Seleno-organic Compound Research Articles

PZ-51 (Ebselen) in vivo protection against Adriamycin-induced mouse cardiac and hepatic lipid peroxidation and toxicity

Adriamycin ® (Adr)-induced cardiotoxicity occurs most likely via an oxidative mechanism of action. Moderation of this activity may result in an improved therapeutic index for this compound. PZ-51, 2-phenyl-1,2-benzoisoselenazol-3(2H)-one, is a selenoorganic compound with thiol-dependent, peroxidase-like activity. We tested this compound alone and in combination with N-acetylcysteine (NAC) for its effect on Adr-induced in vivo toxicity in Balb/c mice. These studies demonstrated that PZ-51 protects against Adr-induced lipid peroxidation in heart and liver tissue and Adr-induced toxicity in general, as measured by total serum creatine kinase activity and body weight.

The influence of ebselen on the toxicity of cisplatin in LLC-PK 1 cells

LLC-PK 1 cells have been used as an in vitro model to study the nephrotoxicity of the antitumor drug cisplatin. A concentration-dependent cytotoxicity of cisplatin, measured as lactate dehydrogenase leakage and amount of protein remaining attached to the culture plate, was observed. At a cisplatin concentration of 0.4 mM cell viability was reduced to 21% after 72 hr. Ebselen, a selenoorganic compound capable of forming selenol intermediates through reaction with thiols, was found to protect LLC-PK 1, cells against cisplatin-induced toxicity at low concentrations (5–15 μM). The ebseleninduced protection against cisplatin toxicity in this in vitro test system apparently correlates well with a similar protection previously observed in vivo in mice and rats.

Ebselen affects calcium homeostasis in human platelets

Ebselen (PZ 51, 2-phenyl-l,2-benzoisoselenazol-3-(2H)-one) is a selenoorganic compound with anti-inflammatory properties. Its pharmacological action is thought to originate from its peroxidase activity which could lower the peroxide tonus required for cyclooxygenase and lipoxygenase activations. From experiments with aspirin-treated human platelets we now present evidence that ebselen also affects intracellular calcium homeostasis by inhibiting the agonist-triggered increase in intracellular calcium. Using Mn 2+ entry to quench the fura-2 fluorescence after cell stimulation, we could exclude an interaction of ebselen with receptor-operated calcium channels and therefore an inhibition of extracellular calcium influx. It became evident from whole cell experiments and by using isolated platelet microsomal vesicles that ebselen inhibits the inositol 1,4,5-trisphosphate (IP 3) induced calcium release. Besides this inhibitory effect of ebselen on the calcium release higher concentrations of the compound (⩾ = 5 μM) induced a calcium release from our microsomal vesicles which also could be reversed by dithiothreitol. An activation of inflammatory cells is usually associated with increased cytosolic calcium concentrations. An inhibition of such calcium movements by ebselen may account for an up to now unidentified antiinflammatory mechanism of ebselen action which is linked to a direct effect of this compound rather than to its peroxidase-like activity.

Interaction of the anti-inflammatory seleno-organic compound ebselen with acid secretion in isolated parietal cells and gastric H +/K +-ATPase

The effects of the anti-inflammatory seleno-organic compound ebselen on gastric H +/K +-ATPase, H +/K +-ATPase-mediated proton transport and on parietal cell HCl production was studied. Ebselen inhibited K +-stimulated ATPase activity in leaky gastric membranes ( ic 50:0.15 μM) and H +/ K +-ATPase-mediated proton transport in intact gastric membrane vesicles ( ic 50:0.7μM). Histamine- and dibutyryl-cAMP-stimulated HCl production in isolated and enriched guinea-pig parietal cells was inhibited with an ic 50 value of 12 μM. The mercaptan dithioerythritol and the nucleotide ATP prevents the H +/K +-ATPase against inactivation and dithioerythritol was found to restore already inhibited enzyme activity and ATPase mediated H + transport. Furthermore, dithioerythritol could prevent ebselen-induced inhibition of HCl production in the parietal cell preparation. It is concluded that ebselen inhibits acid secretion in the parietal cell by interference with SH groups of the gastric proton pump, the H +/K +-ATPase. Therefore ebselen can be regarded as an anti-inflammatory drug for which in vitro anti-secretory properties can be demonstrated.

Acute antiinflammatory and gastric effects of the seleno-organic compound ebselen

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a seleno-organic compound with glutathione peroxidase-like activity in vitro, was compared with indomethacin, BW 755C, and levamisole as an inhibitor of carrageenan- and CVF (cobra venom factor)-induced paw oedema in the rat. The antiinflammatory potency of ebselen against CVF-induced oedema (ED50 = 56 mg/kg p.o.) was similar to that of BW 755C, while indomethacin was weakly active in this model, and levamisole exerted stronger activity. In the carrageenan model, ebselen exhibited weak inhibitory potency, like BW 755C, while indomethacin markedly inhibited this inflammatory response, and levamisole was inactive. Unlike cyclooxygenase inhibitors, ebselen produced almost no gastric irritation in rats up to 316 mg/kg p.o. Moreover, ebselen inhibited significantly diclofenac-induced gastric intolerance at 31.6 and 316 mg/kg p.o. Thus, ebselen represents a new tool for antiinflammatory therapy.

Mechanism for the inhibitory effect of a seleno-organic compound, Ebselen, and its analogues on superoxide anion production in guinea pig polymorphonuclear leukocytes.

Effects of Ebselen and its analogs (PZ-25, NAT06-123, NAT02-761, NAT02-801, NAT06-099, and NAT06-513) on superoxide anion (O2-) production induced by tetradecanoyl phorbol acetate (TPA) were examined in intact guinea pig polymorphonuclear leukocytes (PMNL). Four compounds having a structure of 1,2 benzoisoselenazol-3-(2H) one (Ebselen, NAT06-123, and NAT02-761) and its sulfur-substituted analog (PZ-25), had a potent inhibitory effect on O2- production as compared with others. Ebselen and NAT06-123 also markedly inhibited nicotinamide adenine dinuclestide phosphate (NADPH) oxidase activity, which is responsible for O2- production in intact cells, and in a particulate fraction prepared from TPA-stimulated PMNL, whereas PZ-25 inhibited this enzyme weakly and NAT02-761 did not. On the other hand, Ebselen and PZ-25 had the same degree of potent inhibitory effect on protein kinase C which was involved in the regulation of NADPH oxidase activation. Thus, it is plausible that inhibition of O2- production in intact PMNL by these compounds were due not only to direct inhibition of NADPH oxidase but also to inhibition of protein kinase C.

Effects of ebselen on arachidonate metabolism by ocular and non-ocular tissues

The formation of cyclooxygenase products in rabbit and rat ocular and non-ocular tissues in vitro, detected by radio-thin-layer chromatography, was inhibited in a concentration-dependent manner by ebselen (PZ 51), an anti-inflammatory seleno-organic compound which has glutathione peroxidase and anti-oxidant activities. The exception was prostaglandin F 2α(PGF 2α) formation in the rabbit irisciliary body which was stimulated by ebselen in the concentration range 2–10 μM. These observations were confirmed by gas chromatography-mass spectrometry. The concentration that inhibited 50% of prostaglandin biosynthesis ic 50 in the rabbit iris-ciliary body was 9.3 μM. Ebselen also inhibited the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) in rabbit and rat ocular tissues and rabbit platelets. The ic 50 in the rabbit cornea was 4 μM, whereas higher concentrations were generally required to achieve similar inhibition in other tissues. The formation of 12-HETE by rabbit spleen, however, was not decreased by ebselen at concentrations that were inhibitory in other tissues.

急性胃粘膜障害に対するｓｅｌｅｎｏ‐ｏｒｇａｎｉｃ ｃｏｍｐｏｕｎｄ（Ｅｂｓｅｌｅｎ）の効果

急性胃粘膜障害に対するｓｅｌｅｎｏ‐ｏｒｇａｎｉｃ ｃｏｍｐｏｕｎｄ（Ｅｂｓｅｌｅｎ）の効果

Antimalarial properties of ebselen.

The seleno-organic compound ebselen showed anti-malarial activity in vitro against the murine Plasmodium berghei and the human P. falciparum. In P. berghei, the uptake and incorporation of [3H]-methionine and [3H]-adenosine was inhibited and the infectivity of plasmodia was reduced. Ebselen affects the development of asexual stages of chloroquine-resistant and -sensitive P. falciparum strains. Its IC50 for P. falciparum was about 14 mumol/l and that for P. berghei, about 10 mumol/l. The growth of P. falciparum was blocked by ebselen at all stages, including the invasion of erythrocytes by merozoites. In a human hepatoma cell line and in mouse peritoneal macrophages, no cytostatic or cytotoxic effects were found, indicating selective inhibition of plasmodia by ebselen. Its in vitro inhibitory effect is discussed in relation to its possible reactivity with thiol groups and its lack of an anti-malarial effect in infected mice.

A novel biologically active selenooorganic compound—VII: Biotransformation of ebselen in perfused rat liver

Ebselen (PZ 51) is a selenoorganic compound with antioxidant and antiinflammatory properties, and its metabolism was studied in isolated perfused rat liver (hemoglobin-free, open system). 75Se-labelled ebselen was taken up into liver cells and radioactivity was excreted into bile. Biliary excretion of 75Se-compounds reached maximal values of 4 nmol/min per g wet wt. HPLC analysis of bile and effluent perfusate as well as identification of separated metabolites by mass spectrometry were carried out. The biliary metabolites were (a) an interesting novel Se-glucuronide, 2-glucuronylselenobenzanilide, (metabolite IV), as the major metabolite, and (b) an O-glucuronide, N-(4′-glucuronyloxyphenyl)-2-methylselenobenzanilide (metabolite III). The major effluent perfusate metabolites were Se-methylated derivatives (metabolites I and II). There was no evidence for sulfated metabolites. The selenodisulfide with glutathione, S-(2-phenyl-carbamoyl-phenylselenyl)-glutathione, was not detected, probably because of low steady-state concentrations and/or its biochemical lability. The selenium in ebselen is not bioavailable (e.g. for the synthesis of glutathione peroxidase), in contrast to selenite, for example, thus explaining the very low ebselen toxicity. However, the enzymatic steps in Se-methylation could be similar to those in the metabolism of selenite which include hydrogen selenide methylation. Se-glucuronides constitute a novel category of compounds in addition to the O-, N-, C- and S-glucuronide classes known in biology.

N-acetylcysteine and glutathione-dependent protective effect of PZ51 (Ebselen) against diquat-induced cytotoxicity in isolated hepatocytes

The glutathione peroxidase (GSH-Px)-like reduction of H 2O 2 by the selenoorganic compound 2-phenyl-1,2-benzoisoselenazol-3(H)-one (PZ51: Ebselen) was studied using glutathione (GSH) and the therapeutic agent N-acetylcysteine (NAC) to provide reducing equivalents. In a purely chemical system containing H 2O 2 and in an enzymatic system of glucose/glucose oxidase-generated H 2O 2 Ebselen alone did not reduce H 2O 2. Ebselen in combination with either GSH (1 mM) or NAC (1 mM) was capable of reducing H 2O 2 in both systems. In these non-cellular systems GSH was a more effective source of reducing equivalents than NAC. The GSH-Px-like activity of Ebselen was further investigated in a cellular system. The redox-cycling bipyridylium compound diquat generates active oxygen species, depletes intracellular glutathione, and is cytotoxic in isolated hepatocytes pretreated with the glutathione reductase inhibitor 1,3-bis(Z-chloro-ethyl)-1-nitrosourea (BCNU). Ebselen alone did not ameliorate diquat cytotoxicity, but in combination with either GSH (1 mM) or NAC (1 mM) it produced a significant delay in diquat-induced cytotoxicity. Further additions of either GSH (0.5 mM) or NAC (0.5 mM) at 30 min intervals provided significantly more protection against diquat-induced cytotoxicity and intracellular GSH depletion than the single 1 mM addition. Thus, the combination of Ebselen and NAC may provide an effective antidote in cases of overexposure to bipyridylium herbicides, such as diquat and paraquat.

Antioxidant activity in cells and organs.

Antioxidant activity in cells occurs at several levels. The present work focuses on some aspects of enzymatic antioxidant defense. In particular, the so-called ancillary enzymes and transport systems are presented. Recent aspects on the control of the levels of antioxidant enzymes include the oxyR regulon in microorganisms as well as the response to DNA hypomethylation in livers of mice. Ebselen is a novel selenoorganic compound with antioxidant activity capable of conferring protection against oxidative challenge. Its activity to catalyse the GSH-peroxidase reaction is thought to mediate the effect in intact cells. The nonenzymatic antioxidant defense (e.g., vitamins E and C and other natural or chemically synthesized compounds) form an important complementary part of cellular antioxidant defense.

Inhibition of superoxide anion production in guinea pig polymorphonuclear leukocytes by a seleno-organic compound, ebselen.

Production of superoxide anion (O2-) induced by tetradecanoyl phorbol acetate (TPA) in intact guinea pig polymorphonuclear leukocytes (PMNL) was markedly inhibited by a seleno-organic compound, 2-phenyl-1,2-benzisoselenazol-3(2H)-one (Ebselen), with glutathione peroxidase-like activity. The compound almost completely inhibited O2- production by a particulate fraction prepared from TPA-treated PMNL at a concentration as low as 250 nM.

Synthetic seleno-organic compound with glutathione peroxidase-like activity in the chick

The glutathione peroxidase activity catalyzed by the seleno-organic anti-inflammatory drug Ebselen (registered under the trademark of the Natterman Corp. Cologne, FRG) [PZ51, 2-phenyl-1,2-benzisoselenazol-3(2 H)on], as measured by NADPH oxidation, was inhibited in vitro by the selenium-dependent glutathione peroxidase (SeGSHpx) inhibitors aurothioglucose and D-(−)penicillamine HCl. Vitamin E- and selenium-deficient chicks were given 0, 80 or 320 ppm PZ51 in diets devoid of vitamin E and supplemented with low levels of sodium selenite (0.04 ppm selenium added to the basal diet containing ca. 0.015 ppm selenium) when a small number of chicks ( ca. 13%) had exudative diathesis (ED). By 24 hr, the high PZ51 dose (320 ppm) delayed the onset of ED compared to untreated controls. Similarly, vitamin E-deficient chicks fed diets containing 0, 80, 160, 320, 640 or 1280 ppm PZ51 and supplemented with 0.04 ppm selenium showed ED in inverse proportion to log PZ51 dose. Plasma and liver post-mitochondrial supernatant samples from these chicks also exhibited log-linear relationships between dietary PZ51 level and selenium content or SeGSHpx-like activity. The amount of SeGSHpx-like activity for chicks given PZ51 above that determined for untreated chicks was extractable into ethanol, indicating that those PZ51-associated increases were not due to protein-bound selenium or SeGSHpx. This suggests that selenium from PZ51 was not available to support synthesis of SeGSHpx. Dietary PZ51 (1280 ppm) or selenium (0.1 ppm) alone or in combination decreased the acute lethalities of nitrofurantoin or paraquat in vitamin E-adequate chicks. The results indicate that SeGSHpx-like activity in selenium-deficient chicks is increased by oral administration of PZ51, which appears to mimic the true enzyme by affording protection against clinical signs of selenium deficiency (i.e. ED) and proxidant drug lethality.

A novel biologically active seleno-organic compound—VI: Protection by ebselen (PZ 51) against galactosamine/ endotoxin-induced hepatitis in mice

Male albino NMRI mice were given 700 mg/kg galactosamine and 33 μg/kg salmonella endotoxin intraperitoneally. After 9 hr, serum sorbitol dehydrogenase activity had risen from 60 to 7320 U/1, SGOT from 90 to 5580, and SGPT from 70 to 10,440. When a similar dose of galactosamine alone or endotoxin alone was given, no significant liver injury was found. Animals pre-treated with an oral dose of ebselen (600 mg/kg 1–3 hr before galactosamine/endotoxin administration) were fully protected against this type of hepatitis. When pretreated 1 hr before intoxication with different doses of ebselen, significant dose-dependent reduction of serum enzyme activities was observed at doses higher than 1 mg/kg. After pre-treatment with 6 mg/kg ebselen, no biochemical or histological signs of liver lesions were detectable 36 hr after intoxication. In order to comparatively evaluate the model used, several established anti-inflammatory drugs were administered at doses which showed 50% effectiveness in preventing carageenan paw edema. A dose of 200 μg/kg dexamethasone, or 9 mg/kg indomethacin abolished galactosamine/endotoxin-induced enzyme release in our animals, as did the lipoxygenase pathway inhibitor diethylcarbamazine (78 mg/kg). In contrast, administration of cyclooxygenase pathway inhibitors such as aspirin (220 mg/kg) or ibuprofen (45 mg/kg) failed to prevent hepatitis. The effect of ebselen was also investigated in four different models of acute drug-induced liver damage. A dose of 600 mg/kg of the organic selenium compound was ineffective or weakly active in benzo(α)pyrene- or phenobarbital-treated mice which were intoxicated by intraperitoneal administration of 350 or 400 mg/kg body weight of paracetamol. Similarly negative results were obtained against bromobenzene-induced hepatotoxicity (520 mg/kg bromobenzene i.p.), carbon tetrachloride intoxication (3.2 g/kg), or allyl alcohol-induced liver damage (60 mg/kg). The selective efficacy of ebselen against galactosamine/endotoxin induced liver damage is interpreted in terms of its recently recognized ability to inhibit the formation of leukotrienes.

Inhibitory effects of ebselen on lipid peroxidation in rat liver microsomes.

The effects of ebselen(2-phenyl-1,2-benzoisoselenazol-3(2H)-one), a synthetic seleno-organic compound with glutathione peroxidase-like activity were investigated on lipid peroxidation in rat liver microsomes. Ebselen inhibited malondialdehyde production coupled to the lipid peroxidation stimulated by either ADP-iron-ascorbate or CCl4. The inhibitory activity of ebselen on each system was strongly increased by a 5-min preincubation with liver microsomes; the IC50 values against ADP-Fe-ascorbate-stimulated and CCl4-stimulated lipid peroxidation were 1.6 microM and 70 microM respectively. Ebselen also inhibited the endogenous lipid peroxidation with a NADPH-generating system, but it slightly stimulated the endogenous activity of ADP-Fe-ascorbate-stimulated lipid peroxidation (without a NADPH-generating system). Furthermore, ebselen inhibited oxygen uptake coupled to the lipid peroxidation by ADP-Fe-ascorbate and NADPH-ADP-iron; the IC50 values were 2.5 microM and 20.3 microM respectively. Ebselen also prolonged the lag-time of onset of ADP-Fe-ascorbate-stimulated lipid peroxidation significantly, but not that observed with NADPH-ADP-Fe-stimulated lipid peroxidation. These findings suggest that ebselen penetrates into the membrane lipid and acts as an effective antioxidant, and that there may be some differences between the modes of inhibitory action on the several types of lipid peroxidation.

A novel biologically active seleno-organic compound—V: Inhibition by ebselen (PZ 51) of rat peritoneal neutrophil lipoxygenase

Suspensions of rat peritoneal PMNLs elicited with glycogen were stimulated by calcium and an ionophore to produce leukotrienes from endogenous arachidonic acid. We investigated the effect of the non-toxic, anti-inflammatory seleno-organic compound, ebselen (PZ 51). When ethanolic extracts of the medium of stimulated cells were analysed by HPLC, a dose-dependent inhibition by ebselen of LTB 4 formation with a concomitant decrease of 5-HETE production was found. Half-maximum inhibition was observed at 20 μmoles/1 ebselen. Similar findings were obtained after analysis of chloroform extracts of both cells and medium using a different HPLC system. Under these conditions, enhanced 5-HETE formation was associated with reduced production of LTB 4 and other di-HETE isomers, when purified glutathione peroxidase + GSH were present. We conclude that the reported GSH peroxidase-like activity of ebselen, catalysing the reduction of 5-HPETE to 5-HETE, can not account for our findings. Therefore, the lipoxygenase reaction itself represents the site of inhibition of LTB 4 formation by ebselen.

A novel biologically active selenoorganic compound—IV: Protective glutathione-dependent effect of PZ 51 (Ebselen) against ADP-Fe induced lipid peroxidation in isolated hepatocytes

PZ 51 (Ebselen) is capable of inhibiting ADP-Fe-induced lipid peroxidation in isolated hepatocytes, assessed by generation of low-level chemiluminescence, and the formation of alkanes (ethane, n-pentane) and malondialdehyde. The sulphur analog PZ 25 is much less active. PZ 51 is ineffective in glutathione-depleted hepatocytes that were obtained after phorone pretreatment of the animals. However, other antioxidants, like (+)-cyanidanol-3 or diethyldithiocarbamate are effective in inhibiting lipid peroxidation also in glutathione-depleted cells. The results are attributed to a GSH peroxidase-like activity of PZ 51. Addition of the chemically synthetized compound, PZ 51-SG. a glutathione adduct of PZ 51, does not provide extra protection in GSH-depleted cells in comparison to the parent compound, PZ 51. Dithioerythritol exhibits a protective effect, and PZ 51 is more efficient than in the absence of this thiol reductant. Dithioerythritol also exerts an effect in glutatioone-depleted cells. A peroxidase-like activity using DTE as reductant is demonstrated in vitro.

Oxidative stress: Excited oxygen species and enzyme activity

The metabolic role of aldehydes, hydroperoxides, and quinones was investigated with emphasis on oxidative transitions involving oxygen free radicals and associated with enzymatic activities. The oxidative metabolism of aldehydes (originating either from ethanol oxidation, or monoamine oxidase activity, or oxidative breakdown of lipid hydroperoxides during lipid peroxidation) is a source of alkane production and low-level chemiluminescence. Since both parameters reflect cellular oxidative conditions, it can be inferred that side-products of aldehyde oxidase activity might participate in the link between the initial enzymatic oxidation of aldehyde and the occurrence of oxidizing species leading to chemiluminescence and alkane production. The metabolism of hydroperoxides was considered under two different aspects: first, the hydroperoxide reduction, within the frame of a detoxication mechanism, as mediated by a selenoorganic compound PZ-51 that displays glutathione peroxidase-like activity and an antioxidant activity; second, the enzyme-catalyzed disproportionation of hydroperoxides as a source of a potent oxidizing equivalent, singlete molecular oxygen. The cytotoxicity of quinones, utilized in therapeutic agents such as anticancer drugs, is believed to be related to oxidative stress due to the formation of the superoxide radical and subsequent more reactive oxygen species. The enzyme-catalyzed one-electron reduction of menadione seems to play a substantial role in the development ofcytotoxic effects, at variance with the 2-electron reduction of the quinone. The observation of low-level chemiluminescence under conditions which favor the one-electron reduction process or which diminished the two-electron reduction process indicates the practicability of low-level chemiluminescence measurements in monitoring changes in quinone metabolism and related cytotoxic effects.

A novel biologically active seleno-organic compound—1: Glutathione peroxidase-like activity in vitro and antioxidant capacity of PZ 51 (Ebselen)

A synthetic seleno-organic compound, 2-phenyl-1,2-benzoisoselenazol-3(2 H)-one (PZ 51), exhibits GSH peroxidase-like activity in vitro, in contrast to its sulfur analog, PZ 25. In addition, PZ 51 behaves as an antioxidant shown by a temporary protection of rat liver microsomes against ascorbate/ ADP-Fe-induced lipid peroxidation, an effect also elicited by PZ 25 but to a smaller extent. This protection against lipid peroxidation is independent of GSH and of P-450 monooxygenase activity.