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`Supplemental material to this article can be found at:
`http://dmd.aspetjournals.org/content/suppl/2012/04/10/dmd.112.045450.DC1.html
`
`1521-009X/12/4007-1345–1356$25.00
`DRUG METABOLISM AND DISPOSITION
`Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
`DMD 40:1345–1356, 2012
`
`Vol. 40, No. 7
`45450/3776821
`
`Characterization of the In Vitro and In Vivo Metabolism and
`Disposition and Cytochrome P450 Inhibition/Induction Profile of
`Saxagliptin in Human□S
`
`Hong Su, David W. Boulton, Anthony Barros Jr., Lifei Wang, Kai Cao, Samuel J. Bonacorsi Jr.,
`Ramaswamy A. Iyer, W. Griffith Humphreys, and Lisa J. Christopher
`
`Departments of Pharmaceutical Candidate Optimization (H.S., A.B., L.W., R.A.I., W.G.H., L.J.C.), Discovery Medicine and
`Clinical Pharmacology (D.W.B.), and Radiochemistry (K.C., S.J.B.), Bristol-Myers Squibb Research, Princeton, New Jersey
`
`Received March 2, 2012; accepted April 10, 2012
`
`ABSTRACT:
`
`Saxagliptin is a potent dipeptidyl peptidase-4 inhibitor approved
`for the treatment of type 2 diabetes mellitus. The pharmacokinetics
`and disposition of [14C]saxagliptin were investigated in healthy
`male subjects after a single 50-mg (91.5 ␮Ci) oral dose. Saxagliptin
`was rapidly absorbed (Tmax, 0.5 h). Unchanged saxagliptin and
`5-hydroxy saxagliptin (M2), a major, active metabolite, were the
`prominent drug-related components in the plasma, together ac-
`counting for most of the circulating radioactivity. Approximately
`97% of the administered radioactivity was recovered in the excreta
`within 7 days postdose, of which 74.9% was eliminated in the urine
`and 22.1% was excreted in the feces. The parent compound and
`M2 represented 24.0 and 44.1%, respectively, of the radioactivity
`recovered in the urine and feces combined. Taken together, the
`excretion data suggest that saxagliptin was well absorbed and was
`
`subsequently cleared by both urinary excretion and metabolism;
`the formation of M2 was the major metabolic pathway. Additional
`minor metabolic pathways included hydroxylation at other posi-
`tions and glucuronide or sulfate conjugation. Cytochrome P450
`(P450) enzymes CYP3A4 and CYP3A5 metabolized saxagliptin and
`formed M2. Kinetic experiments indicated that the catalytic effi-
`ciency (Vmax/Km) for CYP3A4 was approximately 4-fold higher than
`that for CYP3A5. Therefore, it is unlikely that variability in expres-
`sion levels of CYP3A5 due to genetic polymorphism will impact
`clearance of saxagliptin. Saxagliptin and M2 each showed little
`potential to inhibit or induce important P450 enzymes, suggesting
`that saxagliptin is unlikely to affect the metabolic clearance of
`coadministered drugs that are substrates for these enzymes.
`
`Introduction
`The dipeptidyl peptidase-4 (DPP4) inhibitors are promising re-
`cent additions to the arsenal of therapies available for the treatment
`of type 2 diabetes mellitus (Scheen, 2012). The DPP4 enzyme is
`
`Parts of this work were previously presented as follows: Christopher LJ, Su H,
`Barros A Jr, Wang L, Cao K, Bonacorsi S Jr, Iyer RA, and Humphreys WG (2009)
`Identification of the enzymes involved in the oxidative metabolism of saxagliptin
`and kinetics of formation of its major hydroxylated metabolite; Abstract 289. 16th
`North American Regional International Society for the Study of Xenobiotics Meet-
`ing; 2009 Oct 18–22; Baltimore, MD.
`International Society for the Study of
`Xenobiotics, Washington, DC; Su H, Christopher LJ, Iyer RA, Cao K, Bonacorsi S
`Jr, and Humphreys W (2011) In vivo disposition and pharmacokinetics and in vitro
`inhibition and induction profiles of [14C]saxagliptin, a potent inhibitor of dipeptidyl
`peptidase 4, in human. Abstract P236. 17th North American Regional Interna-
`tional Society for the Study of Xenobiotics Meeting; 2011 Oct 16–20; Atlanta, GA;
`International Society for the Study of Xenobiotics, Washington, DC.
`Article, publication date, and citation information can be found at
`http://dmd.aspetjournals.org.
`http://dx.doi.org/10.1124/dmd.112.045450.
`□S The online version of this article (available at http://dmd.aspetjournals.org)
`contains supplemental material.
`
`responsible for degrading and inactivating glucagon-like peptide-1
`(GLP-1) and glucose-dependent insulinotropic peptide, incretins
`that regulate blood glucose levels. GLP-1 is released postprandi-
`ally and stimulates meal-induced insulin secretion and contributes
`to glucose homeostasis (Kieffer and Habener, 1999; Gorrell,
`2005). By inhibiting the DPP4 enzyme, GLP-1 is sustained,
`thereby leading to increased activity and improved glycemic con-
`trol
`in patients with type 2 diabetes (McIntosh et al., 2005).
`Because this mechanism results in a glucose-dependent release of
`insulin, DPP4 inhibitors are expected to offer important advantages
`over traditional diabetes treatments including low risk for hypo-
`glycemia and weight gain (Gallwitz, 2008).
`Saxagliptin (Onglyza; Bristol-Myers Squibb, Princeton NJ and
`AstraZeneca, Wilmington, DE) (Fig. 1) is an orally administered,
`small molecule, reversible DPP4 inhibitor approved for the treatment
`of type 2 diabetes mellitus. It was specifically designed for enhanced
`potency and selectivity and to provide extended inhibition of the
`DPP4 enzyme (Augeri et al., 2005). The ability of saxagliptin to affect
`reductions in glycosylated hemoglobin (HbA1C) and fasting plasma
`glucose in type 2 diabetes patients has been demonstrated in multiple
`
`ABBREVIATIONS: DPP4, dipeptidyl peptidase-4; GLP-1, glucagon-like peptide-1; P450, cytochrome P450; HPLC, high-performance liquid
`chromatography; HLM, human liver microsomes; LSC, liquid scintillation counting; LC-MS/MS, liquid chromatography with tandem mass
`spectrometry; MRM, multiple reaction monitoring; AUC, area under plasma concentration-time curve; T-HALF, terminal phase half-life; CLR, renal
`clearance; DMSO, dimethyl sulfoxide; 3-MC, 3-methylcholanthrene; PB, phenobarbital; RIF, rifampicin; CT, threshold cycle.
`
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`SU ET AL.
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`FIG. 1. Primary pathways for biotransformation of
`[14C]saxagliptin in humans. The C-14 label was
`evenly distributed between the carbonyl carbon and
`the adjacent carbon, as indicated by asterisk (ⴱ) on
`the saxagliptin structure. The percentage of admin-
`istered radioactivity recovered in urine and feces as
`saxagliptin and M2 is indicated underneath the struc-
`tures. The estimated flux through each pathway (also
`reported as percentage of administered radioactivity)
`is indicated next to the corresponding arrow. D1, a
`degradant known to form in solution, was found in
`small quantities in all samples.
`
`Phase III clinical trials, both as a single agent and in combination
`regimens with metformin, a sulfonylurea or a thiazolidinedione (Ka-
`nia et al., 2011). The most commonly used clinical dose of saxagliptin
`in adults is 5 mg, once daily (United States prescribing information for
`Onglyza, http://www.packageinserts.bms.com/pi/pi_onglyza.pdf).
`In nonclinical pharmacokinetic studies, saxagliptin was rapidly
`absorbed and showed good oral bioavailability in rats (75%), dogs
`(76%), and monkeys (51%). A significant portion (33– 60%) of the
`administered dose was excreted as unchanged drug in the urine in
`these species. Formation of 5-hydroxy saxagliptin (M2) was a major
`metabolic pathway, and this metabolite was a major circulating me-
`tabolite in all species (Fura et al., 2009). Metabolite M2 was phar-
`macologically active, with an in vitro DPP4 inhibitory activity ap-
`proximately half that of saxagliptin (Augeri et al., 2005; Fura et al.,
`2009).
`The purpose of the current study was to investigate the in vivo
`disposition of saxagliptin and to determine its major metabolic path-
`ways in healthy male subjects after administration of a single 50-mg
`(91.5 ␮Ci) p.o. dose of [14C]saxagliptin. In addition, a series of in
`vitro studies were conducted to gain insight regarding possible cyto-
`chrome P450 (P450)-based drug-drug interactions between saxaglip-
`tin and potential comedications. These included the identification of
`enzymes involved in the metabolism of saxagliptin and formation of
`M2 and the determination of the potential of saxagliptin and M2 to
`inhibit or induce P450 enzymes.
`
`Materials and Methods
`Chemicals. [14C]Saxagliptin (radiochemical purity 99.86%, specific activity
`1.83 ␮Ci/mg) with the C-14 label distributed between the carbonyl carbon and the
`adjacent carbon (Fig. 1) and stable-labeled 13C4,15N-saxagliptin, and 13C4,15N-5-
`hydroxy saxagliptin [internal standards for high-performance liquid chromatogra-
`phy (HPLC) analysis] were synthesized by the Radiochemistry Group of the
`Department of Chemical Synthesis, Bristol-Myers Squibb Research (Princeton,
`
`NJ) (Cao et al., 2007). Unlabeled saxagliptin (P, (1S,3S,5S)-2-((S)-2-amino-2-(-3-
`hydroxyadamantan-1-yl)acetyl)-2-azabicyclo[3.1.0]hexane-3-carbonitrile); and
`reference standards for 5-hydroxy saxagliptin, (M2, (1S,3S,5S)-2-((S)-2-amino-2-
`((1r,3R,5S,7S)-3,5-dihydroxyadamantan-1-yl)acetyl)-2-azabicyclo[3.1.0]hexane-
`3-carbonitrile); degradant (D1, (1aS,4S,6aR,7aS)-4-(-3-hydroxyadamantan-1-yl)-
`6-iminohexahydro-1H-cyclopropa[4,5]pyrrolo[1,2-a]pyrazin-3(1aH)-one); the
`S,R,S,S and S,S,S,R diastereomers of saxagliptin (Supplemental Fig. S1) were
`supplied by the Departments of Chemical Development or Chemical Synthesis
`(Bristol-Myers Squibb).
`Selective chemical inhibitors of P450 enzymes for reaction phenotyping
`experiments were obtained from Sigma-Aldrich (St. Louis, MO), with the
`exception of montelukast, which was purchased from Sequoia Research Prod-
`ucts (Pangbourne, UK), and benzylnirvanol (BD Biosciences, Woburn, MA).
`Chemical inducers, inhibitors, substrates, and metabolites of P450 enzymes
`and internal standards used in experiments to evaluate whether saxagliptin and
`M2 were inhibitors or inducers of P450 enzymes were procured by CellzDirect
`(Pittsboro, NC). All chemicals were of the highest purity available.
`Human liver microsomes (HLM; 19 donors male/female) and individual
`human cDNA-expressed cytochrome P450 enzymes were purchased from BD
`Biosciences. Individual lots (n ⫽ 16) of HLM, for which the vendor had
`determined the activities of various P450 enzymes, were purchased as a
`Reaction Phenotyping kit (version 7) from XenoTech, LLC (Lenexa, KS).
`Monoclonal antibodies with inhibitory activity for specific P450 enzymes were
`obtained from Kristopher W. Krausz at the Laboratory of Metabolism, Na-
`tional Institutes of Health (Bethesda, MD).
`Ecolite scintillation cocktail was obtained from MP Biomedicals (Irvine,
`CA), and Emulsifier-Safe and Permofluor E⫹ scintillation fluid were obtained
`from PerkinElmer Life and Analytical Sciences (Waltham, MA). Deionized
`water was prepared using a MilliQ ultrapure water system (Millipore Corpo-
`ration, Billerica, MA). All organic solvents were HPLC grade, and other
`regents were reagent grade or better.
`Clinical Study Design, Dosing, and Sample Collection. The clinical phase
`of the study was conducted at the Bristol-Myers Squibb Clinical Research
`Center (Hamilton, NJ). This was an open-label, nonrandomized single dose
`study. Six healthy males participated in the study. The mean age was 29 years
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`METABOLISM AND DISPOSITION OF SAXAGLIPTIN IN HUMANS
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`(range, 25 to 37 years), and the mean body mass index was 23.6 kg/m2 (range,
`19.7 to 28.1 kg/m2). The study was conducted in accordance with the Decla-
`ration of Helsinki and guidelines on Good Clinical Practice. Before study
`initiation, the study protocol and informed consent documents were approved
`by the Institutional Review Board of the New England Institutional Review
`Board (Wellesley, MA). All study participants provided written informed
`consent before the initiation of study-specific procedures.
`After a 10-h fast, each subject received a single 50-mg p.o. solution dose of
`[14C]saxagliptin containing 91.5 ␮Ci of radioactivity, immediately followed
`by 240 ml of water on day 1. Blood samples were collected at selected time
`points via an indwelling catheter or direct venipuncture into Vacutainers (BD
`Biosciences Medical Supplies, Franklin Lakes, NJ) containing K3EDTA and
`were centrifuged to obtain plasma for pharmacokinetic and biotransformation
`analysis. The total urine and fecal output was collected for the duration of the
`study (0 –168 h). On the morning of day 6, a single 30-ml oral dose of Milk of
`Magnesia was administered to each subject to facilitate defecation before
`release from the clinical facility. All subjects were released on the morning of
`day 8.
`Blood samples (6 ml total per time point) for the plasma pharmacokinetic
`analysis of saxagliptin, M2, and radioactivity were collected predose and at
`0.25, 0.5, 0.75, 1, 1.5, 2.0, 2.5, 3, 4, 6, 8, 12, 24, 36, 48, 72, 96, 120, 144, and
`168 h postdose. Additional aliquots (10 ml per time point) for metabolite
`profiling were collected in conjunction with the pharmacokinetic samples at
`predose and at 1, 2, 4, 8, 12, 24, 48, 96, 144, and 168 h postdose.
`Cumulative urine was collected predose and over 0 to 12 h, 12 to 24 h, and
`thereafter in 24-h intervals through 168 h for determining saxagliptin, M2, and
`radioactivity concentrations and for metabolite profiling. Feces were collected
`predose and over 24-h intervals postdose for the measurement of radioactivity
`concentrations and for metabolite profiling. All samples were stored at ⫺20°C
`or below until analysis.
`Radioactivity Analysis. Radioactivity in plasma, urine, and feces was
`measured by liquid scintillation counting (LSC) on a Model LS 6500 liquid
`scintillation counter (Beckman Coulter, Inc., Fullerton, CA). Plasma and urine
`were mixed with Emulsifier-Safe scintillation fluid (PerkinElmer Life and
`Analytical Sciences) and counted directly. Water was added to each fecal
`sample to form an approximately 20% (w/w) feces mixture, which was
`homogenized using a probe-type homogenizer (Kinematica Polytron model no.
`PT 45-80; Brinkman Instruments, Westbury, NY). Aliquots of fecal homoge-
`nate were then combusted using a sample oxidizer before counting by LSC, as
`described previously (Christopher et al., 2008).
`Quantification of Saxagliptin and M2 in Plasma and Urine Samples.
`The concentrations of saxagliptin and M2 in individual plasma and urine were
`determined with validated liquid chromatography/tandem mass spectrometry
`(LC-MS/MS) methods. In brief, after the addition of stable-labeled internal
`standards (13C4,15N-saxagliptin and 13C4,15N-5-hydroxy saxagliptin) to each
`plasma or urine sample, the analytes were isolated by solid-phase extraction
`(Waters Oasis HLB, 10 mg; Waters, Milford, MA). The eluates were evapo-
`rated to dryness, reconstituted in mobile phase, and then applied to an Atlantis
`dC18, 2.1 ⫻ 50 mm, 5-␮m HPLC column (Waters). The LC-MS system used
`for plasma samples consisted of LC10AD delivery pumps (Shimadzu Corpo-
`ration, Columbia, MD) and a Series 200 Autosampler (PerkinElmer Life and
`Analytical Sciences). The HPLC system was interfaced to either a Quattro
`Premier mass spectrometer (Waters Corporation, Manchester, UK) for the
`plasma method or an API3000 mass spectrometer (AB Sciex, Foster City, CA)
`for the urine method. The mass spectrometers were operated in positive ion
`electrospray mode, and analytes were monitored by multiple reaction moni-
`toring (MRM) with transitions that were characteristic for each analyte. For
`saxagliptin and M2, the standard curve ranges were 5 to 1000 and 10 to 2000
`ng/ml, respectively, for the plasma method and 25 to 5000 and 50 to 10,000
`ng/ml, respectively, for the urine method.
`Pharmacokinetic Analysis of Saxagliptin, M2, and Total Radioactivity.
`The noncompartmental pharmacokinetic parameters of saxagliptin, M2, and
`total radioactivity were determined from plasma concentration versus time
`profiles and urine concentrations with cumulative urinary excretion volumes
`using noncompartmental methods with Kinetica 4.2 in eToolbox (Thermo
`Fisher Scientific, Waltham, MA). The single-dose pharmacokinetic parameters
`determined included the following: maximum observed concentration (Cmax);
`time of maximum concentration (Tmax); area under the plasma concentration-
`
`time curve (AUC) between time 0 and the last quantifiable concentration
`[AUC(0-T)]; and AUC between time 0 to infinity AUC(INF), terminal phase
`half-life (T-HALF), renal clearance (CLR), and percentage of urinary excre-
`tion. The percentage of fecal excretion was determined for total radioactivity
`only, and the calculation was based on cumulative fecal weights and fecal total
`radioactivity concentrations. For the percentage of dose excreted in urine and
`feces, the actual dose of saxagliptin administered to each subject was deter-
`mined by subtracting the weight of the dosing syringe (in grams) after dosing
`from the weight of the dosing syringe (in grams) before dosing and multiplying
`by the density of the dosing solution (1.0 g/ml) and the concentration of the
`dosing solution (5 mg/ml).
`Preparation of Samples for Biotransformation Profiling and Identifi-
`cation of Metabolites. Representative pools of plasma, urine, and feces were
`prepared for metabolite profiling and identification experiments. Plasma sam-
`ples were segregated by collection time (i.e., 1, 2, 4, and 8 h), and equal
`volumes from all subjects were combined. Plasma samples collected after 8 h
`were not analyzed because the radioactivity in these samples was too low to
`produce meaningful profiles. Urine and fecal homogenate pools (0 –168 h)
`were prepared across all subjects by combining a percentage of the volume
`(urine) or weight (fecal homogenate) proportional to the total amount excreted
`over each interval.
`Pooled plasma and fecal homogenate samples were each extracted with
`three volumes of methanol/acetonitrile 50:50 (v/v). After centrifugation at
`2500g for 40 min, the pellets were extracted an additional two times with
`methanol/acetonitrile/water (25:25:50, v/v/v). The supernatants from each ex-
`traction step were combined and evaporated to dryness under nitrogen. The
`dried residues were reconstituted in methanol/acetonitrile/water (⬃10:20:70,
`v/v/v), and the resulting supernatants were analyzed by HPLC with offline
`radioactivity detection or LC-MS/MS. The recovery of radioactivity from
`extracted plasma and fecal samples was approximately 100%. Pooled urine
`samples were centrifuged at 11,000g to remove any particulates and analyzed
`without additional processing.
`LC-Radiochromatographic and LC-MS/MS Methods for Metabolite
`Profiling of In Vivo Samples and Identification of Metabolites. Samples for
`metabolite profiling were analyzed on a Shimadzu LC-10AD HPLC system
`(Shimadzu Corporation), equipped with two 10AD VP pumps, a SIL-10AD
`autoinjector, a model SCL-10A system controller, and an SPD-M10A photo-
`diode array detector. A Zorbax 4.6 ⫻ 250 mm, 5-␮m, RX-C8 column (Agilent
`Technologies, Santa Clara, CA) maintained at 30°C was used to separate
`drug-related components. The mobile phase consisted of two solvents:
`1) mobile phase (A) 0.1% formic acid and 1% acetonitrile in water and
`2) mobile phase (B) 0.1% formic acid in acetonitrile. The mobile phase flow
`rate was 0.5 ml/min. The gradient program used for sample elution was as
`follows: hold isocratic at 0% B (0 –5 min); linear gradient from 0 to 20% B
`(5–35 min); hold isocratic at 20% B (35– 42 min); linear gradient from 20 to
`30% B (42– 45 min); hold isocratic at 30% B (45–50 min); linear gradient from
`30 to 40% B (50 –52 min); linear gradient from 40 to 80% B (52–55 min); hold
`isocratic at 80% B (55– 60 min); return to 0% B (60 – 62 min); re-equilibrate
`at 0% B for 10 min before the next injection.
`For quantification of metabolites by radioactivity, the HPLC eluate was
`collected in 0.25-min intervals on Wallac ScintiPlate-96-well plates with a
`Gilson Model FC 204 fraction collector (Gilson, Middleton, WI). The
`plates were evaporated to dryness on a Savant Speed-Vac (Savant Instru-
`ments Inc., Holbrook, NY) and counted for 10 min/well with a PerkinElmer
`1450 MicroBeta Wallac TRILUX Liquid Scintillation and Luminescence
`Counter (PerkinElmer Life Sciences, Turku, Finland) to quantify radioac-
`tivity. Radioprofiles were prepared by plotting the net counts per minute
`values obtained from the MicroBeta versus time after injection using
`Microsoft Excel (Microsoft Corporation, Redmond, WA). The metabolites
`were quantified based on the percentage of total radioactivity in each peak
`relative to the entire radiochromatogram.
`Mass spectral analysis was performed on a Finnigan LCQ Deca XP ion trap
`mass spectrometer equipped with an electrospray ionization probe (Thermo
`Fisher Scientific). Analyses were performed in the positive ion mode. Samples
`were introduced into the mass-spectrometer after chromatographic separation,
`using the same HPLC method used for radioprofiling. High purity nitrogen was
`used as the sheath and the auxiliary gas with levels at 60 and 10 (relative flow
`rate), respectively. The capillary temperature was 350°C. The nitrogen gas
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`1348
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`flow rate, spray current, and voltages were adjusted as required to achieve
`maximum sensitivity or optimal fragmentation of drug-related components.
`Reference standards, available for saxagliptin, M2, D1, M13, and the S,R,S,S
`and S,S,S,R epimers of saxagliptin were used to confirm the retention time and
`mass-spectral fragmentation patterns of these analytes.
`Identification of Enzymes Involved in the Metabolism of Saxagliptin
`and in the Formation of M2. [14C]Saxagliptin (10 ␮M) was incubated at
`37°C with pooled HLM (1 mg/ml protein) and individually expressed human
`P450 enzymes (500 pmol/ml each, including CYP1A2, CYP2A6, CYP2B6,
`CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and
`CYP3A5) in 100 mM phosphate buffer (pH 7.4), fortified with 1 mM NADPH
`to evaluate which enzymes were capable of metabolizing the compound and
`forming the major metabolite, M2. After the incubation period (30 min for
`expressed enzymes; 60 min for HLM), the reactions were terminated by adding
`1 to 2 volumes of ice-cold acetonitrile. The samples were then centrifuged, and
`the resulting supernatants were analyzed by LC-MS/MS and off-line radio-
`analysis. The analytical methodology was similar to that described for bio-
`transformation profiling and metabolite identification of in vivo samples, with
`the exception that drug-related components were separated on a YMC
`ODS-AQ S-3 120A column, maintained at 30°C, and the mobile phase gradient
`was modified, with a shorter run time (48 min).
`A correlation analysis between the formation of M2 and P450 activity was
`conducted by incubating saxagliptin, at concentrations of 1 and 10 ␮M, in
`singlicate with individual lots of HLM from 16 different donors. For each lot,
`the activities of CYP1A2, -2A6, -2B6, -2C8, -2C9, -2C19, -2D6, -2E1, -3A4/5,
`and -4A11 had been determined by the vendor using marker substrates specific
`for each enzyme (Technical Information for Reaction Phenotyping Kit version
`7, ref. 0510189; XenoTech, LLC). The reaction mixtures contained 0.25 mg/ml
`of HLM, 1 or 10 ␮M saxagliptin, 1 mM NADPH, and 0.1 M phosphate buffer
`with 2 mM MgCl2 (pH 7.4). Incubations (final volume, 0.5 ml) were conducted
`for 30 min at 37°C in a shaking water bath. Reactions were stopped with the
`addition of ice-cold acetonitrile. The concentration of M2 in each sample was
`then determined by analyzing an aliquot of the resulting supernatant by
`LC-MS/MS with multiple reaction monitoring as described below. Plots of M2
`versus marker substrate activity were prepared, and r values were calculated
`with Microsoft Excel (Office 2003; Microsoft Corporation).
`The metabolism of saxagliptin to M2 by specific P450 enzymes was also
`investigated with HLM in the presence of specific chemical or monoclonal
`antibody inhibitors of P450 enzymes. Chemical inhibitors included direct
`inhibitors, tranylcypromine (2 ␮M, CYP2A6), montelukast (3 ␮M, CYP2C8),
`sulfaphenazole (10 ␮M, CYP2C9), benzylnirvanol (1 ␮M, CYP2C19), quin-
`idine (1 ␮M, CYP2D6), ketoconazole (1 ␮M, CYP3A4/5) and time-dependent
`inhibitors, furafylline (10 ␮M, CYP1A2), orphenadrinie (50 ␮M, CYP2B6),
`diethyldithiocarbamate (50 ␮M, CYP2E1), troleandomycin (20 ␮M, CYP3A4/
`5), and 1-aminobenzotriazole (1000 ␮M, all P450s). Anti-P450 monoclonal
`antibodies included anti-CYP1A2, anti-CYP2B6, anti-CYP2C8, anti-
`CYP2C19, anti-CYP2D6, and anti-CYP3A4/5. A final concentration of 5 to 7
`␮l of antibody mixture was used per incubation. The antibody solutions were
`used as received; the concentration of each of the antibodies was not provided.
`Saxagliptin, at concentrations of 1 and 10 ␮M, was incubated in triplicate
`with pooled HLM (0.25 mg/ml), 1 to 2 mM NADPH, and 2 mM MgCl2 in 0.1
`M phosphate buffer (pH 7.4), in the presence or absence of chemical or
`anti-P450 monoclonal antibody inhibitors. The final volume of the reaction
`mixtures was 1 ml for the chemical inhibitor experiments and 0.25 ml for the
`anti-P450 antibody experiments. For incubations with direct chemical inhibi-
`tors, all ingredients except NADPH were added to the incubation tubes, and the
`samples were equilibrated at 37°C for 2 to 3 min before incubation. Then,
`NADPH was added to initiate the reactions. For incubations with time-
`dependent chemical inhibitors, all ingredients except saxagliptin were added to
`the incubation tubes. The samples were equilibrated at 37°C for 2 to 3 min, and
`then 1 mM NADPH was added to initiate a 15-min preincubation with the
`inhibitors. After the preincubation period, saxagliptin and an additional 1 mM
`NADPH were added to initiate the reactions. For incubations with anti-P450
`antibodies, the antibodies were preincubated with HLM in phosphate buffer on
`ice for 20 min, and then warmed at 37°C for 10 min. Saxagliptin and NADPH
`were added to the incubation mixtures to initiate the reactions. To establish the
`initial rate of metabolism of saxagliptin to M2 in HLM, incubations without
`chemical inhibitors or with antibodies against egg lysozyme (Hy-Hei-9) rather
`
`than anti-P450 antibodies were conducted. The rate of M2 formation in other
`incubations was normalized to the appropriate control incubation. Negative
`control incubations were carried out in the same manner, but they either lacked
`NADPH or contained heat-inactivated microsomes (boiled for 5 min).
`After the appropriate equilibrations and preincubation periods, HLM incu-
`bations were carried out for 30 min at 37°C. An equivalent volume of ice-cold
`acetonitrile was added to stop each reaction. Samples were vortex mixed and
`centrifuged to precipitate proteins. The concentration of M2 in each sample
`was then determined by analyzing an aliquot of the resulting supernatant by
`LC-MS/MS.
`Concentration-Dependent Metabolism of Saxagliptin to M2. The kinet-
`ics for the formation of M2 were determined in pooled HLM and expressed
`CYP3A4 and CYP3A5. Incubations (0.25 ml total volume, in triplicate)
`contained 1 mM NADPH, 2 mM MgCl2, 0.1 mM phosphate buffer (pH 7.4),
`saxagliptin, and HLM (0.25 mg protein/ml), or expressed CYP3A4 or
`CYP3A5 (10 pmol P450 enzyme/ml). Twelve concentrations of saxagliptin
`from 1 to 800 ␮M were evaluated. The HLM incubations were conducted at
`37°C. After the designated incubation period (30 min for HLM, 10 min for
`CYP3A4 and CYP3A5), reactions were quenched by adding an equal volume
`of ice-cold acetonitrile (0.25 ml). The quenched reaction mixtures were vor-
`texed to mix and centrifuged. The concentration of M2 in each sample was
`then determined by analyzing an aliquot of the resulting supernatant by
`LC-MS/MS.
`LC-MS/MS Method for Quantification of M2 in In Vitro Samples.
`Internal standard, 13C4,15N-5-hydroxy saxagliptin, was added to the quenched
`reaction mixtures from the in vitro incubations before LC-MS/MS analysis.
`The LC/MS system used for quantitation of M2 in in vitro samples consisted
`of two 10AD-VP pumps, a model SCL system controller and a degasser
`(Shimadzu Corporation), a LEAP HTC PAL autosampler equipped with a
`cooling stack maintained at 10°C (CTC Analytics, Carrboro, NC), and a
`Micromass Quattro Ultima triple quadrupole mass spectrometer (Waters).
`MassLynx software (version 4.0 or 4.1; Waters) was used to control the
`instrumentation and acquire data. Chromatographic separation of M2 from
`other mono-hydroxylated metabolites was achieved on an Agilent Zorbax
`SB-C8 column (4.6 ⫻ 75 mm, 3.5-␮m particle size) (Agilent, Wilmington,
`DE) maintained at ambient temperature. The mobile phase consisted of two
`solvents: mobile phase (A), 0.1% formic acid in water; and mobile phase (B),
`0.1% formic acid in acetonitrile. The mobile phase flow rate was 0.3 ml/min.
`The linear gradient program used for elution of the sample components was as
`follows: hold isocratic at 15% B (0 – 0.1 min); from 15 to 30% B (0.1–3 min);
`from 30 to 38% B (3– 4 min); from 38 to 40% B (4 –5 min); from 40 to 80%
`B (5–5.5 min); hold isocratic at 80% B (5.5– 6.5 min); return to 15% B (6.5–7
`min); re-equilibrate at 0% B for 5 min before the next injection.
`The Micromass Quattro Ultima mass spectrometer was operated in positive
`electrospray ionization mode. Ultrahigh purity nitrogen was used for the
`nebulizing and desolvation gases at flow rates of approximately 85 and 1000
`l/h, respectively. The capillary voltage was 3.5 kV, the cone voltage was 36V,
`and the collision energy was 45 eV. The desolvation temperature was 300°C,
`and the source temperature was 150°C. Detection of 5-hydroxysaxagliptin and
`its internal standard were achieved through MRM. The individual selected
`reaction monitoring transitions were 332 3 196 for M2 and 335 3 196 for the
`internal standard.
`Assessment of Potential of Saxagliptin and M2 to Inhibit P450 En-
`zymes. The potential for saxagliptin and M2 to inhibit P450 enzymes in a
`direct or time-dependent manner was assessed with HLM (n ⫽ 15 donors,
`mixed gender pool; CellzDirect, Durham, NC). IC50 values for nine enzymes
`were determined using probe substrates specific for each of the enzymes
`evaluated. The metabolic reactions monitored and probe substrate concentra-
`tions used were phenacetin O-deethylation (50 ␮M, CYP1A2), coumarin
`7-hydroxylation (1 ␮M, CYP2A6), bupropion hydroxylation (20 ␮M,
`CYP2B6), paclitaxel 6-hydroxylation (5 ␮M, CYP2C8), tolbutamide hydroxy-
`lation (140 ␮M, CYP2C9), S-mephenytoin 4⬘-hydroxylation (50 ␮M,
`CYP2C19), bufuralol 1⬘-hydroxylation (40 ␮M, CYP2D6), chlorzoxazone
`6-hydroxylation (50 ␮M, CYP2E1), midazolam 1⬘-hydroxylation and testos-
`terone 6␤-hydroxylation (5 and 50 ␮M, respectively, CYP3A). The final
`concentration of each probe substrate was near the experimentally determined
`Km value for the indicated enzyme.
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`METABOLISM AND DISPOSITION OF SAXAGLIPTIN IN HUMANS
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`To evaluate whether saxagliptin or M2 were competitive inhibitors of P450
`enzymes, saxagliptin (at concentrations of 0, 0.1, 1, 5, 20, and 50 ␮M), M2 (at
`concentrations of 0, 0.1, 1, 10, 50, and 200 ␮M), or prototypical P450
`inhibitors (positive controls) were mixed with HLM, and the probe substrates
`in 100 mM phosphate buffer (pH 7.4) in a total volume of approximately 0.5
`ml. After a 3-min equilibration at 37°C, 1 mM NADPH was added to initiate
`the reactions. The reactions were carried out using previously established
`conditions to ensure linearity with respect to protein concentration and incu-
`bation time. Incubations were stopped with addition of organic solvents. To
`assess the time-dependent inhibition, saxagliptin, M2, or positive control
`time-dependent inhibitors were preincubated for 15 min at 37°C with pooled
`human liver microsomes in the presence and absence of 1 mM
`NADPH. After the preincubation, P450-specific probe substrates were added
`to the incubation mixtures at the same concentrations used above. Metabolite
`formation in incubations with test compounds and control inhibitors was
`assessed with validated LC-MS/MS methods for each of the reaction products
`as described in Supplemental Table S1. Then,
`the percentage remaining
`activity was determined by comparison of probe substrate metabolism in
`incubations containing NADPH but without test compounds or control inhib-
`itors. If inhibition reached significant levels (i.e., the percentage remaining
`activity was ⬍50%), IC50 values were reported.
`Assessment of Potential of Saxagliptin and M2