Enantiomeric disposition of inhaled, intravenous and oral racemic-salbutamol in man — no evidence of enantioselective lung metabolism

Abstract

Aims

To establish whether enantioselective metabolism of racemic (rac)-salbutamol occurs in the lungs by determining its enantiomeric disposition following inhalation, in the absence and presence of oral charcoal, compared with that following the oral and intravenous routes.

Methods

Fifteen healthy subjects (eight male) were randomized into an open design, crossover study. Plasma and urine salbutamol enantiomer concentrations were measured for 24 h following oral (2 mg) with or without oral charcoal (to block oral absorption), inhaled (MDI; 1200 μg) with or without oral charcoal and intravenous (500 μg) rac-salbutamol. Systemic exposure (plasma AUC(0,∞) and urinary excretion (Au_24h) of both enantiomers were calculated, and relative exposure to (R)-salbutamol both in plasma (AUC_(R)-/AUC_(S)-) and urine (Au_(R)-/Au_(S)-) was derived for each route. Relative exposure after the inhaled with charcoal and oral routes were compared with the intravenous route.

Results

AUC_(R)-/AUC_(S)- [geometric mean (95% CI)] was similar following the intravenous [0.32 (0.28, 0.36)] and inhaled with charcoal rates [0.29 (0.24, 0.36); P = 0.046], but was far lower following oral dosing [0.05 (0.03, 0.07); P < 0.001]. Similar results were found when relative exposure was analysed using Au_24h.

Conclusions

These results show no evidence of significant enantioselective presystemic metabolism in the lungs, whilst confirming it in the gut and systemic circulation, indicating that the (R)- and (S)-enantiomers are present in similar quantities in the airways following inhaled rac-salbutamol.

Introduction

Salbutamol is a potent and selective β₂-adrenoceptor agonist widely used in the treatment of asthma [1]. Like other β₂-adrenoceptor agonists, salbutamol is a chiral compound with (R)- and (S)-enantiomers, and its pharmacological activity resides almost entirely with the (R)-enantiomer [2]. Commercial preparations of salbutamol (like Ventolin™ and Proventil™) can be administered by the oral, intravenous or inhaled routes, and all contain racemic (rac-) salbutamol.

The systemic clearance of salbutamol is by renal excretion, partly as unchanged drug and partly as its 4,O-sulphate metabolite, with the relative importance of these two mechanisms dependent on the route of administration [3]. Sulphate conjugation is catalysed by the monoamine (M) form of the phenolsulphotransferases. It is highly stereoselective for the active (R)-enantiomer over the (S)-enantiomer and systemically it occurs in the liver and platelets [4] leading to excessive exposure to the inactive (S)-enantiomer following intravenous administration [5]. Following oral dosing, salbutamol undergoes extensive presystemic metabolism [6]. In vitro studies have shown stereoselective sulphation in the human jejunum [4], leading to speculation that presystemic metabolism occurs mainly in the intestinal wall. In vivo studies of enantiomeric disposition of rac-salbutamol in man have also shown that there is excess exposure to the inactive (S)-enantiomer following oral administration [7], indicative of the high level of presystemic enantioselective metabolism [5].

Since salbutamol is mainly given via the inhaled route for the treatment of asthma, it is relevant to investigate its enantiomeric disposition after inhalation and to determine the potential for enantioselective metabolism in the lungs. Recently, it has been suggested that the (S)-enantiomer of salbutamol, instead of being essentially inert, may increase airway hyperreactivity in asthmatic patients [8], although this effect has not been reproduced when more clinically relevant doses were examined [9]. If enantioselective metabolism occurred locally in the lungs, the active (R)-enantiomer would be preferentially metabolized and cleared from the airways following inhaled administration of rac-salbutamol. In vitro, the sulphoconjugation and intrinsic clearance of the (R)-enantiomer has been shown to exceed that of the (S)-enantiomer by 11-fold in human lung cell-free cytosol, and similar results have been found in a bronchial epithelial cell line [10]. In contrast, in urine samples collected from healthy subjects in the first 30 min after inhalation of rac-salbutamol, the ratio of (S)-: (R)-enantiomers was found to be close to unity, suggesting insignificant enantioselective metabolism of salbutamol in the lungs immediately following inhalation [11]. However, an increased ratio was observed at later time points.

The aim of the present study was to investigate the enantiomeric disposition of rac-salbutamol and to determine the potential for enantioselective lung metabolism. An activated oral charcoal block was used to prevent confounding oral absorption of the swallowed fraction of the inhaled dose. As this method has previously been validated for terbutaline [12], but not for salbutamol, an oral administration with charcoal was included. Plasma and urine concentrations of the (R)-and (S)-enantiomers of salbutamol, and their respective 4,O-sulphate metabolites, were measured using a sensitive chiral method [13], with greater sensitivity to previously used methods [14, 15] and pharmacokinetics were compared following oral (positive control for presystemic metabolism), intravenous (negative control for presystemic metabolism) and inhaled administration of rac-salbutamol.

Methods

Materials

The following medications were supplied by Glaxo Wellcome Research & Development Ltd (Greenford, UK), from commercially available stocks: Ventolin™ pressurized metered dose inhalers (MDI; 100 μg rac-salbutamol sulphate per actuation), Ventolin™ injection 1 ml ampoules (containing 500 μg rac-salbutamol sulphate/ml) and 10 ml sterile saline ampoules (0.9% w/v sodium chloride) for use as diluent for Ventolin™ injection. Broncovaleas™ tablets (containing 2 mg rac-salbutamol sulphate per tablet) were obtained from Valeas SpA (Milan, Italy) and Carbomix™ activated charcoal was obtained from Penn Pharmaceuticals (Gwent, UK).

Subjects

Fifteen nonsmoking healthy subjects [seven female; age range of 23–40 (female) and 21–43 (male) years; weight range of 50–90 kg (females) and 55–95 kg (males)] were required to undergo a full physical examination, blood and urine analysis before admission to the study. All subjects were in good health, free from significant disease and not taking any other medication, except for the oral contraceptive pill. They were also required to demonstrate a good MDI inhalation technique. The protocol was approved by the local Independent Ethical Review Committee (Glaxo Wellcome SpA, Verona, Italy), and all volunteers gave written informed consent prior to participation. Procedures were carried out in accordance with the Declaration of Helsinki.

Study design

This was a single centre, randomized, open design, five-way crossover study. Subjects were randomized to receive: 1200 μg inhaled rac-salbutamol with or without oral activated charcoal, 2 mg oral rac-salbutamol with or without activated oral charcoal and 500 μg intravenous rac-salbutamol. Each subject received all 5 treatments, separated by a minimum washout of 7 days. Inhaled rac-salbutamol was administered as 12 inhalations at 20 s intervals from an MDI (100 μg per actuation), with a 10 s breath-holding pause following each inhalation. Oral rac-salbutamol was administered as a tablet and swallowed with a 50 ml drink of water. Intravenous rac-salbutamol was administered as an 8 ml infusion, delivered by IVAC infusion pump over 5 min. Charcoal was administered as a suspension of 5 g in 50 ml water, given 2 min before and 2 min after the inhaled and oral treatments. A further 10 g activated charcoal in 100 ml water was given at 1, 2 and 3 h after the treatment.

The schedule for each study day was identical. Subjects were institutionalized and required to refrain from caffeine-containing and alcoholic beverages for 12 h prior to, and for 24 h following each dose. Subjects were also fasted for 12 h prior to and for 4 h after each dose. Blood samples were taken predose, and at t = 1, 2, 5, 7, 10, 15, 20, 30, 45, 60, 90 min, and at 2, 3, 4, 6, 8, 10, 12, 24 h postdose. Time zero was defined as the end of the last breath-holding pause (inhaled salbutamol), the end of the infusion (intravenous salbutamol) or the time the tablet was swallowed (oral salbutamol). Samples were immediately placed on ice and then centrifuged at 3000 g for 10 min at 4° C and plasma was separated and stored at −80° C until bioanalysis. Urine was collected in six samples (−0–30, 30–120 min; 2–4, 4–6, 6–8 and 8–24 h postdose), into plastic bottles without preservative. After weighing and thorough mixing of the collection, a 20 ml aliquot was removed and stored at −80° C until bioanalysis. Ten minutes prior to dosing, subjects were asked to void their bladder and were then required to consume 300 ml water. A further 200 ml water was consumed by the subjects immediately following collection of the 0–30 min urine samples, and at 2.5 h postdose.

Bioanalysis

The concentration of (R)-and (S)-salbutamol enantiomers and their sulphated metabolites were determined in plasma and urine by validated chiral methods involving solid phase extraction, followed by chiral high performance liquid chromatography with tandem mass spectrometric detection using a teicoplanin-based chiral stationary phase and selected reaction monitoring [13]. The lower limit of quantification (coefficient of variation) was 0.1 ng ml⁻¹ (<7%) for (R) and (S)-salbutamol and 5 ng ml⁻¹ (<11%) for their metabolites [13]. All analyses were carried out in the department of International Bioanalysis, Glaxo Wellcome Research & Development (Ware, UK) and the bioanalyst was blinded to the randomization code whilst plasma analytes were measured.

Data analysis

The following parameters were calculated for each enantiomer, and their respective 4,O-sulphate metabolite, following each route of administration: The maximum concentration (C_max) and associated time (t_max); both 24 h plasma exposure (AUC(0,24h)) and plasma exposure extrapolated to infinity (AUC(0,∞)); apparent bioavailability (F); terminal phase half-life (t_1/2); total plasma (CL_P) and renal clearance (CL_R). Total amounts of enantiomers and metabolites excreted in urine over the 24 h (Au_24h) were calculated by summing the amounts found in each collection period. Each of these parameters was summarized for the study population as the geometric mean and the associated 95% confidence intervals (CI), except t_max which was summarized as the median and the associated range. The following pharmacokinetic parameters were derived from the above parameters: relative proportion of (R)-salbutamol in plasma (AUC_(R)-/AUC_(S)-), calculated both from AUC(0,24h) and AUC(0,∞); relative proportion of (R)-salbutamol in the urine (Au_(R)-/Au_(S)-). Pharmacokinetic parameters were calculated using PEARS, a validated PC based nonparametric pharmacokinetic software package (Department of Clinical Pharmacology, Glaxo Wellcome Research & Development, Greenford, UK), using standardized pharmacokinetic methods.

The primary statistical comparison to investigate if enantioselective lung metabolism occurs following inhalation was between the inhaled with charcoal and intravenous routes. To confirm that presystemic enantioselective metabolism occurs following oral dosing a comparison was also made between the oral and intravenous routes. These statistical comparisons were made on the relative proportion of the (R)-enantiomer in both plasma and urine. These proportions were log-transformed prior to analysis, to fulfil the assumption of constant variance [16], and analysed using analysis of variance allowing for effects due to subject, period and treatment. Estimates of treatment effect were based on the ratio of the least square means and presented on the untransformed scale with the associated 90% CI. Treatment differences were assessed using a two-sided significance test, using SAS (SAS v 6.12, SAS Institute, Cary, NC). Comparisons where P < 0.05 were considered significant.

Results

Validation of oral charcoal block

Following oral rac-salbutamol and activated charcoal (R)-and (S)-salbutamol concentrations could not be reproducibly detected in plasma, with only 3/15 and 5/15 subjects having detectable (R)-and (S)-salbutamol concentrations in a few plasma samples, respectively. This was consistent with oral charcoal blocking the absorption of oral rac-salbutamol, and prevented the calculation of any plasma pharmacokinetic parameters. In the urine, small amounts of (R)-and (S)-salbutamol were detected in 8/15 and 14/15 subjects, respectively. Comparison with oral dosing alone (Table 2) suggested that coadministration of activated charcoal prevents at least 92–98% of the oral absorption of a 2 mg dose of rac-salbutamol.

Table 2.

Apparent bioavailability (F), plasma exposure (AUC(0,∞)) and total urinary excretion (Au_24h) derived from (R)- and (S)-salbutamol plasma and urine concentrations in 15 healthy subjects. Data are geometric means (95% CI).

Pharmacokinetics of (R)-and (S)-salbutamol

Mean plasma concentration-time profiles of (R)-and (S)-salbutamol following intravenous, oral and inhaled rac-salbutamol, with or without oral charcoal are shown in Figure 1, and derived pharmacokinetic parameters are shown in Tables 1 and 2.

Figure 1.

Plasma concentration-time profiles for (R)-salbutamol (open circles) and (S)-salbutamol (solid circles) after (a) 500 μg intravenous (b) 2 mg oral (c) 1200 μg inhaled with oral charcoal and (d) 1200 μg inhaled rac-salbutamol. Mean values±s.e. mean in 15 healthy subjects.

Table 1.

Summary of pharmacokinetic parameters derived from (R)- and (S)-salbutamol plasma and urine concentrations in 15 healthy subjects. Data are geometric means (95% CI).

Following inhaled dosing with oral charcoal, median t_max was measured approximately 5–10 min after dosing for both enantiomers (Table 1). Following inhaled dosing without charcoal, t_max tended to be later than the corresponding t_max after inhaled dosing with charcoal, particularly for (S)-salbutamol. t_max was greatest for both (R)-and (S)-salbutamol following oral dosing.

Apparent bioavailability (F) was also calculated for each enantiomer following oral, inhaled and inhaled dosing with charcoal (Table 2). (S)-salbutamol F was slightly higher following oral dosing (68.7%) than inhaled dosing (60.0%) and considerably lower after inhaled dosing with charcoal (18.7%). However (R)-salbutamol bioavailability was slightly higher following inhaled dosing (23.8%) than inhaled dosing with charcoal (19.5%), and considerably lower following oral dosing (9.4%).

Mean t_1/2 was approximately 2.5 and 5 h for (R)- and (S)-salbutamol, respectively (Table 1). CL_p and CL_R were approximately 2–3 fold higher for (R)- than (S)-salbutamol. CL_R appeared to account for 85% of CL_p for (S)-salbutamol, but only 50% of CL_p for (R)-salbutamol (Table 1).

Administration of rac-salbutamol led to higher systemic exposure to (S)-salbutamol compared to (R)-salbutamol, regardless of the route of administration (Table 3). Following intravenous dosing mean AUC_(R)-/AUC_(S)- and Au_(R)-/Au_(S)- were 0.32 and 0.61, respectively, whilst after oral dosing the values were 0.05 and 0.15, far lower than after intravenous dosing (P < 0.001). This was confirmed by measurements of apparent bioavailability, which were far higher for (S)-salbutamol than for (R)-salbutamol (Table 2). Following inhaled dosing with oral charcoal, relative plasma exposure was 0.29, with a borderline significant difference (P = 0.046). However, if proportions were calculated using AUC (0,24h), comparison of intravenous and inhaled dosing with charcoal was not significantly different (P = 0.219; data not shown). Findings were similar in urine with an Au_(R)-/Au_(S)- value of 0.50, which was not statistically significantly different to that after intravenous dosing (P = 0.327), and confirmed by the similar apparent bioavailabilities of (R)-and (S)-salbutamol (Table 2). Following inhaled dosing the plasma and urine ratios (0.13 and 0.28) fell between those found following inhaled dosing with oral charcoal and oral dosing, again confirmed by the higher apparent bioavailability of (S)- compared with (R)-salbutamol (Table 2).

Table 3.

Relative proportion of (R)-salbutamol in plasma (AUC_(R)-/AUC_(S)-) and urine (Au_(R)-/Au_(S)-), and associated statistical comparisons in 15 healthy subjects.

Pharmacokinetics of (R)- and (S)-salbutamol metabolites

Pharmacokinetic parameters, derived from plasma and urine concentrations of (R)- and (S)-salbutamol 4,O-sulphate metabolites, are listed in Table 4. The 4,O-sulphate metabolites were only reproducibly detectable in plasma following oral dosing, and in a few subjects following inhaled dosing. This was due to the higher limit of quantification for the metabolites assay (5 ng ml⁻¹) compared with that for the parent (0.1 ng ml⁻¹). Following oral rac-salbutamol, median t_max was approximately 4 h for both metabolites, and C_max, AUC(0,∞) and Au₂₄ were higher for the (R)-salbutamol metabolite than for the (S)-salbutamol metabolite. Mean t_1/2 of the (R)-salbutamol metabolite was shorter than that of the (S)-salbutamol metabolite. From the quantifiable concentrations measured, it was impossible to investigate the conversion of the enantiomers into the sulphated metabolites over the same time-course, and no further information could be provided on their disposition. Both (R)- and (S)-salbutamol metabolites were reproducibly detected in the urine following each treatment, allowing Au_24h to be measured (Table 4). In each case Au was greater for the (R)-salbutamol metabolite than the (S)-salbutamol metabolite.

Table 4.

Summary of pharmacokinetic parameters derived from (R)-and (S)-salbutamol 4,O-sulphate plasma and urine concentrations in 15 healthy subjects. Data are geometric means (95% CI).

Mass balance of rac-salbutamol-related materials

Total 24 h urinary excretion of salbutamol-related materials (calculated as the sum of the individual parent enantiomers and their 4, O-sulphate metabolites) was approximately 440 μg following intravenous administration, 2.0 mg following oral dosing, and 380 μg and 950 μg following inhaled dosing with or without oral charcoal, respectively. Regardless of the route of administration, each enantiomer contributed approximately 50% of the total 24 h excretion of salbutamol-related materials.

Discussion

Despite the wide use of salbutamol in clinical practice, pharmacokinetic information following inhalation has not been comprehensive. In addition the disposition of inhaled drugs is complex with only a small proportion of the drug deposited in the lungs, whilst the rest is swallowed [17], hence plasma kinetics reflect both the absorption of the swallowed fraction and the lung absorption of the inhaled fraction. To explore enantiomeric disposition from the lungs, confounding oral absorption was prevented by coadministration of oral charcoal. The validity of this procedure for rac-salbutamol was confirmed with charcoal preventing 92–98% of the absorption of both enantiomers, as with terbutaline where absorption was reduced by 97% [12].

The mean apparent bioavailability of rac-salbutamol, calculated as the mean of the individual enantiomers, following oral dosing (39%) was consistent with values found in previous studies [5, 6, 18], and comparable with that following inhaled dosing (42%). However, bioavailability of the active (R)-enantiomer was far lower following oral (9%) than inhaled dosing (24%), indicating the importance of conducting chiral pharmacokinetic analysis with salbutamol. The mean apparent bioavailability of rac-salbutamol following inhaled dosing with charcoal was approximately 20% confirming deposition in, and absorption from, the lungs of approximately 20% of an inhaled dose [17]. For all routes of administration, total urinary recovery of salbutamol-related material comprised approximately equal proportions of (R)- and (S)-salbutamol-related material, indicating that equal amounts of (R)- and (S)-salbutamol were delivered to and absorbed from the lungs and intestinal tract, and confirming previous observations of insignificant chiral interconversion [7].

t_max for both enantiomers tended to be later following inhaled dosing than after inhaled dosing with charcoal, consistent with an initial rapid absorption phase of salbutamol from the lungs followed by a slower absorption of the swallowed fraction of the inhaled dose. Slow oral absorption was confirmed by a t_max of 2–3 h for both enantiomers following oral dosing, consistent with the results of previous nonchiral [18] and chiral [7] studies.

Terminal half life was approximately 2.5 and 5 h for (R)- and (S)-salbutamol, respectively, accordingly CL_p and CL_R were both approximately 2–3 fold higher for (R)-salbutamol, consistent with the results of a previous chiral study [5]. The reason for stereoselective renal clearance of (R)-salbutamol is not clear. Previous studies have shown no stereoselectivity in V_ss following intravenous rac-salbutamol [5], indicating insignificant enantioselective tissue binding. Enantioselective plasma protein binding of (S)-salbutamol is also likely to be insignificant as only a small fraction (8%) of rac-salbutamol is protein-bound [3]. As the renal clearance of both enantiomers is higher than predicted creatinine clearance, suggesting renal secretion, another possible explanation may be stereoselective active secretion.

Following intravenous dosing there was greater systemic plasma exposure to (S)- compared with (R)-salbutamol. This was consistent with the results of a previous study [5], although comparison of absolute exposure values was not possible due to the different doses employed and the different observation periods. It is likely that enantioselective systemic metabolism of (R)-salbutamol occurs in both the liver and platelets, which both show preferential metabolism in vitro [4], although other cell types cannot be excluded. After oral dosing the relative proportion of (R)-salbutamol in plasma and urine was far lower than after intravenous dosing. These findings were consistent with previous studies following intravenous [5] and oral [5, 7] dosing with rac-salbutamol, and are thought to reflect the high level of presystemic enantioselective metabolism, which is also evident in vitro in human jejunal preparations [4]. This indicates that the likely site of presystemic metabolism in vivo is in the intestinal wall, although first pass metabolism may also occur in the liver. Following inhalation, either with or without a concomitant oral charcoal block, there was also greater systemic exposure to (S)-salbutamol. A comparison of the relative proportion of (R)-salbutamol in plasma and urine following intravenous and inhaled rac-salbutamol with charcoal was used to examine whether there was enantioselective presystemic metabolism in the lungs. A small difference was observed which was of borderline significance using AUC(0,∞), but was not statistically significant when using AUC(0,24h) or Au_24h. This is most likely to have been due to presystemic metabolism in the intestinal tract of the small percentage of the swallowed fraction of the inhaled dose, whose absorption was not prevented by charcoal. Alternatively it may indicate a clinically insignificant low level of enantioselective presystemic lung metabolism. The former conclusion is supported by the quantitative similarity of the mean apparent bioavailability of (S)- and (R)-salbutamol following inhaled dosing with charcoal. The relative proportion and bioavailability of (R)-salbutamol was clearly very different following oral dosing (i.e. positive control for presystemic metabolism), indicating insignificant enantioselective presystemic lung metabolism in healthy subjects.

The lack of evidence of enantioselective presystemic lung metabolism is consistent with previous observations in both nonchiral [19] and chiral studies [11]. These results in vivo question the relevance of in vitro findings that sulphoconjugation of (R)-salbutamol exceeds that of (S)-salbutamol by 11-fold in human lung cell-free cytosol [10], a value similar to the one obtained in jejunal cells [4]. In intact human bronchial epithelial cells in vitro, the overall rate of clearance of (R)-salbutamol was much lower than in human lung cytosol [10], indicating caution when extrapolating metabolic rates assessed in broken cell preparations to the in vivo situation.

Our results also indicate that following inhaled dosing, approximately 60% of systemic bioavailability of rac-salbutamol is from the swallowed fraction of the inhaled dose. However, the difference was more marked for the (S)-salbutamol (70%) than (R)-salbutamol (20%), indicating preferential bioavailability of (S)-salbutamol from the intestinal tract. Accordingly, the relative proportion of (R)-salbutamol in plasma and urine was lower after inhaled dosing than after inhaled dosing with charcoal, but higher than after oral dosing.

Metabolites were only reproducibly detected in plasma following oral dosing. Plasma concentrations of (R)-salbutamol 4,O-sulphate were higher than levels of (S)-salbutamol 4,O-sulphate at all time points, a result reflected by urinary excretion and indicating the high level of enantioselective metabolism. However, as the metabolite clearance kinetics could not be determined in this study, further interpretation of urinary kinetics were not appropriate. It is likely that metabolites could not reproducibly be detected in the plasma following dosing by the other routes due to the comparatively low sensitivity of the analytical method. t_max was later for both metabolites than corresponding times for the parent enantiomers, confirming previous studies [6]. Mean t_1/2 of the (R)-salbutamol metabolite tended to be shorter than that for the (S)-salbutamol metabolite, although there were overlapping confidence intervals, and was similar to t_1/2 measured for the parent enantiomers, consistent with the previous observations that parent and metabolite are cleared at similar rates [18].

In conclusion, we have described the enantiomeric disposition of (R) and (S)-salbutamol and their respective 4,O-sulphate metabolites following oral, intravenous and inhaled dosing with rac-salbutamol in healthy volunteers. In particular, we have found no evidence of enantioselective metabolism of (R)-salbutamol in the lungs of healthy subjects, whilst confirming it in the intestinal tract and systemic circulation. Our results suggest that following inhaled dosing of rac-salbutamol products, both the active (R)- and inactive (S)-enantiomers will be present in similar concentrations in the airways.