DPP-4 inhibitor and alpha-glucosidase inhibitor equally improve endothelial function in patients with type 2 diabetes: EDGE study

Background Alpha glucosidase inhibitor (GI) attenuates postprandial hyperglycemia (PPH) and reduces the risk of cardiovascular events in patients with impaired glucose tolerance or type 2 diabetes. Dipeptidyl peptidase 4 (DPP-4) inhibitors also attenuate PPH. PPH is one of the factors leading to endothelial dysfunction which is an early event in the pathogenesis of atherosclerosis. Furthermore, DPP-4 inhibitors protect endothelial function through a GLP-1-dependent mechanism. However, the impact of these two types of drugs on endothelial dysfunction in patients with type 2 diabetes has not been fully elucidated. We compared the effects of sitagliptin, a DPP-4 inhibitor, and voglibose, an alpha GI, on endothelial function in patients with diabetes. Methods We conducted a randomized prospective multicenter study in 66 patients with type 2 diabetes who did not achieve the treatment goal with sulfonylurea, metformin or pioglitazone treatment; 31 patients received sitagliptin treatment and 35 patients, voglibose treatment. The flow-mediated dilatation (FMD) of the brachial artery was measured in the fasting state at baseline and after 12 weeks of treatment. The primary endpoint was a change in FMD (ΔFMD) from the baseline to the end of follow-up. The effects of sitagliptin and voglibose on FMD were assessed by ANCOVA after adjustment for the baseline FMD, age, sex, current smoking, diabetes duration and body mass index. Secondary efficacy measures included changes in HbA1c, GIP, GLP-1, C-peptide, CD34, lipid profile, oxidative stress markers, inflammatory markers and eGFR and any adverse events. Results ΔFMD was significantly improved after 12 weeks of treatment in both groups, and there was no significant difference in ΔFMD between the two groups. There were no significant differences in changes in HbA1c, GIP, GLP-1, C-peptide, lipid profile, oxidative stress marker, inflammatory marker and eGFR between the two groups. Compared with voglibose, sitagliptin significantly increased the circulating CD34, a marker of endothelial progenitor cells. Adverse events were observed in 5 patients in only the voglibose group (diarrhea 1, nausea 1, edema 2 and abdominal fullness 1). Conclusions Sitagliptin improved endothelial dysfunction just as well as voglibose in patients with type 2 diabetes. Sitagliptin had protective effects on endothelial function without adverse events. Trial registration registered at http://www.umin.ac.jp/ctrj/ under UMIN000003951


Introduction
Postprandial hyperglycemia (PPH) plays a major role in cardiovascular complications in patients with type 2 diabetes [1] and impaired glucose tolerance (IGT) [2]. PPH is one of the main factors leading to endothelial dysfunction, which is an early event in the pathogenesis of atherosclerosis [3,4].
Alpha glucosidase inhibitor (GI) prevents the digestion of carbohydrates including starch and table sugar, attenuates postprandial hyperglycemia [5] and delays the development of type 2 diabetes in patients with IGT [6]. Miglitol, an alpha GI, improves endothelial dysfunction assessed by the response of forearm blood flow to reactive hyperemia and flow-mediated dilatation (FMD) in patients with type 2 diabetes and coronary artery disease [7,8]. Acarbose, an alpha GI, improves postprandial endothelial dysfunction in patients with type 2 diabetes [9,10] and reduces the risk of cardiovascular events in patients with type 2 diabetes [11] and IGT [12,13].
We conducted a randomized prospective multicenter study to compare the effects of sitagliptin, a DPP-4 inhibitor, and voglibose, an alpha GI, on endothelial function assessed by FMD in patients with type 2 diabetes. Liao et al. reported that number of circulating endothelial progenitor cells (EPCs) in patients with type 2 diabetes was significantly lower than that in the healthy subjects, treatment with metformin significantly increased EPCs and the EPCs number was related to endothelial function assessed by FMD [20]. Fadini et al. reported that sitagliptin increased circulating EPCs in type 2 diabetic patients [21]. We also compared the effects of sitagliptin and voglibose on number of circulating EPCs assessed by measurement of CD34, a maker of EPCs [22], postive cells in patients with type 2 diabetes in this study.
The primary endpoint in this study was a sitagliptinor voglibose-induced change in FMD (ΔFMD) from baseline to the end of follow-up. Secondary efficacy measures included changes in HbA1c, gastric inhibitory peptide (GIP), GLP-1, C-peptide, CD34, lipid profile, adiponectin, oxidative stress markers including MDA-LDL and urine 8-OHdG, inflammatory markers including hs-CRP and PTX-3, and estimated glomerular filtration rate (eGFR) and any adverse events.

Study populations
We conducted a randomized prospective multicenter study in patients with type 2 diabetes who did not achieve the treatment goal with diet, exercise, sulfonylurea, metformin or pioglitazone treatment. We recruited 66 patients (men and women) who were from 20 to 85 years of age. Thirty-one patients received the sitagliptin (50 mg/day) treatment and 35 patients, the voglibose (0.6 mg/day) treatment. The doses of the two drugs used in this study are recommended therapeutic doses for Japanese [26,27] and the doses are covered by the Japanese National Health Insurance. The exclusion criteria were as follows: treatment with insulin, alpha GI or glinide, type 1 diabetes, HbA1c ≥ 9.0%, systolic blood pressure ≥ 160 mmHg and serum creatinine ≥ 1.5 mg/dL at baseline. The study protocol was approved by the Ethics Committee of Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, and written informed consent was obtained from all patients before any study procedure was undertaken.

Study protocol
The patients were followed for at least 8 weeks to confirm that they did not achieve the treatment goal with diet, exercise, sulfonylurea, metformin or pioglitazone treatment. The patients were prospectively randomly assigned to additional treatment with either sitagliptin (50 mg/day) or voglibose (0.6 mg/day) for 12 weeks (Figure 1). The flow-mediated dilatation (FMD) of the brachial artery was measured in the fasting state at baseline and after 12 weeks of the treatment. Blood and urine tests were also performed at baseline and at the end of the study. The patients' antihypertensive, antihyperlipidemic and antidiabetic drugs were not changed and anti-oxidant drugs, including vitamin C and E, were not added throughout the study period.

Measurements of biochemical parameters
The following parameters were measured at baseline and after 12 weeks of treatment: complete blood count, liver function test including measurement of AST, ALT and LDH, renal function test including measurement of BUN, creatinine, Na, K and Cl, HbA1c, gastric inhibitory peptide (GIP), GLP-1, C-peptide, CD34, lipid profile including total cholesterol, triglyceride, and high-density lipoprotein (HDL-C), adiponectin, oxidative stress markers including MDA-LDL and urine 8-OHdG, inflammatory markers including hs-CRP and PTX-3, and estimated glomerular filtration rate (eGFR). HbA1c levels were measured using high-performance liquid chromatography. The number of CD34+ cells was determined by flow cytometry using FITC-labeled CD45 and phycoerythrin (PE)-labeled CD34 antibodies (BD Biosciences). eGFR (mL/min/1.73 m 2 ) was determined by the modified Modification of Diet and Renal Disease study formula (MDRD) for Japanese: eGFR = 194 × (age -0.287 ) × (serum creatinine -1.094 ) × (0.739 if female) [28]. These measurements were performed by SRL Company, Ltd. (Tokyo, Japan).

Flow-mediated dilation (FMD)
Endothelium-dependent dilation was assessed as a parameter of vasodilation according to the guidelines for ultrasound assessment of FMD of the brachial artery in the fasting state [29]. Using a 10-MHz linear-array transducer probe (Unex Company Ltd., Nagoya, Japan), longitudinal images of the brachial artery at baseline were recorded with a stereotactic arm, and measurements of the artery diameter were made after supine rest for ≥5 min as previously described [30][31][32]. The diameter of the artery was measured from clear anterior (media-adventitia) and posterior (intima-media) interfaces, which were manually determined. Then, suprasystolic compression (50 mmHg higher than systolic blood pressure) was performed at the right forearm for 5 min, and measurements of the artery diameter were made continuously from 30 sec before to ≥2 min after cuff release. Maximum vasodilation was then evaluated from the change in artery diameter after the release of occlusion (%FMD). FMD is known to be affected by a wide range of biological, environmental, and methodological factors [33]. To quantify inter-and intra-observer reproducibility, baseline brachial diameter and FMD were measured by three individuals in Okayama University Hospital. Interand intra-observer coefficients were high (r > 0.90).

Statistical analysis
The results are expressed as mean ± SD. We assumed that FMD increased by 2% in sitagliptin group and decreased by 0.5% in voglibose group with a standard deviation of 3%. A minimum sample size of 24 participants in each group was required to detect statistical differences in FMD with a power of 80% and α error of 5%. The effects of sitagliptin and voglibose on FMD were assessed by ANCOVA after adjustment for the baseline FMD, age, sex, current smoking, diabetes duration and body mass index (BMI). Differences in age, sex, BMI and abdominal girth were compared using Student's t-test. The duration of diabetes was Figure 1 Study protocol. The patients were followed for at least 8 weeks to confirm that they did not achieve the treatment goal. The patients were prospectively, randomly assigned to additional treatment with either sitagliptin (50 mg/day) or voglibose (0.6 mg/day) for 12 weeks. Measurements of flow-mediated dilatation (FMD) of the brachial artery and blood and urine tests were performed in the fasting state at baseline and after 12 weeks of treatment. compared using the Wilcoxon rank sum test and categorical variables, using Fisher's exact test. Differences in secondary efficacy measures between baseline and 12 weeks were compared using the paired Student's ttest. Values of P < 0.05 were considered to be statistically significant.

Baseline characteristics
Although 66 patients with type 2 diabetes were enrolled in this study between September 2010 and December 2011, 6 patients were excluded from this study (4 protocol violation at baseline and 2 withdrawal of consent). During the follow-up period, 5 patients with hypertension dropped out of the study due to change of concomitant antihypertensive drugs. The follow-up study was completed in 55 (83%) of the patients; 24 patients received the sitagliptin treatment and 31 patients, the voglibose treatment. The data collection was completed in July 2012. Baseline characteristics, including age, sex, BMI and the type of anti-diabetic medication are shown in Table 1. It has been reported that endotheliumdependent vasodilation assessed by FMD declines with advance of age [34,35]. We recruited men and women who were from 20 to 85 years of age. The age range was quite wide in our study, but there was no significant difference in age or baseline FMD between the two groups (age, sitagliptin: 66.6 ± 11.9, >70 years: 12, median 69.5, range: 37 -84 versus voglibose: 68.4 ± 9.2, >70 years: 14, median 70.0, range: 48 -80 years old, P = 0.529, Table 1) (baseline FMD, sitagliptin: 5.41 ± 2.25 versus voglibose: 4.96 ± 2.16%, P = 0.450, Table 2). We did not exclude patients with severe dyslipidemia, but there were no significant differences in use of antihyperlipidemic drugs (Table 1) and baseline total cholesterol, triglyceride and HDL-C (Table 3) between the two groups. No patients died, developed cardiovascular events or were admitted to the hospital during the course of the study.

Adverse events
Adverse events were observed in 5 patients in the voglibose group (Table 4). There were no serious side effects in either group.

Discussion
In summary, ΔFMD was significantly improved after 12 weeks of treatment in both the sitagliptin and voglibose groups, and there was no significant difference in ΔFMD between the two groups.
Alpha GI improves postprandial endothelial dysfunction in patients with type 2 diabetes [9,10]. We showed that not only voglibose, an alpha GI, but also sitagliptin, a DPP-4 inhibitor, improves endothelial function as assessed by FMD in patients with type 2 diabetes. The impact of a DPP-4 inhibitor on endothelial dysfunction in patients with type 2 diabetes is controversial. A recent study showed that sitagliptin improved endothelial dysfunction assessed by the reactive hyperemia arterial tonometry index (RHI) after 6 months of treatment in patients with diabetes and coronary artery disease [24]. We also showed that sitagliptin improved endothelial dysfunction assessed by another method, FMD. A prospective observational single arm trial also showed that the treatment with sitagliptin for 12 weeks increased FMD in patients with type 2 diabetes [36]. Here, we conducted a randomized prospective multicenter study in patients with type 2 diabetes and revealed improvement of FMD after the 12-week treatment with sitagliptin. In contrast, another short-term study showed that DPP-4 inhibitors, including sitagliptin or alogliptin, attenuated endothelial function as evaluated by FMD after 6 weeks of treatment in patients with diabetes [19]. Furthermore, voglibose, an alpha GI, did not affect FMD in the study. Therefore, short-term treatment with either DPP-4 inhibitors or alpha GI does not ameliorate endothelial dysfunction in patients with diabetes. Our study showed that 12-week treatment with either sitagliptin or voglibose ameliorated endothelial function. Thus, at least 12 weeks of treatment is needed to improve endothelial function.
The STOP-NIDDM trial showed that an alpha GI reduces the risk of cardiovascular events in patients with IGT [12]. DPP-4 inhibitors are also expected to have beneficial cardiovascular effects [37,38]. However, two recent trials with DPP-4 inhibitors, SAVOR-TIMI 53 and EXAMINE trials, showed that saxagliptin and alogliptin did not increase or decrease ischemic events in patients with type 2 diabetes [39,40]. These trials demonstrated the safety of DPP-4 inhibitors but did not demonstrate cardiovascular benefits. Patients at high risk patients for cardiovascular disease were enrolled in those studies. In the SAVOR-TIMI 53 trial, about 78% of the patients had established cardiovascular disease, and the EXAMINE trial consisted of patients with acute coronary syndrome. Mean HbA1c levels were about 8.0 in patients of those studies. Our study included relatively low-risk patients. Only about 25% of the patients in our study had established cardiovascular diseases. Mean HbA1c levels were about 7.0. ΔFMD was significantly improved after 12 weeks of treatment in both the sitagliptin and voglibose groups in our study. The clinical implication of this study is the demonstration of beneficial effects of both sitagliptin and voglibose on endothelial dysfunction in low-risk and mild-risk patients with type 2 diabetes. Therefore, sitagliptin and voglibose might have cardiovascular benefits in low-risk and mild-risk patients with type 2 diabetes. Further studies are needed to clarify the long-term benefits for cardiovascular disease [37].
Iwamoto et al. reported that once-daily sitagliptin monotherapy (50 mg/day) showed greater efficacy and better tolerability than did thrice-daily voglibose (0.6 mg/day) over 12 weeks in Japanese patients with type 2 diabetes [41], but there was no significant difference in changes in HbA1c between the sitagliptin (50 mg/day) and voglibose (0.6 mg/day) groups in our  Values are means ± SD or the least square means (95% CI). *The least square means (95% CIs) were derived from ANCOVA adjusted for the baseline FMD, age, sex, current smoking, diabetes duration and BMI.   Values are the least square means (95% CI). The least square means (95% CIs) were derived from ANCOVA adjusted for the baseline FMD, age, sex, current smoking, diabetes duration and BMI. ΔFMD was significantly improved after 12 weeks of treatment in both groups, and there was no significant difference in ΔFMD between the two groups.
artery disease and uncontrolled diabetes [24]. Lipid profiles and eGFR did not change after treatment with sitagliptin in that study as well as in our study. Our study showed no significant difference in changes in hs-CRP after treatment with sitagliptin. Furthermore, Sakamoto et al. reported that sitagliptin improved blood pressure and lipid profiles in patients with type 2 diabetes [42]. These different results might be due to the small sample size, short-term treatment or study in low risk patients with type 2 diabetes. Fadini et al. reported that sitagliptin increased circulating EPCs in type 2 diabetic patients with concomitant upregulation of stromal-derived factor-1α (SDF-1α) [21]. SDF-1α is a chemokine that stimulates bone marrow mobilization of EPCs and is one of the substrates of DPP-4. We also showed that sitagliptin significantly increased circulating CD34, but voglibose did not increase CD34. Therefore, this is an ancillary effect of sitagliptin and it is independent from blood glucose levels. Liao et al. reported that the number of EPCs was an independent risk factor for FMD [20]. Thus, this effect might be favorable for the preservation of endothelial function in the future. Further long-term studies are needed to clarify this point.
A recent study revealed that alogliptin, a DPP-4 inhibitor, did not increase the frequency of serious adverse events, including hypoglycemia, cancer, pancreatitis, and initiation of dialysis in patients with type 2 diabetes who had a recent acute coronary syndrome [40]. A pooled analysis of 25 randomized clinical trials did not indicate that treatment with sitagliptin increases cardiovascular risk in patients with type 2 diabetes mellitus [43]. Our study also showed that sitagliptin did not cause serious adverse events. Thus, DPP-4 inhibitors may be safe drugs for patients with type 2 diabetes.

Study limitations
The first limitation of this study was the small sizes of the groups. The second was heterogeneity of the population in this study. The age range was quite wide in our study. The third limitation was that our study was a short-term study. Our findings need to be confirmed in a large cohort of patients with type 2 diabetes and with a long period.
In conclusion, sitagliptin improves endothelial dysfunction just as well as voglibose in patients with type 2 diabetes. Furthermore, sitagliptin increases the level of circulating CD34. Sitagliptin has protective effects on endothelial function without adverse events. These effects have potential favorable cardiovascular implications for patients with type 2 diabetes.