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Pemigatinib, a selective FGFR1-3 inhibitor, has demonstrated antitumor activity in FIGHT-202, a phase II study in patients with cholangiocarcinoma harboring FGFR2 fusions/rearrangements, and has gained regulatory approval in the United States. Eligibility for FIGHT-202 was assessed using genomic profiling; here, these data were utilized to characterize the genomic landscape of cholangiocarcinoma and to uncover unique molecular features of patients harboring FGFR2 rearrangements. The results highlight the high percentage of patients with cholangiocarcinoma harboring potentially actionable genomic alterations and the diversity in gene partners that rearrange with FGFR2 . Clinicogenomic analysis of pemigatinib-treated patients identified mechanisms of primary and acquired resistance. Genomic subsets of patients with other potentially actionable FGF/FGFR alterations were also identified. Our study provides a framework for molecularly guided clinical trials and underscores the importance of genomic profiling to enable a deeper understanding of the molecular basis for response and nonresponse to targeted therapy. SIGNIFICANCE: We utilized genomic profiling data from FIGHT-202 to gain insights into the genomic landscape of cholangiocarcinoma, to understand the molecular diversity of patients with FGFR2 fusions or rearrangements, and to interrogate the clinicogenomics of patients treated with pemigatinib. Our study highlights the utility of genomic profiling in clinical trials. This article is highlighted in the In This Issue feature, p. 211 ., (©2020 American Association for Cancer Research.)

Background: Epacadostat is a potent inhibitor of the immunosuppressive indoleamine 2,3-dioxygenase 1 (IDO1) enzyme. We present phase 1 results from a phase 1/2 clinical study of epacadostat in combination with ipilimumab, an anti-cytotoxic T-lymphocyte-associated protein 4 antibody, in advanced melanoma (NCT01604889)., Methods: Only the phase 1, open-label portion of the study was conducted, per the sponsor's decision to terminate the study early based on the changing melanoma treatment landscape favoring exploration of programmed cell death protein 1 (PD-1)/PD-ligand 1 inhibitor-based combination strategies. Such decision was not related to the safety of epacadostat plus ipilimumab. Patients received oral epacadostat (25, 50, 100, or 300 mg twice daily [BID]; 75 mg daily [50 mg AM, 25 mg PM]; or 50 mg BID intermittent [2 weeks on/1 week off]) plus intravenous ipilimumab 3 mg/kg every 3 weeks., Results: Fifty patients received ≥1 dose of epacadostat. As of January 20, 2017, 2 patients completed treatment and 48 discontinued, primarily because of adverse events (AEs) and disease progression (n = 20 each). Dose-limiting toxicities occurred in 11 patients (n = 1 each with epacadostat 25 mg BID, 50 mg BID intermittent, 75 mg daily; n = 4 each with epacadostat 50 mg BID, 300 mg BID). The most common immune-related treatment-emergent AEs included rash (50%), alanine aminotransferase elevation (28%), pruritus (28%), aspartate aminotransferase elevation (24%), and hypothyroidism (10%). Among immunotherapy-naive patients (n = 39), the objective response rate was 26% by immune-related response criteria and 23% by Response Evaluation Criteria in Solid Tumors version 1.1. No objective response was seen in the 11 patients who received prior immunotherapy. Epacadostat exposure was dose proportional, with clinically significant IDO1 inhibition at doses ≥25 mg BID., Conclusions: When combined with ipilimumab, epacadostat ≤50 mg BID demonstrated clinical and pharmacologic activity and was generally well tolerated in patients with advanced melanoma., Trial Registration: ClinicalTrials.gov identifier, NCT01604889 . Registration date, May 9, 2012, retrospectively registered.

Objective: Indoleamine 2,3-dioxygenase-1 (IDO1) is a key regulator of immune tolerance in ovarian cancer. This study investigated efficacy and safety of the IDO1 enzyme inhibitor epacadostat versus tamoxifen in patients with biochemical-only recurrence (CA-125 elevation) following complete remission after first-line chemotherapy for advanced epithelial ovarian, primary peritoneal, or fallopian tube cancer., Methods: In this open-label, phase 2 study (NCT01685255), patients were randomised 1:1 to epacadostat 600mg or tamoxifen 20mg twice daily for successive 28-day cycles and stratified by time since completion of first-line chemotherapy to first CA-125 elevation (3 to <12 or ≥12months). The primary endpoint was investigator-assessed progression-free survival (PFS; RECIST v1.1). Secondary endpoints included CA-125 response (Gynecologic Cancer InterGroup criteria), overall survival, safety, and tolerability., Results: The study was terminated primarily due to slow accrual and lack of evidence of superiority. Median PFS was 3.75months for epacadostat (n=22) versus 5.56months for tamoxifen (n=20; HR, 1.34 [95% CI, 0.58-3.14]; P=0.54). Of evaluable patients, 1 (5.0%) epacadostat and 3 (15.8%) tamoxifen patients had confirmed CA-125 responses. The most common treatment-emergent adverse event was fatigue (epacadostat, 36.4%; tamoxifen, 40.0%). Immune-related adverse events, observed with epacadostat only, were primarily rash (18.2%) and pruritus (9.1%). Epacadostat pharmacokinetics/pharmacodynamics were consistent with its known mechanism of action. IDO1 expression was observed in 94% of archival tumour samples., Conclusions: This first report of immunotherapy evaluation in biochemical-only relapse ovarian cancer and of IDO1 inhibitor monotherapy in ovarian cancer found no significant difference in efficacy between epacadostat and tamoxifen. Epacadostat was generally well tolerated., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose: Indoleamine 2,3-dioxygenase-1 (IDO1) catalyzes the degradation of tryptophan to N-formyl-kynurenine. Overexpressed in many solid malignancies, IDO1 can promote tumor escape from host immunosurveillance. This first-in-human phase I study investigated the maximum tolerated dose, safety, pharmacokinetics, pharmacodynamics, and antitumor activity of epacadostat (INCB024360), a potent and selective inhibitor of IDO1. Experimental Design: Fifty-two patients with advanced solid malignancies were treated with epacadostat [50 mg once daily or 50, 100, 300, 400, 500, 600, or 700 mg twice daily (BID)] in a dose-escalation 3 + 3 design and evaluated in 28-day cycles. Treatment was continued until disease progression or unacceptable toxicity. Results: One dose-limiting toxicity (DLT) occurred at the dose of 300 mg BID (grade 3, radiation pneumonitis); another DLT occurred at 400 mg BID (grade 3, fatigue). The most common adverse events in >20% of patients overall were fatigue, nausea, decreased appetite, vomiting, constipation, abdominal pain, diarrhea, dyspnea, back pain, and cough. Treatment produced significant dose-dependent reductions in plasma kynurenine levels and in the plasma kynurenine/tryptophan ratio at all doses and in all patients. Near maximal changes were observed at doses of ≥100 mg BID with >80% to 90% inhibition of IDO1 achieved throughout the dosing period. Although no objective responses were detected, stable disease lasting ≥16 weeks was observed in 7 of 52 patients. Conclusions: Epacadostat was generally well tolerated, effectively normalized kynurenine levels, and produced maximal inhibition of IDO1 activity at doses of ≥100 mg BID. Studies investigating epacadostat in combination with other immunomodulatory drugs are ongoing. Clin Cancer Res; 23(13); 3269-76. ©2017 AACR ., (©2017 American Association for Cancer Research.)

We conducted a phase 2 study of ruxolitinib in patients with relapsed/refractory leukemias. Patients with acceptable performance status (0-2), adequate organ function, and no active infection, received ruxolitinib 25 mg orally twice a day for 4 weeks (1 cycle). Response was assessed after every 2 cycles of treatment, and patients who completed 2 cycles were allowed to continue treatment until disease progression. Dose escalation to 50 mg twice daily was permitted in patients demonstrating a benefit. Thirty-eight patients, with a median age of 69 years (range, 45-88), were treated. The median number of prior therapies was 2 (range, 1-6). Twelve patients had JAK2V617F mutation. Patients received a median of 2 cycles of therapy (range, 1-22). Three of 18 patients with postmyeloproliferative neoplasm (MPN) acute myeloid leukemia (AML) showed a significant response; 2 achieved complete remission (CR) and one achieved a CR with insufficient recovery of blood counts (CRi). The responding patients with palpable spleens also had significant reductions in spleen size. Overall, ruxolitinib was very well tolerated with only 4 patients having grade 3 or higher toxicity. Ruxolitinib has modest antileukemic activity as a single agent, particularly in patients with post-MPN AML. The study was registered at www.clinicaltrials.gov as NCT00674479.

Purpose: The c-MET receptor tyrosine kinase plays important roles in the formation, progression, and dissemination of human cancer and presents an attractive therapeutic target. This study describes the preclinical characterization of INCB28060, a novel inhibitor of c-MET kinase., Experimental Design: Studies were conducted using a series of in vitro and in vivo biochemical and biological experiments., Results: INCB28060 exhibits picomolar enzymatic potency and is highly specific for c-MET with more than 10,000-fold selectivity over a large panel of human kinases. This inhibitor potently blocks c-MET phosphorylation and activation of its key downstream effectors in c-MET-dependent tumor cell lines. As a result, INCB28060 potently inhibits c-MET-dependent tumor cell proliferation and migration and effectively induces apoptosis in vitro. Oral dosing of INCB28060 results in time- and dose-dependent inhibition of c-MET phosphorylation and tumor growth in c-MET-driven mouse tumor models, and the inhibitor is well tolerated at doses that achieve complete tumor inhibition. In a further exploration of potential interactions between c-MET and other signaling pathways, we found that activated c-MET positively regulates the activity of epidermal growth factor receptors (EGFR) and HER-3, as well as expression of their ligands. These effects are reversed with INCB28060 treatment. Finally, we confirmed that circulating hepatocyte growth factor levels are significantly elevated in patients with various cancers., Conclusions: Activated c-MET has pleiotropic effects on multiple cancer-promoting signaling pathways and may play a critical role in driving tumor cell growth and survival. INCB28060 is a potent and selective c-MET kinase inhibitor that may have therapeutic potential in cancer treatment.

Introduction: The aberrantly upregulated c-mesenchymal-epithelia transition factor (c-MET) signaling pathway has been considered to be an attractive target for cancer intervention owing to the important roles it plays in tumor formation, progression, metastasis, angiogenesis and drug resistance. Based on the historical preclinical evidence, a number of c-MET pathway targeted agents are being developed in the clinic, and recent clinical data have begun to provide some insight into which tumor types and patient populations a c-MET pathway inhibitor may be beneficial for., Areas Covered: Through reviewing recent publications in the literature and information disclosed in other public forums, we describe the current understanding of c-MET biology in human malignancies and discuss the latest progress in the development of c-MET pathway inhibitors for cancer treatment., Expert Opinion: The c-MET pathway inhibitors currently being evaluated in the clinic have demonstrated compelling evidence of clinical activity in different cancer types and may provide significant therapeutic opportunities. The challenges, however, are to identify the tumor types and patient populations that benefit most, and find the most effective combinations of therapies while minimizing potential toxicity.

JAKs are required for signaling initiated by several cytokines (e.g., IL-4, IL-12, IL-23, thymic stromal lymphopoietin (TSLP), and IFNγ) implicated in the pathogenesis of inflammatory skin diseases such as psoriasis and atopic dermatitis (AD). Direct antagonism of cytokines, such as IL-12 and IL-23 using ustekinumab, has proven effective in randomized studies in psoriasis patients. We hypothesized that local inhibition of cytokine signaling using topical administration of INCB018424, a small molecule inhibitor of JAK1 and JAK2, would provide benefit similar to systemic cytokine neutralization. In cellular assays, INCB018424 inhibits cytokine-induced JAK/signal transducers and activators of transcription (STAT) signaling and the resultant production of inflammatory proteins (e.g., IL-17, monocyte chemotactic protein-1, and IL-22) in lymphocytes and monocytes, with half-maximal inhibitory concentration values <100  nM. In vivo, topical application of INCB018424 resulted in suppression of STAT3 phosphorylation, edema, lymphocyte infiltration, and keratinocyte proliferation in a murine contact hypersensitivity model and inhibited tissue inflammation induced by either intradermal IL-23 or TSLP. Topical INCB018424 was also well tolerated in a 28-day safety study in Gottingen minipigs. These results suggest that localized JAK1/JAK2 inhibition may be therapeutic in a range of inflammatory skin disorders such as psoriasis and AD. Clinical evaluation of topical INCB018424 is ongoing.

This report describes the design and synthesis of a series of CCR2 antagonists incorporating novel non-aryl/heteroaryl RHS (right hand side) motifs. Previous SAR in the area has suggested an aryl/heteroaryl substituent as a necessary structural feature for binding to the CCR2 receptor. Herein we describe the SAR with regards to potency (binding to hCCR2), dofetilide activity and metabolic stability (in vitro HLM) for this series. The resulting outcome was the identification of compounds with excellent properties for the investigation of the role of CCR2 in disease., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Inhibiting signal transduction induced by inflammatory cytokines offers a new approach for the treatment of autoimmune diseases such as rheumatoid arthritis. Kinase inhibitors have shown promising oral disease-modifying antirheumatic drug potential with efficacy similar to anti-TNF biologics. Direct and indirect inhibition of the JAKs, with small molecule inhibitors like CP-690,550 and INCB018424 or neutralizing Abs, such as the anti-IL6 receptor Ab tocilizumab, have demonstrated rapid and sustained improvement in clinical measures of disease, consistent with their respective preclinical experiments. Therefore, it is of interest to identify optimized JAK inhibitors with unique profiles to maximize therapeutic opportunities. INCB028050 is a selective orally bioavailable JAK1/JAK2 inhibitor with nanomolar potency against JAK1 (5.9 nM) and JAK2 (5.7 nM). INCB028050 inhibits intracellular signaling of multiple proinflammatory cytokines including IL-6 and IL-23 at concentrations <50 nM. Significant efficacy, as assessed by improvements in clinical, histologic and radiographic signs of disease, was achieved in the rat adjuvant arthritis model with doses of INCB028050 providing partial and/or periodic inhibition of JAK1/JAK2 and no inhibition of JAK3. Diminution of inflammatory Th1 and Th17 associated cytokine mRNA levels was observed in the draining lymph nodes of treated rats. INCB028050 was also effective in multiple murine models of arthritis, with no evidence of suppression of humoral immunity or adverse hematologic effects. These data suggest that fractional inhibition of JAK1 and JAK2 is sufficient for significant activity in autoimmune disease models. Clinical evaluation of INCB028050 in RA is ongoing.

Indoleamine 2,3-dioxygenase-1 (IDO1; IDO) mediates oxidative cleavage of tryptophan, an amino acid essential for cell proliferation and survival. IDO1 inhibition is proposed to have therapeutic potential in immunodeficiency-associated abnormalities, including cancer. Here, we describe INCB024360, a novel IDO1 inhibitor, and investigate its roles in regulating various immune cells and therapeutic potential as an anticancer agent. In cellular assays, INCB024360 selectively inhibits human IDO1 with IC(50) values of approximately 10nM, demonstrating little activity against other related enzymes such as IDO2 or tryptophan 2,3-dioxygenase (TDO). In coculture systems of human allogeneic lymphocytes with dendritic cells (DCs) or tumor cells, INCB024360 inhibition of IDO1 promotes T and natural killer (NK)-cell growth, increases IFN-gamma production, and reduces conversion to regulatory T (T(reg))-like cells. IDO1 induction triggers DC apoptosis, whereas INCB024360 reverses this and increases the number of CD86(high) DCs, potentially representing a novel mechanism by which IDO1 inhibition activates T cells. Furthermore, IDO1 regulation differs in DCs versus tumor cells. Consistent with its effects in vitro, administration of INCB024360 to tumor-bearing mice significantly inhibits tumor growth in a lymphocyte-dependent manner. Analysis of plasma kynurenine/tryptophan levels in patients with cancer affirms that the IDO pathway is activated in multiple tumor types. Collectively, the data suggest that selective inhibition of IDO1 may represent an attractive cancer therapeutic strategy via up-regulation of cellular immunity.

Successfully developed target-based therapies have significantly changed cancer treatment. Among many targets, the c-MET receptor tyrosine kinase and its ligand hepatocyte growth factor have recently gained considerable attention. The c-MET pathway is dysregulated in most human malignancies, and regulates tumor formation, progression and dissemination, and numerous c-MET pathway inhibitors are currently being evaluated in the clinic. Although some studies have shown impressive evidence of antitumor activity, the data should be interpreted with caution because of the distinct properties of these agents and diverse patient populations studied. Furthermore, in tumor types where patients might benefit from c-MET inhibition, rational combination treatments might ultimately provide maximal clinical benefit. Here, we review the evidence linking c-MET activation to cancer, and discuss the latest progress, opportunities and challenges in the clinical development of c-MET pathway inhibitors.

The chemokine receptor 2 (CCR2) directs migration of monocytes and has been proposed to be a drug target for chronic inflammatory diseases. INCB3344 was first published as a small molecule nanomolar inhibitor of rodent CCR2. Here, we show that INCB3344 can also bind human CCR2 (hCCR2) with high affinity, having a dissociation constant (K(d)) of approximately 5nM. The binding of the compound to the receptor is rapid and reversible. INCB3344 potently inhibits hCCR2 binding of monocyte chemoattractant protein-1 (MCP-1) and MCP-1-induced signaling and function in hCCR2-expressing cells, including ERK phosphorylation and chemotaxis, and is competitive against MCP-1 in vitro. INCB3344 also blocks MCP-1 binding to monocytes in human whole blood, with potency consistent with in vitro studies. The whole blood binding assay described here can be used for monitoring pharmacodynamic activity of CCR2 antagonists in both preclinical models and in the clinic.

Background: In many human cancers, c-MET is activated via receptor overexpression, amplification, mutation and/or a ligand-dependent autocrine/paracrine loop. These biochemical and genetic abnormalities have been correlated with poor clinical outcomes and drug resistance in cancer patients. Preclinical studies suggest that targeting aberrant c-MET signaling could be an attractive therapy in cancer, but this notion has only recently been tested in the clinic., Objectives: To describe the biological aspects of the c-MET signaling pathway and to discuss recent progress and possible future trends in the development of agents that target the c-MET pathway, with an emphasis on small-molecule c-MET kinase inhibitors., Method: A review of relevant publications, including published articles in literature, reports at scientific meetings, and information available through the Internet., Results/conclusion: The dysregulated c-MET pathway represents a promising target for cancer drug development. The agents that target the c-MET pathway have demonstrated impressive evidence of early clinical activity and may have a significant therapeutic potential.

Potent and selective inhibitors of tumor necrosis factor-alpha converting enzyme (TACE) were discovered with several new heterocyclic P1' groups in conjunction with cyclic beta-amino hydroxamic acid scaffolds. Among them, the pyrazolopyridine provided the best overall profile when combined with tetrahydropyran beta-amino hydroxamic acid scaffold. Specifically, inhibitor 49 showed IC(50) value of 1 nM against porcine TACE and 170 nM in the suppression of LPS-induced TNF-alpha of human whole blood. Compound 49 also displayed excellent selectivity over a wide panel of MMPs as well as excellent oral bioavailability (F%>90%) in rat n-in-1 PK studies.

Novel ((2-substituted-1H-benzo[d]imidazol-1-yl)methyl)benzamides were found to be excellent P1' substituents in conjunction with unique constrained beta-amino hydroxamic acid scaffolds for the discovery of potent selective inhibitors of TNF-alpha Converting Enzyme (TACE). Optimized examples proved potent for TACE, exceptionally selective over a wide panel of MMP and ADAM proteases, potent in the suppression of LPS-induced TNF-alpha in human whole blood and orally bioavailable.

Two novel oxaspiro[4.4]nonane beta-benzamido hydroxamic scaffolds have been synthesized in enantio- and diasteriomerically pure form. These templates proved to be exceptional platforms that have led to the discovery of potent inhibitors of TACE that are active in a cellular assay measuring suppression of LPS-induced TNF-alpha. Furthermore, these inhibitors are selective against related MMPs, demonstrate permeability in a Caco-2 assay, and display good oral bioavailability.

Selective inhibitors of TNF-alpha Converting Enzyme (TACE) based on (1R,2S)-cyclopentyl, (3S,4S)-pyrrolidinyl, and (3R,4S)-tetrahydrofuranyl beta-benzamido hydroxamic acids have been synthesized and evaluated. This study has led to the discovery of novel inhibitors whose profiles include activity against TACE in an enzyme assay, potency in the suppression of LPS-stimulated TNF-alpha in human whole blood, selectivity against a panel of MMPs and oral bioavailability.

Beta-benzamido hydroxamic acids were discovered as potent TACE inhibitors. A computer model was constructed to help understanding the binding activities and guiding SAR study. SAR optimization led to the discovery of compound 30 which met all in vitro and in vivo criteria for the program and was selected for further evaluation.

DPC 333 ((2R)-2-((3R)-3-amino-3{4-[2-methyl-4-quinolinyl) methoxy] phenyl}-2-oxopyrrolidinyl)-N-hydroxy-4-methylpentanamide)) is a potent and selective inhibitor of tumor necrosis factor (TNF)-alpha-converting enzyme (TACE). It significantly inhibits lipopolysaccharide-induced soluble TNF-alpha production in blood from rodents, chimpanzee, and human, with IC(50) values ranging from 17 to 100 nM. In rodent models of endotoxemia, DPC 333 inhibited the production of TNF-alpha in a dose-dependent manner, with an oral ED(50) ranging from 1.1 to 6.1 mg/kg. Oral dosing of DPC 333 at 5.5 mg/kg daily for 2 weeks in a rat collagen antibody-induced arthritis model suppressed the maximal response by approximately 50%. DPC 333 was distributed widely to tissues including the synovium, the site of action for antiarthritic drugs. Pharmacokinetic and pharmacodynamic studies in chimpanzee revealed a systemic clearance of 0.4 l/h/kg, a V(ss) of 0.6 l/kg, an oral bioavailability of 17%, and an ex vivo IC(50) for the suppression of TNF-alpha production of 55 nM (n = 1). In a phase I clinical trial with male volunteers after single escalating doses of oral DPC 333, the terminal half-life was between 3 and 6 h and the ex vivo IC(50) for suppressing TNF-alpha production was 113 nM. Measurement of the suppression of TNF-alpha production ex vivo may serve as a good biomarker in evaluating the therapeutic efficacy of TACE inhibitors. Overall, the pharmacological profiles of DPC 333 support the notion that suppression of TNF-alpha with TACE inhibitors like DPC 333 may provide a novel approach in the treatment of various inflammatory diseases including rheumatoid arthritis, via control of excessive TNF-alpha production.

DPC168, a benzylpiperidine-substituted aryl urea CCR3 antagonist evaluated in clinical trials, was a relatively potent inhibitor of the 2D6 isoform of cytochrome P-450 (CYP2D6). Replacement of the cyclohexyl central ring with saturated heterocycles provided potent CCR3 antagonists with improved selectivity against CYP2D6. The favorable preclinical profile of DPC168 was maintained in an acetylpiperidine derivative, BMS-570520.

A new P1' group for TACE inhibitors was identified by eliminating the oxygen atom in the linker of the original 4-(2-methylquinolin-4-ylmethoxy)phenyl P1' group. Incorporation of this 4-(2-methylquinolin-4-ylmethyl)phenyl group onto different beta-aminohydroxamic acid cores provided compound 18, which demonstrated potent porcine TACE (p-TACE) and human whole blood activity, excellent PK properties, and good selectivity against a variety of MMPs.

Purpose: ErbB receptor signaling pathways are important regulators of cell fate, and their dysregulation, through (epi)genetic alterations, plays an etiologic role in multiple cancers. ErbB ligands are synthesized as membrane-bound precursors that are cleaved by members of the ADAM family of zinc-dependent metalloproteases. This processing, termed ectodomain shedding, is essential for the functional activation of ErbB ligands. Recent studies suggest that elevated levels of ErbB ligands may circumvent the effectiveness of ErbB-targeted therapeutics. Here, we describe the discovery and preclinical development of potent, selective inhibitors of ErbB ligand shedding., Experimental Design: A series of biochemical and cell-based assays were established to identify selective inhibitors of ErbB ligand shedding. The therapeutic potential of these compounds was assessed in multiple in vivo models of cancer and matrix metalloprotease-related toxicity., Results: INCB3619 was identified as a representative selective, potent, orally bioavailable small-molecule inhibitor of a subset of ADAM proteases that block shedding of ErbB ligands. Administration of INCB3619 to tumor-bearing mice reduced ErbB ligand shedding in vivo and inhibited ErbB pathway signaling (e.g., phosphorylation of Akt), tumor cell proliferation, and survival. Further, INCB3619 synergized with clinically relevant cancer therapeutics and showed no overt or compounding toxicities, including fibroplasia, the dose-limiting toxicity associated with broad-spectrum matrix metalloprotease inhibitors., Conclusions: Inhibition of ErbB ligand shedding offers a potentially novel and well-tolerated therapeutic strategy for the treatment of human cancers and is currently being evaluated in the clinic.

Linear unselective CCR3 antagonist leads with IC(50) values in the 200 nM range were converted into low nM binding compounds selective at CCR3 by moving the piperidine nitrogen substituent to the carbon at the 2-position of the ring. Substitution of the piperidine nitrogen with simple alkyl and acyl groups was found to improve the selectivity of this new compound class. In particular, N-{3-[(2S, 4R)-1-(propyl)-4-(4-fluorobenzyl)piperidinyl]propyl}-N'-(3-acetylphenyl)urea exhibited single digit nanomolar IC(50) values for CCR3 with >100-fold selectivity against an extensive counter screen panel.

We describe here the existence of a heregulin-HER3 autocrine loop, and the contribution of heregulin-dependent, HER2-mediated HER3 activation to gefitinib insensitivity in non-small cell lung cancer (NSCLC). ADAM17 protein, a major ErbB ligand sheddase, is upregulated in NSCLC and is required not only for heregulin-dependent HER3 signaling, but also for EGFR ligand-dependent signaling in NSCLC cell lines. A selective ADAM inhibitor, INCB3619, prevents the processing and activation of multiple ErbB ligands, including heregulin. In addition, INCB3619 inhibits gefitinib-resistant HER3 signaling and enhances gefitinib inhibition of EGFR signaling in NSCLC. These results show that ADAM inhibition affects multiple ErbB pathways in NSCLC and thus offers an excellent opportunity for pharmacological intervention, either alone or in combination with other drugs.

CC chemokine receptor (CCR) 3 is a chemokine receptor implicated in recruiting cells, particularly eosinophils (EPhi), to the lung in episodes of allergic asthma. To investigate the efficacy of selective, small molecule antagonists of CCR3, we developed a murine model of EPhi recruitment to the lung. Murine eotaxin was delivered intranasally to mice that had previously received i.p. injections of ovalbumin (OVA), and the effects were monitored by bronchoalveolar lavage. A selective eosinophilic influx was produced in animals receiving eotaxin but not saline. Furthermore, the number of EPhi was concentration- and time-dependent. Although anti-CCR3 antibody reduced the number of EPhi, the effect of eotaxin in OVA-sensitized mice was not a direct chemotactic stimulus because mast cell deficiency (in WBB6F1-Kitw/Kitw-v mice) significantly reduced the response. Two representative small molecule CCR3 antagonists from our program were characterized as being active at mouse CCR3. They were administered p.o. to wild-type mice and found to reduce eotaxin-elicited EPhi selectively in a dose-dependent manner. Pump infusion of one of the inhibitors to achieve steady-state levels showed that efficacy was not achieved at plasma concentrations equivalent to the in vitro chemotaxis IC90 but only at much higher concentrations. To extend the results from our recruitment model, we tested one of the inhibitors in an allergenic model of airway inflammation, generated by adoptive transfer of OVA-sensitive murine T helper 2 cells and aerosolized OVA challenge of recipient mice, and found that it inhibited EPhi recruitment. We conclude that small molecule CCR3 antagonists reduce pulmonary eosinophilic inflammation elicited by chemokine or allergenic challenge.

The HER-2 receptor tyrosine kinase is an important regulator of cell proliferation and survival, and it is a clinically validated target of therapeutic intervention for HER-2 positive breast cancer patients. Its extracellular domain (ECD) is frequently cleaved by protease(s) in HER-2 overexpressing breast cancer patients, rendering the remaining membrane-bound portion (p95) a constitutively activated kinase. The presence of both serum ECD and cellular p95 protein has been linked to poor clinical outcome as well as reduced effectiveness of some therapeutic treatments. We have identified a series of potent, selective small molecule inhibitors of ADAM proteases, exemplified here by INCB003619, and demonstrate that these inhibitors effectively block HER-2 cleavage in HER-2 overexpressing human breast cancer cell lines. Intriguingly, when used in combination, INCB003619 dramatically enhances the antiproliferative activity of suboptimal doses of the anti-HER-2 antibody, trastuzumab, in HER-2 overexpressing/shedding breast cancer cell lines, accompanied by reduced ERK and AKT phosphorylation. Furthermore, INCB003619, in combination with trastuzumab, augments the pro-apoptotic and antiproliferative effects of the chemotherapeutic agent paclitaxel. Consistent with these in vitro data, INCB003619 reduces serum ECD levels and enhances the antitumor effect of trastuzumab in a xenograft tumor model derived from the HER-2 overexpressing BT-474 breast cancer cell line. Collectively, these findings suggest that blocking HER-2 cleavage with selective ADAM inhibitors may represent a novel therapeutic approach for treating HER-2 overexpressing breast cancer patients.

A novel series of achiral TNF-alpha converting enzyme (TACE) inhibitors has been discovered. These compounds exhibited activities from 0.35 to 11nM in a porcine TACE assay and inhibited TNF-alpha production in an LPS-stimulated whole blood assay with an IC(50) value of 23nM for the most potent one. They also have excellent selectivities over related metalloproteases including aggrecanases.

This report describes the characterization of INCB3344, a novel, potent and selective small molecule antagonist of the mouse CCR2 receptor. The lack of rodent cross-reactivity inherent in the small molecule CCR2 antagonists discovered to date has precluded pharmacological studies of antagonists of this receptor and its therapeutic relevance. In vitro, INCB3344 inhibits the binding of CCL2 to mouse monocytes with nanomolar potency (IC(50) = 10 nM) and displays dose-dependent inhibition of CCL2-mediated functional responses such as ERK phosphorylation and chemotaxis with similar potency. Against a panel of G protein-coupled receptors that includes other CC chemokine receptors, INCB3344 is at least 100-fold selective for CCR2. INCB3344 possesses good oral bioavailability and systemic exposure in rodents that allows in vivo pharmacological studies. INCB3344 treatment results in a dose-dependent inhibition of macrophage influx in a mouse model of delayed-type hypersensitivity. The histopathological analysis of tissues from the delayed-type hypersensitivity model demonstrates that inhibition of CCR2 leads to a substantial reduction in tissue inflammation, suggesting that macrophages play an orchestrating role in immune-based inflammatory reactions. These results led to the investigation of INCB3344 in inflammatory disease models. We demonstrate that therapeutic dosing of INCB3344 significantly reduces disease in mice subjected to experimental autoimmune encephalomyelitis, a model of multiple sclerosis, as well as a rat model of inflammatory arthritis. In summary, we present the first report on the pharmacological characterization of a selective, potent and rodent-active small molecule CCR2 antagonist. These data support targeting this receptor for the treatment of chronic inflammatory diseases.

A disintegrin and metalloproteases (ADAMs) are zinc-dependent trans-membrane metalloproteases that shed the extracellular domains of membrane-bound growth factors, cytokines and receptors. Key functions of ADAMs have emerged in ErbB signalling pathways as being sheddases for multiple ErbB ligands. As the ErbB pathway is a validated target for anti-cancer drugs, the upstream activators of ErbB ligands, their sheddases, now enter the spotlight as new drug targets in the ErbB pathway. ADAMs are involved not only in tumour cell proliferation but also in angiogenesis and metastasis. Therefore, strategies targeting ADAMs might be an important complement to existing anti-ErbB approaches.

Starting with our previously described(20) class of CC chemokine receptor-3 (CCR3) antagonist, we improved the potency by replacing the phenyl linker of 1 with a cyclohexyl linker and by replacing the 4-benzylpiperidine with a 3-benzylpiperidine. The resulting compound, 32, is a potent and selective antagonist of CCR3. SAR studies showed that the 3-acetylphenyl urea of 32 could be replaced with heterocyclic ureas or heterocyclic-substituted phenyl ureas and still maintain the potency (inhibition of eotaxin-induced chemotaxis) of this class of compounds in the low-picomolar range (IC(50) = 10-60 pM), representing some of the most potent CCR3 antagonists reported to date. The potency of 32 for mouse CCR3 (chemotaxis IC(50) = 41 nM) and its oral bioavailability in mice (20% F ) were adequate to assess the efficacy in animal models of allergic airway inflammation. Oral administration of 32 reduced eosinophil recruitment into the lungs in a dose-dependent manner in these animal models. On the basis of its overall potency, selectivity, efficacy, and safety profile, the benzenesulfonate salt of 32, designated DPC168, entered phase I clinical trials.

Replacement of the amide functionality in IM491 (N-hydroxy-(5S,6S)-1-methyl-6-[[4-(2-methyl-4-quinolinylmethoxy)anilinyl]carbonyl]5-piperidinecarboxamide) with a sulfonyl group led to a new series of alpha,beta-cyclic and beta,beta-cyclic gamma-sulfonyl hydroxamic acids, which were potent TNF-alpha converting enzyme (TACE) inhibitors. Among them, inhibitor 4b (N-hydroxy-(4S,5S)-1-methyl-5-[[4-(2-methyl-4-quinolinylmethoxy)phenyl]sulfonylmethyl]-4-pyrrolidinecarboxamide) exhibited IC50 values of < 1 nM and 180 nM in porcine TACE (pTACE) and cell assays, respectively, with excellent selectivity over MMP-1, -2, -9 and -13 and was orally bioavailable with an F value of 46% in mice.

Purpose: Semaphorin 4A (Sema4A) is a member of the transmembrane class 4 family of semaphorins. It has recently been shown to participate in cell-cell communication in the immune system. High levels of sema4A are also present in brain and eye, but its function in the central nervous system has not been studied. To investigate the function of Sema4A, we generated mice deficient in this transmembrane signaling molecule., Methods: An embryonic stem (ES) cell clone with a retroviral gene-trap insertion in the sema4A gene was used to generate mice lacking this transmembrane semaphorin. Fundus photography, fluorescein angiography, and electroretinography were used to evaluate retinal anatomy and physiology in mice lacking Sema4A. Electron microscopy and immunohistochemistry with cell-type-specific markers were used to characterize retinal development. In situ hybridization with sema4A-specific riboprobes was used to localize expression of this gene in the developing and adult eye., Results: Fundus photography performed at 14 weeks of age revealed severe retinal degeneration, attenuated retinal vessels, and depigmentation in mice lacking Sema4A. At this age, the outer nuclear layer was reduced to a single row of photoreceptor cells, and the outer plexiform layer was thin and disorganized. Disruption of Sema4A also compromised the physiological function of both rod and cone photoreceptors. Developmental studies in Sema4A-deficient mice revealed abnormal morphology of photoreceptor outer segments during the time at which they establish contacts with apical microvilli of the retinal pigment epithelium (RPE). Sema4A is expressed in the inner retina and RPE during the time at which photoreceptor outer segments elongate., Conclusions: These findings identify a previously unknown function of Sema4A in the developing visual system and provide a useful model for understanding cell-cell interactions that occur between photoreceptors and the RPE.

In this communication we describe the design, synthesis, and evaluation of novel sultam hydroxamates 4 as MMP-2, -9, and -13 inhibitors. Compound 26 was found to be an active inhibitor (MMP-2 IC(50) = 1 nM) with 1000-fold selectivity over MMP-1 and good oral bioavailability (F = 43%) in mouse. An X-ray crystal structure of 26 in MMP-13 confirms the key hydrogen bonds and prime side binding in the active site.

The discovery of novel and selective small molecule antagonists of the CC Chemokine Receptor-3 (CCR3) is presented. Simple conversion from a 4- to 3-benzylpiperidine gave improved selectivity for CCR3 over the serotonin 5HT(2A) receptor. Chiral resolution and exploration of mono- and disubstitution of the N-propylurea resulted in several 3-benzylpiperidine N-propylureas with CCR3 binding IC(50)s under 5 nM. Data from in vitro calcium mobilization and chemotaxis assays for these compounds ranged from high picomolar to low nanomolar EC(50)s and correlated well with antagonist binding IC(50)s.

Modifications of the lead TACE inhibitor 1 (N-hydroxy-trans-2-[[4-(4-quinolinyloxymethyl)anilinyl]carbonyl]-1-cyclohexanecarboxamide) at the cyclohexyl ring and the quinoline moiety led to the identification of a series of piperidine containing TACE inhibitors with potent activity in the inhibition of TNF-alpha release in the whole blood assay (WBA). The most potent analogue IM491 [N-hydroxy-(5S,6S)-1-methyl-6-[[4-(2-methyl-4-quinolinylmethoxy)anilinyl]carbonyl]-5-piperidinecarboxamide] exhibited an IC(50) value of 20 nM in WBA with excellent selectivity over MMP-1, -2 and -9 and is orally bioavailable with an F value of 43% in beagle dogs.

Rational design based on the broad spectrum MMP inhibitor CGS 27023A led to the identification of a novel series of cyclic succinate TACE inhibitors. As a mixture of two enantiomers, the lead compound 17b exhibited potent enzyme activity (IC(50)=8 nM) in the inhibition of porcine TNF-alpha converting enzyme (pTACE) and excellent selectivity over aggrecanase and MMP-1, -2 and -9.

The matrix components responsible for cartilage mechanical properties, type II collagen and aggrecan, are degraded in osteoarthritis through proteolytic cleavage by matrix metalloproteinases (MMPs) and aggrecanases, respectively. We now show that aggrecan may serve to protect cartilage collagen from degradation. Although collagen in freeze-thawed cartilage depleted of aggrecan was completely degraded following incubation with MMP-1, collagen in cartilage with intact aggrecan was not. Using interleukin-1-stimulated bovine nasal cartilage explants where aggrecan depletion occurs during the first week of culture, followed by collagen loss during the second week, we evaluated the effect of selective MMP and aggrecanase inhibitors on degradation. A selective MMP inhibitor did not block aggrecan degradation but caused complete inhibition of collagen breakdown. Similar inhibition was seen with inhibitor addition following aggrecan depletion on day 6-8, suggesting that MMPs are not causing significant collagen degradation prior to the second week of culture. Inclusion of a selective aggrecanase inhibitor blocked aggrecan degradation, and, in addition, inhibited collagen degradation. When the inhibitor was introduced following aggrecan depletion, it had no effect on collagen breakdown, ruling out a direct effect through inhibition of collagenase. These data suggest that aggrecan plays a protective role in preventing degradation of collagen fibrils, and that an aggrecanase inhibitor may impart overall cartilage protection.

New inhibitors of tumor necrosis factor-alpha converting enzyme (TACE) were discovered using an N-hydroxy-2-(2-oxo-3-pyrrolidinyl)acetamide scaffold. The series was found to be potent in a porcine TACE (pTACE) assay with IC(50)s typically below 5 nM. For most compounds, selectivity for pTACE relative to MMP-1,-2, and -9 is at least 300-fold. Compound 2o was potent in inhibition of TNFalpha production in a human whole blood assay (WBA) with an IC(50) of 0.42 micro M.

Elevated levels of tumor necrosis factor-alpha (TNF-alpha) have been associated with several inflammatory diseases, and therefore, strategies for its suppression have become important targets in drug discovery. Our efforts to suppress TNF-alpha have centered on the inhibition of TNF-alpha converting enzyme (TACE) through the use of hydroxamate inhibitors. Starting from broad-spectrum matrix metalloproteinase (MMP) inhibitors, we have designed and synthesized novel benzothiadiazepines as potent and selective TACE inhibitors. The benzothiadiazepines were synthesized with variation in P1 and P1' in order to effect potency and selectivity. The inhibitors were evaluated versus porcine TACE (pTACE), and the initial selectivity was assessed with counterscreens of MMP-1, -2, and -9. Several potent and selective inhibitors were discovered with compound 41 being the most active against pTACE (K(i) = 5 nM) while still maintaining good selectivity versus the MMP's (at least 75-fold). Most compounds were assessed in the human peripheral blood mononuclear cell assay (PBMC) and the human whole blood assay (WBA) to determine their ability to suppress TNF-alpha. Compound 32 was the most potent compound in the PBMC assay (IC(50) = 0.35 microM), while compound 62 was the most active in the WBA (IC(50) = 1.4 microM).

Anti-succinate hydroxamates with cyclic P1 motifs were synthesized as aggrecanase inhibitors. The N-methanesulfonyl piperidine 23 and the N-trifluoroacetyl azetidine 26 were the most potent aggrecanase inhibitors both having an IC(50)=3nM while maintaining >100-fold selectivity over MMP-1, -2, and -9. The cyclic moieties were also capable of altering in vivo metabolism, hence delivering low clearance compounds in both rat and dog studies as shown for compound 14.

Objective: To study the production of aggrecanase 1 (ADAM-TS4) in monolayer chondrocytes, capsular fibroblasts, and cartilage., Methods: Bovine nasal and articular cartilage, monolayer chondrocytes, and capsular fibroblasts were incubated in the absence and presence of interleukin-1 (IL-1). ADAM-TS4 production was evaluated by immunofluorescence or by Western blot analysis. Aggrecanase activity was measured in cells grown on an immobilized peptide substrate, and peptide cleavage was monitored by enzyme-linked immunosorbent assay., Results: There was constitutive production of ADAM-TS4 in both cells and tissue. The protein was associated with the extracellular matrix based on the observation that the staining could be reduced following treatment of chondrocytes with heparin or exposure to chondroitinase ABC. Interestingly, there was no detectable change in the abundance of ADAM-TS4 in response to IL-1. Western blot analysis of cell lysates from IL-1-stimulated chondrocytes showed no evidence of increased ADAM-TS4 production, but resulted in activation of ADAM-TS4. The activation was associated with an increased generation in the aggrecanase neoepitope NITEGE in nasal cartilage in response to IL-1. These data suggest that induction of aggrecanase activity both in cells and in cartilage by IL-1 may involve the stimulation of an activator of ADAM-TS4. Consistent with this observation, culture of chondrocytes on a solid support containing a peptide substrate resulted in the generation of aggrecanase-mediated cleavage that could be blocked by selective inhibitors of ADAM-TS4., Conclusion: These data support the hypothesis that ADAM-TS4 is constitutively produced in these cells and tissue, and that stimulation by IL-1 results in aggrecanase activation. Thus, the activator could be a potential target by which to control aggrecanase-mediated degradation in arthritic diseases.

New gamma-lactam TACE inhibitors were designed from known MMP inhibitors. A homology model of TACE was built and examined to identify the S1' site as the key area for TACE selectivity over MMPs. Rational exploration of the P1'-S1' interactions resulted in the discovery of the 3,5-disubstituted benzyl ether as a TACE-selective P1' group. Further optimization led to the discovery of IK682 as a selective and orally bioavailable TACE inhibitor.

ADAM-TS5 (aggrecanase 2), one of two cartilage aggrecanases is a member of the ADAM protein family. Like ADAM-TS4 (aggrecanase 1) the enzyme cleaves cartilage aggrecan at the Glu(373)-Ala(374) bond, a marker of aggrecanase activity. In this study we have characterized the substrate specificity of ADAM-TS5 and compared it with that of ADAM-TS4. The recombinant human ADAM-TS5, like ADAM-TS4 cleaves aggrecan at Glu(1480)-Gly(1481), Glu(1667)-Gly(1668), Glu(1771)-Ala(1772) and Glu(1871)-Leu(1872) bonds more readily than at the Glu(373)-Ala(374) bond. In addition, ADAM-TS5 exhibited an additional site of cleavage in the region spanning residues Gly(1481) and Glu(1667), representing a unique cleavage of ADAM-TS5. ADAM-TS5 cleaved aggrecan approximately 2-fold slower than ADAM-TS4. Neither ADAM-TS5 nor ADAM-TS4 was able to cleave the extracellular matrix proteins fibronectin, thrombospondin, type I collagen, type II collagen, gelatin or general protein substrates such as casein and transferrin. Finally, the zymogen of stromelysin (MMP-3) was not activated by either ADAM-TS4 or ADAM-TS5., (Copyright 2002 Elsevier Science B.V. and International Society of Matrix Biology)

Structure-activity relationship (SAR) studies of initial screening hits from our corporate library of compounds and a structurally related series of CCR1 receptor antagonists were used to determine that an N-(alkyl)benzylpiperidine is an essential pharmacophore for selective CCR3 antagonists. Further SAR studies that introduced N-(ureidoalkyl) substituents improved the binding potency of these compounds from the micromolar to the low nanomolar range. This new series of compounds also displays highly potent, in vitro functional CCR3-mediated antagonism of eotaxin-induced Ca(2+) mobilization and chemotaxis of human eosinophils.

CCR3 antagonist leads with IC(50) values in the microM range were converted into low nM binding compounds that displayed in vitro inhibition of human eosinophil chemotaxis induced by human eotaxin. In particular, 4-benzylpiperidin-1-yl-n-propylureas and erythro-3-(4-benzyl-2-(alpha-hydroxyalkyl)piperidin-1-yl)-n-propylureas (obtained via Beak reaction of N-BOC-4-benzylpiperidine) exhibited single digit nanomolar IC(50) values for CCR3.