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Cancer Drug Discovery, Pfizer Global Research and Development, Groton Connecticut 06340
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The in-growth of host microvasculature into a solid tumor, referred to as angiogenesis, is now widely appreciated to be a necessary event for tumor growth beyond a few mm3 in volume (1 , 2) . Therefore, it is expected that drugs that block the molecular events responsible for tumor angiogenesis will be effective against a broad spectrum of tumor types. Angiogenesis is a highly regulated process, and although essential for embryogenesis, this process is restricted in adults to ovulation, cyclical endometrial proliferation, and wound repair (3) . Therefore, inhibitors of angiogenesis are predicted to be better tolerated than conventional cytotoxic cancer therapies that affect all rapidly growing cells. Because the target is the invading normal host vasculature, another potential attribute of an antiangiogenesis approach may be the avoidance of drug resistance commonly associated with conventional anticancer modalities, which target genetically unstable tumor cells (4) . Moreover, with p.o. or i.v. delivery, neovascular endothelial cells will be the first cell types encountered by extravasating drugs, providing the possibility that tumor penetration by these agents may not be necessary. Angiogenesis is the hallmark of many vascular proliferative disorders other than cancer, including psoriasis, rheumatoid arthritis, diabetic retinopathy, and age-related macular degeneration (5, 6, 7) . Therefore, it is reasonable to expect that well-tolerated antiangiogenesis agents will be efficacious in these disease states as well.
Angiogenesis is a complex biological process that relies on a variety of growth factors and signaling cascades to stimulate the migration and proliferation of the component cell types and to establish functional blood vessels. Many factors have been shown to affect endothelial proliferation resulting in angiogenesis in vivo, such as angiopoietins, FGFs4 (especially bFGF, PDGF, platelet-derived endothelial cell growth factor, EGF, angiogenin, and VEGF; Refs. 8, 9, 10, 11, 12, 13 ). Of these factors, research has focused on VEGF, attributable in part to its endothelial cell specificity, and its temporal and spatial expression at times of physiological and pathological blood vessel growth (14) . VEGF was originally identified as a tumor-secreted vascular permeability factor (15) , subsequently shown to be secreted by many other cell types, including macrophages and folliculostellate cells (16) . VEGF is regulated by many physiological conditions commonly associated with tumors and ischemic tissues, such as hypoxia and hypoglycemia (17) . There are two high-affinity receptors for VEGF that appear to be exclusively expressed and functional on endothelial cells, fms-like TK receptor 1 (also VEGFR-1) and Flk-1 (also kinase domain receptor or VEGFR-2; Refs. 18 , 19 ). Like VEGF, the expression of these receptors is spatially and temporally regulated on endothelium in times of vasculogenesis and angiogenesis, and is up-regulated in response to hypoxia and hypoglycemia (20 , 21) . The binding of VEGF to its receptors induces receptor dimerization and subsequent activation of the TK domains; these then serve as docking sites for a wide variety of molecules involved in the propagation of the signaling cascade (12 , 14) .
A large body of evidence suggests that blocking VEGF activity will result in the inhibition of tumor growth in murine models. An antibody against VEGF that interrupts VEGF signaling (Bevacizumab; Genentech) has proven to be effective in inhibiting tumor growth and is currently being assessed in clinical trials (22 , 23) . Other biological approaches have shown promise in preclinical models as well, such as ribozymes and anti-VEGF receptor antibodies (24 , 25) . Several reports using small molecule receptor TK inhibitors as single agents have also described inhibition of VEGFR-2 autophosphorylation that translated to an impressive inhibition of VEGF-induced angiogenesis and tumor growth in multiple murine models (26 , 27) . Interestingly, when these anti-VEGF or anti-VEGFR approaches are used in combination with either ionizing radiation or conventional chemotherapeutics in preclinical models, the antitumor responses are often times better than either approach alone, without added toxicity (28 , 29) . Thus, disrupting VEGFR-2 signaling is an attractive target to prevent tumor angiogenesis, leading to the inhibition of tumor growth and metastasis. A few of these agents have made the transition from preclinical testing to evaluation in human clinical trials.5
This article provides data characterizing a small molecule VEGFR inhibitor suitable for p.o. administration. CP-547,632 is an ATP-competitive kinase inhibitor that blocks VEGFR-2 kinase autophosphorylation (IC50 = 11 nM) and VEGF-induced VEGFR-2 phosphorylation in VEGFR-2-transfected endothelial cells (IC50 = 6 nM). In vivo, CP-547,632 inhibits tumor-associated VEGFR-2 phosphorylation resulting in decreased microvascular density and significant tumor growth inhibition in a number of tumor models. This compound is a well-tolerated, potent inhibitor of VEGFR-2 currently in clinical trials for the treatment of cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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. The methanesulfonic acid salt form of this compound was used for the studies reported here.
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Cell-Based Phospho-VEGFR-2 Inhibition Assay.
Porcine aortic endothelial cells stably expressing full-length VEGFR-2 have been described previously (30)
. Cells were seeded at 1.6 x 105
cells/ml in 2-ml growth medium (Ham’s F-12 medium supplemented with 10% FBS, 50,000 units each penicillin and streptomycin, and 500 µg/ml gentamicin) per well in six-well plates. On day 2, the growth medium was replaced with serum-depleted medium (as above, but with 0.1% FBS and 0.1% BSA), and cells were incubated overnight. Immediately before compound addition, the medium was replaced with serum-depleted medium without BSA. Compounds were diluted in 100% DMSO, added to the cells at a final DMSO concentration of 0.25% v/v, and incubated at 37°C for 1 h. The cells were then stimulated with 500 ng/ml VEGF (Becton Dickinson, prepared in serum-depleted medium supplemented with 10 mM NaVO4) and incubated as above for an additional 8 min. The medium was removed and the cells washed once with PBS supplemented with 1 mM NaVO4, then lysed with 1 ml of immunoprecipitation assay buffer [10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 25 mM EDTA, 1% NP40, 0.25% sodium deoxycholate, 2 mM NaVO4, and 1 EDTA-free complete protease inhibitor tablet per 25 ml]. Cell lysates were centrifuged at 14,000 rpm to pellet cellular debris, transferred to a new tube containing 4 µg anti-Flk-1 (Santa Cruz Biotechnology Laboratories; C20), and incubated with agitation overnight at 4°C. The antibody-protein complex was captured with protein A agarose beads (Santa Cruz Biotechnology Laboratories) for 30 min at 4°C and the protein boiled off in the presence of DTT. After electrophoresis and transfer to Immobilon-P membranes, the blots were probed with antibodies recognizing either the protein (monoclonal anti-Flk-1; Santa Cruz Biotechnology Laboratories; A3) or anti-PY-HRP. After incubation of the blot in enhanced chemiluminescence reagent (Amersham), bands were visualized on film or using the Lumi-ImagerF1 (Roche).
HUVEC Mitogenesis.
HUVECs were obtained from Clonetics and grown according to the manufacturer’s instructions. Cells (104
cells/ml) were seeded on Collagen Type I coated 24-well plates in 1 ml of growth medium [Endothelial Growth Media (EGM) with BulletKit; CC-3124]. After 24 h the growth medium was replaced with 1 ml of serum-free medium. Compounds were added 24 h later (diluted in DMSO to a final concentration of 0.2% v/v). Cells were then stimulated with VEGF or FGF (10 ng/well), diluted in HBSS. After 36 h, 1 µCi [3
H]thymidine diluted in HBSS was added to each well and cells allowed to grow an additional 3 h. The cells were then rinsed twice with ice-cold HBSS, and proteins were precipitated in two washes with cold 10% trichloroacetic acid (TCA). Protein was solubilized with 0.1 N NaOH, and an equal volume 0.1 N HCl was used to neutralize the base. Cell mitogenesis was determined by the incorporation of [3
H]thymidine in the cells detected in a scintillation counter.
Animals for in Vivo Studies.
Athymic female mice (CD-1 nu/nu, 
20 grams) were used for all of the in vivo tumor growth inhibition studies. Mice were obtained from Charles River Laboratories (Wilmington, MA) and housed in specific pathogen-free conditions, according to the guidelines of the Association for the Assessment and Accreditation for Laboratory Animal Care, International. Sprague Dawley rats [Crl:CD(SD)BR, 150–175 g], also obtained from Charles River, were used to evaluate angiogenesis inhibition in long bones. All of the in vivo studies were carried out under approved institutional experimental animal care and use protocols.
Ex Vivo ELISA.
Female athymic mice were injected with 1 x 106 NIH-3T3/H-Ras cells (NIH3T3 cells transfected with full-length activated H-ras oncogene) on day 1. When tumors were 300 mm3, the mice received compound or vehicle (5% Gelucire 44/14 in sterile water, Gattefossé, Saint-Priest, France) p.o. One to 3 h after dosing, the tumors were excised and frozen in liquid N2. For the pharmacokinetic/pharmacodynamic analysis, the tumors were removed at the various times postdose. The tumors were homogenized in 1 ml of lysis buffer per 200 mg tumor [lysis buffer: 50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1% Glycerol, 1% Triton X-100, 1.6 mM Na3VO4, 10 mM NaF, 25 mg/liter Soy Bean Trypsin Inhibitor, and EDTA-free complete Protease Inhibitor Tablets]. The tumors were homogenized using a dounce homogenizer mounted on a standing drill press. Homogenates were spun for 5 min at 14,000 rpm, and the supernatant was aliquoted to 96-well polypropylene plates or tubes on dry ice. Total protein concentration was determined using BCA protein assay (Pierce). Protein A coated plates (96-well; Pierce) were blocked with 100 µl/well cold blocking buffer (Tris-buffered saline, 0.1% Tween 20, and 3% BSA) for 60 min on a plate shaker at room temperature. The blocking buffer was replaced with 0.5 µg anti-Flk-1 in 100 µl cold blocking buffer per well and incubated for 60 min, room temperature, on a plate shaker. The plates were washed with Tris-buffered saline and 0.1% Tween 20 using a Skatron Plate washer. Tumor lysate (100 µl diluted to 5 mg/ml total protein in lysis buffer without protease inhibitors) was added to the plate and incubated for 3 h, room temperature, on a plate shaker. The plates were washed as above, then incubated with 15 ng of HRP-PY54 per well (in blocking buffer) for 30 min at room temperature. The plates were washed as above and PY quantitated using TMB as described above.
Plate IP and Western Blot Analysis.
Tumor lysates were incubated on protein A plates as described for the ex vivo ELISA. The plates were then washed as above. We added 45 µl 3x Laemmli loading buffer to the wells, and the plate was placed on a plate shaker for 5 min with vigorous shaking. The plate was heated 8 x 30 s in a microwave oven. After each 30-s heating the plate was removed and tapped gently to cover the wells with the loading buffer. Complete heating was determined by the color change in the loading buffer. The plate was then shaken vigorously on a plate shaker for 5 min. The samples were loaded onto Novex NuPAGE 4–12% Bis-Tris Gels, and electrophoresis was carried out with 3-[N-morpholino]propanesulfonic acid buffer running buffer with antioxidant added to the inner chamber. Proteins were transferred to polyvinylidene fluoride membranes in Tris-Glycine Transfer Buffer with 10% methanol. PY levels were detected using anti-PY-HRP diluted in blocking buffer. VEGFR-2 levels were detected using anti-VEGFR-2 antibody followed by antirabbit HRP reporter antibody. Enhanced chemiluminescence (Amersham) was used according to the manufacturer’s instructions. The VEGFR-2 band was quantitated using the MasterScan Densitometer.
Sponge Angiogenesis Assay.
Surgical sponges were cut into 5 x 5 mm squares and soaked with 1 µg of either bFGF or VEGF (Becton Dickinson) per sponge in Matrigel. One of each sponge was implanted s.c. bilaterally 1 cm from the central incision on the abdomen of female athymic mice. Three days after surgical implantation, animals received qd oral doses of 100 mg/kg CP-547,632. After 5 days, animals were sacrificed, and sponges were removed. Sponges were ground in 0.1 ml ddH2O, samples were centrifuged at 15,000 x g for 5 min, and hemoglobin content was quantitated by adding 50 µl TMB to 50 µl of the supernatant. The reaction was stopped as described above and absorbance read at 450 nM. Inhibition was calculated as a percentage of the angiogenesis for each growth factor present in the vehicle-treated sponges.
Tumor Growth Inhibition Studies.
NIH3T3/Hras cells were cultured in DMEM with 10% FBS, and penicillin and streptomycin, in the presence of 0.4 mg/ml G418 (Life Technologies, Inc.). Colo-205 and DLD-1 human colon adenocarcinoma, and MDA-MB-231 breast carcinoma cells were obtained from American Type Culture Collection (Rockville, MD). All of the human cancer cells were propagated by standard tissue culture procedures in the medium suggested by American Type Culture Collection. Exponentially growing cells were trypsinized and resuspended in sterile PBS, and inoculated s.c. (3T3/Hras at 1 x 106 cells/mouse and human tumor cells at 5 x 106 cells/mouse in 200 µl) into the right flank of mice. Animals bearing tumors of approximately 75–150 mm3
in size were divided into groups receiving either vehicle (5% Gelucire) or CP-547,632 (diluted in vehicle) and dosed by oral gavage. Animal body weight and tumor measurements were obtained every 2–5 days. Tumor volume (mm3
) was measured with Vernier calipers and calculated using the formula: length (mm) x width (mm) x width (mm) x 0.5. Percentage of growth of an individual tumor was calculated using the following formula: % growth = [(tumor volume (mm3
) on final day - tumor volume of day 1) x 100]/tumor volume on day 1. The percentage of growth inhibition was calculated using the following formula: % growth inhibition = {100 - [(% growth of treated/% growth of control) x 100]}.
Microscopy and Immunohistochemistry of Tumor Microvasculature.
At the end of the tumor efficacy studies, tumors were isolated and fixed in 4% paraformaldehyde at 4°C for 18–24 h, then processed into paraffin, sectioned at 4 µm, and immunostained for CD31 using an avidin-biotin-peroxidase technique. Tissue sections were counterstained with Meyer’s hematoxylin and examined using an Olympus BX40 microscope. All discreet, positively stained vascular profiles, with or without lumina, were counted in 10 x200 fields from one or two sections of each tumor. Where possible, fields were chosen in areas of highest MVD. Stromal vessels were included in the count, but capsular and preexisting vessels were excluded. Data were analyzed using simple linear regression to statistically relate microvessel counts to dose of CP-547,632.
Microscopic Evaluation of Angiogenesis Inhibition in Long Bones.
Rats were dosed daily with CP-547,632 and at various postdose intervals, stifle joints were surgically excised, fixed in 10% neutral-buffered formalin, decalcified with formic acid, and processed into paraffin blocks. The tissues were sectioned, stained with H&E, then examined by light microscopy. The proximal tibia were used for semiquantitative assessment of growth plate changes.
Corneal Pellet Angiogenesis Assay.
BALB/c male mice were purchased from Charles River. Erythromycin ophthalmic ointment (Fougera Melville, NY) and 0.5% Proparacaine Hydrochloride Ophthalmic Solution (Bausch & Lomb, Tampa, FL) were used as antibiotic and local anesthetic for the corneas. Microtainer Plasma Separator Tubes (Becton Dickinson) were used to isolate plasma from whole blood. CP-547,632 was dissolved in 5% Gelucire and dosed p.o. VEGF, Hydron NCC (Hydro Med Sciences), sucrose octasulfate-aluminum complex (Sigma), and #3–300/50 Nylon mesh (Tetko) were used to formulate sustained release pellets. Metofane inhalant anesthetic (Schering-Plough Animal Health, Union, NJ) and Nembutal were used for anesthesia. Hydron pellets of <5 µl containing 12.5 ng of VEGF were formulated as described previously (31)
. The pellets were implanted in corneal micropockets of anesthetized mice (Nembutal, 50 mg/kg i.p.) at a distance of 0.5–1 mm from the limbus. Compound was subsequently administered by oral gavage qd. The eyes were examined on day 5 by slit lamp microscopy, and results were photographed. Blood was collected at 2 h after the last oral dose corresponding to the approximate Tmax. The contiguous circumferential zone of neovascularization was measured as clock hour, and inhibition was ranked relative to vessel length and clock hour of corneas in the vehicle-treated group.
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RESULTS AND DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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. This compound is an isothiazole inhibitor of VEGFR-2. Kinetic analyses demonstrate that CP-547,632 is an ATP competitive inhibitor of human VEGFR-2, as increasing amounts of ATP in the reaction were able to restore the kinase Vmax in the presence of compound (Fig. 2)
. The enzymatic selectivity of CP-547,632 was assessed by measuring the inhibitory activity of the compound on other purified recombinant human enzymes. Kinase assays were routinely run at ATP concentrations approximating the measured Km for the respective enzymes. CP-547,632 is approximately equipotent (i.e., <10-fold selective) against VEGFR-2, bFGF receptor, and EGFR/Tie-2 chimera, the receptor for Angiopoietin. However, this compound is 250-1000-fold less potent for platelet-derived growth factor receptor ß, insulin and the EGF receptor TKs (Table 1)
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. This difference may be because of the stronger proliferative stimulus of bFGF on endothelial cells relative to VEGF. It is unclear whether the modest differences in VEGF, bFGF, and Tie-2 enzyme and cell inhibition are pharmacologically relevant in vivo. We have not pursued in vivo inhibition of Tie-2 with CP-547,632.
To confirm that CP-547,632 inhibits receptor signaling in the cell-based assays, inhibition of VEGF-stimulated VEGFR-2 autophosphorylation was measured in porcine aortic endothelial cells transfected with VEGFR-2. After a 1-h incubation with cells, CP-547,632 inhibited VEGF-stimulated VEGFR-2 phosphorylation in a dose-dependent fashion, with an IC50 value of 6 nM (Fig. 3, A and B)
. Concentrations as high as 1 µM did not affect either cell viability or VEGFR-2 protein amounts as measured by MTT assay and Western blotting, respectively, indicating that the compound effect was likely mediated by selective kinase inhibition. Furthermore, the CP-547,632 concentrations required to inhibit VEGF-stimulated signaling (IC50 = 6 nM) are in agreement with concentrations required to inhibit VEGF-stimulated proliferation of HUVEC (IC50 = 14 nM).
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demonstrating that this compound inhibits murine, as well as human VEGFR-2 phosphorylation. A single oral dose of 50 mg/kg yielded plasma concentrations above 500 ng/ml for 12 h. This resulted in inhibition of 50% of measurable VEGFR-2 phosphorylation for 3 h and reductions of at least 30% for up to 12 h. This inhibition corresponded to a Caverage of 590 ng/ml total drug or 24 ng/ml free drug (12 nM), correlating well with the cellular IC50. Taken together, these data suggest a correlation among inhibition of VEGFR-2 enzyme activity, in vitro cellular phosphorylation/proliferation, and in vivo tumor vascular VEGFR-2 phosphorylation.
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. After implantation of the VEGF-hydron pellet, animals received qd doses of CP-547,632 for 5 days. Eyes were examined by slit lamp microscopy, and blood was taken 2 h after the last oral dose. This time point approximates the Cmax after oral delivery of CP-547,632. A dose of 25 mg/kg, corresponding to a plasma concentration of 1067 ng/ml total drug or 42.7 ng/ml free drug (C2 h), resulted in 75% inhibition of VEGF-induced angiogenesis relative to vehicle control. CP-547,632 was able to completely inhibit the VEGF-induced corneal angiogenesis at a dose of 100 mg/kg (1884 ng/ml total or 75 ng/ml free drug, C2 h). These data together with in vitro and in vivo VEGFR-2 phosphorylation inhibition in xenografts confirm the mechanism-based activity of CP-547,632.
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Tumor Growth and MVD Inhibition in Vivo.
The oral antitumor activity of CP-547,632 was additionally examined in several human xenograft models in athymic mice. Mice bearing tumors (75–150 mm3
in size) were randomized into various groups (7–10 animals each) and received CP-547,632 p.o. (6.25–100 mg/kg, qd) for the given number of days. CP-547,632 caused a dose-dependent inhibition of growth in Colo-205, DLD-1, and MDA-MB-231 xenografts (Fig. 6)
. Oral treatment with 100 mg/kg of CP-547,632 for 9 and 12 days resulted in 69% and 85% inhibition of DLD-1 and Colo-205 tumor growth, respectively. Daily oral administration of CP-547,632 at doses of 100 mg/kg for 24 days was well tolerated (no weight loss, morbidity, or deaths) and produced 80% inhibition of MDA-MB-231 tumor growth. Oral antitumor efficacy of CP-547,632 was also observed in animals bearing EBC-1 and H460, human non-small cell lung carcinoma tumors. Approximately 50% tumor growth inhibition was achieved with 100 mg/kg, qd, oral dosing with CP-547,632 (data not shown). In the models described here, administration of CP-547,632 as a single agent did not yield tumor regressions. However, when CP-547,632 was administered in combination with conventional cytotoxic drugs, tumor regressions were observed (data not shown).
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, additional investigations were undertaken to examine whether this compound affected microvascular density in the human tumor models. To this end, DLD-1 and Colo-205 tumors from growth inhibition studies were processed for immunohistochemical detection of endothelium using CD31 Abs (Fig. 7)
. The data demonstrate a statistically significant decrease of 38% in the number of microvessels in vehicle-treated versus drug-treated DLD-1 tumors (100 mg/kg was the dose that resulted in 69% tumor growth inhibition reported above). It should be noted that the modifications to tumor vascular morphology and function in response to VEGFR inhibitors can be complex. Using PTK787, it was found that although vascular density was reduced in tumors, there was an intriguing increase in partial blood volume; and whereas vascular permeability was decreased significantly, there were no measurable changes in extravasation (35)
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. Treatment of rats with CP-547,632 for 30 days at doses 
10 mg/kg produced widening of the growth plate. The hypertrophic zone of the epiphyseal cartilage was affected predominantly, exhibiting up to 10-fold increases in thickness. Similar observations have also been made with ZD-6474, a small molecule VEGFR inhibitor (37)
, as well as with bevacizumab (38)
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In this article, we describe the identification and characterization of a novel TK inhibitor, CP-547,632. This compound is an ATP-competitive and reversible inhibitor of the VEGFR-2 kinase that potently inhibits receptor phosphorylation in vitro and in vivo. Plasma concentrations of this compound that result in 50% inhibition of receptor tyrosine phosphorylation in tumor xenografts achieve 50% inhibition of VEGF-induced angiogenesis in the murine corneal implant model. Additionally, this compound potently inhibits both VEGF- and bFGF-induced angiogenesis. Finally, qd oral administration of this compound was able to significantly inhibit human tumor growth in murine models resulting in significant inhibition of tumor microvascular density in vivo. Taken together, these results demonstrate CP-547,632 to be a potent angiogenesis inhibitor with good oral bioavailability and in vivo antitumor efficacy. CP-547,632 represents an exciting new chemotype for clinical development in the area of VEGFR inhibitors. Clinical testing with this and other inhibitors will be required to relate potency, duration of pharmacokinetic/pharmacodynamic effect, and the spectrum of enzymatic selectivity to the safety and efficacy profile that provides optimal benefit for cancer patients.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This article is dedicated to the memory of Dr. Edward A. Glazer, a member of the Pfizer team and a driving force in the discovery of CP-547,632, who passed away in 2002. 
2 To whom requests for reprints should be addressed, at Pfizer Global Research and Development, Eastern Point Road, MS 8118-209, Groton, CT 06340. Phone: (860) 441-8478; Fax: (860) 686-2382; E-mail: w_gregory_roberts{at}groton.pfizer.com
3 Internet address: http://www.cancer.org.
4 The abbreviations used are: FGF, fibroblast growth factor; bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; FBS, fetal bovine serum; Flk-1, fetal liver kinase receptor 1 (murine vascular endothelial growth factor receptor 2); HRP, horseradish peroxidase; HUVEC, human umbilical vein endothelial cell; MVD, microvessel density; PY, phosphotyrosine; TK, tyrosine kinase; TMB, 3,3',5,5', tetramethylbenzidine dihydrochloride; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; IP, immunoprecipitation; FGFR; qd, once daily.
5 Internet address: http://www.cancer.gov/clinicaltrials.
Received 2/ 5/03. Revised 6/ 6/03. Accepted 8/15/03.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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