The differential impact of PDE4 subtypes in human lung fibroblasts on cytokine-induced proliferation and myofibroblast conversion.

Lung fibroblast proliferation and differentiation into myofibroblasts are pathological key events during development of lung fibrosis. Cyclic nucleotide signaling is described as a negative modulator of these cellular processes, and cyclic nucleotide degrading type 4 phosphodiesterases (PDE4) are important regulators of these pathways. In this study, we elucidated expression and the role of individual subtypes of PDE4 in primary normal human lung fibroblast (NHLF) in controlling cytokines-induced proliferation and conversion to myofibroblasts by short-interfering RNAs (siRNAs) induced knockdown. We verified the expression of PDE4A, B, and D, while PDE4C was only minor or even not expressed in NHLF. An efficient liposome-mediated transfection method for mRNA silencing and a knockdown of the expressed PDE4 subtypes was achieved in these cells. This knockdown was further validated by PDE4 protein expression analysis and PDE4 activity measurements. Functionally, the knockdown of PDE4A and PDE4B inhibited proliferation induced by the cytokine combination of bFGF and IL-1ß, whereas knockdown of PDE4D was ineffective. In contrast, TGF-ß induced differentiation into myofibroblasts was affected by knockdown of PDE4B and PDE4D, but not by PDE4A knockdown. In summary, our data allow to assign different PDE4 subtypes to distinct functions of human lung fibroblasts and highlight the predominant role of PDE4B in controlling pathophysiological processes of human lung fibroblasts. This provides a scientific rationale for focused therapeutic targeting of PDE4B to treat respiratory diseases with fibrotic lesions in the lung.

Phosphodiesterase 2 mediates redox-sensitive endothelial cell proliferation and angiogenesis by thrombin via Rac1 and NADPH oxidase 2.

Cyclic nucleotide phosphodiesterases (PDEs) control the levels of the second messengers cAMP and cGMP in many cell types including endothelial cells. Although PDE2 has the unique property to be activated by cGMP but to hydrolyze cAMP, its role in endothelial function is only poorly understood. Reactive oxygen species (ROS) have been recognized as signaling molecules controlling many endothelial functions. We thus investigated whether PDE2 would link to ROS generation and proliferative responses in human umbilical vein endothelial cells in response to thrombin. Thrombin stimulated the GTPase Rac1, known to activate NADPH oxidases, and enhanced ROS formation, whereas PDE2 inhibition or depletion by short hairpin (sh)RNA prevented these responses. Similar observations were made with 8-Br-cGMP or atrial natriuretic peptide. In agreement, thrombin elevated cGMP but decreased cAMP levels, whereas db-cAMP or forskolin diminished Rac1 activity and ROS production. Subsequently, PDE2 overexpression activated Rac1, increased ROS generation, and enhanced proliferation and in vitro capillary formation. These responses were not observed in the presence of inactive Rac1 or shRNA against the NADPH oxidase subunit NOX2. Inhibition or depletion of PDE2 also prevented thrombin-induced proliferation and capillary formation. Importantly, downregulation of PDE2 by lentiviral shRNA or PDE2 inhibition prevented vessel sprouting from mouse aortic explants and in vivo angiogenesis in a mouse model, respectively. In summary, PDE2 promotes activation of NADPH oxidase-dependent ROS production and subsequent endothelial proliferation and angiogenesis. Targeting PDE2 may provide a new therapeutic approach in diseases associated with endothelial dysfunction, oxidative stress, vascular proliferation, and angiogenesis.

Phosphodiesterase inhibitors: history of pharmacology.

The first pharmacological investigations of phosphodiesterase (PDE) inhibitors were developed with the clinical efficacies of drugs isolated from coffee, cacao and tea but only later their relevant ingredients were identified as xanthines that act as PDE. With its diuretic, inotropic and bronchodilating clinical efficacy, use of theophylline anticipated the clinical goals, which were later approached with the first-generation of weakly selective PDE inhibitors in the period from 1980 to 1990. Pharmacological and clinical research with these early compounds provided a vast pool of information regarding desired and adverse actions - although most of these new drugs had to be discontinued due to severe adverse effects. The pharmacological models for cardiac, vascular and respiratory indications were analysed for their PDE isoenzyme profiles, and when biochemical and molecular biological approaches expanded our knowledge of the PDE superfamily, the purified isoenzymes that were now available opened the door for more systematic studies of inhibitors and for generation of highly selective isoenzyme-specific drugs. The development of simple screening models and clinically relevant indication models reflecting the growing knowledge about pathomechanisms of disease are summarised here for today's successful application of highly selective PDE3, PDE4 and PDE5 inhibitors. The interplay of serendipitous discoveries, the establishment of intelligent pharmacological models and the knowledge gain by research results with new substances is reviewed. The broad efficacies of new substances in vitro, the enormous biodiversity of the PDE isoenzyme family and the sophisticated biochemical pharmacology enabled Viagra to be the first success story in the field of PDE inhibitor drug development, but probably more success stories will follow.

Pharmacology, clinical efficacy, and tolerability of phosphodiesterase-4 inhibitors: impact of human pharmacokinetics.

Since more than two decades anti-inflammatory effects of inhibitors of phosphodiesterase-4 have been described in numerous cellular and animal studies and were finally confirmed in clinical trials. The path from an early, pioneering study with Ro20-1724 showing reduction of psoriatric plaque size in 1979 to modern PDE4 inhibitors such as oral apremilast in development for psoriasis, the inhaled PDE4 inhibitor GSK256066 in development for asthma and COPD and finally roflumilast, the first PDE4 inhibitor approved and currently marketed as an oral, once-daily remedy for severe COPD was marked by large progress in chemical optimization based on improved understanding of PDE4 biology and drug-like properties determining the appropriate pharmacokinetic profile. In this chapter aspects of the pharmacology and clinical efficacy of PDE4 inhibitors, which have been in clinical development over the years are summarized with specific emphasis on their clinical pharmacokinetic properties.

Cytokine-dependent balance of mitogenic effects in primary human lung fibroblasts related to cyclic AMP signaling and phosphodiesterase 4 inhibition.

Interleukin-1beta (IL-1beta) and basic fibroblast growth factor (bFGF) are important regulators of proliferation, and their expression is increased in lungs of patients with asthma, idiopathic pulmonary fibrosis (IPF), or chronic obstructive pulmonary disease (COPD). We investigated the effect of IL-1beta and bFGF on proliferation of human lung fibroblasts and the role of COX-2, PGE(2), and cAMP in this process. Furthermore, the effect of phosphodiesterase (PDE) 3 and 4 inhibition was analyzed. In primary human lung fibroblasts low concentrations of IL-1beta (<10 pg/ml) potentiated the bFGF-induced DNA synthesis, whereas higher concentrations revealed antiproliferative effects. Higher concentrations of IL-1beta-induced COX-2 mRNA and protein associated with an increase in PGE(2) and cAMP, and all of these parameters were potentiated by bFGF. The PDE4 inhibitor piclamilast concentration-dependently reduced proliferation by a partial G1 arrest. The PDE3 inhibitor motapizone was inactive by itself but enhanced the effect of the PDE4 inhibitor. This study demonstrates that bFGF and IL-1beta act in concert to fine-tune lung fibroblast proliferation resulting in amplification or reduction. The antiproliferative effect of IL-1beta is likely attributed to the induction of COX-2, which is further potentiated by bFGF, and the subsequent generation of PGE(2) and cAMP. Inhibition of PDE4 inhibition (rather than PDE3) may diminish proliferation of human lung fibroblasts and therefore could be useful in the therapy of pathological remodeling in lung diseases.

The preclinical pharmacology of roflumilast--a selective, oral phosphodiesterase 4 inhibitor in development for chronic obstructive pulmonary disease.

After more than two decades of research into phosphodiesterase 4 (PDE4) inhibitors, roflumilast (3-cyclopropylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide) may become the first agent in this class to be approved for patient treatment worldwide. Within the PDE family of 11 known isoenzymes, roflumilast is selective for PDE4, showing balanced selectivity for subtypes A-D, and is of high subnanomolar potency. The active principle of roflumilast in man is its dichloropyridyl N-oxide metabolite, which has similar potency as a PDE4 inhibitor as the parent compound. The long half-life and high potency of this metabolite allows for once-daily, oral administration of a single, 500-microg tablet of roflumilast. The molecular mode of action of roflumilast--PDE4 inhibition and subsequent enhancement of cAMP levels--is well established. To further understand its functional mode of action in chronic obstructive pulmonary disease (COPD), for which roflumilast is being developed, a series of in vitro and in vivo preclinical studies has been performed. COPD is a progressive, devastating condition of the lung associated with an abnormal inflammatory response to noxious particles and gases, particularly tobacco smoke. In addition, according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD), significant extrapulmonary effects, including comorbidities, may add to the severity of the disease in individual patients, and which may be addressed preferentially by orally administered remedies. COPD shows an increasing prevalence and mortality, and its treatment remains a high, unmet medical need. In vivo, roflumilast mitigates key COPD-related disease mechanisms such as tobacco smoke-induced lung inflammation, mucociliary malfunction, lung fibrotic and emphysematous remodelling, oxidative stress, pulmonary vascular remodelling and pulmonary hypertension. In vitro, roflumilast N-oxide has been demonstrated to affect the functions of many cell types, including neutrophils, monocytes/macrophages, CD4+ and CD8+ T-cells, endothelial cells, epithelial cells, smooth muscle cells and fibroblasts. These cellular effects are thought to be responsible for the beneficial effects of roflumilast on the disease mechanisms of COPD, which translate into reduced exacerbations and improved lung function. As a multicomponent disease, COPD requires a broad therapeutic approach that might be achieved by PDE4 inhibition. However, as a PDE4 inhibitor, roflumilast is not a direct bronchodilator. In summary, roflumilast may be the first-in-class PDE4 inhibitor for COPD therapy. In addition to being a non-steroid, anti-inflammatory drug designed to target pulmonary inflammation, the preclinical pharmacology described in this review points to a broad functional mode of action of roflumilast that putatively addresses additional COPD mechanisms. This enables roflumilast to offer effective, oral maintenance treatment for COPD, with an acceptable tolerability profile and the potential to favourably affect the extrapulmonary effects of the disease.

Effects of roflumilast, a phosphodiesterase-4 inhibitor, on hypoxia- and monocrotaline-induced pulmonary hypertension in rats.

Phosphodiesterase type 4 (PDE4) is involved in the hydrolysis of cAMP in pulmonary vascular smooth muscle (PA-SMC) and immune inflammatory cells. Given that intracellular cAMP accumulation inhibits contraction and growth of PA-SMCs as well as inflammatory cell functions, we investigated the effects of the PDE4 inhibitor 3-cyclopropylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide (roflumilast), on pulmonary hypertension (PH) in rats. Treatment with roflumilast (0.5 or 1.5 mg x kg(-1) day(-1)) from day 1 to day 21 after monocrotaline (MCT) injection (60 mg x kg(-1) s.c.) attenuated PH development: pulmonary artery pressure, right ventricular hypertrophy, and muscularization of distal vessels on day 21 were decreased compared to control MCT-treated rats. Roflumilast (1.5 mg x kg(-1) day(-1)) also reduced the increases in interleukin-6 and monocyte chemotactic protein-1 mRNAs observed in lung tissue on day 21 without affecting the rise in interleukin-1beta mRNA on days 1 and 21. Roflumilast (1.5 mg x kg(-1) day(-1)) from day 21 to day 42 after MCT reversed established PH, almost normalizing pulmonary artery pressure and structure, and suppressing proliferating cell nuclear antigen-positive cells in pulmonary vascular walls. Treatment with roflumilast similarly attenuated PH development due to chronic hypoxia. Treatment of human PA-SMCs with roflumilast N-oxide, the active metabolite of roflumilast, at concentrations up to 10(-6) M, potentiated PA-SMC growth inhibition induced by prostacyclin (10(-6) M) or interleukin-1beta (10 ng x ml(-1)) but was inactive on its own. In conclusion, the PDE4 inhibitor roflumilast significantly attenuates pulmonary vascular remodeling and hypertension induced by chronic hypoxia or MCT and reverses established PH after MCT administration.

Phosphodiesterase 2 inhibition diminished acute lung injury in murine pneumococcal pneumonia.

OBJECTIVE: Severe pneumococcal pneumonia frequently causes respiratory failure. Both pathogen factors and an uncontrolled host response may contribute to acute lung injury by impairing microvascular barrier function. Phosphodiesterase 2 (PDE2) was examined as a potential target in pneumonia-induced lung microvascular hyperpermeability. DESIGN: Controlled, in vitro, ex vivo, and in vivo laboratory study. SUBJECTS: Female Balb/C and C57Bl/6 mice, 8-12 weeks old. INTERVENTIONS: Human umbilical vein endothelial cells and isolated mouse lungs were challenged with the pneumococcal exotoxin pneumolysin in the presence or absence of the selective PDE2 inhibitors 9-(6-phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6one (PDP) or hydroxy-PDP. Transcellular electrical resistance or human serum albumin leakage in bronchoalveolar lavage fluid was determined, respectively. In addition, we induced pneumococcal pneumonia in mice and treated with hydroxy-PDP via continuous subcutaneous application by osmotic pumps. Human serum albumin leakage in bronchoalveolar lavage fluid was measured 48 hours after transnasal infection, and lung specimens were analyzed by TaqMan real-time polymerase chain reaction and Western blot for PDE2 gene and protein expression. MEASUREMENTS AND MAIN RESULTS: In isolated perfused mouse lungs and in human umbilical vein endothelial cell monolayers, selective inhibition of PDE2 markedly decreased pneumolysin-induced hyperpermeability. Furthermore, in murine pneumococcal pneumonia, pulmonary PDE2-mRNA and -protein expression was significantly increased, and pneumonia-induced vascular permeability was distinctively reduced by PDE2 inhibition. CONCLUSIONS: PDE2 inhibition diminished microvascular leakage in pneumococcal pneumonia, and pulmonary PDE2 upregulation may play a crucial role in this respect. Selective PDE2 inhibitors thus may offer a promising therapeutic approach in severe pneumococcal pneumonia.

Celecoxib dilates guinea-pig coronaries and rat aortic rings and amplifies NO/cGMP signaling by PDE5 inhibition.

OBJECTIVE: Celecoxib carries a smaller cardiovascular risk for myocardial infarction and hypertension than other cyclooxygenase-2 (COX-2)-selective non-steroidal anti-inflammatory drugs NSAIDs ("coxibs") and may ameliorate endothelial dysfunction. We aimed to determine which mechanism possibly accounts for the beneficial effect by investigating its vascular action in different in vitro preparations in comparison with other coxibs and reference phosphodiesterase-5 (PDE5) inhibitors. METHODS: To uncover potential effects on coronary flow, the effects of celecoxib in comparison with other NSAIDs and the PDE5 inhibitors, sildenafil and zaprinast, were investigated in guinea-pig Langendorff heart. This was supported by studies for vasorelaxation, interaction with the NO/cGMP pathway, and measurement of cyclic nucleotide amounts released from rat aortic rings, and inhibition of human PDE5 as well as PDE4 activity. RESULTS: Bolus injections of sildenafil, celecoxib, and zaprinast (at 100 nmol) into the Langendorff heart increased coronary flow by approximately 100, 65, and 25%, respectively, while rofecoxib, lumiracoxib, parecoxib, and diclofenac, except valdecoxib (>100 nmol), failed to increase coronary flow up to 300 nmol. In rat aorta, sildenafil, celecoxib and zaprinast caused endothelium-dependent relaxation with -log[EC(50)]M values of 8.90, 6.66 and 5.56, respectively; their rank order of potency corresponds to their coronary dilatory effect. Celecoxib-induced relaxation of aorta was attenuated by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) and by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ, 10(-5) M). In aortic rings, celecoxib (3x10(-5) M) caused a fivefold increase in the cGMP level and potentiated that induced by sodium nitroprusside (5x10(-7) M). Celecoxib and valdecoxib inhibited human PDE5A1 with an IC(50) of 1.6x10(-5) and 1x10(-4) M, respectively, whereas other coxibs were without inhibitory effect. CONCLUSION: Celecoxib caused coronary vasodilatation in guinea-pig hearts and relaxation of rat aorta and had a potentiating effect on the NO/cGMP signaling pathway in rat aorta through specific blockade of PDE5. These unexpected findings clearly support the notion that celecoxib possesses an as yet undisclosed molecule-specific property that possibly compensates a decrease of prostacyclin-dependent cAMP generation by concomitantly increasing cGMP levels resulting from inhibition of PDE5.

Characterization of inhibitors of phosphodiesterase 1C on a human cellular system.

Different inhibitors of the Ca(2+)/calmodulin-stimulated phosphodiesterase 1 family have been described and used for the examination of phosphodiesterase 1 in cellular, organ or animal models. However, the inhibitors described differ in potency and selectivity for the different phosphodiesterase family enzymes, and in part exhibit additional pharmacodynamic actions. In this study, we demonstrate that phosphodiesterase 1C is expressed in the human glioblastoma cell line A172 with regard to mRNA, protein and activity level, and that lower activities of phosphodiesterase 2, phosphodiesterase 3, phosphodiesterase 4 and phosphodiesterase 5 are also present. The identity of the phosphodiesterase 1C activity detected was verified by downregulation of the mRNA and protein through human phosphodiesterase 1C specific small interfering RNA. In addition, the measured K(m) values (cAMP, 1.7 microm; cGMP, 1.3 microm) are characteristic of phosphodiesterase 1C. We demonstrate that treatment with the Ca(2+) ionophore ionomycin increases intracellular Ca(2+) in a concentration-dependent way without affecting cell viability. Under conditions of enhanced intracellular Ca(2+) concentration, a rapid increase in cAMP levels caused by the adenylyl cyclase activator forskolin was abolished, indicating the involvement of Ca(2+)-activated phosphodiesterase 1C. The reduction of forskolin-stimulated cAMP levels was reversed by phosphodiesterase 1 inhibitors in a concentration-dependent way. Using this cellular system, we compared the cellular potency of published phosphodiesterase 1 inhibitors, including 8-methoxymethyl-3-isobutyl-1-methylxanthine, vinpocetine, SCH51866, and two established phosphodiesterase 1 inhibitors developed by Schering-Plough (named compounds 31 and 30). We demonstrate that up to 10 microm 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine had no effect on the reduction of forskolin-stimulated cAMP levels by ionomycin, whereas the more selective and up to 10 000 times more potent phosphodiesterase 1 inhibitors SCH51866, compound 31 and compound 30 inhibited the ionomycin-induced decline of forskolin-induced cAMP at nanomolar concentrations. Thus, our data indicate that SCH51866 and compounds 31 and 30 are effective phosphodiesterase 1 inhibitors in a cellular context, in contrast to the weakly selective and low-potency phosphodiesterase inhibitors 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine. A172 cells therefore represent a suitable system in which to study the cellular effect of phosphodiesterase 1 inhibitors. 8-Methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine seem not to be suitable for the study of phosphodiesterase 1-mediated functions.

Inhibition of TGF-beta induced lung fibroblast to myofibroblast conversion by phosphodiesterase inhibiting drugs and activators of soluble guanylyl cyclase.

Pulmonary fibroblast to myofibroblast conversion is a pathophysiological feature of idiopathic pulmonary fibrosis and COPD. This conversion is induced by transforming growth factor (TGF)-beta derived from epithelial cells as well as activated macrophages that have infiltrated the lung. Preventing this conversion might be a favourable therapeutic approach. Within this study we examined the activity of different members of the phosphodiesterase (PDE) family in primary human lung fibroblasts and various lung fibroblast cell lines both before and after TGF-beta induced differentiation to myofibroblasts as reflected by the expression of alpha-smooth muscle actin. We showed that the predominant PDE activities in lung fibroblasts are attributed to PDE5, PDE1 and to a smaller extent to PDE4. cyclic GMP (cGMP)-hydrolyzing activity declines by about half after differentiation to myofibroblasts in all pulmonary fibroblasts investigated, which is accompanied by a down-regulation of PDE5 protein. Lung fibroblast to myofibroblast differentiation is blocked by treatment with the PDE4 inhibitor piclamilast alone, depending on the TGF-beta concentration applied, and in combination with prostaglandin E(2) (PGE(2)) in a synergistic manner. Despite the high PDE5 activity the PDE5 inhibitor sildenafil by itself as well as in combination with brain natriuretic peptide or the nitric oxide-donor DETA-NONOate shows no inhibiting effects. However, combining sildenafil with the guanylyl cyclase (GC) activator BAY58-2667 and ODQ (which sensitizes GC for activation by BAY58-2667) suppressed TGF-beta induced differentiation. In summary, our data indicate that drugs interfering with the cyclic AMP (cAMP)-as well as with the NO-cGMP-pathway offer the therapeutic opportunity to prevent the differentiation of pulmonary fibroblasts to myofibroblasts in lung fibrosis.

The effect of Sildenafil on human platelet secretory function is controlled by a complex interplay between phosphodiesterases 2, 3 and 5.

Human platelets contain the cyclic nucleotide-hydrolyzing phosphodiesterases (PDEs) 2, 3 and 5. The cGMP-PDE5 inhibitors Sildenafil and Zaprinast have been demonstrated to potentiate the anti-platelet aggregatory effect of NO donors like sodium nitroprusside (SNP) in vitro but the mechanisms of Sildenafil's action on the secretory function of human platelets have not been analysed in detail. In the present paper, we show (1) that both compounds potentiate the SNP-induced increase in cGMP in human platelets concentration-dependently. (2) However, whereas Sildenafil plus SNP treatment only partially inhibits thrombin-induced release of serotonin, the less selective Zaprinast plus SNP cause a complete inhibition. (3) The inhibition mediated by Sildenafil plus SNP is limited to low compound concentrations at which cAMP levels are increased, probably due to cGMP-mediated inhibition of PDE3. (4) High concentrations of Sildenafil (plus SNP) neither affect cAMP levels, likely due to the activation of PDE2, nor inhibits the release of serotonin. Thus, increases in both cyclic nucleotides seem to control platelet function. (5) Accordingly, treatment with increasing concentrations of Sildenafil plus SNP and a selective PDE2 inhibitor, which by its own has no effect, induced a concentration-dependent increase in cAMP and complete inhibition of platelet activation. In summary, our data indicate that Sildenafil inhibits secretory function of human platelets at least in part due to the cGMP-mediated effects on intracellular cAMP and that entire inhibition of serotonin release from thrombin-activated platelets is controlled by both cyclic nucleotides.

Novel selective PDE4 inhibitors. 3. In vivo antiinflammatory activity of a new series of N-substituted cis-tetra- and cis-hexahydrophthalazinones.

The synthesis and biological activities of a series of N-substituted cis-4a,5,6,7,8,8a-hexa- and cis-4a,5,8,8a-tetrahydro-2H-phthalazin-1-ones are described. It was found that compounds bearing a cycloalkyl group at the 2-position exhibit the highest PDE4 inhibitory activities (pIC(50) = 8.6-9.4). The N-cycloheptyl- and N-adamantanyltetrahydrophthalazinones (7h, 8, 10, 11) show high in vivo antiinflammatory activities after oral application. Additionally, some phthalazinones were found to exhibit potent suppression of LPS-induced TNFalpha release and show moderate potency against fMLP-stimulated production of ROS.

Novel selective phosphodiesterase (PDE4) inhibitors. 4. Resolution, absolute configuration, and PDE4 inhibitory activity of cis-tetra- and cis-hexahydrophthalazinones.

Recently, we reported that 4-catechol-substituted cis-(+/-)-4a,5,6,7,8,8a-hexa- and cis-(+/-)-4a,5,8,8a-tetrahydro-2H-phthalazin-1-ones show potent inhibition of phosphodiesterase (PDE4) activity, while the corresponding trans racemic mixtures exhibit only weak to moderate activity. To determine the absolute configuration and PDE4 inhibitory activity of the individual cis-enantiomers, several optically active phthalazinones have been synthesized. The enantiomers of the various gamma-keto acids, used as starting materials, were resolved in a classical way by the formation of diastereomeric salts, and each was converted to optically active phthalazinone in an enantioselective manner. The absolute configuration of the (+)-enantiomer of cis-hexahydrophthalazinone (+)-12 was determined by X-ray crystallography. The carbon atoms at the 4a and 8a positions were found to have the S- and R-configuration, respectively. In the present series of hexa- and tetrahydrophthalazinones, stereoselectivity for PDE4 inhibition is observed; the cis-(+)-enantiomers of the phthalazinones display high inhibitory activity, whereas their (-)-counterparts exhibit only weak to moderate activity. It is likely that all cis-(+)-phthalazinones have a (4aS,8aR)-configuration and vice versa for the cis-(-)-analogues. In the current series, the N-adamantan-2-yl analogue (+)-14 shows the most potent inhibition of PDE4 (pIC(50) = 9.3); the corresponding (-)-enantiomer is 250-fold less active. In addition, the N-substituted tetrahydrophthalazinones under study were investigated for their in vivo antiinflammatory activities by examining the suppression of arachidonic acid (AA) induced mouse ear edema formation. In this assay analogues (+)-14 and (+)-15 were found to be potent antiinflammatory agents showing about 50% inhibition at 30 micromol/kg po.

Synthesis and structure-activity relationships of cis-tetrahydrophthalazinone/pyridazinone hybrids: a novel series of potent dual PDE3/PDE4 inhibitory agents.

In this study, the synthesis and in vitro and in vivo pharmacological investigations of a new series of phthalazinone/pyridazinone hybrids with both PDE3 and PDE4 inhibitory activities are described. These compounds combine the pharmacophores of recently discovered 4a,5,8,8a-tetrahydro-2H-phthalazin-1-one-type inhibitors of PDE4 and the well-known 2H-pyridazin-3-one-type PDE3 inhibitors such as the tetrahydrobenzimidazoles. Most of the synthesized compounds are pharmacologically spoken PDE3/PDE4 hybrids. All hybrids show potent PDE4 inhibitory activity (pIC(50) = 7.0-8.7), whereas the pIC(50) values for inhibition of PDE3 vary from 5.4 to 7.5. In general, analogues with a 5-methyl-4,5-dihydropyridazinone moiety exhibit the highest PDE3 inhibitory activities. The highest in vivo antiinflammatory activity is displayed by phthalazinones 43 and 44 showing, at a dose of 30 micromol/kg po, 46% inhibition of arachidonic acid (AA) induced mouse ear edema. No correlation was found between the in vitro PDE3 and/or PDE4 inhibitory activity and the in vivo antiinflammatory capacity after oral dosing.

Phosphodiesterase isoenzyme families in human osteoarthritis chondrocytes--functional importance of phosphodiesterase 4.

We studied whether selective inhibitors of cyclic nucleotide hydrolysing phosphodiesterase (PDE) isoenzymes influence IL-1beta-induced nitric oxide (NO) release from human articular chondrocytes. In addition, the pattern of PDE isoenzymes contributing to cyclic nucleotide hydrolysis in human chondrocytes was characterized. Chondrocytes were isolated from human osteoarthritic cartilage and cultured in alginate beads. IL-1beta-induced chondrocyte products (nitric oxide and prostaglandin E(2)) were measured in culture supernatants after 48 h incubation time. PDE activities were assessed in chondrocyte lysates. Inducible nitric oxide synthase (iNOS) and PDE4A-D proteins were detected by immunoblotting. The selective PDE4 inhibitors Piclamilast and Roflumilast partially attenuated IL-1beta-induced NO production whereas selective inhibitors of PDE2 (EHNA), PDE3 (Motapizone) or PDE5 (Sildenafil) were inactive. Indomethacin reversed the reduction of IL-1beta-induced NO by PDE4 inhibitors. It was shown that autocrine prostaglandin E(2) (PGE(2)) enabled PDE4 inhibitors to reduce IL-1beta-induced NO in this experimental setting. Major PDE4 and PDE1 activities were identified in chondrocyte lysates whereas only minor activities of PDE2, 3 and 5 were found. IL-1beta and cyclic AMP-mimetics upregulated PDE4 activity and this was associated with an augmentation of PDE4B2 protein. Based on the view that nitric oxide contributes to cartilage degradation in osteoarthritis our study suggests that PDE4 inhibitors may have chondroprotective effects.

The new phosphodiesterase 4 inhibitor roflumilast is efficacious in exercise-induced asthma and leads to suppression of LPS-stimulated TNF-alpha ex vivo.

Roflumilast is a new phosphodiesterase 4 (PDE4) inhibitor developed by Byk Gulden Pharmaceuticals for the treatment of chronic obstructive pulmonary disease and asthma. A placebo-controlled, randomized, double-blind, two-period crossover study was performed to investigate the safety and efficacy of roflumilast in 16 patients with exercise-induced asthma. The patients received placebo or roflumilast (500 microg/day) for 28 days, each according to the randomly determined treatment sequences roflumilast/placebo and placebo/roflumilast. In both study periods, exercise challenge was performed 1 hour after dosing on days 1, 14, and 28. FEV1 was measured before exercise challenge, immediately after the end of exercise challenge, and then at 1, 3, 5, 7, 9, and 12 minutes after the end of challenge. Blood samples for the determination of lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNF-alpha) in whole blood ex vivo as a surrogate marker for the inhibition of inflammatory cell activation were taken predose on days 1 and 28. Serial safety measurements were performed during both study periods. Analysis of variance for the crossover design showed a significant superiority of roflumilast over placebo on day 28. The mean percentage fall of FEV1 after exercise was reduced by 41% as compared to placebo (p = 0.021). An improvement of lung function during roflumilast treatment was also observed on days 1 and 14. The median TNF-alpha level decreased by 21% (p = 0.009) during roflumilast treatment but remained essentially constant under placebo. It is concluded that roflumilast is effective in the treatment of exercise-induced asthma. This result was accompanied by a significant reduction of TNF-alpha levels ex vivo. Treatment with roflumilast was safe and well tolerated.

Inhibition of PDE3, PDE4 and PDE7 potentiates glucocorticoid-induced apoptosis and overcomes glucocorticoid resistance in CEM T leukemic cells.

Stimulation of the cAMP signaling pathway has been shown to induce apoptosis and augment the effects of glucocorticoids in inducing apoptosis in leukemic cells. We recently reported that in primary B cell chronic lymphocytic leukemic (B-CLL) cells, apoptosis could be induced by stimulating the cAMP signaling pathway with a phosphodiesterase4 (PDE4) inhibitor alone; while in contrast, in the CEM T leukemic cell line, PDE4 inhibitors alone were ineffective, and concurrent stimulation of adenylyl cyclase was required to see effects [Tiwari et al. (2005)]. We report here that in the CEM and Jurkat T leukemic cell lines, the most abundantly expressed PDEs are PDE3B, PDE4A, PDE4D, PDE7A, and PDE8A. Selective inhibition of PDE3, PDE4 or PDE7 alone produces little effect on cell viability, but inhibition of all three of these PDEs together dramatically enhances glucocorticoid-induced apoptosis in CEM cells, and overcomes glucocorticoid resistance in a glucocorticoid-resistant CEM cell line. These studies indicate that for some leukemic cell types, a desired therapeutic effect may be achieved by inhibiting more than one form of PDE.

Differential expression and function of phosphodiesterase 4 (PDE4) subtypes in human primary CD4+ T cells: predominant role of PDE4D.

Type 4 phosphodiesterases (PDE4) are critical regulators in TCR signaling by attenuating the negative constraint of cAMP. In this study, we show that anti-CD3/CD28 stimulation of human primary CD4(+) T cells increases the expression of the PDE4 subtypes PDE4A, PDE4B, and PDE4D in a specific and time-dependent manner. PDE4A and PDE4D mRNAs as well as enzyme activities were up-regulated within 5 days, PDE4B showed a transient up-regulation with highest levels after 24 h. The induction was shown to be independent of different stimulation conditions and was similar in naive and memory T cell subpopulations. To elucidate the functional impact of individual PDE4 subtypes on T cell function, we used PDE4 subtype-specific short-interfering RNAs (siRNAs). Knockdown of either PDE4B or PDE4D inhibited IL-2 release 24 h after stimulation (time point of maximal IL-2 concentrations) to an extent similar to that observed with the panPDE4 inhibitor RP73401 (piclamilast). Substantial amounts of IFN-gamma or IL-5 were measured only at later time points. siRNA targeting PDE4D showed a predominant inhibitory effect on these cytokines measured after 72 h. However, the inhibition of all cytokines was most effective when PDE4 siRNAs were applied in combination. Although the effect of PDE4 inhibition on T cell proliferation is small, the PDE4D-targeting siRNA alone was as effective as the panPDE4 inhibitor, whereas PDE4A or PDE4B siRNAs had hardly an effect. In summary, individual PDE4 subtypes have overall nonredundant, but complementary, time-dependent roles in propagating various T cell functions and PDE4D is the form likely playing a predominant role.

PKCtheta and PKA are antagonistic partners in the NF-AT transactivation pathway of primary mouse CD3+ T lymphocytes.

We here investigate the crosstalk of PKC and PKA signaling during primary CD3(+) T-lymphocyte activation using pharmacologic inhibitors and activators in combination with our established panel of PKC isotype-deficient mouse T cells in vitro. PKCtheta and PKA inversely affect the CD3/CD28-induced IL-2 expression, whereas other PKC isotypes are dispensable in this signaling pathway. Gene ablation of PKCtheta selectively results in a profound reduction of IL-2 production; however, complete abrogation of IL-2 production in these PKCtheta(-/-) T cells was achieved only by simultaneous coactivation of the cAMP/PKA pathway in CD3(+) T cells. Conversely, the reduced IL-2 production in PKC inhibitor-treated T cells can be rescued by inhibition of the cAMP/PKA pathway in wild-type but not in PKCtheta(-/-) T cells. Mechanistically, the cAMP/PKA and PKCtheta pathways converge at the level of NF-AT, as shown by DNA binding analysis. The combined increase in PKA and decrease in PKCtheta activity leads to an enhanced inhibition of nuclear NF-AT translocation. This PKCtheta/PKA crosstalk significantly affects neither the NF-kappaB, the AP-1, nor the CREB pathways. Taken together, this opposite effect between the positive PKCtheta and the negative cAMP/PKA signaling pathways appears rate limiting for NF-AT transactivation and IL-2 secretion responses of CD3(+) T lymphocytes.