Biomedicine & Pharmacotherapy 141 (2021) 111823

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Non-clinical safety profile and pharmacodynamics of two formulations of

the anti-sepsis drug candidate Rejuveinix (RJX)
Fatih M. Uckun a, b, *, Cemal Orhan c, Joy Powell a, Emre Sahin c, Ibrahim H. Ozercan d,
Michael Volk a, Kazim Sahin c
a
Drug Discovery Program, Reven Pharmaceuticals, Westminster, CO 80234, USA
b
Department of Developmental Therapeutics, Immunology, and Integrative Medicine, Ares Pharmaceuticals, St. Paul, MN 55110, USA
c
Department of Animal Nutrition, Faculty of Veterinary, Firat University, Elazig 23119, Turkey
d
Department of Pathology, Faculty of Medicine, Firat University, Elazig 23119, Turkey

A R T I C L E I N F O A B S T R A C T

Keywords: Here, we demonstrate that the two distinct formulations of our anti-sepsis drug candidate Rejuveinix (RJX), have
Cancer a very favorable safety profile in Wistar Albino rats at dose levels comparable to the projected clinical dose levels.
Sepsis 14-day treatment with RJX-P (RJX PPP.18.1051) or RJX-B (RJX-B200702-CLN) similarly elevated the day 15
Pneumonia
tissue levels of the antioxidant enzyme superoxide dismutase (SOD) as well as ascorbic acid in both the lungs and
ARDS
Acute lung injury (ALI)
liver in a dose-dependent fashion. The activity of SOD and ascorbic acid levels were significantly higher in tissues
Multi-organ dysfunction (MODS) of RJX-P or RJX-B treated rats than vehicle-treated control rats (p < 0.0001). There was no statistically signif­
Cytokine release syndrome (CRS) icant difference between tissue SOD activity or ascorbic acid levels of rats treated with RJX-P vs. rats treated with
COVID-19 RJX-B (p > 0.05). The observed elevations of the SOD and ascorbic acid levels were transient and were no longer
detectable on day 28 following a 14-day recovery period. These results demonstrate that RJX-P and RJX-B are
bioequivalent relative to their pharmacodynamic effects on tissue SOD and ascorbic acid levels. Furthermore,
both formulations showed profound protective activity in a mouse model of sepsis. In agreement with the PD
evaluations in rats and their proposed mechanism of action, both RJX-P and RJX-B exhibited near-identical
potent and dose-dependent anti-oxidant and anti-inflammatory activity in the LPS-GalN model of ARDS and
multi-organ failure in mice.

1. Introduction
Sepsis represents a strong inflammatory response to an infection with
a potentially fatal outcome due to its complications, including acute
respiratory distress syndrome (ARDS), septic shock, multiple organ
dysfunction syndrome (MODS), and disseminated intravascular coa­
gulopathy. Our anti-sepsis drug candidate, Rejuveinix (RJX), is a ratio­
nally designed formulation of naturally occurring antioxidants and anti-
inflammatory compounds, and it is being evaluated for its clinical
impact potential for COVID-19 associated viral sepsis in a placebo-
controlled, double-blind Phase II study RPI015 [1–3]. The composi­
tion, mode of action, and recently published favorable clinical safety
profile of RJX [1–3] make it an attractive anti-inflammatory drug
candidate for the prevention and treatment of sepsis. We recently
completed a Phase 1, double-blind, placebo-controlled, randomized,
two-part, ascending dose-escalation study that evaluated its safety and
tolerability in healthy volunteers (Protocol No. RPI003; ClinicalTrials.
gov Identifier: NCT03680105) [3]. No deaths, serious adverse events
(SAEs), or Grade 3–4 adverse events (AEs) were observed in any of 57
healthy volunteers treated with RJX at dose levels ranging from 0.024
mL/kg to 0.759 mL/kg [3].
The primary objective of the present non-clinical study was to
compare the toxicity profiles and pharmacodynamics features of two
formulations of RJX that are in clinical development using Wistar Albino
rats.

* Corresponding author at: Drug Discovery Program, Reven Pharmaceuticals, Westminster, CO 80234, USA.
E-mail addresses: fatih.uckun@reven.com (F.M. Uckun), corhan@firat.edu.tr (C. Orhan), joy.powell@reven.com (J. Powell), esahin@bingol.edu.tr (E. Sahin),
ozercanih@firat.edu.tr (I.H. Ozercan), michael.volk@reven.com (M. Volk), ksahin@firat.edu.tr (K. Sahin).

https://doi.org/10.1016/j.biopha.2021.111823
Received 1 January 2021; Received in revised form 25 May 2021; Accepted 11 June 2021
Available online 17 June 2021
0753-3322/© 2021 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
F.M. Uckun et al. Biomedicine & Pharmacotherapy 141 (2021) 111823

Table 1 2. Materials and methods

Composition of Vial A (Formulation RJX-P = RJX PPP.18.1051).
Test Specification Test results 2.1. RJX formulations
Appearance Brownish-orange/reddish-orange conforms
pH 2.2–3.8 3.0 Formulation RJX-P (RJX PPP.18.1051) and RJX-B (RJX-B200702-
Sterility USP <71> Sterile CLN) are two distinct intravenous formulations of RJX. Both formula­
Endotoxin USP <85>, NMT 17.5 EU/mL – <2.5000 EU/mL tions contain ascorbic acid, cyanocobalamin, thiamine hydrochloride,
MVD = 1:3500 riboflavin 5′ phosphate, niacinamide, pyridoxine hydrochloride, and
Particulate USP <788>, The average number ≥10 µm: 15 particles/
Matter of particles present in the units container; ≥ 25 µm:
calcium D-pantothenate. Each formulation has a Part 1 (Vial A) and a
tested does not exceed 6000 per 0 particles/container Part 2 (Vial B). Vial A contains the active ingredients, and Vial B is the
container ≥10 µm and does not buffer solution that is mixed with Vial A prior to administration. The
exceed 600 per container ≥25 µm composition of Vial A for RJX-P and RJX-B are detailed in Tables 1–4.
Assay Target Lower Upper Measured % of
The preparation of doses of RJX formulations was given in Table 5.
(% Limit (% Limit (% % content Specified
content content content Target Table 6 compares the quantitative composition of RJX-P and RJX-B.
by by by Content
weight) weight) weight)
Ascorbic Acid 8.9933 8.094 9.893 9.011 100.2
2.2. Dose level justification
Niacinamide 1.188 1.069 1.307 1.186 99.8
Pyridoxine HCl 1.188 1.069 1.307 1.191 100.3 The purpose of the rat toxicity study is to compare the toxicity of two
Riboflavin 5′ 0.0253 0.023 0.028 0.025 98.8 RJX formulations to rule out unexpected, unusual severe toxicities at the
Phosphate
clinically applicable dose levels. The toxicity of RJX in rodents has been
Thiamine HCl 0.6333 0.57 0.697 0.643 101.5
Calcium 0.0293 0.026 0.032 0.028 95.6 extensively studied [1,3]. The no observed adverse effect level (NOAEL)
Pantothenate was >5 mL/kg in both Sprague Dawley rats (when administered via
Cyanocobalamin 0.0193 0.017 0.021 0.019 98.4 intra-arterial route daily for 21 days) and Beagle dogs (when adminis­
Magnesium 8.080 7.272 8.888 8.176 101.2
tered via intra-arterial route daily × 28 days) [3]. The HED of the >5
Sulfate
mL/kg NOAEL in rats is 0.8 mL/kg, and the HED of the ≥ 5 mL/kg
Notes: RJX = Rejuveinix; USP = United States Pharmacopeia; EU = endotoxin NOAEL in dogs is 2.7 mL/kg. The safety of RJX in human subjects was
Units; MVD = maximum valid dilution. examined in a Phase 1 study in healthy human volunteers, and the MTD
was determined to be 0.5 mL/kg [3]. In a clinical study in COVID-19
patients, RJX is being used at a fixed dose level of 20 mL/dose/patient
Table 2
(Protocol No. RPI015; ClinicalTrials.gov Identifier: NCT04708340). This
Composition of Vial A (Formulation RJX-B = RJX-B200702-CLN).
corresponds to an estimated average per kg dose level of 0.25–0.33
Test Specification Test results mL/kg for an adult patient weighing 60–80 kg. The selected higher dose
Appearance Brownish-orange/reddish-orange conforms level in rats is 0.5 mL (~2.5 mL/kg), which corresponds to a human
pH 2.2–3.8 3.0 equivalent dose of 2.5 × 0.16 mL/kg = 0.4 mL/kg. The dose is selected
Sterility USP <71> Sterile
as a dose level ~20% higher than the estimated average clinical dose.
Endotoxin USP <85>, NMT 17.5 EU/mL – <2.5000 EU/mL
MVD = 1:3500 The selected lower dose level in rats is 0.25 mL (1.25 mL/kg), which
Particulate USP <788>, The average number ≥10 µm: 123 particles/ corresponds to a human equivalent dose of 0.2 mL/kg. This dose was
Matter of particles present in the units container; ≥25 µm: 1 chosen as a dose level ~20% lower than the estimated low dose level in
tested does not exceed 6000 per particle/container the clinical study for an 80 kg adult patient. Both dose levels are sub­
container ≥10 µm and does not
exceed 600 per container ≥25 µm
stantially higher than the 0.7 mL/kg (i.e., 4.2 mL/kg of a 1:6 diluted
Assay Target Lower Upper Measured % of solution) effective dose level at which RJX was shown to both prevent
(% Limit (% Limit (% % content Specified and reverse cytokine storm, acute lung injury (ALI), and multi-organ
content content content Target dysfunction in a model of LPS-galactosamine induced sepsis, septic
by by by Content
shock, ARDS, and multi-organ failure [3]. The same dose levels were
weight) weight) weight)
Ascorbic Acid 7.6443 6.880 8.409 7.751 101.4 selected for the testing of RJX-B for comparison purposes. As the con­
Niacinamide 1.010 0.909 1.111 1.017 100.7 centration of the active ingredients is lower in RJX-B, the safety profile
Pyridoxine HCl 1.010 0.909 1.111 1.025 101.5 of this new formulation was postulated to be similar or better than the
Riboflavin 5′ 0.0215 0.019 0.024 0.022 102.3 safety profile of RJX-P.
Phosphate
Thiamine HCl 0.5383 0.484 0.592 0.547 101.6
Calcium 0.0249 0.022 0.027 0.023 92.4 2.3. Animals for toxicity studies
Pantothenate
Cyanocobalamin 0.0164 0.015 0.018 0.017 103.7
Magnesium 6.868 6.181 7.555 6.867 100.0 A total of 100 (50 males/50 females) Wistar albino rats with eight
Sulfate weeks were purchased from Bingol University Experimental Research
Centre, Bingol, Turkey. After one week adaptation period, rats were
Notes: RJX = Rejuveinix; USP = United States Pharmacopeia; EU = endotoxin
Units; MVD = maximum valid dilution. housed in cages of three to five same-sex rats with a 12 light-12 h dark
cycle at constant temperature and humidity. The research was carried
out at the Bingol University Experimental Animal Center with the
approval of the Animal Care and Use Committee (IACUC) at the Bingol
University (23092020-E-2310) following the Directive 2010/63/EU of
the European Parliament and of The Council with relevant ethical
guidelines for laboratory animal use and care. All animals were given ad

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F.M. Uckun et al. Biomedicine & Pharmacotherapy 141 (2021) 111823

Table 3
Composition of Vial B (Formulation RJX-P = RJX PPP.18.1051).
Test Specification Test results

Appearance Clear, colorless, free of particulate conforms

pH 6.2 – 8.4 8.3
Sterility USP <71> Sterile
Endotoxin USP <85>, NMT 17.5 EU/mL – MVD = 1:3500 <0.0500 EU/mL
Particulate Matter USP <788>, The average number of particles present in the units tested does not exceed 6000 per ≥10 µm: 10 particles/container; ≥ 25 µm: 3
container ≥ 10 µm and does not exceed 600 per container ≥25 µm particles/container
Assay Target (% content by Lower Limit (% content by Upper Limit (% content by Measured % % of Specified Target
weight) weight) weight) content Content
Sodium 8.40 7.56 9.24 8.80 104.8
Bicarbonate

Notes: RJX = Rejuveinix; USP = United States Pharmacopeia; EU = endotoxin Units; MVD = maximum valid dilution.

Table 4
Composition of Vial B (Formulation RJX-B = RJX-B200702-CLN).
Test Specification Test results

Appearance Clear, colorless, free of particulate conforms

pH 6.2 – 8.4 8.2
Sterility USP <71> Sterile
Endotoxin USP <85>, NMT 17.5 EU/mL – MVD = 1:3500 <0.0500 EU/mL
Particulate Matter USP < 788 >, The average number of particles present in the units tested does not exceed 6000 per ≥10 µm: 59 particles/container; ≥25 µm: 2
container ≥10 µm and does not exceed 600 per container ≥25 µm particles/container
Assay Target (% content by Lower Limit (% content by Upper Limit (% content by Measured % % of Specified Target
weight) weight) weight) content Content
Sodium 8.40 7.56 9.24 8.44 100.5
Bicarbonate

Notes: RJX = Rejuveinix; USP = United States Pharmacopeia; EU = endotoxin Units; MVD = maximum valid dilution.

libitum to feed and water.

Table 5
Dose preparation of RJX formulations. 2.3.1. Experimental design of a 2-week repeated dose toxicity study with
Preparation Each type of dose formulation (RJX-P and RJX-B) for the high and without a 2-week recovery period
dose group was prepared first by mixing the test article part 1 The 2-week repeated toxicity study of RJX-P and RJX-B formulation
and part 2 (Vial A + Vial B) at a 1:1 (v/v) ratio. Then, for the low
dose group, an aliquot of the dose formulations was further
was performed according to European Medicines Agency [4,5] and
diluted with saline in a 1:1 ratio OECD guidelines No. 420 with minor modifications [6]. A total of 100,
Frequency The test article dose formulation was prepared daily and used 20 rats per group (main study: 5 rats/sex/group, for recovery: 5 rats/­
within 4 h of preparation sex/group) were randomly divided into two cohorts (Table 7) as follows:
Correction No correction factor was used
(i) Rats were treated with RJX-P and RJX-B formulations intraperito­
Factor
Storage The prepared test article formulation was stored ambient and neally (i.p.) for 14 days and sacrificed on day 15, (ii) Rats were treated
Conditions protected from light until dose administration with RJX-P and RJX-B formulations i.p. for 14 days and sacrificed on day
Disposition The remaining dose formulations were discarded after 28 following a two week recovery period. In each cohort, there were 5
completion of the dose administration treatment groups, including one control group and 4 RJX treatment
Notes: RJX = Rejuveinix; RJX-P = RJX PPP.18.1051, RJX-B = RJX-B200702- groups as follows (Table 7): (i) Control (saline, i.p.), (ii) RJX-P-high dose
CLN. RJX-P (2.5 mL/kg/day RJX-P, i.p), (iii) RJX-P-low dose (1.25
mL/kg/day RJX-P, i.p), (iv) RJX-B - high dose (2.5 mL/kg/day RJX-B, i.
p), (v) RJX-B-low dose (1.25 mL/kg/day RJX-B, i.p.). Rats in Cohort 1 or
Table 6 Cohort 2 were observed for mortality, behavioral changes, clinical signs
Quantitative composition of RJX-P and RJX-B. of toxicity daily, weighed biweekly, and sacrificed on day 15 (n = 10) or
Actual mg/mL in Vial mg/mL in 1:1 A:B day 28 (n = 10) to determine the toxicity of RJX by examining their
measured % A blood chemistry profiles, blood counts, gross pathology and histopa­
content by thology of tissue specimens.
weight

Lot number RJX- RJX- RJX- RJX- RJX-P RJX-B 2.3.2. Test system justification for toxicity studies
P B P B
According to the European Medicines Agency, the rat is a standard
Ascorbic Acid 9.011 7.751 90.11 77.51 45.055 38.755 rodent species for general toxicity study [4]. Intraperitoneal dosing
Thiamine 0.643 0.547 6.43 5.47 3.215 2.735
achieves the expected systemic exposure of the drug for toxicity evalu­
Magnesium Sulfate 8.176 6.867 81.76 68.67 40.88 34.335
Cyanocobalamin 0.019 0.017 0.19 0.17 0.095 0.085
ation. The number of animals per group (5/sex/group) was the minimal
Niacinamide 1.186 1.017 11.86 10.17 5.93 5.085 number for each cohort, allowing for sufficient data point collection and
Pyridoxine 1.191 1.025 11.91 10.25 5.955 5.125 statistical analysis of several endpoints to determine systemic effects [4].
Riboflavin 0.025 0.022 0.25 0.22 0.125 0.11 The experimental design for each cohort group was explained in detail in
5′ Phosphate
Table 7.
Calcium D- 0.028 0.023 0.28 0.23 0.14 0.115
Pantothenate

Notes: RJX = Rejuveinix; RJX-P = RJX PPP.18.1051, RJX-B = RJX-B200702-

CLN.

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F.M. Uckun et al. Biomedicine & Pharmacotherapy 141 (2021) 111823

Table 7
Experimental design for each cohort.
Group Dose assignment Number of animals Dose Conc. (mg/mL) Dose level (mL/kg/day) Dose volume (mL/animal/day)/Administration route

1 Control (Saline) Cohort 1 5 M/5 F 0 0 0.5 (0.5 mL NS) / i.p.

Cohort 2 5 M/5 F
2 RJX-P - High Dose Cohort 1 5 M/5 F * 2.5 0.5 (0.5 mL RJX-P) / i.p.
Cohort 2 5 M/5 F
3 RJX-P - Low Dose Cohort 1 5 M/5 F * 1.25 0.5 (0.25 mL RJX-P + 0.25 mL NS) / i.p.
Cohort 2 5 M/5 F
4 RJX-B - High Dose Cohort 1 5 M/5 F * 2.5 0.5 (0.5 mL RJX-B) / i.p.
Cohort 2 5 M/5 F
5 RJX-B - Low Dose Cohort 1 5 M/5 F * 1.25 0.5 (0.25 mL RJX-B +0.25 mL NS) / i.p.
Cohort 2 5 M/5 F

Notes: RJX = Rejuveinix; M = male; F = female; Conc. = concentration, i.p. = intraperitoneally NS = normal saline, RJX-P = RJX PPP.18.1051, RJX-B = RJX-B200702-
CLN. * The test article is a solution formulated with multiple components of varying content concentration levels, at fixed ratios. See Tables 1 and 2 for details of target
formulation content.

Fig. 1. Effects of treatment with RJX-P or RJX-B via intraperitoneal injections on body weight of Wistar albino rats (Panel A: Male, Panel B: Female). Groups of 20
Wistar albino rats were treated with i.p injections of RJX-P (1.25 and 2.50 mL/kg/day) or RJX-B (1.25 and 2.50 mL/kg/day) or vehicle (NS) for 14 days. Groups of 10
animals in cohort 1 were sacrificed on day 15, while groups of 10 animals in cohort 2 sacrificed on day 28 following a 2 week recovery period. The bar represents
mean and SEM. ANOVA and Tukey’s post-hoc test were used to compare the results among different treatment groups.

2.4. Biochemical analysis
Blood was collected into serum-separating tubes after decapitation
by cervical dislocation. For the hematological parameters, the blood
samples were collected in EDTA coated vials and analyzed for red blood
cells (RBCs) count, white blood cells (WBCs) count, platelet count, and
hemoglobin (Hb) content were determined using an Exigo Hematology
Analyzer (Exigo Boule. Medical AB, Stockholm). For biochemical ana­
lyses, the blood samples of all rats were centrifuged at 3000 rpm for 10
min, and obtained serums were stored at − 80 ◦ C until analysis day.
Serum concentrations of glucose, total protein (TP), albumin (ALB),
globulin (GLB), aspartate aminotransferase (AST), alanine

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F.M. Uckun et al. Biomedicine & Pharmacotherapy 141 (2021) 111823

Table 8
Effects of 14-day treatment with RJX-P or RJX-B via intraperitoneal injections on safety pharmacology laboratory parameters of Wistar albino rats.
Parameters Control (NS) RJX-P (0.5 mL) RJX-P (0.25 mL) RJX-B (0.5 mL) RJX-B (0.25 mL)

Glucose, mg/dL M 116.60 ± 3.67 115.60 ± 4.80 115.40 ± 3.01 117.20 ± 4.33 119.20 ± 3.76
F 123.00 ± 3.35 120.20 ± 3.38 118.20 ± 3.64 119.20 ± 3.6 114.60 ± 2.38
BUN, mg/dL M 20.04 ± 1.14 22.52 ± 0.78 23.56 ± 1.14 21.82 ± 0.62 22.82 ± 0.87
F 22.76 ± 1.28 22.96 ± 0.93 22.44 ± 0.84 24.90 ± 1.63 21.58 ± 0.53
TP, g/dL M 7.06 ± 0.31 7.78 ± 0.08 7.20 ± 0.36 7.54 ± 0.18 7.48 ± 0.07
F 7.28 ± 0.35 7.58 ± 0.11 7.78 ± 0.35 7.66 ± 0.21 7.50 ± 0.14
ALB, g/dL M 3.44 ± 0.07 3.64 ± 0.07 3.34 ± 0.08 3.62 ± 0.13 3.48 ± 0.10
F 4.08 ± 0.08 3.72 ± 0.07 3.96 ± 0.11 3.74 ± 0.12 3.70 ± 0.09
GLOB, g/dL M 3.62 ± 0.19 3.80 ± 0.23 3.42 ± 0.14 3.64 ± 0.07 3.70 ± 0.08
F 3.82 ± 0.15 3.88 ± 0.12 4.12 ± 0.22 3.98 ± 0.06 3.78 ± 0.08
ALT, U/L M 127.20 ± 6.46 135.80 ± 4.92 128.60 ± 1.44 144.00 ± 3.78 134.40 ± 5.53
F 123.80 ± 7.16 130.80 ± 5.39 134.40 ± 2.66 117.00 ± 2.63 136.00 ± 4.70
AST, U/L M 151.40 ± 7.49 162.20 ± 7.61 133.20 ± 3.76 147.80 ± 22.65 151.00 ± 4.93
F 137.60 ± 11.60 163.00 ± 7.16 167.00 ± 2.55 161.80 ± 17.01 178.20 ± 14.29
ALP, U/L M 258.60 ± 10.58 274.00 ± 18.8 253.60 ± 10.82 273.40 ± 13.17 265.60 ± 8.45
F 265.00 ± 16.06 267.00 ± 16.64 274.20 ± 5.77 261.00 ± 14.74 219.00 ± 16.81
TBIL, mg/dL M 0.24 ± 0.02 0.22 ± 0.01 0.22 ± 0.01 0.27 ± 0.01 0.26 ± 0.01
F 0.22 ± 0.03 0.21 ± 0.003 0.23 ± 0.03 0.22 ± 0.01 0.19 ± 0.01
Creatinine, mg/dL M 37.80 ± 1.80 39.00 ± 1.26 36.20 ± 1.43 38.40 ± 1.44 38.80 ± 0.97
F 35.60 ± 1.44 37.20 ± 1.53 35.80 ± 0.66 39.00 ± 1.67 38.80 ± 0.73
WBC(x103/µL) M 12.08 ± 2.15 10.30 ± 1.59 11.98 ± 1.13 10.96 ± 0.72 10.86 ± 0.31
F 9.62 ± 1.44 9.24 ± 0.66 12.76 ± 1.75 11.70 ± 1.28 9.40 ± 0.57
ALC(x103/µL) M 8.32 ± 1.06 7.40 ± 1.26 8.18 ± 0.73 7.52 ± 0.51 7.00 ± 0.43
F 6.80 ± 1.53 6.52 ± 0.49 9.62 ± 1.48 8.20 ± 0.55 7.18 ± 0.46
MON#(x103/µL) M 0.70 ± 0.11 0.68 ± 0.10 0.80 ± 0.06 0.54 ± 0.02 0.72 ± 0.08
F 0.64 ± 0.18 0.48 ± 0.10 0.70 ± 0.04 0.58 ± 0.04 0.42 ± 0.04
ANC (x103/µL) M 3.00 ± 0.41 1.88 ± 0.31 2.68 ± 0.40 2.48 ± 0.18 2.76 ± 0.43
F 1.38 ± 0.27 1.86 ± 0.12 1.94 ± 0.26 2.46 ± 0.84 1.14 ± 0.12
EOS#(x103/µL) M 0.54 ± 0.13 0.34 ± 0.10 0.32 ± 0.05 0.42 ± 0.05 0.38 ± 0.06
F 0.32 ± 0.14 0.38 ± 0.09 0.50 ± 0.14 0.46 ± 0.08 0.66 ± 0.17
LYM% M 68.40 ± 3.01 71.08 ± 1.54 68.90 ± 2.03 68.40 ± 1.28 64.76 ± 4.27
F 71.76 ± 3.39 70.62 ± 1.54 74.92 ± 1.80 71.74 ± 3.93 76.20 ± 0.77
MON% M 6.18 ± 0.59 6.36 ± 0.41 6.14 ± 0.40 4.96 ± 0.21 6.22 ± 0.58
F 5.20 ± 0.47 4.54 ± 0.71 5.48 ± 0.47 4.60 ± 0.3 4.26 ± 0.16
NEU% M 21.46 ± 2.73 18.74 ± 0.93 22.24 ± 1.51 22.72 ± 0.82 25.46 ± 3.54
F 19.42 ± 2.63 20.78 ± 1.86 15.70 ± 1.56 19.88 ± 4.42 12.82 ± 1.26
EOS% M 3.96 ± 0.72 3.82 ± 1.18 2.72 ± 0.45 3.92 ± 0.34 3.56 ± 0.54
F 3.62 ± 1.73 4.06 ± 0.64 3.90 ± 0.89 3.78 ± 0.65 6.72 ± 1.55
RBC (x106/µL) M 7.77 ± 0.23 8.65 ± 0.12 7.44 ± 0.65 8.43 ± 0.07 8.38 ± 0.23
F 7.89 ± 0.24 7.40 ± 0.29 7.94 ± 0.22 7.24 ± 0.84 8.11 ± 0.10
HGB, g/dL M 17.08 ± 0.65 15.92 ± 2.10 15.42 ± 1.13 17.76 ± 0.22 17.32 ± 0.46
F 16.36 ± 0.34 16.34 ± 0.33 16.92 ± 0.59 15.46 ± 1.72 17.4 ± 0.16
HCT, % M 50.86 ± 1.60 51.92 ± 1.11 46.94 ± 1.55 48.36 ± 0.83 52.04 ± 3.14
F 46.12 ± 1.19 46.62 ± 0.46 47.86 ± 1.32 44.06 ± 5.16 49.82 ± 0.55
MCV, fL M 65.56 ± 2.26 59.96 ± 0.62 64.58 ± 4.53 57.32 ± 0.56 62.42 ± 4.86
F 58.52 ± 1.32 63.42 ± 2.89 60.34 ± 1.24 60.82 ± 0.81 61.46 ± 0.80
MCH, pg M 21.98 ± 0.38 20.66 ± 0.20 20.86 ± 0.39 21.04 ± 0.09 20.70 ± 0.45
F 20.76 ± 0.35 22.14 ± 0.54 21.34 ± 0.39 21.46 ± 0.33 21.44 ± 0.20
MCHC, g/Dl M 33.72 ± 1.48 34.50 ± 0.22 32.70 ± 1.57 36.70 ± 0.22 33.88 ± 2.42
F 35.54 ± 0.3 35.08 ± 0.93 35.42 ± 1.14 35.28 ± 0.38 34.90 ± 0.13
RDWa, fL M 47.84 ± 3.79 42.56 ± 0.54 53.04 ± 5.33 45.04 ± 3.96 49.70 ± 6.11
F 39.84 ± 1.25 46.38 ± 4.54 43.94 ± 2.80 44.16 ± 1.87 41.42 ± 0.47
RDW, % M 17.02 ± 0.62 16.96 ± 0.22 18.20 ± 0.40 16.34 ± 0.07 17.12 ± 0.53
F 16.24 ± 0.17 17.14 ± 0.82 16.90 ± 0.38 16.50 ± 0.29 15.82 ± 0.15
PLT(x103/µL) M 672.20 ± 49.23 770.00 ± 96.55 699.00 ± 64.86 684.40 ± 55.26 689.60 ± 41.66
F 774.00 ± 56.95 835.00 ± 56.50 875.40 ± 57.45 760.40 ± 59.12 756.6 ± 25.51
MPV, fL M 6.62 ± 0.02 6.60 ± 0.07 6.28 ± 0.18 6.64 ± 0.12 6.48 ± 0.11
F 6.90 ± 0.2 6.52 ± 0.06 6.82 ± 0.18 6.88 ± 0.26 6.84 ± 0.20

Notes: Data show the mean ± SEM values for the depicted laboratory parameters. ANOVA and Tukey’s post-hoc test were used for comparing the results among
different treatment groups. M: Male, F: Female, RJX: Rejuveinix, RJX-P: RJX PPP.18.1051, RJX-B: RJX-B200702-CLN, BUN: blood urea nitrogen, TP: total protein,
ALB: albumin, GLOB: globulin, ALT: alanine aminotransferase, AST: aspartate aminotransferase, ALP: alkaline phosphatase, TBIL: total bilirubin, WBC: white blood
cell count, ALC: absolute lymphocyte count, MON: monocytes, ANC: absolute neutrophil count, EOS: eosinophil, LYM: lymphocytes, NEU: neutrophils, RBC: red blood
cell, HGB: hemoglobin, HCT: hematocrit, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration,
RDW: red cell distribution width, PLT: platelet, MPV: mean platelet volume.

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F.M. Uckun et al. Biomedicine & Pharmacotherapy 141 (2021) 111823

Table 9
Effects of 14-day treatment with RJX-P or RJX-B via intraperitoneal injections followed by a 14-day recovery period on safety pharmacology laboratory parameters of
Wistar albino rats.
Parameters Control (NS) RJX-P (0.5 mL) RJX-P (0.25 mL) RJX-B (0.5 mL) RJX-B (0.25 mL)

Glucose, mg/dL M 113.60 ± 2.86 113.40 ± 3.40 115.60 ± 3.25 111.60 ± 3.40 115.80 ± 3.64
F 117.00 ± 2.79 116.00 ± 3.27 115.20 ± 2.65 114.60 ± 2.79 115.00 ± 3.39
BUN, mg/dL MF 21.58 ± 1.36 21.94 ± 0.52 22.30 ± 1.05 23.52 ± 0.89 22.92 ± 0.74
20.98 ± 1.04 24.06 ± 0.4 23.80 ± 1.14 23.56 ± 0.57 21.92 ± 0.52
TP, g/dL MF 6.86 ± 0.23 7.34 ± 0.18 6.76 ± 0.18 7.02 ± 0.21 7.18 ± 0.15
7.06 ± 0.28 7.42 ± 0.23 7.72 ± 0.19 7.48 ± 0.15 7.28 ± 0.17
ALB, g/dL MF 3.82 ± 0.18 3.52 ± 0.10 3.36 ± 0.12 3.68 ± 0.09 3.56 ± 0.12
3.90 ± 0.15 3.42 ± 0.11 3.66 ± 0.13 3.72 ± 0.09 3.54 ± 0.11
GLOB, g/dL M 3.48 ± 0.19 3.78 ± 0.14 3.54 ± 0.13 3.66 ± 0.05 3.58 ± 0.09
F 3.74 ± 0.14 3.68 ± 0.09 3.82 ± 0.16 3.80 ± 0.12 3.68 ± 0.09
ALT, U/L M 117.20 ± 6.87 137.60 ± 2.20 125.60 ± 3.19 125.80 ± 3.83 130.80 ± 3.38
F 112.80 ± 6.75 132.00 ± 3.46 134.00 ± 3.7 122.20 ± 5.42 131.80 ± 2.29
AST, U/L M 144.40 ± 2.20 162.20 ± 7.61 164.20 ± 11.36 163.60 ± 10.29 180.00 ± 12.34
F 146.40 ± 6.44 183.80 ± 6.38 183.40 ± 9.17 177.80 ± 22.83 186.00 ± 11.87
ALP, U/L M 251.00 ± 6.92 262.20 ± 8.91 302.20 ± 5.86 296.00 ± 17.74 263.00 ± 9.57
F 256.00 ± 6.87 295.00 ± 10.58 268.20 ± 5.68 250.60 ± 11.35 249.80 ± 12.34
TBIL, mg/dL M 0.24 ± 0.01 0.22 ± 0.01 0.23 ± 0.01 0.26 ± 0.01 0.26 ± 0.01
F 0.20 ± 0.01 0.22 ± 0.01 0.23 ± 0.01 0.23 ± 0.003 0.22 ± 0.01
Creatinine, mg/dL M 38.00 ± 1.45 38.40 ± 1.12 36.20 ± 1.11 40.20 ± 0.92 39.20 ± 1.11
F 39.80 ± 1.11 40.80 ± 0.73 36.00 ± 1.05 38.80 ± 0.86 38.40 ± 1.12
WBC(x103/µL) M 14.06 ± 0.82 12.16 ± 1.72 12.02 ± 0.95 10.80 ± 1.93 10.86 ± 0.69
F 9.16 ± 1.17 8.38 ± 1.07 8.54 ± 1.46 10.08 ± 1.42 8.58 ± 0.43
ALC(x103/µL) M 9.26 ± 0.63 8.94 ± 1.40 8.20 ± 0.55 7.87 ± 1.69 7.26 ± 0.69
F 6.40 ± 0.62 5.98 ± 0.80 6.46 ± 1.22 7.33 ± 1.19 6.33 ± 0.28
MON#(x103/µL) M 0.86 ± 0.10 0.70 ± 0.08 0.82 ± 0.07 0.67 ± 0.09 0.70 ± 0.06
F 0.50 ± 0.09 0.50 ± 0.05 0.50 ± 0.06 0.55 ± 0.05 0.43 ± 0.06
ANC (x103/µL) M 3.46 ± 0.39 2.24 ± 0.34 2.50 ± 0.35 1.87 ± 0.15 2.48 ± 0.39
F 1.86 ± 0.60 1.84 ± 0.32 1.30 ± 0.20 1.65 ± 0.34 1.43 ± 0.23
EOS#(x103/µL) M 0.48 ± 0.11 0.28 ± 0.14 0.50 ± 0.09 0.40 ± 0.06 0.42 ± 0.06
F 0.40 ± 0.09 0.35 ± 0.14 0.28 ± 0.07 0.55 ± 0.05 0.40 ± 0.12
LYM% M 66.02 ± 2.20 72.92 ± 1.53 68.62 ± 1.06 71.93 ± 2.40 66.64 ± 3.87
F 70.74 ± 2.83 71.06 ± 2.01 75.14 ± 1.67 72.10 ± 3.51 74.18 ± 3.48
MON% M 5.68 ± 0.38 5.54 ± 0.46 6.34 ± 0.20 5.70 ± 0.85 6.06 ± 0.47
F 5.28 ± 0.21 5.84 ± 0.36 5.36 ± 0.60 5.23 ± 0.43 4.65 ± 0.43
NEU% M 24.88 ± 2.44 18.76 ± 0.64 20.74 ± 1.50 18.07 ± 1.56 23.18 ± 3.52
F 19.42 ± 3.29 22.64 ± 1.82 15.74 ± 1.4 16.63 ± 3.26 16.63 ± 2.16
EOS% M 3.42 ± 0.78 2.78 ± 1.68 4.30 ± 0.79 4.30 ± 0.45 4.12 ± 0.45
F 4.56 ± 1.27 3.48 ± 1.77 3.76 ± 0.62 6.05 ± 0.63 4.55 ± 1.14
RBC (x106/µL) M 7.81 ± 0.23 8.71 ± 0.06 7.47 ± 0.64 7.97 ± 0.5 7.98 ± 0.51
F 7.24 ± 0.44 7.46 ± 0.40 8.02 ± 0.09 7.70 ± 0.52 8.36 ± 0.12
HGB, g/dL M 17.32 ± 0.65 18.74 ± 0.21 15.66 ± 1.15 17.03 ± 1.08 16.58 ± 0.95
F 15.14 ± 0.95 16.22 ± 0.67 17.28 ± 0.31 16.55 ± 0.73 17.23 ± 0.23
HCT, % M 50.74 ± 2.06 53.22 ± 0.71 48.88 ± 1.89 51.43 ± 0.82 52.74 ± 1.83
F 43.82 ± 0.92 45.70 ± 1.93 48.44 ± 0.96 46.55 ± 1.99 48.63 ± 0.84
MCV, fL M 65.14 ± 2.91 61.04 ± 0.63 66.70 ± 4.05 65.07 ± 4.91 66.96 ± 4.29
F 61.26 ± 3.37 61.56 ± 1.64 60.34 ± 0.58 60.85 ± 1.91 58.23 ± 1.47
MCH, pg M 22.18 ± 0.38 21.52 ± 0.25 21.06 ± 0.36 21.4 ± 0.23 20.82 ± 0.41
F 20.90 ± 0.20 21.84 ± 0.56 21.56 ± 0.16 21.63 ± 0.67 20.58 ± 0.38
MCHC, g/Dl M 34.34 ± 1.74 35.22 ± 0.27 31.94 ± 1.38 33.23 ± 2.32 31.60 ± 1.95
F 34.44 ± 1.69 35.5 ± 0.14 35.68 ± 0.14 35.55 ± 0.18 35.40 ± 0.25
RDWa, fL M 47.82 ± 4.81 43.4 ± 0.98 56.28 ± 4.84 50.87 ± 8.56 50.64 ± 5.11
F 44.12 ± 5.12 44.96 ± 3.43 41.72 ± 0.56 43.43 ± 2.88 39.38 ± 0.91
RDW, % M 17.10 ± 0.74 16.82 ± 0.33 20.00 ± 0.82 18.30 ± 1.65 17.34 ± 0.41
F 16.90 ± 0.82 17.24 ± 0.83 16.36 ± 0.17 16.83 ± 0.46 16.08 ± 0.19
PLT(x103/µL) M 681.20 ± 54.81 776.80 ± 88.9 713.20 ± 79.21 732.33 ± 116.09 719.40 ± 48.20
F 823.40 ± 86.31 801.8 ± 69.1 892.6 ± 26.41 1026.75 ± 60.18 956.5 ± 70.71
MPV, fL M 6.48 ± 0.04 6.60 ± 0.21 7.24 ± 0.25 7.03 ± 0.36 6.84 ± 0.09
F 6.78 ± 0.1 6.96 ± 0.23 6.48 ± 0.05 6.85 ± 0.27 6.90 ± 0.19

Notes: Data show the mean ± SEM values for the depicted laboratory parameters. ANOVA and Tukey’s post-hoc test were used for comparing the results among
different treatment groups. M: Male, F: Female, RJX: Rejuveinix, RJX-P: RJX PPP.18.1051, RJX-B: RJX-B200702-CLN, BUN: blood urea nitrogen, TP: total protein,
ALB: albumin, GLOB: globulin, ALT: alanine aminotransferase, AST: aspartate aminotransferase, ALP: alkaline phosphatase, TBIL: total bilirubin, WBC: white blood
cell count, ALC: absolute lymphocyte count, MON: monocytes, ANC: absolute neutrophil count, EOS: eosinophil, LYM: lymphocytes, NEU: neutrophils, RBC: red blood
cell, HGB: hemoglobin, HCT: hematocrit, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration,
RDW: red cell distribution width, PLT: platelet, MPV: mean platelet volume.

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Table 10
Histopathology findings in Wistar albino rats treated with 14 days of RJX-P or RJX-B via intraperitoneal injections. Use summary table.
Treatment Normal Saline RJX-P RJX-P RJX-B RJX-B

Treatment Dose 0.5 mL (~2.5 mL/kg) 0.5 mL (~2.5 mL/kg) 0.25 mL (~1.25 mL/kg) 0.5 mL (~2.5 mL/kg) 0.25 mL (~1.25 mL/kg)
Time of Sacrifice (in days) 15 15 15 15 15
#Rats/Group 10 10 10 10 10
Tissues No. Exam % No. Exam % No. Exam % No. Exam. % No. Exam. %
Liver
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Pale liver 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Lung
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Congestion and atelectasis 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Hemorrhage and localized lesions 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Atelectasis 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Hemorrhage 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Kidney
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Spleen
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Splenomegaly 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Heart
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Pancreas
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Brain
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Cerebellum
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Intestine, Large
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Intestine, Small
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Stomach
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Testes
Within normal limits 5/5 100 5/5 100 5/5 100 5/5 100 5/5 100
Not examined 0/5 0 0/5 0 0/5 0 0/5 0 0/5 0
Notable or abnormal 0/5 0 0/5 0 0/5 0 0/5 0 0/5 0
Uterus
Within normal limits 5/5 100 5/5 100 5/5 100 5/5 100 5/5 100
Not examined 0/5 0 0/5 0 0/5 0 0/5 0 0/5 0
Notable or abnormal 0/5 0 0/5 0 0/5 0 0/5 0 0/5 0
Skeletal Muscle
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Skin
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0

Notes: RJX = Rejuveinix, RJX-P = RJX PPP.18.1051, RJX-B = RJX-B200702-CLN.

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Table 11
Day 28 Histopathology findings in Wistar albino rats treated with 14 days of RJX-P or RJX-B via intraperitoneal injections followed by a 14-day recovery period.
Treatment Normal Saline RJX-P RJX-P RJX-B RJX-B

Treatment Dose 0.5 mL (~2.5 mL/kg) 0.5 mL (~2.5 mL/kg) 0.25 mL (~1.25 mL/kg) 0.5 mL (~2.5 mL/kg) 0.25 mL (~1.25 mL/kg)
Time of Sacrifice (in days) 28 28 28 28 28
#Rats/Group 10 10 10 10 10
Tissues No. Exam % No. Exam % No. Exam % No. Exam. % No. Exam. %
Liver
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Pale liver 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Lung
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Congestion and atelectasis 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Hemorrhage and localized lesions 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Atelectasis 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Hemorrhage 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Kidney
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Spleen
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Splenomegaly 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Heart
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Pancreas
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Brain
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Cerebellum
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Intestine, Large
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Intestine, Small
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Stomach
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Testes
Within normal limits 5/5 100 5/5 100 5/5 100 5/5 100 5/5 100
Not examined 0/5 0 0/5 0 0/5 0 0/5 0 0/5 0
Notable or abnormal 0/5 0 0/5 0 0/5 0 0/5 0 0/5 0
Uterus
Within normal limits 5/5 100 5/5 100 5/5 100 5/5 100 5/5 100
Not examined 0/5 0 0/5 0 0/5 0 0/5 0 0/5 0
Notable or abnormal 0/5 0 0/5 0 0/5 0 0/5 0 0/5 0
Skeletal Muscle
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Skin
Within normal limits 10/10 100 10/10 100 10/10 100 10/10 100 10/10 100
Not examined 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0
Notable or abnormal 0/10 0 0/10 0 0/10 0 0/10 0 0/10 0

Notes: RJX = Rejuveinix, RJX-P = RJX PPP.18.1051, RJX-B = RJX-B200702-CLN.

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Fig. 2. The day-15 histopathology of liver tissue in rats treated with 14 days of RJX-P or RJX-B via intraperitoneal injections. Groups of 10 Wistar albino rats were
treated with i.p injections of RJX-P (1.25 and 2.50 mL/kg/day) or RJX-B (1.25 and 2.50 mL/kg/day), or vehicle (NS) for 14 days. Animals were sacrificed on day 15
for histopathologic examinations. No toxic lesions were detected. H&E, 200x.

Fig. 3. The day-15 histopathology of lung tissue in rats treated with 14 days of RJX-P or RJX-B via intraperitoneal injections. Groups of 10 Wistar albino rats were
treated with i.p injections of RJX-P (1.25 and 2.50 mL/kg/day) or RJX-B (1.25 and 2.50 mL/kg/day), or vehicle (NS) for 14 days. Animals were sacrificed on day 15
for histopathologic examinations. No toxic lesions were detected. H&E, 400x.

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F.M. Uckun et al. Biomedicine & Pharmacotherapy 141 (2021) 111823

Fig. 4. The day-15 histopathology of kidney tissue in rats treated with 14 days of RJX-P or RJX-B via intraperitoneal injections. Groups of 10 Wistar albino rats were
treated with i.p injections of RJX-P (1.25 and 2.50 mL/kg/day) or RJX-B (1.25 and 2.50 mL/kg/day), or vehicle (NS) for 14 days. Animals were sacrificed on day 15
for histopathologic examinations. No toxic lesions were detected. H&E, 200x.

Fig. 5. The day-15 histopathology of heart tissue in rats treated with 14 days of RJX-P or RJX-B via intraperitoneal injections. Groups of 10 Wistar albino rats were
treated with i.p injections of RJX-P (1.25 and 2.50 mL/kg/day) or RJX-B (1.25 and 2.50 mL/kg/day), or vehicle (NS) for 14 days. Animals were sacrificed on day 15
for histopathologic examinations. No toxic lesions were detected. H&E, 400x.

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F.M. Uckun et al.
Fig. 6. The day-15 histopathology of brain tissue in rats treated with 14 days of RJX-P or RJX-B via intraperitoneal injections. Groups of 10 Wistar albino rats were
treated with i.p injections of RJX-P (1.25 and 2.50 mL/kg/day) or RJX-B (1.25 and 2.50 mL/kg/day), or vehicle (NS) for 14 days. Animals were sacrificed on day 15
for histopathologic examinations. No toxic lesions were detected. H&E, 200x.
aminotransferase (ALT), alkaline phosphatase (ALP), and total bilirubin
(TBIL), blood urea-N (BUN) were assayed using an automated chemistry
analyzer (Samsung LABGEO PT10, Samsung Electronics Co., Suwon,
Korea). Tissue activity of superoxide dismutase (SOD), and tissue
ascorbic acid levels were also determined to examine the effects of RJX-
P and RJX-B. To determine SOD levels, the tissues (liver and lung) were
homogenized in 10 mL cold buffer (20 mM HEPES, 1 mM EGTA, 210 mM
mannitol, and 70 mM sucrose) and centrifuged in a refrigerated
centrifuge at 4 ◦ C, 1500 rpm for 5 min. The activities of superoxide
dismutase (SOD) were determined by using a commercially available
SOD assay kit (Cayman Chemical, Ann Arbor, MI, USA, Catalog No:
706002) according to the manufacturer’s instruction via a microplate
reader (Bio-Tek Elx800 Universal, Bio-Tek Instruments, Inc, Winooski,
USA) [3]. The intra- and inter assays coefficients of variation were 3.2%
and 3.7%. For the determination of ascorbic acid levels, tissue samples
were homogenized KCl (1.15%). Homogenate (0.2 mL) was mixed with
five-fold 80 mM Tris-maleate buffer. Then 400 µL of ascorbic acid and
400 µL of iron(II) sulfate were added, incubated in a 37 ◦ C water bath.
Subsequently, 26 mM thiobarbituric acid, 0.92 M trichloroacetic acid,
and 0. 8 mM hydrochloric acid were added into the mixture and held for
approximately 15 min in boiling water. Ascorbic acid concentrations
measured by HPLC system (Shimadzu UV–VIS SPD-10 AVP detector
equipped with CTO-10 AS VP column). The mobile phase was KH2 PO4
+methanol (82.5% + 17.5%), and the flow rate was 1.2 mL/min [7].
2.5. Necropsy and histopathologic study
Rats were subjected to gross necropsy, including examining the
thoracic organs, an external surface, and all the internal organs for any
type of abnormalities. At the time of necropsy, vital tissues (liver, lung,
kidney, spleen, heart, pancreas, brain, cerebellum, intestine, stomach,
testes, uterus, skeletal muscle, and skin) were collected within 15 min
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Biomedicine & Pharmacotherapy 141 (2021) 111823
after sacrifice for gross pathological and histopathological examina­
tions. Organs were preserved in 10% neutral phosphate-buffered
formalin and processed for histologic sectioning. For histopathologic
studies, formalin-fixed tissues were dehydrated and embedded in
paraffin by routine methods. Glass slides with affixed 4–5 µm tissue
sections were prepared and stained with hematoxylin and Eosin (H&E).
Sections were examined by light microscopy with an Olympus BX-51
microscope (Olympus, Tokyo, Japan).
2.6. Statistical analysis
The sample size was figured out by the G* Power program (Version
3.1.9.2) with alpha error 0.05% and 89% power with effect size 0.40.
Shapiro-Wilk test was performed for normality with a significance level
of 0.05. Levene’s test was performed for homogeneity with a signifi­
cance level of 0.05. One-way analysis of variance (ANOVA) was per­
formed and Tukey’s multiple comparisons were used as a post hoc test to
detect alterations among the groups using the SPSS statistical program
(IBM, SPPS Version 21). P-values <0.05 were considered significant.
GraphPad Prism Software (8.02, La Jolla, CA, USA) was used for
generating the graphs.
2.7. LPS-GalN Model of fatal cytokine storm and ARDS
The ability of the two formulations of RJX to prevent fatal shock,
ARDS, and multi-organ failure was examined in the well-established
LPS-GalN model, using previously published procedures [3]. LPS was
combined with GalN, which further sensitizes mice to LPS-induced
systemic inflammatory syndrome and multi-organ failure [21]. The
care and treatment of the animals were in accordance with the Guide for
the Care and Use of Laboratory Animals. They were approved by the
Animal Care and Use Committee of Firat University (Project No.
F.M. Uckun et al. Biomedicine & Pharmacotherapy 141 (2021) 111823

Fig. 7. Comparison of the dose-dependent pharmacodynamic effects of 14-day treatment with RJX-P vs. RJX-B on liver tissue superoxide dismutase (SOD) and
ascorbic acid levels. The depicted Whisker plots represent the median and values for the liver SOD (Panel A: Male, Panel C: Female) and ascorbic acid (Panel B: Male,
Panel D: Female) levels. The error bars represent the min and max values. (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 < 0.05; **p < 0.01; ***p< 0.001; ****p< 0.0001 compared as Control group and,
#p < 0.05; ##p < 0.01 compared as RJX-P (0.5 mL) group and, $p < 0.05; $$p < 0.01; $$$p < 0.001 pairwise comparisons between the groups).

04052020-391-046). Groups of 7 BALB/C mice were treated with i.p
injections of RJX-P and RJX-B (6-fold diluted, 4.2 mL/kg or 2.1 mL/kg,
0.5 mL/mouse), or vehicle two hours before and two hours
post-injection of LPS-GalN. Untreated normal control mice did not
receive any treatments. Vehicle control mice were treated with 500 µL
normal saline (NS), i.e., an aqueous solution of 0.9% NaCl with no RJX.
NS was administered i.p 2 h before and 2 h after the i.p injection of
LPS-GalN.
Serum and tissue cytokine levels, tissue activity levels of superoxide
dismutase (SOD), tissue ascorbic acid levels, as well as tissue concen­
trations of malondialdehyde (MDA) were determined to examine the
effects of the two formulations of RJX on the inflammatory process
caused by LPS-GaN. IL-1β, IL-6 and TNF-α levels in mouse serum samples
were measured by quantitative enzyme-linked immunosorbent assays
(ELISA) using the commercially available “Mouse IL-1 beta Quantikine
ELISA Kit” (Sensitivity for mouse IL-1β: 4.8 pg/mL, Assay Range:
12.5–800 pg/mL), “Mouse IL-6 Quantikine ELISA Kit” (Sensitivity for
mouse IL-6: 1.6 pg/mL, Assay Range: 7.8–500 pg/mL) and “Mouse TNFa
Quantikine ELISA Kit” (Sensitivity for mouse TNFα: 7.21 pg/mL, Assay
Range: 10.9–700 pg/mL), respectively, and an absorbance microplate
reader (Bio-Tek Elx800 Universal Microplate Reader, Bio-Tek In­
struments, Inc, Winooski, USA) according to the manufacturer’s in­
structions (R&D Systems, Minneapolis, MN, USA). Samples and
standards were analyzed in duplicate. The Kaplan-Meier method, log-
rank chi-square test was used to analyze the 24 h survival outcomes of
mice in the different treatment groups. At the time of death or elective
sacrifice at 24 h, lungs and liver from all mice per group were harvested,
fixed in 10% buffered formalin and processed for histopathologic

12
F.M. Uckun et al. Biomedicine & Pharmacotherapy 141 (2021) 111823

Fig. 8. Comparison of the dose-dependent pharmacodynamic effects of 14-day treatment with RJX-P vs. RJX-B on lung tissue superoxide dismutase (SOD) and
ascorbic acid levels. The depicted Whisker plots represent the median and values for the lung SOD (Panel A: Male, Panel C: Female) and ascorbic acid (Panel B: Male,
Panel D: Female) levels. The error bars represent the min and max values. (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< 0.01; ***p < 0.001; ****p < 0.0001 compared as Control group and, #p < 0.05;
##p < 0.01 compared as RJX-P (0.5 mL) group and, $p < 0.01; $$p < 0.01; $$$p < 0.001 pairwise comparisons between the groups).

examination. 3. Results

2.8. Statistical analyses for non-clinical studies in the LPS-GalN mouse
model of cytokine storm, ARDS and multi-organ failure
The Decision statistical structure includes analysis of variance
(ANOVA) using SPSS statistical program (IBM, SPPS Version 21) and/or
covariance (ACOVA), nonparametric analysis of variance, simple t-tests,
Tukey’s Test for homogeneity of variance, and pairwise tests by the
Dunnett’s test for parametric and nonparametric data. The Tukey’s
multiple comparisons detected alterations among groups. Furthermore,
the Kaplan–Meier method, log-rank chi-square test, was used to inves­
tigate survival and fatality in each group.
3.1. Toxicity in rats
There were no visible toxicity signs or symptoms, including tremors,
salivation, diarrhea in both male and female rats treated with RJX-P and
RJX-B during the study. All rats survived, and no behavioral changes
were observed. The majority of the RJX-P or RJX-B-treated rats gained
weight during the 0–14 day, 14–28 day and, 0–28 day observation
period. The average weight gain in male and female rats were 2.69 ±
0.27 g and 1.55 ± 0.33 g for vehicle-treated control rats, and 3.13 ±
0.19 g and 1.19 ± 0.20 g or 3.16 ± 0.18 g and 1.44 ± 0.40 g for male and
female rats treated with the higher dose of RJX-P or RJX-B for a 0–28
day period (Fig. 1A and B). The body weight changes were not

13
F.M. Uckun et al. Biomedicine & Pharmacotherapy 141 (2021) 111823

Fig. 9. Comparison of the dose-dependent pharmacodynamic effects of 14-day treatment with RJX-P vs. RJX-B and sacrificed on day 28 following a 2 week recovery
period on liver tissue superoxide dismutase (SOD) and ascorbic acid levels. The depicted Whisker plots represent the median and values for the liver SOD (Panel A:
Male, Panel C: Female) and ascorbic acid (Panel B: Male, Panel D: Female) levels. The error bars represent the min and max values. (ANOVA and Tukey’s post-hoc test
were used for comparing the results among different treatment groups).

statistically different among the groups (p > 0.05).
The differences in blood chemistry and hematology parameter levels
between the groups in male and female rats were not statistically sig­
nificant (p > 0.05), and these results did not suggest any system-specific
toxicity. RJX-P or RJX-B-treated male and female rats showed: (a)
normal serum AST, ALT TBIL and BUN, creatinine levels consistent with
the absence of liver and renal toxicity in rats of both sexes (Tables 8 and
9; p > 0.05); and (b) normal WBC, ANC, ALC, and RBC counts consistent
with the absence of hematologic toxicity (Tables 8 and 9; p > 0.05).
Similarly, no significant alteration was detected in the serum glucose,
ALP, TP, ALB, GLOB levels of treated rats in both sexes compared to the
control group (Tables 8 and 9; p > 0.05). The results of hematological
tests showed that all the parameters in RJX-P and RJX-B-treated and
control groups were within the normal range [8], which clearly
indicates RJX-P and RJX-B at low- or high-dose levels do not affect the
hematological parameters to a significant extent.
There were no abnormalities found on macroscopic examination of
the liver, lung, kidney, spleen, heart, pancreas, brain, cerebellum, in­
testine, stomach, testes, uterus, skeletal muscle, and skin in RJX-P or
RJX-B-treated rats on day 15 group (Table 10) or day 28 groups
(Table 11). In addition, histopathological examination of multiple tis­
sues from RJX-P or RJX-B -treated rats in the day 15 group (Figs. 2–6) or
day 28 groups showed normal organ tissue architecture similar to the
control group. Thus, RJX-P and RJX-B were not toxic to rats at dose
levels up to 2.5 mL/kg/day.

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F.M. Uckun et al. Biomedicine & Pharmacotherapy 141 (2021) 111823

Fig. 10. Comparison of the dose-dependent pharmacodynamic effects of 14-day treatment with RJX-P vs. RJX-B and sacrificed on day 28 following a 2 week re­
covery period on lung tissue superoxide dismutase (SOD) and ascorbic acid levels. The depicted Whisker plots represent the median and values for the lung SOD
(Panel A: Male, Panel C: Female) and ascorbic acid (Panel B: Male, Panel D: Female) levels. The error bars represent the min and max values. (ANOVA and Tukey’s
post-hoc test were used for comparing the results among different treatment groups).

3.2. Pharmacodynamics in rats
14-day treatment with RJX-P and RJX-B similarly elevated the Day
15 tissue activities of the antioxidant enzyme superoxide dismutase
(SOD) as well as ascorbic acid levels in both the lungs and liver in a dose-
dependent fashion (Figs. 7 and 8). The activity of SOD was significantly
higher in liver tissues of male (Fig. 7 A) and female (Fig. 7 C) rats in RJX-
P (2.5 mL/kg) or RJX-B (2.5 mL/kg) groups than in liver tissues of
vehicle-treated control rats (p < 0.001). Also, the RJX-B (2.5 mL) group
of male rats had higher SOD activity in the liver when compared to RJX-
P (1.25 mL) and RJX-B (1.25 mL) (p < 0.05). The ascorbic acid levels of
the liver in both male (Fig. 7B) and female (Fig. 7D) rats were elevated in
RJX-P (2.5 mL/kg) and RJX-B (2.5 mL/kg) groups when compared to
the control group (p < 0.001). The activity of SOD was significantly
higher in lung tissues of male (Fig. 8 A) and female (Fig. 8 C) rats in RJX-
P (2.5 mL/kg) or RJX-B (2.5 mL/kg) groups when compared to vehicle-
treated control rats (p < 0.001). Also, rats of both sexes in the RJX-B
(2.5 mL) group had higher SOD activity in the lungs when compared
to rats in the RJX-P (1.25 mL) and RJX-B (1.25 mL) groups. The ascorbic
acid levels of lungs in both male (Fig. 8B) and female (Fig. 8D) rats were
elevated in RJX-P (2.5 mL) and RJX-B (2.5 mL) groups when compared
to the control group (p < 0.001). There was no statistically significant
difference in SOD activity or ascorbic acid tissue levels of rats treated
with the same dose levels of RJX-P vs. RJX-B (p > 0.05).
The observed elevations of the SOD activity and ascorbic acid levels
in the liver and lung were transient and were no longer detectable on
day 28 following a 14-day recovery period (Figs. 9 and 10). These results
demonstrate that RJX-P and RJX-B are bioequivalent relative to their
pharmacodynamic effects on tissue SOD activity and ascorbic acid
levels.

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F.M. Uckun et al. Biomedicine & Pharmacotherapy 141 (2021) 111823

Fig. 11. In vivo protective activity of

two Rejuveinix (RJX) related pharma­
ceutical compositions (RJX-P and RJX-
B) in the LPS-GalN mouse model of
fatal cytokine storm, sepsis, systemic
inflammation, ARDS and multiorgan
failure. Groups of 7 BALB/C mice were
treated with i.p injections of RJX-P and
RJX-B (6-fold diluted, 4.2 mL/kg or
2.1 mL/kg, 0.5 mL/mouse), and vehicle
(NS, 0.5 mL/mouse) two hours before
and two hours 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-galactos­
amine) 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 anal­
ysis (Panel B of the different treatment
groups.

3.3. Dose-dependent in vivo protective activity of prophylactic RJX
treatments in the LPS-GalN mouse model of ARDS and multiorgan failure
Seven of 7 untreated control mice remained alive throughout the
experiment, whereas 7 of 7 LPS-GalN injected mice died at a median of
4.63 h (Fig. 11). Mice treated with RJX-P or RJX-B prior to the LPS-GalN
challenge had a significantly improved survival outcome. 28.6% of mice
treated with 2.1 mL/kg of 6-fold diluted RJX-P (n = 7) or RJX-B (n = 7)
remained alive, and the median survival time was prolonged to
6.91–7.08 h (Fig. 11). Notably, 57.1% of mice treated with 4.2 mL/kg of
6-fold diluted RJX-P (n = 7) or RJX-B (n = 7) remained alive, and the
median survival time was prolonged to ≥ 24 h. The 4.2 mL/kg dose of a
6-fold diluted RJX corresponds to a human equivalent dose of 0.05 mL/
kg (0.3 mL/kg:6), which is 10-times lower than the 0.5 mL/kg recom­
mended Phase 2 dose (RP2D) level of RJX [3]. Hence, RJX-P and RJX-B
exhibited near-identical potent and dose-dependent activity in the
LPS-GalN model of ARDS and multi-organ failure in mice.
In LPS-GalN challenged control mice not receiving any RJX treat­
ments, serum interleukin 1 beta (IL-1β) interleukin 6 (IL-6), and tumor
necrosis factor-alpha (TNF-α) levels were drastically increased at the
time of death which is consistent with a cytokine storm and marked
systemic inflammation (P < 0.0001 for IL-1β, IL-6, and TNF-α when
compared to control mice not treated with LPS-GalN) (Fig. 12). RJX
treatments had a statistically significant and dose-dependent lowering
effect on the serum levels of the proinflammatory cytokines IL-1β, IL6,
and TNF-α (Fig. 12). The results obtained with the two formulations of
RJX were near identical. These results demonstrate that both formula­
tions of RJX decreased the proinflammatory cytokine responses of mice
injected with LPS-GalN, which is consistent with their ability to improve
the survival time of LPS-GalN challenged mice in a dose-dependent

16
F.M. Uckun et al.
(caption on next column)
17
Biomedicine & Pharmacotherapy 141 (2021) 111823
Fig. 12. In vivo protective activity of two Rejuveinix (RJX)-related pharma­
ceutical compositions (RJX-P and RJX-B) on proinflammatory cytokine
response in a mouse model of fatal cytokine storm, sepsis, systemic inflam­
mation, ARDS and multiorgan failure. Groups of 7 BALB/C mice were treated
with i.p injections of RJX-P or RJX-B (6-fold diluted, 4.2 mL/kg or 2.1 mL/kg,
0.5 mL/mouse), and vehicle (NS, 0.5 mL/mouse) two hours before and two
hours 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 depicted Whisker plots represent the median and
values. In A and B, Welch’s ANOVA and Games-Howell post-hoc test were used
for comparing the results among different treatment groups. In 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 as compared to control group, ###p < 0.001;
####p < 0.0001 as compared to LPS/GalN+NS group, +p < 0.05;
++p < 0.01; +++p < 0.001; ++++p < 0.0001 as compared to LPS/
GalN+RJX-P (4.2 ml/kg) group, and $p < 0.05; $$p < 0.01; $$$$p < 0.0001 as
compared to LPS/GalN+RJX-B (4.2 ml/kg) group.
fashion.
The lung and liver malondialdehyde (MDA) levels measuring lipid
peroxidation were markedly elevated and the levels of the antioxidant
enzyme SOD as well as ascorbic acid were markedly reduced in LPS-
GalN treated mice when compared to healthy control mice, consistent
with severe oxidative stress in the lung and liver tissue (Fig. 13). Both
formulations of RJX equally decreased the lung as well as liver MDA
levels, and increased in a dose-dependent manner the reduced levels of
the antioxidant enzyme SOD and ascorbic acid (Fig. 13).
Histopathological examination of hematoxylin-eosin (H/E)-stained
lung tissues from LPS-GalN injected mice showed histological changes
consistent with severe acute ALI, including alveolar hemorrhage,
thickening of alveolar wall, edema/congestion, and leukocyte infiltra­
tion (Fig. 14). These changes were not observed in the lung tissues of
mice in the control group that was not injected with LPS-GaIN. Notably,
both formulations of RJX were equally effective in attenuating the LPS-
GalN induced ALI in a dose-dependent fashion, as evidenced by signif­
icantly less damage in the lungs of RJX-P and RJX-B treated mice that
were treated at the 2.1 and 4.2 mL/kg dose levels (Fig. 14). The alveolar
wall thickness as a measure of pulmonary edema was substantially
decreased to near normal values at these dose levels of RJX (Fig. 14).
Thus, both formulations of RJX prevented the development of pulmo­
nary edema in LPS-GalN challenged mice. The lung histopathological
scores of ALI depicted in Fig. 16A show a dose-dependent protective
effect of RJX-P and RJX-B with a highly statistically significant and near-
identical reduction of the ALI scores at the two dose levels of RJX-P and
RJX-B that were tested.
Histopathologic examination of the livers from LPS-GalN treated
mice showed massive generalized hepatocyte vacuolation and necrosis
corresponding to >25% necrosis and destruction of the liver paren­
chyma, severe lobular inflammation involving > 50% of the liver pa­
renchyma, and severe portal inflammation involving more than > 50%
of portal tracts (Fig. 15). The histopathological scores documenting the
LPS-GalN caused liver damage in various treatment groups are depicted
in Fig. 16B to demonstrate that both formulations of RJX were equally
able to mitigate the liver damage in a dose-dependent manner.
4. Discussion
Patients with high-risk COVID-19 are in urgent need of effective
treatment strategies that can prevent the development of ARDS and
multi-organ failure [1–3,9]. Recently, we demonstrated that RJX pre­
vents in a mouse model of sepsis the marked increase of each of proin­
flammatory cytokines like IL-6, TNF-α, and TGF-β in the serum as well as
lungs and liver when treatments are initiated prophylactically prior to
the injection of a fatal dose of LPS-GalN [3]. Of the active ingredients of
RJX, ascorbic acid (Vitamin C), cyanocobalamin (Vitamin B12),
F.M. Uckun et al. Biomedicine & Pharmacotherapy 141 (2021) 111823

Fig. 13. Tissue-level in vivo anti-oxidant activity of two Rejuveinix (RJX) related pharmaceutical compositions (RJX-P and RJX-B) in a mouse model of fatal cytokine
storm, sepsis, systemic inflammation, ARDS and multiorgan failure. Groups of 7 BALB/C mice were treated with i.p injections of RJX-P or RJX-B (6-fold diluted,
4.2 mL/kg or 2.1 mL/kg, 0.5 mL/mouse), and vehicle (NS, 0.5 mL/mouse) two hours before and two hours 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 depicted Whisker plots represent the
median and values. In (A–D), ANOVA and Tukey’s post-hoc test were used for comparing the results among different treatment groups. In (E) and (F), Welch’s
ANOVA and Games-Howell post-hoc test were used for comparing the results among different treatment groups. Statistical significance between groups is shown by
**p < 0.01; ***p < 0.001; ****p < 0.0001 as compared to control group, ###p < 0.001; ####p < 0.0001 as compared to LPS/GalN+NS group, ++p < 0.01;
+++p < 0.001; ++++p < 0.0001 as compared to LPS/GalN+RJX-P (4.2 ml/kg) group, and $$p < 0.01; $$$p < 0.001; $$$$p < 0.0001 as compared to LPS/
GalN+RJX-B (4.2 ml/kg) group.

18
F.M. Uckun et al.
thiamine hydrochloride (Vitamin B1), riboflavin 5′ phosphate (Vitamin
B2), niacinamide (Vitamin B3), and pyridoxine hydrochloride (Vitamin
B6) have been proposed as potential anti-sepsis drugs [3]. RJX is
currently being evaluated in hospitalized COVID-19 patients with viral
sepsis to test the hypothesis that it will contribute to a faster resolution
of respiratory failure and a reduced case mortality rate [2]
(Clinicaltrial.gov identifier: NCT04708340; https://clinicaltrials.gov/ct
2/show/NCT04708340). RJX could favorably affect the clinical course
of high-risk COVID-19 and reduce its case mortality rate [1,2].
The current non-clinical study was carried out to compare the safety,
PD features, and efficacy of two formulations of RJX, namely RJX-P and
RJX-B. Our results presented herein demonstrate that the two formula­
tions of RJX are bioequivalent relative to their pharmacodynamic effects
on tissue SOD and ascorbic acid levels as well as safety profile in rodents.
Specifically, the results showed that the administration of RJX-P and
19
Biomedicine & Pharmacotherapy 141 (2021) 111823
Fig. 14. The protective effects of two Rejuvei­
nix (RJX) related pharmaceutical compositions
(RJX-P and RJX-B) on A ALI and inflammation
in the LPS-GalN mouse model of fatal cytokine
storm, sepsis, systemic inflammation, ARDS and
multiorgan failure. Groups of 7 BALB/C mice
were treated with i.p injections of RJX-P and
RJX-B (6-fold diluted, 4.2 mL/kg, or 2.1 mL/kg,
0.5 mL/mouse), and vehicle (NS, 0.5 mL/
mouse) two hours before and two hours post-
injection of LPS-GalN. Except for untreated
control mice (Control, Panel A), each mouse
received 0.5 mL of LPS-GalN (consisting of
100 ng of LPS plus 8 mg of D-galactosamine) i.
p. The lung histopathological ALI scores ranged
from 3 to 4 for the LPS-GalN+NS group (Panel
B), from 2 to 3 for LPS-GalN+RJX-P (4.2 ml/kg)
group (Panel C), from 1 to 3 for LPS-GalN+RJX-
B (4.2 ml/kg) group (Panel D), from 2 to 3 for
LPS-GalN+RJX-P (2.1 ml/kg) group (Panel E),
and LPS-GalN+RJX-B (2.1 ml/kg) group (Panel
F). Depicted are microscopic images of lung
tissues of representative mice from treatment
groups. While the ALI scores median for the
depicted mice treated with RJX-P (4.2 ml/kg)
and/or RJX-B (4.2 ml/kg) were 2 for each, mice
treated with RJX-P (2.1 ml/kg) and/or RJX-B
(2.1 ml/kg) were 3 for each, and the ALI score
median for the LPS-GaIN+NS treated mouse
was 4. White arrow: inflammatory cell infiltra­
tion; Black arrow (short): Exudate, edema;
Black arrow (Long): hemorrhage; Black double-
headed arrow: the thickness of the alveolar
wall. H&E X400.
RJX-B at their lowest and highest tested dosage of 1.25 and 2.5 mL/kg/
day were tolerable to animals, as it did not produce any changes in
mortality, behavior or clinical symptoms, body weight, macroscopic and
histopathological changes of the liver, lung, kidney, spleen, heart,
pancreas, brain, cerebellum, intestine, stomach, testes, uterus, skeletal
muscle, and skin except SOD and ascorbic acid levels increases in lung
and liver. The daily injection of RJX-P and RJX-B for 14 days did not
cause any morbidity, mortality, and adverse changes in safety pharma­
cology laboratory parameters, including ALT, AST, ALP, BUN, and cre­
atine. in male or female rats. The data from the present study
demonstrate that a 14-day treatment of rats with the clinically appli­
cable dose levels of the RJX formulations RJX-P or RJX-B does not
exhibit toxic effects that are associated with morbidity or mortality.
Both formulations increased tissue levels of ascorbic acid and SOD. RJX-
P and RJX-B were bioequivalent relative to their anti-oxidant properties,
F.M. Uckun et al.
as documented by the near-identical pharmacodynamic effects on tissue
SOD activity and ascorbic acid levels.
IL-6, TNF-a, and TGF-β are important pro-inflammatory cytokines
that contribute to the pathophysiology of the cytokine release syndrome
(CRS), ARDS and multi-organ failure in critically sick adult COVID-19
patients [1,3,9]. Recently, we demonstrated that RJX prevents in the
LPS-GalN mouse model of sepsis the marked increase of each of these
cytokines in the serum as well as lungs and liver [3]. Here we extend our
previous study and provide experimental evidence that two distinct
formulations of RJX showed profound protective activity in this pre­
clinical sepsis model. Specifically, our results presented herein provide
experimental evidence that these two distinct formulations of RJX are
20
Biomedicine & Pharmacotherapy 141 (2021) 111823
Fig. 15. The protective effects of two Rejuvei­
nix (RJX) related pharmaceutical compositions
(RJX-P and RJX-B) on liver injury and inflam­
mation in the LPS-GalN mouse model of fatal
cytokine storm, sepsis, systemic inflammation,
ARDS and multiorgan failure. Groups of 7
BALB/C mice were treated with i.p injections of
RJX-P and RJX-B (6-fold diluted, 4.2 mL/kg, or
2.1 mL/kg, 0.5 mL/mouse), and vehicle (NS,
0.5 mL/mouse) two hours before and two hours
post-injection of LPS-GalN. Except for untreated
control mice (Control, Panel A), each mouse
received 0.5 mL of LPS-GalN (consisting of
100 ng of LPS plus 8 mg of D-galactosamine) i.
p. The liver histopathological scores ranged
from 3 to 4 for the LPS-GalN+NS group (Panel
B), from 1 to 3 for LPS-GalN+RJX-P (4.2 ml/kg)
group (Panel C), from 1 to 3 for LPS-GalN+RJX-
B (4.2 ml/kg) group (Panel D), from 2 to 3 for
LPS-GalN+RJX-P (2.1 ml/kg) group (Panel E),
and LPS-GalN+RJX-B (2.1 ml/kg) group (Panel
F). Depicted are microscopic images of liver
tissues of representative mice from treatment
groups. While the liver histopathological scores
median for the depicted mice treated with RJX-
P (4.2 ml/kg) and/or RJX-B (4.2 ml/kg) were 2
for each, mice treated with RJX-P (2.1 ml/kg)
and/or RJX-B (2.1 ml/kg) were 3 for each, and
the liver histopathological score median for the
LPS-GaIN+NS treated mouse was 3. Black
arrow (short): Inflammatory cell infiltration;
Black arrow (long): Congestion; White arrow
(long): Necrosis; White arrow (short): Hydropic
degeneration. H&E 200x.
bioequivalent in their ability to prevent the inflammatory cytokine
response, ALI, and fatal multi-organ failure. In agreement with the PD
evaluations in rats, both formulations exhibited favorable and
near-identical PD effects on oxidative stress in mice challenged with
LPS-GalN. SOD is an important antioxidant enzyme, and its activity is
reduced by oxidative stress that is a hallmark of severe inflammation of
sepsis, including severe viral sepsis in COVID-19 [3]. Both formulations
of RJX exhibited potent anti-oxidant activity, as reflected by signifi­
cantly decreased tissue MDA levels and normalization of the tissue levels
of SOD as well as ascorbic acid. Both formulations of RJX are anticipated
to shorten the time to resolution of ARDS by eliminating the contribu­
tions of pro-inflammatory cytokines to the systemic and pulmonary
F.M. Uckun et al. Biomedicine & Pharmacotherapy 141 (2021) 111823

Fig. 16. Tissue-level in vivo protective activity of two Rejuveinix (RJX) related pharmaceutical compositions (RJX and RJX-B) on lung and liver histopathological
scores in a mouse model of fatal cytokine storm, sepsis, systemic inflammation, ARDS and multiorgan failure. Groups of 7 BALB/C mice were treated with i.p in­
jections of RJX-P and RJX-B (6-fold diluted, 4.2 mL/kg, or 2.1 mL/kg, 0.5 mL/mouse), and vehicle (NS, 0.5 mL/mouse) two hours before and two hours 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-galac­
tosamine) i.p. The depicted Whisker plots represent the median and values. In (A), the lung histopathological score (“lung injury score”) was graded according to a 5-
point scale from 0 to 4 as follows: 0, l, 2, 3, and 4 represented no damage, mild damage, moderate damage, severe damage, and very severe damage, respectively. In
(B), the liver histopathological score (“liver injury score”) was graded according to a 5-point scale from 0 to 4 as follows: 0, l, 2, 3, and 4 represented no damage, mild
damage, moderate damage, severe damage, and very severe damage, respectively. The Kruskal-Wallis test and Mann-Whitney U test were used to compare the results
among different treatment groups. Statistical significance between groups is shown by #p < 0.05; ##p < 0.01 as compared to LPS/GaIN+NS group.

inflammatory process as well as mitigating sepsis-associated oxidative sponsor.

stress.
Author contribution
Each author (F.M.U., C.O., J.P., E.S., I.H.O., M.V., K.S.) has made
significant and substantive contributions to the study, reviewed and
revised the manuscript, provided final approval for submission of the
final version. No medical writer was involved. F.M.U. conceived the
study, designed the evaluations reported in this paper, directed the data
compilation and analysis, analyzed the data, and prepared the initial
draft of the manuscript. Each author had access to the source data used
in the analyses I.H.O. performed the necropsies and histopathologic
examinations on rats.
Funding/Support
This study was funded by Reven Pharmaceuticals, LLC, USA, a
wholly-owned subsidiary of Reven Holdings Inc., USA.
Role of the funder/sponsor
The sponsor did not participate in the collection of data. Three of the
authors (F.M.U, J.P., and M.V.) who participated in the analysis and
decision to submit the manuscript for publication are affiliated with the
Conflict of interest statement
F.M.U., J.P. and M.V. are employees/contractors of Reven Pharma­
ceuticals, the sponsor for the clinical development of RJX. C.O., E.S., I.H.
O, and K.S. declare no current competing financial interests.
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