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 对 DPP-4 具有强效抑制作用，从 Caco-2 细胞提取物中提取的 IC50 为 19 nM[1]。Sitagliptin phosphate 通过 cAMP/PKA/Rac1 激活途径减少离体脾脏 CD4 T 细胞的体外迁移[2]。Sitagliptin phosphate 通过一种不依赖于 DPP-4、蛋白激酶 A 和 MEK-ERK1/2 的途径发挥一种新的直接作用，刺激肠 L 细胞分泌 GLP-1。Sitagliptin phosphate 能降低自身免疫对移植物存活率的影响[3]。

Sitagliptin phosphate 对自由采食的汉-Wistar 大鼠血浆中 DPP-4 活性的抑制 ED50 值为剂量后 7 小时 2.3 mg/kg和剂量后 24 小时 30 mg/kg[1]。链脲佐菌素诱导的 1 型糖尿病小鼠模型显示血浆中的 DPP-4 水平升高，而Sitagliptin phosphate 饮食可大幅抑制这种升高。这可能是通过延长胰岛移植存活时间对高血糖的调节产生积极影响而实现的[4]。Sitagliptin phosphate 在大鼠体内的血浆清除率和分布容积（40-48 mL/min/kg，7-9 L/kg）高于狗（9 mL/min/kg，3 L/kg）；在大鼠体内的半衰期为 2 小时，短于狗的 4 小时[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.