Absorption, Metabolism, and Excretion of [C]Vildagliptin, a Novel Dipeptidyl Peptidase 4 Inhibitor, in Humans

The absorption, metabolism, and excretion of (1-[[3-hydroxy-1adamantyl) amino] acetyl]-2-cyano-(S)-pyrrolidine (vildagliptin), an orally active and highly selective dipeptidyl peptidase 4 inhibitor developed for the treatment of type 2 diabetes, were evaluated in four healthy male subjects after a single p.o. 100-mg dose of [C]vildagliptin. Serial blood and complete urine and feces were collected for 168 h postdose. Vildagliptin was rapidly absorbed, and peak plasma concentrations were attained at 1.1 h postdose. The fraction of drug absorbed was calculated to be at least 85.4%. Unchanged drug and a carboxylic acid metabolite (M20.7) were the major circulating components in plasma, accounting for 25.7% (vildagliptin) and 55% (M20.7) of total plasma radioactivity area under the curve. The terminal half-life of vildagliptin was 2.8 h. Complete recovery of the dose was achieved within 7 days, with 85.4% recovered in urine (22.6% unchanged drug) and the remainder in feces (4.54% unchanged drug). Vildagliptin was extensively metabolized via at least four pathways before excretion, with the major metabolite M20.7 resulting from cyano group hydrolysis, which is not mediated by cytochrome P450 (P450) enzymes. Minor metabolites resulted from amide bond hydrolysis (M15.3), glucuronidation (M20.2), or oxidation on the pyrrolidine moiety of vildagliptin (M20.9 and M21.6). The diverse metabolic pathways combined with a lack of significant P450 metabolism (1.6% of the dose) make vildagliptin less susceptible to potential pharmacokinetic interactions with comedications of P450 inhibitors/inducers. Furthermore, as vildagliptin is not a P450 inhibitor, it is unlikely that vildagliptin would affect the metabolic clearance of comedications metabolized by P450 enzymes. Dipeptidyl peptidase 4 (DPP-4, DPP-IV) is a highly specialized aminopeptidase that is present in plasma, the kidney, and the intestinal brush-border membranes, as well as on the surface of capillary endothelial cells, hepatocytes, and a subset of T lymphocytes (Deacon et al., 1995; Mentlein, 1999). DPP-4 is responsible for the rapid inactivation of the incretin glucagon-like peptide 1 (GLP-1) and glucosedependent insulinotropic peptide. GLP-1, which is released postprandially, stimulates meal-induced insulin secretion and contributes to glucose homeostasis (Gutniak et al., 1997; Kieffer and Habener, 1999). Circulating GLP-1 is rapidly degraded and inactivated by DPP-4 (Deacon et al., 1995; Mentlein, 1999). With the inhibition of the DPP-4 enzyme activity, GLP-1 activity increases markedly, improving glycemic control in experimental and human studies (Balkan et al., 1999; Ahrén et al., 2002, 2004; Reimer et al., 2002). Therefore, administration of a DPP-4 inhibitor to diabetic patients augments endogenous GLP-1 activity, which in turn produces a clinically significant lowering of diabetic glycemia comparable with that observed when GLP-1 is administered by direct infusion (Gutniak et al., 1992; Drucker, 2003; Mest and Mentlein, 2005). Vildagliptin (Galvus, Novartis, East Hanover, NJ; (1-[[3-hydroxy1-adamantyl) amino] acetyl]-2-cyano-(S)-pyrrolidine) is a potent, orally active, highly selective inhibitor of DPP-4 (Villhauer et al., 2003) and is marketed as an antidiabetic drug in this novel class of action mechanisms (He et al., 2007b). Based on an in vitro recombinant DPP-4 assay, the IC50 for vildagliptin is 2 nM. In humans, the efficacy of vildagliptin against the DPP-4 enzyme also shows a low in vivo inhibitory constant (IC50 4.5 nM), a value that suggests higher potency than that reported for another DPP-4 inhibitor, sitagliptin (IC50 26 nM) (Herman et al., 2005; He et al., 2007b). Vildagliptin has shown the ability to inhibit DPP-4, increase plasma concentrations of intact GLP-1 and glucose-dependent insulinotropic peptide, decrease fasting and postprandial glucose, and suppress plasma glucagons in clinical trial in patients with type 2 diabetes. The pharmacokinetics and pharmacodynamics of vildagliptin after various dosing regimens in healthy volunteers and patients with type 2 diabetes have been previously reported (He et al., 2007a,b, 2008; Sunkara et al., 2007). The purpose of this study was to investigate the disposition and Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.108.023010. ABBREVIATIONS: DPP-4, dipeptidyl peptidase 4; GLP-1, glucagon-like peptide 1; vildagliptin, (1-[[3-hydroxy-1-adamantyl) amino] acetyl]-2cyano-(S)-pyrrolidine; [C]vildagliptin, (1-[3-hydroxy-adamant-1-yl-amino)-acetyl]-pyrrolidine-2(S)-carbonitrile; LSC, liquid scintillation counting; LC/MS/MS, liquid chromatography/tandem mass spectrometry; IS, internal standard; ESI, electrospray ionization; HPLC, high-performance liquid chromatography; LC/MS, liquid chromatography/mass spectrometry; DMSO, dimethyl sulfoxide; CID, collision-induced dissociation; P450, cytochrome P450; UGT, UDP glucuronosyltransferase; AUC, area under the curve; amu, atomic mass unit. 0090-9556/09/3703-536–544$20.00 DRUG METABOLISM AND DISPOSITION Vol. 37, No. 3 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 23010/3441254 DMD 37:536–544, 2009 Printed in U.S.A. 536 at A PE T Jornals on M ay 1, 2017 dm d.aspurnals.org D ow nladed from biotransformation of vildagliptin in healthy male volunteers after a single 100-mg (47 Ci) p.o. dose of [C]vildagliptin [(1-[3-hydroxyadamant-1-yl-amino)-acetyl]-pyrrolidine-2(S)-carbonitrile]. A daily dose of 100 mg is the recommended human efficacious dosing regimen for vildagliptin, and no pharmacokinetic gender difference has been observed (He et al., 2007b, 2008). [C]Vildagliptin has been shown to be highly absorbed in both rats and dogs (He et al., 2009). Vildagliptin was mainly metabolized before excretion in rats and dogs. One major metabolite in excreta involved hydrolysis at the cyano moiety to yield a carboxylic acid metabolite (M20.7) in rats and dogs. Another predominant metabolic pathway included the hydrolysis of the amide bond (M15.3) in the dog. Materials and Methods Study Drug. [C]Vildagliptin (specific activity 0.47 Ci/mg, radiochemical purity 99%) was synthesized by the Isotope Laboratory of Novartis Pharmaceuticals Corporation (East Hanover, NJ). The chemical structure of vildagliptin and the position of the radiolabel are shown in Fig. 1. Metabolites. Synthetic standards of metabolites M20.2, M20.7, and M15.3 were also obtained from Novartis Pharmaceuticals Corporation. Human Studies. The study protocol and the informed consent document were approved by an independent institutional review board. The written informed consent was obtained from all the subjects before enrollment. Four healthy, nonsmoking, male white subjects, age 18 to 45 years, with weights ranging from 77 to 93 kg, participated in the study. Subjects were confined to the study center for at least 20 h before administration of the study drug until 168 h (7 days) postdose. After an overnight fast, the subjects were given a single p.o. 100-mg dose of [C]vildagliptin as a 250-ml drinking solution. The radioactive dose given per subject was 47 Ci (1.85 MBq). After administration, the subjects continued to abstain from food for an additional 4 h. Blood was collected at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, 24, 36, 48, 72, 96, 120, 144, and 168 h postdose by either direct venipuncture or an indwelling cannula inserted in a forearm vein. Eighteen milliliters of venous blood was collected at each time point in heparinized tubes. Plasma was separated from whole blood by centrifugation, transferred to a screw-top polypropylene tube, and immediately frozen. Urine samples were collected at predose and at 0 to 4, 4 to 8, 8 to 12, 12 to 16, 16 to 24, 24 to 36, 36 to 48, 48 to 72, 72 to 96, 96 to 120, 120 to 144, and 144 to 168 h postdose. Feces were collected as passed from time of dosing until at least 168 h postdose. All of the samples were stored at 20°C or less until analysis. Radioactivity Analysis of Blood, Plasma, Urine, and Feces Samples. Radioactivity was measured in plasma and blood by liquid scintillation counting (LSC) on a liquid scintillation analyzer (Tri-CARB 2500; Canberra Industries, Meriden, CT). Plasma was mixed with scintillant and counted directly; whole blood samples were digested with tissue solubilizer (Soluene 350; PerkinElmer Life and Analytical Sciences, Waltham, MA), decolorized with hydrogen peroxide, stored in the dark to reduce luminescence, and then counted. Radioactivity in urine and feces was also assessed by LSC. Urine was mixed with liquid scintillant and counted directly. Feces was homogenized in water (approximately 1 2, w/v). Aliquots of feces homogenates were then combusted with a biological oxidizer (Packard Oxidizer 306; PerkinElmer Life and Analytical Sciences) before LSC. The total radioactivity given with the dose was set to 100%. The radioactivity at each sampling time for urine and feces was defined as the percentage of dose excreted in the respective matrices. The radioactivity measured in plasma was converted to nanogram-equivalents of vildagliptin based on the specific activity of the dose. Analysis of Unchanged Vildagliptin. Amounts of unchanged vildagliptin in plasma and urine were measured quantitatively using a validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay. Aliquots of plasma (200 l) or human urine (100 l diluted with 100 l of water) and 200 l of internal standard (IS) solution (C5 N-vildagliptin) were transferred to individual wells in a 1-ml, 96-well polypropylene plate. Extraction of the samples was performed using a Quadra-96 model 320 workstation (TomTec, Hamden, CT). Before extraction of samples, a 10-mg Oasis HLB 96-well solid-phase extraction plate (Waters, Milford, MA) was conditioned with 300 l of methanol, followed by 300 l of water. The samples were applied to the preconditioned extraction plate. The plate was washed with 300 l of 5% methanol (containing 2% ammonium hydroxide), 300 l of 20% methanol (containing 2% ammonium hydroxide), and 300 l of water. After vacuumdrying each well, the samples were eluted with 2 75 l of 80% methanol (containing 0.1% trifluoroacetic acid) and evaporated under nitrogen (35°C) to a volume of 50 l using an Evaporex solvent evaporator (Apricot Designs, Monrovia, CA). The samples were diluted with 50 l of 15% methanol (containing 0.5% ammonium hydroxide) and mixed before injection. Samples were analyzed on a Micromass Quattro LC (Waters) operated in multiple reaction monitoring mode with electrospray ionization (ESI ) as an interface. Vildagliptin and IS were separated on a Polaris 5m C18-A 50 2.0-mm column (45°C) (Metachem Technologies, Torrance, CA) with isocratic elution. The mobile phase of A/B (1:3, v/v) was used, where A was methanol/10 mM ammonium acetate, pH 8.0 (5:95, v/v), and B was acetonitrile/methanol (10:90, v/v). The flow rate was maintained at 0.2 ml/min with an injection volume of 10 l. Multiple reaction monitoring transitions for the drug and IS were m/z 304.2 3 m/z 154.1 and m/z 310.3 3 m/z 160.0, respectively. The dynamic range of the assay was from 1.93 to 2020 ng/ml for plasma and 5.13 to 5010 ng/ml for urine. Sample Preparation of Plasma, Urine, and Feces for Metabolite Investigation. Semiquantitative determination of main and trace metabolites was obtained for plasma, urine, and feces (based on peak areas) using highperformance liquid chromatography (HPLC)-radiodetection with off-line microplate solid scintillation counting and structural characterization by liquid chromatography/mass spectrometry (LC/MS). Plasma samples (3.5–4.5 ml) from each subject at 0.5, 1, 2, 3, 6, 12, 16, and 24 h postdose were proteinprecipitated with acetonitrile/ethanol (90:10 v/v) containing 0.1% acetic acid and removed by centrifugation. Recoveries of radioactivity after plasma sample preparation averaged 95%. The supernatant was evaporated to near dryness under a stream of nitrogen using the Zymark Turbo-Vap LV (Zymark Corp., Hopkinton, MA), and the residues were reconstituted in acetonitrile/5 mM ammonium acetate containing 0.1% trifluoroacetic acid (10:90 v/v). Aliquots (80–85 l) of concentrated plasma extracts were injected onto the HPLC column. For urine analysis, a pool of equal percent volume from the 0to 48-h fractions (10% of urine volume from each time point, e.g., 0–24 and 24–48 h) was prepared for each subject. An aliquot was centrifuged, and 100 l was injected onto the HPLC column without further purification. Recoveries of radioactivity after centrifugation of urine samples were 100%. Feces homogenates were pooled from 0 to 96 h at equal percent weight for each subject (10% of feces homogenates from each time point, e.g., 0–24, 24–48, and 48–72 h) and extracted twice with methanol by vortexing and centrifugation. The average recovery of sample radioactivity in the methanolic extracts was 87%. Aliquots of combined supernatant (5 ml) were evaporated to dryness under a stream of nitrogen using the Zymark Turbo-Vap LV, and the residues were reconstituted in 0.2 ml of acetonitrile/5 mM ammonium acetate containing 0.1% trifluoroacetic acid (10:90 v/v). Aliquots (60–80 l) of concentrated fecal extracts were injected onto the HPLC column. HPLC Instrumentation for Metabolite Pattern Analysis. Vildagliptin and its metabolites in urine, plasma, and feces were analyzed by HPLC with off-line radioactivity detection using a Waters Alliance 2690 HPLC system equipped with a Phenomenex (Torrance, CA) Synergy Hydro-RP column O H N H