Ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], a selenoorganic compound with glutathione peroxidase-like activity is used in clinical trials against stroke. Human and bovine TrxR catalyzed the reduction of ebselen to ebselen selenol by NADPH with an apparent K M-value of 2.5 M and a kcat of 588 min ؊1 . The addition of thioredoxin (Trx) stimulated the TrxR-catalyzed reduction of ebselen several-fold. This result was caused by a very fast oxidation of reduced Trx by ebselen with a rate constant in excess of 2 ؋ 10 7 M ؊1 s ؊1 . This rate is orders of magnitude faster than the reaction of dithiol Trx with insulin disulfides. Ebselen competed with disulfide substrates for reduction by Trx and, therefore, acted as an inhibitor of protein disulfide reduction by the Trx system. The inherent H2O2 reductase activity of mammalian TrxR dependent on its active-site selenocysteine residue was stimulated 10-fold by 2 M ebselen and 25-fold in the additional presence of 5 M Trx. Furthermore, the apparent KM-value of TrxR for H2O2 was lowered 25-fold to about 100 M. Our results demonstrate that ebselen is a TrxR peroxidase which, in the presence of Trx, acted as a mimic of a peroxiredoxin. The activity with TrxR and oxidation of reduced Trx offer mechanistic explanations for the in vivo effects of ebselen as an antioxidant and anti-inflammatory agent. Our results demonstrate that the mechanism of action of ebselen may be predominantly via the Trx system rather than via glutathione.selenol ͉ inflammation ͉ signal transduction ͉ oxidative stress ͉ redox regulation E bselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one] is a lipidsoluble seleno-organic compound that exhibits a weak glutathione peroxidase (GPx)-like activity in vitro (1-6). The specificity for substrates ranges from H 2 O 2 to smaller organic hydroperoxides and includes membrane-bound phospholipid and cholesterylester hydroperoxides. Because ebselen is effective against membrane hydroperoxides, it inhibits both nonenzymatic and enzymatic lipid peroxidation in cells and has anti-inflammatory activity in various animal models (5, 6). Ebselen also will directly inhibit inflammation-related enzymes such as 5-lipoxygenase, nitric oxide synthases, NADPH oxidase, protein kinase C, and ATPase by chemically modifying an SH-group forming a selenosulfide complex (5, 6).Because of its anti-inflammatory properties, ebselen has been used in the treatment of patients with acute ischemic stroke (7,8) or delayed neurological deficits after aneurysmal subarachnoid hemorrhage (9). The results of these clinical trials indicate the benefit of ebselen as a neuroprotective agent. Also, recent animal studies show neuroprotective, antioxidant, and antiinflammatory actions of ebselen in a rodent model of permanent middle as well as transient cerebral artery occlusion (10-13).Thioredoxin (Trx) reductase (TrxR) is a dimeric FADcontaining enzyme that catalyzes the NADPH-dependent reduction of the active-site disulfide in oxidized Trx (Trx-S 2 ) to give a dithiol in reduced Trx [Trx-(SH) 2 ] (14...

Although reactive oxygen species (ROS) have long been considered to play pathogenic roles in various disorders, this classic view is now being challenged by the recent discovery of their physiological roles in cellular signaling. To determine the immunological consequence of pharmacological disruption of endogenous redox regulation, we used a selenium-containing antioxidant compound ebselen known to modulate both thioredoxin and glutaredoxin pathways. Ebselen at 5–20 μM inhibited Con A-induced proliferation and cytokine production by the HDK-1 T cell line as well as the LPS-triggered cytokine production by XS52 dendritic cell (DC) line. Working with the in vitro-reconstituted Ag presentation system composed of bone marrow-derived DC, CD4+ T cells purified from DO11.10 TCR-transgenic mice and OVA peptide (serving as Ag), we observed that 1) both T cells and DC elevate intracellular oxidation states upon Ag-specific interaction; 2) ebselen significantly inhibits ROS production in both populations; and 3) ebselen at 5–20 μM inhibits DC-induced proliferation and cytokine production by T cells as well as T cell-induced cytokine production by DC. Thus, Ag-specific, bidirectional DC-T cell communication can be blocked by interfering with the redox regulation pathways. Allergic contact hypersensitivity responses in BALB/c mice to oxazolone, but not irritant contact hypersensitivity responses to croton oil, were suppressed significantly by postchallenge treatment with oral administrations of ebselen (100 mg/kg per day). These results provide both conceptual and technical frameworks for studying ROS-dependent regulation of DC-T cell communication during Ag presentation and for testing the potential utility of antioxidants for the treatment of immunological disease.

Background and Purpose-The neuroprotective efficacy of an intravenous formulation of the antioxidant ebselen has been comprehensively assessed with specific regard to conventional quantitative histopathology, subcortical axonal damage, neurological deficit, and principal mechanism of action. Methods-Transient focal ischemia (2 hours of intraluminal thread-induced ischemia with 22 hours of reperfusion) was induced in the rat. Ebselen (1 mg/kg bolus plus 1 mg/kg per hour IV) or vehicle was administered at the start of reperfusion and continued to 24 hours. Neurological deficit was assessed 24 hours after ischemia. Gray matter damage was evaluated by quantitative histopathology. Axonal damage was determined with amyloid precursor protein immunohistochemistry used as a marker of disrupted axonal flow and Tau-1 immunohistochemistry to identify oligodendrocyte pathology. Oxidative damage was determined by 8-hydroxy-2Ј-deoxyguanosine (8-OHdG) and 4-hydroxynonenal (4-HNE) immunohistochemistry. Results-Ebselen

1 The aim of this study was to investigate whether delayed treatment with the anti-oxidant and antiin¯ammatory agent ebselen reduces the volume of infarction in a rodent model of permanent focal cerebral ischaemia. 2 Ebselen (10 or 30 mg kg 71 ) or vehicle was administered by gavage 30 min and 12 h after the induction of cerebral ischaemia by permanent occlusion of the left middle cerebral artery (MCA). Animals were killed 24 h following MCA occlusion, and the volumes of ischaemic damage in the ebselen and control groups were evaluated by quantitative histopathology. 3 Ebselen was quickly absorbed following oral (gavage) administration and reached peak levels in the plasma by 1 h post-administration (plasma selenium level of 0.68+0.04 and 0.84+0.1 mg ml 71 for 10 and 30 mg kg 71, respectively, compared to control level of 0.51+0.02 mg kg 71). 4 Treatment with the lower dose of ebselen (10 mg kg 71 ) signi®cantly (P50.01) reduced the volume of infarction in the cerebral hemisphere and cerebral cortex (by 31.8% and 36.7%, respectively compared with the placebo group). 5 The neuroprotective ecacy of the higher dose ebselen (30 mg kg 71 ) was less than that of the lower dose ebselen (10 mg kg 71 ). The volume of ischaemic damage in the cerebral hemisphere was reduced by 23.7% (P50.02), and cerebral cortex by 27.5% (P50.01). 6 Both doses of ebselen (10, 30 mg kg 71) had no therapeutic ecacy on the caudate nucleus, where ischaemia was most severe, in this model. 7 Free radical-mediated injury is normally associated with reperfusion of ischaemic tissue. The present results suggest that oxidative injury is also a signi®cant contributor to brain damage in models of maintained (permanent) ischaemia and that ebselen is eective in attenuating this free radical-induced damage.

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