Treatment with a VEGFR-2 antibody results in intra-tumor immune modulation and enhances anti-tumor efficacy of PD-L1 blockade in syngeneic murine tumor models.

Prolonged activation of vascular endothelial growth factor receptor-2 (VEGFR-2) due to mis-regulation of the VEGF pathway induces aberrant blood vessel expansion, which supports growth and survival of solid tumors. Therapeutic interventions that inhibit the VEGFR-2 pathway have therefore become a mainstay of cancer treatment. Non-clinical studies have recently revealed that blockade of angiogenesis can modulate the tumor microenvironment and enhance the efficacy of concurrent immune therapies. Ramucirumab is an FDA-approved anti-angiogenic antibody that inhibits VEGFR-2 and is currently being evaluated in clinical studies in combination with anti-programmed cell death (PD-1) axis checkpoint inhibitors (pembrolizumab, durvalumab, or sintilimab) across several cancer types. The purpose of this study is to establish a mechanistic basis for the enhanced activity observed in the combined blockade of VEGFR-2 and PD-1-axis pathways. Pre-clinical studies were conducted in murine tumor models known to be responsive to anti-PD-1 axis therapy, using monoclonal antibodies that block mouse VEGFR-2 and programmed death-ligand 1 (PD-L1). Combination therapy resulted in enhanced anti-tumor activity compared to anti-PD-L1 monotherapy. VEGFR-2 blockade at early timepoints post-anti-PD-L1 therapy resulted in a dose-dependent and transient enhanced infiltration of T cells, and establishment of immunological memory. VEGFR-2 blockade at later timepoints resulted in enhancement of anti-PD-L1-driven immune cell infiltration. VEGFR-2 and PD-L1 monotherapies induced both unique and overlapping patterns of immune gene expression, and combination therapy resulted in an enhanced immune activation signature. Collectively, these results provide new and actionable insights into the mechanisms by which concurrent VEGFR-2 and PD-L1 antibody therapy leads to enhanced anti-tumor efficacy.

Dichotomous impact of affinity on the function of T cell engaging bispecific antibodies.

BACKGROUND: Bispecific T cell engagers represent the majority of bispecific antibodies (BsAbs) entering the clinic to treat metastatic cancer. The ability to apply these agents safely and efficaciously in the clinic, particularly for solid tumors, has been challenging. Many preclinical studies have evaluated parameters related to the activity of T cell engaging BsAbs, but many questions remain. MAIN BODY: This study investigates the impact of affinity of T cell engaging BsAbs with regards to potency, efficacy, and induction of immunomodulatory receptors/ligands using HER-2/CD3 BsAbs as a model system. We show that an IgG BsAb can be as efficacious as a smaller BsAb format both in vitro and in vivo. We uncover a dichotomous relationship between tumor-associated antigen (TAA) affinity and CD3 affinity requirements for cells that express high versus low levels of TAA. HER-2 affinity directly correlated with the CD3 engager lysis potency of HER-2/CD3 BsAbs when HER-2 receptor numbers are high (~200 K/cell), while the CD3 affinity did not impact potency until its binding affinity was extremely low (<600 nM). When HER-2 receptor numbers were lower (~20 K/cell), both HER-2 and CD3 affinity impacted potency. The high affinity anti-HER-2/low CD3 affinity BsAb also demonstrated lower cytokine induction levels in vivo and a dosing paradigm atypical of extremely high potency T cell engaging BsAbs reaching peak efficacy at doses >3 mg/kg. This data confirms that low CD3 affinity provides an opportunity for improved safety and dosing for T cell engaging BsAbs. T cell redirection also led to upregulation of Programmed cell death 1 (PD-1) and 4-1BB, but not CTLA-4 on T cells, and to Programmed death-ligand 1 (PD-L1) upregulation on HER-2HI SKOV3 tumor cells, but not on HER-2LO OVCAR3 tumor cells. Using this information, we combined anti-PD-1 or anti-4-1BB monoclonal antibodies with the HER-2/CD3 BsAb in vivo and demonstrated significantly increased efficacy against HER-2HI SKOV3 tumors via both combinations. CONCLUSIONS: Overall, these studies provide an informational dive into the optimization process of CD3 engaging BsAbs for solid tumors indicating that a reduced affinity for CD3 may enable a better therapeutic index with a greater selectivity for the target tumor and a reduced cytokine release syndrome. These studies also provide an additional argument for combining T cell checkpoint inhibition and co-stimulation to achieve optimal efficacy.

ERK Inhibitor LY3214996-Based Treatment Strategies for RAS-Driven Lung Cancer.

RAS gene mutations are the most frequent oncogenic event in lung cancer. They activate multiple RAS-centric signaling networks among them the MAPK, PI3K, and RB pathways. Within the MAPK pathway, ERK1/2 proteins exert a bottleneck function for transmitting mitogenic signals and activating cytoplasmic and nuclear targets. In view of disappointing antitumor activity and toxicity of continuously applied MEK inhibitors in patients with KRAS-mutant lung cancer, research has recently focused on ERK1/2 proteins as therapeutic targets and on ERK inhibitors for their ability to prevent bypass and feedback pathway activation. Here, we show that intermittent application of the novel and selective ATP-competitive ERK1/2 inhibitor LY3214996 exerts single-agent activity in patient-derived xenograft (PDX) models of RAS-mutant lung cancer. Combination treatments were well tolerated and resulted in synergistic (ERKi plus PI3K/mTORi LY3023414) and additive (ERKi plus CDK4/6i abemaciclib) tumor growth inhibition in PDX models. Future clinical trials are required to investigate if intermittent ERK inhibitor-based treatment schedules can overcome toxicities observed with continuous MEK inhibition and-equally important-to identify biomarkers for patient stratification.

ERK Inhibitor LY3214996 Targets ERK Pathway-Driven Cancers: A Therapeutic Approach Toward Precision Medicine.

The ERK pathway is critical in oncogenesis; aberrations in components of this pathway are common in approximately 30% of human cancers. ERK1/2 (ERK) regulates cell proliferation, differentiation, and survival and is the terminal node of the pathway. BRAF- and MEK-targeted therapies are effective in BRAF V600E/K metastatic melanoma and lung cancers; however, responses are short-lived due to emergence of resistance. Reactivation of ERK signaling is central to the mechanisms of acquired resistance. Therefore, ERK inhibition provides an opportunity to overcome resistance and leads to improved efficacy. In addition, KRAS-mutant cancers remain an unmet medical need in which ERK inhibitors may provide treatment options alone or in combination with other agents. Here, we report identification and activity of LY3214996, a potent, selective, ATP-competitive ERK inhibitor. LY3214996 treatment inhibited the pharmacodynamic biomarker, phospho-p90RSK1, in cells and tumors, and correlated with LY3214996 exposures and antitumor activities. In in vitro cell proliferation assays, sensitivity to LY3214996 correlated with ERK pathway aberrations. LY3214996 showed dose-dependent tumor growth inhibition and regression in xenograft models harboring ERK pathway alterations. Importantly, more than 50% target inhibition for up to 8 to 16 hours was sufficient for significant tumor growth inhibition as single agent in BRAF- and KRAS-mutant models. LY3214996 also exhibited synergistic combination benefit with a pan-RAF inhibitor in a KRAS-mutant colorectal cancer xenograft model. Furthermore, LY3214996 demonstrated antitumor activity in BRAF-mutant models with acquired resistance in vitro and in vivo. Based on these preclinical data, LY3214996 has advanced to an ongoing phase I clinical trial (NCT02857270).

Aurora A-Selective Inhibitor LY3295668 Leads to Dominant Mitotic Arrest, Apoptosis in Cancer Cells, and Shows Potent Preclinical Antitumor Efficacy.

Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform-selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A-selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition-associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A-selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent.

The Folate Pathway Inhibitor Pemetrexed Pleiotropically Enhances Effects of Cancer Immunotherapy.

PURPOSE: Combination strategies leveraging chemotherapeutic agents and immunotherapy have held the promise as a method to improve benefit for patients with cancer. However, most chemotherapies have detrimental effects on immune homeostasis and differ in their ability to induce immunogenic cell death (ICD). The approval of pemetrexed and carboplatin with anti-PD-1 (pembrolizumab) for treatment of non-small cell lung cancer represents the first approved chemotherapy and immunotherapy combination. Although the clinical data suggest a positive interaction between pemetrexed-based chemotherapy and immunotherapy, the underlying mechanism remains unknown. EXPERIMENTAL DESIGN: Mouse tumor models (MC38, Colon26) and high-content biomarker studies (flow cytometry, Quantigene Plex, and nCounter gene expression analysis) were deployed to obtain insights into the mechanistic rationale behind the efficacy observed with pemetrexed/anti-PD-L1 combination. ICD in tumor cell lines was assessed by calreticulin and HMGB-1 immunoassays, and metabolic function of primary T cells was evaluated by Seahorse analysis. RESULTS: Pemetrexed treatment alone increased T-cell activation in mouse tumors in vivo, robustly induced ICD in mouse tumor cells and exerted T-cell-intrinsic effects exemplified by augmented mitochondrial function and enhanced T-cell activation in vitro. Increased antitumor efficacy and pronounced inflamed/immune activation were observed when pemetrexed was combined with anti-PD-L1. CONCLUSIONS: Pemetrexed augments systemic intratumor immune responses through tumor intrinsic mechanisms including immunogenic cell death, T-cell-intrinsic mechanisms enhancing mitochondrial biogenesis leading to increased T-cell infiltration/activation along with modulation of innate immune pathways, which are significantly enhanced in combination with PD-1 pathway blockade.See related commentary by Buque et al., p. 6890.

Inhibition of Sphingosine Phosphate Receptor 1 Signaling Enhances the Efficacy of VEGF Receptor Inhibition.

Inhibition of VEGFR signaling is an effective treatment for renal cell carcinoma, but resistance continues to be a major problem. Recently, the sphingosine phosphate (S1P) signaling pathway has been implicated in tumor growth, angiogenesis, and resistance to antiangiogenic therapy. S1P is a bioactive lipid that serves an essential role in developmental and pathologic angiogenesis via activation of the S1P receptor 1 (S1P1). S1P1 signaling counteracts VEGF signaling and is required for vascular stabilization. We used in vivo and in vitro angiogenesis models including a postnatal retinal angiogenesis model and a renal cell carcinoma murine tumor model to test whether simultaneous inhibition of S1P1 and VEGF leads to improved angiogenic inhibition. Here, we show that inhibition of S1P signaling reduces the endothelial cell barrier and leads to excessive angiogenic sprouting. Simultaneous inhibition of S1P and VEGF signaling further disrupts the tumor vascular beds, decreases tumor volume, and increases tumor cell death compared with monotherapies. These studies suggest that inhibition of angiogenesis at two stages of the multistep process may maximize the effects of antiangiogenic therapy. Together, these data suggest that combination of S1P1 and VEGFR-targeted therapy may be a useful therapeutic strategy for the treatment of renal cell carcinoma and other tumor types.

Targeting the TGFß pathway with galunisertib, a TGFßRI small molecule inhibitor, promotes anti-tumor immunity leading to durable, complete responses, as monotherapy and in combination with checkpoint blockade.

BACKGROUND: TGFß signaling plays a pleotropic role in tumor biology, promoting tumor proliferation, invasion and metastasis, and escape from immune surveillance. Inhibiting TGFß's immune suppressive effects has become of particular interest as a way to increase the benefit of cancer immunotherapy. Here we utilized preclinical models to explore the impact of the clinical stage TGFß pathway inhibitor, galunisertib, on anti-tumor immunity at clinically relevant doses. RESULTS: In vitro treatment with galunisertib reversed TGFß and regulatory T cell mediated suppression of human T cell proliferation. In vivo treatment of mice with established 4T1-LP tumors resulted in strong dose-dependent anti-tumor activity with close to 100% inhibition of tumor growth and complete regressions upon cessation of treatment in 50% of animals. This effect was CD8+ T cell dependent, and led to increased T cell numbers in treated tumors. Mice with durable regressions rejected tumor rechallenge, demonstrating the establishment of immunological memory. Consequently, mice that rejected immunogenic 4T1-LP tumors were able to resist rechallenge with poorly immunogenic 4 T1 parental cells, suggesting the development of a secondary immune response via antigen spreading as a consequence of effective tumor targeting. Combination of galunisertib with PD-L1 blockade resulted in improved tumor growth inhibition and complete regressions in colon carcinoma models, demonstrating the potential synergy when cotargeting TGFß and PD-1/PD-L1 pathways. Combination therapy was associated with enhanced anti-tumor immune related gene expression profile that was accelerated compared to anti-PD-L1 monotherapy. CONCLUSIONS: Together these data highlight the ability of galunisertib to modulate T cell immunity and the therapeutic potential of combining galunisertib with current PD-1/L1 immunotherapy.

Preclinical assessment of galunisertib (LY2157299 monohydrate), a first-in-class transforming growth factor-ß receptor type I inhibitor.

Transforming growth factor-ß (TGFß) is an important driver of tumor growth via intrinsic and extrinsic mechanisms, and is therefore an attractive target for developing cancer therapeutics. Using preclinical models, we characterized the anti-tumor activity of a small molecule inhibitor of TGFß receptor I (TGFßRI), galunisertib (LY2157299 monohydrate). Galunisertib demonstrated potent and selective inhibition of TGFßRI with corresponding inhibition of downstream signaling via inhibition of SMAD phosphorylation (pSMAD). Galunisertib also inhibited TGFß-induced pSMAD in vivo, which enabled a pharmacokinetic/pharmacodynamic profile in Calu6 and EMT6-LM2 tumors. Galunisertib demonstrated anti-tumor activity including inhibition of tumor cell migration and mesenchymal phenotype, reversal of TGFß-mediated immune-suppression, and tumor growth delay. A concentration-effect relationship was established with a dosing schedule to achieve the optimal level of target modulation. Finally, a rat model demonstrated a correlation between galunisertib-dependent inhibition of pSMAD in tumor tissues and in PBMCs, supporting the use of PBMCs for assessing pharmacodynamic effects. Galunisertib has been tested in several clinical studies with evidence of anti-tumor activity observed in subsets of patients. Here, we demonstrate that galunisertib inhibits a number of TGFß-dependent functions leading to anti-tumor activity. The enhanced understanding of galunisertib provides rationale for further informed clinical development of TGFß pathway inhibitors.

MET-targeting antibody (emibetuzumab) and kinase inhibitor (merestinib) as single agent or in combination in a cancer model bearing MET exon 14 skipping.

Purpose Approximately 3% of lung cancer bears mutations leading to MET exon 14 skipping, an oncogenic driver which is further evidenced by case reports of patient response to MET kinase inhibitor treatment. Approximately 15% of tumors harboring MET exon14 skipping have concurrent MET amplification. Experimental Design Merestinib is a type II MET kinase inhibitor. Emibetuzumab, a bivalent anti-MET antibody, internalizes MET receptor. Each single agent and the combination were evaluated in the Hs746t gastric cancer line bearing MET exon14 skipping and MET amplification. Results Merestinib inhibited Hs746t cell proliferation (IC50=34 nM) and totally eliminated pMET at 100nM. Emibetuzumab showed little anti-proliferative activity against Hs746t cells (IC50>100nM), did not reduce pMET, and slightly reduced cell surface MET. In the Hs746t xenograft model, dose dependent differences in durability of response were seen with merestinib including durable tumor regression (91.8%) at 12 mg/kg qd. Emibetuzumab treatment (10mg/kg qw) provided transient tumor regression (37.7%), but tumors re-grew while on treatment. Concurrent combination of merestinib (6 mg/kg qd) and emibetuzumab resulted in 85% tumor regression, while a sequential combination (initiating merestinib first) resulted in longer duration of treatment response. Conclusions Data in this study support a clinical evaluation of merestinib in patients with MET exon 14 skipping (NCT02920996). As a type II MET kinase inhibitor, merestinib may provide a therapeutic option to treatment naïve patients or to patients who progress on type I MET inhibitor treatment. Data also support clinical evaluation of the sequential combination of merestinib with emibetuzumab when patients progress on single agent merestinib.

LY3009120, a panRAF inhibitor, has significant anti-tumor activity in BRAF and KRAS mutant preclinical models of colorectal cancer.

Activating mutations in the KRAS and BRAF genes, leading to hyperactivation of the RAS/RAF/MAPK oncogenic signaling cascade, are common in patients with colorectal cancer (CRC). While selective BRAF inhibitors are efficacious in BRAFmut melanoma, they have limited efficacy in BRAFmut CRC patients. In a RASmut background, selective BRAF inhibitors are contraindicated due to paradoxical activation of the MAPK pathway through potentiation of CRAF kinase activity. A way to overcome such paradoxical activation is through concurrent inhibition of the kinase activity of both RAF isoforms. Here, we further examined the effects of LY3009120, a panRAF and RAF dimer inhibitor, in human models of CRC with various mutational backgrounds. We demonstrate that LY3009120 induced anti-proliferative effects in BRAFmut and KRASmut CRC cell lines through G1-cell cycle arrest. The anti-proliferative effects of LY3009120 in KRASmut CRC cell lines phenocopied molecular inhibition of RAF isoforms by simultaneous siRNA-mediated knockdown of ARAF, BRAF and CRAF. Additionally, LY3009120 displayed significant activity in in vivo BRAFmut and KRASmut CRC xenograft models. Examination of potential resistance to LY3009120 demonstrated RAF-independent ERK and AKT activation in the KRASmut CRC cell line HCT 116. These findings describe the preclinical activity of a panRAF inhibitor in a BRAFmut and KRASmut CRC setting.

Activating the Wnt/ß-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 ß-catenin expression occurs with melanoma progression and that nuclear ß-catenin levels are inversely proportional to cellular proliferation, suggesting that activation of the Wnt/ß-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 GSK3ß isoforms. In a panel of melanoma cell lines, nM concentrations of LY2090314 stimulated TCF/LEF TOPFlash reporter activity, stabilized ß-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 ~10 nM after 72hr of treatment) in contrast to other solid tumor cell lines (IC50 >10 uM) 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 ß-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 ß-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.

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.

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).

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.

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.

The 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 2 nM, a pharmacodynamic residence time (Koff) of 0.00132 min(-1) and t1/2 of 525 min. 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).

Preclinical bridging studies: understanding dried blood spot and plasma exposure profiles.

BACKGROUND: Understanding the distribution of the analyte between the cellular and noncellular (plasma) components of the blood is important, especially in situations where dried blood spot (DBS) data need to be compared with plasma data, or vice versa. RESULTS: Pearson's coefficient, Lin's coefficient and the Bland-Altman analysis are appropriate to evaluate the concordance between DBS and plasma data from bridging studies. Percent recovery plots generated using the ex vivo blood:plasma ratio and the regression equations demonstrate the best approach for predicting plasma concentrations from DBS. CONCLUSION: Statistical analysis of bridging study data is needed to characterize the relationship or concordance between blood (DBS) and plasma. The outcomes also provide guidance on selecting the most appropriate approach to transform DBS data to plasma, or vice versa. However, the biological and statistical evidence must be weighed together when deciding if DBS is suitable for preclinical and/or clinical development.

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.

Pharmacological characterization of cGMP regulation by the biarylpropylsulfonamide class of positive, allosteric modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

The biarylpropylsulfonamide class of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) potentiators represented by N-2-(4-(4-cyanophenol)phenol)propyl-2-propanesulfonamide (LY404187) and (R)-4'-[1-fluoro-1-methyl-2-(propane-2-sulfonylamino)-ethyl]-biphenyl-4-carboxylic acid methylamide (LY503430) are positive, allosteric AMPA receptor activators, which enhance AMPA receptor-mediated neurotransmission by reducing desensitization of the ion channel. Although these compounds have efficacy in in vivo rodent models of cognition, depression, and Parkinson's disease, little is known about biochemical pathways activated by these agents. Given the well established regulation of the nitric oxide/cGMP pathway by excitatory neurotransmission, the current study characterized AMPA receptor potentiator-mediated cGMP response in mouse cerebellum. Acute treatment by both LY404187 and LY503430 [2.0, 5.0, or 10 mg/kg subcutaneously (s.c.)] elevated basal cerebellar cGMP levels in a dose-dependent manner. Pretreatment with the noncompetitive, allosteric AMPA receptor-selective antagonist 7H-1,3-dioxolo[4,5-h][2,3]benzodiazepine-7-carboxamide, 5-(4-aminophenyl)-8,9-dihydro-N,8-dimethyl-monohydrochloride-(9CI) (GYKI 53655) [3.0 mg/kg intraperitoneally (i.p.)], completely blocked the effect of LY404187, demonstrating that activation of AMPA receptors induces cGMP levels. Interestingly, pretreatment with the N-methyl-d-aspartate (NMDA) open channel blocker dizocilpine (0.3 and 1.0 mg/kg i.p.) also abolished the AMPA receptor potentiator-mediated cGMP accumulation, indicating that activation of AMPA receptors leads to NMDA receptor-mediated transmission involved in cGMP regulation. Pharmacological augmentation of the endogenous glutamate tone via the alkaloid harmaline (20-60 mg/kg i.p.) synergized with AMPA potentiator activity and provided further direct evidence of in vivo allosteric activation of AMPA receptors by LY404187. The synergism between harmaline and LY404187 was specific, since cGMP accumulation induced by foot-shock stress was not augmented by the AMPA receptor potentiator. Taken together, these data indicate that the cGMP system may play an important role in pharmacological efficacy of the biarylpropylsulfonamide class of AMPA receptor potentiators.