Karl-Heinz Thierauch

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‐β 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.

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.

Abstract 1666: Regorafenib (BAY 73-4506): preclinical pharmacology and clinical identification and quantification of its major metabolites

Regorafenib is a novel diphenylurea oral multikinase inhibitor of angiogenic, stromal, and oncogenic kinases with potent preclinical antitumor activity and long-lasting antiangiogenic activity (as measured by dynamic contrast-enhanced magnetic resonance imaging). Regorafenib treatment has recently shown to result in a 31% partial response (PR) rate and 50% stabilization rate in patients with metastatic renal cell carcinoma in a Phase II study. Promising clinical activity was shown in a Phase I study in 27 evaluable patients with advanced refractory colorectal cancer (median 4 prior regimens) with a 74% overall disease control rate (PR + stable disease). The biotransformation of regorafenib was investigated in vitro with liver microsomes and hepatocytes, and in vivo in the plasma of several species. In man, the N-oxide (M-2) and the demethylated N-oxide (M-5) appear significant as both metabolites show systemic exposure (area under the curve [AUC], mg*h/L) at steady state similar to regorafenib (the parent compound) in patients at a dose of 160 mg in a 3 weeks on/1 week off Phase I study. Characterization of both M-2 and M-5 demonstrated potent pharmacologic activities. In biochemical kinase assays, M-2 and M-5 showed an inhibition profile similar to but distinct from regorafenib. In cellular assays, M-2 and M-5 inhibited key targets such as vascular endothelial growth factor (VEGF) receptor 2, TIE-2, and mutant and wild-type c-KIT and B-RAF, with IC50 values very similar to regorafenib for M-2 and somewhat higher for M-5. Regorafenib and both M-2 and M-5 dosed iv at 1 mg/kg showed significant activity in a rat VEGF hypotensive pharmacodynamic model. Both metabolites dosed orally exhibited potent dose-dependent tumor growth inhibition (TGI) in preclinical murine HT-29 colorectal and MDA-MB-231 breast cancer xenografts, achieving significant TGI of 62/58% and 54/50%, respectively, compared with vehicle controls at 10 mg/kg. Regorafenib revealed 66-77% TGI at 10 mg/kg in these models. On oral administration of regorafenib to mice, the N-oxide (M-2) plasma level accounted for ∼20% of the AUC of regorafenib; conversely, on administration of M-2, regorafenib exposure reached ∼20% of M-2 exposure, indicating that oxidation to and reduction of the N-oxide are metabolic pathways in vivo. In summary, these data demonstrate that the metabolites M-2 and M-5, exhibiting similar exposure as regorafenib, their parent compound, in man, are pharmacologically active and therefore most likely contribute to the clinical antitumor activity of regorafenib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1666.

Abstract 4509: BAY 87-2243 targets hypoxia-induced activation and stabilization of HIF-1α: A novel approach to overcome resistance mechanisms in cancer therapy

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The development of resistance against radio- or chemotherapy is one of the main causes for relapse after treatment and ultimately cancer progression. Local hypoxia within tumors is associated with radio- and chemoresistance as well as aggressive tumor growth and invasion (Vaupel et al., 2004). The transcription factor HIF-1α (hypoxia-inducible factor) is stabilized under low oxygen tension and heterodimerizes with HIF-1s to regulate the expression of a plethora of genes involved in cellular energy metabolism, neoangiogenesis, anti-apoptotic and pro-proliferative mechanisms promoting tumor progression and metastasis (Calzada et al. 2007). Because hypoxia-induced upregulation of HIF-1α appears to be of pivotal importance in tumor resistance mechanisms during cancer treatment, we screened for inhibitors of hypoxia-induced HIF-1 activation. A HCT116 cell line containing 4X-hypoxia response element-luciferase reporter was used in high-throughput screening of small molecule inhibitors under hypoxic condition (1% O2). Lead optimization resulted in the identification of BAY 87-2243, a highly selective and potent inhibitor of hypoxia-induced HIF-1α stabilization and activation. In vitro characterization showed that BAY 87-2243 specifically suppressed HIF-1 regulated target genes as assayed by qPCR. Analyses addressing the mode of action revealed that BAY 87-2243 acts upstream of VHL and PHD because the compound did not suppress HIF-1α protein stabilization and HIF target gene expression either in the presence of a PHD inhibitor in H460 cells or in VHL-null RCC4 cells. In preclinical animal models, BAY 87-2243 dosed orally was well tolerated at therapeutic doses up to 15 mg/kg and showed moderate to high anti-tumor growth inhibitory activity as monotherapy in various subcutaneous and orthotopic xenograft models. Analysis of tumor samples demonstrated a decrease of nuclear HIF-1α protein level by immunohistochemistry as well as a specific suppression of HIF-1 target genes. These data indicate that specific inhibition of hypoxia-induced HIF-1 activation is achievable with small molecule inhibitors and is a novel approach to cancer therapy. Vaupel P, Mayer A, Hockel M (2004) Tumor hypoxia and malignant progression. Methods Enzymol 381:335-354 M. J. Calzada, L. del Peso, Hypoxia inducible factors and cancer. Clin. Transl. Oncol. 2007, 9(5), 278-289: Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4509. doi:10.1158/1538-7445.AM2011-4509

Abstract B4: Regorafenib: a new oral multikinase inhibitor of angiogenic, stromal and oncogenic (receptor tyrosine) kinases with potent preclinical antitumor activity

Angiogenesis is a critical driver of tumor growth and progression, and is controlled by a number of interconnected signaling pathways. In these pathways, the two tyrosine kinase receptors, vascular endothelial growth factor receptor (VEGFR) 2 and tyrosine kinase with immunoglobulin and EGF homology domain 2 (TIE2) play crucial roles in the neovascularization, stabilization, and maintenance of normal and tumor vasculature. Regorafenib, a novel oral multikinase inhibitor, potently inhibits both VEGFR2 and TIE2 in biochemical and cellular kinase phosphorylation assays. It inhibits additional angiogenic kinases ie VEGFR1, VEGFR3, platelet‐derived growth factor receptor‐ , and fibroblast growth factor receptor‐1 and the mutant oncogenic kinases KIT and RET, which play a role in human gastrointestinal stromal and thyroid cancers, respectively, and B‐RAF. The mechanism of action of regorafenib on the tumor vasculature was demonstrated in vivo by dynamic contrast‐enhanced magnetic resonance imaging using Gadomer as contrast agent. Regorafenib significantly decreased tumor perfusion and extravasation of the contrast agent in a rat GS9L glioblastoma tumor xenograft model grown i.m. in the rat leg compared to rat muscle tissue after a single oral administration of 10 mg/kg. The pharmacodynamic effects persisted for 48 hours after the last drug administration and correlated with the tumor growth inhibition in a QDx4 once daily oral dosing study. A significant reduction in tumor microvessel area was observed in a human colorectal xenograft after QDx5 daily oral dosing at 10 and 30 mg/kg of regorafenib in tumor‐bearing mice. Regorafenib exhibited potent dose‐dependent tumor growth inhibition in various preclinical murine xenograft models including breast (MDA‐MB‐231), NSCLC (NCI‐H460), renal (786‐O), and several colorectal (Colo‐205, HCT‐15, HT‐29) carcinoma, some of which carry mutant K‐RAS and/or B‐RAF. Tumor shrinkage was observed in the breast MDA‐MB‐231 and the renal 786‐O carcinoma models. The compound was well tolerated and no significant weight loss or lethality was observed. Immunohistochemical analyses of the breast xenograft tumor revealed a strong reduction in staining of the proliferation marker Ki‐67 and of the phosphorylated extracellular regulated kinases 1 and 2. Non‐clinical metabolism studies have detected two pharmacokinetically stable regorafenib metabolites, with similar but distinct kinase profiles. These data demonstrate that regorafenib is a well‐tolerated, orally active multikinase inhibitor with a unique target profile that may have therapeutic benefit in human malignancies. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B4.