Frank Hilberg

BIBF 1120: Triple Angiokinase Inhibitor with Sustained Receptor Blockade and Good Antitumor Efficacy

Inhibition of tumor angiogenesis through blockade of the vascular endothelial growth factor (VEGF) signaling pathway is a novel treatment modality in oncology. Preclinical findings suggest that long-term clinical outcomes may improve with blockade of additional proangiogenic receptor tyrosine kinases: platelet-derived growth factor receptors (PDGFR) and fibroblast growth factor receptors (FGFR). BIBF 1120 is an indolinone derivative potently blocking VEGF receptor (VEGFR), PDGFR and FGFR kinase activity in enzymatic assays (IC(50), 20-100 nmol/L). BIBF 1120 inhibits mitogen-activated protein kinase and Akt signaling pathways in three cell types contributing to angiogenesis, endothelial cells, pericytes, and smooth muscle cells, resulting in inhibition of cell proliferation (EC(50), 10-80 nmol/L) and apoptosis. In all tumor models tested thus far, including human tumor xenografts growing in nude mice and a syngeneic rat tumor model, BIBF 1120 is highly active at well-tolerated doses (25-100 mg/kg daily p.o.), as measured by magnetic resonance imaging of tumor perfusion after 3 days, reducing vessel density and vessel integrity after 5 days, and inducing profound growth inhibition. A distinct pharmacodynamic feature of BIBF 1120 in cell culture is sustained pathway inhibition (up to 32 hours after 1-hour treatment), suggesting slow receptor off-kinetics. Although BIBF 1120 is rapidly metabolized in vivo by methylester cleavage, resulting in a short mean residence time, once daily oral dosing is fully efficacious in xenograft models. These distinctive pharmacokinetic and pharmacodynamic properties may help explain clinical observations with BIBF 1120, currently entering phase III clinical development.

Design, synthesis, and evaluation of indolinones as triple angiokinase inhibitors and the discovery of a highly specific 6-methoxycarbonyl-substituted indolinone (BIBF 1120).

Inhibition of tumor angiogenesis through blockade of the vascular endothelial growth factor (VEGF) signaling pathway is a new treatment modality in oncology. Preclinical findings suggest that blockade of additional pro-angiogenic kinases, such as fibroblast and platelet-derived growth factor receptors (FGFR and PDGFR), may improve the efficacy of pharmacological cancer treatment. Indolinones substituted in position 6 were identified as selective inhibitors of VEGF-, PDGF-, and FGF-receptor kinases. In particular, 6-methoxycarbonyl-substituted indolinones showed a highly favorable selectivity profile. Optimization identified potent inhibitors of VEGF-related endothelial cell proliferation with additional efficacy on pericyctes and smooth muscle cells. In contrast, no direct inhibition of tumor cell proliferation was observed. Compounds 2 (BIBF 1000) and 3 (BIBF 1120) are orally available and display encouraging efficacy in in vivo tumor models while being well tolerated. The triple angiokinase inhibitor 3 is currently in phase III clinical trials for the treatment of nonsmall cell lung cancer.

Nintedanib: from discovery to the clinic.

Nintedanib (BIBF1120) is a potent, oral, small-molecule tyrosine kinase inhibitor, also known as a triple angiokinase inhibitor, inhibiting three major signaling pathways involved in angiogenesis. Nintedanib targets proangiogenic and pro-fibrotic pathways mediated by the VEGFR family, the fibroblast growth factor receptor (FGFR) family, the platelet-derived growth factor receptor (PDGFR) family, as well as Src and Flt-3 kinases. The compound was identified during a lead optimization program for small-molecule inhibitors of angiogenesis and has since undergone extensive clinical investigation for the treatment of various solid tumors, and in patients with the debilitating lung disease idiopathic pulmonary fibrosis (IPF). Recent clinical evidence from phase III studies has shown that nintedanib has significant efficacy in the treatment of NSCLC, ovarian cancer, and IPF. This review article provides a comprehensive summary of the preclinical and clinical research and development of nintedanib from the initial drug discovery process to the latest available clinical trial data.

CD44 Suppresses TLR-Mediated Inflammation

The cell adhesion molecule CD44, which is the major hyaluronan receptor, has been implicated in the binding, endocytosis, and metabolism of hyaluronan. Previous studies have revealed that CD44 plays crucial roles in a variety of inflammatory diseases. In recent years, TLRs, which are ancient microbial pattern recognition receptors, have been shown to initiate an innate immune response and have been linked to a variety of inflammatory diseases. The present study shows that CD44 negatively regulates in vivo inflammation mediated by TLRs via NF-κB activation, which leads to proinflammatory cytokine production. Furthermore, our results show that CD44 directly associates with TLR2 when stimulated by the TLR2 ligand zymosan and that the cytoplasmic domain of CD44 is crucial for its regulatory effect on TLR signaling. This study indicates that CD44 plays a protective role in TLR-mediated inflammation and is the first to demonstrate a direct association between CD44 and a TLR.

Selective inhibition of the epidermal growth factor receptor tyrosine kinase by BIBX1382BS and the improvement of growth delay, but not local control, after fractionated irradiation in human FaDu squamous cell carcinoma in the nude mouse.

Purpose: To investigate the effect of BIBX1382BS, an inhibitor of the epidermal growth factor receptor tyrosine kinase, on proliferation and clonogenic cell survival of FaDu human squamous cell carcinoma in vitro, and on tumour growth and local tumour control after fractionated irradiation over 6 weeks in nude mice. FaDu human squamous cell carcinoma is epidermal growth factor receptor positive and significant repopulation during fractionated irradiation was demonstrated in previous experiments. Materials and methods: Receptor status, receptor phosphorylation, cell cycle distribution, cell proliferation and clonogenic cell survival after irradiation were assayed with and without BIBX1382BS (5 µM) in vitro. Tumour volume doubling time, BrdUrd and Ki67 labelling indices and apoptosis were investigated in unirradiated tumours growing in NMRI nude mice treated daily with BIBX1382BS (50 mg kg−1 body weight orally) or carrier. Tumour growth delay and dose–response curves for local tumour control were determined after irradiation with 30 fractions within 6 weeks. Results: BIBX1382BS blocked radiation‐induced phosphorylation of the epidermal growth factor receptor and reduced the doubling time of FaDu cells growing in vitro by a factor of 4.9 (p=0.008). Radiosensitivity in vitro remained unchanged after incubation with BIBX1382BS for 3 days and decreased moderately after 6 days (p=0.001). BIBX1382BS significantly reduced the volume doubling time of established FaDu tumours in nude mice by factors of 2.6 when given over 15 days (p<0.001) and 3.7 when applied over 6 weeks (p<0.001). When given simultaneously to fractionated irradiation, growth delay was significantly prolonged by an average of 33 days (p=0.003). Local tumour control was not improved by BIBX1382BS. The radiation doses necessary to control 50% of the tumours locally were 63.6 Gy (95% confidence interval 55; 73) for irradiation alone and 67.8 Gy (60; 77) for the combined treatment (p=0.5). Conclusions: Despite clear antiproliferative activity in rapidly repopulating FaDu human squamous cell carcinoma and significantly increased tumour growth delay when combined with fractionated irradiation, local tumour control was not improved by BIBX1382BS. The results do not disprove that epidermal growth factor receptor inhibition might enhance the results of radiotherapy. However, the results imply that further preclinical investigations using relevant treatment schedules and appropriate endpoints are necessary to explore the mechanisms of action and efficacy of such combinations.

Bortezomib, Dexamethasone, and Fibroblast Growth Factor Receptor 3–Specific Tyrosine Kinase Inhibitor in t(4;14) Myeloma

Purpose: Novel drugs including targeted approaches have changed treatment paradigms for multiple myeloma (MM) and may also have therapeutic potential in the poor-prognosis t(4;14) subset; t(4;14) results in overexpressed and activated fibroblast growth factor receptor 3 (FGFR3). Blocking this receptor tyrosine kinase (RTK) induces apoptosis in t(4;14)+ MM cells and decreases adhesion to bone marrow stromal cells (BMSC). Using combinations of novel drugs, we investigated potential enhancement of single-agent activities within the tumor cells, targeting of the marrow micromilieu, or circumvention of drug resistance in t(4;14)+ MM. Experimental Design: We tested effects on apoptosis and related signaling pathways in the t(4;14)+ MM subset, applying drug combinations including a FGFR3 tyrosine kinase inhibitor (RTKI), the proteasome inhibitor bortezomib, and dexamethasone. Results: RTKI, bortezomib, and dexamethasone were active as single agents in t(4;14)+ MM. RTK inhibition triggered complementary proapoptotic pathways (e.g., decrease of Mcl-1, down-regulation of p44/42 mitogen-activated protein kinase, and activation of proapoptotic stress-activated protein/c-Jun NH2-terminal kinases). Synergistic or additive effects were found by combinations of RTKI with dexamethasone or bortezomib. In selected cases of t(4;14)+ MM, triple combinations were superior to dual combinations tested. Prevention from MM cell apoptosis by BMSC or exogenous interleukin-6 was circumvented by drug combinations. In t(4;14)+, N-ras–mutated NCI-H929 cells, resistance to RTKI was overcome by addition of dexamethasone. Notably, the combination of RTKI and dexamethasone showed additive proapoptotic effects in bortezomib-insensitive t(4;14)+ MM. Conclusions: Combining novel drugs in poor-prognosis t(4;14)+ MM should take into account at least bortezomib sensitivity and probably Ras mutational status.

Triple angiokinase inhibition, tumour hypoxia and radiation response of FaDu human squamous cell carcinomas

BACKGROUND AND PURPOSE To test the effect of BIBF 1120, a novel small molecule inhibitor of multiple angiogenic receptor tyrosine kinases, on the hypoxia and radiation response of tumours. MATERIALS AND METHODS Poorly differentiated human squamous cell carcinoma FaDu growing in nude mice was treated with BIBF 1120 and investigated by functional histology. To test the effect of BIBF 1120 on the radiobiological hypoxic fraction (rHF), the number and intrinsic radiation sensitivity of tumour stem cells and the outcome after fractionated irradiation, a series of local tumour control assays were performed. RESULTS BIBF 1120 significantly reduced the vessel area, vessel area with a perfusion signal and tumour growth rate but did not affect tumour hypoxia or the number and intrinsic radiation sensitivity of tumour stem cells. Concurrent BIBF 1120 had no effect on local tumour control after fractionated irradiation. CONCLUSION Triple angiokinase inhibition resulted in a clear-cut decrease of angiogenesis, vessel area with a perfusion signal and tumour growth but did not change tumour hypoxia or radiation response of tumour stem cells. Further experiments into mechanisms of interaction between anti-angiogenic strategies and irradiation appear to be necessary to better define and exploit the potential of this strategy to improve local tumour control after fractionated radiotherapy.

Nintedanib is a highly effective therapeutic for neuroendocrine carcinoma of the pancreas (PNET) in the Rip1Tag2 transgenic mouse model

Purpose: Pancreatic neuroendocrine tumors (PNET) represent a rare but challenging heterogeneous group of cancers with an increasing incidence over the last number of decades. Herein, we report an in-depth evaluation of the new antiangiogenic small-molecule tyrosine kinase inhibitor (TKI) nintedanib in the preclinical Rip1Tag2 transgenic mouse model of neuroendocrine carcinoma of the pancreas (insulinoma). Experimental Design: We have assessed the antiangiogenic and antitumor activity of nintedanib, in comparison with other antiangiogenic TKI, by treating Rip1Tag2 transgenic mice with different treatment schedules complemented with histopathologic, cell biologic, and biochemical analyses. Results: Prolonged nintedanib treatment of Rip1Tag2 mice has led to a strong suppression of angiogenesis, accompanied by a reduced tumor burden, which translated into a significant prolongation of survival. Despite nintedanibs inhibitory action on perivascular cells, the blood vessels remaining after therapy displayed a considerably mature phenotype with tight perivascular cell coverage and preserved perfusion. Nintedanib treatment did not increase local tumor invasiveness or metastasis to the liver and pancreatic lymph nodes—a phenomenon that has been observed with antiangiogenic treatments of Rip1Tag2 transgenic mice in other laboratories. In contrast with the strong reduction in blood microvessel densities, nintedanib did not have any impact on tumor lymphangiogenesis. Conclusions: Based on our findings, we propose the clinical evaluation of the antiangiogenic drug nintedanib as a new treatment modality for PNET patients, notably in a direct comparison with already established therapeutic regimens, such as sunitinib. Clin Cancer Res; 21(21); 4856–67. ©2015 AACR.

The triple angiokinase inhibitor nintedanib directly inhibits tumor cell growth and induces tumor shrinkage via blocking oncogenic receptor tyrosine kinases

The triple-angiokinase inhibitor nintedanib is an orally available, potent, and selective inhibitor of tumor angiogenesis by blocking the tyrosine kinase activities of vascular endothelial growth factor receptor (VEGFR) 1–3, platelet-derived growth factor receptor (PDGFR)-α and -β, and fibroblast growth factor receptor (FGFR) 1–3. Nintedanib has received regulatory approval as second-line treatment of adenocarcinoma non–small cell lung cancer (NSCLC), in combination with docetaxel. In addition, nintedanib has been approved for the treatment of idiopathic lung fibrosis. Here we report the results from a broad kinase screen that identified additional kinases as targets for nintedanib in the low nanomolar range. Several of these kinases are known to be mutated or overexpressed and are involved in tumor development (discoidin domain receptor family, member 1 and 2, tropomyosin receptor kinase A (TRKA) and C, rearranged during transfection proto-oncogene [RET proto oncogene]), as well as in fibrotic diseases (e.g., DDRs). In tumor cell lines displaying molecular alterations in potential nintedanib targets, the inhibitor demonstrates direct antiproliferative effects: in the NSCLC cell line NCI-H1703 carrying a PDGFRα amplification (ampl.); the gastric cancer cell line KatoIII and the breast cancer cell line MFM223, both driven by a FGFR2 amplification; AN3CA (endometrial carcinoma) bearing a mutated FGFR2; the acute myeloid leukemia cell lines MOLM-13 and MV-4-11-B with FLT3 mutations; and the NSCLC adenocarcinoma LC-2/ad harboring a CCDC6-RET fusion. Potent kinase inhibition does not, however, strictly translate into antiproliferative activity, as demonstrated in the TRKA-dependent cell lines CUTO-3 and KM-12. Importantly, nintedanib treatment of NCI-H1703 tumor xenografts triggered effective tumor shrinkage, indicating a direct effect on the tumor cells in addition to the antiangiogenic effect on the tumor stroma. These findings will be instructive in guiding future genome-based clinical trials of nintedanib.

Abstract B09: Nintedanib inhibits tumor and vessel growth and leads to vascular normalization in A549-NSCLC-xenografts

Angiogenesis plays a major role in the growth and progression of non-small-cell lung cancer (NSCLC). The triple angiokinase inhibitor nintedanib is a potent inhibitor of the receptor tyrosine kinases FGFR-1, 2, 3, PDGFR-α and β, VEGFR-1, 2, 3. and Flt-3. as well as of non-receptor tyrosine kinases like Src, Lyn and Lck by occupying the intracellular ATP-binding pocket. In the pivotal LUME-Lung 1 trial Nintedanib plus docetaxel has proven an overall survival benefit over docetaxel monotherapy in second-line treatment of non-small-cell lung cancer of adenocarcinoma. The aim of this study was to analyse treatment induced vascular normalization of nintedanib, bevacizumab and docetaxel alone versus the respective combinations with docetaxel. Athymic NCRNU-M-F nude mice with subcutaneous A549-NSCLC xenografts were treated with nintedanib (50mg/kg), bevacizumab (10mg/kg) and docetaxel (10mg/kg) alone versus the combination of nintedanib (50mg/kg) + docetaxel (10mg/kg) or bevacizumab (10mg/kg) + docetaxel (10mg/kg)Treatment started seven days after tumor cell inoculation, when the mean tumor volume was ∼200 mm3, and lasted two weeks. The tumor sizes were measured three times a week with calipers. Vessel densities (anti-CD31), cellular proliferation (anti-Ki67) and lymphangiogenesis (anti-LYVE1) were assessed morphometrically by immunohistochemistry. Microvascular corrosion casting was used to analyze and evaluate two- and three-dimensionally the morphology and architecture of blood vessels. Tumor volumes were significantly reduced under nintedanib treatment whereas docetaxel resulted in non-significant tumor growth differences as compared to the controls. The bevacizumab-treated groups were just in-between. This was also true for the vessel densities: significant lower vascular densities were seen only in the nintedanib and combi treatment groups. Docetaxel alone had no significant impact on vessel densities. Microvascular corrosion casting and 3D-morphometry showed significantly qualitative and quantitative differences in the treatment groups. The intervascular distances and interbranching distances were significantly altered towards more control values, evident also in the microscopic appearance of a normalized microvascular architecture. Docetaxel alone had also an effect on these parameters, however, to a far lesser extent. The same is true for the vessel diameters. The absolute vascular volume was significantly lower in the nintedanib and combi treatment groups whereas the relative percentual vascular volume did not show significant differences. Quantitative analyses of anti-ki67 stained sections showed significantly less proliferation in the nintendanib single and combi treatment groups. In summary, we conclude a significant positive therapeutic effect of nintedanib on the microvascular architecture and tumor growth inhibition in A549-NSCLC-xenografts. The vascular architecture in nintedanib treated tumors was characterized by a strong normalization of vasculature. This emerging concept of vascular normalization might be mainly attributable for the significant anti-tumorigenic effects of combinatorial treatment with nintedanib and docetaxel in the NSCLC-xenografts. Citation Format: Maximilian Ackermann, Frank Hilberg, Moritz Anton Konerding. Nintedanib inhibits tumor and vessel growth and leads to vascular normalization in A549-NSCLC-xenografts. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B09.