Dipeptidyl Peptidase-4 Inhibitors Attenuate Endothelial Function as Evaluated by Flow-Mediated Vasodilatation in Type 2 Diabetic Patients

Study 1: Comparison of Sitagliptin and Voglibose—Study Design and Population

This study consisted of a 4-week prestudy observation period, followed by 2 sets of a 6-week treatment period. Study subjects were either those who had been treated with oral antidiabetic drugs at Japan Self Defense Force Maizuru Hospital or those who had been referred to the hospital from 7 Medical Service Units in the Maizuru area because of impaired glucometabolic parameters discovered during annual health checkups and eventually diagnosed to have T2DM or borderline T2DM. Inclusion criteria were age ≥20 and <75 years, hemoglobin A1c (HbA1c) >6.2%, fasting blood glucose level >110 mg/dL despite medical therapy including lifestyle or/and pharmacological interventions for ≥8 weeks. Exclusion criteria included (1) type 1 diabetes; (2) secondary diabetes; (3) poorly controlled diabetes (HbA1c ≥10.0%); (4) history of stroke, acute coronary syndrome, or any cardiovascular diseases requiring inpatient treatment within 6 months, end-stage renal disease, hepatic dysfunction (either level of aspartate aminotransaminase or alanine aminotransferase >3 times normal limits), malignancies, or inflammatory diseases. To investigate the effect of sitagliptin, a DPP-4I, we compared it with an α-glucosidase inhibitor, voglibose, which reportedly improves endothelial function.^18–19 After the observation period, 26 eligible patients (mean age, 46±5 years) were randomized to either sitagliptin (50 mg/day) or voglibose (0.9 mg/day, TID) treatment with crossover to the other drug. Measurement of FMD/nitroglycerin (NTG)–mediated vasodilatation (NMD) and blood/urine sampling were performed at baseline and after 6 weeks on the respective treatments. Thereafter, the treatments were switched, and the same protocol was applied for another 6 weeks.

Study 2: Comparison of Sitagliptin and Alogliptin—Study Design and Population

This study also consisted of a 4-week prestudy observation period, followed by 2 sets of a 6-week treatment period. A washout period (4 weeks) was inserted between the first and second treatment periods. Study subjects were recruited among outpatients of the National Defense Medical College Hospital, among whom some were already being treated for T2DM with oral antidiabetic drugs. Other subjects were those referred from other clinics because they had potential T2DM. Inclusion criteria were age ≥20 and <75 years, HbA1c >6.5%, fasting blood glucose levels >126 mg/dL, or postprandial glucose levels >200 mg/dL despite medical therapy including lifestyle change/medications for ≥8 weeks. Exclusion criteria were the same as those for the study comparing sitagliptin and voglibose. After the observation period, 45 eligible patients (mean age, 66±7 years) were randomized to either sitagliptin (50 mg/day) or alogliptin (25 mg/day) treatment with crossover to the other drug. Measurement of FMD and blood/urine sampling were performed at baseline and after 6 weeks on the treatments. Thereafter, the treatments were ceased for 4 weeks and then switched, and the same protocol was applied for another 6 weeks.

Other medications, including antidiabetic, antihypertensive, or lipid-lowering drugs, were maintained throughout both studies. They were approved by the ethics committee of the National Defense Medical College, and written informed consent was obtained from each subject.

Sample Size Computations

For study 1, the sample size was calculated on the basis of a previous study comparing the effect on FMD of voglibose and miglitol, another α-glucosidase inhibitor,¹⁸ because of limited information on the effect of DPP-4Is on FMD. Assuming that DPP-4Is have a effect similar to miglitol and expecting a 2.3% difference with a standard deviation of 2.0 between sitagliptin and voglibose, a total of 26 subjects would be required at a 5% significance level and 80% power.

We calculated the sample size for study 2 on the basis of the observation that in study 1, prescription of sitagliptin caused a reduction in FMD. On this basis, a total of 40 patients were required to detect a reduction of 2.3% in FMD after treatment, with a standard deviation of 3.6, with 80% power and statistical significance of 5%. Sample-size calculations were performed using SAS PROC POWER procedure (SAS Institute Inc, Cary, NC).

Assessment of Endothelial Function

Endothelial function was assessed by FMD of the brachial artery. After measurement of systolic and diastolic blood pressure, FMD was measured noninvasively using a high-resolution ultrasound apparatus with a 7.5-MHz linear array transducer (Aplio SSA-770A, Toshiba Co Ltd) according to the guidelines of the International Brachial Artery Reactivity Task Force.²⁰ All measurements were performed in the morning from 9 am to 11 am, before taking the drugs, in a temperature-controlled room (25°C) with the subject in a fasting, resting, and supine state. Electrocardiograms were monitored continuously. The subject's dominant arm (right) was immobilized comfortably in the extended position to allow consistent access to the brachial artery for imaging. The vasodilatation responses of the brachial artery were observed using a previously validated technique.²¹ For each subject, optimal brachial artery images were obtained between 2 and 10 cm above the antecubital fossa. First, baseline 2-dimensional (2D) images were obtained, and after measurement of the baseline artery diameter, a narrow-width blood pressure cuff was inflated on the most proximal part of the forearm to an occlusive pressure (200 mm Hg) for 5 minutes to induce hyperemia. The position of the ultrasound transducer was carefully maintained throughout the procedure. The cuff was then deflated rapidly, and 2D images of the artery were obtained for 30 to 120 seconds after deflation. In the study comparing sitagliptin and voglibose, we measured endothelium-independent vasodilatation due to administration of NTG (0.3 mg) using the same method. NMD was measured before (baseline) and 240 to 300 seconds after NTG administration. Throughout the study, FMD and NMD were examined by cardiologists (1 in study 1 and 2 in study 2) who were blinded to the treatment regimen of each subject, using the same ultrasound apparatus and probe set for all measurements. Each subject was examined by the same examiner throughout the study. All images were recorded as movie files on a hard-disk recorder for later analysis. To manually measure vasodilator responses in each patient's artery, movies were played back and a 10- to 20-mm segment was identified for analysis using anatomic landmarks. To select images reproducible for the same point in the cardiac cycle, images at peak systole were identified, and the diameter of the artery was digitized using a caliper function of the ultrasound apparatus. For each condition (baseline, FMD, baseline before NMD, and after NMD), 3 separate images from 3 different cardiac cycles were digitized and their average segment diameters determined. Both FMD and NMD are expressed as percentage change from baseline to peak dilation. The intra- and interobserver variability (coefficient of covariance) for repeated diameter measurements at baseline and reactive hyperemia or NMD in the brachial artery were both <3%.²¹

Blood/Urine Sampling

Venous blood and urine samples for measurement of biochemical parameters were obtained in the morning after an overnight fast before taking the treatment drugs. The subjects were advised to take the DPP-4Is after breakfast. For measurement of GLP-1 levels, plasma was obtained in vacutainers containing a DPP-4I (Millipore, Bedford, MA) according to the manufacturer's instructions. The vacutainers were immediately transferred on ice after sampling, centrifuged, isolated, and stored at −70°C until further analyses.

Biochemical Analyses

Serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL)-C, glucose, and creatinine levels were determined by standard enzymatic methods. LDL-C levels were calculated using the Friedewald formula. Hemoglobin A1c (HbA1c) was determined using high-performance liquid chromatography with calibration using Japan Diabetes Society (JDS) Lot 2.²² The equation used for conversion from HbA1c (JDS) to HbA1c (National Glycohemoglobin Standardization Program [NGSP]) values was as follows: NGSP (%)=JDS (%)+0.4%. Serum glycated albumin was determined by enzymatic methods using albumin-specific protease, ketoamine oxidase, and albumin assay reagents (Lucica GA-L; Asahi Kasei Pharma, Tokyo, Japan; CV=0.63% to 0.93% intra-assay, 0.56% to 0.67% interassay).²³ Plasma insulin levels were measured by chemiluminescent enzyme immunoassay. Serum malondialdehyde-modified LDL (MDA-LDL) levels were determined by enzyme-linked immunosorbent assay (ELISA; CV=6.2% to 9.5% intra-assay, 2.6% to 11.8% interassay).²⁴ Serum high-sensitivity C-reactive protein (hsCRP) levels were measured using a BNII nephelometer (Dade Behring, Germany). Serum 8-hydroxy-2′-deoxyguanosine (8-OHdG) and nitrate/nitrite (NOx) levels were determined by ELISA and a colorimetric assay using commercially available kits—JaICA (Fukuroi, Japan; CV=1.8% to 5.5% intra-assay, 2.8% to 7.9% interassay) and Calbiochem (La Jolla, CA), respectively—and sera filtered though a Microcon Centrifugal Filter Device YM-10 (Millipore, Billerica, MA). Serum asymmetric dimethylarginine (ADMA) was determined by ELISA (Immundiagnostik, Bensheim, Germany; CV=6.5% to 7.0% intra-assay, 6.5% to 7.0% interassay). Serum DPP-4 activity was measured using a commercially available kit (Enzo Life Sciences, Tokyo, Japan). Urinary albumin was determined by a turbidimetric immunoassay and the urinary creatinine concentration measured by a standard laboratory method. Urinary albumin excretion was estimated by calculating the albumin:creatinine ratio (ACR). To determine the levels of active GLP-1(9-37)amide in DPP-4I-treated plasma, C18 reverse-phase extraction columns (MicroSpin C18; GL Science, Tokyo, Japan) were used. The eluent from the extraction columns was evaporated and reconstituted with distilled water, followed by determination of active GLP-1 using an ELISA kit (ALPCO, Salem, NH; CV=4.7% to 10.7% intra-assay, 9.6% to 17.6% interassay).

Statistical Analyses

Data are presented as mean with standard deviation (SD) or median with interquartile range for continuous variables and as frequency with percentage for categorical variables. Baseline characteristics were summarized by treatment sequences and compared using an unpaired t test for continuous variables and Fisher's exact test for categorical variables.

Treatment effects on body weight, blood pressure, biochemical parameters, and endothelial functions were assessed using a linear mixed model. Period and sequence were included in the model as fixed effects. Patients within a sequence were included in the model as a random effect. The model was adjusted for sex, age, smoking status, medication with antihypertensive drugs (β-blockers, calcium channel blockers, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and diuretics), statins, and antidiabetic drugs (biguanides, sulfonylureas, pioglitazone, and α-glucosidase inhibitors). Hypertension and dyslipidemia were excluded from the model because of interaction between these diseases and drug prescription. Tests for carryover effect and period effect were also performed.

In study 2, a relationship between the change in FMD and each biochemical parameter before and after treatment with DPP-4Is was assessed using a linear mixed model including patient number as a random effect. The model was adjusted for sex, age, smoking status, and concomitant medications.

For all analyses, a 2-sided P<0.05 was considered statistically significant. All statistical analyses were performed with SAS version 9.3 (SAS Institute Inc, Cary, NC).

Study 1: Comparison Between Sitagliptin and Voglibose

Study 2: Comparison Between Sitagliptin and Alogliptin

Biochemical parameters including those of diabetic status and endothelial function

Table 4 shows the effects of sitagliptin and alogliptin on various parameters. The primary end points of this study—diabetic status, serum lipids, and FMD—were comparable between the 2 drugs after treatment, except for HDL-C levels, which were reduced after alogliptin compared with sitagliptin. Alogliptin tended to reduce TC and LDL-C levels compared with alogliptin. We also analyzed the effects of the DPP-4Is on other biochemical parameters. Among them, the only difference was seen regarding plasma GLP-1 levels, with sitagliptin raising levels more than alogliptin. Next, we analyzed changes in various clinical parameters before and after treatment with the DPP-4Is and found that neither sitagliptin nor alogliptin affected body weight, blood pressure, or heart rate. Both drugs significantly reduced HbA1c, GA, and fasting glucose levels. Despite improving glycemic control, both sitagliptin and alogliptin attenuated FMD, by 39.6% and 31.7%, respectively (Figure 1). As expected, serum DPP-4 activity was markedly reduced by both sitagliptin and alogliptin. Although neither treatment affected serum insulin levels, sitagliptin, but not alogliptin, significantly increased plasma GLP-1 levels. Most other parameters—including blood pressure, body weight, serum lipids, ACR, MDA-LDL, 8-OHdG, hsCRP, and NOx—were unchanged, although alogliptin significantly reduced TC, LDL-C, and HDL-C levels. Levels of ADMA, an endogenous inhibitor of NOS, were significantly increased by sitagliptin, but not by alogliptin. For each clinical/biological parameter, we have included interaction terms for sex and treatment in the model as independent variables. P values for interaction terms were not significant in any model.

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Figure 1.

Individual changes in flow-mediated vasodilatation (FMD) of the brachial artery before and after treatment with sitagliptin and alogliptin. *P<0.001 vs baseline.