Antioxidant properties of calcium dobesilate in ischemic/reperfused diabetic rat retina.

Calcium dobesilate possesses antioxidant properties and protects against capillary permeability by reactive oxygen species in the rat peritoneal cavity, but whether a similar action can take place in the diabetic rat retina is unknown. We investigated the oral treatment of diabetic rats with calcium dobesilate on the prevention of free radical-mediated retinal injury induced by ischemia/reperfusion (90 min ischemia followed by 3 min and/or 24 h of reperfusion). Streptozotocin-induced diabetic rats were orally treated with 50 and 100 mg/kg of calcium dobesilate for 10 days (n=12 in each group). In the first series of studies, calcium dobesilate was found to significantly reduce the maldistribution of ion content in diabetic ischemic/reperfused rat retina. Thus, in diabetic rats treated with 100 mg/kg/day calcium dobesilate, ischemia/reperfusion provoked: (i) 27.5% increase in retinal Na(+) content compared to 51.8% in the vehicle-treated group (P<0.05), and (ii) 59.6% increase in retinal Ca(2+) content compared to 107.1% in vehicle-treated animals (P<0.05). In the second series of studies, calcium dobesilate was found to significantly protect diabetic rat retina against inhibition of Na(+)/K(+)-ATPase and Ca(2+)/Mg(2+)-ATPase activities by ischemia/reperfusion (54% and 41% reduction, respectively, with 100 mg/kg of calcium dobesilate) and also against changes in retinal ATP, reduced glutathione (GSH), and oxidized glutathione (GSSG) contents. In the third series of experiments, rats treated with 100 mg/kg of calcium dobesilate reduced the hydroxyl radical signal intensity to 41% (measured by electron paramagnetic resonance), induced by ischemia/reperfusion in diabetic rat retina. Finally, 100 mg/kg calcium dobesilate significantly reduced retinal edema (measured by the thickness of the inner plexiform layer) in diabetic rats. In conclusion, oral treatment with calcium dobesilate significantly protected diabetic rat retina against oxidative stress induced by ischemia/reperfusion. Whether the antioxidant properties of calcium dobesilate explain, at least in part, its beneficial therapeutic effects in diabetic retinopathy deserves further investigation.

Calcium dobesilate: pharmacological profile related to its use in diabetic retinopathy.

Calcium dobesilate (Doxium) is used in the treatment of diabetic retinopathy. Clinical studies show a slowdown of the progression of the disease after long-term oral treatment. The main action of the drug is related to a reduction of microvascular permeability as measured by different parameters and methods (vitreous fluorophotometry, retinal haemorrhages, skin capillary resistance, blood albumin leakage, blood viscosity) leading to improved visual acuity. The pharmacological activity may be explained in part by the antioxidant properties of calcium dobesilate and its action on endothelium through the synthesis of nitric oxide, increasing the endothelium-dependent relaxation. The antioxidant effect was demonstrated in different in vitro and in vivo models by decreasing the peritoneal permeability in rats induced by pro-oxidant substances. Moreover, vascular leakage was also decreased by calcium dobesilate in a reperfusion model in streptozotocin induced diabetic rats after ischaemia of the central artery of the retina. Doxium may also preserve vascular endothelial function by acting directly as antioxidant to protect lipids from peroxidation.

Apoptosis and oxidative status in peripheral blood mononuclear cells of diabetic patients.

We have compared the concentrations of intracellular glutathione (GSH), glutathione-dependent antioxidative enzymes, the cell death rate and immunophenotype profile of peripheral blood mononuclear cells (PBMC) from healthy donors and from patients with insulin-dependent type II (NIDDM) diabetes mellitus. The IDDM and NIDDM patients had above-normal absolute lymphocyte counts, whereas the percentages of CD3, CD4 adn CD8 T lymphocytes were significantly reduced. In contrast, the absolute number and percentage of B lymphocytes was higher in diabetic patients than in healthy donors. The low intracellular reduced glutathione(GSH) and the unbalanced profile of key enzymes involved in GSH metabolism, gamma-glutamyltransferase (gamma-GT) and glutathione-S-transferase (GST), account for the increased oxidative status of PBMC from diabetic patients. The plasma membranes of PBMC for diabetic patients were less permeable to propidium iodide than those of PBMC from healthy donors, indicating that the apoptotic cell death rate was lower in the cells from diabetic patients. These differences are potentially useful markers of pathogenic metabolic changes which occur during clinical diabetes and if they are confirmed could be use dot identify the onset of diabetes.

Angioprotective action of calcium dobesilate against reactive oxygen species-induced capillary permeability in the rat.

Calcium dobesilate possesses antioxidant properties in vitro, but the in vivo significance and putative angioprotective role of these properties are undefined. Here, calcium dobesilate was tested in a newly developed in vivo model of microvascular permeabilization induced by reactive oxygen species in the rat peritoneal cavity. In this model, microvascular permeabilization is equated to the rate of Evans blue extravasation toward the peritoneal cavity. Basal Evans blue extravasation (rate constant values ke = 0.0176 +/- 0.0015 h-1) was markedly and significantly increased by reactive oxygen species generated in situ, with: (i) phenazine methosulfate/NADH (delta ke(phenazine methosulfate) = 0.0419 +/- 0.0043 h-1) and (ii) xanthine/xanthine oxidase (delta ke(xo) = 0.0383 +/- 0.0010x h-1). These actions of reactive oxygen species were abolished by locally injected superoxide dismutase (i.p., 300 units/kg). Intraperitoneally given calcium dobesilate (100 mg/kg) inhibited 75-100% of reactive oxygen species-induced Evans blue extravasation. By the intravenous route, calcium dobesilate i.v. (1-50 mg/kg) dose dependently inhibited phenazine methosulfate-induced Evans blue extravasation with an ID50 of 2-5 mg/kg (full inhibition was reached at 20-50 mg/kg). After single oral administration, calcium dobesilate (5-500 mg/kg) dose dependently inhibited phenazine methosulfate-dependent Evans blue extravasation with an ID50 of 50-100 mg/kg (81% inhibition at 500 mg/kg, P < 0.003). After 7 days of oral calcium dobesilate (50 mg/kg once/day) phenazine methosulfate-induced Evans blue peritoneal extravasation was significantly reduced by half. These effects of calcium dobesilate were similar to those observed with a comparative antioxidant molecule, rutin. In conclusion, rat peritoneal microvascular permeability was strongly increased by reactive oxygen species, an effect that was significantly reduced by intraperitoneal, intravenous and oral calcium dobesilate. These results support the hypothesis that the antioxidant properties of calcium dobesilate could play a role in its angioprotective properties in vivo.

In vitro antioxidant properties of calcium dobesilate.

Calcium dobesilate, a vascular protective agent, was tested in vitro for its scavenging action against oxygen free radicals. Calcium dobesilate was as potent as rutin to scavenge hydroxyl radicals (IC50 = 1.1 vs 0.7 microM, respectively). It was also able to scavenge superoxide radicals, but with 23 times less potency than rutin (IC50 = 682 vs 30 microM, respectively). Calcium dobesilate significantly reduced platelet activating factor (PAF)-induced chemiluminescence in human PMN cells and lipid peroxidation by oxygen free radicals in human erythrocyte membranes, although these actions required calcium dobesilate concentrations > or = 50 microM. Finally, in cultured bovine aortic endothelial cells, magnesium dobesilate reduced the increase in cytosolic free calcium induced by hydrogen peroxide and inhibited phenazine methosulfate-induced cell potassium loss. In conclusion, calcium dobesilate was effective in scavenging hydroxyl radicals in vitro, at therapeutically relevant concentrations. Conversely, higher concentrations of the compound were required to scavenge superoxide radicals or to protect the cells against the deleterious effects of intracellular reactive oxygen species. Further studies in vivo are required to determine if these antioxidant properties of calcium dobesilate can play a role in its vascular protective mechanisms.

Oral administration of HSP-containing E. coli extract OM-89 has suppressive effects in autoimmunity. Regulation of autoimmune processes by modulating peripheral immunity towards hsp's?

OM-89 (Subreum) is an E. coli extract used for oral administration in the treatment of rheumatoid arthritis. It contains bacterial heat shock proteins, namely hsp60 and hsp70, which were shown to be major immunogenic constituents of the drug. Immunity to bacterial heat-shock antigens was shown to be a means of immunomodulation of (experimental) autoimmune disease and possibly inflammation in general. This was demonstrated for mycobacterial hsp60 respectively hsp70 in autoimmune disease models for arthritis, diabetes and encephalitis. Parallel to the effects displayed by immunisation with hsp, oral administration of hsp-containing OM-89 was found to modify autoimmune disease in a number of animal models, such as for arthritis, diabetes and SLE. In rats immunisation with OM-89 was found to lead to proliferative T cell responses to hsp60 and hsp70 of both E. coli and mycobacterial origin. Conversely, immunisation with hsp antigens could induce T cell reactivity specific for OM-89. Given this and the autoimmune disease modulating properties of both hsp and OM-89 it is argued that OM-89 acts via the same mechanism as proposed for hsp: that peripheral tolerance is induced at the level of regulatory T cells with specificity for heat-shock proteins. This may constitute one mode of action for OM-89 as an arthritis suppressive oral drug in man.

Calcium Dobesilate protects human peripheral blood mononuclear cells from oxidation and apoptosis.

The antioxidant effects of Calcium Dobesilate (CD, Doxium) were investigated in relation to the oxidative status, apoptosis and in vitro proliferation of human peripheral blood mononuclear cells (PBMC) isolated from healthy donors. CD alone did not modify cell growth in vitrountil 10 microM. This molecule counteracted oxidative damages generated by the high reducing sugar dR and was shown to reduce apoptosis by delaying both membrane permeability changes and DNA fragmentation. CD 10 microM affected in a time-dependent dynamics several parameters representative of the cellular oxidative status. In particular, CD significantly increased the activity of glutathione S-transferase (GST) after three days of treatment and also, but to a lower extent, the activity of gamma-glutamyltransferase (gamma-GT). Both enzymes are known to be involved in the glutathione (GSH) metabolic cycle. This enzymatic behaviour was reversed at seven days of treatment, with a significant GST decrease and a gamma-GT activation. After seven days of CD exposure, the intracellular GSH content was enhanced and this resulted in a dramatic decrease in lipid peroxidation, underlining the powerful antioxidant properties of CD in human PBMC.

Experimental immunization with anti-rheumatic bacterial extract OM-89 induces T cell responses to heat shock protein (hsp)60 and hsp70; modulation of peripheral immunological tolerance as its possible mode of action in the treatment of rheumatoid arthritis (RA).

OM-89 is a bacterial (Escherichia coli) extract used for oral administration in the treatment of RA. Given the evidence that immunity to bacterial heat shock antigens plays a critical role in the immunomodulation of arthritis and possibly inflammation in general, the purpose of the present studies was to evaluate the presence and immunogenicity of hsp in OM-89. Furthermore, we studied the effects of OM-89 in an experimental arthritis, where hsp are known to have a critical significance in disease development. In rats immunization with OM-89 was found to lead to proliferative T cell responses to hsp60 and hsp70 of both E. coli and mycobacterial origin. Conversely, immunization with hsp antigens was also found to induce T cell reactivity specific for OM-89. Based on this and the antigen specificity analysis of specific T cell lines, hsp70(DnaK) turned out to be one of the major immunogenic constituents of OM-89. Parenteral immunization with OM-89 was found to reduce resistance to adjuvant arthritis (AA), whereas oral administration was found to protect against AA. Given the arthritis-inhibitory effect of oral OM-89 in AA, it is possible that peripheral tolerance is induced at the level of regulatory T cells with specificity for hsp. This may also constitute a mode of action for OM-89 as an arthritis-suppressive oral drug.

Animal models in autoimmune disease in immunotoxicity assessment.

To assess the immunotoxicological potential of chemicals or drugs in autoimmune processes, different autoimmune disease models are available in animals. They may provide good examples of the same immunological processes which are involved in natural human autoimmune diseases. Some models, such as those of arthritis, lupus, diabetes, thyroiditis and encephalitis, are discussed. Unfortunately, the diversity of their different possibilities of immunoregulation and outcome makes it very difficult to standardise a model for the purpose of the immunotoxicology study. Some drugs show controversial activities in different models. Indeed, in some models, the autoimmune disease occurs spontaneously, in some others it has to be induced. The incidence and severity can change from one species to the other and varies with sex and strain. Stress and infection can also exert a strong influence. Until now, it is impossible to suggest one ideal animal model to study autoimmune toxicity of chemicals or drugs.

The effect of OM-89 (Subreum) on the murine model of systemic lupus erythematosus MRL-lpr/lpr.

OM-89 (Subreum), an E. coli extract, is used clinically in the treatment of rheumatoid arthritis. In this study, the authors examined the effect of OM-89 on some aspects of SLE in the murine model MRL-lpr/lpr. Animals were given OM-89 orally at a dose of 400 mg/kg weight (40 mg active substance) 5 d a week from six weeks old. It was found that mice receiving the drug reached the point of 55% (6/11) survival at the age of 33 weeks compared with 27 weeks for the control group (54%; 7/13). There was a significant increase in the delay before developing alopecia in the treated group (P < 0.01). The increase in proteinuria in the control group was significantly higher than in the treated group (P < 0.03). In a second set of experiments sacrificing the animals at week 22, a significant decrease in anti-dsDNA auto-antibodies was also found in the treated group (P < 0.05), histopathologically a less severe tubular destruction in the kidney was observed in the treated group. It can be concluded that the oral treatment of OM-89 can significantly reduce the severity of SLE in this strain of mice. It can be postulated that the administration of the bacterial extract could modulate the immune response, modifying the Th1 and Th2 balance and inducing oral tolerance.

The immunomodulator OM-89 induces ACTH and glucocorticoid secretion in rats through an IL-1 dependent pathway.

The immunomodulator OM-89 (bacterial extract from E. coli), known to act on the immune system by modulating both humoral and cellular responses, significantly increases ACTH and glucocorticoids secretion in normal Wistar rats. A comparative administration of IL-1 displays a similar pattern of release. Moreover, OM-89-induced responses are blocked by IL-1ra, the soluble receptor antagonist of IL-1. The effect of OM-89 can thus be explained, at least in part, via IL-1 which directly enhances the secretion of both stress hormones. Finally, a comparative experiment between control and stressed rats (by immobilization) shows that the responses to OM-89 measured in this study (ACTH and corticosterone secretion) are stress-modulated.

Antirheumatic E. coli extract OM-89 induces T cell responses to HSP60 and 70.

Oral administration of E. coli extract OM-89 is used in treating RA. It has been shown that immune reactivity to heat-shock proteins (hsp) is involved in immunomodulation of arthritis. We evaluated the postulated presence and immunogenicity of hsp's in OM-89. The effects of OM-89 in experimental arthritis were analyzed. Proliferative T cell responses to bacterial hsp60 and hsp70 were found in rats immunized with OM-89. And conversely, immunization with hsp antigens induced OM-89-specific T cell responses. Hsp70 (DnaK) was found to be a major immunogenic constituent of OM-89. Parenteral immunization with OM-89 reduces resistance to adjuvant arthritis (AA), whereas oral administration protects against AA. Given the arthritis inhibitory effect of oral OM-89 in AA our findings suggest peripheral tolerance induced by hsp-specific regulatory T cells as a mode of action for OM-89 as an arthritis suppressive oral drug.

[Prospective placebo-controlled double-blind study using a bacterial lysate in infections of the respiratory tract and ENT region in children]. / Prospektive placebo-kontrollierte Doppelblindstudie mit einem Bakterienlysat bei Infektionen der Atemwege und des ORL-Bereiches im Kindesalter.

94 children suffering from frequent infections of the respiratory tract and of the ear, nose and throat were treated under double-blind conditions with either a bacterial lysate (n = 45) or a placebo (n = 49). During the 6 months of the trial both treatments brought about a significant decrease in the incidence and duration of these infections as well as in the duration of concomitant antibiotherapy in comparison to the corresponding prior 6-month reference period. As these positive results recorded under the bacterial lysate and the placebo could not be differentiated statistically, the influence of meteorological and epidemiological factors as well as of the age of the children is discussed.

Double-blind cross-over study of the effect of sultosilic acid piperazine salt (A-585) and bezafibrate in primary hyperlipoproteinemia.

Sultosilic acid piperazine salt (A-585), a new lipid lowering drug, was compared with the action of bezafibrate. In 20 patients suffering from primary hyperlipoproteinemia a double-blind cross-over study was carried out. A series of parameters of the lipoprotein metabolism, as well as of fibrinolysis and platelet function, was compared before and after administration of the drugs. There was a statistically significant diminution of total cholesterol, triglycerides, beta- and pre-beta-cholesterol and an increase of alpha-cholesterol by both drugs. Furthermore, both drugs caused a significant shortening of the euglobulin lysis time and a significant diminution of platelet adhesiveness. In patients who were under oral anticoagulants the thrombotest levels were not influenced by A-585 but were frequently below the therapeutic range during bezafibrate therapy. The difference in the thrombotest during treatment with the 2 drugs was significant. A slightly elevated gamma-GT activity normalized during bezafibrate therapy but was not influenced by A-585. Other parameters did not show significant differences between the 2 drugs.