Promising Bifunctional Chelators for Copper 64-PET imaging: Practical ⁶⁴Cu Radiolabeling and High In Vitro and In Vivo Complex Stability

Abstract

Positron emission tomography (PET) using copper-64 is a sensitive and non-invasive imaging technique for diagnosis and staging of cancer. A bifunctional chelator that can present rapid radiolabeling kinetics and high complex stability with ⁶⁴Cu is a critical component for targeted PET imaging. Bifunctional chelates 3p-C-NE3TA, 3p-C-NOTA, and 3p-C-DE4TA were evaluated for complexation kinetics and stability with ⁶⁴Cu in vitro and in vivo. Hexadentate 3p-C-NOTA and heptadentate 3p-C-NE3TA possess a smaller TACN-based macrocyclic backbone, while nonadentate 3p-C-DE4TA is constructed on a larger CYCLEN-based ring. The frequently explored chelates of ⁶⁴Cu, octadentate C-DOTA and hexadentate C-NOTA were also comparatively evaluated. Radiolabeling kinetics of bifunctional chelators with ⁶⁴Cu was assessed under mild conditions. All bifunctional chelates instantly bound to ⁶⁴Cu in excellent radiolabeling efficiency at room temperature. C-DOTA was less efficient in binding ⁶⁴Cu than all other chelates. All ⁶⁴Cu-radiolabeled bifunctional chelates remained stable in human serum without any loss of ⁶⁴Cu for 2 days. When challenged by an excess amount of EDTA, ⁶⁴Cu complexes of 3p-C-NE3TA and 3p-C-NOTA were shown to be more stable than ⁶⁴Cu-C-DOTA and ⁶⁴Cu-C-DE4TA. 3p-C-NE3TA and 3p-C-NOTA displayed comparable in vitro and in vivo complex stability to ⁶⁴Cu-C-NOTA. In vivo biodistribution result indicates that the ⁶⁴Cu-radiolabeled complexes of 3p-C-NOTA and 3p-C-NE3TA possess excellent in vivo complex stability, while ⁶⁴Cu-3p-C-DE4TA was dissociated as evidenced by high renal and liver retention in mice. The results of in vitro and in vivo studies suggest that the bifunctional chelates 3p-C-NOTA and 3p-C-NE3TA offer excellent chelation chemistry with ⁶⁴Cu for potential PET imaging applications.

Introduction

Positron emission tomography (PET) is a nuclear imaging technique that has been used for diagnostics and staging of various cancers [1–3]. Highly energetic photons released from positron-electron annihilation process make acquisition of PET images in high resolution feasible using a molecular probe in a picomolar range [3]. Fluorine-18 (t_1/2 = 110 min) has been extensively explored as a positron probe for PET imaging, and ¹⁸F-fluoro-2-deoxy-glucose (¹⁸F-FDG) is the most frequently used drug for PET imaging of cancer [1–3]. However, the short half-life of ¹⁸F and synthetic challenges in preparation of the ¹⁸F-radiolabeled molecular tracers remain limitations for broad applications of ¹⁸F-PET imaging. Among metallic radionuclides for PET imaging, copper-64 (t_1/2 = 12.7 h; E_max^β+ = 0.655 MeV; E_max^β− = 0.573 MeV; E_max^γ = 0.511 MeV) is of great interest due to its favorable physical properties [4–6]. ⁶⁴Cu can be readily produced in large quantities using a cyclotron and has longer half-life (t_1/2 = 12.7 h) than fluorine-18, and its β⁻ emission can be utilized for radiotherapy. The coordination chemistry of copper has been well studied and applied for the design of adequate chelates for efficient radiolabeling with ⁶⁴Cu [7–10]. Macrocyclic chelates including DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N‴-tetraacetic acid), and CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid) have been evaluated for ⁶⁴Cu-based PET imaging [7–9]. However, the currently available chelates of ⁶⁴Cu present limitations for practical use in PET applications including preparation of ⁶⁴Cu-radiolabeled chelates under harsh radiolabeling conditions and premature dissociation of ⁶⁴Cu from the complexes in vivo [7,9–13]. NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) is a TACN (1,4,7-Triazacyclononane)-based hexadentate chelate that is known to form a stable complex with ⁶⁴Cu in vitro and in vivo [14]. We previously reported NE3TA (7-[2-[carboxymethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid)-based heptadentate chelates containing both the macrocyclic TACN backbone and a pendant arm. The acyclic binding moiety appended to the TACN backbone was proposed to enhance complexation kinetics of the chelates by rapidly binding ⁶⁴Cu, which can be subsequently solvated by the donors in the TACN backbone to form a stable complex. The bifunctional NE3TA analogues rapidly formed a stable complex with ⁶⁴Cu [15–17]. The bifunctional chelates C-NE3TA (7-[2-[carboxymethyl)amino]-3-(4-nitrophenyl)propyl]-1,4,7-triazacyclononane-1,4-diacetic acid) and N-NE3TA (7-[2-[(carboxymethyl)amino]-2-(4-nitrophenyl)methyl]-1,4,7-triazacyclononane-1,4-diacetic acid) were shown to have superior in vivo stability than ⁶⁴Cu-C-DOTA complex.[15] Both C-NE3TA and N-NE3TA conjugated to a tumor targeting biomolecule (transferrin) rapidly bound to ⁶⁴Cu at room temperature [18]. N-NE3TA-trasnferrin conjugate was slightly more efficient in binding ⁶⁴Cu than C-NE3TA-transferrin conjugate. ⁶⁴Cu-radiolabeled N-NE3TA-transferrin conjugate remained in human serum for 48 h and displayed rapid blood clearance and increasing tumor uptake in PC-3 tumor bearing mice over a 24 h period [18].

In this study, the potential bifunctional chelates 3p-C-NOTA, 3p-C-NE3TA, and 3p-C-DE4TA (Figure 1) along with the standard chelates C-NOTA and C-DOTA were comparatively evaluated for radiolabeling kinetics and in vitro and in vivo complex stability with ⁶⁴Cu. TACN-based chelates 3p-C-NOTA and 3p-C-NE3TA contains six and seven coordinating groups, respectively. 3p-C-DE4TA with nine donors is constructed on relatively larger macrocyclic cyclen (1,4,7,10-tetraazacyclododecane) backbone. All of the chelates studied contain a longer propyl chain which connects the chelating backbone to the functional group (para-nitro-phenyl) for conjugation to a biomolecule. In contrast, C-NOTA and C-DOTA (2-(4-nitrobenzyl)-1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid) contains a methyl spacer. The relatively long alkyl spacer of the present chelates was proposed to reduce steric hindrance in metal complexation of the chelate conjugated to a protein. The bifunctional chelates were evaluated for radiolabeling kinetics with ⁶⁴Cu, and in vitro stability of ⁶⁴Cu-radiolabeled complexes was evaluated in human serum and excess EDTA solution. In vivo stability of ⁶⁴Cu-radiolabeled complexes was also assessed by conducting a biodistribution study in mice.

Figure 1. Bifunctional Chelators of ⁶⁴Cu in preclinical evaluation for targeted PET imaging.

Experimental

Instruments and materials

Analytical HPLC was performed on an Agilent 1200 equipped with a diode array detector (λ = 254 and 280 nm), thermostat set at 35 °C, and a Zorbax Eclipse XDB-C18 column (4.6 × 150 mm, 80 Å). Ultrapure ammonium acetate was purchased from Aldrich. Ultrapure HCl was purchased from Fisher Scientific. Radio-TLC was performed on a TLC scanner (Bioscan, B-FC-1000). ⁶⁴Cu in the chloride form (specific activity of 248 mCi/μg) was obtained from Washington University (Saint. Louis, MO).

Complexation formation kinetics of chelates with ⁶⁴Cu

Bifunctional chelates 3p-C-NOTA, 3p-C-NE3TA, 3p-C-DE4TA were prepared as previously reported [19]. C-DOTA were purchased from Macrocyclics (Dallas, TX). C-NOTA in the form of NH₂ was prepared by the method reported in the literature [20]. All HCl solutions were prepared from ultrapure HCl (Fisher Scientific). For metal-free radiolabeling, plasticware including pipette tips, tubes, and caps was soaked in 0.1M HCl overnight and washed thoroughly with Milli-Q (18 MΩ) water, and air-dried overnight. Ultrapure ammonium acetate was used to prepare buffer solutions (0.25 M, pH 5.5). 0.25M NH₄OAc buffer solution (pH 5.5) using 0.1M and 1M HCl solution was prepared and chelexed, shaken overnight at room temperature, and filtered through 0.22μM filter (Corning) prior to use. The stock solution of the radioisotopes (0.013 μL, 9 mCi) was diluted to 125 μL by adding 0.05M HCl solution (112 μL). TLC plates (6.6 × 1 cm or 6.6 × 2 cm, Silica gel 60 F₂₅₄, EMD Chemicals Inc., #5554-7) with the origin line drawn at 0.6 cm from the bottom were prepared. To a buffer solution (0.25M NH₄OAc, pH 5.5) in a capped microcentrifuge tube (1.5 mL) was sequentially added a solution of the chelator in the buffer (30 μg) and ⁶⁴Cu in 0.05M HCl (90 μCi). The total volume of the resulting solution was 60 μL. The reaction mixture was agitated on the thermomixer (Eppendorf, 5355) set at 1,000 rpm at room temperature for 30 min. The labeling efficiency was determined by TLC eluted with 20 mM EDTA/0.15M NH₄OAc as the mobile phase. A solution of radiolabeled complexes (2.0 μL) was withdrawn at the designated time points (1 min, 10 min, and 30 min), spotted on a TLC plate, and then eluted with the mobile phase. After completion of elution, the TLC plate was warmed and dried on the surface of a hot plate maintained at 35 °C and scanned using TLC scanner (Bioscan, B-FC-1000). Bound and unbound copper-64 appeared 30~40 mm (R_f = 0.54) and 48~55 mm (R_f = 0.94).

In vitro serum stability of ⁶⁴Cu-radiolabeled complexes

Human serum was purchased from Gemini Bioproducts (#100110). ⁶⁴Cu-radiolabeled complexes were prepared by reaction of the chelators (30 μg) with ⁶⁴Cu (90 μCi) in 0.25M NH₄OAc buffer (pH 5.5) for 1 h at room temperature or 90 °C (for C-DOTA). The total volume of the final solution was 10 μL. The labeling efficiency of the radiolabeled complexes were found to be ~100% as determined by TLC. The freshly prepared radiolabeled complexes were directly used for serum stability studies without further purification. ⁶⁴Cu-radiolabeled complex (~75 μCi, ~ 10 μL) was added to human serum (90 μL) in a microcentrifuge tube. ⁶⁴Cu-3p-C-DE4TA were prepared by reaction of the chelators (10 μg) with ⁶⁴Cu (30 μCi) in 0.25M NH₄OAc buffer (pH 5.5) for 1 h at room temperature. The total volume of the final solution was 20 μL. The labeling efficiency of the radiolabeled complexes were found to be ~100% as determined by TLC. ⁶⁴Cu-radiolabeled complex (~7 μCi, ~ 20 μL) was added to human serum (60 μL) in a microcentrifuge tube. The stability of ⁶⁴Cu-radiolabeled complexes in human serum was evaluated at 37 °C for 2 days using TLC and HPLC (solvent A: 0.1% TFA in H₂O, solvent B: 0.1% TFA in CH₃CN, 0–100% B/15 min, flow rate: 1 mL/min). ⁶⁴Cu-EDTA complex was eluted early (t_R = ~ 2.5 min), while ⁶⁴Cu-radiolabeled complexes of the chelators have retention time at 8~8.3 min.

EDTA Challenge

⁶⁴Cu-radiolabeled complexes were prepared by reaction of each chelator (10 μg) with ⁶⁴Cu (30 μCi) in 0.25M NH₄OAc buffer (pH 5.5) for 1 h at room temperature. The total volume of the resulting solution was 20 μL. The freshly prepared radiolabeled complexes were directly used for EDTA challenge experiment without further purification. A solution of the ⁶⁴Cu-radiolabeled complex containing each chelator (1 mM) in 0.25M NH₄OAc buffer was mixed with a solution of EDTA (100 mM, H₂O, pH 5.0) at a 100-fold molar excess. The resulting mixture was incubated for 24 h at 37 °C. The stability of ⁶⁴Cu-radiolabeled complexes in the solution was evaluated using TLC (20 mM EDTA in 0.15M NH₄OAc). Bound and unbound radioisotope appeared 30~40 mm (R_f = 0.54) and 48~55 mm (R_f = 0.94) from the bottom of the TLC plate, respectively. Stability of the complexes was also evaluated at 25 h time point by HPLC (solvent A: 0.1% TFA in H₂O, solvent B: 0.1% TFA in CH₃CN, 0–100% B/15 min, flow rate: 1 mL/min). ⁶⁴Cu-EDTA complex was eluted early (t_R = ~ 2.5 min). ⁶⁴Cu-radiolabeled complex of 3p-C-NE3TA, 3p-C-NOTA, 3p-C-DE4TA, C-NOTA (in the NH₂ form), and C-DOTA have the respective retention time (t_R = 8.2, 8.0, 7.8, 4.2, 7.5 min).

Biodistribution

All animal experiments were conducted in accordance with the guidelines established by the Animal Care and Use Committee of the University of Missouri and the Harry S. Truman Memorial Veterans’ Hospital Subcommittee for Animal Studies. Six to eight week old CF-1 mice were obtained from Charles River Laboratories and housed one week prior to the studies. An aliquot of ⁶⁴Cu-radiolabeled complex (60 μCi) was prepared as described above and intravenously injected via the tail vein in phosphate-buffered saline (100 μL). At 1 h, 4 h and 24 h post-injection, mice were sacrificed and blood, liver, kidney, muscle and bone were collected, weighed and counted in a gamma counter. The radioactivity from each tissue/organ was decay-corrected by a known aliquot of the injected dose, and the percent-injected dose per gram of tissue (% ID/g) was calculated. Values are presented as mean ± SD for each group of 3 mice.

Results and Discussion

The new chelates 3p-C-NE3TA, 3p-C-NOTA, and 3p-C-DE4TA were evaluated for complexation with ⁶⁴Cu. Among the known bifunctional chelators of ⁶⁴Cu, C-DOTA and C-NOTA were selected for the present study on comparative evaluation for radiolabeling with ⁶⁴Cu at room temperature. C-NOTA rapidly forms a stable complex with ⁶⁴Cu and possesses superior radiolabeling kinetics and complex stability with ⁶⁴Cu than C-DOTA [14,15]. C-NOTA analogues have been extensively explored for development of ⁶⁴Cu-based radiopharmaceuticals [21–23]. Radiolabeling efficiency of the chelates in binding ⁶⁴Cu was measured at room temperature in duplicate using TLC and HPLC (Table 1). The new chelates 3p-C-NE3TA, 3p-C-NOTA, and 3p-C-DE4TA instantly bound to ⁶⁴Cu at room temperature and pH 5.5, and radiolabeling was almost complete at the beginning of the measurement (>99% radiolabeling efficiency, 3 μCi/μg specific activity, 1 min, RT). As expected, C-NOTA was extremely rapid in binding ⁶⁴Cu, while C-DOTA display slower radiolabeling kinetics when compared to all other chelates studied (Table 1). 3p-C-NETA, 3p-C-NOTA, and C-NOTA completely bound to Cu-64 at 30 min as evidenced by both TLC and HPLC analysis. However, radiolabeling of C-DOTA with ⁶⁴Cu was incomplete at 30 min time point. The noted difference in Cu-64 radiolabeling efficiency of C-DOTA measured by TLC (95.4%) and HPLC (88.6%) seems to be related to formation of less stable ⁶⁴Cu-C-DOTA complexes that can be dissociated during TLC and HPLC analysis.

Table 1.

Evaluation of bifunctional ligands for radiolabeling efficiency (%) with ⁶⁴Cu (room temperature, 0.25M NH₄OAC, pH 5.5) using TLC and HPLC (parentheses).⁺

Bound 64Cu complex (%)
Time	3p-C-NOTA	3p-C-NE3TA	3p-C-DE4TA	C-NOTA*	C-DOTA
1 min	99.7 ± 0.2	99.9 ± 0.0	99.6 ± 0.3	99.8 ± 0.1	92.4 ± 0.7
10 min	99.9 ± 0.1	99.8 ± 0.1	99.9 ± 0.1	99.8 ± 0.1	94.6 ± 0.4
30 min	100.0 ± 0.1 (99.1 ± 0.8)	100.0 ± 0.1 (100.0 ± 0.0)	100.0 ± 0.1	99.9 ± 0.1 (100.0 ± 0.1)	95.4 ± 0.1 (88.6 ± 0.4)

⁺Radiolabeling efficiency was measured in duplicate;

^*C-NOTA in the NH₂ form

The ⁶⁴Cu-radiolabeled chelates were further evaluated for complex stability in human serum and excess EDTA solution. Copper-64 complexes of 3p-C-NOTA, 3p-C-NE3TA, 3p-C-DE4TA, and C-NOTA were readily prepared by reaction of each chelator with ⁶⁴Cu for 1 h at room temperature (pH 5.5, 100% radiolabeling efficiency, TLC). However, complete radiolabeling of C-DOTA with ⁶⁴Cu required significantly longer time (5 h) at room temperature. Therefore, ⁶⁴Cu-C-DOTA for the in vitro complex stability studies was prepared from reaction of C-DOTA with ⁶⁴Cu for 1 h at 90 °C.

Human serum stability studies of ⁶⁴Cu-radiolabeled chelates were performed to determine if the complexes remained stable without loss of ⁶⁴Cu (37 °C, pH 7). Stability was assessed by measuring the transfer of ⁶⁴Cu from the complex to serum proteins over the course of 2 days using TLC and HPLC (Table 2 and Supporting Information). All ⁶⁴Cu-radiolabeled chelates remained stable in human serum without ⁶⁴Cu being released into the serum over 2 days. Stability of ⁶⁴Cu-radiolabeled complexes was further evaluated by challenging the complexes with EDTA at 100-fold molar excess (Table 3, Figure 2, and Supporting Information). ⁶⁴Cu-3p-C-NOTA, ⁶⁴Cu-3p-C-NE3TA, and C-NOTA remained intact in the EDTA solution for 24 h of incubation. The ⁶⁴Cu complex of 3p-C-DE4TA with nine donors was not able to hold ⁶⁴Cu stably, and a significant amount of ⁶⁴Cu was transchelated from the chelate to EDTA over a 24 h incubation time (~55%). Copper-64-C-DOTA released >5% of ⁶⁴Cu into the EDTA solution.

Table 2.

Evaluation of ⁶⁴Cu-radiolabeled complexes for in vitro stability in human serum (37 °C, pH 7) using TLC and HPLC (parentheses).⁺

Bound 64Cu complex (%)
Time	3p-C-NE3TA	3p-C-NOTA	3p-C-DE4TA	C-NOTA*	C-DOTA
0 h	99.9 ± 0.1 (99.8 ± 0.3)	99.9 ± 0.1 (99.8 ± 0.1)	99.9 ± 0.0 (99.9 ± 0.1)	99.9 ± 0.0 (100.0 ± 0.0)	99.8 ± 0.1 (100.0 ± 0.0)
24 h	99.8 ± 0.3 (99.9 ± 0.1)	100.0 ± 0.1 (99.9 ± 0.1)	100.0 ± 0.0 (99.5 ± 0.3)	99.8 ± 0.1 (99.9 ± 0.2)	99.8 ± 0.2 (99.7 ± 0.1)
48 h	100.0 ± 0.1 (99.9 ± 0.1)	99.9 ± 0.0 (99.9 ± 0.2)	99.9 ± 0.1 (100.0 ± 0.0)	99.9 ± 0.1 (99.9 ± 0.1)	100.0 ± 0.1 (99.6 ± 0.1)

⁺bound ⁶⁴Cu complex (%) was measured in duplicate;

^*C-NOTA in the NH₂ form

Table 3.

EDTA challenge of chelators with ⁶⁴Cu (37 °C, 0.25M NH₄OAC, pH 5.5) using TLC and HPLC (parentheses).⁺

Bound 64Cu complex (%)
Time	3p-C-NE3TA	3p-C-NOTA	3p-C-DE4TA	C-NOTA*	C-DOTA
0 h	99.9 ± 0.1	100.0 ± 0.1	99.9± 0.2	99.9 ± 0.0	99.9 ± 0.0
1 h	99.9 ± 0.1	100.0 ± 0.1	98.0 ± 0.8	99.9 ± 0.1	99.6 ± 0.0
24 h	99.2 ± 0.1 (97.4 ± 0.4)	99.6 ± 0.4 (98.0 ± 0.2)	56.5 ± 1.1 (50.5 ± 0.9)	99.7 ± 0.0 (99.0 ± 0.2)	97.0 ± 0.5 (94.3 ± 0.4)

⁺bound ⁶⁴Cu complex (%) was measured in duplicate;

^*C-NOTA in the NH₂ form

Figure 2. HPLC chromatograms of ⁶⁴Cu-radiolabeled complexes in a solution of EDTA at 100-fold molar excess at 24 h post-incubation.

(eluent: 0.1% TFA in H₂O, 100% to 0%/0.1% TFA in ACN, 0% to 100%).

The in vivo stability of ⁶⁴Cu-radiolabeled chelates was evaluated by a biodistribution study using non-tumor bearing CF-1 mice (i.v. injection, n = 3). After injection of ⁶⁴Cu-radiolabeled chelates, radioactivity accumulation in harvested organs and blood was measured at 1 h, 4 h, and 24 h post-injection (Figure 3 and Supporting Information). ⁶⁴Cu-3p-C-NE3TA almost completely disappeared from the blood and all organs at 24 h post injection. The level of the radioactivity accumulated in the blood and in the organs was negligible (<0.2% ID/g) at 24 h post injection. The highest uptake of the tracer was observed in the kidney at 1 h post-injection (3.0% ID/g). The liver (2.5% ID/g at 1 h) cleared quickly, having less than 0.1% ID/g remaining at 24 h. No considerable accumulation of the tracer was detected in blood, muscle, and bone during the period of study. Copper-64-3p-C-NOTA displayed its highest uptake in the liver at 1 h (3.6% ID/g). However, the tracer rapidly disappeared by 24 h (<0.6% ID/g). The radioactivity retained in the kidney at 1 h (1.1% ID/g) cleared out by 24 h. Uptake of radioactivity in the blood and in the other organs was not significant at any time point. Significant radioactivity was released from ⁶⁴Cu-3p-C-DE4TA and accumulated in liver and kidney at 1 h (14.3% and 8.2%, respectively), and the radioactivity was retained in these organs at 24 h (11.1% and 5.7%, respectively). Blood, bone, and muscle had low levels of the tracer at 24 h (1.5%, 1.4%, and 0.5%, respectively). The biodistribution data indicate that 3p-C-NOTA and 3p-C-NE3TA cleared well in vivo but ⁶⁴Cu-3p-C-DE4TA showed substantially higher uptake and retention in the key organs. The instability of the DE4TA complex to EDTA challenge leads us to believe that the chelate dissociated in vivo.

Figure 3. Biodistribution of ⁶⁴Cu-radiolabeled bifunctional chelates in non-tumor bearing CF-1 mice.

Cu(II) with the prevalent oxidation state of +2 has a small ionic radius (73 pm) for coordination number 6 [24]. The borderline hard and soft Cu(II) is reported to adopt five or six coordination and display high affinity for nitrogen, oxygen, or sulfur donors such as aromatic nitrogens, thiol, amide, aminocarboxylate, and hydroxyl group [7,25.26].

The macrocyclic chelate NOTA with three nitrogens and three oxygen donors forms a stable hexacoordinate complex [25,27]. Hexa-coordination of Cu(II) with the four nitrogens in the cyclen backbone and two pendant carboxylate oxygens in DOTA produced Cu(II)-DOTA complex with high thermodynamic stability [28]. Copper-64-NOTA was reported to have more favorable complex stability and resistance to transchelation than ⁶⁴Cu-DOTA in vivo [15,21–23,29]. It is speculated that the higher kinetic stability of ⁶⁴Cu-NOTA relative with ⁶⁴Cu-DOTA stems from more favorable size-fit of Cu(II) to the smaller ring TACN.

The complexation radiolabeling and EDTA challenge data confirm that C-DOTA with a larger macrocyclic cavity and higher denticity forms less stable complex with Cu(II) than the TACN-based hexadentate C-NOTA. It should be noted that ⁶⁴Cu-C-DOTA complex for the present studies of in vitro stability was prepared from radiolabeling of C-DOTA with ⁶⁴Cu at 90 °C for 1 h, while all other chelates were well labeled with ⁶⁴Cu at RT. Under the harsh radiolabeling condition, C-DOTA was expected to form thermodynamically more stable complex as reaction of C-DOTA at room temperature could produce less stable intermediate ⁶⁴Cu-C-DOTA complexes [30]. However, ⁶⁴Cu-C-DOTA was found to be less stable than C-NOTA and other TACN-based chelates 3p-C-NOTA and 3p-C-NE3TA when challenged by excess EDTA.

In our previous studies, C-DOTA and C-NOTA were comparatively evaluated for in vivo complex stability with ⁶⁴Cu, and C-NOTA displayed superior in vivo stability than C-DOTA [15]. Copper-64-labeled C-DOTA was shown to have higher uptake in all tissues at all time points than ⁶⁴Cu-C-NOTA (1, 4, and 24 h post-injection, n =4, female SCID mice). Particularly, significant radioactivity was retained in the liver for ⁶⁴Cu-C-DOTA (78% at 24 h when compared to 1 h), while ⁶⁴Cu-C-NOTA displayed lower uptake in the liver at all time points (3% retention at 24 h when compared to 1 h) [15].

C-NOTA is built on a small macrocyclic cavity which is expected to be less dynamic in binding a metal than acyclic chelators [20,31]. 3p-C-NE3TA contains the same TACN backbone as NOTA [19]. In virtue of the acyclic bidentate arm, 3p-C-NE3TA was expected to form a complex with Cu(II) more rapidly. However, the predicted dynamic binding of 3p-C-NE3TA with Cu(II) using seven donors could form a less stable complex than C-NOTA. The in vitro and in vivo data indicate that 3p-C-NE3TA was extremely rapid in binding Cu(II) and formed a stable complex with Cu(II), and ⁶⁴Cu-3p-C-NE3TA was well tolerated in the EDTA challenge and produced excellent biodistribution profile in mice. ⁶⁴Cu-3p-C-NE3TA displayed rapid clearance from the liver and kidney and negligible retention in all organs at 24 time point. 3p-C-NE3TA displayed comparable chelation chemistry with ⁶⁴Cu to C-NOTA. As expected, the CYCLEN-based 3p-C-DE4TA with nine donors rapidly bound to Cu(II) but formed a complex with very low kinetic stability as evidenced by EDTA challenge and biodistribution data. Both C-DOTA and 3p-C-DE4TA are structured on the same macrocyclic backbone (CYCLEN). C-DOTA with 8 donors was found to form kinetically more inert complex with ⁶⁴Cu than 3p-C-DE4TA with 9 donors. ⁶⁴Cu-C-DOTA was more resistant to transchelation with EDTA than ⁶⁴Cu-3p-C-DE4TA. Higher retention of ⁶⁴Cu in the liver and kidney was observed with ⁶⁴Cu-3p-C-DE4TA (11.0% and 5.7% ID/g, respectively, Figure 3 and Supporting Information) when compared to ⁶⁴Cu-C-DOTA (3.8% and 2.4%, respectively) [15]. It appears that the enhanced kinetic stability of ⁶⁴Cu-C-DOTA relative to ⁶⁴Cu-3p-C-DE4TA is partly attributed to denticity of the chelates.

Conclusion

The bifunctional chelators 3p-C-NE3TA, 3p-C-NOTA, and 3p-C-DE4TA were evaluated for their potential use in ⁶⁴Cu-based PET imaging applications. All the chelators instantly and almost completely bound to ⁶⁴Cu at room temperature. The corresponding ⁶⁴Cu-radiolabeled chelates showed high in vitro serum stability, releasing no considerable radioactivity over 2 days. Copper-64 complexes of hexadentate (3p-C-NOTA) and heptadentate (3p-C-NE3TA) remained quite stable when challenged with EDTA. However, copper-64 complexes of C-DOTA (octadentate) and 3p-C-DE4TA (nonadentate) released a considerable amount of ⁶⁴Cu, which was gradually transchelated to EDTA. ⁶⁴Cu-3p-C-NOTA and ⁶⁴Cu-3p-C-NE3TA produced excellent biodistribution profiles and displayed low retention in all organs and blood over the entire period of the study. ⁶⁴Cu-radiolabeled 3p-C-DE4TA with nine donors was shown to undergo dissociation in vivo and have high retention in the liver. The in vitro and in vivo results suggest that 3p-C-NE3TA and 3p-C-NOTA possess superior chelation chemistry with ⁶⁴Cu relative to 3p-C-DEPTA and C-DOTA structured on the cyclen backbone and great promise for development of molecular PET imaging probes.