The design and synthesis of three 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) derivatives bearing linkers with terminal thiol groups and a preliminary evaluation of their potential for use in assembling redox-sensitive Magnetic Resonance Imaging (MRI) contrast agents are reported. The linkers were selected based on computational docking with a crystal structure of human serum albumin (HSA). Gd(III)-DO3A and Eu(III)-DO3A complexes were synthesized, and the structure of one complex was established by X-ray crystallographic analysis. The binding to HSA of a Gd(III)-DO3A complex bearing a thiol-terminated 3,6-dioxanonyl chain was competitively inhibited by homocysteine and by the corresponding Eu chelate. Binding to HSA was abolished when the terminal thiol group of this complex was absent. The longitudinal water-proton relaxivities (r 1 ) of the three Gd(III)-DO3A complexes and of two Gd(III)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complexes were measured in saline at 7 Tesla. The DO3A complexes exhibited smaller r 1 values, in both bound and free states, than the DOTA complexes.
Gd-LC6-SH is a thiol-bearing DOTA complex of gadolinium designed to bind plasma albumin at the conserved Cys(34) site. The binding of Gd-LC6-SH shows sensitivity to the presence of competing thiols. We hypothesized that Gd-LC6-SH could provide magnetic resonance imaging (MRI) enhancement that is sensitive to tumor redox state and that the prolonged retention of albumin-bound Gd-LC6-SH in vivo can be exploited to identify a saturating dose above which the shortening of MRI longitudinal relaxation time (T(1)) of tissue is insensitive to the injected gadolinium dose. In the Mia-PaCa-2 pancreatic tumor xenograft model in SCID mice, both the small-molecule Gd-DTPA-BMA and the macromolecule Galbumin MRI contrast agents produced dose-dependent decreases in tumor T(1). By contrast, the decreases in tumor T(1) provided by Gd-LC6-SH at 0.05 and 0.1 mmol/kg were not significantly different at longer times after injection. SCID mice bearing Mia-PaCa-2 or NCI-N87 tumor xenografts were treated with either the glutathione synthesis inhibitor buthionine sulfoximine or the thiol-oxidizing anticancer drug Imexon, respectively. In both models, there was a significantly greater increase in tumor R(1) (=1/T(1)) 60 minutes after injection of Gd-LC6-SH in drug-treated animals relative to saline-treated controls. In addition, Mercury Orange staining for nonprotein sulfhydryls was significantly decreased by drug treatment relative to controls in both tumor models. In summary, these studies show that thiol-bearing complexes of gadolinium such as Gd-LC6-SH can serve as redox-sensitive MRI contrast agents for detecting differences in tumor redox status and can be used to evaluate the effects of redox-active drugs.
The synthesis and structure-activity relationships of a homologous series of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid gadolinium(III) complexes bearing thiol-terminated alkyl sidechains from three to nine carbons in length are reported. The observed binding with human serum albumin (HSA) of the compounds having C-3 through C-7 sidechain lengths was inhibited by homocysteine in a manner consistent with single-site binding. The observed binding with HSA of the compounds having C-8 and C-9 sidechain lengths was only partly inhibited by homocysteine, consistent with multi-site binding. The binding affinity of the C-7 compound could be related to the HSA oxidation state. 2D 1H–1H NMR TOCSY provided evidence of covalent binding of the europium analog of the C-6 compound to HSA-Cys34. The longitudinal water-proton MRI relaxivities of the gadolinium complexes at 7 Tesla increased upon binding to HSA. Based on these results, the C-6 and C-7 compounds were identified as promising redox-sensitive MRI contrast agents.
Induction of oxidative stress is a key component of cancer therapy. Pro-oxidant drugs have been demonstrated to enhance the efficacy of radiotherapy and chemotherapy. An emerging concept is that therapeutic outcomes are dictated by the differential redox buffering reserve in subpopulations of malignant cells, indicating the need for noninvasive biomarkers of tumor redox that can be used for dose identification and response assessment in a longitudinal setting. Magnetic resonance imaging (MRI) enhanced with the thiol-binding contrast agent Gd-LC6-SH, and hemodynamic response imaging (HRI) in combination with hypercapnia and hyperoxia were investigated as biomarkers of the pharmacodynamics of the small molecule pro-oxidant imexon (IMX). Human multiple myeloma cell lines 8226/S and an IMX-resistant variant, 8226/IM10, were established as contralateral tumors in SCID mice. T1slope, an MRI measure of the washout rate of Gd-LC6-SH, was significantly lower post-IMX therapy in 8226/S tumors compared with vehicle controls, indicating treatment-related oxidization of the tumor microenvironment, which was confirmed by analysis of tumor tissue for thiols. T1slope and ex vivo assays for thiols both indicated a more reduced microenvironment in 8226/IM10 tumors following IMX therapy. HRI with hypercapnia challenge revealed IMX inhibition of vascular dilation in 8226/S tumors but not 8226/IM10 tumors, consistent with decreased immunohistochemical staining for smooth muscle actin in treated 8226/S tumors. MRI enhanced with Gd-LC6-SH, and HRI coupled with a hypercapnic challenge provide noninvasive biomarkers of tumor response to the redox modulator imexon.
RRx-001 is a member of the novel dinitroazetidine class of anticancer agents that profoundly perturbs the thiol redox potential of cancer cells while inducing damaging ROS/RNS. A Phase 1 study investigating the safety and tolerability of RRx-001 has been completed and Phase 2 studies are ongoing. Pre-clinical studies indicate that RRx-001 selectively alkylates glutathione and a specific thiol on hemoglobin, resulting in a pro-oxidant effect in tumors. We have previously reported the development of thiol-containing DOTA-based chelates of gadolinium as redox-sensitive MRI contrast agents. In the present preclinical MRI study we investigated the pharmacodynamics and mechanism of action of RRx-001 using the novel thiol-bearing contrast agent Gd-LC7-SH. SCID mice were inoculated in the flank with either CHP-100, HT-29, or PANC-1 cells. Mice were imaged on a 7 Tesla Bruker Biospec® small animal MRI scanner when tumors had grown to 250-400 mm3 in size. Mice were anesthetized using isoflurane and cannulated at the tail vein for injections using a zero dead volume i.v. line for administration of Gd-LC7-SH or RRx-001. Longitudinal relaxation time (T1) maps of the tumor were acquired pre-contrast and at various times post-contrast to 60 min post-injection of 0.05 mmol/kg Gd-LC7-SH. Mice were imaged before treatment and at 1h, 24 h and 72 h post-treatment with 10 mg/kg RRx-001. Gd-LC7-SH spontaneously binds to thiol targets following i.v. administration. The fraction of gadolinium that is bound to macromolecular targets such as plasma albumin and exofacial protein thiols (EPTs) is protected from renal clearance, and produces a prolonged decrease in tumor T1 on MRI. We have quantified this decrease in tumor T1 by the parameter -T1, calculated as pre-contrast tumor T1 minus tumor T1 at 40-60 min post Gd-LC7-SH. The larger the magnitude of -T1, the greater is the retention of Gd-LC7-SH in the tumor. In all 3 tumor types, the tumor -T1 at 1 h post-drug was significantly smaller than pre-drug tumor -T1 (p<0.02). In the HT-29 and PANC-1 tumors the -T1 at 72 h post-drug remained smaller than baseline -T1 (p<0.05). These observations indicate decreased tumor retention of Gd-LC7-SH following treatment with RRx-001, which is consistent with a decrease in availability of reduced albumin and EPTs in the tumor. The previously reported redox activity of RRx-001 together with its very short half-life in vivo suggests an indirect effect on albumin and exofacial thiols that manifests as smaller -T1 values on Gd-LC7-SH MRI imaging. The anti-proliferative activity of RRx-001 may not only be due to glutathione depletion and NO release under hypoxia, but also to an increase in intratumoral ROS burden leading to a direct redox modulation of exofacial thiols integral to tumor protein function. Additional studies are planned to confirm this postulate. Citation Format: Natarajan Raghunand, Jan Scicinski, Bryan Oronsky, Gerald Guntle, Elizabeth Bruckheimer, Ron Korn. RRx-001 oxidation of redox sensitive protein thiols in tumors measured by Gd-LC7-SH enhanced MRI In preclinical tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2068. doi:10.1158/1538-7445.AM2014-2068
RRx-001 is an anticancer agent that subjects cancer cells to reactive oxygen/nitrogen species (ROS/RNS) and acts as an epigenetic modifier. We have used a thiol-bearing MRI contrast agent, Gd-LC7-SH, to investigate the pharmacodynamics of RRx-001 in CHP-100 Ewing's Sarcoma, HT-29 colorectal carcinoma, and PANC-1 pancreatic carcinoma xenografts in SCID mice. Binding of Gd-LC7-SH to the Cys34 residue on plasma albumin prolongs retention in the tumor microenvironment and increases tumor enhancement on MRI. Mice were imaged by MRI and in vivo T1 maps acquired 50 min (T150 min) after injection of 0.05 mmol/kg Gd-LC7-SH (i.v.) at baseline and 1, 24, and 72 h post-treatment with 10 mg/kg RRx-001 (i.v.). Consistent with an indirect thiol-modifying activity of RRx-001, tumor T150 min at 1 h post-drug was significantly longer than pre-drug tumor T150 min in all three tumor models, with the T150 min remaining significantly longer than baseline through 72 h post-drug in the HT-29 and PANC-1 tumors. The T150 min of CHP-100 tumors recovered to baseline by 24 h post-drug, suggesting a robust anti-oxidant response to the RRx-001 challenge that was presaged by a marked increase in perfusion at 1 h post-drug measured by DCE-MRI. MRI enhanced with Gd-LC7-SH provides a mechanistically rational biomarker of RRx-001 pharmacodynamics.
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