The Development of Copper Radiopharmaceuticals for Imaging and Therapy

Abstract

The increasing use of positron emission tomography in preclinical and clinical settings has widened the demand for radiopharmaceuticals with high specificity that can image biological phenomena in vivo. While many PET tracers have been developed from small organic molecules labeled with carbon-11 or fluorine-18, the short half-lives of these radionuclides preclude their incorporation into radiotracers, which can be used to image biological processes that are not induced immediately after system perturbation. Additionally, the continuing development of targeted agents, such as antibodies and nanoparticles, which undergo extended circulation, require that radionuclides with half-lives that are complimentary to the biological half-lives of these molecules be developed. Copper radionuclides have received considerable attention since they offer a variety of half-lives and decay energies and because the coordination chemistry of cooper and its role in biology is well understood. However, in addition to the radiometal chelate, a successful copper based radiopharmaceutical depends upon the chemical structure of the entire radiotracer, which may include a biologically important molecule and a chemical linker that can be used to deliver the copper radionuclide to a specific target and modulate its in vivo properties, respectively. This review discusses the development of copper radiopharmaceuticals and the importance of factors such as chemical structure on their pharmacokinetics in vivo.

INTRODUCTION

Advances in the biological, physical and engineering sciences have allowed molecular imaging to emerge as an important scientific discipline that has quickly made significant contributions to pharmaceutical discovery. Specifically, it has allowed drug manufacturers to rapidly evaluate potential drug candidates while reducing research and development expenditures. Additionally, clinical medicine has also benefited from the use of molecular imaging technologies since they allow physicians to diagnose illness and monitor treatment non-invasively. Computed tomography (CT), magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT) and positron emission tomography (PET) are currently used in the pharmaceutical industry and clinical care. While the former two are primarily anatomical imaging techniques, the latter techniques are nuclear medicine imaging modalities, which provide physiological information. When compared to SPECT, PET has greater advantages with respect to sensitivity and resolution and has been gaining in clinical popularity, with approximately 3.2 million PET based studies being performed in 2010 [1]. In addition, basic researchers are utilizing PET with greater frequency because of technological advances including the small biomedical cyclotron and the dedicated small animal PET scanner. Accordingly, this has increased the demand for numerous PET isotopes including carbon-11 (¹¹C) and fluorine-18 (¹⁸F), which can be introduced into small organic molecules using traditional organic synthetic techniques. However, these radionuclides have short half-lives that preclude using them to investigate biological processes, which occur after long induction periods, or limits the ability to ship them over long distances. Consequently, their use is limited to only the largest PET centers, which have the resources for dedicated radiochemistry facilities that can produce these isotopes on demand.

Over the last 25 years, PET radionuclides such as ^94mTc, ^66/68Ga, ⁸⁶Y and ^60/61/62/64Cu have been evaluated as useful alternatives to ¹¹C and ¹⁸F [2]. Of these, the copper radionuclides have received considerable attention since they offer a variety of half-lives and decay energies and because the coordination chemistry of cooper and its role in biology is well understood [3–7]. Recently, several exhaustive reviews have been published that encompass over 40 years of research and describe the coordination chemistry, production and application of the copper radionuclides to molecular imaging [3,8–10]. In contrast, the goal of this review will be to discuss the development of copper radiopharmaceuticals in a manner that would be relevant to the medicinal chemistry community. Accordingly, this review is organized into six sections with the first section briefly discussing copper coordination chemistry, and the second section describing the production methods of the most medically relevant copper isotopes. The third will describe the use of small inorganic copper complexes as copper radiotracers. The fourth section will describe the development of bifunctional chelators for copper radiopharmaceuticals and the resulting stability of the radiometal complexes. The fifth section will discuss the use of chemical linkers in creating copper radiotracers, and the final section will discuss the development of targeting peptides in copper radiopharmaceutical development.

COPPER COORDINATION CHEMISTRY

Copper, a first row transition metal exists in three distinct oxidation states. Cu(I), the lowest oxidation state has a diamagnetic d¹⁰ electron configuration and reacts readily with relatively soft, polarizable donor ligands such as thiolates and thioethers, phosphines and nitrogen containing ligands demonstrating significant sp² hybridization [4]. Linear, trigonal planar and tetrahedral geometries are preferred with the coordination number determined by the size and conformation of the complexing ligands. Cu(I) complexes are biologically relevant with some being able to reductively activate molecular oxygen (O₂). However, due to their labile nature, they typically lack sufficient kinetic stability for radiopharmaceutical applications.

Copper (II) exists as a d⁹ metal center, which has an ionic radius of 57–73 pm [9]. Its water-exchange rate has been found to be very rapid compared to most first-row transition metal cations and as a result it has relatively facile substitution chemistry. It exhibits some crystal-field stabilization, which is usually ascribed to the Jahn-Teller distortion that elongates one or more of its coordinated ligands. A cation of borderline hardness, it favors amine, imine and bidentate ligands such as phenanthroline, which allow it to adopt square planar, distorted square planar, trigonal pyramid, square pyramidal and distorted octahedral geometries. Its ability to form thermodynamically stable and kinetically inert complexes with a variety of acyclic and macrocyclic ligand systems has made it extremely well suited for incorporation into copper radiopharmaceuticals.

Copper (III) adopts a square planar geometry due to its d⁸ electron configuration, but is a relatively difficult oxidation state to attain. Strong π donating ligands such as anionic amides are necessary to stabilize this oxidation state in solution. Additionally, these systems usually undergo facile proton exchange in solution requiring the use of highly basic solvents. Discussions detailing the chemistry of this oxidation state have been reported [11,12].

PRODUCTION OF COPPER RADIOMETALS

The production of copper radiometals continues to be an area of intense research because they offer a variety of half-lives and decay energies, which can be found in Table 1 and since they decay by both β ⁺ and β ⁻ emission they are well suited for diagnostic imaging and radiotherapeutic applications. However, their incorporation into radiopharmaceuticals for diagnostic imaging and therapy requires a thorough understanding of the nuclear reactions and decay schemes available for their production. Moreover, their processing methods rely upon an intimate knowledge of their aqueous solution chemistries, and this next section will briefly discuss the current production methods utilized in preparing the most medically relevant copper radionuclides.

Table 1.

Decay Characteristics of Copper Radionuclides [41]

Isotope	T1/2 (h)	Methods of Production	Decay Mode	Eβ+ (keV)	Eβ− (keV)
60Cu	0.4	cyclotron, 60Ni(p,n)60Cu	β+ (93%) EC (7%)	39200, 3000 2000
61Cu	3.3	cyclotron, 61Ni(p,n)61Cu	β+ (62%) EC (38%)	1220, 1150 940, 560
62Cu	0.16	62Zn/62Cu generator	β+ (98%) EC (2%)	2910
64Cu	12.7	cyclotron, 64Ni(p,n)64Cu	β+ 19(%) EC (41%) β− (40%)	656
67Cu	62.01	accelerator 67Zn(n,p)	β− (100%)		577, 484,395

Copper-60 (t_½ = 0.4 h, β⁺: 93% E_β⁺_max: 3.9 MeV and 3.0 MeV; EC: 7%, E_{γ max}: 2.0 MeV) is a potentially useful copper radionuclide since high quality images can be obtained due to the high percentage of positron decay [13]. McCarthy et al. have produced approximately 17 GBq of ⁶⁰Cu using 14.7 MeV protons and the ⁶⁰Ni(p,n)⁶⁰Cu nuclear reaction on a biomedical cyclotron [14]. The use of enriched nickel target material dramatically reduced the production of impurities allowing for a more straightforward separation involving anion exchange chromatography. However, the increased production cost associated with the enriched target material has hindered the cost effective production of this radiometal.

Copper-61 (t_½ = 3.3 h, β⁺: 62% E_{β+ max}: 1.2 MeV and 1.15 MeV; EC: 38%, E_{γ max}: 940 MeV and 960 MeV) is also suitable for biomedical imaging applications because of its favorable positron decay and its relatively long half-life making it suitable for imaging biological processes that occur between 1 and 4 hours post-injection [13]. McCarthy et al. produced 3.7 GBq of ⁶¹Cu on a biomedical cyclotron using either 14.7 MeV protons or 8.1 MeV deuterons and the ⁶¹Ni(p,n)⁶¹Cu or ⁶¹Ni(d,n)⁶¹Cu nuclear reactions, respectively [14]. Similar to the production of ⁶⁰Cu, an enriched Ni target was used to reduce impurities, but contributed to increased production costs. Additional production methods of ⁶¹Cu involve the bombardment of ⁵⁹Co foils with 40 MeV alpha particles [15], but the purity of the final product was observed to be highly dependent upon the quality of the cobalt foil used in production. A second process involves the bombardment of natural zinc targets with 22 MeV protons on a medical cyclotron, and was reported to have significantly less impurities. However, the processing of the Zn target required more than one half-life reducing the total available activity at the end of the production [16].

Copper-62 (t_½ = 0.16 h, β⁺: 98% E_β⁺_max: 2.19 MeV; EC: 2%) can be produced in a small cyclotron [17], and is the only generator-produced copper radionuclide, which results from the decay of its parent, ⁶²Zn. Zinc-62 is produced by irradiation of an enriched Cu target with protons according to the nuclear reactions ⁶³Cu(p,n)⁶²Zn or ⁶⁵Cu(p,4n)⁶²Zn [18]. Typically, ⁶²Cu is eluted from a generator in a suitable form for radiopharmaceutical production using a buffered eluent or other aqueous and organic solvent mixture [19,20]. While the lifespan of a ⁶²Cu generator is only 24 h, a clinically useful dose can be prepared every 30 minute and represents an economical alternative for hospitals that do not have access to an onsite cyclotron or the resources for dedicated radiopharmaceutical production facilities. Accordingly, significant research has been invested in developing systems, which can produce enough generators to meet demand. For example, Fukumura et al. have described a remotely controlled dispensing system that is capable of preparing four shielded ⁶²Zn/⁶²Cu generators in about 2 h [21]. Each generator consists of a Waters^® Accell cartridge containing the parent radionuclide, and elution of the cartridge with a 200 mM glycine solution results in the isolation of 68 GBq of ⁶²Cu containing less than 1 ppm of radioactive contaminants.

Copper-64 (t_½ =12.7 h, β⁺: 19% E_{β+ max}, 0.656 MeV; EC: 41%; β ⁻: 40%) is the most widely studied copper radionuclide and can be prepared in a nuclear reactor using the ⁶³Cu(γn,γ)⁶⁴Cu or the ⁶⁴Zn(n,p)⁶⁴Cu nuclear reactions. However, only the latter process results in a product without natural isotopic impurities, often referred to as a carrier-free state. Additional production methods to produce ⁶⁴Cu in a carrier-free state involve the nuclear reactions ⁶⁴Ni(p,n)⁶⁴Cu and ⁶⁴Ni(d,2n)⁶⁴Cu, which can be carried out on a biomedical cyclotron and require the use of an enriched nickel target. The high cost of the enriched target material however, has spurred researchers to find cheaper target materials or alternative nickel complexes such as ⁶⁴NiO to increase ⁶⁴Ni recovery [22]. Additional production methods have also been investigated using ⁶⁸Zn and ⁶⁴Zn, but the coproduction of radionuclidic impurities has prompted the need for rigorous purification techniques and hindered the wide spread acceptance of these processes [23–26]. For example, Kozempel and colleagues reported the production of no-carrier-added (NCA) ⁶⁴Cu using the ⁶⁴Zn(d,2pn)⁶⁴Cu reaction with a 19.5 MeV deuteron beam [27]. After irradiation the radioactive target material was subjected to dual ion exchange chromatography using a strong cation exchange resin to remove any Ga-based impurities. The second step involves anion exchange chromatography, which retains the ⁶⁴Cu and ⁶⁴Zn target material, while allowing other impurities such as ²⁴Na and ⁵⁸Co to flow through the column. Finally, the ⁶⁴Cu is eluted in 2 M HCl while the Zn fraction is eluted in neutral water.

Copper-67 (t_½ = 62.01 h, β⁻: 100% E_β⁻_max, 0.577 MeV; E_{γ max}: 0.185 MeV) decays completely by β⁻ emission, and is an attractive radiometal for radioimmunotherapy with intact antibodies since the β⁻ particles emitted have sufficient energy to penetrate small tumors and its medium range half-life of 62 hours compliments the time needed for non-specific antibody clearance in vivo. It can be produced on a cyclotron or high energy accelerator using the nuclear reactions ⁶⁵Zn(p,2p)⁶⁷Cu and ⁶⁸Zn(p,2p)⁶⁷Cu or ⁶⁵Zn(n,p)⁶⁷Cu, respectively [28], but ⁶⁸Zn targets are preferred since their irradiation leads to substantial increases in ⁶⁷Cu yields [29,30]. Typically, production yields are relatively low, due to the numerous contaminants produced during the target bombardment, which need to be removed using multiple chromatographic steps [29,31]. Additionally, the need for a high energy cyclotron or accelerator, which increases production costs has also contributed to its limited availability and application as a radiotherapeutic agent [32–40].

SMALL COPPER COMPLEXES AS COPPER RADIOPHARMACEUTICALS

Successful development of a copper based radiopharmaceutical requires a thorough understanding of the biological target and how the chemical structure of the radiopharmaceutical influences its behavior in vivo. Additionally, traditional medicinal chemistry techniques can be applied to radiopharmaceutical development, but it is important to recognize that the criteria used to develop a successful radiopharmaceutical is very different from those used in traditional drug development. In contrast to the latter process, the goal of the former is to develop an agent, which exhibits fairly rapid plasma clearance and only localizes in the target tissue of interest so that maximum image contrast can be achieved [42]. In addition, unlike radiopharmaceuticals developed with ¹⁸F or ¹¹C, few copper based radiopharmaceuticals are “small molecules” though; those that have been developed have been utilized successfully [43–54]. For example, Fujibayashi et al. first reported ⁶²Cu-diacetyl bis(N⁴-methylthiosemicarbazone) (Fig. (1) (ATSM)) as a hypoxia imaging agent after observing its retention in tissues where the intracellular environment was observed to be in a reductive state [52].

Fig. (1).

Crystal Structure of Cu(II)-ATSM rendered as a ball and stick model. hydrogens are omitted for clarity. Atom color code: Cu, brown; S, yellow; N, blue; C, gray. Adapted with permission from reference 9. Copyright 2010 American Chemical Society.

However, rapid efflux was observed in tissues with normal intracellular oxygen concentrations; a result independent of blood flow. Since hypoxia is implicated in several disease processes including the development of therapeutically resistant malignancies, significant research efforts have been expended to develop successful hypoxia imaging agents based on the Cu(II)-ATSM complex. For example, Dearling and coworkers prepared a series of Cu(II)-ATSM complexes with increasing alkylation on the diimine backbone or the terminal amine positions within the ATSM ligand to determine the optimal structure for hypoxia selectivity [55–57]. Based upon these studies, the authors concluded that hypoxia selectivity was strongly dependent on the redox potential of the Cu(II)-ATSM complex, but the complex’s lipophilicity, while important for cell membrane penetration, did not correlate significantly with hypoxia selectivity. Additionally, while greater alkyl substitution of the diimine backbone directly correlated with greater reduction potential, it was observed to be only a very crude predictor of lipophilicity. Once the relationship between hypoxia selectivity and chemical structure was understood, numerous reports appeared in the literature describing detailed synthetic methods to prepare novel ATSM ligands [58–63], their asymmetric Cu(II) complexes [60], and ATSM ligands containing fluorescent molecules or ligands capable of being simultaneously radiolabeled with two different radionuclides [64–70]. Despite these advances, Cu-ATSM complexes demonstrated substantial liver and non-target organ uptake due to their lipophilicity when evaluated in vivo. To reduce non-specific liver uptake, Holland et al. developed an ATSM-glucose conjugate, which could be labeled with ⁶⁴Cu [60,71]. Using small animal PET, the authors demonstrated that the addition of the glucose moiety did not significantly alter the hypoxia selectivity of this complex, but did reduce its lipophilicity, which was evident by the increased accumulation of activity in the bladder and decreased accumulation of activity in the liver and other lipophilic compartments of the animals being imaged [71].

Simultaneously, numerous clinical and preclinical studies have been undertaken to evaluate the efficacy of Cu(II)-ATSM as a radiopharmaceutical, and it has been used to identify tumor cells, which exhibit a cancer stem cell like phenotype and high colony forming potential [72,73]. It has also been used to evaluate the efficacy of radiotherapy [74,75], and correlated to multi-drug resistance [49], topoisomerase II expression [76], and fatty acid synthase expression [77]. Clinically, Cu-ATSM has been used to image lung and cervical cancers [78–83]. For example, Dehdashti et al. demonstrated that ⁶⁰Cu-ATSM in conjunction with PET, was a reliable way to predict therapeutic response in patients with non small cell lung cancer [80], and the same authors used ⁶⁰Cu-ATSM to predict which cervical cancer patients were at greater risk of relapse based upon the extent of tumor hypoxia [83,84]. Finally, Dietz and coworkers further expanded the utility of Cu-ATSM imaging by observing similar trends in colorectal cancer patients [85].

THE STABILITY OF THE RADIOMETAL CHELATE IN COPPER RADIOPHARMACEUTICAL DESIGN

While significant success has been achieved using simple, inorganic complexes such as Cu(II)-ATSM whose utility is derived from radiochelate instability, the stability of the radiocopper complex in vivo is a critical design consideration for achieving high uptake of the copper radionuclide in the tissue or organ of interest while minimizing the non-selective binding or incorporation into non-target organs or tissues [9,86]. In general, ligands that have rapid complexation kinetics, but form radiocopper complexes with superior kinetic inertness to Cu(II) decomplexation (proton-assisted as well as transchelation or transmetallation) are ideal since this is more significant than thermodynamic stability after the radiocopper complex is injected into a living organism [87,88]. In addition, the reduction of Cu(II) to Cu(I), subsequent ligand reorganization upon reduction and Cu(I) loss must also be considered since it is believed that Cu(I) formation leads to transchelation of radiocopper to protein in vivo and reduced image quality [89,90]. Although beneficial in hypoxia imaging, it has an adverse effect on the in vivo imaging properties of most copper radiopharmaceuticals. Accordingly, designing bifunctional chelators (BFCs) that form reduction resistant radiocopper complexes, which can be easily attached to the targeting vector through facile chemistry is important (Fig. (2)) [89].

Fig. (2).

Cartoon of a radiopharmaceutical showing a bifunctional chelator to complex the radioactive metal ion that is attached to a targeting group or a molecule of pharmacological importance (small molecule, peptide or protein). Adapted with permission from reference 9. Copyright 2010 American Chemical Society.

Cyclic Polyaminocarboxylates

Early attempts to develop stable radiocopper complexes using BFCs relied upon acyclic ligands because of their high stability [91–98], but serum stability measurements involving these radiolabeled ligands revealed that they were not stable in human serum for long periods [97]. These observations led to the use of numerous macrocyclic ligands, such as the macrocyclic polyaminocarboxylate ligands, which include 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA (3)) and 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA (7)) shown in Fig. (3). These systems have been thoroughly investigated, and in vitro and in vivo testing have shown them to be superior to acyclic chelating agents for radiocopper due to the greater geometrical constraint incorporated into the macrocyclic ligand that enhances the kinetic inertness and thermodynamic stability of their resulting radiocopper complexes [99–101]. While DOTA has been used as a BFC for radiocopper complexation, its non-selective metal binding properties and its relative instability when compared to TETA have made it only a marginal chelator for copper radiopharmaceuticals [102–107]. The tetraazamacrocyclic ligand TETA however, has been used extensively as a copper chelating ligand in radiopharmaceutical development, and successful derivatization of this ligand has allowed researchers to conjugate it to antibodies, proteins, and peptides [97,108–116].

Fig. (3).

Representative examples of polyaminocarboxylate ligands used in Cu radiopharmaceutical development.

Although radiocopper complexes containing the macrocyclic chelator TETA are more stable than those using the macrocyclic chelator DOTA or acyclic ligands, they are not optimal in vivo. For example, Bass et al. demonstrated that when ⁶⁴Cu-TETA-OC (octreotide) was injected into normal Sprague-Dawley rats, nearly 70% of the ⁶⁴Cu from ⁶⁴Cu-TETA-OC was transchelated to a 35 kDa species believed to be superoxide dismutase (SOD) in the liver 20 h post-injection [117]. Additionally, Mirick et al. reported the dissociation of ⁶⁷Cu from TETA to ceruloplasmin during radioimmunotherapy trials in lymphoma patients [39,118], and clinical PET studies using ⁶⁴Cu-TETA-OC also resulted in slow blood clearance and accumulation of ⁶⁴Cu in the liver over time [119]. To overcome this instability in vivo, several other classes of chelators have been designed and evaluated as radiocopper ligands [8,9,86,120].

Derivatives of TACN (10), which contain carboxymethyl pendant arms such as 1,4,7-triazacyclononane-N,N’,N”,N’”-tetraacetic acid (NOTA (11)) are polyamincarboxylate ligands that have been commonly used as a BFC’s for ⁶⁸Ga [121], but whose use as radiocopper chelators has increased because of their ability to stably complex Cu(II) [122–124]. For example, Prasaphanich and coworkers evaluated ⁶⁴Cu-NOTA-8-Aoc-BN(7–14) in mice bearing human prostate PC-3 xenografts [123]. This radiopharmaceutical, which targets the gastrin releasing peptide receptor (GRPR) demonstrated uptake in receptor positive tissues and tumor xenografts, and lower liver accumulation than what is usually observed with similar polyaminocarboxylates. This inertness is thought to be a direct result of the enhanced kinetic stability imparted to the radiotracer by the NOTA chelator.

The Triaminocyclohexane Ligands

Ligands based upon the cis, cis-1,3,5-triaminocyclohexane scaffold (Fig. (4)), which contain thiophene, pyridyl and imidazole rings have been evaluated as potential radiocopper chelators [125–132]. Park et al. studied the natural Cu(II) complexes of N,N’,N”-tris-(2-pyridylmethyl)-1,3,5-cis,cis-triaminocyclohexane (tachpyr (13)) and N, N’, N”-tris-(6-methyl-2-pyridylmethyl-1,3,5-cis,cis-triaminocyclohexane (tachpyr-(6-Me) (15)) in solution, in the solid state and examined their serum stability as copper chelators using ^64/67Cu [132]. Although both ligands demonstrated facile complexation to ^64/67Cu, they exhibited differing stability in human serum with ⁶⁷Cu-tachpyr ion being stable over a period of 7 days, while significant dissociation of ⁶⁷Cu from the ⁶⁷Cu-(tachpyr-(6-Me)) complex occurred after only an hour at physiological temperature.

Fig. (4).

1,3,5-cis,cis-triaminocyclohexane ligands, which were developed as radiocopper chelators.

In a second report, Ma et al. synthesized and evaluated several new tachpyr analogs containing imidazole rings yielding the ligand cis,cis-1,3,5-triaminocyclohexane-N,N’,N”-tris(2-methyl-N- methyl imidazole) (IM (14)) and its derivatives [133]. The authors reported variable formation kinetics when each ligand was radiolabeled with ^64/67Cu. Facile and complete complexation was observed when the aliphatic secondary amines remained unsubstituted in these ligand systems, but when they were alkylated to form tertiary amines, radiocopper complexation was slow and incomplete even after 2 h at 37°C. When all of the complexes were screened against an excess of EDTA, only those complexes synthesized with secondary aliphatic amines in the ligand structure or those synthesized without methyl groups in the 6-position of the pyridyl rings were stable to transchelation by EDTA. In cross-ligand exchange experiments using ⁶⁷Cu-TETA, tachpyr was able to transchelate 92% of ⁶⁷Cu originally coordinated to TETA after 96 h, while IM was able to transchelate 95% of ⁶⁷Cu associated with TETA after the same period. Conversely, TETA was able to transchelate only 30% of the ⁶⁷Cu in ⁶⁷Cu-tachpyr and only 3% of the ⁶⁷Cu in ⁶⁷Cu-IM after 96 h. In addition, human serum stability experiments demonstrated that no activity was transferred to serum over a period of 14 days confirming the kinetic inertness of the radiocopper complexes. While these initial reports described them as promising ligands for radiocopper chelation, significant effort to modify these ligand systems was needed in order to generate useful BFC’s for conjugation to proteins or peptides or other small molecules [131,134]. Moreover, loss of molecular symmetry, complications due to cross-linking and introduction of useful functional groups at specific locations on the ligand system have remained as challenges that impede their use as Cu radioligands for imaging and radiotherapy [133].

The Hexaamine Sarcophagine Chelators

Another class of ligands that has gained attention as potential radiocopper chelators are the hexaazamacrobicyclic cage type ligands, whose syntheses were first described by Sargeson and co-workers (Fig. (5)) [135,136]. Reaction of the inert tris-ethylenediamine cobalt (III) complex with formaldehyde followed by reaction with ammonia/formaldehyde or nitromethane/formaldehyde under basic conditions to generate the sepulchrate or sarcophagine (Sar) ligands, respectively. Use of the chiral Co(III) tris-ethylenediamine complex, allows for the formation of only one of the possible 16 isomers to form in the reaction, but the cobalt (III) metal center must be removed using cyanide ion or concentrated HBr to make the free ligand available as a chelator for other metal ions. Sar analogs substituted at the apical bridgehead carbons with a variety of functional groups including NH₂, OH, Cl, and NO₂ or other alkyl or aryl organic groups have also been synthesized. Thermodynamic stability studies suggested that the hexaazamacrobicyclic complexes resist metal dissociation from the cage ligand as a function of the ligand structure, and it was hypothesized that when one of the chelating nitrogen atoms of the cage dissociates from the metal center, the topological constraint induced by the ligand ensures its facile re-coordination [136,137].

Fig. (5).

Sarcophagine and sepulchrate ligands.

Reports that describe the complexation, stability, and biodistribution of the ⁶⁴Cu sarcophagine complexes revealed that all complexes are stable in human serum after 7 days while biodistribution data, collected using ⁶⁴Cu-Sar (17), ⁶⁴Cu-diamSar (18) and ⁶⁴Cu-SarAr (19) in Balb/c mice, demonstrated that all three complexes cleared from the blood rapidly and exhibited low accumulation in most major organs [138]. Liver clearance was observed to be good over the 30-minute time course of this study demonstrating that the ⁶⁴Cu complexes are initially stable in vivo, but clearance of all three ⁶⁴Cu complexes is much slower through the kidney. Activity levels increased in the case of the ⁶⁴Cu-Sar complex, though this type of accumulation is not uncommon for positively charged complexes. These chelators have also been incorporated into successful imaging agents [139–142]. For example, Smith et al. covalently attached the SarAr ligand to whole and fragmented B72.3 murine antibodies and then labeled it with ⁶⁴Cu for imaging of mice bearing LS174T colon carcinoma tumors [143]. High tumor-to-blood ratios were achieved with effective localization at the tumor suggesting these radioimmunoconjugates are stable in vivo. Additionally, Voss et al. was able to conjugate SarAR to ch14.18, an antibody that targets disialogangliosides, which are over expressd on neuroblastomas and melanoma, and radiolabel it with ⁶⁴Cu. Biodistribution studies in mice bearing M21 melanoma xenografts demonstrated appreciable uptake within the tumors and low non-specific tissue accumulation. Small animal PET corroborated the results of biodistribution studies revealing excellent tumor targeting after 48 h further validating this ligand as a useful chelator in copper radiopharmaceutical development [141].

Cross-Bridged Cyclicpolyaminocarboxylate Ligands

A final class of ligands that will be discussed in this review are the ethylene cross-bridged cyclam and cyclen ligands and their pendant armed derivatives, which were first conceived of and synthesized by Weisman and coworkers in the 1990’s (Fig. (6)) [144,145]. These highly basic tetraamine macrobicyclic ligands were designed to complex several cations including Cu²⁺ within their clamshell-like clefts, and available structural data has confirmed a cis-folded conformation, which is adopted by these chelators upon copper complexation (Fig. (7)). Additional derivatives have been prepared with two carboxymethyl pendant arms to give 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2] hexadecane (CB-TE2A (23)), which further ensure complete envelopment of a six-coordinate Cu(II). Although thermodynamic studies have demonstrated comparable stabilities between Cu-CB-cyclam (log K_f = 27.1) and unbridged Cu-cyclam (log K_f = 27.2) and related complexes [146], the kinetic inertness, especially in aqueous solution, of the Cu(II)-CB-cyclam complexes has been shown to be truly exceptional [89]. For example, proton-assisted decomplexation studies conducted under high acid concentrations (5 M HCl), demonstrated remarkable resistance of the Cu-CB complexes towards demetallation [89]. With respect to ease of Cu(II)/Cu(I) reduction, cyclic voltammetric studies of Cu(II) complexes of a variety of tetraazamacrocyclic complexes revealed that Cu-CB-TE2A has a relatively negative reduction potential, which was observed to be quasi-reversible, and suggests the innate ability of the cross-bridged cyclam ligand to adapt to a geometry suitable for Cu(I) coordination [89].

Fig. (6).

Cross-bridged cyclic polyamine and cross-bridged derivatives.

Fig. (7).

Crystal structures of Cu(II)-TETA (left) and Cu(II)-CB-TE2A (right). The ethylene cross-bridge imparts a cis-folded conformation to the resulting copper complex. Atom color code: Cu, brown; N, blue; O, red; C, gray. Adapted with permission from reference 9. Copyright 2010 American Chemical Society.

This promising in vitro data led Sun et al. to label a series of CB-macrocycles with ⁶⁴Cu [146], and stability experiments performed in rat serum indicated the ⁶⁴Cu-CB-TE2A complex was the most stable undergoing rapid clearance from the blood, liver, and kidney. Boswell et al. elaborated on these studies by directly comparing the in vivo stability of CB-TE2A and 4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane (CB-DO2A (22)) to their non-cross-bridged analogues TETA and DOTA, respectively. Biodistribution studies in normal rats revealed that ⁶⁴Cu-CB-DO2A and ⁶⁴Cu-CB-TE2A had significantly lower amounts of activity associated with the blood, liver and kidney after 24 hrs than the non cross-bridged analogues [118]. For example, 20 h after injection, ⁶⁴Cu-TETA was 92% dissociated as compared to ⁶⁴Cu-CB-TE2A, which was only 24% dissociated. Additional metabolism studies revealed significantly less transchelation of ⁶⁴Cu to proteins such as SOD and metallothienein in the rat liver samples taken from rats receiving ⁶⁴Cu-CB-DO2A or ⁶⁴Cu-CB-TE2A. When comparing the two cross-bridged analogues, greater in vivo stability was observed with ⁶⁴Cu-CB-TE2A suggesting that the cyclam macrocycle in conjunction with the cross bridge provided greater enhancement of in vivo stability. Further validation of CB-TE2A as an excellent radiocopper chelator came from Sprague and colleagues when they compared the two similar radiopharmaceuticals ⁶⁴Cu-TETA-Y³-TATE and ⁶⁴Cu-CB-TE2A-Y³-TATE to evaluate the effects of the cross-bridged chelator on radiopharmaceutical performance in vivo [147]. Receptor binding studies demonstrated that both complexes had similar affinity for the somatostatin subtype 2 receptor (SSTR2). However, the cross-bridged analogue was able to bind 10 fold more receptor sites and observed to undergo more rapid internalization, which was attributed to the change in chelator geometry from trans (TETA) to cis (CB-TE2A). Biodistribution studies revealed that both complexes underwent rapid blood clearance, but injection of ⁶⁴Cu-CB-TE2A-Y³-TATE resulted in better tumor visualization, which was attributed to increased tumor uptake and lower non-specific uptake in non-target tissues.

Despite its enhanced stability in vivo, CB-TE2A requires rigorous labeling conditions for radiocopper incorporation, which precludes its use with a variety of biomolecules such as large proteins and intact antibodies [148]. To address this issue, further research has been directed at developing CB-TE2A analogues, which can be easily incorporated into large biomolecules or exhibit much faster reaction kinetics without sacrificing in vivo stability [149–151]. For example, Stigers et al. reported the synthesis and characterization of the phosphonate pendant armed cross-bridged cyclam chelator bis(phosphonylmethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2P (24)) and its related Cu(II) complex [152]. Cyclic voltammetry revealed a quasi-reversible reduction, while acid inertness studies demonstrated the half-life of the complex to be 4 h when studied in 5M HCl and at 90°C. Although approximately 35 fold less stable than Cu-CB-TE2A, it was observed to be significantly more inert than Cu-DOTA and Cu-TETA. The authors also reported facile radiolabeling with ⁶⁴Cu, and biodistribution studies in normal rats revealed that the ⁶⁴Cu-CB-TE2P complex underwent rapid blood clearance with only residual uptake in dose limiting organs such as the marrow suggesting it and its derivatives may allow for the development of protein and antibody based copper radiopharmaceuticals using these cross-bridged chelators.

CHEMICAL LINKERS IN RADIOPHARMACEUTICAL DEVELEOPMENT

Often during the course of radiopharmaceutical development initial study results indicate the addition of the radiometal chelate adversely affects the in vitro or in vivo properties of the radiopharmaceutical and modifications to the radiopharmaceutical may be needed to restore the desired attributes. Rather than attempting to modify the targeting ligand or radiometal chelate and risk reducing the affinity or stability of the radiopharmaceutical, a chemical modifier such as polyethylene glycol (PEG), an aliphatic carbon chain or polypeptide is introduced between the radiometal complex and the targeting biomolecule to minimize the negative effects imparted by attachment of the radiometal chelate [153–155]. This next section will discuss how chemical spacer can influence the pharmacological properties of a copper radiopharmaceutical using the development of bombesin radiopharmaceuticals as an example.

Bombesin (BN) a 14 aa peptide, which is an analogue of the human gastrin releasing peptide (GRP), consists of 27 amino acids, belongs to a family of brain gut peptides that share very similar activities and occur primarily in the central nervous system [156]. Since GRP receptors have been observed on a wide variety of human cancers including prostate and breast [156], they represent attractive targets for radiopharmaceutical development, and several BN analogues have been developed for radiopharmaceutical applications [157–169]. Rogers et al. described the preparation and evaluation of the ⁶⁴Cu labeled BN analogue, ⁶⁴Cu-DOTA-Aoc-BN(7–14) (Fig. (8)), which was observed to have a K_d of 6.1 nm [170]. Biodistribution studies in athymic nude mice bearing human prostate PC-3 tumors demonstrated excellent tumor uptake, which in some cases, surpassed the uptake of analogous radiopharmaceuticals containing other radiometals. Small animal PET imaging performed in the same mouse model demonstrated excellent GRP receptor mediated tumor uptake. However, enthusiasm was quickly tempered by the increased accumulation of radioactivity in non-target tissues such as the liver. Additional studies were deemed necessary to understand how the chemical structure of the linker between the radiometal conjugate and the BN peptide affected the in vivo biodistribution properties of this radiopharmaceutical. Structure activity studies, were later completed by Parry and coworkers when they evaluated the accumulation of radioactivity in the liver tissue of mice, which were injected with ⁶⁴Cu labeled BN analogues containing 4, 5, 6, 8, and 12 carbon linkers as shown in Fig. (8) [171]. Initial competition assays to evaluate the binding affinity of each analogue for the GRPR, demonstrated that DOTA-Aoc-BN(7–14) and DOTA-Ahx-BN(7–14) had the highest affinities of 7 and 16 nM respectively, while the DOTA-Ava-BN(7–14) conjugate had the worst GRPR affinity of 79 nM. Internalization studies, which were also performed on all complexes except those with the worst binding affinity, demonstrated significant internalization for all of the radiolabeled conjugates tested. However, the ⁶⁴Cu-DOTA-Ado-BN(7–14) complex underwent significantly less internalization than the other radiolabeled analogues, which was attributed to the length of the Ado linker being able to adopt a conformation within the GRPR that allowed for receptor binding but inhibited internalization. Biodistribution studies in SCID mice bearing T-47D human breast cancer xenografts revealed rapid blood clearance, for all complexes tested. Mice receiving ⁶⁴Cu-DOTA-Aoc-BN(7–14) had significantly more tumor associated activity than mice injected with the other radiolabeled BN analogues, but they also had greater liver associated activity, as well. Small animal PET studies revealed GRPR mediated tumor uptake with all of the conjugates tested, but also indicated that the conjugates containing smaller carbon spacers had lower tumor uptake leading to reduced image contrast. Additional attempts by Parry et al. to reduce radioactivity accumulation in the liver involved the incorporation of polypeptide linkers into the BN radiopharmaceutical architecture as shown in Fig. (8) [166]. Conjugates containing tripeptide linkers consisting of the amino acids glycine (G) serine (S) and glutamic acid (E) were prepared, radiolabeled with ⁶⁴Cu and evaluated in vitro and in vivo. Cell binding studies demonstrated that conjugates containing glutamic acid residues had significantly lower (worse) affinity for the GRP receptor, which was hypothesized to result from the disruption of the BN(7–14) peptide’s secondary structure by its interaction with the negatively charged glutamic acid residues at physiological pH. Biodistribution studies in SCID mice bearing PC-3 tumors revealed appreciable tumor uptake and reduced liver uptake in animals receiving the radiolabeled conjugates containing serine residues, and was attributed to the increased hydrophilicity they imparted to the radiopharmaceutical. Small animal PET/CT corroborated the results obtained during biodistribution studies and demonstrated good tumor delineation with lower accumulation of radioactivity in non-target tissues suggesting these radiopharmaceuticals are worthy of further study as GRPR targeting radiopharmaceuticals. Moreover, these studies provide an excellent example of how the in vivo properties of a radiopharmaceutical can be affected by the chemical structure of the incorporated linker.

Fig. (8).

Chemical linkers used to modify the in vivo characteristics of bombesin radiopharmaceuticals labeled with ⁶⁴Cu.

THE INFLUENCE OF PEPTIDE STRUCTURE ON COPPER RADIOPHARMCEUTICALS

While both proteins and peptides have been incorporated into copper based radiopharmaceuticals, peptide based radiotracers have increased in popularity in recent years since they offer superior clearance, in vivo biokinetics and metabolic stability when compared to larger biomolecules such as intact antibodies [172]. Additionally, their tolerance toward a diverse set of reaction conditions, chemical modifications, and their facile synthesis using solid phase methodologies has led researchers to incorporate a wide variety of peptide sequences into copper based radiopharmaceuticals [9,173,174]. Although a discussion of all of the peptide based Cu radiopharmaceutical that have currently been developed, is beyond the scope of this text, this final section will discuss the development of copper radiopharmaceuticals designed to target the somatostatin receptor (SSTR).

The SSTR is present in many different normal organ systems such as the central nervous system (CNS), the gastrointestinal tract, and the exocrine and endocrine pancreas [175], and currently five different somatostatin receptor subtypes have been discovered. These receptors are also expressed on several human tumors of the neuroendocrine system, CNS, breast and lung with receptor subtype expression being dependent on the origin of the tumor. Somatostatin is a 14 amino acid peptide, which is involved in the regulation and release of several hormones and is the natural ligand for the SSTR. However, this ligand has a short biological half-life, which has led to the development of several analogues that demonstrate increased resistance to degradation in vivo [156]. While initially these analogues were developed as therapeutics [176], their potential as diagnostic imaging agents was quickly realized leading to the evaluation of somatostatin analogues in preclinical and clinical studies using SPECT and PET [177–179].

Early development of somatostatin targeting copper radiopharmaceuticals focused on the in vitro and in vivo evaluation of ⁶⁴Cu-labeled octreotide (OC) derivatives [109–111,180] (Fig. (9)). For example, ⁶⁴Cu-TETA-OC was compared with ¹¹¹In-DTPA-D-Phe¹-OC (¹¹¹In-DTPA-OC; Octreoscan^®), a clinically approved SPECT imaging agent in humans (8 subjects) and to ¹¹¹In-DTPA-OC with gamma scintigraphy and SPECT imaging [111]. ⁶⁴Cu-TETA-OC and PET imaged more tumors in two patients compared to ¹¹¹In-DTPA-OC and SPECT, but in one patient ¹¹¹In-DTPA-OC and SPECT weakly imaged a lung lesion that was not detected with ⁶⁴Cu-TETA-OC. Overall, ⁶⁴Cu-TETA-OC and PET showed greater sensitivity for imaging neuroendocrine tumors, in part due to the greater sensitivity of PET compared to SPECT.

Fig. (9).

Somatostatin analogues used in somatostatin receptor targeting radiopharmaceuticals. R denotes chelator attachment.

In addition to these reports, Lewis et al. labeled the four somatostatin analogues TETA-D-Phe¹-octerotide (TETA-OC), TETA-Y³-OC, TETA-TATE and the second generation analogue TETA-Y³-TATE with ⁶⁴Cu to examine their targeted tissue uptake. Additionally, the studies were conducted to evaluate the effect that the single amino acid substitution of tyrosine (Tyr) for phenylalanine (Phe) at position 3 of the peptide sequence and the oxidation of the c-terminal threonine alcohol side chain to a carboxylic acid would have upon the in vivo properties of these ⁶⁴Cu-radiopharmceuticals [181]. All four conjugates demonstrated high binding affinity for the somatostatin subtype 2 receptor (SSTR2) and internalization, which suggested that the oxidation of the alcohol side chain may contribute to high affinity binding and G-protein coupled receptor (GPCR) activation due to enhanced hydrogen bonding within the receptor’s active site. Biodistribution studies in normal mice bearing AR42J rat pancreatic carcinoma tumors revealed that the tyrosine substitution and the modification at the c-terminus had a synergistic effect on uptake in vivo. Of the four conjugates tested, ⁶⁴Cu-TETA-Y³-TATE was observed to have the best in vivo properties balancing target tissue accumulation with non-specific uptake, which led to its further utilization as an imaging agent [147,182,183]. Additional modifications of the Y³-TATE sequence by Ginj and coworkers resulted in a more hydrophobic peptide, which was incorporated into the PET radiopharmaceutical ⁶⁴Cu-CB-TE2A-sst₂-ANT [184,185]. This radiotracer demonstrated high affinity binding for SSTR(2) but underwent minimal internalization, suggesting that the peptide conjugate acts as an antagonist (ANT) within the lipid rich environment of the receptor. Biodistribution studies in normal rats bearing SSTR(2) positive AR42J tumors revealed that animals injected with ⁶⁴Cu-CB-TE2A-sst₂-ANT had significant amounts of activity associated with the tumors, which cleared slowly over time. However, radiopharmaceutical clearance from non-target tissue such as the liver and kidney was much slower when compared to the agonist, ⁶⁴Cu-CB-TE2A-Y³-TATE. Despite this slow clearance from non-target tissue, small animal PET/CT using the same animal model revealed excellent tumor to background contrast 4 h after injection making this conjugate worthy of further study as a SSTR(2) targeting PET radiopharmaceutical, and illustrates how small changes in peptide structure can have a relatively large effect on the properties of the associated radiopharmaceutical.

CONCLUSION

The use of copper radionuclides for diagnostic imaging and radiotherapy has greatly increased over the last 25 years. However, effective copper radiotracers must be thoughtfully designed to optimize stability and targeting in vivo. For example, radiocopper chelate stability is paramount when developing tumor targeting radiopharmaceuticals so that optimum image quality can be achieved. However, the opposite is true when considering radiocopper radiopharmaceuticals for hypoxia imaging. This requires a thorough understanding of the biological process being targeted and how the chemical structure of the radiopharmaceutical being developed will influence its performance in vivo. The successful use of medicinal chemistry techniques in copper radiopharmaceutical development allows researchers to tailor a specific copper radionuclide with the biokinetics of a particular biological targeting molecule and manipulate its pharmacological properties. The result of these endeavors is the development of diagnostic and therapeutic radiopharmaceuticals, which can be tailored to individual disease processes and eventually translated to clinical applications.

ABBREVIATIONS

%IDG

Percent injected dose per gram

μA

Microamp

aa

Amino acid

ATSM

Diacetyl bis(N⁴-methylthiosemicarbazone)

BBB

Blood brain barrier

BFC

Bifunctional chelator

BN

Bombesin

CB

Cross-bridged

CB-DO2A

4,10-bis(Carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane

CB-TE2A

4,11-bis(Carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane

Ci

Curie

CN

Coordination number

CT

Computed tomography

diamSar

3,6,10,13,16,19-Hexaazabicyclo[6.6.6] eicosane-1,8-diamine

DOTA

1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid

DTPA

Diethylenetriaminepentaacetic acid

EDTA

Ethylenediaminetetraacetic acid

FDG

[¹⁸F]-Fluorodeoxy glucose

GBq

Gigabecquere

GRP

Gastrin releasing peptide

GRPR

Gastrin releasing peptide receptor

h

Hour(s)

hplc

High performance liquid chromatography

IM

cis,cis-1,3,5-Triamino-cyclohexane-N,N’,N”-tris(2-methyl-N-methyl imidazole)

kBq

Kilobecquerel

kDa

Kilodalton

keV

Kiloelectron volt

MBq

Megabecquerel

mCi

Millicurie

MeV

Megaelectron volt

mM

Milimolar

mmole

Millimole

MRI

Magnetic resonance imaging

MTD

Maximum tolerated dose

NCA

No carrier added

NET

Neuroendocrine tumor

NIR

Near infrared

OC

Octreotide

p.i.

Post injection

PEG

Polyethylene glycol

PET

Positron emission tomography

PTSM

Pyruvaldehyde bis(N⁴-methylthiosemicarbazone)

RIT

Radioimmunotherapy

RP-hplc

Reversed phase high performance liquid chromatography

Sar

3,6,10,13,16,19-Hexaazabicyclo[6.6.6]eicosane

SarAr

1-N-(4-Aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane-1,8-diamine

SCID

Severly combined immunodeficiency

SOD

Superoxide dismutase

SPECT

Single photon emission computed tomography

SST

Somatostatin

sst₂-ANT

Somatostatin receptor subtype 2 antagonist

SSTR

Somatostatin receptor

SUV

Standard uptake value

tachpyr

N,N’,N”-tris-(2-Pyridylmethyl)-1,3,5-cis,cis-triamino-cyclohexane

tachpyr-(6-Me)

N, N’, N”-tris-(6-Methyl-2-pyridylmethyl-1,3,5-cis,cis-triamino-cyclohexane

TATE

Octreotate

TE2A

1,4,8,11-Tetraazacyclotetradecane-1,8-diacetic acid

TETA

1,4,8,11-Tetraazacyclotetradecane-1,4,8,11-tetraacetic acid

V

Volts

Y³-OC

Tyrosine-3-octreotide

Y³-TATE

Tyrosine-3-octreotate

β⁻

beta - Particle

β⁺

Positron