Prevention_of_Covid-19_Infection
Journal of
Personalized
Medicine
Article
Prevention of Covid-19 Infection and Related Complications by
Ozonized Oils
Alberto Izzotti 1,2,* , Enzo Fracchia 3, William Au 4, Monica Colombo 2, Ulrich Pfeffer 2, Laura Emionite 2,
Simone Pavan 5, Daniele Miotto 5, Paola Lova 6, Elena Grasselli 7, Emanuela Faelli 1, Ruggeri Piero 1,
Micaela Tiso 8and Alessandra Pulliero 9
Citation: Izzotti, A.; Fracchia, E.; Au,
W.; Colombo, M.; Pfeffer, U.;
Emionite, L.; Pavan, S.; Miotto, D.;
Lova, P.; Grasselli, E.; et al.
Prevention of Covid-19 Infection and
Related Complications by Ozonized
Oils. J. Pers. Med. 2021,11, 226.
https://doi.org/10.3390/jpm11030226
Academic Editor: Philip P. Foster
Received: 21 January 2021
Accepted: 18 March 2021
Published: 22 March 2021
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4.0/).
1Department of Experimental Medicine, University of Genoa, 16132 Genoa, Italy;
emanuela.faelli@unige.it (E.F.); ruggeri@unige.it (R.P.)
2IRCCS Ospedale Policlinico San Martino, 16132 Genoa, Italy; monica.colombo@hsanmartino.it (M.C.);
ulrich.pfeffer@hsanmartino.it (U.P.); laura.emionite@hsanmartino.it (L.E.)
3Galliera Hospital, 16132 Genoa, Italy; enzo.fracchia@galliera.it
4Faculty of Medicine, Pharmacy, Science and Technology, The George Emil Palade University,
540142 Targu Mures, Romania; wau@stu.edu.cn
5BWH Graphic Solutions, 28001 Madrid, Spain; pavan@bwhenergysolutions.com (S.P.);
miotto@bwhenergysolutions.com (D.M.)
6Department of Chemistry and Industrial Chemistry, University of Genoa, 16132 Genoa, Italy;
paola.lova@unige.it
7Department of Earth Sciences, University of Genoa, 16132 Genoa, Italy; elena.grasselli@unige.it
8MICAMO Spin-Off Department of Earth Sciences, University of Genoa, 16132 Genoa, Italy;
micaela.tiso@unige.it
9Department of Health Sciences, University of Genoa, 16132 Genoa, Italy; alessandra.pulliero@unige.it
*Correspondence: izzotti@unige.it
Abstract:
Background: The COVID-19 pandemic continues to ravage the human population; there-
fore, multiple prevention and intervention protocols are being rapidly developed. The aim of our
study was to develop a new chemo-prophylactic/-therapeutic strategy that effectively prevents
COVID-19 and related complications. Methods: In
in vitro
studies, COVID-19 infection-sensitive
cells were incubated with human oropharyngeal fluids containing high SARS-CoV-2 loads. Levels of
infection were determined via intra-cellular virus loads using quantitative PCR (qPCR). Efficacies for
infection prevention were determined using several antiviral treatments: lipid-encapsulated ozonized
oil (HOO), water-soluble HOO (HOOws), UV, and hydrogen peroxide. In
in vivo
studies, safety and
efficacy of HOO in fighting COVID-19 infection was evaluated in human subjects. Results: HOO in
combination with HOOws was the only treatment able to fully neutralize SARS-CoV-2 as well as
its capacity to penetrate and reproduce inside sensitive cells. Accordingly, the feasibility of using
HOO/HOOws was tested
in vivo
. Analysis of expired gas in healthy subjects indicates that HOO
administration increases oxygen availability in the lung. For our human studies, HOO/HOOws
was administered to 52 cancer patients and 21 healthy subjects at high risk for COVID-19 infection,
and all of them showed clinical safety. None of them developed COVID-19 infection, although an
incidence of at least 11 cases was expected. Efficacy of HOO/HOOws was tested in four COVID-19
patients obtaining recovery and qPCR negativization in less than 10 days. Conclusions: Based on our
experience, the HOO/HOOws treatment can be administered at standard doses (three pills per day)
for chemo-prophylactic purposes to healthy subjects for COVID-19 prevention and at high doses
(up to eight pills per day) for therapeutic purposes to infected patients. This combined prevention
strategy can provide a novel protocol to fight the COVID-19 pandemic.
Keywords:
COVID-19; SARS-CoV-2; chemoprophylaxis; prevention; oxidative stress; COVID-19
challenge test
J. Pers. Med. 2021,11, 226. https://doi.org/10.3390/jpm11030226 https://www.mdpi.com/journal/jpm
J. Pers. Med. 2021,11, 226 2 of 16
1. Introduction
With waves and waves of COVID-19 infections around the world, it is urgent to
develop novel and effective prevention and intervention programs against the pandemic
and as rapidly as possible. The aim of our study was, therefore, to develop a novel
chemo-prophylactic approach which would generate immediate preventive efficacy and
would also have therapeutic capacities. The investigation was conducted to evaluate
efficacy in using ozonized oils (HOO) to neutralize the SARS-CoV-2 virus. Our interest
in this approach was triggered by the serendipitous observation that no COVID-19 case
was detected in cancer patients treated with HOO administered for prevention of cancer
relapses. Indeed, cancer stem cells are addicted to antioxidants making them able to escape
the therapeutic effects of chemo-radiotherapies.
Our therapeutic substance is an ozonized oil (HOO) which would be developed to
release ozone intracellularly. There were several reasons to choose ozone. As a disinfec-
tant, ozone is well known for killing viruses, especially RNA viruses [
1
,
2
]. In addition, a
multiomics-based characterization of COVID-19’s vulnerability showed that ozone was
a potentially effective drug [
3
]. Other reports also proposed its use for COVID-19 ther-
apy
[4–8]
. The proposed therapeutic mechanisms included (a) inhibition of NFkB- and
IL-1/6-driven inflammation [
4
]; (b) improvement of gas exchange and tissue respiration;
and prevention of hypoxemia and multiorgan failure [
7
]. The recommended administration
route was by auto-hemo-transfusion after blood ozonization. However, due to volatility
of ozone, the timespan for antiviral efficacy would be limited. Furthermore, this invasive
approach may not be suitable for use in healthy subjects nor in some infected patients.
HOO is an oil-based ozone vector, which has been used for a long time in topical
applications [
9
]. When administered
in vivo
by the oral route, it would complex with
lipoprotein in the liver and then be distributed via the blood circulation with lungs as
the first target organ. On the other hand, infection by COVID-19 occurs through the
upper respiratory epithelium and nasopharyngeal mucosae. Therefore, to target these
virus-entry tissues directly, we developed a novel hydrophilic preparation of HOO which
would allow its delivery by aerosol and nasal sprays. This latter preparation is referred
to as water soluble high ozonide oil (HOOws). Therefore, our systemic administration of
HOO/HOOws for intracellular release of ozone represents a novel chemo-prophylactic tool
to prevent COVID-19 infection in healthy subjects and a therapeutic tool to fight COVID-19
infection in affected patients. This tool is unspecific, thus being potentially active on all
virus variants independently from SARS-CoV-2 antigen specificity.
On the virus side, SARS-CoV-2 is highly sensitive to oxidation [
10
]. The sensitivity
is related to the vulnerability lipid envelope of the virus, which is devoid of antioxidant
defenses. Therefore, a key mechanism to efficacy is to take advantage of this viral deficiency
and to kill the virus intracellularly, like what we have investigated using HOO and HOOws.
The efficacy of this approach was initially tested
in vitro
in cells sensitive to SARS-
CoV-2 infection using the standard qPCR test to evaluate viral penetration inside the cells.
Although existing COVID-19 assays are valuable, they have limitations. For example, rapid
antigenic tests are fast but not entirely specific nor sensitive. PCR analyses for portions of
viral RNA may identify degraded products rather than active infections. Furthermore, not
all patients with a COVID-19 diagnosis have a positive qPCR-COVID-19 after two months
for the same diagnosis. Consequently, infectious capacity of asymptomatic subjects can be
underestimated by the existing tests, which contributes to epidemic spreading. Therefore,
to enhance the determination of efficacy, a biological challenge test was developed by us to
detect virus infectivity (or lack thereof) rather than presence or absence of the viral RNA.
This assay can also be used to identify the biological capacity of COVID-19 to infect other
subjects and to spread an active disease, such as poorly symptomatic but infective subjects
who would play a key role in maintaining the epidemic.
In this study, we used the biological challenge test to test the efficacy of HOO and
HOOws to neutralize SARS-CoV-2 and to prevent its penetration inside sensitive cells.
J. Pers. Med. 2021,11, 226 3 of 16
Thereafter, the safety and efficacy of this approach was evaluated in human subjects and
COVID-19 patients.
2. Methods
2.1. In Vitro Studies
2.1.1. Cell Culture for the Viral Challenge Experiments
The VERO C1008 (E6) African green monkey kidney cells (Vero) were certified by
IZSLER (code BSCL87, Experimental Zoo-prophylactic Institute of Lombardia and Emilia
Romagna Region, Ministry of Health, Brescia, Italy). These cells expressed much higher
levels of angiotensin-converting enzyme 2 (ACE) on their outer membrane than most other
cell types, e.g., human bronchial cells [
11
]. Routinely, these cells were maintained in semi-
confluence cultures in our standard laboratory. The culture medium was a DMEM/fetal
calf serum/Hepes buffer, and the cultures were kept inside 37
◦
C incubators with 5% CO
2
.
For the challenge experiments, the culture medium for the Vero cells was changed into
modified DMEM/fetal calf serum/Hepes buffer which allowed the cells to grow without
CO
2
[
12
], and the culture flasks were transferred from our routine laboratory to the BLS3
lab of the Research Center of the San Martino Hospital. For the challenge, SARS-CoV-2
containing oropharyngeal swab samples were taken from the freezer, thawed under a
biosafety hood in a negative pressure room, and used for the challenge experiment in the
BLS3 lab.
2.1.2. SARS-CoV-2 Challenge Experiments
An aliquot (0.5 mL) of the SARS-CoV-2 containing swap sample was dropped into the
culture medium (DMEM/Hepes/FCS) of the Vero culture, each flask was gently mixed for
1 min and then incubated for 12 h at 37
◦
C. The 12 h time span was selected because it has
been reported as the time when the highest level of virus penetrance into the cells would
occur [
13
]. Oropharyngeal samples which were devoid of SARS-CoV-2 (qPCR negativity
>40 amplification cycles) were used as negative controls.
After 12 h, the flasks were transferred from the incubator into a heated-hybridization
oven (Bibby Stuart, Staffordshire ST15, OSA, UK) at 60
◦
C for 30 min to inactivate virus
infectivity without altering viral RNA integrity and to detach cells from the flask. After
inactivation, each flask was transferred to the biosafety hood where the cell-containing
medium (12 mL) was collected into sterile tubes and the tubes were centrifuged at 3000
×
g
for 15 min. The supernatant was discarded. Each cell pellet was resuspended, washed
in molecular-grade physiological solution, and centrifuged twice. For each sample, the
amount of RNA in each cell pellet was quantified by Qubit fluorescent probe analysis
(Qubit 3.0 Fluorimeter, Life technologies, Qubit 3.0 Fluorimeter, Thermo Fisher Scientific,
Carlsbad, CA, USA) and a standard RNA amount equal to those of the Cv19+ reference
sample was used for RNA extraction and qPCR analyses. Each pellet was resuspended in
RNAase-free molecular-grade water (1 mL) and frozen at −20 ◦C until RNA extraction.
2.1.3. RNA Extraction and qPCR Analyses
The presence of viral RNA inside the challenged Vero cells was tested by qPCR using
the SARS-CoV-2 RT-qPCR Reagent Kit (Perkin Elmer, Wathman, MA, USA). Samples were
prepared using the automated Janus G3 workstation (Perkin Elmer, Wathman, MA, USA).
Thawed samples (300
µ
L), composed of resuspended Vero cells, were mixed with a solution
containing poly(A)RNA buffer and proteinase K solution (14
µ
L). RNA extraction was
performed using the automated Chemagic workstation (Perkin Elmer, Wathman, MA,
USA) and the magnetic-beads-based CheMagic extraction kit.
Three qPCR Taqman probes were used for testing a house-keeping gene (Ribonuclease
P/MRP Subunit P30 [RPP30]) as internal control and the SARS-CoV-2 viral genes Orf1ab
(Vic labeled) and N (FAM labeled). Purified RNA underwent PCR amplification according
to the following cycles: 50
◦
C
×
15 min, 95
◦
C
×
2 min, 45 cycles at 95
◦
C
×
3 s, and 60
◦
C
J. Pers. Med. 2021,11, 226 4 of 16
×
30 s. PCR reactions were performed by Light Cycler 480II robotic machine (Roche) in a
final volume of 20 µL.
2.1.4. Prevention of SARS-CoV-2 Infection in Cell Culture
Various antivirus methods were tested for their ability to hamper SARS-CoV-2 pen-
etration into the Vero cells. For each test, an aliquot (0.5 mL) of the same SARS-CoV-2
-containing samples which were used for the challenge test was treated with one of the
following conditions:
(a)
UV-C 254 nm radiation generated by LED, power 0.3 mW/cm
2
(measured by Ref-
erenz Radiometer, Epigap Optoelekronik, GmbH, Bergkirchen, Germany) for 15 min,
corresponding to a dose of 270 mJ/cm2.
(b)
Hydrogen peroxide: Analytical grade sterile hydrogen peroxide (Sigma, Milan City,
Italy) was added at a final concentration of 1% vol/vol and incubated at room tem-
perature for 15 min.
(c)
HOO (O3zone, GS Pharma, La Valletta, Malta): 0.5 mL was added to cultures and
incubated at room temperature for 15 min. This ozonized oil was selected because
(a) it had the highest level of ozonide available, i.e., >900 ozonides, and (b) it was the
only ozonized oil among those tested which was able to penetrate inside pulmonary
A549 cells (see below).
(d)
HOOws (O3zone spray, GS Pharma, La Valletta, Malta): It contained water, lecithin,
polysorbate 20, and ozonized peanuts oil. In addition, 0.5 mL of HOOws was added
to cultures and incubated at room temperature for 15 min.
(e)
HOO and HOOws in combination (1/1 vol/vol) incubated at room temperature for
15 min.
After the various treatments (or no treatment), samples were transferred into flasks
containing Vero cells, incubated at 37
◦
C for a 12 h and then processed as previously
reported for the challenge test.
For this experiment, the negative and positive samples were untreated Vero E6 cells
and nontreated medium containing the COVID-19 oropharyngeal swab, respectively. All
experiments were performed in three independent replicates.
2.1.5. Evaluation of Anti-Inflammatory Capacity of Ozonized Oils
Pulmonary alveolar macrophages can be activated inside the lung of COVID-19
infected patients triggering inflammation. Macrophage activation could cause compli-
cations, e.g., development of thromboembolic pneumonitis consequent to the release of
pro-thrombotic factors, especially Thromboxane A2, from these cells when activated [
14
].
Since ozonized oils have anti-inflammatory capacity [
15
], this capacity was tested using
HOO in immortalized murine macrophages (RAW264.7, IRCCS San Martino Biobank,
Genoa, Italy). These cells were cultured for 24 in DMEM 75% v/vand FCS 25% v/v, and
then activated by incubation with 10 ug/m E. coli lipo-polysaccharidic antigen (Lps, Sigma,
Milan City, Italy) according to a procedure [
16
]. Cells were then either exposed to HOO
(2 h pretreatment, 10% v/v) or, as sham control, to sunflower seed oil (2 h pretreatment,
10% v/v). Macrophage activation was determined by analyzing changes in morphology
using standard microscopy.
2.1.6. Evaluation of Ozonized Oil Penetration Inside Cultured Cells
SARS-CoV-2 replications occur intracellular; therefore, it is important to determine that
HOO would also reach the intracellular compartment. This issue was explored by tracing
labeled HOO (with red Nile dye, Sigma, Milan City, Italy) into cell cytoplasm as visualized
by fluorescence microscopy. In this experiment, A549 human alveolar basal epithelial cells
(ATCC CCL-185) were maintained in Dulbecco’s modified Eagle’s medium/F12 containing
10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 mg/mL) and treated
with labeled sunflower seed oil (sham-control, 2 h,10% v/v) or labeled HOO (2 h, 10% v/v).
J. Pers. Med. 2021,11, 226 5 of 16
2.2. In Vivo Studies
2.2.1. Evaluation of Increased Oxygen Availability in the Lungs after HOO Treatment
In two healthy subjects, HOO effects on respiratory capacity before (T0) and after (T1)
1 week of HOO oral administration (3 cps
×
2
×
day) were evaluated. The maximal oxygen
uptake (VO
2
max), VO
2
at anaerobic threshold (VO
2
@AT), and the percentage of VO
2
max at
anaerobic threshold (%VO
2
@AT) were used as criteria to assess oxygen availability. These
subjects were healthy males, age 56 and 57 years, sedentary lifestyle, no drug consumption,
and nonsmokers.
The subjects participated in a cardiopulmonary exercise test (CPET) to determine a)
VO
2
max (L/min and mL/kg/min; absolute and relative value, respectively); (b) VO
2
@AT
(L/min and mL/kg/min); and (c) %VO
2
@AT. As a warm-up for the CPET, they were asked
to run on a treadmill for 5 min at 7 km/h speed at 1% grade. Then, a strenuous exercise was
performed by running with an increasing speed from 8 km/h with increments of 1 km/h
for each minute till exhaustion. In addition, they performed the CPET with calibrated ergo-
spirometer (Sensormedics, Viasys, CA, USA) to obtain cardiorespiratory parameters during
the bouts, from warm-up to the end of the exercise. Expired gas samples were collected
and analyzed breath by breath. VO
2
max was considered to be reached when at least 3 of
the 4 following criteria were fulfilled: (i) a steady state of VO
2
despite increasing running
velocity (change in VO
2≤
150 mL/min at VO
2
max); (ii) final respiratory-exchange ratio
(RER) exceeded 1.1; (iii) visible exhaustion; or (iv) a heart rate (HR) at the end of exercise
(HRmax) equal to the predicted maximum (210—(0.65
×
age)) [
17
]. The CPET tests were
performed before (T0) the HOO administration and 1 week after (T1) the administration.
2.2.2. Evaluation of Safety and Efficacy of Ozonized Oils in Human Patients
Study Design and Participants
The
in vivo
study was performed on 77 individuals including cancer patients without
the infection (observational study 1), healthy subjects devoid of but at risk for COVID-19
infection (observational study 2), and individuals with the infection (intervention study).
Under the approved protocol, three groups of subjects with our specific requirements
(see inclusion and exclusion criteria) were randomly recruited from our community. All
subjects had provided their consent to participate, undergone standard tests for COVID-19
infection status, and answered standard survey questions on age, gender, health conditions,
etc. From all subjects, nose–mouth pharyngeal swabs were collected using the E-Swab
collection kit (Copan, Brescia, Italy), placed into a vial individually which contained the
company-supplied buffer, stored in our designated
−
20
◦
C freezers, and used within
2 weeks.
Observational study 1 included 52 cancer patients treated with HOO as integrative
support to anticancer therapies; inclusion criteria were previous cancer diagnosis (see
Table 1), both genders, good health, age between 18 and 70 years, lack of participation in
other clinical trials; exclusion criteria were uncontrolled cancer growth with a prognosis <6
months survival, hospitalization, cachexia, severe clinical condition, bone marrow failure,
and liver failure.
Observational study 2 included 21 healthy subjects treated for COVID-19 chemo-
preventive purposes. Inclusion criteria were both genders, good health, age between 18
and 70 years, and lack of participation in other clinical trials. Exclusion criteria were
pregnancy and breastfeeding, BMI
≥
35 kg/m
2
, liver failure, chronic hepatitis, cirrhosis,
and cholestatic liver diseases, any severe medical condition, alcohol and drug abuse, or
history of known allergy to peanuts.
Intervention study included four COVID-19-affected patients treated for therapeutic
purposes. Inclusion criteria (in addition to those previously reported) were SARS-Cov-
2 qPCR positivity, COVID-19 severity score
≤
5; exclusion criteria (in addition to those
previously reported) were severe and critical COVID-19 pneumonia (COVID-19 severity
score >5), patient connected to the ventilator, and blood oxygen saturation (SaO
2)
less than
80%.
J. Pers. Med. 2021,11, 226 6 of 16
Table 1. Evaluation of increased oxygen availability in human lung after ozonized oil intake.
T0 PRE-TREATMENT TEST VO2 Max
(mL/kg/min)
VO2
(L/min)
VO2 Threshold
(mL/kg/min)
VO2 Threshold
(L/min)
% VO2 Max
in Threshold
Subject 1 39.4 3.19 32.5 2.62 82
Subject 2 36.6 2.42 30.6 2.07 86
T1 POST-TREATMENT TEST
Subject 1 40.9 3.27 35.6 2.94 90
Subject 2 38.9 2.63 33.4 2.08 87
T1-T0 delta after HOO TREATMENT
Subject 1 +1.5 +0.08 +2.9 +0.32 +8
Subject 2 +2.3 +0.21 +2.8 +0.01 +1
Consumptions of HOO and HOOws for these studies have been approved by the
Health Ministry of Malta (approval number 0075/2020 according to EC1924/2006 issued
on 17 March 2020). Within a 3-month duration, HOO was administered orally (1–3 cps
×
3
×
day), HOOws was administered by intranasal spray, two puffs (i.e., 100
µ
L) per nostril
every 4 h.
Statistical Analyses
Differences between continuous numeric variables were tested by the nonparametric
Mann–Whitney-U and Kruskal–Wallis tests. Differences between frequencies were tested
by Chi-square analyses. Statistical significance of differences in disease incidence were
evaluated by Mantel–Cox analysis. All statistical analyses were performed using the Stat
view software (Statview, SAS Institute, Abacus Concept Inc., Berkeley, CA, USA).
3. Results
3.1. In Vitro Studies
3.1.1. SARS-Cov-2 Challenge Test and qPCR Analyses
qPCR detection of the house-keeping gene, which was used as internal control and
as expression standard, was consistently present in all Vero cultures (untreated, treated
with virus-negative swab samples, and treated with virus-positive swab samples cultures).
For the Orf1 and N viral genes, the virus-negative controls did not reveal any positive
amplification, whereas the virus-positive cultures showed amplifications for both genes
at the 22th and 24th cycles (average 23th cycle), respectively. The data indicate successful
infection of the Vero cells by SARS-Cov-2.
3.1.2. Efficacy in Preventing SARS-Cov-2 Infection
The efficacy of hydrogen peroxide, UV, HOO, and HOOws in preventing SARS-CoV-2
penetration inside Vero cells was determined calculating the qPCR positivity threshold by
quantifying intracellular load of viral-RNA. Efficacy was indicated by the delay in amplifi-
cation positivity thresholds at qPCR for both the Orf1 and N genes as compared to the virus-
positive cultures. The differences in qPCR threshold-detection cycles between the virus
positive cultures and those treated with antiviral treatments were: hydrogen peroxide (four
cycles), UV (five cycles), HOO (eight cycles), HOOws (nine cycles), and HOO+HOOws
(23 cycles). These results are reported in Figure 1. According to these data, the rankings
of efficacies in prevention of infection, in increasing order, were hydrogen peroxide, UV,
HOO, HOOws, HOO pre-treatment, and HOO+HOOws. Indeed, HOO+HOOws was the
only protocol which was capable of completely neutralizing COVID-19. The statistical
significance of differences between qPCR cycles was calculated both versus negative and
positive controls. The lack of difference between treated samples and negative control indi-
cate a complete efficacy of the preventive treatment. This situation was achieved only by
HOO/HOOws. Conversely, all preventive treatments significantly decreased intracellular
J. Pers. Med. 2021,11, 226 7 of 16
viral load as compared to the positive control. Because no remarkable difference between
results obtained for N and Orf1 probes was observed, statistical analyses considered the
mean of these results for each experimental condition.
J. Pers. Med. 2021, 11, x FOR PEER REVIEW 7 of 16
controls. The lack of difference between treated samples and negative control indicate a
complete efficacy of the preventive treatment. This situation was achieved only by
HOO/HOOws. Conversely, all preventive treatments significantly decreased intracellular
viral load as compared to the positive control. Because no remarkable difference between
results obtained for N and Orf1 probes was observed, statistical analyses considered the
mean of these results for each experimental condition.
Figure 1. Number of qPCR positive amplification cycles for two SARS-CoV-2 viral genes (N, Orf1) under different pre-
vention treatment protocols. Horizontal black line indicates the positivity threshold; samples negative at the 40th qPCR
cycles were negative. Columns height is inversely related to the amount of SARS-CoV-2 penetrated inside Vero cells. All
preventive treatments tested significantly decreased intracellular viral load. The only treatment able to restore, despite
SARS-CoV2 presence in cell culture; the negative results obtained with negative control were HOO+HOOws.
3.1.3. Evaluation of Anti-Inflammatory Capacity of Ozonized Oils
Inflamed activated macrophages in culture usually demonstrate prismatic shape
with pseudopods (Figure 2A). However, these events did not occur after macrophages
were treated with HOO, despite the presence of high amounts (10 ug/mL) of the Lps acti-
vator. Indeed, these treated macrophages maintained their usual rounded shape without
the emission of pseudopods (Figure 2B).
Figure 1.
Number of qPCR positive amplification cycles for two SARS-CoV-2 viral genes (N, Orf1) under different prevention
treatment protocols. Horizontal black line indicates the positivity threshold; samples negative at the 40th qPCR cycles were
negative. Columns height is inversely related to the amount of SARS-CoV-2 penetrated inside Vero cells. All preventive
treatments tested significantly decreased intracellular viral load. The only treatment able to restore, despite SARS-CoV2
presence in cell culture; the negative results obtained with negative control were HOO+HOOws.
3.1.3. Evaluation of Anti-Inflammatory Capacity of Ozonized Oils
Inflamed activated macrophages in culture usually demonstrate prismatic shape with
pseudopods (Figure 2A). However, these events did not occur after macrophages were
treated with HOO, despite the presence of high amounts (10 ug/mL) of the Lps activator.
Indeed, these treated macrophages maintained their usual rounded shape without the
emission of pseudopods (Figure 2B).
3.1.4. Evaluation of Ozonized Oil’s Capacity to Penetrate inside Cultured Cells
After exposing pulmonary cells to un-ozonized (labeled) peanuts oil, no penetration
was observed, as indicated by the lack of red dye intracellularly (Figure 2C). Conversely, a
high abundance of red droplets was visible when cells were treated with labeled HOO. The
results indicate that HOO was highly efficient in reaching the intracellular compartment
(Figure 2D).
J. Pers. Med. 2021,11, 226 8 of 16
J. Pers. Med. 2021, 11, x FOR PEER REVIEW 8 of 16
Figure 2. Left panel. Inhibition of pulmonary alveolar macrophage-activation by HOO. Panel (A), macrophages are acti-
vated in presence of Lps bacterial endotoxins changing their shape and emitting long pseudopods. Panel (B), macrophage
activation does not occur despite the presence of Lps when cells are pretreated with ozonized oil (HOO). Right panel.
Intracellular delivery of HOO (red labeled) in pulmonary cells. Panel (C), no penetration of red-labeled peanuts oil occurs
in lung cells blue-stained for their nucleus and green-stained for their cytoplasmic membranes. Panel (D), Abundant pen-
etration in cytoplasm of red-labeled ozonized oil (HOO) in lung cells.
3.1.4. Evaluation of Ozonized Oil’s Capacity to Penetrate inside Cultured Cells
After exposing pulmonary cells to un-ozonized (labeled) peanuts oil, no penetration
was observed, as indicated by the lack of red dye intracellularly (Figure 2C). Conversely,
a high abundance of red droplets was visible when cells were treated with labeled HOO.
The results indicate that HOO was highly efficient in reaching the intracellular compart-
ment (Figure 2D).
3.2. In Vivo Studies
Evaluation of Increased Oxygen Availability in the Lungs after Ozonized Oil Intake
In the examined subjects, VO
2
max values indicate an increase (T0: 38 ± 1.98; T1: 39.9
± 1.41 mL/kg/min) after 1 week of HOO intake per os. Similarly, VO
2
values at anaerobic
threshold (VO
2
@AT) indicate an increase of 3.4% (T0: 31.55 ± 1.34; T1: 34.50 ± 1.56
mL/kg/min). Finally, percentage of VO
2
max at anaerobic threshold (%VO
2
@AT) increased
by 4%. The data (means ± SD) are reported in Table 1.
3.3. In Vivo Safety in Human Patients
During the past 6 months, 52 cancer patients received HOO oral treatment as integra-
tive support to their anticancer therapies. During the treatment, liver functions (transami-
nase, bilirubin, etc.) and other standard haematochemical analyses were monitored, and no
abnormal values were observed. No other obvious side effects were either observed or
Figure 2.
Left panel. Inhibition of pulmonary alveolar macrophage-activation by HOO. Panel (
A
), macrophages are activated
in presence of Lps bacterial endotoxins changing their shape and emitting long pseudopods. Panel (
B
), macrophage
activation does not occur despite the presence of Lps when cells are pretreated with ozonized oil (HOO). Right panel.
Intracellular delivery of HOO (red labeled) in pulmonary cells. Panel (
C
), no penetration of red-labeled peanuts oil occurs
in lung cells blue-stained for their nucleus and green-stained for their cytoplasmic membranes. Panel (
D
), Abundant
penetration in cytoplasm of red-labeled ozonized oil (HOO) in lung cells.
3.2. In Vivo Studies
Evaluation of Increased Oxygen Availability in the Lungs after Ozonized Oil Intake
In the examined subjects, VO
2
max values indicate an increase (T0: 38
±
1.98; T1:
39.9
±
1.41 mL/kg/min) after 1 week of HOO intake per os. Similarly, VO
2
values at
anaerobic threshold (VO
2
@AT) indicate an increase of 3.4% (T0: 31.55
±
1.34; T1: 34.50
±
1.56 mL/kg/min). Finally, percentage of VO
2
max at anaerobic threshold (%VO
2
@AT)
increased by 4%. The data (means ±SD) are reported in Table 1.
3.3. In Vivo Safety in Human Patients
During the past 6 months, 52 cancer patients received HOO oral treatment as in-
tegrative support to their anticancer therapies. During the treatment, liver functions
(transaminase, bilirubin, etc.) and other standard haematochemical analyses were moni-
tored, and no abnormal values were observed. No other obvious side effects were either
observed or reported. The only minor adverse response was the rarely reported meteorism
during the first 2 days of treatment in four patients. The inflammatory profile was mea-
sured analyzing C-reactive protein and speed of blood red-cell sedimentation after 1 h.
For the C-reactive protein, values were at T0 (before HOO treatment) 0.9
±
0.5 and at T1
(after HOO treatment) 0.3
±
0.2 mg/100 mL (max normal value 0.5 mg/100 mL) (T1 vs.
T0 p< 0.01). For the speed of blood red-cell sedimentation after 1 h, values were at T0
21.8
±
3.1 and at T1 12.0
±
1.4 mm (max normal value 16 mm) (T1 vs. T0 p< 0.01). These
results are in line with the established anti-inflammatory capacity of ozone derivatives [
15
].
J. Pers. Med. 2021,11, 226 9 of 16
Anti-Viral Efficacy in Human Patients
In the same 52 cancer patients, the efficacy of HOO in preventing COVID-19 infection
was evaluated retrospectively. No infection was detected in the 6-month duration of
the follow-up (Table 2). On the other hand, an infection incidence of 20% was expected,
corresponding to at least 10 cases among the cancer patients. The expected frequency
was estimated based on the actual incidence of COVID-19 infection in Italy and the high
sensitivity of cancer patients to the infection. This difference (0% vs. 20% in 52 subjects)
was statistically significant (p< 0.01).
Table 2.
Subjects undergoing ozonized oil (HOO) treatment for either chemo-prophylactic (n= 52 cancer patients +21
normal individuals) or therapeutic purposes (n= 4 infected patients) of COVID-19 infection.
Gender Age Previous Diseases COVID-19 Infection Clinical Outcome
Therapeutic purpose
Female 22 None Yes Recovery
Male 55 COPD, Vascular ischemia Yes Recovery
Female 54 None Yes Recovery
Female 52 None Yes Recovery
Chemo-prophylactic
purpose. Cancer patients
Male 47 Brain cancer (glioblastoma) No No Covid-19 infection
Male 40 Brain cancer (glioblastoma) No No Covid-19 infection
Male 19 Brain cancer (glioblastoma) No No Covid-19 infection
Female 11 Brain cancer (glioblastoma) No No Covid-19 infection
Male 48 Brain cancer (glioblastoma) No No Covid-19 infection
Male 33 Brain cancer (glioblastoma) No No Covid-19 infection
Male 40 Brain cancer (glioblastoma) No No Covid-19 infection
Female 37 Brain cancer (glioblastoma) No No Covid-19 infection
Female 40 Brain cancer (glioblastoma) No No Covid-19 infection
Female 50 Breast cancer No No Covid-19 infection
Female 56 Breast cancer No No Covid-19 infection
Female 60 Breast cancer No No Covid-19 infection
Female 69 Breast cancer No No Covid-19 infection
Female 65 Breast cancer No No Covid-19 infection
Female 55 Breast cancer No No Covid-19 infection
Female 52 Breast cancer No No Covid-19 infection
Female 70 Breast cancer No No Covid-19 infection
Male 57 Colon cancer No No Covid-19 infection
Female 61 Colon cancer No No Covid-19 infection
Male 53 Colon cancer No No Covid-19 infection
Female 57 Colon cancer No No Covid-19 infection
Male 78 Kidney cancer No No Covid-19 infection
Male 73 Bladder cancer No No Covid-19 infection
Female 82 Non-Hodgkin Lymphoma No No Covid-19 infection
Male 54 Non-Hodgkin Lymphoma No No Covid-19 infection
Male 81 Lung cancer (NSCLC) No No Covid-19 infection
Male 58 Lung cancer (NSCLC) No No Covid-19 infection
Female 55 Lung cancer (SCLC) No No Covid-19 infection
Male 27 Lung cancer (SCLC) No No Covid-19 infection
Male 79 Lung cancer (NSCLC) No No Covid-19 infection
Male 76 Lung cancer (NSCLC) No No Covid-19 infection
Female 74 Ovarian cancer No No Covid-19 infection
Female 75 Ovarian cancer No No Covid-19 infection
Female 66 Ovarian cancer No No Covid-19 infection
Female 28 Womb cancer No No Covid-19 infection
Female 62 Pancreas cancer No No Covid-19 infection
Female 78 Pancreas cancer No No Covid-19 infection
J. Pers. Med. 2021,11, 226 10 of 16
Table 2. Cont.
Gender Age Previous Diseases COVID-19 Infection Clinical Outcome
Male 72 Pancreas cancer No No Covid-19 infection
Female 58 Pancreas cancer No No Covid-19 infection
Male 63 Pancreas cancer No No Covid-19 infection
Female 79 Pancreas cancer No No Covid-19 infection
Male 60 Pancreas cancer No No Covid-19 infection
Male 67 Pancreas cancer No No Covid-19 infection
Male 71 Prostate cancer No No Covid-19 infection
Male 80 Prostate cancer No No Covid-19 infection
Male 83 Prostate cancer No No Covid-19 infection
Male 58 Prostate cancer No No Covid-19 infection
Male 61 Prostate cancer No No Covid-19 infection
Female 92 Skin cancer (basal cell carcinoma) No No Covid-19 infection
Male 70
Oral cancer (squamous cell carcinoma)
No No Covid-19 infection
Male 89 Skin cancer (basal cell carcinoma) No No Covid-19 infection
Male 77 Skin cancer (angiosarcoma) No No Covid-19 infection
Chemo-prophylactic
purpose. Healthy subjects
Female 32 None No No Covid-19 infection
Female 12 None No No Covid-19 infection
Female 18 None No No Covid-19 infection
Male 72 None No No Covid-19 infection
Female 45 None No No Covid-19 infection
Female 32 None No No Covid-19 infection
Female 38 None No No Covid-19 infection
Male 45 None No No Covid-19 infection
Male 59 None No No Covid-19 infection
Male 64 None No No Covid-19 infection
Female 49 None No No Covid-19 infection
Female 93 None No No Covid-19 infection
Male 61 None No No Covid-19 infection
Male 52 None No No Covid-19 infection
Male 34 None No No Covid-19 infection
Female 36 None No No Covid-19 infection
Female 48 None No No Covid-19 infection
Male 62 None No No Covid-19 infection
Male 46 None No No Covid-19 infection
Female 51 None No No Covid-19 infection
Male 80 None No No Covid-19 infection
The efficacy of HOO in preventing COVID-19 infection was evaluated in 21 normal
subjects who consumed HOO for 2 months as an integrative food supplement, and they
carried on their normal daily activities. Due to the incidence of COVID-19 infection in Italy
during the monitored period (i.e., 15% prevalence per 100 diagnostic tests performed), at
least three of them would be expected to be infected within our study timeframe. However,
none of the test subjects experienced COVID-19 infection, as demonstrated by the lack of
any symptoms (fever, olfactory and test failure, cough, etc.), and by the negative antigen
and molecular tests for COVID-19 infection. This difference (0% vs. 15% in 21 subjects)
was statistically significant (p< 0.05).
Among the same 21 normal subjects, a strong evidence for the antiviral efficacy was
shown in a frail 93-year-old female. She resided in a nursing home where COVID-19
outbreak did occur. Among the residents in the nursing home, one person in the same
room died, and three others experienced severe pneumonitis and complications. Despite
the extensive exposure to COVID-19 infection of this fragile subject, no symptoms occurred
to her, and the weekly PCR molecular tests were also negative.
J. Pers. Med. 2021,11, 226 11 of 16
Finally, therapeutic efficacy of HOO against COVID-19 was evaluated in four patients
who were either diagnosed with having the infection by both clinical symptoms and
molecular tests.
The first patient was a 22-year-old female with infection on 15 August 2020. Her
clinical symptoms included fever (39
◦
C), severe cough, thorax pain on cough, and loss
of olfactory and taste function. Pharyngeal swab confirmed the diagnosis by detecting a
high SARS-CoV-2 load with early cycle (<25th) qPCR positivity. She was administered
four pills of HOO twice per day per os. After 5 days of treatment, all reported symptoms
disappeared. In addition, thorax Rx confirmed the lack of any lung complications. The
early recovery of olfactory and taste functions was unexpected, given the fact that these
symptoms often persist for months after recovery. The second pharyngeal swab performed
at 14 days since HOO treatment beginning was negative based on qPCR results.
The second patient was a frail 55-year-old male who had existing complications with
COPD-related respiratory failure, obesity, and severe cardiovascular disease. He contracted
COVID-19 infection together with pneumonitis, cough, fever (38.7
◦
C), and decreased O
2
blood saturation down to 84%. After 4 days of HOO treatment, the patient’s fatigue and
fever disappeared together with recovery of olfactory and taste capacities. Importantly,
O
2
blood saturation was restored to 98%. qPCR tests for SARS-CoV-2 performed 7 and
14 days after beginning HOO treatment were negative.
The third patient was a 54-year-old female who was the wife of the second patient
being therefore heavily exposed to the SARS-CoV-2 virus. HOO treatment was started
2 days after her husband was diagnosed with the infection. Although she had no clinical
symptoms for the infection at that time, thorax Rx revealed the presence of asymptomatic
lung pneumonitis amenable to COVID-19 features. After the HOO treatment, the molecular
test for COVID-19 diagnosis was negative, and no symptoms for COVID-19 infection
appeared. Therefore, her infection with COVID-19 was possibly prevented.
The fourth patient was a 52-year-old female working in the nursing home where our
abovementioned 93-year-old female lived and where the COVID-19 outbreak occurred.
She had moderate COVID-19 infection symptoms, which were confirmed by positive qPCR
molecular test. HOO was started immediately, and after 5 days, all symptoms disappeared.
The qPCR molecular test performed 10 days after beginning HOO treatment provided
negative result.
In summary, a total of 77 subjects received HOO administration either for chemo-
prophylaxis in uninfected subjects (n= 73, 52 cancer patients and 21 healthy subjects) or
for therapeutic purposes in COVID-19 infected patients (n= 4). The results indicate that
no COVID-19 infection was detected in the uninfected subjects, and complete recovery
with negative qPCR tests was observed in the four infected patients. These results are
summarized in Table 2.
4. Discussion
With the escalating COVID-19 pandemic, a variety of effective prevention and inter-
vention protocols is being developed and is urgently needed to combat the disease. Our
investigations, using cell culture, normal subjects, cancer patients, and COVID-19-infected
patients, indicate that ozonized oil can be used as novel chemoprophylaxis and therapy
against COVID-19 infection. Large controlled studies and clinical trials are required to
substantiate these findings.
For our investigation, the virus challenge test was developed and was shown to be
sensitive and specific in detecting the ability of SARS-CoV2 to infect sensitive (i.e., ACE2
expressing) cells. For prevention, HOO when used in combination with HOOws was more
effective than some common antiviral treatments. The combined treatment was so effective
that it fully neutralized SARS-CoV2 infectivity because no virus was detected inside the
cells despite their exposure to a very high dose of viral load. This finding was obtained in
pulmonary cells, i.e., SARS-Cov-2 target cells. However, it could be also proven in other
different type of cells to corroborate this result.
J. Pers. Med. 2021,11, 226 12 of 16
HOO is an oil-based ozone vector. When administered
in vivo
by the oral route, it is
complexed with lipoprotein in the liver and then distributed via the general circulation,
with lungs as the first target organ. However, infection from COVID-19 is mainly via the
upper respiratory epithelium. Indeed, the first entry tissue for SARS-Cov-2 infection is
represented by nasal mucosa [
18
]. To directly target these entry tissues, a novel hydrophilic
preparation of HOO was developed by us and is labeled as water soluble high ozonide oil
(HOOws). Specifically, the preparation allows its delivery by aerosol and nasal spray.
Despite our limited sample sizes, our data provide valuable and novel evidence to
indicate that the combination of HOO and HOOws was highly effective in preventing
COVID-19 infection. The prevention mechanisms are mediated by neutralizing the virus
both in intracellular and extracellular environments, inhibiting intracellular viral replication
and blocking extracellular spreading of virions, without obvious side-effects.
The observed effectiveness of HOO is most likely based on the unique structure,
morphology, and composition of the SARS-CoV2 virus. SARS-Cov-2 sensitivity to UV
disinfection is controversial because RNA viruses do not contain thymine (T), the main
molecular target of UV-C, but uracil (U). Indeed, UV-C genotoxicity is exerted by forming
intrastrand T-T cyclobutane dimers [
19
]. However, SARS-Cov-2 has been reported to
be very sensitive to 222 nm and, to a lesser extent, 254 nm UVC radiations [
20
,
21
]. It
should be noted that, besides being genotoxic, UV light is also an oxidizing agent. Human
coronaviridae were reported to be sensitive to hydrogen peroxide disinfection in the same
levels of Glutardialdehyde [
10
]. One reason for the sensitivity of SARS-CoV-2 to oxidizing
agents is that the virus lacks any antioxidant defenses. Another is the presence of chemical
structures very sensitive to oxidation at the terminal part of the spike-protein which binds
the ACE2 cell receptor. The terminal region of the spike-protein is enriched with thiol-rich
amino-acids, i.e., cysteine, whose sulfhydryl (–SH) sites are highly sensitive to oxidation [
7
].
These structures of the viral-spike proteins can be neutralized by oxidizing agents, such as
HOO. This neutralization would hamper its virus binding with the ACE2 receptor thus
blocking intracellular virus penetration (Figure 3A).
Under our experimental conditions, hydrogen peroxide was not particularly effective
in neutralizing SARS-CoV-2. This situation is amenable to the peculiar characteristic of this
virus and to its high lipophilicity, making it a difficult target for hydrophilic disinfectant
such as hydrogen peroxide. Indeed, the ratio between the protein content (spike proteins)
and the lipid content (envelope) is dramatically low in SARS-CoV-2 19 as compared to
other RNA viruses. As an example, in the external surface of flu Orthomyxovirus, there are
1 protein per 100 nm
2
of membrane lipid surface. Due to the high density of outside proteins
(hemagglutinin and neuraminidase), this virus is referred to as “chestnut hedgehog” virus.
By comparison, this ratio dramatically drops by 10-fold in SARS-CoV-2 19 virus to 1 protein
per 1000 nm
2
only [
22
]. Furthermore, SARS-CoV-2 19 spike proteins are highly flexible
and can fold and move along exposing to the outside large sections of the underlying
lipid envelope [
23
]. Such a structure explains the high lipophilicity of SARS-CoV-2 which
exposes large sections of the envelope for the interaction with target cells. Accordingly,
SARS-CoV-2 interacts earlier and more readily with nerves, neurons, and the central
nervous system, which are typically highly lipophilic, than other airborne RNA viruses
such as Orthomyxoviridae [
24
,
25
]. The early neurotropism of SARS-CoV-2 19 causes the
early symptoms which are related to its penetration into olfactory nerve (to cause loss of
olfactory and taste capabilities) and across haemato-encephalic barrier [26].
Since SARS-CoV-2 exposes a wide portion of its lipid envelope to the outside, without
a screen provided by a dense layer of spike proteins, it becomes highly sensitive to ethanol-
containing disinfectants. Indeed, ethanol, which usually fixes but does not kill viruses, due
to its lipid-solvent actions, is the most effective disinfectant against SARS-CoV-2 [10].
The unique structure of SARS-CoV-2, as described earlier, indicates its high sensitivity
to oxidizing disinfectants that are also lipophilic. This situation is clearly demonstrated by
our use of HOO and HOOws. Their effectiveness is likely based on the following scenario:
Due to their lipid component (saturated fatty acids), these compounds easily target the
J. Pers. Med. 2021,11, 226 13 of 16
unscreened lipophilic envelope of the virus. When the target is reached, both HOO and
HOOws would release ozone and reactive oxygen species to induce lipid peroxidation in
the sensitive virus that is devoid of any antioxidant defenses. These activities would destroy
the envelope and neutralize the virus, thus eliminating infectivity and consequences from
the infection. This mechanism of action is reported in Figure 3B.
J. Pers. Med. 2021, 11, x FOR PEER REVIEW 13 of 16
Figure 3. (A–C). Possible mechanisms for sensitivity of SARS-CoV-2 19 virus to HOO. Panel (A)
Neutralization of spike proteins; HOO oxidation blocks the sites of the spike protein used by
SARS-Cov-2 to bind cell receptor ACE2; this situation is highlighted by the darkening of spike
protein when treated with HOO (light blue circles). Panel (B) Peroxidation of the lipid viral enve-
lope; due to the low spike-protein density, and wide sections of the SARS-Cov-2 lipid envelope are
exposed to the interaction with HOO; this situation results in the peroxidation of the viral lipid
envelope, as envisaged by the darkening of this structure when interacting with HOO (light blue
circles). Panel (C) HOO has a unique ability to penetrate inside cell cytoplasm where the viral rep-
lication cycle occurs hidden from extracellular disinfectants; HOO is able to neutralize intracellu-
lar viral assembly oxidizing viral components inside the intracellular environment (darkening of
intracellular viral fragments when interacting with HOO light-blue circles). From left to right: nor-
mal cell, cell infected by SARS-Cov-2, and cell infected by SARS-Cov-2 treated with HOO.
Under our experimental conditions, hydrogen peroxide was not particularly effective
in neutralizing SARS-CoV-2. This situation is amenable to the peculiar characteristic of
this virus and to its high lipophilicity, making it a difficult target for hydrophilic disin-
fectant such as hydrogen peroxide. Indeed, the ratio between the protein content (spike
proteins) and the lipid content (envelope) is dramatically low in SARS-CoV-2 19 as com-
pared to other RNA viruses. As an example, in the external surface of flu Orthomyxovirus,
there are 1 protein per 100 nm
2
of membrane lipid surface. Due to the high density of
outside proteins (hemagglutinin and neuraminidase), this virus is referred to as “chestnut
hedgehog” virus. By comparison, this ratio dramatically drops by 10-fold in SARS-CoV-2
19 virus to 1 protein per 1000 nm2 only [22]. Furthermore, SARS-CoV-2 19 spike proteins
are highly flexible and can fold and move along exposing to the outside large sections of
the underlying lipid envelope [23]. Such a structure explains the high lipophilicity of
SARS-CoV-2 which exposes large sections of the envelope for the interaction with target
cells. Accordingly, SARS-CoV-2 interacts earlier and more readily with nerves, neurons,
and the central nervous system, which are typically highly lipophilic, than other airborne
RNA viruses such as Orthomyxoviridae [24,25]. The early neurotropism of SARS-CoV-2 19
causes the early symptoms which are related to its penetration into olfactory nerve (to cause
loss of olfactory and taste capabilities) and across haemato-encephalic barrier [26].
Figure 3.
(
A
–
C
). Possible mechanisms for sensitivity of SARS-CoV-2 19 virus to HOO. Panel (
A
)
Neutralization of spike proteins; HOO oxidation blocks the sites of the spike protein used by SARS-
Cov-2 to bind cell receptor ACE2; this situation is highlighted by the darkening of spike protein
when treated with HOO (light blue circles). Panel (
B
) Peroxidation of the lipid viral envelope; due
to the low spike-protein density, and wide sections of the SARS-Cov-2 lipid envelope are exposed
to the interaction with HOO; this situation results in the peroxidation of the viral lipid envelope, as
envisaged by the darkening of this structure when interacting with HOO (light blue circles). Panel
(
C
) HOO has a unique ability to penetrate inside cell cytoplasm where the viral replication cycle
occurs hidden from extracellular disinfectants; HOO is able to neutralize intracellular viral assembly
oxidizing viral components inside the intracellular environment (darkening of intracellular viral
fragments when interacting with HOO light-blue circles). From left to right: normal cell, cell infected
by SARS-Cov-2, and cell infected by SARS-Cov-2 treated with HOO.
Another possible explanation for the anti-COVID efficacy of HOO is the ability of this
compound to interfere with the formation and dynamics of lipid vacuoles which protect
the virus during the intracellular production and assembly of the whole virions [
27
]. As
demonstrated in our above-reported
in vitro
studies, HOO has a unique ability to penetrate
inside cell cytoplasm where the viral replication cycle occurs hidden from extracellular
disinfectants. Thus, HOO is able to neutralize intracellular viral assembly oxidizing viral
components inside the intracellular environment. This mechanism of actions is summarized
in Figure 3C.
This mechanism also explains the synergistic effects between HOO and HOOws:
with HOO being lipophilic and targeting SARS-CoV-2 in both extra- and intra-cellular
compartments; with HOOws being hydrophilic and targeting SARS-CoV-2 19 virus in the
J. Pers. Med. 2021,11, 226 14 of 16
extracellular compartment. Indeed, the combined administration of HOO and HOOws
was found to be highly effective in preventing and attenuating COVID-19 infection in
the 77 normal individuals and patients without adverse or side effects. It is important
to point out that our group of subjects is composed of many high-risk individuals: four
COVID-infected patients, 52 cancer patients who are susceptible to infection [
28
], and
normal senior citizens in an infectious environment.
A highly relevant observation is the disappearance of symptoms in just 5 days after
HOO treatment and of negative qPCR results after 7–10 days among the four COVID-
infected patients. The quick recovery was encouraging but unexpected because COVID-19
patients usually display positive qPCR results up to 60 days after recovery [
29
]. On the
other hand, the early disappearance of clinical symptoms was likely due to HOO’s anti-
inflammatory and macrophage-inhibiting effects, which also explains the lack of thrombo-
embolic complications among our patients. In addition, HOO’s capacity to increase oxygen
availability in the lungs would counter pulmonary damage from COVID-19, as demon-
strated in the fragile patient. Such an increased oxygen supply would also be of great
benefit to COVID-19 patients who are also affected by severe pneumonia. Indeed, COVD-19
infection cause an interstitial pneumonia [
30
] hampering oxygen absorption by the alveolar
endothelium despite the delivery of oxygen through the respiratory system, as performed
for therapeutic purposes. Accordingly, the possibility of directly increasing oxygen tissue
availability as performed by HOO is of relevance to improve the prognosis of COVID-19
patients. The limit of our study is that analysis of oxygen tissue availability and aerobic
threshold has been performed in healthy subjects and not in COVID-19 patients. Unfor-
tunately, COVID-19 patients cannot undergo the physical endurance activity required to
perform the aerobic threshold analysis. However, the findings obtained in COVID-19 pa-
tients indicate that blood oxygen saturation is remarkably increased after HOO treatment.
To our knowledge, this is the first study to develop an innovative carrier for ozone which
would release ozone intracellularly and to evaluate its clinically relevant antiviral activities
and efficacy.
Our study has limitations. The findings are not validated for a double-blind clinical
study but an observational study. In this study efforts have been focused on demonstrat-
ing the mechanisms explaining the antiviral efficacy of ozonized oils against COVID-19.
Further clinical studies from other clinical centers are required to substantiate the herein
presented clinical results. The setting up of a double-blind randomized clinical trial as
performed in hospital ward assisting COVID-19 patients is required to prove or deny the
clinical efficacy of the proposed approach in an adequate number of patients.
5. Conclusions
Our novel and effective HOO/HOOws treatment protocol against COVID-19 is highly
encouraging. Due to their naturally nontoxic status, HOO/HOOws can be used as chemo-
prophylactic treatment against COVID-19 infection in different infectious environments:
occupational (medical doctors, nurses) or familiar (relatives or cohabitants) conditions. In
addition, for infected patients, HOO/HOOws can be used as complimentary therapeutic
treatment for COVID-19 infection, without the need for any modifications of the established
standard therapeutic protocols. This complimentary treatment is potentially helpful to
(a) decrease the severity of the diseases, thus lowering the number of patients requiring
high-intensity therapies and for (b) faster recovery and time spent in hospitals. Therefore,
clinicians may adopt the use of our protocol for prevention and intervention of COVID
infection. Randomized controlled clinical trials will be set up to definitively determine the
effectiveness of this treatment in preventing SARS-Cov-2 infection and COVID-19 complica-
tions. With the collection of additional clinical results, efficacy of HOO/HOOws treatment
will be better understood, and enhanced protocol will be used against the pandemic.
J. Pers. Med. 2021,11, 226 15 of 16
Author Contributions:
Conceptualization, A.I. and A.P.; methodology, U.P.; L.E.; M.C.; E.F. (Emanuela
Faelli); E.G.; P.L.; M.T. software, A.I.; validation, M.T.; A.P.; E.F. (Emanuela Faelli); formal analysis,
S.P.; and D.M.; investigation, A.I.; A.P.; resources, A.I.; data curation, R.P.; E.F. (Emanuela Faelli); A.I.;
E.F. (Enzo Fracchia), Writing—Original draft preparation, A.I., and A.P.; Writing—Review and edit-
ing. W.A.; A.I.; visualization, W.A.; supervision, A.I. and A.P.; project administration, A.I.; funding
acquisition, A.I. All authors have read and agreed to the published version of the manuscript.
Funding:
This research received no external funding. This research was funded by University of
Genoa Italy.
Institutional Review Board Statement:
The study was conducted according to the guidelines of th
Declaration of Helsinki, and approved by the Health Ministry of Malta (approval number 0075/2020
according to EC1924/2006 issued on 17 March 2020).
Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.
Data Availability Statement:
The datasets used and/or analysed during the current study are
available from the corresponding author on reasonable request.
Acknowledgments:
We thank Giancarlo Icardi and Andrea Orsi (University of Genoa, Italy) for their
valuable support in the project.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
HOO: ozonized oils at high ozonides; HOOws: water-soluble ozonized oils at high ozonides.
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