Introduction

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Propranolol is a nonspecific -blocker used, as a racemic mixture of (S)-()- and (R)-(+)-propranolol, for the treatment of hypertension, angina, and cardiac arrhythmias (Ridell et al., 1987). The pharmacodynamic profiles of the propranolol enantiomers present some striking differences; for instance, (S)-()-propranolol is approximately 100 times more potent as a -blocker than its antipode. On the other hand, both (S)-()- and (R)-(+)-propranolol exert class II antiarrhythmic activity (Barrett and Cullum, 1968; Rahn, 1983; Stoschitzky and Lindner, 1990). The kinetics of propranolol enantiomers are also stereoselective (Olanoff et al., 1986; Lalonde et al., 1988; Stoschitzky et al., 1992; Egginger et al., 1994; Walle et al., 1983a) and, as a result, plasma concentrations of (S)-()-propranolol are higher than those of the antipode, i.e. the ratio of (S)-()-propranolol to (R)-(+)-propranolol AUC₀ values is >1. The mechanism underlying the stereoselective elimination of propranolol enantiomers remains poorly understood. Stereoselective elimination appears to be influenced by the route of administration of propranolol. For instance, after the iv injection of 5 mg of racemic propranolol the kinetics of the propranolol enantiomers were almost identical, but when 40 mg were given po the ratio of (S)-()-propranolol to (R)-(+)-propranolol AUC₀ values averaged 1.48 (Von Bahr et al., 1982). Olanoff et al. (1984) reported that, after the iv administration of a 0.1 mg/kg dose of a racemate of propranolol, the average ratio of (S)-()-propranolol to (R)-(+)-propranolol AUC₀ values was 1.17. On the other hand, when racemic propranolol was administered po, in single doses ranging from 40 mg to 160 mg or in multiple doses to steady state, the average ratio of (S)-()-propranolol to (R)-(+)-propranolol AUC₀ values ranged from 1.32 to 1.77 (Olanoff et al., 1986; Lalonde et al., 1988; Stoschitzky et al., 1992; Egginger et al., 1994). These data suggest that, in addition to route dependence, the stereoselective elimination of propranolol enantiomers appears to be modulated by the dose of racemic propranolol administered.

The apparent route- and dose-dependent stereoselective kinetics of propranolol enantiomers could be associated with stereoselective presystemic metabolism in the epithelial cells of the intestine and/or in the liver (Krishna and Klotz, 1994; du Souich et al., 1995). This hypothesis is based on the fact that propranolol is subjected to important first-pass metabolism that takes place in these two organs (du Souich et al., 1995). The present study aimed to document in vivo and in vitro whether the intestinal mucosa and/or the liver is responsible for the stereoselective kinetics of propranolol enantiomers. The effects of increasing doses of propranolol on the stereoselective kinetics of its enantiomers were also assessed.

	Materials and Methods

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In Vivo Studies. Pharmacokinetic studies were performed with male New Zealand rabbits weighing approximately 2.7 kg (Ferme Cunicole, Mirabel, Québec, Canada). Rabbits were selected as the animal model because the metabolism of propranolol in this species closely resembles that in humans (Walpole et al., 1990). The rabbits were acclimatized in their cages for at least 1 week before any experimental procedure was undertaken. The central artery and the lateral vein of an ear were cannulated with a Butterfly-25 cannula (Venisystems; Abbot Ireland, Sligo, Ireland), which contained 0.02% of heparin dissolved in a 0.9% NaCl solution to maintain patency.

Binding of Propranolol Enantiomers to Plasma Proteins. To rule out stereoselective differences in plasma protein binding, the binding of (S)-()- and (R)-(+)-propranolol to plasma proteins was assessed by ultrafiltration. Nine samples (15 ml) of pooled plasma from rabbits were spiked with 11.25, 5.625, or 1.40 µg of the individual enantiomers (final plasma concentrations of the enantiomers, 750, 375, and 94 ng/ml, respectively) or with 22.5, 11.25, or 2.8 µg of the racemate (final plasma concentrations of propranolol, 1500, 750, and 188 ng/ml, respectively). The samples were incubated for 20 min in a shaking bath at 37°C, and 1 ml of plasma was used to determine the concentrations of propranolol enantiomers. The remaining plasma was centrifuged at 3500 rpm, in Centriplus system devices (Amicon; W.R. Grace & Co., Beverly, MA), for 45 min at 25°C. The concentrations of unbound enantiomers were assayed in 1 ml of the resulting ultrafiltrate.

In Vitro Studies. The in vitro studies aimed to support the in vivo results by demonstrating the ability of extrahepatic organs and liver to metabolize propranolol. Rabbits (N = 6) were sacrificed by cervical dislocation, and the first 30 cm of the intestine, the right lobe of the liver, the kidneys, and the lungs were removed. The epithelial cells of the small intestine were isolated by gentle scraping of the mucosa. The medulla was removed from the kidneys. The tissues and intestinal mucosa cells were carefully washed and diluted to 25% with 1.15% KCl/0.05 M phosphate buffer (pH 7.4) and were finally homogenized with a Polytron homogenizer (Brinkman, Rexdalle, Sweden). The homogenates were centrifuged at 10,000g, and 1 ml of the supernatant was used to assess the ability of these tissues to metabolize propranolol, after addition of 0.5 ml of an aqueous solution with 3.75 µg/ml of the individual enantiomers (final concentration, 2.9 µM; Sigma) or 0.5 ml of a solution with 7.5 µg/ml of the racemate (final concentration, 5.8 µM). The mixture was incubated at 37°C, and the reaction was started by addition of 1 ml of 0.05 M phosphate buffer containing NADP (1.3 µM; Sigma), glucose-6-phosphate (20 µM; Sigma), nicotinamide (100 µM; Sigma), and MgCl₂ (50 µM). Identical in vitro studies were carried out with a 37.5 µg/ml of the individual enantiomers (final concentration, 29 µM) or 75 µg/ml of (RS)-propranolol (final concentration, 58 µM). The choice of propranolol concentrations was based on the fact that, after po administration, propranolol concentrations in the liver are 20-50-fold greater than those in plasma (Hayes and Cooper, 1971). Propranolol enantiomers were assayed in samples of 100 µl that were withdrawn every 1 min from liver homogenates and every 30 min from the other tissue homogenates.

Assay of Propranolol Enantiomers. (S)-()- and (R)-(+)-Propranolol were separated by HPLC after derivatization with (R)-PEIC² (Fluka, Switzerland), as described elsewhere (Spanh-Langguth et al., 1991), with some modifications. Briefly, 100 µl of a solution containing the internal standard (50 µg/ml metoprolol in methanol), 1 ml of carbonate buffer (pH 10.5), and 10 ml of diethyl ether were added to 0.5- or 1.0-ml aliquots of plasma, in centrifuge tubes with Teflon-coated, sealed, screw-caps. After mixing for 20 min and centrifugation at 2000 rpm for 5 min, 9 ml of the organic phase were transferred to test tubes and evaporated under a nitrogen stream at ambient temperature. The residue was reconstituted in 200 µl of methanol with 50 µl of (R)-PEIC (0.1% in dichloromethane), and the mixture was incubated at 4°C for 20 min. After evaporation under a nitrogen stream, the residue was reconstituted in 200 µl of mobile phase and 20-80 µl were injected into the HPLC system.

Data and Statistical Analyses. The (S)-()- and (R)-(+)-propranolol AUC₀ values were estimated by means of the trapezoidal method. Standard noncompartmental equations were used to calculate the systemic clearance, terminal t_1/2, and predicted apparent volume of distribution at steady state. Assuming that the absorption was complete, the apparent po clearance (Cl_o) values for (S)-()- and (R)-(+)-propranolol were calculated using the equation CL_o = D_o/AUC_o, where D_o is the po dose of the enantiomers and AUC_o is the AUC₀ value for the enantiomers given po (Gibaldi and Perrier, 1982).

	Results

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In Vivo Studies. After the po administration of 40, 80, or 120 mg/kg racemic propranolol, peak plasma concentrations of both enantiomers were observed at about 20 min (fig. 1) and then declined with similar terminal t_1/2 values. Compared with the 40 mg/kg dose, the po administration of 80 and 120 mg/kg doses of the racemate generated (S)-()- and (R)-(+)-propranolol plasma concentrations higher than predicted. Consequently, the AUC₀ values normalized to the 40, 80, and 120 mg/kg doses for (S)-()-propranolol were 33.3, 65.4, and 144.2 ng·min·kg/ml/mg, and for (R)-(+)-propranolol were 30.5, 41.1, and 86.7 ng·min·kg/ml/mg, respectively.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Mean plasma concentration-time curves for (S)-()-propranolol () and (R)-(+)-propranolol () after the po administration of 40, 80, and 120 mg/kg doses of racemic propranolol to conscious rabbits (N = 4 or 5/dose). Vertical bars, SE.

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View larger version (20K): [in this window] [in a new window]   Fig. 2.   Mean plasma concentration-time curves for (S)-()-propranolol () and (R)-(+)-propranolol () after the iv administration of 0.5 and 10 mg/kg doses of racemic propranolol to conscious rabbits (N = 5/dose). Vertical bars, SE.

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Binding of Propranolol Enantiomers to Plasma Proteins. The fraction of unbound (R)-(+)-propranolol in plasma (0.33 ± 0.01) was not different from that of (S)-()-propranolol (0.34 ± 0.01), and the increase in the plasma concentrations of propranolol enantiomers (i.e. from 93.75 to 750 ng/ml) was not associated with elevations in their unbound fractions. When the enantiomers were added as a racemic mixture, their unbound fractions increased slightly, without stereoselective differences [0.39 ± 0.02 and 0.40 ± 0.02 for (R)-(+)-propranolol and (S)-()-propranolol, respectively]. High concentrations of the racemic drug (i.e. 1500 ng/ml) did not modify the unbound fractions of propranolol enantiomers.

In Vitro Studies. Intestinal mucosa and liver homogenates, but not lung and renal cortex supernatants, metabolized (RS)-propranolol. After incubation of the racemate at 5.8 µM with epithelial cells of the intestine, propranolol enantiomer concentrations were diminished by 84%; the rate constant of elimination for (R)-(+)-propranolol was faster (p < 0.05) than that for (S)-()-propranolol (fig. 3A). This difference persisted when the racemate was incubated at 58 µM, although the decrease in propranolol enantiomer concentrations was 45% and the rate of elimination of the enantiomers was almost 10 times slower than at the concentration of 5.8 µM. When the enantiomers were incubated individually with the intestinal mucosa supernatant, the rate constant of elimination for (R)-(+)-propranolol was almost 3-fold faster (p < 0.05) than that for (S)-()-propranolol at both concentrations (fig. 3A). The rate constant of elimination for (R)-(+)-propranolol was greater when (R)-(+)-propranolol was incubated individually than when the racemate was used (fig. 3A).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Rate constants of elimination for (S)-()-propranolol () and (R)-(+)-propranolol () in the 10,000g supernatant of homogenates of epithelial cells of the intestinal mucosa (A) and the liver (B) after incubation of 5.8 or 58 µM concentrations of racemic propranolol or 2.9 or 29 µM concentrations of the individual enantiomers. Vertical bars, SE. *, p < 0.05, compared with (S)-()-propranolol.

	Discussion

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The present study demonstrates that, at low po (40 mg/kg) or iv (0.5 mg/kg) doses of (RS)-propranolol, the kinetics of the enantiomers are very similar. When po doses of (RS)-propranolol are increased to 80 and 120 mg/kg, there is a nonlinear increase in the AUC₀ values for both enantiomers. However, the increase in the AUC₀ value for (S)-()-propranolol is greater than that for (R)-(+)-propranolol, indicating that propranolol enantiomers exhibit stereoselective kinetics after high po doses. Similar results have been reported for humans receiving an iv dose of 5 mg or a po dose of 40 mg, i.e. only after the po dose, the AUC for (S)-()-propranolol was greater than that for (R)-(+)-propranolol (Von Bahr et al., 1982). The iv dose of 10 mg/kg (RS)-propranolol generates a nonlinear increase in the AUC₀ values for both enantiomers but, in this case, the AUC₀ for (R)-(+)-propranolol is greater than that for the antipode. In vitro, at 5.8 µM (RS)-propranolol, the rate of elimination of (R)-(+)-propranolol is faster than that of (S)-()-propranolol with epithelial cells of the intestine but not liver cells. At 58 µM, the elimination of propranolol enantiomers is stereoselective in both the intestine and the liver, although the rates of elimination are reduced. These results strongly suggest that the stereoselective kinetics of the enantiomers of propranolol are route- and dose-dependent.

Several factors, such as plasma protein binding and blood flow to the organs, may influence the systemic clearance of drugs. In the present study, (RS)-propranolol plasma protein binding was not stereoselective, and the findings of stereoselective elimination of propranolol enantiomers in vitro confirm that plasma protein binding is not at the origin of the stereoselective elimination. This latter argument can also be applied to eliminate changes in blood flow as a cause for the stereoselective elimination of propranolol enantiomers.

Three mechanisms could explain the stereoselective metabolism of propranolol in the intestine and in the liver, i.e. differences in the intrinsic clearances of the enantiomers, competitive inhibition of (S)-()-propranolol elimination by its antipode, or preferential saturation of the metabolism of (S)-()-propranolol over that of (R)-(+)-propranolol. In intestinal epithelial cells, the rate of elimination of (S)-()-propranolol was not influenced by the presence of (R)-(+)-propranolol and (R)-(+)-propranolol was always eliminated more quickly than (S)-()-propranolol, suggesting that in the intestine the intrinsic clearance of (R)-(+)-propranolol is greater than that of its antipode. In the liver, with 5.8 µM racemate, the rates of elimination of the two enantiomers were similar. On the other hand, with 58 µM, the rate of elimination of (S)-()-propranolol was slower than that of (R)-(+)-propranolol, suggesting that in the liver (RS)-propranolol stereoselectivity is associated with greater metabolic saturation of (S)-()-propranolol, compared with (R)-(+)-propranolol. Supporting this premise, it has been shown that an immediate-release po formulation of verapamil generates higher plasma concentrations of (S)-()-verapamil than those measured when the racemate is administered in a sustained-release formulation, indicating that the rate of absorption of (RS)-verapamil determines differences in verapamil plasma concentrations great enough to produce a preferential saturation of (S)-()-verapamil elimination and therefore stereoselective kinetics (Karim and Piergies, 1995).

In the present study, the stereoselective clearance of (RS)-propranolol is apparent only when high po doses of the racemate are administered. In vitro, in epithelial cells of the small intestine the kinetics of propranolol enantiomers are stereoselective at concentrations of the racemate lower than those required in the liver. However, the ability of the liver to biotransform the enantiomers of propranolol is >10 times greater than that of the small intestine (du Souich et al., 1995). Hence, even if at low po doses of (RS)-propranolol the intestine might generate stereoselective kinetics of the enantiomers, the stereoselectivity would not be apparent systemically, because the liver, at that dose, does not contribute to the stereoselective elimination of propranolol enantiomers. To reveal stereoselective kinetics of propranolol enantiomers in vivo, high po doses and consequently high hepatic concentrations are needed.

The present results show that the ratio of the AUC₀ for (S)-()-propranolol to the AUC₀ for (R)-(+)-propranolol increased from 1.00 after the iv injection of 0.5 mg/kg to 1.14, 1.32, and 1.66 after the po administration of 40, 80, or 120 mg, respectively. These ratios, reflecting the amplitude of stereoselectivity, were estimated at concentrations included in the range of those observed clinically in humans (Genco and Green, 1986). In addition, the results of the present study are similar to those reported for humans; for instance, after iv doses of 5-10 mg of (RS)-propranolol, the ratio of the AUC₀ for (S)-()-propranolol to the AUC₀ for (R)-(+)-propranolol is smaller than that estimated after the po administration of 40 mg of (RS)-propranolol (Olanoff et al., 1986; Lalonde et al., 1988; Stoschitzky et al., 1992; Egginger et al., 1994; Von Bahr et al., 1982; Silber et al., 1986). We are tempted to speculate that in humans, as in the present study, dose-dependent differences in stereoselectivity result from the route of administration, i.e. the iv doses of (RS)-propranolol are insufficient to saturate the metabolism of (S)-()-propranolol in the liver.

Compared with the 0.5 mg/kg iv dose of (RS)-propranolol, the 10 mg/kg dose reduced the apparent volume of distribution of (R)-(+)-propranolol. Because binding of (R)-(+)-propranolol to plasma proteins is concentration-independent up to 750 ng/ml, we must assume that, after the iv injection of (RS)-propranolol into rabbits, at high plasma concentrations the binding of (R)-(+)-propranolol to tissues is more rapidly saturated than that of the antipode.

The iv injection of 10 mg/kg (RS)-propranolol significantly decreased the clearance of both enantiomers, implying that at this dose level the kinetics of propranolol enantiomers are zero-order. Surprisingly, the ratio of the (S)-()-propranolol AUC₀ to the (R)-(+)-propranolol AUC₀ was 0.82. The differences in the propranolol enantiomer stereoselectivity produced by the different routes of administration may tentatively be explained by the heterogeneous distribution of isozymes in the liver. The rates of sulfo- and glucuronoconjugation (Pang and Terrell, 1980; Pang et al., 1994), deethylation (Pang et al., 1988), and hydrolysis (Pang et al., 1991) in the periportal region differ from those observed in the perihepatic artery region of the liver. Therefore, it is conceivable that, depending upon the route of arrival (portal vein or hepatic artery), one enantiomer might be preferentially metabolized over its antipode. Supporting such an hypothesis, conjugation is rather selective for (S)-()-propranolol (Silber et al., 1986; Walle et al., 1983b), and the formation of desisopropylpropranolol is R-(+)-enantioselective at low substrate concentrations and S-()-enantioselective at high concentrations (Marathe et al., 1994). Indeed, additional studies are required to explain why high iv doses reverse propranolol stereoselectivity.

In conclusion, the in vitro studies show that in the intestine the stereoselective kinetics of (RS)-propranolol are secondary to differences in the intrinsic clearances of the enantiomers, whereas in the liver there is preferential saturation of the metabolism of (S)-()-propranolol. In vivo, low doses of (RS)-propranolol administered iv or po do not generate stereoselective kinetics of the enantiomers of propranolol, possibly because the contribution of the liver overshadows the contribution of the intestine. On the other hand, without changing the protein binding, high po doses generate zero-order kinetics and more profoundly depress (S)-()-propranolol metabolism, producing stereoselective differences in the rates of elimination of the enantiomers. Finally, high iv doses, generating zero-order kinetics, induce small stereoselective differences in the elimination of the enantiomers of propranolol, but in this case (S)-()-propranolol is eliminated more rapidly than (R)-(+)-propranolol.