Frédéric Baribaud

Discovery and Pharmacological Characterization of a Novel Rodent-Active CCR2 Antagonist, INCB3344

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 (IC50 = 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 disease module in the interactome explains disease heterogeneity, drug response and captures novel pathways and genes in asthma

Recent advances in genetics have spurred rapid progress towards the systematic identification of genes involved in complex diseases. Still, the detailed understanding of the molecular and physiological mechanisms through which these genes affect disease phenotypes remains a major challenge. Here, we identify the asthma disease module, i.e. the local neighborhood of the interactome whose perturbation is associated with asthma, and validate it for functional and pathophysiological relevance, using both computational and experimental approaches. We find that the asthma disease module is enriched with modest GWAS P-values against the background of random variation, and with differentially expressed genes from normal and asthmatic fibroblast cells treated with an asthma-specific drug. The asthma module also contains immune response mechanisms that are shared with other immune-related disease modules. Further, using diverse omics (genomics, gene-expression, drug response) data, we identify the GAB1 signaling pathway as an important novel modulator in asthma. The wiring diagram of the uncovered asthma module suggests a relatively close link between GAB1 and glucocorticoids (GCs), which we experimentally validate, observing an increase in the level of GAB1 after GC treatment in BEAS-2B bronchial epithelial cells. The siRNA knockdown of GAB1 in the BEAS-2B cell line resulted in a decrease in the NFkB level, suggesting a novel regulatory path of the pro-inflammatory factor NFkB by GAB1 in asthma.

Gene Expression Profiling and Response Signatures Associated With Differential Responses to Infliximab Treatment in Ulcerative Colitis

OBJECTIVES:Infliximab has been shown to induce clinical response and remission in ulcerative colitis (UC). To characterize the biological response of patients to infliximab, we analyzed the mRNA expression patterns of mucosal colonic biopsies taken from UC patients enrolled in the Active Ulcerative Colitis Trial 1 (ACT1) study.METHODS:Biopsies were obtained from 48 UC patients before treatment with 5 or 10 mg/kg infliximab, and at 8 and 30 weeks after treatment (n=113 biopsies). Global gene expression profiling was performed using Affimetrix GeneChip Human Genome U133 Plus 2.0 arrays. Expression profiling results for selected genes were confirmed using qPCR.RESULTS:Infliximab had a significant effect on mRNA expression in treatment responders, with both infliximab dose and duration of treatment having an effect. Genes affected are primarily involved with inflammatory response, cell-mediated immune responses, and cell-to-cell signaling. Unlike responders, non-responders do not effectively modulate TH1, TH2, and TH17 pathways. Gene expression can differentiate placebo and infliximab responders.CONCLUSIONS:Analysis of mRNA expression in mucosal biopsies following infliximab treatment provided insight into the response to therapy and molecular mechanisms of non-response.

A Transcriptome-driven Analysis of Epithelial Brushings and Bronchial Biopsies to Define Asthma Phenotypes in U-BIOPRED

Rationale: Asthma is a heterogeneous disease driven by diverse immunologic and inflammatory mechanisms. Objectives: Using transcriptomic profiling of airway tissues, we sought to define the molecular phenotypes of severe asthma. Methods: The transcriptome derived from bronchial biopsies and epithelial brushings of 107 subjects with moderate to severe asthma were annotated by gene set variation analysis using 42 gene signatures relevant to asthma, inflammation, and immune function. Topological data analysis of clinical and histologic data was performed to derive clusters, and the nearest shrunken centroid algorithm was used for signature refinement. Measurements and Main Results: Nine gene set variation analysis signatures expressed in bronchial biopsies and airway epithelial brushings distinguished two distinct asthma subtypes associated with high expression of T‐helper cell type 2 cytokines and lack of corticosteroid response (group 1 and group 3). Group 1 had the highest submucosal eosinophils, as well as high fractional exhaled nitric oxide levels, exacerbation rates, and oral corticosteroid use, whereas group 3 patients showed the highest levels of sputum eosinophils and had a high body mass index. In contrast, group 2 and group 4 patients had an 86% and 64% probability, respectively, of having noneosinophilic inflammation. Using machine learning tools, we describe an inference scheme using the currently available inflammatory biomarkers sputum eosinophilia and fractional exhaled nitric oxide levels, along with oral corticosteroid use, that could predict the subtypes of gene expression within bronchial biopsies and epithelial cells with good sensitivity and specificity. Conclusions: This analysis demonstrates the usefulness of a transcriptomics‐driven approach to phenotyping that segments patients who may benefit the most from specific agents that target T‐helper cell type 2‐mediated inflammation and/or corticosteroid insensitivity.

Genes associated with intestinal permeability in ulcerative colitis: Changes in expression following infliximab therapy†

Background: Alterations in intestinal permeability have been implicated in ulcerative colitis (UC). Infliximab, a monoclonal anti‐tumor necrosis factor alpha (TNF&agr;) antibody, can induce clinical response in UC. Gene expression in colonic biopsies taken from responders and nonresponders to infliximab can provide insight into the mechanisms of the altered intestinal permeability at a molecular level. Methods: Colonic biopsies (n = 18 anti‐TNF&agr; naïve UC patients; n = 8 normal controls; n = 80 Active Ulcerative Colitis Trial [ACT] 1 patients) were analyzed for mRNA expression using gene expression microarrays. Computational reverse causal reasoning was applied to build causal network models of UC and response and nonresponse of UC to treatment. Quantitative reverse‐transcription polymerase chain reaction (qPCR) was used to confirm differentially expressed genes. Results: Reverse causal reasoning on mRNA expression data from anti‐TNF&agr;‐naïve UC and normal samples provided a mechanistic disease model of the biology of gene expression observed in UC. mRNA expression data from the ACT 1 study enabled construction of a mechanistic model describing the biology of nonresponders to infliximab, including evidence for increased intestinal permeability compared with normal and responder samples. Gene expression changes identified as central to intestinal permeability dysregulation were confirmed in normal, UC, and infliximab‐treated patients by qPCR analysis. Gene expression returned toward normal levels in infliximab responders, but not in nonresponders. Conclusion: Gene expression analysis and causal network modeling in combination showed that aberrant mRNA expression of genes involved in intestinal epithelial permeability for infliximab responders was restored toward levels observed in normal samples. Infliximab nonresponders showed no equivalent restoration in the expression of these genes. (Inflamm Bowel Dis 2012)

Validated and longitudinally stable asthma phenotypes based on cluster analysis of the ADEPT study.

BackgroundAsthma is a disease of varying severity and differing disease mechanisms. To date, studies aimed at stratifying asthma into clinically useful phenotypes have produced a number of phenotypes that have yet to be assessed for stability and to be validated in independent cohorts. The aim of this study was to define and validate, for the first time ever, clinically driven asthma phenotypes using two independent, severe asthma cohorts: ADEPT and U-BIOPRED.MethodsFuzzy partition-around-medoid clustering was performed on pre-specified data from the ADEPT participants (n = 156) and independently on data from a subset of U-BIOPRED asthma participants (n = 82) for whom the same variables were available. Models for cluster classification probabilities were derived and applied to the 12-month longitudinal ADEPT data and to a larger subset of the U-BIOPRED asthma dataset (n = 397). High and low type-2 inflammation phenotypes were defined as high or low Th2 activity, indicated by endobronchial biopsies gene expression changes downstream of IL-4 or IL-13.ResultsFour phenotypes were identified in the ADEPT (training) cohort, with distinct clinical and biomarker profiles. Phenotype 1 was “mild, good lung function, early onset”, with a low-inflammatory, predominantly Type-2, phenotype. Phenotype 2 had a “moderate, hyper-responsive, eosinophilic” phenotype, with moderate asthma control, mild airflow obstruction and predominant Type-2 inflammation. Phenotype 3 had a “mixed severity, predominantly fixed obstructive, non-eosinophilic and neutrophilic” phenotype, with moderate asthma control and low Type-2 inflammation. Phenotype 4 had a “severe uncontrolled, severe reversible obstruction, mixed granulocytic” phenotype, with moderate Type-2 inflammation. These phenotypes had good longitudinal stability in the ADEPT cohort. They were reproduced and demonstrated high classification probability in two subsets of the U-BIOPRED asthma cohort.ConclusionsFocusing on the biology of the four clinical independently-validated easy-to-assess ADEPT asthma phenotypes will help understanding the unmet need and will aid in developing tailored therapies.Trial registrationNCT01274507 (ADEPT), registered October 28, 2010 and NCT01982162 (U-BIOPRED), registered October 30, 2013.

Pharmacological characterization of INCB3344, a small molecule antagonist of human CCR2

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.

Severe asthma exists despite suppressed tissue inflammation: findings of the U-BIOPRED study

The U-BIOPRED study is a multicentre European study aimed at a better understanding of severe asthma. It included three steroid-treated adult asthma groups (severe nonsmokers (SAn group), severe current/ex-smokers (SAs/ex group) and those with mild–moderate disease (MMA group)) and healthy controls (HC group). The aim of this cross-sectional, bronchoscopy substudy was to compare bronchial immunopathology between these groups. In 158 participants, bronchial biopsies and bronchial epithelial brushings were collected for immunopathologic and transcriptomic analysis. Immunohistochemical analysis of glycol methacrylate resin-embedded biopsies showed there were more mast cells in submucosa of the HC group (33.6 mm−2) compared with both severe asthma groups (SAn: 17.4 mm−2, p<0.001; SAs/ex: 22.2 mm−2, p=0.01) and with the MMA group (21.2 mm−2, p=0.01). The number of CD4+ lymphocytes was decreased in the SAs/ex group (4.7 mm−2) compared with the SAn (11.6 mm−2, p=0.002), MMA (10.1 mm−2, p=0.008) and HC (10.6 mm−2, p<0.001) groups. No other differences were observed. Affymetrix microarray analysis identified seven probe sets in the bronchial brushing samples that had a positive relationship with submucosal eosinophils. These mapped to COX-2 (cyclo-oxygenase-2), ADAM-7 (disintegrin and metalloproteinase domain-containing protein 7), SLCO1A2 (solute carrier organic anion transporter family member 1A2), TMEFF2 (transmembrane protein with epidermal growth factor like and two follistatin like domains 2) and TRPM-1 (transient receptor potential cation channel subfamily M member 1); the remaining two are unnamed. We conclude that in nonsmoking and smoking patients on currently recommended therapy, severe asthma exists despite suppressed tissue inflammation within the proximal airway wall. Severe asthma exists despite suppressed tissue inflammation in proximal airways when on current recommended therapy http://ow.ly/1rb6303haKP

Systemic inflammatory profile and response to anti-tumor necrosis factor therapy in chronic obstructive pulmonary disease

BackgroundChronic obstructive pulmonary disease (COPD) is characterized by progressive worsening of airflow limitation associated with abnormally inflamed airways in older smokers. Despite correlative evidence for a role for tumor necrosis factor-alpha in the pathogenesis of COPD, the anti-tumor necrosis factor-alpha, infliximab did not show clinical efficacy in a double-blind, placebo-controlled, phase II clinical trial. This study sought to evaluate the systemic inflammatory profile associated with COPD and to assess the impact of tumor necrosis factor neutralization on systemic inflammation.MethodsSerum samples (n = 234) from the phase II trial were collected at baseline and after 24 weeks of placebo or infliximab. Additionally, baseline serum samples were obtained from an independent COPD cohort (n = 160) and 2 healthy control cohorts (n = 50; n = 109). Serum concentrations of a broad panel of inflammation-associated analytes were measured using a 92-analyte multiplex assay.ResultsTwenty-five proteins were significantly elevated and 2 were decreased in COPD, including highly elevated CD40 ligand, brain-derived neurotrophic factor, epidermal growth factor, acute-phase proteins, and neutrophil-associated proteins. This profile was largely independent of smoking status, age, and clinical phenotype. The majority of these associations of serum analytes with COPD are novel findings. Increased serum creatine kinase-muscle/brain and myoglobin correlated modestly with decreased forced expiratory volume at 1 second, suggesting cardiac involvement. Infliximab did not affect this systemic inflammatory profile.ConclusionsA robust systemic inflammatory profile was associated with COPD. This profile was generally independent of disease severity. Because anti-tumor necrosis factor-alpha did not influence systemic inflammation, how to control the underlying pathology beyond symptom suppression remains unclear.Trial RegistrationClinicalTrials.gov, No.: NCT00056264.

Identification and Characterization of INCB9471, an Allosteric Noncompetitive Small-Molecule Antagonist of C-C Chemokine Receptor 5 with Potent Inhibitory Activity against Monocyte Migration and HIV-1 Infection

C-C chemokine receptor 5 (CCR5) is a clinically proven target for inhibition of HIV-1 infection and a potential target for various inflammatory diseases. In this article, we describe 5-[(4-{(3S)-4-[(1R,2R)-2-ethoxy-5-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-3-methylpiperazin-1-yl}-4-methylpiperidin-1-yl)carbonyl]-4,6-dimethylpyrimidine dihydrochloride (INCB9471), a potent and specific inhibitor of human CCR5 that has been proven to be safe and efficacious in viral load reduction in phase I and II human clinical trails. INCB9471 was identified using a primary human monocyte-based radioligand competition binding assay. It potently inhibited macrophage inflammatory protein-1β-induced monocyte migration and infection of peripheral blood mononuclear cells by a panel of R5-HIV-1 strains. The results from binding and signaling studies using incremental amounts of INCB9471 demonstrated INCB9471 as a noncompetitive CCR5 inhibitor. The CCR5 residues that are essential for interaction with INCB9471 were identified by site-specific mutagenesis studies. INCB9471 rapidly associates with but slowly dissociates from CCR5. When INCB9471 was compared with three CCR5 antagonists that had been tested in clinical trials, the potency of INCB9471 in blocking CCR5 ligand binding was similar to those of 4,6-dimethyl-5-{[4-methyl-4-((3S)-3-methyl-4-{(1R0–2-(methyloxy)-1-[4-(trifluoromethyl) phenyl]ethyl}-1-piperazingyl)-1-piperidinyl]carbonyl}pyrimidine (SCH-D; vicriviroc), 4-{[4-({(3R)-1-butyl-3-[(R)-cyclohexyl(hydroxyl)methyl]-2, 5-dioxo-1,4,9-triazaspiro[5.5]undec-9-yl}methyl)phenyl]oxy}benzoic acid hydrochloride (873140; aplaviroc), and 4,4-difluoro-N-((1S)-3-{(3-endo)-3-[3-methyl-5-(1-methylethyl)-4H-1,2,4-triazol-4-yl]-8-azabicyclo[3.2.1]oct-8-yl}-1-phenylpropyl)cyclohexanecarboxamide (UK427857; maraviroc). Its inhibitory activity against CCR5-mediated Ca2+ mobilization was also similar to those of SCH-D and 873140. Further analysis suggested that INCB9471 and UK427857 may have different binding sites on CCR5. The significance of two CCR5 antagonists with different binding sites is discussed in the context of potentially overcoming drug-resistant HIV-1 strains.