High-affinity Near-infrared Fluorescent Small-molecule Contrast Agents for In Vivo Imaging of Prostate-specific Membrane Antigen

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.

An HPLC/mass spectrometry platform for the development of multimodality contrast agents and targeted therapeutics: prostate‐specific membrane antigen small molecule derivatives

“…The results, shown in Table II, confirm that GPI conjugates have affinity in the nM range, which is consistent with previously published reports of GPI conjugated to NIR fluorophores [1].…”

“…As described in detail previously [1], GPI conjugates compete for the active site of the PSMA enzyme with phosphate and other anions. For this reason, our studies were conducted in non-phosphate containing medium.…”

“…To demonstrate the utility of the purification platform described above, we focused on a family of highly anionic PSMA-specific small molecules described previously by our group [1]. The GPI molecule, in particular, is especially difficult to work with since it has three carboxylic acids and a phosphinic acid on a mass of only 311 Da, leading to a high mass to charge ratio (Table II).…”

“…To avoid internalization of the radioligand due to constitutive endocytosis [1], live cell binding was performed at 4°C. Cells were washed 2 times with ice-cold tris-buffered saline (TBS), pH 7.4 and incubated for 20 min at 4°C with 100 µCi [ 99m Tc-MAS 3 ]-GPI in the presence or absence of the agent being tested.…”

“…Recently, we described a family of highly anionic small molecules specific for prostate-specific membrane antigen (PSMA [1]). PSMA is a 100 kDa type II transmembrane glycoprotein whose expression in the systemic circulation is abundant in, and highly restricted to, normal and malignant prostate epithelial cells.…”