Long lasting response of bIL-8-Luc reporter construct.

<p><b>A</b>) Representative images of groups of mice (n = 3 per group) transiently transgenized with bIL-8-Luc or Empty vector DNA and intratracheally instilled with LPS, TNF-α or vehicle (saline solution) at 8, 32, 45 and 60 days post transgenization. Mice were monitored at 3 hours post stimulation by BLI by drawing a region of interest (ROI) over the chest. <b>B</b>) Light intensity quantification of the ROI using the LivingImage software. The experiment was repeated three times and each point represents the mean ± standard deviation of 9 animals. <b>C</b>) Data were expressed as folds of induction over vehicle-treated mice (Saline) and statistical differences were tested by One Way ANOVA followed by Dunnet’s post hoc test for group comparisons. Results are reported as mean ± SD and significance attributed when <i>P</i><0.05 (*) or <i>P</i><0.01(**).</p

<i>In vitro</i> characterization of bIL-8-Luc reporter construct.

<p><b>A</b>) Schematic diagram (not to scale) of the <i>Bos taurus</i> chromosome 6 with the IL-8 gene locus comprising promoter and exons annotated by numbers. The 2 kb IL-8 promoter sequence, comprising the putative TATA box, the transcriptional start site (+1) and the UTR (red underlined) was cloned into the pGL3 basic vector in front of the open reading frame of the Luciferase reporter. <b>B</b>) RT-PCR products of TLRs from 1 to 9 and their corresponding amplicon size. GAPDH was used as an internal control and the amplification performed in the absence of reverse transcriptase (-RT) as a negative control. <b>C</b>) RT-PCR products of TNF-αRI,II and their corresponding amplicon size. GAPDH was used as an internal control and the amplification performed in the absence of reverse transcriptase (-RT) as a negative control. D) IL-8 promoter activation after treatment of bIL-8-Luc transfected LA-4 cells with LPS or TNF-α, along with the untreated control (Untr.). Each experiment was done in quadruplicate, and each point represents the mean ± standard deviation of three experiments. Data were expressed as folds of induction (2,1 and 2,8 for LPS and TNF-α respectively) over vehicle-treated cells and statistical differences were tested by One Way ANOVA followed by Dunnet’s post hoc test for group comparisons. Results are reported as mean ± SD and significance attributed when <i>P</i><0.05 (*) or <i>P</i><0.01(**).</p

White blood cells and neutrophils infiltration.

<p>Cellular infiltration into the lung of mice intratracheally instilled with LPS, TNF-α or vehicle. The amount of White blood cells (WBC), Neutrophils (Neut) and Monocytes (Mono) found in BALF was expressed as number of cells per μl at 3 (A) and 24 hours (B) post treatment. The experiment was repeated three times and each point represents the mean ± standard deviation of 9 animals. Data were expressed as folds of induction over vehicle-treated mice (Saline) and statistical differences were tested by One Way ANOVA followed by Dunnet’s post hoc test for group comparisons. Results are reported as mean ± SD and significance attributed when <i>P</i><0.05 (*) or <i>P</i><0.01(**).</p

bIL-8-Luc reporter construct response <i>in vivo</i>.

<p><b>A</b>) Representative images of groups of mice (n = 3 per group) transiently transgenized with bIL-8-Luc or Empty vector DNA and intratracheally instilled with LPS, TNF-α or vehicle (saline solution). Mice were monitored at 3, 5 and 24 hours post stimulation by BLI drawing a region of interest (ROI) over the chest. <b>B</b>) Light intensity quantification of the ROI using the LivingImage software. The experiment was repeated three times and each point represents the mean ± standard deviation of 9 animals. <b>C</b>) Data were expressed as folds of induction over vehicle-treated mice (Saline) and statistical differences were tested by One Way ANOVA followed by Dunnet’s post hoc test for group comparisons. Results are reported as mean ± SD and significance attributed when <i>P</i><0.05 (*) or <i>P</i><0.01(**).</p

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 (<i>K</i>_i) 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 <b>17</b> as a potent ICS agonist with a PK profile showing an adequate lung retention and low systemic exposure in vivo. Finally, compound <b>17</b> proved to be more potent than budesonide in a rat model of acute pulmonary inflammation

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 (<i>K</i>_i) 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 <b>17</b> as a potent ICS agonist with a PK profile showing an adequate lung retention and low systemic exposure in vivo. Finally, compound <b>17</b> proved to be more potent than budesonide in a rat model of acute pulmonary inflammation

Atropisomerism and Conformational Equilibria: Impact on PI3Kδ Inhibition of 2‑((6-Amino‑9<i>H</i>‑purin-9-yl)methyl)-5-methyl-3‑(<i>o</i>‑tolyl)quinazolin-4(3<i>H</i>)‑one (IC87114) and Its Conformationally Restricted Analogs

IC87114 [compound <b>1</b>, (2-((6-amino-9<i>H</i>-purin-9-yl)­methyl)-5-methyl-3-(<i>o</i>-tolyl)­quinazolin-4­(3<i>H</i>)-one)] is a potent PI3K inhibitor selective for the δ isoform. As predicted by molecular modeling calculations, rotation around the bond connecting the quinazolin-4­(3<i>H</i>)-one nucleus to the <i>o</i>-tolyl is sterically hampered, which leads to separable conformers with axial chirality (i.e., atropisomers). After verifying that the a<i>S</i> and a<i>R</i> isomers of compound <b>1</b> do not interconvert in solution, we investigated how biological activity is influenced by axial chirality and conformational equilibrium. The a<i>S</i> and a<i>R</i> atropisomers of <b>1</b> were equally active in the PI3Kδ assay. Conversely, the introduction of a methyl group at the methylene hinge connecting the 6-amino-9<i>H</i>-purin-9-yl pendant to the quinazolin-4­(3<i>H</i>)-one nucleus of both a<i>S</i> and a<i>R</i> isomers of <b>1</b> had a critical effect on the inhibitory activity, indicating that modulation of the conformational space accessible for the two bonds departing from the central methylene considerably affects the binding of compound <b>1</b> analogues to PI3Kδ enzyme

Atropisomerism and Conformational Equilibria: Impact on PI3Kδ Inhibition of 2‑((6-Amino‑9<i>H</i>‑purin-9-yl)methyl)-5-methyl-3‑(<i>o</i>‑tolyl)quinazolin-4(3<i>H</i>)‑one (IC87114) and Its Conformationally Restricted Analogs

IC87114 [compound <b>1</b>, (2-((6-amino-9<i>H</i>-purin-9-yl)­methyl)-5-methyl-3-(<i>o</i>-tolyl)­quinazolin-4­(3<i>H</i>)-one)] is a potent PI3K inhibitor selective for the δ isoform. As predicted by molecular modeling calculations, rotation around the bond connecting the quinazolin-4­(3<i>H</i>)-one nucleus to the <i>o</i>-tolyl is sterically hampered, which leads to separable conformers with axial chirality (i.e., atropisomers). After verifying that the a<i>S</i> and a<i>R</i> isomers of compound <b>1</b> do not interconvert in solution, we investigated how biological activity is influenced by axial chirality and conformational equilibrium. The a<i>S</i> and a<i>R</i> atropisomers of <b>1</b> were equally active in the PI3Kδ assay. Conversely, the introduction of a methyl group at the methylene hinge connecting the 6-amino-9<i>H</i>-purin-9-yl pendant to the quinazolin-4­(3<i>H</i>)-one nucleus of both a<i>S</i> and a<i>R</i> isomers of <b>1</b> had a critical effect on the inhibitory activity, indicating that modulation of the conformational space accessible for the two bonds departing from the central methylene considerably affects the binding of compound <b>1</b> analogues to PI3Kδ enzyme

Potency (EC₅₀ and CI) of the various TRPA1 agonists in different non-neuronal cell types of the human respiratory tract.

<p>Potency (EC₅₀ and CI) of the various TRPA1 agonists in different non-neuronal cell types of the human respiratory tract.</p

TRPA1 mediates IL-8 release by acrolein from human airway/lung cells in primary culture.

<p>Overnight exposure to acrolein (ACR) induces IL-8 release from small airway epithelial cells (SAEC) (<b>A</b>), normal human lung fibroblasts (NHLF) (<b>B</b>) and human bronchial smooth muscle cells (HBSMC) (<b>C</b>) in a concentration–dependent manner. IL-8 release evoked by ACR is reduced by HC-030031 (HC, 30 µM) and AP18 (10 µM). Each column represents mean ± SEM of at least 3 independent experiments. ^§<i>P</i><0.05 vs. Basal group or Veh/Veh-ACR; ^*<i>P</i><0.05 vs. Veh/ACR.</p