During this “coronavirus era”, fake news is creating a historical confusional state.
Our goal is to clarify the effectiveness of ozone therapy vs Covid-19, reporting all the authentic scientific evidence of any problem related to the use of ozone.

Ozone is a double-faceted gas. It has a crucial protective relevance in partially blocking mutagenic and carcinogenic UV radiations emitted by the sun (wavelengths of 100-280 nm) in the stratosphere [a], while its increasing concentration in the troposphere causes severe pulmonary damage and increased mortality [b,c].

In spite of this drawback, there are growing experimental and clinical evidences about the medical use of ozone [d-e]. Since XVI Century, Paracelsus had ingeniously guessed that “all things are poison and nothing is without poison and only the right dose differentiates a poison from a remedy”.

In 2005, Timbrell reiterated the concept in his book: “The poison paradox; chemicals as friends and foes” [f]. During the Earth evolution, harnessing oxygen by metazoans has allowed a fantastic biodiversity and growth but it has also created a slow acting “poison”.

It is reasonable to believe that the antioxidant system slowly evolved and specialized during the last two billion years for counteracting the daily formation (3-5 g in humans) of anion superoxide in the mitochondria and the release of H2O2 by ubiquitous NADPH oxidases.

However, there is a general consensus that the physiological production of H2O2 is essential for life. Olivieri et al. [g] and Wolff [h] were the first to describe the effect of either low concentrations of radioactive thymidine or of a very low dose of radiation inducing an adaptive response in human cells in comparison to a high dose.

Goldman [i] introduced the term “hormesis” to mean “the beneficial effect of a low level exposure to an agent that is harmful at high levels”. It goes to the merit of Calabrese to have experimentally controlled this concept and to have presented a number of examples of stimulatory responses following stimuli below the toxicological threshold.

Until 2002 ozone therapy was pharmacologically conceived as a therapy where low ozone doses were stimulatory, while high doses were inhibitory. This conception, reflecting the classical idea that a low antigen dose is stimulatory, where an antigen overdose is inhibitory, was vague and unsuitable because ozone acts in a complex way and a high dose can still be effective but accompanied by side-effects.

Indeed, one of us in 2002 amply delineated the sequence of biochemical reactions elicited ex vivo after the addition of a certain volume of O2-O3 gas mixture to an equal volume of human blood [m].

First of all, mixing blood with an oxidant implies a calculated and precise oxidative stress, i.e. a homeostatic change with production of highly reactive messengers. The oxidative stress, like many others, induces a biological response leading to an adaptive phenomenon.

The teleological significance of this response is universal, from bacteria to plants and Mammals, and small repetitive stresses induce an extremely useful adaption response represented by the revival of critical defense mechanisms [m-n].

At the same time, Calabrese and Baldwin described the “overcompensation stimulation hormesis” (OCSH) as the result of a compensatory biological process following an initial disruption in homeostasis [l].

After a reviewer’s information also Relater on had expressed this possibility [o]. Ozone presents some subtle differences that will be explained by clarifying the biochemical reactions occurring between the organic compounds of plasma and this gas.

Ozone (O3) is the triatomic allotrope form of oxygen, its oxidant potency is the third after fluorine and persulfate and it is higher than O₂ [15].

Ozone therapy (OT), in the medical setting, employs a gas mixture of O₂/O₃, obtained from the modification of medical-grade oxygen using an ozone generator device that has to be administered in situ because of ozone’s short half-life (at 20 °C the O3 concentration is halved within 40 min, at 30 °C within 25 min) [15].

Usual clinical O₃ concentrations range from 10 to 60 µg/mL (using O₂ as vehicle) which, on a percentage basis, may range in a mixture of O3 (0.5–0.05%) and O2 (95–99.5%) [16].

The main mechanism of O2/O3 on human physiology fits the concept of oxidative preconditioning [17].

This concept has now been demonstrated at both the proteomic and genomic level [18], in in vitro studies and in clinical trials [19]. A calibrated oxidant stimulus by O2/O3 can modulate the endogenous antioxidant system and aid in the control of different pathological conditions [20].

The modulation of O2/O3 at the Keap1/Nrf2/ARE pathway and the reduction of IL-6 and IL-1β are involved in the mechanism of action of ozone [21]. This implies that the cytoprotective effect observed during the O₂/O₃ treatment may impact clinical conditions caused by SARS-CoV-2.

This review is focused on the cytoprotective effect of O₂/O₃ in different tissues, primarily though the modulation of the NF-κB /Nrf2 pathways and the IL-6 and IL-1 β cytokines.

There is preclinical and clinical evidence to support the potential role of OT in the prevention and management of cytotoxicity induced by different drugs and diseases including viral diseases [21–24].

The main mechanism is related to the modulation of the oxidative stress and pro-inflammatory cytokines. The Evidence Acquisition Terms included in the information search were:

COVID-19, SARS-CoV-2, SARS, ozone, OT, viral pneumonia. Bibliographic databases consulted: MEDLINE/PubMed, SciELO, LILACS, PAHO, EMBASE, ZOTERO ISCO3, WHO International Clinical Trials Registry Platform and NIH. U.S. National Library of Medicine.

The type of documents reviewed were published between 1980 to 2020, in Russian or English and included: Original articles, published thesis, clinical reports, ongoing clinical trials and bibliographic reviews. The exclusion criteria were the lack of free access to complete text due to financial constraints and/or, studies presenting inadequate scientific evidence.

Potential Therapeutic Actions of Ozone in Viral Diseases

Ozone can inactivate viruses via direct oxidation of its components [25]. However, the viricidal activity in vivo becomes uncertain when viruses are in biological fluids or, even worse, when they are intracellular (pneumocytes, hepatocytes, epithelia, CD4+ lymphocytes, monocytes, glial and neuronal cells) because the cell’s potent antioxidant system protects viral integrity [15].

That is why it is irrational to use direct I.V. injection of gas or other non-recommended methods of application of ozone [16,26].

OT represents a useful adjunctive and complementary therapy but neither ozone, nor H2O2 (one of the main O3 mediators) can reach sufficient concentrations in tissues because the plasma antioxidant capacity protects free pathogens and intracellular viruses are inaccessible [27].

In order to explore the efficacy of OT in viral diseases, Bocci and Paulesu [28] explained the possibility of how ozone may act in vivo. The following mechanisms may have some relevance:

1. A prolonged ozone therapeutic treatment appears able to induce an adaptation to oxidative stress, hence a re-equilibration of the cellular redox state, which is a fundamental process for inhibiting viral replication. The paradoxical mechanism by which ozone (a potent oxidant) can induce an antioxidant response, is currently demonstrated not only at a proteomic level, but also at a genomic one. Ozone applied at a therapeutic dose modulates the nuclear factor Nrf2 and NF-κB and induces the homeostasis of the antioxidant environment [18,29–32]. Oxidative stress and innate immunity have a key role in lung injury pathways that control the severity of acute lung cytotoxicity during viral infections like SARS [33].
2. The induction of antiviral cytokines such as IFN and the modulation of pro-inflammatory cytokines as IL-6, have been demonstrated by ozonating blood such as major autohemotherapy (MAH). Although ozone is a weak inducer, reinfused lymphocytes and monocytes during mayor autohemotherapy (MAH), can migrate through the lymphoid system, and activate other cells that, in time, will lead to the stimulation of the immune system [32,34]. This may represent an important process because it is known that an acute viral disease becomes more severe because the virus is particularly virulent, the heterogeneous viral population evolves rapidly and escapes immune control, or because the immune system becomes tolerant to viral antigens and becomes unable to counteract the infection. Moreover, besides the induction of HO-1 [18], a protective enzyme, there is also the release of some heat shock proteins (HSP) such as HSP60, HSP70 and HSP90 that also have an influence on viricidal activity. These proteins are potent activators of the innate immune system, and are able to induce the monocyte-macrophage system and the activation of antigen- presenting cells [15,35]. The evidence shows that, during the COVID-19 epidemic, the severe deterioration of some patients has been closely related to a dysregulated inflammatory process referred to as “the cytokine storm” [36,37].
3. Oxygen-ozone therapy improves oxygenation [38,39], especially in poorly oxygenated tissues [40]. Patients with SARS are prone to have mild non-specific hepatitis [41], lung fibrosis[42] and renal failure [43]. OT stabilizes hepatic metabolism and tend to normalize fibrinogen and prothrombin plasma levels in infected patients, suggesting an improvement of the hepatic protein synthesis [15]. There is extensive research demonstrating the cytoprotective effect of ozone to prevent oxidative damage to the heart [44,45], liver [46,47], lungs [48] and renal tissues [49]. The authors have described in a recent review, the cytoprotective effect of ozone to prevent and even to treat chemotherapy-induced damage in heart, liver, renal and lung tissue [22].
4. During blood ozonation ex vivo for the minor autohemotherapy, using ozone concentrations near 90 µg/mL per mL of blood, it may be feasible to induce the oxidation of free viral components, which could theoretically represent an inactivated and immunogenic vaccine [15,50,51].
5. Ozonized Saline Solution (O3SS). This method was formalized by the Ministry of Health of the Russian Federation in the early 1980s and has been officially implemented in public health hospitals, specifically for the specialties of orthopedics, dermatology, gynecology and obstetrics [16,52]. In 2004, it was also officially recognized in Ukraine. The beneficial effects of this therapy are supported by a large amount of scientific papers and strong clinical experience. [53]. The method consists of bubbling and saturating a physiological solution (0.9%) with ozone-oxygen mixture at concentrations that are calculated depending on the patient’s weight (ranging 1–5 µg/kg b.w.). Its administration takes about 20–30 min. Unlike MAH, the O3SS has been used as complementary therapy in viral diseases such as Epstein Barr, Cytomegalovirus, Papillomavirus, HIV, Herpes zoster, Herpes simplex, etc. Since the saline solution is a plasma expander, O3SS represents a greater amount of blood being treated than MAH and therefore, theoretically, the number of sessions could be reduced.Korolev B.A., Boyarinov G.A. and Sokolov V.V.[54,55] showed that when an O3SS was used during cardiopulmonary bypass, the cells of the patient’s organs use more glucose compared to basal levels. Therefore, it is concluded that the therapeutic effects of ozonated physiological solutions, is determined by the dissolved O2/O3 mixture, free radicals, hydrogen peroxide and hexagonal aqueous structures formed during the bubbling of aqueous NaCl solutions with a mixture of O2/O3 gas.

Ozone Therapy as Redox Modulator

During a systemic application of O2/O3 (mainly MAH, O3SS, vaginal and rectal insufflation), part of the O3 is removed by the antioxidants of the medium. Further reaction of O3 with biomolecules generates aldehyde (e.g., 4-hydroxynonenal (4-HNE)) and peroxide (H2O2 and organic peroxides).

These byproducts of the reaction act as secondary messengers and induce a further adaptive hormetic responses [56–58]. Ozone at a therapeutic dose “only acts” as a modulator or pro-drug and, by inducing secondary messengers, will enhance subsequent adaptive responses [21].

Mediators such as 4-HNE and H2O2 are among the most relevant secondary messengers producing beneficial effects during medical applications, they induce a gradual oxidative stimulus, that produces the synthesis of endogenous antioxidants such as SOD, CAT and GPx [18,59].

This fact implies that O2/O3 is a paradoxical pro-oxidant therapy that invokes an endogenous antioxidant response. Moreover, low quantities of H₂O₂ formed as consequence of O₂/O₃ have a key role in the molecular mechanism. H2O2 is crucial and a common activator of the modulation of NF-κB and Nrf2 pathways [60–62].

In addition, 4-HNE also sends a signal of transient oxidative stress and its effects depend on concentration as well as cell/tissue origin. This pathway can activate the synthesis of several substances such as: γ-glutamyl transferase, γ-glutamyl transpeptidase, HSP-70, HO-1, and antioxidant enzymes such as SOD, GPx, CAT and glucose-6-phosphate dehydrogenase [21].

In addition, these pluripotent effects of 4-HNE can be explained by its concentration-dependent interactions with the cytokine networks and complex cellular antioxidant systems also showing cell and tissue specificities [59,63].

Experimental results demonstrated that ozone at therapeutic dosages ex vivo or in vivo can activate Nrf2 [20,32] that involve an indirect modulation (inhibition) of the NF-κB pathway.

In addition, Nrf2 suppresses NF-κB activity by eliminating ROS, which may cause NF-κB activation via antioxidative protein induction, such as HO-1 and NQO1. Moreover, Nrf2 suppresses NF-κB activity through some protein–protein interactions, and also suppresses an inflammatory cytokine gene expression through binding to their gene promoter directly [64]. NF-κB pathway activates the release of pro-inflammatory cytokines like: TNFα, IFNγ, IL1β, IL6, IL8, as well as pro-inflammatory genes likecyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) [65].

It is well known that the regulation of both pathways, NF-κB and Nrf2, involved a crosstalk to bring a coordinated inflammatory response [66,67]. The modulation of the inflammatory response by ozone was evident in a clinical trial l on patients with multiple sclerosis (ME) treated with O2/O3 by rectal insufflation for 30 days [19].

Taking the original data of this trail and recalculating the values in terms of the ratio Nrf2 phosphorylation (as expression of activation of Nrf2 pathway) and IL-1β (as marked of the NF- κB pathway) this modulation became evident (Figure 1).

In patients with ME without treatment, the balance Nrf2/NF-κB favor the inflammatory process, and O2/O3 restores the balance of those pathways.

An individual analysis of the variable assayed in this study shown a significant (p < 0.05) increase in Nrf2 values in ozone treated patients compared to control group (0.93 vs. 0.75 densitometry unit, respectively), that restore the significant downregulated values of Nrf2 at basal level in ME patients (0.56 densitometric unit).

The increment in Nrf2 was in line with the significant reduction in 61 % of the level of the pro inflammatory cytokine IL-1β in ME-treated patients compared with basal levels, even IL-1β levels in ME remain 94 % higher than values in normal subjects.

An imbalance between Nrf2/NF-κB has been proposed in other diseases like diabetic neuropathy

[68] in which ozone experimentally demonstrated its efficacy equilibrating this disruption [62]. Similar trends were found in viral diseases, NF-κB pathways can support influenza A virus infection and promote pneumonia.

Through the Activation of the Nrf2 signaling some drug as emodin can increased the survival rate, reduce lung edema, pulmonary viral titer and inflammatory cytokines, and improve lung histopathological changes [69].

In addition, it has been shown that the rabbit hemorrhagic disease virus (that causes lethal fulminant hepatitis in rabbits) has a pathological mechanism that involves is the repression of Nrf2 pathway [70].

Ozone Therapy and Cytoprotection

Antioxidants are important for the maintenance of cellular integrity and cytoptotection. Modulating the balance Nrf2/NF-κB, O₂/O₃ not only increases the endogenous antioxidant system but also modulates the expression of pro-inflammatory cytokines and has an impact in cytoprotection.

COVID-19 infects the upper and lower respiratory tracts and causes mild to highly acute respiratory syndrome with consequent over-expression of pro-inflammatory cytokines, including interleukin IL- 1β and IL-6.

Activation of toll-like receptors by SARS Cov-2 RNA lead to the release of pro-IL-1β which is cleaved by caspase-1, followed by inflammatory activation and production of active mature IL-1β which is a mediator of lung inflammation, fever and fibrosis [71]. However, suppression but not depletion of the pro-inflammatory IL-1 family and IL-6 have been shown to have a therapeutic effect in many inflammatory diseases, including viral infections for instance, Mice lacking IL-1 signaling expression, elevated viral replication of coronavirus [72].

In addition, IL-6-deficient mice infected with influenza virus exhibited a higher lethality, more body weight loss and had higher fibroblast accumulation and lower extracellular matrix (ECM) turnover in the lungs than their wild- type counterparts [73].

The inflammasome, a cytosolic protein complex that mediates the processing and secretion of pro-inflammatory cytokines, is one of the first responders during viral infection.

The cytokines secreted, following inflammasome activation, regulate cells of both the innate and adaptive immune system, guiding the subsequent immune responses. Therefore, not suppressive but a modulator of cytokines may impact efficiently in vital cytotoxicity. A representative data about the downregulated of cytokines IL-1β, IL-6, IL-8 and TNF-α are shown in Table 1.

Table 1. Effect of ozone therapy as modulator of pro-inflammatory cytokines.

The expression of cytokine responses to a previous signal is closely connected with the action of nuclear factors. An in vitro experiment, conducted in cardiomyocytes and skin fibroblasts, analysed the role of ozone at the level of Nrf2 and NF-κB induced by doxorubicin [44]. The authors analysed the individual role of different doses of ozone in this model.

A re-analysis of this data, calculating the rate NF-κB/ Nrf2, showed the evident downregulation of the effect of ozone but not suppression (Figure 2). The same trend was observed also in skin fibroblasts cultures treated with doxorubicin and O2/O3.

An analysis of the individual values showed that the restoration of the equilibrium NF-κB/Nrf2 was reached essentially by the preservation of the level of Nrf2 in ozone-treated cells (0.8 vs. 0.9 fold of chance, in control group, respectively), compared with the depleted values of Nrf2 observed in doxorubicin-treated cells (0.5, fold of chance, with respect to control cell culture).

This maintenance of Nrf2 levels avoids the 100% increment in NF-κB that takes place during the doxorubicin treatment. The experiment showed that the intervention with ozone, preserve Nrf2 essential facts to avoid and up-regulate NF-κB.

The hormetic response, oxidative preconditioning or the adaptation to the chronic oxidative stress, during OT, has been now demonstrated experimentally [78].

The concept of doses is very remarkable in OT, the administration route and clinical protocols are also important.

At higher doses the described effect of ozone could drastically change. High doses of ozone induce the gene transcription of the pro-inflammatory cytokine, its receptor, and inflammatory proteins. At the same time, they invoke a negative regulation of type 1 Interferon and the response to viral infections pathways [79].

The recommended systemic administration routes are: O3SS, MAH and Extracorporeal Blood Oxygenation-Ozonation (EBOO).

Clinical protocols should comply with the standard doses and procedures defined in the Madrid Declaration of OT [16]. At least three clinical trials using major autohemotherapy are in progress in China and more clinical trials are needed to confirm the efficacy of OT as complementary therapy in the treatment of COVID-19 diseases.

It is a complementary therapy because, while the infected patient is treated with allopathic medicine, at the same time the patient will also receive the complementary proposed treatment.

It should be noted that, even if ozone has no effect on the virus infection, the demonstrated modulation of oxidative stress and inflammatory cytokines by ozone therapy could offer a relevant and beneficial clinical effect. Additionally, a small impact in the requirements of inpatients days, especially on intensive care units, could lead to a high benefit in the current critical situation that many countries are suffering.

The Biochemical and Pharmacological Properties of Ozone

General Aspects of Ozone Mechanisms of Action and Effects on Cell Metabolism
The observed pharmacological effects of ozone underlie its chemical and biochemical properties.

The pharmacological properties of ozone are depending on ozone being a triatomic oxygen molecule, reacting with organic compounds containing double bonds and adding the three oxygen atoms to the unsaturated bond with the formation of ozonides.

This reaction is of great importance, since ozone causes the split of the double bonds with a reaction called ozonolysis. In an aqueous medium (i.e., blood), the ozonides are immediately transformed into stable hydroperoxides having the ability to release oxygen when the pH increases, as it occurs in protonic environments.

Such a physical-chemical characteristic is typical of degenerative processes and/or ischemic conditions. The lipoperoxides derived from the breaking of a chain of ozonides lose the hydrophobicity characteristic of lipids and become soluble in water since they are short-chain lipid compounds.

It is generally understood that the toxic effects of ozone are mediated through free radical reactions, although ozone is not a radical species per se [206]. Two different mechanisms may be advocated to explain the ozone-derived radical formation: a direct mechanism involving the oxidation of biomolecules to give classical radical species (hydroxyl radical) and a mechanism involving the radical-dependent production of cytotoxic, nonradical species (aldehydes) [207].

However, in order to fully elucidate the biochemical basis underlying the pharmacological effects of ozone, it is important to illustrate its effects on various coenzymes which are responsible for ozone cell metabolism regulation.

To this regard, one of the main effects of ozone is the acceleration of glycolysis; as is known, in fact, a fundamental condition for guaranteeing the continuity of this process is the constant reoxidation of NADH as it occurs following ozone exposure.

As far as concerning protein metabolism, ozone intervenes mainly due to its remarkable affinity towards sulfhydryl groups such that it occurs when reacting with glutathione.

Similarly, ozone reacts with essential amino acids such as methionine, tryptophan, and other amino acids containing sulfur (i.e., cysteine). In this case, the amino acids are protected from ozone inactivation by two reactions that prevent their degradation: first the oxidation of glutathione and then the oxidation of the coenzymes NADH and NADPH, key reactions in the biochemical mechanism of ozone.

Finally, ozone reacts directly with unsaturated fatty acids, which have a double carbon bond and are therefore available for an oxidative reaction, leading to the formation of peroxides following hydrolytic cleavage of the lipid chains. The lipid chains are thus fragmented with a loss of their hydrophobic character and are transformed into hydrophilic components.

Ozone Therapy and Pulmonary Diseases

Previous studies showed that ozone exhibits controversial effects in pulmonary disease. In particular, Leroy et al. [208] showed that the exposure of human subjects to ozone (200 ppb) results in a significant increase in the expression of various genes involved in the wound healing process (i.e., osteopontin).

The authors included in their study nineteen subjects, with and without asthma, exposed to clean air (0 ppb), low (100 ppb), and high (200 ppb) ambient levels of ozone for 4 h on three separate occasions in a climate-controlled chamber.

Successively, the subjects recruited for the study were treated with bronchoscopy with bronchoalveolar lavage (BAL) 20 h after the end of exposure. In order to further explain the controversial effects of ozone in pulmonary diseases, we further analyzed the authors’ datasets from the NCBI GEO (Available online: http://www.ncbi.nlm.nih.gov/geo/) databank under accession number GSE58682 (GPL6244), in order to delineate a gene expression profile able to determine an analysis of gene ontology.

In particular, we focused our attention to the data of healthy subjects and compared 18 subjects exposed to clean air (0 ppb) vs. 19 subjects to high (200 ppb) ambient levels of ozone.

By restricting the threshold level of significance to p < 0.01 and the log10 fold change to 0.7 for the upregulated genes and −0.7 for the downregulated, we identified 29 upregulated and 309 downregulated genes in subjects exposed to 200 ppb of ozone, compared to the subjects exposed to clean air.

Our Gene Ontology (GO) analysis was performed using the web utility GeneMANIA (Available online: http://genemania.org/) [209] and the GHATER (Gene Annotation Tool to Help Explain Relationships) (Available online: http://changlab.uth.tmc.edu/gather/) [10], allowing us to assemble all the available interaction data in the dataset by creating large networks, which captures the current knowledge on the functional modularity and interconnectivity of genes in a cell.

During our analysis, we identified 29 upregulated and 309 downregulated genes significantly expressed in the BAL of healthy subjects exposed to 200 ppb of ozone for 4 h compared to exposed only to clean air.

The GO analysis showed surprising results. Indeed, following ozone exposure, the alveolar parenchyma responds with the activation of genes involved in immuno-activation. Processes such as chemotaxis (cell chemotaxis, leucocyte chemotaxis, and granulocyte chemotaxis), responses to cytokines (cytokine receptor binding), and inflammation were significantly triggered (Figures 1A and 2A–C).

On the other hand, the downregulation of several genes involved in the antiviral response and in the regulation of type I interferons could, in the long term, predispose to a susceptibility to opportunistic infections (Figure 1B, Figure 2D–F, and Figure 3). Our findings are consistent with previous reports [211], showing that ozone exposure reduces the ability of mice to survive a K. pneumoniae infection by reducing the phagocytic ability of alveolar macrophages.

Ozone Therapy and CNS

Recent studies have reported that the nervous system is affected by ozone exposure. In particular, an increase in slow wave sleep time and a decrease in the total time of paradoxical sleep have been found in cats exposed to 1 ppm (parts per million) of O3 [212].

Similarly, changes in mental performance, fatigue, headaches, and lethargy have been referred to in humans exposed to this gas [213]. To this regard, several reports suggest that ozone leads to increased oxidative stress in the central nervous system (CNS), which in turns leads to significant brain dysfunction as measured by cognitive and motor activity impairment (Figure 4).

In particular, Rivas-Arancibia et al. showed that rats exposed to various ozone concentrations (0, 0.1, 0.2, 0.5, or 1 ppm) exhibited deterioration of their long-term memory in the passive avoidance test and decreased motor activity [214].

Furthermore, the authors suggested that such functional impairment was also accompanied by increased Cu/Zn SOD levels, thus suggesting that ozone exposure affects long-term memory possibly in association with oxidative stress (Figure 4).

Consistently with this hypothesis, Guerrero et al. [215] demonstrated that vitamin E (50 mg/Kg) prevented ozone exposure-mediated lipid peroxidation and mitigated brain dysfunction as measured by short- and long-term memory improvement in a passive avoidance task.

Consistently, Avila-Costa showed that ozone inhalation led also to morphological changes in the brain with particular regards to the hippocampus CA1 region [216]. In particular, the authors showed that ozone exposure (1 ppm) resulted in a significant reduction in spine density in the pyramidal neurons of the hippocampus and that this may account for the previous described memory impairment.

Similarly, other areas of the CNS are particularly involved in ozone-mediated toxicity, including the central respiratory areas (i.e., nucleus tractus solitaries and ventrolateral medulla).

Ozone exposure leads to increased oxidative stress in these regions and, as a result of such oxidative insult, astroglial cells increased the vascular endothelial growth factor (VEGF) expression as a potential protective mechanism persisting following the ozone exposure cessation (Figure 4).

Consistently, Gackière et al. [217] showed that the exposure of rats to various concentrations of ozone (0.5 to 2 ppm) for a time interval included between 1.5 and 120 h determined a significant time- and dose-dependent neuronal activation in the dorsolateral regions of the nucleus tractus solitarius.

Interestingly, the authors also showed neuronal activation in other interconnected central structures, including the caudal ventrolateral medulla, the parabrachial nucleus, the central nucleus of the amygdala, the bed nucleus of the stria terminalis, and the paraventricular hypothalamic nucleus.

The existence of a possible lung–brain axis in ozone-mediated cytotoxicity was further supported by Mumaw CL et al. [18], demonstrating that inhaled ozone increased microglial activation via the MAC1 receptor independently from circulating proinflammatory cytokine release and in an age-dependent manner.

On the other hand, it should be reported that there exists strong scientific and clinical evidence supporting the beneficial effects of ozone for the treatment of some neurological disorders.

However, such results are not contradicting previous mentioned studies since in most of the cases, ozone was administered locally and since animals and/or patients were not exposed to ozone by inhalation.

The first evidence of the beneficial effects of ozone in neurological disorders refers to the successful treatment of a case of intractable pain in the head and face associated with pathological changes in the optic thalamus [219].

Since then, ozone has been used to treat neuropathic pain allodynia, and hyperalgesia is defined as an increased sensitivity to normally painful stimuli [220,221]. The use of ozone for the treatment of neuropathic pain has been proposed for years based on its antioxidant and anti-inflammatory potential; however the clear molecular cut underlying the analgesic effect of this gas on such clinical conditions has not been fully elucidated.

Interestingly, Fuccio et al. [222] showed that a subcutaneous injection of ozone (90 or 180 µg/kg) decreased mechanical allodynia and normalized the mRNA caspase-1, caspase-12, and caspase-8 gene levels in an animal model of the neuropathic model.

Interestingly, a subcutaneous injection of ozone translated also in significant pharmacological effects in the orbito-frontal cortex, normalizing the expression of pro-inflammatory caspases and reducing IL-1β staining.

Such results were extensively confirmed in a clinical setting and provided significant evidence for the efficacy and safety of this treatment in various cohorts of patients. In particular, a multicenter randomized, doubled-blind, simulated therapy-controlled trial in patients suffering with acute low back pain showed that intramuscular lumbar paravertebral injections of an oxygen–ozone mixture was safe and effective in relieving pain and reduced both disability and the intake of analgesic drugs.

Other beneficial effects of ozone therapy have been associated with a significant improvement in cognitive functions and mood status.

In particular, Coppola et al. [23] empirically found that geriatric patients with peripheral occlusive disease and undergoing the reinfusion of autologous blood exposed to ozone exhibited significant improvements in mood tones within hours with no relation to the general clinical condition.

Furthermore, the authors evidenced that such an improvement was also accompanied by a significant increase of the brain-derived neurotrophic factor. These results were further confirmed in an experimental model of stroke demonstrating a significant reduction of the neuronal injury and radical formation [24]. Finally, rectal insufflation of ozone exhibited a significant anti-inflammatory effect in an experimental model of multiple sclerosis demonstrating a pharmacological efficacy comparable to methyl-prednisolone [25].

Ozone Therapy and Skin Diseases
Because of its safety, ozone therapy has been used for many years as a method ancillary to basic treatment, especially in those cases in which traditional treatment methods do not give satisfactory results, e.g., skin loss in non-healing wounds, ulcers, pressure sores, fistulae, etc. [226,227].

The first evidence dealing with the beneficial effects of ozone in skin disease was provided by Shpektorova [28] in 1964. Consistently, Białoszewski et al. [229] treated ozone patients with heavy, chronic, antibiotic-resistant septic complications after trauma, surgical procedures, and secondary skin infections, showing that in the wounds of the all experienced patients, the inhibition of septic processes and wound healing was much faster than normal.

A secondary result which should be taken into due account is that this method also lowers the cost of antibiotic therapy.
In order to fully elucidate the mechanisms underlying the pharmacological effects of ozone in the skin, some important anatomical and functional aspects should be taken into due account.

In particular, ozone has a low penetration potential into cutaneous tissues since it rapidly reacts with polyunsaturated fatty acids and water of the stratum corneum leading to the formation of ROS and lipooligopeptides (LOP) which in turn are readily scavenged by skin antioxidants and/or partially absorbed via the venous and lymphatic capillaries (Figure 5).

The safety of non-inhaling ozone therapy is also substantiated by clinical evidence obtained with healthy volunteers following exposure to a mixture of oxygen and ozone using a closed cabin allowing a temporary exposure of the skin to concentrations as low as 0.9 µg mL−1 [230].

Interestingly, the authors showed that under such clinical condition, there was a significant increase of pO2 and of peroxidation products in the venous plasma with a concomitant feeling of well-being in the next few days.

A different approach to exploit the beneficial effect of ozone overcoming the instability of ozone in pharmaceutical preparation is the ozonide. Such preparations are of possible clinical interest since they are stable for 2 years at 4 ◦C and may be used in the treatment of various infections of cutaneous and mucosal areas.

Ozonated oil is currently used topically for wounds repair, anaerobic and herpetic infections, trophic ulcers, burns, cellulitis, abscesses, anal fissures, fistulae, gingivitis, and vulvovaginitis [231].

Consistently with these observations, Matsumoto et al. showed that ozonated oil is effective in almost all enrolled patients in treatment for fistulae and chronic surgical wounds without side effects [232].

Similarly, exposure to ozone significantly reduced the severity of radiodermatitis lesions in patients with cancer [233].

Furthermore, Kim et al. [34] suggested that ozonated oil promotes acceleration in acute cutaneous wound repairs by the increased expression of PDGF, TGF-β1, and VEGF (Figure 5).

Finally, it was reported that ozone exposure is associated with the activation of transcription factor NF-κB, playing a pivotal role in the inflammatory response regulation and wound healing mechanisms [235–238].

Furthermore, previous reports showed that significant PDGF and TGF-β1 levels were released from platelets in the heparinized plasma of a limb ischemia patient after ozonation (Figure 5) [231,239].

Consistently, a previous study has shown that hydrogen peroxide (H2O2) potently induced the VEGF expression in human keratinocytes, which can stimulate wound healing [240].

From these previous studies, the authors hypothesized that ozone might enhance acute cutaneous wound healing, and this could be associated with growth factors such as FGF, PDGF, TGF-β, and VEGF (Figure 5).

Similarly, Krkl et al. [241] showed that ozonated olive oil improved neovascularization in rats when it was topically applied on skin flaps via VEGF upregulation. In addition, ozonated oil reduced doxorubicine-induced skin necrosis and injury as measured by a significant reduction of TNF-α, SOD, GSH-Px, and IL-1β [242] and by the enhanced flap viability in a rat model [243].

Ozone Therapy and CVD

Cardiovascular diseases (CVD) are a major factor in mortality rates around the world [244]. Increasing evidence has highlighted the roles of oxidative stress and inflammation in the promotion of CVD [245], leading to impaired endothelial function, a process which promotes atherosclerotic lesion or fatty streak formation (foam cells) associated with ischemic heart disease, stroke, and major debilitating events [246,247]. In this context, ozone therapy may represent a possible approach for correcting oxidative stress and thus improve the prognosis of many patients [48,49].

Ozone, by acting on multiple targets, is indirectly involved in recovering functional activities impaired by chronic disease. On the basis of the mechanisms of action, ozone therapy induces a biological response including the improvement of blood circulation and oxygen delivery to ischemic tissue as a result of the concerted effect of NO and CO, an increase in intraerythrocytic 2,3-DPG level (Figure 6) [250,251], an increase in reduced glutathione, an enhancement in basal metabolism through improved oxygen delivery, an upregulation of cellular antioxidant enzymes, the induction of HO-1 and HSP-70 [252,253], the induction of a mild activation of the immune system, and the increased release of growth factors in the absence of acute or late adverse effects (Figure 6).

Therefore, these properties suggest that ozone properties may be exploited in the treatment of CVD. In fact, several in vitro and in vivo studies, as well as some clinical trials, have shown a positive effect in cardiovascular disorders such as coronary artery disease (CAD), chronic heart failure (CHF), myocardial infarction, and peripheral artery diseases.

To this regard, a clinical trial conducted by Martinez-Sanchez et al. demonstrated that integrative therapy with ozone resulted in a beneficial treatment of CAD and its complications [54].

In particular, this study showed that the beneficial effect of ozone therapy was related to a reduction of SOD and catalase activities, advanced oxidation protein products, malondialdehyde, peroxidation potential, and total hydroperoxides with a concomitant increase in GSH levels and the ferric reducing ability of plasma, leading to a low production of O2•− and reduced oxidative stress.

Similarly, several studies showed that ozone therapy in patients with ischemic heart disease or had suffered an acute myocardial infarction (AMI) resulted in favorable effects in terms of pain and prognosis.

Furthermore, in a case report of a 76-year-old patient suffering from a myocardial infarction with parkinsonism, hypertension, chronic renal disease, and dyslipidemia as comorbidities, the authors showed that large autologous ozonized blood infusions for 18 months resulted in an improved myocardial contractility and in the protection against the risk of subsequent recurrences of AMI (Ozone Therapy 2017; volume 2:6745).

In order to elucidate the possible pharmacological mechanisms of ozone therapy, in a previous study, the authors exposed rats to an oxygen/ozone mixture prior to an acute ischemia/reperfusion injury of the myocardium, demonstrating a significant decrease of the myocardial infarcted area associated with an increased recruitment of endothelial progenitor cells (EPCs) within the myocardium [255] (Figure 6). Furthermore, in this study, the protective effect of oxygen/ozone was closely related to the increase in cardiac eNOS expression and activity.

In addition, Barone et al. demonstrated that ozone therapy reduced restenosis 30 days following percutaneous transluminal coronary angioplasty with metal stent implantations in pigs [256].

The authors suggested that ozone autohemotransfusion upregulated the innate detoxifying and antioxidant system (i.e., thioredoxins), preventing post-stenting neointima hyperplasia and an inflammatory reaction, thus supporting successive re-endothelization which may be dependent on the increased glycolysis rate, stimulation of NO synthesis, and induction of growth factors [257,258].

Moreover, several studies demonstrated that in patients with occlusive peripheral arterial disease (PAD), the reinfusion of autologous blood after ozonation lead to correcting the altered hemostatic-hemorheological parameters improving blood flow and the release of O2 from hemoglobin into the tissues [259–261].

It was also demonstrated that autohemotransfusion with ozonized blood per se does not significantly influence the fibrinolytic balance [262].

However, ozone therapy was more effective than prostacyclin in treating skin lesions in PAD patients, resulting also in a significant improvement of patient general conditions [263]. Furthermore, more studies showed that in patients with peripheral occlusive atherosclerotic disease (POAD), the ozone therapy improved the symptoms of intermittent claudication [264,265].

Non-recommended routes of application in ozone therapy, a critical review

Ozone used within the determined therapeutic windows is absolutely safe and more effective than golden standard medications in numerous pathologies.

However, there are practitioners who for the interest of increasing cost effectiveness and increasing speed in  treatments, pretend to cure chronic diseases applying alternative administration ways, using high ozone doses, which are neither standardized, nor supported by pre-clinical / clinical data, nor evaluated toxicologically.¹⁰¹

The Madrid Declaration on Ozone Therapy,¹⁰² is an international consensus document, not legally binding instruments. The Declaration draws its authority from the degree to which it has been codified in, or influenced, national or regional legislation and regulations. This document emphasizes the following stamens:

“It is absolutely necessary to work with specific objectives and in a unified way to assure a practice with great precision and safety.

“There is variance that the medical community wishes to standardize, and that progress already has been made, that it should be taken into account; it is necessary to continue with the development of medical definitions of procedures and protocols determining the best applications where it is necessary, as well as a code of good practice, in order to overcome more efficiently the possibility of malpractice.”

In addition, this document is in line with two general principles of the medicine:

1. Primum non nocere: Before anything else, not to do any harm.
2. The ethical principles in medicine.¹⁰³ These general principles should be taken into consideration in the clinical practice of the ozone therapy. As a consequence, any recommended application of ozone should be documented by pre-clinical and clinical trials.

Hence, the aim of this manuscript is to emphasize the rules for a secure practice of ozone therapy in the hope that ozone therapy will be used under scientific evidence base to avoid irreversible damage to the patients.

The search included a review of scientific articles and experimental results papers in the MEDLINE and Zotero ISCO3 Ozone Database, between the years 1980-2018. Descriptors: ozone therapy, toxicology, side effects were used.

The primary (original articles) sources of information were located. Additionally, official documents of ISCO3 were consulted (In particular ISCO3/LEG/00/10 Not recommended application routes).¹⁰⁴

Practitioners, devises and protocol, the key of a secure medical ozone therapy

Practitioner

As defined by the Madrid Declaration on Ozone Therapy¹⁰² to carry out any procedure is required technically qualified personnel. Professionals should attend post-graduate formation courses which include at least the basic contents defined by ISCO3,¹⁰⁵ or similar contents under the supervision of a local university or a scientific association of ozone therapy.

Practitioners should limit their practice to the field of their basic professional formation. This means: physicians will be in charge of human medical treatment or clinical trials; veterinarians should treat diseases, disorders and injuries in non-human animals; dentists should treat diseases and conditions of the oral cavity.

Biochemists, pharmacists, biologists will participate  in the molecular, preclinical and clinical research (in case of clinical research the direct interaction with patients will be responsibility of a physician). Nurses and technicians will act following the instruction of the corresponding doctor.

Devices / disposables

Generators used should be in line with the recommendations of ISCO3⁶ and the oxygen-ozone gas mixture must pass an antimicrobial sterile filter (< 20 µm) before injection. All materials used must be disposable and ozone resistant: glass, silicone probes, catheters and silicone tubes, connections of Kynar or stainless steel 316, and siliconized syringes.²

Protocols

Clinical protocols should be based in evidences (pre-clinical / clinical) that must be conformed  to generally accepted scientific / ethic principles; hinged on thorough knowledge of the scientific literature and other relevant sources of information, adequate laboratories and, as appropriate, animal experimentation.

New routes of application of ozone therapy imply a clinical research. The design and performance of each research study involving human subjects must be clearly described and justified in a research protocol.

Any clinical research in the area of ozone therapy must fit the same criteria set up for a regular drug. The proposed new routes of application must demonstrate some advantage over available treatment, such as:

• Showing superior effectiveness
• Avoiding serious side effects of an available treatment
• Improving the diagnosis of a serious disease where early diagnosis result in an improved outcome
• Decreasing a clinically significant toxicity of an available treatment
• Addressing an expected public health need

Basic pharmacology / toxicology mechanism of the ozone

Dose effect relationship

Higher ozone concentrations are not necessarily better, in the same way that it occurs with all the medicines. Ozone therapy has in general two main action mechanisms 1) Direct oxidation, with immediate effect of O₃ (e.g. inactivation of microorganism or pain mediator) 2) Surrogate effects, that involve the activation of a nuclear effectors (Nrf2 or NFkB) to induce a pharmacological response.

In both, there is a therapeutic window. The knowledge of the therapeutic windows for each application route is described in the Madrid Declaration of Ozone Therapy¹⁰² and it represents a summary extracted from the clinical experience of the main schools of ozone therapy, derived from the clinical practice, or from the experimental research.

The hormetic response of ozone is not a hypothesis, is a fact demonstrated clinically and experimentally.^107,108 The interaction of ozone mediators (mainly H₂O₂ and 4-hydroxy-2,3- transnonenal (HNE) with nuclear factor induces a therapeutic respond is now well established by scientific data.^109-111

Low doses of ozone is capable of utilizing known molecular redox master switches such as Nrf2/Keap1 or NF-kB/IkB to effect adaptive resistance. In that way, low doses stimulate cell protective pathways and nuclear transcription without altering cell viability;¹¹¹ on the contrary high doses can be genotoxic.^112-115

The use of non-appropriate range doses of ozone in clinic may originate serious side effects, from tissue necrosis^116,117 to a potential cancer that may develop during chronic exposition or during high doses exposition.¹¹⁸

Modalities of ozone administration

Ozone can be administered with great flexibility by different routes (e.g. extravascular blood oxygenation-ozonation, subcutaneous, intramuscular, intradiscal, intravaginal, intrauretral, vesical, etc.); but it should never be used:

.1. By inhalation. Ozone oxidizes available antioxidants and reacts instantaneously with surfactant’s polyunsaturated fatty acids (PUFA) present at the air – epithelial lining fluid interface to form reactive oxygen species that damages the respiratory system.¹¹⁹ Immediately after the exposition the first symptoms (headache, cough, dry throat, heavy chest, shortness of breath) became evident, and urgent aid measures should be taken.¹²⁰

There are two exceptions of administration of ozone or derivates by inhalation.

• Small volume of ozone gas (O₂/O₃) at low concentration (6 µg/mL), only in apnea and conducted by a well-trained medical doctor, to treat sinus diseases.^121,122

• Volatile organic compounds generated by ozone. There are different methods, such as the bubbling of essential oils (e.g. essential oil of pine, thymus, eucalyptus, tea tree) or fixed oils (sunflower oil or olive oil), which generate derivatives that can potentially be used from the therapeutic point of view.^123,124 In this case, the inhaled vapor is not O₂/O₃, but terpenes or other organic compounds

.2. Directly injected (intra-arterial injection or intra venous injection) as a gas mixture in the circulatory vessels because of the risk of provoking oxygen embolism, given the fact that the gas mixture never contains less than 95% oxygen.¹¹⁹

Its application is strongly discouraged due to the risk of gas embolism which can occur even in the case of using a slow infusion pump and volumes of 20 mL. The complications of stroke ranges from a simple axillary bubbling sensation, then cough, a feeling of retrosternal weight, dizziness, to changes in vision (amblyopia), hypotensive crisis, with signs of cerebral ischemia (paresis of the members) and to death.

It is important to note that at least five patients died as a result of a gas embolism after administration of ozone by direct intravenous injection.^125-128 It ought to be kept in mind that oxygen solubility at 37 °C is only about 0.23 mL per 100 mL of plasmatic water, and therefore, venous plasma cannot dissolve oxygen quickly enough, leading to the formation of a gas embolus.¹⁰²

Additionally, ozone is a very unstable gas, as the minutes pass, the concentration is lost. If it starts with 20 µg/mL, at the end of the 5 min, the concentration will end up in (10-14) µg/mL or even less.

Taking into account the current scientific knowledge, the use of DIV (direct intra venous application) involves an unnecessary risk, which should not be done outside of a clinical trial, nor in a center with no capacity to solve potential complications.

On the other hand, scientific societies should officially make clear their position against it (outside of its registered studies), to safeguard the image of ozone therapy in the event of accidents.

It would be enough that only one iatrogenic result produced for using DIV would originate a chaos in the ozone therapy professional community. So, it is of utmost importance to avoid in daily medical practice interventions that put the patient life in risk, especially if the procedure has not been scientifically proved. Any bad result would be detrimental for the ozone therapy.

A German paper describe a method use O₂ administration by i.v. According to this method they use a flow of 1 mL / min to about 3 mL / min in the vein. The starting dose is (10-20) mL and increases in the next 3 weeks by 10 mL each week.

According to the authors, apart from the general improvement in oxygen availability, i.v. oxygen therapy causes eosinophilia, which can be valued as an increase in undetermined cellular immunological resistance.

Furthermore, rheological qualities of the blood as well as diuresis are improved, the release of oxygen into  the tissue is increased, and the blood pH is normalized. In addition, author suggest that compared with hyperbaric oxygen therapy, i.v. oxygen therapy seems to have less side effects.

Application is less complicated, less expensive but probably of higher efficacy.¹²⁹ However, result was limited to the analysis of 20 patients and can’t be reproduced by any other clinical trials. Because the potential risk of embolic event, potential benefit of this method should be verified in pre-clinical studies.

This study was done only using O₂ nor O₃, promoter of i.v. O₃ use this paper to extrapolate the results and confuse the people. Is well know that the biological effects of O₂ or O₃ are completely different.

Emerging therapies in ozone therapy

With the term “emerging therapies” in ozone therapy, can be summarized a series of methods non-supported by scientific evidences which do not take into consideration any of the issues concerning the secure practice of ozone therapy.

Nor even a “case report” can be found nor a pre-clinical data that support those aberrant variants. The only channels for the diffusion of the “success” of the therapy are Facebook, YouTube, personal web sites, testimonials from patients, etc.

Therefore, in most cases, they constitute serious cases of human experimentation that skip the elementary medical ethical standards.¹⁰³ In most cases, the methods are advertised as “curative” or “resolving” chronic or terminal diseases (e.g. cancer or HIV). However, the list of diseases that “cure” these methods is endless.

In contrast to appear as more effective methods than the traditional ones, there are reports of deaths and severe adverse events among patients who have been treated with emerging therapies. There is a permanent report in social media about serious side effect after those practice (e.g. in Table 1 and Fig. 1).

The most representative of the emerging therapies are: direct intra venous application (DIV), Robins method of direct intravenous ozone therapy℠ (RMDIV ℠), Hyperbaric (HBO3) multi passes method, (3-10, or more passes) with 200 mL blood + 200 mL O₃ at 70 µg/mL at 1 bar pressure, and (2000- 25 000) IU heparin per pass, high dose ozone therapy (HDO), and Intraperitoneal Ozone (IPO3).

Direct intra venous application (DIV)

DIV has not any scientific evidence. A simulation of the effect of DIV in a preclinical study using mouse and rabbit models, get this conclusion: “The preclinical results obtained provide evidence that the implementation of direct intravenous ozone is highly risky, because of the severe adverse effects and the mortality that can lead, so its use is not justified in humans¹³⁰

Due to the lack of homogeneity in the terminology used in ozone therapy, a bibliographic search using the keyword “intravenous ozone” may lead to the appearance 15 papers.^131-145

However, the reading of the “materials and methods” section, evidences that the authors have called “intravenous ozone” to the classic major authohemotherapy (MAHT) or to the administration of ozonated saline solution (O3SS).

In any case they do not use the ozone gas directly into the vein. In clinic, the only report claims the benefit of the use of DIV appeared in 2016¹⁴⁶, this article report the use of DIV and ozone by other ways in 3 patients suffering from Ebola Virus Disease (EVD) during the epidemic in Sierra Leona.

Gaseous embolism symptoms are evident in patients undergoing DIV. Despite the theoretical discussion, on whether oxygen (the main component of O₂/O₃) may be embolic gas or not, the fact is that there are reports of deaths by the application of this method.^125-127

Hyperbaric (HBO3) multi passes method

Hyperbaric multi passes method uses extra-doses of ozone and extra-doses of heparin. As  DIV, HBO3 has not any pre-clinical or scientific clinical evidence. According to anecdotical evidences from patients (Table 1) or practitioners the main side effects are: loss of vision, lung disturbances, colored urine (red, brown).

Is well know, that the association of heparin with ozone increases the activations of platelets.^147,148 This is the reason why the MAHT uses a citrate based anticoagulant. In a typical 10 passes (200 mL blood + 200 mL O₃ at 70 µg/mL + 2000 U of heparin) the patient receives a total dose of 140 mg of ozone (In MAHT 100 mL of O₃ at 40 µg/mL, the patients receive 4 mg) and 20 000 U of heparin.

The dose of heparin is too high for  a patient without coagulation disorder, and can exacerbate the main side effect of heparin: thrombocytopenia, mild pain, hematoma, hemorrhage, local irritation, erythema, increased liver aminotransferase, anaphylaxis and immune hypersensitivity reaction.¹⁴⁹ The observed side effects during de HBO3 multi pass are indicative of the toxicity of high ozone.

Intraperitoneal (i.p.) Ozone

It is being said that in case of mesothelioma, peritoneal carcinomatosis or peritonitis, endoperitoneal or endopleural injection of up to 2.5 L of gaseous mixture with an ozone concentration of 10-20 μg/mL can be performed.¹⁰⁴ This modality is rarely used and must be performed by a specialist.¹⁰¹

There is not any clinical trial documented its benefits. However, the use of this therapy in cancer is supported by a pre-clinical study.^150-152 The administration of drug in pre-clinical study by intraperitoneal (i.p.) way is usual, because the difficult approach to the animal’s veins.

I.p. is mainly considered an experimental way. The experimental model of cancer in rabbits, is done by the implantation of the tumor in the rabbit ear, as consequence the marginal ear vein cannot be used for drug administration.

It is mean that result observed in preclinical trial probably does not depend of the administration way.

The use of i.p. in humans is not frequent, and as it involves a very invasive method needs quirophan condition. Therefore, the benefits of ozone as adjuvant in cancer should be reached using other way as the MAHT,¹⁵³ with low side effects, low cost and low invasiveness compared with the i.p. (Intraperitoneal hemorrhage, pain, etc.).

Any intervention on cancer should be approved by the patient and consulted with an oncologist. The only fact available today about the role of ozone in cancer, is it role as adjuvant,¹⁵⁴ not as a cure. Promising or creating expectations of healing a patient with cancer is a serious lack of medical ethics.

Comparative analysis of various methods

A comparative table shows the differences between well-established methods of application of ozone and “the emergent methods” (Table 1). As shown the evidence, there are an important differences between the number of scientific evidence between regular ways of ozone applications and the “emergent methods”.

Table 1. Evidence basis comparison of some regular and “emergent” methods of applications of ozone therapy.

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