, we selected the chelator with longer arms, AAZTHAG, as the best complexing agent for 89Zr presenting a stability of 86.4 5.5% in human serum (HS) for at least 72 h. Small animal PET/CT static scans acquired at different time points (up to 24 h) and ex vivo organ distribution studies were then carried out in healthy nude mice (n = 3) to investigate the stability and biodistribution in vivo of this new 89Zr-based complex. High stability in vivo, with low accumulation of free 89Zr in bones and kidneys, was measured. Furthermore, an activated ester functionalized version of AAZTHAG was synthesized to allow the conjugation with biomolecules such as antibodies. The bifunctional chelator was then conjugated to the human anti-HER2 monoclonal antibody Trastuzumab (Tz) as a proof of principle test of conjugation to biologically active molecules. The final 89Zr labeled compound was characterized via radio-HPLC and SDS-PAGE followed by autoradiography, and its stability in different solutions was assessed for at least 4 days

Towards synthesis of a novel chelator for zirconium-89

The radioisotope zirconium-89 has attracted attention in recent years as a candidate for clinical use in positron emission tomography (PET), due to its favourable properties. Zirconium-89 has a long half-life of 78.4 hours, rendering it suitable for use in conjugation to monoclonal antibodies (mAB), therefore giving potential applications in personalised medicines. it also has favourable emission characteristics, such as low energy positron emissions. Furthermore, it can be produced at low cost. These factors combine to support zirconium-89 as a cheap and high-resolution radioisotope for PET imaging. The most widely used chelator of zirconium-89, desferrioxamineB (DFO), forms an insufficiently stable complex with zirconium-89 and undergoes some demetallation in vivo, leading to ablation of zirconium-89 to bone tissues. While recent efforts have focussed on developing novel chelators with more stable properties, B9Zr-DFO is the only complex which has been examined in a clinical setting, and novel zirconium chelators have required either difficult synthetic procedures or are insufficiently soluble. it is hypothesised that the ideal chelator for zirconium-89 will: bind via hydroxamate groups in an octadentate manner; be bifunctional; and be simple to synthesise. The novel chelators 8 and 10 were designed based on these criteria and to examine the feasibility of a zirconium-89 chelator incorporating a-amino acids. Synthesis of both compounds was attempted via a modular solution-phase peptide synthesis. The molecular subunits 4a and 4b were synthesised with overall yields of 21% and 48% respectively. However, during the latter stages of the synthesis intramolecular degradation of compounds 7 and 8 resulted in the formation of an alternative product: compound 8.1. Analytical evidence obtained through 1HNMR and HRMS suggested this occurred by a mechanism akin to aspartimide formation, as seen in solid-phase peptide synthesis. To examine this hypothesis, an alternative chelator 14 was designed, incorporating ~-alanine in place of glycine. This compound was successfully synthesised via solid-phase peptide synthesis and identified with HRMS and 1HNMR. As the monohydroxamate compound 8.1 was isolated as a pure product, it was of interest to assess its binding affinity for the radiometals zirconium-89 and gallium-68: The chelator was compared to DFO and subject to challenge assays to assess resistance to competitive metal ions, transchelation by EDTA, and non-specific protein binding. While it was evident that compound 8.1 was inferior to DFO as a chelator of zirconium-89 under all conditions, it did display significantly increased stability than previously reported for monohydroxamate chelators. However, both compound 8.1 and DFO displayed low stability when complexed with gallium-68.Thesis (MPhil) -- University of Adelaide, School of Physical Sciences, 202

Octadentate zirconium(IV)-loaded macrocycles with varied stoichiometry assembled from hydroxamic acid monomers using metal-templated synthesis

Published: February 28, 2017The reaction between Zr(IV) and the forward endo-hydroxamic acid monomer 4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoic acid (for-PBH) in a 1:4 stoichiometry in the presence of diphenylphosphoryl azide and triethylamine gave the octadentate Zr(IV)-loaded tetrameric hydroxamic acid macrocycle for-[Zr(DFOT₁)] ([M + H]⁺ calc 887.3, obs 887.2). In this metal-templated synthesis (MTS) approach, the coordination preferences of Zr(IV) directed the preorganization of four oxygen-rich bidentate for-PBH ligands about the metal ion prior to ring closure under peptide coupling conditions. The replacement of for-PBH with 5-[(5-aminopentyl) (hydroxy)amino]-5-oxopentanoic acid (for-PPH), which contained an additional methylene group in the dicarboxylic acid region of the monomer, gave the analogous Zr(IV)-loaded macrocycle for-[Zr(PPDFOT₁)] ([M + H]⁺ calc 943.4, obs 943.1). A second, well-resolved peak in the liquid chromatogram from the for-PPH MTS system also characterized as a species with [M + H]⁺ 943.3, and was identified as the octadentate complex between Zr(IV) and two dimeric tetradentate hydroxamic acid macrocycles for-[Zr(PPDFOT1D)₂]. Treatment of for-[Zr(PPDFOT₁)] or for-[Zr(PPDFOT1D)₂] with EDTA at pH 4.0 gave the respective hydroxamic acid macrocycles as free ligands: octadentate PPDFOT₁ or two equivalents of tetradentate PPDFOT1D (homobisucaberin, HBC). At pH values closer to physiological, EDTA treatment of for-[Zr(DFOT₁)], for-[Zr(PPDFOT₁)], or Zr(IV) complexes with related linear tri- or tetrameric hydroxamic acid ligands showed the macrocycles were more resistant to the release of Zr(IV), which has implications for the design of ligands optimized for the use of Zr(IV)-89 in positron emission tomography (PET) imaging of cancer.William Tieu, Tulip Lifa, Andrew Katsifis, and Rachel Cod