The major metabolic pathway of vildagliptin in mice, rats, dogs, and humans is hydrolysis at the cyano group to produce a carboxylic acid metabolite M20.7 (LAY151), whereas the major metabolic enzyme of vildagliptin has not been identified. In the present study, we determined the contribution rate of dipeptidyl peptidase-4 (DPP-4) to the hydrolysis of vildagliptin in the liver. We performed hydrolysis assay of the cyano group of vildagliptin using mouse, rat, and human liver samples. Additionally, DPP-4 activities in each liver sample were assessed by DPP-4 activity assay using the synthetic substrate H-glycyl-prolyl-7-amino-4-methylcoumarin (Gly-Pro-AMC). M20.7 formation rates in liver microsomes were higher than those in liver cytosol. M20.7 formation rate was significantly positively correlated with the DPP-4 activity using Gly-Pro-AMC in liver samples (r = 0.917, P < 0.01). The formation of M20.7 in mouse, rat, and human liver S9 fraction was inhibited by sitagliptin, a selective DPP-4 inhibitor. These findings indicate that DPP-4 is greatly involved in vildagliptin hydrolysis in the liver. Additionally, we established stable single expression systems of human DPP-4 and its R623Q mutant, which is the nonsynonymous single-nucleotide polymorphism of human DPP-4, in human embryonic kidney 293 (HEK293) cells to investigate the effect of R623Q mutant on vildagliptin-hydrolyzing activity. M20.7 formation rate in HEK293 cells expressing human DPP-4 was significantly higher than that in control HEK293 cells. Interestingly, R623Q mutation resulted in a decrease of the vildagliptin-hydrolyzing activity. Our findings might be useful for the prediction of interindividual variability in vildagliptin pharmacokinetics. IntroductionDipeptidyl peptidase-4 (DPP-4; CD26, EC 3.4.14.5), a serine protease belonging to type II transmembrane glycoproteins, is widely expressed on the surface of epithelial cells of diverse tissues, including liver, kidney, and intestine; on endothelial cells of blood vessels; and on lymphoid cells (Mentlein, 1999;Gorrell et al., 2001). In addition to the integral membrane form, a soluble form of DPP-4 presents in serum (Durinx et al., 2000). By cleaving dipeptides from the N-terminal end of peptides and polypeptides with proline or alanine in the second position, DPP-4 controls the activity of many bioactive molecules, including incretins, cytokines, chemokines, and neuropeptides (Boonacker and Van Noorden, 2003).Vildagliptin (LAF237) is a potent, orally active inhibitor of DPP-4 for the treatment of type 2 diabetes mellitus (Villhauer et al., 2003). DPP-4 inhibitors, so-called incretin enhancers, are attracting attention among therapeutic agents for type 2 diabetes mellitus, because they improve glucose control with a low risk of hypoglycemia (Scheen, 2010a;Deacon, 2011). Although most DPP-4 inhibitors allow one single oral administration per day for management of type 2 diabetes mellitus, twice-daily administration is recommended for vildagliptin because of its shorter half-life (Deacon, 20...

Vildagliptin is a potent, orally active inhibitor of dipeptidyl peptidase-4 (DPP-4) for the treatment of type 2 diabetes mellitus. It has been reported that vildagliptin can cause hepatic dysfunction in patients. However, the molecular-mechanism of vildagliptin-induced liver dysfunction has not been elucidated. In this study, we employed an expression microarray to determine hepatic genes that were highly regulated by vildagliptin in mice. We found that pro-inflammatory S100 calcium-binding protein (S100) a8 and S100a9 were induced more than 5-fold by vildagliptin in the mouse liver. We further examined the effects of vildagliptin and its major metabolite M20.7 on the mRNA expression levels of S100A8 and S100A9 in human hepatoma HepG2 and leukemia HL-60 cells. In HepG2 cells, vildagliptin, M20.7, and sitagliptin – another DPP-4 inhibitor – induced S100A9 mRNA. In HL-60 cells, in contrast, S100A8 and S100A9 mRNAs were significantly induced by vildagliptin and M20.7, but not by sitagliptin. The release of S100A8/A9 complex in the cell culturing medium was observed in the HL-60 cells treated with vildagliptin and M20.7. Therefore, the parental vildagliptin- and M20.7-induced release of S100A8/A9 complex from immune cells, such as neutrophils, might be a contributing factor of vildagliptin-associated liver dysfunction in humans.

Nitrilase, which is found in plants and many types of bacteria, is known as the enzyme that catalyzes hydrolysis of a wide variety of nitrile compounds. While human nitrilase-like protein (NIT), which is a member of the nitrilase superfamily, has two distinct isozymes, NIT1 and NIT2, their function has not been well understood. In this study, we investigated whether human NIT1 and NIT2 are involved in the hydrolysis of drugs using vildagliptin as a substrate. We performed Western blot analysis using human liver samples to examine protein expression of human NIT in the liver, finding that human NIT1 and NIT2 were highly expressed in the liver cytosol. We established stable single expression systems of human NIT1 and NIT2 in HEK293 cells to clarify the contribution of human NIT to hydrolysis of vildagliptin. Although the formation of a carboxylic acid metabolite of vildagliptin (M20.7) was observed in human liver samples, M20.7 was not formed by incubating vildagliptin with HEK293 cells expressing human NIT1 or NIT2. This suggests that human NIT1 or NIT2 is not involved in the metabolism of vildagliptin. Further investigation using other drugs is needed to clarify the contribution of human NIT to drug metabolism.

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The main route of elimination of vildagliptin, which is an inhibitor of dipeptidyl peptidase-4 (DPP-4), in humans is cyano group hydrolysis to produce a carboxylic acid metabolite M20.7. Our in vitro study previously demonstrated that DPP-4 itself greatly contributed to the hydrolysis of vildagliptin in mouse, rat, and human livers. To investigate whether hepatic DPP-4 contributes to the hydrolysis of vildagliptin in vivo, in the present study, we conducted in vivo pharmacokinetics studies of vildagliptin in mice coadministered with vildagliptin and sitagliptin, which is another DPP-4 inhibitor, and also in streptozotocin (STZ)-induced diabetic mice. The area under the plasma concentration-time curve (AUC) value of M20.7 in mice coadministered with vildagliptin and sitagliptin was significantly lower than that in mice administered vildagliptin alone (P < 0.01). Although plasma DPP-4 expression level was increased 1.9-fold, hepatic DPP-4 activity was decreased in STZ-induced diabetic mice. The AUC values of M20.7 in STZ-induced diabetic mice were lower than those in control mice (P < 0.01). Additionally, the AUC values of M20.7 significantly positively correlated with hepatic DPP-4 activities in the individual mice (Rs = 0.943, P < 0.05). These findings indicated that DPP-4 greatly contributed to the hydrolysis of vildagliptin in vivo and that not plasma, but hepatic DPP-4 controlled pharmacokinetics of vildagliptin. Furthermore, enzyme assays of 23 individual human liver samples showed that there was a 3.6-fold interindividual variability in vildagliptin-hydrolyzing activities. Predetermination of the interindividual variability of hepatic vildagliptin-hydrolyzing activity might be useful for the prediction of blood vildagliptin concentrations in vivo.