Targeting prostate cancer cells with PSMA inhibitor-guided gold nanoparticles.

Prostate-specific membrane antigen (PSMA) is a notable biomarker for diagnostic and therapeutic applications in prostate cancer. Gold nanoparticles (AuNPs) provide an attractive nanomaterial platform for combining a variety of targeting, imaging, and cytotoxic agents into a unified device for biomedical research. In this study, we present the generation and evaluation of the first AuNP system functionalized with a small molecule phosphoramidate peptidomimetic inhibitor for the targeted delivery to PSMA-expressing prostate cancer cells. The general approach involved the conjugation of streptavidin-coated AuNPs with a biotin-linked PSMA inhibitor (CTT54) to generate PSMA-targeted AuNPs. In vitro evaluations of these targeted AuNPs were conducted to determine PSMA-mediated and time-dependent binding to PSMA-positive LNCaP cells. The PSMA-targeted AuNPs exhibited significantly higher and selective binding to LNCaP cells compared to control non-targeted AuNPs, thus demonstrating the feasibility of this approach.

PSMA-targeted SPECT agents: mode of binding effect on in vitro performance.

BACKGROUND: The enzyme-biomarker prostate-specific membrane antigen (PSMA) is an active target for imaging and therapeutic applications for prostate cancer. The internalization of PSMA has been shown to vary with inhibitors' mode of binding: irreversible, slowly reversible, and reversible. METHODS: In the present study, PSMA-targeted clickable derivatives of an irreversible phosphoramidate inhibitor DBCO-PEG(4) -CTT-54 (IC(50) = 1.0 nM) and a slowly reversible phosphate inhibitor, DBCO-PEG(4) -CTT-54.2 (IC(50) = 6.6 nM) were clicked to (99m) Tc(CO)(3) -DPA-azide to assemble a PSMA-targeted SPECT agent. The selectivity, percent uptake, and internalization of these PSMA-targeted SPECT agents were evaluated in PSMA-positive and PSMA-negative cells. RESULTS: In vitro studies demonstrated that PSMA-targeted SPECT agents exhibited selective cellular uptake in the PSMA-positive LNCaP cells compared to PSMA-negative PC3 cells. More importantly, it was found that (99m) Tc(CO)(3) -DPA-DBCO-PEG(4) -CTT-54 based on an irreversible PSMA inhibitor core, exhibited greater uptake and internalization than (99m) Tc(CO)(3) -DPA-DBCO-PEG(4) -CTT-54.2 constructed from a slowly reversible PSMA inhibitor core. CONCLUSIONS: We have demonstrated that a PSMA-targeted SPECT agent can be assembled efficiently using copper-less click chemistry. In addition, we demonstrated that mode of binding has an effect on internalization and percent uptake of PSMA-targeted SPECT agents; with the irreversible targeting agent demonstrating superior uptake and internalization in PSMA+ cells. The approach demonstrated in this work now supports a modular approach for the assembly of PSMA-targeted imaging and therapeutic agents.

A phosphoramidate-based prostate-specific membrane antigen-targeted SPECT agent.

BACKGROUND: Prostate-specific membrane antigen (PSMA) remains an active target for imaging and therapeutic applications for prostate cancer. METHODS: In the present study, an irreversible phosphoramidate inhibitor, CTT-54 (IC50 = 14 nM), has been modified to deliver 99mTc-(CO)3-DTPA as a SPECT imaging payload to PSMA+ cells in vivo and in vitro. Percent uptake, competitive binding, and internalization will evaluate the imaging agent in vitro. Preliminary biodistribution and imaging will be utilized for in vivo evaluation. RESULTS: In vitro studies demonstrate that the radiotracer 99mTc-(CO)3-DTPA-CTT-54 exhibits increasing cellular uptake in the PSMA+ LNCaP cells over time. More importantly, it was found that 99mTc-(CO)3-DTPA-CTT-54 is rapidly internalized into LNCaP cells, presumably through the PSMA enzyme-inhibitor complex. In a pilot biodistribution study, increasing accumulation of the radiotracer in LNCaP xenografts was observed from 2 to 4 hr and significant clearance from non-target tissues. CONCLUSIONS: While DTPA may not represent the ideal chelate structure for 99mTc(CO)3, the data provides proof-of-concept support for the development of a next-generation phosphoramidate-based PSMA inhibitor-conjugates for use as SPECT imaging agents.

Targeting prostate cancer cells with a multivalent PSMA inhibitor-guided streptavidin conjugate.

Prostate-specific membrane antigen (PSMA), a type II membrane glycoprotein, its high expression is associated with prostate cancer progression, and has been becoming an active target for imaging or therapeutic applications for prostate cancer. On the other hand, streptavidin-biotin system has been successfully employed in pretargeting therapy towards multiple cancers. Herein, we describe the synthesis of bifunctional ligands (biotin-CTT54, biotin-PEG(4)-CTT54, and biotin-PEG(12)-CTT54) possessing two functional motifs separated by a length-varied polyethylene glycol (PEG) spacer: one (CTT54) binds tumor-marker PSMA and the other (biotin) binds streptavidin or avidin. All three compounds exhibited high potencies (IC(50) values: 1.21, 2.53, and 10nM, respectively) and irreversibility; but only biotin-PEG(12)-CTT54 demonstrated specifically labeling PSMA-positive prostate cancer cells in a two-step pretargeting procedure. Additionally, the pre-formulated complex between biotin-PEG(12)-CTT54 and Cy5-streptavidin displayed the improved inhibitory potency (IC(50)=1.86 nM) and irreversibility against PSMA and rapid uptake of streptavidin conjugate into PSMA-positive prostate cancer cells through PSMA-associated internalization. Together, all these results supported a proof-concept that combination of streptavidin and PSMA's biotinylated inhibitor may lead to development of a novel strategy of tumor-targeting imaging or drug delivery towards prostate cancer.

Spacer length effects on in vitro imaging and surface accessibility of fluorescent inhibitors of prostate specific membrane antigen.

Prostate-specific membrane antigen (PSMA), a type II transmembrane protein, has been becoming an active target for imaging and therapeutic applications for prostate cancer. Recently, the development of its various chemical inhibitor scaffolds has been explored to serve as carriers for therapeutic or diagnostic payloads targeted to PSMA-positive tumor cells. However, there have been few efforts to definitively determine the optimal length of linker between PSMA inhibitor cores and their payload molecules with regard to the affinity to PSMA and in vitro performance. In our present model study, three spacer-length varied fluorescent inhibitors (FAM-CTT-54, FAM-X-CTT-54 and FAM-PEG(8)-CTT-54) were synthesized, and further enzymatic inhibition studies displayed linker length-dependent changes in: inhibitory potency (IC(50)=0.41 nM, 0.35 nM, 1.93 nM), modes of binding (reversible, slowly reversible, irreversible), respectively. Furthermore, cell-labeling imaging revealed the spacer length-related change of fluorescence intensity (FAM-X-CTT-54>FAM-PEG(8)-CTT-54>FAM-CTT-54). These results suggest that selection of linkers and their lengths will be important considerations in the development of next-generation prostate tumor-targeted imaging probes and therapeutic agents that specifically home to PSMA on tumor cells.

Detection of prostate-specific membrane antigen on HUVECs in response to breast tumor-conditioned medium.

Prostate-specific membrane antigen (PSMA), a well-known biomarker of prostate cancer, has also been found to be highly expressed in the neovasculature of multiple non-prostatic solid tumors. As a consequence, it has the potential to become a biomarker for tumor-associated vasculature. Herein, we describe an in vitro model for assessing PSMA expression associated with tube formation by primary human umbilical vein endothelial cells (HUVECs) cultured in Matrigel and induced by tumor-conditioned medium (TCM) derived from human breast cancer cells (MDA-MB-231). In contrast to vascular endothelial growth factor (VEGF)-containing endothelial cell medium, TCM induced higher expression of PSMA in HUVECs. The vessel-like tubes were detected by imaging with fluorescent PSMA inhibitors. Consequently, this in vitro model is expected to enable subsequent studies aimed at determining the role of PSMA in angiogenesis and factors that induce it.

Assessment of an 18F-labeled phosphoramidate peptidomimetic as a new prostate-specific membrane antigen-targeted imaging agent for prostate cancer.

UNLABELLED: Prostate-specific membrane antigen (PSMA) is a transmembrane protein commonly found on the surface of late-stage and metastatic prostate cancer and a well-known imaging biomarker for staging and monitoring therapy. Although (111)In-labeled capropmab pendetide is the only approved agent available for PSMA imaging, its clinical use is limited because of its slow distribution and clearance that leads to challenging image interpretation. A small-molecule approach using radiolabeled urea-based PSMA inhibitors as imaging agents has shown promise for prostate cancer imaging. The motivation of this work is to explore phosphoramidates as a new class of potent PSMA inhibitors to develop more effective prostate cancer imaging agents with improved specificity and clearance properties. METHODS: N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB) was conjugated to S-2-((2-(S-4-amino-4-carboxybutanamido)-S-2-carboxyethoxy)hydroxyphosphorylamino)-pentanedioic acid (Phosphoramidate (1)), yielding S-2-((2-(S-4-(4-(18)F-fluorobenzamido)-4-carboxybutanamido)-S-2-carboxyethoxy)hydroxyphosphorylamino)-pentanedioic acid (3). In vivo studies were conducted in mice bearing either LNCaP (PSMA-positive) or PC-3 (PSMA-negative) tumors. PET images were acquired at 1 and 2 h with or without a preinjection of a nonradioactive version of the fluorophosphoramidate. Tissue distribution studies were performed at the end of the 2 h imaging sessions. RESULTS: Phosphoramidate (1) and its fluorobenzamido conjugate (2) were potent inhibitors of PSMA (inhibitory concentration of 50% [IC(50)], 14 and 0.68 nM, respectively). PSMA-mediated tumor accumulation was noted in the LNCaP versus the PC-3 tumor xenografts. The LNCaP tumor uptake was also blocked by the administration of nonradioactive (2) prior to imaging studies. With the exception of the kidneys, tumor-to-tissue and tumor-to-blood ratios were greater than 5:1 at 2 h. The strong kidney uptake may be due to the known PSMA expression in the mouse kidney, because significant reduction (>6-fold) in kidney activity was seen in mice injected with (2). CONCLUSION: (18)F-labeled phosphoramidate (3) is a representative of a new class of PSMA targeting peptidomimetic molecules that shows great promise as imaging agents for detecting PSMA+ prostate tumors.