CHAPTER24

OZONETHERAPY IN VARIOUS PATHOLOGIES

"Natura inflrmttatts humanae tardiora sunt remedia quam mala " Tacitus (-54-120 A.D.), The Life ofGnaeus Julius Agricola, Chapter 3 (From the nature ofhuman frailty, remedies work more slowly than illnesses) What Tacitus asserted remains true even today with modem medicine, although he could not know that some diseases (atherosclerosis, cancer) take years to set in. The title of the 14th Annual Euro Meeting (March 5-8, 2002, Basel) is "The Patient is Waiting", meaning that "At the start of the 21'1 century only a relatively small proportion ofall diseases can be adequately treated or even cured and only a small proportion of all patients have access to medicinal products at affordable prices". Although this seems a pretty harsh judgement, it is true that for some diseases we do not yet have a rational drug treatment, that diseases are often poorly treated , and that too many patients in poor countries do not receive any benefit or even worse are the prey of quacks . However, we can forget neither the value of antibiotics and vaccines nor the long effort to fulfil Paul Erhlich's dream of the magic bullet, which , in spite of enormous expenses, has so far yielded meagre results and remains amirage. I often complain that basic and clinical research on ozonetherapy has been too little and too slow . Yet, therapeutic progress in cancer therapy has also not been as fast and positive as had been predicted. Three decades have already passed since President Richard Nixon dec1ared war on cancer and, although knowledge about tumorigenesis has shown an incredible expansion, the mortality rate has barely decreased. I very much hope that the new generation of drugs, like the specific inhibitor 2-phenylamino pyrimidine (STI-571), which precisely targets the AbI tyrosine kinase in chronic myeloid leukemia cells, lives up to expectations and does not disappo int us as several potential miracle eures have done (Gorre et a1., 2001). Indeed there always appears to be unforeseen toxicity observed during prolonged treatment, as occurred for the HAART (Hruz et al., 2001 ; Fellay et a1. , 2001) which in 1996 seemed to have resolved the problem ofHIV disease. All the hype during the last decade about research on gene therapy of tumours and more recently therapeutic angiogenesis (Isner et al., 1996; Patterson and Runge, 2000; Simons, 2001), once again obtained at huge cost, has indeed generated new knowledge and allowed authors to publish interesting papers in the best medical journals. Yet, all ofthis has , so far, yielded only minimal practical results and even a few deaths . Recent developments in autoimmune diseases have highlighted potentially useful new therapies with an anti-TNFa monoclonal IgG 1 antibody and a protein made of two chains ofp75 TNF receptor monomer fused to the Fe domain of IgG 1 (Hanauer

243

V. Bocci, Oxygen-Ozone Therapy © Springer Science+Business Media Dordrecht 2002 244 CHAPTER24 and Oassopoulos, 2001). However, it remains uneertain whether they are simply able to alleviate symptoms for a while or, more importantly, to modify the course of these diseases. If I believed aneedotal results of ozonetherapy, obtained with minimal resourees and manpower, I would say that perhaps ozonetherapy is not as bad or obsolete as orthodox medicine depiets it. Yet rather than resting on false laureIs, I would incite ozonetherapists to work hard and seriously; the sooner we clarify the validity of ozonetherapy the better it will be for everyone. As will be discussed in this chapter, the medical applications ofozonetherapy are innumerable (Table 19) and this fact exposes the approach to medical derision. Is it possible that ozone acts as a panacea or the ill-famed Theriaca? Aecording to tradition, Andromachus (who was Nero's quack) invented Theriaea, a very complex mixture able to neutralize poisoning and eure every illness! Indeed, he wrote a poem in 175 verses (Oe Theriaca) to describe and praise it.

Table 19. Ozonetherapy is used in thefollowing medical spectalities.

Angiology Gerontology Oncology Cardiology Gynaeeology Orthopaedies Cosmetology Hepatology Pneumology Oentistry Infectivology Rheumatology Oermatology Intensive therapy Surgery Gastroenterology Neurology Urology

The answer is a decisive NO and only a superficial observer or a sareastie skeptic would say YES : In reality, ozonetherapy seems to exert beneficial effeets in so many, and somewhat unrelated, pathologies beeause ozone aets at abasie level on several blood eomponents with different funetions. Moreover, the generated ROS and LOPs not only have the most powerful disinfeetant aetivity but ean aet either loeally or systemieally in praetieally all eells of an organism. The most important ROS are listed in Chapter 9 (Table 6), while only the most signifieant LOPs (we know very little about them as ozone effeetor molecules) are mentioned in Chapters 9 and 10. Target cells vary from bacteria, fungi, parasites to blood and all eells ofthe body. The reason why I decided to continue working in this controversial field is that I believe, in contrast to the erroneous (that is only my opinion!) axiom that "ozone is toxie any way you deal with it", that oxygen-ozone, if properly used, can act contemporaneously as a disinfectant, an oxygen donor, an immunomodulator, an inducer ofantioxidants (this is a real paradoxl) a metabolie enhancer and perhaps as an activator of resident stern cells . It is difficult to figure out how beneficial effects can develop during ozonetherapy, but Figure 90 attempts to give a visual image of how this can happen. The centre of action is always the plasma, where ozone dissolves and generates all the effector moleeules that interaet with erythrocytes (ER), BMC, granulocytes (GRAN), platelets (PLAT) and the Endothelium. All of OZONETHERAPY IN VARIOUS PA THOLOGIES 245

these cells will be more or less affected depending on the ozone concentration and the ozonization modality, which in any case must be absolutely risk-free.

Figure 90. The multivaried biological response ofthe organism to ozonized blood can he envisaged hy considering that ozonized hlood cells and compounds interact with a number of organ s. Some ofthese represent real targets (liver in chronic hepatitis, vascular systemfor vasculopathies), while other argan s are prohahly involved in restoring normal homeostasis

This aspect is important to understand the consequent effects that will be directed to twelve different organs or functional sectors. The plasma and the extracellular fluid are the media interconnecting the ozone generated compounds, cell released moleeules (cytokines, growth factor, autacoids, PAF, metabolites, etc.) and a multitude of target cells. Therefore, the ozonetherapeutic stimulus simultaneously triggers many functional activities, thus allowing a multivaried therapeutic response, but also prornotes a calculated, transitory oxidative stress which, unlike chronic endogenous oxidative stress, has the value of a therapeutic "shock". As an exaggerated example, somewhat like the dreaded 246 CHAPTER24 e1ectroshock that we used to inf1ict on schizophrenic patients when I was a medical student! In our case , the difference is substantial as we do not harm the patient and yet we induce one of the most important body responses, i.e. upregulation of the antioxidant system which is far more than the simple adaptation to COS (Fig . 88). If this is true, and we can prove or disprove it experimentally, ozone may stimulate and correct the natural resources gone astray, in accordance with the old saw "Medicus curat, natura sanat" (The physician treats, nature heals). There is a profound difference between modern orthodox medicine and ozonetherapy. On the basis ofbiochemical, physiological and pharmacological data, the former tries to develop a specific drug able to correct the cause of the disease, and if this succeeds the result is wonderful. Unfortunately this does not always happen or the success is not complete because a disease generally causes many dysfunctions and the use of a reductionist approach is inherently disadvantageous. Although ozonetherapy has limited specificity, it has the considerable advantage that "the hodgepodge of ozonized products" can mobilize natural resources that eventually may resolve the overall problem. With this, I am not saying that ozonetherapy is preferable; my deep feeling is that, in any circumstance, we must first offer the patient the best treatment that orthodox medicine provides. Only ifthis does not work, or has serious side effects, or the patient does not comply with it, can we offer the option of ozonetherapy if it is applicable to the disease.I would also take this opportunity to reject the term "alternative" because ozonetherapy, as empirical as it is today, cannot be antithetical but only complementary. This is the correct term. It would certainly be a wonderful achievement if, one day, official medicine would state that "for this disease. ozonetherapy is the therapy (4' choice", This would mean that ozonetherapy has gained respect but, I guess that plenty of water will pass under the bridge before that day arrives. In spite ofimportant advances, conventional medicine is still unable to provide a definitive improvement in some pathologies. Thus it is reasonable and ethically correct to take advantage of ozonetherapy when the best orthodox treatment has failed. As typical examples, I will report treatment of a) suppurative infections refractory to all antibiotics, b) III and IV grade hind-limb ischaemia facing amputation, and c) chronic hepatitis patients either resistant to or intolerant of IFNa. Occasionally, I may only touch upon the etiopathogenesis of a disease; the reader can fare better by consulting Harrison's "Principles of Internal Medicine", Nevertheless, the ozonetherapist must know all the pros and cons of all conventional therapeutic modalities before suggesting the use ofozonetherapy. If they have been unsuccessful or are unavailable, ozonetherapy can then be considered.

l. INFECTIOUS DJSEASES (BACTERIAL, VIRAL, FUNGAL, PARASITIC)

It is intuitive that ozone can have an important therapeutic role in various types of 0 infections because it generates ROS (02 ', OH', HzOz, NO' and HOCI), also produced by granulocytes and macrophages during an infectious process (Badwey and Kamowsky, 1980; Chanock et al., 1994; Anderson et al., 1997; Saran et al., 1999; OZONETHERAPY IN VARIOUS PA THOLOGIES 247

Titheradge, 1999; Babior, 2000) . Moreover, neutrophils have a wealth ofantimierobial proteins in their granules and release eytokines and autaeoids whieh, by exerting a variety ofeffeets, eause tissue damage as weil (Witko-Sarsat et al., 2000) . We observe that, owing to diffuse antibiotic-resistant baeteria, rieh eountries continue to use often useless, expensive antibiotics, while poor countries, by sheer necessity, use ozone whieh is quite aetive and so far has hardly induced resistance. Ozone is profitably employed either as agas (02 + 0 3), which must be weil contained in an ozone-resistant bag saturated with water vapour, or as ozonized saline (to be used only topieally), or better as ozonized bidistilled water, or as ozonized oils for the treatment of war wounds, anaerobie infections, trophic ulcers and bums (Miroshin and Kontorshikova, 1995). Abscesses, anal fissures, deeubitus (bed sores), fistulae, fungal diseases, furunculosis, gingivitis, inveterate osteomyelitis, peritonitis, sinusitis, stomatitis, vulvovaginitis and wound healing disturbances have been shown to improve rapidly beeause ozonized solutions display a c1eansing effeet and act as a powerful disinfectant, whieh kills even antibiotic-resistant or anaerobic baeteria. On the whole, ozonized solutions eontrol the bleeding, improve the metabolism and reduee the infection (Payr, 1935; Aubourg, 1940; Rokitansky, 1982; Werkmeister, 1995; Shaschova et al., 1995; Filippi and Kirschner, 1995; Wasser, 1995; Bulinin et al., 1995; Kudravcev et al., 1995; Kasumjan et a1. , 1995; Steinhart et al., 1999). In poor countries (I repeat), by sheer necessity, physieians have had to devise all sorts of ways to employ the gas, or more easily the ozonized water, to avoid environmental contamination. In Western countries, we still need to create the mental attitude to profitably use ozone. Yet, I am eonvinced that, once medieal personnel realize the advantages, it will be put into general use, to the benefit of patients. Moreover, with the eurrent inerease in medieal costs, ozonetherapy deserves attention because it reduces hospital assistance and is extremely cheap. Obviously we will need to explain how ozone works and show what ozone concentrations are appropriate for the particular infeetion or lesion. The sehe me reported in Figure 71 (Chapter 16) shows that a concentration of 80 ug/ml (as gas) can be used only during the first phase, in which there is pus, bacteria and necrotie tissue. The wound must be cleaned and exposed to the gas for only 10-15 min. Bidistilled water ozonized with 80 ug/ml has an effective content ofabout 20 ug/ml 0 3 and is far more practieal for cleansing the wound and changing the moisted compress throughout the day . Ozonized oil can be applied for the night. As the infection regresses, ozone concentrations must be lowered to 2-5 ug/ml to avoid cytotoxieity and to activate local metabolism, cell proliferation and synthesis of cytokines (POGF, bFGF, TGFß I, EGF, KOF), so as to promote synthesis of the intercellular matrix and the healing process (Beck et al., 1993; Pierce et al., 1995; Sporn and Roberts, 1993; Schmid et al., 1993; Slavin, 1996; Martin, 1997). Topical treatment is easy to perform because daily observation ofthe wound is a good guide; however, it helps to know that time, patience and complianoe are good allies . The problem is more complex in systemic infeetions (peritonitis, large abscesses, pleural empyema), possibly complieated with toxie and septic shock. Removal of purulent material and rapid washing with ozonized water is useful , particularly combined with 0 3-AHT whieh can be earried out 1-3 times a day at low 0 3 248 CHAPTER 24

concentrations (20-30 ug/ml per ml ofblood). OJ-AHT is intended to improve tissue oxygenation and metabolism but not to increase production of pro-intlammatory cytokines, which are already superinduced by toxins. It is also not intended to sterilize blood: although bacteria and viruses suspended in water are sensitive to ozone, they become fairly resistant in plasma beeause of the proteetion exerted by endogenous antioxidants. Direet IV injeetion of gas, similar to the sterilization of drinking water in a aqueduet, is simply a mad idea and is proseribed. OJ-AHT (2-3 sessions weekly) must eomplement topical treatment of uleers and wounds beeause the synergism leads to more rapid healing. I will briefly deseribe three eases that have eonvineed me that ozone has great therapeutic aetivity in suppurative infeetions: • The first ease was reported by Dr. Salvatore Mieeli, of the Hospital at Corleone (Palermo), during the I st Ozonetherapy Meeting in Palermo (Mieeli, 1999). The patient was a woman, 51 years old, born with a eleft spine and sueeessive paraplegia of the hind limbs.A previous infected deeubitus probably eaused osteomyelitis of the right coxo-femoral joint with a fistula releasing a foul­ smelling seeretion (Fig . 91 a,b; Fig. 92) . For about six weeks, she was treated intensively with several wide-spectrum antibiotics to no avail ; in the meantime, she developed septie fever (-39 Oe), progressive caehexia, lethargy and profound debilitation. With her permission, ozonetherapy was carried by : 1) 0 3-AHT (30 ug/rnl per ml of blood) every day during the first week and then onee weekl y, and, most importantly, 2) repeated direet insuftlation (via a polyethylene catheter introduced as far as it could go) of 02-0J at a concentration of 40 ug/ml every 5 min for 1 hour. After only about 12 hours, the fever receded and purulent secret ion ceased. Loeal gas treatment was then repeated every day for one week and then twiee weekly for three months, when total resolution of the osteomyelitis was shown radiologieally (Fig . 93). The general condition of the patient returned almost to normal shortl y after ozonetherapy, without antibiotie support. After six months (Fig . 94), the patient was perfeetly healthy. • Another ease of osteomyelitis was referred to me but this eolleague had no radiological records. It appeared in a multiple myeloma patient und er eytostatie therapy at the level of implantation of a eoxo-femoral prosthesis. The physieian also used the dual approach: 0 rAHT twice weekly, but laeking a fistula he repeatedly infiltrated the area surrounding the lesion with 50 ml of gas at a coneentration of 20 ug/ml thrice weekly. He elaimed to have resolved the problem after 5 weeks. Osteomyelitis can easily become a ehronic infeetion and I feel that the timely use ofozone should be weil kept in mind . • The third ease was a 67 year-old woman in dialysis at our Hospital since 1977 . The problem started with an initial deeubitus in the eoceygeal area . In spite of conventional therapy, the infeetion spread and she presented an extensive necrotizing faseiitis in both legs and at the site of primary deeubitus (Fig. 95 ). Antibioties and con ventional loeal therapy carried out by a dermatologist were ineffective and the patient worsened, with septie fever and a semieomatose state . Ozonetherapy was carried out in the Nephrology Unit , using the combined OZONETHERAPY IN V ARIOUS PATHOLOGIES 249

treatment. In this case, it was the EBOO approach three times weekly in conjunction with intensive local therapy consisting in the continuous change of compresses soaked in freshly ozonized water during the day, substituted with ozonized oil at night. The therapeutic activity of ozonized oil is simply unbelievable. The patient returned practically to new after two months . I personally followed this case and I was amazed at her rapid recovery (Di Paolo et al., manuscript in preparation).

Figure 91. (June 14. 1999). a)Radiograph ofthe rightfemoral head and iliac bone: the aspect ofthefemoral neck indicates the presence ofan osteomyelitic process. b) The external aspect ofthefistula 250 CHAPTER 24

Figure 92. (June 14. 1999) Radiographie image 0/the osteomyelitic process visualized after injection ofradiopaque material via a catheter. The catheter was also used to insufflate ozone into the purulent cavity

Figure 93. (September 25. 1999) After Figure 94. (April 11. 20(0) The topical ozone application and OJ-ART. the radiograph shows an intense radiograph shows a striking improvement osteometaplasia with disappearance of and a markedreduction ofthe abscess. the abscess cavity and resolution of'the There are signs ofosteoblastic osteomyelitis hyperactivity OZONETHERAPY IN VARIOUS PATHOLOGIES 251

Figure 95. The amazing results obtained in one patient with necrotizingfasciitis treated with parenteral (EBOO) and topical (ozonized water and oil) treatments. Extensive necrotic lesions were present between the buttocks , Oll the legs and heels. Before (left) and after (right) the treatment 252 CHAPTER24

1.1. The Special Case 0/Helicobacter Pylori (Hp) Hp is a gram negative, microaerophilic bacterium that is acquired in childhood; it infects the stomach of about 50-80% of children and remains for life (Rowland, 2000) . In about halfof subjects, Hp may cause peptic ulcer disease, chronic gastritis and possibly gastric adenocarcinoma and gastric B cell lymphoma. Hp thrives in the acid environment of the stomach by activating its own cytoplasmic urease, which

converts urea into CO2 and ammonia (NH) . The ammonia neutralizes the gastric acid that otherwise would diffuse into the periplasm and kill the bacterium or inhibit its colonisation. A protein denominated Ure I, a member of the amidoporin family, has the crucial role of allowing up to a 300-fold passage of urea into the cytoplasm of Hp (Weeks et al., 2000). Tombola et al., (2001) have also shown that the cytotoxin VacA increases the transepithelial flux ofurea. There is evidence suggesting that Hp infeetion is associated with inflamed gastric mucosal epithelium accompanied by a marked local enhancement of CD4' Th I-type response. This may explain why even eradication of the infeetion does not always improve the dyspeptic symptoms. However, the current therapeutie approach aims at eradicating the infection, which is possible in about 80% of patients after treatment with omeprazole (a protonic pump inhibitor) and a combination of two antibiotics chosen among amoxicillin, clarithromycin and metronidazole (the latter two seem most effective). However, there are already problems of bacterial resistance, lirnited patient compliance, high costs and the Iikelihood of reinfection about I month after discontinuing the treatment. It appears to me that Hp colonisation is a prototypie case in which the use of ozonetherapy would be ideal: the bacilli are localized in the deep portions of the mucus gel layer and in between this layer and the apical surfaces of the gastric epithelial cells. They do not invade the mucosa and are rarely situated between adjacent epithelial cells. Thus, there is nothing better than ozone and the ROS caseade to transitorily create a hostile environment to Hp, since it is sensitive to ozone . I would Iike to propose a protocol investigating the effeet of ozonized water and ozonized oil on the model already used in experiments on Cryptosporidiosis and Giardiasis. It may be sufficient to ingest a teaspoon of ozonized oil in the moming on an empty stomaeh, followed by two-three glasses (200-300 ml) of freshly ozonized water (final 0) concentration about I0 ug/ml) I hour before breakfast. The oil is a bit disgusting but the water is easily drinkable. I would suggest continuing this treatment for four weeks and then repeating all the tests (Hahn et al., 2000), possibly the non-invasive ones (Hp antigens in faeces, urea breath test, whole blood antibody tests) during the exploratory phase . A possible substitute of ozonized oil is to drink a glass of ozonized full cream milk rieh in PUFAs. It may weil have a similar effect and it is more palatable. Is there any danger? No adverse effects have been reported in children with Giardiasis. Moreover, ozonized water is promptly distributed over the large mucosal surface and reacts instantaneously with mucoproteins, which at least partly neutralize it. Hp bacilli are fully exposed to the oxidative stress and will be eliminated. Obviously the enthusiastic patient should not exceed in drinking too OZONETHERA,PY IN VARIOUS PATHOLOGIES 253 much ozonized water to avoid the loss of cytoprotective mucus, with the risk of insulting epithelial cells. There is a practical disadvantage since the patient must take freshly prepared ozonized water or perhaps milk from the hospital pharrnacy at least every other day (for four weeks). Ozonized oil is quite stable in the refrigerator. I have no doubt that an effective vaccine will eventually be developed; this mayaiso solve the problem in poor countries where Hp infection is widespread.

1.2. Viral Infections (HIV-1 Infection) Most lipid-enveloped viruses in aqueous media are sensitive to ozone because it easily oxidizes glycoproteins and lipoproteins of the external envelope (Akey and Walton, 1985; Shinriki et al., 1988; Vaughn et al., 1990; We11s et al., 1991; Carpendale and Freeberg, 1991). However, the virucidal activity becomes less certain when viruses are in biological .fluids or, even worse, when they are intracellular (hepatocytes, epithelia, CD4+ lymphocytes, monocytes, glial and neuronal cells, etc.), where the potent antioxidant system protects viral integrity. This emphasizes once again the irrationality ofdirect IV injection ofgas. Moreover, by means of some viral components, e.g. HIV-1 trans-activator oftranscription (Tat protein), HIV and HCV are able to inhibit or downregulate the synthesis of antioxidant enzymes such as SOD and GSH-Px. This induces an intracellular chronic oxidative stress (increase of0/', OHO), which favours viral replication and, by accelerating cell death, enhances expansion of the disease (Ho, 1997). There are unequivocal experimental data (Westendorp et al., 1995; Oe Maria et al., 1996; Ranjbar and Holmes, 1996; Schwarz , 1996: Akaike et al., 1998; Larrea et al., 1998; Romero et al., 1998; Rubartelli et al., 1998) that fully agree with the fact that an excess ofNAC, GSH and cystamine suppresses in vitro HIV replication (Roederer et al., 1990; Kalebic et al., 1991; Bergamini et al., 1994), while a GSH deficiency impairs survival (Herzenberg et al., 1997). The increased release of extracellular Tat, associated with circulating IFNa, also suppresses immune ce11 activation and inhibits the production ofC-C chemokines , leading to immune co11apse (Zagury et al., 1998). Since 1993, the mass media have misinforrned the public, boasting that ozonetherapy could eure HIV infection . I was so perplexed about these rumours that I wrote to Dr. A. Fauci and I appreciated his concern that ozone could promote further oxidative stress in HIV patients (Chapter 2). The spreading offalse, sensational news is a typical but reprehensible propensity of complementary medicine, particularly ozonetherapy, favoured by quacks to exploit anguished patients and by some producers and salesmen ofozone generators. In the period 1993-95, the epidemie was mounting, AZT monotherapy was hardly useful and yet the wenderful results with ozone had not been published in scientific journals, except for a pilot study of its safety and efficacy by Garber et al. (1991). Unfortunately, this work was very badly conceived: although it showed neither efficacy nor toxicity, it could not support any valid conclusion and I am still puzzled as to how it was accepted for publication. Can you imagine the scientific validity of 254 CHAPTER24

a test using 10 rnl ofblood (HIV infected) treated with an unknown 0 3 concentration plus heat (?) plus irradiation with IV (?) and then reinjected IM. In 1994, I feit as if I was between the devil and the deep blue sea: many patients at the hospital refused AZT and other therapies and solicited me to perform ozonetherapy. Garber's study was uninformative but news from Germany was c1aiming exceIlent results and I wondered what was true. I must confess that even in these days, when I receive news that ozone (direct IV injection or hyperbaric 0 3 !!) works weIl, I get confused and I start to wonder ifI am mistaken. I had mixed feelings when I tried to evaluate the pros and cons (Bocci , I994a, b, 1996a) in order to elaborate a rational approach: I) By oxidizing the viral gp 120 or gp41, ozone may inactivate some free viruses in plasma, but the required concentration is between 40 and 80 ug/rnl gas per ml ofblood. 2) If it were true that infected leukocytes have a decreased content ofantioxidant enzymes, we might even induce their death, but this could lead to further viral dissemination. In any case, the ozonization of 250 mI blood (about 1/20 of the blood mass) would have only a negligible impact on the total viral load. 3) It remained uncertain whether the free inactivated viruses may either induce tolerance or may act as an endogenous immunogen and/or as an activator of cell-rnediated immunity. While an increase of antibodies is hardly helpful, the activation of cytotoxic T Iymphocytes (CTL) could be. 4) By acting on BMC, ozone may stirnulate the production of irnmunoregulatory cytokines; in 1994, we hoped that CD8 + T Iyrnphocytes present in long-term survivors might either release the phantom cell antiviral factor (CAF; Walker and Levy, 1989) or Th l -type cytokines, such as IFNy and IL-2, to block the shift towards the production of Th2-type cytokines (IL-4, 5, 6, 10) (Clerici and Shearer, 1993, 1994). At that time, we did not know that CTL could release ß-chemokines (MIP-Ia, MIP-Iß and Rantes) , which by binding to the second receptor (CCR5) on CD4 4 T Iymphocytes (the first receptor is known as CD4) impede the infection of ceIls by HIV-I (Cocchi et al., 1995; Feng et al., 1996; Alkhatib et al., 1996; Deng et al., 1996; Zagury et al., 1998). On the other hand, release of GM-CSF and TNF-a may have increased viral replication and accelerate the progression of the disease (Pemo et al., 1989; Mellors et al., 1991). 5) It was difficult to predict if improved oxygenation and activation of rnetabolism could have exerted a prevalently beneficial or negative effect. 6) One great hope was the possibility of inducing the adaptation to COS and I don 't think that the daily oral antioxidant supplement could inhibit the process. 7) Activation of psychosomatic factor might have been helpful but, at the same time, an increase of ACTH-cortisol release with a reduced DHEA secretion may have enhanced imrnunosuppression (Clerici et al., 1994; Corley, 1995). 8) A pitfall was that in 1995 we were using PVC bags for autotransfusion, which we now know may cause immunosuppression and PVC toxicity, to the patients' disadvantage. In July 1995, we began a trial on 10 patients (8 men, 2 wornen), aJl of them also HCV positive. The patients had CD4 + T cell counts of about 260 cells /ul and an average plasrna HIV-I RNA level of 138,000 copies per ml. After a few sessions, the women refused to continue owing to emotional stress and one man was very depressed and gave up because his girlfriend had left hirn. OZONETHERAPY IN V ARIOUS PATHOLOGIES 255

They were between 26 and 37 years old and, after talking with them often, I realised how unhappy and strained they were. They were very grateful for what we were trying to do and occasionally when I apologized for an imperfect venous puncture, they were most kind and said: don 't worry , we have been so stupid to inject and drug ourselves so many times , throwing away our lives, that you are always perfect. Obviously I wished very much that the treatment would be beneficial, but in any case their gratitude was so sincere that I rarely feit more rewarded. The patients had never been treated, because they refused AZT and other complementary therapies, and they signed an informed consent form for ozonetherapy. The trial ended in February 1996 and three patients underwent as many as 54 OJ-AHT, receiving an overall ozone dose of 1080 mg evenly distributed in 16.2 I of blood. Although the study analysed a limited number of patients, repeated measurements of relevant virological markers indicated that ozonetherapy carried out with great care neither improves nor worsens the dynamics of HIV-1 replication. CD4+ lymphocytes slightly increased (p=0.066) from 272±99 to 341±133. Therapy was stopped in one patient after two months because the viral load in plasma showed a marked increase. Plasma HIV-I DNA remained stable (-57,000 copies/Iü" CD4) and HIV-I RNA levels also remained practically unvaried, except in one case . Serum ß2-micro-globulin increased significantly, possibly as a result of OJ-AHT-mediated immunological enhancement. Analysis of the three long-term ozone-treated patients at week 24 confirmed sustained CD4 counts and a stable viral load . While in the lay press there have been many undocumented claims that OJ-AHT is effective in HIV -I infection, we could not document any substantial advantage, even though no patient reported side effects, haematology parameters remained stable and some patients reported a feeling of well-being and a decreased incidence of oral candidosis and herpes labialis. The full report has been published (Bocci et al., 1998c). In any event, against the most pessimistic predictions of distinguished scientists, ozonetherapy did not harm the patients and it is possible that the adaptation to COS induced by ozonetherapy countered the COS established by the virus . Indeed in two patients, we measured a significant increase oferythrocytic SOD after 4 and 5 OJ-AHT (Bocci, 1996a). Even in these days, I continue to ask myself if I was wrong in selecting the ozone concentration (-68 ug/ml per ml blood), or the schedule, or the use of PVC bags or what else? I also very much regret that I was unable to retrace these patients and see how they fared, but the physicians in charge at the hospital did not bother to help me. Needless to say, I have often been solicited to perform 03-AHT in the occasional patient, but the ID unit at the Polyclinic is not willing to perform a study. One good reason is that HAART (no venous punctures) can be done at horne and is usually very effective. This therapy has been able to inhibit HIV-1 replication, resulting in undetectable levels of free viruses in plasma in about two-thirds of patients for at least 3 years, and thus has been a great success (Pomerantz, 1999; Gulick et al., 2000 ; Montaner and Mellors, 200 I) because it has reduced morbidity and mortality. Unfortunately, the initial hope to totally eradicate the virus has not come true because the virus remains hidden in resting CD4+ T cells and in sanctuaries (Chun and Fauci, 1999); as soon as HAART is stopped, plasma viremia becomes detectable in about 3 weeks (Chun et al., 1999). The benefit of complementing the therapy 256 CHAPTER24

with SC administration of IL-2 (Levy et al., 1999; Davey et a\., 2000) or with the promising option of "structured intermittent therapy" (Ruiz et a\., 2001) remains to be assessed, but it is now eertain that eontinuous HAART is toxie (Hruz et a\., 2001 ; Fellay et a\., 2001), diffieult to adhere to and very expensive, even for Amerieans (Steinbrook, 2001) . Does it make any sense today to think that ozonetherapy eould help HIV patients? My answer is: yes and no! No, if we want to substitute HAART with ozone . The former is in eontinuous evolution and there is great hope of having even more potent and less toxie drugs, thus redueing treatment failures due to the induetion ofresistanee or poor eomplianee (Weller and Williams, 2001) . Despite the news I reeeive from quaeks, I am eonvineed that ozone eannot match HAART in removing HIV from the plasma, when we know that blocking viral replication is a fundamental step. There is no need to eomment about the belief that HIV is not the erucial eulprit. However, ozonetherapy may be useful as a eomplementary therapy for the following reasons: a) Now, with the new option of BOEX (or at least RI), we have a praetieal , inexpensive and above all non-invasive approach (no venous puncture or risk of infeetion). b) Using a gradual increase of ozone eoneentrations (from low to medium : 20-40 ug/rnl), we may aehieve: • adaptation to COS, hence a re-equilibration of the eellular redox state, which is a fundamental proeess for inhibition of HIV replication; • eorrection of hyperlipidemia and peripheral lipodystrophy . With the EBOO proeedure, we have already eorreeted two serious eases ofMadelung's disease; • a eorreetion of the wasting syndrome instead of administering reeombinant GH and DHEA (Murphy and Longo, 2000) ; • a feeling ofeuphoria, counteraeting asthenia and depression.

The same objeetives can be aehieved using EBOO or OJ-AHT (in very preearious patients, even using allogeneic AHT with LD blood), but these approaches are technically more eomplex , invasive and more expensive. It is not yet elear what will be the most profieient strategy for intermittent therapy, i.e. HAART either on a monthly or a seven-day-on-off sehedule, with ozonetherapy performed during the periods ofHAART interruptions. I would Iike to elose this section by offering my enthusiastic eollaboration to anyone seriously interested in eondueting a eontrolled study . I cannot do it here beeause I have neither funds nor support . Yet I would bet that official medicine will disregard my offer and eontinue to test IL-2 and hormones because , eoneeptually, the injection of drugs is preferred. Nonetheless, I insist in saying that if we want to assess whether ozonetherapy has any value, we must eonduet appropriate studies in eollaboration with expert infectivologists, virologists, pathologists and statistieians. OZONETHER..-\PY IN VARIOUS PATHOLOGIES 257

1.3. Chronic Hepatitis (HBV, HCVand HDV) It has been estimated that 300 million people in the world are chronically infected with HCV and about 2 million live in Italy. Chronic hepatitis diseases are somewhat less dramatic than HIV but are certainly very serious ailments from a socio-economic point of view. In Italy about 80% of infected people have a chronic disease but, luckily about 60% remain with a mild infection. On the other hand, 20% develop an aggressive disease leading to cirrhosis and liver cancer with some 20,000 deaths yearly. Current knowledge about the potential usefulness of ozonetherapy is inconclusive. In the recent past, I failed to elicit the interest of five of the best Italian hepatologists. Because of my inability to produce experimental evidence or asound rationale, they all dedined my request to perform a RCT using OrAHT. Is there any valid reason to justify the study?

1) The demonstration of a virucidal effect against HAV in vitro (Vaughn et al., 1990) has little relevance in vivo because hepatitis viruses are more resistant to ozone than HIV and they may be protected by the plasma antioxidant capacity. 2) The oxidation ofviral particle components during blood ozonization may generate an inactivated and immunogenic vaccine, but this idea remains hypothetical. 3) The induction of cytokine synthesis (IFNß, IFNy, IL-4, IL-6, IL-8 and TNFa) in ozonized blood incubated in vitro has been demonstrated (Bocci and Paulesu, 1990; Paulesu et al., 1991; Bocci et al., 1993a,b, 1994b, 1998b). Yet the amount of cytokines released is small and the evidence that this happens in vivo upon reinfusion of ozonized blood is limited to the demonstration that the Mx protein is induced in leukocytes (Bocci et al., 1994a). It is unknown whether the ozonization process leads to activation of CD4+ helper T cells, (CD8 +) CTL responses (Cemy and Chisari, 1999; Lechner et al., 2000) and NK cells . We also do not know whether infected hepatocytes increase the expression ofMHC class II on the membrane, which is an important co-signal for their identification and subsequent clearance. In the past, it was claimed that ozonetherapy increases IgG production (Washuttl et al., 1988), but there is no clear evidence of increased antibody-dependent cellular cytotoxicity. The possible involvement of Kupffer and Ito ceIls (O'Farrelly and Crispe, 1999) with infiltrating CD4+T lymphocytes and CTL in destroying infected hepatocytes remains another possibility. Obviously, if the cellular immune response is weak because of insufficient quantities of cytokines, or because the heterogeneous viral population evolves rapidly and becomes IFN-insensitive, or the immune system becomes tolerant to HCV antigens, the infection is not eradicated and becomes chronic (Bendinelli et al., 1999; Cemy and Chisari, 1999; Farci et al., 2000) . 4) We always say that OrOJ therapy improves oxygenation and hepatic metabolism, but this is more a cliche than a proven reality. Indirect, but rather tenuous, evidence of this has been discussed in Chapter 19 (Rectal insufflation): after OrAHT or EBOO, fibrinogen and prothrombin plasma levels tend to normalize. 5) It is weIl documented that hepatic viral infections induce a COS, to the advantage ofthe virus, and the release ofcytokines (TNFa and TGFß) enhances the process (De Maria et al., 1996; Schwarz, 1996; Romero et al., 1998; Akaike 258 CHAPTER24

et al., 1998). In line with this ftnding is the fact that administration of NAC (plus IFN) reduces the plasma level of transaminases (Larrea et al., 1998). The local release of TGFß is doubly deleterious because it inhibits the synthesis of MnSOD (while IL-I and IFNy increase it) and enhances hepatic fibrogenesis (Poynard et al., 1997; Poli and Parola, 1997). It can be hypothesized that prolonged ozonetherapy may be able to reverse the COS by upregulating antioxidant enzymes in the liver. This would be an interesting and positive result, but it remains to be demonstrated. It is also unknown whether ozonetherapy can induce the release of growth-stimulating factors (mainly hepatocyte growth factor, HGF, and TGFo.) and/or growth-inhibitory factors to regulate liver homeostasis during infections, which implies continuous destruction and regeneration ofliver tissue (Fausto et al., 1995; Ankoma-Sey, 1999).

1.3.1. 1s There Any Clinical Evidence That Ozonetherapy is Useful in Chronic Hepatitis? This is a sore point and it is shameful that neither in Germany nor in Italy have we been able to produce serious documentation. In practice, we are still at the stage of vague, insignificant oral communications by Dorstewitz, Konrad , Mattassi (1985) and Knock et al. (1987) who reported "more than satisfactory results" (?) in patients with chronic HBV infection. The only diagrammatic example of treatment, reported by Viebahn (1999) as a "diamond", is shown in Figure 96. --- Bill .------GOT ---y-GT

200 150 ~:r! ;1t'lJj!! !~ !j1 j! 1J

100 2 \ I I

'J I,r \ ' 50 I " ".,, '\

25/08 24/09 15/10 29110 04/12 04/02 27/05 28/06 1980/81

Figure 96. In the ozonetherapy literature, there is only this diagram showing the improvement 0/three markers in a patient with chronic HB V hepatitis. 21 DrAHT were perform ed between August 25. 1980 andJune 1981. The bilirubin (BILl) level increased during theflrst month and then declined to normal values (Viehahn. 1994). OZONETHERAPY IN VARIOUS PATHOLOGIES 259

Apparently after 21 0 3-AHT carried out discontinuously in about 9 months, there was normalization of the levels of two hepatic enzymes and bilirubin. Apparently two to five 0 3-AHT were already effeetive in reducing HCV RNA levels in plasma (Yamamoto et al., 1996); these results are at variance with our data. The 0 3eoneentrations used are uneertain, but a famous German ozonetherapist onee told me that either 20 ug/ml or sometimes 100-150 (?) ug/ml produce "good results" and "patients fee! mueh better". Any eomment appears superfluous. Regretfully, we also have done very little. As I mentioned previously, my seminars in Rome , Florence, etc. proved useless. Eventually, I was lueky enough that Dr. Giuseppe Amato, one of the most re!iable ozonetherapists, who works at the hospital at Conegliano Veneto, agreed to evaluate our protoeol in the period 1996-98. We planned to evaluate the treatment of ehronie HCV patients with 0 3-AHT: 250 rnl blood eolleeted in ACD using at first an 0 3 eoneentration of40 ug/ml per ml blood (total dose : 10 mg) twiee weekly for 5 months (about 40 treatments). One big problem at that time was that we were using PVC bags and the protoeol was stopped around Fall 1998 when we realized the risk of the release of plastic mieroparticles and phthalates. Of twe!ve patients, only nine received the full treatment while the other three reeeived between 17 and 31 treatments. However, the results were very disappointing: none of the nine patients showed any normalization of hepatie transaminases, while the viral load was not measured. At least no side effeets were reeorded. It was then decided to begin a study (June 1999) with new patients using the atoxie system (giass bottles) and testing the 0 3 eoncentration of 70 ug/ml per rnl of blood (225 ml) eollected in Na citrate (25 ml) to prevent any risk of plate!et aggregation (total 0 3 dose : 15.75 mg). Fifteen patients were treated but only 14 were evaluated beeause one woman withdrew after the third treatment. The schedule followed was somewhat unusual: three 0 3-AHT weekly for the first 3 weeks and then onee a month for a total of 20­ 21 sessions. The sehedule had to be adjusted aecording to the hospital labour supply! In addition to the initial sampie prior to therapy, 13 sampies were colleeted per patient (about every month) to evaluate liver enzymes, namely serum aspartate aminotransferase (SGOT), alanine aminotransferase (SGPT) and y-Glutamyl transpeptidase (GGT), and the viral pattern (anti-HCV, HCV-RNA-PCR, anti-HBs, anti-HBC, anti-Hbe, HbeAg, HbsAg) beeause four patients were also HBV positive. Figure 97 shows that all three hepatie enzymes deereased progressive!y and were eventually within the normal range (the statistical signifieanee between the pretreatment values and the last values was p

does not overcome the problem of lack of funding to pay for laboratory exams and medical personnel and we are now at astandstill. 120 .,------, 100 - \ ···..··SGOT .., \ - ·SGPT . -~, 80 .,...... " --GGT ...... , 60 "­ " .'.•.. '-. .... -----~ 40 ...... ---- .--~~ ~. -. :::- ....-...... & _-~---- .. .. "': 20 • '* '* ..

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Sampie number

Figure 97. The pattern oftransaminase plasma levels ofpatients with chronic Hel' hepatitis treated with OrAHTthroughout one year. Ordinate: lU/mi enzymes. All three decreased levels are statistically significant (p<.O.01) (Amato et al.• 2000)

I must mention that, at the Siena congress, areport was presented by Luongo et al. (2000) regarding 0 3-AHT in 82 HCV patients. They c1aimed to have monitored and recorded the redox potential of cell membranes (?) with a non-invasive method for each patient so that, on the basis of the ongoing measurements, they could modify and optimize the 0 3 concentration. This was not specified but presumably ranged from 0 to 60 ug/ml, according to the redox potential. My comment was that the results sounded too good to be true, but the proof of the pudding is in the eating. Several types of the redox potent ial apparatus appear often in the field of complementary medicine; while their real validity remains questionable, the fee of the "physician" goes up. Nonetheless, I have the duty to summarize their data: a) 30% of patients initially presented between 250,000 and 1,000,000 HCV copies/ml. b) 40% of patients initially presented between 1,000,000 and 5,000,000 HCV copies/mI. c) 20% of patients initially presented between 5,000,000 and 7,000,000 HCV copies/ml. d) 10% of patients initially presented between 7,000,000 and 10,200,000 HCV copies/ml The HCV-RNA was quantitatively assessed by the Reverse PCR method. OZONETHERAPY IN V ARIO US PA THOLOGIES 261

The amazing results are that 99% of the 82 patients treated for 3 to 6 months (schedule unknown) showed a more than 80% reduction of the viral load, with 9 patients achieving negativity between 4 and 16 weeks. Biochemical and histological responses were not mentioned. Patients previously pretreated with IFN or diabetic subjects were particularly resistant to the treatment and this is not surprising. I very much hope that this group will publish these data in an international, peer­ reviewedjournal, because, iftrue, they will represent a significant advancement. In conclusion, ozonetherapy may be useful in chronic HCV hepatitis, but firm evidence ofthis is stilliacking. Therefore, the refusal of orthodox hepatologists to test this approach is comprehensible. It is more so because, since the 1980s, IFNs (particularly u type) have proved to be useful, although not always resolutive. Indeed even after intensive 6-12 months therapy, up to 50% of patients may show a good clinical response, but about 1/3 of them soon relapse. Particularly during the first month of therapy, most patients report significant and well known side effects, which partially recede later on. Moreover, elderly patients may show a worrisome depressive state (Bocci, 1988a; Spat-Schwalbe et al., 2000; Malaguarnera et al., 200 I; Musselman et al., 2001 ). About 20% of patients do not tolerate IFN therapy, while about 70% experience acceptable side effects and a lucky 10% have no problems. Patients with extensive liver fibrosis or thrombocytopenia or anaemia have to be treated with great caution. Resistance to IFN is frequent in older patients, in those with a highly chronicized disease or with high serum viral load or with HCV genotypes land 4 (while genotypes 2 and 3 are favourable), in men more than women, in black patients, in alcoholic or immunodeficient (HIV) patients, or in hepatitis complicated by cryoglobulinemia, vasculitis, membranoproliferative glomerulonephritis, arthritis, etc. (Johnson et al., 1994). Hundreds ofgood studies (Dusheiko, 1995; Hoofnagle and Bisceglie, 1997) have been performed, mostly using IFNu, and to make a long story short, IFN is now considered the treatment of choice. The use of Peg IFNu-2a (with a very long half­ life, so that only one dose per week is required to maintain effective levels in the blood) in combination with ribavirin (1.0-1.2 g a day) for at least six months appears effective in about 40% of patients, although half of them may continue to have viremia once the treatment is stopped. (Heathcote et al., 2000; Zeuzem et al., 2000; Manns et al., 2001). Ribavirin, an oral purine nucleoside analogue, is modestly effective on its own, but it seems to develop asynergie effect with either IFNu alone or Peg IFNu (Reichard et al., 1998; McHutchison et al., 1998; Poynard et al., 1998; Andreone et al., 1999; Cummings et al., 200 I; Younossi et al., 200 I). Ribavirin may induce haemolytic anaemia, which occasionally is severe enough to require the discontinuation oftreatment. Even though Peg IFNu-2a is a "retard" IFN, it induces adverse effects similar to those with the unpegylated counterpart. Chronic HBV infection affects more than 350 million people worldwide, Like HCV infection, it carries the risk of developing cirrhosis and, once viral DNA has been integrated into hepatocytes, liver cancer. The therapeutic aim is to stimulate a valid immune response, which with time may lead to viral clearance and reduce liver 262 CHAPTER24

inflammation, necrosis and cancerogenesis in vivo . Orthodox medicine is now providing new effective therapeutic strategies based on IFNa, whieh has anti viral and immunomodulatory properties, and several nucleoside/nucleotide analogues, name1y lamivudine, famciclovir, adefovir dipivoxil, etc., which inhibit HBV polymerase. Vaccines and antisense oligonucleotides complete the armament, whieh is promising particularly because it combines drugs with different mechanisms of action (Boni et al., 1998; Dianzani, 1999; Pianko and McHutchison, 1999). The optimal dose of IFN is somewhat contentious, but usually 8-10 megaunits three times a week for 4-6 months e1icit a positive response in about 40% of patients, as documented by lack ofviral replication and improvement of liver histology. What about the remaining 60% of patients? Clearly the remarkable scientifie progress in this area must be translated into improved clinical praetices for all patients, particularly those with the associated delta agent who are at a high risk. In reality some hepatitis patients seek complementary treatments: an interesting review has evaluated the effects and toxicity of frequently used herbaI produets while ozonetherapy was not even mentioned (Seeffet al., 2001) This digression has two purposes: the first is to inform the ozonetherapist of the state of the art, because she/he has the duty to inform the patient thoroughly about IFN therapy. The second is to point out that orthodox rnedicine reeeives plenty of funding from national agencies and multinational pharmaeeutical industries, which are interested in developing drugs to recover their investment and making a profit. In comparison, ozonetherapy is like an ant to an elephant: no funding , no laboratories, no clinics and total disorganization; the gap is too big to be closed. Moreover, there is another huge disadvantage: IFN therapy is intrinsieally expensive, but the patient, once instructed, can do it casily at horne and visit the hospital every three months for acheck up. In eontrast, except for the very empirieal RI, OrAHT, EBOO and BOEX requ ire a day hospital and expensive (for the National Health Service) medical assistance. Because it is performed privately, the patient must bear the financial burden out of his own pocket. Moreover, medical personnel are hostile to both ozone and the handl ing of infected blood. Thus, although it is likely that ozonetherapy represents a money-saving approach, we cannot evaluate the costlbenefit ratio beeause the benefits have not yet been demonstrated. In spite of enormous difficulties, I still believe that we should clarify whether ozonetherapy has some merits. This can only be done by RCTs , comparing OrO.. therapy against O2 alone, because the relevance ofspontaneous remissions must be c1arified. The most suitable and practical methods are I) OJ-AHT; 2) RI and 3) BOEX, but we could start with the first one and then proceed depending on the results. Among ehronic hepatitis diseases, we eould examine hepatitis C with defined HCV genotype, possibly without any previous treatment beeause of refusal of IFN. Patients should be of both sexes, between 30 and 50 years old . Informed eonsent is needed. OZONETHERAPY IN VARIOUS PATHOLOGIES 263

1.3.2. Proposal ofa Protocol The most practical schedule seems twice a week (M and Th or Tu and F): 225 ml blood in 3.8% Na citrate (25 ml) plus 225 ml O2alone or 02-0J. Use of citrate instead of heparin may reduce ozone's effectiveness but avoids possible complications due to dyscoagulation and potential formation ofminiclots.

ISI week: 30 ug/ml for a total OJ dose of6.75 mg per treatment, 2nd week: 40 ug/ml for a total OJ dose of9.00 mg per treatment, 3rd week: 50 ug/ml for a total OJ dose of 11.25 mg per treatment, 4lh week: 60 ug/ml for a total OJ dose of 13.50 mg per treatment, t h week: 70 ug/ml for a total OJ dose of 15.75 mg per treatment, to be continued for 24 weeks (48 sessions) unless a problem arises. Therapy may be continued once a week during the second semester depending on the results. Patients should take the usual daily.oral antioxidant supplement. Evaluation of therapeutic effectiveness should consider the following end-points: a) permanent serum HCV RNA clearance, tested with the most precise system . Viral load should be assessed before treatment, after 3 and 6 months therapy and then after a further 3 months . b) normalization of hepatic biochemistry (SGOT , SGPT, GGT, bilirubin levels). Test as in (a). c) liver histological results, whenever possible before and 3 months after the 6­ month course . If liver biopsy is refused, a surrogate test to indirectly evaluate liver fibrosis may be used. Moreover, in addition to all the routine biochemical tests, TAS, TBARS and PTG should be measured every 3 months. Of particular interest is the evaluation of cholesterol, LOL, HOL, albumin, fibrinogen, prothrombin and CRP.

Patients with HIV, autoantibodies, autoimmune hepatitis, hypergammaglobulinemia, haemochromatosis, liver metastasis, incipient cirrhosis, extrahepatic manifestation of HCV infection should be excIuded. Treatment is obviously cost-free and control patients have the right to be treated with 02-0J after the first semester. This switch-over might actually be interesting to clearly demonstrate the role of ozone. It would be very important to have the results of this study and I would be glad to collaborate with anyone seriously interested in conducting it. If the results are clearly negative, we should forget about ozonetherapy. If they show that at least 50% of patients are good responders, ozonetherapy could be useful in patients who do not tolerate IFN, in elderly patients particularly sensitive to psychotic effects, in hepatitis C patients with normal serum aminotransferase levels but with viremia (Hirsch and Wright, 2000), in patients after liver transplantation and in patients who cannot afford the cost ofIFN. As the current best conventional combination (Peg IFNa-2a with ribavirin) is not entirely satisfactory, it could be supplemented with one OJ-AHT treatment per week, which may reduce the severity of adverse effects and enhance immunoactivation. 264 CHAPTER24

Moreover, as I doubt that a brief course of ozonetherapy can reduce the viral load (as observed with HIV infection), we could test a hybrid approach: firstly, knock down the viral load with a short (1-2 weeks) intensive treatment with IFNa (Neumann et al., 1998) or IFN-ß (Ikeda et al., 2000) followed by 0 3-AHT according to the schedule described above. ladmit that these may be hopeless speculations. Yet, for many reasons, hepatitis C is a very heterogeneous and difficult disease, for which ozonetherapy might be useful in complementing conventional therapies to achieve a favourable outcome. Finally, a sort of minor plasma autotherapy can be envisaged and utilized for priming and activating natural immunity: it may be worth while to evaluate a protocol based on repeated, weekly IM injections of 1-2 ml autologous plasma heavily ozonized to produce a viral immunogenic vaccine.

1.4. Herpetic 1nfections and Herpes Zoster Herpes simplex viruses (HSV-I and HSV-2) cause human infections involving mucocutaneous surfaces, the CNS and possibly visceral organs in immunosuppressed patients. HSV-I is mostly responsible for causing oral-facial herpes, but it can spread to give an herpetic eye infection that may lead to corneal blindness. HSV-2 is mostly responsible for lesions on the genitalia, and it recurs petiodically. HSV infection of the finger (herpetic whitlow) usually represents a complication of oral or genital herpes. Although these infections are usually limited, their frequent recurrence compromises the patient's quality oflife (Arvin and Prober, 1997). Current antiviral chemotherapy is prevalently based on systemic (oral andJor IV) administration of nucleoside analogues: aciclovir, famciclovir and valaciclovir. These drugs are not always effective because ofaciclovir-resistant strains . Control of HSV infection may be achieved by a vaccine, which has been late in coming and has showed effectiveness only in women not previously infected with HSV-I (Stephenson, 2000) . A promising therapy for genital herpes is the local use of a gel containing an immune response modifier called resiquimod, which is able to stimulate antibody and cytokine production (Bishop et al., 2001) . Herpetic cheratitis can be treated with ophthalmic IFNa or IFNß plus aciclovir. Herpes zoster (HZ), or shingles, is a distressing disease affecting about 1% of the over-60 population. It is caused by the varicella-zoster virus, which remains in a quiescent state in the nerve root ganglia after recovery from chicken pox. The virus may be reactivated during an immunosuppressive state. It causes a unilateral dermatomal, vesicular rash associated with severe pain. The frequency of location is: trigeminal (16%), thoracic (50'%), cervical (14%) and lumbar (12%) dermatomers. If the disease goes untreated, the pain can last for months and can be complicated by post-herpetic neuralgia (PHN). This complication is rare in young middle-age patients (30-50 years) but is frequent in elderly patients. PHN should be prevented by intensive therapy as early as possible. Unfortunately, the incidence of this complication increases with age and with immune depression. It seems that OZONETHERAPY IN V ARIOUS PATHOLOGIES 265 microinfusion of anaesthetics via the peridural route, initiated no later than I week from the appearance of the cutaneous exanthema, may reduce the incidence and minimize the pain . By blocking the axonplasmatic transport, local anaesthetics can prevent diffusion of the HZ virus to neurones in the spinal cord, thus reducing neuronal death and the consequent allodynia and abnormal sensations. The anti­ epileptic, gabapentin, is widely used, but is not always effective. The sooner an appropriate treatment is started, the better. Prophylaxis in patients over 60 and at risk has been partially accomplished by the administration ofspecific zoster immune globulin (ZIG) or by shingles vaccine (NIAID, Bethesda, USA, 1999). Antiviral chemotherapy is based on aciclovir, valaciclovir or, probably even better, famciclovir with or without prednisolone (Wood et aI., 1994, Whitley and Roizman, 200 I), but they have little effect on the healing of skin lesions or pain. The use of corticosteroids is controversial: although they reduce inflammation, they inhibit healing and enhance immunosuppression, which is exactly what favours the virus. Administration of amitryptline (25 mg for 3 months) seems to reduce the pain (Dworkin, 1999). Taking drugs continuously can reduce or suppress herpetic infections, but it is expensive, can cause adverse effects and induce viral resistance. This is what official medicine offers today, but it cannot necessarily satisfy all patients. Although these diseases are not deadly, they are serious and we must foster a sense ofconcern and the need ofbetter therapeutics. As this book is aimed at critically evaluating ozonetherapy, I have the duty to report the usual litany of wonderful results obtained with ozonetherapy. The problem is that these results have been reported at best in an abstract form, are very difficult to trace and analyse, and can be considered anecdotaI. It seems that Mattassi (1981 , 1983) treated 20 patients, of which ll presented herpes simplex and 9 had HZ. I believe the patients were treated with 5 to 12 IV injections of 02-03! After a few injections, all patients overcame the infectious episode and only a few had a recurrence over several years. None of the patients had side effects. Mattassi (1981) stated that results were incredibly rapid and that to be successful the therapy should be started as soon as the lesion appears. Dr. J. Delgado, of the Center of Medical and Surgical Research in Havana, treated 15 patients suffering from HZ with daily IM infections ofOr03 and topical applications ofozonized sunflower oil. He noted a marked improvement after a few days and alI patients were cured after two weeks, without showing any relapse. He concluded that "the low cost, the easy ava ilability and simple application made ozonetherapy the treatment of choice".I believe that Dr. H. Konrad works in Sao Paulo (Brazil); he has reported (1995, 2001) that 03-AHT was effective in both herpetic infections and was able to minimize the complication of PHN evaluated in 55 patients. I think it is important to report the experience of Dr. Giuseppe Amato, already cited for the study performed in chronic HCV infection. I believe that bis work in treating PHN patients is outstanding; it has been performed at the Hospital "DeGironcoli" at Conegliano Veneto in Italy during the last decade. Although this is an open study, it is praiseworthy and regards 180 patients (84 men and 96 women) between 40 and 85 years ofage : 266 CHAPTER24

• age 40-50 : 30 patients (16.7%); • age 51-70: 60 patients (33.3%); • age 71-80 : 54 patients (30%) ; • age 81-85 : 36 patients (20%);

The location ofHZ was as folIows:

• Ocular region: 18 patients. • Head, neck and anns: 30 patients. • Thorax : 30 patients. • Lumbar region: 48 patients. • Limbs : 54 patients.

Patients always arrived at the hospital with some delay when previous physicians feit unable to deal with the intense pain of acute HZ infection. Evaluation of pain was carried out with the visual analogue scale (VAS). On the basis of previous experience, Dr. Amato decided to abandon all conventional medication and examine ozonetherapy associated with the microinfusion of anaesthetics (usually 12 ml of marcaine at 0.25% daily) mostly via the epidural route to block the sympathetic system in relation to the dennatome presenting the cutaneous rash. The concomitant use of two therapies or the lack of a control is usually open to criticism, but in the case of PHN it had be done for ethical reasons in order to reduce the pain. Dr. Amato proceeded systematically to perfonn: a) 03-AHT (150 ml of blood collected in Na citrate and a total ozone dose of 10.5 mg or 70 ug/ml) every day for 4 consecutive days and then every other day for 2 weeks (at least 10 treatments). b) Local treatment using compresses moistened with ozonized water during the day. The applicatlon of ozonized oil at night is effective and most irnportant. c) Sympathicolysis ofthe stellate ganglion or other ganglia at various levels .

Owing to the fact that patients below 50 years rarely develop PHN, they underwent only ozonetherapy. Pain disappeared after 2-3 days (i.e. after 2-3 0 3­ AHT) and the exanthema also improved very rapidly. Three patients (out of 30) developed PHN after 2 months and they were promptly treated with anaesthetics. However, in the subjects over 50 (150 patients), Dr. Amato believed it ethically correct to practise both therapies on a prophylactic basis, because they are at areal risk ofdeveloping PHN. Anaesthetic treatment was perfonned daily for no more than 10 days at the level ofthe stellate ganglion and for no more than 20 days in other locations . . On average, pain disappeared after the first application:

• for about 12 hours in 36 patients. OZONETHERAPY IN VARIOUS PATHOLOGIES 267

• for about 18 hours in 114 patients. • and for 24 hours in 30 patients.

After 3-4 days, the pain disappeared in about 90% of patients; although further treatment seemed unnecessary, it continued for up to 20 days in order to prevent PHN later on. All patients were followed for 2 to 5 years : of 99 patients older than 50 and treated as indicated above in the first week, only 12 developed mild PHN that was successfully treated with both therapies . Of the remaining 51 patients treated with a delay longer than one week, the percentage increased and was in relation to the delay. In conclusion, it appears that the combination of ozonetherapy with anaesthetic intervention is most effective in preventing PHN in patients older than 50. In view of the difficulty of managing PHN, the results appear impressive. By sheer necessity, they lack controls (02 only) and, in this regard, I must report another surprising study. Olwin et al. (1997) found that minor AHT (10 ml of blood not treated with O2-03 or O 2) was effective in eliminating clinical sequelae in 8 of 12 (66%) patients with thoracic HZ, in 9 of9 (100%) patients with ophthalmic HZ and in I with lurnbar-thigh HZ. They claimed (data not presented) that IFNa., IFNß and IL-4 levels were increased in the patients within 24 hours after the IM blood injection. They also mentioned that another 25 cases ofherpes infections ofvarious types yielded favourable results, noting that the rate of success depends on early intervention. A delay of 2-13 months between the first symptoms and treatment yields negative results. As this report originates from reliable institutions (Rush Presbyterian St. Luke's Medical Center and Life Seiences Department, IIT Research Institute, Chicago, USA), the data ought to be true. If they are, they partly support Amato's data; yet they totally refute the value of oxygen-ozone, Moreover, if they are true, Health Authorities and official Medicine have the obligation to verify them: irrespective of the skepticism toward ozone, it is outrageous that we intoxicate HZ patients with expensive, but modestly effective, pills when a few trivial injections of autologous blood into the patient's buttock could relieve awful pain in 2 to 8 days. However, authoritative scientists and clinicians obviously do not bother to believe, or to read, papers published in the Journal of Alternative and Complementary Medicine and prefer to administer expensive drugs. This is even more reproachable because simple AHT is an old medical practice (Maddox and Back, 1935; Hardwick , 1940; Martindale and Capper, 1952); even I performed it in 1953 when I was an intern in Clinical Medicine! If dermatologists and neurologists, as weil as general practitioners, ever read this section, they might think that everything I have written is false and it would be better to forget everything. I have no direct experience, but I fully trust Dr. Amato and I am inclined to believe that, bearing in mind that orthodox medicine does not offer much to highly distressed patients, we should attempt to prove or disprove the validity of either simple AHT or 0 3-AHT. As soon as I fmish writing this book, I will revive my proposal of a protocol evaluating herpetic infections and HZ, which in 1999 was refused by the Professor of Dermatology . In spite of everything , I am still optimistic that many physicians are not aware of ozonetherapy and that they may be willing to try it. I am certainly ready to help anyone. 268 CHAPTER24

In Chapter 16, I extensively discussed the approaches of the so-called "rnajor" OrAHT and "rninor" OJ-AHT. If venous access is lacking, we can use the option of RI or BOEX . Minor AHT, without or with 0 2-0J, is in my opinion an interesting immunoenhancer approach and it is easy, simple, inexpensive and rapid to perform. Starting with a low dose and gradually increasing it, we can ozonize 5 ml blood (30 ug /rnl and upward) followed by IM injection three times a week, and then slow down as soon as the lesions are healed and the pain is gone . Local treatment is also important and effective when combined with OrAHT. It can be performed easily by applying and repeatedly changing a compress moistened with ozonized water (or ozonized oil at night) . Vaginal or rectal suppositories of ozonized oil can be employed in genital-anorectal herpes . We must try to start the treatment as soon as cutaneous lesions and pain appear; the viral reactivation should be suppressed as soon as possible because it reduces the PHN complication. It appears necessary to alert all GPs to send HZ patients to the special PHN unit at the hospital as soon as they make the diagnosis.

1.5. Papillomavirus Infections (HPV) HPV infects the epithelium of skin or mucous membranes and may produce warts, or benign and malignant neoplasias. Common warts (Verrucae vulgaris and plana) may be present in children, while plantar warts (Verrucae plantaris) are painful and fairly common in young adults. The incidence of venereal warts (Condyloma acurninatum) has risen, particularly in women, and represents a common sexually transmitted disease (Cannistra and Niloff, 1996). Viral genotypes 6 and 11 carry a low risk and may cause modest dysplasia of the uterine cervical epithelium, known as cervical intra-epithelial neoplasia (CIN I). Viral genotypes 16, 18, 31, 33 and 35 are more carcinogenic and can induce a CIN II or the more severe form, CIN rn (Liaw et al., 1999). Laryngeal papillomas are typical of children and may produce life-threatening airway obstruction. Anogenital warts (venereal warts) can reach monstrous proportions and may be associated with cervical cancer. Effective conventional therapies include cryosurgery, surgical excision and ablation with a laser. Topical treatments with antimetabolites and podophyllum preparations are scarcely resolutive because the virus is widespread in the basal cell layer and persists if the immune system is unable to destroy infected cells. The use ofboth IFNa and IFNß have been successful for laryngeal papillomatosis and partly useful (30-40% response) in preventing venereal HPV recurrences even after prolonged treatment (Friedman-Kien et al., 1988; Kirby et al., 1988; Weck et al., 1988; Bocci et al., 1990). Both the cost and the adverse effects of IFNs reduce the compliance. The fact that HPV infection is an important risk factor for carcinoma is weil known and several HPV vaccines are undergoing trials. However, ozonetherapy could be useful as a complementary therapy. To the best of my knowledge, there are no data, but it may be worthwhile evaluating a protocol in the hope of eradicating cervical-vaginal infections. Therapy should combine parenteral approaches, such as major or minor OrAHT, RI or BOEX, with local treatment. After the basic surgical treatment, always important to remove the bulk of infected tissue, there are several OZONETHERAPY IN VARIOUS PATHOLOGIES 269 possibilities: one is intralesional injections ofsmall volurnes ofOr03 (from 10 to 20 ug/ml) . The infiltrating injections of gas must be done slowly and with great care, possibly at the base of the wart; as reported for IFNß, they are painful and the patient may get discouraged. Intravaginal insufflation of Or03 (concentration: 30­ 50 ug /ml) for a few seconds is more acceptable, as noted during treatment of bacterial and fungal vaginitis. Instillation of ozonized water (final 0 3 concentration - 20 ug/rnl) for 5-10 min can be done at home and application of an ozonized oil pessary every night is practical and certainly far less expensive than an IFNß gel. The benefit of ozonetherapy remains to be ascertained, but there is no risk, no side effects and certainly a low cost. The possibility of minimizing viral shedding, thus reducing the potential of transmission to sexual partners is not a trivial advantage.

1.6. The Common Cold The well-known manifestations of the common cold, i.e. rhinorrhea, nasal congestion, lachrymation and sneezing, are commonly experienced each year. Sore throat, malaise and headache are also frequently present. Although the common cold resolves without sequelae in 4 to 9 days, it is a very bothersome infection. Normal individuals do not need particular treatment, but immunosuppressed patients are at risk ofpulmonary infections and can be prophylactically treated with IFNa or IFNß. Enormous funds have been spent in the hope that a few applications of IFNs sprayed at the appearance of the first symptoms would abort the infection. As a matter of fact, the applications are always too late : in order to establish the antiviral state , the IFN should bind to the cell receptors a few hours before the viral invasion. The IFN approach has been a financial blunder because the local adverse effects of IFN are worse than the infection itself. I wish a small part of the money would be spent on evaluating the effect of ozone! I would like to give an example of the versatility ofozone, and I am not pretending to be too serious! Ozone as agas is toxic for the nasal and respiratory mucosa and must not be used . However, in our lab, during the last five years we have prepared a lot of ozonized bidistilled water every day. It is ready after 5 min of bubbling ozone (concentration 80 ug/rnl) in water. The final 0 3 concentration is about 20 ug/ml and ifit stored in a glass bottle with a tight teflon tap, it keeps for two days, even though the ozone concentration progressively decreases. If anyone thinks he has caught a cold, he can aspirate the ozonized water into the nostrils 3-4 times a day and can take the bottle home for further use . Water passes into the rhinopharynx and is eliminated, but it is not harmful if swallowed. It also helps to gargle the ozonized water at the same time . Although nasal aspiration ofozonized water causes transient irritation (10-15 sec) , it is unbelievable how rapidly the nasal congestion, sinusal oedema and pharyngodynia disappear rapidly for 3-5 hours, after which it is necessary to repeat the procedure. The infection resolves in 3-4 days, but it is far more tolerable than if it went untreated. If I have time, I also perform 03-AHT on myselfto reduce the feeling offatigue. 270 CHAPTER 24

This approach is quite empirical and it is difticult to produee praetieal applications, so pharmaeeutieal eompanies have no reason to worry about it!

1.7. Fungal and Parasitic Infections

Most of these diseases are present in hot-humid countries and are seen less frequently in Europe, either as opportunistic infections or after a trip to the tropies . Among fungal infections, those that have been treated with ozone are onychomycosis (tinea pedis or athlete's foot) and eandidiasis. Owing to the US embargo against Cuba, scientists and physieians in Havana have used ozone , as necessity is the mother of invention. At the XII IOA Congress (LiIle 1995), it was reported that treatment of a sick nail with 1-2 drops of ozonized sunflower oil for several days led to a complete eure in 69% of patients (the remaining 31'% showed marked improvement). In contrast, in the group treated with tolnaftate solution twiee a day, only 7% were eured, 25'% improved and there was no change in 68'%, probably owing to drug resistance (Menendez et al., 1995). Using ozonized olive oil topically, we have achieved incredible results; in fact, we now believe that this simple preparation is really effective because it is amply disinfectant and able to stimulate the healing proeess.I predict that, as soon as prejudiees disappear and physieians beeome aware of this fact and try ozonized oil with good results, it wiIl become widely used worldwide with great patient satisfaction. Candidiasis and any other fungal infection can obviously be treated with systemic and topical antifungals, but I can guarantee that gas insufflation (whenever possible), ozonized water and ozonized oil applied topically have equal , if not superior, effectiveness. Giardiasis is a parasitic infeetion eaused by the protozoan Giardia lamblia, common in areas with poor sanitation and present even in the United States . Cryptosporidiosis is also a diarrhoeal disease, caused by protozoa of the genus Cryptosporidium. Good drugs Iike metronidazole are effective but have some side effects. In Cuba, at first they used to drink ozonized water, at least four of five glasses per day on an empty stornach for repeated periods of 10 days separated by a I week interval. According to Sardina et al. (1991), up to 48% of patients became asymptomatic after the second cycle . Ingestion of ozonized oil seems more effective, but it is hard to swaIlow. An improved administration is represented by capsules filled with ozonized oil. A 10-day cycle "cured" 79% of children, while the remaining 21% showed a marked improvement of symptoms but still had cysts or trophozoites in the faeces (Menendez et al., 1995). No side effects were reported. There is no need to report other studies because the therapeutic modality is the same . However, it is certainly worth keeping this approach in mind for use in poor countries ofAfrica, Asia and South America affected by several fungal and parasitic diseases. Areas lacking electricity cannot produee ozone and ozonized water . Thus the World Health Organization (WHO) ought to promote a standard and very eeonomical production of ozonized oil (which keeps weil) and distribute it where needed.I should try to promote this enterprise, although it may have little value unless we ean reduce the rate of infection by improving sanitation in all directions. OZONETHERAPY IN VARIOUS PATHOLOGIES 271

Just a few .words about malaria, which remains another scourge of our time, exacting atoll of I million deaths each year . Unfortunately, both the mosquitoes and the protozoan Plasmodium falciparum have become resistant, the former to insecticides and the latter to drugs. Almost 20 years ago, Dockrell and Playfair showed in mice that H202 is able to kill Plasmodium yoelii. At the XV 10A Congress (London, September 10-15, 200 I), Viebahn-Hansler et al. reported that parasite growth can be inhibited by ozone at a concentration of 80 ug/ml after ozonization of a blood cell suspension. However, in contrast to the sarcastic opinion of many scientists that ozone is a panacea, I doubt that ozonetherapy would ever be useful because parasites are well protected by the plasma and cellular antioxidant system, as weil as being hidden in the spleen and other sanctuaries. Moreover, 0 3­ AHT or EBOO are demanding approaches and would be difficult to organize in tropical countries for the treatment of millions of people. I feel pessimistic about wasting our meagre resources on diseases such as HIV and malaria for which the administration of oral drugs or a long-sought vaccine appear rational and could be more useful on a large scale.

2. AUTOIMMUNE DISEASES Autoimmune diseases are areal thomy problem because serious diseases such as rheumatoid arthritis (RA), Sjögren's syndrome, vaseulitis, multiple sc1erosis (MS), systemic lupus erythematosus (SLE), Crohn's disease, systemic sc1erosis (SSc) should have attracted our attention a long time ago, instead of treating unaesthetic, albeit very lucrative, lipodystrophy. Since 1983, this has been the almost exclus ive interest of Italian ozonetherapists, which explains why today ozonetherapy is regarded so poorly. It is even worse that at the annual meetings, two or three ozonetherapists claim, with great rhetoric, that they are able to obtain such wonderful results with a few OrAHT (ozone concentrations and schedule are always uncertain) so that invalid patients are able to rise frorn the wheelchair and go bicycling or dancing . In obedience to holistic therapy and to the patient's advantage (so they say), they invariably mix ozonetherapy with magnetotherapy, phytotherapy, homeopathy, chelation therapy, etc., so that it becomes impossible to c1arifythe role of ozonetherapy . Assuming that this exerts areal effect, it would be important to confirm the results with a proper RCT in order to understand what mechanisms have been activated, how relevant is the placebo effect and how long the improvement lasts. The aetiology of these diseases remains hypothetical, while the pathogenetic mechanisms are fairly common, obviously with different locations and with a strong prevalence in women. Different tissues (articular, gut mucosa, myelin, etc.) are infiltrated by macrophages, neutrophils and CTL, responsible for an abnormal release of ROS and proinflammatory cytokines (IL-I ß, IL-2, IL-8, IL-12, IL-15, IL­ 18, TNFa, IFNy), while inhibitory cytokines (lL-IO, IL- I I, TGFß1) are largely suppressed (Kuruvilla et al., 1991; Brandes et al., 1991; Taga et al., 1993; Akdis et al., 1998; Letterio and Roberts , 1998; Mclnnes and Liew, 1998; Pizarro et al., 1999; Perdue, 1999; Dinarello, 1999; Herrrnann et al., 2000) . 272 CHAPTER24

Activation of enzymes, such as phospholipase A2 (PLA 2), metalloproteinases (collagenases, elastases), cathepsins Band D, and plasminogen activators causes the release of compounds leading to cell death and disintegration of the intercellular matrix and/or myelin, thus perpetuating negative involution. Local release of substance P, calcitonin grp (gene related peptide), bradikynin, leukotrienes, LTB4 (a potent chemotactic and hyperpermeabilizing factor) , PGE2, PGD 2, PG12 (vasodilatators), TxA 2, and Frisoprostanes (both vasoconstrictors) wreak further havoc and elicit oedema and pain (Cracowski et al., 2000). Interestingly, these eicosanoids (2-series PGs and 4-series LTs) derive from AA (20:4n-6), while 5­ series LTs and 3-series PGs, deriving from 5, 8, 11, 14, 17 eicosapentaenoic acid (EPA, 20:5n-3) and from 4,7, 10, 13, 16, 19 docosahexaenoic acid (DHA, 22:6n-3), are far less phlogogenic but are practically absent (Purasiri et al., 1997). EPA and DHA competitively inhibit the conversion of AA to PGs, thus exerting useful inhibitory effects on inflammation and inappropriate immune responses (Calder, 1998). That is why a diet rich in n-3 PUFAs has been advocated for the treatment of various chronic inflammatory conditions typical of autoimmune diseases (Belluzzi et al., 1996; Rose and Connolly, 1999). Throughout the years, with the progressive understanding of pathogenetic mechanisms, orthodox medicine has striven hard to offer the most effective therapy. Yet it has succeeded only in part, owing to adverse effects and unforeseen complications. Nonetheless, the ozonetherapist has the duty to present all the possible options. For the sake of space, 1can only give a few brief examples, but the reader can find a wealth ofdetails in Hanauer (1996) and Hanauer and Dassopoulos (2001) :

2.1. Current Therapies for Inflammatory Bowel Disease

• Sulfasalazine, oral and topical with slow release preparation. • Corticosteroids, among which budesonide is a new compound with high mucosal potency (enema formulation) and low systemic activity. I mention these two compounds because they are specific inhibitors of NFKB (Chapter 14, Leukocytes), which allows the synthesis of IL-113 and TNFu (Auphan et al., 1995; Wahl et al., 1998). • Antibiotics, such as metronidazole and ciprofloxacin, used alone or in combination . • Immunomodulatory drugs: azathioprine, 6-mercaptopurine, methotrexate, mycophenolate mofetil, cyclosporine, tacrolimus (FK 506), thalidomide. They have different mechanisms of action , but substantially inhibit the production of pro-inflammatory cytokines (lL-l, lI-2, lI-8, IL-12, TNFu). • Imunosuppressive cytokines to inhibit the Th I-type » Th2-type excessive response. lI-IO and Il-Ll seem to suppress effector functions and Th I-type cytokine production (Taga et al., 1993; Akdis and Blaser, 2001) . A few trials have shown the safety and tolerance , but the ultimate efficacy remains unknown. An interesting possibility, so far evaluated in mouse colitis, is the increased release of IL-lO into the gut lumen by genetically engineered bacteria. IL-IO may be absorbed via a paracellular route and may downregulate T cell activation OZONETHERAPY IN VARIOUS PATHOLOGIES 273

in the submucosa (Steidler et al., 2000). TGFßl mayaiso be efficacious but has not yet been tested. The usefulness of IFNa remains equivocal. • Oral tolerance. If the responsible autoantigens ean be identified, their oral administration eould induee an immune tolerance and represent a rational treatment. • Probiotics. In Chapter 19, the critical role of genetic susceptibility of the gut mieroflora and its interaction with enterocytes and the MALT was discussed. There are promising, yet unsubstantiated, results after administration of laetobaeilli, bifidobacteria, Streptococcus thermophilus, ete. This complementary approach should not be disregarded, since it is non-toxie and may become more useful by modifying the luminal environment by intermittent hydrocolon therapy. A correet ecologieal environment can be restored by microflora administered via enema . • Dietetic support. As previously mentioned, a diet enriehed with n-3 PUFAs present in fish oil generates (via cyclooxygenase and lipoxygenase) 3-series PGs and 5-series LTs, which are anti-inflammatory and may re-equilibrate the Th l­ Th2 pattern (Hodgson, 1996). n-3 PUFAs can easily be taken in eapsules (Belluzzi et al., 1996) or emulsionated with milk. Although this approach is probably not sufficient to solve the problem, it is recommended and obviously has to be continued for life. •SC administration 0/growth hormone for four months (Slonim et al., 2000). The optimal dose, schedule and duration ofresponse remain to be defined . • The last approach regards a biotechnological "jewel", i.e. the licensing in the USA and Europe of infliximab, an antibody against TNFa (anti-TNFa). This chimeric antibody is about 25% mouse and 75% human and is still immunogenic. A humanised antibody, CDP571 (only 5% mouse and 95% human), is probably better, but is not yet available. RCTs involving the IV infusion of these antibodies have shown clinical benefits in about 65% of patients with severe Crohn's disease and fistulae. Four trials have examined a total of 306 patients (Targan et al., 1997; Present et al., 1999) and a very informative commentary has been published in which Bell and Kamm (2000) conclude that "although treatment with anti-TNFa has improved the quality 0/ life for some patients with Crohn's disease, knowledge ofits proper clinical role will come only with time". Another geneticallyengineered product is the soluble TNFa receptors consisting of two identical chains of the extracellular human p75 TNF reeeptor monomer fused to the Fe domain of human IgG I. This is known as etanercept and is administered SC twice a week. These new products are now limited by their cost, not only in Europe but also in the U.S.

2.2. Current Therapies o/RA Beeause of similarities, I will now briefly report eurrent therapies of RA (Choy and Panayi, 200 I), in whieh degeneration of cartilage and erosion ofjuxta-articular bone seems mainly due to the presence ofTNFa (Feldmann and Maini, 2001). 274 CHAPTER 24

Orthodox therapy aims to relieve pain, reduce and possibly resolve inflammation and enhance healing.

• Non-steroidal anti-lrflammatory drugs (NSAlD). Besides the old aspirin , they inc1ude ibuprofen, indomethacin, naproxen, sulindac, etc. They are all associated with at least gastric irritation. The latest-generation cyclooxygenase I1 inhibitors seem to be effective and have less adverse effects (Fitzgerald and Patrono, 2001) . • Glucocorticoid therapy. Except occasionally prednisone to control symptoms, this therapy is now proscribed. • Immunosuppressive therapy. Azathioprine, cyc1ophosphamide, methotrexate at an interrnittent low-dose may be useful, but attention must be given to adverse effects. • Disease-modifying drugs , such as D-penicillamine, sulfasalazine, gold compounds, are partly useful , but there is minimal evidence that they delay bone erosion or allow real healing. • The new drugs are the previously mentioned anti-TNFa antibodies and the soluble TNFa R fusion protein etanercept. IV administered antibodies appear to regulate the production of IL-6, IL-8, MCP-I and VEGF, reduce the influx of inflammatory cells into joints and the blood levels of rnatrix metalloproteinases. As has been observed in Crohn's disease, in a few RCTs, about two-thirds ofRA patients have shown a c1ear beneficial response for up to 2 years (Elliott et al., 1994; Maini et al., 1999). However, in RA, infliximab has been associated with methotrexate. Similarly, the combination of etanercept and methotrexate seems to provide greater c1inical benefit than methotrexate alone (Lovell et al., 2000; Weinblatt et al., 2000) .

Lets us now consider the official therapeutic arsenal of Crohn's disease and RA versus ozonetherapy. Because someone may think that I am biased against official medicine, I invite everyone to read Sartor's commentary (2000) about new approaches to Crohn's disease and the commentaries ofO'Dell (1999) and Pisetsky (2000) about RA. Although all three authors agree that a new era of improved treatment has arrived, they caution that a multitude ofbad effects may appear. Sartor (but also Bell and Kamm, 2000) is particularly critical. Possible problems include : allergic reactions and serum-sickness-like reaction, induction of antibodies to double-stranded DNA, occurrence of lymphoma (shown already in 6 patients) and development of tubercolosis (Keane et al., 200 I). Use of infliximab costs about 12,000 pounds per year. About 33% of patients are unresponsive and some patients are not suitable for immunological therapy. All previous therapies , as reviewed by Sartor, are both scarcely effective and present serious adverse effects . Regarding ozonetherapy, besides a few unbelievable anecdotes or scanty results by Knock and Klug (1990) , there is only the study performed at Cuba's Institute of Rheumatology in 1988 on 17 patients treated with IM injections of Os-O, (total dose of ozone : 700 ug) for 8 weeks combined with NSAID . Apparently about 25% of ozone-treated patients scored 25% better than controls (Menendez et al., 1989). It is OZONETHERAPY IN VARIOUS PATHOLOGIES 275 reasonable to conclude that we do not know today if ozonetherapy may be useful. Nevertheless, in spite ofa few charlatans' tales, we should not dismiss it a priori. How and why could ozonetherapy be beneficial? a) Using either 03-AHT or BOEX or even the more complex EBOO approach, we may be able to enhance immunosuppression and increase the production of IL-10, IL-II, TGFß and perhaps IL-I Receptor antagonist (IL-I Ra). The immunological investigation carried out in patients should aim at clarifying if ozonetherapy induces anergy of the T cells that are cytotoxic. The RI approach, associated with the parenteral ones, may be helpful in inducing immunosuppression in the gut (see Chapter 19). RI mayaiso inhibit the bacterial flora that is partly responsible for Crohn's disease . However, the possibility ofinducing immunosuppression has yet to be tested, although a hypothetical rationale and a tentative protocol have been proposed in Chapter 14 (Leukocytes, Point 3). b) Generalized and localized induction ofantioxidant enzymes, thus eliminating the excess of ROS production. This is a reasonable proposition, which already has experimental support and may be achieved by inducing the adaptation to COS. c) Inhibition of the release of inflammatory enzymes, metalloproteinases etc., with a progressive decrease of plasma levels of PAF, LTB4, PGE2, TxA2 and isoprostanes. The chronie inflammatory process can be slowly tumed off only if we can, with 6 months oftherapy, implement points a) and b). d) Fistulae and abscesses in Crohn's diseases can be dealt with by insufflation of ozone or ozonized water and oil. An RCT could be performed in one or more hospitals, but this is beyond our means and dreams at the moment. It will be indispensable to have a control arm because the placebo effect may be relevant.

Who can pay the cost of endoscopic, radiological, histologieal, biochemical and clinical exams? We are not backed by any pharmaceutical and/or biotechnological firm because ozonetherapy does not produce profits. However, if ozonetherapy proved to be useful, it would save money for the National Health Service. Yet, I doubt that the Ministry of Health, biased and myopie as it has proved to be in the past, will ever support this research.

2.3. Therapies ofMS The same problems will apply to a RCT planned for MS. This is a tragic disease because it very often disables young adults just when they are about to show their merit. I worked enthusiastically on IFN research, but in the meantime 1 was longing to evaluate if ozonetherapy would be useful for these patients. All physicians know that MS is a T cell-mediated autoimmune disease that can either relapse (relapsing­ remitting MS) or be very aggressive (progressive MS) . Good reviews of the topic are available (Rudick et al., 1997; Karp et al., 2000; Polman and Uitdehaag, 2000; Wingerchuk et al., 2001) . Orthodox medical therapy is based on a) corticosteroids, b) immunosuppressive drugs, namely azathioprine, methotrexate, cyclophosphamide and cyc1osporine, and more or less c) experimental biologicals, such as IV immune globulins, copolymer I 276 CHAPTER24

(COP) 01' glatiramer acetate (Duda et al., 2000; Kipnis et al., 2000; Neuhaus et a\., 2000), which induces a shift from a Th-I to a Th-2 cytokine profile in COP-treated patients. The present treatment of choice (for r-rMS) is IFNß-Ia (glycosylated) and IFNß-Ib (a mutein). All of these drugs can cause immunosuppression to different degrees, and particularly those indicated in a) and b) may cause serious adverse effects. Despite their biochemical difference, both fonns of IFNß (approved by US and European regulatory authorities) have a positive elinical effect, characterized by a elear reduction of both the frequency and severity of exacerbations (Amason, 1993; Rudick et al., 1997; Polman and Uitdehaag, 2000). IFNßs are fairly weil tolerated. Unfortunately, owing to striking phannacokinetic and pharmaeodynamic differenees (Bocci, 1981b; 1987b; 1988a; 1990a; Bocci et al., 1988), IFNa-2a, which eould be therapeutically useful, causes adverse events that negatively affect the already poor quality of life of these patients (Nortvedt et al., 1999). IFNsß are now in wide use, but problems such as the optimal dose and sehedule, the appearance of neutralising antibodies (mostly to IFNß-I b) that may jeopardize efficacy (AntoneIli et al., 1998), a possible relapse when stopping therapy and the eonsiderable cost, provide a glimmer of hope that a serious RCT based on ozonetherapy is still meaningful. In the ease of MS, nothing serious has been done and my attempt to interest three neurologists was in vain beeause they were weil sponsored by finns producing IFNß . Two ozonetherapists (one in Turin and another in Milan) reported to me that they had achieved "good rcsults" treating MS patients with OJ-AHT eombined with either magnetotherapy 01' ehelation therapy. No comment! Thus very \ittle hope remains and it also seems useless to speculate that in these diseases, as weil as in others cited at the beginning of this section, it might have been useful to combine 01' to alternate cyeles of IFNß 01' anti-TNFa antibodies with ozonetherapy. An old adage says: "Be not afraid of going slowly, be afraid only of standing still". The problem of Raynaud's phenomenon (Block and Sequeira, 2001) in selerodenna patients will be considered in the next section.

3. ISCHAEMIC DISEASES (HlND-LIMB ISCHAEMIA, CEREBRAL AND HEART ISCHAEMIA, VENOUS STASIS)

It appears logical to think that if blood briefly exposed to oxygen-ozone can exert a benefit, this would be best noted in isehaemic tissues. Even a partial obstruction of limb arteries due to atheroselerosis (Lusis, 2000) 01' diabetes 01' Buerger's disease (thromboangiitis obliterans) leads to a progressive reduction of blood flow to the feet. Lack ofperfusion leads to tissue ischaemia and possibly cell death. Any minor trauma, nonnally irrelevant, facilitates the fonnation of an ulcer, which will not heal beeause oxygen , nutrients and soluble mediators involved in the repair process are laeking. Aeute \imb ischaemia is frequently eaused by aeute thrombotie oeclusion of a pre-existing stenosis 01' by an embolus; it requires immediate surgieal 01' medieal OZONETHERAPY IN VARIOUS PATHOLOGIES 277 attention . Chronic limb ischaemia becomes progressively evident. In Europe, we distinguish four stages, as classified by Fontaine:

Stage J: Feeling of cold or nurnbness in the foot and toes. Skin temperature is reduced. The foot is pale and frequently becomes cyanotic. Stage JI: Paresthesia and hypoesthesia, firstly localized and successively diffused to the whole foot. Hyporeflexia. This is the phase with incipient neurological defect. Intermittent claudication. Pain may cease with rest. Stage IJI: Pain at rest with noctumal exacerbation. Cyanosis becomes weil evident in one or several toes , with an incipient trophic lesion or a frank uleer. (Rate of amputation is - 15%). Stage IV: Partial or total necrosis of one or several toes. Pain often becomes unbearable (Rate of amputation is - 50%) .

The angiologist has several, precise non-invasive techniques to objectively assess the severity of POAD. Extensive epidemiological studies have shown that these patients have practically the same relative risk of death from cardiovascular causes as do patients with a history of cerebrovascular or coronary disease. A useful predictive value is given by the ankle-brachial index (ABI). The normal range of values is 0.91-1.30, which decreases to 0.41-0.90 in mild to moderate POAD and to below 0.40 in severe POAD. Patients with ABI below 0.40 are at high risk of a cardiovascular event and present an annual mortality of about 25%. As I cannot go into details, I suggest reading two excellent review articles (Dorrnandy and Rutheford, 2000; Hiatt, 200 I). Each patient must be evaluated for any possible revascularization and there are several operative procedures with a high success rate . Lurnbar sympathectomy is no longer performed because it does not increase blood flow to muscle. Spinal-cord stimulation also does not prevent amputation (Klomp et al., 1999). At the extreme, Taylor et al. (1999) have shown that distal venous arterialisation is a unique procedure with promising possibilities for salvage of critically ischaemic, inoperable limbs (stage IV). Besides surgery, orthodox medicine offers several therapeutic options, including useful supportive measures, such as quitting smoking, proper diet, exercise (Davies, 2000), and pharrnacological treatments as folIows :

1) Vasodilatators must be able to improve collateral blood flow and avoid "stealing" blood away from underperfused muscle. Pentoxyfylline may enhance oxygenation in ischaemic tissues by increasing blood flow to the microcirculation. lt may improve blood rheology by decreasing blood viscosity and enhance erythrocyte flexibility. However, arecent double-blind RCT showed no significant difference in healing rates of pure venous uleers between patients taking pentoxyfilline and those taking placebo (Dale et al., 1999). In 1999, the FDA approved cilostazol, an inhibitor of phosphodiesterase type 3, which by increasing the concentration of cAMP causes vasodilatation and reduces claudication. PGE 1 and a stable prostacyclin analogue (iloprost) have been infused in patients with critical leg ischaemia. Both cilostazol and iloprost 278 CHAPTER24

improve POAD, but also cause frequent headaches, palpitations and dizziness and should not be used with patients who also have heart failure. 2) Progression of atherosclerosis may be delayed by treatment of hypercholesterolemia and platelet aggregation inhibitors (aspirin, ticlopidine, clopidogrel), while thrombolytic intervention does not help POAD patients. Propionyl levocamitine improves muscle metabolism and seems useful in improving the quality of life, but certainly does not solve the central problem. Needless to say, diabetic patients must be weil under control (although it hardl y helps), the homocysteine serurn concentration must be lowered and hypertension must be treated with caution. The prognosis of POAD patients is dirn, with progressive deterioration that limits their ability to perform daily activities.

In these circumstances, patients look for a treatment that may real1y improve their condition. Thus oxygen-ozone therapy has attracted much attention. Whether it deserves it and is really better than the multi form traditional therapy remains to be seen, because most ofthe following data are questionable. In the ozonetherapy field, the work by Rokitansky, who was president of the Austrian Society ofOzonetherapy, is revered as the best. In 1981, he presented data for 152 patients treated with OJ-AHT between 1974 and 1980.

Table 20. Results in POAD patients after ozonetherapy.

Fontaine stage: II III IV Patient number: 62 51 39 Very good improvement: 87.1 % 70.6 % 53.8 % Walking distance: > 1000 m > 800 m > 500 m no pain at rest gangrene healed Improved: 9.7 % 21.6 % 25.6 % Walking distance: > 400-500 m > 300-400 m Amputation oftoes occasional pain with healing of stump No improvement or progression: 3.2 % 7.8% 20.6%

He examined 2,3-DPG levels in erythrocytes in 11 patients and reported a marked increase in 9, no change in land a decrease in another (who had a very high level before ozonetherapy). This study was without any contro\. At a subsequent meeting (1983), Rokitansky presented the following table (Table 21) : two groups of patients (232 versus 140), roughly similar in age and stage, were treated with either O2-0 3 administered by intra-arterial injection into the femoral artery plus local treatment with gas, or conventional (C) vasodilatation therapy (a sort ofcontrol). OZONETHERA.PY IN VARIOUS PATHOLOGIES 279

Table 21. Comparison ofresults in two groups ofPOAD patients.

Stage Nutnber of Marked Modest Progression p"tients improvement (%) improvement (%) 0 3 C 0 3 C 0 3 C 0 3 C 11 105 73 80.0 43.8 11.4 19.2 8.5 37.0 III 72 46 70.8 39.1 19.4 17.4 9.7 43.5 IV 55 21 50.9 28.6 21.8 19.0 27 .3 54.0

The rate of amputations declined from 15 to 10% for stage III and from 50 to 27% for patients (Stage IV) treated with IA Ozone plus topical bagging. This study is of no use today because IA injection of ozone has been abandoned and the traditional treatment at that time is no match for the modern integrated approach. In 1987, Mattassi et al., working at the Vascular Surgical Unit at Garbagnate Hospital near Milan, compared the old approach of IA injection ofozone (20 ml, 0 3 concentration: 40 ug/ml; 3 times per week or every day) to the c1assical 03-AHT (200 ml blood in citrate, 0 3 concentration not specified, probably 30-40 ug/mI; one session per week for a total of five sessions). They found that that OrAHT yielded slightly better results than IA ozone (Table 22). They also reported (it is unclear if they pooled the data) a significant increase of HDL-cholesterol and triglycerides.

Tahle 22. Comparison between IA ozone administration (IA03) and OrAHTin 101 patients (II stage). 53 patients (III stage) and 65 patients (IV stage).

Stage Optimal Result (%) Good Result (%) No Result (%) /AOI DrAHT IA0 3 DrAHT IA0 3 DrAHT 11 16 27 59 49 25 24 III 7 32 67 36 26 32 IV 11 11 42 33 47 56

In 1988, Romero et al. perfonned a study on 60 patients at the National Institute of Angiology and Vascular Surgery in Havana, comparing IA03, 03-AHT and traditional (?) medical treatment. It is unclear if the IA administration of 0 3 was better than O]-AHT, but ozonetherapy apparently produced a 20-35% improvement over conventional therapy. Three Polish studies (published in Polish) in 1990, 1991 and 1992 continued to examine the effect of IA03 in POAD atherosclerotic and diabetic patients. On the whole, the conclusion was that IAO} was "valuable and safe" and markedly improved the intermittent c1audication distance measured before and after the treatment. In one study, an increase of ABI was also shown. The measurement of some biochemical parameters led the authors to say that IA03 caused "a significant decrease in blood cholesterol levels and a marked reduction in blood and plasma viscosity". 280 CHAPTER24

After Rokitansky et al. (1981) asserted that ozone increases 2,3-DPG, lowers fibrinogen and plasma viscosity, and reduces plasma cholesterol, it has become a regular ritual to confirm these data. The aim of orthodox therapy is to improve blood rheology by changing the lifestyle (no smoking, almost a vegetarian diet, exercise, etc.) and using several drugs aimed at different targets . Yet, in spite of a great effort, the success has been meagre . As discussed in Chapter 14 (Erythrocytes), it is hard to believe that ozone can produce all these magie results , because during the last 6 years of c1inical work (with 0 3-AHT and EBOO) we have not been able to confirm them. We have not observed a decrease of cholesterol plasma levels, nor a change of LDL; if anything, the fibrinogen level tends to increase slightly. In our ARMD study (about 5,000 0 3­ AHT), often with 13-14 sessions , we observed a slight increase of2,3-DPG only in a few patients who had a very low level prior to therapy . This result was also obtained by Mattassi (1985) before uso Romero et al. (1993) conducted a useful study, comparing the relative efficacy of ozone adrninistered to POAD patients by: a) IM gas injection, b) OrAHT, c) RI, and d) standard conventional treatment. Irrespective of the administration route, ozonetherapy markedly improved the ABI and reduced c1audication. Thus the authors reached the conclusion that RI was as effective but far easier to perform than 03-AHT. As I am not enthusiastic about RI, I was surprised when Mattassi confirmed, at the Verona Congress (1999), that among IA, OJ-AHT and RI, the last approach is as effective as the first two and concluded that we must bear in mind this finding because the patient who owns a generator can perform a self-administration at horne. Russian c1inicians working at the Ozonetherapy Centre in Nizhni Novgorod reported at the XII 10A Congress in Lille (1995) that ozone administered by various techniques to 132 elderly patients (IV and SC injections of O2-03, IV infusion of ozonized rheomacrodex solution and 3-4 0 3-AHT) is "highly effective" and induces "significant improvement ofpatients' weil being". Amato's observations (2000) on the effect of 03-AHT as a unique therapy for angina abdominis (AA) cannot be overlooked. AA is arare, painful abdominal syndrome that manifests itself after a meal, probably owing to a localized transitory ischaemia of the gut. Surgical vascular correction normally solves the problern. but in the three elderly patients studied by Amato it was not feasible . A cycle of 10 Or AHT (150 ml of blood treated with 0 3: 20-40 ug/rnl per ml of blood) followed by maintenance therapy (one treatment every month) resolved the problem very weil and patients, no Ionger afraid to have a meal, showed a marked improvement without any side effects. The oldest patient, a woman of 87 years, has undergone this therapy since 1994! Who says that ozone is toxic? A final remark can be made regarding the extraordinary capacity of combining 03-AHT with topical therapy (either gas or, better, ozonized water and oil) to allow healing of awful decubitus or neerotie ulcers in the limbs (also see Chapter 14, Platelets, and this Chapter). It takes some time but they do heal. Figure 98, taken from Werkmeister's work (1995), shows that extensive lesions after X irradiation took up to 190 days with only loeal treatment, but I believe that association with parenteral treatment would have shortened the resolution time. Regarding ulcers on limbs, irrespective of the aetiology (atherosclerosis, Buerger's disease, diabetes, OZONETHERAPY IN VARIOUS PATHOLOGIES 281

Raynaud's phenomenon), they do heal, even in the two exceptional cases described by De Monte and van der Zee (2001). 160 r------, 140 _120 N E 100 ~ c:: 80 ­ o :3 60 ..J 40 20

oL:::::::!;s;:::;:e:~ä3I;~~A--~--_..,._--~, , o 50 100 150 200 250 Days

Figure 98. The diagram reports the time necessary/01' the healing 0/decubitus ulcers in 8 patients after only topical hypobaric application o/OrOJ. according to Werkmeister (1995). The ulcer q{153 cm' took about 190 days to heal. This time can be markedly shortened by combining the parenteral (DrAHT) and topical (ozonized water and oil) treatments

In the first, a woman was initially treated with a percutaneous chemical (phenol) lumbar sympathectomy, supplemented with a continuous infusion (0.5 ml/hour) of bupivacaine 0.15% via an epidural catheter; this treatment only controlled the pain. The second case was a man with painful bilateral leg ulcerations due to a vaseulitis. A lumbar epidural catheter delivering 0.5 ml/hour of bupivacaine 0.20% and 0.125 mg/hour ofmorphine (3 mg/day) bare1y controlled the pain.and the ulcers worsened. In both cases, healing was achieved by removing the catheters and perfonning 40 and 45 03-AHT, respectively, plus topical treatment with ozone. Uleers from venous stasis have been treated and they also heal rapidly with the combined treatment. However, phlebopathies have attracted less interest than arteriopathies. If venous hypertension cannot be compensated by physiological mechanisms, it leads to increased penneability at the level of the microcirculation, Iymphatic hypertension, oedema and possibly torpid ulcers. I can report only one investigation (Lo Prete, 2000) perfonned in patients with extended varicosity, which examined subjective parameters (phlebalgia, feeling of orthostatic weight or pain, fonnication and paresthesia), objective parameters (evening oedema, constant oedema, haemosiderinic dermitis, fibrous hypodennitis, eczema, skin ulcerations) and instrumental parameters (plethysmography, videocapillaroscopy and evaluation of circumference at the calf and at the ankle-malleolus), There were 15 patients (14 women and 1 man), from 20-60 years old, with marked varicosity complicated by chronic venous insufficiency. Ozonetherapy was perfonned by SC and perivenous injection ofup to 300 ml ofgas (O~ + Oj) at an ozone concentration of 8 ug/ml in 60 282 CHAPTER24 sites (5 ml per site) . There were two treatments per week, repeated for 12 weeks (total 24 sessions). The results are presented in the following tables:

Table 23a. Subjective parameters.

Before treatment Aper treatment Phlebalgia 8(15) 53% 0(15) 0 Feeling oforthostatic weight 15 (15) 100% 4 (15) 26.6%. Orthostatic pain 15(15) 100% I (15) 6.6'% Formication 9 (15) 60% 0(15) 0 Paresthesia 7 (15) 40% o(15) 0

Table 23h. Objective parameters.

Before treatment After treatment Evening oedema 15 (15) 100% 2 (15) 13.3% Constant oedema 4 (15) 26.6'Yo 2 (15) 13.3% Haemosiderinic dermitis 5(15) 33.3% 1 (15) 6.6%. Fibrous hypodermitis 6 (15) 40% 4 (15) 26.6%. Eczema 4 (15) 26.6% 0(15) 0 Skin ulcers 2 (15) 13.3% o(15) 0

Table 23c. Instrum ental parameter s.

Before treatment After treatm ent Plethysmography - 18.0" 22" Evaluation ofcircumference (cm) : calf 34.3 33.1 ankle (malleolus) 25.2 24.5

There was a marked reduction of the peripheral venous stasis, likely due to improved microcirculation. The SC and perivenous administration of gas caused modest but transitory pain. No more than 5 ml per site ought to be injected. There are no other adverse effects . Simultaneous topical treatment enhances the healing oftorpid ulcers. The association with OrAHT may further improve the treatment.

Let us now try to reach some conclusions. It is unfortunate that the possibility of improving tissue ischaemia with ozonetherapy has interested only a few vascular surgeons. Conceptually, ozonetherapy is the warhorse of this approach and appears to be very useful in skeletal muscle, myocardial and cerebraI ischaemia, because it may : increase oxygen and glucose delivery by several mechanisms (Chapters 12 to 14), b) enhance angiogenesis via activation of resident stern cells, c) induce the preconditioning phenomena by upregulating the expression of antioxidant enzymes and HSPs, and d) trigger a neuro-humoral response to improve the quality of life . This is exciting, but objectively we can conclude that none of the several studies is OZONETHERAPY IN V ARIOUS PA THOLOGIES 283 satisfactory because of inappropriate administration routes, unknown or uncertain 0 3 concentrations, too brief schedules and lack of areal contro\. The only available RCT , in line with the rules of orthodox medicine, is that perforrned by Kraft et al. (1998), who examined the effect of03-AHT on patients with mild hypertension. The original abstract folIows: "Autohemotherapy with ozone is widely applied by German therapists who prefer complementary medicine, in eider patients with cardiovascular problems in order to improve various symptoms and the quality of Iife. The strong oxidant ozone may further increase the already high oxidative potential of these patients and thereby counteract positive effects of antioxidants. In the present study, the effects of autohemotherapy with ozone on cardiovascular risk factors were evaluated in 17 patients, with mild hypertension. In a randomised, double-blind, placebo-controlled crossover-study, 24 h ambulatory blood pressure and blood pressure load were significantly reduced after a cycle of ten autohemotherapies with ozone, but not after a cycle with placebo (oxygen) (p < 0.05). The initial blood pressure level was restored after the end of the therapy within 4 months. Aggregation of thrombocytes was significant and the malondialdehyde concentration was c1early increased, serum HDL concentration was reduced, and the rate of side effects was higher during the cycle with ozone . The reduction of blood pressure may be due to an increased oxygen supply of the vascular endothelium, but an activation of the oxidative potential of polymorphonuclear leukocytes and/or macrophages cannot be excluded . Considering the small reduction of blood pressure, the potentially noxious oxidative mechanisms and the rather high expenses, autohemotherapy with ozone cannot be recommended as a useful alternative to pharmacological treatment ofpatients with mild hypertension." Clearly Kraft et a\. are very critical and, in my opinion, they have verified the validity of ozonetherapy on the wrong disease. Indeed mild hypertension is a life­ long ailment and there are optimal conventional remedies (Iow-sait diet, statins, ACE-inhibitors, etc.) to keep it under control, so that the use of ozonetherapy is unjustified. Moreover, the conclusions of these authors have been influenced by prejudice and an incomplete knowledge ofthe potentials ofozonetherapy. With regard to the standard set by official Medicine, we are practically back to square one and if we really want to define the value of ozonetherapy in POAD patients we must first define standard conditions and then perforrn serious RCTs. The following guidelines are suggested:

I) among the four approaches, 03-AHT, RI, BOEX and EBOO, we can select the first or the second because they are easy to perforrn and inexpensive. IA, IV or SC injection of ozone as gas should be excluded. After a convenient wash-out period, patient groups may switch over to compare 0rAHT and RI in the same patient. For OrAHT, suitable ozone concentrations may range from an initial 20 ug/ml per ml of blood up to 40 ug/rnl at the beginning of the 3'd week. Two treatments per week for a total of 16 treatments (8 weeks). In our experience a shorter schedule is not valid. For RI, ozone concentrations may range frorn an initial 5 J.lg up to 30 ug/rnl, increasing the gas volume progressively frorn 150 ml to 600 ml in 2 weeks. Five treatments per week for 8 weeks. This schedule is not practical for patients. 2) Groups of POAD patients belonging to stage 11, III and IV should be as homogeneous as possible. 284 CHAPTER24

3) Objective assessment of the stage and severity of the disease should be detennined by the best and most precise non-invasive techn iques before treatment, after 8 weeks and again two months thereafter. Evaluation should be perfonned by independent c1inicians. 4) Patients should be randomized into two groups: one receiving O2-03 and one O2 only, with the same technical modalities. A third group treated with the best conventional treatment could be included for a comparison of the efficacy. If the treatment is valid, the control group has the right to be switched over and receive the ozonized treatment. These data may strengthen the results . 5) A specialized statistician should evaluate all the data . 6) The same protocol can be tentatively explored in patients with Raynaud's phenomenon secondary to systemic sclerosis.A comparison can be made with an historicalor a new control with infusion of iloprost, which has proved to be effective for short-tenn palliation (Wigley et al., 1994; Block and Sequeira, 2001).

Two RCTs, one dealing with POAD and another with terminal cardiopathic patients, will evaluate the toxicity and efficacy of EBOO according to the optimized methodology (reported in Chapter 17) at the Siena and Modena University clinics. We hope to be able to report the results within 2003. Owing to the systemic nature of atherosclerosis, both the heart and CNS are at high risk in POAD, and there is a rather high incidence of myocardial infarction or ischaemic stroke and/or terminal stage lirnb ischaemia. That is why we are testing the validity of EBOO in end-stage cardiopathic patients when either transplantation or surgical revascularization is not feasible . Our preliminary study (Di Paolo et al., 2000) on three patients yielded results that are encouraging but regarded as anecdotal because angiocardiographic examination could not be repeated after the treatment. This project is ongoing and we have already treated 15 patients (manuscript in preparation). For the time being, there are two studies: the Russian trial was carried out in 39 patients with advanced coronary atherosclerosis. They underwent five daily infusions (for 20 days) of ozonized saline solution.I believe that ozonization was carried out at a very low ozone concentration (perhaps 2-3 ug/rnl), so the levels of HOCI were not too high and thus not too caustic! I must say that I am dead against this procedure. However, Zhulina et al. (1993) concluded that the treatment was effective because angina attacks decreased from an average of 6 to about 2 per day . There were no controls with either oxygenated saline or simple saline, which may have shown a significant placebo effect. The second study is one ofthe best reported in the field and I am glad to say that the editor of "Free Radicals in Biology and Medicine", who strongly opposes ozone in Medicine (Chapter 2), decided to publish this paper. Hemandez et al. (1995) quite correctly performed 03-AHT, five days per week for a total of 15 treatments, in 22 cardiopathic patients. They found a significant decrease in plasma cholesterol and LDL levels (we shall see ifwe can confinn this finding after EBOO) and an increase of erythrocytic GSH-Px and G-6PD , which is in line with the phenomenon of adaptation to COS paradoxically induced by ozone. It is fair to mention that the great hope of modem medicine is to use gene therapy to elicit therapeutic angiogenesis in patients with chronic myocardial OZONETHERAPY IN VARIOUS PATHOLOGIES 285 ischaemia (Patterson and Runge, 2000; Jackson et al., 2001) . However, while this new approach matures, I do not see anything wrong in evaluating ozonetherapy. In about 80% of patients, ischaemic stroke results from atherothrombotic or thromboembolic processes. Stroke can strike relatively young persons at the peak of their intellectual activity and, if not fatal, can be highly debilitating. Fortunately, Handel, Pasteur and Roosevelt, to cite a few, were able to make great contributions to music, science and politics in spite of suffering a stroke . Modem medicine has developed prophylactic measures able to reduce the risk of transient ischaemic attacks (TIAs) or of stroke in prone individuals by 20-30% (Gubitz and Sandercock, 2000). Moreover, anti-atherosclerotic drugs and, if necessary, carotid endarcterectomy appear beneficial. In case of an acute stroke, therapy must begun within the shortest possible time (from 0.5 to 2-3 hours) to reperfuse the ischaemic penumbra surrounding the core of a cerebral infarction . Time deIays are predominantly in the pre-hospital phase and can be fatal. Hypoxia induces a cascade of metabolic disorders, such as tissue acidosis, reduction of ATP 2 levels, Ca + overload, activation of glutamate receptors, N-methyl-D-aspartate (NMDA) channel opening and release ofproteinases, leading to neuronal death. The interested reader will appreciate the complex sequence of events carefully analyzed by Besson and Bogousslavsky (1995) , Back (1998), Small et al. (1999) and Rosenberg (1999) . Since the 1990s, IV thrombolysis using recombinant tissue plasminogen activator (Tpa), with due caution to avoid cerebral haemorrhage, has been applied to reduce the time ofreperfusion and neuronal damage . In Cuba, where there is a lack of Tpa (owing to the embargo), many hospital emergency units have ozone generators at hand and patients with stroke are luckily treated as soon as possible with oxygen-ozone therapy . Thus, we again face the old dilemma, which need not exist if we have been able to weigh the risks and advantages of the two approaches. To my knowledge, only a preliminary study has been reported by Wasser, a German ozonetherapist, who has treated stroke patients privately, with all possible inherent disadvantages. He reported at the XII IOA Congress (Lilie, 1995) that he had treated several patients some time after they suffered an acute stroke. In spite of this limitation, the use of 0 3-AHT every day seems to have improved the outcome, in the sense that no patient died and they apparently recovered very rapidly. A1though the study has obvious limitations, Dr. Wasser (1995b) has to be congratulated for his adventurous enterprise, which deserves to be pursued. At my University, I have found great disinterest; neurologists do not want to risk what they consider a reasonably valid treatment (thrombolysis) for the uncertainty ofozonetherapy. For what it is worth, my opinion is that a controlled study using either Tpa or ozonetherapy, or a combination of the two, performed at the earliest possible time after a stroke would be very informative. Indeed it would probably save lives or reduce the disability. On the basis ofsome experimental findings and on the uncertain validity ofthe cited c1inical open studies, I believe that ozonetherapy could be useful in vascular diseases, particularly in poor countries. However, it must be validated by RCTs in developed countries. Regretfully, no support from Health Authorities or private organizations is foreseeable and thus we have to continue the struggle and try to do our best. 286 CHAPTER24

4. RETINAL DEGENERATIVE DISORDERS A. Diadori and V. Bocci 4.1. Retina: Anatomical Aspects

The retina, a thin transparent membrane lining the interior of the eye , is the initial receptor for visual stimuli. Its outer face is in contact with Bruch's membrane of the vascular choroid; its inner surface is in contact with the vitreous body (Fig . 99) .

Sclera

Choroid

.'=~~'t 's membrane

Pholo receplo rs

Relinal vessels

Figure 99. Image ofthe ocularfundus (left side) with the macula lutea at the centre. Schematic drawing (right side) 0/the several retinal layers, from the internal limiting membrane to the sclera . RPE: retinal pigment epithelium

The neurosensorial retina is separated into 10 layers, with the photoreceptors (rods and cones) located in the outer layer and the axons of the ganglion cells (second-order neurons) collecting on the inner layer to form the optic nerve . At the extreme outer layer of the retina, there is the retinal pigment epithelium (RPE), a monolayer of cells resting on Bruchs membrane (which separates the RPE from the choriocapillaris), ftmctionally connected to the photoreceptors. The RPE has several important functions : it phagocytoses and processes the tips of the outer segment of the photoreceptors, recycles vitamin A, transmits oxygen and other nutrients from the choroid to the photoreceptors and outer retina, and differentiates into macrophages and other types ofcells in response to various stimuli . The central area of the retina (c1inically defined as the area within the temporal vascular arcade) is referred to as the macula; the highest visual acuity (ability to see fine details) resides in the cone-rich centre of the macula, the fovea. Retina! vesse!s are absent in the fovea, rendering the fovea critically dependent on the choriocapillaris for its metabolic requirements. OZONETHERAPY IN VARIOUS PATHOLOGIES 287

Several ophthalmologie disorders of the retina and the optie nerve, of multifactorial aetiology, lead inevitably to peripheral and/or eentral loss of vision by degeneration ofthe neurosensorial eells (0'Amico, 1994). Reeent studies on the meehanisms mediating neuronal damage suggest that apoptosis might be implieated in the death of neurosensorial eells in the retina and optie nerve (Xu et al., 1996; Gregory and Bird, 1995; Lo et al., 1995; Adler 1996; Fraser et al., 1996). In partieular, advanees in the understanding of eellular meehanisms of retinal ganglion eell and nerve axonal damage in isehaemie injury suggest an isehaemie eomponent; seeondary effeets (seeondary neuronal degeneration) are eaused by a toxie environment produeed by the dying eells, resuIting in free radical produetion and further tissue damage . There is an inereasing amount of experimental evidenee that neuronal degeneration is mediated by numerous processes, including exeitatory amino acid (espeeially glutamate) toxieity , bursts of reactive oxygen species (free­ radicaJ damage, oxidative stress), nitric oxide perturbation and Ca2+-indueed damage (intracellular calcium influx), whieh interact to provide a fmal common pathway for eell vulnerability. By intervening in such processes, neuroprotective agents have produced beneficial effeets in animal models ofretinaI ganglion cell damage and optic neuropathy. These data open a novel pathway toward really innovative pharmacologieal research, aimed at protecting the retina and optie nerve from neurotoxic injury (Steinberg, 1994; Orago et al., 1993; Nayak et al., 1993; La Vail et al., 1992). In such an effort, ozonetherapy eould play a role, as a new, reliable, eomplementary treatment for the management of several neurodegenerative disorders . Indeed there is experimental evidenee that ozone ean have important biological effeets (Coppola et al., 1992; Romero Valdes et al., 1993; Hernandez et al., 1995; Bocci, 1996), such as:

• improvedperfusionand eellular oxygenation(aetivationof erythrocytefimction) • vasodilatation by release ofNO and CO; • upregulation ofthe enzymatic antioxidant system; • induetion and release ofcytokines and growth factors.

These effects eould eooperate beneficially in an attempt to increase the defence meehanisms of eells against isehaemie and neurotoxic injury, thus preventing eell death or reducing tissue injury in reversibly damaged cells. It has been speculated that several irreversible degenerative disorders of the retina and optie nerve, for which no therapy has proven effective, could benefit from ozonetherapy (Riva Sanseverino et al., 1990; Moraleda, 1995; Marmer and Parker 1998; Diadori et al., 1996, 2000):

• age-related maeular degeneration, • degenerative myopia, • retinal vascular disorders (such as diabetes) , • retinal inherited-degenerative disorders (such as retinitis pigmentosa), 288 CHAPTER 24

• ischaemic optic neuropathies, • glaucoma.

We report our experience on the efficacy of OJ-AHT in the treatment of age­ related macular degeneration (ARMD). Other disorders are still under investigation.

4.2. Age-Related Macular Degeneration ARMD is a progressive disabling bilateral condition affecting central vision. In developed countries, it is the leading cause of irreversible loss of vision in people over 50 years of age (Bressler et al., 1988); its incidence increases with age, varying from 1.6% in patients 50 to 60 years of age to 20-30 % of people over the age of65 (Framingham Eye Study, 1980; Pauleikoffand Koch, 1995). Since this sector of the population is expected to increase during the next century, the social and economic consequences of ARMD-related visual impairment and blindness are destined to increase unless successful means of prevention and treatment can be found . Therefore, ARMD is a public health problem ofsevere and growing proportions (Evans and Wormald, 1996).

4.2.1. Clinieal Presentation The most frequent signs of ARMD are:

I) drusen. These lesions are ophthalmoscopically visible as pale yellow spots that may occur individually or in clusters throughout the macula. They consist of an accumulation of amorphous material between the RPE and Bruch's membrane, resulting in a microscopic elevation of the RPE. Although their exact origin remains unknown, current theories favour the accumulation oflipofuscin and other cellular debris derived from cells ofthe RPE that are compromised by age or other factors. 2) disturban ee ur the RPE, which may appear disrupted into small areas of hypo- and hyperpigmentation (pigmentary changes) or may become absent, forming large areas ofatrophy (areolar [geographie) atrophy). 3) ehuroidal neovascularization. In response to as yet undefined stimuli , choroidal vessels proli ferate across Bruch's membrane under the RPE and, in certain cases, continue their extension into the subretinal space .

According to recent studies, neovascularization could be stimulated and mediated by VEGF , produced by the RPE cells under hypoxic conditions (Frank, 1997; Pournaras et al., 1997). Copious leakage from these neovascular membranes may result in exudative detachment of the RPE or haemorrhages, which may be confined to the area under the RPE or may extend under the retina. The natural course ofthis process is fibrotic evolution, with formation ofa disciform sear. Clinically, two forms or stages ofARMD are usually described: OZONETHERAPY IN VARIOUS PATHOLOGIES 289

• "dry" or atrophie form (the most eommon, aeeounting for 80-95% of eases), eharaeterized by disturbanee of the RPE with varying degrees of drusen (hard, soft, mixed) and areolar (geographie) atrophy, in whieh loss of the RPE is aeeompanied by fallout of photoreeeptors and ehorioeapillaris. The visual deterioration is usually slow and gradual, and beeomes really important only in 5-10% of eases, depending on the extent and loeation of the area ofatrophy. • "Wet" or exudative-neovaseular form, eharaeterized by detaehment ofthe RPE (serous or haemorrhagic), subretinal neovaseularization, and flbrovascular disciform searring. It is less eommon (5-20% ofeases), but is associated with an even worsevisual prognosis(severe loss of eentral vision in 70-80% of eases).

The most frequent symptoms ofthese alterations ofthe maeula are:

deereased visual aeuity (loss of eentral vision, eolour vision, ability to see fine details) metamorphopsia (distortion ofthe shape ofobjeets in view) paraeentral-central seotoma

Loss ofvision in ARMD is the result ofphotoreeeptor death, oeeurring when RPE eells, with whieh they are assoeiated, deteriorate and die. The loss of vision resulting from drusen and pigmentary ehanges (early stages of the disease) is highly variable: most patients are asymptomatic or experienee only a small visual loss or metamorphopsia . With the progressive development of larger areas of atrophy of the RPE involving the fovea, visual aeuity deereases abruptly and relative or absolute seotomas appear within the eentral 10 degrees of the visual field. Sudden substantial loss of eentral vision, over a larger area and often at an earlier age, is generally the result ofehoroidal neovaseularization, with serous or haemorrhagie detaehment ofthe RPE. The natural elinieal eourse of ARMD is progressive and the final visual aeuity is usually < 20/200 (Piguet et al., 1992; Sarks et al., 1988; Barondes et al., 1990; Klein et al., 1993, 1997).

4.2.2. Pathophysiology and Risk Faetors The eauses and faetors related to the onset and progression of ARMD remain unknown, but it now appears likely that ARMD is a multifaetorial disease, triggered by environmental faetors in those who are genetieally predisposed . Although multiple possible riskfactors for ARMD have been identified, the importanee ofeaeh remains uncertain. Those thought to be important include:

ageing (> 55 years); genetic predisposition [familial trait], (Silvestri et al., 1994; Silvestri, 1997; Allikmets et al., 1997); 290 CHAPTER24

smoking (Vingerling et al., 1996); exposure to sunlight [photo-oxidative stress], (Cruickshanks et al., 1993; Darzins et al., 1997); blue iris (Sandberg et al., 1994); hyperopia (Broker et al., 1993); systemic hypertension, cardiovascular disease, and vasculopathic trait (Vingerling et al., 1995); nutritional factors, such as a low zinc and antioxidants intake (Newsome et al., 1988; Sperduto et al., 1990; Seddon et al., 1994; West et al., 1994;Chew, 1995).

The cause and pathogenesis of ARMD is unclear (Young, 1987), but an ensemble of nutritional, degenerative, oxidative (phototoxic), haemodynamic and genetic factors, appear responsible and are being actively investigated. Three main theories can be distinguished:

• primary dysfunction of the RPE (genetic predisposition or environmental exposure). There is a consensus that the crucial lesion in ARMD involves the RPE cells. Light damage, ageing and a variety of dietary deficiencies have been invoked as possible primary causes of RPE failure in ARMD , with secondary atrophy ofthe choroid. There are data suggesting that the outer retina, in particular photoreceptors and RPE cells, may be particularly susceptible to the oxidation process, which leads to the formation of free radicals and highly reactive singlet oxygen within the cells, causing cell death . Animal studies suggest that exposure to intense sunlight or UV radiation may cause changes in the RPE similar to those seen in ARMD . The high metabolic rate of the outer retina and chronic exposure to light (photochemical damage) may contribute to an underlying oxidative stress (Organisciak et al., 1997; Rapp et al., 1997, Tate et al., 1995). The retina contains a wide variety ofprotective mechanisms (e.g. antioxidants) against such damage under normal conditions, but these natural defences may decrease with age, or may be inadequate ifenvironmental exposure to oxidants increases, or ifthere is a genetic deficiency ofsuch enzymes. • Haemodynamic dysfunction in the choriocapillaris. The vascular theory (Friedman, 1997) postulates that ARMD is caused by a progressive decrease in the compliance of the sclera (a higher coefficient of scleral rigidity was found in patients with ARMD) and choroidal vessels, leading to an increase in the resistance of the choroid to the flow of blood. This process initiates with the deposition of lipids in the sclera and Bruch's membrane, and results in decreased choroidal perfusion, with reduced supply of oxygen and glucose to the retina (ischaemic damage) , and higher pressure in the choroidal vessels . According to this haemodynamic model, the decompensation of the RPE is the result of its incapacity to transport OZONETHERAPY IN VARIOUS PATHOLOGIES 291

debris and metabolites against progressively unfavourable hydrostatic and osmotic gradients. • Primary dysfunction ofphotoreceptors (ABCR mutation). ABCR mutation was found in patients with ARMD, especially in those affected by the "dry" form. Since the ABCR protein is a transporter protein found in the outer segment of rod cells, its mutation could cause degraded material to accumulate, thus interfering with retinal cell function.

4.2.3. Prevention and Treatment At present useful therapy to prevent the natural history of ARMD remains controversial. On the basis of the role of oxidative stress in the pathogenesis of the disease, the protective effect of several food supplements (minerals and antioxidant vitamins) have been investigated, but conflicting results have emerged (Newsome et al., 1988; Sperduto et al., 1990; Prashar et al., 1993; Cohen et al., 1994; West et al., 1994; De La Paz et al., 1996; Larkin, 2001) . There is no known treatment for the "atrophic" form of ARMD, which accounts for most cases ofARMD . Most potential therapies are addressed to the wet form ofARMD, with the aim of reducing the neovascularization . However, these treatments do not improve sight; they only help to slow visual deterioration, so they are unlikely to have an important impact on the blindness caused by ARMD. Laser photocoagulation (Macular photocoagulation group, 1986, 1991) is indicated only for selected patients with well defined extrafoveal and juxtafoveal neovascular membranes, but it is often followed by a decrease in visual acuity and it does not prevent the long-term recurrence of neovascularization (Lambert et al., 1992; Thomas et al., 1992). Photodynarnic therapy looks more promising, since it is more selective (it does not affect the RPE, photoreceptors and choriocapillaris), but it is only suitable for 10-15% ofpatients with wet ARMD and it is expensive. Low-dose radiation therapy (teletherapy) has been proposed for the treatment of subfoveal neovascular membranes that are unsuitable for laser treatment, but its effectiveness has not been demonstrated. Despite initial enthusiasm about their potential angiogenetic inhibitory effects, antivasoproliferative substances, such as IFNo. (Fung, 1991; Ezekowitz et al., 1992), have shown little benefit in the treatment of age-related subretinal neovascularization. There are now far more prornising anti-angiogenetic compounds under investigation (O'Reilly et al., 1994; Aiello et al., 1995; Robinson et al., 1996). Subretinal surgery, aiming to directly remove the offending neovascular membrane, is very appealing, but not free from risks; moreover, in ARMD the disturbances of the RPE make the process more complicated, and the results may be disappointing. 292 CHAPTER24

4.3. ARMD: Personal Experience with Ozonetherapy Owing to the lack of an effective therapy, it was decided to conduct a preliminary investigation of the efficacy of 0 3-AHT in the dry form of ARMD; this was carried out in the Department of Ophthalmology and Neurosurgery, in cooperation with the Institute ofGeneral Physiology, University ofSiena. 0 3-AHT is a safe complementary approach widely used in the treatment of ischaemic vascular disorders , such as peripheral chronic arterial occlusive disease. It has been shown to activate erythrocytic metabolism and oxygen delivery to hypoxic tissues and to upregulate the expression of antioxidant enzymes, possibly leading to the correction ofan endogenous oxidative stress (Bocci 1996a,c,d). The purpose of our study was to check its c1inical efficacy in patients with dry ARMD, compared with an age-matched control group (prospective RCT) . The study population consisted ofpatients referred to the outpatient c1inic of the Department ofOphthalmology and Neurosurgery of the University of Siena. All patients signed an informed consent statement prior to inclusion in the study. All patients were affected by the "dry" form of ARMD, documented by fluorescein angiography, and had at least 10 ophthalmoscopically visible macular drusen, characteristic pigmentary alterations, or geographic atrophy; one eye was considered for each patient, the functionally best one in the bilateral forms . Subjects whose visual loss may have been secondary to media opacities or any other ocular disease besides ARMD were excluded. The following were carried out before the first treatment, after the last treatment, and then every 3 months for up to I year: a fully corrected near and distance visual acuity (V .A.) measurement; a complete biomicroscopic and ophthalmoscopic examination, in which the refractive error, iris colour, lens status (aphakia, pseudophakia or initial cataract), disc and macula morphology, and intra-ocular pressure values (measured by applanation tonometry) were specifieally reeorded for eaeh eye. The experimental group consisted of 50 "dry" ARMD patients, who underwent 12-13 sessions ofOrAHT within 6.5-7.5 weeks. Standardized 03-AHT was carried out by exposing about 250 g blood in eitrate-phosphate-dextrose (CPD) to agas mixture eomposed of about 96% O2 and 4% 0 3, with a final 0 3 concentration of 40-70 ug/rnl gas per g of blood (starting with 40 Ilg/m1 and gradually increasing by 5 ug/ml each session to reach 70 ug/ml by the 7th treatment). After about 5 min of thorough mixing, eaeh blood sampIe was reinfused into the donor. This procedure was repeated twiee a week for 12-13 times . As a "control" group, we treated 30 "dry" ARMD patients by exposing the same amount ofblood to O2 alone (the same number ofsessions twice a week).

Funetional ophthalmologie examination included: Mean distance visual acuity (ETDRS charts). Macula threshold test (Humphrey automated perimeter). Self-assessment test ofthe quality ofvision (Carta et al., 1998). OZONETHERAPY IN VARIOUS PATHOLOGIES 293

General examination included:

Blood pressure. Haematochemical data (blood cell count, plasma proteins, plasma lipids, coagulation and fibrinolysis tests) .

These data were recorded at the baseline time (before starting the therapy), at the end of the last 03-AHT sessions, and then every 3 months for up to 1 year. In all the follow-up examinations, the examiners were blind to the results of each patient's initial examination.

4.4. Results 4.4.1. Ophthalmologie Results Change in visual acuity from baseline at each examination was the primary parameter used to verify the response, if any, to 03-AHT. Mean distance visual acuity (ETDRS charts) was significantly improved in ARMD patients (p<0.05), while no significant improvement was observed in the control group. This data was confirmed by the results of the visual field (increased mean sensitivity and foveal threshold in 54% oftreated patients and in 18% ofcontrols) and the self-assessment test of quality of vision (improved quality of vision in 60% of the treated patients and 23% ofcontrols) . In the treatment group, the improvement remained stable in the first quarter and declined slightly in the next six months after the treatment (Fig. 100); at 12 months, up to 41.6% showed either a regression to their pre-treatment values (33.3%) or had worse V.A. (8.3%).

0.30

200 0,25 • • 175 ä 0,20 '" :; ~'" 150 .

0,15 12S

100 0,10 Pr. Post 6 9 12 Pr. Post 6 9 12 Months

Figure 100. Modifications ofvisual acuity before, during and after (3, 6, 9 and 12 months) one cycle 0//3-14 OrAHTsessions. The left diagram reports the actual change, expressed as a percentage ofthe right diagram. 294 CHAPTER24

4.4.2. Laboratory and Biochemical Results

The laboratory resuIts reported in Table 24a show that OrAHT did not cause significant modifications ofcritical parameters measured just before and at the end of the treatment. The levels oftypicalliver enzymes were also unmodified.

Tahle 24a. Laboratory tests carried out in 34 ARAfD patients before and after 12-13 sessions ofOJ-AHT.

PRE FINAL Blood cells: RBC (M / ~I) 4.58 ± 0.7 4.50 ± 0.7 HGB (g/dl) 14± 0.2 13.6 ± 0.2 HCT (%) 41.7 ± 0.6 40.6 ± 0.7 MCV (0) 91 ± 0.8 9 1.4 ± 0.8 MCH (pg) 30.5 ± 0.3 30.7 ± 0.3 MCHC (g/dl) 33.5 ± 0.1 33.5 ± 0.1 PLT (K1~1) 232.2 ± 9.2 237.4 ± 9.8 WBC (KfI.,t1) 6.3 ± 0.3 6.4 ± 0.3 Coagulation tests: Fibrinogen (rng/dl) 293 .6± 12.5 327.6 ± 14.7 F.VIIIvV (%) 151.6±12.8 153.6± 14.0 Fl+2 (nM/I) 1.42±0.14 1.15 ± 0.11 AT III (%) 100.9 ± 3.6 100.9 ± 2.6 PT(%) 96.2 ± 3.1 96.1 ± 1.8 a PTT (sec) 31.7 ± 0.7 30.3 ± 0.6 TT (sec) 19.4±0.7 19.6 ± 0.3 Fibrinolysis tests: t-PA (ng/ml) 11.2 ± 0.8 10.4 ± 0.9 PAli (IU/ml) 11.2 ± 1.4 13.1±1.6 FM test (uz/ml) lO.1 ± 1.2 13.5 ± 3.0 FDP (ug/ml) 6.4 ± 0.8 7.6 ± 2.4 D dimer (ng/ml) 111.3 ± 5.5 114.5 ± 8.4 Lp (a) (rng/dl) 43.8± 10.4 35.7 ± 8.2 Plate/et tests: PF4 (lU/mi) 4.6 ± 0.8 3.8 ± 0.5 ß-TG (IU/ml) 27.1 ±2.0 29.2 ± 2.9 Plasma proteins: Proteinemia (g/dl) 6.8 ± 0.4 6.9 ± 0.5 Plasminogen (g/l) 0.12 ± 0.4 0.12 ± 0.6 Fibronectin (mg/dl) 43.3 ± 1.5 45.4 ± 2.2 Plasma lipids: HOL (mgf/dl) 60.2 ± 2.6 54.9 ± 2.8 Cholesterol (mg/dl) 285.5 ± 8.9 278.9 ± 8.5 Triglycerides (mg/dl) 119.9 ± 13.9 114.4± 10.5 ÜZONETHERAPY IN VARIOUS PATHOLOGIES 295

Table 24b reports the data on TBARS, 2,3-DPG and SüD content measured in the blood of the ARMD patients before and at the 5th, 9th and 13th Ü3-AHT treatment. Unfortunately, these determinations could be reliably carried out in only some of the patients. It appears that there is no increased peroxidation and 2,3-DPG levels remained constant. SüD levels increased after the first five sessions and then retumed to normal. The slight increase ofG-6PD was also not significant.

Table 24b. Evaluation ofplasma TBARS, 2.3-DPG content, SOD and G-6PD activities before (0), at the 5th (5), 9th (9) and 13th (13) OrAHTtreatment in ARMD patients.

Parameters Treatment () 5 9 13 TBARS 32.6±8.10 (15) 28.4±8.9 (15) 36.4±13.7 (16) 26 .1±6.4 (15)

2,3-DPG 1.95±O.06 (25) 2.07±O.09 (25) 1.95±O.10 (23) 1.86±O.08 (22)

SüD 1.51±0.20 (14) 1.83±O.22 (14) 1.37±O.Ol (16) 1.45±0.2 (13)

G-6PD 7.46±O.89 (9) 7.76±O.62 (9) Values are expressed as mean ± S.E. No significant differences were observed. Number of observations in brackets .

4.4.3. Side-effects and compliance We did not observe any important side effects during or after the treatment; thus we can state that, in our experience, the procedure was safe . Actually, most of the patients reported an improvement of their general condition, particularly in terms of increased strength, mental concentration and memory. The on1y noticeable problem was that a few patients had poor venous access and this often required more than one venipuncture. Compliance to the treatment was excellent, even in the patients who showed no improvement and it must be emphasised that most of the patients undertook two long journeys week1y to receive the treatment. On the other hand, adherence to the se1ected follow-up examination was not fully satisfactory.

4.4.4. Conclusions

At the present time, there is no effective medical therapy for dry ARMD: the use of minerals and antioxidant vitamins, while harmless, may delay but does not recover vision loss. Most ARMD patients, still physically and mentally active, are very concemed about the lack of an effective treatment and although there are now interesting avenues of research, it will take time before the results appear on the pharmacist's shelf. On this basis, ozonetherapy could be proposed to ARMD patients as a reliable cornplementary therapy able to offer, in most patients, a significant 296 CHAPTER24

general improvement of the quality of vision, so that they can experience a better lifestyle.

5. DERMATOLOGICAL DISEASES In 1989, the Director of the Institute of Dermatology of Siena University surprisingly asked me to help study the value of O)-AHT in psoriasis, because one of his colleagues was privately performing this therapy and c1aiming great success. Unfortunately, at that time I had no practical experience. Firstly, I noted that an assistant 'collected ozone in a syringe and, without closing the tip,' walked a long corridor before insufflating it into the blood bottle. Secondly, they assured me that the ozone concentration must be very 10w (probably 2-5 ug/ml) and it was not really very important because blood would become "very red" in any case. The surprising result was that after 7-10 treatments, one patient showed extraordinary improvement, another was slightly better and three patients remained the same. They tried to publish a paper but it was rejected because there were no controls. Thus my first c1inical experience was very disconcerting. Since then, I have heard several other anecdotes of splendid results and 11eave the judgement to the readers. In spite of this puzzling story, it seems that Russian dermatologists and a German ozonetherapist (Kief, 1993b) also succeeded in treating various forms of eczema and atopic dermatitis (AD), which, to my knowledge, is challenging and frustrating to treat. I had been interested in this disease from an immunological point of view : the hallmark of AD is a Th l/Th2 imbalance (Bohn and Bauer, 1997; Campbell et al., 1999) with a reduced production of IFNy and an elevated release of IL-4 and IL-5, which favours IgE production and eosinophilia, a typical disorder of atopic diathesis (Beltrani, 1999; Leung, 1999) . Prophylactic measures such as avoidance of irritants, allergic food (eggs, soy, peanuts, etc.), contact with house-dust mites or other aeroallergens, are helpful but the mainstays of therapy have been and still are topical corticosteroids. More recently, in severe forms , phototherapy, cyclosporin A, azathioprine and tacrol imus ointment appear to be effective but with some side effects (Rudikoff and Lebwohl , 1998; Hanifin and Tofte, 1999; Fleischer Jr., 1999). Does ozonetherapy have any future in dermatology? If seriously performed, 0 )­ AHT may be useful and my "gut feeling" is that we should progressively try from medium (40-50 ug/ml) to high ozone concentrations (80 ug/ml), as discussed in Chapter 14 (Leukocytes), to readjust the Thl/Th2 balance. However, it should be remembered that this is a proposal that may not be effective. The BOEX procedure, combining systemic and cutaneous treatment, may be an ideal approach. However, patients with these diseases are often very distressed and understandably anxious to receive the most effective treatment immediately. Therefore, they are not interested in our controversies and this rnakes it awkward to recruit patients for a RCT . Ozonetherapy may yield some benefit at a slow pace and patients will accept it only if, at least in the initial period, they are assisted with the proven topical anti-inflammatory options. OZONETHERAPY IN VARIOUS PATHOLOGIES 297

6. PULMONARY DISEASES (EMPHYSEMA, ASTHMA, COPD AND ARDS)

This secnon is dedicated 10 the memory ofDr. Maria Trusso. Ozonetherapy has not yet been tested in pulmonary disease, probably because everybody knows that breathing air polluted with ozone is toxic to the respiratory system (Kelly et al., 1995). Two or three papers on this topic are published every week in international journals and this problem has been discussed in Chapters 3 and 5. However, an almost irrelevant episode that occurred about four years aga suggests that this fact has misled us. Among our numerous ARMD patients treated with 0 3­ AHT, one, with emphysema, told us that after about fourteen sessions his dyspnea was alleviated and he could walk up to the third floor of his apartment with little effort. I sensed that he had given us a good tip and I took him to the Pneumology Unit where the specialist, Dr. Maria Trusso, was bewildered by the result. Actually, at first she imagined that the treatment for ARMD was based on breathing ozone and the proposal to continue this sort of treatment appeared crazy to her. After 1 explained that we simply ozonized and reinfused the patient's blood, she became interested and correctly asked how ozonized blood eould improve lung funetion. That was a good question and I had to use the best ofmy imagination to provide a few answers: the simple one was improved blood oxygenation. Yet this is not quite valid because, although we reinfuse hyperoxygenated blood (p02 is easily at 500 mmHg), the infusion rate is so small (about 15 ml per minute, compared with a cardiae output of about 5 L) that the p02 of venous blood reaehing the lungs is hardly modified. However, if ozonetherapy enhanees the delivery of oxygen at the tissue level, metabolie conditions may improve, even though ideas such as decreased blood viscosity and increased 2,3-DPG levels in erythrocytes have not been definitively demonstrated.I advaneed the hypothesis that perhaps ozonized blood acting on endothelium could aetivate the release of prostacyclin. Only last year, we deteeted an enhanced release of NO in HUVECs in vitro, but we have not yet evaluated prostaeyclin. It appears that release ofNO and NOthiols may represent a mechanism for vasodilatation, although NO could also be a double-edged sword (Barnes and Liew, 1995; Warren and Higenbottam, 1996; Jindal and Dellinger, 2000) . A eommon denominator of asthma, emphysema, chronie obstructive pulmonary disease (COPD) and aeute respiratory distress syndrome (ARDS) is oxidative stress. It is demonstrated by an increase ofROS (H202)and 8-isoprostane, activation ofNF-KB with inereased synthesis ofTNF-a, IL-6, IL-8, and inaetivation (by oxidative damage) of o l-antitrypsin and leukoproteinase inhibitor, unable to counteraet elastase, cathepsins and matrix metalloproteinases (Smith et al., 1997; Barnes, 2000). In 1996, we postulated that the paradoxieal action of ozone in inducing an adaptation to COS could be critical in readjusting the oxidant-antioxidant balance. Finally, we searehed for other eases of emphysema and COPD . We found two patients, a man and a woman, who after two eycles of therapy had noticed an improvement in their performance of daily activities . This response was subjective and could have been due to a placebo effect, but it encouraged us to make a protocol. The Director of the Pneumology Unit agreed although he was somewhat skeptical, but not so much as the University's Professor of Pneumology, who was 298 CHAPTER24

absolutely antagonistic. The protocol was then prepared, submitted to the Ethical Committee and, after revision, approved after about seven months. Unfortunately, the health of Dr. Trusso deteriorated (she had a metastatie breast tumour) and she died shortly afterward, leaving four young children practically alone. We lost a very nice, energetic woman, who after accepting the idea became very enthusiastic to try this unusual therapy . I hoped that one of her colleagues would accept the challenge, but he thought that the project was too laborious to develop and uncertain, so I found myself back where I started. Nothing has been done, but luckily there has been some progress in conventional Medicine, since COPD morbidity and mortality is increasing due to the use oftobacco. Indeed it may soon become the third most common cause ofdeath. In addition to rehabilitation with exercise training, anti-smoking measures and domiciliary oxygen therapy, new bronchodilators and appropriate antibiotics can control acute exacerbations. After a long incubation (1957), surgical removal of the most emphysematous parts of the lung has come of age; when the operation is successful, short-term results are good, with marked improvement of the quality of life (Hillerdal 1997; Bames, 2000). However, some of the patients do not benefit from surgery (National Emphysema Treatment Trial Research Group , 2001) and the value and cost­ effectiveness of the volume reduction surgery remain uncertain in the long run , Moreover, medieal expenditures to treat COPD, associated with invalidity , represent a significant economic and social burden for Health Authorities and society in general. I believe that these are sufficiently good reasons to justify serious and wide­ ranging experimentation with ozonetherapy. We can take advantage of OJ-AHT and even more of BaEX and RI (non-invasive procedures), always starting with a low dose (20 ug/rnl) with gradual dose escalation up to 35-40 ug/rnl. Needless to say, supporting measures, particularly oxygen therapy, remain available to the patient.

7. RENAL DISEASES Prof. N. Di Paolo is the head physician of the Nephrology and Dialysis Unit at the Siena Polyclinic. He is a good friend of mine and his role in developing EBOO has been crucial. However, when I proposed to evaluate ozonetherapy in not too advanced dialysis patients, he answered that the kidney does not have the regenerative ability of liver and there is no hope to improve the function of a sclerotic kidney . He may weil be right in regard to terminal patients but my idea was to limit progression, possibly achieving regression of initial chronic diseases . A few recent reports have further convinced me that during infective glomerulo-nephritis or at the initial stage ofrenal failure, ozonetherapy could have a beneficial influence . It is not worth repeating the usual mechanisms, but I wish to re-emphasize the possibility of adaptation to COS, typically present in chronic renal failure (Ceballos­ Picot et al., 1996b; Witko-Sarsat et al., 1998). It is weil established that haemodialysis in itself generates RaS and consequently enhances oxidative stress. I feel that it is wrong to passively accept the concept of unavoidable irreversibility because, in addition to potentiating the antioxidant system, ozonetherapy may switch on angiogenesis or favour the release of unknown nephropoietins. Unscientifically, OZONETHERAPY IN VARIOUS PATHOLOGIES 299

I believe that ozone is a stimulator of untapped natural resources and we know that if we can just give a little help to Nature, she may respond in ways that are wonderful and unsuspected by our little minds. Einstein used to say that "imagination is more important than knowledge". Ruggenenti et al. (200 I) have given a moral boost to my belief because they expertly say that today "nephropathies lack a specific treatment and progress relentlessly to end-stage renal disease". In order to delay or avoid dialysis (which would be a great success), nephrologists should develop a combined approach to renal diseases by pharmacologically controlling blood pressure and loss of proteins. I now like the idea of adding ozonetherapy performed at low-rnedium ozone concentrations for long periods. Selected patients could undergo 1-2 EBOO treatments every week during the final hour of dialysis. The ozonizer could be simply placed after the dialysis filter with blood exposed to very low ozone concentration (1-2 ug/ml). The lack of side effects, the modest adjunctive cost, the control of hyperoxidative state and the feeling of wellness are the most obvious and eloquent advantages. I agree that the study of gene and stem-cell biology will favour the advancement ofscience and produce important therapeutic innovations, but common sense also suggests that the possibility of stimulating natural resources should not be neglected. Moreover, we should keep in mind that a combination of therapeutic approaches, and not the reductionist simplification of substituting a gene, is most likely the best way to effectively treat the multiform manifestations of a chronie disease.

8. HAEMATOLOGICAL DISEASES ß thalassaemia major, fairly frequent in Italy, and sickle cell anaemia (SCA), affecting black populations, are genetic diseases leading to oxygen blood deficiency and other serious manifestations. Ozonetherapy cannot correct the gene alteration but in SCA it can apparently reduce the frequency of vessel occ1usive crises with related infarctions. SCA involves a modified Hb (Hbs), which tends to crystallize during deoxygenation ; this leads to a change in the shape ofthe erythrocytes, which aggregate and cause vessel occ1usion. At least conceptually, ozonetherapy may increase oxygenation and, as often c1aimed, may improve cell pliability, although I am not convinced about this change. A RCT, which must be reported because they are so rare in this field, was performed in 55 SCA patients (25 control and 30 experimental) at the National Center for Scientific Research at Havana. Ozone was administered daily (5 days per week) for 3 weeks in 30 patients via the rectal route. The control group received only analgesics, vasodilatators and IV saline infusion. The results showed that the ozone-treated group displayed a rise in arterial blood pOz,the frequency and severity ofpainful crises was significantly reduced (by about 50%) and there were no adverse reactions (Gomez et al., 1995). This work was supported by a firm producing ozone generators and it is surprising that no further experimentation has been reported. 300 CHAPTER24

SCA is a serious disease and it suffices to say that only 2% of about 120,000 affected babies born in Africa survive to the age of five . What can official Medicine do to help patients in poor countries? Practically nothing, because transplantation can only be perfonned in about 1% of patients: in 1999, the number of bone marrow transplants was only 100 and 800 for SCA and ß-thalassaemia, respectively. Moreover, 10-15% ofpatients do not survive. For SCA, it would be important to have an effective and atoxic oral drug that could be widely and easily used . So far, hydroxyurea appears useful as it reduces Hbs and increases the percentage of HbF, but the drug is mutagenic and somewhat toxic (Steinberg, 1999). Clotrimazole, a specific Ca2+-activated K-I- channel (Gardos channel) inhibitor able to reduce the deleterious dehydration of sickle erythrocytes, is being tested and seems promising (Brugnara et al., 1996) . Morris et al. (2000) have reported that oral arginine administration may benefit SCA patients by increasing NO production during a vaso-occlusive crisis. Clearly, these approaches are experimental and only partly satisfactory and the promise of gene therapy is far from being materialized. Thus, I do not see anything wrong in using ozone; with small generators, patients (after careful instruction) could do horne autotreatment using RI. However, ozonetherapy has the serious drawback that ozone must be generated and used extempore. The unavailability of generators, medical oxygen, electricity and the need ofan almost daily use for life makes ozonetherapy a solution that cannot be practically proposed in poor countries for SCA, malaria and HIV infection. As always, the possibility of ozonetherapy is never mentioned by official Medicine and 1 must presume that it is unknown. This is one of our weak points, which can only be overcome by presenting good controlled results to peer-reviewed international journals. However, someone has written that hydroxyurea advocates are covering up its long-term carcinogenic potential, but I want to think that this cannot be true . I hope that this book will serve to make this topic known and promote unbiased clinical trials to evaluate the validity ofozonetherapy. Finally, I must mention that I often receive calls frorn desperate people because a relative is affected by a haematological malignancy.I have great respect for what haematologists are able to achieve in these diseases and unless we can produce good experimental evidence, I am not certain that the addition of ozonetherapy would be useful. This aspect will be discussed further in the section "Cancer", although the pathophysiology of solid tumours is immensely different from haematological ones. For the inexpert ozonetherapist, I would like to remark that ozone cannot displaya direct cytotoxic effect on malignant cells.

9. NEURODEGENERATIVE DISEASES

The progressive prolongation of the human life-span is accompanied by an increase ofdegenerative diseases, and those of the CNS are very crippling. There is substantial evidenee that the combination of genetie predisposition, life-Iong oxidative damage, an excessive or poorly balanced diet , exposure to transition metal ions, alcohol and tobaceo smoke intoxieation, lack of physical exercise and diabetes may be responsible for neurodegenerative disorders such as Parkinson', Menkes' OZONETHERAPY IN VARIOUS PATHOLOGIES 301 and Wilson's disease, senile dementia, amyotrophic lateral sclerosis, optic nerve dysfunction, primary open angle glaueoma, neurosensorial bilateral hypoacusia and maeulopathies (Halliwell et al., 1992; Halliwell, 2001; Yu, 1994; Ames et al., 1993; Cohen et al., 1994; Jenner, 1994; Bondy, 1995; Carlsson et al., 1995; Jaeschke, 1995; Pardo et al., 1995; Yoritaka et al., 1996; Simonian and Coyle, 1996; Rotilio et al., 2000; Poli and Schaur, 2000; Rotilio, 2001). Authoritative scientists, such as Ames, Halliwell, Gutteridge, Pryor, Cross, Packer, Rotilio, ete., have suggested that neurodegenerative diseases triggered by an uneertain primary eause are perpetuated as the cellular redox system goes awry. The pathophysiology is quite variable: in some cases, there is ehronic inflammation with the release of ROS and pro-inflammatory cytokines; in other cases, we ean observe a biochemical defect such as low GSH content or a deerease of antioxidant enzymes (GSH-Pxs, SOD, catalase) associated with improper metal binding; in other cases, there is an excessive release of NO' (hence ONOO) or of noradrenaline from presynaptic terminals or of glutamate with 2 Ca + influx and activation of protein kinases, phospholipases, ete. (Pardo et al., 1995; Nakao et al., 1995; Ceballos-Picot et al., 1996a; Markesbery 1997; Aejmelaeus et al., 1997; Sagara et al., 1998; Floyd 1999; Li et al., 1999; Perry et a\., 2000; Rotilio, 2001). Ozonetherapists must know that there is intense research activity trying to find drugs able to delay or block the neuronal degeneration and death: the usual hydrophilie and lipophilie antioxidants taken in appropriate amounts via os are not harmful but are minimally effeetive (McCall and Frei, 1999), also because only a small percentage reach the CNS. Metal chelators may help by reducing free transition metals and OH' formation, but one must pay attention not to exceed with the dose. Moreover, several inhibitors ofthe reuptake of doparnine, ofNO' synthesis and of ionotropic reeeptors to block glutamate neurotoxicity are being tested. The more biologically oriented approaches are attempting to use neurotrophic factors or to transplant dopaminergic foetal cells (or, probably very soon, stern cells) into seleeted areas (Weber and Buteher, 2001). Among neurodegenerative diseases, Parkinson's disease is the ideal one, because the degeneration is fairly restricted to partieular areas ofdopaminergic neurons (Lang and Lozano, 1998a,b). In spite of all this tremendous effort and biIlions of dollars spent in basic and clinieal research, we can eonc1ude that pharmaeologicaI therapy is certainly useful (levodopa is still the most effective therapy after three decades !) but only for a limited time and it does not arrest progression of the disease. The combination of several experimental therapies promises to improve on the present limitations, but still we are fighting a virtually lost war beeause neurodegenerative diseases are projected to surpass even cancer as the second eause of death by the year 2040 (Lilienfeld and Perl, 1993). Can ozonetherapy be of any use? At first glance, it seems irrational to propose a treatment of neurodegenerative diseases based on aseries of brief and ealculated oxidative stress (therapeutie "shock"). However, this approach (which is not a panacea) may paradoxically break and stabilize an otherwise irreversible situation. The idea (already discussed in Chapter 22) is that a gradual escalation ofthe ozone dose (from 15 to 40 ug/ml) may 302 CHAPTER24

be able to induce adaptation to COS, which in practical tenns means that, by gradually receiving trace amounts of LOPs, neuronal cells under oxidative stress may reactivate the depressed synthesis of antioxidant enzymes. Today there is no phannacological approach able to achieve this objective, which instead can be realized, without any biotechnological trick, simply by ozonizing blood for a few minutes. More than ever, I persevere in my idea that if neurodegeneration is not due to an irreversible genetic defect (like amyotrophic lateral sclerosis (ALS) or Lou Gehrig's disease, for example), judicious administration of ozone can be helpful. In Chapter 2, I related the awful story I had with the Editor of FRBM, who refused to send me the offensive referees' comments on my minireview proposing the concept of adaptation to COS . More than ever, I am convinced that excessive dogmatism in biology and medicine can be very dull. While I am aware, and I repeat to everyone, that ozone is intrinsically toxic and must be used with care, I do not see any risk in evaluating this problem with 0 3-AHT, or RI, or BOEX. At worst, we will not obtain any positive result but patients will not be harmed. Actually, if it can be believed and in support of my stubbomness, as early as 1993 in Cuba , Rodriguez et al. performed a double blind RCT on 60 patients affected by senile dementias: group A (30 patients) was treated with O2-03 by daily RI (50 ug/ml) for 21 days and group B with O2 only . This was an important pioneering study . Using several psychometric tests (mental condition, capacity for self-administered medication and evaluation of daily activities), it demonstrated that 73-90% of ozone-treated patients showed marked improvement without any adverse effect. In 1995, at the XII IOA Congress in Lilie, Gomez Moraleda summarized several studies carried out in Cuba since 1990. Use of OrAHT in optic nerve dysfunction, primary open angle glaucoma, cochleo-vestibular syndrome and ischaemic cerebro-vascular disease yielded an improvement ranging frorn 50 to 100%. Therefore, in Europe and the USA, we are already a decade too late and if ozonetherapy is really useful , to how many patients have we denied this possibility? Our inadequacy and short-sightedness is unforgivable! If we are ever able to perform a study , it will be important not only to evaluate the clinical effects but also to c1arify the mechanism of action. At the moment, I can only advance a few speculations: ozonetherapy can simultaneously improve blood flow and oxygen supply to hypoxie tissue, thus stimulating aerobic glycolysis in hypofunctional cells, which by resuming normal metabolism might restore the normal ATP content and GSH /GSSG ratio . LOPs generated during lipoperoxidation of plasma or absorbed from the rectal mucosa (R!) or the skin (BOEX) will be diluted in the plasma pool and in part can pass through the blood-brain barrier to reach the sites of neurodegeneration and upregulate the cellular synthesis of antioxidant enzymes, which is the crucial step to readjust the impaired cell redox system. The release of neuronal growth honnones and the activation of resident stern cells remain speculative, but they are not too far-fetched ideas . The possibility that Alzheimer's disease, associated with adeposition of insoluble ß-amyloid aggregates, reflects an NO·/superoxide imbalance has been entertained by Thomas et al. (1996) . The therapeutic implication is that a prevalence of NO· over superoxide is advantageous and may inhibit aggregation. This may be achieved by OZONETHERAPY IN VARIOUS PATHOLOGIES 303 the administration of exogenous SOD mimetics andJor antioxidants but, interestingly, 0 3-AHT could correct the imbalance by inducing SOD and the production of NO' at the same time . Two cautionary annotations appear to be in order: the first is that functional recovery may be achieved only in initial or not too advanced patients, and secondly an optimal 03-AHT schedule has not yet been worked out, although it appears reasonable to start with a low 0 3 concentration (15­ 20 ug/rnl) and slowly raise it (in 3-4 weeks) to 35-40 ug/ml per ml ofblood. For RI, I would suggest beginning with 5 ug/ml and proceed to a maximum of30 ug/ml and a volume of 600 ml gas. In this case, I think that the concentration (50 ug/ml) used constantly by Rodriguez et al. (1993) is excessive and frequently causes intestinal cramps. If an improvement really occurs, it may be necessary to continue the treatment (once weekly or biweekly?) for life. It must be explained and understood that one cycle of ozonetherapy cannot be the "eure": all cells have a more or less long biochemical memory and must be stimulated by LOPs at intervals of 1-2 weeks . Our study on ARMD has been very instructive in this sense. Retinal maculopathies deserve a short discussion on their own. Open studies carried out using OrAHT in both ARMD and retinitis pigmentosa showed an improvement of visual activity in about 2/3 of patients (Gomez Moraleda, 1995; Diadori et al, unpublished). Our study is described in a previous section. However, arecent evaluation of 10 patients with retinitis pigmentosa performed in Cuba with a regimen of electrical stimulation, 0 3-AHT and ocular surgery has not validated this multi-technique approach. Actually it suggested that, in comparison to simple vitamin A supplementation, this complex intervention may worsen the course of the disease (Berson et al., 1996; Weleber, 1996). Thus, this problem remains open, since the study examined a genetic disease and used a too complex protocol, in which it was not possible to clarify the exclusive role of03-AHT. Patients with neurodegenerative diseases undergoing ozonetherapy must receive oral antioxidant supplementation (as has been specified in Chapter 22) because they are frequently undernourished and may have a low TAS.

10. CANCER Although a number of haematological cancers are now being treated successfully, the common solid cancers, which are the great majority, continue to be a problem for mankind (Bailar III and Gornik, 1997). Owing to earlier diagnoses and some therapeutic advances, for the first time in western European countries, the total cancer mortality was moderate1y reduced for both sexes in the period 1990-1994 (Levi et al., 1999). However, due to prolongation of the life-span, the figures for overall mortality from cancer (in Italy about 160,000 and in the USA about 520,000 in 1993) are still dramatic. Moreover, in the same period, cancer mortality was still increasing in eastern European countries. This is not likely to change ovemight because a highly desirable improvement of chemotherapeutic compounds, so far rather unspecific and toxic , may come too slowly . An appropriate cancer prevention campaign, aimed at early detection and the use of an appropriate diet rich in fibre and antioxidants (Dreher and Junod , 1996; Bailar III and Gamick, 1997; Kramer and 304 CHAPTER24

Klausner, 1997), may help up to a point. Yet, on the whole, smoking is not decreasing and has partly shifted from men to women and to Third World countries. At least theoretically, immunotherapy (the fifth modality of cancer treatment) aims specifically at destroying only neoplastic cells, but unfortunately these cells are poorly immunogenic and diabolically equipped to evade or suppress the immune system. In addition to showing the multiform conventional anti-neoplastic therapy, Figure 101 indicates that since 1980 a considerable effort has been made to develop new and efficient immunotherapeutic approaches, which however have failed to achieve substantial advances (Rosenberg et al., 1987; Rosenberg, 200 I; Bocci, 1985, 1987b, 1990b; Kim et al., 1996; Fenton et al., 1996; Wemer and Jolles , 1996; Reddy et al., 1997; Ernst, 1997; Motzer et al., 2001) .

SURGERY 11 RADIOTHERAPY 1 ICHEMOTHERAPY I I HORMONE THERAPY

1) Hlgh-Intenslty chemotherapy, regtonal RECENT DEVELOPMENTS adminIstration wlth Inhibitors of I\~- •. I chemoreslstance and wlth granulopoletlns rescue

~ 1 2) Hyperthermla Therapy w ith anti sen se oligodeoxynucleolides '------' I 3) Photodynamlc therapy I Therapy with ribozymes I 4) Bone marrow transplantation I

S) Immunotherapy: a) wlthlmmunomodulatory compounds (thymJc ( Anliangiogenetic therapy ) hormones, melatonln, etc .) b) wlth Inducers or cytoklnes c) wlth exegenuus cytcklnes (lFN ,IL-2, -12, TNFa) wlth or wlthout adoptlve Immunotherapy (LAI(, T1L) d) wlth gene therapy e) wlth twnour vacctnes and dendrltlc cetls (APe) FUTURE DEVELOPMENTS o wlth dllTerenUaUng agents (all trans RA. tamoxlfen) g) wlth radloactlve and toxle 8I1tlbodles (the old maglc bulletI)

Figure J0J. The various aspects ofconventional antineoplastic therapy

Immunological gene therapy works weil in experimental murine tuu.ours, but so far has been disappointing in patients (Anderson, 1992; Bubenick, 1996; Roth and Cristiano, 1997; Parmiani et al., 2000) . The greatest hurdle for successful cancer therapy is a thorough understanding ofthe several mechanisms used by tumour cells to evade the immune attack . The latest disappointment has been anti-angiogenic therapy; in spite ofa perfect rationale (Carmeliet and Jain, 2000), it works very weil in mice (O'Reilly et aI., 1997; Boehm et al., 1997; Perletti et al., 2000) but not, as we hoped, in human tumours, even though angiogenic inhibitors (Oehier and Bicknell, 2000) combined with other drugs may still play an important role. Thus, after all the untimely and deleterious propaganda of the mass media, it is not surprising that desperate patients are always looking for other possibilities, OZONETHERAPY IN VARIOUS PATHOLOGIES 305 particularly in the vast field 'of complementary medical practices (Cassileth and Chapman, 1996; Burstein et al., 1999) such as diet, nutrition and lifestyle changes, therapeutic touch (Rosa et al., 1998), mind-body control (Flach and Seachrist, 1994) and anthroposophie medicine based on the use of mistletoe lectins (Bocci, 1993b; Ernst, 2001; Steuer-Vogt et al., 2001). In June 1995, the National Institutes of Health (NIH, Bethesda, MD, USA) included the use of oxidizing agents (ozone, hydrogen peroxide) in class 5, among chelation and metabolie therapies, cell treatment and anti-oxidizing agents. It is noteworthy that H20 2 has been evaluated as an anti-neoplastic agent by Zanvil Cohn at the Rockefeller University (Nathan et al., 1979; Nathan and Cohn, 1981). Other studies have been performed by Sasaki et al. (1967) and Samoszuk et al. (1989). At an earlier stage, ozone was tested in cancer by Varro (1966, 1974, 1983) and Zabel (1960). Thus, although ozonetherapy is more than 40 years old, it has been carried out in a few private clinics in central Europe but it has never been accepted by official Medicine and is currently despised in France, England, the USA and barely tolerated in Italy. Several reasons, mostly right but partly wrong, have been discussed at length previously (Chapters 2 and 11). Is ozonetherapy useful in cancer? Varro (1983) claimed that, after undergoing surgery, chemotherapy and radiotherapy, most of his private cancer patients benefited from ozonetherapy, as their quality of life improved and they survived for a long period. However, these statements were not validated by statistical data and have no scientific value. There are other anecdotal reports of major or minor autohaemotherapy having beneficial effects: for example, Beyerle (1996) treated prostate cancer with "phenornenal" (?) results. For other types of cancer (throat, ovarian, colon and breast), he comments: "We are seeing patients who were bedridden two years ago and sent home to die. They are becoming ambulatory. Their energy level is coming up. They are gaining weight. And we see these spontaneous fractures in the spine are gradually disappearing. Strength is retuming to the musculature. There is no spinal pain". It is unclear why Dr. Beyerle has not reported the data in a peer-reviewed medical journal, because as presented they are worthless. His comments were actually recorded by a journalist (Null, 1996) during an interview published, fancy that!, in Penthouse, where certainly you can admire beautiful women! Another confusing example was the abstract entitled "Ozonetherapy in oncology", surprisingly selected for presentation at the 12th Ozone World Congress in Lille in May 1995 (Baltin). If one reads the abstract, it becomes clear why ozonetherapy has such a low reputation in the medical field. Kief (1993a), at his clinic at Ludwigshafen (Germany), has used Auto-homologous Immunetherapy (AHIT) to treat a variety of malignancies . AHIT was administered daily for aperiod of four months and he claimed that it is: "cost-effective, individually-oriented, has no-side effects, decreases pain in 70% of alI cases and increases the life-qualityand vitality in approximately 90% ofthe cancer patients", What AHIT really was remains a mystery (apparently a mixture of the patient's blood and urine treated with ozone!) and, to the best of my knowledge, the German Health Authorities have now prohibited its use. My personal experience is very 306 CHAPTER24 limited, mostly because I have found that oncologists are very reluctant to evaluate ozonetherapy. The only three cases that I had a chance to follow (two terminal lung carcinoma and one metastatic and ulcerated breast carcinoma) did not show, as expected, any objective improvement after repeated o.,-AHT. My feeling is that once the disease has reached the point ofno return, any therapy becomes practically useless. In conclusion, today there is no evidence that ozonetherapy can be beneficial to cancer patients because:

• Randomized, double-blind clinical trials have not been performed as they should have been done (Ernst and Resch, 1996). • It is unclear whether biological and/or clinical effects, if any, are due to either oxygen or ozone or to both, or simply to blood retransfusion. • The relevance ofthe placebo effect is unknown. • Too often ozonetherapy is carried out together with other conventional or natural therapies, so that any result remains questionable.

In spite of these negative conclusions, let me enumerate (Table 25) and discuss some biological mechanisms of action that potentialIy could be activated by ozonetherapy.

Tahle 25. Possible mechanisms ofaction ofozonetherapy in cancer.

I) Direct effect ofoxygen-ozone on cancer cells in vitro and in vivo. 2) Improved oxygenation and metabolism. 3) Potential upregulation ofthe antioxidant enzymatic system with improvement ofthe celIular redox potential. 4) Effects on the immune system. 5) Effects on the CNS and endocrine system. Therapy ofcancer-related fatigue .

I) Direct effect ofoxygen-ozone on cancer cells in vitro and in vivo. Solid tumours generally tend to create an ideal microenvironment for their growth, characterized by hypoxia, increased glycolysis and a high intracellular level of ascorbic acid. Hypoxia varies widely for different neoplasias but, on average, oxygen partial pressure can be between 1 and 10 mmHg (normal cells have a p02 between 40 and 50 mmHg) depending on the distance from a nutrient vessel and the rate ofblood flow. CelIs beyond the O2 diffusion limit are anoxic and die, but hypoxic celIs remain viable, clonogenic and resistant to therapy . Indeed the rate ofmalignancy is positively corre1ated with the over-expression of hypoxia-inducible factor l« (HIF-I) which, by upregulating the expression of transferrin, VEGF, endothelin-I and inducible NO synthase, enhances vasodilatation, neoangiogenesis and tumour metastasis (Brown and Giaccia, 1998; Zhong et al., 1999). Interestingly, glioblastoma multi forme, "the terminator", expresses HIF-Ia very strongly. Warburg's work in the 1920s demonstrated that, even in hypoxia, cancer celIs intensely convert glucose to OZONETHERAPY IN V ARIOUS PATHOLOGIES 307

lactic acid , but unless they are in anoxia their intracellular pH remains neutral (pH 7.0-7.2) while the pH is slightly acidic (6.8) in the interstitial fluid . It is also noteworthy that epithelial and haematopoietic tumour cell lines actively take up DHA and, by reductase activity, reduce it to AR (Agus et al., 1999). Vitamin C has many functions (Chapter 12) but the administration of megadoses, as often done by charlatans, may even give a metabolic advantage to malignant cells . Throughout the years, I have found reviews on tumour hypoxia very stimulating (Coleman 1988; Brown, 1999; Vaupel and Hockei , 2000; Hockel and Vaupel, 2001). In conclusion, improving tumour oxygenation by significantly and constantly increasing O2 availability and the microcirculation may slow down tumour growth and inhibit metastatization. Can ozone be of any help? It has been shown (Sweet et al., 1980; Van der Zee et al., 1987; Washuttl et al., 1990) that the growth of human cancer is inhibited by ozone in culture, suggesting that cancer cells have an impaired defence system against ozone damage. The fact that cancer cells live better in a hypoxie environment may imply that they have a rudimentary antioxidant system to get rid of ROS . However, it remains uncertain whether this is true for all human tumour cells in vivo , as they have high levels ofAR and produce large amounts of H20 2 (Szatrowski and Nathan, 1991). It also remains controversial whether lipid peroxidation is low or high in tumours and when 4-HNE induces cell proliferation or cell death (Dianzani 1993; Kondo et al., 1999). This is an important point because ozonization ofblood produces LOPs which, upon blood reinfusion, could exert important cytotoxie effects on neoplastic cells if they have a poor defensive system. Zanker and Kroczek (1990) found that incubating neoplastic cells in the continuous presence of a low dose ofozone «0.5 ppm) for 24 h was distinctly cytotoxic. Moreover, ozone was able to potentiate the cytotoxicity of 5-fluorouracil (5-FU) and to increase the sensitivity in a 5-FU­ resistant colon carcinoma variant in vitro . However, I do not feel that the direct cytotoxic action of ozone in tissue culture is important and may be misleading because cancer cells never come in direct contact with ozone, not even when a mad charlatan injects the gas intravenously. In practice, a cytotoxic effect could be obtained only by intratumoural injection of0 2-0J. Cutaneöus malignancies could be infiltrated with gas or ozonized H20 or oil. Hepatic metastasis could be embolized with OrOJ via the hepatic artery (Chapter 16). 2) Improved oxygenation and metabolism This topic received attention in Chapter 14 (Erythrocytes) and again in this chapter when we examined the problem in ischaemic tissues after reinfusion of ozonized blood. Oxygenation ofblood during ozonization has a negligible value, but the biochemical modifications induced by ozone on erythrocytes seem important because ischaemic muscles and perhaps hypoxie tumours will receive more oxygen. In the case of muscles, after ozonetherapy, the venous p02 decreased to 10-15 mmHg instead of 35-40, suggesting that oxygen was delivered to the ischaemic tissue (Rokitansky et al., 1981) . If indeed ozone is able to induce new generations of "supergifted erythrocytes", with an increased content of ATP, 2,3-DPG, antioxidant enzymes as well as an 308 CHAPTER24

elevated G6PD (Hemandez et al., 1995; Viebahn-Hansler, 1996; Bocci, 1996c), by continuing ozonetherapy for several months (actually for life), it may be possible to profoundly modify their funct ional activities so that delivery of oxygen increases and tumour tissues pass from a hypoxie to a normoxic state. If this happens, it will dramatically change the tumour microenvironment, probably leading neoplastic cells to a dormant or a very vulnerable state, Vaupel and Hockel (2000) examined several possibilities to improve the oxygen availability to neoplastic tissue: normobaric hyperoxia (100% O2plus carbogen), hyperbaric hyperoxia, modification of 02-Hb affinity (shift to the right of the Hb02 sigmoid curve), anaemia correction with transfusions and/or erythropoietin administration. All of these ways are correct but more or less impractical and transitory. Obviously, ozonetherapy was not inc1uded among these strategies, because it is a poorly known approach. It must be c1ear that a few OJ-AHT treatments are practically useless and that we cannot permanently change the biochemistry of mature circulating erythrocytes. I feel that this a crucial point to keep in mind. It is only during the phase of erythroblast differentiation in the bone marrow that we can modify the cellular biochemical machinery by means of the LOP messengers produced during ozonization of blood just before reinfusion. Considering the erythrocyte half-life, a schedule of two sessions per week ofOJ-AHT (225 ml blood and maximum OJ concentration of40 ug/ml per ml of blood) and the dilution and catabolism of LOPs, it may be possible in 6 months (48 sessions) to substitute the normal erythrocyte population with a majority of "supergifted" erythrocytes able to normalize oxygen levels in neoplastic tissues. After each 03-AHT, a new cohort of young erythrocytes will appear in the blood pool , replacing old and less effective erythrocytes. Moreover, by acting on endothelial cells, LOPs enhance NO and NOthiol formation, which may further increase the O 2 supply by improving the tumour microcirculation. The consequence of a desirable downregulation of HIF-I will be an inhibition ofangiogenesis and tumour metastasis. An additional (and not trivial) effect may be a generalized metabolic improvement involving the immune and neuro-endocrine system, with a tolerable quality of life for the patient. This is not a wild hypothesis: it already has some experimental support and the possibiJity of being fully tested by measuring tissue p02 and biochemical parameters of erythrocytes. The ideal methodological approach is OUf EBOO and secondarily BOEX and 03-AHT (Chapter 16). 3) Potential upregulation of the antioxidant enzymatic system with improvement of the cellular redox potential. Ozone is a strong oxidizer and decomposes in a few seconds when it com es in contact with blood. Oxygen per se is practically inactive: it does saturate Hb but although there is an increase of solubilized O2 in the blood water, no formation of lipoperoxides has been detected. In contrast, when ozone dissolves in the plasmatic water, it gives rise immediately to a cascade of ROS and LOPs (Chapter 13). All of these compounds are unselective and in theory can damage blood cel1 components. This is probably the reason why conventional Medicine considers ozonetherapy a dangerous approach, more Iikely to be toxie than therapeutical1y advantageous. However, it must be considered that if ozonization OZONETHERAPY IN VARIOUS PATHOLOGIES 309

of blood is carried out when the ozone dose per gram of blood is precisely known, the reservoir of non-enzymatic and enzymatic antioxidants is capable of minirnizing any possible damage to blood cell components. It has already been explained (Chapters 13 and 14) that during the ozonization procedure a "calculated" oxidative stress must occur in order to generate a certain amount of ROS and LOPs, which act as crucial signalling molecules to elicit biochemical and immunological responses i.e. the therapeutic "shock". This means that the oxidative stress ought to be strong enough to trigger signals above the threshold level (as otherwise they would be ineffective) but also be abated in a very short time by the antioxidant system. Thus ozonetherapy is a procedure that involves a "calculated", very transient oxidative stress capable of inducing cellular responses without adverse effects. The exciting novelty is that ozonetherapy, cautiously carried out with a scaling-up ofozone doses, stimulates the increase of cellular antioxidant enzymes (SOD, GSH-Pxs) capable of inhibiting chronic oxidative stress. What now seems a paradoxical effect ofozone has been described since 1984 as an adaptive response. Olivieri et al. (1984) were probably the first to observe that human lymphocytes undergoing very low doses of ionizing radiation become resistant to a high dose of x-rays, Other studies cited in Chapter 22 clearly showed that animals kept in a hyperoxygenated environment could survive by upregulating the expression and activity ofantioxidant enzymes. This interesting new phenomenon of oxidative stress adaptation may explain, at least in part, why ozonetherapy has a therapeutic activity in ischaemic, degenerative and autoimmune diseases, and possibly in cancer in which a persistent oxidative stress has been noted as a factor favouring the progression of invasion and metastasis (Toyokuni et al., 1995). 4) Effects on the immune system. It is obvious that, due to its strong disinfectant effect, ozone kills bacteria, viruses, fungi, etc., thus facilitating their phagocytosis by leucocytes. The next step was to understand how ozone activates both the humoral and cell-mediated immune system. We discovered that ozone acts as a mild inducer of cytokines because the generated H20 2 crosses the cell membrane freely and activates the cytoplasmic NFKB, ultimately causing the transcription of mRNAs of several cytokines (Los et al., 1995; Sen and Packer, 1996). Since the production of several ILs, TNFa and IFNs is very small and transient , it appears unlikely that induction occurs via stimulation of a membrane lectin. In fact, in the same experimental conditions, the use of amitogen (PHA) that persistently activates a cascade of protein kinases allows the synthesis of cytokines in amounts 1000 times higher than when using ozone. Similarly, the proliferation index of BMC barely increases after blood exposure to ozone. This interpretation is in line with the results of Schreck et al. (1991), who found that human lymphocytes can express specific mRNAs after a transient exposure to 30-100 uM H20 2• It has taken several years of research to understand how ozone works and why even a small activation of BMC can be useful in an immune-depressed patient. If ozone acted as amitogen upon blood reinfusion, causing the massive release of cytokines, we would have noted a frightening clinical response similar to that observed after intravenous injection of LPS, characterized by shivering, 310 CHAPTER24

hyperthennia, hypotension and malaise (Mackensen et al., 1991). Moreover, the disadvantage ofa toxic syndrome that few patients are able to tolerate is associated with the disruption ofthe cytokine network, which is usually deleterious . In contrast, ozonetherapy never causes any side effects, only very rarely a transient tiredness usually followed by a sense of well-being. Nonetheless, after homing in their microenvironments, activated BMC may prime or stimulate neighbouring cells, thus slowly upregulating the immune system . Obviously a single session has no effect, and I would like to propose the same protocol suggested previously to induce "supergifted" erythrocytes. Even a simplistic calculation justifies this way of thinking. The immune system comprises about 1012 cells dispersed in various organs . Yet for each treatment, we can at best transiently activate only about 6 x IOR cells, for which we do not know the life­ span or the pattern of redistribution. It is useful to recall that in the course of 10 11 adoptive immunotherapy, a fairly critical number of about 10 - LAK have to be reinfused after exposure to IL-2 ex vivo (Rosenberg et al., 1987). An aspect deserving some attention is the possibility that, during exposure of the patient's blood ex vivo to ozone, circulating neoplastic cells (from breast, gastric, prostate and colon cancers) could undergo oxidation and become a potential autovaccine. This hypothesis is not too far-fetched because a) tumour cells have been detected in blood (Pantel et al., 1999; Riethmuller et al., 1999); b) they are more sensitive to ROS and LOPs than normal blood cells, and c) they could be taken up by APe. Moreover, HSP-peptide complexes may elicit a CTL response and tumour immunity (Tamura et al., 1997; Wells and Malkovsky, 2000) . 5) Effect on the CNS and endocrine system. Therapy ofcancer-related fatigue. On the basis ofcasual observations, a functional interaction between the nervous system , the endocrine glands and the immune cells has been suspected for decades. Immunological and neuroendocrinological studies (Blalock, 1984; Payne and Krueger, 1992; Reichlein, 1993; Jones and Kennedy, 1993) have c1early shown that these apparently distinct systems are indeed highly integrated. This topic is beyond the scope of this review, but I cannot fail to comment about the pleasant feeling of euphoria and well-being reported by the majority of patients with chronic hepatitis and AMRD during OrAHT. This effect must be kept in mi nd because cancer patients are orten plagued by fatigue. At long last this severe complication is receiving attention and "Cancer" has published a supplement on the topic (92, n.6, September 15, 200 I). Would this effect also occur in cancer patients? Does ozonetherapy trigger a psychoneuroimmunological effect via the release of a cascade of endocrine secretions, namely of CRH, ACTH, cortisol, DHEA, growth honnone, endorphins, melatonin, etc.? Could this effect be due to the withdrawal ofa large blood volume or to the reinfusion of ozonized and oxygenated blood with the stimulatory effect ofLOPs on the endothelial bed? It is not difficult to envisage that a change in the homeostatic balance is bound to evoke a multi-organ response that could positively influence the psychological status ofthe patient, hence the immune response (Fig. 102) (Flach and Seachrist, 1994). How important is the placebo effect (Chapter 25) remains undetennined because even a slight increase of cortisol may improve the mood of the patient OZONETHERAPY IN VARIOUS PATHOLOGIES 311

(Coleman, 1992). Aseries of circumstances, e.g. being attentively cared for and observing the AHT steps with "energized" blood being reinfused, compose the so-called "Hawthorne's effect", which may have a boosting effect and evoke a psycho-hormonal-immune response, the significance of which cannot be under­ estimated (Cassileth et a1., 1991; Cassileth and Chapman, 1996; Trussel, 1999). It is weil documented that a few cancer patients have been miraculously healed after a pilgrimage to Lourdes!

COGNmvE STIMUlI (phys lcll , ehomlell, omDthle> D.

Figure 102. Modern psychoneuroimmunology is based on the interactions among the CNS, endocrine system and immune system. Release ofcytokines by the immune system influences both the CNS and endocrine system. Thus it has become possible to understand the genesis and relevance ofthe placebo effect 312 CHAPTER24

Another important question is whether ozonetherapy should be carried out in the morning or in the aftemoon. Intuitively, I would favour the aftemoon (4-8 PM) because the normal circadian rhythm ought to be least disturbed (Bocci, 1985b). However, only experimental data can define the optimal time of the day, although there is the practical problem that most ofthe work must be done in the moming .

As exemplified in Figure 103, the war on cancer is won when all cells have been killed. There is no doubt that tumour debulking with surgery or other therapies (Fig. 10I) is essential, because a large tumour load or extensive metastasis enhances the anergie state (Elgert et al., 1998) and reduces the chance of a eure . Figure 103 schematically indicates that the primary tumour could be either eradicated or more or less extensively removed. The former case is rare because haematogenous dissemination of individual tumour cells occurs at early stages ofthe malignancy, as has been clearly shown by immunocytochemical detection ofepithelial tumour cells in bone marrow (Riethmuller et al., 1999). death

.:- detection _·...... level ------_. .· . .. . relapse .' .. ·. .. minimal residual disease

o cornplete ablation and eure Time

Figure J03. Tumoural massreduction by cytoreductivetherapy

Thus, if only about 104 neoplastic cells have been disseminated, there is hope of either destroying them or preventing metastatic growth ifthe surveillance ofthe immune system remains active. Today conventional medicine offers several approaches OZONETHERAPY IN VARIOUS PATHOLOGIES 313 attempting to achieve this goal (Fig. 10I). The most promising appear to be immunotherapy and the various forms of gene therapy. However, all these approaches are still experimental and it may take several years before they are validated. If metastases are present, the problem is far more complex and chemotherapy is widely used with mixed results; indeed the side effects frequently impoverish the quality oflife. Can ozonetherapy be more useful than chemotherapy in metastatic cancer? It is very difficult to answer this question because the few anecdotal reports are not valid . Only unbiased, randomized, double-blind clinical studies for several cancer types, possibly carried out in several oncological institutions, can ultimately prove whether ozonetherapy can really be useful . Due to the lack of serious biological and clinical research in the past, this approach remains in limbo today and is totally disregarded by conventional oncology, particularly by chemotherapists. This is very unsatisfactory, mostly because, in spite of some progress, the death rate remains high and real breakthroughs are not yet in sight . Because I feel that this is one ofthe most important issues, I have tried to objectively review several, albeit hypothetical, reasons to pursue the evaluation of ozonetherapy, not as a procedure able to eure the neoplasia but rather as a means to stabilize its progression, particularly in elderly patients susceptible to the serious side effects ofhigh-dose chemotherapy. In the last few years, I have made a useless effort to explain that this approach has a rational basis and can be carried out in a scientific and reproducible fashion. The ozone dose can be precisely adjusted to the blood volume or patient's weight, and optimal ozone concentrations for the proposed methods are based on experimental data and not on homoeopathic or imaginary beliefs. Almost needless to say, a lot of basic work remains to be done, particularly in order to define the molecular and immunological modifications of erythrocytes and leukocytes . Analysis of the adaptation to COS may weil be able to show that ozone can profoundly modify the biochemistry and functionality of these ceUs in order to create an environment hostile to cancer cells. In my opinion, this a new line of thought stating that the cell malignancy can be tamed simply through the use of a potent biological modifier . However, only reliable c1inical data can ultimately inform us about the validity of the approach; indeed we have often observed that an improvement of immunological parameters is not necessarily paralleled by a complete response and prolonged survival (Bocci, 1987b; 1990b; Reddy et a1., 1997). What will be the future ofthis approach? As usual, we are facing the same story . It looks unpromising unless we carry out controlled c1inical trials. At the moment, on the basis of my frustrating experience, I doubt very much that we will be able to perform them, due to the disinterest and skepticism of oncologists. Obviously the pressure of pharmaceutical companies does not allow them to experiment with anything other than cytotoxic drugs. Although I have a great admiration for the scientific strides in biology and medicine, I feet that the biased attitude of most oncologists towards ozonetherapy is wrong and unjustified. On the other hand, most physicians performing ozonetherapy in private practice are unable to perform a c1inical t)-(al and the habit of mixing other therapies makes any conclusion impossible.Donsequently, the only hope is that serious and concerted efforts will be made in the next few years . For the moment, however, it seems that, because of the 314 CHAPTER 24

laek of dialogue and eooperation, a potential therapeutie advantage will eont inue to remain in Iimbo, perhaps to the patient's disadvantage.

11. ORTHOPAEDIC DISEASES. THE PROBLEM OF BACK-ACHE

In the last deeade, a number of orthopaedie surgeons (Riva Sanseverino, 1989; Siemsen, 1995) have begun to treat aeute and ehronie polyarthritis (osteoarthritis of the hip, knee, interphalangeal joints, saeroiliae joint, ete.), tendinitis, myofaseial pain, epicondilitis and earpal tunnel syndrome with intra-artieular or peri-articular insufflation of small volumes of Or03 (5-10 ml in one or three sites with 0 3 eoneentrations from 5 to 15 ug/ml) with very eneouraging results . In Morton's disease (neurorna), up to six infiltrations of O2-03 (4 ml eaeh at 20 ug/rnl have yielded great pain relief. In a review article , Siemsen (1995) reported that applieation of medieal ozone in aeute and ehronie painful diseases ofthe joints is a eomplementary method of treatment to obtain rapid pain relief, deeongestion, disappearanee ofoedema, reduetion of loeal temperature and inereased mobility. If performed by an expert orthopaedie surgeon, the treatment is not risky and eauses only transitory loeal pain that disappears in 5-10 min without any other adverse effeet. The pathophysiology of these diseases is eomplex and eharacterized by the softening and even destruetion of the artieular eartilage, with inereased matrix degradation due to eollagenase and proteoglyeanases. The enzymes may be seereted by aetivated ehondroeytes and monoeytes, whieh release IL-I and TNFo ; Synthesis of POs inereases several fold and there is a natural attempt to maintain a biomeehanieally adequate matrix. In eontrast to RA, pannus does not develop. Joint pain may be aggravated by eoneomitant synovitis. Drug therapy is symptomatie, aiming to reduee pain and disability. Inhibitors of eyelooxygenase I are in wide use, with aceompanying gastritis, ete. and are being substituted with inhibitors of eyclo H. Loeal injeetion ofglueoeortieoids into a given joint ean be earried out no more than twiee per year. Beeause eonventional medieine does not provide a "eure", patients seareh for eomplementary therapies. On the basis ofthe pathology, ozonetherapy should be the last treatment to perform, beeause ozone (a potent oxidant) injeeted into the synovial spaee should elicit further inflammation or degeneration. Therefore, it is incredible that, after initial but tolerable pain, ozone produees great relief for a long time. By now, innumerable patients have been treated and we eannot doubt the results. Obviously ozone is not a "miraculous" medicine and we must try to understand how it aets. On several oeeasions, I have asked orthopaedie surgeons to eollaborate with us beeause I think it would be interesting to examine the synovial eontent before and after ozonetherapy. So far, this has not been possible, either beeause most patients are treated privately or beeause it is difficult to eolleet sampIes. Thus I can only advanee a few speeulations. Onee ozone dissolves in the synovial fluid , it reaets with free proteins, enzymes, proteoglyeans and ehondroeytes and may elicit: OZONETHERAPY IN V ARIOUS PATHOLOGIES 315 a) Inactivation and inhibition of the release of proteolytic enzymes and of endogenous ROS. b) Stimulation of the proliferation of chondrocytes (probably via H202) and fibroblasts, with increased synthesis of matrix and possibly of articular cartilage. Induction of the synthesis of antioxidant enzymes (SOD, GSH-Pxs and catalase) may be a crucial event as an adaptive response to COS and to ozone. That is the reason why I would start infiltrating ozone at low doses. c) Release ofbradykinin and synthesis of inflamrnatory PGs is probably inhibited, with reabsorption ofoedema and pain relief. d) An increased release of IL-I soluble receptor or of other soluble receptors and antagonists able to neutralize IL-I, IL-8, IL-12, IL-15 and TNFa.,just to name a few possible culprits. e) Conversely the release of imrnunosuppressive cytokines, such as TGF-ßI and IL­ 10, may inhibit inflammation. Among several growth factors, TGFß I is interesting because it modulates the expression of integrins and stimulates the synthesis of matrix proteins such as collagen and glycosaminoglycans (TrippeI, 1995; Qi and Scully, 1997). 1fthis is the case, the long period ofremission can be explained.

These are just hypothetical ideas, which shouldbe verified by examining the synovial fluid and bioptic fragments to c1arify these really paradoxical ozone effects. Ozone never ceases to surprise us! In RA, the use of03-AHT at high doses has been suggested (Chapter 14). Yet it remains untested whether the association of one treatment (per week) of 03-AHT at a low-medium ozone dose would improve the outcome in osteoarthritis. Low back pain is a very disturbing symptom that can affect, at least for a while, up to about 80% of the worId's population. Luckily, in most cases, physicaI therap ies (exercise, manipulation therapy, etc.) can solve the problem (Cherkin et al., 1998; Samanta and Beardsley, 1999). However, if a herniated disc (protrusion of the nucleus puIposus through the annulus fibrosus) is present and causes considerable pain, it must be removed with the least invasive procedure. Up to the I970s, the typical orthopaedic operation removed the compression but often destabilized the mechanical and functional stability of the vertebral colurnn. Thus it has been substituted by a mini-invasive intervention. This trend was accentuated by chemonucleolysis, introduced by Smith in 1969. However, the intradiscal injection of chymopapain and collagenase, potent enzymes able to digest the components of the nucleus pulposus, has been abandoned because of occasional risk of allergie reactions and the exorbitant cost of the pure enzymes. Subsequently, Onik et al. (1987) introduced the alternative concept of aspirating the degenerated disc including part of the herniated material, thus reducing the abnormal pressure and relieving the nerve root compression. This technique is still in use with a success rate of about 75% (Bocchi et al., 1998). There are other variants ofthis type ofapproach, the latest being nucleoplasty. In 1988, Verga , a private ozonetherapist, noted pain relief after infiltrating trigger points in myalgias with O2-03 and proposed to use an indireet technique by injecting the gas into the points localizable in the paravertebral muscle (locus 316 CHAPTER24

dolendi) corresponding to the metamer of the hemiated disc , This approach is now widely used by many ozonetherapists in Italy and it can be defined as the indirect approach, or as I call it: "chemical acupuncture" (Bocci, 1998a). The "chymopapain model" probably inspired a neurosurgeon, Jucopilla et al. (1995), to test whether intradiscal injection of ozone would be nucleolytic and beneficial. This can be defined as the direct intradiscal injection of 0 2-03' More recently, another indirect variant has been introduced by the epidural injection of O2­ 0 3 in correspondence to the lesion. This is being performed by anaesthesiologists and seems promising. The use ofO2-0 3 to treat back pain syndrome is now widely used in Italy, while it is unknown abroad. As it is a minimally invasive treatment with a negligible cost and rare side effects, it is worth trying before surgical intervention . At our University, on the basis of our protocol , over 100 patients have been treated and about 80% have shown marked improvement (Bocchi et al., 2000). Thus there are as many as three technical approaches, which are exemplified in Figure 104.

EP CA ID

Figure J04. Schematic view ofa transverse section ofthe lumbarregion: NP: nucleus pulposus. MC: medullarycana!. Thearrows indicate the three possible routes O!Or03 administration. ID: intradiscal; CA: "chemical acupuncture" in theparavertebral muscle, PM and EP: epidural injection OZONETHERAPY IN V ARIOUS PATHOLOGIES 317

The Direct Approach A clear view of the L4-L5 intersomatic space with the needle just before direct insufflation ofO2-0 3 is shown in Figure 105 (Andreula, 2001; Simonetti et al., 2001). The direct approach is carried out under radioscopic control and an expert can do it in about 10min. After a rest of 10-15 min, the patient can get up and often he/she is amazed by the disappearance ofthe pain, as occurs after nucleoplasty. Ifnecessary, the application can be repeated a.second time before changing the approach.

Figure 105. The intradiscal approachfor direct injection o/OrOJ into the nucleus pulposus. The radiograph (above) shows the correct positioning 0/the needle in afrontal scan (Andreula , 2001). Discographie view (below) 0/a transverse lesion in the fibres ofthe annulus in disc L4-L5. in continuity with ascending disc herniation (Simonetti et al.. 2001)

Good results have been obtained after either intradiscalor intraforaminal injection of a variable volume (3-15 ml) of gas at an 0 3 concentration of 27-30 318 CHAPTER24 ug/rnl. Several thousand patients have been treated, with a success rate of 54-86'% (Alexandre and Fumo, 1998; Jucopilla et a1., 2000; Bonetti et a1., 200 I; Fabris et al., 200 I; Leonardi et al., 2001a; Petralia et al., 200 I). It remains unc1ear how ozone acts. One real possibility, previously discussed at length (Bocci 1998a, 1999), is that ozone dissolves in the interstitial water and reacts immediately, generating a cascade of ROS, among which HzOz and possibly the hydroxyl radical, OHo, whieh is most reactive. The hydroxyl radieal appears to react with carbohydrates and amino acids composing proteoglycans and collagen type land 11, major components of the degenerate nuc1eus pulposus, leading to its breakdown (MeCord, 1974; Curran et a1. , 1984; Hawkins and Davies, 1996; Bocci et al., 2001b; Leonardi et a1. , 2001b). These studies, as weil as those performed on human blood, have been carried out using the Electron Paramagnetie Resonance (EPR) spin trapping technique (Ueno et al., 1998). Consequently, reabsorption of hydrolytic products and water may lead to progressive shrinkage and disappearance of the hemiated material. Reduced mechanical irritation decreases the sensitivity of nerve axons, but nociceptors are also excited by endogenous algesie substances released during perineural ischaemia or neural inflammation present in the spinal ganglion and neural roots (WiIIis, 1995). Thus, more than the mechanical compression as primum movens, it is the inflammatory reaction that sustains chronic pain by releasing PLAz, several proteinases and cytokines. The continued release of ROS, PGEz, serotonin, bradykinin, cathepsins, IL-I , IL-6, substance P, etc., causes oedema, possibly demyelination and increased excitability of nociceptors (Fields, 1986). Indeed , it has been observed that even a large hemia can be painless. Moreover, the hemia may remain after an operation (as seen radiographically), but the pain disappears once the inflammatory disorder dies down . Interestingly, epidural injections of the anti­ inflammatory methylprednisolone transitorily improve leg pain and sensory deficits in patients with sciatica due to a hemiated disc (Carette et al., 1997). So, how does ozone act? We are again facing the ozone paradox : although OHocan degrade the degenerated material and reduce pressure, it often exerts a rapid "anti­ inflammatory action", partieularly because only a few ml of gas can be introduced inside the nucleus pulposus and most ofthe gas invades the intraforaminal space . This may mean that ozone rapidly blocks inflammatory reactants and stimulates the restitutio ad integrum. What is even more surprising is that this change remains stable (unlike corticosteroids) and it does not necessarily coincide with the disappearance of the herniated material. In fact, CAT or NMR controls in 612 patients, 5 months after treatment, showed that the hemia disappeared in 226 (37%), was reduced in 251 (41%) and was unmodified in 135 (22%). After another 5 months, CAT/NMR controls were performed again in 200 (of251) patients in whom the hernia was reduced : a further reduction and improvement was noted in 44 patients (22%) . In 120 patients (of 135) in whom the hernia was unmodified, there was an improvement in 11.6'% (14 of 120) (Alexandre et al., 2000) . Thus the ozone effect is deployed in successive phases: there is an initial rapid change, probably with disappearance ofoedema and improvement of circulatory and metabolic conditions, followed by stasis and then a further improvement possibly due to release of TGFß1 and bFGF (Silver and Glasgold, 1995; TrippeI, 1995), OZONETHERAPY IN V ARIOUS PATHOLOGIES 319 favouring the reorganization ofthe residual nucleus pulposus with incipient fibrosis. So far, attempts to examine the histopathological changes have been inconclusive. A few problems have been reported. In young patients, it is often very difficult to introduce more than 1-2 rnl of gas inside the nucleus pulposus, so that the gas is released into the intraforaminal space. I have been wondering if, in these cases, a preliminary aspiration of the nucleus followed by the gas introduction might improve the result. Apparently, the intraforaminal administration of gas yields good results even in the case ofsclerotic hernias (Fabris et a1., 2001). Side effects are very rare: one patient had a transient lipothymia and one reported by Alexandre et a1. (1999) presented amaurosis fugax (bilateral blindness which reversed after about 24 hours) after cervical discolysis in a young athlete (Chapter 21).

The Indirect Approach, or " Chemical Acupuncture " Use of the paravertebral muscles as a route for infiltration of Or03 is shown in Figure 106 (taken from Tabaracci, 2001).

Figure 106.The iliac crests are palpated and the transiliac line is determined to identify the L4 spinous process, the interspinous spaces are identified by selecting the space corresponding 10 the herniated disc. Roughly 2 cm are calculated bilaterally to the spinous process (above). Once the needle is inserted througb thefasciae, material is aspirated while holding the needle still and a 20 ug/ml concentration 0/an oxygen-ozone mixture is injected velY slowly up to a maximum 0/10 ml per infiltration . Aspiration is repeated during infiltration (below) (Tabaracci, 2001) 320 CHAPTER24

This approach, which seems technically simple, has become very popular in Italy . Indeed some physicians think they can become ozonetherapists ovemight and start to inject a patient with an excessive dose ofozone, which might kill hirn owing to a complex neurovegetative over-reaction. This has happened once and that is why it is important to have precise guidelines and mies for the practice ofozonetherapy. In reality, it is an easy approach consisting in one or several (up to four) injections of 5-1 0 rn1 of O2-03 per site. The ozone concentration must not exceed 15-20 IJglml because it is painful. At first, it is wise to test the patient's reactivity with an injection of sterile saline and then start with 10 ug/ml ozone. The injection must be done very slowly into the trigger points corresponding to the metamers of the hemiated disk. The length of the needle varies (from G22 to G25) depending on the patient's obesity. Usually two symmetrical injections (total dose 10-20 ml gas with at most 200-400 ug ozone) repeated twice per week for about 5-6 weeks (I0-12 sessions) are sufficient; ifnot, the patient is unresponsive to this approach. I repeat that injection ofO2-03 e1icits a sharp pain lasting a few minutes and the injection must be done very slowly to avoid any risk of embolization. If we act carefully, we can avoid serious adverse effects, such as sud den hypotension, bradycardia, mydriasis, intense perspiration and cardiac arrest (vasovagal reflex). Any serious ozonetherapist must be prepared for this emergency, which is very rare but can happen (see Chapter 36) . The results of a number of studies vary somewhat (Cinnella and Brayda-Bruno, 2001), but they can be summarized as : about 40% optimal, 35-40'% marked improvement 15-25% minimal or no result. Gionovich et al. (2001) compared three approaches:

A) Paravertebral injection of02-03 75% Satisfactory response B) Peridural injections with desamethasone 55% Satisfactory response C) Paravertebral injection of'buvipicaine 0.25% 70% Satisfactory response

The term "chemical acupuncture" was coined (Bocci, 1998a) because we must c1arify the role of the needle, oxygen and ozone. It was proposed to compare this procedure against a waiting-list control, two placebo controls (one with oxygen alone and another without any gas) and a standard-treatment control. Gionovich et al. have now shown that, as expected, even an anaesthetic has some effect. Owing to an unexpected, unintentional incorrect use of the medical generator (delivering medical oxygen only), we can now give a reasonable answer to the above-mentioned uncertainty. Torri et al. (1999) treated a group of 66 patients with O2-03 and a group of 30 patients with oxygen alone. Interestingly, excellent or good responses were observed in 86% of patients of both groups but the O2-03 group showed a statistically significant improvement of some c1inical parameters. This suggests that the needle and oxygen together already have a therapeutic role, wh ich is potentiated by the addition ofozone. Then the question is: how does ozone injected intramuscularly work? The gas infiltrates the musele and after 24 hours some gas bubbles move towards the vertebral canal (as seen radiologically). It was postulated that ozone will reach the site of the hemiated material and will Iyse it. This is an untenable idea : ozone OZONETHERAPY IN V ARIOUS PATHOLOGIES 321

dissolves rapidly into the interstitial water of the muscle and will generate H202 within a few minutes; by inhibiting amyelinic fibres (C-nociceptors), the H202 will activate the anti-nociceptive system via the descending antinociceptive system (Fig . 107). As occurs during acupuncture (Ceccherelli et al., 1995), the introduction ofthe needle, reinforced by the pressure ofO2-03, induces strong inhibition ofnociceptors, perhaps a prolonged stunning due to H202• It is known that an algic stimulation of the skin and muscles can reduce pain through the mechanism of diffuse noxious inhibitory control (DNIC). That is why the needle + H202 + oxygen pressure can be translated into chemical acupuncture.

Figure J07. The scheme indicates the mechanismsfor the control ofalgesie signals. By releasing endorphins (End.), the enkephalinergic interneuron may inhibit the presynaptic connection ofa neurocyte (C) ofa spinal ganglion which, under compression ofa herniated disc, stimulates the release ofsubstance P (SP). Endorphins ean inhibit the transmission of the algesie signal to neuron D. hence to the ascending spinal-thalamicfibres. The monoam inergic 01' serotoninergic neuron A. as a component ofantinociceptive descending fibres , ean reinforce the analgesie effect ofneuron B 322 CHAPTER24

This mechanism is likely correct because too low 0 3 concentrations (3-10 ug/rnl) or gas volumes (1-2 ml) are ineffective, whereas too high 0 3 concentrations (above 20 ug/rnl) or gas volumes can cause lipothymia . It is unclear whether pre-infiltration with an anaesthetic reduces the effect of ozone. We do not know whether the generated H20 2 causes irreversible damage and death of the nociceptors, with a consequent increase of the activation threshold. Furthermore, it is not known if this means a blocked release ofalgesie compounds, with a simultaneous release ofendorphins . In conclusion, the probable mechanisms playing a role are the following : a) release of endorphins blocks transmission of the noxious signal to the thalamus and cortex. b) Hypostimulation (elevation of the activation threshold) linked to the oxidative degeneration ofC-nociceptors.H20 2 and LOPs may act like capsaicin. c) Activation ofthe descending antinociceptive system. d) Simultaneous psychogenic stimulation of the central analgesie system induced by the gas injection (elicitation ofa placebo effect). e) The localized oxygenation and analgesia permit muscle relaxation and vasodilatation, and thus a reactivation of muscle metabolism, by favouring 2 oxidation of lactate, neutralization of acidosis, increased synthesis of ATP, Ca ; reuptake and reabsorption ofoedema.

Once again, by reactivating natural defence mechanisms, the use of oxygen­ ozone surprisingly seems to solve a painful problem. We have prepared a protocol proposing to evaluate the loeal effect (paravertebral muscles) ofa solution ofH20 2 diluted in a 5% glucose solution. We may be able to ascertain if H20 2 is the compound that acts on nociceptors and evokes the analgesie response. Samanta and Beardsley (1999) wondered what was the best way forward to treat low back pain, but they did not know and eould not mention the O2-0 , approach. If American and English orthopaedic surgeons read this book and try this approach, they may produce new and interesting results, useful for science and above all for patients.

12. CHRONIC FATIGUE SYNDROME (CFS) AND FIBROMYALGIA I am grateful to Prof. R. Mareolongo, Director of the Institute of Rheumatology, Siena University, for his broad-rnindedness and interest in evaluating ozonetherapy in RA, CFS and fibromyalgia. Some three years ago, we submitted a eomplex protocol to the Ethical Committee, which approved it after a few revisions. However, the study on RA never took off because I was personally very doubtful about the ozone dose and because Prof. Marcolongo did not agree to suspend conventional medication in these patients. CFS and fibromyalgia are diseases charaeterized by poorly understood signs and symptoms, with severe fatigue and a eontinuous flu-like syndrome that profoundly disables patients. Since 1990, I have fotlowed the abundant medicalliterature on CFS and I have realized that I could not even attempt to give a short synthesis of an ambiguous aetiology and OZONETHERAPY IN V ARIOUS PATHOLOGIES 323 pathophysiology. Recent papers have added some new ideas and the interested reader may consult them (Komaroff, 2000; Manu, 2000; Reid et al., 2000; Spence et al., 2000; Natelson, 2001; Powell et al., 2001; Prins et al., 2001; Wessely, 2001). Conventional treatment is based on antidepressants, low-dose glucocorticoids, exercise (in contrast, prolonged rest seems harmful), immunotherapy and oral nicotinamide adenine dinueleotide to increase the generation of ATP. Benefits are limited and there are adverse effects . Cognitive behavioural therapy performed by skilled therapists appears to be an effective intervention without harmful effects. In this book, I have reported that 03-AHT often yields a feeling of well-being and euphoria. This is quite true, even though we can only speculate about the reasons for these positive effects . In search of a good therapy , CFS patients go from one physician to another. Last year, at the hospital of Conegliano Veneto, they treated six patients diagnosed with CFS. 03-AHT was carried out twice per week for 8 weeks and the physician in charge assured me that four patients showed a "remarkable improvement". He could not give information about the follow-up. In our hospital, Dr. Cosentino treated one patient with a modest outcome. I apologize for such a crude report but, given the severity of the disease, I would not hesitate one second to do 03-AHT if the patient wanted to try it. I would suspend any other drug and c1early explain that ozonetherapy may readjust the metabolie, immunological and hormonal derangements, causing only the discomfort of the venous puncture. Fibromyalgia is another mysterious disease . The American College of Rheumatology (ACR) has established a procedure for examining 18 tender points on the patient. A tentative diagnosis can be made ifat least 11 ofthese points elicit pain when pressed. In Italy, fibromyalgia is considered a disease causing considerable socio-economic problems, since it affects about 6 million people between 30 and 60 years of age. The aetiology is unknown but initially seems caused by a psychosomatic factor later complicated by biochemical modifications in the musele, possibly similar to those found in CFS (Fulle et al., 2000), and neuro-psycho­ immune endocrine disorder.' Two studies have been carried out in Italy and they were reported at the IMOS congress last year at Siena University (November 2-4, 2000). Unfortunately, they have not yet been published and I will give a short summary. From 1988 to 2000, Dr. Salvatore Loconte (Andria, Bari) has treated 150 patients by infiltrating 5 mI gas directly on the trigger points (03 concentration: 5-10 ug/ml) and perforrning a cycle of 03-AHT with about 150 ml blood and a total ozone dose of4500 Ilg (30 ug/ml), He is a private ozonetherapist and cannot do a control but he has elaimed to achieve total remission in about 60% ofpatients and partial improvement in 15%. A RCT has been performed in the Institute of Rheurnatology of our University on 40 women (age 30-50) diagnosed as having fibromyalgia on the basis ofthe ACR criteria . The scope ofthe study was to evaluate the effect ofA) AHT with O2-03 (20 patients, with 0 3 concentrations scaling up from 20 to 40 ug/ml , twice per week for a total of 16 treatments), B) AHT with O2 alone (10 patients), and C) simple AHT without gas (10 patients). Several standard end-points were tested before treatment, after 8 weeks and I month thereafter. 324 CHAPTER24

Patients of group C did not show any improvement and are now under Or03 treatment. Three patients of group B (30%) showed good improvement. Seven patients ofgroup A (35%) showed excellent improvement, while one (5%) had good improvement. Cosentino et al. (2000) conc1uded that 03-AHT has therapeutic validity and no side effects. However, in comparison to conventional automedication, it is time-consuming for hospital personnel.

13. TRAUMNBURN INJURIES AND EMERGENCY SURGERY. OZONETHERAPY BEFORE TRANSPLANTAnON OR BEFORE ELECTIVE SURGERY.

Regarding the first topic, I never managed to convince the chief doctor of intensive therapy of the potential usefulness of AHT performed with O2 and 0 3 at low concentrations (15-25 ug/ml of blood) in patients with permanently cannulated central or peripheral veins . They are mostly concemed about the legal aspect of using a non -validated and somewhat controversial therapy in high-risk patients. When I visited Russian hospitals, I was told that they do not worry about it and use ozonetherapy to disinfect traumatic and war wounds, bums (due most frequently to flames) , radiation injuries and abdominal surgery after stomach or intestinal perforations. Disinfection with ozonized bidistilled water and application of ozonized oil has been found to be most useful in bums. It is unfortunate that they abundantly use ozonized saline instead ofozonized blood for systemic treatment. On this point, our opinions are greatly divergent. I cannot agree with their assertion that ozonized saline is as effective as blood, because on several occasions we have noted the multidirectional potentialities of ozonized blood. Serious trauma, bums and peritonitis lead more or less rapidly to systemic alterations of several organs, particularly the cardiopulmonary (AROS), coagulative (DIC) and renal systems. Because of an adverse series of metabolic impairments, these alterations cause the patient's death . Thus, using all the most appropriate conventional supporting therapies combined with OrAHT (every 3-4 hours throughout the day), I "feel" that we could save some lives. The second topic is less tragic, but no less serious. I have often wondered if a cardiac patient waiting for a heart transplant might gain increased resistance to infections and to immunesuppression (unavoidably linked to deep anaesthesia and surgery) ifhe could undergo two OJ-AHT per week (at low 0J concentrations: 20 to 40 ug/ml) for 3-8 weeks before transplantation. This strategy is all too obvious and may induce a sort of ischaemic preconditioning or, to use language comprehensible to most people, the adaptation to chronic oxidative stress (Chapter 22). During heart transplantation, all organs (particularly the CNS, retina and kidneys) undergo a bland ischaemia-reperfusion syndrome, which in unlucky cases may have dire consequences even if the operation is technically perfect. Thus prophylactic ozonetherapy, with little effort and expense, might be useful. The final point worth pursuing involves the scheduled operation for application of a prosthesis, particularly joint implants. In particular, as a precaution, coxo­ femoral surgery requires the collection of I or 2 standard units of blood from the patient. Discussing this problem with several orthopaedic surgeons, J found that at OZONETHERAPY IN V ARIOUS PATHOLOGIES 325 least three were interested in evaluating whether performing at least four 03-AHT (ozone at low concentrations) during the 2 weeks before the operation and then every day immediately after it for 4-5 days (using the predeposits as weil) would reduce the complications by enhancing healing and the patient's mood.I presented a protocol to our Ethical Committee, which was approved. However, no trial has started as yet because the orthopaedic surgeons do not have supporting personnel to do AHT.

In summary, I regret that this long and dreary chapter is rather inconclusive. On the whole, I will feel great iftwo out often proposed clinical trials begin and if we can finish one study . The pace is too slow and I wish that we had already made incisive advances so that I could indicate optimal doses and schedules. Nevertheless, we have some ideas and a working hypothesis on how to proceed. It is almost unnecessary to repeat that our good will is not sufficient and unless there is a concerted effort by official Medicine and govemment authorities, we will remain at the stage of "if", "perhaps" and "speculations". This is sad, not so much for me because I have always tried to do my best, but for the patients who will not have the advantage of recovering more rapidly and improving their health. National health authorities, which are always complaining about the increasing costs of medical assistance, could have an economical advantage if ozonetherapy was widespread and organized in a systematic way in all public hospitals. The savings could be dedicated to improving other areas of medical assistance. Even a child could understand this simple reasoning. Although I have no hard data to support my contention, I am convinced that the benefit ofozonetherapy does outweigh its cost. Obviously pharmaceutical giants or other private organizations would make less profit, but they would continue to thrive just the same . An obvious comment is that although the Communis' creed has failed in many ways, it has succeeded in Cuba in truly improving health assistance for everyone. A good part of the merit, however, has to be attributed to the versatility ofozone .