EGFR is over-expressed in several solid tumors including breast, prostate, pancreas, and lung cancers and is correlated to the metastasic potential of the tumor. Anti-EGFR receptor-binding peptidomimetics (AERP) were examined to assess the small molecule's potential use as tumor-specific imaging agents. The aim of this work was to design and characterize the binding specificity of the radiolabeled peptidomimetics to EGFR over-expressing cell lysate and to A431 xenograft tumors. Our newly designed peptidomimetic, AERP, was conjugated to DTPA and labeled with (99m)Tc. The in vivo tumor accumulation of [(99m)Tc] DTPA-AERP-2 was 1.6 +/- 0.1 %ID/g and tumor to muscle ratio was 5.5. Our studies suggest that this novel peptidomimetic, AERP-2, warrants further development as an EGFR specific tumor-imaging agent. (C) 2011 Elsevier Ltd. All rights reserved.

Angiogenesis is crucial for solid tumor growth. By secreting angiogenic factors, tumor cells induce angiogenesis. However, targeting these angiogenic factors for cancer therapy is not always successful, suggesting that other factors may be involved in tumor angiogenesis. This work shows that 25 protein spots were differentially expressed by two-dimensional gel electrophoretic analysis when HepG2 cells induced endothelial cell differentiation to tube in vitro, and most of them were upregulated. Twenty-one proteins were identified with MALDI-TOF-MS, and the other four were identified by LTQ-MS/MS. Keratins were identified as one class of these upregulated proteins. Further study indicated that the expression of keratin 17 in cultured endothelial cells is likely microenvironment regulated, because its expression can be induced by HepG2 cells and bFGF as well as serum in culture media. Increased expression of keratins in endothelial cells, such as keratin 17, may contribute to the angiogenesis induced by HepG2 cells.

OBJECTIVE To investigate the effect of keratin 17 (K-17) on the migration, proliferation and tube formation of human umbilical vein endothelial cell (HUVEC), and to realize the role of K-17 in angiogenesis. METHODS After HUVEC were cultured in DMEM medium supplemented with 10%FBS overnight, K-17-siRNA-mixture (experimental group) and control-siRNA-mixture (negative control group) were added into HUVEC, respectively, by Lipofectamine 2000 transfection assay, and the final concentration of the siRNA was 50 nmol/L. Lipofectamine 2000 alone was used as the control. After the cells were cultured for 36 hours, the cell proliferation ability was detected by cell counting. After 30-hour culture, the cell's abilities of migration and differentiation to tube were detected by 24-well Millicell units and the collagen gel assay, respectively. In addition, non-siRNA-treated HUVEC were cultured for 24 hours in DMEM medium supplemented with 10%FBS (group A), 2%FBS (group B) and 2%FBS+10 ng/mL bFGF (group C), respectively, and then the expression of K-17 in HUVEC was detected by RT-PCR and Western blot. RESULTS After the treatment with K-17-siRNA for 36 hours, HUVEC exhibited no significant difference in the proliferation, compared with both control and negative control groups (P > 0.05). After transfected with K-17-siRNA for 30 hours, the number of HUVEC in the experimental group which migrated from the upper chamber to the lower chamber of Millicell wells within 24 hours (3719.0 +/- 319.0) was smaller than both control (7 437.5 +/- 212.0) and negative control (7 356.3 +/- 795.7) groups, with significant difference (P < 0.01). However, there was no significant difference between the control group and the negative control group (P > 0.05). After HUVEC were transfected with K-17-siRNA for 30 hours, the number of tubes in the experimental group, the negative control group and the control group in 24 hours was (1.1 +/- 0.5), (3.6 +/- 0.5) and (3.2 +/- 0.6) per field, respectively. The experimental group was significantly different from both control and negative control groups (P < 0.01), and there was no significant difference between the negative control group and the control group (P > 0.05). The expression of K-17 protein in HUVEC in groups A, B and C was 0.25 +/- 0.02, 0.08 +/- 0.01 and 0.72 +/- 0.03, respectively. There was significant difference among these three groups (P < 0.01). CONCLUSION K-17 has no impact on cell proliferation, but may augment endothelial cell migration, which may facilitate angiogenesis.

AbstractThe relationship of structural characters of the tripeptidic amine–bisamido‐thiol (N3S type) chelators with the lipophilicity, configuration, and stability of four [99mTcO]N3S and one [ReO]N3S chelates is studied here. The results show that the hydroxymethyl group on the two N3S chelators, RP294 and RP435, has inhibited neither the formation nor interconversion of syn and anti stereoisomers of the chelates, while the tert‐butyl group on RP455 and RP535 has prevented the anti isomer from converting to the syn one both in acidic and neutral solutions. The interconversion rates of a stereoisomer can be accelerated at higher pH. [99mTcO]RP455 is stable in pH 7.4 aqueous solution, while [99mTcO]RP535 undergoes decomposition at the same medium, suggesting the influence of a larger side‐chain on the stability of the chelate. Unlike [99mTcO]RP535, [ReO]RP535 is stable even in 0.1 N NaOH for 3 h without change. Combining factors of medium pH values, nature of substituents on a chelator's backbone, size of side‐chains, and property of central metal ions together determine the lipophilicity, configuration, and stability of [99mTcO]N3S and [ReO]N3S chelates. This information may be useful for a further design of [99mTcO]N3S or [186ReO]N3S or [188ReO]N3S chelates with predictable physicochemical properties favorable for the quality diagnosis of cancers. Copyright © 2008 John Wiley & Sons, Ltd.

Detection of tumor hypoxia is of importance because hypoxic cancer cells can resist radiation therapy and also induce molecules to help them surviving and metastasizing. Our previous study has reported the development of technetium-99m labeled amine-oxime compounds containing 2-nitroimidazoles to target hypoxic cells. In order to develop new generation of Tc-99m, Cu-64, and Cu-67 based amine-oxime chelates with improved selectivity on tumor hypoxia in the future, it is important to understand the correlation of some structural parameters such as lipophilicity and polarity of the backbone-constituents of a radiochelate with its serum protein binding potential and cellular-uptake-level. The correlation is investigated in this work by using five Tc-99m-amine-oxime chelates containing 2-NI group and another five containing aniline group (as control). The results indicate that the level of protein-and cellular-binding of a radiochelate increased with their lipophilicity which generally correlates with the lengthening of the alkyl chains on the backbone of the radiochelates. The greater lipophilicity of a radiochelate, the higher percentage it bound to serum proteins and cellular membrane. Our study also indicates that, in addition to lipophilicity, polarity of the constituents is also an important factor of determining the levels of serum protein binding and cellular accumulation of a radiochelate. Copyright (c) 2007 John Wiley & Sons, Ltd.

The p185HER2/nue receptors which regulate the growth of cancer cells and are overexpressed in 20-30% of the cancers can be the markers for cancer diagnosis. This work describes the DTPA conjugation, Indium(111) complexes characterization, Tc-99m labeling, and Biacore test of the peptides such as [Cys(6)-Cys(12)]H-GAGGYCDGFYACYMDV-CONH2 (anti-HER2/neu-peptidomics, (Ala 2)AHNP), [Cys(6)-Cys(12)]H-G(Abu)GGYCDGFYACYMDV-CONH2 ((Abu(2))AHNP), and H-dYCALTYYDYECdFAY-CONH2 (EP6, EGFR selective peptide) binding affinity to the pl85HER2/neu proteins. The results show the evidence of EP6 binding to the immobilized p185HER2/neu proteins. The labeled EP6, [Tc-99m]DTPA-EP6, had displayed greater association percentage to the lysate of T6-17 cells expressing p185HER2/neu proteins compared to the labeled AHNPs. The uptake (in terms of ID%/g) of [Tc-99m]DTPA-EP6 in animal xenografted tumor was seven folds greater than that of the [Tc-99m](Ala(2))AHNP. The nature of the dual-receptors (EGFR and HER2/neu) binding of EP6 is a merit for the peptide to target cancer. Copyright (c) 2006 John Wiley & Sons, Ltd.

The level of αVβ3 integrins on endothelial cells is elevated in angiogenesis. The high binding specificity to αVβ3 integrins of peptides containing Arg-Gly-Asp (RGD) residues suggests that the radiolabeled RGD peptides may be useful as tumor specific imaging agents. In this research, cyclised peptides containing Arg-Gly-Asp (RGD) and Arg-Gly-Glu (RGE, as control) residues were conjugated with HYNIC and labeled with 99mTc. Objective: The goal was to evaluate the influence of co-ligand, either tricine or ethylenediamine-N,N′-diacetic acid (EDDA) on protein and integrin binding and on cellular uptake in culture. Methods: The n-octanol/water partition coefficient, binding to bovine serum albumin (BSA) and human umbilical vein endothelial (HUVE) cells, and cell lysate distributions of the radiolabeled peptides were evaluated. Results: The co-ligands had a significant effect on the labeling efficiency of the HYNIC conjugates and on certain properties of the 99mTc complexes. The labeling efficiency with tricine was 10 fold higher and BSA binding was over 8 fold greater compared to EDDA. Both RGD labels showed higher (6 to 28 fold) binding to HUVE cells than that of the RGE labels, indicating binding specificity. After cell-lysis, only a small percentage of the total RGD label that accumulated in the cells was found bound to cellular proteins (9% of RGD/tricine and 5% of RGD/EDDA), implying that over 90% of the radiolabeled peptides were internalized for both radiolabeled RGDs. The number of the RGD molecules bound to proteins was estimated to be approximately three per cell, suggesting that only a small number of αVβ3 integrin proteins are expressed on the cells. Conclusions: Apart from the differences in radiolabeling, the only important effect of substituting EDDA for tricine as co-ligand on the HYNIC-peptides was the lower degree of serum protein binding. In spite of the lower serum protein binding potential, in vivo tumor accumulation of the RGD/EDDA may not be improved compared to RGD/tricine since quantitation of the cell binding results suggests that the number of αVβ3 integrin proteins per cell might be limited.

Three amino acids residues, Arg-Gly-Asp (RGD), in vitronectin and fibronectin show affinity for alpha(v)beta(3) integrins expressed in vascular endothelial cells. That tumor growth can upregulate the expression of these integrins on tumor cells for invasion and metastasis and in tissue neovasculature suggests the potential of developing radiolabeled RGD peptides as antagonists of alpha(v)beta(3) integrins for broad spectrum tumor specific imaging. The polypeptide RGD-4C, which contains four cysteine residues for cyclization, has shown preferential localization on integrins at sites of tumor angiogenesis. Both RGD-4C and RGE (Arg-Gly-Glu)-4C (as control) were purchased and conjugated with 6-hydrazinopyridine-3-carboxylic acid (HYNIC) for Tc-99m radiolabeling. After purification of the conjugated peptides by a C18 Sep-Pak cartridge with 20% methanol, both peptides were radiolabeled using tricine. For cell binding studies, both Tc-99m peptides were further purified by SE HPLC. High specific radioactivity of labeled cyclized RGD/E (cyclized RGD/E will be simplified as RGD/E through out the text) of about 20 Ci/mumol was achieved. Both Tc-99m complexes were stable in the labeling solution for over 24 h at room temperature. In the human umbilical vein endothelial (HUVE) cell studies, the binding at 1 h of radiolabeled RGD/E was determined at 4 degreesC and at concentrations in the picomolar to nanomolar range. Under these conditions, cell accumulation of 99mTc in the case of RGD was as much as 16 times greater than the control RGE. As a check on specificity, 7 nM of native cyclized RGD blocked 50% of the binding of Tc-99m-labeled RGD to cells. The binding percentage of Tc-99m-labeled RGD to purified alpha(v)beta(3) integrin protein, as determined by SE HPLC, increased with the concentration of the integrin while Tc-99m-labeled RGE showed no binding. The association constant for Tc-99m-RGD was modest at 7 x 10(6) M-1. In both human renal adenocarcinoma (ACHN) and human colon cancer cell line (LS174T) nude mouse tumor models, the accumulation of Tc-99m-labeled RGD/E exhibited no statistical difference. In conclusion, possibly because of limited numbers Of alpha(v)beta(3) integrin receptors per tumor cell and low binding affinity, radiolabeled RGD peptides may have limitations as tumor imaging agents.

A method is described to amplify the delivery of 111In to human breast cancer cells utilizing a novel human serum albumin-human EGF (HSA-hEGF) bioconjugate substituted preferentially in the HSA domain with multiple DTPA metal chelators for 111In. 111In-DTPA-HSA-hEGF exhibited a lower receptor-binding affinity than 111In-DTPA-hEGF but was rapidly and specifically bound, internalized and translocated to the nucleus in EGFR-positive MDA-MB-468 breast cancer cells. 111In-DTPA-HSA-hEGF was cytotoxic in vitro mainly through the emission of short-range Auger electrons and partially through the effects of the hEGF moiety to MDA-MB-468 cells overexpressing EGFR (1–2 × 106 receptors/cell) but not towards MCF-7 breast cancer cells with a 100-fold lower level of EGFR on their surface. The cytotoxicity in vitro against MDA-MB-468 cells of 111In-DTPA-HSA-hEGF substituted with nine DTPA chelators was enhanced 4-fold compared to 111In-DTPA-hEGF monosubstituted with DTPA. Studies are planned to further evaluate 111In-DTPA-HSA-hEGF in vivo as a new imaging and targeted radiotherapeutic agent for breast cancer.

The vascular endothelial cell is believed to be a major target cell of radiation-induced injury to the central nervous system. Dysfunction of the blood-brain barrier is associated with radiation-induced white matter lesions. The aim of this study was to determine the role of hypoxia in radiation-induced blood-brain barrier disruption. Adult rats were irradiated with graded single doses of 0-22 Gy to the cervical spinal cord. At various times up to 28 weeks after radiation, blood-spinal cord barrier (BSCB) permeability was assessed using immunohistochemistry with antialbumin antibody and gamma counting of (99m)Tc-diethylenetriamine pentaacetic acid. Expression of vascular endothelial growth factor (VEGF) was assessed using immunohistochemistry and in situ hybridization. Hypoxia was assessed using two 2-nitroimidazole markers, [(125)I]iodoazomycin arabinodise and 2-(2-nitro-1H-imidazol-l-yl)-N-(2,2,3,3,3,-pentafluoropropyl) acetamide (EF5), with binding in the rat spinal cord measured using gamma counting and immunohistochemistry, respectively. In the nonirradiated rat spinal cord, there was no evidence of BSCB disruption or VEGF expression. After 16-22 Gy, there was a dose-dependent increase in albumin staining and (99m)Tc-diethylenetriamine pentaacetic acid activity beginning at 16 weeks, consistent with barrier breakdown. A similar dose-dependent increase in white matter astrocytes that showed immunoreactivity and in situ hybridization signals for VEGF was observed. No increase in VEGF-positive cells was observed in gray matter. By 20 weeks after 20-22 Gy, animals developed white matter necrosis associated with diffuse albumin staining. Irradiated rat spinal cord showed a dose (16-22 Gy)- and time-dependent (16-20 weeks after 22 Gy) increase in [(125)I]iodoazomycin arabinodise accumulation compared to nonirradiated controls. A similar pattern of dose- and time-dependent EF5 immunoreactivity was also observed in white matter. Areas of EF5 expression and VEGF in situ signals colocalized with areas of albumin immunoreactivity. It is concluded that there is a dose-dependent temporal and spatial association of hypoxia, VEGF up-regulation, and radiation-induced BSCB dysfunction. Hypoxia may provide the signal for VEGF up-regulation and perpetuate endothelial permeability damage in the central nervous system after ionizing radiation.

Tumor hypoxia is an important prognostic factor for response to therapy. Radiolabeled 2-nitroimidazoles have been used for imaging hypoxia, and the octanol/water partition coefficient (P) of these compounds appears to play a crucial role in their suitability for imaging. A series of 11 2-nitroimidazoles coupled to peptidic chelators for (99m)Tc with divergent P was developed and evaluated in an in vitro system. Two classes of N(3)S chelators were used: dialkyl-Gly-Ser-Cys-linker-2-nitroimidazole (Class I) and dialkyl-Gly-Lys(2-nitroimidazole)-Cys (Class II). The chelators were prepared by automated solid-phase peptide synthesis. Xanthine oxidase was able to reduce the 2-nitroimidiazole moiety on the ligands, but the rate of reduction varied 5-fold among the different chelators. The chelators were labeled by transchelation from [(99m)Tc]gluconate at temperatures between 22 and 100 degrees C. The reaction mixtures were analyzed by HPLC and their P values determined. The accumulation of each complex in suspension cultures of Chinese hamster ovary cells incubated under aerobic or extremely hypoxic conditions was determined. Radiochemical yields ranged from 5 to 80% for the 11 compounds. HPLC showed that some of the compounds formed two complexes with (99m)Tc, possibly syn and anti conformations with respect to the Tc=O bond. In general, the Class I chelators labeled more readily than the class II chelators. The P values of the (99m)Tc complexes varied from 0.0002 to 5 and were generally in accordance with predictions based on structure. There were also differences in P as a function of pH; the free acids had a lower P at pH 7.4 than at pH 2.0 due to ionization, whereas the amides did not show this effect. Accumulation levels in aerobic cells were related to P but varied over a narrow range. Four of the 11 compounds showed selective accumulation in hypoxic cells. The peptidic class of 2-nitroimidazoles, with flexible design and convenient solid-phase synthesis, deserves further study as agents for imaging hypoxia in tumors.

The potential clinical use of technetium-99m labeled sestamibi (Tc-MIBI) and tetrofosmin (Tc-Tfos) to image tumours is currently being evaluated. In this study, the accumulation and efflux of Tc-MIBI and Tc-Tfos in the nasopharyngeal carcinoma cell line CNE-1 were examined in the presence or absence of various inhibitors of P-glycoprotein (PGP) and/or multidrug resistance associated protein (MRP) activity [GG918, PSC833, verapamil (Vrp), cyclosporin A (CsA) and buthionine sulfoximine (BSO)]. Reverse-transcriptase polymerase chain reaction analysis and immunodetection of the CNE-1 cells detected expression of MRP, MRP1 and MRP2 but not PGP. Tc-MIBI and Tc-Tfos accumulation was increased (P<0.0001) and efflux decreased (P<0.05) in the presence of BSO, CsA, Vrp and PSC833 but not GG918, which is a specific inhibitor of PGP. The absolute accumulation of Tc-MIBI was approximately twofold higher than that seen with Tc-Tfos, whereas the addition of inhibitors caused a much greater suppression of Tc-Tfos transport (>2 times greater than for Tc-MIBI). However, no qualitative differences in inhibitors were seen between Tc-MIBI and Tc-Tfos. These results suggest that both Tc-MIBI and Tc-Tfos are substrates for the MRP transporter and that PSC833, Vrp, CsA and BSO but not GG918 can inhibit MRP activity. These results indicate that Tc-MIBI and Tc-Tfos may be suitable imaging agents for detecting MRP-mediated drug resistance in human cancers.

A novel amine-dioxime chelator for Tc-99m has been developed. It offers the advantages of ease of synthesis and flexibility in alteration of lipophilicity. Labeling by stannous reduction of pertechnetate takes place rapidly and efficiently at room temperature and is stable for 24 h. The Tc-99m:ligand ratio is believed to be 1:2. Seven different alkyl moieties were used to achieve a range of lipophilicities. Three series of compounds were prepared: 2-nitroimidazoles as potential hypoxia-targeting agents, 4-nitroimidazoles as a less easily reduced isomer, and untargeted anilines. In an in vitro model of cellular hypoxia, the 2-nitroimidazole compounds all showed selective accumulation whereas 4-nitroimidazoles showed variable selectivity and aniline showed no selectivity. These experiments demonstrate the potential utility of the 2-nitroimidazole derivatives of the amine-dioxime class of chelator as hypoxia-targeting agents.

The presence of hypoxic cells in solid tumors is a marker for therapy-resistant, aggressive disease. The noninvasive detection of hypoxic cells in tumors by radiolabeled 2-nitroimidazoles is a diagnostic technique under current evaluation. Two peptidic agents, dimethylglycyl-L-seryl-L-cysteinyl-lysyl {N-epsilon-[1-(2-nitro-1H-imidazolyl)acetamido]}glycine (RP435) and dimethylglycyl-tert-butylglycyl-L-cysteinyl-glycine-[2-(2-nitro-1H-imidazolyl)ethyl]amide (RP535) have been synthesized. Both age nts contain an N3S class chelator for Tc-99m and Re and a 2-nitroimidazole group which can be enzymatically reduced and selectively trapped in cells under hypoxic conditions. Two isomers of (TcO)-Tc-99m-RP435, which are assumed to be syn and anti conformations, were observed on HPLC analysis. The interconversion of the two isomers in aqueous solution was investigated. In contrast, RP535 chelated Tc-99m to form a single isomer and no conversion to its counterpart has been observed on HPLC analysis. The tert-butyl group on the chelator may inhibit the formation and interconversion of the syn and anti isomers of (TcO)-Tc-99m-RP535. Both tracers showed a significant degree of hypoxia-specific accumulation in an in vitro assay, with (TcO)-Tc-99m-RP535 showing higher selectivity for hypoxic cells than (TcO)-Tc-99m-RP435. These results suggest that (TcO)-Tc-99m-RP535 represents a lead compound worthy of further investigation as an agent for imaging hypoxia in tumors.