An integrative approach in the treatment of chronic obstructive pulmonary disease: The role of the ozone therapy

Abstract

A pilot study has been performed on fifty COPD patients: besides using effective drugs, half of the patients have been treated also with major ozonated autohaemotherapy. This treatment has been evaluated during the last two decades and is absolutely atoxic: the treated patients have shown a significant improvement of the six minute walking test and Saint George Respiratory Questionnaire total score. Surprisingly their quality of life was also much improved. Orthodox medications appears to be greatly potentiated when integrated by the autologous infusion of ozonated blood. This combination deserves to be evaluated in an ample clinical trial.

Full text

American Journal of Clinical and Experimental Medicine
2014; 2(2): 9-13
Published online March 10, 2014 (http://www.sciencepublishinggroup.com/j/ajcem)
doi: 10.11648/j.ajcem.20140202.11
Oxygen ozone therapy in the treatment of chronic
obstructive pulmonary disease: An integrative approach
Emma Borrelli
1, *
, Velio Bocci
2
1
Department of Medical Biotechnologies, University of Siena, Italy
2
Department of Biotechnologies, University of Siena, Italy
Email address:
Ebitaly2007@libero.it (E. Borrelli), bocci@unisi.it (V. Bocci)
To site this article:
Emma Borrelli, Velio Bocci. Oxygen Ozone Therapy in the Treatment of Chronic Obstructive Pulmonary Disease: An Integrative
Approach. American Journal of Clinical and Experimental Medicine. Vol. 2, No. 2, 2014, pp. 9-13.
doi: 10.11648/j.ajcem.20140202.11
Abstract:
A pilot study has been performed on fifty COPD patients: besides using effective drugs, half of the patients
have been treated also with major ozonated autohaemotherapy. This treatment has been evaluated during the last two
decades and is absolutely atoxic: the treated patients have shown a significant improvement of the six minute walking test
and Saint George Respiratory Questionnaire total score. Surprisingly their quality of life was also much improved.
Orthodox medications appears to be greatly potentiated when integrated by the autologous infusion of ozonated blood. This
combination deserves to be evaluated in an ample clinical trial.
Keywords:
Ozonetherapy, Chronic Obstructive Pulmonary Disease, Oxygen Radicals
1. Introduction
Chronic obstructive pulmonary disease (COPD) is a
multi component and heterogeneous disease, with patients
differing in terms of clinical presentation and rate of
disease progression. Although mainly categorized by
airflow limitation that is progressive and not fully
reversible, latest severity categorization also includes
exacerbation frequency and symptom burden as key
features. Moreover, in many patients the disease seems to
be associated with several extra-pulmonary
manifestations.[1,2].What is unclear at present is whether
these manifestations are directly related to COPD or are
just an independent consequence of the exposure to
common causal effects such as tobacco smoking and
inactivity. The most widely recognized manifestations
include the presence of concomitant cardiovascular
disease, skeletal muscle dysfunction, osteoporosis, clinical
depression and anxiety[3-5]. A major goal in the
management of this disease is to ensure that its burden is
minimized, such that patients have the best possible
health-related quality of life [6]. An ample range and type
of drugs such as salmeterol/fluticasone, tiotropium,
theophilline, beclometasone, formoterol etc have been tried
with useful but variable results. Recurrent pulmonary
infections are treated with antibiotics. However it is now
clear that COPD is fundamentally a disease inducing a
chronic inflammation with a remarkable increase of
reactive oxygen species (ROS), of circulating
pro-inflammatory cytokines (IL-1β,IL-6,IL-8,IL-18 and
TNFα) as well as acute-phase proteins such as C reactive
protein (CRP) and serum amyloid.
Therefore it is clear that COPD is a very complex disease
where the chronic inflammation of the lungs [7,8] extends
to the whole body with deranged metabolic cardiovascular
and bone diseases. Beside smoking cessation, which is a
must, an anti-inflammatory treatment associated to a
suppression of pulmonary inflammation appears essential
to slow down the progression of the disease [8]. During the
last two decades the biological and molecular mechanism
of action of oxygen ozone therapy have been fully clarified
[9 -12] and its rationale is clarified in the next session. It
has been emphasized what this treatment has unique
properties in reducing inflammation and upregulating the
cellular antioxidant system. Consequently the optimal anti
COPD medical therapy integrated by ozonetherapy is
suggested here as novel approach.
The rationale of ozone therapy
The chronic oxidative stress can be corrected by using
ozone-therapy
Among the numerous complementary methods, only one
is specifically able to correct the chronic oxidative stress.

10 Emma Borrelli and Velio Bocci: Oxygen Ozone Therapy in the Treatment of Chronic Obstructive Pulmonary Disease:
An Integrative Approach
Among the numerous complementary methods, only one is
specifically able to correct the chronic oxidative stress.
Preclinical and clinical studies performed with
ozone-therapy have been performed in many countries and
in Germany more than one million sessions are successfully
held every year without any side effects.
Ozone, like other gases as NO,CO,H
2
S and H
2
, although
intrinsically toxic, when properly used in well determined
small concentrations, has become important medical gas
[13-14]. Usually 100-150 ml of blood of the patient,
anticoagulated with Na Citrate, are collected in a sterile
glass ozone-resistant bottle and an equal volume of a gas
mixture (96% O
2
-4%O
3
) is added at a precisely determined
ozone concentration within 0.21-0.84 mM per ml of
blood .The ozonated blood is then infused into the donor
patients within 2-3 min. The initial dose is minimal and, for
avoiding any side effects, is slowly upgraded up to 0.84
mM throughout the necessary 30-40 sessions, twice weekly.
The axiom:”start low, go slow” proves to be ideal and
under these conditions, ozone behaves as an acceptable
stressor. Indeed ozone therapy induces a series of graduated
small oxidative stress acting on all organs and able to
reactivate the potent defense system, which counteracts the
deleterious chronic oxidative stress induced by COPD .
The high reactivity and solubility of ozone in the water
of plasma allows its exhaustion in one min while it
generates two crucial messengers such as H
2
O
2
and
4-hydroxynonenal (4-HNE) finally responsible for eliciting
the well defined biochemical and molecular reactions
responsible for the biological activities. 4-HNE readily
forms an adduct with the Cys34 of albumin or with
glutathione and this compound is consequently be able to
reach most of cells of the body and to reactivate the
antioxidant defenses.
The real molecular mechanisms of antioxidant activation
Alkenal adducts are able to react with the Kelch-like
ECH-associated protein 1 (Keap 1)-NF-E2-related factor 2
(Nrf2) system that is present in the cell cytosol with the
role of antagonizing oxidative and electrophilic insults. In
detail, keap1 is a protein molecule with many –SH groups
that are important for the repression of Nrf2 activity.
Normally, the complex Nrf2-Keap1 has a half life of about
20 minutes because keap 1 is readlily ubiquitinated and
digested in the proteasome. However, the alkenal
interaction with both Cys 272 and 288 of Keap-1 allows the
release of Nrf2, which escapes proteosomal degradation
and traslocates into the nucleus, heterodimerizes with a
small Maf protein and binds to the antioxidant Response
Element (ARE or EHRE) on DNA [15].
On this basis, it is clear that Nrf2 is now correctly
recognized as the key cellular defense system against
oxidative and xenobiotic stresses. Such a crucial event is
able to upregulate the synthesis of relevant enzymes.
The reactivation of innate defense system leads to the
synthesis of a number of antioxidant enzymes able to
counteract the chronic oxidative stress:
a) activation of the gamma-glutamyl-cysteine ligase and
of GSH synthase allows a marked increase of the GSH
intracellular level. The enhanced GSH/GSSG ratio allows
an important protection against ROS ;
b) activation of the synthesis of antioxidant enzymes able
to detoxify an excess of ROS such as catalase,
SOD,GSH-peroxidases,GSH-reductase,NADPH-quinoneox
idoreductase(NQO1),cytochrome P450 monooxygenase
system and HSP70 ;
c) the upregulation of Heme-oxygenase 1 (HO-1) is also
very protective and the trace of CO in combination with
NO allows vasodilatation of ischemic tissues;
d) the enhancement of the synthesis and levels of phase
II enzymes such as GSH S-transferases,
UDP-glucuronosyltransferases, N-acetyltransferases ans
sulfotransferases ;
e) inhibition of cytokine mediated inflammation via the
induction of leukotriene B4 reductase ;
f) reducing iron overload and subsequent oxidative stress
induced via elevated ferritin and bilirubin as a lipophilic
antioxidant ;
g) the repetition of graduated small oxidative stresses
induces a multiform adaptative response. Moreover, during
the ozone therapy sessions there is an increased release of
adrenocorticothropic hormone, followed by cortisol from
the cortex of the adrenal glands as a consequence of the
liberation of corticotrophin releasing factor from the
hypothalamus. Such a response is responsible for an
improved feeling of well being reported by the majority of
patients.
The efficacy of the mentioned orthodox drugs associated
with the safe and valid support of the ozonated
autohaemotherapy proves that the concept of integrated
medicine is the best combination because it correctly
associates suitable drugs with the critical stimulus of
reactivating the natural defenses.
The absolute lack of toxicity of ozone-therapy
Gaseous ozone can be harmful at even low
concentrations of part per million, affecting especially the
eyes and respiratory systems. Administration of ozone by
aerosol is toxic and must be avoided, as well as the
intravenous administration of the oxygen-ozone gas
mixture. On the contrary, very small and precisely
determined ozone dosages during ozone therapy do not
procure any acute or late side effects. In fact addition of
ozone to blood happens ex vivo and the minimal amount of
ozone acts as a pro drug and within 2-3 min is exhausted in
small parts by the plasma antioxidants by generation of
H
2
O
2
and alkenals .
The performance of ozonetherapy twice weekly for at
least five months ( about 40 sessions) also allows to
improve the vascular system and to enhance the
oxygenation of ischemic tissues. Moreover, such a bio
oxidative procedure, can be continued with a weekly
session for life with an excellent compliance of patients .
On this basis it was decided to start a preliminary clinical
investigation on COPD patients.

American Journal of Clinical and Experimental Medicine 2014, 2(2): 9-13 11
2. Subjects and Methods
In this study we enrolled 50 patients affected by
moderate/ severe COPD, which was diagnosed according to
the current guidelines [16]. All of them were ex smokers
and referred a score 2-3 according to MRC classification.
They were observed in a stable phase of the disease, free
from exacerbations in the 4 weeks before starting the study
protocol and under regular pharmacologic therapy ( inhaled
long acting beta2 agonists/ corticosteroids LABA/ICS
and/or tiotropium in all patients).
Before starting the study each patient underwent a
clinical assessment, chest X-ray, detailed pulmonary
function test (PFT), resting arterial blood gas analysis.
Exclusion criteria were unstable ischemia and uncontrolled
pathology.
Patients were enrolled in two groups: group A ( 25
patients) underwent a cycle of oxygen ozonetherapy
( major ozonated autohaemotherapy, O
3
-AHT) twice a
week for the first 5 weeks, thereafter a single treatment
every week for other 10 weeks. Group B ( 25 patients)
served as control and not received treatments otherwise
the standard therapy with inhaled beta2 long acting
bronchodilators and/or corticosteroids .In all patients we
measured before and after the end of the study: 1)
Pulmonary FunctionTest 2 )Resting Arterial blood gas 3)
exercise tolerance by 6 min walking test (6MWT) 4)
dyspnea index by Borg dyspnea scale 5) health status,
evaluate by the St. George Respiratory questionnaire
( SGRQ). The patient’s characteristics at the start of the
study are reported in table 1.
Table 1. Baseline characteristics (mean ± SD) of the patients enrolled in
Group A ( ozone treatments) and Group B ( control group).
GROUP A GROUP B
Number 25 25
Gender (M/F) 15/10 17/8
BMI 26.8 ± 3.1 26.2 ± 2.7
FEV1(% pred) 51.1± 18 50.7± 17.4
FVC (% pred) 76.2± 16.4 78 ± 15.2
RV(% pred) 130± 37.1 132± 40
TLC(% pred) 106.6±20.3 107.1±21
COPD severity *
Moderate 15 17
Severe 10 8
FEV1= forced expiratory volume in one second; FVC= forced vital
capacity; RV= residual volume ; TLC= total lung content; * according to
GOLD classification. All comparison between group was not statistically
significant.
An informed consent for the oxygen ozone therapy was
obtained in patients of group A. The study was approved by
the Siena University Hospital Ethical Committee, and it
was conducted in accordance to the ethical consideration of
the World Medical Association ( Helsinki Declaration).
Oxygen ozonetherapy protocol
Oxygen ozonetherapy procedure O3-AHT was carried
out as follows: 225mL of blood were drawn by vacuum
from an antecubital vein into a sterile glass bottle
(Ozonosan, Iffezeim, Germany) in which 25mL of 3.8% Na
citrate solution (Galenica Senese Industries, Siena, Italy) as
an anticoagulant, had been previously added so that the
blood/citrate volume ratio was 9:1. After blood withdrawal,
the bottle was momentarily disconnected leaving the
venous access open by a saline infusion. The use of 225 mL
of blood was unrelated to the subject's body mass, rather, it
was related to the dROM values in plasma and to
previously reported protocols [12]. A corresponding
volume (225mL) of gas was immediately added with an O
3
concentration of 20 micrograms/ml for the first four
treatments, thereafter the concentration of ozone was 40
micrograms/mL of gas for the other 16 treatments.
Ozone was produced by an Ozonline 80 E generator
(Medica srl, Bologna, Italy), in which O
3
concentration was
measured photometrically in real time and checked by
iodometric titration according to the rules established by
the International Ozone Association.
The gas was immediately and continuously mixed with
the blood in the bottle for at least 5 minutes and with gentle
rotating movement to avoid foaming. Due to the blood
viscosity, the gas mixture does not instantaneously come
into contact with the whole blood mass, thus this mixing
time is necessary. Indeed, the pO
2
value only after this
mixing period usually reaches a plateau level (of about
500mmHg). During these 5 minutes of mixing the ozone
totally reacted with both the potent antioxidants of plasma
and the unsaturated lipids bound to albumin, generating a
small amount of hydrogen peroxide and alkenals. These
two messengers were responsible for eliciting crucial
biochemical reactions on both erythrocytes and within cells
when the hyper-oxygenated ozonated blood was re-infused
into the patient. At this point, the hyper-oxygenated
ozonated blood was re-infused by promptly substituting the
saline infusion with it. Reinfusion was accomplished in
about 15-20 minutes and the whole procedure was carried
out in approximately 40 minutes. O
3
-AHT was carried
out in an out-patient setting twice weekly (on Tuesday and
on Friday) for the first 5 weeks, thereafter a single
treatment a week for other 10 weeks.
3. Statistical Analysis
Statistical analyses were performed using statistical
package SPSS and the Student t test for the intragroup
( before and after the end of study) and intergroup ( before
study, group A vs group B and after the end of study, group
A vs group B) comparison of parameters. P<0.01 was
considered significant.
4. Results
In the group A there was a significant increase in 6MWT
( 417.5 +/- 107.4 m before the study vs 493.8 +/- 106 m
at the end of study, p< 0.01) and a significant decrease in
the degree of dyspnea measured by Borg scale after the

12 Emma Borrelli and Velio Bocci: Oxygen Ozone Therapy in the Treatment of Chronic Obstructive Pulmonary Disease:
An Integrative Approach
6MWT ( 4.1 +/- 2.4 vs 3 +/- 2.1, p<0.01) and in SGRQ
activity impact and total scores ( activity: 65.4 +/- 16.3% vs
54.2 +/- 15% p<0.01; impact: 38.4 +/- 18.2% vs 30.1 +/-
16.4% , p<0.01; total score: 48.9 +/- 16.0% vs 32.2 +/-
12.1%, p<0.01) (Table 2). No statistical difference were
observed in the pulmonary function test and resting arterial
blood gas before and after ozone therapy.
Table 2. Modification of parameters in Group A and Group B before and
after the study.
Group A Group B p
6MWT before (m) 417.5±107.3 411.4±109.2
ns
6MWT after (m) 493.8±105.4 407.3±104.4
P<0.01 a, b
BORG SCALE before
4.1±2.4 4.3±2.1 ns
BORG SCALE After 3±1.2 4.3±2.1 P<0.01 a
SGRQ
Activity before (%) 65.4±16.3 66.1±17.4 ns
Activity after (%) 54.2±15 62.4±16.6 P<0.01a
Impact before (%) 38.4±18.2 37.6±17.1 ns
Impact after (%) 30.1±16.4 34.5±17.9 P<0.01 a
Total score before(%) 48.9±16 47.3±16.4 ns
Total score after(%) 32.2±12.1 49.9±14.3 P<0.01 a, b
6MWT= six minutes walking test; Borg SCALE= Borg dyspnea
scale;SGRQ= Saint George Respiratory Questionnaire (items activity,
impact,total score);before=value before study;after= value at the end of
the study; a=intragroup comparison before versus after study, group A;
b= intergroup comparison Group A versus Group B, after study.
In the control group, no significant difference were
observed before and after the study in all parameters
recorded. The intergroup comparison in 6MWT and SGRQ
total score at the end of study showed a significant
difference ( 6MWT at the end of study: group A 493.8 +/-
106 m vs group B 407.3 +/- 104.4 m, p<0.01 ; SGRQ total
score at the end of study :group A 32.2± 12.1 vs group B
49.9± 14.3, p<0.01).
Side Effects and compliance
We have observed only temporary face redness in a small
percentage of patients (3%) during the treatment with
oxygen ozone therapy. Patients reported an improvement of
their general conditions, particularly in terms of increased
efficiency, mental concentration and memory.
5. Discussion
Traditionally, the severity of COPD was equated with air
flow limitation, as measured by impairment in forced
expiratory volume in one second (FEV1) and treatment and
management of the disease was also largely based on
spirometric assessment. However, because COPD is a
multicomponent disorder, structural and functional changes
take place in other organs, as well as in the lungs
[17].Therefore, airflow limitation alone does not reflects
the full burden of COPD such as dyspnea, exercise
intolerance and impairment of health related quality of life
[18].Oxygen ozone therapy represents a systemic therapy
with a deep impact with many organs, and it is not
surprising that it was a significant improvement in patient’s
quality of life and exercise tolerance.
Three years ago we performed the ozonated
autohaemotherapy in two COPD patients also affected by
age-related macular degeneration, who showed an
unexpected improvement. Consequently, it was decided to
more accurately examine the outcome in this pilot trial. The
patients themselves were enthusiastic about their improved
quality of life. It appears evident how the most suitable
medical treatment integrated by ozonated
autohaemotherapy was responsible for the improvement.
Now one autohaemotherapy per week is continued with the
satisfaction of patients. It must be clarified that the minimal
dosage of ozone (20µg/mL or 0.42 µmol/mL gas) acts as a
pro-drug because, in less than five minutes, is totally
exhausted during mixing ex vivo in the glass bottle. Its
relevant messengers are H
2
O
2
, which acts biochemically
mostly on the erythrocytes shifting the oxyhaemoglobin
curve to the right, plus an improved NO production by the
endothelium. Even more relevant is the effect caused by
submicromolar levels of 4-HNE with hydroperoxides that,
after infusion of the ozonated blood, act as a well tolerated
oxidative stress on a variety of body cells reactivating both
the defence system consisting of the up regulation of
antioxidant system plus phase-2 enzymes. The progressive
improvement of the redox homeostasis appears the critical
factors justifying the improvement of the quality of life.
Indeed the patients refer to feel much better after receiving
the first 7-12 infusions, that is from 1575 to 2700 ml of
ozonated blood. Naturally ozone as a gas is neither inhaled,
nor directly infused because ozone is so reactive to be
partly neutralized by plasma antioxidants and partly
consumed during the peroxidation reaction of
polyunsaturated lipids occurring ex vivo. This must be said
because by 2006 the dogma that ozone is always toxic has
been fully refuted. Consequently it appears useful to inform
that the ozonetherapeutic integration can represent a novel
approach for the COPD patients: it would be most
important to verify our preliminary data in an ample
clinical trial.
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