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CEP-7055

A Novel, Orally Active Pan Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases with Potent Antiangiogenic Activity and Antitumor Efficacy in Preclinical Models¹

Departments 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 of Medical 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

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Inhibition of the vascular endothelial growth factor VEGF-VEGF receptor (VEGF-R) kinase axes in the tumor angiogenic cascade is a promising therapeutic strategy in oncology. CEP-7055 is the fully synthetic orally active N,N-dimethyl glycine ester of CEP-5214, a C3-(isopropylmethoxy) fused pyrrolocarbazole with potent pan-VEGF-R kinase inhibitory activity. CEP-5214 demonstrates IC₅₀ values of 18 nM, 12 nM, and 17 nM against human VEGF-R2/KDR kinase, VEGF-R1/FLT-1 kinase, and VEGF-R3/FLT-4 kinase, respectively, in biochemical kinase assays. CEP-5214 inhibited VEGF-stimulated VEGF-R2/KDR autophosphorylation in human umbilical vein endothelial cells (HUVECs) with an IC ₅₀ of 10 nM and demonstrated an equivalent inhibition of murine FLK-1 autophosphorylation in transformed SVR endothelial cells. Evaluation of the antiangiogenic activity of CEP-5214 revealed a dose-related inhibition of microvessel growth ex vivo in rat aortic ring explant cultures and in vitro on HUVEC capillary-tube formation on Matrigel at low nanomolar concentrations. The antiangiogenic activity of CEP-5214 in these bioassays was observed in the absence of apparent cytotoxicity. Single-dose p.o. or s.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 phosphorylation in murine lung tissues. Administration p.o. of CEP-7055 at 2.57 to 23.8 mg/kg/dose b.i.d. resulted in dose-related reductions in neovascularization 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) inhibition of 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 23.8 mg/kg/dose b.i.d. resulted in significant inhibition (50–90% maximum inhibition relative to controls) in the growth of a variety of established murine and human s.c. tumor xenografts in nude mice, including A375 melanomas, U251MG and U87MG glioblastomas, CALU-6 lung carcinoma, ASPC-1 pancreatic carcinoma, HT-29 and HCT-116 colon carcinomas, MCF-7 breast carcinomas, and SVR angiosarcomas. Significant antitumor efficacy was observed similarly against orthotopically implanted LNCaP human prostate carcinomas in male nude mice and orthotopically implanted renal carcinoma (RENCA) tumors in BALB/c mice, in terms of a significant reduction in the metastatic score and the extent of pulmonary metastases. These antitumor responses were associated with marked increases in tumor apoptosis, and significant reductions in 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 independent of initial tumor volume (in the ASPC-1 pancreatic carcinoma model) and reversible on withdrawal of treatment. Chronic p.o. administration of CEP-7055 in preclinical efficacy studies for periods of up to 65 days was well tolerated with no apparent toxicity or significant morbidity. Orally administered CEP-7055 has entered Phase I clinical trials in cancer patients.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Angiogenesis, the development of new blood vessels from the endothelium of a preexisting vasculature, is a critical process required by most solid tumors to support their localized growth and metastatic dissemination within the host (1, 2, 3, 4) . The autocrine, paracrine, and amphicrine interactions of the vascular endothelium with its surrounding stromal components, as well as with the proangiogenic and angiostatic cytokines and growth factors orchestrating physiological angiogenesis, are tightly regulated both spatially and temporally. In contrast, the pathological angiogenesis necessary for active tumor growth is sustained and persistent, with the initial acquisition of the angiogenic phenotype being a common mechanism for the development of a variety of solid and hematopoietic tumor types (1, 2, 3, 4) .

Among the known angiogenic growth factors and cytokines implicated in the modulation of normal and pathological angiogenesis, the VEGF³ family (VEGF-A, VEGF-B, VEGF-C, VEGF-D) and their corresponding receptor tyrosine kinases [VEGF-R1 (FLT-1), VEGF-R2 (FLK-1, KDR), and VEGF-R3 (FLT-4)] play a paramount and indispensable role in regulating the multiple facets of the angiogenic and lymphangiogenic processes [VEGF-R3 (FLT-4)], as well as the induction of vascular permeability and inflammation (1 , 2 , 5, 6, 7) . In contrast to pleiotrophic angiogenic factors and cytokines, the VEGF family has a relatively narrow target cell specificity and exert their mitogenic, chemotactic, and thrombogenic effects, primarily on endothelial cells implicated in hemangiogenesis (VEGF-A, VEGF-B) and lymphangiogenesis (VEGF-C and VEGF-D; 2 , 5, 6, 7 ). The endotheliotropic activities of the VEGF family are mediated through the receptor tyrosine kinases: VEGF-R1/FLT-1, VEGF-R2/KDR, and VEGF-R3/FLT-4, the expression of which is up-regulated on vascular endothelial cells during embryonic and tumor angiogenesis (2, 3, 4, 5) . The receptor VEGF-R2/KDR is the principal one through which VEGFs exerts their mitogenic, chemotactic, and vascular permeabilizing effects on the host vasculature (7, 8, 9, 10) . Increased expression of VEGFs by tumor cells and VEGF-R2/KDR and VEGF-R1/FLT-1 by the tumor-associated vasculature are a hallmark of a variety of human and rodent tumors in vivo and correlated with tumor growth rate, microvessel density/proliferation, tumor metastatic potential, and poorer patient prognosis in a variety of malignancies (1, 2, 3, 4, 5, 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, 10, 11, 12) , mediating chemotactic activity in monocytes and macrophages (13 , 14) , regulating extracellular matrix proteolytic activity (15) and the release of tissue factor and nitric oxide in endothelial cells and trophoblasts, respectively (12 , 13) , and in mediating placental growth factor-induced recruitment and mobilization of bone-marrow derived endothelial and hematopoietic stem cells in normal and pathological (tumor-associated) angiogenesis (16 , 17) . Similarly, a growing body of evidence implicates VEGF-R3/FLT-4 and its ligands, VEGF-C and VEGF-D, in the induction of tumor lymphangiogenesis and lymphatic metastases in multiple solid tumor types (18, 19, 20, 21, 22) . These collective findings and the critical and nonredundant role of VEGF in tumor-associated angiogenesis (23 , 24) support the importance of the selective abrogation VEGF-mediated signaling events through all three VEGF-R subtypes as an optimal therapeutic strategy in the management and treatment of a variety of solid and hematopoietic tumors.

Antiangiogenesis therapies directed against the VEGF-VEGF-R kinase axes through a variety of approaches have been a promising and well-validated therapeutic approach under active evaluation for their safety and efficacy in multiple clinical trials (25, 26, 27) . The preclinical biochemical, pharmacological and in vivo efficacy profile of antibody-, soluble receptor-, and ribozyme-based antiangiogenic therapies currently under clinical evaluation have been described extensively (see 25, 26, 27, 28, 29, 30, 31, 32, 33 ). In addition, several orally active small molecule inhibitors of specific VEGF-R kinases (see Ref. 34 ), including PTK-787 (vatalanib; Refs. 35, 36, 37, 38 ), PKC-412 (midostaurin; Ref. 39 ), ZD 6474 (40 , 41) , SU-11248 (42 , 43) , and CP-547,632 (44) are currently under clinical evaluation.

In this report, we describe the biochemical and pharmacological profile of CEP-7055, the N,N-dimethyl glycine ester prodrug of CEP-5214, a novel, orally active and fully synthetic low nanomolar inhibitor of all three VEGF-R kinases (Table 1) . Its ester derivative, CEP-7055, was prepared to increase aqueous solubility and to facilitate p.o. delivery. CEP-7055 demonstrates a broad acting preclinical antitumor and antiangiogenic efficacy and a tolerability profile amenable for chronic p.o. administration. CEP-7055 is currently in Phase I trials in patients with solid tumors.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Lines
The majority of rodent and human tumor cell lines used in these studies were obtained from the American Type Culture Collection (Manassas, VA). The Dunning G/VEGF₁₆₅ rat prostate carcinoma and LNCaP human prostate carcinoma cell lines were obtained from Dr. Roberto Pili (Johns Hopkins University, Baltimore, MD), and cultures of human U251MG and SF 767 glioblastoma cells were obtained from the Brain Tumor Research Center Tissue Bank (University of California, San Francisco, CA). All of the cell lines were MAP-16- and Mycoplasma-tested by a commercial laboratory (Bio Reliance Corp., Rockville, MD) and were deemed suitable for in vivo studies. HUVECs were obtained from Clonetics (San Diego, CA) and cultured in endothelial cell basal medium (EBM-2; Clonetics) with 2% heat-inactivated fetal bovine serum (Life Technologies, Inc., Grand Island, NY), 50 µg/ml endothelial cell growth supplement, 50 µg/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, Wilmington, MA) were maintained five/cage in microisolator units on a standard sterilizable 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 6–8 weeks of age were obtained from Charles River (Wilmington, MA) and female out-bred Tuck Original mice were obtained from Harlan (Oxon, United Kingdom). The animals were housed in groups of 12 mice per cage in a room with a 12-h light/dark cycle. The mice were fed ad libitum normal mouse chow pellets. Male Sprague Dawley rats (250–300 g) were obtained from Charles River and housed five/cage in a conventional vivarium facility. All mice and rats were quarantined 1 week before experimental manipulation. All of the animal studies were conducted under protocols approved by the Institutional Animal Care and Use Committees (IACUC) of Cephalon and Sanofi-Synthelabo.

Recombinant Proteins and Biochemical Kinase Assays
Enzymes and Substrates.
The cytoplasmic domains of recombinant human tyrosine kinases (epidermal growth factor receptor, FGF-R1, FLT-3(ITD1), PDGF-Rß, TIE-2, TRKA, VEGF-R1, VEGF-R2, VEGF-R3) and serine/threonine kinases (CHK1, CDK1/cyclinB, CDS1, DLK, JNK1ß1, MLK1, MLK2, MLK3) were expressed in a baculovirus insect cell system. Recombinant ß-insulin receptor kinase was purchased from Stratagene (La Jolla, CA). Purified rat brain protein kinase C (mixture of Ca⁺²-dependent isozymes , ß, and ) and activated recombinant human GST-p38 were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). PLC- was generated 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), respectively. Time-resolved fluorescence experiments were performed using Eu-N1 antiphosphotyrosine antibody, Eu-N1 antiphosphothreonine antibody, Eu-N1 antirabbit IgG, and DELFIA enhancement solution purchased from Perkin-Elmer Life Sciences (Gaithersburg, MD). Phospho-specific antibodies to ATF-2 (Thr71), Cdc25c (Ser216), and Rb (Ser795) were obtained from New England Biolabs, Inc. (Beverly, MA).

Receptor-linked Tyrosine Kinase Assays.
Enzyme-inhibition studies were performed using a modification of the ELISA described for TRKA kinase (48) . Briefly, the 96-well microtiter plate (FluoroNUNC or Costar High Binding) was coated with 10 µg/ml recombinant human PLC-/GST. Kinase assays were performed in 100 µl reaction mixtures containing 50 mM HEPES (pH 7.4), K_m level of ATP, 10 mM MnCl₂, 0.1% BSA, 2% DMSO, and various concentrations of CEP-5214 or CEP-7055. The reaction was initiated by adding baculoviral recombinant human enzyme (epidermal growth factor receptor, FGF-R1, ßI-RK, PDGF-Rß, FLT-3, TIE-2, TRKA, VEGF-R1, VEGF-R2, or VEGF-R3) and was allowed to proceed for 15 min at 37°C. The detection antibody, Eu-N1 antiphosphotyrosine (PT66) antibody was added. After a 1-h incubation at 37°C, 100 µl of enhancement solution was added and the plate was gently agitated. After 5 min, the fluorescence of the resulting solution was measured using the Victor² Multilabel Counter (Model 1420-018). The activities of the serine/threonine kinases listed in Table 1 were measured using either a radioactive Multiscreen TCA "in-plate" assay (49) or an ELISA similar to that described above for the tyrosine kinases.

Data Analysis.
Inhibition curves for compounds were generated by plotting percentage control activity versus log₁₀ of the concentration of compound. IC₅₀ values were calculated by nonlinear regression using the sigmoidal dose-response (variable slope) equation in GraphPad Prism. IC₅₀ values were reported as the average of at least three separate determinations.

Human and Murine Endothelial Cell-based Receptor Phosphorylation Assays
Subconfluent HUVECs were serum starved by replacing medium with EBM-2 (endothelial cell basal medium, serum-free; Clonetics) containing 0.05% BSA for 1 h at 37°C, during which time, a range of concentrations of CEP-5214 or DMSO (control) was added to the cells. Human VEGF (Clonetics) was then added to HUVECs at a concentration of 10 ng/ml for 5 min. Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing 1 mM activated sodium vanadate and protease inhibitors (Protease Inhibitor Cocktail Set III; Calbiochem, San Diego, CA), sheared with a 27-gauge syringe, and then centrifuged at 12,000 x g for 15 min. Clarified cell lysates were immunoprecipitated with anti-VEGF-R2 antibody (CEP-133) for 1 h, followed by incubation with Protein A-Sepharose for another hour at 4°C and analysis by SDS-PAGE as detailed previously (49 , 50) . Phosphorylated proteins were visualized using enhanced chemiluminescence (Amersham, Piscataway, NJ). The percentage inhibition was calculated by analyzing scanned autoradiographs on a densitometer. Scores were based on decrease in protein band density 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%. Murine SVR-transformed endothelial cells (51) were treated in the same way as HUVECs with the following modifications: subconfluent cells were serum starved for 1 h at 37°C with DMEM containing 0.05% BSA; in addition, autophosphorylation of murine FLK-1 was measured after stimulation for 5 min 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 a capillary tube formation assay using HUVECs cultured on a synthetic basement membrane matrix (52, 53, 54, 55) . Under these conditions, HUVECs are capable of morphological differentiation into an extensive network of capillary-like structures composed of highly organized three-dimensional cords (55) . Forty-eight-well Nunclon (Fisher Scientific, Newark, DE) plates were coated with a 200-µl (2 mg) layer of the synthetic basement membrane substrate Matrigel (Collaborative Research, Bedford, MA) at 10 mg/ml concentration and were incubated at 37°C for 30 min to promote gelling. HUVECs (Clonetics) were cultured in EMB-2 medium (Clonetics) with 2% fetal bovine serum, and cells between passages 3 and 8 were seeded (at 3 x 10⁴ cells in 200 µl of medium) in each of the Matrigel-coated wells. CEP-5214 was diluted in DMSO to a final DMSO concentration in the treated and untreated wells of 0.02%. After an 18-h incubation at 37°C and 5% CO₂ humidified atmosphere, HUVECs were aspirated from the medium and were fixed and stained using a modified Wright-Giemsa staining protocol according to the manufacturer’s recommendations (Diff-Quik Stain Set; Baxter Healthcare Corp., McGraw Park, IL). Complete capillary tube networks within a designated area of a low magnification (x10) field were counted under light microscopy, and the data were expressed as percentage of complete capillary tube formation relative to untreated HUVEC control cultures incubated under the same conditions (54 , 55) . All of the assays were done in quadruplicate in three independent experiments. Statistical analysis of the inhibition of tube formation relative to control cultures was done by the Dunnet’s multiple-comparison test, with P < 0.05 designated as significant. Visual inspection of cultures on a routine basis was used to assess the potential cytotoxicity of CEP-5214 relative to control cultures.

Ex Vivo Rat Aortic Ring Explant Assay in Collagen Gel Matrices
Rat aortic ring explant cultures were prepared by a modification of protocols previously described (54 , 56, 57, 58) . This assay enables a quantitative assessment of microvessel growth, branching, and remodeling; and vessel regression in a primary explant culture system in which endothelial cell-adventitial cell interactions critical for angiogenesis in vivo can be effectively modeled in a more physiologically relevant ex vivo setting. CEP-5214 was dissolved in DMSO and mixed with serum-free EBM immediately before the addition or replacement of media to collagen-embedded aortic ring explant cultures in quadruplicate. 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 and control 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 female CD-1 mice and female athymic nude mice of 6–8 weeks of age. A dose of 1.19 mg/kg CEP-7055 is equivalent to 1.0 mg/kg dose of CEP-5214. Trunk blood from mice that were given the HCl salt of CEP-7055 p.o. was collected after decapitation using heparin as the anticoagulant. Plasma was prepared for high-performance liquid chromatography (HPLC)/mass spectrometric analysis by protein precipitation with two volumes of acetonitrile (200 µl) per 100-µl sample of plasma. Pharmacokinetic parameters were determined by WinNonLin software.

Pharmacodynamic Effects of CEP-7055 on VEGF-R2/FLK-1 Phosphorylation 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 nude mice bearing established VEGF-R2 expressing (52) SVR angiosarcomas (250 mm³) were administered a single p.o. or s.c. dose of CEP-7055 at a 23.8-mg/kg/dose, and tumors were excised 30–45 min later. Tumor lysates were prepared and the tyrosine autophosphorylation of the VEGF-R2/FLK-1 receptor was analyzed by immunoprecipitation with SC-504 and SC-315G anti-FLK-1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and by Western blotting with 4G10 antiphosphotyrosine antibody as described above, which confirmed the detection of a M_r 220,000 protein of the expected size. Immunoabsorbtion with increasing concentrations of blocking peptides to these antisera confirmed the specificity of the immunoprecipitation for murine FLK-1 receptor in SVR cells and tumors. In related studies, athymic mice received a single p.o. dose of CEP-7055 at 59 mg/kg/dose or CEP-5214 directly at a 50-mg/kg/dose. At 20, 40, and 60 min after dosing, animals were given murine VEGF at 3 ng/mouse i.v. and were sacrificed 5 min later; protein extracts were then prepared from lungs. Tyrosine phosphorylation of FLK-1 was evaluated by Western blotting as above, using PC460, a phospho FLK-1 antibody. The abundance of VEGF-R2/FLK-1 in each sample lysate was assessed by Western blotting, and the samples were evaluated densitometrically.

PAEC-VEGF/bFGF-induced Matrigel Implant in Vivo Angiogenesis Model in Nude Mice
The Matrigel plug implantation assay used in these studies was a modification of that described previously (59 , 60) . Briefly, PAECs were grown to confluency in Ham’s F-12 medium supplemented with 10% fetal bovine serum. Cells were used between passages 5 and 10. Nude mice were given bilateral s.c. injections of 0.5 ml Matrigel synthetic basement membrane (Collaborative Research) containing 1 x 10⁶ PAECs/plug and recombinant murine VEGF and bFGF (R&D Systems) at 20 ng/ml and 250 ng/ml, respectively, final concentrations per plug. Mice bearing PAEC-VEGF/bFGF-Matrigel implants were randomized into groups (10/group) and were given doses 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 been reported to be directly proportional to the degree of neovascularization in each plug (59) and was determined as described previously (60) . Contralateral plugs were evaluated histologically for vessel morphology. Results from duplicate in vivo experiments are expressed as mean g/dl of hemoglobin ± SE. Statistical analyses of the data were done using the paired Student’s t test, with P < 0.05 deemed significant.

Chronic Inflammation-induced in Vivo Angiogenesis Model in Mice
Angiogenesis was induced in female out-bred Tuck Original mice (25 g initial body weight) with a chronic granulomatous reaction to Freund’s complete adjuvant in croton oil as described previously (61) . Briefly, granulomatous air pouches were induced by the s.c. injection of 3 ml of air into each anesthetized mouse and by the injection of 0.5 ml of Freund’s complete adjuvant with 0.1% croton oil 4–5 h later. Doses of CEP-7055 were administered p.o. b.i.d. in 1% aqueous acetic acid over a 6-day period, and the degree of inflammation and vascular density were evaluated on day 7. The vascular content was assessed by the formation of vascular casts incorporating carmine. Mice were anesthetized with pentobarbital (60 mg/kg/dose, i.p.) and peripheral vasodilatation 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 granulomatous air pouch linings were dissected, and the tissues were dried, enzymatically digested, 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 sodium pentobarbital at 60 mg/kg/dose i.p. Animals were given i.v. injections of Evans blue dye (15 mg/kg/dose), and, 5 min later, saline solution (NaCl 0.9%) or VEGF (3 ng) were injected intradermally (0.1 ml/site). Four paired injections of saline solution and VEGF were performed on the back of each rat. Measurements of the vertical and the horizontal diameter of the blue area were performed for each injection point, and the area of plasma extravasation was calculated (radius vertical x radius horizontal x ). Basal plasma extravasation (0.9% saline solution) was not modified by the treatment with CEP-7055 at any time or at any dose tested. Consequently, for each VEGF/saline solution pair, the basal extravasation area corresponding to the injection of saline solution was subtracted from the extravasation area that resulted from the VEGF injection. Administration p.o. of CEP-7055 or vehicle (1% aqueous acetic acid) was done over a 6-h time course. For dose-response studies, CEP-7055 was administered 60 min before the measurement of local plasma extravasation at the indicated doses (see Fig. 7 ). The corrected areas of the CEP-7055 treated group were compared with the vehicle group with Student’s unpaired t test, with P < 0.05 deemed significant.

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Fig. 7. Effects of p.o. administration of CEP-7055 on VEGF-induced plasma extravasation 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-Dawley rats (n = 10/group) were evaluated using Evans blue dye (15 mg/kg) injected i.v. Five min after VEGF injection, saline solution (NaCl, 0.9%) or VEGF (3 ng) were injected intradermally (0.1 ml/site). Measurement of the vertical and the horizontal diameter of the Evan’s blue dye area in the skin was performed for each injection point and the area of plasma extravasation was calculated (radius vertical x radius horizontal x ). For each VEGF/saline solution pair the basal extravasation area corresponding to injection of saline solution was subtracted from the extravasation area resulting from VEGF injection. A, time course of VEGF-induced plasma extravasation after p.o. administration of 20 mg/kg/dose of CEP-7055. B, p.o. CEP-7055 dose-response relationship for inhibition of VEGF-induced plasma extravasation. Doses of CEP-7055 were administered 60 min before the measurement of local plasma extravasation at the indicated doses. *, P < 0.05; **, P < 0.01; *** P < 0.001 by Student’s t test.

Orthotopic Prostate and Renal Carcinoma Tumor Models in Mice
The antitumor efficacy of CEP-7055 was further evaluated in orthotopic models 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 on primary, local invasive, and metastatic tumor growth in both immunocompromised and immunocompetent mice, respectively. Briefly, exponentially growing human androgen-sensitive LNCaP prostate carcinoma cells were harvested from tissue culture plates by trituration. LNCaP cells were injected orthotopically (2 x 10⁶ cells/20 µl per mouse) into the prostate of athymic male nude mice (6–8 weeks old). Treatment with CEP-7055 was initiated 1 day after orthotopic implantation of LNCaP cells and continued for 21 days, at which time 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 murine renal 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 x 10⁵ in 50 µl of sterile 1x PBS) were injected into the renal subcapsular space of the left kidney of female BALB/c mice. Two days after recovery from surgical implantation, groups of mice were randomized into treatment groups and were given CEP-7055 over a 21- to 26-day period. Human recombinant IL-2 (Proleukin; Chiron Corp, Emeryville, CA) was used as a reference standard in the RENCA model using previously established dosing protocols of 30,000 units/dose i.p. for 5 days/week (64) . Mice were necropsied for gross assessment of local and disseminated tumor burden (lungs, regional lymph nodes) and histopathological analysis of selected tissues. A metastatic scoring system was 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 nodules in the lung; III, primary mass and too numerous to count (TNTC) nodules in the lung but still normal areas of lung visible; IV, primary mass with TNTC nodules in the lung with no normal lung visible. Statistical analyses of tumor and lung weights were done by the Mann-Whitney rank-sum test, with P < 0.05 deemed significant.

Tumor Histological and Immunohistochemical Analyses of Microvessel Density
In situ evaluation of IMD in tumor xenografts was determined immunohistochemically by evaluating expression of the endothelium-associated antigen, von Willebrand factor (Factor VIII) and CD34 immunostaining for confirmation) by modification of methods described previously (24 , 65) . Tissue sections were processed, deparaffinized, rehydrated, and quenched for endogenous peroxidase activity as described previously (65) . After a 10-min permeabilization step with proteinase K, murine vessels in tumor xenografts were immunostained with rabbit anti-von Willebrand factor (DAKO Corp., Carpenteria, CA) at 20 µg/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 goat antirabbit IgG after incubation with streptavidin-horseradish peroxidase (VectaStain Elite; Vector Laboratories, Burlingame, CA). Sections were developed after washing with diaminobenzidine (DAB) as a chromogen and counterstained with 1% methyl green. For quantitation of IMD, 10 fields (97,500 µm²/field) of five tumors were evaluated in a blinded fashion at x100 magnification, and the percentage inhibition of IMD relative to vehicle-treated control tumors was determined. Statistical analyses of IMD in tumor xenografts from CEP-7055-treated mice compared with vehicle-treated mice were done using a 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 previously (62) in a 96-well format to assess direct effects of CEP-5214 at 24- and 48-h incubation on the proliferative fraction of select tumor cell lines. Plates were read on a Cytofluor 2300 fluorescence plate reader at various sensitivities with an excitation wavelength of 485 nm and emission wavelength of 530 nm. Negative control wells contained medium, but no cells, and were assayed with calcein, as described above. All of the studies were conducted twice and in triplicate for each sample concentration.

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Kinase Inhibition Profile of CEP-7055 and CEP-5214
The effects of CEP-5214 and its pro-drug CEP-7055 (Fig. 1) were evaluated in enzyme- based assays for the inhibition of VEGF-R2 kinase activity. The primary assay for identifying inhibitors of the VEGF-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 for CEP-7055, it was first necessary to evaluate the rate at which the prodrug converts to CEP-5214 in aqueous solution under conditions representative of the kinases assays. CEP-7055 was incubated in 50 mM HEPES buffer at pH 7.2 for 15 min, and samples were analyzed by high-performance liquid chromatography for the presence of CEP-5214. No detectable hydrolysis of the ester was observed (data not shown). These results demonstrate that CEP-5214 is produced from CEP-7055 at an insignificant rate during the course of the in vitro assays and indicate that the reported IC₅₀ values reflect the inhibitory activity of CEP-7055.

View larger version (10K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Chemical structure of CEP-7055 and CEP-5214.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Inhibition of VEGF-R2/KDR by (A) CEP-5214 and (B) CEP-7055. Kinase inhibition plots for human VEGF-R2 by CEP-5214 (A) and CEP-7055 (B) were determined in an ELISA-based format in a 96-well FluoroNUNC MaxiSorp plate with a time-resolved fluorescence readout as detailed in "Materials and Methods." Recombinant human baculovirus-expressed VEGF-R2 cytoplasmic domain was prephosphorylated in the presence of 30 µM ATP and 10 mM MnCl2 for 1 h at 4°C before use and was added to the assay at a final concentration of 150 ng/ml.

Effects of CEP-5214 on Angiogenesis in in Vitro and ex Vivo Bioassays
On the basis of its potent and selective inhibition of the VEGF-R kinases and pronounced inhibition of VEGF-R2 phosphorylation in human and rodent endothelial cells, the activity of CEP-5214 was evaluated in in vitro and ex vivo bioassays of angiogenesis. The rat aortic ring explant model in three-dimensional collagen gel matrices and the HUVEC capillary tube formation assay on a Matrigel synthetic basement membrane matrix are two widely used ex vivo and in vitro systems to model effectively the distinct temporal and spatial events underlying angiogenesis in vivo (52, 53) . Both assays are sensitive to the angiogenic effects of VEGF and to the antiangiogenic effects of inhibitors of the VEGF-R, VEGF-R2/KDR (35 , 57 , 57) . CEP-5214 displayed statistically significant dose-related inhibition of complete HUVEC capillary tube formation on a Matrigel synthetic basement membrane matrix in the absence of apparent endothelial cell cytotoxicity 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, respectively (Fig. 3) . The antiangiogenic response to CEP-5214 was further evaluated ex vivo in rat aortic ring explant cultures over a 19-day time course in the absence of exogenous VEGF stimulation. Dose-related inhibitory effects on microvessel growth were observed at 4, 20, 40, 100, and 400 nM of CEP-5214 (Fig. 4) . Statistically significant effects were observed at 40 nM CEP-5214 at days 7 and 11 with a 65% (P < 0.01) and 53% (P < 0.05) inhibition of microvessel growth, respectively, in the absence of apparent cytotoxicity to endothelial cells, fibroblasts, and pericytes in these primary aortic ring explants. Stimulation of rat aortic explant cultures with murine VEGF resulted in a more robust angiogenic response relative to unstimulated cultures, but the growth kinetics of aortic ring explants were comparable with that observed in the absence of exogenous VEGF stimulation. Under these experimental conditions, the administration of CEP-5214 resulted in significant (P < 0.05) inhibition of microvessel growth at 40 nM, relative to control cultures at day 7 during the peak of microvessel growth.

View larger version (94K): [in this window] [in a new window] [Download PPT slide]   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 of HUVECs (passage 5) after 18-h incubation on Matrigel basement membrane. The final DMSO concentration in the compound-treated and -untreated wells was 0.02%. HUVECs were aspirated from media and were fixed and stained using a modified Wright-Giemsa staining protocol as described in "Materials and Methods." Complete capillary tube networks within a designated area of a low-magnification (x10) field were counted under light microscopy, and the data were expressed as percentage inhibition of complete capillary tube formation relative to untreated HUVEC control cultures incubated under the same conditions. Photo micrographs (x100) of HUVEC capillary tube formation in vehicle-treated cultures (top panel) and cultures incubated with 100 nM CEP-5214 (bottom panel).

View larger version (44K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Effects of CEP-5214 on microvessel growth in primary rat aortic ring collagen gel explant cultures ex vivo. A, dose-related antiangiogenic effects of increasing concentrations of the pan-VEGF-R kinase inhibitor CEP-5214 in serum-free collagen gel cultures of rat aortic ring explants maintained in serum-free MCDB 131 medium in the absence of exogenous VEGF. Values are mean + SE of microvessel outgrowths, n = 6 replicates total per time point from three experiments. The final DMSO concentration in the CEP-5214-treated and -untreated explant cultures was 0.02%; medium was replenished every day. *, P 0.05; **, P = 0.01; ***, P 0.01, relative to untreated (DMSO in serum-free medium) controls by Student-Newman-Keuls method. B, photomicrographs (x100) of rat aortic ring explants at day 8 during the peak phase of microvessel sprouting. Untreated cultures (top panel) and cultures treated with 100 nM CEP-5214 (bottom panel).

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   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-µl volume). Six time points were collected using four mice per time point, and plasma samples were analyzed by LCMSMS. Data shown is mean ± S.E.M. values.

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 or CEP-5214.

Effects of CEP-7055 Administration p.o. in in Vivo Angiogenesis Models
Efficacy of p.o. CEP-7055 Administration in the PAEC-VEGF/bFGF-Matrigel Implant Model in Nude Mice.
To determine whether p.o. administration of CEP-7055 could inhibit VEGF-mediated angiogenic responses in vivo independent of its potential antitumor activity, we conducted a series of studies in models of normal and pathological 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 in athymic 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 a dose-related inhibition of neovascularization in the absence of apparent toxicity or morbidity, with an 82% inhibition (P < 0.01) relative to vehicle-treated control mice, with a MED for significant antiangiogenic activity of 7 mg/kg/dose of CEP-7055 p.o. b.i.d. These in vivo findings provided additional corroboration of in vitro and ex vivo data demonstrating potent and significant p.o. antiangiogenic activity of CEP-7055 on the host vasculature.

View larger version (56K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. Dose-related effect of p.o. administration of CEP-7055 on angiogenesis in the PAEC-VEGF/bFGF-Matrigel implant model in nude mice. Female nude mice (n = 10/group) were implanted s.c. bilaterally with Matrigel pellets (100-µl volume) containing PAECs 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 was determined colorimetrically (Drabkin method) as detailed in "Materials and Methods." **, P 0.01, relative to vehicle-treated control mice. The minimum effective dose for inhibition of neovascularization in vivo is 7.1 mg/kg/dose p.o. b.i.d. B, the reduction in neovascularization (hemoglobin content) in the implants from mice given CEP-7055 was confirmed histologically in 0.25% glutarylaldehyde-fixed implants stained with H&E and Trichrome-Masson to evaluate vascular morphology. Histological section of Matrigel implant from vehicle-treated mouse (top panel) and CEP-7055 (11.9 mg/kg/dose p.o. b.i.d.)-treated mouse (bottom panel).

Effects of p.o. CEP-7055 Administration on VEGF-induced Plasma Extravasation in Rats.
The vascular permeability activity of VEGF is one of its characteristic functions that distinguishes it from a number of other angiogenic cytokines (6 , 7 , 14) . To further confirm that p.o. administration of CEP-7055 directly inhibits VEGF-R2/KDR-dependent events in the vasculature, we assessed its ability to attenuate VEGF-induced vascular permeability in rat skin using a modification of the Miles/Evan’s Blue assay. The administration of CEP-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 plasma extravasation (as measured by Evan’s Blue dye release) in the skin of male 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) . Basal plasma extravasation (0.9% saline solution without VEGF) was not effected by CEP-7055 administration at any time point evaluated. Similarly, p.o. administration of CEP-7055 resulted in a significant (P < 0.05 or greater) dose-related inhibition of VEGF (3 ng)-induced plasma extravasation in rats when administered 1 h before measurement of local plasma extravasation, with an ED₅₀ of 20 mg/kg/dose CEP-7055. Basal plasma extravasation (0.9% saline solution without VEGF) was not effected by CEP-7055 administration at any dose evaluated.

Collectively, these in vivo data in three distinct angiogenesis models demonstrate that p.o. administration of CEP-7055 exhibits potent and significant dose-related inhibition of VEGF-mediated biological processes involving various components of normal and pathological angiogenesis in vivo.

Antitumor Efficacy of Chronic p.o. CEP-7055 Administration on the Growth of Human and Murine s.c. Xenografts in Nude Mice and in the Inhibition of Tumor-associated Angiogenesis.
The antitumor efficacy and tolerability of chronic p.o. CEP-7055 administration were investigated in a therapeutic context against a series of established human and rodent tumor xenografts implanted s.c. in athymic nude or syngeneic mice. These data are summarized in Table 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 growth inhibition of multiple human and rodent s.c. tumor xenografts ranging from 50–90%, relative to vehicle-treated control mice at doses of 11.9–23.8 mg/kg/dose in 9 of the 10 s.c. tumor xenograft models evaluated, including CALU-6 lung carcinomas; HCT-116, HT-29, and COLO 205 colon carcinomas; U251MG glioblastomas; and MCF-7 breast carcinoma xenografts (Table 2) . The MED for significant antitumor efficacy was observed at 3.57 mg/kg/dose in several s.c. human tumor models, including the A375 melanoma, U87MG glioblastoma, and ASPC-1 pancreatic ductal carcinoma, with partial tumor regressions observed in the U87MG model at doses of CEP-7055 at 11.9 mg/kg/dose b.i.d. (Fig. 8) . The plasma concentrations (C_max) of CEP-5214 associated with antitumor efficacy in these tumor models (and those described below) in normal or immunocompromised mice were in the range of 50–200 nM.

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Fig. 8. Dose-related effects of chronic p.o. administration of CEP-7055 on the growth of select established human s.c. tumor xenografts in athymic nude mice. Tumor xenografts were established after the s.c. implantation of tumor cells in Matrigel (1:1, v/v) into the flank of female athymic nude mice. On tumors achieving volumes of 80–180 mm3, tumor-bearing mice were randomized into treatment groups (n = 10/group) and p.o. b.i.d. (BID) administration of CEP-7055 was initiated at the doses shown in a vehicle of 1% aqueous acetic acid (100 µl/dose) for the period of time show. Tumor volumes were normalized to individual tumor volumes (relative tumor volumes ± SE) at day 1, the initiation of dosing as detailed in "Materials and Methods." Statistical analyses of tumor data were done using the Mann-Whitney rank-sum test, or when appropriate 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 and sustained inhibition of tumor growth was observed at doses of 3.57 mg/kg/dose or greater in A375 and U87MG tumors, and 1.19 mg/kg/dose or greater in ASPC-1 tumors as detailed in "Results" and "Discussion." Partial tumor regressions were observed in U87MG tumors at p.o. doses greater than 11.9 mg/kg/dose b.i.d.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   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 varying initial volumes. Female nude mice bearing established s.c. ASPC-1 human pancreatic ductal carcinoma xenografts were subdivided into groups (n = 10 mice/group) with different mean tumor volumes (150-mm3, 400-mm3, and 900-mm3 absolute volumes) as tumor xenograft growth progressed and administered CEP-7055 p.o. b.i.d. at 11.9 mg/kg/dose for the durations of time indicated. Arrows, the initiation of CEP-7055 administration. Statistical analyses are detailed in "Results" and "Discussion." CE-7055 administration inhibited ASPC-1 xenograft growth significantly relative to volume-matched controls independently of initial tumor xenograft volume. Values shown are mean ± S.E.M.

Effects of CEP-7055 on IMD in Vivo and Tumor Cell Proliferation in Vitro.
IMD was evaluated immunohistochemically as an indirect assessment of effects on tumor angiogenesis in several human tumor xenografts from CEP-7055-treated mice and compared with the IMD from corresponding vehicle-treated mice. The percentage reduction in IMD (as assessed by Factor VIII and CD34 immunostaining) in CEP-7055-treated A375 melanoma and U87MG glioblastoma xenografts after 30 days of p.o. administration relative to oral-vehicle-treated control mice was 20–22% (P < 0.05). Chronic CEP-7055 administration also resulted in a 36% reduction (P < 0.05) in both the mean IMD (as assessed by CD34 immunostaining) and the mean microvessel density per mm² in pancreatic tumor xenografts relative to vehicle-treated tumors, and a 23% reduction in CD34 immunostaining in orthotopic RENCA tumor pulmonary metastases (data not shown).

Further evidence to suggest that the antitumor efficacy of CEP-7055 is attributable predominantly to its inhibition of VEGF-mediated angiogenic processes was the absence of antiproliferative or proapoptotic activity of the active moiety, CEP-5214, on tumor cells directly in vitro. CEP-5214 had no effect on the proliferation or viability of these tumor cell lines at concentrations up to 3 µM. These findings lend additional support to the absence of direct antitumor activity of this compound at concentrations demonstrating significant and sustained antitumor and antiangiogenic efficacy in vivo.

Effects of p.o. Administration of CEP-7055 on Primary Tumor Growth and Metastatic Profile in Orthotopic Models of Human Prostate Carcinoma and Murine RENCA.
The antitumor efficacy of CEP-7055 administration on local and distant metastatic tumor growth was also examined in more clinically relevant orthotopic tumor models of hormone-sensitive human prostate carcinoma (in athymic nude mice) and murine RENCA in BALB/c syngeneic mice. In the LNCaP orthotopic prostate carcinoma model, treatment was initiated 1 day after orthotopic implantation of LNCaP cells in male athymic nude mice. Administration of CEP-7055 or vehicle was performed for 21 days, at which time animals were euthanized, and orthotopically grown prostate tumors were evaluated. The take rate for the prostate tumors was 100% (10 of 10 mice) in the vehicle-treated control group. Administration p.o. of CEP-7055 had a significant effect on the growth (tumor wet weight) of orthotopically implanted LNCaP 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 days after recovery from surgery to implant RENCA cells beneath the renal capsule, groups of mice were randomized into treatment groups and received 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 µl/dose). The administration of CEP-7055 for 21–26 days had no significant effect on the weight of primary orthotopic tumor-bearing kidneys, 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 overall metastatic burden (pulmonary and lymph node metastases) in the RENCA model, as manifested in both the severity of metastatic scores obtained and the weights of lung tissue with metastatic nodules (Fig. 10) , with values approaching those observed in normal, non-RENCA-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. Administration p.o. of CEP-7055 resulted in a larger percentage of mice having a lower overall metastatic score from their orthotopically implanted RENCA, relative to control mice, but did not result in completely eliminating a high metastatic burden in all of the tumor-bearing animals. These results obtained with p.o. CEP-7055 at 23.8 mg/kg/dose p.o. b.i.d. surpassed those obtained using a clinically relevant dosing regimen of recombinant human IL- 2 (Proleukin) in this model (Fig. 10, B and C) .

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Fig. 10. Effects of p.o. administration of CEP-7055 on the growth of primary and metastatic lesions from orthotopically implanted murine RENCA xenografts. Effects of p.o. CEP-7055 administration at 3.57 and 23.8 mg/kg/dose b.i.d. on pulmonary metastases (wet weights); and p.o. CEP-7055 [23.8 mg/kg/dose b.i.d. (BID) versus recombinant human (rh)IL-2 (Proleukin) on pulmonary metastases (B) and total metastatic score (C) as defined in "Materials and Methods:" in the murine RENCA model. Data with rhIL-2 shown as a reference for effects of treatment on metastatic score. Treatment was initiated 2 days after implantation of RENCA cells into the subcapsular space of syngeneic Balb/c mice (n = 10/group) and continued for 26 days total. Lung weights of normal, non-RENCA implanted mouse lungs are shown for a reference (A). *, P < 0.05, **, P < 0.01.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 significant and dose-related inhibition of VEGF-mediated neovascularization (PAEC-VEGF/bFGF-Matrigel implants) by up to 82%, relative to controls, and inhibits significantly both vascularity and granuloma formation in a murine model of chronic inflammation-induced angiogenesis, a process in which VEGF has been demonstrated to play a salient role (61 , 67, 68, 69) . In addition to the activity of CEP-7055 in inhibiting VEGF-mediated neovascularization and inflammation-induced angiogenesis, p.o. administration of CEP-7055 results in significant and sustained (>6 h) dose-related inhibition of VEGF-induced dermal vascular permeability in rats (Evan’s dye assay) with an ED₅₀ of 20 mg/kg/dose. Earlier studies demonstrated that acute and chronic p.o. administration of CEP-7055 to rats inhibited intravitreal VEGF-induced retinal vascular permeability and leakage significantly (62 and 52%, respectively), and completely inhibited diabetes-induced retinal vascular permeability, although having no effects on basal vascular permeability (70) . Collectively, these data indicate that p.o. administration of CEP-7055 at doses of 11.9–23.8 mg/kg/dose b.i.d. inhibits VEGF-mediated signaling and angiogenesis directly in vivo under both physiological and pathological conditions in the absence 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 3.57–23.8 mg/kg/dose b.i.d. results in dose-related inhibition of s.c. tumor xenograft growth ranging from 50–90%, relative to vehicle-treated mice against a broad range of human and rodent tumors varying in their histological origin, latency and growth rate, and responsiveness to conventional cytotoxic agents. There are a number of influences that may account in part for differences in the range and magnitude of antitumor responses observed on chronic CEP-7055 administration. These influences include the distinct growth profiles of the varied tumor xenografts examined; the role of the tumor microenvironment, i.e., the extent of localized hypoxia and ischemia within a given tumor xenograft, over the time course of treatment on tumor response; the varied dependency of particular tumor types on VEGFs versus other angiogenic cytokines at specific stages of their vascular growth and maturation; and tumor-specific differences in the temporal expression and/or activation of key intracellular mediators of VEGF-induced angiogenesis, e.g., AKT/phosphatidylinositol 3'-kinase, p44/42 mitogen-activated protein kinase, specific integrins and adherens (70, 71, 72, 73, 74, 75) . This tumor inhibition profile was observed in both immunocompromised and immunocompetent mice when given CEP-7055 for periods ranging from 10 to 65 days. In several distinct tumor xenograft models, including the A375 human melanoma, U87MG human glioblastoma, ASPC-1 pancreatic carcinoma, and SVR murine angiosarcoma, the minimum effective p.o. dose for sustained and significant antitumor efficacy was 3.57 mg/kg/dose b.i.d. In the case of the U87MG 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 tumor regressions (50–90% reduction of initial tumor volume). Partial or complete tumor regressions in select tumor xenograft models have been observed with monotherapy administration of several other VEGF-R kinase inhibitors, including PTK-787 in CWR-22 prostate carcinomas (35) , ZD6474 in PC-3 prostate carcinomas (41) , and SU11248 (a VEGF-R2/PDGF-RB kinase inhibitor), in the A431 epidermoid and COLO 205 colon carcinoma models (42) . Of note is the fact that the administration of CEP-7055 resulted in significant and sustained tumor growth inhibition in ASPC-1 pancreatic carcinoma xenografts, independent of initial tumor volumes, even in tumors of 1.0 cm³ . Similar findings have been reported with ZD6474 in established CALU-6 lung tumor xenografts (40 , 41) and could have significant implications for the clinical administration of these antitumor agents.

The extent to which the weaker inhibitory activities of CEP-5214 or its prodrug, CEP-7055, against c-Kit, FGF-R1 kinase and PDGF-Rß (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 evaluation, possess one or more of these activities (34 , 42 , 44 , 76, 77, 78) .

The antitumor efficacy of chronic p.o. administration of CEP-7055 was not confined to s.c. tumor xenografted tumors but extended to orthotopically implanted human prostatic carcinomas (60) and murine RENCA xenografts in both immunocompetent and immunocompromised mice. This renal carcinoma model has been reported to be sensitive to the administration of several antiangiogenic agents, including TNP-470 (64) and, in particular, PTK-787 (35, 36, 37) . Curiously, although CEP-7055 inhibited the extent of metastases in the RENCA model, as well as the growth of s.c. implanted RENCA tumors, marginal antitumor activity was observed against primary RENCA tumor growth within the renal capsule. Similar findings for more pronounced activity of antiangiogenic agents (e.g., PTK-787) against metastases versus primary tumor growth in the RENCA model have been reported (35) . The homogeneous and extensive vascularization throughout these tumors suggests that, in rapidly growing RENCA tumors, the tumor cell proliferation rate is superior to that of tumor-associated endothelial cells and results in partial angiogenesis-independent tumor growth (36) . This fact, and the proximity of orthotopically implanted RENCA tumors to an abundant vascular supply in the kidney, could account for the marginal effect of CEP-7055 on primary RENCA tumor growth, despite its significant effects on the seeding and subsequent growth of distant lung metastases and on the inhibiting of s.c. implanted RENCA xenograft growth, processes that would be predicted to be more angiogenesis-dependent. An additional explanation for the significant antimetastatic efficacy of CEP-7055 in the murine RENCA model may be the potent inhibitory activity of CEP-5214 against VEGF-R3/FLT-4, the VEGF receptor subtype implicated in the induction of tumor lymphangiogenesis and lymphatic metastases in multiple solid tumor types (18, 19, 20, 21) .

The magnitude of human and rodent tumor xenograft growth inhibition observed with chronic CEP-7055 administration across multiple organ-specific tumor types observed at total daily p.o. doses of 23.8–47.6 mg/kg/dose is comparable with, or lower than, that observed with chronic p.o. administration of several VEGF-R kinase inhibitors currently under clinical evaluation. Several orally active VEGF-R kinase inhibitors have demonstrated significant and comparable antitumor efficacy preclinically in s.c. tumor xenografts at effective total daily p.o. doses of 50–100 mg/kg/dose (PTK-787; Refs. 35, 36, 37, 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 observed reductions in IMD (20–36% relative to control tumors depending on tumor type) on its chronic administration in several tumor xenograft models, and the significant in vivo efficacy observed with chronic CEP-7055 administration in a variety of VEGF-mediated angiogenesis models described in this report, are consistent with primarily indirect, i.e., antiangiogenesis-related, antitumor effects of this pan VEGF-R kinase inhibitor rather than with a direct proapoptotic effect on individual tumor cells per se. In support of this conclusion, is the fact that 3–10-µM concentrations of CEP-5214 were required to achieve significant antiproliferative or proapoptotic effects against a panel of human tumor cell lines in vitro, significantly higher than the plasma concentrations (50–200 nM) at which in vivo efficacy has been observed with CEP-7055 administration in preclinical murine models.

In summary, we have identified and are developing CEP-7055, the prodrug of CEP-5214, an orally active, low-nanomolar pan VEGF-R kinase inhibitor that demonstrates significant and durable antiangiogenic efficacy in vitro, ex vivo, and in vivo, and antitumor efficacy against s.c. and orthotopically implanted human and rodent tumors in vivo. The pharmacological, pharmacokinetic, and tolerability profile of this agent in preclinical studies is compatible with chronic administration against a variety of disease states in which VEGF-mediated angiogenesis plays a salient role. CEP-7055 is currently in Phase I clinical trials as an orally administered therapy in patients with a variety of solid tumors.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ The pharmacological development of CEP-7055 is being pursued in the context of a partnership 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: bruggeri{at}cephalon.com

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 factor receptor; PLC-, phospholipase C-; GST, glutathione S-transferase; RENCA, (murine) renal cell carcinoma (model); IMD, intratumoral microvessel density; MED, minimum effective dose; TCA, trichloroacetic acid; PAEC, porcine aortic endothelial cell; IL, interleukin.

Received 2/14/03. Revised 6/ 3/03. Accepted 6/30/03.

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