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Resistin has been originally identified as an adipokine that links obesity to insulin resistance in mice. In our previous studies in spontaneously hypertensive rats (SHR) expressing a nonsecreted form of mouse resistin ( Retn) transgene specifically in adipose tissue (SHR- Retn), we have observed an increased lipolysis and serum free fatty acids, ectopic fat accumulation in muscles, and insulin resistance. Recently, brown adipose tissue (BAT) has been suggested to play an important role in the pathogenesis of metabolic disturbances. In the current study, we have analyzed autocrine effects of transgenic resistin on BAT glucose and lipid metabolism and mitochondrial function in the SHR- Retn vs. nontransgenic SHR controls. We observed that interscapular BAT isolated from SHR- Retn transgenic rats compared with SHR controls showed a lower relative weight (0.71 ± 0.05 vs. 0.91 ± 0.08 g/100 g body wt, P < 0.05), significantly reduced both basal and insulin stimulated incorporation of palmitate into BAT lipids (658 ± 50 vs. 856 ± 45 and 864 ± 47 vs. 1,086 ± 35 nmol/g/2 h, P ≤ 0.01, respectively), and significantly decreased palmitate oxidation (37.6 ± 4.5 vs. 57 ± 4.1 nmol/g/2 h, P = 0.007) and glucose oxidation (277 ± 34 vs. 458 ± 38 nmol/g/2 h, P = 0.001). In addition, in vivo microPET imaging revealed significantly reduced 18F-FDG uptake in BAT induced by exposure to cold in SHR- Retn vs. control SHR (232 ± 19 vs. 334 ± 22 kBq/ml, P < 0.05). Gene expression profiles in BAT identified differentially expressed genes involved in skeletal muscle and connective tissue development, inflammation and MAPK and insulin signaling. These results provide evidence that autocrine effects of resistin attenuate differentiation and activity of BAT and thus may play a role in the pathogenesis of insulin resistance in the rat.

Wilson's disease is a genetic disorder that causes excessive accumulation of copper in the body, leading to toxic damage, especially in the liver and nervous system. The current treatment cause burdensome side effects. We describe the use of chemically modified biopolymer carriers based on microcrystalline cellulose and chitosan containing the highly specific copper chelator 8-hydroxyquinoline as a new type of therapy for Wilson's disease. The chelators can scavenges copper ions released from food during digestion and copper ions present in secretions in the gastrointestinal tract. Because the chelator is covalently bound to indigestible biopolymer carriers (crosslinked chitosan or modified cellulose), it is not taken up by the gastrointestinal tract and it can be eliminated through the feces, avoiding unwanted side effects. This concept was tested on Wistar rats, which received a radioactive 64CuCl2 solution together with the polymers with covalently bound 8-hydroxyquinoline through a gastric probe. 64Copper complex uptake from the gastrointestinal tract was significantly inhibited by both chelating polymers. With the modified polymers, the presence of 64Cu was detected mostly in the gastrointestinal tract, not in the internal organs. These findings indicate modified cellulose and crosslinked chitosan, with covalently bound 8-hydroxyquinoline exhibited the potential to be excellent therapeutics for treating Wilson's disease.

The purpose of this study was to investigate whether (44)Sc-labeled puromycin can be utilized for imaging of protein synthesis in vivo.For micro-positron emission tomographic (μPET) studies, 20-25 MBq of [(44)Sc]-DOTA-puromycin was administered to tumor-bearing rats, and animals were scanned for 1 h dynamically. Results were further validated by dissecting organs and tissues of the animals after the measurement and in vitro blocking experiments using puromycin or cycloheximide to block protein synthesis.μPET images of tumor-bearing rats showed significant tumor uptake of [(44)Sc]-DOTA-puromycin and a clear-cut tumor visualization. In both blocking experiments, cellular uptake of [(44)Sc]-DOTA-puromycin ([(44)Sc]-DOTA-Pur) could be suppressed by blocking protein synthesis.We report for the first time successful μPET imaging with (44)Sc obtained from a (44)Ti/(44)Sc generator, as well as noninvasive μPET imaging of ribosomal activity, respectively protein synthesis, with a puromycin-based radiopharmaceutical and the direct correlation between cellular uptake of [(44)Sc]-DOTA-Pur and protein synthesis.

Objectives Nimotuzumab (h-R3) is a humanized monoclonal antibody (mAb) which recognizes the external domain of the epidermal growth factor receptor (EGFR) with high specificity. It was demonstrated that h-R3 has a unique clinical profile for immunotherapy of adult gliomas and pediatric pontine gliomas. The aim of this work was to evaluate the conjugate 177 Lu-h-R3 as a potential radioimmunoconjugate for radioimmunotherapy (RIT) of tumors overexpressing EGFR. Methods h-R3 was modified with the macrocylcic ligand S -2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid ( p -SCN-Bn-DOTA) and the acyclic ligand S -2-(4-Isothiocyanatobenzyl)-diethylenetriamine pentaacetic acid ( p -SCN-Bn-DTPA); the immunoconjugates were labeled with no-carried added 177 Lu. Specificity and affinity were tested using radioimmunoassays in a cell line overexpressing EGFR. Biodistribution in mice, healthy or bearing A431 epithelial carcinoma xenografts, was performed for 11 days. Tumor uptake, the influence of the nature of the chelate and the way of administration were studied. Absorbed dose in tumor and selected organs was calculated using the OLINDA/EXM software; the data from the animals was extrapolated to humans. Results 177 Lu-h-R3 conjugates were obtained with specific activity up to 915 MBq/mg without significant loss of immunoreactivity. The binding of 177 Lu-h-R3 conjugates to A431 cells showed to be EGFR specific, and the affinity was similar to native h-R3. Tumor uptake reached a maximum value of 22.4±3.1 %ID/g at 72 h and remained ∼20% ID/g over 1 week. Locoregional application showed better tumor/nontumor ratios than intravenous application. Conclusions 177 Lu-h-R3 should be considered for further evaluations as a potential radiopharmaceutical for RIT of tumors overexpressing EGFR.

The humanized monoclonal antibody Nimotuzumab (h-R3) has demonstrated an exceptional and better clinical profile than other monoclonal antibodies for immunotherapy of epidermal growth factor receptor-overexpressing tumors. This work deals with the preparation and radiolabeling optimization of (177)Lu-Nimotuzumab and their preclinical evaluation.Nimotuzumab was conjugated with S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA), testing different molar ratios. The immunoconjugates were characterized. The radiolabeling with (177)Lu was optimized. Radioimmunoconjugates stability was tested in 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA) excess and human serum. In vitro studies were performed in tumor model cell lines. Receptor-specific binding was tested by competitive inhibition. (177)Lu-Nimotuzumab in vivo studies were conducted in healthy and xenograft animals.Nimotuzumab conjugates were obtained with high purity. Radiolabeling yield and specific activities ranged from 63.6% to 94.5% and from 748 to 1142 MBq/mg, respectively. The stability in DTPA excess and human serum was 95.9% and 93.2% after 10 days, respectively. The radioimmunoconjugate showed specific receptor binding in tumor cell lines. Biodistribution in healthy animals showed the typical behavior of the immunoconjugates based on monoclonal antibodies. The study in xenografts mice demonstrated uptake of (177)Lu-Nimotuzumab in the tumor and reticuloendothelial organs.(177)Lu-Nimotuzumab was obtained with high purity and specific activities under optimal conditions without significant loss in immunoreactivity and might be a potential radioimmunoconjugate for radioimmunotherapy of tumors with epidermal growth factor receptor overexpression.

Puromycin has played an important role in our understanding of the eukaryotic ribosome and protein synthesis. It has been known for more than 40 years that this antibiotic is a universal protein synthesis inhibitor that acts as a structural analog of an aminoacyl-transfer RNA (aa-tRNA) in eukaryotic ribosomes. Due to the role of enzymes and their synthesis in situations of need (DNA damage, e.g., after chemo- or radiation therapy), determination of protein synthesis is important for control of antitumor therapy, to enhance long-term survival of tumor patients, and to minimize side-effects of therapy. Multiple attempts to reach this goal have been made through the last decades, mostly using radiolabeled amino acids, with limited or unsatisfactory success. The aim of this study is to estimate the possibility of determining protein synthesis ratios by using (68)Ga-DOTA-puromycin ((68)Ga-DOTA-Pur), [(3)H]tyrosine, and 2-fluoro-[(3)H]tyrosine and to estimate the possibility of different pathways due to the fluorination of tyrosine.DOTA-puromycin was synthesized using a puromycin-tethered controlled-pore glass (CPG) support by the usual protocol for automated DNA and RNA synthesis following our design. (68)Ga was obtained from a (68)Ge/(68)Ga generator as described previously by Zhernosekov et al. (J Nucl Med 48:1741-1748, 2007). The purified eluate was used for labeling of DOTA-puromycin at 95°C for 20 min. [(3)H]Tyrosine and 2-fluoro-[(3)H]tyrosine of the highest purity available were purchased from Moravek (Bera, USA) or Amersham Biosciences (Hammersmith, UK). In vitro uptake and protein incorporation as well as in vitro inhibition experiments using cycloheximide to inhibit protein synthesis were carried out for all three substances in DU145 prostate carcinoma cells (ATCC, USA). (68)Ga-DOTA-Pur was additionally used for μPET imaging of Walker carcinomas and AT1 tumors in rats. Dynamic scans were performed for 45 min after IV application (tail vein) of 20-25 MBq (68)Ga-DOTA-Pur.No significant differences in the behavior of [(3)H]tyrosine and 2-fluoro-[(3)H]tyrosine were observed. Uptake of both tyrosine derivatives was decreased by inhibition of protein synthesis, but only to a level of 45-55% of initial uptake, indicating no direct link between tyrosine uptake and protein synthesis. In contrast, (68)Ga-DOTA-Pur uptake was directly linked to ribosomal activity and, therefore, to protein synthesis. (68)Ga-DOTA-Pur μPET imaging in rats revealed high tumor-to-background ratios and clearly defined regions of interest in the investigated tumors.Whereas the metabolic pathway of (68)Ga-DOTA-Pur is directly connected with the process of protein synthesis and shows high tumor uptake during μPET imaging, neither [(3)H]tyrosine nor 2-fluoro-[(3)H]tyrosine can be considered useful for determination of protein synthesis.

The epidermal growth factor receptor (EGFR) is a rational target of anticancer therapies due to its overexpression in a variety of malignant epithelial tumors. Nevertheless, this antigen is also present in normal tissues. Consequently, monoclonal antibodies which selectively bind to EGFR-overexpressing tumors will be choice drug candidates for development of radioimmunoconjugates (RIC). Nimotuzumab (h-R3) and trastuzumab are monoclonal antibodies (mAbs) which would preferentially target tissues with EGFR and HER2 overexpression, respectively. In this chapter, we describe preparation and evaluation of the targeting properties of RIC formed by (177)Lu/(90)Y and monoclonal antibodies which selectively target EGFR- and HER2/c-neu-overexpressing tissues. mAbs were labeled with n.c.a. (177)Lu/(90)Y using bifunctional chelating agents. RIC binding properties and toxicity were evaluated in vitro using cell lines with varying antigen expression. In vivo tumor targeting properties of RIC were evaluated in mice bearing colorectal (SNU-C2B) and A431 tumor xenografts. RICs were prepared with specific activities up to 2 GBq/mg without significant loss in biological activity. (90)Y-h-R3/trastuzumab increased cell growth inhibition compared with unmodified mAbs or (90)YCl(3) alone in cell lines with overexpression of the target antigen. (177)Lu-h-R3 showed significantly higher uptake in A431 (22.8 ± 3.1% ID/g) than in SNU-C2B (8.8 ± 4.1% ID/g) xenografts at 72 h post injection, indicating strong association between tumor uptake and EGFR expression levels.

The epidermal growth factor receptor (EGFR) is a rational target of anticancer therapies due to its overexpression in a variety of malignant epithelial tumors. Nevertheless, this antigen is also present in normal tissues. Consequently, monoclonal antibodies which selectively bind to EGFR-overexpressing tumors will be choice drug candidates for development of radioimmunoconjugates (RIC). Nimotuzumab (h-R3) and trastuzumab are monoclonal antibodies (mAbs) which would preferentially target tissues with EGFR and HER2 overexpression, respectively. In this chapter, we describe preparation and evaluation of the targeting properties of RIC formed by 177Lu/90Y and monoclonal antibodies which selectively target EGFR- and HER2/c-neu-overexpressing tissues. mAbs were labeled with n.c.a. 177Lu/90Y using bifunctional chelating agents. RIC binding properties and toxicity were evaluated in vitro using cell lines with varying antigen expression. In vivo tumor targeting properties of RIC were evaluated in mice bearing colorectal (SNU-C2B) and A431 tumor xenografts. RICs were prepared with specific activities up to 2 GBq/mg without significant loss in biological activity. 90Y-h-R3/trastuzumab increased cell growth inhibition compared with unmodified mAbs or 90YCl3 alone in cell lines with overexpression of the target antigen. 177Lu-h-R3 showed significantly higher uptake in A431 (22.8 ± 3.1% ID/g) than in SNU-C2B (8.8 ± 4.1% ID/g) xenografts at 72 h post injection, indicating strong association between tumor uptake and EGFR expression levels.

Cold exposure of rats leads to ameliorated glucose and triglyceride utilization with females displaying better adaptation to a cold environment. In the current study, we used hairless rats as a model of increased thermogenesis and analyzed gender-related effects on parameters of lipid and glucose metabolism in the spontaneously hypertensive (SHR) rats. Specifically, we compared hairless coisogenic SHR-Dsg4 males and females harboring mutant Dsg4 (desmoglein 4) gene versus their SHR wild type controls. Two way ANOVA showed significant Dsg4 genotype (hairless or wild type) x gender interaction effects on palmitate oxidation in brown adipose tissue (BAT), glucose incorporation into BAT determined by microPET, and glucose oxidation in skeletal muscles. In addition, we observed significant interaction effects on sensitivity of muscle tissue to insulin action when Dsg4 genotype affected these metabolic traits in males, but had little or no effects in females. Both wild type and hairless females and hairless males showed increased glucose incorporation and palmitate oxidation in BAT and higher tissue insulin sensitivity when compared to wild type males. These findings provide evidence for gender-related differences in metabolic adaptation required for increased thermogenesis. They are consistent with the hypothesis that increased glucose and palmitate utilization in BAT and muscle is associated with higher sensitivity of adipose and muscle tissues to insulin action.