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Dept of Molecular Pharmacology, University Centre for Pharmacy, Groningen, The Netherlands
CORRESPONDENCE: J. Zaagsma, Dept of Molecular Pharmacology, University Centre for Pharmacy, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands. Fax: 31 503636908. E-mail: j.zaagsma@rug.nl
Keywords: Airway hyperreactivity, allergic asthma, enantiomers, inflammation, salbutamol
Received: September 10, 2003
Accepted December 27, 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study evaluated the bronchoprotective effects of inhalation of therapeutically relevant doses of the racemate and individual enantiomers in guinea pigs.
It was found that basal airway reactivity to histamine was similarly reduced 30 min after inhalation of equivalent doses of RS- and R-salbutamol; this protective effect disappeared within 3 h. Inhalation of RS- and R-salbutamol 30 min before and 5.5 h after allergen challenge suppressed allergen-induced airway hyperreactivity to histamine after the early and late asthmatic reaction, completely inhibiting the early asthmatic reaction and tending to reduce the development of the late asthmatic reaction. At 5 h after allergen challenge, the inhibition of airway hyperreactivity was more pronounced in animals treated with R-salbutamol compared to racemate-treated animals. Both basal airway reactivity and allergen–induced hyperreactivity were not affected by S-salbutamol. Inflammatory cell infiltration was not affected by the racemate or the individual enantiomers.
In conclusion, inhalation of therapeutically relevant doses of R- and RS-salbutamol effectively suppress allergen-induced airway reactivity after the early and late asthmatic reactions, the R-enantiomer being slightly more potent with respect to early airway reactivity than the racemate. No adverse effects were observed for the S-enantiomer.
Although ß2-agonists, including salbutamol, are the most effective bronchodilators available for the treatment of asthma, long-term use of these drugs has been associated with adverse effects, including the development of airway hyperreactivity (AHR) and tolerance to ß2-agonist-induced bronchoprotection against allergic and pharmacological stimuli 1–4. These adverse effects are thought to be caused by desensitisation of ß2-adrenoceptors on airway smooth muscle as well as on inflammatory cells 5–7.
Salbutamol is a mixture of R- and S-salbutamol. Potent agonist-induced bronchodilator properties have been ascribed to R-salbutamol, whereas the S-enantiomer was, for a long time, thought to be pharmacologically inactive 8. However, recent studies have suggested that S-salbutamol may exhibit the potential to exert adverse effects, counteracting the bronchoprotective properties of the R-enantiomer. Thus, in mild asthmatics, a single inhalation of S-salbutamol was found to increase airway responsiveness to methacholine 3 h after administration 9, although this was not invariably found by others 10, 11. In vitro, S-salbutamol was shown to increase the contractile response to carbachol in guinea pig tracheal smooth muscle preparations 12, intracellular calcium levels in bovine airway smooth muscle cells 13, and histamine and interleukin-4 production in immunoglobulin (Ig)E-stimulated murine mast cells 14. If S-salbutamol has biological activities, counteracting the bronchodilator properties of the R-enantiomer, enantiomerically pure formulations might be recommended in asthma therapy.
To date, the effects of the individual salbutamol enantiomers on allergen-induced early (EAR) and late (LAR) asthmatic reactions, AHR and airway inflammation have not been established. Therefore, in this study, a guinea pig model of allergic asthma was used to compare the protective effects of inhaled racemic salbutamol and the equivalent doses of the R- and S-enantiomers on the magnitudes of the EAR and LAR, AHR to histamine after both the EAR and LAR, and airway inflammation after the LAR. In addition, the acute protective effects against histamine-induced bronchoconstriction were assessed in both normoreactive and hyperreactive airways, after both the EAR and the LAR.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Measurement of airway function
Airway function was assessed by on-line measurement of pleural pressure (Ppl) under unrestrained conditions as described previously 15. In brief, a small fluid-filled latex balloon connected to a saline-filled cannula was surgically implanted inside the thoracic cavity. The free end of the cannula was driven subcutaneously to and permanently attached in the neck of the animal. The pleural balloon was connected to a pressure transducer (Ohmeda DTXTM; Spectramed, Bilthoven, The Netherlands) via an external fluid-filled cannula. Ppl was measured continuously using an on-line computer system. Using a combination of flow measurement with a pneumotachograph, implanted inside the trachea, and pressure measurement with the pleural balloon, it has previously been shown that changes in Ppl are linearly correlated with changes in airway resistance 15. In this way, airway function can be monitored repeatedly and continuously for prolonged periods of time, with the animals unaware of the measurements being taken. However, since guinea pigs are nose breathers, changes in Ppl may possibly also reflect upper airway resistance. In a carefully designed study, Finney and Forsberg 16 quantified the involvement of the nose, using a technique that allowed the separate provocation and measurement of respiratory function in the nasal and lower airways of anaesthetised guinea pigs. It was found that aerosols of histamine, carbachol and ovalbumin delivered to the nose of sensitised animals did not affect nasal conductance, even at doses 100-times higher than that required to reduce pulmonary conductance. Thus, nasal resistance is unaffected by the aerosol drugs studied. The nasal contribution to the changes in Ppl was, therefore, considered negligible.
Provocation procedures
Ovalbumin and histamine provocations were performed by inhalation of aerosolised solutions produced by a DeVilbiss nebuliser (DeVilbiss 646; DeVilbiss, Somerset, PA, USA), driven by an airflow of 8 L·min–1, resulting in an output of 0.33 mL·min–1. The provocations were performed in a specially designed perspex cage with a volume of 9 L, in which the animals could move freely 15. The animals were habituated to the experimental conditions on 2 sequential days 
1 week after the surgery, when preoperative weight was restored. On the 1st day, the animals were placed into the provocation cage unconnected to the pressure transducer. After an adaptation period of 30 min, three consecutive provocations with saline were performed, each provocation lasting 3 min, and separated by 7-min intervals. The next day, this procedure was repeated with the animals attached to the measurement system.
On the experimental days following the habituational procedure, allergen and histamine provocations were performed as described below. All provocations were preceded by an adaptation period of 30 min, followed by two consecutive provocations with saline as described above. Baseline Ppl was calculated by averaging the Ppl of the last 20 min of the adaptation period. In order to assess airway reactivity to histamine, histamine provocations were performed with an initial concentration of 25 µg·mL–1 in saline, followed by increasing dosage steps of 25 µg·mL–1. Each provocation lasted 3 min and provocations were separated by 7-min intervals. Animals were challenged until Ppl was increased by >100% for 3 min consecutively. Ppl returned to baseline within 15 min after the last histamine provocation. Airway reactivity to histamine was expressed as the provocative concentration of histamine causing a 100% increase in Ppl (PC100), which was calculated by linear intrapolation of the concentration–Ppl response curve.
Allergen provocations were performed by increasing aerosol concentrations of 0.1, 0.3 and 0.5 mg·mL–1 ovalbumin in saline for 3 min, each separated by 7-min intervals. Allergen inhalations were discontinued when an increase in Ppl of >100% was observed. Using these conditions, none of the animals developed anaphylactic shock after allergen provocation.
Bronchoalveolar lavage
At 25 h after the ovalbumin provocation, bronchoalveaolar lavage (BAL) was performed in all animals as described previously 17. In short, the animals were anaesthetised with 20 mg·kg body weight–1 Brietal® (methohexital sodium; Eli Lilly, Nieuwegein, The Netherlands), 35 mg·kg body weight–1 Ketalar® (ketamine hydrochloride; Parke-Davis, Hoofddorp, The Netherlands) and 6 mg·kg body weight–1 Rompun® (2-(2,6-xylidino)-5,6-dihydro-4H-1,3-thiazine-hydrochloride, methylparaben; Bayer, Leverkusen, Germany), administered intraperitoneally. The trachea was exposed and cannulated, and the lungs were lavaged gently using 5 mL of sterile saline at 37°C, followed by three subsequent 8-mL aliquots of saline. The recovered lavage samples were cooled on ice and centrifuged at 200xg for 10 min at 4°C. The pellets were combined and resuspended in phosphate-buffered saline at a final volume of 1 mL and total cell numbers were counted using a Coulter counter. For cytological examination, cytospin preparations were stained with May–Grünwald and Giemsa stains (Sigma). Cell differentials were determined by counting 400 cells in duplicate.
Experimental protocols
Effects of RS-, R- and S-salbutamol on basal airway reactivity
In the first group of animals, the effect and duration of action of inhaled racemic salbutamol, R-salbutamol and S-salbutamol (gifts from Sepracor, Inc., Marlborough, MA, USA) on basal airway reactivity were assessed. At 30 min after the assessment of basal PC100, aerosols of salbutamol enantiomers (1.25 mM for 3 min) or the equivalent concentration of racemate (2.5 mM for 3 min) were inhaled, and PC100 was measured again after 30 min and 1.5, 3, 6, 12 and 24 h. The nebuliser concentrations of the drugs were selected on the basis of preliminary experiments with RS-salbutamol in order to provide a clinically relevant three-to-four-fold shift in histamine reactivity.
Bronchoprotective effects of RS-, R- and S-salbutamol
In the second series of animals, the effects of racemic salbutamol and the individual enantiomers on allergen-induced EAR and LAR, AHR after the EAR and LAR, and airway inflammation were determined. In the 1st week, all guinea pigs inhaled vehicle (saline) for 3 min, 30 min before and 5.5 h (between the EAR and LAR) and 23.5 h (after the LAR) after ovalbumin provocation.
In the 2nd week, the animals were subdivided into four groups, inhaling either saline (control group), or RS-salbutamol (2.5 mM), R-salbutamol (1.25 mM) or S-salbutamol (1.25 mM) for 3 min at the time points indicated above. In each animal, allergen provocations performed in weeks 1 and 2 were identical with respect to the ovalbumin dose used.
Basal PC100 was determined 24 h before each allergen provocation, and 30 min later either saline or (in the 2nd week) RS-, R- or S-salbutamol were inhaled, and a second PC100 determined after another 30 min in order to determine the effects on basal airway reactivity. On the next day, PC100 were assessed again 5 h (after the EAR) and 23 h (after the LAR) after ovalbumin provocation in all animals in order to determine allergen-induced AHR at these time points. In order to investigate acute bronchoprotective effects after the EAR and LAR, respectively, aerosols of saline or the drug were again inhaled 5.5 and 23.5 h, and PC100 were redetermined 6 and 24 h, after ovalbumin provocation, respectively.
A schematic illustration of the protocol is given in figure 1
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Data analysis
The magnitudes of the allergen-induced EAR and LAR were expressed as the area under the Ppl time–response curve (AUC) between 0 and 5 h after allergen provocation for the EAR, and between 8 and 23 h after provocation for the LAR. Ppl was expressed as percentage change from baseline, and the AUC was calculated by trapezoid integration over discrete (5 min) time periods. Based on saline control provocations, threshold values of AUC (mean+2SD (95% confidence interval)) were defined as 1,185%·5 min–1 for a positive EAR and 2,790%·5 min–1 for a positive LAR, respectively 17. Using these criteria, animals were characterised as single early responders and dual responders (i.e. animals expressing both an EAR and a LAR). Inherent to the research question, only dual-responding animals (72% of the animals) were included in the present study.
Changes in airway reactivity towards histamine after allergen or saline challenge were expressed as the pre-/post-treatment PC100 ratio. PC100 before and after inhalation of allergen, salbutamol or saline, as well as the changes in EAR and LAR, were compared using the Wilcoxon signed-rank test.
Statistical analysis of the changes in basal airway reactivity at several time points after salbutamol inhalation was performed using the Kruskal–Wallis ANOVA on ranks. When significance was observed (p<0.05), a complementary Newman–Keuls test was performed. For comparison between groups, an unpaired Mann–Whitney rank-sum test was used.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and b). Both protective effects disappeared within 3 h, the effect of R-salbutamol, but not that of the racemate, still being significant 1.5 h after inhalation (p<0.05). In contrast to R- and RS-salbutamol, the S-enantiomer exhibited no effect on PC100 (fig. 2c). Figure 2 also shows that, during the repeated histamine challenges, no tachyphylaxis developed.
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. In the saline-treated group of animals, the magnitudes of the EAR and LAR were similar in weeks 1 and 2. The magnitudes of the EAR in animals that inhaled RS-salbutamol or R-salbutamol 30 min prior to ovalbumin were 1,781.7±566.3%·5 min–1 and 1,225.5±523.7%·5 min–1, respectively, which do not differ significantly from the threshold value for a positive EAR (1,185%·5 min–1), indicating that the EAR is suppressed (fig. 3a). This suppression of the EAR in week 2 by RS- and R-salbutamol is significant when compared to the control EAR (saline-treated) in week 1 (p<0.01 and p<0.05, respectively). In contrast, the S-enantiomer did not affect the magnitude of the EAR.
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). Both RS- and R-salbutamol, but not S-salbutamol, strongly suppressed the peak increase in Ppl during the EAR (both p<0.01; fig. 4).
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Effects of R-, S- and RS-salbutamol on allergen-induced airway hyperreactivity
In week 1, all groups of animals received saline and developed comparable AHR to histamine, at both 5 h (after the EAR) and 23 h (after the LAR) after allergen challenge (fig. 5). In week 2, inhalation of RS- and R- salbutamol, given 30 min before and 5.5 h after ovalbumin challenge, caused significant inhibition of allergen-induced AHR at 5 h (fig. 6a) and 23 h (fig. 6b) after the challenge compared to the saline control inhalation (p<0.05 and p<0.01, respectively). Inhibition of AHR was more pronounced in animals treated with R-salbutamol compared to racemate-treated animals (from 3.51±0.82-fold in controls to 1.29±0.11-fold in RS- versus 0.81±0.03-fold in R-salbutamol-treated animals; p<0.01) 5 h after challenge. Both 5 and 23 h after allergen challenge, S-salbutamol exhibited no significant effect on allergen-induced AHR (fig. 6).
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shows the acute effects of RS-, R- and S-salbutamol or saline on PC100 24 h before and 5 and 23 h after allergen challenge, demonstrating similar bronchoprotective effects of RS- and R-salbutamol at all time points, whereas S-salbutamol was ineffective.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). No protection by S-salbutamol (1.25 mM) was observed, nor was there an increase in basal histamine reactivity at the dose used. Similar levels of bronchoprotection against methacholine 11 and against methacholine and adenosine monophosphate 10 by racemic salbutamol and the equivalent dose of the R-enantiomer have also been reported in asthmatic patients following single-dose inhalations of RS-, R- and S-salbutamol; notably, in the former study, 10-fold higher doses of the racemate and the individual enantiomers were applied. These studies also found no adverse effects of the distomer, and no difference in the duration of action between the racemate and the eutomer, with respect to both bronchoprotection 11 and bronchodilatation 10, 11. A recent study in asthmatic patients, in which a wide range of cumulatively administered doses were compared, also showed identical bronchodilatory effects at all equivalent doses of RS- and R-salbutamol 18.
The above-described clinical findings were all obtained in mildly to moderately severe asthmatics. The aim of the present study was, therefore, to investigate the acute bronchoprotective effects of salbutamol and its enantiomers not only in normoreactive but also in hyperreactive guinea pigs, after both the EAR and the LAR. Surprisingly, however, when administered 30 min before and 5.5 h after the allergen challenge, RS- and R-salbutamol effectively inhibited the allergen-induced AHR towards histamine at both 5 h, after the EAR, and 23 h, after the LAR. With respect to AHR after the EAR, the protective effect of the R-enantiomer was slightly but significantly greater than that of the racemate. This inhibition of the allergen-induced AHR observed 5 and 23 h after challenge was unexpected, since the PC100 measurements were performed 5.5 and 17.5 h after the latest inhalation of the ß-agonist, respectively, whereas the duration of action of the acute (bronchoprotective) effects of R- and RS-salbutamol on basal histamine reactivity was <3 h (fig. 2). This strongly points to the involvement of indirect effects of R- and RS-salbutamol, unrelated to smooth muscle relaxation or acute inhibition of mediator release from mast cells, which suppresses the EAR (fig. 3), in particular the immediate rise in Ppl following antigen challenge (fig. 4).
It could be argued, however, that the reduction in AHR after the EAR and LAR might yet be related to inhibition of histamine release during the EAR, since, in the same animal model, histamine H1 receptor blockade using mepyramine was found to suppress both early and late AHR and to dampen the severity not only of the EAR but also of the LAR 19. However, although mepyramine inhalation had also reduced inflammatory cell infiltration 24 h after allergen challenge 19, both salbutamol and its eutomer were without any effect on BAL fluid cell content and composition (fig. 8) as monitored at the time at which they had effectively suppressed late AHR. This would imply that inhibition of activation, rather than of influx of inflammatory cells, is involved. Indeed, RS- and R-salbutamol were found to suppress superoxide anion production and eosinophil peroxidase release from activated human eosinophils 20, 21. It was recently observed that the generation of superoxide anions is associated with the AHR of guinea pigs after the LAR 22, whereas eosinophil-derived polycations, such as major basic protein, are importantly involved in the AHR after the EAR 23. Hence inhibition of inflammatory cell activation by RS- and R-salbutamol may well be associated with the observed suppression of early and late AHR. Presumably, this may also underlie the tendency of inhibition, by both the racemate and the eutomer, to reduce the magnitude of the LAR, starting in guinea pigs 7–8 h after allergen challenge 17; however, this reduction did not reach significance (fig. 3).
It should be mentioned that, contrary to the general belief that short-acting ß-agonists do not suppress the LAR, occasional reports have indicated that nebulised salbutamol is able to reduce the development of the LAR in asthmatic patients, not only at high 24 but also at normal doses 25.
The acute bronchoprotective effects of salbutamol and its enantiomers were measured before and 5 and 23 h after allergen provocation. Two consecutive PC100 determinations were performed for this purpose at 1-h intervals, the second one preceded, 30 min before, by inhalation of the ß-agonist. As illustrated in figure 7, the equivalent doses of RS- and R-salbutamol provided very similar levels of bronchoprotection at all time points, whereas the S-enantiomer was without effect, irrespective of the time point. This latter result is at variance with the enhanced responsiveness to histamine observed in anaesthetised guinea pigs after intravenous administration of S-salbutamol 26. As discussed elsewhere, acute administration of the S-enantiomers of ß-agonists has been associated with enhanced airway reactivity of guinea pigs towards spasmogens, especially if the animals are sensitised 10. Beside direct pro-inflammatory effects 14, S-salbutamol has been reported to increase the cytosolic calcium concentration in airway smooth muscle cells through a phospholipase C-dependent mechanism 13. Obviously, such properties might enhance airway reactivity.
However, in unanaesthetised freely moving sensitised guinea pigs, no changes in airway responsiveness to histamine were observed as a result of S-salbutamol inhalation in either normoreactive (before allergen challenge) or hyperreactive airways, after the EAR and the LAR (fig. 7).
Interestingly, Keir et al. 27 have recently shown that long-term continuous treatment of guinea pigs, using osmotic minipumps, with S- and also with RS-salbutamol for 10 days evoked AHR to several bronchoconstrictive agonists that was not observed after chronic treatment with the R-enantiomer. Evidence was presented that, in the development of this AHR, which was obviously unrelated to ß2-adrenenoceptor desensitisation, sensory nerves played an important role, since capsaicin-induced neurokinin depletion, performed prior to RS- and S-salbutamol treatment, prevented the AHR to an important extent.
A most interesting finding of the present study was that salbutamol (both the racemate and the eutomer), inhaled at a low therapeutically relevant concentration before allergen provocation, suppresses the development of AHR after the EAR as well as after the LAR (fig. 6). Although, in animal models, such effects have not been reported before with short-acting ß-agonists, only one study has addressed the question as to whether salbutamol is able to influence the allergen-induced AHR in asthmatic patients. Using a high dose (2.5 mg) of nebulised salbutamol, Twentyman et al. 24 found not only complete abolition of the EAR, but also inhibition of the LAR, as well as suppression of the attendant AHR to histamine by mechanisms that could not be accounted for by bronchodilatation or functional antagonism of bronchoconstriction.
In conclusion, the present study has shown that inhalation of equivalent and clinically relevant doses of RS- and R-salbutamol exhibit similar bronchoprotective properties towards histamine in unchallenged guinea pigs, and effectively suppress allergen-induced airway hyperreactivity, after both the early and the late asthmatic reaction, through mechanisms not related to direct smooth muscle relaxation; R-salbutamol was slightly more efficacious towards the early airway hyperreactivity than the racemate. No adverse effects were observed for the individual S-enantiomer.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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: week 1 (saline); 
: week 2 (treatment)). Data are presented as mean±SEM (n = 5–7 animals). AUC: area under the pleural pressure time–response curve. *: p<0.05; **: p<0.01 versus saline (Mann–Whitney rank-sum test).
) and S-salbutamol (
) inhalation on bronchoalveolar lavage fluid cell counts 25 h after ovalbumin challenge (