2. Animal Models
Animal models more closely resemble the clinical state -o f note is that some studies in experimental animal models have indicated that ozone itself can exert “indirect action” on the progression of some tumor types.
In 2008, two different preclinical studies in mice were published in the same article. In the first study, cells of Ehrlich ascitic tumor and sarcoma 37 tumor were implanted in the ocular plexus of mice. After implantation, the animals were treated with ozone via rectal insufflation over 12 sessions using different ozone concentrations. In both tumors, a significant decrease in the numbers of lung metastases was observed, with lower numbers of tumor cells per mice at higher ozone concentrations (2). In the second preclinical study, varying ozone concentrations were applied intraperitoneally for 15 days. Twenty-four hours after the last ozone treatment, Lewis lung carcinoma cells were inoculated via subcutaneous route. Relative to the control group, all the ozone pretreated groups showed a delay in tumor volume increase and the kinetics of tumor development, with a trend to better results when lower ozone concentrations had been used. Additionally, at 16 days after tumor cell inoculation, all animals in the control group had tumor development, while in the ozone-treated groups there were animals without signs of tumor growth, even after 35 days (2).
Also in 2008, an article was published using a model of carcinoma metastases of squamous cells in rabbits. The results showed that tumor growth tended to be produced at the site of inoculation (usually the ears), together with lung metastases. The O3T was administered intraperitoneally, i.e., the route that is frequently employed in small animals as being an approximation to the intravenous route in humans. In the group receiving O3T, 7 of the 14 rabbits survived and, of them, all but one showed complete response (complete disappearance of the tumor). Conversely, in the sham group (no ozone gas administered), of the 13 rabbits in the study, 3 survived and only 2 of them showed complete disappearance of the tumor. The ozone did not enter into direct contact with the tumor cells and, as such, the action of the ozone must have had an “indirect effect”. In the second part of the same study, the authors did not observe the same outcomes when immunosuppressors had been administered. This would suggest that the effect was mediated by boosting/stimulating the immune system . Four years later, the same authors described ozone as having induced the synthesis of prostacyclins at the systemic level. Years earlier the antimetastases effect of prostacyclins had been described.
A subsequent study by the same research group showed the indirect antitumor effect of ozone more clearly . Employing the same rabbit experimental model, the authors observed that the animals receiving intraperitoneal ozone had a greater and statistically significant percentage regression of the tumor, relative to the sham group. Also, the tumor regression was associated with a significant increase in the intratumor infiltration of CD3+ T lymphocytes. Further, when new rabbits with induced tumors had received leukocytes from peripheral blood from rabbits that had tumor regression previously, 60% of the new animals had tumor regression. Conversely, when leukocytes from rabbits that had tumor progression were injected into the new rabbits, no antitumor effect was observed. This study demonstrates that, at least in this model, the ozone could exercise an indirect antitumor effect via modulation of the immune system.
The action of O3T as having a potential “indirect effect” has been clinically confirmed in several studies by Bocci et al. since the 1990s. The studies demonstrate that ozone can modulate the production of various cytokines (such as interleukins and interferon) and, as such, modulate the activity of the immune system which is responsible for the defense of tumor cells.
It is evident that the immune system plays a primordial role in the defense of the organism against infection and against cancer. Thanks to some recent clinical trials, the role of immune modulation as an antitumor strategy has been clearly established. As a result, monoclonal antibodies targeting cytotoxic T lymphocyte-associated antigen 4 (CTLA4), the programmed death-1 receptor (PD-1), and its ligand (PD-L1) have been approved by the European Medicines Agency (EMA) and/or the Food and Drug Administration (FDA) for the treatment of several tumors, especially melanoma and non-small cell lung cancer.
Immune modulation produced by O3T is nonspecific. The mode of action differs in relation to the activity of different types of lymphocytes and on the production of different types of cytokines. The extent of activity depends on environment, functional status, and ozone concentration. Indeed, several years ago Bocci et al. proposed the hypotheses that low-medium ozone concentrations could upregulate cytokines produced by CD4+ TH1 lymphocytes enhancing TH1/TH2 ratio while higher ozone concentrations could decrease this ratio. Additionally, there could be considerable clinical gain in combining monoclonal antibody therapy and O3T.
Another study in an animal model (tumor-bearing mouse model with rectal cancer) showed an antitumor effect of intratumor injection of ozonated water. The effect was probably mediated by a local immunomodulation effect induced by ozone. However, this method of ozone administration does not have high clinical application.
In addition to the indirect effect on the tumor, ozone potentiation of CT and RT could be of higher clinical relevance. A few experimental studies in animals with induced cancers have evaluated the effect of O3T in combination with RT. The results have been promising.
In 1974 Hernuss et al., studying Walker carcinosarcoma of the rat, reported that RT combined with O3T produced significantly better outcomes than RT alone. Tumor remission was 39% in the RT + ozone group versus 0% in RT group without ozone. Indeed, 6 months later, 17% of the ozone-treated animals remained alive without recurrences or metastases.
Conversely, in 1976 the same journal published several studies by Grundner et al. which did not find a radiosensitizing effect of ozone administered after RT in animals with Ehrlich-ascite carcinoma cells, despite the authors having previously observed an enhancing effect in vitro.
Later, in 2015, with the same Ehrlich-ascite tumor cell model, intraperitoneal ozone was described as being effective (administered alone or concurrently with RT) with respect to antiedema and antitumor effects and with longer survival times. The effects were ozone concentration dependent.
More recently, in 2018, another study from Turkey evaluated the impact of ozone alone and also when administered concurrently with RT in an experimental rat model of tongue cancer. The study described an antitumor effect as well as improvement in survival in the ozone group compared to the cancer-group without any treatment. Additionally, the most remarkable observation was that tumor response and survival rates were significantly higher in rats treated with RT + O3T compared to those treated with RT alone. The median survival rates were 49 and 3.5 days, respectively.
All the experimental studies described above would warrant more investigation of the use of ozone in combination with RT and CT.