Sulfenamide and Sulfonamide Derivatives of Metformin – A New Option to Improve Endothelial Function and Plasma Haemostasis

Type 2 diabetes mellitus (T2DM) is a multi-factorial disease which can cause multiple organ dysfunction, including that of the vascular endothelium. The aim of the present study was to evaluate the effects of metformin, and its sulfenamide and sulfonamide derivatives (compounds 1–8) on the selected markers of endothelial function and blood coagulation. The integrity of endothelial cells(ECs) was examined using the real-time cell electric impedance system. Tissue Factor(TF) production, the release of von Willebrand Factor (vWF) and tissue plasminogen activator(t-PA) from ECs were determined using immunoenzymatic assays, while the process of platelet thrombus formation using the Total Thrombus-Formation Analysis System. Sulfenamide with n-butyl alkyl chain(3) does not interfere with ECs integrity, and viability (nCI(24h) = 1.03 ± 0.03 vs. 1.06 ± 0.11 for control), but possesses anticoagulation properties manifested by prolonged platelet-dependent thrombus formation (Occlusion Time 370.3 ± 77.0 s vs. 286.7 ± 65.5 s for control) in semi-physiological conditions. Both p- and o-nitro-benzenesulfonamides (compounds7,8) exhibit anti-coagulant properties demonstrated by decreased vWF release and prolonged parameters of platelet thrombus formation and total blood thrombogenicity. In conclusion, chemical modification of metformin scaffold into sulfenamides or sulfonamides might be regarded as a good starting point for the design and synthesis of novel biguanide-based compounds with anticoagulant properties and valuable features regarding endothelial function.

The blood samples were stored at room temperature up to 3 hours before the measurements.

8. Quantification of TF in HUVECs
Standard samples of TF at the concentration 0 -400 pg/mL or test samples (50 μL) were added on a 96-well plate precoated with TF specific antibody and incubated for 2 hours. The plate was washed five times with washing buffer (200 μL), and a TF specific biotinylated detection antibody was added (50 μL), following by 2 hours incubation and another set of washing. Then streptavidin-peroxidase conjugate was added and unbound conjugates were washed away with wash buffer. The next step included addition of chromogenic substrate to visualize streptavidin-peroxidase enzymatic reaction. After 10 minutes incubation at room temperature the reaction was stopped by addition of acidic stop solution (50 μL), and the absorbance was read at 450 nm (iMARK, Bio-Rad). The density of yellow colour is directly proportional to the amount of TF captured in plate. The amount of TF in HUVECs was calculated using calibration curve (R 2 = 0.964); the test was conducted in quadruplicate (n = 4).
To further elucidate whether tested biguanides might directly interact with TF additional experiments using human plasma were conducted. Compounds were incubated for 6 hours with twice diluted citrate plasma, and afterwards the samples (50 μL) were analysed using abovedescribed method.

9. Von Willebrand Factor release from HUVECs
Standard samples of vWF (range 0 -1500 pg/mL) or test samples in a volume of 100 μL were added on a 96-well plates precoated with specific vWF antibody and incubated for 90 minutes at room temperature with gentle shaking. Then the plates were washed with provided wash buffer working solution (200 μL), and appropriately diluted biotin-labeled detection antibody working solution was added (100 μL), followed by 1 hour incubation at 37 °C and another set of washing. Streptavidin-HRP working solution was added into each well, incubated for another 45 minutes at 37 °C, and washed once again. 100 μL of chromogenic substrate was added, and the colour turned into blue. The plates were incubated for 30 minutes at 37 °C in the dark, and the acidic stop solution (100 μL) was added. The measurements were taken at 450 nm using microplate reader (iMARK, Bio-Rad). The amount of vWF released from HUVECs was calculated using calibration curve (R 2 = 0.969); the number of replicates in the test was 4-6 (n = 4-6).

10. Tissue Plasminogen Activator (t-PA) release from HUVECs
A t-PA specific antibody has been precoated onto 96-well plates and blocked. Standard samples (0 -1 ng/mL) of t-PA or test samples in a volume of 50 μL were added and incubated for 2 hours. The plate was washed five times with washing buffer (200 μL), and subsequently a t-PA specific biotinylated detection antibody was added followed by washing with wash buffer. Then Streptavidin-Peroxidase Conjugate was added and unbound conjugates were washed away with wash buffer. Chromogenic substrate (TMB; 3,3',5,5'-tetramethylbenzidine) was used to visualize enzymatic reaction (blue color product). The last step incuded addition of acidic stop solution and immediate absorbance measurements at the wavelength of 450 nm (iMARK, Bio-Rad). The density of yellow coloration was proportional to the amount of t-PA captured in plate. The amount of t-PA in HUVECs supernatant was calculated using calibration curve (R 2 = 0.983); the number of replicates in the test was 4 (n = 4). 1.16 ± 0.09 1.03 ± 0.07* 0.06 ± 0.02*** 0.00 ± 0.01*** 0.00 ± 0.01*** 12 h

Supplementary
Supplementary Table S5. The effects of biguanides on the expression of CD54 (ICAM-1) on the surface of endothelial cells.
[ Endothelial cells were treated with various concentrations of biguanides for 24 hours followed by staining with PE-CD54 antibody. The concentration for the studies were chosen on the basis of results collected in viability and integrity test. 1 -Cells gathered within the gate B reflect the % of acquired events existing as single cells (detection using FSC-A/FSC-H plots); 2the cells gathered in gate B were stained with CD54. The results are presented as mean ± standard deviation (SD), n = 3-6. The values given in bold represent statistically significant (*p < 0.05; ** p < 0.01; *** p < 0.001) changes versus respective controls. The blood samples were incubated for 15 minutes with indicated concentrations of biguanide derivatives, and subsequently analysed by T-TAS system (PL-Chips). The results are presented as mean ± standard deviation (SD), n = 3. The values given in bold represent statistically significant (p < 0.05) changes versus respective controls. T10the onset of platelet thrombus formation, counted as the time for the flow pressure to increase to 10 kPa from baseline due to a partial occlusion of the capillary; OT -occlusion time, the time for the flow pressure to increase by 60 kPa from baseline owing to near complete occlusion of the capillary; CT -Clotting timethe difference between OT and T10; AUC10area under the flow pressure curve for 10 minutes.