Treatment of Radiofibrosis with Liposomal Superoxide Dismutase. Preliminary Results of 50 Cases

Well and long established radio-fibroses have been treated successfully with a liposomal encapsulated bovine copper superoxide dismutase. After a short treatment (three weeks intramuscular injection of 5 mg twice a week) regression of the fibrosis is stable. The average size is reduced by one third and significant softening occurs in 82% of the cases. Efficiency is independent of the time between radiotherapy (origin of the fibrosis) and treatment with liposomal SOD. Complete regression even after this limited treatment is seen in cases of chronic prefibrotic inflammatory syndromes and prophylactic action in cases where the probability of fibrosis formation is certain appears to be successful. The roles of superoxide and superoxide dismutase are discussed.     

Treatment of Brain Trauma with Liposomal Superoxide Dismutase

Brain trauma was induced in rats by impact of a steel bar on the head with a force such that damage (as measured by neurological scoring) was reversible in fourteen days. Systemic treatment (intraperitoneal injections) with free bovine copper superoxide dismutase or a liposomal form of the enzyme considerably shortened recovery time to less than half Tests included cranial nerves - cornean and aural reflexes. and sensorial motricity functions — gripping reflexes. displacement reactions, recovery and flexion reflexes, equilibrium tests and spontaneous mobility. Normalisation of EEG recordings was also greatly accelerated in the case of treated animals. No changes of brain glutathione peroxidase, glutathione transferase or Mn superoxide dismutase in traumatized animals were observed. However a slight decrease in Cu-SOD occurs. Cerebral lipoperoxidation is increased in the traumatized animals compared with controls. This increase is reduced on treatment of the rats with liposomal SOD (or the free enzyme). Very small amounts of the exogenous SOD pass the brain barrier. the permeability of which is increased in traumatized animals. The enzyme is particularly concentrated in the cortex. Despite apparent total neurological recovery at 15 days for untreated traumatized animals. significant differences in EEG recordings, in percentage cerebral water content and in histological examination of brain tissue of these controls compared with treated animals were observed with a net improvement in the latter case. The results obtained with this model suggest that clinical treatment of coma states and brain traumas with liposomal superoxide dismutase may have certain advantages over orthodox treatments     

Treatment of Brain Trauma with Liposomal Superoxide Dismutase

Brain trauma was induced in rats by impact of a steel bar on the head with a force such that damage (as measured by neurological scoring) was reversible in fourteen days. Systemic treatment (intraperitoneal injections) with free bovine copper superoxide dismutase or a liposomal form of the enzyme considerably shortened recovery time to less than half Tests included cranial nerves - cornean and aural reflexes. and sensorial motricity functions — gripping reflexes. displacement reactions, recovery and flexion reflexes, equilibrium tests and spontaneous mobility. Normalisation of EEG recordings was also greatly accelerated in the case of treated animals. No changes of brain glutathione peroxidase, glutathione transferase or Mn superoxide dismutase in traumatized animals were observed. However a slight decrease in Cu-SOD occurs. Cerebral lipoperoxidation is increased in the traumatized animals compared with controls. This increase is reduced on treatment of the rats with liposomal SOD (or the free enzyme). Very small amounts of the exogenous SOD pass the brain barrier. the permeability of which is increased in traumatized animals. The enzyme is particularly concentrated in the cortex. Despite apparent total neurological recovery at 15 days for untreated traumatized animals. significant differences in EEG recordings, in percentage cerebral water content and in histological examination of brain tissue of these controls compared with treated animals were observed with a net improvement in the latter case. The results obtained with this model suggest that clinical treatment of coma states and brain traumas with liposomal superoxide dismutase may have certain advantages over orthodox treatments     

Antifibrotic effects of aldehyde dextran modified superoxide dismutase in experimental silicosis]

The present paper dwells on biomedical study of aldehyde dextran modified superoxide dismutase. Pharmacokinetic data demonstrated that modification of superoxide dismutase increased its half-time. A rat model of experimental silicosis showed that aldehyde dextran modified superoxide dismutase inhibited evolving fibrosis in the lungs. The same dose of native enzyme produced no therapeutic effect. Thus, superoxide dismutase can be considered as a potential agent for treatment of fibrosis due to its modification.     

Effects of Liposomal Superoxide Dismutase on Human Neutrophil Activity

Study of the effects of liposomal bovine copper superoxide dismutase on human polymorphonuclear neutrophils with respect to production of active oxygen species, chemotaxis and random migration, or bacterial killing show that no significant interference with neutrophil function is observed at levels far exceeding the clinical doses used in the treatment of various pathologies.     

Paraquat Suicide in a Young Woman: Results of Therapy Directed Against the Superoxide Radical

The clinical course of a young woman following two separate suicide attempts using the herbicide paraquat is reported. The patient survived an intramuscular injection of paraquat almost asymptomatically, but later exhibited a typical fatal course with fulminant proliferative pulmonary fibrosis after an intravenous injection. Fibrosis and death occurred despite a therapeutic regimen based upon a known action of paraquat, the generation of superoxide (O₂[unk]), using superoxide dismutase, α-tocopherol, and ascorbic acid in conjunction with forced diuresis and prednisone. While treatment failed explanations for the failure of therapy in this case and current therapeutic alternatives are discussed so that they may be considered when future cases are encountered.     

Superoxide dismutase and free radical pathology

Different defense systems against oxidative damage leading to pathological conditions are described. The superoxide radical plays a primary role in initiating and sustaining biological damage and is responsible for the production of other free radicals and lipoperoxides. Certain pathologies are associated with these events such as post-irradiation necrosis, or the Spanish Toxic Oil Syndrome. Superoxide dismutase has been used clinically with considerable success, particularly the liposomal form, to treat various diseases in which it has been shown that the superoxide radical plays an important role. Although the mechanism of the enzymic reaction catalysed by superoxide dismutase is now well defined, a complete explanation of the anti-inflammatory properties in vivo of the enzyme has not yet been established.     

Nebulization of liposomal rh-Cu/Zn-SOD with a novel vibrating membrane nebulizer

Liposomes are potential drug carriers for pulmonary drug delivery: They can be prepared from phospholipids, which are endogenous to the respiratory tract as a component of pulmonary surfactant, and at an appropriate dose liposomes do not pose a toxicological risk to this organ. Among the various categories of drug that benefit from liposomal entrapment is the anti-inflammatory enzyme superoxide dismutase, thus prolonging its biological half-life. The delivery of liposomes by nebulization is hampered by stability problems, like physical and chemical changes that may lead to chemical degradation and leakage of the encapsulated drug. Here we present data of liposomes aerosolized with a novel electronic nebulizer based on a vibrating membrane technology (PARI eFlow), which amends drawbacks like liposomes degradation and product release. The data acquisition included aerosol properties such as aerodynamic particle size, nebulization efficiency, and liposome leakage upon nebulization. In conclusion, this study shows the ability of the PARI eFlow to nebulize high amounts of liposomal recombinant human superoxide dismutase with reduced vesicle disruption tested in an enclosing experimental protocol.     

Nebulization of Liposomal rh-Cu/Zn–SOD with a Novel Vibrating Membrane Nebulizer

Liposomes are potential drug carriers for pulmonary drug delivery: They can be prepared from phospholipids, which are endogenous to the respiratory tract as a component of pulmonary surfactant, and at an appropriate dose liposomes do not pose a toxicological risk to this organ. Among the various categories of drug that benefit from liposomal entrapment is the anti-inflammatory enzyme superoxide dismutase, thus prolonging its biological half-life. The delivery of liposomes by nebulization is hampered by stability problems, like physical and chemical changes that may lead to chemical degradation and leakage of the encapsulated drug. Here we present data of liposomes aerosolized with a novel electronic nebulizer based on a vibrating membrane technology (PARI eFlow?), which amends drawbacks like liposomes degradation and product release. The data acquisition included aerosol properties such as aerodynamic particle size, nebulization efficiency, and liposome leakage upon nebulization. In conclusion, this study shows the ability of the PARI eFlow? to nebulize high amounts of liposomal recombinant human superoxide dismutase with reduced vesicle disruption tested in an enclosing experimental protocol.     

Involvement of hydrogen sulfide and homocysteine transsulfuration pathway in the progression of kidney fibrosis after ureteral obstruction

Hydrogen sulfide (H₂S) produced by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in the transsulfuration pathway of homocysteine plays a number of pathophysiological roles. Hyperhomocysteinemia is involved in kidney fibrosis. However, the role of H₂S in kidney fibrosis remains to be defined. Here, we investigated the role of H₂S and its acting mechanism in unilateral ureteral obstruction (UO)-induced kidney fibrosis in mice. UO decreased expressions of CBS and CSE in the kidney with decrease of H₂S concentration. Treatment with sodium hydrogen sulfide (NaHS, a H₂S producer) during UO reduced UO-induced oxidative stress with preservations of catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD) expression, and glutathione level. In addition, NaHS mitigated decreases of CBS and CSE expressions, and H₂S concentration in the kidney. NaHS treatment attenuated UO-induced increases in levels of TGF-β1, activated Smad3, and activated NF-κB. This study provided the first evidence of involvement of the transsulfuration pathway and H₂S in UO-induced kidney fibrosis, suggesting that H₂S and its transsulfuration pathway may be a potential target for development of therapeutics for fibrosis-related diseases.     

Analysis of extracellular superoxide dismutase in fibroblasts from patients with systemic sclerosis

Systemic sclerosis (SSc) is a chronic disease of connective tissue characterized by vascular damage, autoantibody production and extensive fibrosis of skin, skeletal muscles, vessels and visceral organs. Fibrosis is a biological process involving inflammatory response and reactive oxygen species (ROS) accumulation leading to fibroblast activation. Extracellular superoxide dismutase (SOD3), a copper and zinc superoxide dismutase, which is expressed in selected tissues, is secreted into the extracellular space and catalyzes the dismutation of superoxide radical to hydrogen peroxide and molecular oxygen. Moreover, SOD3 is associated to inflammatory responses in some experimental models. In this paper we analysed, by RT-PCR and immunofluorescence, SOD3 expression and intracellular localization in dermal fibroblasts from both healthy donors and patients affected by diffuse form of SSc. Moreover, we determined SOD3 enzymatic activity in fibroblast culture medium with the xanthine/xanthine oxidase method. Increased expression of SOD3 mRNA was detected in systemic sclerosis fibroblasts (SScF), as compared to control healthy fibroblasts (HF), and SOD3 immunofluorescence staining displayed a characteristic pattern of secretory proteins in both HF and SScF. Superoxide dismutase assay demonstrated that SOD3 enzymatic activity in SScF culture medium is four times more than in HF culture medium. These data suggest that an alteration in SOD3 expression and activity could be associated to SSc fibrosis.     

Topical superoxide dismutase reduces post-irradiation breast cancer fibrosis

Fibrosis following breast radiotherapy for mammary cancer is a frequent undesired effect with objective (esthetic) and subjective (pain) consequences. Forty-four patients with clinical radiofibrosis following conservative treatment of breast cancer were evaluated for the local antifibrosis effect of copper zinc superoxide dismutase [SOD(Cu/Zn)]. Extracted SOD(Cu/Zn) in a concentration of 3,600 units/mg was applied as ointment to the fibrotic affected area, b.i.d. for 90 days, in a total dose of 40 mg. The radiofibrosis intensity was scored on the basis of clinical criteria (pain and the fibrosis area) before and after SOD(Cu/Zn) treatment. SOD(Cu/Zn) was found to be effective in reducing radiation induced fibrosis by a lowering pain score in 36/39 patients and a decrease of the fibrotic area size in half cases, after 6 months. The intensity and changes of breast fibrosis were assessed also by mammography and, for the topographical distribution of subcutaneous temperature, by infrared thermography. Mammography density suggested decreased fibrosis in one third of patients. Thermography showed that fibrosis was accompanied by two zones clinically indistinctive: a central area with maximum thermal activity, called "Maximal Thermic zone" (MTZ) and a peripheral area with less thermal activity but higher than in the surrounding normal tissue, "Transitional Thermic Zone" (TTZ). Both MTZ and TTZ were significantly decreased in 36/44 patients after SOD(Cu/Zn) treatment. Clinical changes persisted all along the study. Treatment was well tolerated except for one case of local allergic reaction, and no important side effects. Molecular mechanisms involved are discussed. Further studies are running to confirm and explain these results.     

Iron-containing superoxide dismutase from Crithidia fasciculata. Purification, characterization, and similarity to Leishmanial and trypanosomal enzymes

Leishmania tropica, Trypanosoma brucei, Trypanosoma cruzi, and Crithidia fasciculata have superoxide dismutases which are insensitive to cyanide and sensitive to peroxide and azide, properties characteristic of iron-containing superoxide dismutase. Studies on the superoxide dismutase of C. fasciculata have revealed that: 1) the enzyme is located in the cytosol; 2) isozymes exist; 3) the major superoxide dismutase isozyme (superoxide dismutase 2) has Mr approximately equal to 43,000 and consists of two equal-sized subunits, each of which contains 1.4 atoms of iron. Comparisons of the amino acid content of this crithidial superoxide dismutase with those of superoxide dismutases from other sources suggests that the crithidial enzyme is closely related to bacterial Fe-containing superoxide dismutases, and only distantly related to human Mn- and Cu,Zn-containing superoxide dismutases and to Euglena Fe-containing superoxide dismutase. Attempts are now underway to develop specific inhibitors of the trypanosomatid superoxide dismutase which may be of use in the treatment of leishmaniasis or trypanosomiasis.     

Effect of ethanol on superoxide dismutase activity in cultured neural cells

Superoxide dismutase (EC 1.15.1.1) activity was investigated in several types of neural cells cultivated in the presence of 100 mM ethanol. Superoxide dismutase was inhibited by acute treatment with ethanol. Chronic treatment with ethanol specifically inhibited superoxide dismutase in glial cells. In all instances withdrawal of ethanol produced a quick return to control values. Inhibition of superoxide dismutase by ethanol may increase toxic oxygen radicals in nervous tissue.     

A rapid whole-cell assay for superoxide dismutase

A procedure is described for the intact-cell assay of superoxide dismutase(s). The technique involves the use of toluene which renders the cells permeable to the necessary components of a photochemical assay for superoxide dismutase. Whole-cell superoxide dismutase activities from a number of procaryotic and eucaryotic microorganisms compare with cell-free activities and with activities reported in the literature. Using this procedure, changing levels of superoxide dismutase are readily monitored under conditions known to modulate superoxide dismutase activity assayed in vitro. In whole cells of Escherichia coli, exogenous methyl viologen causes a marked increase in superoxide dismutase activity, whereas in the cyanobacterium, Microcystis aeruginosa, such treatment leads to a marked, light-dependent loss of whole-cell superoxide dismutase activity.     

Copper complexes of 1,10-phenanthroline and related compounds as superoxide dismutase mimetics

In a preliminary study we tested CuSO4.5H2O, (Cu(II]2[3,5-diisopropylsalicylate]4.2H2O and a number of copper complexes of substituted 1,10-phenanthrolines for superoxide anion dismutase activity. It appeared that this activity depends on the ligands involved and might be governed by the redox potential of the Cu(I) complex/Cu(II) complex couple. The strong superoxide anion dismutase activity of Cu(II)[DMP]2 complex can be expected considering its high redox potential. Rather surprisingly is the superoxide anion dismutase activity of the Cu(I)[DMP]2 complex since it involves oxidation to Cu(II)[DMP]2 complex. From regression analysis it was established that steric and field effects of the substituents of the investigated phenanthrolines play an important role in SOD activity and therefore it is concluded that complex formation is important for the superoxide dismutase-like activity.     

Photosynthetic apparatus of chilling-sensitive plants. XI. Reversibility by light of cold- and dark-induced inactivation of cyanide-sensitive superoxide dismutase activity in tomato leaf chloroplasts

(1) The inactivation of cyanide-sensitive, copper- and zinc-containing superoxide dismutase activity in chloroplasts following cold and dark storage of both detached leaves and growing tomato plants is accompanied by a decrease in copper and zinc content in both chloroplast preparations and butanol extracts of the enzyme. In contrast, this treatment of chloroplast preparations affects neither superoxide dismutase activity nor copper and zinc content. (2) Copper- and zinc-containing superoxide dismutase is not reactivated following the 2–3 h illumination of cold- and dark-stored detached leaves. However, prolonged illumination of growing seedlings results in the restoration of both the enzyme activity and copper and zinc content in chloroplasts. (3) The data suggest that the dissociation of copper, and probably of zinc, from the enzyme during cold and dark treatment of either detached leaves or growing plants and reincorporation of the metals following the illumination of intact plants are responsible for the reversible inactivation of chloroplast cyanide-sensitive superoxide dismutase of chilling-sensitive plants.     

The effect of bacterial endotoxin on synthesis of (Cu,Zn)superoxide dismutase in lungs of oxygen-exposed rats

Administration of bacterial endotoxin to rats exposed to greater than 95% O2 results in increased lung superoxide dismutase activity, decreased O2-induced lung damage, and a 3- to 4-fold improvement in survival rate (Frank, L., Yam, J., and Roberts, R. J. (1978) J. Clin. Invest, 61, 269-275). Antibodies to rat liver (Cu,Zn) superoxide dismutase were prepared and utilized to investigate the mechanism by which endotoxin treatment leads to increased lung superoxide dismutase activity. Assay of enzyme activity and of immunodetectable enzyme showed that the increased activity is due to an increase in the number of enzyme molecules rather than activation of existing enzyme. Compared to air controls, lung slices from rats exposed to greater than 95% O2 and treated with endotoxin have elevated rats of synthesis of (Cu,Zn)superoxide dismutase (51%) and of total protein (100%). Lung slices from untreated rats exposed to greater than 95% O2 have no such elevations. Endotoxin treatment thus appears to stimulate lung protein synthesis, leading to greater (Cu,Zn)superoxide dismutase activity due to an increased number of enzyme molecules.     

Permeability of bilayer lipid membranes for superoxide (O2-.) radicals

Egg yolk phosphatidylcholine monolamellar liposomes (1000 A in diameter) loaded with cytochrome c were placed into an external solution, in which superoxide radicals, O2-., were generated by a xanthine-xanthine oxidase system. The penetration of the superoxide radicals across the liposomal membrane was detected by cytochrome c reduction in the inner liposome compartment. The effects of modifiers and temperature on this process were studied. The permeability of liposomal membrane for O2-. (P'O-2 = (7.6 +/- 0.3) X 10(-8) cm/s), or HO.2 (P'HO.2 = 4.9 X 10(-4) cm/s) were determined. The effect of the transmembrane electric potential (K+ concentration gradient, valinomycin) on the permeability of liposomal membranes for O2-. were investigated. It was found that O2-. can penetrate across liposomal membrane in an uncharged form. The feasibility of penetration of superoxide radicals through liposomal membrane, predominantly via anionic channels, was demonstrated by the use of an intramolecular cholesterol-amphotericin B complex.     

A new approach for extracellular spin trapping of nitroglycerin-induced superoxide radicals both in vitro and in vivo

Anti-ischemic therapy with nitrates is complicated by the induction of tolerance that potentially results from an unwanted coproduction of superoxide radicals. Therefore, we analyzed the localization of in vitro and in vivo, glyceryl trinitrate (GTN)-induced formation of superoxide radicals and the effect of the antioxidant vitamin C and of superoxide dismutase (SOD). Sterically hindered hydroxylamines 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidin (PP-H) can be used for in vitro and in vivo quantification of superoxide radical formation. The penetration/incorporation of CP-H or PP-H and of their corresponding nitroxyl radicals was examined by fractionation of the blood and blood cells during a 1-h incubation. For monitoring in vivo, GTN-induced (130 microg/kg) O2*- formation CP-H or PP-H were continuously infused (actual concentration, 800 microM) for 90 to 120 min into rabbits. Formation of superoxide was determined by SOD- or vitamin C-inhibited contents of nitroxide radicals in the blood from A. carotis. The incubation of whole blood with CP-H, PP-H, or corresponding nitroxyl radicals clearly shows that during a 1-h incubation, as much as 8.3% of CP-H but only 0.9% of PP-H is incorporated in cytoplasm. Acute GTN treatment of whole blood and in vivo bolus infusion significantly increased superoxide radical formation as much as 4-fold. Pretreatment with 20 mg/kg vitamin C or 15,000 U/kg superoxide dismutase prevented GTN-induced nitroxide formation. The decrease of trapped radicals after treatment with extracellularly added superoxide dismutase or vitamin C leads to the conclusion that GTN increases the amount of extracellular superoxide radicals both in vitro and in vivo.