Showing papers by "Xiaoyuan Chen published in 2006"

Peptide-Labeled Near-Infrared Quantum Dots for Imaging Tumor Vasculature in Living Subjects

Abstract: We report the in vivo targeting and imaging of tumor vasculature using arginine-glycine-aspartic acid (RGD) peptide-labeled quantum dots (QDs). Athymic nude mice bearing subcutaneous U87MG human glioblastoma tumors were administered QD705-RGD intravenously. The tumor fluorescence intensity reached maximum at 6 h postinjection with good contrast. The results reported here open up new perspectives for integrin-targeted near-infrared optical imaging and may aid in cancer detection and management including imaging-guided surgery.

In Vivo Visualization of Embryonic Stem Cell Survival, Proliferation, and Migration After Cardiac Delivery

Abstract: Background— Recent studies have shown that stem cell therapy can promote tissue regeneration; however, monitoring stem cells in vivo remains problematic owing to limitations of conventional histological assays and imaging modalities. Methods and Results— Murine embryonic stem (ES) cells were stably transduced with a lentiviral vector carrying a novel triple-fusion (TF) reporter gene that consists of firefly luciferase, monomeric red fluorescence protein, and truncated thymidine kinase (fluc-mrfp-ttk). ES cell viability, proliferation, and differentiation ability were not adversely affected by either reporter genes or reporter probes compared with nontransduced control cells (P=NS). Afterward, 1×107 of ES cells carrying the TF reporter gene (ES-TF) were injected into the myocardium of adult nude rats (n=20). Control animals received nontransduced ES cells (n=6). At day 4, the bioluminescence and positron emission tomography signals in study animals were 3.7×107±5.8×106 photons · s−1 · cm−2 per steradian (s...

Quantitative PET Imaging of Tumor Integrin αvβ3 Expression with 18F-FRGD2

Abstract: The development of noninvasive methods to visualize and quantify integrin αvβ3 expression in vivo appears to be crucial for the success of antiangiogenic therapy based on integrin antagonism. Precise documentation of integrin receptor levels will allow appropriate selection of patients who will most likely benefit from an antiintegrin treatment regimen. Imaging can also be used to provide an optimal dosage and time course for treatment based on receptor occupancy studies. In addition, imaging integrin expression will be important to evaluate antiintegrin treatment efficacy and to develop new therapeutic drugs with favorable tumor targeting and in vivo kinetics. We labeled the dimeric RGD peptide E[c(RGDyK)]2 with 18F and evaluated its tumor-targeting efficacy and pharmacokinetics of 18F-FB–E[c(RGDyK)]2 (18F-FRGD2). Methods: E[c(RGDyK)]2 was labeled with 18F by conjugation coupling with N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) under a slightly basic condition. The in vivo metabolic stability of 18F-FRGD2 was determined. The diagnostic value after injection of 18F-FRGD2 was evaluated in various xenograft models by dynamic microPET followed by ex vivo quantification of tumor integrin level. Results: Starting with 18F− Kryptofix 2.2.2./K2CO3 solution, the total reaction time for 18F-FRGD2, including final high-performance liquid chromatography purification, is about 200 ± 20 min. Typical decay-corrected radiochemical yield is 23% ± 2% (n = 20). 18F-FRGD2 is metabolically stable. The binding potential extrapolated from graphical analysis of PET data and Logan plot correlates well with the receptor density measured by sodium dodecyl sulfate polyacrylamide electrophoresis and autoradiography in various xenograft models. The tumor-to-background ratio at 1 h after injection of 18F-FRGD2 also gives a good linear relationship with the tumor tissue integrin level. Conclusion: The dimeric RGD peptide tracer 18F-FRGD2, with high integrin specificity and favorable excretion profile, may be translated into the clinic for imaging integrin αvβ3 expression. The binding potential calculated from simplified tracer kinetic modeling such as the Logan plot appears to be an excellent indicator of tumor integrin density.

PET of Vascular Endothelial Growth Factor Receptor Expression

Abstract: For solid tumors and metastatic lesions, tumor vascularity is a critical factor in assessing response to therapy. Here we report the first example, to our knowledge, of 64Cu-labeled vascular endothelial growth factor 121 (VEGF121) for PET of VEGF receptor (VEGFR) expression in vivo. Methods: VEGF121 was conjugated with 1,4,7,10-tetraazadodecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA) and then labeled with 64Cu for small-animal PET of mice bearing different sized U87MG human glioblastoma xenografts. Blocking experiments and ex vivo histopathology were performed to confirm the in vivo results. Results: There were 4.3 ± 0.2 DOTA molecules per VEGF121, and the VEGFR2 binding affinity of DOTA-VEGF121 was comparable to VEGF121. 64Cu labeling of DOTA-VEGF121 was achieved in 90 ± 10 min and the radiolabeling yield was 87.4% ± 3.2%. The specific activity of 64Cu-DOTA-VEGF121 was 3.2 ± 0.1 GBq/mg with a radiochemical purity of >98%. Small-animal PET revealed rapid, specific, and prominent uptake of 64Cu-DOTA-VEGF121 in small U87MG tumors (high VEGFR2 expression) but significantly lower and sporadic uptake in large U87MG tumors (low VEGFR2 expression). No appreciable renal clearance of 64Cu-DOTA-VEGF121 was observed, although the kidney uptake was relatively high likely due to VEGFR1 expression. Blocking experiments, immunofluorescence staining, and western blot confirmed the VEGFR specificity of 64Cu-DOTA-VEGF121. Conclusion: Successful demonstration of the ability of 64Cu-DOTA-VEGF121 to visualize VEGFR expression in vivo may allow for clinical translation of this radiopharmaceutical for imaging tumor angiogenesis and guiding antiangiogenic treatment, especially patient selection and treatment monitoring of VEGFR-targeted cancer therapy.

Anti-Angiogenic Cancer Therapy Based on Integrin αvβ3 Antagonism

Abstract: Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a fundamental process during cancer progression. Anti-angiogenic strategies have been pursued for cancer treatment and prevention of cancer recurrence and metastasis. Integrins are a family of cell adhesion molecules consisting of two non-covalently bound transmembrane subunits (alpha and beta). Much research has demonstrated that integrin signaling plays a key role in tumor angiogenesis and metastasis. Integrin alphavbeta3 is highly expressed on activated endothelial cells and tumor cells but is not present in resting endothelial cells and most normal organ systems, which makes it a suitable target for anti-angiogenic cancer therapy. In this review we will focus on cancer therapy targeting integrin alphavbeta3 while other integrins (such as alpha5beta1, alphaIIbbeta3, alphavbeta5, alpha6beta4) will only be briefly mentioned when relevant. MEDI-522 (a humanized anti-human integrin alphavbeta3 monoclonal antibody) and Cilengitide (cyclic peptidic integrin alphavbeta3/alphavbeta5 antagonist) are currently in clinical trials for anti-angiogenic cancer therapy. Small interfering RNA (siRNA) that specifically silences integrin alphav and/or beta3 was reported to cause tumor shrinkage in preclinical xenograft models. Combination of anti-integrin alphavbeta3 therapy and other therapeutic approaches (such as chemotherapy, radiotherapy and gene therapy) has also been applied for cancer treatment. Mounting evidence suggests that there is potentially synergistic effect of combined therapeutic approaches over single modality alone. Lastly, integrin targeted delivery (drugs, genes, and radioisotopes) and imaging (optical, MRI, ultrasound, SPECT, and PET) is discussed in detail.

In vitro and In vivo Characterization of 64Cu-Labeled AbegrinTM, a Humanized Monoclonal Antibody against Integrin αvβ3

Abstract: Abegrin (MEDI-522 or Vitaxin), a humanized monoclonal antibody against human integrin alpha(v)beta(3), is in clinical trials for cancer therapy. In vivo imaging using Abegrin-based probes is needed for better treatment monitoring and dose optimization. Here, we conjugated Abegrin with macrocyclic chelating agent 1,4,7,10-tetra-azacylododecane N,N',N'',N'''-tetraacetic (DOTA) at five different DOTA/Abegrin ratios. The conjugates were labeled with (64)Cu (half-life = 12.7 hours) and tested in three human (U87MG, MDA-MB-435, and PC-3) and one mouse (GL-26) tumor models. The in vitro and in vivo effects of these (64)Cu-DOTA-Abegrin conjugates were evaluated. The number of DOTA per Abegrin varied from 1.65 +/- 0.32 to 38.53 +/- 5.71 and the radiolabeling yield varied from 5.20 +/- 3.16% to 88.12 +/- 6.98% (based on 2 mCi (64)Cu per 50 microg DOTA-Abegrin conjugate). No significant difference in radioimmunoreactivity was found among these conjugates (between 59.78 +/- 1.33 % and 71.13 +/- 2.58 %). Micro-positron emission tomography studies revealed that (64)Cu-DOTA-Abegrin (1,000:1) had the highest tumor activity accumulation (49.41 +/- 4.54% injected dose/g at 71-hour postinjection for U87MG tumor). The receptor specificity of (64)Cu-DOTA-Abegrin was confirmed by effective blocking of MDA-MB-435 tumor uptake with coadministration of nonradioactive Abegrin. (64)Cu-DOTA-IgG exhibited background level tumor uptake at all time points examined. Integrin alpha(v)beta(3)-specific tumor imaging using (64)Cu-DOTA-Abegrin may be translated into the clinic to characterize the pharmacokinetics, tumor targeting efficacy, dose optimization, and dose interval of Abegrin and/or Abegrin conjugates. Chemotherapeutics or radiotherapeutics using Abegrin as the delivering vehicle may also be effective in treating integrin alpha(v)beta(3)-positive tumors.

How molecular imaging is speeding up antiangiogenic drug development.

Abstract: Drug development is a long process that generally spans about 10 to 15 years. The shift in recent drug discovery to novel agents against specific molecular targets highlights the need for more robust molecular imaging platforms. Using molecular probes, molecular imaging can aid in many steps of the drug development process, such as providing whole body readout in an intact system, decreasing the workload and speeding up drug development/validation, and facilitating individualized anticancer treatment monitoring and dose optimization. The main focus of this review is the recent advances in tumor angiogenesis imaging, and the targets include vascular endothelial growth factor and vascular endothelial growth factor receptor, integrin alpha(v)beta(3), matrix metalloproteinase, endoglin (CD105), and E-selectin. Through tumor angiogenesis imaging, it is expected that a robust platform for understanding the mechanisms of tumor angiogenesis and evaluating the efficacy of novel antiangiogenic therapies will be developed, which can help antiangiogenic drug development in both the preclinical stage and the clinical settings. Molecular imaging has enormous potential in improving the efficiency of the drug development process, including the specific area of antiangiogenic drugs.

18F-Labeled Bombesin Analogs for Targeting GRP Receptor-Expressing Prostate Cancer

Abstract: The gastrin-releasing peptide receptor (GRPR) is found to be overexpressed in a variety of human tumors. The aim of this study was to develop 18F-labeled bombesin analogs for PET of GRPR expression in prostate cancer xenograft models. Methods: [Lys3]Bombesin ([Lys3]BBN) and aminocaproic acid-bombesin(7–14) (Aca-BBN(7–14)) were labeled with 18F by coupling the Lys3 amino group and Aca amino group, respectively, with N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) under slightly basic condition (pH 8.5). Receptor-binding affinity of FB-[Lys3]BBN and FB-Aca-BBN(7–14) was tested in PC-3 human prostate carcinoma cells. Internalization and efflux of both radiotracers were also evaluated. Tumor-targeting efficacy and in vivo kinetics of both radiotracers were examined in male athymic nude mice bearing subcutaneous PC-3 tumors by means of biodistribution and dynamic microPET imaging studies. 18F-FB-[Lys3]BBN was also tested for orthotopic PC-3 tumor delineation. Metabolic stability of 18F-FB-[Lys3]BBN was determined in mouse blood, urine, liver, kidney, and tumor homogenates at 1 h after injection. Results: The typical decay-corrected radiochemical yield was about 30%–40% for both tracers, with a total reaction time of 150 ± 20 min starting from 18F−. 18F-FB-[Lys3]BBN had moderate stability in the blood and PC-3 tumor, whereas it was degraded rapidly in the liver, kidneys, and urine. Both radiotracers exhibited rapid blood clearance. 18F-FB-[Lys3]BBN had predominant renal excretion. 18F-FB-Aca-BBN(7–14) exhibited both hepatobiliary and renal clearance. Dynamic microPET imaging studies revealed that the PC-3 tumor uptake of 18F-FB-[Lys3]BBN in PC-3 tumor was much higher than that of 18F-FB-Aca-BBN(7–14) at all time points examined (P

A Thiol-Reactive 18F-Labeling Agent, N-[2-(4-18F-Fluorobenzamido)Ethyl]Maleimide, and Synthesis of RGD Peptide-Based Tracer for PET Imaging of αvβ3 Integrin Expression

Abstract: The cell adhesion molecule integrin αvβ3 plays a key role in tumor angiogenesis and metastasis. A series of 18F-labeled RGD peptides have been developed for PET of integrin expression based on primary amine-reactive prosthetic groups. In this study we introduced a new method of labeling RGD peptides through a thiol-reactive synthon, N-[2-(4-18F-fluorobenzamido)ethyl]maleimide (18F-FBEM). Methods:18F-FBEM was synthesized by coupling N-succinimidyl 4-18F-fluorobenzoate (18F-SFB) with N-(2-aminoethyl)maleimide. After high-pressure liquid chromatography purification, it was allowed to react with thiolated RGD peptides, and the resulting tracers were subjected to receptor-binding assay, in vivo metabolic stability assessment, biodistribution, and microPET studies in murine xenograft models. Results: Conjugation of monomeric and dimeric sulfhydryl-RGD peptides with 18F-FBEM was achieved in high yields (85% ± 5% nondecay-corrected on the basis of 18F-FBEM). The radiochemical purity of the 18F-labeled peptides was >98% and the specific activity was 100∼150 TBq/mmol. Noninvasive microPET and direct tissue sampling experiments demonstrated that both 18F-FBEM-SRGD (RGD monomer) and 18F-FBEM-SRGD2 (RGD dimer) had integrin-specific tumor uptake in subcutaneous U87MG glioma and orthotopic MDA-MB-435 breast cancer xenografts. Conclusion: The new tracer 18F-FBEM-SRGD2 was synthesized with high specific activity via 18F-FBEM and the tracer exhibited high receptor-binding affinity, tumor-targeting efficacy, metabolic stability, as well as favorable in vivo pharmacokinetics. The new synthon 18F-FBEM developed in this study will also be useful for radiolabeling of other thiolated biomolecules.

Discovery of Small Molecule Integrin αvβ3 Antagonists as Novel Anticancer Agents

Abstract: Integrin alphavbeta3 has been implicated in multiple aspects of tumor progression and metastasis. Many tumors have high expression of alphavbeta3 that correlates with tumor progression. Therefore, alphavbeta3 receptor is an excellent target for drug design and delivery. We have discovered a series of novel alphavbeta3 antagonists utilizing common feature pharmacophore models. Upon validation using a database of known alphavbeta3 receptor antagonists, a highly discriminative pharmacophore model was used as a 3D query. A search of a database of 600 000 compounds using the pharmacophore Hypo5 yielded 832 compounds. On the basis of structural novelty, 29 compounds were tested in alphavbeta3 receptor specific binding assay and four compounds showed excellent binding affinity. A limited SAR analysis on the active compound 26 resulted in the discovery of two compounds with nanomolar to subnanomolar binding affinity. These small-molecule compounds could be conjugated to paclitaxel for selective delivery to alphavbeta3 positive metastatic cancer cells.

Near-Infrared Fluorescence Imaging of Tumor Integrin αvβ3 Expression with Cy7-Labeled RGD Multimers

Abstract: Purpose Cell adhesion molecule integrin αvβ3 is an excellent target for tumor interventions because of its unique expression on the surface of several types of solid tumor cells and on almost all sprouting tumor vasculatures. Here, we describe the development of near-infrared (NIR) fluorochrome Cy7-labeled RGD peptides for tumor integrin targeting.

In Vivo Near-Infrared Fluorescence Imaging of Integrin αvβ3 in an Orthotopic Glioblastoma Model

Abstract: Purpose Expression of cell adhesion molecule integrin αvβ3 is significantly up-regulated during tumor growth, and sprouting of tumor vessels and correlates well with tumor aggressiveness. The purpose of this study was to visualize tumor integrin αvβ3 expression in vivo by using near-infrared fluorescence (NIRF) imaging of Cy5.5-linked cyclic arginine–glycine–aspartic acid (RGD) peptide in an orthotopic brain tumor model.

Imaging Chemically Modified Adenovirus for Targeting Tumors Expressing Integrin αvβ3 in Living Mice with Mutant Herpes Simplex Virus Type 1 Thymidine Kinase PET Reporter Gene

Abstract: Recombinant adenovirus (rAd) has been widely used as an attractive gene delivery vehicle to mammalian cells because of its unparalleled efficacy in accomplishing gene transfer in vivo (1,2). There are, however, some limitations associated with Ad for gene delivery. One such disadvantage is related to the wide native tropism of Ad, which often results in high accumulation in nontargeting tissues (3–9). Another major disadvantage for using Ad vector in vivo is that administration of Ad to a host leads to the host immune response against Ad (10–12). Vector targeting to a specific tissue of cell type would enhance gene therapy efficacy and permit the delivery of lower doses, which would consequently result in reduced toxicity. Cell-specific gene delivery via Ad vectors has been achieved by both genetic (13–15) and chemical (16–22) approaches. Chemical modification is a nongenetic strategy to modify the surface of a viron by covalently attaching a polymer containing the targeting ligand with the lysine residues on the surface of Ad. Modification of Ad vectors with poly(ethylene glycol) (PEG) prolongs persistence in the blood and circumvents inflammatory and humoral immune responses (16–22). However, the PEGylation of Ad vectors also leads to loss of infectivity due to the steric hindrance induced by PEG chains. To overcome the decreased efficiency of infection of PEGylated Ad, vectors containing functional molecules on the tip of PEG restore target-specific infectivity. Lanciotti et al. reported targeted Ad vectors using heterofunctional PEG and FGF-2 (22). Ogawara et al. (17) reported PEGylated Ad vectors containing E-selectin–specific antibody or αv integrin–specific RGD peptide for targeting activated endothelial cells. Ad vectors coated with polymers other than PEG have also been developed. Fisher et al. used a multivalent hydrophilic polymer based on poly[N-(2-hydroxypropyl) methacrylamide] to modify Ad vectors (23). Although improved pharmacokinetic properties of polymer-coated Ad vectors without ligands have been reported, those of polymer-coated Ad vectors with ligands have not been reported in detail. Most studies performed in animal models to optimize vector targeting and functional gene delivery use protocols that require the animals to be sacrificed to monitor reporter gene expression. Recently, however, several noninvasive technologies for monitoring reporter gene expression in vivo have been described (24). The most commonly used PET reporter gene is herpes simplex virus 1 thymidine kinase (HSV1-tk). Many of the enzyme substrates have been labeled with 18F or 124I to image HSV1-tk gene expression. Two major substrates for HSV1-tk enzyme/PET include 9-[4-18F-fluoro-3-(hydroxymethyl)butyl]guanine (18F-FHBG) and 124I- or 18F-labeled 2′-fluoro-2′-deoxy-5′-iodo-1-β-D-arabinofuranosyluracil (124I-FIAU or 18F-FIAU) (25–27). It has also been reported that the mutant HSV1-sr39tk is more effective than the wild-type HSV1-tk at using acycloguanosines as substrates. We have demonstrated that the utility of HSV1-sr39tk and 18F-FHBG is able to monitor the reporter gene expression over a 3-mo period after somatic gene transfer (26,28). In this study, a replication-deficient Ad was modified with bifunctional PEG and then conjugated with cyclic RGD peptide (Fig. 1). We hypothesized that surface modification of Ad fiber knobs with PEGylated-RGD peptide will ablate the normal tropism and reduce transduction of nontarget tissues in vivo; incorporation of integrin αvβ3–specific RGD peptides will enhance the gene delivery to tumor neovasculature and integrin-positive tumor cells. To monitor the localization and expression of the chemically modified virus we packed the HSV1-sr39tk mutant as a reporter gene under the control of cytomegalovirus (CMV) promoter. The homing selectivity and transgene expression after intravenous administration of integrin-directed adenovirus were studied by microPET using 18F-FHBG as a reporter probe. FIGURE 1 Schematic representation of Ad vector modified with RGD-PEG. Synthesis of RGD-PEG-NHS (where NHS = N-hydroxysuccinimide) is shown on the top and coupling is shown on the bottom. Where Adtk is the first-generation Ad vector encoding the HSV1-sr39tk gene; ...

Longitudinal MicroPET Imaging of Brain Tumor Growth with F-18-labeled RGD Peptide

Abstract: EMD 121974, a potent cyclic RGD peptide inhibitor of α v-integrins, demonstrated effectiveness in suppressing brain tumor growth in both preclinical models and phases I/II clinical trials. The ability to non-invasively evaluate α v-integrin expression provides a novel and unique way to better understand brain tumor angiogenesis in relationship to α v-integrin expression, and allow for direct assessment of anti-integrin treatment efficacy. We developed a F-18-labeled RGD peptide [F-18]FB-RGD and performed serial microPET imaging scans to follow brain tumor growth and angiogenesis as a function of time in an orthotopic U87MG glioblastoma xenograft model in athymic nude mice. The tumor was barely visible on microPET at the size of ≤1.5 mm diameter at which time no angiogenesis was evident on histological examination. When tumor started to grow exponentially by day 35 the activity accumulation in the brain tumor also increased accordingly, with best tumor-to-brain contrast seven weeks after inoculation of 105 U87MG cells into the mice forebrain. Longitudinal microPET imaging and [F-18]FB-RGD provides the sensitivity and resolution to visualize and quantify anatomical variations during brain tumor growth and angiogenesis, most likely through interaction with α v-integrins expressed on tumor cells and angiogenic tumor vessels.

Significance of one-bead-one-compound combinational chemistry.

Abstract: One-bead-one-compound combinatorial peptidomimetic libraries, in conjunction with a high-stringency screening method, are a powerful tool for screening peptide and peptidomimetic ligands for target proteins. Picomolar-affinity peptidomimetics for the integrin α4β1 have now been developed and have been successfully used to image α4β1-expressing tumors in living mice.

Effect of chlorination on estrogenicity in chlorinated treated effluent

Abstract: Effluents from sewage treatment plants can be discharged into rivers with estrogenic contaminants at levels sufficient to induce adverse reproductive and fertility developments in humans and wildlife. Of great concern in recent years are the estrogenic activities of chlorinated by-products (CBPs) in effluents. Simplified cell proliferation tests using the human estrogen receptor-positive MCF-7 ATCC breast cancer cells (E-SCREEN) were performed to investigate the influence of chlorination on the estrogenicity in effluents. It was found that the increase of chlorine dosages from 0 mg/L to 4 mg/L led to the decrease of total content of estrogenic activity: the 17β-estradiol equivalent concentration (EEQ) from 22.40 to 8.35 ng/L. However, the increase of contact time from 10-30 minutes increased EEQ from 14.04 to 29.97 ng/L. Furthermore, increasing TOC level using humic acid from 5 mg/L to 15 mg/L in effluents correspondingly resulted in an increase of EEQ from 19.69-27.20 ng/L; it was thus confirmed that chlorination of humic acid could produce estrogenic by-products.