Study setting and participants
A total number of 72 participants with mild-to-moderate depression (referred to symptoms of liver stagnation and spleen deficiency) and 36 healthy participants will be recruited at the five trial sites in china, including Peking university sixth hospital, the first affiliated hospital of Jinan university(Guangzhou overseas Chinese hospital), the affiliated brain hospital of Guangzhou medical university, Dongfang Hospital (Beijing University of Chinese Medicine second affiliated hospital) and Beijing Anding hospital Capital medical university. Participants will be informed of details about the study which are purpose, duration, procedures, and key contacts, as well as risks and potential benefits. Participants may withdraw their consent for any reason without any consequences at any time. Eligible 72 participants with mild-to-moderate depression will be randomly allocated to Xiaoyao pill group (Group1) and placebo group (Group2), who will be required to take Xiaoyao pills and placebos respectively twice daily for four consecutive weeks. 36 healthy participants will be allocated to Normal group (Group 3) without taking any drug. The flow chart is listed in Figure 1.
Eligibility criteria {10}
Inclusion criteria
Participants meeting the following criteria will be included:
1. Meet the Diagnostic Statistics Manual of Mental Disorders (DSM-5) regarding the diagnosis of mild to moderate depression;
2. A score between 20 and 35 on HAMD;
3. Meet the TCM criteria of liver stagnation and spleen deficiency syndrome;
4. Aged between 16 - 18 years old, both genders;
5. Patients agree to participate in this trial and assign the informed consent;
6. Capable of reading and follow-up treatment, and permanently live locally.
Exclusion criteria
Participants meeting one or more of the following criteria will be excluded:
1. Bipolar depression, treatment-resistant depression and severe suicidal risk;
2. History of bipolar disorder, schizophrenia, obvious psychotic symptoms and depression disorder caused by non-addictive substances;
3. Combine with severe cardiovascular diseases, cerebrovascular diseases, hepatic diseases, renal diseases, hematological disease, cancer, or other severe primary diseases;
4. Pregnant or lactating women;
5. Inability to finish the compliance test, judge the efficacy and have complete data;
6. Involved with any other clinical trial at the time of consent.
Who will take informed consent? {26a}
All depressed subjects enrolled in the study as well as healthy controls will sign a paper version of informed consent prior to study initiation. Two copies of the informed consent were kept by the researchers and the subjects respectively.
Additional consent provisions for collection and use of participant data and biological specimens {26b}
Standard clinical laboratory findings (including complete blood count, liver and kidney function tests) as indicators of safety evaluation were routinely collected before and after the enrollment of patients to ensure the safety of subjects. Participants' urine samples and blood samples will be monitored by liquid chromatography-mass spectrometry (LC-MS) technology to explore the mechanism of action of the metyrapone at baseline and at 4 weeks.At the same time, participants' blood samples will be used to separate the total RNA. All the samples required for the study do not need to sign additional informed consent, because the Committee has already been approved by the ethics committee and can use the data after signing the informed consent.
Interventions
Explanation for the choice of comparators {6b}
Placebos were manufactured by Jiuzhitang Co., Ltd. Placebos are similar to the physical traits of Xiaoyao pills in size, flavor, scent, and color without the key ingredients. The placebos are composed of corn starch, Pregelatinized starch, maltose, Caramel color and water.
Intervention description {11a}
Participants randomized to treatment Group 1 will take Xiaoyao pills, which are composed of ChaiHu(Radix Bupleuri)-100g, DangGui(Radix Angelicae Sinensis)-100g, BaiShao(Radix Paeoniae Alba)-100g, BoHe(Mentha)-20g, ShengJiang(Ginger)-100g,BaiZhu (Atractylodes macrocephala Koidz) - 100 g, FuLing (Poria cocos Wolf) -100g, ZhiGanCao (Glycyrrhiza uralensis Fisch)-80g.The action and batch number of each herb is summarized in Table 1. Xiaoyao pills are water honey pills (Z20013060) manufactured by Jiuzhitang Co., Ltd. Participants in Group 2 will take placebos which are manufactured by Jiuzhitang Co., Ltd. Placebos are similar to the physical traits of Xiaoyao pills in size, flavor, scent, and color without the key ingredients. The placebos are composed of corn starch, Pregelatinized starch, maltose, Caramel color and water. Participants will be required to take the medicine twice daily for four consecutive weeks and the dosage is 9g twice a day. The normal group will be not given any drug.
Criteria for discontinuing or modifying allocated interventions {11b}
Shedding criteria:
Cases that have been enrolled but do not complete the clinical protocol should be considered drop outs in the following circumstances:
1. Patients withdrew from the trial on their own;
2. Lost to follow-up;
3. Poor adherence;
4. Because some diseases were considered by the study physician to be amenable to withdrawal from the trial.
The case should be explained. If the baseline pharmacodynamic data are available, the results of the last major outcome can be transferred to the final result for statistical analysis, and the research records should be kept for reference.
Discontinuing criteria:
The entire trial was completely stopped in multi-centers for the following reasons:
1. Serious safety concerns identified by the investigator;
2. There were major lapses in the program;
3. Reasons for funding or management by the sponsor;
4. Withdrawal of the trial from administration.
Total discontinuation of the trial can be temporary or permanent. When discontinuing a trial, full trial records should be retained.
Rejection criteria
Cases that have been enrolled but meet one of the following should be removed:
1. Cases were incorrectly diagnosed and incorrectly included;
2. Met the exclusion criteria;
3. One medication was not used;
4. Without any record of detection;
5. Due to the use of some prohibited medication, it was not possible to evaluate drug efficacy.
Rejected cases should state the reason, and their original medical records should be retained for statistical analysis of efficacy.
Strategies to improve adherence to interventions {11c}
To guarantee subject compliance, the study will set up a clinical research coordinator. Distribute drug use record cards to allow patients to record their medication status and follow-up time.
Relevant concomitant care permitted or prohibited during the trial {11d}
Concomitant treatments and forbidden drugs
1. Avoid cold and greasy foods that are difficult to digest.
2. During the period of taking the medicine, keep optimistic and avoid getting angry.
3. People with severe chronic diseases such as high blood pressure, heart disease, liver disease, diabetes, and kidney disease should take it under the guidance of a physician.
4. Normal menstruation, sudden excessive menstrual flow, prolonged menstrual period, or oligomenorrhea, wrong menstrual period, or irregular vaginal bleeding should go to the hospital for treatment.
5. People who are allergic to this product should not use it with caution.
6. It is forbidden to use this product when its properties change.
7. If you are using other drugs, please consult your physician or pharmacist before using this product.
Provisions for post-trial care {30}
There are no expected harms resulting from the trial. After the trial, patients were provided with guidance on specialized treatment protocols in psychiatry.
Outcomes {12}
Primary outcome measures
Hamilton Depression Scale (HAMD)
The HAMD is a tool developed for clinical evaluation of depression and is widely used in studies. It is composed of 17 questions. Each question is scored from 1 to 4 points, where a higher score indicates severe symptoms; 0 to 7 point is normal, 7 to17 point may have depression, 17 to 24 point definitely have depression, and more than 24 points means severe depression. Measurements are taken at the baseline, 1 week, 2 weeks, and 4 weeks after treatment.
Secondary outcome measures
Traditional Chinese Medicine (TCM) Syndrome Scale
TCM Syndrome Scale is used as a measure of depression. It consists of 1 primary symptom and 9 second diagnostic symptoms. The participants will be required with mental depression, and needs to have more than 4 other concurrent symptoms of the main symptoms at the same time, and the symptoms should last for a 4-week session. Moreover, participant will be provided with symptoms of stagnation of liver qi and spleen deficiency as follows: (1) Suspicious; (2) Fullness of chest and thigh; (3) Chest tightness; (4) Easy to sigh;(5) Complexion is chlorosis; (6) Stomach fullness; (7) Abdominal pain;(8) Bloating; (9)Nausea; (10)Bowel; (11) Loose stools;(12) Foreign body sensation in the pharynx; (13) Light tongue, white tongue coating; (14) Pulse string is thin or slippery. Participant who is involved with more than 5 symptoms can be diagnosed. Measurements are taken at the baseline, 1 week, 2 weeks, and 4 weeks after treatment.
Exploratory outcome
Urine and blood of participants will be monitored to explore the mechanism of Xiaoyao pills at baseline and 4 weeks by liquid chromatograph-mass spectrometer (LC-MS) technology. Meanwhile, total RNA will be isolated from blood sample using TRIzol (Life Technologies). Differential expression will be analyzed by using TopHat method. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes will be implemented by Methylmion Specific PCR. And the exploratory outcomes will be expected by Network pharmacology to conduct correlation analysis with the differential genes.
Participant timeline {13}
Participant timeline is shown in Table 3.
Sample size {14}
The sample size calculation was performed using the central random distribution system and drug random system provided by BioZi. It is planned to recruit 60 depressive subjects with liver depression and spleen deficiency and 36 healthy subjects. Meanwhile, taking into account a dropout of 15%, we concluded that a total of 108 participants with 36 for each group would need to be recruited to ensure statistically significant results.
Recruitment {15}
A total number of 72 participants with mild-to-moderate depression (referred to symptoms of liver stagnation and spleen deficiency) and 36 healthy participants will be recruited at the five trial sites in china. The number of registrations at each facility is monitored by the monitoring committee via Electronic data capture (EDC).
Assignment of interventions: allocation
Sequence generation {16a}
The study adopts the method of central stratified block randomization. According to the given seed number and segment length, the random number table of 72 subjects was generated by an independent statistician using SAS 9.4 statistical software according to the ratio of 1:1 of the Xiaoyao pills group and the placebo group.
Concealment mechanism {16b}
Not applicable
Implementation {16c}
Not applicable
Assignment of interventions: Blinding
Who will be blinded {17a}
The study used a double-blind design and drugs were coded and packaged blind according to a generated random number table by personnel unrelated to this trial. The drugs in each group were uniformly packaged, while guaranteeing that the Xiaoyao pills and placebos were not different in appearance. The centers dispensed the drugs sequentially with the assigned drug number and in the order of subject enrollment. The blind bottom was kept in duplicate by the principal investigator and the sponsor after sealing, and the blind bottom could not be removed during the trial.
Procedure for unblinding if needed {17b}
Not applicable.
Data collection and management
Plans for assessment and collection of outcomes {18a}
The results and baseline of all participants will be collected and evaluated on EDC.
Plans to promote participant retention and complete follow-up {18b}
A subject follow-up record form was set up for this study, and the follow-up of subjects was managed by the clinical research coordinator.
Data management {19}
The sponsor will collect paper version CRFs of patient data for proper storage as study raw materials. All data will be entered into the EDC, any traces of entries, modifications, deletions etc. in the EDC will be retained in the log showing who and when they were changed.
Confidentiality {27}
All patients' data are kept confidential and not disclosed. Only study physicians granted authority by the sponsor have access to the EDC by account number and password, and can only enter and review patient data at their site. The statistician and sponsor have access to the data for all participants.
Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33}
After signing an informed consent form, patients will be given a baseline blood sample and retention urine sample, in which blood routine, liver and kidney function and urine routine are safety evaluation indicators. Meanwhile, a portion of blood samples will be used for DNA methylation detection and a portion of urine samples will be used for metabolomics detection. These blood and urine tests will be performed again when the patient is out of the group.
Blood and urine samples will be stored properly and will not be used by any other route than the study. Meanwhile, existing as well as further sample specific studies will be conducted with ethics committee approval.
Statistical methods
Statistical methods for primary and secondary outcomes {20a}
Investigators will establish the electronic case into the EXCEL form when making follow-up observations by interviewing the participants. Paper will be entered into a database by those investigators in order to ensure data validity. All analyses will be performed using SPSS software version 21.0. Differences are considered to be statistically significant for two-sided P<0.05.Comparisons between groups will be conducted by using an analysis of covariance (ANCOVA). The significance of the cure rate, improvement rate and unhealed rate differences between the groups will be compared using ridit analysis and chi-square test.
Interim analyses {21b}
Security monitoring will follow through the study at all times. Adverse events are reported immediately to the principal investigator, who is required to report them to the clinical trial monitoring committee within the prescribed period of time, depending on the severity. The clinical trial monitoring committee decided on a case by case basis whether to terminate the study.
No efficacy interim analysis was set up in this study.
Methods for additional analyses (e.g. subgroup analyses) {20b}
Not applicable.
Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c}
Participants with missing primary or secondary outcome data will be excluded.
Plans to give access to the full protocol, participant level-data and statistical code {31c}
This study protocol is registered with the registration number ISRCTN12746343 on the International Standard Randomized Controlled Trial ( http://www.isrctn.com/ISRCTN12746343).
Oversight and monitoring
Composition of the coordinating centre and trial steering committee {5d}
There are several clinical study coordinators at each study center. And a clinical research monitoring board is set up to monitor the safety of the study, the authenticity and integrity of the data. The clinical study monitor is held by the sponsor and a third party other than the investigator.
Composition of the data monitoring committee, its role and reporting structure {21a}
Not applicable.
Adverse event reporting and harms {22}
1. Once an adverse event occurs, the investigator shall state to the subject that the subject is required to report truthfully the changes in his condition following the medication. Physicians are to avoid inducing questions.
2. While observing efficacy, pay close attention to observing adverse events or unanticipated toxic side effects (including symptoms, signs, laboratory tests), analyze the causes, make judgment, and follow-up observations and records.
3. For adverse events occurring during the study period, their symptoms, extent, time of appearance, duration, handling measures, experience, etc. should be recorded on a case report form, evaluated their relevance to the study drug, and recorded in detail by the investigator, signed and dated.
4. When an adverse event is identified, the observing physician may decide whether to discontinue observation based on the condition, and cases who discontinue because of adverse effects should be followed up with a detailed record of the handling and outcome.
5. In the event of a serious adverse event in a study, the unit that assumes the responsibility for the clinical study must take immediate measures to protect the safety of subjects, must report to the project leader and the Research Center for clinical trials of drugs, should report within 24 hours to the drug administration, the subject responsible unit and the ethics committee. Investigator to sign and date on report. The subject responsible unit will guarantee reporting procedures that meet all legal and regulatory requirements.
6. When urgent breaking of blinding is required for a serious adverse event to occur in a clinical study, the blinding should be broken jointly by the project leader, investigator, clinical monitor.
Frequency and plans for auditing trial conduct {23}
The sponsor and the study center will meet regularly to ensure that the study is being conducted in accordance with the study protocol.
Plans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees) {25}
If the study protocol is amended, it must be approved by the ethics committee of Beijing University of Chinese medicine, and patients will also be provided written informed consent to inform the modification.
Dissemination plans {31a}
The results of this study will be published in a scientific journal.