Rationale for ozone-therapy as an adjuvant therapy in COVID-19

Abstract

Coronavirus disease 2019 (COVID-19) is the respiratory disease caused by the novel severe acute respiratory syndrome-coronavirus-2 and is characterized by clinical manifestations ranging from mild, flu-like symptoms to severe respiratory insufficiency and multi-organ failure. Patients with more severe symptoms may require intensive care treatments and face a high mortality risk. Also, thrombotic complications such as pulmonary embolisms and disseminated intravascular coagulation are frequent in these patients. Indeed, COVID-19 is characterized by an abnormal inflammatory response resembling a cytokine storm, which is associated to endothelial dysfunction and microvascular complications. To date, no specific treatments are available for COVID-19 and its life-threatening complication. Immunomodulatory drugs, such as hydroxychloroquine and interleukin-6 inhibitors, as well as antithrombotic drugs such as heparin and low molecular weight heparin, are currently being administered with some benefit. Ozone therapy consists in the administration of a mixture of ozone and oxygen, called medical ozone, which has been used for over a century as an unconventional medicine practice for several diseases. Medical ozone rationale in COVID-19 is the possibility of contrasting endothelial dysfunction, modulating the immune response and acting as a virustatic agent. Thus, medical ozone could help to decrease lung inflammation, slow down viral growth, regulate lung circulation and oxygenation and prevent microvascular thrombosis. Ozone-therapy could be considered a feasible, cost-effective and easy to administer adjuvant therapy while waiting for the synthesis of a therapy or the development of the vaccine.

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134
REVIEW
INTRODUCTION
Coronavirus disease 2019 (COVID-19) is a disease caused
by the novel severe acute respiratory syndrome (SARS)-
coronavirus-2 (SARS-CoV-2), a virus belonging to the same
family of the viruses causing SARS and middle-eastern
respiratory syndrome (MERS), which was rst reported in
Wuhan in December 2019, in the province of Hubei, China.1
COVID-19 shows a wide spectrum of clinical manifestations,
ranging from mild, u-like symptoms to severe interstitial
pneumonia.
Patients with more severe symptoms may require intensive
care treatment due to acute respiratory failure, needing
mechanical ventilation and high positive end-expiratory
pressure, and face high mortality risk.2 COVID-19 can
cause an abnormal inammatory response, which indeed
resembles a “cytokine storm” characterized by increased
plasma concentrations of C-reactive proteins, ferritin, pro-
inammatory cytokines (interleukin (IL)-1β, IL-6, IL-12,
tumor necrosis factor, interferon-γ) and decreased numbers of
CD16+ and CD56+ lymphocytes.3 This inammatory pattern is
different from that of severe SARS patients4 and it is similar
to that at the basis of adaptive immunity.5 Immunomodulatory
agents such as anti-rheumatoid arthritis drugs (chloroquine,
hydroxychloroquine and tocilizumab, a humanized monoclonal
antibody targeting IL-6) are currently prescribed as an adjuvant
treatment of COVID-19, in addition to antiviral drugs.6
Immune activation is associated to endothelial dysfunction
and microvascular complications,7 with signicant septal
capillary injury, characterized by mural and luminal
brin deposition, permeation of the interalveolar septa by
neutrophils and signicant deposits of terminal complement
components consistent with sustained, systemic activation
of the alternative and lectin-based complement pathways,
associated with procoagulant state.8 COVID-19 can also
determine thrombotic complications: 1.4% of dead patients
meet the International Society on Thrombosis and Haemostasis
criteria for disseminated intravascular coagulation, as proven
by the increased levels of D-dimer and brinogen, with lower
anti-thrombin levels and spontaneous increase of the length
of international normalized ratio,9 while only 0.6% of patients
who survive meet these criteria10; pulmonary congestion with
microvascular thrombosis and occlusion is the most relevant
aspect on pathology.3 Pathological ndings include “spotty
lungs,” where hyperemic/hemorrhagic areas co-exist with
areas of normal lung. There is also vascular hypertrophy,
with enlarged (up to 20 times) and tubular pulmonary vessels,
with microthrombi, followed by reduction of caliber. In the
alveoli, there are typical ndings of diffuse alveolar damage,
with desquamation of pneumocytes, formation of hyaline
membranes and brotic exudate.11 To suggest the systemic
origin of the coagulative disorder, there is increasing evidence
of central line thrombosis and vascular occlusive events (e.g.,
ischemic limbs).12 Moreover, there is increasing evidence of
Rationale for ozone-therapy as an adjuvant therapy in
COVID-19: a narrative review
Giovanni Tommaso Ranaldi1, Emanuele Rocco Villani2, *, Laura Franza3
1 Unità Operativa Semplice Dipartimentale Farmacologia Clinica e Sperimentazione Clinica, Azienda Sanitaria, Potenza, Italy
2 Department of Geriatrics, Università Cattolica del Sacro Cuore, Rome, Italy
3 Department of Emergency Medicine, Fondazione Policlinico Universitario “A. Gemelli” IRCCS, Rome, Italy
*Correspondence to: Emanuele Rocco Villani, MD, emanuele.rocco.villani@gmail.com.
orcid: 0000-0001-8813-2419 (Emanuele Rocco Villani)
Coronavirus disease 2019 (COVID-19) is the respiratory disease caused by the novel severe acute respiratory syndrome-coronavirus-2 and
is characterized by clinical manifestations ranging from mild, u-like symptoms to severe respiratory insufciency and multi-organ failure.
Patients with more severe symptoms may require intensive care treatments and face a high mortality risk. Also, thrombotic complications
such as pulmonary embolisms and disseminated intravascular coagulation are frequent in these patients. Indeed, COVID-19 is character-
ized by an abnormal inammatory response resembling a cytokine storm, which is associated to endothelial dysfunction and microvascular
complications. To date, no specic treatments are available for COVID-19 and its life-threatening complication. Immunomodulatory drugs,
such as hydroxychloroquine and interleukin-6 inhibitors, as well as antithrombotic drugs such as heparin and low molecular weight heparin,
are currently being administered with some benet. Ozone therapy consists in the administration of a mixture of ozone and oxygen, called
medical ozone, which has been used for over a century as an unconventional medicine practice for several diseases. Medical ozone rationale
in COVID-19 is the possibility of contrasting endothelial dysfunction, modulating the immune response and acting as a virustatic agent.
Thus, medical ozone could help to decrease lung inammation, slow down viral growth, regulate lung circulation and oxygenation and pre-
vent microvascular thrombosis. Ozone-therapy could be considered a feasible, cost-effective and easy to administer adjuvant therapy while
waiting for the synthesis of a therapy or the development of the vaccine.
Key words: COVID-19; cytokine storm; endothelial dysfunction; immunomodulation; medical ozone; ozone therapy; ozone; SARS-COV-2;
virustatic
doi: 10.4103/2045-9912.289462
How to cite this article: Ranaldi GT, Villani ER, Franza L. Rationale for ozone-therapy as an adjuvant therapy in COVID-19: a narrative
review. Med Gas Res. 2020;10(3):134-138.
Abstract
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Ranaldi et al. / Med Gas Res www.medgasres.com
other SARS-CoV2-related vasculitis such as skin purpura8
and cerebrovascular accidents.13
The use of heparin has, indeed, proven useful in these
patients: high-dose heparin is being used with success
in patients who develop moderate to severe forms of
COVID-19.14 Other treatments being investigated include
debrotide, a mixture of single-stranded oligonucleotides
showing anti-clotting properties and used to treat veno-
occlusive disease (NCT04335201), and tissue-plasminogen
activators.15 Yet, even though these approaches seem to work,
they are symptomatic treatments and do not act on the causes
of the endothelial dysfunction.
In this scenario, it is imperative to nd new therapeutic
options. An interesting option could be ozone therapy. In the
present short review, we describe potential benets of ozone
therapy in COVID-19. A literature search was conducted on
PubMed and Embase including “ozone,” “medical ozone,”
“endothelial dysfunction,” “cytokine storm,” “shock,” and
“ARDS” as search terms. An additional search was conducted
on ClinicalTrial.gov including “ozone,” “COVID-19,”
“SARS-CoV-2” and “oxygen therapy.”
WHAT IS OZONE THERAPY?
Ozone is an allotropic form of oxygen, with a molecule made
up by three oxygen atoms. Ozone is a powerful oxidant, di-
rectly acting on the cells through lipidic peroxidation, amino
acids oxidations and DNA irreversible damage, leading to cell
death.16 Being one of the strongest oxidants, it is extremely tox-
ic. Ozone therapy consists in the preparation of an extemporary
mixture of ozone (5% maximum concentration) and oxygen
(95% minimum concentration), so-called medical ozone (MO).
MO has several actions and has been applied on a wide range
of pathologies as an unconventional medicine practice.17 MO
can be administered systemically by adding it to a sample of
patient’s blood which is then reinfused (auto-hemo-infusion)
or by adding it to saline solution. It can also be administered
locally, by subcutaneous/intramuscular injection, by inhalation
or by exposing the skin or other bodily cavities (i.e., rectal,
nasal) to an air mixture containing MO.18 The biological effects
of ozone are mainly mediated by antioxidant systems.19 The
anti-inammatory, immuno-modulator and virustatic effects
of ozone as well as its direct effect on coagulation and micro-
circulation improvement are particularly important.
MECHANISMS OF ACTION
Biochemical reactions
MO reacts within biological liquids – especially in the blood –
with a wide range of substrates, involved in several metabolic
pathways. These complex reactions occur very quickly, and
MO’s half-life lasts only milliseconds. MO mainly reacts with
polyunsaturated fatty acids, bound to albumin and present in
most lipids and phospholipids, as well as with antioxidants,
proteins and carbohydrates.20 Most of MO is involved in the
reaction of “addition of polyunsaturated fatty acids to double
carbon bonds,” known as “Criegée reaction.”20 This reaction
involves the formation of a primary ozonide, which splits into
a lipid peroxidation product whose structure is α-hydroxy-
hydroperoxide and its aldehyde. Lipid peroxidation products
oxidizing power is lower than other peroxides and in an aque-
ous solution it is degraded into hydrogen peroxide, a reactive
oxygen species. Among the aldehydes that are formed in this
process, the most active is the 4-hydroxy-2,3 trans-nonenal,
fundamental in cellular signal-transduction, by upregulating
the antioxidant system in a controlled way on numerous cells
of the organism.21 This cascade of reactions ends with the ex-
haustion of MO. In other words, ozone is an unstable molecule
and causes oxidative reactions but, if administered properly
(i.e., MO), it generates a “controlled and transient oxidative
stress,” which stimulates the cell’s antioxidant system. This
is the paradox that a molecule having oxidizing activity can
be the basis of a complex antioxidant mechanism with several
metabolic effects, as summarized in Figure 1.22
Figure 1: Biological effects of oxygen-ozone.
Note: LOP: Lipid peroxidation product; NO: nitric oxide; O3: ozone; ROS: reactive
oxygen species. Adapted from Bocci et al.22
Anti-inflammatory and immunomodulatory effects
The anti-inammatory and immunomodulatory effect of MO
is expressed through the activation or inhibition of different
molecular pathways, involved in systemic inammation.
For instance, MO inhibits the nuclear factor-kappaB (NF-
κB) pathway, whose activation promotes the transcription
of proinammatory cytokine genes such as tumor necrosis
factor-α, IL-1β, IL-8.23 The underlying reasons for the anti-
inammatory efcacy of MO therapy can therefore be found
in a systemic reduction of inammatory parameters such as
IL-1.24 On the other hand, MO stimulates the activation of
the nuclear factor erythroid 2-related factor 2 pathway,25 an
intracellular transcription factor, binding to the anti-oxidant
response elements nuclear regions encoding for antioxidants
enzymes such as superoxide dismutase, catalase and heme
oxygenase-1. Heme oxygenase-1 is a microsomal enzyme
that catalyzes the degradation of haeme and produces carbon
monoxide, which is another inhibitor of the NF-κB pathway.26
Moreover, heme oxygenase-1 directly activates anti-inamma-
tory cytokines,25 and increases the number of progenitor cells
of the endothelium.27,28 Given the above-mentioned reasons,
one might wonder if ozone can have an immunosuppressant
effect. On the contrary, ozone application in addition to an-
tibiotic therapy can have protective effects on septic injuries
of lungs, by lowering the lipopolysaccharide-induced NF-κB
hyperexpression.16,29 Yet, even though a few studies have been
carried out to evaluate effects of ozone on sepsis, no clear
benets have been found.30-32
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Effects on microcirculation
MO is mostly applied where microcirculation is impaired,
as in peripheral arteriopathies (i.e., diabetic foot or systemic
sclerosis).33 Ozone therapy improves blood ow in poorly
perfused territories, promoting revascularization, vascular
compliance, rheology and blood-tissues gas exchanges. Re-
vascularization can be assessed by video capillaroscopy with
optical probe, with the restoration of the three-dimensional
capillary bed framework,28 if the capillary damage has not
already caused a severe capillary loss.32 Revascularization
depends on a non-chaotic neo-angiogenesis, favored by an
appropriate synthesis of vascular endothelial growth factor,
due to an increased production of hydrogen peroxide.34 In ad-
dition, MO induces nitric oxide synthase with the consequent
formation of nitric oxide and other reactive nitrogen species.
Reactive nitrogen species play a central role as modulators of
the physiological signals of the cardiovascular system and in
particular of vasodilator mechanisms; nitric oxide also acts as
a neuromodulator, and inhibitor of platelet aggregation, vas-
cular adhesion of leukocytes and the proliferation of smooth
muscle cells.35 At an erythrocyte level, the molecular pathways
determined by MO lead to an increase in the concentration
of 2,3-diphosphoglicerate, which determines a right shift in
the dissociation curve of oxyhemoglobin and, therefore, an
increased exchange of oxygen to the peripheral tissues. In ad-
dition, the temporary lipid peroxidation action makes the red
blood cell membrane more deformable. Thus, in the smaller
capillaries, the red cells, instead of moving in disorder, align
themselves and proceed along the axis of the vessel, arranging
in a pile (Fahraeus-Lindquist effect) and facilitating metabolic
exchanges.36 MO has a benecial action in the granulation
and healing processes through lowering production of tissue
plasminogen activation factor and inducing greater quantities
of its inhibitors, favoring the processes of brinolysis to those
of deposition of brin.37
The net result of MO in the damage of microcirculation is to
favor the regeneration of the microcirculation and the gaseous
exchanges (through the increase of the blood ow, decrease
of the blood viscosity and of the platelet aggregation, of the
erythrocyte deformability and of the release of oxygen) and,
at the same time, to reduce thrombotic and brotic processes.
Virustatic effects
Ozone directly inactivates some viruses. To successfully pen-
etrate cells, various viruses (e.g., hepatitis A, human immuno-
deciency virus, Ebola) require that membrane glycoproteins
have sulfhydryl groups in the reduced form. The Ebola virus
has regions on the envelope rich in cysteine, whose altera-
tion blocks the growth properties of the virus.38 Ozone can
permanently oxidize the thiol residues of cysteine in vitro.39
Virustatic ozone against Ebola has also been tested in vivo,
with encouraging results but on a limited sample.40
OZONE IN cOVID-19
MO could play a benecial role in the patient suffering from
COVID-19 on various levels. To foster the use of ozone
therapy in COVID-19 there is the similarity between the
microvascular damage in the peripheral arterial diseases41
and in the diabetic retinopathy42 with the microvascular
(arterioles, precapillary arterioles, capillaries, postcapillary
venules, and venules) damage at lung level in COVID-19.
The latter is evidenced by CT imaging of tubular and en-
larged pulmonary vessels followed by a sudden reduction of
their caliber43 and by pathological ndings of microthrombi
and capillary aneurisms. All these diseases share vascular
features of microhemorrhages, microthrombi, microaneu-
rysms, pericapillary and tissue edema, as demonstrated both
by video capillaroscopy with optical probe and by autopsy
ndings. The action of MO on microcirculation in terms of
organized neo-angiogenesis, increase in blood ow, decrease
in blood viscosity and platelet aggregation, and increase in
red blood cells deformability and gas exchanges was previ-
ously discussed. Overall, MO therapy has given excellent
benets in diabetic foot, trophic or pressure ulcers, systemic
sclerosis.17,33,34 Positive responses in terms of reduction of
ischemic-hemorrhagic damage of the microcirculation could
be obtained in COVID-19 pneumonia and on other paren-
chyma by an early MO auto-hemo-infusion. These positive
responses could be mediated both by increasing the produc-
tion of surfactant and the elasticity of the alveoli and by a
reduction of endothelial damage, through platelet adhesion
reduction and through blockage of the coagulation cascade
preventing microthrombi formation.44 In addition, this effect
would also occur at a peripheral level, by reducing formation
of deep venous thrombi and consequent pulmonary embolism.
Both micro and macrothrombi lead to the formation of pul-
monary shunt and pulmonary hypertension, that are typical
features of hypoxemia refractory to ventilatory therapy.45 To
our knowledge, no studies have been published regarding the
development of pulmonary hypertension in COVID-19 or its
severity in those with a history of pulmonary hypertension.
In the authors’ opinion, MO should not be administered in a
context of advanced microvascular lung damage with massive
capillary loss, similar to what occurs in systemic sclerosis
with pulmonary hypertension46; in this context ozone could
determine a worsening of lung damage, inammation and
edema in patients with pulmonary hypertension,47 or it could
have no effect due to the capillary loss. Yet, an anti-hypoxemic
action was observed by the MO auto-hemo-infusion in pa-
tients suffering from chronic obstructive bronchitis.48 Further-
more, in an animal model with a healthy lung, ozone seems to
have a protective action on the genesis of pulmonary edema,
thanks to the ability to stimulate the pulmonary sympathetic
nervous system, and block microvascular responses to acetyl-
choline and substance P at a lung level.49 Interestingly, when
applied to the patient with severe respiratory insufciency,
requiring mechanical ventilation with long-term elevated
positive end-expiratory pressure (as in severe COVID-19
pneumonia), inhaled ozone improved gas exchanges in the
lung, increased surfactant production and lung compliance
in a small sample of patients.44
Another potential benet of MO auto-hemo-infusion could
be on the “cytokine storm” in the pathogenesis of respiratory
failure, disseminated intravascular coagulation and multi-
organ failure in COVID-19. The protective immunomodula-
tory effects of ozone in septic lung injury have already been
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Ranaldi et al. / Med Gas Res www.medgasres.com
discussed previously. “Cytokine storm” hemodynamic conse-
quences are similar to those of toxic shock.50 In toxic shock,
as in septic shock, microcirculatory abnormalities are cardinal
features, being dissociated from that of systemic hemody-
namic and more severe among non-survivors.51 Therefore,
microcirculatory alterations may persist despite correction of
systemic hemodynamic variables with vasopressors drugs.52
Ozone preconditioning has been demonstrated to be as potent
as dexamethasone in reducing tumor necrosis factor-α level
during lipopolysaccharide-induced endotoxic shock and
oxidative stress in a murine model.31 In addition to primary
“cytokine storm,” a superinfection and a secondary septic
shock are prevalent among patients with COVID-19.31,53
Ozone has both a direct bactericidal action, through lipid
peroxidation product and reactive oxygen species,54 and an
indirect one, through an increase in neutrophilic chemotaxis
and myeloperoxidase activity.55 Thus, MO could both reduce
the microcirculatory alterations induced by the “cytokine
storm” and the occurrence of bacterial superinfections and
septic shock.
Like in the case of Ebola virus, another potential action
of ozone is the inactivation of the spike protein that coro-
naviruses need to infect guest cells. The spike protein is
rich in cysteine residues, permanently oxidized by ozone,
and these residues are preserved during the evolution of the
viral strains.56 Therefore, SARS-CoV-2 could potentially be
inactivated by ozone-mediated oxidation during the viremic
phase (if administered early via auto-hemo-infusion) and at
the level of the colonized nasopharyngeal mucosa of healthy
carriers through topic formulations.57
Only one clinical trial about ozone therapy in patients
affected by severe COVID-19 related pneumonia has been
conducted to date (NCT04370223). Preliminary data showed
that the patients treated with MO presented both shorter time
to clinical improvement and a signicantly higher proportion
of patients achieving 14-day clinical improvement compared
to those receiving supportive care.58 Yet, it was a single-center
study enrolling few patients and larger randomized clini-
cal trials are needed. On the other hand, three trials about
hyperbaric oxygen therapy have already been conducted
(NCT04370223, NCT04343183, and NCT04332081). This
preliminary data suggest potential benet of hyperbaric
oxygen therapy in COVID-19.59 This nding is also of in-
terests because, in a murine model of pulmonary damage,
a combination of hyperbaric oxygen therapy and MO was
demonstrated to be more effective than hyperbaric oxygen
therapy regarding serum IL-1β, lung glutathione storages
and histologic outcome.29
cONTRAINDICATIONS FOR OZONE THERAPY
Regarding MO systemic therapy, there are some contraindi-
cations and warnings. First, MO should not be administered
directly in the blood and should not be mixed in solution
with other drugs but saline solution, because of its oxidizing
effect. Second, there are no studies about ozone-therapy and
pregnancy; hence it should be avoided in this case. Another
absolute contraindication is glucose-6-phosphate dehydroge-
nase deciency in systemic administration,60 the same warning
concerning antimalarials.61 Potentially, MO administration in
patients suffering from hyperthyroidism could be harmful for
the lungs, as shown in murine models.62
cONCLUSIONS
The adjuvant use of ozone-therapy in COVID-19 through auto-
hemo-infusion could better oxygenate the tissues, decrease
lung inammation and regulate the immune response, avoiding
the “cytokine storm,” slow down viral growth, regulate lung
microcirculation and avoid or slow down vascular hypertrophy
and the consequent hyperemia, especially in the initial stages,
by contrasting endothelial damage, in analogy with what hap-
pens in peripheral arterial pathologies. An association with
other currently available treatments is mandatory, to avoid or
limit the use of intubation and, ultimately, allowing a shorten-
ing of healing times with the possibility of greater replacement
in the intensive care, to date the real limiting factor.
If auto-hemo-infusion is not administrable, MO could be
delivered through dilution in saline solution or rectal infu-
sion. Endonasal application could be administered to inac-
tivate colonizing SARS-CoV2 in the asymptomatic carriers.
Furthermore, since there are no major side effects and it can
be synergistic with other therapies, it is a candidate to be
an essential therapy in home care, given its easy execution
and low cost. Ozone-therapy could be considered a feasible,
cost-effective and easy to administer adjuvant therapy while
waiting for the synthesis of a therapy or the development of
the vaccine.
Author contributions
Manuscript concepts and preparation: GTR; search strategy and manu-
script editing: GTR, ERV; manuscript review: ERV, LF. All authors
read and approved the nal version of manuscript for publication.
Conflicts of interest
The authors declare no conicts of interest.
Financial support
The authors declare they did not receive nancial support for the
present review.
Copyright license agreement
The Copyright License Agreement has been signed by all authors
before publication.
Plagiarism check
Checked twice by iThenticate.
Peer review
Externally peer reviewed.
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the terms of the Creative Commons Attribution-NonCommercial-
ShareAlike 4.0 License, which allows others to remix, tweak, and
build upon the work non-commercially, as long as appropriate credit
is given and the new creations are licensed under the identical terms.
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Received: May 10, 2020
Accepted: May 11, 2020
Published Online: July 13, 2020
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