Objective: The natural molecule α-lipoic acid has been shown to be partially cytoprotective through antioxidant and antiapoptotic mechanisms. To obtain an initial assessment of the safety and potential efficacy of a synthetic derivative, CMX-2043, in preventing ischemic complications of percutaneous coronary intervention (PCI) we conducted the Subjects Undergoing PCI and Perioperative Reperfusion Treatment (SUPPORT-1) trial, the first patient experience with this agent. Methods and Results: SUPPORT-1 was a phase 2a, 6-center, international, placebo-controlled, randomized, double-blind trial. A total of 142 patients were randomized to receive a single intravenous bolus dose of drug or placebo administered 15–60 minutes before PCI. Cardiac biomarker assessments included serial measurements of creatine kinase myocardial band (CK-MB) at 6, 12, 18, and 24 hours after PCI and a single measurement of troponin T (TnT) at 24 hours. Peak concentrations of CK-MB and TnT were significantly reduced in the 2.4 mg/kg group compared with placebo (P = 0.05 and 0.03, respectively). No subject administered 2.4 mg/kg of CMX-2043 had an increase of CK-MB to ≥3X upper limit of normal versus 16% for placebo (P = 0.02); 16% of the 2.4-mg/kg dose group developed an elevation of TnT to ≥3X upper limit of normal versus 39% in the placebo group (P = 0.05). No drug-related serious adverse events were observed in any group. Conclusion: These data suggest that CMX-2043 may reduce PCI periprocedural myonecrosis and support further clinical evaluation of this novel agent for its potential cytoprotective effects.

α-Lipoic acid has been shown to provide cytoprotection in some tissues through antioxidant and antiapoptotic mechanisms. We have enhanced these properties by synthetic modification, resulting in a new chemical entity, CMX-2043, with proven efficacy in an animal model of cardiac ischemia-reperfusion injury. The present studies compare cytoprotective cellular pathways of R-α-lipoic acid and CMX-2043. Biochemical and cellular assays were used to compare antioxidant potency, tyrosine kinase activation, and protein kinase B (Akt) phosphorylation. CMX-2043 was more effective than lipoic acid in antioxidant effect, activation of insulin receptor kinase, soluble tyrosine kinase, and Akt phosphorylation. Activation of insulin-like growth factor 1 receptor was similar for both. CMX-2043 stimulation of Akt phosphorylation was abolished by the phosphatidylinositide 3-kinase inhibitor LY294002. Consistent with Akt activation, CMX-2043 reduced carbachol-induced calcium overload. The S-stereoisomer of CMX-2043 was less active in the biochemical assays than the R-isomer. These results are consistent with cytoprotection through activation of Akt and antioxidant action. CMX-2043 may thus provide a pharmacological approach to cytoprotection consistent with established anti-apoptotic mechanisms.

α-Lipoic acid (LA) has been shown to offer protection against ischemia–reperfusion injury (IRI) in multiple organ systems. N-[(R)-1,2-dithiolane-3-pentanoyl]-L-glutamyl-L-alanine (CMX-2043), a novel analogue of LA, was studied as part of a preclinical development program intended to identify safe and efficacious drug candidates for prevention or reduction in myocardial IRI. This study was designed to evaluate the efficacy of CMX-2043 in an animal model of myocardial IRI and to establish effective dosing conditions. CMX-2043 or placebo was administered at different doses, routes, and times in male Sprague-Dawley rats subjected to 30-minute left coronary artery ligation. Fluorescent microsphere injection defined the area at risk (AR). Animals were euthanized 24 hours after reperfusion, and the hearts were excised, sectioned, and stained with triphenyltetrazolium. Cytoprotective effectiveness was determined by comparing the unstained myocardial infarction zone (MI) to the ischemic AR. The reduction in the MI–AR ratio was used as the primary measure of drug efficacy relative to placebo injections. Treatment with CMX-2043 reduced myocardial IRI as measured by the MI–AR ratio and the incidence of arrhythmia. The compound was effective when administered by injection, both before and during the ischemic injury and at reperfusion. The most efficacious dose was that administered 15 minutes prior to the ischemic event and resulted in a 36% ( P < .001) reduction in MI–AR ratio compared to vehicle control.

CMX-2043 is an a-lipoic acid analogue targeted to reduction of cellular injury and organ damage due to ischaemia– reperfusion injury (IRI). It has been shown to be effective in a rat model of cardiac IRI. The studies here reported evaluate its safety and pharmacokinetic profile in preparation for human clinical studies in procedures associated with IRI. Safety and tolerability were tested in standard pre-clinical in vitro and animal models and in a Phase 1 human clinical trial. CMX-2043 did not bind to a wide range of receptors and specific targets at approximately 4 lg/mL (10 lM). It was not mutagenic by Ames assay, did not produce chromosome aberrations in Chinese hamster ovary (CHO) cells, and was negative for clastogenic potential. Toxicological studies in rats including both single and 14-day repeat intravenous doses and in dogs (single intravenous dose) with a 2-week recovery period were conducted. The NOAEL in rats and dogs was 30 and >10 mg/kg, respectively. No serious adverse events were reported in a placebo-controlled, sequential dose escalation Phase 1 clinical trial. The low toxicity in the pre-clinical studies and the absence of adverse events in the Phase 1 trial have supported investigation of CMX-2043 in a human efficacy trial. Prevention and treatment of ischaemia–reperfusion injury (IRI) is a major unmet medical need. In such injury, local inflammation, production of free oxygen radicals, alterations in calcium homeostasis and initiation of apoptotic cell death are all believed to contribute to potentially serious pathological and functional consequences [1,2]. IRI occurs in many organ systems, including heart, kidney, liver and brain. Although numerous treatment options have been evaluated, none has attained clinical acceptance [3]. Thus, there remains a need for an easy to use, safe and efficacious therapeutic agent for prevention or reduction of IRI. CMX-2043 (a-N-[(R)-1,2-dithiolane-3-pentanoyl]-L-glutamyl-L-alanine) is an a-lipoic acid (LA) analogue targeted to IRI. The LA moiety was used as the backbone structure to confer lipophilicity and antioxidant properties, and for its known ability to activate cellular protective mechanisms [4,5]. CMX-2043 reduces cardiac IRI in a rat model and is more effective in this model than LA itself. Its mechanism of action is consistent with established cell survival pathways (Lader AS, BaguisiA, Casale R, Kates SA, Beeuwkes III R, manuscripts in preparation) . The safety of the CMX-2043 parent molecule LA has been extensively validated. It is well tolerated in human beings and has been shown to be extremely safe in multiple in vitro and animal studies [6]. Consistent with its attractive safety profile, LA in oral (600 mg tablet [7]) and parenteral (Thioctacid 600 T [8]) formulations has been found to be effective for complications associated with diabetes [9,10]. This background supported the development and testing of a family of LA analogues as potential therapeutic agents. From these, based on biochemical and animal efficacy studies [11], CMX2043 was selected as the lead candidate for further investigation. Results of pre-clinical safety studies and a Phase 1 human trial are reported here. Methods and Materials The chemical structure and properties of CMX-2043 and lipoic acid are listed in table 1. Dose solution preparation and analysis. For non-clinical studies, the free acid of CMX-2043 was added to a solution that included sufficient 0.5 N NaOH for its neutralization, together with water and NaCl in quantities calculated to produce isotonicity and desired concentration. Analysis of dosing solution samples was conducted in compliance with GLP regulations by BASi (Evansville, IN, USA) using a high-performance reversed-phase liquid chromatography with UV detection (HPLC-UV). The concentration of CMX-2043 in the dosing solution was determined using a CMX-2043 external working standard. The cycle/run time for the HPLC analysis was an 8-min. isocratic gradient. CMX-2043 eluted in this HPLC method at ~3.5 min. Prior to testing for rat and dog toxicology studies, analysis of the dosing solution showed that measured doses ranged from 91% to 94% and from 97% to 100%, respectively, of nominal range. For clinical studies, CMX-2043 drug product was prepared under cGMP conditions at Bio-Concept Laboratories, Inc. (Salem, NH, USA) at a concentration of 10 mg/mL in a buffered sodium phosphate saline solution in a pH range of 6.8–7.6. CMX-2043 was added to a solution containing sterile water for injection, sodium chloride, sodium phosphate dibasic and sodium hydroxide, and the pH of the solution was adjusted using 1 N sodium hydroxide or 1 N hydrochloric acid to 7.1 0.1. The concentration of CMX-2043 in the drug product was Author for correspondence: Steven A. Kates, Ischemix LLC, 63 Great Road, Maynard, MA 01754, USA (fax 978 897 4952, e-mail skates@ischemix.com). © 2014 Ischemix LLC. Basic & Clinical Pharmacology & Toxicology published by John Wiley & Sons Ltd on behalf of [Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society). This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Basic & Clinical Pharmacology & Toxicology Doi: 10.1111/bcpt.12254

CMX ‐2043 is an α‐lipoic acid analogue targeted to reduction of cellular injury and organ damage due to ischaemia–reperfusion injury ( IRI ). It has been shown to be effective in a rat model of cardiac IRI . The studies here reported evaluate its safety and pharmacokinetic profile in preparation for human clinical studies in procedures associated with IRI . Safety and tolerability were tested in standard pre‐clinical in vitro and animal models and in a Phase 1 human clinical trial. CMX ‐2043 did not bind to a wide range of receptors and specific targets at approximately 4 μg/mL (10 μM). It was not mutagenic by Ames assay, did not produce chromosome aberrations in Chinese hamster ovary ( CHO ) cells, and was negative for clastogenic potential. Toxicological studies in rats including both single and 14‐day repeat intravenous doses and in dogs (single intravenous dose) with a 2‐week recovery period were conducted. The NOAEL in rats and dogs was 30 and >10 mg/kg, respectively. No serious adverse events were reported in a placebo‐controlled, sequential dose escalation Phase 1 clinical trial. The low toxicity in the pre‐clinical studies and the absence of adverse events in the Phase 1 trial have supported investigation of CMX ‐2043 in a human efficacy trial.

Crotoxin (CTX), the major component of the venom from the South American rattlesnake (Crotalus durissus terrificus) has diverse toxic effects, including pre-synaptic neurotoxicity. Among these, the effect of CTX in the heart is the least understood. In this study, we explored the effect(s) of CTX on the electrophysiological activity of neonatal rat cardiomyocytes (NRCM). By using patch, voltage-, and current-clamping techniques, we found that, in NRCM, CTX strongly potentiates L-type Ca2+ currents, an important contributor to the cardiac action potential (AP) and excitation-contraction (EC) coupling. External addition of CTX produced a rapid increase in L-type Ca2+ currents. Addition of verapamil (10 mu M) an L-type Ca2+ channel blocker, completely blocked the CTX-induced increase in the currents. A detailed kinetic analysis of AP in NRCM revealed that CTX caused both an elongation of AP duration (APD) and an increase of its amplitude, while a decrease of firing frequency. In addition, increasing concentrations of CTX completely blocked the AP as well as the beating of NRCM. The data indicate that CTX is a potent modulator of L-type Ca2+ currents, which provides a possible mechanistic correlation for the cardiotoxicity of the toxin, and may offer potential clinical implications in the control of cardiac function. (C) 2010 Elsevier Ltd. All rights reserved.

Non-small cell lung cancer (NSCLC) is a difficult disease to treat. The c-Met receptor is an attractive potential target for novel therapeutic inhibition in human cancers. We provide strong evidence that c-Met is overexpressed, activated, and sometimes mutated in NSCLC cell lines and tumor tissues. Expression of c-Met was found in all (100%) of the NSCLC tumor tissues examined (n = 23) and most (89%) of the cell lines (n = 9). Sixty-one percent of tumor tissues strongly expressed total c-Met, especially adenocarcinoma (67%). Specific expression of phospho-Met (p-Met) [Y1003] and [Y1230/1234/1235] was seen by immunohistochemistry. p-Met expression was preferentially observed at the NSCLC tumor invasive fronts. c-Met alterations were identified within the semaphorin domain (E168D, L299F, S323G, and N375S) and the juxtamembrane domain (R988C, R988C + T1010I, S1058P, and alternative splice product skipping entire juxtamembrane domain) of a NSCLC cell line and adenocarcinoma tissues. We validated c-Met as potential therapeutic target using small interfering RNA down-regulation of the receptor expression by 50% to 60% in NSCLC cells. This led to inhibition of p-Met and phospho-AKT and up to 57.1 +/- 7.2% cell viability inhibition at 72 hours. The selective small molecule inhibitor of c-Met SU11274 inhibited cell viability in c-Met-expressing NSCLC cells. SU11274 also abrogated hepatocyte growth factor-induced phosphorylation of c-Met and its downstream signaling. Here, we provide first direct evidence by small interfering RNA targeting and small molecule inhibitor that c-Met is important in NSCLC biology and biochemistry. These results indicate that c-Met inhibition will be an important therapeutic strategy against NSCLC to improve its clinical outcome.

1875 c-MET is believed to be an attractive target for molecular therapeutic inhibition in cancers associated with overexpression or activating mutations of c-MET. Since non-small cell lung cancer (NSCLC) is a difficult disease to treat, we have examined the expression of c-MET in 15 NSCLC cell lines using standard immunoblotting with anti-human c-MET antibody. There was overexpression of c-MET in most of the cell lines, including A549, H1838, H2170, SW-900, H358, H1993, Calu-3, Calu-1 and H596; while SK-NES-1, SW-1573, SK-LU-1, and Calu-6 had a lower c-MET expression. H661 did not express c-MET, and H520 expressed c-MET only minimally. Utilizing standard immunoperoxidase staining technique with anti-c-MET antibody and also phosphospecific c-MET antibodies (pY1230/1234/1235 auto-phosphorylation sites; and pY1003 juxtamembrane c-Cbl binding site), we show that expression of both total c-MET and also phosphorylated activated-MET are present in various subtypes of NSCLC tumor tissues. Specifically, strong activated-MET immunoperoxidase staining was seen in the invasion fronts of the tumor tissues. Also, up to 80% or more of the various NSCLC tumor tissue subtypes, including adenocarcinoma, squamous cell, large cell, examined expressed pY1003-Met and pY1230/1234/1235-Met. Since we have recently identified novel somatic missense mutations and alternatively spliced transcripts of c-MET in SCLC, we have begun to screen for mutations of c-MET in NSCLC cell lines (n=8) and also adenocarcinoma tumor tissues (n=127). Most interestingly, preliminary analysis of the sequencing results show clustering of mutations within the Sema (semaphorin) and JM (juxtamembrane) domains; however no mutations of the tyrosine kinase domain have been identified. We have now identified two novel sema domain missense mutations, S323G and N375S, in 3 different tumor tissue samples. In addition, we found several interesting JM mutations in the various cDNA samples. These include the R988C JM mutation, simultaneous JM mutations of R988C and T1010I, and also an alternative splice form with 47 amino acid deletion of the entire exon 14 (JM domain) of c-MET. Utilizing the adenocarcinoma A549 cell line that overexpresses c-MET to study signal transduction, we further show that HGF/c-MET pathway is functional and responsive to HGF (40 ng/ml) stimulation in a time-dependent fashion, with significant HGF-induced phosphorylation of c-MET, AKT, and p70-S6K. Also, there was HGF-induced tyrosine phosphorylation of the pY1230/1234/1235 sites demonstrated with the immunofluorescent staining using the phosphospecific antibodies. c-MET appears to be an important therapeutic target in NSCLC, and inhibitory strategies developed to target the receptor tyrosine kinase would be important to improve the therapeutic outcome.