IL-33 Expression Is Lower in Current Smokers at both Transcriptomic and Protein Levels.

Rationale: IL-33 is a proinflammatory cytokine thought to play a role in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). A recent clinical trial using an anti-IL-33 antibody showed a reduction in exacerbation and improved lung function in ex-smokers but not current smokers with COPD. Objectives: This study aimed to understand the effects of smoking status on IL-33. Methods: We investigated the association of smoking status with the level of gene expression of IL-33 in the airways in eight independent transcriptomic studies of lung airways. Additionally, we performed Western blot analysis and immunohistochemistry for IL-33 in lung tissue to assess protein levels. Measurements and Main Results: Across the bulk RNA-sequencing datasets, IL-33 gene expression and its signaling pathway were significantly lower in current versus former or never-smokers and increased upon smoking cessation (P < 0.05). Single-cell sequencing showed that IL-33 is predominantly expressed in resting basal epithelial cells and decreases during the differentiation process triggered by smoke exposure. We also found a higher transitioning of this cellular subpopulation into a more differentiated cell type during chronic smoking, potentially driving the reduction of IL-33. Protein analysis demonstrated lower IL-33 levels in lung tissue from current versus former smokers with COPD and a lower proportion of IL-33-positive basal cells in current versus ex-smoking controls. Conclusions: We provide strong evidence that cigarette smoke leads to an overall reduction in IL-33 expression in transcriptomic and protein level, and this may be due to the decrease in resting basal cells. Together, these findings may explain the clinical observation that a recent antibody-based anti-IL-33 treatment is more effective in former than current smokers with COPD.

Efficacy and Safety of Itepekimab in Patients with Moderate-to-Severe Asthma.

BACKGROUND: Monoclonal antibodies targeting IgE, interleukin-4 and -13, and interleukin-5 are effective in treating severe type 2 asthma, but new targets are needed. Itepekimab is a new monoclonal antibody against the upstream alarmin interleukin-33. The efficacy and safety of itepekimab as monotherapy, as well as in combination with dupilumab, in patients with asthma are unclear. METHODS: In a phase 2 trial, we randomly assigned, in a 1:1:1:1 ratio, adults with moderate-to-severe asthma receiving inhaled glucocorticoids plus long-acting beta-agonists (LABAs) to receive subcutaneous itepekimab (at a dose of 300 mg), itepekimab plus dupilumab (both at 300 mg; combination therapy), dupilumab (300 mg), or placebo every 2 weeks for 12 weeks. After randomization, LABA was discontinued at week 4, and inhaled glucocorticoids were tapered over weeks 6 through 9. The primary end point was an event indicating a loss of asthma control, assessed in the itepekimab group and the combination group, as compared with the placebo group. Secondary and other end points included lung function, asthma control, quality of life, type 2 biomarkers, and safety. RESULTS: A total of 296 patients underwent randomization. By 12 weeks, an event indicating a loss of asthma control occurred in 22% of the patients in the itepekimab group, 27% of those in the combination group, and 19% of those in the dupilumab group, as compared with 41% of those in the placebo group; the corresponding odds ratios as compared with placebo were as follows: in the itepekimab group, 0.42 (95% confidence interval [CI], 0.20 to 0.88; P = 0.02); in the combination group, 0.52 (95% CI, 0.26 to 1.06; P = 0.07); and in the dupilumab group, 0.33 (95% CI, 0.15 to 0.70). As compared with placebo, the forced expiratory volume in 1 second before bronchodilator use increased with the itepekimab and dupilumab monotherapies but not with the combination therapy. Itepekimab treatment improved asthma control and quality of life, as compared with placebo, and led to a greater reduction in the mean blood eosinophil count. The incidence of adverse events was similar in all four trial groups. CONCLUSIONS: Interleukin-33 blockade with itepekimab led to a lower incidence of events indicating a loss of asthma control than placebo and improved lung function in patients with moderate-to-severe asthma. (Funded by Sanofi and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT03387852.).

Safety and efficacy of itepekimab in patients with moderate-to-severe COPD: a genetic association study and randomised, double-blind, phase 2a trial.

BACKGROUND: Genetic data implicate IL-33 in asthma susceptibility. Itepekimab, a monoclonal antibody targeting IL-33, demonstrated clinical activity in asthma, with potential in chronic obstructive pulmonary disease (COPD). In this study we first aimed to test the hypothesis that genetic variants in the IL-33 pathway were also associated with COPD. On the basis of the strong association of IL-33 pathway genes with pulmonary diseases like asthma and COPD, we conducted this phase 2a trial to assess the safety and efficacy of itepekimab in patients with moderate-to-severe COPD on a stable regimen of triple-inhaled or double-inhaled background maintenance therapy. METHODS: In this two-part study, genetic analyses of loss-of-function and gain-of-function variants in the IL-33 pathway, previously associated with asthma risk, were initially characterised for COPD. We then did a double-blind, phase 2a trial comparing itepekimab with placebo in patients with moderate-to-severe COPD despite standard therapy, at 83 study sites in ten countries. Patients aged 40-75 years who were current or former smokers, had been diagnosed with COPD for at least 1 year, and were on a stable regimen of triple-inhaled or double-inhaled background maintenance therapy, were randomly assigned (1:1) to receive itepekimab 300 mg or placebo, administered as two subcutaneous injections every 2 weeks for 24-52 weeks. The primary endpoint of the phase 2a trial was annualised rate of moderate-to-severe acute exacerbations of COPD during the treatment period. The key secondary outcome was change in prebronchodilator FEV1 from baseline to weeks 16-24. Prespecified subgroup analyses were done for each of the endpoints, including by smoking status. Efficacy and safety analyses were done in all participants who received at least one dose of assigned treatment (modified intention-to-treat population). This trial is registered at ClinicalTrials.gov (NCT03546907). FINDINGS: Genetic analyses demonstrated association of loss of function in IL33 with reduced COPD risk, and gain of function in IL33 and IL1RL1 variants with increased risk. Subsequent to this, in the phase 2 trial, 343 patients were randomly assigned to placebo (n=171) or itepekimab (n=172) from July 16, 2018, to Feb 19, 2020. Annualised rates of acute exacerbations of COPD were 1·61 (95% CI 1·32-1·97) in the placebo group and 1·30 (1·05-1·61) in the itepekimab group (relative risk [RR] 0·81 [95% CI 0·61-1·07], p=0·13), and least squares mean prebronchodilator FEV1 change from baseline to weeks 16-24 was 0·0 L (SD 0·02) and 0·06 L (0·02; difference 0·06 L [95% CI 0·01-0·10], p=0·024). When analysis was restricted to former smokers, treatment with itepekimab was associated with nominally significant reductions in acute exacerbations of COPD (RR 0·58 [95% CI 0·39-0·85], p=0·0061) and FEV1 improvement (least squares mean difference 0·09 L [0·02-0·15], p=0·0076) compared with placebo. Current smokers treated with itepekimab showed no treatment benefit versus placebo for exacerbations (RR 1·09 [0·74-1·61], p=0·65) or FEV1 (least squares mean difference 0·02 [-0·05 to 0·09], p=0·54). Treatment-emergent adverse events (TEAEs) occurred in 135 (78%) patients in the itepekimab group and 136 (80%) in the placebo group. The most common TEAEs were nasopharyngitis (28 [16%] in the itepekimab group vs 29 [17%] in the placebo group), bronchitis (18 [10%] vs 14 [8%]), headache (14 [8%] vs 23 [13%]), and upper respiratory tract infection (13 [8%] vs 15 [9%]). INTERPRETATION: The primary endpoint in the overall population was not met, subgroup analysis showed that itepekimab reduced exacerbation rate and improved lung function in former smokers with COPD. Two phase 3 clinical studies are ongoing to confirm the efficacy and safety profile of itepekimab in former smokers with COPD. FUNDING: Sanofi and Regeneron Pharmaceuticals.

Discovery and Pharmacokinetic and Pharmacological Properties of the Potent and Selective MET Kinase Inhibitor 1-{6-[6-(4-Fluorophenyl)-[1,2,4]triazolo[4,3-b]pyridazin-3-ylsulfanyl]benzothiazol-2-yl}-3-(2-morpholin-4-ylethyl)urea (SAR125844).

The HGF/MET pathway is frequently activated in a variety of cancer types. Several selective small molecule inhibitors of the MET kinase are currently in clinical evaluation, in particular for NSCLC, liver, and gastric cancer patients. We report herein the discovery of a series of triazolopyridazines that are selective inhibitors of wild-type (WT) MET kinase and several clinically relevant mutants. We provide insight into their mode of binding and report unprecedented crystal structures of the Y1230H variant. A multiparametric chemical optimization approach allowed the identification of compound 12 (SAR125844) as a development candidate. In this chemical series, absence of CYP3A4 inhibition was obtained at the expense of satisfactory oral absorption. Compound 12, a promising parenteral agent for the treatment of MET-dependent cancers, promoted sustained target engagement at tolerated doses in a human xenograft tumor model. Preclinical pharmacokinetics conducted in several species were predictive for the observed pharmacokinetic behavior of 12 in cancer patients.

Discovery of (2S)-8-[(3R)-3-methylmorpholin-4-yl]-1-(3-methyl-2-oxobutyl)-2-(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one: a novel potent and selective inhibitor of Vps34 for the treatment of solid tumors.

Vps34 (the human class III phosphoinositide 3-kinase) is a lipid kinase involved in vesicle trafficking and autophagy and therefore constitutes an interesting target for cancer treatment. Because of the lack of specific Vps34 kinase inhibitors, we aimed to identify such compounds to further validate the role of this lipid kinase in cancer maintenance and progression. Herein, we report the discovery of a series of tetrahydropyrimidopyrimidinone derivatives. Starting with hit compound 1a, medicinal chemistry optimization led to compound 31. This molecule displays potent activity, an exquisite selectivity for Vps34 with excellent properties. The X-ray crystal structure of compound 31 in human Vps34 illustrates how the unique molecular features of the morpholine synthon bestows selectivity against class I PI3Ks. This molecule exhibits suitable in vivo mouse PK parameters and induces a sustained inhibition of Vps34 upon acute administration. Compound 31 constitutes an optimized Vps34 inhibitor that could be used to investigate human cancer biology.

The selective intravenous inhibitor of the MET tyrosine kinase SAR125844 inhibits tumor growth in MET-amplified cancer.

Activation of the MET/HGF pathway is common in human cancer and is thought to promote tumor initiation, metastasis, angiogenesis, and resistance to diverse therapies. We report here the pharmacologic characterization of the triazolopyridazine derivative SAR125844, a potent and highly selective inhibitor of the MET receptor tyrosine kinase (RTK), for intravenous administration. SAR125844 displayed nanomolar activity against the wild-type kinase (IC50 value of 4.2 nmol/L) and the M1250T and Y1235D mutants. Broad biochemical profiling revealed that SAR125844 was highly selective for MET kinase. SAR125844 inhibits MET autophosphorylation in cell-based assays in the nanomolar range, and promotes low nanomolar proapoptotic and antiproliferative activities selectively in cell lines with MET gene amplification or pathway addiction. In two MET-amplified human gastric tumor xenograft models, SNU-5 and Hs 746T, intravenous treatment with SAR125844 leads to potent, dose- and time-dependent inhibition of the MET kinase and to significant impact on downstream PI3K/AKT and RAS/MAPK pathways. Long duration of MET kinase inhibition up to 7 days was achieved with a nanosuspension formulation of SAR125844. Daily or every-2-days intravenous treatment of SAR125844 promoted a dose-dependent tumor regression in MET-amplified human gastric cancer models at tolerated doses without treatment-related body weight loss. Our data demonstrated that SAR125844 is a potent and selective MET kinase inhibitor with a favorable preclinical toxicity profile, supporting its clinical development in patients with MET-amplified and MET pathway-addicted tumors.

A highly potent and selective Vps34 inhibitor alters vesicle trafficking and autophagy.

Vps34 is a phosphoinositide 3-kinase (PI3K) class III isoform that has attracted major attention over the recent years because of its role in autophagy. Herein we describe the biological characterization of SAR405, which is a low-molecular-mass kinase inhibitor of Vps34 (KD 1.5 nM). This compound has an exquisite protein and lipid kinase selectivity profile that is explained by its unique binding mode and molecular interactions within the ATP binding cleft of human Vps34. To the best of our knowledge, this is the first potent and specific Vps34 inhibitor described so far. Our results demonstrate that inhibition of Vps34 kinase activity by SAR405 affects both late endosome-lysosome compartments and prevents autophagy. Moreover, we show that the concomitant inhibition of Vps34 and mTOR, with SAR405 and the US Food and Drug Administration-approved mTOR inhibitor everolimus, results in synergistic antiproliferative activity in renal tumor cell lines, indicating a potential clinical application in cancer.

MET genetic abnormalities unreliable for patient selection for therapeutic intervention in oropharyngeal squamous cell carcinoma.

BACKGROUND: Identification of MET genetic alteration, mutation, or amplification in oropharyngeal squamous cell carcinoma (OPSCC) could lead to development of MET selective kinase inhibitors. The aim of this study was to assess the frequency and prognostic value of MET gene mutation, amplification, and protein expression in primary OPSCC. METHODS: A retrospective chart review was conducted of patients treated for single primary OPSCC between January 2007 and December 2009. Pre-treatment OPSCC tissue samples were analyzed for MET mutations, gene amplification, and overexpression using Sanger sequencing, FISH analysis, and immunohistochemistry respectively. Univariate and multivariate analyses were used to analyze correlations between molecular abnormalities and patient survival. RESULTS: 143 patients were included in this study. Six cases (4%) were identified that had a genetic variation, but previously described mutations such as p.Tyr1235Asp (Y1235D) or p.Tyr1230Cys (Y1230C) were not detected. There were 15 high polysomy cases, and only 3 cases met the criteria for true MET amplification, with ≥10% amplified cells per case. Immunohistochemistry evaluation showed 43% of cases were c-MET negative and in 57% c-MET was observed at the tumor cell level. Multivariate analysis showed no significant association between MET mutation, amplification, or expression and survival. CONCLUSIONS: Our study shows a low frequency of MET mutations and amplification in this cohort of OPSCC. There was no significant correlation between MET mutations, amplification, or expression and patient survival. These results suggest that patient selection based on these MET genetic abnormalities may not be a reliable strategy for therapeutic intervention in OPSCC.

Tricyclic series of heat shock protein 90 (Hsp90) inhibitors part I: discovery of tricyclic imidazo[4,5-c]pyridines as potent inhibitors of the Hsp90 molecular chaperone.

A novel class of heat shock protein 90 (Hsp90) inhibitors was developed after a low throughput screen (LTS) of a focused library containing approximately 21K compounds selected by virtual screening. The initial [1-{3-H-imidazo[4-5-c]pyridin-2-yl}-3,4-dihydro-2H-pyrido[2,1-a]isoindole-6-one] (1) compound showed moderate activity (IC(50) = 7.6 µM on Hsp82, the yeast homologue of Hsp90). A high-resolution X-ray structure shows that compound 1 binds into an "induced" hydrophobic pocket, 10-15 Å away from the ATP/resorcinol binding site. Iterative cycles of structure-based drug design (SBDD) and chemical synthesis led to the design and preparation of analogues with improved affinity. These optimized molecules make productive interactions within the ATP binding site as reported by other Hsp90 inhibitors. This resulted in compound 8, which is a highly potent inhibitor in biochemical and cellular assays (K(d) = 0.35 nM on Hsp90; IC(50) = 30 nM on SKBr3 mammary carcinoma cells) and in an in vivo leukemia model.