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Objective: Oxidative stress plays an important role in human disease, but antioxidant therapies are limited. Under physiological conditions superoxide is controlled by the enzyme superoxide dismutase. A recombinant human Cu/Zn superoxide dismutase (rhSOD) might open new therapeutic possibilities., Methods: Safety profile and pharmacokinetics in plasma and urine were assessed in an open label phase I study with dose-escalation. 18 healthy male volunteers received a single intravenous 10-minute infusion of 150, 300, or 600 mg rhSOD, respectively (n = 6 per dose group)., Results: rhSOD was well tolerated. Peak plasma concentrations (cmax; mean ± SD) were reached at the end of infusion, with 32.96 ± 10.31, 51.60 ± 8.23, and 103.90 ± 19.02 μg/ ml, respectively. Non-compartmental halflife was 1.06 ± 0.37, 1.59 ± 0.64, and 1.63 ± 0.28 hours. Urinary excretion (10 h) showed dose-dependent relative increases with 11.28 ± 6.46 (7.5%), 54.93 ± 15.25 (18.3%), and 191.81 ± 104.60 mg (32.0%)., Conclusions: Our results show a good safety profile and predictable pharmacokinetics of rhSOD, suggesting that therapeutic exploratory studies might be safely conducted in humans.

Reactive oxygen species (ROS) are dangerous intermediates of cellular oxygen metabolisms, and are involved in pathogenesis of a wide range of diseases. Superoxide Dismutases (SODs) are an important part of antioxidant defense systems in mammalian cells capable of reducing the harmful effect of ROS on human tissues. Unfortunately, intravenously administered SOD shows a biological half-life of a few minutes, and enteral administration fails due to biodegradation of the enzyme in the gastrointestinal system. The aim of our study was to improve biological half-life of recombinant human Cu/Zn SOD (rhSOD) within systemic circulation by liposomal encapsulation and aerosolization into the lungs. We studied the feasibility of a "needle-free" route of drug administration via the lungs combined with the sustained release effect of liposomes in an experimental pig model. We studied 14 anesthetized pigs separated into three groups. The first group (n = 5) received 15 mg aerosolized liposomal rhSOD. The second group (n = 4) received 15 mg intravenously injected liposomal rhSOD. The third group (n = 5) served as an untreated control. We determined rhSOD concentration as well as activity within the lungs by the use of bronchoalveolar lavages (BALs). RhSOD plasma concentrations were determined by blood sampling. In animals treated with aerosolized liposomal rhSOD plasma concentration of the enzyme increased and formed a plateau ranging from 19 to 21 ng/mL over the whole observational period (5 h). At the end of the experiment 5 h after completion of aerosol administration 95.2% of peak plasma concentration was found in this group. Three and 5 h after completion of aerosolization leucocytes (p = 0.54, 0.40) in BALs as well as PaO2 (p = 0.44, 0.35), PaCO(2) (p = 0.83, 0.75), and pH (p = 0.07, 0.07) in arterial blood remained unchanged compared to baseline. In animals treated with intravenously injected liposomal rhSOD, plasma concentration of the enzyme substantially increased to 3987 ng/mL but rapidly decreased over the observational period (5 h). At the end of the experiment 14.1% of peak plasma concentration was found in this group. Aerosolization of liposomal rhSOD leads to long-term and uniform uptake into systemic circulation without acute deleterious effects on respiratory tract. Compared to intravenously administered liposomal rhSOD, biologic half-life within systemic circulation was substantially prolonged in aerosol-treated animals. It could be a feasible strategy for administration of radical scavenging enzymes for treatment of systemic diseases.

A computer-based technique using a 2((5-2)) fractional factorial design was applied for screening the factors affecting the penetration effectiveness of liposomal recombinant human-Cu/Zn-superoxide dismutase. Unilamellar liposomes, containing recombinant rh-Cu/Zn-SOD in the aqueous core, are aimed at enhancing the penetration of the drug applied topically. Factors that mainly influence the chemical and physical characteristics of liposomes such as charge, molar content of cholesterol, size, surfactant and lipid were evaluated at two levels. In vitro skin penetration studies with pigskin were carried out in Franz-type diffusion cells over a period of 4 and 8h. The response variables, namely the amounts of rh-Cu/Zn-SOD penetrated into the different skin layers, were analyzed by ELISA (enzyme linked immunosorbent assay). Analysis of variance showed that the size and the cholesterol content of liposomes as well as the duration of the penetration studies have a statistically significant influence on the amount of protein found in deeper skin layers.

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.

Objective: To demonstrate the efficacy and safety of a topical gel containing liposomally encapsulated recombinant human Superoxide Dismutase (lrhSOD) in the treatment of painful Peyronie's Disease. The theoretical background is that lrhSOD, by scavenging of free oxygen radicals, might interrupt inflammatory cascades and thereby limit further disease progression., Methods: In a placebo-controlled randomized clinical trial, 39 patients with Peyronie's Disease and significant pain symptoms were treated with lrhSOD or placebo for a 4 week period. At this time, statistical evaluation of pain resolution was performed as primary study endpoint. Patients then were continued in a cross-over study design to ensure a total of 8 weeks of lrhSOD therapy for all study participants. Pain, plaque and curvature assessment was performed at study entry and every 4 weeks until week 12., Results: LrhSOD treatment resulted in a statistically significant reduction of pain (p=0.017) compared to placebo already after 4 weeks. At week 12 pain was significantly reduced in 89% of patients who all had received 8 weeks of lrhSOD therapy at that time. Response to other disease parameters was assessed at week 12: plaque size was reduced in 47% of patients, as was plaque consistence in 38%. Penile curvature was improved at 5-30 degrees in 23% of patients. The expected spontaneous disease progression rate of up to 40%, as reported by several investigators, was significantly reduced to <10% under lrhSOD therapy, and patients satisfaction was high, also consequent to the lack of therapy-related side effects observed in the present study., Conclusion: LrhSOD is an easily administrable, safe and effective local therapeutic for the painful phase of Peyronie's Disease.

Highly reactive metabolites, such as oxygen free radicals, initiate a cascade of inflammatory processes in thermally damaged skin, leading to enhanced tissue loss and delayed wound healing. The extent of tissue necrosis in the zone of stasis is of prognostic significance in the wound healing process. In this study, the effect of oxygen free radical removal by recombinant human-Cu/Zn-superoxide dismutase, given in three different formulations during the inflammatory postburn phase and wound repair, was examined. Recombinant human superoxide dismutase was either injected directly into the lesions, spread as enzyme-containing gel onto the burned tissue, or encapsulated into liposomes consisting of 1,2 dipalmitoy-sn-glycero-3-phosphocholine, cholesterol and stearylamine, suspended into a hydrophilic gel and administered to burned animals immediately after trauma. Controls were treated with plain gel or kept untreated. Edema formation, size of lesions, deepening of necrosis, and reepithelialization were examined. Results indicate that superoxide dismutase treatment resulted in reduced and faster recruitment of edema formation, smaller wound sizes, and minor tissue necrosis compared to the controls, thus resulting in significantly faster reepithelialization after 3 weeks. These animal studies on the efficacy of liposomal oxygen free radical scavenger showed accelerated wound healing in all parameters tested.

The current market position of liposomes as drug carriers is still being discussed with regard to large scale production, product characterisation and the stability of the dispersions. In this study, long term stability of liposomal suspensions with encapsulated rh-Cu/Zn-superoxide dismutase was tested according to the International Conference on Harmonisation (ICH) recommendations. The guidelines of the ICH provide general requirements for stability testing for registration and export in particular. The Institute of Applied Microbiology has examined a process to produce large amounts of pharmaceutical-grade liposomes for the treatment of inflammatory diseases by topical application. For the evaluation of its long-term storage stability, liposomal stability and protein stability were tested under appropriate conditions. Therefore, size alterations of the vesicles, protein release and protein activity were evaluated. During the observation period, neither significant alterations of the liposomes nor any protein degradation could be detected. In the light of these findings our liposomal formulations seem to provide chemical, physical and biological stability according to the definitions of the ICH. Appropriate lipid compounds and environmental factors, in combination with an optimised process and adequate storage conditions, facilitate the production of liposome dispersions suitable as drug carriers on the market.

Methods for encapsulation of a drug into liposomes should preferably result in a high encapsulation efficiency and a high encapsulation capacity. Our studies were focussed on the establishment of an efficient encapsulation procedure of the radical scavenging protein, rh-Cu/Zn-SOD, into liposomes with the cross flow injection method. Limitations to increase the encapsulation efficiency are caused by the enclosed aqueous volume, by the lipid concentration, the aspired vesicle size and the final ethanol concentration. Our research was performed to maximize the encapsulation following several strategies of injecting higher lipid concentrations into the aqueous phase. The one way triple technique, a sophisticated preparation procedure is presented, which enables three times higher encapsulation rates in comparison to standard procedures. Additionally, scalability studies demonstrate reproducibility independent of the preparation volume. Vesicle size distribution and encapsulation efficiency remain constant. Furthermore, special attention is paid on reproducibility of prepared liposomes, scale-up and on long term stability of the lipid vesicles.

Objective: To assess the efficacy and safety of liposomally encapsulated recombinant human superoxide dismutase (lrhSOD) for the treatment of Peyronie's disease., Methods: In an uncontrolled phase-2 study, 20 patients with Peyronie's disease were treated with a gel containing lrhSOD (1.5 mg/g). Patients with penile deviation of >45 degrees or plaque calcifications of >5 mm were regarded as candidates for surgical correction and excluded from this study., Results: Elimination of pain was observed in 7/13 patients (in 2 patients after only 3 days of therapy), and an almost complete resolution of pain was reported by the remaining 6/13 patients. Plaque size was reduced in 8/14 patients. Minimal improvement of penile deviation was observed in 3/12 patients. Post-therapeutic improvement of sexual function, mainly due to cessation of pain, was reported by 12/15 patients. No systemic or local side effects were observed., Conclusion: In the present study, 100% pain relief as well as a plaque size reduction in 56% of Peyronie's disease patients were observed after a maximum of 6 weeks of lrhSOD therapy. The convenience and safety of lrhSOD gel therapy were superior compared to other current regimens. The present results suggest that lrhSOD gel is a promising treatment for patients with early stage Peyronie's disease. Early institution of lrhSOD therapy may prevent disease progression to penile deviation. The present preliminary results are the basis of a placebo-controlled randomized study.

The overproduction of biochemical mediators, and activation of leukocytes and endothelial cells, generated in thermally injured tissue, gives rise to both local and distant effects. The formation of short-lived, highly reactive metabolites, such as oxygen free radicals, increases with increasing tissue ischemia, and causes further cell damage. Human recombinant Cu/Zn-superoxide dismutase (rh-Cu/Zn-SOD), an enzyme which captures these radicals, may have a beneficial effect on the postburn inflammation processes. In this study, the influence of rh-Cu/Zn-SOD application to thermally injured tissue of rabbit backskin was examined. Three different delivery strategies were compared, pure or liposomally encapsulated enzyme, or intralesionally injected rh-Cu/Zn-SOD. For control, one animal group was treated with plain gel and another group was kept untreated. At 24 h following trauma a statistically significant difference in lesion sizes between the enzyme treated and control groups was observed. After 72 h tissue swelling had diminished significantly more in the rh-Cu/Zn-SOD treated groups as compared to the control animals. The best results were achieved by spreading liposomes encapsulating the enzyme onto the wounds. Our results suggest that local treatment of burn wounds with enzymatic radical scavengers such as rh-Cu/Zn-SOD has a beneficial effect on the extent of the postburn damage.

Human recombinant superoxide dismutase (SOD), purified to homogeneity, is resolved by both conventional isoelectric focusing and immobilized pH gradients into three bands, with isoelectric points (pIs) in the pH range 4.8 to 5.1, the pI 4.80 form representing the minor component. Due to the fact that this enzyme is expressed in E. coli, N-terminal acetylation or glycosylation should be ruled out. When purified by small-scale preparative isoelectric focusing in immobilized pH gradient gels, it was found that, upon subsequent analysis, the pI 5.07 form would band in the same position, but the intermediate pI 4.92 band would split into the upper (pI 5.07) and the lower (pI 4.80) species, in nearly the same amounts, whereas the lowest pI component would always generate both the intermediate and upper forms. Enzymatic essays pointed out that these three isoforms had nearly the same specific activity, slightly higher than that of the starting material. Metal analysis indicated that all three forms contained the same metal/protein ratio, approaching the value Cu2Zn2-SOD, as reported in the literature. Circular dichroism spectra of the pI 4.80 and 5.07 forms showed the same profile in the 190-240 nm range, but marked differences in the 250-350 nm region. Treatment with EDTA produces 1-2 additional, slightly higher pI isoforms, whereas treatment with KCN generates a number of higher pI components, reaching pI values as high as pH 7, with nearly complete disappearance of the three major SOD isoforms. It is concluded that these three isoforms could represent interconvertible species, the highest pI component representing the most stable conformer.

Flocculation using pellicular charged flocculants was investigated as an alternative process to conventional chromatography in purification of recombinant proteins using human recombinant superoxide dismutase expressed in E. coli as a model system. The removal of pyrogens, proteins, debris and the yield were determined. At laboratory scale, the starting conditions were optimized to yield a stable solution and the flocculation process fitted into a purification scheme. 100 L fermentation broth was the initial volume at pilot scale. The process parameters determined at the laboratory scale were tested and the results were compared to the pilot scale. The method was also compared to an ion-exchange chromatography.