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author:("cease, rennie")
1.  64Cu-Labeled Inhibitors of Prostate-Specific Membrane Antigen for PET Imaging of Prostate Cancer 
Journal of Medicinal Chemistry  2014;57(6):2657-2669.
Prostate-specific membrane antigen (PSMA) is a well-recognized target for identification and therapy of a variety of cancers. Here we report five 64Cu-labeled inhibitors of PSMA, [64Cu]3–7, which are based on the lysine–glutamate urea scaffold and utilize a variety of macrocyclic chelators, namely NOTA(3), PCTA(4), Oxo-DO3A(5), CB-TE2A(6), and DOTA(7), in an effort to determine which provides the most suitable pharmacokinetics for in vivo PET imaging. [64Cu]3–7 were prepared in high radiochemical yield (60–90%) and purity (>95%). Positron emission tomography (PET) imaging studies of [64Cu]3–7 revealed specific accumulation in PSMA-expressing xenografts (PSMA+ PC3 PIP) relative to isogenic control tumor (PSMA– PC3 flu) and background tissue. The favorable kinetics and high image contrast provided by CB-TE2A chelated [64Cu]6 suggest it as the most promising among the candidates tested. That could be due to the higher stability of [64Cu]CB-TE2A as compared with [64Cu]NOTA, [64Cu]PCTA, [64Cu]Oxo-DO3A, and [64Cu]DOTA chelates in vivo.
doi:10.1021/jm401921j
PMCID: PMC3983358  PMID: 24533799
2.  Heterobivalent Agents Targeting PSMA and Integrin-αvβ3 
Bioconjugate Chemistry  2014;25(2):393-405.
Differential expression of surface proteins on normal vs malignant cells provides the rationale for the development of receptor-, antigen-, and transporter-based, cancer-selective imaging and therapeutic agents. However, tumors are heterogeneous, and do not always express what can be considered reliable, tumor-selective markers. That suggests development of more flexible targeting platforms that incorporate multiple moieties enabling concurrent targeting to a variety of putative markers. We report the synthesis, biochemical, in vitro, and preliminary in vivo evaluation of a new heterobivalent (HtBv) imaging agent targeting both the prostate-specific membrane antigen (PSMA) and integrin-αvβ3 surface markers, each of which can be overexpressed in certain tumor epithelium and/or neovasculature. The HtBv agent was functionalized with either 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or the commercially available IRDye800CW. DOTA-conjugated HtBv probe 9 bound to PSMA or αvβ3 with affinities similar to those of monovalent (Mnv) compounds designed to bind to their targets independently. In situ energy minimization experiments support a model describing the conformations adapted by 9 that enable it to bind both targets. IRDye800-conjugated HtBv probe 10 demonstrated target-specific binding to either PSMA or integrin-αvβ3 overexpressing xenografts. HtBv agents 9 and 10 may enable dual-targeted imaging of malignant cells and tissues in an effort to address heterogeneity that confounds many cancer-targeted imaging agents.
doi:10.1021/bc4005377
PMCID: PMC4112557  PMID: 24410012
3.  AEG-1 promoter-mediated imaging of prostate cancer 
Cancer research  2014;74(20):5772-5781.
We describe a new imaging method for detecting prostate cancer, whether localized or disseminated and metastatic to soft tissues and bone. The method relies on the use of imaging reporter genes under the control of the promoter of AEG-1 (MTDH), which is selectively active only in malignant cells. Through systemic, nanoparticle-based delivery of the imaging construct, lesions can be identified through bioluminescence imaging and single photon emission-computed tomography in the PC3-ML murine model of prostate cancer at high sensitivity. This approach is applicable for the detection of prostate cancer metastases, including bone lesions for which there is no current reliable agent for non-invasive clinical imaging. Further, the approach compares favorably to accepted and emerging clinical standards, including positron emission tomography with [18F]fluorodeoxyglucose and [18F]sodium fluoride. Our results offer a preclinical proof of concept that rationalizes clinical evaluation in patients with advanced prostate cancer.
doi:10.1158/0008-5472.CAN-14-0018
PMCID: PMC4234089  PMID: 25145668
molecular-genetic imaging; bioluminescence; SPECT; metastasis; nanoparticle; PC3
4.  A red-shifted fluorescent substrate for aldehyde dehydrogenase 
Nature communications  2014;5:3662.
Selection of cells positive for aldehyde dehydrogenase (ALDH) activity from a green fluorescent background is difficult with existing reagents. Here we report a red-shifted fluorescent substrate for ALDH, AldeRed 588-A, for labeling viable ALDHpos cells. We demonstrate that AldeRed 588-A successfully isolates ALDHhi human hematopoietic stem cells from heterogeneous cord blood mononuclear cells. AldeRed 588-A can be used for multi-color applications to fractionate ALDHpos cells in the presence of green fluorophores including the ALDEFLUOR™ reagent and cells expressing eGFP. AldeRed 588-A stains ALDHpos murine pancreatic centroacinar and terminal duct cells, as visualized by fluorescent microscopy. AldeRed588-A provides a useful tool to select stem cells or study ALDH within a green fluorescent background.
doi:10.1038/ncomms4662
PMCID: PMC4063304  PMID: 24759454
5.  Synthesis and Biological Evaluation of Substrate-Based Imaging Agents for the Prostate-Specific Membrane Antigen 
Macromolecular research  2013;21(5):565-573.
Prostate-specific membrane antigen (PSMA) is an attractive target for the imaging of prostate cancer (PCa) due to the elevated expression on the surface of prostate tumor cells. Most PSMA-targeted low molecular weight imaging agents are inhibitors of PSMA. We have synthesized a series of substrate-based PSMA-targeted imaging agents by mimicking poly-γ-glutamyl folic acid, an endogenous substrate of PSMA. In vitro the γ-linked polyglutamate conjugates proved to be better substrates than the corresponding α-linked glutamates. However, in vivo imaging studies of γ-ray-emitting and γ-linked glutamates did not demonstrate selective uptake in PSMA-pos-itive over PSMA-negative tumors. Subsequent chromatographic studies and in silico molecular dynamics simulations indicated that hydrolysis of the substrates is slow and access to the enzymatic active site is limited. These results inform the design of future substrate-based imaging agents for PSMA.
doi:10.1007/s13233-013-1050-5
PMCID: PMC4198338  PMID: 25328507
molecular imaging; prodrug; prostate cancer; SPECT; PSMA
6.  Synthesis and Biological Evaluation of Low Molecular Weight Fluorescent Imaging Agents for the Prostate-Specific Membrane Antigen 
Bioconjugate chemistry  2012;23(12):2377-2385.
Targeted near-infrared (NIR) optical imaging can be used in vivo to detect specific tissues, including malignant cells. A series of NIR fluorescent ligands targeting the prostate-specific membrane antigen (PSMA) was synthesized and each compound was tested for its ability to image PSMA+ tissues in experimental models of prostate cancer. The agents were prepared by conjugating commercially available active esters of NIR dyes, including IRDye800CW, IRDye800RS, Cy5.5, Cy7, or a derivative of indocyanine green (ICG) to the terminal amine group of (S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido)pentanedioic acid 1, (14S,18S)-1-amino-8,16-dioxo-3,6-dioxa-9,15,17-triazaicosane-14,18,20-tricarboxylic acid 2 and (3S,7S)-26-amino-5,13,20-trioxo-4,6,12,21-tetraaza-hexacosane-1,3,7,22-tetracarboxylic acid 3. The Ki values for the dye-inhibitor conjugates ranged from 1 to 700 pM. All compounds proved capable of imaging PSMA+ tumors selectively to varying degrees depending on the choice of fluorophore and linker. The highest tumor uptake was observed with IRDye800CW employing a poly(ethylene glycol) or lysine-suberate linker, as in 800CW-2 and 800CW-3, while the highest tumor to nontarget tissue ratios were obtained for Cy7 with these same linkers, as in Cy7-2 and Cy7-3. Compounds 2 and 3 provide useful scaffolds for targeting of PSMA+ tissues in vivo and should be useful for preparing NIR dye conjugates designed specifically for clinical intraoperative optical imaging devices.
doi:10.1021/bc3003919
PMCID: PMC4131203  PMID: 23157641
7.  Effect of Chelators on the Pharmacokinetics of 99mTc-Labeled Imaging Agents for the Prostate-Specific Membrane Antigen (PSMA) 
Journal of medicinal chemistry  2013;56(15):6108-6121.
Technetium-99m, the most commonly used radionuclide in nuclear medicine, can be attached to biologically important molecules through a variety of chelating agents, the choice of which depends upon the imaging application. The prostate-specific membrane antigen (PSMA) is increasingly recognized as an important target for imaging and therapy of prostate cancer (PCa). Three different 99mTc-labeling methods were employed to investigate the effect of the chelator on the biodistribution and PCa tumor uptake profiles of 12 new urea based PSMA-targeted radiotracers. This series includes hydrophilic ligands for radiolabeling with the [99mTc(CO)3]+ core (L8-10), traditional NxSy-based chelating agents with varying charge and polarity for the 99mTc-oxo core (L11-18), and a 99mTc-organohydrazine-labeled radioligand (L19). 99mTc(I)-Tricarbonyl-labeled [99mTc]L8 produced the highest PSMA+ PC3 PIP to PSMA− PC3 flu tumor ratios, and demonstrated the lowest retention in normal tissues including kidney after 2 h. These results suggest that choice of chelator is an important pharmacokinetic consideration in the development of 99mTc-labeled radiopharmaceuticals targeting PSMA.
doi:10.1021/jm400823w
PMCID: PMC3773988  PMID: 23799782
8.  Heterobivalent Agents Targeting PSMA and Integrin-αvβ3 
Bioconjugate chemistry  2014;25(2):393-405.
Differential expression of surface proteins on normal vs malignant cells provides the rationale for the development of receptor-, antigen-, and transporter-based, cancer-selective imaging and therapeutic agents. However, tumors are heterogeneous, and do not always express what can be considered reliable, tumor-selective markers. That suggests development of more flexible targeting platforms that incorporate multiple moieties enabling concurrent targeting to a variety of putative markers. We report the synthesis, biochemical, in vitro, and preliminary in vivo evaluation of a new heterobivalent (HtBv) imaging agent targeting both the prostate-specific membrane antigen (PSMA) and integrin-αvβ3 surface markers, each of which can be overexpressed in certain tumor epithelium and/or neovasculature. The HtBv agent was functionalized with either 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or the commercially available IRDye800CW. DOTA-conjugated HtBv probe 9 bound to PSMA or αvβ3 with affinities similar to those of monovalent (Mnv) compounds designed to bind to their targets independently. In situ energy minimization experiments support a model describing the conformations adapted by 9 that enable it to bind both targets. IRDye800-conjugated HtBv probe 10 demonstrated target-specific binding to either PSMA or integrin-αvβ3 overexpressing xenografts. HtBv agents 9 and 10 may enable dual-targeted imaging of malignant cells and tissues in an effort to address heterogeneity that confounds many cancer-targeted imaging agents.
doi:10.1021/bc4005377
PMCID: PMC4112557  PMID: 24410012
9.  Evaluation of Prostate-Specific Membrane Antigen as an Imaging Reporter 
Genetic reporters provide a noninvasive method to monitor and evaluate a population of cells. The ideal properties of a gene reporter-probe system include biocompatibility, lack of immunogenicity, low background expression or signal, and high sensitivity of detection. The prostate-specific membrane antigen (PSMA) is an attractive candidate for a genetic reporter as it is a human trans-membrane protein with a selective expression pattern, and there are several PSMA imaging agents available for clinical and preclinical applications. We evaluated the use of PSMA as a genetic imaging reporter by comparison to 2 clinically established reporters, the mutant herpes simplex virus type I thymidine kinase and the human sodium-iodide symporter.
Methods
Adenoviruses expressing each reporter were constructed and validated in vitro for expression and function. To compare PSMA with existing imaging reporters, a bilateral Matrigel suspension model was established with nude mice bearing cells equally infected with each reporter or control adenovirus. Dynamic PET was performed, and time–activity curves were generated for each reporter-probe pair.
Results
A comparison of peak target-to-background ratios revealed that PSMA offered the highest ratio relative to the control Matrigel suspension as well as muscle. Further, as proof of concept, PSMA was applied as an imaging reporter to monitor adenoviral liver transduction with both nuclear and optical imaging probes.
Conclusion
These preliminary studies support further development of PSMA as a noninvasive genetic reporter.
doi:10.2967/jnumed.113.134031
PMCID: PMC4074907  PMID: 24700883
PSMA; molecular-genetic imaging; DCFPyL; positron emission tomography; optical imaging
10.  PET Imaging in Prostate Cancer: Focus on Prostate-Specific Membrane Antigen 
Prostate cancer (PCa) is the second leading cause of cancer-related death in American men. Positron emission tomography/computed tomography (PET/CT) with emerging radiopharmaceuticals promises accurate staging of primary disease, restaging of recurrent disease, detection of metastatic lesions and, ultimately, for predicting the aggressiveness of disease. Prostate-specific membrane antigen (PSMA) is a well-characterized imaging biomarker of PCa. Because PSMA levels are directly related to androgen independence, metastasis and progression, PSMA could prove an important target for the development of new radiopharmaceuticals for PET. Preclinical data for new PSMA-based radiotracers are discussed and include new 89Zr- and 64Cu-labeled anti-PSMA antibodies and antibody fragments, 64Cu-labeled aptamers, and 11C-, 18F-, 68Ga-, 64Cu-, and 86Y-labeled low molecular weight inhibitors of PSMA. Several of these agents, namely 68Ga-HBED-CC conjugate 15, 18F-DCFBC 8, and BAY1075553 are particularly promising, each having detected sites of PCa in initial clinical studies. These early clinical results suggest that PET/CT using PSMA-targeted agents, especially with compounds of low molecular weight, will make valuable contributions to the management of PCa.
PMCID: PMC4067736  PMID: 23590171
DCFBC; molecular imaging; positron emission tomography; PSMA; radiopharmaceutical
11.  Structural Optimization, Biological Evaluation and Application of Peptidomimetic Prostate Specific Antigen Inhibitors 
Journal of medicinal chemistry  2013;56(11):4224-4235.
Prostate-Specific Antigen (PSA) is a serine protease produced at high levels by normal and malignant prostate epithelial cells that is used extensively as a biomarker in the clinical management of prostate cancer. To better understand PSA’s role in prostate cancer progression we prepared a library of peptidyl boronic acid based inhibitors. To enhance selectivity for PSA vs. other serine proteases, we modified the P1 site of the inhibitors to incorporate a bromopropylglycine group. This allowed the inhibitors to participate in halogen bond formation with the serine found at the bottom of the specificity pocket. The best of these Ahx-FSQn(boro)Bpg had PSA Ki of 72 nM and chymotrypsin Ki of 580 nM. In vivo studies using PSA-producing xenografts demonstrated that candidate inhibitors had minimal effect on growth but significantly altered serum levels of PSA. Biodistribution of 125I labeled peptides showed low levels of uptake into tumors compared to other normal tissues.
doi:10.1021/jm301718c
PMCID: PMC3740768  PMID: 23692593
Prostate cancer; PSA inhibitors; boronic acids; peptidyl-boronic acid; peptidomimetics; serine protease inhibition; chymotrypsin inhibition
12.  Biodistribution, Tumor Detection, and Radiation Dosimetry of 18F-DCFBC, a Low-Molecular-Weight Inhibitor of Prostate-Specific Membrane Antigen, in Patients with Metastatic Prostate Cancer 
Prostate-specific membrane antigen (PSMA) is a type II integral membrane protein expressed on the surface of prostate cancer (PCa) cells, particularly in androgen-independent, advanced, and metastatic disease. Previously, we demonstrated that N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-18F-fluorobenzyl-Lcysteine (18F-DCFBC) could image an experimental model of PSMA-positive PCa using PET. Here, we describe the initial clinical experience and radiation dosimetry of 18F-DCFBC in men with metastatic PCa.
Methods
Five patients with radiologic evidence of metastatic PCa were studied after the intravenous administration of 370 MBq (10 mCi) of 18F-DCFBC. Serial PET was performed until 2 h after administration. Time- activity curves were generated for selected normal tissues and metastatic foci. Radiation dose estimates were calculated using OLINDA/EXM 1.1.
Results
Most vascular organs demonstrated a slow decrease in radioactivity concentration over time consistent with clearance from the blood pool, with primarily urinary radiotracer excretion. Thirty-two PET-positive suspected metastatic sites were identified, with 21 concordant on both PET and conventional imaging for abnormal findings compatible with metastatic disease. Of the 11 PET-positive sites not identified on conventional imaging, most were within the bone and could be considered suggestive for the detection of early bone metastases, although further validation is needed. The highest mean absorbed dose per unit administered radioactivity (µGy/MBq) was in the bladder wall (32.4), and the resultant effective dose was 19.9 ± 1.34 µSv/MBq (mean ± SD).
Conclusion
Although further studies are needed for validation, our findings demonstrate the potential of 18F-DCFBC as a new positron-emitting imaging agent for the detection of metastatic PCa. This study also provides dose estimates for 18F-DCFBC that are comparable to those of other PET radiopharmaceuticals such as 18F-FDG.
doi:10.2967/jnumed.112.104661
PMCID: PMC3742115  PMID: 23203246
prostate-specific membrane antigen; prostate cancer; 18F; urea; PET/CT
13.  [32P]ATP inhibits the growth of xenografted tumors in nude mice 
Cell Cycle  2012;11(10):1878-1882.
The search for new therapeutic agents that are effective against cancer has been difficult and expensive. The activity of anticancer candidate agents against human cancer-derived cell lines in immunocompromised mice is an important tool in this search. Because ATP is a naturally occurring small molecule, its radiolabeled form poses many advantages as a potential anticancer therapeutic agent. We previously found that a single, low-dose intravenous injection of [32P]ATP inhibited the growth of xenografted tumors in nude mice for up to several weeks. The current study describes the biodistribution and the results and advantages of multi-dose administration of this potential drug. Future studies should investigate the mechanism involved in the possible use of [32P]ATP as a cytotoxic agent that homes naturally to the tumor microenvironment.
doi:10.4161/cc.19955
PMCID: PMC3359117  PMID: 22544324
mice; tumorinhibition; xenografts; [32P]ATP
15.  2-(3-{1-Carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid, [18F]DCFPyL, a PSMA-based PET Imaging Agent for Prostate Cancer 
Purpose
We have synthesized and evaluated in vivo 2-(3-{1-carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid, [18F]DCFPyL, as a potential imaging agent for the prostate-specific membrane antigen, PSMA. PSMA is upregulated in prostate cancer epithelia as well as in the neovasculature of most solid tumors.
Experimental Design
[18F]DCFPyL was synthesized in two steps from the p-methoxybenzyl (PMB) protected lys-C(O)-glu urea precursor using 6-[18F]fluoronicotinic acid tetrafluorophenyl ester ([18F]F-Py-TFP) for introduction of 18F. Radiochemical synthesis was followed by biodistribution and imaging with PET in immunocompromised mice using isogenic PC3 PSMA+ and PSMA− xenograft models. Human radiation dosimetry estimates were calculated using OLINDA/EXM 1.0.
Results
DCFPyL displays a Ki value of 1.1 ± 0.1 nM for PSMA. [18F]DCFPyL was produced in radiochemical yields of 36-53% (decay corrected) and specific radioactivities of 340 – 480 Ci/mmol (12.6 – 17.8 GBq/μmol, n = 3). In an immunocompromised mouse model [18F]DCFPyL clearly delineated PSMA+ PC3 PIP prostate tumor xenografts on imaging with PET. At 2 h post-injection, 39.4 ± 5.4 percent injected dose per gram of tissue (%ID/g) was evident within the PIP tumor, with a ratio of 358:1 of uptake within PIP to PSMA− PC3 flu tumor placed in the opposite flank. At or after 1 h post-injection, minimal non-target tissue uptake of [18F]DCFPyL was observed. The bladder wall is the dose-limiting organ.
Conclusions
These data suggest [18F]DCFPyL as a viable, new positron-emitting imaging agent for PSMA-expressing tissues.
doi:10.1158/1078-0432.CCR-11-1357
PMCID: PMC3243762  PMID: 22042970
PSMA; PET; molecular imaging; prostate cancer; PC3
16.  Novel substrate-based inhibitors of human glutamate carboxypeptidase II with enhanced lipophilicity 
Journal of medicinal chemistry  2011;54(21):7535-7546.
Virtually all low molecular weight inhibitors of human glutamate carboxypeptidase II (GCPII) are highly polar compounds that have limited use in settings where more lipophilic molecules are desired. Here we report the identification and characterization of GCPII inhibitors with enhanced liphophilicity that are derived from a series of newly identified dipeptidic GCPII substrates featuring non-polar aliphatic side chains at the C-terminus. To analyze the interactions governing the substrate recognition by GCPII, we determined crystal structures of the inactive GCPII(E424A) mutant in complex with selected dipeptides and complemented the structural data with quantum mechanics/molecular mechanics calculations. Results reveal the importance of non-polar interactions governing GCPII affinity towards novel substrates as well as formerly unnoticed plasticity of the S1′ specificity pocket. Based on those data, we designed, synthesized and evaluated a series of novel GCPII inhibitors with enhanced lipophilicity, with the best candidates having low nanomolar inhibition constants and clogD > -0.3. Our findings offer new insights into the design of more lipophilic inhibitors targeting GCPII.
doi:10.1021/jm200807m
PMCID: PMC3222833  PMID: 21923190
PSMA; NAALADase; GCPII; zinc peptidase; folate hydrolase; inhibition; quantum mechanics/molecular mechanics (QM/MM)
18.  High-Throughput Screen Identifies Novel Inhibitors of Cancer Biomarker α-Methylacyl Coenzyme A Racemase (AMACR/P504S) 
Molecular cancer therapeutics  2011;10(5):825-838.
Alpha-methylacyl coenzyme A racemase (AMACR) is a metabolic enzyme whose over-expression has been shown to be a diagnostic indicator of prostatic adenocarcinoma as well as other solid tumors. Here we confirm that attenuation of AMACR expression diminishes the growth of prostate cancer cell lines using stably expressed shRNA constructs. This observation strongly suggests that the AMACR enzyme may be a target for therapeutic inhibition in prostate cancer. To this end, we report here a novel assay capable of screening libraries of diverse small molecules for inhibitors of AMACR activity. This assay facilitated the screening of approximately 5,000 unique compounds and the discovery of seven distinct chemical entities capable of inhibiting AMACR at low micromolar concentrations. The most potent inhibitor discovered is the seleno-organic compound ebselen oxide (IC50:0.80 μM). The parent compound, ebselen (IC50:2.79 μM), is a covalent inactivator of AMACR (KI(inact):24 μM). Two of the AMACR inhibitors appear selectively toxic to prostate cancer cell lines (LAPC4/LNCaP/PC3) that express AMACR compared to a normal prostate fibroblast cell line (WPMY1) that does not express the protein. This report demonstrates the first high-throughput screen for the discovery of novel AMACR inhibitors, characterizes the first non-substrate based inhibitors, and validates that AMACR is a viable chemotherapeutic target in-vitro.
doi:10.1158/1535-7163.MCT-10-0902
PMCID: PMC3423201  PMID: 21441411
High-Throughput; Inhibitor; AMACR; Prostate Cancer; Racemase; Screen; Assay; Imaging
19.  68Ga-Labeled Inhibitors of Prostate-Specific Membrane antigen (PSMA) for Imaging Prostate Cancer 
Journal of Medicinal Chemistry  2010;53(14):5333-5341.
Gallium-68 is a generator-produced radionuclide for positron emission tomography (PET) that is being increasingly used for radiolabeling of tumor-targeting peptides. Compounds [68Ga]3 and [68Ga]6 are high-affinity, urea-based inhibitors of the prostate-specific membrane antigen (PSMA) that were synthesized in decay-uncorrected yields ranging from 60 – 70% and radiochemical purities of more than 99%. Compound [68Ga]3 demonstrated 3.78 ± 0.90 percent injected dose per gram of tissue (%ID/g) within PSMA+ PIP tumor at 30 min post-injection, while [68Ga]6 showed a two hour PSMA+ PIP tumor uptake value of 3.29 ± 0.77%ID/g. Target (PSMA+ PIP) to non-target (PSMA− flu) ratios were 4.6 and 18.3, respectively, at those time points. Both compounds delineated tumor clearly by small animal PET. The urea series of imaging agents for PSMA can be radiolabeled with 68Ga, a cyclotron-free isotope useful for clinical PET studies, with maintenance of target specificity.
doi:10.1021/jm100623e
PMCID: PMC3341619  PMID: 20568777
gallium; molecular imaging; positron emission tomography; prostate-specific membrane antigen; radiopharmaceutical
20.  Synthesis and Evaluation of Technetium-99m- and Rhenium-Labeled Inhibitors of the Prostate-Specific Membrane Antigen (PSMA) 
Journal of Medicinal Chemistry  2008;51(15):4504-4517.
The prostate-specific membrane antigen (PSMA) is increasingly recognized as a viable target for imaging and therapy of cancer. We prepared seven 99mTc/Re-labeled compounds by attaching known Tc/Re chelating agents to an amino-functionalized PSMA inhibitor (lys-NHCONH-glu) with or without a variable length linker moiety. Ki values ranged from 0.17 to 199 nM. Ex vivo biodistribution and in vivo imaging demonstrated the degree of specific binding to engineered PSMA+ PC3 PIP tumors. PC3-PIP cells are derived from PC3 that have been transduced with the gene for PSMA. Despite demonstrating nearly the lowest PSMA inhibitory potency of this series, [99mTc(CO)3(L1)]+ (L1 = (2-pyridylmethyl)2N(CH2)4CH(CO2H)-NHCO-(CH2)6CO-NH-lys-NHCONH-glu) showed the highest, most selective PIP tumor uptake, at 7.9 ± 4.0% injected dose per gram of tissue at 30 min postinjection. Radioactivity cleared from nontarget tissues to produce a PIP to flu (PSMA-PC3) ratio of 44:1 at 120 min postinjection. PSMA can accommodate the steric requirements of 99mTc/Re complexes within PSMA inhibitors, the best results achieved with a linker moiety between the ε amine of the urea lysine and the chelator.
doi:10.1021/jm800111u
PMCID: PMC3336105  PMID: 18637669
21.  A Modular Strategy to Prepare Multivalent Inhibitors of Prostate-Specific Membrane Antigen (PSMA) 
Oncotarget  2011;2(12):1244-1253.
We have developed a modular scaffold for preparing high-affinity, homo-multivalent inhibitors of the prostate-specific membrane antigen (PSMA) for imaging and therapy of prostate cancer (PCa). Our system contains a lysine-based (∝-, ε-) dialkyne residue for incorporating a PSMA binding Lys-Glu urea motif exploiting click chemistry and a second lysine residue for subsequent modification with an imaging or therapeutic moiety. The utility of the multivalent scaffold was examined by synthesizing bivalent compounds 2 and 3 and comparing them with the monovalent analog 1. Determination of inhibition constants (Ki) revealed that bivalent 2 (0.2 nM) and 3 (0.08 nM) are significantly more potent (~ 5 fold and ~ 11 fold, respectively) inhibitors of PSMA than monovalent 1 (0.9 nM). A single photon emission computed tomography (SPECT)-CT imaging study of [111In]3 demonstrated high and specific uptake in PSMA+ PC-3 PIP tumor until at least 48 h post-injection, with rapid clearance from non-target tissues, including kidney. A biodistribution study revealed that [111In]3 demonstrated 34.0 ± 7.5 percent injected dose per gram of tissue in PSMA+ tumor at 24 h post-injection and was capable of generating target-to-non-target ratios of ~ 379 in PSMA+ PC-3 PIP tumors vs. isogenic PSMA-negative PC3-flu tumors in vivo. The click chemistry approach affords a convenient strategy toward multivalent PSMA inhibitors of enhanced affinity and superior pharmacokinetics for imaging.
PMCID: PMC3282081  PMID: 22207391
Prostate-specific membrane antigen (PSMA); NAALADase; bivalent urea inhibitor; molecular imaging; multivalency; SPECT imaging
22.  Improved Syntheses of Precursors for PET Radioligands [18F]XTRA and [18F]AZAN 
Tetrahedron letters  2010;51(40):5333-5335.
Improved syntheses of 7-methyl-2-exo-[3′-(2-bromopyridin-3-yl)-5′-pyridinyl]-7-azabicyclo[2.2.1]heptanes (3) and 7-methyl-2-exo-[3′-(6-bromopyridin-2-yl)-5′-pyridinyl]-7-azabicyclo[2.2.1]heptanes (4), precursors for PET radioligands [18F]XTRA (1) and [18F]AZAN (2), involving a key Stille coupling step followed by deprotection of Boc group and N-methylation are described. The new synthetic procedures provided the title compounds in more than 40% overall yields.
doi:10.1016/j.tetlet.2010.08.001
PMCID: PMC2936105  PMID: 20835363
nAChR; PET radioligand; Stille coupling; N-methylation
23.  OPTIMIZATION OF PEPTIDE-BASED INHIBITORS OF PROSTATE-SPECIFIC ANTIGEN (PSA) AS TARGETED IMAGING AGENTS FOR PROSTATE CANCER 
Bioorganic & medicinal chemistry  2009;17(14):4888-4893.
Prostate-Specific Antigen (PSA) is a serine protease biomarker that may play a role in prostate cancer development and progression. The inhibition of PSA’s enzymatic activity with small molecule inhibitors is an attractive and, as of yet, unexploited target. Previously, we reported a series of peptidyl aldehyde and boronic acid based inhibitors of PSA. In this study, the structural requirements in the P2 and P3 positions of peptide based PSA inhibitors are explored through the substitution of a series of natural and unnatural amino acids in these positions. This analysis demonstrated a preference for hydrophobic residues in the P2 position and amino acids with the potential to hydrogen bond in the P3 position. Using this information, a peptide boronic acid inhibitor with the sequence Cbz-Ser-Ser-Gln-Nle-(boro)-Leu was identified with a Ki for PSA of 25 nM. The attachment of a bulky metal chelating group to the amino terminal of this peptide did not adversely affect PSA inhibition. This result suggests that a platform of PSA-inhibitor chelates could be developed as SPECT or PET-based imaging agents for prostate cancer.
doi:10.1016/j.bmc.2009.06.012
PMCID: PMC3087300  PMID: 19541487
24.  Synthesis and in vivo Evaluation of N-[N-[(S)-1,3-Dicarboxypropyl]carbamoyl]-4-[18F]fluorobenzyl-L-cysteine, [18F]DCFBC: a New Imaging Probe for Prostate Cancer 
Previously we demonstrated successful imaging of xenografts that express the prostate-specific membrane antigen (PSMA) using small animal positron emission tomography (PET) and the radiolabeled PSMA inhibitor N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-S-[11C]methyl-L-cysteine, [11C]DCMC. Herein we extend that work by preparing and testing a PSMA inhibitor of the same class labeled with fluorine-18 to facilitate clinical use.
Methods
N-[N-[(S)-1,3-Dicarboxypropyl]carbamoyl]-4-[18F]fluorobenzyl-L-cysteine, [18F]DCFBC was prepared by reacting 4-[18F]fluorobenzyl bromide with the corresponding thiol precursor. SCID mice bearing subcutaneous PSMA+ PC-3 PIP and PSMA- PC-3 FLU tumors were administered [18F]DCFBC via tail vein injection for ex vivo biodistribution and in vivo imaging. For biodistribution, mice were sacrificed and tumor, blood, and major organs were harvested, weighed, and radioactivity was counted. Imaging was performed using small animal PET.
Results
The radiochemical yield for [18F]DCFBC averaged 16 ± 6% (n = 8) from 4-[18F]fluorobenzyl bromide. Specific radioactivities averaged 52 GBq/μmol (1,392 Ci/mmol, n = 6). Biodistribution and imaging studies showed high uptake of [18F]DCFBC in the PIP tumors with little to no uptake in FLU tumors. The maximum PIP tumor uptake was 8.16 ± 2.55 %ID/g, achieved at 60 min after injection, which decreased to 4.69 ± 0.89 at 120 min. The PIP tumor/muscle ratio was 20 at 120 min postinjection. Based on the mouse biodistribution, the dose-limiting organ is kidney (human estimated absorbed dose: 0.05 mGy/MBq; 0.2 rad/mCi).
Conclusion
[18F]DCFBC localizes to PSMA+ expressing tumors in mice, permitting imaging by small animal PET. This new radiopharmaceutical is an attractive candidate for further studies of PET imaging of prostate cancer.
doi:10.1158/1078-0432.CCR-07-1517
PMCID: PMC3078104  PMID: 18483369
positron emission tomography; prostate-specific membrane antigen; xenograft; PC-3; molecular imaging
25.  Bioisosterism of Urea-Based GCPII Inhibitors: Synthesis and Structure-Activity Relationships Studies 
We report a strategy based on bioisosterism to improve the physicochemical properties of existing hydrophilic, urea-based GCPII inhibitors. Comprehensive structure-activity relationship studies of the P1’ site of ZJ-43- and DCIBzL-based compounds identified several glutamate-free inhibitors with Ki values below 20 nM. Among them, compound 32d (Ki = 11 nM) exhibited selective uptake in GCPII-expressing tumors by SPECT-CT imaging in mice. A novel conformational change of amino acids in the S1’ pharmacophore pocket was observed in the X-ray crystal structure of GCPII complexed with 32d.
doi:10.1016/j.bmcl.2009.10.061
PMCID: PMC2818328  PMID: 19897367
PSMA; glutamate carboxypeptidase II; molecular imaging; radiopharmaceutical; SPECT

Results 1-25 (28)