2.1 Study design
This protocol is designed as a randomized and placebo-controlled trial. Participants and investigators are blinded. A total of 60 subjects will be recruited at the Section II of Endocrinology & Nephropathy department of Dongzhimen Hospital affiliated to Beijing University of Chinese Medicine. They will be randomized in a 1:1 ratio to an experiment group (RG formula) and a control group (placebo). Patients in both groups are given basic blood glucose control. Only the use of insulin and insulin secretion promoters(such as thiazolidinedione and sulfonylurea) can be allowed as basic control in both groups, which have no significant influence on gut microbiota according to the available research. The trial flow diagram is illustrated in Fig. 1. The SPIRIT Checklist is shown in Supplemental file 1[27].
2.2 Participants
2.2.1 Diagnostic criteria
The diagnostic criteria for this trial will be set based on the American Diabetes Association(ADA)
guidelines in 2018[28]:(1) Patients with typical diabetes symptoms and random blood glucose ≥11.1mmol/L or random venous plasma glucose ≥11.1mmol/L. Typical symptoms of diabetes include polydipsia, polyphagia, polyuria, dysphoria, and weight loss without other triggers; (2) FBG or venous plasma glucose≥7.0mmol/L , which is defined as no calorie intake for at least 8 hours; (3) PBG after OGTT test ≥11.1mmol/L; (4) HbA1c ≥6.5%.
2.2.2 Inclusion criteria.
Patients who meet the following criteria will be considered for enrollment:(1) Age 18-65;(2) Meet the diabetes diagnostic criteria of ADA;(3) FBG is stable at less than 7mmol/L and PBG is stable at less than 10mmol/L.(4) Informed and consented to volunteer. The process of obtaining informed consent shall comply with GCP regulations.
2.2.3 Exclusion criteria.
Patients are excluded from participation in the study if any of the exclusion criteria are met: (1) Age >=65 years old; (2) Patients with serious heart, liver, kidney and other complications or with severe cataclysmic disease or mental illness; (3) Pregnant or lactating women; (4) Patients who can not be in conjunction with the diet and medication as prescribed; (5) Inflammatory bowel disease; (6) recent use of antibiotics(within three month); (7) Those who refuse to participate in the study or cannot sign the informed consent.
2.2.4 Withdrawal criteria.
Participants who meet any of the following conditions will be removed from the study: (1) Those who cannot adhere to or automatically abandon the prescribed treatment during the study observation; (2) Incomplete data or poor compliance are found during the study observation, which affected the judgment of efficacy; (3) Severe cardiovascular and cerebrovascular complications and infections occurred during the study observation; (4) Serious adverse events occurred during the study observation and it is inappropriate to continue the study;(5) Severe changes of primary disease in the course of observation.
2.3 Study setting
The treatment will be conducted at Dongzhimen Hospital affiliated to Beijing University of Chinese Medicine. The study will enroll 60 individuals in total, with 30 individuals in each
group. Patients who are included in the study are required to sign an informed consent form and will participate in the study on a voluntary basis. The researchers will obtain written informed consent from each participant before screening. The informed consent form is provided in Supplemental file 2. An overview of specific measurements and time points of data collection are provided in Table 1.
Table 1 Measurement items and point of data capture
Visit project
|
|
Screening period/
baseline
|
Visit 1
|
Visit 2
|
Visit 3
|
visit 4-6
|
Visiting time
|
|
–7 to 0 days
|
Medication
week 4
|
Medication
week 8
|
Medication
week 12
|
follow-up
week 4,8,12
|
Collect basic medical history
|
|
|
|
|
|
Sign informed consent
|
√
|
|
|
|
|
Fill in general information
|
√
|
|
|
|
|
Medical history and treatment history
|
√
|
|
|
|
|
Determine inclusion and exclusion criteria
|
√
|
|
|
|
|
Physical examination
|
√
|
√
|
√
|
√
|
√
|
Comorbidity and medication records
|
√
|
√
|
√
|
√
|
√
|
Effectiveness observation
|
|
|
|
|
|
Glycosylated Hemoglobin
|
√
|
|
|
√
|
√
|
Fasting blood glucose
|
√
|
|
|
√
|
√
|
Postprandial blood glucose
|
√
|
|
|
√
|
√
|
Fasting insulin
|
√
|
|
|
√
|
|
Fasting C-peptide
|
√
|
|
|
√
|
|
inflammatory factors
|
√
|
|
|
√
|
|
Other work
|
|
|
|
|
|
Random grouping
|
√
|
|
|
|
|
Fecal specimen
|
√
|
|
|
√
|
√
|
liver and kidney function
|
√
|
|
|
√
|
√
|
Adverse event
|
√
|
√
|
√
|
√
|
√
|
2.4 Participant recruitment.
The trial Subjects will be recruited from the clinic or the ward in the Section II of Endocrinology & Nephropathy department. Patients recruited by advertisement from other hospitals need to undergo an observation period of 1 week. After the observation, The patients who meet the inclusion criteria will be allowed to enter the clinical trial. The following basic personal information will be collected: sex, age, body mass index (BMI), educational background, occupation, marital status, related past health history, etc. These details about the participants
will be maintained by the data monitoring committee (DMC) and will never be revealed to any individual or organization not connected to the study.
2.5 Randomization and blinding
This clinical research adopts randomized, double-blind, parallel-group study method. Patients who meet the criterion of Type 2 Diabetes are randomly divided into control group and experimental group. Randomization is realized by using the random number table method, which is generated by Strategic Applications Software by a statistician blinded to the treatment and data collection.
The group name will be written on a card and sealed in an opaque envelope. The sequence numbers will be written on the envelopes, and the envelopes will be numbered sequentially.
The sequence number table should be made in triplicate and kept by the project leader, the sponsor and the data analyst. The seal of envelope should not be broken. When the blind is broken, the three copies of the random assignment table should be unsealed face to face at the same time. All experimental drugs of each subject are put into a drug bag and numbered outside the drug bag (1,2,3...). According to the sequence number of the random allocation table, different drugs will be loaded into the corresponding drug bag by a third party. In this trial, we use the simulation agent made of dextrin and condiment as the placebo. Both RG formula and placebo are manufactured by
Kang Taisheng Pharmaceutical Co., Ltd. with the same specification, appearance and flavor.The blinding will be monitored and assessed by independent statisticians responsible for data monitoring and by the Ethics Committee of the study center. The blind code will be disclosed after completion of the statistical analysis.
2.6 Intervention
Patients in both groups are given diabetes education and lifestyle intervention. After basic blood glucose control, the fasting blood glucose is stable at less than 7mmol/L and postprandial blood glucose is stable at less than 10mmol/L. In this process, metformin, GSDI (α-glucosidase inhibitor), DPP-4(dipeptidyl peptidse 4) inhibitor and GLP-1 (glucagon-likepeptide-1) analogue are avoided. Insulin and insulin secretion promoters are allowed. On the basis of standard treatment, The experimental group is given TCM granule packet. Each packet contains 11g RG formula, which include 10g coptis root and 1g ginseng. Patients will take RG formula at least for 3 months, 2 times a day, 11g each time. The control group is given the same amount of Chinese medicine simulation agent which is taken by the same way. The simulation agent contains dextrin and condiment, which looks and tastes exactly like RG formula. The course of intervention is 12 weeks, and medicine is given every 4 weeks. Serum and stool samples are collected at baseline and at week 12 for relevant indicators. During the trial, the subjects are forbidden not to use other TCM drugs (including proprietary Chinese medicine, TCM injections, etc.) in combination. For patients who have chronic complicated diseases and need to take drugs for a long time, which do not conflict with the above inclusion and exclusion criteria, their combined diseases and medication conditions shall be recorded in detail in the Case Report Form (CRF). Follow-up observation is continued after 12 weeks of administration to determine the subsequent effects of the drug. This study has been approved by the Ethics Committee of Dongzhimen Hospital
and all patients will sign the informed consent after understanding the content, process and significance of the study.
2.7 Outcome measures
The physical condition of patients should be recorded at baseline, week 4, week 8 and week 12.
At the start of the trial, we will collect the patient's general information including name, gender, age, education, marriage condition, BMI and etc. The complications of diabetes such as diabetic retinopathy, diabetic nephropathy, diabetic peripheral neuropathy and diabetic peripheral vascular disease should be especially recorded. The other diseases, the corresponding treatment, symptoms, tongue and pulse condition of TCM should also be collected. At week 4 and week 8, two follow-up visits are conducted to collect TCM related indicators such as symptoms, tongue signs, pulse signs and symptoms diagnosis. At week 0 and week 12, fecal samples and blood samples are collected for quantitative determination of gut microbiota, serum biochemical indicators and inflammatory indicators.
2.7.1 Primary outcomes
The primary outcomes in this study is the mean change in Glycosylated Hemoglobin(HbA1c) from baseline to 12 weeks intervention and 12 weeks follow-up intervention between the two groups and within groups. HbA1c is a critical indicator for the diagnosis of T2DM, which can reflect average levels of glycemia during the preceding 3months and is widely used for diabetic complication assessment [29]. It is considered as the gold standard for predicting glycaemia-associated risks for the microvascular and macrovascular complications of diabetes mellitus over 5–10 years[30]. Therefore, The value of HbA1c in the care of patients with T1DM and T2DM is unassailable[31]. Professional societies, public health organizations, regulatory agencies, patients, and clinicians have focused on HbA1c levels to gauge the quality of diabetes care and over time, HbA1c level has supplanted other indicators of the quality of diabetes care, such as blood glucose levels and symptoms of hyperglycemia, despite being a surrogate rather than a direct marker of glycemic control[32]. In addition, careful regulation of blood glucose concentrations in patients with diabetes mellitus normalized HbA1c over 6 weeks[33], so it has also excellent reliability and validity and is sensitive enough for clinical and research practice.
2.7.2 Secondary outcomes
The mean changes in FBG, PBG, FIL, C-P, IRI, inflammatory factors and species abundance of gut microbiota before and after 12 weeks treatment are secondary outcomes. FBG, PBG, FIL and C-P will be measured to further assess and compare change in insulin resistance status. PBG is obtained by rapid detection of fingertip blood. FBG, FIL and C-P is obtained by venous blood sample which will be collected after an overnight fast by a trained medical officer/investigator and transported to a standard tertiary care laboratory for analysis. Laboratory technicians are blinded to the study participants. The insulin resistance index of homeostasis model is based on the formula developed by Matthews[34]. The inflammatory factors include Interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) and C-reactive protein (CRP), which will be determined by investigator by ELISA. The species abundance of gut microbiota will be based on the second-generation high-throughput sequencing technology, in which the V3-V4 region of 16S rRNA gene in the samples is sequenced without isolation and culture of microorganisms. The OTU clustering, species annotation, α-diversity, β-diversity and LDA Effect Size difference score will be analyzed with bioinformatics analysis method.
2.7.3 Safety indicator
Laboratory tests including blood routine and urine routine, liver and kidney function are measured before and after the study. Adverse events, such as hypoglycemic reactions, constipation, and abdominal pain, are recorded in detail and their association with medication is determined. The trial can be terminated if necessary.
2.7.4. Safety assessment
During the study period, the Adverse Event Record Form is completed to record the duration, occurrence time, severity, measures and outcome of the adverse event. The severity of the adverse events is divided into mild, moderate and severe. Mild means mild discomfort, which is tolerable and do not require special treatment; moderate is intolerable and require special treatment; severe discomfort require timely termination of the trial and immediate emergency treatment. Researchers will report it to the person in charge of the trial, the sponsoring unit and the medical ethics committee in time, and even report it to the adverse reaction center according to the relevant regulations of the State Medical Products Administration. Ethics committee have the right to make the final decision to terminate the trial.
2.8 Sample size
Sixty participants(n = 60) will be recruited after run-in period and allocated to RG group (n = 30) and placebo group (n = 30). The sample size was calculated considering the expected change of glycosylated hemoglobin (HbA1c) (primary outcome) of the participants by software PASS15.0. Previous studies have reported that formula including Coptis Root and Ginseng had statistically and clinically significant reduction of absolute HbA1c by about 0.72% [35,36]. Sample size was calculated to have 80% power to detect a moderate 0.65-SD difference in HbA1c, with an alpha/α value of 0.05. Additional participants will be recruited until the required number is achieved (n = 30) per group to account for the estimated drop-out rate of 15%. Therefore, we take the sample size as 30 in both groups.
2.9 Patient and public involvement
Patients in this trial will not be involved in the design, recruitment or conduction of the study. After the measure of the samples, the patients will be informed of the results of indicator data in the form of report. The doctor in charge of the patient will be also informed of the results to optimize treatment and the rehabilitation scheme.
2.10 Adherence to study medication
Strategies to improve adherence to intervention protocols include:
(1)Providing professional lifestyle guidance, 2 weeks of TCM treatment and laboratory index containing liver function, kidney function, HbA1c, fasting blood glucose, postprandial blood glucose and gut microbiota for free. The outcome of HbA1c, fasting blood glucose and postprandial blood glucose are instant available.
(2)Subsidizing the cost of patients' travel to the hospital for visits.
(3)Adding the patient's network contact address, communicating and solving the patients’ problems at any time, requiring the patient to monitor blood glucose at home regularly and upload blood glucose data, continuing to conduct diabetes education.
(4)Checking the number of medicine bags left by the patients at each return visit, as well as confirming the situation of medication.
2.11 Quality control and data collection
In order to maintain the high quality of the trial and ensure compliance with the protocol, all researchers and drug administrators participating in the study will receive rigorous training in accordance with the standardized operating procedures (SOP) manual. Researchers should accurately and clearly record patients’ information in accordance with the requirements of the CRF. The correction of error and its time will be marked. The quality of the study will be managed through the Dongzhimen Hospital Data Monitoring Committee(DMC), where managers regularly check data sheets for omissions and errors to monitor the integrity of test data. The DMC consists of endocrinology clinical experts, methodology experts and biostatisticians, which are independent of the sponsor and have no conflict of interest with research team. Original CRFs will be reserved at the research center in Dongzhimen hospital for 5 years for reference by researchers after completion of the study.
2.12 Statistical analysis
SPSS 20.0 software was used for statistical analysis of data. Measurement data were described as mean (M)±standard deviation (SD). Normality of continues data was evaluated using Kolmogorov–Smirnov test and Q–Q plot. If the paired samples were in accordance with
normal distribution, the t-test of the paired samples was used for pairwise comparison. If not, the nonparametric test was adopted. If the two independent samples were in accordance with normal
distribution and homogeneity test of variances, the t-test of independent sample was used. The t'-test was used when variance was not uniform and the nonparametric test was adopted when the samples were in not accordance with normal distribution. Enumeration data like gender and medical history was described statistically by frequency. Chi-square test and Wilcoxon rank sum test were used to determine whether there was comparability between the two groups. All statistical tests are bilateral, and a P value≤0.05 would be considered statistically significant.