Pharmacologic activity and pharmacokinetics of metabolites of regorafenib in preclinical models.

Regorafenib is an orally administered inhibitor of protein kinases involved in tumor angiogenesis, oncogenesis, and maintenance of the tumor microenvironment. Phase III studies showed that regorafenib has efficacy in patients with advanced gastrointestinal stromal tumors or treatment-refractory metastatic colorectal cancer. In clinical studies, steady-state exposure to the M-2 and M-5 metabolites of regorafenib was similar to that of the parent drug; however, the contribution of these metabolites to the overall observed clinical activity of regorafenib cannot be investigated in clinical trials. Therefore, we assessed the pharmacokinetics and pharmacodynamics of regorafenib, M-2, and M-5 in vitro and in murine xenograft models. M-2 and M-5 showed similar kinase inhibition profiles and comparable potency to regorafenib in a competitive binding assay. Inhibition of key target kinases by all three compounds was confirmed in cell-based assays. In murine xenograft models, oral regorafenib, M-2, and M-5 significantly inhibited tumor growth versus controls. Total peak plasma drug concentrations and exposure to M-2 and M-5 in mice after repeated oral dosing with regorafenib 10 mg/kg/day were comparable to those in humans. In vitro studies showed high binding of regorafenib, M-2, and M-5 to plasma proteins, with unbound fractions of ~0.6%, ~0.9%, and ~0.4%, respectively, in murine plasma and ~0.5%, ~0.2%, and ~0.05%, respectively, in human plasma. Estimated free plasma concentrations of regorafenib and M-2, but not M-5, exceeded the IC50 at human and murine VEGFR2, suggesting that regorafenib and M-2 are the primary contributors to the pharmacologic activity of regorafenib in vivo.

Inhibition of hypoxia-induced gene transcription by substituted pyrazolyl oxadiazoles: initial lead generation and structure-activity relationships.

The transcription factors hypoxia-inducible factor-1 and -2 (HIF-1 and HIF-2) orchestrate a multitude of processes that allow tumor cells to survive under conditions of low oxygen and nutrients, and that lead to resistance to some apoptotic pathways and facilitate invasion and metastasis. Therefore, inhibition of transactivation by HIF has become an attractive target in cancer research. Herein we present the results of a cell-based screening approach that led to the discovery of substituted 1H-pyrazole-3-carboxamides. Chemical optimization of the hit class with respect to potency and metabolic stability is described; it resulted in novel 5-(1H-pyrazol-3-yl)-1,2,4-oxadiazoles that inhibit the hypoxia-induced accumulation of HIF-1α and HIF-2α. The HIF inhibitory potency in the screening cell system was improved from IC50 190 to 0.7 nM, and significant parts of the SAR are disclosed. For a key compound, the ability to suppress the hypoxia-induced expression of HIF target genes was studied in A549 human lung adenocarcinoma cells. The same compound shows a favorable pharmacokinetic profile in rats after i.v. and p.o. administration.

Regorafenib (BAY 73-4506): antitumor and antimetastatic activities in preclinical models of colorectal cancer.

Regorafenib, a novel multikinase inhibitor, has recently demonstrated overall survival benefits in metastatic colorectal cancer (CRC) patients. Our study aimed to gain further insight into the molecular mechanisms of regorafenib and to assess its potential in combination therapy. Regorafenib was tested alone and in combination with irinotecan in patient-derived (PD) CRC models and a murine CRC liver metastasis model. Mechanism of action was investigated using in vitro functional assays, immunohistochemistry and correlation with CRC-related oncogenes. Regorafenib demonstrated significant inhibition of growth-factor-mediated vascular endothelial growth factor receptor (VEGFR) 2 and VEGFR3 autophosphorylation, and intracellular VEGFR3 signaling in human umbilical vascular endothelial cells (HuVECs) and lymphatic endothelial cells (LECs), and also blocked migration of LECs. Furthermore, regorafenib inhibited proliferation in 19 of 25 human CRC cell lines and markedly slowed tumor growth in five of seven PD xenograft models. Combination of regorafenib with irinotecan significantly delayed tumor growth after extended treatment in four xenograft models. Reduced CD31 staining indicates that the antiangiogenic effects of regorafenib contribute to its antitumor activity. Finally, regorafenib significantly delayed disease progression in a murine CRC liver metastasis model by inhibiting the growth of established liver metastases and preventing the formation of new metastases in other organs. In addition, our results suggest that regorafenib displays antimetastatic activity, which may contribute to its efficacy in patients with metastatic CRC. Combination of regorafenib and irinotecan demonstrated an increased antitumor effect and could provide a future treatment option for CRC patients.

Angiopoietin-2 drives lymphatic metastasis of pancreatic cancer.

Lymphatic metastasis constitutes a critical route of disease dissemination, which limits the prognosis of patients with pancreatic ductal adenocarcinoma (PDAC). As lymphangiogenesis has been implicated in stimulation of lymphatic metastasis by vascular endothelial growth factor-C (VEGF-C) and VEGF-D, we studied the effect of the angioregulatory growth factor angiopoietin-2 (Ang-2) on PDAC progression. Ang-2 was found to be expressed in transformed cells of human PDAC specimens, with corresponding Tie-2 receptors present on blood and lymphatic endothelium. In vitro in PDAC cells, Ang-2 was subject to autocrine/paracrine TGF-ß stimulation (2-fold induction, P=0.0106) acting on the -61- to +476-bp element of the human Ang-2 promoter. In turn, Ang-2 regulated the expression of genes involved in cell motility and tumor suppression. Orthotopic PDAC xenografts with forced expression of Ang-2, but not Ang-1, displayed increased blood and lymphatic vessel density, and an enhanced rate of lymphatic metastasis (6.7- to 9.1-fold, P<0.01), which was prevented by sequestration of Ang-2 via coexpression of soluble Tie-2. Notably, elevated circulating Ang-2 in patients with PDAC correlated with the extent of lymphatic metastasis. Furthermore, median survival was reduced from 28.4 to 7.7 mo in patients with circulating Ang-2 ≥ 75th percentile (P=0.0005). These findings indicate that Ang-2 participates in the control of lymphatic metastasis, constitutes a noninvasive prognostic biomarker, and may provide an accessible therapeutic target in PDAC.

LDH-A influences hypoxia-inducible factor 1α (HIF1 α) and is critical for growth of HT29 colon carcinoma cells in vivo.

Serum lactate dehydrogenase (LDH) is a well-known clinical surrogate parameter. A high activity of LDH is associated with a poor prognosis in different tumor types. Here we demonstrate by a gene silencing approach that LDH-A is critical for in vivo but not in vitro growth of HT29 colon carcinoma cells. We provide evidence that the suppression of the LDH-A gene leads to an increased level of hypoxia inducible factor 1α (HIF1α) but in consequence not to an increase of HIF1 regulated proteins such as carbonic anhydrase IX (CAIX), vascular endothelial growth factor (VEGF), prolyl-hydroxylase 2 (PHD2), and factor-inhibiting HIF (FIH) in cell cultures and tumor lysates. This effect is independent of LDH activity in vivo. We conclude that LDH-A has an influence on the activity of HIF1α and thus on the adaptation of cells to a hypoxic tumor microenvironment in HT29 colon cells. We suggest the use of LDH-M as a potential therapeutic target for anticancer treatment.

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

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

Inflammatory corneal (lymph)angiogenesis is blocked by VEGFR-tyrosine kinase inhibitor ZK 261991, resulting in improved graft survival after corneal transplantation.

PURPOSE: To analyze whether tyrosine kinase inhibitors blocking VEGF receptors (PTK787/ZK222584 [PTK/ZK] and ZK261991 [ZK991]) can inhibit not only hemangiogenesis but also lymphangiogenesis and whether treatment with tyrosine kinase inhibitors after corneal transplantation can improve graft survival. METHODS: Inflammatory corneal neovascularization was induced by corneal suture placement. One treatment group received PTK/ZK, and the other treatment group received ZK991. Corneas were analyzed histomorphometrically for pathologic corneal hemangiogenesis and lymphangiogenesis. The inhibitory effect of tyrosine kinase inhibitors on lymphatic endothelial cells (LECs) in vitro was analyzed with a colorimetric (BrdU) proliferation ELISA. Low-risk allogeneic (C57Bl/6 to BALB/c) corneal transplantations were performed; the treatment group received ZK991, and grafts were graded for rejection (for 8 weeks). RESULTS: Treatment with tyrosine kinase inhibitors resulted in a significant reduction of hemangiogenesis (PTK/ZK by 30%, P < 0.001; ZK991 by 53%, P < 0.001) and lymphangiogenesis (PTK/ZK by 70%, P < 0.001; ZK991 by 71%, P < 0.001) in vivo. Inhibition of proliferation of LECs in vitro was also significant and dose dependent (PTK/ZK, P < 0.001; ZK991, P < 0.001). Comparing the survival proportions after corneal transplantation, treatment with ZK991 significantly improved graft survival (68% vs. 33%; P < 0.02). CONCLUSIONS: Tyrosine kinase inhibitors blocking VEGF receptors are potent inhibitors not only of inflammatory corneal hemangiogenesis but also lymphangiogenesis in vivo. Tyrosine kinase inhibitors seem to have the ability to restrain the formation of the afferent and efferent arm of the immune reflex arc and are therefore able to promote graft survival after corneal transplantation.

Vascular endothelial growth factor-D induces lymphangiogenesis and lymphatic metastasis in models of ductal pancreatic cancer.

The presence of lymphatic metastases is a strong indicator for poor prognosis in patients with ductal pancreatic cancer. In order to better understand the mechanisms controlling lymphatic growth and lymph node metastasis in human ductal pancreatic cancer, we analyzed the expression pattern of the vascular endothelial growth factor-D (VEGF-D), its receptor VEGF-receptor-3 (VEGFR-3) and the lymphatic endothelium-specific hyaluronan receptor LYVE-1 in a panel of 19 primary human ductal pancreatic tumors and 10 normal pancreas specimens. We further addressed the biological function of VEGF-D for induction of lymphatic metastasis in a nude mouse xenograft model using two human ductal pancreatic cancer cell lines with overexpression of VEGF-D. Compared to normal human pancreas, pancreatic cancer tissue showed overexpression of VEGF-D and VEGFR-3 in conjunction with a high lymphatic vascularization as determined by immunohistochemistry and in situ hybridization. Tumors derived from VEGF-D-overexpressing cells had a higher microvessel density compared to their mock-controls, as determined based on CD31 immunohistochemistry. Importantly, these tumors also revealed a significant induction of intra- and peritumoral lymphatics, as judged from immunohistochemical detection of LYVE-1 expression. This was associated with a significant increase in lymphatic vessel invasion by tumor cells and an increased rate of lymphatic metastases, as indicated by pan-cytokeratin reactive cells in lymph nodes. Our results suggest that VEGF-D plays a pivotal role in stimulating lymphangiogenesis and lymphatic metastasis in human ductal pancreatic cancer, and therefore represents a novel therapeutic target for this devastating disease.

PTK 787/ZK 222584, a tyrosine kinase inhibitor of all known VEGF receptors, represses tumor growth with high efficacy.

The angiogenesis inhibitor PTK 787/ZK 222584 (PTK/ZK) blocks all known VEGF receptor (VEGFR) tyrosine kinases, including the lymphangiogenic VEGFR3, in the lower nanomolar range. From a panel of 100 kinases only PDGFR, c-kit, and c-fms are inhibited beyond those in the nanomolar range. PTK/ZK functions as a competitive inhibitor at the ATP-binding site of the receptor kinase as shown here in kinetic experiments. The VEGF signal blockade in microvascular endothelial cells (MVEC) results in a blockade of MVEC proliferation (IC50=30 nM), without affecting the proliferation of normal tissue cells and tumor cells. The efficacy of PTK/ZK depends on its continuous presence within the endothelial target cells. Early removal attenuates its antiproliferative activity in vitro. Growth inhibition of endothelial cells is fully reversible as demonstrated by "washout" experiments. Without inhibiting tumor cell proliferation directly, PTK/ZK results in a significant retardation of tumor growth in a number of experimental tumor models of different tissue origin. Combination of PTK/ZK with an antiandrogen revealed additive effects on tumor-growth inhibition. Treatment efficacy was monitored both by tumor weight and by the determination of serum concentrations of the surrogate marker PSA. PTK/ZK is currently being investigated in patients with different solid tumor types for its therapeutic utility. Preliminary data from phase I/II clinical trials of PTK/ZK as a monotherapy suggested a positive safety and tolerability profile, which we interpret to be a consequence of the high selectivity of the drug for a limited number of kinases. Preliminary response, time to progression, and overall survival data were promising.1 Based on these encouraging results, PTK/ZK is currently in Phase III clinical trials for metastatic colorectal cancer.

EphB4 signaling is capable of mediating ephrinB2-induced inhibition of cell migration.

Signaling between the ligand ephrinB2 and the respective receptors of the EphB class is known to play a vital role during vascular morphogenesis and angiogenesis. The relative contribution of each EphB receptor type present on endothelial cells to these processes remains to be determined. It has been shown that ephrinB2-EphB receptor signal transduction leads to a repulsive migratory behavior of endothelial cells. It remained unclear whether this anti-migratory effect can be mediated by EphB4 signaling alone or whether other EphB receptors are necessary. It also remained unclear whether the kinase activity of EphB4 is pivotal to its action. To answer these questions, we developed a cellular migration system solely dependent on ephrinB2-EphB4 signaling. Using this system, we could show that EphB4 activation leads to the inhibition of cell migration. Furthermore we identified PP2, a known inhibitor of kinases of the Src family, and PD 153035, a known inhibitor of EGF receptor kinase, as inhibitors of EphB4 kinase activity. Using PP2, the inhibition of cell migration by ephrinB2 could be relieved, demonstrating that the kinase function of EphB4 is of prominent importance in this process. These results show that EphB4 activation is not only accompanying ephrinB2 induced repulsive behavior of cells, but is capable of directly mediating this effect.

Potent inhibition of angiogenesis by D,L-peptides derived from vascular endothelial growth factor receptor 2.

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and plays a central role in angiogenesis and vasculogenesis. Therefore, VEGF and its receptors VEGFR-1 and VEGFR-2 are prime targets for anti-angiogenic intervention which is thought to be one of the most promising approaches in cancer therapy. Recently, we have discovered a VEGFR-2-derived peptide ((247)RTELNVGIDFNWEYP(261)) representing a potential binding site to VEGF. Using the spot synthesis technique, systematic D-amino acid substitutional analyses of this peptide were conducted and the resulting D,L-peptides inhibit VEGF binding to VEGFR-2 at half maximal concentration of 30 nM. The serum-stable D,L-peptides further inhibited autophosphorylation of the VEGFR-2 at nanomolar concentrations. Testing of the peptides in a spheroid-based angiogenesis assay demonstrated a potent anti-angiogenic effect in vitro. The rational design of potent and stable anti-angiogenic peptide inhibitors from their parent receptors provides a feasible route to develop novel leads for anti-angiogenic medicines.

CRIM1 is involved in endothelial cell capillary formation in vitro and is expressed in blood vessels in vivo.

In endothelial cells that form capillary-like structures in vitro a variety of genes is upregulated as we have demonstrated previously. In addition to well known genes, we also identified genes never described in endothelial cells before. Here, we report the further characterization of one selected gene called cysteine-rich motor neuron 1 (CRIM1). CRIM1 is strongly upregulated in endothelial cells during tube formation and is expressed by a variety of adherent growing cell lines whereas cell lines grown in suspension do not express CRIM1. By using antisense technology we were able to inhibit CRIM1 expression and demonstrate impaired formation of capillary-like structures in vitro in transfected endothelial cells. Furthermore, we show that CRIM1 is a glycosylated type I transmembrane protein, that accumulates at sites of close cell-to-cell contact upon stimulation. Finally, we found CRIM1 protein to be expressed by endothelial cells of the inner lining of blood vessels in vivo. Taken together our results imply a possible role of CRIM1 in capillary formation and maintainance during angiogenesis.

Anthranilic acid amides: a novel class of antiangiogenic VEGF receptor kinase inhibitors.

Two readily synthesized anthranilamide, VEGF receptor tyrosine kinase inhibitors have been prepared and evaluated as angiogenesis inhibitors. 2-[(4-Pyridyl)methyl]amino-N-[3-(trifluoromethyl)phenyl]benzamide (5) and N-3-isoquinolinyl-2-[(4-pyridinylmethyl)amino]benzamide (7) potently and selectively inhibit recombinant VEGFR-2 and VEGFR-3 kinases. As a consequence of their physicochemical properties, these anthranilamides readily penetrate cells and are absorbed following once daily oral administration to mice. Both 5 and 7 potently inhibit VEGF-induced angiogenesis in an implant model, with ED(50) values of 7 mg/kg. In a mouse orthotopic model of melanoma, 5 and 7 potently inhibited both the growth of the primary tumor as well as the formation of spontaneous peripheral metastases. The anthranilamides 5 and 7 represent a new structural class of VEGFR kinase inhibitors, which possess potent antiangiogenic and antitumor properties.