Efficacy And Safety Of Medical Ozone Therapy In Patients With Chronic Hepatitis B A Randomized, Three Arm Controlled, Open-label And Multi-center Clinical Trial

BackgroundHepatitis B virus is one of the most serious and prevalent health problems, affecting more than2billion people worldwide. Although highly effective vaccines against hepatitis B virus have been available since1982, there are still more than350million chronic carriers,75%of whom reside in the Asia Pacific region. People with hepatitis B are at increased risk of developing hepatic decompensation, cirrhosis, and hepatocellularcarcinoma. The estimated worldwide mortality is0.5to1.2million deaths a year. In China, there are93million people with HBV infection, out of which20million people show chronic hepatitis B virus infection. Chronic infection remains a challenging global health problem.An improved understanding of hepatitis B virology, immunology, and the natural course of chronic infection, has identified hepatitis B virus replication as the key driver of immune-mediated liver injury and disease progression. Current consensus guidelines from Asia, Europe, and the United States recommend nucleoside analogue or nucleotide analogue, or conventional and pegylated interferon alfa for the treatment of chronic hepatitis B, and monotherapy with these drugs greatly suppresses virus replication, reduces hepatitis activity, and halts disease progression. The interferon is the first antiviral drug that be approved to treat chronic hepatitis. It has been lasted more than20years. Interferon is effective in a minority of patients and has frequent side effects that limit its tolerability. The approval of potent oral antiviral agents has revolutionized hepatitis B treatment sincel998. Nucleoside analogues or nucleotide analogues (lamivudine, adefovir, telbivudine and entecavir etc.) are widely authorized treatments. They have the advantages of oral administration and excellent safety profiles. They can suppress HBV replication and result in an improvement in liver histological during therapy. However, the efficacy of lamivudine is limited by the emergence of drug-resistant hepatitis B virus (HBV) mutants, restricting its usefulness as a long-term therapy [11-13]. Adefovir is associated with a low incidence of resistance, but its antiviral effect is not optimal [14-16]. Entecavir has a high antiviral effect and is well tolerated [17,18], but its long-term efficacy and resistance profile are not yet determined. Therefore the development of new drugs and strategies is needed to improve treatment outcomes.In recent years, medical ozone for the treatment of hepatitis B has become a new kind of exploration. Medical ozone is a gas mixed with ozone and medical oxygen, which can be used to treat a variety of diseases such as rheumatism, ulcerative colitis and protrusion of intervertebral disc. The reaction of ozone with human whole blood could generate reactive oxygen species (ROS, mainly H2O2) and lipid peroxidation products (LOPs), the role of these two products is to separately act on the various components of the blood (monocytes, platelets, red blood cell, WBC) and endothelial cells and other organs, resulting in a variety of effects. In recent years, hydrogen peroxide (H2O2) is considered an important intracellular messenger in animal and plant cells, so ozone plays an indirect role through its reaction products. At the end of2004in mainland China ozone therapy was firstly used in treatment of patients with chronic hepatitis B and has shown initial signs of efficacy, medical ozone treatment of chronic hepatitis B is expected to become a new strategy.ObjectiveTo verify the efficacy and safety of medical ozone for the treatment of hepatitis B through a randomized, controlled, open-label, multi-center clinical study and to provide the basis for evidence-based medicine for the treatment of CHB with medical ozone in clinical application.Methods and materials1. This randomized, controlled, open-label, multi-center clinical study was conducted at3sites in China. The study was conducted in compliance with the Declaration of Helsinki and with the principles of Good Clinical Practice. All patients gave written informed consent. Patients were randomly assigned in a1:1:1ratio to receive medical ozone therapy with instrument made in TianYi three times a week, medical ozone therapy with humares three times a week, or150mg of Diammonium glycyrrhizinate Capsules three times daily. The total course of treatment is12weeks.Inclusion/exclusion criteria:Adults were eligible if they had been positive for hepatitis B surface antigen (HBsAg) for at least6months, were negative for antibodies to HBsAg (anti-HBs antibodies), had an HBV DNA level of more than10,000copies per mililiter, had a serum alanine aminotransferase level that was greater than2but less than or equal to10times the upper limit of the normal range, and serum bilirubin level that was less than80μmol/L. Exclusion criteria included decompensated or serious metabolic liver disease, a coexisting serious medical or psychiatric illness, such as cardio-vascular, kidney events, hyperthyroidism or serious electrolyte disturbance etc, a history of hemorrhagic or hemolysis disease, a history of carcinoma, or finding suggestive of possible hepatocellular carcinoma (HCC), or AFP over than100ng/ml, a history of alcohol or drug abuse within half a year before entry, and coinfection with hepatitis C or D virus or human immunodeficiency virus. Treatment with immunosuppressive agent for long time, including patient has a history of organ transplantation, difficulty to draw blood through veins, pregnancy or enrollment in any other clinical trials are also excluded. Previous treatment for chronic hepatitis B was permitted, but not within the six months before the study.2. EFFICACY MEASURESEfficacy analyses included all randomized patients who received at least8weeks of study treatment. The primary measures of efficacy assessed after12weeks of treatment:suppression of HBV DNA to levels below1000internation unit per milliliter (IU/ml) or decreased100times. The secondary efficacy measures assessed after12weeks of treatment included, HBeAg seroconversion (defined by the loss of HBeAg and the presence of anti-HBe antibody), normalization of ALT (defined by the levels of alanine minotransferase below upper normal limit). Serum HBeAg, anti-HBe antibody and HBV DNA were measured at a central laboratory with the use of the AxSYM test (Abbott) and the Cobas Amplicor HBV Monitor Test (Roche Diagnostics).3. SAFETY ANALYSISMeasures of safety included adverse events, hematologic measurements, clinical chemical measurements, vital signs and safety and stability of medical apparatus. The severity of adverse events was graded on a three-point scale (mild, moderate, and severe), and causality was determined by the investigator. Safety was assessed at baseline; weeks4,8and12throughout treatment. Safety analyses included all patients who underwent randomization and received at least once a time of study treatment and who underwent at least one safety assessment after the baseline assessment.4. STATISTICAL ANALYSISFrom previous experiencet of the effectiveness of ozone treatments and Diammonium glycyrrhizinate Capsules, the estimated virological response rate for12weeks treatment course were22%and3%respectively. The design is balance. The proportion in three groups is1:1:1. The special software nQuery7.0was used to get accurate sample size and power. Based on the trade off between power attainable for the desired effect size and sample size feasibility, determine that the sample size for the study in each sequence group is at least57. The sample size was increased to63patients to allow for10%withdrawals. A sample size of189patients per treatment group provided the study with a statistical power of at least80percent at the0.025level of significance, with a two-sided test, to detect a difference in HBV DNA response rates (suppression below1000IU per milliliter or decreased100times) of22percent versus3percent. Descriptive analysis was conducted among the baseline data of three groups including demographic characteristics. For each treatment group, response rates were computed with corresponding95percent confidence intervals. The Pearson χ2testing was used to determine the differences in the binary qualitative variables among groups. Fisher’s exact test was used when appropriate. The total significant differences in the quantitative variables among groups were compared by two way anova, post hoc testing were used to evaluate the significance of subgroup differences by bonferroni methods. Response rates were calculated for all patients who received at least eight weeks of study treatment according to the intention-to-treat principle. The Pearson χ2testing was used to determine the differences in side effects among three groups.Patients and resultsPatientsSince May2010to Dec2012,189legal patients with compensated chronic hepatitis B had been enrolled and of them completed the entire course of treatment by3study center hospitals located in Guangzhou, Shanghai and Shenzhen city of mainland China. Patients with hepatitis B were divided equally and randomly into three groups A to C. Patients in group A and group B were treated by ozone major autohaemotherapy with two different medical ozone generators. Patients in group C were treated with Diammonium glycyrrhizinate Capsules, a common used liver protective herb drug. The term of therapy was12weeks. Virology response and biochemistry response were analyzed at the end of12weeks treatment.Results1. Evaluation of the therapeutic effectsThe virological response rates of groups A to C were22.4%(13/58),16.4%(10/61) and10.7%(6/50) respectively; There was no remarkable difference between group A and B (p=0.408), group A and C(p=0.095), group B and C(p=0.374). The biochemical response rate is33.9%(19/58),35.5%(21/61)in the two medical ozone therapy groups and26.8%(15/55)in the oral drug group at the end of treatment. There was no remarkable difference between group A and B (p=0.860), group A and C(p=0.165), group B and C(p=0.311). The HBeAg seroconversion rates of three groups were12.1%(4/29),2.4%(1/40) and2.2%(1/45) respectively; There was no remarkable difference between group A and B (p=0.165), group A and C(p=0.155), group B and C(p=0.096).2. Evaluation of the side effectsThe rates of adverse reaction were1.7%(1/58),3.3%(2/61)in the medical ozone therapy groups and7.3%(4/55)in the oral drug group at the end of treatment. There was no remarkable difference between group A and B (p=0.1.0), group A and C(p=0.198), group B and C(p=0.421).Conclusions1. The clinical study results showed that medical ozone therapy with two ozone therapy systems (TY-CZ-9999system and Humares system) demonstrated similar activity against hepatitis B virus to a certain degree (p=0.408). They are similar to Diammonium glycyrrhizinate Capsules, a common used liver protective herb drug. The clinical study results also showed that Diammonium glycyrrhizinate Capsules had antiviral effects to a certain degree.2. Medical ozone therapy was used in the treatment of chronic hepatitis B. The anti-inflammatory effect (the biochemical response rate) of medical ozone is similar to Diammonium glycyrrhizinate Capsules. Medical ozone therapy had also showed effect of reducing alanine aminotransferase.3. A12-weeks medical ozone therapy was well tolerate and showed potent antiviral effect.