Benzyl derivatives of 2,1,3-benzo- and benzothieno[3,2-a]thiadiazine 2,2-dioxides: First phosphodiesterase 7 inhibitors

The synthesis of a new family of benzyl derivatives of 2,1,3-benzo- and benzothieno[3,2-a]-thiadiazine 2,2-dioxides was achieved. The biological data revealed the first heterocyclic family of compounds with PDE 7 inhibitory properties appearing to be a new objective for the treatment of T-cell- dependent disorders. The IC50 values or percent inhibition values of the compounds against PDE 7 were calculated by testing them against human recombinant PDE 7 expressed in S. cerevisiae. In this expression system the only cyclic nucleotide hydrolyzing activity present in cell extracts corresponded to human PDE 7. Isoenzyme selectivity PDE 7 versus PDE 4 and PDE 3 was also measured. Considering simultaneously inhibition of the three different isoenzymes, monobenzyl derivatives 15 and 23 showed interesting PDE 7 potency (around 10 μM); although not statistically significant, a trend toward selectivity with respect to PDE 3 and PDE 4 was obtained. Benzothiadiazine 16, although less potent at PDE 7 (IC50 = 25 μM), also showed a trend of selectivity toward PDE 3 and PDE 4. These compounds are considered the best leads for further optimization.Peer Reviewe

Pharmacological Characterization of Abediterol, a Novel Inhaled ␤ 2 -Adrenoceptor Agonist with Long Duration of Action and a Favorable Safety Profile in Preclinical Models

ABSTRACT Abediterol is a novel potent, long-acting inhaled ␤ 2 -adrenoceptor agonist in development for the treatment of asthma and chronic obstructive pulmonary disease. Abediterol shows subnanomolar affinity for the human ␤ 2 -adrenoceptor and a functional selectivity over ␤ 1 -adrenoceptors higher than that of formoterol and indacaterol in both a cellular model with overexpressed human receptors and isolated guinea pig tissue. Abediterol is a full agonist at the human ␤ 2 -adrenoceptor (E max ϭ 91 Ϯ 5% of the maximal effect of isoprenaline). The potency and onset of action that abediterol shows in isolated human bronchi (EC 50 ϭ 1.9 Ϯ 0.4 nM; t 1 ⁄2 onset ϭ 7-10 min) is not significantly different from that of formoterol, but its duration of action (t 1 ⁄2 ϳ 690 min) is similar to that of indacaterol. Nebulized abediterol inhibits acetylcholine-induced bronchoconstriction in guinea pigs in a concentration-dependent manner, with higher potency and longer duration of action (t 1 ⁄2 ϭ 36 h) than salmeterol (t 1 ⁄2 ϭ 6 h) and formoterol (t 1 ⁄2 ϭ 4 h) and similar duration of action to indacaterol up to 48 h. In dogs, the bronchoprotective effect of abediterol is more sustained than that of salmeterol and indacaterol at doses without effects on heart rate, thus showing a greater safety margin (defined as the ratio of dose increasing heart rate by 5% and dose inhibiting bronchospasm by 50%) than salmeterol, formoterol, and indacaterol (5.6 versus 3.3, 2.2, and 0.3, respectively). In conclusion, our results suggest that abediterol has a preclinical profile for once-daily dosing in humans together with a fast onset of action and a favorable cardiovascular safety profile

Inhibition of eotaxin-mediated human eosinophil activation and migration by the selective cyclic nucleotide phosphodiesterase type 4 inhibitor rolipram

1. The effect of the selective type 4 phosphodiesterase (PDE 4) inhibitor rolipram on human eosinophil activation and migration mediated by eotaxin was investigated. 2. Studies were performed with human freshly isolated eosinophils from peripheral blood of healthy donors by a magnetic cell separation (MACS) technique to a purity>99%. To test the effect of rolipram, eosinophils were stimulated with recombinant human eotaxin and the cell surface activation markers CD11b and L-selectin were analysed by flow cytometry. Furthermore, eotaxin mediated eosinophil migration was measured in a transendothelial chemotaxis assay. 3. Our results indicate that rolipram inhibited eotaxin-induced CD11b up-regulation up to 60.6±7.6% at the highest tested dose (10 μM), whereas transendothelial chemotaxis was partially inhibited reaching a plateau of approx. 30% at a rolipram concentration of 0.1 μM. 4. We conclude that the selective PDE 4 inhibitor rolipram decreases eotaxin mediated eosinophil activation, an observation that may contribute to elucidate the mechanism by which PDE 4 inhibitors reduce antigen-induced eosinophil infiltration in different animal models of allergic inflammation

Functional and biochemical evidence for diazepam as a cyclic nucleotide phosphodiesterase type 4 inhibitor

1. The responses of the electrically-driven right ventricle strip of the guinea-pig heart to diazepam were recorded in the absence and in the presence of different selective cyclic nucleotide phosphodiesterase (PDE) inhibitors. 2. Diazepam, at concentrations ranging from 1 μM to 100 μM, was devoid of effect on the contractile force in this preparation. 3. Conversely, diazepam (5 μM–100 μM) produced a consistent positive inotropic response in the presence of a concentration (1 μM), that was without effect in the absence of diazepam, of either of the selective PDE 3 inhibitors milrinone or SK&F 94120, but not in the presence of the selective PDE 4 inhibitor rolipram. 4. This effect of diazepam was not γ-aminobutyric acid (GABA)-dependent, since it was neither mimicked nor potentiated by GABA, and was not affected by either a high concentration (5 μM) of the antagonists of the benzodiazepine/GABA/channel chloride receptor complex, picrotoxin, flumazenil and β-carboline-3-carboxylic acid methyl ester (βCCMe), or by the inverse agonists, β-carboline-3-carboxylic acid N-methylamide (βCCMa) and methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM, 0.1 μM). Furthermore, a specific antagonist of the peripheral benzodiazepine receptors, PK 11195 (5 μM), did not influence the effect of diazepam. 5. Biochemical studies with isolated PDEs, confirmed that diazepam selectively inhibits type 4 PDE from guinea-pig right ventricle rather than the other PDEs present in that tissue. The compound inhibited this enzyme in a non-competitive manner. Diazepam was also able to inhibit PDE 5, the cyclic GMP specific PDE absent from cardiac muscle, with a potency close to that shown for PDE 4. 6. Diazepam displaced the selective type 4 PDE inhibitor, rolipram from its high affinity binding site in rat brain cortex membranes, and also potentiated the rise in cyclic AMP levels induced by isoprenaline in guinea-pig eosinophils, where only type 4 PDE is present. 7. The PDE inhibitory properties of diazepam were shared, although with lower potency, by other structurally-related benzodiazepines, that also displaced [(3)H]-rolipram from its high affinity binding site. The order of potency found for these compounds in these assays was not related to their potencies as modulators of the GABA receptor through its benzodiazepine binding site. 8. The pharmacological and biochemical data presented in this study indicate that diazepam behaves as a selective type 4 PDE inhibitor in cardiac tissue and this effect seems neither to be mediated by the benzodiazepine/GABA/channel chloride receptor complex nor by peripheral type benzodiazepine receptors

Characterization of 5-HT receptors on human pulmonary artery and vein: functional and binding studies

1. This study aimed to investigate the 5-hydroxytryptamine (5-HT) receptors mediating contraction of ring preparations isolated from human pulmonary arteries and veins. In functional studies, the responses to 5-HT, sumatriptan, ergotamine, serotonin-O-carboxymethyl-glycyl-tyrosinamide (SCMGT), α-methyl 5-HT (α-Me) and 2-methyl 5-HT (2-Me) were studied with WAY100635, GR127935, ritanserin, zacopride and SB204070 as antagonists. 2. All agonists produced concentration-dependent contractions of human pulmonary artery and vein preparations. The order of potency (−log EC(50) values) was ergotamine (6.88)>5-HT (6.41)⩾SCMGT (6.20)=sumatriptan (6.19) ⩾α-Me (6.04) in the artery, and ergotamine (7.84)>5-HT (6.96)>sumatriptan (6.60)=α-Me (6.56)>SCMGT (6.09) in the vein. The potency of each agonist, except for SCMGT, was greater in vein than in artery preparations. Contractile responses to 5-HT were similar in intact and endothelium-denuded preparations but responses to sumatriptan were enhanced in artery rings without endothelium. 3. GR127935 (1 nM to 0.5 μM) produced an unsurmountable antagonism of the response to 5-HT, sumatriptan, ergotamine and SCMGT. Ritanserin (1 nM to 1 μM) also reduced the maximum contractile responses to 5-HT, ergotamine and α-Me in artery and vein preparations without affecting those to sumatriptan and SCMGT. In endothelium-denuded preparations, surmountable antagonism of sumatriptan by GR127935 (in the presence of ritanserin) and of α-Me by ritanserin (in the presence of GR127935) allowed for the calculation of the apparent pK(B) values of GR127935 (9.17±0.11 in artery and 9.11±0.05 in vein) and ritanserin (8.82±0.09 in artery and 8.98±0.12 in vein). 4. WAY100635 (1 nM to 1 μM), zacopride (1 nM to 1 μM), or SB204070 (1 nM) did not significantly alter the concentration-response curves for 5-HT, sumatriptan, ergotamine, SCMGT or 2-Me in human pulmonary artery or vein thus indicating that 5-HT(1A), 5-HT(3) and 5-HT(4) receptors are presumably not involved in the contractile response to these agonists. 5. Binding studies using selective radioligands for different 5-HT receptors could not detect the presence of 5-HT(1A) receptor binding in human pulmonary blood vessels whereas the 5-HT(1B/1D) radioligand [(3)H]-5-CT significantly labelled a population of specific binding sites in both vessel types. The presence of 5-HT(2A) receptors could also be inferred from the level of binding of [(3)H]-ketanserin to membranes obtained from human pulmonary vessels, although significance could not be reached for arteries. 5-HT(4) specific receptor binding was scarce in veins and absent in the case of arteries. 6. These findings indicate that the human pulmonary artery and vein have a mixed functional population of 5-HT(1B/1D) and 5-HT(2A) receptors mediating the contractile response to 5-HT which is consistent with results of the binding studies

Validated and longitudinally stable asthma phenotypes based on cluster analysis of the ADEPT study

BACKGROUND: Asthma is a disease of varying severity and differing disease mechanisms. To date, studies aimed at stratifying asthma into clinically useful phenotypes have produced a number of phenotypes that have yet to be assessed for stability and to be validated in independent cohorts. The aim of this study was to define and validate, for the first time ever, clinically driven asthma phenotypes using two independent, severe asthma cohorts: ADEPT and U-BIOPRED. METHODS: Fuzzy partition-around-medoid clustering was performed on pre-specified data from the ADEPT participants (n = 156) and independently on data from a subset of U-BIOPRED asthma participants (n = 82) for whom the same variables were available. Models for cluster classification probabilities were derived and applied to the 12-month longitudinal ADEPT data and to a larger subset of the U-BIOPRED asthma dataset (n = 397). High and low type-2 inflammation phenotypes were defined as high or low Th2 activity, indicated by endobronchial biopsies gene expression changes downstream of IL-4 or IL-13. RESULTS: Four phenotypes were identified in the ADEPT (training) cohort, with distinct clinical and biomarker profiles. Phenotype 1 was "mild, good lung function, early onset", with a low-inflammatory, predominantly Type-2, phenotype. Phenotype 2 had a "moderate, hyper-responsive, eosinophilic" phenotype, with moderate asthma control, mild airflow obstruction and predominant Type-2 inflammation. Phenotype 3 had a "mixed severity, predominantly fixed obstructive, non-eosinophilic and neutrophilic" phenotype, with moderate asthma control and low Type-2 inflammation. Phenotype 4 had a "severe uncontrolled, severe reversible obstruction, mixed granulocytic" phenotype, with moderate Type-2 inflammation. These phenotypes had good longitudinal stability in the ADEPT cohort. They were reproduced and demonstrated high classification probability in two subsets of the U-BIOPRED asthma cohort. CONCLUSIONS: Focusing on the biology of the four clinical independently-validated easy-to-assess ADEPT asthma phenotypes will help understanding the unmet need and will aid in developing tailored therapies. TRIAL REGISTRATION: NCT01274507 (ADEPT), registered October 28, 2010 and NCT01982162 (U-BIOPRED), registered October 30, 2013