Human Nitrilase-like Protein Does Not Catalyze the Hydrolysis of Vildagliptin

Asakura, Mitsutoshi; Hayashida, Kohei; Fujii, H.; Nakajima, Miki; Atsuda, Koichiro; Itoh, Tomoo; Fujiwara, Ryoichi

doi:10.2133/dmpk.dmpk-14-rg-027

Cited by 5 publications

(7 citation statements)

References 14 publications

(13 reference statements)

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“…Rabbit polyclonal anti-actin antibody (A2103), bis(p-nitrophenyl) phosphate (BNPP), and ethopropazine were purchased from Sigma-Aldrich (St. Louis, MO). Vildagliptin was synthesized in our laboratory using the standard technique (Asakura et al, 2014). Vildagliptin carboxylic acid metabolite (M20.7) was purchased from Santa Cruz Biotechnology (Dallas, TX).…”

Section: Methodsmentioning

confidence: 99%

“…The final concentration of mouse, rat, and human liver microsomes and S9 fraction of HEK293 cells expressing human DPP-4 or its mutants was 0.5 mg/mL, respectively. Mouse liver S9 fraction, cytosol, and microsomes were prepared previously (Asakura et al, 2014). The reaction was initiated by adding vildagliptin after a 5-minute preincubation at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…In our previous study, we investigated whether human nitrilase-like protein (NIT)1 and NIT2, which are members of the nitrilase superfamily, are involved in the hydrolysis of vildagliptin, because the hydrolysis of the cyano group of vildagliptin is similar to nitrilase reaction (Asakura et al, 2014). However, the result of measurement of M20.7 formation rate in human embryonic kidney 293 (HEK293) cells expressing human NIT1 or NIT2 suggested that human NIT1 or NIT2 was not involved in the metabolism of vildagliptin.…”

Section: Introductionmentioning

confidence: 99%

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Dipeptidyl Peptidase-4 Greatly Contributes to the Hydrolysis of Vildagliptin in Human Liver

et al. 2015

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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...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Dipeptidyl Peptidase-4 Greatly Contributes to the Hydrolysis of Vildagliptin in Human Liver

et al. 2015

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“…Chemicals and Reagents. Vildagliptin was synthesized in our laboratory using the standard technique (Asakura et al, 2014). Vildagliptin carboxylic acid metabolite (M20.7) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).…”

Section: Methodsmentioning

confidence: 99%

“…It has been reported that the parental vildagliptin and its main metabolite M20.7 account for the majority of vildagliptin-related materials in human plasma (approximately 26% and 56%, respectively) and that the liver has an M20.7 formation activity (He, et al, 2007(He, et al, , 2009a. Cytochrome P450s (CYPs) and nitrilase-like proteins did not exhibit the formation of M20.7 (He et al, 2009a;Asakura et al, 2014). Although the main metabolic enzyme responsible for vildagliptin cyano group hydrolysis in humans was unknown, our in vitro study previously demonstrated that DPP-4, which is the pharmacological target of vildagliptin, greatly contributed to the cyano group hydrolysis of vildagliptin in human liver (Asakura et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Hepatic Dipeptidyl Peptidase-4 Controls Pharmacokinetics of Vildagliptin In Vivo

et al. 2016

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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.

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MicroRNA expression in the vildagliptin-treated two- and three-dimensional HepG2 cells

Yamashita

Asakura

Mitsugi

et al. 2016

Drug Metabolism and Pharmacokinetics

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Human Nitrilase-like Protein Does Not Catalyze the Hydrolysis of Vildagliptin

Cited by 5 publications

References 14 publications

Dipeptidyl Peptidase-4 Greatly Contributes to the Hydrolysis of Vildagliptin in Human Liver

Dipeptidyl Peptidase-4 Greatly Contributes to the Hydrolysis of Vildagliptin in Human Liver

Hepatic Dipeptidyl Peptidase-4 Controls Pharmacokinetics of Vildagliptin In Vivo

MicroRNA expression in the vildagliptin-treated two- and three-dimensional HepG2 cells

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