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Respiratory Medicine (2007) 101, 845 849

Comparison of bronchodilator responses of levosalbutamol and salbutamol given via a pressurized metered dose inhaler: A randomized, double blind, single-dose, crossover study
A. Jantikara, B. Brashiera, M. Maganjia, A. Raghupathya, P. Mahadikb, P. Gokhaleb, J. Gogtayb, S. Salvia,
a b

Chest Research Foundation, Marigold Premises, Survey No. 15, Vadgaonsheri, Kalyaninagar, Pune 411 014, India Cipla Pharmaceuticals Ltd, Mumbai, India

Received 28 October 2005; accepted 24 February 2006

KEYWORDS
Asthma; Bronchodilator; Levosalbutamol; pressurized metered dose inhaler; salbutamol

Summary Background: Salbutamol, the most widely used short-acting b2-agonist, consists of a racemic mixture of equal amounts of two enantiomers, (R)-salbutamol and (S)-salbutamol. The bronchodilator effects of salbutamol are attributed entirely to (R)-salbutamol (levosalbutamol), while (S)-salbutamol has been shown to possess bronchospastic and pro-inammatory effects both in vitro and in vivo studies. Levosalbutamol, the (R)enantiomer of salbutamol is currently available only in a liquid formulation for use via a nebulizer. Recently, levosalbutamol to be administered via a pressurized metered dose inhaler (pMDI) has been developed. Aims: To compare the time-dependent bronchodilator responses of single doses of 100 mcg levosalbutamol and 200 mcg racemic salbutamol administered via a pMDI in subjects with stable mild-to-moderate bronchial asthma over a period of 6 h. Methods: Single doses of 100 mcg levosalbutamol, 200 mcg salbutamol and placebo were administered with a pMDI in 30 stable asthmatic subjects in a randomized, double-blind, placebo-controlled, three-way cross over study. Forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) were measured at baseline, and over 6 h post-study drug administration.

Abbreviations: ANOVA Analysis of variance; ATS American Thoracic Society; AUC Area under curve; CI Condence interval; COPD Chronic obstructive pulmonary disease; FEV1 Forced expiratory volume in 1 s; FVC Forced vital capacity; L Liters; Min Minutes; mL Milliliters; pMDI pressurized metered dose inhaler Corresponding author. Tel.: +91 20 27035361; fax: +91 20 27035371. E-mail address: ssalvi@crndia.com (S. Salvi). 0954-6111/$ - see front matter & 2006 Published by Elsevier Ltd. doi:10.1016/j.rmed.2006.02.020

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846 A. Jantikar et al. Results: Levosalbutamol and salbutamol produced signicantly better bronchodilator responses than placebo. Both the drugs showed equivalent time-dependent bronchodilator responses as measured by area under curve for percent change in FEV1 and FVC over 6 h. The time to onset of action, mean maximum bronchodilator response and duration of bronchodilator response were similar between levosalbutamol and salbutamol. Conclusion: A single dose of 100 mcg levosalbutamol administered by a pMDI produced a similar bronchodilator response as salbutamol when measured over 6 h in subjects with stable, mild-tomoderate bronchial asthma. & 2006 Published by Elsevier Ltd.

Introduction
Salbutamol (Albuterol) is the most widely used short-acting b2-agonist in the symptomatic relief of asthma and chronic obstructive pulmonary disease (COPD). In all formulations, salbutamol consists of a racemic mixture of equal amounts (50:50) of (R)- and (S)-isomers.1 Although these isomers are chemically identical, they differ in conformation, being exact non-superimposable (mirror) images of one another, or stereoisomers.2 (R)-salbutamol has been shown to have a 2-fold greater binding afnity than racemic salbutamol and a 100-fold greater binding afnity than (S)-salbutamol for the b2-adrenergic receptor.3 As a result, the bronchodilator property of racemic (R,S)-salbutamol is attributed entirely to (R)-salbutamol. Clinical studies, especially in children have shown that levosalbutamol produces a similar bronchodilator response as racemic salbutamol even when administered at one-half or one-fourth the dose.49 (S)-salbutamol has no clinically meaningful ability to relax airway smooth muscle. Pre-clinical studies have suggested that (S)-salbutamol might antagonize the smooth muscle relaxing actions of (R)-salbutamol by increasing intracellular Ca2+ levels.10 (S)-salbutamol given alone has also been reported to enhance airway hyperresponsiveness to spasmogens in vitro, promote eosinophil recruitment and activation, increase the production of histamine, and increase airway smooth muscle contractility.11 These divergent properties of (R)- and (S)-salbutamol suggest that administration of only (R)-salbutamol would have a better therapeutic index than racemic salbutamol. Although levosalbutamol [(R)-salbutamol] has been introduced in the management of asthma and COPD since 1999, it is available only in a liquid formulation to be administered by a nebulizer. The pressurized metered dose (pMDI) inhaler is the most widely used device for drug delivery in patients with asthma. It is convenient, cheap, easy to use and effective.12 Salbutamol administered via pMDI is widely used in the management of symptomatic relief of acute asthma, and when administered via a spacer, is as effective as that administered via a nebulizer in patients with acute severe exacerbations.13 Recently, levosalbutamol has been developed to be delivered via a pMDI (50 mcg per puff). The aim of this study was to compare the time-dependent bronchodilator effects of single doses of 100 mcg levosalbutamol and 200 mcg racemic salbutamol, both administered by a pMDI in subjects with chronic stable mild-to-moderate asthma.

Study design
Male and female stable asthmatic subjects between the age groups of 1865 years were recruited into the study. Asthma was dened according to American Thoracic Society (ATS) criteria (history suggestive of asthma, improvement in forced expiratory volume in 1 s (FEV1) of at least 12% and 200 mL with 200 mcg racemic salbutamol).14 All the study subjects were non-smokers and none of them received oral steroids or had an acute asthma exacerbation 4 weeks prior to the start of the study. Those subjects who were on regular (at least 4 weeks duration) inhaled corticosteroids were allowed to continue with their medication at the same dose throughout the entire duration of the study. All subjects had a baseline FEV1 of at least 60% predicted [calculated as per European Community for Coal and Steel (ECCS) X 0.9]15 and underwent assessment for the proper use of a pMDI. Pregnant and lactating women were exempted from the study. Peripheral venous blood was analyzed for routine hemogram and biochemistry during the screening visit to rule out other associated disorders. The study was approved by the institutional ethics committee and a written informed consent was obtained from all study subjects. Single doses of 100 mcg levosalbutamol, 200 mcg racemic salbutamol and placebo (manufactured by Cipla Ltd. India) were administered on three separate study days, at least 3 days apart, in a randomized, double-blinded, crossover manner via a pMDI and a non-static spacer (ZerostatTM spacer, Cipla Ltd., India). The propellant used in this study was CFC (Chlorouorocarbons) comprising of trichlorouromethane and dichlorouromethane. Care was taken to ensure that the subjects had avoided short-acting b2-agonists for at least 8 h and long-acting b2-agonists and oral theophyllines for at least 24 h prior to the start of the study visits. Lung function parameters (FEV1 and forced vital capacity (FVC)) were measured before (baseline values) and 5, 15, 30, 60, 120, 240 and 360 min after the study drug administration using a volume-based bellows Gold Standard Vitalographs Spirometer. The lung function tests were performed by a trained lung function technician using ATS guidelines.16 The highest FEV1 value at each time point was considered for analysis. The difference of up to 12% in the baseline FEV1 between the two study visits was accepted. Blood was collected for the measurement of serum potassium levels before and 1 h after the administration of the study medications. Pulse rate was measured before, 1 and 6 h after the administration of study drug medications.

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Bronchodilator effect of pMDI levosalbutamol The primary efcacy parameter was the mean difference in area under the curve (AUC) for percent change in FEV1 and FVC from baseline to 6 h. The secondary efcacy parameters were the mean maximum FEV1 and FVC change from baseline, time to onset of response, time to maximum response and duration of response. The time to onset of bronchodilator response was dened as an increase in FEV1 of 12% and 200 mL from the baseline value, while the duration of action was taken as the previous time point when the fall in FEV1 was at least 200 mL from the baseline value. The safety parameters assessed were mean changes in pulse rate and serum potassium levels from baseline values to 1 h after study medications. Sample size estimation for this study was done using a PS (Power and Sample Size) Software Version 2.1.31 (Vanderbilt, Canada) considering the mean maximum change in FEV1 following salbutamol administration from our previous study. A sample size of 24 was required to detect differences between study medications with 80% power to show equivalence at a signicance level of 5%. Spirometric data (FEV1, FVC) for each study treatment were analyzed using students t-test for paired variables. The mean response to all treatments was compared by multi-factorial analyses of variance (ANOVA) to determine the overall effect of interventions. Duncans multiple range testing with 95% condence interval (CI) was used where the differences were signicant (Po0.05). The AUC for FEV1 was calculated for each subject during each study treatment using the trapezoidal rule. The mean difference in the AUC for the two treatment groups were analyzed using ANOVA approach for Schuirmanns two one-sided test of equivalence. Equivalence was accepted if the mean difference with 95% CIs of the study variables were contained within the set limits. An independent statistician who was blinded to the codes for the study medications performed the statistical analysis. The medication codes were broken at the end of the complete analysis. 847

Table 1 Variables

Demographic details of study subjects. Mean (SD) 44.8 (11.0) 17 males, 13 females 1.70 (0.55) 2.73 (0.86) 70.54 (5.73) 17.61 (4.87)

Mean age (years) Sex Mean FEV1 (SD) L Mean FVC (SD) L Mean FEV1/FVC (SD) FEV1 reversibility (SD) as percentage

(FEV1: forced expiratory volume after 1 s, FVC: forced vital capacity, L: litres, SD: standard deviation).

40 35 Mean % change in FEV1 30 25 20 15 10 5 0 Baseline -5 5

Levosalbutamol Racemic salbutamol Placebo n=30 p0.05

15

30 60 120 Time (in minutes)

240

360

Figure 1 Mean percentage change in FEV1 values over a period of 6 h following single dose administration of levosalbutamol, racemic salbutamol and placebo.

Results
Forty-two subjects were screened for the study and 30 were randomized (11 subjects did not meet the criteria for either airow reversibility or percent FEV1 predicted, while one subject withdrew consent). All the subjects completed the study without any signicant adverse effects. The mean age of the study population was 44.8 (711.0) years. The summary of the demographic details is shown in Table 1. There was no signicant difference in the baseline FEV1 values on the 2 study days (mean7SD for levosalbutamol was 1.7570.63 and racemic salbutamol was 1.7770.66; P 0:28). Single doses of 100 mcg levosalbutamol and 200 mcg racemic salbutamol produced a signicantly better timedependent bronchodilator response as measured by AUC percentage change for FEV1 and FVC from baseline to 6 h when compared to placebo (P values o0.0001 for FEV1 and o0.0001 for FVC) (Fig. 1). The mean differences in AUC for percent change in FEV1 and FVC between racemic salbutamol and levosalbutamol from baseline to 6 h were not signicant [(FEV1 AUC06 h: 7486.3 versus 7493.6 for levosalbutamol and salbutamol respectively; P40:05) (FVC

AUC06 h: 3814.8 versus 3463.2 for levosalbutamol and salbutamol, respectively; P40:05)] (Fig. 1 showing FEV1% change), suggesting that 100 mcg levosalbutamol and 200 mcg racemic salbutamol produced equivalent bronchodilator responses over 6 h. The mean maximum difference for FEV1 from baseline was 537 mL with levosalbutamol and 538 mL with racemic salbutamol, while the mean maximum difference for FVC was 456 and 417 mL, respectively (Fig. 2). These differences were not statistically signicant, suggesting that 100 mcg levosalbutamol and 200 mcg racemic salbutamol produced an equivalent mean maximum increase in FEV1 and FVC. Levosalbutamol took 58.3 and 52.6 min to reach the maximum FEV1 and FVC response, while racemic salbutamol took 67.5 and 64.3 min, respectively. These differences were not statistically signicant when compared to each other (P 0:94). The time to onset for the bronchodilator response (increase of at least 200 mL and 12% in FEV1 from baseline) for levosalbutamol was 12 min, while that for racemic salbutamol was 18 min and the differences between the two were not signicant. The mean duration of response (difference between onset and termination of effect) for levosalbutamol was 208 min and for racemic salbutamol was 212 min. This difference was not statistically signicant (P 0:75), suggesting that there was no

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848
n=30 700 Mean maximum increase in FEV1 (mL) 600 500 400 300 200 100 0 Placebo Salbutamol Levosalbutamol p>0.05

A. Jantikar et al. mol in subjects with asthma lie entirely with the (R)-isomer, with the (S)-isomer being inert. Moreover, in vitro cellular data have implicated (S)-salbutamol as a possible cause of airway hyperreactivity, bronchoconstriction or inammation,17,18 perhaps induced by stimulating intracellular calcium accumulation and inhibiting adenyl cyclase.6,19,20 These pre-clinical ndings have been conrmed in some clinical studies that show a greater bronchodilation with levosalbutamol compared to salbutamol when given either as a long-term therapy6 (4 weeks) or when given in a single dose.19 This leads to an increase in the therapeutic index of levosalbutamol when compared to racemic salbutamol. These divergent pharmacologic properties form the scientic rationale for the potential advantages of levosalbutamol over racemic salbutamol in the treatment of asthma and other airway disorders. Until recently, optically pure isomers of b2-agonists were not available or were not thought necessary for optimum clinical safety or efcacy in acute asthma treatment. The availability of puried (R)-salbutamol (levosalbutamol) and the United States Food and Drug Administrations position to quantify the safety of racemic drugs have generated renewed interest in the role of inert isomers. In this study, we compared the bronchodilator effects of single doses of 100 mcg levosalbutamol and 200 mcg racemic salbutamol administered via a pMDI in subjects with asthma over a period of 6 h. These dosages were used to simulate those used in routine clinical practice. To our knowledge, this is the rst reported study that has investigated the efcacy and safety of a single dose of levosalbutamol administered via a pMDI. Our study demonstrates that 100 mcg levosalbutamol administered in a pMDI formulation is as effective as 200 mcg racemic salbutamol, when measured by percentage changes in FEV1 and FVC AUCs from baseline to 6 h, time to onset of bronchodilator response, mean maximum change in FEV1 and FVC and duration of the bronchodilator effect. The results of this study conrm earlier reports that the bronchodilator effects of salbutamol are entirely attributable to its (R)-enantiomer. Although earlier studies 19,20 had suggested that levosalbutamol might show lesser clinical side effects than the racemic form, we did not nd any difference in pulse rates and decreases in serum potassium levels between levosalbutamol and racemic salbutamol. These observations are in accordance with Lotvall et al.5 who have demonstrated that the adverse effects of racemic salbutamol on heart rate and potassium levels are mediated mainly by the (R)-enantiomer. Cockcroft et al. have reported that nebulized levosalbutamol produced similar effects on heart rate as racemic salbutamol, suggesting that tachycardia caused by salbutamol is attributed to (R)-salbutamol. Pancu et al.21 have reported that (R)-salbutamol and racemic salbutamol are equally effective in lowering serum potassium levels. This study has therefore shown that the bronchodilator effect and systemic side effects of salbutamol reside with the (R)-enantiomer. We did not assess tremors. Since we used single doses of 100 mcg levosalbutamol and 200 mcg of racemic salbutamol we did not expect to nd signicant numbers of patients experiencing tremors at this small dose. Also tremor assessment is a subjective observation, which has its own drawbacks.

Figure 2 Mean maximum increase in FEV1 from baseline after administration of placebo, racemic salbutamol and levosalbutamol.

difference in the duration of bronchodilator response between the two study drugs. Both levosalbutamol and racemic salbutamol produced a signicant decrease in serum potassium level 1 h after study drug administration compared to their respective baseline values, (4.614.25; Po0.001 and 4.394.13; P 0:004, respectively). The placebo treatment did not show any signicant decline in serum potassium levels (4.474.37; P 0:19). Racemic salbutamol and levosalbutamol 60 min post-drug administration both produced a signicant increase in pulse rate versus their baseline, (73.977.8 beats/ min; Po0.05 and 72.477.3 beats/min; Po0.05, respectively). There was no signicant difference in the pulse rate 6 h post-drug administration compared to baseline values (data not shown).

Discussion
In this randomized, double-blind, placebo-controlled, crossover study, we have demonstrated for the rst time that a single dose of 100 mcg of levosalbutamol administered via a pMDI produced a similar time-dependent bronchodilator response as that of 200 mcg racemic salbutamol in subjects with mild-to-moderate stable asthma. The time to onset of bronchodilator response, mean maximum change in FEV1 and FVC, time to reach the mean maximum FEV1 and FVC responses and the duration of responses were similar between the two study medications. Both the drugs increased pulse rate and reduced serum potassium levels equally, when measured 1 h after administration. Salbutamol is the most widely used inhaled short-acting b2-agonist in the symptomatic relief of asthma and COPD. It consists of a racemic mixture of equal amounts of (R)- and (S)-enantiomers. These enantiomers have similar physical and chemical properties, but have different receptor specicity and therefore have different pharmacological and physiological effects. The bronchodilator properties of racemic salbutamol (R,S) have been shown to be attributed entirely to the (R)-enantiomer. Human epinephrine produced by the adrenal gland occurs only in the (R)-isomer form. Clinical studies have demonstrated that the bronchodilator and bronchoprotective effects of racemic salbuta-

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Bronchodilator effect of pMDI levosalbutamol In summary, a single dose of levosalbutamol administered in a pMDI formulation produced an equivalent timedependent bronchodilator response over 6 h as racemic salbutamol at half the dose. Thus, levosalbutamol administered in a pMDI formulation may be used routinely for acute symptomatic relief in subjects with asthma. Further studies involving larger numbers of patients and for longer duration are required to determine long-term safety and efcacy of levosalbutamol administered via a pMDI. 849
10. Mitra S, Ugur M, Ugur O, Goodman HM, McCullough JR, Yamaguchi H. (S)-Albuterol increases intracellular free calcium by muscarinic receptor activation and a phospholipase C-dependent mechanism in airway smooth muscle. Mol Pharmacol 1998;53(3):34754. 11. Johansson F, Rydberg I, Aberg G, Andersson RG. Effects of albuterol enantiomers on in vitro bronchial reactivity. Clin Rev Allergy Immunol 1996;14(1):5764. 12. Fink JB. Metered-dose inhalers, dry powder inhalers, and transitions. Respir Care 2000;45(6):62335. 13. Cates CC, Bara A, Crilly JA, Rowe BH. Holding chambers versus nebulisers for beta-agonist treatment of acute asthma. Cochrane Database Syst Rev 2003(3):CD000052. 14. American Thoracic Society. Medical Section of the American Lung Association. Guidelines for the evaluation of impairment/ disability in patients with asthma. Am Rev Respir Dis 1993; 147(4):105661. 15. Quanjer PH, Tammeling GJ, Cotes JE, Pedersen OF, Peslin R, Yernault JC. Lung volumes and forced ventilatory ows. Report working party standardization of lung function tests, European Community for Steel and Coal. Ofcial Statement of the European Respiratory Society. Eur Respir J Suppl 1993;16: 540. 16. American Thoracic Society. Standardization of spirometry, 1994 update. Am J Respir Crit Care Med 1995;152(3):110736. 17. Baramki D, Koester J, Anderson AJ, Borish L. Modulation of Tcell function by (R)- and (S)-isomers of albuterol: antiinammatory inuences of (R)-isomers are negated in the presence of the (S)-isomer. J Allergy Clin Immunol 2002; 109(3):44954. 18. Jafarian A, Handley DA, Biggs DF. Effects of RS-albuterol on the development of antigen-mediated airway hyperreactivity in guinea pigs. Clin Rev Allergy Immunol 1996;14(1):91100. 19. Gawchik SM, Saccar CL, Noonan M, Reasner DS, DeGraw SS. The safety and efcacy of nebulized levalbuterol compared with racemic albuterol and placebo in the treatment of asthma in pediatric patients. J Allergy Clin Immunol 1999;103(4): 61521. 20. Milgrom H, Skoner DP, Bensch G, Kim KT, Claus R, Baumgartner RA. Low-dose levalbuterol in children with asthma: safety and efcacy in comparison with placebo and racemic albuterol. J Allergy Clin Immunol 2001;108(6):93845. 21. Pancu D, LaFlamme M, Evans E, Reed J. Levalbuterol is as effective as racemic albuterol in lowering serum potassium. J Emerg Med 2003;25(1):136.

References
1. Dhand R, Goode M, Reid R, Fink JB, Fahey PJ, Tobin MJ. Preferential pulmonary retention of (S)-albuterol after inhalation of racemic albuterol. Am J Respir Crit Care Med 1999; 160(4):113641. 2. Berger WE. Levalbuterol: pharmacologic properties and use in the treatment of pediatric and adult asthma. Ann Allergy Asthma Immunol 2003;90(6):58391. 3. Penn RB, Frielle T, McCullough JR, Aberg G, Benovic JL. Comparison of R-, S-, and RS-albuterol interaction with human beta 1- and beta 2-adrenergic receptors. Clin Rev Allergy Immunol 1996;14(1):3745. 4. Handley DA, Tinkelman D, Noonan M, Rollins TE, Snider ME, Caron J. Doseresponse evaluation of levalbuterol versus racemic albuterol in patients with asthma. J Asthma 2000; 37(4):31927. 5. Lotvall J, Palmqvist M, Arvidsson P, Maloney A, Ventresca GP, Ward J. The therapeutic ratio of R-albuterol is comparable with that of RS-albuterol in asthmatic patients. J Allergy Clin Immunol 2001;108(5):72631. 6. Nelson HS, Bensch G, Pleskow WW, DiSantostefano R, DeGraw S, et al. Improved bronchodilation with levalbuterol compared with racemic albuterol in patients with asthma. J Allergy Clin Immunol 1998;102(6 Pt 1):94352. 7. Nelson HS. Clinical experience with levalbuterol. J Allergy Clin Immunol 1999;104(2 Pt 2):S7784. 8. Nowak R. Single-isomer levalbuterol: a review of the acute data. Curr Allergy Asthma Rep 2003;3(2):1728. 9. Pereira A, Mendes E, Ferreira T, Wanner A. Effect of inhaled racemic and (R)-albuterol on airway vascular smooth muscle tone in healthy and asthmatic subjects. Lung 2003;181(4): 20111.