The Antiangiogenic Activity in Xenograft Models of Brivanib, A

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 Factor Receptor-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 Tumor angiogenesis is a complex and tightly regulated network mediated by various proangiogenic factors. The fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) family of growth factors, and associated tyrosine kinase receptors have a major influence in tumor growth and dissemination and may 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 antitumor activity in a broad range of xenograft models over multiple dose levels and that brivanib alaninate shows dose-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 reduced tumor 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 VEGF or basic FGF alone, or combined. Dynamic contrast-enhanced magnetic resonance imaging, used to assess the effects 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 that brivanib alaninate is an effective antitumor agent in preclinical models across a range of doses, and that efficacy is accompanied by changes in cellular and vascular activities. Mol Cancer Ther; 9(2); 369–78. ©2010 AACR.

Introduction of growth factors and associated tyrosine kinase receptors (VEGF receptor, VEGFR) has been shown to have a Angiogenesis or neovascularization, the process by major influence on tumor angiogenesis, and anti-VEGF which new blood vessels are formed from preexisting agents, including bevacizumab, have been developed vessels, is critical to the growth, survival, and dissemina- and shown to provide therapeutic advantage in some tion of tumors. Initiation of tumor angiogenesis is gener- tumor types (3, 4). VEGF-A binds preferentially to ally mediated by secretion of a variety of growth factors VEGFR-1 and VEGFR-2, the latter being recognized as in response to local environmental triggers, as yet not ful- a primary mediator of vascular permeability and a pro- ly understood but thought to include hypoxia (1). Prom- moter of tumor-related endothelial cell migration and inent among these mediators are vascular endothelial proliferation. The implications for the survival and growth growth factor ligand A (VEGF-A) and fibroblast growth of tumor cells make the therapeutic targeting of VEGFR-2 factor (FGF)-1 (acidic FGF) and FGF-2 [basic FGF especially attractive (2, 5); however, there is growing evi- (bFGF)]. dence of VEGFR-1 promoting angiogenesis (2). The identification and characterization of tumor- The FGF family is also known to be influential in the derived angiogenic factors, their corresponding endothe- stimulation of endothelial cell proliferation and migra- lial cell receptors, and complex signaling networks tion (6, 7) and therefore has potential as an additional an- provides novel opportunities for the therapeutic develop- tineoplastic target (8). FGF receptor (FGFR)-1 is the ment of new antineoplastic agents (2). The VEGF family predominant FGFR to be expressed on endothelial cells, with FGFR-2 also present to a lesser extent. FGF/FGFR signaling is thought to contribute to both the early and Authors' Affiliation: Bristol-Myers Squibb Research & Development, Princeton, New Jersey late stages of tumor angiogenesis and to affect both tu- Corresponding Author: Joseph Fargnoli, Bristol-Myers Squibb, Re- mor cell invasion and migration (6, 7). Some evidence search & Development, Route 206 and Province Line Road, Princeton, suggests that stimulation of the FGF signaling network NJ 08540. Phone: 609-252-5268. E-mail: [email protected] can induce a proangiogenic effect creating a favorable en- doi: 10.1158/1535-7163.MCT-09-0472 vironment for vasculature growth through the modula- ©2010 American Association for Cancer Research. tion of endothelial cell proliferation and migration, the

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production of proteases, and the promotion of integrin Quarantine was ensured for 7 d before tumor implanta- and cadherin receptor expression (7). tion and efficacy testing. All animal studies were done Recent research has pointed to the importance of cross- under the approval of the Bristol-Myers Squibb Animal talk between bFGF and VEGF, which may reflect temporal Care and Use Committee and in accordance with the differences in their role. Synergistic vascularization act- American Association for Accreditation of Laboratory ivity is seen when the pair is coexpressed simultaneously Animal Care. in athymic mouse xenograft models, resulting in fast- growing tumors with high vessel density patency and Xenograft Tumor Models permeability (9). In mouse models, the administration of Human tumor xenografts used included L2987 (non– VEGFR-2 antagonists has been found to block both VEGF- small-cell lung carcinoma), H3396 (breast carcinoma), and bFGF-induced angiogenesis and endothelial cell pro- and HCT116/VM46 (paclitaxel-resistant colon carcino- liferation. These findings led to the hypothesis that ma), which were established at Bristol-Myers Squibb. endogenous VEGF is required for the in vitro angiogenic Tumors were implanted s.c. as small fragments of ≤0.1 effect induced by exogenous bFGF (10, 11). The expression to 0.2 mm3 using a 13-g trocar. Tumors were allowed to of dominant-negative FGFR-1 and FGFR-2 in glioma cells grow to an approximate size of 100 mm3 before the initi- has been shown to result in decreased tumor vasculariza- ation of treatment. Treatment and control groups con- tion paralleled by VEGF downregulation (12). These sisted of 8 to 10 mice. Tumor size was measured twice findings indicate that tumors may be able to evade ini- weekly. Tumor volume was calculated by measuring per- tial VEGF inhibition by altering the regulation of other pendicular tumor diameters with Vernier scale calipers, growth factors involved in angiogenesis and tumor cell and using the formula: 1/2 [length × (width)2]. The proliferation. The complexity of the regulation of tumor health of the mice was closely monitored and the mice angiogenesis suggests that the targeting of more than were immediately euthanized if any signs of distress one pathway may be beneficial (4). were observed. Brivanib is a small-molecule VEGFR/FGFR tyrosine Antitumor efficacy was expressed as percentage tumor kinase inhibitor that is currently undergoing clinical in- growth inhibition (% TGI) and therefore represents the vestigation. Due to the low aqueous solubility of briva- maximum effect. TGI was calculated as follows: nib, a prodrug strategy was pursued that resulted in a prodrug with high aqueous solubility, brivanib alaninate % TGI ¼f1 ½ðTt T0Þ=ðCt C0Þg Â 100 (13). Brivanib, the parent compound, has previously been C 3 shown to be a selective inhibitor of FGFR-1 and FGFR-2, in which t is the median tumor volume (mm ) of vehicle C – t T and VEGFR-1, VEGFR-2, and VEGFR-3 in vitro (14, 15). control ( ) treated mice at time ; t is the median tumor 3 T t C Additionally, brivanib has also shown antitumor activity volume (mm ) of treated mice, , at time ; and 0 is the 3 in vivo against the H3396 human breast tumor xenograft median tumor volume (mm ) of vehicle control–treated model (14). Here, we report the in vivo antiangiogenic mice at time 0. and tumor growth-inhibitory effects of brivanib and its Tumor volume doubling time was measured over the prodrug, brivanib alaninate, in human xenograft mouse linear growth range of the tumor, generally between 250 models across a range of tumor types. and 1,000 mm3 tumor size. TGI of ≥50% over one tumor volume doubling time was considered an active antitu- Materials and Methods mor response.

Reagents Immunohistochemistry Brivanib alaninate is the L-alanine ester prodrug of bri- L2987 tumor xenografts were excised, fixed in 10% vanib to improve the low aqueous solubility of brivanib neutral buffered formalin, processed for paraffin embed- at equilibrium (14, 15). In this series of studies, brivanib ding, and sectioned at 5 μm. Slides were allowed to dry alaninate was administered at a volume of 0.01 mL/g of and immunohistochemistry was done. Cell proliferation mouse with sodium citrate buffer, with a final pH of 3.5 was evaluated using an anti-Ki67 antibody (Santa Cruz used as vehicle for the dosing solution. Brivanib was dis- Biotechnology, Inc.). Vascular density was visualized solved in a 7:3 solution of PEG400/H2O and was admin- through an anti-CD34 antibody (BD Pharmingen). The istered at a volume of 0.01 mL/g. Both compounds were dilution of both antibodies was 1:100 in a serum-free orally dosed using disposable 20-g oral gavage needles blocking agent (DAKO Cytomation). Tissue was blocked (Popper and Sons, Inc.) on a daily schedule with a gen- for endogenous peroxidase by immersion in 3% H2O2 for eral duration of 14 d. 8 min. Antigen retrieval was done using Citra Plus in a pressure cooker (Biocare Medical decloaking chamber; Animals Biocare Medical). Tissue was heated to 98°F for 3 min Female BALB/c athymic mice (nu/nu) were purchased and then allowed to cool for a further 15 min before rins- at 5 to 6 wk of age from Harlan Sprague-Dawley Co. ing in immunohistochemistry wash buffer (DAKO). This Mice were maintained in an ammonia- and pathogen-free buffer was used as a rinse between all subsequent steps. environment and were fed water and food ad libitum. Primary antibodies were applied to tissue sections and

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Figure 1. Effects of brivanib (A-C) and brivanib alaninate (D) on tumor growth rate of human H3396 breast cancer xenograft (A), human HCT116/VM46 colon 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. were allowed to incubate overnight at 4°C in a humidi- Matrigel In vivo Assays and Histology. The effects of fied chamber. For the anti–CD34-treated tissues, an anti- brivanib alaninate, bevacizumab (Genentech), and rat polymer probe (Biocare Medical) was applied and DC101 (BioXCell) on endothelial cells were assessed in incubated for 20 min. After rinsing, horseradish peroxi- Matrigel plugs in nude mice. Matrigel (BD, growth factor dase complex (Biocare Medical) was applied and in- reduced), stored frozen at −80°C, was thawed on ice at cubated for 20 min. For the anti–Ki67-treated tissues, a 4°C overnight. To prepare the Matrigel for injection, ei- secondary polymer probe conjugated to peroxidase (En- ther 100 ng/μL VEGF (PeproTech) alone, 250 ng/μL Vision, DAKO Cytomation) was applied for 20 min. 3,3′- bFGF(PeproTech)alone,orbothincombination Diaminobenzidine (Biocare Medical) was applied for (100 ng/μL VEGF and 250 ng/μL bFGF) were sus- 2 min each slide. Following the developing step, tissues pended in the gel and mixed by gentle inversion. Ap- were counterstained with Mayer's hematoxylin for 20 s, proximately 24 h after implant, mice were treated with rinsed in tap water, put through a series of graded al- vehicle (PBS), brivanib alaninate at 50 and 100 mg/kg, cohols to xylene, and coverslipped. Quantification of DC101 at 1.63 mg/dose and 3.25 mg/dose, or bevacizu- proliferation and vascular density was done using an mab at 4 mg/kg over a period of 10 d. At the end of the Olympus BX-10 microscope (Tokyo) and Image-Pro Plus dosing period, Matrigel plugs were excised and fixed in software (Media Cybernetics, Inc.), without prior knowl- 10% neutral buffered formalin before processing for par- edge of groups. Proliferation was analyzed by a cell count affin embedding. They were then sectioned at 5 μm and of positively stained cells. All fields of view were quanti- stained with H&E. Identification of the endothelial cell fied at ×100 magnification. Vascular density (defined as population using immunohistochemical approaches was the number of vessels per unit area) was done in 25 fields based on positive staining of CD34, a well characterized of view at ×200 magnification. Statistical analysis was marker of endothelial cells (16). The Ariol image analy- done using the SAS JMP software (SAS Institute). sis system (Applied Imaging Corp.; Genetix) was used

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to analyze the Matrigel plugs. Endothelial cells were tion of 11.8 s for three contiguous 2-mm slices through counted at a magnification of ×200 and 10 fields of view the tumor and one through the leg muscle (reference were used for each animal. Raw data were transferred tissue) using a spin echo sequence with repetition time to a statistical software package, SAS JMP, and the mean TR = 110 ms, echo time I = 10 ms, and 128 × 128 matrix. number of cells migrating was calculated for each Five DCE-MRI sets were collected before the administra- group. A Student's t test was used to show significance. tion of contrast. The next 35 images were collected after bolus injection of 0.3 mmol/kg gadopentetate dimeglu- Dynamic Contrast Enhanced-Magnetic Resonance mine (Magnevist, Berlex Labs) through a syringe pump Imaging with an infusion rate of 2 mL/min. A manual 220-μL The in vivo activity of brivanib alaninate was assessed saline wash was done over 5 s to flush the catheter line pharmacodynamically in L2987 tumor-bearing mice us- (180 μL line volume) and to ensure the complete delivery ing dynamic contrast enhanced-magnetic resonance im- of the contrast agent. aging (DCE-MRI). All DCE-MRI experiments were T1 and DCE were initially processed using zero-filling done in accordance with the Bristol-Myers Squibb Ani- and two-dimensional Fourier transformation to yield mal Care and Use Committee Guidelines. 256 × 256 data points. Images were analyzed as a pixel Athymic mice (18–28 g) bearing s.c. implanted L2987 average of the regions of interest (ROI) by image se- human lung tumor cells (5 × 106) on the lateral portion quence analysis (Bruker Biospin). ROIs were drawn near the hind limb were included in the study when tu- around the entire tumor area for each tumor slice exclud- 3 mor size reached 100 mm . Mice were anesthetized with ing the surrounding skin and muscle using the T1 images. isoflurane (AERRANE, Baxter Healthcare) and the tail A separate ROI was drawn in the contralateral leg for veins were catheterized for administration of MRI con- muscle T1 values. These ROIs were transferred to the trast agent. Mice were dosed orally with brivanib alani- DCE images with the same geometry. Individual T1 va- nate after baseline DCE-MRI measurements were made. lues were calculated for each ROI for both tumor and All animals received three doses of brivanib alaninate: muscle using the image sequence analysis T1sat function immediately after the baseline DCE-MRI assessment and were used to normalize DCE images with their andthenat24and48h.ThesecondandthirdDCE- corresponding T1 values. The first five preinjection MR imaging were done within 2 h of dosing. images were averaged to increase signal-to-noise ratio DCE-MRI images were acquired on a Bruker Pharma- and subtracted from the postinjection images to provide Scan 4.7 T horizontal magnet with a 16-cm bore (Bruker contrastenhancementimagesofthetumorandmuscle Biospin). After initial axial scout images, a precontrast T1 ROIs. T1 timecoursesfortheROIsoftumorandmus- measurement was done by saturation recovery for the cle were then obtained from the respective enhancements slices that were to be used for dynamic imaging. The and baseline T1 values and were translated to gadopente- images were acquired with a variable repletion time tate concentration using gadopentetate relaxivity. The ini- (TR; 3,000, 1,200, 900, 600, 300, and 152.18 ms) and an tial area under the contrast uptake curve for the first 60 s echo time of 15 ms. A series of 40 dynamic-contrast T1- (AUC60) after the contrast agent administration was then weighted echoes were obtained with a temporal resolu- integrated and the tumor values were normalized to their

Figure 2. Representative photographs of the gross morphology of excised L2987 tumors from athymic mice treated with brivanib alaninate at two different dose levels. Brivanib alaninate suppressed tumor growth and vascularization.

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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 with anti–Ki-67) in L2987 lung xenografts. A significant dose-dependent decrease in the number of proliferating (Ki-67 positive) cells was observed following brivanib alaninate treatment (D). corresponding muscle values to eliminate the animal-to- tivity of brivanib in chemoresistant HCT116/VM46 hu- animal variability in the arterial input function (17, 18). man colon carcinoma xenografts was also shown All data were tested for variance using the Student's (Fig. 1B). Brivanib was shown to have greater inhibitory t test two-tailed paired analysis, which is used for test- effects on tumor growth at doses of 15 to 45 mg/kg twice ing the significance between different groups of treat- a day for 14 days than 36 mg/kg paclitaxel i.v. every ment. Because the same animal was used for baseline 2 days for 10 days (maximum tolerated dose), which and posttreatment imaging, each served as its own was ineffective in inhibiting tumor growth. control. Reproducibility experiments performed in the Both brivanib and its ester prodrug brivanib alaninate same mice 24 h apart showed that the 95% limit of showed potent antitumor activity against L2987 human change was −18% to +22% for a group of nine mice; lung carcinoma xenografts over a variety of doses normalization to muscle improved the reproducibility (Fig. 1C and D). The inhibition of tumor growth relative of results (19). The within-subject coefficient of varia- to control as measured by the treated/control ratio fol- tion was 24%. lowing 9 days of brivanib treatment was 0.85, 0.40, and 0.36 at doses of 30, 60, and 90 mg/kg once a day, respec- Results tively. Furthermore, inhibition of tumor growth relative to control was also seen with the administration of briva- Antitumor Activity in Xenograft Tumor Models nib alaninate, with treated/control ratios of 0.88, 0.40, In xenograft models, brivanib shows potent antitumor and 0.28 at doses of 53, 80, and 107 mg/kg once a day, activity in vivo against human H3396 breast tumor xeno- respectively, after 14 days of treatment. grafts. The inhibition of tumor growth relative to control Gross assessment of removed tumors from mice as measured by the TGI was 30%, 37%, and 88% after treated with brivanib alaninate also clearly showed a 14 days of treatment with brivanib at doses of 7.5, 15, dose-dependent reduction in tumor growth and vascu- and 30 mg/kg twice a day, respectively (Fig. 1A). The ac- larization (Fig. 2).

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Effects on Tumor Cell Proliferation in Xenograft chemical approaches was based on positive staining of Tumor Models CD34 (data not shown). Treatment with brivanib alaninate significantly inhib- As illustrated in Fig. 4A, a significant reduction in en- ited tumor cell proliferation in L2987 lung tumor xeno- dothelial cell number was observed in plugs containing grafts, as shown by a dose-dependent decrease in the either cytokine alone or when both cytokines were com- number of Ki-67–positive cells on staining with anti–Ki- bined followed by treatment with brivanib alaninate. In 67 (Fig. 3). A statistically significant reduction in Ki-67 addition, as summarized in Fig. 4B, a lower dose of bri- staining was observed at a dose of 36 mg/kg (45% reduc- vanib alaninate, which generally results in limited effica- tion; P < 0.001 versus control), but a much more dramatic cy in tumor xenograft studies, was still effective in reduction (76%; P < 0.001 versus control) was observed at inhibiting angiogenesis in plugs containing VEGF, bFGF, a dose of 107 mg/kg, which is a highly efficacious dose in or both cytokines. In contrast, treatment with the xenograft models when given daily. VEGFR-2–selective inhibitor DC101, a monoclonal antibody specifically directed at the murine host VEGFR-2, Effects of Brivanib on both VEGF- and or bevacizumab, a commercially marketed monoclonal bFGF-Stimulated Angiogenesis In vivo antibody directed at the human VEGF ligand with mini- The Matrigel plug in vivo assay was used to assess the mal to no neutralizing activity against murine VEGF (20), ability of brivanib alaninate and other known angiogen- resulted in no inhibition of either bFGF- or VEGF/bFGF- esis inhibitors to inhibit angiogenesis stimulated by stimulated angiogenesis. In plugs containing only VEGF, VEGF or bFGF alone or in combination. Identification bevacizumab, when delivered at a dose level and sched- of the endothelial cell population using immunohisto- ule that results in maximal preclinical antitumor activity,

Figure 4. A, effects of vehicle, brivanib alaninate (100 mg/kg), DC101 (3.25 mg/dose), and bevicizumab (4 mg/kg) on endothelial cell population as assessed by the Matrigel plug assay. B, effects of brivanib alaninate (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 in inhibiting endothelial cells in plugs containing either VEGF, bFGF, or both cytokines.

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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 significant dose-dependent decrease in vascular density was observed following brivanib alaninate treatment (D). showed significant inhibition consistent with its direct ef- agent with brivanib alaninate 107 mg/kg when com- fect on the VEGF ligand that is known to stimulate both pared with the 36 mg/kg dose (Fig. 6A) in the tumor. Bri- VEGFR-1 and VEGFR-2 (21). vanib alaninate at a dose of 107 mg/kg was shown to be more efficacious than the lower dose level of 36 mg/kg. Effects on Vascular Density in Xenograft There was a significant reduction in AUC60 of 54% and Tumor Models 64% at 24 and 48 hours, respectively, at the 107 mg/kg The effects of brivanib alaninate on vascular density dose (n = 9). In contrast, there was no significant differ- within the L2987 tumor xenografts are shown in Fig. 5. ence at 36 mg/kg (n = 10; Fig. 6B and C). There was a dose-dependent reduction in CD34 staining for endothelial cells compared with vehicle control at Discussion doses of 36 mg/kg (21% reduction) and 107 mg/kg (76% reduction), with a statistically significant difference Brivanib, the parent compound of the prodrug briva- observed only at the 107 mg/kg dose (P < 0.001 versus nib alaninate, has previously been identified as a selec- control). tive dual inhibitor of FGF and VEGF signaling, with in vitro activity encompassing FGFR-1, FGFR-2, VEGFR-1, Effects on Tumor Microcirculation Assessed by VEGFR-2, and VEGFR-3 (14, 15). Brivanib demonstrates DCE-MRI good cellular selectivity as shown by the specific inhibi- DCE-MRI was used to assess the effect of brivanib ala- tion of human umbilical vascular endothelial cell prolifer- ninate (36 mg/kg, n = 10; and 107 mg/kg, n = 9) on tu- ation when these cells were stimulated with VEGF mor microcirculation in the L2987 lung xenograft model. or bFGF, but not by epidermal growth factor (EGF) or DCE-MRI analysis of coronal single-slice images through platelet-derived growth factor-β (14). The dual inhibition the tumor and the muscle of the hind limb area before of VEGFR and FGFR activity with brivanib alaninate has and 24 hours after the administration with brivanib ala- previously been shown in preclinical studies in the GEO- ninate showed a markedly reduced uptake of contrast and bevacizumab-resistant HT-29 colon cancer models

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(22). This distinctive inhibitory profile provides addition- against multiple tumor models resulting in tumor stasis al potential for enhanced antitumor activity, drawing on are consistent with literature reports in preclinical models the suggested synergistic mechanisms of VEGF and bFGF with other agents targeting VEGFR-2 (23–25). on tumor vasculature. Brivanib alaninate also clearly has an effect on endo- This series of in vivo studies show that brivanib and its thelial cells in a Matrigel plug assay when using VEGF prodrug brivanib alaninate have a broad spectrum of and bFGF alone or in combination as stimulators of an- antitumor activity over multiple dose levels and sche- giogenesis. Using this model, we show that brivanib ala- dules. The studies showed that antitumor activity encom- ninate not only effectively inhibits angiogenesis driven passes breast, colon, and lung human tumor xenografts by either VEGF or bFGF alone but also robustly inhibits as well as in a model of chemoresistant disease consistent angiogenesis driven by a combination of these cytokines. with its antiangiogenic activity. Additionally, the prodrug As shown in Fig. 4B, the combination of VEGF and bFGF brivanib alaninate was equally efficacious in vivo against resulted in a doubling of the endothelial cell population the L2987 human lung tumor model when dose levels compared with either of these cytokines alone, and both were adjusted for the active parent moiety. The robust dose levels of brivanib significantly reduced angiogenesis antitumor effects in the L2987 lung tumor model, which driven by the combined growth factors. Importantly, al- does not express VEGFR in vivo but still showed a though bevacizumab was effective in inhibiting angio- marked antitumor response to brivanib alaninate, strong- genesis driven by VEGF alone, it had no impact on ly suggest activity against a host-driven angiogenic angiogenesis when VEGF was supplemented with bFGF. mechanism. Furthermore, the results obtained in vivo Therefore, the inhibition of VEGF signaling seems to be

Figure 6. A, DCE-MRI analysis of coronal single slice images through the tumor and the muscle of the hind limb area before and 24 h after administration with brivanib alaninate in the L2987 lung xenograft model showed a markedly reduced uptake of contrast agent with 107 mg/kg brivanib alaninate compared with the 36 mg/kg dose. The ROI is indicated in

the figure. B and C, AUC60 values for tumor/muscle ratio by DCE-MRI analysis following 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) was observed in contrast to the 36 mg/kg (n = 10) dose. Arrows, brivanib alaninate dosing time.

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insufficient to inhibit angiogenesis in the presence of DCE-MRI effects. In another experiment to examine the bFGF signaling in the Matrigel plug assay. Consequently, time course of response, the serial reduction in AUC60 we propose that the inhibition of VEGF- and bFGF- at 2, 4, and 24 hours after a single dose of brivanib alani- driven angiogenesis requires targeting of both signaling nate at 107 mg/kg was 24% (P = 0.009), 27% (P = 0.02), pathways, as is the case with brivanib. Whether blockade and 16% (P = 0.16), respectively, and then returned to of only the FGF pathway will lead to the inhibition of baseline by 48 hours (data not shown). VEGF- and bFGF-driven angiogenesis will require the These results show the utility of DCE-MRI to measure identification and testing of a highly specific compound the effect of changes in tumor microcirculation induced targeting only bFGF signaling. Interestingly, DC101 did by the antiangiogenic effects of brivanib alaninate in a not seem to inhibit angiogenesis in plugs containing only dose-dependent manner. DCE-MRI is now widely used VEGF despite use of a dose that is well above the maxi- in the clinic on a relatively routine basis and seems to mum efficacious level determined in preclinical antitu- be a suitable approach for use as a biomarker for asses- mor models (26). It is possible that a longer period of sing the antitumor activity of brivanib alaninate, both on treatment might be required for revealing an effect based the evidence of this study and from other agents target- on the in vivo antitumor regimen; alternatively, the ing VEGFR-2 in the clinic (30–32). The preclinical DCE- blockade of VEGFR-2 alone may be insufficient to obtain MRI changes described here seem to highly correlate a response in this assay in light of the additional angio- with imaging findings from patients receiving brivanib genic activities that are mediated by VEGFR-1 and alaninate in an ongoing phase 1 study in which DCE- which would not be inhibited by DC101 as suggested MRI also showed a dose-dependent decrease in AUC60 by Ferrara et al. (27). in tumors (33). The robust antiangiogenic activity of brivanib alaninate Brivanib has similar potency against VEGFR-2 as the is also shown by the significant reduction in tumor vascu- platelet-derived growth factor β/VEGFR inhibitor suni- lar density and in tumor blood flow (as measured by tinib and has higher potency against VEGFR-2 than DCE-MRI). Pharmacodynamic measurements in tumor- the currently approved VEGFR-2/c-Kit/raf inhibitor in vivo bearing mice were used to assess activity using sorafenib (IC50 values: 25 nmol/L with brivanib versus DCE-MRI to measure contrast agent uptake, which is de- 10 nmol/L with sunitinib versus 90 nmol/L with sorafe- pendent on a combination of blood flow, vessel surface nib; refs. 14, 34, 35). However, unlike sunitinib and sora- area, and permeability. This technique has been used pre- fenib, brivanib targets the FGFR signaling pathway, viously with a variety of compounds targeting the VEGF which is critical for angiogenesis. Together with the data pathway in preclinical models (28, 29). Several other reported in the current study, this provides a strong ratio- agents targeting the VEGFR pathway have also shown nale for the clinical investigation of brivanib in solid tumors. DCE-MRI changes, although the relationship of DCE- Based on these promising findings, brivanib alaninate has MRI changes to clinical benefit still awaits validation (30). now progressed to phase 2/3 clinical investigation. A significant reduction in AUC60 with brivanib alaninate as measured by DCE-MRI over 48 hours was ob- Disclosure of Potential Conflicts of Interest served using a dose of 107 mg/kg. This dose produces complete cytostasis in this tumor model in athymic mice All authors are employees of Bristol-Myers Squibb Research & whendosedfrom10to30daysandwasdesignated Development. as the efficacious dose. At the subefficacious dose of Grant Support 36 mg/kg, tumor growth was not significantly different from that in controls and the mean reduction in AUC60 Editorial support was provided by M. English, PhD, of PAREXEL, and did not vary significantly from baseline. The use of refer- was funded by Bristol-Myers Squibb. ence muscle tissue for normalization allowed the assess- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ment of tumor microcirculation, expressed as AUC60, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate withoutthemeasurementofarterialinputfunction. this fact.

Therefore, in this tumor model, the dose response for Received 6/1/09; revised 11/16/09; accepted 12/7/09; published antitumor efficacy is in the same dose range as that for OnlineFirst 1/26/10.

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378 Mol Cancer Ther; 9(2) February 2010 Molecular Cancer Therapeutics

The Antiangiogenic Activity in Xenograft Models of Brivanib, a Dual Inhibitor of Vascular Endothelial Growth Factor Receptor-2 and Fibroblast Growth Factor Receptor-1 Kinases

Rajeev S. Bhide, Louis J. Lombardo, John T. Hunt, et al.

Mol Cancer Ther 2010;9:369-378. Published OnlineFirst January 26, 2010.

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