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64Cu-labeled lissamine rhodamine B: a promising PET radiotracer targeting tumor mitochondria.

ABSTRACT: Enhanced mitochondrial potential in carcinoma cells is an important characteristic of cancer. It is of great current interest to develop a radiotracer that is sensitive to mitochondrial potential changes at the early stage of tumor growth. In this report, we present the synthesis and evaluation of (64)Cu-labeled Lissamine rhodamine B (LRB), (64)Cu(DOTA-LRB) (DOTA-LRB = 2-(6-(diethylamino)-3-(diethyliminio)-3H-xanthen-9-yl)-5-(N-(2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclo-dodecan-1-yl)acetamido)ethyl)sulfamoyl)benzenesulfonate) as a new radiotracer for imaging tumors in athymic nude mice bearing U87MG human glioma xenografts by positron emission tomography (PET). We also explored its localization mechanism using Cu(DOTA-LRB) as the fluorescent probe in both the U87MG human glioma cell line and the cultured primary U87MG glioma cells. It was found that (64)Cu(DOTA-LRB) had the highest tumor uptake (6.54 ± 1.50, 6.91 ± 1.26, 5.68 ± 1.13, 7.58 ± 1.96, and 5.14 ± 1.50%ID/g at 0.5, 1, 2, 4, and 24 h postinjection, respectively) among many (64)Cu-labeled organic cations evaluated in the same animal model. The cellular staining study indicated that Cu(DOTA-LRB) was able to localize in mitochondria of U87MG glioma cells due to the enhanced negative mitochondrial potential. This statement is completely supported by the results from decoupling experiment with carbonylcyanide-m-chlorophenylhydrazone (CCCP). MicroPET data showed that the U87MG glioma tumors were clearly visualized as early as 30 min postinjection with (64)Cu(DOTA-LRB). (64)Cu(DOTA-LRB) remained stable during renal excretion, but underwent extensive degradation during hepatobiliary excretion. On the basis of the results from this study, it was concluded that (64)Cu(DOTA-LRB) represents a new class of promising PET radiotracers for noninvasive imaging of the MDR-negative tumors.

SUBMITTER: Zhou Y

PROVIDER: S-EPMC3148415 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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64Cu-labeled lissamine rhodamine B: a promising PET radiotracer targeting tumor mitochondria.

Zhou Yang Y Kim Young-Seung YS Yan Xin X Jacobson Orit O Chen Xiaoyuan X Liu Shuang S

Molecular pharmaceutics 20110524 4

Enhanced mitochondrial potential in carcinoma cells is an important characteristic of cancer. It is of great current interest to develop a radiotracer that is sensitive to mitochondrial potential changes at the early stage of tumor growth. In this report, we present the synthesis and evaluation of (64)Cu-labeled Lissamine rhodamine B (LRB), (64)Cu(DOTA-LRB) (DOTA-LRB = 2-(6-(diethylamino)-3-(diethyliminio)-3H-xanthen-9-yl)-5-(N-(2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclo-dodecan-1-y ...[more]

PMID: 21545131

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Project description:In this study, we evaluated Cu(L1) in two xenografted tumor-bearing (U87MG and MDA-MB-435) animal models to prove the concept that Cu(II)-labeled rhodamine derivatives, Cu(L) (L = L1 - L4) are useful as selective fluorescent probes for tumor imaging. We found that both multidrug resistance (MDR) negative U87MG gliomas and MDR-positive MDA-MB-435 breast tumors could be visualized. Because of tissue attenuation, accurate quantification of tumor uptake was difficult by optical methods. Therefore, (64)Cu(L) (L = L1 - L4) were evaluated to compare their biodistribution properties. It was found that all four (64)Cu radiotracers had a high glioma uptake ((64)Cu(L1): 5.71± 1.43 %ID/g; (64)Cu(L2): 5.98 ± 2.75 %ID/g; (64)Cu(L3): 4.28 ± 1.45 %ID/g; and (64)Cu(L4): 6.25 ± 3.42 %ID/g) with (64)Cu(L1) showing the highest tumor/background ratios. In athymic nude mice bearing MDA-MB-435 breast cancer xenografts, (64)Cu(L4) showed almost identical normal organ uptake to that in the glioma-bearing animals, but its breast tumor uptake (1.26 ± 0.10% ID/g) was significantly lower (p < 0.001) than that in the glioma (6.25 ± 3.42% ID/g) because of MDR Pgps (P-glycoproteins) and MRPs (multidrug resistance-associated proteins) overexpressed in the xenografted MDA-MB-435 breast tumors. Results from cellular staining assays showed that both Cu(L2) and Cu(L4) were able to localize in mitochondria of U87MG cells, and their tumor selectivity was caused by the elevated negative mitochondrial potential in U87MG glioma cells as compared to that in human fibroblast cells. On the basis of these results, it was concluded that Cu(L) (L = L1 - L4) are useful as selective fluorescent probes for cellular staining assays and optical tumor imaging while (64)Cu(L) (L = L1 - L4) have the potential as PET radiotracers for tumor imaging. This study represents a good example of dual modality imaging (PET and optical) using two agents, (64)Cu(L) and Cu(L), with identical chemical composition. Future research will focus on developing new fluorescent probes with longer wavelength and reduced liver uptake.

Project description:Radiolabeled organic cations, such as triphenylphosphonium (TPP), represents a new class of radiotracers for imaging cancers and the transport function of multidrug resistance P-glycoproteins (particularly MDR1 Pgp) by single photon emission computed tomography (SPECT) or positron emission tomography (PET). This report presents the synthesis and biological evaluation of (64)Cu-labeled 2-(diphenylphosphoryl)ethyldiphenylphosphonium (TPEP) cations as novel PET radiotracers for tumor imaging. Biodistribution studies were performed using the athymic nude mice bearing subcutaneous U87MG human glioma xenografts to explore the impact of linkers, bifunctional chelators (BFCs), and chelates on biodistribution characteristics of the (64)Cu-labeled TPEP cations. Metabolism studies were carried out using normal athymic nude mice to determine the metabolic stability of four (64)Cu radiotracers. It was found that most (64)Cu radiotracers described in this study have significant advantages over (99m)Tc-Sestamibi for their high tumor/heart and tumor/muscle ratios. Both BFCs and linkers have significant impact on biological properties of (64)Cu-labeled TPEP cations. For example, (64)Cu(DO3A-xy-TPEP) has much lower liver uptake and better tumor/liver ratios than (64)Cu(DO3A-xy-TPP), suggesting that TPEP is a better mitochondrion-targeting molecule than TPP. Replacing DO3A with DO2A results in (64)Cu(DO2A-xy-TPEP) (+), which has a lower tumor uptake than (64)Cu(DO3A-xy-TPEP). Substitution of DO3A with NOTA-Bn leads to a significant decrease in tumor uptake for (64)Cu(NOTA-Bn-xy-TPEP). The use of DOTA-Bn to replace DO3A has little impact on the tumor uptake, but the tumor/liver ratio of (64)Cu(DOTA-Bn-xy-TPEP) (-) is not as good as that of (64)Cu(DO3A-xy-TPEP), probably due to the aromatic benzene ring in DOTA-Bn. Addition of an extra acetamido group in (64)Cu(DOTA-xy-TPEP) results in a lower liver uptake, but tumor/liver ratios of (64)Cu(DOTA-xy-TPEP) and (64)Cu(DO3A-xy-TPEP) are comparable due to a faster tumor washout of (64)Cu(DOTA-xy-TPEP). Substitution of xylene with the PEG 2 linker also leads to a significant reduction in both tumor and liver uptake. MicroPET imaging studies on (64)Cu(DO3A-xy-TPEP) in athymic nude mice bearing U87MG glioma xenografts showed that the tumor was clearly visualized as early as 1 h postinjection with very high T/B contrast. There was very little metabolite (<2%) detectable in the urine and feces samples for (64)Cu(DO3A-xy-TPEP), (64)Cu(DOTA-Bn-xy-TPEP)(-), and (64)Cu(NOTA-Bn-xy-TPEP). Considering both tumor uptake and T/B ratios (particularly tumor/heart, tumor/liver, and tumor/muscle), it was concluded that (64)Cu(DO3A-xy-TPEP) is a promising PET radiotracer for imaging the MDR-negative tumors.

Project description:This purpose of this study is to determine the efficacy of a 45-amino acid Gp2 domain, engineered to bind to epidermal growth factor receptor (EGFR), as a positron emission tomography (PET) probe of EGFR in a xenograft mouse model. The EGFR-targeted Gp2 (Gp2-EGFR) and a nonbinding control were site-specifically labeled with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. Binding affinity was tested toward human EGFR and mouse EGFR. Biological activity on downstream EGFR signaling was examined in cell culture. DOTA-Gp2 molecules were labeled with 64Cu and intravenously injected (0.6-2.3 MBq) into mice bearing EGFRhigh (n = 7) and EGFRlow (n = 4) xenografted tumors. PET/computed tomography (CT) images were acquired at 45 min, 2 h, and 24 h. Dynamic PET (25 min) was also acquired. Tomography results were verified with gamma counting of resected tissues. Two-tailed t tests with unequal variances provided statistical comparison. DOTA-Gp2-EGFR bound strongly to human (KD = 7 ± 5 nM) and murine (KD = 29 ± 6 nM) EGFR, and nontargeted Gp2 had no detectable binding. Gp2-EGFR did not agonize EGFR nor antagonize EGF-EGFR. 64Cu-Gp2-EGFR tracer effectively localized to EGFRhigh tumors at 45 min (3.2 ± 0.5%ID/g). High specificity was observed with significantly lower uptake in EGFRlow tumors (0.9 ± 0.3%ID/g, p < 0.001), high tumor-to-background ratios (11 ± 6 tumor/muscle, p < 0.001). Nontargeted Gp2 tracer had low uptake in EGFRhigh tumors (0.5 ± 0.3%ID/g, p < 0.001). Similar data was observed at 2 h, and tumor signal was retained at 24 h (2.9 ± 0.3%ID/g). An engineered Gp2 PET imaging probe exhibited low background and target-specific EGFRhigh tumor uptake at 45 min, with tumor signal retained at 24 h postinjection, and compared favorably with published EGFR PET probes for alternative protein scaffolds. These beneficial in vivo characteristics, combined with thermal stability, efficient evolution, and small size of the Gp2 domain validate its use as a future class of molecular imaging agents.

Project description:Lung cancer accounts for 17% of cancer-related deaths worldwide, and most patients present with locally advanced or metastatic disease. Novel PET imaging agents for assessing vascular endothelial growth factor receptor-2 (VEGFR-2) expression can be used for detecting VEGFR-2-positive malignancies and subsequent monitoring of therapeutic response to VEGFR-2-targeted therapies. Here, we report the synthesis and characterization of an antibody-based imaging agent for PET imaging of VEGFR-2 expression in vivo.Ramucirumab (named RamAb), a fully humanized IgG1 monoclonal antibody, was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with (64)Cu. Flow cytometry analysis and microscopy studies were performed to compare the VEGFR-2 binding affinity of RamAb and NOTA-RamAb. PET imaging and biodistribution studies were performed in nude mice bearing HCC4006 and A549 xenograft tumors. Ex vivo histopathology was performed to elucidate the expression patterns of VEGFR-2 in different tissues and organs to validate in vivo results.Flow cytometry examination revealed the specific binding capacity of fluorescein isothiocyanate-RamAb to VEGFR-2, and no difference in VEGFR-2 binding affinity was seen between RamAb and NOTA-RamAb. After being labeled with (64)Cu, PET imaging revealed specific and prominent uptake of (64)Cu-NOTA-RamAb in VEGFR-2-positive HCC4006 tumors (9.4 ± 0.5 percentage injected dose per gram at 48 h after injection; n = 4) and significantly lower uptake in VEGFR-2-negative A549 tumors (4.3 ± 0.2 percentage injected dose per gram at 48 h after injection; n = 3). Blocking experiments revealed significantly lower uptake in HCC4006 tumors, along with histology analysis, further confirming the VEGFR-2 specificity of (64)Cu-NOTA-RamAb.This study provides initial evidence that (64)Cu-NOTA-RamAb can function as a PET imaging agent for visualizing VEGFR-2 expression in vivo, which may also find potential applications in monitoring the treatment response of VEGFR-2-targeted cancer therapy.

Project description:A novel recombinant disintegrin, vicrostatin (VCN), displays high binding affinity to a broad range of human integrins in substantial competitive biological advantage over other integrin-based antagonists. In this study, we synthesized a new 64Cu-labeled VCN probe and evaluated its imaging properties for prostate cancer in PC-3 tumor-bearing mice. Macrocyclic chelating agent 1,8-diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosine (DiAmSar) was conjugated with PEG unit and followed by coupling with VCN. The precursor was then radiolabeled with positron emitter 64Cu (t1/2 = 12.7 h) in ammonium acetate buffer to provide 64Cu-Sar-PEG-VCN, which was subsequently subjected to in vitro studies, small animal PET, and biodistribution studies. The PC-3 tumor-targeting efficacy of 64Cu-Sar-PEG-VCN was compared to a cyclic RGD peptide-based PET probe (64Cu-Sar-RGD). 64Cu labeling was achieved in 75% decay-corrected yield with radiochemical purity of > 98%. The specific activity of 64Cu-Sar-PEG-VCN was estimated to be 37 MBq/nmol. MicroPET imaging results showed that 64Cu-Sar-PEG-VCN has preferential tumor uptake and good tumor retention in PC-3 tumor xenografts. As compared to 64Cu-Sar-RGD, 64Cu-Sar-PEG-VCN produces higher tumor-to-muscle (T/M) imaging contrast ratios at 2 h (4.66 ± 0.34 vs. 2.88 ± 0.46) and 24 h (4.98 ± 0.80 vs. 3.22 ± 0.30) post-injection (pi) and similar tumor-to-liver ratios at 2 h (0.43 ± 0.09 vs. 0.37 ± 0.04) and 24 h (0.57 ± 0.13 vs. 0.52 ± 0.07) pi. The biodistribution results were consistent with the quantitative analysis of microPET imaging, demonstrating good T/M ratio (2.73 ± 0.36) of 64Cu-Sar-PEG-VCN at 48 h pi in PC-3 tumor xenografts. For both microPET and biodistribution studies at 48 h pi, the PC-3 tumor uptake of 64Cu-Sar-PEG-VCN is lower than that of 64Cu-Sar-RGD. 64Cu-Sar-PEG-VCN has the potential for in vivo imaging of prostate cancer with PET, which may provide a unique non-invasive method to quantitatively localize and characterize prostate cancer.

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64Cu-labeled lissamine rhodamine B: a promising PET radiotracer targeting tumor mitochondria.

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64Cu-labeled lissamine rhodamine B: a promising PET radiotracer targeting tumor mitochondria.

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