Sitagliptin (phosphate)

Kinase experiment:

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].

Cell experiment:

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.

Animal experiment:

Mice: Overnight fasted C57BL/6J mice are challenged 45 min after compound administration with an oral glucose load (2 g/kg). Blood samples for glucose measurement are obtained by tail bleed predose and at serial time points after the glucose load. To evaluate the duration of the effect on glucose tolerance, vehicle or DPP-4 inhibitors are administered 16 h before the glucose challenge.

References:

[1]. Thomas, L., et al. (R)-8-(3-amino-piperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-2-ylm ethyl)-3,7-dihydro-purine-2,6-dione (BI 1356), a novel xanthine-based dipeptidyl peptidase 4 inhibitor, has a superior potency and longer duration of acti
[2]. Kim, S.J., et al., Dipeptidyl peptidase IV inhibition with MK0431 improves islet graft survival in diabetic NOD mice partially via T-cell modulation. Diabetes, 2009. 58(3): p. 641-51.
[3]. Sangle, G.V., et al., Novel biological action of the dipeptidylpeptidase-IV inhibitor, sitagliptin, as a glucagon-like peptide-1 secretagogue. Endocrinology, 2012. 153(2): p. 564-73.
[4]. Kim, S.J., et al., Inhibition of dipeptidyl peptidase IV with sitagliptin (MK0431) prolongs islet graft survival in streptozotocin-induced diabetic mice. Diabetes, 2008. 57(5): p. 1331-9.
[5]. Beconi, M.G., et al. Disposition of the dipeptidyl peptidase 4 inhibitor sitagliptin in rats and dogs. Drug Metab Dispos, 2007. 35(4): p. 525-32.