1521-009X/44/2/209–219$25.00 http://dx.doi.org/10.1124/dmd.115.067868DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:209–219, February 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Pharmacokinetics and Disposition of the Thiouracil DerivativePF-06282999, an Orally Bioavailable, Irreversible Inactivator of

Myeloperoxidase Enzyme, Across Animals and Humans

Jennifer Q. Dong, Manthena V. Varma, Angela Wolford, Tim Ryder, Li Di, Bo Feng,Steven G. Terra, Kazuko Sagawa, and Amit S. Kalgutkar

Pfizer Inc., Cambridge, Massachusetts (J.Q.D., A.S.K.); and Pfizer Inc., Groton, Connecticut (M.V.V., A.W., T.R., L.D., B.F., S.G.T., K.S.)

Received October 14, 2015; accepted November 23, 2015

ABSTRACT

The thiouracil derivative PF-06282999 [2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide] is an irrevers-ible inactivator of myeloperoxidase and is currently in clinical trialsfor the potential treatment of cardiovascular diseases. Concernsover idiosyncratic toxicity arising from bioactivation of the thiouracilmotif to reactive species in the liver have been largely mitigatedthrough the physicochemical (molecular weight, lipophilicity, andtopological polar surface area) characteristics of PF-06282999,which generally favor elimination via nonmetabolic routes. To testthis hypothesis, pharmacokinetics and disposition studies wereinitiated with PF-06282999 using animals and in vitro assays, withthe ultimate goal of predicting human pharmacokinetics and elim-ination mechanisms. Consistent with its physicochemical proper-ties, PF-06282999 was resistant to metabolic turnover from livermicrosomes and hepatocytes from animals and humans andwas devoid of cytochrome P450 inhibition. In vitro transport

studies suggested moderate intestinal permeability and minimaltransporter-mediated hepatobiliary disposition. PF-06282999 dem-onstratedmoderate plasmaprotein binding across all of the species.Pharmacokinetics in preclinical species characterized by low to mod-erate plasma clearances, good oral bioavailability at 3- to 5-mg/kgdoses, and renal clearance as the projected major clearancemechanism in humans. Human pharmacokinetic predictions usingsingle-species scaling of dog and/or monkey pharmacokineticswere consistent with the parameters observed in the first-in-human study, conducted in healthy volunteers at a dose range of20–200 mg PF-06282999. In summary, disposition characteristics ofPF-06282999 were relatively similar across preclinical species andhumans, with renal excretion of the unchanged parent emerging asthe principal clearance mechanism in humans, which was antici-pated based on its physicochemical properties and supported bypreclinical studies.

Introduction

The hemoprotein myeloperoxidase (MPO; EC 1.11.1.7), produced inthe bone marrow, is one of the major neutrophil bactericidal proteins.MPO has recently emerged as a critical modulator of inflammatoryconditions including rheumatoid arthritis (Odobasic et al., 2014),chronic obstructive pulmonary disease (Churg et al., 2012), acutekidney injury (Odobasic et al., 2007), Parkinson’s disease (Choi et al.,

2005), and cardiovascular disease (Mayyas et al., 2014). Highconcentrations of MPO have been detected in atheroscleroticplaques (Hazell et al., 1996) and elevated MPO levels are associatedwith risk of cardiovascular disease in otherwise healthy individuals(Meuwese et al., 2007). Studies in MPO knockout mice havedemonstrated protection from cardiac remodeling and impaired leftventricular function after surgically induced myocardial infarction(Penn, 2008).The identification ofMPO as a central player in inflammation renders

this enzyme as a potential target for therapeutic interventions. Ourlaboratory recently described the design, synthesis, and preclinicalcharacterization of some novel N1-substituted-6-aryl-2-thiouracil ana-logs as mechanism-based inactivators of MPO, which culminated inthe discovery of the clinical candidate PF-06282999 [2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)

This research was sponsored by Pfizer. At the time of data generation, allauthors were employees and stockholders of Pfizer Inc. Pfizer Inc. providedsupport in the form of salaries for the authors but did not have any additional rolein the study design, data collection and analysis, decision to publish, orpreparation of the manuscript.

dx.doi.org/10.1124/dmd.115.067868.

ABBREVIATIONS: AUC, area under plasma concentration-time curve; CLb, blood clearance; CLp, plasma clearance; CLrenal, renal clearance;CLrenal,u, unbound renal clearance; CL/F, apparent oral clearance; CP-628374, [(2E)-3-(4-{[(2S,3S,4S,5R)-5-{(1E)-N-[(3-chloro-2,6-difluorobenzyl)oxy]ethanimidoyl}-3,4-dihydroxytetrahydrofuran-2-yl]oxy}-3-hydroxyphenyl)-2-methyl-N-[(3aS,4R,5R,6S,7R,7aR)-4,6,7-trihydroxyhexahydro-1,3-benzodioxol-5-yl]prop-2-enamide; F, oral bioavailability; GFR, glomerular filtration rate; HBSS, Hanks’ balanced salt solution; HEK, humanembryonic kidney; hOAT, human organic anion transporter; hOCT, human organic cation transporter; HPLC, high-performance liquidchromatography; KBF, kidney blood flow; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; m/z, mass-to-chargeratio; MDCKII-LE, low-efflux Madin-Darby canine kidney; MDR1, multidrug-resistant gene 1; MPO, myeloperoxidase; P450, cytochrome P450;Papp, apparent permeability; Papp,AB, apical-to-basolateral permeability; PF-06282999, 2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide; PF-06428486, 2-(6-(5-chloro-2-methoxyphenyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide;SCHH, sandwich culture human hepatocyte; t1/2, half-life; Tmax, time of first occurrence of Cmax; tR, retention time; Vdss, apparent steady-state distribution volume; Vz/F, apparent volume of distribution.

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acetamide] (Fig. 1) (Ruggeri et al., 2015). PF-06282999 is a suicidesubstrate of MPO with a high degree of selectivity relative to otherperoxidases such as thyroid peroxidase. The inhibitory mechanismlikely involves oxidation of the thiouracil sulfur atom in PF-06282999by the compound I intermediate of oxidized MPO to yield a thiylradical, which covalently attaches to the heme moiety, causing enzymeinactivation (Ruggeri et al., 2015). The mode of inhibition is similar tothe one described with the recently reported 2-thioxanthine class ofMPO inactivators (e.g., compound TX5 in Fig. 1) (Tidén et al., 2011;Geoghegan et al., 2012).From a drug design perspective, thiouracils and related analogs

(e.g., thioureas and thioamides) are structural alerts, which can bemetabolically activated by MPO, cytochrome P450 (P450 en-zymes), and flavin monooxygenases to electrophilic sulfenic acidand sulfonate intermediates, which are capable of covalentlymodifying proteins and eliciting a toxicological outcome (Hanzlikand Cashman, 1983; Henderson et al., 2004). For instance, immune-mediated hepatotoxicity and agranulocytosis associated with theclinical use of the antithyroid agent propylthiouracil has been causallylinked to the oxidative bioactivation of thiouracil functionality byMPO to yield protein-reactive propylthiouracil-2-sulfenic acid andpropylthiouracil-2-sulfonate metabolites, which induce the formationof the antineutrophil antibodies in patients with propylthiouracil-induced agranulocytosis (Lee et al., 1988; Waldhauser and Uetrecht,1991). The risk of wayward electrophilic species arising from MPO-catalyzed oxidation of PF-06282999 was principally mitigated on thebasis of its low partition ratio (approximately 7.0) forMPO inactivation,which is in contrast with the very high partition ratio of 75 obtained withpropylthiouracil (Ruggeri et al., 2015). Correspondingly, no glutathi-one conjugates of PF-06282999 were detected in incubations withrecombinant human MPO/hydrogen peroxide incubations (Ruggeriet al., 2015). Likewise, reductions in lipophilicity and molecular weightin the thiouracil class of MPO inhibitors ensured that the compoundswere latent toward oxidative metabolism (including bioactivation) inthe liver (Ruggeri et al., 2015).

A thorough understanding of major clearance pathways and accurateprediction of human pharmacokinetics of drug candidates is a criticalendeavor in drug discovery, given the intrinsic links with clinicalpharmacology and/or safety, which continue to dominate attrition ratesin drug development (Kola and Landis, 2004; Hay et al., 2014).Consequently, in preparation for preclinical toxicological and first-in-human clinical studies for assessment of safety, tolerability, andpharmacokinetics, the disposition of PF-06282999 was examined inanimals and in vitro human models. This work summarizes thepreclinical absorption, distribution, metabolism, and excretion profileof PF-06282999. Results from the preclinical studies were used topredict human pharmacokinetics including the identification of antic-ipated clearance pathways. The collective findings are discussed againstthe backdrop of the single-dose pharmacokinetics and major elimina-tion mechanism of PF-06282999 in healthy human volunteers after oral(p.o.) administration.

Materials and Methods

Chemicals and Materials. PF-06282999 [chemical purity . 99% by high-performance liquid chromatography (HPLC) nuclear magnetic resonance]and internal standard CP-628374 ([(2E)-3-(4-{[(2S,3S,4S,5R)-5-{(1E)-N-[(3-chloro-2,6-difluorobenzyl)oxy]ethanimidoyl}-3,4-dihydroxytetrahydrofuran-2-yl]oxy}-3-hydroxyphenyl)-2-methyl-N-[(3aS,4R,5R,6S,7R,7aR)-4,6,7-trihydroxyhexahydro-1,3-benzodioxol-5-yl]prop-2-enamide) were synthesizedat Pfizer Worldwide Research and Development (Groton, CT). Commerciallyobtained chemicals and solvents were of HPLC or analytical grade.

Plasma Protein Binding. The extent of binding of PF-06282999 to rat (fivemales/five females), mouse (30males/30 females), dog (fivemales/five females),monkey (five males/five females), and human (at least three males and threefemales) plasma proteins was determined using a disposable rapid equilibriumdialysis device with prepacked inserts (ThermoScientific, West Palm Beach,FL). Plasma samples were prepared by mixing PF-06282999 with plasma toproduce a final incubation concentration of 2 mM for PF-06282999. Aliquots(220 ml; n = 4) of spiked plasma were added to the donor wells, followed by theaddition of phosphate-buffered saline (350 ml; n = 4) to the receiver wells. Thedevice was covered with a gas permeable membrane and agitated on an orbitalshaker at approximately 450 rpm within a humidified incubator (75% relativehumidity, 5% CO2) operating at 37�C for 4 hours. Control plasma samples (t = 0,15 ml, n = 2) were prepared from the remaining spiked plasma solution. Eachsample was matrix matched with buffer (45 ml) and plasma (15 ml). The sampleswere precipitated with a 120-ml acetonitrile solution containing 100 ng/ml CP-628374 as the internal standard. All samples for analysis were prepared in a96-well plate, centrifuged (1900 � g, 5 minutes, room temperature), and thesupernatants were transferred and analyzed by liquid chromatography coupled totandemmass spectrometry (LC-MS/MS). The fraction unbound of PF-06282999to plasma proteins was calculated as the ratio of concentration of analyte in bufferover concentration of analyte in plasma.

Blood Cell Partitioning. Blood to plasma partitioning of PF-06282999 wasdetermined in fresh rat, mouse, dog, monkey, and human whole blood andplasma to which PF-06282999 was added to yield a final concentration of 1 mM(n = 3). Samples were incubated on an orbital shaker in a humidified CO2

incubator for 60 minutes at 37�C. After incubation, whole blood samples wereeither centrifuged to obtain plasma or matrix matched with appropriate blankplasma. Aliquots (20ml) of plasma sample incubations and either plasma isolatedfrom whole blood or matrix-matched whole blood were mixed with acetonitrile(200 ml) containing CP-628374 (100 ng/ml, internal standard) and centrifuged.Supernatants were analyzed using LC-MS/MS. A blood/plasma partitioning ratiofor PF-06282999 was calculated from the concentrations in blood and plasma.

In Vitro Permeability Studies. Apparent permeability (Papp) of PF-06282999was examined in Caco-2 and low-efflux Madin-Darby canine kidney (MDCKII-LE)cell lines in triplicate using previously published protocols (Frederick et al., 2009;Di et al., 2011; Varma et al., 2011). MDCKII-LE cells were established in houseand the cell culture and experimental procedures were discussed in detailpreviously (Di et al., 2011). Caco-2 cells, obtained from the American TypeCulture Collection (Manassas, VA), were seeded in 24-well Falcon multiwellplates (polyethylene terephthalate membranes, pore size 1mm) (Thomas Scientific,

Fig. 1. Structure of PF-06282999 and mechanism of MPO inactivation by 2-thioxanthines.

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Swedesboro, NJ) at 4 � 104 cells/well. Caco-2 absorptive permeability wasassessed at apical/basal pH of 6/7.4 and 7.4/7.4. The effect of calcium-free mediumand efflux inhibitor cocktail, cyclosporin A (7 mM) and verapamil (40 mM), onabsorptive permeability (pH 7.4/7.4) of PF-06282999 (1 mM) was alsoevaluated. Permeability of nadolol, a low permeability marker, was measured toassess the cell monolayer integrity.

Human MDCKII-MDR1 Efflux Assay. MDCKII–multidrug-resistant gene1 (MDR1) cells were obtained from Dr. Piet Borst (Netherland Cancer Institute,Amsterdam, The Netherlands). The cells were seeded at a cell density of 2.5 �105 cells/ml in complete minimum Eagle’s medium/a medium on Falcon/BD96-well membrane inserts and incubated (37�C, 95% humidity, 5% CO2) for 5days. A transwell assay analogous to the one previously described (Feng et al.,2008a) was used. For apical-to-basolateral permeability (Papp,AB), medium wasremoved from the insert on day 5. Seventy-five microliters of fresh Hanks’balanced salt solution (HBSS) with added components (Life Technologies, GrandIsland, NY) containing PF-06282999 (2 mM) was added on the apical side and250ml fresh buffer was added on the basolateral side. The plate was then incubatedat 37�C for 2 hours. For basolateral-to- apical permeability, medium from the insertwas replaced with 75 ml fresh buffer and 250 ml fresh buffer containing PF-06282999 (2 mM) was added on the basolateral side, followed by incubation for 2hours at 37�C. All studies with PF-06282999 were conducted in triplicate.

The following equations were used to calculate Papp values and the MDR1efflux ratio:

Papp ¼ 1area •CDð0Þ •

dMr

dt

Efflux  ratio ¼ Papp;BAPapp;AB

where area is the surface area of the transwell insert, CD(0) is the initialconcentration of compound applied to the donor chamber, t is incubation time,Mr is the mass of the compound in the receiver compartment, dMr/dt is the flux ofthe compound across the cell monolayer, and Papp,BA is the basolateral-to-apicalpermeability.

Liver Microsomal Stability. Pooled male Wistar-Han rat, male CD-1mouse, male beagle dog, male cynomolgus monkey, and male and femalehuman liver microsomes (pool of 50 livers) were purchased from BD Gentest(Woburn, MA). Half-life (t1/2) assessments in liver microsomes was determinedin duplicate after incubation of PF-06282999 (1 mM) with liver microsomes(P450 concentration = 0.25 mM) in 0.1 M potassium phosphate buffer (pH 7.4)containing 1 mMMgCl2 at 37�C. The reaction mixture was prewarmed at 37�Cfor 2 minutes before adding NADPH (1.3 mM). Aliquots of the reaction mixtureat 0, 5, 10, 15, 30, 45, and 60 minutes were added to acetonitrile containing aninternal standard (100 ng/ml CP-628374), and the samples were centrifugedbefore LC-MS/MS analysis of substrate disappearance. For control experiments,NADPH and/or liver microsomes were omitted from these incubations. For thepurposes of metabolite identification studies, the concentration of PF-06282999in the microsomal incubations was raised to 10 mM and the microsomal proteinconcentration increased to 1 mg/ml. The duration of the incubation time was45 minutes. Separate incubations of PF-06282999 (10 mM) were also conductedin human liver microsomes containing NADPH (1.3 mM) and glutathione(1 mM) for the purposes of trapping reactive species arising from the oxidativemetabolism of PF-06282999.

Stability in Cryopreserved Hepatocytes. Cryopreserved Wistar-Han rat(male, pool of 12 donors), CD-1 mouse (male, pool of 12 donors), beagle dog(male, pool of five donors), cynomolgus monkey (male, pool of five donors), andhuman (pool of 10 donors of mixed sex) hepatocytes were purchased from Celsis(Chicago, IL). Williams’ E media was prepared by adding 26 mM sodiumbicarbonate and 50 mM HEPES, followed by 0.2-mm filtration and then30 minutes of CO2/O2 bubbling at 37�C. This medium was used for thawingsuspension of hepatocytes. Incubations were conducted in a 96-well flat-bottompolystyrene plate in duplicate. Hepatocytes were suspended at 0.5 � 106 viablecells/ml of Williams’ E media and prewarmed at 37�C for 30 minutes.Incubations were initiated with the addition of PF-06282999 (final concentration= 1 mM) and were conducted at 37�C, 75% relative humidity, and 5% CO2. Thereaction was stopped at 0, 3, 15, 30, 60, 90, or 120 minutes by the addition ofacetonitrile (135 ml) containing an internal standard. The samples werecentrifuged at approximately 1900 � g for 10 minutes before LC-MS/MS

analysis for the disappearance of PF-06282999. For the purposes of qualitativemetabolite identification studies, the concentration of PF-06282999 in thehepatocyte incubations was increased to 10 mM.

For examination of PF-06282999 depletion in cryopreserved human hepato-cytes via the relay method (Di et al., 2013), the hepatocyte plate was incubated at37�C with 95% O2/5% CO2 and 75% relative humidity for 4 hours daily for5 days. Aliquots (25 ml) of the total suspension were sampled at 0, 4, 8, 12, 16,and 20 hours and the rest of the hepatocyte incubation plate was centrifuged(approximately 1900 � g, 10 minutes, room temperature) at the end of eachrelay. Aliquots (50 ml) of supernatant were sampled for quantification ofconcentration and 300ml supernatant was transferred to a clean 24-well plate andstored at 280�C until the next relay experiment. For the second relayexperiment, the supernatant plate was warmed to 37�C for 30 minutes andhepatocytes were added to the samples to give a final cell density of 0.5 �106 cells/ml. The plates were incubated at 37�C for 4 hours, sampled, andprocessed as described above. All of the samples were quenched with 2� volumeof acetonitrile containing an internal standard, centrifuged, and transferred for LC-MS/MS analysis. Five relays were performed to give a total incubation time of20 hours, and t1/2 was calculated as described in the microsomal stability section.The following equations were used to calculate apparent intrinsic clearance, scaledintrinsic clearance, and hepatic blood clearance (Di et al., 2012):

Hepatocyte CLint;app ¼ ln 2 •1

t1=2 ðminÞ •ml incubation0:5 M cells

•1000 ml

ml

¼ ml=min per million cells

Hepatocyte CLint;scaled ¼ ln 2 •1

t1=2 ðminÞ •ml incubation

0:5 million cells

•million cellsg liver

•g liverkg

Well-stirred model CLb ¼Q • ðfup=RbÞ •CLu;int

Qþ ðfup=Rb=fumicÞ •CLu;int

where CLint,app is apparent intrinsic clearance, CLint,scaled is scaled intrinsicclearance, CLb is hepatic blood clearance, Q is human hepatic blood flow(20 ml/min/kg), fup is unbound fraction in plasma, Rb is blood to plasma ratio CLu,int is unbound intrinsic clearance, and fumic is unbound fraction in liver microsomes.

Transport Studies Using Sandwich Culture Human Hepatocytes.Cryopreserved human hepatocyte lot Hu4241 was obtained from Life Technol-ogies. The sandwich culture human hepatocyte (SCHH) methodology appliedhere was described previously (Bi et al., 2006). Briefly, cryopreserved hepato-cytes were thawed and plated with a cell density of 0.75 � 106 cells/ml. Theplates were overlaid with 0.25 mg/ml matrigel on the second day and the cultureswere maintained. On day 5, the hepatocytes were preincubated for 10 minuteswith Ca2+ HBSS buffer in the absence or presence of rifamycin SV (100mM) fordetermination of apparent intrinsic uptake clearance. For determination ofapparent intrinsic biliary clearance, the cells were preincubated with or withoutCa2+ HBSS buffer for 10 minutes. After aspiration of the preincubation buffer,the reactions were initiated by addition of PF-06282999 (1 mM) and wereterminated at predetermined time points (0.5, 1, 2, 5, 10, and 15 minutes) bywashing the cells three times with ice-cold HBSS. Cells were then lysed with100% methanol containing terfenadine as the internal standard and the sampleswere analyzed by LC-MS/MS. Calculation of the apparent intrinsic uptakeclearance, passive diffusion, apparent intrinsic biliary clearance, and biliaryexcretion index was described in detail previously (Kalgutkar et al., 2007).

P450 Inhibition Studies. Reversible inhibition of major human P450isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, andCYP3A4/5) by PF-06282999 was evaluated via incubation of standard markersubstrates [phenacetin (CYP1A2), bupropion (CYP2B6), paclitaxel (CYP2C8),diclofenac (CYP2C9), S-mephenytoin (CYP2C19), dextromethorphan (CYP2D6),testosterone (CYP3A4/5), midazolam (CYP3A4/5), and nifedipine (CYP3A4/5)]of human P450 isozymes with pooled human liver microsomes in the presence ofan NADPH-regenerating system [NADP (1 mM), glucose-6-phosphate (5 mM),and glucose-6-phosphate dehydrogenase (1 U/ml)] in 50 mM potassiumphosphate buffer (pH 7.4) containing 3.3 mM MgCl2 and 1 mM EDTA at 37�Copen to air. The incubation volume was 0.2 ml. Microsomal proteinconcentrations, substrate concentrations, incubation times, and reaction termi-nation solvents for each activity have been described in detail previously

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(Walsky and Obach, 2004). Incubation mixtures contained PF-06282999 atconcentrations ranging from 0.1 to 100 mM. Stock solutions of PF-06282999were prepared in methanol. The final concentration of methanol in the incubationmixtures was 1% (v/v).

To examine the ability of PF-06282999 to act as a time- and/or NADPH-dependent inhibitor of major human P450 isoforms, PF-06282999 (0–100 mM)was preincubated at 37�C, in duplicate, with human liver microsomes (proteinconcentration 50–100 mg/ml, depending on which isozyme was assessed) in50 mM potassium phosphate buffer (pH 7.4) containing 3.3 MgCl2 and 1 mMEDTA at 37�C in the presence and absence of an NADPH-regenerating systemfor approximately 30 minutes. After preincubation, a portion of the incubationmixture (20 ml) was added to a mixture containing a probe P450 isozymesubstrate (concentration of approximately the Km value) (Walsky and Obach,2004) in 50mMpotassium phosphate buffer (pH 7.4) containing 3.3 mMMgCl2,1 mM EDTA, and the NADPH-regenerating system at 37�C. At the end of theincubation period (30 minutes), acetonitrile containing an internal standard wasadded, and the mixtures were centrifuged (920 � g, 10 minutes, 10�C). Thesupernatants were analyzed by LC-MS/MS for metabolites of probe P450substrates using validated bioanalytical conditions established previously(Walsky and Obach, 2004). IC50 values for inhibition of P450 isozymes wereestimated using Galileo software (version 3.3; ThermoFisher Scientific Inc.,Philadelphia, PA).

Human Renal Transporter Substrate Studies. Human embryonic kidney(HEK) 293 cells transfected with the human renal transporter proteins organiccation transporter 2 (hOCT2), organic anion transporter 1 (hOAT1), or organicanion transporter 3 (hOAT3) were generated at Pfizer. Wild-type HEK 293 cellswere cultured in Dulbecco’s modified Eagle’s medium supplemented with 10%heat-inactivated fetal bovine serum. HEK-hOAT1, HEK-hOAT3, and HEK-hOCT2 cells were cultured using growth media containing hygromycin B(60 mg/ml). Cells were maintained at 37�C, 5% CO2, and 95% relative humidity.Uptake studies used our previously published protocol (Feng et al., 2008b).Briefly, cells were seeded at a density of 1.12� 105 cells/well in 24-well poly(D-lysine)–coated plates andwere cultured for 48 hours. Uptake buffer was preparedusing HBSS, supplemented with 20 mMHEPES, at pH 7.4. Uptake was initiatedby the addition of 0.1 ml uptake buffer containing varying concentrations(0.137–300 mM) of PF-06282999. The plates were then incubated for 4 minutesat 37�C with shaking at 150 rpm. The experiment was stopped by washing thecells four times with 0.2 ml ice-cold uptake buffer/well, and then lysed with0.2 ml internal standard solution in 100% methanol. The lysates were analyzedby LC-MS/MS for PF-06282999.

Animal Pharmacokinetics. All experiments involving animals were con-ducted in our AAALAC-accredited facilities andwere reviewed and approved bythe Pfizer Institutional Animal Care and Use Committee. Male CD-1 mice(25–35 g), jugular vein/carotid artery double cannulated Wistar-Han rats(approximately 250 g; obtained from Charles River Laboratories, Wilmington,MA), male beagle dogs (approximately 12 kg), and male cynomolgus monkeys(approximately 7 kg) were used for these studies. Animals were fasted overnightand through the duration of the study (1 or 2 hours), whereas access to water wasprovided ad libitum. PF-06282999 was administered intravenously (i.v.) ineither 12% sulfobutylether-b-cyclodextrin with or without 1% dimethylsulf-oxide or 30% hydroxypropyl-b-cyclodextrin containing 5% dimethylsulfoxidevia the tail vein (mice, n = 3), carotid artery (rats, n = 3), saphenous vein (dogs,n = 3), or femoral vein (monkeys, n = 2) at a dose of 1 mg/kg in a dosing volumeof 5 ml/kg (mice), 2 ml/kg (rats), 0.5 ml/kg (dogs), and 1ml/kg (monkeys). Serialblood samples were collected before dosing and 0.083, 0.25, 0.5, 1, 2, 4, 7, and24 hours after dosing. PF-06282999 was also administered by p.o. gavage tomice (5 mg/kg at 5 ml/kg in 0.5% methylcellulose), rats (5 mg/kg at 2.5 ml/kg in0.5% methylcellulose), dogs (3 mg/kg at 3 ml/kg in 0.5% methyl cellulose), andmonkeys (3 mg/kg at 5 ml/kg in 5% hydroxypropyl methylcellulose in 20 mMTris/0.5% hydroxypropyl methylcellulose acetate succinate). Blood sampleswere taken prior to p.o. administration then serial samples were collected at 0.25,0.5, 1, 2, 4, 7, and 24 hours after dosing. Blood samples from the variouspharmacokinetic studies were centrifuged to generate plasma. All plasmasamples were kept frozen until analysis. Urine samples (0–7 and 7–24 hours;pooled 0–24 hours for mice) were also collected after i.v. administration to mice,rats, dogs, and monkeys. For mouse samples, aliquots of plasma (10 ml) or urine(2 ml mixed with 8 ml plasma) were extracted using liquid/liquid extraction withmethyl-t-butyl ether containing an internal standard. For rat, dog, or monkey

samples, aliquots of plasma or urine (20–25 ml) were transferred to 96-wellblocks and acetonitrile (150–200 ml) containing an internal standard was addedto each well. The supernatant was dried under nitrogen and reconstituted with100 ml acetonitrile/water (1:1 with 0.1% formic acid for mice; 20:80 for dogs) oradded to 100 ml water without evaporation. After extraction, the samples werethen analyzed by LC-MS/MS and concentrations of analyte in plasma and urinewere determined by interpolation from a standard curve.

Pharmacokinetics Studies in Humans. The phase I first-in-human studywas an investigator- and subject-blinded (sponsor-open), randomized, placebo-controlled crossover study of ascending single p.o. doses of PF-06282999administered sequentially, at least 1 week apart, in healthy adult subjects. Thestudy protocol and informed consent documents were approved by theinstitutional review board. All subjects signed an approved written informedconsent form before any study-related activities for this clinical study. Allsubjects were male, and the majority were white (n = 7 of 8, 87.5%) and ofHispanic or Latino descent. Age ranged from 22 to 46 years. Weight ranged from70.5 to 98.2 kg, and body mass index ranged from 23 to 30.3 kg/m2 across thetreatment groups. No dose-limiting tolerability was noted over the dose range of20–200 mg. PF-06282999 was administered as a solution [1% (w/v) methylcel-lulose, 0.5% (w/v) hydroxypropyl methylcellulose acetate succinate-HF, and 80mM tromethamine with a small amount of sodium hydroxide] to one cohort ofeight fasted male subjects at doses of 20, 50, 125, and 200 mg in a four-period,four-sequence crossover treatment design with placebo substitution (Table 1).Within each treatment period, six subjects were randomized to the active studydrug and two were randomized to a matching placebo. A standardized meal wasprovided 4 hours after dosing. Blood samples were collected at intervals for up to48 hours postdose for quantitation of circulating levels of PF-06282999. Twourine samples were collected from 0 to 24 hours pre- and postdose, the volumewas measured, and aliquots were examined quantitatively for PF-06282999.

Determination of Pharmacokinetic Parameters. Pharmacokinetic param-eters in animals and humans were determined using noncompartmental analysis(a Pfizer internally validated system of noncompartmental analysis for humandata; Watson version 7.4 was used for animal data; Thermo Scientific, Waltham,MA). Maximum plasma concentrations (Cmax) of PF-06282999 in plasma afterp.o. dosing in preclinical species and humans were determined directly from theexperimental data, with Tmax defined as the time of first occurrence of Cmax. Thearea under the plasma concentration-time curve (AUC) from t = 0 to 24 hours(AUC0–24) and t = 0 to infinity (AUC0–‘) was estimated using the lineartrapezoidal rule. In animals, systemic plasma clearance (CLp) was calculated asthe intravenous dose divided by AUC0–‘

i.v.. In humans, apparent p.o. clearance(CL/F) was calculated as the p.o. dose divided by AUC0–‘

p.o.. The terminal rateconstant (kel) was calculated by a linear regression of the log-linearconcentration-time curve, and the terminal elimination t1/2 was calculated as0.693 divided by kel. Apparent steady-state distribution volume (Vdss) in animalsand apparent volume of distribution (Vz/F) in humans were determined as the i.v.or p.o. dose divided by the product of AUC0–‘ and kel. The absolutebioavailability (F) of the p.o. doses in animals was calculated by using thefollowing equation: F = AUC0–‘

p.o./AUC0–‘i.v. � dosei.v./dosep.o.. The percent-

age of unchanged PF-06282999 excreted in urine over 24 hours [Ae,urine,(0–24 h)]was calculated using the following equation: amount (in milligrams) ofPF-06282999 in urine over the 24-hour interval postdose/actual amount ofPF-06282999 dose administered (in milligrams) � 100%. The renal clearance(CLrenal) was derived as the ratio of amount (in milligrams) of PF-06282999 inurine over the 24-hour interval postdose/AUC0–24 h.

LC-MS/MS Analysis for Quantitation of PF-06282999. Concentrations ofPF-06282999 were determined on an AB Sciex model API-4000 LC-MS/MStriple quadrupole mass spectrometer (AB Sciex, Framingham, MA). Analytes

TABLE 1

Clinical study treatment assignment for PF-06282999

Cohort Sequence Subjects Period 1 Period 2 Period 3 Period 4

n mg1 1 2 Placebo 50 125 200

2 2 20 Placebo 125 2003 2 20 50 Placebo 2004 2 20 50 125 Placebo

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were chromatographically separated using an Eksigent Express HT (EksigentTechnologies, Dublin, CA) or Shimadzu LC-20AD (Shimadzu ScientificInstruments, MD) pumps. A CTC PAL autosampler (Agilent, Santa Clara,CA) was programmed to inject 20 ml on a Luna C8 (3 m) 30� 2 mm for rats or akinetex C18 (2.6 m) 30 � 3 mm for mice, using a mobile phase consisting of10 mM ammonium formate buffer containing 0.1% (v/v) formic acid (solvent A)and acetonitrile (solvent B) at a flow rate of 0.5 ml/min. Ionization was conductedin the positive ion mode at the ionspray interface temperature of 400�C, usingnitrogen for nebulizing and heating gas. The ion spray voltage was 5.0 kV and thedeclustering potential was optimized at 41 eV. PF-06282999 was detected usingelectrospray ionization in the multiple reaction monitoring mode monitoring for amass-to-charge ratio (m/z) transition 326→155. PF-06282999 standards were fit byleast-squares regression of their areas to a weighted linear equation, fromwhich theunknown concentrations were calculated. The dynamic range of the assay was1–2000 ng/ml. Assay performance was monitored by the inclusion of qualitycontrol samples with acceptance criteria of 630% target values.

Bioanalytical Methodology for Metabolite Identification. Qualitativeassessment of the metabolism of PF-06282999 in liver microsomes andhepatocytes was conducted using a Thermo Finnegan Surveyor photodiodearray plus detector, a Thermo Acela pump, and a Thermo Acela Autosampler(ThermoScientific). Chromatography was performed on a Phenomenex HydroRP C18 (4.6 mm� 150mm, 3.5mm) column (Phenomenex, Torrance, CA). Themobile phase was composed of 5 mM ammonium formate buffer with 0.1%formic acid (pH 3) (solvent A) and acetonitrile (solvent B) at a flow rate of1 ml/min. The binary gradient was as follows: the solvent A to solvent B ratio washeld at 95:5 (v/v) for 3 minutes and then adjusted to 55:45 (v/v) from 0 to 35minutes, 30:70 (v/v) from 35 to 45 minutes, and 5:95 (v/v) from 45 to 52 minutes,where it was held for 3 minutes and then returned to 95:5 (v/v) for 6 minutes beforethe next analytical run. Identification of the metabolites was performed on aThermo Orbitrap mass spectrometer operating in positive ion electrospray mode.The spray potential was 4 V and the heated capillary was at 275�C. Xcalibursoftware (version 2.0; Thermo Scientific) was used to control the HPLC–massspectrometry system. Product ion spectra were acquired at a normalized collisionenergy of 65 eV with an isolation width of 2 atomic mass units. Metabolites fromliver microsomes or hepatocyte incubations were identified in the full-scan mode(from m/z 100 to 850) by comparing t = 0 samples with t = 45 minute (livermicrosomes) 240-minute (hepatocyte) samples or through comparison withsynthetic standards, and structural information was generated from the collision-induced dissociation spectra of the protonated molecular ions (MH+).

Pharmacokinetics Projections to Humans. The single-species allometricmethod was used to predict human pharmacokinetic parameters of PF-06282999(Frederick et al., 2009). CLp and Vdss observed in preclinical species werecorrected for species differences in plasma protein binding and then scaled tohumans (Frederick et al., 2009). This approach uses a fixed allometric exponentof 0.75 and 1 for clearance and volume, respectively. Renal clearance in humanswas predicted from rats, dogs, and monkeys using the direct correlationmethodology reported previously (Paine et al., 2011). Rat, dog, or monkeyCLrenal values corrected for plasma protein binding and kidney blood flow (KBF)differences were used to predict human CLrenal using the following equation:

CLrenal;human ¼ CLrenal;animals•

fu;human

fu;animal

!•

KBFhuman

KBFanimal

!

where fu is fraction unbound in plasma. Values used for rat, dog, monkey, andhuman KBF were 52 (Hsu et al., 1975), 22 (Keil et al., 1989), 28 (Davies andMorris, 1993), and 16 ml/min per kilogram (Wolf et al., 1993), respectively.

Results

Plasma Protein Binding and Blood Cell Partitioning of PF-06282999. In vitro plasma protein binding of PF-06282999 wasevaluated by equilibrium dialysis in rat, mouse, dog, monkey, andhuman plasma. The mean fraction unbound in mouse rat, dog, monkey,and human plasma was 0.451, 0.447, 0.460, 0.536, and 0.376,respectively, indicating that PF-06282999 is moderately bound toplasma proteins across preclinical species and humans. PF-06282999was stable in mouse, rat, dog, monkey, and human plasma for more than

4 hours at 37�C at the concentrations used in the plasma free fractiondetermination. The blood/plasma ratios for PF-06282999 were 1.1, 1.1,0.91, 1.2, and 0.94 in mice, rats, dogs, monkeys, and humans,respectively, suggesting that PF-06282999 was equally distributed intoplasma and red blood cells.Metabolic Stability Studies with PF-06282999. PF-06282999

(1 mM) did not show metabolic turnover in NADPH-supplementedliver microsomes (t1/2 . 120 minutes) and cryopreserved hepatocytes(t1/2. 240 minutes) from rats, mice, dogs, monkeys, and humans in thetime course of incubation (60 minutes for liver microsomes and 120minutes for hepatocytes). Furthermore, no metabolic turnover wasnoted in PF-06282999 (1 mM) incubations in the cryopreserved humanhepatocyte assay under relay conditions (t1/2 . 2400 minutes;CLint,scaled , 1.46 ml/min per kilogram, hepatic blood clearance, 0.57 ml/min per kilogram) (Di et al., 2013). Consistent with thesefindings, qualitative examination of metabolites in liver microsomesand hepatocytes from rats, mice, dogs, monkeys, and humans byHPLC-UV (l = 254 nm) revealed only trace levels of three metabolites,designated as M1, M2, andM3 (Table 2). In liver microsomes, the threemetabolites were formed in a NADPH-dependent fashion, suggestiveof a role for P450 or flavin monooxygenase enzymes in the oxidativemetabolism of PF-06282999.PF-06282999 had a retention time (tR) of approximately 29.0

minutes on HPLC and showed an exact mass of m/z 326.0361(MH+). Its MS2 product ion spectrum showed diagnostic fragmentions atm/z 281.0144, 222.0315, and 155.0256, respectively. The ions atm/z 281 and 222 resulted from neutral losses of formamide andisothiocyanic acid, respectively, whereas the ion at m/z 155 resultedfrom the formation of a tropylium ion from the chloromethoxybenzenemoiety.Metabolite M1 had a tR of approximately 21.9 minutes on HPLC and

an exact mass of m/z 296.0433, which was 30 Da less than that ofPF-06282999. The mass spectrum contained fragment ions at m/z251.0214, 235.0265, and 208.0156. The ion at m/z 208, which was 14Da less than the parent product ion at m/z 222, suggested a loss of amethyl group from the aromatic ether motif. The fragment ion at m/z251, which was 30 Da less than the parent ion, suggested loss of amethyl group (214 Da) and replacement of the sulfur (232 Da) with anoxygen atom (+16 Da). This information suggested that M1 resultedfrom oxidative desulfurization and O-demethylation of the chlorome-thoxyphenyl moiety.Metabolite M2 had a tR of approximately 24.1 minutes on HPLC and

an exact mass of m/z 312.0204, which was 14 Da less than that ofPF-06282999. Themass spectrum contained fragment ions atm/z 266.9986and 208.0157. The ions at m/z 267 and 208, which were 14 Da less thenparent product ions at m/z 281 and 222, suggested that M2 was derivedfrom an O-demethylation of the methoxyphenyl group in PF-06282999.Metabolite M3 had a tR of approximately 25.7 minutes on HPLC and

an exact mass of m/z 310.0589 (MH+), which was 16 Da less than thatof PF-06282999. The mass spectrum contained fragment ions at m/z265.0372, 222.0314, and 155.0253. The ions at m/z 222 and 155 werealso observed in the mass spectrum of the parent compound, indicatingthat the 6-aryl substituent was unaltered. The fragment ion at m/z 265,which was 16 Da less than the parent product ion m/z 281, suggestedoxidative desulfurization of the thiouracil motif. An authentic standard ofthe proposed metabolite PF-06428486 [2-(6-(5-chloro-2-methoxyphenyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide] was found to have anidentical tR and mass spectrum (data not shown).Because of the lack of metabolic turnover of PF-06282999 in human

liver microsomes, human P450 enzymes responsible for oxidativemetabolism of PF-06282999 could not be identified using prototypicmethodology (e.g., use of selective P450 enzyme inhibitors or

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recombinant P450 enzymes). Overall, the qualitative biotransformationstudy suggests that the metabolic routes of PF-06282999 are similar inall species with no evidence of human-specific metabolites. Finally, noglutathione conjugates were detected in human liver microsomalincubations of PF-06282999 in the presence of NADPH and excessglutathione, suggesting that no reactive species were released in thecourse of oxidative metabolism of PF-06282999.Human P450 Isozyme Inhibition with PF-06282999. PF-

06282999 exhibited no relevant reversible (IC50 . 100 mM) and time-or NADPH-dependent (IC50 . 100 mM) inhibitory effects againstCYP1A2 (phenacetin-O-dealkylation), CYP2B6 (bupropion hydroxyl-ation), CYP2C8 (paclitaxel-6a-hydroxylation), CYP2C9 (diclofenac-49-hydroxylation), CYP2C19 (S-mephenytoin-49-hydroxylation),CYP2D6 (dextromethorphan-O-demethylation), or CYP3A4/5 (testos-terone-6b-hydroxylase, midazolam-19-hydroxylase, and nifedipineoxidase) activities. IC50 values for reversible and time-dependentinhibition could not be calculated because PF-06282999 did not inhibitany P450 activity more than 30% at the highest concentration tested.Under the present experimental conditions, known P450 isozyme-specific inhibitors a-naphthoflavone, orphenadrine, montelukast,sulfaphenazole, modafinil, quinidine, and ketoconazole demonstratedpotent competitive inhibition of CYP1A2, CYP2B6, CYP2C8,

CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5 activity, respectively,in human liver microsomes (Pfizer data on file). Likewise, metabolism-dependent inhibition of P450 activity was also noted with the respectivepositive control compounds [furafylline (CYP1A2), phencyclidine(CYP2B6), gemfibrozil glucuronide (CYP2C8), tienilic acid(CYP2C9), (S)-fluoxetine (CYP2C19), paroxetine (CYP2D6),and troleandomycin (CYP3A4/5)] in a time- and NADPH-dependent fashion (Pfizer data on file).Absorptive Permeability of PF-06282999. The Papp value of PF-

06282999 across the Caco-2 cell monolayer (Table 3) in the apical-to-basolateral direction was 2.7 � 1026 cm/s, which is comparable todrugs exhibiting moderate oral absorption in humans (Varma et al.,2012). Under identical experimental conditions, the secretory (B-to-A)permeability of PF-06282999 was 10 � 1026 cm/s (efflux ratio = 3.8),suggesting that PF-06282999 is a substrate for efflux transport. As such,PF-06282999 was found to be a substrate for MDR1 based onbidirectional transport study using the MDCKII-MDR1 cell monolayerwith a low efflux ratio of 3.3. This finding is consistent with thereduction in efflux ratio to 1.1 noted in the Caco-2 assay in the presenceof the MDR1 inhibitors of verapamil and cyclosporin A.Papp,AB in Caco-2 was not affected by the apical pH, with Papp,AB at

pH 6/7.4 and 7.4/7.4 estimated to be approximately 2.7� 1026 cm/s. In

TABLE 2

Mass spectral characteristics of the in vitro metabolites of PF-06282999

All metabolites were observed in liver microsomes and hepatocytes from preclinical species and humans.

Metabolite MH+ Retention Time (tR) Structure Fragment Ions

min

Parent 326.0361 29 281.0144, 222.0315, 155.0256

M1 296.0433 21.9 251.0214, 235.0265 (-S), 208.0156

M2 312.0204 24.1 266.9986, 208.0157

M3a 310.0589 25.7 265.0372, 222.0314, 155.0253

aThe structure of M3 was unambiguously confirmed by comparison of its LC-MS/MS characteristics with an authentic standard.

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the absence of calcium in the media, the Papp,AB value of PF-06282999increased by 4.4-fold to 12 � 1026 cm/s. The Papp,AB values forPF-06282999 in the Caco-2 assay are consistent with its measuredpermeability of 2.2 � 1026 cm/s in MDCKII-LE cells. Nadolol, alow-permeability paracellular marker, showed absorptive permeabilityof about 0.9 � 1026 cm/s at apical pH of both 6 and 7.4 in the Caco-2studies. Overall, PF-06282999 demonstrates moderate permeability viaboth paracellular and transcellular pathways and is a substrate ofMDR1with a low efflux ratio.Hepatobiliary Transport of PF-06282999 in SCHH. The hep-

atobiliary transport of PF-06282999 was examined in SCHHs (Bi et al.,2006). Compared with rosuvastatin, PF-06282999 exhibited low totaluptake clearance and biliary intrinsic clearance (Table 4). Thecorresponding mean biliary excretion index for rosuvastatin andPF-06282999 was 49% and 11%, respectively. Rifamycin SV, a paninhibitor of transporters, had a small effect (approximately 25%) on theuptake of PF-06282999; by contrast, rifamycin SV inhibited approx-imately 90% of rosuvastatin uptake. These observations suggest that thecontribution of transporter-mediated hepatobiliary disposition to theoverall clearance of PF-06282999 is likely minimal.PF-06282999 Uptake by Major Human Renal Transporters.

Substrate affinity to the major human renal anion (hOAT1 and hOAT3)and cation (hOCT2) transporters was evaluated using HEK 293 cellstransfected with hOAT1 , hOAT3 , and hOCT2 transporters. Nosignificant difference in PF-06282999 (0.1–900 mM) uptake by thesetransporters versus wild-type HEK 293 cells was observed (data notshown), indicating that PF-06282999 is not transported by the majorhuman renal uptake transporters.Pharmacokinetics of PF-06282999 after Single Doses to

Animals. The pharmacokinetic parameters describing the dispositionof PF-06282999 after i.v. and p.o. administration in preclinical speciesare shown in Table 5. PF-06282999 demonstrated low plasma clearancein mice (10.1 ml/min per kilogram), dogs (3.39 ml/min per kilogram),

and monkeys (10.3 ml/min per kilogram) andmoderate clearance in rats(41.8 ml/min per kilogram). PF-06282999 was well distributed withVdss ranging from 0.5 to 2.1 l/kg in mice, rats, dogs, and monkeys. Theterminal elimination t1/2 ranged from 0.75 to 3.3 hours in the fourspecies. After p.o. administration at a single p.o. dose of 3 or 5 mg/kg,PF-06282999 was rapidly (Tmax = 0.7–1.70 hours) and well absorbed inmice, rats, dogs, and monkeys, with F values of 100%, 86%, 75%, and76%, respectively.Table 6 depicts data for renal excretion of unchanged PF-06282999

in preclinical species after i.v. administration. The unbound renalclearance (CLrenal,u) in mice and dogs was lower than the glomerularfiltration rate (GFR), suggesting lack of active tubular secretion.However, in rats and monkeys, the unbound CLrenal,u values of 28.3ml/min per kilogram and 3.70 ml/min per kilogram exceeded the GFRof 8.7 ml/min per kilogram and 2 ml/min per kilogram (Lin, 1995) byapproximately 3.20- and 1.86-fold, respectively, implying thatPF-06282999 is subjected to active tubular secretion mediated bytransporters in these species.Prediction of Human Pharmacokinetics. PF-06282999 was re-

sistant to metabolic turnover in human liver microsomes and hepato-cytes under relay conditions, suggesting that metabolism would beinconsequential as a clearance mechanism in human. Furthermore,SCHH studies indicated that hepatobiliary disposition of unchangedPF-06282999 in humans is likely to be insignificant as an eliminationmechanism. We therefore speculated that PF-06282999 would becleared in humans via renal excretion in unchanged form. Theobservation that all preclinical species exhibited some degree of renalexcretion and the similarity in physicochemical properties ofPF-06282999 with literature drugs (e.g., acetazolamide, furosemide,captopril, and methotrexate) conducive to renal elimination (Varmaet al., 2009) further supported this hypothesis. Consequently, humanpharmacokinetics (CLp and Vdss) (Table 7) were predicted from single-species scaling of animal pharmacokinetics (corrected for plasmaprotein binding differences) (Frederick et al., 2009), and estimates ofhuman CLrenal (Table 7) were obtained from rat, dog, or monkey CLrenal

data normalized for plasma protein binding and KBF differences asreported previously (Paine et al., 2011). Overall, this exercise led topredicted parameters comprising low to moderate CLp (1.8–8.6 ml/minper kilogram or 126 –600 ml/min for a 70-kg adult) and Vdss (0.4–1.8l/kg or 28–126 l for a 70-kg adult) and a relatively short predicted t1/2 of2.4–2.7 hours, derived from the equation t1/2 = 0.693 � Vdss/CLp. Weassumed that the high oral bioavailability of PF-06282999 observed inanimals (F $ 75%) would also be translated in humans especially atrelatively low doses (20–200 mg) in which absorption would not benegatively affected by solubility and permeability. Considering thatPF-06282999 was metabolically stable in human hepatocytes (under relay

TABLE 3

Permeability and transporter data of PF-06282999 in Caco-2, MDCKII-LE, and MDCKII-MDR1 cell monolayers

Compound A-to-B Papp B-to-A Papp Efflux RatioFold Change fromA to B at pH 7.4

Caco-2 � 1026 cm/s

PF-06282999 (pH 7.4) 2.7 10 3.8PF-06282999 (pH 6.0) 2.7 1PF-06282999 (pH 7.4, Ca2+-free) 12 4.4PF-06282999 (pH 7.4) + MDR1

inhibitors2.9 7.6 2.6 1.1

MDCKII-LEPF-06282999 (pH 7.4) 2.2

MDCKII-MDR1PF-06282999 (pH 7.4) 0.6 2.1 3.3

A, apical; B, basolateral.

TABLE 4

Hepatobiliary disposition of PF-06282999 in SCHHs

Compound

ApparentIntrinsicUptake

Clearance

PassiveUptake

ClearanceaActiveUptake

Apparent IntrinsicBiliary Clearance

BiliaryExcretionIndex

ml/min per mg protein % ml/min per mgprotein

%

PF-06282999 2.3 1.7 25 0.2 11Rosuvastatin 8.5 0.9 89 3.5 49

aUptake was measured in the presence of rifamycin SV (100 mM).

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conditions), first-pass metabolism was not anticipated to contributesignificantly as a deterrence to oral absorption.Clinical Pharmacokinetics of PF-06282999. A total of eight

subjects were randomized and treated at each of the four periods witha single p.o. dose of PF-06282999 or placebo. Mean plasmaPF-06282999 concentration-time profiles across the studied doses arepresented in Fig. 2. Plasma pharmacokinetic parameters from thenoncompartmental analysis are summarized in Table 8. After single p.o.doses of up to 200 mg PF-06282999 in solution, oral absorption ofPF-06282999 was rapid with mean peak plasma concentrations achievedwithin 1 hour postdose (Fig. 2). Exposures as estimated by AUC andCmax after an p.o. dose were dose proportional up to 200 mg (Fig. 3A),and the overall mean plasma elimination t1/2 was comparable at all doselevels with mean values of approximately 4.3–4.4 hours (Table 8). Renalexcretion of PF-06282999 was dose proportional and appeared to be thepredominant elimination pathway, with approximately 75% of doseexcreted as parent within 24 hours postdose at the 200-mg dose (Fig. 3B;Table 9). The mean unbound renal clearance of .300 ml/min (renalclearance ranging from 127 to 151 ml/min) exceeded the GFR of 106618 ml/min per 1.73 m2 reported for healthy adults (Soares et al., 2013),indicating potential active tubular secretion of PF-06282999. The t1/2(4.3–4.4 hours) observed in the clinic was within 2-fold of the predictedvalue (2.4–2.7 hours).

Discussion

PF-06282999 is a low molecular weight (326 Da), hydrophilic(cLogP = 0.49, LogD7.4 = 0.81, topological polar surface area = 84.66Å2), weak acid (pKa = 7.82) (Ruggeri et al., 2015). Consistent with itsphysicochemical attributes, PF-06282999was virtually resistant towardmetabolic turnover in NADPH-supplemented liver microsomes andhepatocytes from preclinical species and humans, was devoid of

glutathione conjugate formation in human liver microsomes andhepatocytes, and caused no reversible or time- and NADPH-dependent inhibition of major human P450 isoforms. Because tracelevels of oxidative metabolites were noted in human liver microsomaland hepatocyte incubations of PF-06282999 (Table 2), we initiallyspeculated that a proportion of the clearance mechanism ofPF-06282999 in humans would be dictated by slow metabolism.Metabolic turnover rates for slowly metabolized compounds cannot beestimated in standard hepatocyte assays involving short incubationperiods. Recently, a novel human hepatocyte suspension relay assaywas used to extend drug residence time in cryopreserved hepatocytes,which provided reliable estimates of in vitro intrinsic clearance for slowlymetabolized compounds (Di et al., 2013). Even under extendedincubations, PF-06282999 was stable toward metabolic turnover, whichimplied that metabolic elimination would be inconsequential as anelimination mechanism in humans. Furthermore, the possibility ofhepatobiliary disposition of unchanged PF-06282999mediated by uptakeand efflux transporters was explored in human hepatocytes undersandwich culture conditions (Table 4). Rifamycin SV, a pan inhibitorof transporters, had minimal effect on the hepatic uptake of PF-06282999, and the biliary excretion index, an indication of extent ofbiliary secretion, was found to be low. Collectively, hepatic clearanceinvolvingmetabolizing enzymes and drug transporters has a minimal rolein the systemic clearance of PF-06282999.On the basis of physicochemical properties and membrane perme-

ability, PF-06282999 fit into extended clearance classification systemclass 3A, where renal clearance is predicted to be the major clearancemechanism in humans (Varma et al., 2015). Consistent with this,preclinical findings implied that PF-06282999 would be clearedunchanged via renal clearance, which was subsequently confirmed inthe first-in-human study (e.g., approximately 75% of the dose excretedunchanged in urine at the 200 mg p.o. dose). Against this backdrop, it is

TABLE 5

Preclinical pharmacokinetics of PF-06282999

All experiments involving animals were conducted in our AAALAC-accredited facilities and were reviewed and approved by the Pfizer Institutional Animal Careand Use Committee. Pharmacokinetic parameters were calculated from plasma concentration-time data and are reported as means6 S.D. for n = 3 and maximum valuesfor n = 2. All pharmacokinetics were conducted in male sex of each species (Wistar rats, CD-1 mice, beagle dogs, and/or cynomolgus monkeys). Intravenous doses forPF-06282999 were 1 mg/kg and were administered as a solution. Oral doses for PF-06282999 were 3 or 5 mg/kg and were formulated in 5% HPMC in 20 mM Tris/0.5% HPMCAS.

Species Cmaxa Tmax

a Oral AUC0–‘a CLp Vdss t1/2 Oral Fb

ng/ml h ng×h/ml ml/min per kg l/kg h %

Mouse 3650 6 325 1.0 6 0.1 24,800 6 1010 10.1 6 0.83 0.90 6 0.06 1.36 6 0.12 .100Rat 596 6 282 1.1 6 0.88 1780 6 287 41.8 6 9.65 2.13 6 1.08 0.75 6 0.19 86Dog 4090 6 979 0.7 6 0.3 11,800 6 3480 3.39 6 1.13 0.54 6 0.04 2.2 6 0.5 75Monkey 771 (1040, 501) 1.5 (1.0, 2.0) 3720 (4130, 3300) 10.3 (10.5,10.1) 1.09 (1.17, 1.01) 3.32 (5.22, 1.42) 76

aObtained from p.o. dose; HPMC, hydroxypropyl methylcellulose; HPMCAS = hydroxypropyl methylcellulose acetate succinate.

TABLE 6

Renal excretion of unchanged PF-06282999 in preclinical species after i.v. administration

Species Renal Excretion Total CLp Total CLrenala Unbound CLrenal

b Unbound CLrenal/GFR Ratioc

% of total dose ml/min per kg

Mouse 44.2 10.1 4.03 9.8 1Rat 30.5 41.8 11.6 28 3.2Dog 26 3.39 0.96 1.9 0.5Monkey 19.5 10.3 1.67 3.7 1.86

aTotal CLrenal in blood was estimated by multiplying the total CLb with fraction of total i.v. dose of unchanged PF-06282999 excreted inurine. CLb was obtained by dividing CLp by the blood to plasma ratio in the individual animal species (mouse B/P = 1.1, rat B/P = 1.1, dogB/P = 0.91, monkey B/P = 1.2).

bUnbound CLrenal in blood was estimated by normalizing total CLrenal with the unbound free fraction in blood (mouse = 0.41, rat = 0.41,dog = 0.51, monkey = 0.45), which in turn was obtained by dividing the unbound plasma free fraction by the blood to plasma ratio.

cGFR values in rats, mice, dogs, and monkeys are 8.7, 10, 4, and 2 ml/min per kilogram, respectively.

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noteworthy to point out that the percentage of the PF-06282999 doseexcreted in the urine after i.v. administration in preclinical species wasless than 50%, despite the lack of metabolic turnover detected in thetime course of the in vitro assays using animal hepatic tissue (30minutesof incubation for liver microsomes and 120 minutes for hepatocytes).This may suggest additional in vivo clearance pathways forPF-06282999, such as slow metabolism (low turnover rates in vitrobut a relatively higher extent of metabolism in vivo) (Chan et al., 2013)

and/or biliary elimination in the preclinical species, especially the rat,where the systemic CLp (41.8 ml/min per kilogram) is in the moderaterange. An alternative clearance mechanismmay be a possibility to somedegree in humans as well. For example, elimination of approximately75% of the dose in urine at the 200-mg dose suggests that the remainder(25%) of the dose could be cleared through metabolic or biliaryexcretion routes. However, the possibility remains that 25% of the p.o.dose is not orally absorbed. Radiolabeled mass balance/excretion routestudies and absolute oral bioavailability studies with PF-06282999should shed additional light on the similarities/differences in excretionprofile between animals and humans.The predominantly renal elimination pathway of PF-06282999 in

humans is not altogether surprising. The physicochemical attributes ofPF-06282999 coupled with the measured thermodynamic solubility(crystalline form) of 0.184 mg/ml in phosphate-buffered saline at pH6.5 and 0.166 mg/ml in unbuffered water, and moderate passiveMDCKII-LE permeability (approximately 2.2 � 1026 cm/s), wouldultimately manifest into a solubility-limited and/or permeability-limitedabsorption depending on the dose and CLrenal as the likely clearancemechanism (Varma et al., 2009, 2012).Renal transporters including hOAT1, hOAT3, and hOCT2 are major

transporters expressed on the basolateral membrane involved in theactive renal secretion, whereas MDR1, breast cancer resistant protein,multidrug-resistant–associated proteins, and multidrug and toxic com-pound extrusion 1/2K transporters can potentially mediate secretionacross the apical membrane (Masereeuw and Russel, 2001). PF-06282999 is not a substrate to hOAT1, hOAT3, and hOCT2 but is asubstrate of MDR1. Consequently, the .3-fold increase in humanCLrenal,u (.300ml/min) of PF-06282999 over the humanGFR could beaccounted for by an active secretion process in the renal elimination ofPF-06282999, mediated by efflux transporter MDR1 coupled withunknown renal uptake transporters. If so, then it is tempting to speculatethat species differences in MDR1 expression in the kidney and/or PF-06282999 affinity toward MDR1 would partially account for thedifferences in CLrenal,u exceeding GFR in rats (3.2-fold of GFR),monkeys (1.86-fold of GFR), and humans (3.3-fold of GFR), but not indogs and mice. Although quantitative information on species differenceof MDR1 expression is limited, fairly good correlation was found in theMDR1 efflux activity between humans and monkeys, which could beattributable to high homology of the amino acid sequences (96%similarity) (Takeuchi et al., 2006). In addition, efflux activity of humanMDR1 showed a relatively better correlation with rat Mdr1a andMdr1bcompared with the correlation with mouse or canine efflux activity,although the substrate affinity was maintained across the species.Interestingly, these observations are consistent with the PF-06282999net secretion noted in humans, rats, and monkeys but not in mice anddogs. Nevertheless, further understanding of the mechanisms involvedin the renal secretion and the observed species difference is warranted.Although transporters can exhibit significant species differences in

terms of expression level and functional activity (Chu et al., 2013), it isnoteworthy to point out that human CLrenal of 36 structurally diversedrugs was reasonably well predicted using dog CLrenal after correctingfor plasma protein binding and KBF (Paine et al., 2011). The clinicalpharmacokinetics of PF-06282999 after p.o. administration (20–200 mg)(Table 8) were characterized by a low systemic CL/F (186–197ml/min or2.6–2.8 ml/min per kilogram for a 70-kg adult) and a moderate Vz/F(68.9–73.7 l or 0.98–1.05 l/kg for a 70-kg adult), which led to arelatively short t1/2 (4.3–4.4 hours). Normalization of CL/F values forthe 20- to 200-mg dose range shown in Table 8, with approximateF values corresponding to the percentage of dose excreted in urine in a24-hour period at these doses (Table 9), yielded CLp ranging fromapproximately 124 to 149 ml/min or 1.8–2.1 ml/min per kilogram

TABLE 7

Prediction of human pharmacokinetics and CLrenal for PF-06282999

Predicted HumanPharmacokinetics Parametera

Rat Dog Monkey

CLp (ml/min per kilogram) 8.6 1.8 3.4Vdss (l/kg) 1.8 0.4 0.8t1/2 (h) 2.4 2.6 2.7Total CLrenal (ml/min per kg)b 3.0 0.6 0.7

aObserved pharmacokinetics in humans was as follows: CLp = 1.8–2.1 ml/min per kilogramand Vdss = 0.65–0.78 l/kg (20–200 mg p.o. doses) obtained from the respective CL/F and Vz/Fvalues normalized for F values corresponding to the percentage of dose eliminated in unchangedform in urine in a 24-hour period at the respective p.o. doses. Human t1/2 was estimated to be 3.9–4.2 hours (20–200 mg p.o. doses) and was calculated using the equation 0.693 � Vdss/CLp.

bObserved total CLrenal in humans was1.8–2.1 ml/min per kilogram (20–200 mg p.o. doses).

Fig. 2. (A and B) Mean plasma PF-06282999 concentration-time profiles aftersingle-dose oral administration of solution formulation of PF-06282999 at 20, 50,125, and 200 mg to human subjects (n = 6) in linear (A) and log-linear (B) scales.Data are presented as means 6 S.D.

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(for a 70-kg adult), which were reasonably well predicted (62-fold)from the single-species scaling of dogs (CLp approximately 1.8 ml/min per kilogram) and monkeys (CLp approximately 3.4 ml/min perkilogram) (Table 7). By contrast, rat CLp overpredicted the humanCLp by approximately 4-fold, which implies that clearance mecha-nisms in addition to renal elimination are operative in the rat and willneed additional investigations via metabolic stability assessments inrat hepatocytes under relay conditions and biliary excretion studies.In a similar fashion, p.o. Vz/F (68.9–73.7 l) values were normalizedfor F equal to the percentage of dose excreted in urine, yielding Vdssvalues ranging from 45.9 to 54.8 l or 0.65 to 0.78 l/kg, which werepredicted from single-species scaling of dog (0.4 l/kg) and monkey

(0.8 l/kg) Vdss (Table 7). Despite the subtle nuances in CLp and Vdsspredictions from single-species scaling of animal pharmacokineticsdata, it is interesting to note that all preclinical species ultimatelypredicted a short t1/2 (2.4–2.7 hours) for PF-06282999 in humans,which is in close proximity to the observed values (3.9–4.2 hours)generated from the CLp and Vdss estimates at the various dose ranges.Although the human pharmacokinetics predictions utilizing the dog

or monkey were fairly accurate, prediction of human CLrenal, thepredominant clearance mechanism of PF-06282999, using dog and/ormonkey CLrenal normalized for plasma free fraction and KBF differ-ences and the direct correlations methodology of Paine et al. (2011), ledto .2.5-fold to 3-fold underprediction (predicted human CLrenal =0.6– 0.7 ml/min per kilogram for dogs and monkeys, respectively;observed p.o. CLrenal in humans = 127–151 ml/min or 1.81–2.15 ml/minper kilogram). By contrast, rat CLrenal after correction for plasmaunbound fraction and KBF differences led to a predicted human CLrenal

value of 3 ml/min per kilogram, which was in close proximity to theobserved value. Suffice to say, with the exceptions of some anioniccompounds, human CLrenal was also reasonably well predicted from ratCLrenal in the work of Paine et al. (2011). In addition, CLrenal,u exceedingGFR in rats (3.2-fold of GFR) similar to that in humans (3.3-fold ofGFR), but not in dogs, could explain a better prediction of humanCLrenal from rat CLrenal but an underprediction using dog CLrenal.On the basis of an analysis of MDCKII-LE permeability versus

human intestinal absorption for 105 marketed drugs, compounds withPapp of .5 � 1026 cm/s are most likely to demonstrate complete oralabsorption when solubility is not a limiting factor, whereas a Papp rangeof 2–5 � 1026 cm/s represent compounds with human intestinalabsorption in the moderate range (approximately 30%–80%) (Varmaet al., 2012). In alignment with the in vitro permeability data, oralbioavailability studies were indicative of good oral absorption (oral F.75%) in preclinical species at relatively low dose. For the preliminaryclinical pharmacokinetics studies at the 20- to 200-mg dose range,PF-06282999 was solubilized in Tris buffer (pH adjusted with NaOH)containing 0.5% hydroxypropyl methylcellulose acetate succinate toprevent precipitation to eliminate any potential solubility-limited

TABLE 8

Summary of plasma pharmacokinetics of PF-06282999 in healthy fasted male subjects after single p.o. dosesadministered as solution

Values are presented as follows: geometric means 6 S.D. AUC, Cmax, CL/F, and Vz/F; medians (ranges) for Tmax; and arithmeticmeans 6 S.D. for t1/2.

Dose AUC0–‘ Cmax Tmax t1/2 CL/F Vz/F

mg ng×h/ml ng/ml h h ml/min liters

20 1792 6 363 402 6 131 0.75 (0.5, 1.1) 4.3 6 0.7 186 6 44 68.9 6 10.850 4294 6 664 920 6 232 0.5 (0.5, 1.0) 4.4 6 0.6 194 6 34 73.6 6 11.5125 10,730 6 1950 2659 6 631 0.5 (0.5, 1.0) 4.4 6 0.6 194 6 40 73.7 6 7.5200 16,950 6 2880 3799 6 1156 0.5 (0.5, 2.0) 4.3 6 0.4 197 6 32 72.6 6 7.2

Fig. 3. (A) Dose proportionality of pharmacokinetic parameters AUC and Cmax

observed after single-dose oral administration of 20–200 mg PF-06282999 to humansubjects (n = 6). (B) Urinary excretion of PF-06282999 as unchanged drug(percentage of dose) after single-dose oral administration of 20–200 mgPF-06282999 to human subjects (n = 6). Data are presented as means 6 S.D.

TABLE 9

Summary of urinary excretion of PF-06282999 in healthy fasted male subjects aftersingle p.o. doses administered as solution

Geometric means 6 S.D. are presented for Ae, urine, (0–24 h) and CLrenal.

Dose Ae,urine,(0–24 h) Ae,urine,(0–24 h) CLrenal CLrenal,ua

mg mg % of dose ml/min

20 13.3 6 1.7 66.7 6 8.5 127 6 18 33850 36.3 6 3.2 72.4 6 6.4 144 6 14 383125 86.9 6 8.3 69.5 6 6.6 138 6 26 367200 151.1 6 12.6 75.5 6 6.2 151 6 21 402

aCLrenal,u was estimated by dividing CLrenal by human plasma unbound fraction of 0.376.

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absorption. PF-06282999was rapidly absorbed (Tmax = 0.5–0.75 hours)in humans when administered orally as a solution, which suggestedgood permeation in the upper gastrointestinal tract. Likewise, on thebasis of the renal excretion data (approximately 75% of the 200-mgdose excreted unchanged in urine), it is safe to assume that the oral F ofPF-06282999 is at least 75% at the 200-mg dose.In conclusion, this study provides key information regarding the

disposition characteristics of PF-06282999, a novel mechanism-basedinactivator of MPO, in preclinical species and humans. Renal clearanceof unchanged PF-06282999 emerged as the principal clearancemechanism in humans, a phenomenon that was consistent with thephysicochemical and permeability characteristics as defined by theextended clearance classification system. Furthermore, the humanpharmacokinetics of PF-06282999 were reasonably well predictedfrom single-species scaling of dog and/or monkey pharmacokineticsdata, corrected for plasma free fraction differences. Selective inhibitionof MPO relative to other peroxidases (e.g., thyroid peroxidases), renalelimination with minimal hepatic oxidative metabolism, and absence ofmechanism-based P450 inhibition by PF-06282999 provides a strongrationale for mitigating idiosyncratic toxicity and drug–drug interactionrisks with a drug candidate containing a structural alert.

Acknowledgments

The authors thank Samuel Bell, Karen Atkinson, Yi-An Bi, Renato Scialis,and Charles Rotter for assistance with the formulation support and the executionof metabolic stability and transporter studies. The authors also thank Vu Le forproviding statistical guidance.

Authorship ContributionsParticipated in research design: Dong, Wolford, Terra, Kalgutkar.Conducted experiments: Dong, Wolford, Ryder.Contributed new reagents or analytic tools: Di.Performed data analysis: Dong, Wolford, Kalgutkar.Wrote or contributed to the writing of the manuscript: Dong, Varma, Di,

Feng, Terra, Sagawa, Kalgutkar.

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Address correspondence to: Amit S. Kalgutkar, Pharmacokinetics, Dynamics,and Metabolism Department, Worldwide Research and Development, Pfizer Inc.,610 Main Street, Cambridge, MA 02139. E-mail: amit.kalgutkar@pfizer.com

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