Evaluation of the potential of Rejuveinix plus dexamethasone against sepsis

Abstract

Aim: Our main objectives were to compare the effects of Rejuveinix (RJX), dexamethasone (DEX) and their combination on the severity of sepsis and survival outcome in an animal model of fatal sepsis. Methods: We used the LPS plus D-galactosamine mouse model of sepsis to compare the anti-inflammatory activities of RJX, dexamethasone and a combination of RJX plus DEX. Additionally, we examined the clinical feasibility and tolerability of combining RJX with DEX in COVID-19 patients in a clinical phase I study. Data were analyzed using standard methods. Results & conclusion: RJX exhibited potent anti-inflammatory activity in the murine sepsis model. The combination of RJX plus DEX was more effective than either agent alone, decreased the inflammatory cytokine responses and associated organ damage, and improved the survival outcome in mice. In the phase I clinical study, RJX plus DEX was well tolerated by COVID-19 patients.

Trial registration: ClinicalTrials.gov NCT03680105.

Keywords: COVID-19; LPS; RJX; inflammation; sepsis; vitamins.

Figure 1.. Therapeutic use of low-dose RJX plus supratherapeutic high-dose DEX combination after onset of systemic inflammation and lung injury reverses inflammatory cytokine response and systemic inflammation in the LPS-GalN mouse model of fatal cytokine storm and sepsis.

Depicted are the effects of RJX, DEX and RJX + DEX combination treatments on serum levels of IL-6 (A), TNF-α (B) and LDH (C) [23]. Groups of six BALB/c mice were treated with intraperitoneal injections of RJX (sixfold diluted, 4.2 ml/kg, 0.5 ml/mouse), DEX (6 mg/kg, 0.5 ml/mouse), RJX + DEX (0.5 ml/mouse) or vehicle (NS, 0.5 ml/mouse) 2 h post-injection of LPS-GalN. Except for untreated control mice (control), each mouse received 0.5 ml of LPS-GalN (consisting of 100 ng of LPS plus 8 mg of D-galactosamine) intraperitoneally. The depicted whisker plots represent the median and values. Welch's analysis of variance and Tamhane's T2 post hoc test were used for comparing the results among different treatment groups. Statistical significance between groups is shown by ****p < 0.0001 as compared with control group; ^###p < 0.001; ^####p < 0.0001 as compared with LPS/GaIN (2 h kill) group; ^$$$p < 0.001; ^$$$$p < 0.0001 as compared with LPS/GaIN + NS and ⁺p < 0.05; ⁺⁺p < 0.01; ⁺⁺⁺p < 0.001; ⁺⁺⁺⁺p < 0.0001 pairwise comparisons between the groups. DEX: Dexamethasone; GalN: Galactosamine; LDH: Lactate dehydrogenase; LPS: Lipopolysaccharide; NS: Normal saline; RJX: Rejuveinix.

Figure 2.. Tissue-level in vivo antioxidant activity of RJX, DEX and RJX + DEX in a mouse model of fatal cytokine storm, sepsis, systemic inflammation, ARDS and multiorgan failure.

BALB/c mice were treated with intraperitoneal injections of RJX (n = 20, sixfold diluted, 4.2 ml/kg, 0.5 ml/mouse), DEX (n = 10, 6 mg/kg, 0.5 ml/mouse), RJX + DEX (n = 20, 0.5 ml/mouse) or vehicle (NS, 0.5 ml/mouse) 2 h post-injection of LPS-GalN. We used previously published methods and statistical analysis tools [17,21]. Except for untreated control mice (control, n = 10), each mouse received 0.5 ml of LPS-GalN (consisting of 100 ng of LPS plus 8 mg of D-galactosamine) intraperitoneally. The depicted whisker plots represent the median and values. (A, C, E & F), Welch's analysis of variance and Games–Howell post hoc test were used for comparing the results among different treatment groups. (B & D) Analysis of variance and Tukey's post hoc test were used for comparing the results among different treatment groups. Statistical significance between groups is shown by ****p < 0.0001 as compared with control group; ^##p < 0.01; ^####p < 0.0001 as compared with LPS/GaIN + NS group;; ⁺⁺⁺⁺p < 0.0001 as compared with LPS/GaIN + DEX group and ^$$p < 0.01; ^$$$p < 0.001; ^$$$$p < 0.0001 pairwise comparisons between the groups. ARDS: Acute respiratory distress syndrome; DEX: Dexamethasone; GalN: Galactosamine; LPS: Lipopolysaccharide; NS: Normal saline; RJX: Rejuveinix; SOD: Superoxide dismutase.

Figure 3.. In vivo treatment activity of low-dose RJX, supratherapeutic high-dose DEX and their combination on lung and liver histopathological scores in the LPS-GalN mouse model of fatal cytokine storm and sepsis.

We used previously published methods and statistical analysis tools [17,21]. Groups of six BALB/c mice were treated with intraperitoneal injections of RJX (sixfold diluted, 4.2 ml/kg, 0.5 ml/mouse), DEX (6.0 mg/kg, 0.5 ml/mouse) or vehicle (NS, 0.5 ml/mouse) 2 h post-injection of LPS-GalN. Except for untreated control mice (control), each mouse received 0.5 ml of LPS-GalN (consisting of 100 ng of LPS plus 8 mg of D-galactosamine) intraperitoneally. The tissue damage in the (A) lungs and (B) liver were scored using previously published grading scales [17,21]. Mann–Whitney U test was used for comparing the results among different treatment groups. Statistical significance between groups is shown by ^#p < 0.05; ^##p < 0.01 as compared with LPS/GaIN (2 h kill) group, ^$p < 0.05; ^$$p < 0.01 as compared with LPS/GaIN + NS group. DEX: Dexamethasone; GalN: Galactosamine; LPS: Lipopolysaccharide; NS: Normal saline; RJX: Rejuveinix.

Figure 4.. In vivo treatment activity of RJX, DEX and RJX + DEX on lung and liver histopathological scores in the LPS-GalN mouse model of fatal cytokine storm, sepsis, systemic inflammation, ARDS and multiorgan failure.

BALB/c mice were treated with intraperitoneal injections of RJX (n = 20, sixfold diluted, 4.2 ml/kg, 0.5 ml/mouse), DEX (n = 10, 0.6 mg/kg, 0.5 ml/mouse), RJX + DEX (n = 20, 0.5 ml/mouse) or vehicle (NS, 0.5 ml/mouse) 2 h post-injection of LPS-GalN. Except for untreated control mice (control, n = 10), each mouse received 0.5 ml of LPS-GalN (consisting of 100 ng of LPS plus 8 mg of D-galactosamine) intraperitoneally. The depicted whisker plots represent the median and values. The tissue damage in the (A) lungs and (B) liver were scored using previously published grading scales [17,21]. Kruskal–Wallis test and Mann–Whitney U test were used for comparing the results among different treatment groups. (C) For severe damage, lung and liver histological scores were compared by Pearson's Chi-Square or Fisher's Exact test. ^†Fisher's exact Chi-Square test used. Statistical significance between groups is shown by ^##p < 0.01; ^###p < 0.001; ^####p < 0.0001 as compared with LPS/GaIN + NS group; ⁺p < 0.05 as compared with LPS/GaIN + DEX group and ^$p < 0.05; ^$$$p < 0.001 pairwise comparisons between the groups. ARDS: Acute respiratory distress syndrome; DEX: Dexamethasone; RJX: Rejuveinix.

Figure 5.. In vivo treatment activity of low-dose RJX, standard-dose DEX and their combination in the LPS-GalN mouse model of fatal cytokine storm, sepsis, systemic inflammation, ARDS and multiorgan failure.

BALB/c mice were treated with intraperitoneal injections of RJX (n = 20, sixfold diluted, 4.2 ml/kg, 0.5 ml/mouse), DEX (n = 10, 0.6 mg/kg, 0.5 ml/mouse), RJX + DEX (n = 20, 0.5 ml/mouse) or vehicle (NS, 0.5 ml/mouse) 2 h post-injection of LPS-GalN. Except for untreated control mice (control, n = 10), each mouse received 0.5 ml of LPS-GalN (consisting of 100 ng of LPS plus 8 mg of D-galactosamine) intraperitoneally. The cumulative proportion of mice remaining alive (survival, %) is shown as a function of time after the LPS-GalN challenge. Depicted are the Kaplan–Meier survival curves (A) and survival data with statistical analysis (B & C) of the different treatment groups. ARDS: Acute respiratory distress syndrome; DEX: Dexamethasone; LPS: Lipopolysaccharide; NS: Normal saline; RJX: Rejuveinix.