No abstract

Surgical resection remains a definitive treatment for prostate cancer. Yet, prostate cancer surgery is performed without image guidance for tumor margin, extension beyond the capsule and lymph node positivity, and without verification of other occult metastases in the surgical field. Recently, several imaging systems have been described that exploit near-infrared (NIR) fluorescent light for sensitive, real-time detection of disease pathology intraoperatively. In this study, we describe a high-affinity (9 nM), single nucleophile-containing, small molecule specific for the active site of the enzyme PSMA. We demonstrate production of a tetra-sulfonated heptamethine indocyanine NIR fluorescent derivative of this molecule using a high-yield LC/MS purification strategy. Interestingly, NIR fluorophore conjugation improves affinity over 20-fold, and we provide mechanistic insight into this observation. We describe the preparative production of enzymatically active PSMA using a baculovirus expression system and an adenovirus that co-expresses PSMA and GFP. We demonstrate sensitive and specific in vitro imaging of endogenous and ectopically expressed PSMA in human cells and in vivo imaging of xenograft tumors. We also discuss chemical strategies for improving performance even further. Taken together, this study describes nearly complete preclinical development of an optically based small-molecule contrast agent for image-guided surgery.

Small-molecule ligands specific for prostate-specific membrane antigen (PSMA) have the potential to improve prostate cancer imaging. However, highly charged ligands are difficult to label with 99m Tc and to purify. In this study, we present an adamantanetrimerized small molecule that has nanomolar binding to PSMA and also has 12 negative charges. Methods: To convert this molecule into a clinically viable SPECT diagnostic, we have developed a simple, cartridge-based, solid-phase prelabeling strategy that, within 25 min, converts readily available and inexpensive 99m Tc-pertechnetate into a chemically pure complex, with a reactive N-hydroxysuccinimide (NHS) ester, in neat organic solvent. This stable intermediate can label any aminecontaining small molecule or peptide with 99m Tc in 1 step, with high specific activity and without the need for high-performance liquid chromatography (HPLC). Results: Solid-phase conversion of 99m Tc-pertechnetate to 99m Tc-MAS 3 -NHS (MAS3 is S-acetylmercaptoacetyltriserine) could be completed in 25 min, with .99% radiochemical purity and with no coligands present. This intermediate was then conjugated to adamantane-trimerized GPI (2[(3-amino-3-carboxypropyl)(hydroxy)(phosphinyl)-methyl]pentane-1,5-dioic acid) in 1 step with .95% yield and no need for HPLC purification. The final molecule bound specifically to living human tumor cells expressing PSMA on their surface. Quantitative comparison was made among GPI monomer, GPI trimer, and their 99m Tc-derivatives. Conclusion: Our study describes a simple cartridge-based conversion of 99m Tcpertechnetate to a useful, preloaded NHS ester intermediate that takes only 25 min to prepare and results in .99% radiochemical purity. Using this chemistry, we produced a highspecific-activity, 99m Tc-labeled, PSMA-targeted small molecule and demonstrate g-ray radioscintigraphic imaging of living human prostate cancer cells.

Adamantane scaffolds for affinity maturation of prostate cancer specific ligands of low molecular mass are described. These scaffolds are modular and can be used for conjugation of up to three ligands and an additional effector molecule by standard peptide coupling techniques. The potential of the scaffolds is demonstrated with the multimerization of GPI 1, a prostate cancer specific small molecule. A detailed study of multimerized GPI conjugates with NIR-fluorophores and their binding properties to different prostate cancer cell lines shows the specific binding of these conjugates to cell types positive for prostate specific membrane antigen (PSMA). We demonstrate that these conjugates allow the sensitive imaging of prostate cancer cells with NIR methodology and suggest that our adamantane scaffolds might be generally useful for affinity maturation of small molecules targeting cell surface epitopes.