0090-9556/09/3703-536–544$20.00
`DRUG METABOLISM AND DISPOSITION
`Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics
`DMD 37:536–544, 2009
`
`Vol. 37, No. 3
`23010/3441254
`Printed in U.S.A.
`
`Absorption, Metabolism, and Excretion of [14C]Vildagliptin, a Novel
`Dipeptidyl Peptidase 4 Inhibitor, in Humans
`
`Handan He, Phi Tran, Hequn Yin, Harold Smith, Yannick Batard, Lai Wang, Heidi Einolf,
`Helen Gu, James B. Mangold, Volker Fischer, and Dan Howard
`
`Departments of Drug Metabolism and Pharmacokinetics (H.H., P.T., H.Y., H.S., L.W., H.E., H.G., J.B.M., D.H.) and Exploratory
`Clinical Development (Y.B.), Novartis Pharmaceuticals Corporation, East Hanover, New Jersey; and Departments of Drug
`Metabolism and Pharmacokinetics/Bioanalysis, Abbott, Abbott Park, Illinois (V.F.)
`
`Received June 19, 2008; accepted December 12, 2008
`
`ABSTRACT:
`
`The absorption, metabolism, and excretion of (1-[[3-hydroxy-1-
`adamantyl) 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
`[14C]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 remain-
`der 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), glucu-
`ronidation (M20.2), or oxidation on the pyrrolidine moiety of vilda-
`gliptin (M20.9 and M21.6). The diverse metabolic pathways com-
`bined 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. Fur-
`thermore, 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 endo-
`thelial cells, hepatocytes, and a subset of T lymphocytes (Deacon et
`al., 1995; Mentlein, 1999). DPP-4 is responsible for the rapid inacti-
`vation of the incretin glucagon-like peptide 1 (GLP-1) and glucose-
`dependent insulinotropic peptide. GLP-1, which is released postpran-
`dially, 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, im-
`proving glycemic control in experimental and human studies (Balkan
`et al., 1999; Ahre´n 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 sig-
`nificant lowering of diabetic glycemia comparable with that observed
`
`Article, publication date, and citation information can be found at
`http://dmd.aspetjournals.org.
`doi:10.1124/dmd.108.023010.
`
`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-hydroxy-
`1-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 recombi-
`nant 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
`
`ABBREVIATIONS: DPP-4, dipeptidyl peptidase 4; GLP-1, glucagon-like peptide 1; vildagliptin, (1-[[3-hydroxy-1-adamantyl) amino] acetyl]-2-
`cyano-(S)-pyrrolidine; [14C]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.
`
`536
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`ABSORPTION, METABOLISM, AND EXCRETION OF [14C]VILDAGLIPTIN
`
`537
`
`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 radioac-
`tivity 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 (13C5
`15N-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 vacuum-
`drying 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 5-␮m C18-A 50 ⫻
`2.0-mm column (45°C) (Metachem Technologies, Torrance, CA) with iso-
`cratic 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 acetoni-
`trile/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 Inves-
`tigation. Semiquantitative determination of main and trace metabolites was
`obtained for plasma, urine, and feces (based on peak areas) using high-
`performance liquid chromatography (HPLC)-radiodetection with off-line mi-
`croplate 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 protein-
`precipitated with acetonitrile/ethanol (90:10 v/v) containing 0.1% acetic acid
`and removed by centrifugation. Recoveries of radioactivity after plasma sam-
`ple 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 0- to 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 homog-
`enates 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 contain-
`ing 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
`
`N
`
`O
`
`*
`
`N
`
`NH
`
`O H
`
`* indicates position of C-14 label
`
`FIG. 1. Chemical structure of [14C]vildagliptin.
`
`biotransformation of vildagliptin in healthy male volunteers after a
`single 100-mg (47 ␮Ci) p.o. dose of [14C]vildagliptin [(1-[3-hydroxy-
`adamant-1-yl-amino)-acetyl]-pyrrolidine-2(S)-carbonitrile]. A daily
`dose of 100 mg is the recommended human efficacious dosing regi-
`men for vildagliptin, and no pharmacokinetic gender difference has
`been observed (He et al., 2007b, 2008). [14C]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 hydroly-
`sis of the amide bond (M15.3) in the dog.
`
`Materials and Methods
`Study Drug. [14C]Vildagliptin (specific activity 0.47 ␮Ci/mg, radiochem-
`ical 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 [14C]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 count-
`ing (LSC) on a liquid scintillation analyzer (Tri-CARB 2500; Canberra Indus-
`tries, 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
`
`Page 2 of 9
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`538
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`HE ET AL.
`
`(4.6 ⫻ 150 mm, 4 ␮m, maintained at 30°C) and a guard column of the same
`type. The mobile phase consisted of 5 mM ammonium acetate containing 0.1%
`trifluoroacetic acid, pH 2.3 (solvent A), and acetonitrile (solvent B), and a
`gradient method was used. The mobile phase was initially composed of solvent
`A (100%) and held for 4 min. The mobile phase composition was then linearly
`programmed to solvent A/solvent B (87:13) over 26 min and held for 2 min
`and to solvent A/solvent B (40:60) in 0.5 min and held for 4 min. A short
`gradient was programmed to solvent A/solvent B (5:95) over 0.5 min, and
`these conditions were held for 4 min. The mobile phase condition was returned
`to the starting solvent mixture over 0.5 min. The system was allowed to
`equilibrate for 10 min before the next injection. A flow rate of 1.0 ml/min was
`used for all the analyses. The HPLC effluent was fractionated into a 96-deep-
`well Lumaplate (PerkinElmer Life and Analytical Sciences) using a fraction
`collector (FC 204; Gilson Inc., Middleton, WI) with a collection time of 8.4
`s/well. Samples were dried under a stream of nitrogen, sealed, and counted for
`1 to 15 min/well on a TopCount microplate scintillation counter (PerkinElmer
`Life and Analytical Sciences).
`The amounts of metabolites of parent drug in plasma or excreta were
`derived from the radiochromatograms (metabolite patterns) by dividing the
`radioactivity in original sample in proportion to the relative peak areas. Parent
`drug or metabolites were expressed as concentrations (in nanogram-equivalent
`per milliliter) in plasma or as percentage of dose in excreta. These values are
`to be considered as semiquantitative only, unlike those determined by the
`validated quantitative LC/MS/MS assay.
`In Vitro Human Blood Distribution and Protein Binding. A single 10-␮l
`aliquot of the stock solution in ethanol containing [14C]vildagliptin (35 ⫻ 106
`dpm) and known amounts of unlabeled vildagliptin was spiked to 1 ml of
`human fresh blood or plasma (n ⫽ 3) to achieve final concentrations of 10 to
`10,000 ng/ml.
`For the blood distribution study, triplicate determinations of the hematocrit
`in blood were made, and single blood samples (1 ml) were prepared from each
`of the three male human volunteers. After gentle mixing, a single 200-␮l
`aliquot of blood containing [14C]vildagliptin was pipetted for radioactivity
`analysis. Then the blood samples were incubated at 37°C for 30 min and
`centrifuged at ⬃3000g for ⬃15 min at 37°C. The resultant plasma was
`analyzed for radioactivity using a single 200-␮l aliquot.
`For the protein binding study, single plasma samples (1 ml) were prepared
`from each of the three male human volunteers. The pH was adjusted to 7.4 by
`adding 10 ␮l of 0.1 N HCl/ml plasma and gently vortexing the sample. After
`a single aliquot of plasma containing [14C]vildagliptin (200 ␮l) was pipetted
`for radioactivity analysis, each sample (⬃0.8-ml aliquot) was transferred to the
`sample reservoir of individual micropartition centrifuge tube (Centrifree Mi-
`cropartition Centrifuge Tube; Millipore Corporation, Billerica, MA). The
`membrane had a molecular mass cutoff of 30,000 Da. Samples were centri-
`fuged for 20 min at ⬃1000g at 37°C. The ultrafiltrate contained the free
`fraction, and 200-␮l aliquots were analyzed for radioactivity. Nonspecific
`binding studies were conducted in 0.2 M sodium phosphate buffer, pH 7.4,
`under the same conditions described above.
`For radioactivity analysis of blood or plasma samples in the blood distri-
`bution study, 200 ␮l of blood was pipetted onto individual Combusto-Pads
`(PerkinElmer Life and Analytical Sciences), air-dried, and combusted in a
`Packard 308 oxidizer before counting in a liquid scintillant (PerkinElmer Life
`and Analytical Sciences). For radioactivity analysis in the protein binding
`study, aliquots (200 ␮l) of plasma samples and filtrates were mixed with 2 ml
`of a liquid scintillant (NEN Formula 989; PerkinElmer Life and Analytical
`Sciences) in a vial for direct counting. The radioactivity in all the samples was
`determined by LSC in a Packard spectrometer (Hewlett Packard, Palo Alto,
`CA). M20.7 human protein binding was also determined over the concentra-
`tion range from 10 to 1000 ng/ml using the same ultrafiltration method as
`described above.
`In Vitro Metabolism in Human Liver Slices. [14C]Vildagliptin was incu-
`bated with liver slice preparations from one human subject. The human tissue
`was obtained through the Association of Human Tissue Users (Tucson, AZ).
`Each of the human organs had been perfused with Belzer’s University of
`Wisconsin solution but was rejected for transplantation. The incubations were
`carried out at 5 and 20 ␮M substrate concentrations for 1, 2, 4, 10, 18, and
`24 h. The incubates were analyzed by HPLC with online radioactivity detec-
`tion. Metabolites formed from the incubations were characterized by LC/MS.
`
`Human liver slices with a diameter of approximately 200 ⫾ 25 ␮m were
`prepared from 8-mm diameter tissue cores using a Vitron (Tucson, AZ) sterile
`tissue slicer. The individual organs were cored transversely and sliced using a
`Vitron sterile tissue slicer in ice-cold oxygenated (95% O2/5% CO2) V7
`preservation media. The viability of the human liver slices was assessed by
`determining the intracellular K⫹ content and measurement of ATP content in
`0.1% dimethyl sulfoxide (DMSO) and vildagliptin-exposed slice incubates.
`The slices were placed onto roller culture inserts and maintained at 37°C in
`Dulbecco’s modified Eagle’s/F-12 media without phenol red (Invitrogen,
`Carlsbad, CA) and supplemented with 10 ml/l Antibiotic AntiMycotic solution
`(Invitrogen), 10% Nu Serum, and Mito/Serum Extender, 1 ml/l (BD Bio-
`sciences, Franklin Lakes, NJ). After a preincubation period of 90 min, fresh
`media containing [14C]vildagliptin in 0.1% DMSO were added. At the various
`time points, the slice and media were transferred to separate vials, and the
`roller culture vial and insert were bathed in methanol (3.0 ml), which was then
`collected. Before HPLC analysis, the human liver slices were disrupted by
`homogenization with MeOH/H2O (50:50) followed by brief sonication. The
`incubation media were extracted with methanol, and the methanol wash was
`evaporated to dryness. All the fractions were pooled, and the protein was
`pelleted at approximately 40,000g for 10 min at 20°C. The pellet was re-
`extracted with methanol, and the resultant supernatant was evaporated to
`dryness and combined with the pooled sample.
`Structural Characterization of Metabolites by LC/MS/MS. Metabolite
`characterization was conducted with a Finnigan LCQ ion-trap mass spectrom-
`eter (Thermo Fisher Scientific, Waltham, MA) equipped with an ESI source.
`The effluent from the HPLC column was split, and approximately 500 ␮l/min
`was introduced into the atmospheric ionization source after diverting to waste
`during the first 4 min of each run to protect the mass spectrometer from
`nonvolatile salts. The electrospray interface was operated at 5000 V, and the
`mass spectrometer was operated in the positive ion mode. Collision-induced
`dissociation (CID) studies were performed using helium gas at the collision
`energy of 35% (arbitrary unit).
`Metabolism of Vildagliptin in Human Liver Microsomes and by Re-
`combinant Cytochromes P450. The metabolism of [14C]vildagliptin (specific
`activity of 154.5 ␮Ci/mg) was examined in pooled human liver microsomes
`(n ⫽ 46 donors, mixed gender) and in microsomal preparations from baculo-
`virus-infected insect cells expressing recombinant human cytochrome P450
`(P450) enzymes (BD Gentest, Woburn, MA). The recombinant P450 enzymes
`examined in this study were CYP1A1, CYP1A2, CYP1B1, CYP2A6,
`CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1,
`CYP2J2, CYP3A4, CYP3A5, and CYP4A11. Human liver microsomes (1 mg
`of microsomal protein/ml) or recombinant P450 enzymes (100 pmol of P450/
`ml) were preincubated with [14C]vildagliptin (47 ␮M, 0.5% final DMSO
`concentration, v/v) in 100 mM potassium phosphate buffer, pH 7.4, and 5 mM
`MgCl2, final concentrations, at 37°C for 3 min. The reactions were initiated by
`the addition of NADPH (1 mM, final concentration) and incubated for 30 min;
`reactions were then quenched by the addition of half the reaction volume of
`cold acetonitrile. The precipitated protein was removed by centrifugation, and
`an aliquot of each sample was analyzed by HPLC with in-line radioactivity
`detection as described above.
`P450 Inhibition Assessment by Vildagliptin and M20.7. The ability of
`vildagliptin and its metabolite M20.7 to inhibit P450 enzyme activity was
`assessed using pooled human liver microsomes (n ⫽ 50 donors, mixed gender;
`XenoTech, LLC, Lenexa, KS). To determine individual P450 activities, several
`probe substrate reactions were used that are known to be P450 enzyme-
`selective. The reactions used and corresponding probe substrate concentrations
`included phenacetin O-deethylation (5 ␮M, CYP1A2), bupropion hydroxyla-
`tion (25 ␮M, CYP2B6), paclitaxel 6␣-hydroxylation (10 ␮M, CYP2C8),
`diclofenac 4⬘-hydroxylation (5 ␮M, CYP2C9), S-mephenytoin 4⬘-hydroxyla-
`tion (15 ␮M, CYP2C19), bufuralol 1⬘-hydroxylation (5 ␮M, CYP2D6), chlor-
`zoxazone 6-hydroxylation (10 ␮M, CYP2E1), and midazolam 1⬘-hydroxyla-
`tion (5 ␮M, CYP3A4/5). The probe concentrations used were less than or
`approximately equal to their reported Km values. The conditions for each probe
`reaction were previously established to ensure linearity with time and protein
`concentration and to limit probe substrate turnover to ⬃⬍10% (results not
`shown). Increasing concentrations of the vildagliptin or authentic synthetic
`M20.7 (up to 100 ␮M) were incubated at 37°C individually with human liver
`microsomes (0.2 or 0.5 mg/ml) and one probe substrate in (final concentration)
`
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`539
`
`TABLE 1
`Pharmacokinetic parameters (mean ⫾ S.D.) after a single 100-mg p.o. dose of
`关14C兴vildagliptin
`
`Pharmacokinetic Parametersa
`
`Cmax (ng/ml) or (ngEq/ml)
`tmax (h)
`AUC0-t (ng 䡠 h/ml) or
`(ngEq 䡠 h/ml)d
`AUC0-⬁ (ng 䡠 h/ml) or
`(ngEq 䡠 h/ml)
`t1/2 (h)
`CL/F (l/h)
`Vz/F (l)
`
`Plasma
`Vildagliptinb
`397 ⫾ 92
`1.1 ⫾ 0.6
`1620 ⫾ 460
`
`Plasma
`Radioactivityc
`594 ⫾ 153
`2.1 ⫾ 1.3
`
`Blood
`Radioactivityc
`470 ⫾ 87
`2.0 ⫾ 1.4
`
`1610 ⫾ 460
`
`6000 ⫾ 1610
`
`3850 ⫾ 1580
`
`2.8 ⫾ 1.0
`65.2 ⫾ 15.5
`269 ⫾ 125
`
`4.6 ⫾ 0.3
`
`5.1 ⫾ 2.5
`
`a The abbreviation definitions for pharmacokinetic parameters, e.g., Cmax, tmax, AUC, t1/2,
`CL/F, and Vz/F are denoted in the Pharmacokinetic Analysis under Materials and Methods.
`b Vildagliptin was determined by validated LC/MS/MS.
`c Total radioactivity was determined by LSC.
`d t was 24 or 48 h.
`
`centrifuged at ⬃4000g (10 min). A 25-␮l aliquot was analyzed by LC/MS. The
`LC/MS method was as described above (see UGT involvement).
`Pharmacokinetic Analysis. The following pharmacokinetic variables were
`determined by fitting the concentration-time profiles to a noncompartmental
`model with an iterative nonlinear regression program (WinNonlin software
`version 4.0; Pharsight, Mountain View, CA): area under the blood or plasma
`drug concentration-time curve between time 0 and time t (AUC0-t); AUC until
`time infinity (AUC0-⬁); highest observed blood or plasma drug concentration
`(Cmax); time to highest observed drug concentration (tmax); apparent terminal
`half-life (t1/2); apparent volume of distribution of parent drug (Vz/F) calculated
`as dose/(AUC 䡠 ␭
`
`z), where F is bioavailability and ␭z is the terminal rate
`constant; and apparent clearance (CL/F), calculated as dose/AUC0-⬁.
`
`Results
`In Vitro Human Blood Distribution and Protein Binding. The
`mean human blood/plasma ratio (Cb/Cp) and fraction of [14C]vilda-
`gliptin bound to red blood cells (fBC) were 1.0 and 0.44, respectively,
`indicating approximately equal distribution between plasma and blood
`cells. The blood distribution was independent of concentration be-
`tween 10 and 10,000 ng/ml.
`The mean plasma protein binding of vildagliptin in humans was
`low (9.3%) and also independent of concentration. The nonspecific
`binding of the compound to centrifuge tubes and/or membranes was
`low (⬍12%), suggesting that ultrafiltration is a suitable method. In
`addition, M20.7 showed no plasma protein binding in humans over
`the concentration range of 10 to 1000 ng/ml and was independent of
`concentration.
`Absorption. The absorption of vildagliptin was rapid after oral
`administration, with the peak plasma concentration of vildagliptin
`observed at an average of 1.1 h (range, 0.5–2 h). The percentage of
`drug absorbed was estimated to be at least 85.4%, because this amount
`of the radioactivity was recovered in urine.
`Pharmacokinetics of Vildagliptin and Total Radioactivity. The
`mean plasma concentration-time profiles and pharmacokinetic param-
`eters of total radioactivity and unchanged vildagliptin in healthy male
`volunteers after a single oral dose of [14C]vildagliptin are shown in
`Fig. 2 and Table 1, respectively. The highest concentrations in plasma
`(Cmax) were achieved at 2.1 h postdose with the mean value 594
`ng-Eq/ml (total radioactivity) and at 1 h postdose with the mean value
`of 397 ng/ml (vildagliptin) in all four subjects. Radioactivity and
`parent levels at 48 h were below the limit of quantification. The
`terminal elimination half-life (t1/2) of radioactivity and vildagliptin
`averaged 4.6 and 2.8 h, respectively. Based on AUC0-⬁ values, ap-
`proximately 25.7 and 55% of the circulating radioactivity were attrib-
`utable to unchanged vildagliptin and its major metabolite M20.7,
`
`100 mM potassium phosphate buffer, pH 7.4, 1 mM NADPH, 5 mM MgCl2,
`1 mM EDTA, and 0.2% DMSO. After incubation, the reactions were quenched
`by addition of an equal volume of cold acetonitrile. Probe substrate turnover
`was determined by LC/MS/MS analysis (PE Sciex API300 mass spectrometer;
`Applied Biosystems, Foster City, CA; Shimadzu LC, Shimadzu, Kyoto, Japan)
`of metabolite formation. Reference standards for probe metabolites were
`obtained from commercial sources as follows: acetaminophen (Sigma-Aldrich,
`St. Louis, MO); 1⬘-hydroxybufuralol, hydroxybupropion, 6-hydroxychlorzoxa-
`zone, 4⬘-hydroxy-S-mephenytoin, and 1⬘-hydroxymidazolam (Ultrafine Chem-
`icals, Manchester, UK); and 6-hydroxypaclitaxel and 4⬘-hydroxydiclofenac
`(BD Biosciences, San Jose, CA). Chromatographic separation was achieved on
`a Supelco (Bellefonte, PA) Discovery DP-Amide C16 column (50 ⫻ 2.1 mm,
`4 ␮m, 0.3 ml/min flow rate, 25°C). The chromatographic solvents were as
`follows: A ⫽ 0.1% formic acid in 10 mM ammonium acetate, pH ⬃4.7, B ⫽
`acetonitrile; the gradient elution program (%B) was 031 min (5%), 134 min
`(from 5% to 95%), 436 min (95%), 636.5 min (from 95% to 5%). Probe
`metabolites derived from phenacetin, bupropion, midazolam, bufuralol, and
`paclitaxel were analyzed using ESI in positive ion mode, whereas the metab-
`olites of the remaining probes (diclofenac, chlorzoxazone, S-mephenytoin)
`were analyzed in negative ion mode.
`UDP Glucuronosyltransferase Enzyme Involvement in the Glucu-
`ronidation of Vildagliptin. The enzymes involved in the glucuronidation of
`vildagliptin to form M20.2 were determined using a panel of recombinant
`human UDP glucuronosyltransferase (UGT) enzymes, including UGT1A1,
`UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10,
`UGT2B4, UGT2B7, UGT2B15, and UGT2B17 (BD Gentest). In the initial
`assessment, incubations (100 ␮l, 37°C) consisted of (final concentrations):
`vildagliptin (20 ␮M), UDP-glucuronic acid (5 mM), alamethicin (0.25 mg/mg
`protein), enzyme protein (1 mg/ml), MgCl2 Tris-HCl buffer (pH 7.6, 50 mM),
`and acetonitrile (⬍0.2%). The enzyme protein had been preincubated with the
`alamethicin for 15 min on ice immediately before the experiments. The
`reactions were initiated by the addition of UDP-glucuronic acid after a 3-min
`preincubation and terminated after 60 min by the addition of acetonitrile (200
`␮l). The incubation samples were evaporated, reconstituted in 100 ␮l of 10:90
`(v/v) acetonitrile/water, and centrifuged at ⬃4000g (10 min). A 25-␮l aliquot
`was analyzed by LC/MS (Finnigan hybrid LTQ ion trap, ESI, positive mode).
`Chromatographic separation was achieved using an Ace3 C18 (50 ⫻ 3 mm, 3.5
`␮m, 0.25 ml/min flow rate, 35°C). The chromatographic solvents were as
`follows: A ⫽ 0.1% formic acid in 10 mM ammonium acetate, pH ⬃4.7, B ⫽
`acetonitrile; the gradient elution program (%B) was 031 min (10%), 135 min
`(from 10 to 30%), 536 min (from 30 to 98%), 637 min (98%), and 738 min
`(from 98 to 10%).
`DPP-4-Catalyzed Formation of M20.7. Vildagliptin (0.1 ␮M) was incu-
`bated with human recombinant DPP-IV (expressed in Sf9 cells; Sigma-Al-
`drich) in Tris-HCl buffer (50 mM, pH 8.0) at 37°C. After 60 min, the reaction
`was terminated by the addition of acetonitrile (200 ␮l). The incubation samples
`were evaporated, reconstituted in 100 ␮l of 10:90 (v/v) acetonitrile/water, and
`
`800
`
`700
`
`600
`
`500
`
`400
`
`300
`
`200
`
`100
`
`0
`
`Concentration (ngEq/mL or ng/mL)
`
`0
`
`5
`
`10
`
`15
`
`20
`Time (h)
`
`25
`
`30
`
`35
`
`40
`
`vildagliptin
`
`total radioactivity
`
`FIG. 2. Plasma concentrations of radioactivity (squares) and vildagliptin parent
`drug (triangles) after a single 100-mg p.o. dose of [14C]vildagliptin to humans.
`
`Page 4 of 9
`
`

`
`540
`
`HE ET AL.
`
`TABLE 2
`Cumulative excretion of 14C radioactivity in urine and feces after a single p.o.
`100-mg dose of 关14C兴vildagliptin to humans, mean ⫾ S.D.
`
`Time Period (h)
`
`Urine
`
`h
`
`0–24
`0–48
`0–72
`0–96
`0–168
`
`% dose
`72.7 ⫾ 4.8
`81.6 ⫾ 4.2
`83.8 ⫾ 4.4
`84.7 ⫾ 4.4
`85.4 ⫾ 4.4
`
`Feces
`
`% dose
`1.37 ⫾ 2.0
`9.93 ⫾ 8.0
`13.2 ⫾ 4.8
`14.3 ⫾ 3.7
`14.8 ⫾ 3.5
`
`Total
`
`% dose
`
`100 ⫾ 1
`
`respectively. In addition, mean blood-to-plasma ratios of radioactivity
`calculated at specific time points (between 0.25 and 1 h) averaged
`near 1.1, indicating that vildagliptin distributed almost equally be-
`tween blood cells and plasma as the main circulating component was
`the parent drug at the early time points, consistent with the in vitro
`finding (Cb/Cp ratio of vildagliptin ⬃1). However, the total radioac-
`tivity AUCb/AUCp ratio was 0.64, suggesting that metabolite(s)
`should be distributed more to plasma than blood cells. The CL/F and
`Vz/F values of vildagliptin were 6