Bertil Waldeck

On the predictive value of experiments in vitro in the evaluation of the effect duration of bronchodilator drugs for local administration.

Six bronchodilating beta-adrenoceptor agonists, clinically documented with respect to the duration of action after inhalation, were included in this study in vitro on the guinea-pig trachea. Relaxation of carbachol contracted trachea strip preparations and inhibition of contraction of a vagus nerve-tracheal tube preparation were measured. The relaxing effects of salbutamol and fenoterol (both with relatively short duration in man) were rapid in onset and easily reversed by washing in a drug-free medium. The relaxation by salmeterol (long duration) and D2343 (intermediate duration) developed more slowly, resisted washing but was reversed by propranolol. Formoterol (long duration) and salmefamol (intermediate duration) showed properties between these two extremes. All test compounds inhibited the vagally-induced contractions of tracheal concentration dependently. The EC50 values for the hydrophilic compounds salbutamol and fenoterol were higher with intra- as compared with extratracheal administration. For the more lipophilic compounds formoterol, salmefamol, salmeterol and D2343, this difference was less pronounced. A high lipophilicity and a retention by the tissue in vitro of a beta-adrenoceptor agonist may be factors contributing to a long effect duration after inhalation but a further selection has to be made in vivo since metabolic and circulatory effects may influence the effect kinetics.

The interaction between salmeterol and β2-adrenoceptor agonists with higher efficacy on guinea-pig trachea and human bronchus in vitro

1 In guinea‐pig tracheal preparations precontracted with 1 μmol 1−1 carbachol, formoterol, procaterol, fenoterol, salmefamol, salbutamol and terbutaline (in that order of potency) caused a concentration‐dependent and almost complete, relaxation. However, under these conditions, the maximum relaxation by salmeterol was approximately 30% of the maximum attainable relaxation. 2 We have therefore explored the ability of salmeterol to inhibit the relaxant response to β2‐adrenoceptor agonists of different chemical structure and relatively higher efficacy in smooth muscle preparations from guinea‐pig trachea and human bronchus. 3 With 1 μmol 1−1 salmeterol in the organ bath, the concentration‐effect curves for the other agonists were shifted to the right in a variable way by 1.8‐2.8 log units, fenoterol and salbutamol being the extremes. 4 When 20 μmol 1−1 sulfonterol, another low efficacy β2‐adrenoceptor agonist, was substituted for salmeterol, the difference in the magnitude of the rightward shift between fenoterol and salbutamol was eliminated. 5 In the human bronchus, formoterol and terbutaline had a higher apparent efficacy than salmeterol. With 1 μmol 1−1 salmeterol in the organ bath, the concentration‐effect curve for formoterol was shifted 2.7 log units to the right. 6 Salmeterol inhibits, competitively, relaxant responses to β2‐adrenoceptor agonists with higher efficacy. The degree of inhibition seems to be dependent on the agonist used. This contrasts with results obtained with sulfonterol and suggests that salmeterol interacts with the β2‐adrenoceptor in a complex way.

Enantiomers of bronchodilating β2-adrenoceptor agonists : Is there a cause for concern?

All bronchodilating beta2-adrenoceptor agonists in current clinical use are derivatives of adrenaline and are available as racemates. Whereas the vast majority of the pharmacologic and clinical documentation has been made with the racemates, there are a few studies with the individual enantiomers. It thus appears that all established pharmacologic effects of racemic beta2-agonists reside in the (R)-enantiomer, with the (S)-enantiomer being virtually inactive. In recent years the suspicion has been raised that the (S)-enantiomer of the beta2-agonists is responsible for induction of airway hyperreactivity. This suspicion is based primarily on results obtained in guinea pigs exposed to (S)-enantiomers of beta2-agonists. A number of experiments in vitro have been undertaken to find a mechanism of action for these observations in vivo. Most of the results obtained are equivocal. However, the observation that (S)-salbutamol may cause mobilization of intracellular Ca2+, apparently by means of a cholinergic mechanism, deserves further investigation. The clinical studies are focused on the enantiomers of salbutamol. They confirm the preclinical findings that the pulmonary, as well as the extrapulmonary, effects of salbutamol reside in the (R)-enantiomer. The studies available so far do not convincingly show clinically significant airway hyperreactivity after exposure to the (S)-enantiomer. Further studies are needed to settle this issue.

Some pharmacodynamic aspects on long-acting β-adrenoceptor agonists

1. 1. Formoterol and salmeterol are the first members of a new generation of long-acting β2-adrenoceptor agonists for inhalation. The discovery of the long effect duration of formoterol was made by chance, while the development of salmeterol appeared to follow a purposeful research strategy. 2. 2. Preclinical evaluation predictive of the clinical duration of effect of long-acting bronchodilators is not straightforward. Experiments in vitro may give false positive results, while experiments in vivo may show false negative results. 3. 3. Once the principle of a long duration of effect was established, a number of novel long-acting β2-adrenoceptor agonists of various chemical structure have emerged. 4. 4. There are two alternative models for the explanation of the long duration of effect: the exosite binding explaining the mode of action of salmeterol, and the more general diffusion microkinetic model applicable for both formoterol and salmeterol. 5. 5. Long-acting β-adrenoceptor agonists with a relatively low efficacy like salmeterol may, under certain circumstances, inhibit competitively the relaxing effect of agonists with higher efficacy like formoterol and salbutamol. 6. 6. Like all other β2-adrenoceptor agonists in current clinical use, formoterol and salmeterol comprise racemic mixtures. Only the RR- and R-enantiomers are pharmacologically active. The experimental compounds TA-2005 and picumeterol have been developed as pure RR- and R-enantiomers, respectively.

Neuropeptide Y: prejunctional inhibition of vagally induced contractions in the guinea pig trachea

The effects of neuropeptide Y (NPY) on the contractile response to vagus nerve stimulation at different frequencies was studied in an isolated tracheal tube preparation from guinea pig. NPY had no effect on basal smooth muscle tension or on the contractile effect of carbachol, but inhibited vagally induced contractions in a concentration-dependent manner with a greater inhibition at low frequencies than at high. We suggest that the effect is exerted prejunctionally.

Formoterol and salmeterol are both long acting compared to terbutaline in the isolated perfused and ventilated guinea-pig lung

An isolated, perfused and ventilated guinea-pig lung was used to compare the duration of effect of the bronchodilating beta 2-adrenoceptor agonists formoterol, salmeterol and terbutaline. Lung conductance was measured real time with the aid of a computer. Bronchoconstriction was induced in the preparation every 10 min by bolus injections of acetylcholine into the pulmonary artery. Lung conductance was reduced by about 70% after acetylcholine. The test compounds or the vehicle was administered for 1 min as aerosols generated from solutions: formoterol (10 mumol/l), salmeterol (100 mumol/l) and terbutaline (1000 mumol/l). This treatment inhibited the response to acetylcholine by 50-60% within the first 10 min for all three test compounds. The onset of action appeared to be slower for salmeterol than for formoterol and terbutaline. The inhibitory effect of terbutaline disappeared completely during the next 20 min of continuous perfusion (single pass), while both formoterol and salmeterol displayed a significant inhibitory effect 40 min after their administration. Formoterol, when inhaled at a higher dose (100 mumol/l), caused a 90% inhibition of the response to acetylcholine. This effect was completely reversed by 0.1 mumol/l propranolol in the perfusion medium. There were in general no major changes in the basal conductance measured between the acetylcholine provocations.

Steric aspects of formoterol and terbutaline: Is there an adverse effect of the distomer on airway smooth muscle function?

Experiments were made on isolated tissues from guinea-pig to test the hypothesis that the distomers of rac-beta 2-adrenoceptor agonists induce airway hyperreactivity. Tracheal strip preparations were contracted with carbachol. Both rac- and (R;R)-formoterol (2 and 1 mumol/l, respectively) produced an immediate relaxation, followed by a slow recovery of tone. (S;S)-Formoterol (2 mumol/l) had no effect on smooth muscle tone. Similar results were obtained with the enantiomers of terbutaline. In other strip preparations of the trachea or the main bronchi, cholinergic or nonadrenergic/noncholinergic (NANC) excitatory responses were evoked by electrical field-stimulation. The eutomers, (R;R)-formoterol and (R)-terbutaline, inhibited concentration-dependently both cholinergic and NANC-induced contractions. The distomers, (S;S)-formoterol and (S)-terbutaline, showed qualitatively the same effects but were about 1,000 times less potent than the corresponding eutomer. In a third series of experiments, either enantiomer of formoterol was administered to an electrically stimulated vagus nerve-trachea tube preparation. The nerve-induced contractions were inhibited by both enantiomers, but (S;S)-formoterol was about 1,000 times less potent than (R;R)-formoterol. For both enantiomers of formoterol, about tenfold higher concentrations was required to obtain the same degree of inhibition when given intratracheally as compared with administration in the external medium. There was no indication in any of the experimental approaches that (S;S)-formoterol or (S)-terbutaline might enhance the response to cholinergic or NANC-related stimuli.

Comparison of the effects of forskolin and isoprenaline on tracheal, cardiac and skeletal muscles from guinea-pig

Forskolin, a potent activator of adenylate cyclase, and isoprenaline, an unselective beta-adrenoceptor agonist, were studied in vitro on tissues from guinea-pig with respect to relaxation of the carbachol-contracted trachea, increase in the force of contraction of the papillary muscle and depression of subtetanic contractions of the soleus muscle, three well-characterized beta-adrenoceptor-mediated effects. Forskolin and isoprenaline relaxed the trachea and increased the force of contraction of the papillary muscle. Isoprenaline but not forskolin caused a depression of the subtetanic contraction of the soleus muscle. Forskolin did not seem to potentiate the effects of isoprenaline on the tissues studied; the combined effects appeared to be a mere addition. Forskolin did not increase the efficacy of the partial agonist prenalterol either. It is concluded that there is no simple relation between c-AMP generation and the functional response to beta-adrenoceptor agonists. Forskolin should not be used uncritically to probe beta-adrenoceptor-mediated effects.

THE INTERACTION OF EPHEDRINE WITH β‐ADRENOCEPTORS IN TRACHEAL, CARDIAC AND SKELETAL MUSCLES

1. Three β‐adrenoceptor mediated effects were measured in vitro on tissues from guinea‐pig: (a) relaxation of the trachea (mainly β2), (b) increase in the force of contraction of the papillary muscle (β1), and (c) depression of the subtetanic contractions of the soleus muscle (β2).

On the interaction of threo-3H-α-methylnoradrenaline with the uptake and storage mechanism of the adrenergic neuron

Abstract Threo-3H-α-methylnoradrenaline (3H-α-Me-NA) was given i.v. to mice. The amine was efficiently taken up by the heart. Blockade of the granular storage-mechanism by reserpine did not inhibit the uptake of the amine but increased the rate of disappearance. Pretreatment with protriptyline, which blocks the amine transport mechanism at the level of the cell membrane of the adrenergic neuron, blocked the uptake of threo-3H-α-Me-NA completely. In comparison with erythro-3H-α-Me-NA the threo isomer disappeared more rapidly from the heart. Reserpine, but not protriptyline, proved efficient in releasing threo-3H-α-Me-NA taken up by the heart. The principal metabolite of threo-3H-α-Me-NA appeared to be threo-3H-α-methylnormetanephrine. It is concluded that the two diastereoisomers of 3H-α-Me-NA, although different in receptor affinity, have almost the same relation to the amine uptake- and storage- mechanisms of the adrenergic neuron.