salmeterol

Salmeterol

Malcolm Johnson Glaxo Research 6 Development Ltd., Uxbridge, Middlesex, U.K. UBl l IBT

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Design of Long-Acting P,-Agonists . . .

111. The Salmeterol Molecule . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Salmeterol Interaction with the P-Adre

A. P-Adrenoceptor Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V. Pharmacology of Salmeterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. P,-Adrenoceptor Selectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Chemical Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.........................

B. P-Receptor Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B. Affinity and Efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Intracellular Mediators .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Onset and Duration of Action . . . E. Pharmacology of Salmeterol Enantiomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. p-Receptor Desensitization .................... ..........................

VI. Bronchodilator Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Airways Smooth .......................

VII. Nonbronchodilator Properties . . . . . . . . . . . . . ....................... A. Mast Cells and Inflammatory Mediator B. Endothelial Cells; Vascular Permeability C . Inflammatory Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Bronchial Epithelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgments ....................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8. Bronchodilation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

................................. . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

225 228 228 233 234 234 235 240 240 241 242 243 244 246 247 247 248 248 249 249 250 25 1 253 254 255 255

I. INTRODUCTION

Adrenaline has been known for over a century as an adrenal hormone which produces a wide range of biological actions, many of which are mimicked by the sympathetic nervous system. Indeed, at one time it was believed that adrenaline was the sympathetic neurotransmitter substance, a view which was only proved incorrect with the discovery of noradrenaline. It was clear that the pharmacology of these two closely related amines differed in a number of ways, but it was not until Ahlquist proposed the existence of two separate classes of adrenoceptor (a- and p-), that such differences could be rationalized.1 While this classification of adrenoceptors into a- and p-types was crucial to our under- standing of the actions of the natural catecholamines, and is a classification still used today, it is inadequate to explain all of the actions of this class of compounds. This was obvious first with p-adrenoceptors, where both noradrenaline and adrenaline were potent agonists in the heart and gastro-intestinal smooth muscle, and in inducing lipolysis in fat cells, whereas only adrenaline was potent in relaxing airway and vascular smooth muscle, and in inducing glycolysis. Lands and his colleagues2 explained these divergent observations by proposing the existence of subtypes of p-adrenoceptors, termed PI- and &-adrenoceptors.

Medicinal Research Reviews, Vol. 15, No. 3, 225-257 (1995) 0 1995 John Wiley & Sons, Inc. CCC 0198-6325/95/030225-33

226 JOHNSON

Bronchial asthma is now recognized as a complex multifactorial disease characterized by episodic bronchoconstriction, airway hyperreactivity, inflammation, and muco-ciliary abnormalities.3 For many years, adrenaline was used as a bronchodilator for the treatment of asthma, but the discovery of a- and P-adrenoceptors opened up the possibility of developing a more p-selective compound to avoid some of side effects of adrenaline. Isoprenaline was the first of such compounds, and as a highly p-selective adrenoceptor agonist, proved to be a significant advance over adrenaline in the treatment of airways constriction.4 However, it did not discriminate between PI and p,-receptors, and although an effective bronchodilator, its use was associated with unacceptable extrapulmonary side effects. Like adrenaline, isoprenaline is also a catecholamine, and this class of compounds is both chemically and metabolically unstable, and therefore short- acting. The development of the resorcinol analogue, orciprenaline,5 was an important further advance in that this compound shared the pharmacological profile of isoprenaline, yet was substantially more stable. However, like isoprenaline, orciprenaline suffered from associated cardiac side effects. It was the timely subclassification proposed by Lands and his colleagues2 that made possible the development of agonists selective for P,-adrenoceptors, which produced marked bronchodilatation without the concomi- tant tachycardia associated with the earlier compounds. The prototype P,-adrenoceptor agonist was salbutamol,6 showing a > 500-fold separation in activity at p2 over p1 receptors.* Despite the subsequent development of many other such compounds such as terbutaline, fenoterol, clenbuterol, and procaterol, (Fig. l), salbutamol remains the most clinically widely used of this class of drugs.

The next stage in improving the profile of p-agonists would reasonably have been to further reduce side effects, such as vasodepression and skeletal muscle tremor. How- ever, it appears that relaxation of bronchial smooth muscle, relaxation of vascular smooth muscle, and enhancement of physiological tremor are all mediated by p2-adrenoceptors, and despite considerable efforts, no convincing evidence for further subclassification of p2-adrenoceptors has been reported.7 There is, therefore, no indication, at the present time, that a lung-selective p2-agonist is an achievable target, except by topical administration, i.e., by inhalation.

One clear shortcoming of the potent, selective P2-adrenoceptor agonists developed in the late 1960s and 1970s, and exemplified by salbutamol, terbutaline, and fenoterol, is their duration of action. In each case, the bronchodilator activity resulting from a single administration of the drug, whether by inhalation or by mouth, has no more than 4-6 h duration,s and this is clearly inadequate for protection from bronchospasm through the night, or to provide convenient maintenance therapy for the asthmatic patient. Al- though other p,-agonists, such as pirbuterol and clenbuterol, were reported to possess a significantly longer duration of action than salbutamol, in the case of pirbuterol, this difference was at best, marginal, and clenbuterol is only longer acting after oral administration.9 A more recently developed compound, bambuterollo does exhibit an extended

Malcolm Johnson graduated in pharmacy, and obtained his Ph.D. in pharmacologyfrom the University of Newcastle-upon-Tyne. He was a postdoctoral research associate at Stanford University, California, and a lecturer at Georgetown University Medical Center, Washington, D.C., before joining ICI Pharmaceuticals as a senior scientist in 1975. He was appointed Head of Cardiovascular and Respiratory Pharmacology at Glaxo Group Research in 1985, and is currently Director of Respiratory Science.

*Salbutamol was introduced by Allen & Hanbury's as Ventolinm in 1969.

SALMETEROL

OH

227

OH

HMe no Adrenaline

OH HowNHpp on

Orciprenaline

NHBu' no

no Salbutamol

on

OH

OH

Fenoterol

Et

no h0 I

OH n Procatcrol

no nowNn pr'

Isoprenaline

NHBU'

OH

Terbutaline

OH

NHBd

no Pirbuterol

NHBu'

H l N cw CI

GNnPp Clenbuterol

OH

Quinprenaline

-0

no Salmeterol

Formoterol

Figure 1. Structures of representative p-adrenoceptor agonists.

228 JOHNSON

duration of action, but this is a prodrug of terbutaline, only effective after oral administration, and suffers from all of the side effects of systemically administered P,-adrenoceptor agonists.

The challenge was to develop a drug which would be effective for > 12 h. In the early 1980s, work in a number of pharmaceutical companies was underway with the target of developing an inherently long-acting drug, that would be suitable for twice-a-day treatment. A prolonged duration of action of systemically administered drugs can be achieved with an appropriate pharmacokinetic profile, i.e ., resistance to metabolism and low plasma clearance. Indeed, clenbutero19 is such a compound, and does have a prolonged duration of action after oral administration. Alternatively, a relatively labile drug may be formulated in such a way as to delay systemic release, and salbutamol has been prepared as a delayed release formulation.* Finally, a drug may be prepared as a prodrug, i.e., a drug which is inactive in its own right, but which is slowly metabolized to an active entity, and both bitolterolll and bambutero1,lO terbutaline prodrugs, have achieved an extended duration of action. The main problem with orally administered P,-agonists is their side-effect liability, with physiological tremor and tachycardia being particular problems. It is for this reason that a different approach to an inherently long-acting P,-agonist was required.

11. DESIGN OF LONG-ACTING &-AGONISTS

A research programme was initiated at Glaxo Group Research in the U.K. in 1980, with the concept that it would be possible to design a chemical entity which would have a long duration of action by virtue of increased specific binding in the vicinity of the P-adrenoceptor. The new molecule would have two major sites of interaction; one at the active site of the p-receptor and the other at a nearby, specific site, possibly in an adjacent nonpolar region of the cell membrane or in a hydrophobic domain of the receptor protein.12 Such a molecule would persist at its additional binding site(s) through an anchoring mechanism, while the functional center repeatedly stimulated the active site of the receptor. If successful, given binding of sufficient affinity, the new p-agonist would therefore be retained at its side of action on the P,-adrenoceptor protein and the resulting drug-receptor complex that catalyzes the cellular response would, provided it retained its efficacy, be effective for longer.13

A. The Chemical Approach

The chemical rationale adopted was to introduce large, lipophilic N-substituents into saligenin ethanolamines, in an attempt to engage any exo-receptor binding sites, as well as to increase potency and selectivity (Fig. 2). Saligenins were selected as the molecular configuration of the new compound, based on the fact that low toxicity was associated with this structural type, from the experience with salbutamol, and that well-defined synthetic methods already existed for these compounds.14

Before describing the chemical approach to long acting p,-agonists in detail, it is necessary to discuss briefly the biological strategy employed to identify such compounds. The primary in vitro screen was superfused, isolated, guinea pig tracheal strips (Fig. 3; Ref. 15). In the early studies, the tissue was contracted with the spasmogen, prostaglandin F,, (PGF,,). p,-stimulants, as typified by isoprenaline, cause a dose- related inhibition of the contractile response [Fig. 3(a)]. Isoprenaline and the test com-

*Sustained release salbutamol was marketed as Volmaxe.

SALMETEROL

OH

229

A 0

Figure 2. Hypothesis for the design of a long-acting p,-adrenoceptor agonist.

pound were infused until equilibrium was obtained for a range of concentrations. Poten- cy (response magnitude) and duration of action (time to 50% recovery) were measured for each concentration in the same experiment. Potency values were expressed as equipotent concentrations (isoprenaline = 1) while duration values (RtS0) are time for 50% recovery from a response of 50% of the maximum produced by isoprenaline. The PGF,,- induced tone tended to fade during the experiment and spontaneous activity, often observed, made interpretation of results difficult. Consequently, a new method, whereby the tissue was electrically stimulated to contract every 2 min, was developed and was

Krebs flow 1 to strain gauge

to stimulator + silicon rubber tubing

stainless steel tubing

latch

stainless steel hook

platinum electrodes

0 perspex chamber

(a) PGFp-Contracted

+ w- 12 40 120nMlOmin contraction ' O F 0 If

1.2 4 (0)

lsoprenaline

(b) Electrically-Stimulated

of U

force 1 contraction 0

3 10 30 lOOnM 10 min (0)

lsoprenaline

Figure 3. Guinea pig tracheal preparation. Contractions induced by (a) PGF, and @) electrical stimulation. (For details see Refs. 15, 16).

230 JOHNSON

TABLE I Modifications to the Aryl Ether Function in Saligenin Ethanolamines

OH

Compound X

Duration of Actiona

&-Po tencya (Min) Log P

1 Salbutamol 2.0 3.7 0.66 2 OCH, (Salmefamol) 2.7 7.3 1.68 3 O(CH,),OC,H, 2.1 6.7 1.85 4 O(CH,),CH, 35.0 >50 3.96 5 OPh 11.0 22.5 3.96

aPGF,,-contracted guinea pig trachea.

increasingly used [Fig. 3(b), Ref. 161 as the project progressed. The contractile responses of these preparations were highly reproducible for 10-12 h. Values of potency and duration (Rt,,) were calculated as for the PGF,,-stimulated tissue.

There was already evidence to support the strategy of incorporating lipophilic, bulky substituents into a saligenin to obtain long-acting compounds (Table I). Salmefamol (compound 2), which contains a branched phenethyl substituent was found to be longer acting (6 h) than salbutamol as a bronchodilator in man.17 Comparison of calculated log P values revealed that salmefamol had a log P one unit greater than salbutamol. Modifica- tion of the aryl ether group in salmefamol gave compounds (4 and 5) with significantly increased durations of action, but with much reduced potencies at p,-receptors in vitro (Table I). Despite the significant reduction in potency, the enhanced duration of action observed within this series of compounds was sufficiently encouraging for other spacer groups between the N-atom and the terminal aryl ring to be examined. One such group was the hexyloxyethyl unit. Introduction of this spacer into the saligenin afforded compound 6, which was equipotent with isoprenaline, but with a considerably longer duration of action on the electrically stimulated guinea pig tracheal preparation (Table 11). The calculated log P of this compound was 2.8 units. Sequential extension of the chain gave further analogues (e.g., compound 7) with comparable potencies but, more importantly, with enhanced durations of action.

A considerable amount of structure-activity investigation was carried out around the series.14 In Table 111, the effect of modifying the length of the alkyl functionality adjacent to the side-chain ether group is shown. Two observations can be made from this data. Firstly, to maintain potency, (m) should equal 5 or 6 carbon atoms and (n) should be 2-4 carbon atoms. Secondly, to maintain a long duration of action, the calculated log P value should be within the range 3.3-4.5 units. Log P values < 3.3 invariably resulted in short- acting compounds in vitro. These compound variants also indicated the importance of the position of the 0 atom in the side chain. For example, in compound 27 (Table IV), with the oxygen placed towards the nitrogen, the duration of action was only 2.7 min; in compound 30, when the 0 atom was positioned close to the terminal phenyl ring, again

SALMETEROL 231

TABLE I1 Substitution of Spacer Groups Between (N) and Terminal Aryl Ring

OH

Duration of P,-Potencya Action”

Compound n (Isoprenaline = 1) Log P

6 2 0.5 189 2.8 7 3 1.2 >400 3.35 8 4 0.9 >420->720 3.88

~~

a Electrically stimulated guinea pig trachea preparation.

TABLE I11 Structure-Activity Relationships in the Saligenin Ethanolamine Series

OH

Duration of &-Potency Action

Compound m n Log P (Isoprenaline = 1)

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

4 4 4 4 5 5 5 5 6 6 6 6 6 6 7

3 4 5 6 2 3 4 5 1 2 3 4 5 6 3

2.3 2.83 3.3 3.88 2.3 2.83 3.35 3.88 2.3 2.83 3.35 3.88 4.41 4.8 3.88

100.0 17.1 10.5 6.5 1.2 3.6 0.5 1.4

10.5 1.1 2.4 0.9 5.6 7.8 8.8

6.8 20.5

>30.0 >60.0

5.0 >14.0 >30.0

>400.0 6.5

10.7 >35.0

>720.0 >300.0

290.0 >300.0

232 JOHNSON

TABLE IV Effect of Position of (0) Atom in the Side Chain on Duration of Action

Duration of Actiona

Compound m n Log r (Min)

25 4 6 3.88 >60.0 26 5 5 3.88 >400.0 27 2 8 3.88 2.7 28 8 2 3.88 >30.0 29 6 4 3.88 >720.0 30 9 1 3.88 47.6

a Electrically-stimulated guinea pig trachea preparation.

the duration of action was limited to < 50 min (Table IV). Optimal activity (duration of action > 720 min) appeared in compound 29 (Table IV) with a side chain skeleton comprising 11 atoms, in a (CH,),O(CH,), configuration, together with a terminal phenyl group (Fig. 4). This compound is salmeterol, with a calculated log P of 3.88. Note that the reversed isomer (compound 12) of salmeterol, (i.e., the compound where m = 4 and n = 6), is some ten-fold weaker than salmeterol in vitro (Table 111). This compound, although less potent, has a comparable duration of action to salmeterol possibly by virtue of its identical log P value.

A necessary adjunct to the saligenin research was the evaluation of the most promis- ing right-hand side substituents with left-hand sides of other commercially available &-stimulants and those in the patent literature. Incorporation of the salmeterol right- hand chain onto a resorcinol or soterenol subunit also furnished &-agonists with high potency and long duration of action.'*

In summary, from structure-activity relationships in a series of N-aralkyloxyalkyl analogues of salbutamol, it emerged that there were several factors which determined duration of action as p-agonists: the overall lipophilicity of the molecule and both the length of the side chain, and the presence and position of an 0 atom. As the size of the side chain was increased, there was a prolongation in the activity of the P-agonist molecules in relaxing airways smooth muscle. It was also clear that although the associated increase in lipophilicity was important, it was not the only critical factor, since further increases in chain length and lipophilicity resulted in an decrease in duration of action

HOH ,C

HOG!: CH, NH CH,CH,CH,CH, CH,CH,OCH,CH,CH,CH,

Figure 4. Structure of salmeterol.

SALMETEROL 233

SALBUTAMOL 0

11 A

SALMETEROL

25 I I

HOH &, OH

Figure 5. Comparative molecular dimensions of salmeterol and salbutamol.

(Table 111). Salmeterol, in the form of the hydroxynaphthoate salt was selected for development by Glaxo in 1981.*

111. THE SALMETEROL MOLECULE

The molecular structure of a P-adrenoceptor agonist determines its physicochemical properties. The salmeterol molecule (Fig. 5) is 25 A in length, with the side chain being 17A, compared with 11 A for salbutamol. It is > 10,000 times more lipophilic than salbutamol, with an octanol-water partition coefficient of 7600 and a partition coefficient in biological membranes of 22,500.18 The lipophilicity of the p-agonist determines the degree of partitioning into the cell membrane and subsequent diffusion kinetics in interacting with the P-adrenoceptor. In the case of salbutamol, which is hydrophilic in nature, the molecule remains in the extracellular aqueous compartment. The drug accesses the active site of the p-adrenoceptor directly (Fig. 6), but rapidly re-equilibrates with aqueous phase and therefore its residency time at the active site is limited and its duration of action is short. The use of low-angle neutron diffraction techniques19 to study the interaction of salmeterol with the cell membrane, has indicated that as a result of its lipophilic properties, salmeterol partitions rapidly (< 1 min) into the outer phospholipid monolayer by a factor approaching 30,000:1.18 Molecular modeling suggests that the orientation is such that the saligenin moiety is the same plane as the polar head groups, with the side chain in close association with hydrophobic tails of the phospholipids (Fig. 7). It is of interest that the 17A side chain of salmeterol, which was found to be optimal for duration of action (Tables I11 and IV), is the same as the depth of the phospholipid monolayer (Fig. 7). There is no evidence that salmeterol "flip-flops" from the outer to the inner monolayers of the surface phospholipids, but instead the molecule diffuses laterally to approach the active site of the p-adrenoceptor through the membrane (Fig. 6; Ref. 18). This translocation process appears to be slow (> 30 min.). Although salmeterol less readily re-equilibrates with the extracellular aqueous phase, on washing, it does dissoci-

*Salmeterol (Serevent@) was launched in the U.K. in 1990.

234 JOHNSON

* I FORMOTEROL

Biophase Aqumum Lipid

- I SALBUTAMOL

Bilayer

p,- adrenoceptor

btive Site 3 Binding

translocation !!IN Figure 6. Model for the interaction of salmeterol with the P,-adrenoceptor.

ate from membranes with a half life of approximately 30 min.,ls suggesting that there is another mechanism involved in the long duration of action (> 7 h) of the compound.

IV. SALMETEROL INTERACTION WITH THE P-ADRENOCEPTOR

A. p-Adrenoceptor Structure

One of the most exciting recent developments has been a cloning of p-adrenoceptors and the single amino acid mutation studies, which have given a great deal of information as to how P-adrenoceptors function. It is now established that p-adrenoceptors are members of the family of G-protein coupled, receptors related to bacterial rhodopsin, which was used for the early structural work and has been fully characterized.20.21 The

Figure 7. Likely orientation of the salmeterol molecule in the cell membrane.

SALMETEROL 235

human p,-adrenoceptor is believed to compromise 413 amino acid residues, and to have a molecular weight of approximately 46,480.21 Analysis of the receptor revealed seven domains of hydrophobic amino acids that are of sufficient length to span the lipid bilayer, suggesting that the receptor is composed of seven transmembrane segments connected by alternating intracellular and extracellular loops. The primary sequences of the hydrophobic regions of various G-coupled proteins are highly conserved whereas the hydrophilic loops are more divergent.

Site-directed mutagenesis has been used to identify regions of the P,-adrenoceptor protein important for ligand and G-protein coupling.2 Deletion mutagenesis experiments have shown that the hydrophilic loops connecting the seven hydrophobic domains are not required for ligand binding, indicating that the ligand binding domain of the &-adrenoceptor must involve amino acids within the hydrophobic core of the protein. Point mutation experiments have identified key amino acids that seem to be involved in agonist and/or antagonist binding. Although not all such studies have arrived at precisely the same conclusions, it is generally agreed that there are residues of critical importance with respect to agonist binding, namely the aspartate (Asp)-residue 113 (counted from the extracellular or N-terminus end) of the third domain, two serine (Ser) residues, 204 and 207, which are both on the fifth domain and two phenylalanines (Phe), 259 and 290, on the sixth domain.23 Thus, a mode124 has emerged for the agonist binding site of the &-adrenoceptor in which the ligand is bound within the hydrophobic core of the protein, intercalated among the transmembrane helices, and anchored by specific molecular interactions between amino acid residues in the receptor and functional groups on the ligand. Asp binds to the nitrogen of the P-adrenoceptor agonist molecule, while the two Ser residues interact with the hydroxyl groups on the phenyl ring.25 Other residues may also be important; for example, there is evidence that Asp residue 79 on the second domain and threonine (Thr) 164 are involved in agonist recognition.26

B. P-Receptor Modeling

At Glaxo, a model of the &-receptor was also constructed.27 While the primary sequence homology between the &-receptor and bacteriorhodopsin is limited, mammalian opsins which are more homologous to the p,-receptor may be used in conjunction for sequence alignment. We have used bovine opsin as an intermediary to align bacteriorhodopsin and the &-receptor. Electron diffraction 3D-coordinates of bacteriorhodopsin were taken as a template for the back bone of the @,-receptor with the side-chain substituted to that of the p,-receptor. Poor steric interactions were removed within the programme, DISCOVER. The model places the majority of the polar, charged residues on the inside of the receptor core as expected, with the binding site located 1SA into the core.

The important aspect of the modeling work was to examine the potential modes of binding of salmeterol at the receptor. Assuming salmeterol binds to the same functional groups at the agonist binding site as adrenaline, the long fle~ible-(CH,),O(CH~)~Ph chain can, in principle, fit in the receptor in three orientations (Fig. 8; Ref. 14). In these orientations, there are serine or threonine residues in proximity of the ether oxygen linkage that could form hydrogen bonds ( n r l l 0 , sr120, and Ser165 for "up," "down," and "membrane" orientations, respectively). If hydrogen bonding with the side-chain ether oxygen is an important component in the binding of salmeterol at the receptor, then the SAR observed in Table IV can be rationalized. Should (m) or (n ) fall outside the optimum range presumably the ether oxygen cannot then form an efficient hydrogen bond with the appropriate amino acid.

236

" ~ ~ ~ n " binding of S d l m C t C r O l H-bond Of -0- u i t h Serl:@

H5

Figure 8. Possible interactions of salmeterol with the P,-adrenoceptor.

S ALMETEROL 237

In the “up” position, the end aromatic group of salmeterol extends out into the extracellular boundary. In the “down” position, the end aromatic group could reach Asp,,, a residue shown by mutagenesis experiments to be involved in agonist binding.26 In the “membrane” orientation, the chain extends out into the lipid membrane region and may form hydrophobic interactions with phospholipid molecules (Fig. 8).

The “up” orientation may be the least likely, since in this position the highly lipophilic side chain of salmeterol would be located in regions of the receptor containing hydrophilic amino acids.27 The membrane orientation of salmeterol is, however, of particular interest. Herbette et d.18 have shown that salmeterol is avidly absorbed into artificial membranes and that salmeterol may therefore access the active site of the receptor via the lipid bilayer as depicted in Figure 6 . The tail remains within the membrane, thus anchoring the molecule. Is the bulk lipid of the membrane, in effect, the exo-site? This hypothetical mode of binding would explain why structurally similar compounds to salmeterol, but with lower log P values, are not long acting. These compounds cannot approach the active site in the same way since their lower lipophilicity precludes initial membrane uptake. Such compounds must access the receptor directly from the extracellular fluid and it is possible that then they cannot adopt the membrane orientation of salmeterol. However, functional evidence indicates that the long duration of action of salmeterol may be unique to p2-adrenoceptor-containing tissues (see Ref. 31 and 58). This suggests that the “down“ orientation, where the salmeterol molecule is located within the receptor protein structure, but with the side chain in a region of hydrophobic amino acids, may be the preferred conformation. Clearly, this interpretation of the mode of salmeterol binding is speculative and can only be confirmed by further experimentation.

Analysis of P-agonist binding kinetics indicates that while the binding of salbutamol and other p-agonists to lung membranes is competitive in nature, and the molecules readily dissociate from their binding sites (TV2 < 10 min), the binding kinetics of salmeterol are noncompetitive, and the dissociation rate is very slow (TVz > 300 min).28 This difference in binding characteristics is reflected in the comparative pharmacological activity of salmeterol. Salmeterol was originally identified as being extremely long-acting as a P,-adrenoceptor agonist in isolated airway preparations, such as the guinea pig trachea and human bronchus, where after a single exposure of the tissue to the agonist, even continuous washing for periods in excess of 10 h failed to cause any decline in the P,-adrenoceptor agonist activity.29 Such persistence of action, although clearly p-receptor mediated, does not result from irreversible binding to the active site of the P,-adrenoceptor itself, since rapid and complete reversal of the effects of salmeterol can be achieved by relatively low concentrations of p-adrenoceptor blocking drugs, such as propranolol and sotalol, indicating a competitive interaction with the p-receptor.30 An important and interesting observation is, however, that following washout of the p-adrenoceptor blocking drug, and without administration of further salmeterol, the p2-agonist relaxant effects are reasserted and this pattern of reversal and reassertion can be repeated several times [Fig. 9(a)]. Similarly, while salmeterol had no effect in airways preparations, in the presence of a p-blocker, it exerts normal relaxant activity many hours later, following termination of the antagonist administration [Fig. 9(b)]. None of these properties are demonstrated by other P-agonists. Taken together, the data suggests that salmeterol persists in tissues, but in a specific manner that enables the molecule to interact freely and reversibly with the active site of the @-adrenoceptor.

The major metabolite of salmeterol which is also a lipophilic molecule, is a potent p-agonist, some three-fold more potent than salmeterol itself, but has a duration of action of < 20 mins. Molecular modeling suggests that the hydroxylated side-chain of

238

Salmeterol . JOHNSON

111111111111111111111111L 2h 4h 6h

Sotald t

Salmeterol 4 *

0 - 10 min

Sotalol

2h 4h 6h 4 b

Sotalol

l l l l l l l l l l l l l l l

7 Solatol

Figure 9. Dynamic interactions between salmeterol and the p-adrenoceptor antagonist, sotalol, in airways smooth muscle.

this molecule would not adopt the same orientation as salmeterol in the p-adrenoceptor protein. In addition, work on a series of human cloned P-receptor subtypes in trans- fected cells, and in intact tissue, shows salmeterol to be both long-acting and to possess the phenomenon of reassertion of activity after P-blocker reversal only at P2 receptors.31 Finally, if the salmeterol sidechain is added to a P-blocker such as the p,-selective antagonist, atenolol, which has an approximately 100-fold selectivity for P1 over p2 receptors, the hybrid molecule becomes a long-acting, p,-selective compound. This experiment has been repeated with a number of different types of p-receptor antagonists.32 Lipophilicity alone, as suggested by Anderson et a1.,33 cannot, therefore, explain the duration of action of the molecule, salmeterol. Instead, the data suggest that the nature of the side chain in some way evokes a specific interaction with 9,-adrenoceptors.

In order to rationalize the experimental findings that the receptor binding of salmeterol is only slowly reversible and noncompetitive, whereas functional responses

SALMETEROL

I II 111 IV v VI VII

EX0 - SITE?

239

1

Figure 10. Predicted conformation of salmeterol in the p,-adrenoceptor and interaction with the exo-site.

to the molecule are both fully reversible and competitive, the "exo-site" hypothesis was proposed.*3 The original concept34 was that the long-side chain of the molecule inter- acted with a nonpolar region in the cell membrane, the "exo-site," in the vicinity of the P-receptor. High affinity binding of the side-chain to the exo-site then allowed the saligenin head to repeatedly activate the receptor, enabling salmeterol to be long-acting without causing desensitization or tachyphylaxis. From the molecular modeling studies,27 it has been predicted that there is a preferred "down" conformation of the molecule in the receptor protein, whereby the saligenin head binds to the active site in an analogous position to that of salbutamol, and the long, flexible side-chain is located deep into a hydrophobic core domain of the receptor (Fig. 10). The latter may then represent the specific "exo-site" for salmeterol, an integral part of the P2-adrenoceptor protein itself; the current exo-site model is shown in Figure 11.

The mechanism of action of salmeterol is therefore believed to involve the interaction of the side chain of the salmeterol with an auxiliary binding site (exo-site), which may be

Figure 11. Proposed mechanism of action of salmeterol.

240 JOHNSON

a domain of hydrophobic amino acids or peri-receptor lipid, adjacent to the active site of the P,-adrenoceptor. When the side chain is in association with the exo-site, the molecule is prevented from dissociating from the p,-adrenoceptor, but the saligenin head can freely engage and disengage the active site by the Charniere (hinge) principle, flexion being about the 0 atom in the side-chain, the position of which the SAR showed to be critical for duration of action (Table IV). Although definitive proof of the exo-site hypothesis has still to be produced, it does provide the most convincing explanation to account for the @,-agonist profile of salmeterol.

V. PHARMACOLOGY OF SALMETEROL

The novel mechanism of action of salmeterol, which is not shared by other p-agonists, provides the drug with a unique profile of pharmacological activity. The first example of this is in the P-adrenoceptor selectivity of the molecule, which is an important factor in the safety of the p-agonists.

A. P,-Adrenoceptor Selectivity

p-adrenoceptors have now been further subclassified into at least three distinct groups-+,, p2, and P3-and there are reports of a fourth subgroup.35 The basis of p,/P,-receptor selectivity may well be in the differences in the amino acid sequences of p1 and P,-adrenoceptors, which have approximately 20% heterogeneity (depending on species).36 However, considerably more work will be required before it is clear which amino acids are important, and their role in determining P-adrenoceptor selectivity. P,-adrenoceptors are mainly located in the heart, whereas P,-adrenoceptors are more widespread, including airways and vascular smooth muscle, leucocytes, endothelial cells, epithelial cells, and mast cells.37 The aim of bronchodilator therapy should be to produce an optimal airways response, with minimal systemic adverse effects. The latter may be divided into those which are p,-adrenoceptor mediated and include skeletal muscle tremor, hypokalaemia, and ECG changes, and those such as heart rate which appear to be the result of direct stimulation of both PI and P,-adrenoceptors.38

P,-adrenoceptor agonists not only vary widely in potency, but their degrees of selectivity for p,-adrenoceptors with respect to pl- and P,-adrenoceptors also differ markedly. For example, at p,-adrenoceptors in airways smooth muscle, salmeterol is ca. %fold more potent than isoprenaline and significantly more potent than salbutamol (Table V). In contrast, at cardiac p,-adrenoceptors although all p,-agonists are less potent than isoprenaline, this ranges from 20-fold for fenoterol to >10,000-fold for salmeterol (Table V). Salbutamol and salmeterol also have low potency at P3-adrenoceptors, while fenoterol and formoterol are only 15- and 50-fold less potent than isoprenaline (Table V). There is now considerable information on the p2:p1 selectivity (determined by dividing the relative potencies at each receptor subtype) of a wide range of &-adrenoceptor agonists;39 in contrast, data on p2:p3 selectivity are more limited. Fenoterol appears to exhibit the lowest selectivity compared with isoprenaline and salmeterol is the most functionally selective P,-adrenoceptor agonist identified to date, being 50-fold more selective than salbutamol itself (Table V).

Salmeterol behaves pharmacologically as a conventional @,-agonist, in that all of its effects at concentrations up to approximately 100 nM can be prevented with both non- selective and p,-selective antagonists.40 The molecule is, however, highly lipophilic in nature. At concentrations >1 pM, salmeterol exhibits non-@-receptor effects, which are rapidly reversed on washing41 and not antagonized by p-blocking drugs.42 Such effects

SALMETEROL

TABLE V Comparative p-Adrenoceptor Selectivity of Salmeterol

241

P,-Adrenoceptors P,-Adrenoceptors P,-Adrenoceptors

(Atria: Inotropic Activity)

P-Agonist Relative Potency

(Bronchus: Relaxant Activity)

Relative Potency

(Adipocytes: Lipolytic Activity)

Relative Potency

Isoprenaline 1.0 Salbutamol O.OOO4 Fenoterol 0.005 Formoterol 0.05 Salmeterol o.oO01

1.0 0.55 0.6

20.0 8.5

1.0 0.002 0.02 0.065 0.009

Selectivity Ratio

P2:P1 P 2 : P 3

1.0 1.0 1,375 275

120 30 400 305

85,000 945

Selectivity ratios were derived by dividing the relative potency at P,-adrenoceptors by the corresponding potency at p1 or P,-adrenoceptors.

may occur as a result of the molecule concentrating in cell membranes. These responses are not observed at low concentrations, and are unlikely to play a role in the effects of the drug at therapeutic doses.

B. Affinity and Efficacy

The affinity of a ligand is a measure of the avidity of its binding to its receptor. Affinity may be determined from binding studies, where if a labeled version of the agonist is available, it may be measured directly, but more usually, it is calculated in terms of its ability to compete with the binding of a suitable radio-labeled ligand, and in the case of P-adrenoceptors, most commonly [125I]-cyanopindolol.

Interestingly, most p-adrenoceptor agonists appear to have a rather low affinity for their receptors. Adrenaline, the natural p,-adrenoceptor ligand, has an affinity value of approximately 1 pM, whereas even that of isoprenaline is only approximately 10-fold higher. Few p,-agonists have been shown to have a much higher affinity than isoprenaline and, indeed, salbutamol, has a relatively low affinity (2.5 pM) for p,-adrenoceptors.43 In contrast, salmeterol has high affinity for the p-adrenoceptor, with a Ki of 53 nM, compared with 200 and 2500 nM for isoprenaline and salbutamol, respectively.44

p-adrenoceptor potency is a function, not only of receptor affinity, but also of receptor efficacy, as well as being influenced by the tissue-related factors such as receptor density and efficiency of G-protein coupling (see Ref. 22). In comparing p-agonist potencies on a particular tissue, the tissue factors will be effectively constant, leaving affinity and efficacy as the prime determinants of potency. It is also important to appreciate that the difference between an p-agonist and a (3-antagonist is not absolute, but rather one of degree; thus both will have affinity for the receptor, but different efficacies. A full agonist will have a high efficacy, while a pure antagonist will have low or zero efficacy. The majority of p,-adrenoceptor agonists have an intermediate efficacy, and if tissue factors permit, they will behave as full agonists, but if receptor density is too low, or coupling is inadequate, the p-agonist may behave in a partial manner, i.e., it will be incapable of achieving the same maximum effect as an agonist of higher efficacy, and it may even behave as an antagonist. A simple way of assessing whether a p,-adrenoceptor agonist is of high or low efficacy is to compare the maximum relaxant response obtained with that of the standard, usually isoprenaline, to give a value termed intrinsic activity.45 Intrinsic

242 JOHNSON

activity is a function of efficacy and is useful in ranking different p,-agonists. Inter- estingly, there is little evidence for any synthetic P,-adrenoceptor agonist having a higher intrinsic activity than isoprenaline. Examples of compounds of high efficacy (approximately equivalent to isoprenaline) are procaterol and formoterol; whereas most saligenins and resorcinols, salbutamol and terbutaline, for example, tend to be of moderate efficacy (65-85%), and the efficacy of the dichloroaniline, clenbuterol, is low (40%). Salmeterol has an efficacy at p,-adrenoceptors in airways smooth muscle of ca. 65%.& Low efficacy in a p,-adrenoceptor agonist does not, however, compromise its clinical effectiveness as a bronchodilator drug. The efficacy of p,-agonists at extrapulmonary p-adrenoceptors, may, however, be of clinical importance. For example, fenoterol and formoterol have the same efficacy as isoprenaline at cardiac PI-adrenoceptors (Table VI), whereas salbutamol is a partial agonist (14% of isoprenaline) and salmeterol has very low efficacy (4%). Clinical studies have recently confirmed these differences between fenoterol, formoterol, and salbutamol.47

C. Intracellular Mediators

P-Adrenoceptors are widely distributed and there is a substantial body of evidence that they are commonly linked through a trimeric Gs-protein to the enzyme, adenylyl cyclase. All of the effects of P-adrenoceptor agonists are, therefore, thought to be mediated by increases in intracellular levels of cyclic 3’,5’-AMP (CAMP).& Thus, P-adrenoceptor agonist activity can be mimicked by stable analogues of CAMP, such as dibutyryl CAMP, and by other agents which are believed to produce their effects through elevation in cAMP levels, e.g., activators of adenylyl cyclase, such as forskolin, and inhibitors of cAMP phosphodiesterase, such as the methylxanthines.49 P-Adrenoceptor agonists can also be shown to increase intracellular levels of cAMP in preparations in which they produce their cellular responses. The mechanism by which cyclic AMP induces smooth muscle cell relaxation is not fully understood, but it is believed that cAMP catalyzes the activation of protein kinase A, which in turn results in inhibition of CaZ+ release from intracellular stores, reduction of membrane Ca2+ entry, and sequestration of intracellular Ca2+, leading to relaxation of the smooth muscle.50

McCrea and Hill51 have shown that the increment in cAMP in cultured smooth muscle cells is rapid with isoprenaline and salbutamol, whereas salmeterol increases intracellular cAMP more slowly, consistent with the hypothesis of the membrane access of the molecule to the p-adrenoceptor. In addition, the maximum elevation of cAMP to salmeterol achieves only 45% of that to isoprenaline, confirming the partial agonist nature of the response.& However, whereas cAMP responses to isoprenaline and

TABLE VI Comparative Efficacy at P-Adrenoceptor Subtypes

Efficacy (%)

P-Agonist P,-Receptors P,-Receptors

Isoprenaline 100 Salbutamol 14 Fenoterol 100 Formoterol 100 Salmeterol 4

100 86

100 100 63

SALMETEROL 243

salbutamol are transient, and rapidly reversed towards basal levels by washing the cells, salmeterol induces a sustained (>120 min) elevation of intracellular cAMP.52

Although it has been the accepted dogma, since the 1960s that P-adrenoceptors are coupled to adenylyl cyclase, and induce their effects entirely through increases in intracellular cAMP levels, more recently, an invariable association between P-adrenoceptors and cAMP has been questioned. This has followed the observation that some, although not all, effects of p-adrenoceptor agonist stimulation may be inhibited by charybdotoxin and iberiotoxin, inhibitors of high conductance, calcium-activated K+-channels (maxi-K channels).53 In support of the involvement of K+-fluxes in P-adrenoceptor agonist activity, is the observation that in bovine tracheal smooth muscle cells, isoprenaline and salbutamol both depolarize the cell membrane and cause the opening of K+-channels as indicated by an increase in rubidium efflux.54 It is interesting, however, that although this effect is clearly P-adrenoceptor mediated, only isoprenaline and salbutamol appear to cause depolarization and induce Rb-efflux, salmeterol apparently being without effect, although all three P,-agonists relax the preparation.55 It is difficult to reconcile these data, but it suggests that K+-channel activation is not obligatory to the effects of salmeterol on airway smooth muscle relaxation.

D. Onset and Duration of Action

In addition to differences in receptor affinity, efficacy and selectivity, the novel mechanism of action of salmeterol has resulted in a pharmacodynamic profile (rate of onset and duration of action) which is clearly different from other p,-agonists. For example, in isolated airway preparations, containing p,-adrenoceptors, such as the guinea pig trachea and human bronchial smooth muscle in vitro, the onset of action of isoprenaline, salbutamol, terbutaline, and fenoterol is rapid (<4 min), that of formoterol is somewhat delayed (>6 min), and responses to salmeterol are slow to each equilibrium under these conditions (>30 min).56 These differences are not so apparent clinically, however, where, for example, the time to 15% increase in FEV, following a 50 pg dose of salmeterol is only a few minutes slower than with a standard 200 pg dose of salbutamol.57 In contrast, there are marked differences in the duration of action of p,-agonists. While isoprenaline has a short duration of action, and equipotent concentrations of salbutamol, fenoterol, and formoterol are similar (<20 min), the relaxant effects of salmeterol on airways smooth muscle are long-lasting (>7 h). The duration of action of p,-agonists against spasmogen-induced, neuronally mediated, and inherent tone in the human bronchus is in the order: salmeterol >> formoterol 3 salbutamol 2 terbutaline > fenoterol (Table VII). In contrast, in tissues containing predominantly PI- or P,-adrenoceptors, salmeterol is short-acting,5* suggesting that the mechanism of action (exosite?) may be specific to &-receptors. Indeed, salmeterol has the longest-acting relaxant effects on human airways smooth muscle of any p-agonist, but despite this long duration of action, there is no evidence of tachyphylaxis.59

Analysis of the pharmacodynamic data, has revealed a further difference with salmeterol from other p,-agonists. Whereas the duration of action of salbutamol and formoterol can be increased by increasing the concentration applied to the tissue (Fig. 12), salmeterol appears to be inherently long-acting, in that its effects are independent of the concentration applied to the tissue (Fig. 12). For example, a low concentration (4 nM) producing only 40% relaxation, still persists for more than 4 h (Fig. 12).

The pharmacodynamic profile of slower onset in action, and an inherently long duration of effect is consistent with the proposed exo-site mechanism of action of salmeterol.

244

SalbtMmol (nM)

0 10 0 loo A low

JOHNSON

TABLE VII Pharmacodynamics of Salmeterol: Duration of Action in Human Airways

Smooth Muscle in vitro

Duration of Action (h4in)a

PGF,,-Contracted Electrical Stimulation Inherent Tone

Isoprenaline <2.0 4.0 2.2 Salbutamol 3.0 11.0 6.9 Formoterol 5.8 30.5 6.6 Salmeterol > 60 >480 >240

aTime to 50% recovery.

This profile also allows us to differentiate between p-agonists like salbutamol which are used for the rapid relief of acute bronchospasm, and salmeterol, which is aimed at twice- daily maintenance therapy, and where the slower onset of action is not a factor, and the key element is the long duration of effect. However, in clinical practice, it is likely that, on occasions, both drugs will be used together-for example, in the asthmatic patient taking salmeterol who requires use of a short-acting p-agonist to control breakthrough symptoms. It is therefore important to demonstrate that in the presence of an ongoing response to salmeterol, there is a normal and full relaxation of human airway tissue to salbutamol. In such studies the response to salbutamol was the same both before and after salmeterol (Fig. 13). This experimental finding has been confirmed in the extensive clinical trial programme on salmetero1.m

E. Pharmacology of Salmeterol Enantiomers

All P-adrenoceptor agonists have an asymmetric center due to the presence of the p-OH group on the ethanolamine function. The presence of an asymmetric center results in the molecule existing as a pair of optical isomers (or mirror images), referred to as the R and S [or (-) and (+)I enantiomers, in a racemic mixture. In fact, some agonists-for

s E

6 t - 4 0

r 2 0

- .-

100

80

60

40

20

0

100

80

60

40

20

0

Salmelerol (nM) F-. a 0 60 120 180 240

Time (min) lime (rnin) lime (rnin) - 2

Figure 12. Effect of concentration on the duration of action of P,-agonists in the human bronchus.

S ALMETEROL 245

. _ : . 0 4 4 0 40nmdes 0 2 2 0 2Onmoles 40nmoles 1 1 1 1 1 1 1

t wash

Figure 13. Normal human airway relaxant response to salbutamol in the presence of salmeterol.

example, fenoterol, formoterol, and procaterol-have two asymmetric centers, and there are 4 enantiomers, RR, SS, RS, and SR present. It is a feature of most biological systems that they are stereospecific, and this is true of ligandheceptor interactions. Where the individual enantiomers of P,-adrenoceptor agonists have been resolved and tested, it is clear that the activity lies predominantly in the R-enantiomer.61 For salbutamol, for example, the R-enantiomer is at least 100-fold more potent as a p,-agonist than the S-enantiomer, whereas this difference is greater than 1000-fold for the RR and SS forms of formoterol (Table VIII). It is often difficult, however, to establish the true potency of the S-enantiomer of p-agonists with any certainty, as it is technically demand- ing to prepare a sample with no contamination by the R-form. Clearly, even the presence of 1% of the more active R-enantiomer will provide significant p,-agonist activity, and will hinder the evaluation of activity in the S-form, particularly if it is a hundred or more times weaker. In the case of salmeterol, where enantiomerically pure samples have been prepared, there is still significant p,-agonist activity in the S-enantiomer, which is only 40-fold less potent than the R-form and 15-fold weaker than the racemic mixture (Table VIII). Interestingly, both the R and S-enantiomers of salmeterol are long-acting.

There is no evidence of the S-isomer of salmeterol antagonizing the effects of the corresponding R-form, or of the S-enantiomer having pharmacological effects different to those of the racemic mixture.

TABLE VIII Relative Potency of Enantiomers of P,-Agonists

EC, (nM)

(R)-Enantiomer (S)-Enantiomer Potency Ratio

Salbutamol 3.6 1,070 300 Terbutaline 52.0 174,000 3,350 Clenbuterol 2.9 >50,000 >lO,OOo Carbuterol 7.0 3,000 430 Salmefamol 2.0= 7,2Wb 3,600 Formoterol 0.2a 200b LOO0 Salmeterol 1.7 80.7 47.5

a RR-Isomer. b SS-Isomer.

246 JOHNSON

F. p-Receptor Desensitization

Associated with p-adrenoceptor activation is the autoregulatory process of receptor desensitization.62 This process operates as a safety device to prevent overstimulation of receptors in the face of excessive p-agonist exposure. Desensitization occurs in response to the association of receptor with agonist molecule, and will actually be prevented by the interaction of the receptor with an antagonist. The mechanisms by which desensitization can occur appears to consist of three main processes: (1) uncoupling of the receptors from adenylate cyclase, (2) internalization of uncoupled receptors, and (3) phosphorylation of internalized receptors.62 The extent of desensitization will depend on the degree and duration of the p-adrenoceptor p-agonist response. Thus, simple uncoupling is a transient process, and may be reversed within minutes of removal of the agonist. Internalization takes longer to reverse, but full reversal normally occurs within hours. However, phosphorylation may or may not be reversible, and is dependent on either dephosphorylation or de ~ O D O synthesis of new p-adrenoceptors. The process of desensitization to p-agonists may differ markedly from tissue to tissue. It is clear, for example, that human lymphocytes desensitize very rapidly on exposure to p-adrenoceptor agonists, whereas human bronchial smooth muscle is more resistant. This type of difference is manifested in the well-documented decline in the side effects associated with P-adrenoceptor agonist therapy (e.g., tachycardia and physiological tremor) in asthmatic patients, but the maintenance of bronchodilatation despite regular treatment for prolonged periods.63 As desensitization results from agonist occupancy, and can be inhibited by antagonists, it follows that a partial agonist would be less prone to induce receptor desensitization than a full agonist. Indeed this has been demonstrated to be the case with P-adrenoceptor agonists in vivo.64

The study of relaxation of airways preparations in vitro has demonstrated that pharmacologically there is no evidence of a loss of a functional relaxation response to salmeterol for up to 15 h (Fig. 14). This applies whether a relatively high concentration producing 70% relaxation of the tissue, or a low concentration, producing only 20% relaxation (Fig. 14), is used. Similarly, the increase in intracellular cAMP in airways smooth muscle, induced by the addition of salmeterol, is sustained (see Ref. 51), whereas with isoprenaline, there is a progressive decline in cAMP with time back to baseline.52 Clinical studies over a number of years have confirmed a sustained bronchodilator response to salmeterol, without tolerance, in asthmatic patients.65 There is also no evi-

100

9 0 1

30(nM) i 50 - d 40

10 (nM)

::: 9 12 3(nM) 15 10 0 0 3 6

TIME (H)

Figure 14. Lack of desensitization in the relaxant response of salmeterol on human airways smooth muscle.

S ALMETEROL 247

dence that the nonbronchodilator effects of salmeterol on mast cells (see Refs. 92 and 102) both in vitro and in vivo are subject to tachyphylaxis.

In both pharmacological and biochemical terms, therefore, rapid p-receptor desensitization to salmeterol does not seem to occur.

It is interesting to speculate on the mechanism(s) that enables salmeterol to produce prolonged effects without desensitizing the (3-adrenoceptor. If the concept behind the exo-site hypothesis is valid, then the salmeterol molecule does not appear to bind irre- versibly to the active site of the receptor in order to be long-acting. If this was the case, then rapid desensitization would probably occur. Preliminary experiments that have been carried out52 indicate that, at least up to 2-4 h, the cell appears to be able to follow repeated receptor stimulation with salmeterol, with a cAMP response. The molecular and intracellular mechanism(s) behind this observation are not yet clear. It will be of interest, for example, to look at the p-ARK and (3-arrestin system to see whether this molecule, perhaps because of the way in which it interacts with the receptor, evokes a different response from the cell. One other possibility is that because salmeterol is a partial agonist with respect to the degree of cAMP elevation, the level of cAMP is kept below that at which key intracellular phosphorylation events, which lead to receptor desensitization, occur.& Further experimentation is necessary in order to investigate receptor cycling in response to salmeterol.

VI. BRONCHODILATOR ACTIVITY

Episodic bronchoconstriction and airway hyper-reactivity are two of the cardinal features of bronchial asthma. Bronchoconstriction can arise by a number of mechanisms, including direct activation of bronchial smooth muscle by locally generated mediators, stimulation of neurogenic pathways in the airways, and extravasation of plasma proteins into lung tissue, leading to oedema formation and narrowing of the airway lumen.

To date, p,-agonists have been the most successful approach to controlling bronchospasm in asthma. Salbutamol,6 and other selective p,-adrenoceptor agonists, have proven to be highly efficacious and well tolerated over many years of clinical use. As discussed previously, the major drawback with the first generation drugs is their duration of action of 4-6 h, which limits their usefulness in controlling the symptoms of nocturnal asthma and in providing convenient maintenance therapy for the patient. This problem has been overcome with the identification of the new generation of long-acting p,-agonists, of which salmeterol is the prototype drug.

A. Airways Smooth Muscle

In isolated airways preparations, such as the guinea pig trachea and bronchus, cat bronchus and bovine trachea, all p-agonists relax the tissue either when tone is induced with a spasmogen (prostaglandin (PG)F,,, histamine, carbachol) or by electrical field stimulation.67 Contractions may result from direct stimulation of the muscle or be of cholinergic, sympathetic or nonadrenergic/noncholinergic (NANC) in origin, in which case the P-agonist may exert its relaxant effects at the pre- and/or postjunctional level. In isolated guinea pig airways smooth muscle, salmeterol is of similar potency to isoprenaline in relaxing PGF,,-contracted preparations, while salbutamol is at least 10 times less potent.@ The maximal degree of relaxation induced by salmeterol is consistently less than that of isoprenaline and salbutamol indicating the partial agonist nature of the response.68 Salbutamol, fenoterol, and formoterol have short durations of action, where-

248 JOHNSON

as salmeterol has a duration of effect in excess of 7 h. These differences between salmeterol and other p-agonists are also apparent in human airways.

B. Bronchodilatation

The evaluation of P-agonists against basal airway tone in experimental animals, in general, is difficult, and therefore antibronchoconstrictor activity is normally measured. This refers to protection against bronchoconstriction induced in nonsensitized animals, either by directly acting spasmogens (histamine, 5HT, PGF,,) given as i.v. injec- tion/infusion, by the inhaled route, or following vagal or NANC stimulation.

Nebulized aerosol administration of p-agonists causes dose-related protection against histamine-induced bronchoconstriction in the conscious guinea pig.69 At equivalent threshold effective doses, salbutamol, salmeterol, and formoterol result in similar maximal degrees of bronchodilatation. Salbutamol has a duration of action of less than 1.5 h, and even after administration of doses 10-fold greater, this is still less than 3 h under these conditions.68 Formoterol is longer-acting than salbutamol, its activity again appear- ing to be dose-related. In contrast, as predicted from its in vitro activity on isolated airways smooth muscle (Fig. 12), the bronchodilator effects of salmeterol are apparent for at least 6 h at all dose levels (Fig. 15). These differences between the profile of action of salmeterol and other p,-agonists have been extrapolated to man and to the asthmatic patient in a number of extensive clinical trials.65

VII. NONBRONCHODILATOR PROPERTIES

The pharmacological activity of p,-agonists is not restricted to airways smooth muscle and they also exhibit a range of nonbronchodilator properties. For example, there is experimental evidence that P-agonists can inhibit acute inflammatory responses in the lung, subcutaneous tissues, and the skin following local, topical or oral administration; data arising from studies both in animals70 and in man.71 Clinical experience, however, has not shown currently available p,-agonists to have significant anti-inflammatory activity in the lung. Indeed, whereas it is generally recognized that drugs such as salbutamol and terbutaline are effective against immediate bronchospasm resulting from allergen challenge, they have little, if any, effect on the late response72 or the accompanying increase in bronchial hyper-reactivity.73 Similar findings have been reported in the skin, where although P,-agonists inhibit the early weal and flare response (WFR), these agents

100

80

9 60 ' 40

e

c 20

n

A Salmelerol 0.05mg/ml Salmelerol 0.5mglml

. . I i 3 6 9 1 2 24

lime (hours)

Figure 15. Bronchodilator effects of salmeterol in the conscious guinea pig.

SALMETEROL 249

have minimal activity on the late cutaneous reaction LCR), which has many similarities to the delayed response in the lung.74 Recent evidence from preclinical and clinical studies,75 however, suggests that the lack of anti-inflammatory activity of first generation p,-agonists may be a consequence of their short duration of action, since even the processes involved in acute inflammation can be slow to develop and resolve.

The second generation of p,-adrenoceptor agonists, represented by salmeterol, are both more potent, and more importantly, longer-acting than the first generation compounds such as salbutamol and terbutaline, and appear to exhibit more significant nonbronchodilator activity.

A. Mast Cells and Inflammatory Mediator Release

Functional p-adrenoceptors have been detected in lung mast cells and p,-agonists share the property of inhibiting spasmogenic and inflammatory mediator (histamine, leukotrienes, prostaglandins) release from these cells.76 Salmeterol is potent in inhibiting antigen-induced histamine, leukotriene C4 and D4 prostaglandin D, release from human lung tissue in vitro, with a 50% inhibitory concentration range of 1-3 nM. The rank order of potency is: formoterol > salmeterol 3 isoprenaline > salbutamol, but all of the drugs are capable of producing complete inhibition of mediator release. 77 Importantly, the concentration range for this effect of salmeterol is the same as that which relaxes human airway smooth muscle.78 Interestingly, by comparison with its bronchodilator effects, the onset of inhibition of mast cell activation is rapid (<3 min). However, as in airway preparations, and in marked contrast to short-acting p,-agonists which are active for only up to 4 h, salmeterol has a sustained effect, suppressing inflammatory mediator release for >20 h, while the duration of action of formoterol (8 h) is intermediate between that of salmeterol and salbutamol.77 The effects of salmeterol and the other p,-agonists on mast cell mediator release are antagonized in a competitive manner by propranolol,77 confirming an action mediated by p-adrenoceptors.

An effect of p,-agonists on mast cell-derived mediator release has also been demonstrated in vivo. However, in early studies, salbutamol was only shown to inhibit the acute rise in circulating histamine and neutrophil chemotactic factor79 or urinary leukotriene E480 following antigen-challenge of sensitized subjects, and in the context of a potential anti-inflammatory activity of P,-agonists in the lung, a long duration of mediator inhibition may be of paramount importance in down-regulating acute inflammatory processes. In a recent report,8* exercise-induced increases in circulating plasma histamine (of mast cell and basophil origin) in mild asthmatics were shown to be inhibited by salmeterol(50 pg) for up to 12 h, whereas the effect of salbutamol (200 pg) was lost by 6 h.

B. Endothelial Cells: Vascular Permeability

Inflammatory mediators interact with specific receptors on vascular endothelial cells, particularly in postcapillary venules, to induce contraction and the opening of the tight junctions between adjacent cells.82 Plasma leakage then occurs from the vascular into the tissue compartment, and the extravasated protein may directly cause tissue oedema by an osmotic effect, or indirectly, by the local activation of complement, kinin, and coagu- lation cascades. A number of groups have shown p,-agonists to inhibit increases in vascular permeability in the lung and skin, elicited by histamine, bradykinin, and substance P, by an action which does not appear secondary to vasodilatation.83 In the hamster cheek-pouch microvascular preparation, topically applied p,-agonists inhibit LTB4-induced leakage of fluoresceinated protein,a and Gudgeon and Martin85 have

250 JOHNSON

demonstrated a direct inhibitory effect of P-agonists on protein transit through endothelial cell monolayers in culture. Salmeterol exhibits a concentration-dependent inhibition of thrombin-stimulated albumin transport, with an EC,, of 2.6 nM, compared with 100 nM for isoprenaline.86 This activity was prevented by propranolol and the P2- selective antagonist, ICI 118551, indicating the activation of P,-adrenoceptors on these cells. P-Agonists, like salmeterol, by a functional antagonist effect mediated by increased intracellular cyclic AMP, may maintain the integrity of the vascular endothelium, thereby limiting plasma protein extravasation.

Salmeterol, given at bronchodilator doses by the inhaled route, inhibits vascular permeability in the lung in viv087 and similar findings have been reported for salbutamol, terbutaline, and formoterol.88 This effect can clearly be shown to be P-adrenoceptor- mediated, since prior treatment of animals with propranolol reverses the inhibitory effect.87 Similarly, in the skin, inhibition of histamine and bradykinin-induced vascular permeability by intradermal administration of salbutamol, salmeterol or formoterol has been reported.89 Again, differences in duration of action for the effect of p,-agonists on vascular permeability in both the lung and skin were observed, in the rank order salmeterol > formoterol > salbutamol with the effects of salmeterol being apparent for up to 8 h.89 Increased vascular permeability can be a process which is rapid in onset and of short duration, or alternatively, dependent on granulocyte activation and accumulation,90 in which case, the response is slower to develop and resolve, particularly where the chemotaxin for the granulocytes has to be generated in response to the inflammatory stimulus. However, the limiting step in resolving tissue oedema is not only the rate of extravasation, but the rate of clearance of the interstitial protein.91 The duration of action of p,-agonists in inhibiting vascular permeability may therefore be of critical importance in determining their activity against this aspect of acute inflammation. A transient reduction in plasma protein extravasation, such as would result from the action of a short- acting p-agonist, would not have a substantial anti-oedema effect. In contrast, a long- acting p,-agonist like salmeterol, which inhibits vascular permeability changes in the lung for up to 8 h after a single inhaled dose,87 may be capable of limiting protein and water leakage from the vasculature for a sufficient period to allow tissue clearance mechanisms to be effective, and therefore oedema to resolve. Importantly, in the context of P,-agonists as anti-inflammatory drugs, inhibition of vascular permeability must not be subject to tachyphylaxis. With salmeterol, twice daily dosing for 5 days did not lead to a loss of the inhibitory response in the guinea pig lung.92

C. Inflammatory Cells

In experimental studies, the property of long-acting P,-agonists, such as salmeterol, which most distinguishes them from the first generation short-acting drugs is their ability to inhibit the accumulation of granulocytes at sites of acute inflammation. Local administration of low concentrations of salmeterol (10 nM) inhibits both plasma exuda- tion and leukocyte emigration induced by inflammatory stimuli in the intact microcir- cu1ation.a At bronchodilator doses in the guinea pig in vim, salmeterol inhibits both neutrophil accumulation in the airways in response to lipopolysaccharide, and attenu- ates eosinophil infiltration into the lung, which occurs 24 h following challenge with platelet activating factor93 or antigen,9* while salbutamol is without effect. Possibly as a result of this inhibition of eosinophil recruitment, salmeterol prevents the associated PAF-induced airway hyper-reactivity, an effect which occurs at doses which have no bronchodilator activity95 Importantly, the inhibitory effects of salmeterol on cell accu-

SALMETEROL 251

mulation are not restricted to the guinea pig, but equally apply to the rat,96 a species which interestingly does not readily bronchodilate in response to P,-agonists. These effects can be demonstrated either when the P,-agonist is given before, or up to 1 h after, challenge and are P-adrenoceptor-mediated, in that they are reversed by the P-blocker, DL-propranolol, but not by the non-P-blocker, D-pr~pranolol .~~ Oral or intradermal salmeterol, also inhibits zymosan (complement-mediated)-induced neutrophil accumulation in the skin and the associated granulocyte-dependent-oedema, while the short-acting P,-agonist, salbutamol is again ineffective.89

The inhibitory effects of salmeterol on cell accumulation is unlikely to be due to a direct action on inflammatory cells, since they are relatively insensitive to inhibition with this type of drug, requiring the use of high concentrations (>10-6M). Although inhibitory effects have been reported against LTB, synthesis in neutrophils,97 ECP release from eosinophils,98 TxB, formation by alveolar macrophages,99 and anti-CD,-induced lym- phocyte proliferation,lW these effects are only modest and are not susceptible to inhibition with propranolol, indicating a non-P-adrenoceptor mediated effect. Instead, the differential effects of salmeterol and salbutamol on granulocyte accumulation in vivo, may reflect differences in cell kinetics, with neutrophils being recruited early ((4 h) whereas eosinophils enter sites of inflammation much later (>24 h). Alternatively, an effect on the vascular endothelium, in addition to attenuating plasma protein extravasation, may also explain the observed actions of salmeterol on granulocyte accumulation, since inflammatory cells diapedese into tissues through the same tight junctions between endothelial cells, and the inhibition of these two acute inflammatory processes may therefore be through a common mechanism. Indeed, P,-agonists, like salbutamol and salmeterol, increase intracellular cyclic AMP in endothelial cells in culture.101 The rank order of potency is: salmeterol > isoprenaline > salbutamol, although both salmeterol and salbutamol appear to be partial agonists, with respect to isoprenaline. Cyclic AMP responses to salmeterol are antagonized by both propranolol, and by the p,-antagonist, ICI 118551, but not by the p,-selective antagonist, atenolol, indicating stimulation of p,-adrenoceptors coupled adenylate cyclase in these cells.101 By increasing intracellular cyclic AMP, it is possible that salmeterol may act as a functional antagonist to prevent the shape change induced by inflammatory stimuli, thereby maintaining the integrity of the endothelium and inhibiting the recruitment of eosinophils and neutrophils. Support for this hypothesis is again provided by studies in the microcircula- tion, where topical administration of salmeterol inhibits the LTB,-induced emigration of leukocytes from the vascular compartment.= Importantly, there is no evidence that the effect of the long-acting P,-agonists on inflammatory cell recruitment is subject to tachyphylaxis. Twice-daily dosing with salmeterol for 4 days did not lead to loss of the inhibition of eosinophil accumulation in the lung in response to antigen challenge.102

D. Bronchial Epithelium

The bronchial epithelium also contains functional P-adrenoceptors103 and long-acting p,-agonists, like salmeterol, may, therefore, also be effective in modulating some of the consequences of acute inflammation at the level of the epithelial cell. Cilia play a major role in preserving the functional integrity of the airways, particularly as part of the clearance mechanisms for excessive airway secretions which occur in response to inflammatory stimuli. In experimental animals and man, studies have shown that efficient mucus clearance is dependent on co-ordinated patterns of ciliary activity and the total numbers of cilia involved.104 There is a clinical evidence of both abnormal mucus pro-

252 JOHNSON

duction and defects in muco-ciliary clearance in bronchial asthma and other respiratory diseases.105 For example, in patients colonized by Pseudornonas aeruginosa, there is slow- ing of ciliary beating, possibly due to the release of a range of ciliotoxins such as pyocyanin.106 Mechanistic studies have suggested that neurohormones and neurotransmit- ters also have a significant influence on muco-ciliary clearance by moddying cilial beat frequency (CBF) through changes in epithelial cell calcium and cyclic AMP.

Until recently, little attention has been paid to the effects of p,-adrenoceptor agonists on cilial function. However, salbutamol and salmeterol have now both been shown to increase cilial beat frequency (CBF) in human nasal and bronchial epithelial cells in culture, with salmeterol being more potent than salbutamol.107 The effects of salbutamol are modest and transient (<2 h) in nature, whereas salmeterol produced an increment in CBF in bronchial epithelium from 9.2 to 10.9Hz, at 100-fold lower concentrations, and its effect was sustained for 15-20 h.107 Salmeterol exhibits a concentration-dependent inhibition of the ciliary dyskinesia induced by prolonged contact with pyocyanin or rhamn- olipid.108 In addition, a protective effective of salmeterol against the cellular damage induced by ciliotoxins has also been reported. 109 These effects are P-adrenoceptor mediated in that they are reversed by propranolol, and associated with an increase in intracellular cyclic AMP.108

p,-agonists also have been reported to be effective in inducing Cl-/water secretion from the human airway mucosa, by an effect on the luminal rather than on the serosal membrane of the epithelial cell.110 Electrophysiological intracellular recordings indicate, for example, that salmeterol results in sustained activation of C1- /secretion in amiloride- pretreated (Na+-channel blocked) membranes, by an action on Cl-/conductance.lll This effect may again be a consequence of increased intracellular cyclic AMP.110 Salmeterol is at least as potent as isoprenaline in stimulating cyclic AMP formation in human epithelial cells in culture, EC,, concentrations being 5 nM and 20 nM, respectively. However, salmeterol again appears to be a partial agonist in this system, with ca. 80% of the maximum response to isoprenaline. The rate of onset of action of salmeterol in increasing epithelial cell cyclic AMP is similar to that of isoprenaline (<5 min), but the effects of isoprenaline reach a plateau more rapidly (ca. 30 min). If these in vitro effects of salmeterol on the bronchial epithelium can be confirmed in man, this may suggest an additional therapeutic benefit, particularly for the use of long-acting p,-agonists, in a range of lung diseases.

Experimentally, therefore, in addition to its long-lasting effects on airways smooth muscle, salmeterol, by acting as a functional antagonist, can be shown to inhibit a number of processes involved in acute inflammation (Fig. 16), a profile which was not

Inflammatory stimulus

1 \ #/ Inflammatory cell activation

K Inflammatory

mediator release

Inllammalory cell recruitment

m e m a A Figure 16. Possible effects of salmeterol on acute inflammation.

SALMETEROL 253

predicted at the outset of the research programme in 1980. Its sustained effects on mediator release, cell recruitment and vascular permeability are more apparent than those of drugs with reported anti-inflammatory activity such as ketotifen, theophylline, and sodium cromoglycate and may have important implications for the use of salmeterol in prophylactic therapy. However, the profile of activity of salmeterol is likely to be different from that of corticosteroids, which are more effective against chronic inflammation. Salmeterol should be seen, therefore, not as a replacement, but rather as potentially complementary to steroids. Indeed, a synergistic inhibitory action between salmeterol and dexamethasone has been reported for cytokine release from human mononuclear cells in vitrol12 and against LPS-induced neutrophil accumulation in the lung in viv0.123

VIII. CLINICAL EXPERIENCE WITH SALMETEROL

Although it is not the purpose of this report to review the clinical development programme with salmeterol in detail, a brief analysis of the extent to which the preclinical predictions made for salmeterol have been borne out would be of interest.

The long-acting p,-adrenoceptor agonists were developed specifically for inhaled use as twice daily maintenance therapy in the treatment of reversible airways obstruction. Clinical studies for up to 5 years duration, in many thousands of patients, have confirmed the pharmacological predictions made for salmeterol. It is highly efficacious in bronchial asthma, producing sustained bronchodilatation after aerosol admini~trat ion.~~ The onset of bronchodilatation is slower (by 3-10 min) with salmeterol(50 pg) than with salbutamol (200 pg), although this may be of little clinical importance, as salmeterol is intended for maintenance therapy and not as symptomatic relief. Peak bronchodilatation at 3 h is of similar magnitude to salbutamol, but the duration of action of salmeterol is much longer, FEVl remaining >15% over baseline values for 14 h, compared with 4-6 h for salbutamol.6 Salmeterol, at 50 and 100 pg doses, protected against methacholine or histamine challenge for >12 h.114 Similarly, in a study in which exercise challenge was employed,115 whereas salbutamol was effective at 1 h, but not at 6 and 12h, salmeterol was active for at least 12 h. This was also observed in paediatric studies. Multicenter European dose-ranging studies using 12.5-200 pg, have shown that salmeterol, 50 pg twice daily, is the optimum therapeutic dosage in the treatment of mild to moderate asthma, reducing diurnal variation and leading to a progressive improvement in lung function, with 100 pg twice daily providing further benefit in patients with more severe disease.116 There was no indication of tachyphylaxis to the pulmonary effect of salmeterol following administration for up to 1 year. Similarly, no evidence of rebound deterioration of lung function was observed after withdrawal of salmeterol treatment.

One of the objectives in developing long-acting p-agonists was to be able to control the symptoms of nocturnal asthma. In patients, who were experiencing approximately 80% weekly awakenings due to asthma, both 50 and 100 pg of salmeterol has a significant effect, with the higher dose eliminating the symptoms of nocturnal asthma within 7 days of starting treatment.117

Recent analysis of long-term studies (>12 months) has revealed that asthma exacerba- tions are less in salmeterol-treated patients, and remain low whether or not inhaled corticosteroids were taken concurrently. There is also evidence that salmeterol improves the quality of life in asthmatic patients, compared with their existing therapy.118

Clinical pharmacological studies have also investigated the potential nonbronchodilator properties of salmeterol. The late response to allergen is often taken as reflecting acute inflammatory processes in the lung. Currently available P-adrenoceptor agonists

254 JOHNSON

such as salbutamol and terbutaline, however, do not inhibit the late response, except at doses several times higher than the therapeutic dose, and are not therefore regarded as having clinical significant anti-inflammatory activity. However, unlike salbutamol and terbutaline, in two clinical studies by Twentyman and colleagues119 and Dahl and Pe- dersen,120 salmeterol has been shown not only to inhibit the early, but also the late response to inhaled allergen, which has been associated with inflammatory changes in the airways. The interpretation of such findings as an action of salmeterol on the under- lying acute inflammatory processes has been controversial, and an alternative explanation of functional antagonism by long-lasting bronchodilatation has been proposed.121 Importantly, however, in both studies,l19,*20 salmeterol also inhibited the bronchial hyper-responsiveness which followed allergen challenge, at a time (24-48 h) when there was no evidence of residual bronchodilator activity. Similarly, Gronneberg et al.1" have shown that intradermal salmeterol, administered into the forearm of nonatopic subjects, inhibited the early WFR and had a marked effect against the LCR. This, together with the other reports supports the hypothesis75 that long-acting p2-agonists, like salmeterol, may have the potential for inhibition of acute inflammation. The previous failure to demonstrate clinically useful anti-inflammatory activity may then reflect the pharmacodynamics of the first generation drugs, a problem that salmeterol appears to have overcome. Further clinical work is, however, clearly necessary before the clinical signifi- cance of the nonbronchodilator effects of salmeterol in bronchial asthma can be evalu- ated.

IX. CONCLUSIONS

Although as a class, P-adrenoceptor agonists share a number of pharmacological properties, there are marked differences in receptor affinity, efficacy, selectivity, and duration of action between individual compounds. The optimal pharmacological profile is represented by a drug which is lipophilic, in order to increase occupancy at the P-adrenoceptor, and to promote retention in the lung, leading to decreased systemic absorption and therefore less extrapulmonary side effects. It should be both potent and highly p,-adrenoceptor selective, and ideally, have an inherently long duration of action at P-adrenoceptors, without causing tachyphylaxis.

The objective of developing a drug with therapeutically significant, sustained bronchodilator activity for the treatment of reversible airways obstruction, when administered by the inhaled route, has been achieved in the new generation of long-acting p,-agonists, represented by salmeterol. The working hypothesis (Fig. 17) is that the saligenin head of the molecule, which evokes potency and high P,-selectivity, and the kinetics of the drug at the receptor, which is governed by the side chain and its interaction with the exo-site, combine to give salmeterol a pharmacodynamic profile (Fig. 19, unlike that of any other p-agonist. As a result of potent and prolonged activation of P,-adrenoceptors in airways smooth muscle cells, endothelial cells, mast cells, and epithelial cells, salmeterol produces bronchodilatation for at least 12 h, inhibition of vascular permeability and inflammatory cell recruitment into the lung, suppression of inflammatory mediator release, and the stimulation of cilia1 function and promotion of ion/water transport across the bronchial mucosa. This pharmacological activity arises from the novel mechanism of action of salmeterol, whereby as a direct consequence of the design features of the molecule, it is believed to be localized in a specific domain of the P,-receptor protein, from where it can continuously stimulate the active site of the receptor.

SALMETEROL 255

Receptor Pharmacodynamics

Receptor Pharmacology

Receptor Pharmacokinetics

Figure 17. Salmeterol: Molecular determinants of action

Clinical studies have confirmed that salmeterol is well tolerated and highly efficacious in man, providing bronchodilatation in asthmatic patients, for at least 12 h. Serevent@ was launched in the U.K. in December 1990, and is now available in over 50 countries.

The author gratefully acknowledges the contribution of the following to the work described in this review: J. Bradshaw, H. Finch, X. Lewell, L. H. C. Lunts, R. T. Brittain, D. Jack, R. A. Coleman, I. Kennedy, A. T. Nials, S. Sanjar, I. Skidmore, M. J. Sumner, C. J. Vardey, C. J. Wallis, and C. J. Whelan and to Mrs. B. Gummer for typing the manuscript.

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