The Antiangiogenic Activity in Xenograft Models of...

Published OnlineFirst January 26, 2010; DOI: 10.1158/1535-7163.MCT-09-0472

Research Article Molecular
Cancer
Therapeutics
The Antiangiogenic Activity in Xenograft Models of Brivanib,a Dual Inhibitor of Vascular Endothelial Growth FactorReceptor-2 and Fibroblast Growth Factor Receptor-1 Kinases
Rajeev S. Bhide, Louis J. Lombardo, John T. Hunt, Zhen-wei Cai, Joel C. Barrish, Susan Galbraith,Robert Jeyaseelan, Sr., Steven Mortillo, Barri S. Wautlet, Bala Krishnan, Daniel Kukral,Harold Malone, Anne C. Lewin, Benjamin J. Henley, and Joseph Fargnoli

Abstract

Authors' APrinceton,

Corresponsearch & DNJ 08540.

doi: 10.115

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Tumor angiogenesis is a complex and tightly regulated network mediated by various proangiogenic fac-tors. The fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) family of growth fac-tors, and associated tyrosine kinase receptors have a major influence in tumor growth and dissemination andmay work synergistically to promote angiogenesis. Brivanib alaninate is the orally active prodrug of brivanib,a selective dual inhibitor of FGF and VEGF signaling. Here, we show that brivanib demonstrates antitumoractivity in a broad range of xenograft models over multiple dose levels and that brivanib alaninate showsdose-dependent efficacy equivalent to brivanib in L2987 human tumor xenografts. Brivanib alaninate(107 mg/kg) reduced tumor cell proliferation as determined by a 76% reduction in Ki-67 staining and reducedtumor vascular density as determined by a 76% reduction in anti-CD34 endothelial cell staining. Furthermore,Matrigel plug assays in athymic mice showed that brivanib alaninate inhibited angiogenesis driven by VEGFor basic FGF alone, or combined. Dynamic contrast-enhanced magnetic resonance imaging, used to assess theeffects of brivanib alaninate on tumor microcirculation, showed a marked decrease in gadopentetate dime-glumine contrast agent uptake at 107 mg/kg dose, with a reduction in area under the plasma concentration-time curve from time 0 to 60 minutes at 24 and 48 hours of 54% and 64%, respectively. These results show thatbrivanib alaninate is an effective antitumor agent in preclinical models across a range of doses, and that ef-ficacy is accompanied by changes in cellular and vascular activities. Mol Cancer Ther; 9(2); 369–78. ©2010 AACR.

Introduction

Angiogenesis or neovascularization, the process bywhich new blood vessels are formed from preexistingvessels, is critical to the growth, survival, and dissemina-tion of tumors. Initiation of tumor angiogenesis is gener-ally mediated by secretion of a variety of growth factorsin response to local environmental triggers, as yet not ful-ly understood but thought to include hypoxia (1). Prom-inent among these mediators are vascular endothelialgrowth factor ligand A (VEGF-A) and fibroblast growthfactor (FGF)-1 (acidic FGF) and FGF-2 [basic FGF(bFGF)].The identification and characterization of tumor-

derived angiogenic factors, their corresponding endothe-lial cell receptors, and complex signaling networksprovides novel opportunities for the therapeutic develop-ment of new antineoplastic agents (2). The VEGF family

ffiliation: Bristol-Myers Squibb Research & Development,New Jersey

ding Author: Joseph Fargnoli, Bristol-Myers Squibb, Re-evelopment, Route 206 and Province Line Road, Princeton,Phone: 609-252-5268. E-mail: [email protected]

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of growth factors and associated tyrosine kinase recep-tors (VEGF receptor, VEGFR) has been shown to have amajor influence on tumor angiogenesis, and anti-VEGFagents, including bevacizumab, have been developedand shown to provide therapeutic advantage in sometumor types (3, 4). VEGF-A binds preferentially toVEGFR-1 and VEGFR-2, the latter being recognized asa primary mediator of vascular permeability and a pro-moter of tumor-related endothelial cell migration andproliferation. The implications for the survival and growthof tumor cells make the therapeutic targeting of VEGFR-2especially attractive (2, 5); however, there is growing evi-dence of VEGFR-1 promoting angiogenesis (2).The FGF family is also known to be influential in the

stimulation of endothelial cell proliferation and migra-tion (6, 7) and therefore has potential as an additional an-tineoplastic target (8). FGF receptor (FGFR)-1 is thepredominant FGFR to be expressed on endothelial cells,with FGFR-2 also present to a lesser extent. FGF/FGFRsignaling is thought to contribute to both the early andlate stages of tumor angiogenesis and to affect both tu-mor cell invasion and migration (6, 7). Some evidencesuggests that stimulation of the FGF signaling networkcan induce a proangiogenic effect creating a favorable en-vironment for vasculature growth through the modula-tion of endothelial cell proliferation and migration, the

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production of proteases, and the promotion of integrinand cadherin receptor expression (7).Recent research has pointed to the importance of cross-

talk between bFGF andVEGF, whichmay reflect temporaldifferences in their role. Synergistic vascularization act-ivity is seen when the pair is coexpressed simultaneouslyin athymic mouse xenograft models, resulting in fast-growing tumors with high vessel density patency andpermeability (9). In mouse models, the administration ofVEGFR-2 antagonists has been found to block both VEGF-and bFGF-induced angiogenesis and endothelial cell pro-liferation. These findings led to the hypothesis thatendogenous VEGF is required for the in vitro angiogeniceffect induced by exogenous bFGF (10, 11). The expressionof dominant-negative FGFR-1 and FGFR-2 in glioma cellshas been shown to result in decreased tumor vasculariza-tion paralleled by VEGF downregulation (12). Thesefindings indicate that tumors may be able to evade ini-tial VEGF inhibition by altering the regulation of othergrowth factors involved in angiogenesis and tumor cellproliferation. The complexity of the regulation of tumorangiogenesis suggests that the targeting of more thanone pathway may be beneficial (4).Brivanib is a small-molecule VEGFR/FGFR tyrosine

kinase inhibitor that is currently undergoing clinical in-vestigation. Due to the low aqueous solubility of briva-nib, a prodrug strategy was pursued that resulted in aprodrug with high aqueous solubility, brivanib alaninate(13). Brivanib, the parent compound, has previously beenshown to be a selective inhibitor of FGFR-1 and FGFR-2,and VEGFR-1, VEGFR-2, and VEGFR-3 in vitro (14, 15).Additionally, brivanib has also shown antitumor activityin vivo against the H3396 human breast tumor xenograftmodel (14). Here, we report the in vivo antiangiogenicand tumor growth-inhibitory effects of brivanib and itsprodrug, brivanib alaninate, in human xenograft mousemodels across a range of tumor types.

Materials and Methods

ReagentsBrivanib alaninate is the L-alanine ester prodrug of bri-

vanib to improve the low aqueous solubility of brivanibat equilibrium (14, 15). In this series of studies, brivanibalaninate was administered at a volume of 0.01 mL/g ofmouse with sodium citrate buffer, with a final pH of 3.5used as vehicle for the dosing solution. Brivanib was dis-solved in a 7:3 solution of PEG400/H2O and was admin-istered at a volume of 0.01 mL/g. Both compounds wereorally dosed using disposable 20-g oral gavage needles(Popper and Sons, Inc.) on a daily schedule with a gen-eral duration of 14 d.

AnimalsFemale BALB/c athymic mice (nu/nu) were purchased

at 5 to 6 wk of age from Harlan Sprague-Dawley Co.Mice were maintained in an ammonia- and pathogen-freeenvironment and were fed water and food ad libitum.

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Quarantine was ensured for 7 d before tumor implanta-tion and efficacy testing. All animal studies were doneunder the approval of the Bristol-Myers Squibb AnimalCare and Use Committee and in accordance with theAmerican Association for Accreditation of LaboratoryAnimal Care.

Xenograft Tumor ModelsHuman tumor xenografts used included L2987 (non–

small-cell lung carcinoma), H3396 (breast carcinoma),and HCT116/VM46 (paclitaxel-resistant colon carcino-ma), which were established at Bristol-Myers Squibb.Tumors were implanted s.c. as small fragments of ≤0.1

to 0.2 mm3 using a 13-g trocar. Tumors were allowed togrow to an approximate size of 100 mm3 before the initi-ation of treatment. Treatment and control groups con-sisted of 8 to 10 mice. Tumor size was measured twiceweekly. Tumor volume was calculated by measuring per-pendicular tumor diameters with Vernier scale calipers,and using the formula: 1/2 [length × (width)2]. Thehealth of the mice was closely monitored and the micewere immediately euthanized if any signs of distresswere observed.Antitumor efficacy was expressed as percentage tumor

growth inhibition (% TGI) and therefore represents themaximum effect. TGI was calculated as follows:

% TGI ¼ f1� ½ðTt � T0Þ=ðCt � C0Þ�g � 100

in which Ct is the median tumor volume (mm3) of vehiclecontrol (C)–treated mice at time t; Tt is the median tumorvolume (mm3) of treated mice, T, at time t; and C0 is themedian tumor volume (mm3) of vehicle control–treatedmice at time 0.Tumor volume doubling time was measured over the

linear growth range of the tumor, generally between 250and 1,000 mm3 tumor size. TGI of ≥50% over one tumorvolume doubling time was considered an active antitu-mor response.

ImmunohistochemistryL2987 tumor xenografts were excised, fixed in 10%

neutral buffered formalin, processed for paraffin embed-ding, and sectioned at 5 μm. Slides were allowed to dryand immunohistochemistry was done. Cell proliferationwas evaluated using an anti-Ki67 antibody (Santa CruzBiotechnology, Inc.). Vascular density was visualizedthrough an anti-CD34 antibody (BD Pharmingen). Thedilution of both antibodies was 1:100 in a serum-freeblocking agent (DAKO Cytomation). Tissue was blockedfor endogenous peroxidase by immersion in 3% H2O2 for8 min. Antigen retrieval was done using Citra Plus in apressure cooker (Biocare Medical decloaking chamber;Biocare Medical). Tissue was heated to 98°F for 3 minand then allowed to cool for a further 15 min before rins-ing in immunohistochemistry wash buffer (DAKO). Thisbuffer was used as a rinse between all subsequent steps.Primary antibodies were applied to tissue sections and

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were allowed to incubate overnight at 4°C in a humidi-fied chamber. For the anti–CD34-treated tissues, an anti-rat polymer probe (Biocare Medical) was applied andincubated for 20 min. After rinsing, horseradish peroxi-dase complex (Biocare Medical) was applied and in-cubated for 20 min. For the anti–Ki67-treated tissues, asecondary polymer probe conjugated to peroxidase (En-Vision, DAKO Cytomation) was applied for 20 min. 3,3′-Diaminobenzidine (Biocare Medical) was applied for2 min each slide. Following the developing step, tissueswere counterstained with Mayer's hematoxylin for 20 s,rinsed in tap water, put through a series of graded al-cohols to xylene, and coverslipped. Quantification ofproliferation and vascular density was done using anOlympus BX-10 microscope (Tokyo) and Image-Pro Plussoftware (Media Cybernetics, Inc.), without prior knowl-edge of groups. Proliferation was analyzed by a cell countof positively stained cells. All fields of view were quanti-fied at ×100 magnification. Vascular density (defined asthe number of vessels per unit area) was done in 25 fieldsof view at ×200 magnification. Statistical analysis wasdone using the SAS JMP software (SAS Institute).

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Matrigel In vivo Assays and Histology. The effects ofbrivanib alaninate, bevacizumab (Genentech), andDC101 (BioXCell) on endothelial cells were assessed inMatrigel plugs in nude mice. Matrigel (BD, growth factorreduced), stored frozen at −80°C, was thawed on ice at4°C overnight. To prepare the Matrigel for injection, ei-ther 100 ng/μL VEGF (PeproTech) alone, 250 ng/μLbFGF (PeproTech) alone, or both in combination(100 ng/μL VEGF and 250 ng/μL bFGF) were sus-pended in the gel and mixed by gentle inversion. Ap-proximately 24 h after implant, mice were treated withvehicle (PBS), brivanib alaninate at 50 and 100 mg/kg,DC101 at 1.63 mg/dose and 3.25 mg/dose, or bevacizu-mab at 4 mg/kg over a period of 10 d. At the end of thedosing period, Matrigel plugs were excised and fixed in10% neutral buffered formalin before processing for par-affin embedding. They were then sectioned at 5 μm andstained with H&E. Identification of the endothelial cellpopulation using immunohistochemical approaches wasbased on positive staining of CD34, a well characterizedmarker of endothelial cells (16). The Ariol image analy-sis system (Applied Imaging Corp.; Genetix) was used

Figure 1. Effects of brivanib (A-C) and brivanib alaninate (D) on tumor growth rate of human H3396 breast cancer xenograft (A), human HCT116/VM46colon carcinoma xenograft (B), and human L2987 lung cancer xenograft lines (C and D) compared with vehicle-treated mice. bid, twice a day; qd,once a day; q2d x 5, every two days times five.

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to analyze the Matrigel plugs. Endothelial cells werecounted at a magnification of ×200 and 10 fields of viewwere used for each animal. Raw data were transferredto a statistical software package, SAS JMP, and the meannumber of cells migrating was calculated for eachgroup. A Student's t test was used to show significance.

Dynamic Contrast Enhanced-Magnetic ResonanceImagingThe in vivo activity of brivanib alaninate was assessed

pharmacodynamically in L2987 tumor-bearing mice us-ing dynamic contrast enhanced-magnetic resonance im-aging (DCE-MRI). All DCE-MRI experiments weredone in accordance with the Bristol-Myers Squibb Ani-mal Care and Use Committee Guidelines.Athymic mice (18–28 g) bearing s.c. implanted L2987

human lung tumor cells (5 × 106) on the lateral portionnear the hind limb were included in the study when tu-mor size reached 100 mm3. Mice were anesthetized withisoflurane (AERRANE, Baxter Healthcare) and the tailveins were catheterized for administration of MRI con-trast agent. Mice were dosed orally with brivanib alani-nate after baseline DCE-MRI measurements were made.All animals received three doses of brivanib alaninate:immediately after the baseline DCE-MRI assessmentand then at 24 and 48 h. The second and third DCE-MR imaging were done within 2 h of dosing.DCE-MRI images were acquired on a Bruker Pharma-

Scan 4.7 T horizontal magnet with a 16-cm bore (BrukerBiospin). After initial axial scout images, a precontrast T1

measurement was done by saturation recovery for theslices that were to be used for dynamic imaging. Theimages were acquired with a variable repletion time(TR; 3,000, 1,200, 900, 600, 300, and 152.18 ms) and anecho time of 15 ms. A series of 40 dynamic-contrast T1-weighted echoes were obtained with a temporal resolu-



tion of 11.8 s for three contiguous 2-mm slices throughthe tumor and one through the leg muscle (referencetissue) using a spin echo sequence with repetition timeTR = 110 ms, echo time I = 10 ms, and 128 × 128 matrix.Five DCE-MRI sets were collected before the administra-tion of contrast. The next 35 images were collected afterbolus injection of 0.3 mmol/kg gadopentetate dimeglu-mine (Magnevist, Berlex Labs) through a syringe pumpwith an infusion rate of 2 mL/min. A manual 220-μLsaline wash was done over 5 s to flush the catheter line(180 μL line volume) and to ensure the complete deliveryof the contrast agent.T1 and DCE were initially processed using zero-filling

and two-dimensional Fourier transformation to yield256 × 256 data points. Images were analyzed as a pixelaverage of the regions of interest (ROI) by image se-quence analysis (Bruker Biospin). ROIs were drawnaround the entire tumor area for each tumor slice exclud-ing the surrounding skin and muscle using the T1 images.A separate ROI was drawn in the contralateral leg formuscle T1 values. These ROIs were transferred to theDCE images with the same geometry. Individual T1 va-lues were calculated for each ROI for both tumor andmuscle using the image sequence analysis T1sat functionand were used to normalize DCE images with theircorresponding T1 values. The first five preinjectionimages were averaged to increase signal-to-noise ratioand subtracted from the postinjection images to providecontrast enhancement images of the tumor and muscleROIs. T1 time courses for the ROIs of tumor and mus-cle were then obtained from the respective enhancementsand baseline T1 values and were translated to gadopente-tate concentration using gadopentetate relaxivity. The ini-tial area under the contrast uptake curve for the first 60 s(AUC60) after the contrast agent administration was thenintegrated and the tumor values were normalized to their

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Figure 2. Representativephotographs of the grossmorphology of excised L2987tumors from athymic mice treatedwith brivanib alaninate at twodifferent dose levels. Brivanibalaninate suppressed tumor growthand vascularization.

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corresponding muscle values to eliminate the animal-to-animal variability in the arterial input function (17, 18).All data were tested for variance using the Student'st test two-tailed paired analysis, which is used for test-ing the significance between different groups of treat-ment. Because the same animal was used for baselineand posttreatment imaging, each served as its owncontrol. Reproducibility experiments performed in thesame mice 24 h apart showed that the 95% limit ofchange was −18% to +22% for a group of nine mice;normalization to muscle improved the reproducibilityof results (19). The within-subject coefficient of varia-tion was 24%.

Results

Antitumor Activity in Xenograft Tumor ModelsIn xenograft models, brivanib shows potent antitumor

activity in vivo against human H3396 breast tumor xeno-grafts. The inhibition of tumor growth relative to controlas measured by the TGI was 30%, 37%, and 88% after14 days of treatment with brivanib at doses of 7.5, 15,and 30 mg/kg twice a day, respectively (Fig. 1A). The ac-



tivity of brivanib in chemoresistant HCT116/VM46 hu-man colon carcinoma xenografts was also shown(Fig. 1B). Brivanib was shown to have greater inhibitoryeffects on tumor growth at doses of 15 to 45 mg/kg twicea day for 14 days than 36 mg/kg paclitaxel i.v. every2 days for 10 days (maximum tolerated dose), whichwas ineffective in inhibiting tumor growth.Both brivanib and its ester prodrug brivanib alaninate

showed potent antitumor activity against L2987 humanlung carcinoma xenografts over a variety of doses(Fig. 1C and D). The inhibition of tumor growth relativeto control as measured by the treated/control ratio fol-lowing 9 days of brivanib treatment was 0.85, 0.40, and0.36 at doses of 30, 60, and 90 mg/kg once a day, respec-tively. Furthermore, inhibition of tumor growth relativeto control was also seen with the administration of briva-nib alaninate, with treated/control ratios of 0.88, 0.40,and 0.28 at doses of 53, 80, and 107 mg/kg once a day,respectively, after 14 days of treatment.Gross assessment of removed tumors from mice

treated with brivanib alaninate also clearly showed adose-dependent reduction in tumor growth and vascu-larization (Fig. 2).

Figure 3. Effects of vehicle control (A) and brivanib alaninate [36 mg/kg (B) and 107 mg/kg (C)] on tumor cell proliferation (quantified by staining withanti–Ki-67) in L2987 lung xenografts. A significant dose-dependent decrease in the number of proliferating (Ki-67 positive) cells was observed followingbrivanib alaninate treatment (D).




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Effects on Tumor Cell Proliferation in XenograftTumor ModelsTreatment with brivanib alaninate significantly inhib-

ited tumor cell proliferation in L2987 lung tumor xeno-grafts, as shown by a dose-dependent decrease in thenumber of Ki-67–positive cells on staining with anti–Ki-67 (Fig. 3). A statistically significant reduction in Ki-67staining was observed at a dose of 36 mg/kg (45% reduc-tion; P < 0.001 versus control), but a much more dramaticreduction (76%; P < 0.001 versus control) was observed ata dose of 107 mg/kg, which is a highly efficacious dose inxenograft models when given daily.

Effects of Brivanib on both VEGF- andbFGF-Stimulated Angiogenesis In vivoThe Matrigel plug in vivo assay was used to assess the

ability of brivanib alaninate and other known angiogen-esis inhibitors to inhibit angiogenesis stimulated byVEGF or bFGF alone or in combination. Identificationof the endothelial cell population using immunohisto-



chemical approaches was based on positive staining ofCD34 (data not shown).As illustrated in Fig. 4A, a significant reduction in en-

dothelial cell number was observed in plugs containingeither cytokine alone or when both cytokines were com-bined followed by treatment with brivanib alaninate. Inaddition, as summarized in Fig. 4B, a lower dose of bri-vanib alaninate, which generally results in limited effica-cy in tumor xenograft studies, was still effective ininhibiting angiogenesis in plugs containing VEGF, bFGF,or both cytokines. In contrast, treatment with theVEGFR-2–selective inhibitor DC101, a monoclonal anti-body specifically directed at the murine host VEGFR-2,or bevacizumab, a commercially marketed monoclonalantibody directed at the human VEGF ligand with mini-mal to no neutralizing activity against murine VEGF (20),resulted in no inhibition of either bFGF- or VEGF/bFGF-stimulated angiogenesis. In plugs containing only VEGF,bevacizumab, when delivered at a dose level and sched-ule that results in maximal preclinical antitumor activity,

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Figure 4. A, effects of vehicle,brivanib alaninate (100 mg/kg),DC101 (3.25 mg/dose), andbevicizumab (4 mg/kg) onendothelial cell population asassessed by the Matrigel plugassay. B, effects of brivanibalaninate (50 and 100 mg/kg),DC101 (1.63 and 3.25 mg/dose),and bevacizumab (4 mg/kg;assessed by Matrigel plug assay).A lower dose of brivanib alaninate(50 mg/kg) was also effective ininhibiting endothelial cells in plugscontaining either VEGF, bFGF, orboth cytokines.

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showed significant inhibition consistent with its direct ef-fect on the VEGF ligand that is known to stimulate bothVEGFR-1 and VEGFR-2 (21).

Effects on Vascular Density in XenograftTumor ModelsThe effects of brivanib alaninate on vascular density

within the L2987 tumor xenografts are shown in Fig. 5.There was a dose-dependent reduction in CD34 stainingfor endothelial cells compared with vehicle control atdoses of 36 mg/kg (21% reduction) and 107 mg/kg(76% reduction), with a statistically significant differenceobserved only at the 107 mg/kg dose (P < 0.001 versuscontrol).

Effects on Tumor Microcirculation Assessed byDCE-MRIDCE-MRI was used to assess the effect of brivanib ala-

ninate (36 mg/kg, n = 10; and 107 mg/kg, n = 9) on tu-mor microcirculation in the L2987 lung xenograft model.DCE-MRI analysis of coronal single-slice images throughthe tumor and the muscle of the hind limb area beforeand 24 hours after the administration with brivanib ala-ninate showed a markedly reduced uptake of contrast



agent with brivanib alaninate 107 mg/kg when com-pared with the 36 mg/kg dose (Fig. 6A) in the tumor. Bri-vanib alaninate at a dose of 107 mg/kg was shown to bemore efficacious than the lower dose level of 36 mg/kg.There was a significant reduction in AUC60 of 54% and64% at 24 and 48 hours, respectively, at the 107 mg/kgdose (n = 9). In contrast, there was no significant differ-ence at 36 mg/kg (n = 10; Fig. 6B and C).

Discussion

Brivanib, the parent compound of the prodrug briva-nib alaninate, has previously been identified as a selec-tive dual inhibitor of FGF and VEGF signaling, within vitro activity encompassing FGFR-1, FGFR-2, VEGFR-1,VEGFR-2, and VEGFR-3 (14, 15). Brivanib demonstratesgood cellular selectivity as shown by the specific inhibi-tion of human umbilical vascular endothelial cell prolifer-ation when these cells were stimulated with VEGFor bFGF, but not by epidermal growth factor (EGF) orplatelet-derived growth factor-β (14). The dual inhibitionof VEGFR and FGFR activity with brivanib alaninate haspreviously been shown in preclinical studies in the GEO-and bevacizumab-resistant HT-29 colon cancer models

Figure 5. Effects of vehicle (A) and brivanib alaninate [36 mg/kg (B) and 107 mg/kg (C)] on vascular cell density in L2987 lung xenografts. A significantdose-dependent decrease in vascular density was observed following brivanib alaninate treatment (D).




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(22). This distinctive inhibitory profile provides addition-al potential for enhanced antitumor activity, drawing onthe suggested synergistic mechanisms of VEGF and bFGFon tumor vasculature.This series of in vivo studies show that brivanib and its

prodrug brivanib alaninate have a broad spectrum ofantitumor activity over multiple dose levels and sche-dules. The studies showed that antitumor activity encom-passes breast, colon, and lung human tumor xenograftsas well as in a model of chemoresistant disease consistentwith its antiangiogenic activity. Additionally, the prodrugbrivanib alaninate was equally efficacious in vivo againstthe L2987 human lung tumor model when dose levelswere adjusted for the active parent moiety. The robustantitumor effects in the L2987 lung tumor model, whichdoes not express VEGFR in vivo but still showed amarked antitumor response to brivanib alaninate, strong-ly suggest activity against a host-driven angiogenicmechanism. Furthermore, the results obtained in vivo



against multiple tumor models resulting in tumor stasisare consistent with literature reports in preclinical modelswith other agents targeting VEGFR-2 (23–25).Brivanib alaninate also clearly has an effect on endo-

thelial cells in a Matrigel plug assay when using VEGFand bFGF alone or in combination as stimulators of an-giogenesis. Using this model, we show that brivanib ala-ninate not only effectively inhibits angiogenesis drivenby either VEGF or bFGF alone but also robustly inhibitsangiogenesis driven by a combination of these cytokines.As shown in Fig. 4B, the combination of VEGF and bFGFresulted in a doubling of the endothelial cell populationcompared with either of these cytokines alone, and bothdose levels of brivanib significantly reduced angiogenesisdriven by the combined growth factors. Importantly, al-though bevacizumab was effective in inhibiting angio-genesis driven by VEGF alone, it had no impact onangiogenesis when VEGF was supplemented with bFGF.Therefore, the inhibition of VEGF signaling seems to be

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Figure 6. A, DCE-MRI analysis of coronalsingle slice images through the tumor andthe muscle of the hind limb area beforeand 24 h after administration with brivanibalaninate in the L2987 lung xenograftmodel showed a markedly reduceduptake of contrast agent with 107 mg/kgbrivanib alaninate compared with the36 mg/kg dose. The ROI is indicated inthe figure. B and C, AUC60 values fortumor/muscle ratio by DCE-MRI analysisfollowing brivanib alaninate treatment.A significant reduction in AUC60 of 54%and 64% at 24 and 48 h, respectively,at a dose of 107 mg/kg (n = 9) wasobserved in contrast to the 36 mg/kg(n = 10) dose. Arrows, brivanib alaninatedosing time.


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insufficient to inhibit angiogenesis in the presence ofbFGF signaling in the Matrigel plug assay. Consequently,we propose that the inhibition of VEGF- and bFGF-driven angiogenesis requires targeting of both signalingpathways, as is the case with brivanib. Whether blockadeof only the FGF pathway will lead to the inhibition ofVEGF- and bFGF-driven angiogenesis will require theidentification and testing of a highly specific compoundtargeting only bFGF signaling. Interestingly, DC101 didnot seem to inhibit angiogenesis in plugs containing onlyVEGF despite use of a dose that is well above the maxi-mum efficacious level determined in preclinical antitu-mor models (26). It is possible that a longer period oftreatment might be required for revealing an effect basedon the in vivo antitumor regimen; alternatively, theblockade of VEGFR-2 alone may be insufficient to obtaina response in this assay in light of the additional angio-genic activities that are mediated by VEGFR-1 andwhich would not be inhibited by DC101 as suggestedby Ferrara et al. (27).The robust antiangiogenic activity of brivanib alaninate

is also shown by the significant reduction in tumor vascu-lar density and in tumor blood flow (as measured byDCE-MRI). Pharmacodynamic measurements in tumor-bearing mice were used to assess in vivo activity usingDCE-MRI to measure contrast agent uptake, which is de-pendent on a combination of blood flow, vessel surfacearea, and permeability. This technique has been used pre-viously with a variety of compounds targeting the VEGFpathway in preclinical models (28, 29). Several otheragents targeting the VEGFR pathway have also shownDCE-MRI changes, although the relationship of DCE-MRI changes to clinical benefit still awaits validation (30).A significant reduction in AUC60 with brivanib alani-

nate as measured by DCE-MRI over 48 hours was ob-served using a dose of 107 mg/kg. This dose producescomplete cytostasis in this tumor model in athymic micewhen dosed from 10 to 30 days and was designatedas the efficacious dose. At the subefficacious dose of36 mg/kg, tumor growth was not significantly differentfrom that in controls and the mean reduction in AUC60

did not vary significantly from baseline. The use of refer-ence muscle tissue for normalization allowed the assess-ment of tumor microcirculation, expressed as AUC60,without the measurement of arterial input function.Therefore, in this tumor model, the dose response forantitumor efficacy is in the same dose range as that for



DCE-MRI effects. In another experiment to examine thetime course of response, the serial reduction in AUC60

at 2, 4, and 24 hours after a single dose of brivanib alani-nate at 107 mg/kg was 24% (P = 0.009), 27% (P = 0.02),and 16% (P = 0.16), respectively, and then returned tobaseline by 48 hours (data not shown).These results show the utility of DCE-MRI to measure

the effect of changes in tumor microcirculation inducedby the antiangiogenic effects of brivanib alaninate in adose-dependent manner. DCE-MRI is now widely usedin the clinic on a relatively routine basis and seems tobe a suitable approach for use as a biomarker for asses-sing the antitumor activity of brivanib alaninate, both onthe evidence of this study and from other agents target-ing VEGFR-2 in the clinic (30–32). The preclinical DCE-MRI changes described here seem to highly correlatewith imaging findings from patients receiving brivanibalaninate in an ongoing phase 1 study in which DCE-MRI also showed a dose-dependent decrease in AUC60

in tumors (33).Brivanib has similar potency against VEGFR-2 as the

platelet-derived growth factor β/VEGFR inhibitor suni-tinib and has higher potency against VEGFR-2 thanthe currently approved VEGFR-2/c-Kit/raf inhibitorsorafenib (IC50 values: 25 nmol/L with brivanib versus10 nmol/L with sunitinib versus 90 nmol/L with sorafe-nib; refs. 14, 34, 35). However, unlike sunitinib and sora-fenib, brivanib targets the FGFR signaling pathway,which is critical for angiogenesis. Together with the datareported in the current study, this provides a strong ratio-nale for the clinical investigationof brivanib in solid tumors.Based on these promising findings, brivanib alaninate hasnow progressed to phase 2/3 clinical investigation.

Disclosure of Potential Conflicts of Interest

All authors are employees of Bristol-Myers Squibb Research &Development.

Grant Support

Editorial support was provided by M. English, PhD, of PAREXEL, andwas funded by Bristol-Myers Squibb.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received 6/1/09; revised 11/16/09; accepted 12/7/09; publishedOnlineFirst 1/26/10.

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2010;9:369-378. Published OnlineFirst January 26, 2010.Mol Cancer Ther Rajeev S. Bhide, Louis J. Lombardo, John T. Hunt, et al. Receptor-2 and Fibroblast Growth Factor Receptor-1 KinasesDual Inhibitor of Vascular Endothelial Growth Factor The Antiangiogenic Activity in Xenograft Models of Brivanib, a

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