Sitagliptin (MK0431), a new oral hypoglycemic (anti-diabetic drug), is a new dipeptidyl peptidase-4 (DPP-4) inhibitor. This enzyme-inhibiting drug is used either alone or in combination with metformin or thiazolidinedione for treatment of type 2 diabetes mellitus. The drug can competitively inhibit a protein/enzyme and DPP-4, that leads to an incremental amount of active incretins (GLP-1 and GIP), the diminished amount of release of glucagon and increased release of insulin.

DPP4:18 nM

Sitagliptin phosphate demonstrates a potent inhibitory effect on dipeptidyl peptidase-4 (DPP-4), achieving an IC50 of 19 nM in Caco-2 cell extracts[1]. It also inhibits in vitro migration of isolated splenic CD4 T-cells through cAMP/PKA/Rac1 pathway activation[2]. Furthermore, sitagliptin directly enhances GLP-1 secretion from intestinal L cells via a pathway that is independent of DPP-4, but relies on protein kinase A and MEK-ERK1/2 activation. Additionally, it mitigates the impact of autoimmunity on graft survival[3].

In vivo studies demonstrate that the ED50 (effective dose for 50% of the population) of sitagliptin phosphate, which inhibits DPP-4 activity in plasma, is estimated to be 2.3 mg/kg seven hours post-administration and 30 mg/kg twenty-four hours post-administration in freely fed Han-Wistar rats[1]. In the streptozotocin-induced type 1 diabetes mouse model, characterized by elevated plasma DPP-4 levels, sitagliptin phosphate supplementation notably reduces these levels, effectively regulating hyperglycemia and potentially enhancing islet graft longevity[4]. Moreover, pharmacokinetic profiles reveal that both plasma clearance and the volume of distribution for sitagliptin phosphate are significantly higher in rats (40-48 mL/min/kg, 7-9 L/kg) compared to dogs (9 mL/min/kg, 3 L/kg), with a half-life of 2 hours in rats versus 4 hours in dogs[5].

DPP-4 is extracted from confluent Caco-2 cells. After 5 minutes of incubation at room temperature with lysis buffer (10 mM Tris-HCl, 150 mM NaCl, 0.04 U/mL aprotinin, 0.5% Nonidet P40, pH 8.0), cells are centrifuged at 35,000 g at 4°C for 30 minutes, and the supernatant is stored at -80°C. Assays are performed by mixing 20 μL of appropriate compound dilutions with 50 μL of the substrate for the DPP-4 enzyme, H-Ala-Pro-7-amido-4-trifluoromethylcoumarin (final concentration in the assay, 100 μM) and 30 μL of the Caco-2 cell extract (diluted 1000-fold with 100 mM Tris-HCl, 100 mM NaCl, pH 7.8). Plates are incubated at room temperature for 1 hour, and fluorescence is measured at excitation/emission wavelengths of 405/535 nm using a SpectraMax GeminiXS. Dissociation kinetics of inhibitors from the DPP-4 enzyme is determined after a 1-hour preincubation of Caco-2 cell extracts with high inhibitor concentrations (30 nM for BI 1356, 3 μM for vildagliptin). The enzymatic reaction is started by adding the substrate H-Ala-Pro-7-amido-4-trifluoromethylcoumarin after a 3000-fold dilution of the preincubation mixture with assay buffer. Under these conditions, the difference in DPP-4 activity at a certain time point in the presence or absence of an inhibitor reflects the amount of this inhibitor still bound to the DPP-4 enzyme. Maximal reaction rates (fluorescence units/seconds ×1000) at 10-minute intervals are calculated using the SoftMax software of the SpectraMax and corrected for the rate of an uninhibited reaction [(vcontrol-vinhibitor)/vcontrol].

CD4T-cells are plated on membrane inserts in serum-free RPMI 1640, and cell migration is assayed using Transwell chambers (Corning), in the presence or absence of purified porcine kidney DPP-4 (32.1 units/mg; 100 mU/mL final concentration) and DPP-4 inhibitor (100 μM). After 1 hour, cells on the upper surface are removed mechanically, and cells that have migrated into the lower compartment are counted. The extent of migration is expressed relative to the control sample.

DMSO: 170 mg/mL (417.37 mM), Sonication is recommended.

H2O: < 1 mg/mL (insoluble)

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 2 mg/mL (4.91 mM), Sonication is recommended.