Preclinical evaluation of a 64Cu-labeled disintegrin for PET imaging of prostate cancer.

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.

Size-Optimized Ultrasmall Porous Silica Nanoparticles Depict Vasculature-Based Differential Targeting in Triple Negative Breast Cancer.

Rapid sequestration and prolonged retention of intravenously injected nanoparticles by the liver and spleen (reticuloendothelial system (RES)) presents a major barrier to effective delivery to the target site and hampers clinical translation of nanomedicine. Inspired by biological macromolecular drugs, synthesis of ultrasmall (diameter ≈12-15 nm) porous silica nanoparticles (UPSNs), capable of prolonged plasma half-life, attenuated RES sequestration, and accelerated hepatobiliary clearance, is reported. The study further investigates the effect of tumor vascularization on uptake and retention of UPSNs in two mouse models of triple negative breast cancer with distinctly different microenvironments. A semimechanistic mathematical model is developed to gain mechanistic insights into the interactions between the UPSNs and the biological entities of interest, specifically the RES. Despite similar systemic pharmacokinetic profiles, UPSNs demonstrate strikingly different tumor responses in the two models. Histopathology confirms the differences in vasculature and stromal status of the two models, and corresponding differences in the microscopic distribution of UPSNs within the tumors. The studies demonstrate the successful application of multidisciplinary and complementary approaches, based on laboratory experimentation and mathematical modeling, to concurrently design optimized nanomaterials, and investigate their complex biological interactions, in order to drive innovation and translation.

Simultaneous Preclinical Positron Emission Tomography-Magnetic Resonance Imaging Study of Lymphatic Drainage of Chelator-Free 64Cu-Labeled Nanoparticles.

BACKGROUND: Hybrid positron emission tomography (PET)-magnetic resonance imaging (MRI) systems have been taken in use as new clinical diagnostic tools including detection and therapy planning of cancer. To reduce the amount of contrast agents injected in patients while fully benefitting both modalities, dual-modality probes are required. MATERIAL AND METHODS: This study was first aimed at developing a hybrid PET-MRI probe by labeling superparamagnetic iron oxide nanoparticles (SPIONs) with 64Cu using a fast and chelator-free conjugation method, and second, to demonstrate the ability of the agent to target sentinel lymph nodes (SLNs) in vivo using simultaneous PET-MRI imaging. RESULTS: High labeling efficiency of 97% produced within 10-15 min was demonstrated at room temperature. 64Cu-SPIONs were chemically stable in mouse serum for 24 h and after intradermal injection in the hind paw of C57BL/6J mice, demonstrated specific accumulation in the SLN. Simultaneous PET-MRI clearly demonstrated visualization of 64Cu-SPIONs, in dynamic and static imaging sequences up to 24 h after administration. CONCLUSION: The use of a single hybrid probe and simultaneous hybrid imaging provides an efficient, complementary integration of quantitation and is expected to improve preoperative planning and intraoperative guidance of cancer treatments.

CuS Nanodots with Ultrahigh Efficient Renal Clearance for Positron Emission Tomography Imaging and Image-Guided Photothermal Therapy.

Translation of nanoparticles (NPs) into clinical practice has been limited by toxic effects induced by nonspecific accumulation of NPs in healthy organs after systemic administration. The ideal NPs should accumulate in the target site, carry out their function, and then ultimately be eliminated from the body. Here, we show a single-compartment, multifunctional ultrasmall copper sulfide nanodot (CuS ND) that is rapidly cleared from the body. These CuS NDs have a hydrodynamic diameter of <6 nm, can efficiently absorb near-infrared light for photothermal ablation therapy, and stably incorporate the copper-64 radioisotope for noninvasive positron emission tomography (PET). Importantly, ∼95% of CuS NDs are excreted intact through the renal-urinary system within 24 h with minimal retention in the liver and the spleen. The ultrasmall CuS NDs accumulate in 4T1 tumors in Balb/c mice, as monitored by PET imaging, and mediate tumor ablation when combined with near-infrared light irradiation. As a first example of PET-visible, renal-clearable inorganic nanomaterials with peak absorption in the near-infrared region, CuS NDs represent a robust platform for cancer imaging and therapy.

Dose painting based on tumor uptake of Cu-ATSM and FDG: a comparative study.

BACKGROUND: Hypoxia and increased glycolytic activity of tumors are associated with poor prognosis. The purpose of this study was to investigate differences in radiotherapy (RT) dose painting based on the uptake of 2-deoxy-2-[(18) F]-fluorodeoxyglucose (FDG) and the proposed hypoxia tracer, copper(II)diacetyl-bis(N(4))-methylsemithiocarbazone (Cu-ATSM) using spontaneous clinical canine tumor models. METHODS: Positron emission tomography/computed tomography scans of five spontaneous canine sarcomas and carcinomas were obtained; FDG on day 1 and (64)Cu-ATSM on day 2 and 3 (approx. 3 and 24 hours pi.). Sub-volumes for dose escalation were defined by a threshold-based method for both tracers and five dose escalation levels were formed in each sub-volume. Volumetric modulated arc therapy plans were optimized based on the dose escalation regions for each scan for a total of three dose plans for each dog. The prescription dose for the GTV was 45 Gy (100%) and it was linearly escalated to a maximum of 150%. The correlations between dose painting plans were analyzed with construction of dose distribution density maps and quality volume histograms (QVH). Correlation between high-dose regions was investigated with Dice correlation coefficients. RESULTS: Comparison of dose plans revealed varying degree of correlation between cases. Some cases displayed a separation of high-dose regions in the comparison of FDG vs. (64)Cu-ATSM dose plans at both time points. Among the Dice correlation coefficients, the high dose regions showed the lowest degree of agreement, indicating potential benefit of using multiple tracers for dose painting. QVH analysis revealed that FDG-based dose painting plans adequately covered approximately 50% of the hypoxic regions. CONCLUSION: Radiotherapy plans optimized with the current approach for cut-off values and dose region definitions based on FDG, (64)Cu-ATSM 3 h and 24 h uptake in canine tumors had different localization of the regional dose escalation levels. This indicates that (64)Cu-ATSM at two different time-points and FDG provide different biological information that has to be taken into account when using the dose painting strategy in radiotherapy treatment planning.

Preclinical studies of [61Cu]ATSM as a PET radiopharmaceutical for fibrosarcoma imaging.

[61Cu]diacetyl-bis(N4-methylthiosemicarbazone) ([61Cu] ATSM) was prepared using in house-made diacetyl-bis(N4-methylthiosemicarbazone) (ATSM) ligand and [61Cu]CuCl2 produced via the natZn(p, x)61Cu (180 muA proton irradiation, 22 MeV, 3.2 h) and purified by a ion chromatography method. [61Cu]ATSM radiochemical purity was >98 %, as shown by HPLC and RTLC methods. [61Cu]ATSM was administered into normal and tumor bearing rodents for up to 210 minutes, followed by biodistribution and co-incidence imaging studies. Significant tumor/non-tumor accumulation was observed either by animal sacrification or imaging. [61Cu]ATSM is a positron emission tomography (PET) radiotracer for tumor hypoxia imaging.

Basic characterization of 64Cu-ATSM as a radiotherapy agent.

64Cu-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM) is a promising radiotherapy agent for the treatment of hypoxic tumors. In an attempt to elucidate the radiobiological basis of 64Cu-ATSM radiotherapy, we have investigated the cellular response patterns in vitro cell line models. Cells were incubated with 64Cu-ATSM, and the dose-response curves were obtained by performing a clonogenic survival assay. Radiation-induced damage in DNA was evaluated using the alkali comet assay and apoptotic cells were detected using Annexin V-FITC and propidium iodide staining methods. Washout rate and subcellular distribution of 64Cu in cells were investigated to further assess the effectiveness of 64Cu-ATSM therapy on a molecular basis. A direct comparison of subcellular localization of Cu-ATSM was made with the flow tracer analog Cu-pyruvladehyde-bis(N4-methylthiosemicarbazone). In this study, 64Cu-ATSM was shown to reduce the clonogenic survival rate of tumor cells in a dose-dependent manner. Under hypoxic conditions, cells took up 64Cu-ATSM and radioactive 64Cu was highly accumulated in the cells. In the 64Cu-ATSM-treated cells, DNA damage by the radiation emitted from 64Cu was detected, and inhibition of cell proliferation and induction of apoptosis was observed at 24 and 36 h after the treatment. The typical features of postmitotic apoptosis induced by radiation were observed following 64Cu-ATSM treatment. The majority of the 64Cu taken up into the cells remained in the postmitochondrial supernatant (the cellular residue after removal of the nuclei and mitochondria), which indicates that the beta- particle emitted from 64Cu may be as effective as the Auger electrons in 64Cu-ATSM therapy. These data allow us to postulate that 64Cu-ATSM will be able to attack the hypoxic tumor cells directly, as well as potentially affecting the peripheral nonhypoxic regions indirectly by the beta- particle decay of 64Cu.

Comparison of copper-67- and iodine-125-labeled anti-CEA monoclonal antibody biodistribution in patients with colorectal tumors.

UNLABELLED: Copper-67 has comparable beta-particle emissions to that of 131I, but it displays more favorable gamma emission characteristics for application in radioimmunotherapy (RIT). This study investigates the potential of 67Cu-labeled monoclonal antibody (MAb) 35 for RIT of colorectal carcinoma. METHODS: Biokinetics of simultaneously injected 67Cu- and 125I-labeled MAb35 were studied in six patients scheduled for surgery of primary colorectal cancer. RESULTS: Whole-body clearance (T 1/2) of 67Cu, estimated from sequential anterior and posterior whole-body scans and corrected for decay of 67Cu, was 41 hr. Serum clearance of 67Cu was faster (27.41 hr) than that of 125I (38.33 hr). Mean tumor uptake of the 67Cu-labeled compound (0.0133% ID/g) exceeded that of 125I (0.0095% ID/g), and tumor-to-blood ratios were higher for 67Cu than for 125I, with averages of 6.07 and 2.41, respectively. The average 67Cu/125I ratio was 1.9 for tumor uptake, 0.7 for blood and 2.6 for tumor-to-blood ratios. Nonspecific liver uptake of 67Cu as calculated from whole-body scans was high in four patients, up to 25% of residual whole-body activity at 48 hr, but did not increase with time. We also observed some nonspecific bowel activity, as well as moderate to high uptake in benign polyps. CONCLUSION: Copper-67-labeled MAb35 is more favorable than its radioiodine-labeled counterpart for RIT of colorectal carcinoma due to higher tumor-to-blood ratios, but the problem of nonspecific liver and bowel uptake must first be overcome. The absolute accumulation of activity in tumor remains low, however, so the probability of cure with this compound alone is questionable. The use of 67Cu as one component of a multimodality adjuvant treatment seems to remain the most appropriate application for RIT.

Copper-64 metabolism in two patients with non-Wilsonian movement disorders and copper deficiency.

Copper-64 studies are presented of 2 patients with non-Wilsonian movement disorder and with abnormal copper handling. Both patients differed from the usual phenotype of non-Wilsonian low copper movement disorder as they had choreiform movement disorders with an onset in the first decade; one patient lacked significant intellectual impairment. Both patients had reduced serum total copper and marginal free copper and caeruloplasmin levels, and both patients were capable of incorporating 64Cu2+ into caeruloplasmin but the second case did so at markedly reduced level. Both showed slightly increased basal and stimulated urinary copper loss compared to normal controls with the rate in patient 1 being capable of leading to copper depletion. Liver copper content was normal in both cases. These 2 patients add to the reports of cases with copper deficiency and movement disorder in whom copper chelation therapy is unlikely to be beneficial.

Distribution of 64Cu in Saccharomyces cerevisiae: kinetic analyses of partitioning.

The cell association of copper in the yeast Saccharomyces cerevisiae can involve both binding to the cell wall and the accumulation of copper within the cell. The former process requires the concurrent generation of H2S by the cell via the reduction of sulphate. The contributions of each of these processes to the uptake of 64Cu by wild type and met3-containing (ATP sulphurylase-deficient) strains have been kinetically dissected. The Michaelis constant for uptake (4 microM) is independent of the type of cell association which is occurring, suggesting, although not requiring, that both processes are associated with a common kinetic intermediate. The time dependence of the cell-association of 64Cu also suggests the presence of this intermediate pool of bound copper. The Vmax for uptake includes a constant contribution from accumulation of 64Cu within the plasmalemma [0.1 nmol min-1 (mg protein)-1] plus that fraction of the 64Cu within the intermediate pool which diffuses away and is trapped on the cell wall as a metal sulphide. This latter contribution to Vmax can be two- to three-times greater than the intracellular uptake depending on the amount and type of sulphur supplementation provided in the 64Cu2+ uptake buffer. Both processes are energy-dependent although the sulphide-dependent periplasmic accumulation is somewhat more sensitive to metabolic inhibition. This can be attributed to the ATP required for the activation of sulphate prior to its reduction to the level of sulphite and then sulphide. Periplasmic 64Cu accumulation is strongly inhibited by Zn2+ and Ni2+. This inhibition is due to competition for cell-generated sulphide; in the presence of 65Zn2+, the decrease in 64Cu bound is quantitatively related to the amount of 65Zn which becomes cell-associated. In contrast, intracellular 64Cu uptake is not inhibited by these two metals (at 50 microM) showing that the copper translocation pathway is metal-specific. These observations suggest a model for the way newly arrived copper is handled at the cell membrane and is partitioned for intracellular uptake.

Further characterization of the process of in vitro uptake of radiolabeled copper by the rat brain.

We have previously demonstrated that hypothalmic slices obtained from adult male rats accumulate 67Cu by two ligand-dependent, saturable processes: a high and low affinity process. To further establish the generality of these uptake processes, we defined the ligand requirements and the saturation kinetics of 67Cu uptake by tissue slices obtained from the newborn hypothalamus (HT); adult male hypothalamus, hippocampus, cortex, median eminence, and caudate nucleus; hypothalamus and hippocampus of castrated (14 days) males and of pregnant (19 days) and ovariectomized (14 days) females. It was found that ionic 67Cu2+ was poorly taken up by newborn HT and adult caudate, complexation with His enhanced 67Cu uptake 3-4-fold, and complexation with albumin inhibited 67Cu uptake. These ligand requirements are identical to those we have previously shown for the adult HT. When 67Cu uptake was evaluated under conditions optimal for the high or the low affinity process, for each process the dose response curves generated from these various tissues were very similar. In addition, we assessed the uptake of both components of the CuHis2 complex by incubating tissues with 67Cu3 H-His2 and found that the tissue ratio of 67Cu:3H was a sigmoidal function of the concentration of the Cu complex such that at greater than 5 microM, the ratio was about 3-fold greater than the medium ratio; indicating preferential uptake of 67Cu relative to 3H-His. The changes in isotope ratios were observed in newborn HT and adult HT, as well as caudate. These similarities in the ligand requirements and saturation kinetics of 67Cu uptake establish the generality of these two processes of in vitro uptake of copper in the rat brain.