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1 Pharmaceutical Development Laboratories, Kirin Brewery Co. Ltd., Takasaki, Gunma and 2 Division of Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan
Requests for reprints: Kazuhide Nakamura, Pharmaceutical Development Laboratories, Kirin Brewery Co. Ltd., 3 Miyahara, Takasaki, Gunma 370-1295, Japan. Phone: 81-27-346-9423. Fax: 81-27-347-5280. E-mail: ka-nakamura{at}kirin.co.jp
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Abstract |
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Key Words: KRN633 • oral administration • quinazoline urea derivative • VEGF • VEGFR-2
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Introduction |
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Vascular endothelial growth factor (VEGF) is the angiogenic factor that is most closely associated with aggressive disease in numerous solid tumors. Overexpression of VEGF by tumor cells frequently occurs in response to hypoxia (3, 4), loss of tumor suppressor gene function (5, 6), and oncogene activation (7). Elevated VEGF levels are correlated with increased microvessel counts and poor prognosis in many human cancers (8–10). This correlation is attributed to the ability of VEGF to stimulate endothelial cell proliferation, protease expression, cell migration, and the formation of capillary tubes (11–13). Furthermore, VEGF functions as a potent prosurvival (antiapoptotic) factor for endothelial cells in newly formed blood vessels (14–16). In addition to the effects on preexisting vessels, VEGF also influences the mobilization and differentiation of bone marrow–derived endothelial cell progenitors that can contribute to the formation of new blood vessels (17, 18).
The receptor tyrosine kinase (RTK) VEGF receptor (VEGFR)-2 is almost exclusively located on endothelial cells (19). Its expression levels are low in normal tissues and only increase in pathologic states when neovascularization occurs. VEGFR-2 has an extracellular VEGF-binding domain, a single membrane-spanning domain, and an intracellular split tyrosine kinase domain. Binding of VEGF and VEGFR-2 induces receptor tyrosine phosphorylation and stimulates the phospholipase C
-protein kinase C-mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) pathway, as well as the phosphatidylinositol 3'-kinase/AKT pathway (20, 21). VEGFR-2 is therefore thought to provide both mitogenic and survival signals and to be a major signaling component in angiogenesis. Consequently, blockade of VEGF signaling is a highly attractive therapeutic strategy. An ideal agent would be a low molecular weight compound with the ability to cross membranes, bind specifically to VEGFR-2, and inhibit its tyrosine kinase. Such a compound should be effective when administered p.o. because it is likely that continuous blockade of the VEGF pathway would be required to control tumor growth.
In this study, we describe a novel quinazoline urea derivative, KRN633, which strongly and selectively inhibits VEGFR-2 tyrosine kinase and intracellular VEGF signaling. We also report the effect of the p.o. administration of KRN633 on the in vivo growth of human tumor xenografts in mice and rats.
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Materials and Methods |
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Cell-Free and Cellular Kinase Assays
Cell-free kinase assays were done to obtain IC50 values against a variety of recombinant receptor and non-RTKs. KRN633 was tested from 0.3 nmol/L to 10 µmol/L. All assays were done in quadruplicate with 1 µmol/L ATP.
For the cellular assays, the cells were cultured in the following media: EGM-2 (Cambrex) for the HUVECs; DMEM (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum (FBS) and 200 µg/mL Geneticin (G418) for the NIH3T3-Flt-1 cells; RPMI 1640 (Sigma-Aldrich) containing 10% FBS for the Ku812-F cells; FGM-2 (Cambrex) for the normal human dermal fibroblasts; and DMEM containing 10% FBS for the A431 cells. All cells were serum starved for 16 to 24 hours in their respective basal media with 0.5% FBS. KRN633 was then added to the cells and they were incubated for 1 hour. The cells were stimulated with 50 ng/mL VEGF, 100 ng/mL stem cell factor, 50 ng/mL platelet-derived growth factor (PDGF)-BB, 20 ng/mL epidermal growth factor (PeproTech EC Ltd, London, United Kingdom), 25 ng/mL basic fibroblast growth factor (bFGF, Upstate Biotechnology, Inc., Lake Placid, NY), or 50 ng/mL hepatocyte growth factor/scatter factor (BD Biosciences Discovery Labware, Bedford, MA) at 37°C. Receptor phosphorylation was induced for 5 minutes, except for c-Kit and c-Met, which were induced for 15 and 10 minutes, respectively.
Cells were lysed with lysis buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/mL phenylmethylsulfonyl fluoride, 1 mmol/L Na3VO4, and 3% aprotinin in PBS). The receptors were immunoprecipitated, subjected to SDS-PAGE, and transferred to polyvinylidene fluoride microporous membranes. All antibodies for immunoprecipitation were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The membranes were probed with phosphotyrosine antibody 4G10 (Upstate Biotechnology). Phosphorylation was detected with peroxidase-conjugated anti–immunoglobulin G and enhanced chemiluminescence reagent (Amersham Biosciences Inc., Piscataway, NJ). Blots of the receptors were scanned and the density was quantified using the public domain software package Scion Image Beta 4.02 for Windows (Scion Corporation, Frederick, MD). IC50 values were calculated by nonlinear regression analysis using GraphPad Prism (GraphPad Software, Inc., San Diego, CA).
MAP Kinase Activation
After serum starvation (0.5% FBS), HUVECs were incubated with KRN633 for 1 hour and stimulated with either 50 ng/mL VEGF or 25 ng/mL bFGF. Cell lysates were subjected to SDS-PAGE. Immunoblotting of phosphorylated MAP kinases was done using phospho-p44/42 MAP kinase antibody (Cell Signaling Technology Inc., Beverly, MA).
Endothelial Cell Proliferation
Endothelial cell-proliferation assays were done as described previously (23). Briefly, HUVECs were seeded in collagen-coated 96-well plates at 4,000 cells per 200 µL/well in M-199 (Invitrogen Corp., Carlsbad, CA) containing 5%FBS. After 24 hours, KRN633 was added followed by 20 ng/mL VEGF or 10 ng/mL bFGF, and the cells were cultured for 78 hours. [3H]thymidine (1 µCi/mL) was added and the cells were cultured for a further 14 hours. They were then harvested and their radioactivity was measured using a liquid scintillation counter (Wallac 1205 Beta Plate; Perkin-Elmer Life Sciences, Boston, MA).
Capillary Tube Formation
Capillary tube formation of endothelial cells was measured by coculture with normal human dermal fibroblasts. Normal human dermal fibroblasts in FGM-2 were plated in 24-well plates at 5 x 104 cells/well and cultured for 24 hours. HUVECs were then added at 3,000 cells/well. After 24 hours, KRN633 was added followed by 10 ng/mL VEGF. The cells were cocultured for 9 days, fixed in ice-cold 70% ethanol for 30 minutes, and the HUVECs visualized by immunocytochemical detection of von Willebrand factor (TCS Biologicals Ltd., Buckingham, United Kingdom). The areas, lengths, paths, and joints of the stained tubelike structures were measured using an angiogenesis image analyzer (Kurabo, Osaka, Japan) in five different fields for each condition.
Cytotoxicity Assays
Cancer cells were plated in media with 10% FBS and antibiotics, at densities known to permit exponential growth over the assay period. The details were as follows: A549 in DMEM at 200 cells/well; Ls174T and DU145 in Eagle's MEM (Invitrogen) with 2 mmol/L L-glutamine (Invitrogen) at 3,000 cells/well; HT29 in McCoy's 5a (Invitrogen) at 3,000 cells/well; LNCap in RPMI 1640 (Sigma-Aldrich) at 3,000 cells/well; and PC-3 in Ham's F12K medium (Invitrogen) with 2 mmol/L L-glutamine at 3,000 cells/well. The cells were cultured for 24 hours before adding KRN633 (0.01 to 10 µmol/L) or vehicle (0.1% DMSO in medium) and then grown for a further 96 hours. Cell viability was measured using WST-1 reagent (Roche Applied Science, Indianapolis, IN). The percentage viability was determined relative to the untreated control.
Tumor-Xenograft Models
All in vivo experiments were conducted in accordance with the guidelines of the Kirin Animal Care and Use Committee. Athymic mice (BALB/cA, Jcl-nu) and athymic rats (F344/N, Jcl-rnu) were obtained from CLEA Japan Inc. (Tokyo, Japan). Mice and rats were housed in a barrier facility with a 12-hour light/dark cycle, and provided with sterilized food and water ad libitum.
A549, Ls174T, HT29, DU145, LNCap, and PC-3 cells were cultured in the appropriate media and implanted s.c. into the hind flanks of mice or rats. To facilitate tumor grafting, DU145, LNCap, and PC-3 were suspended in Matrigel (BD Biosciences Discovery Labware) and diluted 1:1 in the relevant medium before implantation. LC-6-JCK tumor xenografts were established in the hind flank by s.c. implantation of a cubic tumor fragment of 
2-mm diameter. Mice were grouped randomly when the tumors had reached an average volume of 100 to 260 mm3 for standard models (day 0) and 500 to 670 mm3 for well-established models. Rats were grouped randomly when tumors had reached an average volume of 162 to 618 mm3. The animals were then given KRN633 by p.o. gavage (as described in the figure and table legends). Tumor volume was measured twice weekly using Vernier calipers and was calculated as length x width x height x 0.5. Relative tumor volume (RTV) was calculated using the following formula: RTV at day X = (tumor volume at day X)/(tumor volume at day 0). Percentage tumor growth inhibition (TGI%) was calculated as follows: TGI% at day X = [(RTV of vehicle-treated group at day X – RTV of KRN633-treated group at day X)/(RTV of vehicle-treated group at day X – 1)] x 100. P values were determined by comparing mean tumor size in the treated group with mean tumor size in the vehicle-treated group using Dunnett's test.
Immunohistochemistry
A549 tumor xenografts were established in athymic rats as described above. Drug treatments were initiated when the tumor volumes were 440 mm3. Rats received KRN633 (as described in the figure and table legends). Tumor tissues were harvested and cryosections of 4 µm were prepared. Immunofluorescence staining was done using biotinylated anti-rat CD31 antibody (BD Biosciences PharMingen, San Diego, CA) and TSA fluorescence systems (Perkin-Elmer Life Sciences). To identify viable regions of the tumor, each serial section was stained with H&E. The areas of CD31+ endothelial cells within the viable regions were measured by imaging sections digitally and processing five random 0.2122-mm2 fields per slide at x200 magnification with LSM510 systems (Version 2.01; Carl Zeiss MicroImaging, Inc., Thornwood, NY).
Tumor Vascular Permeability Assays
Tumor blood vessel leakage was determined using the Evans Blue dye perfusion technique (24) with some modifications. Rats with A549 tumor xenografts were i.v. injected with 12.5 mL/kg of Evans Blue dye solution (10 mg/mL). After 30 minutes, they were sacrificed and the A549 tumors were harvested immediately. Dye was extracted from the tumors and measured spectrophotometrically.
Determination of Serum Concentrations after Oral Administration
At the allotted times, blood samples were collected from the heart or tail vein of athymic mice and rats, respectively. Serum samples were analyzed by reverse phase high-performance liquid chromatography (HPLC) using the tandem mass spectrometry method. Briefly, KRN633 and internal standard were extracted from serum samples using methyl-t-butylether. The organic phase was evaporated to dryness, reconstituted with the mobile phase, and then analyzed directly. KRN633 concentrations were determined from the peak area ratio of KRN633 and internal standard. The quantification range was 0.4 to 200 ng/mL.
Pharmacokinetic Analysis
Serum concentration-time data were analyzed by a noncompartmental pharmacokinetic method using WinNonlin version 2.1 (Pharsight Corporation, Mountain View, CA) to determine the area under the serum concentration-time curve extrapolated to infinity (AUC
), the apparent terminal-elimination half-life (t1/2), oral clearance (CL/F), and the apparent distribution volume (Vd/F). The maximum serum concentration (Cmax) and the time at which Cmax was achieved (Tmax) were obtained directly from the serum concentration data. The serum concentration-time profiles after repeated administration were simulated by WinNonlin using the pharmacokinetic parameters obtained from compartment-model analysis of the profile after single p.o. administration at a dose of 20 mg/kg.
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Results |
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and -ß, c-Kit, breast tumor kinase, and tunica interna endothelial cell kinase tyrosine kinases (IC50 = 965, 9,850, 4,330, 9,200, and 9,900 nmol/L, respectively). The IC50 values for EGFR, EphB2 and -B4, insulin-like growth factor-1 receptor, fibroblast growth factor receptor (FGFR)-1, -3 and -4, Abl, erbB4, fme-like tyrosine kinase-3, insulin receptor, Janus kinase 2, c-Met, muscle-specific RTK, Wee1, Src, and focal adhesion kinase tyrosine kinases were all >10 µmol/L. The cellular assays used the appropriate normal and cancer cell lines, and phosphorylation of the RTKs was stimulated with their cognate ligands. The effect of KRN633 was measured by immunoblotting with antiphosphotyrosine antibody after immunoprecipitation. As shown in Table 1, the phosphorylation of VEGFR-2 was potently inhibited by exposure to KRN633 for 1 hour prior to stimulation with VEGF (IC50 = 1.16 nmol/L). KRN633 also inhibited the phosphorylation of VEGFR-1 (IC50 = 11.7 nmol/L), c-Kit, and PDGFR-ß (IC50 = 8.01 and 130 nmol/L, respectively), both of which contain a large insert within the kinase domain. KRN633 did not block the phosphorylation of FGFR-1, EGFR, or c-Met, even at a concentration of 10 µmol/L.
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KRN633 Suppresses Capillary Tube Formation of Endothelial Cells In vitro
To investigate the effect of KRN633 on in vitro tube formation by endothelial cells, HUVECs were cocultured with fibroblasts in the presence of KRN633. The formation of capillary-like structures induced by 10 ng/mL VEGF was inhibited by KRN633 (Fig. 3A). The average area of the tubes, cell overcrowding, total length of the tubes, and number of capillary connections and paths per field were measured (Fig. 3B). There was limited formation of capillary-like structures without VEGF stimulation. VEGF increased the average area, total tube length, number of capillary connections, and number of paths per field by 2.9-, 3.4-, 39-, and 7.4-fold, respectively. KRN633 at a concentration of 3 nmol/L approximately halved the increase in the number of capillary connections and paths. At a concentration of 10 nmol/L, it also inhibited the increase in average area and total tube length by 50%. At a concentration of 30 nmol/L or more, the parameters of tube formation were reduced to below their basal levels. These findings suggest that KRN633 can block survival signaling by VEGF and trigger the apoptosis of HUVECs at sufficiently high concentrations.
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500 to 700 mm3. Twice-daily administration of KRN633 at 100 mg/kg induced 90% growth inhibition of HT29 tumors and caused the regression of A549 and Du145 tumors by 35% and 60%, respectively (Table 2B). The antitumor effect in each established tumor model was superior to that in the corresponding standard (less well established) tumor model. This suggests that the antitumor activity of KRN633 is less affected by initial tumor size. In the periodic intermittent-dosing model, the resumption of treatment led to the regression of DU145 tumors that had regrown after treatment had ceased; this indicates that tumors are less likely to acquire resistance to KRN633 (Fig. 4).
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KRN633 Inhibits Tumor Angiogenesis and Vascular Permeability
To determine whether the inhibition of tumor growth was associated with a reduction in tumor vessel formation, we examined the histology of implanted A549 tumors in athymic rats. Treatment with 2, 10, and 50 mg/kg KRN633 twice daily p.o. for 14 days reduced the numbers of CD31+ cells in viable regions of the tissue by 15%, 53%, and 76%, respectively (Fig. 5). These doses also increased the percentage of necrotic areas within the A549 tumors, and inhibited tumor growth by 58%, 95%, and >100%, respectively. These findings reflect the antitumor activity in the athymic rat xenograft models (data not shown).
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Pharmacokinetics of KRN633 after p.o. Administration to Mice and Rats
The pharmacokinetic parameters of KRN633 after single p.o. administration to athymic mice and rats are shown in Table 4. KRN633 was absorbed after p.o. administration with a Tmax of 4 to 5 hours in all animals. The Cmax of mice was 2-fold greater than that of rats, and the t1/2 of mice was about one-third of the value in rats. The serum concentration in mice declined relatively rapidly in a monoexponential manner.
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Discussion |
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KRN633 is a novel quinazoline urea derivative that targets the VEGF signaling pathway in endothelial cells by inhibiting the catalytic activity of VEGFR-2 tyrosine kinase. Although our results showed that this activity was relatively weak in the cell-free assay (IC50 = 160 nmol/L), it was highly potent in the cellular assay (IC50 = 1.16 nmol/L). In comparison with other reported VEGFR inhibitors (30–34), KRN633 is a powerful inhibitor of VEGFR-2-mediated signaling in endothelial cells. KRN633 also potently inhibited the MAP kinase activation and proliferation induced by VEGF, but not by bFGF, which coincided with its selectivity for the respective RTKs. Furthermore, KRN633 inhibited tube formation by endothelial cells in an in vitro angiogenesis assay. There is now general agreement that VEGFR-2 is the major mediator of the mitogenic and angiogenic effects of VEGF (35). It is therefore clear that the primary mechanism by which KRN633 blocks VEGF-induced endothelial cell responses and in vitro angiogenesis is the inhibition of VEGFR-2 phosphorylation.
The present study found that KRN633 inhibited VEGFR-1 phosphorylation (IC50 = 11.7 nmol/L). Despite the higher affinity for VEGF, the VEGFR-1 tyrosine kinase is not as active as that of VEGFR-2. Gene-targeting analyses suggested that it acted as a ligand-trapping molecule in embryonic development (36, 37). However, recent reports have shown elevated levels of specific ligands of VEGFR-1, such as placental growth factor and VEGF-B, in some human cancers (38, 39). Placental growth factor–overexpressing Lewis lung carcinoma cells were reported to grow much faster in wild-type mice than in Flt-1 tyrosine kinase domain–deficient mice (40). Furthermore, placental growth factor deficiency in mice can inhibit angiogenesis in many pathologic disorders, including cancer (41). VEGFR-1 activation has been shown to cause the intermolecular transphosphorylation of VEGFR-2, as well as VEGF/placental growth factor heterodimer–activated intramolecular VEGFR cross-talk, through the formation of VEGFR-1/VEGFR-2 (42). VEGFR-1 also plays an important role in the VEGF-dependent migration of macrophages (36, 43, 44), which produce several proangiogenic cytokines and growth factors in tumors (45). Taken together, these findings suggest that VEGFR-1 is involved in pathologic angiogenesis and that the inhibition of VEGFR-1 signaling might contribute to the antiangiogenic and antitumor activities of KRN633.
VEGFR-3 tyrosine kinase is also inhibited by KRN633. VEGFR-3 was initially thought to be restricted to the lymphatic endothelium (46) and, therefore, to be of less relevance to tumor angiogenesis. However, recent data suggest that its level can be elevated in tumor blood vessels during neovascularization and that tumor VEGF-C expression correlates with lymphatic invasion, increased metastasis, and relatively poor clinical prognosis (47, 48). Hence, the KRN633-induced inhibition of VEGFR-3 tyrosine kinase and its signaling in cells could potentially impart a therapeutic benefit.
It should be noted that KRN633 exhibited modest potency versus ligand-induced PDGFR-ß phosphorylation despite its high potency versus VEGFR-2 phosphorylation. According to the IC50 comparison in the cellular assay, KRN633 seemed to be more than 100-fold less potent against PDGFR-ß than VEGFR-2. Although several VEGFR-2 tyrosine kinase inhibitors have been reported (29–34), few are likely to show such highly selectivity. This unique property of KRN633 might lead to different in vivo efficacy and toxicity compared with other VEGFR-2 tyrosine kinase inhibitors, although this remains to be confirmed experimentally.
Based on VEGF signaling inhibition, KRN633 was expected to prevent the formation and survival of new vessels in tumors. Accordingly, we showed a reduction in the microvessel density of A549 tumor xenografts in rats, a decrease in the number of viable regions on the periphery of tumors, and an increase in the avascular and necrotic area in the center of tumors. KRN633 also decreased the vascular permeability of tumors after only 3 days of treatment. This was probably due to the inhibition of VEGF signaling by KRN633 because VEGF functions as a potent vascular permeability factor (49). Vascular permeability and the introduction of a provisional plasma-derived matrix precede and accompany the onset of endothelial cell division and new blood vessel formation in tumors (50). Therefore, the reduction of vascular permeability might be partly responsible for the inhibition of angiogenesis by KRN633.
Taken together, our results suggest that the antitumor effects of KRN633 are secondary to its antiangiogenic action based on the inhibition of VEGFRs. KRN633 is clearly suitable for the treatment of a wide range of solid tumors; it showed in vivo antitumor activities against various cancer cell lines, although it did not substantially inhibit their in vitro proliferation. Interestingly, there were differences in the effects of KRN633 on tumor growth among cancer cell lines, including those derived from the same tissue. This suggests that there might be differences in the mechanism of angiogenesis induction and/or in the reliance on vascular supply for tumor maintenance and growth among these cell lines. Further investigation of the factors affecting susceptibility will be required not only for the design, scheduling, and monitoring of antiangiogenic therapies in clinical settings but also for interpreting the results obtained from such therapies.
Pharmacokinetic analysis revealed that KRN633 was well absorbed after p.o. administration, although the t1/2 was relatively short in mice in particular. Higher trough serum concentrations in simulations and more potent antitumor efficacy of KRN633 were observed in rats. In addition, twice-daily administration, which increased the trough serum concentrations of KRN633 during treatment in simulations, generally resulted in more potent activity than once-daily administration of a similar dose. Therefore, the trough concentration seems to be more significant for antitumor activity than the Cmax. We propose that a target serum concentration exists at which KRN633 can sufficiently inhibit VEGF signaling in tumors. Our findings suggest that the antitumor activity of KRN633 depends on the length of time that the serum concentration remains above this target concentration rather than on the value of Cmax.
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Footnotes |
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Received 3/24/04; revised 8/ 9/04; accepted 10/18/04.
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and DNA synthesis in vascular endothelial cells. EMBO J 2001;20:2768–78.[CrossRef][Medline]
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  O. Kazanskaya, B. Ohkawara, M. Heroult, W. Wu, N. Maltry, H. G. Augustin, and C. Niehrs The Wnt signaling regulator R-spondin 3 promotes angioblast and vascular development Development, November 15, 2008; 135(22): 3655 - 3664. [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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 M. Murakami, S. Iwai, S. Hiratsuka, M. Yamauchi, K. Nakamura, Y. Iwakura, and M. Shibuya Signaling of vascular endothelial growth factor receptor-1 tyrosine kinase promotes rheumatoid arthritis through activation of monocytes/macrophages Blood, September 15, 2006; 108(6): 1849 - 1856. [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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). The cells were then cultured for 78 hours followed by incubation with [3H]thymidine (1 µCi/mL) for 14 hours. The incorporated radioactivity of the cells was measured using a liquid scintillation counter. Points, means (n = 12); bars, SE.
), or vehicle (•), were initiated when tumors reached an average of 107 to 110 mm3. After 10 days, the dosing was stopped to monitor tumor regrowth. KRN633 administration resumed on day 20 and was terminated on day 29. Administration recommenced on day 40 and finally ended on day 49. Points, mean tumor volume per group (n = 8); bars, SE.
