Rejuveinix Shows a Favorable Clinical Safety Profile in Human Subjects and Exhibits Potent Preclinical Protective Activity in the Lipopolysaccharide-Galactosamine Mouse Model of Acute Respiratory Distress Syndrome and Multi-Organ Failure

Abstract

Background: New treatment platforms that can prevent acute respiratory distress syndrome (ARDS) or reduce its mortality rate in high-risk coronavirus disease 2019 (COVID-19) patients, such as those with an underlying cancer, are urgently needed. Rejuveinix (RJX) is an intravenous formulation of anti-oxidants and anti-inflammatory agents. Its active ingredients include ascorbic acid, cyanocobalamin, thiamine hydrochloride, riboflavin 5' phosphate, niacinamide, pyridoxine hydrochloride, and calcium D-pantothenate. RJX is being developed as an anti-inflammatory and anti-oxidant treatment platform for patients with sepsis, including COVID-19 patients with viral sepsis and ARDS. Here, we report its clinical safety profile in a phase 1 clinical study (ClinicalTrials.gov Identifier: NCT03680105) and its potent protective activity in the lipopolysaccharide galactosamine (LPS-GalN) mouse model of ARDS. Methods: A phase 1, double-blind, placebo-controlled, randomized, two-part, ascending dose-escalation study was performed in participating 76 healthy volunteer human subjects in compliance with the ICH (E6) good clinical practice guidelines to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of RJX (Protocol No. RPI003; ClinicalTrials.gov Identifier: NCT03680105). The ability of RJX to prevent fatal shock, ARDS, and multi-organ failure was examined in the well-established LPS-GalN mouse model of sepsis and ARDS. Standard methods were employed for the statistical analysis of data in both studies. Findings: In the phase 1 clinical study, no participant developed serious adverse events (SAEs) or Grade 3-Grade 4 adverse events (AEs) or prematurely discontinued participation in the study. In the non-clinical study, RJX exhibited potent and dose-dependent protective activity, decreased the inflammatory cytokine responses (interleukin-6, tumor necrosis factor alpha, transforming growth factor beta), and improved survival in the LPS-GalN mouse model of sepsis and ARDS. Histopathological examinations showed that RJX attenuated the LPS-GalN induced acute lung injury (ALI) and pulmonary edema as well as liver damage. Conclusion: RJX showed a very favorable safety profile and tolerability in human subjects. It shows potential to favorably affect the clinical course of high-risk COVID-19 by preventing ARDS and its complications.

Keywords: COVID-19; acute lung injury; cancer; cytokine release syndrome; multi-organ dysfunction; pneumonia; sepsis.

FIGURE 1

Plasma Pharmacokinetic Parameters for Ascorbic Acid (Panel A); Cyanocobalamin (Panel B); Niacinamide (Panel C); Thiamine (Panel D) after Single Ascending Dose of IV Infusion of Rejuveinix in Healthy Adult and Elderly Volunteers. See text for discussion.

FIGURE 2

Dose-Dependent In Vivo Protective Activity of Prophylactic Rejuveinix (RJX) Treatments in the Lipopolysaccharide-Galactosamine (LPS-GalN) Model of acute respiratory distress syndrome (ARDS) and Multiorgan Failure. Groups of 10 BALB/C mice were treated with i.p injections of RJX (0.5, 1.05, 2.1, 4.2 ml/kg of 6‐fold diluted RJX, 0.5 ml/mouse) or vehicle (NS) 2 h before or 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) i.p. 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 (Panel A) and survival data with statistical analysis (Panels B,C) of the different treatment groups. The RJX dose levels (in ml/kg) are indicated in parentheses.

FIGURE 3

Rejuveinix (RJX) Prevents the Surge of Inflammatory Markers in the Blood Following Lipopolysaccharide-Galactosamine (LPS-GalN) Challenge in a Mouse Model of acute respiratory distress syndrome (ARDS) and Multi-organ Failure. Mice were treated with i.p injections of RJX (0.5, 1.05, 2.1, 4.2 ml/kg of 6‐fold diluted RJX, 0.5 ml/mouse) or normal saline (NS) 2 h before and 2 h post-injection of LPS-GalN. Except for untreated mice (Control), each mouse received 0.5 ml of LPS-GalN (consisting of 100 ng of LPS plus 8 mg of D-galactosamine) i.p. The RJX dose levels (in ml/kg) are indicated in parentheses. See text for discussion of results. The depicted lines represent the mean and standard deviation for the indicated inflammatory markers, namely serum IL‐6 (Panel A), TNF‐a (Panel B), and LDH levels (Panel C). ANOVA 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 compared as control group and, #p < 0.05; ##p < 0.01; ####p < 0.0001 compared as LPS/GaIN + NS group).

FIGURE 4

Rejuveinix (RJX) Prevents Pro-inflammatory Cytokine Response in the Lungs of Mice Challenged with Lipopolysaccharide-Galactosamine (LPS-GalN). Mice were treated with i.p injections of RJX (0.5, 1.05, 2.1, 4.2 ml/kg, 0.5 ml/mouse) or normal saline (NS) 2 h before and 2 h post-injection of LPS-GalN. Except for untreated mice (Control), each mouse received 0.5 ml of LPS-GalN (consisting of 100 ng of LPS plus 8 mg of d-galactosamine) i.p. The RJX dose levels (in ml/kg) are indicated in parentheses. See text for discussion of results. Depicted are the results of Western blot analyses of cytokine expression in the pooled lung tissue samples from mice in various treatment groups. Panel A: Lung IL‐1b levels; Panel B: Lung TNF‐a levels; Panel C: Lung IL‐6 levels; D: Lung TGF‐b levels; E: Immunoblot. Results are expressed as percent of control with the expression level in the lung tissue sample from untreated control mice taken as 100% for comparisons. The bar represents mean and standard deviation. Immunoblotting with an anti-actin antibody was used to ensure equal protein loading. (ANOVA and Tukey’s post-hoc test. Statistical significance between groups is shown by: ***p < 0.001; ****p < 0.0001 compared as control group and, #p < 0.05; ###p < 0.001; ####p < 0.0001 compared as LPS/GaIN + NS group).

FIGURE 5

Tissue-Level In Vivo Anti-Inflammatory and Anti-Oxidant Activity of Rejuveinix (RJX) in the Lipopolysaccharide-Galactosamine (LPS-GalN) Mouse Model of acute respiratory distress syndrome (ARDS) and Multi-organ Failure. Mice were treated with i.p injections of RJX (0.5, 1.05, 2.1, 4.2 ml/kg, 0.5 ml/mouse) or normal saline (NS) 2 h before and 2 h post-injection of LPS-GalN. Except for untreated mice (Control), each mouse received 0.5 ml of LPS-GalN (consisting of 100 ng of LPS plus 8 mg of D-galactosamine) i.p. The RJX dose levels (in ml/kg) are indicated in parentheses. See text for discussion of results. These assays were performed on the lungs of mice that died after the LPS-GalN challenge. The lungs of the mice were harvested at the time of death which occurred within 24 h for all mice. The depicted lines represent the mean and standard deviation for the indicated parameters. In (A), the score was graded according to a 5-point scale from 0 to 4 as follows: 0, l, 2, three and four represented no damage, mild damage, moderate damage, severe damage and very severe damage, respectively. Statistical significance between groups is shown by: ****p < 0.0001 compared as control group and, ##p < 0.01; ####p < 0.0001 compared as LPS/GaIN + NS group, Kruskal-Wallis test and Mann Whitney test. In (B–F), ANOVA 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 compared as control group and, #p < 0.05; ##p < 0.01; ####p < 0.0001 compared as LPS/GaIN + NS group).

FIGURE 6

Rejuveinix (RJX) Prevents Acute Lung Injury and Inflammation in the Lipopolysaccharide-Galactosamine (LPS-GalN) Mouse Model of acute respiratory distress syndrome (ARDS) and Multi-organ Failure. Mice were treated with i.p injections of RJX (0.5, 1.05, 2.1, 4.2 ml/kg, 0.5 ml/mouse) or normal saline (NS) 2 h before and 2 h post-injection of LPS-GalN. Except for untreated mice (Control), each mouse received 0.5 ml of LPS-GalN (consisting of 100 ng of LPS plus 8 mg of D-galactosamine) i.p. The RJX dose levels (in ml/kg) are indicated in parentheses. Yellow arrow: inflammatory cell infiltration; Blue arrow: Exudate; Orange arrow: hemorrhage; Green block: thickness of alveolar wall. Panel B: Blue arrow: Exudate (see B1 of Supplementary Figure S7 for an enlarged inset); Yellow arrow: inflammatory cell infiltration (see B2 of Supplementary Figure S7 for an enlarged inset); Green block: thickness of alveolar wall (see B3 of Supplementary Figure S7 for an enlarged inset). Orange arrow: hemorrhage (see B4 of Supplementary Figure S7 for an enlarged inset); H&E ×400.

FIGURE 7

Rejuveinix (RJX) Prevents Acute Liver Injury and Inflammation in the Lipopolysaccharide-Galactosamine (LPS-GalN) Mouse Model of acute respiratory distress syndrome (ARDS) and Multi-organ Failure. Mice were treated with i.p injections of RJX (0.5, 1.05, 2.1, 4.2 ml/kg, 0.5 ml/mouse) or normal saline 2 h before and 2 h post-injection of LPS-GalN. Except for untreated mice (Control), each mouse received 0.5 ml of LPS-GalN (consisting of 100 ng of LPS plus 8 mg of D-galactosamine) i.p. The RJX dose levels (in ml/kg) are indicated in parentheses. Panel A: Treatment‐naive control mouse; Panel B: Mouse challenged with LPS‐GalN and treated with vehicle (placebo) (i.e., NS); Panel C: Mouse challenged with LPS‐GalN and treated with 0.5 ml/kg 6‐fold diluted RJX; Panel D: Mouse challenged with LPS‐GalN and treated with 1.05 ml/kg 6‐fold diluted RJX; Panel E: Mouse challenged with LPS‐GalN and treated with 2.1 ml/kg 6‐fold diluted RJX; Panel D: Mouse challenged with LPS‐GalN and treated with 4.2 ml/kg 6‐fold diluted RJX; Panel B: White arrow (long): necrosis (see B1 of Supplementary Figure S11 for an enlarged inset); Black arrow: inflammatory cell infiltration (see B2 of Supplementary Figure S11 for an enlarged inset); White arrow (short): hemorrhage (see B3 of Supplementary Figure S11 for an enlarged inset). See text for a discussion of the findings. H&E ×400

FIGURE 8

In Vivo Protective Activity of Delayed-Onset Rejuveinix (RJX) Treatments in the Lipopolysaccharide-Galactosamine (LPS-GalN) Model of acute respiratory distress syndrome (ARDS) and Multiorgan Failure. Groups of 6 BALB/C mice were treated with i.p. injections of 6-fold diluted RJX (4.2 ml/kg, 0.5 ml/mouse) or vehicle [normal saline (NS)] 2 and 3 h post-injection of LPS-GalN. Each mouse received 0.5 ml of LPS-GalN (consisting of 100 ng of LPS plus 8 mg of Dgalactosamine) i.p. Percent (%) survival for each treatment group is shown as a function of time after the LPS-GalN challenge. Depicted are the survival curves for each group along with the median survival times and the log-rank p-value for the comparison of LPS-GalN + RJX group vs. the LPS/GaIN + NS group.