YLTZ in the treatment of primary hyperlipidemia (phlegm-turbid obstruction type): study protocol for randomized, double-blind, parallel dose control and multi-center phase II a dose exploration

Backgrounds: Yinlan Tiaozhi Capsule (YLTZ) is a prescription of traditional Chinese medicine (TCM) based on clinical experience. YLTZ have been used for the treatment of primary hyperlipidemia (HLP) in China, performing the disorder of blood lipids level, but few clinical studies have been conducted to assess its ecacy and safety in the treatment of primary HLP. Here, we designed a clinical trial combining Western Chinese medicine and TCM evaluation systems to evaluate the ecacy and safety of primary HLP. Methods: The study is designed as a randomized, double-blind, parallel dose control and multi-center clinical trial. Eligible subjects (n=120) will be allocated after satisfying the criteria (western medicine). Subjects will be randomized to receive YLTZ, or a placebo for 12-week treatment and with follow-up after treatment to record symptoms and signs and to collect serum samples for detecting the lipids level. At the same time, the syndrome differentiation criteria of TCM, such as body mass index, furred tongue and palpitation, will be recorded as determined by doctors of both Western and Chinese medicine. Participants will be instructed to comply with the protocol and to keep a daily record of symptoms. The primary and secondary outcomes and safety indicators will be used to evaluate the ecacy and safety of YLTZ in the treatment of primary HLP based on both Western Chinese medicine and TCM evaluation systems. Discussion: Previous studies were expected to evaluate whether the addition of YLTZ to standard routine treatment would enhance the treatment effectiveness and improve the primary HLP. However, this trial is focused on the outcome of lipids level, and we chose a range of outcome measurements to assess the improvement of relevant symptoms and signs. This trial is the rst study designed to dene and optimize the outcome measurements of lipids level of YLTZ in the treatment of patients with primary HLP. Trial registration: The trial has also been registered with the Center for Drug Evaluation (CDE, CTR20190061) and the China Clinical Trials Registry (ChiCTR1900021326). Scr, Serum creatinine; TBIL, Total Bilirubin; TC, total cholesterol; TG, Triglyceride; TT, Plasma thrombin time; TLC, Therapeutic life-style change; CK, Creatine Kinase.


Introduction
Background and rationale HLP is a chronic metabolic disease caused by abnormal metabolism of lipids, which mainly refers to a disorder of blood lipid metabolism in which total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-C) and/or high density lipoprotein-cholesterol (HDL-C) in plasma are too high and/or too low 1 .
Numerous studies have demonstrated a consistent and linear association between the magnitude and the duration of the exposure to high LDL-C levels and the risk of developing atherosclerotic cardiovascular diseases (ASCVD) which is noted as a high-risk fatal diseases [2][3][4] . How to carry out lipidlowering therapy has become an important topic in the eld of treatment of ASCVD 5 6 .
Generally, it was classi ed into two type, including primary HLP and secondary HLP 7 . In perspective of diagnosis of HLP, except that bad lifestyles (such as high energy, high fat, high sugar diet, excessive drinking, etc.) are associated with dyslipidemia, most primary HLP is caused by single or multiple gene mutations which has family aggregation and obvious hereditary tendency, so it is usually called familial HLP in clinic 7 . Secondary HLP refers to dyslipidemia caused by systemic diseases, such as diabetes mellitus, nephrotic syndrome hypothyroidism and polycystic ovary syndrome, etc 8 .
However, there is no conception of HLP in TCM. According to its clinical manifestations, it can be classi ed as "vertigo", "chest obstruction", "stroke", "blood stasis" and "phlegm-dampness" 9 10 . Most TCM physicians believe that the basic pathological changes of HLP belong to the de ciency of the liver, spleen and kidney, and the de ciency of phlegm and blood stasis 10 12 . Hence, lipid-regulating therapy should be based on the principles of smoothing the liver, invigorating the spleen and tonifying the kidney. Recently, in terms of the analysis of literature reports, many clinical practices and experimental studies of TCM have proved that it has unique advantages in regulating lipid 13 14 : it can regulate the function of viscera, enhance the metabolism of the body to clear the source of the root, and has no drawbacks of the decline of western medicine, the gradual increase of drug withdrawal and many side effects.
YLTZ, consisted of Pummelo Peel, Ginkgo biloba, Gynostemma Pentaphyllum and propolis, was clinically used to treat HLP for ten years. It was comprehensively considered to replenish qi to invigorate the spleen, promote blood circulation and remove blood stasis and eliminate phlegm and dampness based on TCM theory, and previous pharmacological experiments evidenced these functions [15][16][17] , thereby resolving the root causes and symptoms of HLP in the theory of TCM 18 . In addition, it was approved by the State Food and Drug Administration for clinical trial in 2012, the batch number is 2012L01011.
At present, this trial is planned to carry out a phase IIa exploration, Beijing Hospital of Traditional Chinese Medicine a liated to Capital Medical University is the head of the clinical trial group, coupled with other 7 quali ed research hospitals. The e cacy and safety of YLTZ in the treatment of primary HLP (phlegm turbidity obstruction type) are preliminarily evaluated, and the optimal clinical dose is explored to provide the basis for the next phase of clinical trial.
According to the Regulations on Drug Registration Management, GCP, Technical Requirements for Clinical

Research of New Chinese Medicines and Guiding Principles for Clinical Research of New Chinese
Medicines, this clinical trial plan was formulated based on the data of pre-clinical research, main components of prescriptions and main functions of YLTZ.

Objetives
With LDL-C as the main evaluation index, the e cacy and safety of YLTZ in the treatment of primary HLP (phlegm turbidity obstruction syndrome) were evaluated by randomized, double-blind, dose-parallel control and multi-center clinical trial design.

Trail design
This study is a double-blind, randomized, parallel-dose, placebo-controlled, multi-center clinical trial comparing YLTZ and placebo capsules (PC) in people with HLP. A brief ow chart of the study protocol is shown in gure1. The study complies with the principles of the Declaration of Helsinki and Good Clinical Practice guidelines. Its reporting will be guided by the CONSORT statement and the relevant extensions related to herbal medicine interventions.
The trial will be lead and organized by Beijing Traditional Chinese Medicine Hospital A liated to Capital Medical University (Beijing, China), and will be conducted together with other 7 hospitals. A total of 120 participants will be recruited. After acquiring consent from the participants and their parents/legal guardian by investigators, the participants will be enrolled for a trial period of 16 weeks and will be required to attend a total of 5 visits during the trial. The study consists of a 4-week run-in period and a 12week treatment period. The duration for the run-in and treatment was decided based on our previous systematic reviews.
Potential participants will undergo preliminary screening for eligibility during visit 1 by investigators, which will include a registered medical practitioner. Initial assessments for baseline data collection will be conducted if the participants are eligible, which includes the Chinese Medicine Questionnaire, measurement of primary outcome, second outcome and safety assessments. A daily diary will then be given to record the occurrence of adverse events and use of topical treatments during the 4-week run-in period. The treatment period will start after that, participants will be randomly assigned to either the treatment (YLTZ) groups or the control (PC) group. During the treatment period, the outcome measurements will be collected once per 4 weeks, and participants will also be required to record their medication intake, including trial interventions and occurrence of adverse events to assist with compliance monitoring and acceptability of intervention.

Settings and participants
We will recruit participants in two ways. Under normal diet, the serum lipid level after fasting for 12 hours was measured, which met the following criteria of blood lipid level strati cation. TC or TG increased, or HDL-C decreased.   Tongue and pulse images are only described and not scored.

Inclusion criteria
(1) Age between 18 and 75 years old; (2) TCM syndrome differentiation of phlegm and turbidity obstruction syndrome; (3) The introduction period conforms to the diagnostic criteria of Western medicine for primary HLP, and 3.4 mmol/L (130 mg/dl) = LDL-C < 4.9 mmol/L (190 mg/dl); (4) Therapeutic life-style change(TLC) intervention for four weeks during the introduction period (maintaining a balanced diet and living habits, and discontinuing all drugs affecting blood lipid, see attached. Record 1) The fasting blood lipids were still in accordance with the above diagnostic criteria after the end of the import period, and the difference between the import period and the baseline period was not more than 12% (based on the higher value); (5) Those whose baseline dietary score was less than 5 points; (6) Voluntary signing of informed consent. Note: The total score < 3 is qualified; the total score is 3-5 is mild malnutrition; the total score > 6 is severe malnutrition.

Exclusion criteria
(1) Patients who suffered from acute myocardial infarction, cerebrovascular accident, severe trauma or major surgery within six months; (2) Patients with HLP caused by other diseases, such as nephrotic syndrome, hypothyroidism, gout, severe hepatobiliary diseases (viral hepatitis, cholecystitis, cholelithiasis), etc., have been diagnosed as secondary HLP. Patients with HLP and known homozygous hypercholesterolemia caused by drugs (phenothiazines, beta-blockers, adrenocorticosteroids and some contraceptives, etc.); (3) Patients who are using hormones, thyroxine and other drugs affecting blood lipid metabolism and those who have taken other lipid-lowering measures in the past two weeks; (4) Patients who have undergone PCI and CABG in one year and a half Patients, patients with acute coronary syndrome, patients with cardiac function of three or more levels within six months; (5) Allergic constitution or allergic to known drug ingredients; (6) Patients whose blood pressure (> 180/110 mmHg) has not been controlled by systemic hypotension therapy; (7) Patients with triglyceride (> 5.7 mmol/L) (500 mg/dl); (8) Patients who smoked more than ve cigarettes a day in the rst three months of screening, or more than ve cigarettes a day during the experiment; (9) Patients with abnormal liver and kidney function (ALT and/or AST (> 1.5 times of the upper limit of normal value, Scr > the upper limit of normal value) and their fabrication. Patients with severe diseases of the blood system; (10) Patients (including male patients's pouses) had pregnancy plans, pregnant or lactating women during the trial period and within 6 months after the last medication; (11) Patients requiring long-term anticoagulation and adrenocorticosteroids; (12) Type 1 diabetes mellitus and type 2 diabetes mellitus (systemic hypoglycemic therapy, random blood sugar (> 11.0 mmol/L), glycosylation after the introduction period. Patients with hemoglobin (> 9%); (13) Patients with malignant tumors, chronic alcoholism, drug dependence or mental illness; (14) Those who have participated in or are participating in other clinical trials in the past three months; (15) Those who are not considered suitable to participate in clinical trials by researchers.

Suspension criteria
In the course of the study, the whole experiment will be terminated in many centers for the following reasons: (1) The researchers nd serious safety problems; (2) The e cacy is too poor to continue the experiment; (3) The scheme has major failures; (4) The bidder has major nancial or management reasons; (5) The administrative department could cancel the experiment, and all the experiments could be stopped in the middle of the study;

Randomization and blinding
The method of strati ed block randomization was adopted, and the center was taken as strati ed factor for random grouping. Professional statisticians use SAS statistical software to generate random number grouping tables and complete the blindness of experimental drugs.
In this experiment, a double-blind design was used to package YLTZ and PC in the same way. The experiment was designed by two-stage blind method. The random coding table is set up by the statistical unit and sealed in two copies. It is submitted to the responsible unit of clinical trial and the bidding unit for proper preservation. Each trial drug has an emergency letter with the corresponding drug code, which is distributed to the centers. Emergency letters can only be opened when an emergency occurs in the subjects, and the emergency management must depend on the understanding of the type of test drug, so that it can be broken blindly. All emergency letters can be retrieved after the end of the trial.

Sample size
According to the "Drug Registration Management Measures" and the requirements of CDE, it is decided that this experiment is an exploratory study, and the number of cases drafts into the group is 120, inclusive of 20% dropout compensation. Among them, there are 30 cases in high dose, middle dose, low dose and PC group.

Trial interventions
The treatment interventions are YLTZ and matching PC, which are identical in appearance, taste and odor. The capsule of drug and placebo are both produced by Guangdong Yi Fang Pharmaceutical Co., Ltd, and the drug is provided by Guangdong Province Engineering Technology Research Institute of T.C.M.
The concrete administration method includes introduction period and treatment period.
Introduction period for 4 weeks: PC were given orally, 6 capsules per time, 3 times per day. Introducing period: 4 weeks. Treatment period: 12 weeks of continuous medication.
All drugs will be taken orally after meals. All the drugs are concealed in the same sealed and opaque packages. The label of the package contains the drug name, the approval number, functions, usage, dosage, storage conditions, expiration date, and the name of the manufacturer. Participants will be informed that they would be randomly assigned to receive either YLTZ or PC. They will be encouraged to contact the investigators if they have any uncomfortable feeling or if they think the drugs are not helpful.
Participants in both groups can continue their prior routine treatments, with the exception of Chinese herbal medicine. The details of these routine treatments will be recorded in the case report forms (CRFs).
During the trial, the following drugs will be prohibited. Once the prohibited drugs are used, the subjects will be informed to drop out of the study. Any natural and synthetic statins (such as lovastatin, etc.), beta (such as feno brate), nicotinic acid (such as nicotinic acid), cholic acid chelating agent (such as colenemide), ezymeb, probucol, n-3 fatty acid preparations, other traditional Chinese medicines with phlegm-resolving effects, Chinese herbal tablets or Chinese patent medicines that have the same or similar functions as YLTZ, as well as pharmaceuticals and herbal decoctions containing phlegmresolving effects.

Outcomes measures
The primary outcome measures include the evaluation of the relative baseline changes of LDL-C in 12th weeks of administration.

Safety evaluation
The indices of vital signs, cardiopulmonary signs and electrocardiography will be compared before and after the treatment to assess for safety. Additionally, research interviewers will keep in touch with each participant to monitor safety.
Any adverse events will be recorded in a CRF with details, including occurrence time, severity, duration, effective measures and the outcomes. According to the judgement of severity, the investigators will decide whether the participants should be suspended or withdrawn from the trial. When SAEs occur, such as severe haemorrhage, hepatic failure, renal failure or death, the investigator will report to the principal unit and ethics committee, and the investigator can open the patient's emergency envelope to determine the group allocation..

Statistical analysis
All statistical analyses will be performed by an independent statistician blinded to the allocation, using the Statistical Analysis System (SAS, Version 9.4, Institute Inc., Cary, NC, USA). The main contents of the statistics include the following parts: the actual number of subjects enrolled in each group, the situation of dropped and excluded cases, demographic and other baseline characteristics, compliance, e cacy analysis and safety analysis.
All statistical tests are carried out bilaterally, and the difference between the two tests will be considered statistically signi cant if the P value is less than or equal to 0.05. The description of quantitative indicators will calculate the mean, standard deviation, median, minimum, maximum, lower quartile (Q1), upper quartile (Q3), and classi cation indicators will describe all kinds of cases and percentages. The comparison of general situation among groups will be analyzed by appropriate methods according to the types of indicators. The comparison of quantitative data between groups will be conducted by group t test or Wilcoxon rank sum test, the classi cation data by chi-square test or exact probability method, the rank data by Wilcoxon rank sum test or CMH test, and the comparison of multiple groups of data by variance analysis or Kruskal-Wallis test.
Full analysis set (FAS) refers to the ideal set of subjects as close as possible to the intentional analysis principle (the main analysis should include all randomized subjects). This data set is obtained after all randomized subjects are eliminated with the smallest and reasonable method, including all randomized subjects who have used the research drug once. For the estimation of the missing values of the main variables, the results closest to the rst observation (last observation carry forward) are used to carry forward to the missing place of the test data. Per Protocol Set (PPS) is a subset of FAS data set that is more compliant with the protocol, requirements refer to the case set that conforms to the inclusion criteria, does not conform to the exclusion criteria, and completes the treatment protocol, that is, there is no serious violation of the protocol (including the inclusion criteria), good compliance (compliance between 80% and 120%), and complete the CRF requirements (no lack of main e cacy indicators) for PP analysis. Safety data set (SS), which is treated at least once, has actual data recorded by safety indicators. Safety missing value does not need to be carried forward.

Discussion
This study protocol was designed to investigate the e cacy and safety of YLTZ in the treatment of primary HLP (phlegm turbidity obstruction syndrome), and would be conducted under the principle of random, double-blind, parallel dose control and multiple-center.
In this trial, combined with the outcome measurement of biochemical indexes, dietary evaluation and quantitative grading of TCM syndromes, we mainly investigate the effect of YLTZ on primary HLP which is also characterized as phlegm and turbidity obstruction syndrome, coupled with the LDL-C level is between 3.4 mmol/L (130 mg/dl) and 4.9 mmol/L (190 mg/dl).
For statistical analysis, PP analysis and FAS analysis were carried out for the primary outcome at the same time. Covariance analysis model was used to estimate LSmean and 95% CI of LDL-C before and after treatment. 95% con dence interval of the difference of LDL-C between high, medium and low dose groups and control groups was calculated. In order to investigate the consistency among the centers, a covariance analysis model with center-grouping interaction terms will be considered on the basis of the above-mentioned covariance analysis model, and whether the interaction terms are meaningful will be judged at 0.10 level. Secondary outcomes, including TC relative baseline change value, TG relative baseline change value, HDL-C relative baseline change value, non-HDL-C relative baseline change value, TCM syndromes e cacy and TCM single symptoms e cacy, will be compared between groups using the above general statistical methods on the basis of descriptive statistics.
Nevertheless, there are some challenges. The enrollment will meet some di culties, because the run-in period is su ciently long for patients to lower their lipid levels by taking exercise and balancing diet. In addition, 6 capsules per time might increase the reluctance and therefore decreasing the compliance. Hence, the measurements we probably take is expanding the source of patient recruitment, such as community hospitals, and increase the frequency of formal explanation of the danger of primary HLP to enhance the voluntariness of taking medicine.
The results of this trial will provide clinical data on the e cacy and safety of YLTZ in decreasing the content of LDL-C and improving the quality of life of patients with HLP. Positive results from the trial can lead to a better management of primary HLP to help patients. This investigation will also contribute to the understanding and treatment of primary HLP from Chinese medicine perspectives.

Quality control
Quality control will be performed as follows

Clinical trial records
Researchers should ll in all cases according to the design requirements of "original medical record form".
The original medical record is the original record and cannot be changed. When making any correction, the original record should not be blacked out but only a horizontal line shall be drawn at the modi ed place, and the reason should be explained by adding a description. The doctor participating in the clinical trial shall sign and date it.
Laboratory data in clinical trials should be recorded and the original report (or copy) should be attached to the original medical record. The signi cant deviation or data beyond the clinical acceptable range should be veri ed, and the doctors participating in the clinical trial should make necessary explanation.

Training researchers
The personnel involved in clinical trials must have professional quali cations, receive training on relevant work of the trial, and have su cient experience to complete the tasks they are responsible for. Before the start of clinical trial, the personnel participating in the clinical trial shall be trained uniformly. The training documents of clinical trial (one set for each person) include: the researcher's manual, clinical trial protocol, informed consent, original medical records, trial drug use record form, and standard operating procedures formulated or modi ed for speci c research projects, so as to enable the personnel to understand clinical trial protocol well.

Veri cation of data
The researcher should verify the signi cant deviation or data beyond the acceptable range, and the researcher shall make necessary explanation and sign to determine whether it has clinical signi cance.
Each test item must indicate the unit of measurement adopted. All observations and ndings in clinical trials should be veri ed to ensure the reliability of the data and ensure that all conclusions in clinical trials are derived from the original data.

Laboratory quality control requirements
The laboratory should establish standard operating procedures and quality control procedures. When the main indicators may be subject to subjective in uence, consistency test should be carried out. When the test results of each central laboratory are signi cantly different or the normal reference value range is different, the normal value range of each center should be checked. Content for the schedule of enrolment, interventions, assessments and adverse events.

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