Drug Metab Dispo0

Excretion and Metabolism of Lersivirine (5-{[3,5-Diethyl-1-(2-hydroxyethyl)(3,5-14C2)-1H-pyrazol-4-yl]oxy}benzene-1,3-

dicarbonitrile), a Next-Generation Non-Nucleoside ReverseTranscriptase Inhibitor, after Administration of [14C]Lersivirine to

Healthy Volunteers

Manoli Vourvahis, Michelle Gleave, Angus N. R. Nedderman, Ruth Hyland, Iain Gardner,Martin Howard,1 Sarah Kempshall, Claire Collins, and Robert LaBadie

Pfizer Global Research and Development, New London, Connecticut (M.V., R.L.); and Pharmacokinetics, Dynamics andMetabolism, Pfizer Global Research and Development, Sandwich, United Kingdom (M.G., A.N.R.N., R.H., I.G., M.H., S.K., C.C.)

Received November 19, 2009; accepted February 2, 2010

ABSTRACT:

Lersivirine [UK-453,061, 5-((3,5-diethyl-1-(2-hydroxyethyl)(3,5-14C2)-1H-pyrazol-4-yl)oxy)benzene-1,3-dicarbonitrile] is a next-generation non-nucleoside reverse transcriptase inhibitor, with aunique binding interaction within the reverse transcriptase bindingpocket. Lersivirine has shown antiviral activity and is well toleratedin HIV-infected and healthy subjects. This open-label, Phase Istudy investigated the absorption, metabolism, and excretion of asingle oral 500-mg dose of [14C]lersivirine (parent drug) and char-acterized the plasma, fecal, and urinary radioactivity of lersivirineand its metabolites in four healthy male volunteers. Plasma Cmaxfor total radioactivity and unchanged lersivirine typically occurredbetween 0.5 and 3 h postdose. The majority of radioactivity wasexcreted in urine (80%) with the remainder excreted in the feces

(20%). The blood/plasma ratio of total drug-derived radioactivity[area under the plasma concentration-time profile from time zeroextrapolated to infinite time (AUCinf)] was 0.48, indicating that ra-dioactive material was distributed predominantly into plasma. Le-rsivirine was extensively metabolized, primarily by UDP glucurono-syltransferase- and cytochrome P450-dependent pathways, with22 metabolites being identified in this study. Analysis of precipi-tated plasma revealed that the lersivirine-glucuronide conjugatewas the major circulating component (45% of total radioactivity),whereas unchanged lersivirine represented 13% of total plasmaradioactivity. In vitro studies showed that UGT2B7 and CYP3A4 areresponsible for the majority of lersivirine metabolism in humans.

The AIDS epidemic has reached pandemic proportions, culminat-ing in the death of more than 2 million people in 2007 alone andresulting in 33 million people currently estimated as living withHIV/AIDS worldwide (UNAIDS/WHO, http://data.unaids.org/pub/epislides/2007/2007_epiupdate_en.pdf). The widespread usage ofhighly active antiretroviral therapy since 1996 resulted in a substantial

reduction in the mortality and morbidity of people infected with HIV(Palella et al., 1998). However, the emergence of drug-resistant vi-ruses in patients treated with highly active antiretroviral therapy,together with the increasing transmission of these viruses in newlyinfected patients, has increased the demand for further therapeuticimprovements (Cane et al., 2005; Daar and Richman, 2005). It isunfortunate that the three currently approved first-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) agents (efavirenz,nevirapine, and delavirdine) and the next-generation NNRTI etra-virine all have side effects and/or drug interactions that limit their usefor the treatment of HIV. In addition, all first-generation NNRTIs aresusceptible to rapid-resistance generation through single-point muta-tions (Turpin, 2003).

This research was sponsored by Pfizer Inc. and was conducted at CharlesRiver Clinical Services Ltd., Edinburgh, United Kingdom.

1 Current affiliation: Drug Metabolism and Pharmacokinetics, PharmaceuticalDivision, F. Hoffman-La Roche Ltd., Basel, Switzerland.

Article, publication date, and citation information can be found athttp://dmd.aspetjournals.org.

doi:10.1124/dmd.109.031252.

ABBREVIATIONS: NNRTI, non-nucleoside reverse transcriptase inhibitor; lersivirine/UK-453,061, 5-((3,5-diethyl-1-(2-hydroxyethyl)-1H-pyrazol-4-yl)oxy)benzene-1,3-dicarbonitrile; P450, cytochrome P450; ADME, absorption, distribution, metabolism, and excretion; [14C]lersivirine, 5-{[3,5-diethyl-1-(2-hydroxyethyl)(3,5-14C2)-1H-pyrazol-4-yl]oxy}benzene-1,3-dicarbonitrile; HPLC, high-performance liquid chromatography; AUCinf,area under the plasma concentration-time profile from time zero extrapolated to infinite time; MS/MS, tandem mass spectrometry; AE, adverseevent; rUGT, recombinant human UDP-glucuronosyltransferase; PF-04580552, 2-[4-(3,5-dicyanophenoxy)-3,5-diethyl-1H-pyrazol-1-yl]ethylb-D-glucopyranosiduronic acid; PF-03139905, 5-{[3-ethyl-5-(1-hydroxyethyl)-1-(2-hydroxyethyl)-1H-pyrazol-4-yl]oxy}benzene-1,3-dicarbonitrile;UK-533,713, [4-(3,5-dicyanophenoxy)-3,5-diethyl-1H-pyrazol-1-yl]acetic acid; UK-508,550, 5-{[5-ethyl-1,3-bis(2-hydroxyethyl)-1H-pyrazol-4-yl]oxy}benzene-1,3-dicarbonitrile; PF-03230716, 5-{[5-ethyl-3-(1-hydroxyethyl)-1-(2-hydroxyethyl)-1H-pyrazol-4-yl]oxy}benzene-1,3-dicarboni-trile; JNJ-10198409, 3-Fluoro-N-(6,7-dimethyoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl)phenylamine.

0090-9556/10/3805-789800$20.00DRUG METABOLISM AND DISPOSITION Vol. 38, No. 5Copyright 2010 by The American Society for Pharmacology and Experimental Therapeutics 31252/3577279DMD 38:789800, 2010 Printed in U.S.A.

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Lersivirine (formerly UK-453,061), an NNRTI with a uniquebinding interaction within the reverse transcriptase binding pocket(Phillips et al., 2007), is currently in clinical development for thetreatment of HIV-1 infection. Lersivirine has shown potent in vitroactivity against both wild-type and clinically relevant drug-resis-tant viruses (Mori et al., 2008). Furthermore, lersivirine has shownsynergy in vitro with other classes of compounds, particularlythose in the nucleoside reverse transcriptase inhibitor class. In vitrostudies suggest that lersivirine has good membrane permeability(Allan et al., 2008) and is predominantly cleared by metabolism,via glucuronidation and cytochrome P450 (P450)-mediated oxida-tion (Vourvahis et al., 2009). Furthermore, the clinical safety,tolerability, and pharmacokinetics of lersivirine have been studiedin both HIV-infected and healthy subjects (Davis et al., 2007;Fatkenheuer et al., 2009). In a 7-day monotherapy study, lersi-virine was found to be well tolerated at all the doses used, achiev-ing significant mean viral load reductions of 1.62 log10 fordosing regimens of 500 mg once daily, 750 mg once daily, and 500mg b.i.d. in HIV-infected patients (Fatkenheuer et al., 2009).

This open-label Phase I study investigated the absorption, distribu-tion, metabolism, and excretion (ADME) of a single oral 500-mg doseof [14C]lersivirine (parent drug) to characterize the plasma, fecal, andurinary radioactivity of lersivirine and its metabolites in healthymale subjects. In addition, in vitro data identifying the major drug

metabolism enzymes responsible for the metabolism of lersivirineare described.

Materials and Methods

Chemicals and Reagents. [14C]Lersivirine was synthesized by GE Health-care (Little Chalfont, Buckinghamshire, UK) at a specific activity of 3.7

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FIG. 1. MS/MS spectrum of lersivirine (A) and proposed fragmen-tation of lersivirine (B).

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FIG. 2. The mean blood and plasma concentration-time profiles for drug-derivedtotal radioactivity and lersivirine (parent drug) after a single oral administration of500 mg of [14C]lersivirine.

790 VOURVAHIS ET AL.

kBq/mg and radiochemical purity of 97.6% as assessed by high-performanceliquid chromatography (HPLC) (Alliance 2695 Separations Module with 2487Dual Absorbance Detector; Waters, Milford, MA). Authentic, nonradiolabeledlersivirine was supplied by Pfizer Global Research and Development; puritywas 99.3% as assessed by HPLC. Authentic standards of known metabolites oflersivirine [PF-04580552 (M15), PF-03139905 (M17), and UK-533,713(M19)] were synthesized at Pfizer Global Research and Development (Sand-wich, UK). Aquasafe 500 Plus liquid scintillation fluid was obtained fromZinsser Analytic (Maidenhead, UK). Carbo-Sorb CO2 absorbing solution andPermafluor E scintillation fluid, used in conjunction with the Packard Tri-Carb 307 Automatic Sample Oxidizer, were supplied by PerkinElmer Life andAnalytical Sciences (Waltham, MA). Spec-Check-14C, used to estimate com-bustion efficiency, was also from PerkinElmer Life and Analytical Sciences.Luna C18 (for radiopurity; Phenomenex, Torrance, CA) and HIRPB (for urine,feces, and plasma profiling; Hichrom Ltd., Theale, UK) columns were used forHPLC analyses. All the other commercially available chemicals and solventswere of analytical grade where available.

Clinical ADME Study Design. Study design. This study, conducted atCharles River Clinical Services Ltd. (Edinburgh, UK), was an 8-day, open-label, Phase I study of lersivirine in four healthy male subjects. All the subjectsgave written informed consent; the study was conducted in accordance with theDeclaration of Helsinki and was approved by an accredited institutional ethicscommittee. After an overnight fast, all the subjects received a single oral500-mg dose of [14C]lersivirine as a 5-ml suspension in aqueous Avicel RC591, containing a maximum of 62 Ci of [14C]; this amount of radioactivitywas chosen to comply with the International Commission on RadiologicalProtection category IIa guidelines (radioactive exposure not exceeding 1 mSv),followed by 240 ml of water. Participants remained in an upright position andrefrained from eating or drinking for 4 h postdose.

Sample collection. Blood samples (12 ml) were collected into nonbeadedlithium-heparinized tubes immediately predose and at 0.5, 1, 2, 3, 4, 6, 8, 10,12, 18, 24, 36, 48, 72, 96, 120, 144, and 168 h postdose. One milliliter of wholeblood per sample was transferred into a nonheparinized tube and stored at 4Cfor subsequent liquid scintillation counting. The remaining samples wereprocessed for plasma via centrifugation at 1500g for 10 min at 4C; sampleswere then divided into three aliquots for HPLC tandem mass spectrometry(HPLC/MS/MS) analysis of lersivirine (1 ml), liquid scintillation counting (1ml), and metabolite profiling (approximately 3 ml).

Urine samples were collected at 12-h intervals postdose until day 4 and thenat 24-h intervals until 168 h postdose. Urine was stored at 4C until the end ofthe collection period. A 10-ml aliquot of each sample was withdrawn forscintillation counting, and 2 50-ml aliquots were frozen at 20C forsubsequent metabolite profiling.

Fecal samples were collected in polypropylene containers at 24-h intervalsuntil 168 h postdose and stored at 20C. After each collection, samples werehomogenized in water. Duplicate portions of the homogenate (0.4 g each) weretaken for combustion in oxygen, and a separate 100-g homogenate aliquot wasused for metabolite profiling. Sample collection was stopped when 90% ofadministered radioactivity had been recovered in the combined urine and fecalsamples or when 1% of radioactivity was recovered in 24 h.

Determination of total radioactivity. Whole blood, plasma, urine, and fecalsamples were assayed for drug-derived total radioactivity. Liquid scintillation

TABLE 1Lersivirine pharmacokinetic parameter values (geometric means and intersubject variability)

PK Parameter (Units) Lersivirine (Plasma) Total Drug-Related Radioactivity (Plasma) Total Drug-Related Radioactivity (Whole Blood)n 4 4 4AUCinf (ng h/ml)a,b 4100 (19) 39,130 (4) 18,650 (7)Cmax (ng/ml)a,b 1010 (40) 6042 (21) 2767 (27)Tmax (h)c 1.50 (0.503.00) 1.50 (1.003.00) 1.50 (1.003.00)t1/2 (h)d 5.83 (26) 9.41 (30) 9.91 (19)CL/F (l/h)b 122 (18) 12.78 (4) 26.81 (7)Vz/F (liter)b 994 (24) 169.4 (26) 37,804 (15)

AUCinf, area under the plasma concentration-time profile from time zero extrapolated to infinite time; CL/F, apparent clearance; Cmax, maximum plasma concentration; t1/2, terminal half-life;Tmax, time to reach maximum concentration; VZ/F, apparent volume of distribution.

a Units for radioactive compound are ng-Eq h/g for AUCinf and ng-Eq/g for Cmax.b Geometric mean (percentage coefficient of variation).c Median (range).d Arithmetic mean (percentage coefficient of variation).

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FIG. 3. Excretion of total radioactivity after a single oral administration of 500 mgof [14C]lersivirine in urine (A), feces (B), and in total (C).

791EXCRETION AND METABOLISM OF LERSIVIRINE

counting was used for both urine (2300 TR; Canberra Industries, Meriden, CT)and plasma (Guardian; PerkinElmer Life and Analytical Sciences) samplesafter mixing with scintillation mixture. Whole-blood samples were first mixedwith SOLVABLE (PerkinElmer Life and Analytical Sciences), EDTA, andH2O2 before mixing with scintillation mixture and subjected to liquid scintil-lation counting (Guardian; PerkinElmer Life and Analytical Sciences). Fecalsamples were homogenized in water using a Waring (Stamford, CT) industrialblender, combusted in a biological oxidizer (model 307 MK2 Tri-Carb;PerkinElmer Life and Analytical Sciences), and the evolved 14CO2 wastrapped and measured by liquid scintillation counting in Permafluor Escintillation mixture (PerkinElmer Life and Analytical Sciences). A 1-mlaliquot of each plasma sample was analyzed by scintillation counting. Thedetection limit was 2.5 dpm above background. Samples of plasma (0.2 g)were mixed with scintillation mixture (16 ml, Starscint; PerkinElmer Lifeand Analytical Sciences) before liquid scintillation counting (Guardian;PerkinElmer Life and Analytical Sciences). Total radioactivity (percentage ofdose) was determined for urine, feces, and their combination.

Plasma assay. Plasma samples were analyzed for lersivirine concentrationsusing a validated HPLC/MS/MS method (Allan et al., 2008). The method was

linear over the range of 1 to 2000 ng/ml. The lower limit of quantification was 1ng/ml.

Metabolite profiling. Urine samples (036 h) were prepared for metaboliteprofiling by centrifugation (3500 rpm, 15 min, 4C); resultant pools (2 ml/subject) were profiled by HPLC using PU-2080 Plus pumps coupled to anHIRPB column (250 7.75 mm; Hichrom Ltd.). Separation was achievedwith a binary solvent gradient at 2 ml/min, comprising methanol and 0.1 Mammonium acetate, pH 5. The gradient consisted of 30% methanol held for 10min, increased to 50% over 1 min, held at 50% for 14 min, increased to 80%methanol over 20 min and held for 5 min, before returning to 30% methanolover 1 min, which was maintained for a further 4 min. Fractions (6.88 s) werecollected into 96-well Scintiplates (PerkinElmer Life and Analytical Sciences),which were then dried and counted on a Microbeta scintillation counter(PerkinElmer Life and Analytical Sciences). Composite plasma samples (024h time-normalized pools) (Hamilton et al., 1981; Hop et al., 1998) were treated1:4 with methanol, centrifuged, and resultant supernatants were collected,dried, and profiled by HPLC in a manner similar to that of the urine samples.Drug-related material was extracted from composite fecal homogenate samples(096 h) by mixing with methanol, followed by sonication. The mixture

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FIG. 4. Representative radiochromatograms showing lersivirinemetabolites in the plasma (A), urine (B), and feces (C) of onehealthy male subject after a single oral (500 mg) administration of[14C]lersivirine.

792 VOURVAHIS ET AL.

was centrifuged at 3500 rpm for 15 min, and the supernatant was collected.This extraction procedure was repeated with a mixture of methanol (1 ml) andTris buffer (0.1 M, pH 6, 24 ml) and a mixture of methanol (23 ml) and Trisbuffer (0.1 M, pH 9, 2 ml). The extracts were combined and reduced to drynessunder nitrogen at 37C in a Turbovap (Zymark, Hopkinton, MA), and theresidue was suspended in mobile phase for HPLC analyses. The fecal extractswere profiled using the same HPLC system as urine and plasma; radiochro-matographic peaks were isolated manually via on-line radiochemical detection(-RAM; IN/US Systems, Tampa, FL) and reduced to dryness under nitrogenat 37C in a Turbovap (Zymark).

Metabolite identification. Initial identification of drug-related metabolites iso-lated from human plasma and excreta was determined by direct-infusion MS on aSciex API 4000 Q Trap mass spectrometer (Applied Biosystems, Foster City, CA)using appropriate precursor and neutral loss experiments based on the MS/MSfragmentation of lersivirine (Fig. 1, A and B). The mass spectrometer was operatedwith a Turbo Spray source and controlled by Analyst 1.4.1 software (AppliedBiosystems). For MS/MS experiments, the collision energy was typically 40 V;declustering potential was set to 50 V; and the ion spray voltage was set to 5000eV. Additional confidence in structural assignments was obtained by HPLC/MS/MS comparison with authentic standards and by the acquisition of additionalMS3 and accurate mass data using a Thermo LTQ Orbitrap mass spectrometer(Thermo Fisher Scientific, Waltham, MA). The mass spectrometer was operated inpositive ion mode with data-dependent acquisition, typically consisting of full-scanaccurate mass data (100800 mass units) triggering MS/MS experiments from aparent ion list and subsequently MS3 experiments on the two largest ions in theMS/MS spectrum. The Fourier transform MS resolution was set at 15,000. BothMS/MS and MS3 experiments were performed with a normalized collision energy

of 40 V and an activation time of 30 ms. The HPLC/MS system consisted ofAgilent Technologies (Santa Clara, CA) 1100 binary pumps coupled to a SunfireC18 column (3.5 m, 100 2.1 mm; Waters), with a binary solvent gradient (200l/min) of formic acid (0.1% aqueous) and acetonitrile (plus 0.1% formic acid)held at 5% acetonitrile for 1 min, increased to 98% over 7 min, held for 1 min, thenreturned to 5% over 0.1 min and held for 3.9 min.

Glucuronide conjugates isolated from human urine were subjected to deconju-gation using Helix pomatia extract (Sigma-Aldrich, St. Louis, MO) to provideadditional structural information on these metabolites. Dried isolates were recon-stituted in water and incubated with an equal volume of H. pomatia extract for upto 24 h. Incubations were terminated by addition of acetonitrile, diluted with 0.1 Mammonium acetate, pH 5, and centrifuged (3500 rpm, 15 min, 4C) to enableanalysis of each sample on the same HPLC system used for urine profiling.Regions of interest were then analyzed by HPLC/MS/MS using a Thermo LTQOrbitrap MS (Thermo Fisher Scientific) as described previously.

Safety and tolerability. Blood pressure, pulse rate, ECG, and physical exams,urine drug screening, and monitoring of adverse events (AEs) were reviewed on anongoing basis throughout the study. The investigator obtained and recorded all theobserved or volunteered AEs, the severity (mild, moderate, or severe) of theevents, and the investigators opinion of the relationship to lersivirine.

Statistics. Pharmacokinetic and radioactivity parameters were calculated foreach subject by using noncompartmental analysis of lersivirine and totalradioactivity plasma and blood concentration versus time profiles. Values weresummarized using descriptive statistics.

In Vitro Preclinical Metabolism Studies. Pooled human liver micro-somes and microsomes prepared from baculovirus-infected insect cellsengineered to individually express recombinant human UDP-glucuronosyl-

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transferase (rUGT) isoforms UGT1A1, UGT1A3, UGT1A4, UGT1A6,UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15,and UGT2B17 were obtained from BD Gentest (Woburn, MA). Humanrecombinant P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4,and CYP3A5) were obtained from PanVera Corp. (Madison, WI), whereasisozymes CYP2C8 and CYP2B6 were obtained from BD Gentest.

For glucuronidation studies, lersivirine (50 M) was incubated withhuman liver microsomes (0.5 mg/ml) or each of the rUGTs (0.5 mg/ml) forup to 120 min at 37C. Incubations comprised 50 mM Tris-HCl (pH 7.4 at25C), 5 mM saccharolactone, alamethicin at 50 g/mg protein, 10 mMMgCl2, and 5 mM UDP-glucuronic acid. Microsomes and rUGTs wereactivated and incubated as described previously (Hyland et al., 2009). Thereaction was terminated by adding 3 volume ice-cold acetonitrile atspecific times throughout the duration of the incubation, centrifuged at3000 rpm for 45 min at 4C, and analyzed for lersivirine and lersivirineglucuronide on a Sciex API 3000 mass spectrometer (Applied Biosystems).For UGT, enzyme kinetic study incubations were initially conducted tooptimize incubation time and protein concentration before the kineticstudy, which was conducted over a lersivirine range of 10 to 1500 M.Rates of glucuronide formation were quantified against an authentic stan-dard and used to obtain values for Km and Vmax.

For P450 studies, lersivirine (1 M) was incubated with each of therecombinant P450 isozymes (150 pmol/ml) for 60 min. Incubations comprised50 mM phosphate buffer, pH 7.4, 5 mM MgCl2, and 1 mM NADPH, regen-erated in situ from an isocitric acid/isocitric acid dehydrogenase regeneratingsystem (Youdim et al., 2008). Reactions were terminated by the addition ofacetonitrile containing midazolam (as internal standard), followed by centrif-ugation at 3000 rpm for 45 min at 4C. Samples were then analyzed forlersivirine and two of the major oxidized products identified from the humanADME study (M19 and M17) on a Sciex API 4000 mass spectrometer(Applied Biosystems).

ResultsClinical ADME Study. Study subjects. Four healthy white male

subjects completed the study; mean age (S.D.) was 37.3 years(7.4; range, 3148 years), and mean body weight was 81.3 kg(12.8).

Pharmacokinetics. The mean plasma concentration-time profilesfor total drug-derived radioactivity and lersivirine are shown in Fig. 2,and the geometric means and intersubject variability for pharmacoki-netic parameters for lersivirine and total drug-derived radioactivity areshown in Table 1. There were appreciable differences in the Cmax andAUCinf of total drug-derived radioactivity and lersivirine, indicatingthat extensive metabolism of lersivirine had occurred. Tmax for totaldrug-derived radioactivity in plasma and blood and lersivirine inplasma occurred between 0.5 and 3 h postdose, and the mean blood/plasma ratio of total drug-derived radioactive material AUCinf was0.48.

Mass balance. The majority of radioactivity was excreted in urine,accounting for 80% of the dose, whereas 23% was recovered in feces(Fig. 3C). Mean total recovery of radioactivity (urine plus feces) was103.7%. By 120 h postdose, all the radioactivity had been excreted,with the largest recovery occurring within the first 12 h for urine andfirst 48 h for fecal samples (Fig. 3, A and B).

Metabolite profiling. HPLC profiling of precipitated plasma (024h) after time-normalized pooling, pooled urine (036 h), and pooledfecal homogenate (096 h) using radiochemical detection showedextensive metabolism for all the matrices in all the subjects (Fig. 4).Based on cochromatography, unchanged lersivirine was a major com-ponent of human plasma but only a minor component in excreta.

Metabolite identification. MS analysis of components isolated fromhuman plasma and excreta identified 22 metabolites of lersivirine(Fig. 5, A and B). The MS/MS spectrum and proposed fragmentationof lersivirine are shown in Fig. 1, A and B, with major ions at m/z 293and 267 representing loss of water and loss of C2H4O, respectively.Analysis of precipitated plasma (024 h; Table 2) revealed that theglucuronide conjugate (M15) was the major circulating component(45% of total radioactivity). The MS/MS spectrum and proposedfragmentation of M15 (m/z 487) is shown in Fig. 6, A and B, showingthe major fragment ion at m/z 311, representing the characteristic lossof the glucuronic acid moiety (176 atomic mass units). As expected,

TABLE 2Relative quantitation of [14C]lersivirine metabolites in human plasma and excreta

Metabolite m/z %Circulating Radioactivity%Dose

Urine Feces Total

mean (range) mean (range) mean (range) mean (range)M1 345 1 (N.D.3) 1 (N.D.1) N.D. (N.A.) 1 (N.D.1)M2 459 2 (23) 2 (12) 1 (N.D.1) 2 (23)M3 503 2 (12) 1 (N.A.) N.D. (N.A.) 1 (N.A.)M4 521 2 (12) 1 (N.A.) N.D. (N.A.) 1 (N.A.)M5 343 N.D. (N.A.) N.D. (N.A.) 1 (N.D.2) 1 (N.D.2)M6 503 3 (23) 2 (N.A.) N.D. (N.A.) 2 (N.A.)M7 459 1 (N.A.) 1 (N.A.) N.D. (N.A.) 1 (N.A.)M8 503 4 (35) 3 (34) N.D. (N.A.) 3 (34)M9 443 6 (47) 5 (36) 5 (37) 10 (911)M10 501 2 (N.A.) 2 (N.A.) N.D. (N.A.) 2 (N.A.)M11 503 1 (12) 2 (12) N.D. (N.A.) 2 (12)M12 341 N.D. (N.A.) N.D. (N.A.) 1 (11) 1 (11)M13 330 N.D. (N.A.) N.D. (N.A.) 4 (35) 4 (35)M14 501 N.D. (N.A.) 1 (12) N.D. (N.A.) 1 (12)M15 487 45 (4149) 54 (4966) N.D. (N.A.) 54 (4966)M16 286 N.D. (N.A.) N.D. (N.A.) 1 (N.D.2) 1 (N.D.2)M17 327 8 (610) 1 (N.A.) 1 (N.D.1) 2 (12)M18 391 N.D. (N.A.) 1 (N.A.) 1 (12) 2 (23)M19 325 3 (23) 3 (23) 6 (47) 8 (610)M20 325 1 (12) N.D. (N.A.) N.D. (N.A.) N.D. (N.A.)M21 267 1 (N.D.1) N.D. (N.A.) 1 (12) 1 (12)M22 283 N.D. (N.A.) N.D. (N.A.) 1 (N.D.2) 1 (N.D.2)Lersivirine 311 13 (1016) 1 (N.A.) 1 (N.D.1) 1 (N.D.1)Total 92 (9092) 77 (6989) 22 (1427) 99 (96103)

N.A., not available; N.D., not detected. Data from all the subjects were included in the calculation of the mean, N.D. being equal to zero for the calculation. Where there is no range stated itis because there was no variability between subjects.

794 VOURVAHIS ET AL.

the MS3 spectrum of m/z 311 is essentially identical to the MS/MSspectrum of lersivirine. A product of mono-oxidation of one of theethyl side chains (M17) accounted for 8% of the total radioactivity,whereas unchanged lersivirine accounted for 13%. The MS/MS spec-trum and proposed fragmentation of M17 (Fig. 7, A and B) showed amajor ion at m/z 309, consistent with loss of water. The MS3 spectrumindicated a further loss of water to yield m/z 291 and a loss of C2H4Oto give m/z 265. Nine additional products of glucuronidation weredetected; metabolites M3, M6, M8, and M11 were derived by glucu-ronidation in conjunction with monohydroxylation, and each ac-counted for between 1 and 4% of the total radioactivity; metabolitesM2 and M7 were produced by glucuronidation in conjunction withN-dealkylation and mono-oxidation, both accounting for 3% of cir-culating radioactivity; M9 was a product of N-dealkylation and glu-curonidation (6%); M4 was a product of oxidation, glucuronidation,and hydrolysis of one of the nitrile moieties to form an amide (2%);whereas M10 formation was postulated to involve oxidation to forma ketone on one of the ethyl side chains and glucuronidation (2%).

Additional characterization of four of the glucuronic acid conju-gates was achieved by enzymatic hydrolysis with -glucuronidasefollowed by HPLC/MS/MS analysis of the deconjugated analog.Figure 8, A and B, shows the MS/MS spectrum and proposed frag-mentation of M3, whereas Fig. 8C shows the radiochromatogram forM3 before and after hydrolysis together with chromatographic com-parison with the mono-oxidized authentic standard UK-508,550. Theapproach resulted in identification of M3 as a product of oxidation onthe terminal carbon of one of the ethyl side chains followed byglucuronidation. The same approach was used to provide furtherstructural information for M6, M8, and M11, which shows that M6was a glucuronic acid conjugate of the hydroxylated metabolite PF-03230716, whereas M8 and M11 were both glucuronides of thehydroxy metabolite M17, involving conjugation on both alcohol moi-eties. The MS/MS data for the glucuronic acid metabolite M9 showedthe characteristic loss of 176 mass units to yield a major fragment ionat m/z 267 (Fig. 9, A and B). The specific site of glucuronidation forM9 was not determined but is postulated as a product of glucuronida-

A

B

NN

O

O

N

N

O

OHHO

HO

OH

O311

293

267

150 200 250 300 350 400 450 500m/z

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Rel

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311.19

312.21 32.96423.392 51.15492.762 33.35350.951141.03 399.17 411.49210.24 235.13

Rel

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ance

(%)

80 100 120 140 160 180 200 220 240 260 280 300 320m/z

0

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100 293.21

267.26

137.19124.22

150.23 265.32109.18 294.16278.30239.28153.23 42.76122.59 210.23 02.11361.38180.26

FIG. 6. MS/MS and MS3 spectrum of M15 (A) and proposedfragmentation of M15 (B).

795EXCRETION AND METABOLISM OF LERSIVIRINE

tion of the pyrazole ring. Further low-level drug-related products ofN-dealkylation, hydroxyethyl side chain oxidation, glucuronidation,nitrile group hydrolysis, and ethyl side chain oxidation were alsodetected in the plasma (Fig. 4A).

Profiling of human urine and extracted fecal homogenates alsorevealed similar and extensive metabolism in all four subjects (Fig. 4,A and B). The major components were the glucuronide of lersivirine,M15, accounting for 54% of the dosed radioactivity; a metaboliteinvolving N-dealkylation and glucuronidation (M9, 10%); and M19, aproduct of oxidation of the hydroxyethyl moiety to a carboxylic acid(8%). The MS/MS data for the carboxylic acid metabolite (M19) areshown in Fig. 10, A and B, with the major ion at m/z 279 representingloss of CH2O2. Sulfation of the parent compound and nitrile grouphydrolysis were also detected in excreta, whereas unchanged lersi-virine accounted for 1% of the dose in both urine and feces.

Safety. There were no serious AEs and no discontinuations as aresult of AEs after administration of radioactive lersivirine (ADMEstudy). The only AE reported during the ADME study was mildheadache, which was not considered treatment-related and resolvedduring the course of the study.

In Vitro Preclinical Metabolism Studies. Of the UGTs investi-gated only rUGT2B7 was able to metabolize lersivirine. Furtherinvestigations through enzyme kinetic experiments performed in hu-man liver microsomes and rUGT2B7 showed that formation of gluc-uronide was linear with time up to 60 min and protein up to 1 mg/ml.Kinetic studies were performed at 0.5 mg/ml for 20 min in bothrUGT2B7 and human liver microsomes over a substrate concentrationrange of 10 to 1500 M (Fig. 11).

Data analysis indicated that in human liver microsomes glucu-ronidation exhibited typical Michaelis-Menten kinetics, characterizedby a Km of 224.7 56.7 M and a Vmax of 1583.3 468.0pmol/min/mg protein. Enzyme kinetics in rUGT2B7 also exhibitedMichaelis-Menten kinetics with a Km of 120.2 31.8 M and a Vmaxof 725.1 64.8 pmol/min/mg protein.

In incubations with recombinant P450 enzymes, CYP3A4 metab-olized lersivirine most rapidly with an intrinsic clearance of 0.9l/pmol P450/min. The only other enzyme shown to metabolizelersivirine based on a substrate depletion approach was CYP3A5, witha greater than 10-fold lower rate of metabolism (0.08 l/pmolP450/min). The in vitro clearance in rCYP3A4 was scaled to activity

A

B

m/z

Rel

ativ

e ab

und

ance

(%)

80 100 120 140 160 180 200 220 240 260 280 300 320 340

309.18

310.27283.24 291.25265.10166.29 32.15204.62292.141 181.17123.280

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Rel

ativ

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(%)

m/z

291.19

265.20

148.18122.17 263.19166.16107.12 236.17 276.17 294.15208.15181.1595.12 309.07

80 100 120 140 160 180 200 220 240 260 280 300 3200

10

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40

50

60

70

80

90

100

NN

O

HO

N

N

HO309

291

265

-C2H4O

FIG. 7. MS/MS and MS3 spectrum of M17 (A) and proposed frag-mentation of M17 (B).

796 VOURVAHIS ET AL.

150 200 250 300 350 400 450 500

327.14

328.14309.17 485.35381.36369.22291.09 12.76459.80402.262 444.39198.98 243.47161.16 514.02

80 100 120 140 160 180 200 220 240 260 280 300 320 340

309.17

310.28291.23265.10153.20 62.11352.63231.221 165.94 219.26209.87100.23

O

NN

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OH

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OHHO

HO

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327 309-H2O

m/z

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M3

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mins

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500.0

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800.0

Co

unts

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10.012.014.016.018.020.022.024.026.028.030.032.034.0

Co

unts

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ute

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mm:ss

0.0100.0200.0300.0400.0500.0600.0700.0800.0900.0

1000.01100.0

mV

C

FIG. 8. MS/MS and MS3 spectrum of M3 (A), proposed fragmentation of M3 (B), and radiochromatogram for M3 before and after hydrolysis and comparison withUK-508,550 (C).

A

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80 100 120 140 160 180 200 220 240 260 280 300 320 340m/z

020406080

100

Rel

ativ

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ance

(%) 309.17

310.20291.15265.19153.21136.16122.06 236.24225.93209.66109.35 186.47166.08 320.6295.18

O

NN

HO

OH

NN

309

FIG. 9. MS/MS and MS3 spectrum of M9 (A) and proposed frag-mentation of M9 (B).

797EXCRETION AND METABOLISM OF LERSIVIRINE

in human liver microsomes using a relative activity factor of 0.28(determined in-house) and a CYP3A4 abundance of 120 pmolP450/mg microsomal protein. The scaled recombinant P450 clearanceof 8.55 l/min/mg liver microsomal protein compared well with thehuman liver microsomal clearance of 8.23 l/min/mg. In addition,CYP3A4 was shown to be capable of forming the oxidative metabo-lites seen in vivo (M19 and M17).

DiscussionIn this human ADME study, recovery of the oral [14C]lersivirine

dose was complete (103.7%) and occurred within 5 days after inges-tion in healthy volunteers. The high urinary excretion of radioactivity(80.4%) is evidence that lersivirine is well absorbed after oral dosingin humans. After absorption lersivirine was extensively metabolized,with unchanged lersivirine constituting 13% of total plasma radioac-tivity (AUCinf) and 1% of excreted radioactivity.

The blood/plasma ratio of total drug-derived radioactivity AUCinf was0.48, indicating limited distribution of radiolabeled material to red bloodcells. This result likely reflects limited penetration of the polar lersivirine

glucuronide (the major circulating metabolite in plasma) into red bloodcells because in vitro studies with [14C]lersivirine show a higher blood/plasma ratio of 0.77 (Pfizer Inc., data on file).

Characterization of the radioactivity in plasma and excreta ofhumans dosed with [14C]lersivirine showed that at least 22 differentmetabolites were present and indicated that glucuronidation is theprimary metabolic pathway of lersivirine. In both plasma and excretathe major metabolite was a glucuronide conjugate of lersivirine(M15). In vitro studies showed that of the isozymes studied onlyUGT2B7 was capable of forming lersivirine glucuronide. Further-more, 9 of the 22 metabolites identified in both plasma and excretawere products of glucuronidation.

The renal clearance of M15 was 2 ml/min/kg. Although the plasmaprotein binding of M15 has not been determined, given the greaterpolarity of M15 compared with lersivirine and that the plasma proteinbinding of lersivirine is itself low to moderate (fraction unbound 0.424), M15 would be expected to have minimal plasma proteinbinding. In this case, the unbound renal clearance of M15 would beessentially equivalent to glomerular filtration rate.

A

B

150 200 250 300 350 400 450

267.11

425.13309.12159.02141.05 407.25268.12239.20226.82 382.27 434.15340.42 353.30176.66

60 80 100 120 140 160 180 200 220 240 260 280

124.10

123.09239.11

109.02

212.11 238.37183.9096.08 195.14 250.18156.04 167.0680.54141.18 267.25

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OH

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2392H4

0

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m/z

FIG. 10. MS/MS spectrum of M19 (A) and proposed fragmentationof M19 (B).

798 VOURVAHIS ET AL.

Although M15 is the major circulating metabolite in humans, itdoes not circulate in the plasma of rat, mouse, or dog, but it is formedin the rat and excreted into bile. This glucuronide is significantly lesspotent than the parent compound (see later in Discussion) and doesnot contribute to the activity of lersivirine in vivo. As an unreactive,inactive ether glucuronide M15 does not raise any particular safetyconcerns, and in fact this type of glucuronide metabolite is specificallyexempted in the Food and Drug Administration Human Metabolites inSafety Testing guidance (http://www.fda.gov/downloads/Drugs/Guid-anceComplianceRegulatoryInformation/Guidances/ucm079266.pdf).

The formation of a pyrazole N-glucuronide (M9) has previouslybeen identified for the investigative anticancer agent JNJ-10198409 inhuman, rat, and monkey liver microsomes (Yan et al., 2006). M9 wasresistant to hydrolysis with -glucuronidase, as was observed for themajor pyrazole N-glucuronide of JNJ-10198409 (Yan et al., 2006).

A number of oxidative metabolites of lersivirine were identified, ofwhich metabolites with oxidation of the alcohol to a carboxylic acidgroup (M19) and a hydroxylated metabolite (M17) were the mostabundant. In vitro, CYP3A4 was found to be the predominant P450isozyme tested that metabolized lersivirine. In addition, CYP3A4 wasshown to be capable of forming the major oxidative metabolitesformed in vivo (M19 and M17). Although these data are consistentwith previous observations that CYP3A4 is the primary contributor tooxidative metabolism of lersivirine, in a previous study, using slightlydifferent buffers and reagents, other P450 enzymes were able tometabolize lersivirine (Allan et al., 2008). This result most likelyreflects the different source of the P450 isozymes used in the twostudies and the inherent metabolic capabilities of the two systems.

CYP3A5*1, a polymorphic-expressed enzyme in which the allelicfrequency of wild-type is only 10 to 15% in the white population(increasing to approximately 50% in blacks) (Daly, 2006), was alsoassessed in this study. Although it is not currently possible to make aquantitative extrapolation from recombinant CYP3A5 data because ofthe lack of an appropriate CYP3A5 probe substrate for the generationof relative activity factors, the low rate of metabolism of lersivirine byCYP3A5 compared with CYP3A4 suggests that no dosage adjustmentwill be needed in populations in which the expression of this poly-morphic enzyme is high.

Compared with the parent compound (IC50, 2 nM), the metabolitesM17 (138 nM), M15 (5 M), and M19 (15.8 M) are significantlyless potent against the laboratory-adapted HIV-1 strain NL 4-3. Infact, the potency of either glucuronide could be accounted for by 0.1%parent in the glucuronide standard or by a small amount of degrada-tion of M15 to parent in the culture. When the average for each of themetabolites is compared with the IC50 of these four components, theratio is 325, 3, 0.5, 0.01 for lersivirine, M17, M15, and M19, respec-tively, suggesting that the antiviral activity is mainly associated withthe lersivirine parent molecule.

In addition to glucuronidation and oxidative metabolism, metabolicpathways of lersivirine also involve sulfation and nitrile hydrolysis. Itis well recognized that the extent of a drug-drug interaction as a resultof enzyme inhibition will depend on the fraction of the substratemetabolized by that enzyme (Brown et al., 2005; Ito et al., 2005). Thebalanced clearance of lersivirine is expected to reduce the interindi-vidual variability and drug-drug interaction liability of the compoundby reducing the reliance on a single enzyme to clear the compound.Furthermore, the predominant clearance pathway in humans in vivo isUGT2B7-mediated glucuronidation. In general, glucuronidation is ahigh-capacity low-affinity metabolic pathway, and drug-drug interac-tions mediated through UGTs are known to be small in magnitude(Williams et al., 2004; Kiang et al., 2005). Coadministration withvalproic acid, a potent inhibitor of UGT2B7, results in increasedexposure of a number of compounds that undergo glucuronidation viaUGT2B7, including zidovudine and lamotrigine; however, the mag-nitude of the changes in exposure is low [1.8-fold (Lertora et al.,1994) and 2.6-fold (Morris et al., 2000), respectively]. It is notexpected that the plasma levels of lersivirine will increase dramati-cally on coadministration with medications that inhibit UGT metab-olism because only a 1.3-fold increase in lersivirine AUC was ob-served after coadministration with valproic acid (Langdon et al.,2008).

ConclusionIn conclusion, this open-label Phase I study has shown that lersi-

virine is almost completely absorbed, extensively metabolized by bothUGT- and P450-dependent pathways, and radiolabeled material iseliminated entirely by renal and fecal routes. The major humancirculating metabolites examined thus far do not appear to contributeto the antiviral activity of lersivirine.

Acknowledgments. We thank Drs. Kristin Larsen and ClemenceHindley of Complete Medical Communications for editorial support(funded by Pfizer Inc.).

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00 2 4

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tein

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A

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799EXCRETION AND METABOLISM OF LERSIVIRINE

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Address correspondence to:Manoli Vourvahis, Pfizer Global R&D, 50 PequotAvenue, New London, CT 06320. E-mail: [email protected]

800 VOURVAHIS ET AL.