The in vitro and in vivo profile of aclidinium bromide in comparison with glycopyrronium bromide.

This study characterised the in vitro and in vivo profiles of two novel long-acting muscarinic antagonists, aclidinium bromide and glycopyrronium bromide, using tiotropium bromide and ipratropium bromide as comparators. All four antagonists had high affinity for the five muscarinic receptor sub-types (M1-M5); aclidinium had comparable affinity to tiotropium but higher affinity than glycopyrronium and ipratropium for all receptors. Glycopyrronium dissociated faster from recombinant M3 receptors than aclidinium and tiotropium but more slowly than ipratropium; all four compounds dissociated more rapidly from M2 receptors than from M3 receptors. In vitro, aclidinium, glycopyrronium and tiotropium had a long duration of action at native M3 receptors (>8 h versus 42 min for ipratropium). In vivo, all compounds were equi-potent at reversing acetylcholine-induced bronchoconstriction. Aclidinium, glycopyrronium and ipratropium had a faster onset of bronchodilator action than tiotropium. Aclidinium had a longer duration of action than glycopyronnium (time to 50% recovery of effect [t½ offset] = 29 h and 13 h, respectively); these compare with a t½ offset of 64 h and 8 h for tiotropium and ipratropium, respectively. Aclidinium was less potent than glycopyrronium and tiotropium at inhibiting salivation in conscious rats (dose required to produce half-maximal effect [ED50] = 38, 0.74 and 0.88 µg/kg, respectively) and was more rapidly hydrolysed in rat, guinea pig and human plasma compared with glycopyrronium or tiotropium. These results indicate that while aclidinium and glycopyrronium are both potent antagonists at muscarinic receptors with similar kinetic selectivity for M3 receptors versus M2, aclidinium has a longer dissociation half-life at M3 receptors and a longer duration of bronchodilator action in vivo than glycopyrronium. The rapid plasma hydrolysis of aclidinium, coupled to its kinetic selectivity, may confer a reduced propensity for systemic anticholinergic side effects with aclidinium versus glycopyrronium and tiotropium.

Pharmacological characterization of abediterol, a novel inhaled ß(2)-adrenoceptor agonist with long duration of action and a favorable safety profile in preclinical models.

Abediterol is a novel potent, long-acting inhaled ß(2)-adrenoceptor agonist in development for the treatment of asthma and chronic obstructive pulmonary disease. Abediterol shows subnanomolar affinity for the human ß(2)-adrenoceptor and a functional selectivity over ß(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 ß(2)-adrenoceptor (E(max) = 91 ± 5% of the maximal effect of isoprenaline). The potency and onset of action that abediterol shows in isolated human bronchi (EC(50) = 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.

Discovery of novel quaternary ammonium derivatives of (3R)-quinuclidinyl carbamates as potent and long acting muscarinic antagonists.

Novel quaternary ammonium derivatives of N,N-disubstituted (3R)-quinuclidinyl carbamates have been identified as potent M(3) muscarinic antagonists with long duration of action in an in vivo model of bronchoconstriction. These compounds have also presented a high level of metabolic transformation (human liver microsomes). The synthesis, structure-activity relationships and biological evaluation of these compounds are reported.

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 (M(1)-M(5)). [(3)H]Aclidinium dissociated slightly faster from M(2) and M(3) receptors than [(3)H]tiotropium but much more slowly than [(3)H]ipratropium. Its association rate for the M(3) receptor was similar to [(3)H]ipratropium and 2.6 times faster than [(3)H]tiotropium. Residence half-life of [(3)H]aclidinium at the M(2) receptor was shorter than at the M(3) receptor, demonstrating kinetic selectivity for the M(3) 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 (t(1/2) = 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.

Discovery and characterization of 4'-(2-furyl)-N-pyridin-3-yl-4,5'-bipyrimidin-2'-amine (LAS38096), a potent, selective, and efficacious A2B adenosine receptor antagonist.

A novel series of N-heteroaryl 4'-(2-furyl)-4,5'-bipyrimidin-2'-amines has been identified as potent and selective A(2B) adenosine receptor antagonists. In particular, compound 5 showed high affinity for the A(2B) receptor (Ki = 17 nM), good selectivity (IC(50): A(1) > 1000 nM, A(2A) > 2500 nM, A3 > 1000 nM), displayed a favorable pharmacokinetic profile in preclinical species, and showed efficacy in functional in vitro models.

Synthesis and structure-activity relationships of novel histamine H1 antagonists: indolylpiperidinyl benzoic acid derivatives.

A series of indolylpiperidinyl derivatives were prepared and evaluated for their activity as histamine H(1) antagonists. Structure-activity relationship studies were directed toward improving in vivo activity and pharmacokinetic profile of our first lead (1). Substitution of fluorine in position 6 on the indolyl ring led to higher in vivo activity in the inhibition of histamine-induced cutaneous vascular permeability assay but lower selectivity toward 5HT(2) receptor. Extensive optimization was carried out within this series and a number of histamine H(1) antagonists showing potency and long duration of action in vivo and low brain penetration or cardiotoxic potential were identified. Within this novel series, indolylpiperidines 15, 20, 48,51 and 52 exhibited a long half-life in rat and have been selected for further preclinical evaluation.

Aclidinium bromide, a novel long-acting muscarinic antagonist for COPD with improved preclinical renal and urinary safety profile.

AIMS: Aclidinium bromide is a novel, long-acting, inhaled muscarinic antagonist currently in registration phase for the treatment of chronic obstructive pulmonary disease. Since urinary difficulty and retention have been reported for anticholinergic agents such as tiotropium and ipratropium, it is important to examine the preclinical urinary and renal safety profile of aclidinium. MAIN METHODS: The effect of aclidinium on urine and electrolyte excretion, renal function and voiding cystometry was analysed in conscious water-loaded Wistar rats (10-1000 µg/kg, s.c.), anaesthetised Beagle dogs (1000 µg/kg, i.v.) and anaesthetised guinea pigs (3-100µg/kg, intratracheally), respectively. Aclidinium plasma levels were determined in an independent study. Active comparators were tiotropium (all studies) and ipratropium (cystometry only). KEY FINDINGS: Aclidinium 1000 µg/kg had no effect on urine excretion in rats, in contrast to tiotropium 100 µg/kg which significantly decreased this parameter (p<0.05). Aclidinium 1000 µg/kg also had no effect on renal function in Beagle dogs. In guinea pigs, aclidinium 3-100 µg/kg had no effect on urinary bladder function, whereas tiotropium and ipratropium 100 µg/kg decreased the peak micturition pressure (p<0.05), increased the volume of urine retained in the bladder (p<0.01) and showed a trend to decrease the volume of urine excreted. SIGNIFICANCE: Aclidinium had no significant effect on urinary and renal function in the animal models studied. These results, together with the rapid plasma clearance of aclidinium reported previously, suggest a lower propensity to induce urinary retention in humans than tiotropium and ipratropium.

Synthesis and biological activity of pyrido[3',2':4,5]furo[3,2-d]pyrimidine derivatives as novel and potent phosphodiesterase type 4 inhibitors.

A series of pyrido[3',2':4,5]furo[3,2-d]pyrimidines (PFP) were synthesized and tested for phosphodiesterase type 4 (PDE4) inhibitory activity, with the potential to treat asthma and chronic obstructive pulmonary disease (COPD). Structure-activity relationships within this series, leading to an increase of potency on the enzyme, is presented. Both gem-dimethylcyclohexyl moieties fused to the pyridine ring and the substitution at the 5 position of the PFP scaffold, proved to be key elements in order to get a high affinity in the enzyme.

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.

Synthesis and biological activity of pyrido[3',2':4,5]thieno[3,2-d]pyrimidines as phosphodiesterase type 4 inhibitors.

A series of pyrido[3',2':4,5]thieno[3,2-d]pyrimidines (PTP) has been synthesized and tested as phosphodiesterase IV inhibitors (PDE4), a target for the treatment of asthma and chronic obstructive pulmonary disease (COPD). Structure-activity relationships within this series, leading to an increase of potency on the enzyme, are presented. The gem-dimethylcycloalkyl moiety fused to the pyridine ring proved to be a key element of the scaffold in order to get a higher affinity in the enzyme.

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.

A new chemical tool (C0036E08) supports the role of adenosine A(2B) receptors in mediating human mast cell activation.

Asthma is a chronic inflammatory disease of the airways that involves many cell types, amongst which mast cells are known to be important. Adenosine, a potent bronchoconstricting agent, exerts its ability to modulate adenosine receptors of mast cells thereby potentiating derived mediator release, histamine being one of the first mediators to be released. The heterogeneity of sources of mast cells and the lack of highly potent ligands selective for the different adenosine receptor subtypes have been important hurdles in this area of research. In the present study we describe compound C0036E08, a novel ligand that has high affinity (pK(i) 8.46) for adenosine A(2B) receptors, being 9 times, 1412 times and 3090 times more selective for A(2B) receptors than for A(1), A(2A) and A(3) receptors, respectively. Compound C0036E08 showed antagonist activity at recombinant and native adenosine receptors, and it was able to fully block NECA-induced histamine release in freshly isolated mast cells from human bronchoalveolar fluid. C0036E08 has been shown to be a valuable tool for the identification of adenosine A(2B) receptors as the adenosine receptors responsible for the NECA-induced response in human mast cells. Considering the increasing interest of A(2B) receptors as a therapeutic target in asthma, this chemical tool might provide a base for the development of new anti-asthmatic drugs.

Phosphodiesterase 7A1 is expressed in human CD4+ naïve T cells at higher levels than in CD4+ memory cells and is not required during their CD3/CD28-dependent activation.

PDE7A1 is a cAMP-hydrolyzing phosphodiesterase expressed in lymphoid tissue, where its possible role during T cell activation remains unclear. We have characterized the functional relevance of PDE7A1 in the naïve (CD4+CD45RA+) and memory (CD4+CD45RO+) subsets of human peripheral CD4+ T cells during CD3/CD28-dependent stimulation. Our results indicate that PDE7A1 is expressed in resting naïve CD4+ T cells at higher levels than in the corresponding memory cells and that levels of PDE7A1 mRNA are not upregulated upon CD3/CD28 mediated stimulation of these T cell subsets. Treatment with a selective inhibitor of PDE7A1 does not impair CD3/CD28 induced activation of naïve or memory CD4+ T cells, nor does it increase intracellular cAMP in CD4+ T cells. We conclude that PDE7A1 is not required during CD3/CD28-dependent activation of naïve and memory CD4+ T cells, but cannot rule out other regulatory roles of PDE7A1 during maturation of CD4+ T cells.

New pyrrolopyrimidin-6-yl benzenesulfonamides: potent A2B adenosine receptor antagonists.

A new series of 4-(1,3-dialkyl-2,4-dioxo-2,3,4,5-tetrahydro-1H-pyrrolo[3,2-d]pyrimidin-6-yl)benzenesulfonamides has been identified as potent A2B adenosine receptor antagonists. The products have been evaluated for their binding affinities for the human A2B, A1 and A3 adenosine receptors. 6-(4-{[4-(4-Bromobenzyl)piperazin-1-yl]sulfonyl}phenyl)-1,3-dimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-dione (16) showed a high affinity for the A2B adenosine receptor (IC50=1 nM) and selectivity (A1: 183x; A3: 12660x). Synthesis and SAR of this novel class of compounds showing improved absorption properties is presented herein.

Synthesis and structure-activity relationships of piperidinylpyrrolopyridine derivatives as potent and selective H1 antagonists.

The synthesis and structure-activity relationships of piperidinylpyrrolopyridines as potent and selective H(1) antagonists are discussed. It was found that the nature of the acid chain bonded to piperidine was a key feature for maintaining both the duration of action in vivo and lack of sedative properties.

Synthesis and evaluation of some pyrazolo[3,4-d]pyridazinones and analogues as PDE 5 inhibitors potentially useful as peripheral vasodilator agents.

A series of pyrazolo[3,4-d]pyridazinones and analogues, potentially useful as peripheral vasodilators, were synthesized and evaluated as inhibitors of PDE5 extracted from human platelets. Several of them showed IC50 values in the range 0.14-1.4 microM. A good activity and selectivity profile versus PDE6 was found for compound 11e (6-benzyl-3-methyl-1-isopropyl-4-phenylpyrazolo[3,4-d] pyridazin-7(6H)-one). Structure-activity relationship studies demonstrated the essential role played by the benzyl group at position-6 of the pyrazolopyridazine system. Other types of pyridazinones fused with five and six membered heterocycles (pyrrole, isoxazole, pyridine and dihydropyridine), as well as some open models were prepared and evaluated. Besides the pyrazole, the best fused systems proved to be isoxazole and pyridine.