anti-inflammatory properties of a novel n-phenyl...

Anti-Inflammatory Properties of a Novel N-Phenyl PyridinoneInhibitor of p38 Mitogen-Activated Protein Kinase:Preclinical-to-Clinical Translation

Heidi R. Hope, Gary D. Anderson, Barry L. Burnette, Robert P. Compton,Rajesh V. Devraj, Jeffrey L. Hirsch, Robert H. Keith,1 Xiong Li, Gabriel Mbalaviele,Dean M. Messing, Matthew J. Saabye, John F. Schindler, Shaun R. Selness,Loreen I. Stillwell,2 Elizabeth G. Webb, Jian Zhang, and Joseph B. MonahanDiscovery Biology (H.R.H., G.D.A., B.L.B., R.P.C., J.L.H., R.H.K., X.L., G.M., M.J.S., J.F.S., L.I.S., E.G.W., J.Z., J.B.M.),Medicinal Chemistry (R.V.D., S.R.S.), and Pharmacokinetics, Dynamics, and Metabolism (D.M.M.), Inflammation Research,Pfizer Global Research and Development, Chesterfield, Missouri

Received July 17, 2009; accepted August 28, 2009

ABSTRACTSignal transduction through the p38 mitogen-activated pro-tein (MAP) kinase pathway is central to the transcriptionaland translational control of cytokine and inflammatory me-diator production. p38 MAP kinase inhibition hence consti-tutes a promising therapeutic strategy for treatment ofchronic inflammatory diseases, based upon its potential toinhibit key pathways driving the inflammatory and destruc-tive processes in these debilitating diseases. The presentstudy describes the pharmacological properties of the N-phenyl pyridinone p38 MAP kinase inhibitor benzamide [3-[3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2-oxo-1(2H)-pyridinyl]-N,4-dimethyl-, (�)-(9CI); PH-797804].PH-797804 is an ATP-competitive, readily reversible inhibitorof the � isoform of human p38 MAP kinase, exhibiting a Ki �5.8 nM. In human monocyte and synovial fibroblast cellsystems, PH-797804 blocks inflammation-induced produc-tion of cytokines and proinflammatory mediators, such asprostaglandin E2, at concentrations that parallel inhibition of

cell-associated p38 MAP kinase. After oral dosing, PH-797804 effectively inhibits acute inflammatory responses in-duced by systemically administered endotoxin in both ratand cynomolgus monkeys. Furthermore, PH-797804 demon-strates robust anti-inflammatory activity in chronic diseasemodels, significantly reducing both joint inflammation andassociated bone loss in streptococcal cell wall-induced ar-thritis in rats and mouse collagen-induced arthritis. Finally,PH-797804 reduced tumor necrosis factor-� and interleu-kin-6 production in clinical studies after endotoxin adminis-tration in a dose-dependent manner, paralleling inhibition ofthe target enzyme. Low-nanomolar biochemical enzyme in-hibition potency correlated with p38 MAP kinase inhibition inhuman cells and in vivo studies. In addition, a direct corre-spondence between p38 MAP kinase inhibition and anti-inflammatory activity was observed with PH-797804, thusproviding confidence in dose projections for further humanstudies in chronic inflammatory disease.

Rheumatoid arthritis (RA) is an aggressive autoimmunedisease involving complex interactions among T cells, mac-rophages, synoviocytes, and other immune cells (Firestein,2003). Analysis of synovial fluid and tissue from RA patientsimplicated key cytokines, including TNF-�, IL-1�, and IL-6in the pathogenesis of the disease (Firestein et al., 1990).Further studies shed light on the complex networks withinwhich these cytokines function and the delicate balance be-tween a pro- or an anti-inflammatory outcome (McInnes and

This study was sponsored by Pfizer Inc.Portions of this work were presented at the following conference: Monahan

J, Hope H, Schindler J, Jungbluth G, Burnette B, Guzova J, Hirsch H, SaabyeM, Compton R, Zhang J, et al. (2009) Anti-inflammatory properties of a novelN-phenyl pyridinone inhibitor of p38 MAP kinase: preclinical to clinical trans-lation. Annual European Congress of Rheumatology; 2009 Jun 10–13; Copen-hagen, Denmark, EULAR, Zurich, Switzerland.

1 Current affiliation: Millipore Corporation, St. Charles, Missouri.2 Current affiliation: Monsanto Company, St. Louis, Missouri.Article, publication date, and citation information can be found at

http://jpet.aspetjournals.org.doi:10.1124/jpet.109.158329.

ABBREVIATIONS: RA, rheumatoid arthritis; TNF, tumor necrosis factor; IL, interleukin; COX, cyclooxygenase; MAP, mitogen-activated protein;ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; PH-797804, benzamide, 3-[3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2-oxo-1(2H)-pyridinyl]-N,4-dimethyl-, (�)- (9CI); SCW, streptococcal cell wall; LPS, lipopolysaccharide; MKK, mitogen-activated proteinkinase kinase; EGFRP, epidermal growth factor receptor peptide; GST, glutathione transferase; RASF, rheumatoid arthritis synovial fibroblast(s);MK-2, mitogen-activated protein kinase-activated protein kinase 2; PG, prostaglandin; ELISA, enzyme-linked immunosorbent assay; M-CSF,macrophage–colony-stimulating factor; TRAP, tartrate-resistant acid phosphatase; TRAP�, tartrate-resistant acid phosphatase-positive; HSP,heat shock protein; Arry-797, Arry-371797 (N-substituted-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxamide); CIA, collagen-inducedarthritis; CII, chick type II collagen; LC-MS/MS, liquid chromatography-tandem mass spectrometry; SB203580, 4-(4-fluorophenyl)-2-(4-methyl-sulfinylphenyl)-5-(4-pyridyl)1H-imidazole; RANKL, receptor activator for nuclear factor-�B ligand; DFG, Asp168-Phe169-Gly170.

0022-3565/09/3313-882–895$20.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 331, No. 3Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 158329/3530045JPET 331:882–895, 2009 Printed in U.S.A.

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Schett, 2007). TNF-�, IL-1�, and IL-6 are macrophage- andfibroblast-derived proteins that induce expression of inflam-matory mediators such as COX-2, inducible nitric-oxide syn-thase, adhesion molecules, and metalloproteinases, resultingin synovial inflammation and associated cartilage and bonedestruction. Evidence generated in both animal models andin human studies support the role of TNF-�, IL-1, and IL-6 inthe pathogenesis of RA (Dayer et al., 2001; Scott and Kings-ley, 2006; Hennigan and Kavanaugh, 2008). Treatment withRemicade, Humira, Enbrel, Kineret, and Tocilizumab reducejoint pain and swelling, and retard progression of bone loss inpatients whose disease is unsatisfactorily controlled by con-ventional disease-modifying antirheumatic drugs (Okamotoet al., 2008). However, these biologic agents are limited dueto the requirement for parenteral administration, difficultyin dose titration, poor reversibility, induction of host neutral-izing antibody response, high production costs, and a signif-icant population of patient refractory to the treatment.

An alternative approach for the modulation of RA criticalproinflammatory cytokines is the manipulation of the signal-ing cascades involved in their production. It is interesting tonote that a marked overexpression of the phosphorylated(activated) form of p38 MAP kinase was demonstrated in RAsynovium (Schett et al., 2000). p38 kinase along with ERKand JNK are key MAP kinase signaling enzymes that cellsuse to adapt to inflammatory and stressful conditions. Patho-gens or inflammatory stimuli initiate a phosphorylation cas-cade mediated by p38 kinase that leads to the transcriptionand translation of inflammatory response-associated genesthat encode proteins such as TNF-�, IL-1, IL-6, and IL-8.Activation of the p38 pathways also leads to the production ofanti-inflammatory cytokines such as IL-10 (Chanteux et al.,2007), activation of negative feedback loops initiated by thephosphorylation of transforming growth factor �-activatedkinase-1-binding protein (Shin et al., 2009) as well as up-regulation of MAP kinase phosphatases that dephosphory-late and inactivate p38 kinase, ERK, and JNK (Wadgaonkaret al., 2004). These multiple positive and negative pathwaycomponents highlight the complex feedback and feedforwardaspects of the p38 signaling cascade (Hu et al., 2008).

The p38 MAP kinase family consists of four isoforms: �, �,�, and �. These kinases show a high degree of sequencehomology, with 60 to 75% overall sequence identity and�90% within the kinase domain (Kumar et al., 1997). The �and � isoforms are ubiquitously expressed, with the � isoformbeing the better characterized in terms of its role in inflam-mation. Although studies using a chemical genetic approachin mice indicate that the � isoform does not play a role in theregulation of inflammation (O’Keefe et al., 2007), it may havea role in pain (Svensson et al., 2005). The � and � isoforms areexpressed in a tissue-restricted manner, with � expressed inskeletal muscle (Li et al., 1996) and � expressed in lungs,kidneys, testis, pancreas, and intestines (Kumar et al., 1997).

Due to the apparent role of p38� kinase in inflammation,its therapeutic potential has been extensively studied. Sev-eral small-molecule inhibitors of p38 kinase have been gen-erated and characterized. These include VX-745, VX-702,SCIO-323, SCIO-469, AMG-548, BIRB 796, and pamapimod(Pettus and Wurz, 2008; Cohen et al., 2009). A substantialnumber of these inhibitors have progressed into human clin-ical studies, and development was discontinued due to unac-ceptable safety profiles (Pettus and Wurz, 2008). Common

side effects include skin rash, elevated liver enzymes, andgastrointestinal disorders. Because these adverse effectsvary with chemotype, and each of these compounds havedistinct kinase selectivity patterns, the toxicities observedmay be structure- rather than mechanism-based.

PH-797804 is a highly selective inhibitor of p38� kinase.In cell-based assays as well as animal models of acuteinflammation, PH-797804 blocked the production of cyto-kines and proinflammatory mediators. Furthermore, PH-797804 demonstrated robust anti-inflammatory activity inchronic disease models, significantly reducing both jointinflammation and associated bone loss in streptococcal cellwall-induced arthritis in rats and mouse collagen-inducedarthritis. Results from an endotoxin challenge model inhumans demonstrated the ability of PH-797804 to modu-late lipopolysaccharide (LPS)-stimulated cytokine produc-tion in a dose-dependent and concentration-dependentmanner.

Materials and MethodsPreparation of PH-797804

PH-797804 was prepared by the Pfizer Discovery Medicinal Chem-istry Department (St. Louis, MO).

In Vitro Assays

Kinase Activation. The activation of p38� kinase by mitogen-activated protein kinase kinase (MKK) 6 was carried out in thepresence of 300 M ATP, 10 mM magnesium acetate, and 25 mMHEPES, pH 7.5. Constitutively active MKK6 was used for activationof p38� kinase at a molar ratio for p38�:MKK6 ranging from 50:1 to100:1. The reaction mixture was allowed to incubate at 30°C for 1 h.The activation of JNK2 by MKK7 was carried out at 30°C for 1 h inthe presence of 500 M ATP, 10 mM magnesium acetate, and 25 mMHEPES, pH 7.5. The constitutively active form of MKK7 was addedto yield a 50:1 M ratio of JNK2/MKK7. The enzyme was either usedimmediately or measured (in aliquots) and stored at �80°C.

Kinase Activity Assay. A resin capture assay method was usedto determine the phosphorylation of epidermal growth factor recep-tor peptide (EGFRP) or GST-c-Jun by p38 kinases or JNK2, respec-tively. Reactions mixtures contained 25 mM HEPES, pH 7.5, 10 mMmagnesium acetate, ATP (at the indicated concentration), 0.05 to 0.3Ci of [�-33P]ATP, 0.8 mM dithiothreitol, and either 200 M EGFRPor 10 M GST-c-Jun for p38� kinase or JNK2 reactions, respectively.The reaction was initiated by the addition of either 25 nM p38�kinase or 100 nM JNK2 to give a final volume of 50 l. The JNK2 andp38� kinase reactions were incubated at 25°C for either 20 or 30 min,respectively. Under these conditions, the formation of product forboth p38� kinase and JNK2 was linear with time. The reaction wasstopped, and the unreacted [�-33P]ATP was removed by the additionof 150 l of AG 1 8 ion exchange resin in 900 mM sodium formate,pH 3.0. Once thoroughly mixed, solutions were allowed to stand for5 min. A 50-l aliquot of head volume containing the phosphorylatedsubstrate was removed from the mixture and transferred to a 96-wellplate. MicroScint-40 scintillation cocktail (150 l; PerkinElmer Lifeand Analytical Science, Boston, MA) was added to each well and theradioactivity quantitated using a TopCount NXT microplate scintil-lation and luminescence counter (PerkinElmer Life and AnalyticalScience, Boston, MA).

Inhibition Studies. The initial velocities in the presence andabsence of PH-797804 were obtained using ATP/[�-33P]ATP as thevaried substrate. The peptide (EGFRP) or protein substrate (GST-c-Jun) was fixed at a single concentration. The initial velocities weredetermined in triplicate using the assay described above. The inhi-bition data were fit to the competitive inhibition (eq. 1), noncompet-

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itive inhibition (eq. 2), or uncompetitive inhibition (eq. 3) by usingGraFit version 4.0 service pack (Erithacus Software, Surrey, UK).

v � Vmax�S�/ Km 1 � �I�/Kis� � �S�� (1)

v � Vmax�S�/ Km 1 � �I�/Kis� � �S� 1 � �I�/Kii)) (2)

v � Vmax�S�/ Km � �S� 1 � �I�/Kii)) (3)

In these equations, Vmax is the maximum velocity, Km is the Michae-lis-Menten constant for the varied substrate (ATP), S is the concentra-tion of the varied substrate, I is the concentration of the inhibitor, andKis and Kii are the dissociation constants for the interaction of inhibitorwith the free enzyme or the substrate-bound enzyme, respectively.

Ligand Exchange Reaction. The competition between inhibi-tors and a fluorescent probe for binding to nonphosphorylated, non-active p38� kinase was monitored in real-time by using stopped flowinstrumentation (TgK Scientific Limited, Bradford-on-Avon, UK)equipped with a fluorescence detector. The fluorescence resonanceenergy transfer signal from probe binding to nonactive p38� kinasewas measured by exciting the sample at 295 nm and monitoring thefluorescence intensity using a 420 nm cut-off filter. The buffer usedfor all ligand exchange reactions was 25 mM HEPES, pH 7.5, with10% dimethyl sulfoxide. In the first ligand exchange reaction, 100nM nonactive p38� kinase is equilibrated with 250 nM probe (Kd �13 nM) and then diluted into solution containing the unlabeledinhibitor (at time 0). In the second reaction, 100 nM nonactive p38�kinase is equilibrated with the unlabeled inhibitor and then dilutedinto solution containing the probe (at time 0). The final concentra-tions of reactants are held constant so that both reactions have thesame response at equilibrium (Morelock et al., 1995).

Progress Curve Analysis. The binding constants of PH-797804were determined using a competition assay as described previously(Pargellis et al., 2002). The binding constants were calculated basedon a one-step binding model using the numerical integration soft-ware DYNAFIT (Kuzmic, 1996).

Cell-Based Assays

Cell Lines and Primary Human Monocytes. The U937 humanpremonocytic cell line was obtained from the American Type CultureCollection (Manassas, VA). These cells were differentiated to a mono-cytic/macrophage phenotype as described by Burnette et al. (2009).Rheumatoid arthritis synovial fibroblasts (RASF) were derived fromthe inflamed synovium of a female RA patient who was undergoingtotal knee replacement. Synovial tissue was teased away from adja-cent cartilage and dispersed into single cells with collagenase. Cellswere expanded and banked. RASF were further cultured as de-scribed by Burnette et al. (2009). Primary human monocytes wereobtained from venous blood of donors collected anonymously withinformed consent at an on-site clinic into sodium heparin tubes.Monocytes were isolated as described previously (Burnette et al.,2009).

Stimuli-Induced MAP Kinase Activation. Cells (differentiatedU937 cells) pretreated with or without inhibitors for 1 h, were stim-ulated with LPS (0.1 g/ml) for 30 min, washed twice with ice-coldphosphate-buffered saline, and lysed with 150 mM NaCl, 20 mMTris, pH 7.5, 1% Triton X-100, 5 mM EDTA, 50 mM NaF, 10%glycerol, and 1 mM Na3VO4 and protease inhibitors (BoehringerMannheim, Indianapolis, IN) for immunoprecipitation in vitro ki-nase assays.

Immunoprecipitation in Vitro Kinase Assay. The protein con-tent of the U937 cell lysates was determined by Bradford assay(Bradford, 1976). Lysates (750 g) were immunoprecipitated withanti-MK-2 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz,CA) and protein A/G-agarose (2–3 h; 4°C). Immune complexes werewashed three times with lysis buffer, once with kinase assay buffer(50 mM HEPES, 10 mM MgCl2, 5 mM MnCl2, and 1 mM dithiothre-itol), and resuspended in kinase assay buffer with 4 g of HSP27substrate and 0.5 Ci of [�-33P]ATP. After incubation at 30°C for 15

min, the reaction was stopped by adding an equal volume of 2SDS-sample buffer, and heated for 3 min. Reaction mixtures wereresolved by 4 to 20% SDS-polyacrylamide gel electrophoresis, andradiolabeled substrate was visualized by autoradiography. The la-beled bands were excised from the gel, scintillation cocktail wasadded, and the bands were counted on an LS6000IC counter (Beck-man Coulter, Fullerton, CA). MK-2 is selectively activated by p38kinase in U937 cells, and its phosphorylation of HSP27 is thereby anindirect measurement of p38 kinase activity.

LPS-Induced Cytokine (TNF-� and IL-1�) Production. Dif-ferentiated U937 cells or purified human monocytes were seeded into96-well tissue culture plates (200,000 cells/well) in complete media.After 24 h, the cells were pretreated for 60 min in the presence orabsence of PH-797804 and then stimulated with LPS (0.1 g/ml) for4 h (U937 cells) or 16 h (monocytes). Culture media were thencollected for determination of TNF-� or IL-1� levels by Luminexbead array technology (Linco/Millipore, Billerica, MA). Cytokine con-centrations were extrapolated from recombinant protein standardcurves using a BioAssay Solver macro with a four-parameter logisticmodel and solving for IC50 after iterating to the best least-squares fit.

LPS-Induced TNF-� and IL-1� Production in Human WholeBlood. Venous blood was collected with informed consent as de-scribed above. Assay of LPS-induced cytokine production was per-formed as described by Burnette et al. (2009) with the exception thatthe final LPS concentration was 10 g/ml, the incubation period forIL-1� production was 24 h, and cytokine analysis was done using theLuminex bead array technology (Linco/Millipore).

IL-1�-Induced PGE2 Production. RASF were seeded into 96-well tissue culture plates (5 104 cells/well) in complete growthmedium. After 24 h, the medium was replaced with fresh growthmedium containing 1% fetal bovine serum. Cells were treated withserial concentrations (10,000–0.01 nM) of PH-797804 or dimethylsulfoxide vehicle control for 1 h, stimulated with 1 ng/ml IL-1� (R&DSystems, Minneapolis, MN) for 18 to 20 h at 37°C, and then condi-tioned media were collected. PGE2 levels the in cultured media werequantitated by ELISA (Cayman Chemical, Ann Arbor, MI).

Osteoclast Formation from Rat Bone Marrow Cells. MaleLewis rats weighing 250 to 300 g were obtained from Harlan Indus-tries (Houston, TX). Rat bone marrow cells were collected fromfemora and tibiae of the animals. Cells were washed twice withserum-free �-minimal essential medium (Invitrogen, Carlsbad, CA)containing 100 U/ml penicillin and 100 g/ml streptomycin (Invitro-gen). Cells were plated at a density of 1.7 106/cm2 in �-minimalessential medium supplemented with 10% (v/v) fetal bovine serum(Invitrogen), incubated at 37°C, in an atmosphere of 95% air and 5%CO2, and then treated with a combination of recombinant mouseRANKL (100 ng/ml) and recombinant mouse macrophage–colony-stimulating factor (M-CSF) (10 ng/ml) (R&D Systems) along withPH-797804 at various concentrations (0.0001–1 M). The cytokineconcentrations used had been shown to induce maximal osteoclastformation in previous studies (data not shown). After 2 days ofculture, half of the culture medium was gently removed, to minimizeloss of nonadherent cells, and replaced with an equal volume of freshculture media containing cytokines and p38 kinase inhibitors. Onday 3, the medium was removed, and the cells were fixed by theaddition of a 60% (v/v) acetone solution prepared in sodium citratebuffer, pH 5.4, for 30 s. The fixed cells were washed twice withdistilled water and air-dried. Tartrate-resistant acid phosphatase(TRAP)-positive (TRAP�) cells were detected using a TRAP stainingkit (Sigma-Aldrich, St. Louis, MO). Osteoclasts, defined as cellsTRAP� multinucleated cells with three or more nuclei were countedunder a light microscope by using computer-assisted AnalySiS Prosoftware (Soft Imaging System, Munster, Germany). The IC50 valuedeterminations were made using a four-parameter logistic analysis.

Cell Viability Assay. Cell viability was determined as describedpreviously (Burnette et al., 2009).

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In Vivo Studies

Animal Use. The Pfizer Institutional Animal Care and Use Com-mittee reviewed and approved the animal use in these studies. Theanimal care and use program is fully accredited by the Associationfor Assessment and Accreditation of Laboratory Animal Care, Inter-national. Adult male Lewis rats (225–250 g; Harlan, Indianapolis,IN) or adult male cynomolgus monkeys (6–8 kg; Charles RiverLaboratories, Houston, TX) were used in the LPS-induced TNF�production studies. Female Lewis rats (125–140 g; Harlan) wereused in the SCW arthritis model and DBA/1 mice (8–12 weeks old;The Jackson Laboratory, Bar Harbor, ME) were used in the collagen-induced arthritis (CIA) model.

LPS-Induced TNF-� Production in Rats. Rats were fasted18 h before oral dosing and were allowed free access to waterthroughout the experiment. Each treatment group consisted of fiveanimals. PH-797804 was prepared as a suspension in a vehicleconsisting of 0.5% methylcellulose (Sigma-Aldrich) and 0.025%Tween 20 (Sigma-Aldrich). The compound or vehicle was adminis-tered by oral gavage in a volume of 1 ml. Two vehicle groups wereused per experiment to control for intraexperiment variability, andtwo experiments were performed. LPS (Escherichia coli serotype0111:B4; Sigma-Aldrich) was administered 4 h after compound in-travenous injection at a dose of 1 mg/kg in 0.5 ml of sterile saline(Baxter Healthcare, Deerfield, IL), a dose determined previously tobe optimal (data not shown). Blood was collected in serum separatortubes via cardiac puncture 90 min after LPS injection, a time pointcorresponding to maximal TNF-� production (data not shown). Afterclotting, serum was withdrawn and stored at �20°C until it wasassayed for TNF-� by ELISA (Burnette et al., 2009).

LPS-Induced TNF-� Production in Cynomolgus Monkeys.The study was conducted at Charles River Laboratories in accor-dance with the institutional guidelines for humane treatment ofanimals. Adult male cynomolgus monkeys were fasted with freeaccess to water for at least 18 h before testing. TNF-� was induced incynomolgus monkeys (n � 3) by a single intravenous aqueous injec-tion of LPS (E. coli serotype 055:B5) (Sigma-Aldrich) at a dose of 10g/kg in a volume of 0.5 ml of sterile saline. This dose of LPS hadbeen shown previously to produce nanogram per milliliter levels ofTNF-� in rats (data not shown). PH-797804 was delivered through anasogastric tube in a 1-ml volume 2 h before LPS in an aqueousvehicle of 0.5% methylcellulose (w/v) and 0.1% Tween 80 (v/v). Bloodwas collected into lithium heparin Vacutainer tubes just before LPSinjection and 0.5, 0.75, 1, 1.25, and 2 h after LPS injection. PlasmaTNF-� was determined using a human TNF-� ELISA kit (BD Bio-sciences Pharmingen, San Diego, CA) that detected human TNF-�with a sensitivity of 7.5 pg/ml. A crossover design was used wherebyeach of three monkeys received 0.03, 0.1, 0.3, and 1.0 mg/kg PH-797804 with 3-week intervals between treatments. Vehicle responsefor each monkey was determined as the mean LPS-stimulatedTNF-� levels from four nondrug studies. The four nondrug studieswere done 2 to 9 months before the PH-797804 studies, with aminimum interval of 3 weeks between studies. The PH-797804 dose,maximal plasma concentration (Cmax), and are under the curve0–24h

required to produce 50 and 80% inhibition of peak TNF-� levels weredetermined using a four-parameter logistic model with two param-eters fixed to 0% for minimum and 100% for maximum.

Streptococcal Cell Wall-Induced Arthritis in Rats. Arthritiswas induced in female Lewis rats by a single intraperitoneal admin-istration of peptidoglycan-polysaccharide complexes isolated fromgroup A SCW (15 g/g body weight). The SCW preparation waspurchased from Lee Laboratories (Grayson, GA). The disease courseis biphasic in which an acute inflammatory arthritis develops withindays 1–3 (non–T-cell-dependent phase) followed by a chronic erosivearthritis (T-cell-dependent phase) developing on days 14 to 28(Kuiper et al., 1998). Only animals developing the acute phase weretreated with PH-797804 from days 10 to 21 after SCW injection. Pawvolume was measured on day 21 by using a water displacement

plethysmometer. PH-797804 was prepared as an aqueous suspen-sion in 0.5% methylcellulose and 0.025% Tween 20 (Sigma-Aldrich).The compound was administered by oral gavage in a volume of 0.5 mlbeginning on day 10 post-SCW injection and continuing daily untilday 21. Animals were dosed between 0.015 and 5 mg/kg b.i.d. withPH-797804. Methylcellulose/Tween 20 vehicle was used for compar-ison. Group size was four to eight animals per group. Two pawvolumes were taken for each animal. Paw volume was measured onday 21 by using a water displacement plethysmometer. Three to fourpaws from each treatment group were scanned for bone densityevaluation. Plasma samples were collected on day 21 for determina-tion of compound levels.

Bone Density Determination. Hind paws obtained from (day21) arthritic rats were submitted for bone density analysis. Pawswere stored at �80°C until use. Bone density was determined usingLunar PixiMus densitometer (GE Medical Systems, Madison, WI)(Nagy and Clair, 2000). Bone density was evaluated in hind pawsfrom the heel through half of the length of the paw and values wereexpressed as grams per square millimeter.

Collagen-Induced Arthritis in Mice. Arthritis was induced inDBA/1 mice by injection of 100 l of 50 g of chick type II collagen(CII) (provided by Dr. M. Griffiths, University of Utah, Salt LakeCity, UT) in Complete Freund’s adjuvant (Sigma-Aldrich) at the baseof the tail on day 0. The mice were boosted on day 21 as describedabove. Compound administered in chow was mixed to deliver theapproximate dose per day indicated in Fig. 7. The dosing of com-pound was based on a 20- to 25-g mouse consuming 4 g of chow perday. The mix for 4, 12, 40, and 120 mg/kg/day was 25, 76, 250, and760 ppm, respectively. The chow was given ad libitum on days 21through 56. Dose groups consisted of 10 animals per group. Animalswere evaluated several times each week for signs of arthritis for upto 8 weeks. Any animal with paw redness or swelling was counted asarthritic. Scoring of severity was carried out using a score of 1 to 3 foreach paw (maximal score of 12/mouse). Animals displaying any red-ness or swelling of digits or the paw were scored as 1, gross swellingof the whole paw or deformity was scored as 2, and ankylosis of jointswas scored as 3. Any animal demonstrating a score of 1 or more wasconsidered arthritic. Statistical analysis comparing the percentage ofarthritis between groups was done using Fisher’s exact test.

Human Endotoxin-Induced Inflammatory Response Model.This was a single-center, randomized, subject- and investigator-blind/sponsor-open placebo-controlled study. This study was con-ducted in compliance with the ethical principles derived from theDeclaration of Helsinki and in compliance with the InstitutionalReview Board and informed consent regulations. The study wasconducted at the MDS Pharma Services Clinical Center (New Or-leans, LA). Included in this study were 36 healthy male subjects fromthe ages of 18 to 55 years. After an 8-h fast, subjects were adminis-trated a single oral dose of study medication (1, 2, 4, 13, or 30 mg ofPH-797804 or placebo) with 300 ml of water (day 1). Twenty-fourhours later (day 2), a bolus intravenous injection of 2 ng/kg LPS wasadministered to all subjects. Based on pharmacokinetic data ob-tained after administration of single oral doses from 0.3 to 30 mg, itwas determined that relatively constant plasma concentrations arepresent from 24 to 48 h after dosing. Thus, the timing of the LPSadministration was chosen to be 24 h after the dose of PH-797804.Blood samples to determine the plasma pharmacokinetics of PH-797804 were collected immediately before study drug administrationas well as 24, 29, and 48 h after study drug administration. PlasmaPH-797804 concentrations were determined using liquid chromatog-raphy-tandem mass spectrometry (LC-MS/MS). The analytical rangeof the assay was 0.02 to 20 ng/ml. Blood samples for measurementsof cytokines in the plasma were collected just before study drugadministration, just before LPS administration, and serially up to5 h after LPS administration. Blood samples for measurement ofMK-2 activity in whole blood were collected at just before LPSadministration up to 5 h after LPS administration. TNF-� in plasmawas assayed by ELISA, with a dynamic range of 15 to 1000 pg/ml.


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IL-6 in plasma was assayed using Luminex multiplex ELISA tech-nology, with a dynamic range of 3.2 to 10,000 pg/ml. MK-2 activity inwhole blood lysates was determined as described by Burnette et al.(2009). In brief, whole blood samples were lysed in a buffer contain-ing protease and phosphatase inhibitors. The lysates were processedusing QiaShredder tubes (QIAGEN, Valencia, CA), and the resultingsupernatants were used for the assay. The assay was a radiometricassay consisting of capture of MK-2, in vitro phosphorylation ofbiotin-tagged HSP27 peptide, and quantitation of the radiolabeledphospho-HSP27 by capture on a streptavidin-coated flashplate andscintillation counting (Burnette et al., 2009).

Measurement of PH-797804 in Rat and Monkey Plasma.PH-797804 concentrations in rat and monkey plasma were deter-mined using an extraction procedure followed by LC-MS/MS. PH-797804 standards were prepared by spiking a stock solution indimethyl sulfoxide (Mallinckrodt Baker Inc., Phillipsburg, NJ) intonormal plasma and performing serial dilutions to achieve a nine-point calibration curve. For rat, 20 to 40 l of plasma was extractedby adding acetonitrile (Honeywell Burdick & Jackson, Muskegon,MI) containing analytical internal standard to a final volume of 200l, and the samples were vortex-mixed and centrifuged to precipitateproteins. For monkey, 50 l of plasma was combined with acetoni-trile containing analytical internal standard to a final volume of 200l, and the samples were extracted using solid phase extraction.Monkey plasma samples were applied to an Oasis HLB solid-phaseextraction 96-well plate (Waters, Milford, MA) that had been preac-tivated with 100% methanol followed by 2% acetic acid in water andthen washed with two 400-l aliquots of methanol/2% acetic acid inwater [30:70 (v/v)] followed by two 150-l elutions of the analyteswith 100% methanol. The combined eluate was dried down undernitrogen, reconstituted in 100 l of acetonitrile/0.1% formic acid inwater [25:75 (v/v)], and vortex-mixed. A volume of each extract wasinjected into the LC-MS/MS system using a Chiracel OJ-RH column(Chiral Technologies, Inc., Exton, PA) or Thermo Aquasil C18 col-umn (Thermo Fisher Scientific, Waltham, MA) and a 1% formic acid(Mallinckrodt Baker Inc.) and acetonitrile gradient. An API 4000Sciex mass spectrometer (Applied Biosystems, Foster City, CA) withturbo-ionspray source was operated in the multiple reaction-moni-toring mode to detect PH-797804 and the internal standard in thesamples. PH-797804 peak area ratios were determined using the m/z4773127 transition of PH-797804 versus the m/z transition of theinternal standard, and plasma concentrations were calculated usinglinear regression analysis of the calibration samples. The detectionlimits of the assay were 0.076 to 5000 ng/ml PH-797804.

Determination of PH-797804 Plasma Protein Binding. Theprotein binding of PH-797804 in rat, monkey and human plasma wasdetermined in vitro. Plasma was fortified with PH-797804 to achieveconcentrations in the projected therapeutic range and also 10- and100-fold higher (0.1, 1.0, and 10 g/ml). Triplicate aliquots of plasmamatrix standards underwent equilibrium dialysis against an equalvolume of phosphate-buffered saline in an in-house-devised 96-wellapparatus similar to that described by Kariv et al. (2001) by usingdialysis membranes with a molecular weight cut-off of 12 to 14 kDa.The dialysis was performed in a 37°C shaking incubator for 6 h. Thefraction unbound was calculated from the concentrations of PH-797804 in the protein-rich and buffer matrices using the LC-MS/MSmethod described above. The following equation was used to calcu-late the fraction bound at the end of the incubation period:

(Ct � Cf) (volume of plasma after incubation/150)(Ct � Cf) (volume of plasma after incubation/150) � Cf

where Ct is concentration of analyte in plasma after incubation andCf is concentration of analyte in buffer after incubation.

The following equation was used to calculate the fractionunbound:

fu � 1 � Fraction Bound

The plasma protein binding of PH-797804, averaged over therange of 0.1 to 10 g/ml, was 97.8, 93.4, and 96.7% in rat, monkey,and human, respectively, and fraction unbound was 2.2, 6.6, and3.4%, respectively.

ResultsPHA-797804 Is a Potent and Selective Inhibitor of

p38� Kinase. PH-797804 (Fig. 1) is a novel pyridinone in-hibitor of the MAP kinase signaling enzyme p38� kinase.Binding, enzyme kinetics, and crystallographic analyses sup-port the ATP-competitive nature of PH-797804 inhibition ofp38� kinase activity as well as its high degree of selectivity.PH-797804 inhibited p38� kinase, with an IC50 value of 26nM (Table 1), and the inhibition is best described by a com-petitive model as shown in Fig. 2A. Nonlinear fit of the initialvelocity data to an equation describing competitive inhibition(see eq. 1 under Materials and Methods) are shown in Fig.2A. The competitive nature of PH-797804 is apparent in theplot (Fig. 2B), which depicts a family of lines intersecting onthe 1/v axis, indicative of competitive inhibition. The averageenzymatic competitive inhibition constant (Ki) for PH-797804toward p38� kinase is 5.8 � 0.3 nM (Table 1). In contrast,PH-797804 did not inhibit the related MAP kinase JNK2 atconcentrations up to 200 M.

Ligand exchange reactions were used to determine bindingassociation and dissociation rate constants as indicators ofthe time dependence and reversibility for PH-797804. Thefluorescence resonance energy transfer signal from a fluores-cent probe was monitored in the presence of inhibitor todetermine the nature of the interaction of PH-797804 withunactivated p38� kinase. PH-797804 demonstrates rapid as-sociation and dissociation rates in these experiments. Theassociation rate (Kon) and dissociation rate (Koff) value forPH-797804 is 1.53 107 � 2.88 106 M�1 � s�1 and 0.058 �0.008 s�1, respectively. In addition, the t1/2 value for disso-

Fig. 1. Structure of PH-797804.

TABLE 1PH-797804 affinity and potency for inhibiting recombinant p38 andJNK2 kinasesData are expressed as the mean value of three independent experiments � S.E.M.

Assaya PH-797804

p38� Kinase IC50 26 � 8 nMp38� Kinase Ki 5.8 � 0.3 nMp38� Binding Ki 3.9 � 0.3 nMKon (M�1 � s�1) 1.53 107 � 2.88 106

Koff (s�1) 0.058 � 0.008t1/2 12.6 � 1.9 sp38� Kinase IC50 102 � 38 nMp38� Kinase Ki 40 � 24 nMJNK2�1 Binding Ki �200 MJNK2 �1/p38� Binding Ki ratio �50,000

a Assays are as described under Materials and Methods.

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ciation was determined to be 12.6 � 1.9 s. The binding Ki

value determined in these ligand exchange experiments was3.9 � 0.3 nM, which is in good agreement with the enzymaticKi of 5.8 � 0.3 nM (Table 1). These results suggest thatPH-797804 acts as a reversible inhibitor of p38� kinase withfast on and fast off kinetics and suggests that PH-797804binds to both activated and unactivated p38� kinase withsimilar affinity.

The profile of PH-797804 for inhibition of other proteinkinases and nucleotide binding proteins suggests remarkableselectivity for p38 kinase. PH-797804 inhibited the activity ofthe closely related p38� kinase isoform, with a Ki value of 40nM, resulting in a p38� kinase to p38� kinase selectivityratio of 6.9-fold (Table 1). No significant inhibition of theremaining two human p38 isoforms, p38� and p38� kinases,or other MAP kinases, JNK1, JNK2, JNK3, and ERK2, wereobserved at concentrations of PH-797804 up to 200 M(Table 2). The selectivity of PH-797804 against the JNK2kinase (binding Ki ratio �50,000-fold) is significantly greaterthan that observed for the p38 kinase inhibitor SB203580(16-fold binding Ki ratio; data not shown). In addition to thekinases noted above, PH-797804 was profiled against approx-imately 65 kinase and nucleotide binding proteins, and nocross-reactivity was observed (selectivity ratio �500-fold; Ta-ble 2) (Xing et al., 2009).

PH-797804 Blocks Inflammation-Induced Produc-tion of Cytokines and Proinflammatory Mediators inCellular Assays Relevant to Rheumatoid Arthritis andInflammation. PH-797804 was potent and efficacious inblocking cellular p38 kinase activity along with biologicalresponses in human cells associated with inflammation inthe arthritic joint. LPS-stimulated TNF-� production andp38 kinase activity as measured by immune complex kinaseassay of the downstream kinase MK-2 in the human mono-cytic U937 cell line were inhibited, with comparable IC50

values of 5.9 and 1.1 nM, respectively (Table 3; Fig. 3A).Although PH-797804 was shown to inhibit cellular p38 ki-nase activity as assessed by either MK-2 immune complexkinase assay or p38 kinase-dependent phosphorylation ofHSP27, no inhibitory effect was observed on either the JNK

pathway (c-Jun phosphorylation) or ERK pathway (ERKphosphorylation) in U937 cells at concentrations up to 1 M(Xing et al., 2009).

In addition to a critical role in inflammatory cytokine pro-duction, PH-797804 also regulates COX-2 induction andPGE2 production. RASF stimulated with IL-1� producedlarge amounts of PGE2, with maximal production occurringafter 20 h. Complete concentration-dependent inhibition ofPGE2 production was observed with RASF treated with PH-797804 (IC50 � 1.5 nM) (Table 3; Fig. 3B). Cell viability wasunaffected over the time course studied (data not shown). Itis interesting to note that PH-797804 did not affect recombi-nant COX-2 or COX-1 activity at the concentrations thatblock PGE2 while inhibiting IL-1� up-regulation of COX-2mRNA (data not shown), suggesting a unique mechanism forprostaglandin blockade by p38 kinase inhibitors.

The cellular potency of PH-797804 correlates well with itsinhibition of recombinant enzyme activity, consistent with ap38 kinase mechanism of action. The U937 cellular activity ofPH-797804 was confirmed and extended using LPS-stimu-lated human monocytes and human whole blood. In mono-cytes, LPS-stimulated production of both TNF-� and IL-1�was inhibited by PH-797804 in a concentration-dependentmanner, with an IC50 value 3.4 nM for each cytokine (Table3; Fig. 3C), whereas in whole blood, a cellular system thatmimics the physiological in vivo milieu, TNF-� and IL-1�production was inhibited, with an IC50 value of 85 and 37.5nM, respectively (Table 3; Fig. 3D). The lower potency ofPH-797804 in human whole blood compared with monocytesand U937 cells is consistent with the plasma protein bindingcharacteristics of the inhibitor (human plasma protein bind-ing, 96.7%) and the hypothesis that only unbound compoundis available to penetrate cells. The IC50 values for PH-797804inhibition of LPS-induced TNF-� and IL-1� production basedon the free fraction of the compound in human whole bloodwere 2.8 and 1.2 nM, respectively, which corresponds withvalues from both U937 cells and human monocytes.

MAP kinase pathways are also implicated in bone andcartilage destruction in RA by modulating the productionand signaling functions of cytokines such as TNF-�, IL-1�,

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Fig. 2. Competitive inhibition pattern for PH-797804 versus p38� kinase. Initial velocities were obtained with ATP/[�-33P]ATP as the varied substrate(50, 100, 200, 500, 1000, and 2000 M). The EGFRP was held constant at 200 M. The concentrations of PH-797804 were 0 (E), 6 (F), 12 (�), 25 (f),and 50 nM (‚). A, nonlinear fit of data to a competitive inhibition model using GraFit 4.0 service pack. B, double reciprocal plot of the data shown inFig. 1A. In this experiment, the enzymatic competitive inhibition constant for PH-797804 equals 6.06 � 0.8 nM (S.E.M.) in this representativeexperiment.


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and receptor activator of nuclear factor-�B ligand (RANKL).It is now known that TNF-� and IL-1� function together withRANKL, leading to the propagation of inflammation andbone erosion (Mbalaviele et al., 2006). Using primary ratbone marrow cells, osteoclast formation was induced after 3days of coculture with RANKL and M-CSF (Fig. 4). PH-797804 inhibited this RANKL- and M-CSF-induced oste-oclast formation in a concentration-dependent manner, withIC50 � 3 nM (Table 3; Fig. 4). Because RANKL and M-CSFare required for osteoclast differentiation in vivo, these datasuggest that PH-797804 has the potential to inhibit bonedestruction associated with inflammatory arthritis.

PH-797804 Inhibits Acute Inflammatory ResponsesInduced by Intravenously Administered Endotoxin.Biochemical models were established to evaluate the oralefficacy and potency of PH-797804 as a blocker of cytokineproduction in vivo. These models included LPS-inducedTNF-� production in rats and cynomolgus monkeys. Intrave-nous administration of LPS into Lewis rats produced a rapidand transient elevation of TNF-� levels in plasma thatpeaked 1 to 2 h after LPS injection (data not shown). Theextent of inhibition of TNF-� production by PH-797804 wasdetermined by quantifying plasma TNF-� levels of treatedand untreated animals. Treatment of rats with PH-797804resulted in dose-dependent inhibition of LPS-induced TNF-�production (Fig. 5). Plasma levels of PH-797804 were deter-mined 5.5 h after compound administration in parallel withTNF-� concentration determination, and efficacy correspond-ing to compound concentrations at this time point was deter-mined. Nonlinear, four-parameter analysis of the dose-re-sponse and concentration-response data resulted in an ED50

value of 0.07 mg/kg and an EC50 value of 8.6 ng/ml forPH-797804. ED80 and EC80 values were calculated as 0.142mg/kg and 26.7 ng/ml, respectively (Table 4; Fig. 5).

PH-797804 was also orally effective in an analogous modelof LPS-induced TNF-� production in cynomolgus monkeyswhen administered 2 h before LPS challenge (Table 4; Fig. 5).Dose-response analysis resulted in ED50 and ED80 values of0.095 and 0.349 mg/kg, respectively. The peak exposure ofPH-797804 (Cmax) required to produce 50% inhibition of LPS-

TABLE 2Selectivity profile of PH-797804

PH-797804 Kinase Selectivity (IC50)a Panlab in Vitro Pharmacology Screen PH-797804Selectivity (IC50)a

M M

p38� 0.021 GSK3� �10 PKC �10p38� 0.101 CK2 �10 Fyn �10p38� �200 NIM1 �10 HER2 �10p38� �200 PDGFR �10 PKC� �10JNK1 �200 IR �10 PKCg �10JNK2 �200 PAK4 �10 Ca�2/calmodulin protein kinase �10JNK3 �200 AUR2 �10 ERK1 �10ERK2 �200 PKA� �10 ATPase, Na�/K� �10IKK2 �200 SULU1 �10 ATPase, H�/K� �10IKK1 �200 PKC� �10 Phosphodiesterase PDE1 �10IKKi �200 VEGFR2 �10 Phosphodiesterase PDE2 �10TBK �200 VEGFR3 �10 Phosphodiesterase PDE3 �10MKK6 �100 RET �10 Phosphodiesterase PDE4 �10MKK7 �100 Lyn �10 Phosphodiesterase PDE5 �10MK-2 �200 FGFR1 �10 Purinergic P2x �10MK-3 �200 EGFR1 �10 Purinergic P2y �10PRAK �200 c-MET �10 Adenosine A1 �10RSK2 �200 LCK �10 Adenosine A2A �10MNK1 �200 PDK1 �10 Adenosine A3 �10MSK1 134 IGFR1 �10 5-HT receptor �10CDK2A �200 PLK1 �10 5-HT transporter �10AKT1 �10 STLK �10 GLP-1 �10CDC7 �10 c-ABL �10 Glucocorticoid receptor �10CHK1 �10 ZAP70 �10 Monoamine oxidase MAOA �10

Monoamine oxidase MAOB �10

5-HT, 5-hydroxytryptamine; c-Abl, Abelson murine leukemia viral oncogene homolog; CDC7, cell division cycle protein kinase 7; CHK1, checkpoint kinase-1;CDK2A, cyclin A-associated cyclin-dependent kinase 2; CK2, casein kinase-2; c-MET, mesenchymal-epithelial transition factor receptor; EGFR1, epidermal growthfactor receptor; FGFR1, fibroblast growth factor receptor 1; Fyn, fyn tyrosine kinase proto-oncogene; GLP-1, glucagon-like peptide 1; GSK3�, glycogen synthasekinase-3�; HER2, human epidermal growth factor receptor 2; IGFR1, insulin-like growth factor receptor; IKK1, I� B kinase 1; IKK2, I� B kinase 2; IKKi, inhibitorof � light polypeptide gene enhancer in B cells, kinase � -inducible; IR, insulin receptor; Lck, lymphocyte-specific protein tyrosine kinase; Lyn, v-yes-1 Yamaguchisarcoma viral related oncogene homolog; MNK1, mitogen-activated protein kinase-interacting kinase 1; MSK1, mitogen/stress kinase 1; NIM1, new inducer of mitosis1; PAK4, p21 activated kinase-4; PDGFR, platelet-derived growth factor receptor; PDK1, 3-phosphoinositide-dependent protein kinase-1; PKA�, 3�:5�-AMP-dependentprotein kinase �; PKC�, protein kinase C �; PKC�, protein kinase C �; PKC �, protein kinase C �; PLK1, polo-like kinase; PRAK, p38-regulated/activated proteinkinase; Ret, rearranged during transfection proto-oncogene; Rsk2, ribosomal protein S6 kinase 2; STLK, Mst3 and SOK1-related kinase; SULU1, Caenorhabditiselegans kinase homolog; TBK, TANK binding kinase; VEGFR2, vascular endothelial growth factor receptor 2; VEGFR3, vascular endothelial growth factor receptor3; ZAP-70, 70-kDa TCR z-chain-associated protein kinase.

a IC50 values are based on enzymatic activity.

TABLE 3Cellular activity of PH-797804 on cytokine production, p38 activity,and PGE2 production—cell types relevant to rheumatoid arthritis andinflammation

Assay PH-797804

nM

LPS-stimulated human U937 Cell TNF-�, IC50 5.9 � 1.3LPS-stimulated human U937 cell p38 kinase

activity, IC50

1.1 � 0.6

Human rheumatoid synovial fibroblastIL-1�-stimulated PGE2, IC50

1.5 � 0.4

LPS-stimulated human monocyte TNF-�, IC50 3.4 � 2.0LPS-stimulated human monocyte IL-1�, IC50 3.4 � 0.5LPS-stimulated human whole blood TNF-�, IC50 85 � 21LPS-stimulated human whole blood IL-1�, IC50 37.5 � 3.8Rat osteoclast differentiation, IC50 3

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induced TNF-� production was 7 ng/ml. The dose- and con-centration-response analyses demonstrate comparable effi-cacy and potency for PH-797804 in both the rat andcynomolgus monkey studies (Fig. 5).

PH-797804 Suppresses Chronic Inflammation in RatStreptococcal Cell Wall-Induced Arthritis and inMouse Collagen-Induced Arthritis Models. The ability ofPH-797804 to suppress chronic inflammation was demon-strated in both an inflammatory arthritis model in rats in-duced by SCW extract and in the widely used mouse collagen-induced arthritis model. The SCW model is characterized byan acute phase (hemorrhage and fibrin deposition in thesynovial space, accumulation of activated macrophages inthe soft tissue, and mild osteolysis) from day 1 to day 5,followed by a more severe and chronic phase (intense cell

infiltration, joint inflammation, and bone destruction) thatoccurs from day 10 to day 21. A role for TNF-� and IL-1� inthe chronic phase has been demonstrated with neutralizingTNF-� and IL-1 antibodies (Kuiper et al., 1998).

PH-797804 was highly effective in attenuating SCW-in-duced inflammation (Table 4; Fig. 6A). PH-797804 treatmentresulted in dose-dependent inhibition of paw swelling whenadministered daily from day 10 to day 21 (a time whenuntreated control rats exhibited profound joint inflamma-tion), with a maximal efficacy of �95% and an ED50 � 0.186mg/kg/ EC50 (Cmax) � 203 ng/ml/ EC50 (Cmin) � 32 ng/ml.Doses of 0.5 to 5 mg/kg PH-797804 produced almost completeprotection of the joint against inflammation-related bonedensity loss, whereas doses of 0.05 and 0.15 mg/kg werepartially efficacious (Fig. 6B).

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Fig. 3. PH-797804 inhibition of cytokine, PGE2 production, and p38 MAP kinase activity in human monocytic cells, human whole blood, andrheumatoid arthritis synovial fibroblasts. A, LPS-challenged U937 cells (human monocytic cell line). Data are from a single representative experiment(duplicate determinations for each data point) in which U937 cells were stimulated with LPS for 4 h after a 60-min incubation in the presence orabsence of the indicated concentrations of PH-797804. Culture supernatants were collected and assayed for TNF-� by Luminex multiplex TNF-� kits.Fitting the data to a standard curve generated with known amounts of recombinant TNF-� protein yielded an IC50 value of 7.35 nM. The control value(�LPS) for this experiment was 1.96 ng/ml TNF-� for the designated time. Data from three separate experiments yielded an IC50 of 5.9 � 1.3 nM(mean � S.E.M.; Table 3). The inhibition of p38 kinase activity was measured indirectly by evaluating the effect of PH-797804 on the ability of adownstream enzyme (MK-2) to phosphorylate its substrate HSP27. U937 cells were stimulated with LPS for 30 min, after a 60-min preincubation inthe presence or absence of the indicated concentrations of PH-797804. Cell lysates were prepared, MK-2 was immunoprecipitated, and activity wasdetermined as described under Materials and Methods. Data are from two independent experiments (IC50 � 1.05 � 0.64; mean � S.D.). B, IL-1�challenged rheumatoid arthritis synovial fibroblasts. RASF were incubated with PH-797804 for 1 h. IL-1� (1 ng/ml) was added to the assay plate andincubated for 20 h at 37°C with 5% CO2. PGE2 levels in cultured media were determined by ELISA. Data plotted are from a single representativeexperiment from a total of six experiments each performed in duplicate. C, LPS-challenged isolated human monocytes. Data are from a singlerepresentative experiment preformed in duplicate in which purified human monocytes were stimulated with LPS for 16 h after a 60-min incubationin the presence or absence of the indicated concentrations of PH-797804. Culture supernatants were collected and assayed for TNF-� and IL-1� byLuminex multiplex cytokine kits. Fitting the data to a standard curve generated with known amounts of recombinant TNF-� and IL-1� protein yieldedIC50 values of 3.0 nM each. Control values (�LPS) for this experiment were 9.4 and 1.76 ng/ml for TNF-� and IL-1�, respectively. Data from threeseparate experiments yielded IC50 values of 3.4 � 2.0 and 3.4 � 0.5 nM for TNF-� and IL-1�, respectively (mean � S.E.M.) (see Table 3).D, LPS-challenged human whole blood. Human whole blood was incubated with PH-797804 for 1 h. LPS (10 g/ml) was added to the mixture andincubated for 4 h at 37°C as described under Materials and Methods. Plasma TNF-� and IL-1� levels were determined by Luminex. TNF-� data areexpressed as the mean � S.E.M. from four separate experiments performed in duplicate, and IL-1� data are expressed as mean � S.D. from twoseparate experiments performed in duplicate. IC50 values from these experiments are shown in Table 3.


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The mouse CIA model is characterized by the appearanceof proliferative synovitis as assessed by paw swelling andredness approximately 3 weeks after immunization withchick type II collagen in Complete Freund’s adjuvant. Histo-logical analysis shows infiltration of polymorphonuclear and

mononuclear cells into the synovium, cartilage degradation,pannus formation, and bone destruction (Williams, 2007).Treatment of mice from day 21 to day 56 with vehicle dem-onstrated a 90% incidence of arthritis observed on day 56(Fig. 6C). Treatment of the animals with PH-797804 at 40

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Fig. 4. Morphological view and quantitative analysis of PH-797804 effects on osteoclast formation. Rat bone marrow cells were cultured without (topleft) or with RANKL (receptor activator of nuclear factor-�B ligand) (100 ng/ml) and M-CSF (10 ng/ml) (top right) for 3 days in the presence of 0.1 MPH-797804. At the end of the culture period, cells were stained for TRAP activity. Microphotographs of representative cultures were taken under lightmicroscope at the same magnification. The formation of osteoclasts TRAP� multinucleated cells with three or more nuclei (arrows) that was inducedby RANKL and M-CSF (middle) was blocked by the addition of PH-797804. Rat bone marrow cells were cultured in the presence or absence ofRANKL (100 ng/ml) and M-CSF (10 ng/ml). Various concentrations of PH-797804 (0.0001–1 M) were added to the cultures and the end of theculture period (3 days), cells were stained for TRAP activity, and the numbers of osteoclasts (TRAP�-multinucleated cells with three or morenuclei) were counted. PH-797804 inhibited osteoclast formation in a dose-dependent manner. Results shown are the mean � S.E.M. of threeindependent experiments.

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Fig. 5. Effect of PH-797804 on LPS-induced TNF-� production in rats and cynomolgus monkeys as a function of dose (A) and plasma-free fraction (B).Rat: adult male Lewis rats (�225–250 g) were orally dosed with PH-797804 or vehicle (0.5% methylcellulose and 0.025% Tween 20) 4 h before anintravenous administration of LPS (1 mg/kg). Blood was collected 90 min after LPS challenge. Serum TNF-� levels were quantified by ELISA.Compound blood levels were quantified by liquid chromatography-mass spectrometry. The ED50 value was calculated to be 0.07 mg/kg (A), with anEC50 value of 8.6 ng/ml (18.0 nM). Adjusting for free fraction gives a final EC50 value of 0.4 nM (B). Data are from three separate experiments withfive animals per group and are expressed as the mean � S.E.M.. Monkey: cynomolgus monkeys (n � 3) were dosed intragastrically with PH-797804or vehicle. Two hours later (time 0), LPS (10 g/kg) was administered by intravenous injection. Blood samples were taken at 0.75 to 1 h after LPSadministration. TNF-� concentrations in the plasma were determined using ELISA. Values are the mean of the percentage of control of peak TNF-�in plasma. Plasma concentrations of PH-797804 were determined by liquid chromatography-mass spectrometry. The ED50 value was calculated to be0.095 mg/kg (A), with an EC50 value of 7 ng/ml (15.0 nM). Adjusting for free fraction gives a final EC50 value of 0.97 nM (B). Data are expressed asmean values � S.E.M.

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and 120 mg/kg/day in chow resulted in a significant decreasein incidence, with only 20% exhibiting any clinical signs ofarthritis (Fig. 6C). This level of inhibition was similar tostudies using anti-TNF-� antibody therapy using a similarprotocol (Graneto et al., 2007). Lower doses, 4 and 12 mg/kg/day, were less effective in affecting incidence. A dose-depen-dent decrease in the severity of arthritis was observed at theend of the study, with the 12, 40, and 120 mg/kg/day doses.All three doses were significantly different from vehicle (p �0.05, ANOVA) (Fig. 6D).

A compilation of enzymatic, cellular, and in vivo data gen-erated on PH-797804 from binding studies to rat SCW-in-duced arthritis, adjusting for free fraction when appropriate,is shown in Fig. 7. The biochemical efficiency, as defined asthe relationship between a functional response (IC50) and thebinding affinity of the drug for the target (Ki) (Swinney,2006), shows a nearly 1:1 correlation across all assays andmodels. This translatability from enzyme to cell to animal,coupled with human pharmacokinetic projections, resulted ina high level of confidence in the dosage determination ofPH-797804 for human clinical studies.

PH-797804 Suppresses LPS-Induced Cytokine Re-lease in Healthy Human Subjects. A double-blind, placebo-controlled, single-dose study was done to assess the dose andconcentration response of PH-797804 on LPS-induced cytokinerelease in healthy human subjects. In this study, the endotoxinchallenge is administered as an intravenous bolus at a finaldose of 2 ng/kg. The endotoxin produces a transient endotox-emia evoking mild “flu-like” symptoms that resolve within a fewhours. Endotoxin-induced cytokines can be monitored as a mea-sure of the inflammatory response. The objectives of the endo-toxemia study were to describe the response of five single-dose

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Fig. 6. PH-797804 dose-dependently reduces paw inflammation (A) and bone mineral density loss (grams per square centimeter) (B) in ratSCW-induced arthritis and incidence (C) and severity (D) in mouse collagen-induced arthritis. A, rat SCW-induced paw swelling. Administration ofPH-797804 from day 10 to day 21 resulted in a dose-dependent decrease in paw swelling. Inhibition of edema was determined by a four-parameterlogistical model. ED50 and ED80 values were calculated to be 0.186 and 1.610 mg/kg, respectively. Data are expressed as the mean value obtained fromfour to eight animals per group, with each of four experiments plotted separately. B, rat SCW-induced bone mineral density loss. Administration ofPH-797804 from day 10 to day 21 resulted in a dose-dependent decrease in bone loss measured on day 21. Data are expressed as mean � S.E.M. froma single experiment with five rats per group. C, disease incidence (mouse CIA). Data are expressed as percentage of animals that demonstrated pawswelling at the termination of the experiment (a severity score of 1 or greater on day 56). D, disease severity (mouse CIA). Animals were evaluatedseveral times per week for signs of arthritis. Scoring of severity was carried out using a score of 1 to 3 for each paw as described under Materials andMethods (maximal score of 12/mouse). Data are the cumulative score determined at the end of the study. Any animal demonstrating a score of 1 ormore was considered arthritic. Dose groups consisted of 10 animals per group. �, p � 0.05 (by ANOVA), statistically different from control as describedunder Materials and Methods.

TABLE 4In vivo efficacy of PH-797804 in acute and chronic animal models:LPS-induced TNF-� production in rats and cynomolgus monkeys andrat SCW-induced arthritis

Assay PH-797804

Lewis rat LPS-induced TNF-�, ED50/ED80 (mg/kg) 0.07/0.142Lewis rat LPS-induced TNF-�, EC50/EC80 �C(5.5); ng/ml� 8.6/26.7Cyno LPS-induced TNF-�, ED50/ED80 (mg/kg) 0.095/0.349Cyno LPS-induced TNF-�, EC50/EC80 (Cmax; ng/ml) 7/22Rat SCW arthritis paw swelling, ED50/ED80 (mg/kg) 0.186/1.610Rat SCW arthritis paw swelling, EC50/EC80

(Cmax; ng/ml)203/1880

Rat SCW arthritis paw swelling, EC50/EC80(Cmin; ng/ml)

32/222


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levels of PH-797804 (1, 2, 4, 13, and 30 mg) versus placebo onTNF-� and IL-6 blood levels and p38 activity levels.

TNF-� concentrations were measured before study drugdosing on day 1; before LPS administration on day 2; and at1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 3, and 5 h after LPS adminis-tration. The highest mean TNF-� concentration occurred ateither 1.25 or 1.5 h after LPS administration for all groups,with the mean maximal TNF-� concentration for each treat-ment group being 327.9 pg/ml (placebo), 208.8 pg/ml (1 mg),188.7 pg/ml (2 mg), 105.8 pg/ml (4 mg), 62.9 pg/ml (13 mg),and 44.5 pg/ml (30 mg). The highest concentrations of TNF-�occurred in the placebo treatment group, consistent withthis established human experimental endotoxemia model,whereas PH-797804 induced a dose-dependent inhibition ofTNF-� response relationship (Fig. 8A). The data were welldescribed by an inhibitory effect Emax model [E � Emax (1 � C/(C � EC50))], resulting in EC50 � 2.9 � 1.2 (0.5–5.3)ng/ml and Emax � 311 � 35 (240–382) pg/ml [estimate � S.E.(95% CI)]. The observed data (closed circles) and the pre-dicted concentration-response relationship (solid line) areshown in Fig. 8A. The EC50 concentration along with one

approximated S.E. are shown as vertical solid and dashedlines, respectively. As expected in this model, there was amoderate amount of variability (uncertainty over parame-ter estimate) as reflected by the 40% coefficient of varia-tion on the EC50 parameter estimate.

The affects of PH-797804 administration on LPS-inducedIL-6 production and MK-2 activity were assessed as anotherindication of cytokine modulation and as target modulation,respectively. IL-6 concentrations were measured at pre-LPSadministration and 1, 1.5, 2, 2.5, 3, and 5 h after LPS admin-istration. The mean maximal IL-6 concentrations were 620.3pg/ml (placebo), 370.3 pg/ml (1 mg), 392.2 pg/ml (2 mg), 273.5pg/ml (4 mg), 289.5 pg/ml (13 mg), and 123.7 pg/ml (30 mg),with the largest mean concentrations occurring at 2 h (pla-cebo and 1, 2, and 30 mg) and 2.5 h (4 and 13 mg) after LPSadministration. A concentration response with PH-797804was observed (Fig. 8B). MK-2 activity, a biomarker for p38kinase activity, also correlated inversely with PH-797804dose and showed maximal inhibition at the highest doses andcompound blood levels (Fig. 8B). Both IL-6 and MK-2 activ-ity-response relationships correlated well with that of

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Fig. 7. Correlation of functional re-sponses to target binding affinity for PH-797804. A, data for p38� kinase binding,U937 TNF-� production, and humanmonocyte TNF-� production are each ex-pressed as the mean value of three inde-pendent experiments � S.E.M. Data forU937 p38� kinase activity is as de-scribed in Fig 3. B, data for humanmonocyte IL-1� production and oste-oclast formation are each expressed asthe mean value of three independentexperiments � S.E.M. Data for RASFPGE2 production are expressed as themean of six independent experi-ments � S.E.M. C, data for humanwhole blood TNF-� production and hu-man whole blood IL-1� production arethe mean/average of four and two inde-pendent experiments � S.E.M./S.D. re-spectively. Data for rat and cynomolgusTNF-� production and rat SCW pawswelling are as described in Fig 5.

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TNF-�, suggesting that administration of PH-797804 24 hbefore LPS challenge resulted in lower concentrations (rela-tive to placebo) of TNF-�, IL-6, and MK-2 activity in a dose-and concentration-dependent manner.

DiscussionThe novel N-phenylpyridinone PH-797804 is a highly se-

lective, readily reversible, ATP-competitive inhibitor of hu-man p38� kinase. Binding potency is primarily driventhrough two key interactions: 1) engagement of the activesite adjacent hydrophobic pocket by the 2,4-difluorophenylmoiety and 2) the formation of a bidentate H-bond betweenthe PH-797804 pyridinone and the M109-G110 peptide back-bone in the kinase crossover region (Xing et al., 2009). Therelative uniqueness across the human kinome of the largehydrophobic pocket gatekeeper residue (T106) in p38� kinasecoupled with the induced peptide backbone flip at G110 re-quired for bidentate H-bond formation are key structuralfeatures driving kinase selectivity for PH-797804 (Xing et al.,2009). The p38� kinase isoform-restricted expression inmonocytes, U937 cells, and RASF coupled with the highselectivity observed with PH-797804 suggests that the bio-logical activity observed with this compound is driventhrough modulation of p38� in these cells.

PH-797804 exhibits rapid binding kinetics, consistent witha binding mode that does not engage the allosteric siteformed upon the movement of the conserved Asp168-Phe169-Gly170 (DFG) to the “out” conformation. Inhibitors that en-gage this DFG pocket in p38� exhibit significantly slowerassociation and dissociation binding kinetics (Pargellis et al.,2002) relative to the classical ATP competitive inhibitors.Interactions with this DFG-out pocket in p38�, althoughgenerally enhancing compound affinity, may result in de-creased selectivity for kinase inhibitors (Angell et al., 2008).Interpretation of p38 kinase-driven biology by using highlyselective inhibitors such as PH-797804 is more conclusivethan studies using earlier generation p38 inhibitors in whichsignificant kinase crossover was observed (Karaman et al.,2008). Furthermore, the selectivity observed for PH-797804coupled with a human pharmacokinetic profile exhibiting alow peak/trough ratio (�1.8) resulted in a superior safetyprofile both preclinically and clinically.

The consistent cellular potency of PH-797804, PH-797804exhibited by IC50 values in the low nanomolar range acrosscell types, inflammatory mediator production, and p38 ki-nase activity measurements is made more interesting by thecorrelation with the inhibitory potency for the purified en-zyme in biochemical test systems. There is essentially a 1:1correlation between biochemical and cellular potency for PH-797804 across several markers of inflammation (Fig. 7). Thishigh level of biochemical efficiency (Swinney, 2006) isachieved despite the high physiological levels of cellular ATPand the observation that PH-797804 is an ATP-competitiveinhibitor. This apparent dichotomy can be rationalizedthrough examination of the relative affinities of ATP andPH-797804 for activated and unactivated p38 kinase. It hasbeen demonstrated previously that ATP binds to activatedp38 kinase with much higher affinity than unactivated ki-nase (Frantz et al., 1998), whereas PH-797804 binds withcomparable affinity to both forms of the enzyme. The bindingof PH-797804 to the unactivated enzyme in the absence ofATP competition decreases the activation rate of p38 kinase,resulting in the observed biochemical efficiency near unity. Asurvey of drugs receiving regulatory approval during thecurrent decade indicates that a significant majority exhibitbinding mechanisms predicted to achieve high biochemicalefficiency, thereby translating to lower dose levels needed toachieve efficacy and resulting in a larger therapeutic index(Swinney, 2006). The potency, selectivity and biochemicalefficiency demonstrated by PH-797804 supports this com-pound as a viable drug candidate for the treatment of inflam-matory diseases.

Three of the key hallmarks of RA are pain, joint destruc-tion, and inflammation. We developed cellular and in vivomodels to examine the impact of PH-797804 on these keydisease features. First, PGE2 is a mediator of inflammatorypain in the arthritic joint. PH-797804 effectively antagonizedIL-1�-induced PGE2 production from RA joint synoviocytes,consistent with a potential analgesic role in nociceptive pain.Brief reports of two other p38 kinase inhibitors SCIO-469(Tong et al., 2004) and Arry-797 (Remmers et al., 2008)described analgesic activity in human clinical studies of den-tal pain, further supporting the potential utility of p38 kinaseinhibitors in this clinical indication.

The second key disease feature investigated is joint de-struction. Destruction of bone and cartilage is a hallmark of

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Fig. 8. PH-797804 inhibits LPS-induced TNF-�, IL-6, and MK-2 activityin a dose- and concentration-dependent manner in a human endotoxinchallenge model. A, maximal TNF-� concentration versus plasma PH-797804 concentration: individual subject data (closed circles) and pre-dicted response profile (solid line). TNF-� concentrations were maximallevels at each dose group at a time point between 1 h and 3 h after LPSadministration. B, cytokine and MK-2 activity inhibition profiles as afunction of plasma concentration. Cytokine values are maximal concen-trations � S.E.M.


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progressive RA and result in debilitation and morbidity. Inaddition to attenuating paw edema, significant protection ofjoint bone mineral density was observed with PH-797804 inthe SCW-induced arthritis model, consistent with studies onearlier p38 kinase inhibitors (Mbalaviele et al., 2006). Stud-ies using rat bone marrow osteoclasts suggested that themechanism by which PH-797804 exerts its bone protectiveeffect may be through inhibition of osteoclast differentiation,the cell type primarily responsible for bone resorption (Rood-man, 1999). Treatment of rat bone marrow cells with PH-797804 resulted in a concentration-dependent inhibition ofosteoclast formation induced by the inflammatory cytokinesRANKL and M-CSF, with a potency comparable with itsability to inhibit TNF-� production and p38 kinase activity inother cell-based assays.

Third, from an inflammation standpoint, the significantefficacy of anticytokine biologic therapy in RA has demon-strated a role for cytokines such as TNF-�, IL-1, and IL-6 ascentral mediators of the inflammation associated with thedisease. Numerous studies have demonstrated that p38 ki-nase is a key enzyme in the intracellular networks thattransmit an inflammatory signal from the cell surface totrigger cytokine production. PH-797804-induced blockade ofTNF-� and IL-1� production from human monocytes andwhole blood support a role for p38 kinase in the biosynthesisand release of these cytokines. The inhibitory potency ofPH-797804 is consistent across cell models and cytokine mea-surements, with IC50 values in the 3 to 6 nM range (takinginto account nonprotein bound drug in human whole blood asthe active fraction). Furthermore, the corresponding inhibi-tion of p38 kinase activity in these cells is consistent with amechanism of action associated with specific antagonism ofthis kinase.

PH-797804 efficacy in modulating cytokine production inendotoxin challenge models in the rat and cynomolgus mon-key was comparable across these two species and corre-sponded to efficacy observed in human cellular models. Inaddition, concentration-response analysis of these data dem-onstrated compound potency comparable to human cell ac-tivity of PH-797804. EC50 values for rat and cynomolgusmonkey inhibition of endotoxin induced TNF-� productionwere 0.34 and 0.92 nM, respectively, based upon drug-freefraction (PH-797804 plasma protein binding was 98.1 and93.7% in these two species, respectively). The similar potencyobserved across cellular and acute in vivo models providedconfidence in the human efficacious dose projection basedupon concentrations of PH-797804 needed modulate p38 ki-nase activity and TNF-� production in these models.

Further evidence supporting anti-inflammatory proper-ties of PH-797804 was demonstration of efficacy in block-ing paw edema in two rodent models of RA, rat SCW andmouse CIA, after oral dosing. It is interesting that in ratSCW, effect on paw edema directly correlated with Cmin

values for PH-797804, defined as blood concentration ofdrug 12 h after administration of compound (Fig. 8C; inthis model, PH-797804 is dosed twice a day for 10 days).These observations suggest two pharmacodynamic proper-ties of PH-797804. 1) Persistent inhibition of p38 kinaseactivity is required to achieve anti-inflammatory efficacyin the SCW arthritis model, and 2) the potency of PH-797804 for achieving anti-inflammatory efficacy in a sub-

chronic model of RA is comparable to that required inacute in vivo and cellular models.

The potency of PH-797804 in nonclinical efficacy studiestogether with human pharmacokinetic projections was usedto determine the doses of PH-797804 for the human endotox-emia study. Doses bracketed the range projected to generateblood concentrations of drug that spanned 0 to 95% p38kinase inhibition and consequent cytokine modulation. As aresult of slow drug absorption and long half-life, endotoxinchallenge was administered 24 h after PH-797804 dosing, atime in which maximal drug levels were observed. A com-plete dose and concentration response was achieved, result-ing in a maximal 80 to 90% inhibition of TNF-� and IL-6. Asexpected, inhibition of p38 kinase activity was complete atthe higher doses and correlated with cytokine regulation. Theeffect of p38 kinase inhibition on cytokine production in hu-man endotoxemia has been reported previously (Branger etal., 2002; Faas et al., 2002); however, experiments describedhere are the first that demonstrate quantitative preclinical-to-clinical pharmacokinetic and pharmacodynamic transla-tion of inhibitor effect on the target (p38 kinase) and keyinflammatory mediators. These experiments further demon-strate the utility of cellular and/or acute inflammation mod-els as predictors of human activity.

The potential anti-inflammatory therapeutic utility of p38kinase inhibition remains an open question. Although thefirst wave of p38 kinase inhibitors that were evaluated clin-ically demonstrated development-limiting toxicities, reportsof newer generation, more selective compounds suggest lessof an issue with adverse events. It is interesting that recentreports suggest that the anti-inflammatory efficacy of p38kinase inhibition in RA was not robust or sustained(Genovese et al., 2008; Cohen et al., 2009; Damjanov et al.,2009). Lack of information on pharmacokinetic and targetcoverage information on these compounds precludes a mech-anistic understanding of this observation. Overall, the po-tency, selectivity, biochemical efficiency, in vivo efficacy, andpharmacokinetic properties of PH-797804 make it a strongcandidate to definitively evaluate the role of p38 kinase inhuman inflammatory disease. Toward that end, PH-797804is under evaluation in phase 2 clinical studies of RA andchronic obstructive pulmonary disease.

Acknowledgments

We thank Gail Jungbluth and Marie-Pierre Hellio Le Graverand-Gastineau for contributions to the clinical studies and Robert Chottfor technical assistance.

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Address correspondence to: Dr. Heidi R. Hope, Pfizer Global Research andDevelopment, 700 Chesterfield Parkway West BB4A, Chesterfield, MO 63017.E-mail: [email protected]


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