Chinese Herbal Medicine Combined with Entecavir to Reduce the Off-therapy Recurrence Riks in HBeAg-positive Chronic Hepatitis B Patients: a Multi-center, Double-blind, Randomized Controlled Trial in China

Background: Nucleos(t)ide analogs (NAs) are the first-line options against chronic hepatitis B (CHB). NAs produce a potent suppression of viral replication but are associated with tiny opportunity of HBsAg seroclearance and high risks of virological relapse after discontinuation. The combined therapy of NAs plus traditional Chinese medicine (TCM) is widely accepted and has been recognized as a prospective alternative approach in China. Based on preliminary works, this study is designed to observe the therapeutic effect of TCM plus entecavir (ETV) against HBeAg-positive chronic hepatitis B with respect to reducing the recurrence risk after NAs withdrawal. Methods/design: The study is a nationwide, multi-center, double-blind, randomized, placebo-controlled trial with a designed duration of 120 weeks. A total of 18 hospitals and 490 eligible Chinese HBeAg-positive CHB patients will be enrolled and randomly allocated into the Experimental group and Control group in a 1:1 ratio. Patients in the experimental group will be prescribed with TCM formulae (Tiao-Gan-Jian-Pi-Jie-Du granule) plus ETV 0.5mg per day for consolidation therapy of 96 weeks. Patients in the control group will be prescribed with TCM granule placebo plus ETV 0.5mg per day for the same course. After the consolidation therapy, all patients will discontinue their trial drugs and closely monitored in the next 24 weeks. Once clinical recurrence (CR) occurs, ETV treatment will be restarted. The primary outcome is the accumulative rate of CR at the end of this trial. Conclusion: This study is the first of its kind to observe therapeutic effects in respect of reducing recurrence after drug withdrawals after a unified integrative consolidation therapy in CHB population.

the same course. After the consolidation therapy, all patients will discontinue their trial drugs and closely monitored in the next 24 weeks. Once clinical recurrence (CR) occurs, ETV treatment will be restarted. The primary outcome is the accumulative rate of CR at the end of this trial.

Conclusion:
This study is the first of its kind to observe therapeutic effects in respect of reducing recurrence after drug withdrawals after a unified integrative consolidation therapy in CHB population.

Background
Chronic hepatitis B (CHB) has been one of the most concerning diseases worldwide. Every year, approximately 1 million people die of CHB-related cirrhosis and hepatocellular carcinoma 1 .
Nucleos(t)ide analogues (NAs) therapy is widely applied in patients with chronic hepatitis B virus (HBV) infection. Currently, six NAs are approved for the treatment of CHB infection: lamivudine, telbivudine, entecavir (ETV), adefovir, and tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide (TAF), of which ETV and TDF are recommended as first-line therapy [2][3][4] . NAs inhibit the reverse transcriptase activity of the HBV polymerase and thus suppress viral replication. However, 5 NAs exert little effect on the viral covalently closed circular DNA (cccDNA) in the nucleus 5 . Hence they can not permanently eradicate the virus. Currently, hepatitis B surface antigen (HBsAg) loss is the most widely accepted endpoint to guide cessation of NA treatment. However, as HBsAg loss is an infrequent event, this strategy entails an indefinite therapeutic duration that could be lifelong for the vast majority of NAs-treated patients 6 .Therefore, the benefit of long-term treatment should be weighed against the burden of life-long medication, monitoring, and adherence. In addition, the longterm risk for the development of resistance and adverse events remains unclear.
In recent years, some scholars had attempted to find approaches for preventing relapses after NAs withdrawal. A previous study observed the outcomes after the cessation of ETV therapy among patients who fulfilled the stopping rules of the Asia-Pacific Association for the Study of the Liver (APASL). The 2-year cumulative rates of virological and clinical relapse were 41.3% and 33% in hepatitis B e antigen (HBeAg)-positive patients, and 3-year cumulative rates of virological and clinical relapse were 62.7% and 48.3% in HBeAg-negative patients, respectively. The risk is alarming after discontinuation of the therapy.
Traditional Chinese medicine (TCM) therapy has a long history and definite curative effect treating various chronic liver diseases, and abundant data and experience have been accumulated in longterm clinical practices and scientific research. Although TCM therapy alone has no explicit antiviral effect, it has some advantages in improving clinical symptoms, alleviating liver inflammation, antifibrosis, and regulating immune function. In China, supported by major national special research projects funded by the Ministry of Health, the treatment of chronic hepatitis B by the combination of TCM and NAs has achieved some research results, the advantages of TCM therapy in the treatment of chronic hepatitis B have been preliminarily clarified, 7 the integrated TCM and NAs therapeutic schedule has been developed, 8 and it has been proved that the combination of TCM and NAs can significantly enhance the negative conversion ratio of HBeAg, including that of refractory diseases. 9,10 Based on the preliminary work, this study carries out research of integrated TCM and NAs for HBeAgpositive chronic hepatitis B. Through national multi-center double-blind randomized controlled study, the effect of integrated TCM and NAs in reducing the recurrence rate after drug withdrawal is to be verified.

Registration
This multi-center, double-blind, randomized placebo-controlled trial was registered before recruitment The case inclusion obeyed the principle of geographical balance nationwide. Volunteers were mainly recruited from outpatient clinics. The inclusion and exclusion criteria were listed below. The criteria for premature withdrawal from the study included protocol deviation, pregnancy, or investigator discretion. Participants were also entitled to halt the study any time.

Exclusion Criteria
Any of the following cases will be excluded: (1)

Sample Size Estimation
We calculated the samples size by the equation: N = (Uα + Uβ)2 × 2P × (1 -P) / (P1 -P0) 2. We referred to a study published in 2016, in which the recurrence rate was 32% CHB patients after withdrawal NAs at 24weeks. In this study, we assumed to reduce the recurrence rate by 10% to 22%. The certainty was set at 80%, considering 20% of sample lose. The sample size was set to 245 for each group, 490 in total.

Therapeutic Regimen
Patients who meet the inclusion criteria will be allocated in a 1:1 ratio to either the experimental group or the control group. Patients in the experimental group will receive TGBXJD Granule along with ETV. Patients in the control group will receive placebo along with ETV. The treatment duration is 96 weeks. All patients will accept drug withdrawal and observe for 24 weeks after treatment course.
Once clinical recurrence occurs, antiviral treatment is suggested. (Figure 1)

Randomization and Blind
Stratified block randomization is conducted by the third party (Chinese Academy of Chinese Medicine Sciences, CACMS) with their online central randomization system. Clinical physicians in sub-centers oversaw recruitment. They would apply for an ID through a web interface provided by CACMS for each patient, by which the patients would be randomly allocated to each group at a ratio of 1:1 via a central randomization system. Each trial drug was assigned to a patient with a unique number, which would be dispatched by the online system. All experimental drugs and corresponding placebo were consistent in appearance and taste. The grouping information of each participant was concealed to all research personnel and data analyzers until the trial ended.

Interviews
From 1st day to the observation point of week 96, patients would be interviewed once every 24 weeks (± 3 days). Corresponding drugs were dispatched each visit. From week 96 to week 120, interviews were conducted every 4 weeks (± 3 days). Medications will be reintroduced in patients who had a relapse during the period of observation.

Assessments
To ensure safety and reveal the curative effects of trail drugs, assessments were adopted regularly throughout the whole trial. (Table 1)

Primary Outcomes
The primary outcome measurement will be the recurrence rate after drug withdrawal.

Secondary Outcomes
Secondary outcome measurements include the virus serological indicator, HBV DNA, liver function, liver biopsy, cccDNA, HBcrAg, HBV-pgRNA, L-HBsAg, symptomatic score of TCM, SF-36 and the CLDQ, which will be measured at the intervention.

Safety Monitoring
Primary vital signs, physical examinations, and some laboratory tests will be performed for safety assessment daily. Primary vital signs include body temperature, blood pressure, heart rate, and respiratory rate. Laboratory tests include routine blood, urine, and stool tests, along with fecal occult blood tests, ECG, abdominal B ultrasound scan. In particular, the relapse after drug discontinuation was defined HBV DNA levels > 2,000 IU/mL as while serum ALT > 2-fold of the upper limit of the normal.

Adverse events
All unexpected AEs occurring during the intervention period will be reported, and the causality related to the TGBXJD will be analyzed. If any adverse event occurs, the study managers will ensure that the participant receives adequate treatment. The AEs will be immediately reported to the principal investigator and to the ethics committee to decide whether the participant should withdraw from the trial. If any symptoms aggravate, the patient will be withdrawn from the study and will be referred for further treatment.

Statistics Analysis
The statistical report is made by an independent statistical analysis team using the proposed blind method in the statistical analysis plan and then submitted to the personnel responsible for summarizing clinical research data for comprehensive analysis. The statistical analysis plan covers objectives, indicators, analysis types, hypothesis testing, significance level, statistical software, analysis, and result expression methods, curative effect analysis, safety evaluation, and analysts, etc.
The statistical analysis team conducts intention-to-treat (ITT) analysis of the main effect indicators.
Data description: The enumeration data is described by the constituent ratio, the measurement data is represented by mean ± standard deviation, and non-normal distribution is described by median and interquartile range.
Comparison of baseline data: Pearson chi-square test or Fisher test is used for comparison of enumeration data, group variance test is used for the comparison of normal distribution measurement data, and non-parametric analysis and test is used for the comparison of non-normal distribution measurement data.
Comparison of curative effect: The total effect of the scale scores is tested by Wilcoxon rank-sum test.
Variance test is adopted for the baseline comparison of symptom score and rate, the comparison after treatment and the difference comparison among groups before and after treatment, and paired t-test is used for the comparison before and after treatment within groups.
Safety evaluation: The comparison of quantitative data in the laboratory is made using the variance test. Meanwhile, all the laboratory data is classified according to whether they are normal or not. The number of normal and abnormal cases before and after treatment is respectively described according to different intervention groups. The adverse events are described using incidence, and the specific performance and degree of all the adverse events in each group and their relationship with drugs are described in detail.

Discussion
HBV infection remains a major global health concern, as the disease itself and its complications, mainly hepatocellular carcinoma (HCC) and cirrhosis, caused 887,000 deaths in 2015 alone 11 .
Currently, therapeutic drugs for CHB are limited to two types of NAs and interferon. Nucleotide drugs can inhibit virus replication for a long time, but it cannot prevent the synthesis of cccDNA 12 13 . Therefore, lifelong medication is required for most patients.However, long-term treatment by nucleotide drugs still has problems that cannot be ignored. Although the drug resistance rate of the currently recommended Entecavir and TDF is low, the forward incidence rate is unclear. Meanwhile, the hygienic economic burden brought by long-term oral drugs is a vital trade-off factor. The withdrawal of NAs can significantly reduce expenditures for medical care and public health. Also, the side effects of NAs can not be neglected 14 , such as the renal toxicity and skeletal toxicity of the first-line therapeutic drug TDF. The new drug TAF can solve this problem, but its price is high and thus the drug has not been widely applied in many countries.
In recent years, a number of scholars have proposed the concept of immune control, namely, the host's immune system still exerts significant and explicit control over HBV after drug withdrawal, HBV DNA is in an immeasurable level, and no virology or clinical relapse will occur a long time after drug withdrawal (above 6 months). Therefore, the long-term virology inhibition after the withdrawal of nucleotide drugs can be regarded as the possible endpoint of therapy. According to the Asia Pacific Association for the Study of the Liver (APASL) Guidelines, HBeAg-positive patients can try to discontinue drugs under strict follow-up visits after serological conversion of e-antigen and consolidation therapy for 12 months 15 .
Current studies show that the risk of relapse after NAs withdrawal is high. A prospective study included 178 CHB patients, and 59.5% of the patients suffered virological relapse within two years, and 48% suffered clinical relapse. However, the safety of drug withdrawal for HBeAg-positive patients seems to be higher. Liu F et al. 16 conducted a long-course observational study on drug withdrawal. A total of 223 patients were included in the study. The results showed that the 10-year relapse rate of long-term effect is better than NAs monotherapy without compromising the safety profile.
Interestingly, certain herbal combinations could act as 'immune-incubation agent' or 'synergist of NAs' against HBV infection, particularly in HBeAg positive CHB patients 8 . This is the first time that integrated Chinese and western medicine has been used to reduce the off-drug recurrence risks in NAs treated population. The results will provide patients with alternative options, especially in those who had undergone long-term NAs treatment and are unwilling to take NAs lifelong.

Trial Status
The

Ethics approval and consent to participate
The study protocol was approved by Ethics Committee of Dongzhimen Hospital, affiliated to Beijing University of Chinese Medicine, before recruiting participants (Ethical approval No.DZMEC-KY-2018-61). and we will not begin recruiting at other centers in the trial until local ethical approval has been obtained. The purpose, procedures, and potential risks of the RCT will be explained clearly to the participants. All participants will give their written informed consent to the research assistant before joining the RCT.

Consent for publication
Not applicable.

Availability of data and materials
Data are all contained within the paper. And we will share the clinical research data in the system of Chinese Clinical Trials Registry (No. ChiCTR1900021232, 2019/02/02, http://www.chictr.org.cn/showproj.aspx?proj=35297)

Competing interests
The authors declare that they have no competing interests.

Funding
This study is funded by China National Science and Technology major projects 13th 5-year plan, No.2018ZX10725505.

Author Contributions
Xiaoke