An investigation into the structure-activity relationships associated with the systematic modification of the ß(2)-adrenoceptor agonist indacaterol.

The synthesis of a series of indacaterol analogues in which each of the three structural regions of indacaterol are modified in a systematic manner is described. Evaluation of the affinity of these analogues for the ß(2)-adrenoceptor identified the 3,4-dihydroquinolinone and 5-n-butylindanyl analogues to demonstrate the most similar profiles to indacaterol. An α-methyl aminoindane analogue was discovered to be 25-fold more potent than indacaterol, and functional studies revealed an atypical ß(2)-adrenoceptor activation profile for this compound consistent with that of a slowly dissociating 'super agonist'.

Solubility-driven optimization of phosphodiesterase-4 inhibitors leading to a clinical candidate.

The solubility-driven optimization of a series of 1,7-napthyridine phosphodiesterase-4 inhibitors is described. Directed structural changes resulted in increased aqueous solubility, enabling superior pharmacokinetic properties with retention of PDE4 inhibition. A range of potent and orally bioavailable compounds with good in vivo efficacy in animal models of inflammation and reduced emetic potential compared to previously described drugs were synthesized. Compound 2d was taken forward as a clinical candidate for the treatment of COPD.

Sphingosine-1-phosphate-induced airway hyper-reactivity in rodents is mediated by the sphingosine-1-phosphate type 3 receptor.

There is a need to better understand the mechanism of airway hyper-reactivity, a key feature of asthma. Evidence suggests that sphingosine-1-phosphate (S1P) could be a major player in this phenomenon. The purpose of this work was to define the S1P receptor responsible for this phenomenon. We have studied, in the rat, the effect of two S1P synthetic receptor ligands, 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol (FTY720) (which in its phosphorylated form is a potent agonist at each S1P receptor except S1P(2)) and 3-[[2-[4-phenyl-3-(trifluoromethyl)phenyl]-1-benzothiophen-5-yl]methylamino]propanoic acid (AUY954) (a selective S1P(1) agonist) on lung function in vivo. This was complemented by in vitro studies using isolated trachea from the rat, the S1P(3) receptor-deficient mouse, and its wild-type counterpart. After oral administration, FTY720 induced a generalized airway hyper-reactivity to a range of contractile stimuli. This was observed as early as 1 h postdosing, lasted for at least 24 h, and was not subject to desensitization. In both rat and wild-type mouse isolated trachea, preincubation with the active phosphorylated metabolite of FTY720 induced hyper-responsiveness to 5-hydroxytryptamine. This effect was not seen in the isolated tracheas from S1P(3) receptor-deficient mice. AUY954, did not mimic the effect of FTY720 either in vivo or in vitro. Our data are consistent with activation of the S1P pathway inducing a generalized airway hyper-reactivity in rats and mice that is mediated by the S1P(3) receptor. S1P(3) receptor antagonists might prove to be useful as new therapeutic strategies aimed at blocking the airway hyper-reactivity observed in asthma.

A role for sensory nerves in the late asthmatic response.

BACKGROUND: In allergic asthma, exposure to relevant antigens leads to an early asthmatic response (EAR) followed, in certain subjects, by a late asthmatic response (LAR). Although many subjects with asthma consider LAR to be one of the defining symptoms of their disease, and despite its widespread use in the clinical assessment of new therapeutic entities, the mechanism underlying the LAR remains unclear. METHOD: A study was undertaken using ovalbumin-sensitised and challenged Brown Norway rat and C57BL/6J mouse models which recapitulate phenotypic features of allergic asthma including the LAR and its susceptibility to clinically effective agents. RESULTS: In conscious animals an EAR was followed by a LAR. The LAR was subjectively evidenced by audible (wheeze) and visual signs of respiratory distress associated with quantifiable changes in non-invasive lung function assessment. Treatments that attenuated the EAR failed to impact on the LAR and, while anaesthesia did not impact on EAR, it abolished LAR. A key role for airway sensory neuronal reflexes in the LAR was therefore hypothesised, which was confirmed by the blockade observed after administration of ruthenium red (non-selective cation channel blocker), HC-030031 (TRPA1 inhibitor) and tiotropium bromide (anticholinergic) but not JNJ-17203212 (TRPV1 inhibitor). CONCLUSION: These results suggest that LAR involves the following processes: allergen challenge triggering airway sensory nerves via the activation of TRPA1 channels which initiates a central reflex event leading to a parasympathetic cholinergic constrictor response. These data are supported by recent clinical trials suggesting that an anticholinergic agent improved symptoms and lung function in patients with asthma.

Progress towards novel adenosine receptor therapeutics gleaned from the recent patent literature.

IMPORTANCE OF THE FIELD: The principle of treating disease with selective adenosine receptor ligands has been demonstrated with drugs on the market, while the lesser understood receptor subtypes are still being probed with new and drug-like pharmaceutical tools. The field of adenosine receptor research is, therefore, highly important as an emerging and proven point of intervention in disease. AREAS COVERED IN THIS REVIEW: From 2008 to 2009, > 120 primary patent applications have claimed adenosine receptor ligands, which we analyze by applicant and target. Particularly significant disclosures are described in detail, paying particular attention to the biological data marshalled to support the case. WHAT THE READER WILL GAIN: The first published disclosure of new compounds, compound uses or drug targets is often in the patent literature, which can be difficult to trawl, interpret and verify as it is not subject to peer review. We have critically reviewed this area and share our conclusions regarding progress, trends and identification of early tool compounds or compounds of potential clinical significance ahead of peer-reviewed publication. TAKE HOME MESSAGE: Adenosine receptor research is a thriving field with continuing claims of exciting new compounds with high specificity and intriguing examples of new uses for such ligands.

The identification of indacaterol as an ultralong-acting inhaled beta2-adrenoceptor agonist.

Following a lipophilicity-based hypothesis, an 8-hydroxyquinolinone 2-aminoindan derived series of beta(2)-adrenoceptor agonists have been prepared and evaluated for their potential as inhaled ultralong-acting bronchodilators. Determination of their activities at the human beta(2)-adrenoceptor receptor showed symmetrical substitution of the 2-aminoindan moiety at the 5- and 6-positions delivered the targeted intermediate potency and intrinsic-efficacy profiles relative to a series of clinical reference beta(2)-adrenoceptor agonists. Further assessment with an in vitro superfused electrically stimulated guinea-pig tracheal-strip assay established the onset and duration of action time courses, which could be rationalized by considering the lipophilicity, potency, and intrinsic efficacy of the compounds. From these studies the 5,6-diethylindan analogue indacaterol 1c was shown to possess a unique profile of combining a rapid onset of action with a long duration of action. Further in vivo profiling of 1c supported the long duration of action and a wide therapeutic index following administration to the lung, which led to the compound being selected as a development candidate.

Synthesis and evaluation of two series of 4'-aza-carbocyclic nucleosides as adenosine A2A receptor agonists.

The synthesis of two series of 4'-aza-carbocyclic nucleosides are described in which the 4'-substituent is either a reversed amide, relative to the carboxamide of NECA, or an N-bonded heterocycle. Using established purine substitution patterns, potent and selective examples of agonists of the human adenosine A(2A) receptor have been identified from both series. The propionamides 14-18 and the 4-hydroxymethylpyrazole 32 were determined to be the most potent and selective examples from the 4'-reversed amide and 4'-N-bonded heterocyclic series, respectively.

Bleomycin-induced lung injury assessed noninvasively and in spontaneously breathing rats by proton MRI.

PURPOSE: To apply proton magnetic resonance imaging (MRI) techniques to assess noninvasively and in spontaneously breathing rats, structural changes following a single intratracheal administration of bleomycin (BLM). MATERIALS AND METHODS: Rats were scanned by MRI prior to BLM or vehicle administration and at six hours, 24 hours, week 1, and at weeks 2, 3, 6, and 8 after treatment. Bronchoalveolar lavage (BAL) fluid and histological analyses were performed at 24 hours, and at weeks 1 and 8 (histology only). RESULTS: Prominent MRI fluid signals were detected in the lungs of BLM-treated rats one week after challenge. These signals correlated with increased inflammatory parameters in BAL fluid and with marked perivascular and parenchymal infiltration with inflammatory cells in histological slices. At week 2 the MRI signals due to edema resolved, but nevertheless an increase in MRI signal intensity from the lung parenchyma was apparent. In some areas of the right lung the MRI signal intensity in the parenchyma decreased between weeks 2 and 8. These observations were in line with histology demonstrating collagen deposition and atelectasis (hallmarks of fibrosis) at week 1 and a partial recovery of the lung parenchyma at week 8. CONCLUSION: The data demonstrate the ability of proton MRI to detect BLM-induced lung fibrosis as well as the acute inflammatory response caused by the agent.

Lung inflammation and vascular remodeling after repeated allergen challenge detected noninvasively by MRI.

Magnetic resonance imaging (MRI) has been used previously to follow noninvasively inflammatory processes in rat acute models of lung inflammation. Here the technique was applied to a model involving repeated intratracheal administration of ovalbumin (OA). Anatomical MRI was performed at different time points with respect to a single or multiple OA challenges in Brown Norway rats actively sensitized to the allergen. Vascular permeability was assessed using dynamic contrast-enhanced MRI (DCE-MRI). Bronchoalveolar lavage (BAL) fluid analysis and histology were performed to validate the MRI data. The time course of MRI signals after a single OA challenge reached a maximum at 48 h and decreased significantly at 96 h. After the second and subsequent challenges, the maximum signal occurred at 6 h with a time-dependent decline over the remainder of the time course. A reduction of the inflammatory response following repeated administration of OA was also detected by BAL fluid analysis. The decrease in vascular permeability assessed by DCE-MRI in repeatedly OA-challenged rats was consistent with the thickening of the vascular wall for vessels of diameter up to 300 microm revealed by histology. Angiogenesis of vessels smaller than 30 microm was also detected histologically. These results suggest that MRI can be used to detect the inflammatory response and vascular remodeling associated with chronic airway inflammation in rat models involving repeated administration of allergen. As the contrast agent used in the DCE-MRI experiments is approved for clinical use, there is potential to translate the approach to patients.

Proton MRI as a noninvasive tool to assess elastase-induced lung damage in spontaneously breathing rats.

Elastase-induced changes in lung morphology and function were detected in spontaneously breathing rats using conventional proton MRI at 4.7 T. A single dose of porcine pancreatic elastase (75 U/100 g body weight) or vehicle (saline) was administered intratracheally (i.t.) to male Brown Norway (BN) rats. MRI fluid signals were detected in the lungs 24 hr after administration of elastase and resolved within 2 weeks. These results correlated with perivascular edema and cellular infiltration observed histologically. Reductions in MRI signal intensity of the lung parenchyma, and increases in lung volume were detected as early as 2 weeks following elastase administration and remained uniform throughout the study, which lasted 8 weeks. Observations were consistent with air trapping resulting from emphysema detected histologically. In a separate experiment, animals were treated daily intraperitoneally (i.p.) with all-trans-retinoic acid (ATRA; 500 microg/kg body weight) or its vehicle (triglyceride oil) starting on day 21 after elastase administration and continuing for 12 days. Under these conditions, ATRA did not elicit a reversal of elastase-induced lung damage as measured by MRI and histology. The present approach complements other validated applications of proton MRI in experimental lung research as a method for assessing drugs in rat models of respiratory diseases.

Identification with MRI of the pleura as a major site of the acute inflammatory effects induced by ovalbumin and endotoxin challenge in the airways of the rat.

Inflammatory effects in the rat lung have been investigated, non-invasively by MRI, at early time points (3 and 6 h) after ovalbumin (OA) or endotoxin (LPS) challenges. Six hours after challenge with OA, a strong, even inflammatory signal was present around the periphery of the lung in a region corresponding to the pleura. Histological analysis confirmed the presence of marked edema associated with the pleural cavity of OA-treated animals. Lower levels of pleural edema were observed in MRI and histological evaluation of LPS-treated animals and no abnormality was observed in actively sensitized and naïve, saline-treated groups. Diffuse edematous signals were detected in the lung 3 and 6 h after challenge with OA or LPS; the signal volumes were larger at both time points following OA instillation. Bronchoalveolar lavage (BAL) fluid analysis performed 6 h after challenge revealed increased levels of protein and greater cellular activation in OA- than in LPS-treated animals. Furthermore, increased levels of peribronchial edema were found by histology 6 h after OA. BAL fluid and histological assessments demonstrated that the inflammatory signals were due to edema and not mucus as no significant changes in BAL mucin concentrations or differences in goblet cells were identified between OA or LPS challenge and their respective vehicle groups. Our data show that MRI is able to detect, non-invasively, inflammatory signals in both the lung and the pleura in spontaneously breathing animals, highlighting its potential to study the consequences of pulmonary insults on both sites.

In vitro and in vivo pharmacological characterization of 5-[(R)-2-(5,6-diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one (indacaterol), a novel inhaled beta(2) adrenoceptor agonist with a 24-h duration of action.

Here, we describe the preclinical pharmacological profile of 5-[(R)-2-(5,6-diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one (indacaterol), a novel, chirally pure inhaled beta(2) adrenoceptor agonist, in comparison with marketed drugs. Indacaterol is close to a full agonist at the human beta(2) adrenoceptor (E(max) = 73 +/- 1% of the maximal effect of isoprenaline; pEC(50) = 8.06 +/- 0.02), whereas salmeterol displays only partial efficacy (38 +/- 1%). The functional selectivity profile of indacaterol over beta(1) human adrenoceptors is similar to that of formoterol, whereas its beta(3) adrenoceptor selectivity profile is similar to that of formoterol and salbutamol. In isolated superfused guinea pig trachea, indacaterol has a fast onset of action (30 +/- 4 min) similar to formoterol and salbutamol, and a long duration of action (529 +/- 99 min) comparable with salmeterol. In the conscious guinea pig, when given intratracheally as a dry powder, indacaterol inhibits 5-hydroxytryptamine-induced bronchoconstriction for at least 24 h, whereas salmeterol, formoterol, and salbutamol have durations of action of 12, 4, and 2 h, respectively. When given via nebulization to anesthetized rhesus monkeys, all of the compounds dose-dependently inhibit methacholine-induced bronchoconstriction, although indacaterol produces the most prolonged bronchoprotective effect and induces the lowest increase in heart rate for a similar degree of antibronchoconstrictor activity. In conclusion, the preclinical profile of indacaterol suggests that this compound has a superior duration of action compatible with once-daily dosing in human, together with a fast onset of action and an improved cardiovascular safety profile over marketed inhaled beta(2) adrenoceptor agonists.

A new orally bioavailable dual adenosine A2B/A3 receptor antagonist with therapeutic potential.

The synthesis and SAR of 5-heterocycle-substituted aminothiazole adenosine receptor antagonists is described. Several compounds show high affinity and selectivity for the A2B and A3 receptors. One compound (5f) shows good ADME properties in the rat and as such may be an important new compound in testing the current hypotheses proposing a therapeutic role for a dual A2B/A3 antagonist in allergic diseases.

The receptor mechanism mediating the contractile response to adenosine on lung parenchymal strips from actively sensitised, allergen-challenged Brown Norway rats.

Parenchymal strips prepared from lungs removed from actively sensitised Brown Norway rats challenged with allergen show hyperresponsiveness to adenosine. The response is mast cell mediated and a preliminary pharmacological analysis suggested the involvement of a receptor (or receptors) that could not be classified as any of the known adenosine receptor subtypes. We present a further analysis of the response. Male Brown Norway (BN) rats, actively sensitised to ovalbumin (OA), were challenged intratracheally with OA and killed 3 h later to provide parenchymal strip preparations. The augmented contractile responses to adenosine were partially blocked by the 5-HT receptor antagonist, methysergide, or the A(1) receptor antagonist, DPCPX, and abolished in the presence of both antagonists. Responses to high concentrations of the A(1) receptor agonist, CPA were, like those to adenosine, augmented on tissues from allergen-challenged animals and blocked by a combination of methysergide and DPCPX. The A(3) receptor agonist, Cl-IB-MECA, did not contract the tissue, but partially blocked the response to adenosine. A combination of Cl-IB-MECA and methysergide induced a similar degree of blockade to that seen with either drug given alone. Combination of Cl-IB-MECA and/or methysergide with DPCPX abolished the response to adenosine. The effects of the A(3) receptor agonist, inosine, were augmented on tissues from allergen-challenged animals and markedly inhibited by disodium cromoglycate, methysergide or Cl-IB-MECA. Responses to adenosine were abolished when parenchymal strips were taken from rats pretreated 48 h previously with pertussis toxin. 8-SPT, CGS 15943, XAC, MRS 1754, DPCPX and theophylline, at concentrations which inhibit the A(1) A(2A) and/or A(2B) receptors but have negligible affinity for the rat A(3) receptor, inhibited responses to adenosine, but high concentrations were required and blockade was incomplete. MRS 1523 and MRS 1191, which are antagonists at the rat A(3) receptor, had no effect on the response to adenosine. The present results support and clarify our earlier conclusion that an atypical receptor mechanism mediates contraction of the parenchymal strip prepared from the lungs of actively sensitised BN rats challenged with allergen to adenosine. The response arises from a combined effect of adenosine on the A(1) receptor and a receptor with similarities to the A(3) receptor, but where Cl-IB-MECA behaves as an antagonist and MRS 1523 and MRS 1191 are inactive at concentrations that substantially exceed their affinities for the rat A(3) receptor.

A locally active antiinflammatory macrolide (MLD987) for inhalation therapy of asthma.

One of the characteristic features of asthma is a persistent pulmonary inflammation, with increased numbers of eosinophils and activated T-lymphocytes in the airways. T-helper cells of the Th2 phenotype play a pivotal role in the pathogenesis of asthma, and they are believed to orchestrate the asthmatic response by releasing a wide repertoire of cytokines. Herein, we describe the design, synthesis, and evaluation in models of allergic asthma of a locally active T-cell modulator, MLD987 (1). Compound 1 is a potent immunosuppressant that inhibits the activation, proliferation, and release of cytokines from T-cells with IC(50) values in the low nanomolar range. In a Brown-Norway rat model of allergic asthma, 1, when given into the airways by intratracheal administration (ED(50) = 1 mg/kg) or by inhalation (ED(50) = 0.4 mg/kg), potently reduced the influx of leukocytes into bronchoalveolar lavage fluid samples obtained from antigen-challenged animals. In contrast, 1 had an appreciably weaker activity in this model when given orally or intravenously. Pharmacokinetic evaluation in rat and rhesus monkey showed that 1 had both a low oral (2-4%) and a low pulmonary (7%, monkey) bioavailability. These findings are consistent with a local site of action of the compound and rule out that its antiinflammatory activity in the lung was caused by systemically absorbed material, which had been swallowed during inhalation or which had entered the circulation via the airways. Local administration and the metabolically soft structure of 1, which favors rapid systemic metabolism to less immunosuppressive metabolite 2, are the main reasons for the low exposure and weak systemic activity of the compound. Administration of a locally active compound such as 1, by inhalation, should reduce systemic side effects. Our results indicate that 1 has the potential to serve as an alternative to inhaled glucocorticosteroids for the long-term therapy of asthma of all grades of severity.

Interaction between adenosine and allergen or compound 48/80 on lung parenchymal strips from actively sensitized Brown Norway rats.

We have investigated the effect of mast cell activation induced by immunological and non-immunological stimuli on the sensitivity to adenosine of parenchymal strips prepared from lungs removed from Brown Norway (BN) rats actively sensitized to ovalbumin. Strips responded to ovalbumin with a biphasic contractile response. Responses to adenosine were markedly increased 30 min after ovalbumin. The first phase of the response to ovalbumin was abolished by the 5-hydroxytryptamine (5-HT)2 receptor antagonist, methysergide and unaffected by the cysteinyl leukotriene receptor antagonist, iralukast. The second phase was abolished by iralukast and unaffected by methysergide. The response to adenosine was markedly reduced by methysergide but not significantly altered by iralukast. Compound 48/80 (condensation product of N-methyl-p-methoxyphenylethylamine with formaldehyde) induced methysergide-sensitive contractions of the parenchymal strip and potentiated adenosine; the augmented response to adenosine was blocked by methysergide. Thus, activation of mast cells in the lung by either immunological or non-immunological stimuli results in augmentation of the mast cell-mediated contractile response to adenosine.

Synthesis and biological properties of novel glucocorticoid androstene C-17 furoate esters.

A series of novel corticosteroid derivatives featuring C-17 furoate ester functionality have been synthesised. Profiling in vitro and in vivo has resulted in the identification of a compound with a longer duration of action and a lower oral side effect profile in rodents compared to budesonide.

New highly potent and selective adenosine A(3) receptor antagonists.

A class of potent, selective adenosine A(3) receptor antagonists was obtained via optimisation of the screening hit N-[4-(4-methoxyphenyl)-thiazol-2-yl]-acetamide. Structural modifications of this hit revealed very quickly that a 5-(pyridin-4-yl) substituent on the 2-aminothiazole ring was optimal for high potency at the adenosine A(3) receptor. Structure activity relationship studies led to both potent and selective A(3) receptor antagonists, including N-[5-pyridin-4-yl-4-(3,4,5-trimethoxyphenyl)-thiazol-2-yl]-acetamide, a highly potent aden-osine A(3) receptor antagonist with greater than 100- fold selectivity against the related adenosine receptors. As well as demonstrating selective in vitro binding on the human A(3) adenosine receptor, this compound was also shown to selectively block the rat A(3) receptor in vivo. This important new compound can be readily synthesised in four steps from commercially available starting materials.

Effects of a mitogen-activated protein kinase inhibitor on allergic airways inflammation in the rat studied by magnetic resonance imaging.

We recently described a new model to study non-invasively with magnetic resonance imaging (MRI) the effects of compounds to prevent and/or resolve airway inflammation induced by ovalbumin in the lungs of actively sensitised rats. We report here the effects of 4-(4-fluorophenyl)-2-(1-methylpiperidin-4-yl)-5-(2-(1-(S)-phenylethyl)amino-4-pyridinyl)thiazole fumarate (Compound 1), which exhibits inhibitory activity against p38alpha and p38beta2 and residual activity on c-Jun amino-terminal kinase (JNK)2 mitogen-activated protein (MAP) kinases, on the oedematous signals detected by MRI and generated by antigen challenge in the lungs of sensitized rats. Compound 1 (10 mg kg(-1)) given orally 1 h prior to allergen challenge significantly reduced the oedematous signal measured at 24 h. Similar effects were seen with a synthetic corticosteroid, mometasone furoate (0.3 mg kg(-1)), given intratracheally 3 h prior to challenge. For both compounds, inhibition of the oedematous signal was accompanied by reductions in the inflammatory parameters in the bronchoalveolar lavage fluid measured 24 h after challenge with ovalbumin. Compound 1 (10 mg kg(-1)) administered 24 h after challenge with ovalbumin did not change the rate of resolution of the signal detected by MRI in the lungs. In contrast, mometasone furoate (0.3 mg kg(-1)) significantly increased resolution of these signals, which was evident 3 h after drug administration and maintained to 48 h post challenge. Collectively, our data suggest that the p38 MAP kinase inhibitor Compound 1 shows a different profile than glucocorticosteroids since its ability to resolve existing inflammation is limited.