Hybrid Chelator-Based PSMA Radiopharmaceuticals: Translational Approach

Abstract

(1) Background: Prostate-specific membrane antigen (PSMA) has been extensively studied in the last decade. It became a promising biological target in the diagnosis and therapy of PSMA-expressing cancer diseases. Although there are several radiolabeled PSMA inhibitors available, the search for new compounds with improved pharmacokinetic properties and simplified synthesis is still ongoing. In this study, we developed PSMA ligands with two different hybrid chelators and a modified linker. Both compounds have displayed a promising pharmacokinetic profile. (2) Methods: DATA^5m.SA.KuE and AAZTA⁵.SA.KuE were synthesized. DATA^5m.SA.KuE was labeled with gallium-68 and radiochemical yields of various amounts of precursor at different temperatures were determined. Complex stability in phosphate-buffered saline (PBS) and human serum (HS) was examined at 37 °C. Binding affinity and internalization ratio were determined in in vitro assays using PSMA-positive LNCaP cells. Tumor accumulation and biodistribution were evaluated in vivo and ex vivo using an LNCaP Balb/c nude mouse model. All experiments were conducted with PSMA-11 as reference. (3) Results: DATA^5m.SA.KuE was synthesized successfully. AAZTA⁵.SA.KuE was synthesized and labeled according to the literature. Radiolabeling of DATA^5m.SA.KuE with gallium-68 was performed in ammonium acetate buffer (1 M, pH 5.5). High radiochemical yields (>98%) were obtained with 5 nmol at 70 °C, 15 nmol at 50 °C, and 60 nmol (50 µg) at room temperature. [⁶⁸Ga]Ga-DATA^5m.SA.KuE was stable in human serum as well as in PBS after 120 min. PSMA binding affinities of AAZTA⁵.SA.KuE and DATA^5m.SA.KuE were in the nanomolar range. PSMA-specific internalization ratio was comparable to PSMA-11. In vivo and ex vivo studies of [¹⁷⁷Lu]Lu-AAZTA⁵.SA.KuE, [⁴⁴Sc]Sc-AAZTA⁵.SA.KuE and [⁶⁸Ga]Ga-DATA^5m.SA.KuE displayed specific accumulation in the tumor along with fast clearance and reduced off-target uptake. (4) Conclusions: Both KuE-conjugates showed promising properties especially in vivo allowing for translational theranostic use.

Keywords: hybrid chelator; prostate specific membrane antigen PSMA; radionuclide diagnosis and therapy.

Figure 1

Bifunctional derivatives of the hybrid chelators AAZTA⁵ and DATA^5m.

Figure 2

Asymmetric amidation of SADE with different amines. The reactions are driven by change in aromaticity of the different intermediates.

Figure 3

Synthesis of DATA^5m: (a) tert-butyl bromoacetate, Na₂CO₃, MeCN, 96%; (b) Pd/C, EtOH, formic acid, H₂, 98%; (c) paraformaldehyde, 2-nitrocyclohexanone, MeOH, 77%; (d) Raney^®-Nickel, EtOH, H₂, 72%; (e) tert-butyl bromoacetate, DIPEA, MeCN, 62%; (f) Formalin (37%), AcOH, NaBH₄, ACN, 74%; (g) LiOH, dioxane/H₂O, 84%; (h) tertbutyl(2aminoethyl)carbamate, HATU, DIPEA, ACN, 94%; (i) TFA/DCM, 1:1.

Figure 4

Synthesis of the PSMA inhibitor lysine-urea-glutamate-squaric acid monoester: (a) N(ε)-benzoyloxycarbonyl-l-lysine, triphosgene, triethylamine DCM, 0 °C; (b) l-glutamic acid di-tert-butyl ester hydrochloride, triethylamine, DCM, 41%; (c) Pd/C, MeOH, H₂, 96%; (d) 3,4-dibutoxycyclobut-3-en-1,2-dione, 0.5 M phosphate buffer pH 7, ethyl acetate, 77%; (e) TFA/DCM, 1:1, 83%.

Figure 5

Synthesis of DATA^5m.SA.KuE (14) 0.5 M phosphate buffer pH 9, 10%.

Figure 6

(A) Kinetic studies of ⁶⁸Ga-radiolabeling of DATA^5m.SA.KuE for various amounts of precursor and different temperatures. Labeling of 15 nmol at 50 °C, 5 nmol at 70 °C and 60 nmol at RT resulting in quantitative RCYs after one minute. Radiolabeling of 10 nmol at 50 °C results in a RCY of 56% after 15 minutes. (B) Radio-HPLC of [⁶⁸Ga]Ga-DATA^5m.SA.KuE. t_R (free gallium-68) = 2.0 min; t_R ([⁶⁸Ga]Ga-DATA^5m.SA.KuE) = 8.8 min. Radio-HPLC confirmed purity and high RCY of [⁶⁸Ga]Ga-DATA^5m.SA.KuE. (C) Stability studies for [⁶⁸Ga] Ga-DATA^5m.SA.KuE complex in human serum (HS) and phosphate buffered saline (PBS) of intact conjugate at different time points.

Figure 7

Inhibition curve of AAZTA⁵.SA.KuE, DATA^5m.SA.KuE and PSMA-11. cpm: counts per minute.

Figure 8

Internalization ratio of [⁶⁸Ga]Ga-DATA^5m.SA.KuE and [⁴⁴Sc]Sc-AAZTA⁵.SA.KuE with [⁶⁸Ga]Ga-PSMA-11 as reference; % injected dose per 10⁶ LNCaP cells.

Figure 9

Ex vivo biodistribution data of [⁴⁴Sc]Sc-AAZTA⁵.SA.KuE, [¹⁷⁷Lu]Lu-AAZTA⁵.SA.KuE, [⁶⁸Ga]Ga-DATA^5m.SA.KuE and [⁶⁸Ga]Ga-PSMA-11 in LNCaP tumor-bearing Balb/c nude mice 1 h p.i. %ID/g: % injected dose per gram. Values are mean ± SD.

Figure 10

Ex vivo biodistribution of [⁶⁸Ga]Ga-DATA^5m.SA.KuE compared to organ accumulation after co-injection with an access PMPA.

Figure 11

Maximum intensity projections of µPET scans 1 h p.i. of (a) [⁶⁸Ga]Ga-PSMA-11, (b) [⁶⁸Ga]Ga-DATA^5m.SA.KuE, (c) [⁴⁴Sc]Sc-AAZTA⁵.SA.KuE and (d) co-injection of [⁴⁴Sc]Sc-AZTA⁵.SA.KuE and PMPA (tumor circled).

Figure 12

Time–activity curves of the uptake of [⁶⁸Ga]Ga-DATA^5m.SA.KuE and [⁶⁸Ga]Ga-PSMA-11 in the kidneys (a) and the tumor (b) over the total period of the µPET scan.