EMD 1214063 and EMD 1204831 Constitute a New …...Cancer Therapy: Preclinical EMD 1214063 and EMD 1204831 Constitute a New Class of Potent and Highly Selective c-Met Inhibitors Friedhelm

Cancer Therapy: Preclinical

EMD 1214063 and EMD 1204831 Constitute a New Class ofPotent and Highly Selective c-Met Inhibitors

Friedhelm Bladt, Bettina Faden, Manja Friese-Hamim, Christine Knuehl, Claudia Wilm, Claus Fittschen,Ulrich Gr€adler, Michael Meyring, Dieter Dorsch, Frank Jaehrling, Ulrich Pehl, Frank Stieber, Oliver Schadt, andAndree Blaukat

AbstractPurpose: The mesenchymal–epithelial transition factor (c-Met) receptor, also known as hepatocyte

growth factor receptor (HGFR), controls morphogenesis, a process that is physiologically required for

embryonic development and tissue repair. Aberrant c-Met activation is associated with a variety of human

malignancies including cancers of the lung, kidney, stomach, liver, and brain. In this study, we investigated

the properties of two novel compounds developed to selectively inhibit the c-Met receptor in antitumor

therapeutic interventions.

Experimental Design: The pharmacologic properties, c-Met inhibitory activity, and antitumor effects of

EMD 1214063 and EMD 1204831 were investigated in vitro and in vivo, using human cancer cell lines and

mouse xenograft models.

Results: EMD 1214063 and EMD 1204831 selectively suppressed the c-Met receptor tyrosine kinase

activity. Their inhibitory activity was potent [inhibitory 50% concentration (IC50), 3 nmol/L and 9 nmol/L,

respectively] and highly selective, when compared with their effect on a panel of 242 human kinases.

Both EMD 1214063 and EMD 1204831 inhibited c-Met phosphorylation and downstream signaling in a

dose-dependent fashion, but differed in the duration of their inhibitory activity. In murine xenograft

models, both compounds induced regression of human tumors, regardless of whether c-Met activation was

HGFdependent or independent. Both drugswerewell tolerated and induced no substantial weight loss after

more than 3 weeks of treatment.

Conclusions: Our results indicate selective c-Met inhibition by EMD 1214063 and EMD 1204831 and

strongly support clinical testing of these compounds in the context of molecularly targeted anticancer

strategies. Clin Cancer Res; 19(11); 2941–51. �2013 AACR.

IntroductionThe mesenchymal–epithelial transition factor (c-Met)

has emerged as a promising target in the development ofanticancer therapeutics because of its low level of expressioninnormal tissues and its aberrant activation inmanyhumancancers. The c-Met oncoprotein encodes a prototypic mem-ber of the receptor tyrosine kinase superfamily, which bindsto the hepatocyte growth factor (HGF), identified as its onlyhigh-affinity ligand (1). c-Met engagement by HGF resultsin receptor dimerization and autophosphorylation, whichin turn activatesmultiple signaling cascades involved in cell

survival, motility, and proliferation (e.g., the PI3K/Akt andMAPK/ERK pathways; ref. 2).

Targeted deletion of c-Met or HGF severely compromisesembryonic development, organ morphogenesis, and cellmotility, resulting in early embryonic lethality (3, 4). Inadult tissues, c-Met and HGF are detectable only at lowlevels, with epithelial and mesenchymal cells preferentiallyexpressing c-Met and HGF, respectively. However, upontissue damage, upregulation of both c-Met and HGF isreportedly associated with tissue regeneration and damagerepair (5, 6). Interestingly, c-Met activation can induce atransdifferentiation program known as epithelial–mesen-chymal transition, which results in loss of E-cadherin–mediated tight junctions, thereby conferring on epithelialcells’ mesenchymal characteristics, such as motility andinvasiveness (7).

Molecular epidemiologic analyses of patient sampleshave revealed different types of c-Met alterations, includingactivating point mutations, gene amplification, and aber-rant expression or overexpression (8). Notably, activatingpoint mutations of c-Met have been identified in hereditaryand sporadic papillary renal carcinoma, whereas gene

Authors' Affiliation: EMD Serono and Merck Serono Research andDevelopment, Merck KGaA, Darmstadt, Germany

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Friedhelm Bladt, Merck KGaA, Frankfurter Str.250, Darmstadt 64293, Germany. Phone: 49-0-6151-725069; Fax: 49-0-6151-727398; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-3247

�2013 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 2941

on August 13, 2020. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst April 3, 2013; DOI: 10.1158/1078-0432.CCR-12-3247

http://clincancerres.aacrjournals.org/

amplification has been found in lung cancers with acquiredresistance to EGF receptor inhibitors (9–11). In a murinemodel of glioblastoma, evidence suggesting a crucial rolefor an autocrine c-Met–activating loop mediated by HGF,with the therapeutic efficacy of c-Met inhibition, hasemerged (12, 13).

Several strategies have been used to inhibit c-Met activity,including c-Met– or HGF-specific antibodies or small-mol-ecule inhibitors. In the generation of small-molecule inhi-bitors targeting the ATP-binding pockets of the c-Met pro-tein kinase, a major obstacle has been the high degree ofsequence identity within the ATP-binding clefts of canon-ical protein kinases (14). Indeed, all currently approvedsmall-molecule protein kinase inhibitors exhibit promiscu-ous binding, targeting multiple enzymes.

Our studies define the pharmacologic and preclinicalcharacteristics of 2 novel compounds, EMD 1214063 andEMD 1204831, which selectively inhibit c-Met. Both com-pounds exhibited high tolerability in vivo and effectivelyinhibited growth of human tumors in murine xenograftmodels irrespectively of whether c-Met activation resultedfrom HGF-dependent or -independent mechanisms.

Materials and MethodsCompounds

EMD 1214063 (3-(1-(3-(5-(1-Methylpiperidin-4-ylmethoxy)-pyrimidin-2-yl)-benzyl)-1,6-dihydro-6-oxo-pyridazin-3-yl)-benzonitrile) and EMD 1204831 (4-(2-(2-(3-(3-(1-Methyl-1Hpyrazol-4-yl)-6-oxo-1,6-dihydro-pyridazin-1-ylmethyl)-phenyl)-pyrimidin-5-yloxy)-eth-yl)-morpholine) were originally developed at MerckSerono, and were synthesized at Merck Serono.

Cell lines and antibodiesHuman cancer cell lines were purchased from the Amer-

ican Type Culture Collection (A549, NCI-H441, U87MG,

Hs746T, HT-29, LoVo, HCT-116, NCI-H1975, NCI-1437,H460), Riken Cell Bank (EBC-1, KP-4), and "DeutscheSammlung von Mikroorganismen und Zellkulturen"(MKN-45), and maintained according to the manufac-turer’s recommendations. The epitope specificity, clonedesignation, and commercial source of the antibodies usedare indicated in Supplementary Table S1.

c-Met in vitro kinase assayKinase inhibition by EMD 1214063 or EMD 1204831 (1

and 10 mmol/L) was assessed in vitro using a panel of 242different kinases. Biochemical activity was measured in aflash-plate assay. His6-tagged recombinant human c-Metkinase domain (Aa 974–end; 20 ng) and biotinylated poly-Ala-Glu-Lys-Tyr (6:2:5:1; 500 ng) were incubated with orwithout the test compound for 90 minutes at room tem-perature in 100mL buffer containing 0.3 mCi 33P-ATP, 2.5 mgpolyethylene glycol 20.000, and 1% dimethyl sulfoxide(DMSO), as previously described (15). Radioactivity wasmeasured with a TopCount microplate scintillation andluminescence counter (Packard BioScience BV). Inhibitory50% concentration values (IC50) were calculated by non-linear regression analysis using the RS/1 software program.

Phospho-c-Met-capture ELISATotal c-Met phosphorylation was assessed by c-Met–

capture ELISA in Nunc-Immuno MicroWell 96-well solidplates (Sigma-Aldrich). A549 human lung cancer cells wereseeded 2 days before treatment, serum-starved for 20 hours,and treated on day 3 with different concentrations of EMD1214063 or EMD 1204831 or 0.2% DMSO for 45 minutesat 37�C, 5% CO2. Upon stimulation with 100 ng/mL HGFfor 5 minutes, cells were lysed with 70 mL per well ice-coldlysis buffer [20 nmol/LHEPES, pH7,4; 10% (V/V)Glycerol;150 nmol/LNaCl; 1% (V/V) Triton-X-100; 2 nmol/L EDTA]supplementedwith protease andphosphatase inhibitors. Inthe wash-out experiments, A549 were treated with EMD1214063 or EMD 1204831 for 45 minutes, washed, andincubated in serum-free medium for 14 hours, beforestimulation with HGF (100 ng/mL).

In the ELISA, the capture antibody was specific for the c-Met extracellular domain, whereas an antiphosphotyrosinebiotin-labeled antibody was used for detection. Tyrosinephosphorylation was revealed using a streptavidin peroxi-dase conjugate and chemiluminescence read-out.

Biochemical analysisPhosphorylation of c-Met, Gab-1, Akt, and Erk1/2 was

analyzed by Western blot analysis in EBC-1 cells. In brief,cells were seeded at a density of 3� 106 cells per well, serum-starved for 20hours, and lysedonday3after incubationwithEMD 1214063. Proteins were separated by SDS-PAGE andblotted onto nitrocellulose membranes. Membranes wereblocked with Tris-buffered saline and incubated in primaryantibody solution (anti-pMet, anti-pAkt, anti-pERK1/2, anti-Gab1) at 4�C overnight. Proteins were detected by chemi-luminescence,withVersaDocMP5000 imaging system(Bio-Rad) equipped with Quantity One 1-D analysis software.

Translational RelevanceThe primary objective of anticancer therapeutic inter-

ventions is to selectively target cancer cells while notdamaging healthy tissues. To this end, the c-Met receptortyrosine kinase has been regarded as an interesting targetfor anticancer therapy because it is physiologicallyexpressed at low levels in most adult tissues, but isoverexpressed and aberrantly activated in a number ofsolid tumors. Pathologic triggering of c-Met is associatedwith activation of signaling pathways leading to cellproliferation, survival, migration, invasion, metastasis,and drug resistance. Here, we report the development oftwo smallmolecules, EMD1214063 and EMD1204831,with the characteristics of c-Met inhibitors. We provideevidence that these compounds are selective for the c-Met receptor tyrosine kinase and suppress c-Met activa-tion triggered by both ligand-dependent and -indepen-dent mechanisms.

Bladt et al.

Clin Cancer Res; 19(11) June 1, 2013 Clinical Cancer Research2942




Wound healing test and proliferation assaysWound healing tests were carried out as previously

described (16). In brief, a scratch was produced with asterile pipette tip on a monolayer of NCI-H441 lung cancercells. The effect of EMD 1210463 and EMD 1204831 onclosure of the cell gap was monitored over 24 hours in thepresence or absence of 100 ng/mL HGF. All proliferationand colony formation assays were conducted in 4 replicatesand included 4 DMSO vehicle controls. IC50 values weredetermined by 4PL fitting in GraphPad Prism v5.

Pharmacokinetic and pharmacodynamic studiesPlasma and tumor drug concentrations were measured

using high-performance liquid chromatography (HPLC)andmass spectrometry (MS). In brief, protein precipitationwas carried out in methanol for plasma samples, and inethanol/water 80:20 (v/v) using a Precellys 24 homogenizer(Bertin Technologies) for homogenized tumor samples.The HPLC/tandem mass spectrometry (MS-MS) systemconsisted of an Agilent 1100 Series HPLC system with aCTC HTC PAL Autosampler coupled to an Applied Biosys-tems API4000 mass spectrometer. HPLC separation wasachieved on a reversed-phase column (Chromolith Spee-dROD RP-18e, 50–3mm) using gradient elution (eluent A:formic acid 0.1%; eluent B: acetonitrile). Selectivity wasachieved usingmultiple reactionmonitoring (MRM) for theMS/MS detection of the compounds.For the in vivo pharmacodynamic studies, all animal

studies were conducted according to standard proceduresapproved by local animal welfare authorities. Mice wereinjected subcutaneously with 5 � 106 Hs746T cells (100mL). Once the tumor volume had reached 600 to 1,000mm3, mice were randomized into different experimentalgroups, receiving a single oral dose of 3, 10, 30, and 100mg/kg of EMD 1214063, EMD 1204831, or vehicle. Tumorand plasma samples were collected at 3, 6, 12, 24, 48, 72,and 96 hours after treatment. Each experimental groupcomprised 4 mice per dose and time point. Samples of thetumor tissue were snap-frozen for pharmacokinetic andbiomarker analyses, or formalin-fixed for immunohisto-chemical analysis (see below).

Pharmacodynamic markers on ex vivo tumor samplesc-Met autophosphorylation was investigated by Western

blot analysis on frozen ex vivo tumor samples. The tumortissue was mechanically homogenized, lysed using Pre-cellys 24 homogenizer, or Precellys ceramic lysing tubes(PEQLab Ltd) according to the manufacturer’s instruc-tions. Further preparation of lysates and protein separationby SDS-PAGE were conducted as already described forEBC-1 cells.Histone H3 phosphorylation and biomarkers of cell-

cycle arrest and apoptosis (cyclin D1, p27, and cleaved,activated capase-3) were analyzed by immunohistochem-istry (IHC) on formalin-fixed, paraffin-embedded sections.IHC was conducted using Discovery staining instruments(Ventana Medical Systems, Inc), with the OmniMap Kit(Ventana Medical Systems, Inc), according to the manufac-

turer’s instructions. Sections were counterstained withhematoxylin.

Xenograft models of antitumor efficacyThe antitumor efficacy of EMD 1214063 or EMD

1204831 was investigated in mouse xenograft models.CD-1 or BALB/C nude mice (Charles River Laboratories)were injected subcutaneously with human cancer cell linesKP-4,U87MG: 10� 106 cells in 100mL,Hs746T, EBC-1: 5�106 cells in 100 mL. As soon as the tumor reached the lineargrowth phase (70–150 mm3), tumor-bearing mice (10mice/group) were injected daily with the indicated dosesof EMD1214063 or EMD1204831, or vehicle. Bodyweightand tumor size [length (L) and width (W)] were measuredtwice weekly. The tumor volume was calculated using theformula L � W2/2. Statistical significance was determinedby one-way ANOVA (Kruskal–Wallis, Dunn posttest). P �0.05 were considered significant.

ResultsEMD 1214063 and EMD 1204831 are competitive andselective inhibitors of c-Met kinase activity

EMD 1214063 and EMD 1204831 were synthesized tospecifically interactwith c-Met and inhibit its kinase activity.The biochemical activity of these compoundswasmeasuredin a flash-plate assay using recombinant human c-Metkinase domain and a biotinylated peptide substrate. Underthese conditions, EMD 1204831 inhibited c-Met kinaseactivity with an average IC50 of 9 nmol/L, whereas EMD1214063 exhibited an average IC50 of 3 nmol/L.

The development of selective inhibitors of c-Met activityhas been hampered by the high sequence and structuralhomology of the ATP-binding pocket of protein tyrosinekinases. Indeed, most of the inhibitors currently evaluatedin clinical trials show ahigh degree of interactionwith otherkinases. To assess the selectivity of EMD 1214063 and1204831 for c-Met kinase activity, these compounds wereprofiled against a protein kinase panel of 242 humankinases (Fig. 1A and B). At 10 mmol/L, a concentrationapproximately 1,000-fold above the IC50 of both inhibitors,only 5 of the other 241 kinases were inhibited by EMD1214063 by more than 50%, namely IRAK4, TrkA, Axl,IRAK1, and Mer. Formal IC50 determinations conductedwith these kinases resulted in IC50 values ranging from 615to 2,272nmol/L. Suchhigh IC50 valuesmake it unlikely thatEMD 1214063 will achieve in vivo pharmacologically rele-vant inhibitory activity on IRAK4, TrkA,Axl, IRAK1, andMer(proposed as IC90 for 24 hours).

EMD 1204831 showed an exceptionally high level ofkinase selectivity toward c-Met with an inhibitory activity ofmore than 3,000-fold in comparison with the other 241human kinases tested, as none of these kinases was inhib-ited bymore than 50% at 10 mmol/L concentration of EMD1204831. Such selectivity was reflected both in vitro and invivo. Treatment with EMD 1214063 and EMD 1204831resulted in a decrease in tumor cell viability in vitro (Sup-plementary Table S2). In in vivo mouse xenograft models,

EMD 1214063 and EMD 1204831, a New Class of c-Met Inhibitors

www.aacrjournals.org Clin Cancer Res; 19(11) June 1, 2013 2943




administration of either compound induced a reduction intumor growth of c-Met–"addicted" human cell lines, butonly negligible effects on c-Met–independent cell lines(Supplementary Table S3). Taken together, our data showthat EMD 1214063 and EMD 1204831 are highly selective,potent, and reversible inhibitors of c-Met. Interestingly,increasing concentrations of ATP suppressed inhibition ofc-Met phosphorylation by EMD 1214063, indicating thatthis compound act via an ATP-competitive mechanism(Supplementary Fig. S1).

EMD 1214063 and EMD 1204831 inhibit c-Metphosphorylation and downstream signaling pathways

We next investigated whether EMD 1214063 and EMD1204831 could inhibit c-Met phosphorylation induced intumor cells by different mechanisms. To this end, we usedthe A549 and EBC-1 cancer cell lines, in which c-Metphosphorylation is respectively triggered by HGF bindingor by c-Met gene amplification and ligand-independentactivation.

Exposure of A549 cells to EMD 1214063 and EMD1204831 resulted in inhibition of HGF-induced c-Metphosphorylation, with an average IC50 of 6 and 15nmol/L, respectively (Fig. 2A and B).

Treatment with these compounds induced a markedreduction of c-Met–constitutive phosphorylation in EBC-1cells with IC50 of 9 nmol/L for EMD 1214063 (Fig. 2C and

Supplementary Fig. S2A and S2B) and 12 nmol/L for EMD1204831 (Fig. 2D and Supplementary Fig. S2E and S2F), asassessed byWestern blot analysis. Similarly, a strong inhib-itory effectwas observed in the gastric cancer cell linesMKN-45 andHs746T (data not shown), inwhich c-Met is inducedin a ligand-independent manner.

Upon HGF-induced or ligand-independent c-Met phos-phorylation, several adapter and effector proteins, such asGrb2, Gab1, Sos, PLCg , and phosphoinositide 3-kinase(PI3K) are recruited and ultimately orchestrate the activa-tion of downstream signal transduction pathways, such asthe Akt and the ERK/MAPK cascades (7). To assess the effectof EMD 1214063 and EMD 1204831 on the activation ofsignaling pathways downstream of c-Met, the phosphory-lation of Gab-1, Akt, and Erk1/2 was monitored. Bothinhibitors effectively blocked phosphorylation of thesemajor downstream effectors of the c-Met enzyme in EBC-1(EMD 1214063: Fig. 2C and Supplementary Fig. S2A–S2D;EMD 1204831: Fig. 2D and Supplementary Fig. S2E–S2H),MKN-45, and Hs746T cells in the range of 1 to 10 nmol/L(data not shown). Taken together, these data are compatiblewith the hypothesis that EMD 1214063 and EMD 1204831effectively block c-Met–mediated signaling pathwaysinvolved in tumor cell growth and survival.

We next defined the efficiency of the cellular uptake andretention of EMD 1214063 and EMD 1204831. A series ofwash-out studies was conducted, in which A549 cells were

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Figure 1. EMD1204831 and EMD1214063 selectively inhibit c-Met kinase activity. The selectivity of kinase inhibition by EMD1204831 and EMD1214063wasassessed by kinaseprofiling. Inhibition levels between 80%and100%and60%and80%are indicated asbig and small red circles, respectively. The IC50wasspecifically determined for kinases exhibiting the highest levels of inhibition. EMD 1204831 (A) and EMD 1214063 (B) displayed high selectivity for c-Met evenat concentrations more than 1,000-fold the IC50. The data shown are cumulative of 3 independent experiments.

Bladt et al.





incubated for 30 to 45 minutes in the presence of differentconcentrations of EMD 1214063 and EMD 1204831,washed, stimulated with HGF, and subsequently assessedfor c-Met phosphorylation.Our findings indicate that c-Met phosphorylation was

inhibited upon exposure to EMD 1214063 and lasted formore than 14 hours, with an average IC50 of 5 nmol/L(Fig. 2A). These data show cellular retention of EMD1214063, accompanied by sustained c-Met inhibition.Interestingly, the inhibitory effect of EMD 1214063 onHGF-induced c-Met phosphorylation was only moderatelyaffected by the presence of 10% (v/v) murine or humanserum, resulting in average IC50 values of 21.0 and 23.0nmol/L, respectively. Higher serum concentrations couldnot be used in this test, as they suppressed the HGF-induced c-Met phosphorylation, probably because of HGFbinding to serum proteins or HGF inactivation by serumproteases.In contrast, in the case of EMD 1204831, incubation

with the compound followed by removal and HGF stim-ulation led to a transient, unsustained inhibition of li-gand-induced c-Met phosphorylation. Similarly to EMD1214063, the effect of EMD 1204831 was not affected bythe presence of 10% (v/v) murine or human serum, result-ing in average IC50 values of 26.0 and 25.0 nmol/L,respectively (Fig. 2B).Taken together, our data show that both EMD 1214063

and EMD 1204831 have excellent cellular activity andmoderate binding to serum proteins. Furthermore, theyindicate that the effect of EMD 1214063 lasts longer thanthat of EMD 1204831.

EMD 1214063 and EMD 1204831 inhibit tumor cellproliferation and migration in vitro

c-Met can increase the proliferation rate of tumor cellsand support their survival under stress conditions, partic-ularly in the cells with c-Met gene amplification. To inves-tigate the effect of EMD 1214063 and EMD 1204831 ontumor cell viability in vitro, MKN-45 gastric cancer cells wereincubatedwith increasing concentrations of both inhibitorsand the metabolic activity of cells was assessed. EMD1214063 and EMD 1204831 incubation for 72 hours con-siderably inhibited the viability of MKN-45 cells with IC50

values of less than 1 and 52 nmol/L, respectively. In con-trast, other gastric cancer cells with normal c-Met gene copynumber, such as SNU-16, were less sensitive to c-Metinhibitors with IC50 values higher than 10 mmol/L (Sup-plementary Table S2 and data not shown).

The alternative name ofHGF, scatter factor, indicates thatHGF via c-Met activation can have a pronounced effect oncell motility, migration, and invasion. To address the effectof both c-Met inhibitors on these HGF-mediated phenom-ena, in vitro "wound healing" tests were conducted. Treat-ment with EMD 1214063 and EMD 1204831 (as low as 0.1nmol/L) inhibited HGF-induced NCI-H441 cell migration,whereas concentrations of 100 nmol/L to 1 mmol/L almostcompletely prevented it (Supplementary Fig. S3).

EMD 1214063 and EMD 1204831 inhibit in vivo c-Metphosphorylation

To define the in vivo activity of different doses of EMD1214063 and EMD 1204831, we investigated the relation-ship between drug exposure (pharmacokinetic) and

Figure 2. Dose-dependentinhibition of c-Met phosphorylationand downstream signaling pathwaysby EMD 1214063 and EMD1204831.A and B, A549 lung cancer cells arecharacterized by c-Met expressionand ligand dependence. In vitroA549culture was carried out under serum-free conditions or in the presence of10% human or mouse serum. UponHGF stimulation, inhibition of total c-Met phosphorylation by EMD1214063 (A) or EMD 1204831 (B) wasassessed by c-Met capture ELISAusing a pan-phospho-Tyr antibody.The levels of total phospho-c-Metwere also assessed after wash-out ofthe inhibitors, as described in theMaterials and Methods section. Cand D, inhibition of c-Metautophosphorylation (Y1234/Y1235)and downstream signalingmolecules by EMD 1214063 (C) orEMD 1204831 (D) were assessed byWestern blotting in c-Met–amplified,ligand-independent, EBC-1 non–small cell lung cancer cells. Resultsare representative of 2 independentexperiments.

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pharmacodynamic effects in vivo in a cancer xenograftmodel, using Hs746T gastric cancer cells. In line with itshigh volume of distribution of more than 8 L/kg in mice,EMD 1214063 concentration was higher in the tumor thanin the plasma (Fig. 3A). In both matrices, concentrationsand exposures seemed to increase as a function of the dose.Kinetic studies indicated that, within the tumor, the activepharmacologic range identified in vitro was reached at alltime points tested (3–96 hours) upon administration ofdoses of EMD 1214063 of 10 mg/kg or more. EMD1214063-treated tumors exhibited strongly reduced c-Metphosphorylation. At the lowest dose of EMD 1214063 (3mg/kg), 90% inhibition of c-Met phosphorylation wasreached at 6 hours, and decreased to 30% at 24 hours(Supplementary Fig. S4A). At doses of 10 mg/kg or more,EMD 1214063 resulted in more than 90% inhibition of c-Met phosphorylation for a period of at least 72 hours(Supplementary Fig. S4A).

For EMD1204831, comparable drug concentrationsweredetected in plasma and tumor, in agreementwith its volumeof distribution of around 1 L/kg in this rodent species (Fig.3B). Overall, the exposure in both matrices increased withthe dose, but not in a strictly proportional fashion as in thecase of EMD 1214063. Similarly to EMD 1214063, theactive pharmacologic range identified in vitro was reachedwith doses of 10 mg/kg or more, and complete targetinhibition was induced. c-Met phosphorylation was con-sistently reduced in all EMD 1204831-treated tumors. Thelowest dose of EMD 1204831 (3 mg/kg) induced transientc-Met inhibition, which reached maximal levels (55%) at 3hours, and subsequently returned to baseline levels (12% at12 hours). Administration of 10 mg/kg resulted in a 90%inhibition of c-Met phosphorylation that declined after 6hours, whereas doses of 30 mg/kg or more induced pro-longed c-Met inhibition,with 58% inhibition still present at

96 hours after drug administration (Supplementary Fig.S4B).

To rule out that the reduction of c-Met phosphorylation isdue to a decrease in c-Met expression, c-Met expression andphosphorylation were analyzed in vitro in U87MG cells(Supplementary Fig. S5) and ex vivo in KP4 tumors (Sup-plementary Fig. S6). Thesemodelswere selected because therelatively moderate expression of c-Met would enable tobetter detect small changes upon treatment with EMD1214063 and EMD 1204831. Our data indicate that EMD1214063 and EMD 1204831 decrease c-Met phosphoryla-tion (Y1234/Y1235; Y1349) in the absence of alterations in c-Met expression.

Taken together, these findings establish a correlationbetween the inhibitor concentration in the plasma and inthe tumor, indicating the ability of these compounds toinhibit c-Met phosphorylation in vivo. Furthermore, thesedata provide pharmacodynamic evidence that indicate thattwice daily dosing of EMD 1204831 may be required toobtain continuous and efficient target inhibition, whereasless frequent administrations may be feasible for EMD1214063.

EMD 1214063 and EMD 1204831 interfere with tumorcell proliferation in vivo

To elucidate the molecular changes occurring in tumorsupon in vivo administration of EMD 1210463 or EMD1204831, we assessed c-Met phosphorylation and investi-gated a series of biomarkers indicative of cell-cycle arrestand apoptosis induction. The levels of phospho–c-Metweredramatically reduced upon treatment with a single dose of30 and 100mg/kg of EMD1214063.While high doses (100mg/kg) of EMD1214063 resulted in persistent inhibition ofc-Met phosphorylation, lower doses (30 mg/kg) engen-dered a transient inhibitory effect that returned to baseline

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Figure 3. Dose-dependent inhibition of c-Met auto-phosphorylation in vivo. Mice bearing established tumors, derived from subcutaneous injection of Hs746Tgastric cancer cells, received a single dose of 30 mg/kg EMD 1214063 (A) or EMD 1204831 (B). The pharmacokinetics of EMD 1214063 and EMD 1204831were assessed by analysis of the compound levels in plasma and tumor tissue. The pharmacodynamic effects were determined by Western blot analyses ofc-Met autophosphorylation (Y1234/Y1235). The lower limits of detection for EMD 1214063 and EMD 1204831 were, respectively, 0.007 mmol/L (4 ng/mL)and 0.014 mmol/L (8 ng/mL) in the plasma, and 0.037 mmol/L (20 ng/mL) and 0.014 mmol/L (8 ng/mL) in tumor samples. Each data point represents themean�SD of 3 or more values obtained from experimental groups composed of 4 mice. Further analyses were conducted upon in vivo injection of different dosesof EMD 1214063 and EMD 1204831. The results obtained from this set of experiments are depicted in Supplementary Fig. S5.

Bladt et al.





after 96 hours (Fig. 4A). Further analysis revealed suppres-sion of cyclin D1 expression, which reverted to baselinelevels at 24, 48, or 96 hours, for doses of 3, 10, or 30mg/kg,respectively. In contrast, more than 50% reduction of cyclinD1 expression persisted after 96 hours upon treatment withdoses of 100mg/kg (Fig. 4B and data not shown). Finally, atransient induction of p27 was observed upon treatmentwith 3, 10, 30, and 100 mg/kg of EMD 1214063. Maximalincrease of p27 was detected at 24 to 48 hours, whereasbaseline levels were restored after 72 to 96 hours, with theexception of the samples treated with 100 mg/kg, in whichp27 remained elevated even after 96 hours (Fig. 4C and datanot shown).

The inhibition of c-Met phosphorylation following EMD1204831 administration did not last as long as that inducedby EMD 1214063. The phosphorylation of c-Met and his-tone H3 as well as the levels of cyclin D1 were reduced in adose-dependent manner (Fig. 4A and B and data notshown). A transient increase in the levels of p27 wasobserved upon treatment with 3, 10, 30, and 100 mg/kgof EMD 1204831. Maximal increase of p27 was detected at12 to 24 hours, whereas baseline levels were restored after48 to 96 hours (Fig. 4C and data not shown).

Finally, a transient increase of cleaved caspase-3 wasobserved in tumor samples from mice treated with a singledose of EMD 1214063 or EMD 1204831 (Supplementary

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Figure 4. EMD1214063 andEMD1204831 effectively inhibit tumor c-Met auto-phosphorylation and cyclin D1 expressionwhile inducing p27 expression.Micebearing established tumors, derived from subcutaneous injection of Hs746T gastric cancer cells, received a single dose of 30mg/kg EMD1214063 (A–C, top)or 100 mg/kg EMD 1204831 (A–C, bottom). The levels of c-Met autophosphorylation (Y1234/Y1235; A), cyclin D1 expression (B), and p27 expression (C) wereassessed by immunohistochemistry at 1 to 6, 12, 24, 48, and 96 hours after treatment. Data are representative of 4 mice per experimental group.






Fig. S7), suggesting that these compoundsmay also increasethe rate of tumor cell apoptosis.

Collectively, these data provide evidence that EMD1214063 and EMD 1204831 are capable of inhibiting invivo c-Met phosphorylation, thereby inducing cell-cyclearrest and apoptosis of cancer cells.

EMD 1214063 and EMD 1204831 induce tumorregression in xenograft models

We next investigated the impact of EMD 1214063 andEMD 1204831 on the growth of HGF-dependent and HGF-independent c-Met–amplified human tumors grafted ontonude mice (Fig. 5 and Supplementary Fig. S8). To bettermimic the clinical occurrence of c-Met–driven tumors, wetested the effect of the 2 compounds on 2 sets of tumor cells:the Hs746T gastric carcinoma and EBC-1 non–small celllung cancer tumor cell line, in which c-Met is amplified,overexpressed, and activated in a ligand-independent fash-ion (17, 18); on the other hand, the U87MG glioblastomaand KP-4 pancreatic carcinoma cell line in which the coex-pression of HGF results in ligand-dependent c-Met activa-tion (19, 20).

Daily administration of 5 or 15 mg/kg of EMD 1214063to EBC-1 tumor-bearingmice resulted in effective inhibitionor complete tumor regression, respectively (Fig. 5A). Sim-ilarly, EMD 1204831 administered at a dose of 25 mg/kgtwice daily resulted in partial remissions in 50% of tumor-bearing mice. Interestingly, a single dose of 50 mg/kg wasless effective than 2 daily doses of 25 mg/kg administered 6to 7hours apart,whichdidnot induce tumor regression, butonly tumor growth inhibition (Fig. 5B).

EMD 1214063 and EMD 1204831 also induced dose-dependent tumor growth inhibition inmice bearinghumanpancreatic carcinoma KP-4 tumors (Fig. 5C and D). Dailyadministration of EMD 1214063 at 200 mg/kg resulted inpartial tumor regressions in 60% of tumor bearing mice,whereas twice daily administration of 200 mg/kg EMD1214831 induced growth arrest of KP-4 tumors.

Effective tumor growth inhibition and regression ofestablished tumors by EMD 1214063 or EMD 1204831were also observed in mice bearing ligand-independentHs746T andHGF-dependent U87MG tumors (Supplemen-tary Fig. S8A–S8D).

In all studies, both inhibitors were well tolerated. Treat-ment-related toxic effects, as indicated by severe bodyweight loss or death of mice, were not observed for any ofthe doses studied.

Taken together, our studies indicate that EMD 1214063and EMD1204831 are effective in treatment ofHGF-depen-dent and –independent tumors in vivo xenograft models.

DiscussionThis report provides preclinical evidence that EMD

1214063 and EMD 1204831 effectively inhibit c-Met acti-vation and suggests that they may be promising candidatesfor cancer treatment. Our data biochemically define EMD1214063 and EMD 1204831 as ATP-competitive c-Metinhibitors, characterized by high potency, selectivity, andreversibility. Finally, EMD 1214063 and EMD 1204831efficiently inhibited c-Met phosphorylation and down-stream signaling in vivo and induced regression of estab-lished tumors in xenograft models.

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Figure 5. EMD 1214063 and EMD1204831 display antitumor activityin HGF-dependent and HGF-independent xenograft tumormodels. Mice bearingsubcutaneous tumors derived fromthe HGF-independent human lungcancer cell line EBC-1 (A and B) orthe human HGF-dependentpancreatic carcinomacell lineKP-4(C and D) were administered dailythe indicated doses of EMD1214063 (A and C), EMD 1204831(B and D), or vehicle for theindicated treatment duration. Eachdata point represents the mean �SEM of the calculated tumorvolumes observed in experimentalgroups including 10 mice.Statistical significance wasevaluated by one-way ANOVA(Kruskal–Wallis, Dunn posttest).Asterisks indicate statisticallysignificant differences betweentreated and control experimentalgroups (P � 0.05). The resultsobtained from additional testing inother in vivo xenograft models areshown in Supplementary Fig. S8.bid, twice daily; NSCLC, non–smallcell lung cancer; qd, daily.

Bladt et al.





The rationale behind developing c-Met kinase inhibitorsfor the treatment of cancer is based on multiple lines ofpreclinical evidence showing that aberrant activation of theHGF/c-Met signaling pathway plays a pivotal role in cancerprogression andmetastasis by promoting cell proliferation,survival, and motility (2). Dysregulation of the c-Met/HGFpathway can occur by ligand-dependent or -independentmechanisms. Indeed, overexpression of wild-type c-Met orengagement by HGF in an autocrine or paracrine fashioninduces tumor transformation (21, 22). In this context, c-Met activation is dynamic, depends on altered environmen-tal conditions, and is controlled bymultiple, nonredundantsignaling cross-talk. On the other hand, mutations in c-Metcytoplasmic domains canprolong the duration of activatingsignals, releasing inhibitory constraints, enhancing c-Metsensitivity to activating stimuli, or preventing kinase deg-radation. Recent studies have shown that ligand-indepen-dent c-Met activation can also occur through mutationsleading to continuous c-Met endocytosis/recycling,impaired degradation, and chronic activation (23).In this context, constitutive c-Met activity produces a

phenomenon defined as "oncogene addiction," which ischaracterized by high steady-state signaling due to chronicreceptor activation. The strong reliance of the tumor on thecontinued activity of c-Met makes it very sensitive to kinasedeprivation, which is likely to induce strong clinicalresponses.Small-molecule kinase inhibitors have a broad therapeu-

tic efficacy and their development is becoming increasinglyfeasible as a result of the improved understanding of thestructure and molecular mode of action of kinases. Small-molecule c-Met inhibitors impede c-Met activity irrespec-tively of the initial mechanism responsible for the activa-tion. This property provides an advantage over blockingantibodies specific for HGF or c-Met, because the latter areexclusively active on ligand-dependent tumors andmay notinterfere with c-Met–active dimers and intracellular fusionproteins, such as TPR-Met.EMD 1214063 and EMD 1204831 are type I small-mol-

ecule kinase inhibitors, as they bind c-Met in a U-shapegeometry through interactions with both hinge and activa-tion loop residue Y1230. Such binding interferes with ATP-binding and distinguishes these compounds from type II c-Met inhibitors (e.g., XL880 and XL184), which adopt amore extended orientation, binding protein conformationswith varying degrees of "DFG-out" character. Unlike type IIc-Met inhibitors, which target multiple kinases, type I c-Metinhibitors are more selective (24). Indeed, broad kinaseprofiling with 242 kinases highlighted the strong selectivityof EMD 1214063 and EMD 1204831 for c-Met. Such a highgrade of selectivity is comparable with that of 2 other type Iinhibitors, INCB28060 and JNJ-38877605, which are cur-rently under evaluation in phase I clinical trials (25).From a therapeutic point of view, it remains controversial

as to whether single- or multiple-targeted inhibitors aremost advantageous. Clinical evidence suggests that multi-ple-targeted inhibitors may be more often associated withdose-limiting effects, whereas single-targeted kinase inhi-

bitors can be used at maximal dosing level without causingtoxic effects. Indeed, XL880 and XL184, which target mul-tiple, non-family-related kinases including VEGF receptor,are associated with dose-limiting toxicities that may not beattributed to c-Met inhibition. On the other hand, theachievement of therapeutic effects with EMD 1204831 andEMD 1214063 may require thorough patient screening toidentify a responsive subset, in which the tumor is sensitiveto selective disruption of the c-Met signaling pathway.

Our preclinical data show that EMD 1204831 and EMD1214063 block both HGF-dependent and constitutivephosphorylation of c-Met, thereby interfering with survival,anchorage-independent growth, and HGF-induced migra-tion of susceptible tumor cells. Such observations are inagreement with reports on the effects of JNJ-38877605 andINCB28060, which also belong to the category of type I c-Met inhibitors (25).

From a pharmacokinetic point of view, EMD 1214063andEMD1204831have different properties.Our data showthat the effect of EMD 1214063 on c-Met is sustained,whereas that of EMD 1204831 is less prolonged. In vivopharmacokinetic/pharmacodynamics studies evaluatedsuch differences in depth. We showed that a single dose ofEMD 1214063, due to its high volume of distribution,resulted in higher drug concentrations in the tumor thanin the plasma. Notably, a single dose of EMD1214063 at 10or 30 mg/kg resulted in a complete and long-lasting inhi-bition of c-Met phosphorylation in the tumors.

In contrast, a single dose of EMD 1204831 at 30 mg/kgresulted in equal drug concentrations in the tumor andplasma and in complete but transient inhibition of c-Metphosphorylation in the tumors. The reduced duration ofEMD 1204831 activity on the target may have the disad-vantage of requiring more frequent dosing to achieve con-tinuous target inhibition, but may offer the advantage of agreater flexibility in the administration schedule in combi-nationwithother drugs. In light of their different properties,EMD 1214063 may be the best candidate to achieve highlevels of continuous target inhibition and provide a ther-apeutic advantage not only over EMD 1204831, but alsoover other c-Met inhibitors such as JNJ-38877605 (26, 27)and INCB28060 (16).

Our immunohistochemical studies indicated that treat-ment with EMD 1214063 and EMD 1204831 results in asubstantial reduction in the levels of phospho-histone H3,in the suppression of cyclinD1 expression, and in increasedlevels of p27. Taken together, these findings indicate thattreatment with EMD 1214063 or EMD 1204831 is effectivein inducing cell-cycle arrest and inhibiting tumor cellproliferation.

In vivo, both inhibitors displayed similar antitumor activ-ity, when the dose and schedule were adjusted according totheir respective pharmacokinetic properties (i.e., daily orevery other day for EMD 1214063 and twice daily for EMD1204831). Both compounds induced a marked tumorgrowth delay and, eventually, regression ofHGF-dependentand HGF-independent c-Met–amplified tumors graftedonto nude mice. Our data are in agreement with previous






reports on the activity of JNJ-38877605 (26) andINCB28060 (16), but differ from those obtained withMetMab, a single-armed monoclonal antibody against c-Met, which was only active in HGF-dependent tumor mod-els (14, 28). Evaluation of different schedules of adminis-tration revealed that continuous target inhibition is neces-sary to achieve maximal antitumor activity. EMD 1214063and EMD 1204831 were well tolerated at doses as high asdaily or twice-daily 200 mg/kg, as indicated by the lack ofsubstantial weight loss in treated mice. Antitumor activitywas observed at several dose levels for EMD 1214063 andEMD 1204831, at doses as low as 6 mg/kg and 10 mg/kg,respectively, indicating a large therapeutic index for bothc-Met inhibitors.

In conclusion, our preclinical data show that EMD1214063 and EMD 1204831 are selective and potent invitro and in vivo inhibitors of the c-Met kinase, therebysupporting their clinical development as a potentially effec-tive oral treatment for human cancers.

Disclosure of Potential Conflicts of InterestF. Bladt is employed (other than primary affiliation; e.g., consulting) as a

Director, Biomarker Discovery by Merck KGaA. C. Fittschen has ownershipinterest (including patents) in EMD 1214063 and EMD 1204831. D. Dorschhas ownership interest (including patents) in WO2009/006959. U. Pehl isemployed (other than primary affiliation; e.g., consulting) as a Laboratoryhead by Small Molecules Platform. O. Schadt has ownership interest(including patents) in WO2009006959. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: F. Bladt, M. Friese-Hamim, U. Pehl, F. Stieber,A. BlaukatDevelopment of methodology: F. Bladt, B. Faden, M. Friese-Hamim,C. Wilm, C. Fittschen, U. Pehl, F. StieberAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): F. Bladt, M. Friese-Hamim, C. Knuehl, C. Wilm,C. Fittschen, D. Dorsch, F. Jaehrling, U. Pehl, O. SchadtAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): F. Bladt, B. Faden, C. Knuehl, C. Wilm,F. Jaehrling, U. Pehl, O. SchadtWriting, review, and/or revision of the manuscript: F. Bladt, B. Faden,C. Wilm, C. Fittschen, U. Graedler, M. Meyring, D. Dorsch, F. Jaehrling,F. Stieber, O. Schadt, A. BlaukatAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): F. BladtStudy supervision: F. Bladt, C. Wilm, C. Fittschen, A. Blaukat

AcknowledgmentsThe authors thank M. Eggermont, PhD, TRM Oncology (The Hague,

Netherlands) for editorial assistance and medical writing.

Grant SupportThis work was supported by funding from EMD Serono, Merck Serono

Research and Development, and Merck KGaA.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 17, 2012; revised March 13, 2013; accepted March 18,2013; published OnlineFirst April 3, 2013.

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2013;19:2941-2951. Published OnlineFirst April 3, 2013.Clin Cancer Res Friedhelm Bladt, Bettina Faden, Manja Friese-Hamim, et al. and Highly Selective c-Met InhibitorsEMD 1214063 and EMD 1204831 Constitute a New Class of Potent

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EMD 1214063 and EMD 1204831 Constitute a New …...Cancer Therapy: Preclinical EMD 1214063 and EMD 1204831 Constitute a New Class of Potent and Highly Selective c-Met Inhibitors Friedhelm