Preclinical Evaluation of a New Series of Albumin-Binding 177Lu-Labeled PSMA-Based Low-Molecular-Weight Radiotherapeutics

Abstract

Prostate-specific membrane antigen (PSMA)-based low-molecular-weight agents using beta(β)-particle-emitting radiopharmaceuticals is a new treatment paradigm for patients with metastatic castration-resistant prostate cancer. Although results have been encouraging, there is a need to improve the tumor residence time of current PSMA-based radiotherapeutics. Albumin-binding moieties have been used strategically to enhance the tumor uptake and retention of existing PSMA-based investigational agents. Previously, we developed a series of PSMA-based, β-particle-emitting, low-molecular-weight compounds. From this series, ¹⁷⁷Lu-L1 was selected as the lead agent because of its reduced off-target radiotoxicity in preclinical studies. The ligand L1 contains a PSMA-targeting Lys-Glu urea moiety with an N-bromobenzyl substituent in the ε-amino group of Lys. Here, we structurally modified ¹⁷⁷Lu-L1 to improve tumor targeting using two known albumin-binding moieties, 4-(p-iodophenyl) butyric acid moiety (IPBA) and ibuprofen (IBU), and evaluated the effects of linker length and composition. Six structurally related PSMA-targeting ligands (Alb-L1-Alb-L6) were synthesized based on the structure of ¹⁷⁷Lu-L1. The ligands were assessed for in vitro binding affinity and were radiolabeled with ¹⁷⁷Lu following standard protocols. All ¹⁷⁷Lu-labeled analogs were studied in cell uptake and selected cell efficacy studies. In vivo pharmacokinetics were investigated by conducting tissue biodistribution studies for ¹⁷⁷Lu-Alb-L2-¹⁷⁷Lu-Alb-L6 (2 h, 24 h, 72 h, and 192 h) in male NSG mice bearing human PSMA+ PC3 PIP and PSMA- PC3 flu xenografts. Preliminary therapeutic ratios of the agents were estimated from the area under the curve (AUC_0-192h) of the tumors, blood, and kidney uptake values. Compounds were obtained in >98% radiochemical yields and >99% purity. PSMA inhibition constants (K_is) of the ligands were in the ≤10 nM range. The long-linker-based agents, ¹⁷⁷Lu-Alb-L4 and ¹⁷⁷Lu-Alb-L5, displayed significantly higher tumor uptake and retention (p < 0.001) than the short-linker-bearing ¹⁷⁷Lu-Alb-L2 and ¹⁷⁷Lu-Alb-L3 and a long polyethylene glycol (PEG) linker-bearing agent, ¹⁷⁷Lu-Alb-L6. The area under the curve (AUC_0-192h) of the PSMA+ PC3 PIP tumor uptake of ¹⁷⁷Lu-Alb-L4 and ¹⁷⁷Lu-Alb-L5 were >4-fold higher than ¹⁷⁷Lu-Alb-L2, ¹⁷⁷Lu-Alb-L3, and ¹⁷⁷Lu-Alb-L6, respectively. Also, the PSMA+ PIP tumor uptake (AUC_0-192h) of ¹⁷⁷Lu-Alb-L2 and ¹⁷⁷Lu-Alb-L3 was ~1.5-fold higher than ¹⁷⁷Lu-Alb-L6. However, the lowest blood AUC_0-192h and kidney AUC_0-192h were associated with ¹⁷⁷Lu-Alb-L6 from the series. Consequently, ¹⁷⁷Lu-Alb-L6 displayed the highest ratios of AUC(tumor)-to-AUC(blood) and AUC(tumor)-to-AUC(kidney) values from the series. Among the other agents, ¹⁷⁷Lu-Alb-L4 demonstrated a nearly similar ratio of AUC(tumor)-to-AUC(blood) as ¹⁷⁷Lu-Alb-L6. The tumor-to-blood ratio was the dose-limiting therapeutic ratio for all of the compounds. Conclusions: ¹⁷⁷Lu-Alb-L4 and ¹⁷⁷Lu-Alb-L6 showed high tumor uptake in PSMA+ tumors and tumor-to-blood ratios. The data suggest that linker length and composition can be modulated to generate an optimized therapeutic agent.

Keywords: albumin; linker; prostate cancer; prostate-specific membrane antigen; radiopharmaceutical therapy.

Figure 1

Structures of clinically studied PSMA-targeted albumin-binding agents (¹⁷⁷Lu-PSMA-ALB-56, ¹⁷⁷Lu-EB-PSMA-617, ¹⁷⁷Lu-CTT1403), ¹⁷⁷Lu-PSMA-617, PSMA I&T, ¹⁷⁷Lu-L1, and ¹⁷⁷Lu-L14.

Figure 2

Structures of new agents studied in this report are derived from Structure A (Alb-L1, Alb-L2, and Alb-L3) and Structure B (Alb-L4, Alb-L5, and Alb-L6).

Figure 3

Cell uptake and internalization (mean ± SD, n = 3) of ¹⁷⁷Lu-Alb-L1–¹⁷⁷Lu-Alb-L6 in PSMA+ PC3 PIP cells and PSMA− PC3 flu cells (~1 million) at 37 °C. PSMA expression blockade studies in PSMA+ PC3 PIP cells of ¹⁷⁷Lu-Alb-L1–¹⁷⁷Lu-Alb-L6 were performed at 2 h post-incubation. The cells (~1 million) were pre-incubated with a known PSMA inhibitor, ZJ43 (10 µM final concentration), for 30 min before the corresponding radioactive dose was added to the medium.

Figure 4

In vitro clonogenic assay. Clonogenic survival of PSMA+ PC3 PIP cells and PSMAPC3 flu cells treated with increasing concentrations of ¹⁷⁷Lu-Alb-L2, ¹⁷⁷Lu-Alb-L5, and ¹⁷⁷Lu-L1 for 48 h at 37 °C.

Figure 5

(A) Tissue biodistribution data of ¹⁷⁷Lu-Alb-L2–¹⁷⁷Lu-Alb-L6 and ¹⁷⁷Lu-L1 are shown as the percentage of injected dose per gram of tissue, mean ± SD, n = 3–4 mice, dose: 1.87 MBq (intravenous injection via tail-vein), tumor model: PSMA+ PC3 PIP, and PSMA− PC3 flu tumor-bearing male NSG mice. (B) The tumor-to-blood (T/B), tumor-to-kidney (T/K), and tumor-to-salivary (T/Sal) ratios were obtained from the biodistribution data of ¹⁷⁷Lu-L1 and ¹⁷⁷Lu-Alb-L2–¹⁷⁷Lu-Alb-L6. Biodistribution data of ¹⁷⁷Lu-L1 were obtained from [27].

Figure 6

(A) Areas under the curves (AUCs) of tumor, blood, and kidney uptakes for ¹⁷⁷Lu-Alb-L2–¹⁷⁷Lu-Alb-L6 and ¹⁷⁷Lu-L1 were calculated. (B) AUC values of ¹⁷⁷Lu-Alb-L2–¹⁷⁷Lu-Alb-L6 and ¹⁷⁷Lu-L1 are compared. (C) AUC_0-192h(tumor-to-blood) and AUC_0-192h(tumor-to-kidney) were analyzed. AUC_0-192h(tumor-to-blood) is ~867 and AUC_0-192h(tumor-to-kidney) is ~8, respectively, for ⁷⁷Lu-L1, removed from the graph to provide clarity. The estimated maximum tumor-absorbed doses of ¹⁷⁷Lu-Alb-L2–¹⁷⁷Lu-Alb-L6 were analyzed, assuming the maximum absorbed dose for blood 2 Gy and kidney 28 Gy, respectively. p-values lower than 0.05 (p < 0.05), p < 0.01, p < 0.001, and p < 0.0001 were referred to one (*), two (**), or four (****) asterisks, respectively.