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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
1 Cancer Bioscience, 2 Safety Assessment, and 3 Discovery DMPK, AstraZeneca, Alderley Park, Macclesfield, Cheshire, United Kingdom; and 4 AstraZeneca Pharma, Centre de Recherches, Z.I. La Pompelle, Chemin de Vrilly, Reims, France
Requests for reprints: Stephen R. Wedge, Cancer and Infection Research, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, United Kingdom. Phone: 44-1625-513236; Fax: 44-1625-513624; E-mail: steve.wedge{at}astrazeneca.com.
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Introduction |
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VEGF binds to the second and third immunoglobulin-like domains of its specific transmembrane receptors Flt-1 (VEGFR-1) and KDR (VEGFR-2) on endothelial cells, initiating receptor homodimerization or heterodimerization (16, 17). The conformational change induced in the receptor complex stimulates intrinsic kinase activity which transphosphorylates tyrosine residues within the cytoplasmic domains. These phosphorylated peptide sequences serve as recognition sites for Src homology 2 domain-binding proteins that subsequently propagate intracellular signaling. Activated KDR has been shown the major stimulator of angiogenesis and vascular permeability (18, 19). KDR signaling responses include (i) mitogenic signaling via activation of a phospholipase C-
/protein kinase C/Raf/mitogen-activated protein kinase (MAPK) pathway (20), (ii) motogenic signaling through phosphorylation of FAK and paxillin (21), and (iii) survival signaling, in a complex with VE-cadherin, ß-catenin, and phosphoinositide 3'-kinase (22).
With the exceptions of longitudinal bone extension during growth and cyclical changes in the female reproductive tissues, angiogenesis does not occur in healthy adults. Chronic inhibition of this process may therefore be tolerated and provide a means by which to prevent tumor progression (23). Given that KDR transduces the angiogenic effects of VEGF, and tissue expression of this receptor is largely confined to the endothelium, inhibition of KDR signaling should provide a means for achieving selective therapeutic intervention.
A number of small molecule approaches to inhibit the intrinsic tyrosine kinase activity of KDR have been described previously, with a range of nanomolar potencies, selectivities, and pharmacokinetic properties (24–27). Here we describe AZD2171, a highly potent (subnanomolar IC50) inhibitor of KDR tyrosine kinase and VEGF-induced signaling in endothelial cells. This compound has pharmacokinetic properties that make it suitable for chronic once-daily oral dosing. AZD2171 prevents VEGF-induced angiogenesis in vivo and shows dose-dependent activity in a range of human tumor xenografts in mice; statistically significant inhibition of tumor growth being evident with doses as low as 0.75 to 1.5 mg per kg per day. Furthermore, we show that inhibition of tumor growth is not merely a consequence of preventing new vessel formation; vascular regression can be observed in tumors following AZD2171 treatment.
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Materials and Methods |
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, PDGFR-ß, CSF-1R, Flt-3, FGFR1, Src, Abl, epidermal growth factor receptor (EGFR), ErbB2, Aur-A, and Aur-B] using ELISA methodology described previously (28). Selectivity versus CDK2 and CDK4 serine/threonine kinases was examined using scintillation proximity assays with a retinoblastoma substrate and [-33P]ATP (29). Activity versus the dual specificity kinase MAPK kinase (MEK), was determined with a MAPK substrate, [-33P]ATP, and paper capture/scintillation counting (29). Microcal Origin software (vs. 3.78, Microcal Software, Inc., Northhampton, MA) was used to interpolate IC50 values by nonlinear regression.
Inhibition of growth factor–stimulated receptor phosphorylation. Inhibition of receptor phosphorylation within cells was determined using ELISA (KDR and EGFR) or Western blotting (c-Kit, PDGFR- and PDGFR-ß, or Flt-3) methods, or methods incorporating a fluorescent immunocytochemical end point (CSF1-R or ErbB2). Parental cell lines were purchased from the American Type Tissue Collection (ATCC, Manassas, VA), except for MonoMac6 cells, which were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). With the exception of experiments examining erbB2, cells were serum starved overnight, incubated with AZD2171 for 60 to 120 minutes, and stimulated, where required, with the relevant ligand: VEGF165 (100 ng/mL) for 7 minutes, SCF (50 ng/mL) for 10 minutes, PDGF-AA or PDGF-BB (50 ng/mL) for 5 minutes, EGF (1 µg/mL) for 3 minutes, or CSF-1 (40 ng/mL) for 10 minutes. VEGF, SCF, EGF, and CSF-1 were obtained from R&D Systems, Inc. (Abingdon, United Kingdom) and PDGF-AA and PDGF-BB from Sigma-Aldrich (Poole, United Kingdom). Inhibition of KDR phosphorylation was determined in human umbilical vein endothelial cells (HUVEC) by ELISA at ProQinase. EGFR phosphorylation was examined using a sandwich ELISA in KB human oral squamous tumor cells, with an sc-120 capture antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and detection via horseradish peroxidase immunoconjugated to a 4G10 phosphotyrosine antibody (Upstate Biotechnology).
Western blotting was done using standard SDS-PAGE methods, loading 50 to 75 µg of protein per lane, with detection by enhanced chemiluminescence. Total and phosphorylated c-Kit was measured in NCI-H526 human small cell lung tumor cells, using antibodies to c-Kit (Santa Cruz Biotechnology) and phospho-c-Kit (Cell Signaling Technology, Inc., Beverly, MA). PDGFR- and PDGFR-ß phosphorylation was examined in MG63 human osteosarcoma cells, using antibodies to total (R&D Systems) and phosphorylated (Santa Cruz Biotechnology and Cell Signaling Technology) receptors. Inhibition of Flt-3 phosphorylation was investigated in MonoMac6 human acute myeloid leukemia cells, which contain an activating mutation in the juxtamembrane sequence of the receptor, using Flt-3 (Cell Signaling Technology) and phospho-Flt-3 (Cell Signaling Technology) antibodies.
ErbB2 phosphorylation was examined in MCF-7 breast carcinoma cells stably expressing constitutively activated wild-type erbB2 receptor. Cells were incubated with AZD2171 for 4 hours at 37°C, fixed in 3.3% formaldehyde/PBS, and immunostained with a phospho-erbB2 primary antibody (Santa Cruz Biotechnology) followed by an Alexa-Fluor 488 secondary antibody (Molecular Probes, Paisley, United Kingdom). ErbB2 phosphorylation was quantitated using an Acumen Explorer fluorescence laser-scanning plate reader (TTP Labtech Ltd., Royston, United Kingdom). CSF-1R phosphorylation was detected in 3T3 murine fibroblasts stably transfected with human CSF-1R. Cells were fixed in 4% formaldehyde and immunostained using a primary antibody to phosphorylated CSF-1R (Cell Signaling Technology). CSF-1R phosphorylation was quantitated using a Cellomics ArrayScan HCS Reader (Cellomics, Inc., Pittsburgh, PA).
Inhibition of KDR phosphorylation was also examined in HUVEC by Western blotting. HUVEC were serum starved overnight, incubated with AZD2171 for 90 minutes and stimulated with VEGF (50 ng/mL) for 8 minutes. Human VEGF165 was generated as described previously (29). Antibodies against pKDR (rabbit polyclonal generated to phosphorylated Y1214), KDR (Santa Cruz Biotechnology), pMAPK (Cell Signaling Technology), or MAPK (Cell Signaling Technology) were used.
Inhibition of growth factor–mediated cellular proliferation. HUVEC proliferation in the presence and absence of growth factors was evaluated following a 4-day incubation by 3H-thymidine incorporation, as described previously (28). Proliferation of MG63 osteosarcoma cells was induced by PDGF-AA, which selectively activates PDGFR- homodimer signaling. Cells were cultured in DMEM without phenol red (Sigma-Aldrich) containing 1% charcoal stripped FCS, 2 mmol/L glutamine, and 1% nonessential amino acids (Invitrogen, Paisley, United Kingdom) for 24 hours. AZD2171 or vehicle was added with PDGF-AA ligand (50 ng/mL; Sigma-Aldrich) and plates reincubated for 72 hours. Cellular proliferation was determined using a bromodeoxyuridine ELISA (Roche Diagnostics Ltd., Lewes, United Kingdom).
Inhibition of tumor cell proliferation in vitro. The human tumor cell lines, Calu-6 (lung carcinoma), SW620 (colorectal carcinoma), MDA-MB-231 (mammary gland adenocarcinoma), PC-3 (prostate adenocarcinoma), and SKOV-3 (ovarian adenocarcinoma) were obtained from ATCC. Cell proliferation was determined over a 3-day period as described previously (30).
Human in vitro angiogenesis assay. HUVECs and human diploid fibroblasts were obtained (day 0) as cocultures in 24-well plates (AngioKit, TCS CellWorks Ltd., Buckinghamshire, United Kingdom) and the medium immediately replaced with MCDB 131 medium (Life Technologies, Paisley, United Kingdom) containing 2% FCS, 1% glutamine, and 1% penicillin/streptomycin (Sigma, Poole, United Kingdom). AZD2171 or vehicle (0.01% DMSO in MCDB 131) was added to the cocultures. Medium, with AZD2171 or vehicle, was replenished on days 3, 6, and 8. Tubule formation was examined at day 10 following fixing and staining of tubules for CD31 (platelet/endothelial cell adhesion molecule 1) according to the manufacturer's instructions. Comparative treatment with either an antihuman VEGF neutralizing antibody or a control immunoglobulin G antibody (30 µg/mL, R&D Systems) was also examined.
To quantify tubule growth, a novel whole-well image analysis method was developed using a Zeiss KS400 3.0 Image Analyser (Imaging Associates Ltd., Bicester, United Kingdom) and a bespoke computer program. Tubule formations within each well were measured excluding a rim of 100-µm depth to avoid edge retraction artifact. The computer program segmented the images using a gray level threshold tool to select the stained cells of interest. The resultant binary images were skeletonized and nodal junctions (branch points) removed to determine the total length of an individual tubule. The branch points were counted and the total area of CD31 staining determined from the original binary image but with a correction for cells that had not migrated (labeled cells with an area of <20 µm).
Inhibition of vascular endothelial growth factor–induced angiogenesis in vivo. A Matrigel plug assay done by Vasculogen (Minneapolis, MN) was used to examine VEGF-induced angiogenesis directly. Briefly, Matrigel (0.5 mL) containing heparin (20 units/mL; Sigma Chemicals, St. Louis, MO) was diluted 3:1 in HBSS or recombinant human VEGF165 (150 ng/mL; R&D systems) and injected s.c. into female, BALB/c athymic (nu/nu genotype) mice of 6 to 8 weeks of age (five per group). AZD2171 (1.5 or 6 mg per kg per day) or vehicle was given orally, from the day of Matrigel implantation, for 7 days. Matrigel plugs were recovered on day 8, one half being snap-frozen in liquid nitrogen using Tissue-Tek, ornithine carbamyl transferase compound (Fisher Scientific, Pittsburgh, PA) and the other fixed in neutral formalin and embedded in paraffin for H&E staining. Frozen sections were stained with a rat monoclonal antibody reactive to mouse CD31 conjugated to phycoerythrin (BD Biosciences PharMingen, San Diego, CA). Two sections were cut from the Matrigel samples at different levels and analyzed for vessel density. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) and immunofluorescent images (n = 7-10) of CD31 staining obtained from each sample at random. Each area was photographed under both red and UV filters. Morphometric analysis of vessel density and architecture was carried out after converting the images into a binary image using Adobe Software. Images were skeletonized using a program that converts binary data into single pixel density tracings (31). This enabled the number of vessel ends, vessel branch points (nodes), and total vessel length to be quantified with an Image Processing Toolkit (RGI, Inc., Raleigh, NC).
Effect of AZD2171 on bone growth and luteal development. Young female Alderley Park rats (6 weeks of age, Wistar derived, n = 5) were dosed orally, once daily for 28 days with AZD2171 (1.25-5 mg per kg per day) or vehicle. Additional rats (five per group) were treated with AZD2171 (5 mg per kg per day) or vehicle for 28 days and maintained for a further 28 days without treatment, to examine the effect of compound withdrawal. Histologic paraffin wax sections of the femorotibial joints and ovaries were stained with H&E. Morphometric image analysis of femorotibial sections was done (29), with growth plate areas from both the femur and tibia in each joint being combined for an analysis of the effect of compound treatment. The area of corpora lutea in H&E-stained ovary sections was similarly determined by morphometric analysis (Joyce-Loebl Magiscan Image Analyser, Applied Imaging Ltd., Newcastle upon Tyne, United Kingdom).
In vivo tumor models. Protocols for establishing s.c. PC-3, Calu-6, SKOV-3, MDA-MB-231, and SW620 tumors in female nude (nu/nu genotype) mice were as described previously (29, 30). When tumors reached a volume of 0.1 to 0.5 cm3, mice were randomized (6-12 per group) and AZD2171 (0.75-6 mg per kg per day) or vehicle given once daily by oral gavage. Tumor volumes were assessed by bilateral Vernier caliper measurement at least twice weekly and calculated using the formula (length x width) x 
(length x width) x (
/6), where length was taken to be the longest diameter across the tumor and width the corresponding perpendicular. Growth inhibition was calculated from the start of treatment by comparison of the mean change in tumor volume for control and treated groups. To remove any size dependency before statistical evaluation (the variance in mean tumor volume data increases proportionally with volume and is therefore disproportionate between groups), data was log-transformed before statistical evaluation using a one-tailed two-sample t test.
Histologic assessment of tumor vasculature in response to AZD2171 therapy. Mice bearing established Calu-6 human lung tumor xenografts (0.2 ± 0.01 cm3, mean volume ± SE) were selected (day 0) and treated chronically with AZD2171 (6 mg per kg per day, p.o.) or vehicle. Tumors were collected (6-15 per group) 4 hours after the last dose of AZD2171 or vehicle, on days 1, 2, 7, 14, and 21. CD31 was then detected in sections using a chromagen end point or fluorescent immunostaining. CD31 staining with a chromagen end point was done on tumor specimens fixed in zinc fixative (PharMingen) using methodology described previously (29). These were analyzed blind to treatment assignment using a KS400 instrument (Imaging Associates). CD31-positive vessel number and total CD31 staining area/5,000 µm2 viable tumor area were calculated for each section. For CD31 fluorescent immunostaining, formalin-fixed sections were incubated with a CD31 antibody (Santa Cruz Biotechnology) in serum block followed by an immunoglobulin G conjugated to Alexa Fluor 488 (Molecular Probes) and counterstained using ProLong Gold anti-fade reagent with DAPI (Molecular Probes). Fluorescent CD31 staining was visualized using an Axiovert S100 fluorescent microscope (Carl Zeiss SMT, Inc., Thornwood, NY) and images analyzed using MetaMorph version 6.1 software (Universal Imaging Co., Downingtown, PA).
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Results |
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3 nmol/L). The inhibitory activity of AZD2171 was also examined against each recombinant PDGFR-related kinase in vitro because of their structural similarity to the VEGF family of receptors. The IC50 values for inhibition of c-Kit and PDGFRß tyrosine kinase (2 and 5 nmol/L, respectively) were in a range similar to that measured versus Flt-1 and Flt-4. However, when compared with KDR kinase inhibition, AZD2171 selectivity versus the remaining PDGFR-related members ranged from >36-fold (PDGFR-) to >1,000-fold (Flt-3). Excellent selectivity for KDR was evident versus a range of unrelated tyrosine and serine/threonine kinases, including EGFR (>1,600-fold selectivity) and MEK (>10,000-fold selectivity; Table 1). Furthermore, in addition to the data presented, no inhibition of enzyme activity was detected when 10 µmol/L of AZD2171 was examined with 100 µmol/L ATP against AMPK, ChK1, c-jun NH2-terminal kinase, MAPK2, MSK-1, PKA, Akt/PKB, PKC, Rock II, SAPK2b, SAPK2c, SGK, CSK, and PI 3-kinase (data not shown).
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and PDGFR-ß phosphorylation in MG63 osteosarcoma cells. In accordance with the kinase selectivity profile, AZD2171 showed a high degree of selectivity (420- to >20,000-fold) for inhibition of KDR phosphorylation versus the remaining PDGFR family members, CSF-1R and Flt-3, and versus the erb family members, EGFR and erbB2 (Table 2).
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–dependent cellular proliferation (stimulated by PDGF-AA) was examined in MG63 cells, and an IC50 value of 0.04 µmol/L determined. This concentration is 100-fold greater than that required for comparable inhibition of VEGF-induced proliferation in HUVEC. The data indicate that AZD2171 can selectively inhibit VEGFR-dependent proliferation, in contrast to proliferation mediated by FGFR1 or EGFR, and that appreciable functional selectivity is also evident versus PDGFR.
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AZD2171 inhibits tubule sprouting in vitro at subnanomolar concentrations and prevents VEGF-induced angiogenesis in vivo. Although the complex angiogenic phenotype cannot be completely replicated in vitro, assays can be used to model vascular sprouting. We modified a commercially available fibroblast/endothelial cell coculture system (AngioKit, TCS CellWorks) so that exogenous bFGF was omitted from the incubation medium. Under these conditions, tubule growth is regulated more directly by VEGF, production of which increases during the course of the assay.5 When compared with treatment with an isotype control antibody (Fig. 3B, iv) that did not affect tubule growth, a VEGF-neutralizing antibody inhibited vessel development significantly (Fig. 3B, v). Consistent with potent activity against VEGF signaling, AZD2171 inhibited vessel branching, length, and area (Fig. 3) with IC50 values (mean ± SE; five independent experiments) of 0.0001 ± 0.00004, 0.0001 ± 0.00003, and 0.0002 ± 0.00007 µmol/L, respectively. To examine inhibition of angiogenesis in vivo that was selectively driven by VEGF, Matrigel plugs containing VEGF were implanted s.c. in mice and vessel development examined over the course of 8 days. AZD2171 completely abolished VEGF-induced vessel formation. The data obtained with 6 mg per kg per day AZD2171 versus number of vessel ends, branch points and total vessel length was not significantly different to that observed with 1.5 mg per kg per day (Fig. 4; P > 0.05 by one-tailed t test). Collectively, these data show that AZD2171 is a potent inhibitor of VEGF-induced angiogenesis.
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The effect of AZD2171 treatment on bone growth plate and ovary is supportive of inhibition of VEGF-induced angiogenesis within complex physiologic settings.
AZD2171 shows broad-spectrum activity in human tumor models at doses that are well tolerated. Once-daily oral administration of AZD2171 (1.5 mg per kg per day) inhibited growth of all human tumor xenografts examined in nude mice, irrespective of the histologic type (Table 4; Fig. 6). Statistically significant growth inhibition was obtained with 0.75 mg per kg per day AZD2171 in three of five tumor models examined, and the growth of each tumor model inhibited by >90% following administration of 6 mg per kg per day. AZD2171 treatment was well tolerated; following continuous administration of 6 mg per kg per day for 3 weeks (data accumulated from all experiments over 21 days), body weights were comparable with pretreatment values and only 1.5 % different to those of control animals (Fig. 6B). This is particularly notable when the weight of the additional tumor burden in control animals is considered. The broad-spectrum antitumor activity observed with AZD2171 may be attributed to a common effect on tumor vasculature, and its good tolerability to a comparatively selective profile in vivo.
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6-fold over the 21-day period examined, and AZD2171 treatment was found to inhibit this tumor growth by 68% (Fig. 7A).
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With acute AZD2171 treatment (day 1: two doses of AZD2171 given 28 and 4 hours before sampling) neither vascular variable was affected significantly. However, following an additional dose of AZD2171 (i.e., day 2: AZD2171 given at 52, 28, and 4 hours before sampling), a 47% reduction in vessel number was observed (P = 0.02), accompanied by a 40% reduction in CD31 area that was just outside statistical significance (P = 0.06). By day 21, vessel number and CD31 area were reduced by 
70% (P < 0.005). These data indicate that AZD2171 can cause significant vascular regression in tumors. The rapid onset of vessel regression is most probably due to a direct effect of AZD2171 on tumor endothelium, which is likely to be derived from potent inhibition of VEGF signaling.
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Discussion |
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AZD2171 also showed activity at low nanomolar concentrations in enzyme assays, versus the kinases associated with the two other VEGF receptor family members, Flt-1 and Flt-4. The exact role of Flt-1 signaling in physiologic and pathologic angiogenesis remains unclear. Flt-1 has a relatively weak signaling capacity in vitro and, in contrast to KDR, a number of reports suggest that its stimulation has minimal effects on mitogenic, motogenic, or permeability responses in endothelial cells (33–35). Flt-1 with a deleted kinase domain has also been shown to support normal vascular development in mice, suggesting that Flt-1–stimulated angiogenic sprouting may be mediated by ligand sequestration, to regulate the spatial availability of VEGF, rather than via an intrinsic signaling response (36). Despite these observations, Flt-1 has been shown to co-operate directly with KDR via heterodimerization, and Flt-1 homodimers may transactivate KDR through cross-talk (37). Furthermore, Flt-1 alone can bind two additional VEGF homologues, PlGF and VEGF-B, and the former has been shown, under experimental in vitro conditions, to induce differential signaling through Flt-1 when compared with VEGF itself (37). These findings support the possibility that direct inhibition of Flt-1 tyrosine kinase may be beneficial. In contrast to Flt-1, Flt-4 does not bind VEGF, PlGF, or VEGF-B but only binds the homologues VEGF-C and VEGF-D. This receptor has a critical role in lymphangiogenesis (38) and a prognostic link with expression of VEGF-C and/or VEGF-D and nodal metastasis has been identified for different tumor types (39, 40). Experimentally, lymph node metastasis can be promoted by expression of VEGF-C (41) and inhibited with an antibody to Flt-4 (42). Direct inhibition of Flt-4 signaling may therefore also have therapeutic benefit in limiting subsequent tumor dissemination. However, it has recently been suggested that KDR heterodimerization (induced by VEGF-C or VEGF-D) is required for ligand-stimulated phosphorylation of Flt-4 and its associated cellular signaling (43); a dependency that would be perturbed directly by a KDR tyrosine kinase inhibitor. Determining the full relevance of AZD2171 activity against Flt-1 and Flt-4 kinase will necessitate additional investigation of receptor signaling in cells.
The selectivity of an ATP-competitive kinase inhibitor in vivo is dependent upon the comparative potency of the compound against a target, the degree of inhibition required to prevent a given phenotypic effect and the level of plasma/tissue exposure attained at the evaluated dose. Collectively, the enzyme, receptor phosphorylation, and cellular proliferation data obtained with AZD2171 indicate that it has selectivity for inhibition of VEGF signaling but also suggest that it may have relevant activity versus c-Kit tyrosine kinase which could provide added therapeutic benefit in the treatment of c-Kit–dependent tumors (44, 45). That large concentrations of AZD2171 were required to inhibit the growth of tumor cells directly in vitro, and that AZD2171 was particularly well tolerated in tumor xenograft models at doses that proved highly efficacious, is in further support of a selective inhibitory profile.
AZD2171 combines potent activity versus KDR tyrosine kinase and selectivity with pharmacokinetic properties that are appropriate for oral once-daily administration. In female rat, studies examining i.v. (5 mg/kg) and oral (30 mg/kg) dosing of AZD2171 revealed a terminal plasma half-life of 9 hours, a relatively low clearance of 0.8 L per hour per kg, an oral bioavailability of 60%, and an unbound plasma fraction of 6% (data not shown). For this reason, all preclinical in vivo work was conducted by daily oral gavage.
AZD2171 treatment inhibited physiologic processes that are critically dependent upon VEGF signaling and angiogenesis. During endochondral bone formation, VEGF expression by hypertrophic chondrocytes regulates metaphyseal angiogenesis (46), with capillaries that invade the cartilage expressing both Flt-1 and KDR (47). This vascular invasion is critical to enable the terminal differentiation and apoptosis of growth plate chondrocytes, matrix resorption by osteoclasts, and mineralization by osteoblasts. The survival of osteoclasts (48) and migration and differentiation of osteoblastic cells (49, 50), both of which express Flt-1 and KDR, can also be influenced directly by VEGF signaling. Furthermore, chondrocyte survival has recently been suggested to have a VEGF dependency (51). Consistent with a fundamental regulatory role for VEGF signaling in bone morphogenesis, AZD2171 perturbed endochondral ossification in growing rats significantly and produced a marked growth plate hypertrophy; a phenotype also induced by VEGF sequestration (52) or conditional knockout of VEGF in cartilage (53). Whereas this phenotype has also been reported with other VEGF receptor tyrosine kinase inhibitors (26, 30), the doses of AZD2171 that produce significant effects are comparatively much lower. The magnitude of hypertrophy induced by AZD2171 has also not been shown previously. This phenotype was reversed completely following an additional 28-day period of AZD2171 withdrawal, indicating that chronic inhibition of VEGF signaling is required to maintain an effect in vivo. In addition to inhibiting ossification in bone growth plate, AZD2171 also markedly reduced corpora luteal area in rat ovary. The cyclic corpus luteum of the ovary is considered the site of strongest physiologic angiogenesis, with rapid luteal development involving distinct phases of VEGF-dependent blood vessel growth and maturation, followed by vessel regression during luteolysis that involves endothelial cell detachment (54). This is the first study to report the effects of a VEGF receptor kinase inhibitor on follicular development, although a soluble VEGF receptor construct has been previously found to have inhibitory effects on corpora luteal angiogenesis and development in mice (55) and a KDR blocking antibody shown to significantly delay follicular development in rhesus monkey (56). Analogous changes in bone growth plate and ovary have also been observed in primate following treatment with the VEGF-neutralizing antibody, bevacizumab (57).
AZD2171 (1.5 and 6 mg per kg per day) completely abolished VEGF-dependent angiogenesis in vivo, in a s.c. Matrigel plug assay, suggesting that it might also inhibit tumor growth at comparatively small doses. Doses of 6 mg per kg per day AZD2171 and lower were examined for activity in a panel of established, histologically distinct (colon, lung, prostate, breast, and ovary), human tumor xenograft models in athymic mice. Statistically significant inhibition was obtained with 1.5 mg per kg per day AZD2171 in all models and with 0.75 mg per kg per day in a number of models. The broad-spectrum antitumor profile observed with AZD2171 is consistent with an effect on tumor vasculature, a common growth-limiting target in all models, as opposed to tumor cell targets that are subject to variable expression and dependency. AZD2171 was efficacious at doses that are significantly lower than reported with other VEGFR tyrosine kinase inhibitors, which require administration within the 20 to 100 mg per kg per day range to achieve significant inhibition of tumor growth in mice (24–26).
To investigate temporal changes in tumor vessel growth and survival following AZD2171 treatment, a Calu-6 lung tumor xenograft model was examined, which is known to be growth inhibited by VEGF inhibition but does not regress in response to treatment (29). Vascular density in control Calu-6 tumors did not change significantly during 21 days of growth. In contrast, a comparison of vascular density in AZD2171-treated tumors with corresponding time-matched controls revealed a 47% reduction after 52 hours of treatment and a 73% reduction following 3 weeks of treatment. These data indicate that progressive vascular regression occurs in human tumor xenografts treated chronically with AZD2171, demonstrating that inhibition of VEGF signaling can have a potent antivascular effect. A number of preclinical studies with VEGF sequestering agents have also shown evidence of reduced vascular density in tumors following treatment. These include regression of existing tumor vessels with a VEGFR-Fc fusion construct in tumor bearing RIP-Tag2 transgenic mice (58), a KDR blocking antibody in squamous cell tumor xenografts (59), and a VEGF-neutralizing antibody in LS174T colon tumor xenografts (60). Primitive vessels are believed to be susceptible to VEGF blockade, because their survival is highly dependent upon KDR mediated Akt/PKB signaling (8). These data serve to emphasize that the consequences of inhibiting VEGF signaling in pathologic conditions may extend beyond simply preventing new vessel growth.
That inhibition of VEGF signaling can provide clinical benefit to patients with solid tumors, has been confirmed recently by bevacizumab (Avastin, Genentech, South San Francisco, CA), a monoclonal antibody to VEGF-A. In combination with irinotecan, 5-fluorouracil and leucovorin, bevacizumab imparts a significant prolongation of survival and response variables in patients with first-line metastatic colorectal cancer (61). An additional small preoperative biomarker study has also suggested antivascular effects in rectal carcinoma patients, following bevacizumab treatment (62). Direct inhibition of KDR tyrosine kinase activity with a small molecule, such as AZD2171, is an alternative therapeutic approach that may afford advantages to sequestration of VEGF. Inhibiting the kinase domain of KDR is mechanistically different, in that it should prevent receptor signaling irrespective of the activating ligand. In addition to VEGF-A, the fully proteolytically cleaved forms of the related VEGF gene family members VEGF-C and VEGF-D can also bind to and activate KDR (63, 64). In particular, VEGF-C can induce proliferative, migratory, tubulogenic, and permeability responses through KDR (63, 65). Chronic administration of a small molecule to generate steady state plasma levels may also provide a pharmacokinetic advantage, because the distribution and clearance of a large biopharmaceutical may possibly be influenced more highly by heterogeneous tumor anatomy and variable tumor blood flow (66).
In summary, AZD2171 is a highly potent inhibitor of KDR tyrosine kinase that has pharmacokinetic properties suitable for once-daily oral administration. VEGF-induced angiogenesis, neovascular survival, and growth of human tumor xenografts, are inhibited significantly by AZD2171, at comparatively low doses. AZD2171 is currently being evaluated in clinical trials as a once-daily oral therapy for the treatment of a variety of malignancies. Encouragingly, these clinical studies indicate that AZD2171 retains a good pharmacokinetic profile in man, with dose-proportional increases in plasma exposure and a mean terminal plasma half-life of 20 hours (67).
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Acknowledgments |
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We thank Karen Malbon, Claire Barnes, Minwei Ye, Stephen Brightwell, John Stawpert, and Sandra Oakes for the technical assistance in selectivity testing; Kevin Randal for morphometric image analysis; David Blowers, Ian Taylor, Hazel Weir, and Rick Davies for protein production and purification; Mick Shaw for the isolation and supply of HUVECs; and Michel Vautier for doing the initial synthesis of AZD2171.
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Footnotes |
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Received 12/ 9/04. Revised 2/16/05. Accepted 3/ 2/05.
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 E. Chen, D. Jonker, I. Gauthier, M. MacLean, J. Wells, J. Powers, and L. Seymour Phase I Study of Cediranib in Combination with Oxaliplatin and Infusional 5-Fluorouracil in Patients with Advanced Colorectal Cancer Clin. Cancer Res., February 15, 2009; 15(4): 1481 - 1486. [Abstract] [Full Text] [PDF]
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S. J. Crabb, D. Patsios, E. Sauerbrei, P. M. Ellis, A. Arnold, G. Goss, N. B. Leighl, F. A. Shepherd, J. Powers, L. Seymour, et al. Tumor Cavitation: Impact on Objective Response Evaluation in Trials of Angiogenesis Inhibitors in Non-Small-Cell Lung Cancer J. Clin. Oncol., January 20, 2009; 27(3): 404 - 410. [Abstract] [Full Text] [PDF]
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 B. M. Johnson, A. V. Kamath, J. E. Leet, X. Liu, R. S. Bhide, R. W. Tejwani, Y. Zhang, L. Qian, D. D. Wei, L. J. Lombardo, et al. Metabolism of 5-Isopropyl-6-(5-methyl-1,3,4-oxadiazol-2-yl)-N-(2-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amine (BMS-645737): Identification of an Unusual N-Acetylglucosamine Conjugate in the Cynomolgus Monkey Drug Metab. Dispos., December 1, 2008; 36(12): 2475 - 2483. [Abstract] [Full Text] [PDF]
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D. D. Hu-Lowe, H. Y. Zou, M. L. Grazzini, M. E. Hallin, G. R. Wickman, K. Amundson, J. H. Chen, D. A. Rewolinski, S. Yamazaki, E. Y. Wu, et al. Nonclinical Antiangiogenesis and Antitumor Activities of Axitinib (AG-013736), an Oral, Potent, and Selective Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases 1, 2, 3 Clin. Cancer Res., November 15, 2008; 14(22): 7272 - 7283. [Abstract] [Full Text] [PDF]
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 K J Williams, B A Telfer, A M Shannon, M Babur, I J Stratford, and S R Wedge Inhibition of vascular endothelial growth factor signalling using cediranib (RECENTINTM; AZD2171) enhances radiation response and causes substantial physiological changes in lung tumour xenografts Br. J. Radiol., October 1, 2008; 81(Special_Issue_1): S21 - S27. [Abstract] [Full Text] [PDF]
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T. P. Padera, A. H. Kuo, T. Hoshida, S. Liao, J. Lobo, K. R. Kozak, D. Fukumura, and R. K. Jain Differential response of primary tumor versus lymphatic metastasis to VEGFR-2 and VEGFR-3 kinase inhibitors cediranib and vandetanib Mol. Cancer Ther., August 1, 2008; 7(8): 2272 - 2279. [Abstract] [Full Text] [PDF]
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 C. A. Heckman, T. Holopainen, M. Wirzenius, S. Keskitalo, M. Jeltsch, S. Yla-Herttuala, S. R. Wedge, J. M. Jurgensmeier, and K. Alitalo The Tyrosine Kinase Inhibitor Cediranib Blocks Ligand-Induced Vascular Endothelial Growth Factor Receptor-3 Activity and Lymphangiogenesis Cancer Res., June 15, 2008; 68(12): 4754 - 4762. [Abstract] [Full Text] [PDF]
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F. Hilberg, G. J. Roth, M. Krssak, S. Kautschitsch, W. Sommergruber, U. Tontsch-Grunt, P. Garin-Chesa, G. Bader, A. Zoephel, J. Quant, et al. BIBF 1120: Triple Angiokinase Inhibitor with Sustained Receptor Blockade and Good Antitumor Efficacy Cancer Res., June 15, 2008; 68(12): 4774 - 4782. [Abstract] [Full Text] [PDF]
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J. O. Curwen, H. L. Musgrove, J. Kendrew, G. H.P. Richmond, D. J. Ogilvie, and S. R. Wedge Inhibition of Vascular Endothelial Growth Factor-A Signaling Induces Hypertension: Examining the Effect of Cediranib (Recentin; AZD2171) Treatment on Blood Pressure in Rat and the Use of Concomitant Antihypertensive Therapy Clin. Cancer Res., May 15, 2008; 14(10): 3124 - 3131. [Abstract] [Full Text] [PDF]
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S. A. Laurie, I. Gauthier, A. Arnold, F. A. Shepherd, P. M. Ellis, E. Chen, G. Goss, J. Powers, W. Walsh, D. Tu, et al. Phase I and Pharmacokinetic Study of Daily Oral AZD2171, an Inhibitor of Vascular Endothelial Growth Factor Tyrosine Kinases, in Combination With Carboplatin and Paclitaxel in Patients With Advanced Non-Small-Cell Lung Cancer: The National Cancer Institute of Canada Clinical Trials Group J. Clin. Oncol., April 10, 2008; 26(11): 1871 - 1878. [Abstract] [Full Text] [PDF]
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J. Cummings, C. Hodgkinson, R. Odedra, P. Sini, S. P. Heaton, K. E. Mundt, T. H. Ward, R. W. Wilkinson, J. Growcott, A. Hughes, et al. Preclinical evaluation of M30 and M65 ELISAs as biomarkers of drug induced tumor cell death and antitumor activity Mol. Cancer Ther., March 1, 2008; 7(3): 455 - 463. [Abstract] [Full Text] [PDF]
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 K. J. Higgins, S. Liu, M. Abdelrahim, K. Vanderlaag, X. Liu, W. Porter, R. Metz, and S. Safe Vascular Endothelial Growth Factor Receptor-2 Expression Is Down-Regulated by 17{beta}-Estradiol in MCF-7 Breast Cancer Cells by Estrogen Receptor {alpha}/Sp Proteins Mol. Endocrinol., February 1, 2008; 22(2): 388 - 402. [Abstract] [Full Text] [PDF]
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 S. Choi and J.N. Myers Molecular Pathogenesis of Oral Squamous Cell Carcinoma: Implications for Therapy Journal of Dental Research, January 1, 2008; 87(1): 14 - 32. [Abstract] [Full Text] [PDF]
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N. Korsisaari, J. Ross, X. Wu, M. Kowanetz, N. Pal, L. Hall, J. Eastham-Anderson, W. F. Forrest, N. Van Bruggen, F. V. Peale, et al. Blocking Vascular Endothelial Growth Factor-A Inhibits the Growth of Pituitary Adenomas and Lowers Serum Prolactin Level in a Mouse Model of Multiple Endocrine Neoplasia Type 1 Clin. Cancer Res., January 1, 2008; 14(1): 249 - 258. [Abstract] [Full Text] [PDF]
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 B. E. Johnson, C. M. Rudin, and R. Salgia Novel and Targeted Agents for Small Cell Lung Cancer ASCO Educational Book, January 1, 2008; 2008(1): 363 - 367. [Abstract] [Full Text] [PDF]
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N. R. Smith, N. H. James, I. Oakley, A. Wainwright, C. Copley, J. Kendrew, L. M. Womersley, J. M. Jurgensmeier, S. R. Wedge, and S. T. Barry Acute pharmacodynamic and antivascular effects of the vascular endothelial growth factor signaling inhibitor AZD2171 in Calu-6 human lung tumor xenografts Mol. Cancer Ther., August 1, 2007; 6(8): 2198 - 2208. [Abstract] [Full Text] [PDF]
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F. Gomez-Rivera, A. A. Santillan-Gomez, M. N. Younes, S. Kim, D. Fooshee, M. Zhao, S. A. Jasser, and J. N. Myers The Tyrosine Kinase Inhibitor, AZD2171, Inhibits Vascular Endothelial Growth Factor Receptor Signaling and Growth of Anaplastic Thyroid Cancer in an Orthotopic Nude Mouse Model Clin. Cancer Res., August 1, 2007; 13(15): 4519 - 4527. [Abstract] [Full Text] [PDF]
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J. Drevs, P. Siegert, M. Medinger, K. Mross, R. Strecker, U. Zirrgiebel, J. Harder, H. Blum, J. Robertson, J. M. Jurgensmeier, et al. Phase I Clinical Study of AZD2171, an Oral Vascular Endothelial Growth Factor Signaling Inhibitor, in Patients With Advanced Solid Tumors J. Clin. Oncol., July 20, 2007; 25(21): 3045 - 3054. [Abstract] [Full Text] [PDF]
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 N. Korsisaari, I. M. Kasman, W. F. Forrest, N. Pal, W. Bai, G. Fuh, F. V. Peale, R. Smits, and N. Ferrara From the Cover: Inhibition of VEGF-A prevents the angiogenic switch and results in increased survival of Apc+/min mice PNAS, June 19, 2007; 104(25): 10625 - 10630. [Abstract] [Full Text] [PDF]
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R. W. Wilkinson, R. Odedra, S. P. Heaton, S. R. Wedge, N. J. Keen, C. Crafter, J. R. Foster, M. C. Brady, A. Bigley, E. Brown, et al. AZD1152, a Selective Inhibitor of Aurora B Kinase, Inhibits Human Tumor Xenograft Growth by Inducing Apoptosis Clin. Cancer Res., June 15, 2007; 13(12): 3682 - 3688. [Abstract] [Full Text] [PDF]
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M. Takeda, T. Arao, H. Yokote, T. Komatsu, K. Yanagihara, H. Sasaki, Y. Yamada, T. Tamura, K. Fukuoka, H. Kimura, et al. AZD2171 Shows Potent Antitumor Activity Against Gastric Cancer Over-Expressing Fibroblast Growth Factor Receptor 2/Keratinocyte Growth Factor Receptor Clin. Cancer Res., May 15, 2007; 13(10): 3051 - 3057. [Abstract] [Full Text] [PDF]
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G. Giaccone The Potential of Antiangiogenic Therapy in Non-Small Cell Lung Cancer Clin. Cancer Res., April 1, 2007; 13(7): 1961 - 1970. [Abstract] [Full Text] [PDF]
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K. J. Williams, B. A. Telfer, A. M. Shannon, M. Babur, I. J. Stratford, and S. R. Wedge Combining radiotherapy with AZD2171, a potent inhibitor of vascular endothelial growth factor signaling: pathophysiologic effects and therapeutic benefit Mol. Cancer Ther., February 1, 2007; 6(2): 599 - 606. [Abstract] [Full Text] [PDF]
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C. Cao, J. M. Albert, L. Geng, P. S. Ivy, A. Sandler, D. H. Johnson, and B. Lu Vascular Endothelial Growth Factor Tyrosine Kinase Inhibitor AZD2171 and Fractionated Radiotherapy in Mouse Models of Lung Cancer Cancer Res., December 1, 2006; 66(23): 11409 - 11415. [Abstract] [Full Text] [PDF]
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 A. P. Hall Review of the Pericyte during Angiogenesis and its Role in Cancer and Diabetic Retinopathy Toxicol Pathol, October 1, 2006; 34(6): 763 - 775. [Full Text] [PDF]
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 R.A. Goodlad, A.J. Ryan, S.R. Wedge, I.T. Pyrah, D. Alferez, R. Poulsom, N.R. Smith, N. Mandir, A.J. Watkins, and R.W. Wilkinson Inhibiting vascular endothelial growth factor receptor-2 signaling reduces tumor burden in the ApcMin/+ mouse model of early intestinal cancer Carcinogenesis, October 1, 2006; 27(10): 2133 - 2139. [Abstract] [Full Text] [PDF]
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K. Nakamura, E. Taguchi, T. Miura, A. Yamamoto, K. Takahashi, F. Bichat, N. Guilbaud, K. Hasegawa, K. Kubo, Y. Fujiwara, et al. KRN951, a Highly Potent Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases, Has Antitumor Activities and Affects Functional Vascular Properties. Cancer Res., September 15, 2006; 66(18): 9134 - 9142. [Abstract] [Full Text] [PDF]
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 K. J. Higgins, S. Liu, M. Abdelrahim, K. Yoon, K. Vanderlaag, W. Porter, R. P. Metz, and S. Safe Vascular Endothelial Growth Factor Receptor-2 Expression Is Induced by 17{beta}-Estradiol in ZR-75 Breast Cancer Cells by Estrogen Receptor {alpha}/Sp Proteins Endocrinology, July 1, 2006; 147(7): 3285 - 3295. [Abstract] [Full Text] [PDF]
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 A. Morabito, E. De Maio, M. Di Maio, N. Normanno, and F. Perrone Tyrosine Kinase Inhibitors of Vascular Endothelial Growth Factor Receptors in Clinical Trials: Current Status and Future Directions Oncologist, July 1, 2006; 11(7): 753 - 764. [Abstract] [Full Text] [PDF]
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N. Amino, Y. Ideyama, M. Yamano, S. Kuromitsu, K. Tajinda, K. Samizu, H. Hisamichi, A. Matsuhisa, K. Shirasuna, M. Kudoh, et al. YM-359445, an Orally Bioavailable Vascular Endothelial Growth Factor Receptor-2 Tyrosine Kinase Inhibitor, Has Highly Potent Antitumor Activity against Established Tumors Clin. Cancer Res., March 1, 2006; 12(5): 1630 - 1638. [Abstract] [Full Text] [PDF]
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A. P. Hall, F. R. Westwood, and P. F. Wadsworth Review of the Effects of Anti-Angiogenic Compounds on the Epiphyseal Growth Plate Toxicol Pathol, February 1, 2006; 34(2): 131 - 147. [Abstract] [Full Text] [PDF]
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N. Matsunaga, K. Nakamura, A. Yamamoto, E. Taguchi, H. Tsunoda, and K. Takahashi Improvement by solid dispersion of the bioavailability of KRN633, a selective inhibitor of VEGF receptor-2 tyrosine kinase, and identification of its potential therapeutic window Mol. Cancer Ther., January 1, 2006; 5(1): 80 - 88. [Abstract] [Full Text] [PDF]
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K. D. Miller, M. Miller, S. Mehrotra, B. Agarwal, B. H. Mock, Q.-H. Zheng, S. Badve, G. D. Hutchins, and G. W. Sledge Jr. A Physiologic Imaging Pilot Study of Breast Cancer Treated with AZD2171 Clin. Cancer Res., January 1, 2006; 12(1): 281 - 288. [Abstract] [Full Text] [PDF]
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, 0.75 mg per kg per day; 
, 1.5 mg per kg per day; 
, 3 mg per kg per day; 
, 6 mg per kg per day) or vehicle (
) on growth of MDA-MB-231 human breast tumor xenografts. Xenografts were established s.c. in athymic mice and allowed to reach a volume of 0.2 ± 0.01 cm3 (mean ± SE) before treatment. Once-daily oral administration of AZD2171 or vehicle then commenced and was continued for the duration of the experiment. Points, mean from 10 to 11 mice; bars, SE in one direction. B, percentage change in mouse body weight over a 21-day period, when treated chronically with vehicle (
). At intervals (arrows) tumors (n = 6-15) were collected from each group, 4 hours after dosing with vehicle or AZD2171, to enable CD31 to be determined by immunohistochemistry. B, mean CD31 area per 5,000 µm2 of tumor and (C) mean vessel number per 5,000 µm2 of tumor, for vehicle-treated (open columns) and AZD2171-treated (blue columns) tumors. The statistical significance of AZD2171 treatment was examined versus the respective time-matched control using a one-tailed t test. While CD31 area and vessel number (both normalized to area) remain relatively constant in control tumors, a