Amplified delivery of indium-111 to EGFR-positive human breast cancer cells.

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 x 10(6) 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.

Hypoxia in radiation-induced blood-spinal cord barrier breakdown.

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.

A novel amine-dioxime chelator for technetium-99m: synthesis and evaluation of 2-nitroimidazole-containing analogues as markers for hypoxic cells.

A novel amine-dioxime chelator for (99m)Tc 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 (99m)Tc: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.

Targeting hypoxia in tumors using 2-nitroimidazoles with peptidic chelators for technetium-99m: effect of lipophilicity.

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.

Comparison of the accumulation and efflux kinetics of technetium-99m sestamibi and technetium-99m tetrofosmin in an MRP-expressing tumour cell line.

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, MRPI 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.

Synthesis and evaluation of two technetium-99m-labeled peptidic 2-nitroimidazoles for imaging hypoxia.

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 -im idazolyl)acetamido]}glycine (RP435) and dimethylglycyl-tert-butylglycyl-L-cysteinyl-glycine-[2-(2-ni tro-1H-im idazolyl)ethyl]amide (RP535) have been synthesized. Both agents contain an N(3)S class chelator for (99m)Tc and Re and a 2-nitroimidazole group which can be enzymatically reduced and selectively trapped in cells under hypoxic conditions. Two isomers of (99m)TcO-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 (99m)Tc 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 (99m)TcO-RP535. Both tracers showed a significant degree of hypoxia-specific accumulation in an in vitro assay, with (99m)TcO-RP535 showing higher selectivity for hypoxic cells than (99m)TcO-RP435. These results suggest that (99m)TcO-RP535 represents a lead compound worthy of further investigation as an agent for imaging hypoxia in tumors.

Synthesis, characterization and biodistribution of the cationic technetium(II) 1,10-phenanthroline chelate.

[99Tc(phen)3]Cl2 was synthesized by reduction of pertechnetate with sodium borohydride in the presence of an excess of 1,10-phenanthroline and characterized by FAB mass spectrometry, magnetic susceptibility measurements, and i.r./vis/u.v. spectrophotometry. For biodistribution evaluation in mice [99mTc(phen)3]Cl2 could be obtained in a radiochemical purity of 92.5% using a similar procedure. The rather hydrophilic chelate was predominantly excreted by the kidneys. Its low blood retention resulted in a heart/blood activity ratio of 4.2 at 100 min post-injection.