Daniel Menezes

In vivo Target Modulation and Biological Activity of CHIR-258, a Multitargeted Growth Factor Receptor Kinase Inhibitor, in Colon Cancer Models

Purpose: To evaluate the therapeutic and biological effects of CHIR-258, an orally bioavailable, potent inhibitor of class III-V receptor tyrosine kinases, in colon cancer models. Experimental Design: The pharmacologic activity of CHIR-258 was characterized by monitoring target modulation as well as by evaluating the antitumor and antiangiogenic effects in human colon xenograft models. Results: CHIR-258 inhibits vascular endothelial growth factor receptor 1/2, fibroblast growth factor receptor 1/3, and platelet-derived growth factor receptor β (PDGFRβ) and shows both antitumor and antiangiogenic activities in vivo. Treatment of KM12L4a human colon cancer cells with CHIR-258 resulted in a dose-dependent inhibition of vascular endothelial growth factor receptor 1 and PDGFRβ phosphorylation and reduction of phosphorylated extracellular signal-regulated kinase (ERK) levels, indicating modulation of target receptors and downstream signaling. In vivo administration of CHIR-258 resulted in significant tumor growth inhibition and tumor regressions, including large, established tumors (500-1,000 mm3). Immunohistochemical analysis showed a reduction of phosphorylated PDGFRβ and phosphorylated ERK in tumor cells after oral dosing with CHIR-258 compared with control tumors. These changes were accompanied by decreased tumor cell proliferation rate and reduced intratumoral microvessel density. CHIR-258 inhibited the phosphorylation of PDGFRβ and ERK phosphorylation in tumors within 2 hours following dosing and the inhibitory activity was sustained for >24 hours. Significant antitumor activity was observed with intermittent dosing schedules, indicating a sustained biological activity. Conclusion: These studies provide evidence that biological activity of CHIR-258 in tumors correlates with efficacy and aids in the identification of potential biomarkers of this multitargeted receptor tyrosine kinase inhibitor. CHIR-258 exhibits properties that make it a promising candidate for clinical development in a variety of solid and hematologic malignancies.

Identification of NVP-BKM120 as a Potent, Selective, Orally Bioavailable Class I PI3 Kinase Inhibitor for Treating Cancer

Phosphoinositide-3-kinases (PI3Ks) are important oncology targets due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein we describe the structure guided optimization of a series of 2-morpholino, 4-substituted, 6-heterocyclic pyrimidines where the pharmacokinetic properties were improved by modulating the electronics of the 6-position heterocycle, and the overall druglike properties were fine-tuned further by modification of the 4-position substituent. The resulting 2,4-bismorpholino 6-heterocyclic pyrimidines are potent class I PI3K inhibitors showing mechanism modulation in PI3K dependent cell lines and in vivo efficacy in tumor xenograft models with PI3K pathway deregulation (A2780 ovarian and U87MG glioma). These efforts culminated in the discovery of 15 (NVP-BKM120), currently in Phase II clinical trials for the treatment of cancer.

CHIR-124, a Novel Potent Inhibitor of Chk1, Potentiates the Cytotoxicity of Topoisomerase I Poisons In vitro and In vivo

Purpose: Chk1 kinase is a critical regulator of both S and G2-M phase cell cycle checkpoints in response to DNA damage. This study aimed to evaluate the biochemical, cellular, and antitumor effects of a novel Chk1 inhibitor, CHIR124. Experimental Design: CHIR-124 was evaluated for its ability to abrogate cell cycle checkpoints, to potentiate cytotoxicity, and to inhibit Chk1-mediated signaling induced by topoisomerase I poisons in human tumor cell line and xenograft models. Results: CHIR-124 is a quinolone-based small molecule that is structurally unrelated to other known inhibitors of Chk1. It potently and selectively inhibits Chk1 in vitro (IC50 = 0.0003 μmol/L). CHIR-124 interacts synergistically with topoisomerase poisons (e.g., camptothecin or SN-38) in causing growth inhibition in several p53-mutant solid tumor cell lines as determined by isobologram or response surface analysis. CHIR-124 abrogates the SN-38–induced S and G2-M checkpoints and potentiates apoptosis in MDA-MD-435 breast cancer cells. The abrogation of the G2-M checkpoint and induction of apoptosis by CHIR-124 are enhanced by the loss of p53. We have also shown that CHIR-124 treatment can restore the level of cdc25A protein, which is normally targeted by Chk1 for degradation following DNA damage, indicating that Chk1 signaling is suppressed in the presence of CHIR-124. Finally, in an orthotopic breast cancer xenograft model, CHIR-124 potentiates the growth inhibitory effects of irinotecan by abrogating the G2-M checkpoint and increasing tumor apoptosis. Conclusions: CHIR-124 is a novel and potent Chk1 inhibitor with promising antitumor activities when used in combination with topoisomerase I poisons.

CHIR-258 : a potent inhibitor of FLT3 kinase in experimental tumor xenograft models of human acute myelogenous leukemia

Purpose: Fms-like tyrosine kinase 3 (FLT3) encodes a receptor tyrosine kinase (RTK) for which activating mutations have been identified in a proportion of acute myelogenous leukemia (AML) patients and associated with poor clinical prognosis. Given the relevance of FLT3 mutations in AML, we investigated the activity of CHIR-258, an orally active, multitargeted small molecule, with potent activity against FLT3 kinase and class III, IV, and V RTKs involved in endothelial and tumor cell proliferation in AML models. Experimental Design: CHIR-258 was tested on two human leukemic cell lines in vitro and in vivo with differing FLT3 mutational status [MV4;11 cells express FLT3 internal tandem duplications (ITD) versus RS4;11 cells with wild-type (WT) FLT3]. Results: Antiproliferative activity of CHIR-258 against MV4;11 was ∼24-fold greater compared with RS4;11, indicating more potent inhibition against cells with constitutively activated FLT3 ITD. Dose-dependent down modulation of receptor phosphorylation and downstream signaling [signal transducer and activator of transcription 5 (STAT5) and extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase] in MV4;11 cells with CHIR-258 confirmed the molecular mechanism of action. Target modulation of phospho-FLT3, phospho-STAT5, and phospho-ERK in MV4;11 tumors was achieved at biologically active doses of CHIR-258. Tumor regressions and eradication of AML cells from the bone marrow were shown in s.c. and bone marrow engraftment leukemic xenograft models. Tumor responses were characterized by decreased cellular proliferation and positive immunohistochemical staining for active caspase-3 and cleaved poly(ADP-ribose) polymerase, suggesting cell death was mediated in part via apoptosis. Conclusions: Our data indicate that CHIR-258 may be an effective therapy in FLT3-associated AML and warrants clinical trials.

Design, structure-activity relationships and in vivo characterization of 4-amino-3-benzimidazol-2-ylhydroquinolin-2-ones: a novel class of receptor tyrosine kinase inhibitors.

The inhibition of key receptor tyrosine kinases (RTKs) that are implicated in tumor vasculature formation and maintenance, as well as tumor progression and metastasis, has been a major focus in oncology research over the last several years. Many potent small molecule inhibitors of vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) kinases have been evaluated. More recently, compounds that act through the complex inhibition of multiple kinase targets have been reported and may exhibit improved clinical efficacy. We report herein a series of potent, orally efficacious 4-amino-3-benzimidazol-2-ylhydroquinolin-2-one analogues as inhibitors of VEGF, PDGF, and fibroblast growth factor (FGF) receptor tyrosine kinases. Compounds in this class, such as 5 (TKI258), are reversible ATP-competitive inhibitors of VEGFR-2, FGFR-1, and PDGFRbeta with IC(50) values <0.1 microM. On the basis of its favorable in vitro and in vivo properties, compound 5 was selected for clinical evaluation and is currently in phase I clinical trials.

Design, structure-activity relationship, and in vivo characterization of the development candidate NVP-HSP990.

Utilizing structure-based drug design, a novel dihydropyridopyrimidinone series which exhibited potent Hsp90 inhibition, good pharmacokinetics upon oral administration, and an excellent pharmacokinetic/pharmacodynamic relationship in vivo was developed from a commercial hit. The exploration of this series led to the selection of NVP-HSP990 as a development candidate.

Abstract LB-237: BMS-986012, a fully human anti-fucosyl-GM1 antibody has potent in vitro and in vivo antitumor activity in preclinical models of small cell lung cancer

Fucosyl-GM1, is a sphingolipid monosialoganglioside and tumor-associated antigen with a high prevalence in human small cell lung cancer (SCLC), while its expression is minimal in most normal tissue. Here, we report results of our preclinical studies with BMS-986012, a novel fully human, non-fucosylated, IgG1 monoclonal antibody that specifically binds to FucGM1. The binding affinity of BMS-986012 was evaluated in Biacore® and cell-based assays. BMS-986012 binds with a high affinity to FucGM1 and demonstrates no significant binding to other closely related gangliosides. The antibody was engineered in a non-fucosylated IgG1 format, resulting in a 40-fold increased affinity for FcγRIIIa (CD16). In in vitro assays, BMS-986012 binds to FucGM1 on cancer cells and activates antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-mediated phagocytosis (ADCP). The antitumor activity of BMS-986012 was profiled in a panel of SCLC lung cancer models in immunodeficient mice with varying FucGM1 target expression levels qualified using immunohistochemistry. Dose-dependent antitumor activity was observed in a panel of SCLC xenograft models with tumor regressions occurring at BMS-986012 doses greater than 0.3 mg/kg against DMS79 tumors. BMS-986012 efficacy generally correlated with FucGM1 target expression in tumor models. In mice, the antibody was well tolerated, with no adverse events observed in long term efficacy studies. Preclinical studies also explored combinations of BMS-986012 with standard-of-care (SOC) chemotherapeutics, including cisplatin, etoposide, topotecan and irinotecan. Combination therapy of BMS-986012 with SOC resulted in significantly enhanced antitumor activity compared to monotherapy. Finally, studies conducted with BMS-986012 in combination with anti-CD137 antibody resulted in significant improvement in efficacy. These data provide preclinical support for evaluation of BMS-986012 in SCLC. BMS-986012 is currently in a phase I trial. Citation Format: Bing Chen, Chin Pan, Paul Ponath, Miho Oyasu, Daniel Menezes, Pina Cardarelli. BMS-986012, a fully human anti-fucosyl-GM1 antibody has potent in vitro and in vivo antitumor activity in preclinical models of small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-237.

CHAPTER 9:Discovery and Selection of NVP-HSP990 as a Clinical Candidate

This chapter describes the discovery of NVP-Hsp990, a potent, orally bioavailable Hsp90 inhibitor currently in early clinical development. The program strategy for the discovery of Hsp990 is detailed from hit identification to in vivo pre-clinical evaluation. This approach relied heavily on the application of structure-based drug design to rapidly optimize biochemical potency, and identify opportunities for fine-tuning the in vitro and in vivo properties. A significant protein rearrangement is described, which enabled the identification of a highly potent inhibitor series. PK/PD/efficacy relationships are described that guided dose and schedule optimization for the clinical candidate.