Recombinant Human Superoxide Dismutase Research Articles

Purification and characterization of recombinant human superoxide dismutase integrated with resilin-like polypeptide

Human superoxide dismutase (hSOD1) plays an important role in the aerobic metabolism and free radical eliminating process in the body. However, the production of existing SOD faces problems such as complex purification methods, high costs, and poor product stability. This experiment achieved low-cost, rapid, and simple purification of hSOD1 through ammonium sulfate precipitation method and heat resistance of recombinant protein. We constructed a recombinant protein hSOD1-LR containing a resilin-like polypeptide tag and expressed it. The interest protein was purified by ammonium sulfate precipitation method, and the results showed that the purification effect of 1.5 M (NH4)2SO4 was the best, with an enzyme activity recovery rate of 80 % after purification. Then, based on its thermal stability, further purification of the interest protein at 60 °C revealed a purification fold of up to 24 folds, and the purification effect was similar to that of hSOD1-6xHis purified by nickel column affinity chromatography. The stability of hSOD1-LR showed that the recombinant protein hSOD1-LR has better stability than hSOD-6xHis. hSOD1-LR can maintain 76.57 % activity even after 150 min of reaction at 70 °C. At same time, hSOD1-LR had activity close to 80 % at pH < 5, indicating good acid resistance. In addition, after 28 days of storage at 4 °C and 40 °C, hSOD1-LR retained 92 % and 87 % activity, respectively. Therefore, the method of purifying hSOD1-LR through salt precipitation may have positive implications for the study of SOD purification.

Application of Recombinant Human Superoxide Dismutase in Radical Concurrent Chemoradiotherapy for Cervical Cancer to Prevent and Treat Radiation-induced Acute Rectal Injury: A Multicenter, Randomized, Open-label, Prospective Trial

PurposeTo evaluate the efficacy of recombinant human superoxide dismutase (rhSOD) enemas in radiation-induced acute rectal injury (RARI) in patients with locally advanced cervical cancer. MethodsIn this phase III, randomized, open-label trial (NCT04819685) conducted across 14 medical centers in China from June 2021 to August 2023, all patients received concurrent chemoradiotherapy (CCRT). The experimental group was treated with a rhSOD enema during chemoradiotherapy, and the control group had no enema. The Common Terminology Criteria for Adverse Events (version 5.0) was used to evaluate radiotherapy-induced side effects. Endoscopic appearance was assessed using the Vienna Rectoscopy Score. The primary endpoint in the acute phase was the occurrence rate and duration of grade ≥1 (≥G1) diarrhea during CCRT. Secondary endpoints included the occurrence rate and duration of ≥G2 and ≥G3 diarrhea; ≥G1 and ≥G2 diarrhea lasting at least 3 days; and damage to the rectal mucosa due to radiotherapy measured by endoscopy. ResultsTwo-hundred-and-eighty-three patients were randomly divided into the experimental (n = 141) or control group (n = 140). The mean number of ≥G1 and ≥G2 diarrhea days were significantly lower in the experimental group than in the control group (3.5 and 0.8 days vs. 14.8 and 4.5 days, respectively; P<0.001). The incidence of ≥G2 diarrhea decreased from 53.6% to 24.1% when rhSOD enemas were used. Use of antidiarrheals was lower in the experimental group (36.2% vs. 55.7%, P<0.001). Three patients felt intolerable or abdominal pain following rhSOD enema. RARI grades in the experimental group tended to be lower than those in the control group (P=0.061). Logistic regression analysis revealed that rhSOD enema was associated with a lower occurrence rate of ≥G1/2 diarrhea for at least 3 days (P<0.001). ConclusionThe results of this study suggest that rhSOD enema is safe and significantly reduces the incidence, severity, and duration of RARI, protecting the rectal mucosa.

Cloning and expression of recombinant human superoxide dismutase 1 (hSOD1) in Bacillus subtilis 1012.

We aimed to clone and express the human Cu, Zn superoxide dismutase (hSOD1) in Bacillus subtilis 1012. Also, we investigated the expression level of hSOD1 under different induction conditions. As an essential member of the antioxidant defense system in vivo, hSOD1 has become a therapeutic agent against host diseases, such as oxygen toxicity, acute inflammation, and radiation injury. The recombinant hSOD1 was successfully secreted extracellularly into B. subtilis 1012. The expression conditions were optimized, including inoculum size, different media, temperatures, and inducer concentrations. Finally, the highest level of hSOD1 was produced as a soluble form in Super rich medium by 2% inoculum with 0.2 mM of IPTG at 37°C after the induction for 24h. Besides, 20g/L of lactose also displayed the same inductive effect on hSOD1 expression as that of IPTG (0.2 mM). Finally, the specific activity of purified hSOD1 was determined to be 1625U/mg in the presence of 800μM of Cu2+ and 20μM of Zn2+. We propose that the B.subtilis 1012-hSOD1 strain system has great potential in future industrial applications.

Influence of pharmacological composition with gel, containing superoxide dismutase and silver nanocluster monomers, and recombinant human superoxide dismutase on blood microcirculation in the gums of rats under experimental pathology

It is shown that in experiments on narcotic male rats of the Wistar line using laser Doppler flometry (LDF) under conditions of induced (ligature) experimental periodontitis (EP), indicators of blood microcirculation in the gum mucosa in the lower incisors is reduced compared with intact periodontium. When using traditional drug therapy (TDT) in the conditions of EP in the selected localization, observed a significant promotion in blood microcirculation after a 12-day period of treatment. However, on the 74th day of the study, it decreases. The use of TDT in combination with gel, containing cluster silver in the form of micelle monomers and cluster monomers of the AgK+ type and superoxide dismutase, and a new injectable form, which is a recombinant human superoxide dismutase, under EP conditions in the selected field of study both after applying the pharmacological composition for twelve days, and on the 74th day of the experiment caused an increase in microcirculation to the original values.

Effect of Traditional Drug Therapy and its Combination with Soderm®-Forte and New Injectable Form of Rexod® on the Antioxidant-prooxidant Red Blood Cell System in Rats with Experimental Periodontitis

Background.The development of periodontitis is strongly linked with both periodontopathogenic microflora and antioxidant-proxidant system disorders, which determines the need for the combined use of antimicrobial and antioxidant agents.Objectives.A comparative evaluation of the antioxidant-prooxidant system of red blood cells in rats with experimental periodontitis against the background of the traditional drug therapy and its combination with Soderm®-Forte and new injectable form of Rexod®.Methods.The authors conducted a comparative study of the effect of traditional drug therapy (TDT) and its combination with Soderm®-Forte gel containing silver nanoparticles, recombinant human superoxide dismutase (Rexod®) and new injectable form (NIF) of Rexod® on the condition of the antioxidant-prooxidant system of red blood cells in rats with experimental periodontitis (EP). The experiments were performed on Wistar male rats of 210–230 g. EP was induced by ligature method. The rats were randomly divided into 5 comparable groups of 12 animals: group 1 — with intact periodontium; group 2 — with EP; group 3 — with EP, where TDT was used, including oral irrigation with chlorhexedine (0.05% solution) and application of Septo-Pack dento-gingival dressing; group 4 — with EP, TDT and Soderm®-Forte gel (applied into the periodontal pockets of the lower incisors); group 5 — with EP, TDT in combination with Soderm®-Forte gel and NIF of Rexod®, which was injected intraperitoneally at a dose of 8000 U/kg. Treatment of rats with EP (groups 3–5) was carried out for 12 days. The observation period for all animals lasted 42 days. The biochemical tests and the antioxidant-prooxidant index were used to assess the condition of antioxidant-prooxidant system. Statistical analysis of the obtained results was carried out with parametric and non-parametric methods of Microsoft Excel (Microsoft, USA), Analysis package and AtteStat, as well as Statistica 8.0 (StatSoft, USA) software.Results.The application of TDT against the developed EP caused a moderate positive correction of the indicators of Antioxidant protection system (APS). The enrichment of TDT for the EP animals with Soderm®-Forte and especially Soderm®-Forte in combination with NIF of Rexod® to enhance the APS potential caused more significant positive dynamics of the balance in the antioxidant-prooxidant system compared to TDT.Conclusion.The combination of TDT with Soderm®-Forte and NIF of Rexod® has the most significant positive corrective effect on the condition of antioxidant-prooxidant system of red blood cells in rats with EP in comparison with the complex of TDT with Soderm®-Forte and TDT used separately.

Impact of new Rexod® injection form on blood microcirculation in rat skin

Background. Free radical oxidation underlies many morbid processes in various organs and tissues, including skin. A major antioxidant preventing the free radical impact is superoxide dismutase (SOD). A particularly valued SOD-containing agent is recombinant human SOD, Rexod, possessing a wide spectrum of medical applications in form of lyophilisate. Marketing of a new Rexod® injection preparation in form of solution requires research into its properties, including the impact on blood microcirculation in skin, especially with the lack of relevant clinical trials.Objectives. Evaluation of anticipated positive effects of the new injection form of Rexod® on blood microcirculation in rat skin.Мethods. The new Rexod® preparation impact on blood microcirculation in skin was studied with laser Doppler fl uometry by recording the following non-oscillatory parameters of the basal blood fl ow: microcirculation (MC), mean squared deviation (MSD) and coeffi cient of variation (CV). Blood fl ow fl uctuations were measured in a wavelet analysis at different frequency bands: 0.0095–0.02, 0.02–0.046, 0.07–0.15, 0.15–0.4 and 0.8–0.16 Hz corresponding to endothelial (Ae), neurogenic (An), myogenic (Am), respiratory (Ar) and pulse (Ap) rhythm amplitudes, respectively.Results. The new Rexod® injection preparation at a dose of 8000 U/kg after 15 min of intraperitoneal administration in rats caused a statistically signifi cant (p &lt; 0.05) increase in the blood fl ow fl uctuation amplitudes Ae (43.1%), An (43.4%), Am (60.8%), Ar (58.3%) and Ap (32.0%) compared to the control group. Because the Ae fl uctuations coincide with nitric oxide (NO) emission episodes, such a growth indicates an elevated NO excretion by endothelial cells leading to endothelium-dependent vasodilation. The observed changes in blood microcirculation in skin are also associated with higher integral values of the basal blood fl ow, MC (33.4% increase), MSD (14.0%) and CV (27.6%), although only MC and CV values were statistically signifi cant (p &lt; 0.05). The results obtained suggest that the new injection form of Rexod® stimulates endothelial NO excretion, exerts adrenergic relaxation in smooth muscle cells of arteriolae and arteriovenular anastomoses, reduces precapillary sphincter and arteriolar contractility through Ca2+-dependent muscle relaxation, affects respiratory modulation of the venular blood microcirculatory compartment and vegetative cardiac support, intensifi es arterial blood fl ow by increasing the cardiac output.Conclusion. Application of the new Rexod® injection form improves blood microdynamics in rat skin via stimulating endothelium-dependent and independent vasodilatation and endothelial metabolism, decelerating adrenergic vasomotor activity and peripheral microvascular resistance. These processes in coupling improve blood fl ow to nutritive microvascular bed and normalise venular outfl ow. Our results provide further insights into pharmacodynamics of recombinant human SOD (Rexod).

Hybrid Modeling and Intensified DoE: An Approach to Accelerate Upstream Process Characterization.

Process characterization is necessary in the biopharmaceutical industry, leading to concepts such as design of experiments (DoE) in combination with process modeling. However, these methods still have shortcomings, including large numbers of required experiments. The concept of intensified design of experiments (iDoE) is proposed, that is, intra-experimental shifts of critical process parameters (CPP) that combine with hybrid modeling to more rapidly screen a particular design space. To demonstrate these advantages, a comprehensive experimental design of Escherichia coli (E. coli) fed-batch cultivations (20L) producing recombinant human superoxide dismutase is presented. The accuracy of hybrid models trained on iDoE and on a fractional-factorial design is evaluated, without intra-experimental shifts, to simultaneously predict the biomass concentration and product titer of the full-factorial design. The hybrid model trained on data from the iDoE describes the biomass and product at each time point for the full-factorial design with high and adequate accuracy. The fractional-factorial hybrid model demonstrates inferior accuracy and precision compared to the intensified approach. Moreover, the intensified hybrid model only required one-third of the data for model training compared to the full-factorial description, resulting in a reduced experimental effort of >66%. Thus, this combinatorial approach has the potential to accelerate bioprocess characterization.

Recombinant Human Superoxide Dismutase and N-Acetylcysteine Addition to Exogenous Surfactant in the Treatment of Meconium Aspiration Syndrome.

This study aimed to evaluate the molecular background of N-acetylcysteine (NAC) and recombinant human superoxide dismutase (rhSOD) antioxidant action when combined with exogenous surfactant in the treatment of meconium aspiration syndrome (MAS), considering redox signalling a principal part of cell response to meconium. Young New Zealand rabbits were instilled with meconium suspension (Mec) and treated by surfactant alone (Surf) or surfactant in combination with i.v. NAC (Surf + NAC) or i.t. rhSOD (Surf + SOD), and oxygen-ventilated for 5 h. Dynamic lung-thorax compliance, mean airway pressure, PaO2/FiO2 and ventilation efficiency index were evaluated every hour; post mortem, inflammatory and oxidative markers (advanced oxidation protein products, total antioxidant capacity, hydroxynonenal (HNE), p38 mitogen activated protein kinase, caspase 3, thromboxane, endothelin-1 and secretory phospholipase A2) were assessed in pulmonary tissue homogenates. rhSOD addition to surfactant improved significantly, but transiently, gas exchange and reduced levels of inflammatory and oxidative molecules with higher impact; Surf + NAC had stronger effect only on HNE formation, and duration of treatment efficacy in respiratory parameters. In both antioxidants, it seems that targeting reactive oxygen species may be strong supporting factor in surfactant treatment of MAS due to redox sensitivity of many intracellular pathways triggered by meconium.

Liquid chromatography–tandem mass spectrometry of recombinant human extracellular superoxide dismutase (rhSOD3) in mouse plasma and its application to pharmacokinetic study

The antioxidant enzyme human extracellular superoxide dismutase (SOD3) is a promising biopharmaceutical candidate for the treatment of various diseases. To support the early development of SOD3 as a biopharmaceutical, a simple, sensitive, and rapid liquid chromatography tandem mass spectrometry procedure was developed and validated for the determination of SOD3 levels in the plasma of ICR mice. After purification with Ni-NTA magnetic beads and digestion with trypsin, SOD3 signature peptides and internal standard signature peptide (ISP) were separated via high performance liquid chromatography using a Zorbax C18 column (2.1 × 50 mm, 3.5 μm) and a mobile phase consisting of 10 mM ammonium formate, 0.1% formic acid, and acetonitrile. The analyte and ISP were detected via a tandem mass spectrometer in electrospray ionization and multiple reaction monitoring modes to select both the signature peptide for SOD3 at m/z 669 to 969 and the ISP at m/z 655 to 941 in the positive ion mode. The calibration curves were linear (r > 0.99) between 5 and 1000 μg/mL with a lower limit of quantification of 5 μg/mL. The relative standard deviation ranged from 3.08 to 8.84% while the relative error ranged from -0.13 to -9.56%. This method was successfully applied to a preclinical pharmacokinetic study of SOD3 in male ICR mice.

A Study of the Activity of Recombinant Mn-Superoxide Dismutase in the Presence of Gold and Silver Nanoparticles

Superoxide dismutase (SOD) is one of the best characterized enzyme maintaining the redox state in the cell. A bacterial expression system was used to produce human recombinant manganese SOD with a His-tag on the C-end of the protein for better purification. In addition, gold and silver nanoparticles were chemically synthesized in a variety of sizes, and then mixed with the enzyme for immobilization. Analysis by dynamic light scattering and scanning transmission electron microscopy revealed no aggregates or agglomerates of the obtained colloids. After immobilization of the protein on AuNPs and AgNPs, the conjugates were analyzed by SDS-PAGE. It was determined that SOD was adsorbed only on the gold nanoparticles. Enzyme activity was analyzed in colloids of the gold and silver nanoparticles bearing SOD. The presence of a nanoparticle did not affect enzyme activity; however, the amount of protein and size of the gold nanoparticle did influence the enzymatic activity of the conjugate. Our findings confirm that active recombinant human superoxide dismutase can be produced using a bacterial expression system, and that the enzyme can be immobilized on metal nanoparticles. The interaction between enzymes and metal nanoparticles requires further investigation.

Bronchopulmonary dysplasia: Pathogenesis and treatment.

Bronchopulmonary dysplasia (BPD) is a form of chronic lung disease of infancy, which mostly affects premature infants with significant morbidity and mortality. Premature infants who require to be treated for conditions including respiratory distress syndrome have a higher risk of developing BPD. In spite of the improvement in clinical methods, the incidence of BPD has not reduced. In the present review, the pathogenesis of BPD is described along with the treatments available at present and the role of nursing in the management of BPD. Emerging preventive therapies for BPD are also discussed, including the use of recombinant human superoxide dismutase, which has been proven effective in reducing respiratory injury and its long-term effects.

Current and Future Treatments for Persistent Pulmonary Hypertension in the Newborn.

Persistent pulmonary hypertension in newborn (PPHN) is a serious and possibly fatal syndrome characterized by sustained foetal elevation of pulmonary vascular resistance at birth. PPHN may manifest secondary to other conditions as meconium aspiration syndrome, infection and congenital diaphragmatic hernia. This MiniReview provides the reader with an overview of current and future treatment options for patients with PPHN without congenital diaphragmatic hernia. The study is based on systematic searches in the databases PubMed and Cochrane Library and registered studies on Clinicaltrials.gov investigating PPHN. Inhaled nitric oxide (iNO) is well documented for treatment of PPHN, but 30% fail to respond to iNO. Other current treatment options could be sildenafil, milrinone, prostaglandin analogues and bosentan. There are several ongoing trials with sildenafil, but evidence is lacking for the other treatments and/or for the combination with iNO. Currently, there is no evidence for effect in PPHN of other treatments, for example tadalafil, macitentan, ambrisentan, riociguat and selexipag used for pulmonary arterial hypertension in adults. Experimental studies in animal models for PPHN suggest effect of a series of approaches including recombinant human superoxide dismutase, L-citrulline, Rho-kinase inhibitors and peroxisome proliferator-activated receptor-γ agonists. We conclude that iNO is the most investigated and the only approved pulmonary vasodilator for infants with PPHN. In the iNO non-responders, sildenafil currently seems to be the best alternative either alone or in combination with iNO. Systematic and larger clinical studies are required for testing the other potential treatments of PPHN.

In Vitro Reconstitution of the Highly Active and Natively Folded Recombinant Human Superoxide Dismutase 1 Holoenzyme

AbstractSOD1 is an antioxidant enzyme that exists as a highly stable dimer in healthy humans. Each subunit contains an intramolecular disulfide bond and coordinates one zinc and one copper ion. The dimer is destabilized in the absence of the ions and disruption of the disulfide bond, which leads to the formation of small oligomers and subsequently larger insoluble aggregates. An acquired toxic function of destabilized SOD1 is postulated to be associated with amyotrophic lateral sclerosis (ALS), which is a neurodegenerative disease characterized by peripheral and central paralysis and by 3‐ to 5‐year median survival after diagnosis. In this study, we present a protocol for heterologous expression of human SOD1 in E. coli and total reconstitution of the holoenzyme, which exhibits the highest reported specific activity (four‐fold higher) of recombinant hSOD1. Biophysical characterization confirms the native state of this protein. The presented protocol provides highly active hSOD1 that will benefit in vitro investigations of this protein.

Cloning, purification and enzymatic characterization of recombinant human superoxide dismutase 1 (hSOD1) expressed in Escherichia coli.

Superoxide dismutase 1 (SOD1) is a metalloenzyme that catalyzes the disproportionation of superoxide into molecular oxygen and hydrogen peroxide. In this study, the human SOD1 (hSOD1) gene was cloned, expressed and purified. The hSOD1 gene was amplified from a pool of Bxpc3 cell cDNAs by PCR and cloned into expression vector pET-28a (+). The recombinant soluble hSOD1 was expressed in E. coli BL21 (DE3) at 37°C and purified using nickel column affinity chromatography. Soluble hSOD1 was produced with a yield of 5.9 μg/mL medium. As metal ions can have a certain influence on protein structure and activity, we researched the influences of different concentrations of Cu2+ and Zn2+ on hSOD1 activity at induction and the time of activity detection. The results implied that Cu2+ and Zn2+ do not enhance SOD1 expression and solubility; they can, however, improve the catalytic activity at induction. Meanwhile, Cu2+ and Zn2+ also enhanced the enzyme activity at the time of detection. Furthermore, most other bivalent cations had the potential to replace Zn2+ and Cu2+, and also improved enzyme activity at the time of detection.

Modified porcine surfactant enriched by recombinant human superoxide dismutase for experimental meconium aspiration syndrome

AimsCombination of exogenous surfactant with antioxidant enzyme recombinant human superoxide dismutase (rhSOD) was tested in the treatment of experimental meconium aspiration syndrome as oxidative processes play key role in its pathogenesis. Material and methodsYoung New Zealand rabbits were instilled by saline (Sal group) or by meconium suspension (Mec group). Some of meconium-instilled animals were treated by surfactant alone (Surf group) or surfactant in combination with rhSOD (Surf + SOD group) and oxygen-ventilated for 5 h. PaO2/FiO2, oxygenation index, oxygen saturation, PaCO2, ventilation efficiency index and alveolar-arterial gradient were evaluated every hour; post mortem, cells in bronchoalveolar lavage were counted, inflammatory and oxidative markers were assessed using ELISA in lung tissue homogenates. Key findingsExogenous surfactant combined with rhSOD improved oxygenation during the first hour after the treatment more than surfactant alone (p = 0.039 to 0.0001 vs. Mec and Surf group). Amelioration was also seen in CO2 elimination (p = 0.049 to 0.0096 vs. Mec group), alveolar-arterial gradient diminution (p = 0.024 to 0.0019 vs. Mec and Surf group), prevention of oxidative damage and cytokine production (p = 0.049 to 0.002 vs. Mec group). SignificanceIt seems that inhibition of oxidative signalization may be strong supporting factor in surfactant treatment of MAS.

Cloning, Purification, and Characterization of Recombinant Human Extracellular Superoxide Dismutase in SF9 Insect Cells.

A balance between production and degradation of reactive oxygen species (ROS) is critical for maintaining cellular homeostasis. Increased levels of ROS during oxidative stress are associated with disease conditions. Antioxidant enzymes, such as extracellular superoxide dismutase (EC-SOD), in the extracellular matrix (ECM) neutralize the toxicity of superoxide. Recent studies have emphasized the importance of EC-SOD in protecting the brain, lungs, and other tissues from oxidative stress. Therefore, EC-SOD would be an excellent therapeutic drug for treatment of diseases caused by oxidative stress. We cloned both the full length (residues 1–240) and truncated (residues 19–240) forms of human EC-SOD (hEC-SOD) into the donor plasmid pFastBacHTb. After transposition, the bacmid was transfected into the Sf9-baculovirus expression system and the expressed hEC-SOD purified using FLAG-tag. Western blot analysis revealed that hEC-SOD is present both as a monomer (33 kDa) and a dimer (66 kDa), as detected by the FLAG antibody. A water-soluble tetrazolium (WST-1) assay showed that both full length and truncated hEC-SOD proteins were enzymatically active. We showed that a potent superoxide dismutase inhibitor, diethyldithiocarbamate (DDC), inhibits hEC-SOD activity.

Expression, subcellular localization, and enzyme activity of a recombinant human extra-cellular superoxide dismutase in tobacco (Nicotiana benthamiana L.)

Human extracellular superoxide dismutase (hEC-SOD) is an enzyme that scavenges reactive oxygen species (ROS). Because of its antioxidant activity, hEC-SOD has been used as a therapeutic protein to treat skin disease and arthritis in mammalian systems. In this study, codon-optimized hEC-SOD was expressed in tobacco (Nicotiana benthamiana L.) via a plant-based transient protein expression system. Plant expression binary vectors containing full-length hEC-SOD (f-hEC-SOD) and modified hEC-SOD (m-hEC-SOD), in which the signal peptide and heparin-binding domain were deleted, were constructed for the cytosolic-, endoplasmic reticulum (ER)-, and chloroplast-localizations in tobacco leaf mesophyll cells. The results demonstrated that f-hEC-SOD was more efficiently expressed in the cytosolic fractions than in the ER or chloroplasts of tobacco cells. Our data further indicated that differently localized f-hEC-SOD and m-hEC-SOD displayed SOD enzyme activities, suggesting that the hEC-SODs expressed by plants may be functionally active. The f-hEC-SOD was expressed up to 3.8% of the total leaf soluble protein and the expression yield was calculated to be 313.7 μg f-hEC-SOD per g fresh weight of leaf. Overall, our results reveal that it was possible to express catalytically active hEC-SODs by means of a transient plant expression system in tobacco leaf cells.

Molecular Design of Bisphosphonate-Modified Proteins for Efficient Bone Targeting In Vivo.

To establish a rational molecular design for bisphosphonate (BP)-modified proteins for efficient bone targeting, a pharmacokinetic study was performed using a series of alendronate (ALN), a nitrogen-containing BP, modified proteins with various molecular weights and varying degrees of modification. Four proteins with different molecular weight—yeast glutathione reductase (GR; MW: 112,000 Da), bovine serum albumin (BSA; MW: 67,000 Da), recombinant human superoxide dismutase (SOD; MW: 32,000 Da), and chicken egg white lysozyme (LZM; MW: 14,000 Da)—were modified with ALN to obtain ALN-modified proteins. Pharmacokinetic analysis of the tissue distribution of the ALN-modified and unmodified proteins was performed after radiolabeling them with indium-111 (111In) by using a bifunctional chelating agent. Calculation of tissue uptake clearances revealed that the bone uptake clearances of 111In-ALN-modified proteins were proportional to the degree of ALN modification. 111In-GR-ALN and BSA-ALN, the two high-molecular-weight proteins, efficiently accumulated in bones, regardless of the degree of ALN modification. Approximately 36 and 34% of the dose, respectively, was calculated to be delivered to the bones. In contrast, the maximum amounts taken up by bone were 18 and 13% of the dose for 111In-SOD-ALN(32) and LZM-ALN(9), respectively, because of their high renal clearance. 111In-SOD modified with both polyethylene glycol (PEG) and ALN (111In-PEG-SOD-ALN) was efficiently delivered to the bone. Approximately 36% of the dose was estimated to be delivered to the bones. In an experimental bone metastasis mouse model, treatment with PEG-SOD-ALN significantly reduced the number of tumor cells in the bone of the mice. These results indicate that the combination of PEG and ALN modification is a promising approach for efficient bone targeting of proteins with a high total-body clearance.

Evaluation of lecithinized human recombinant super oxide dismutase as cardioprotectant in anthracycline-treated breast cancer patients.

Anthracycline-induced cardiotoxicity is (partly) mediated by free radical overload. A randomized study was performed in breast cancer patients to investigate whether free radical scavenger super oxide dismutase (SOD) protects against anthracycline-induced cardiotoxicity as measured by changes in echo, electrocardiography and an array of biomarkers. Eighty female, chemotherapy-naïve breast cancer patients (median age 49, range 24-67 years) scheduled for four or five courses of adjuvant 3 weekly doxorubicin plus cyclophosphamide (AC) chemotherapy, were randomly assigned to receive 80 mg PC-SOD (human recombinant SOD bound to lecithin) or placebo, administered intravenously (i.v.) immediately prior to each AC course. The primary end point was protection against cardiac damage evaluated using echocardiography, QT assessments and a set of biochemical markers for myocardial function, oxidative stress and inflammation. Assessments were performed before and during each course of chemotherapy, and at 1, 4 and 9 months after completion of the chemotherapy regimen. In all patients cardiac effects such as increases in NT-proBNP concentration and prolongation of the QTc interval were noticed. There were no differences between the PC-SOD and placebo-treated patients in systolic or diastolic cardiac function or for any other of the biomarkers used to assess the cardiac effects of anthracyclines. PC-SOD at a dose of 80 mg i.v. is not cardioprotective in patients with breast carcinoma treated with anthracyclines.

Experience of Using the Recombinant Human Superoxide Dismutase Drug in Neurological and Ophthalmological Practice

Recsod? has been used for treating epilepsy and ophthalmological disease. Oxidative stress has been demonstrated to be a pathogenic chain of these diseases. Parameters of pro- and antioxidant systems were studied in all the patients treated. Recsod? drug was shown to produce positive effect in all the patients. Improvement of patients’ clinical condition correlated with an increase in antioxidant activities. Antioxidants, in particular, the recombinant human SOD drug, proved to be effective in treatment of some neurological and ophthalmological diseases.