Agnieszka Ciarka
email: aciarka@ulb.ac.be

Philippe Van De Borne

Jean-Louis Vincent

The effects of dopamine on the respiratory system: Friend or foe?

Dopamine (DA) is an immediate precursor of noradrenaline that has stimulatory or inhibitory effects on a variety of adrenergic receptors. DA is primarily used in the management of circulatory shock for its combined vasopressor and inotropic effects, but it may also exert significant effects on the respiratory system Although the respiratory effects of intravenous DA attract less attention than its hemodynamic effects, there is evidence that DA affects ventilation, pulmonary circulation, bronchial diameter, neuromodulation of sensory pulmonary nerves and lung water clearance. Through these complex mechanisms, DA may exert beneficial as well as detrimental effects on respiration.

DA may have beneficial effects on the respiratory system by decreasing oedema formation and improving respiratory muscle function, but can also have deleterious effects, by inhibiting ventilation.

Hence, DA may be beneficial in lung oedema, but harmful in cases of difficult weaning from mechanical ventilation. DA should be used with caution in patients with heart failure during weaning from mechanical respiration, however, critically ill patients with chronic obstructive pulmonary disease (COPD) do not show this negative effect of DA on ventilatory drive.

Introduction

Dopamine (DA) is an immediate metabolic precursor of noradrenaline and adrenaline [START_REF] Hardman | Goodman and Gilman's The Pharmacological Basis of Therapeutics[END_REF]. It has an intrinsic capacity to activate peripheral adrenergic and dopaminergic receptors [START_REF] Hardman | Goodman and Gilman's The Pharmacological Basis of Therapeutics[END_REF]. In physiologic conditions, DA increases cardiac output and aortic pressure, reduces renal vascular resistance and increases glomerular filtration rate and urinary output [START_REF] Mcdonald Rh | EFFECT OF DOPAMINE IN MAN: AUGMENTATION OF SODIUM EXCRETION, GLOMERULAR FILTRATION RATE, AND RENAL PLASMA FLOW[END_REF]. At low doses (5 µg/kg/min), which do not have important hemodynamic effects, DA causes modest diuresis [START_REF] Varriale | The benefit of low-dose dopamine during vigorous diuresis for congestive heart failure associated with renal insufficiency: does it protect renal function?[END_REF]. DA is used in patients suffering from circulatory shock (primarily septic and cardiogenic) [START_REF] Goldberg | Dopamine--clinical uses of an endogenous catecholamine[END_REF], for its combined inotropic and vasopressor effects. Although the dopaminergic actions may theoretically be beneficial for renal function, the renal protective effects of DA are seriously questioned and there is ongoing discussion about the place of DA in the intensive care unit [START_REF] Holmes | Bad medicine: low-dose dopamine in the ICU[END_REF].

DA can exert various effects on the respiratory system (Table 1). First, DA has an established role in the control of ventilation. Dopaminergic receptors are located pre-and postsynaptically on the carotid glomus cells [START_REF] Mir | Direct biochemical and neuropharmacological identification of dopamine D2-receptors in the rabbit carotid body[END_REF] and high concentrations of DA have been measured in the carotid bodies [START_REF] Fidone | Putative neurotransmitters in the carotid body: the case for dopamine[END_REF].

DA inhibits the chemosensory discharge rate in various animal species and there is clearcut evidence from animal and human studies that low dose DA infusion decreases the ventilatory response to hypoxia [START_REF] Hanbauer | The regulation of dopamine and noradrenaline in the rat carotid body and its modification by denervation and by hypoxia[END_REF][START_REF] Ide | Effects of a continuous infusion of dopamine on the ventilatory and carotid body responses to hypoxia in cats[END_REF][START_REF] Lahiri | Interaction of dopamine and haloperidol with O2 and CO2 chemoreception in carotid body[END_REF][START_REF] Llados | Effects of dopamine analogues and antagonists on carotid body chemosensors in situ[END_REF][START_REF] Nishino | Effects of dopamine on chemoreflexes in breathing[END_REF][START_REF] Olson | Ventilatory responsiveness to hypercapnic hypoxia during dopamine infusion in humans[END_REF][START_REF] Ward | Reduction of hypoxic ventilatory drive by dopamine[END_REF][START_REF] Welsh | Depression of ventilation by dopamine in man. Evidence for an effect on the chemoreceptor reflex[END_REF]. Second, DA provokes a reduction in arterial oxygen saturation at a given alveolar oxygen tension by impairing regional ventilation/perfusion matching in the lung [START_REF] Huckauf | Dopamine induced hypoxemia in patients with left heart failure[END_REF]. When administered to patients with circulatory failure, DA may be deleterious for ventilation and pulmonary gas exchange, especially in hypoxemic patients with compromised respiratory function [START_REF] Van De Borne | Dopamine depresses minute ventilation in patients with heart failure[END_REF]. Third, DA can also reduce pulmonary vascular resistance, resulting in a decrease in pulmonary hypertension [START_REF] Gorman | Vasodilator response to dopamine in the ferret pulmonary circulation[END_REF][START_REF] Loick | The effect of dopamine on pulmonary hemodynamics and tissue damage after inhalation injury in an ovine model[END_REF][START_REF] Mccormack | Effects of dopexamine hydrochloride on hypoxic pulmonary vasoconstriction in isolated rat lung[END_REF][START_REF] Polak | Effect of fenoldopam on preconstricted isolated salt-perfused rat lungs[END_REF][START_REF] Polak | Manipulation of dopamine receptors alters hypoxic pulmonary vasoconstriction in isolated perfused rat lungs[END_REF]. Fourth, DA may promote lung oedema clearance by activation of epithelial Na+ /K+-ATPase [START_REF] Barnard | Dopamine stimulates sodium transport and liquid clearance in rat lung epithelium[END_REF][START_REF] Barnard | Stimulation of the dopamine 1 receptor increases lung edema clearance[END_REF][START_REF] Lecuona | Ventilatorassociated lung injury decreases lung ability to clear edema in rats[END_REF][START_REF] Saldias | Dopamine restores lung ability to clear edema in rats exposed to hyperoxia[END_REF][START_REF] Saldias | Dopamine increases lung liquid clearance during mechanical ventilation[END_REF] and epithelial sodium channels [START_REF] Helms | Dopamine regulation of amiloride-sensitive sodium channels in lung cells[END_REF]. Fifth, DA is involved in the regulation of airway diameter [START_REF] Bonnert | Dual D2-receptor and beta2adrenoceptor agonists for the treatment of airway diseases. 1. Discovery and biological evaluation of some 7-(2-aminoethyl)-4-hydroxybenzothiazol-2(3H)one analogues[END_REF][30][31] and in neuromodulation of pulmonary sensory nerves [32,33]. These effects have primarily been tested in animal studies. There is some clinical evidence that dopamine may improve respiratory muscle function in respiratory failure [34]. A randomised, placebocontrolled human study, based on the observation that DA receptor activation inhibits rapidly adapting stretch receptors in the lungs, gave disappointing results. Indeed, the DA-2 and beta2 adrenergic This paper focuses on the effects of DA on the respiratory system, to evaluate whether DA can be safely used in acutely ill patients with respiratory failure.

.

Clinical pharmacology

DA exerts complex actions on peripheral and central adrenergic and dopaminergic receptors

[37]. In the peripheral nervous system, DA is present in postganglionic sympathetic nerve endings and is an immediate precursor of noradrenaline. Intravenous DA does not cross the brain-blood barrier, so that it does not have central effects. Although the respiratory effects of intravenous DA have attracted less attention than its hemodynamic actions, there is evidence that DA affects ventilation [START_REF] Huckauf | Dopamine induced hypoxemia in patients with left heart failure[END_REF][START_REF] Hanbauer | The regulation of dopamine and noradrenaline in the rat carotid body and its modification by denervation and by hypoxia[END_REF][START_REF] Ide | Effects of a continuous infusion of dopamine on the ventilatory and carotid body responses to hypoxia in cats[END_REF][START_REF] Lahiri | Interaction of dopamine and haloperidol with O2 and CO2 chemoreception in carotid body[END_REF][START_REF] Llados | Effects of dopamine analogues and antagonists on carotid body chemosensors in situ[END_REF][START_REF] Nishino | Effects of dopamine on chemoreflexes in breathing[END_REF][START_REF] Olson | Effect of a dopamine antagonist on ventilation during sustained hypoxia in mice[END_REF][START_REF] Ward | Reduction of hypoxic ventilatory drive by dopamine[END_REF][START_REF] Welsh | Depression of ventilation by dopamine in man. Evidence for an effect on the chemoreceptor reflex[END_REF], pulmonary circulation [START_REF] Gorman | Vasodilator response to dopamine in the ferret pulmonary circulation[END_REF][START_REF] Loick | The effect of dopamine on pulmonary hemodynamics and tissue damage after inhalation injury in an ovine model[END_REF][START_REF] Mccormack | Effects of dopexamine hydrochloride on hypoxic pulmonary vasoconstriction in isolated rat lung[END_REF][START_REF] Polak | Effect of fenoldopam on preconstricted isolated salt-perfused rat lungs[END_REF][START_REF] Polak | Manipulation of dopamine receptors alters hypoxic pulmonary vasoconstriction in isolated perfused rat lungs[END_REF], bronchial diameter [START_REF] Bonnert | Dual D2-receptor and beta2adrenoceptor agonists for the treatment of airway diseases. 1. Discovery and biological evaluation of some 7-(2-aminoethyl)-4-hydroxybenzothiazol-2(3H)one analogues[END_REF][30][31], neuromodulation of sensory pulmonary nerves [32,33], and lung water clearance [START_REF] Barnard | Dopamine stimulates sodium transport and liquid clearance in rat lung epithelium[END_REF][START_REF] Barnard | Stimulation of the dopamine 1 receptor increases lung edema clearance[END_REF][START_REF] Lecuona | Ventilatorassociated lung injury decreases lung ability to clear edema in rats[END_REF][START_REF] Saldias | Dopamine restores lung ability to clear edema in rats exposed to hyperoxia[END_REF][START_REF] Saldias | Dopamine increases lung liquid clearance during mechanical ventilation[END_REF][START_REF] Helms | Dopamine regulation of amiloride-sensitive sodium channels in lung cells[END_REF]. The exact mechanisms of certain pulmonary actions of DA are not entirely understood. Recently, DA-2 and DA-1 receptors were identified in the lung arteries. Interestingly, these receptors were found within the tunica adventitia in human pulmonary arteries and within the tunica media of large and medium sized intrapulmonary arteries of the rabbit [START_REF] Kim | Localization of dopamine D1 and D2 receptor mRNAs in the rat systemic and pulmonary vasculatures[END_REF][START_REF] Kobayashi | Autoradiographic localization of dopamine D1-like receptors in the rabbit pulmonary circulation[END_REF][START_REF] Kobayashi | Localisation of dopamine D2-like receptors in pulmonary artery of the human and rabbit but not of the rat[END_REF][START_REF] Kobayashi | Autoradiographic localization of dopamine D2-like receptors in the rabbit pulmonary vascular tree[END_REF][START_REF] Kobayashi | Localization of dopamine receptors in the rabbit lung vasculature[END_REF][START_REF] Kobayashi | Localisation of dopamine D1-like and D2-like receptors in the pulmonary vasculature[END_REF]. No dopaminergic receptor has been found in the bronchial tree yet, despite the described bronchodilating action of DA in healthy subjects during histamine administration [31]. Studies on mechanisms of lung water clearance have shown that in alveolar cells, DA increases the activity of basolateral Na-K-ATPase and the activity of epithelial sodium channels [START_REF] Barnard | Stimulation of the dopamine 1 receptor increases lung edema clearance[END_REF][START_REF] Helms | Dopamine regulation of amiloride-sensitive sodium channels in lung cells[END_REF]. DA-1 and DA-2 receptors have been found in the carotid bodies of experimental animals [START_REF] Bairam | Expression of dopamine D1-receptor mRNA in the carotid body of adult rabbits, cats and rats[END_REF][START_REF] Bairam | Autoreceptor mechanism regulating carotid body dopamine release from adult and 10-day-old rabbits[END_REF], providing an argument for a modulatory role of DA in the control of ventilation.

Clinical use

DA is primarily used for its vasopressor and inotropic actions in the management of critically ill patients with circulatory failure [START_REF] Goldberg | Dopamine--clinical uses of an endogenous catecholamine[END_REF][START_REF] Goldberg | Cardiovascular and renal actions of dopamine: potential clinical applications[END_REF]. By combined beta-adrenergic and dopaminergic effects, low doses of DA (typically <5 µg/kg/min) can decrease renal vascular resistance, increase renal blood flow, glomerular filtration rate and sodium excretion in animals and healthy humans [START_REF] Mcdonald Rh | EFFECT OF DOPAMINE IN MAN: AUGMENTATION OF SODIUM EXCRETION, GLOMERULAR FILTRATION RATE, AND RENAL PLASMA FLOW[END_REF]. However, the renal protective effects of DA have not been demonstrated in critically ill humans and randomised control trials did not provide direct proof that DA confers protection from renal dysfunction in critically ill patients at risk of renal failure [START_REF] Holmes | Bad medicine: low-dose dopamine in the ICU[END_REF]. Furthermore, the place of DA as a therapeutic agent is

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debated [START_REF] Friedrich | Meta-analysis: low-dose dopamine increases urine output but does not prevent renal dysfunction or death[END_REF][START_REF] Debaveye | Is there still a place for dopamine in the modern intensive care unit?[END_REF], with many clinicians prefering the use of noradrenaline with or without dobutamine in the treatment of shock states [START_REF] Sakr | Does dopamine administration in shock influence outcome? Results of the Sepsis Occurrence in Acutely Ill Patients (SOAP) Study[END_REF].

DA is not used clinically for its effects on respiration. Neverthless, it does exert effects on the respiratory system, including depression of ventilatory drive, change in bronchial diameter, and increase in lung water clearance, which will be discussed further.

THE EFFECTS OF DOPAMINE ON RESPIRATORY FUNCTION

Effects on respiration

Respiration is largely regulated by chemoreflex mechanisms [START_REF] Berger | Regulation of respiration (first of three parts)[END_REF]. Peripheral chemoreceptors located in the carotid bodies respond primarily to hypoxia and provoke an increase in sympathetic nerve activity [START_REF] Somers | Contrasting effects of hypoxia and hypercapnia on ventilation and sympathetic activity in humans[END_REF] and ventilation [START_REF] Prabhakar | Peripheral chemoreceptors in health and disease[END_REF]. Human and animal type I cells (glomus cells) of the carotid bodies contain abundant quantities of DA [START_REF] Chiocchio | Catecholamines in the carotid body of the cat[END_REF][START_REF] Dearnaley | The identification of dopamine in the rabbit's carotid body[END_REF][START_REF] Hellstrom | Selective decrease of dopamine content in rat carotid body during exposure to hypoxic conditions[END_REF][START_REF] Steele | Catecholamines and 5-hydroxytryptamine in the carotid body in vascular, respiratory, and other diseases[END_REF][START_REF] Zapata | Chemical, electron microscopic and physiological observations on the role of catecholamines in the carotid body[END_REF], with a 5 to 1 ratio of DA to noradrenaline [START_REF] Vicario | Characterization of the synthesis and release of catecholamine in the rat carotid body in vitro[END_REF]. DA intervenes in the regulation of respiration not only at the level of the carotid bodies, but it is also present in the neurons of the nucleus tractus solitarius, where afferents from the carotid bodies end [START_REF] Bairam | Autoreceptor mechanism regulating carotid body dopamine release from adult and 10-day-old rabbits[END_REF][START_REF] Bisgard | Carotid body mechanisms in acclimatization to hypoxia[END_REF]. Hypoxia increases carotid body tyrosine hydroxylase activity [START_REF] Gonzalez | Effects of hypoxia on tyrosine hydroxylase activity in rat carotid body[END_REF], resulting in increased DA synthesis [START_REF] Fidone | Putative neurotransmitters in the carotid body: the case for dopamine[END_REF] and release [START_REF] Hanbauer | The regulation of dopamine and noradrenaline in the rat carotid body and its modification by denervation and by hypoxia[END_REF], which suggests that DA can play a role in the regulation of ventilation.

In addition, many studies in animals and healthy humans have demonstrated that low dose DA is associated with decreased carotid sinus drive and diminished ventilatory response to hypoxia. In healthy subjects, low dose DA blunts the ventilatory response to isocapnic [START_REF] Ward | Reduction of hypoxic ventilatory drive by dopamine[END_REF][START_REF] Welsh | Depression of ventilation by dopamine in man. Evidence for an effect on the chemoreceptor reflex[END_REF] and hypercapnic [START_REF] Olson | Ventilatory responsiveness to hypercapnic hypoxia during dopamine infusion in humans[END_REF] hypoxia, an effect which is abolished by the dopaminergic receptor blocker, haloperidol [START_REF] Bainbridge | Effect of haloperidol on ventilatory responses to dopamine in man[END_REF].

These effects of DA on respiration can be detrimental in seriously ill patients with limited breathing reserve. This detrimental effect has been shown in patients with heart failure, where low dose DA decreased minute ventilation by more than 1 L/min during normoxic breathing; this did not occur in age-matched control subjects [START_REF] Van De Borne | Dopamine depresses minute ventilation in patients with heart failure[END_REF]. Heart failure patients often have only mild arterial blood oxygen desaturation as a result of low cardiac output and pulmonary vascular congestion. DA also impairs regional ventilation/perfusion matching in the lungs [START_REF] Shoemaker | Comparison of hemodynamic and oxygen transport effects of dopamine and dobutamine in critically ill surgical patients[END_REF]. Thus, there are two synergistic

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mechanisms which can affect ventilation in heart failure: first, direct impairment of gas exchange [START_REF] Shoemaker | Comparison of hemodynamic and oxygen transport effects of dopamine and dobutamine in critically ill surgical patients[END_REF],

and second, impaired compensatory ventilation [START_REF] Van De Borne | Dopamine depresses minute ventilation in patients with heart failure[END_REF]. Both mechanisms can be counterbalanced by the administration of supplemental oxygen [START_REF] Johnson | Low-dose dopamine and oxygen transport by the lung[END_REF] so that these effects of DA would not have important consequences, especially in mechanically ventilated patients. Sleep disordered breathing, frequently observed in patients with heart failure [START_REF] Javaheri | Central sleep apnea-hypopnea syndrome in heart failure: prevalence, impact, and treatment[END_REF][START_REF] Mansfield | Sleep apnea and congestive heart failure[END_REF], could be another factor contributing to the detrimental actions of DA in these patients. During sleep, central and obstructive apnoeas decrease arterial oxygen saturation and this may play a pivotal role in the genesis of cardiac arrhythmias. Both hypoxia and hypercapnia activate chemoreceptors [START_REF] Somers | Contrasting effects of hypoxia and hypercapnia on ventilation and sympathetic activity in humans[END_REF] and induce short lasting arousals that terminate obstructive apnoeas. Chemoreflex inhibition by DA could be particularly deleterious in this condition, as endapnoeic desaturations, combined with potential DA arrhythmogenicity, could promote life-threatening arrhythmias.

In patients with heart failure, DA may have dual effects during weaning from mechanical ventilation. Patients receiving DA would experience reduced discomfort associated with hypoxia, but DA would further decrease ventilatory drive and diminish arterial blood oxygen saturation [START_REF] Van De Borne | Dopamine depresses minute ventilation in patients with heart failure[END_REF], with the potential danger of precipitating respiratory failure and problems during weaning from mechanical ventilation.

We recently investigated the possible impact of DA on weaning from mechanical ventilation in patients with COPD [START_REF] Ciarka | Effects of low-dose dopamine on ventilation in patients with chronic obstructive pulmonary disease[END_REF]: we observed that DA did not attenuate ventilation and had no effect on arterial blood gases [START_REF] Ciarka | Effects of low-dose dopamine on ventilation in patients with chronic obstructive pulmonary disease[END_REF]. This discrepancy between patients with COPD and those with heart failure may be explained by differences in the peripheral chemoreceptor drive for ventilation, which is reduced [START_REF] Erbland | Interaction of hypoxia and hypercapnia on respiratory drive in patients with COPD[END_REF][START_REF] Fahey | Won't breathe" vs "can't breathe". Detection of depressed ventilatory drive in patients with obstructive pulmonary disease[END_REF] in COPD patients and heightened in patients with heart failure [START_REF] Ponikowski | Peripheral chemoreceptor hypersensitivity: an ominous sign in patients with chronic heart failure[END_REF]. We speculate that the low peripheral chemoreceptor drive cannot be further inhibited in COPD, and therefore DA cannot further decrease ventilatory drive in these patients.

Effects on the respiratory muscles

Improvement in blood flow can increase the force of contraction of the respiratory muscles, and an early study suggested that DA may improve diaphragmatic function in COPD patients [34].

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Effects on the pulmonary circulation DA can reduce pulmonary vascular tone and thus decrease pulmonary hypertension [START_REF] Gorman | Vasodilator response to dopamine in the ferret pulmonary circulation[END_REF][START_REF] Loick | The effect of dopamine on pulmonary hemodynamics and tissue damage after inhalation injury in an ovine model[END_REF][START_REF] Mccormack | Effects of dopexamine hydrochloride on hypoxic pulmonary vasoconstriction in isolated rat lung[END_REF][START_REF] Polak | Effect of fenoldopam on preconstricted isolated salt-perfused rat lungs[END_REF][START_REF] Polak | Manipulation of dopamine receptors alters hypoxic pulmonary vasoconstriction in isolated perfused rat lungs[END_REF].

This has been shown in patients with COPD [START_REF] Paganelli | Hemodynamic effects of bilevel nasal positive airway pressure ventilation in patients with heart failure[END_REF]. Several mechanisms have been proposed. First, DA-1 receptors are present in the tunica intima and media of large and medium pulmonary arteries, while DA-2 receptors are found in the adventitia of extrapulmonary arteries and in medium and large intrapulmonary arteries. All these findings were made using histochemical techniques [START_REF] Kobayashi | Autoradiographic localization of dopamine D1-like receptors in the rabbit pulmonary circulation[END_REF][START_REF] Kobayashi | Localisation of dopamine D2-like receptors in pulmonary artery of the human and rabbit but not of the rat[END_REF][START_REF] Kobayashi | Autoradiographic localization of dopamine D2-like receptors in the rabbit pulmonary vascular tree[END_REF][START_REF] Kobayashi | Localization of dopamine receptors in the rabbit lung vasculature[END_REF][START_REF] Kobayashi | Localisation of dopamine D1-like and D2-like receptors in the pulmonary vasculature[END_REF], while in situ hybridisation further confirms the presence of DA-1 receptors in the tunica media of the aorta and the pulmonary artery [START_REF] Kim | Localization of dopamine D1 and D2 receptor mRNAs in the rat systemic and pulmonary vasculatures[END_REF]. Second, dopaminergic receptors may also induce vasodilation through relaxation of the pulmonary endothelium.

Effects on extravascular lung water

Pulmonary oedema can be viewed as an imbalance between the biological mechanisms that drive water into the alveolar space and the forces assuring its removal. It can result from a change in the hydrostatic and/or oncotic pressure gradients across the pulmonary circulation or from increased alveolar permeability.

Several studies have reported that DA can increase lung oedema clearance by increasing active sodium transport (via epithelial Na/K-ATP-ase stimulation). In rats, experiments with DA were performed using three different models of pulmonary oedema (hyperoxia, mechanical ventilation with high tidal volume, and increase in hydrostatic pressure). DA increased lung oedema clearance in rats breathing room air and rats exposed to 100 % oxygen (such exposure to oxygen results in severe lung injury and death due to respiratory failure) [START_REF] Saldias | Dopamine restores lung ability to clear edema in rats exposed to hyperoxia[END_REF]. Moreover, rat alveolar type II cells incubated with DA present abundant quantities of the alpha1 subunit protein of Na K-ATPase in the basolateral membranes, indicating that DA may recruit Na pumps from intracellular pools [START_REF] Saldias | Dopamine restores lung ability to clear edema in rats exposed to hyperoxia[END_REF]. In line with this hypothesis, colchicine, which inhibits the microtubular transport system, blocks the stimulatory effect of DA on active Na+ transport [START_REF] Saldias | Dopamine restores lung ability to clear edema in rats exposed to hyperoxia[END_REF]. The same effect of DA on lung oedema clearance was observed in an isolated perfused lung rat model, where ouabain, a specific inhibitor of Na K-ATP-ase, abolished

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the dose dependent increase in liquid clearance exerted by DA [START_REF] Barnard | Dopamine stimulates sodium transport and liquid clearance in rat lung epithelium[END_REF]. Furthermore, it was demonstrated that the lung oedema clearance was mediated by D1 receptor activation [START_REF] Barnard | Stimulation of the dopamine 1 receptor increases lung edema clearance[END_REF].

Sznajder and co-workers developed an animal model of lung oedema, induced by mechanical ventilation with high tidal volume, with resulting impairment of active Na+ transport and lung liquid clearance [START_REF] Lecuona | Ventilatorassociated lung injury decreases lung ability to clear edema in rats[END_REF]. In this model, DA increased alveolar fluid reabsorption in nonventilated control rats by 60 %, rats ventilated with low tidal volume by 55 % and rats ventilated with high tidal volume by 200 % [START_REF] Saldias | Dopamine increases lung liquid clearance during mechanical ventilation[END_REF]. These effects of DA were abolished by administration of colchicine, which supports previous observations that DA increases Na+-K+-ATPase mobilisation from the intracellular pool [START_REF] Saldias | Dopamine increases lung liquid clearance during mechanical ventilation[END_REF].

Finally, there are studies on the effects of DA in animal models of hydrostatic pulmonary oedema, where active Na+ transport and alveolar fluid reabsorption are decreased [START_REF] Rutschman | Active transport and passive liquid movement in isolated perfused rat lungs[END_REF]. Similar to other reports on different models of lung oedema, DA increased alveolar fluid reabsorption in isolated rat lungs with increased left atrial pressure, and this effect was abolished by the administration of the specific D1 receptor antagonist, ouabain (a Na K-ATPase inhibitor) and colchicine [START_REF] Azzam | Catecholamines increase lung edema clearance in rats with increased left atrial pressure[END_REF].

In contrast to these studies, Tibayan et al failed to show that DA given intravenously (5-10 µg/kg/min), or intra-alveolarly (10-4 M), affected alveolar liquid clearance [START_REF] Tibayan | Dobutamine increases alveolar liquid clearance in ventilated rats by beta-2 receptor stimulation[END_REF], while dobutamine increased alveolar liquid clearance by 50 %. Moreover, this effect was abolished by a potent and specific beta 2 receptor blocker [START_REF] Tibayan | Dobutamine increases alveolar liquid clearance in ventilated rats by beta-2 receptor stimulation[END_REF]. This results are in line with the hypothesis that alveolar epithelial sodium transport is mediated by beta 2 receptor stimulation [START_REF] Tibayan | Dobutamine increases alveolar liquid clearance in ventilated rats by beta-2 receptor stimulation[END_REF].

Effects on the airways

There is growing evidence that DA is involved in the regulation of airway diameter. The control of the bronchial diameter by the autonomic nervous system is highly complex and involves neural and humoral factors [START_REF] Barnes | Neural control of human airways in health and disease[END_REF]. Adrenergic and cholinergic systems are involved in this regulation, as well as inhibitory and excitatory non-adrenergic, non-cholinergic nerves (i-NANC/e-NANC) [START_REF] Widdicombe | Autonomic regulation. i-NANC/e-NANC[END_REF].

Adrenergic control is carried out by the release of noradrenaline from the nerve endings and by the release of adrenaline from the adrenal medulla. Adrenaline and noradrenaline can produce bronchodilation by the stimulation of β2 adrenergic receptors, but they can also cause bronchoconstriction, by stimulation of α1 and α2 adrenergic receptors, if pathological disturbances

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exist in the airways [88]. Non-adrenergic/non-cholinergic nervous control of airway smooth muscle is exerted by neurotransmitters, such as substance P and neurokinin A (contraction of the airway smooth muscle) and neuropeptide Y (relaxation of the neural smooth muscle) [START_REF] Cabezas | The actions of dopamine on the airways[END_REF]. Thus, local reflexes regulate airway diameter through nerves that release neuropeptides as well as by other substances released by epithelial and interstitial cells [START_REF] Barnes | Pharmacology of airway smooth muscle[END_REF][START_REF] Spina | Epithelium smooth muscle regulation and interactions[END_REF]. Some studies have demonstrated that DA may also exert a modulatory role on airway diameter. DA inhibits the bronchoconstriction produced by increasing doses of histamine, infused or inhaled in healthy subjects as well as in bronchospastic patients (Table 2) [31]. However, DA had no effect on the bronchial tree when administered to subjects with a history of bronchial asthma but no bronchospasm at the time of the investigation (Table 2) [START_REF] Cabezas | The actions of dopamine on the airways[END_REF][START_REF] Thomson | Effect of dopamine on airways conductance in normals and extrinsic asthmatics[END_REF]. Moreover, the DA2 receptor blocker, metoclopramide, had no effect on asthmatic patients without bronchospasm (Table 2) [START_REF] Cabezas | The actions of dopamine on the airways[END_REF]. One animal study reported that it is possible to inhibit bronchcoconstriction induced by electrical field stimulation and this effect may be prevented by blocking DA1 receptors [30].

No DA receptor has yet been identified in the human bronchial tree. However, one recent animal study reported that stimulation of DA2 receptors by a compound similar to DA produces a bronchodilatatory effect on the airways in dogs and guinea pigs [START_REF] Bonnert | Dual D2-receptor and beta2adrenoceptor agonists for the treatment of airway diseases. 1. Discovery and biological evaluation of some 7-(2-aminoethyl)-4-hydroxybenzothiazol-2(3H)one analogues[END_REF].

Thus, DA could be potentially beneficial in clinical states associated with bronchoconstriction, but further randomised, placebo controlled clinical studies are needed to investigate its potential to alleviate bronchospasm in humans.

DA may also act by neuromodulation of pulmonary sensory nerves. Sensory nerve fibres in the airways control events such as bronchoconstriction, airway plasma leakage and cough [START_REF] Birrell | Effect of dopamine receptor agonists on sensory nerve activity: possible therapeutic targets for the treatment of asthma and COPD[END_REF]. There are two classes of sensory nerve fibres, which control sensory nerve reflexes: myelinated, rapidly adapting stretch receptors (RARs) and non-myelinated, C-fibres [START_REF] Birrell | Effect of dopamine receptor agonists on sensory nerve activity: possible therapeutic targets for the treatment of asthma and COPD[END_REF]. RARs are located along the entire tracheobronchial tree and are activated by chemical (histamine), mechanical, and osmotic stimuli [START_REF] Dougall | Dual dopamine D2 receptor and beta2adrenoceptor agonists for the treatment of chronic obstructive pulmonary disease: the pre-clinical rationale[END_REF]. They elicit defensive actions such as cough, mucus production, and rapid shallow breathing [START_REF] Dougall | Dual dopamine D2 receptor and beta2adrenoceptor agonists for the treatment of chronic obstructive pulmonary disease: the pre-clinical rationale[END_REF]. Activation of myelinated as well as non myelinated fibres, elicits cough, bronchoconstriction and mucus secretion via an afferent central reflex pathway [START_REF] Karlsson | Afferent neural pathways in cough and reflex bronchoconstriction[END_REF][START_REF] Karlsson | A role for capsaicin sensitive, tachykinin containing nerves in chronic coughing and sneezing but not in asthma: a hypothesis[END_REF]. It was, therefore, postulated that DA receptor agonists would alleviate the debilitating symptoms of COPD, and this hypothesis was tested in a randomised, placebo controlled trial, which is discussed later.

Clinical application in COPD

Patients with COPD present dyspnoea, cough, and excess sputum production, which decrease their quality of life [START_REF] Van Der Molen | Development, validity and responsiveness of the Clinical COPD Questionnaire[END_REF]. The underlying mechanisms are complex, but reflex activation of sensory afferent nerves by irritants, which in the normal organism serves to clear the airways and is an important mechanism of self-defence, is known to induce these symptoms.. Since sensory RAR fibres are implicated in the control of cough, bronchospasm and sputum production [START_REF] Dougall | Dual dopamine D2 receptor and beta2adrenoceptor agonists for the treatment of chronic obstructive pulmonary disease: the pre-clinical rationale[END_REF], and DA2 receptors are present in RARs [33], and DA infusion in animal models inhibited histamine-induced activation of RARs [START_REF] Dougall | Dual dopamine D2 receptor and beta2adrenoceptor agonists for the treatment of chronic obstructive pulmonary disease: the pre-clinical rationale[END_REF], it has been suggested that the dual DA-2 and β 2 receptor agonist, sibenadet, could have beneficial effects on debilitating symptoms in COPD patients [36,[START_REF] Dougall | Dual dopamine D2 receptor and beta2adrenoceptor agonists for the treatment of chronic obstructive pulmonary disease: the pre-clinical rationale[END_REF]. Although the role of beta2 receptor blockers has been well established in the treatment of COPD, dopamine agonism is a completely new approach in this disease.

An initial study [35] aimed to compare the effects of sibenadet, placebo and common bronchodilator therapies (salbutamol and ipratropium bromide) on key symptoms of COPD evaluated by the Breathlessness, Cough and Sputum Scale (BCSS) [START_REF] Leidy | Evaluating symptoms in chronic obstructive pulmonary disease: validation of the Breathlessness, Cough and Sputum Scale[END_REF]. This patient-reported scale is a validated and reliable outcome measure of symptom severity [START_REF] Leidy | Evaluating symptoms in chronic obstructive pulmonary disease: validation of the Breathlessness, Cough and Sputum Scale[END_REF]. Early clinical evaluation of sibenadet was very promising [35]. Sibenadet improved the symptoms of breathlessness, cough and sputum in this 28-day trial, while standard bronchodilators provided only limited symptomatic improvement [35].

The positive effects of sibenadet were dose-related, and the dose of 500 µg, delivered in two equal However, long term studies have not supported a clinical benefit of sibenadet on the symptoms of COPD [36]. The efficacy of sibenadet (at a dose 500 µg) in comparison with placebo was assessed in studies of 3-and 6-month duration. The primary endpoints were defined as changes in BCSS and forced expiratory volume in one second (FEV1) from baseline. Health-related quality of life, perception of treatment efficacy and adverse effects were also recorded. Although there was an immediate reduction in BCSS at the beginning of the study, there was a gradual increase in symptom scores among the patients treated with sibenadet, in comparison to placebo. By the end of the study, the differences in BCSS between the placebo and sibenadet group were not statistically significant.

The fact that initial improvements in symptomatology tailed off over time, could reflect a tolerance phenomenon. FEV1 measurement clearly demonstrated a significant improvement in the sibenadet group, which was attributed to the vasodilatatory effects of the drug. However, the magnitude of these increases diminished over time, which could be attributed to tachyphylaxis, a recognised potential effect of regular b2-agonist use. No significant difference between the two groups was detected in the health-related quality of life score, although a higher proportion of investigators and patients rated sibenadet as more effective than placebo. The proportion of patients experiencing at least one exacerbation was similar in placebo and sibenadet groups. There was no difference in the frequency of adverse effects between the placebo and sibenadet groups and a 1-year safety study also demonstrated that sibenadet was well tolerated [START_REF] Hiller | Long-term use of Viozan (sibenadet HCl) in patients with chronic obstructive pulmonary disease: results of a 1-year study[END_REF].

Therefore, despite encouraging preclinical and early clinical data, the long-term clinical trial demonstrated that sibenadet does not provide long-term symptomatological benefit. One possible explanation is that early symptomatic benefit was beta2 rather than D2 driven and diminished secondary to tachyphylaxis. Moreover, it is possible that beta2 tachyphylaxis diminished dual beta2/D2 actions and masked such mechanisms as dopamine2 receptor agonism on the cough component [START_REF] Hiller | Long-term use of Viozan (sibenadet HCl) in patients with chronic obstructive pulmonary disease: results of a 1-year study[END_REF].

Conclusions

A c c e p t e d m a n u s c r i p t

In summary, DA is still used in the intensive care unit for its vasopressor and inotropic effects, combined with dopaminergic effects that may protect renal and splanchnic blood flow. DA can also have both favourable and detrimental effects on pulmonary function. Low dose DA can depress ventilation in patients with heart failure, thus it must be used cautiously in these patients during weaning from mechanical ventilation. However, DA is safe in patients with COPD, because it did not alter ventilation either in patients during weaning, or in hypoxemic, spontaneously breathing COPD patients.

Some anecdotal reports show that DA can improve diaphragmatic function and respiratory muscle contraction, as well as decrease pulmonary hypertension in COPD. Nevertheless, no placebo controlled, randomised trial, has been conducted to confirm these observations.

DA has potentially beneficial effects on the respiratory system, such as an increase in lung oedema clearance or inhibition of bronchoconstriction. However, the impact of DA on lung oedema has not been tested in human studies and clinical trials on the effects of DA on airway diameter have proved disappointing, unless administered during an acute asthmatic exacerbation.

Finally, the dopaminergic receptor agonist, sibenadet, did not induce sustained clinical benefits in patients with COPD, despite evidence from animal studies that DA inhibits sensory nerve fibres (activation of which elicits cough, bronchoconstriction and mucus production).

Thus, despite many years of widespread use, the role of dopaminergic support in respiratory disease remains poorly defined. We lack direct, convincing human studies in favour of DA's beneficial effects on the respiratory system, but we have evidence of a detrimental impact of low dose DA on ventilation in healthy humans and patients with heart failure. DA should, therefore,be used with caution in critically ill hypoxemic patients during weaning from mechanical ventilation.

A c c e p t e d m a n u s c r i p t

  receptor agonist, sibenadet, did not alleviate breathlessness, cough or excess sputum in patients with chronic obstructive pulmonary disease (COPD)[35, 36].

A c c e p t e d m a n u s c r i p t

  The different pharmacological actions of DA are effected through dose-dependent activation of α-adrenergic, β-adrenergic and dopaminergic receptors. Activation of β 1 , β 2 , and α 1 adrenergic receptors in the heart results in an increase in heart rate and contractility, while activation of presynaptic α 2 receptors has an inhibitory effect on further release of noradrenaline from nerve terminals. In the vasculature, the activation of postsynaptic α 1 and α 2 receptors mediates vasoconstriction, while β 2 receptors mediate vasodilatation. Peripheral DA receptors belong to two classes. The DA-1 receptors activate adenyl cyclase, with a consequent increase in intracellular cAMP, while DA-2 receptors inhibit adenyl cyclase and result in activation of K+ channels, inhibition of Ca 2+ channels, and the exchange of phospatidyl inositol. DA-1 receptors provoke renal, coronary, and mesenteric vasodilatation and a natriuretic response. Stimulation of DA-2 receptors is associated with noradrenaline release from sympathetic nerve endings and inhibition of prolactin release.DA influences these receptors in a dose dependent manner. Low doses of DA (1-2 µg/kg/min) act primarily on the DA-1 receptors, resulting in selective vasodilatation in the renal, mesenteric, cerebral, and coronary beds. Vasodilatation can be balanced by an increase in stroke volume, so that arterial pressure remains relatively stable. Higher doses (2-5 µg/kg/min) activate not only the dopaminergic but also the β-adrenergic receptors, resulting in increased cardiac output, due to combined increases in stroke volume and heart rate[START_REF] Lollgen | Use of inotropes in the critical care setting[END_REF]. Doses of 5-10 µg/kg/min have predominant β-adrenergic effects and start to have α-adrenergic effects, resulting in combined increases in cardiac output and blood pressure. Higher doses (10-20 µg/kg/min) provoke a predominantly α-adrenergic activation, with vasoconstriction and increased systemic vascular resistance. The dose-dependent effects of DA are, however, not entirely predictable because of significant interindividual variability in enzymatic DA inactivation. In practical terms, DA should be titrated to a desired physiologic effect, because weight-based administration of DA can achieve quite different concentrations among individuals[START_REF] Macgregor | Pharmacokinetics of dopamine in healthy male subjects[END_REF].

A c c e p t e d m a n u s c r i p t

 t DA2 receptor mRNA is present in the sensory ganglia of rat airways [33]. It has been demonstrated that DA receptor activation inhibits RAR activation in vivo [32] and inhibits neuropeptide release from the peripheral endings of airway sensory neurons [33]. In dogs pretreated with propranolol and phentolamine (to prevent the β and the α adrenergic effects of DA, respectively), DA infusion inhibited histamine-induced stimulation of RARs [32]. This action was prevented by the DA-2 antagonist, sulpiride [32].

A c c e p t e d m a n u s c r i p t

  fractions, proved to have the highest efficacy without compromising safety, and was subsequently used in large scale studies[36].

  . Kamikawa Y, Shimo Y. Inhibitory effects of catecholamines on cholinergically and non-cholinergically mediated contractions of guinea-pig isolated bronchial muscle. J Pharm Pharmacol 1990; 42:131-134. [31]. Michoud MC, Amyot R, Jeanneret-Grosjean A. Dopamine effect on bronchomotor tone in vivo. Am Rev Respir Dis 1984; 130:755-758. [32]. Jackson DM, Simpson WT. The effect of dopamine on the rapidly adapting receptors in the dog lung. Pulm Pharmacol Ther 2000; 13:39-42. [33]. Peiser C, Trevisani M, Groneberg DA, Dinh QT, Lencer D, Amadesi S, Maggiore B, Harrison S, Geppetti P, Fischer A. Dopamine type 2 receptor expression and function in rodents sensory neurons projecting to the airways. Am J Physiol Lung Cell Mol Physiol 2005. [34]. Aubier M, Murciano D, Menu Y, Boczkowski J, Mal H, Pariente R. Dopamine effects on diaphragmatic strength during acute respiratory failure in chronic obstructive pulmonary disease. Ann Intern Med 1989; 110:17-23. [35]. Ind PW, Laitinen L, Laursen L, Wenzel S, Wouters E, Deamer L, Nystrom P. Early clinical investigation of Viozan (sibenadet HCl), a novel D2 dopamine receptor, beta2-adrenoceptor agonist for the treatment of chronic obstructive pulmonary disease symptoms. Respir Med 2003; 97 Suppl A:S9-21. [36]. Laursen LC, Lindqvist A, Hepburn T, Lloyd J, Perrett J, Sanders N, Rocchiccioli K. The role of the novel D2/beta2-agonist, Viozan (sibenadet HCl), in the treatment of symptoms of chronic obstructive pulmonary disease: results of a large-scale clinical investigation. Respir Med 2003; 97 Suppl A:S23-S33.

A c c e p t e d m a n u s c r i p t Table 2 .

 2 Summary of studies on the effects of dopamine (DA) on bronchial diameter. DA was administered either intravenously or by inhalation and the effects were tested in healthy subjects, patients with a past history of asthma, and patients with acute bronchospasm. One study tested effects of the DA receptor blocker, metoclopramide, on bronchial airway diameter. (FVC= forced vital capacity, FEV1= forced expiratory volume in the first second, FEFmax= maximal forced expiratory flow, FEF50= forced expiratory flow at 50 % of vital capacity).

Table 1 .

 1 Published beneficial and detrimental effects of dopamine in the respiratory system.

	Beneficial effects of dopamine	Detrimental effects of dopamine
	• decrease in pulmonary vascular	• modulation of chemosensitivity
	resistance and in pulmonary	and decreased ventilation in
	hypertension	hypoxic conditions
	• increase in lung oedema clearance	• increase in ventilation/perfusion
	by activation of epithelial ATP-	mismatch in the lungs
	ase and epithelial sodium
	channels
	• improvement of diaphragmatic
	function in chronic obstructive
	pulmonary disease
	• inhibition of bronchoconstriction
	during bronchospasm
	• inhibition of rapidly adapting
	stretch receptors in the lung,
	which control sensory nerve
	reflexes

Reference

Subjects and patients Drug and dose tested Effects on bronchial diameter Cabezas


O Noga

U Petzold

G Kunkel

R Otto-Knapp

F Conrad

S Hösch

P Metzenauer

J Maus

Efficacy and safety of formoterol delivered through the Novolizer ᭨ , a novel dry powder inhaler (DPI) compared with a standard DPI in patients with moderate to severe asthma

Efficacy and safety of formoterol delivered through the Novolizer, a novel dry powder inhaler (DPI) compared with a standard DPI in patients with moderate to severe asthma

Introduction

Long-acting β 2 adrenoceptor agonists (LABAs) such as formoterol are established in the treatment of moderate and severe asthma [START_REF] Bousquet | Global initiative for asthma (GINA) and its objectives[END_REF] and in the treatment of chronic obstructive pulmonary disease (COPD) at the GOLD [START_REF] Fabbri | Global strategy for the diagnosis, management and prevention of COPD: 2003 update[END_REF] stages 2 and 3 [START_REF] Tashkin | The role of long-acting bronchodilators in the management of stable COPD[END_REF]. LABAs reduce asthma exacerbations and subsequent hospitalisation in adults [START_REF] Andersson | Adding formoterol to budesonide in moderate asthma--health economic results from the FACET study[END_REF][START_REF] Pauwels | Effect of inhaled formoterol and budesonide on exacerbations of asthma. Formoterol and Corticosteroids Establishing Therapy (FACET) International Study Group[END_REF] and exert additional antiinflammatory effect with long-term use [START_REF] Li | An antiinflammatory effect of salmeterol, a long-acting beta(2) agonist, assessed in airway biopsies and bronchoalveolar lavage in asthma[END_REF]. The bronchodilatory effect over 12 hours [e.g. 7] results in a better asthma control and improved quality of life [START_REF] Wenzel | Efficacy, safety, and effects on quality of life of salmeterol versus albuterol in patients with mild to moderate persistent asthma[END_REF][START_REF] Juniper | Asthma quality of life during 1 year of treatment with budesonide with or without formoterol[END_REF]. Daily administration of LABAs in patients with asthma or COPD reduces health costs [START_REF] Miravitlles | Costs of chronic bronchitis and COPD: a 1-year follow-up study[END_REF][START_REF] Antonicelli | Asthma severity and medical resource utilisation[END_REF].

After intensive discussions on the use of environmentally damaging chlorofluorocarbons as propellants in inhaler devices, the European Union [START_REF]Note for Guidance: Replacement of chloro-fluoro-carbones (CFCs) in metered dose inhalation products[END_REF] instructed in 1993 to replace CFC-containing devices by environmentally friendly propellants. Development of dry powder inhalers (DPI), which can dispense active substances as a dry-powder, was started in the following.

The Novolizer ® (VIATRIS, Frankfurt am Main, Germany) is one of such devices. This inhaler is a novel multi-dose dry powder inhaler (MDPI) with low to medium airflow resistance (0.0260 kPa 0.5 x min x L -1 [START_REF] Fyrnys | Stability and Performance characteristics of a Budesonide Powder for Inhalation with a Novel Dry Powder Inhaler Device[END_REF]) and visual, acoustic and sensory feedback mechanisms that indicate a correct inhalation manoeuvre. Sufficient flows can be generated easily even by patients with impaired lung function and low inspiratory capacity [START_REF] Berg | Asthmatic children generate significantly different peak inspiratory[END_REF]15,16,20]. Drug deposition in vivo at medium (60 L/min) and high (90 L/min) flow rates is at least as effective as that of the higher resistance (0.0367 kPa 0.5 x min x l -1 ) device Turbuhaler ® (Pulmicort ® , AstraZeneca) [17], The threshold flow rate for the Novolizer ® is 35-50 L/min; thus a comparison at lower flow rates such as 30 L/min is not meaningful. Nevertheless, findings of an in-vitro study showed that the fine particle dose (FPD) of budesonide delivered through the Novolizer ® The Aerolizer ® is a single dose device that is widely used to administer formoterol (Foradil ® P) as well as budesonide (Miflonide ® ). Single dose devices are known to deliver constant doses of medication with disadvantages in their handling comfort.. A multidose device such as the Novolizer ® with easily achievable high flow rates, comfortable handling and multiple feedback should be a very valuable alternative.

This study aimed to prove the therapeutic equivalence of inhaled formoterol delivered through the Novolizer ® in comparison to the Aerolizer ® in patients with moderate to severe asthma.

Material and Methods

Study design

This clinical trial was performed as a randomised, double-blind, double-dummy multicentre multinational (throughout Europe) parallel-group study. During a run-in period of 1-2 weeks, any long-acting β 2 adrenoceptor agonist was discontinued.

Patients received a salbutamol pMDI in case bronchodilator therapy was required.

The use of anti-inflammatory therapy was allowed during the study if applied before study entry and kept at constant dosages. After the washout period, eligible patients were randomised to one of two treatment groups. Both groups used both study and formoterol per day through the Novolizer ® and 2 x 1 puff placebo through the Aerolizer ® inhaler at the same time. Group 2 used the reference device for inhalation of formoterol and the Novolizer ® for application of placebo in the same way.

The study protocol and the patient informed consent were reviewed and approved by the competent independent local ethic committees. Written informed consent was obtained from each patient prior to enrolment.

Methods

Forced expiratory volume in the first second (FEV 1 ) at the end of treatment phase, i.e. after 4 weeks of treatment, was the primary variable to evaluate the efficacy of the trial medication. Secondary variables were: forced vital capacity (FVC) and peak expiratory flow rate (PEF). Pulmonary function tests were performed by means of spirometry before the start of study treatment (baseline, week 0), as well as 2 and 4

weeks after the start of study treatment, always at approximately the same time in the morning, between 8:00 and 10:00 a.m., and always prior to the intake of study medication. Three readings were taken and the highest value was documented.

Patients were instructed to avoid short-acting and long-acting β 2 adrenoceptor agonists 6 and 12 hours, respectively, before testing.

During the study, patients recorded their PEF using a pocket peak flow meter (Mini Wright ® ) provided by the study centre. Measurements were carried out daily prior to the intake of asthma medication in the morning and in the evening. Three measurements were recorded in the patient's diary and the highest value was used for evaluation.

A c c e p t e d m a n u s c r i p t

The intensity of important asthma symptoms (cough, wheezing, shortness of breath, morning and evening dyspnoea) was scored once daily by the patient using a 4-point rating scale (0: symptom free, 1: mild, 2: moderate, 3: severe). In addition, the number of nocturnal awakenings and the use of rescue medication (salbutamol MDI)

were documented twice per day by the patient.

Patients' and physicians' overall impression of the therapeutic effect of the study medication was documented at the end of the treatment period using a 5-point rating scale.

Treatment safety was evaluated based on the incidence of adverse events (AEs) and changes in vital signs, ECG and laboratory variables (haematology, clinical chemistry). Occurrence of cough, dyspnoea or paradoxical bronchospasm, defined as a decrease in FEV 1 by at least 15% within 15 minutes after inhalation, was documented after pulmonary function testing at each visit. A global assessment on safety rated by the patient and by the physician on a 5-grade rating scale gave an overall impression of the tolerability of the study drug. For further information regarding study plan and methodology, please refer to the flow chart ( Subgroup analyses for those patients treated with ICS in addition were conducted.

The safety population (SAF) consisted of all patients who were exposed to study medication.

Results

After screening of 412 patients, 393 patients were randomised and 392 patients received at least one dose of study medication (safety population). 199 patients received formoterol via the Novolizer ® and 193 patients via the reference inhaler Aerolizer ® . 366 patients (93% of randomised patients) completed the study without major protocol violations. 26 patients had to be excluded from the PP population, 2 due to inadequate compliance and 24 due to major protocol violations (n = 18: too short or too long treatment with study medication, n = 4: FEV 1 predicted too high, n = 2: intake of prohibited medication during run-in phase). Distribution of protocol violations was similar across both treatment groups (Tables 1 and5). Demographic and baseline data (Table 2) did not show relevant differences between both treatment groups. Regarding the use of concomitant anti-inflammatory medication, its use was similar in both treatment groups with n=317 patients administering ICS (Novolizer ® n=161, Aerolizer ® n=156), n=39 patients using cromoglycate sodium or nedocromil (Novolize ® r n=14, Aerolizer ® n=25), and n=16 patients applying leukotriene-receptor-antagonists (Novolizer ® n=10, Aerolizer ® n=6). A comparison of

A c c e p t e d m a n u s c r i p t

the numbers of patients using low, medium or high dosages of ICS in the Novolizer ® with the Aerolizer ® group does not show differences between both treatment groups (low daily dose n=23 vs. n=21, medium or high dose n=138 vs.n=135, for Novolizer ® and Aerolizer ® , respectively). No ICS was used by n=37 patients in the Novolizer ® and n=36 patients in the Aerolizer ® group.

Efficacy

Mean (± standard deviation, SD) FEV 1 at baseline as well as 2 and 4 weeks after study treatment is presented in Figure 1. For FEV 1 after 4 weeks of treatment the one-sided t-test showed non-inferiority (p-value <0.0001) of formoterol therapy using the Novolizer ® versus the Aerolizer ® at a lower limit of equivalence set at 0.25 L. After Testing of FEV 1 in the PP population (p=0.0003) confirmed non-inferiority. Subset analysis showed that the results in 26 children aged 6 to 11 years and 25 adolescents aged 12 to 18 years were similar to those in adults.

Secondary pulmonary function test variables as well as the need of rescue medication and the frequency of asthma symptoms including nocturnal awakening did not reveal any relevant differences between both groups (Table 3).

One patient in the formoterol Novolizer ® group and three of the Aerolizer ® group had to discontinue the study due to asthma exacerbations. The global assessment of drug efficacy by the patient and the investigator showed an equal pattern without significant differences. 86.8 % of patients in the formoterol Novolizer ® group evaluated their condition as improved, whereas 83.1 % of the reference group did so.

Safety

Among the patients receiving at least one dose of study medication 85 adverse events (AEs, 38 in the formoterol Novolizer ® group and 47 of reference group) were documented. There was no significant difference between formoterol Novolizer ® group and reference group in the incidence of AEs (Table 4, all adverse events were included that appeared in more than two patients.). Four AEs were rated by the investigator as probably related to the study drug including 1 case of headache

A c c e p t e d m a n u s c r i p t

(formoterol Novolizer ® ), 2 cases of cough (Aerolizer ® ), and 1 case of paradoxical bronchospasm (Aerolizer ® ). Two cases were rated by the investigator as definitely related to study drug: 1 case of paradoxical bronchospasm and 1 case of cough (both Aerolizer ® ). Please consider that both the formulation containing the active ingredient and the inhaler device are integral parts of the study drug, i.e. the finished inhaler products.

One patient of the reference group was admitted to a hospital due to serious asthma exacerbation. This event was recorded as a serious adverse event (SAE) considered as unlikely to be related to the study drug.

No notable changes in laboratory values, vital signs and ECGs were observed.

The overall assessment of tolerability by the patient and by the investigator showed an equal pattern without significant differences. 75.9 % of the formoterol Novolizer ® patients evaluated the treatment as very well tolerated, whereas 73.6% of the Aerolizer ® population did so.

Discussion

In this multicentre study, formoterol delivered through the Novolizer ® was therapeutically equivalent to Aerolizer ® , one of the standard devices in asthma therapy. After 4 weeks of treatment, formoterol treatment through the Novolizer ® was as effective as through the reference inhaler. With both treatments similar increases in FEV1, FVC and PEF were recorded. The decrease in asthma symptoms and the reduction in the frequency of rescue inhalations were also similar. Safety analysis did not show important differences between the two study groups. The only serious adverse event occurred in the reference inhaler group which was likely to be related to the underlying disease. None of the adverse events were judged to be likely related to the inhaler products. The formoterol Novolizer ® treatment was well

A c c e p t e d m a n u s c r i p t

  compared to that delivered through the Turbuhaler ® at a flow rate of 30 L/min was 12.6% and 12.8% of the nominal dose, respectively, whereas the reduction in the coefficient of variation of the FPD was statistically significant (p=0.013) in favour of the Novolizer ® [18]. The Novolizer ® 's therapeutic equivalence to the Turbuhaler ® delivering the corticosteroid budesonide has been shown in a randomised open multicentre study with 315 asthmatic patients during a 12-week treatment period [19].

A c c e p t e d m a n u s c r i p t

  reference device sets (double-blind double-dummy technique) twice daily (morning and evening) for a treatment period of 4 weeks. Group 1 received 2 x 12 µg

A c c e p t e d m a n u

  s c r i p t were calculated. All primary and secondary variables were analysed for the intentionto-treat (ITT, patients with FEV 1 at baseline and at least one more after randomisation) and the per-protocol (PP) population. The primary analysis was based on the ITT population, the analysis based on the PP population being supportive. Missing values were replaced by the last value carried forward principle.

A c c e p t e d m a n u s c r i p t

  tolerated by the patients of this study (96.5% good and very good) as well as the reference treatment (95.9% good and very good). This compares well to a previous study in which the tolerability of treatment with budesonide Novolizer ® was rated as good or better by more than 90% of patients according to a questionnaire about various details in the handling of the device[19].The European Community banned the chlorofluorocarbon (CFC) containing pressurised metered dose inhalers[23]. Alternative inhalation devices became necessary. The Novolizer ® as a multidose refillable breath-actuated dry powder inhaler is a useful alternative for the inhalation of anti-asthmatic substances such as formoterol.

Figure 1

 1 Figure 1Time course of FEV 1 (l) at baseline, after 2 and 4 weeks of

A c c e p t e d m a n u s c r i p t

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A c c e p t e d m a n u s c r i p t

Figure 1 Figure 2

 12 Figure 1Time course of FEV 1 (l) at baseline, after 2 and 4 weeks of treatment ITT (n = 198 Novolizer ® ; n=192 Aerolizer ® ), mean ± SD

A c c e p t e d m a n u s c r i p t negligible

  fraction of smokers in each treatment group: Novolizer ® 5.1%, Aerolizer ® 6.3% (p=0.835 with Fisher's exact test).Exclusion criteria were: known allergic reactions to investigational products, pregnancy/nursing or risk of pregnancy, pre-existing acute or serious disease, contraindications to LABAs, and inability to use the provided inhalation devices properly. It was not recorded how many screening failures occurred due to inability to use the devices correctly. However, it is already proven that all patients aged 6 years or more are able to use the Novolizer correctly[20, 15, 16, 17].

	The trial was carried out in accordance
	with current laws and guidelines (German Drug Law and national laws of the
	participating centres, Declaration of Helsinki, and the ICH E6 Guidance on Good
	Clinical Practice).
	Patients
	Patients were eligible if they met the following inclusion criteria: 6 to 75 years of age,
	moderate to severe asthma requiring long-term treatment with inhaled β 2
	adrenoceptor agonists or theophylline or daily use of short-acting β 2 adrenoceptor
	agonists, stable disease for at least one month prior to enrolment (no exacerbation,
	no hospitalisation), reversibility of airway obstruction of ≥12% after inhalation of two
	puffs of 100 µg salbutamol at the screening visit, and a baseline FEV 1 of 40-80%
	predicted at randomisation. Inhaled corticosteroids (ICS) were allowed co-medication
	if they were applied before study entry and they could be taken during the study
	period provided they were used in unchanged dose. Most patients of both treatment
	groups used ICS before the study (Novolizer ® 81.3%, Aerolizer ® 81.3%, p=1 with
	Fisher's exact test). There was no restriction regarding the daily dose. The results of
	sub-analyses on all patients using ICS as concomitant medication confirm that the
	concomitant therapy with ICS had no influence on the study results. There was only a

Table 5

 5

	Aerolizer ® . Primary endpoint for efficacy evaluation was the FEV 1 measurement after
	4 weeks of treatment. A number of 350 (175 per group) evaluable patients was
	considered as adequate to demonstrate non-inferiority by means of a one-sided t-test
	with 90% power. The lower limit for equivalence was set to 0.25 L and the

):

Analysis

Statistical analyses were done by means of the SAS ® software, Version 8.01. The hypothesis was that formoterol treatment via Novolizer ® is not inferior to that via significance level to α = 0.025. Evaluation of secondary efficacy variables and safety variables was performed by t-test or by χ 2 -test, the appropriate confidence intervals

Table 3

 3 Spirometric measures, total asthma symptom score, and use of rescue medication (salbutamol) at baseline, and after 2 and 4 weeks of treatment ITT (n = 198 Novolizer ® ; n=192 Aerolizer ® )

	Variable	Baseline NOV AER	Week 2 NOV AER	Week 4 NOV AER
	FEV 1 [L] 1	2.05	1.99	2.32	2.32	2.34	2.31
	±0.55	±0.61	±0.67	±0.74	±0.73	±0.72
		0.261 3			-0.117 to 0.053 4
	FEV 1 [% pred.] 1	69.0	66.4	78.2	78.1	78.9	78.1
		±8.8	±9.9	±14.2	±16.6	±16.4	±17.4
		0.007 3			-3.78 to 1.75 4
	FEV 1 /FVC [%] 1	73.1	71.9	75.1	75.2	74.9	74.9
	±11.9	±12.5	±11.9	±12.6	±11.9	±11.9
		0.337 3			-2.317 to 0.783
	PEF [L/s] 1	4.6	4.5	5.5	5.4	5.5	5.4
		±1.6	±1.7	±1.8	±1.9	±1.9	±1.9
		0.684 3			-0.251 to 0.260 4
	FVC [L] 1	2.9	2.9	3.1	3.1	3.2	3.1
		±0.9	±1.0	±0.9	±1.0	±1.0	±1.1
		0.786 3			-0.103 to 0.094 4
	Total asthma score 2	2.9	3.1	2.1	2.1	1.8	1.9
		±2.2	±2.4	±2.2	±2.1	±2.2	±2.0
	Rescue inh. [n/d] 2	3.2	3.1	1.7	1.4	1.5	1.3
		±2.7	±3.0	±2.6	±2.3	±2.4	±2.4
	Displayed are means ±SD;
	1 from spirometry at clinic
	2 weekly means from diaries
	3 p-value from t-test
	4 two-sided 95% confidence interval for difference NOV-AER derived from baseline adjusted
	ANCOVA with factors treatment and centre

Table 4

 4 Incidence of adverse events during 4 weeks of treatment. Safety population (n = 199 Novolizer ® ; n=193 Aerolizer ® )

	Adverse events	Total	Novolizer ®	Aerolizer ®	P*
		n = 392	n = 199	n = 193
		n	%	n	%	n	%
	Total	64	16.3	29	14.6	35	18.1	-
	Hyperglycaemia	11	2.8	4	2.0	7	3.6 0.374
	Upper respiratory tract	7	1.8	4	2.0	3	1.6 1.000
	infections
	Coughing	6	1.5	1	0.5	5	2.6 0.117
	Exacerbation of disease	6	1.5	3	1.5	3	1.6 1.000
	Influenza-like symptoms	6	1.5	2	1.0	4	2.1 0.443
	Increase in hepatic enzymes	4	1.0	1	0.5	3	1.6 0.366
	Paradoxical bronchospasm	3	0.8	0	0.0	3	1.6 0.118
	Headache	3	0.8	2	1.0	1	0.5 1.000
	Rhinitis	3	0.8	3	1.5	0	0.0 0.248

*Calculated with Fisher's exact test

Table 5

 5 Flow chart (NOV= Novolizer ® ; AER=Aerolizer ® )

	Randomisation Enrollment		Not eligible n=19 Erroneously randomised; did not receive treatment n=1
	SAF Population	No more visits after start of treatment n=1	AER group n=193 No more visits after start of treatment n=1
	ITT-Population	NOV group n=198 Major protol violations n=12	Major protol violations n=12
	PP-Population	NOV group n=186	AER group n=180

Per protocol (PP) population n=366

Acknowledgements

This study was supported by a study grant of Fujisawa GmbH, München, Germany, and VIATRIS GmbH & Co. KG, Frankfurt, Germany, study code FORAFORM 2000.

The authors would like to thank the following investigators who participated in the study: Gebhardt R, Guensberg K, Linnhoff A, Ponitz HH, Possner S, von Versen L, Berlin, Germany; Malolepszy J, Wroclaw, Stelmasiak T, Lublin, Nalepa P and Guzik-Adamek T, Krakow, Swierczynska B, Bielsko-Biala, Poland; Feshcenko YI and Yashina LA, Kiev, Ukraine; Blaziene A, Vilnius, Norvaisas GA, Klaipeda, Lithuania; Iacomi A, Cernatescu I, Rodica RJ, Bumbacea R, Bisca N, Nemes R, Bogdan M, Bucaresti, Romania.


MD Kai Richter

MD Lars Grönke

Stanislawa Janicki

MD Joachim Maus

PhD Rudolf A Jörres
email: rudolf.joerres@med.uni-muenchen.de

MD Helgo Magnussen

Effect of azelastine, montelukast, and their combination on allergen-induced bronchoconstriction in asthma

Keywords: Anti-histamine, leukotriene receptor antagonist, bronchial allergen response, early phase response, late phase response, bronchoprotection

published or not. The documents may come

Effect of azelastine, montelukast, and their combination on allergen-induced bronchoconstriction in asthma

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t INTRODUCTION

In patients with allergic asthma the lung function response to an inhaled allergen often shows a typical time course comprising an early allergen reaction (EAR) occurring within 30 minutes, a late allergen reaction (LAR) delayed by several hours, and often both, i.e. a dual reaction [START_REF] Varner | The early and late asthmatic response to allergen[END_REF][START_REF] O´byrne | State of Art: Late asthmatic response[END_REF].

These responses are quite common, with a prevalence of 9-53, 7-50, and 18-84 %, respectively [START_REF] Varner | The early and late asthmatic response to allergen[END_REF].

The LAR is considered particularly important, as it is associated with prolonged inflammation involving eosinophils, lymphocytes, mast cells and basophiles [START_REF] Gratziou | Inflammatory and Tcell profile of asthmatic airways 6 hours after local allergen provocation[END_REF][START_REF] Gauvreau | Increased numbers of both airway basophils and mast cells in sputum after allergen inhalation challenge of atopic asthmatics[END_REF]. Based on this it is believed that blocking of allergen responses is beneficial for both the clinical state and the course of the asthmatic disease.

In vitro and in vivo studies have shown that histamine and cysteinyl leukotrienes play a major role during EAR and LAR [START_REF] Björck | Leukotrienes and histamine mediate IgE-dependent contractions of human bronchi: pharmacological evidence obtained with tissues from asthmatic and nonasthmatic subjects[END_REF][START_REF] Drazen | Asthma therapy with agents preventing leukotriene synthesis or action[END_REF][START_REF] Lordan | H 1 -antihistamines in asthma[END_REF] in patients with asthma and rhinitis. As a consequence, antihistamines are capable of providing protection against allergen inhalation-induced bronchoconstriction, particularly during the EAR. In particular, the H 1 -receptor antagonist azelastine has been shown to be effective against allergen inhalation, in addition to other bronchoconstrictor stimuli [START_REF] Ollier | The effect of a single and multiple dose therapy with azelastine on the immediate asthmatic response to allergen provocation testing[END_REF][START_REF] Rafferty | The inhibitory effect of azelastine hydrochloride on histamine-and allergen-induced bronchoconstriction in atopic asthma[END_REF][START_REF] Motojima | Effects of azelastine on allergen-and exercise induced asthma[END_REF][11][12].

Similarly, anti-leukotrienes, such as zafirlukast and montelukast, afford protection against allergen-induced bronchoconstriction [13][14][15][16]. As the protection is not complete, ranging from 50 to 75%, the question is raised whether the combination with an anti-histamine can further attenuate allergen responses. Indeed, concurrent treatment with zafirlukast and the anti-histamine loratadine was capable of enhancing the protection against allergen-induced bronchoconstriction, compared to the individual agents [17]. However, the dose of the anti-leukotriene employed in this study was four-times greater than that recommended for clinical use.

As it is not known whether the enhanced protection also occurs with the doses that are clinically used, the aim of our study was to investigate the effect of a combination of daily recommended

PATIENTS AND METHODS

Patients

Seventeen patients with a history of allergic asthma and a forced expiratory volume in EAR and LAR at a second screening challenge. Thirtythree patients were screened for this study.

Sixteen of them did not meet the criteria for EAR and LAR at screening and were not randomized.

The allergic status of each individual was confirmed by a skin-prick test comprising a panel of 21 allergens (ALK, Hamburg, Germany). Patients receiving treatment for asthma, other than shortacting β 2 -agonists, were excluded. Patients were also required to be nonsmokers and not to suffer from an acute respiratory tract infection within the last three weeks prior to inclusion. The study was approved by the local Ethics Committee and all patients gave their written informed consent.

Study design

This was a randomized, double-blind, cross-over study involving a screening and a treatment phase. The screening phase comprised three visits, during which the patient's history of asthma was confirmed, skin prick test and radio-allergo-sorbent test (RAST) were performed, and lung function, as well as PC 20 FEV 1 of methacholine, PD 20 FEV 1 of allergen and LAR were assessed. At screening allergen provocation was repeated after ≥2 weeks, to confirm reproducibility of EAR and LAR.

A c c e p t e d m a n u s c r i p t

Two or more weeks later the cross-over treatment phase comprising three treatment periods per patient was started. Patients were randomly assigned to receive either 4 mg azelastine twice daily plus montelukast placebo once daily, or 10 mg montelukast once daily plus azelastine placebo twice daily, or 4 mg azelastine twice daily plus 10 mg montelukast once daily. After treatment for one week (day 1-7), allergen provocation was repeated two hours after the last intake of study medication in the morning of day 8. This cycle of a two-week wash-out period, followed by treatment for one week and subsequent allergen challenge, was then repeated twice for a total of three treatment periods per patient.

Study medication

The study was performed in a double-blind design. For this purpose the commercially available tablets for montelukast and azelastine were covered with an inert gelatin capsule. The placebo capsules contained only inert material. Due to this procedure all capsules appeared identical to the investigator and patients. Study medication was provided by Asta Medica, Frankfurt, Germany.

Pulmonary function tests and inhalation challenges

Spirometry was performed according to the ATS recommendations [START_REF] Thoracic | Standardization of spirometry: 1994 update[END_REF]. Baseline FEV 1 was measured at least three times, until reproducibility within 5 %, and the highest value was selected.

Provocation with inhaled methacholine was performed following a standard protocol [START_REF] Chai | Standardization of bronchial inhalation challenge procedures[END_REF] and the results were expressed as concentration causing a 20 % fall in FEV 1 (PC 20 FEV 1 ).

Dependent on the findings of prick and RAST tests, patients were challenged with individual allergen preparations (ALK, Hamburg, Germany), each of which was used over the course of the entire study. Allergen content of lyophilised, purified allergen extract is expressed in SQ-units correlating with the amount of the specific major allergen. During the second screening visit, allergen responsiveness was determined using increasing doses of allergen, whereby FEV 1 was measured up to 10 minutes following each inhalation. After achieving a 20 % drop in FEV 1 ,

A c c e p t e d m a n u s c r i p t

measurements were repeated at 15 minutes intervals for up to 2 hours, to determine the EAR, and at 30 minutes intervals thereafter for up to 9 hours after the challenge, to determine the LAR. If the FEV 1 dropped by more than 10 % during the LAR, measurements were performed every 15 minutes until recovery was achieved. The cumulative dose of allergen which led to a 20 % fall in FEV 1 (PD 20 FEV 1 ) was calculated for each patient individually and was used as a single dose in all subsequent allergen challenges. Details on allergen type used for the individual patient and EAR and LAR at screening are given in table 1.

Data analysis

Mean values and standard deviations (SD) or standard errors of mean (SEM) were computed for all relevant variables. The primary efficacy variable used was the maximal percentage fall in FEV 1 compared to baseline during 0-2 hours after allergen challenge (EAR) as well as 2-9 hours after allergen challenge (LAR). Statistical significance was assumed for p<0.05. Data were analyzed using Friedman's nonparametric ANOVA, whereby EAR and LAR after single-dose provocation at screening were compared with the respective responses after treatment. In case of significant treatment effects, differences between treatments were assessed by the Wilcoxon matched-pairs signed-ranks test.

RESULTS

Bronchial provocation with allergen led to a dual EAR and LAR in all patients, which was highly reproducible in terms of the fall in FEV 1 . Mean (range) EAR at the two screening visits was 33.7 (20.4-57.1) and 30.3. (19.8-49.9) %, and mean (range) LAR was 29.2 (14.7-59.7) and 29.4

(13.1-66.9) % (Table 1).

A c c e p t e d m a n u s c r i p t

Early allergen reaction

All three treatments significantly attenuated allergen-induced EAR, as indicated by the maximal fall of FEV 1 compared with that observed during the second screening challenge (p<0.001; Table 2, Figure 1). The combination of azelastine and montelukast achieved about 89 % protection, montelukast 75 %, and azelastine 46 %. According to pair-wise comparisons, the combination of azelastine and montelukast was superior to both azelastine (mean±SEM, ∆=12.5±1.6 %; p<0.001) and montelukast (∆=3.8±1.1 %; p=0.003) alone, and montelukast was superior to azelastine (∆=8.7±1.8 %; p<0.001).

Late allergen reaction

All three treatments also protected against allergen-induced LAR, compared with the screening visit (p<0.004; Table 1, Figure 1). The level of protection ranked in the order of the combination of azelastine and montelukast (78 %), montelukast (59 %), and azelastine (43 %). Pair-wise comparisons confirmed that the combination of azelastine and montelukast was superior to both azelastine (∆=10.3±2.8 %; p<0.001) and montelukast (∆=5.5±2.0 %; p=0.017) alone, whereas the efficacy of azelastine and montelukast was not significantly different (∆=4.8±2.3 %; p=0.080).

DISCUSSION

The present study demonstrated significant protection against allergen-induced early and late phase reactions after treatment for one week with clinically recommended oral doses of azelastine and montelukast, either alone or in combination. The combined treatment was superior to the single treatments. Furthermore, montelukast was more effective than azelastine in diminishing the early, but not the late phase response.

A c c e p t e d m a n u s c r i p t

Regarding the effects of montelukast or azelastine as single drugs, our findings are in accordance with previous results. In dose-finding experiments, 4-8 mg azelastine were found to protect against EAR after allergen challenge [START_REF] Ollier | The effect of a single and multiple dose therapy with azelastine on the immediate asthmatic response to allergen provocation testing[END_REF][START_REF] Rafferty | The inhibitory effect of azelastine hydrochloride on histamine-and allergen-induced bronchoconstriction in atopic asthma[END_REF]. Other studies found azelastine to attenuate bronchoconstriction by 24-37 % during EAR and by 36-100 % during LAR [START_REF] Motojima | Effects of azelastine on allergen-and exercise induced asthma[END_REF]11]. Similarly, montelukast, when given alone, is known to protect against allergen-induced responses in asthma One starting point for our study was that the dose of zafirlukast as previously used (160 mg daily) was four times greater than the clinically recommended daily dose. The question was whether enhanced protection by combining the two drugs also occurred at doses that are recommended and acceptable for daily treatment. To facilitate the comparison of results, we employed a similar study design. Comparability was further emphasized by the fact that responses in terms of FEV 1 were reproducible in both studies and that there were no carry-over effects. Our results demonstrated that the combined treatment enhanced protection against allergen-induced bronchoconstriction also at doses of the anti-leukotriene that are clinically introduced.

The role of cysteinyl leukotrienes as mediators of allergic responses is well documented [START_REF] Drazen | Asthma therapy with agents preventing leukotriene synthesis or action[END_REF][START_REF] Drazen | Treatment of asthma with drugs modifying the leukotriene pathway[END_REF],

and studies have demonstrated montelukast to be a potent and selective leukotriene D 4 (cysLT 1 )

receptor antagonist [START_REF] De Lepeleire | Montelukast causes prolonged, potent leukotriene D 4 -receptor antagonism in the airways of patients with asthma[END_REF], which is capable of inhibiting a variety of effects, among which The same applies for anti-histamines, though their anti-inflammatory effect seems to be less pronounced than that of anti-leukotrienes. In addition to, or as a consequence of their anti-histaminic activity, anti-histamines might also reduce the release of other mediators, including leukotrienes [START_REF] Church | Non H 1 -receptor effects of antihistamines[END_REF][START_REF] Walsh | New insights into the second generation antihistamines[END_REF]. Azelastine shows many of these properties [START_REF] Conti | The anti-inflammatory actions of azelastine[END_REF][START_REF] Katayama | Effect of azelastine on the release and action of leukotrienes C 4 and D 4[END_REF][START_REF] Acterracth-Tuckermann | Inhibition of cysteinylleukotriene production by azelastine and its biological relevance[END_REF], among which decreased activation of mast cells coupled with decreased release of cysteinyl leukotrienes seems to be of particular relevance for the present study.

Table 1 demonstrates that azelastine caused a two-fold reduction of the maximal response of FEV 1 during EAR, and montelukast an about four-fold reduction. When both were combined, azelastine attenuated the response relative to montelukast alone again by a factor of about two. A similar pattern of protection emerged during the LAR. These observations do not suggest a true synergism but a multiplicative effect of their combination, indicating an in-series action of both compounds. This conclusion sounds reasonable when considering the mechanisms underlying the action of both drugs. Both interfere with the effects of mediators released within short periods of time after allergen inhalation, and both have similar cell types, such as mast cells, basophils and airway smooth muscle, as primary targets [START_REF] Björck | Leukotrienes and histamine mediate IgE-dependent contractions of human bronchi: pharmacological evidence obtained with tissues from asthmatic and nonasthmatic subjects[END_REF][START_REF] Drazen | Asthma therapy with agents preventing leukotriene synthesis or action[END_REF][START_REF] Lordan | H 1 -antihistamines in asthma[END_REF].

The protection provided by all three types of treatment during LAR was about the same as that observed previously using loratadine and zafirlukast [17], but there seemed to be stronger effects during EAR. This was true particularly for the antihistamines. Probably this stronger effect promoted the significantly greater protection by the combined compared to single drugs in our study. One might argue that with high doses of the drugs involved, potential multiplicative effects are likely to be reduced by plateau-forming of the dose-response curve. Such effects should be most apparent at low doses and would suggest that our results on an additive action are to some extent predictable from existing data. However, the protection achieved by the compounds used, protection by azelastine on the EAR was also observed when combined with montelukast. Whether the combination of an anti-leukotriene and an anti-histamine is a realistic therapeutic option in patients with allergic airway diseases, remains, however, to be proven [START_REF] Lordan | H 1 -antihistamines in asthma[END_REF][START_REF] Meltzer | Concomitant montelukast and loratadine as treatment for seasonal allergic rhinitis: a randomized, placebo-controlled clinical trial[END_REF]29].

In conclusion, our results demonstrate that azelastine, montelukast and their combination can exhibit considerable efficacy in the attenuation of early and late phase responses to allergen in patients with allergic asthma. This was achieved with clinically recommended doses of both preparations, whereby the combination of the two drugs was superior to each drug alone. The findings indicate a multiplicative action of both compounds and are compatible with an additive inhibitory mechanism involving mast cell-and basophil-derived mediators. 2.

A c c e p t e d m a n u s c r i p t

A c c e p t e d m a n u s c r i p t

  doses of montelukast (10 mg) and azelastine (8 mg) on allergen-induced bronchoconstriction in patients with allergic asthma.

[

  [13][14][15][16]. For example, pre-treatment with 10 mg montelukast, given 36 and 12 hours before and 12 hours after provocation with house dust mite, significantly reduced EAR and LAR between 0-3 and 3-8 hours, respectively[16].At present, one other study is available which has investigated the effect of combined treatment with an anti-histamine and an anti-leukotriene on EAR and LAR in asthma[17]. Treatment was performed for one week with zafirlukast 80 mg twice daily, loratadine 10 mg twice daily, or a combination of the two. The combination led to about 75 % protection during both EAR and LAR, compared to 25 and 40 %, respectively, by loratadine, and 62 and 55 % by zafirlukast alone.

A c c e p t e d m a n u s c r i p t

  bronchoconstriction, airway smooth muscle hyperresponsiveness and inflammation are particularly important during EAR and LAR in asthma[START_REF] Drazen | Asthma therapy with agents preventing leukotriene synthesis or action[END_REF]16,[START_REF] Drazen | Treatment of asthma with drugs modifying the leukotriene pathway[END_REF][START_REF] De Lepeleire | Montelukast causes prolonged, potent leukotriene D 4 -receptor antagonism in the airways of patients with asthma[END_REF].

A c c e p t e d m a n u s c r i p t

  Richter et al. Effect of azelastine, montelukast, and their combination........ 10 either alone or in combination, was at least as high as that achieved previously, probably due to the different potency of the compounds used. Compared to zafirlukast and loratadine [17], the stronger

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t 11 .A c c e p t e d m a n u s c r i p tLegend to figure 1

 111 Rafferty P, Phillips G, Clough J, Church MK, Aurich R, Ollier S, Holgate ST. The inhibitory actions of azelastine hydrochloride on the early and late bronchoconstrictor response to inhaled allergen in atopic asthma. J Allergy Clin Immunol 1989;84:649-57. 12. McTavish D, Sorkin EM. Azelastine: A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential. Drugs 1989;38:778-800. 13. Taylor IK, O'Shaughnessy KM, Fuller RM, Dollery CT. Effect of cysteinyl-leukotriene receptor antagonist ICI 204,219 on allergen-induced bronchoconstriction and airway hyperreactivity in atopic subjects. Lancet 1991;337:691-4. 14. O'Shaughnessy KM, Taylor IK, O'Connor B, O'Connell F, Thomson H, Dollery, CT. Potent leukotriene D 4 receptor antagonist ICI 204,219 given by the inhaled route inhibits the early but not the late phase of allergen-induced bronchoconstriction. Am Rev Respir Dis 1993;147:1431-5. 15. Dahlén B, Zetterström O, Björck T, Dahlén SE. The leukotriene-antagonist ICI 204,219 inhibits the early airway reaction to cumulative bronchial challenge with allergen in atopic asthmatics. Eur Respir J 1994;7:324-31. 16. Diamant Z, Grootendorst DC, Veselic-Charvat M, Timmers MC, De Smet M, Leff JA, Seidenberg BC, Zwinderman AH, Peszek I, Sterk PJ. The effect of montelukast (MK-0476), a cysteinyl leukotriene receptor antagonist, on allergen-induced airway responses and sputum cell counts in asthma. Clin Exp Allergy 1999;29:42-51. 17. Roquet A, Dahlén B, Kumlin M, Ihre E, Anstrén G, Binks S, Dahlén SE. Combined antagonism of leukotrienes and histamine produces predominant inhibition of allergen-induced early and late phase airway obstruction in asthmatics. Am J Respir Crit Care Med 1997;155:1856-63. 18. Quanjer PH, Tammeling GJ, Cotes JE, Pedersen OF, Peslin R, Yernault JC. Lung volumes and forced ventilatory flows. Report working party standardization of lung function tests, Richter et al. Effect of azelastine, montelukast, and their combination........ 14 29. Reicin A, White R, Weinstein SF, Finn AF Jr, Nguyen H, Peszek I, Geissler L, Seidenberg BC. Montelukast, a leukotriene receptor antagonist, in combination with loratadine, a histamine receptor antagonist, in the treatment of chronic asthma. Arch Intern Med 2000;160:2481-8. Comment in: Arch Intern Med. 2001;161:1920. Richter et al. Effect of azelastine, montelukast, and their combination........ 15 Mean values of the time course of FEV 1 after single-dose allergen inhalation challenge at the second screening challenge and after 1-week treatment with either azelastine or montelukast or the combination of both. The single allergen dose chosen was the cumulative dose which had caused a 20 % fall of FEV 1 in the first screening challenge. Within the first 2 hours after inhalation, lung function was measured every 15 min, and afterwards every 30 min. The early phase response (EAR) was assessed as maximum % fall in FEV 1 between 0 and 120 min after inhalation and the late phase response (LAR) as maximum % fall between 120 and 540 min, relative to baseline. The horizontal line is the common line of reference (baseline) to which curves have been shifted to facilitate the comparison. Absolute baseline values of FEV 1 are given in Table

A c c e p t e d m a n u s c r i p t

  mean, *geometric mean and SEM (to be interpreted as a factor relative to the geometric mean), PC 20 FEV 1 = provocative concentration of methacholine (Mch) for eliciting a 20 % decrease in FEV 1 versus baseline, PD 20 FEV 1 = allergen dose for eliciting a 20 % decrease of FEV 1 during EAR. Der p1 = Dermatophagoides pteronyssinus, Bet v1 = birch, Sec c5 = rye, Phl p5 = timothy, SQ-Units = standardized allergen content according to the manufacturer (ALK-Scherax, Germany)

A c c e p t e d m a n u s c r i p t

Fig 1

 1 Fig 1

Table 1 -Patients' demographics and early (EAR) and late phase (LAR) responses to allergen upon screening (1 st test dose-response, 2 nd test single dose)

 1

	screening test
	1 st

Table 2 -Baseline values and early (EAR) and late phase (LAR) responses to allergen at screening and after randomized, three-way, cross-over treatment

 2 Mean values ± SEM are given. Each test was performed in n=17 patients according to the crossover design.

		FEV 1	EAR	LAR
		baseline	0-120 min	120-540 min
		prior to challenge	after challenge	after challenge
		(L)	(% fall in FEV 1 )	(% fall in FEV 1 )
	Screening (2 nd challenge)	4.03 ± 0.16	30.3 ± 2.4	29.4 ± 3.9
	Azelastine	4.00 ± 0.15	15.9 ± 1.7	16.8 ± 2.3
	Montelukast	4.03 ± 0.16	7.2 ± 0.8	12.0 ± 2.2
	Azelastine+Montelukast	4.14 ± 0.18	3.4 ± 0.9	6.5 ± 1.9