METABOLISM, EXCRETION, AND PHARMACOKINETICS OF DULOXETINE IN HEALTHYHUMAN SUBJECTS

R. J. LANTZ, T. A. GILLESPIE, T. J. RASH, F. KUO, M. SKINNER, H-Y. KUAN,1 AND M. P. KNADLER

Departments of Drug Disposition (R.J.L., T.A.G., T.J.R., F.K., M.P.K.) and Pharmacokinetics (H-Y.K.), Lilly Research Laboratories,Eli Lilly and Company, Indianapolis, Indiana; and Clinical Pharmacology, Eli Lilly and Company, and Indiana University School of Medicine,

Indianapolis, Indiana (M.S.)

(Received March 10, 2003; accepted June 6, 2003)

This article is available online at http://dmd.aspetjournals.org

ABSTRACT:

Duloxetine is a potent and balanced dual inhibitor of serotonin andnorepinephrine reuptake being investigated for the treatment of de-pression and urinary incontinence. The disposition of duloxetine wasstudied in four healthy human subjects after a single 20.2-mg (100.6�Ci) oral dose of [14C]duloxetine in an enteric-coated tablet. Themean total recovery of radioactivity (� S.E.M.) after 312 h was 90.5%(�0.4%) with 72.0% (�1.1%) excreted in the urine. Duloxetine wasextensively metabolized to numerous metabolites primarily excretedinto the urine in the conjugated form. The major biotransformationpathways for duloxetine involved oxidation of the naphthyl ring ateither the 4-, 5-, or 6- positions followed by further oxidation, meth-ylation, and/or conjugation. The major metabolites found in plasma

were glucuronide conjugates of the following: 4-hydroxy duloxetine(M6), 6-hydroxy-5-methoxy duloxetine (M10), 4, 6-dihydroxy dulox-etine (M9), and a sulfate conjugate of 5-hydroxy-6-methoxy du-loxetine (M7). The major metabolites found in plasma were alsofound in the urine, but the urine contained many additional metabo-lites. In addition to duloxetine, 4-hydroxy duloxetine (M14) and anunidentified polar metabolite were observed in feces. Following[14C]duloxetine administration, Cmax was reached at a median of 6 hfor both duloxetine and total radioactivity. Duloxetine accounted forless than 3% of the circulating radioactivity based on mean areaunder the curve values. The elimination half-life of total radioactivity(120 h) was substantially longer than that of duloxetine (10.3 h).

Duloxetine [LY248686, (�)-N-methyl-3-(1-naphthalenyloxy)-2-thiophenepropanamine] is a potent and balanced inhibitor of thereuptake of serotonin (5-hyroxytryptamine, 5HT2) and norepinephrine(NE) in vitro and in vivo (Fig. 1). Duloxetine has demonstrated arelatively evenly balanced and potent inhibition of both the 5HT andNE reuptake at the transport sites and a weak effect on dopaminereuptake in both in vitro and in vivo studies (Pitsikas, 2000; Wong,1998). Duloxetine lacks significant affinity for muscarinic, histamineH1, �1-adrenergic, dopamine D2, 5HT1A, 5HT1B, 5HT1D, 5HT2A,5HT2C, and opioid receptors (Wong et al., 1993). The combinedaction on more than one monoamine neurotransmitter could result ina more favorable clinical outcome when compared with current se-lective serotonin reuptake inhibitors (Wong and Bymaster, 2002).

Preclinical studies in animals have shown that duloxetine enhancesthe release of 5HT and NE in limbic areas of the rat brain (Rueter etal., 1998) and, under irritated bladder conditions in cats, increasesbladder capacity and periurethral striated sphincter electromyographicactivity (Thor, 1995). Based upon these data, duloxetine is being

studied clinically for use in the treatment of major depressive disor-ders and stress urinary incontinence. Not only has duloxetine shownefficacy in these two disorders, but it has been shown to be safe andwell tolerated (Sharma et al., 2000; Detke et al., 2002; Goldstein et al.,2002; Norton et al., 2002). At doses of 20, 30, or 40 mg b.i.d.administered to healthy male subjects, the mean oral clearance, ap-parent volume of distribution, and half-life for duloxetine were 114l/h, 1943 liters, and 12.5 h, respectively (Sharma et al., 2000).

The safety and pharmacokinetics of duloxetine have been evaluatedextensively in healthy subjects. This study was conducted in fourhealthy participants to understand the adsorption, disposition, metab-olism, and excretion of duloxetine following a single oral dose ofduloxetine hydrochloride in an enteric-coated tablet.

Materials and Methods

Reference Compounds and Chemicals. The following nonlabeled andlabeled compounds were synthesized at Eli Lilly and Company: duloxetineHCl, [14C]duloxetine HCl, and 13CD3 duloxetine HCl; 4-, 5-, and 6-hydroxyduloxetine (M14, M12, and M13); N-desmethyl duloxetine (M23); 6-hydroxy-5-methoxy duloxetine (M15); 5-hydroxy-6-methoxy duloxetine (M16); thienylalcohol (M26); dihydrodiol duloxetine (M2); 4,6-dihydroxy duloxetine (6);glucuronide conjugate 4,6-dihydroxy duloxetine (M9); glucuronide conjugateof 6-hydroxy-5-methoxy duloxetine (M10); sulfate and glucuronide conjugatesof 5-hydroxy-6-methoxy duloxetine (M7, M3); glucuronide conjugates of 4-and 6-hydroxy duloxetine (M6, M8); and glucuronide conjugate of dihydrodiolduloxetine (M1). Reagents and solvents were of analytical grade and wereobtained from commercial sources.

Synthesis of Primary Metabolites of Duloxetine. The syntheses of someof the duloxetine metabolites M14 to M16 were accomplished by condensingthe thiophene side chain 1 with the corresponding fluoronaphthols, 2 to 5 (Fig.

1 Current address: Ligand Pharmaceuticals, 10275 Science Center Drive, SanDiego, CA.

2 Abbreviations used are: 5HT, hydroxytryptamine (serotonin); NE, norepi-nephrine; LC/MS-MS, liquid chromatography/tandem mass spectrometry; HPLC,high performance liquid chromatography; SPE, solid phase extraction; AUC, areaunder the curve; amu, atomic mass unit(s).

Address correspondence to: Dr. Mary Pat Knadler, Drug Disposition, Eli Lillyand Company, Lilly Corporate Center, DC0710 Indianapolis, IN 46285. E-mail:Knadler_Mary_Pat@Lilly.com

0090-9556/03/3109-1142–1150$7.00DRUG METABOLISM AND DISPOSITION Vol. 31, No. 9Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 1113/1090842DMD 31:1142–1150, 2003 Printed in U.S.A.

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2). The hydroxyl groups were protected as ketals or as acetals, under theconditions for the synthesis of duloxetine (R1 � R2 � R3 � H) (Wheeler et al.,1995). The protecting groups were then removed by acetic acid. The glucuro-nide conjugates M6 and M10 were synthesized by O-alkylation of M14 andM15 with acetobromo-�-D-glucuronic acid methyl ester followed by saponi-fication of the ester group. Compound M9 was synthesized by enzymaticglucuronidation of 6. The glucuronidation was accomplished by addition of 6to two cultures, Streptomyces sp. and Actinoplanes missouriensis, which wereprepared with expression (bioconversion) medium before incubation. Thecultures were incubated for 3 days at 30°C with 165-rpm shaking beforefiltration and isolation. The sulfate conjugate, M7, was synthesized as shownin Fig. 2. [NMR spectra (in ppm): Compound M13 (in CD3OD): 8.2 (d, 1 H),7.38 (d, 1 H), 7.28 (d, 1 H), 7.03 (dd, 1 H), 7.01 (d, 1 H), 6.52 (s, 2 H), 5.66(t, 1 H, methine next to thiophine), 2.85 (m, 2 H, CH2N), 2.42 and 2.25 (m, 1H each, CH2), 2.42 (s, 3 H, NCH3). Compound M6 (in CD3OD/D2O): 8.33 (dd,1 H), 8.18 (dd, 1 H), 7.50 (dd, 2 H), 7.30 (d, 1 H), 7.10 (dd, 2 H), 6.95 (dd,1 H), 6.81 (d, 1 H), 5.75 (d, 1 H methine next to thiophene), 4.97 (d, 1 H ofacetal on sugar ring), 3.50 to 3.80 (cluster, 4 H on sugar ring), 3.21 (m, 1 H,CH2N) and 3.10 (m, 1 H, CH2N), 2.70 (s, 3 H, NCH3), 2.49 (m, 1 H) and 2.39(m, 1 H, CH2). Compound M10 (in CD3OD/D2O): 8.05 (1 H, d), 7.59 (d, 1 H),7.44(d, 1 H), 7.31 (d, H), 7.29 (d, 1H), 7.10 (d, 1 H), 6.86 (dd, 1 H), 6.83 (d,1 H), 5.82 (t, 1 H, methine next to thiophene), 5.12 (d, 1 H of acetal on sugarring), 3.96 (s, 3 H, OCH3), 3.60 to 3.80 (cluster, 4 H on sugar ring), 2.75 and2.70 (m, 1 H each, CH2N), 2.41 and 2.22 (m, 1 H each, CH2), 2.32 (s, 3 H,NCH3). Compound M7 (CD3OD/D2O): 8.0 (d, 1 H), 7.6 (d, 1 H), 7.26 (d, 1 H),7.15 (m, 2 H), 6.97 (d, 1 H), 6.80 (dd, 1 H), 6.68 (d, 1 H), 5.68 (t, 1 H methinenext to thiophene), 3.80 (s, 3 H, OCH3), 2.60 and 2.52 (m, 1 H each, CH2N),2.25 and 2.05 (m, 1 H each, CH2), 2.15 (s, 3 H, NCH3).

Study Design. This open-label, single-dose metabolic inpatient study wasconducted with four participants (three males and one female) ranging in agefrom 44 to 48 years (Table 1). Study participants were selected on inclusion/exclusion criteria, medical history, physical examination, and other proceduresoutlined in the protocol. Before the study started, an institutional reviewcommittee approved the protocol and the informed consent document. Studyparticipants gave written informed consent prior to the study. Participants werenot involved in the administration of an investigational new drug or a radio-active investigational new drug within 30 days or 12 months, respectively,prior to the start of the study. Prior to dosing, the participants were also testedto determine their metabolic phenotype to CYP2D6 to determine whether theywere poor metabolizers or extensive metabolizers. The study was conducted atLilly Laboratories for Clinical Research, Indiana University Hospital andOutpatient Center, Indianapolis, Indiana.

Dose Administration. [14C]Duloxetine hydrochloride was synthesized atEli Lilly and Company (Wheeler et al., 1995), and the 14C label was located atthe chiral center of the molecule. Purity of the unlabeled and labeled drug was�99%.

Study drug was supplied as an enteric-coated tablet formulation containing20.2 mg of unlabeled and radiolabeled duloxetine with an activity of approx-imately 100.6 �Ci. The specific activity of the material was approximately 5�Ci/mg. The dose was administered with 180 ml of water. The subjects fasted

from 12 midnight the night before drug administration, and they fasted for anadditional 4 h after dosing.

Biologic Sample Collection. Blood samples (20 ml) were drawn from eachsubject within 0.5 h before dosing (0-h sample), and at approximately 1, 2, 4,6, 8, 10, 12, 16, 20, 24, 36, 48, 72, 96, 120, 144, 192, 216, and 240 h afterdosing in Vacutainer tubes containing sodium heparin. Plasma samples wereobtained after centrifugation and aliquots were analyzed for radioactivity. Theplasma samples were stored at approximately �20°C for future analyses ofduloxetine and metabolites. Since it had been shown previously that radioac-tivity does not partition into the cellular fraction of human blood, whole bloodwas not analyzed for radioactivity in this study.

Urine samples were collected in plastic containers. They were collectedprior to dosing and at the following time points: 0 to 4, 4 to 8, 8 to 16, 16 to24, 24 to 36, 36 to 48, and every 24 h out to 312 h. Samples were stored onice until the end of the collection period. Aliquots were taken for radioanalysisand the urine samples were stored at approximately �20°C.

Fecal samples were collected in plastic bags and homogenized after theaddition of water (if necessary to create a homogeneous slurry). Aliquots ofeach homogenate sample were combusted and then analyzed for trapped14CO2. The remaining homogenized samples were stored in plastic containersat approximately �20°C.

A sample of expired breath was collected from each subject for analysis of14CO2 within approximately 0.5 h before dosing (0-h sample), and at approx-imately 1, 2, 4, 6, 8, 10, 12, 16, 20, and 24 h after dosing. Subjects exhaled intoa solution containing a trapping agent (benzethonium hydroxide) for thecollection of expired CO2 and 14CO2.

Radioanalysis. Plasma, urine, and expired breath samples were preparedfor analysis of radioactivity by adding Ready Protein� (Beckman Coulter, Inc.,Fullerton, CA) liquid scintillation cocktail to a known volume of sample.Radioactivity analysis was performed using a Packard Tri-Carb liquid scintil-lation spectrometer (model 2300; PerkinElmer Life Sciences, Boston, MA).Aliquots of the fecal homogenates were weighed into pretared combustioncones and allowed to dry overnight. The samples were combusted with aPerkinElmer Life Sciences Tri-Carb sample oxidizer (model 307) followed byliquid scintillation counting in Carbo-Sorb E/Permafluor E� (PerkinElmerLife Sciences). Determination of [14C]duloxetine radioequivalents, data acqui-sition and storage, and statistical handling of the data were performed using theADME/WINPET system (Eli Lilly and Co.) application and a validated Lab-oratory Information Management System (ADME/LIMS), based on an avail-able software package (PerkinElmer Nelson’s SQL�LIMS; PerkinElmer In-struments, Norwalk, CT). Subject number, sample weights or volumes,specific activity, and dpm results were used for the determination of percentageof dose excreted.

Analysis of Duloxetine. Plasma concentrations of the parent compoundwere determined using a validated LC/MS-MS assay. The assay was validatedin the range of 0.5 to 100 ng/ml. Duloxetine and the stable isotope-labeledinternal standard (13CD3 duloxetine HCl) were extracted from plasma byautomated solid phase extraction using Ansys Spec Plus 96-well C8 extractionplates (Thames Restek UK Ltd., Windsor, UK) on a robotic liquid handlingsystem (Multiprobe 204 Liquid Handling System, Canberra Industries, Pang-borne, UK). The cartridges were conditioned with methanol followed by waterprior to the addition of the plasma samples. The cartridges were then washedwith water and 60% methanol in water (v/v). The samples were eluted with100% methanol for direct injection by the autosampler. The compounds wereseparated chromatographically using an HI-RPB (30 mm � 3.2 mm i.d.)analytical column (Hichrom Ltd., Theale, Berkshire, UK) with mobile phaseconsisting of 55% acetonitrile/45% 10 mM ammonium acetate, pH 5 (v/v). Theextracts were analyzed on a PerkinElmerSciex API III� LC/MS-MS systemusing Turbo Ionspray (PerkinElmerSciex Instruments, Boston, MA). Tandemmass spectrometry was used to monitor the transition of m/z 298.1344.0 forduloxetine and 302.1348.0 for the internal standard. Standard curves andquality control samples were analyzed along with the samples. The overallpercentage CV (precision) and percentage relative error (accuracy) of the assayafter three validation runs were �8%. Duloxetine was stable in human plasmafor at least 1 year when stored at approximately �20°C or �70°C.

Metabolite Profiling and Identification by HPLC. Plasma samples werepooled and extracted by precipitation with acetonitrile or by solid phaseextraction (SPE) using a C8 cartridge (100 mg; Varian Medical Systems, Palo

FIG. 1. Structure of duloxetine.

The asterisk denotes the position of the 14C label.

1143METABOLISM AND EXCRETION OF DULOXETINE IN HUMANS

Alto, CA). A 1-ml plasma sample was precipitated by adding 3 ml of aceto-nitrile followed by centrifugation. The aqueous/organic layer was transferredto a silanized glass tube, evaporated under nitrogen, and reconstituted with50% methanol/50% water (containing 0.1% trifluoroacetic acid) (v/v) andinjected for analysis. The same reconstitution fluid was used with all of theplasma, urine, and fecal extractions. The extraction recovery of radioactivitywas approximately 81%. For SPE, 1 ml of plasma was added to a precondi-tioned C8 SPE cartridge (1 ml of methanol followed by 1 ml of water) on avacuum system. The plasma sample was slowly eluted through the cartridgeand the cartridge was then washed with 1 ml of water. The metabolites wereeluted with 2 ml of methanol into a silanized glass tube, evaporated undernitrogen, and reconstituted for injection. The extraction recovery of radioac-tivity was approximately 71%.

Urine samples were either directly injected (for those samples containinghigh amounts of radioactivity) or extracted using a C8 SPE cartridge prior toanalysis. For SPE, 1 to 2 ml of urine was extracted using the same procedureas described for plasma. The extraction recovery of radioactivity was approx-imately 84%.

Plasma and urine samples were hydrolyzed by incubating a 1:1 mixture ofsample and 0.2 M acetate buffer (pH 4.7) in a 37°C water bath overnight with�-glucuronidase containing sulfatase (type H-2 from Helix pomatia). Thesamples were then processed for analysis as described previously.

Fecal homogenates were extracted by adding approximately 5 ml of meth-anol to 1 to 2 g of sample. The sample was vortexed, mixed on a rotator forapproximately 15 min, and centrifuged. The extract was transferred to asilanized tube, and the extraction procedure was repeated. The extracts were

TABLE 1

Subject demographics

SubjectIdentification Gender Age Height Weighta Origin Smoking

StatusSmokingHistory

CYP2D6Genotype

yr cm kg yr

1 Male 46 173 73.9 White Y 28 EM2 Male 44 188 71.7 White Y 25 EM3 Male 44 175 83.5 Black N 0 EM4 Female 48 155 66.7 White N 0 EM

EM, extensive metabolizer.a Screening weight.

FIG. 2. Synthesis scheme for the metabolites of duloxetine.

1144 LANTZ ET AL.

combined into one tube, evaporated under nitrogen, and reconstituted forinjection. The extraction recovery of radioactivity was approximately 81%.

Duloxetine and its metabolites were separated on a Discovery C18 column(4.6 mm � 150 mm, 5 �m; Supelco, Bellefonte, PA) after passing through a0.2-�m prefilter attached prior to the analytical column. A gradient separationwas used to separate the metabolites. Mobile phase A consisted of 5%acetonitrile and 95% water (containing 0.1% trifluoroacetic acid) (v/v). Mobilephase B had an acetonitrile content of 75%. The gradient consisted of thefollowing steps: 0 to 25 min, linear gradient from 100% A to 70% A; 25 to 35min, isocratic at 70% A; 35 to 45 min, linear gradient to 0% A; 45 to 49 min,isocratic at 0% A; 49 to 50 min, linear gradient to 100% A; 50 to 60 min,isocratic at 100% A. HPLC flow was at 1 ml/min. Detection of the metaboliteswas by UV (232 nm) and radiodetection (IN/US �-RAM; IN/US Systems, Inc.,Tampa, FL). The radiodetector utilized a 1,000-�l liquid cell with the flow rateof liquid scintillant (Ready Protein�) to mobile phase at a ratio of 4:1.

Due to the number of metabolites, HPLC mobile phase and gradientconditions were later improved to separate metabolites that coeluted in theurine samples. Mobile phase A consisted of 5% acetonitrile and 95% water(containing 0.025% trifluoroacetic acid and 0.075% formic acid, pH 2.5) (v/v).Mobile phase B had an acetonitrile content of 75%. The gradient consisted ofthe following steps: 0 to 12 min, linear gradient from 100% A to 85% A; 12to 25 min, linear gradient to 80% A; 25 to 32 min, isocratic at 80% A; 32 to35 min, linear gradient to 70% A; 35 to 40 min, isocratic at 70% A; 40 to 50min, linear gradient to 0% A; 50 to 54 min, isocratic at 0% A; 54 to 55 min,linear gradient to 100% A; 55 to 65 min, isocratic at 100% A. HPLC flow wasat 1 ml/min.

Metabolite Identification by Mass Spectrometry and NMR. The LC/MSanalyses were performed using a Finnigan TSQ-7000 mass spectrometer(ThermoFinnigan, San Jose, CA). HPLC conditions (column and mobilephases) were identical to those used for metabolite profiling. However, differ-ent HPLC conditions were utilized for analysis of the sulfate metabolites.Mobile phase A consisted of 95% 10 mM ammonium acetate and 5% aceto-nitrile. Mobile phase B was 5% 10 mM ammonium acetate and 95% acetoni-trile. The gradient consisted of the following steps: 0 to 5 min, hold at 100%A; 5 to 39 min, linear gradient to 20% A; 39 to 40 min, linear gradient to 0%A; 40 to 41 min, linear gradient to 100% A. HPLC flow rates for bothconditions were at 1 ml/min. Approximately 25% of the eluant was diverted tothe mass spectrometer via a 1:4 T splitter, and the remaining eluant (75%) waspassed to a radiochemical detector (LB507; EG&G Berthold, Bad Wildbad,Germany). The radiodetector utilized a 500-�l liquid cell with a scintillant(Ultima Flo M; PerkinElmer Life Sciences) flow rate of 3 ml/min. Thisprovided simultaneous detection of radioactivity and MS data. Mass spectralanalysis was performed with both positive and negative ion electrosprayionization. The capillary temperature was 250°C. MS-MS analyses wereperformed using a collision energy of �20 eV and a collision cell pressureusing argon gas at 2.0 mTorr.

A Varian 400-MHz NMR instrument was used for synthesized standardstructural characterization. Spectra were recorded at room temperature (lockedat CD3OD signal) with Me4Si at 0 ppm as a reference. The metabolitestructures were confirmed on either a Varian 600-MHz or 500-MHz NMRinstrument. Either flow LC with column trapping or probe experiments wereutilized for the various metabolites.

Pharmacokinetic Analysis. Plasma concentration versus time data forduloxetine and total radioactivity were analyzed using noncompartmentalpharmacokinetic methods. Maximum observed plasma concentration and thecorresponding sampling time were designated as Cmax and Tmax, respectively.Concentration-time data were plotted on a semilogarithmic scale, and theterminal log-linear phase was identified by visual inspection. The eliminationrate constant (�z) was determined as the slope of the linear regression for theterminal log-linear portion of the concentration-time curve. A terminal half-lifevalue (t1/2) was calculated as 0.693/�z. A predicted concentration (C) at the lastsampling time at which the assay value was above the limit of quantificationwas calculated from the regression equation. Area under the plasma concen-tration versus time curve (AUC0-t) was calculated by the trapezoidal methodand extrapolated to infinite time as AUC0-� � AUC0-t � C/�z.

Apparent plasma clearance (CLp/F) and volume of distribution (Vz/F) dur-ing the terminal elimination phase of duloxetine were calculated as CLp/F �Dose/AUC0-� and Vz/F � Dose/(�z � AUC0-�), respectively.

Results

Clinical Signs. Duloxetine, administered orally as an enteric-coated 20.2-mg tablet, was well tolerated by all three of the malesubjects in this study. The one female subject experienced nausea,retching (no actual emesis), headache, somnolence, and sweatingwithin 3 h after duloxetine administration. Direct observation of thissubject at the time of occurrence suggested that the phlebotomyprocedure itself may have played a role in her symptoms. Regardlessof their cause, these adverse events were mild to moderate andtransient in nature. Electrocardiograms, laboratory reports, and vitalsigns showed no evidence of any safety problems related to dulox-etine.

Excretion of Radioactivity in Urine and Feces. After oral admin-istration of [14C]duloxetine in a 20.2-mg enteric-coated tablet, thetotal mean � S.E.M. recovery of radioactivity at 312 h postdose was90.5 � 0.4% with 72.0 � 1.1% excreted in urine and 18.5 � 0.9%excreted in feces (Fig. 3). Radioactivity was not detected in theexpired breath samples. The majority (87.2 � 1.1%) of radioactivitywas excreted within 120 h after dosing. The total recovery of radio-activity from individual subjects ranged from 89.5% to 91.1%.

Pharmacokinetic Evaluation. The mean (SD) plasma concentra-tion-time profiles of duloxetine and total radioactivity following oraladministration of [14C]duloxetine in all four subjects are depicted inFig. 4. Table 2 summarizes selected plasma pharmacokinetic param-eters of duloxetine and total radioactivity in these individuals.Whereas the median Tmax was 6 h for both duloxetine and totalradioactivity, the mean Cmax and AUC for duloxetine were approxi-mately 9% and 3%, respectively, of the values for total radioactivity.The t1/2 of total radioactivity was 12-fold longer compared with thatof duloxetine.

Metabolism. Plasma samples from each subject were pooled persampling time point prior to extraction for metabolite analysis. Du-loxetine, the glucuronide conjugate of 4-hydroxy duloxetine (M6), thesulfate conjugate of 5-hydroxy-6-methoxy duloxetine (M7), the gluc-uronide conjugate of 4, 6-dihydroxy duloxetine (M9), and the gluc-uronide conjugate of 6-hydroxy-5-methoxy duloxetine (M10) wereidentified from the extracted plasma samples by comparison to syn-thetic standards and mass spectral data. The major metabolite inplasma was the glucuronide conjugate of 4-hydroxy duloxetine (M6).The second most abundant metabolite in plasma was the sulfateconjugate of 5-hydroxy-6-methoxy duloxetine (M7). Radiochromato-graphic peak area percentages of duloxetine and the plasma metabo-lites are shown in Table 3. Figure 5 illustrates a radiochromatographic

FIG. 3. Mean (�S.E.M.) cumulative elimination of radioactivity in urine andfeces following a single oral 20.2-mg dose of [14C]duloxetine.

1145METABOLISM AND EXCRETION OF DULOXETINE IN HUMANS

profile of plasma in addition to the profiles of urine and feces.Although only the 10-h plasma profile is shown in Fig. 5, the profilesof samples analyzed out to 48 h were similar.

Urine samples were analyzed for metabolites only out to 72 h, sincethe levels of radioactivity in samples past 72 h were low and/ornondetectable. Approximately 94% (mean, n � 4) of the radioactivityexcreted in the urine from each subject was eliminated by 72 h. Table4 lists the individual and mean results of the percentage of total doseeliminated by 72 h for each metabolite based on radioactive peakareas. Results show that the glucuronide conjugate of 4-hydroxyduloxetine (M6) was the predominant metabolite, and it accounted forapproximately 16.9% of the mean total dose. The sulfate conjugate of5-hydroxy-6-methoxy duloxetine (M7) was the second most abundantmetabolite, and it accounted for approximately 12.5% of the meantotal dose. Duloxetine was not detectable in the urine by HPLC/radiochemical detection.

A fecal sample with the highest amount of radioactivity from eachsubject was extracted for metabolite analysis. The samples were eitherfrom the 48- to 72-h or 72- to 96-h collections. Two of three radio-active peaks were identified by mass spectrometry as duloxetine and4-hydroxy duloxetine (M14), and one early eluting peak remains

unknown. The radioactive peak area percentages of duloxetine and4-hydroxy duloxetine were used to calculate the percentage of totaldose for each sample (Table 5).

Metabolite Structure Elucidation by MS and NMR. Duloxetinewas identified in plasma and feces. The retention time and product ionmass spectrum of the duloxetine peak matched that of the authenticduloxetine standard. The product ion mass spectrum of the protonatedmolecular ion m/z 298 produced the characteristic product ions at m/z44 and m/z 154 corresponding to C2H6N and the loss of naphthol,respectively. These results confirmed the identification of the dulox-etine peak in plasma and feces. The characteristic product ions of m/z44 and m/z 154 were observed in the majority of the metabolites andwere utilized as “characteristic markers” of duloxetine metabolites. Inmany instances these were the only fragment ions observed in theproduct ion mass spectrum along with an aglycone ion in some of theglucuronide and sulfate conjugates of duloxetine.

The glucuronide conjugate of 4-hydroxy duloxetine (M6) wasfound in plasma and urine producing a protonated molecular ion atm/z 490. This ion is 192 amu greater than that of duloxetine andindicates that M6 is a glucuronide conjugate of hydroxy duloxetine.The product ion mass spectrum of m/z 490 produced the product ionsat m/z 44 and m/z 154. These ions suggest that the oxidation andsubsequent glucuronidation of duloxetine has occurred on the naph-thol ring. This peak was not present in the urine after hydrolysis with�-glucuronidase. Comparison of the retention time, product ion massspectrum, and NMR spectrum with a synthesized standard of theglucuronide conjugate of 4-hydroxy duloxetine confirmed the identi-fication of M6.

Metabolite M7 was found in plasma and urine. Both positive andnegative electrospray ionization were utilized to determine the pro-tonated and deprotonated molecular ions at m/z 424 and m/z 422,respectively. The characteristic ions for duloxetine at m/z 44 and m/z154 were observed in the positive product ion mass spectrum of m/z424. In addition, the product ions at m/z 344 and m/z 342 show the lossof 80 amu from m/z 424 and m/z 422, respectively. This loss ischaracteristic for sulfate conjugates. A product ion at m/z 314 indi-cates an addition of at least one oxygen moiety to duloxetine. How-ever, this peak was present in urine after hydrolysis with �-glucuron-idase. The synthesis of a standard and further detailed analysis withNMR confirmed the structural assignment of M7 as the sulfate con-jugate of 5-hydroxy-6-methoxy duloxetine.

Metabolite M9 was also found in plasma and urine. The protonatedmolecular ion at m/z 506 did not produce the characteristic productions of duloxetine at m/z 44 and m/z 154. However, the product ion atm/z 330 shows a loss of 176 amu from m/z 506, indicative of aglucuronide conjugate. This peak was not observed in urine followingincubation with �-glucuronidase. A synthesized standard of 4,6-dihydroxy duloxetine was incubated with cell cultures to produce theglucuronide conjugates for comparison. Two different cell cultures(Streptomyces sp. and Actinoplanes missouriensis) produced the twoglucuronide conjugates of 4,6-dihydroxy duloxetine. The M9 metab-olite peak matched the retention time and product ion mass spectrumof a peak produced in the Streptomyces sp. cell culture incubation.This peak’s structural assignment was determined with NMR analysis(Table 6). The proton chemical shifts for sites 2 and 3 on the naphthylring of the 4,6-dihydroxy duloxetine standard are magnetically equiv-alent and form a singlet at 6.63 ppm. In the case of metabolite M9,sites 2 and 3 are nonequivalent and form a doublet (6.99 ppm and 6.77ppm), and have significantly shifted downfield (0.36 ppm and 0.14ppm) compared with the 4,6-dihydroxy standard. In addition, theproton chemical shifts for sites 7 and 8 on the naphthyl ring formetabolite M9 (7.15 ppm and 8.15 ppm) and the 4,6-dihydroxy

FIG. 4. Mean (SD) duloxetine concentrations and total radioactivity in plasmafollowing oral administration of [14C]duloxetine.

TABLE 2

Pharmacokinetic parameters of duloxetine and total radioactivity in subjectsreceiving an oral dose of 14Cduloxetine

Parameter

Arithmetic Mean (%CV)

Duloxetine(n � 4)

Radioactivity(n � 4)

Duloxetine/RadioactivityRatio

%

Tmaxa 6.0 6.0

(h) (4.0–16.0) (6.0–6.0)Cmax 23.5 274 8.5(ng/ml) (60) (6) (61)AUC0-t 236 7774 2.8(ng � h/ml) (71) (25) (52)AUC0-� 257 8770 2.7(ng � h/ml) (71) (25) (52)t1/2

b 10.3 120(h) (6.75–13.6) (104–141)CLp/F 119(l/h) (68)Vz/F 1787(liters) (71)

n � number of subjects included in means.a Median (range).b Harmonic mean (range).

1146 LANTZ ET AL.

standard (7.13 ppm and 8.11 ppm) are insignificant (0.02 ppm and0.04 ppm). The assignment of the structure of metabolite M9 wasconsistent with the observation that protons 2 and 3 shift downfieldwhen the glucuronide residue was attached through the oxygen atomat position 4. Based on the MS and NMR data, metabolite M9 wasconfirmed as 4-O-glucuronide-6-hydroxy duloxetine (glucuronide

conjugate of 4,6-dihydroxy duloxetine). M10 was found in plasmaand urine and produced a protonated molecular ion at m/z 520. Thecharacteristic product ions at m/z 44 and m/z 154 are observed. Theproduct ion at m/z 344 shows a loss of 176 amu from m/z 520,indicative of a glucuronide conjugate. This peak was not found inurine after hydrolysis with �-glucuronidase and was converted to

TABLE 3

Radiochromatographic HPLC peak area percentages of duloxetine and duloxetine metabolites in pooled plasma

CompoundPeak Area %

4 h 6 h 10 h 24 h

Glucuronide conjugate of 4-hydroxy duloxetine (M6) 49 42 47 56Sulfate conjugate of 5-hydroxy-6-methoxy duloxetine (M7) 31 24 21 8Glucuronide conjugate of 4, 6-dihydroxy duloxetine (M9) 5 18 14 7Glucuronide conjugate of 6-hydroxy-5-methoxy duloxetine (M10) 12 12 13 12Duloxetine 3 4 4 11

FIG. 5. HPLC-radiochemical profiles for plasma, urine, and feces.

1147METABOLISM AND EXCRETION OF DULOXETINE IN HUMANS

6-hydroxy-5-methoxy duloxetine. The comparison of the retentiontime, product ion mass spectrum, and NMR analysis of M10 with thatof a synthesized standard confirmed this metabolite as the glucuronideconjugate of 6-hydroxy-5-methoxy duloxetine.

Similar structural analyses were performed for M2, M3, M4, M8,M12 to M16, M23, and M26 and compared with synthesized stan-dards. The retention time, product ion mass spectra, and NMR spectrawere consistent with the structural assignments for these metabolites(Fig. 6). In addition, product ion mass spectra were obtained for M1and M11. Both of these metabolites were not observed in urine afterincubation with �-glucuronidase. Comparison of the retention timeand product ion mass spectra with that of the other identified metab-olites (and synthesized standards) produced the structural assignmentof these two conjugates of duloxetine.

Discussion

The biotransformation and disposition of duloxetine and its metab-olites after administration of a single enteric-coated tablet containing20.2 mg or 100.6 �Ci of 14C-labeled duloxetine were characterized inthis study. The mean total recovery of radioactivity was approxi-mately 91%. The radioactivity was excreted primarily in urine (72%),with about 19% excreted in feces. The elimination pattern was similarbetween all the subjects. Figure 6 illustrates a schematic of thebiotransformation pathways for duloxetine in humans.

Duloxetine was rapidly and extensively metabolized to form mul-tiple oxidative and conjugated metabolites. Duloxetine accounted foronly a small portion (approximately 3% for AUC and approximately9% for Cmax) of the circulating radioactivity in plasma. The peakconcentrations of total radioactivity occurred at the same time as thatfor duloxetine, with a Tmax of approximately 6 h. The late time of peakconcentrations was presumably due to the enteric coating of the

TABLE 4

Duloxetine metabolites in urine analyzed from 0 to 72 h expressed as individual and mean percentage of total dose

CompoundTotal % of Dose (0–72 h)a

Mean % of Total Dose(0–72 h, n � 4)

Subject 1 Subject 2 Subject 3 Subject 4

Glucuronide conjugate of dihydrodiol duloxetine (M1) 1.7 N.D. 0.4 0.7 0.7Dihydrodiol duloxetine (M2) 3.8 1.2 0.6 2.2 1.9Glucuronide conjugate of 5-hydroxy-6-methoxy duloxetine (M3) 2.3 1.1 2.0 1.1 1.6Glucuronide conjugate of 5-hydroxy duloxetine (M4) 0.9 1.0 0.4 N.D. 0.6Glucuronide conjugate of dihydroxy duloxetine (M5)b 2.6 1.5 2.4 2.7 2.3Glucuronide conjugate of 4-hydroxy duloxetine (M6) 13.2 21.4 17.5 15.5 16.9Sulfate conjugate of 5-hydroxy-6-methoxy duloxetine (M7) 9.7 10.5 16.2 13.5 12.5Glucuronide conjugate of 6-hydroxy duloxetine (M8) 1.6 1.7 2.6 2.2 2.0Glucuronide conjugate of 4,6-dihydroxy duloxetine (M9)b 5.7 2.5 5.1 3.7 4.3Glucuronide conjugate of 6-hydroxy-5-methoxy duloxetine (M10) 4.1 3.5 6.4 6.2 5.0Sulfate conjugate of 4-hydroxy duloxetine (M11) 4.1 3.5 6.4 6.2 5.0Sum of the mean % identified metabolites over 72 h: 52.9Mean % recovery of radioactivity excreted in urine over 72 h: 67.9

N.D., the metabolite was not detected by HPLC/radiochemical detection.a Urine samples were not analyzed past 72 h post dose due to low amounts of radioactivity. Results are based on 84% extraction efficiency.b This compound exists in different isomeric forms that show different retention times on HPLC, but have the same mass.

TABLE 5

Percentage of total dose of duloxetine and its metabolites in feces

Compound

Percentage of Dosea

Subject 172–96 h

Subject 272–96 h

Subject 348–72 h

Subject 448–72 h

Duloxetine 0.2 0.1 4.1 3.44-Hydroxy duloxetine (M14) 0.3 0.5 2.3 1.7Unknown metabolite(s) 4.6 4.9 2.3 4.7Sum 5.1 5.5 8.7 9.8Percentage of dose excreted within the 24-h time period 6.3 6.8 10.8 12.0

a Selected fecal samples with sufficient radioactivity from each subject were profiled for metabolites. The percentage of dose results were based on an extraction efficiency of 81%.

TABLE 61H NMR data comparison of M9

Site Type LY248686 4,6-Dihydroxyduloxetine

Metabolite M9: 4-O-Glucuronide-6-hydroxy

duloxetine

ppm ppm ppm

2 �CH- 7.02 6.63 6.993 �CH- 7.33 6.63 6.774 �CH- 7.48 NP NP5 �CH- 7.85 7.35 7.526 �CH- 7.54 NP NP7 �CH- 7.56 7.13 7.158 �CH- 8.31 8.11 8.159 �CH-O 5.97 5.79 5.87

11 �CH- 7.33 7.32 7.3312 �CH- 6.97 6.95 6.9613 �CH- 7.19 7.11 7.1514 -CH2- 3.30, 3.20 3.26, 3.16 3.26, 3.1615 -CH2- 2.65, 2.41 2.56, 2.37 2.58, 2.3816 -CH3 2.70 2.65 2.60

NP, no proton.

1148 LANTZ ET AL.

formulation used for duloxetine. In addition, the elimination half-lifeof total plasma radioactivity was much longer than that of duloxetine(120 h versus 10.3 h). These results suggest that the metabolism ofduloxetine may occur on first-pass after oral administration and thatradioactivity was eliminated more slowly than for duloxetine. Thelonger half-life of radioactivity could not be attributed to a singleevent (formation, distribution, or elimination) or to the elimination ofa single metabolite. Duloxetine also appears to have a large apparent

volume of distribution. The apparent plasma clearance, apparent vol-ume of distribution, and half-life for duloxetine obtained in this studyagree well with the previously published data on the pharmacokineticsof duloxetine (Sharma et al., 2000).

In plasma, the radioactivity was primarily glucuronide conjugatesof 4-hydroxy duloxetine (M6), 6-hydroxy-5-methoxy duloxetine(M10), and 4,6-dihydroxy duloxetine (M9) or a sulfate conjugate of5-hydroxy-6-methoxy duloxetine (M7). The major metabolite in

FIG. 6. A schematic of the biotransformation pathways for duloxetine in humans.

The asterisk denotes the position of the 14C label on duloxetine.

1149METABOLISM AND EXCRETION OF DULOXETINE IN HUMANS

plasma was the glucuronide conjugate of 4-hydroxy duloxetine (M6).The unconjugated forms of 4-hydroxy duloxetine (M14), 6-hydroxy-5-methoxy duloxetine (M15), and 4,6-dihydroxy duloxetine were notdetected in plasma using the radiodetection and mass spectrometryconditions used to identify the metabolites, although these compoundswould be formed prior to conjugation. Plasma samples analyzed fromother clinical studies in which duloxetine was dosed daily at 60 mgb.i.d. have shown a similar profile of circulating metabolites in plasmaand confirm that the unconjugated forms are rapidly conjugated.Therefore, if these compounds circulate in plasma, they are veryminor metabolites. The only difference between this radiolabeledstudy and the data at steady state was the presence of trace amountsof N-desmethyl duloxetine (M23) in the samples collected at steadystate. The synthesized metabolites that were identified in both plasmaand urine were tested for their affinity to human monoamine trans-porters and receptors, and they were found to be pharmacologicallyinactive (data on file).

The radioactivity was eliminated primarily in the urine within thefirst 96 h as glucuronide or sulfate conjugates of oxidative metabolitesof duloxetine. This indicated that duloxetine was well absorbed, butextensively metabolized. The majority of the radioactivity found infeces was eliminated 48 h or more postdosing, suggesting that fecalradioactivity was probably due more to biliary excretion than to poorabsorption of the compound. The profile of fecal radioactivity showedan unknown metabolite of duloxetine and 4-hydroxy duloxetine(M14) accounting for the majority of the fecal radioactivity.

All of the metabolites circulating in plasma were also detected inurine. Urine contained at least 11 different metabolites of duloxetine,with duloxetine itself being undetectable. All but one of these metab-olites were glucuronide or sulfate conjugates. The major plasmametabolites were also the major urinary metabolites. Multiple bio-transformation reactions occurred prior to elimination of the radioac-tivity. The initial biotransformation of duloxetine appears to be oxi-dation at either the 4-, 5-, or 6- position of the naphthyl ring. Thesehydroxyl compounds can then be conjugated or they can undergofurther oxidation to form a catechol intermediate or another dihy-droxy. The catechol can then undergo methylation to form a methylcatechol, which undergoes sulfation and glucuronidation. A minorpathway for 5- or 6-hydroxy duloxetine is the formation of a dihy-drodiol, which is then glucuronidated. An intermediate in the forma-tion of the dihydrodiol is an epoxide. The epoxide intermediate waschemically unstable and was short-lived since it was not detected inthe samples, nor was a cysteine conjugate of the epoxide found in thesamples. These data indicated the very rapid formation of the dihy-drodiol from the postulated epoxide intermediate.

A very minor pathway was the cleavage of duloxetine at the chiralcenter to form a thienyl alcohol and naphthol. Since the molecule waslabeled at the chiral center, once the cleavage occurred, the naphtholmoiety would no longer contain the radiolabel and would not bedetected. The thienyl alcohol (M26) and naphthol were detected inurine with the initial chromatographic/MS system at very low con-centrations but were not detectable when the chromatographic andmass spectrometry conditions were optimized for metabolite separa-tion and identification.

The clearance of duloxetine is mainly through the elimination of its

metabolites. As stated previously, approximately 3% of the radioac-tivity in plasma was unchanged duloxetine. The main route of me-tabolism for duloxetine is the oxidation of the naphthol ring followedby conjugation. In vitro studies in human liver microsomes wereperformed to determine which human cytochrome P450 enzymeswere responsible for the biotransformation of duloxetine to 4-, 5-, and6-hydroxy duloxetine. Results of the studies have shown CYP2D6 andCYP1A2 to be the primary enzymes responsible for the oxidativemetabolism at the 4-, 5-, or 6- position of the naphthyl ring (B. J. Ringet al., unpublished data). All four subjects in the study were found tobe extensive metabolizers of CYP2D6. Further evidence of the in-volvement of CYP2D6 in the metabolism of duloxetine was obtainedin a separate study in which coadministration of the potent CYP2D6inhibitor, paroxetine, caused a 1.6-fold increase in the steady-stateCmax and AUC of duloxetine (Skinner et al., 2003). Differences inmetabolism were not observed in smokers versus nonsmokers (two offour subjects smoked), although this study was too small to determinethe effects of smoking on CYP1A2 expression.

In conclusion, duloxetine, administered orally as an enteric-coated20.2-mg tablet, was shown to be safe and well tolerated by thesubjects in this study. Duloxetine was extensively metabolized tonumerous conjugative metabolites primarily excreted into the urine.Duloxetine accounted for a small percentage (�3%) of the circulatingradioactivity based on mean AUC values.

Acknowledgments. We recognize and thank the following peoplewhose work contributed to the manuscript. Steve Brooks analyzedplasma samples for duloxetine by LC/MS-MS and David Jacksonhelped with metabolite confirmation on the NMR. Milton Zmijewskiprepared cell culture incubations for metabolite identification. Wealso thank Barb Ring, Jennifer Gillespie, John Bernstein, and QiminLi for their work in determining which cytochrome P450 enzymeswere responsible for duloxetine metabolism.

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1150 LANTZ ET AL.