Karen E. Linder

Tuftsin Binds Neuropilin-1 through a Sequence Similar to That Encoded by Exon 8 of Vascular Endothelial Growth Factor

Tuftsin, Thr-Lys-Pro-Arg (TKPR), is an immunostimulatory peptide with reported nervous system effects as well. We unexpectedly found that tuftsin and a higher affinity antagonist, TKPPR, bind selectively to neuropilin-1 and block vascular endothelial growth factor (VEGF) binding to that receptor. Dimeric and tetrameric forms of TKPPR had greatly increased affinity for neuropilin-1 based on competition binding experiments. On endothelial cells tetrameric TKPPR inhibited the VEGF165-induced autophosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) even though it did not directly inhibit VEGF binding to VEGFR-2. Homology between exon 8 of VEGF and TKPPR suggests that the sequence coded for by exon 8 may stabilize VEGF binding to neuropilin-1 to facilitate signaling through VEGFR-2. Given the overlap between processes involving neuropilin-1 and tuftsin, we propose that at least some of the previously reported effects of tuftsin are mediated through neuropilin-1.

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.

Selective in vitro targeting of GRP and NMB receptors in human tumours with the new bombesin tracer 177Lu-AMBA

PurposeTo investigate the in vitro binding properties of a novel radiolabelled bombesin analogue, 177Lu-AMBA, in human neoplastic and non-neoplastic tissues selected for their expression of the bombesin receptor subtypes GRP-R, NMB-R and BRS-3.MethodsIn vitro receptor autoradiography was performed in cancers expressing the various bombesin receptor subtypes. The novel radioligand 177Lu-AMBA was used and compared with established bombesin radioligands such as 125I-Tyr4-bombesin and 125I-[D-Tyr6,β-Ala11,Phe13,Nle14]-bombesin(6–14). In vitro incidence of detection of each of the three bombesin receptor subtypes was evaluated in each tumour.Results177Lu-AMBA identified all GRP-R-expressing tumours, such as prostatic, mammary and renal cell carcinomas as well as gastrointestinal stromal tumours. 177Lu-AMBA also identified all NMB-expressing tumours, but did not detect BRS-3-expressing tumours or BRS-3-expressing pancreatic islets. GRP-R-expressing peritumoural vessels were heavily labelled with 177Lu-AMBA. In contrast to the strongly GRP-R-positive mouse pancreas, the human pancreas was not labelled with 177Lu-AMBA unless chronic pancreatitis was diagnosed. In general, the sensitivity was slightly better with 177Lu-AMBA than with the conventional bombesin radioligands.ConclusionThe present in vitro study suggests that 177Lu-AMBA may be a very useful in vivo targeting agent for GRP-R-expressing tumours, NMB-R-expressing tumours and GRP-R-expressing neoangiogenic vessels.

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.

Isolation of a 177Hf complex formed by beta-decay of a 177Lu-labeled radiotherapeutic compound and NMR structural elucidation of the ligand and its Lu and Hf complexes.

(177)Lu-AMBA (AMBA = DO3A-CH(2)CO-G-[4-aminobenzoyl]-QWAVGHLM-NH(2)) is being developed for the radiotherapeutic treatment of tumors that express the gastrin-releasing peptide receptor (GRP-R). In this study we investigated the fate of the (177)hafnium ((177)Hf) that forms upon the decay of (177)Lu while the latter is complexed with AMBA. When decayed solutions of (177)Lu-AMBA were analyzed, it was found that (177)Hf is retained in the DO3A monoamide chelator, forming a pair of interconverting isomers. We report the synthesis and full characterization of (nat)Lu-AMBA and the studies performed to demonstrate its correspondence to radioactive (177)Lu-AMBA. We also report the synthesis and characterization of Hf-AMBA and, by NMR studies, show structural analogies between Hf-AMBA, its parent compound Lu-AMBA, and the unmetallated AMBA ligand. In the NMR spectra of both the metallated and unmetallated AMBA ligand, a stacking interaction between the amino benzoyl residue in the linker and a tryptophan in the truncated bombesin [BBN(7-14)-NH(2)] peptide targeting group was found.