CEP-7055: A Novel, Orally Active Pan Inhibitor of Vascular … · CEP-7055, was prepared to increase aqueous solubility and to facil-itate p.o. delivery. CEP-7055 demonstrates a broad

[CANCER RESEARCH 63, 5978–5991, September 15, 2003]

CEP-7055: A Novel, Orally Active Pan Inhibitor of Vascular Endothelial GrowthFactor Receptor Tyrosine Kinases with Potent Antiangiogenic Activity andAntitumor Efficacy in Preclinical Models1

Bruce Ruggeri,2 Jasbir Singh, Diane Gingrich, Thelma Angeles, Mark Albom, Hong Chang, Candy Robinson,Kathryn Hunter, Pawel Dobrzanski, Susan Jones-Bolin, Lisa Aimone, Andres Klein-Szanto, Jean-Marc Herbert,Francoise Bono, Paul Schaeffer, Pierre Casellas, Bernard Bourie, Roberto Pili, John Isaacs, Mark Ator,Robert Hudkins, Jeffry Vaught, John Mallamo, and Craig DionneDepartments of Chemistry [J. S., D. G., R. H., J. M.], Biochemistry [T. A., M. Al., M. At.], Pharmacology [L. A.] and Oncology [B. R., H. C., C. R., K. H., P. D., S. J. B., C. D.],Discovery Research [J. V.], Cephalon, Inc., West Chester, Pennsylvania 19380; Johns Hopkins University Oncology Center [R. P., J. I.], Baltimore, Maryland 21231; Division ofMedical Oncology, Fox Chase Cancer Center [A. K-S.], Philadelphia Pennsylvania 19111; Department of Immunology-Oncology [P. C., B. B.], Sanofi-Synthelabo, Montpellier,Cedex 04 France; and Department of Cardiovascular-Thrombosis Research [J-M. H., F. B., P. S.], Sanofi-Synthelabo, 31036 Toulouse, France

ABSTRACT

Inhibition of the vascular endothelial growth factor VEGF-VEGF re-ceptor (VEGF-R) kinase axes in the tumor angiogenic cascade is a prom-ising therapeutic strategy in oncology. CEP-7055 is the fully syntheticorally active N,N-dimethyl glycine ester of CEP-5214, a C3-(isopropylme-thoxy) fused pyrrolocarbazole with potent pan-VEGF-R kinase inhibitoryactivity. CEP-5214 demonstrates IC50 values of 18 nM, 12 nM, and 17 nM

against human VEGF-R2/KDR kinase, VEGF-R1/FLT-1 kinase, andVEGF-R3/FLT-4 kinase, respectively, in biochemical kinase assays. CEP-5214 inhibited VEGF-stimulated VEGF-R2/KDR autophosphorylation inhuman umbilical vein endothelial cells (HUVECs) with an IC 50 of �10 nM

and demonstrated an equivalent inhibition of murine FLK-1 autophos-phorylation in transformed SVR endothelial cells. Evaluation of the an-tiangiogenic activity of CEP-5214 revealed a dose-related inhibition ofmicrovessel growth ex vivo in rat aortic ring explant cultures and in vitroon HUVEC capillary-tube formation on Matrigel at low nanomolar con-centrations. The antiangiogenic activity of CEP-5214 in these bioassayswas observed in the absence of apparent cytotoxicity. Single-dose p.o. ors.c. administration of CEP-7055 or CEP-5214 to CD-1 mice at 23.8 mg/kg/dose b.i.d. resulted in a reversible inhibition of VEGF-R2/FLK-1 phos-phorylation in murine lung tissues. Administration p.o. of CEP-7055 at2.57 to 23.8 mg/kg/dose b.i.d. resulted in dose-related reductions in neo-vascularization in vivo in porcine aortic endothelial cell (PAEC)-VEGF/basic fibroblast growth factor-Matrigel implants in nude mice (maximum,82% inhibition), significant reductions in granuloma formation (30%)and granuloma vascularity (42%) in a murine chronic inflammation-induced angiogenesis model, and significant and sustained (6 h) inhibitionof VEGF-induced plasma extravasation in rats, with an ED50 of 20 mg/kg/dose. Chronic p.o. administration of CEP-7055 at doses of 11.9 to 23.8mg/kg/dose b.i.d. resulted in significant inhibition (50–90% maximuminhibition relative to controls) in the growth of a variety of establishedmurine and human s.c. tumor xenografts in nude mice, including A375melanomas, U251MG and U87MG glioblastomas, CALU-6 lung carci-noma, ASPC-1 pancreatic carcinoma, HT-29 and HCT-116 colon carci-nomas, MCF-7 breast carcinomas, and SVR angiosarcomas. Significantantitumor efficacy was observed similarly against orthotopically im-planted LNCaP human prostate carcinomas in male nude mice andorthotopically implanted renal carcinoma (RENCA) tumors in BALB/cmice, in terms of a significant reduction in the metastatic score and theextent of pulmonary metastases. These antitumor responses were associ-ated with marked increases in tumor apoptosis, and significant reductionsin intratumoral microvessel density (CD34 and Factor VIII staining) of

22–38% relative to controls depending on the specific tumor xenograft.The antitumor efficacy of chronic CEP-7055 administration was inde-pendent of initial tumor volume (in the ASPC-1 pancreatic carcinomamodel) and reversible on withdrawal of treatment. Chronic p.o. adminis-tration of CEP-7055 in preclinical efficacy studies for periods of up to 65days was well tolerated with no apparent toxicity or significant morbidity.Orally administered CEP-7055 has entered Phase I clinical trials in cancerpatients.

INTRODUCTION

Angiogenesis, the development of new blood vessels from theendothelium of a preexisting vasculature, is a critical process requiredby most solid tumors to support their localized growth and metastaticdissemination within the host (1–4). The autocrine, paracrine, andamphicrine interactions of the vascular endothelium with its surround-ing stromal components, as well as with the proangiogenic and an-giostatic cytokines and growth factors orchestrating physiologicalangiogenesis, are tightly regulated both spatially and temporally. Incontrast, the pathological angiogenesis necessary for active tumorgrowth is sustained and persistent, with the initial acquisition of theangiogenic phenotype being a common mechanism for the develop-ment of a variety of solid and hematopoietic tumor types (1–4).

Among the known angiogenic growth factors and cytokines impli-cated in the modulation of normal and pathological angiogenesis,the VEGF3 family (VEGF-A, VEGF-B, VEGF-C, VEGF-D) andtheir corresponding receptor tyrosine kinases [VEGF-R1 (FLT-1),VEGF-R2 (FLK-1, KDR), and VEGF-R3 (FLT-4)] play a paramountand indispensable role in regulating the multiple facets of the angio-genic and lymphangiogenic processes [VEGF-R3 (FLT-4)], as well asthe induction of vascular permeability and inflammation (1, 2, 5–7). Incontrast to pleiotrophic angiogenic factors and cytokines, the VEGFfamily has a relatively narrow target cell specificity and exert theirmitogenic, chemotactic, and thrombogenic effects, primarily on en-dothelial cells implicated in hemangiogenesis (VEGF-A, VEGF-B)and lymphangiogenesis (VEGF-C and VEGF-D; 2, 5–7). The endo-theliotropic activities of the VEGF family are mediated through thereceptor tyrosine kinases: VEGF-R1/FLT-1, VEGF-R2/KDR, andVEGF-R3/FLT-4, the expression of which is up-regulated on vascularendothelial cells during embryonic and tumor angiogenesis (2–5). Thereceptor VEGF-R2/KDR is the principal one through which VEGFsexerts their mitogenic, chemotactic, and vascular permeabilizing ef-Received 2/14/03; revised 6/3/03; accepted 6/30/03.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 The pharmacological development of CEP-7055 is being pursued in the context of apartnership agreement between Cephalon, Inc. (West Chester, PA) and Sanofi-Synthelabo(Gentilly, France).

2 To whom requests for reprints should be addressed, at Division of Oncology,Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380. Phone: (610) 738-6637; Fax: (610) 344-0065; E-mail: [email protected].

3 The abbreviations used are: VEGF, vascular endothelial growth factor; VEGF-R,VEGF receptor; FGF, fibroblast growth factor; FGF-R, FGF receptor; bFGF, basic FGF;HUVEC, human umbilical vein endothelial cell; PDGF-R, platelet-derived growth factorreceptor; PLC-�, phospholipase C-�; GST, glutathione S-transferase; RENCA, (murine)renal cell carcinoma (model); IMD, intratumoral microvessel density; MED, minimumeffective dose; TCA, trichloroacetic acid; PAEC, porcine aortic endothelial cell; IL,interleukin.

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fects on the host vasculature (7–10). Increased expression of VEGFsby tumor cells and VEGF-R2/KDR and VEGF-R1/FLT-1 by thetumor-associated vasculature are a hallmark of a variety of human androdent tumors in vivo and correlated with tumor growth rate, mi-crovessel density/proliferation, tumor metastatic potential, and poorerpatient prognosis in a variety of malignancies (1–6).

Evidence exists for the importance of VEGF-R1 and its ligands,VEGF and placental growth factor (PIGF), in modulating KDR/VEGF-R2 activity (9–12), mediating chemotactic activity in mono-cytes and macrophages (13, 14), regulating extracellular matrix pro-teolytic activity (15) and the release of tissue factor and nitric oxide inendothelial cells and trophoblasts, respectively (12, 13), and in me-diating placental growth factor-induced recruitment and mobilizationof bone-marrow derived endothelial and hematopoietic stem cells innormal and pathological (tumor-associated) angiogenesis (16, 17).Similarly, a growing body of evidence implicates VEGF-R3/FLT-4and its ligands, VEGF-C and VEGF-D, in the induction of tumorlymphangiogenesis and lymphatic metastases in multiple solid tumortypes (18–22). These collective findings and the critical and nonre-dundant role of VEGF in tumor-associated angiogenesis (23, 24)support the importance of the selective abrogation VEGF-mediatedsignaling events through all three VEGF-R subtypes as an optimaltherapeutic strategy in the management and treatment of a variety ofsolid and hematopoietic tumors.

Antiangiogenesis therapies directed against the VEGF-VEGF-Rkinase axes through a variety of approaches have been a promisingand well-validated therapeutic approach under active evaluation fortheir safety and efficacy in multiple clinical trials (25–27). The pre-clinical biochemical, pharmacological and in vivo efficacy profile ofantibody-, soluble receptor-, and ribozyme-based antiangiogenic ther-apies currently under clinical evaluation have been described exten-sively (see 25–33). In addition, several orally active small moleculeinhibitors of specific VEGF-R kinases (see Ref. 34), including PTK-787 (vatalanib; Refs. 35–38), PKC-412 (midostaurin; Ref. 39), ZD6474 (40, 41), SU-11248 (42, 43), and CP-547,632 (44) are currentlyunder clinical evaluation.

In this report, we describe the biochemical and pharmacologicalprofile of CEP-7055, the N,N-dimethyl glycine ester prodrug of CEP-5214, a novel, orally active and fully synthetic low nanomolar inhib-itor of all three VEGF-R kinases (Table 1). Its ester derivative,CEP-7055, was prepared to increase aqueous solubility and to facil-itate p.o. delivery. CEP-7055 demonstrates a broad acting preclinicalantitumor and antiangiogenic efficacy and a tolerability profile ame-nable for chronic p.o. administration. CEP-7055 is currently in PhaseI trials in patients with solid tumors.

MATERIALS AND METHODS

Compound Synthesis

CEP7055, N,N-dimethylglycine 3-{5,6,7,13-tetrahydro-9-[(1-methyle-thoxy)methyl]-5-oxo-12H-indeno(2,1-a)pyrrolo(3,4-c)carbazol-12-yl}propylester, the ester prodrug of CEP-5214, was synthesized in the Department ofChemistry at Cephalon, Inc. (West Chester, PA). The synthetic routes forCEP-7055 and CEP-5214 have been described elsewhere (45, 46). For all invivo experiments described with CEP-7055, the HCl salt was used at �97%purity. A dose of 1.19 mg/kg CEP-7055 prodrug is equivalent to 1.0 mg/kgdose of CEP-5214.

Cell Lines

The majority of rodent and human tumor cell lines used in these studieswere obtained from the American Type Culture Collection (Manassas, VA).The Dunning G/VEGF165 rat prostate carcinoma and LNCaP human prostatecarcinoma cell lines were obtained from Dr. Roberto Pili (Johns Hopkins

University, Baltimore, MD), and cultures of human U251MG and SF 767glioblastoma cells were obtained from the Brain Tumor Research CenterTissue Bank (University of California, San Francisco, CA). All of the cell lineswere MAP-16- and Mycoplasma-tested by a commercial laboratory (BioReliance Corp., Rockville, MD) and were deemed suitable for in vivo studies.HUVECs were obtained from Clonetics (San Diego, CA) and cultured inendothelial cell basal medium (EBM-2; Clonetics) with 2% heat-inactivatedfetal bovine serum (Life Technologies, Inc., Grand Island, NY), 50 �g/mlendothelial cell growth supplement, 50 �g/ml heparin, 10 mM HEPES, and 2 mM

L-glutamine. Cells between passages 3 and 8 were used as described below(“Human and Murine Endothelial Cell-based Receptor Phosphorylation Assays”and “In Vitro Capillary Tube Formation Assay with HUVECs on Matrigel”).

Animals

Female and athymic nu/nu mice (6–8 weeks old; Charles River, Wilming-ton, MA) were maintained five/cage in microisolator units on a standardsterilizable laboratory diet (Teklad Labchow; Harlan Teklad, Madison, WI).Animals were housed under humidity- and temperature-controlled conditions,and the light/dark cycle was set at 12-h intervals. Out-bred Balb/C mice of 6–8weeks of age were obtained from Charles River (Wilmington, MA) and femaleout-bred Tuck Original mice were obtained from Harlan (Oxon, United King-dom). The animals were housed in groups of 12 mice per cage in a room witha 12-h light/dark cycle. The mice were fed ad libitum normal mouse chowpellets. Male Sprague Dawley rats (250–300 g) were obtained from CharlesRiver and housed five/cage in a conventional vivarium facility. All mice andrats were quarantined 1 week before experimental manipulation. All of the animalstudies were conducted under protocols approved by the Institutional Animal Careand Use Committees (IACUC) of Cephalon and Sanofi-Synthelabo.

Recombinant Proteins and Biochemical Kinase Assays

Enzymes and Substrates. The cytoplasmic domains of recombinant hu-man tyrosine kinases (epidermal growth factor receptor, FGF-R1, FLT-3(ITD1), PDGF-R�, TIE-2, TRKA, VEGF-R1, VEGF-R2, VEGF-R3) andserine/threonine kinases (CHK1, CDK1/cyclinB, CDS1, DLK, JNK1�1,MLK1, MLK2, MLK3) were expressed in a baculovirus insect cell system.Recombinant �-insulin receptor kinase was purchased from Stratagene (LaJolla, CA). Purified rat brain protein kinase C (mixture of Ca�2-dependentisozymes �, �, and �) and activated recombinant human GST-p38� wereobtained from Upstate Biotechnology, Inc. (Lake Placid, NY). PLC-� wasgenerated as a fusion protein with GST following the procedure of Rotin et al.(47). Histone H-1 and MBP were purchased from Fluka Chemical Corp.(Milwaukee, WI) and Upstate Biotechnology, Inc. (Lake Placid, NY), respec-tively. Time-resolved fluorescence experiments were performed using Eu-N1antiphosphotyrosine antibody, Eu-N1 antiphosphothreonine antibody, Eu-N1antirabbit IgG, and DELFIA enhancement solution purchased from Perkin-Elmer Life Sciences (Gaithersburg, MD). Phospho-specific antibodies toATF-2 (Thr71), Cdc25c (Ser216), and Rb (Ser795) were obtained from NewEngland Biolabs, Inc. (Beverly, MA).

Receptor-linked Tyrosine Kinase Assays. Enzyme-inhibition studieswere performed using a modification of the ELISA described for TRKA kinase(48). Briefly, the 96-well microtiter plate (FluoroNUNC or Costar HighBinding) was coated with 10 �g/ml recombinant human PLC-�/GST. Kinaseassays were performed in 100 �l reaction mixtures containing 50 mM HEPES(pH 7.4), Km level of ATP, 10 mM MnCl2, 0.1% BSA, 2% DMSO, and variousconcentrations of CEP-5214 or CEP-7055. The reaction was initiated byadding baculoviral recombinant human enzyme (epidermal growth factorreceptor, FGF-R1, �I-RK, PDGF-R�, FLT-3, TIE-2, TRKA, VEGF-R1,VEGF-R2, or VEGF-R3) and was allowed to proceed for 15 min at 37°C. Thedetection antibody, Eu-N1 antiphosphotyrosine (PT66) antibody was added.After a 1-h incubation at 37°C, 100 �l of enhancement solution was added andthe plate was gently agitated. After 5 min, the fluorescence of the resultingsolution was measured using the Victor2 Multilabel Counter (Model 1420-018). The activities of the serine/threonine kinases listed in Table 1 weremeasured using either a radioactive Multiscreen TCA ”in-plate“ assay (49) oran ELISA similar to that described above for the tyrosine kinases.

Data Analysis. Inhibition curves for compounds were generated by plot-ting percentage control activity versus log10 of the concentration of compound.IC50 values were calculated by nonlinear regression using the sigmoidal

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CEP-7055, THE PRODRUG OF CEP-5214



dose-response (variable slope) equation in GraphPad Prism. IC50 values werereported as the average of at least three separate determinations.

Human and Murine Endothelial Cell-based ReceptorPhosphorylation Assays

Subconfluent HUVECs were serum starved by replacing medium withEBM-2 (endothelial cell basal medium, serum-free; Clonetics) containing0.05% BSA for 1 h at 37°C, during which time, a range of concentrations ofCEP-5214 or DMSO (control) was added to the cells. Human VEGF (Clonet-ics) was then added to HUVECs at a concentration of 10 ng/ml for 5 min. Cellswere lysed in radioimmunoprecipitation assay (RIPA) buffer containing 1 mM

activated sodium vanadate and protease inhibitors (Protease Inhibitor CocktailSet III; Calbiochem, San Diego, CA), sheared with a 27-gauge syringe, andthen centrifuged at 12,000 � g for 15 min. Clarified cell lysates were immu-noprecipitated with anti-VEGF-R2 antibody (CEP-133) for 1 h, followed byincubation with Protein A-Sepharose for another hour at 4°C and analysis bySDS-PAGE as detailed previously (49, 50). Phosphorylated proteins werevisualized using enhanced chemiluminescence (Amersham, Piscataway, NJ).The percentage inhibition was calculated by analyzing scanned autoradio-graphs on a densitometer. Scores were based on decrease in protein banddensity compared with VEGF-stimulated control (no inhibitor) as follows: 0,no decrease; 1, 1–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%. MurineSVR-transformed endothelial cells (51) were treated in the same way asHUVECs with the following modifications: subconfluent cells were serumstarved for 1 h at 37°C with DMEM containing 0.05% BSA; in addition,autophosphorylation of murine FLK-1 was measured after stimulation for 5min with recombinant murine VEGF (R&D Systems, Minneapolis, MN).

In Vitro Capillary Tube Formation Assay with HUVECs on Matrigel

The ability of CEP-5214 to inhibit angiogenesis in vitro was evaluated in acapillary tube formation assay using HUVECs cultured on a synthetic base-ment membrane matrix (52–55). Under these conditions, HUVECs are capableof morphological differentiation into an extensive network of capillary-likestructures composed of highly organized three-dimensional cords (55). Forty-eight-well Nunclon (Fisher Scientific, Newark, DE) plates were coated with a200-�l (2 mg) layer of the synthetic basement membrane substrate Matrigel(Collaborative Research, Bedford, MA) at 10 mg/ml concentration and wereincubated at 37°C for 30 min to promote gelling. HUVECs (Clonetics) werecultured in EMB-2 medium (Clonetics) with 2% fetal bovine serum, and cellsbetween passages 3 and 8 were seeded (at 3 � 104 cells in 200 �l of medium)in each of the Matrigel-coated wells. CEP-5214 was diluted in DMSO to a finalDMSO concentration in the treated and untreated wells of 0.02%. After an18-h incubation at 37°C and 5% CO2 humidified atmosphere, HUVECs wereaspirated from the medium and were fixed and stained using a modifiedWright-Giemsa staining protocol according to the manufacturer’s recommen-dations (Diff-Quik Stain Set; Baxter Healthcare Corp., McGraw Park, IL).Complete capillary tube networks within a designated area of a low magnifi-cation (�10) field were counted under light microscopy, and the data wereexpressed as percentage of complete capillary tube formation relative tountreated HUVEC control cultures incubated under the same conditions (54,55). All of the assays were done in quadruplicate in three independent exper-iments. Statistical analysis of the inhibition of tube formation relative tocontrol cultures was done by the Dunnet’s multiple-comparison test, withP � 0.05 designated as significant. Visual inspection of cultures on a routinebasis was used to assess the potential cytotoxicity of CEP-5214 relative tocontrol cultures.

Ex Vivo Rat Aortic Ring Explant Assay in Collagen Gel Matrices

Rat aortic ring explant cultures were prepared by a modification of protocolspreviously described (54, 56–58). This assay enables a quantitative assessmentof microvessel growth, branching, and remodeling; and vessel regression in aprimary explant culture system in which endothelial cell-adventitial cell inter-actions critical for angiogenesis in vivo can be effectively modeled in a morephysiologically relevant ex vivo setting. CEP-5214 was dissolved in DMSOand mixed with serum-free EBM immediately before the addition or replace-ment of media to collagen-embedded aortic ring explant cultures in quadru-plicate. The final DMSO concentration in treated and control cultures was

0.02%. Cultures were incubated at 35.5°C in a humidified CO2 atmosphere,and the medium was replaced daily over the course of the 8- to 10-day studies.Visual counts of microvessel outgrowths from replicate explant cultures (n � 8)were done under bright-field microscopy following an established protocol (57,58). Experiments were done three times, and microvessel counts in treated andcontrol cultures were analyzed by one-way ANOVA and the Student-Newman-Keuls multiple-comparison test, with P � 0.05 deemed significant.

Pharmacokinetics of p.o. CEP-7055 Administration in Mice

Single-dose p.o. administration of CEP-7055 was performed in femaleCD-1 mice and female athymic nude mice of 6–8 weeks of age. A dose of 1.19mg/kg CEP-7055 is equivalent to 1.0 mg/kg dose of CEP-5214. Trunk bloodfrom mice that were given the HCl salt of CEP-7055 p.o. was collected afterdecapitation using heparin as the anticoagulant. Plasma was prepared forhigh-performance liquid chromatography (HPLC)/mass spectrometric analysisby protein precipitation with two volumes of acetonitrile (200 �l) per 100-�lsample of plasma. Pharmacokinetic parameters were determined by WinNon-Lin software.

Pharmacodynamic Effects of CEP-7055 on VEGF-R2/FLK-1Phosphorylation in Vivo in Mice

Pharmacodynamic effects of CEP-7055, or direct administration of CEP-5214, were evaluated as described by others (43, 44). Briefly, athymic nudemice bearing established VEGF-R2 expressing (52) SVR angiosarcomas(�250 mm3) were administered a single p.o. or s.c. dose of CEP-7055 at a23.8-mg/kg/dose, and tumors were excised 30–45 min later. Tumor lysateswere prepared and the tyrosine autophosphorylation of the VEGF-R2/FLK-1receptor was analyzed by immunoprecipitation with SC-504 and SC-315Ganti-FLK-1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and byWestern blotting with 4G10 antiphosphotyrosine antibody as described above,which confirmed the detection of a Mr 220,000 protein of the expected size.Immunoabsorbtion with increasing concentrations of blocking peptides tothese antisera confirmed the specificity of the immunoprecipitation for murineFLK-1 receptor in SVR cells and tumors. In related studies, athymic micereceived a single p.o. dose of CEP-7055 at 59 mg/kg/dose or CEP-5214directly at a 50-mg/kg/dose. At 20, 40, and 60 min after dosing, animals weregiven murine VEGF at 3 ng/mouse i.v. and were sacrificed 5 min later; proteinextracts were then prepared from lungs. Tyrosine phosphorylation of FLK-1was evaluated by Western blotting as above, using PC460, a phospho FLK-1antibody. The abundance of VEGF-R2/FLK-1 in each sample lysate was assessedby Western blotting, and the samples were evaluated densitometrically.

PAEC-VEGF/bFGF-induced Matrigel Implant in Vivo AngiogenesisModel in Nude Mice

The Matrigel plug implantation assay used in these studies was a modifi-cation of that described previously (59, 60). Briefly, PAECs were grown toconfluency in Ham’s F-12 medium supplemented with 10% fetal bovineserum. Cells were used between passages 5 and 10. Nude mice were givenbilateral s.c. injections of 0.5 ml Matrigel synthetic basement membrane(Collaborative Research) containing 1 � 106 PAECs/plug and recombinantmurine VEGF and bFGF (R&D Systems) at 20 ng/ml and 250 ng/ml, respec-tively, final concentrations per plug. Mice bearing PAEC-VEGF/bFGF-Matrigel implants were randomized into groups (10/group) and were givendoses of CEP-7055 or vehicle (1% aqueous acetic acid) p.o. b.i.d. for 8 days.The hemoglobin content of the PAEC-VEGF/bFGF-Matrigel plugs has beenreported to be directly proportional to the degree of neovascularization in eachplug (59) and was determined as described previously (60). Contralateral plugswere evaluated histologically for vessel morphology. Results from duplicate invivo experiments are expressed as mean g/dl of hemoglobin � SE. Statisticalanalyses of the data were done using the paired Student’s t test, with P � 0.05deemed significant.

Chronic Inflammation-induced in Vivo Angiogenesis Model in Mice

Angiogenesis was induced in female out-bred Tuck Original mice (25 ginitial body weight) with a chronic granulomatous reaction to Freund’s com-plete adjuvant in croton oil as described previously (61). Briefly, granuloma-

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tous air pouches were induced by the s.c. injection of 3 ml of air into eachanesthetized mouse and by the injection of 0.5 ml of Freund’s completeadjuvant with 0.1% croton oil 4–5 h later. Doses of CEP-7055 were admin-istered p.o. b.i.d. in 1% aqueous acetic acid over a 6-day period, and the degreeof inflammation and vascular density were evaluated on day 7. The vascularcontent was assessed by the formation of vascular casts incorporating carmine.Mice were anesthetized with pentobarbital (60 mg/kg/dose, i.p.) and peripheralvasodilatation induced by placing mice on a heated pad at 40°C for 10 min.The cast was formed by the i.v. injection of one ml of 5% carmine red in 10%gelatin into the warmed mice. The carcasses were chilled, the granulomatousair pouch linings were dissected, and the tissues were dried, enzymaticallydigested, and analyzed spectrophotometrically as described previously (61).

VEGF-induced Plasma Extravasation Model in Rat Skin

Male Sprague Dawley rats (270 to 320 g) were anesthetized with sodiumpentobarbital at 60 mg/kg/dose i.p. Animals were given i.v. injections of Evansblue dye (15 mg/kg/dose), and, 5 min later, saline solution (NaCl 0.9%) orVEGF (3 ng) were injected intradermally (0.1 ml/site). Four paired injectionsof saline solution and VEGF were performed on the back of each rat. Meas-urements of the vertical and the horizontal diameter of the blue area wereperformed for each injection point, and the area of plasma extravasation wascalculated (radius vertical � radius horizontal � �). Basal plasma extravasa-tion (0.9% saline solution) was not modified by the treatment with CEP-7055at any time or at any dose tested. Consequently, for each VEGF/saline solutionpair, the basal extravasation area corresponding to the injection of salinesolution was subtracted from the extravasation area that resulted from theVEGF injection. Administration p.o. of CEP-7055 or vehicle (1% aqueousacetic acid) was done over a 6-h time course. For dose-response studies,CEP-7055 was administered 60 min before the measurement of local plasmaextravasation at the indicated doses (see Fig. 7). The corrected areas of theCEP-7055 treated group were compared with the vehicle group with Student’sunpaired t test, with P � 0.05 deemed significant.

Human and Murine s.c. Tumor Xenograft Models in Nude Mice

The p.o. antitumor efficacy of CEP-7055 was evaluated in multiple murineand human tumor xenograft models on therapeutic dosing regimens, i.e.,administration of CEP-7055 to athymic nude mice bearing established palpables.c. tumor xenografts. Female athymic nu/nu mice (6 to 8 weeks old) weremaintained five/cage in microisolator units on a standard sterilizable laboratorydiet (Teklad Labchow). Mice were quarantined 1 week before experimentalmanipulation. Tumor growth was initiated after the s.c. implantation of sub-confluent cultures into the right flank of female athymic nude mice in theirrespective serum-free media along with Matrigel (Collaborative Research)synthetic basement membrane (1:1 v/v). The following cell lines at the indi-cated cell densities were implanted in vivo: A375 human melanoma (5 � 106);U251MG and SF767 human glioblastomas (3 � 106); U87MG human glio-blastoma (5 � 106); ASPC-1 human pancreatic ductal carcinoma (5 � 106);CALU-6 human non-small cell lung adenocarcinoma (5 � 106); HT-29,HCT-116, and COLO 205 human colon carcinomas (2 � 106); MCF-7 breastcarcinoma (1 � 106); Dunning G/VEGF165 rat androgen-insensitive prostatecarcinoma (4 � 106); and SVR murine angiosarcoma (1 � 106). Uponachieving volumes of 80–180 mm3, tumor-bearing nude mice for each xe-nograft were randomized into treatment groups (usually 10 mice/group) basedon a power analysis of group sizes required for obtaining statistically mean-ingful data sets. Administration p.o. of CEP-7055 b.i.d. was initiated at dosesranging from 0.35 to 23.8 mg/kg/dose in a vehicle of 1% aqueous acetic acidat 100 �l/dose. Tumor volumes were determined with vernier calipers every3–4 days by measuring the largest perpendicular diameters using the formula:V(mm3) � 0.5236 � length(mm) � width(mm) [(length(mm) � width(mm)]/2, as described previously (62). Tumor measurements were expressed asabsolute volumes, as well as normalized to individual tumor volumes at day 1,the initiation of dosing (relative tumor volumes) to assess changes in the rateof tumor growth relative to treatment. Statistical analyses of tumor data weredone using the Mann-Whitney rank-sum test, or when appropriate for the dataset, by one-way ANOVA and the Dunnet’s multiple-comparison test, withP � 0.05 deemed significant. Animal body weights were determined andanalyzed over a similar time course.

Orthotopic Prostate and Renal Carcinoma Tumor Models in Mice

The antitumor efficacy of CEP-7055 was further evaluated in orthotopicmodels of hormone-dependent human prostate carcinoma in male nude mice(60) and in the RENCA in BALB/c mice (35, 36, 63) to assess its efficacy onprimary, local invasive, and metastatic tumor growth in both immunocompro-mised and immunocompetent mice, respectively. Briefly, exponentially grow-ing human androgen-sensitive LNCaP prostate carcinoma cells were harvestedfrom tissue culture plates by trituration. LNCaP cells were injected orthotopi-cally (2 � 106 cells/20 �l per mouse) into the prostate of athymic male nudemice (6–8 weeks old). Treatment with CEP-7055 was initiated 1 day afterorthotopic implantation of LNCaP cells and continued for 21 days, at whichtime animals were euthanized by CO2 asphyxiation and tumors were analyzed(mean wet tumor weight in mg � SE).

Administration p.o. of CEP-7055 was evaluated in an orthotopic murinerenal carcinoma (RENCA) metastasis model in syngeneic BALB/c mice,essentially as described for the evaluation of other antiangiogenic agents (35,36, 63). For antitumor efficacy studies, RENCA cells (1 � 105 in 50 �l ofsterile 1� PBS) were injected into the renal subcapsular space of the leftkidney of female BALB/c mice. Two days after recovery from surgicalimplantation, groups of mice were randomized into treatment groups and weregiven CEP-7055 over a 21- to 26-day period. Human recombinant IL-2(Proleukin; Chiron Corp, Emeryville, CA) was used as a reference standard inthe RENCA model using previously established dosing protocols of 30,000units/dose i.p. for 5 days/week (64). Mice were necropsied for gross assess-ment of local and disseminated tumor burden (lungs, regional lymph nodes)and histopathological analysis of selected tissues. A metastatic scoring systemwas used to evaluate the local and metastatic tumor burden in these studies: I,primary mass with 0–10 nodules in the lung; II, primary mass; 10–100 nodulesin the lung; III, primary mass and too numerous to count (TNTC) nodules inthe lung but still normal areas of lung visible; IV, primary mass with TNTCnodules in the lung with no normal lung visible. Statistical analyses of tumorand lung weights were done by the Mann-Whitney rank-sum test, withP � 0.05 deemed significant.

Tumor Histological and Immunohistochemical Analyses ofMicrovessel Density

In situ evaluation of IMD in tumor xenografts was determined immunohis-tochemically by evaluating expression of the endothelium-associated antigen,von Willebrand factor (Factor VIII) and CD34 immunostaining for confirma-tion) by modification of methods described previously (24, 65). Tissue sectionswere processed, deparaffinized, rehydrated, and quenched for endogenousperoxidase activity as described previously (65). After a 10-min permeabili-zation step with proteinase K, murine vessels in tumor xenografts wereimmunostained with rabbit anti-von Willebrand factor (DAKO Corp., Carpen-teria, CA) at 20 �g/ml or anti-CD34 (DAKO Corp.) at 1:2000 for 18 h at 4°C.Sections were incubated 30 min at room temperature with biotin-labeled goatantirabbit IgG after incubation with streptavidin-horseradish peroxidase (Vec-taStain Elite; Vector Laboratories, Burlingame, CA). Sections were developedafter washing with diaminobenzidine (DAB) as a chromogen and counter-stained with 1% methyl green. For quantitation of IMD, 10 fields (97,500�m2/field) of five tumors were evaluated in a blinded fashion at �100magnification, and the percentage inhibition of IMD relative to vehicle-treatedcontrol tumors was determined. Statistical analyses of IMD in tumor xenograftsfrom CEP-7055-treated mice compared with vehicle-treated mice were done usinga two-tailed Student’s t test, with P � 0.05 deemed significant.

Calcein-AM Cell Proliferation Assays

Calcein-AM cell proliferation assays were conducted as described previ-ously (62) in a 96-well format to assess direct effects of CEP-5214 at 24- and48-h incubation on the proliferative fraction of select tumor cell lines. Plateswere read on a Cytofluor 2300 fluorescence plate reader at various sensitivitieswith an excitation wavelength of 485 nm and emission wavelength of 530 nm.Negative control wells contained medium, but no cells, and were assayed withcalcein, as described above. All of the studies were conducted twice and intriplicate for each sample concentration.

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RESULTS

Kinase Inhibition Profile of CEP-7055 and CEP-5214

The effects of CEP-5214 and its pro-drug CEP-7055 (Fig. 1) wereevaluated in enzyme- based assays for the inhibition of VEGF-R2kinase activity. The primary assay for identifying inhibitors of theVEGF-R2 kinase activity uses an ELISA-based assay with time-resolved fluorescence readout and uses recombinant human PLC-�1/GST fusion protein as a substrate. To interpret the inhibition data forCEP-7055, it was first necessary to evaluate the rate at which theprodrug converts to CEP-5214 in aqueous solution under conditionsrepresentative of the kinases assays. CEP-7055 was incubated in 50mM HEPES buffer at pH 7.2 for 15 min, and samples were analyzedby high-performance liquid chromatography for the presence of CEP-5214. No detectable hydrolysis of the ester was observed (data notshown). These results demonstrate that CEP-5214 is produced fromCEP-7055 at an insignificant rate during the course of the in vitroassays and indicate that the reported IC50 values reflect the inhibitoryactivity of CEP-7055.

As described in Table 1, CEP-5214 is a potent, low-nanomolar paninhibitor of the human VEGF-R tyrosine kinase family (VEGF-R1/FLT-1 IC50, 16 nM; VEGF-R2/KDR IC50, 8 nM; VEGF-R3/FLT-4IC50, 4 nM). The same rank order of inhibition was observed forCEP-7055, although the ester is slightly less potent than the parentcompound (VEGF-R1/FLT-1 IC50, 74 nM; VEGF-R2/KDR IC50, 18nM; VEGF-R3/FLT-4 IC50, 8 nM). The Hill slopes for CEP-5214 andCEP-7055 inhibition of human VEGF-R2/KDR kinase are shown inFig. 2. CEP-5214 demonstrates a somewhat weaker inhibition of thehuman c-Kit, PDGF-RB, and FGF-R1 kinases, with IC 50 values of 57,128, and 162 nM, respectively, and is a potent inhibitor of MLK 1, 2, 3,and 6, with IC 50 values of 13, 46, 7, and 240 nM, respectively. CEP-5214is largely inactive against the remainder of the tyrosine and serine/threonine kinases evaluated in this enzyme-based assay (Table 1).

Effects of CEP-5214 on VEGF-induced VEGF-RPhosphorylation in Human and Murine Endothelial Cells

The VEGF-R kinase inhibitory activity of CEP-5214 was evaluatedfor its dose-related inhibition of VEGF-R2/KDR autophosphorylationin cell-based assays using HUVECs and murine SVR endothelial cellsand monitoring the inhibition of VEGF-induced stimulation of VEGF-R2/KDR phosphorylation. The estimated IC50 of CEP 5214 in thesecellular assays was �10 nM with both HUVEC and SVR cells andcompared closely with its observed IC50 in enzyme-based assays(4–14 nM; Table 1), indicating that this compound is highly cellpermeable and essentially equivalent in its inhibition of human andmurine VEGF-R2 receptor kinase activity. At concentrations of CEP-5214 of 100 nM or greater, there was complete inhibition of VEGF-stimulated VEGF-R2 phosphorylation within a 15-min exposure ofboth human and murine endothelial cells in vitro. In a wash-out assayconducted to determine the duration of the inhibitory effects of

CEP-5214 on VEGF-stimulated VEGF-R2 phosphorylation, inhibi-tion of VEGF-R2/KDR phosphorylation in vitro was sustained max-imally for 1 h after CEP-5214 exposure, was still apparent 3 h afterexposure, and declined to basal levels by 6 h postexposure; resultswere consistent with competitive and reversible inhibition of VEGF-R2/KDR kinase activity in vitro.

Effects of CEP-5214 on Angiogenesis in in Vitro andex Vivo Bioassays

On the basis of its potent and selective inhibition of the VEGF-Rkinases and pronounced inhibition of VEGF-R2 phosphorylation inhuman and rodent endothelial cells, the activity of CEP-5214 wasevaluated in in vitro and ex vivo bioassays of angiogenesis. The rataortic ring explant model in three-dimensional collagen gel matricesand the HUVEC capillary tube formation assay on a Matrigel syn-thetic basement membrane matrix are two widely used ex vivo and invitro systems to model effectively the distinct temporal and spatialevents underlying angiogenesis in vivo (52–53). Both assays aresensitive to the angiogenic effects of VEGF and to the antiangiogeniceffects of inhibitors of the VEGF-R, VEGF-R2/KDR (35, 57, 57).CEP-5214 displayed statistically significant dose-related inhibition ofcomplete HUVEC capillary tube formation on a Matrigel syntheticbasement membrane matrix in the absence of apparent endothelial cellcytotoxicity based on trypan blue exclusion. VEGF-induced capillary-tube formation was inhibited by 21% (P � 0.01), 75% (P � 0.001),and 89% (P � 0.001) at 40, 100, and 400 nM of CEP-5214, respec-tively (Fig. 3). The antiangiogenic response to CEP-5214 was furtherevaluated ex vivo in rat aortic ring explant cultures over a 19-day timecourse in the absence of exogenous VEGF stimulation. Dose-relatedinhibitory effects on microvessel growth were observed at 4, 20, 40, 100,

Fig. 1. Chemical structure of CEP-7055 and CEP-5214.

Table 1 Kinase-inhibitory activities of CEP-5214 and CEP-7055

The inhibitory activities of CEP-5214 and CEP-7055 were evaluated against theVEGF-R kinase family and a panel of tyrosine kinases and serine-threonine kinases asdetailed in “Materials and Methods.” Assays for the tyrosine kinases were performed ina 96-well plate precoated with substrate (GST-PLC-�). Phosphorylation of the substrateby a specific tyrosine kinase was detected with an antiphosphotyrosine antibody in anELISA-based format with time-resolved fluorescence readout. The activities of the serine-threonine kinases were evaluated using either a radioactive Multiscreen TCA in-plateassay or an ELISA-based assay similar to the tyrosine kinases. Inhibition studies wereperformed by varying the concentration of CEP-5214 at the Km level of ATP for a specifictarget. Inhibition curves were generated by plotting percentage control activity versuslog10 of the concentration of compound. IC50 values were calculated by nonlinearregression using the sigmoidal dose-response (variable-slope) equation in GraphPad Prismand were reported as the average of three or four separate determinations. Values aremean � SD.

Molecular target

IC50 (nM) Mean � SD

CEP-5214 CEP-7055

VEGF-R1 16 � 1 74 � 1VEGF-R2 8 � 2 18 � 1VEGF-R3 4 � 2 8 � 1TIE2 �10,000 �10,000FLT3 1.6 � 0.7 5 � 1FGF-R1 162 � 37 485 � 42PDGF-R� 406 � 76 252 � 10Kit 57 � 5 NDb

TRKA �3,000 �10,000�IRK �10,000 �10,000MLK1 13 � 3 74 � 15MLK2 46 � 2 170 � 38MLK3 7 � 1 20 � 4DLK 119 � 24 1441 � 480JNK1�1 448 � 102 1021 � 125CHK1 �3,000 �3,000CDS1 �3,000 �10,000CDK1/cyclinB �10,000 �10,000Rat brain PKCa (�, �, � isozymes) �10,000 �10,000p38� �10,000 �10,000

a PKC, protein kinase C.b ND, not determined

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and 400 nM of CEP-5214 (Fig. 4). Statistically significant effects wereobserved at 40 nM CEP-5214 at days 7 and 11 with a 65% (P � 0.01) and53% (P � 0.05) inhibition of microvessel growth, respectively, in theabsence of apparent cytotoxicity to endothelial cells, fibroblasts, andpericytes in these primary aortic ring explants. Stimulation of rat aorticexplant cultures with murine VEGF resulted in a more robust angiogenicresponse relative to unstimulated cultures, but the growth kinetics ofaortic ring explants were comparable with that observed in the absence ofexogenous VEGF stimulation. Under these experimental conditions, theadministration of CEP-5214 resulted in significant (P � 0.05) inhibitionof microvessel growth at 40 nM, relative to control cultures at day 7during the peak of microvessel growth.

Pharmacokinetic and Pharmacodynamic Profile of OrallyAdministered CEP-7055 in Mice

Pharmacokinetic studies in rats and nude mice demonstrated that afterp.o. administration of CEP-7055, only CEP-5214 is detected in theplasma compartment; no CEP-7055 is detectable within the shortest timeframe (�5 min postdosing) evaluated (data not shown). Moreover, p.o.administration of CEP-7055 produced similar or better overall systemicexposure to CEP-5214 relative to molar-equivalent dosages of CEP-5214(Fig. 5). Dose-proportional plasma levels of CEP-5214 were observedbetween the 3.57- and 11.9-mg/kg dose of orally administered CEP-7055.The p.o. bioavailability of CEP-7055 in nude mice (measured as CEP-5214) was �15%, a value comparable with that observed in CD-1 miceas well. The protein binding of CEP-5214 to mouse plasma �-1 acidicglycoprotein (AGP) was 39% at 10 �M, as assessed by quinolidine reddisplacement.

Single-dose p.o. administration of CEP-7055 or CEP-5214 directlyto nude mice bearing VEGF-R2 expressing SVR tumor xenograftsresulted in a time-dependent inhibition of VEGF-R2/FLK-1 phospho-rylation in tumor lysates. Inhibition peaked at �30 min postadminis-

tration, was still apparent at 3 h, and returned to baseline levels ofreceptor phosphorylation by 4 h, results consisted with the plasmaprofile of CEP-5214 after p.o. CEP-7055 administration in mice (datanot shown). Similar observations for the inhibition of VEGF-R2/FLK-1 phosphorylation were obtained after p.o. administration ofCEP-7055 or CEP-5214 directly in murine lung tissue lysates fromVEGF-stimulated mice.

Collectively, these biochemical efficacy data examining VEGF-R2/FLK-1 phosphorylation profiles in vivo confirm the molecular target-directed inhibitory activity of orally administered CEP-7055 orCEP-5214.

Effects of CEP-7055 Administration p.o. in in VivoAngiogenesis Models

Efficacy of p.o. CEP-7055 Administration in the PAEC-VEGF/bFGF-Matrigel Implant Model in Nude Mice. To determinewhether p.o. administration of CEP-7055 could inhibit VEGF-medi-

Fig. 2. Inhibition of VEGF-R2/KDR by (A) CEP-5214 and (B) CEP-7055. Kinaseinhibition plots for human VEGF-R2 by CEP-5214 (A) and CEP-7055 (B) were deter-mined in an ELISA-based format in a 96-well FluoroNUNC MaxiSorp plate with atime-resolved fluorescence readout as detailed in “Materials and Methods.” Recombinanthuman baculovirus-expressed VEGF-R2 cytoplasmic domain was prephosphorylated inthe presence of 30 �M ATP and 10 mM MnCl2 for 1 h at 4°C before use and was addedto the assay at a final concentration of 150 ng/ml.

Fig. 3. Effect of CEP-5214 on HUVEC capillary-tube formation on Matrigel in vitro.Dose-related effect of CEP-5214 on VEGF (5 ng/ml)-induced capillary-tube formation ofHUVECs (passage 5) after 18-h incubation on Matrigel basement membrane. The finalDMSO concentration in the compound-treated and -untreated wells was 0.02%. HUVECswere aspirated from media and were fixed and stained using a modified Wright-Giemsastaining protocol as described in “Materials and Methods.” Complete capillary tubenetworks within a designated area of a low-magnification (�10) field were counted underlight microscopy, and the data were expressed as percentage inhibition of completecapillary tube formation relative to untreated HUVEC control cultures incubated under thesame conditions. Photo micrographs (�100) of HUVEC capillary tube formation in vehicle-treated cultures (top panel) and cultures incubated with 100 nM CEP-5214 (bottom panel).

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ated angiogenic responses in vivo independent of its potential antitu-mor activity, we conducted a series of studies in models of normal andpathological angiogenesis similar to those reported previously (60).The effect of p.o. administration of CEP-7055 on neovascularization

was examined in the PAEC-VEGF/bFGF-Matrigel implant model inathymic nude mice. As shown in Fig. 6, p.o. administration of a 0.35-to 23.8-mg/kg/dose of CEP-7055 b.i.d. for 8 days resulted in adose-related inhibition of neovascularization in the absence of appar-ent toxicity or morbidity, with an 82% inhibition (P � 0.01) relativeto vehicle-treated control mice, with a MED for significant antiangio-genic activity of �7 mg/kg/dose of CEP-7055 p.o. b.i.d. These in vivofindings provided additional corroboration of in vitro and ex vivo datademonstrating potent and significant p.o. antiangiogenic activity ofCEP-7055 on the host vasculature.

Efficacy of p.o. CEP-7055 Administration on Inflammation-induced Angiogenesis in Vivo. Given the involvement of VEGF anda number of angiogenic cytokines in inflammatory angiogenesis andleukocyte adhesion to microvessels (66–69), the efficacy of p.o.CEP-7055 administration was evaluated in a murine chronic granu-lomatous tissue model of chronic inflammation (61) in which effectson both granuloma formation and vascularity (based on carmine reddye content) could be assessed. Administration p.o. b.i.d. of a 23.8-mg/kg/dose of CEP-7055 for 6 days resulted in a 30% reduction ingranuloma mass (P � 0.01) relative to vehicle-treated control mice,and a 42% reduction (P � 0.01) in granuloma vascularity. These dataindicate that, in addition to its direct antiangiogenic activities, CEP-7055 demonstrates significant anti-inflammatory activity in a murinemodel of chronic inflammatory angiogenesis, consistent with its in-hibition of VEGF-R-mediated activity in vivo.

Effects of p.o. CEP-7055 Administration on VEGF-inducedPlasma Extravasation in Rats. The vascular permeability activity ofVEGF is one of its characteristic functions that distinguishes it froma number of other angiogenic cytokines (6, 7, 14). To further confirmthat p.o. administration of CEP-7055 directly inhibits VEGF-R2/KDR-dependent events in the vasculature, we assessed its ability toattenuate VEGF-induced vascular permeability in rat skin using amodification of the Miles/Evan’s Blue assay. The administration ofCEP-7055 at the 23.8-mg/kg/dose p.o. resulted in a significant(P � 0.01) and sustained inhibition of VEGF (3 ng)-induced plasmaextravasation (as measured by Evan’s Blue dye release) in the skin ofmale Sprague Dawley rats over a 6-hour period after a single p.o.dose, compared with vehicle (1% aqueous acetic acid) controls,achieving a maximum 66% inhibition at 1 h postdose (Fig. 7). Basalplasma extravasation (0.9% saline solution without VEGF) was noteffected by CEP-7055 administration at any time point evaluated.Similarly, p.o. administration of CEP-7055 resulted in a significant

Fig. 4. Effects of CEP-5214 on microvessel growth in primary rat aortic ring collagengel explant cultures ex vivo. A, dose-related antiangiogenic effects of increasing concen-trations of the pan-VEGF-R kinase inhibitor CEP-5214 in serum-free collagen gel culturesof rat aortic ring explants maintained in serum-free MCDB 131 medium in the absence ofexogenous VEGF. Values are mean � SE of microvessel outgrowths, n � 6 replicatestotal per time point from three experiments. The final DMSO concentration in theCEP-5214-treated and -untreated explant cultures was 0.02%; medium was replenishedevery day. �, P 0.05; ��, P � 0.01; ��, P 0.01, relative to untreated (DMSO in serum-freemedium) controls by Student-Newman-Keuls method. B, photomicrographs (�100) of rataortic ring explants at day 8 during the peak phase of microvessel sprouting. Untreatedcultures (top panel) and cultures treated with 100 nM CEP-5214 (bottom panel).

Fig. 5. Plasma levels of CEP-5214 in nude mice after p.o. administration of CEP-7055.Female nude mice (8 weeks, 20–22 g) were fasted overnight before single-dose p.o.administration of CEP-7055 in 1% aqueous acetic acid (100-�l volume). Six time pointswere collected using four mice per time point, and plasma samples were analyzed byLCMSMS. Data shown is mean � S.E.M. values.

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(P � 0.05 or greater) dose-related inhibition of VEGF (3 ng)-inducedplasma extravasation in rats when administered 1 h before measure-ment of local plasma extravasation, with an ED50 of 20 mg/kg/doseCEP-7055. Basal plasma extravasation (0.9% saline solution without

VEGF) was not effected by CEP-7055 administration at any doseevaluated.

Collectively, these in vivo data in three distinct angiogenesis mod-els demonstrate that p.o. administration of CEP-7055 exhibits potentand significant dose-related inhibition of VEGF-mediated biologicalprocesses involving various components of normal and pathologicalangiogenesis in vivo.

Antitumor Efficacy of Chronic p.o. CEP-7055 Administrationon the Growth of Human and Murine s.c. Xenografts in NudeMice and in the Inhibition of Tumor-associated Angiogenesis.The antitumor efficacy and tolerability of chronic p.o. CEP-7055administration were investigated in a therapeutic context against aseries of established human and rodent tumor xenografts implanteds.c. in athymic nude or syngeneic mice. These data are summarized inTable 2 and illustrated in Figs. 8 and 9. Administration of p.o. b.i.d.CEP-7055 for periods of 10–60 days resulted in dose-related growthinhibition of multiple human and rodent s.c. tumor xenografts rangingfrom 50–90%, relative to vehicle-treated control mice at doses of11.9–23.8 mg/kg/dose in 9 of the 10 s.c. tumor xenograft modelsevaluated, including CALU-6 lung carcinomas; HCT-116, HT-29, and

Fig. 6. Dose-related effect of p.o. administration of CEP-7055 on angiogenesis in thePAEC-VEGF/bFGF-Matrigel implant model in nude mice. Female nude mice (n � 10/group) were implanted s.c. bilaterally with Matrigel pellets (100-�l volume) containingPAECs and VEGF (20 ng/ml) and bFGF (100 ng/ml). CEP-7055 was administered p.o.b.i.d. for 8 days postimplantation. Hemoglobin content of the excised Matrigel plugs wasdetermined colorimetrically (Drabkin method) as detailed in “Materials and Methods.” ��,P 0.01, relative to vehicle-treated control mice. The minimum effective dose for inhibitionof neovascularization in vivo is �7.1 mg/kg/dose p.o. b.i.d. B, the reduction in neovas-cularization (hemoglobin content) in the implants from mice given CEP-7055 wasconfirmed histologically in 0.25% glutarylaldehyde-fixed implants stained with H&E andTrichrome-Masson to evaluate vascular morphology. Histological section of Matrigelimplant from vehicle-treated mouse (top panel) and CEP-7055 (11.9 mg/kg/dose p.o.b.i.d.)-treated mouse (bottom panel).

Fig. 7. Effects of p.o. administration of CEP-7055 on VEGF-induced plasma extrav-asation in rats: kinetics and dose-response. The time- and dose-related effects of p.o.administration of CEP-7055 on VEGF-induced plasma extravasation in Sprague-Dawleyrats (n � 10/group) were evaluated using Evans blue dye (15 mg/kg) injected i.v. Five minafter VEGF injection, saline solution (NaCl, 0.9%) or VEGF (3 ng) were injectedintradermally (0.1 ml/site). Measurement of the vertical and the horizontal diameter of theEvan’s blue dye area in the skin was performed for each injection point and the area ofplasma extravasation was calculated (radius vertical � radius horizontal � �). For eachVEGF/saline solution pair the basal extravasation area corresponding to injection of salinesolution was subtracted from the extravasation area resulting from VEGF injection. A,time course of VEGF-induced plasma extravasation after p.o. administration of 20mg/kg/dose of CEP-7055. B, p.o. CEP-7055 dose-response relationship for inhibition ofVEGF-induced plasma extravasation. Doses of CEP-7055 were administered 60 minbefore the measurement of local plasma extravasation at the indicated doses. �, P � 0.05;��, P � 0.01; �� P � 0.001 by Student’s t test.

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COLO 205 colon carcinomas; U251MG glioblastomas; and MCF-7breast carcinoma xenografts (Table 2). The MED for significantantitumor efficacy was observed at �3.57 mg/kg/dose in several s.c.human tumor models, including the A375 melanoma, U87MG glio-blastoma, and ASPC-1 pancreatic ductal carcinoma, with partial tu-mor regressions observed in the U87MG model at doses of CEP-7055at �11.9 mg/kg/dose b.i.d. (Fig. 8). The plasma concentrations (Cmax)of CEP-5214 associated with antitumor efficacy in these tumor mod-els (and those described below) in normal or immunocompromisedmice were in the range of 50–200 nM.

The effects of initial tumor volume on the responsiveness to p.o.CEP-7055 administration were evaluated in the highly aggressiveASPC-1 pancreatic carcinoma xenograft model. Chronic p.o. b.i.d.administration of CEP-7055 at 11.9 mg/kg/dose to nude mice bearingASPC-1 pancreatic carcinoma xenografts of various initial tumorvolume (�150 mm3, �400 mm3, and �900 mm3) for periods of30–35 days duration resulted in significant inhibition of tumor xeno-graft growth over a comparable time course of treatment, independentof initial tumor volume (Fig. 9). No overt toxicity or significantmorbidity (body weight loss) was observed on chronic p.o. adminis-tration of CEP-7055 in these in vivo studies. Evaluation of gastroin-testinal histopathology in mice receiving p.o. CEP-7055 furtherrevealed no significant findings, although mild focal mucosal inflam-mation with lymphoid Peyer’s patch hyperplasia was associated withp.o. administration of the dosing vehicle in several cases.

Effects of CEP-7055 on IMD in Vivo and Tumor Cell Prolifer-ation in Vitro. IMD was evaluated immunohistochemically as anindirect assessment of effects on tumor angiogenesis in several humantumor xenografts from CEP-7055-treated mice and compared with theIMD from corresponding vehicle-treated mice. The percentage reduc-tion in IMD (as assessed by Factor VIII and CD34 immunostaining)in CEP-7055-treated A375 melanoma and U87MG glioblastoma xe-nografts after 30 days of p.o. administration relative to oral-vehicle-treated control mice was 20–22% (P � 0.05). Chronic CEP-7055administration also resulted in a 36% reduction (P � 0.05) in both themean IMD (as assessed by CD34 immunostaining) and the meanmicrovessel density per mm2 in pancreatic tumor xenografts relativeto vehicle-treated tumors, and a 23% reduction in CD34 immuno-staining in orthotopic RENCA tumor pulmonary metastases (data notshown).

Further evidence to suggest that the antitumor efficacy of CEP-7055 is attributable predominantly to its inhibition of VEGF-mediatedangiogenic processes was the absence of antiproliferative or proapo-ptotic activity of the active moiety, CEP-5214, on tumor cells directlyin vitro. CEP-5214 had no effect on the proliferation or viability ofthese tumor cell lines at concentrations up to 3 �M. These findingslend additional support to the absence of direct antitumor activity ofthis compound at concentrations demonstrating significant and sus-tained antitumor and antiangiogenic efficacy in vivo.

Effects of p.o. Administration of CEP-7055 on Primary TumorGrowth and Metastatic Profile in Orthotopic Models of HumanProstate Carcinoma and Murine RENCA. The antitumor efficacyof CEP-7055 administration on local and distant metastatic tumorgrowth was also examined in more clinically relevant orthotopictumor models of hormone-sensitive human prostate carcinoma (inathymic nude mice) and murine RENCA in BALB/c syngeneic mice.In the LNCaP orthotopic prostate carcinoma model, treatment wasinitiated 1 day after orthotopic implantation of LNCaP cells in maleathymic nude mice. Administration of CEP-7055 or vehicle wasperformed for 21 days, at which time animals were euthanized, andorthotopically grown prostate tumors were evaluated. The take rate forthe prostate tumors was 100% (10 of 10 mice) in the vehicle-treatedcontrol group. Administration p.o. of CEP-7055 had a significanteffect on the growth (tumor wet weight) of orthotopically implantedLNCaP human prostate carcinoma xenografts, resulting in a 52%reduction in prostate tumor wet weight (P � 0.05) (data not shown).

In a series of experiments in the orthotopic RENCA model, 2 daysafter recovery from surgery to implant RENCA cells beneath the renalcapsule, groups of mice were randomized into treatment groups andreceived CEP-7055 at 3.57 mg/kg/dose p.o. b.i.d. and at 23.8 mg/kg/dose p.o. b.i.d. or 1% aqueous acetic acid vehicle p.o. b.i.d. (100�l/dose). The administration of CEP-7055 for 21–26 days had nosignificant effect on the weight of primary orthotopic tumor-bearingkidneys, despite inhibiting significantly the growth of RENCA s.c.implanted tumor xenografts (Table 2). A significant effect of CEP-7055 administration was observed, however, on reducing the overallmetastatic burden (pulmonary and lymph node metastases) in theRENCA model, as manifested in both the severity of metastatic scoresobtained and the weights of lung tissue with metastatic nodules (Fig.10), with values approaching those observed in normal, non-RENCA-

Table 2 Effects of chronic oral b.i.d. administration of CEP-7055 on the growth of s.c. human and rodent tumor xenografts in athymic nude mice

Tumor growth was initiated after the s.c. implantation of subconfluent cultures into the right flank of female athymic nude mice (6–8 weeks old) or in Balb/c female mice (6–8weeks old, for RENCA cells) in their respective serum-free media along with Matrigel synthetic basement membrane (1:1, v/v). Cell densities implanted in vivo were U251 MG andSF767 human glioblastomas (3 � 106); CALU-6 human non-small cell lung adenocarcinoma (5 � 106); HT-29, HCT-116, and COLO 205 human colon carcinomas (2 � 106); MCF-7breast carcinoma (1 � 106); Dunning G/VEGF165 rat prostate carcinoma (4 � 106); SVR murine angiosarcoma (1 � 106) and murine RENCA (3 � 105). Upon achieving volumesof 80–180 mm3, tumor-bearing mice for each xenograft were randomized into treatment groups (n � 10 mice/group) and were given CEP-7055 at the doses indicated in a vehicle of1% aqueous acetic acid (100 �l volume/dose). Tumor absolute volumes were normalized to individual tumor volumes at day 1, the initiation of dosing (relative tumor volumes) to assesschanges in the rate of tumor growth, relative to treatment. Comparable results were obtained for absolute and relative tumor volumes in the xenografts described. Statistical analysesare detailed in “Materials and Methods.”

Tumor type andimplantation site Dosing regimen and duration in mice

Magnitude of antitumor efficacy relative to vehiclecontrols

CALU-6 human NSCLa CA 3.57 and 11.9 mg/kg/dose b.i.d. (30 days) 35% and 60% inhibition of tumor growth vs. controls;NS, P � 0.01, respectively

SVR murine angiosarcoma 0.35 to 23.8 mg/kg/dose b.i.d. (10 days) Dose-related inhibition of tumor growth; 70%maximum at 23.8 mg/kg vs. controls, P � 0.01

Dunning G rat prostate CA 11.9 mg/kg/dose b.i.d. (21 days) 72% inhibition of tumor growth vs. controls, P � 0.01MCF-7 human breast CA 23.8 mg/kg/dose b.i.d. (26 days) 65% inhibition of tumor growth vs. controls, P � 0.01U251MG human GBM 11.9 mg/kg/dose b.i.d. (65 days) 50% inhibition of tumor growth vs. controls, P � 0.01SF767 human GBM 11.9 mg/kg/dose b.i.d. (30 days) NS inhibition vs. controlsRENCA murine renal CA 11.9 mg/kg/dose b.i.d. (15 days) 50% inhibition of tumor growth vs. controls, P � 0.05HCT-116 human colon CA 11.9 and 23.8 mg/kg/dose b.i.d. (30 days) 40% inhibition of tumor growth vs. controls at both

dose levels, P � 0.05HT-29 human colon CA 11.9 and 23.8 mg/kg/dose b.i.d. (30 days) NS inhibition at 11.9-mg/kg dose; 42% inhibition at

23.8-mg/kg dose, P � 0.05COLO205 human colon CA 11.9 and 23.8 mg/kg/dose b.i.d. (30 days) NS inhibition at 11.9-mg/kg dose; 46% inhibition at

23.8-mg/kg dose, P � 0.05a NSCL, non-small cell lung; CA, carcinoma; GBM, glioblastoma; NS, not statistically significant, P � 0.05.

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implanted BALB/c mouse lungs of similar body weight (Fig. 10A).Significant reductions in tumor microvessel density (CD34 staining)were also observed in metastatic lesions as noted above. Administra-tion p.o. of CEP-7055 resulted in a larger percentage of mice having

a lower overall metastatic score from their orthotopically implantedRENCA, relative to control mice, but did not result in completelyeliminating a high metastatic burden in all of the tumor-bearinganimals. These results obtained with p.o. CEP-7055 at 23.8 mg/kg/

Fig. 8. Dose-related effects of chronic p.o. administration of CEP-7055 onthe growth of select established human s.c. tumor xenografts in athymic nudemice. Tumor xenografts were established after the s.c. implantation of tumorcells in Matrigel (1:1, v/v) into the flank of female athymic nude mice. Ontumors achieving volumes of 80–180 mm3, tumor-bearing mice were random-ized into treatment groups (n � 10/group) and p.o. b.i.d. (BID) administrationof CEP-7055 was initiated at the doses shown in a vehicle of 1% aqueous aceticacid (100 �l/dose) for the period of time show. Tumor volumes were normal-ized to individual tumor volumes (relative tumor volumes � SE) at day 1, theinitiation of dosing as detailed in “Materials and Methods.” Statistical analysesof tumor data were done using the Mann-Whitney rank-sum test, or whenappropriate for the data set, by one-way ANOVA and the Dunnet’s multiple-comparison test. A, A375 human melanoma; B, U87MG human glioblastoma;C, ASPC-1 human pancreatic ductal carcinoma. Statistically significant andsustained inhibition of tumor growth was observed at doses of 3.57 mg/kg/doseor greater in A375 and U87MG tumors, and 1.19 mg/kg/dose or greater inASPC-1 tumors as detailed in “Results” and “Discussion.” Partial tumorregressions were observed in U87MG tumors at p.o. doses greater than 11.9mg/kg/dose b.i.d.

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dose p.o. b.i.d. surpassed those obtained using a clinically relevantdosing regimen of recombinant human IL- 2 (Proleukin) in this model(Fig. 10, B and C).

DISCUSSION

In this report, we describe the biochemical and pharmacologicalactivity profile of CEP-7055, the prodrug of CEP-5214, a low-nano-molar, orally active inhibitor of all three VEGF-R kinase receptorsubtypes (VEGF-R1/FLT-1 IC50, 16 nM; VEGF-R2/KDR IC50, 8 nM;VEGF-R3/FLT-4 IC50, 4 nM). The parent compound, CEP-5214,displays potential therapeutic value in oncology and other VEGF-mediated angiogenic disease states based on its low nanomolar inhi-bition of all three VEGF-R kinases, its antiangiogenic activity inmultiple in vitro, ex vivo, and in vivo models, and its p.o. antitumorefficacy against a variety of aggressive rodent and human tumorxenograft models in athymic nude mice in the absence of apparentmorbidity or toxicity. Its ester derivative, CEP-7055, was prepared toincrease aqueous solubility and to facilitate p.o. delivery. CEP-7055 isalso a pan-VEGF-R kinase inhibitor with activity in vitro that is nearlyequivalent to that of CEP-5214. An increasing body of evidence forthe role of multiple FLT-1/VEGF-R1 and FLT-4/VEGF-R3-mediatedbiological activities in normal and pathological angiogenesis (12–22)lend strong support for the therapeutic utility of an orally active paninhibitor of these VEGF receptor subtypes in addition to KDR/VEGF-

R2, through which the VEGFs exerts their mitogenic, chemotactic,and vascular permeabilizing effects on the vascular endothelium. Wehave demonstrated in in vitro and ex vivo biochemical and biologicalassays that CEP-5214 reversibly inhibits VEGF-induced phosphoryl-ation of all three VEGF-R kinases with a cellular IC50 of �10 nM inboth murine and human endothelial cells. Single-dose p.o. or s.c.administration of CEP-7055 to CD-1 mice at 20 mg/kg/dose b.i.d.equivalents resulted in a significant and reversible inhibition ofVEGF-R2/FLK-1 phosphorylation in murine SVR tumors or murinelung tissues for 2–3 h postdose and returned to baseline VEGF-R2/FLK-1 phosphorylation by 4 h. Similar types of pharmacodynamicapproaches to assess in vivo biochemical efficacy have been reportedfor the evaluation of orally active VEGF-R kinase inhibitors preclini-cally, and have demonstrated a comparable time course for the inhi-bition of VEGF-R2/FLK-1 phosphorylation after single-dose admin-istration in vivo (44).

The p.o. administration of CEP-7055 at doses of 11.9–23.8 mg/kg/dose b.i.d. to normal or immunocompromised mice results in signif-icant and dose-related inhibition of VEGF-mediated neovasculariza-tion (PAEC-VEGF/bFGF-Matrigel implants) by up to 82%, relative tocontrols, and inhibits significantly both vascularity and granulomaformation in a murine model of chronic inflammation-induced angio-genesis, a process in which VEGF has been demonstrated to play asalient role (61, 67–69). In addition to the activity of CEP-7055 ininhibiting VEGF-mediated neovascularization and inflammation-induced angiogenesis, p.o. administration of CEP-7055 results insignificant and sustained (�6 h) dose-related inhibition of VEGF-induced dermal vascular permeability in rats (Evan’s dye assay) withan ED50 of 20 mg/kg/dose. Earlier studies demonstrated that acute andchronic p.o. administration of CEP-7055 to rats inhibited intravitrealVEGF-induced retinal vascular permeability and leakage significantly(62 and 52%, respectively), and completely inhibited diabetes-induced retinal vascular permeability, although having no effects onbasal vascular permeability (70). Collectively, these data indicate thatp.o. administration of CEP-7055 at doses of 11.9–23.8 mg/kg/doseb.i.d. inhibits VEGF-mediated signaling and angiogenesis directly invivo under both physiological and pathological conditions in theabsence of toxicity or pronounced morbidity.

The most salient feature of the pharmacological profile of CEP-7055 is the observation that chronic p.o. administration of 3.57–23.8mg/kg/dose b.i.d. results in dose-related inhibition of s.c. tumor xe-nograft growth ranging from 50–90%, relative to vehicle-treated miceagainst a broad range of human and rodent tumors varying in theirhistological origin, latency and growth rate, and responsiveness toconventional cytotoxic agents. There are a number of influences thatmay account in part for differences in the range and magnitude ofantitumor responses observed on chronic CEP-7055 administration.These influences include the distinct growth profiles of the variedtumor xenografts examined; the role of the tumor microenvironment,i.e., the extent of localized hypoxia and ischemia within a given tumorxenograft, over the time course of treatment on tumor response; thevaried dependency of particular tumor types on VEGFs versus otherangiogenic cytokines at specific stages of their vascular growth andmaturation; and tumor-specific differences in the temporal expressionand/or activation of key intracellular mediators of VEGF-inducedangiogenesis, e.g., AKT/phosphatidylinositol 3�-kinase, p44/42 mito-gen-activated protein kinase, specific integrins and adherens (70–75).This tumor inhibition profile was observed in both immunocompro-mised and immunocompetent mice when given CEP-7055 for periodsranging from 10 to 65 days. In several distinct tumor xenograftmodels, including the A375 human melanoma, U87MG human glio-blastoma, ASPC-1 pancreatic carcinoma, and SVR murine angiosar-coma, the minimum effective p.o. dose for sustained and significant

Fig. 9. Effects of chronic orally administered CEP-7055 (11.9 mg/kg/dose p.o. b.i.d.)on the growth of established ASPC-1 human pancreatic carcinoma xenografts of varyinginitial volumes. Female nude mice bearing established s.c. ASPC-1 human pancreaticductal carcinoma xenografts were subdivided into groups (n � 10 mice/group) withdifferent mean tumor volumes (�150-mm3, �400-mm3, and �900-mm3 absolute vol-umes) as tumor xenograft growth progressed and administered CEP-7055 p.o. b.i.d. at11.9 mg/kg/dose for the durations of time indicated. Arrows, the initiation of CEP-7055administration. Statistical analyses are detailed in “Results” and “Discussion.” CE-7055administration inhibited ASPC-1 xenograft growth significantly relative to volume-matched controls independently of initial tumor xenograft volume. Values shown aremean � S.E.M.

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antitumor efficacy was 3.57 mg/kg/dose b.i.d. In the case of theU87MG model, monotherapy with CEP-7055 at doses of 11.9 mg/kg/dose b.i.d. or above resulted in a 50% incidence of partial tumorregressions (50–90% reduction of initial tumor volume). Partial orcomplete tumor regressions in select tumor xenograft models havebeen observed with monotherapy administration of several otherVEGF-R kinase inhibitors, including PTK-787 in CWR-22 prostatecarcinomas (35), ZD6474 in PC-3 prostate carcinomas (41), andSU11248 (a VEGF-R2/PDGF-RB kinase inhibitor), in the A431 epi-dermoid and COLO 205 colon carcinoma models (42). Of note is thefact that the administration of CEP-7055 resulted in significant andsustained tumor growth inhibition in ASPC-1 pancreatic carcinomaxenografts, independent of initial tumor volumes, even in tumors of�1.0 cm3. Similar findings have been reported with ZD6474 inestablished CALU-6 lung tumor xenografts (40, 41) and could havesignificant implications for the clinical administration of these anti-tumor agents.

The extent to which the weaker inhibitory activities of CEP-5214 orits prodrug, CEP-7055, against c-Kit, FGF-R1 kinase and PDGF-R�(Table 1) contribute to the in vivo pharmacological profile of CEP-7055 is not clear, although several antiangiogenic agents, currently(CP-547,632; SU11248) and previously (SU6668) under clinical eval-uation, possess one or more of these activities (34, 42, 44, 76–78).

The antitumor efficacy of chronic p.o. administration of CEP-7055

was not confined to s.c. tumor xenografted tumors but extended toorthotopically implanted human prostatic carcinomas (60) and murineRENCA xenografts in both immunocompetent and immunocompro-mised mice. This renal carcinoma model has been reported to besensitive to the administration of several antiangiogenic agents, in-cluding TNP-470 (64) and, in particular, PTK-787 (35–37). Curi-ously, although CEP-7055 inhibited the extent of metastases in theRENCA model, as well as the growth of s.c. implanted RENCAtumors, marginal antitumor activity was observed against primaryRENCA tumor growth within the renal capsule. Similar findings formore pronounced activity of antiangiogenic agents (e.g., PTK-787)against metastases versus primary tumor growth in the RENCA modelhave been reported (35). The homogeneous and extensive vascular-ization throughout these tumors suggests that, in rapidly growingRENCA tumors, the tumor cell proliferation rate is superior to that oftumor-associated endothelial cells and results in partial angiogenesis-independent tumor growth (36). This fact, and the proximity oforthotopically implanted RENCA tumors to an abundant vascularsupply in the kidney, could account for the marginal effect of CEP-7055 on primary RENCA tumor growth, despite its significant effectson the seeding and subsequent growth of distant lung metastases andon the inhibiting of s.c. implanted RENCA xenograft growth, pro-cesses that would be predicted to be more angiogenesis-dependent. Anadditional explanation for the significant antimetastatic efficacy of

Fig. 10. Effects of p.o. administration of CEP-7055 on thegrowth of primary and metastatic lesions from orthotopicallyimplanted murine RENCA xenografts. Effects of p.o. CEP-7055administration at 3.57 and 23.8 mg/kg/dose b.i.d. on pulmonarymetastases (wet weights); and p.o. CEP-7055 [23.8 mg/kg/doseb.i.d. (BID) versus recombinant human (rh)IL-2 (Proleukin) onpulmonary metastases (B) and total metastatic score (C) asdefined in “Materials and Methods:” in the murine RENCAmodel. Data with rhIL-2 shown as a reference for effects oftreatment on metastatic score. Treatment was initiated 2 daysafter implantation of RENCA cells into the subcapsular space ofsyngeneic Balb/c mice (n � 10/group) and continued for 26days total. Lung weights of normal, non-RENCA implantedmouse lungs are shown for a reference (A). *, P � 0.05,**, P � 0.01.

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CEP-7055 in the murine RENCA model may be the potent inhibitoryactivity of CEP-5214 against VEGF-R3/FLT-4, the VEGF receptorsubtype implicated in the induction of tumor lymphangiogenesis andlymphatic metastases in multiple solid tumor types (18–21).

The magnitude of human and rodent tumor xenograft growth inhi-bition observed with chronic CEP-7055 administration across multi-ple organ-specific tumor types observed at total daily p.o. doses of23.8–47.6 mg/kg/dose is comparable with, or lower than, that ob-served with chronic p.o. administration of several VEGF-R kinaseinhibitors currently under clinical evaluation. Several orally activeVEGF-R kinase inhibitors have demonstrated significant and compa-rable antitumor efficacy preclinically in s.c. tumor xenografts ateffective total daily p.o. doses of 50–100 mg/kg/dose (PTK-787;Refs. 35–38), 25–100 mg/kg/dose (ZD 6474; Refs. 40, 41), 50 mg/kg/dose (CP-547,632; Ref. 44), and 40 mg/kg/dose (SU11248; Ref.42). This broad antitumor efficacy profile of CEP-7055, the observedreductions in IMD (20–36% relative to control tumors depending ontumor type) on its chronic administration in several tumor xenograftmodels, and the significant in vivo efficacy observed with chronicCEP-7055 administration in a variety of VEGF-mediated angiogene-sis models described in this report, are consistent with primarilyindirect, i.e., antiangiogenesis-related, antitumor effects of this panVEGF-R kinase inhibitor rather than with a direct proapoptotic effecton individual tumor cells per se. In support of this conclusion, is thefact that 3–10-�M concentrations of CEP-5214 were required toachieve significant antiproliferative or proapoptotic effects against apanel of human tumor cell lines in vitro, significantly higher than theplasma concentrations (50–200 nM) at which in vivo efficacy has beenobserved with CEP-7055 administration in preclinical murine models.

In summary, we have identified and are developing CEP-7055, theprodrug of CEP-5214, an orally active, low-nanomolar pan VEGF-Rkinase inhibitor that demonstrates significant and durable antiangio-genic efficacy in vitro, ex vivo, and in vivo, and antitumor efficacyagainst s.c. and orthotopically implanted human and rodent tumors invivo. The pharmacological, pharmacokinetic, and tolerability profileof this agent in preclinical studies is compatible with chronic admin-istration against a variety of disease states in which VEGF-mediatedangiogenesis plays a salient role. CEP-7055 is currently in Phase Iclinical trials as an orally administered therapy in patients with avariety of solid tumors.

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[CANCER RESEARCH 63, 7542–7544, November 1, 2003]

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7542

Corrections

In the article by T. Wissniowski et al., titled “Activation of Tumor-specificT Lymphocytes by Radio-Frequency Ablation of the VX2 Hepatoma in Rab-bits,” which appeared in the October 1, 2003 issue of Cancer Research (pp.6496–6500), the names of the first two authors were misspelled. The correctauthor list is as follows: Thaddaus Till Wissniowski, Johannes Hansler, DanielNeureiter, Markus Frieser, Stefan Schaber, Birgit Esslinger, Reinhard Voll,Deike Strobel, Eckhart Georg Hahn, and Detlef Schuppan.

In the article by B. Ruggeri et al., titled “CEP-7055: A Novel, Orally ActivePan Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Ki-nases with Potent Antiangiogenic Activity and Antitumor Efficacy in Preclin-ical Models,” which appeared in the September 15, 2003 issue of CancerResearch (pp. 5978–5991), two co-authors, Shi Yang (Department of Bio-chemistry, Cephalon, Inc.) and Sonya Pritchard (Department of Oncology,Cephalon, Inc.), were inadvertently omitted from the list of authors. Thecorrect list is as follows: Bruce Ruggeri, Jasbir Singh, Diane Gingrich, ThelmaAngeles, Mark Albom, Shi Yang, Hong Chang, Candy Robinson, KathrynHunter, Pawel Dobrzanski, Susan Jones-Bolin, Sonya Pritchard, Lisa Aimone,Andres Klein-Szanto, Jean-Marc Herbert, Francoise Bono, Paul Schaeffer,Pierre Casellas, Bernard Bourie, Roberto Pili, John Isaacs, Mark Ator, RobertHudkins, Jeffrey Vaught, John Mallamo, and Craig Dionne.

In the article by Y-T Tai et al., titled “Insulin-like Growth Factor-1 InducesAdhesion and Migration in Human Multiple Myeloma Cells via Activation of�1-Integrin and Phosphatidylinositol 3’-Kinase/AKT Signaling,” which ap-peared in the September 15, 2003 issue of Cancer Research (pp. 5850–5858),the Western blotting in the bottom “IGF-1R” panel of figure 3B is incorrect.The correct figure appears below:

Fig. 3. IGF-I stimulates association of IGF-IR and �1 integrin, as wellas activation of PI3-K/AKT and ERK pathways. A, serum-starvedMM.1S or OPM6 cells were treated with IGF-I for the indicated times.Cell lysate (500 �g) was immunoprecipitated with anti-IGF-IR andanti-IRS-1 Abs and then immunoblotted with anti-pTyr mAb. Mem-branes were stripped and reprobed with anti-IGF-IR, anti-�1 integrin,and anti-p85PI3-K Abs. A total of 60 �g of each lysate was also resolvedby 8% SDS-PAGE, and immunoblotted with anti-pAKT and anti-pERKAbs. �-tubulin serves as loading controls. B, serum-starved cells wereincubated with IGF-I (100 ng/ml) or PMA (100 nM) for 10 min orpretreated with �IR3 (5 �g/ml) for 30 min before incubation with IGF-I(100 ng/ml) for 10 min. One mg of cell lysates was immunoprecipitatedwith 4 �g of anti-�1 integrin Abs. Immunoprecipitates were analyzed byimmunoblotting with anti-IGF-IR and anti-�1 integrin Abs.

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ANNOUNCEMENTS

In the article by G. Yousef et al., titled “Human Kallikrein 5: A PotentialNovel Serum Biomarker for Breast and Ovarian Cancer,” which appeared inthe July 15, 2003 issue of Cancer Research (pp. 3958–3965), figure 4 wasprinted incorrectly. Below is the correct figure.

Fig. 4. Fractionation of three biological fluids (serum, ascites fluid from an ovariancancer patient, and breast milk) by size-exclusion liquid chromatography. The elutionprofile of molecular mass standards is denoted by arrows. In serum, hK5 elutes as twoimmunoreactive peaks, one with an apparent molecular mass of 50 kDa (fractions 37–39)and one with an apparent molecular mass of approximately 150–180 kDa (fractions31–33). The elution profile of another kallikrein with a similar theoretical molecular mass,hK6, is also shown by dashed lines. This kallikrein elutes at a molecular mass of �35kDa, corresponding to free hK6. In ascites fluid, the same comments apply as for serum.In breast milk, hK5 elutes mainly as a single immunoreactive peak. hK6 elutes as twodistinct peaks, one at a molecular mass of �35 kDa and another one at a molecular massof �100 kDa.

7544

ANNOUNCEMENTS

2003;63:5978-5991. Cancer Res Bruce Ruggeri, Jasbir Singh, Diane Gingrich, et al. Preclinical ModelsPotent Antiangiogenic Activity and Antitumor Efficacy inEndothelial Growth Factor Receptor Tyrosine Kinases with CEP-7055: A Novel, Orally Active Pan Inhibitor of Vascular

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CEP-7055: A Novel, Orally Active Pan Inhibitor of Vascular … · CEP-7055, was prepared to increase aqueous solubility and to facil-itate p.o. delivery. CEP-7055 demonstrates a broad