Jason Manro

LY2801653 is an orally bioavailable multi-kinase inhibitor with potent activity against MET, MST1R, and other oncoproteins, and displays anti-tumor activities in mouse xenograft models.

SummaryThe HGF/MET signaling pathway regulates a wide variety of normal cellular functions that can be subverted to support neoplasia, including cell proliferation, survival, apoptosis, scattering and motility, invasion, and angiogenesis. MET over-expression (with or without gene amplification), aberrant autocrine or paracrine ligand production, and missense MET mutations are mechanisms that lead to activation of the MET pathway in tumors and are associated with poor prognostic outcome. We report here preclinical development of a potent, orally bioavailable, small-molecule inhibitor LY2801653 targeting MET kinase. LY2801653 is a type-II ATP competitive, slow-off inhibitor of MET tyrosine kinase with a dissociation constant (Ki) of 2xa0nM, a pharmacodynamic residence time (Koff) of 0.00132xa0min−1 and t1/2 of 525xa0min. LY2801653 demonstrated in vitro effects on MET pathway-dependent cell scattering and cell proliferation; in vivo anti-tumor effects in MET amplified (MKN45), MET autocrine (U-87MG, and KP4) and MET over-expressed (H441) xenograft models; and in vivo vessel normalization effects. LY2801653 also maintained potency against 13 MET variants, each bearing a single-point mutation. In subsequent nonclinical characterization, LY2801653 was found to have potent activity against several other receptor tyrosine oncokinases including MST1R, FLT3, AXL, MERTK, TEK, ROS1, DDR1/2 and against the serine/threonine kinases MKNK1/2. The potential value of MET and other inhibited targets within a number of malignancies (such as colon, bile ducts, and lung) is discussed. LY2801653 is currently in phase 1 clinical testing in patients with advanced cancer (trial I3O-MC-JSBA, NCT01285037).

LY2875358, a Neutralizing and Internalizing Anti-MET Bivalent Antibody, Inhibits HGF-Dependent and HGF-Independent MET Activation and Tumor Growth

Purpose: MET, the receptor for hepatocyte growth factor (HGF), has been implicated in driving tumor proliferation and metastasis. High MET expression is correlated with poor prognosis in multiple cancers. Activation of MET can be induced either by HGF-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional upregulation or by HGF-dependent autocrine or paracrine mechanisms. Experimental Design/Results: Here, we report on LY2875358, a novel humanized bivalent anti-MET antibody that has high neutralization and internalization activities, resulting in inhibition of both HGF-dependent and HGF-independent MET pathway activation and tumor growth. In contrast to other bivalent MET antibodies, LY2875358 exhibits no functional agonist activity and does not stimulate biologic activities such as cell proliferation, scattering, invasion, tubulogenesis, or apoptosis protection in various HGF-responsive cells and no evidence of inducing proliferation in vivo in a monkey toxicity study. LY2875358 blocks HGF binding to MET and HGF-induced MET phosphorylation and cell proliferation. In contrast to the humanized one-armed 5D5 anti-MET antibody, LY2875358 induces internalization and degradation of MET that inhibits cell proliferation and tumor growth in models where MET is constitutively activated. Moreover, LY2875358 has potent antitumor activity in both HGF-dependent and HGF-independent (MET-amplified) xenograft tumor models. Together, these findings indicate that the mechanism of action of LY2875358 is different from that of the one-armed MET antibody. Conclusions: LY2875358 may provide a promising therapeutic strategy for patients whose tumors are driven by both HGF-dependent and HGF-independent MET activation. LY2875358 is currently being investigated in multiple clinical studies. Clin Cancer Res; 20(23); 6059–70. ©2014 AACR.

Utility of Oatp1a/1b-Knockout and OATP1B1/3-Humanized Mice in the Study of OATP-Mediated Pharmacokinetics and Tissue Distribution: Case Studies with Pravastatin, Atorvastatin, Simvastatin, and Carboxydichlorofluorescein

Although organic anion transporting polypeptide (OATP)–mediated hepatic uptake is generally conserved between rodents and humans at a gross pharmacokinetic level, the presence of three major hepatic OATPs with broad overlap in substrate and inhibitor affinity, and absence of rodent-human orthologs preclude clinical translation of single-gene knockout/knockin findings. At present, changes in pharmacokinetics and tissue distribution of pravastatin, atorvastatin, simvastatin, and carboxydichlorofluorescein were studied in oatp1a/1b-knockout mice lacking the three major hepatic oatp isoforms, and in knockout mice with liver-specific knockin of human OATP1B1 or OATP1B3. Relative to wild-type controls, oatp1a/1b-knockout mice exhibited 1.6- to 19-fold increased intravenous and 2.1- to 115-fold increased oral drug exposure, due to 33%–75% decreased clearance, 14%–60% decreased volume of distribution, and ≤74-fold increased oral bioavailability, with the magnitude of change depending on the contribution of oatp1a/1b to pharmacokinetics. Hepatic drug distribution was 4.2- to 196-fold lower in oatp1a/1b-knockout mice; distributional attenuation was less notable in kidney, brain, cardiac, and skeletal muscle. Knockin of OATP1B1 or OATP1B3 partially restored control clearance, volume, and bioavailability values (24%–142% increase, ≤47% increase, and ≤77% decrease vs. knockout, respectively), such that knockin pharmacokinetic profiles were positioned between knockout and wild-type mice. Consistent with liver-specific humanization, only hepatic drug distribution was partially restored (1.3- to 6.5-fold increase vs. knockout). Exposure and liver distribution changes in OATP1B1-humanized versus knockout mice predicted the clinical impact of OATP1B1 on oral exposure and contribution to human hepatic uptake of statins within 1.7-fold, but only after correcting for human/humanized mouse liver relative protein expression factor (OATP1B1 = 2.2, OATP1B3 = 0.30).

Activating the Wnt/β-Catenin Pathway for the Treatment of Melanoma – Application of LY2090314, a Novel Selective Inhibitor of Glycogen Synthase Kinase-3

It has previously been observed that a loss of β-catenin expression occurs with melanoma progression and that nuclear β-catenin levels are inversely proportional to cellular proliferation, suggesting that activation of the Wnt/β-catenin pathway may provide benefit for melanoma patients. In order to further probe this concept we tested LY2090314, a potent and selective small-molecule inhibitor with activity against GSK3α and GSK3β isoforms. In a panel of melanoma cell lines, nM concentrations of LY2090314 stimulated TCF/LEF TOPFlash reporter activity, stabilized β-catenin and elevated the expression of Axin2, a Wnt responsive gene and marker of pathway activation. Cytotoxicity assays revealed that melanoma cell lines are very sensitive to LY2090314 in vitro (IC50 ~10nM after 72hr of treatment) in contrast to other solid tumor cell lines (IC50 >10uM) as evidenced by caspase activation and PARP cleavage. Cell lines harboring mutant B-RAF or N-RAS were equally sensitive to LY2090314 as were those with acquired resistance to the BRAF inhibitor Vemurafenib. shRNA studies demonstrated that β-catenin stabilization is required for apoptosis following treatment with the GSK3 inhibitor since the sensitivity of melanoma cell lines to LY290314 could be overcome by β-catenin knockdown. We further demonstrate that in vivo, LY2090314 elevates Axin2 gene expression after a single dose and produces tumor growth delay in A375 melanoma xenografts with repeat dosing. The activity of LY2090314 in preclinical models suggests that the role of Wnt activators for the treatment of melanoma should be further explored.

Inhibition of Tumor Growth and Metastasis in Non–Small Cell Lung Cancer by LY2801653, an Inhibitor of Several Oncokinases, Including MET

Purpose: Lung cancer is the leading cause of cancer-related death worldwide. Sustained activation, overexpression, or mutation of the MET pathway is associated with a poor prognosis in a variety of tumors, including non–small cell lung cancer (NSCLC), implicating the MET pathway as a potential therapeutic target for lung cancer. Previously, we reported on the development of LY2801653: a novel, orally bioavailable oncokinase inhibitor with MET as one of its targets. Here, we discuss the evaluation of LY2801653 in both preclinical in vitro and in vivo NSCLC models. Experimental Design/Results: Treatment with LY2801653 showed tumor growth inhibition in tumor cell lines and patient-derived tumor xenograft models as a single agent (37.4%–90.0% inhibition) or when used in combination with cisplatin, gemcitabine, or erlotinib (66.5%–86.3% inhibition). Mechanistic studies showed that treatment with LY2801653 inhibited the constitutive activation of MET pathway signaling and resulted in inhibition of NCI-H441 cell proliferation, anchorage-independent growth, migration, and invasion. These in vitro findings were confirmed in the H441 orthotopic model where LY2801653 treatment significantly inhibited both primary tumor growth (87.9% inhibition) and metastasis (64.5% inhibition of lymph node and 67.7% inhibition of chest wall). Tumor-bearing animals treated with LY2801653 had a significantly greater survival time (87% increase compared with the vehicle-treated mice). In the MET-independent NCI-H1299 orthotopic model, treatment with LY2801653 showed a significant inhibition of primary tumor growth but not metastasis. Conclusions: Collectively, these results support clinical evaluation of LY2801653 in NSCLCs and suggest that differences in the MET activation of tumors may be predictive of response. Clin Cancer Res; 19(20); 5699–710. ©2013 AACR.

How well do lipophilicity parameters, MEEKC microemulsion capacity factor, and plasma protein binding predict CNS tissue binding?

Brain fraction unbound (Fu) is critical to understanding the pharmacokinetics/dynamics of central nervous system (CNS) drugs, thus several surrogate predictors have been proposed. At present, correlations between brain Fu, microemulsion electrokinetic chromatography capacity factor (MEEKC k), plasma Fu, octanol-water partition coefficient (clogP), and LogP at pH 7.4 (clogD(7.4) ) were compared for 94 diverse molecules, and additionally for 587 compounds. MEEKC k was a better predictor of brain Fu (r(2) = 0.74) than calculated lipophilicity parameters (clogP r(2) = 0.51-0.54, clogD(7.4) r(2) = 0.41-0.44), but it was not superior to plasma Fu (r(2) = 0.74-0.85) as a predictor of brain Fu. MEEKC k did not predict plasma Fu(r(2) = 0.58) as well as brain Fu, and the extent of improvement over clogP or clogD(7.4) (r(2) = 0.41-0.49) was less pronounced. Although log-log-correlation analysis supported seemingly strong prediction of brain Fu both by MEEKC k and by plasma Fu (r(2) ≥ 0.74), analysis of prediction error estimated a 10-fold and 6.9-8.6-fold prediction interval for brain Fu estimated using MEEKC k and plasma Fu, respectively. Therefore, MEEKC k and plasma Fu can predict the log order of CNS tissue binding, but they cannot provide truly quantitative brain Fu predictions necessary to support in-vitro-to-in-vivo extrapolations and pharmacokinetic/dynamic data interpretation.

Patient-derived acute myeloid leukemia (AML) bone marrow cells display distinct intracellular kinase phosphorylation patterns

Multiparametric analyses of phospho-protein activation in patients with acute myeloid leukemia (AML) offers a quantitative measure to monitor the activity of novel intracellular kinase (IK) inhibitors. As recent clinical investigation with FMS-like tyrosine-3 inhibitors demonstrated, targeting IK with selective inhibitors can have a modest clinical benefit. Because multiple IKs are active in patients with AML, multikinase inhibitors may provide the necessary inhibition profile to achieve a more sustained clinical benefit. We here describe a method of assessing the activation of several IKs by flow cytometry. In 40 different samples of patients with AML we observed hyper-activated phospho-proteins at baseline, which is modestly increased by adding stem cell factor to AML cells. Finally, AML cells had a significantly different phospho-protein profile compared with cells of the lymphocyte gate. In conclusion, our method offers a way to determine the activation status of multiple kinases in AML and hence is a reliable assay to evaluate the pharmacodynamic activity of novel multikinase inhibitors.

Abstract 2738: c-Met antibody LY2875358 (LA480) has pre-clinical enhanced efficacy with gastric cancer standard-of-care in vitro and in vivo

cMet is a member of the receptor tyrosine kinase family and is the receptor for hepatocyte growth factor (HGF). cMet has been implicated in the initiation and progression of cancer due to the range of activities that cMet stimulates including proliferation, migration, morphogenesis, and survival. Inappropriate activation of c-Met can be induced by ligand-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional upregulation, or by ligand-dependent autocrine or paracrine mechanisms. Indeed, amplification of the c-Met gene, with consequent protein overexpression and constitutive kinase activation, has been reported in a number of human cancers, including gastric, esophageal and non-small-cell lung carcinomas. It has been reported that ∼10-20% of gastric tumors have increased copy numbers of the MET gene and overexpression of c-Met significantly correlates with poor prognosis in gastric cancer. c-Met antibody LY2875358 treatment reduces proliferation of gastric cancer cell lines with ligand-independent activation of c-Met resulting from gene amplification. The ability of LY2875358 to internalize and deplete cell surface c-Met is implicated in its activity against ligand-independent driven gastric cell lines. Here, we demonstrate that the pre-clinical combination of c-Met antibody LY2875358 with gastric cancer standard-of-care treatment has better efficacy than either treatment alone, both in vitro and in vivo. These data suggest that LY2875358 in combination with standard-of-care may be a promising treatment for gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2738. doi:1538-7445.AM2012-2738

Abstract 2734: c-Met antibody LY2875358 (LA480) shows differential antitumor effects in non-small cell lung cancer

c-Met is a member of the receptor tyrosine kinase family and is the receptor for hepatocyte growth factor (HGF). c-Met is involved in many mechanisms of cancer proliferation and metastasis. Inappropriate activation of c-Met can be induced by ligand-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional up-regulation, or by ligand-dependent autocrine or paracrine mechanisms. Lung cancer is the leading cause of cancer death worldwide. Despite the successful development of EGFR- or EML4-ALK-targetd therapies, treatment options remain limited for patients with advanced lung cancer, making the identification of new therapeutic targets essential. c-Met expression was reported in 41-72% non-small cell lung cancer (NSCLC), amplification of c-Met occurs in 5-10 % of patients, and c-Met mutations have been detected in 8-13% of patients. We have developed a bi-valent c-Met antibody LY2875358 (LA480), which blocks ligand-dependent and ligand-independent c-Met activations. It is currently in clinical development. Here, we have demonstrated that LY2875358 alone or in combination with standard-of-care (SOC) affected cell proliferation, migration and signal transduction in NSCLC cells with c-Met gene amplification, mutations and overexpression. In vitro, LY2875358 induces wild type and mutant c-Met internalization and degradation. In vivo, LY2875358 alone shows a marked antitumor activity in Met amplification NSCLC xenograft models. The combination of LY2875358 with SOC chemotherapeutics treatments has better efficacy than either treatment alone, both in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2734. doi:1538-7445.AM2012-2734

Abstract 4973: Discovery of LY3214996, a selective and novel ERK1/2 inhibitor with potent antitumor activities in cancer models with MAPK pathway alterations

The RAS/MAPK pathway is dysregulated in approximately 30% of human cancers, and the extracellular-signal-regulated kinases (ERK1 and ERK2) serves as key central nodes within this pathway. The feasibility and clinical impact of targeting the RAS/MAPK pathway has been demonstrated by the therapeutic success of BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma. However, resistance develops frequently through reactivation of the pathway. Therefore, simultaneous targeting of multiple effectors such as RAF, MEK and ERK in this pathway, offers a potential for enhanced efficacy while delaying and overcoming resistance. LY3214996 is a highly selective inhibitor of ERK1 and ERK2, with IC50 of 5 nM for both enzymes in biochemical assays. It potently inhibits cellular phospho-RSK1 in BRAF and RAS mutant cancer cell lines. In an unbiased tumor cell panel sensitivity profiling for inhibition of cell proliferation, tumor cells with MAPK pathway alterations including BRAF, NRAS or KRAS mutation are generally sensitivity to LY3214996. In tumor xenograft models, LY3214996 inhibits PD biomarker phospho-p90RSK1 in tumors and the PD effects are correlated with compound exposures and anti-tumor activities. LY3214996 shows either similar or superior anti-tumor activity as compared to other published ERK inhibitors in BRAF or RAS mutant cell lines and xenograft models. Oral administration of single-agent LY3214996 significantly inhibits tumor growth in vivo and is well tolerated in BRAF or NRAS mutant melanoma, BRAF or KRAS mutant colorectal, lung and pancreatic cancer xenografts or PDX models. Therefore, LY3214996 can be tailored for treatment of cancers with MAPK pathway alteration. In addition, LY3214996 has anti-tumor activity in a vemurafenib-resistant A375 melanoma xenograft model due to MAPK reactivation, may have potential for treatment of melanoma patients who have failed BRAF therapies. More importantly, LY3214996 can be combined with investigational and approved agents in preclinical models, particularly KRAS mutant models. Combination treatment of LY3214996 and CDK4/6 inhibitor abemaciclib was well tolerated and results in potent tumor growth inhibition or regression in multiple in vivo cancer models, including KRAS mutant colorectal and non-small cell lung cancers. Here, we first report the preclinical characterization of LY3214996, a novel small molecule ERK1/2 inhibitor currently in Phase I clinical trials in patients with advanced and metastatic cancers (NCT02857270). Citation Format: Shripad V. Bhagwat, William T. McMillen, Shufen Cai, Baohui Zhao, Matthew Whitesell, Lisa Kindler, Robert S. Flack, Wenjuan Wu, Karen Huss, Bryan Anderson, Xiu-Juan Yuan, Susan Jaken, Denis McCann, Brian Mathes, Andrew J. Dropsey, Jason Manro, Jennie Walgren, Eunice Yuen, Xueqian Gong, Michael J. Rodriguez, Jianping Huang, Ramon V. Tiu, Sajan Joseph, Sheng-Bin Peng. Discovery of LY3214996, a selective and novel ERK1/2 inhibitor with potent antitumor activities in cancer models with MAPK pathway alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4973. doi:10.1158/1538-7445.AM2017-4973