C.J. Vardey

The pharmacology of salmeterol

Salmeterol was developed to provide prolonged bronchodilatation to control nocturnal symptoms and improve maintenance therapy in asthmatic patients. Salmeterol is > 10,000 times more lipophilic than salbutamol and has greater affinity for the beta 2-adrenoceptor. Membrane binding is non-competitive and dissociation is slow so that its effects last for many hours. Despite this, salmeterol does not accumulate in tissues. Its mechanism of action can be explained by binding to a specific exo-site domain of the beta 2-receptor protein to produce continuous stimulation of the active site of the receptor, which gives salmeterol a profile of pharmacological activity unlike that of other beta 2-agonists. Due to its potent and prolonged activation of beta 2-adrenoceptors in airway smooth muscle cells, endothelial cells, mast cells and epithelial cells, salmeterol induces prolonged bronchodilatation, reduced vascular permeability, inhibition of inflammatory mediators, stimulation of ciliary function and modulation of ion and water transport across the bronchial mucosa.

Functional characterization of three adenosine receptor types

1 The purpose of the present study was to classify adenosine receptors into A1 and A2 subtypes in a wide range of isolated tissues and cell types (rat adipocytes and atria, guinea‐pig ileum and atria (A1); guinea‐pig aorta, dog coronary artery and human platelets and neutrophils (A2)) using the R‐ and S‐diastereoisomers of N‐phenylisopropyladenosine (PIA), N‐cyclopentyladenosine (CPA), the novel compound, N‐[(1S,trans)‐2‐hydroxycyclopentyl]adenosine (GR79236), N‐[(2‐methylphenyl)methyl]adenosine (metrifudil), 2‐(phenylamino)adenosine (CV1808), and 2‐[[2‐[4‐(2‐carboxyethyl)phenyl]ethyl]amino]‐N‐ethylcarboxamidoadenosine (CGS21680); N‐ethylcarboxamidoadenosine (NECA) was used as a standard. 2 Results obtained in all tissue preparations previously reported to contain A1‐receptors could be described by a single rank order of agonist potency: CPA ≥ GR79236, R‐PIA ≥ NEC A > > S‐PIA ≥ metrifudil ≥ CV1808, CGS21680. 3 In contrast, two distinct rank orders of agonist potency were observed in preparations previously reported to contain A2‐receptors. In dog coronary artery, human neutrophils and platelets the rank order of potency was: CV1808, CGS21680 ≥ NECA > R‐PIA ≥ metrifudil ≥ CPA > GR79236, S‐PIA. However, in guinea‐pig aorta the rank order was: NECA > metrifudil > R‐PIA, CPA > CV1808, GR79236 ≥ S‐PIA, CGS21680. 4 The results of this study are consistent with the existence of three types of adenosine receptor: A1‐and two subtypes of A2‐receptor. The receptor present in dog coronary artery, human platelets and neutrophils, probably corresponds to the A2a subtype, whilst that present in the guinea‐pig aorta may be of the A2b subtype.

A COMPARISON OF THE ANTI-ANAPHYLACTIC ACTIVITIES OF SALBUTAMOL AND DISODIUM CROMOGLYCATE IN THE RAT, THE RAT MAST CELL AND IN HUMAN LUNG TISSUE

1 Salbutamol and disodium cromoglycate were compared for anti‐anaphylactic activity against passive anaphylaxis in rat skin and peritoneum in vivo and in rat mast cells and human lung fragments in vitro. 2 Salbutamol administered intravenously to rats inhibited cutaneous anaphylaxis, but also inhibited cutaneous responses to histamine and 5‐hydroxytryptamine. Salbutamol administered intraperitoneally inhibited the release of slow reacting substance of anaphylaxis (SRS‐A) but not the release of histamine in the peritoneum. It was a very weak inhibitor of histamine release from rat mast cells in vitro. 3 Disodium cromoglycate administered intravenously to rats inhibited cutaneous anaphylaxis. Disodium cromoglycate administered intraperitoneally to rats inhibited the release of histamine and, to a lesser extent, SRS‐A in the peritoneum. It was an effective but short‐acting inhibitor of histamine release from rat mast cells in vitro. 4 Salbutamol was a potent inhibitor of the anaphylactic release of histamine and SRS‐A from fragments of human lung. 5 Disodium cromoglycate was a weak inhibitor of the anaphylactic release of histamine and SRS‐A from fragments of human lung. The inhibition was variable and not dose‐related. 6 The concentration of salbutamol required to inhibit anaphylaxis in human lung is of the same order as that required to relax human bronchial muscle. It is suggested that salbutamol may be more effective in allergic asthma if given in a prophylactic regimen.

Formoterol on airway smooth muscle and human lung mast cells: a comparison with salbutamol and salmeterol

Formoterol, like salbutamol and salmeterol, relaxed isolated preparations of guinea-pig trachea and human bronchus, and inhibited antigen-induced mediator release from human lung fragments in a concentration-related fashion. In each case, these actions were mediated through beta 2-adrenoceptors, with formoterol being 50-120-fold more potent than salbutamol, and 2-27-fold more potent than salmeterol. The duration of action of formoterol was longer than that of salbutamol in all preparations, but was markedly shorter than that of salmeterol, whose actions persisted for many hours despite continuous or extensive washing of the tissues. In conscious guinea-pigs, inhaled formoterol, salbutamol and salmeterol all caused dose-related inhibition of histamine-induced bronchoconstriction. Formoterol was again more potent (10-20-fold) than either salbutamol or salmeterol. However, while the actions of a threshold-effective dose of formoterol persisted for less than 3 h, somewhat longer than those of salbutamol (< 1.5 h), an equivalent dose of salmeterol was active for at least 6 h. Therefore, while formoterol is a potent beta 2-adrenoceptor agonist in vitro and in vivo, and is consistently longer-acting than salbutamol, its duration of action is markedly shorter than that of salmeterol.

CHARACTERIZATION OF THE RECEPTOR MEDIATING THE ANTI-ANAPHYLACTIC EFFECTS OF β-ADRENOCEPTOR AGONISTS IN HUMAN LUNG TISSUE in vitro

1 The rank order of potency of six β‐adrenoceptor agonists as inhibitors of the anaphylactic release of histamine from fragments of passively sensitized human lung in vitro was (‐)‐isoprenaline > (‐)‐adrenaline > (±)‐salbutamol > (‐)‐noradrenaline > R0363 > H133/22. 2 The β‐adrenoceptor antagonists, propranolol, atenolol and H35/25, blocked the response to both (‐)‐isoprenaline and (±)‐salbutamol competively. Each antagonist gave similar pA2 values with both agonists. pA2 values were consistenly at the high end of the range expected for interaction at a β2‐adrenoceptor. 3 Practolol did not antagonize isoprenaline in a competitive manner but was a competitive antagonist of salbutamol with a pA2 at the high end of the range expected for interaction at a β2‐adrenoceptor. 4 Data obtained with agonists are consistent with the receptor being of the β2‐subtype. Data obtained with antagonists indicate a consistently higher affinity for the receptor than observed for the β2‐subtype in other tissues but do not suggest a novel β‐adrenoceptor subtype on the mast cell of the human lung.

Exosites: their current status, and their relevance to the duration of action of long-acting β2-adrenoceptor agonists

The four beta 2-adrenoceptor agonists, clenbuterol, bambuterol, formoterol and salmeterol, are all long-acting bronchodilators, that have distinct mechanisms for their extended durations of action. Various theories have been put forward in an attempt to explain these mechanisms. In this respect, there is strong evidence for the existence of specific additional binding sites (exosites) for salmeterol and related compounds, and that exosites exist on non-ligand recognition regions of the beta 2-adrenoceptor protein. Here, Robert Coleman and colleagues compare and contrast the profiles of action of these long-acting beta 2-adrenoceptor agonists, particularly as they relate to the role of exosites.

Characterization of the inhibitory prostanoid receptors on human neutrophils

1 We have evaluated the effects of various prostanoid agonists on the release of leukotriene B4 (LTB4) and superoxide anions (O2−) from human neutrophils stimulated with opsonized zymosan (OZ) and formyl‐methionyl‐leucyl‐phenylalanine (FMLP), respectively. 2 Prostaglandin E2 (PGE2) and PGD2 inhibited both OZ‐induced LTB4 release (EC50 0.72 μm and 0.91 μm respectively), and FMLP‐induced O2− release (EC50 0.42 μm and 0.50 μm respectively). PGF2α, the TP‐receptor agonist, U46619, and the IP‐receptor agonist, iloprost, were also active, but were all at least an order of magnitude less potent than PGE2 and PGD2. 3 The EP2/EP3‐receptor agonist, misoprostol, and the selective EP2‐agonist, AH13205, were both effective inhibitors of LTB4 release, being approximately equipotent with and 16‐times less potent than PGE2, respectively. In contrast, the EP1/EP3‐receptor agonist, sulprostone, had no inhibitory activity at concentrations of up to 10 μm. 4 The selective DP‐receptor agonist, BW245C, inhibited LTB4 release, (EC50 0.006 μm) being approximately 50 times more potent than PGD2. BW245C also inhibited O2− release, and this inhibition was antagonized competitively by the DP‐receptor blocking drug, AH6809 (pA2 6.6). 5 These data indicate the presence of both inhibitory EP‐ and DP‐receptors on the human neutrophil. The rank order of potency of EP‐receptor agonists suggest that the EP‐receptors are of the EP2‐subtype.;

Characterization of the adenosine receptor responsible for the inhibition of histamine and SRS‐A release from human lung fragments

1 The inhibitory effects of a range of natural and synthetic derivatives of adenosine on the antigen‐induced release of histamine and slow reacting substance of anaphylaxis (SRS‐A) from human lung has been studied. 2 The nucleotides ATP, ADP and AMP appear to act by being converted to adenosine. 3 The rank order of inhibitory potency of the synthetic analogues indicates that these compounds act at an extracellular A2/Ra purinoceptor. 4 The xanthines, 1, 3‐diethyl‐8‐phenylxanthine, 8‐phenyltheophylline and theophylline antagonized the inhibitory action of N‐ethyl‐carboxamideadenosine competitively. Theobromine was inactive. This supports the view that the inhibitory receptor is of the A/R type. 5 Hexobendine and dipyridamole, reported to antagonize the uptake of adenosine, failed to modify the response of human lung fragments to adenosine. 6 The P site agonist 2′, 5′ dideoxyadenosine inhibited the release of histamine and SRS‐A. This effect was not prevented by the inhibitors of uptake, hexobendine and dipyridamole, nor was it antagonized by 8‐phenyltheophylline.

The duration of action of non-β2-adrenoceptor mediated responses to salmeterol

To investigate further the mechanism of the long duration of action of the selective β2‐adrenoceptor agonist, salmeterol, we have determined the duration of action of some responses to salmeterol which are not mediated throughβ2‐adrenoceptors. In the presence of propranolol (1 μm), salmeterol (1–30 μm) caused concentration‐related relaxation of superfused, pre‐contracted strips of guinea‐pig gastric fundus. On washing the tissues, these relaxant responses were rapidly lost, the time to 50% recovery being approximately 30 min. In human neutrophils, salmeterol (1–100 μm) caused concentration‐related inhibition of FMLP‐induced O2− release. Propranolol (1 μm) had little or no effect on the inhibitory activity of salmeterol. Washing the cells twice over a 40 min period caused a marked reduction of the inhibitory activity of salmeterol. In guinea‐pig superfused trachea, in the absence of propranolol, infusions of (RS)‐salmeterol (10–30 nm) and the less potent (S)‐enantiomer of salmeterol (300–3000 nm) inhibited electrically‐induced contractile responses. When the infusion was stopped, there was no recovery from the inhibitory responses within 200 min. In the presence of propranolol (1 μm), infusions of (RS)‐salmeterol (10 μm) and (S)‐salmeterol (10–100 μm) also inhibited the contractile responses, but, in contrast, on stopping the infusions differences were observed in recovery times. Thus no appreciable recovery was observed from the responses to (RS)‐salmeterol, whereas a rapid loss of inhibition was observed on stopping the infusion of (S)‐salmeterol, the time to 50% recovery being 30–35 min. These relatively short‐lasting effects of salmeterol which are not mediated through β2‐adrenoceptors, contrast with the persistence of the responses which are mediated through β2‐adrenoceptors seen in a variety of tissues, but are similar to the rate of dissociation of salmeterol observed from artificial membranes. These observations suggest that the sustained agonist activity of salmeterol is peculiar to responses mediated by β2‐adrenoceptors.