The Efficacy and Safety of Chinese Herbal Medicine Jinlida as Add-On Medication in Type 2 Diabetes Patients Ineffectively Managed by Metformin Monotherapy: A Double-Blind, Randomized, Placebo-Controlled, Multicenter Trial

Background Metformin plays an important role in diabetes treatment. Studies have shown that the combined use of oral hypoglycemic medications is more effective than metformin monotherapy. In this double-blind, randomized, placebo-controlled, multicenter trial, we evaluated whether Jinlida, a Chinese herbal medicine, enhances the glycemic control of metformin in type 2 diabetes patients whose HbA1c was ineffectively controlled with metformin alone. Methods A total of 186 diabetes patients were enrolled in this double-Blind, randomized, placebo-controlled, multicenter trial. Subjects were randomly allocated to receive either Jinlida (9 g) or the placebo TID for 12 consecutive weeks. All subjects in both groups also continuously received their metformin without any dose change. During this 12-week period, the HbA1c, FPG, 2h PG, body weight, BMI were assessed. HOMA insulin resistance (HOMA-IR) and β-cell function (HOMA- β) were also evaluated. Results At week 12, compared to the HbA1c level from week 0, the level of the Jinlida group was reduced by 0.92 ± 1.09% and that of the placebo group was reduced by 0.53 ± 0.94%. The 95% CI was 0.69 - 1.14 for the Jinlida group vs. 0.34 - 0.72 for the placebo group. There was a very significant HbA1c reduction between the two groups after 12 weeks (p < 0.01). Both FG and 2h PG levels of the Jinlida group and placebo group were reduced from week 0. There were a very significant FG and 2h PG level reductions between the two groups after 12 weeks (both p < 0.01). The Jinlida group also showed improved β-cell function with a HOMA-β increase (p < 0.05). No statistical significance was observed in the body weight and BMI changes. No serious adverse events were reported. Conclusion Jinlida significantly enhanced the hypoglycemic action of metformin when the drug was used alone. This Chinese herbal medicine may have a clinical value as an add-on medication to metformin monotherapy. Trial Registration Chinese Clinical Trial Register ChiCTR-TRC-13003159

There are more diabetic patients, and diabetes can cause damage to vital organs of human body, which severely impact quality of life and limit patient's life, and greatly increase health care costs. Therefore, diabetes has posed a common threat to global human life and survival. Due to limited medical standards, diabetes cannot be cured recently. Therefore it should pay close attention to lifelong health of diabetic patients.
The immediate goal of diabetic treatment is to control symptoms of diabetes, control blood sugar, and prevent acute metabolic complications; long-term goal is to achieve good metabolic control, prevent chronic complications, and improve quality of life of diabetic patients.
Metformin is currently the preferred drug treatment of patients with type 2 diabetes.
According to requirements of "Type 2 Diabetes Prevention Guide in China, 2010 Edition", Metformin should remain in treatment programs for patients without contraindications. However, during clinical treatment, when blood sugar is not within reference range, is to continue to increase the dose, or change to a multi-drug combination therapy, and when is the time to make a change, all of these are lack of evidence-based medical research evidence. Doctors often make judgments based on personal experience, resulting in poor treatment.
Data shows [1] that blood sugar begins to rise at 6 months to a year after Metformin monotherapy. A simple increase in single-dose can increase adverse reactions, but not improve efficacy. According to 2011 national survey on type 2 diabetes treated with oral medication, there was 10.7% patients taking Metformin monotherapy and 33.5% taking combination therapy with Metformin as basic drug in a total of 9872 patients being surveyed, but the overall compliance rate of glycated hemoglobin (<6.5%) was only 28%.
The data indicates that it is insufficient to confirm Metformin as preferred therapeutic drug or basic drug in combination therapy for type 2 diabetes, meanwhile Metformin can easily cause hypoglycemia and weight gain with existing antidiabetic drugs such as sulfonylureas / glinides in combination drug treatment, which affects tolerability and compliance of patients, so that the effect is limited.
Traditional Chinese medicine can improve clinical symptoms associated with diabetes, improve quality of life, lower blood sugar and urine sugar, and reduce complications. So Chinese medicine collaterals theory has some advantages to guide treatment of diabetes and reduce its complications.
Collaterals theory suggests that the major pathogenesis of diabetes is unbalanced distribution and usage of body fluid and metabolic disturbance caused by splenic transfusion dysfunction, so it should make emphasis on nourishing Yin, benefiting qi, dissipating dampness, clearing heat, dredging collaterals and promoting splenic transfusion, and take "nourishing spleen and transfusing body fluid" as therapeutic guide. According to literature, the difference is 0.5% in reducing glycosylated hemoglobin for combination of Metformin and Jinlida Granule comparing with single administration of Metformin, and standard deviation is 1.1%, as calculating with a ratio of 1:1 between two groups, taking α=0.05 and β=0.20, and sample size of 77 cases in each group (a total of 154 case). The total number of cases is 192 with consideration of expulsion rate of 20%.
Note: sample size is calculated with PASS2008 software.

Randomized design
This study takes randomized grouping design. Subjects are grouped at a ratio of 1:1 for trial group and control group, which can maximize the advantages of double-blind trial, and eliminate bias produced by unbalanced allocation ratio. This kind of allocation can enhance curative balance for each group, and control other factors that may affect the test results.
The biometric experts who are independent of the data management and statistical analysis use SAS statistics software package on the computer to produce the codes. By employing the stratified randomization method, the codes for the trial group and the control group are generated at random according to a 1:1 ratio. The length of the selected block and the seed parameter of random numbers initial value shall be sealed in the blind codes as confidential data. On the basis of this random number, the statistician who is independent of the trial will encode the drugs. The clinical trial centers will use the drugs according to the assigned drug code and the patient's order in the group. The blind codes are in duplicate and shall be kept in the national clinical research base for drug where the sponsor and the major investigator come from.

Blinding level
This research adopts double-blind design that researchers, patients and supervisors are unknown of the drug subjects being taken, and this method can greatly eliminate bias produced during clinical research and explanation of clinical results.

Emergency unblinding
In double blind study, each blind code is set up with an emergency letter, the content of which includes the group this numbered subject belongs to and situation of medical treatment. In the meanwhile of the drug encoding, the biometric experts who are in charge of generating of the blind codes will prepare an emergency letter for each drug code. The group which the drug code actually belongs to is sealed in the letter. The emergency envelopes are sent to the centers along with the corresponding drugs. The chief investigator in that center is responsible for keeping the envelope. When a serious adverse event occurs, there is an urgent need for unblinding. At this time, the chief investigator in that center shall open the emergency envelope corresponding to its drug code. Once the envelope is opened, the case is considered as drop-out case. However, if there are any adverse reactions, this case shall be recorded in the adverse reaction analysis. The double-blind research will be invalid when the blind codes is leaked or when the opening and reading rate of the emergency envelopes exceeds 20% in the process of clinical research.

Unblinding provision
In this study, subjects of trial group and control group are at a ratio of 1:1, and two-unblinding method is adopted. Data files will be locked after blind review and being confirmed with reliability and accuracy, and then the first unblinding will be carried out.
The first unblinding only states the group that each case belongs to (such as group A or group B), but does not indicate which is the trial group or control group. Results of the first unblinding should be entered computer by statistical professionals, and statistically analyzed after connecting with the data file. The second unblinidng will be carried out after statistical analysis to clear the treatment scheme received by each group.

Diagnostic criteria
Type 2 diabetic disease is diagnosed according to standards established by WHO Committee of Diabetic Experts in 1999.
(1) Symptoms of diabetes (typical symptoms include polydipsia, polyuria and unexplained weight loss) and 1) random blood glucose (level of blood glucose at any time without consideration of the last meal) be and more than 11.1mmol/L (200mg/dl), or 2) fasting blood glucose (fasting state refers to the state without adding calories for at least 8 hours) be and more than 7.0mmol/L (126mg/dl), or 3) at 2 hours after glucose load, blood glucose be and more than 11.1mmol/L (200mg/dl).
(2) The subjects, who are without diabetic symptoms, should detect blood glucose at another day to make a definite diagnosis.

Population selection
(1) The patient, who takes administration of Metformin at a stable dosage for more than 3 months, and underwent strict diet and exercise control, is with blood glucose and gycosylated hemoglobin out of reference range.

Appendix:
① Diet control method: take balanced and reasonable dietary guidance, calculate required total calories according to height, weight and activity level of patient, calculate ideal calories with total intake calorie and actual consumed calorie, balance dietary by nutritionist, and take low-fat diet. Then nutritionist develops recipes and varieties of food, which should be carried out by patients. Calories provided by common fat do not exceed 30% of total calories of diet, acceptable daily intake of saturated fat should not exceed 10% of total calories, and do not eat fried foods or whole milk foods; calorie provided by carbohydrates should take 55% to 60% of total calories, mainly are complex carbohydrates, which should be equally distributed in three meals; calories provided by protein takes 15% to 20% of total calories, mainly is high-quality protein. Quit smoking and limit alcohol, the salt intake should be less than 6 g for hypertensive patients. Take 1600 calorie as example, distribution ratio of three major nutrients is that protein takes 15% to provide 240 calorie, and equivalents to 60 g protein; carbohydrate takes 60% to provide 960 calories, and equivalents to 240 g carbohydrate; fat takes 25% to provide 400 calories, and equivalents to 100 g fat. According to Chinese Food Composition Table   or Food Exchange List, clinical nutrition is calculated, and exchanged to specific amount of daily food. Calorie distribution ratio of three meals is 1/5, 2/5 and 2/5 for breakfast, lunch and dinner of diabetic patients, respectively.

Inclusion criteria
(1). Meet WHO diagnostic criteria for type 2 diabetes mellitus.

Major index
Glycated hemoglobin (being detected in Central Laboratory)

Minor index
(1) Insulin resistance index, insulin sensitivity index and β cell function index for two groups at 12 weeks after treatment; (2) Fasting glucose and 2-hour post-meal blood glucose for two groups at 12 weeks after treatment; (3) Clinical symptoms of patients in two groups at 12 weeks after treatment; (4) Body mass index (BMI); (5) Waistline. Changes of fasting blood glucose and 2-hour post-meal blood glucose are compared between two groups by corresponding statistical method at 12 weeks after medication.

Criteria on clinical curative effect
Disappear: clinical symptom disappears before treatment, and score is 0.
Improve: clinical symptom improves before treatment, and score decrease, but not 0.
Invalid: clinical symptom without improvement or even becoming worse, and score increase.

Body mass index and waistline
Changes of body mass index and waistline are compared between two groups by corresponding statistical method at 12 weeks after medication.

Withdraw decided by researcher
Subject withdrew the study indicates that the enrolled subject cannot continue research at certain condition, and researcher decides the subject to withdraw from research process.
(1) During research, subject has severe acute and chronic complications of diabetes (diabetic ketoacidosis, hyperosmolar nonketotic syndrome, lactic acidosis, hypoglycemic coma, acute myocardial infarction, acute stroke, etc) or special physiological changes (positive HCG) that he or she is inadequate to participate in research continuously.
(2) During research, subject is with poor compliance, dosage of medication is less than 80% or more than 120% of specified amount.
(3) During research, subject violates program rules, and takes oral administration of other hypoglycemic drugs without approval of researcher.
(4) After drug administration of 4 weeks, the subject, who is with measured value of fasting blood glucose higher than 13.9 mmol/L at consecutive 2 times within a week, should withdraw from study.

Subject withdraw research voluntarily
According to informed consent, subject has the right to withdraw research; if the subject does not put forward the idea of withdrawing research clearly, but losses to follow up for refusal taking medication or detection, he or she also withdraws researcher (or "off" (1) If there is serious security problem, the research must discontinue immediately.
(2) If the drug is with poor curative efficacy, or even ineffective, or without clinical significance, the research should discontinue, to avoid delay for effective treatment of subjects or unnecessary economic loss.
(3) If the clinical research program has a major mistake, and is difficult to evaluate drug effects; or a better designed program has significant deviation during implementation, and cannot continue to carry out to evaluate drug efficacy, the research should discontinue. Property: its content is brown to black brown granule, and tastes bitter.

Packaging and label
Jinlida Granule is a kind of granule preparation; its placebo has the same appearance.
Outer packaging is as follows, and each small box has 9 bags.

Drug inventory and recovery
At each visit, observe doctor should record receive amount, dosage and return amount of drug in detail, to determine the compliance of patient, which should be recorded in case report If the patient meets all eligible criteria of this scheme, he or she should complete all examinations. The patient, who meets inclusion criteria and exclusion criteria, should be randomized grouped.
Visit 2: Randomized treatment stage (at the end of the fourth week): (1) Inquire symptoms to determine subject continue or discontinue study; (2) Fasting blood-glucose, 2-hour post-meal blood glucose, glycated hemoglobin, fasting insulin detection; (3) Blood sample for detection of glycated hemoglobin should be preserved in JINLIDA GRANULE AND METFORMIN APPLIED TO TREATMENT OF TYPE 2 DIABETES MELLITUS 21 refrigerator at -20℃ in Central Laboratory; (3) Laboratory safety detection (blood routine test, routine urine test) and ECG; (4) Record clinical symptoms table; (5) Inquire and record concomitant medication and adverse events; (6) Dispense drug, and complete drug dispensation record.

Visit 3: Randomized treatment stage (at the end of the eighth week):
(1) Inquire symptoms to determine subject continue or discontinue study; (2) Fasting blood-glucose; (3) Record clinical symptoms table; (4) Inquire and record concomitant medication and adverse events; (5) Dispense drug, and complete drug dispensation record.

Visit 4: At the end of research (at the end of the 12th week)
(1) Fasting blood-glucose, 2-hour post-meal blood glucose, glycated hemoglobin, fasting insulin detection; (2) Blood sample for detection of glycated hemoglobin should be preserved in refrigerator at -20℃ in Central Laboratory; (3) Laboratory safety detection (blood routine test, routine urine test) and ECG; (4) Record clinical symptoms table; (5) Inquire and record concomitant medication and adverse events; (6) Fill in drug dispensation and recovery record, and make an inventory of recovery drug; (7) Fill in research completion record.

Visit 5: (required follow-up and visit)
The patient, who had persist adverse events, laboratory abnormity with clinical significance, abnormal vital signs, etc, should take necessary follow-up and obtain detection results at follow-up visit. Researcher can take follow-up or telephone visit according to specific situation.

Provision of basic medication Metformin
At research period, Metformin should take original variety and dosage, and cannot change; if there is hyperglycemia, the subject should withdraw research. If there is hypoglycemia, the subject should take lower dosage of Metformin. Adverse events should be recorded.

Combination therapy
(1) The patient should take reasonable diet and exercise for diabetic patients to change lifestyle. Besides of research drugs, the patient should not take other hypoglycemic drugs, insulin or Chinese and Western medicines for treating diabetes, or the drug greatly influencing blood glucose (as corticosteroids, thyroxine, tricyclic antidepressants, weight-reducing drugs etc.).
(2) The drugs, which have administrated to treat other concomitant diseases, should not change the variety and dosage in principle.
( (2) Significant adverse event: besides severe adverse event, any adverse event happens and causes the use of pointed medical measures (e.g. drug withdrawal, reducing dosage and expectant treatment) and hematological and/or other laboratorial anomaly.

Criteria for Judging Intensity of Adverse Event
All clinical adverse events occurred in this clinical research will be recorded on the adverse events page of CRF and classified according to the intensity. In order to have a unified standard, the intensity grade of events is as follows: Slight perceptible malaise but not affect daily activities Moderate stronger malaise leading to affect or reduce daily activities Severe malaise that unable to work or carry out daily activities It needs to pay attention to distinguish the severity and intensity of adverse events.
Severity is used to describe intensity and it may not be a serious adverse event (SAE).
For example, the expression of headache is possibly severe in intensity, but it can't be regarded as SAE unless it meets the criterion of SAE.

Judging standard of the relation between adverse events and investigational drug
The causality analysis of the relation between all adverse events and the investigational drug is judged according to five grades, i.e. certainly related, probably related, possibly related, possibly unrelated and definitely unrelated. The first three grades are judged drug adverse reaction. In the causality analysis we consider the following five aspects. (2) Clinical events which haven't cause death, life risk or admission, but after proper medical judgment, it is believed that the events can cause the potential harm to the patient or it is necessary to give the patient drug treatment or surgery so as to avoid causing the above states should be also identified as serious adverse event.

Follow-up and adverse event record
Adverse events, especially the events which have a connection with the investigational drugs, should be followed up until they return to the baseline or tend to be steady. If  If there is any problem, data administrator should inform supervisor immediately, and ask researcher to make an explanation. Query table should be used to record various questions and answers, and kept properly for reference.
After completion of above works, main researchers, sponsor unit, trail statistical worker and data administrator should conduct blind review, to confirm analysis set each case belonging to, treatment of missing value, determination of outlier, etc. After the blind review and confirming that a correct data base has been established, the data should be locked. The first unblinding is conducted, and blind answer and database should hand over to statistical professionals to conduct statistical analysis. Baseline comparative analysis and curative effect analysis are conducted based on FAS and PPS, and safety evaluation is conducted according to SS.

Statistical analysis method
Statistical analysis is made by SPSS19.0 software. All statistical tests should be performed by two-sided test. P value less than or equal to 0.05 are considered as statistical difference.
Descriptive analysis: enumeration data is represented as constituent ratio; measurement data is expressed as mean, standard deviation, median, minimum value and maximum value.
Statistical analysis: appropriate method should be taken according to type of index. (2) After Informed Consent being signed by subjects, clinical researcher should get research drugs from "research drug administrator", and dispenses to subject according to visit order.

Relevant information should be recorded in Clinical Research Drug Usage Record.
(3) Provision of detecting glycated hemoglobin in Central Laboratory: if the glycated hemoglobin is 7.0% or more detected in branch center, the patient can be grouped with consideration of other indexes. The designated person is responsible for taking venous blood; anticoagulant vacuum blood vessels are used to collect double blood samples at 3 ml, which should be kept in low temperature freezer at -20℃. After completion of tasks in hospital, supervisor should contact cold chain company, and one sample is sent to Central Laboratory.
The detection results by Central Laboratory are as the criterion for statistical analysis, and the other sample is as backup copy.
(4) Provision of standard meal: to reduce difference of 2-hour post-meal blood glucose detection, research group should provide instant noodles at the same specification and brand to each research center, and oil bag should be discarded  Table   should be filled in, there is without null term or arbitrary alteration.
(3) Significant deviation and data out of acceptable range should be verified, and investigator should make necessary instructions.
(4) Each test item should be marked with unit of measurement. (2) The patient, who is with poor efficacy and cannot take medication on time, should be followed up by researcher commonly.
(3) Auditor should appoint supervisor, who should ensure the benefit of subjects, accuracy and completion of research record and report data, and the research follows the approved clinical scheme, drug quality management practice and regulations.
14. Ethics-related Matters Make a detail record of tongue and pulse situations, but not give a score.

Standard Operating Procedure for Preparation, Detection and Cold Chain
Transportation of Blood Sample

Objective
Standardize preservation, transportation, received condition and process of blood sample.

Scope of application
A randomized double-blind placebo control multicentre clinical research on combination of Jinlida Granule and Metformin applied to treatment of type 2 diabetes mellitus Glycated hemoglobin is detected in Central Laboratory, which is responsible for preparation, detection and transportation of blood sample.

Preparation and preservation
Each center should prepare blood sample within 2 hours after collection according to standard operating procedure for preparation and preservation of blood sample. Blood sample should be kept in freeze pipe. Two pages of self-adhesive label with drug No.
should be attached to freeze pipe, and the other is attached in reserved sample record (Table 1), which should be fill as requirement.
Preparation of blood sample: 2) Patient preparation: venous blood is collected at sitting position from Am 8:00 to 9:00 after remaining fasting for more than 12 hours.
3) Preparation of blood sampling: take two anticoagulant blood vessels to collect 6 ml venous blood, to avoid hemolysis and lipemia (please make a note, if there is).
Each vessel should contain more than 3 ml blood, one for detection in central laboratory and the other as reserved sample. Both vessels should be marked with same No. as that in record table (Table 1), including name, sex, age, sample collecting time and collect unit. 4) Transportation and preservation of sample: whole blood frozen sample vessel should be kept in refrigerator at -20℃, and the longest preservation period is 6 months. The sample should be kept in ice box during transportation. Refrigerator temperature should be recorded for reference each day.

Transportation
After researchers are enrolled, supervisor is responsible for calling Shanghai Express Company should sent samples to Central Laboratory within 24 hours.

Receive
On the day of service, the blood samples should be checked, numbered and put in

Detection
Central Laboratory should complete detection within 10 working days after receiving samples, and submits the detection results to the sponsor and supervisors.
Within a week after receiving the detection results, the supervisor should provide feedback results to corresponding research center.