0090-9556/07/3504-533–538$20.00
`DRUG METABOLISM AND DISPOSITION
`Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics
`DMD 35:533–538, 2007
`
`Vol. 35, No. 4
`13136/3187368
`Printed in U.S.A.
`
`Metabolism And Excretion of the Dipeptidyl Peptidase 4 Inhibitor
`[14C]Sitagliptin in Humans
`
`Stella H. Vincent, James R. Reed, Arthur J. Bergman, Charles S. Elmore, Bing Zhu, Shiyao Xu,
`David Ebel, Patrick Larson, Wei Zeng, Li Chen, Stacy Dilzer, Kenneth Lasseter,
`Keith Gottesdiener, John A. Wagner, and Gary A. Herman
`
`Departments of Drug Metabolism (S.H.V., J.R.R., C.S.E., B.Z., S.X.) and Clinical Pharmacology (D.E., P.L., K.G., J.A.W.,
`G.A.H.), Merck Research Laboratories, Rahway, New Jersey; Department of Drug Metabolism, Merck Research Laboratories,
`West Point, Pennsylvania (A.J.B., W.Z., L.C.); and Clinical Pharmacology Associates, Miami, Florida (S.D., K.L.)
`
`Received September 27, 2006; accepted January 3, 2007
`
`ABSTRACT:
`
`The metabolism and excretion of [14C]sitagliptin, an orally active,
`potent and selective dipeptidyl peptidase 4 inhibitor, were investi-
`gated in humans after a single oral dose of 83 mg/193 ␮Ci. Urine,
`feces, and plasma were collected at regular intervals for up to 7
`days. The primary route of excretion of radioactivity was via the
`kidneys, with a mean value of 87% of the administered dose re-
`covered in urine. Mean fecal excretion was 13% of the adminis-
`tered dose. Parent drug was the major radioactive component in
`plasma, urine, and feces, with only 16% of the dose excreted as
`metabolites (13% in urine and 3% in feces), indicating that sitaglip-
`tin was eliminated primarily by renal excretion. Approximately 74%
`of plasma AUC of total radioactivity was accounted for by parent
`
`drug. Six metabolites were detected at trace levels, each repre-
`senting <1 to 7% of the radioactivity in plasma. These metabolites
`were the N-sulfate and N-carbamoyl glucuronic acid conjugates of
`parent drug, a mixture of hydroxylated derivatives, an ether gluc-
`uronide of a hydroxylated metabolite, and two metabolites formed
`by oxidative desaturation of the piperazine ring followed by cy-
`clization. These metabolites were detected also in urine, at low
`levels. Metabolite profiles in feces were similar to those in urine
`and plasma, except that the glucuronides were not detected in
`feces. CYP3A4 was the major cytochrome P450 isozyme respon-
`sible for the limited oxidative metabolism of sitagliptin, with some
`minor contribution from CYP2C8.
`
`Dipeptidyl peptidase 4 (DPP-4) is a ubiquitous proline-specific
`serine protease responsible for the rapid inactivation of incretins,
`including glucagon-like peptide 1 (GLP-1) and glucose-dependent
`insulinotropic peptide (Gorrell, 2005). GLP-1, which is released upon
`nutrient ingestion, stimulates meal-induced insulin secretion and con-
`tributes to glucose homeostasis (Kieffer and Habener, 1999). Stabili-
`zation of GLP-1 via DPP-4 inhibition is a new therapeutic approach
`for type 2 diabetes (Drucker, 2003; Holst, 2004; Mest and Mentlein,
`2005; Nielsen, 2005).
`Sitagliptin (Januvia), also known as MK-0431 (Fig. 1), is an orally
`active, potent and selective DPP-4 inhibitor with an IC50 value of 18
`nM (Kim et al., 2005). Sitagliptin has been shown to inhibit plasma
`DPP-4 activity in a dose-dependent manner and to enhance active
`GLP-1 levels in normal volunteers (Bergman et al., 2005, 2006;
`Herman et al., 2005b) and patients with type 2 diabetes (Herman et al.,
`2004). Furthermore, in patients with type 2 diabetes, single doses of
`sitagliptin enhanced insulin and C-peptide release, decreased gluca-
`gon secretion, and reduced plasma glucose levels after an oral glucose
`
`Article, publication date, and citation information can be found at
`http://dmd.aspetjournals.org.
`doi:10.1124/dmd.106.013136.
`
`tolerance test (Herman et al., 2004), whereas 12-week treatment with
`sitagliptin significantly reduced HbA1c and fasting plasma glucose
`(Herman et al., 2005a; Scott et al., 2005).
`The metabolism and excretion of [14C]sitagliptin were studied in
`male human volunteers after oral administration of 83 mg/193 ␮Ci. In
`preclinical species, [14C]sitagliptin was shown to be eliminated by
`biliary and/or renal excretion of parent drug (Beconi et al., 2007).
`Metabolism was minimal, and it involved N-sulfation (M1), N-car-
`bamoyl glucuronidation (M4), hydroxylation (M6) followed by ether
`glucuronidation (M3), and oxidative desaturation followed by cycliza-
`tion (M2 and M5) (Fig. 1). Synthetic standards of metabolites M1,
`M2, and M5 were tested for DPP-4 inhibition and shown to be ⬃300-,
`1000-, and 1000-fold less active, respectively, than parent drug.
`
`Materials and Methods
`Chemicals and Dose Preparation. [14C]Sitagliptin was synthesized as the
`phosphate salt with a specific activity of 2.36 ␮Ci/mg free base (1.9 ␮Ci/mg
`salt) by the Labeled Compound Synthesis Group [Merck Research Laborato-
`ries (MRL), Rahway, NJ]. The chemical purity was 99.7%, as determined by
`HPLC. The dose was prepared as a capsule formulation containing 20 mg/48.3
`␮Ci [14C]sitagliptin (25 mg of phosphate salt). The 2,5-difluoro analog of
`sitagliptin used to saturate nonspecific binding sites on the solid phase extrac-
`tion cartridges was provided by Process Research (MRL, Rahway, NJ). The
`
`ABBREVIATIONS: DPP-4, dipeptidyl peptidase 4; GLP-1, glucagon-like peptide-1; LC-MS/MS, liquid chromatography-tandem mass spec-
`(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-
`trometry; LC-MS, LC-mass spectrometry; MRL, Merck Research Laboratories; sitagliptin,
`[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine); HPLC, high-performance liquid chromatography; MRM, multiple
`reaction monitoring; AUC, area under the curve; ADME, absorption, distribution, metabolism, and excretion.
`
`533
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`534
`
`VINCENT ET AL.
`
`FIG. 1. Main biotransformation pathways for [14C]sitagliptin in humans
`
`synthetic standard of metabolite M1 was synthesized by the Labeled Com-
`pound Synthesis Group (MRL, Rahway, NJ), and M2 and M5 were synthe-
`sized by Basic Chemistry (MRL, Rahway, NJ). The solid phase extraction
`cartridges, Varian C18 Bond Elut columns, were purchased from Varian Inc.
`(Harbor City, CA).
`Subjects and Dose Administration. The study was conducted at Clinical
`Pharmacology Associates in Miami, FL, in six healthy male volunteers, 27 to
`43 years old, weighing 60 to 95 kg. Subjects were admitted to the clinical
`research unit the evening before dosing and remained in the unit until the
`completion of all laboratory collections for the duration of the study (approx-
`imately 7 days). Subjects abstained from food and drink except water from
`midnight the evening before dosing, and consumed approximately 240 ml of
`water approximately 2 h before drug administration. Water was restricted 1 h
`before and 1 h after drug administration. Each subject ingested four capsules
`containing a total of 83 mg/193 ␮Ci [14C]sitagliptin with approximately 240
`ml of water. A standardized lunch was given at approximately 4 h postdose, a
`standardized dinner was given at approximately 10 h postdose, and a snack was
`allowed in the evening. Blood was collected in EDTA-coated tubes at selected
`time points up to and including 7 days postdose, and spun in a centrifuge to
`obtain plasma. Urine and feces were collected daily for 7 days. Plasma was
`
`stored at ⫺70°C, and urine and feces at ⫺20°C. Safety and tolerability were
`assessed by clinical and laboratory evaluations prestudy, predose, postdose,
`and post-study. Vital signs and ECGs were also evaluated at selected intervals.
`Radioactivity excretion data from one subject (AN 803) showed that there
`was substantially lower overall recovery compared with the other five subjects,
`suggesting incomplete collection of radioactivity. This subject was subse-
`quently re-dosed with a nonlabeled 100-mg dose of sitagliptin, followed by
`collection of blood and urine samples for 72 h postdose.
`Determination of Radioactivity and Sample Processing for Metabolite
`Profiling. The concentration of radioactivity in aliquots (0.5 ml) of plasma
`taken at 0, 0.5, 1.5, 2, 3, 5, 6, 10, 15, 24, 36, 48, 60, 72, 96, 120, 144, and 168 h
`after dosing was determined by liquid scintillation counting. For metabolite
`profiling, approximately 4 ml of plasma taken at 1, 4, 8, 12, and 18 h from the
`six subjects were pooled for each time point. The resulting pools were mixed
`with 4 ml of 8 M urea and applied to 20-g Varian C18 Bond Elut columns,
`using the following procedure: 1) two washes with 15 ml of methanol; 2) two
`washes with 15 ml of water; 3) equilibration with 30 ml of 50 ␮M 2,5-difluoro
`analog of sitagliptin (to saturate sites of nonspecific adsorption); 4) washes
`with methanol (two times, 15 ml) and water (two times, 15 ml); 5) loading of
`the plasma; 6) three washes with 15 ml of water; and 7) elution with 12 ml of
`
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`[14C]SITAGLIPTIN HUMAN ADME
`
`535
`
`TABLE 1
`Excretion of radioactivity in human urine and feces after an oral dose of [14C]sitagliptin
`Six healthy volunteers received 83 mg/193 ␮Ci of 关14C兴sitagliptin in four dry-filled capsules. Radioactivity in fecal homogenates and urine was determined and expressed as percentage of
`administered dose. Subject 0803* was considered an outlier and data from this subject were not used for the calculation of the mean and standard deviation values.
`
`Urine
`0–6 h
`6–12 h
`12–24 h
`24–168 h
`0–168 h
`Feces
`0–24 h
`24–48 h
`48–168 h
`0–168 ha
`Total
`
`0800
`
`43
`17
`13
`9.4
`82
`
`2.1
`15
`0.4
`18
`100
`
`0801
`
`38
`17
`16
`11
`82
`
`0.1
`18.2
`1.4
`20
`102
`
`0802
`
`44
`26
`14
`9.4
`93
`
`0.3
`3.1
`6.9
`10
`103
`
`Percentage of Dose by Subject
`
`0803*
`
`16
`4.2
`3.4
`1.4
`25
`
`4.4
`0.5
`⬍0.1
`4.9
`30
`
`0804
`
`46
`18
`12
`8.3
`85
`
`0.3
`5.4
`3.0
`8.9
`94
`
`0805
`
`47
`21
`14
`7.7
`90
`
`0.3
`6.9
`1.6
`8.7
`99
`
`Mean
`
`S.D.
`
`n ⫽ 5
`
`44
`20
`14
`9.2
`87
`
`0.6
`9.8
`2.7
`13
`100
`
`3.7
`3.9
`1.5
`1.4
`5.2
`
`0.9
`6.6
`2.6
`5.3
`4.1
`
`a Includes radioactivity on fecal wipes.
`
`methanol containing 10% formic acid. Previous studies had shown that sita-
`gliptin and its metabolites were stable under acidic conditions (5% HClO4).
`The column eluates were evaporated under N2 and the samples were recon-
`stituted in 0.3 ml of water/methanol/acetic acid (90:10:0.1, by volume) and
`analyzed by LC-MS/MS and radiometric detection.
`Concentrations of radioactivity and percentage of radioactive dose excreted
`into the urine and feces were determined at Charles River Laboratories (Wil-
`mington, MA). Feces were homogenized with water (4 ml/g feces) and
`weighed aliquots were analyzed by combustion followed by liquid scintillation
`counting of the trapped 14CO2. Radioactivity in weighed aliquots of urine was
`determined directly by liquid scintillation counting.
`For metabolite profiling, 0- to 168-h pools of urine and 0- to 96-h pools of fecal
`homogenates were prepared for each subject based on the volume recovered at
`each time point. Aliquots from each urine or feces pool were treated with an equal
`volume of acetonitrile. The mixtures were placed on melting ice for 10 min and
`centrifuged at 3000 rpm for 10 min. The resulting supernatants were dried under
`N2 at 30°C overnight, reconstituted in 350 ␮l (urine) or 600 to 750 ␮l (feces) of
`water/methanol/acetic acid (90:10:0.1, by volume), and analyzed by LC-MS/MS
`coupled with radiometric detection. Also, representative metabolite profiles were
`generated by analyzing samples consisting of equal volumes of urine or feces
`extracts from five subjects (excluding subject 803) or all six subjects. For confir-
`mation of metabolites by MRM transition monitoring and MS/MS fragmentation,
`fecal samples were subjected to solid phase extraction, using a procedure similar
`to the method described for plasma.
`Qualitative LC-MS/MS Analysis. LC-MS analysis was conducted on a PE
`Sciex API 3000 mass spectrometer (PerkinElmerSciex Instruments, Boston,
`MA), which was interfaced with a PerkinElmer HPLC system (PerkinElmer
`Life and Analytical Sciences, Boston, MA) equipped with two Series 200
`micro pumps and a PerkinElmer Series 200 autosampler. A Thermo Hypersil
`Fluophase PFP column (3.0 ⫻ 150 mm, 5 ␮m; Thermo Electron Corporation,
`Waltham, MA) was used for chromatographic separation. The column was
`eluted with a mixture of 5 mM ammonium acetate in water plus 0.05% acetic
`acid (mobile phase A) and 5 mM ammonium acetate in methanol plus 0.05%
`acetic acid (mobile phase B). The gradient was begun with 18% B for 2 min,
`and increased linearly to 80% B over 33 min and then to 95% B in 5 min,
`followed by a hold at 95% B for 5 min. The effluent from the HPLC, pumped
`at a rate of 0.6 ml/min, was diverted at a 5:1 ratio into the radiometric flow
`detector and into the mass spectrometer, respectively. Scintillation cocktail
`(Packard Ultima Flo-M, Downers Grove, IL) was pumped at a rate of 1.2
`ml/min in the radiometric detector. The contributions of parent drug and
`metabolites were calculated from the amount of radioactivity eluting in each
`peak relative to the total radioactivity in the HPLC chromatogram. LC-MS and
`LC-MS/MS experiments were carried out using the TurboIonSpray interface
`operated in the positive ion mode. The source voltage was 3500 V and the
`probe temperature, 300°C. Metabolites were identified by selective ion mon-
`itoring of the following precursor3product
`transitions (MRM): M1, m/z
`488.23408.2 and m/z 488.23193.0; M2, M3, and M5, m/z 406.23174 and
`m/z 4063191; M4, m/z 628.23408 and m/z 628.23452; and M6, m/z
`
`424.23406.1 and m/z 424.23191. The presence of parent drug and metabo-
`lites was confirmed by a signal of the MRM transition for each metabolite at
`least 2- to 3-fold above background at the correct retention time. Also, product
`ion scan experiments were carried out to compare the MS/MS fragmentation of
`M1 with that of the synthetic standard.
`Identification of P450 Isozymes Involved in the Metabolism of Sitaglip-
`tin. Sitagliptin (25 ␮M) was incubated with cell membranes containing singly
`expressed P450 isoforms (CYP2A6, 2B6, C8, C9, C19, 2D6, and 3A4),
`cytochrome b5, and an NADPH-regenerating system in 0.05 M potassium
`phosphate buffer. The final concentration of the P450 isoforms and cyto-
`chrome b5 in the incubation mixture was 0.5 ␮M. The relative contribution of
`individual P450s to the metabolism of sitagliptin was determined by preincu-
`bating human liver microsomes (2 mg protein/ml) with monoclonal antibodies
`against human CYP3A4 and 2C8 for 1 h at room temperature, followed by the
`addition of 10 ␮M sitagliptin. Control incubations were carried out using a
`control antibody. The mixtures were incubated at 37°C for 5 min, NADPH was
`added, and incubations continued for another 30 min. The reactions from both
`sets of incubations were quenched by the addition of acetonitrile containing
`2% formic acid, and supernatants were analyzed by LC-MS/MS, for the
`formation of metabolites M2, M5, and M6 using MRM.
`Quantitative LC-MS/MS Analysis. Concentrations of sitagliptin in plasma
`were determined by direct on-line LC-MS/MS analysis using a Cohesive
`Technologies (Franklin, MA) high turbulence liquid chromatography system,
`as described in more detail elsewhere (Bergman et al., 2006). Analyte and
`internal standard were detected using selected reaction monitoring with Tur-
`boIonSpray interface in the positive ion mode. The lower limit of quantifica-
`tion for the plasma assay was 0.5 ng/ml (1.23 nM) and the linear calibration
`range was 0.5 to 1000 ng/ml (1.23–2455 nM).
`Pharmacokinetic Calculations. Plasma concentrations of sitagliptin and
`radioactivity were converted into molar units (nM or nM Eq) using the
`molecular weight of 407.321 before pharmacokinetic analysis. Area under the
`plasma concentration-time curve to the last time point where radioactivity was
`above the lower limit of quantitation (AUC0-last) was calculated for both
`sitagliptin concentrations and radioactivity using the linear trapezoidal method
`for ascending concentrations and the log trapezoidal method for descending
`concentrations. Sitagliptin and radioactivity plasma Cmax and Tmax were ob-
`tained by inspection of the plasma concentration data.
`
`Results
`Excretion of Radioactivity. The excretion of radioactivity in hu-
`man urine and feces after a single 83 mg/193 ␮Ci oral dose of
`[14C]sitagliptin is summarized in Table 1. The results indicated that
`most of the radioactive dose was excreted via the kidneys, with a
`mean value of ⬃87% of the administered dose recovered within 7
`days in urine (range: ⬃83–94%) in five of six subjects who partici-
`pated in this study. Fecal excretion averaged ⬃13% (n ⫽ 5) of the
`
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`536
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`VINCENT ET AL.
`
`picted in Fig. 2, and pharmacokinetic parameters are summarized in
`Table 2. The highest concentrations in plasma (Cmax) were achieved
`at 2 to 4 h postdose and ranged from 706 to 993 nmol-Eq (total
`radioactivity) and 523 to 930 nM (sitagliptin) in five of the six
`subjects (excluding subject AN 803). Radioactivity levels at 60 to
`168 h postdose were below the limit of quantification, ⬃25 nM Eq.
`Plasma AUC0-last values in the five subjects ranged from 7.19 to 9.21
`␮mol-Eq 䡠 h, with a mean value of 8.20 ␮M Eq 䡠 h for total
`radioactivity, and from 5.12 to 6.83 (mean 6.04) ␮M 䡠 h for sitagliptin.
`The mean AUC0-last of radioactivity was 74% of the AUC0-last of
`sitagliptin. Data from subject AN803 were not used in the calculation
`of mean and standard deviation values, because of the low recovery of
`the radioactive dose in this subject.
`Metabolite Profiles in Plasma. Metabolite profiles in plasma
`pooled across subjects at 1 and 8 h are illustrated in Fig. 3. Similar
`profiles were observed at 4, 12, and 18 h. Due to the low levels of
`radioactivity and the limited volume of plasma, it was not possible to
`obtain individual metabolite profiles. Radioactivity in plasma was
`composed primarily of parent drug at all time points examined, with
`approximately 90% of circulating radioactivity detected at 1 h, ap-
`proximately 80% detected at 4, 8, and 18 h, and approximately 78%
`detected at 12 h. Six known metabolites were detected, each account-
`ing for ⬍1 to 8% of the circulating radioactivity between 1 and 18 h
`postdose (Table 3). The most abundant metabolites in plasma were
`M5 (4 –7% of radioactivity) and M2 (1– 6%), both of which are
`formed by oxidative desaturation of the piperazine ring followed by
`cyclization (Fig. 1). Other metabolites include M6 (a group of hy-
`droxylated derivatives; 1– 4%), M1 (N-sulfate conjugate; 2– 4%), M4
`(N-carbamoyl glucuronide conjugate; 1%,) and M3 (ether glucuronide
`conjugate of a hydroxylated derivative; ⬍1%).
`Metabolite Profiles in Urine and Feces. The metabolite profile of
`a pooled sample of urine collected at 0 to 168 h postdose is shown in
`Fig. 4. Similar profiles were obtained for urine collected from indi-
`vidual subjects, including subject AN 803. Parent drug was the major
`radioactive component, comprising ⬃84 to 88% of the urinary radio-
`activity. All six metabolites detected in plasma were excreted in small
`amounts into urine (⬍1–5% of the urinary radioactivity, ⬍1 to ⬃4%
`of the dose, each).
`The metabolite profile of a pooled human fecal sample collected at
`0 to 96 h is illustrated in Fig. 4. Feces collected between 4 and 7 days
`postdose contained negligible amounts of radioactivity and were not
`analyzed. Parent drug was the major radioactive component in feces,
`
`FIG. 2. Mean concentration-time profiles of sitagliptin and total radioactivity in
`plasma after oral administration of [14C]sitagliptin in healthy young men. Healthy
`volunteers received 83 mg/193 ␮Ci [14C]sitagliptin in four dry-filled capsules.
`Radioactivity in plasma was determined by liquid scintillation counting; mean ⫾
`standard deviation values (n ⫽ 5) are shown.
`
`administered dose (range: ⬃9 –20%), with a mean total recovery of
`radioactivity in urine and feces of ⬃100% (range: ⬃94 –105%).
`The recovery of radioactivity in one of the subjects (subject 803)
`was much lower than the average recovery in the other five subjects
`(30 versus 100% of the dose). The data from this subject were not
`used in the calculation of the mean and standard deviation values
`reported in Table 1 and Fig. 2. In a subsequent study with nonradio-
`labeled sitagliptin (100 mg), it was determined that the pharmacoki-
`netics and renal excretion of sitagliptin in this subject were similar to
`historical data. Pharmacokinetic analysis of the plasma and urine
`samples after this dosing revealed that approximately 66% of the
`sitagliptin dose was excreted unchanged in urine over the 72-h col-
`lection period (data not shown). Also, the renal clearance of MK-0431
`was generally similar to that observed in other subjects (439 ml/min).
`These results suggested that the results obtained for this subject after
`the 83.04-mg [14C]sitagliptin were very likely spurious, and therefore,
`this subject was excluded from the primary analysis.
`Radioactivity and Sitagliptin Levels in Plasma. Concentrations
`of radioactivity (expressed as [14C]sitagliptin nmol-Eq) and sitagliptin
`(nM) in human plasma after oral dosing of [14C]sitagliptin are de-
`
`TABLE 2
`Individual plasma pharmacokinetic parameters of sitagliptin and radioactivity after administration of a single oral 83-mg dose of [14C]sitagliptin to
`healthy young men
`Six healthy volunteers received 83 mg/193 ␮Ci 关14C兴sitagliptin in four dry-filled capsules. Concentrations of radioactivity and sitagliptin in plasma were determined by liquid scintillation
`counting and LC-MS/MS, respectively.
`
`Subject
`
`800
`801
`802
`803b
`804
`805
`AMc
`S.D.c
`GMc
`
`Sitagliptin
`
`Radioactivity
`
`Sitagliptin/Radioactivity
`
`a
`AUC0-last
`
`␮M 䡠 h
`5.27
`6.83
`6.67
`3.45
`6.31
`5.12
`6.04
`0.80
`6.00
`
`Cmax
`
`nM
`869
`771
`702
`447
`930
`523
`759
`158
`745
`
`Tmax
`
`h
`2
`2
`5
`1.5
`1.5
`4
`2d
`N.C.
`N.C.
`
`AUC0-last
`
`␮M Eq 䡠 h
`7.30
`8.55
`9.21
`4.64
`8.73
`7.19
`8.20
`0.90
`8.16
`
`Cmax
`
`nM Eq
`955
`833
`888
`572
`993
`706
`875
`113
`869
`
`Tmax
`
`h
`2
`2
`5
`1.5
`2
`3
`2d
`N.C.
`N.C.
`
`AUC0-last Ratio
`
`Cmax Ratio
`
`0.72
`0.80
`0.72
`0.74
`0.72
`0.71
`N.C.
`N.C.
`0.74
`
`0.91
`0.93
`0.79
`0.78
`0.94
`0.74
`N.C.
`N.C.
`0.86
`
`N.C., not calculated.
`a last ⫽ last time point where plasma radioactivity is above the lower limit of quantitation.
`b,c Subject 0803 was considered an outlier and data from this subject were not used for the calculation of the arithmetic mean (AM), geometric mean (GM), or S.D. values.
`d Median.
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`[14C]SITAGLIPTIN HUMAN ADME
`
`537
`
`FIG. 3. HPLC radiochromatograms of human plasma after oral administration of
`[14C]sitagliptin. Healthy volunteers received 83 mg/193 ␮Ci [14C]sitagliptin.
`Plasma pooled across subjects was subjected to solid phase extraction followed by
`LC-MS/MS analysis coupled with radiometric detection.
`
`TABLE 3
`Relative contribution of [14C]sitagliptin and metabolites to the radioactivity in
`human plasma following oral administration
`
`Compound
`
`Sitagliptin
`M1
`M2
`M3
`M4
`M5
`M6
`
`Contribution (Percentage of Radioactivity)
`
`1 h
`
`90
`3
`1
`⬍1
`⬍1
`4
`1
`
`4 h
`
`81
`4
`3
`⬍1
`1
`6
`4
`
`8 h
`
`80
`3
`4
`⬍1
`1
`7
`3
`
`12 h
`
`78
`3
`6
`⬍1
`⬍1
`7
`2
`
`18 h
`
`81
`2
`6
`⬍1
`⬍1
`6
`2
`
`comprising ⬃51 to 86% of the radioactivity. The metabolite profiles
`in feces were similar in all subjects (not shown) and were also similar
`to the profiles in urine and plasma, with the exception that the
`conjugates M3 and M4 were not detected in feces, presumably be-
`cause they had been hydrolyzed to their corresponding aglycones, M6
`and sitagliptin, respectively. Metabolites M1, M2, M5, and M6 were
`detected in small amounts (⬃1–12% of the fecal radioactivity, ⬍1 to
`⬃1.6% of the dose, each).
`Safety Evaluation. There were no clinical or laboratory adverse
`experiences reported in this study. In addition, there were no apparent
`treatment-related clinically relevant changes in vital signs, ECG, or
`laboratory safety parameters.
`Identification of Cytochrome P450 Involved in Sitagliptin Me-
`tabolism. After 1-h incubations of 10 ␮M [14C]sitagliptin with
`
`FIG. 4. HPLC radiochromatogram of pooled human urine (0 –168 h) and feces
`(0 –96 h) after oral administration of [14C]sitagliptin. Healthy volunteers received 83
`mg/193 ␮Ci [14C]sitagliptin. Urine and fecal homogenate extracts pooled across
`subjects were analyzed by LC-MS/MS coupled with radiometric detection.
`
`NADPH-enriched human liver microsomes, ⬃2% turnover was ob-
`served. The only metabolites detected by LC-MS/MS were a hydroxy-
`lated derivative (M6), and the cyclized products M2 and M5 (Beconi
`et al., 2007). Incubations with recombinant P450s indicated that
`CYP3A4 and, to a much smaller extent, CYP2C8 were capable of
`catalyzing the formation of M2, M5, and M6. Due to the low turnover,
`the relative contribution of these P450s could not be determined
`accurately. However, based on LC-MS/MS analysis,
`it could be
`discerned that the formation of M2 and M5 in human liver micro-
`somes could be inhibited to a much larger extent by anti-CYP3A4
`than anti-CYP2C8 antibody. Also, the formation of M6 could be
`inhibited by anti-CYP3A4 only.
`
`Discussion
`After oral administration of [14C]sitagliptin to healthy volunteers,
`the total recovery of radioactivity was approximately 100%. The
`results of this study demonstrate that the primary route of elimination
`of sitagliptin in healthy subjects is via renal excretion of intact drug.
`Approximately 16% of the oral radioactive dose was excreted as
`metabolites (13% in urine, 3% in feces), and ⬃10% of the radioac-
`tivity dose was excreted unchanged in feces. The unchanged sitaglip-
`tin found in feces may represent unabsorbed material, drug cleared by
`biliary excretion, or back-converted M1 (N-sulfate) and/or M4 (N-
`carbamoyl glucuronide). Nonetheless, these results indicated that sita-
`gliptin was well absorbed after oral administration, as ⬃87% of the
`radioactivity was recovered in urine. These data are corroborated by
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`VINCENT ET AL.
`
`the high bioavailability (Bergman et al., 2005) and high recovery of
`parent drug in urine after the administration of unlabeled sitagliptin to
`healthy subjects (Herman et al., 2005a; Bergman et al., 2006).
`Examination of the sitagliptin and radioactivity pharmacokinetic
`data indicate that sitagliptin makes up the majority (74%) of the
`radioactivity in plasma after an oral dose (as determined by the ratio
`of sitagliptin AUC and radioactivity AUC), with the remaining radio-
`activity accounted for by the metabolites shown in Fig. 1. Due to their
`low affinity for the DPP-4 enzyme (M1, M2, and M5) and their low
`levels in plasma, these metabolites would not be expected to contrib-
`ute to the pharmacological activity of sitagliptin.
`Similar observations were made in rats and dogs (Beconi et al., 2007),
`where, as in humans, sitagliptin was eliminated primarily unchanged into
`urine (dog) or urine and bile (rat). Also, all the metabolites observed in
`human plasma, urine, and feces were observed also in rat and/or dog
`plasma, urine, bile, and feces, as well as in incubations in vitro with rat,
`dog, and human liver preparations (Beconi et al., 2007). Results from in
`vitro experiments with recombinant P450s and monoclonal anti-P450
`antibodies indicated that the oxidative metabolism of sitagliptin in human
`liver microsomes is catalyzed primarily by CYP3A4 with some minor
`contribution from CYP2C8. Because sitagliptin is eliminated primarily
`unchanged into urine, it is not expected to be a victim of metabolism-
`based drug interactions.
`
`Acknowledgments. We thank the following individuals, all of
`MRL: Dr. David Liu and Christopher Kochansky for help in the
`qualitative LC-MS/MS analysis, Drs. Dennis Dean and Allen N. Jones
`for overseeing the synthesis and analysis of [14C]sitagliptin, and Drs.
`Ronald Franklin, Greg Winchell, David Evans, and Tom Baillie for
`helpful discussions and support. Also, we acknowledge the contribu-
`tion of Paul Zavorskas and other personnel of Charles River Labora-
`tories (Worcester, MA) in the radiometric analysis of the human feces
`and urine.
`
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`Address correspondence to: Dr. Stella Vincent, Merck Research Labora-
`tories, RY 80-141, P.O. Box 2000, Rahway, NJ 07065. E-mail: Stella_Vincent@
`Merck.com
`
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