Study design
This is a large, prospective, multicenter, centrally randomized, double-blind, placebo-controlled trial. The study is being conducted in accordance with the Declaration of Helsinki[15] and has been approved by the ethics review committees of each participating institution. Meanwhile, this study has been registered with the Chinese Clinical Trial Registry (ChiCTR1900025897). Informed consents are being signed for all patients before enrollment. The treatment will be for 72 weeks. The study’s flow chart is shown in Figure 1.
Patient population
In all, 480 eligible CHB patients is being enrolled and followed up in eleven institutions in China between September 2019 and December 2021. The eleven institutions are eleven academic hospital from eight different provinces in China, including Guangdong Hospital of Chinese Medicine, Shaanxi Provincial Hospital of Traditional Chinese Medicine, Guangzhou Eighth People's Hospital, First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine, Chongqing Hospital of Traditional Chinese Medicine, Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Beijing Ditan Hospital, Xi'an Hospital of Traditional Chinese Medicine, Shanghai municipal Hospital of Traditional Chinese Medicine, Shanxi Provincial Hospital of Chinese Medicine, First Teaching Hospital of Tianjin University of Traditional Chinese Medicine. All enrolled patients are randomly assigned at a 2:1 ratio into two groups: the intervention group treated with HHSYP plusAHP (n=320) and the placebo grouptreated with placebo of HHSYP and AHP (n=160).
Western medicine diagnostic criteria
The patients’ western medicine diagnostic criteria are based on “The guideline of prevention and treatment for chronic hepatitis B (2015 version)”[16] and “Liver fibrosis: consensus recommendations of the Asian Pacific Association for the Study of the Liver”[17].
Diagnostic criteria for TCM syndrome differentiation
The diagnostic criteria of TCM syndrome of liver stagnation, spleen deficiency and blood stasis are based on the Standards of Traditional Chinese Medicine syndrome differentiation for liver fibrosis[7].
Inclusion criteria
The inclusion criteria of this study include: agreeing to participate in this clinical trial and signing the informed consent form; fulfilling the diagnosis criteria for CHB-fibrosis with TCM syndrome of liver stagnation, spleen deficiency and blood stasis; age 18-65 years; patients with alanine aminotransferase (ALT)<two times upper limit of normal (ULN) and liver stiffness measurement (LSM) tested by Transient Elastography (TE)< 9.4 Kpa; patients with liver inflammation grade G≤2 and liver fibrosis stage F<2 at first liver biopsy, if patients with F2, who should sign an another informed consent of refusing antiviral treatment voluntarily; Female patients must fulfill: (a) menopause (defined as no menstruation for at least one year) or surgical sterilization, or (b) fertility and fulfilling: negative result of blood pregnancy examination at baseline, and consent to use of appropriate contraceptive measures and nobreast-feed during the treatment and follow up period.
Exclusion criteria
The exclusion criteria of this study include: patients with allergy to the constituents or excipients of the drug used in this experiment or allergic constitution; co-infection with another hepatitis virus (A, C, D or E) or human immunodeficiency virus; patients with metabolic or autoimmune liver disease, or drug-induced liver damage, or congenital liver disease; patients with a history of alcohol, drug or drug abuse; patients with renal creatinine >one upper limit of normal; patients with evidence of cirrhosis or liver cancer tested by upper abdomen ultrasonography/CT/MR; patients with evidence of decompensation of liver function, such as ascites, portal hypertensive related upper gastrointestinal bleeding, hepatic encephalopathy, primary spontaneous peritonitis, hepatorenal syndrome, etc;patients with serious primary diseases such as heart, lung, kidney and other important organs and hematopoietic system, such as heart failure classified as Grade II, III and IV by the New York Heart Association, heart pacemaker, chronic obstructive pulmonary disease, chronic kidney disease, type 2 diabetes mellitus, etc; patients with neurological or mental disorders who are unable to cooperate or unwilling to cooperate; patients with history of diseases of affecting drug absorption, distribution and metabolism (such as inflammatory bowel disease, gastrointestinal surgery, chronic pancreatitis, glutenin allergy, vagotomy, etc.); patients having participated in other clinical trials in the past three months.
Withdrawal criteria
Any enrolled patients will be withdrawn from this study if they have such conditions as follows: a) taking prohibited drugs or therapies, such as antiviral drugs, other anti-fibrosis TCM drugs, or drugs leading to liver fibrosis and so on; b) having serious adverse events (SAE), such as severe liver dysfunction (ALT>10ULN or PT>15s or TBIL>5ULN), cirrhosis or liver cancer, etc; c) having poor compliance, who do not take medicine for more than 4 weeks according to the protocol; d) lost to follow up; e) wanting to cease or withdrawing the consent. If patients showed ALT>2ULN during treatment, hepatoprotective drugs will be used. If patient receiving hepatoprotective drugs still shows ALT>2ULN level for more than two weeks, antiviral agents will be used. These patients will be dropped from this study too.
Recruitment methods
Patients are being recruited in the following ways: a) inpatients or outpatients in each participating institution, b) recruitment advertisement has been designed and posted to attract potential participants, c) recruitment advertisements are also widely disseminated through WeChat’s circle and hospital WeChat Subscription. All participants are being informed of the benefits and risks of participating in the clinical trial, especially the right to withdraw from the trial at any time.
Randomization and blinding
A center-stratified and permuted blockrandomization sequence is being generated by JMP 6.0 software (SAS Inc., Cary, NC) and was performed by Key Unit of Methodology in Clinical Research of Guangdong Provincial Hospital of Chinese Medicine. The research doctor will enroll and assign participantsrandomly to either intervention group or placebo group at a 2:1 ratio through the interactive web response system which was a verified online randomization facility established by the Key Unit of Methodology in Clinical Research. The randomization schedule has been kept confidential and treatment assignments are being blinded to patients, investigators, outcomes assessors and statistician until the completion of the entire study. Corresponding emergency letter has been designed for each coded trial drug. The emergency letter contains a note recording what the coded trial drug is. The emergency letters have been sealed and sent to each participating situation. Unblinding is only permissible in emergencies, such as SAE.
Interventions
All the enrolled patients are being randomized into two groups. The intervention group is beingtreated with HHSYP (23.6g, twice daily) and AHP (6g, twice daily), while the placebo groupis beingtreated with placebo of HHSYP dummy agent (23.6g, twice daily) and AHP dummy agent (6g, twice daily). The placebo of HHSYP and AHParesimilar in taste, shape and color to HHSYP and AHP, respectively and the main ingredients include pearl barley and grilled germinate barley. Treatment will be conducted over a period of 72 weeks and the follow-up visits last for 72 weeks after the treatment. The time points of treatment for the visits are at baseline, 12 weeks, 24 weeks, 36 weeks, 48 weeks, 60 weeks and 72 weeks post baseline. The time schedule of enrolment, interventions and assessments is shown detailly in Figure 2.
Concomitant care and interventions
Any drugs having good benefits for liver fibrosis, such as interferon, nucleoside (acid) antiviral drugs, colchicine, compound bovine fetal liver extract or anti-fibrosis TCM drugs, are not allowed throughout this trial. Hepatoprotective drugs except for Schisandra medication are permitted in theparticipants with ALT>2ULN during the treatment. However, all the concomitant drugs should be recorded detailly in the case report forms (CRF), which will be analyzed and reported at the end of the trial.Drinking, staying up late or being tiredness, which might aggravate the disease, are also not allowed throughout the trial.
Strategies to improve patients’ compliance
a) Before enrollment, individual health education is being given to the participants by the project doctors. Therefore, participants could understand the necessity and importance of the treatment and adhere to intervention protocols strictly. b) Besides to face-to-face interview at regular visits, the project nurses are also conducting another kind of follow-up via wechat every 2 weeks so as to supervise patients to taking drugs on time. c)At each regular visit, the unused drugs and the empty package of the used drugs must be returned. Meanwhile, compliance is being assessed at each regular visit. If poor compliance is found, the project doctor will conduct a face-to-face interview to find out the causes of low compliance and solve relevant problems.
Outcome measurements
Primary outcomes
Improvement or progression of liver fibrosis by histological assessment
Improvement or progression of fibrosis will be defined as at least 1 stage decrease or increase by METAVIR system, whereas no change or less than one stage change compared to that at baseline will be identified as the absence of improvement or progression.Liver fibrosis is assessed by the METAVIR system for fibrosis stage[18]. The fibrosis stages are as follows: no fibrosis (F0), mild fibrosis (F1), moderate fibrosis (F2), severe fibrosis (F3) and cirrhosis (F4). Liver fibrosis will be assessed before and after treatment (at baseline and week 72) based on patients’ preferences. To ensure the quality of liver specimens, every liver specimenshould have a minimum of 15-mm length and at least ten portal tracts. Liver biopsies in all centers are being performed using 18G MAXCO needles (Bard Co., NJ,USA) and the tissue is being fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin, eosin and Masson. All the biopsy slides will be collected by the independent third-party and three pathologists will be invited to examine the biopsy slidestogether in double-blind manner.The final pathological result can be determined only when the results of two pathologists are consistent. If the results of three experts are inconsistent, discussion should be conducted to determine the final result.
Secondary outcomes
Liver stiffness changes assessed by Transient Elastography (TE)
Liver fibrosis progression assessed by Transient elastography will be reflected by an increase in LSMof at least one fibrosis stage. The relationship between LSM and liver fibrosis stage is as follows[19]: LSM≤7.4Kpa: F0-F1; 7.4≤LSM<9.4: F2; 9.4≤LSM<12.4:F2-F3; 12.4≤LSM<17.5: F3-F4; LSM ≥17.5: F4. Measurements of LSM using TEare being performed by a single trained operator at each participating institution according to the standard operating procedures of TE in “clinical application of transient elastography”[19]. And TE will be performed every 12 weeks for each patient.
Other secondary outcomes include changes of the cumulative TCM symptom score, changes in the scores of the Chronic Liver Disease Questionnaireand the short form healthsurvey SF-36, the occurrence of liver cirrhosis and hepatocellular carcinoma.
Safety outcomes
Safety indicators will be monitored regularly and timely to ensure the safety of each enrolled patients, including routine urine test, routine stool test, routine blood test, liver and renal function test and so on. Safety outcomes include results of. These biological indicators are monitored at baseline, 24 weeks, 48 weeksand 72 weeks post baseline.
Data collection, data and safety monitoring board
The CRF are being completed by the investigators and submitted to submitted to the inspector for verification. The CRF data will be double entered into Electronic Data Capture and checked by the data manager. After the data of each center is confirmed to be correct, blind audit and data locking will be carried out. In order to ensure the consistency of the study implementation at each participating center, the trial progress will be supervised by independent monitoring visits so that the trial will be conducted strictly in accordance with the protocol and standard operating procedures. The data in this study will be collected and entered at each site. The data quality will be checked regularly by investigators and monitors. The data and safety monitoring board consists of three independent investigators. The monitoring board will protectthe ethical interests and safety of each enrolled patients by reviewing safety data. Based on the safety concerns, the board also has the power to recommend termination of the study. Any adverse effects or other unintended effects will be collected, assessed and reported truthfully and detailly through out the trial.
Ethics, consent, confidentiality and dissemination
This current trial has been approved by Guangdong Provincial Hospital of Chinese Medicine Ethics Review Committee (BF2018-175-01) and accepted by the ethics review committees at each participating center. The investigators are responsible for obtaining informed consents from trial participants, who should inform the participants about the objectives, treatment regimens, risks, benefits of the trial clearly, including provisions for collection and use of participant data and biological specimens. Though the results of current trial will be published in peer-reviewed journals or presented in academic conference, the patient's information will be kept confidentially according to the legal requirements, which can be disclosed only through legal proceedings. If any harm is confirmed to come from the trial participation, medical expenses and corresponding economic compensation will be paid according to the laws and regulations.
Modification of the protocol
When the clinical started, the protocol is not allowed to be modified arbitrarily. If the protocol was modified a lot, such as changes of study design, patient population, sample sizes, study procedures and so on, a formal amendment should be agreed upon by the sponsor's delegates and the researchers. This amendment should be also approved by each institutional review board before implementation.
Sample size and statistical methods
Based on results from our previous study, it is expected that the rate of liver fibrosis improvement in intervention group will be increased to 45%, while it will be about 20% in placebo group. Using Pass11.0 (package for encyclopedia of medical statistics), we calculated that the sample size needs to be 164 cases in all and 240 cases if there is a dropout rate of 20%. Moreover, it is estimated that 50% of all enrolled patients will agree to have a second liver biopsy after 72-week treatment. Therefore, a total of 480 patients (360 in intervention group and 120 in placebo group) are needed to achieve a significance level of 0.05 and a power level of 0.90.
SAS 9.2 software (SAS Institute, Cary, NC, USA) will be used to perform the statistical analysis for this study. The adverse events and the number of lost cases will be recorded and calculated. Missing data were imputed by the last observation carried-forward method. At baseline, the demographic data and other essential information will be compared between the intervention group and the placebo group. The intention-to-treat (ITT) analysis set included all participants who received at least one dose of the study drug, while per-protocol (PP) analysis was done in a population who completed the whole study without a main violation of protocol. The primary outcomes will be analyzed in both the per-protocol (PP) population and the intention-to-treat (ITT) population. Categorical variables were presented as counts and percentages. Chi-square test or Fisher’s exact test will be used to compare the rate of liver fibrosis improvement or progression between the two groups or subgroups of patients with different ALT stratification (patients with normal ALT vs patients with abnormal ALT at baseline). Since this study is a multicenter clinical trial, the influence of central effect on primary outcomes should be considered in the analysis. Improvement or progression of liver fibrosis by histological assessment (METAVIR score) will be compared by the Cochran–Mantel–Haenszel test between the two groups. In addition to LSM analyzed in the ITT and PP population, the other secondary outcomes were analyzed on a PP analysis principle. LSM were expressed as the mean and standard deviation, or median and interquartile range. Group t test or nonparametric test (Wilcoxon) will be used for comparison between the two groups, while paired t test or signed rank sum test will be used for intra group comparison. The covariance model will be chosen to evaluate the change in LSM between the two groups with an adjustment for baseline. Statistically significant is being defined as a P-value of less than 0.05 and all P values are two-tailed.