INTRODUCTION

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Racemic albuterol, a specific ₂-agonist, is probably the most commonly used bronchodilator, with millions of doses being inhaled on a daily basis. Whereas single doses of ₂-agonists produce a predictable improvement in lung function, several investigators have reported that their long-term use is associated with a small decline in pulmonary function, and a paradoxical increase in airway hyperresponsiveness (1). Although several mechanisms have been proposed to explain this adverse effect (2), its pathogenesis is unclear.

Racemic albuterol is a 50:50 mixture of an (R)- and (S)- enantiomer (3). In contrast to the (R)-enantiomer, the (S)- form not only lacks ₂-adrenergic activity, but it increases airway hyperreactivity to several bronchoconstrictor stimuli in previously sensitized guinea pigs (4). Gradual accumulation of the (S)-enantiomer in the lung may increase airway reactivity after long-term use of racemic albuterol in patients with asthma (5, 6). However, direct evidence to support this hypothesis does not exist.

When racemic albuterol is administered by the oral, rectal, or intravenous routes, a tenfold faster metabolism of (R)- albuterol than that of (S)-albuterol (7) results in plasma levels of the (R)-enantiomer that are 2 to 3 times lower than those of the (S)-enantiomer (8). A similar profile of (R)- and (S)-enantiomer levels is observed after nebulization of racemic albuterol (6, 11). However, the fate of the enantiomers after inhalation of racemic albuterol from a metered-dose inhaler (MDI) is not known. We hypothesized that the pharmacokinetics of albuterol's enantiomers after inhalation from a MDI differ from those observed after enteral or parenteral administration of racemic albuterol.

Previously, we showed that the inhalation of racemic albuterol from a MDI produces a sharp peak in serum levels within 10 to 15 min, and that serum levels over time provide an estimate of the systemic bioavailability of the drug after inhalation (12). Accordingly, the primary aim of this investigation was to measure the plasma levels of the (R)- and (S)- enantiomers for up to 4 h after inhalation of racemic albuterol from a MDI. Because racemic albuterol contains equal quantities of the (R)- and (S)-enantiomers, differences in the plasma levels of the two enantiomers after inhalation indicate differences in their disposition or metabolism within the lung. Urinary concentrations of albuterol have been used by previous investigators as an indirect measure of systemic bioavailability after inhalation (13, 14). We measured the concentrations of unchanged (R)- and (S)-enantiomers in urine for up to 24 h after inhalation of racemic albuterol to determine whether they reflect the levels in the plasma, and to study the disposition of the enantiomers after the plasma levels had decreased to very low concentrations.

The majority of racemic albuterol administered by a MDI (~ 80% of the nominal dose) deposits in the oropharynx (15), is absorbed from the gastrointestinal tract, and contributes to the plasma levels about 60 min after inhalation. Aerosol deposition in the oropharynx is decreased approximately 14-fold with the use of a MDI and holding chamber versus a MDI alone (16), although pulmonary deposition is not increased (17). If gastrointestinal absorption contributes significantly to the plasma and urinary levels of albuterol, the concentrations achieved with a MDI alone should be higher than those with a MDI and holding chamber. We determined the contribution of gastrointestinal absorption to the plasma levels of albuterol by comparing the pharmacokinetics in healthy subjects who inhaled racemic albuterol from a MDI alone versus a MDI and holding chamber.

	METHODS

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Ten nonsmoking, healthy volunteers (2 female, 8 male) with a mean age of 34 (range, 26 to 47) yr participated in the study. The subjects did not have a history of systemic disease nor were they receiving any other drug therapy before or during the study. A history of cardiac rhythm disturbances, asthma, pneumonia, viral upper respiratory illness in the preceding 6 wk, pregnancy, or lactation were criteria for exclusion from the study. Informed consent was obtained from all subjects, and the study was approved by the Human Studies Subcommittee of Edward Hines Jr. Veterans Affairs Hospital.

Protocol

A randomized, crossover study design was employed. The subjects were studied in the morning after an overnight fast. Each subject was randomized by a computer-generated schedule to receive 6 puffs of racemic albuterol (Goldline Zenith Laboratories, Northvale, NJ; 90 µg/puff) from a MDI or a MDI and holding chamber (11 × 3.5 cm, volume 145 ml; Aerochamber, Monaghan Medical Co., Plattsburgh, NY) on separate occasions. After receiving instructions on the proper use of a MDI alone and with a holding chamber, each subject performed 5 sham trials with the appropriate device and placebo aerosol (Allen and Hanburys, Research Triangle Park, NC) to master the correct technique of inhalation.

Each subject inhaled 6 puffs of albuterol from a MDI by the method to which they were randomized, with 30-s intervals between each puff. A 7-ml sample of venous blood was drawn into heparinized tubes from an indwelling catheter before and at 10, 15, 30, 60, 120, and 240 min after administration of albuterol; patency of the catheter was maintained by bolus injections of saline. No food or water was permitted for at least 2 h after albuterol administration. All blood samples were stored on ice, and were centrifuged at 3,000 × g for 30 min within 4 h of obtaining the sample. The plasma obtained by centrifugation was stored at 70° C until assay. Urine samples were obtained before and at 0.5, 4, 8, and 24 h after the last actuation of albuterol, their volume recorded, and aliquots (50 ml) stored at 70° C until assay. The subjects returned within 1 wk of the initial visit for a repetition of the study using the other randomly assigned inhalation device (MDI or MDI and holding chamber).

Assay of Albuterol's Enantiomers

The (R)- and (S)-enantiomers of albuterol (-[(t-butylamino)- methyl]-4 hydroxy-m-xylene---diol) were a gift from Glaxo Wellcome Research, Inc. (Research Triangle Park, NC). Racemic albuterol and bamethane (-[butylamino] methyl-p-hydroxy benzyl alcohol) were purchased from Sigma (St. Louis, MO). Bond Elute Certify columns for solid phase extraction were obtained from Varian Analytical (Harbor City, CA). Methylene chloride, isopropanol, and methanol were chromatographic grade (Fisher Chemical Co., Itasca, IL). All other chemicals used for analysis were obtained from standard chemical suppliers and were of analytical grade.

Standards and controls. Stock solutions of 50 ng/ml of bamethane, racemic albuterol and (R)- and (S)-enantiomers of albuterol were prepared from weighed-out pure powder. The powder was dissolved in 0.01 M monochloroacetic acid and stored in dark plastic bottles at room temperature. Stock solutions were stable for several months under such conditions. Each day, unextracted standards consisting of 0.5 ng/µl of racemic albuterol and bamethane were dissolved in the mobile phase. A 20-µl sample was injected and analyzed for retention time, peak shape, resolution of the enantiomers, and bamethane. Two separate working solutions of bamethane (30 ng/ml and 600 ng/ml in water) were prepared to provide internal standardization for extracted plasma and urine samples, respectively.

Plasma and urine extraction. Plasma samples of 1 ml plus 0.5 ml (30 ng/ml) of bamethane or urine samples of 1 ml and 0.5 ml (600 ng/ml) of bamethane were diluted with 4 ml of 0.06 M phosphate buffer (pH 9.1) and shaken gently in borosilicate glass tubes. After allowing the samples to set for 10 min, they were centrifuged at 5° C in a refrigerated centrifuge at 2,500 × g for 15 min. Accompanying each sample batch were two in-house controls made by reconstituting albuterol-free plasma with aqueous solutions of 5 ng/ml and 10 ng/ml of racemic albuterol. For urine, control samples contained 100 ng/ml and 150 ng/ ml of racemic albuterol. Specimens and control samples were processed analytically in the same manner. The extraction of albuterol and bamethane on solid-phase extraction columns followed the procedure reported previously (12). Nitrogen-dried extracts were reconstituted with 40 µl of the mobile phase, and 20 µl was injected for analysis.

Chromatography. The high-performance liquid chromatography (HPLC) system consisted of a Varian 9010 solvent delivery system (Varian 9010 LC pump; Varian Instruments, Walnut Creek, CA) and a LC-4B electrochemical detector (Bioanalytic Systems, West Lafayette, IN). The output of the detector was linked to a Star Chromatography Work Station (Varian, Harbor City, CA). A 150 × 4.6 mm chiral-CBH (cellobiohydrolase) column (Crom Tech, Hägersten, Sweden, distributed by Regis, Morton Grove, IL) was used. The column was packed with 5-mm spherical silica particles, on which a stable enzyme (cellobiohydrolase) was immobilized to create the stationary phase. The mobile phase consisted of 35 mM phosphate, 100 µM ethylenediaminetetraacetic acid (EDTA), pH 7.0 containing 0.25% isopropanol in H₂O (final concentration). A Rheodyne injector (Model #7725; Rheodyne LP, Cotati, CA) with an injection loop of 20 µl was used to inject the mobile phase. The flow rate was held at 0.6 ml/min. For electrochemical detection, the oxidizing electrode was held at 0.92 V and the detector sensitivity was set at 20 nanoamperes for full scale (nAMPFS). The solvent was held in the recycling mode to provide a better signal-to-noise ratio.

Creatinine assay. A standard kit which employs the Jaffe picrate photometric calorimetric method with the ILAB-900 chemistry analyzer (Lexington, MA) was used to assay creatinine in urine.

Data Analysis

Data are expressed as mean ± SE. Plasma levels of the unchanged (R)-enantiomer, (S)-enantiomer, and total albuterol were plotted over time. For each enantiomer, the peak plasma concentration (C_max) and the time to reach peak concentration (T_max) were obtained directly from the data. The area under the curve (AUC) of the plasma concentration from 0 to 240 min was determined by standard procedures (18). The ratio of unchanged (R)- and (S)-enantiomers in urine (R/S ratio) was calculated for each time point of urine collected. To determine the effect of using the holding chamber, we compared the values of the various parameters obtained by using a MDI and holding chamber with those obtained by using a MDI alone. The variables were compared by one-way analysis of variance (ANOVA) or Student's t test (2-tailed) using the Microcal Origin 4.10 software (Microcal Software, Inc., Northampton, MA). Significance was established at a p value < 0.05.

	RESULTS

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Validation of Assay

The assay for albuterol was highly sensitive and selective. The retention times of the pure (S)- and pure (R)-enantiomer were 7.42 and 8.32 min, respectively. Distinct peaks of the (R)- and (S)-enantiomers were observed after injection of racemic albuterol. The reliable limit of quantitation was 0.25 ng/ml for each enantiomer. The precision of analysis for 2.5 ng/ml of racemic albuterol in drug-free plasma (n = 12) was 1.3 ± 0.04 and 1.3 ± 0.03 ng/ml for the (R)- and (S)-enantiomers, respectively. For control samples consisting of 5 and 10 ng/ml of racemic albuterol in drug-free plasma (n = 7), the values of (R)- enantiomer on different days of analysis were 2.8 ± 0.2 and 5.6 ± 0.4 ng/ml, respectively, and the values for the (S)-enantiomer were 2.7 ± 0.3 and 5.5 ± 0.4 ng/ml, respectively.

Effect of a Holding Chamber on Enantiomer Disposition

The profiles of the (R)- and (S)-enantiomers were compared after administration of racemic albuterol by a MDI alone versus a MDI and holding chamber. The C_max, t_max, and the systemic bioavailability (AUC) of the enantiomers were similar for both methods of administration (Table 1). It was not possible to reliably calculate the elimination half-life of the (R)- and (S)-enantiomers, because of the contribution by gastrointestinal absorption to the excretion phase of the plasma level profile. When the plasma levels of the (R)- and (S)-enantiomers were followed over time, the pattern of absorption and elimination with a MDI was similar to that with a MDI and holding chamber except that the increases in (R)- and (S)- enantiomer levels observed at 120 min after racemic albuterol inhalation from a MDI were not seen after inhalation from a MDI and holding chamber. For the total 24-h urine collection, the R/S ratio for a MDI alone did not differ from that of a MDI and holding chamber: 0.78 ± 0.09 and 0.80 ± 0.08, respectively (p = 0.87). The R/S ratios at 0.5, 4, or 8 h were also similar for both methods of albuterol administration (Table 2). Therefore, the data from the two study days were pooled for further analysis.

Plasma Levels of Enantiomers

The (R)- and (S)-enantiomers of racemic albuterol were rapidly absorbed and reached peak concentrations within 30 min of administration (Figure 1). The levels of the enantiomers declined up to 60 min. No further decline in the plasma levels occurred, at least up to 240 min (Figure 1). The systemic bioavailability of the (S)-enantiomer was significantly lower than that of the (R)-form: the C_max and AUC for the (S)-enantiomer were approximately half those of the (R)-form (p < 0.05 and p < 0.01, respectively); and the t_max of the (S)-enantiomer was longer than that of the (R)-enantiomer (p < 0.05; Table 3).

View larger version (15K): [in this window] [in a new window]   Figure 1.   Plasma levels of (R)- and (S)-enantiomers in venous blood after administration of 6 puffs (0.54 mg) of albuterol in 10 healthy subjects. Because the profiles of plasma levels with a MDI and with a MDI and holding chamber were similar, data from the two study days were combined, resulting in n = 20. Both the (R)- and the (S)-enantiomer were rapidly absorbed from the lung, with peak plasma levels being attained within 30 min of inhalation. Plasma levels of the (S)-enantiomer (open squares) were lower than those of the (R)-enantiomer (closed squares) for up to 4 h after albuterol administration (p < 0.05). Bars represent SE.

Urinary Excretion of Enantiomers

Thirty minutes after administration of albuterol, the mean value for the ratio of the unchanged (R)- to (S)-enantiomer (R/S) in urine was 1.3, indicating a greater initial excretion of the unsulfated (R)-enantiomer (Figure 2). Within the first 30 min, the urinary clearance of the free (R)-enantiomer was similar to that of the free (S)-enantiomer (1.7 ± 0.4 versus 1.2 ± 0.3 ng/mg creatinine/ml, respectively; p = 0.3). The higher excretion of the unchanged (R)-enantiomer in the urine paralleled the plasma levels, reflecting the greater initial absorption of the (R)-enantiomer into the blood after administration of racemic albuterol with a MDI (Table 3). Over the next 8 h, the R/S ratio declined to < 1 and remained < 1 at 24 h; by 24 h, albuterol was detectable in urine in only four of the 10 subjects.

View larger version (11K): [in this window] [in a new window]   Figure 2.   The ratio of unchanged (R)- and (S)-enantiomer excretion in urine (R/S ratio) over time. Subjects received 6 puffs of albuterol (0.54 mg) with a MDI or MDI and holding chamber. Because the profiles of urinary levels with a MDI and with a MDI and holding chamber were similar, data from the two study days were combined, resulting in n = 20. The R/S ratio exceeded 1 at 0.5 h after albuterol inhalation and was less than 1 at 4, 8, and 24 h. Bars represent SE.

	DISCUSSION

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Following administration of racemic albuterol from a MDI alone or a MDI and holding chamber, the systemic bioavailability of the (S)-enantiomer was approximately one-half that of the (R)-enantiomer for up to 4 h after inhalation (Figure 1); that is, the (R)-enantiomer was rapidly absorbed into the systemic circulation, whereas the (S)-enantiomer was retained preferentially in lung tissues. These findings may help to explain the adverse effects noted after regular use of inhaled albuterol in patients with airway obstruction.

The enantioselective disposition of albuterol has been studied by only a few investigators (6, 19). After oral, rectal, or intravenous administration of racemic albuterol, plasma levels of the (S)-enantiomer are 2 to 3 times higher than those of the (R)-form owing to a 7- to 10-fold faster intestinal and first-pass hepatic metabolism of the latter (8, 10). After nebulization of sequentially doubling doses of racemic albuterol, Lipworth and coworkers (6) found that plasma levels of the (S)-enantiomer were almost double those of the (R)-form, in contrast to our findings. The profiles of the plasma levels after inhalation of albuterol differ depending on whether it is administered by nebulization or a MDI. After administration of nebulized racemic albuterol, Lipworth and coworkers (6) reported peak plasma levels of the (R)- and (S)-enantiomers that were 2 to 40 times lower than the peak levels in our subjects who received racemic albuterol from a MDI, despite a 3-fold smaller dose with the MDI. Moreover, the total albuterol levels after nebulization (6, 20) were 5 to 10 times lower than those after use of a 6- to 10-fold smaller dose with a MDI (12, 21). Peak plasma albuterol levels were 6-fold higher with use of a MDI and spacer than with a nebulizer (22). To reach peak plasma levels comparable to those achieved with inhalation of 0.54 mg of albuterol by a MDI in our study, an average adult would need to receive approximately 10 mg by a nebulizer (23); that is, systemic bioavailability of racemic albuterol is much lower when administered by nebulization versus a MDI. Moreover, the absence of a sharp peak in plasma levels within 30 min of administration by nebulization, coupled with the higher levels of the (S)- relative to the (R)-enantiomer (6, 11, 22), suggest that gastrointestinal absorption of racemic albuterol makes a greater contribution to the plasma levels with nebulizer therapy versus a MDI.

More than 80% of the nominal dose of racemic albuterol from a MDI is deposited in the oropharynx (15) and subsequently absorbed from the gastrointestinal tract (24). Thus, 6 puffs of racemic albuterol (0.54 mg) with a MDI should deliver approximately 0.4 mg to the gastrointestinal tract. Because an oral dose of 2 mg of racemic albuterol achieves peak serum levels of 3 to 4 ng/ml (24), absorption of 0.4 mg of racemic albuterol by the gastrointestinal tract is expected to produce a serum level of less than 1.0 ng/mlconsiderably below the peak total albuterol concentrations of 10 to 15 ng/ml in our study. Gastrointestinal absorption may have been responsible for curbing the decline in plasma levels at 1 h (Figure 1), because peak plasma levels are achieved at 1 to 3 h after oral administration of racemic albuterol (6, 8, 10, 25). That plasma levels of racemic albuterol over the first hour after administration with a MDI are caused by absorption from the lung is supported by our findings with the use of a MDI and holding chamber. Although the use of a holding chamber achieves a 14-fold reduction in gastrointestinal absorption of racemic albuterol compared with a MDI alone (16), it does not influence the profile of plasma levels within the first hour after racemic albuterol inhalation. Similar findings were reported recently by Lipworth and Clark (17). In summary, gastrointestinal absorption does not appear to contribute significantly to the systemic bioavailability of racemic albuterol in the first hour after its administration by a MDI.

After administration of racemic albuterol with a MDI, a sharp peak in the plasma levels occurred at 15 to 30 min, with levels of the (R)-enantiomer being 2 to 3 times higher than those of the (S)-form (Figure 1). The peak plasma levels of the (R)-enantiomer (approximately 8 ng/ml) in the present study correspond to the peak levels of total albuterol in our previous study in healthy subjects given 6 puffs of albuterol with a MDI and holding chamber (12). We also observed similar serum levels after administration of 10 puffs of racemic albuterol to mechanically ventilated patients, in whom the cuffed artificial airway prevents gastrointestinal absorption of the drug (12). Likewise, Anderson and coworkers found an early peak in plasma levels (12.6 ± 2.2 min) after administration of racemic albuterol with a MDI (26). The effects of inhaled albuterol on heart rate, tremor, and serum potassium are mediated solely by the (R)-enantiomer (6), and the time course of these extrapulmonary effects is consistent with the rapid absorption of the (R)-enantiomer into the systemic circulation (Figure 1).

The urinary excretion of unchanged albuterol after inhalation of the racemate can be used to estimate the systemic bioavailability of the drug (13, 14). Hindle and Chrystyn suggested that the drug fraction absorbed from the lung appears in the urine almost immediately, and most of it is excreted within 30 min of inhalation (13, 14). In accordance with earlier findings (8), we observed that the renal clearance of the (R)- and (S)-enantiomers was similar. Therefore, the ratio of the unchanged enantiomers in urine should parallel the plasma levels. After intravenous administration of racemic albuterol, the R/S ratio in urine declined from ~ 1 at 1 h to ~ 0.6 over 8 to 12 h (9, 19). In contrast, the urinary R/S ratio ranged from 0.2 to 0.4 after oral administration of racemic albuterol (7, 9). The urinary R/S ratio after inhalation of racemic albuterol from a MDI has not been reported. We found that the ratio of unsulfated R/S was > 1 in the urine at 0.5 h after inhalation of racemic albuterol (Figure 2), reflecting the higher plasma level of the (R)-enantiomer during this period. The subsequent decrease in the unchanged R/S ratio at 4, 8, and 24 h after racemic albuterol presumably reflects the faster metabolism of the systemically absorbed (R)- compared with the (S)-enantiomer (7). A urinary R/S ratio > 1 at 0.5 h reaffirms our view that the (R)-enantiomer of racemic albuterol is absorbed from the lung into the systemic circulation in greater amounts than the (S)- form.

We can only speculate about the mechanism underlying the difference in the disposition of the (R)- and (S)-enantiomers after inhalation of racemic albuterol from a MDI. Among the possible mechanisms, mucociliary clearance, phagocytosis by macrophages, or particle dissolution are unlikely to explain the rapid development of differences in the plasma levels of the two enantiomers (Figure 1). Preferential binding of the (S)-enantiomer to plasma proteins is unlikely, because such an effect is not observed with intravenous racemic albuterol (8), and because racemic albuterol shows minimal binding to plasma proteins (27). The (R)-enantiomer may interfere with the transfer of the (S)-enantiomer across the epithelial-endothelial barrier in the lung as observed with the clearance of the enantiomers of terbutaline by the kidney (28). However, we found no difference in the renal clearance of the (R)- and (S)-enantiomers. Finally, the enantiomers of propranolol, a -adrenoceptor antagonist, exhibit differences in their tissue binding (29), and the enantiomers of several -agonists exhibit differences in their affinity to bind -receptors in various tissues of the body (30). In sum, a greater binding of the (S)- enantiomer to lung tissues is probably responsible for the lower plasma levels of the (S)-enantiomer compared with the (R)-form after inhalation of racemic albuterol.

Administration of single doses of (S)-albuterol to previously sensitized guinea pigs increases airway reactivity to a variety of bronchoconstrictor stimuli (4). In patients with asthma, the effects of (S)-albuterol are less clear-cut. An increase in bronchial hyperreactivity after inhalation of a single dose of (S)-albuterol was reported in a pilot investigation (31), but not confirmed in a double-blind, randomized, four-way crossover trial by Cockcroft and Swystun (5). The latter investigators (5) expressed concerns, however, that their (S)-albuterol may have been contaminated with the (R)-enantiomer. Moreover, the development of adverse effects may require regular use of racemic albuterol for several weeks (1). An increase in airway reactivity due to the (S)-enantiomer may not be detectable soon after commencing albuterol treatment because the effect is masked by the bronchodilator action of the (R)- enantiomer in the racemate. Previous investigators believed that preferential metabolism of the (R)-enantiomer by phenolsulfotransferase enzymes would lead to a progressive increase in the proportion of the (S)-enantiomer, such that the (S)-form might be increased 10-fold after regular administration of racemic albuterol (32). Racemic terbutaline, however, promotes increased susceptibility to histamine (33), even though its (S)-enantiomer is about twice as susceptible to sulfate conjugation as is the (R)-enantiomer (34). Despite its lack of a pathway for sulfate conjugation, racemic albuterol induces airway hyperresponsiveness in guinea pigs (35). Accordingly, differences in enantiomer metabolism are unlikely to explain fully the development of airway hyperreactivity after long-term use of racemic albuterol. Our study suggests an alternative explanation: the development of increased airway hyperreactivity could be due to preferential retention of the (S)-enantiomer leading to its gradual accumulation in the lung with long-term use of racemic albuterol.

In summary, we have shown for the first time that after the administration of racemic albuterol with a MDI, the disposition of the (S)-enantiomer differs from that of the (R)-enantiomer. The plasma levels of the (S)-enantiomer were significantly lower than those of the (R)-enantiomer for up to 4 h. We infer that the decrease in systemic bioavailability of the (S)-enantiomer to approximately half that of the (R)-enantiomer after inhalation of racemic albuterol in healthy subjects is caused by a preferential retention of the (S)-enantiomer in the lung. Further investigations are needed to determine if the increased airway hyperreactivity after regular use of racemic albuterol in patients with asthma is the result of a gradual accumulation of the (S)-enantiomer in the lung.