CP-673,451 is a potent inhibitor of platelet-derived growth factor β-receptor (PDGFR-β) kinase- and PDGF-BB-stimulated autophosphorylation of PDGFR-β in cells (IC50 = 1 nmol/L) being more than 450-fold selective for PDGFR-β versus other angiogenic receptors (e.g., vascular endothelial growth factor receptor 2, TIE-2, and fibroblast growth factor receptor 2). Multiple models have been used to evaluate in vivo activity of CP-673,451 and to understand the pharmacology of PDGFR-β inhibition and the effect on tumor growth. These models include an ex vivo measure of PDGFR-β phosphorylation in glioblastoma tumors, a sponge model to measure inhibition of angiogenesis, and multiple models of tumor growth inhibition. Inhibition of PDGFR-β phosphorylation in tumors correlates with plasma and tumor levels of CP-673,451. A dose of 33 mg/kg was adequate to provide >50% inhibition of receptor for 4 hours corresponding to an EC50 of 120 ng/mL in plasma at Cmax. In a sponge angiogenesis model, CP-673,451 inhibited 70% of PDGF-BB-stimulated angiogenesis at a dose of 3 mg/kg (q.d. × 5, p.o., corresponding to 5.5 ng/mL at Cmax). The compound did not inhibit vascular endothelial growth factor- or basic fibroblast growth factor-induced angiogenesis at concentrations which inhibited tumor growth. The antitumor efficacy of CP-673,451 was evaluated in a number of human tumor xenografts grown s.c. in athymic mice, including H460 human lung carcinoma, Colo205 and LS174T human colon carcinomas, and U87MG human glioblastoma multiforme. Once-daily p.o. × 10 days dosing routinely inhibited tumor growth (ED50 33 mg/kg). These data show that CP-673,451 is a pharmacologically selective PDGFR inhibitor, inhibits tumor PDGFR-β phosphorylation, selectively inhibits PDGF-BB-stimulated angiogenesis in vivo, and causes significant tumor growth inhibition in multiple human xenograft models.

The ingrowth 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 cubic millimeters in volume (1, 2). 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. Owing to the invading normal host vasculature being the target, another potential attribute of an antiangiogenesis approach may be the avoidance of drug resistance, commonly associated with conventional anticancer modalities that target genetically unstable tumor cells (4). Moreover, with oral 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 a complex biological process that requires a variety of factors and signaling pathways to stimulate the migration and proliferation of the component cell types and to establish functional blood vessels (5).

Platelet-derived growth factor (PDGF) was originally identified in platelets and serum as a potent mitogen for smooth muscle cells and fibroblasts in vitro(6, 7). The PDGF family consists of four polypeptides, A-D, forming dimeric proteins that signal through two tyrosine kinase receptors, PDGFR-αand PDGFR-β. The ligands and receptors can form homodimers or heterodimers depending on cell type, receptor expression, and ligand availability (8). PDGFR-β is a split kinase transmembrane tyrosine kinase receptor closely related to c-kit and CSF-1R (9). Signaling through PDGFR-β has been shown to initiate endothelial, pericyte, and smooth muscle cell migration and proliferation in vitro and in vivo(10). The PDGF-B and PDGFR-β system is critical for the migration and proliferation of pericytes and the development of a functional vasculature (11, 12). Disruption of PDGF-B or PDGFR-β genes in mice is lethal, resulting in microvascular hemorrhage and edema with kidney, cardiovascular, and hematologic abnormalities (13–16) PDGF-B−/− murine embryos are totally devoid of microvascular pericytes (17). Endothelial cells and pericytes are co-dependent and although pericytes are commonly associated with capillaries, they are most abundant on venules (18), the vessel subtype most commonly responsive to angiogenic stimuli. Through secretion of growth factors and modulation of the extracellular matrix, endothelial-pericyte interactions are critical for vascular maturation, remodeling, and maintenance (19). Pericytes have classically been believed to be absent from tumor vasculature, but recent evidence shows that they are common on tumor vasculature although their morphology and permeability function are perturbed (20, 21).

PDGF growth factors and receptors are involved in both autocrine and paracrine stimulation in many tumor types, in situ and in human xenografts. Evaluation of surgical specimens of human breast carcinoma, colorectal adenocarcinoma, and lung carcinoma revealed PDGFR-β staining in periepithelial stroma, but absent in epithelial cells, whereas PDGF-BB was observed in tumor cells, suggesting paracrine stimulation (22–24). Autocrine stimulation has been observed in human prostate adenocarcinoma and astrocytic tumors, where PDGF-A or PDGF-B and their cognate receptors were expressed in both stromal and tumor epithelial cells (6, 25). Although PDGF ligands A and B, as well as PDGFR-α, are expressed by all stages of astrocytic tumors, PDGFR-β is expressed only on the tumor vasculature (26, 27). PDGFR inhibition is an antiangiogenic approach potentially affecting all solid tumors due to tumor vasculature having a pericyte coverage. However, depending on the tumor type, a PDGFR inhibitor would also be expected to inhibit tumor growth by directly targeting those tumor cells driven by a PDGF autocrine loop as well as by affecting tumor vasculature.

This manuscript describes the pharmacologic activity of CP-673,451, a potent inhibitor of PDGFR kinase that is being investigated for use as an anticancer agent. This compound inhibits both PDGFR-β and PDGFR-α kinase (IC50 = 1 and 10 nmol/L, respectively) but is >200-fold selective versus a variety of other kinases [e.g., c-kit, vascular endothelial growth factor receptor (VEGFR)-2, TIE-2, fibroblast growth factor receptor (FGFR)-2, epidermal growth factor receptor (EGFR), erbB2, and src]. CP-673,451 also inhibits PDGF-BB-stimulated autophosphorylation of dimeric PDGFR-β in transfected porcine aortic endothelial (PAE) cells with an IC50 value of 1 nmol/L. In vivo, oral administration of CP-673,451 significantly inhibits PDGF-BB-induced angiogenesis as evaluated using a sponge implant model. CP-673,451 also potently inhibits the growth of multiple tumor xenografts despite a lack of PDGFR expression in tumor cells. Inhibition of angiogenesis or tumor growth is correlated with plasma and tumor concentration and inhibition of phospho-PDGFR in vivo.

Chemical Synthesis. CP-673,451 was prepared according to the procedures described in PCT patent application WO 2001040217. The structure and physical properties of this compound are shown in Fig. 1. The tosylate salt form of this compound was used for the studies reported here.

Figure 1.

Chemical features and physical properties. Chemical name: 1-{2-[5-(2-methoxy-ethoxy)-benzoimidazol-1-yl]-quinolin-8-yl}-piperidin-4-ylamine. Molecular formula: C24H27N5O2. Molecular weight: 417.52 (free base); 589.71 (tosylate salt). Physical properties: white crystalline solid (tosylate salt). Melting point: 216°C (tosylate salt). pKa: 9.16, 4.88 (free base). Log P: 3.66 (free base). Log D (pH 7.4): 2.92 (free base). Solubility: >30 mg/mL in SGF; >1 mg/mL in PBS (tosylate salt).

Figure 1.

Chemical features and physical properties. Chemical name: 1-{2-[5-(2-methoxy-ethoxy)-benzoimidazol-1-yl]-quinolin-8-yl}-piperidin-4-ylamine. Molecular formula: C24H27N5O2. Molecular weight: 417.52 (free base); 589.71 (tosylate salt). Physical properties: white crystalline solid (tosylate salt). Melting point: 216°C (tosylate salt). pKa: 9.16, 4.88 (free base). Log P: 3.66 (free base). Log D (pH 7.4): 2.92 (free base). Solubility: >30 mg/mL in SGF; >1 mg/mL in PBS (tosylate salt).

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Kinase Inhibition Assay and Enzyme Kinetics. A glutathione S-transferase–tagged kinase domain construct of the intracellular portion of the PDGFR-β (amino acids 693-1,401, accession no. J03278) was expressed in Sf-9 cells (baculovirus expression system, Invitrogen, Carlsbad, CA). Enzyme kinetics were determined by incubating the enzyme with increasing concentrations of ATP in phosphorylation buffer [50 mmol/L HEPES (pH 7.3), 125 mmol/L NaCl, 24 mmol/L MgCl>2 in Nunc Immuno MaxiSorp 96-well plates previously coated with 100 μL of 100 μg/mL poly-Glu-Tyr (4:1 ratio) diluted in PBS. After 10 minutes, the plates were washed (PBS, 0.1% Tween 20), incubated with anti-phosphotyrosine-horseradish peroxidase antibody, and diluted in PBS, 0.05% Tween 20, 3% BSA for 30 minutes at room temperature. The plates were washed as above and incubated with 3,3′,5,5′-tetramethylbenzidine. The reaction was stopped by adding an equal volume of 0.09 N H2SO4. The phosphotyrosine-dependent signal was then quantitated on a plate reader at 450 nm. For routine enzyme assays, the enzyme was incubated with 10 μmol/L (final) ATP in the presence of compound diluted in DMSO (1.6% v/v DMSO assay final) for 30 minutes at room temperature in plates, as above, previously coated with 100 μL of 6.25 μg/mL poly-Glu-Tyr. The remainder of the assay was carried out as above, and IC50 values were calculated as percent inhibition of control. Selectivity assays were done as described above using purified recombinant enzyme (generated as described above) and ATP concentrations at or up to 3× above the Km for each enzyme. CP-673,451 was also tested at a concentration of 100 nmol/L in KinaseProfiler (Upstate Cell Signaling, Waltham, MA). All enzymes in this panel are recombinant human and are run at enzyme Km.

Cell-Based Phospho-PDGFR Inhibition Assay. PAE cells stably expressing full-length PDGFR and VEGFR have been previously described (28). For cell-based selectivity assays, PAE cells were transfected with full-length human PDGFR-α, PDGFR-β, or VEGFR-2. Cells were seeded at 4 × 105 cells/mL in 50 μL growth medium (Ham's F-12 media supplemented with 10% fetal bovine serum, 50,000 units each penicillin and streptomycin, and 500 μg/mL gentamicin) per well in 96-well plates. After 6 to 8 hours, the growth medium was replaced with 50 μL serum-depleted medium (as above, but with 0.1% fetal bovine serum) and cells were incubated overnight. Immediately before compound addition, the medium was replaced with 95 μL serum-depleted medium. 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 10 minutes. Cells were stimulated with the appropriate ligand (Becton Dickinson, Franklin Lakes, NJ, prepared in serum-depleted supplemented medium) and incubated as above for an additional 8 minutes. The medium was removed and the cells washed once with PBS, then lysed with 50 μL HNTG buffer [20 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 2% Triton X-100, 10% glycerol, 5 μmol/L EDTA, 2 mmol/L NaVO4, and 1 EDTA-free complete protease inhibitor tablet per 25 mL] for 5 minutes at room temperature. Lysates were then diluted with 50 μ L HG buffer [20 mmol/L HEPES (pH 7.5), 10% glycerol]. The diluted cell lysates were mixed thoroughly, 50 μL of supernatant were transferred to the ELISA capture plate, and incubated at room temperature for 2 hours with agitation. ELISA capture plates were prepared by coating 96-well ReactiBind goat-antirabbit plates (Pierce, Rockville, IL) with 100 μL/well of 5 μg/mL rabbit anti-human PDGFR-β, anti-PDGFR-α, or anti-VEGFR-2 antibody (Santa Cruz, Santa Cruz, CA) for 60 to 90 minutes. At the end of the 2-hour incubation the plates were washed (PBS, 0.1% Tween 20) before incubation with anti-phosphotyrosine-horseradish peroxidase antibody (diluted in PBS, 0.05% Tween 20) for 30 minutes at room temperature. The plates were washed again, then incubated with tetramethylbenzidine and evaluated as described above. IC50 values were calculated as percent inhibition of control. Cellular activity was also evaluated by Western blotting. For PDGFR-β activity, PAE-β cells stimulated with PDGF-BB were used. For c-kit selectivity, H526 small cell lung cancer cells stimulated with stem cell factor were used (29). For both assays, cells were starved overnight and treated the following day with increasing concentrations of CP-673,451 for 30 minutes at 37°C. During the final 5 to 8 minutes of incubation, either stem cell factor (50 ng/mL) or PDGF-BB (500 ng/mL) was added. Unstimulated cells were used as phosphorylation controls. Cells were washed, lysed, and equivalent protein levels were separated by PAGE. Following transfer to nitrocellulose, membranes were blotted to detect phospho-PDGFR (pY857 antibody, Santa Cruz) or phospho-c-kit (pY719 antibody, Cell Signaling) antibody at 1: 200 overnight at 4°C. Densitometry of bands and IC50 calculation were measured using a LumiImager (Roche, Indianapolis, IN).

Animals for In vivo Studies. Athymic female mice (CD-1 nu/nu, ∼20 grams) were used for all in vivo 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. All 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 × 106 C6 rat glioblastoma cells on day 1. On day 9, when tumors were approximately 300 mm3, the mice received compound or vehicle (5% Gelucire 44/14 in sterile water, Gattefossé, Paramus, NJ) orally. For pharmacokinetic/pharmacodynamic analysis, blood and tumor samples were collected from each animal (n ≥ 4 mice/group/time point) into heparinized vacutainers and liquid N2, respectively, at the indicated times post-dose. Blood and tumors were harvested for evaluation of drug levels and tumor-associated phospho-PDGFR-β. Plasma concentrations of CP-673,451 were determined using reverse-phase high-performance liquid chromatography with mass spectrometric (MS/MS) detection. Tumors were homogenized in 1 mL lysis buffer per 200 mg tumor [lysis buffer: 50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 1% glycerol, 1% Triton X-100, 1.6 mmol/L Na3VO4, 10 mmol/L NaF, 25 mg/L soy bean trypsin inhibitor, EDTA-free complete protease inhibitor tablets], spun for 5 minutes at 14,000 rpm, and the supernatant aliquoted to 96-well polypropylene plates on dry ice. Total protein concentration was determined using bicinchoninic acid protein assay (Pierce). Ninety-six-well ReactiBind goat-antirabbit plates (Pierce) were blocked with 100 μL/well cold blocking buffer (TBS, 0.1% Tween 20, 3% BSA) for 60 minutes on a plate shaker at room temperature. The blocking buffer was replaced with 0.5 μg anti-PDGFR-β in 100 μL cold blocking buffer per well and incubated for 60 minutes at room temperature with agitation. Plates were washed with TBS-T (TBS, 0.1% Tween 20) before addition of tumor lysate (100 μL diluted to ∼5 mg/mL total protein in lysis buffer without protease inhibitors) and incubated for 2 hours at room temperature with agitation. The plates were washed as above, then incubated with 15 ng of anti-phosphotyrosine-horseradish peroxidase per well (in blocking buffer) for 30 minutes at room temperature. The plates were washed as above and phosphotyrosine quantitated using tetramethylbenzidine as described above.

Tumor lysates were also evaluated by Western blotting for measurement of phospho-PDGFR-β. C6 tumor-bearing animals received doses of 10, 33, or 100 mg/kg (p.o.) and tumors were harvested 1.5 hours post-dose (∼Tmax). Tumors were lysed and equivalent amounts of protein were separated by PAGE, transferred to nitrocellulose, and blotted for phospho-PDGFR-β. Band density was quantitated using a LumiImager. Multiple tumors were evaluated at each dose.

Prediction of Efficacious Concentration. Blood and tumor samples were collected at each time point post-administration of CP-673,451 for determination of plasma drug concentration and PDGFR phosphotyrosine reduction. The relationship between CP-673,451 concentration and PDGFR phosphotyrosine reduction has been explored in pharmacologic models (tumor-bearing athymic mice) with pooled experimental data from 12 individual studies. PDGFR phosphotyrosine reduction correlates well with plasma concentrations of CP-673,451 in athymic mice and follows a simple Emax pharmacodynamic model:

\[\mathit{E}\ =\ \frac{\mathit{E}_{max}\ {\times}\ \mathit{C}}{\mathit{EC}_{50}\ {+}\ \mathit{C}}\]

where E is the measured response (PDGFR phosphorylation reduction), Emax is the maximum response, EC50 is the concentration of CP-673,451 required for half-maximal reduction of PDGFR phosphorylation, and C is the plasma concentration of CP-673,451.

The predicted maximal inhibition of PDGFR tyrosine phosphorylation is about 70% and the plasma concentration to achieve 35% inhibition of PDGFR phosphotyrosine is about 28 ng/mL. The predicted efficacious concentration of CP-673,451 to inhibit 50% PDGFR tyrosine phosphorylation using this model was calculated to be about 80 ng/mL (Fig. 4A).

In vivo Sponge Angiogenesis Assay. Lyophilized growth factors were reconstituted in HBSS + 1% BSA and admixed with growth factor reduced Matrigel 1:1 before adding to surgical gelatin sponges (4 × 4 × 2 mm). Mice were anesthetized with isoflurane and a 5-mm incision was made in the midline of the abdomen. For the dose response experiments, one PDGF-BB sponge was used per animal. Animals received doses of 3, 10, or 33 mg/kg q.d. p.o. × 5 days. For the selectivity experiments, each mouse received basic FGF (bFGF), VEGF, PDGF-BB, and control (no GF) sponges. Sponges were placed s.c. in the axillary and inguinal areas. Incisions were stapled shut and 2 days later mice began b.i.d. oral dosing with 5 mg/kg CP-673,451 (10 mg/kg total daily dose) for a total of 5 days. Following the last oral dose of compound, animals were sacrificed and sponges were removed. Sponges were homogenized in double-distilled water (100 μL/sponge) and lysates were transferred to a 96-well plate. Hemoglobin content was quantitated by measuring the colorimetric change following addition of tetramethylbenzidine. The plates were evaluated as described above. Four mice per dose group were used. A Student's t test was used to determine P value.

Tumor Growth Inhibition Studies. All tumor cell lines were obtained from American Type Culture Collection (ATCC, Rockville, MD) and propagated by standard tissue culture procedures in the medium as suggested by the supplier. Exponentially growing cells were trypsinized and resuspended in sterile PBS and inoculated s.c. (1 × 106 cells/mouse in 200 μL) into the right flank of mice. Animals bearing tumors of approximately 150 mm3 in size were divided into groups receiving either vehicle (5% Gelucire) or CP-673,451 (diluted in vehicle), and dosed by oral gavage. Animal body weight and tumor measurements were obtained every 2 days. Paclitaxel was diluted in saline and delivered i.p. at a dose of 10 mg/kg/d for 5 consecutive days (30, 31). Equivalent volumes of saline were injected for controls. Tumor volume (mm3) was measured with Vernier calipers and calculated using the formula: length (mm) × width (mm) × width (mm) × 0.5. Percent growth inhibition of an individual tumor was calculated using the following formula: % growth inhibition = (1−[(TLT1)/(CLC1)] × 100%), where TL and CL are the treated and control tumor volumes on day last, and T1 and C1 are treated and control tumor volumes on day 1. For all tumor growth inhibition experiments, 8 to 10 mice per dose group were used. A Student's t test was used to determine P value.

Microscopy and Immunohistochemistry of Tumor Microvasculature. Mice bearing Colo205 tumors were treated for 3 or 5 days with 5 mg/kg CP-673,451 (b.i.d., p.o.). Following treatment, tumors were excised and quick frozen in OCT media. Sections of 7 μm were cut and processed for immunohistochemical detection of microvasculature using rat anti-murine endothelial MECA32 antibody (anti-endoglin, PharMingen, San Diego, CA). Tissue sections were counterstained with hematoxylin or methyl green and examined using a Zeiss Axiophot microscope at 20× with a reticule grid. All discreet, positively stained vascular profiles, with or without lumina, were counted in 10 fields (200×) from multiple sections of each tumor. Fields were randomly chosen throughout the entire section. For each time point, four mice were evaluated. A Student's t test was used to determine P value.

Selective Inhibition of PDGFR Kinase by CP-673,451. CP-673,451 is a highly selective benzimidazole inhibitor of PDGFR-α and PDGFR-β tyrosine kinases (Fig. 1). Increasing concentrations of ATP resulted in a Michaelis-Menten saturation with a calculated Km of 3.3 μmol/L (not shown). The compound shows concentration-dependent inhibition of enzyme activity consistent with competitive inhibition of ATP (Fig. 2). Although CP-673,451 is approximately equipotent between PDGFR-α and PDGFR-β kinase (IC50 = 10 and 1 nmol/L, respectively), it is greater than 250× selective for PDGFR-β kinase relative to c-kit kinase (Table 1). Moreover, CP-673,451 shows 450-fold to more than 5,000-fold selectivity for PDGFR-β compared with other angiogenic tyrosine kinases, including VEGFR-1, VEGFR-2, TIE-2, and FGFR-2. Importantly, CP-673,451 is 1,000× to 10,000× selective relative to many other receptor tyrosine kinases (Tables 1 and 2). In cell-based assays using PAE cells stably transfected with recombinant human receptor and stimulated by the cognate ligand, CP-673,451 shows the same 10× selectivity for PDGFR-β relative to PDGFR-α seen in the kinase assays. Interestingly, this compound is >1,000× selective for PDGFR-β relative to VEGFR-2 when tested in PAE cell-based assays. Cell activity of CP-673,451 was also visualized in immunoblots (Fig. 3). PDGFR-β in PAE-β cells was inhibited with an IC50 of 6.4 nmol/L, comparable to results in the more quantitative cell-based ELISA (Fig. 3A). H526 cells expressing endogenous c-kit were also evaluated (Fig. 3B). CP-673,451 incubation with these cells resulted in an IC50 of 1.1 μmol/L against c-kit, showing ∼180× selectivity between these targets at the cell level mimicking kinase selectivity. Levels of total PDGFR and c-kit remained unchanged with compound treatment (data not shown). CP-673,451 was also submitted to the KinaseProfiler at 100 nmol/L (Table 2). These results show the extent of kinase selectivity of this compound against multiple kinases and kinase families.

Figure 2.

Kinetic analysis of PDGFR-β enzymatic activity in the presence of varied concentrations of ATP and PDGFR inhibitor CP-673,451. Increasing concentrations of the kinase inhibitor resulted in a concentration-dependent inhibition of enzyme activity. The Vmax could be restored by increasing the concentration of the ATP substrate. This enzymatic profile describes the competitive inhibition of ATP in the presence of the PDGFR inhibitor CP-673,451 [0.37 μmol/L (*); 0.1 μmol/L (×); 0.04 μmol/L (▴); 0.01 μmol/L (▪); 0.0 μmol/L (♦)].

Figure 2.

Kinetic analysis of PDGFR-β enzymatic activity in the presence of varied concentrations of ATP and PDGFR inhibitor CP-673,451. Increasing concentrations of the kinase inhibitor resulted in a concentration-dependent inhibition of enzyme activity. The Vmax could be restored by increasing the concentration of the ATP substrate. This enzymatic profile describes the competitive inhibition of ATP in the presence of the PDGFR inhibitor CP-673,451 [0.37 μmol/L (*); 0.1 μmol/L (×); 0.04 μmol/L (▴); 0.01 μmol/L (▪); 0.0 μmol/L (♦)].

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Table 1.

Kinase and cell selectivity

TargetKinase (IC50, nmol/L)Cell*
*

ELISA-based cell assay.

(IC50, nmol/L)
Cell

Immunoblotting-based cell assay.

(IC50, nmol/L)
PDGFR-β 
PDGFR-α 10 14  
c-kit 252 ND >1100 
VEGFR-2 450 10,600  
VEGFR-1 450 ND  
Lck >1,000 ND  
Src >2,000 ND  
v-abl >5,000 ND  
TIE-2 >5,000 ND  
bFGFR >5,000 ND  
EGFR >10,000 ND  
erbB2 >10,000 ND  
c-met >10,000 ND  
IGF-1R >10,000 ND  
TargetKinase (IC50, nmol/L)Cell*
*

ELISA-based cell assay.

(IC50, nmol/L)
Cell

Immunoblotting-based cell assay.

(IC50, nmol/L)
PDGFR-β 
PDGFR-α 10 14  
c-kit 252 ND >1100 
VEGFR-2 450 10,600  
VEGFR-1 450 ND  
Lck >1,000 ND  
Src >2,000 ND  
v-abl >5,000 ND  
TIE-2 >5,000 ND  
bFGFR >5,000 ND  
EGFR >10,000 ND  
erbB2 >10,000 ND  
c-met >10,000 ND  
IGF-1R >10,000 ND  

NOTE: All IC50 data are a mean of three assays run with triplicate wells on five- or six-point curves, except for PDGFR-α and PDGFR-β kinase and cell, which were run more than 20 times with triplicate wells and five- or six-point IC50 curves.

Table 2.

Broad panel kinase selectivity

Target% Inhibition at 100 nmol/LTarget% Inhibition at 100 nmol/L
Aurora-A GSK3α 12 
Bmx GSK3β 11 
BTK IKKβ 14 
CaMKIV JNK1α 
CDK1/cyclinB 12 MAPK1 
CDK2/cyclinA MEK1 
CDK2/cyclinE 14 MKK7β 
CDK3/cyclinE NEK2 
CDK5/p35 20 p70S6K 12 
CDK6/cyclinD3 PKA 
CDK7/cyclinH PKBα 
CHK1 10 PKCα 
CHK2 11 PKCβII 
CK2 12 PKC𝛉 
c-RAF PKCζ 
EphB2 ROCK-II 
Fes SAPK2a 
FGFR3 Trk B 
Fyn 21 Yes 
Target% Inhibition at 100 nmol/LTarget% Inhibition at 100 nmol/L
Aurora-A GSK3α 12 
Bmx GSK3β 11 
BTK IKKβ 14 
CaMKIV JNK1α 
CDK1/cyclinB 12 MAPK1 
CDK2/cyclinA MEK1 
CDK2/cyclinE 14 MKK7β 
CDK3/cyclinE NEK2 
CDK5/p35 20 p70S6K 12 
CDK6/cyclinD3 PKA 
CDK7/cyclinH PKBα 
CHK1 10 PKCα 
CHK2 11 PKCβII 
CK2 12 PKC𝛉 
c-RAF PKCζ 
EphB2 ROCK-II 
Fes SAPK2a 
FGFR3 Trk B 
Fyn 21 Yes 

NOTE: CP-673,451 was tested in a commercially available kinase screen of multiple kinases at 100 nmol/L. All enzymes are run at their Km and are human recombinant.

Figure 3.

A, effect of CP-673,451 on PDGF-BB-stimulated PAE-PDGFR-β cells. Increasing concentrations of compound result in a dose-dependent inhibition of phosphorylation resulting in an IC50 of 6.4 nmol/L in this assay readout. B, effect of CP-673,451 on stem cell factor–stimulated H526 small cell lung cancer cells. Compound concentrations above 1100 nmol/L are necessary to inhibit 50% of phospho-c-kit signal, showing that CP-673,451 is >180× selective for PDGFR-β compared with c-kit in a cell-based assays. Total amounts of PDGFR-β and c-kit remained unchanged following cell exposure to CP-673,451 (data not shown).

Figure 3.

A, effect of CP-673,451 on PDGF-BB-stimulated PAE-PDGFR-β cells. Increasing concentrations of compound result in a dose-dependent inhibition of phosphorylation resulting in an IC50 of 6.4 nmol/L in this assay readout. B, effect of CP-673,451 on stem cell factor–stimulated H526 small cell lung cancer cells. Compound concentrations above 1100 nmol/L are necessary to inhibit 50% of phospho-c-kit signal, showing that CP-673,451 is >180× selective for PDGFR-β compared with c-kit in a cell-based assays. Total amounts of PDGFR-β and c-kit remained unchanged following cell exposure to CP-673,451 (data not shown).

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Inhibition of PDGFR Phosphorylation in Tumor Xenografts. In order to evaluate the pharmacologic modulation of phosphorylated receptor in vivo, rat C6 glioblastoma xenograft models were used. These tumors consistently express phosphorylated PDGFR. All experiments evaluate total PDGFR and phospho-PDGFR in the ELISA to ensure consistent and reproducible endogenous expression. Glioblastoma tumors are among the few solid tumor types which express PDGFR on the tumor itself providing sufficient signal-to-noise for an ex vivo ELISA-based evaluation of phospho-PDGFR levels in the tumor.

Plasma levels generally increased with increasing dose from 3 to 100 mg/kg with a mean Tmax of 1 to 2 hours and a half-life of 2 to 3 hours in mice. The highest dose tested was 100 mg/kg. A concomitant decrease of tumor-associated phosphorylated PDGFR-β was measured with a calculated EC50 of 80 ng/mL (Fig. 4A). Phosphorylated PDGFR-β was reduced >50% for ∼4 hours following a single 50 mg/kg oral dose (Fig. 4B). CP-673,451 Cmax plasma levels following a single oral dose of 50 mg/kg never reached the cellular VEGFR-2 IC50 (10,600 nmol/L = 4425 ng/mL; Fig. 4C). The IC50 for PDGFR-α and β kinase activity are 4 and 0.4 ng/mL, respectively. Tumor lysates (2-3 tumors/dose) were evaluated in immunoblots (Fig. 4D). The ED50 following a single dose of CP-673,451 at the ∼Tmax was 10 mg/kg. Levels of total PDGFR remained unchanged with compound treatment (data not shown).

Figure 4.

A,ex vivo inhibition of phospho-PDGFR-β in C6 tumors versus plasma concentration of CP-673,451 at multiple points post-dose compiled from multiple experiments. B, inhibition of PDGFR-β pY following a single dose of 50 mg/kg. C, plasma concentrations of CP-673,451 versus time following a single oral dose of 50 mg/kg. Dotted line,in vivo PDGFR EC50 calculated from A. Note that the plasma concentration never reaches the concentrations required to inhibit VEGFR-2 in vitro (10,600 nmol/L = 4,425 ng/mL). For reference, PDGFR-β cell IC50 is 0.4 ng/mL and PDGFR-α cell IC50 is 5.6 ng/mL (depicted in similar units). Representative of >10 pharmacokinetic/pharmacodynamic and ex vivo experiments. LLOQ, lower limit of detection. D, dose-dependent decrease of phospho-PDGFR in CP-673,451-treated C6 tumors evaluated using immunoblotting. Total PDGFR-β amount remained unchanged with CP-673,451 treatment (data not shown).

Figure 4.

A,ex vivo inhibition of phospho-PDGFR-β in C6 tumors versus plasma concentration of CP-673,451 at multiple points post-dose compiled from multiple experiments. B, inhibition of PDGFR-β pY following a single dose of 50 mg/kg. C, plasma concentrations of CP-673,451 versus time following a single oral dose of 50 mg/kg. Dotted line,in vivo PDGFR EC50 calculated from A. Note that the plasma concentration never reaches the concentrations required to inhibit VEGFR-2 in vitro (10,600 nmol/L = 4,425 ng/mL). For reference, PDGFR-β cell IC50 is 0.4 ng/mL and PDGFR-α cell IC50 is 5.6 ng/mL (depicted in similar units). Representative of >10 pharmacokinetic/pharmacodynamic and ex vivo experiments. LLOQ, lower limit of detection. D, dose-dependent decrease of phospho-PDGFR in CP-673,451-treated C6 tumors evaluated using immunoblotting. Total PDGFR-β amount remained unchanged with CP-673,451 treatment (data not shown).

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CP-673,451 Selectively Blocks PDGF-BB Induced Angiogenesis. Unlike FGFR-2 and VEGFR-2, the critical nature of PDGFR signaling in angiogenesis is not as well established. To confirm that a selective PDGFR inhibitor can inhibit angiogenesis, a sponge angiogenesis model was used. Two experimental designs were used to evaluate dose response (Fig. 5A) and selectivity (Fig. 5B). Following 5 days of dosing (p.o., q.d.), 3.3 mg/kg was sufficient to inhibit PDGF-induced angiogenesis by 70% corresponding to a Cmax of 5.5 ng/mL. In order to better evaluate selectivity in vivo, four sponges containing either VEGF, bFGF, PDGF-BB, or saline were implanted into different abdominal quadrants of the same mouse. Following 5 mg/kg p.o., b.i.d. administration of CP-673,451, sponges were examined for hemoglobin content. Absolutely no inhibition of VEGF- or bFGF-induced angiogenesis was observed. In contrast, PDGF-BB-induced angiogenesis was inhibited by 70% relative to untreated animals (Fig. 5B).

Figure 5.

In vivo evaluation of antiangiogenesis using the sponge model. A, PDGF-BB-induced angiogenesis was inhibited by 70-90% following 5 daily oral doses of 3-30 mg/kg CP-673,451. Corresponding Cmax plasma concentrations taken after the last dose were 5.5-419 ng/mL. B, selective inhibition of PDGF-BB versus bFGF- or VEGF165-induced angiogenesis was evaluated in a second experiment. Animals had four sponges implanted: control, PDGF-BB, bFGF, and VEGF165. Each animal received 5 daily oral doses of 5 mg/kg CP-673,451 and angiogenesis was evaluated relative to the internal control sponge. A Student's t test was used to determine the P between PDGF +/− CP. Columns, averages of 10 animals; bars, SD.

Figure 5.

In vivo evaluation of antiangiogenesis using the sponge model. A, PDGF-BB-induced angiogenesis was inhibited by 70-90% following 5 daily oral doses of 3-30 mg/kg CP-673,451. Corresponding Cmax plasma concentrations taken after the last dose were 5.5-419 ng/mL. B, selective inhibition of PDGF-BB versus bFGF- or VEGF165-induced angiogenesis was evaluated in a second experiment. Animals had four sponges implanted: control, PDGF-BB, bFGF, and VEGF165. Each animal received 5 daily oral doses of 5 mg/kg CP-673,451 and angiogenesis was evaluated relative to the internal control sponge. A Student's t test was used to determine the P between PDGF +/− CP. Columns, averages of 10 animals; bars, SD.

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Tumor Growth Inhibition and Decreased Microvascular Density. Having shown that CP-673,451 inhibited PDGFR-β phosphorylation in vivo and PDGF-BB-induced angiogenesis, tumor growth inhibition studies were undertaken. Three of four of the xenograft models used (Colo205 and LS174T, human colon carcinomas, H460, and human lung carcinoma) do not express PDGFR-β on the tumor cells, minimizing the possibility for a tumor cell-based effect. These cell lines do secrete PDGF-BB, which can then induce receptor phosphorylation on the invading murine tumor vasculature. Therefore, any phospho-PDGFR inhibition observed in these models with CP-673,451 would be due to an effect on stromal expression, especially pericytes and endothelia. Of the models used, only U87MG human glioblastoma xenografts express PDGFR on the tumor cells; thus, inhibition could be due to a direct antitumor effect as well as an antiangiogenic effect. Once s.c. tumors reached >150 mm3, animals received CP-673,451 q.d. or b.i.d. orally for 10 to 11 days. Colo205 tumor growth was inhibited in a dose-dependent fashion up to 87% with 50 mg/kg (p.o., b.i.d.; Fig. 6A). Following multiday dosing at 100 mg/kg of H460 xenografts, tumor mass is still measurable, but there are significant areas of gross necrosis and fluid (Fig. 6B). Similar tumor growth inhibition experiments completed on LS174T, H460, and U87MG xenografts resulted in comparable inhibition of tumor growth ranging from 64% to 80% with 33 to 100 mg/kg total daily dose (Table 3). At all doses where tumor growth was inhibited, there were no signs of morbidity or weight loss.

Figure 6.

A, tumor growth inhibition study with Colo205 human colon adenocarcinoma xenografts and CP-673,451. Compound was dosed twice daily (b.i.d.) to achieve total daily doses of 10, 33, and 100 mg/kg orally for 10 days. B, representative tumors from another tumor growth inhibition study using H460 human lung adenocarcinoma xenografts following 10 once-daily oral doses of 100 mg/kg. Note that the treated tumors are hemorrhagic and avascular relative to vehicle-treated control tumors. A Student's t test was used to evaluate the P for CP-673,451-treated relative to vehicle-treated tumors. Vehicle treated (♦), 5 mg/kg b.i.d. (▪), 16.5 mg/kg b.i.d. (▴), 50 mg/kg b.i.d. (×). Points, averages of 5 animals; bars, SE. #, P < 0.05; *, P < 0.01, relative to vehicle treated.

Figure 6.

A, tumor growth inhibition study with Colo205 human colon adenocarcinoma xenografts and CP-673,451. Compound was dosed twice daily (b.i.d.) to achieve total daily doses of 10, 33, and 100 mg/kg orally for 10 days. B, representative tumors from another tumor growth inhibition study using H460 human lung adenocarcinoma xenografts following 10 once-daily oral doses of 100 mg/kg. Note that the treated tumors are hemorrhagic and avascular relative to vehicle-treated control tumors. A Student's t test was used to evaluate the P for CP-673,451-treated relative to vehicle-treated tumors. Vehicle treated (♦), 5 mg/kg b.i.d. (▪), 16.5 mg/kg b.i.d. (▴), 50 mg/kg b.i.d. (×). Points, averages of 5 animals; bars, SE. #, P < 0.05; *, P < 0.01, relative to vehicle treated.

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Table 3.

Tumor growth inhibition

Tumor xenograft typeDosing regimenTotal daily dose (mg/kg)% Inhibition
U87MG human glioblastoma p.o., b.i.d. × 10 d 100 80, P < 0.01 
LS174T human colon p.o., b.i.d. × 10 d 50 64, P < 0.01 
H460 human lung p.o., q.d. × 11 d 33 80, P < 0.01 
Colo205 human colon p.o., q.d. × 10 d 33 75, P < 0.01 
Tumor xenograft typeDosing regimenTotal daily dose (mg/kg)% Inhibition
U87MG human glioblastoma p.o., b.i.d. × 10 d 100 80, P < 0.01 
LS174T human colon p.o., b.i.d. × 10 d 50 64, P < 0.01 
H460 human lung p.o., q.d. × 11 d 33 80, P < 0.01 
Colo205 human colon p.o., q.d. × 10 d 33 75, P < 0.01 

Tumor vessels in Colo205 xenografts were detected immunohistochemically using a pan-endothelial antibody. Tumors were examined following 3 or 5 days of treatment with CP-673,451 (5 mg/kg, p.o., b.i.d.; Fig. 7). Following 3 or 5 days of dosing with CP-673,451, microvessel density was reduced by 35% or 47%, respectively, relative to vehicle-treated control tumors (P < 0.01). A representative microphotograph of Colo205 tumors treated for 5 days shows the decrease in vessel number as well as collapsed and smaller vascular profiles (arrows).

Figure 7.

Immunohistochemical evaluation of microvascular density using a pan-endothelial antibody (MECA32) on Colo205-bearing mice dosed with CP-673,451 for 3 or 5 days (twice daily doses of 5 mg/kg). Representative micrographs from 5-day cohorts are shown with stained vessels (arrows). Microvascular density in treated tumors was inhibited 35% by day 3, which increased to 47% by day 5 compared with vehicle-treated control tumors. A Student's t test was used to determine the P of treated relative to control tumors. Inhibition of microvessel density: 3-day dosing, 35%, P < 0.01; 5-day dosing, 47%, P < 0.01.

Figure 7.

Immunohistochemical evaluation of microvascular density using a pan-endothelial antibody (MECA32) on Colo205-bearing mice dosed with CP-673,451 for 3 or 5 days (twice daily doses of 5 mg/kg). Representative micrographs from 5-day cohorts are shown with stained vessels (arrows). Microvascular density in treated tumors was inhibited 35% by day 3, which increased to 47% by day 5 compared with vehicle-treated control tumors. A Student's t test was used to determine the P of treated relative to control tumors. Inhibition of microvessel density: 3-day dosing, 35%, P < 0.01; 5-day dosing, 47%, P < 0.01.

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Combination Chemotherapy Efficacy. Combination studies with paclitaxel were undertaken to examine whether CP-673,451 complemented the efficacy of a commonly used cytotoxic agent (Fig. 8). Paclitaxel (10 mg/kg, i.p., q.d. × 5 days) resulted in 54% tumor growth inhibition of LS174T human tumor xenografts. This dose and regimen has been previously shown to provide moderate efficacy and, more importantly, is well tolerated in tumor-bearing athymic nude mice (internal experience and refs. 30, 31). CP-673,451 (33 mg/kg, p.o., q.d. × 10 days) resulted in a comparable inhibition of 47%. When the two regimens were combined the tumor growth inhibition increased to 88% by the end of the 10-day experiment.

Figure 8.

Combination therapy with paclitaxel and CP-673,451 in mice bearing LS174T human colon adenocarcinoma xenografts. Paclitaxel was dosed once daily on days 1 to 5 whereas CP-673,451 was dosed once daily for 10 days. The combination of the two agents together resulted in substantially more tumor growth inhibition than either agent alone. Vehicle treated (♦), 33 mg/kg CP-673,451 (▴), 10 mg/kg paclitaxel (×), 33 mg/kg CP-673451, and 10 mg/kg paclitaxel (•). Points, averages of 8 animals; bars, SE. *, P < 0.05 versus vehicle-treated animals.

Figure 8.

Combination therapy with paclitaxel and CP-673,451 in mice bearing LS174T human colon adenocarcinoma xenografts. Paclitaxel was dosed once daily on days 1 to 5 whereas CP-673,451 was dosed once daily for 10 days. The combination of the two agents together resulted in substantially more tumor growth inhibition than either agent alone. Vehicle treated (♦), 33 mg/kg CP-673,451 (▴), 10 mg/kg paclitaxel (×), 33 mg/kg CP-673451, and 10 mg/kg paclitaxel (•). Points, averages of 8 animals; bars, SE. *, P < 0.05 versus vehicle-treated animals.

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Antiangiogenesis approaches to cancer therapy have generated much enthusiasm over the years and some targeted therapies have shown promise (32). Significant research has focused on associating specific receptor signal transduction pathways with downstream angiogenic processes. Owing to most angiogenic receptors being critically important in normal embryonic development, genetic knockout approaches are limited in elucidating the role of these receptors in tumor vascular biology. Other approaches to block the pathways are often hampered due to inability to deliver the agent in vivo or systemically (33, 34). The discovery of small-molecule inhibitors has provided an opportunity to better define the roles of angiogenic receptors in tumor biology. However, poor pharmacologic selectivity for the receptors by these tyrosine kinase inhibitors does not allow one to tease apart the relative impact of each receptor. Most first-generation antiangiogenesis tyrosine kinase inhibitors have varying degrees of potency and specificity and often include some activity toward PDGFR (35, 36). For example, SU-5416 and SU-6668 have been used to distinguish between PDGFR- and VEGFR-mediated effects in tumor vascular biology (37), however, these compounds are potent inhibitors of many kinases, including c-kit, FGFR-1, PDGFR, VEGFR-1, VEGFR-2, and VEGFR-3 (38–40). These compounds and others do not show pharmacologic selectivity for a single receptor or family of receptors, complicating the interpretation of results and understanding of the precise role of the receptors. Few tyrosine kinase inhibitors are absolutely selective if plasma concentrations are sufficiently elevated, as is commonly achieved with i.p. dosing. Therefore, it is critical to evaluate a given pharmacodynamic response in the context of the corresponding pharmacokinetic data for a given agent.

CP-673,451 is a novel, reversible, ATP-competitive inhibitor of PDGFR. In kinase assays, this compound does not show substantial potency against any other kinase tested, including c-kit, VEGFR, bFGFR, and TIE-2, among others. Although the compound has a modest 10× selectivity for PDGFR-β versus PDGFR-α, it is unlikely to distinguish between these two receptors in vivo at efficacious doses due to potency and plasma levels achieved. However, this selectivity profile is unique among a number of small-molecule multitarget kinase inhibitors with PDGFR activity (41, 42). To obtain the most accurate comparison, cell activity was characterized in the same cell background for PDGFR-β, PDGFR-α, and VEGFR-2.

Activity against PDGFR in vivo was evaluated using C6 rat glioblastoma xenografts, which express PDGFR-β on the cells. Although these tumor cells express rat PDGFR, the inhibitory effect of CP-673,451 on murine (NIH3T3 and tissues), rat (C6), dog (MDCK), and human (transfected PAE and tumor cells) PDGFR is indistinguishable (data not shown). Given the sequence similarities across species, these data are not surprising (43, 44). The advantage to this model is that it provides sufficient signal-to-noise in a quantitative ELISA-based measure of phosphorylated PDGFR in vivo. CP-673,451 has an EC50 of 80 ng/mL against the receptor, commonly achieved with a 33 mg/kg p.o. dose. Furthermore, a single oral dose of 50 mg/kg resulted in plasma concentrations above the EC50 for ∼4 hours. Notably, the IC50 for inhibition of phosphorylated VEGFR-2 in vitro is 10× higher than the Cmax of a 50 mg/kg dose and >40× higher than the in vivo EC50 for PDGFR-β, showing significant pharmacologic selectivity in vivo at this relatively high dose.

Sponge angiogenesis models were used to assess functional inhibition of specific angiogenic growth factor pathways. This model was used in two separate sets of experiments. In one, only PDGF-BB sponges were used in a dose escalation study of CP-673,451. Surprisingly, low doses and plasma levels were able to substantially inhibit PDGF-BB-induced angiogenesis, likely reflecting the sensitivity of a single growth factor angiogenesis system compared with a more complex tumor-based model. The other experimental design evaluated multiple angiogenic growth factors in the same animal, allowing for direct comparison of CP-673,451 on angiogenesis stimulated by each growth factor. The dose and regimen for this experiment was identical to what was used in the Colo205 tumor growth inhibition experiments that provided 55% tumor growth inhibition (Fig. 6A). Therefore, doses that result in tumor growth inhibition do not inhibit VEGF- or bFGF-induced angiogenesis. A representative photomicrograph of H460 human lung adenocarcinoma tumors removed following a tumor growth inhibition experiment visually shows the magnitude of the antitumor response (Fig. 6B). Although a measurable tumor is observed in the treated cohort, much of the tumor mass is fluid-filled and necrotic. This phenomenon may result in underpredicting the magnitude of the antitumor response when inhibition is only measured by decreases in volume.

Tumor growth inhibition studies were completed on a number of different tumor xenografts with comparable results. Although regressions were not typically observed, all models showed growth inhibition with PDGFR inhibition, regardless of tumor cell PDGFR status. Tumor growth inhibition was dose-responsive with no accumulation of compound with repeated dosing. All studies were initiated after tumors reached 100 to 200 mm3 and were in their exponential growth phase. All tumor inhibition experiments were carried out past two to three doublings of the control tumors. Pharmacokinetic/pharmacodynamic experiments showed that single oral doses of 33 mg/kg resulted in, at most, 4-hour inhibition of ≥50% phosphorylated PDGFR. Interestingly, doses which inhibited tumor growth (5 mg/kg, p.o., q.d. × 10 days) resulted in less than 50% inhibition of phospho-PDGFR at Tmax when given as a single dose. This indicates that as little as 30% to 50% inhibition of phospho-PDGFR for 3 to 6 hours per day is sufficient to inhibit tumor growth ≥50% in multiple models. This somewhat surprising efficacy, given the selective inhibition of PDGFR, may be indicative of the importance of pericytes (and PDGFR) on neovascular endothelium or the sensitivity of our tumor models to angiogenesis inhibition (20, 45–47). However, the antitumor efficacy observed in Colo205 with CP-673,451 is comparable to published reports with other multitarget angiogenesis inhibitors (40). It is also possible that repeated daily or twice daily dosing used in the tumor growth inhibition experiments results in accumulated non-PDGFR-mediated effects that a single dose experiment (as in the pharmacokinetic/pharmacodynamic experiments) underrepresents. For example, in glioblastoma multiforme where EGFR and PDGFR are both overexpressed and can be amplified (48), a coordination of multiple growth factor receptor pathways may result in PDGFR transactivation of EGFR (49). In this circumstance, it is possible that repeated inhibition of PDGFR-β results in greater tumor growth inhibition due to additional downstream inhibitory affects on another receptor due to PDGFR cross-talk. Similar complementary effects can occur with other growth factors and receptors in tumor stroma, such as PDGFR inhibition of VEGF production (50). There were no signs of morbidity or weight loss at any efficacious dose in the tumor growth inhibition studies, suggesting that selective PDGFR inhibition is well tolerated at efficacious doses.

To further show a measurable antiangiogenesis effect with CP-673,451, tumors were evaluated for microvessel density following a multiday dosing regimen. Tumors at the end of a typical 10-day tumor growth inhibition experiment were necrotic and unsatisfactory for immunohistochemical analysis of microvessel density. Therefore, tumors were removed and evaluated after 3 or 5 days of dosing. Interestingly, doses that resulted in 47% inhibition of microvessel density on day 5 yielded a 55% tumor growth inhibition with continued dosing by day 10.

It is likely that targeted agents such as CP-673,451 will be combined with conventional chemotherapeutic agents. It is anticipated that the mechanism of action of each agent will be complementary, toxicities will not be additive, and combination of the agents will result in greater efficacy (51). In testing this hypothesis, CP-673,451 was combined with paclitaxel in a tumor growth inhibition study on LS174T human xenografts. The plasma concentration of CP-673,451 was unchanged regardless of coadministration with paclitaxel, showing the increased tumor growth inhibition in combination is not due to increased CP-673,451 plasma levels. Although paclitaxel concentrations were not measured, there is a low probability of drug-drug interaction with CP-673,451 because of the compound having greater than 40 μmol/L IC50 against all the major cytochrome P450 enzymes (data not shown). Single daily doses of CP resulted in comparable tumor growth inhibition (49%) to paclitaxel (54%). Paclitaxel was not used at the maximally tolerated dose in order to better evaluate any additive efficacy and toxicities. When CP-673,451 was combined with paclitaxel, 88% tumor growth inhibition was observed with no increase in morbidity or weight loss versus vehicle- or single agent-treated animals. When given together, these compounds were roughly additive.

In this report, data were presented on CP-673,451 showing it is a highly selective PDGFR tyrosine kinase inhibitor in vitro and in vivo. At doses and plasma concentrations where tumor growth inhibition was observed in multiple models, there was no inhibition of VEGF- or bFGF-stimulated angiogenesis. At all efficacious doses, the compound was well tolerated in tumor-bearing athymic mice. The significant selectivity of this compound versus VEGFR, in particular, should allow dissection of angiogenesis signal transduction pathways in vitro and in vivo as well as increase our understanding of the role of pericytes in tumor vascular biology. The oral availability of this compound allows for a meaningful evaluation of the role of PDGFR in tumor biology as well as other pathologies where PDGFR figures prominently, such as restenosis, lung fibrosis, atherosclerosis, or glomerulonephritis (10, 52, 53). The oral dosing regimen has the added benefit of avoiding an extremely high Cmax often observed with i.p. dosing, resulting in cross-inhibition of multiple kinases. Finally, the true benefit of having a pharmacologically selective PDGFR inhibitor must be appreciated in the clinic to determine whether selectivity can avoid unwanted side effects, such as hypertension, associated with other targets unable to be avoided with nonselective multitarget tyrosine kinase inhibitors (54).

Note: G. Moraski, J.P. Lyssikatos, V. Pollack, and W. Barth are former Pfizer employees.

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.

We thank Jim Moyer, Jean Beebe, and Rick Connell for comments and for review of the manuscript.

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