Phosphodiesterase 4 inhibitors as novel anti-inflammatory agents.

Preclinical and clinical studies of phosphodiesterase 4 inhibitors have shown that these agents may find utility in a wide range of inflammatory disorders, including asthma, chronic obstructive pulmonary disease, atopic dermatitis, rheumatoid arthritis, multiple sclerosis and various neurological disorders. The future of this class of drugs will depend upon the ability to demonstrate a reasonable safety margin against emesis and other typical phosphodieserase (PDE4) side effects, as well as in identification of the inflammatory disorder(s) most relevant to PDE4 inhibition.     

Phosphodiesterase 4 conformers: preparation of recombinant enzymes and assay for inhibitors.

Cyclic nucleotides are key regulators of many cellular processes. Their immediate action is terminated through the activity of phosphodiesterases, a diverse family of enzymes. This diversity has given rise to drug discovery opportunities, and assay technology is therefore of key importance. Inhibitors of the cyclic-AMP-specific phosphodiesterases (the PDE4 family) are drug candidates for a variety of inflammatory disorders. However, PDE4 inhibitors, besides their immunomodulatory effects, also cause side effects including nausea and emesis. Recently, it has been suggested that PDE4 exists in two different conformations with respect to inhibition by the prototypical compound rolipram. Inhibition of the low-affinity conformer is thought to give rise to anti-inflammatory effects, and inhibition of the high-affinity conformer to side effects. Therefore, a selective inhibitor of the low-affinity conformer may have clinical utility. Methods are described to prepare recombinant forms of PDE4B that allow screening for compounds that could preferentially inhibit the low-affinity conformer. Furthermore, conditions for an efficient, scintillation proximity, microtiter plate-based assay are described, providing a considerable advance over previous assays in terms of throughput and automatability.     

Reactive oxygen species: current knowledge and applications in cancer research and therapeutic

Reactive oxygen species (ROS) are natural products inevitably generated along cellular metabolism. Due to their highly reactive nature, which can damage DNA, proteins and lipids, cells utilize antioxidative or defense systems to balance these toxic products to keep the cells in a state of redox homeostasis. However, under the situation of imbalance in redox status, depending on the magnitude of ROS encountered, high levels of ROS can induce apoptosis, whereas chronic low levels of ROS promote vascular diseases such as arteriosclerosis. Although ROS seem to be catastrophic to life, accumulating evidence points to the beneficial roles of ROS by virtue of the ability as chemotherapeutic agents to cure human diseases. Many anti-cancer drugs have been developed in this way which can generate ROS and cause oxidative stress-induced apoptosis in cancer cells. The effects of ROS are paradoxical because they can act as both disease culprits and chemotherapeutic agents. In this review, the current knowledge of ROS and the potential applications of ROS in cancer therapeutic will be discussed.     

Phosphodiesterase 4 inhibitors modulate beta2-adrenoceptor agonist-induced human airway hyperresponsiveness

Chronic exposure of human isolated bronchi to beta2-adrenergic agonists, especially fenoterol, potentiates smooth muscle contraction in response to endothelin-1 (ET-1), a peptide implicated in chronic inflammatory airway diseases. 5'-Cyclic adenosine monophosphate (cAMP) pathways are involved in fenoterol-induced hyperresponsiveness. The present study investigated whether chronic elevation of intracellular cAMP by other pathways than beta2-adrenoceptor stimulation provokes bronchial hyperresponsiveness. Samples from eighteen human bronchi were sensitized to ET-1 by prolonged incubation with 0.1 microM fenoterol (15 h, 21 degrees C), or, under similar conditions, were incubated with a selective type-3 phosphodiesterase inhibitor (1 microM siguazodan), two selective type-4 phosphodiesterase inhibitors (0.1 microM rolipram and 0.1 microM cilomilast), a combination of fenoterol and rolipram (0.1 microM each) or of fenoterol and cilomilast (0.1 microM each). Rolipram and cilomilast, but not siguazodan, induced hyperresponsiveness (p < 0.01 and p < 0.05 vs. paired controls, respectively) similar to the fenoterol effect. Fenoterol-induced bronchial hyperresponsiveness was significantly enhanced by coincubation with cilomilast (p < 0.05 vs. fenoterol alone) but not with rolipram. Our results suggest that prolonged activation of intracellular cAMP through phosphodiesterase 4 inhibition induces hyperresponsiveness to ET-1 in human isolated bronchi. However, differences in subcellular localization of phosphodiesterase 4 may provoke divergent responsiveness patterns when human bronchi are continuously exposed to selective phosphodiesterase inhibitors with or without beta2-adrenergic agonists.     

Phosphodiesterase 4 inhibitors as airways smooth muscle relaxant agents: synthesis and biological activities of triazine derivatives

A series of triazine derivatives was synthesized. The compounds were evaluated for tracheal smooth muscle relaxant and type 4 phosphodiesterase inhibitory activities. A highly significant correlation was observed between the two effects. Two compounds exhibited potent relaxant activity (EC50: 17 and 24 nM) and might be useful for the treatment of asthma.     

Phosphodiesterase inhibitors. Part 5: Hybrid PDE3/4 inhibitors as dual bronchorelaxant/anti-inflammatory agents for inhaled administration

(?)-6-(7-Methoxy-2-(trifluoromethyl)pyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (KCA-1490) exhibits moderate dual PDE3/4-inhibitory activity and promises as a combined bronchodilatory/anti-inflammatory agent. N-alkylation of the pyridazinone ring markedly enhances potency against PDE4 but suppresses PDE3 inhibition. Addition of a 6-aryl-4,5-dihydropyridazin-3(2H)-one extension to the N-alkyl group facilitates both enhancement of PDE4-inhibitory activity and restoration of potent PDE3 inhibition. Both dihydropyridazinone rings, in the core and extension, can be replaced by achiral 4,4-dimethylpyrazolone subunits and the core pyrazolopyridine by isosteric bicyclic heteroaromatics. In combination, these modifications afford potent dual PDE3/4 inhibitors that suppress histamine-induced bronchoconstriction in vivo and exhibit promising anti-inflammatory activity via intratracheal administration.     

Macular degeneration: recent advances and therapeutic opportunities

The central retina mediates high acuity vision, and its progressive dysfunction due to macular degeneration is the leading cause of visual disability among adults in industrialized societies. Here, we summarize recent progress in understanding the pathophysiology of macular degeneration and the implications of this new knowledge for treatment and prevention. The past decade has witnessed remarkable advances in this field, including the development of new, non-invasive retinal imaging technologies, the development of animal models for macular disease, and the isolation of many of the genes responsible for both early- and late-onset macular diseases. These advances have set the stage for the development of effective mechanism-based therapies.     

Expression and Activity of Phosphodiesterase Isoforms during Epithelial Mesenchymal Transition: The Role of Phosphodiesterase 4

Epithelial–mesenchymal transition (EMT) has emerged as a critical event in the pathogenesis of organ fibrosis and cancer and is typically induced by the multifunctional cytokine transforming growth factor (TGF)-β1. The present study was undertaken to evaluate the potential role of phosphodiesterases (PDEs) in TGF-β1-induced EMT in the human alveolar epithelial type II cell line A549. Stimulation of A549 with TGF-β1 induced EMT by morphological alterations and by expression changes of the epithelial phenotype markers E-cadherin, cytokeratin-18, zona occludens-1, and the mesenchymal phenotype markers, collagen I, fibronectin, and α-smooth muscle actin. Interestingly, TGF-β1 stimulation caused twofold increase in total cAMP-PDE activity, contributed mostly by PDE4. Furthermore, mRNA and protein expression demonstrated up-regulation of PDE4A and PDE4D isoforms in TGF-β1-stimulated cells. Most importantly, treatment of TGF-β1 stimulated epithelial cells with the PDE4-selective inhibitor rolipram or PDE4 small interfering RNA potently inhibited EMT changes in a Smad-independent manner by decreasing reactive oxygen species, p38, and extracellular signal-regulated kinase phosphorylation. In contrast, the ectopic overexpression of PDE4A and/or PDE4D resulted in a significant loss of epithelial marker E-cadherin but did not result in changes of mesenchymal markers. In addition, Rho kinase signaling activated by TGF-β1 during EMT demonstrated to be a positive regulator of PDE4. Collectively, the findings presented herein suggest that TGF-β1 mediated up-regulation of PDE4 promotes EMT in alveolar epithelial cells. Thus, targeting PDE4 isoforms may be a novel approach to attenuate EMT-associated lung diseases such as pulmonary fibrosis and lung cancer.     

Phosphodiesterase 4 inhibitors and db-cAMP inhibit TNF-alpha release from human mononuclear cells. Effects of cAMP and cGMP-dependent protein kinase inhibitors

We investigated the effects of specific inhibitors of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) on the inhibitory activity of phosphodiesterase (PDE) type 4 inhibitors and of the cell permeable analogue of cAMP, db-cAMP on LPS-induced TNF-alpha release from human mononuclear cells. Incubation from 30 min of mononuclear cells with dbcAMP (10(-5) to 10(-3) M), rolipram (10(-9) M to 10(-5) M) or Ro 20-1724 (10(-9) M to 10(-5) M) significantly inhibited LPS-induced TNF-alpha release. When mononuclear cells were preincubated for 30 min with the selective PKA inhibitor, H89 (10(-4) M), but not with the selective PKG inhibitor, Rp-8-pCPT-cGMPs (10(-4) M), a significant reduction of the inhibitory effect of db-cAMP was noted. Thirty min incubation of mononuclear cells with Rp-8-pCPT-cGMPs induced a significant reduction of the inhibitory activities of both rolipram and Ro 20-1724 (10(-9) to 10(-5) M) on LPS-induced TNF-alpha release, whereas H89 elicited a moderate, but significant inhibition. The present data indicate that db-cAMP inhibits TNF-alpha release from human mononuclear cells through a PKA-dependent mechanism. In contrast, PDE 4 inhibitors elicit their in vitro anti-inflammatory activities via a PKG-dependent rather than PKA-dependent activation.     

Miniature bioreactors: current practices and future opportunities

This review focuses on the emerging field of miniature bioreactors (MBRs), and examines the way in which they are used to speed up many areas of bioprocessing. MBRs aim to achieve this acceleration as a result of their inherent high-throughput capability, which results from their ability to perform many cell cultivations in parallel. There are several applications for MBRs, ranging from media development and strain improvement to process optimisation. The potential of MBRs for use in these applications will be explained in detail in this review. MBRs are currently based on several existing bioreactor platforms such as shaken devices, stirred-tank reactors and bubble columns. This review will present the advantages and disadvantages of each design together with an appraisal of prototype and commercialised devices developed for parallel operation. Finally we will discuss how MBRs can be used in conjunction with automated robotic systems and other miniature process units to deliver a fully-integrated, high-throughput (HT) solution for cell cultivation process development.     

Lipids and drugs of abuse

Drug abuse continues to take an enormous economic and social toll on the world. Among the costs are reduced productivity, increased need for medical services and stress on families. Treatments that allow affected individuals to reduce compulsive drug use are lacking and novel approaches to their development will likely come from increased understanding of the consequences of chronic exposure to reinforcing drugs. The purpose of this review is to explore the role of lipids in drug abuse and to present a rationale for an increased focus on the interactions between drugs of abuse and lipids in the brain. Small molecular weight lipids function as neuromodulators in the brain and, as such, play a role in the synaptic plasticity that occurs following exposure to drugs of abuse. In addition, the membrane lipid bilayer consists of lipid subdomains and emerging evidence suggests that protein function can be altered by transient associations with these subdomains. Finally, lipidomics is a very new field devoted to the exploration of changes in cellular lipid constituents during phenotypic alterations. Enhanced research in all of these areas will likely provide useful insights into and, perhaps, therapeutic targets for the treatment of drug abuse.     

Orally active PDE4 inhibitors with therapeutic potential

Based on the successful results in the clinical trial of Ariflo, further optimization of the spatial arrangement of the three pharmacophores (carboxylic acid moiety, nitrile moiety and 3-cyclopentyl-4-methoxyphenyl moiety) in the structure of Ariflo 1 was attempted using a bicyclo[3.3.0]octane template instead of a cyclohexane template. As a result, 2a, 7a and 7b were found to be orally active and were predicted to have an improved therapeutic potential based on evaluation by cross-species and same-species comparisons. Structure-activity relationships (SARs) of these compounds are also discussed.     

Phosphodiesterase inhibitors. Part 1: Synthesis and structure-activity relationships of pyrazolopyridine-pyridazinone PDE inhibitors developed from ibudilast

Ibudilast [1-(2-isopropylpyrazolo[1,5-a]pyridin-3-yl)-2-methylpropan-1-one] is a nonselective phosphodiesterase inhibitor used clinically to treat asthma. Efforts to selectively develop the PDE3- and PDE4-inhibitory activity of ibudilast led to replacement of the isopropyl ketone by a pyridazinone heterocycle. Structure-activity relationship exploration in the resulting 6-(pyrazolo[1,5-a]pyridin-3-yl)pyridazin-3(2H)-ones revealed that the pyridazinone lactam functionality is a critical determinant for PDE3-inhibitory activity, with the nitrogen preferably unsubstituted. PDE4 inhibition is strongly promoted by introduction of a hydrophobic substituent at the pyridazinone N(2) centre and a methoxy group at C-7′ in the pyrazolopyridine. Migration of the pyridazinone ring connection from the pyrazolopyridine 3′-centre to C-4′ strongly enhances PDE4 inhibition. These studies establish a basis for development of potent PDE4-selective and dual PDE3/4-selective inhibitors derived from ibudilast.     

Highly potent PDE4 inhibitors with therapeutic potential

The hypothesis that the dose-limiting side effects of PDE4 inhibitors could be mediated via the central nervous system prompted us to design and synthesize a hydrophilic piperidine analog to improve the side effect profile of Ariflo 1, which is an orally active second-generation PDE4 inhibitor. During evaluation of various water-soluble piperidine analogs, 2a-b, 11b-14b, and 17a showed therapeutic potential in cross-species comparison studies. The following three findings were obtained: (1) The hydroxamic acid group, a well known metal chelator, caused a marked increase of inhibitory activity. (2) Water-soluble piperidine analogs lacked the configurational isomerism of Ariflo 1 without loss of inhibitory activity. (3) Replacement of the 4-methoxy residue with a difluoromethoxy residue led to an increase of in vivo potency. Structure-activity relationships are presented. Single-dose rat pharmacokinetic data for 11b, 12b, and 17a are also presented.     

Highly potent PDE4 inhibitors with therapeutic potential

Based on the hypothesis that the dose-limiting side effects of PDE4 inhibitors could be mediated via the central nervous system (CNS), design and synthesis of a hydrophilic analogue is considered to be one approach to improving the side-effect profile of Ariflo 1. Water-soluble piperidine derivatives were found to possess therapeutic potential.     

N-palmitoyl-ethanolamine: Biochemistry and new therapeutic opportunities

Although its presence in mammalian tissues has been known since the 1960s, N-palmitoyl-ethanolamine (PEA) has emerged only recently among other bioactive N-acylethanolamines as an important local pro-homeostatic mediator which, due to its chemical stability, can be also administered exogenously as the active principle of current anti-inflammatory and analgesic preparations (e.g. Normast^®, Pelvilen^®). Much progress has been made towards the understanding of the mechanisms regulating both the tissue levels of PEA under physiological and pathological conditions, and its pharmacological actions. Here we review these new developments in PEA biochemistry and pharmacology, and discuss novel potential indications for the therapeutic use of this compound and of synthetic tools that selectively retard its catabolism, such as the inhibitors of the recently cloned N-acylethanolamine-hydrolyzing acid amidase.     

Viral peptide immunogens: current challenges and opportunities.

Synthetic peptide vaccines have potential to control viral infections. Successful experimental models using this approach include the protection of mice against the lethal Sendai virus infection by MHC class I binding CTL peptide epitope. The main benefit of vaccination with peptide epitopes is the ability to minimize the amount and complexity of a well-defined antigen. An appropriate peptide immunogen would also decrease the chance of stimulating a response against self-antigens, thereby providing a safer vaccine by avoiding autoimmunity. In general, the peptide vaccine strategy needs to dissect the specificity of antigen processing, the presence of B-and T-cell epitopes and the MHC restriction of the T-cell responses. This article briefly reviews the implications in the design of peptide vaccines and discusses the various approaches that are applied to improve their immunogenicity.