Showing papers by "Xiaoyuan Chen published in 2004"

In vivo Near-Infrared Fluorescence Imaging of Integrin αvβ3 in Brain Tumor Xenografts

Abstract: Noninvasive visualization of cell adhesion molecule alpha(v)beta(3) integrin expression in vivo has been well studied by using the radionuclide imaging modalities in various preclinical tumor models. A literature survey indicated no previous use of cyanine dyes as contrast agents for in vivo optical detection of tumor integrin. Herein, we report the integrin receptor specificity of novel peptide-dye conjugate arginine-glycine-aspartic acid (RGD)-Cy5.5 as a contrast agent in vitro, in vivo, and ex vivo. The RGD-Cy5.5 exhibited intermediate affinity for alpha(v)beta(3) integrin (IC(50) = 58.1 +/- 5.6 nmol/L). The conjugate led to elevated cell-associated fluorescence on integrin-expressing tumor cells and endothelial cells and produced minimal cell fluorescence when coincubated with c(RGDyK). In vivo imaging with a prototype three-dimensional small-animal imaging system visualized subcutaneous U87MG glioblastoma xenograft with a broad range of concentrations of fluorescent probe administered via the tail vein. The intermediate dose (0.5 nmol) produces better tumor contrast than high dose (3 nmol) and low dose (0.1 nmol) during 30 minutes to 24 hours postinjection, because of partial self-inhibition of receptor-specific tumor uptake at high dose and the presence of significant amount of background fluorescence at low dose, respectively. The tumor contrast was also dependent on the mouse viewing angles. Tumor uptake of RGD-Cy5.5 was blocked by unlabeled c(RGDyK). This study suggests that the combination of the specificity of RGD peptide/integrin interaction with near-infrared fluorescence detection may be applied to noninvasive imaging of integrin expression and monitoring anti-integrin treatment efficacy providing near real-time measurements.

Pegylated Arg-Gly-Asp peptide: 64Cu labeling and PET imaging of brain tumor alphavbeta3-integrin expression.

Abstract: UNLABELLED The alphav-integrins, cell adhesion molecules that are highly expressed on activated endothelial cells and tumor cells but not on dormant endothelial cells or normal cells, present an attractive target for tumor imaging and therapy. We previously coupled a cyclic Arg-Gly-Asp (RGD) peptide, c(RGDyK), with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) and labeled the RGD-DOTA conjugate with 64Cu (half-life, 12.8 h; 19% beta+) for solid tumor targeting, with high tumor-to-background contrast. The rapid tumor washout rate and persistent liver and kidney retention of this tracer prompted us to optimize the tracer for improved pharmacokinetic behavior. In this study, we introduced a polyethylene glycol (PEG; molecular weight, 3,400) moiety between DOTA and RGD and evaluated the 64Cu-DOTA-PEG-RGD tracer for microPET imaging in brain tumor models. METHODS DOTA was activated in situ and conjugated with RGD-PEG-NH2 under slightly basic conditions. alphavbeta3-Integrin-binding affinity was evaluated with a solid-phase receptor-binding assay in the presence of 125I-echistatin. Female nude mice bearing subcutaneous U87MG glioblastoma xenografts were administered 64Cu-DOTA-PEG-RGD, and the biodistributions of the radiotracer were evaluated from 30 min to 4 h after injection. microPET (20 min of static imaging at 1 h after injection) and then quantitative autoradiography were used for tumor visualization and quantification. The same tracer was also applied to an orthotopic U87MG model for tumor detection. RESULTS The radiotracer was synthesized with a high specific activity (14,800-29,600 GBq/mmol [400-800 Ci/mmol]). The c(RGDyK)-PEG-DOTA ligand showed intermediate binding affinity for alphavbeta3-integrin (50% inhibitory concentration, 67.5 +/- 7.8 nmol/L [mean +/- SD]). The pegylated RGD peptide demonstrated rapid blood clearance (0.57 +/- 0.15 percentage injected dose [%ID]/g [mean +/- SD] at 30 min after injection and 0.03 +/- 0.02 %ID/g at 4 h after injection). Activity accumulation in the tumor was rapid and high at early time points (2.74 +/- 0.45 %ID/g at 30 min after injection), and some activity washout was seen over time (1.62 +/- 0.18 %ID/g at 4 h after injection). Compared with (64)Cu-DOTA-RGD, this tracer showed improved in vivo kinetics, with significantly reduced liver uptake (0.99 +/- 0.08 %ID/g vs. 1.73 +/- 0.39 %ID/g at 30 min after injection and 0.58 +/- 0.07 %ID/g vs. 2.57 +/- 0.49 %ID/g at 4 h after injection). The pegylated RGD peptide showed higher renal accumulation at early time points (3.51 +/- 0.24 %ID/g vs. 2.18 +/- 0.23 %ID/g at 30 min after infection) but more rapid clearance (1.82 +/- 0.29 %ID/g vs. 2.01 +/- 0.25 %ID/g at 1 h after injection) than 64Cu-DOTA-RGD. The integrin receptor specificity of this radiotracer was demonstrated by blocking of tumor uptake by coinjection with nonradiolabeled c(RGDyK). The high tumor-to-organ ratios for the pegylated RGD peptide tracer (at 1 h after injection: tumor-to-blood ratio, 20; tumor-to-muscle ratio, 12; tumor-to-liver ratio, 2.7; and tumor-to-kidney ratio, 1.2) were confirmed by microPET and autoradiographic imaging in a subcutaneous U87MG tumor model. This tracer was also able to detect an orthotopic brain tumor in a model in which U87MG cells were implanted into the mouse forebrain. Although the magnitude of tumor uptake in the orthotopic xenograft was lower than that in the subcutaneous xenograft, the orthotopic tumor was still visualized with clear contrast from normal brain tissue. CONCLUSION This study demonstrated the suitability of a PEG moiety for improving the in vivo kinetics of a 64Cu-RGD peptide tracer without compromising the tumor-targeting ability and specificity of the peptide. Systematic investigations of the effects of the size and geometry of PEG on tumor targeting and in vivo kinetics will lead to the development of radiotracers suitable for clinical applications such as visualizing and quantifying alphav-integrin expression by PET. In addition, the same ligand labeled with therapeutic radionuclides may be applicable for integrin-targeted internal radiotherapy.

MicroPET imaging of breast cancer alphav-integrin expression with 64Cu-labeled dimeric RGD peptides.

Abstract: Purpose Alphavβ3 and αvβ5 integrins are cell adhesion molecules that play a vital role in tumor angiogenesis and metastasis. The ability to visualize and quantify integrin expression in vivo will foster our understanding of the role of integrins αvβ3 and αvβ5 in tumor angiogenesis and allow for direct assessment of anti-angiogenic treatment efficacy based on integrin antagonists. This study compared the tumor targeting characteristics of two dimeric 64Cu-labeled RGD peptide agonists of αvβ3 integrin. Procedures Dimeric RGD peptides E[c(RGDyK)]2 and E[c(RGDfK)]2 were conjugated with 1,4,7,10-tetraazadodecane-N,N′,N″,N″′-tetraacetic acid (DOTA) and labeled with positron emitter 64Cu(t1/2 = 12.8 h, β+ = 19%). Both 64Cu-DOTA-E[c(RGDyK)]2 and 64Cu-DOTA-E[c(RGDfK)]2 were used in biodistribution, microPET imaging and whole-body autoradiography studies in athymic female nude mice with orthotopically growing MDA-MB-435 breast carcinoma xenografts. Results At all time points, activity accumulation of 64Cu-DOTA-E[c(RGDyK)]2 in tumors was significantly higher compared to the D-Phe analog. Liver uptake of the D-Tyr derivative was lower than the D-Phe derivative at early time points but the difference became marginal with time. Overall, 64Cu-DOTA-E[c(RGDyK)]2 yielded better position emission tomography (PET) images in orthotopic MDA-MB-435 bearing mice than did 64Cu-DOTA-E[c(RGDfK)]2. Both radiotracers had αv-integrin specific tumor activity accumulation, as demonstrated by significant reduction of uptake with a coinjected blocking dose of c(RGDyK). Conclusions The radiolabeled dimeric RGD peptides 64Cu-DOTA-E[c(RGDyK)]2 and 64Cu-DOTA-E[c(RGDfK)]2 have high and specific tumor uptake in a human breast cancer tumor xenograft, with the D-Tyr derivative showing better in vivo kinetics than the D-Phe derivative, most likely due to the increased hydrophilicity of the D-Tyr. Both dimeric peptides showed better tumor retention than the previously tested monomeric RGD counterparts, presumably because of bivalency and increase in apparent molecular size.

MicroPET imaging of brain tumor angiogenesis with 18F-labeled PEGylated RGD peptide.

Abstract: We have previously labeled cyclic RGD peptide c(RGDyK) with fluorine-18 through conjugation labeling via a prosthetic 4-[18F]fluorobenzoyl moiety and applied this [18F]FB-RGD radiotracer for αv-integrin expression imaging in different preclinical tumor models with good tumor-to-background contrast. However, the unfavorable hepatobiliary excretion and rapid tumor washout rate of this tracer limit its potential clinical applications. The aims of this study were to modify the [18F]FB-RGD tracer by inserting a heterobifunctional poly(ethylene glycol) (PEG, M.W. =3,400) between the 18F radiolabel and the RGD moiety and to test this [18F]FB-PEG-RGD tracer for brain tumor targeting and in vivo kinetics. [18F]FB-PEG-RGD was prepared by coupling the RGD-PEG conjugate with N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) under slightly basic conditions (pH=8.5). The radiochemical yield was about 20–30% based on the active ester [18F]SFB, and specific activity was over 100 GBq/μmol. This tracer had fast blood clearance, rapid and high tumor uptake in the subcutaneous U87MG glioblastoma model (5.2±0.5%ID/g at 30 min p.i.). Moderately rapid tumor washout was observed, with the activity accumulation decreased to 2.2±0.4%ID/g at 4 h p.i. MicroPET and autoradiography imaging showed a very high tumor-to-background ratio and limited activity accumulation in the liver, kidneys and intestinal tracts. U87MG tumor implanted into the mouse forebrain was well visualized with [18F]FB-PEG-RGD. Although uptake in the orthotopic tumor was significantly lower (P<0.01) than in the subcutaneous tumor, the maximum tumor-to-brain ratio still reached 5.0±0.6 due to low normal brain background. The results of H&E staining post mortem agreed with the anatomical information obtained from non-invasive microPET imaging. In conclusion, PEGylation suitably modifies the physiological behavior of the RGD peptide. [18F]FB-PEG-RGD gave improved tumor retention and in vivo kinetics compared with [18F]FB-RGD.

Micro-PET imaging of alphavbeta3-integrin expression with 18F-labeled dimeric RGD peptide.

Abstract: The alphav integrins, which act as cell adhesion molecules, are closely involved with tumor invasion and angiogenesis In particular, alphavbeta3 integrin, which is specifically expressed on proliferating endothelial cells and tumor cells, is a logical target for development of a radiotracer method to assess angiogenesis and anti-angiogenic therapy In this study, a dimeric cyclic RGD peptide E[c(RGDyK)]2 was labeled with 18F (t(1/2) = 1097 min) by using a prosthetic 4-[18F]fluorobenzoyl moiety to the amino group of the glutamate The resulting [18F]FB-E[c(RGDyK)]2, with high specific activity (200-250 GBq/micromol at the end of synthesis), was administered to subcutaneous U87MG glioblastoma xenograft models for micro-PET and autoradiographic imaging as well as direct tissue sampling to assess tumor targeting efficacy and in vivo kinetics of this PET tracer The dimeric RGD peptide demonstrated significantly higher tumor uptake and prolonged tumor retention in comparison with a monomeric RGD peptide analog [18F]FB-c(RGDyK) The dimeric RGD peptide had predominant renal excretion, whereas the monomeric analog was excreted primarily through the biliary route Micro-PET imaging 1 hr after injection of the dimeric RGD peptide exhibited tumor to contralateral background ratio of 95 +/- 08 The synergistic effect of polyvalency and improved pharmacokinetics may be responsible for the superior imaging characteristics of [18F]FB-E[c(RGDyK)]2

microPET and Autoradiographic Imaging of GRP Receptor Expression with 64Cu-DOTA-[Lys3]Bombesin in Human Prostate Adenocarcinoma Xenografts

Abstract: Overexpression of gastrin-releasing peptide (GRP) receptor (GRPR) in both androgen-dependent (AD) and androgen-independent (AI) human neoplastic prostate tissues provides an attractive target for prostate cancer imaging and therapy. The goal of this study was to develop 64Cu-radiolabeled GRP analogs for PET imaging of GRPR expression in prostate cancer xenografted mice. Methods: [Lys3]bombesin ([Lys3]BBN) was conjugated with 1,4,7,10-tetraazadodecane-N,N′,N″,N‴-tetraacetic acid (DOTA) and labeled with the positron-emitting isotope 64Cu (half-life = 12.8 h, 19% β+). Receptor binding of DOTA-[Lys3]BBN and internalization of 64Cu-DOTA-[Lys3]BBN by PC-3 prostate cancer cells were measured. Tissue biodistribution, microPET, and whole-body autoradiographic imaging of the radiotracer were also investigated in PC-3 (AI)/CRW22 (AD) prostate cancer tumor models. Results: A competitive receptor- binding assay using 125I-[Tyr4]BBN against PC-3 cells yielded a 50% inhibitory concentration value of 2.2 ± 0.5 nmol/L for DOTA-[Lys3]BBN. Incubation of cells with the 64Cu-labeled radiotracer showed temperature- and time-dependent internalization. At 37°C, >60% of the tracer was internalized within the first 15 min and uptake remained constant for 2 h. Radiotracer uptake was higher in AI PC-3 tumor (5.62 ± 0.08 %ID/g at 30 min after injection, where %ID/g is the percentage of injected dose per gram) than in AD CWR22 tumor (1.75 ± 0.05 %ID/g at 30 min after injection). Significant accumulation of the activity in GRPR-positive pancreas was also observed (10.4 ± 0.15 %ID/g at 30 min after injection). Coinjection of a blocking dose of [Lys3]BBN inhibited the activity accumulation in PC-3 tumor and pancreas but not in CWR22 tumor. microPET and autoradiographic imaging of 64Cu-DOTA-[Lys3]BBN in athymic nude mice bearing subcutaneous PC-3 and CWR22 tumors showed strong tumor-to-background contrast. Conclusion: This study demonstrates that PET imaging of 64Cu-DOTA-[Lys3]BBN is able to detect GRPR-positive prostate cancer.

Micro-PET Imaging of α v β 3 -Integrin Expression with 18 F-Labeled Dimeric RGD Peptide

Abstract: The αv integrins, which act as cell adhesion molecules, are closely involved with tumor invasion and angiogenesis. In particular, αvβ3 integrin, which is specifically expressed on proliferating end...

Drug ADME‐associated protein database as a resource for facilitating pharmacogenomics research

Abstract: Increasing effort and interest have been directed at pharmacogenomics for elucidating the mechanism of individual differences in drug response and the design of personalized drugs and dosages. Knowledge about drug absorption, distribution, metabolism, and excretion (ADME) proteins and drug targets is useful for facilitating the search and evaluation of polymorphisms and other pharmacogenomic-contributing factors. A database of drug ADME-associated proteins, at http://bidd.nus.edu.sg/group/admeap/admeap.asp, serves as a resource for comprehensive information about proteins potentially responsible for pharmacogenetic effects of pharmacokinetic origin. This database currently contains entries for 316 ADME-associated proteins, 1,070 substrates and inhibitors, 1,337 known polymorphisms in 121 proteins, and 327 drugs reported to have altered responses linked to an ADME-associated protein. It also provides physiological function of each protein, pharmacokinetic effect, ADME classification, direction, and driving force of disposition, location and tissue distribution, synonyms, and gene name. Information in this database and its usefulness for facilitating pharmacogenomic research is discussed. Drug Dev. Res. 62:134–142, 2004. © 2004 Wiley-Liss, Inc.

In vivo Near-Infrared Fluorescence Imaging of Integrin v3 in Brain Tumor Xenografts

Abstract: Noninvasive visualization of cell adhesion molecule v3 integrin expression in vivo has been well studied by using the radionuclide imaging modalities in various preclinical tumor models. A literature survey indicated no previous use of cyanine dyes as contrast agents for in vivo optical detection of tumor integrin. Herein, we report the integrin receptor specificity of novel peptide-dye conjugate arginine-glycine-aspartic acid (RGD)-Cy5.5 as a contrast agent in vitro, in vivo, and ex vivo. The RGD-Cy5.5 exhibited intermediate affinity for v3 integrin (IC50 58.1 5.6 nmol/L). The conjugate led to elevated cell-associated fluorescence on integrin-expressing tumor cells and endothelial cells and produced minimal cell fluorescence when coincubated with c(RGDyK). In vivo imaging with a prototype three-dimensional small-animal imaging system visualized subcutaneous U87MG glioblastoma xenograft with a broad range of concentrations of fluorescent probe administered via the tail vein. The intermediate dose (0.5 nmol) produces better tumor contrast than high dose (3 nmol) and low dose (0.1 nmol) during 30 minutes to 24 hours postinjection, because of partial self-inhibition of receptor-specific tumor uptake at high dose and the presence of significant amount of background fluorescence at low dose, respectively. The tumor contrast was also dependent on the mouse viewing angles. Tumor uptake of RGDCy5.5 was blocked by unlabeled c(RGDyK). This study suggests that the combination of the specificity of RGD peptide/integrin interaction with near-infrared fluorescence detection may be applied to noninvasive imaging of integrin expression and monitoring anti-integrin treatment efficacy providing near real-time measurements.