Evaluation of the effects of the R- and S-enantiomers of salbutamol on equine isolated bronchi

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Abstract

Background

Equine obstructive pulmonary disease, also known as heaves or recurrent airway obstruction (RAO) is a common equine pulmonary disease with some similarities to human asthma and COPD, which represents a major cause of morbidity and loss of lung performance. Salbutamol has been widely used for the treatment of human airway diseases and has usually been prepared as the racemic form of the drug. However, recently the R-enantiomer of salbutamol has been introduced into clinical practice in the treatment of asthma in humans and this has been suggested to be an improvement on the racemic form of the drug; therefore thus the S-enantiomer has been demonstrated to have adverse effects in the lung and thus using the R-enantiomer may improve the therapeutic ratio. However, little is known about the properties of the R- and S-enantiomers of salbutamol in equine airways and the present study has evaluated the relaxant effects of racemic β2-agonists in comparison with the R- and S-enantiomers in isolated equine isolated bronchi, as well as the bronchoprotective effects of these drugs on cholinergic and histaminergic pathway.

Methods

We have studied the effects of the R- and S-enantiomers of salbutamol on bronchi isolated from RAO-affected or unaffected horses. The first study assayed the relaxant effects of R- and S-salbutamol on isolated bronchial rings contracted with carbachol or histamine at a sub-maximal concentration (EC70). A second study evaluated the effects of R- and S-salbutamol on semi-logarithmic cumulative concentration-response curves induced by carbachol or histamine. Specific software was used to calculate statistical significance and the appropriate sigmoidal curve-fitting model.

Results

Neither enantiomers of salbutamol caused a relaxant effect on the sub-maximal plateau contractile effects of carbachol; in fact, both R- and S-salbutamol induced a slight, but significant contraction (P ≤ 0.05) compared to the controls. In contrast, R-salbutamol induced a significant relaxation of bronchi pre-contracted with histamine (RAO-unaffected: 92.06% ± 2.00; RAO-affected 100.20 ± 3.99; P ≤ 0.01). S-salbutamol induced a weak relaxation (RAO-unaffected: 15.81% ± 5.65; RAO-affected 12.36 ± 5.15) when compared to that induced by papaverine. The incubation with either R- or S-salbutamol shifted rightward (P ≤ 0.001) the carbachol contraction curve in RAO-unaffected bronchi, but not in RAO-affected bronchi, compared to control tissues. R-salbutamol induced a reduction in Emax values (C: 9.07gr ± 0.68; R-salb.: 6.36gr ± 0.21; P ≤ 0.01) in normal bronchi. On the contrary it reduced the histamine potency in RAO-affected bronchi (EC50 7.10 μM ± 0.35, P < 0.001). The incubation with S-salbutamol shifted leftward the histamine concentration curve in both normal bronchi (C: 7.00 μM ± 0.29; S-salb.: 2.25 μM ± 0.19; P ≤ 0.001) and bronchi from RAO-affected horses (C: 2.80 μM ± 0.26; S-salb.: 1.50 μM ± 0.80; P ≤ 0.05).

Conclusion

Our studies have demonstrated that S-salbutamol elicited a modest increase in contraction of equine airway smooth muscle induced by carbachol and induced a significant hyperresponsiveness to histamine. These results confirm the ability of the S-enantiomer of salbutamol to potentiate the contractile effect of certain spasmogens on airway smooth muscle. Such an adverse effect would be determined in the airways of horses with RAO and suggest that if salbutamol is to be used in the treatment of symptoms of RAO in horses, the R-enantiomer, rather than the racemic mixture should be considered.

Introduction

There is an increasing amount of evidence demonstrating an association of inhaled β2-agonists with a rise in asthma-related deaths and life-threatening experiences, as well as an ability of β2-agonists to induce bronchial hyperresponsiveness when used clinically, particularly when administered as monotherapy [1]. The mechanisms contributing to the adverse effects in the airways are not known, but it has been suggested that some of these undesirable effects may be due to the S-enantiomer as most commonly available β2-agonists are produced as racemic mixtures of R- and S-enantiomers in equal ratios [2]. The S-enantiomer of salbutamol has been considered to be largely inert or at best to have weak β2-agonist activities. However, recent studies have documented that S-salbutamol can potentiate the effects of spasmogens in airway smooth muscle from both guinea pigs and humans, with a number of clinical studies also reporting worsening of airways hyperresponsiveness in animals [3] and in subjects with asthma [4]. It has also been documented that the metabolic clearance of S-salbutamol occurs much less rapidly than R-salbutamol [5], and thus the paradoxical responses may persist after the beneficial effects of (R)-salbutamol have waned [2], particularly after chronic use of the drug. S-salbutamol has also been reported to have pro-inflammatory effects that could negatively affect the airways [6].

Chemically, all β-agonists are racemates, or drugs composed of two non-superimposable mirror image molecules in a 50/50 ratio [7]. These molecules are classified as R- or S- according to their molecular configuration, and as ‘lev’ or ‘dex’ based on the rotation of polarized light as it passes through the molecule.

Equine obstructive pulmonary disease, also known as heaves or recurrent airway obstruction (RAO) is a common equine pulmonary disease with certain similarities to human asthma and COPD and represents a major cause of morbidity and loss of lung performance in this species [8], [9].

Whilst β2-agonists are used in the treatment of RAO, little is known about the properties of the enantiomers of salbutamol in the horse. The present study has therefore evaluated effects of racemic β2-agonists in isolated equine bronchi in comparison with the pure R- and S-enantiomers.

Section snippets

Tissue preparations

Equine lungs, derived from healthy horses following humane killing in an abattoir, were obtained from six RAO-unaffected horses (3 male and 3 female; aged 2.3 ± 0.1 years; weighted kg 375 ± 54.3) and four RAO-affected horses (2 male and 1 female; aged 2.5 ± 0.2 years; weighted kg 388 ± 46.5). Lungs were rapidly transported to our laboratory in a Krebs–Henseleit solution (KH) (composition, mM: NaCl 117.5. KCl 5.60. MgSO4 1.18. CaCl2 2.50. NaH2P04 1.28. NaHCO3 25.00 and glucose 5.5, pregassed

Protocol 1: relaxant effect of salbutamol enantiomers on sub-maximal contractions induced by carbachol or histamine

The baseline tension of bronchial tissues did not change throughout the experiments carried out with S-Salbutamol or the racemic mixture.

In the control bronchi, papaverine completely relaxed equine bronchial rings contracted with a sub-maximal concentration of carbachol (CCh: +4.37gr ± 0.42; P: −4.64gr ± 0.45) or histamine (His: +6.59gr ± 0.98; P: −7.93gr ± 0.85). In contrast, salbutamol enantiomers did not show direct relaxant effects on the sub-maximal plateau contractile response induced by

Discussion

Inhaled β2-agonists remain the first line treatment of acute symptoms of airway obstructive diseases in horses. Most commonly available β2-agonist preparations are racemic, i.e. they are composed of an equimolar mixture of R- and S-stereoisomers. Traditionally the S-enantiomers were considered inert or at best weak β2-agonists [10], but it is now recognized that the S-enantiomer can have adverse effect in the airways [2]. Thus, it has been documented in in vitro and in vivo experimental studies

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