Design, Synthesis, and Evaluation of DFO-Em: A Modular Chelator with Octadentate Chelation for Optimal Zirconium-89 Radiochemistry

Abstract

Zirconium-89 has quickly become a favorite radionuclide among academics and clinicians for nuclear imaging. This radiometal has a relatively long half-life, which matches the biological half-life of most antibodies, suitable decay properties for positron emission tomography (PET), and efficient and affordable cyclotron production and purification. The "gold standard" chelator for [ 89 Zr]Zr 4+ is desferrioxamine B (DFO), and although it has been used both preclinically and clinically for immunoPET with great success, it has revealed its suboptimal stability in vivo. DFO can only bind to [ 89 Zr]Zr 4+ through its six available coordination sites made up by three hydroxamic acid (HA) moieties, which is not sufficient to saturate the coordination sphere . In this study, we have designed, synthesized, and characterized a new octadentate chelator we have called DFO-Em, which is an improved derivative of our previously published dodecadentate chelator DFO2. This octadentate DFO-Em chelator is smaller than DFO2 but still satisfies the coordination sphere of zirconium-89 and forms a highly stable radiometal−chelator complex. DFO-Em was synthesized by tethering a hydroxamic acid monomer to commercially available DFO using glutamic acid as a linker, providing an octadentate chelator built on a modular amino acid-based synthesis platform. Radiolabeling performance and radiochemical stability of DFO-Em were assessed in vitro by serum stability, ethylenediamine tetraacetic acid (EDTA), and hydroxyapatite challenges. Furthermore, [ 89 Zr]Zr-(DFO-Em) and [ 89 Zr]Zr-DFO were injected in healthy mice and measured in vivo by PET/CT imaging and ex vivo biodistribution. Additionally, the coordination of DFO-Em with Zr(IV) and its isomers was studied using density functional theory (DFT) calculations. The radiolabeling studies revealed that DFO-Em has a comparable radiolabeling profile to the gold standard chelator DFO. The in vitro stability evaluation showed that [ 89 Zr]Zr-(DFO-Em) was significantly more stable than [ 89 Zr]Zr-DFO, and in vivo both had similar clearance in healthy mice with a small decrease in tissue retention for [ 89 Zr]Zr-(DFO-Em) at 24 h post injection. The DFT calculations also confirmed that Zr-(DFO-Em) can adopt highly stable 8-coordinate geometries, which along with NMR characterization suggest no fluxional behavior and the presence of a single isomer. The modular design of DFO-Em means that any natural or unnatural amino acid can be utilized as a linker to gain access to different chemistries (e.g., thiol, amine, carboxylic acid, azide) while retaining an identical coordination sphere to DFO-Em.

DFO-Km: A Modular Chelator as a New Chemical Tool for the Construction of Zirconium-89-Based Radiopharmaceuticals

“…Synthetic procedures and characterization data for compounds 1−3 are detailed in our previous publication on DFO-Em. 30 4-((Benzyloxy)(methyl)amino)-4-oxobutanoic Acid (4). Compound 4 was synthesized according to a published procedure with modifications.…”

“…32,53,54 In our previous work, we reported the synthesis and evaluation of a glutamic acid-based octadentate chelator we named DFO-Em. 15,19,30,31 We attempted synthesis of the bifunctional version of DFO-Em via N-hydroxysuccinimide (NHS) ester activation of the glutamic acid side chain to enable conjugation to antibodies and other vectors. However, this method was not successful due to intramolecular cyclization resulting from reaction of hydroxamate groups with the activated ester.…”

“…Simply switching the amino acid linker in DFO-Em from glutamic acid to lysine in DFO-Km would retain an We previously performed a similar DFT evaluation of geometric isomers for Zr-(DFO-Em). 30 DFO-Km was successfully bifunctionalized by reacting the side chain primary amine (ε-amino group) of the lysine with pphenylene diisothiocyanate to form its p-isothiocyanatophenylderivative (p-SCN-Ph-DFO-Km). This new bifunctional chelator and the current clinical "gold standard" p-SCN-Ph-DFO were conjugated to a nonspecific human IgG antibody via thiourea linkage, a common strategy used for bioconjugation of chelators to antibodies.…”

Radiochemical, Computational, and Spectroscopic Evaluation of High-Denticity Desferrioxamine Derivatives DFO2 and DFO2p toward an Ideal Zirconium-89 Chelate Platform

“…Despite the apparent instability of [ 89 Zr]­Zr-DFO in mice, human immuno-PET imaging studies using zirconium-89 do not appear to experience the same level of bone uptake; however, an optimized chelation system could still have value. ,− There is a need to develop chelators with improved radiolabeling performance, greater Zr­(IV) binding affinity and kinetic inertness, and greater in vivo stability. − The high denticities and large structures of DFO2/DFO2p provide much flexibility and present metal ions with multiple options for binding denticities and geometries. To this end, several promising new [ 89 Zr]­Zr 4+ chelators have been proposed. ,,− ,,,− Many of these promising new chelators utilize the DFO scaffold. However, the denticity of DFO is not high enough to bind larger therapeutic radiometals such as actinium-225 or thorium-227, and so higher-denticity derivatives of DFO with minimum coordination numbers of 8 (e.g., DFO*) are of potential utility beyond zirconium-89. ,,− …”

Synthesis of Modular Desferrioxamine Analogues and Evaluation of Zwitterionic Derivatives for Zirconium Complexation

“…Various other Zr(IV)‐chelators have been developed to overcome the shortcomings of DFOB and to increase the complex stability [17] . In this context, hexadentate macrocyclic hydroxamates of the fusarinine and the desferrichrome family, [18] octadentate desferrichrome‐derivatives like Orn4‐hx, [18b] DFO‐HOPO, [19] DFO*, [20] DFOcyclo*, [21] DFO2, [22] DFO−Em, [23] H 3 DFOSqOEt [24] and macrocyclic octadentate analogues are notable examples [25] . However, many in vivo experiments with the corresponding targeted analogues showed comparable radioactive bone‐uptake to DFOB despite improved complex stability as measured in vitro [18b,26] .…”