Novel Bifunctional Cyclic Chelator for 89Zr Labeling–Radiolabeling and Targeting Properties of RGD Conjugates

Within the last years 89Zr has attracted considerable attention as long-lived radionuclide for positron emission tomography (PET) applications. So far desferrioxamine B (DFO) has been mainly used as bifunctional chelating system. Fusarinine C (FSC), having complexing properties comparable to DFO, was expected to be an alternative with potentially higher stability due to its cyclic structure. In this study, as proof of principle, various FSC-RGD conjugates targeting αvß3 integrins were synthesized using different conjugation strategies and labeled with 89Zr. In vitro stability, biodistribution, and microPET/CT imaging were evaluated using [89Zr]FSC-RGD conjugates or [89Zr]triacetylfusarinine C (TAFC). Quantitative 89Zr labeling was achieved within 90 min at room temperature. The distribution coefficients of the different radioligands indicate hydrophilic character. Compared to [89Zr]DFO, [89Zr]FSC derivatives showed excellent in vitro stability and resistance against transchelation in phosphate buffered saline (PBS), ethylenediaminetetraacetic acid solution (EDTA), and human serum for up to 7 days. Cell binding studies and biodistribution as well as microPET/CT imaging experiments showed efficient receptor-specific targeting of [89Zr]FSC-RGD conjugates. No bone uptake was observed analyzing PET images indicating high in vivo stability. These findings indicate that FSC is a highly promising chelator for the development of 89Zr-based PET imaging agents.


■ INTRODUCTION
Over the last years the positron-emitter 89 Zr has attracted considerable interest for molecular imaging applications using positron emission tomography (PET). In comparison to the commonly used radionuclides for PET ( 18 F, 68 Ga, 11 C, etc.), 89 Zr with its relatively long half-life of 78.4 h is particularly suited for in vivo imaging at late time-points. With a mean positron energy of 0.395 MeV, which is between the positron energies of 18 F (0.250 MeV) and 68 Ga (0.836 MeV), it allows for high resolution PET images for small animal imaging applications. 1 For imaging applications at late time points 89 Zr has to be stably bound to a chelator to minimize dissociation in vivo, as free 89 Zr can accumulate in the bone or associate with plasma proteins. Over the years, several chelators, such as diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), and 1,4,7,10-tetraazacyclododecane N,N′,N″,N‴tetraacetic acid (DOTA), have been evaluated with limited success. 2 Today desferrioxamine B (DFO) is the most prominent chelator for 89 Zr labeled biomolecules ( Figure 1).
[ 89 Zr]DFO mAb-conjugates have been used in a number of clinical trials for in vivo tracking and quantification of monoclonal antibodies (mAbs) by PET with good imaging quality. However, there are concerns about the stability of the complexes in vivo not only in relation to image quality but in particular to radiation dose. Several preclinical studies reported bone accumulation of dissociated 89 Zr ranging from 3 to 15% after 3 to 7 days. 3−8 Alternative chelators for 89 Zr 4+ that could eliminate the release of osteophilic 89 Zr 4+ and lead to safer PET procedures with reduced radiation dose and optimal image quality are required for the development of novel targeted PETtracers.
Several studies have focused on the conjugation moiety 6,9,10 and recently also on the ligand itself. 11−15 Considering that DFO is a linear ligand and exposed to endogenous competitive cations and natural chelators in vivo that may challenge the stability of the metal chelate, macrocyclic structures may favor a higher kinetic inertness of their metallic complexes. 16 Recently we demonstrated that fusarinine C (FSC), a representative of the class of hydroxamate siderophores, is a promising bifunctional chelator for 68 Ga-radiolabeling ( Figure  1). 17,18 FSC has three primary amines, which can be derivatized in a number of ways also applying the concept of multivalency. This is the combination of several targeting units in one single molecule. By attaching a cyclic RGD peptide, binding to α v β 3 integrins that are expressed during angiogenesis, via a succinic acid linker, stable [ 68 Ga]FSC(succ-RGD) 3 with excellent receptor binding properties and in vivo targeting was prepared.
[ 68 Ga]FSC(succ-RGD) 3 showed superiority to monomeric cyclic RGD peptides, such as [ 18 F]Galacto-RGD and [ 68 Ga]-NODAGA-RGD. FSC has a similar structure like DFO with three hydroxamate groups providing six oxygen donors for metal binding. We postulated that FSC could be an alternative for 89 Zr labeling with complex formation properties comparable to DFO but potentially higher stability due to its cyclic structure. Thus, increased image contrasts at delayed time points are possible. In an initial study we could demonstrate that cyclic siderophores can be labeled with 89 Zr displaying comparable properties to its 68 Ga counterparts. 19 In this study triacetylfusarinine C (TAFC), the triacetylated form of FSC, was investigated, and the labeling procedure and in vitro stability were compared with [ 89 Zr]DFO. Additionally, the feasibility of different FSC conjugation strategies, where the RGD peptides have been bound either directly or through a linker, has been evaluated. Moreover, radiolabeling properties, stability, and in vivo behavior of [ 89 Zr]FSC(succ-RGD) 3 in comparison to its 68 Ga labeled counterpart were compared. The study also included microPET/CT imaging in mice bearing α v β 3 integrin expressing tumor xenografts. ■ EXPERIMENTAL SECTION General. All substances described were of reagent grade and were used without further purification. Human melanoma M21 and M21-L cells were a kind gift from D. A. Cheresh, Departments of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, CA, USA. 89 Zr was purchased from PerkinElmer, Inc. (USA). Analytical reversedphase high performance liquid chromatography (RP-HPLC) analysis was performed using a Vydac 218 TP5215, 150 × 3.0 mm column (SRD, Vienna, Austria) at a flow rate of 1.0 mL/ min with the following acetonitrile (CH 3 CN)/H 2 O/0.1% trifluoroacetic acid (TFA) gradients: 0−0.5 min 0% CH 3 CN, 0.5−7.0 min 0−55% CH 3 CN. Radiolabeling efficiency and radiochemical purity were determined by instant thin-layer chromatography (ITLC) on ITLC silica gel strips (Agilent Technologies) with 50 mM EDTA (pH 7, adjust pH using 10 M NaOH) as mobile phase. The strips were scanned using a mini-scan radio TLC scanner with flow-count detector (LabLogic, Sheffield, UK).
[Fe]FSC (10 mg, 12.8 μmol) and an excess of 5 equiv of succ-c(RGDfK) (Pbf, OtBu) (64.9 mg, 64.2 μmol) or c(RGDfE) (Pbf, OtBu) (58.5 mg, 64.2 μmol) were dissolved in 2 mL of DMF. After addition of 24.4 mg of HATU (64.2 μmol) and 8.7 mg of HOAt (64.2 μmol) and adjustment of the pH to 9 using DIPEA, the reaction mixture was stirred for 72 h at room temperature (RT). Hereafter, the volume of the solvent was reduced in vacuo, and the conjugate was precipitated using water. The crude [Fe]FSC(succ-RGD) 3 (Pbf, OtBu)) and [Fe]FSC(RGDfE) 3 (Pbf, OtBu) were dissolved in 2 mL of a solution composed of TFA/H 2 O/triisopropylsilan in a ratio of 38:1:1. The reaction mixture was allowed to react for 1 h at RT. Subsequently, the solvent was reduced, and the crude product was obtained by precipitation using diethyl ether. Finally, the product was dried and purified via preparative RP-HPLC.
[Fe]FSC(RGDfE) 3  SAT(PEG) 4 -RGD. c(RGDfK) (107 mg, 0.13 mmol) in 2 mL of DMF was mixed with 855 μL of N-succidimidyl-Sacetyl(thiotetraethylene glycol) (SAT(PEG) 4 ) stock solution in dimethyl sulfoxide (DMSO; 0.16 mmol, 1.2 equiv). The reaction mixture was alkalized to a pH of 9 using DIPEA and stirred for 2 h at RT. Subsequently, the solvent was reduced, and the conjugate was precipitated by addition of diethyl ether. Then, SAT-(PEG) 4 -RGD was washed thrice with diethyl ether and was dried in vacuo to give the product as yellow oil with honey-like consistency. Yield 122 mg (0.13 mmol).
HS-(PEG) 4 -RGD. SAT-(PEG) 4 -RGD (12 mg, 55 μmol) was dissolved in 0.5 mL of phosphate buffered saline (PBS, pH 7.4), and 2 mL of 0.5 M hydroxylamine solution was added. The pH was adjusted to 6.0. After the mixture was stirred for 2 h at RT, HS-(PEG) 4   The pH of the reaction vial was measured to be sure that it was between 6.8 and 7.2. The solution was allowed to react at RT for 90 min. The reaction was monitored via ITLC ( 89 Zrcomplexes remained at the origin and free 89 Zr migrates with the solvent front, see Supporting Information, Figure S1) and confirmed by HPLC. For animal experiments, after radiolabeling 60 μL of CaCl 2 (0.5 M) was added to the reaction vial, which resulted in a precipitate of Ca-oxalate. The solution was passed through a 0.2 μm filter to remove Ca-oxalate. The filtrate was diluted to an appropriate volume using saline for further evaluation.   3 (approximately 1.5 × 10 6 cpm), the cells were incubated in triplicates with either PBS with 0.5% BSA (150 μL, total series) or with 10 μM c(RGDyV) in PBS/0.5% BSA (150 μL, nonspecific series) at 37°C for 90 min. After the incubation time the tubes were centrifuged, and incubation was stopped by removal of the medium. Then the cells were washed twice with ice-cold TRIS-buffered saline. Subsequently, the cells were incubated two times in acid wash buffer (20 mM acetate buffer, pH 4.5) at 37°C for 5 min. Then the tubes were centrifuged, and the supernatant was collected in plastic vials (membrane bound activity). In a last step the cells were lysed by addition of 2 M NaOH, and the radioactivity associated with cells was collected in plastic vials (internalized radioligand fraction). Protein content in the NaOH fraction was determined using a spectrometric method (Bradford assay). The internalized activity was calculated and expressed as percentage of total activity per milligram protein.  3 female, athymic BALB/c nude mice (Charles River Laboratories) were used. For the induction of tumor xenografts mice were injected subcutaneously with 5 × 10 6 α v β 3 integrin positive M21 cells into the right hind limb and with 5 × 10 6 α v β 3 integrin negative M21-L cells (negative control) into the left hind limb of the same mouse (n = 4). The tumors were allowed to grow until they had reached a volume of 0.3 to 0.6 cm 3 . On the day of the experiment [ 89 Zr]FSC-(succ-RGD) 3 (∼0.5 MBq/mouse, ∼0.5 μg of peptide) was intravenously injected in the lateral tail vein. Mice were sacrificed by cervical dislocation at 1, 2, and 4 h after injection. Organs (spleen, pancreas, stomach, intestine, kidney, liver, heart, and lung), blood, muscle tissue, bone, and tumors were dissected and weighted. As a comparison, four tumor-bearing mice were intravenously injected in the tail vein with

Molecular Pharmaceutics
Article which was used to protect the complexing moiety, from [Fe]FSC-RGD conjugates was carried out using 40-fold molar excess of EDTA solution at pH 4 within 25 to 60 min with high yield (>90%). The rapid disappearance of the intense red color of the solution indicated the successful removal of [Fe]. After HPLC purification the chemical purity of the final compounds was >95%. 89 Zr Radiolabeling. Labeling of DFO, TAFC, and the FSC-RGD conjugates with 89 Zr-oxalate was performed with slight modifications as described recently. 22 Radiolabeling was carried out in HEPES buffer at RT at pH 6.8 to 7.2. FSC could acquire 89 Zr quantitatively from 89 Zr-oxalate at RT with incubation times between 30 and 90 min depending on the concentration of precursor. Moreover, 1 μg of TAFC could be quantitatively labeled with 30 MBq of 89 Zr-oxalate leading to a specific activity of 25 GBq/μmol (without optimization). The time course of 89 Zr-complexation of FSC(succ-RGD) 3 (30 μg, 58 μM) is shown in Figure 2. Oxalate, which could be toxic due to the production of solid calcium oxalate causing kidney failure, was readily removed by preprecipitation with CaCl 2 and filtration using a 0.2 μm filter for in vivo experiments.
In  3 of total activity per milligram protein (% cpm/mg). The corresponding activities were reduced to 0.2 to 0.3% cpm/mg of the reference activity via addition of excess of c(RGDyV), which demonstrated receptor-specific binding.
A microPET/CT imaging study with tumor-bearing nude mice was performed in order to investigate the in vivo pharmacokinetics of [ 89 Zr]FSC(succ-RGD) 3 ( Figure 5). Again images confirmed receptor-specific activity accumulation

Molecular Pharmaceutics
Article in the α v β 3 integrin positive M21 tumors, and more importantly, no bone uptake was observed, confirming high in vivo stability of the compound. The M21 tumors could be clearly visualized over the whole monitoring period of 24 h, whereas the α v β 3 integrin negative M21-L tumors showed no uptake confirming the high specificity of [ 89 Zr]FSC(succ-RGD) 3 . Besides the M21 tumors, only kidneys were visible, which is related to excretion and kidney retention.

■ DISCUSSION
The positron-emitting radionuclide 89 Zr is increasingly used for molecular imaging with PET; its clinical application is currently limited to conjugates using DFO as chelator. Even though several antibodies conjugated to DFO and radiolabeled with 89 Zr achieved some successful results, dissociation of 89 Zr in vivo has been observed indicating limited in vivo stability. Therefore, increasing efforts are made to design and synthesize novel robust and stable chelators for 89 Zr. The design of an ideal chelator is affected by many factors, such as the type and number of coordinated atoms, acyclic vs cyclic constructs, ring size, etc. Even the charge of the metal-complex may influence the biodistribution of a corresponding tracer labeled with a radiometal.
Based on the knowledge on structure, synthesis, and labeling of DFO-conjugates, the natural siderophore FSC was chosen as the starting point for our research. FSC is produced by fungi for acquiring iron from the environment and has been proven to be an excellent bifunctional chelator for 68 Ga in our laboratory. 17 Therefore, FSC, having three hydroxamic acid moieties similar to those of DFO, embedded in a cyclic structure, was expected to be an interesting alternative for 89 Zr labeling. In our study, TAFC was initially chosen to explore the labeling procedure and to evaluate the in vitro stability. It was labeled with 89 Zr-    Zr-oxalate at pH 6.8−7.2 and reaction times between 1 to 2 h, 11 quantitatively form complexes at acidic pH (1−5) within 5 min (data not shown). This indicates that the chloride form of 89 Zr could be the better choice for labeling reactions. Moreover, it would avoid the use of highly concentrated solutions of oxalate, which is potentially toxic.
One of the main attractions of FSC-based constructs for 89 Zr radiolabeling lies in its excellent stability in challenging solution.
[ 89 Zr]TAFC remained stable toward challenge with 1000-fold molar excess of EDTA and only limited transchelation was found after days of incubation with 1000-fold molar excess of DFO. In comparison [ 89 Zr]DFO revealed rapid transchelation toward EDTA even at pH 7 and complete transmetalation toward TAFC (both 1000-fold molar excess) within hours. This high stability of the [ 89 Zr]FSC structure was further confirmed by the studies with the [ 89 Zr]FSC-RGD conjugates. Additionally, as the pH in tumors (approximately pH 6.5) is lower than in healthy tissue, the observed high stability of [ 89 Zr]TAFC in slightly acidic conditions (pH 6) may be beneficial in particular for cancer imaging.
Inspired by the promising results found for [ 89 Zr]TAFC, the potential use of FSC conjugated to targeting vectors was investigated. For several reasons cyclic RGD pentapeptides were chosen as model vectors. First, RGD-peptides are very stable in vitro and in vivo and, more important, tolerate a variety of modifications at position 5 in the amino acid sequence without losing binding affinity. 24 Second, rapid predominantly renal excretion with low background activity is described for a big variety of different tracer based on this pentapeptide allowing an easier monitoring of the distribution of fragments based on rapid degradation in the body and especially in the bone. 25 Third, it is a well-known class of tracer where a great set of data is available. 26,27 Fourth, it is known that binding affinity and imaging properties of this tracer class benefit from the so-called multimerization approach. 28 Due to the fact that FSC includes three primary amine functions, this chelator allows conjugation of up to three ligands making them, besides working as chelator for the radiometal, an ideal scaffold for such approaches. Thus, this option additionally increases potential applications of FSC and derivatives. Anyway, it has to be pointed out that only small peptides may benefit from such a multimerization approach and not antibodies, which is the main field of application of 89 Zr-labeling. Nevertheless, it has been demonstrated by using, e.g., 64 Cu-NODAGA-RGD that tumorto-background ratios further improve with prolonged imaging time making labeling of this peptide class with 89 Zr not per se uninteresting. 29 As a proof of concept, different cyclic RGD pentapeptides were synthesized, and versatile conjugation strategies were explored. FSC(succ-RGD) 3 was synthesized using a succinic acid linker by amidation with a yield of 40%. In another approach c(RGDfE) was directly conjugated via the glutamic acid carboxylate to the chelator. This approach reduces the synthesis steps but also resulted in a lower yield possibly related to steric effects. In a third approach the maleimide−thiol strategy was introduced. This is a well-established technique and allows fast and mild reaction conditions in aqueous solution, facilitating the site specific conjugation of peptides and antibodies, which might be not stable under harsh conditions. Thus, the PEG-modified RGD-peptide could be coupled with even higher yield as found for the other two derivatives under mild conditions with the FSC derivative.
Biological properties of [ 89 Zr]FSC(succ-RGD) 3 were comparable to the properties of [ 68 Ga]FSC(succ-RGD) 3 , which has clearly shown the advantage of multivalency through remarkable improvement of internalized activity in vitro and tumor uptake in vivo in our previous study. 17 An exception was the lower activity concentration in blood resulting in better tumor/blood ratios for the 89 Zr compound in comparison with the 68 Ga-labeled analogue, indicating an excellent in vivo stability of [ 89 Zr]FSC(succ-RGD) 3 . The high stability was confirmed by microPET/CT imaging using the same murine tumor model. These studies clearly demonstrated no uptake in bone and high uptake in α v ß 3 integrin positive M21 tumors. A reason for the superior properties of [ 89 Zr]FSC(succ-RGD) 3 compared with [ 68 Ga]FSC(succ-RGD) 3 could partially be to the difference in the charge, having been reported to potentially influence excretion patterns of such biomolecules. 30 The preferred coordination number for 89 Zr is eight, which was confirmed recently. 31 Certain developments aim on the design and synthesis of novel both octadentate and oxygen-rich Zr 4+ chelates to achieve superior stabilities to DFO. Both Deri et al. and Patra et al. reported two kinds of linear octadentate chelators named HOPO and DFO*. 11,12 Gueŕard et al. also described a cyclic octadentate tetrahydroxamic acid chelator (C7). 13 In vitro studies of all three octadentate ligands showed improved stability. However, HOPO and C7 lack the pendant group for coupling it to a targeting molecule. DFO* was linked to the bombesin peptide analogue [Nle 14 ]BBS (7−14), but was only investigated in vitro. [ 89 Zr]TAFC and C7, both have a 36membered ring but different coordination numbers for 89 Zr. Nevertheless, transchelation found in an EDTA challenging experiment was lower for TAFC (3−5% TAFC vs 13% C7 after 7 days) despite its lower coordination number (6 vs 8).
Although further research is warranted, our study shows that the use of macrocyclic structure leads to considerably improved

Molecular Pharmaceutics
Article in vitro stability compared to DFO. A limitation of the study is that DFO cannot be used to generate a trimeric structure. Thus, in vivo comparison of both chelating systems is difficult because not only the chelator system would change but also the number of targeting moieties. Based on these changes the in vivo properties are not only correlated to the different chelators but also are influenced by the different structures. Therefore, it was decided for this proof of principle study to restrict comparison to in vitro assays where it is assumed that these differences have only minor effects. Anyway, to our knowledge this is the first report where a 89 Zr-labeled macrocyclic bifunctional chelating system conjugated to a target vector is studied in vivo including biodistribution and PET imaging. Both, the in vitro and in vivo studies of [ 89 Zr]TAFC and [ 89 Zr]FSC(succ-RGD) 3 demonstrated very promising results. However, small peptides, even though an excellent model for this evaluation based on their rapid pharmacokinetics, are certainly not the most suitable application for this concept. Nevertheless, for larger targeting vectors (such as antibodies or antibody fragments), due to their slower pharmacokinetics, FSC system could be a very interesting alternative for 89 Zr-labeling to currently used bifunctional chelators. At the moment, mono-and diacetylated FSC are under investigation, which may, due to the reduced free valences, better suit the application with higher molecular weight targeting vector systems.

■ CONCLUSION
Versatile conjugation strategies of FSC were explored using RGD as model peptide and quantitative labeling with 89 Zr was achieved successfully. Excellent in vitro and in vivo stability of [ 89 Zr]TAFC and [ 89 Zr]FSC-RGD conjugates were demonstrated. These results led us to conclude that FSC is a novel promising chelator for the development of 89 Zr-based PET imaging agents.
■ ASSOCIATED CONTENT * S Supporting Information Figure S1 showing the representative chromatograms of ITLC of 89