Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease affecting >220 million individuals worldwide.1 Numerous different antidiabetic drugs have been developed to treat T2DM according to the pathogenesis in individual patients. Glucagon-like peptide 1 (GLP-1) is a gut-derived incretin that stimulates insulin and suppresses glucagon secretion, inhibits gastric emptying and β-cell apoptosis, and reduces appetite and food intake.2,3 GLP-1 has been proposed for the treatment of patients with T2DM; however, native GLP-1 can be degraded within 2–3 minutes.3 The development of GLP-1 receptor agonists resistant to this degradation is necessary.4 Exenatide, a long-acting GLP-1 receptor agonist that improves insulin secretion and increases β-cell mass, is a potentially important drug in the treatment of T2DM.5 Furthermore, exenatide can improve glycemic control and weight loss.6,7 Although the antidiabetic activity of exenatide has been widely reported,8–10 the therapy can cause significant adverse events, such as nausea, vomiting and diarrhea.11,12
Biphasic insulin aspart (BIA) is a man-made insulin. BIA treatment can improve glycemic control in patients with T2DM in whom the condition is poorly controlled by other medications.13 In a previous study,14 a larger proportion of patients with T2DM achieved glycemic goals when treated with BIA than when treated with exenatide. Furthermore, the insulin-treated groups had an increased incidence of minor hypoglycemic events and weight gain. BIA was found to be more effective for the treatment of patients with a high baseline HbA1c level, enabling most patients to achieve glycemic control.14 BIA has additionally been shown to improve glycemic control subsequent to the failure of diet, exercise, and oral antidiabetic agents15; however, the side effects of the drug are evident, and BIA therapy can increase the risk of daytime hypoglycemia.16
Metformin (MET) is an oral antidiabetic drug and the first-line agent for the treatment of T2DM, particularly in overweight and obese individuals17; however, MET-treated patients are known to experience a progressive deterioration in glycemic control. Furthermore, evidence that MET modifies β-cell deterioration is limited.18
The above drugs all have significant side effects; however, the use of a combination therapy may enhance the efficacy and reduce the dose-related side effects of the medicines by a reduction in the dose of each type of drug. The combination of exenatide and MET has been shown to improve weight loss and glycemic control and to increase C-peptide levels, β-cell function index, and adiponectin levels.19 The efficacy of a triple combination therapy of exenatide, MET and BIA, however, has never been reported. The efficacy and safety of the triple combination therapy for T2DM were therefore explored in this study.
MATERIALS AND METHODS
The clinical protocol of the study was approved by the Institutional Review Board of Shandong Yanzhou Mining Group General Hospital (Jining, China), and all steps were conducted in accordance with the principles described in the World Medical Association Declaration of Helsinki.20 A total of 480 patients with T2DM, who had experienced T2DM therapy failure, were recruited between November 2008 and October 2011.
The subjects would be considered if they (1) had experienced T2DM therapy failure after the use of a single type of medicine (exenatide, MET, or BIA) for 3–12 months; (2) had a hemoglobin A1c (HbA1c) level of ≤10.5%; (3) had a body mass index (BMI) of >27 and <40 kg/m2; (4) had stable weight for ≥3 months before the study; and (5) had no clinically significant abnormal findings.
The subjects would be excluded if they (1) had experienced more than 3 episodes of severe hypoglycemia within the 6 months before screening; (2) had used any prescription drug to obtain weight loss within the 3 months preceding the study; (3) had been treated with insulin, thiazolidinediones, meglitinides, or other diabetes medicine (with the exception of exenatide, MET, and BIA) for >2 weeks within the 3 months preceding the study.
Clinical variables and validation
BMI was calculated by dividing weight (kilogram) by the square of height (square meters). The main questionnaire requested that the waists and hips of the patients were measured at the point of greatest circumference while the participants stood in a relaxed stance. Self-reported weight has been shown to be valid in this cohort. Certain factors, including age, sex, drinking and smoking status, BMI, and hypertension, were taken into consideration to reduce multiple factors interfering with the results of the groups. Furthermore, all subjects were from the city of Jining and were of a similar religious and economic background. Subsequent to analyzing the answers to the survey, 200 cases were considered to be consistent with the selection criteria. These cases were randomly divided into the low-dose (0.5 μg exenatide, 0.05 U·kg−1·d−1 BIA30, and 0.01 g MET twice daily) and normal-dose (2 μg exenatide, 0.2 U·kg−1·d−1 BIA30, and 0.05 g MET twice daily) groups and treated for 48 weeks.
Subsequent to obtaining informed consent, all patients were measured for capillary whole-blood glucose levels. The participants with a random capillary glucose level (CG) of >5.6 mM were considered to be at a higher risk of diabetes21 and received further measures. The levels of glucose and HbA1c were measured 2 hours after a 75-g oral glucose load. The participants received no formal dietary preparation. Serum glucose levels were determined using a glucose oxidase method by a dry chemistry analyzer (Ningbo Scientz Biotechnology Co, Ltd, Ningbo, China). HbA1c levels were determined by affinity chromatography (GE Healthcare, Piscataway, NJ). Levels of high-density lipoprotein–cholesterol were assessed using a commercial kit (Boehringer Mannheim GmbH, Mannheim, Germany), and levels of low-density lipoprotein–cholesterol were measured using the formula proposed by Friedewald et al.22 Triglyceride levels were measured using a colorimetric method involving lipoprotein lipase and glycerol kinase (Bayer RA-1000 triglyceride kit; Bayer SAS, Puteaux, France). The serum insulin concentration was tested by enzyme-linked immunosorbent assay (ELISA) (Abnova Corp, Walnut, CA). Triglyceride levels were measured using enzyme colorimetry (China Certification and Inspection Group Shanghai Co, Shanghai, China). Fasting blood glucose (FBG) levels were automatically analyzed on an automatic random access biochemistry analyzer (Hitachi 917; Hitachi, Ltd, Tokyo, Japan). Serum levels of fasting insulin (FINS) were measured using radioimmunoassays (Tongji Hospital, Shanghai, China). All measurements were conducted in triplicate. The homeostatic model assessment of estimated insulin resistance (HOMA-IR) index was calculated using the following formula: HOMA-IR = FBG × FINS/22.5. The homeostatic model assessment of estimated insulin secretory function (HOMA-IS) was calculated using the equation HOMA-IS = 20 × FINS/(FBG-3.5).
Human ELISA kits for C-reactive protein (CRP; cat. no. ab99995), tumor necrosis factor–α (TNF-α; cat. no. ab46087), adiponectin (cat. no. ab99968), and resistin (cat. no. ab100634) were purchased from Abcam (Hong Kong) Ltd (Hong Kong SAR, China). All other cell factors were also measured with ELSIA kits.
Safety and tolerability were analyzed by investigating adverse events using a fingerstick glucose measurement to identify the symptoms of hypoglycemia. Hypoglycemic events were classified as “mild” (CG < 3.5 mmol/L), “severe” (CG < 2.8 mmol/L), and “nocturnal hypoglycemia” (hypoglycemia occurring between falling asleep in the evening and breakfast).23 Before the administration of the triple combination treatment and 48 weeks after the treatment initiation, the occurrence of the following symptoms and conditions was assessed: nausea, vomiting, nasopharyngitis, diarrhea, influenza, headache, back pain, anorexia, dyspepsia, arthralgia, bronchitis, depression, pain in the extremities, anorexia/decreased appetite, injection site reactions, cardiac disorders, hypertension, neoplasms, accidental falls, serious adverse events and mortality. All the symptoms were diagnosed by doctors from Shandong Yanzhou Mining Group General Hospital.
The treatment effects were assessed at a 2-sided significance level of 0.05. Fisher exact tests were used for the comparisons based on categorical variables (eg, adverse events, incidence of hypoglycemia). Predefined analyses were completed to determine the effect of baseline characteristics, sulfonylurea dose reduction, and antibody status on changes in HbA1c and FBG levels. Two analyses (associations between weight loss and gastrointestinal side effects and between sulfonylurea dose decrease and rates of hypoglycemia) were added to the statistical plan (post hoc). Results are presented as the mean ± SD of the mean.
Characteristics of patients
A total of 480 patients who met the inclusion criteria were recruited for the study. At the end of the run-in period, 200 patients were randomly divided into the 2 groups (Figure 1). After the 48-week trial, 82 and 90 individuals from the low-dose and normal-dose groups, respectively, completed the study (Figure 1). Table 1 shows the baseline characteristics of the patients. The mean age of the participants was 59 ± 9.2 years, ranging from 48 to 68 years. There were 68 male and 104 female patients (female:male ratio, ∼3:2). No significant differences in the baseline demographic and metabolic characteristics of the patients were found between the low-dose and normal-dose groups (P > 0.05).
Effect of triple combination therapy on the metabolic characteristics of the patients
Among the total study population, 85% of participants were living with a spouse. After the 48-week trial with the combination therapy, the patients in the low-dose and normal-dose groups had significantly lower mean glucose values at the prebreakfast (P = 0.017 and 0.001, respectively), prelunch (P = 0.022 and 0.003, respectively), and 3:00 PM (P = 0.032 and 0.010, respectively) timepoints. Furthermore, all cases had lower 2-hour postbreakfast (P < 0.05) and postsupper (P < 0.05) blood glucose concentrations. In addition, the normal-dose triple combination therapy reduced the incidence of postprandial glucose fluctuations after the morning (P < 0.05), mid-day (P < 0.05), and evening (P < 0.05) meals to a greater extent than the low-dose triple combination therapy. Other parameters also showed the normal-dose triple combination therapy to be a more efficacious diabetes treatment than the low-dose therapy (Table 2).
Glycemic and body weight control
The changes in HbA1c levels after the 48-week trial are shown in Table 2. The primary objective of the study was achieved, with the normal-dose triple combination therapy demonstrating superiority to the low-dose therapy with respect to the change in HbA1c after the 48-week treatment period. In addition, the 2 groups exhibited significant reductions in the HbA1c level at week 48 versus the level at the baseline (P < 0.01) (Table 2). Patients in the normal-dose group were able to achieve better glycemic control than those in the low-dose group (HbA1c changes of −2.5 ± 0.19% and −0.8 ± 0.07%, respectively).
The change in body weight is also shown in Table 2. The primary objective of the study was achieved, with the triple combination therapy demonstrating superiority to the double combination therapy regarding the change in body weight. The patients in the 2 groups exhibited significant reductions in body weight from baseline to week 48 (P < 0.01) (Table 2), with maximum weight decreases of 4.0 and 0.9 kg in the normal-dose and low-dose group, respectively (Table 2).
Effect of triple combination therapy on the function of β cells and insulin sensitivity
The patients in the normal-dose group showed a significant baseline-to-endpoint improvement in β-cell function, as evaluated by the HOMA-B. The HOMA-B index increased from 48.8 ± 2.0% (baseline) to 67.6 ± 3.2% (endpoint) (P < 0.01). The HOMA-IS index did not change significantly. In comparison, the patients in the low-dose group also exhibited a statistically significant baseline-to-endpoint improvement in β-cell function, as assessed by HOMA-B. The HOMA-B index increased from 47.8 ± 1.7% (baseline) to 77.3 ± 4.3% (endpoint) (P < 0.01). The HOMA-IS index did not change significantly.
Effect of triple combination therapy on the changes in adiponectin, TNF-α, CRP, and resistin
Adiponectin, a hormone secreted from fat cells, has insulin-sensitizing and anti-inflammatory properties. Low levels of adiponectin in the plasma have been reported to be associated with insulin resistance, manifesting as obesity and diabetes.24 Adiponectin levels in the blood of diabetic individuals are lower than those in normal individuals.25 In this study, the normal-dose treatment caused a greater increase in adiponectin levels than the low-dose therapy (P < 0.05) (Table 2). TNF-α is a cytokine involved in systemic inflammation and plays an important role in the mechanism of insulin resistance associated with obesity.26 TNF-α has been reported to be widely expressed in adipose tissue.26 In this study, the normal-dose treatment caused a greater decrease in the TNF-α level than the low-dose therapy at week 48 (P < 0.05) (Table 2).
CRP is a protein that can be found in the blood. The levels of CRP increase after the occurrence of inflammation, and baseline CRP is an important biomarker for the risk of diabetes. High levels of CRP are closely associated with the development of diabetes.27 In this study, the normal-dose treatment caused a greater decrease in the CRP levels than the low-dose treatment (P < 0.05) (Table 2). Resistin is a hormone that potentially links obesity to diabetes.28 Levels of resistin are increased in diet-induced and genetic forms of obesity, and the administration of an anti-resistin antibody improves blood sugar levels and insulin action in mice with diet-induced obesity.28 In this study, the normal-dose treatment caused a greater decrease in resistin levels than the low-dose treatment (P < 0.05) (Table 2). These results in combination suggest that the normal-dose treatment has a superior antidiabetic effect to the low-dose treatment.
Safety and clinical laboratory findings
Compared with the incidence of adverse events recorded before the 48-week trial, the triple combination treatment was observed to reduce the incidence significantly. The incidence of gastrointestinal adverse events was higher in the normal-dose group than that in the low-dose group (Table 3), but no significant difference between the groups was found for that or for the incidence of any other adverse event (P > 0.05) with the exception of accidental falls. Nausea (30% incidence during the 48 weeks) and vomiting (15% incidence) were mostly mild or moderate in severity. Of the total study population, 2% of patients withdrew because of gastrointestinal-related adverse events. With regard to the other symptoms, the triple combination treatment caused fewer side effects in the normal-dose group than in the low-dose group (P > 0.05) (Table 3). No drug-related mortalities were recorded.
This study investigated the efficacy of the triple combination therapy of exenatide, MET, and BIA for T2DM, which, to the best of our knowledge, has not been previously reported. The most significant findings were associated with weight reduction, enhanced glycemic control, and the reduction in HbA1c values (Table 2) and showed that the combination therapy could significantly improve the clinical parameters of T2DM (Table 2).
The results of this study suggest that the investigated triple combination treatment is effective for glucose control. These data have provided additional information, which may enhance the clinical decision-making process for patients who have been unable to achieve the appropriate glycemic control for a long period. The study showed that the normal-dose triple combination treatment was superior to the low-dose treatment. It should be taken into consideration that all patients were treated with a triple combination therapy, whereas only a double combination has been used in the previous premixed trials; however, since both the double and triple combination therapies involve subcutaneous injections of the agents at the same times twice daily and have the potential to favorably affect fasting and postprandial glucose, they can be considered to be suitable comparators. Exenatide has been shown to improve glycemic control in patients with T2DM who failed to achieve glycemic control with maximally effective MET doses.29
Consistent with the results of recent clinical trials with exenatide, a significant decrease in body weight was observed in this study in the normal-dose triple combination therapy group (Table 2); this decrease was significantly greater than that observed in the low-dose treatment group. Furthermore, the HbA1c levels were reduced significantly in the normal-dose triple combination treatment group compared with the levels before the trial (Table 2); since this decrease was consistent with the reduction in body weight (Table 2), it was indicated that the observed weight reduction was sufficient to affect glycemic control. From all changing trends between low-dose and normal-dose treatments, it appeared that a significant cause of the weight reduction observed in patients receiving triple combination therapy (Table 2). It should be noted that the long-term implications of the progressive weight reduction observed in the patients treated with the triple combination therapy, with respect to the glycemic control and overall systemic effects, were not fully elucidated in the study; however, given the central role of obesity in the pathophysiology of T2DM,30 it is suggested that agents acting to lower 73 blood glucose may prevent this risk factor.
The strength of this study was its long duration. There were, however, a number of limitations to the study design due to the lack of control groups treated with placebo or a single type of medicine. All experiments were omitted mainly considering most T2DM patients failing to achieve glycemic control with maximally effective exenatide, MET, or BIA doses.14,31,32 All controls will be tested for the large number of recruited patients in future. In conclusion, this study has been the first, to the best of our knowledge, to investigate the use of a triple combination therapy of exenatide, MET, and BIA for T2MD. Glycemic control was achieved in the normal-dose and low-dose groups. The normal-dose triple combination treatment caused a significantly greater increase in the level of adiponectin and significantly greater reductions in the levels of CRP, resistin, and TNF-α compared with the low-dose combination treatment (P < 0.05). This triple combination treatment therefore provides a potential alternative for the safe and effective treatment of T2MD. There were certain limitations to this study design, due to the lack of control groups treated with placebo and a single type of medicine, but these limitations will be rectified in future studies.
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Keywords:Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.
biphasic insulin aspart; exenatide; metformin; triple combination therapy; type 2 diabetes mellitus