Novel ⁶⁴Cu-Radiolabeled Bile Acid Conjugates for Targeted PET Imaging

Abstract

A promising bifunctional chelate (N-NE3TA) was conjugated to bile acids, cholic acid (CA), deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA) as tumor targeting vectors. Bile acid conjugates of N-NE3TA (CA-N-NE3TA, DCA-N-NE3TA, and CDCA-N-NE3TA) were comparatively evaluated for complexation with ⁶⁴Cu, an imaging probe for positron emission tomography (PET). N-NE3TA-bile acid conjugates were evaluated for radiolabeling kinetics with ⁶⁴Cu, and the corresponding ⁶⁴Cu-radiolabeled conjugates were screened for complex stability in human serum and EDTA solution. The NE3TA-bile acid conjugates instantly bound to ⁶⁴Cu with excellent radiolabeling efficiency at room temperature. All NE3TA-bile acid conjugates radiolabeled with ⁶⁴Cu remained inert in human serum for 2 days without releasing a considerable amount of the radioactivity. The ⁶⁴Cu-radiolabeled complexes were further challenged by EDTA in a 100-fold molar excess. Bile acid-N-NE3TA conjugates radiolabeled with ⁶⁴Cu were quite stable with a minimal transfer of ⁶⁴Cu to EDTA at 4 h time point. The in vitro data indicate that the bile acid-N-NE3TA conjugates deserve further biological evaluation for ⁶⁴Cu-based targeted PET imaging applications.

A sensitive diagnostic modality, positron emission tomography (PET) has been demonstrated to give highly sensitive detection and staging of cancers.^1–3 Metallic radionuclides such as ⁶⁴Cu, ⁶⁸Ga, and ⁸⁶Y have been explored for PET imaging. Among the radionuclides, ⁶⁴Cu (t_1/2 = 12.7 h; E_max^β+ = 0.655 MeV; E_max^β− = 0.573 MeV; E_max^γ = 0.511 MeV) possesses half-life and decay property suitable for PET imaging with extended imaging window.^1,4,5 For development of clinically viable ⁶⁴Cu-based radiopharmaceuticals for targeted PET imaging, it is essential to employ a bifunctional chelate that can rapidly form a stable complex with Cu(II).^5–7 Rapid radiolabeling of ⁶⁴Cu with a short half-life by a bifunctional chelate attached to a sensitive biomolecule such as antibodies is required for practical preparations of biologically active ⁶⁴Cu-radiolabeled complexes. ⁶⁴Cu-radiolabeled complex must be stable in vivo without undergoing transchelation with other metal-binding proteins or biologically important metals. Cu(II) has a relatively small ionic radius (73 ppm) and is known to display a high affinity for nitrogen and oxygen donor atoms. Various acyclic and macrocyclic polyaminocarboxylate-based chelates including DTPA (diethylenetriamine pentaacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane tetraacetic acid), and TETA (2-[1,4,8,11-tetraazacyclotetradecane tetraacetic acid) have been explored for PET imaging applications using ⁶⁴Cu.^1,4,5

We previously reported a bifunctional chelate N-NE3TA (Figure 1) containing both acyclic and macrocyclic binding moieties as a promising chelate of ⁶⁴Cu.⁸ N-NE3TA rapidly bound to ⁶⁴Cu under mild conditions, and in vitro and in vivo stability of ⁶⁴Cu-N-NE3TA was favorably compared to ⁶⁴Cu-radiolabeled complex of C-DOTA, one of the most frequently used chelate for PET imaging.⁸ Encouraged by the complexation kinetics and stability profile of N-NE3TA with ⁶⁴Cu, we were interested in utilizing the bifunctional chelate for targeted PET imaging using a tumor targeting vector. The primary bile acids (cholic acid and chenodeoxycholic acid) and secondary bile acid (deoxycholic acid) are known to target bile acid receptors or carriers overproduced in hepatic and colorectal cancers.^9–12 The amphifacial bile acids were shown to form helical globular aggregates and enter into the cancer cells due to their great cell permeability and have been explored as a delivery shuttle of anti-cancer agents.^10–12

Figure 1.

Structure of N-NE3TA and N-NE3TA-Bile Acid Conjugates

We herein report synthesis of bile acid conjugates of N-NE3TA and evaluation of the corresponding bile acid-NE3TA conjugates for complexation with ⁶⁴Cu for targeted PET imaging. The bifunctional chelate N-NE3TA was conjugated to tumor-targeting bile acids, cholic acid (CA), deoxycholic acid (DCA), or chenodeoxycholic acid (CDCA). The bile acid conjugates were evaluated for radiolabeling kinetics with ⁶⁴Cu for PET imaging application. ⁶⁴Cu-radiolabeld bile acid conjugates were evaluated for complex stability in human serum and a solution of EDTA.

Synthesis of bifunctional N-NE3TA analogue 5 and N-NE3TA-bile acid conjugates 10-12 is shown in Scheme 1. Compound 2 was readily prepared from Swern oxidation of 1¹³, and reductive amination of 2 with 3¹⁴ provided the key precursor macrocyclic compound 4¹³. The nitro group in 4 was converted to the amino group in 5 which further was reacted with an activated bile acid analogue 6a, 6b, or 6c which were prepared from reaction of bile acid with 2-mercaptothiazoline as reported previously.¹⁵ tert-Butyl N-NE3TA-NH₂ (5) was reacted with the preactivated cholic acid analogue (CA, 6a) in the presence of triethylamine under reflux to provide N-NE3TA-CA conjugate 7. Similarly, N-NE3TA-DCA and N-NE3TA-CDCA analogues 8 and 9 were prepared from reaction of 5 with 6b and 6c, respectively. The removal of tert-butyl protecting groups in 7-9 using 4M HCl (g) in 1,4 dioxane provided N-NE3TA-CA (10), N-NE3TA-DCA (11) and N-NE3TA-CDCA (12), respectively.

Scheme 1.

Synthesis of N-NE3TA analogues and N-NE3TA-bile acid conjugates

The new N-NE3TA-bile acid conjugates were evaluated for radiolabeling reaction kinetics with ⁶⁴Cu at room temperature (Table 1, Figure 2, and Supporting Information). Each conjugate (20 μg) in 0.25M NH₄OAc (pH 5.5) was radiolabeled with ⁶⁴Cu (60 μCi) at room temperature. During the reaction time (30 min), the components were withdrawn at the designated time points (1 min, 10 min, and 30 min), and the radiolabeling efficiency (%) was determined using ITLC (20 mM EDTA in 0.15M NH₄OAc). ⁶⁴Cu-EDTA migrated with the solvent front on TLC (R_f = 0.88), while ⁶⁴Cu-radiolabeled chelator complexes travel slower on the TLC (R_f = 0.54). The ⁶⁴Cu-radiolabeled complexes of the conjugates and ⁶⁴Cu-EDTA were well separated on the ITLC. All N-NE3TA bile acid conjugates instantly bound to ⁶⁴Cu with excellent radiolabeling efficiency (>90%, 1 min time point, Table 1, NFigure 2a) at room temperature. -NE3TA-DCA was slightly slower in binding ⁶⁴Cu as compared to N-NE3TA-CA and N-NE3TA-CDCA, although radiolabeling of the conjugate with ⁶⁴Cu was nearly complete at 30 min time point. All ⁶⁴Cu-radiolabeled complexes were shown to be stable against EDTA present in the eluent of TLC (Supporting Information).

Table 1.

^* Evaluation of bile acid conjugates for radiolabeling efficiency (%) with ⁶⁴Cu (RT, 0.25M NH₄OAC, pH 5.5) using ITLC.

Time	N-NE3TA-CA	N-NE3TA-DCA	N-NE3TA-CDCA
1 min	99.7 ± 0.3	89.6 ± 3.8	97.6 ± 0.3
10 min	99.9 ± 0.1	97.0 ± 0.4	98.2 ± 0.4
30 min	99.9 ± 0.1	98.9 ± 0.5	99.2 ± 0.1

^*Radiolabeling efficiency (mean ± standard deviation%) was measured in triplicate using ITLC (eluent: 20 mM EDTA in 0.15M NH₄OAc)

Figure 2.

(a) TLC chromatogram of ⁶⁴Cu-radiolabeled complexes at 1 min time point of radiolabeling; (b) TLC chromatogram of ⁶⁴Cu-radiolabeled complexes in a solution of EDTA at 100-fold molar excess (1 h time point); (c) TLC chromatogram of ⁶⁴Cu-radiolabeled complexes in a solution of EDTA at 100-fold molar excess at 24 h; (d) HPLC chromatogram of ⁶⁴Cu-radiolabeled complexes in human serum at 48 h.

In vitro serum stability of the radiolabeled N-NE3TA-bile acid conjugates was performed to determine if the ⁶⁴Cu-radiolabeled conjugates remained stable without loss of the radioactivity in human serum. This was assessed by measuring the transfer of ⁶⁴Cu from the complex to human serum proteins using radio-HPLC (Table 2, Figure 2d, and Supporting Information). A fresh solution of ⁶⁴Cu-radioabeled conjugates were readily prepared from the reactions of N-NE3TA-bile acid conjugates with ⁶⁴Cu at room temperature and directly used for serum stability studies (pH 7, 37 °C) using radio-HPLC by radio-HPLC analysis (solvent A: 0.1% TFA in H₂O, solvent B: 0.1% TFA in CH₃CN, 0–100% B/15 min, flow rate: 1 mL/min). The trace related to ⁶⁴Cu bound to serum (t_R = 2.5 min) was clearly distinguished from the peaks of the ⁶⁴Cu-N-NE3TA-bile acid conjugate (t_R = 11–21 min). All ⁶⁴Cu-radiolabeled conjugates remained quite stable in human serum for 2 days as evidenced by radio-HPLC. While a tiny amount of ⁶⁴Cu (<1.0%) was detected from ⁶⁴Cu-N-NE3TA-CA and ⁶⁴Cu-N-NE3TA-CDCA over 2 days, ⁶⁴Cu-N-NE3TA-DCA released ~ 3% of the radioactivity at 48 h time points (Figure 2d) and appears to be least stable in serum among the ⁶⁴Cu-radioabeled conjugates tested.

Table 2.

^* Evaluation of ⁶⁴Cu-radiolabeld bile acid conjugates for in vitro stability in serum (37 °C, pH 7) using radio-HPLC.

Day	N-NE3TA-CA	N-NE3TA-DCA	N-NE3TA-CDCA
0	98.0 ± 0.3	97.3 ± 0.8	97.7 ± 0.2
1	97.7 ± 0.3	95.5 ± 1.3	98.7 ± 0.1
2	99.0 ± 0.4	97.0 ± 1.0	99.7 ± 0.3

^*bound complex (mean ± standard deviation%) was measured in duplicate using radio-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-radiolabeled N-NE3TA-bile acid conjugates were further challenged for complex stability in an excess amount of EDTA solution. ⁶⁴Cu-radiolabeled complexes were treated with a solution of EDTA at a 100-fold molar excess, and the resulting solution (pH 5.5) was incubated at 37 °C for 24 h. A sample was withdrawn at different time points (0 h, 1 h, 4 h, and 24 h) and analyzed using ITLC (Table 3, Figures 2b and 2c, and Supporting Information). All ⁶⁴Cu-radiolabeled conjugates remained bound against EDTA challenge up to 4 h time point (Supporting Information), although slow release of the radioactivity was observed over 24 h. A small portion of the activity (<5%) was transferred from the complexes to EDTA (99.4% for N-NE3TA-CA, 97.9% for N-NE3TA-CDCA, 97.5% for N-NE3TA-DCA) at 1 h time point (Figure 2b). ⁶⁴Cu-N-NE3TA-DCA was shown to be less tolerant of EDTA treated. ~20% of ⁶⁴Cu was dissociated from the complex at 24 h time point (Figure 2c). Approximately 10% of the radioactivity was leaked from ⁶⁴Cu-N-NE3TA-CA and ⁶⁴Cu-N-NE3TA-CDCA at 24 h time point (Figure 2c).

Table 3.

^* Stability of ⁶⁴Cu-radiolabeled complexes against EDTA at a 100-fold molar excess (37 °C).

Time	N-NE3TA-CA	N-NE3TA-DCA	N-NE3TA-CDCA
0 h	100.0 ± 0.1	99.8 ± 0.1	99.5 ± 0.6
1 h	99.4 ± 0.0	97.5 ± 0.1	97.9 ± 0.3
4 h	98.5 ± 0.4	95.2 ± 0.9	97.1 ± 0.2
24 h	88.4 ± 0.4	80.4 ± 1.3	88.9 ± 0.4

^*bound complex (mean ± standard deviation%) was measured in duplicate using ITLC (eluent: 20mM EDTA in 0.15M NH₄OAc)

In summary, N-NE3TA-bile acid conjugates were evaluated for complexation kinetics and stability with ⁶⁴Cu for potential use in targeted PET imaging. All N-NE3TA-bile acid conjugates rapidly and almost completely bound to ⁶⁴Cu. The corresponding ⁶⁴Cu-radiolabeled complexes remained quite stable in human serum, and no considerable release of the radioactivity was observed with the complexes. When rigorously challenged by excess amount of EDTA at 37 °C for 24 h, a small amount of the radioactivity (>10%) was dissociated from ⁶⁴Cu-radiolabeled N-NE3TA-bile acid conjugates. The in vitro complexation kinetics and stability data suggest that the N-NE3TA-bile acid conjugates can be further evaluated for targeted PET imaging using animals.