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Dengue virus (DENV) is the most prevalent arthropod-borne viral disease of humans and has a major impact on global public health. There is no clinically approved drugs for DENV infection. Since intracellular VEGFR2 is increased in DENV infected patients, we thus hypothesized that VEGFR2 participated DENV proliferation and its inhibitors could be served as antivirals against DENV. Actually our results showed that VEGFR2 was induced by DENV infection. Also the agonist of VEGFR2, VEGF-A, promoted DENV proliferation. Therefore, we screened the inhibitors of VEGFR2 and found that brivanib alaninate (brivanib) showed the best anti-DENV ability with the lowest cellular cytotoxicity. Mechanically, our results indicated VEGFR2 directly interacted with PTP1B to dephosphorylate AMPK to provide lipid environment for viral replication. However, this effect could be inhibited by brivanib, which significantly reversed the reduction of AMPK phosphorylation caused by DENV infection, thus improving the cellular lipid environment. Moreover, the antiviral effect of brivanib could be reversed by AMPK inhibitor, Compound C. In addition, oral administration of brivianib (20–50 mg/kg/day) clearly improved the survival rate of DENV2 infection, and this effect was abolished in accompanied with Compound C (10mg/kg/day). Collectively, our study disclosed the mechanism of VEGFR2 in DENV2 and evaluated the antiviral ability of brivanib, which deserved more attention for clinical usage in DENV infection.

Application of in-line real-time process monitoring using a process analytical technology for granule size distribution can enable quality-by-design development of a drug product and enable attribute-based monitoring and control strategies. In this study, an in-line laser focused beam reflectance measurement (FBRM) C35 probe was used to investigate the effect of formulation and process parameters on the granule growth profile over time during the high shear wet granulation of a high drug load formulation of brivanib alaninate. The probe quantitatively captured changes in the granule chord length distribution (CLD) with the progress of granulation and delineated the impact of water concentration used during granulation. The results correlated well with offline particle size distribution measured by nested sieve analyses. An end point indication algorithm was developed that was able to successfully track the process time needed to reach the target CLD. Testing of the brivanib alaninate granulation through 25-fold scale-up of the batch process indicated that the FBRM CLD profile can provide a scale-independent granule attribute-based process fingerprint. These studies highlight the ability of FBRM to quantitate a granule attribute of interest during wet granulation that can be used as an attribute-based scale-up and process monitoring and control parameter.

The era of molecular-targeted agents, particularly bevacizumab and cetuximab, has revolutionized the treatment paradigm for metastatic colorectal cancer (mCRC). Amongst the multikinase inhibitors (MKIs) examined, regorafenib was the first to establish its role in mCRC. Despite its modest efficacy, this finding had reignited interest in exploring MKIs with the hope of maximizing their therapeutic potential in mCRC.This review summarizes the previous studies of MKIs in mCRC, targeting two signaling pathways activated through vascular endothelial growth factor receptors and epidermal growth factor receptors. The article provides discussion with a focus on: the challenges encountered when combining MKI with chemotherapy, the lack of predictive markers, and strategies utilized to address escape pathways through combining MKIs with other targeted agents.Clinical progress using MKIs in mCRC has been disappointing due to their limited efficacy. The exact role of regorafenib, apart from in chemo-refractory disease setting, requires further delineation. The role of MKIs in combination with other targeted agents or chemotherapy and in the maintenance setting is still considered experimental and warrants further investigation. The broader role of the current generation of MKIs will depend upon the accurate identification of patients with specific molecular phenotypes and better pharmacodynamic understanding of these agents to minimize toxicity.

Real-time monitoring and control of high shear wet granulation (HSWG) using process analytical technologies is crucial to process design, scale-up, and reproducible manufacture. Although significant progress has been made in real-time measurement of granule size distribution using focused beam reflectance measurement (FBRM), real-time in-line assessment of granule densification remains challenging. In this study, a drag force flow (DFF) sensor was developed and used to probe wet mass consistency in real-time. In addition, responses from FBRM and DFF sensors were compared to assess complementarity of information on granulation progress from the two probes. A placebo and a brivanib alaninate formulation were granulated with different concentrations of binder or water, respectively, while measuring granule size growth, densification, and DFF sensor response. The DFF sensor was able to quantitatively characterize with high resolution a response of wet mass consistency distinct from granule size distribution. The wet mass consistency parameter correlated well with granule densification, which was shown as a critical material attribute that correlated with tablet dissolution. In addition, application of DFF sensor to scale-up of granulation was demonstrated. These results showed the value of wet mass consistency measurement using DFF for WG monitoring and control.

A thermal runaway potential was identified for the conversion of a tertiary alcohol to a hydroxypyrrolotriazine intermediate in the synthesis of brivanib alaninate. A continuous process was developed to mitigate the potential thermal runaway and allow for safer scale-up. This paper describes the hazard analysis, process development, reactor development, reaction engineering model development, and scale-up of the continuous process. The process includes three separate and stable feed streams that are mixed in distinct order using in-line static mixers. Heat exchangers are arranged and connected to facilitate a “plug flow” reactor scheme allowing sufficient residence time for reaction completion. The process has been scaled-up to the pilot plant and to manufacturing.

This manuscript describes the control strategy for the commercial process to manufacture brivanib alaninate. The active pharmaceutical ingredient is a prodrug which is susceptible to hydrolysis. In addition to controlling hydrolysis, a robust strategy was required in order to control input and process-related impurities. Three significant aspects of control include understanding of the reaction parameters in order to minimize the regioisomer during the alkylation with (R)-propylene oxide, development of a design space through statistical models to control impurity formation, and the use of in situ FT-IR to monitor the hydrogenolysis of the Cbz protecting group.

Background and Aim Vascular endothelial (VEGF) and fibroblast growth factor (FGF)-induced hepatic stellate (HSCs) and liver endothelial cells (LECs) activation accelerates hepatic fibrogenesis and angiogenesis, and hemodynamic dysarrangements in cirrhosis. VEGF targeting agents had been reported as potential drugs for cirrhosis. However, the evaluation of effects of dual VEGF/FGF targeting agent in cirrhosis is still limited. Methods Using hemodynamic parameters, blood chemistry, primary isolated HSCs and LECs, histology, and digital imaging, we assess the effects of 2-week brivanib alaninate, a dual VEGFR/FGFR inhibitor, treatment in the pathophysiology of bile duct-ligated-cirrhotic rats. Results Fibrogenic and angiogenic markers in the serum and liver of bile duct-ligated-cirrhotic rats, including hydroxyproline, transforming growth factor-β1, angiopoietin-1, VEGF, FGF-2, endocan and phosphorylated-VEGFR2/VEGFR2, and phosphorylated-FGFR/FGFR together with hepatic CD31/angiopoietin-1 expressions (immunohistochemistry staining), angiogenesis (micro-computed tomography scan), microcirculatory dysfunction (in vivo miscroscopy and in situ liver perfusion study), portal hypertension, and hyperdynamic circulations (colored microsphere methods) were markedly suppressed and ameliorated by brivanib alaninate treatment. In in vitro study, acute brivanib alaninate incubation inhibited the transforming growth factor-β1-induced HSCs contraction/migration and VEGF-induced LECs angiogenesis. Concomitantly, the overexpression of various fibrogenic and angiogenic markers in HSCs and LECs, and in their culture media, was increased in parallel and these changes were suppressed by acute brivanib alaninate incubation. Conclusions This study demonstrated that brivanib alaninate targeting multiple mechanisms and working in the different pathogenic steps of the complications of cirrhotic rats with portal hypertension.

Background and Aims: Assessing treatment responses in hepatocellular carcinoma (HCC) is challenging, and alternative radiologic methods of measuring treatment response are required. Modified Response Evaluation Criteria in Solid Tumors (mRECIST) for HCC and alpha-fetoprotein (AFP) levels were assessed in a post hoc analysis of a phase II study of brivanib, a selective dual inhibitor of fibroblast growth factor and vascular endothelial growth factor signaling. Methods: HCC patients were treated with first-line (cohort A; n = 55) or second-line (cohort B; n = 46) brivanib alaninate 800 mg once daily. Outcomes were compared between World Health Organization (WHO) criteria and (retrospectively by) mRECIST by independent review. The relationship between on-study AFP changes and outcome was analyzed in patients with elevated AFP at baseline. Results: Response rates were higher with mRECIST versus WHO criteria in cohorts A (25.5% vs. 7.3%) and B (10.9% vs. 4.3%). Progressive disease (PD) as assessed by mRECIST was associated with a very short median overall survival (OS; cohort A, 2.8 months; cohort B, 5.3 months); PD as assessed by WHO criteria reflected a mixed population of patients with better outcomes. mRECIST responders tended to have a>50% AFP decrease during therapy. In cohorts A and B pooled, an early AFP response (>20%or >50% decline from baseline within the first 4 weeks) was not associated with longer median OS. Conclusions: Tumor response as assessed by mRECIST differed from that by WHO criteria, with mRECIST possibly identifying true nonresponders with a poor prognosis. Many patients had AFP decreases correlating with tumor shrinkage, yet an association with long-term benefit was unclear. mRECIST and on-treatment AFP levels are being explored further with brivanib in HCC.

The development of a practical, commercial process for the preparation of 4-fluoro-2-methyl-indol-5-ol and its subsequent coupling with a pyrrolotriazine to form an advanced intermediate of the oncology therapy brivanib alaninate is described. A key aspect is the multikilogram-scale preparation of the fluoroindole intermediate from trifluoronitrobenzene and the subsequent coupling while achieving impurity minimalization. As brivanib alaninate is a high-dose drug, the synthesis of high-quality API with low levels of impurities is critical.

The development of new agents in oncology has focused on disrupting key pathways in oncogenesis. Both malignant angiogenesis and peptide growth factor signaling have been studied extensively and have been validated for cancer treatment. While antibody-directed therapeutics offer increased specificity, small-molecule tyrosine kinase inhibitors often have the ability to hit multiple targets. Brivanib alaninate (BMS582664) is an oral, potent selective inhibitor of both the FGF and VEGF family of receptors. It is a first-in-class FGF/VEGF inhibitor now in late-phase clinical trials. Besides its antiangiogenic activity from blocking VEGF receptor 1–3, its ability to disrupt FGF receptors 1–3 has been suggested to add additional antiangiogenic activity, overcome resistance from VEGF blockade, and block FGF-dependent tumor proliferation. In this review, we will discuss the preclinical science driving brivanib’s development and the clinical data generated to date.

Brivanib alaninate is the orally available prodrug of brivanib, a dual inhibitor of fibroblast growth factor and vascular endothelial growth factor signaling pathways that is under therapeutic investigation for various malignancies. Brivanib alaninate inhibits CYP3A4 in vitro, and thus there is potential for drug-drug interaction with CYP3A4 substrates, such as midazolam. The present study evaluated pharmacokinetic parameters and safety/tolerability upon coadministration of brivanib alaninate and midazolam. Healthy participants received intravenous (IV) or oral midazolam with and without oral brivanib alaninate. Blood samples for pharmacokinetic analysis were collected up to 12 hours after midazolam and up to 48 hours after brivanib alaninate. Twenty-four participants were administered study drugs; 21 completed the trial. No clinically relevant effect of brivanib alaninate on the overall exposure to midazolam following IV or oral administration was observed. Orally administered brivanib alaninate was generally well tolerated in the presence of IV or oral midazolam. The lack of a pharmacokinetic interaction between brivanib and midazolam indicates that brivanib alaninate does not influence either intestinal or hepatic CYP3A4 and confirms that brivanib alaninate may be safely coadministered with midazolam and other CYP3A4 substrates.

Brivanib alaninate, the L-alanine ester prodrug of brivanib, is currently being developed as an anticancer agent. In humans, brivanib alaninate is rapidly hydrolyzed to brivanib. Prominent biotransformation pathways of brivanib included oxidation and direct sulfate conjugation. A series of in vitro studies were conducted to identify the human esterases involved in the prodrug hydrolysis and to identify the primary human cytochrome P450 and sulfotransferase (SULT) enzymes involved in the metabolism of brivanib. Brivanib alaninate was efficiently converted to brivanib in the presence of either human carboxylesterase 1 or carboxylesterase 2. Because esterases are ubiquitous, it is likely that multiple esterases are involved in the hydrolysis. Oxidation of brivanib in human liver microsomes (HLM) primarily formed a hydroxylated metabolite (M7). Incubation of brivanib with human cDNA-expressed P450 enzymes and with HLM in the presence of selective chemical inhibitors and monoclonal P450 antibodies demonstrated that CYP1A2 and CYP3A4 were the major contributors for the formation of M7. Direct sulfation of brivanib was catalyzed by multiple SULT enzymes, including SULT1A1, SULT1B1, SULT2A1, SULT1A3, and SULT1E1. Because the primary in vitro oxidative metabolite (M7) was not detected in humans after oral doses of brivanib alaninate, further metabolism studies of M7 in HLM and human liver cytosol were performed. The data demonstrated that M7 was metabolized to the prominent metabolites observed in humans. Overall, multiple enzymes are involved in the metabolism of brivanib, suggesting a low potential for drug-drug interactions either through polymorphism or through inhibition of a particular drug-metabolizing enzyme.

Rationale: Hepatocellular carcinoma (HCC) is one of the most frequent causes of cancer-related death worldwide. Its poor prognosis is due to the high invasiveness of the disease and limited efficacy of available treatments. Patient concerns: We reported an HCC patient who developed lung metastases 1 year after HCC resection. Sorafenib was then initiated; however, disease progression was noted 3 months later. Sorafenib therapy was initially maintained due to lack of effective alternatives, but disease progression continued. Diagnoses: HCC patient with lung metastases, and pulmonary portal, and mediastinal lymph node metastases (stage IVB). Interventions: Brivanib alaninate was used alone as second-line therapy. Outcomes: All metastases showed increased size on radiographic imaging approximately 3 months after brivanib alaninate was initiated. However, 2.5 months later, the lung metastases significantly decreased in size or disappeared. The pulmonary portal, and mediastinal lymph node metastases also significantly decreased in size. At 9.5 months after brivanib alaninate initiation, the pulmonary portal, and mediastinal lymph node metastases nearly disappeared, and the lung metastases continued to decrease in size. Alpha fetoprotein (AFP) level showed the same change pattern as the tumor-response observed on radiographic imaging. The total duration of brivanib alaninate treatment was 11 months, which was stopped due to repeated grade 2 thrombocytopenia. The other side effects were tolerable. Fifteen months after initiation of brivanib alaninate, the patient remained in very good condition without evidence of disease progression. Lessons: Brivanib alaninate alone as second-line therapy showed excellent antitumor efficacy for an HCC patient with numerous lung and lymph node metastases. It may exert its antitumor effects in a delayed-onset fashion. We suggest that patients receive brivanib alaninate for a long duration to fully determine its efficacy.

Introduction: Angiogenesis inhibition represents a rational therapeutic strategy in the management of solid tumors. Brivanib is a dual tyrosine kinase inhibitor with selectivity against VEFGR-2 and FGFR. Areas covered: This review provides an updated summary of preclinical and clinical experience with brivanib in cancer. Data presented in abstract form from international conferences or journal articles found with a PubMed search of published literature up to December 2010 are described in this review. Expert opinion: Brivanib appears tolerable and exhibits favorable pharmacokinetic and pharmacodynamic profiles with evidence of target inhibition in surrogate tissues. Clinical and pharmacodynamic data support an oral once daily administration at 800 mg. Brivanib shows promising activity as single agent in hepatocellular carcinoma and in combination with cetuximab in colorectal cancer. Further evaluations with cytotoxic chemotherapy and in other solid tumors are currently ongoing.

The interruption of tyrosine kinase vascular endothelial growth factor receptor-2 (VEGFR-2) signaling by the binding of a small molecule inhibitor, for example, Brivanib, to VEGFR-2 kinase domain has been shown as an effective method of slowing angiogenesis and tumor progression. [14C]Brivanib, 13 and its prodrug [14C]Brivanib Alaninate, 15 were prepared to support preclinical and clinical studies. Their respective stable isotope-labeled versions, [13CN2]Brivanib, 21 and [13CN2]Brivanib Alaninate, 28, were also prepared to support bioanalytical LC-MS analyses of clinical samples. All of the four title compounds were synthetically derived from the respective isotopically labeled common pyrrolotriazinone intermediate, 6 or 16. This labeled central core pyrrolotriazinone was also conveniently used to synthesize other structurally related drug discovery candidates. Copyright © 2011 John wiley & Sons, Ltd.

Brivanib [(R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[1,2,4]triazin-6-yloxy)propan-2-ol, BMS-540215] is a potent and selective dual inhibitor of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) signaling pathways. Its alanine prodrug, brivanib alaninate [(1R,2S)-2-aminopropionic acid 2-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-1-methylethyl ester, BMS-582664], is currently under development as an oral agent for the treatment of cancer. This study describes the in vivo biotransformation of brivanib after a single oral dose of [(14)C]brivanib alaninate to intact rats, bile duct-cannulated (BDC) rats, intact monkeys, BDC monkeys, and humans. Fecal excretion was the primary route of elimination of drug-derived radioactivity in animals and humans. In BDC rats and monkeys, the majority of radioactivity was excreted in bile. Brivanib alaninate was rapidly and completely converted via hydrolysis to brivanib in vivo. The area under the curve from zero to infinity of brivanib accounted for 14.2 to 54.3% of circulating radioactivity in plasma in animals and humans, suggesting that metabolites contributed significantly to the total drug-related radioactivity. In plasma from animals and humans, brivanib was a prominent circulating component. All the metabolites that humans were exposed to were also present in toxicological species. On the basis of metabolite exposure and activity against VEGF and FGF receptors of the prominent human circulating metabolites, only brivanib is expected to contribute to the pharmacological effects in humans. Unchanged brivanib was not detected in urine or bile samples, suggesting that metabolic clearance was the primary route of elimination. The primary metabolic pathways were oxidative and conjugative metabolism of brivanib.

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 dimeglumine 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

Purpose: Hepatocellular carcinoma (HCC) is the fifth most common primary neoplasm; surgery is the only curative option but 5-year survival rates are only 25% to 50%. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) are known to be involved in growth and neovascularization of HCC. Therefore, agents that target these pathways may be effective in the treatment of HCC. The aim of this study was to determine the antineoplastic activity of brivanib alaninate, a dual inhibitor of VEGF receptor (VEGFR) and FGF receptor (FGFR) signaling pathways. Experimental Design: Six different s.c. patient-derived HCC xenografts were implanted into mice. Tumor growth was evaluated in mice treated with brivanib compared with control. The effects of brivanib on apoptosis and cell proliferation were evaluated by immunohistochemistry. The SK-HEP1 and HepG2 cells were used to investigate the effects of brivanib on the VEGFR-2 and FGFR-1 signaling pathways in vitro. Western blotting was used to determine changes in proteins in these xenografts and cell lines. Results: Brivanib significantly suppressed tumor growth in five of six xenograft lines. Furthermore, brivanib–induced growth inhibition was associated with a decrease in phosphorylated VEGFR-2 at Tyr1054/1059, increased apoptosis, reduced microvessel density, inhibition of cell proliferation, and down-regulation of cell cycle regulators. The levels of FGFR-1 and FGFR-2 expression in these xenograft lines were positively correlated with its sensitivity to brivanib-induced growth inhibition. In VEGF-stimulated and basic FGF stimulated SK-HEP1 cells, brivanib significantly inhibited VEGFR-2, FGFR-1, extracellular signal-regulated kinase 1/2, and Akt phosphorylation. Conclusion: This study provides a strong rationale for clinical investigation of brivanib in patients with HCC.

A series of amino acid ester prodrugs of the dual VEGFR-2/FGFR-1 kinase inhibitor 1 (BMS-540215) was prepared in an effort to improve the aqueous solubility and oral bioavailability of the parent compound. These prodrugs were evaluated for their ability to liberate parent drug 1 in in vitro and in vivo systems. The l-alanine prodrug 8 (also known as brivanib alaninate/BMS-582664) was selected as a development candidate and is presently in phase II clinical trials.

The process of neovascularization from preexisting blood vessels, referred to as angiogenesis, plays a critical role in both tumor growth and dissemination in multiple cancer types. Currently, there exists a need to identify biomarkers that can both indicate biological activity and predict efficacy at the molecular level for antiangiogenesis drugs which are anticipated to result in tumor stasis rather than regression. To identify such biomarkers, athymic mice bearing L2987 human tumor xenografts were treated with the antiangiogenic agent brivanib alaninate, which is currently under clinical evaluation. This is an orally available and selective tyrosine kinase inhibitor that targets the key angiogenesis receptors vascular endothelial growth factor receptor 2 (VEGFR-2) and fibroblast growth factor receptor 1. In the described studies, tumor samples were collected from these xenografts and RNA was extracted for gene expression profiling on Affymetrix 430A mouse GeneChips. Statistical analysis was done using a defined set of genes identified to be coexpressed with VEGFR-2 from a clinical tumor gene expression profiling database and between tumor samples isolated from brivanib alaninate–treated and untreated mice. Tyrosine kinase receptor 1 (Tie-1), collagen type IV α1 (Col4a1), complement component 1, q subcomponent receptor 1 (C1qr1), angiotensin receptor–like 1 (Agtrl1), and vascular endothelial-cadherin (Cdh5) were all identified to be significantly modulated by treatment with brivanib alaninate. These genes, which may be potentially useful as markers of brivanib alaninate activity, were further studied at the protein level in two separate in vivo human colon tumor xenograft models, HCT116 and GEO, using immunohistochemistry-based approaches. [Cancer Res 2007;67(14):6899–906]

Metastatic colorectal cancer (mCRC) is still one of the tumor types with the highest incidence and mortality. In 2012, colorectal cancer was the second most prevalence cancer among males (9%) and the third among females (8%). In this disease, early diagnosis is important to improve treatment outcomes. However, at the time of diagnosis, about one quarter of patients already have metastases, and overall survival of these patients at 5-years survival is very low. Because of these poor statistics, the development of new drugs against specific targets, including the pathway of angiogenesis, has witnessed a remarkable increase. So, targets therapies through epidermal growth factor and its receptor and also KRAS pathways modulation acquired a main role whether in association with standard chemotherapy and radiotherapy. With the current knowledge in the field of molecular biology, including genetic mutations and polymorphisms, we know better why patients respond so differently to the same treatments. So, in the future we can develop increasingly personalized treatments to the patient and not the disease. This review aims to summarize some molecular pathways and their relation to tumor growth, as well as novel targeted developing drugs and recently approved for mCRC. (C) 2014 Baishideng Publishing Group Inc. All rights reserved. info:eu-repo/semantics/publishedVersion

Purpose The antiepidermal growth factor receptor monoclonal antibody cetuximab has improved survival in patients with metastatic, chemotherapy-refractory, wild-type K-RAS colorectal cancer. The addition of brivanib, a tyrosine kinase inhibitor targeting vascular endothelial growth factor receptor and fibroblast growth factor receptor, to cetuximab has shown encouraging early clinical activity. Patients and Methods Patients with metastatic colorectal cancer previously treated with combination chemotherapy were randomly assigned 1:1 to receive cetuximab 400 mg/m2 intravenous loading dose followed by weekly maintenance of 250 mg/m2 plus either brivanib 800 mg orally daily (arm A) or placebo (arm B). The primary end point was overall survival (OS). Results A total of 750 patients were randomly assigned (376 in arm A and 374 in arm B). Median OS in the intent-to-treat population was 8.8 months in arm A and 8.1 months in arm B (hazard ratio [HR], 0.88; 95% CI, 0.74 to 1.03; P = .12). Median progression-free survival (PFS) was 5.0 months in arm A and 3.4 months in arm B (HR, 0.72; 95% CI, 0.62 to 0.84; P < .001). Partial responses observed (13.6% v 7.2%; P = .004) were higher in arm A. Incidence of any grade ≥ 3 adverse events was 78% in arm A and 53% in arm B. Fewer patients received ≥ 90% dose-intensity of both cetuximab (57% v 83%) and brivanib/placebo (48% v 87%) in arm A versus arm B, respectively. Conclusion Despite positive effects on PFS and objective response, cetuximab plus brivanib increased toxicity and did not significantly improve OS in patients with metastatic, chemotherapy-refractory, wild-type K-RAS colorectal cancer.

Hepatic impairment may impede tyrosine kinase inhibitor metabolism. This phase I study compared the pharmacokinetics of brivanib in patients with hepatocellular carcinoma (HCC) and varying levels of hepatic impairment with those with non-HCC malignancies and normal liver function. Patients were assigned to the following groups: Groups A, B, and C (HCC plus mild, moderate, or severe hepatic impairment, respectively) and Group D (non-HCC malignancy and normal hepatic function). Brivanib alaninate (brivanib prodrug) doses were 400 mg in Groups A, B, and D and 200 mg in Group C. Brivanib exposure was determined on day 1 (single dose) and day 28 (multiple doses). Twenty-four patients participated in the study. After a single brivanib alaninate dose, brivanib exposure was comparable between Groups A, B, and D. Area under the concentration–time curve was 50 % higher in Group C versus Group D. There were not enough data to draw conclusions on multiple doses. Safety profile in Groups A, B, and D was consistent with previous brivanib monotherapy experience. Tolerability could not be assessed in Group C because of dose interruptions and discontinuations, generally due to the disease natural history. Brivanib exposure was similar in patients with HCC and mild or moderate hepatic impairment (Child-Pugh [CP] A or B status) and those with non-HCC malignancies and normal hepatic function, suggesting dose adjustment is unnecessary with CP A or B status. Experience with HCC and severe hepatic impairment (CP C status) is insufficient to recommend brivanib use in this population.

Brivanib alaninate is an orally administered alanine prodrug of brivanib, a dual inhibitor of the vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) signaling pathways. It is currently in clinical trials for the treatment of hepatocellular carcinoma and colorectal cancer. Brivanib has a single asymmetric center derived from a secondary alcohol. The potential for chiral inversion was investigated in incubations with liver subcellular fractions and in animals and humans after oral doses of brivanib alaninate. Incubations of [¹⁴C]brivanib alaninate with liver microsomes and cytosols from rats, monkeys, and humans followed by chiral chromatography resulted in two radioactive peaks, corresponding to brivanib and its enantiomer. The percentage of the enantiomeric metabolite relative to brivanib in microsomal and cytosolic incubations of different species in the presence of NADPH ranged from 11.6 to 15.8 and 0.8 to 3.1%, respectively. The proposed mechanism of inversion involves the oxidation of brivanib to a ketone metabolite, which is subsequently reduced to brivanib and its enantiomer. After oral doses of brivanib alaninate to rats and monkeys, the enantiomeric metabolite was a prominent drug-related component in plasma, with the percentages of area under the curve (AUC) at 94.7 and 39.7%, respectively, relative to brivanib. In humans, the enantiomeric metabolite was a minor circulating component, with the AUC3% of brivanib. Pharmacological studies indicated that brivanib and its enantiomer had similar potency toward the inhibition of VEGF receptor-2 and FGF receptor-1 kinases. Because of low plasma concentration in humans, the enantiomeric metabolite was not expected to contribute significantly to target-related pharmacology of brivanib. Moreover, adequate exposure in the toxicology species suggested no specific safety concerns with respect to exposure to the enantiomeric metabolite.

Objectives Drug-excipient binding can affect in-vitro drug release. Literature suggests that drug-excipient ionic binding interaction that is not disrupted by physiological salt concentration in the dissolution medium can impact a drug's oral bioavailability. We investigated whether nondisruption of interaction by physiological salt concentration was an adequate predictor of its biorelevance using the binding of a model amine high dose drug brivanib alaninate (BA) to croscarmellose sodium (CCS) as an example. Methods BA was formulated into an immediate release tablet using CCS as disintegrant by a wet granulation process. In-vitro drug release was carried out as a function of pH and buffer concentration of the medium. BA-CCS binding was studied in buffer solution and data fitted to a Langmuir isotherm. A simulation model and an isothermal titration calorimetry method were developed to assess the bioavailability risk and strength of drug-excipient binding interaction, independent of physiological salt concentration consideration. Key findings BA-CCS binding was pH-dependent, reversible, ionic, and not disrupted by increasing the buffer concentration in the dissolution medium. Absorption simulation predictions of no effect of CCS binding on BA's bioavailability were confirmed by a monkey pharmacokinetic study. Conclusions A pH-dependent and reversible weak drug-excipient binding interaction is unlikely to affect the oral bioavailability of high dose drugs.

Background: The objectives of this phase I study were to determine the safety, pharmacokinetics (PK), pharmacodynamics and efficacy of brivanib combined with full-dose cetuximab in patients with advanced gastrointestinal malignancies. Methods: Patients with advanced gastrointestinal malignancies who had failed prior therapies received brivanib (320, 600 or 800 mg daily) plus cetuximab (400 mg m–2 loading dose then 250 mg m–2 weekly). Assessments included adverse events, PK, tumour response, 2[18F]fluoro-2-deoxyglucose positron-emitting tomography and K-Ras mutation analyses. Results: Toxicities observed were manageable; the most common treatment-related toxicities (>10% of patients) were fatigue, diarrhoea, anorexia, increase in aspartate aminotransferase and alanine aminotransferase, acneiform dermatitis, headache, mucosal inflammation, nausea, dry skin, vomiting, hypertension, pruritus, proteinuria and weight loss. Of 62 patients, 6 (9.7%) had objective radiographic partial responses, with an overall response rate of 10%. Median duration of response was 9.2 months; median progression-free survival was 3.9 months. Conclusions: The acceptable toxicity profile and efficacy of brivanib observed in this study were promising. These findings are being further evaluated in a phase III study of brivanib plus cetuximab vs cetuximab alone in patients previously treated with combination chemotherapy for K-Ras wild-type advanced metastatic colorectal cancer.

Background: This study was designed to determine the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of brivanib in patients with advanced/metastatic solid tumors. Patients and methods: Ninety patients enrolled in this two-part, phase I open-label study of oral brivanib alaninate. The primary objectives of this study were (in part A) dose-limiting toxicity, maximum tolerated dose (MTD) and the lowest biologically active dose level and (in part B) the optimal dose/dose range. The secondary objectives of this study were preliminary evidence of antitumor activity, PK and PD. Results: Across part A (open-label dose escalation and MTD) and part B (open-label dose optimization), 68 patients received brivanib alaninate. Brivanib demonstrated a manageable toxicity profile at doses of 180–800 mg. Most toxic effects were mild. Systemic exposure of the active moiety brivanib increased linearly £1000 mg/day. The MTD was 800 mg/day. Forty-four patients were treated at the MTD: 20 with 800 mg continuously, 11 with 800 mg intermittently and 13 with 400 mg b.i.d. doses. Partial responses were confirmed in two patients receiving brivanib ‡600 mg. Dynamic contrast-enhanced magnetic resonance imaging demonstrated statistically significant decreases in parameters reflecting tumor vascularity and permeability after multiple doses in the 800-mg continuous q.d. and 400-mg b.i.d. dose cohorts. Conclusion: In patients with advanced/metastatic cancer, brivanib demonstrates promising antiangiogenic and antitumor activity and manageable toxicity at doses £800 mg orally q.d., the recommended phase II study dose.

Brivanib alaninate, an orally available prodrug of brivanib, is currently under evaluation for the treatment of several malignancies. This study aimed to (1) investigate effects of a high-fat meal on single-dose pharmacokinetics of brivanib in subjects with advanced/metastatic solid tumors and (2) assess the safety and preliminary efficacy of single and multiple doses of brivanib alaninate in this population.A two-part study was conducted consisting of a single-dose phase (Part A) and a multiple-dose phase (Part B). In Part A, subjects received a single dose of brivanib alaninate (800 mg) either in a fasting state or following ingestion of a high-fat meal (approximately 951 kcal [15% protein, 33% carbohydrate, 52% fat]); serial blood samples were collected for pharmacokinetic analysis up to 48 h post-dosing. In Part B, subjects received brivanib alaninate (800 mg) once daily until discontinuation. Throughout both phases, subjects were evaluated for adverse events (AEs) and best clinical response.No clinically significant differences in brivanib exposure were observed between fed and fasting subjects in Part A; C (max) was unchanged and AUC(INF) decreased marginally when administered in a fed versus fasted state. In Part A, the incidence of treatment-emergent AEs was broadly similar in a fed or fasted state. Brivanib alaninate was generally well tolerated throughout the study and showed preliminary evidence of antitumor activity.Consumption of a high-fat meal had no significant effect on brivanib pharmacokinetics. The study further demonstrates the acceptable safety/tolerability profile and antitumor potential of brivanib in patients with advanced malignancies.

The goal of this study was to evaluate the pharmacokinetics, mass balance, metabolism, routes and extent of elimination, and safety of a single oral dose of (14)C-labeled brivanib alaninate and the safety and tolerability of brivanib after multiple doses in patients with advanced or metastatic solid tumors. This was a two-part, single-center, open-label, single oral-dose (part A) followed by multiple-dose (part B) study in patients with advanced or metastatic solid tumors. In part A, patients received a single dose of [(14)C]brivanib alaninate and in part B patients received 800 mg of nonradiolabeled brivanib alaninate every day. Four patients (two white, two black: two with non-small-cell lung cancer, one with ovarian cancer, and one with renal cell carcinoma) were treated in both parts. The median time to reach the maximal plasma concentration of brivanib was 1 h, geometric mean maximal plasma concentration was 6146 ng/ml, mean terminal half-life was 13.8 h, and geometric mean apparent oral clearance was 14.7 l/h. After a single oral dose of [(14)C]brivanib alaninate, 12.2 and 81.5% of administered radioactivity was recovered in urine and feces, respectively. Brivanib alaninate was completely converted to the active moiety, brivanib, and the predominant route of elimination was fecal. Renal excretion of unchanged brivanib was minimal. Brivanib was well tolerated; fatigue was the most frequent adverse event occurring in all patients and the most frequent treatment-related adverse event in three (75%). The best clinical response in one patient was stable disease; the other three had progressive disease. Brivanib alaninate was rapidly absorbed and extensively metabolized after a single 800-mg oral dose; the majority of drug-related radioactivity was excreted in feces.

Purpose Tamoxifen, a selective oestrogen receptor modulator (SERM), and brivanib alaninate, a vascular endothelial growth factor receptor 2 (VEGFR-2) inhibitor, are two target specific agents that result in a substantial decrease in tumour growth when given alone. Tamoxifen activates SERM stimulated breast and endometrial tumour growth. Tamoxifen and brivanib alaninate have side-effects that can affect therapeutic outcomes. The primary goal of the current study was to evaluate the therapeutic effects of lower doses of both agents when given in combination to mice with SERM sensitive, oestrogen stimulated tumour xenografts (MCF-7 E2 tumours). Experiments were conducted to evaluate the response of SERM stimulated breast (MCF-7 Tam, MCF-7 Ral) and endometrial tumours (EnCa 101) to demonstrate the activity of brivanib alaninate in SERM resistant models. Experimental design In the current study, tumour xenografts were minced and bi-transplanted into the mammary fat pads of athymic, ovariectomised mice. Preliminary experiments were conducted to determine an effective oral dose of tamoxifen and brivanib alaninate that had minimal effect on tumour growth. Doses of 125 μg of tamoxifen and 0.05 mg/g of brivanib alaninate were evaluated. An experiment was designed to evaluate the effect of the two agents together when started at the time of tumour implantation. An additional experiment was done in which tumours were already established and then treated, to obtain enough tumour tissue for molecular analysis. Results Brivanib alaninate was effective at inhibiting tumour growth in SERM sensitive (MCF-7 E2) and SERM stimulated (EnCa 101, MCF-7 Ral, MCF-7 Tam) models. The effect of the low dose drug combination as an anti-tumour strategy for SERM sensitive (MCF-7 E2) in early treatment was as effective as higher doses of either drug used alone. In established tumours, the combination is successful at decreasing tumour growth, while neither agent alone is effective. Molecular analysis revealed a decreased phosphorylation of VEGFR-2 in tumours that were treated with brivanib alaninate and an increase in VEGFA transcription to compensate for the blockade of VEGFR-2 by increasing the transcription of VEGFA. Tamoxifen increases the phosphorylation of VEGFR-2 and this effect is abrogated by brivanib alaninate. There was also increased necrosis in tumours treated with brivanib alaninate. Conclusion Historically, tamoxifen has a role in blocking angiogenesis as well as the blockade of the ER. Tamoxifen and a low dose of an angiogenesis inhibitor, brivanib alaninate, can potentially be combined not only to maximise therapeutic efficacy but also to retard SERM resistant tumour growth.

Brivanib alaninate is a prodrug of brivanib (BMS-540215), a potent oral VEGFR-2 inhibitor and is currently in development for the treatment of hepatocellular and colon carcinomas. In vitro and in vivo studies were conducted to characterize the preclinical pharmacokinetics and disposition of brivanib and brivanib alaninate, and antitumor efficacy in mice bearing human xenografts.In vitro studies were conducted in liver and intestinal fractions, plasma and Caco-2 cells to assess the metabolic stability. Pharmacokinetics of brivanib were determined in preclinical species after administration of single intravenous or oral doses of both brivanib and brivanib alaninate. The antitumor efficacy was assessed at equimolar doses in nude mice bearing human tumor xenografts. Human efficacious dose was predicted based on projected human pharmacokinetic parameters and exposure at efficacious doses in the mouse efficacy models.In vitro and in vivo studies indicated that brivanib alaninate was efficiently converted to brivanib. Brivanib showed good brain penetration in rats consistent with its high intrinsic permeability and lack of active efflux in Caco-2 cells. The oral bioavailability of brivanib varied among species (22-88%) and showed dissolution rate-limited absorption even when combined with organic co-solvents. Administration of brivanib as brivanib alaninate allowed completely aqueous vehicles, and an improvement in the oral bioavailability (55-97%) was observed. The clearance of brivanib in humans is anticipated to be low to intermediate (hepatic extraction ratio0.7), while its volume of distribution is expected to be high. The minimum efficacious dose of brivanib alaninate was determined to be 60 mg/kg per day.Brivanib alaninate is rapidly and efficiently converted to the parent, brivanib, as demonstrated both in vitro and in vivo and offers an excellent mode to deliver brivanib orally.

A series of substituted 4-(4-fluoro-1H-indol-5-yloxy)pyrrolo[2,1-f][1,2,4]triazine-based inhibitors of vascular endothelial growth factor receptor-2 kinase is reported. Structure-activity relationship studies revealed that a methyl group at the 5-position and a substituted alkoxy group at the 6-position of the pyrrolo[2,1-f][1,2,4]triazine core gave potent compounds. Biochemical potency, kinase selectivity, and pharmacokinetics of the series were optimized and in vitro safety liabilities were minimized to afford BMS-540215 (12), which demonstrated robust preclinical in vivo activity in human tumor xenograft models. The l-alanine prodrug of 12, BMS-582664 (21), is currently under evaluation in clinical trials for the treatment of solid tumors.

3572 Notice of Retraction: "Analysis of plasma biomarkers potentially associated with antiangiogenic resistance in NCIC CTG/AGITG CO.20: A phase III randomized trial of cetuximab (CET) plus either brivanib alaninate (BRIV) or placebo in patients (pts) with chemotherapy refractory, k-RAS wild-type (WT), metastatic colorectal carcinoma (mCRC)." Abstract 3572, published in the 2012 Annual Meeting Proceedings Part I, a supplement to the Journal of Clinical Oncology, has been retracted by Jeremy Shapiro, MBBS, and Lillian Siu, MD, on behalf of the NCIC, AGITG, and all authors of the abstract. The authors have been unable to finalize their data presentation due to the complexity of the analysis, and difficulties with the statistical interpretation of the significance of several possible predictive biomarkers. Background: In the CO.20 trial, the addition of BRIV, a tyrosine kinase inhibitor targeting vascular endothelial and fibroblast growth factor receptors (VEGFR2,3 and FGFR 1,2,3), to CET, increased objective response rate and progression free survival, but did not significantly increase overall survival. Previous clinical studies demonstrated modulation of circulating angiogenic factors (CAF) in CRC which occur on therapy or upon progression (Kopetz JCO 2010). Methods: CO.20 pts were randomized 1:1 to CET plus either BRIV (Arm A) or placebo (ARM B) in a double-blind design. Pts may have had 1 prior anti-VEGF based therapy, but no prior anti-EGFR therapy. Primary endpoint was overall survival (OS). Baseline pre-treatment plasma samples were analyzed using commercial Multi-Analyte ELISAs to measure CAF, immunologic, and growth factors, with an initial discovery and subsequent validation data set. Results: 750 pts were randomized (376 in Arm A and 374 in Arm B). Median OS in the intent-to-treat population was 8.8 months in Arm A and 8.1 months in Arm B, hazard ratio (HR)=0.88; 95%CI=0.74-1.03; p=0.12. In an exploratory subgroup analysis, there is a statistically non-significant trend favoring the effect of brivanib on OS among the 41% pts who received prior anti-VEGF therapy versus those who did not.Baseline plasma samples were collected from 96% of pts, and analysis is ongoing. Results of the largest circulating biomarker analysis will be presented. CAF results will be compared with response and survival data to look for potential patient subgroups that may benefit more from the combination treatment. Conclusions: This large scale analysis will provide insights on the potential use of CAF or CAF profiles as predictive markers in CRC.

3504 Background: The anti-EGFR monoclonal antibody CET has improved survival in pts with chemotherapy refractory, K-RAS WT mCRC. BRIV is a potent inhibitor of multiple receptor tyrosine kinases including both VEGFR and FGFR. The combination of CET and BRIV targets tumor growth and angiogenesis and demonstrated encouraging activity in an early phase clinical trial. Methods: Pts with mCRC previously treated with combination chemotherapy were randomized 1:1 to receive CET 400 mg/m2 IV loading dose followed by weekly maintenance of 250 mg/m2 plus either BRIV 800 mg PO daily (Arm A) or placebo (Arm B). Pts may have had 1 prior anti-VEGF, but no prior anti-EGFR therapy. Primary endpoint was overall survival (OS). Results: From 02/2008 to 02/2011, 750 pts were randomized (376 in Arm A and 374 in Arm B). Demographics: median age=64 (range 27-88); male=64%; ECOG 0:1:2 (%)=32:58:10; >3 prior chemotherapy regimens=92%; prior anti-VEGF therapy=41%; K-RAS WT=97%. Primary analysis was conducted per protocol after 536 deaths were observed, with median OS of 8.8 months in Arm A and 8.1 months in Arm B, hazard ratio (HR)=0.88; 95% CI=0.74 to 1.03; p=0.12. Median progression-free survival (PFS) was 5.0 months in Arm A and 3.4 months in Arm B, HR=0.72; 95% CI=0.62 to 0.84; p

542 Background: The CO.20 trial randomized pts with K-RAS WT chemotherapy refractory mCRC to receive CET + BRIV (Arm A) or CET + placebo (Arm B). Although overall survival (primary endpoint) was not significantly improved (HR=0.88, p=0.12) in this heavily pre-treated population, progression free survival (PFS) favoured Arm A (HR=0.72 p Methods: Patients with K-RAS WT mCRC previously treated with or with contraindications to a fluoropyrimidine, irinotecan, and oxaliplatin were randomized to a loading dose of IV CET followed by weekly IV CET + BRIV 800 mg PO daily or CET + placebo daily. QoL, a secondary endpoint, was assessed using the EORTC QLQ-C30 at baseline and at 2, 4, 6, 8, 12, 16 and 24 weeks until progression or clinical deterioration. Co-primary QoL endpoints were defined a priori as definitive deterioration (first worsening from baseline of ≥10 points) on the physical function (PF) and Global scales. Time to QoL deterioration (DET) was measured from randomization using the Hochberg procedure to adjust for multiple testing. Results: 721 (358 Arm A) of 750 randomized pts were assessable for QoL. QoL compliance did not differ by arm and declined in follow-up from 87% to 47% over time. Baseline Global and PF scores did not differ by arm. Median time to QoL DET was 1.6 vs 1.1 mo for Global (p=0.02) and 5.6 vs 1.7 mo. for PF (p Conclusions: Despite a PFS benefit, the combination of CET + BRIV worsened time to QoL DET on the PF and Global scales of EORTC QLQ-C30 in pts with chemotherapy refractory K-RAS WT mCRC. This result may be due to higher rates of fatigue and gastrointestinal adverse events observed with the combination.

386 Background: The anti-EGFR monoclonal antibody CET has improved survival in pts with MET, chemotherapy refractory, K-RAS wild type (WT) CRC. The addition of BRIV, a tyrosine kinase inhibitor targeting vascular endothelial and fibroblast growth factor receptors (VEGFR/FGFR), to CET has shown encouraging activity in an early phase clinical trial. Methods: Pts with MET CRC previously treated with combination chemotherapy were randomized 1:1 to receive CET 400 mg/m2 IV loading dose followed by weekly maintenance of 250 mg/m2 plus either BRIV 800 mg PO daily (Arm A) or placebo (Arm B). Pts may have had 1 prior anti-VEGF, but no prior anti-EGFR therapy. The trial was amended shortly after opening to enrol K-RAS WT pts. Primary endpoint was overall survival (OS). Results: From 02/2008 to 02/2011, 750 pts were randomized (376 in Arm A and 374 in Arm B). Demographics: median age=64 (range 27-88); male=64%; ECOG 0:1:2 (%)=32:58:10; >3 prior chemotherapy regimens=92%; prior anti-VEGF therapy=41%; K-RAS WT=97%. Median OS in the intent-to-treat population was 8.8 months in Arm A and 8.1 months in Arm B, hazard ratio (HR)=0.88; 95% CI=0.74 to 1.03; p=0.12. Median progression-free survival (PFS) was 5.0 months in Arm A and 3.4 months in Arm B, HR=0.72; 95% CI=0.62 to 0.84; p Conclusions: Despite positive effects on PFS and objective response, the combination of CET+BRIV did not significantly improve OS in pts with MET, chemotherapy refractory, K-RAS WT CRC. AEs were consistent with those reported for each drug given as monotherapy.

TPS163 Background: While anti-EGFR monoclonal antibody, CET, improves survival in KRAS WT chemotherapy refractory aCRC, dual biologic strategies may overcome observed resistance to therapy. BRIV is...

3095 Background: Brivanib alaninate is the L-alanine ester prodrug of brivanib, an oral selective dual inhibitor of vascular endothelial growth and fibroblast growth pathway receptors. It is curren...