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.

Studies on the interaction between formoterol and salmeterol in guinea-pig trachea in vitro.

The actions of and interaction between formoterol and salmeterol were studied on guinea-pig trachea in vitro. Tracheal strip preparations were contracted by 1 mumol/l carbachol giving a near maximal contraction. Salmeterol in concentrations from 0.1 to 3 mumol/l relaxed the tracheal smooth muscle by about 30 per cent of the maximum relaxation produced by theophylline. Formoterol caused a concentration-dependent and almost complete relaxation with a pD2 of 8.56. In the presence of salmeterol there was a rightward shift of the concentration-response curve for formoterol. The pA2 for salmeterol was estimated to 7.42. Similar experiments with isoprenaline indicated that salmeterol has a low affinity for beta 1-adrenoceptors. Formoterol and salmeterol both inhibited in a concentration-dependent manner the contractions evoked by stimulation of the vagus nerve in a tracheal tube preparation. The degree of inhibition decreased with increasing stimulation frequency. Complete inhibition was attained with salmeterol, but not with formoterol, at the highest frequency employed (45 Hz). The inhibiting effect of 10 mumol/l salmeterol was not blocked by 10 mumol/l sotalol, a beta-adrenoceptor antagonist. It is concluded that salmeterol, in comparison to formoterol, is a partial beta 2-adrenoceptor agonist and has, at high concentrations, an additional unspecific inhibitory action.

Hydrogen peroxide-induced epithelial damage increases terbutaline transport in guinea-pig tracheal wall: implications for drug delivery.

An isolated vagus nerve-tracheal tube preparation from guinea-pig was treated intraluminally with hydrogen peroxide (H2O2) at various concentrations. Exposure to, 100 mmol/L H2O2 for 20 min was chosen for further experiments since it appeared to cause selective damage to the epithelium. Thus the subepithelial layers of the tracheal wall appeared intact as judged by light microscopic examination. The response to nerve stimulation (increase in intratracheal pressure) was attenuated by only about 20%. Terbutaline administration into the tracheal lumen caused a concentration-dependent inhibition of the response to nerve stimulation. In tracheal preparations pretreated with 100 mmol/L H2O2 there was a 20-fold decrease in the EC50 for terbutaline. The EC50 for terbutaline added to the external medium was not changed by the H2O2 pretreatment. The efflux of 3H-terbutaline from the tracheal lumen into the external medium was three times higher in H2O2-treated than in control preparations. It is concluded that in the H2O2-damaged epithelium the absorption of terbutaline is enhanced resulting in a better availability of the drug in the smooth muscle layer after intraluminal administration.

Pharmacodynamic and pharmacokinetic aspects on the transport of bronchodilator drugs through the tracheal epithelium of the guinea-pig.

An isolated vagus nerve-trachea tube preparation from guinea-pig was used to study the effect kinetics of bronchodilating beta-adrenoceptor agonists. The test compounds were added either into the fluid-filled lumen or into the external medium and they all inhibited, dose-dependently and completely, the vagally induced contractions. The hydrophilic compounds isoprenaline, salbutamol and terbutaline were much less potent when administered intratracheally as compared with extratracheal administration indicating a slow transport through the epithelial layer. For the lipophilic compound, D2489 (the resorcinol derivative of salmeterol), this difference was less pronounced. When terbutaline was administered as its lipophilic diisobutyrate ester prodrug, ibuterol, the difference between the routes of administration was largely eliminated. The inhibitory effect of terbutaline, but not D2489, was readily reversed by washing. Measurements of terbutaline and D2489 in the tracheal tissue and in the external medium after the intratracheal administration of the compounds support the view that a hydrophilic compound slowly passes the epithelium and is not retained in the tissue, whereas a lipophilic compound rapidly passes the epithelium and is retained by the tissue. The isolated vagus nerve-trachea tube preparation of the guinea-pig is well suited for the concommitant study of pharmacodynamic and pharmacokinetic properties of bronchodilator drugs.

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.

Interaction between bambuterol and physostigmine: aspects on cholinesterase inhibition and neuromuscular transmission in the smooth and skeletal muscles of the guinea-pig.

Bambuterol, the bis-dimethyl carbamate prodrug of terbutaline, and physostigmine were examined with respect to their ability to interfere with the neuromuscular transmission in an isolated vagus nerve-trachea preparation, a phrenic nerve-diaphragm preparation and the transmurally stimulated extensor digotorum longus (EDL) isolated from the guinea-pig. Physostigmine increased the contractile response of the trachea to stimulation of the vagus nerve. Bambuterol had an opposite effect in this respect and inhibited the effect of physostigmine. Both compounds, in high concentrations, increased the tension of the unstimulated tracheal smooth muscle. Physostigmine, but not bambuterol, caused a threefold increase in the twitch tension of the indirectly stimulated diaphragm. Bambuterol counteracted this increase almost completely. In the EDL, physostigmine caused a concentration-dependent and curare-sensitive increase in the force of both twitches and subtetanic contractions. This increase was completely inhibited by bambuterol which had no effect per se on the contractions. Both enantiomers of bambuterol appeared to be equally potent in counteracting the effect of physostigmine on the EDL. It is concluded that bambuterol, in concentrations which selectively and completely block the butyrylcholinesterase, has no effect on the neuromuscular transmission. In higher concentrations, at which bambuterol might interfere with acetylcholinesterase, it counteracts the effects of the unselective inhibitor of cholinesterases, physostigmine.

Partial agonism and functional selectivity: a study on beta-adrenoceptor mediated effects in tracheal, cardiac and skeletal muscle.

Colterol, procaterol, sulfonterol, terbutaline and three monophenolic derivatives of terbutaline were examined with respect to their ability to react in vitro on beta-adrenoceptors in tissues isolated from guinea-pig. The effects measured were a) relaxation of the tracheal smooth muscle (mostly beta 2), b) depression of subtetanic contractions of the soleus muscle (beta 2), and c) increase in the force of the papillary muscle of the left ventricle (beta 1). Antagonistic effects were measured against isoprenaline as an agonist. The compounds studied showed a wide variation in selectivity, potency and intrinsic activity. All agonists showed a pronounced beta 2-selectivity, in general characterized by a higher intrinsic activity at beta 2- than at beta 1-adrenoceptors, while differences in affinity, as judged from the pA2-values were small. Partial agonists, such as sulfonterol, which did not cause a complete relaxation of a moderately contracted tracheal muscle, produced identical concentration-response curves from the trachea and soleus muscle. It is concluded that partial agonism at beta 1-adrenoceptors is an important factor for functional selectivity of beta 2-adrenoceptor agonists. On the other hand there seems to be no useful differences between the maximum effect elicited by a partial beta 2-adrenoceptor agonist on the skeletal muscle as compared with airway smooth muscle.

Further studies on the cardiomegaly induced by beta-adrenoceptor agonists.

Rats received continuous infusions of beta-adrenoceptor agonists by means of Alzet osmotic minipumps implanted subcutaneously. After 7 days of isoprenaline infusion (2 mg/kg per day) the heart/body weight ratio increased about 40 per cent compared with placebo treatment. The difference persisted after freeze-drying indicating a true hypertrophy and not merely an oedema. When terbutaline (20 mg/kg per day) was substituted for isoprenaline, the increase in wet weight ratio reached only about 5 per cent. Procaterol (2 mg/kg per day) and pirbuterol (20 mg/kg per day) had no effect on the heart weight. It is concluded that in doses expected to cause comparable stimulation of beta 2-adrenoceptors the unselective agonist isoprenaline is able, more than the beta 2-selective agonists terbutaline, procaterol and pirbuterol to cause cardiac hypertrophy thus indicating the involvement of beta 1-adrenoceptors.

Steric aspects of agonism and antagonism at beta-adrenoceptors: experiments with the enantiomers of terbutaline and pindolol.

The enantiomers of terbutaline, a beta 2-selective adrenoceptor agonist, and pindolol, an unselective antagonist with partial agonist activity, were examined with respect to their ability to react in vitro on adrenoceptors in the trachea (mostly beta 2), the soleus muscle (beta 2) and in the papillary muscle of the left ventricle (beta 1) from the guinea-pig (+)-terbutaline was more than 3,000 times less potent than (-)-terbutaline in relaxing the trachea and in depressing subtetanic contractions of the soleus muscle. (+)-terbutaline did not inhibit the effects of (-)-terbutaline in these tissues. The effect of (-)-terbutaline on the papillary muscle was about 200 times weaker than on the soleus. (+)-terbutaline had a negligible inotropic effect on the papillary muscle and it did not inhibit the effect of isoprenaline. The enantiomers of pindolol did not show any consistent agonistic activity under the present experimental conditions. (-)-pindolol inhibited competitively the effect of isoprenaline to the same extent in all three tissues. (+)-pindolol was about 200 times less potent in this respect. Our data do not reveal any qualitative differences in the pharmacological properties between the optical isomers of terbutaline and pindolol, respectively.

Dissociation between the effects of some xanthine derivatives on the tracheal smooth muscle and on the skeletal muscle.

The ability of some xanthine derivatives to relax the trachea, contracted by pilocarpine, and to increase the force of contraction of directly stimulated skeletal muscles from the guinea-pig was studied in vitro. No relationship was found between these two effects. Relaxation of the trachea occurred at lower concentrations and with a different order of potency as compared with the effects on the slow-contracting soleus muscle or on the fast-contracting extensor digitorum longus. One of the compounds, IBMX, 1-methyl-3-isobutyl-xanthine, showed an isoprenaline-like effect on the soleus muscle i.e. it depressed the force and fusion of subtetanic contractions. The relaxing effect of theophylline and IBMX on the trachea was additive to that of terbutaline but no clear potentiation was observed. The depression of the contraction of the soleus muscle elicited by terbutaline was reinforced by IBMX but not by theophylline. Theophylline in concentrations which used alone enhanced the contractions of the soleus muscle inhibited the effect of terbutaline. We conclude that the relative contribution of the various effects of xanthine derivatives differs from compound to compound.

Selective development of tolerance to beta-adrenoceptor agonists in skeletal muscle as compared with airway smooth muscle from the guinea-pig.

1. Guinea-pig were fed with a diet containing terbutaline or placebo for 4--5 days. The trachea, soleus muscle and the extensor digitorum longus (EDL) from these animals were prepared for recording of isometric contractions in vitro. 2. After treatment with terbutaline in vivo, the response of the pilocarpine-contracted trachea to terbutaline and isoprenaline was slightly suppressed with no change in maximum relaxation. 3. After treatment with terbutaline in vivo the maximum depression of the incomplete tetanic contractions of the soleus muscle brought about by terbutaline or isoprenaline was diminished by about 70%. The response of the EDL was also attenuated after previous treatment with terbutaline in vivo. 4. These data indicate a selective development of tolerance to the effects of beta-adrenoceptor agonists in skeletal muscle as compared with tracheal smooth muscle. 5. The present results provide an experimental analogue to the clinical observation that patients being treated with beta-adrenoceptor agonists become tolerant to the tremorogenic rather than to the bronchodilating effect.

The adrenoceptor blocking properties of the new beta 2-selective antagonist, IPS 339 on tracheal smooth muscle and on slow contracting skeletal muscle.

The beta-adrenoceptor blocking properties of IPS 339 and propranolol were studied on isolated preparations of trachea and of the slow-contracting soleus muscle from the guinea-pig. Both compounds antagonized the relaxation of the trachea and the depression of subtetanic contractions of the soleus produced by the beta2-selective agonist, terbutaline. On the soleus muscle the pA2-values for IPS 339 and propranolol were similar and close to those obtained for propranolol on the trachea. However, on the trachea the slope of the Schildplot for IPS 339 against terbutaline became less than one and a reliable pA2-value could not be calculated.