ASP3258, an orally active potent phosphodiesterase 4 inhibitor with low emetic activity

We investigated the pharmacology of a novel phosphodiesterase (PDE) 4 inhibitor, ASP3258 (3-[4-(3-chlorophenyl)-1-ethyl-7-methyl-2-oxo-1,2-dihydro-1,8-naphthyridin-3-yl] propanoic acid), comparing its potency with that of the most advanced PDE4 inhibitors, roflumilast and cilomilast. PDE4 inhibition by ASP3258 (IC(50)=0.28nM) was as potent as that achieved with roflumilast. ASP3258 inhibited lipopolysaccharide-induced tumor necrosis factor (TNF)-α production in rat whole blood cells (IC(50)=8.8 nM) and rat alveolar macrophages (IC(50)=2.6 nM). Orally administered ASP3258, roflumilast, and cilomilast dose-dependently inhibited production of interleukin-4, TNF-α, and cysteinyl leukotrienes, as well as leukocyte infiltration in bronchoalveolar lavage fluid from the airways of ovalbumin-sensitized Brown Norway rats, and these compounds showed almost complete inhibition at doses of 3, 3, and 30 mg/kg, respectively. PDE4 inhibitors induce emesis by mimicking the pharmacological action of α(2)-adrenoceptor antagonist. However, orally administered roflumilast (3mg/kg) and cilomilast (10mg/kg), but not ASP3258 (3mg/kg), inhibited α(2)-adrenoceptor agonist-induced anesthesia in rats and induced emesis in ferrets. Although ASP3258 (3mg/kg) inhibited airway inflammation completely, it had no emetic activity. As such, this compound may be useful in treating airway inflammatory diseases such as asthma and COPD.     

Anti-neutrophilic inflammatory activity of ASP3258, a novel phosphodiesterase type 4 inhibitor

Neutrophil-dominant pulmonary inflammation is an important feature of chronic obstructive pulmonary disease (COPD). Here, we evaluated the in vitro and in vivo anti-neutrophilic inflammatory activities of ASP3258, a novel, orally active, and selective phosphodiesterase (PDE) 4 inhibitor with anti-inflammatory potency comparable to that of second-generation compound roflumilast but with lower emetic activity in vivo. In in vitro experiments using human peripheral blood neutrophils, PDE4 inhibitors ASP3258, cilomilast, and roflumilast inhibited fMLP-induced superoxide production in a concentration-dependent manner with IC50 values of 5.0, 96, and 4.7 nM, respectively. ASP3258, cilomilast, and roflumilast also attenuated fMLP-induced neutrophil chemotaxis in a concentration-dependent manner with IC30 values of 18, 270, and 9.7 nM, respectively. In contrast, the glucocorticoid prednisolone inhibited neither superoxide production nor chemotaxis up to 1 μM. In a rat model of lipopolysaccharide (LPS)-induced lung inflammation, orally administered ASP3258, cilomilast, roflumilast, and prednisolone (at 10 or 30 mg/kg) dose-dependently attenuated pulmonary accumulation of neutrophils. The inhibitory effect of ASP3258 was more potent than cilomilast and almost the same as roflumilast and prednisolone. Treatment with ASP3258 inhibited the elevation of TNF-α in the bronchoalveolar lavage fluid following LPS instillation. Histological examination revealed significant inhibition of neutrophil and macrophage infiltration into alveoli by ASP3258. Overall, these findings suggest that ASP3258 has therapeutic potential for treating neutrophilic inflammation such as COPD, partly through direct inhibition of neutrophil activation as well as possibly through inhibition of the TNF-α-mediated pathway.     

Anti-asthmatic effect of ASP3258, a novel phosphodiesterase 4 inhibitor

ASP3258 is a potent and selective PDE4 inhibitor and exerts a wide-range of anti-inflammatory effects with low emetic potential, a major adverse effect of PDE4 inhibitors. Here, we investigated the anti-asthmatic potency of ASP3258 as compared with those of two representative PDE4 inhibitors: roflumilast and cilomilast. Orally administered ASP3258, roflumilast, and cilomilast all inhibited ovalbumin (OVA)-induced eosinophil infiltration into the airway of sensitized Brown Norway rats with ED(50) values of 0.81, 0.46, and 4.4 mg/kg, respectively. Histological examination also revealed a decreasing trend in inflammatory cell infiltration into the lung following ASP3258 administration. In vitro investigation of bronchodilatory activities showed that these compounds (10(-8)-10(-6) M) concentration-dependently inhibited OVA-induced contraction of trachea isolated from sensitized guinea pigs but had no effect on spasmogen-precontracted tracheal tension prepared from non-sensitized guinea pigs up to 10(-6) M. In vivo experiments using sensitized guinea pigs showed that these orally administered compounds inhibited OVA-induced increases in airway resistance with ED(50) values of 2.2, 0.35, and 12 mg/kg, respectively. Further, orally administered ASP3258 (0.1 and 1 mg/kg), roflumilast (0.1 and 1 mg/kg), and cilomilast (10 mg/kg) significantly suppressed airway hyperresponsiveness caused by OVA exposure. ASP3258's potent inhibition of antigen-induced bronchoconstriction and airway hyperresponsiveness, two characteristic symptoms of bronchial asthma, suggests that this compound will be useful in treating asthma.     

Effects of the new benzimidazole derivative TAS-203, an orally active phosphodiesterase 4 inhibitor, on airway inflammation in rats and emetic responses in ferrets

TAS-203 (2-[3-(cyclopropylmethoxy)-4-methoxyphenyl]-5-(1H-1,2,4-triazol-1-yl)-1H-benzimidazole, CAS 223909-92-0) is a novel phosphodiesterase 4 (PDE4) inhibitor that has been found to have good anti-inflammatory effects and low emetogenic activity in vivo. In the present studies, the anti-inflammatory profile of TAS-203 was examined and compared with that of cilomilast (CAS 153259-65-5), the most advanced PDE4 inhibitor. TAS-203 inhibited the activity of purified human PDE4 with an IC50 value of 88 nM and also the recombinant PDE4 subtypes (4A, 4B, 4C and 4D) with respective IC50 values of 47, 35, 227 and 43 nM. In the experiments using inflammatory cells, TAS-203 concentration-dependently inhibited platelet-activating factor-induced eosinophil chemotaxis and lipopolysaccharide (LPS)-stimulated tumor necrosis factor-a release from human monocytes with respective IC50 values of 250 and 38.5 nM. For airway inflammation, TAS-203 at 10 mg/kg and cilomilast at 30 mg/kg significantly inhibited antigen-induced airway eosinophilia and LPS-induced airway neutrophilia in rats. The emetogenicity of TAS-203 and cilomilast was evaluated in a ferret model of emesis. The maximum dose of TAS-203 not carrying emesis was 100 mg/kg, while that of cilomilast was less than 10 mg/kg. Finally, TAS-203 was found to be poorly distributed to the brain after oral administration of 10 mg/kg TAS-203 in rats. These results indicate that TAS-203 is an orally active PDE4 inhibitor with potent anti-inflammatory activities and low emetogenicity that may be useful in the treatment of airway inflammatory conditions such as asthma and chronic obstructive pulmonary disease.     

Roflumilast inhibits leukocyte-endothelial cell interactions, expression of adhesion molecules and microvascular permeability

BACKGROUND AND PURPOSE: The present study addressed the effects of the investigational PDE4 inhibitor roflumilast on leukocyte-endothelial cell interactions and endothelial permeability in vivo and in vitro. EXPERIMENTAL APPROACH: In vivo, intravital video-microscopy was used to determine effects of roflumilast p.o. on leukocyte-endothelial cell interactions and microvascular permeability in rat mesenteric venules. In vitro, the effects of roflumilast N-oxide, the active metabolite of roflumilast in humans, and other PDE4 inhibitors on neutrophil adhesion to tumour necrosis factor alpha (TNFalpha)-activated human umbilical vein endothelial cells (HUVEC), E-selectin expression and thrombin-induced endothelial permeability was evaluated. Flow cytometry was used to determine the effect of roflumilast on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced CD11b upregulation on human neutrophils. KEY RESULTS: In vivo, roflumilast, given 1 h before lipopolysaccharide (LPS), dose-dependently reduced leukocyte-endothelial cell interactions in rat mesenteric postcapillary venules. It also diminished histamine-induced microvascular permeability. Immunohistochemical analyses revealed that roflumilast prevented LPS-induced endothelial P- and E-selectin expression. In vitro, roflumilast N-oxide concentration-dependently suppressed neutrophil adhesion to TNFalpha-activated HUVEC and CD11b expression on fMLP-stimulated neutrophils. It also reduced TNFalpha-induced E-selectin expression on HUVEC, when PDE3 activity was blocked. HUVEC permeability elicited by thrombin was concentration-dependently suppressed by roflumilast N-oxide. While roflumilast N-oxide was as potent as roflumilast at inhibiting stimulated endothelial cell and neutrophil functions, both compounds were significantly more potent than the structurally unrelated PDE4 inhibitors, rolipram or cilomilast. CONCLUSIONS AND IMPLICATIONS: These findings further support earlier observations on the inhibition of inflammatory cell influx and protein extravasation by roflumilast in vivo.     

Phosphodiesterase inhibitors in airways disease

Phosphodiesterases hydrolyse intracellular cyclic nucleotides, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) into inactive 5' monophosphates, and exist as 11 families. They are found in a variety of inflammatory and structural cells. Inhibitors of PDEs allow the elevation of cAMP and cGMP which lead to a variety of cellular effects including airway smooth muscle relaxation and inhibition of cellular inflammation or of immune responses. PDE4 inhibitors specifically prevent the hydrolysis of cAMP, and PDE4 isozymes are present in inflammatory cells. Selective PDE4 inhibitors have broad spectrum anti-inflammatory effects such as inhibition of cell trafficking, cytokine and chemokine release from inflammatory cells, such as neutrophils, eosinophils, macrophages and T cells. The second generation PDE4 inhibitors, cilomilast and roflumilast, have reached clinical trial stage and have some demonstrable beneficial effects in asthma and chronic obstructive pulmonary disease (COPD). The effectiveness of these PDE4 inhibitors may be limited by their clinical potency using doses that have minimal effects on nausea and vomiting. Topical administration of PDE4 inhibitors may provide a wider effective to side-effect profile. Development of inhibitors of other PDE classes, combined with PDE4 inhibition, may be another way forward. PDE5 is an inactivator of cGMP and may have beneficial effects on hypoxic pulmonary hypertension and vascular remodelling. PDE3 and PDE7 are other cAMP specific inactivators of cAMP. PDE7 is involved in T cell activation and a dual PDE4-PDE7 inhibitor may be more effective in asthma and COPD. A dual PDE3-PDE4 compound may provide more bronchodilator and bronchoprotective effect in addition to the beneficial PDE4 effects.     

Roflumilast inhibits the release of chemokines and TNF-α from human lung macrophages stimulated with lipopolysaccharide

BACKGROUND AND PURPOSE

Lung macrophages are critically involved in respiratory diseases. This study assessed the effects of the PDE4 inhibitor roflumilast and its active metabolite, roflumilast N-oxide on the release of a range of chemokines (CCL2, 3, 4, CXCL1, 8, 10) and of TNF-α, from human lung macrophages, stimulated with bacterial lipopolysaccharide LPS.

EXPERIMENTAL APPROACH

Lung macrophages isolated from resected human lungs were incubated with roflumilast, roflumilast N-oxide, PGE₂, the COX inhibitor indomethacin, the COX-2 inhibitor NS-398 or vehicle and stimulated with LPS (24 h). Chemokines, TNF-α, PGE₂ and 6-keto PGF_1α were measured in culture supernatants by immunoassay. COX-2 mRNA expression was assessed with RT-qPCR. PDE activities were determined in macrophage homogenates.

KEY RESULTS

Expression of PDE4 in lung macrophages was increased after incubation with LPS. Roflumilast and roflumilast N-oxide concentration-dependently reduced the LPS-stimulated release of CCL2, CCL3, CCL4, CXCL10 and TNF-α from human lung macrophages, whereas that of CXCL1 or CXCL8 was not altered. This reduction by the PDE4 inhibitors was further accentuated by exogenous PGE₂ (10 nM) but abolished in the presence of indomethacin or NS-398. Conversely, addition of PGE₂ (10 nM), in the presence of indomethacin restored inhibition by roflumilast. LPS also increased PGE₂ and 6-keto PGF_1α release from lung macrophages which was associated with an up-regulation of COX-2 mRNA.

CONCLUSIONS AND IMPLICATIONS

Roflumilast and roflumilast N-oxide reduced LPS-induced release of CCL2, 3, 4, CXCL10 and TNF-α in human lung macrophages.     

Phosphodiesterase-4 inhibitors in the treatment of inflammatory lung disease

Phosphodiesterases (PDE) belong to an important family of proteins that regulate the intracellular levels of cyclic nucleotide second messengers. Targeting PDE with selective inhibitors may offer novel therapeutic strategies in the treatment of various conditions, and in the context of respiratory disease these include asthma and chronic obstructive pulmonary disease (COPD). The rationale for such an approach stems, in part, from the clinical efficacy of theophylline, an orally active drug that is purportedly a nonselective PDE inhibitor. In addition, intracellular cyclic adenosine monophosphate (cAMP) levels regulate the function of many of the cells thought to contribute to the pathogenesis of respiratory diseases such as asthma and COPD, and these cells also selectively express PDE4. This has offered pharmaceutical companies the opportunity to selectively targeting these enzymes for the treatment of these diseases. Finally, the success of targeting PDE5 in the treatment of erectile dysfunction provides clinical proof of concept for the targeting of PDE in disease. Whether a 'Viagra' of the airways can be found for the treatment of asthma and COPD remains to be seen, but positive results from recent clinical studies examining the efficacy of selective PDE4 inhibitors such as cilomilast and roflumilast offer some optimism. However, one of the major issues to be resolved is the tolerability profile associated with this drug class that is a consequence of PDE4 inhibition. While cilomilast and roflumilast have low emetic potential they are not free from emesis and various strategies are being investigated in the hope of developing a PDE4 inhibitor without this adverse effect.     

Phosphodiesterase 4 inhibitors for the treatment of asthma and COPD

Type 4 cyclic nucleotide phosphodiesterases (PDE4s) are metallo-hydrolases which specifically hydrolyze cAMP to AMP in various cells types. The catalytic core is a bimetallic ion center composed of a tightly bound Zn(2+) and a loosely bound Mg(2+), which plays a dictating role in eliciting cAMP binding and catalysis activation. An invariant glutamine positioned opposite to the ion center serves as the substrate recognition determinant and synergizes the transient Mg-oxo-phosphate interaction in the substrate complex. The Mg(2+) binding is activated by a PKA-mediated serine phosphorylation and modulated through protein-protein interactions, thus, providing efficient mechanisms in the temporal regulation of cAMP signaling. Several PDE4 inhibitors including roflumilast, cilomilast and rolipram also rely on the interaction with the glutamine and metallic ion center for binding, with their affinity enhanced dramatically by the presence of the Mg(2+) ion. Recent studies have provided new insights into the role of this enzyme in inflammatory settings, CFTR regulation, long term potentiation, and its importance in immune surveillance. The major inflammatory cytokines which are modulated with PDE4 inhibitors include TNFalpha, IL-2, IFNgamma, IL-12, GM-CSF and LTB(4). The role of PDE4 inhibitors in modulating cytokines, lipid mediators and in mucociliary clearance, along with clinical efficacy in asthma and/or COPD demonstrated with roflumilast and cilomilast, suggest a broad anti-inflammatory spectrum for these compounds. Presently, the major impediment to approval of these novel therapies has been the mechanism based gastrointestinal adverse events which has limited the dosing and the ultimate efficacy with these novel therapeutic agents.     

Inhibitors of PDE4: a review of recent patent literature

The preregistration of roflumilast and the preapproval by the US Food and Drug Administration of cilomilast highlight the continued, albeit slow, progress towards a selective PDE4 inhibitor for the treatment of inflammatory diseases, such as asthma and chronic obstructive pulmonary disease. Whereas the potential therapeutic utility of PDE4 inhibition has been demonstrated in various preclinical animal models (e.g., in rheumatoid arthritis and multiple sclerosis), clinical evaluation has been restricted by dose-limiting side effects, mainly nausea and emesis. Continued disclosure of novel, selective and chemically diverse inhibitors by pharmaceutical companies demonstrates the great interest in PDE4 research. This review focuses on the PDE4 patent literature regarding clinical developments since 2002, and discusses disclosed PDE4 inhibitors from a medicinal chemistry perspective.     

Roflumilast for the treatment of chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a progressive disease of the airways that is triggered primarily by smoking. It manifests clinically with dyspnea, cough and sputum production, all of which become aggravated with disease progression. The only intervention that can halt the decline in lung function in COPD is smoking cessation--other interventions and therapeutic treatments can only slow down the progression of the disease. Pharmacologic treatment of stable COPD consists primarily of bronchodilators, which are used for relieving symptoms and reducing lung function decline, and corticosteroids, which are used for minimizing the associated inflammation. Methylxanthines are non-selective phosphodiesterase (PDE) inhibitors with bronchodilatory and anti-inflammatory effects; however, their use in COPD and other respiratory conditions is limited by their narrow therapeutic index and poor safety profile. Cilomilast and roflumilast are selective PDE4 inhibitors that are currently in pre-registration and phase III clinical trials, respectively, for the treatment of COPD (cilomilast and roflumilast) and asthma (roflumilast).     

The effects of a novel phosphodiesterase 7A and -4 dual inhibitor, YM-393059, on T-cell-related cytokine production in vitro and in vivo

YM-393059, (+/-)-N-(4,6-dimethylpyrimidin-2-yl)-4-[2-(4-methoxy-3-methylphenyl)-5-(4-methylpiperazin-1-yl)-4,5,6,7-tetrahydro-1H-indol-1-yl]benzenesulfonamide difumarate, is a novel phosphodiesterase (PDE) inhibitor that inhibited the PDE7A isoenzyme with a high potency (IC50=14 nM) and PDE4 with a moderate potency (IC50=630 nM). In a cell-based assay, YM-393059 was found to inhibit anti-CD3 antibody, Staphylococcal enterotoxin B, and phytohaemagglutinin-induced interleukin (IL)-2 production in mouse splenocytes with IC50 values ranging from 0.48 to 1.1 microM. It also inhibited anti-CD3 antibody-induced interferon (IFN)-gamma and IL-4 production in splenocytes with IC50 values of 1.8 and 2.8 microM, respectively. YM-393059's inhibition of anti-CD3 antibody-stimulated cytokine (IL-2, IFN-gamma, and IL-4) production was 20- to 31-fold weaker than that of YM976, a selective PDE4 inhibitor. However, orally administered YM-393059 and YM976 inhibited anti-CD3 antibody-induced IL-2 production equipotently in mice. In addition, YM-393059 inhibited lipopolysaccharide-induced tumor necrosis factor-alpha production in vivo more potently than IL-2 (ED50 values of 2.1 mg/kg and 74 mg/kg). In contrast to YM976, YM-393059 did not shorten the duration of alpha2-adrenoceptor agonist-induced sleep in mice, which is a model for the assessment of the typical side effects caused by PDE4 inhibitors, nausea and emesis. YM-393059 is a novel and attractive compound for the treatment of a wide variety of T-cell-mediated diseases.     

Therapeutic benefit of PDE4 inhibitors in inflammatory diseases

Intracellular levels of cyclic nuclec tides are closely regulated by distinct families of PD Es, which are responsible for the breakdown and degradation of cyclic nucleotides within cells. Type 4 PDEs have the potency to modulate the release of inflammatory mediators through cAMP-dependent and -independent mechanisms. Selective targeting of PDE4 is currently being investigated as a novel therapeutic approach in the treatment of inflammation-associated respiratory diseases such as asthma and COPD. The development of several PDE4 inhibitors, including roflumilast and cilomilast, reflects the success of this approach. In principle, therapeutic intervention of an inflammatory response by PDE4 inhibitors may be extended to other chronic inflammatory disease states such as psoriasis, rheumatoid arthritis and inflammatory bowel diseases (e.g., Crohns disease and ulcerative colitis). This retiiew explores the feasibility of PDE4 inhibitors as a promising alternative for therapeutic intervention in systemic inflammation and inflammation-based disease.     

Effects of SCA40 on human isolated bronchus and human polymorphonuclear leukocytes: comparison with rolipram, SKF94120 and levcromakalim.

1. SCA40 (0.1 nM-0.1 mM) produced concentration-dependent suppression of the spontaneous tone of human isolated bronchus (-log EC50 = 6.85 +/- 0.09; n = 10) and reached a maximal relaxation similar to that of theophylline (3 mM). The potency (-log EC50 values) of SCA40 compared to other relaxants was rolipram (7.44 +/- 0.12; n = 9) > SCA40 > or = levcromakalim (6.49 +/- 0.04; n = 6) > SKF94120 (5.87 +/- 0.10; n = 9). 2. When tested against the activity of the isoenzymes of cyclic nucleotide phosphodiesterase (PDE) isolated from human bronchus, SCA40 proved highly potent against PDE III (-log IC50 = 6.47 +/- 0.16; n = 4). It was markedly less potent against PDE IV (4.82 +/- 0.18; n = 4) and PDE V (4.32 +/- 0.11; n = 4). 3. Human polymorphonuclear leukocytes (PMNs) stimulated with N-formylmethionyl-leucyl-phenylalanine (FMLP) produced a concentration-dependent superoxide anion generation and elastase release. SCA40 (1 nM-10 microM) produced a concentration-related inhibition of FMLP (30 nM approximately EC50)-induced superoxide production (-log IC50 = 5.48 +/- 0.10; n = 6) and elastase release (-log IC50 = 5.50 +/- 0.26; n = 6). Rolipram was an effective inhibitor of superoxide generation and elastase release (-log IC50 values approximately 8) while SKF94120 and levcromakalim were scarcely effective. 4. FMLP (30 nM) and thimerosal (20 microM) induced leukotriene B4 production and elevation of intracellular calcium concentration in human PMNs. The production of leukotriene B4 was inhibited by SCA40 in a concentration-related manner (-log IC50 = 5.94 +/- 0.22; n = 6) but SCA40 was less effective against the elevation of intracellular calcium. Rolipram was an effective inhibitor of leukotriene B4 synthesis (-log IC50 approximately 7) and intracellular calcium elevation (-log IC50 approximately 6) while SKF94120 and levcromakalim were scarcely effective. 5. It is concluded that SCA40 is an effective inhibitor of the inherent tone of human isolated bronchus. The bronchodilatation produced by SCA40 appears mainly related to PDE inhibition since the potency of SCA40 as a relaxant of human isolated bronchus was found to be close to its potency as inhibitor of PDE III activity isolated from human bronchus. In addition, SCA40 exhibited inhibitory effects on human PMN function stimulated by FMLP. These effects may be related to the ability of SCA40 to inhibit PDE IV from human PMNs while the contribution of PDE V inhibition is uncertain. We found no evidence of a role for levcromakalim-sensitive plasmalemmal K+-channels in human PMNs.     

PDE4 inhibition: a novel approach for the treatment of inflammatory bowel disease

Inflammation is a hallmark of inflammatory bowel disease (IBD), and elevation of cAMP levels can inhibit the pro-inflammatory and tissue-destructive properties of leukocytes. Phosphodiesterase 4 (PDE4) is the predominant enzyme that metabolizes cAMP in inflammatory cells, and the anti-inflammatory and immunomodulatory potential of PDE4 inhibitors in human leukocytes, endothelium and epithelium is well documented. Although PDE4 inhibitors have been investigated as treatments for several inflammatory diseases, this has focused mainly on asthma and chronic obstructive disease (COPD). Historically, their clinical utility has been limited by nausea and emesis. However, the PDE4 inhibitors cilomilast and roflumilast have recently shown efficacy in asthma and COPD, with a reduced propensity to cause nausea and emesis. In this review, we summarize for the first time the evidence that PDE4 inhibitors might have therapeutic benefit in IBD, and discuss mechanisms of action beyond the inhibition of inflammatory cells.     

The discovery of tadalafil: a novel and highly selective PDE5 inhibitor. 2: 2,3,6,7,12,12a-hexahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4-dione analogues

Modification of the hydantoin ring in the previously described lead compound 2a has led to the discovery of compound 12a, tadalafil, a highly potent and highly selective PDE5 inhibitor. The replacement of the hydantoin in compound 2a by a piperazinedione ring led to compound cis-11a which showed similar PDE5 inhibitory potency. Introduction of a 3,4-methylenedioxy substitution on the phenyl ring in position 6 led to a potent PDE5 inhibitor cis-11c with increased cellular potency. Optimization of the chain on the piperazinedione ring led to the identification of the racemic cis-N-methyl derivative 11i. High diastereospecificity for PDE5 inhibition was observed in the piperazinedione series with the cis-(6R,12aR) enantiomer displaying the highest PDE5 inhibitory activity. The piperazinedione 12a, tadalafil (GF196960), has been identified as a highly potent PDE5 inhibitor (IC(50) = 5 nM) with high selectivity for PDE5 vs PDE1-4 and PDE6. Compound 12a displays 85-fold greater selectivity vs PDE6 than sildenafil 1. 12a showed profound and long-lasting blood pressure lowering activity (30 mmHg/>7 h) in the spontaneously hypertensive rat model after oral administration (5 mg/kg).     

Inhibitory effects of flavonoids on phosphodiesterase isozymes from guinea pig and their structure-activity relationships

The structure-activity relationships of flavonoids with regard to their inhibitory effects on phosphodiesterase (PDE) isozymes are little known. The activities of PDE1-5 were measured by a two-step procedure using cAMP with [(3)H]-cAMP or cGMP with [(3)H]-cGMP as substrates. In the present results, PDE1, 5, 2, and 4 isozymes were partially purified from guinea pig lungs in that order, and PDE3 was from the heart. The IC(50) values of PDE1-5 were greater than those reported previously for the reference drugs, vinpocetin, EHNA, milrinone, Ro 20-1724, and zaprinast, by 5-, 5-, 7-, 5-, and 3-fold, respectively. As shown in Table 2, luteolin revealed non-selective inhibition of PDE1-5 with IC(50) values in a range of 10-20 microM, as did genistein except with a low potency on PDE5. Daidzein, an inactive analogue of genistein in tyrosine kinase inhibition, showed selective inhibition of PDE3 with an IC(50) value of around 30 microM, as did eriodictyol with an IC(50) value of around 50 microM. Hesperetin and prunetin exhibited more-selective inhibition of PDE4 with IC(50) values of around 30 and 60 microM, respectively. Luteolin-7-glucoside exhibited dual inhibition of PDE2/PDE4 with an IC(50) value of around 40 microM. Diosmetin more-selectively inhibited PDE2 (IC(50) of 4.8 microM) than PDE1, PDE4, or PDE5. However, biochanin A more-selectively inhibited PDE4 (IC(50) of 8.5 microM) than PDE1 or PDE2. Apigenin inhibited PDE1-3 with IC(50) values of around 10-25 microM. Myricetin inhibited PDE1-4 with IC(50) values of around 10-40 microM. The same was true for quercetin, but we rather consider that it more-selectively inhibited PDE3 and PDE4 (IC(50) of < 10 microM). In conclusion, it is possible to synthesize useful drugs through elucidating the structure-activity relationships of flavonoids with respect to inhibition of PDE isozymes at concentrations used in this in vitro study.     

Dopamine D4 receptors inhibit depolarization-induced [3H]GABA release in the rat subthalamic nucleus

We explored the role of dopamine D4 receptors on [3H]GABA release in the subthalamic nucleus. [3H]GABA release was evoked by high K+ in slices of the nucleus. The selective dopamine D4 receptor agonist PD168,077 (N-[[4-(2-cyanophenyl)-1-piperazynil]methyl]-3-methyl-benzamide) inhibited GABA release with greater potency (EC50=3.2 nM) than quinpirole (EC50=200 nM). SKF 21297 (6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide), a dopamine D1-like receptor agonist, had no effect. L-745,870 (3-[[4-(4-chlorophenyl)piperazin-1-yl]methyl]-1-1H-pyrollo[2,3-b] pyridine), a selective dopamine D4 receptor antagonist, reverted the quinpirole inhibition with greater potency (IC50=8.7 nM) than that of the dopamine D2/D3 receptor antagonist sulpiride and raclopride (IC50=4804 and 788 nM, respectively). Both methylphenidate and methamphetamine, dopamine reuptake blockers, inhibited by 30% high K(+)-evoked GABA release; the inhibition was blocked by L-745,870. These results show that dopamine D4 receptors modulate GABA release in the subthalamic nucleus. The results would explain how agents that increase interstitial dopamine like methylphenidate and amphethamine might control locomotor hyperactivity seen in disorders of dopamine D4 receptors.