ABSTRACT
Chemokine receptor 9 (CCR9), a cell surface chemokine receptor which belongs to the G protein-coupled receptor, 7-trans-membrane superfamily, is expressed on lymphocytes in the circulation and is the key chemokine receptor that enables these cells to target the intestine. It has been proposed that CCR9 antagonism represents a means to prevent the aberrant immune response of inflammatory bowel disease in a localized and disease specific manner and one which is accessible to small molecule approaches. One possible reason why clinical studies with vercirnon, a prototype CCR9 antagonist, were not successful may be due to a relatively poor pharmacokinetic (PK) profile for the molecule. We wish to describe work aimed at producing new, orally active CCR9 antagonists based on the 1,3-dioxoisoindoline skeleton. This study led to a number of compounds that were potent in the nanomolar range and which, on optimization, resulted in several possible preclinical development candidates with excellent PK properties.
Subject(s)
Inflammatory Bowel Diseases/drug therapy , Isoindoles/chemistry , Receptors, CCR/antagonists & inhibitors , Administration, Oral , Animals , Biological Availability , Chemistry Techniques, Synthetic , Chemotaxis/drug effects , Colitis/chemically induced , Colitis/drug therapy , Disease Models, Animal , Humans , Isoindoles/administration & dosage , Isoindoles/pharmacokinetics , Male , Mice, Inbred C57BL , Receptors, CCR/metabolism , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacokineticsABSTRACT
Asthma is a chronic inflammatory lung disease with considerable unmet medical needs for new and effective therapies. Cytosolic phospholipase A(2)α (cPLA(2)α) is the rate-limiting enzyme that is ultimately responsible for the production of eicosanoids implicated in the pathogenesis of asthma. We investigated a novel cPLA(2)α inhibitor, PF-5212372, to establish the potential of this drug as a treatment for asthma. PF-5212372 was a potent inhibitor of cPLA(2)α (7 nM) and was able to inhibit prostaglandin (PG)D(2) and cysteinyl leukotriene release from anti-IgE-stimulated human lung mast cells (0.29 and 0.45 nM, respectively). In a mixed human lung cell population, PF-5212372 was able to inhibit ionomycin-stimulated release of leukotriene B(4), thromboxane A(2), and PGD(2) (2.6, 2.6, and 4.0 nM, respectively) but was significantly less effective against PGE(2) release (>301 nM; p < 0.05). In an in vitro cell retention assay, PF-5212372 retained its potency up to 24 h after being washed off. In a sheep model of allergic inflammation, inhalation of PF-5212372 significantly inhibited late-phase bronchoconstriction (78% inhibition; p < 0.001) and airway hyper-responsiveness (94% inhibition; p < 0.001), and isolated sheep lung mast cell assays confirmed species translation via effective inhibition of PGD(2) release (0.78 nM). Finally, PF-5212372 was assessed for its ability to inhibit the contraction of human bronchi induced by AMP. PF5212372 significantly inhibited AMP-induced contraction of human bronchi (81% inhibition; p < 0.001); this finding, together with the ability of this drug to be effective in a wide range of preclinical asthma models, suggests that inhibition of cPLA(2)α with PF-5212372 may represent a new therapeutic option for the treatment of asthma.
Subject(s)
Asthma/drug therapy , Cytosol/enzymology , Enzyme Inhibitors/therapeutic use , Group IV Phospholipases A2/antagonists & inhibitors , Phenylpropionates/pharmacology , Sulfonamides/pharmacology , Animals , Antibodies, Anti-Idiotypic/pharmacology , Bronchoconstriction/drug effects , Calcium Ionophores/pharmacology , Cell Line , Humans , Mast Cells/physiology , Prostaglandin D2/metabolism , SheepABSTRACT
In this Letter we present data for a novel series of ICS for the treatment of asthma. 'Inhalation by design' principles have been applied to a series of highly potent steroidal GR agonists, with a focus on optimising the potential therapeutic index in human. Pharmacokinetic properties were tuned with high intrinsic clearance and low oral bioavailability in mind, to minimise systemic exposure and reduce systemically driven adverse events. High CYP mediated clearance as well as glucuronidation were targeted to achieve high intrinsic clearance coupled with multiple routes of clearance to minimise drug-drug interactions. Furthermore, pharmaceutical properties such as stability, crystallinity and solubility were considered to ensure compatibility with a dry powder inhaler. This work culminated in the identification of the clinical candidate 15, which demonstrates preclinically the desired efficacy and safety profiles confirming its potential as an inhaled agent for the treatment of asthma.
Subject(s)
Adrenal Cortex Hormones/chemical synthesis , Adrenal Cortex Hormones/pharmacokinetics , Anti-Asthmatic Agents/chemical synthesis , Anti-Asthmatic Agents/pharmacokinetics , Asthma/drug therapy , Drug Design , Administration, Inhalation , Adrenal Cortex Hormones/administration & dosage , Adrenal Cortex Hormones/pharmacology , Adrenergic beta-Agonists/administration & dosage , Adrenergic beta-Agonists/therapeutic use , Androstadienes/chemistry , Androstadienes/pharmacology , Animals , Anti-Asthmatic Agents/administration & dosage , Anti-Asthmatic Agents/pharmacology , Asthma/epidemiology , Asthma/physiopathology , Delayed-Action Preparations , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Drug Therapy, Combination , Dry Powder Inhalers , Fluticasone , Hepatocytes , Humans , Liver , Lung , Microsomes, Liver , Neutrophils/metabolism , Randomized Controlled Trials as Topic , Rats , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/bloodABSTRACT
Rhinovirus infections cause the majority of acute exacerbations of airway diseases such as asthma and chronic obstructive pulmonary disease, with increased pro-inflammatory cytokine production by infected bronchial epithelial cells contributing to disease pathogenesis. Theses diseases are a huge cause of morbidity worldwide, and contribute a major economic burden to healthcare costs. Current steroid based treatments are only partially efficient at controlling virus induced inflammation, which remains an unmet therapeutic goal. Although NF-kappaB has been implicated, the precise mechanisms of rhinovirus induction of pro-inflammatory gene expression in bronchial epithelial cells are unclear. We hypothesised that rhinovirus replication and generation of dsRNA was an important process of pro-inflammatory cytokine induction. Using pharmalogical (2-aminopurine and a new small molecule inhibitor) and genetic inhibition of the dsRNA binding kinase protein kinase R, striking inhibition of dsRNA (polyrIC) and rhinovirus induced CCL5, CXCL8 and IL-6 protein was observed. Using confocal microscopy, rhinovirus induced protein kinase R phosphorylation co-located with NF-kappaB p65 nuclear translocation. Focusing on CXCL8, both rhinovirus infection and dsRNA treatment required IkappaB kinase-beta for induction of CXCL8. Analysis of cis-acting sites in the CXCL8 promoter revealed that both rhinovirus infection and dsRNA treatment upregulated CXCL8 promoter activation via NF-kappaB and NF-IL6 binding sites. Together, the results demonstrate the importance of dsRNA in induction of pro-inflammatory cytokines by rhinoviruses, and suggest that protein kinase R is involved in NF-kappaB mediated gene transcription of pro-inflammatory cytokines via IkappaB kinase-beta. These molecules regulating rhinovirus induction of inflammation represent therapeutic targets.
Subject(s)
Bronchi/immunology , Cytokines/metabolism , I-kappa B Kinase/metabolism , Rhinovirus/immunology , Transcription Factor RelA/metabolism , eIF-2 Kinase/metabolism , 2-Aminopurine/pharmacology , Bronchi/chemistry , Bronchi/virology , Cytokines/genetics , Epithelial Cells/chemistry , Epithelial Cells/immunology , Epithelial Cells/virology , Gene Expression Regulation , Humans , NF-kappa B/metabolism , Protein Kinase Inhibitors/pharmacology , RNA, Double-Stranded/metabolism , RNA, Double-Stranded/pharmacology , RNA, Viral/metabolism , RNA, Viral/pharmacology , Transcription Factor RelA/analysis , eIF-2 Kinase/analysis , eIF-2 Kinase/antagonists & inhibitorsABSTRACT
Rhinoviruses (RV) are the major cause of the common cold and acute exacerbations of asthma and chronic obstructive pulmonary disease. Toll-like receptors (TLRs) are a conserved family of receptors that recognize and respond to a variety of pathogen-associated molecular patterns. TLR3 recognizes double-stranded RNA, an important intermediate of many viral life cycles (including RV). The importance of TLR3 in host responses to virus infection is not known. Using BEAS-2B (a human bronchial epithelial cell-line), we demonstrated that RV replication increased the expression of TLR3 mRNA and TLR3 protein on the cell surface. We observed that blocking TLR3 led to a decrease in interleukin-6, CXCL8, and CCL5 in response to poly(IC) but an increase following RV infection. Finally, we demonstrated that TLR3 mediated the antiviral response. This study demonstrates an important functional requirement for TLR3 in the host response against live virus infection and indicates that poly(IC) is not always a good model for studying the biology of live virus infection.
Subject(s)
Membrane Glycoproteins/physiology , Receptors, Cell Surface/physiology , Respiratory Mucosa/immunology , Respiratory Mucosa/virology , Rhinovirus/immunology , Bronchi/cytology , Bronchi/immunology , Bronchi/virology , Cell Line , Chemokine CCL5 , Chemokines, CC/analysis , Chemokines, CXC/analysis , Flow Cytometry , Humans , Interleukin-6/analysis , Membrane Glycoproteins/analysis , Membrane Glycoproteins/genetics , Membrane Proteins/analysis , RNA, Messenger/analysis , Receptors, Cell Surface/analysis , Receptors, Cell Surface/genetics , Respiratory Mucosa/cytology , Reverse Transcriptase Polymerase Chain Reaction , Toll-Like Receptor 3 , Toll-Like ReceptorsABSTRACT
Asthma and chronic obstructive pulmonary disease are highly prevalent and economically important inflammatory airway diseases associated with mucus hypersecretion. Considerable additional morbidity and mortality are related to acute exacerbations, which are associated with further mucus hypersecretion. MUC5AC is a prominent airway mucin; however, the signalling pathways regulating MUC5AC hypersecretion are not fully characterised. We investigated the signalling pathway regulating phorbol 12-myristate 13-acetate (PMA)-induced MUC5AC gene and protein expression in human respiratory epithelial cells. Using NCI-H292 cells, we demonstrated that treatment with PMA increased production of total and MUC5AC-specific mucin proteins. This increase was dependent on de novo MUC5AC gene transcription. We identified a short, proximal region of the MUC5AC promoter essential for this activity containing three specificity protein (Sp) 1 transcription factor-binding sites and a single CACCC site. By chemical inhibition, site-directed promoter mutagenesis and electrophoretic mobility-shift assay (EMSA), we demonstrated that PMA induced proteins binding to all three Sp1 sites and that they were all required for full induction of MUC5AC promoter activity. We then demonstrated a Ras-Raf-MEK/ERK signalling pathway was exclusively activated upstream of Sp1 activating the promoter and confirmed the requirement for matrix metalloproteinase activation leading to a ligand-dependent activation of the epidermal growth factor receptor. Finally, we demonstrated that activation of the novel protein kinase C isoforms delta and theta; was required upstream of the metalloproteinase activation. We have characterised a signalling pathway regulating PMA induction of MUC5AC. Studies such as this identify key signalling intermediates as targets for pharmacological intervention to treat mucus hypersecretion.