Mark A. Giembycz

Experimental design and analysis and their reporting: new guidance for publication in BJP

This Editorial is part of a series. To view the other Editorials in this series, visit: http://onlinelibrary.wiley.com/doi/10.1111/bph.12956/abstract; http://onlinelibrary.wiley.com/doi/10.1111/bph.12954/abstract; http://onlinelibrary.wiley.com/doi/10.1111/bph.12955/abstract and http://onlinelibrary.wiley.com/doi/10.1111/bph.13112/abstract

Identification of cyclic AMP phosphodiesterases 3, 4 and 7 in human CD4+ and CD8+ T-lymphocytes: role in regulating proliferation and the biosynthesis of interleukin-2

1 The cyclic AMP phosphodiesterases (PDE) expressed by CD4+ and CD8+ T‐lymphocytes purified from the peripheral blood of normal adult subjects were identified and characterized, and their role in modulating proliferation and the biosynthesis of interleukin (IL)‐2 and interferon (IFN)‐γ evaluated. 2 In lysates prepared from both subsets, SK&F 95654 (PDE3 inhibitor) and rolipram (PDE4 inhibitor) suppressed cyclic AMP hydrolysis indicating the presence of PDE3 and PDE4 isoenzymes in these cells. Differential centrifugation and subsequent inhibitor and kinetic studies revealed that the particulate fraction contained, predominantly, a PDE3 isoenzyme. In contrast, the soluble fraction contained a PDE4 (∼65% of total activity) and, in addition, a novel enzyme that had the kinetic characteristics of the recently identified PDE7. 3 Reverse transcription‐polymerase chain reaction (RT‐PCR) studies with primer pairs designed to recognise unique sequences in the human PDE4 and PDE7 genes amplified cDNA fragments that corresponded to the predicted sizes of HSPDE4A, HSPDE4B, HSPDE54D and HSPDE7. No message was detected for HSPDE4C after 35 cycles of amplification. 4 Functionally, rolipram inhibited phytohaemagglutinin‐ (PHA) and anti‐CD3‐induced proliferation of CD4+ and CD8+ T‐lymphocytes, and the elaboration of IL‐2, which was associated with a three to four fold increase in cyclic AMP mass. In all experiments, however, rolipram was approximately 60 fold more potent at suppressing IL‐2 synthesis than at inhibiting mitogenesis. In contrast, SK&F 95654 failed to suppress proliferation and cytokine generation, and did not elevate the cyclic AMP content in T‐cells. Although inactive alone, SK&F 95654 potentiated the ability of rolipram to suppress PHA‐ and anti‐CD3‐induced T‐cell proliferation, and PHA‐induced IL‐2 release. 5 When a combination of phorbol myristate acetate (PMA) and ionomycin were used as a co‐mitogen, rolipram did not affect proliferation but, paradoxically, suppressed IL‐2 release indicating that cyclic AMP can inhibit mitogenesis by acting at, or proximal to, the level of inositol phospholipid hydrolysis. 6 Collectively, these data suggest that PDE3 and PDE4 isoenzymes regulate the cyclic AMP content, IL‐2 biosynthesis and proliferation in human CD4+ and CD8+ T‐lymphocytes. However, the ability of rolipram to suppress markedly mitogen‐induced IL‐2 generation without affecting T‐cell proliferation suggests that growth and division of T‐lymphocytes may be governed by mediators in addition to IL‐2. Finally, T‐cells have the potential to express PDE7, although elucidating the functional role of this enzyme must await the development of selective inhibitors.

Inhibition of eosinophil cyclic nucleotide PDE activity and opsonised zymosan‐stimulated respiratory burst by ‘type IV’‐selective PDE inhibitors

1 The cyclic nucleotide phosphodiesterase (PDE) of guinea‐pig eosinophils was partially characterized and the effects of selective inhibitors of PDE isoenzymes upon opsonized zymosan (OZ)‐stimulated respiratory burst were studied. 2 PDE activity in eosinophil lysates appeared to be membrane‐associated, displayed substrate specificity for adenosine 3′: 5′ cyclic monophosphate (cyclic AMP) versus guanosine 3′: 5′ cyclic monophosphate (cyclic GMP) and was insensitive to cyclic GMP or Ca2+ and calmodulin. 3 The non‐selective PDE inhibitor, 3‐isobutyl‐1‐methylxanthine caused a concentration‐dependent inhibition of both OZ‐stimulated hydrogen peroxide (H2O2) generation and cyclic AMP hydrolysis. The type IV‐selective PDE inhibitors, rolipram and denbufylline, also inhibited H2O2 generation and cyclic AMP hydrolysis in a concentration‐dependent manner whilst SK&F 94120 and Org 9935 (type III‐selective) and zaprinast (type Ia or V‐selective) were ineffective. 4 Dibutyryl cyclic AMP, a cell‐permeable, non‐hydrolysable analogue of cyclic AMP, caused a concentration‐dependent inhibition of H2O2 generation stimulated by OZ. Dibutyryl cyclic GMP was ineffective. 5 It is concluded that eosinophil respiratory burst activity induced by OZ can be regulated by intracellular cyclic AMP and that the levels of cyclic AMP are controlled exclusively by a rolipram‐ and denbufylline‐sensitive PDE isoenzyme that resembles a type IV species.

Phosphodiesterase 4 inhibitors and the treatment of asthma: where are we now and where do we go from here?

Research conducted over the last 20 years has established that inflammation of the airways is central to the airway dysfunction that characterises asthma. Typically, the airway wall is infiltrated by a variety of cells including mast cells, eosinophils and T lymphocytes, which have deviated towards a Th2 phenotype. Together, these cells release a plethora of mediators including interleukin (IL)-4, IL-5, granulocyte/macrophage colony-stimulating factor and eotaxin which ultimately cause the histopathology and symptoms of asthma. Glucocorticosteroids are the only drugs currently available that effectively impact upon this inflammation and resolve, to a greater or lesser extent, compromised lung function. However, steroids are nonselective and generally unsuitable for paediatric use. New drugs are clearly required. One group of potential therapeutic agents for asthma are inhibitors of cyclic AMP-specific phosphodiesterase (PDE), of which theophylline may be considered a prototype. It is now known that PDE is a generic term which refers to at least 11 distinct enzyme families that hydrolyse cAMP and/or cGMP. Over the last decade, inhibitors of PDE4 (a cAMP-specific family that negatively regulates the function of almost all pro-inflammatory and immune cells, and exerts widespread anti-inflammatory activity in animal models of asthma) have been developed with the view to reducing the adverse effects profile associated with non-selective inhibitors such as theophylline. Such is the optimism regarding PDE4 as a viable therapeutic target that more than 100 PDE4 inhibitor patent applications have been filed since 1996 by 13 major pharmaceutical companies. This article reviews the progress of PDE4 inhibitors as anti-inflammatory agents, and identifies problems that have been encountered by the pharmaceutical industry in the clinical development of these drugs and what strategies are being considered to overcome them.

Protein kinase C isoenzymes: a review of their structure, regulation and role in regulating airways smooth muscle tone and mitogenesis.

Protein kinase C (PKC) is a multifunctional, cyclic nucleotide-independent protein kinase that phosphorylates serine and threonine residues in many target proteins. This enzyme was identified in bovine cerebellum by Nishizuka and co-workers (Takai et al., 1977; Inoue et al., 1977) as a protein kinase that phosphorylated histone and protamine. Since its discovery, much interest has been shown in PKC and its role in signal transduction. Development (Otte et al., 1991), memory (Alkon, 1989), differentiation (Cutler et al., 1993), proliferation (Murray et al., 1993) and carcinogenesis (Ashendel, 1985) all are processes for which PKC has been implicated. Once thought to be a single protein, PKC is now known to comprise a large family of enzymes that differ in structure, cofactor requirements and function. Indeed, the PKC family is the largest serine/threonine-specific kinase family known (Parker, 1992) to which many cellular responses have been credited (Nishizuka, 1995). This enzyme multiplicity, together with variation in cellular and tissue distribution, and abundance might explain why so many signal transduction functions have been attributed to this kinase. Here we briefly describe the organization and regulation of PKC and review the current understanding of their role in the regulation of airways smooth muscle (ASM) tone and mitogenesis, which have been investigated in some detail.

Long-Acting β2-Adrenoceptor Agonists Synergistically Enhance Glucocorticoid-Dependent Transcription in Human Airway Epithelial and Smooth Muscle Cells

Addition of an inhaled long-acting β2-adrenoceptor agonist (LABA) to an inhaled corticosteroid (ICS) is more effective at improving asthma control and reducing exacerbations than increasing the dose of ICS. Given that LABA monotherapy is not anti-inflammatory, pathways may exist by which LABAs enhance ICS actions. In the current study, the glucocorticoid dexamethasone had no effect on β2-adrenoceptor agonist-induced cAMP-response element-dependent transcription in the human bronchial epithelial cell line BEAS-2B. In contrast, simple glucocorticoid response element (GRE)-dependent transcription induced by dexamethasone, budesonide, and fluticasone was synergistically enhanced by β2-adrenoceptor agonists, including salmeterol and formoterol, to a level that could not be achieved by glucocorticoid alone. This enhancement was mimicked by other cAMP-elevating agents, and a cAMP mimetic, and was blocked by an inhibitor of cAMP-dependent protein kinase (PKA). Thus, β2-adrenoceptor agonists synergistically enhance simple GRE-dependent transcription via the classical cAMP-PKA pathway. Consistent with the clinical situation, the addition of a β2-adrenoceptor agonist to a glucocorticoid is steroid-sparing in that maximal GRE-dependent responses, evoked by glucocorticoid, are achieved at ∼10-fold lower concentrations in the presence of β2-adrenoceptor agonist. Finally, analysis of dexamethasone-inducible genes, including glucocorticoid-inducible leucine zipper (GILZ), aminopeptidase N, FKBP51, PAI-1, tristetraprolin, DNB5, p57KIP2, metallothionein 1X, and MKP-1, revealed enhanced inducibility of some genes by glucocorticoid/β2-adrenoceptor agonist combinations in a manner that was consistent with the GRE-reporter. Because such effects also occur in primary human airway smooth muscle cells, we propose that enhancement of glucocorticoid-inducible gene expression may contribute to the superior efficacy of LABA/ICS combination therapies, over ICS alone, in asthma treatment.

Differential Regulation of Cytokine Release and Leukocyte Migration by Lipopolysaccharide-Stimulated Primary Human Lung Alveolar Type II Epithelial Cells and Macrophages

Bacterial colonization is a secondary feature of many lung disorders associated with elevated cytokine levels and increased leukocyte recruitment. We hypothesized that, alongside macrophages, the epithelium would be an important source of these mediators. We investigated the effect of LPS (0, 10, 100, and 1000 ng/ml LPS, up to 24 h) on primary human lung macrophages and alveolar type II epithelial cells (ATII; isolated from resected lung tissue). Although macrophages produced higher levels of the cytokines TNF-α and IL-1β (p < 0.0001), ATII cells produced higher levels of chemokines MCP-1, IL-8, and growth-related oncogene α (p < 0.001), in a time- and concentration-dependent manner. Macrophage (but not ATII cell) responses to LPS required activation of ERK1/2 and p38 MAPK signaling cascades; phosphorylated ERK1/2 was constitutively up-regulated in ATII cells. Blocking Abs to TNF-α and IL-1β during LPS exposure showed that ATII cell (not macrophage) MCP-1 release depended on the autocrine effects of IL-1β and TNF-α (p < 0.003, 24 h). ATII cell release of IL-6 depended on autocrine effects of TNF-α (p < 0.006, 24 h). Macrophage IL-6 release was most effectively inhibited when both TNF-α and IL-1β were blocked (p < 0.03, 24 h). Conditioned media from ATII cells stimulated more leukocyte migration in vitro than conditioned media from macrophages (p < 0.0002). These results show differential activation of cytokine and chemokine release by ATII cells and macrophages following LPS exposure. Activated alveolar epithelium is an important source of chemokines that orchestrate leukocyte migration to the peripheral lung; early release of TNF-α and IL-1β by stimulated macrophages may contribute to alveolar epithelial cell activation and chemokine production.

Prostanoid receptor antagonists: development strategies and therapeutic applications

Identification of the primary products of cyclo‐oxygenase (COX)/prostaglandin synthase(s), which occurred between 1958 and 1976, was followed by a classification system for prostanoid receptors (DP, EP1, EP2 …) based mainly on the pharmacological actions of natural and synthetic agonists and a few antagonists. The design of potent selective antagonists was rapid for certain prostanoid receptors (EP1, TP), slow for others (FP, IP) and has yet to be achieved in certain cases (EP2). While some antagonists are structurally related to the natural agonist, most recent compounds are ‘non‐prostanoid’ (often acyl‐sulphonamides) and have emerged from high‐throughput screening of compound libraries, made possible by the development of (functional) assays involving single recombinant prostanoid receptors. Selective antagonists have been crucial to defining the roles of PGD2 (acting on DP1 and DP2 receptors) and PGE2 (on EP1 and EP4 receptors) in various inflammatory conditions; there are clear opportunities for therapeutic intervention. The vast endeavour on TP (thromboxane) antagonists is considered in relation to their limited pharmaceutical success in the cardiovascular area. Correspondingly, the clinical utility of IP (prostacyclin) antagonists is assessed in relation to the cloud hanging over the long‐term safety of selective COX‐2 inhibitors. Aspirin apart, COX inhibitors broadly suppress all prostanoid pathways, while high selectivity has been a major goal in receptor antagonist development; more targeted therapy may require an intermediate position with defined antagonist selectivity profiles. This review is intended to provide overviews of each antagonist class (including prostamide antagonists), covering major development strategies and current and potential clinical usage.

Release of granulocyte-macrophage colony stimulating factor by human cultured airway smooth muscle cells: suppression by dexamethasone.

Smooth muscle cells represent a significant percentage of the total cells in the airway but their contribution to the inflammatory response seen in airway disease has not been studied. Hence, we have looked at the release of the cytokine granulocyte‐macrophage colony stimulating factor (GM‐CSF) in response to bacterial lipopolysaccharide (LPS) and the pro‐inflammatory cytokines interleukin‐1β (IL‐1β), tumour necrosis factor α (TNFα) and interferon γ (IFNγ). Human airway smooth muscle (HASM) cells released GM‐CSF under basal conditions (45.4 ± 13.1 pg ml−1) that was significantly enhanced by IL‐lβ and TNFα with a maximal effect seen at 10 ng ml−1 (1.31 ± 0.07 ng ml−1 and 0.72 ± 0.16 ng ml−1, respectively). In contrast, neither LPS nor IFNγ produced a significant increase in GM‐CSF release. However, HASM cells exposed to IL‐1β, TNFα and IFNγ generated more GM‐CSF than that evoked by any cytokine alone (2.2 ± 0.15 ng ml−1). The release of GM‐CSF elicited by the cytokine mixture was inhibited by cycloheximide and dexamethasone. These data suggest that HASM cells might play an active part in initiating and/or perpetuating airway inflammation in addition to controlling airway calibre.

Cilomilast: a second generation phosphodiesterase 4 inhibitor for asthma and chronic obstructive pulmonary disease

Cilomilast (Ariflo™ , SB-207499) is an orally-active, second generation phosphodiesterase (PDE) inhibitor that may be effective in the treatment of asthma and chronic obstructive pulmonary disease (COPD). It has high selectivity for the cyclic AMP-specific, or PDE4, isoenzyme that predominates in pro-inflammatory and immune cells and is ten-fold more selective for PDE4D than for PDE4A, B and C. In vitro, cilomilast suppresses the activity of many pro-inflammatory and immune cells that have been implicated in the pathogenesis of asthma and COPD and is highly active in animal models of these diseases. Cilomilast demonstrates a markedly improved side effect profile over the archetypal PDE4 inhibitor, rolipram, which has been attributed to its inability to discriminate between the high affinity rolipram binding site and the catalytic domain of the enzyme, and the fact that it is negatively charged which at physiological pH should limit its penetration in to the CNS. In humans cilomilast is rapidly absorbed after oral administration, providing dose-proportional systemic exposure up to 4 mg, completely bioavailable, has a half-life of ~ 7 h and is subject to negligible first pass hepatic metabolism. Cilomilast is extensively metabolised with decyclopentylation, acyl glucuronidation and 3-hydroxylation of the cyclopentyl ring representing the principal routes. Most of the drug is excreted in the urine (~ 90%) and faeces (6 - 7%) with unchanged cilomilast accounting for less than 1% of the administered dose. Cilomilast has been evaluated in Phase I, Phase II and Phase III trials and dose-response experiments have demonstrated a clinically significant increase in lung function and a perceived improvement in quality of life in patients with COPD. Trials of cilomilast in asthma have been less impressive although a trend towards improved lung function has been reported. Cilomilast is safe and well-tolerated at doses up to 15 mg in both short- and long-term dosing trials with a low incidence of adverse effects. No evidence for drug-drug interactions with commonly prescribed medications for COPD and asthma such as digoxin, corticosteroids, salbutamol, theophylline or warfarin has been found. Moreover, the pharmacokinetics of cilomilast are essentially the same in smokers and non-smokers, indicating that no dose adjustments of cilomilast will be required in patients with COPD. Thus, cilomilast displays a promising clinical profile in the treatment of inflammatory airway diseases, in particular COPD and the results of further Phase III trials are awaited with interest.