Recently, the somatostatin receptor subtype 2 (SSTR2) selective antagonist sst 2 -ANT was determined to have a high affinity for SSTR2. Additionally, 111 In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-sst 2 -ANT showed high uptake in an SSTR2-transfected, tumor-bearing mouse model and suggested that radiolabeled SSTR2 antagonists may be superior to agonists for imaging SSTR2-positive tumors. This report describes the synthesis and evaluation of 64 Cu-CB-4,11-bis(carboxymethyl)-1, 4,8,11-tetraazabicyclo[6.6.2]hexadecane-sst 2 -ANT ( 64 Cu-CB-TE2A-sst 2 -ANT) as a PET radiopharmaceutical for the in vivo imaging of SSTR2-positive tumors. Methods: Receptor-binding studies were performed to determine the dissociation constant of the radiopharmaceutical 64 Cu-CB-TE2A-sst 2 -ANT using AR42J rat pancreatic tumor cell membranes. The internalization of 64 Cu-CB-TE2A-sst 2 -ANT was compared with that of the 64 Cu-labeled agonist 64 Cu-CB-TE2A-tyrosine 3 -octreotate ( 64 Cu-CB-TE2A-Y3-TATE) in AR42J cells. Both radiopharmaceuticals were also compared in vivo through biodistribution studies using healthy rats bearing AR42J tumors, and smallanimal PET/CT of 64 Cu-CB-TE2A-sst 2 -ANT was performed. Results: The dissociation constant value for the radiopharmaceutical was determined to be 26 6 2.4 nM, and the maximum number of binding sites was 23,000 fmol/mg. 64 Cu-CB-TE2A-sst 2 -ANT showed significantly less internalization than did 64 Cu-CB-TE2A-Y3-TATE at time points from 15 min to 4 h. Biodistribution studies revealed that the clearance of 64 Cu-CB-TE2A-sst 2 -ANT from the blood was rapid, whereas the clearance of 64 Cu-CB-TE2A-sst 2 -ANT from the liver and kidneys was more modest at all time points. Tumor-to-blood and tumor-to-muscle ratios were determined to be better for 64 Cu-CB-TE2A-sst 2 -ANT than those for 64 Cu-CB-TE2A-Y3-TATE at the later time points, although liver and kidney uptake was significantly higher. Small-animal imaging using 64 Cu-CB-TE2A-sst 2 -ANT revealed excellent tumor-to-background contrast at 4 h after injection, and standardized uptake values remained high even after 24 h. Conclusion: The PET radiopharmaceutical 64 Cu-CB-TE2A-sst 2 -ANT is an attractive agent, worthy of future study as a PET radiopharmaceutical for the imaging of somatostatin receptorpositive tumors.

Bombesin is a 14–amino-acid amphibian peptide that binds with high affinity to the gastrin-releasing peptide receptor (GRPR), which is overexpressed on a variety of solid tumors. It has been demonstrated that bombesin analogs can be radiolabeled with a variety of radiometals for potential diagnosis and treatment of GRPR-positive tumors. In this regard, several studies have used different chelators conjugated to the 8 C-terminal amino acids of bombesin(7–14) for radiolabeling with 64Cu. These analogs have demonstrated GRPR-specific small-animal PET of tumors but have various advantages and disadvantages. The objective of this study was to conjugate the previously described (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine) (SarAr) chelator to bombesin (7–14), radiolabel the conjugate with 64Cu, and evaluate in vitro and in vivo. Methods SarAr was synthesized as previously published and conjugated to bombesin(7–14) by solid-phase peptide synthesis using standard Fmoc chemistry. Succinic acid (SA), 8-aminooctanoic acid (Aoc), and Gly-Ser-Gly (GSG) were used as linkers between SarAr and bombesin(7–14) to yield the resulting SarAr-SA-Aoc-bombesin(7–14) and SarAr-SA-Aoc-GSG-bombesin(7–14) peptides. The unlabeled peptides were evaluated in a competitive binding assay using PC-3 prostate cancer cells and 125I-Tyr4-bombesin to determine the inhibitory concentration of 50%. The peptides were radiolabeled with 64Cu and evaluated for internalization into PC-3 cells in vitro and for in vivo tumor uptake in mice bearing PC-3 xenografts using biodistribution and small-animal PET/CT studies. Results The competitive binding assay demonstrated that both SarAr-SA-Aoc-bombesin(7–14) and SarAr-SA-Aoc-GSG-bombesin(7–14) bound with high affinity to GRPR with an inhibitory concentration of 50% of 3.5 and 4.5 nM, respectively. Both peptides were radiolabeled with 64Cu at room temperature without further purification and demonstrated similar internalization into PC-3 cells. In vivo, the radiolabeled peptides demonstrated tumor-specific uptake (13.0 and 8.5 percentage injected dose per gram for 64Cu-SarAr-SA-Aoc-bombesin(7–14) and 64Cu-SarAr-SA-Aoc-GSG-bombesin(7–14), respectively, at 1 h) and imaging that was comparable to, or better than, that of the previously reported 64Cu-labeled bombesin analogs. The 64Cu-SarAr-SA-Aoc-GSG-bombesin(7–14) had more rapid blood clearance and lower tumor and normal-tissue uptake than 64Cu-SarAr-SA-Aoc-bombesin(7–14), resulting in similar tumor-to-blood ratios for each analog (15.1 vs. 11.3 for 64Cu-SarAr-SA-Aoc-bombesin(7–14) and 64Cu-SarAr-SA-Aoc-GSG-bombesin(7–14), respectively, at 1 h). Conclusion These studies demonstrate that 64Cu-SarAr-SA-Aoc-bombesin(7–14) and 64Cu-SarAr-SA-Aoc-GSG-bombesin(7–14) bound with high affinity to GRPR-expressing cells and that these peptides can be used for PET of GRPR-expressing prostate cancer.

The epidermal growth-factor receptor (EGFR) and its ligands have been recognized as critical factors in the pathophysiology of tumorigenesis. Overexpression of the EGFR plays a significant role in the tumor progression of a wide variety of solid human cancers. Therefore, the EGFR represents an attractive target for the design of novel diagnostic and therapeutic agents for cancer. Cetuximab (C225, Erbitux) was the first monoclonal antibody targeted against the ligand-binding site of EGFR approved by the Food and Drug Administration for the treatment of patients with EGFR-expressing, metastatic colorectal carcinoma, although clinical trials showed variability in the response to this treatment. The aim of this study involved using cetuximab to design a positron emission tomography (PET) agent to image the overexpression of EGFR in tumors. Cetuximab was conjugated with the chelator, DOTA, for radiolabeling with the positron-emitter, 64Cu (T(1/2) = 12.7 hours). 64Cu-DOTA-cetuximab showed high binding affinity to EGFR-positive A431 cells (K(D) of 0.28 nM). Both biodistribution and microPET imaging studies with 64Cu-DOTA-cetuximab demonstrated greater uptake at 24 hours postinjection in EGFR-positive A431 tumors (18.49% +/- 6.50% injected dose per gram [ID/g]), compared to EGFR-negative MDA-MB-435 tumors (2.60% +/- 0.35% ID/g). A431 tumor uptake at 24 hours was blocked with unlabeled cetuximab (10.69% +/- 2.72% ID/g), suggesting that the tumor uptake was receptor mediated. Metabolism experiments in vivo showed that 64Cu-DOTA-cetuximab was relatively stable in the blood of tumor-bearing mice; however, there was significant metabolism in the liver and tumors. 64Cu-DOTA-cetuximab is a potential agent for imaging EGFR-positive tumors in humans.

Purpose The goal of this study was to determine the specificity of 64Cu-CB-TE2A-c(RGDyK) (64Cu-RGD) for osteoclast-related diseases, such as Paget's disease or rheumatoid arthritis. Procedures C57BL/6 mice were treated systemically with osteoprotegerin (OPG) for 15 days or RANKL for 11 days to suppress and stimulate osteoclastogenesis, respectively. The mice were then imaged by positron emission tomography/computed tomography using 64Cu-RGD, followed by determination of serum TRAP5b and bone histology. Standard uptake values were determined to quantify 64Cu-RGD in bones and other tissues. Results Mice treated with OPG showed decreased bone uptake of 64Cu-RGD at 1, 2, and 24 h post-injection of the tracer (p<0.01 for all time points) compared to vehicle controls, which correlated with a post-treatment decrease in serum TRAP5b. In contrast, mice treated with RANKL showed significantly increased bone uptake at 2 h post-injection of 64Cu-RGD (p<0.05) compared to the vehicle control group, corresponding to increased serum TRAP5b and OC numbers as determined by bone histology. Conclusions These data demonstrate that 64Cu-RGD localizes to areas in bone with increased osteoclast numbers and support the use of 64Cu-RGD as an imaging biomarker for osteoclast number that could be used to monitor osteoclast-related pathologies and their treatments.

The effect of maturation on the morphological and photosynthetic characteristics, as well as the expression of two genes involved in photosynthesis in the developing, current year foliage of Eastern larch (Larix laricina [Du Roi]) is described. These effects were observed on foliage during the third growing season after grafting of scions from trees of different ages onto 2 year old rootstock. Specific leaf weight (gram dry weight per square meter), leaf cross-sectional area (per square millimeter), and chlorophyll content (milligram per gram dry weight) all increase with increasing age in long shoot foliage from both indoor-and outdoor-grown trees. Net photosynthesis (NPS) (mole of CO2 per square millimeter per second) increases with age on indoor-but not outdoor-grown trees. NPS also increases with increased chlorophyll content, but outdoor-grown scions of all ages had higher chlorophyll content, and chlorophyll does not appear to be limiting for NPS outdoors. To extend these studies of maturationrelated differences in foliar morphology and physiology to the molecular genetic level, sequences were cloned from the cab and rbcS gene families of larch. Both cab and rbcS gene families are expressed in foliage but not in roots, and they are expressed in light-grown seedlings of larch but only at very low levels in dark-grown seedlings (-2% of light-grown seedlings). Steadystate cab mRNA levels are relatively higher (-40%) in newly expanding short shoot foliage from juvenile plants compared to mature plants. Unlike cab, the expression of the rbcS gene family did not seem to vary with age. These data show that the maturation-related changes in morphological and physiological phenotypes are associated with changes in gene expression.