Methods
This single-centre, randomized, open-label, parallel study compared microvascular and hemorheological effects of treatment with either vildagliptin or glimepiride in type 2 diabetic patients pre-treated with metformin. To be considered eligible, patients had to be aged 30–80 years with an HbA1c in the range of 6.5 to 9.5%. The main exclusion criteria were myocardial infarction or stroke within 6 months prior to study enrolment; impaired hepatic or renal function; moderate or proliferative diabetic retinopathy, more than one unexplained episode of severe hypoglycemia within 6 months; pre-treatment with other anti-diabetic drugs with the exception of metformin within the last 3 months and uncontrolled hypertension (systolic blood pressure >160 and/or diastolic blood pressure >90 mmHg).
The study was performed in compliance with Good Clinical Practice and all applicable national laws and regulations. All patients provided written informed consent and the study was approved by an appropriate independent ethics committee.
Eligible patients were randomized to vildagliptin or glimepiride in a 1:1 ratio. Patients received 50 mg vildagliptin twice daily. Glimepiride was administered in the morning with an individual dose titration in the range of 0.5 – 4 mg to achieve best possible glycaemic control as judged by the investigator. At baseline, after 12 and 24 weeks of treatment, patients entered the study site in the morning after an overnight fast of at least eight hours. Fasting blood samples were obtained for the measurement of blood glucose, HbA1c, adiponectin, and the determination of erythrocyte deformability. In addition, all patients underwent retinal fundoscopy and retinal microvascular assessments.
Measurement of Erythrocyte Deformability
Blood cell deformability was measured using a laser-assisted optical rotational cell analyzer by determining the elongation index (EI). Laserdiffractoscopy was performed using the Rheodyn SSD shear stress diffractometer (Myrenne GmbH, Roetgen, Germany). The method of laserdiffractoscopy has been described in detail previously. The applied shear stress was electronically regulated and consists of 8 increasing shear stress ranges (0.3; 0.6; 1.2; 3; 6; 12; 30; 60 Pa). The measurement detects scattered light intensities along orthogonal axes (A, B) of red blood cells within the laser diffraction light cone. The erythrocyte elongation index (EI) was calculated by the following equation: EI(%) = ((A − B)/(A + B)) * 100. To compare the EI over the applied shear stress range between both treatment groups, the area under the curve from 0.3 to 60 Pa (AUC0.3–60) was calculated using the trapezoidal method.
Measurement of Retinal Microvascular Blood Flow (RBF) and Retinal Arteriolar Wall to Lumen Ratio (WLR)
Retinal capillary blood flow was assessed using scanning laser doppler flowmetry at 670 nm (Heidelberg Retina Flowmeter, Heidelberg Engineering, Germany). A retinal sample of 2.56 × 0.64 × 0.30 mm was scanned within 2 seconds at a resolution of 256 points × 64 lines × 128 lines. The confocal technique of the device ensured that only the capillary blood flow of the superficial retinal layer of 300 μm was measured. Measurements were performed in the juxtapapillary area of both eyes, 2 to 3 mm temporally to the optic nerve; the average from 3 singular measurements was taken.
Analysis of perfusion images was performed offline with automatic full-field perfusion imaging analysis. This led to a perfusion map excluding vessels with a diameter of >30 μm, without lines with saccades, and without pixels with inadequate reflectivity. The mean retinal capillary blood flow was calculated in the area of interest and expressed as arbitrary units.
Analysis of vessel diameters was performed offline with automatic full field perfusion imaging analysis (SLDF version 3.7). Outer arteriole diameter (AD) was measured in reflection images, and lumen diameter (LD) was measured in perfusion images. The wall to lumen ratio (WLR) was calculated as (AD-LD)/LD.
The laser scanning records were stored electronically and sent to a central reading centre (Interdisciplinary Centre for Ophthalmic Preventive Medicine and Imaging (IZPI) of the Friedrich-Alexander-University Erlangen-Nürnberg, Germany), for the measurement of retinal microvascular blood flow and the calculation of the retinal wall to lumen ratio (WLR). This reading centre was blinded for all other study procedures.
Laboratory Measurements
Blood glucose levels were determined using an electrochemical biosensor (Hitado, Möhnesee, Germany). Plasma adiponectin was measured using ELISA (total human adiponectin, TECOmedical) and HbA1c was measured by HPLC (Menarini Diagnostics, Neuss, Germany).
Statistical Analysis
This study was designed as an exploratory study aimed to provide new data for thesis generation. No a priory confirmatory sample size estimation has been performed. All study endpoints have been analyzed with equal priority in a non-confirmatory, exploratory sense. RBF and WLR were assessed in a central reading centre in a blinded fashion. All other study endpoints were assessed in an open label approach. All study results were evaluated using primarily descriptive statistics. Inferential statistics was used to compare results from baseline to endpoint within both treatment groups. Differences in means of study endpoints were tested by Student's t-test. In case of not equal variances in the data, the results of the Welch approximation was taken into account as result of the unpaired comparison. Results are presented as mean ± SD. Significance was set at a p-value less than 0.05. Data processing was performed with the software modules of SPSS (Statistical package for analysis in social sciences, release 19.0, SPSS Inc., Chicago, USA).