A multivalent approach of imaging probe design to overcome an endogenous anion binding competition for noninvasive assessment of prostate specific membrane antigen.

2[(3-Amino-3-carboxypropyl)(hydroxy)(phosphinyl)methyl]pentane-1,5-dioic acid) (GPI) is a highly potent inhibitor of prostate specific membrane antigen (PSMA) with a rapid in vivo clearance profile from nontarget organs including kidneys, but its use for imaging of PSMA is impeded by an endogenous anion (serum phosphate) competition, which compromises its specific binding to the antigen. Multipresentation of a targeting molecule on a single entity has been recognized as a practical way for imaging sensitivity enhancement. Herein, we demonstrate a multivalent approach based on a (64)Cu-specific bifunctional chelator scaffold to overcome the endogenous phosphate competition thus enabling the utility of GPI conjugates for in vivo detection of PSMA and imaging quantification. Both monomeric (H2CBT1G) and dimeric (H2CBT2G) conjugates were synthesized and labeled with (64)Cu for in vitro and in vivo evaluations. A 4-fold enhancement of PSMA binding affinity was observed for H2CBT2G as compared to H2CBT1G from the PSMA competitive binding assays performed on LNCaP cells. In vivo PET imaging studies were conducted on mouse xenograft models established with a PSMA(+) cell line, LNCaP, and PSMA(-) PC3 and H2009 cell lines. (64)Cu-CBT2G showed significantly higher LNCaP tumor uptake than (64)Cu-CBT1G at 1, 4, and 24 h postinjection (p.i.) (p < 0.05). In addition, tumor uptake of (64)Cu-CBT2G remained steady out to 24 h p.i. (1.46 ± 0.54, 1.12 ± 0.56, and 1.00 ± 0.50% ID/g at 1, 4, and 24 h p.i., respectively), while (64)Cu-CBT1G showed a great decrease from 1 to 4 h p.i. The PSMA imaging specificity of both H2CBT1G and H2CBT2G was demonstrated by their low uptake in PSMA(-) tumors (PC3 and H2009) and further confirmed by a significant signal reduction in PSMA(+) LNCaP tumors in the blockade study. In addition, the LNCaP tumor uptake (% ID/g) of (64)Cu-CBT2G was found to be in a positive linear correlation with the tumor size (R(2) = 0.92, 0.94, and 0.93 for 1 h, 4 h, and 24 h p.i.). This may render the probe with potential application in the management of patients with prostate cancer.