Jorge Beleta

Investigation into the role of phosphodiesterase IV in bronchorelaxation, including studies with human bronchus

1 We have investigated the role of cyclic nucleotide phosphodiesterase IV (PDEIV) in the relaxation of human bronchus and guinea‐pig trachea in vitro and in guinea‐pigs in vivo. 2 Functional studies showed that the selective PDE IV inhibitors, rolipram and denbufylline, relaxed human and guinea‐pig preparations in vitro. 3 Two clinically used xanthine non‐selective PDE inhibitors, theophylline and pentoxifylline, were also effective in these preparations, but were much less potent than the selective agents used. 4 The rank order of potency for the four PDE inhibitors in both species was similar. 5 Biochemical studies indicated that PDE IV was the major PDE isoform present in the human bronchial tissue. PDEs I, II and V were also identified. 6 Theophylline and pentoxifylline were, as expected, non‐selective inhibitors of the human enzymes, but there was a good correlation between PDE IV inhibitory and bronchorelaxation potencies, suggesting that PDE IV inhibition is important for the clinical bronchodilator activities of the two xanthine compounds. 7 We have confirmed the ability of selective PDE IV inhibitors to cause bronchodilatation in guinea‐pigs in vivo. 8 We conclude that our study has provided further evidence that selective PDE IV inhibitors could act as bronchodilators in the clinic.

Characterization of Aclidinium Bromide, a Novel Inhaled Muscarinic Antagonist, with Long Duration of Action and a Favorable Pharmacological Profile

Aclidinium bromide is a novel potent, long-acting inhaled muscarinic antagonist in development for the treatment of chronic obstructive pulmonary disease. Aclidinium showed subnanomolar affinity for the five human muscarinic receptors (M1–M5). [3H]Aclidinium dissociated slightly faster from M2 and M3 receptors than [3H]tiotropium but much more slowly than [3H]ipratropium. Its association rate for the M3 receptor was similar to [3H]ipratropium and 2.6 times faster than [3H]tiotropium. Residence half-life of [3H]aclidinium at the M2 receptor was shorter than at the M3 receptor, demonstrating kinetic selectivity for the M3 receptor. In isolated guinea pig trachea, aclidinium showed comparable potency to ipratropium and tiotropium, faster onset of action than tiotropium, and duration of action similar to tiotropium and significantly longer than ipratropium. Nebulized aclidinium inhibited bronchoconstriction induced by acetylcholine in guinea pigs in a concentration-dependent manner with an onset of action faster than tiotropium. Duration of action of aclidinium (t1/2 = 29 h) was much longer than ipratropium (8 h) but shorter than tiotropium (64 h). In dogs, aclidinium induced a smaller and more transient increase in heart rate than tiotropium at comparable supratherapeutic doses. Therefore, under these conditions, aclidinium showed a greater therapeutic index than tiotropium (4.2 versus 1.6). These results indicate that aclidinium is a potent muscarinic antagonist with a fast onset of action, a long duration of effect, and a favorable cardiovascular safety profile.

Aclidinium bromide, a new, long-acting, inhaled muscarinic antagonist: in vitro plasma inactivation and pharmacological activity of its main metabolites.

Aclidinium bromide is a novel, long-acting inhaled muscarinic antagonist drug in Phase III clinical trials for chronic obstructive pulmonary disease (COPD). The aims of this study were to evaluate the in vitro stability of the ester drug aclidinium in plasma from various species, and the in vitro and in vivo pharmacological activity of its hydrolysis metabolites. Following incubation of aclidinium in pooled samples of human, rat, guinea pig or dog plasma, the rate of hydrolysis was quantified by reversed phase ultra performance liquid chromatography and mass spectrometry. Tiotropium and ipratropium were used as comparators. The in vitro biochemical profile of the hydrolysis metabolites of aclidinium was assessed in human M(1) to M(5) muscarinic receptors and in a standard selectivity panel (85 G protein-coupled receptors [GPCRs], ion channels and enzymes). The bronchodilator activity of the metabolites of aclidinium bromide was studied in guinea pigs after acetylcholine-induced bronchoconstriction. Aclidinium was rapidly hydrolysed into carboxylic acid and alcohol derivatives in guinea pig, rat, human and dog plasma with half-lives of 38, 11.7, 2.4 and 1.8 min, respectively. In contrast, > or =70% of tiotropium and ipratropium remained unchanged in the plasma after 60 min of incubation. The carboxylic acid and alcohol metabolites had no significant affinity for any of the muscarinic receptors, other GPCRs, ion channels or enzymes studied and showed no relevant antibronchoconstrictory activity in vivo. These results suggest that aclidinium may have a reduced systemic exposure and therefore less propensity for class-related systemic side effects in the clinical setting.

Discovery of novel quaternary ammonium derivatives of (3R)-quinuclidinol esters as potent and long-acting muscarinic antagonists with potential for minimal systemic exposure after inhaled administration: identification of (3R)-3-{[hydroxy(di-2-thienyl)acetyl]oxy}-1-(3-phenoxypropyl)-1-azoniabicyclo[2.2.2]octane bromide (aclidinium bromide).

The objective of this work was to discover a novel, long-acting muscarinic M(3) antagonist for the inhaled treatment of chronic obstructive pulmonary disease (COPD), with a potentially improved risk-benefit profile compared with current antimuscarinic agents. A series of novel quaternary ammonium derivatives of (3R)-quinuclidinol esters were synthesized and evaluated. On the basis of its overall profile, (3R)-3-{[hydroxy(di-2-thienyl)acetyl]oxy}-1-(3-phenoxypropyl)-1-azoniabicyclo[2.2.2]octane bromide (aclidinium bromide) emerged as a candidate for once-daily maintenance treatment of COPD. This compound is a potent muscarinic antagonist, with long duration of action in vivo, and was found to have a rapid hydrolysis in human plasma, minimizing the potential to induce class-related systemic side effects. Aclidinium bromide is currently in phase III development for maintenance treatment of patients with COPD.

Phosphodiesterase inhibitory properties of losartan. Design and synthesis of new lead compounds.

A 4-centre PDE4 pharmacophore search has been carried out in several 3D-databases containing compounds belonging to different therapeutic areas. Losartan, an angiotensin-II antagonist, has been identified as a new lead compound for developing PDE4 inhibitors. New families of compounds derived from losartan has been synthesized and their PDE inhibition has been measured.

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 EC50 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 pKB 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‐HT1A, 5‐HT3 and 5‐HT4 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‐HT1A receptor binding in human pulmonary blood vessels whereas the 5‐HT1B/1D radioligand [3H]‐5‐CT significantly labelled a population of specific binding sites in both vessel types. The presence of 5‐HT2A receptors could also be inferred from the level of binding of [3H]‐ketanserin to membranes obtained from human pulmonary vessels, although significance could not be reached for arteries. 5‐HT4 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‐HT1B/1D and 5‐HT2A receptors mediating the contractile response to 5‐HT which is consistent with results of the binding studies.

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

The effect of the selective type 4 phosphodiesterase (PDE 4) inhibitor rolipram on human eosinophil activation and migration mediated by eotaxin was investigated. 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. Our results indicate that rolipram inhibited eotaxin‐induced CD11b up‐regulation up to 60.6±7.6% at the highest tested dose (10μm), whereas transendothelial chemotaxis was partially inhibited reaching a plateau of approx. 30% at a rolipram concentration of 0.1μm. 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 [3H]‐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.

Pharmacological Characterization of Abediterol, a Novel Inhaled β2-Adrenoceptor Agonist with Long Duration of Action and a Favorable Safety Profile in Preclinical Models

Abediterol is a novel potent, long-acting inhaled β2-adrenoceptor agonist in development for the treatment of asthma and chronic obstructive pulmonary disease. Abediterol shows subnanomolar affinity for the human β2-adrenoceptor and a functional selectivity over β1-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 β2-adrenoceptor (Emax = 91 ± 5% of the maximal effect of isoprenaline). The potency and onset of action that abediterol shows in isolated human bronchi (EC50 = 1.9 ± 0.4 nM; t½ onset = 7–10 min) is not significantly different from that of formoterol, but its duration of action (t½ ∼ 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 (t½ = 36 h) than salmeterol (t½ = 6 h) and formoterol (t½ = 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.

Pharmacological characterization of almotriptan: an indolic 5-HT receptor agonist for the treatment of migraine

Almotriptan (3-[2-(dimethylamino)ethyl]-5-(pyrrolidin-1-ylsulfonylmethyl )-1H-indo le) has been studied in several models predictive of activity and selectivity at 5-HT receptors. Almotriptan showed low nanomolar affinity for the 5-HT(1B) and 5-HT(1D) receptors in several species, including the human, while affinity for 5-HT receptors other than 5-HT(1B/1D) was clearly less. Affinity for 5-HT(7) and 5-HT(1A) receptors was approximately 40 and 60 times lower than that for 5-HT(1B/1D) receptors, respectively. Almotriptan did not exhibit significant affinity for several non-5-HT receptors studied up to 100 microM. Almotriptan inhibited forskolin-stimulated cyclic AMP accumulation in HeLa cells transfected with 5-HT(1B) or 5-HT(1D) human receptors. In this model, almotriptan had the same efficacy as serotonin and an affinity in the low nanomolar range. It induced vasoconstriction in several vessels in which it was compared with sumatriptan. In isolated dog saphenous veins, almotriptan elicited concentration-dependent contractions with an EC(50) of 394 nM. In both these systems, almotriptan behaved as a full agonist. Infusion of almotriptan into the porcine meningeal vasculature induced vasoconstriction. In contrast, in the pig renal and rabbit mesenteric arteries, it had a very low maximal efficacy even at 100 microM, with similar results obtained in the rabbit renal artery. The results suggest that almotriptan is a potent and selective 5-HT(1B/1D) receptor agonist, with selectivity for the cranial vasculature as compared with peripheral vessels.