Am J Cardiovasc Drugs 2011; 11 (2): 73-82

CURRENT OPINION 1175-3277/11/0002-0073/$49.95/0

ª 2011 Adis Data Information BV. All rights reserved.

Ozone: A New Therapeutic Agent in Vascular Diseases

Velio Bocci,1 Iacopo Zanardi2 and Valter Travagli2
1 Department of Physiology, University of Siena, Siena, Italy
2 Department of Pharmaceutical Chemistry and Technology, University of Siena, Siena, Italy

Abstract In this article, we scientifically evaluate the bio-oxidative procedure known as oxygen-ozone therapy.
Research over a decade has established a comprehensive framework for understanding and recommending
this type of autohemotherapy in vascular diseases. In contrast, a non-specific immunomodulation therapy,
using heavily oxidized and denatured blood, has been recently used in studies involving a total of approx-
imately 3000 patients and has led to ‘disappointing’ results. Such a treatment appears to be an inappropriate
example of the so-called minor autohemotherapy, and its poor outcomes may discourage any further
studies. Therefore it appears necessary to clarify that the use of only a minimal ozone dose and a valid
experimental protocol is likely to produce beneficial results. Millions of people suffer from chronic limb,
brain, and heart ischemia, and such patients may benefit if appropriate ozone therapy could be implemented.
Accordingly, we propose the need for a well designed, multicenter, clinical trial to be conducted.

1. Introduction modulation therapy (IMT) using heavily oxidized blood has

Statins, platelet aggregation inhibitors, antidiabetic agents,
anticoagulants, antihypertensives, vasodilators, and antiar-
rhythmic agents are drugs commonly used every day by millions
of patients, while complementary medications recommended
by phytotherapists and homeopaths are far less popular be-
cause their mechanisms of action, safety, and efficacy remain
uncertain. While oxygen is well known as a useful medical
gas,[1] the combination with ozone, namely oxygen-ozone
therapy or simply ozone therapy, has been used less often and
with mixed results. Bio-oxidative approaches to the treatment
of diseases have been known from the beginning of the 20th
century, but UV blood irradiation used in infectious diseases in
the pre-antibiotic era has fallen out of use because it is difficult
to standardize and cumbersome to perform.
Oxygen-ozone therapy has come of age in the last three de-
cades, with ozone representing the therapeutic agent, while
oxygen is simply used as the feeding gas for ozone generation.
Ozone is one of the most reactive gases and has a spontaneous
half-life of 40 minutes at 20C. In spite of the extemporary use,
modern medical ozone generators produce precisely determined
ozone concentrations in real time. The medical literature on
ozone therapy is modest, although it has been shown since 1981
to be more useful than pentoxyfilline and prostanoid analogs in
peripheral arterial disease (PAD).[2-8] In contrast, immuno-
yielded ‘disappointing’ results in the treatment of functional
class II–IV chronic heart failure (CHF).[9]
The present review has four objectives: firstly, to inform
clinical scientists and physicians that using ozone therapy is no
longer an empirical approach, but rather it is based on a
framework built on a sequence of biochemical reactions leading
to well defined biological effects on both blood cells and the
organism. Secondly, careful scrutiny[10] has excluded any po-
tentially toxic effects, by using small ozone dosages within a
precisely determined therapeutic range. Thirdly, by using
standardized procedures, the usefulness of ozone therapy in
PAD has already been shown by several authors.[2-8] Fourthly,
in open contrast, the misuse of an excessive amount of ozone
combined with UV irradiation and heat stress on blood has
totally compromised its efficacy in improving the prognosis of
CHF.[9] Therefore this article may inform readers that ozone
therapy is a rational treatment for vascular diseases provided
that ozone is used as a ‘real drug’ and not as a toxic gas.
2. The Mechanisms of Action of Ozone in
Contact with Blood
Ozone is known chemically as the third most reactive gas,
with an oxidation potential (E) of +2.08 V, while hydrogen
74
peroxide (H2O2), produced by several oxido-reductases known
as nicotinamide adenine dinucleotide phosphate oxidases
(NOXs), has an E of +1.78 V. Moreover, H2O2 is physiologi-
cally generated in the mitochondria following dismutation of
superoxide anion (O2 -) produced by the mitochondrial com-
plex III. By comparison, hypochlorite has an E of +1.48 V.
On this basis, it is not surprising that, until recently, chemists
have considered ozone a toxic gas that should not be used in
medicine.[11,12] Indeed, there is ample literature and knowledge
showing the toxicity of ozone to the pulmonary system.[13-16]
Although this is correct, it has also led to an incorrect con-
clusion. The surface of the human lungs (about 70 m2) is cov-
ered with a very thin liquid film (0.1 mm)[17] consisting of a small
volume (about 20 mL) of epithelial lining fluid (ELF), which
contains a small amount of antioxidants. It is obvious that
individuals inhaling ozone-contaminated air over a period of
months experience a continuous and cumulative toxicity due to
exposure to many grams of ozone, capable of inducing an
enormous production of reactive oxygen species (ROS). These
compounds cause local and generalized damage, by generating
a local inflammation amplified by the release of lipid oxidation
products (LOPs), and pro-inflammatory cytokines (tumor ne-
crosis factor [TNF]-a, interleukin [IL]-1, IL-6, etc.) released by
an excess of macrophages and neutrophils.[18,19] It must also be
clarified that ozone acts only and always on the surface of the
ELF and never enters the epithelial cells of any tissue, because it
reacts in a matter of seconds and only within the superficial
aqueous film.[20] The damaging effect of ozone, linked to the
generated toxic compounds, has led to the belief that ozone is
always toxic, but this is not true when an appropriate ozone
therapy procedure is followed: in this case, a small volume of
gas mixture composed of oxygen (‡95%)-ozone (£5%) comes
into contact, for only 5 minutes, with an equivalent volume of
blood, which in comparison to the ELF has a far higher anti-
oxidant capacity able to easily quench ozone reactivity. An
extensive review of this topic has been previously published.[21]
Among the numerous approaches classified within comple-
mentary medicine, there are several with obscure mechanisms
of action, perhaps because our current analytical method-
ologies are unable to decipher them. On the other hand, ozone
therapy has been recorded among the bio-oxidative therapies
by the National Center for Complementary and Alternative
Medicine of the National Institutes of Health.[22] It has been
possible to elucidate and to show that, once ozone has dissolved
in the aqueous environment of plasma, it gives rise to a series of
reactions, as shown in figure 1, that are well understood bio-
chemically. Moreover, currently two important paradoxes have
been reviewed:[23,24] the first one is that small amounts of gases
ª 2011 Adis Data Information BV. All rights reserved.
Bocci et al.
Ozone
Aqueous environment of plasma
ROS
(early phase)
LOPs
(late phase)
Blood cells Albumin adducts
Fig. 1. A schematic view of ozone reactivity in contact with blood.
LOPs = lipid oxidation products; ROS = reactive oxygen species.
such as nitric oxide (NO), carbon monoxide (CO), and hydro-
gen sulfide (H2S), produced under physiological conditions and
acting as signaling molecules, are considered essential for life
and are categorized as medical gases, together with hydrogen
(H2) and xenon (Xe).[1,25] The second one is that micromolar,
or even submicromolar, amounts of H2O2 and LOPs such as
4-hydroxynonenal (4-HNE), from long-chain n-6 polyunsaturated
fatty acids (PUFAs), and a smaller amount of trans-4-hydroxy-
2-hexenal (4-HHE)[26] are valued as crucial signaling molecules
capable of switching on cell proliferation and differentiation,
the production of some cytokines, and the upregulation of a
number of antioxidant enzymes such as superoxide dismutase
(SOD), catalase, glutathione (GSH)-peroxidases, GSH-reduc-
tase, and GSH-transferases, and of a very protective enzyme
such as heme-oxygenase-1 (HO-1). It follows that the concept
of ozone as a strong oxidant as well as a therapeutic agent has
been accepted by the scientific community.[27]
The following reaction:
3O2 þ 68; 400 Cal#2O3
shows that ozone in the aqueous environment of blood, where it
dissolves very quickly,[28] releases its energy to hematic com-
ponents, and hence into the body during reinfusion into the
blood donor. Ozone never enters into the circulation because it
is totally consumed either ex vivo in the glass bottle, or after
triggering the reaction on the surface of the skin and mucosae.
Most of the basic chemical reactions involved have been clari-
fied over the last 15 years.[27,29-34] The therapeutic range has
been precisely defined to be within ozone concentrations of
20–80 mg/mL (0.42–1.68 mmol/mL of gas [pure O2: 95% and
O3: 5%]) per mL of human blood. An ozone concentration
below 10 mg/mL of gas per mL of blood (0.21 mmol/mL) is in most
cases totally quenched by the hydrosoluble antioxidants and
therefore no biological effects can be elicited and, at best, the
procedure has a placebo effect only. Owing to the potent anti-
oxidant capacity of blood, due to its hydrophilic and lipophilic
antioxidants and cellular enzymes, a small part of the ozone
Am J Cardiovasc Drugs 2011; 11 (2)
Ozone Therapy in Vascular Diseases
dose dissolved in the aqueous environment is instantly
quenched by free antioxidants (mainly uric acid, ascorbic acid,
GSH, cysteine, and albumin), while the bulk of the ozone reacts
with PUFAs mostly present in the three hydrophobic binding
domains of albumin.[10] Lipoproteins are not targeted by ozone.
The fundamental reaction follows:
-R-CH¼CH-R þ H2 O þ O3 #2 RCHO þ H2 O2
Thus the potential energy of ozone is finally transferred into two
fundamental messengers such as H2O2, as a ROS, and aldehydic
molecules, of which 4-HNE and 4-HHE are the relevant LOPs:
Water of plasma þ O3 #H2 O2 þ 4-HNE þ HHE
Given the high reactivity of ozone, these biochemical re-
actions occur in a few seconds, and, in fact, within 5 minutes
of mixing an average of 200 mL of human blood ex vivo in a
sterile glass bottle with 200 mL of gas mixture (O2 + O3), the
ozone is totally exhausted while the ~95% oxygen, dissolved in
the plasma water, fully saturates hemoglobin:
Hb4 O4-6 þ O2 #Hb4 O8
Blood pO2 up to ~400 mmHg in the bottle is useful, but it is
only of small practical relevance because oxygenated-ozonated
blood is normally reinfused via the venous route during the
subsequent 20 minutes and is heavily diluted with venous
blood. Therefore, ozone represents the therapeutic agent while
the oxygen is only necessary for generating the ozone.
2.1 What is the Fate of H2O2?
Our work[10,35,36] and that of Shinriki et al.[37] has verified
that depending upon the ozone dosage, H2O2 in plasma oxi-
dizes most of the ascorbic acid to dehydroascorbate and about
half of the uric acid to allantoin, while bilirubin remains un-
altered. Whether a trace of GSH is oxidized to a sulfonic acid
remains undetermined and, similarly, so far we have not iden-
tified whether the sulfhydryl (SH) group of albumin is oxidized
to sulfenic acid:[38,39]
R-SH þ H2 O2 #RSOH
During the initial rapid multiple reactions of ozone with plas-
ma components, a variable amount of the ozone is obliterated by
the abundance of hydrophilic antioxidants. However, it is note-
worthy that, with the exception of uric acid, dehydroascorbate
and oxidized glutathione (GSSG) are reduced back to their nor-
mal levels in less than 20 minutes. This is achieved through the
efficiency of the recycling system based on a multitude of reducing
molecules including a-lipoate, vitamin E, thioredoxin, and nic-
otinamide adenine dinucleotide phosphate[40,41] (NADPH), and
involving a well coordinated sequence of electron donations.[42]
ª 2011 Adis Data Information BV. All rights reserved.
75
Most importantly, H2O2, which has no electric charge,
rapidly enters the blood cells, and the chemical gradient be-
tween plasma and cells has been measured, with intracellular
levels being about 10% of the extracellular concentration.[43,44]
In other words, when the highest ozone concentration is mixed
with blood, depending upon the modest interindividual vari-
ability of antioxidant potency (1.28–1.83 mmol/L plasma),[45]
the highest H2O2 concentration measured in plasma is ~40 mM,[46]
and therefore inside the cells is at most 4 mM. The sudden influx
of this small amount of H2O2 into blood cells provides the
essential stimulus to activate a series of biochemical reactions.
2.2 Biochemical Reactions in Erythrocytes
As a first consequence, ozone therapy leads to the activation
of glycolysis with an increase in adenosine triphosphate (ATP)
and 2,3-diphosphoglycerate (2,3-DPG).[27] Functionally, the
sigmoidal oxygen-binding curve of hemoglobin shifts to the right
and increases the release of oxygen at the tissue level. The
erythrocytes mop up most of the H2O2 and promptly reduce it to
water through the action of GSH. The sudden formation of
GSSG alters the GSH/GSSG ratio within the cell. However, this
is rapidly corrected by either expelling some of the GSSG or by
reducing it via GSH reductase at the expense of either ascorbic
acid or thioredoxin, which has two free sulfhydryl groups. This
important recycling process occurs within about 3 minutes.[40]
Alternatively, activation of glucose-6-phosphate dehydrogenase
(G6PDH) provides reducing power and activates glycolysis.[34]
2.3 Biochemical Reactions in Leukocytes
Following ozone therapy, neutrophil phagocytic activity is
enhanced.[27] Inside monocytes and lymphocytes, H2O2 acti-
vates a tyrosine kinase with consequent phosphorylation of
IkB, one of the components of the nuclear factor-kB (NF-kB)
trimeric complex.[47] The phosphorylated IkB detaches from
the complex and is degraded in the proteasome. The remaining
heterodimer p50-p65 is transferred into the nucleus where it can
activate up to ~100 genes. Of particular significance is the final
release of a number of cytokines (interferon [IFN]-g and IL-8)
and acute-phase proteins.[27]
2.4 Biochemical Reactions in Platelets
In response to the ozone concentration, the release of platelet-
derived growth factor (PDGF)-AB, transforming growth factor
(TGF) b-1, and IL-8 have been measured.[48] Growth factors
have specific relevance in the enhancement of ulcer healing in PAD.
Am J Cardiovasc Drugs 2011; 11 (2)
76
It must be said that the H2O2 concentration in the cells
(4 mM) is the minimum necessary to switch on cellular responses,
and it probably lasts for a few seconds as GSH peroxidases,
peroxiredoxin, and catalase promptly reduce it to H2O. In
plasma, the H2O2 half-life is less than 1 minute, and it is absent
during blood reinfusion.[32]
2.5 What is the Fate of Lipid Oxidation Products?
Among a variety of LOPs, newly formed lipoperoxide rad-
icals are rapidly reduced to hydroperoxide, while among a va-
riety of alkenals, the bulk is represented by the fairly stable
4-HNE.[35] This compound is an amphipathic molecule react-
ing with free GSH, carnosine, and mainly albumin. Although
4-HNE is a toxic aldehyde generated by ozone, it eventually
acts as a useful messenger through three processes schemati-
cally indicated as: (1) detoxification, (2) dilution, and (3) ex-
cretion. Specifically:
1. A small amount of 4-HNE is broken down immediately by
enzymes such as GSH-S-transferases and aldehyde dehydrogenase
or by other detoxifying enzymes as described by Awasthi
et al.[49]
2. The bulk is bound to the -SH group of Cys34 present in
domain-I of albumin but also to free GSH. Furthermore, 11
nucleophilic residues present in albumin (including Lys199 and
His146) can also bind 11 molecules of 4-HNE.[39] Thus, because
of the large amount of albumin (~120 g intravascular and
160–200 g extravascular), the bound alkenals undergo major
dilution in the body fluids implying a most important loss of
toxicity. 4-HNE has no membrane receptor but the albumin
adduct can facilitate its transport throughout the body.
3. 4-HNE is also excreted into bile and urine after hepatic
detoxification[50] and renal excretion as mercapturic acid
conjugates.[51] An interesting aspect is that albumin can
transport 4-HNE in all body tissues, from liver to endocrine
glands and the CNS. Thus 4-HNE-Cys adducts can be released
at many sites and inform a variety of cells of a transient, acute
oxidative stress. At submicromolar or picomolar levels, 4-HNE
can act as a signaling molecule,[52] able to activate the synthesis
of g-glutamate cysteine ligase, g-glutamyl transferase, g-glutamyl
transpeptidase, HSP-70, HO-1, and antioxidant enzymes such
as SOD, GSH-peroxidase, catalase, and G6PDH, a critical
enzyme electron-donor during erythropoiesis in the bone
marrow. There is a wide consensus on the relevance of the
induction of protective molecules during small but recurrent
oxidative stress.[53-57] In other words, the concept that a
precisely controlled oxidative stress can strengthen the anti-
oxidant defenses is well accepted today. The biological behavior
ª 2011 Adis Data Information BV. All rights reserved.
Bocci et al.
of 4-HNE is an enlightening example of how the normal serum
level of a potentially toxic aldehyde produced by physiological
peroxidation can activate a number of useful signaling path-
ways.[58]
At the time of ozonated blood infusion, 4-HNE-Cys adduct
can also act on the vast expanse of endothelial cells and, by
stimulating endothelial nitric oxide synthase (eNOS), enhance
the biosynthesis of NO via the 5-electron oxidation of L-argi-
nine. This crucial mediator acts on the enzyme-soluble guanyl-
ate cyclase able to catalyze the conversion of guanosine
triphosphate (GTP) to cyclic guanosine monophosphate
(cGMP), which is the second messenger involved in crucial
physiological processes including the regulation of vascular
smooth muscle tone and inhibition of platelet aggregation,
smooth muscle proliferation, and monocyte adhesion to the
vascular endothelial cells.[59,60] Thus, the enhanced endogenous
formation of NO and S-nitrosothiols is one of the bases of
ozone therapy for preventing and treating hypertension, stroke,
and coronary infarction. Indeed, in our clinical trial in PAD it
was shown to be better than the orthodox infusion of iloprost.[8]
NO, S-nitrosothiols, and a trace of CO released with bilirubin
via the upregulation of HO-1 activity allows vasodilation, thus
improving tissue oxygenation in ischemic tissues. Moreover, an
increased production of NO counteracts the excessive endo-
thelial release of O2 - caused by the chronic inflammation
typical of atherosclerosis.
H2S is endogenously synthesized from L-cysteine by cysta-
thionine-b-synthase (CBS) and cystathionine-g-lyase (CSE).
Interestingly this is another gas, a potentially toxic molecule,
that when released in trace amounts becomes a further im-
portant physiological mediator like NO and CO.[1] At physio-
logical concentrations, H2S seems to modulate the K+-ATP
channel opening involving vascular flow regulation.[1,61]
As far as hydrogen treatment is concerned, it significantly
increases antioxidant enzyme activity, decreases lipid peroxide
levels, and decreases circulating proinflammatory cytokine
levels.[1] The therapeutic potential of H2 in correcting ischemia-
reperfusion damage after either coronary infarction or stroke is
potentially enormous.[62,63]
Similarly, the minimal amount of ozone necessary to trigger
useful biological effects fits very well with the concept of the
xenohormesis theory.[64] Another very interesting aspect ob-
served in 67- to 78-year-old subjects affected by the dry form of
age-related macular degeneration is that a majority of them
report a feeling of euphoria and a sense of wellness and physical
energy throughout the ozone therapy cycle of 14–16 treatments
lasting about 2 months.[30] Whether these feelings are simply
due to faith in the medical treatment, i.e. the power of the
Am J Cardiovasc Drugs 2011; 11 (2)
Ozone Therapy in Vascular Diseases
placebo effect,[65] or are caused by the generated ozone mes-
sengers able to modify or improve the hormonal secretion, is
not yet known. Lack of funding has always prevented per-
forming a study in normal volunteers where, before and after
ozone therapy, the complete hormonal pattern and cycling in
the plasma throughout the 24 hours can be determined. This
study would be very informative and helpful for understanding
why the patients experience a feeling of well-being throughout
the ozone therapy course. In our opinion, this may be due to
improved oxygenation and/or enhanced secretion of growth
hormone (GH), cortisol, and dehydroepiandrosterone (DHEA).
Patients with arthrosis pain have also reported marked im-
provement and less pain that may be due to release of ACTH-
cortisol or to a limited stimulation of COX-2 with enhanced
release of prostacyclin (PGI2).[66] It is also possible that LOPs
reaching the hypothalamic area improve the release of neuro-
transmitters such as serotonin, dopamine, and endorphins as
has been observed after intense physical exercise.[67]
By using the above-reported therapeutic range of ozone
concentrations strictly related to the blood volume, it must be
noted that neither acute nor chronic toxicity has ever been
observed during or after ozone therapy. Such a result seems
unbelievable, but further studies[10,33] performed to evaluate
possible hematochemical or biochemical and enzymatic mod-
ifications have clearly demonstrated that ozone concentrations
of 20–80 (even up 160) mg/mL (of gas per mL of whole blood) do
not damage blood cells or other components. Indeed, during
the ozonation process no more than 35% of the total anti-
oxidant capacity is oxidized and as mentioned earlier this is
reconstituted within 20 minutes in vitro and even more rapidly
in vivo. The evaluation of hemolysis after blood ozonation is
one of the most reliable markers for assessing damage to er-
ythrocytes: it always remains below 1%[33] and actually a small
quantity of free heme is useful for inducing the upregulation of
HO-1.
Is it possible that ozone can oxidize the phospholipids and
cholesterol of the cell membrane? The demonstration that the
negative electric charge of the erythrocyte membrane is un-
modified even after ozonation at 160 mg/mL (of ozone per mL
of blood) excludes this possibility.[33] It is well known that
blood cell membranes are always shielded by a cloud of albu-
min molecules able to prevent any ozone attack, as it may occur
as a result of excessive oxidation or may well occur during
ozonation of saline-washed erythrocytes. Unfortunately too
many artificial experiments performed with saline-washed cells
totally deprived of the plasmatic antioxidants have led to in-
correct conclusions about a direct effect of ozone on the cell
membrane phospholipids.[68]
ª 2011 Adis Data Information BV. All rights reserved.
77
3. The Reliable Route of Ozone Administration
Ozone can be administered with great flexibility but it is of
paramount importance to bear in mind that it should never be
injected intravenously as a gas because of the risk of provoking
oxygen embolism, given the fact that the gas mixture always
contains no less than 95% oxygen. So far, the optimal approach
has been major autohemotherapy (AHT) because, on the basis
of the patient’s bodyweight, a predetermined volume of blood
(from 100 mL up to 225 mL) to which has been added either
sodium citrate 3.8% (1 + 9 mL blood) or heparin (20 IU/mL of
blood) can be exposed to an equal volume of gas (O2– O3), with
the ozone concentration precisely determined by using an
ozone-resistant, disposable 500 mL glass bottle under vacuum.
It is a simple, inexpensive (all the necessary disposable materials
cost about $US12) procedure and it has already yielded ther-
apeutic results in vascular diseases that are superior to those
achieved by conventional medicine.[2-8] Moreover, the ther-
apeutic modalities have been extended: they include the quasi-
total body exposure to O2– O3[69] and the extracorporeal blood
circulation against O2– O3 (EBOO).[8] The latter procedure, to
be used in emergency conditions, is rather invasive because
blood collected from a vein circulates through an ozone-
resistant gas exchanger[70] and, with the help of a peristaltic
pump, returns to the circulation via a contralateral vein. On the
other hand, the partial cutaneous exposure to oxygen-ozone
does not need any venous puncture and, owing to the vast ex-
panse of the skin, allows a generalized and beneficial effect.
Clearly, today we can select the most suitable method for dif-
ferent pathologies, their stage, and the patient’s condition.
A separate discussion is needed for minor AHT, which basically
consists of withdrawing 5 mL of blood to be immediately and
vigorously mixed for 1 minute with an equal volume of O2/O3 at
an ozone concentration ranging between 80 and 100 mg/mL of
gas per mL of blood. The slightly oxidized blood, including the
foam, is promptly injected into the gluteus muscle without the
need for any anesthetic. As a non-specific immunomodulatory
approach, it has been widely used during the last two decades
for successfully treating herpetic infections.[30] A slight degree
of hemolysis (1–2%) is purposefully required because the heme
released in the gluteal muscle will stimulate the synthesis of
HO-1.[71]
4. Which Diseases are Suitable for Treatment with
Ozone Therapy?
On the basis of the mechanisms of action, ozone therapy can
induce the following biological responses:
Am J Cardiovasc Drugs 2011; 11 (2)
78
Table I. Results in patients with peripheral arterial disease after ozone therapy[2]
Outcome Fontaine-Leriche stage
II (n = 62)
Very good improvement (% patients) 87.1
walking distance (m) >1000
Improved (% patients) 9.7
walking distance (m) >400–500
No improvement or progression (% patients) 3.2
 Improvement in blood circulation and oxygen delivery to
ischemic tissue as a result of the concerted effect of NO and
CO and an increase in intraerythrocytic 2,3-DPG level;
 enhancement in general metabolism through improved
oxygen delivery;
 upregulation of cellular antioxidant enzymes and induction
of HO-1 and HSP-70;
 induction of mild activation of the immune system and
enhanced release of growth factors;
 excellent disinfectant activity when used topically, but this is
negligible in the circulation because of the blood antioxidant
capacity; this property is of value in all cutaneous and
mucosal infections and ulcers, especially in the diabetic foot;
 absence of acute or late adverse effects;[72]
 a surprising feeling of well-being, probably through stim-
ulation of the neuroendocrine system.
On this basis it appears that ozone, by acting on many tar-
gets, can indirectly help in recovering functional activities af-
fected by chronic disease and, if this interpretation is correct,
ozone therapy acts as a biological response modifier. Although
ozone therapy is now used in many countries, it is mostly used
by private physicians, and the performance of large clinical
trials has been severely hampered by a lack of sponsors and
disinterest of pharmaceutical companies. All these reasons may
explain why ozone therapy remains little known to orthodox
medicine and why it has not been evaluated in cardiovascular
diseases, which represent the ideal pathology. However, a
number of studies have been performed with results as de-
scribed in the following sections.
4.1 Peripheral Obstructive Arterial Diseases
Even a modest obstruction of limb arteries due to athero-
sclerosis, diabetes, or Buerger’s disease (thromboangiitis ob-
literans) leads to a progressive reduction of blood flow to the
feet. Tissue ischemia and any minor trauma facilitate the for-
ª 2011 Adis Data Information BV. All rights reserved.
Bocci et al.
III (n = 51) IV (n = 39)
70.6 53.8
>800 >500
No pain at rest Gangrene healed
21.6 25.6
>300–400 Amputation of toes with healing of stump
Occasional pain
7.8 20.6
mation of an ulcer, which will not heal because oxygen, nu-
trients, and growth factors indispensable for the repair process
are lacking. This pathology is the most suitable for treatment
with major AHT. According to the Fontaine-Leriche classi-
fication,[73] a patient at either stage II (intermittent claudication
and transitory pain) or stage III (continuous pain, cyanosis,
and possibly initial ulcers) will achieve the best results. Stage IV
includes incipient necrosis of the toes and unbearable pain,
leading to surgical amputation that can be avoided with AHT
in about 50% of cases.[2,3] Table I summarizes a study per-
formed between 1974 and 1980 in 152 patients and shows the
beneficial effects of major AHT.[2] In comparison to pentox-
yfilline and prostanoids (the standard of orthodox treatment),
major AHT has proved to be more effective and without ad-
verse effects in patients with ischemic vascular disease. Several
other small studies[3-7,74-76] have amply confirmed these results.
In a small trial using EBOO, 28 patients were randomized to
receive either their own ozonated blood or an intravenous in-
fusion of prostacyclin.[8] All patients continued conventional
treatment with statins, antihypertensive agents, and platelet
aggregation inhibitors. Ozone therapy proved more effective
than prostacyclin in terms of pain reduction and improvement
in quality of life, but no significant difference was seen in vas-
cularization of the lower limbs between the two groups, most
likely due to the short duration of treatment (14 treatments in
7 weeks). More prolonged treatments have led to a satisfactory
healing of ulcers.[4] It is a mistake to stop therapy too early in
these patients because major AHT, as with other conventional
drug therapies, must be continued, albeit less frequently, for life.
Topical therapy performed with ozonated olive oil is extremely
useful when initial dry gangrene or ulcers are present. The fre-
quency of major AHT depends upon the stage of the disease,
and with stages III and IV it can be carried out every day in an
attempt to prevent amputation. How well major AHT works
appears evident from the fact that the excruciating nocturnal
pain disappears after the first two to three treatments,[2,30]
Am J Cardiovasc Drugs 2011; 11 (2)
Ozone Therapy in Vascular Diseases
indicating an improvement in blood flow in the ischemic tissue
and a lack of ‘stealing’ blood away from underperfused muscle.
5. Studies that may have Compromised
the Future of Ozone Therapy
On the basis of very encouraging clinical results achieved
with correctly administered ozone therapy, the possibility that
ozone therapy may help patients with CHF must be considered
because this is a critical and costly pathology. In the past, we have
treated three CHF patients with EBOO. After some 20 treatments,
they showed a marked and consistent improvement, suggesting
the possible induction of neo-angiogenesis after the mobilization
of staminal cells.[7] However, once again we attempted to under-
take a randomized trial but were unable to cover the cost of the
gas-exchange device and insurance, and the study was stopped.
In the meantime, other clinical trials using a device by
Vasogen Inc. (Mississauga, ON, Canada) have progressed be-
cause this company was able to support these studies in multiple
countries. The outcome of this enterprise may have compro-
mised the progress of ozone therapy, although clinical scientists
seem not to have noticed the terribly harsh oxidation of blood
injected into patients with CHF.
In 1997, Bulmer et al.[77] proposed a new procedure that
involved 10 mL of the patient’s anticoagulated venous blood
(+ 2 mL sodium citrate) being exposed to an oxygen/ozone gas
mixture (ozone concentration 15.35 g/m3) delivered into the
blood at a flow rate of 240 mL/min and UV light (253.7 nm) at a
temperature of 42.5C for about 20 minutes. The treated blood
sample was removed from the system and immediately ad-
ministered by intragluteal injection back into the patient.
Later, the procedure used an expensive device (VC7000A
system, Celacade, Vasogen Inc.) that was able to deliver an
enormously toxic dose of ozone (107.5 mg per mL of blood)
plus a further blood oxidation induced by UV irradiation at
42.5C. The final ozone dose is about 15 000-fold higher than
the average ozone dose used during classic major AHT[78] and
the extremely high oxidation of blood causes a complete de-
naturation of blood components.[10] Two treatments were given
on consecutive days, followed by a third on day 14. Subsequent
treatments were given at 4-week (28 days) intervals for at least
22 weeks, for a total of eight injections. The above-described
procedure was invented with the aim of establishing a non-
specific IMT in the hope of reducing the inflammatory process
and chronic oxidative stress present in vascular diseases. It
proved to be ineffective in AIDS trials[79,80] and in a multicen-
ter, randomized, double-blind, placebo-controlled study in 533
patients with symptomatic PAD, called SIMPADICO (Study
ª 2011 Adis Data Information BV. All rights reserved.
79
of Immune Modulation Therapy in Patients with Symptomatic
Peripheral Arterial Disease).[81] It is important to note that this
trial had to be stopped 3 months early because it did not show
any improvement in PAD and because of a significantly higher
rate of malignancies in the IMT group. Although in a pilot
study[82] of 73 patients with heart failure, the IMT seemed to
result in a reduction in mortality, a subsequent multicenter study
in 2426 patients,[9] called the ACCLAIM (Advanced Chronic
Heart Failure Clinical Assessment Of Immune Modulation
Therapy) trial, in chronic heart failure produced ‘disappointing’
results. In this trial no particular cancer predominated ‘‘although
an imbalance was seen in reports of colorectal cancer’’ (nine
patients in the IMT group and three in the placebo group).
The proponents of IMT[77] support the concept that after
intramuscular administration of heavily denatured blood, a
non-specific immunomodulation ensues with an up-regulation
in the production of anti-inflammatory cytokines such as IL-10
and TGF-b and inhibition of proinflammatory cytokines, such
as TNF-a, IL-1, and IL-6.[9] As CHF and chronic limb ischemia
are conditions linked to inflammation and chronic oxidative
stress, in theory the reduction of proinflammatory cytokines
may down-regulate chronic inflammation and delay the prog-
ress of the disease. However, other studies performed using anti-
TNF-a antibodies have failed to improve outcomes in CHF.[9]
The earlier failure of the SIMPADICO trial not only was an
excellent indication of a poor result in CHF, but also indicated
that a strong immune suppression may favor neoplastic growth.
Sliwa and Ansari[83] pointed out several potential problems but
did not comment on the incorrect oxidative technology used.
On the other hand, specific criticisms regarding the irrational
technology and deep dissensions with Vasogen’s hypothesis
have been published.[84-86]
After two decades of studying the mechanisms of action in
blood,[27] the therapeutic range of ozone as a therapeutic agent
(0.42/1.68 mmol/mL of ozone per mL of anticoagulated blood)
has been well defined. Ozone is a particularly reactive gas but, if
judiciously used, it is very useful in vasculopathies because it
enhances vasodilation, it increases the delivery of both oxygen
and growth factors in ischemic tissues, and it up-regulates sev-
eral antioxidant enzymes (above all HO-I).[87] Immunomodulation
may be only a small additional factor. The misinterpretation of
the real mechanisms of action and the obstinate use of an in-
correct approach can explain the ‘disappointing’ results and the
previous failure in treating chronic limb ischemia in the
SIMPADICO trial. Moreover, several case studies have dem-
onstrated the validity and safety of correctly administered
ozone therapy in severe chronic limb ischemia.[2-8,74-76] At
present, the situation is worse than before but, surprisingly
Am J Cardiovasc Drugs 2011; 11 (2)
80 Bocci et al.

enough, practically no attention has been paid to the fact that Table II. Schedule of ozone treatment in proposed clinical trial for chronic
this clamorous failure was due to an excessive use of ozone. The heart failure
Vasogen procedure was very irrational, not only because it used No. of Ozone Total blood Total ozone
an enormous and toxic dose of ozone, but also because it added treatments concentrations volume as well as dose (mg)
an ambiguous UV irradiation of blood further heat-stressed at (mg per mL gas volume (mL)
of gas mixture)
42.5C for 20 minutes, resulting in the blood being so ‘cooked’
that had to be immediately injected. 1st and 2nd 20a 200 4.0
(week 1)
3rd and 4th 30 200 6.0
6. A Schematic Proposal for a Clinical Trial (week 2)
in Patients with Chronic Heart Failure 5th and 6th 40 200 8.0
(week 3)
This section describes the proposed design for a trial of
7th and 8th 50 200 10.0
ozone therapy in patients with CHF. Patients eligible to enter
(week 4)
the trial would be those classified within the New York Heart
9th to 32nd 60 200 12.0
Association (NYHA) Class II and III heart failure with a left-
a Such a low concentration is useful to avoid an initial excessive
ventricular ejection fraction (LVEF) of 30% or less, and who oxidative stress.
have been hospitalized or received intravenous drug therapy for
heart failure within the previous 12 months. The control group shows that oxygen/ozone protects the heart from acute myo-
will be treated with the best orthodox medications, while the cardial infarction and supports the need for performing a
experimental group will also receive ozone therapy, according clinical trial as described here.
to the classic and well-standardized procedure of major AHT.
Briefly, 200 mL of blood plus 22 mL of 3.8% sodium citrate will 7. Concluding Remarks
be exposed in an ozone-resistant glass bottle and gently mixed
for 5 minutes with 200 mL of a gas mixture composed of oxygen In this review, we aimed to draw attention to a rational bio-
and ozone precisely collected in a 50 mL syringe from a precise oxidative therapy, the biological reactions and effects of which
ozone generator with a direct photometric control. By consid- have been evaluated and proven to be valuable when tested in
ering the severity of the clinical conditions, it is proposed that PAD patients. We further aimed to stimulate the established
each patient receiving major AHT will undergo two treatments medical community to evaluate the potential value of ozone
every week for 4 months (i.e. a total of 32 treatments). The first therapy according to the 2008 Declaration of Helsinki. How-
two treatments will be performed with a low ozone concen- ever, it is regretful that several clinical trials to date that have
tration (20 mg/mL) to avoid excessive oxidative stress during the involved approximately 3000 patients have been performed
first week and will then be progressively increased up to using an irrational methodology. Millions of people suffer from
60 mg/mL for treatment after the first month. In other words, chronic limb, brain, and heart ischemia, which collectively
each patient should undergo the auto-reinfusion of a total represent the major cause of death worldwide. Such pathologies
of 6400 mL of ozonated blood over a period of 4 months. have a huge socioeconomic impact, particularly in the devel-
Although this is a minimal size trial, the number of AHT oping world. If the appropriate ozone therapy could be im-
treatments for 40 patients adds up to as many as 1280 auto- plemented in every public hospital, patients may have a better
transfusions, including 1280 sterile glass bottles of 500 mL each. prognosis and an improved quality of life.
The schedule of the ozone treatments is reported in table II.
Biochemical and instrumental tests and assessment of typi- Acknowledgments
cal end-points such as the comparative evaluation of morbidity, No funding was used in the preparation of this article. The authors have
mortality, ejection fraction and chronic inflammation para- no conflicts of interest that are relevant to the content of this article.
meters will be carried out before starting treatments, while final
results will be evaluated during the first 2 weeks of the fifth References
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Correspondence: Prof. Velio Bocci, MD, Emeritus Professor of Physiology at
the University of Siena, Italy, Department of Physiology, University of Siena,
Via Aldo Moro 2, 53100 Siena, Italy; Prof. Valter Travagli, PharmD, Associate
Professor of Pharmaceutical Technology and Chief of the Graduate School of
Hospital Pharmacy at the University of Siena, Italy, Department of Phar-
maceutical Chemistry and Technology, University of Siena, Via Aldo Moro 2,
53100 Siena, Italy.
E-mails: bocci@unisi.it; travagli@unisi.it
Am J Cardiovasc Drugs 2011; 11 (2)