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Pharmacokinetics and Pharmacodynamics of Chlorine Dioxide

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 Pharmacokinetics and Pharmacodynamics of Chlorine Dioxide
Alberto Rubio Casillas1, Pablo Campra Madrid2

1. Clinical Laboratory, Regional Hospital of Autlán, Jalisco health Secretariat, México.

2. Digestive Modeling Research Group AGR152, University of Almería, Spain.

Corresponding author: alberto-rubioc@hotmail.com

The original version in Spanish can be found at:

http://e-cucba.cucba.udg.mx/index.php/e-Cucba/article/view/202

Content
1. Introduction
2. Toxicity
3. Viricidal / antiviral activity
4. Pharmacokinetics
4.1. Degradation in the oral / gastric cavity
4.2. Metabolism
5. Pharmacodynamics
5.1. Interactions between chlorite and peripheral blood cell heme enzymes
5.1.1. Chlorite interaction with hemoglobin
5.1.2. Chlorite is an erythrocyte protective agent
5.1.2.1. Chlorite oxidize hemoglobin to methemoglobin
5.1.2.2. Chlorite degrades methemoglobin
5.1.2.3. Reduces (inactivates) ferril hemoglobin (Fe4+) to methemoglobin (Fe3+)
5.1.2.4. Chlorite-mediated inhibition of myeloperoxidase activivy
5.1.3. Taurine chloramine neutralizes hypochlorous acid-induced toxicity
5.1.4. Chlorite restores redox balance via hormetic mechanisms
6. Conclusion

Abstract
Chlorine dioxide (ClO2) is widely used as a drinking water disinfectant in many countries.
Due to its antibiotic and antiviral capacity, has aroused interest as a potential therapeutic
agent with respect to COVID-19 disease, AIDS and Influenza. As a result of this debate in
scientific and governmental settings, it was deemed highly timely to provide an up-to-date
assessment of the pharmacokinetics and pharmacodynamics of ClO2. The main findings
indicate that, due to its high chemical reactivity, ClO2 is rapidly reduced in oral and gastric
secretions, producing the chlorite ion (ClO2⁻) which becomes the active agent responsible
for its systemic actions. ClO2⁻ also showed potential to act as an oxidant at high
concentrations and as antioxidant at low concentrations. Of particular therapeutic interest
are the findings that, at low concentrations, ClO2⁻ can protect erythrocytes from oxidative
stress while at the same time inhibiting the excessive production of hypochlorous acid
(HClO) induced by myeloperoxidase (MPO), thus reversing inflammatory responses and
macrophage activation. Finally, taurine-chloramine represents the most relevant functional
product formed under the influence of ClO2⁻, said molecule activates erythroid nuclear
factor 2 (Nrf2), (this transcription factor regulates the inducible expression of numerous
genes for enzymes detoxifiers and antioxidants), increases the expression of heme-
oxygenase (HO-1), protects cells from death caused by hydrogen peroxide (H2O2),
improves the expression and activities of antioxidant enzymes, such as superoxide
dismutase, catalase and glutathione peroxidase, and contributes to the resolution of the
inflammatory process.
Key words: chlorine dioxide, chlorite, hormesis, taurine-chloramine, myeloperoxidase.

1. Introduction
ClO2 is a yellow gas that can decompose quickly in the air. Because ClO2 is very reactive, it
can inactivate viruses, bacteria and other microorganisms in the water. About 5% of the
large water-treatment facilities (serving more than 100,000 people) in the United States use
ClO2 to treat drinking water. An estimated 12 million people can be exposed in this way to
ClO2 and ClO2⁻. In the communities that use ClO2 to treat drinking water, ClO2 and its by-
product, the ClO2⁻ ion, may be present at low levels in tap water (US-ASTDR (2004). EPA
(Environmental Protection Agency) has established the maximum concentration in drinking
water at 0.8 milligrams per liter (mg / L) for ClO2 and 1.0 mg / L for the ClO2⁻ ion. The
FDA (Food and Drug Administration) of the United States of America and COFEPRIS
(Federal Commission for Protection against Sanitary Risks) in México state that the
consumption of ClO2 causes kidney and liver failure, and it also destroys red blood cells.
In the paradigm of drug administration, to determine the correct dose of a drug is often a
challenge. Previously, it has been observed that several drugs demonstrate contradictory
effects per se, at high and low concentrations. This duality in the effect of one drug at
different concentrations is known as hormesis (Bhakta-Guha and Efferth, 2015). For
several decades it was believed that drug dosing follows a linear pattern, generating an
enormous ignorance about the responses in the area of low concentrations (Calabrese and
Baldwin, 2001). However, in recent years, several studies have shown an inverse response
to different concentrations of a drug in the same individual, thus completely ruling out
linearity and threshold response models of determination of concentration (Calabrese et al.,
2010). This effect, known as the “biphasic response to concentration”, has shown
importance to establish the administration of a drug (Calabrese, 2001; Huang and Zheng,
2006; Day and Suzuki, 2006; Calabrese, 2014).
It is well documented that mild environmental stress such as exposure to low doses of
stressful stimuli often prompts the adaptive stress response in individuals to maintain
homeostasis (Kuoda and Iki, 2010; Martins et al.,2011). It also complies with the fact that
while higher levels of a toxic substance can be clearly harmful, small doses of it can
promote health, governed by growth and development (Calabrese, 2001; Schumacher,
2009). External effectors (stimuli), such as stressors or aggressors that induce stress in
higher concentrations, are often referred to as hormetins (Menendez et al., 2013; Mattson,
2008). In this conjuncture, it is imperative to establish that the term "stress” can have
multiple implications. In the context of this review, we mean parameter, extrinsic or
intrinsic, which can induce a deviation of the normal physiological processes of the body
(Bhakta-Guha and Efferth, 2015). Exposure to stress often causes pathways designed to
combat the same, which are known as responses to stress (Dattilo et al., 2015). Several of
these responses often require stimulation of survival pathways (Rattan, 2006). The
hormetins can be of biological, physical or chemical origin (Kouda and Iki, 2010;
Richardson, 2009). The generation of the adaptive response to continuous mild exposures
to such stressors is an evolutionarily conserved trait, which in the long run protects an
individual against future attacks of high concentration and stress (Martins et al., 2011). In
this regard, an exhaustive review (Calabrese and Baldwin, 2003) compiled a series of
inorganic agents (inducing hormetic dose-response relationships), including toxic agents of
great environmental and public health interest (for example, arsenic, cadmium, lead,
mercury, selenium and zinc).Therefore, in this article we will review the status of the
experimental knowledge on toxicity, viricidal /antiviral action, pharmacokinetics and
pharmacodynamics of ClO2 / ClO2⁻ with the aim of looking for hormetic mechanisms that
can induce adaptive responses to stress that explain its supposed therapeutic properties.

2. Toxicity
The toxicological profile of ClO2, and of its first reducing product, the chlorite anion
(ClO2⁻), has been extensively studied in animal tests and reviewed in successive technical
reports of the United States administration (US-EPA, 2000; US-ASTDR, 2004). In these
studies, toxic reactions have been reported above different exposure levels, after the oral
and inhalation routes. Adverse reactions consisted of oral and digestive irritation, anemia
and methemoglobinemia, altered thyroid function, neurotoxicity with delayed brain
development in puppies. After a comprehensive review of animal studies, the US-ASTDR
(2004) concluded that the maximum dose tested among all the studies reviewed in which no
adverse effects were observed (NOAEL level) should be set at 3 mg (ClO2 / ClO2⁻) / kg /
day. The lowest level of adverse effects observed in this review was concluded to be 5.7 mg
/ kg / day. These levels were obtained after a final US EPA-mandated animal study (Gill et
al, 2000) in which toxicity was characterized in a 2-generation study to examine
reproductive, developmental, and reproductive end points, neurological and hematological
in rats exposed to sodium chlorite (NaCIO2) in drinking water, including sensitive groups.
Although few clinical reports of toxicity in humans have been reported to date, animal
studies have shown effects of ClO2 and ClO2⁻ that are similar to those seen in people
exposed to very high amounts of these chemicals. In a trial in non-human primates, the
increasing sub-chronic toxicities of oral administration of ClO2, NaCIO2, NaCIO3, and
NH2CI were studied in African green monkeys for 30-60 days. A reversible inhibition of
thyroid metabolism at sub-chronic doses of ClO2 (9 mg / kg / day, corresponding to 100
mg / L) in drinking water was registered, but no effects were observed at 3 mg / kg / day
(Bercz et al., 1982). The ingestion of ClO2⁻ in primates at high doses caused a decrease in
the erythrocyte count, as well as an increase in transaminases. Interestingly, most of the
ClO2 doses induced a self-compensating oxidative stress in hematopoiesis, since a rebound
phenomenon occurred in the synthesis of hemoglobin and red blood cells, suggesting an
hormetic effect that will be discussed later. Furthermore, no thyroid inhibition was detected
after the use of ClO2⁻ at concentrations up to 60 mg / kg / day (Bercz et al., 1982). The
selective thyroid effect of ClO2 was paradoxical since ClO2 was rapidly reduced by oral and
gastric secretions to non-oxidizing species, presumably chloride (Cl⁻).
In one of the first human studies ordered by the US-EPA (Lubbers et al., 1982), acute
tolerance was evaluated at an increasing dose from the oral administration of different
chlorinated water disinfectants. Systemic toxicity was not detected below the maximum
dose of 24 mg / L of ClO2 and 2.4 mg / L of ClO2⁻, ingested twice daily 4 hours apart. In
another sub-chronic toxicity experiment, daily oral ingestion of ClO2 at a concentration of
5 mg / L for 12 consecutive weeks produced no obvious undesirable clinical adverse
effects. The absence of human ClO2⁻ toxicity below this US-ASTDR established NOAEL
level has been reported in recent controlled clinical trials. In a phase I, placebo-controlled
study of safety and tolerability in patients with Amyotrophic Lateral Sclerosis (ALS),
Miller et al., (2014) tested single ascending doses of 0.2, 0.8, 1.6 and 3.2 mg / kg of
intravenous NaCIO2. After treatment, patients were monitored for avariety of safety
variables and clinical status during and for 8 h after infusions, one, four and seven days
after dosing. All doses were generally safe and well tolerated, and there were no serious
treatment-related adverse events. In an additional phase II study in patients with ALS, no
adverse effects were observed when 2 mg / kg / day was administered in repeated sub-acute
exposures monthly (3-5 days) (Miller et al, 2015).
At extreme levels of exposure, adverse events have been described due to suicide attempts
when ingesting massive doses of ClO2⁻. After intoxication by a single 10 g dose of
NaCIO2, approximately 142 mg / kg (Lin and Lim, 1993), excessive oxidative stress caused
irritation in the digestive tract accompanied by nausea, vomiting and kidney failure. In
another suicide attempt by an adult male who ingested about 100 ml of 28% NaCIO2
solution (Gebhardtova et al., 2014), initial laboratory tests revealed 40% methemoglobin
formation and acute kidney failure. The lowest observable toxicity (LOAEL) would be
expected from 5.7 mg kg / day, equivalent to 420 mg / day for an average adult human. In
conclusion, there is no experimental evidence available to support that administering doses
lower than 3 mg / kg / day there is a risk of systemic toxicity or variations in relevant
clinical parameters. This dose is equivalent to 210 mg of ClO2 per day for an average 70 kg
adult.

3. Viricidal / antiviral activity

The viricidal activity of ClO2 in vitro has been described against different human and
animal viruses (Ogata and Shibata, 2008; Sanekata et al, 2010). For example, it was
reported that ClO2 was able to inactivate the human Influenza virus by 99.9% at 15 seconds
using 1 ppm, against the Measles virus this inactivation was achieved at 30 seconds using
10 ppm and against the Herpes virus was achieved at 15 seconds using 10 ppm (Sanekata et
al., 2010). Other researchers demonstrated that ClO2 is capable of destroying the
Poliomyelitis virus using 1-2 ppm, that of Hepatitis A with 7.5 ppm, Rotavirus with 0.2
ppm, 10 ppm for the HIV-1 virus, and 2.1 ppm for the coronavirus that caused SARS (cited
in Miura and Shibata, 2010).
The potential use as a prophylactic against viral infections was demonstrated for influenza
A by inhalation in mice (Ogata and Shibata, 2008). Regarding the direct viricidal action
mechanism of ClO2 / ClO2-, mechanisms have been proposed based on the oxidation of key
amino acid residues in the viral protein envelope, such as denaturation of virus RBD
(receptor binding domain), thereby abolishing its receptor-binding ability (Ogata,
2007,2012). According to studies carried out with radioactive labeling, chlorite has a half-
life of 3.5 hours (Abdel-Rahman et al., 1982), enough time to produce direct plasma
antiviral effects, which would consist of the oxidation of amino acids found in the protein
envelope of viruses. In particular, the oxidation of thiol residues (-SH) of tyrosine,
threonine and tryptophan is important. The formation of disulfide bridges between the
spike protein of SARS COV2 and the ACE2 receptors of human alveolus cells has been
cited (Hati and Bhattacharya, 2020). By reducing oxidative stress as one of the pathogenic
mechanisms of the virus, therapeutic doses of ClO2 / ClO2⁻ could prevent the formation of
disulfide bonds and binding between the ACE2 receptor and the spike protein.
Importantly, the first in vivo trial describing an antiviral effect of ClO2 against avian
coronavirus has recently been reported (Zambrano-Estrada et al., in press). ClO2 treatment
had a marked impact on viral infection. That is, viral titers were 2.4 times lower and
mortality was reduced by half in infected embryos that were treated with ClO2. The
infection caused developmental abnormalities regardless of treatment. Typical lesions of
this virus infection were observed in all inoculated embryos, but gravity tended to be
significantly lower in embryos treated with ClO2. No macroscopic or microscopic evidence
of toxicity caused by ClO2 was found at the doses used in this study (Zambrano-Estrada et
al., in press). In addition, the results of the first clinical study demonstrating the efficacy of
ClO2 for the treatment of COVID-19 disease have recently been published. In this research,
the effect of Cl02 on the clinical evolution of 20 patients with active infection by the Sars-
CoV-2 virus was analyzed. The control group consisted of 20 patients who did not receive
ClO2. The experimental group consisted of 20 patients who received an oral solution of
ClO2 at a concentration of 30 mg / L (well below the NOAEL) for 21 days. When
comparing the experimental group with the control group on the seventh day after the
manifestation of symptoms, a significant difference was found in the experimental group
with respect to the control group for the symptoms: Fever (p: 0000), Cough (p: 0.0000) ,
Chills (p: 0.0000) and Dyspnea (p: 0.0006). When performing the analogous visual
comparison of pain in the control group and in the experimental group, it was found that in
all the items that make up the scale it decreased significantly in this group with respect to
the control group (p: 0.0000; p: 00017). To the day 14, the difference was greater (p: 0.000;
p: 0.0043). When evaluating both groups (Control and Experimental) in the laboratory, a
difference was found for the values of the C-reactive protein (CRP) parameters on day 7 (p:
0.0001) and LDH (Lactate Dehydrogenase) (0.0036) , with higher scores for the
experimental group; D-dimer on day 7 (p: 0.0194) and day 14 (p: 0.0029); difference was
found in all parameters. Overall results (p <0.05) confirmed the hypothesis that chlorine
dioxide is effective in the treatment of COVID-19 (Insignares-Carrione et al., 2021).
ClO2⁻ also produces antiviral / viricidal effects. A study carried out in the 90s aimed to
determine the effects of ClO2⁻ (WF10) on the replication of HIV and on the infectivity of
HIV-free particles (Raffanti et al., 1998). It was shown that the WF10 complex exhibits a
significant antiviral activity against HIV. These investigators demonstrated that the ClO2⁻-
oxygen complex modifies the virion outer envelope (glycoprotein gp120), thus inhibiting
it’s binding to the CD4 molecule on CD4 + T cells. HIV inactivation is mediated by the
oxidative activity of the ClO2⁻-oxygen complex (Raffanti et al., 1998). This oxidative
mechanism is similar to that mediated by ClO2 for the influenza virus; an oxidation of a
tryptophan (W153) residue in hemagglutinin (a spike protein of the virus), thus abolishing
its receptor binding capacity. In other words, this oxidative modification induces structural
changes in the hemagglutinin binding site and disrupts its interaction with the host cell
receptor (Ogata, 2012). The spike protein of the new coronavirus SARS CoV2 contains 54
tyrosine residues, 12 tryptophan and 40 cysteine residues (Tao et al., 2020. In a recent
work, 3D reconstructions were made by computer, use of data through studies in cryo-
electron microscopy and previous work based on augmented reality software (Insignares-
Carrione et al, 2020). Said simulations made it possible to determine the positions of the
amino acids susceptible to being oxidized by ClO2 and which allows inferring their possible
mechanism of action on the SARS-CoV-2 virus. These researchers have postulated that
ClO2 could oxidize the cysteine Cys480-Cys488, which are key to the binding between the
SARS-CoV-2 spike and the ACE2 receptor (Insignares-Carrione et al., 2020).
4. Pharmacokinetics
There are still few studies in humans that allow us to characterize the pharmacokinetics of
ClO2, that is, the processes of digestion, absorption, distribution, metabolism and excretion
to which it is subjected through its passage through the body (US-ASTDR, 2004). In this
work we present a hypothesis based both on the redox properties of this gas and its
metabolites, as well as on studies carried out in animals and in two controlled clinical trials
published in humans (Miller et al., 2014; 2015). Chemically, ClO2 is an unstable free
radical, with an unpaired electron that makes it a strong oxidant, rapidly reducing to ClO 2⁻
in the presence of electron donor species, such as proteins and amino acids, that is, the
processes of digestion, absorption, distribution, metabolism and excretion to which it is
subjected through its passage through the body (US-ASTDR, 2004).
4.1. Degradation in the oral / gastric cavity
The most relevant experimental data to characterize pharmacokinetics come from animal
studies. In vivo studies in mice based on the radioactive labeling of ClO2, Abdel-Rahman
et al (1979a; 1984), using radioactively labeled chlorine (Cl), determined a rapid
gastrointestinal absorption and a wide distribution in tissues, with a plasma peak after one
hour of ingestion. The absorption rate and half-life were estimated at 3.77 and 0.18 hours,
respectively (Abdel-Rahman et al., 1982).
Bercz et al. (1982) studied the oral processing of ClO2 and ClO2⁻ solutions in non-human
primates. Saliva was shown to be a powerful ClO2 reducer, with only 5-12% of the initial
content remaining after 1 minute of in vitro reaction. Human saliva contains a wide range
of biomolecules, many of which are reactive with ClO2, including amino acids, the latter
including cysteine, tryptophan, and methionine (Silwood et al., 1999). Bercz et al. (1982)
also studied the gastric digestion of ClO2 "in vivo", recovering only 8% of the initial
oxidative capacity of ingested ClO2 after 5 minutes of contact with gastric contents.
4.2. Metabolism
The absorbed 36Cl is slowly removed from the blood. After 72h, 80% of the residual 36Cl
found in plasma was in the chloride form, and 20% in the chlorite form. After 72 hours,
chlorine labeled with a radioactive isotope was mostly detected in plasma, intestine and
stomach, as well as in various tissues such as lung and kidney. A small percentage of the
initial radioactive chlorine (0.4-0.8%) was detected in the blood cells, in unidentified
species in the body prior to its excretion, mainly as chloride and chlorite. A 72-hour urinary
excretion rate of 30-35% of the 36Cl that was not fixed by the tissues or remaining in the
plasma was estimated, with a peak between 24 and 48 hours, and a half-life of 44 hours.
-
The 36Cl excreted was found mainly as chloride (Cl ) (87%), chlorite (ClO2-) (1.3%), and to
a lesser degree as chlorate (ClO3-). In this same work, potassium chlorite was reduced by in
vitro plasma to chloride by 100%, and that oral ClO2 was reduced by in vivo plasma by
82.3% to chlorite and 17.6% to chloride. Of the total eliminated, 72% was in urine and 25%
in feces. In urine, the chlorine marked in ClO2 and chlorite was mainly excreted as chloride
ion, 87% and 84% respectively. The formation of trihalomethanes was not detected at the
low doses tested (maximum 1.5mg / kg / day, 100 ppm). No excretion by air was detected.
The absorption and elimination of ClO2 and its metabolites was faster than for hypochlorite
(HClO), the active component of common bleach.
Chlorite is also rapidly absorbed from the gastrointestinal tract. Maximum plasma levels of
radiolabeled chlorine were reached 2 hours after administration of a single 100 mg/L dose
of 36ClO2- (approximately 0.13 mg/kg) to Sprague-Dawley rats. Using 72-hour urinary
excretion data, 35% of the initial dose was estimated to have been absorbed (Abdel-
Rahman et al., 1984). The absorption rate constant and the half-life were 0.198 / hour and
3.5 hours, respectively (Abdel-Rahman et al., 1982). Due to the extreme reactivity of ClO2,
it is rapidly reduced to chlorite in the stomach when it reacts with food, organic substances,
tissues, or other materials that can serve as electron donors. Although ClO2 is unlikely to
survive the stomach environment long enough to be absorbed, the chlorite ion can be
absorbed and enter the blood (Harrington et al., 1989).
These works (Abdel-Rahman et al, 1979, 1982; Bercz et al. 1984) come to support our
hypothesis of the global reduction of ClO2 to ClO2⁻ and this to chloride in blood as the
basis of the mechanism of action, through intermediate redox interactions with plasma
proteins and cellular enzymes. Abdel-Rahman et al., 1979 found that, in aqueous solution,
potassium chlorite was partly reduced to chloride (21%), but to a lesser extent oxidized to
chlorate (7%), a fact to take into account when controlling the quality and stability of
formulations based on chlorite as an active principle. Therefore, and depending on the
redox environment and pH, a rapid interconversion between these metabolites can be
triggered in initial aqueous solutions of pure ClO2, intensified after gastric and intestinal
digestion (Bercz et al, 1982). The final processing of ClO2 and its metabolites would
consist of its excretion via the urinary and fecal routes, mainly as chloride, with smaller
amounts of chlorite and chlorate, as shown in the aforementioned work by Abdel-Rahman
et al, (1979b; 1984).
5. Pharmacodynamics
Similarly, there is limited work that describes the pharmacodynamics of ClO2 and ClO2⁻in
vivo, that is, the biochemical and physiological effects of this molecule, its mechanisms of
action and the relationship between the dosage of the drug solution and its therapeutic
systemic effect. Taking into account the pharmacokinetic data, it can be hypothesized that
orally administered ClO2 is rapidly reduced to ClO2⁻ in the oral cavity through saliva or in
the stomach through acid secretions (Bercz et al, 1982). In the small intestine, ClO2⁻ would
be absorbed through the villi that are abundantly vascularized, being absorbed mainly as
such anion, without ruling out minor amounts of chloride generated by partial reduction.
Subsequently, both ClO2⁻ absorbed by oral exposure and directly intravenously
administered react by oxidizing electron donors from the complex organic matrix of plasma
and blood cells, in particular proteins and amino acids.

5.1. Interactions between chlorite and peripheral blood cell heme enzymes
Schempp et al. (2001) reported that the product of macrophage hemoprotein-catalyzed
chlorite reduction is a species of chloro-oxygen, probably hypochlorous acid (HClO). The
common characteristic of the redox pair "H2O2 / hemoglobin" and "chlorite-hemoglobin" is
the initial oxidation of heme by transfer of 2 electrons, giving rise to an oxidized heme
intermediate called compound I. Therefore, both oxidants are reduced, resulting in the
hydroxide anion for H2O2, while chlorite is reduced to hypochlorous acid (HClO) that is,
ClO2 would be converted by reduction into chlorite, hypochlorous acid and finally into
chloride (Henderson et al, 1999). In addition, HClO can chlorinate sulfur amino acid
residues generating chloramines such as taurine-chloramine (Tau-Cl), which exert a more
attenuated and stable oxidizing action than HClO, thus modulating excessive inflammation
(Schempp et al., 2001).
5.1.1. Chlorite interaction with hemoglobin
Exposure to ClO2⁻ can produce methemoglobinemia, but this effect was recorded only at
high concentrations. For example, early reports documented that chlorite concentrations of
100 mg / L and higher in the rat caused a decrease in the number of red blood cells and
hemoglobin at 30 and 60 days of exposure (Heffernan et al., 1979).
Another study (Moore and Calabrese, 1982) evaluated the toxicity of chlorite in 2 strains of
mice: one with normal levels (A / J) and another with low levels (C57L / J) of Glucose-6-
phosphate dehydrogenase. G6PD-deficient cells have a reduced ability to produce NADPH,
thus they synthesize very little glutathione (the main defense mechanism of erythrocytes
against oxidative stress). The results showed that when they were exposed to a maximum
level of 100 ppm of chlorite for 30 days, there was an increase in osmotic fragility, mean
corpuscular volume, and G6PD levels for both strains. However, methemoglobinemia was
not reported. In the animals that were exposed to 1.0 and 10 mg / L there were none of the
aforementioned anomalies. The administered doses of sodium chlorite (up to 100 mg / L) in
the drinking water did not cause any toxic damage to the kidneys of the animals at 60, 90
and 180 days of exposure (Moore and Calabrese, 1982).
From these data it is clear that chlorite-mediated destruction of erythrocytes was observed
only at high concentrations, revealing that erythrocytes have an efficient anti-oxidant
protection system. However, when a high ClO2⁻ concentration is administered, this system
is no longer able to neutralize the oxidizing agent and hemolysis occurs. Normal red blood
cells (RBCs) are subject to a high level of oxidative stress as a result of the continuous
production of the superoxide anion that accompanies hemoglobin (Hb) autoxidation. The
superoxide anion is dismutated to hydrogen peroxide (H2O2), which is further converted to
the hydroxyl radical through the Fenton reaction in the presence of iron. To cope with
oxidative stress, RBCs are equipped with superoxide dismutase (SOD1), catalase,
glutathione peroxidase 1 (GPx1), and three isoforms of peroxiredoxin (Prx I, Prx II, and
Prx VI). SOD1 converts the superoxide anion to H2O2, which is then removed by catalase,
GPx1 and the Prxs (Rhee and Lee, 2017).
It has been reported that ClO2, ClO2⁻; and C1O3⁻ in drinking water decreased blood
glutathione and changed the morphology of erythrocytes in rat after 2 months (Abdel-
Rahman et al., 1979). However, rats gradually adapted to C102 stress by increasing the
activity of glutathione reductase and catalase (Couri and Abdel-Rahman, 1979). These
findings are consistent with the known protective role of glutation against damage by
oxidants (Hill et al., 1964).

5.1.2. Chlorite is an erythrocyte protective agent

Although most investigations have reported a hemolytic effect of ClO2⁻ at high
concentrations, it was recently shown that ClO2⁻ at low concentrations is capable of
eliminating methemoglobin (Pichert and Arnhold, 2015; Flemming et al., 2016). Oxygen
transport in our body is carried out by tetrameric hemoglobin in red blood cells and
monomeric myoglobin in muscle tissue. In each subunit of both proteins, a protoporphyrin
IX group is present with a central iron ion that can present different oxidation states. In
both proteins, only the iron of the heme group in the ferrous state (Fe2 +) can bind to
oxygen. Therefore, it is important to keep them in a reduced state. In erythrocytes, this is
achieved primarily by methemoglobin reductase, which converts methemoglobin
(containing ferric iron (Fe3 +) to ferrous hemoglobin (Fe2 +). Spontaneous methemoglobin
formation generally occurs at a low rate, without generating methemoglobin yield greater
than 1% (Van Slyke et al., 1946; Siggaard-Andersen et al., 1972).
From clinical trials conducted with the drug WF10, it is known that ClO2⁻ interacts with
hemoproteins (Schempp et al., 2001; Jakopitsch et al., 2008; Jakopitsch et al., 2014; Pichert
and Arnhold, 2015), which are degraded by ClO2⁻ with the loss of heme structure (Pichert
and Arnhold, 2015). The ClO2⁻-based immunomodulatory drug solution (WF10) and the
more dilute form Oxoferin are applied to cushion severe inflammatory conditions and
improve wound healing processes (Raffanti et al., 1998; McGrath et al., 2002; Veerasam et
al., 2006; Yingsakmongkol et al., 2011).
The ClO2⁻ contained in WF10 was shown to react in three different ways with hemoglobin
and oxidized forms of heme (Pichert and Arnhold, 2015).
5.1.2.1. ClO2⁻ oxidizes hemoglobin to methemoglobin
This can be evidenced by the change in absorbance maximum from 414 nm to 406 nm. The
percentage of methemoglobin increased from 0 to 45% in the presence of WF10 when a 1:
3 dilution was used (corresponding to 21 millimoles / L of ClO2⁻, equivalent to 140 mg /
L). When dilutions greater than 1: 200 (corresponding to 315 micromoles / L, equivalent to
21 mg / L) were used, methemoglobinemia did not occur (Pichert and Arhold, 2015).These
results clearly demonstrate that methemoglobinemia only occurs when high concentrations
of ClO2⁻ are administered.
5.1.2.2. ClO2⁻ degrades methemoglobin
As evidenced by the continuous decrease in the Soret band around 406 nm. This effect was
obtained when a 1: 500 dilution was used (corresponding to 126 µmol of ClO2⁻, equivalent
to 8.40 mg / L). This decrease in ClO2⁻-mediated methemoglobin formation, which was
also later reported by Flemming et al. (2016), the ClO2⁻ component of WF10, inhibited
heme-induced hemolysis in a concentration-dependent manner; these authors concluded
that the beneficial effects of WF10 are closely associated with the inactivation of the free
heme. Furthermore, at a high concentration of WF10, iron release and degradation of the
heme porphyrin ring were also observed. A drug dilution as low as 0.1%, corresponding to
a ClO2⁻ concentration of 62.9 µM (4.19 mg / L), completely inhibited the hemolytic effect
elicited by 100 µM heme (Fleming et al., 2016).
Therefore, these data suggest that one mole of ClO2⁻ is capable of inactivating around two
moles of heme, by these mechanisms; heme is inactivated and loses its ability to induce
hemolytic events in intact red blood cells (Flemming et al., 2016). Free heme is highly
toxic to cells and tissues, especially in the spleen, liver and kidney (Schaer et al., 2013)
through the activation of the Toll-like receptor 4, contributes to the activation of endothelial
cells and macrophages and induces inflammatory reactions (Figueiredo et al., 2007;
Belcher et al., 2014). Sepsis can evoke disseminated intravascular coagulation (such as that
found in Covid-19 patients), resulting in multiple organ failure and death (Marchandot et
al., 2020). Heme oxygenase-1 (HO-1) and hemopexin (HPx) can mediate cytoprotective
mechanisms against these deleterious effects (Passainte et al., 2015). We suggest that ClO2⁻
derived from ClO2 metabolism could decrease the activation of disseminated intravascular
coagulation in patients with Covid-19.
Other researchers (Pichert and Arnhold, 2015) found that WF10 (diluted 1: 500) is capable
of efficiently reducing the production of cytotoxic hemoglobin species that can appear after
excessive hemolysis of red blood cells in pathological situations. Since almost identical
changes were recorded when replacing WF10 with ClO2⁻ at the same concentration, these
researchers concluded that ClO2⁻ is the active ingredient in WF10 (Pichert and Arnhold,
2015). Very recently, a group of researchers discovered that elevated glucose levels directly
induce viral replication and the expression of pro-inflammatory cytokines. Glycolysis is
necessary for the replication of SARS-CoV-2 and the production of mitochondrial ROS
(reactive oxygen species) induced by this virus activates the hypoxia-inducible factor (HIF-
α), which in turn upregulates the glycolytic genes and IL-1b expression. Finally, they
showed that SARS-CoV2-infected monocytes promote T-cell dysfunction and lung
epithelial cell death. These data may explain why the uncontrolled blood glucose levels
observed in diabetic patients are a significant risk for the severity of COVID-19 (Codo et
al., 2020). In this sense, it was shown that the ClO2⁻ -based drug (WF10) constantly
reduces glycosylated hemoglobin (A1c) values and improves glucose control in diabetic
patients (Maraprygsavan et al., 2016).
5.1.2.3. ClO2⁻ reduces ferril hemoglobin (Fe4 +) to methemoglobin (Fe3+)
Ferril hemoglobin is capable of oxidizing numerous biological substrates such as lipids,
nucleic acids, proteins, amino acids, and small molecules (Everse and Hsia, 1997; Gebicka
and Banasiak, 2009). These cytotoxic reactions contribute significantly to tissue destruction
in a number of diseases in which massive hemolysis occurs (Rother et al., 2005). In
addition to the plasma protective components haptoglobin and hemopexin, which have the
ability to bind and eliminate free heme species (Chiabrando et al., 2011), there are other
components in the blood and adjacent tissues that act as antioxidants against oxidation
mediated by ferril hemoglobin.
Ascorbate, urate, and flavonoids such as catechin, quercetin, and rutin reduce ferril
hemoglobin to methemoglobin (Gebicka and Banasiak, 2009; Giulivi and Davies, 1990; Jia
and Alayash, 2008). Pichart and Arnhold (2015) showed that ClO2⁻ (WF10) has greater
potency than the natural antioxidants ascorbate and urate to reduce ferril hemoglobin,
especially when it is derived from methemoglobin. This experimental evidence confirms
that a strong oxidant such as ClO2⁻ is capable of acting as an antioxidant when
administered at low concentrations.
5.2. Inhibition of myeloperoxidase activity mediated by ClO2⁻
Klebanoff (1968) discovered that myeloperoxidase (MPO) is an enzyme that contains a
heme group and is mainly found in neutrophils and macrophages. In the presence of
superoxide, hydrogen peroxide, and chloride, MPO catalyzes the formation of HOCl. The
MPO / HOCl system plays an important role in the destruction of microbes by phagocytes.
However, myeloperoxidase can also be released outside the cell, increasing the potential for
damage in atherosclerosis, Alzheimer's disease, ALS (amyotrophic lateral sclerosis),
multiple sclerosis, stroke, rheumatic arthritis, Parkinson and Alzheimer's disease.
(Klebanoff, 2005). HClO is kinetically the most reactive two-electron oxidant produced in
appreciable amounts in our bodies. Its reactivity with most substrates exceeds that of
hydrogen peroxide, hydroperoxides, and peroxynitrite by several orders of magnitude
(Winterbourn, 2008). In addition to these direct toxic effects, HOCl can modulate the
function and activity of various immune cells. For example, in vitro studies demonstrated
that HOCl can activate nuclear factor kB (NF-kB) and tyrosine phosphorylation in T and B
cells, increasing calcium signaling and tumor necrosis factor α production. (TNF-α)
(Schieven et al., 2002; Schoonbroodt et al., 1997).
Because organizations such as the WHO and the FDA have claimed that ClO2 / ClO2⁻ are
highly toxic, hardly anyone has investigated whether they could have a therapeutic effect if
administered at low concentrations. There are very few studies that have investigated the
therapeutic effects of ClO2⁻. The drugs called NP001 and WF10 are formulated based on
NaClO2 / ClO2⁻, respectively. NP001, a highly purified, pH-adjusted stabilized form of
NaClO2 (sodium chlorite), is a new effector molecule that represents a new class of drug for
regulating the function of inflammatory macrophages in both in vitro and in vivo studies
(McGrath et al. al., 2002). ClO2⁻ mediates its anti-inflammatory effect on macrophages by
creating high intracellular levels of taurine chloramine, a factor known to negatively
regulate inflammatory pathways induced by NF-κB (Joo et al., 2009; Giese et al., 2004). In
previous clinical studies with another form of ClO2⁻ (WF10), this regulation reverses
inflammation and causes systemic macrophages to return to a normal phagocytic state
(McGrath et al., 2002). The results of a randomized phase 2 clinical trial of a new immune
regulator, NP001, in amyotrophic lateral sclerosis (ALS) (NCT01281631) were recently
presented. Although the trial results were negative, they showed that NP001-treated
patients whose baseline CRP (C - reactive protein) levels were above the median of the
entire randomized population had a slower progression of ALS than patients who received
placebo (Miller et al., 2015).
There are several diseases related to abnormal inflammatory response and macrophage
activation: ALS, AIDS, Alzheimer and multiple sclerosis (Minagar et al., 2002).
Macrophage activation has been shown to require high intracellular myeloperoxidase
activity (Rodrigues et al., 2002). Several studies showed that MPO-mediated HOCl
formation can serve as a major source of macromolecular oxidative damage (Winterbourn
et al., 1992; Hawkins et al., 2003; Kawai et al., 2004). Clearly, cellular exposure to HOCl
can cause many deleterious effects by altering the cellular redox state (Woods et al., 2009),
so it is important to modulate excessive MPO activity to avoid further HClO production. In
this sense, a seminal study demonstrated that ClO2⁻ (WF10) at low concentration
(micromolar range) effectively inhibits myeloperoxidase activity (Schempp et al., 2001).
This effect was later confirmed by Jakopitsch et al. (2014). This MPO inhibition is directly
related to the anti-inflammatory effect reported for this drug (Schempp et al., 2001; Giese et
al., 2004).
The inhibition of MPO activity with low ClO2⁻ concentrations could have an important
application in medicine. Resolution of inflammation depends on neutrophils undergoing
apoptosis; however, this does not always happen and neutrophils continue to promote the
inflammatory process. An unexpected role for MPO in influencing neutrophil fate and
consequently the duration of inflammation has been reported. By suppressing the
constitutive cell death program (apoptosis), MPO prolonged the lifespan of the neutrophils,
thus delaying the resolution of inflammation (El Kebir et al., 2008). From these data, it is
clear that induction of neutrophil apoptosis would improve the resolution of inflammation.
As ClO2⁻ is an effective MPO inhibitor, many acute and chronic inflammatory conditions
could be mitigated with the use of this molecule.
Resistance to apoptosis is a key characteristic of cancer cells and is believed to be regulated
by nitrosonium ion (NO) -induced S-nitrosylation of apoptotic proteins such as caspase 3.
Nitric oxide (NO), produced by Inducible nitric oxide synthase (iNOS), is used by MPO to
generate NO. It was shown that 65% of the analyzed invasive epithelial ovarian carcinoma
samples express MPO and iNOS in neoplastic cells, without expression in normal ovarian
epithelium (Saed et al., 2010). These authors also demonstrated that the genetic silencing of
MPO or iNOS significantly induced apoptosis, highlighting its role as a redox switch that
regulates apoptosis. ClO2⁻ could induce apoptosis of cancer cells based on its demonstrated
inhibitory effect on MPO activity.
5.3. Taurine-chloramine neutralizes HClO-induced toxicity
When neutrophils ingest and destroy bacteria, HClO is produced within the phagocytic
vacuole that surrounds the bacteria. Although it is a powerful bactericide, it can also be
toxic because it oxidizes various biological molecules (Kettle et al., 2014). To avoid this
toxicity, taurine chemically reacts with HClO in leukocytes to form taurine-chloramine
(tau-clo), a more stable, less reactive, and more selective oxidant than HClO (Weiss et al.,
1982; Schempp et al. ., 2001; Giese et al., 2004). It is commonly accepted that tau-clo
works in biological systems as a general antioxidant: it can specifically protect cells from
self-destruction during processes that generate HClO excessively (Marcinkiewicz et al.,
1995). Pre-treatment of rats with taurine prior to acute ozone exposure results in a decrease
in oxidant-induced lung damage (Schuller-Levis et al., 1994). In addition, taurine may
prevent HOCl / OCl-induced lysis of lung epithelial cells by decreasing the chlorination of
cellular components rather than preventing oxidation of sulfhydryl groups (Cantin, 1994).
Tau-clo also inhibits the generation of inflammatory mediators produced by macrophages,
such as macrophage inflammatory protein-2 (MIP-2), monocyte chemo-attractant protein-1
and 2 (MCP-1 and 2), nitric oxide, nitrites, prostaglandin E2 (PGE2), necrosis factor tumor
alpha (TNF-α) and interleukin 6 (IL-6) (Kim et al., 1996; Schuller-Levis et al., 1994;
Marcinkiewicz et al., 1995, Barua et al., 2001; Schempp et al. , 2001; Quinn et al., 2003).
These down-regulation mechanisms transform monocytes and macrophages from a pro-
inflammatory state to a basal phagocytic state (Lunetta et al., 2017).
In addition, tau-clo activates the nuclear factor erythroid 2 (Nrf2) (this transcription factor
regulates the inducible expression of numerous genes for detoxifying and antioxidant
enzymes), increases the expression of heme-oxygenase (HO-1), protects cells from death
caused by hydrogen peroxide (H2O2), and improves enzyme expression and activities of
antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase
(Jang et al., 2009). The tau-clo-mediated NRf2 activation mechanism involves a biphasic
effect related to HClO concentration. Low to moderate HOCl concentrations elicit robust
Nrf2 activation in cultured mouse macrophages that acts to restore redox balance. Much
higher concentrations elicit second and third level stress responses that can potentially
terminate the expression of the target Nrf2 gene (Woods et al., 2009).
Other studies demonstrated that the transcriptional effects of tau-clo in genes that regulate
the expression of iNOS (inducible nitric oxide synthase) and TNF-α result, in part, from the
reduction of the translocation of NF-κB to the nucleus of activated cells. . The transcription
of the iNOS and TNF-α genes is critically dependent on the signaling pathway of the
transcription factor NF-κB (Murphy, 1999; Liu et al., 2000; Collart et al., 1990). The
genetic expressions of inflammatory cytokines are known to be regulated by NF-κB (Lloyd
and Oppenheim, 1992; Cassatella, 1995). Tau-clo has been shown to inhibit the production
of inflammatory cytokines (Marcinkiewicz et al., 1998; Kontny et al., 2000); the molecular
mechanism of inhibition of tau-clo-induced NF-kB activation consists of the oxidation of
IκBα in methionine45.
5.4. Taurine-chloramine contributes to the resolution of inflammation
The aforementioned information allows us to conclude that the biological function of tau-
clo in the neutrophil is not only the reduction of the cytotoxicity of HClO, but also the
mitigation of an excessive inflammatory reaction (Kanayama et al., 2002). After destroying
the microbes, mature neutrophils undergo constitutive programmed cell death (apoptosis)
that renders them insensitive to chemo-attractants and allows their recognition and
elimination by scavenging macrophages (Savill et al., 1989), leading to resolution of
inflammation (Savill et al., 2002). However, neutrophil survival and apoptosis are
profoundly influenced by the inflammatory environment, and suppression of neutrophil
apoptosis causes chronic inflammation (Simon, 2003; Gilroy et al., 2004). In fact, a marked
decrease in neutrophil apoptosis was detected in patients with inflammatory diseases
(Matute-Bello et al., 1997; Keel et al., 1997). The regulation of neutrophil apoptosis during
the acute phase of inflammation is less well defined; however, it is essential for optimal
expression and resolution of the inflammation. Recent work confirmed that tau-clo
contributes to the resolution of inflammation by stimulating efferocytosis (phagocytic
engulfment of apoptotic neutrophils by macrophages) (Kim et al., 2015) and inhibiting the
release of MPO by neutrophils (Kim et al., 2020). The increase in the intracellular level of
MPO negatively regulates the expression of MIP-2 (a chemokine involved in the
recruitment of neutrophils at the site of inflammation), thus contributing to a negative
feedback mechanism that ensures that neutrophils do not generate an excessive
inflammatory response (Kim et al., 2020). During efferocytosis, macrophages increase their
secretion of the anti-inflammatory and immunoregulatory cytokine interleukin 10 (IL-10)
and decrease the secretion of pro-inflammatory cytokines, tumor necrosis factor-a (TNF-a),
IL-1 and IL-12 (Voll et al. 1997). Tau-clo-stimulated efferocytosis derived from ClO2⁻
metabolism should be studied in depth, as altered efferocytosis has been observed in many
human autoimmune and inflammatory diseases, including cystic fibrosis, chronic
obstructive pulmonary disease, asthma, fibrosis, idiopathic pulmonary disease, rheumatoid
arthritis, systemic lupus erythematosus, glomerulonephritis, and atherosclerosis (Vandivier
et al., 2006).
5.5. ClO2⁻restores redox balance through hormetic mechanisms
ClO2⁻ is able of inducing protective effects through hormesis, which is a dose-response
phenomenon characterized by stimulation at low concentrations and inhibition at high
concentrations (Calabrese and Baldwin, 2001; Mattson, 2008; Calabrese and Baldwin,
2002; Calabrese and Mattson, 2017). Hormesis can occur as a result of direct stimulation or
as an overcompensatory response after a disruption of homeostasis or the induction of low
to moderate toxicity. (Calabrese, 2008; Calabrese, 2013). As previously mentioned, low
concentrations of ClO2⁻ can inhibit methemoglobin production, inactivate ferril
hemoglobin, and inhibit excessive MPO activity. In general terms, these protective effects
are indirectly induced by ClO2⁻, stimulating the production of antioxidant enzymes that
protect erythrocytes from oxidative damage. In addition, ClO2⁻ indirectly modulates the
synthesis of Nrf2, by inhibiting the excessive production of HClO (Woods et al., 2009).
Although we did not find biphasic effects mediated by ClO2-, an indirect stimulation could
result in strengthening of the body's antioxidant systems, thus stabilizing the redox balance
to protect against oxidative damage.
6. Conclusion
We have found that the main bioactive molecule (derived from ClO2 metabolism) within
the body is ClO2⁻, since, due to its high chemical reactivity, ClO2 is unlikely to remain in
the body unreduced for long after its ingestion. ClO2⁻ has a high permanence in plasma and
tissues after its absorption, before its elimination through the urinary and fecal routes,
mainly in the form of chloride. The antiviral activity of ClO2 and ClO2⁻ has been described
in in vitro and in vivo tests, and is based on the ability to oxidize and denature virus capsid
proteins. The fact that ClO2 / ClO2⁻ are not toxic if they are ingested at low concentrations
suggests an interesting therapeutic application. In vitro studies have shown that ClO2 can
inactivate the human influenza virus, measles virus, and herpes virus (Sanekata et al.,
2010). Other researchers have shown that ClO2 is capable of destroying the polio virus,
hepatitis A, HIV-1, and the coronavirus that caused SARS (cited in Miura and Shibata,
2010). Oral ingestion of aqueous solutions of ClO2 at low concentrations (0.3-0.9 mg / kg /
day) could be used for the prevention and treatment of deadly viral infections, such as
influenza, COVID-19, and AIDS.
The most studied processes of immunomodulatory action are the interaction with
hemoproteins of red blood cells, in particular the metabolization of excess methemoglobin
and the regulation of the enzymatic activity of MPO in neutrophils and macrophages,
transforming monocytes and macrophages from a pro-inflammatory to a basal phagocytic
state. A key intermediate in inflammatory control is the generation of tau-clo induced by
ClO2⁻ (Chinake and Simoyi, 1997; Schempp et al., 2001; Giese et al., 2004) as a stable and
moderate oxidizing agent, which neutralizes excess HClO, mitigates the excessive
inflammatory reaction (Giese et al., 2004) and contributes to the resolution of the
inflammatory response by inducing neutrophil apoptosis (Kim et al., 2020). Therefore, tau-
clo represents the most relevant functional product formed under the influence of ClO2⁻
(WF10) (Schempp et al., 2001; Giese et al., 2004). In fact, an in vitro experiment showed
that WF10 inhibits pro-inflammatory activation of the M1 type in macrophages; the results
suggested that ClO2⁻ is the active principle in WF10 since it produced the same changes as
WF10 (Schönberg et al., 2016). Since ClO2⁻ is also the main product of ClO2 metabolism
after its oral ingestion, we suggest that the use of aqueous solutions of ClO2 at low
concentrations could induce the same therapeutic effects as WF10.
These immuno-modulatory effects could have clinical relevance for the treatment of some
diseases, such as Alzheimer, Parkinson, AIDS, rheumatoid arthritis and amyotrophic lateral
sclerosis. Analysis of the literature also revealed the existence of opposite effects mediated
by ClO2, a stimulating or beneficial effect at low concentrations and a toxic or harmful
effect at high concentrations, according to hormetic principles. This contradicts the
accepted paradigm around ClO2 and ClO2⁻, considered only as toxic agents.

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