Introduction: Two 7-fluoroimidazobenzodiazepines (AH114726 and GEH120348), analogs of flumazenil, were labeled with fluorine-18 and evaluated as alternative radioligands for in vivo imaging of the GABA(A)/benzodiazepine receptor by comparing them to [C-11]flumazenil in rhesus monkey.Methods: Radiotracers were prepared from the corresponding nitro-precursors in an automated synthesis module, and primate imaging studies were conducted on a Concorde MicroPET P4 scanner. The brain was imaged for 60 (12 x 5 min frames) or 90 min (18 x 5 min frames), and data was reconstructed using the 3D MAP algorithm. Specificity of [F-18]AH114726 and [F-18]GEH120348 was confirmed by displacement studies using unlabeled flumazenil.Results: [F-18]GEH120348 and [F-18]AH114726 were obtained in 13-24% yields (end of synthesis) with high chemical (>95%) and radiochemical (>99%) purities, and high specific activities (2061 +/- 985 Ci/mmol). The in vivo pharmacokinetics of [F-18]AH114726 and [F-18]GEH120348 were determined in a non-human primate and directly compared with [C-11]flumazenil. Both fluorine-18 radioligands showed time-dependent regional brain distributions that correlated with the distribution of [C-11]flumazenil and the known concentrations of GABA(A)/benzodiazepine receptors in the monkey brain. [F-18]AH114726 exhibited maximal brain uptake and tissue time-radioactivity curves that were most similar to [C-11]flumazenil. In contrast, [F-18]GEH120348 showed higher initial brain uptake but very different pharmacokinetics with continued accumulation of radioactivity into the cortical regions of high GABA/benzodiazepine receptor concentrations and very little clearance from the regions of low receptor densities. Rapid washout of both radiotracers occurred upon treatment with unlabeled flumazenil.Conclusion: The ease of the radiochemical synthesis, together with in vivo brain pharmacokinetics most similar to [C-11]flumazenil, support that [F-18]AH114726 is a suitable option for imaging the GABA(A) receptor. (C) 2013 Elsevier Inc. All rights reserved.

Protease-cleavable peptides containing a suitable fluor/quencher (Fl/Q) pair are optically dark until cleaved by their target protease, generating fluorescence. This approach has been used with many Fl/Q pairs, but little has been reported with IRDye 800CW, a popular near-infrared (NIR) fluor. We explored the use of the azo-bond-containing Black Hole Quencher 3 (BHQ-3) as a quencher for IRDye 800CW and found that IRDye 800CW/BHQ-3 is a suitable Fl/Q pair, despite the lack of proper spectral overlap for fluorescence resonance energy transfer (FRET) applications. Cleavage of IRDye 800CW-PLGLK(BHQ-3)AR-NH(2) (8) and its D-arginine (Darg) analogue (9) by matrix metalloproteinases (MMPs) in vitro yielded the expected cleavage fragments. In vivo, extensive metabolism was found. Significant decomposition of a "non-cleavable" control IRDye 800CW-(1,13-diamino-4,7,10-trioxatridecane)-BHQ-3 (10) was evident in plasma of normal mice by 3 min post injection. The major metabolite showed a m/z and UV/vis spectrum consistent with azo bond cleavage in the BHQ-3 moiety. Preparation of an authentic standard of this metabolite (11) confirmed the assignment. Although the IRDye 800CW/BHQ-3 constructs showed efficient contact quenching prior to enzymatic cleavage, BHQ-3 should be used with caution in vivo, due to instability of its azo bond.

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.

177Lu-AMBA (AMBA = DO3A-CH2CO-G-[4-aminobenzoyl]-QWAVGHLM-NH2) 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 177hafnium (177Hf) that forms upon the decay of 177Lu while the latter is complexed with AMBA. When decayed solutions of 177Lu-AMBA were analyzed, it was found that 177Hf is retained in the DO3A monoamide chelator, forming a pair of interconverting isomers. We report the synthesis and full characterization of natLu-AMBA and the studies performed to demonstrate its correspondence to radioactive 177Lu-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)-NH2] peptide targeting group was found.

The metabolism of Lu-177-AMBA (AMBA = DO3A-CH3CO-G-(4-aminobenzoyl)-QWAVGHLM-NH2), a radio-therapeutic 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 Lu-177-AMBA). The primary metabolites were Lu-DO3A-CH2CO-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 Lu-177-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 Lu-nat. and Lu-177 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 Lu-177-labeled metabolites A-E were performed in PC-3 tumor-bearing mice and the results compared with intact Lu-177-AMBA. IC50 values for unmetalated metabolite ligands A-E were >400 nM in PC-3 cells in competition binding studies against Lu-177- AMBA. No direct binding to PC-3 cells was observed with Lu-177-labeled A-C, confirming IC50 results. Lu-177-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 (similar to 78-87%) within I It. These results demonstrate that the tumor uptake observed with Lu-177-AMBA is due to parent drug and not due to any of its identified metabolites.

Lu-177-DO3A-CH2CO-G-4-aminobenzoyl-Q-W-A-V-G-H-L-M-NH2 (Lu-177-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, Lu-177-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 x 10(5)/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 x 10(3) and 1.2 x 10(4)/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 Lu-177-AMBA in low GRP-R models of prostate cancer and determine how reduced expression affects Lu-177-AMBA radiotherapy efficacy. Methods: The LNCaP and DU145 cell lines were used to determine the binding (K-d), retention, and efflux of Lu-177-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: Lu-177-AMBA binds to GRP-R in these cell lines with high affinity (K-d of LNCaP, 0.65 +/- 0.2 nM; K-d of DU145, 0.53 +/- 0.1 nM). The uptake of Lu-177-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 gamma-images of Lu-177-AMBA identify tumors in vivo. Despite having lower uptake, Lu-177-AMBA demonstrated radiotherapeutic efficacy and decreased proliferation in the LNCaP and DU145 xenografts; in the LNCaP model, Lu-177-AMBA normalized the phenotype of microvasculature, reducing tumoral blood pooling. Conclusion: Lu-177-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.

4216 177Lu-AMBA is a radiolabeled bombesin derivative that is bound and internalized by cells expressing the gastrin-releasing peptide receptor (GRP-R), a G-protein-coupled receptor. In our previous report of radiotherapy studies with immunocompromised mice bearing PC-3 xenografts, 177Lu-AMBA treatment significantly increased survival and reduced tumor growth rates. The PC-3 tumor cell line has a high number of GRP receptors (2.5 x 105 /cell), whereas LNCaP, a prostate cancer metastatic cell line thought to be representative of the early androgen-sensitive stage of prostate cancer, expresses a lower receptor number (5.9 x 103 / cell). While the high number of receptors per cell in the PC-3 line may not be representative of the clinical situation, there is little definitive work on the GRP receptor status of primary prostate tumors and metastases. Therefore we sought to evaluate the binding and efficacy of 177Lu-AMBA in a low GRP receptor number model of prostate cancer, and determine how reduced GRP receptor expression affects 177Lu-AMBA radiotherapy treatment efficacy and survival. Biodistribution studies confirmed the binding of 177Lu-AMBA to LNCaP xenografts in a receptor density-dependent manner when compared to PC-3. At 1h, LNCaP demonstrated 1.52 ± 0.94 % ID/g tumor as compared to 6.35 ± 2.23 %ID/g tumor in PC-3; and 0.83 ± 0.18 %ID/g tumor at 24 h, compared to 3.39 ± 0.85 %ID/g tumor in PC-3 at 24 h. There was no difference in survival between treated and controls for LNCaP at the end of the study (60-day; n=12), in contrast to PC-3 bearing mice (n=32) which demonstrated a two-fold increase in survival over controls with treatment. However, with the same 0.1 mL administered dose of 177Lu-AMBA (1.11 GBq/kg; 27.75 MBq/0.1 mL; peptide mass ~ 41.3 mg/m2), the Mean Time to Progression of tumor growth was increased by 100% in both LNCaP and PC-3 xenografts. Immunohistochemistry for ki67 showed a reduction in proliferation in treated tumors compared to control. In addition, the characteristic extravasation of blood (ecchymosis) of LNCaP xenografts was significantly reduced and confirmed histologically. The LNCaP cell line internalized a 177Lu-AMBA dose sufficient to reduce abnormal vascular phenotypes, mimicking androgen ablation and creating a more normal vessel route for further treatment delivery. These results confirm that 177Lu-AMBA, which has both gamma-emissions suitable for imaging and medium energy beta-emissions for radiotherapy and is currently in Phase I trials, has the potential to be clinically effective as a therapeutic and imaging agent, or as an adjunct to chemotherapy in prostate cancer, even in cases where the relative expression of GRP levels in the primary tumor or metastases is low.

380 Objectives: In this study we investigated the fate of the 177Hafnium (177Hf) that forms upon decay of 177Lu-AMBA, a peptide-derivatized DO3A complex that is being studied for the radiotherapeutic treatment of tumors that express the GRP receptor. Methods: HPLC-purified 177Lu-AMBA (621.6 MBq) in stabilizing buffer was stored for 43 days at -20°C. During this time two new nonradioactive peaks formed that we postulated might be isomers of Hf-AMBA. They were isolated and interconversion studies performed using HPLC. Synthesis of a Hf-AMBA standard was attempted by reaction of HfCl4 with AMBA in various solvents. The Hf-AMBA products formed in DMF were purified by HPLC, characterized (elemental analysis, NMR, MS) and coinjected with the products from 177Lu-AMBA decay. Results: In two HPLC methods, the two Lu-AMBA decay products coeluted with a standard of Hf-AMBA made by heating AMBA with HfCl4 in DMF; Hf-AMBA did not form in H20. Analysis results (CHNF) for the Hf-AMBA standard were consistent with its formulation as Hf(IV)AMBA.3TFA.6H2O. The two peaks formed underwent slow interconversion when isolated. By LC/MS, both had a molecular weight (m/z) consistent with formulation as Hf-AMBA. The Hf-AMBA 1H and 13C NMR spectra had chemical shifts similar to those of Lu-AMBA. The ability to obtain an NMR indicated that the Hf is diamagnetic, consistent with Hf(IV), not Hf(III). Conclusions: To our knowledge, the fate of hafnium that forms on decay of 177Lu while the Lu is complexed to a chelator has not been reported. As a result of this study, we conclude that when 177Lu in 177Lu-AMBA undergoes β- decay in H20, the resulting 177Hf is retained in the DO3A-acetyl chelator, but oxidizes from Hf(III) to Hf(IV), forming a stable pair of interconverting isomers.

267 Objectives: Bombesin receptors are expressed in prostate and other cancers. A novel bombesin receptor binding peptide, AMBA, labeled with Lu177 is in phase 1 clinical trials for the radiotherapy of prostate cancer. This study evaluated the biodistribution in monkeys of Lu177-AMBA preliminary to late radiation toxicity studies. Methods: Lu177-AMBA was prepared via a DO3A-CH2CO-G-4 aminobenzoyl chelate. Radiochemical purity at administration was established by RP-HPLC and averaged 91%. Four male cynomologus monkeys received in a leg vein 1 ug/kg (1 mCi/ug) delivered at 1ug/ml/min. Planar images were obtained every minute for 20 min, then periodically over 3 h and finally at 24 h. Radioactivity accumulations were estimated for major organs by comparison with a set of standard phantoms. SPECT images were acquired for 3 animals in the first 3 h. Results: The early images confirmed the fast renal clearance of Lu177-AMBA. At 20 min, the remaining radioactivity was primarily in the blood pool (heart), liver, pancreas and kidneys. Activity in all four tissues decreased steadily, reaching about 5%, 2-4%, 1-2% and 5-8% at 2.5 h, respectively, while at 24 h neither the liver nor heart was visible. The pancreas was obscured by the kidneys and liver in early planar images so SPECT was used to confirm accumulations in this organ. The pancreas became visible in the planar images at 24 h, since clearance was slower than that of the kidneys. At 24 h whole body radioactivity was about 8%, essentially in intestinal contents and pancreas, and was confirmed by counting urine and faeces that showed about 90% had cleared by this time. Conclusions: Lu177-AMBA in the non-human primate cleared major organs including the kidneys. There was a small percentage in the pancreas, a GRP receptor containing tissue. These results generally support observations in rodents and suggest that the monkey is a reasonable animal model for the study of late radiation toxicity.

A robust formulation was developed for [177Lu]Lu-AMBA (177Lu-DO3A-CH2CO-G-[4-aminobenzoyl]-QWAVGHLM-NH2), 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.

266 Objectives: We compared the biodistribution of 111In-, 67Ga- (a surrogate for 68Ga-), and 177Lu-AMBA in male nude mice xenographed with human (PC-3) prostate cancer cells [AMBA=DO3A-CH2CO-G-[4-aminobenzoyl]-QWAVGHLM-NH2]. This cell line expresses Gastrin-Releasing Peptide receptors (GRP-R), which are over-expressed in many cancers. 111In or 68Ga analogs of Lu-AMBA may have value for GRP-R positive tumor diagnosis, patient selection and evaluation of 177Lu-AMBA radiotherapeutic treatment effects. Methods: Compounds were prepared by reaction of radiometals with excess AMBA at 100oC and HPLC purified. PC-3 tumor-bearing mice (n=4, 4 and 9 per group for In-, Ga- and Lu-AMBA) were administered 5 μCi (0.1 mL) of test compound via i.v. tail vein, sacrificed after 1 and 24 h and tissues counted for residual radioactivity. Results: All compounds exhibited similar biodistributions and uptake in target and non-target tissues. Tumor uptake was favorable (4-6% ID/g) for all agents at 1 h, with 2.5-3% ID/g remaining at 24 h. The route of excretion was renal (~50-60% within 1 h). Kidney retention was low (≦1% ID at 24h) for all three compounds. Conclusions: Based on the comparable biodistribution results obtained for 111In, 67Ga and 177Lu-AMBA in PC-3 tumor bearing mice, and their high uptake and retention in GRP-R positive tissues, we anticipate that 68Ga-AMBA labeled for PET imaging or 111In-AMBA for gamma scintigraphy will demonstrate similar distribution clinically to the radiotherapeutic compound 177Lu-AMBA. The monosubstituted DOTA chelate in the AMBA ligand allows ready substitution of +3 radioisotopes useful for planar, SPECT, PET and radiotherapeutic applications.

To 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.