Mark R. Hopkins

A targeted low molecular weight near-infrared fluorescent probe for prostate cancer.

Prostate-specific membrane antigen (PSMA) remains an active target for imaging and therapeutic applications for prostate cancer. Although radionuclide-based imaging is generally more sensitive and also has been deeply explored, near-infrared fluorescence imaging agents are simple to prepare and compatible with long-term storage conditions. In the present study, a near-infrared fluorescent imaging probe (Cy5.5-CTT-54.2) has been developed by chemical conjugation of Cy5.5N-hydroxysuccinimide ester (Cy5.5-NHS) with a potent PSMA inhibitor CTT-54.2 (IC(50)=144 nM). The probe displays a highly potency (IC(50)=0.55 nM) against PSMA and has demonstrated successful application for specifically labeling PSMA-positive prostate cancer cells in both two and three-dimensional cell culture conditions. These results suggest that the potent, near-infrared Cy5.5-PSMA inhibitor conjugate may be useful for the detection of prostate tumor cells by optical in vivo imaging.

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.

A high-affinity [(18)F]-labeled phosphoramidate peptidomimetic PSMA-targeted inhibitor for PET imaging of prostate cancer.

INTRODUCTION In this study, a structurally modified phosphoramidate scaffold, with improved prostate-specific membrane antigen (PSMA) avidity, stability and in vivo characteristics, as a PET imaging agent for prostate cancer (PCa), was prepared and evaluated. METHODS p-Fluorobenzoyl-aminohexanoate and 2-(3-hydroxypropyl)glycine were introduced into the PSMA-targeting scaffold yielding phosphoramidate 5. X-ray crystallography was performed on the PSMA/5 complex. [(18)F]5 was synthesized, and cell uptake and internalization studies were conducted in PSMA(+) LNCaP and CWR22Rv1 cells and PSMA(-) PC-3 cells. In vivo PET imaging and biodistribution studies were performed at 1 and 4 h post injection in mice bearing CWR22Rv1 tumor, with or without blocking agent. RESULTS The crystallographic data showed interaction of the p-fluorobenzoyl group with an arene-binding cleft on the PSMA surface. In vitro studies revealed elevated uptake of [(18)F]5 in PSMA(+) cells (2.2% in CWR22Rv1 and 12.1% in LNCaP) compared to PSMA(-) cells (0.08%) at 4 h. In vivo tumor uptake of 2.33% ID/g and tumor-to-blood ratio of 265:1 was observed at 4 h. CONCLUSIONS We have successfully synthesized, radiolabeled and evaluated a new PSMA-targeted PET agent. The crystal structure of the PSMA/5 complex highlighted the interactions within the arene-binding cleft contributing to the overall complex stability. The high target uptake and rapid non-target clearance exhibited by [(18)F]5 in PSMA(+) xenografts substantiates its potential use for PET imaging of PCa. ADVANCES IN KNOWLEDGE The only FDA-approved imaging agent for PCa, Prostascint®, targets PSMA but suffers from inherent shortcomings. The data acquired in this manuscript confirmed that our new generation of [(18)F]-labeled PSMA inhibitor exhibited promising in vivo performance as a PET imaging agent for PCa and is well-positioned for subsequent clinical trials. Implications for Patient Care Our preliminary data demonstrate that this tracer possesses the required imaging characteristics to be sensitive and specific for PCa imaging in patients at all stages of the disease.

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 PSMAs biotinylated inhibitor may lead to development of a novel strategy of tumor-targeting imaging or drug delivery towards prostate cancer.

Chemoaffinity capture of pre-targeted prostate cancer cells with magnetic beads

Prostate circulating tumor cells (PCTCs) in circulation are shed from either a primary tumor or metastases, which are directly responsible for most prostate cancer deaths. Quantifying exfoliated PCTCs may serve as an indicator for the clinical management of prostate cancer, isolating and removing of PCTCs could potentially reduce prostate cancer metastasis, and culturing and characterizing captured PCTCs could facilitate the development of personalized treatment options. Prostate‐specific membrane antigen (PSMA) is an established biomarker for prostate cancer being strongly expressed on prostate tumor cells associated with high‐grade primary, androgen independent, and metastatic tumors.

Development of inhibitor-directed enzyme prodrug therapy (IDEPT) for prostate cancer

Prostate cancer (PCa) is the second most common cause of cancer death among American men after lung cancer. Unfortunately, current therapies do not provide effective treatments for patients with advanced, metastatic, or hormone refractory disease. Therefore, we seek to generate therapeutic agents for a novel PCa treatment strategy by delivering a suicide enzyme (yCDtriple) to a cell membrane bound biomarker found on PCa cells (prostate-specific membrane antigen (PSMA)). This approach has resulted in a new PCa treatment strategy reported here as inhibitor-directed enzyme prodrug therapy (IDEPT). The therapeutic agents described were generated using a click chemistry reaction between the unnatural amino acid (p-azidophenylalanine (pAzF)) incorporated into yCDtriple and the dibenzylcyclooctyne moiety of our PSMA targeting agent (DBCO-PEG4-AH2-TG97). After characterization of the therapeutic agents, we demonstrate significant PCa cell killing of PSMA-positive cells. Importantly, we demonstrate that this click chemistry approach can be used to efficiently couple a therapeutic protein to a targeting agent and may be applicable to the ablation of other types of cancers and/or malignancies.

Structure–Activity Relationship of 18F-Labeled Phosphoramidate Peptidomimetic Prostate-Specific Membrane Antigen (PSMA)-Targeted Inhibitor Analogues for PET Imaging of Prostate Cancer

A series of phosphoramidate-based prostate specific membrane antigen (PSMA) inhibitors of increasing lipophilicity were synthesized (4, 5, and 6), and their fluorine-18 analogs were evaluated for use as positron emission tomography (PET) imaging agents for prostate cancer. To gain insight into their modes of binding, they were also cocrystallized with the extracellular domain of PSMA. All analogs exhibited irreversible binding to PSMA with IC50 values ranging from 0.4 to 1.3 nM. In vitro assays showed binding and rapid internalization (80-95%, 2 h) of the radiolabeled ligands in PSMA(+) cells. In vivo distribution demonstrated significant uptake in CWR22Rv1 (PSMA(+)) tumor, with tumor to blood ratios of 25.6:1, 63.6:1, and 69.6:1 for [(18)F]4, [(18)F]5, and [(18)F]6, respectively, at 2 h postinjection. Installation of aminohexanoic acid (AH) linkers in the phosphoramidate scaffold improved their PSMA binding and inhibition and was critical for achieving suitable in vivo imaging properties, positioning [(18)F]5 and [(18)F]6 as favorable candidates for future prostate cancer imaging clinical trials.

Rationally Designed Sulfamides as Glutamate Carboxypeptidase II Inhibitors

Glutamate carboxypeptidase II (GCPII) is a membrane‐bound cell surface peptidase. There is significant interest in the inhibition of GCPII as a means of neuroprotection, while GCPII inhibition as a method to treat prostate cancer remains a topic of further investigation. The key zinc‐binding functional group of the well‐characterized classes of GCPII inhibitors (phosphonates and phosphoramidates) is tetrahedral and negatively charged at neutral pH, while glutamyl urea class of inhibitors possesses a planar and neutral zinc‐binding group. This study explores a new class of GCPII inhibitors, glutamyl sulfamides, which possess a putative net neutral tetrahedral zinc‐binding motif. A small library containing six sulfamides was prepared and evaluated for inhibitory potency against purified GCPII in an enzymatic assay. While most inhibitors have potencies in the micromolar range, one showed promising sub‐micromolar potency, with the optimal inhibitor in this series being aspartyl–glutamyl sulfamide (2d). Lastly, computational docking was used to develop a tentative binding model on how the most potent inhibitors interact with the ligand‐binding site of GCPII.

Abstract A53: Prostate cancer cell capture using immobilized inhibitors of prostate-specific membrane antigen.

Prostate circulating tumor cells (PCTCs) in the peripheral blood are shed from either a primary tumor or its metastases. These disseminated PCTCs are directly responsible for the most cancer deaths and the major cause of prostate cancer-related mortality. Therefore, elucidating the quantity of the exfoliated PCTCs in blood circulation can serve as an indicator for the clinical management in prostate cancer diseases by providing information on the success/failure of therapy. And the isolation and removal of PCTCs from circulation could potentially reduce prostate cancer metastasis. Furthermore, culturing and characterizing captured PCTCs from a patient is an attractive strategy for developing personalized treatment options. It has been established that prostate-specific membrane antigen (PSMA) is up-regulated and strongly expressed on prostate cancer cells associated with high grade primary, androgen independent, and metastatic tumors. While it is known that PSMA expression is heterogeneous in primary prostate tumors and metastases, completely PSMA negative prostate carcinomas are rare. The present study has outfitted streptavidin coated magnetic beads with a unique irreversible small-molecule PSMA inhibitor, Biotin-PEG12-CTT-54, and demonstrated that they can selectively capture PSMA + cells. The results demonstrated prostate cancer cells capturing is very specific and highly efficient, and high recovery and high viability were obtained. Subsequent in vitro propagation of these viable captured lymph node-based metastatic prostate LnCaP cells is possible without removal of magnetic beads on the cell surface. The results suggested that PCTCs enrichment by streptavidin coated magnetic beads with biotinated PSMA inhibitor has a high PCTC detection rate with an easy manual protocol, and can serve as an additional research tool and clinical assay in PCTC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A53.

Abstract A117: Rapid uptake of a streptavidin conjugate into prostate cancer cells: A model of macromolecule targeting delivery.

Introduction: Prostate-specific membrane antigen (PSMA), a type II membrane glycoprotein, has been confirmed to be an important tumor-marker indicating prostate cancer progression, and is becoming an active target for imaging or therapy for prostate cancer. Furthermore, the streptavidin-biotin coupling pair has been successfully employed in pretargeting therapy for multiple cancers. In the present study, we describe the synthesis and in vitro performance evaluation of a biotinylated PSMA inhibitor (biotin-PEG12-CTT54) possessing two functional motifs separated by a PEG linker: 1) a PSMA targeting core (CTT-54) that specifically binds enzyme-biomarker PSMA and 2) biotin for binding streptavidin. Results and Discussion: Biotin-PEG12-CTT54 was confirmed by MALDI-TOF analysis: calculated 1267.52, found 1267.5221. This bifunctional compound was further evaluated for its inhibitory potency against purified PSMA and in vitro cell imaging efficiency. PSMA inhibition study confirmed that conjugation of CTT-54 to biotin through PEG linker (biotinylated inhibitor, IC50 = 10 nM) had no adverse effect on the inhibitory potency of the parent inhibitor core CTT-54 (IC50 = 14 nM). In a two-step pre-targeting imaging experiment, 3-day grown PSMA-positive LNCaP cells was treated with this biotinylated compound, then followed by the incubation with Cy5-conjugated streptavidin, our data demonstrated specifically labeling PSMA-positive prostate cancer cells, but not for cells treated with Cy5-conjugated streptavidin only. Additionally, the complex between the biotinylated compound and Cy5-conjugated streptavidin was prepared and purified prior to cell labeling. The purified pre-formed complex was found to contain three biotinylated inhibitors for each streptavidin using a newly-developed digitally-operated linear quadrupole ion trap orthogonal acceleration time-of-flight mass spectrometer, which is designed to analyze non-covalently bound complex by confidentially maintaining its integrity through the analytical process. The preformed complex was tested for its inhibitory potency against purified PSMA and the cell-labeling of PSMA-positive cells, the experimental results revealed an improved inhibitory potency (IC50 = 1.9 nM) against PSMA and rapid uptake of streptavidin into PSMA-positive prostate cancer cells within 10 min. To better understand the intracellular fate of Cy5-streptavidin once internalized in LNCaP cells, co-localization studies were conducted with transferrin-fluorescein, which can be tracked as an endosomal marker for the ligand-receptor internalization via clathrin-coated pits. Co-localization of internalized streptavidin-Cy5 with transferrin-fluorescein appeared strongly in perinuclear region after 2h-incubation. The pattern of PSMA internalization induced by preformed complex is consistent with antibody or small-molecule inhibitors alone. Conclusions: We have successfully demonstrated that macromolecules such as streptavidin can be specifically delivered into prostate cancer cells. These results support a proof-of-concept that a combination of streptavidin (serving as a drug or imaging agent carrier) and a biotinylated PSMA inhibitor may lead to the development of a novel strategy of tumor-targeting imaging or drug delivery for prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A117.