Synthesis, in vitro evaluation, and in vivo metabolism of fluor/quencher compounds containing IRDye 800CW and Black Hole Quencher-3 (BHQ-3).

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.

Automated synthesis, characterization and biological evaluation of [(68)Ga]Ga-AMBA, and the synthesis and characterization of (nat)Ga-AMBA and [(67)Ga]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.