A low molecular weight PSMA-based fluorescent imaging agent for cancer

We synthesized YC-27 3 to provide a fluorescent imaging agent for the prostate-specific membrane antigen (PSMA), a marker for hormone-independent prostate cancer and tumor neovasculature, with suitable pharmacokinetics for use in vivo. Immediate precursor trifluoroacetate salt of 2-(3-{5-[7-(5-amino-1-carboxy-pentylcarbamoyl)-heptanoylamino]-1-carboxy-pentyl}-ureido)-pentanedioic acid 2 was conjugated with a commercially available near-infrared light-emitting dye (IRDye 800CW) to provide 3 in 72% yield. YC-27 3 demonstrated a PSMA inhibitory activity of 0.37 nM and was capable of generating target-to-nontarget ratios of at least 10 in PSMA-expressing PC3-PIP vs. PSMA-negative PC3-flu tumors in vivo. YC-27 3 may be useful for study of PSMA-expressing tissue in preclinical models or for intraoperative guidance.     

Synthesis and preclinical evaluation of 68Ga-PSMA-BCH for prostate cancer imaging

Prostate specific membrane antigen (PSMA) is a promising target for the diagnosis and therapy of prostate cancer. In this report, a NOTA-conjugated precursor, NOTA-PSMA (also named PSMA-BCH), was synthesized by peptide synthesizer with the chemical purity over 95%. ⁶⁸Ga-PSMA-BCH was obtained by radiolabeling NOTA-PSMA with ⁶⁸GaCl₃ with >99% radiochemical purity and 59–74?GBq/μmol specific activity. In vitro and in vivo study of ⁶⁸Ga-PSMA-BCH showed high stability, high uptake in PSMA-expressing cells and tumor, fast clearance and low non-target uptake. 22Rv1 tumors were clearly observed in micro-PET images of and showed good retention. Compared with ⁶⁸Ga-PSMA-617, ⁶⁸Ga-PSMA-BCH showed comparable tumor uptake and tumor-background ratios. Indicating ⁶⁸Ga-PSMA-BCH is a promising candidate for prostate cancer imaging and worthy of further clinical investigations.     

Synthesis of novel multivalent fluorescent inhibitors with high affinity to prostate cancer and their biological evaluation

Prostate-specific membrane antigen (PSMA) is an important biological target for therapy and diagnosis of prostate cancer. In this study, novel multivalent PSMA inhibitors with glutamate-urea-lysine structures were designed to improve inhibition characteristics. Precursors of the novel inhibitors were prepared from glutamic acid with di-tert-butyl ester. A near-infrared molecular dye, sulfo-Cy5.5, was introduced into the precursors to generate the final PSMA fluorescent inhibitors, compounds 12–14, to visualize prostate cancer. Biological behaviors of the inhibitors were evaluated using in vitro inhibition assays, in vivo fluorescent imaging, and ex vivo biodistribution assays. K_i values from inhibition studies indicated that dimeric inhibitor 13 with a glutamine linker showed approximately 3-fold more inhibitory activity than monomeric inhibitor 12. According to other biological studies using a mouse model of prostate cancer, dimeric inhibitor compounds 13 and 14 had higher tumor accumulation than the monomer. However, glutamine-based dimeric inhibitor 13 showed lower liver uptake than dimeric inhibitor 14, which had a benzene structure. Thus, these studies suggest that glutamine-based dimeric inhibitor 13 can be a promising optical inhibitor of prostate cancer.     

A bispecific diabody directed against prostate-specific membrane antigen and CD3 induces T-cell mediated lysis of prostate cancer cells

Background Although cancer of the prostate is one of the most commonly diagnosed cancers in men, no curative treatment currently exists after its progression beyond resectable boundaries. Therefore, new agents for targeted treatment strategies are needed. Cross-linking of tumor antigens with T-cell associated antigens by bispecific monoclonal antibodies have been shown to increase antigen-specific cytotoxicity in T-cells. Since the prostate-specific membrane antigen (PSMA) represents an excellent tumor target, immunotherapy with bispecific diabodies could be a promising novel treatment option for prostate cancer. Methods A heterodimeric diabody specific for human PSMA and the T-cell antigen CD3 was constructed from the DNA of anti-CD3 and anti-PSMA single chain Fv fragments (scFv). It was expressed in E. coli using a vector containing a bicistronic operon for co-secretion of the hybrid scFv V_HCD3-V_LPSMA and V_HPSMA-V_LCD3. The resulting PSMAxCD3 diabody was purified from the periplasmic extract by immobilized metal affinity chromatography (IMAC). The binding properties were tested on PSMA-expressing prostate cancer cells and PSMA-negative cell lines as well as on Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability test (WST) was used. For in vivo evaluation the diabody was applied together with human peripheral blood lymphocytes (PBL) in a C4-2 xenograft-SCID mouse model. Results By Blue Native gel electrophoresis, it could be shown that the PSMAxCD3 diabody is mainly a tetramer. Specific binding both to CD3-expressing Jurkat cells and PSMA-expressing C4-2 cells was shown by flow cytometry. In vitro, the diabody proved to be a potent agent for retargeting PBL to lyze C4-2 prostate cancer cells. Treatment of SCID mice inoculated with C4-2 tumor xenografts with the diabody and PBL efficiently inhibited tumor growth. Conclusions The PSMAxCD3 diabody bears the potential for facilitating immunotherapy of prostate cancer and for the elimination of minimal residual disease. P. Bühler and P. Wolf equally contributed to the work.     

Synthesis and SAR of 99mTc/Re-labeled small molecule prostate specific membrane antigen inhibitors with novel polar chelates

Prostate specific membrane antigen (PSMA) is recognized as an attractive molecular target for the development of radiopharmaceuticals to image and potentially treat metastatic prostate cancer. A series of novel ^99mTc/Re-tricarbonyl radiolabeled PSMA inhibitors were therefore synthesized by the attachment of glutamate-urea-lysine (Glu-urea-Lys) and glutamate-urea-glutamate (Glu-urea-Glu) pharmacophore to single amino acid chelate (SAAC) where the SAAC ligand was either bis(pyridin-2-ylmethyl)amino (DPA), bis((1-methyl-1H-imidazol-2-yl)methyl)amino (NMI), bis((1-(carboxymethyl)-1H-imidazol-2-yl)methyl)amino (CIM) or bis((1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino (TIM). The in vitro binding affinity of the rhenium complexes was evaluated using PSMA-expressing human prostate cancer LNCaP cells. IC₅₀ values ranged from 3.8 ± 2 to >2000 nM. A linker between the SAAC chelate and pharmacophore was required for high affinity binding. However, extending the length of the linker did not substantially improve binding. PSMA binding was also influenced by the nature of the SAAC chelate. One of the most potent compounds, 23b (IC₅₀ = 4.8 ± 2.7 nM), was radiolabeled with technetium tricarbonyl ({^99mTc(CO)₃}⁺) to afford the {^99mTc(CO)₃}⁺ complex in excellent yield and high purity. This effort has led to the identification of a diverse series of promising high affinity {^99mTc(CO)₃}⁺ radiolabeled PSMA inhibitors.     

Design and synthesis of dye-conjugated hepsin inhibitors

Hepsin is a type II serine protease that is highly expressed in neoplastic prostate. It is an attractive biomarker for imaging metastatic prostate cancer because of its overexpression in advanced prostate cancer and the location of its active site on the cell surface. We designed and synthesized novel hepsin-targeted imaging probes by conjugating the hepsin-binding ligand with near-infrared (NIR) optical dyes. The Leu-Arg dipeptides, attached to BODIPY or SulfoCy7, exhibited strong hepsin-inhibitory activities with K_i values of 21 and 22 nM, respectively. Compound 2 showed selective uptake and retention in hepsin-overexpressing cells. This is the first report of hepsin-targeted optical probes with strong binding affinities and high selectivity over matriptase. Compound 2 has the potential to be used for developing hepsin-based imaging probes and be as a prototype molecule in the design of new hepsin inhibitors.     

Tyrosine-based asymmetric urea ligand for prostate carcinoma: Tuning biological efficacy through in silico studies

In this article, we have explored the chemical interactions of tyrosine-based asymmetric urea ligands in the binding pockets of prostate specific membrane antigen (PSMA) through in silico studies. The S1 pocket of the PSMA protein offers better scope for modifications in the urea ligands to improve the binding affinity. Accordingly, tyrosine-based (S)-2-(3-((S)-1-carboxy-2-(4-(carboxymethoxy)phenyl)ethyl)ureido)pentanedioic acid (CYUE, 3) ligand was designed, synthesized and predicted to possess inhibition constant (Ki) of 55 nM with PSMA protein. The CYUE (3) ligand was further elaborated into a fluorescent diagnostic probe for detection of PSMA⁺ cancers. In vitro studies on human malignant cell lines such as LNCaP and PC-3 were performed to show the efficacy and specificity of the newly synthesized bio-construct. The fluorescent bio-conjugate was found to be very specific to the PSMA protein with an overall binding affinity constant (K_D) of 88 nM.     

Synthesis and biological evaluation of PSMA-targeting paclitaxel conjugates

Prostate cancer (PC) is the second most commonly occurring cancer in men. Conventional chemotherapy has wide variety of disadvantages such as high systemic toxicity and low selectivity. Targeted drug delivery is a promising approach to decrease side effects of therapy. Prostate specific membrane antigen (PSMA) is overexpressed in prostate cancer cells while low level of expression is observed in normal cells. In this study we describe the development of Glu-urea-Lys based PSMA-targeting conjugates with paclitaxel. A series of new PSMA targeting conjugates with paclitaxel was designed and synthesized. The cytotoxicity of conjugates was evaluated against prostate (LNCaP, 22Rv1 and PC-3) and non-prostate (Hek293T, VA13, A549 and MCF-7) cell lines. The most promising conjugate 21 was examined in vivo using 22Rv1 xenograft mice model. It demonstrated good efficiency comparable with paclitaxel, while reduced toxicity. 3D molecular docking study was also performed to understand underlying mechanism of binding and further optimization of the linker substructure and conjugates structure for improving the target affinity. These conjugates may be useful for further design of novel PSMA targeting delivery systems for PC.     

PSMA theragnostics for metastatic castration resistant prostate cancer

There has been tremendous growth in the development of theragnostics for personalized cancer diagnosis and treatment over the past two decades. In prostate cancer, the new generation of prostate specific membrane antigen (PSMA) small molecular inhibitor-based imaging agents achieve extraordinary tumor to background ratios and allow their therapeutic counterparts to deliver effective tumor doses while minimizing normal tissue toxicity. The PSMA targeted small molecule positron emission tomography (PET) agents ¹⁸F-DCFPyL (2-(3-{1-carboxy-5-((6-(18)F-fluoro-pyridine-3-carbonyl)-amino)-pentyl}-ureido)-pentanedioic acid) and Gallium-68 (⁶⁸Ga)-PSMA-11 have been approved by the United States Food and Drug Administration (FDA) for newly diagnosed high risk prostate cancer patients and for patients with biochemical recurrence. More recently, the Phase III VISION trial showed that Lutetium-177 (¹⁷⁷Lu)-PSMA-617 treatment increases progression-free survival and overall survival in patients with heavily pre-treated advanced PSMA-positive metastatic castration-resistant prostate cancer (mCRPC). Here, we review the PSMA targeted theragnostic pairs under clinical investigation for detection and treatment of metastatic prostate cancer.     

Structure-Activity Relationships of 2′,5′-Oligoadenylate Analogue Modifications of Prostate-Specific Membrane Antigen (PSMA) Antagonists

Prostate-specific membrane antigen (PSMA) is an ideal biomarker for prostate cancer. A previously reported 2-5A conjugate RBI1033 (3) showed binding affinity more than 10 times higher than the parent urea-based compound (S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido) pentanedioic acid (1). The purpose of this work is to further optimize the structure of 3 to identify highly selective ligands of PSMA. It was found that conjugates having 2-5A in their structure showed extraordinary improved binding affinity to PSMA compared with compound 1. Removal of 2-5A significantly reduced its biological activity. The results will provide a path to agents for targeted imaging and treatment of prostate cancer.     

Prostate‐specific membrane antigen‐targeted photoacoustic imaging of prostate cancer in vivo

A sensitive, noninvasive method to detect localized prostate cancer, particularly for early detection and repetitive study in patients undergoing active surveillance, remains an unmet need. Here, we propose a molecular photoacoustic (PA) imaging approach by targeting the prostate‐specific membrane antigen (PSMA), which is over‐expressed in the vast majority of prostate cancers. We performed spectroscopic PA imaging in an experimental model of prostate cancer, namely, in immunocompromised mice bearing PSMA+ (PC3 PIP) and PSMA? (PC3 flu) tumors through administration of the known PSMA‐targeted fluorescence agent, YC‐27. Differences in contrast between PSMA+ and isogenic control tumors were observed upon PA imaging, with PSMA+ tumors showing higher contrast in average of 66.07‐fold with 5 mice at the 24‐hour postinjection time points. These results were corroborated using standard near‐infrared fluorescence imaging with YC‐27, and the squared correlation between PA and fluorescence intensities was 0.89. Spectroscopic PA imaging is a new molecular imaging modality with sufficient sensitivity for targeting PSMA in vivo, demonstrating the potential applications for other saturable targets relevant to cancer and other disorders.      

Is prostate-specific membrane antigen a multifunctional protein?

Prostate-specific membrane antigen (PSMA) is a metallopeptidase expressed predominantly in prostate cancer (PCa) cells. PSMA is considered a biomarker for PCa and is under intense investigation for use as an imaging and therapeutic target. Although the clinical utility of PSMA in the detection and treatment of PCa is evident and is being pursued, very little is known about its basic biological function in PCa cells. The purpose of this review is to highlight the possibility that PSMA might be a multifunctional protein. We suggest that PSMA may function as a receptor internalizing a putative ligand, an enzyme playing a role in nutrient uptake, and a peptidase involved in signal transduction in prostate epithelial cells. Insights into the possible functions of PSMA should improve the diagnostic and therapeutic values of this clinically important molecule. prostate cancer; receptor; peptidase; endocytosis     

Dendritic Cell Based PSMA Immunotherapy for Prostate Cancer Using a CD40-Targeted Adenovirus Vector

Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.     

Synthesis and evaluation of novel radioiodinated PSMA targeting ligands for potential radiotherapy of prostate cancer

Radioligand therapy (RLT) using prostate-specific membrane antigen (PSMA) targeting ligands is an attractive option for the treatment of Prostate cancer (PCa) and its metastases. We report herein a series of radioiodinated glutamate-urea-lysine-phenylalanine derivatives as new PSMA ligands in which l-tyrosine and l-glutamic acid moieties were added to increase hydrophilicity concomitant with improvement of in vivo targeting properties. Compounds 8, 15, 19a/19b and 23a/23b were synthesized and radiolabeled with ¹²⁵I by iododestannylation. All iodinated compounds displayed high binding affinities toward PSMA (IC₅₀ = 1–13 nM). In vitro cell uptake studies demonstrated that compounds containing an l-tyrosine linker moiety (8, 15 and 19a/19b) showed higher internalization than MIP-1095 and 23a/23b, both without the l-tyrosine linker moiety. Biodistribution studies in mice bearing PC3-PIP and PC3 xenografts showed that [¹²⁵I]8 and [¹²⁵I]15 with higher lipophilicity exhibited higher nonspecific accumulations in the liver and intestinal tract, whereas [¹²⁵I]19a/19b and [¹²⁵I]23a/23b containing additional glutamic acid moieties showed higher accumulations in the kidney and implanted PC3-PIP (PSMA+) tumors. [¹²⁵I]23b displayed a promising biodistribution profile with favorable tumor retention, fast clearance from the kidney, and 2–3-fold lower uptake in the liver and blood than that observed for [¹²⁵I]MIP-1095. [^125/131I]23b may serve as an optimal PSMA ligand for radiotherapy treatment of prostate cancer over-expressing PSMA.     

Discovery of novel jaspine B analogues as autophagy inducer

A series of 2-alkylaminomethyl jaspine B analogues were synthesized and evaluated for their cytotoxic effects on human lung adenocarcinoma, breast cancer, and prostate cancer cell lines and a mouse melanoma cell line. Most of the compounds exhibited moderate to good activity against the cancer cell lines. Compound 7f showed the best overall cytotoxicity on PC-3 cells (IC₅₀?=?0.85?μM). Further mechanistic studies revealed that compound 7f induced marked changes in PC-3 cell morphology, disrupted the mitochondrial membrane potential, and increased expression of the autophagy proteins beclin-1, LC3, and P62.     

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.     

Tumor target prostate specific membrane antigen (PSMA) and its regulation in prostate cancer

Prostate specific membrane antigen (PSMA), is a unique membrane bound glycoprotein, which is overexpressed manifold on prostate cancer as well as neovasculature of most of the solid tumors, but not in the vasculature of the normal tissues. This unique expression of PSMA makes it an important marker as well as a large extracellular target of imaging agents. PSMA can serve as target for delivery of therapeutic agents such as cytotoxins or radionuclides. PSMA has two unique enzymatic functions, folate hydrolase and NAALADase and found to be recycled like other membrane bound receptors through clathrin coated pits. The internalization property of PSMA leads one to consider the potential existence of a natural ligand for PSMA. In this review we have discussed the regulation of PSMA expression within the cells, and significance of its expression in prostate cancer and metastasis.     

Development of a Ga-68 labeled PET tracer with short linker for prostate-specific membrane antigen (PSMA) targeting

Glu-Urea-Lys (GUL) derivatives have been reported as prostate-specific membrane antigen (PSMA) agent. We developed derivatives of GUL conjugated with NOTA or DOTA via a thiourea linker and tested their feasibility as PSMA imaging agents after labeling with ⁶⁸Ga. NOTA-GUL and DOTA-GUL were synthesized and labeled with ⁶⁸Ga using generator-eluted ⁶⁸GaCl₃ in 0.1?M HCl in the presence of 1?M NaOAc at pH 5.5. The stabilities of ⁶⁸Ga-labeled compounds in human serum were tested at 37.5?°C. A competitive binding assay was performed using the PSMA-positive prostate cancer cell line 22Rv1 and [¹²⁵I]MIP-1072 (PSMA-specific binding agent) as a tracer. Biodistribution and micro-PET studies were performed using 22Rv1-xenograft BALB/c nude mice. The radiolabeling efficiency of NOTA-GUL (>99%) was higher than that of DOTA-GUL (92%). The IC₅₀ of Ga-NOTA-GUL was 18.3?nM. In the biodistribution study, tumor uptake of ⁶⁸Ga-NOTA-GUL (5.40% ID/g) was higher than that of ⁶⁸Ga-DOTA-GUL (4.66% ID/g) at 1?h. Tumor/muscle and tumor/blood uptake ratios of ⁶⁸Ga-NOTA-GUL (31.8 and 135, respectively) were significantly higher than those of ⁶⁸Ga-DOTA-GUL (16.1 and 31.1, respectively). The tumor/kidney uptake ratio of ⁶⁸Ga-NOTA-GUL was 3.4-fold higher than that of ⁶⁸Ga-DOTA-GUL. ⁶⁸Ga-NOTA-GUL showed specific uptake to PSMA positive tumor xenograft and was blocked by co-injection of the cold ligand. In conclusion, we successfully synthesized ⁶⁸Ga-NOTA-GUL and ⁶⁸Ga-DOTA-GUL for prostate cancer imaging. ⁶⁸Ga-NOTA-GUL showed better radiochemical and biodistribution results. ⁶⁸Ga-NOTA-GUL may be a promising PSMA targeting radiopharmaceutical.     

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.