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1 Research Unit EA220, Faculty of Medicine, Versailles University, Hospital Foch, Suresnes, and 3 Dept of Pharmacology, University Victor Segalen Bordeaux 2, Bordeaux, France. 2 Novartis Institute for BioMedical Research, Horsham, UK.
CORRESPONDENCE: M. Molimard, Département de Pharmacologie, CHU de Bordeaux, Université Victor Segalen-INSERM U657, 33076 Bordeaux cedex, France. Fax: 33 557574671. E-mail: mathieu.molimard{at}pharmaco.u-bordeaux2.fr
Keywords: Airways smooth muscle, ß2-adrenoceptor agonists, formoterol, indacaterol, isolated human bronchus, salmeterol
Received: March 7, 2006
Accepted November 21, 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIAL AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Potency (-logEC50), maximal relaxant effect (Emax) and onset of action were determined at resting tone. Duration of action was determined against cholinergic neural contraction induced by electrical field stimulation (EFS).
At resting tone, -logEC50 and Emax values were 8.82±0.41 and 77±5% for indacaterol, 9.84±0.22 and 94±1% for formoterol, 8.36±0.16 and 74±4% for salmeterol, and 8.43±0.22 and 84±4% for salbutamol, respectively.
In contrast to salmeterol, indacaterol did not antagonise the isoprenaline response. Indacaterol's onset of action (7.8±0.7 min) was not significantly different from that of formoterol (5.8±0.7 min) or salbutamol (11.0±4.0 min), but it was significantly faster than that of salmeterol (19.4±4.3 min). EFS-induced contractions were inhibited with -logIC50 values of 6.96±0.13 (indacaterol), 8.96±0.18 (formoterol), 7.18±0.34 (salmeterol) and 6.39±0.26 (salbutamol). Duration of action was >12 h for indacaterol and salmeterol, and 35.3±8.8 and 14.6±3.7 min for formoterol and salbutamol, respectively.
In isolated human bronchi, indacaterol behaved as a long-acting ß2-adrenoceptor agonist with high intrinsic efficacy and fast onset of action.
The introduction of long-acting inhaled ß2-adrenoceptor agonists (LABAs; salmeterol and formoterol) represented an important advance in asthma therapy. Treatment with LABAs provides better control of symptoms and lung function than short-acting ß2-agonists (SABAs) 1, 2. In fact, when combined with moderate doses of inhaled glucocorticosteroids, these drugs have been shown to improve symptoms and lung function more effectively than doubling the dose of inhaled corticosteroids 3–5. They have also been shown to reduce the number of asthma exacerbations during a 12-month treatment period, although to a lesser extent than doubling the dose of inhaled corticosteroids 4. LABAs are also used in the treatment of stable chronic obstructive pulmonary disease (COPD), according to the Global Initiative for Chronic Obstructive Lung Disease guidelines recommendations 6, 7.
The clinical efficacy and duration of action of formoterol and salmeterol appear to be very similar 8, 9. There are, however, some differences between the two compounds in terms of onset of action 10, 11 and interaction with the ß2-receptor 10, 12, 13. The ß2-receptor interaction is characterised in terms of the following factors: the ability to bind to the receptor and induce an intracellular response; potency, which is related to the amount of drug required for a physiological response; and efficacy, which is related to the drug's ability to induce a maximum physiological effect 10, 12, 13.
Indacaterol is a novel inhaled ß2-adrenoceptor agonist under clinical development for the treatment of asthma and COPD. In pre-clinical studies in guinea pigs, indacaterol demonstrated a longer duration of action and faster onset than salmeterol 14. Clinical studies have indicated that indacaterol might be used as a once-daily compound 15, which would be of great interest regarding patient convenience and compliance with treatment.
The present study aimed to evaluate the relaxant effect (potency and maximal efficacy) and the onset and duration of action of indacaterol in comparison with marketed compounds (formoterol, salbutamol and salmeterol) on isolated human bronchial rings. The interaction of indacaterol, formoterol and salmeterol with isoprenaline-induced bronchial relaxation was also studied. Since isoprenaline is a SABA, this permitted the assessment of the potential antagonism of these compounds with the use of SABA rescue medicine.
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MATERIAL AND METHODS |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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20 mm from the malignancy was supplied by the hospital pathologist and submerged in a physiological salt solution (PSS; Kreb's solution, composition in mM: NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 0.6, KH2PO4 1.1, NaHCO3 25.0, glucose 11.7) at 4°C. After removal of adhering lung parenchyma and connective tissue, eight to 24 rings from each bronchus were prepared (4–5 mm lengthx2–4 mm diameter) as previously described elsewhere 16. The preparations were stored overnight at 4°C in PSS equilibrated with 5% CO2 in O2 and the experiments were performed the next day. Previous experience in the laboratory and other published data have demonstrated that overnight storage of tissue does not alter its reactivity 10, 16, 17.
Experimental procedures
Tissue preparation
The total number of rings used in the present study was 411. Bronchial rings were suspended in parallel on tissue hooks in 10-mL organ baths containing PSS, gassed with 5% CO2 in O2 at 37°C (pH 7.4). Each preparation was connected to a force displacement transducer (UF1 Pioden strain gauges; EMKA Technologies, Mitry Mory, France) and isometric tension changes were recorded (I.O.X. recorder system; EMKA Technologies). The preparations were suspended under an initial load of 3 g and equilibrated for 60–90 min with changes in bath PSS every 15–20 min before any pharmacological intervention occurred. At the end of the equilibration period, the resting load was stable at 2–4 g. In all experiments, human bronchi were first contracted maximally with acetylcholine (3 mM). The tissues were then washed and equilibrated for 60 min before beginning the experimental procedure. Under these conditions, responses were optimal and reproducible according to data from the present authors’ laboratory and other studies 10, 16–18.
Potency and efficacy
A total of 110 rings obtained from 10 patients were used for the following experiments and only one concentration–response curve for one compound was recorded in each ring. Within a given set of experiments, the response for each patient was tested in duplicate. Concentration–response curves for indacaterol, formoterol, salmeterol and salbutamol were produced by cumulative addition of the compounds at intervals of 5–30 min to obtain a relaxation plateau on bronchi at resting tone and on bronchi pre-contracted with histamine (10 µM, representing 30% of 3 mM acetylcholine) or carbachol (1 µM, representing 40% of 3 mM acetylcholine). At the end of the experiment, theophylline 3 mM was added to the bath to determine the maximal relaxation.
Antagonism of isoprenaline-induced relaxation
A total of 93 rings obtained from seven patients were used for the following experiments and only one concentration–response curve for isoprenaline was recorded in each ring. Within a given set of experiments, the response for each patient was tested in duplicate. Following the resting period, bronchial rings were contracted with 1 µM carbachol. After the contraction plateau was reached, bronchial rings were incubated for 30 min with PSS (control) or with equi-effective concentrations of formoterol (10–9 M and 3x10–9 M) or indacaterol (10–7 M and 3x10–7 M), which induced 
20 and 35% inhibition of the carbachol-induced contraction. As a result of its partial agonistic activity, salmeterol (10–7 M and 3x10–7 M) did not induce >20% inhibition of the carbachol-induced contraction. Thereafter, concentration–response curves to isoprenaline in the presence of ß2-adrenoceptor agonist drugs or PSS were recorded by applying increasing concentrations of isoprenaline (10–8–10–5 M) at 8–15-min intervals. At the end of the experiment, theophylline (3 mM) was added to the bath to determine maximal relaxation.
Onset of action
A total of 32 rings obtained from four patients were used for the following experiments and only one compound and one concentration were studied in each ring. Within a given set of experiments, the response for each patient was tested in duplicate. The onset of action was measured on resting tone and was calculated as the time (min) to induce 50% of the maximal relaxation observed for the compound 10, 17. Equi-effective drug concentrations (salbutamol, salmeterol and indacaterol 3x10–8 M and formoterol 10–9 M) corresponding to 80% of the maximal relaxation observed for each compound, were used.
Electrical field stimulation
Experiments were performed as previously described elsewhere 19. A total of 176 rings obtained from 13 patients were used for the following experiments and only one compound and one concentration were studied in each ring. Each organ bath was fitted with two platinum plate electrodes (1 cm2) placed alongside the tissue (10 mm apart) for transmural electrical field stimulation (EFS; biphasic pulse width 1 ms, constant current of 320 mA for 10 s at 5 Hz). A first control response was established for all bronchi preparations by adding 3 mM acetylcholine to obtain a plateau of maximal contraction. After washing, bronchi were allowed to equilibrate for 60 min with a change of the PSS every 15 min. For the subsequent duration of the experiment, 1 µM montelukast and 1 µM indomethacin were present in the buffer to avoid the influence on the neuronal responses of indirect effects of leukotrienes and prostaglandins, respectively.
After tension had returned to the baseline tone, the preparation was stimulated every 10 min at 5 Hz, pulse width 1 ms and 320 mA current for 10 s using a stimulator (EMKA Technologies) where the voltage output was adjusted to give a constant current and biphasic rectangular pulse of alternating polarity. These contractions represent 20–50% of the maximal contraction induced by 3 mM acetylcholine. Compounds or vehicle were then added to the bath for 1 h and washed out before the beginning of a second train of stimulations every 20 min for 12 h (fig. 1
).
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Expression and analysis of data
The maximal relaxant effect (Emax) of each ß2-adrenoceptor agonist was expressed as a percentage of the relaxation induced by 3 mM theophylline. Potency was determined by the value of concentration producing 50% of the maximal effect of a given compound (EC50). The onset of action was calculated as the time (min) from compound administration to the attainment of half its maximal relaxation.
The concentration of each ß2-adrenoceptor agonist that induced a diminution of EFS response equal to 50% of the first control response (IC50) was determined. The duration of action was then calculated as the time taken for response to 50% recovery from maximum inhibition.
A statistical significance of p<0.05 was assessed using ANOVA and a t-test for paired or unpaired data. Data are presented as mean±SEM.
Drugs
Indacaterol maleate and formoterol fumarate were synthesised by the Dept of Chemistry (Novartis Horsham Research Centre, Horsham, UK). Salmeterol xinafoate was either synthesised or isolated from clinical dosage forms by the Dept of Chemistry (Novartis Horsham Research Centre) or purchased from Tocris Cookson Ltd (Bristol, UK). Acetylcholine HCl, indomethacin, carbachol (carbamylcholine chloride), salbutamol hemisulphate, theophylline and histamine were purchased from Sigma (Saint Quentin Fallavier, France), montelukast (MK476) from Merck (Paris, France) and isoprenaline HCl from Laboratoires Winthrop (Paris, France). Salmeterol and formoterol were dissolved in distilled water in the presence of HCl and DMSO (3%). Indacaterol was dissolved in distilled water in the presence of acetic acid (2%) and ethanol (20%). Indomethacin and MK476 were dissolved in pure ethanol and then diluted in PSS. Stock solutions (1 mM) were kept at -20°C until use.
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RESULTS |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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salmeterol. The order of maximal efficacy was: formoterol > salbutamol > indacaterol salmeterol (table 1, fig. 2a).
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, fig. 2b). Conversely, carbachol pre-contraction decreased the potency of indacaterol (p<0.01), formoterol (p<0.01) and salbutamol (p<0.01). However, a decrease in the maximal relaxant effect was only observed for formoterol (p<0.01), salmeterol (p<0.001) and salbutamol (p<0.01; table 1, fig. 2c).
Antagonism of isoprenaline-induced relaxation
On preparations pre-contracted with 1 µM carbachol and at concentrations inducing 20 and 35% inhibition, formoterol and indacaterol did not affect the potency of isoprenaline-induced bronchi relaxation. In contrast, in the presence of salmeterol, at a concentration inhibiting the contraction by 20%, a statistically significant decrease in isoprenaline potency was observed (table 2, fig. 3).
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80% of the maximal relaxation at resting tone, the onset of action of indacaterol (3x10–8 M; 7.8±0.7 min, n = 4) was not significantly different from that of formoterol (10–9 M; 5.8±0.7 min, n = 4) and salbutamol (3x10–8 M; 11.0±4.0 min, n = 4) but was significantly faster than that of salmeterol (3x10–8 M; 19.4±4.3 min, n = 4; p<0.05).
Protection against cholinergic neural bronchoconstriction
EFS-induced contractions were inhibited in a concentration-dependent manner by all compounds with the following order of potency: formoterol > salmeterol > indacaterol > salbutamol (fig. 4, table 3). The duration of protection against cholinergic neural bronchoconstriction determined at doses corresponding to their respective IC50 values was >12 h for indacaterol and salmeterol, 35 min for formoterol and 15 min for salbutamol (table 3). At high doses, indacaterol, formoterol and salbutamol induced close to full inhibition of the EFS-induced contraction. At the highest dose tested (10–5 M), the effect of indacaterol lasted 
12 h, whereas formoterol (10–6 M) and salbutamol (10–4 M) showed a 20% reduction of the inhibition of the cholinergic response at 5 and 1.5 h, respectively. Salmeterol (10–5 M) induced a maximal inhibition of the cholinergic response of 80%, followed by a 20% reduction of the inhibition of the cholinergic response at 4 h.
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DISCUSSION |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Under the present experimental conditions, indacaterol at resting tone or in the bronchi contracted with histamine was about 10 times less potent than formoterol but about three times more potent than salbutamol and salmeterol. When the bronchi were pre-contracted with carbachol, the compounds' potencies were moderately (1 log unit for formoterol) or strongly reduced (1.98 log units for salbutamol and 2.61 log units for indacaterol). Salmeterol's potency was not affected when compared with the resting tone condition. This decrease in potency observed in carbachol-treated bronchi could be related to the fact that carbachol induced a greater contraction when compared with histamine (40 versus 30% of the maximal effect seen with acetylcholine, respectively). However, it is difficult to believe that such a minimal difference in the strength of the contraction could explain such a dramatic effect on the potency of the compounds studied. A more likely explanation resides in a functional antagonism between the ß2-adrenoceptor and the muscarinic receptor, probably due to ß2-adrenoceptor or Gs protein phosphorylation by protein kinase C 20, 21 or to the inhibition of adenylate cyclase by the muscarinic M2 receptor-mediated Gi protein 22.
In terms of efficacy, which is related to the ability of the drugs to activate the ß2-adrenoceptor, all four drugs seem to be full agonists on preparations at resting tone. In the presence of histamine, none of the compounds had a statistically significant reduction in maximal effect. However, in the presence of carbachol, the efficacy of salbutamol and salmeterol was moderately to considerably reduced, whereas formoterol and indacaterol were marginally or not affected. This suggests that formoterol and indacaterol are nearly full agonists, whereas salbutamol and salmeterol are partial agonists, as previously demonstrated elsewhere 10, 23, 24. These differences in the intrinsic efficacy of the four drugs were confirmed by the inhibition by salmeterol in the present and other studies 10, 12, 23, and by salbutamol 10, 12, but not by formoterol and indacaterol, of the relaxation induced by isoprenaline. These results are in agreement with receptor theories suggesting that a partial agonist has to occupy more receptors than a full agonist and thereby creates a situation in which functional receptor number becomes limiting in terms of obtaining a maximal response. As a consequence, a partial agonist behaves as an antagonist in the presence of an agonist with higher efficacy acting on the same receptor.
In isolated human bronchi, indacaterol has a fast onset of action, similar to that of salbutamol and formoterol. Although this could seem to be of secondary importance for maintenance therapy, such a profile could still be of interest. Indeed, a rapid improvement in breathlessness could increase a patient's confidence in treatment and, subsequently, increase treatment compliance. Furthermore, a fast onset of action could permit the use of indacaterol as rescue medicine.
The duration of action was evaluated as the protection against cholinergic neural bronchoconstriction induced by EFS and was shown to be markedly higher for indacaterol and salmeterol when compared with formoterol and salbutamol. Although in the present system the duration of action for salmeterol is in line with its known clinical profile, the duration of action of formoterol does not reflect the clinical situation. This discrepancy has been previously reported in human bronchus 25, 26 and a number of theories have been put forward to explain this inconsistency. The most rational explanation for the observed duration of action of formoterol in males almost certainly lies within the high local concentrations achieved after inhalation and the interaction with the membrane lipid bilayer as a key component 27. The present model of EFS-induced contraction does not allow a study for >12 h. Indeed, even under control conditions, a 30–50% loss in bronchial cholinergic response was observed after 12 h of EFS. As a result, the duration of action of indacaterol could not be differentiated from that of salmeterol. However, at the highest concentration tested, indacaterol induced 100% relaxation, with this level being maintained for 12 h, whereas salmeterol induced a maximal relaxation of 80% and demonstrated a loss of 20% of this maximal effect after 4 h of stimulation. This suggests that a longer duration of action can be achieved for indacaterol when compared with salmeterol. Indeed, in clinical studies, indacaterol has demonstrated a profile compatible with once-daily dosing 15.
In conclusion, in the isolated human bronchus indacaterol efficiently prevents the occurrence of bronchial contraction, has a fast onset of action and possesses a very long duration of action. Furthermore, in contrast to salmeterol, indacaterol does not antagonise the effect of rescue medication. All these characteristics, which are promising regarding the use of indacaterol as a very long-acting ß2-adrenoceptor agonist, are now being tested in clinical studies 15.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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REFERENCES |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
: Indacaterol; 
: formoterol; •: salmeterol; 
: salbutamol.
: Physiological salt solution; 
: indacaterol 10–7 M; •: indacaterol 3x10–7 M; 
: formoterol 3x10–9 M; 
: salmeterol 10–7 M; 
: salmeterol 3x10–7 M.
: 10–6 M); c) salbutamol (n = 3;