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.

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.

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(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.

Rationale and Objectives: Light imaging agents can respond to the presence of protease enzymes when formulated as FRET agents. These agents contain a fluorescent imaging probe (Fl) that is rendered optically silent by the molecular proximity of a quencher (Q), which is expected to be effective when its absorption spectrum overlaps the Fl emission spectrum. In use, enzyme is detected when it metabolizes the link between the Fl-Q. IRDye800 is a near-infrared Fl with utility for in vivo optical imaging due to its nearly ideal 800 nm emission. We explored the use of the highly conjugated azo-containing compound BHQ-3 as a quencher for IRDye800 and found unexpectedly that IRDye800/BHQ-3 is a suitable Fl/Q pair, despite the lack of proper spectral overlap for fluorescence resonance energy transfer (FRET) applications. Methods: IRDye800-PLGLK(BHQ-3)AR-NH 2 (1) and a 'non-cleavable' control IRDye800-(1,2,3-diamino-4,7 a ,0-trioxatridecane)-BHQ-3 (2) were prepared and characterized. For in vitro cleavage studies, compounds (5 mmol) were incubated at RT in a 96-well plate for 60 min with MMP bufferAE40ng of various activated matrix metalloproteases. In-growth of Fl signal at 780 nm was monitored in a plate reader. For in vivo metabolism studies, 40 nmol of 1 or 2 was injected via tail vein into normal male Balb/c mice (n¼4). Serial plasma samples were collected from 3-60 min post injection. Plasma and urine extracts were analyzed by LC/MS. Results: Treatment of 1 with MMP-13 caused Fl to rise from 30AE4 RFU at t¼0 to 1723AE492 after 60 min (n¼3), a 60-fold increase in Fl with enzyme treatment. Slower cleavage was seen with other MMPs. Under similar conditions, 2 had values of 119 and 123 at t¼0 and 60 min, revealing incomplete quenching but no metabolism by MMPs. In vivo studies with 1 (n¼3) showed peptide cleavage in normal mice. Most metabolites were fluorescent and did not contain BHQ-3. Significant metabolism of 2 in normal mice (n¼4) was evident by 3 min post injection and nearly complete by 20 min. LC/MS analysis of plasma extracts showed the metabolite had a MW of 1394, consistent with azo bond cleavage in the BHQ-3 moiety. Conclusion: IRDye800/BHQ-3 construct (1) showed good quenching prior to enzymatic cleavage; the compound was unstable in vivo. As spectral overlap is not suitable for FRET, contact quenching is inferred. Detailed metabolism studies with 2 revealed in vivo instability of the azo bond in BHQ-3 quencher. BHQ-3 should be used with caution in vivo. All authors except M.T. certify that they are employees of Bracco Research USA Inc. M.T. has no financial interest in this work. All authors hereby grant permission to publish this abstract or an appropriate summary thereof with the abstract.Session 3: Manganese CMR2009: 3.01 High temporal-resolution contrast-enhanced dynamic scanning using MnCl 2 R. Hvass HansenDepartment of Diagnostic Radiology, Copenhagen University Hospital, Herlev, Denmark Rationale and Objective: The relatively recent (1,2) use of a nonchelated form of manganese as contrast agent with...