The efficacy and safety of Shenzhu Guanxin Recipe Granules for treatment of patients with coronary artery disease: protocol for a double-blind, randomized controlled trial

Coronary artery disease (CAD) is one of the most common types of the cardiovascular diseases, previous pilot trials suggested that Traditional Chinese Medicine brought clinical benefits for patients with CAD. We will conduct this trial to determine the efficacy and safety of Shenzhu Guanxin Recipe Granules for treatment of patients with coronary artery disease. Patients and Methods This randomized controlled trial recruited 194 patients who were diagnosed CAD by clinical manifestations and examinations, in which coronary computed tomography angiography (CCTA) showed 50-70% of stenosis, with soft or mixed plaque. The included participants were randomly assigned to the case group and control group with a 1:1 allocation ratio; patients in the case group received SGR and usual care, and those in the control group received placebo (6 g/day for 6 months) and usual care. The endpoint of the study included calcium coverage score (CCS), C-Reactive protein (CRP), and the levels of blood lipids, tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6), and ATP-binding membrane cassette transporter A1 (ABCA1) were calculated before recruiting at6th month. The indicators were Seattle Angina Questionnaire (SAQ) and TCM syndrome questionnaire scores at 0th, 3rd, and 6th months. This clinical trial may provide a reliable evidence regarding the clinical effectiveness and safety of SGR therapy for patients with CAD by clinical manifestations and examinations, in which coronary computed tomography angiography (CCTA) showed 50-70% of stenosis, with soft or mixed plaque.


Introduction
Coronary artery disease (CAD) is one of the most common types of the cardiovascular diseases [1], resulting in over 9.5 million deaths worldwide [2], that has remarkably increased globally from 5.2 million deaths in 1990 [3]. Intermediate coronary lesions (ICL), which was defined by luminal narrowing with a diameter stenosis of 40-70%, continues to be a therapeutic dilemma for cardiologists [4]. Although previous studies have indicated that diameter stenosis of 40-70% has been vessel dx involving proximal left anterior descending branch (LAD) with either diabetes mellitus (DM) or low ejection fraction (EF) .

Dropout criteria
The included patients have the right to stop treatment and withdraw from the research project for any reason at any time, and the reason why they want to quit the research project will be recorded in their case report form (CRF). Participants who do not complete the research project with the following reasons should be considered as dropped out. (1) The patient chooses to quit the research project.
(2) loss to follow-up. (3) Poor compliance. (4) The participant develops another severe disease that needs to be treated during the study. (5) Patients whose symptom especially chest pain is not relived during the study should exit and undergo revascularization.

Recruitment Strategies
In order to recruit patients, advertisements were placed in a broad range of media outlets, including the flyers within the hospital, as well as website of Chinese Clinical Trial Registry. Patients who were interested in the trial received information about the study. Each potential participant was informed that the participation is fully voluntary and refuse to participate in the research has no negative effect on their treatment. Those who would like to join the study were later assessed to determine whether they meet the inclusion criteria or not.

Randomization and Blinding
A random number generator in SAS 6.12 software (SAS Institute, Cary, NC, USA) was used to generate a random number in a 1:1 randomization ratio by DME (Design, Measurement, and Evaluation in Clinical Research) Center of Guangzhou University of Chinese Medicine (Guangzhou, China). The research team members, except for the clinical research methodology personnel, will be blinded to the treatment and the group assignment. Participants were informed about the information of case group and control group, while were not told their group assignment, thereby allowing blinding of the participants between the treatment groups. Both the SGR and placebo granule were manufactured by the Jiangyin Tianjiang Pharmaceutical Co., Ltd. (Jiangyin, China), and the placebo was identical compared with SGR in color, size, shape, and taste. The study code will not be revealed until the end of the study, unless there is a serious adverse event (AE).

Intervention
After a recruitment period prior to baseline assessment, the included participants were randomized to the case group and control group, in which the patients in case group received SGR and those in the control group received placebo (6 g/day for 6 months). Simultaneously, the included patients also received usual care according to patients' conditions, including aspirin, clopidogrel, angiotensin converting enzyme inhibitors or beta-blockers, calcium channel blockers, and nitrate esters irrespective of the initial randomization assignment. All the treatments were under the responsibility of physicians according to the clinical guidelines. Study medication (including both placebo and SGR) will be dispensed by the Hospital Central Pharmacy as a set of boxes at the beginning of each study month. In order to check the compliance of patients, the patient was asked to bring the box back. The treatment prescriptions and conditions of patients were recorded in the case form. Details of study procedures of the trial are given in Table 2.

Outcome measures
Before starting the intervention, at 3 months intervention, after completing the intervention (6 months after treatment), and 1 year intervention, all the patients had to complete a questionnaire related to quality of life. Additionally, all the patients underwent laboratory examinations and CCTA at baseline (prior to starting either intervention) and at 6 months follow-up. During the follow-up period, any enrolled participants who were unable to continue the study during the treatment remained in their randomized group to perform intention-to-treat (ITT) analysis.

Primary outcomes
The primary endpoint of the study was CCTA, that mainly included calcium coverage score (CCS). CCS represents the percentage of coronary arteries affected by calcific plaque, which was detected with cardiac CT (CCT). CCS has been shown to be reliable, reproducible, and predictive of cardiovascular risk, and also was highly associated with the risk of CAD [14][15][16]. A study revealed that a twofold increase in CCS was associated with a 34% (P 0.001) increase in the risk of a hard CAD event, in addition to a 52% (P 0.001) increase in the risk of any CAD event [17].

Follow-up protocol
The first follow-up was carried out at 3 months after receiving treatment, and the patients' health condition was assessed by inspecting medical records, which were acquired by completing the case report form. The second follow-up was undertaken at 6 months after receiving treatment, in which the eligible participants underwent CCTA and laboratory examinations, and the researchers also needed to assess the participants' physical conditions according to the SAQ scores and scores of TCM syndrome questionnaire. The third follow-up was performed at 12 months, the eligible participants had to complete SAQ and TCM syndrome questionnaire.

Adverse events (AEs)
All drugs may have side effects or allergic reactions, although no adverse reactions of SGR have been reported yet. Any discomforts or unexpected situations that happen during the experiment period were taken as AEs into account regardless of whether they are related to the study intervention or not. All the AEs were recorded in case report form in detail. Serious AEs, including death, lifethreatening or severely or permanently disabling events, were immediately reported to the principal investigator. The ethics committee assessed whether an AE was related to the experimental drug or not.

Data management
To ensure strict adherence to the study protocol and familiarity with the trial administration process, an independent committee will be formed by the principal research members prior to the beginning of the study. Data management personnel of the committee should be qualified, effectively trained and familiar with the functions of data management. A designated person is responsible for the data management of this clinical trial. When the patients are recruited to the research project, the demographic and baseline characteristic data will be collected by researchers. A standard case report form was used to collect data. Before start of recording, data were de-identified. Clinical outcomes, the results of SAQ, TCM syndrome questionnaire, adverse events, and the reasons why participants drop out of the study will be recorded in detail on CRFs. In order to decease errors, data of the CRFs will be entered by two researchers independently. They will check each other's input values and only the consistent data can they be stored in the database. Paper files were kept in a locked filing cabinet in the hospital. With respect to the electronic documents, the results of laboratory tests and CCTA were stored on a password-protected computer, and access was restricted only to the principal investigator.

Determination of the sample size
Firstly, we hypothesized that the expected difference in the primary outcome (coronary artery calcification score) between the SGR group and placebo group was estimated to be 10%. The reason that we considered 10% decrease as the clinically significant effect size was because a coverage probability of 90% for the confidence interval in the case of bioequivalence studies had become the accepted standard when evaluating whether the average values of the pharmacokinetic parameters of two formulations were sufficiently close [20]. Thus, the 95% CI of the difference in the group means within the interval of -10 to +10% was defined as clinical equivalence in the current study. Secondly, to calculate the sample size, we employed the "pwr.t.test" function in R package "pwr" (R package version 1.2-2. https://CRAN.R-project.org/package=pwr) [21]. As an example, say we want to be able to detect a difference of at least 6.2 in the mean CCS (about 10% decreases in CCS) with a common standard deviation of the two groups to be 10. Therefore our effect size is 6.2/10 = 0.62 according to Cohen (1988) [22]. For a desired power of 80%, Type I error tolerance of 0.05, and a hypothesized effect size of 0.62, we should sample at least 84 participants per group, i.e., a total of 168 participants. If assuming there will be a dropout rate of 15% within 6 months, then 194 participants can eventually be recruited.

Statistical analysis
All data analysis will be conducted by qualified statisticians in a double-blind manner according to the intention-to-treat principle. The database will be built by EpiData 3.1 software. In this study, SPSS 22.0 software (IBM, Armonk, NY, USA) was used to perform statistical analysis. Continuous variables were expressed as mean ± standard deviation (SD) or median, and categorical variables were reported as numbers and percentages. Student's t-test was used for making comparison between the two groups as well. Besides, one-way analysis of variance (ANOVA) was applied for making comparison between the groups. Pearson's chi-squared test was applied to sets of categorical data to evaluate how likely it is that any observed difference between the sets arose by chance. P-value < 0.05 was considered statistically significant.

Discussion
Although the treatment of CAD has remarkably advanced, the treatment of ICL has still remained controversial. Chinese herbs had been proved to have definite curative effects on CAD and gradually attracted scholars' attention in clinical trials[23-25]. Furthermore, SGR had been proved to be effective for patients with angina pectoris after PCI, and it can also upregulate the expression of platelet/endothelial cell adhesion molecule-1 (PECAM-1)/CD31 and vascular endothelial growth factor (VEGF), thereby promoting myocardium angiogenesis in myocardial infarction rats [26].In this study, we attempted to further investigate the efficacy and safety of SGR in patients with ICL, in which CCS was used as an indicator to evaluate the efficacy. Blood profile was associated with atherosclerosis, which was one of the most important risk factors of CAD. The serum concentration of high-density lipoprotein-cholesterol (HDL-C) was detected as a strong, independent and inverse predictor of atherosclerotic cardiovascular disease (ASCVD), in particular, CAD [27,28]. Genes had been proved to be associated with the serum concentration of HDL-C, including ABCA1, which can modulate the concentration of HDL-C and catalyze the transfer of lipids from various tissues and cells to apolipoprotein A-I (apo A-I) [29,30]. In this trial, in order to further reveal whether SGR can affect the metabolism of blood lipids, the concentration ABCA1 was also calculated in addition to the concentration of blood lipids.
Numerous studies have reported a relationship between the elevated levels of circulating inflammatory markers and adverse cardiovascular events [31]. TNF-α induces the production of IL-6, that can activate hepatocyte production of CRP [32]. Among myriads of inflammatory markers, CRP was found as a valuable biomarker in refining risk assessment [33,34]. In the present clinical trial, the influence of SGR on inflammatory markers, such as TNF-α, IL-1, IL-6, and CRP were assessed as well.
This study not only evaluated the efficacy and safety of SGR therapy for patients with ICL, but also discussed the potential mechanisms of SGR for the treatment of CAD. To minimize bias of the trial, a rigorous set of methods were conducted, including randomization, in which the blinding method and statistical analysis were carried out according to the ITT.
There are some limitations in this study. Firstly, although we recruited patients with PBSS due to qi deficiency, however, this Chinese medicine syndrome can dynamically change after the intervention.
Secondly, the calculation of sample size was based on a pilot study and clinical observations, in which a larger sample size may result in different achievements. Third, even though several endpoints had been included in our trial to evaluate the efficacy of SGR on patients with CAD, and most of the patients in our study had already finished the cardiac echocardiography. However, the indicators of color doppler echocardiography were not listed as endpoint indicators in our study. In order make the trial to be more rigorous and comprehensive, we should consider to gather and provide other important baseline data such as EF which may affect the treatment protocols significantly in our future studies.
In summary, the present study has provided a solid foundation for the clinical treatment of CAD, in which further evidence can be achieved regarding the application of TCM for the treatment of CAD.

Trial status
This research has been registered on Chinese Clinical Trial Registry (No. ChiCTR1900020501), and the