Preclinical and Clinical Pharmacokinetics of PF-02413873, aNonsteroidal Progesterone Receptor Antagonist

Peter J. Bungay, Sarah Tweedy, David C. Howe, Karl R. Gibson, Hannah M. Jones,and Natalie M. Mount

Pfizer Worldwide Research & Development, Pharmacokinetics, Dynamics and Metabolism (P.J.B., H.M.J.), Worldwide MedicinalChemistry (K.R.G.) and Clinical Research (S.T., D.C.H., N.M.M.), Sandwich, United Kingdom

Received November 12, 2010; accepted April 15, 2011

ABSTRACT:

The recently discovered selective nonsteroidal progesterone recep-tor (PR) antagonist 4-[3-cyclopropyl-1-(methylsulfonylmethyl)-5-methyl-1H-pyrazol-4-yl]oxy-2,6-dimethylbenzonitrile (PF-02413873)was characterized in metabolism studies in vitro, in preclinical phar-macokinetics in rat and dog, and in an initial pharmacokinetic study inhuman volunteers. Clearance (CL) of PF-02413873 was found to behigh in rat (84 ml � min�1 � kg�1) and low in dog (3.8 ml � min�1 � kg�1),consistent with metabolic stability determined in liver microsomesand hepatocytes in these species. In human, CL was low in relation tohepatic blood flow, consistent with metabolic stability in human invitro systems, where identified metabolites suggested predominantcytochrome P450 (P450)-catalyzed oxidative metabolism. Predictionof CL using intrinsic CL determined in human liver microsomes

(HLM), recombinant human P450 enzymes, and single species scaling(SSS) from pharmacokinetic studies showed that dog SSS and HLMscaling provided the closest estimates of CL of PF-02413873 in hu-man. These CL estimates were combined with a physiologicallybased pharmacokinetic (PBPK) model to predict pharmacokineticprofiles after oral suspension administration of PF-02413873 in fastedand fed states in human. Predicted plasma concentration versus timeprofiles were found to be similar to those observed in human over thePF-02413873 dose range 50 to 500 mg and captured the enhancedexposure in fed subjects. This case study of a novel nonsteroidal PRantagonist underlines the utility of PBPK modeling techniques inguiding prediction confidence and design of early clinical trials ofnovel chemical agents.

Introduction

Endometriosis and uterine fibroids are common gynecological con-ditions that affect approximately 20 to 40% of women. These condi-tions are characterized by chronic pelvic pain, cyclical menstrual pain,excessive menstrual blood loss, and infertility. The most widelyprescribed medical therapies for the treatment of endometriosis aregonadotropin releasing hormone analogs and the progestin medroxy-progesterone acetate (Olive, 2003). Gonadotropin releasing hormoneanalogs and progestins are only for short-term use (6 months), becausethey result in bone mineral density loss as well as other side effectssecondary to hypoestrogenism. A body of preclinical and clinical datasuggests the potential utility of selective progesterone receptor (PR)antagonists, acting as functional estrogen antagonists, in the treatmentof endometriosis and uterine fibroids (Grow et al., 1996; Kettel et al.,1996, 1998; Baird et al., 2003; Fiscella et al., 2006). A selective PR

antagonist would be expected to maintain estrogen at mid follicularlevels, hence avoiding the systemic effects of hypoestrogenism.

A selective nonsteroidal PR antagonist, 4-[3-cyclopropyl-1-(meth-ylsulfonylmethyl)-5-methyl-1H-pyrazol-4-yl]oxy-2,6-dimethylbenzo-nitrile (PF-02413873), was recently identified (Gibson et al., 2009).Preclinical pharmacological and toxicological characterization of PF-02413873 suggested that systemic exposure levels in human that arelikely to be required to elicit the desired functional estrogen antago-nism could do so with an acceptable safety margin. Therefore, itspreclinical pharmacokinetic (PK) properties were investigated to as-sess the likelihood that its PK in human would support its progressionas a potential therapeutic agent. The accurate prediction of PK inhuman can be a significant challenge to drug discovery scientists, anda number of approaches have been used with varying degrees ofsuccess (Beaumont and Smith, 2009). PK prediction methods havebeen described and compared in several retrospective analyses ofgroups of compounds (Obach et al., 1997; Ito and Houston, 2005;Jones et al., 2006a; Hosea et al., 2009), but there have been fewexamples reported that show a PK prediction strategy used in practice

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

doi:10.1124/dmd.110.037234.

ABBREVIATIONS: PR, progesterone receptor; PF-02413873, 4-[3-cyclopropyl-1-(methylsulfonylmethyl)-5-methyl-1H-pyrazol-4-yl]oxy-2,6-di-methylbenzonitrile; PK, pharmacokinetics; FIH, first-in-human; CL, clearance; CL/F, oral clearance; t1/2, half-life; PBPK, physiologically basedpharmacokinetic; PBS, phosphate-buffered saline; PF-02339955, 4-[3,5-dicyclopropyl-1-(methylsulfonylmethyl)-1H-pyrazol-4-yl]oxy-2,6-dimeth-ylbenzonitrile; PF-02327888, 4-[3,5-dicyclopropyl-1-(N-methyl-acetamide)-1H-pyrazol-4-yl]oxy-2,6-dimethylbenzonitrile; FaSSIF, fasted statesimulated intestinal fluid; FeSSIF, fed state simulated intestinal fluid; HPLC, high-performance liquid chromatography; LC, liquid chromatography;MS/MS, tandem mass spectrometry; Fu, unbound fraction; CLu, unbound clearance; P450, cytochrome P450; MRM, multiple reaction monitoring;CLint,app, apparent intrinsic clearance; PEG, polyethylene glycol; SSS, single species scaling; BW, body weight; CLp, plasma clearance; AUC, areaunder the curve; Kp, tissue-to-plasma partition coefficient; CLint, intrinsic clearance; ACAT, Advanced Compartmental Absorption Transit; r,recombinant; HLM, human liver microsomes; BPR, blood/plasma ratio; DLM, dog liver microsomes; RLM, rat liver microsomes.

0090-9556/11/3908-1396–1405$25.00DRUG METABOLISM AND DISPOSITION Vol. 39, No. 8Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics 37234/3701715DMD 39:1396–1405, 2011 Printed in U.S.A.

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before initial first-in-human (FIH) clinical studies (Allan et al., 2008;Yamazaki et al., 2011). The prospective approach used in the presentarticle for PF-02413873 was to integrate the knowledge of its in vitroproperties, including metabolic turnover and routes, with preclinical PKto determine likelihood of appropriate PK in human, by applying PKprediction methods and taking into account the weight of evidencepresented in the literature. PK predictions often focus on parameters suchas clearance (CL) and half-life (t1/2) because these are determinants ofdose and duration of action. However, it can also be of value to predictPK concentration versus time profiles (Allan et al., 2008) and the poten-tial for food intake to influence absorption, particularly where drugcandidates possess high lipophilicity and consequent low solubility. Thelatter point is especially relevant to PF-02413873 because its neutral andlipophilic nature was a requirement for its PR binding affinity.

This article describes the preclinical data obtained in in vitro and invivo systems, the human PK prediction methods used, and the humanPK of PF-02413873 subsequently determined in a FIH clinical study.Based on preclinical data, simulations of PK profiles were performedusing physiologically based pharmacokinetic (PBPK) modeling asimplemented in GastroPlus software (Simulations Plus Inc., Lan-caster, CA), which also enabled the potential for food effects after oraldosing to be examined. This is the first reported study of human PKof a nonsteroidal PR antagonist. It additionally serves as a caseexample of the assessment of PK prediction, whereby the accuracy ofPK prediction methods used for PF-02413873 may be viewed in thecontext of retrospective assessment of published PK prediction meth-ods over ranges of compounds.

Materials and Methods

Dulbecco’s phosphate-buffered saline (PBS) without CaCl2 and MgCl2(PBS) was purchased from Sigma-Aldrich (St. Louis, MO). Spectra/Por dial-ysis membranes (120 � 22 mm, molecular weight cutoff 12–14 kDa) werepurchased from Spectrum Laboratories Inc. (Rancho Dominguez, CA). MF3 (2mM ammonium acetate in 10% methanol, 90% water containing 0.027%formic acid) and MF4 (2 mM ammonium acetate in 90% methanol, 10% watercontaining 0.027% formic acid) were purchased from Romil (Cambridge, UK).PF-02413873 was synthesized in the Chemistry Department, Sandwich Lab-oratories, Pfizer Global Research and Development (Sandwich, UK) andwas �95% pure. 4-[3,5-Dicyclopropyl-1-(methylsulfonylmethyl)-1H-pyrazol-4-yl]oxy-2,6-dimethylbenzonitrile) (PF-02339955) and 4-[3,5-dicyclopropyl-1-(N-methyl-acetamide)-1H-pyrazol-4-yl]oxy-2,6-dimethylbenzonitrile (PF-02327888)were analogs of PF-02413873 that were used as internal standards in bioanalyticalassays and were also synthesized at Pfizer Global Research and Development.

logD7.4. logD7.4 was measured by a fully automated shake flask method,performed on a Hamilton Microlab STAR (Hamilton Robotics, Inc., Reno,NV), that determined the partition of PF-02413873 between octanol andphosphate buffer, pH 7.4 (Stopher and McClean 1990).

Caco-2 Transcellular Flux. The transcellular flux of PF-02413873 acrossCaco-2 cell monolayers was determined in both apical-to-basolateral andbasolateral-to-apical directions using an established method (Walker et al.,2005).

Solubility in Simulated Intestinal Fluids. Simulated intestinal fluids rep-resenting the fasted (FaSSIF) and fed (FeSSIF) states were prepared accordingto published recipes (Klein et al., 2005). FaSSIF medium was supplementedwith 3 mM sodium taurocholate and 1 mM lecithin, pH 6.5, and FeSSIF wassupplemented with 15 mM sodium taurocholate and 9 mM lecithin, pH 6.5. Toassess solubility, an excess of PF-02413873 was shaken in medium for 24 h at37°C, and undissolved material was separated by centrifugation at 16,000g for5 min. Medium concentration of PF-02413873 was determined by a high-performance liquid chromatography (HPLC) method.

Plasma Protein Binding. Plasma protein binding of PF-02413873 wasdetermined at two concentrations of PF-02413873 in rat (1.5 and 15 �g/ml),dog (1.0 and 10 �g/ml), and human (0.1 and 1.0 �g/ml) by equilibriumdialysis. In each species, pooled plasma was obtained from at least five donorsubjects with plasma from males and females pooled separately. Equilibrium

dialysis was performed using a 96-well device in which dialysis membranes(12–14 kDa cutoff) were incorporated that had been soaked in PBS for 1 hbefore use. Plasma samples (150 �l) were dialyzed against an equal volume ofPBS for 4 h at 37°C in an atmosphere of 5% CO2 in the device that had beensealed with a breathable membrane. At the end of the incubation, samples ofplasma and buffer were removed and stored at �80°C until analysis. Sampleswere matrix matched with blank reagents before analysis (i.e., 25 �l of blankPBS was added to 25 �l of plasma samples and vice versa). Protein precipi-tation was performed by addition of 200 �l of methanol containing 100 ng/mlinternal standard (PF-02339955) followed by centrifugation at 2000g at 6°Cfor 15 min. Samples of supernatant (150 �l) were dried under a stream ofnitrogen at 40°C, and the residues were reconstituted with 150 �l of water/methanol (1:1, v/v). After vortex mixing, samples were analyzed on a SCIEXAPI 4000 (AB Sciex Instruments, Foster City, CA) LC-MS/MS turbo ion spraysystem operating in positive ion mode under multiple reaction monitoring. Theelution mobile phase was 45% 10 mM ammonium formate, pH 3.5, 55%acetonitrile delivered at 1 ml/min through a Zorbax SB column (C8 3.5 �m,75 � 4.6 mm; MacMod Analytical, Inc., Chadds Ford, PA). A collision energyof 25 eV was used, and transitions of 360.28 to 280.20 and 386.16 to 306.16were monitored for PF-02413873 and internal standard, respectively. Theunbound fraction (Fu) of PF-02413873 was calculated by dividing the con-centration in buffer by the concentration in plasma.

Blood Partitioning. The blood/plasma ratio of PF-02413873 was deter-mined after incubation of PF-02413873 (1 �g/ml) in rat, dog, and human bloodfor 3 h. After incubation, 50-�l aliquots were removed and the remainingblood was centrifuged at 2000g for 10 min, after which 50-�l aliquots ofplasma were removed. Concentrations of PF-02413873 in whole blood andplasma were determined using the LC-MS/MS method described under PF-02413873 Analysis in Rat and Dog Plasma Samples. Separate calibration lineswere determined using blank blood and plasma containing known addedamounts of PF-02413873.

Metabolic Turnover in In Vitro Systems. The in vitro stability of PF-02413873 was studied in microsomes (BD Gentest, San Jose, CA) and cryo-preserved hepatocytes (InVitro Technologies, Baltimore, MD) from rat, dog,and human liver and in human recombinant cytochrome (P450) isozymes(CYP1A2, 2C9, 2C19, 2D6, and 3A4; PanVera, Madison, WI). All incubationswere performed at a concentration of 1 �M PF-02413873. Liver microsomepreparations were diluted to yield 0.5 �M P450, and recombinant P450isozymes were used at 0.1 �M. Incubations (1.3 ml) contained 50 mM KPO4

buffer (pH 7.4) and 1 mM MgCl2 and were supplemented with a reducingequivalent regenerating system (isocitrate/isocitrate dehydrogenase). The assaycocktail was incubated at 37°C for 15 min before the addition of NADP� (1mM) to initiate reactions. Samples (100 �l) were removed at time intervals upto 60 min after addition of NADP�, and the reaction was quenched by additionof each aliquot to 200 �l of acetonitrile containing fluconazole (1 �M) as aninternal standard. Cryopreserved rat, dog, and human hepatocytes were de-frosted and washed according to the supplier’s instructions. Incubations werecarried out at a concentration of 1 �M PF-02413873 with 0.5 � 106 viablehepatocytes/ml in 50 �l Williams E medium. Incubations were carried out for3 h at 37°C in a CO2 incubator, and reactions were terminated at time intervalsof 3, 5, 10, 15, 30, 45, and 60 min with 200 �l of acetonitrile containingfluconazole (1 �M) as an internal standard.

Samples (80 �l) from microsomal and hepatocyte incubations were ana-lyzed by LC-MS/MS using a Sciex API2000 mass spectrometer (PerkinElmer-Sciex Instruments). The API2000 was operated in positive ion mode using theturbo ion spray interface, and data were acquired in the multiple reactionmonitoring (MRM) mode using argon as the collision gas. Chromatographywas performed using a 2.1-mm C18 Opti-lynx special column with a porediameter of 40 �m (Optimize Technologies Inc., Oregon City, OR) and mobilephases A (2 mM ammonium acetate, pH 3, in 90% water, 10% methanol) andB (2 mM ammonium acetate, pH 3, in 10% water, 90% methanol). A stepgradient of 0 to 10 s 100% A, 11 to 78 s 100% B, and 79 to 116 s 100% A wasused with a flow rate of 1 ml/min. Flow was split (1:5) postcolumn beforeentering the mass spectrometer. Ratios of peak areas of PF-02413873 tointernal standard were calculated, and the natural logarithm of peak area ratiowas plotted against time. The apparent first-order rate constant taken from theslopes of the plots were used to calculate apparent intrinsic clearance (CLint,app)and normalized per milligram of microsomal protein or per 106 hepatocytes.

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Microsomal Binding. A human liver microsomal incubation was preparedas described above, but without addition of NADP�, and left at room temper-ature overnight to inactivate metabolizing enzymes. PF-02413873 was addedto the mixture to a final concentration of 1 �M, and 220 �l was used forequilibrium dialysis against blank buffer mix in a rapid equilibrium device(Waters et al., 2008). After incubation at 37°C for 4 h, samples of microsomalincubation (15 �l) and buffer (45 �l) were added to 120 �l of ice-coldacetonitrile containing internal standard. Concentrations of PF-02413873 inmicrosomal and buffer compartments were then assessed using LC-MS/MS.

Identification of Metabolites of PF-02413873 In Vitro. Experimentsinvestigating routes of metabolism were conducted using the same livermicrosome and hepatocyte preparations used to study metabolic turnover.Microsomal and hepatocyte incubations were conducted with PF-02413873 atan initial concentration of 1 �M. Liver microsomal protein concentration wasadjusted to 0.25 �M of total P450, and hepatocyte incubations were performedat 0.5 � 106 viable hepatocytes/ml. Incubations were conducted in a waterbath at 37°C and, in the case of liver microsomes, were performed in thepresence and absence of NADPH. At appropriate time-points, depending onpreviously measured half-lives, reactions were terminated by addition of fivevolumes of ice-cold acetonitrile. Terminated samples were centrifuged, and thesupernatant was removed and evaporated to dryness under a stream of nitro-gen. Dried samples were stored frozen until analysis.

Qualitative analysis to identify potential metabolites of PF-02413873 wasconducted by HPLC-MS/MS. The HPLC aqueous mobile phase component ofthe binary gradient consisted of 0.1% v/v formic acid in water (mobile phaseA), and the organic component consisted of 0.1% v/v formic acid in acetoni-trile (mobile phase B). An Agilent 1100 binary pumping system (AgilentTechnologies, Santa Clara, CA) was used to control the mobile phase gradientat 95% A for the first minute, changing linearly to 2% A at 8 min, followed by1 min held at 2% A, then back to 95% A over 0.1 min, and held for 4 min. Thegradient was delivered to a 2.1 � 100 mm Sunfire C18 3 �m analytical column(Phenomenex, Torrance, CA) via a CTC HTS Pal autosampler (LEAP Tech-nologies, Carrboro, NC). The flow rate was set at 200 �l/min and wasintroduced into the source of the mass spectrometer (Q-ToF Premier; Waters,Milford, MA) with no postcolumn split. The mass spectrometer was coupled tothe HPLC via a Z-Spray interface (Micromass, Altrincham, UK) with adesolvation temperature of 350°C, a source temperature of 120°C, and a conevoltage of 25 V. Leucine-enkephalin was introduced into the ion source via alock-spray inlet to provide a standard for accurate mass calibration. Positiveion mass spectrometry and MS/MS experiments were performed with thetime-of-flight operated in V-mode, and data acquisitions were performed incentroid mode.

Animal Experimentation. All studies involving animals were conducted incompliance with United Kingdom national legislation and subject to localethical review. At all stages, consideration was given to experimental refine-ment, reduction in animal use, and replacement with in vitro techniques.

PK in the Rat. Male Sprague-Dawley rats weighing approximately 250 gwere used in the following studies. At least 48 h before compound adminis-tration, animals were surgically prepared by the placement of a cannula in thejugular vein. Throughout the study, animals were housed in stock boxes withaccess to food (RM1 rodent diet; Special Diet Service, Witham, Essex, UK)and water ad libitum. PF-02413873 was dissolved in 20% polyethylene glycol(PEG) in saline to a final concentration of 0.2 mg/ml. A total of 2 ml/kg wasdosed either intravenously via the tail vein or orally by gavage to give a finaldose of 0.4 mg/kg. Separate groups of rats (n � 3) were used for each doseroute. After dosing by either route, blood (approximately 175 �l) was with-drawn via the jugular vein catheter at various times into heparin-coated tubes.The cannula was flushed with heparinized saline (10 units/ml) after eachsample. Plasma was prepared by centrifugation at 2000g, and the samples werethen stored at �20°C before analysis.

PK in the Dog. One female and one male dog weighing approximately 12kg were used for the intravenous and oral studies conducted 1 week apart.Before the studies, the dogs were given food and water ad libitum. For bothintravenous and oral administration, PF-02413873 was dissolved in dimethylsulfoxide to a concentration of 10 mg/ml, then diluted with PEG 200 to 0.5mg/ml, and finally with saline to a final concentration of 0.1 mg/ml in 1%dimethyl sulfoxide 20% PEG 200 in saline. Intravenous administration was byinfusion via the saphenous vein of 2 ml/kg over 15 min to give a final dose of

0.2 mg/kg, and oral administration was via gavage at 2 ml/kg to give a finaldose of 0.2 mg/kg.

On the day of dosing, the dogs were placed in slings and remained there forthe first 6 h after dosing. This allowed blood samples (approximately 2 ml) tobe collected from the saphenous vein by means of indwelling catheters. Afterthis period, the dogs were returned to their pens and samples were obtainedfrom the cephalic vein by venipuncture. After sampling, blood was centrifugedat 2000g and plasma was removed into separate Eppendorf tubes, which werestored at �20°C until analysis.

PF-02413873 Analysis in Rat and Dog Plasma Samples. PF-02413873was extracted from 50-�l aliquots of rat or dog plasma using a specificliquid-liquid extraction and HPLC method with mass spectrometric detection.Samples were prepared by the addition of 1 ml borate buffer, pH 10, containing10 ng of internal standard PF-02327888, followed by 2 ml of tert-butylmethyl-ester. Samples were vortex mixed, centrifuged at 2000g for 5 min, and 1.8 mlof the tert-butylmethylester layer was removed and blown to dryness undernitrogen at 40°C. The residues were reconstituted in 300 �l of MF3, and 180�l was injected into an LC-MS/MS system for quantitation of analyte. In eachassay, standard calibration lines were constructed using known amounts ofPF-02413873 added into 50-�l aliquots of blank plasma from the appropriatespecies.

Samples were analyzed using a Sciex API 4000 with turbo ion sprayinterface in positive ion multiple reaction monitoring mode with collisionenergy of 25 eV. The MRM transitions monitored were 360 to 280 forPF-02413873 and 358 to 278 for PF-02327888. Each sample was injectedsequentially into the LC-MS/MS system with a CTC PAL autoinjector. Themobile phase (flow rate, 3 ml/min) was split 5:1 postcolumn using an Acurateflow splitter (Presearch Ltd., Hitchin, Hertfordshire, UK).

Clinical Study in Human Volunteers. The randomized, double-blind, thirdparty open, placebo controlled dose-escalation study was conducted in healthymale subjects. The protocol was reviewed and approved by an independentethics committee, and all subjects gave written informed consent. PF-02413873 was administered orally as a micronized suspension. Subjects weredivided into two cohorts: Cohort 1 received 4 escalating doses (0.5, 5, 50, and150 mg) with randomized placebo insertion in the fasted state plus one dose(150 mg) in the fed state; Cohort 2 received 3 doses (500, 1500, and 3000 mg)with randomized placebo insertion in the fed state. Subjects in the fed statereceived a standard high-fat, high-calorie breakfast. Serial blood samples fordetermination of plasma concentrations of PF-02413873 were collected for upto 120 h in Cohort 1 and up to 240 h in Cohort 2. Standard noncompartmentalPK analysis was performed on plasma concentration data.

Analysis of Clinical PK Samples. Plasma samples were analyzed with avalidated assay using PF-02339955 as an internal standard. PF-02413873 andinternal standard were extracted from 100 �l of human plasma using solid-phase extraction. Thawed plasma samples were briefly mixed and centrifugedat 2000g for 10 min at room temperature. One hundred microliters of eachsample was mixed with 100 �l of methanol containing 200 ng/ml PF-02339955. After preconditioning of 15 mg of OMIX C18 tips (Quadra;Tomtec, Hamden, CA) with 2 � 150 �l of methanol followed by 150 �l ofwater, the samples were loaded onto the C18 micropipette tips. The tips werewashed with 2 � 150 �l of water, and the analytes eluted with 2 � 75 �l ofmethanol. Eluates were evaporated under nitrogen at 40°C, reconstituted with200 �l of MF5, and shaken for 10 min. Fifty microliters of each reconstitutedsample was injected onto an LC-MS/MS system containing a 100 � 4.6-mmOnyx Monolithic column (Phenomenex), and analyte was measured byMS/MS detection in an API 3000 triple-quadrupole mass spectrometer (ABSciex Instruments) operating in positive ion MRM mode. The lower and upperlimits of quantification of this assay were 1 and 500 ng/ml PF-02413873,respectively.

Prediction Methods. CL prediction from allometric SSS. PK in rat and dogwere used to predict CL in human using the following equation (Hosea et al.,2009): human CLu � animal CLu � ((human BW/animal BW)0.75), where BWis body weight (kg), assumed to be 0.25 for rat, 12 for dog, and 70 for human,and CLu is the unbound plasma CL with units of ml/min. Then, plasma CL(CLp, ml � min�1 � kg�1) in human is given by the following: human CLp �human CLu � Fu/human BW.

CL prediction from in vitro metabolism. Using human liver microsomalturnover, prediction of in vivo intrinsic CL and, subsequently, hepatic CL

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was carried out using physiologically based scaling factor and the “wellstirred” liver model as described in the literature (Obach et al., 1997;Ito and Houston, 2005). It was assumed that human liver contains45 mg/g microsomal protein and that there are 21 g liver/kg human bodyweight.

Dose prediction. Doses of PF-02413873 predicted to be required to achievea given exposure were calculated from the relationship, assuming 100%

absorption: Dose � CLp/(1 � (CLb/Q)) � AUCeff, where Q � human hepaticblood flow; BPR � blood/plasma ratio; CLb � blood CL, calculated asCLp/BPR; AUCeff � target AUC in plasma at steady state.

Prediction of plasma concentration versus time profiles in GastroPlus. ThePBPK intravenous (disposition) and oral simulations were performed withinthe GastroPlus software using version 5.0. These models have been describedin detail previously (Jones et al., 2009). The choice of GastroPlus softwareoffered the potential to incorporate biorelevant solubility and simulate PKprofiles under fed and fasted conditions. The PBPK model was composed ofseveral compartments corresponding to the different tissues of the body whichare connected by the circulating blood system. Each compartment was definedby a tissue volume and a tissue blood flow rate that is specific for the speciesof interest. Each tissue was assumed to be perfusion rate limited. The liver wasconsidered to be the only site of elimination. Tissue-to-plasma partition coef-ficients (Kps) and intrinsic CL (CLint) predicted from preclinical data wererequired as input to this model. The Kp values were predicted from physico-chemical and in vitro inputs using methods described by Rodgers and Rowland(2006). In brief, these equations account for partitioning of un-ionized druginto neutral lipids and neutral phospholipids, dissolution of drug in tissuewater, and interactions with extracellular protein.

The Advanced Compartmental Absorption Transit (ACAT) model withinGastroPlus was used to predict the rate and extent of oral absorption inhuman and preclinical species. The ACAT model has been described indetail previously (Agoram et al., 2001) and is based on the CAT modeldescribed by Yu and Amidon (1999). The main input parameters for the

FIG. 1. Proposed pathways of metabolism of PF-02413873 in microsomes and hepatocytes from rat, dog, and human liver. Labels next to arrows denote that metabolitewas formed in microsomes from rat (RLM), dog (DLM), and human (HLM) and hepatocytes from rat (rHEP), dog (dHEP), or human (hHEP) liver.

TABLE 1

Input data used in the GastroPlus PBPK model

Parameter Value

Molecular weight (g/mol) 359.5LogD at pH 7.4 3.4CaCo-2 permeability (10�6cm/s) 41Effective human permeability (10�4cm/s) 6.9FaSSIF solubility (mg/ml) 0.012FeSSIF solubility (mg/ml) 0.039Particle radius, micronized suspension (�m) 1.6Blood/plasma ratio in rat, dog, human 0.93, 0.82, 0.61Plasma Fu in rat, dog, human 0.047, 0.036, 0.031CLint HLM (�l � min�1 � mg�1) 29Fu,mic in HLM 0.78Human CLp predicted (from HLM) (ml � min�1 � kg�1) 1.0Human CLp predicted (from dog PK) (ml � min�1 � kg�1) 2.6Fu gut 1

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absorption prediction were as follows: solubility, permeability, particlesize, LogD, and dose (Table 1).

The following PBPK prediction strategy was followed as proposed by Joneset al. (2006a):

1. validation of intravenous disposition prediction in rat and dog2. validation of oral absorption prediction in dog3. simulation of disposition and absorption in human

Results

LogD7.4 of PF-02413873. The geometric mean logD7.4 of PF-02413873 was found to be 3.4 � 0.03 (S.E.M., n � 6), consistent withits nature as a lipophilic, neutral (nonionizable) compound.

Validation of Analytical Methods. The PF-02413873 rat plasmaassay displayed linearity of signal with concentration over a calibra-tion range of 0.2 to 1000 ng/ml. Inaccuracy (6 replicate samples) was1.4, 10.9, 2.8, and 10.7% at 1, 20, 100, and 200 ng/ml, respectively,and the imprecision at the same concentrations was 3.1, 8.7, 1.3, and8.3%. The dog plasma assay displayed linearity of signal with con-centration over a calibration range of 0.2 to 1000 ng/ml. Inaccuracy (6replicate samples) was 1.7 and 3.7% at 5 and 500 ng/ml, respectively,and the imprecision at the same concentrations was 11.7 and 2.9%.The clinical assay was linear over a calibration range of 1 to 400ng/ml and had an inaccuracy at 1, 3, 250, and 400 ng/ml of 7, 7, 2.8,and 0.5%, respectively. At the same concentrations, the imprecisionwas 6.1, 3.9, 2.6, and 6.4%.

Plasma Protein Binding and Blood/Plasma Ratio. In rat, dog,and human plasma, no concentration dependence of binding or dif-ferences between male and female were evident across a concentra-tion range of 1.5 and 15 �g/ml (rat), 1 and 10 �g/ml (dog), and 0.1and 1.0 �g/ml (human) (data not shown). Therefore, a single value ofFu was used for each species based on the overall mean of the dataacross concentration and sexes. Thus, the Fu of PF-02413873 in rat,dog, and human plasma was 0.042, 0.035, and 0.031, respectively.The blood/plasma ratios of PF-02413873 in rat, dog, and human were0.93, 0.82, and 0.61, respectively.

In Vitro Metabolism by Liver Microsomes and RecombinantP450s and Microsomal Binding. In rat liver microsomes, theCLint,app of PF-02413873 was �500 �l � min�1 � mg protein�1 (t1/2] � �2min). In dog and human microsomes, CLint,app values were signifi-cantly lower than in rat, at 36 and 29 �l � min�1 � mg protein�1,respectively (t1/2 � 25 and 30 min, respectively). The unboundfraction of PF-02413873 in human liver microsomes was 0.78. In therecombinant P450 experiments, turnover of PF-02413873 was onlydetectable in the presence of rCYP3A4. The CLint,app value (1.27 �l �min�1 � pmol P450�1) calculated for rCYP3A4 was subsequentlyscaled to human liver, using the appropriate relative activity factor andintersystem extrapolation factor (Youdim et al., 2008). The scaledrCYP3A4 CLint value was 25.9 �l � min�1 � mg protein�1.

In Vitro Metabolism by Hepatocytes. In cryopreserved rat anddog hepatocytes, CLint,app of PF-02413873 was �500 and 20 �l �min�1 � 106 cells�1, respectively (t1/2 � �2 and 35 min, respectively).In human hepatocytes, the turnover of PF-02413873 (t1/2 � �120min) was too low to allow an accurate measurement (�6 �l � min�1 �106 cells�1).

Metabolite Profiles in In Vitro Systems. The main metabolites ofPF-02413873 formed by liver microsomes and hepatocytes are shownin the scheme in Fig. 1. After incubation with human liver micro-somes, the major metabolite of PF-02413873 resulted from mono-oxidation of one of the methyl groups on the dimethylbenzonitrilemoiety (M1). Further minor products of mono-oxidation of the methyland/or cyclopropyl substituents of the pyrazole ring were also de-tected (M2 and M3), along with products of di-oxidation of both ringsystems (M4 and M9). The mono-oxidized metabolite M3 was theonly product formed after incubation of PF-02413873 in humanhepatocytes. In rat and dog liver microsomes and hepatocytes, asimilar range of mono-oxidized (M1, M2, M3) and di-oxidized prod-ucts (M4 and M9) were found. The only product of phase 2 conju-gation reactions that was found was a glucuronide in dog hepatocytes,a minor product that was most likely the result of conjugation to ahydroxylated metabolite (data not shown).

PK in Rat. The PK of PF-02413873 were determined after intra-venous administration at a single dose of 0.4 mg/kg in male rats(Fig. 2; PK parameters in Table 2). The mean elimination half-life was1.1 h and CLp was 84 ml � min�1 � kg�1. After oral dosing, plasmaconcentrations of PF-02413873 were below the level of quantitation.Assuming that hepatic blood flow in the rat is 70 ml � min�1 � kg�1

(Boxenbaum, 1980), the lack of exposure after oral administration isconsistent with the CLp observed after intravenous administration,i.e., CLp � hepatic blood flow.

PK in Dog. The PK of PF-02413873 was determined after intra-venous administration at a single dose of 0.2 mg/kg in dogs. The mean

FIG. 2. PF-02413873 pharmacokinetics after intravenous administration in malerats (0.4 mg/kg). Symbols represent the mean value from three animals (error barsrepresent S.D.).

TABLE 2

Pharmacokinetic parameters of PF-02413873 in rat after intravenous bolus andoral solution administration

Data are mean � S.D. (n � 3).

Parameter Intravenous Oral

n 3 3Dose (mg/kg) 0.4 0.4CLp (ml � min�1 � kg�1) 84 � 21Vdss (l/kg) 5.3 � 1.5t1/2 (h) 1.1 � 0.1MRT (h) 2.4 � 0.6AUCinf (ng � h�1 � ml�1) 83 � 24 0F (%) 0

CLp, plasma clearance; AUCinf, area under plasma concentration vs. time profile from time0 extrapolated to infinite time; t1/2, terminal half-life; Vdss, volume of distribution at steady state;MRT, mean residence time; F, oral bioavailability.

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elimination half-life was 7.6 h, CLp was 3.8 ml � min�1 � kg�1, and Vss

was 2.5 l/kg. The plasma concentrations after oral dosing of PF-02413873 are shown in Fig. 3, and the PK parameters are shown inTable 3. In contrast to rat, the plasma exposure and calculated PKparameters demonstrated high oral bioavailability of PF-02413873 inthe dog and, furthermore, were consistent with complete absorption.

PK in Male Volunteers. The PK profiles of PF-02413873 inCohort 1 (doses of 0.5–150 mg) are shown in Fig. 4, the PK profilesin Cohort 2 are shown in Fig. 5, and the derived PK parameters forboth cohorts are shown in Table 4. In subjects that were fasted,exposure increased slightly less than proportionally with doses be-tween 5 and 150 mg. Exposure at 0.5 mg was too low in relation to thelimit of quantitation to allow satisfactory calculation of all PK param-eters. At 150 mg, dosing in fed subjects increased AUCinf by approx-imately 60% in comparison with fasted subjects, with a corresponding76% increase in Cmax and delay in Tmax from 3 to 6 h. In fed subjectsoverall, exposure increased roughly in proportion to dose between 150and 3000 mg. The terminal half-life of PF-02413873 over the doserange varied between 28 and 44 h, with no obvious pattern of variationthat reflected dose or fed or fasted states. Oral CL ranged between

approximately 4 and 7 ml � min�1 � kg�1, with the lower valuesreflecting higher AUC obtained per unit dose in the fed state.

Prediction of Human PK. The predictions of the Vdss of PF-02413873 in rat, dog, and human using the tissue composition equa-tions developed by Rodgers and Rowland (2006) are shown inTable 5. These equations provided accurate estimates of Vdss in ratand dog. When the Kp values provided by this prediction method werecombined with the observed CL as inputs, simulation of the intrave-nous concentration versus time profiles in rat and dog providedgood approximations to the observed data. Therefore, the Rodgersand Rowland (2006) equations were used for PBPK simulation inhuman.

The ACAT model was able to accurately simulate the dog oralplasma concentration versus time profiles using the in vitro solubility,predicted Kp values, and observed intravenous CLp as inputs to themodel (Fig. 6). Therefore, the ACAT model was used to simulateabsorption in human.

FIG. 3. PF-02413873 pharmacokinetics after intravenous (closed circles) and oralsuspension (open symbols) dosing (0.2 mg/kg) in dogs (n � 2). Symbols representthe mean value from two animals.

FIG. 4. PF-02413873 pharmacokinetics after oral suspension dosing in Cohort 1.Doses were 5 mg, fasted (open circles, n � 7); 50 mg, fasted (open triangles, n �8); 150 mg, fasted (open squares, n � 7); and 150 mg, fed (closed squares, n � 7).The error bars represent S.D.

FIG. 5. PF-02413873 pharmacokinetics after oral suspension dosing in Cohort 2(fed condition). Doses were 500 mg (circles, n � 8), 1500 mg (triangles, n � 8), and3000 mg (squares, n � 2). The error bars represent S.D.

TABLE 3

Pharmacokinetic parameters of PF-02413873 in dogs after intravenous and oralsolution administration

ParameterFemale Male

Intravenous Oral Intravenous Oral

Dose (mg/kg) 0.2 (infusion) 0.2 0.2 (infusion) 0.2CLp (ml � min�1 � kg�1) 4.5 4.7Vdss (l/kg) 2.3 4.8Terminal half-life (h) 6.1 11.6MRT (h) 8.7 17.9AUCinf (ng � h�1 � ml�1) 748 601 705 752F (%) 80 107Cmax (ng/ml) 96.4 109.6Tmax (h) 1.5 3.0Oral half-life (h) 11.4 21.9

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Human CLp was predicted to be 13.5 and 2.6 ml � min�1 � kg�1

when scaled from unbound CLp in rat and dog, respectively. When invitro CLint determined in human liver microsomes (HLM) was used,the predicted human CLp was 1.0 ml � min�1 � kg�1. The predictionof blood CL from rat SSS (CLp/BPR � 22 ml � min�1 � kg�1) wasclose to hepatic blood flow in human (assumed to be 20 ml � min�1 �kg�1). For this reason, CL from rat SSS was not used in subsequentsimulations using GastroPlus.

Because intravenous administration was not undertaken in theclinical study, CLp predicted from preclinical data were comparedwith the range of oral CL (CL/F) determined in the clinical study. Thisdemonstrated an overprediction by rat SSS. Predictions from dog SSSand from scaling of HLM turnover were more comparable with themeasured human CL/F, and they were broadly consistent with char-acterization of PF-02413873 as a low CL molecule in human. DogSSS provided the closest estimates of Cmax and AUC observed in theclinical study and predicted an oral CL of 5.5 ml � min�1 � kg�1,assuming complete absorption.

Separate preclinical pharmacological studies of PF-02413873 pre-dicted that steady-state systemic exposure (AUC0–24) of approxi-mately 6000 ng � h/ml would be required to elicit a pharmacologicaleffect in human (data not shown). This information was combinedwith CL/F predictions to calculate approximate doses of 30 mg (HLMscaling) and 85 mg (dog SSS) that would be expected to be pharma-cologically active and hence inform the range to be explored in earlyhuman studies (5–3000 mg).

GastroPlus simulations of the plasma concentration versus timeprofile of PF-02413873 after micronized suspension doses of 50, 150,and 500 mg in human alongside the actual measured plasma concen-trations are shown in Fig. 7. In general, the shapes of the profiles werepredicted well using the Rodgers and Rowland (2006) Kp values.However, plasma exposures of PF-02413873 (Cmax and AUC) were

generally overpredicted using CLp predicted from HLM (up to 4-fold)and dog PK (up to 3-fold) (Table 6), indicating a potential underpre-diction of CL.

At a dose of 150 mg, using CL predicted from HLM or dog SSS,biorelevant solubility (FaSSIF and FeSSIF), together with alteredphysiologies in the two feeding states, GastroPlus predicted increasesin Cmax (approximately 2-fold) and AUC (approximately 1.2-fold)with food. These compared well with observed increases in Cmax andAUC in the clinical PK study of 1.8- and 1.6-fold, respectively. Themodel also predicted the slight reduction in dose-normalized exposurethat was observed in the fasted state between 50 and 150 mg, consis-tent with saturation in absorption (Table 6). However, as doses in-creased to 500, 1500, and 3000 mg, the model predicted progressivereduction of dose-normalized exposure, while it remained broadlyconstant in the clinical study.

Discussion

A combination of preclinical in vitro metabolism and in vivo PKdata was used to determine the likelihood that the PK of PF-02413873in human would be suitable for its progression to clinical studies. Inhuman in vitro systems, turnover of PF-02413873 was relatively low,so that scaling of HLM CLint to in vivo CL using the appropriatescaling factors and the well stirred liver model predicted low CL inhuman (�20% hepatic blood flow). The PK of PF-02413873 in ratand dog demonstrated that CL was high (greater than hepatic bloodflow) in rat and low in dog (approximately 10% of hepatic bloodflow). This result was in keeping with metabolic stability of PF-02413873 in liver microsomes and hepatocytes obtained from thesespecies. These differences in PK and metabolic stability between ratand dog were reflected in predictions of human CL by SSS (Hosea etal., 2009) from the two species which are widely different; dogpredicted low CL (approximately 20% hepatic blood flow) and ratpredicted high CL (similar to human hepatic blood flow).

Metabolites of PF-02413873 formed in in vitro systems in all threespecies were products of oxidation. Although appearance in urine ofunchanged PF-02413873 was not determined in the PK studies, itsphysicochemical characteristics (lipophilic neutral compound) make itunlikely that renal excretion is a significant CL pathway. Therefore,the major route of CL in vivo is likely to be via oxidative metabolismcatalyzed by hepatic P450 enzymes. In the absence of quantifiablemetabolite samples, rates of metabolite formation were not investi-

TABLE 4

PF-02413873 pharmacokinetic parameters in human male volunteers after oral micronized suspension dosing

Dose n AUC0–24a AUCinf

a Cmaxa Tmax

b t1/2c Vz/Fc CL/Fc

ng � h � ml�1 ng � h � ml�1 ng/ml h h l/kg ml � min�1 � kg�1

Cohort 1Fasted

0.5 mg 8 NC NC 3.74 (28) 0.50 (0.50,1.0) NC NC NC5 mg 7 148 (23) 191 (36) 19.8 (36) 2.0 (0.50,4.0) NC NC NC50 mg 8 1220 (17) 2300 (23) 117 (34) 1.0 (0.50,4.0) 33.7 (27) 15.0 (25) 5.3 (21)150 mg 7 2910 (19) 5290 (21) 242 (17) 3.0 (2.0,4.0) 28.0 (18) 16.4 (23) 6.9 (20)

Fed150 mg 7 4810 (14) 8310 (32) 427 (28) 6.0 (3.0,6.0) 31.4 (31) 11.5 (27) 4.5 (31)

Cohort 2Fed

500 mg 8 14,500 (12) 28,400 (20) 1420 (33) 6.0 (4.0,8.0) 42.7 (22) 15.4 (20) 4.3 (21)1500 mg 8 38,800 (19) 74,500 (16) 3350 (29) 5.0 (4.0,6.1) 43.6 (25) 18.3 (30) 4.4 (16)3000 mg 2 74,500 (18) 151,000 (13) 5420 (20) 4.0 (4.0,4.0) 39.7 (4) 16.3 (10) 3.9 (13)

n, number of subjects assessed; AUC0–24, area under the concentration-time curve from time 0 to 24 h postdose; AUCinf, area under plasma concentration vs. time profile from time 0 extrapolatedto infinite time; Cmax, first incidence of maximal observed plasma concentration; Tmax, time at which Cmax was observed; t1/2, terminal half-life; Vz/F, apparent volume of distribution associatedwith the terminal phase; CL/F, apparent clearance after oral administration; NC, not calculated; CV, coefficient of variation.

a Geometric mean (CV%).b Median (range).c Arithmetic mean (CV%).

TABLE 5

Prediction of Vdss of PF-02413873 in rat, dog, and human using tissuecomposition equations

SpeciesPlasma Volume of Distribution at Steady State (l/kg)

Measured Rodgers and Rowland (2006) Single Species Scaling

Rat 5.1 3.5Dog 2.5 2.8Human 2.6 3.5 (rat) 2.9 (dog)

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gated. However, other studies that monitored disappearance of PF-02413873 showed that it was turned over by recombinant humanP450s, with CYP3A4 displaying the highest turnover of those en-zymes tested. When appropriate scaling factors were applied to thedata based on enzyme abundance in human liver, a CLint value forCYP3A4 turnover of PF-02413873 was calculated that was similar tothat measured in HLM. These data are consistent with CYP3A4 beingthe major P450 isoform involved in metabolism of PF-02413873 inhuman liver. However, further studies in vivo would be required toconfirm the dominance of oxidative pathways in human and also tounderstand the potential pharmacokinetic interactions between PF-

02413873 and other drugs known to be metabolized principally byCYP3A4 or that are inhibitors of this enzyme.

Before constructing a model in GastroPlus for human PK simula-tion, the assumptions of the model were tested by reference to ob-served rat and dog PK profiles (Jones et al., 2006a; DeBuck et al.,2007). This validation exercise showed that the Rodgers and Rowland(2006) equations provided the simulated profiles that most closelyresembled the observed data. The intrinsic absorption of PF-02413873was expected to be good, based on comparison of intravenous and oralPK determined in dog. The simulation of absorption in the dog usingthe ACAT model was found to provide a good approximation to the

FIG. 6. Predicted and observed plasma concen-tration versus time profiles of PF-02413873after intravenous administration to rat (A), anddog (B), and oral solution administration to dog(C). Open circles represent observed data fromindividual animals, and the solid lines representthe profiles predicted from the PBPK model inGastroPlus. Doses were 0.4 mg/kg in rat and0.2 mg/kg in dog.

FIG. 7. Predicted and observed plasma concen-tration versus time profiles of PF-02413873after administration of micronized suspensionto human volunteers. Symbols represent meanobserved data, and error bars represent S.D.The simulated profiles from the PBPK model inGastroPlus are shown, using clearance pre-dicted from dog SSS (solid lines). Doses were50 mg under fasted conditions (A), 500 mgunder fed conditions (B), and 150 mg underboth fasted (closed symbols, solid line) and fed(open symbols, dotted line) conditions (C).

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observed oral PK data (Fig. 6C). Because PF-02413873 possesseshigh lipophilicity (logD � 3.4) and consequent low aqueous solubility(�10 �g/ml in aqueous buffer), the formulation chosen for clinicaluse was a micronized suspension, intended to maximize particlesurface area and, hence, dissolution rate. Thus, the absorption modelused the particle size in the micronized formulation and solubilitydetermined in biorelevant media (FaSSIF and FeSSIF). Estimates ofCL from HLM and dog PK were then combined with the absorptionand distribution model in GastroPlus, and simulations of PK profileswere performed with inputs of oral suspension doses in the plannedFIH study single ascending dose range. Given the range of CLpredictions, some uncertainty in the outcome of the FIH study existed.For instance, taking a Vdss of 2.6 l/kg [Rodgers and Rowland (2006)prediction], effective t1/2 estimates of approximately 8 and 24 h werecalculated from CL predictions made from dog SSS and HLM scaling,respectively, whereas, conversely, prediction from rat SSS was con-sistent with little oral exposure due to a predicted large first pass.However, metabolic turnover rates exhibited by rat and dog livermicrosomes were consistent with their respective CL determined inthese species in vivo, providing some confidence that the low turnoverin HLM would translate to low CL in human. Furthermore, similarityin microsomal CLint in dog and human suggest that the dog, ratherthan rat, may be the most appropriate species for SSS of human CL ofPF-02413873. Therefore, confidence in acceptability of human PKwas considered to outweigh the uncertainty, supporting progression ofPF-02413873 to a FIH study.

The human PK of PF-02413873 determined after oral dosing in thePhase 1 study was characterized by low CL/F (4–7 ml � min�1 � kg�1)and terminal t1/2 of 28 to 44 h, characteristics compatible with highoral bioavailability and a once-daily dose that would be desirable fortherapeutic administration. Of the human CL prediction methodsused, SSS from rat was not found to be a good guide because it greatlyoverpredicted observed CL. The predictions from dog SSS and scalingfrom HLM were much more consistent with the low oral CL exhibitedby PF-02413873, in accordance with the observed metabolic turnoverin liver microsomes (CLint in RLM �� DLM � HLM). Previousretrospective analysis of CL prediction methods suggested that if CLis principally via metabolism catalyzed by P450, HLM scaling was aspredictive of oral CL as SSS based on data from rat, dog, and monkey(Hosea et al., 2009). In the present study, metabolite profiling andscaling of CLint catalyzed by recombinant CYP3A4 were consistentwith P450-mediated CL of PF-02413873. HLM scaling correctlypredicted CL to be low in relation to hepatic blood flow, although thevalue was just below the observed CL range. Although turnover inHLM of lower concentrations of PF-02413873 could indicate poten-tial underestimation of CLint at 1 �M, P450 inhibition data (CYP3A4

IC50 �10 �M; data not shown) were consistent with a CYP3A4 Km

for PF-02413873 of �1 �M. Dog SSS provided the estimate of CLclosest to that observed, and it was a better predictive model than ratin this instance. This corresponds with the retrospective analysis ofHosea et al., 2009, who showed that dog SSS predicted oral CL within2-fold of that observed in 52% of P450-cleared compounds, whereasHLM scaling achieved 30%. Our prospective use of preclinical datashowed the following: 1) two of three prediction methods (HLM anddog SSS) predicted low human CL; 2) CL was likely to be principallyvia metabolism catalyzed by P450; and 3) in vitro CLint and in vivoCL correlated in dog and rat, i.e., low DLM CLint and high RLM CLint

predicted low dog CL and high rat CL, implying P450-mediated CL,and that low HLM CLint is likely to predict low human CL.

Dose escalation in fasted subjects provided approximately dose-proportional increases in AUCinf but less than dose-proportional in-creases in Cmax. Simulations in GastroPlus displayed trends towardoverprediction of Cmax and underprediction of the terminal phasehalf-life, which could be explained by an underprediction of Vdss.This theory was confirmed by increasing the muscle Kp (largest tissuemass), resulting in a lower predicted Cmax and a better prediction ofthe terminal phase (data not shown).

Provision of a standard high-fat, high-calorie food before doseadministration led to an increase in exposure over fasted subjects, asdemonstrated by an increase in AUCinf and Cmax at a dose of 150 mg.Remarkably, exposure was also found to increase largely in propor-tion to dose in fed subjects from 150 to 3000 mg. This suggests thatabsorption was enhanced in fed subjects, presumably as a result ofchanges in increased secretion of bile acids with consequent higherdissolution rate. The increased exposure with food was captured atdoses of 150 and 500 mg using the ACAT model, which accounts forthe increased stomach transit time, the change in stomach pH, and thebiorelevant solubility data that incorporates solubility differences inthe presence and absence of bile salts. Although the model was unableto fully account for the approximately dose-linear exposure increasefrom 500 to 3000 mg under fed conditions, it was able to predict thatdosing PF-02413873 with food would increase exposure at the higherend of its dose range and permit extensive testing of pharmacologi-cally relevant exposures. Other studies have demonstrated the utilityof PBPK methodology and GastroPlus, incorporating biorelevant sol-ubility, for simulating food effects of lipophilic compounds (Jones etal., 2006b; Parrott et al., 2009), although these examples were gen-erally weak bases. In this program, as the modeling provided evidencefor a higher exposure with food, the plan for the subsequent repeatdose part of the clinical study incorporated fed subjects to optimizeexposure for a given dose. Furthermore, the PBPK model in Gastro-Plus allowed simulation of repeat dose scenarios of PF-02413873,

TABLE 6

Predicted and observed PF-02413873 AUC and Cmax in human after oral micronized suspension dosing

Fasted Fed

50 mg 150 mg 150 mg 500 mg 1500 mg 3000 mg

AUCinf (ng � h � ml�1)Mean observed 2300 5290 8310 29,400 74,500 151,000GastroPlus, dog CLp 3579 8722 10,745 32,056 50,392 59,924GastroPlus, HLM CLp 10,920 26,606 32,780 97,790 153,700 182,700

Dose-normalized AUCinf (ng � h � ml�1 � mg�1)Mean observed 46 35 55 59 50 50GastroPlus, dog CLp 72 58 72 64 33 20GastroPlus, HLM CLp 218 177 218 196 102 61

Cmax (ng/ml)Mean observed 117 245 427 1420 3350 5420GastroPlus, dog CLp 324 459 961 1817 2121 2268GastroPlus, HLM CLp 440 652 1310 2620 3170 3448

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targeting pharmacologically relevant exposures that were also withinacceptable limits established in separate preclinical safety studies(e.g., 150 mg per day under fed conditions).

In conclusion, the nonsteroidal PR antagonist PF-02413873 pos-sessed low oral CL in human with a PK profile consistent with a oncedaily dosing schedule. Further studies are required in appropriatesubjects to confirm the duration of desired PR pharmacological effectsthat would lead to amelioration of symptoms in gynecological dis-eases. The PK characteristics, including an increase in exposure in fedsubjects, were predicted well using an ACAT model in GastroPlus andlow CL prediction from HLM scaling and dog SSS. This represents acase study that highlights the prospective use of preclinical datacombined with PBPK modeling techniques to provide predictions thatguide decision making and design of early clinical trials of novelchemical agents.

Acknowledgments

We thank Ben Laverty, Claire Collins, Christine Tyman, Kuresh Youdim,and Michelle Gleave for preclinical work on PF-02413873 and Asadh Miah forbioanalysis of clinical samples.

Authorship Contributions

Participated in research design: Bungay, Tweedy, Howe, Gibson, andMount.

Conducted experiments: Tweedy.Contributed new reagents or analytic tools: Gibson.Performed data analysis: Bungay and Jones.Wrote or contributed to the writing of the manuscript: Bungay, Jones, and

Mount.

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Address correspondence to: Peter Bungay, Pharmacokinetics, Dynamics &Metabolism, Pfizer Worldwide Research & Development, Ramsgate Road, Sand-wich, Kent, CT13 9NJ, UK. E-mail: peter.bungay@pfizer.com

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