Gessica Marchini

Cigarette smoke and its component acrolein augment IL-8/CXCL8 mRNA stability via p38 MAPK/MK2 signaling in human pulmonary cells.

Interleukin-8 (IL-8/CXCL8) is an important neutrophil chemoattractant known to be elevated in the airways of cigarette smokers and in patients with chronic obstructive pulmonary disease (COPD). We examined the acute effect of aqueous cigarette smoke extract (CSE) on IL-8 expression in primary human pulmonary cells, in particular in normal human bronchial smooth muscle cells (HBSMCs). IL-8 mRNA levels increased upon CSE exposure in a concentration- and time-dependent manner, and such an effect was accompanied by IL-8 secretion. CSE-evoked elevation of IL-8 mRNA was mimicked by its component acrolein. Both CSE and acrolein induced p38 mitogen-activated protein kinase (MAPK) phosphorylation, accompanied by the phosphorylation of MAPK-activated kinase 2 (MK2), a known downstream substrate of the p38 MAPK, both in HBSMCs and in human airway epithelial cells. Furthermore, pharmacological inhibition of p38 MAPK or MK2 strongly accelerated the decay of IL-8 mRNA levels upon stimulation with CSE or acrolein and subsequent blockade of mRNA neosynthesis with actinomycin D in pulmonary structural cells (HBSMCs and airways epithelial cells) as well as in human alveolar macrophages. Conversely, pharmacological inhibition of ERK1/2 signaling inhibited CSE-induced steady-state levels of IL-8 mRNA without affecting mRNA stability, thus suggesting inhibition at the transcriptional level. In sum, p38 MAPK/MK2 signaling is an important posttranscriptional mechanism underlying upregulation of IL-8 mRNA levels elicited by CSE and acrolein. Given the pivotal role of IL-8 in neutrophil chemotaxis and activation, our results shed light on the mechanisms through which cigarette smoke can initiate inflammation in the lung.

CHF6001 I: a novel highly potent and selective phosphodiesterase 4 inhibitor with robust anti-inflammatory activity and suitable for topical pulmonary administration.

This study examined the pharmacologic characterization of CHF6001 [(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-oxide], a novel phosphodiesterase (PDE)4 inhibitor designed for treating pulmonary inflammatory diseases via inhaled administration. CHF6001 was 7- and 923-fold more potent than roflumilast and cilomilast, respectively, in inhibiting PDE4 enzymatic activity (IC50 = 0.026 ± 0.006 nM). CHF6001 inhibited PDE4 isoforms A–D with equal potency, showed an elevated ratio of high-affinity rolipram binding site versus low-affinity rolipram binding site (i.e., >40) and displayed >20,000-fold selectivity versus PDE4 compared with a panel of PDEs. CHF6001 effectively inhibited (subnanomolar IC50 values) the release of tumor necrosis factor-α from human peripheral blood mononuclear cells, human acute monocytic leukemia cell line macrophages (THP-1), and rodent macrophages (RAW264.7 and NR8383). Moreover, CHF6001 potently inhibited the activation of oxidative burst in neutrophils and eosinophils, neutrophil chemotaxis, and the release of interferon-γ from CD4+ T cells. In all these functional assays, CHF6001 was more potent than previously described PDE4 inhibitors, including roflumilast, UK-500,001 [2-(3,4-difluorophenoxy)-5-fluoro-N-((1S,4S)-4-(2-hydroxy-5-methylbenzamido)cyclohexyl)nicotinamide], and cilomilast, and it was comparable to GSK256066 [6-((3-(dimethylcarbamoyl)phenyl)sulfonyl)-4-((3-methoxyphenyl)amino)-8-methylquinoline-3-carboxamide]. When administered intratracheally to rats as a micronized dry powder, CHF6001 inhibited liposaccharide-induced pulmonary neutrophilia (ED50 = 0.205 μmol/kg) and leukocyte infiltration (ED50 = 0.188 μmol/kg) with an efficacy comparable to a high dose of budesonide (1 μmol/kg i.p.). In sum, CHF6001 has the potential to be an effective topical treatment of conditions associated with pulmonary inflammation, including asthma and chronic obstructive pulmonary disease.

Discovery of a novel isoxazoline derivative of prednisolone endowed with a robust anti-inflammatory profile and suitable for topical pulmonary administration

A novel glucocorticoids series of (GCs), 6α,9α-di-Fluoro 3-substituted C-16,17-isoxazolines was designed, synthesised and their structure-activity relationship was evaluated with glucocorticoid receptor (GR) binding studies together with GR nuclear translocation cell-based assays. This strategy, coupled with in silico modelling analysis, allowed for the identification of Cpd #15, an isoxazoline showing a sub-nanomolar inhibitory potency (IC50=0.84 nM) against TNFα-evoked IL-8 release in primary human airways smooth muscle cells. In Raw264.7 mouse macrophages, Cpd #15 inhibited LPS-induced NO release with a potency (IC50=6 nM)>10-fold higher with respect to Dexamethasone. Upon intratracheal (i.t.) administration, Cpd #15, at 0.1 μmol/kg significantly inhibited and at 1 μmol/kg fully counteracted eosinophilic infiltration in a model of allergen-induced pulmonary inflammation in rats. Moreover, Cpd #15 proved to be suitable for pulmonary topical administration given its sustained lung retention (t1/2=6.5h) and high pulmonary levels (>100-fold higher than plasma levels) upon intratracheal administration in rats. In summary, Cpd #15 displays a pharmacokinetic and pharmacodynamic profile suitable for topical treatment of conditions associated with pulmonary inflammation such as asthma and COPD.

Discovery and optimization of thiazolidinyl- and pyrrolidinyl- derivatives as inhaled PDE4 inhibitors for respiratory diseases

Phosphodiesterase 4 (PDE4) is a key cAMP-metabolizing enzyme involved in the pathogenesis of inflammatory disease, and its pharmacological inhibition has been shown to exert therapeutic efficacy in chronic obstructive pulmonary disease (COPD). Herein, we describe a drug discovery program aiming at the identification of novel classes of potent PDE4 inhibitors suitable for pulmonary administration. Starting from a previous series of benzoic acid esters, we explored the chemical space in the solvent-exposed region of the enzyme catalytic binding pocket. Extensive structural modifications led to the discovery of a number of heterocycloalkyl esters as potent in vitro PDE4 inhibitors. (S*,S**)-18e and (S*,S**)-22e, in particular, exhibited optimal in vitro ADME and pharmacokinetics properties and dose-dependently counteracted acute lung eosinophilia in an experimental animal model. The optimal biological profile as well as the excellent solid-state properties suggest that both compounds have the potential to be effective topical agents for treating respiratory inflammatory diseases.

Design and synthesis of 4,5,6,7-tetrahydro-1H-1,2-diazepin-7-one derivatives as a new series of Phosphodiesterase 4 (PDE4) inhibitors.

Phosphodiesterase 4 (PDE4) inhibitors have attractive therapeutic potential in respiratory, inflammatory, metabolic and CNS disorders. The present work details the design, chemical exploration and biological profile of a novel PDE4 inhibitor chemotype. A diazepinone ring was identified as an under-represented heterocyclic system fulfilling a set of PDE4 structure-based design hypotheses. Rapid exploration of the structure activity relationships for the series was enabled by robust and scalable two/three-steps parallel chemistry protocols. The resulting compounds demonstrated PDE4 inhibitory activity in cell free and cell-based assays comparable to the Zardaverine control used, suggesting potential avenues for their further development.

Novel Pyrrolidine Derivatives of Budesonide as Long Acting Inhaled Corticosteroids for the Treatment of Pulmonary Inflammatory Diseases

Inhaled corticosteroids (ICSs) represent the first line therapy for the treatment of asthma and are also extensively utilized in chronic obstructive pulmonary disease. Our goal was to develop a new ICS with a basic group, which can allow solid state feature modulation, achieving at the same time high local anti-inflammatory effect and low systemic exposure. Through a rational drug design approach, a new series of pyrrolidine derivatives of budesonide was identified. Within the series, several compounds showed nanomolar binding affinity ( Ki) with GR that mostly correlated with the effect in inducing GR nuclear translocation in CHO cells and anti-inflammatory effects in macrophagic cell lines. Binding and functional cell-based assays allowed identifying compound 17 as a potent ICS agonist with a PK profile showing an adequate lung retention and low systemic exposure in vivo. Finally, compound 17 proved to be more potent than budesonide in a rat model of acute pulmonary inflammation.