Florian Colbatzky

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.

A Novel RAF Kinase Inhibitor with DFG-Out–Binding Mode: High Efficacy in BRAF-Mutant Tumor Xenograft Models in the Absence of Normal Tissue Hyperproliferation

BI 882370 is a highly potent and selective RAF inhibitor that binds to the DFG-out (inactive) conformation of the BRAF kinase. The compound inhibited proliferation of human BRAF–mutant melanoma cells with 100× higher potency (1–10 nmol/L) than vemurafenib, whereas wild-type cells were not affected at 1,000 nmol/L. BI 882370 administered orally was efficacious in multiple mouse models of BRAF-mutant melanomas and colorectal carcinomas, and at 25 mg/kg twice daily showed superior efficacy compared with vemurafenib, dabrafenib, or trametinib (dosed to provide exposures reached in patients). To model drug resistance, A375 melanoma–bearing mice were initially treated with vemurafenib; all tumors responded with regression, but the majority subsequently resumed growth. Trametinib did not show any efficacy in this progressing population. BI 882370 induced tumor regression; however, resistance developed within 3 weeks. BI 882370 in combination with trametinib resulted in more pronounced regressions, and resistance was not observed during 5 weeks of second-line therapy. Importantly, mice treated with BI 882370 did not show any body weight loss or clinical signs of intolerability, and no pathologic changes were observed in several major organs investigated, including skin. Furthermore, a pilot study in rats (up to 60 mg/kg daily for 2 weeks) indicated lack of toxicity in terms of clinical chemistry, hematology, pathology, and toxicogenomics. Our results indicate the feasibility of developing novel compounds that provide an improved therapeutic window compared with first-generation BRAF inhibitors, resulting in more pronounced and long-lasting pathway suppression and thus improved efficacy. Mol Cancer Ther; 15(3); 354–65. ©2016 AACR.

Central Nervous System Demyelination and Remyelination is Independent from Systemic Cholesterol Level in Theiler's Murine Encephalomyelitis

High dietary fat and/or cholesterol intake is a risk factor for multiple diseases and has been debated for multiple sclerosis. However, cholesterol biosynthesis is a key pathway during myelination and disturbances are described in demyelinating diseases. To address the possible interaction of dyslipidemia and demyelination, cholesterol biosynthesis gene expression, composition of the bodys major lipid repositories and Paigen diet‐induced, systemic hypercholesterolemia were examined in Theilers murine encephalomyelitis (TME) using histology, immunohistochemistry, serum clinical chemistry, microarrays and high‐performance thin layer chromatography. TME‐virus (TMEV)‐infected mice showed progressive loss of motor performance and demyelinating leukomyelitis. Gene expression associated with cholesterol biosynthesis was overall down‐regulated in the spinal cord of TMEV‐infected animals. Spinal cord levels of galactocerebroside and sphingomyelin were reduced on day 196 post TMEV infection. Paigen diet induced serum hypercholesterolemia and hepatic lipidosis. However, high dietary fat and cholesterol intake led to no significant differences in clinical course, inflammatory response, astrocytosis, and the amount of demyelination and remyelination in the spinal cord of TMEV‐infected animals. The results suggest that down‐regulation of cholesterol biosynthesis is a transcriptional marker for demyelination, quantitative loss of myelin‐specific lipids, but not cholesterol occurs late in chronic demyelination, and serum hypercholesterolemia exhibited no significant effect on TMEV infection.

Best Practice Approach for Assessment of Microchip-associated Tumors in Preclinical Safety Studies: Position of the Registry of Industrial Toxicology Animal-data (RITA)

Microchip (passive radio-frequency identification device) implantation is a common and widely employed means of animal identification in laboratory animal facilities. However, these devices have been associated with tumors of the skin and subcutis in rodents. While microchip-associated tumors are rare, they pose a challenge for accurate diagnosis and documentation in preclinical toxicity studies. Documentation of these tumors should differentiate microchip-associated lesions with spontaneously occurring or test article–induced tumors. Standardizing criteria for microchip-associated lesions will aid the diagnostic process and allow for preclinical regulatory standardization. To this end, the Registry of Industrial Toxicology Animal-data have developed clear recommendations for diagnosis and documentation of microchip-associated lesions.

Abstract 2344: Modeling 1st- and 2nd-line therapy of BRAF mutant melanoma using a novel BRAF inhibitor with DFG-out binding mode.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The BRAF inhibitor vemurafenib (VEM) was approved for treatment of patients with BRAFV600E melanoma based on an ORR of 48% and a PFS of 5.3 months. Phase III data reported for the BRAF inhibitor dabrafenib (DAB) as well as the MEK inhibitor trametinib (TRA) in BRAFmut melanoma indicate similar outcomes. Phase I/II data published recently for a combination of DAB and TRA suggest improved efficacy compared with single-agent therapy; nevertheless, the short duration of responses even for the combination points to an urgent need for further improvement. BI 882370, a potent and selective B/CRAF inhibitor, binds to the DFG-out (inactive) conformation of BRAF, whereas VEM as well as DAB occupy the DFG-in (active) conformation. BI 882370 inhibited proliferation of BRAFmut melanoma cell lines with about 100x higher potency (EC50 1 - 10 nM) than VEM; BRAFwt cell lines were not affected at 1 μM. For studies in nude mouse xenograft models, VEM and DAB were dosed to provide exposures achieved in patients; TRA was used at the MTD for mice, resulting in exposures at or above the exposures tolerated by humans. In the G361 melanoma model (BRAFV600E), 1st line treatment with 25 mg/kg BI 882370 administered twice daily (bid) resulted in partial regression of all tumors, superior to results achieved by VEM, DAB as well as their combination. To model clinical resistance, a large cohort of A375 melanoma (BRAFV600E) bearing mice was treated with VEM; without exception, all tumors responded with partial regression and their median volume reached a nadir on day 11. Thereafter, the majority of tumors resumed growth in spite of continued treatment, whereas tumors in a subset of animals regressed further. Progressing tumors were randomized to 2nd line therapy on day 36 (median volume 280 mm3). TRA did not show any efficacy in this resistant population. BI 882370 induced tumor regression, however, resistance developed within 3 weeks. BI 882370 in combination with TRA resulted in more pronounced regressions and resistance was not observed until the end of the experiment, 6 weeks after initiation of 2nd line therapy. Importantly, in multiple in vivo studies performed to date, mice treated with BI 882370 at doses of 25 mg/kg bid for several weeks did not show any body weight loss or clinical signs of intolerability. Mice treated for 2 weeks were euthanized and major organs subjected to histological analysis. No changes were observed in any of the animals; in particular, there was no evidence for epidermal hyperplasia. Further, a pilot study in male rats dosed at 25 mg/kg bid for 2 weeks did not result in any toxicologically relevant findings in terms of clinical chemistry, hematology, pathology and toxicogenomics of liver and skin. Our results suggest that compared to 1st generation BRAF inhibitors, BI 882370 may provide an improved therapeutic window, enabling more pronounced and longer-lasting pathway suppression and thus resulting in improved efficacy. Citation Format: Irene C. Waizenegger, Anke Baum, Heinz Stadtmuller, Steffen Steurer, Gerd Bader, Pilar Garin-Chesa, Norbert Schweifer, Andreas Bernthaler, Christian Haslinger, Otmar Schaaf, Sien Mousa, Florian Colbatzky, Gunther R. Adolf. Modeling 1st- and 2nd-line therapy of BRAF mutant melanoma using a novel BRAF inhibitor with DFG-out binding mode. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2344. doi:10.1158/1538-7445.AM2013-2344