Previously published reports indicate that serum copper levels are elevated in prostate cancer (PCa) patients and that increased copper uptake can be used as a means to image prostate tumors. It is unclear, however, to what extent copper is required for PCa cell function as we observed only modest effects of chelation strategies on the growth of these cells in vitro. With the goal of exploiting PCa cell proclivity for copper uptake, we developed a “conditional lethal” screen to identify compounds whose cytotoxic actions were manifest in a copper-dependent manner. Emerging from this screen were a series of dithiocarbamates, which when complexed with copper, induced Reactive Oxygen Species (ROS)-dependent apoptosis of malignant, but not normal, prostate cells. One of the dithiocarbamates identified, Disulfiram (DSF), is an FDA approved drug that has previously yielded disappointing results in clinical trials in patients with recurrent prostate cancer. Similarly, in our studies DSF alone had a minimal effect on the growth of PCa tumors when propagated as xenografts. However, when DSF was coadministered with copper a very dramatic inhibition of tumor growth in models of hormone sensitive and of castrate resistant disease was observed. Furthermore, we determined that prostate cancer (PCa) cells express high levels of CTR1, the primary copper transporter, and additional chaperones that are required to maintain intracellular copper homeostasis. The expression levels of most of these proteins are increased further upon treatment of AR-positive PCa cell lines with androgens. Not surprisingly robust CTR1-dependent uptake of copper into PCa cells was observed; an activity that was accentuated by activation of androgen receptor (AR). Given these data linking AR to intracellular copper uptake, we believe that dithiocarbamate/copper complexes are likely to be effective for the treatment of PCa patients whose disease is resistant to classical androgen ablation therapies.

A wide variety of imaging approaches have been developed in the past few decades for monitoring tumor oxygenation and hypoxia in vivo. In particular, nuclear medicine has seen the development of several radiolabeled hypoxia markers and is the preferred method for imaging of tumor hypoxia. Hypoxia imaging is increasingly being used in the clinical setting and is progressing from a mere detection method to application in individualization of chemoradiotherapy.

[(99m)Tc]-5 and its rhenium congener were synthesized and characterized. In vitro studies showed that the rhenium compound has a high affinity for CA IX and effectively inhibits CA IX activity. In vivo studies revealed a limited tracer accumulation in a CA IX expressing tumor but with increasing tumor-to-blood activity ratios as a function of time.

In a previous study, we evaluated a HER2-specific single domain antibody fragment (sdAb) 2Rs15d labeled with 18F via conjugation of a residualizing prosthetic agent that was synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC). In order to potentially increase overall efficiency and decrease the time required for labeling, we now investigate the use of a strain-promoted azide-alkyne cycloaddition (SPAAC) between the 2Rs15d sdAb, which had been pre-derivatized with an azide-containing residualizing moiety, and an 18F-labeled aza-dibenzocyclooctyne derivative. The HER2-targeted sdAb 2Rs15d and a nonspecific sdAb R3B23 were pre-conjugated with a moiety containing both azide- and guanidine functionalities. The thus derivatized sdAbs were radiolabeled with 18F using an 18F-labeled aza-dibenzocyclooctyne derivative ([18F]F-ADIBO) via SPAAC, generating the desired conjugate ([18F]RL-II-sdAb). For comparison, unmodified 2Rs15d was labeled with N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB), the prototypical residualizing agent for radioiodination. Radiochemical purity (RCP), immunoreactive fraction (IRF), HER2-binding affinity and cellular uptake of [18F]RL-II-2Rs15d were assessed in vitro. Paired label biodistribution of [18F]RL-II-2Rs15d and [125I]SGMIB-2Rs15d, and micro-PET/CT imaging of [18F]RL-II-2Rs15d and the [18F]RL-II-R3B23 control sdAb were performed in nude mice bearing HER2-expressing SKOV-3 xenografts. A radiochemical yield of 23.9 ± 6.9% (n=8) was achieved for the SPAAC reaction between [18F]F-ADIBO and azide-modified 2Rs15d and the RCP of the labeled sdAb was >95%. The affinity (Kd) and IRF for the binding of [18F]RL-II-2Rs15d to HER2 were 5.6 ± 1.2 nM and 73.1 ± 22.5 % (n=3), respectively. The specific uptake of [18F]RL-II-2Rs15d by HER2-expressing BT474M1 breast carcinoma cells in vitro was 14–17% of the input dose at 1, 2, and 4 h, slightly higher than seen for co-incubated [125I]SGMIB-2Rs15d. The uptake of [18F]RL-II-2Rs15d in SKOV-3 xenografts at 1 h and 2 h p.i. were 5.54 ± 0.77% ID/g and 6.42 ± 1.70% ID/g, respectively, slightly higher than those for co-administered [125I]SGMIB-2Rs15d (4.80 ± 0.78% ID/g and 4.78 ± 1.39% ID/g). Micro-PET/CT imaging with [18F]RL-II-2Rs15d at 1–3 h p.i. clearly delineated SKOV-3 tumors while no significant accumulation of activity in tumor was seen for [18F]RL-II-R3B23. With the exception of kidneys, normal tissue levels for [18F]RL-II-2Rs15d were low and cleared rapidly. To our knowledge, this is the first time SPAAC method has been used to label an sdAb with 18F, especially with residualizing functionality.

Serum glutamate/glutamine increased and correlated with its hepatic ratio and fibrosis severity in nonalcoholic steatohepatitis fibrosis. These changes reflect increased glutaminolysis in myofibroblasts and are shown by noninvasive positron emission tomography. Increased glutaminolysis is a potential diagnostic marker and therapeutic target during nonalcoholic steatohepatitis fibrosis progression. BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) occurs in the context of aberrant metabolism. Glutaminolysis is required for metabolic reprograming of hepatic stellate cells (HSCs) and liver fibrogenesis in mice. However, it is unclear how changes in HSC glutamine metabolism contribute to net changes in hepatic glutaminolytic activity during fibrosis progression, or whether this could be used to track fibrogenic activity in NASH. We postulated that increased HSC glutaminolysis marks active scarring in NASH. METHODS: Glutaminolysis was assessed in mouse NASH fibrosis models and in NASH patients. Serum and liver levels of glutamine and glutamate and hepatic expression of glutamine transporter/metabolic enzymes were correlated with each other and with fibrosis severity. Glutaminolysis was disrupted in HSCs to examine if this directly influenced fibrogenesis. 18 Ffluoroglutamine positron emission tomography was used to determine how liver glutamine assimilation tracked with hepatic fibrogenic activity in situ. RESULTS: The serum glutamate/glutamine ratio increased and correlated with its hepatic ratio, myofibroblast content, and fibrosis severity. Healthy livers almost exclusively expressed liver-type glutaminase (Gls2); Gls2 protein localized in zone 1 hepatocytes, whereas glutamine synthase was restricted to zone 3 hepatocytes. In fibrotic livers, Gls2 levels reduced and glutamine synthase zonality was lost, but both Slc1a5 (glutamine transporter) and kidney-type Gls1 were up-regulated; Gls1 protein was restricted to stromal cells and accumulated in fibrotic septa. Hepatocytes did not compensate for decreased Gls2 by inducing Gls1. Limiting glutamine or directly inhibiting GLS1 inhibited growth and fibrogenic activity in cultured human HSCs. Compared with healthy livers, fibrotic livers were 18 F-fluoroglutamine-avid by positron emission tomography, suggesting that glutamine-addicted myofibroblasts drive increased hepatic utilization of glutamine as fibrosis progresses. CONCLUSIONS: Glutaminolysis is a potential diagnostic marker and therapeutic target during NASH fibrosis progression.