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1.  Myocardial substrate and route of administration determine acute cardiac retention and lung bio-distribution of cardiosphere-derived cells 
Quantification of acute myocardial retention and lung bio-distribution of cardiosphere-derived cells (CDCs) following transplantation is important to improve engraftment.
Methods and results
We studied acute(1 hour) cardiac/lung retention in 4 groups (n = 25) of rats (normal—NL, acute ischemia-reperfusion—AI-RM, acute permanent ligation—PL, and chronic infarct by ischemia-reperfusion—CI-R) using intra-myocardial delivery, 1 group using intracoronary delivery (acute ischemia-reperfusion, AI-RC, n = 5) and 1 group using intravenous delivery (acute ischemia-reperfusion, AI-RV, n = 5) of CDCs by PET. Cardiac retention was similar in the NL, AI-RM, CI-R, and A-IRC groups (13.6% ± 2.3% vs 12.0% ± 3.9% vs 9.9 ± 2.8 vs 15.4% ± 5.5%; P = NS), but higher in PL animals (22.9% ± 5.2%; P < .05). Low cardiac retention was associated with significantly higher lung activity in NL and AI-RM groups (43.3% ± 5.6% and 39.9% ± 9.3%), compared to PL (28.5% ± 5.9%), CI-R (20.2% ± 9.3%), and A-IRC (19.9% ± 5.6%) animals (P < .05 vs AI-RM and NL). Lung activity was highest following intravenous CDC delivery (55.1% ± 9.3%, P < .001) and was associated with very low cardiac retention (0.8% ± 1.06%). Two-photon microscopy indicated that CDCs escaped to the lungs via the coronary veins following intra-myocardial injection.
Acute cardiac retention and lung bio-distribution vary with the myocardial substrate and injection route. Intra-myocardially injected CDCs escape into the lungs via coronary veins, an effect that is more pronounced in perfused myocardium.
PMCID: PMC4334365  PMID: 21448759
Stem cells; acute heart retention; PET; lung bio-distribution
2.  Influence of Bioluminescence Imaging Dynamics by D-Luciferin Uptake and Efflux Mechanisms 
Molecular imaging  2012;11(6):499-506.
Bioluminescence imaging (BLI) detects light generated by luciferase-mediated oxidation of substrate and is used widely for evaluating transgene expression in cell-based assays and in vivo in relevant preclinical models. The most commonly used luciferase for in vivo applications is firefly luciferase (fLuc), for which D-luciferin serves as the substrate. We demonstrated previously that the expression of the ABCG2 efflux transporter can significantly reduce BLI signal output and that HhAntag-691 can inhibit the efflux of D-luciferin, thereby enhancing BLI signal. Here we show that an HhAntag-691-sensitive uptake mechanism facilitates the intracellular concentration of D-luciferin and that the BLI dynamics of different cell lines are coregulated by this uptake mechanism in conjunction with ABCG2-mediated efflux. After administration of D-luciferin, the HhAntag-691-sensitive uptake mechanism generates a rapid increase in BLI signal that decreases over time, whereas ABCG2-mediated efflux stably reduces signal output. We implicate SLC22A4 (OCTN1), a member of the organic cation/zwitterion uptake transporter family, as one potential mediator of the HhAntag-691-sensitive D-luciferin uptake. These findings provide insight into mechanisms that contribute to the cellular uptake kinetics and in vivo biodistribution of D-luciferin.
PMCID: PMC4332814  PMID: 23084250
3.  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.
PMCID: PMC3983358  PMID: 24533799
4.  The Evolution of Imaging in Cancer: Current State and Future Challenges 
Seminars in oncology  2011;38(1):3-15.
Molecular imaging allows for the remote, noninvasive sensing and measurement of cellular and molecular processes in living subjects. Drawing upon a variety of modalities, molecular imaging provides a window into the biology of cancer from the subcellular level to the patient undergoing a new, experimental therapy. As signal transduction cascades and protein interaction networks become clarified, an increasing number of relevant targets for cancer therapy—and imaging—become available. Although conventional imaging is already critical to the management of patients with cancer, molecular imaging will provide even more relevant information, such as early detection of changes with therapy, identification of patient-specific cellular and metabolic abnormalities, and the disposition of therapeutic, gene-tagged cells throughout the body—all of which will have a considerable impact on morbidity and mortality. This overview discusses molecular imaging in oncology, providing examples from a variety of modalities, with an emphasis on emerging techniques for translational imaging.
PMCID: PMC4313866  PMID: 21362512
5.  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.
PMCID: PMC4112557  PMID: 24410012
6.  Imaging Axl Expression in Pancreatic and Prostate Cancer Xenografts 
The receptor tyrosine kinase Axl is overexpressed in and leads to patient morbidity and mortality in a variety of cancers. Axl-Gas6 interactions are critical for tumor growth, angiogenesis and metastasis. The goal of this study was to investigate the feasibility of imaging graded levels of Axl expression in tumors using a radiolabeled antibody. We radiolabeled anti-human Axl (Axl mAb) and control IgG1 antibodies with 125I with high specific radioactivity and radiochemical purity, resulting in an immunoreactive fraction suitable for in vivo studies. Radiolabeled antibodies were investigated in severe combined immunodeficient mice harboring subcutaneous CFPAC and Panc1 pancreatic cancer xenografts by ex vivo biodistribution and imaging. Based on these results, the specificity of [125I]Axl mAb was also validated in mice harboring orthotopic Panc1 (Axllow) or CFPAC (Axlhigh) tumors and in mice harboring subcutaneous 22Rv1 (Axllow) or DU145 (Axlhigh) prostate tumors by ex vivo biodistribution and imaging studies at 72 h post-injection of the antibody. Both imaging and biodistribution studies demonstrated specific and persistent accumulation of [125I]Axl mAb in Axlhigh (CFPAC and DU145) expression tumors compared to the Axllow (Panc1 and 22Rv1) expression tumors. Axl expression in these tumors was further confirmed by immunohistochemical studies. No difference in the uptake of radioactivity was observed between the control [125I]IgG1 antibody in the Axlhigh and Axllow expression tumors. These data demonstrate the feasibility of imaging Axl expression in pancreatic and prostate tumor xenografts.
PMCID: PMC3918901  PMID: 24333418
Molecular imaging; ImmunoPET; Receptor tyrosine kinase; Axl; Gas6
7.  Noninvasive Molecular Imaging of Tuberculosis-Associated Inflammation With Radioiodinated DPA-713 
The Journal of Infectious Diseases  2013;208(12):2067-2074.
Background. Increased expression of translocator protein (TSPO) is a feature of microglial and macrophage activation. Since activated macrophages are key components of tuberculosis-associated inflammation, we evaluated radioiodinated DPA-713, a synthetic ligand of TSPO, for in vivo imaging of host response.
Methods. Mice were infected with aerosolized Mycobacterium tuberculosis and evaluated using whole-body [125I]iodo-DPA-713 single-photon emission computed tomography (SPECT). Ex vivo biodistribution and correlative immunofluorescence studies were also performed.
Results. [125I]Iodo-DPA-713 SPECT imaging clearly delineated tuberculosis-associated pulmonary inflammation in live animals. Biodistribution studies confirmed radiotracer specificity for inflamed pulmonary tissues. Immunofluorescence studies demonstrated that TSPO is highly expressed in CD68+ macrophages and phagocytic cells within tuberculosis lesions and that [125I]DPA-713 specifically accumulates within these cells. Coadministration of excess unlabelled DPA-713 abrogated both the SPECT and ex vivo fluorescence signals. Lesion-specific signal-to-noise ratios were significantly higher with [125I]iodo-DPA-713 SPECT (4.06 ± 0.52) versus [18F]fluorodeoxyglucose (FDG) positron emission tomography (PET) (2.00 ± 0.28) performed in the same mice (P = .004).
Conclusions. [125I]Iodo-DPA-713 accumulates specifically in tuberculosis-associated inflammatory lesions by selective retention within macrophages and phagocytic cells. [125I]Iodo-DPA-713 SPECT provides higher lesion-specific signal-to-noise ratios than [18F]FDG PET and may prove to be a more specific biomarker to monitor tuberculosis in situ.
PMCID: PMC3836460  PMID: 23901092
translocator protein; molecular imaging; tuberculosis; PET; pyrazolopyrimidine; macrophage
8.  Nanoparticles for cancer imaging: The good, the bad, and the promise 
Nano today  2013;8(5):454-460.
Recent advances in molecular imaging and nanotechnology are providing new opportunities for biomedical imaging with great promise for the development of novel imaging agents. The unique optical, magnetic, and chemical properties of materials at the scale of nanometers allow the creation of imaging probes with better contrast enhancement, increased sensitivity, controlled biodistribution, better spatial and temporal information, multi-functionality and multi-modal imaging across MRI, PET, SPECT, and ultrasound. These features could ultimately translate to clinical advantages such as earlier detection, real time assessment of disease progression and personalized medicine. However, several years of investigation into the application of these materials to cancer research has revealed challenges that have delayed the successful application of these agents to the field of biomedical imaging. Understanding these challenges is critical to take full advantage of the benefits offered by nano-sized imaging agents. Therefore, this article presents the lessons learned and challenges encountered by a group of leading researchers in this field, and suggests ways forward to develop nanoparticle probes for cancer imaging. Published by Elsevier Ltd.
PMCID: PMC4240321  PMID: 25419228
Nanomedicine; Cancer; Imaging; Detection; Screening
9.  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.
PMCID: PMC4234089  PMID: 25145668
molecular-genetic imaging; bioluminescence; SPECT; metastasis; nanoparticle; PC3
10.  Glycol chitosan nanoparticles as specialized cancer therapeutic vehicles: Sequential delivery of doxorubicin and Bcl-2 siRNA 
Scientific Reports  2014;4:6878.
Conventional chemotherapy is plagued with adverse side effects because cancer treatments are subject to numerous variations, most predominantly from drug resistance. Accordingly, multiple or multistage chemotherapeutic regimens are often performed, combining two or more drugs with orthogonal and possibly synergistic mechanisms. In this respect, glycol chitosan (GC)-based nanoparticles (CNPs) serve as an effective platform vehicle that can encapsulate both chemotherapeutics and siRNA to achieve maximal efficacy by overcoming resistance. Herein, DOX-encapsulated CNPs (DOX-CNPs) or Bcl-2 siRNA-encapsulated CNPs (siRNA-CNPs) exhibited similar physicochemical properties, including size, surface properties and pH sensitive behavior, regardless of the different physical features of DOX and Bcl-2 siRNA. We confirmed that the CNP platform applied to two different types of drugs results in similar in vivo biodistribution and pharmacokinetics, enhancing treatment in a dose-dependent fashion.
PMCID: PMC4217108  PMID: 25363213
11.  Three-Dimensional Amide Proton Transfer MR Imaging of Gliomas: Initial Experience and Comparison with Gadolinium Enhancement 
Journal of magnetic resonance imaging : JMRI  2013;38(5):10.1002/jmri.24067.
To investigate the feasibility of a three-dimensional amide-proton-transfer (APT) imaging sequence with gradient- and spin-echo readouts at 3T in patients with high- or low-grade gliomas.
Materials and Methods
Fourteen patients with newly diagnosed gliomas were recruited. After B0 inhomogeneity correction on a voxel-by-voxel basis, APT-weighted images were reconstructed using a magnetization-transfer-ratio asymmetry at offsets of ±3.5 ppm with respect to the water resonance. Analysis of variance post-hoc tests were used for statistical evaluations, and results were validated with pathology.
In six patients with gadolinium-enhancing high-grade gliomas, enhancing tumors on the post-contrast T1-weighted images were consistently hyperintense on the APT-weighted images. Increased APT-weighted signal intensity was also clearly visible in two pathologically proven, high-grade gliomas without gadolinium enhancement. The average APT-weighted signal was significantly higher in the lesions than in the contralateral normal-appearing brain tissue (P < 0.001). In six low-grade gliomas, including two with gadolinium enhancement, APT-weighted imaging showed iso-intensity or mild punctate hyperintensity within all the lesions, which was significantly lower than that seen in the high-grade gliomas (P < 0.001).
The proposed three-dimensional APT imaging sequence can be incorporated into standard brain MRI protocols for patients with malignant gliomas.
PMCID: PMC3664658  PMID: 23440878
APT imaging; CEST imaging; glioma; tumor grade; gadolinium enhancement
12.  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.
PMCID: PMC4063304  PMID: 24759454
13.  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.
PMCID: PMC4198338  PMID: 25328507
molecular imaging; prodrug; prostate cancer; SPECT; PSMA
14.  Translational Molecular Imaging of Prostate Cancer 
Current radiology reports  2013;1(3):216-226.
Prostate cancer is a heterogeneous disease, and its management is now evolving to become more personalized and to incorporate new targeted therapies. With these new changes comes a demand for molecular imaging techniques that can not only detect disease but also assess biology and treatment response. This review article summarizes current molecular imaging approaches in prostate cancer (e.g. 99mTc bone scintigraphy and 18F-fluorodeoxyglucose positron emission tomography) and highlights emerging clinical and preclinical imaging agents, with an emphasis on mechanism and clinical application. Emerging agents at various stages of clinical translation include radiolabeled analogs of lipid, amino acid, and nucleoside metabolism, as well as agents more specifically targeting prostate cancer biomarkers including androgen receptor, prostate-specific membrane antigen and others. We also highlight new techniques and targeted contrast agents for magnetic resonance imaging and spectroscopy. For all these imaging techniques, a growing and important unmet need is for well-designed prospective clinical trials to establish clear indications with clinical benefit in prostate cancer.
PMCID: PMC3804343  PMID: 24159427
Molecular imaging; prostate cancer; positron emission tomography; magnetic resonance imaging; single photon emission computed tomography; prostate-specific membrane antigen; androgen receptor
15.  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.
PMCID: PMC4131203  PMID: 23157641
16.  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.
PMCID: PMC3773988  PMID: 23799782
18.  Preclinical Evaluation of Novel Glutamate-Urea-Lysine Analogs that Target Prostate Specific Membrane Antigen as Molecular Imaging Pharmaceuticals for Prostate Cancer 
Cancer research  2009;69(17):6932-6940.
Prostate-specific membrane antigen (PSMA) is expressed in normal human prostate epithelium and is highly upregulated in prostate cancer. We previously reported a series of novel small molecule inhibitors targeting PSMA. Two compounds, MIP-1072, (S)-2-(3-((S)-1-carboxy-5-(4–iodobenzylamino)pentyl)ureido)pentanedioic acid and MIP-1095, (S)-2-(3-((S)-1-carboxy-5-(3-(4-iodophenyl)ureido)pentyl)ureido)pentanedioic acid, were selected for further evaluation. MIP-1072 and MIP-1095 potently inhibited the glutamate carboxypeptidase activity of PSMA (Ki = 4.6 ± 1.6 and 0.24 ± 0.14 nM, respectively), and when radiolabeled with 123I exhibited high affinity for PSMA on human prostate cancer LNCaP cells (Kd = 3.8 ± 1.3 and 0.81 ± 0.39 nM, respectively). The association of [123I]MIP-1072 and [123I]MIP-1095 with PSMA was specific; there was no binding to human prostate cancer PC3 cells, which lack PSMA, and binding was abolished by co-incubation with a structurally unrelated NAALADase inhibitor, 2-(phosphonomethyl)pentanedioic acid (PMPA). [123I]MIP-1072 and [123I]MIP-1095 internalized into LNCaP cells at 37 °C. Tissue distribution studies in mice demonstrated 17.3 ± 6.3 (at 1 hr) and 34.3 ± 12.7 (at 4 hr) % injected dose per gram of tissue, for [123I]MIP-1072 and [123I]MIP-1095, respectively. [123I]MIP-1095 exhibited greater tumor uptake but slower washout from blood and non-target tissues compared to [123I]MIP-1072. Specific binding to PSMA in vivo was demonstrated by competition with PMPA in LNCaP xenografts, and the absence of uptake in PC3 xenografts. The uptake of [123I]MIP-1072 and [123I]MIP-1095 in tumor bearing mice was corroborated by SPECT/CT imaging. PSMA-specific radiopharmaceuticals should provide a novel molecular targeting option for the detection and staging of prostate cancer.
PMCID: PMC4114247  PMID: 19706750
prostate cancer; molecular imaging; prostate-specific membrane antigen; NAALADase; SPECT
19.  Development of a High-Throughput Fluorescence Polarization Assay to Identify Novel Ligands of Glutamate Carboxypeptidase II 
Journal of biomolecular screening  2012;17(8):1030-1040.
Glutamate carboxypeptidase II (GCPII) is an important target for therapeutic and diagnostic interventions aimed at prostate cancer and neurologic disorders. Here we describe the development and optimization of a high-throughput screening (HTS) assay based on fluorescence polarization (FP) that facilitates the identification of novel scaffolds inhibiting GCPII. First, we designed and synthesized a fluorescence probe based on a urea-based inhibitory scaffold covalently linked to a Bodipy TMR fluorophore (TMRGlu). Next, we established and optimized conditions suitable for HTS and evaluated the assay robustness by testing the influence of a variety of physicochemical parameters (e.g., pH, temperature, time) and additives. Using known GCPII inhibitors, the FP assay was shown to be comparable to benchmark assays established in the field. Finally, we evaluated the FP assay by HTS of a 20 000–compound library. The novel assay presented here is robust, highly reproducible (Z′ = 0.82), inexpensive, and suitable for automation, thus providing an excellent platform for HTS of small-molecule libraries targeting GCPII.
PMCID: PMC4112551  PMID: 22751730
fluorescence polarization; high-throughput screening; glutamate carboxypeptidase II; prostate-specific membrane antigen; metallopeptidase
20.  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.
PMCID: PMC4112557  PMID: 24410012
21.  [18F]ASEM, a radiolabeled antagonist for imaging the α7-nicotinic acetylcholine receptor (α7-nAChR) with positron emission tomography (PET) 
The α7-nicotinic cholinergic receptor (α7-nAChR) is a key mediator of brain communication and has been implicated in a wide variety of central nervous system disorders. None of the currently available PET radioligands for α7-nAChR are suitable for quantitative PET imaging, mostly due to insufficient specific binding. The goal of this study was to evaluate the potential of [18F]ASEM ([18F]JHU82132) as an α7-nAChR radioligand for PET.
Inhibition binding assay and receptor functional properties of ASEM were assessed in vitro. The brain regional distribution of [18F]ASEM in baseline and blockade were evaluated in DISC1 mice (dissection) and baboons (PET).
ASEM is an antagonist for the α7-nAChR with high binding affinity (Ki = 0.3 nM). [18F]ASEM readily entered the baboon brain and specifically labeled α7-nAChR. The in vivo specific binding of [18F]ASEM in the brain regions enriched with α7-nAChRs was 80–90%. SSR180711, an α7-nAChR selective partial agonist, blocked [18F]ASEM binding in the baboon brain in a dose-dependent manner, suggesting that the binding of [18F]ASEM was mediated by α7-nAChRs and the radioligand was suitable for drug evaluation studies. In the baboon baseline studies, the brain regional volume of distribution (VT) values for [18F]ASEM were 23 (thalamus), 22 (insula), 18 (hippocampus) and 14 (cerebellum), whereas in the binding selectivity (blockade) scan, all regional VT values were reduced to less than 4. The range of regional binding potential (BPND) values in the baboon brain was from 3.9 to 6.6. In vivo cerebral binding of [18F]ASEM and α7-nAChR expression in mutant DISC1 mice, a rodent model of schizophrenia, was significantly lower than in control animals, which is in agreement with previous post-mortem human data.
[18F]ASEM holds promise as a radiotracer with suitable imaging properties for quantification of α7-nAChR in the human brain.
PMCID: PMC4112566  PMID: 24556591
α7-nAChR; nicotine; PET; baboon; [18F]ASEM
22.  Regional brain distribution of translocator protein using [11C]DPA-713 PET in individuals infected with HIV 
Journal of neurovirology  2014;20(3):219-232.
Imaging the brain distribution of translocator protein (TSPO), a putative biomarker for glial cell activation and neuroinflammation, may inform management of individuals infected with HIV by uncovering regional abnormalities related to neurocognitive deficits and enable non-invasive therapeutic monitoring. Using the second-generation TSPO-targeted radiotracer, [11C]DPA-713, we conducted a positron emission tomography (PET) study to compare the brains of 12 healthy human subjects to those of 23 individuals with HIV who were effectively treated with combination antiretroviral therapy (cART). Compared to PET data from age-matched healthy control subjects, [11C]DPA-713 PET of individuals infected with HIV demonstrated significantly higher volume-of-distribution (VT) ratios in white matter, cingulate cortex, and supramarginal gyrus, relative to overall gray matter VT, suggesting localized glial cell activation in susceptible regions. Regional TSPO abnormalities were evident within a sub-cohort of neuro-asymptomatic HIV subjects, and an increase in the VT ratio within frontal cortex was specifically linked to individuals affected with HIV-associated dementia. These findings were enabled by employing a gray matter normalization approach for PET data quantification, which improved test–retest reproducibility, intra-class correlation within the healthy control cohort, and sensitivity of uncovering abnormal regional findings.
PMCID: PMC4078660  PMID: 24567030
NeuroAIDS; HIV-associated neurocognitive disorder; Translocator protein; Neuroinflammation; Microglia; Molecular neuroimaging
23.  Mix to Validate: A Facile, Reversible PEGylation for Fast Screening of Potential Therapeutic Proteins, TRAIL, In Vivo** 
Mix to Validate: To advance the rate of novel protein therapies entering the clinic, we provide researchers a facile tool for protein drug efficacy testing in animal models in a high throughput manner. Here, we utilize the concept of PEGylating proteins through complementary interactions between His-tag and Ni2+ complex of NTA, a well-established practice in protein research, to improve blood half-life of therapeutic protein candidates after systemic administration in vivo.
PMCID: PMC3902477  PMID: 23740787
Histidine tag; in vivo drug screening; pegylation; protein delivery; protein modification
24.  Biomaterial-mediated cancer-specific DNA delivery to liver cell cultures using synthetic poly(beta-amino esters) 
Liver cancer is a leading cause of cancer death. Most patients are treated by arterial injection of chemoembolizing agents, providing a convenient avenue for local treatment by novel therapies, including gene therapy. Poly(beta-amino esters) (PBAEs) were synthesized and used to form nanoparticles for non-viral transfection of buffalo rat hepatoma (MCA-RH7777) and hepatocyte (BRL-3A) lines with eGFP and luciferase DNA. Hepatoma cells were transfected with up to 98±0.4% efficacy with no measurable cytotoxicity. Hepatocytes were transfected with as high as 73±0.4% efficacy with 10±4% non-specific cytotoxicity. In contrast, positive controls (branched polyethylenimine, Lipofectamine™ 2000, and X-tremeGENE® DNA HP) caused 30–90% toxicity in BRL-3A cells at doses required for >50% transfection. Of the 21 optimized PBAE-DNA formulations tested, 12 showed significant specificity for hepatoma cells over hepatocytes in monoculture (P<0.05 for both percentage transfected and eGFP expression intensity). Top polymers from eGFP studies also delivered luciferase DNA with 220±30-fold and 470±30-fold greater specificity for hepatoma cells than hepatocytes. Transfections of co-cultures of hepatoma and hepatocytes with eGFP DNA also showed high specificity (1.9±0.1- to 5.8±1.4-fold more transfected hepatoma cells than hepatocytes, measured by percentage transfected and flow cytometry). By eGFP intensity, up to 530±60-fold higher average expression per cell was measured in hepatoma cells. One top formulation caused 95±0.2% transfection in hepatoma cells and 27±0.2% in hepatocytes (96±9% relative hepatocyte viability). PBAE-based nanoparticles are a viable strategy for liver cancer treatment, delivering genes to nearly 100% of cancer cells while maintaining high biomaterial-mediated specificity to prevent toxic side-effects on healthy hepatocytes.
PMCID: PMC3915782  PMID: 23559534
25.  Focal positive prostate-specific membrane antigen (PSMA) expression in ganglionic tissues associated with prostate neurovascular bundle: Implications for novel intraoperative PSMA-based fluorescent imaging techniques☆ 
Urologic oncology  2011;31(5):572-575.
Prostate specific membrane antigen (PSMA) is primarily expressed in glandular prostatic tissue and is frequently utilized to detect primary or metastatic prostatic adenocarcinoma (CaP). A purported novel application of PSMA detection is the intraoperative real-time identification of CaP using radioimmunoscintigraphy to define the extension of the surgical resection. Considering that PSMA expression has been reported in other tissues, we evaluated its immunoexpression in prostatic neurovascular bundle elements to assess the convenience and safety of the aforementioned procedure.
Materials and methods
Twenty consecutive specimens of radical prostatectomy (RP) were retrieved from our surgical pathology archives. PSMA immunoexpression (Clone 3E6, DAKO) was assessed in a representative section from each specimen containing neurovascular bundle elements.
PSMA expression was documented in 20/20 of examined CaP slides. Most cases exhibited an apical/cytoplasmic or cytoplasmic with membranous accentuation pattern of staining. Focal weak to moderate cytoplasmic staining was detected in associated ganglionic tissue in 3/15 of the examined RP. In all cases, staining was cytoplasmic, less extensive, and weaker than the pattern observed in CaP. None of the peripheral nerve sheath cells or lymphovascular components of the examined neurovascular bundles were positive for PSMA.
We found focal positive PSMA expression in the ganglionic cells of the prostatic neurovascular bundle. Our results suggest that the radioimmunoscintigraphic detection of radiolabeled PSMA antibodies might not be entirely specific for prostatic cells; this observation must be taken into account should an intraoperative PSMA-based fluorescent imaging technique be used to define the extension of the surgical procedure.
PMCID: PMC4074903  PMID: 21640619
Prostate specific membrane antigen (PSMA); Prostate carcinoma; Radioimmunoscintigraphy; Radiolabeled antibodies; Fluorescent imaging technique; Radical prostatectomy

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