ABSTRACT
Copper is required for the proliferation of endothelial cells and copper-lowering therapy reduces tumour growth in animal models. It has been reported that ATN-224, a novel copper chelator, potently inhibits the activity of the copper-dependent enzyme superoxide dismutase 1 (SOD1) in endothelial cells. We performed microarray analysis of gene expression in endothelial cells exposed to ATN-224 which revealed upregulation of stress response genes including heme-oxygenase 1 (HO-1) and differential regulation of several genes previously implicated in angiogenesis including CXCR4, ANGP2, PGES2, RHAMM, ITB4 and AQP1 (p<0.01). These changes were confirmed on qPCR. Treatment of HUVEC with ATN-224 caused increased superoxide levels, phospho-ERK signalling, nuclear NRF1 expression, HO-1 expression and induction of the anti-apoptotic proteins P21, BCL2 and BCLXL. There was also nuclear translocation of SOD1. SOD1 RNA interference replicated the effects of ATN-224 on endothelial cell function but did not cause upregulation of HO-1 or PGES2, suggesting additional mechanisms of action of ATN-224. Downregulation of AQP1, which has been shown to have a role in angiogenesis, was seen with both ATN-224 and SOD1 siRNA. AQP1 expression could be rescued after ATN-224 by added copper. RNA interference to AQP1 inhibited endothelial proliferation and migration, confirming the role of AQP1 in endothelial cell function. Therefore regulation of AQP1 may represent an important action of copper chelation therapy.
Subject(s)
Chelating Agents/pharmacology , Endothelium, Vascular/drug effects , Enzyme Inhibitors/pharmacology , Molybdenum/pharmacology , Neovascularization, Physiologic/genetics , Pericytes/drug effects , Chelation Therapy , Down-Regulation , Endothelium, Vascular/metabolism , Gene Silencing , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Humans , Microarray Analysis , Pericytes/pathology , RNA Interference , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Transfection , Up-RegulationABSTRACT
PURPOSE: Copper chelation reduces the secretion of many angiogenic factors and reduces tumor growth and microvascular density in animal models. ATN-224 is a second-generation analogue of ammonium tetrathiomolybdate. The aim of our phase I study was to reduce serum copper levels, as measured by ceruloplasmin, to 5 to 15 mg/dL (normal 16-60) in 14 to 21 days, to determine the pharmacokinetic profile of ATN-224 and to evaluate dose-limiting toxicities. PATIENTS AND METHODS: Cohorts of patients were treated with escalating oral doses of ATN-224 until copper depletion followed by a titrated maintenance dose. RESULTS: Eighteen patients received 78 cycles of ATN-224. Mean baseline ceruloplasmin was 39.6 mg/dL. The maximum administered dose was 330 mg/d where grade 3 fatigue was dose-limiting. At the maximum tolerated dose of 300 mg/d, the median time to achieve target ceruloplasmin was 21 days, and toxicities included grade 3 anemia, grade 3 neutropenia, fatigue, and sulfur eructation. ATN-224 treatment caused a significant reduction (> 90%) in RBC superoxide dismutase 1 activity and circulating endothelial cells. Pharmacokinetic data indicate greater absorption of ATN-224 and more rapid ceruloplasmin reduction when administered with a proton pump inhibitor. Stable disease of > 6 months was observed in 2 patients. CONCLUSIONS: Oral ATN-224 is a well-tolerated therapy and at a loading dose of 300 mg/d leads to a reduction of serum ceruloplasmin levels in 80% patients within 21 days. A loading dose of 300 mg/d for 2 weeks followed by a titrated maintenance dose will be the recommended starting dose for phase II study.