Strain-Promoted Azide-Alkyne Cycloaddition-Based PSMA-Targeting Ligands for Multimodal Intraoperative Tumor Detection of Prostate Cancer

Abstract

Strain-promoted azide-alkyne cycloaddition (SPAAC) is a straightforward and multipurpose conjugation strategy. The use of SPAAC to link different functional elements to prostate-specific membrane antigen (PSMA) ligands would facilitate the development of a modular platform for PSMA-targeted imaging and therapy of prostate cancer (PCa). As a first proof of concept for the SPAAC chemistry platform, we synthesized and characterized four dual-labeled PSMA ligands for intraoperative radiodetection and fluorescence imaging of PCa. Ligands were synthesized using solid-phase chemistry and contained a chelator for ¹¹¹In or ^99mTc labeling. The fluorophore IRDye800CW was conjugated using SPAAC chemistry or conventional N-hydroxysuccinimide (NHS)-ester coupling. Log D values were measured and PSMA specificity of these ligands was determined in LS174T-PSMA cells. Tumor targeting was evaluated in BALB/c nude mice with subcutaneous LS174T-PSMA and LS174T wild-type tumors using μSPECT/CT imaging, fluorescence imaging, and biodistribution studies. SPAAC chemistry increased the lipophilicity of the ligands (log D range: -2.4 to -4.4). In vivo, SPAAC chemistry ligands showed high and specific accumulation in s.c. LS174T-PSMA tumors up to 24 h after injection, enabling clear visualization using μSPECT/CT and fluorescence imaging. Overall, no significant differences between the SPAAC chemistry ligands and their NHS-based counterparts were found (2 h p.i., p > 0.05), while ¹¹¹In-labeled ligands outperformed the ^99mTc ligands. Here, we demonstrate that our newly developed SPAAC-based PSMA ligands show high PSMA-specific tumor targeting. The use of click chemistry in PSMA ligand development opens up the opportunity for fast, efficient, and versatile conjugations of multiple imaging moieties and/or drugs.

Figure 1

Chemical structures of PSMA-N048 (N48), PSMA-N049 (N49), PSMA-N050 (N50), and PSMA-N051 (N51). Ligands consisting of KuE (blue), linker (black), SPAAC (red), IRDye800CW (green), and MAG3 or DOTA chelator (purple).

Figure 2

In vitro characterization of N48, N49, N50, and N51. (A) IC₅₀ values of ligands as determined in competitive binding assays using LS174T-PSMA cells. IC₅₀ values were determined using a nonradiolabeled ligand (N48, N49, N50, and N51) in competition with ¹¹¹In-labeled PSMA-617. Lipophilicity of ligands expressed in log D values. Internalization ratio as determined in LS174T-PSMA cells. (B) Membrane binding and internalization kinetics of N48, N49, N50, and N51 in LS174T-PSMA-positive and -negative cells. Nonspecific binding was determined by blocking with an excess of 2-PMPA (50 μg). PSMA-617 was added as a positive control.

Figure 3

In vivo pharmacokinetics of ^99mTc-N49 and ¹¹¹In-N48. (A) Biodistribution as determined after dissection of ^99mTc-N49 (3 MBq/mouse) and ¹¹¹In-N48 (10 MBq/mouse) 2, 4, and 24 h p.i. (0.3 nmol, n = 5/group). Biodistribution was determined in mice bearing subcutaneous LS174T-PSMA and LS174T wild-type xenografts. Data are expressed as %ID/g ± SD; ^** indicates p < 0.01. (B) Representative μSPECT/CT scans and fluorescence images of mice with s.c. LS174T-PSMA (left) and wild-type LS174T (right) tumors after i.v. injection of ^99mTc-N49 (3 MBq/mouse) and ¹¹¹In-N48 (10 MBq/mouse) 2, 4, and 24 h p.i.

Figure 4

In vivo comparison of N48, N49, N50, and N51. (A) Biodistribution as determined after dissection and (B) resulting tumor-to-organ ratios of four ¹¹¹In- (10 MBq/mouse) or ^99mTc-labeled (15 MBq/mouse) ligands and positive control PSMA-617 (0.3 nmol, 2 h p.i., n = 5/group). Biodistribution was determined in mice bearing subcutaneous LS174T-PSMA and LS174T wild-type xenografts. Data are expressed as %ID/g ± SD; * indicates p < 0.05, ^** indicates p < 0.01, and ^*** indicates p < 0.001.

Figure 5

All ligands clearly visualize PSMA-positive tumors using both μSPECT/CT and fluorescence imaging. Representative μSPECT/CT scans (A) and fluorescence images (B) of mice with s.c. LS174T-PSMA (right) and wild-type LS174T (left) tumors after i.v. injection of ¹¹¹In- (10 MBq/mouse) or ^99mTc-labeled (15 MBq/mouse) ligands (0.3 nmol, 2 h p.i.).

Figure 6

Multimodal fluorescence and μSPECT/CT imaging of intraperitoneal PSMA-positive tumors using ¹¹¹In-N50. Mouse with several intraperitoneal LS174T-PSMA tumors located at different depths in the peritoneal cavity. (A) Same-scale NIRF images of mouse with several intraperitoneal tumors after i.v. injection of ¹¹¹In-labeled N50 (0.3 nmol, 10 MBq/mouse, 2 h p.i.). (B) NIRF image of removed tumors. (C) Corresponding μSPECT/CT images in supine and left lateral positions.