Randomized, Double-Blind, Phase I Pharmacokinetic Study of Subcutaneous Recombinant Human Superoxide Dismutase (rhSOD) in Healthy Volunteers

Numerous studies have shown that chronic inflammation plays a central role in many of the most challenging diseases of our time. These include inflammatory diseases (e.g., arthritis, rheumatism, chronic inflammatory bowel diseases, joint inflammations), metabolic disorders (e.g., diabetes, obesity), neurological conditions (e.g., Alzheimer's disease), heart diseases, atherosclerosis, cancer, asthma, autoimmune diseases, and chronic fatigue [1‐9]. One of the main pathophysiological characteristics during human inflammation is an excessive production of reactive oxygen species (ROS) by macrophages and neutrophils [10]. Furthermore, a massive production of ROS has been described during chemo- and/or radiotherapy [11‐13]. ROS include, among other oxygen-derived radicals, the highly reactive superoxide anions (\({\text{O}}_{2}^{ \cdot - }\)). Activated neutrophils adhere to endothelial structures and transmigrate to the extravascular space and release free ROS, which damage normal tissue and extracellular matrix proteins. ROS, in turn, increases the activation of neutrophils and the existing inflammatory process is further advanced, and a chain reaction triggered [14]. Superoxide dismutase (SOD) constitutes nature’s most efficient antioxidant defense against elevated ROS levels. Three isoforms of SOD exist in humans, cytosolic Cu/Zn SOD (SOD1), mitochondrial Mn SOD (SOD2) and extracellular Cu/Zn SOD (SOD3)[15]. These enzymes efficiently catalyze the dismutation of \({\text{O}}_{2}^{ \cdot - }\) into molecular oxygen and hydrogen peroxide and decreases \({\text{O}}_{2}^{ \cdot - }\) levels [10]. SOD thereby serves as an inhibitory agent of oxidative stress and tissue damage resulting from neutrophil-mediated inflammation. The protective role of endogenous SOD in counteracting the harmful effects caused by the primary production of superoxide anions is well documented. Nevertheless, some superoxide radicals may evade enzymatic dismutation owing to either elevated production of \({\text{O}}_{2}^{ \cdot - }\) or a reduced availability of the enzyme.

RhSOD supplementation may prove to be a novel therapeutic approach for ROS-dependent tissue damage induced by activated neutrophils, especially in chronic inflammatory diseases. A previously marketed Cu/Zn-SOD of bovine origin was approved for the treatment of chronic inflammatory joint diseases, but anaphylactic reactions led to the withdrawal from the pharmaceutical drug market. The present study was performed with the recombinant human enzyme Cu/Zn-Superoxide Dismutase (rhSOD, SOD1), which is of human origin in order to avoid immunogenic reactions after repeated applications. RhSOD was produced, filled and long-term stability tested under GMP by Polymun Scientific GmbH. The present study was conducted to assess the safety, tolerability, and pharmacokinetics of subcutaneous administration of rhSOD in healthy volunteers.

The data of the present study indicate that rhSOD is well tolerated by healthy volunteers after a single dose and five doses of 40 mg of rhSOD. The VAS Score and objective SEV_ratio were similar between the verum and placebo group. No serious adverse events were documented and all adverse events that were assessed as related to the study drug administration were mild.

This is the first study that investigated the plasma PK of rhSOD following subcutaneous administration. Additionally, a novel state-of-the-art objective skin imaging and analysis method was employed to enhance the assessment of the injectable drug’s safety profile, paving the way for improved traceability and a more accurate evaluation of potential ISRs in future clinical trials. Compared with a study by Schaller et al. that investigated intravenous administration of 150 mg rhSOD in healthy volunteers the elimination t_1/2 and AUC_0-∞ of rhSOD were markedly increased after subcutaneous administration of rhSOD.[17] The mean t_1/2 after administration of a single rhSOD dose was 1.06 ± 0.37 h for intravenous versus 24.1 ± 16.1 h for subcutaneous administration. The AUC_0-∞ after intravenous administration of 150 mg rhSOD was 31,990 ± 5150 h*ng/mL. Assuming dose independent PK, the calculated AUC_0-∞ for an intravenous dose of 40 mg would amount to 8541 ± 1375 h*ng/mL compared with 13,923 ± 7900 h*ng/mL after subcutaneous administration, observed in our study. For this reason, the treatment effect after subcutaneous administration could be expected to be substantially improved compared with intravenous administration. However, previous studies with bovine Cu SOD have indicated that the anti-inflammatory activity does not correlate with the PK parameters of exogenous bovine Cu SOD [19]. As an explanation, it has been hypothesized that SOD binds to the outer cell membrane and exerts its anti-inflammatory action from this binding site, even if free extracellular SOD is removed from the body [16, 19]. It remains to be investigated, if this holds true for rhSOD and further studies are needed to assess its PK and activity in a patient population.

Excessive production of reactive oxygen species, especially highly reactive \({\text{O}}_{2}^{ \cdot - }\), is an important pathophysiological mechanism that is present in many diseases, including cancer, inflammatory diseases, neurodegenerative diseases, and metabolic diseases [1, 5‐7, 9, 10, 13]. Additionally, the decreased SOD activity in the elderly has been suggested to contribute to age-related morbidity and mortality [20, 21]. Therefore, rhSOD could be a promising agent with great therapeutic potential in situations of excessively high \({\text{O}}_{2}^{ \cdot - }\) exposure and/or low endogenous SOD concentration, where reactive oxygen species can no longer be efficiently neutralized by the body. Historically, studies with SOD as a therapeutic agent have been focused on rheumatoid arthritis. Numerous clinical trials were able to show a clinical benefit of intraarticular Cu/Zn-SOD of bovine origin (Orgotein) administration in patients with inflammatory joint conditions comparable to that of intraarticular corticosteroids [22‐26]. Of these, the multicenter, double-blind, placebo controlled study by McIlwain et al. included the largest sample size with 139 patients.[26] A number of clinical studies even showed improved clinical outcomes compared with steroid treatments [27‐29]. However, injection site reactions occurred more often compared with corticosteroid treatments.

It is estimated that roughly two-thirds of DNA damage caused by radiation therapy results from indirect effects through the radiolysis of water, leading to inflammation and cellular damage mediated by ROS, especially \({\text{O}}_{2}^{ \cdot - }\) and hydroxyl radicals [30, 31]. In this context another focus of previously performed studies was the prevention of radiation therapy-induced side effects in patients with bladder cancer. While contradictory results have been published regarding the radioprotective effect, the vast majority of publications showed an advantage of SOD treatment [32‐36]. A recent randomized, open-label trial was able to demonstrate the benefit of a rhSOD enema on radiation-induced acute rectal injury in patients with locally advanced cervical cancer [37]. Furthermore, recent data suggest that microgravity-induced oxidative stress, primarily driven by increased \({\text{O}}_{2}^{ \cdot - }\) levels, may be a key factor underlying the pathophysiological effects observed in astronauts, particularly during extended space missions [38]. Microgravity-induced oxidative stress alters multiple functions, causing bone loss, skeletal muscle atrophy, myocardial and vascular abnormalities, release of pro-inflammatory cytokines, and DNA damage, which are the most observed reactions [39‐42]. Repeated subcutaneous administration of a slow-releasing SOD formulation may present a promising strategy for mitigating these effects and safeguarding astronaut health in future space exploration.

It should be noted that this study alone is insufficient to establish a complete dose–exposure–safety relationship. While in previous studies, higher SOD doses were well tolerated, no additional human data exist for subcutaneous administration of rhSOD. Further studies with varying dosing regimens are therefore required to fully characterize its safety profile. Prior to such trials, in vitro experiments could help identify the plasma concentration range in which antioxidant capacity against superoxide is enhanced. This would enable a more targeted evaluation of different dosing strategies in future clinical studies. Another limitation of the present study is that the measured SOD values must be adjusted for each subject's baseline level owing to naturally occurring SOD activity. This approach assumes that baseline SOD levels remain relatively stable throughout the sampling period. Notably, while fluctuations were observed in the SOD values of placebo patients, no clear correlation with the time of day was detected. Further limitations are the small sample size and that only healthy volunteers were included in the study. Finally, although this study showed a more stable concentration–time profile after subcutaneous than after intravenous administration, the correlation between plasma pharmacokinetics and efficacy remains undetermined.

To conclude, subcutaneous administration of multiple doses of 40 mg of rhSOD was well tolerated by healthy volunteers. Compared with intravenous administration of rhSOD, subcutaneous administration showed markedly improved PK characteristics. This route enabled sustained elevation of circulating SOD levels over an extended duration, aligning more closely with the desired physiological profile to support the effective clearance of reactive oxygen species. The present study provides important safety, tolerability, and pharmacokinetic data to support the further development of rhSOD as a therapeutic agent. Next steps include in vitro analyses of blood samples to determine the antioxidant capacity for reducing superoxide radicals in relation to plasma SOD levels, followed by studies evaluating safety, tolerability, and pharmacokinetics across additional dosing regimens. Ultimately, clinical efficacy trials in patient populations with elevated superoxide levels are warranted to assess the anti-inflammatory effects of subcutaneous rhSOD.