Regorafenib (BAY 73-4506): A new oral multikinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases with potent preclinical antitumor activity


  • Potential conflicts of interest: SMW, LA, GS, KHT and DZ are currently employees of the Bayer HealthCare organisation.


Angiogenesis, a critical driver of tumor development, is controlled by interconnected signaling pathways. Vascular endothelial growth factor receptor (VEGFR) 2 and tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2 play crucial roles in the biology of normal and tumor vasculature. Regorafenib (BAY 73-4506), a novel oral multikinase inhibitor, potently inhibits these endothelial cell kinases in biochemical and cellular kinase phosphorylation assays. Furthermore, regorafenib inhibits additional angiogenic kinases (VEGFR1/3, platelet-derived growth factor receptor-β and fibroblast growth factor receptor 1) and the mutant oncogenic kinases KIT, RET and B-RAF. The antiangiogenic effect of regorafenib was demonstrated in vivo by dynamic contrast-enhanced magnetic resonance imaging. Regorafenib administered once orally at 10 mg/kg significantly decreased the extravasation of Gadomer in the vasculature of rat GS9L glioblastoma tumor xenografts. In a daily (qd)×4 dosing study, the pharmacodynamic effects persisted for 48 hr after the last dosing and correlated with tumor growth inhibition (TGI). A significant reduction in tumor microvessel area was observed in a human colorectal xenograft after qd×5 dosing at 10 and 30 mg/kg. Regorafenib exhibited potent dose-dependent TGI in various preclinical human xenograft models in mice, with tumor shrinkages observed in breast MDA-MB-231 and renal 786-O carcinoma models. Pharmacodynamic analyses of the breast model revealed strong reduction in staining of proliferation marker Ki-67 and phosphorylated extracellular regulated kinases 1/2. These data demonstrate that regorafenib is a well-tolerated, orally active multikinase inhibitor with a distinct target profile that may have therapeutic benefit in human malignancies.

Activation of multiple signaling pathways in the tumor microenvironment, including the receptor tyrosine kinases (RTKs) vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR) and platelet-derived growth factor receptor (PDGFR), controls the initiation of tumor neoangiogenesis.1 Of these, VEGF was the first vascular-specific growth factor to be characterized and is one of the most critical drivers of tumor angiogenesis.2 Of the three closely related members of the VEGFR family, the major effects of VEGF on vessel growth and permeability are mediated via VEGFR2.3, 4 A range of multitargeted kinase inhibitors has been developed, with a focus on targeting VEGFR2 to treat cancer. They include sorafenib, which has been approved for the treatment of renal cell carcinoma (RCC) and hepatocellular carcinoma5 and sunitinib, which has been approved for the treatment of RCC and imatinib-resistant gastrointestinal stromal tumor (GIST).6 Furthermore, the VEGF-intercepting antibody bevacizumab has been shown to be clinically effective in antiangiogenic tumor therapy.7

Many other growth factors and receptors, in addition to VEGF and its receptors, have been shown to work in a complementary and coordinated manner with established pathways to regulate tumor growth and angiogenesis, suggesting that blockade of multiple growth factors and receptor pathways may be needed to increase the efficacy of cancer therapy.8 These pathways include FGFR, which is activated by a range of functionally defined ligands, leading to tumor cell proliferation and differentiation via several downstream signaling pathways9 and PDGFR, which supports vessel stabilization by modulating the recruitment and maturation of pericytes.10

Tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2 (TIE2) is a crucial regulator of angiogenesis11 that is exclusively or predominantly expressed on endothelial cells and is indispensible for the maturation of immature vessels, via interactions with the ligands angiopoietin (Ang) 1, Ang2, VEGF and FGF.8 Ang1 and 2 are angiogenic factors that have divergent effects on binding to TIE2 and are modulated by the presence or absence of VEGF and FGF.12 Constitutive Ang1 expression is found in pericytes, smooth muscle cells, fibroblasts and some tumor cells, while Ang2 expression is tightly controlled and almost exclusively expressed by endothelial cells.11 Expression studies have shown that Ang2 and/or VEGF are upregulated in a variety of human tumor samples, including endometrial adenocarcinoma, bladder cancer and gastric cancer, and suggest that the balance between Ang1 and Ang2 is important in neoangiogenesis.13–15

In addition to upregulation associated with VEGF signaling, FGFR signaling appears to upregulate Ang2 expression and reduce Ang1, thus promoting tumor vascular disruption via TIE2.9 This, in combination with FGFR-stimulated upregulation of VEGF expression in tumors, highlights the potential for FGFR signaling to stimulate the development of abnormal tumor vasculature.

FGFR signaling plays a key role in many functions including cell proliferation, survival and migration, and can promote cancer development by affecting a range of major downstream processes.16 An emerging body of evidence has identified roles for FGFR signaling in oncogenic processes, including proliferation, survival, migration and angiogenesis.16

The receptor tyrosine kinases (RTKs) RET and KIT represent valid targets for cancer therapy. Activating mutations of the RET gene have been identified as driving oncogenic events in subsets of thyroid carcinomas, and the presence of oncogenic KIT mutations in GIST is well documented.17, 18

In this report, we provide data characterizing the biochemical and in vitro cellular activity, and in vivo antitumor efficacy, of regorafenib (BAY 73-4506; Bayer Schering Pharma AG, Berlin, Germany). This novel oral multikinase inhibitor possesses a distinct profile targeting angiogenic, stromal and oncogenic RTKs. Regorafenib demonstrated very robust antitumor and antiangiogenic activity in a panel of tumor models that support its clinical development.


Ang: angiopoietin; ATCC: American Type Culture Collection; ATP: adenosine-5′-triphosphate; BC: breast cancer; BSA: bovine serum albumin; CHO: Chinese hamster ovary; CRC: colorectal cancer; DCE-MRI: dynamic contrast-enhanced magnetic resonance imaging; DMEM: Dulbecco's Modified Eagle Medium; DMSO: dimethylsulfoxide; ECCC: European collection of cell cultures; EDTA: ethylene diamine tetraacetic acid; ELISA: enzyme-linked immunosorbent assay; FBS: fetal bovine serum; FGFR: fibroblast growth factor receptor; GIST: gastrointestinal stromal tumor; HAoSMC: human aortic smooth muscle cell; hiFBS: heat-inactivated fetal bovine serum; HUVEC: : human umbilical vascular endothelial cell; IAUC360: area under the curve of the initial 360 sec; IC50: inhibitory concentration of 50%; IHC: immunohistochemistry; MRI: magnetic resonance imaging; MVA: microvessel area; NCI: National Cancer Institute; nd: not determined; PBS: phosphate-buffered saline; PDGFR: platelet-derived growth factor receptor; pFRS2: phosphorylated FGFR substrate 2; qd: daily; RCC: renal cell carcinoma; RTK: receptor tyrosine kinase; SD: standard deviation; SEM: standard error of the mean; SMCGM: smooth muscle cell growth medium; TEMs: TIE2-expressing monocytes; TGI: tumor growth inhibition; TIE2: tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2; VEGFR: vascular endothelial growth factor receptor; VHL: Von-Hippel Lindau; WB: Western blotting; wt: wild type

Material and methods

Preparation of chemical compounds

Synthesis of regorafenib has been described in the literature.19 Its chemical name is 4-[4-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]-n-methylpyridine-2-carboxamide, and the structural formula is shown in Table 1.

Table 1. Structural formula and biochemical and cellular activities of regorafenib
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For administration to mice, regorafenib was formulated as a solution in either PEG400/125 mM aqueous methanesulfonic acid (80/20) or polypropylene glycol/PEG400/Pluronic F68 (42.5/42.5/15 + 20% Aqua). All compound preparations were stored at room temperature in the dark and used the same day.

Sunitinib and vatalanib were synthesized according to published procedures.20, 21

Paclitaxel was purchased from Bristol-Myers Squibb, Wallingford, CT.

Cell lines and reagents

Cell lines were obtained from the American Type Culture Collection, the National Cancer Institute tumor repository, the European Collection of Cell Cultures, and academic institutions (Supporting Information Table 1). All tumor lines were maintained and propagated in RPMI-1640 media (GIBCO®, Invitrogen Corp., Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (hiFBS, Wallingford, CT; JRH Biosciences, Lenexa, KS) at 37°C and 5% CO2 for ≤6 months. Human umbilical vascular endothelial cells (HUVECs) and human aortic smooth muscle cells (HAoSMCs) were cultured with 2% fetal calf serum (FCS) or 0.1% bovine serum albumin (BSA), respectively.

In vitro

Additional methods are available online (Supporting Information Materials and methods).

Kinase assays

In vitro assays using recombinant VEGFR2 (murine aa785–aa1367), VEGFR3 (murine aa818–aa1363), PDGFR-β (aa561–aa1106), RAF-1 (aa305–aa648) and BRAFV600E (aa409–aa765) kinase domains were performed as previously described.22 Recombinant KIT (aa544-aa976) was purchased from ProQinase GmbH (Freiburg, Germany). Initial in vitro kinase inhibition profiling was performed at Millipore Corporation (Billerica, MA) at a fixed 1 μM compound concentration under Millipore standard conditions [10 μM adenosine-5′-triphosphate (ATP) concentration]. Inhibitory concentration of 50% (IC50) values were determined from selected responding kinases, e.g., VEGFR1 and RET. TIE2 kinase inhibition was measured with a homogeneous time-resolved fluorescence (HTRF) assay using a recombinant fusion protein of glutathione-S-transferase, the intracellular domain of TIE2 and the peptide biotin-Ahx-EPKDDAYPLYSDFG (Biosynthan GmbH, Berlin-Buch, Germany) as substrate (Supporting Information Materials and methods).

Cellular mechanistic phosphorylation assays

VEGFR2 phosphorylation was analyzed by enzyme-linked immunosorbent assay (ELISA) and Western blotting

NIH-3T3 cells transfected with human VEGFR2 were plated at 30,000 cells/well in 96-well plates in Dulbecco's Modified Eagle Medium (DMEM; Sigma-Aldrich, St. Louis, MO) containing 10% FBS; 6 hr after plating, media was changed to 0.1% BSA/DMEM and incubation continued for 24 hr. Cells were treated with vehicle or various concentrations of regorafenib in 0.1% BSA/DMEM/0.1% dimethylsulfoxide (DMSO) for 1 hr at 37°C, prior to stimulation with recombinant VEGF165 at 30 ng/mL final concentration for 5 min. Cells were washed with cold phosphate-buffered saline (PBS) and lysed in 100 μL of lysis buffer (50 mM HEPES, pH 7.2, 1% Triton X-100, 1 mM Na3VO4, 150 mM NaCl, 10% glycerol, 1.5 mM ethylene glycol tetraacetic acid and complete protease inhibitor cocktail). VEGFR2 was captured on plates coated with anti-VEGFR2 monoclonal antibody (#ab9530; Abcam, Cambridge, MA) overnight at 4°C. pVEGFR2 signal was detected with a rabbit polyclonal antibody against pTyr951-VEGFR2 (#44-1040; Invitrogen Corp.) and anti-rabbit IgG peroxidase-linked antibody (#NA934; GE Healthcare). For determination of the cellular off-rate of the compound, cells were treated with compounds as above, washed 5× with 0.1% BSA/DMEM and incubated for 0, 1, 2, 3, 8 and 24 hr without compound before treatment with recombinant VEGF165 for 5 min. Subsequent pVEGFR2 was determined as described above. For comparison, cells were treated similarly but omitting the 5× washing step.

For Western blotting experiments, 1×106 cells/well were plated in 6-well plate in complete growth medium, as described in Wilhelm et al.22 After 6 hr, medium was changed to 0.1% BSA/DMEM and incubation was continued for 24 hr. Cells were preincubated with compound for 30 min followed by stimulation with VEGF165 at 30 ng/mL for 10 min. Cell lysates were prepared and probed with anti-pTyr1054-VEGFR2 (BioSource International, Camarillo, CA) or anti-VEGFR2 antibody (#sc-315; Santa Cruz Biotechnology, Santa Cruz, CA).

Cell proliferation assays

For proliferation assays, GIST 882 and TT cells were grown in RPMI medium containing L-glutamine, and MDA-MB-231, HepG2 and A375 cells in DMEM always containing 10% hiFBS. Cells were trypsinized, plated at 5×104 cells/well in 96-well plates in complete media containing 10% FBS and grown overnight at 37°C. The next day, vehicle or regorafenib serially diluted in complete growth media to between 10 μM and 5 nM final concentrations, and 0.2% DMSO, was added and incubation was continued for 96 hr. Cell proliferation was quantified using CellTitre-Glo™ (Promega Corporation, Madison, WI).

Dynamic contrast-enhanced magnetic resonance imaging

For DCE-MRI experiments, Fischer 344 rats (Charles River GmbH, Sulzfeld, Germany) were inoculated with 3×106 GS9L cells intramuscularly into the left thigh. Treatment was initiated when the tumor reached between 300 and 700 mm3. MRI was performed using a Siemens 1.5T Avanto MRI system equipped with a dedicated animal receiver coil (Siemens Medical Solutions, Erlangen, Germany). Regorafenib was administered orally, either as single administration (daily [qd]×1) or qd×4 at a dose of 10 mg/kg body weight. DCE-MRI examinations were performed using the contrast agent Gadomer-17 (Bayer Schering Pharma AG, Berlin, Germany) before therapy and 4, 8, 24, 48 hr and 4 days after the last regorafenib administration. For MRI, animals were anesthetized using 1.5% Isoflurane (Baxter, Unterschleißheim, Germany) in O2/N2O. Gadomer-17 was injected intravenously at a dose of 50 μmol Gd/kg body weight into the tail vein at a rate of 0.5 mL/s using an automated injection device (Spectris Solaris, Medrad, Volkach, Germany). For DCE-MRI data acquisition, a 2D turbo flash saturation recovery pulse sequence was used with the settings: echo time (TE): 1.63 ms, repetition time (TR): 350 ms, inversion time (TI): 180 ms, flip angle (FL): 10°, four averages, 5 mm slice thickness at 0.8 × 0.8 mm2 in plane resolution. The acquisition time for 1 image was 1.4 s, and 254 images were acquired over ∼6 min. Before contrast agent injection, six images were acquired as baseline. For data evaluation, a region of interest was defined covering the complete tumor on one acquired slice. Signal intensity in the region of interest over time was analyzed. Area under the curve of the initial 360 seconds after Gadomer-17 injection (IAUC360) of MRI signal intensity over time graphs in tumor were normalized to muscle as nonaffected reference tissue in each animal and used for data evaluation. Tumor volume was determined at various time points: at staging (predose), at qd×4 of oral dosing (day 4 post-treatment) and at days 6 and 8 after staging using MRI pulse sequence set at: 3D gradient recalled echo, TE: 9 ms, TR: 16 ms, FL: 40°, one average, 60 slices at 0.7 mm slice thickness and 0.35 × 0.35 mm2 in plane resolution. Volume was calculated by slice per slice tumor area evaluation. Statistical analysis was performed using unpaired two-sided Student's t test.

Examination of microvessel area, Ki-67 and MAPK in tumor xenograft models

Animals with tumors of ∼200 mg were treated orally with regorafenib at 10 and 30 mg/kg on a qd×5 schedule. Subsequently, tumors were harvested, paraffin-embedded, and analyzed by immunohistochemistry (IHC). Tumor endothelial cells were detected using an antibody against CD-31 (#M-20, 1:750; Santa Cruz Biotechnology). Inhibition of cell signaling was assessed using an antibody against pERK1/2 (#9101L, 1:100; Cell Signaling Technology (CST), Danvers, MA), and tumor cell proliferation was analyzed using an antibody against Ki-67 (#A0047, 1:50; Dako, Carpinteria, CA), as previously described.22, 23

For tumor MVA determinations, CD-31 stained slides were coded before analysis. Tissue sections were viewed using a 10× objective magnification (0.644 mm2 per field). Four fields per section were randomly analyzed, excluding peripheral surrounding connective and central necrotic tissues. CD-31-positive areas were quantified using the software Image-Pro Plus version 3.0 (Media Cybernetics, Bethesda, MD) and SIS image analysis (Olympus Soft Imaging Systems GmbH, Münster, Germany). The data are presented as MVA, %. Data were analyzed statistically with one-way analysis of variance on ranks using Kruskal–Wallis, and the Dunnett method was used for comparison with the vehicle group (SigmaStat 3.0; Systat, San Jose, CA); p < 0.05 was considered significant.

Tumor xenograft experiments

Female athymic NCr nu/nu mice (Taconic Farms, Germantown, NY), kept in accordance with Federal guidelines, were subcutaneously inoculated with 5×106 Colo-205 or MDA-MB-231 cells or implanted with 1 mm3 786-O tumor fragments. When tumors reached a volume of ∼100 mm3, regorafenib or vehicle control was administered orally qd×21 in the 786-O model, and qd×9 in the Colo-205 and MDA-MB-231 models, respectively, at doses of 100, 30, 10, and 3 mg/kg. Paclitaxel was administered intravenously at 10 mg/kg in ethanol/Cremophor EL® (BASF, Ludwigshafen, Germany)/saline (12.5%/12.5%/75%) every 2 days × 5. Tumor size (volume) was estimated twice weekly (l×w2)/2, and the percentage of tumor growth inhibition (TGI) was obtained from terminal tumor weights (1-T/C×100). Mice were weighed every other day starting from the first day of treatment. The general health status of the mice was monitored daily.


A distinct biochemical and cellular kinase profile characterizes regorafenib as an antiangiogenic and antitumorigenic agent

During the identification of new kinase inhibitors for cancer treatment, we have identified regorafenib, a novel bi-aryl urea compound (Table I). To characterize regorafenib, we first determined its kinase inhibition profile in vitro, in biochemical assays. It potently inhibited a distinct set of kinases, including the angiogenic and stromal RTKs VEGFR1-3, TIE2, FGFR1 and PDGFR-β, with IC50 values ranging from 4 to 311 nM, and the oncogenic RTKs KIT and RET, along with the intracellular signaling kinases c-RAF/RAF-1 and B-RAF and its V600E mutant, with IC50 values ranging from 1.5 to 28 nM (Table I). Additional kinases such as DDR2, EphA2, PTK5, p38α and p38β were also inhibited with IC50 values below 100 nM in biochemical assays (data not shown); however, these activities have not been tested in cellular assays. No inhibition was observed for kinases of the epidermal growth factor receptor family, the protein kinase C family, cyclin-dependent kinases, insulin and insulin growth factor receptor kinase, MET, MEK, ERK1/2 and AKT, even with concentrations up to 1 μM (data not shown).

Key target kinases were then tested in mechanistic cellular phosphorylation assays. Inhibition of receptor autophosphorylation was measured in cells which express the endogenous kinases FGFR, PDGFR-β, B-RAF or after transfection VEGFR2, VEGFR3, TIE2, after stimulation with respective ligands. For mutant RET and KIT RTKs, we used tumor cells which express constitutively active kinase mutants (cKITK642E, RETC634W). Phosphorylation of kinases was determined by ELISA and/or Western blotting (Table 1, Fig. 1 and Supporting Information Fig. 1). Regorafenib potently inhibited VEGFR2 autophosphorylation in NIH-3T3/VEGFR2 cells with an IC50 of 3 nM, as measured by ELISA (Table 1). Interestingly, similar results were obtained with VEGFR2 pTyr951 detected in ELISA (Table 1) and VEGFR2 pTyr1054 in Western blotting (Fig. 1a). Potent inhibition of TIE2 autophosphorylation (Fig. 1b) was observed in vanadate-stimulated Chinese hamster ovary (CHO)-TIE2 cells with an IC50 of 31 nM, as determined by ELISA (Table 1 and Supporting Information Fig. 1) and by Western blotting following TIE2 immunoprecipitation (Fig. 1b). In HAoSMCs, regorafenib inhibited PDGFR-β autophosphorylation after stimulation with PDGF-BB, with an IC50 of 90 nM by Western blotting (Fig. 1c).

Figure 1.

Regorafenib inhibits key kinase targets in cells expressing VEGFR2, TIE2, PDGFR-β, or FGFR. Transfected cells expressing (a) VEGFR2 (NIH-3T3) or (b) TIE2 (CHO) and nontransfected cells expressing (c) PDGFR-β (HAoSMC) or (d) FGFR (MCF-7) were activated with the corresponding ligands, or with vanadate in the case of TIE2, in the presence of the indicated concentrations of DMSO (control) or regorafenib. Cell lysates were analyzed by Western blotting (WB) with the phospho-specific antibodies against the indicated target proteins, as described in the Material and Methods Section. TIE2 was immunoprecipitated (IP) prior to immunoblotting, and pTIE2 was detected by the anti-phosphotyrosine antibody 4G10 (b). Due to the cross-reactivity of the antibody against pFRS2 with the unphosphorylated FRS2, phosphorylation inhibition is demonstrated by the mobility shift between both proteins during SDS-PAGE (d).

Regorafenib also inhibited FGFR signaling in MCF-7 breast cancer (BC) cells stimulated with FGF10. Inhibition of phosphorylated FGFR substrate 2 (pFRS2) and the downstream signaling kinase pERK1/2 was detected by Western blotting (Fig. 1d). pFRS2 and pERK1/2 were inhibited, with very similar estimated IC50 values of ∼200 nM. Only weak inhibition of pAKT was observed even at the highest dose tested (3 μM). Similar results were seen for pFRS2 and pERK inhibition when MCF-7 cells were stimulated with FGF2 or FGF7 (data not shown). Inhibition of the MAPK signaling pathway was measured using pERK1/2 ELISA in the pancreatic tumor cell BxPC-3 (which expresses wild-type K-RAS and B-RAF; IC50 = 380 nM), the melanoma cell line LOX (B-RAFV600E; IC50 = 272 nM) and the MDA-MB-231 cell line (B-RAFG464V and K-RASG13D; IC50 = 43 nM) (Table 1). Regorafenib very potently inhibited the mutant receptors KITK642E and RETC634W, with IC50 values of ∼20 and ∼10 nM, respectively (Table 1). Constitutive kinase activation of these receptor kinases is reported to lead to tumorigenicity of the GIST cell line GIST 882 and thyroid cell line TT, respectively.24, 25 In general, the level of inhibition observed in the cellular assay closely correlated with that observed in the biochemical assays, except for TIE2, which showed an inhibition ∼10-fold weaker in the biochemical assay compared with the cellular autophosphorylation assay.

We then investigated the antiproliferative effects of regorafenib on vascular and tumor cell lines (Table 1). Consistent with the potent biochemical inhibition of VEGFR2, regorafenib inhibited the proliferation of VEGF165-stimulated HUVECs, with an IC50 of ∼3 nM. Proliferation of FGF2-stimulated HUVECs and of PDGF-BB-stimulated HAoSMCs was observed, with IC50 values of 127 and 146 nM, respectively. Tumor cell proliferation was inhibited to various degrees, depending on the cell line used. In the GIST 882 and thyroid TT cell lines, which are known to express activated oncogenic mutants of KIT and RET receptors, a very potent inhibition of proliferation was observed, with IC50 values of ∼40 nM (Table 1). However, in HepG2, SW620, Colo-205 and A375 tumor cell lines, inhibition of proliferation was in the range of 500–3000 nM (Table 1). Other tumor cell lines originating from breast (e.g., MCF-7), pancreas (e.g., BxPC-3) and lung (e.g., NCI-H460) were inhibited, with an IC50 of 2–5 μM. The antiproliferative effects may in part be mediated by induction of apoptosis as observed in HepG2 cells, which are not driven by activated oncogenes and demonstrated by induction of caspases 3/7 and DNA fragmentation (data not shown).

DCE-MRI reveals prolonged inhibition of extravasation by regorafenib

We assessed the pharmacodynamic effect of regorafenib on the tumor vasculature in vivo, in a rat GS9L glioblastoma xenograft grown intramuscularly in the rat thigh, by DCE-MRI using Gadomer-17 (Fig. 2). Gadomer-17 is a preclinical macromolecular MRI contrast agent which is better suited to display changes in blood vessel permeability than low molecular contrast agents that extravasate more easily.26 Treating tumor-bearing rats with a single oral dose of regorafenib at 10 mg/kg caused a significant decrease in tumor perfusion and extravasation of the contrast agent, as measured by IAUC360 after infusion of the agent, normalized to rat muscle tissue (Fig. 2a). A significant reduction of the normalized IAUC360 was observed by 10 hr (p < 0.001) after regorafenib treatment and persisted for up to 2 days (p < 0.01) when compared with vehicle. The modulation of extravasation by regorafenib was also analyzed after four daily administrations of regorafenib at 10 mg/kg (qd×4 schedule), which was found to correlate with therapeutic antitumor efficacy in this model. A pharmacodynamic effect very similar to the single-dose study was observed, as determined by a statistically significant decrease of the normalized IAUC360, which persisted for up to 2 days after treatment (Fig. 2b). The qd×4 dosing schedule prevented GS9L tumor growth, as measured by MRI pharmacodynamics (Fig. 2c). Interestingly, no tumor regrowth was observed for 4 days after the last dose. Thus, TGI in this rat GS9L glioblastoma model is consistent with the antiangiogenic effect of regorafenib on the tumor vasculature.

Figure 2.

Regorafenib inhibits tumor vasculature and tumor growth in a rat GS9L glioblastoma model: time-course analysis by DCE-MRI. Rats carrying glioblastoma tumors in their thighs were treated at (a) single dose or (b) for 4 consecutive days with regorafenib at 10 mg/kg, and the pharmacodynamic effects were analyzed by DCE-MRI, using Gadomer-17 as a contrast agent. Change of IAUC360 of Gadomer DCE-MRI (normalized to muscle as reference tissue) versus baseline before compound application was plotted over time. (c) Volume of intramuscular rat GS9L tumors pre- (day 0) and post-treatment with regorafenib or vehicle was determined by MRI. Data points are means of n = 8 animals or n = 9 in the regorafenib group treated for 4 days. Error bars indicate SEM. Statistical significance of the difference between regorafenib- and vehicle-treated animals is given by unpaired two-sided Student's t test (*p < 0.01; **p < 0.001; ***p < 0.0001).

Regorafenib inhibits tumor angiogenesis, tumor cell proliferation and MAPK signaling in vivo

The antiangiogenic effect of regorafenib on the vasculature of tumors was analyzed in tumor xenograft tissue sections by measuring MVA by IHC staining with the endothelial cell marker CD-31 (Fig. 3a). MVA was significantly reduced in a human Colo-205 colorectal cancer (CRC) xenograft grown in mice treated with regorafenib at a qd×5 schedule (Fig. 3b). The CD-31 positive area was significantly reduced from 0.39% in untreated animals to 0.05% and 0.04% (p < 0.05) in animals treated with 10 or 30 mg/kg, respectively. A qualitative reduction in vascularity was also observed in murine xenografts of the human MDA-MB-231 BC cell line after a qd×5 treatment with regorafenib at 30 mg/kg (Fig. 4a). In these sections, the reduced CD31 staining was accompanied by tumor necrosis.

Figure 3.

Regorafenib significantly reduces tumor MVA in the Colo-205 CRC xenograft model. (a) Xenograft tissue sections from untreated and vehicle- and regorafenib-treated animals qd × 5 (n = 5/group) were stained by IHC with antibodies against CD-31, as described in the Material and Methods Section. Sections were counterstained with hematoxylin and eosin. CD-31-positive microvessels are indicated by brown staining. Representative vessels are indicated by arrows. (b) MVA was calculated, as described in Materials and Methods Section and plotted. Tumor-bearing mice received a 5-day treatment of regorafenib at the indicated doses after tumors had reached a size of 100–200 mg. *p < 0.05 versus vehicle control.

Figure 4.

Regorafenib exhibits antitumorigenic and antiangiogenic effects in the MDA-MB-231 breast xenograft model. Xenograft tissue sections from vehicle- and regorafenib-treated animals were analyzed by IHC using phospho-specific antibodies against (a) the endothelial cell marker CD-31, (b) the cell proliferation marker Ki-67 and (c) pERK1/2. Positive staining is indicated by a brown color. Blood vessels in (a) are indicated by arrows. Tumors were taken from mice at the end of a 5-day treatment period with 30 mg/kg regorafenib.

As detected by IHC for Ki-67 in the MDA-MB-231 model, tumor cell proliferation appeared to be drastically inhibited. Xenograft tissue sections showed reduced proliferation levels in treated mice compared with untreated controls (Fig. 4b). Furthermore, the level of activated pERK1/2, which was also detected by IHC, was strongly reduced in treated mice (Fig. 4c), indicating that, in vivo, regorafenib effectively inhibited the RAF/MEK/ERK signaling cascade. These data show that regorafenib has a consistent effect on tumor neoangiogenesis in both BC and CRC xenograft models, and that in the BC model, reduced proliferation also contributes to TGI by the compound.

Regorafenib exhibits antitumor activity in multiple murine xenograft models

The in vivo antitumor activity of regorafenib was analyzed in human xenograft models in mice. Regorafenib dosed qd orally inhibited tumor growth in a dose-dependent manner in multiple xenograft models, including models derived from CRC (Colo-205), BC (MDA-MB-231) and RCC (786-O) tumors (Fig. 5 and Supporting Information Table 2). Regorafenib effectively inhibited growth of the Colo-205 xenografts in the dose range of 10–100 mg/kg (Fig. 5a), reaching a TGI of ∼75% at day 14 at the 10 mg/kg dose. A slow regrowth was observed at all doses when treatment was terminated after 9 days.

Figure 5.

In vivo antitumor efficacy of regorafenib. Mice bearing xenografts of (a) the human CRC cell line Colo-205 (B-RAFV600E), (b) the human BC cell line MDA-MB-231 (K-RASG13D, B-RAFG464V) or (c) the human RCC cell line 786-O (Von-HippelLindau gene −/−) were treated with the indicated doses of regorafenib for periods indicated by the solid lines (⟷). For comparison, the 786-O model was treated with intravenous paclitaxel every 2 days × 5. Treatment was initiated when tumors reached 100–200 mg. Tumor weights were calculated, as described in Materials and Methods section, and are given as means of n = 10. Errors are given as SEM.

In the MDA-MB-231 model, regorafenib was highly efficacious at a dose as low as 3 mg/kg, resulting in a significant TGI of 81%, which increased to ∼93% at doses of 10 and 30 mg/kg, where tumor stasis was reached (Fig. 5b). Indeed, one complete and six partial responses (defined as a reduction in tumor volume of at least 50%) were observed in the 10 animals treated with regorafenib at 30 mg/kg. In contrast to the Colo-205 model, a sustained, dose-dependent delay in tumor growth was observed after treatment stopped, which eventually led to tumor regrowth after ∼4–7 days.

Regorafenib also very efficiently inhibited the growth of the 786-O RCC model (Fig. 5c), which is highly vascularized due to a homozygous mutation of the Von-HippelLindau gene, leading to increased synthesis of VEGF. TGI >90% was observed at the end of a 21-day dosing period with regorafenib 10 and 30 mg/kg. Interestingly, clear signs of tumor shrinkage were observed in this model, with six and four partial responses out of 10 animals recorded in the 10 mg/kg and 30 mg/kg groups, respectively. Paclitaxel, a highly potent chemotherapeutic, was weakly active in this model at 10 mg/kg, with TGI of only 21% at day 35. Recent data indicate that regorafenib inhibited not only the growth of syngeneic primary 4T1 breast tumors growing orthotopically in the fat pad, but also inhibited the formation of tumor metastasis in the lung (data not shown).

In general, regorafenib significantly inhibited tumor growth in a wide range of additional xenografts derived from lung, melanoma, pancreatic and ovarian tumor cell lines (Supporting Information Table 2) at doses of 10 and 30 mg/kg, which correspond to clinically efficacious doses.27 Regorafenib was very well tolerated in mice up to 100 mg/kg qd×9 without significant weight loss or animal lethality (Supporting Information Fig. 2), suggesting a high apparent therapeutic index. However, further nonclinical toxicology studies in additional animal species are required for a final assessment of safety and tolerability.


Protein kinase inhibitors from the urea class have been described in the literature since 1996.28 Notably, sorafenib, a diphenylurea, was discovered in the late 1990s and was launched in the USA in 2005 as a new treatment option for advanced RCC. Despite this early success, the high medical need in the management of malignancies requires the development of new and improved therapeutic agents. Regorafenib results from a novel discovery program aimed at the optimization of potency and drug-like properties within the well-established urea class. The structure of regorafenib differs from that of sorafenib by the addition of a fluorine atom in the center phenyl ring (Table 1) and leads to a similar but distinct biochemical profile compared with sorafenib.22, 29, 30 In addition, regorafenib appears to be pharmacologically more potent.

Regorafenib potently inhibits the angiogenic and stromal RTKs VEGFR1, 2, and 3, TIE2 and PDGFR-β that promote tumor neovascularization, vessel stabilization and lymphatic vessel formation and play an important role in the tumor microenvironment, which all contribute to tumor development and metastasis formation.8, 11 Regorafenib demonstrated nanomolar inhibition (3–200 nM) of the RTKs VEGFR2, PDGFR-β and FGFR in biochemical and cellular assays (Table 1 and Fig. 1). The biochemical kinase inhibitory activity of ∼300 nM against recombinant TIE2 was ∼10-fold higher than that found in the vanadate-induced CHO TIE2 autophosphorylation assay. Based on its structure, regorafenib is considered to be a type 2 kinase inhibitor31 which binds to the inactive kinase in a DFG-out conformation, which is supported by preliminary modeling studies (unpublished data). This discrepancy may be partly due to the ratio of these two DFG conformations, which may be different in the biochemical compared with cellular TIE2 assays reported here. Competition for regorafenib binding by ATP is diminished due to a poor binding of ATP to the inactive kinase conformation. In addition, intracellular accumulation of regorafenib (which has not been studied) could contribute to its increased TIE2 activity in cells.

The Ang/TIE2 pathway is regarded as a key angiogenic signaling pathway. Expression of Ang2 associated with high expression of VEGF and enhanced angiogenesis has been observed in tumor types including hepatocellular carcinoma32 and gastric cancer,33 and Ang2 expression in tumors has been linked to invasive and metastatic phenotypes in gliomas and gastric, colon, breast and prostate cancer.13, 34 Neutralizing Ang2 by peptibodies such as AMG 38635 resulted in significant TGI when tested preclinically in tumor xenograft models and was often accompanied by significant reduction of tumor vessels. AMG 386 has advanced into clinical development for cancer therapy, and recent reports on Phase I data demonstrated pharmacodynamic activity measured by changes in Ktrans by DCE-MRI in 10/30 patients treated.36 However, the clinical activity as defined by complete or partial response was restricted to four patients with advanced solid cancer in a Phase I trial when used in combination with oxaliplatin, leucovorin and fluorouracil.37 These data not only confirm the Ang/TIE2 signaling pathway as an attractive antiangiogenic target in cancer therapy but also indicate that Ang/TIE2 selective inhibitors such as AMG 386 may need to be developed in combination with other targeted agents (e.g., bevacizumab or cetuximab) and/or chemotherapy.

We have used DCE-MRI, a preclinically and clinically well-established, noninvasive imaging technology,38 to measure the effects of regorafenib on the tumor vasculature in vivo. We observed a prolonged decrease in the vascular hyperpermeability and blood volume in rat glioblastoma tumors. The reduced total tumor blood volume may be explained by a decrease in the number of microvessels and by a transient normalization of the tumor blood vessels.39 This is supported by the observation of a decrease in tumor microvasculature in vivo after 5 days of oral dosing with regorafenib (Figs. 3 and 4a), suggesting that regorafenib treatment is also accompanied by a loss of neovasculature in glioblastoma tumors. The prolonged pharmacodynamic effects of regorafenib on tumor blood volume/permeability in this model may be explained by the slow off-rate observed in a cellular VEGFR2 autophosphorylation assay (Supporting Information Fig. 3) or the combined inhibition of the VEGFR2 and TIE2 pathways, or both. Additional experiments to further clarify the role of TIE2 inhibition by regorafenib are ongoing.

In general, a combined blockage of VEGFR2 and TIE2 signaling with regorafenib may exert more profound antiangiogenic effects than inhibition of VEGF signaling alone. This is supported by recent preclinical results from Tsai and Lee,40 showing synergistic activity when combining anti-Ang/TIE2 (s-TIE2 Fc fusion) and the multikinase inhibitor sorafenib, which led to increased overall survival in a preclinical model of melanoma.

Combined inhibition of several proangiogenic pathways may prevent resistance or prolong progression-free survival. Indeed, adaptive responses by the tumor and its vasculature to anti-VEGF therapy have been hypothesized.41 Such responses include hypoxia-mediated induction of other proangiogenic factors, such as Ang1 and FGFs,42, 43 or upregulation of PDGF-C expression in tumor-associated fibroblasts.44 Furthermore, TIE2-expressing monocytes (TEMs) appear to play an important role in tumor angiogenesis by secreting proangiogenic signals; interestingly, TEMs are attracted to the tumor by Ang2 and their TIE2 expression is upregulated under hypoxic conditions.45 Inhibition of TIE2 by regorafenib may interfere with the proangiogenic activities of TEMs and may thereby improve antiangiogenic therapy.

Regorafenib also demonstrated potent inhibition (20–40 nM) of the oncogenic RTKs KITK642E and RETC634Win vitro (Table 1), which indicates that regorafenib may have clinical potential in tumor types driven by mutated RET, which occurs in a subset of medullary thyroid tumors, or mutated KIT in GIST.17, 18 Imatinib, a potent KIT inhibitor, is currently used as first-line therapy of GIST patients, followed by sunitinib treatment, when tumors become resistant or are intolerant to imatinib treatment. Recent in vitro evidence indicates that sunitinib inhibited KIT variants containing mutations in the drug/ATP-binding pocket that confers resistance to imatinib, such as T670I and V654A.46 Preliminary characterization of regorafenib demonstrated encouraging KIT inhibitory activity in vitro, with IC50 values ranging from 12 to 130 nM on imatinib-resistant KIT double mutants, which carry a deletion of amino acids 557–558 in the juxtamembrane region (exon 11) and secondary mutations at T670I and V654A in the ATP-binding pocket or D816G, N882K and Y832D in the activation loop.29 Further molecular characterization against imatinib- and sunitinib-resistant KIT mutants is warranted.

In biochemical assays, regorafenib potently inhibited the serine/threonine kinase BRAF, a downstream target of the RAS signaling pathway, and its oncogenic mutant B-RAFV600E (Table 1). However, no correlation was observed between the efficacy of regorafenib in inhibiting either tumor cell proliferation in vitro (Table 1) or tumor growth in vivo (Fig. 5 and Supporting Information Table 2) and the mutation status of either BRAF or K-RAS. Furthermore, inhibition of the MAPK pathway was observed in treated MDA-MB-231 tumors (Fig. 4c), consistent with in vitro MAPK inhibition (Table 1), but no inhibition of basal MAPK phosphorylation was observed in NCI-H460 nonsmall-cell lung cancer tumors treated with regorafenib (data not shown). These results suggest that additional pathways may be involved in MEK/ERK activation; thus, combination of regorafenib with MEK or PI3K inhibitors may enhance the antitumor efficacy of regorafenib.

Taken together, these results demonstrate that regorafenib effectively inhibits a number of key angiogenic and tumorigenic kinases which drive various human cancers and potently prevents growth of a large number of human tumor xenografts in preclinical models, suggesting that it may also be efficacious in man. Promising clinical efficacy and a good tolerability profile were observed in a Phase I study of regorafenib in patients with advanced refractory CRC47 and in a Phase II trial of regorafenib in previously untreated patients with metastatic or unresectable RCC.48 Regorafenib therefore warrants further investigations in additional tumor types (e.g., tyrosine kinase inhibitor-refractory GIST).

Note added in proof

The cooperative activity of VEGFR and TIE2 blockade in tumor growth suppression has been demonstrated by others by genetic delivery of soluble receptors Sallinen H, Anttila M, Gröhn O, Koponen J, Hämäläinen K, Kholova I, Kosma VM, Heinonen S, Alitalo K, Ylä-Herttuala S. Cotargeting of VEGFR1 and -3 and angiopoietin receptor Tie2 reduces the growth of solid human ovarian cancer in mice. Cancer Gene Ther 2010;epub.


The authors wish to thank Debbie Braun, Debbie Celanie, Charles Chen, Susan Gawlak, Claudia Heyer, Hong Rong, Dean Wilkie and Xiaomei Zhang for their excellent technical assistance in these studies. We thank Drs. Bernard Haendler, Arne Scholz and Gerd Siemeister (Global Drug Discovery, Bayer Schering Pharma AG) for critical reading of the manuscript. The authors thank Karen Brayshaw and Eleanor Steele from Complete HealthVizion for medical writing support funded by Bayer Schering Pharma AG.