Jianqing Chen

177Lu-AMBA Biodistribution, Radiotherapeutic Efficacy, Imaging, and Autoradiography in Prostate Cancer Models with Low GRP-R Expression

177Lu-DO3A-CH2CO-G-4-aminobenzoyl-Q-W-A-V-G-H-L-M-NH2 (177Lu-AMBA) is a radiolabeled bombesin derivative that is bound and internalized by cells expressing the G-protein–coupled gastrin-releasing peptide receptor (GRP-R) and is currently in phase I clinical trials. In previous radiotherapy studies with PC-3 xenografted mice, 177Lu-AMBA treatment significantly increased survival and reduced tumor growth rates. The PC-3 tumor cell line has an elevated expression of GRP-Rs (2.5 × 105/cell), whereas LNCaP—a prostate cancer metastatic cell line representing the early androgen-sensitive stage of prostate cancer—and DU145—an androgen-insensitive metastatic line—express lower receptor numbers (5.9 × 103 and 1.2 × 104/cell, respectively). Because of tumor heterogeneity, the high number of receptors in the PC-3 line may not represent the clinical situation, and little definitive work on the GRP-R status of primary prostate tumors and metastases exists. We sought to evaluate the tumor binding and imaging potential of 177Lu-AMBA in low GRP-R models of prostate cancer and determine how reduced expression affects 177Lu-AMBA radiotherapy efficacy. Methods: The LNCaP and DU145 cell lines were used to determine the binding (Kd), retention, and efflux of 177Lu-AMBA. Biodistribution radiotherapy, imaging, and autoradiography studies were performed in LNCaP, DU145, or PC-3 tumor–bearing male nude mice. Immunohistochemistry was used to determine the proliferative state in LNCaP and DU145 models and the vascular phenotype of LNCaP radiotherapy tumors. Results: 177Lu-AMBA binds to GRP-R in these cell lines with high affinity (Kd of LNCaP, 0.65 ± 0.2 nM; Kd of DU145, 0.53 ± 0.1 nM). The uptake of 177Lu-AMBA is at least 10-fold less in LNCaP and DU145 cell lines than it is in the PC-3 cell line. Autoradiography identifies activity concentrated in areas of viable tumor tissue, and γ-images of 177Lu-AMBA identify tumors in vivo. Despite having lower uptake, 177Lu-AMBA demonstrated radiotherapeutic efficacy and decreased proliferation in the LNCaP and DU145 xenografts; in the LNCaP model, 177Lu-AMBA normalized the phenotype of microvasculature, reducing tumoral blood pooling. Conclusion: 177Lu-AMBA is a single radiolabeled agent that combines targeted radiotherapy after imaging dosimetry with the potential for single-agent or multimodality therapy for prostate cancer.

In Vitro and in Vivo Metabolism of Lu-AMBA, a GRP-Receptor Binding Compound, and the Synthesis and Characterization of Its Metabolites

The metabolism of (177)Lu-AMBA (AMBA = DO3A-CH(2)CO-G-(4-aminobenzoyl)-QWAVGHLM-NH(2)), a radiotherapeutic compound in clinical development that binds to GRP and NMB receptors, was studied in vitro (mouse, rat and human plasma, mouse kidney homogenate) and in vivo (by analysis of mouse and rat plasma and urine following IV injection of (177)Lu-AMBA). The primary metabolites were Lu-DO3A-CH(2)CO-G-Abz4-R, where R = -Q-OH (A), -QW-OH (B), and -QWAVGH-OH (C). Minor amounts of (D) where R = -QWAVGHLM-OH and (E) -QWAVGHL-OH were also observed. Clearance of (177)Lu-AMBA and of radioactivity from mouse and rat blood was rapid in vivo. In mouse and rat urine, only metabolites Lu-A and Lu-B were found-no parent drug was excreted. Unmetalated ligands and (nat)Lu and (177)Lu complexes for Lu-AMBA metabolites A-E were synthesized, characterized by HPLC and MS, and used to perform in vitro competition and direct binding studies on GRP receptor-positive PC-3 (human prostate) cancer cells. Biodistribution studies with (177)Lu-labeled metabolites A-E were performed in PC-3 tumor-bearing mice and the results compared with intact (177)Lu-AMBA. IC(50) values for unmetalated metabolite ligands A-E were >400 nM in PC-3 cells in competition binding studies against (177)Lu-AMBA. No direct binding to PC-3 cells was observed with (177)Lu-labeled A-C, confirming IC(50) results. (177)Lu-labeled metabolites A-E showed no uptake in GRP-receptor positive tumor or pancreas in PC-3 tumor bearing mice. All metabolites were rapidly excreted via the renal route (approximately 78-87%) within 1 h. These results demonstrate that the tumor uptake observed with (177)Lu-AMBA is due to parent drug and not due to any of its identified metabolites.

Synthesis, stabilization and formulation of [177Lu]Lu-AMBA, a systemic radiotherapeutic agent for Gastrin Releasing Peptide receptor positive tumors

A robust formulation was developed for [(177)Lu]Lu-AMBA ((177)Lu-DO3A-CH(2)CO-G-[4-aminobenzoyl]-QWAVGHLM-NH(2)), a Bombesin-like agonist with high affinity for Gastrin Releasing Peptide (GRP) receptors. During optimization of labeling, the effect of several radiostabilizers was evaluated; a combination of selenomethionine and ascorbic acid showed superiority over other tested radiostabilizers. The resulting two-vial formulation maintains a radiochemical purity (RCP) of >90% for at least 2 days at room temperature. The method of stabilization should be useful for other methionine-containing peptide radiopharmaceuticals in diagnostic and therapeutic applications.

Automated synthesis, characterization and biological evaluation of [68Ga]Ga-AMBA, and the synthesis and characterization of natGa-AMBA and [67Ga]Ga-AMBA

Ga-AMBA (Ga-DO3A-CH(2)CO-G-[4-aminobenzoyl]-QWAVGHLM-NH(2)) is a bombesin-like agonist with high affinity for gastrin releasing peptide receptors (GRP-R). Syntheses for (nat)Ga-AMBA, [(67)Ga]Ga-AMBA and [(68)Ga]Ga-AMBA were developed. The preparation of HPLC-purified and Sep-Pak purified [(68)Ga]Ga-AMBA were fully automated, using the built-in radiodetector of the Tracerlab FX F-N synthesizer to monitor fractionated (68)Ge/(68)Ga generator elution and purification. The total synthesis time, including the fractional elution of the generator, was 20 min for Sep-Pak purified material and 40 min for HPLC-purified [(68)Ga]Ga-AMBA. Both [(67)Ga]Ga-AMBA and [(177)Lu]Lu-AMBA showed comparable high affinity for GRP-R in the human prostate cancer cell line PC-3 in vitro (k(D)=0.46+/-0.07; 0.44+/-0.08 nM), high internalization (78; 77%) and low efflux from cells at 2 h (2.4+/-0.7; 2.9+/-1.8%). Biodistribution results in PC-3 tumor-bearing male nude mice showed comparable uptake for [(177)Lu]Lu-, [(111)In]In-, [(67)Ga]Ga- and [(68)Ga]Ga-AMBA.