Novel therapy for COVID-19 does intravenous ozonated-saline affect blood and tissue oxygenation?

Ozone therapy has been used in medicine for over a century after it was discovered in the mid-1800’s. It is extensively used in the USA, especially in the pre-antibiotic era for infectious diseases including combat injuries. Most recently reports from China, Italy, Spain and South America report the benefi cial use of ozone therapy in extremely ill patients with COVID-19 associated acute respiratory insuffi ciency [1-8]. Despite the extensive use of ozone among Integrative / Wellness providers in the US and world wide there are very few prospective studies [9-11]. There are multiple proposed mechanisms by which adjuvant ozone therapy for COVID-19 could possibly improve outcomes: 1) Ozone is virucidal; 2) Ozone increases oxygenation of blood and tissues; 3) Ozone stimulates immune function via nuclear factor erythroid 2–related factor 2 (NRF2); 4) Ozone inhibits infl ammatory mediators (interleukins, cytokines, and tumor necrosis factors; 5) Ozone activates endogenous antioxidant immune defenses; 6) Ozone inhibits viral replication; 7) Ozone inhibits micro thrombus formation; 8) Ozone up-regulates HO-1 in endothelial cells and; 9) Ozone stimulates the 2-3 diphosphoglycerate that shifts the oxygen Abstract


Introduction
Ozone therapy has been used in medicine for over a century after it was discovered in the mid-1800's. It is extensively used in the USA, especially in the pre-antibiotic era for infectious diseases including combat injuries. Most recently reports from China, Italy, Spain and South America report the benefi cial use of ozone therapy in extremely ill patients with COVID-19 associated acute respiratory insuffi ciency [1][2][3][4][5][6][7][8]. Despite the extensive use of ozone among Integrative / Wellness providers in the US and world wide there are very few prospective studies [9][10][11]. There are multiple proposed mechanisms by saturation curve to the right, thus delivering more oxygen to the tissues [12]. The purpose of this investigation is to determine if ozone-gas infused saline administered intravenously affects oxygen desaturation and recovery in a hypoxia chamber.

Results
Vital signs remained stable before, during and after the two sequential hypoxia runs. There was a signifi cant increase in duration of time the subject could stay in the hypoxia model after receiving ozone compared with that prior to the ozone therapy ( ANOVA; P < 0.05, F = 7.8, Figure 1). In contrast, three experiments using control demonstrate a signifi cant reduction in time that the subject could remain in the hypoxia chamber in the second run compared to the fi rst and (ANOVA; P = 0.001, F=109, Figure 2). T he percent change of time in the hypoxia chamber after ozone was calculated using the pre-ozone time in the hypoxia chamber as a reference. The null hypothesis was rejected as there was as substantial increase in the percent of time the subject could stay in the hypoxia chamber. (i ncrease by 31%, P < 0.0001, Figure 3). Likewise, in the three control Figure 1: The amount of time that the subject could tolerate the hypoxia chamber before and after intravenous ozonated-saline therapy in four experiment seach with two runs in the hypoxia model. There was a substantial increase in time that the subject could remain in the hypoxia chamber after ozone therapy (ANOVA, F = 6.9, P < 0.05).

Discussion
Intravenous ozonated-saline was associated with clinically and statistically signifi cant improvements in oxygenation. This was evidenced by the duration of time in the hypoxia chamber and by the delay of the lowest oxygen saturation (Figures 1-4). There was a substantial increase in the duration of time in seconds that the subject could remain in the hypoxia chamber after receiving ozone compared with that of the pre-ozone run (ANOVA; P < 0.05, F = 7.8, Figure 1). Remarkably the exact opposite occurred in the three experiments that were performed without ozone, that being a signifi cant decrease in the duration of time in seconds that the subject could remain in the hypoxia chamber in the post-control run compared with that of the pre-control run (ANOVA; P = 0.001, F=109, Figure 2). Similarly, in the 4 experiments using ozone, the proportion of time in the hypoxia model in the post-ozone runs were compared using Figure 3: The proportionate increase of time in the hypoxia chamber after ozone was calculated using the pre-ozone time as a reference. The null hypothesis was rejected as there was as substantial increase in the percent of time the subject could stay in the hypoxia chamber. (31% increase, P < 0.0001).

Figure 4:
The proportionate decrease of time in the hypoxia chamber after the first run without ozone was calculated using the fi rst run in the hypoxia chamber as a reference. The null hypothesis was rejected as there was as substantial decrease in the percent of time the subject could stay in the hypoxia chamber relative to the fi rst run. (43% decrease, P < 0.0001).

Figure 5:
The effect of ozone on the time to the lowest oxygen saturation (nadir) was compared in the run before and after ozone. There was a substantially longer time to reach the oxygen desaturation nadir in the post-ozone run compared to that of the fi rst run before ozone administration. (P < 0.02). the pre-ozone runs as the reference and there was an average 31% increase(P < 0.0001 Figure 3). In striking contrast, in the three experiments using the exact same technique without ozone, there was an average 43% reduction in the proportion of time that the subject could stay in the hypoxia chamber in the second compared to the fi rst run (P < 0.0001, Figure 4). Of great interest to corroborate the fi ndings outlined above there was dramatic improvement in oxygenation measured by the continuous oxygen saturation monitor associated with the four experiments with the runs before and after ozone. There was a striking and consistent increase in time to achieve the lowest oxygen saturation level (nadir) measured by pulse oximetry in the post ozone run compared to that of the pre ozone run ( Figure 5, P < 0.05). Consistent with the patterns observed above, there was a complete opposite effect in the three control experiments in which ozone was not used. Instead of having a longer time to reach the oxygen desaturation nadir in the post ozone run, there was a striking reversal of that pattern in the post control run (without ozone). In the post-control run there was a signifi cant reduction in time to achieve the lowest level of oxygen saturation as measured by pulse oximetry compared to that of the pre control run( Figure 6, P < 0.05).
Independent observers unanimously observed a lessening and delay in hypoxia symptoms observed in the post-ozone run compared with that of the pre-ozone run. To our knowledge this is the fi rst documentation of intravenous ozonatedsaline improving the ability to remain in hypoxia with the same endpoint, that is, severely symptomatic hemoglobin desaturation documented by pulse oximetry. This study did NOT address the effi cacy of adjuvant ozone therapy on COVID-19. However, it does support one of the mechanisms postulated to improve outcomes, that is, the improvement in oxygenation of blood and tissues.
Like oxygen and water, high concentrations of ozone can be hazardous to the lungs if directly inhaled for extended periods of time. There are a variety of modalities of ozone administration that have been extensively utilized [12]. Although controversial, direct ozone gas infusion into the venous blood is the most simple and effi cient ozonation method and is widely used as the route of choice by some experts. In contrast to air, ozone gas infusion intravenously does not pose the same risk since it is not composed of 80% nitrogen and ozone is highly soluble in blood & plasma. Ozone is about 10 to 13-fold more soluble in water than is oxygen [12]. Depending upon the temperature and pressure, about 50 ml of ozone gas dissolves in 100 ml of water compared to only about 4 ml of oxygen. [12]. Many experts prefer the use of extracorporeally ozonated autologous blood transfusion of 100 ml to 200 ml, widely known as "major autohemotherapy" (also referred to as "MAH") [12]. Various protocols for MAH typically use volumes of only 100-200 ml of blood. The drawbacks are that it requires bloodletting, heparinization, and ozonation of the blood followed by autologous transfusion. This process is further complicated by the necessity of heparin with its attendant signifi cant side effects (allergic reactions and heparin induced thrombocytopenia) and also by care providers requiring personal protective equipment for COVID-19. Additionally, MAH could not be "blinded" in future studies. features [14]. As these authors point out the treatment is primarily supportive therapy and they conclude that there is no anti-viral therapy proven to be effective against COVID-19 [14]. A few small-scale studies have claimed benefi t with chloroquine, hydroxychloroquine and other drugs including lopinavir/ritonavir, remdesivir, favipiravir, oseltamivir, ribavirin, interferon beta, tocilizumab and abidol [14]. In a separate publication, Singh et al review innovative technologies that may provide more effective screening and treatments for COVID-19 in the outpatient setting [15].
The limitations of this study are that the experiments were performed in just one subject, using himself as the control.
Another limitation is that the subject was not blinded to the intervention. A rather unique consideration is whether the impressive oxygen desaturations that this subject achieved in the hypoxia chamber were physiologic or pathophysiologic.
The subject has underlying chronic lung disease from a 17-segment spinal fusion for severe scoliosis earlier in life.
Whether or not this contributed to the impressive oxygen desaturations that were achieved is unknown but regardless, this study convincingly demonstrated that ozone therapy was associated with dramatic improvements in oxygenation.
Whether or not young healthy subjects will desaturate as easily will be determined by the follow up phase two study.
Our future planned study will evaluate the use of ozone with a more sophisticated hypoxia "altitude simulator" device in 30 young healthy adults with ozonated-saline administered in a double blinded fashion.
The study supports that intravenous ozonated-saline improves oxygenation of blood and tissues and prolongs the ability of an individual to remain in a hypoxic environment. This study supports the potential benefi t of adjuvant ozone therapy for COVID-19 as observed in China, Spain, Italy, and South Citation: Thorp  America [1][2][3][4][5][6][7][8]. Based upon the fi ndings of this study a large randomized controlled prospective trial should be conducted in the United States. Ultimately a multicenter randomized controlled clinical trial will be necessary to prove whether adjuvant ozone therapy can improve outcomes in COVID-19 patients as purported by observational studies from China, South America, Spain and Italy [18]. Based upon a reduction in the progression of disease to require mechanical ventilation by 50% (from 10% to 5%), a sample size of approximately 1000 patients would achieve a power of greater than 80% to demonstrate this difference (alpha < 0.05, beta < 0.20). We are attempting to organize and fund this trial.