Progress of Coordination and Utilization of Zirconium-89 for Positron Emission Tomography (PET) Studies

Abstract

Radiometals have been commonly used in medical applications, and utilization of such metals continues to be an attractive research area. In particular, a variety of radiometals have been developed and implemented for molecular imaging. For such applications, ⁸⁹Zr has been one of the most interesting radiometals currently used for tumor targeting. Several chemical ligands were developed as ⁸⁹Zr chelators, and new coordinating methods have also been developed more recently. In addition, immuno-positron emission tomography (PET) studies using ⁸⁹Zr-labeled monoclonal antibodies have been performed by several scientists. In this review, recent advances to the coordination of ⁸⁹Zr and the utilization of ⁸⁹Zr in PET studies are described.

Introduction

Zirconium-89 (⁸⁹Zr), with an atomic number of 40, has useful biomedical applications. This is due to its favorable decay characteristics, a half-life of 78.41 h, which make it suitable for labeling biomolecules, such as antibodies, for imaging (Fig. 1, Table 1). Nowadays, ⁸⁹Zr is considered an important positron-emitting radionuclide used for the development of novel radiopharmaceuticals for positron emission tomography (PET). In particular, ⁸⁹Zr has been widely used for immuno-PET studies due to ideal physical characteristics.

Fig. 1.

Zirconium-89 decay

Table 1.

Properties of ⁸⁹Zr

t½ (h)	Methods of production	Decay mode	Eβ+ (keV)	References
78.41	89Y(p,n)89Zr	β+ (22.7%) EC (77%)	909	[1]

Production of ⁸⁹Zr

There are several reaction pathways that produce ⁸⁹Zr, such as the ⁸⁹Y(p,n)⁸⁹Zr reaction, ⁸⁹Y(d,2n)⁸⁹Zr reaction, ^natZr(p,pxn)⁸⁹Zr reaction, ^natSr(α,xn)⁸⁹Zr reaction, and ⁹⁰Zr(n,xn)⁸⁹Zr reactions (Table 2) [5, 6, 12–14]. The first two of these reactions are common pathways to produce ⁸⁹Zr due to the availability of ⁸⁹Y from natural sources. The Zweit group utilized natural yttrium pellets to produce ⁸⁹Zr using the ⁸⁹Y(d,2n)⁸⁹Zr reaction: the starting material was irradiated with a 16–7-MeV optimum energy beam of deuterons and then purified in an ion-exchange column to obtain a 66.6-MBq/μAh yield of ⁸⁹Zr with a minor fraction of long-lived ⁸⁸Zr (0.008%). Using a similar reaction, high-purity ⁸⁹Zr production was experimentally reported by Tang and co-workers and theoretically calculated by the Sadeghi group [3, 15]. Despite the higher yield of the ⁸⁹Y(d,2n)⁸⁹Zr reaction compared to the ⁸⁹Y(p,n)⁸⁹Zr reaction, application of the ⁸⁹Y(d,2n)⁸⁹Zr reaction in medical accelerators is still restricted. This is due to the fact that common small medical cyclotrons are not capable of producing the high-energy deuterons required for the ⁸⁹Y(d,2n)⁸⁹Zr reaction. Although several medical cyclotrons, such as the GE PETtrace 800 or IBA Cyclone 18/9, have two beam currents, the deuteron energy still is not sufficient to produce a high yield of ⁸⁹Zr. Hence, the ⁸⁹Y(p,n)⁸⁹Zr reaction is the more practical approach to the production of ⁸⁹Zr in these kinds of machines.

Table 2.

Several reactions for ⁸⁹Zr production

No.	Nuclear reaction	Target	Product chemical form	Yield (MBq/μAh)	Time of irradiation	Energy (MeV)	Beam current (μA)	Thickness of target	Refs.
1	89Y(d,2n)89Zr	Pellet	Chloride	66.6 ± 5.6	12–20 min	16–7	3–5	240–340 mg cm−2	[2]
2	89Y(d,2n)89Zr	Magnetron sputtering	Chloride	58 ± 5	1 h	13	10–15	25 μm	[3]
3	89Y(p,n)89Zr	Magnetron sputtering	Chloride	44 ± 4	1 h	14	10–30	25 μm	[3]
4	89Y(p,n)89Zr	Foil	Oxalate	38.9	40 min	13	10	286 mg cm−2	[4]
5	89Y(p,n)89Zr	Thin foil	Oxalate	13	2 h	11.4–10	10	57 mg cm−2	[5]
6	89Y(p,n)89Zr	Foil	Oxalate	56.2 ± 4.1	2–5 h	15	15	100 μm	[6]
7	89Y(p,n)89Zr	Foil	Oxalate	12.5 ± 0.5	2 h	18–10	12	150 μm	[7]
8	89Y(p,n)89Zr	Foil	Oxalate	48.9 ± 4.4	1 h	12.8	45	640 μm	[8]
9	89Y(p,n)89Zr	Sputtered layer	Oxalate	48.1	1 h	14	100	25 μm	[9]
10	89Y(p,n)89Zr	Sputtered coin	Oxalate	6.4–18	30 min or 2 h	12.5 or 12.8	10–40	90–250 μm	[10]
11	89Y(p,n)89Zr	Y(NO3)3 solution (2.75 M)	Oxalate	4.36 ± 0.48	2 h	14	40	Liquid target	[11]

The first ⁸⁹Y(p,n)⁸⁹Zr reaction was carried out by Link and co-workers who employed an ⁸⁹Y source on Y foil which was irradiated with 13 MeV protons. After irradiation, the Y foil was dissolved in HCl solution, and ⁸⁹Zr(IV) was extracted via multistep extraction using 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione (TTA) and then HNO₃/HF. Purification by anion exchange with 1 M HCl/0.01 M oxalate resulted in an 80% yield of ⁸⁹Zr (99.99% purification). A similar protocol was reported by the Dejesus group using a thin Y foil [4, 5]. Based on the same starting material of a Y foil target, several studies modified parameters such as foil thickness, time of irradiation, energy, and beam current in the attempt to improve production yields [6–8]. However, the increase of beam energy over 13 MeV inevitably causes the undesirable production of long-lived ⁸⁸Zr via the ⁸⁹Y(p,2n)⁸⁸Zr reaction. Recently, the Queern group worked on the production of ⁸⁹Zr using sputtered yttrium on niobium coin. They found that a reduction of beam energy from 17.8 to 12.8 MeV or 12.5 MeV using a 0.75-mm-thick aluminum degrader yielded good results with no ⁸⁸Zr observed [10].

The use of solid targets can be limited by a lack of facilities, so liquid targets have also been utilized to produce ⁸⁹Zr. For instance, Pandey and co-workers irradiated yttrium (III) nitrate in nitric acid solution. Although their results showed a yield of only 4.4 MBq/μAh for 2 h of irradiation at a 40-μA beam current, which is barely adequate for a solid target, this yield was still better than what has been achieved with conventional liquid targets [11].

Coordination Chemistry and Ligands of ⁸⁹Zr

Desferrioxamine and Its Derivatives

In order to effectively utilize ⁸⁹Zr, coordination chemistry has been applied to study various chelates. The chelate first utilized for ⁸⁹Zr is also currently the widely used: desferrioxamine (DFO). As showed in Fig. 2, DFO, which contains three RCO-N(R′)-OH motifs, is a hydroxamate-type siderophore that chelates with ⁸⁹Zr to form a ⁸⁹Zr-DFO complex, which is used in ⁸⁹Zr-immuno-PET studies. Complexes with ⁸⁹Zr based on the iron-chelator Desferal, DFO (L23), which includes hexadentate coordination of three hydroxamate units, and its derivatives have also been used in ⁸⁹Zr-PET studies. However, ⁸⁹Zr-DFO has been known to have some disadvantages, such as poor stability. Since its hexadentate complex is not saturated by a stably octa-coordinated Zr⁴⁺ sphere, ⁸⁹Zr-DFO instability has been observed in several animal model experiments [6, 16]. Due to the importance of developing ligands for zirconium-89-based radiopharmaceuticals, especially for immuno-PET imaging, several DFO derivatives have been reported (Fig. 3), such as N-(S-acetyl) mercaptoacetyldesferal (SATA-DFO) [17] and 2,3,5,6-tetrafluorphenoxy (TFP)-N-succinyldesferal-Fe [18]. These modifications were prepared for bifunctional mAb coupling; however, both protocols showed several drawbacks. For example, an unstable thioether linker exists between maleimide-mAb and SATA-DFO at physiological pH and a complicated six-step reaction is used to prepare mAb-N-succinyldesferal-⁸⁹Zr, consisting of carboxylation of the amine, protection with Fe(III), activation of the ester, attachment with a mAb, deprotection of Fe(III) from complex, and labeling with ⁸⁹Zr radionuclide [19].

Fig. 2.

Structure of DFO and its ⁸⁹Zr-complex

Fig. 3.

DFO derivatives

A simple two-step synthesis to prepare bifunctional ⁸⁹Zr-labeled mAb via p-isothiocyanatobenzyl-desferrioxamine (DFO-Bz-NCS) was reported by Perk and co-workers more recently. This complex was described to be stable due to the strong and steady thiourea bond between the monoclonal antibodies and the chelator. Although this process proved to be a fast and effective method to acquire ⁸⁹Zr-labeled mAbs, the restricted water solubility of the DFO-Bz-NCS precursor required experimental skill to prevent aggregation and precipitation of the antibody. Also, despite the stability of thiourea linker, it was reported to be easily cleaved by radiation in some buffers that contain chlorinated compounds [20, 21]. Another rapid and specific conjugation between modified-[⁸⁹Zr]Zr-DFO and RGP peptides by the click reaction was described by Gao and co-workers. The modification of DFO at the terminal amine with 2-cyanobenzothiazole (CBT) or 1,2-aminothiol (cys) produced [⁸⁹Zr]Zr-DFO-CBT or [⁸⁹Zr]Zr-DFO-cys, respectively. Luciferin linkage formation from the click reaction of those with their complementary functionality on RGP peptides showed a high stability with an almost intact complex upon cysteine challenge [22].

Octadentate coordination using DFO-1-hydroxy-2-pyridone (DFO-HOPO) was first described by White and co-workers [23]. This study employed the DFO-HOPO ligand as a plutonium(IV) chelator for treatment of plutonium poison. Low toxicity and a stable octadentate coordination complex with Pu(IV) were observed when the 1,2-HOPO compound was introduced to the DFO molecule. Allott adopted this method and utilized DFO-HOPO to evaluate the stability of octadentate as an ⁸⁹Zr chelator. Results showed ⁸⁹Zr-DFO-HOPO to be stable compared to ⁸⁹Zr-DFO with no demetallation during radio-ITLC analysis, and no bone uptake of ⁸⁹Zr was observed within 24 h after ⁸⁹Zr injection. Moreover, ⁸⁹Zr-DFO-HOPO showed inertness to transchelation by EDTA or serum. DFO*, a modification of DFO by adding one more hydroxamic acid part, was reported as the first octadentate chelator for ⁸⁹Zr labeling molecules with improved stability [24, 25]. A few years later, the bifunctional chelator DFO*-pPhe-NCS was prepared as an octadentate chelator with ⁸⁹Zr. ⁸⁹Zr-DFO*-mAb demonstrated greater stability than the previous hexadentate ⁸⁹Zr-DFO-mAb with more than twice the intact tracer when stored at room temperature. Yet, solubility is still a challenge for the thiourea structure [26].

Other Hydroxamate-Type Chelators

To expand the utilization of hydroxamate-type coordination with ⁸⁹Zr, many hydroxamate-containing non-DFO structures have been developed (Fig. 4). Guérard and co-workers reported the simplest structures, acetohydroxamic acid (AHA) and its methylated derivative (Me-AHA), as ligands to coordinate with Zr(IV) and ⁸⁹Zr(IV). Based on X-ray crystallography and potential titration, these studies found a metal to ligand ratio of 1:4 and octadentate coordination with Zr(IV) that was supposedly better than results with DFO. Through an ⁸⁹Zr labeling complexation study, Me-AHA showed a better activity complex than did AHA. This can be explained by a higher electron density of the oxygen atom (N–O) of Me-AHA which forms a strong bond toward the ⁸⁹Zr radionuclide [27]. Recently, two bifunctional tetrahydroxamate ligands were synthesized by Rousseau and co-workers based on an iminodipropionamide scaffold. This work was a modification of their previous study that elongated the aliphatic chain on the main ligand and reduced the distance between ligand and isothiocyanate moiety. Despite improved stability, the biodistribution and PET imaging properties of these ligands showed no significant differences compared to those this group studied previously or to DFO [28].

Fig. 4.

Other hydroxamate-type ligands

Macrocyclic structures including hydroxamate moieties have also been developed, such as triacetylfusarinine C (TFAC), desferrichrome (DFC), and tetrahydroxy octaazacyclohexatriacontan-octaone (CTH36). This type of ligand was reported to form steadier coordination than linear ligands. In addition, as a result of the macrocycle effect, a ligand that has a macrocyclic structure could possess an advantage due to the strong stability of the complex [29–32].

Other Types of Chelators

There is a similar structure between hydroxypyridone (HOPO) and hydroxamate; hence, HOPO was also employed as a polydentate hydroxypyridone ligand. The development of a HOPO ligand for chelating ⁸⁹Zr radionuclides was reported by Deri and co-workers. 3,4,3-(LI-1,2-HOPO), which has four hydroxypyridone moieties (Fig. 5), could make an octadentate ⁸⁹Zr complex which significantly enhances stability compared to DFO in DFT calculations. The ⁸⁹Zr-HOPO complex was inert to transchelation in EDTA and serum challenge tests. In serum, the ⁸⁹Zr complex was an almost intact radiotracer after a 7-day incubation. ⁸⁹Zr-HOPO also possesses satisfactory biological behavior such as rapid renal excretion and low radioactivity in bone tissue. Conjugation of ⁸⁹Zr-HOPO with antibodies to make bifunctional ligands is currently an active area of research [33].

Fig. 5.

Structure of 3,4,3-(LI-1,2-HOPO)

Hydroxyisophthalimide (IAM) ligands, originally used for lanthanides, were also described to produce stable ⁸⁹Zr complexes. Bhatt group investigated two analogs of IAM, including IAM 1 and IAM 2 which differed by one pensile IAM group (Fig. 6). The result showed that the stability of ⁸⁹Zr-IMA 1 was greater than that of ⁸⁹Zr-DFO which was in turn greater than that of ⁸⁹Zr-IMA 2 (with 72%, 41%, and 26% tracer intact, respectively, after a 7-day incubation with DTPA). However, in the amino model, ⁸⁹Zr-IAM 1 and ⁸⁹Zr-IAM 2 accumulated much more in the kidneys, liver, and bone than did ⁸⁹Zr-DFO. ⁸⁹Zr-immuno-PET imaging with ⁸⁹Zr-IAM 1 is currently still under further investigation [34].

Fig. 6.

Structure of hydroxyisophthalimide (IAM) ligands

Ligands containing carboxylate and amino donors, such as EDTA and DTPA, have also been reported to complex with ⁸⁹Zr [35]. Recently, the Wadas group used various kinds of tetraazamacrocycle ligands, namely, DOTA, DOTP, and DOTAM, to react with ⁸⁹ZrCl₄ to form Zr complexes (Fig. 7) [36]. The stability of resulting Zr-complexes (Zr-DOTA, Zr-DOTP, Zr-DOTAM) which were tested with an excess amount of EDTA or a high concentration of metal ions (Fe , Zn , Co , Cu , Mg , Gd , Ga ) was showed as following order: Zr-DOTA >> Zr-DOTP> Zr-DOTAM> Zr-DFO. In additions, they found that Zr-DOTA was stable, showing no change even after 7 days.

Fig. 7.

Structure of tetraazamacrocylic ligands and their ⁸⁹Zr complexes

In in vivo biodistribution experiments, ⁸⁹Zr-DOTAM showed a large amount of radioactivity in the liver and spleen, while ⁸⁹Zr-DOTA showed relatively low radioactivity in the liver, kidneys, and bone. Results from ⁸⁹Zr-DOTP were generally similar to those from ⁸⁹Zr-DOTA, except that high amounts of radioactive material were found in the bone with ⁸⁹Zr-DOTP. Based on these results, dynamic PET imaging studies were conducted using ⁸⁹Zr-DOTA and ⁸⁹Zr-DFO. In contrast, ⁸⁹Zr-DFO accumulates significantly in the kidneys after 4 and until 24 h. However, ⁸⁹Zr-DOTA accumulates less in the kidneys and bones than does ⁸⁹Zr-DFO. A small amount of ⁸⁹Zr-DOTA was observed in the bladder at 4 h, and after 24 h, the radioactivity in the bladder was found to be negligible. Thus, it was found that ⁸⁹Zr-DOTA was easily cleared from the living body over a short period of time. Therefore, we confirmed that ⁸⁹Zr-DOTA could be effectively applied to precision medicine without the disadvantages that come with ⁸⁹Zr-DFO, which is currently used.

Immuno-PET Studies Using ⁸⁹Zr

In order to apply ⁸⁹Zr to precision medicine, immuno-PET studies using ⁸⁹Zr-labeled monoclonal antibodies (mAbs) have been carried out by various researchers (Table 3). For instance, measurements of metastasis in persons with breast cancer have been carried out using trastuzumab [49]. Trastuzumab is a target for human epidermal growth factor receptor 2 (HER2), which has been used to diagnose HER2-positive breast cancer, and thus, treatment with trastuzumab has shown positive results in patients with HER2-positive breast cancer and gastric cancer [50, 51]. In one case, HER2-negative early breast cancer patients were found to have HER2-positive cancer metastases with PET/CT scans using ⁸⁹Zr-trastuzumab (Fig. 8) [49]. In addition, ⁸⁹Zr-trastuzumab PET was used to evaluate the alteration of HER2 expression in patients with HER2-positive breast cancer after they were treated with the anti-angiogenic agent NVP-AUY922, the novel heat shock protein 90 (HSP90) inhibitor. This study suggested that ⁸⁹Zr-immuno-PET can be useful for determining the alteration of antigen expression and for monitoring the response to treatment with anti-cancer agents [52].

Table 3.

Application of ⁸⁹Zr-mAb in clinical oncology studies

Year	mAb	Target	Tumor type	Refs.
2006	Chimeric mAb U36	CD44v6	Head and neck cancer	[37]
2012	Ibritumomab-tiuxetan	CD20	B cell lymphoma	[38]
2013	Bevacizumab	VEGF-A	Breast cancer	[39]
2014	Bevacizumab	VEGF-A	Neuroendocrine tumors	[40]
2015	Fresolimumab	TGF-β	Glioma	[41]
2016	MMOT0530A	MSLN	Pancreatic, ovarian cancer	[42]
2017	Cetuximab	EGFR	Head and neck, lung cancer	[43]
2017	Rituximab	CD20	B cell lymphoma	[44]
2017	Lumretuzumab	HER3	Multiple cancer types	[45]
2017	Bevacizumab	VEGF-A	Metastatic renal cell carcinoma	[46]
2018	Trastuzumab	HER2	Breast cancer	[47]
2018	Atezolizumab	PD-L1	Bladder cancer, non-small cell lung cancer, triple-negative breast cancer	[48]

Fig. 8.

Eighty-three-year-old woman with primary ER-positive/HER2-negative invasive ductal breast carcinoma. a⁸⁹Zr-trastuzumab maximum intensity projection demonstrates several foci of ⁸⁹Zr-trastuzumab avidity that localize to osseous structures. b Axial CT and ⁸⁹Zr-trastuzumab PET/CT demonstrate ⁸⁹Zr-trastuzumab avidity in proximal left femur. Reprinted with permission from ref. [49]. Copyright 2016 Society of Nuclear Medicine

Studies targeting vascular endothelial growth factor A (VEGF-A) have also been conducted using ⁸⁹Zr-labeled mAbs [39, 40, 52–54]. VEGF-A is overexpressed in malignant breast tumors and ductal carcinoma in situ and is known to be associated with various diseases. Bevacizumab has been reported as a monoclonal antibody that targets VEGF-A, and it has been successfully utilized in several studies. In particular, ⁸⁹Zr-bevacizumab PET has been used for various ailments such as breast cancer, pelvic cancer, renal cell carcinoma, and neuroendocrine tumors to effectively identify the biological properties of the tumor and confirm the effectiveness of treatment (Fig. 9).

Fig. 9.

PET images of heterogeneous ⁸⁹Zr-bevacizumab accumulation in tumor lesions of metastatic renal cell carcinoma patients. a Serial ⁸⁹Zr-bevacizumab PET scans of patient with RCC metastases in the pancreas, liver, and thyroid, with associated jugular and portal vein thrombosis at baseline (left) and 2 (middle) and 6 weeks (right) after the start of bevacizumab/IFNα. b Serial ⁸⁹Zr-bevacizumab PET scans of patient with RCC metastases in the lungs, mediastinal lymph nodes, bone, and brain at baseline (left) and 2 (middle) and 6 weeks (right) after the start of sunitinib—that is, after 2 sunitinib-free weeks. Reprinted with permission from ref. 54. Copyright 2015 Society of Nuclear Medicine

EGFR is also another interesting target antigen. Cetuximab is a widely known agent to target EGFR. Attachment of cetuximab to EGFR prohibits binding of growth factor to the receptor, and the receptor tyrosine kinase activity is prevented. Thus, biological events such as cell growth, proliferation and differentiation, and cellular invasiveness and apoptosis can be slowed or stopped. ⁸⁹Zr-cetuximab has been used to evaluate patients with advanced colorectal cancer; tumor uptake was investigated via checking the biodistribution of this labeled antibody [55].

Visualization of metastatic prostate cancer is critical to monitoring the treatment of metastatic prostate cancer. HuJ591 was developed for selectively targeting the extracellular domain of prostate-specific membrane antigen (PSMA), which most prostate cancers express. An immuno-PET imaging study in patients with metastatic prostate cancer using ⁸⁹Zr-huJ591 was performed [56]. In this case, 5 mCi of ⁸⁹Zr-huJ591 was injected into 10 patients, and its distribution, elimination, and lesion accumulation were examined. In the PET image, ⁸⁹Zr-huJ591 was found to accumulate in lesions in the bone and soft tissues more effectively than ^99mTc-MDP or FDG. In particular, when analyzing images using ⁸⁹Zr-huJ591, 11 out of 12 lesions were positive, which proved to be superior to corresponding PET scans using FDG that yielded only 9 positive results.

Another study used ⁸⁹Zr-labeled cmAb U36 to detect head and neck squamous cell carcinoma (HNSCC) tumors in 20 patients. This study suggested that most of primary tumors were identified by ⁸⁹Zr-immuno-PET, and performance results of ⁸⁹Zr-immuno-PET for the detection of lymph node metastasis were no different from those of computed tomography (CT) or magnetic resonance imaging (MRI) [37].

There are no important drug targets for cancers such as pancreatic and ovarian carcinoma. However, it was reported that membrane-bound surface glycoprotein mesothelin (MSLN) is overexpressed in pancreatic and ovarian cancer. Thus, the anti-MSLN antibody MMOT0530A was discovered as a potential imaging biomarker [42, 57]. PET studies using ⁸⁹Zr-MMOT0530A indicated that its tumor uptake in patients with either pancreatic cancer or ovarian cancer could be clearly visualized. IHC studies suggested that MSLN expression levels, determined with IHC scores, were strongly associated with the intensity of tumor uptake of ⁸⁹Zr-MMOT0530A.

Conclusion

These results suggest that further study of ⁸⁹Zr will solve current shortcomings and contribute to molecular imaging research using PET, such as ⁶⁸Ga. In particular, development of new coordinate chemistry for ⁸⁹Zr labeling has led to wider application of ⁸⁹Zr in clinical studies. In oncology, ⁸⁹Zr-immuno-PET techniques have significantly enhanced tumor detection and the efficiency of treatment. Until now, there has been no standard scale for the use of ⁸⁹Zr. Thus, some image processing steps, including measurements of tumor uptake and data analysis, should be validated and standardized for wider usage. Overall, based on previous studies, it can be expected that ⁸⁹Zr will be more successfully applied to the diagnosis and treatment of patients via ⁸⁹Zr-immuno-PET in the future.

Conflicts of Interest

Minh Thanh La, Van Hieu Tran, and Hee-Kwon Kim declare that they have no conflict of interest.

Ethical Approval

This article does not contain any studies with animals or human participants performed by any of the authors.

Informed Consent

None.