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Stereoselective Pharmacokinetics of Bisoprolol After Intravenous and Oral Administration in Beagle Dogs
Stereoselective Pharmacokinetics of Bisoprolol after Intravenous and Oral Administration in Beagle Dogs YUJI HORIKIRIX, TAKEHIKO SUZUKI,
AND
MASAKAZU MIZOBE
Received November 4, 1996, from the Pharmaceutics Research Laboratory, Tanabe Seiyaku Company, Ltd., 16-89, Kashima 3-chome, Yodogawa-ku, Osaka 532, Japan. Accepted for publication February 10, 1997X. Abstract 0 The stereoselective pharmacokinetics of bisoprolol, a highly β1-selective adrenoceptor blocking agent, was studied in dogs. After intravenous and oral administration of the racemate, there was a difference in the plasma concentration between S(−)- and R(+)-bisoprolol. The area under the curve (AUC) of concentration versus time of S(−)-bisoprolol was ∼1.5 times higher than that of R(+)-bisoprolol, and the elimination half−life of S(−)-bisoprolol was ∼1.4 times longer than that of R(+)bisoprolol. However, no differences were observed in the volume of distribution, absolute bioavailability, and renal clearance between the two enantiomers. The plasma protein binding of S(−)-bisoprolol was also the same as that of the R(−)-isomer. No chiral inversion or enantiomer− enantiomer interaction was observed, when enantiomers were solely administered via the intravenous route. The comparison of the oxidative metabolic rate of two enantiomers using dog liver microsomes demonstrated that the metabolite was more slowly formed from S(−)- than from R(+)-bisoprolol. Consequently, we concluded that the stereoselective difference in the metabolic clearance between S(−)- and R(+)-bisoprolol caused the difference in the disposition of bisoprolol enantiomers.
Introduction Bisoprolol hemifumarate, [(]-1-{p-[(2-isopropoxyethoxy)methyl]phenoxy}-3-isopropyl-amino-2-propanol-hemifumarate, is a highly β1-selective adrenoceptor blocking agent without membrane-stabilizing activity or intrinsic sympathomimetic activity.1,2 The results of preclinical studies using several animals and clinical studies including healthy human subjects or patients,3-5 indicate that bisoprolol has a high absolute bioavailability (90% of an oral dose) due to its nearly complete enteral absorption (>90%) and a very small firstpass effect (<10%). A long elimination half-life (10-12 h) allows this drug to be used on a once-a-day dosage regimen. Bisoprolol has a chiral center, which gives rise to two optical isomers. The activity of S(-)-bisoprolol to block the β-adrenergic receptors is ∼30-80 times higher than that of R(+)bisoprolol.6 As with most β-adrenergic blocking agents, bisoprolol is marketed as a racemic mixture. Over the past two decades, many studies have indicated that drug enantiomers may have different pharmacodynamic and pharmacokinetic properties as a consequence of stereoselective interaction with optically active biological macromolecules.7-10 In general, the pharmacological activity of a racemic drug is associated predominantly with one enantiomer of the racemic drug. Therefore, it is necessary to evaluate the pharmacokinetic behavior of each enantiomer rather than that of the racemate, to use a racemic drug effectively and safely. In this study, we examined the stereoselective disposition of bisoprolol enantiomers. Also, we elucidated the pharmacokinetic properties of bisoprolol enantiomers in dogs, which are known to exhibit similar pharmacokinetic profiles of X
Abstract published in Advance ACS Abstracts, April 1, 1997.
560 / Journal of Pharmaceutical Sciences Vol. 86, No. 5, May 1997
racemic bisoprolol, such as bioavailability, metabolic pathways, and urinary excretion, as in humans.3
Experimental Section MaterialssS(-)-, R(+)-, and racemic bisoprolol, and its racemic metabolite (M4; {[(]4-(2-hydroxy-3-isopropylaminopropoxy)benzyloxy}ethanol) were obtained as their hemifumarates from E. Merck (Darmstadt, Germany). The purities of both the S(-)- and R(+)enantiomers were >99%, as confirmed by chiral high-performance liquid chromatography (HPLC) resolution. 1-[p-(Tetrahydro-3-furanyl)phenoxy]-3-isopropylamino-2-propanol, used as the internal standard in the analysis of both bisoprolol enantiomers and M4, was also obtained from E. Merck. HPLC-grade hexane, 2-propanol, and pesticide-grade chloroform were obtained from Katayama Chemical Industries Ltd. (Osaka, Japan). The other chemicals and solvents were of reagent grade and were used without further purification. AnimalssThree male beagle dogs, each weighing 13-18 kg, were used. The animals were fasted for 18 h prior to administration and were fed 8 h after dosing. They were allowed water throughout the course of the experiment. The elapsed interval between two separate studies was at least 2 weeks. Dosing ProceduresOne milliliter of the saline solution containing 2.5 mg of racemic bisoprolol or 1.25 mg of each enantiomer was injected directly into the forearm vein of conscious dogs. For oral administration, 5 mg of racemic bisoprolol dissolved in 20 mL of water was given to dogs by gastric intubation with a rubber cannula. The dogs were administered test agents in fixed order. Sample CollectionsApproximately 5 mL of venous blood was taken from each dog with a heparinized, disposable 10-mL blood collection syringe and an 18-gauge needle at 0, 0.05, 0.1, 0.17, 0.25, 0.5, 1, 2, 3, 4, 6, 8, and 24 h after intravenous (iv) injection, and at 0, 0.5, 1, 2, 3, 4, 6, 8, and 24 h after oral administration. The plasma was separated by centrifugation at 1000 × g for 20 min immediately after collection and stored at -25 °C until analyzed. The voided urine was fractionally collected from a metabolic cage during each interval of 0-2, 2-4, 4-6, 6-8, 8-24, and 24-48 h. In addition, the urine was completely emptied with a urinary catheter at the end of each interval. The total volume of urine of each interval was measured, and two 20-mL aliquots were stored at -25 °C. Analysis of Enantiomers and MetabolitesEach enantiomer of bisoprolol in plasma and urine was analyzed by the HPLC method with a chiral column (Chiralcel OD) according to the procedure described previously.11 One of the oxidative metabolites of bisoprolol, M4 (racemate), was determined by the method we developed,12 using a selective and sensitive reversed-phase HPLC with fluorimetric detection. Briefly, samples (1 mL) were alkalinized with NaOH (1 N) to pH of ∼9 and then M4 was extracted with a mixture of chloroform and 2-propanol (9:1). After the organic layer was evaporated to dryness under a nitrogen stream, the residue was reconstituted with a mobile phase containing the internal standard. The internal standard for analysis of M4 was the same as that used for bisoprolol enantiomers. The mobile phase consisted of 20% methanol in a 0.25% triethylamine solution in water (pH 3). The flow rate of 0.5 mL/min and column temperature of 60 °C were maintained throughout the analysis. An octadecylsilane analytical column (Hypersil 5ODS, Shandon, Cheshire, UK) was used. The detection of M4 was monitored with a fluorimetric detector at the excitation wavelength of 228 nm and the emission wavelength of 298 nm. Pharmacokinetic AnalysissPharmacokinetic parameters were determined from the plasma concentration versus time data. The maximum plasma concentration (Cmax) and the time to reach the
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maximum concentration (Tmax) were read directly from the plasma concentration versus time data. The apparent terminal elimination rate constant, k, was calculated by least squares regression analysis on the terminal portion of the log plasma concentration versus time profile. The terminal half-life (t1/2) was calculated by dividing ln 2 by k. The area under the curve of plasma concentration versus time from time zero to the time of last measurable plasma concentration point (AUC0-t) was determined according to the trapezoidal rule. Extrapolation to time infinity (AUC0-∞) was determined by dividing the last measurable plasma concentration by k and adding the result to the corresponding AUC0-t. The total amount of bisoprolol enantiomers excreted in urine from time zero to 48 h (Ae0-48) was the cumulative amount of enantiomer excreted in each sampling time period. The urinary excretion amount of bisoprolol enantiomers from the last urine sampling time extrapolated to time infinity (Ae48-∞) was estimated by dividing the urinary excretion rate at 48 h by K, where K is the elimination rate constant calculated by least squares regression analysis on the terminal portion of semilogarithmic plot of urinary excretion rate versus a midpoint of the urine collection period. The cumulative urinary excretion amount to time infinity (Ae0-∞) was calculated by adding Ae0-48 to Ae48-∞. The total clearance (CLt) was calculated as dose‚F/AUC0-∞, where F is the absolute bioavailability, which was calculated by dividing the AUC0-∞ of oral administration corrected for oral dose (AUC0-∞(po)/dose(po)) by the AUC0-∞ of iv administration corrected for iv dose (AUC0-∞(iv)/Dose(iv)). The renal clearance (CLr) was calculated as Ae0-∞/AUC0-∞. The difference between CLt and CLr was defined as the metabolic clearance (CLm); that is, CLm) CLt - CLr. Distribution volume (Vd) was calculated as the quotient of CLt and k. All pharmacokinetic data were expressed as the mean ( standard deviation (SD). In Vitro Metabolism StudiessA freshly obtained dog liver was excised and immediately placed in three volumes (per wet weight of liver) of ice-cold 1.15% KCl and then homogenized. The homogenate was centrifuged at 9000 × g for 30 min at 4 °C. The supernatant was then centrifuged at 105000 × g for 60 min at 4 °C. The resultant pellet was suspended in 100 mM phosphate buffer (pH7.4). One milliliter of this suspension contained the pellet from 3 g of wet liver. The protein concentration was determined by the Protein Assay Kit (Bio-Rad, CA). Microsomal oxidation reactions were carried out as follows. The reaction mixture (2.5 mL), containing 75 mM MgCl2, 25 mM glucose-6-phosphate, 5 mM NADPH, and 50 mM nicotinamide in 50 mM phosphate buffer (pH 7.4), was incubated with 10 µM S()-bisoprolol or R(+)-bisoprolol (2.5 mL) at 37 °C. The reaction was initiated by addition of 2.5 mL of the aforementioned microsomal suspension. At 1, 3, and 5 h after incubation, an aliquot (1 mL) was transferred to a tube containing NaOH. The metabolite, M4, was determined by the method described in Analysis of Enantiomers and Metabolite. Protein Binding ExperimentssPlasma protein binding of bisoprolol was studied by an ultrafiltration method with drug-free dog plasma samples spiked with bisoprolol (in vitro) and dog plasma samples obtained at 1 h after oral administration of 10 mg of bisoprolol (ex vivo) to two dogs, which were the same dogs as used for the pharmacokinetic study. The plasma sample was immediately transferred to a centrifuged micropartition system device (MPS-3, Amicon, MA) and centrifuged for 15 min at 1000 × g. Spiked plasma samples (200 ng/mL) were incubated for 15 min at 37 °C prior to centrifugation. Preliminary studies had revealed that there is no loss of bisoprolol during ultrafiltration due to membrane binding. The concentrations of bisoprolol enantiomers in the ultrafiltrate and in plasma or spiked plasma samples prior to centrifugation were determined by the HPLC method for analysis of bisoprolol enantiomers, as already described, and the amount of binding (%) to plasma protein was calculated. Statistical AnalysissComparisons between the S(-)- and R(+)bisoprolol pharmacokinetic parameters observed after administration of racemate were assessed by a t test for paired data. Comparisons of administration of pure enantiomer versus racemate were made by an independent measures t test.
Results Plasma Concentration Profiles after Oral AdministrationsThree beagle dogs were each orally administered 5 mg of racemic bisoprolol. The plasma concentration of S-
Figure 1sPlasma concentration−time profiles of bisoprolol enantiomers after oral administration of 5 mg of racemic bisoprolol to beagle dogs. Each point represents the mean ± SD of three dogs. Key: (b) S(−)-bisoprolol; (O) R(+)-bisoprolol. Table 1sPharmacokinetic Parameters of Bisoprolol Enantiomers after Oral Administration of 5 mg of Racemic Bisoprolol to Beagle Dogsa Parameter
S(−)
R(+)
Cmax (ng/mL) 51.9 ± 9.0 43.3 ± 3.7 Tmax (h) 1.33 ± 0.58 1.67 ± 0.58 AUC0-∞ (ng ‚ h/mL) 585 ± 76 435 ± 70 t1/2 (h) 6.5 ± 1.7 5.3 ± 1.6 Availability (F, %)c 89.8 ± 12.8 93.3 ± 11.6
S/R ratio
Significanceb
1.19 ± 0.12 0.83 ± 0.29 1.35 ± 0.09 1.25 ± 0.16 0.96 ± 0.03
NS NS p < 0.05 NS NS
a Each value represents the mean ± SD of three dogs. b Significance. Based on the paired t test (n ) 3); NS, not significant between S(−)-and R(+)-bisoprolol. c Absolute bioavailability calculated with the iv dose as the reference.
(-)-bisoprolol, a more potent form, was higher than that of R(+)-bisoprolol at each time point in every dog, as shown in Figure 1. The mean (( SD) pharmacokinetic parameters of enantiomers are listed in Table 1. The Cmax of S(-)-bisoprolol (51.9 ( 9.0 ng/mL) was slightly higher, but not statistically different, from that of R(+)-bisoprolol (43.3 ( 3.7 ng/mL). The Tmax of S(-)-bisoprolol was similar to that of the R(+)enantiomer. However, the AUC0-∞ value of S(-)-bisoprolol (585 ( 76 ng ‚ h/mL) was significantly (p < 0.05) higher than that of R(+)-bisoprolol (435 ( 70 ng ‚ h/mL). The S/R ratio of the AUC0-∞ was 1.35 ( 0.09. The mean t1/2 of S(-)bisoprolol was not significantly different from that of R(+)bisoprolol. However, the t1/2 values in dogs 1, 2, and 3 were 5.1, 6.1, and 8.4 h for S(-)-bisoprolol, and 3.6, 5.6, and 6.8 h for R(+)-bisoprolol, respectively, which indicated that the t1/2 of S(-)-bisoprolol was always longer than that of R(+)bisoprolol in each dog. The absolute bioavailability (F) of S()-bisoprolol (89.8 ( 12.8%) was similar to that of R(+)bisoprolol (93.3 ( 11.6%). Both values were close to the bioavailability of racemic bisoprolol reported in dogs (∼80%)3 and in humans (90%)4. Plasma Concentration Profiles after Intravenous AdministrationsThe mean plasma concentration versus time curves of S(-)-bisoprolol and R(+)-bisoprolol after a single iv administration of 2.5 mg of racemic bisoprolol are shown in Figure 2. The pharmacokinetic parameters of the individual enantiomers are presented in Table 2. Similar to the results following oral administration, the plasma concentrations of S(-)-bisoprolol exceeded those of R(+)-bisoprolol throughout the time course. As the AUC0-∞ of S(-)-bisoprolol (326 ( 5 ng ‚ h/mL) was significantly higher than that of R(+)bisoprolol (224 ( 23 ng ‚ h/mL), the total body clearance (CLt) of R(+)-bisoprolol (93.8 ( 10.1 mL/min) was significantly larger than that of S(-)-bisoprolol (63.9 ( 1.1 mL/min). The plasma concentrations of S(-)-bisoprolol decreased more slowly than those of R(+)-bisoprolol. The elimination halflife (t1/2) of S(-)-bisoprolol (7.0 ( 1.4 h) was significantly longer than that of R(+)-bisoprolol (5.0 ( 1.6 h). The S/R ratio of AUC0-∞ (1.47 ( 0.14) was consistent with that of t1/2 (1.42 (
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Table 3sPharmacokinetic Parameters of Bisoprolol Enantiomers after Intravenous Administration of 1.25 mg of Pure Enantiomer to Beagle Dogsa Administration of S(−)
Figure 2sPlasma concentration−time profiles of bisoprolol enantiomers after iv administration of 2.5 mg of racemic bisoprolol to beagle dogs. Each point represents the mean ± SD of three dogs. Key: (b) S(−)-bisoprolol; (O) R(+)-bisoprolol.
Administration of R(+)
Parameter
S(−)
R(+)
S(−)
R(+)
AUC0-∞ (ng ‚ h/mL) t1/2 (h) CLt (mL/min) Vd (L)
413 ± 88 8.0 ± 1.9 51.9 ± 9.9 34.8 ± 4.5
s s s s
s s s s
227 ± 34 5.8 ± 1.5 93.4 ± 14.0 46.0 ± 4.8
a Each value represents the mean ± SD of three dogs; (s) not detected; there were no significant differences in all parameters between administration of pure enantiomer and administration of racemate.
Table 2sPharmacokinetic Parameters of Bisoprolol Enantiomers after Intravenous Administration of 2.5 mg of Racemic Bisoprolol to Beagle Dogsa Parameter
S(−)
R(+)
S/R ratio
Significanceb
AUC0-∞ (ng ‚ h/mL) t1/2 (h) CLt (mL/min) Vd (L)
326 ± 5 7.0 ± 1.4 63.9 ± 1.1 38.4 ± 7.1
224 ± 23 5.0 ± 1.6 93.8 ± 10.1 40.1 ± 9.2
1.47 ± 0.14 1.42 ± 0.21 0.69 ± 0.06 0.97 ± 0.06
p < 0.05 p < 0.01 p < 0.05 NS
a Each value represents the mean ± SD of three dogs. b Significance: based on the paired t test (n ) 3); NS, not significant between S(−)-and R(+)-bisoprolol.
Figure 4sCumulative urinary excretion of bisoprolol enantiomers after oral administration of 5 mg of racemic bisoprolol to beagle dogs. Each point represents the mean ± SD of three dogs. Key: (b) S(−)-bisoprolol; (O) R(+)-bisoprolol. Table 4sUrinary Excretion and Clearances of Bisoprolol Enantiomers after Oral Administration of 5 mg of Racemic Bisoprolol to Beagle Dogsa
Figure 3sPlasma concentration−time profiles of bisoprolol enantiomers after iv administration of 1.25 mg of S(−)-bisoprolol (A) or R(+)-bisoprolol (B) to beagle dogs. Each point represents the mean ± SD of three dogs. Key: (b) S(−)-bisoprolol; (O) R(+)-bisoprolol.
0.21), and was in good agreement with the ratio obtained following oral administration (1.35 ( 0.09). However, no difference was observed in the Vd value between S(-)bisoprolol (38.4 ( 7.1 L) and R(+)-bisoprolol (40.1 ( 9.2 L). To evaluate metabolic inversion, S(-)-bisoprolol and R(+)bisoprolol were administered separately by iv injection. When one of the enantiomers was administered, the other enantiomer was not found at all in plasma (Figure 3). Therefore, the possibility that metabolic inversion caused the stereoselective disposition could be excluded. Also, as shown in Table 3, the pharmacokinetic parameters after iv administration of each pure enantiomer were not significantly different from those after dosing with the racemate. These results suggest that there is no interaction between the enantiomers. Urinary ExcretionsThe cumulative urinary excretion versus time curves of S(-)-bisoprolol and R(+)-bisoprolol are presented in Figure 4. Similar to the plasma concentrations, the urinary excretion of S(-)-bisoprolol predominated in comparison with that of R(+)-bisoprolol at each time segment. The cumulative recovery of S(-)- and R(+)-bisoprolol excreted in urine up to 48 h (Ae0-48) were 35.1 ( 6.9% and 27.6 ( 7.1% 562 / Journal of Pharmaceutical Sciences Vol. 86, No. 5, May 1997
Parameterb
S(−)
R(+)
S/R ratio
Significancec
Ae0-48 (% dose) Ae0-∞ (% dose) CLr (mL/min) CLt (mL/min) CLm (mL/min)
35.1 ± 6.9 35.8 ± 6.7 25.3 ± 1.8 63.9 ± 1.1 38.5 ± 0.7
27.6 ± 7.1 28.1 ± 7.0 26.7 ± 3.3 93.8 ± 10.1 67.1 ± 8.7
1.28 ± 0.10 1.29 ± 0.10 0.96 ± 0.06 0.69 ± 0.06 0.58 ± 0.07
p < 0.05 p < 0.05 NS p < 0.05 p < 0.05
a Each value represents the mean ± SD of three dogs. b Ae, urinary excretion; CLr, renal crearance obtained by Ae0-∞/AUC0-∞; CLt, total body clearance obtained by dose ‚ F/AUC0-∞, where F is the absolute bioavailability; CLm, metabolic clearance obtained by CLt − CLr. c Significance: based on the paired t test (n ) 3); NS: not significant between S(−)-and R(+)-bisoprolol.
of the administered dose, respectively (Table 4). The cumulative recovery of S(-)-bisoprolol extrapolated to time infinity (Ae0-∞, 35.8 ( 6.7%) was significantly larger than that of R(+)bisoprolol (28.1 ( 7.0%). The S/R ratio of Ae0-∞ was 1.29 ( 0.10, which was close to the ratio of the AUC0-∞ (1.35 ( 0.09) obtained from plasma concentrations in the same experiment (Table 1). From the plasma concentration, the urinary excretion rate and absolute bioavailability (F) of S(-)-bisoprolol and R(+)bisoprolol after an oral administration, total body clearance (CLt), and renal clearance (CLr) were estimated for each enantiomer. Bisoprolol is eliminated by two pathways, (i.e., hepatic metabolism and renal excretion3-5), so the difference between CLt and CLr is the metabolic clearance (CLm). These clearance values are summarized in Table 4. The CLt and CLm of S(-)-bisoprolol were significantly different from those of R(+)-bisoprolol, but no significant difference was observed for CLr between S(-)-bisoprolol (25.3 ( 1.8 mL/min) and R(+)bisoprolol (26.7 ( 3.3 mL/min). The CLm of R(+)-bisoprolol (67.1 ( 8.7 mL/min) was ∼1.7 times larger than that of S(-)bisoprolol (38.5 ( 0.7 mL/min). These findings indicate that the difference in stereoselective disposition of bisoprolol enantiomers is caused by the difference in the metabolic clearance between the S(-)- and R(+)-enantiomers.
Table 5sPlasma Protein Binding of Bisoprolol Enantiomers Protein Binding (%) dog
Ex/In Vitro
S(−)
R(+)
S/R ratio
1
in vitroa ex vivob in vitro ex vivo
31.6 40.1 29.8 26.0
29.7 38.2 29.9 23.9
1.06 1.05 1.00 1.09
2
a Bisoprolol was added to blank plasma (final concentration, 200 ng/mL). Plasma was obtained from dogs 1 h after oral administration of 10 mg of bisoprolol. b
Figure 5sTime profiles for M4 formation after addition of bisoprolol enantiomers into dog liver microsomes. Each point represents the mean ± SD of three experiments. Key: (b) M4 formation from S(−)-bisoprolol; (O) M4 formation from R(+)-bisoprolol.
Scheme 1sMetabolic pathways of bisoprolol.
Protein BindingsThe in vitro and ex vivo binding of bisoprolol enantiomers to dog plasma is shown in Table 5. The S/R ratios of protein binding obtained from two dogs, dog 1 and dog 2, were 1.06 and 1.00 in the in vitro study, and 1.05 and 1.09 in the ex vivo study, respectively. Although the protein binding (%) varied slightly in dog 1, the S/R ratio of plasma protein binding was ∼1 both in vitro and ex vivo in the two dogs. Consequently, the plasma protein binding of S(-)- and R(+)-bisoprolol can be considered to be the same. In Vitro MetabolismsThe metabolic fate of racemic bisoprolol in several animals as well as humans following iv and oral administration of studies [14C]bisoprolol has already been reported.3 The structures of the metabolites and the metabolic pathways in dogs shown in Scheme 1 are considered to be the same as those in humans. Metabolites M4 and M1, which are major metabolites in dogs, are formed by Odealkylation of bisoprolol and oxidation of M4, respectively. In the present investigation, we evaluated the formation rate of M4 from S(-)- or R(+)-bisoprolol in dog liver microsomes to examine the stereospecific metabolism. The formation of M4 from each bisoprolol enantiomer is shown in Figure 5. The M4 metabolite was formed more rapidly from R(+)-bisoprolol than from S(-)-bisoprolol. The S/R ratio of the M4 formation after a 5-h incubation was 0.62, which was about the same as the ratio of the metabolic clearance (i.e., 0.58 ( 0.07; Table 4).
Discussion Many studies have shown that enantiomers of some drugs often exhibit different pharmacokinetic and/or pharmacological properties.7,8 The stereoselectivity in pharmacodynamics and pharmacokinetics for β-adrenoceptor antagonists has been extensively studied.13-15 Bisoprolol is used as a racemic mixture, similar to most β-blocking agents. Therefore, we examined the stereoselective distinction between S(-)- and R(+)-bisoprolol in the disposition of bisoprolol enantiomers in the body.
Leopold4 suggested that there was no difference in the metabolism between S(-)- and R(+)-bisoprolol in European subjects. Dutta et al.16 determined the plasma concentration of both S(-)- and R(+)-bisoprolol after administration of racemic bisoprolol in the 5-40-mg range by a method with a chiral derivatizing reagent. The mean AUC of the S(-)isomer was 7% greater than that of the R(+)-isomer, with a 90% confidence interval of [2%,12%]. The power of the test to detect 20% difference was >90%. From these results, the authors16 concluded that no stereoselective difference was observed in the pharmacokinetics of bisoprolol in American subjects. Recently, we conducted pharmacokinetic studies of bisoprolol enantiomers in Japanese subjects after oral administration of 20 mg of racemic bisoprolol,12 using the method we developed with a chiral HPLC column for the direct determination of each bisoprolol enantiomer in plasma and urine.11 Although the difference in the AUC was small (S/R ratio, 1.16), the AUC of S(-)-bisoprolol was significantly (p < 0.05) higher than that of the R(+)-isomer as determined by the paired t test, with n ) 4. Therefore, we suggest that stereoselectivity in the disposition of bisoprolol may exist in humans and animals. In this study, the concentrations of bisoprolol enantiomers in plasma and urine of dogs were determined to clarify the fundamental pharmacokinetic properties of both S(-)- and R(+)-enantiomers. The AUC0-∞ of S(-)-bisoprolol was ∼1.5 times higher than that of R(+)-bisoprolol after both oral and iv administration of racemic bisoprolol to three dogs. These findings indicated that the pharmacokinetics of bisoprolol in dogs was stereoselective, so we examined the cause of the stereoselective disposition of bisoprolol in dogs. The stereoselective first-pass effect of verapamil has been reported.17 Because the absolute bioavailability of S(-)bisoprolol was the same as that of R(+)-bisoprolol, the stereoselective first-pass metabolism is negligible. One cause of the stereoselective disposition of chiral drugs is considered to be metabolic chiral inversion, as observed for ibuprofen.18 In addition, the enantiomers of some chiral drugs are known to interact with one another.19-21 However, no chiral inversion or enantiomer-enantiomer interaction was observed for bisoprolol in this study. Stereoselective plasma protein binding may lead to the difference in kinetic behavior of two enantiomers. Some enantiomers of β-blockers showed stereoselective binding to plasma proteins, such as R1-acid glycoprotein.22,23 However, the stereoselective binding of bisoprolol enantiomers to plasma proteins was not observed (Table 5). The stereoselectivity of renal clearance was also known to affect the disposition of some chiral drugs. For example, the higher AUC of R(+)-pindolol than that of S(-)enantiomer was thought to be due to the smaller renal clearance of R(+)-pindolol than that of the S(-)-form.24 The renal clearance of R(+)-bisoprolol was equal to that of S(-)-
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bisoprolol, which indicates the absence of stereoselectivity in the renal clearance of the bisoprolol enantiomers. On the other hand, significant differences in the metabolic clearance between R(+)- and S(-)-bisoprolol were found. To confirm the stereoselective hepatic metabolism, the preliminary in vitro metabolism study was conducted with dog liver microsomes. As the rate of M4 formation from R(+)-bisoprolol was faster than from S(-)-isomer (Figure 5), we suggest that the stereoselectivity in the metabolic clearance of S(-)- and R(+)-bisoprolol was caused by the difference in the hepatic intrinsic clearance between S(-)- and R(+)-bisoprolol. The stereoselective biliary excretion may cause the difference in the total clearance between the two enantiomers. However, the biliary excretion of unchanged bisoprolol was <5% in rats,3 and the fecal excretion was <20% in rats and <10% in dogs. Furthermore, a small part (17%) of biliary excreta (unchanged + metabolites) was re-absorbed in rats.25 Therefore, the biliary excretion is a very minor route of elimination for bisoprolol. In conclusion, an interesting finding was that the more pharmacologically active enantiomer, S(-)-bisoprolol, had a higher plasma concentration and longer elimination half-life than R(+)-bisoprolol after both oral and iv administration in dogs. The absolute bioavailability, plasma protein binding, and renal clearance of one enantiomer were similar to that of the other enantiomer. Stereoselective disposition of bisoprolol enantiomers was caused by the smaller intrinsic hepatic clearance of S(-)-bisoprolol than that of R(+)-bisoprolol. Further studies to clarify the kinetics of the stereoselective metabolism in dogs and humans are now in progress.
References and Notes 1. Manalan, A. S.; Besch, H. R.; Watanabe, A. M. Circ. Res. 1982, 49, 326-336. 2. Schliep, H.-J.; Harting, J. J. Cardiovasc. Pharmacol. 1984, 6, 1156-1160. 3. Bu¨hring, K. U.; Sailer, H.; Faro, H.-P.; Leopold, G.; Pabst, J.; Garbe, A. J. Cardiovasc. Pharmacol. 1986, 8, S21-S28. 4. Leopold, G. J. Cardiovasc. Pharmacol. 1986, 8, S16-S20.
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5. Kirch, W.; Rose, I.; Demers, H. G.; Leopold, G.; Pabst, J.; Ohnhaus, E. E. Clin. Pharmacokinet. 1987, 13, 110-117. 6. Haeusler, G.; Schliep, H.-J.; Schelling, P.; Becker, K. H.; Klockow, M.; Minck, K. O.; Enenkel, H. J.; Schulze, E.; Bergmann, R.; Schmitges, C.-J.; Seyfried, C.; Harting, J. J. Cardiovasc. Pharmacol. 1986, 8, S2-S15. 7. Drayer, D. E. Clin. Pharmacol. Ther. 1986, 40, 125-133. 8. Arie¨ns, E. J.; Wuis, E. W. Clin Pharmacol. Ther. 1987, 42, 361363. 9. Jamali, F.; Mehvar, R.; Pasutto, F. M. J. Pharm. Sci. 1989, 78, 695-715. 10. Lee, E. J. D.; Williams, K. M. Clin. Pharmacokinet. 1990, 18, 339-345. 11. Suzuki, T.; Horikiri, Y.; Mizobe, M.; Noda, K. J. Chromatogr. Biomed. Appl. 1993, 619, 267-273. 12. Horikiri, Y.; Suzuki, T.; Mizobe, M; Kobayashi, M; Noda, K. Biol. Pharm. Bull., in preparation. 13. Silber, B.; Holford, N. H. G.; Riegelman, S. J. Pharm. Sci. 1982, 71, 699-703. 14. Lennard, M. S.; Tucker, G. T.; Silas, J. H.; Freestone, S.; Ramsay, L. E.; Woods, H. F. Clin. Pharmacol. Ther. 1983, 34, 732-737. 15. Francis, R. J.; East, P. B.; Larmann, J. Eur. J. Clin. Pharmacol. 1982, 23, 529-533. 16. Dutta, A.; Lanc, R.; Begg, E.; Robson, R.; Sia, L.; Dukart, G.; Desjardins, R.; Yacobi, A. J. Clin. Pharmacol. 1994, 34, 829836. 17. Vagelgesang, B.; Echizen, H.; Schmidt, E.; Eichlbaum, M. Br. J. Clin. Pharmacol. 1984, 18, 733-740. 18. Lee, E. J. D.; Williams, K.; Day, R.; Graham, G.; Champion, D. Br. J. Clin. Pharmacol. 1985, 19, 669-674. 19. Williams, K.; Lee, E. Drugs 1985, 30, 333-354. 20. Carr, R. A.; Pasutto, F. M.; Foster, R. T. Biopharm. Drug Dispos. 1994, 15, 109-120. 21. Masubuchi, Y.; Yamamoto, L. A.; Uesaka, M.; Fujita, S.; Narimatsu, S.; Suzuki, T. Biochem. Pharmacol. 1993. 46, 17591765. 22. Kushiya, M. M.; Okada, S.; Kimura, T.; Hasegawa, R. J. Pharm. Pharmacol. 1993, 45, 225-228. 23. Kimura, K.; Mizuno, Y.; Sugihara, K.; Ohbe, Y. Xenobiotic Metabolism and Disposition; Kato, R.; Estabrook, R. W.; Cayen M. N., Eds.; Taylor and Francis: London, 1989; pp 185-192. 24. Hsyu, P. H.; Giacomini, K. H. J. Clin. Invest. 1985, 76, 17201726. 25. Yamada, Y.; Nakahara, M.; Naito, K.; Kohno, M.; Otsuka, M.; Takaichi, O. Xenobiot. Metab. Dispos. 1989, 4, 149-164.
JS960453V
AND
MASAKAZU MIZOBE
Received November 4, 1996, from the Pharmaceutics Research Laboratory, Tanabe Seiyaku Company, Ltd., 16-89, Kashima 3-chome, Yodogawa-ku, Osaka 532, Japan. Accepted for publication February 10, 1997X. Abstract 0 The stereoselective pharmacokinetics of bisoprolol, a highly β1-selective adrenoceptor blocking agent, was studied in dogs. After intravenous and oral administration of the racemate, there was a difference in the plasma concentration between S(−)- and R(+)-bisoprolol. The area under the curve (AUC) of concentration versus time of S(−)-bisoprolol was ∼1.5 times higher than that of R(+)-bisoprolol, and the elimination half−life of S(−)-bisoprolol was ∼1.4 times longer than that of R(+)bisoprolol. However, no differences were observed in the volume of distribution, absolute bioavailability, and renal clearance between the two enantiomers. The plasma protein binding of S(−)-bisoprolol was also the same as that of the R(−)-isomer. No chiral inversion or enantiomer− enantiomer interaction was observed, when enantiomers were solely administered via the intravenous route. The comparison of the oxidative metabolic rate of two enantiomers using dog liver microsomes demonstrated that the metabolite was more slowly formed from S(−)- than from R(+)-bisoprolol. Consequently, we concluded that the stereoselective difference in the metabolic clearance between S(−)- and R(+)-bisoprolol caused the difference in the disposition of bisoprolol enantiomers.
Introduction Bisoprolol hemifumarate, [(]-1-{p-[(2-isopropoxyethoxy)methyl]phenoxy}-3-isopropyl-amino-2-propanol-hemifumarate, is a highly β1-selective adrenoceptor blocking agent without membrane-stabilizing activity or intrinsic sympathomimetic activity.1,2 The results of preclinical studies using several animals and clinical studies including healthy human subjects or patients,3-5 indicate that bisoprolol has a high absolute bioavailability (90% of an oral dose) due to its nearly complete enteral absorption (>90%) and a very small firstpass effect (<10%). A long elimination half-life (10-12 h) allows this drug to be used on a once-a-day dosage regimen. Bisoprolol has a chiral center, which gives rise to two optical isomers. The activity of S(-)-bisoprolol to block the β-adrenergic receptors is ∼30-80 times higher than that of R(+)bisoprolol.6 As with most β-adrenergic blocking agents, bisoprolol is marketed as a racemic mixture. Over the past two decades, many studies have indicated that drug enantiomers may have different pharmacodynamic and pharmacokinetic properties as a consequence of stereoselective interaction with optically active biological macromolecules.7-10 In general, the pharmacological activity of a racemic drug is associated predominantly with one enantiomer of the racemic drug. Therefore, it is necessary to evaluate the pharmacokinetic behavior of each enantiomer rather than that of the racemate, to use a racemic drug effectively and safely. In this study, we examined the stereoselective disposition of bisoprolol enantiomers. Also, we elucidated the pharmacokinetic properties of bisoprolol enantiomers in dogs, which are known to exhibit similar pharmacokinetic profiles of X
Abstract published in Advance ACS Abstracts, April 1, 1997.
560 / Journal of Pharmaceutical Sciences Vol. 86, No. 5, May 1997
racemic bisoprolol, such as bioavailability, metabolic pathways, and urinary excretion, as in humans.3
Experimental Section MaterialssS(-)-, R(+)-, and racemic bisoprolol, and its racemic metabolite (M4; {[(]4-(2-hydroxy-3-isopropylaminopropoxy)benzyloxy}ethanol) were obtained as their hemifumarates from E. Merck (Darmstadt, Germany). The purities of both the S(-)- and R(+)enantiomers were >99%, as confirmed by chiral high-performance liquid chromatography (HPLC) resolution. 1-[p-(Tetrahydro-3-furanyl)phenoxy]-3-isopropylamino-2-propanol, used as the internal standard in the analysis of both bisoprolol enantiomers and M4, was also obtained from E. Merck. HPLC-grade hexane, 2-propanol, and pesticide-grade chloroform were obtained from Katayama Chemical Industries Ltd. (Osaka, Japan). The other chemicals and solvents were of reagent grade and were used without further purification. AnimalssThree male beagle dogs, each weighing 13-18 kg, were used. The animals were fasted for 18 h prior to administration and were fed 8 h after dosing. They were allowed water throughout the course of the experiment. The elapsed interval between two separate studies was at least 2 weeks. Dosing ProceduresOne milliliter of the saline solution containing 2.5 mg of racemic bisoprolol or 1.25 mg of each enantiomer was injected directly into the forearm vein of conscious dogs. For oral administration, 5 mg of racemic bisoprolol dissolved in 20 mL of water was given to dogs by gastric intubation with a rubber cannula. The dogs were administered test agents in fixed order. Sample CollectionsApproximately 5 mL of venous blood was taken from each dog with a heparinized, disposable 10-mL blood collection syringe and an 18-gauge needle at 0, 0.05, 0.1, 0.17, 0.25, 0.5, 1, 2, 3, 4, 6, 8, and 24 h after intravenous (iv) injection, and at 0, 0.5, 1, 2, 3, 4, 6, 8, and 24 h after oral administration. The plasma was separated by centrifugation at 1000 × g for 20 min immediately after collection and stored at -25 °C until analyzed. The voided urine was fractionally collected from a metabolic cage during each interval of 0-2, 2-4, 4-6, 6-8, 8-24, and 24-48 h. In addition, the urine was completely emptied with a urinary catheter at the end of each interval. The total volume of urine of each interval was measured, and two 20-mL aliquots were stored at -25 °C. Analysis of Enantiomers and MetabolitesEach enantiomer of bisoprolol in plasma and urine was analyzed by the HPLC method with a chiral column (Chiralcel OD) according to the procedure described previously.11 One of the oxidative metabolites of bisoprolol, M4 (racemate), was determined by the method we developed,12 using a selective and sensitive reversed-phase HPLC with fluorimetric detection. Briefly, samples (1 mL) were alkalinized with NaOH (1 N) to pH of ∼9 and then M4 was extracted with a mixture of chloroform and 2-propanol (9:1). After the organic layer was evaporated to dryness under a nitrogen stream, the residue was reconstituted with a mobile phase containing the internal standard. The internal standard for analysis of M4 was the same as that used for bisoprolol enantiomers. The mobile phase consisted of 20% methanol in a 0.25% triethylamine solution in water (pH 3). The flow rate of 0.5 mL/min and column temperature of 60 °C were maintained throughout the analysis. An octadecylsilane analytical column (Hypersil 5ODS, Shandon, Cheshire, UK) was used. The detection of M4 was monitored with a fluorimetric detector at the excitation wavelength of 228 nm and the emission wavelength of 298 nm. Pharmacokinetic AnalysissPharmacokinetic parameters were determined from the plasma concentration versus time data. The maximum plasma concentration (Cmax) and the time to reach the
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maximum concentration (Tmax) were read directly from the plasma concentration versus time data. The apparent terminal elimination rate constant, k, was calculated by least squares regression analysis on the terminal portion of the log plasma concentration versus time profile. The terminal half-life (t1/2) was calculated by dividing ln 2 by k. The area under the curve of plasma concentration versus time from time zero to the time of last measurable plasma concentration point (AUC0-t) was determined according to the trapezoidal rule. Extrapolation to time infinity (AUC0-∞) was determined by dividing the last measurable plasma concentration by k and adding the result to the corresponding AUC0-t. The total amount of bisoprolol enantiomers excreted in urine from time zero to 48 h (Ae0-48) was the cumulative amount of enantiomer excreted in each sampling time period. The urinary excretion amount of bisoprolol enantiomers from the last urine sampling time extrapolated to time infinity (Ae48-∞) was estimated by dividing the urinary excretion rate at 48 h by K, where K is the elimination rate constant calculated by least squares regression analysis on the terminal portion of semilogarithmic plot of urinary excretion rate versus a midpoint of the urine collection period. The cumulative urinary excretion amount to time infinity (Ae0-∞) was calculated by adding Ae0-48 to Ae48-∞. The total clearance (CLt) was calculated as dose‚F/AUC0-∞, where F is the absolute bioavailability, which was calculated by dividing the AUC0-∞ of oral administration corrected for oral dose (AUC0-∞(po)/dose(po)) by the AUC0-∞ of iv administration corrected for iv dose (AUC0-∞(iv)/Dose(iv)). The renal clearance (CLr) was calculated as Ae0-∞/AUC0-∞. The difference between CLt and CLr was defined as the metabolic clearance (CLm); that is, CLm) CLt - CLr. Distribution volume (Vd) was calculated as the quotient of CLt and k. All pharmacokinetic data were expressed as the mean ( standard deviation (SD). In Vitro Metabolism StudiessA freshly obtained dog liver was excised and immediately placed in three volumes (per wet weight of liver) of ice-cold 1.15% KCl and then homogenized. The homogenate was centrifuged at 9000 × g for 30 min at 4 °C. The supernatant was then centrifuged at 105000 × g for 60 min at 4 °C. The resultant pellet was suspended in 100 mM phosphate buffer (pH7.4). One milliliter of this suspension contained the pellet from 3 g of wet liver. The protein concentration was determined by the Protein Assay Kit (Bio-Rad, CA). Microsomal oxidation reactions were carried out as follows. The reaction mixture (2.5 mL), containing 75 mM MgCl2, 25 mM glucose-6-phosphate, 5 mM NADPH, and 50 mM nicotinamide in 50 mM phosphate buffer (pH 7.4), was incubated with 10 µM S()-bisoprolol or R(+)-bisoprolol (2.5 mL) at 37 °C. The reaction was initiated by addition of 2.5 mL of the aforementioned microsomal suspension. At 1, 3, and 5 h after incubation, an aliquot (1 mL) was transferred to a tube containing NaOH. The metabolite, M4, was determined by the method described in Analysis of Enantiomers and Metabolite. Protein Binding ExperimentssPlasma protein binding of bisoprolol was studied by an ultrafiltration method with drug-free dog plasma samples spiked with bisoprolol (in vitro) and dog plasma samples obtained at 1 h after oral administration of 10 mg of bisoprolol (ex vivo) to two dogs, which were the same dogs as used for the pharmacokinetic study. The plasma sample was immediately transferred to a centrifuged micropartition system device (MPS-3, Amicon, MA) and centrifuged for 15 min at 1000 × g. Spiked plasma samples (200 ng/mL) were incubated for 15 min at 37 °C prior to centrifugation. Preliminary studies had revealed that there is no loss of bisoprolol during ultrafiltration due to membrane binding. The concentrations of bisoprolol enantiomers in the ultrafiltrate and in plasma or spiked plasma samples prior to centrifugation were determined by the HPLC method for analysis of bisoprolol enantiomers, as already described, and the amount of binding (%) to plasma protein was calculated. Statistical AnalysissComparisons between the S(-)- and R(+)bisoprolol pharmacokinetic parameters observed after administration of racemate were assessed by a t test for paired data. Comparisons of administration of pure enantiomer versus racemate were made by an independent measures t test.
Results Plasma Concentration Profiles after Oral AdministrationsThree beagle dogs were each orally administered 5 mg of racemic bisoprolol. The plasma concentration of S-
Figure 1sPlasma concentration−time profiles of bisoprolol enantiomers after oral administration of 5 mg of racemic bisoprolol to beagle dogs. Each point represents the mean ± SD of three dogs. Key: (b) S(−)-bisoprolol; (O) R(+)-bisoprolol. Table 1sPharmacokinetic Parameters of Bisoprolol Enantiomers after Oral Administration of 5 mg of Racemic Bisoprolol to Beagle Dogsa Parameter
S(−)
R(+)
Cmax (ng/mL) 51.9 ± 9.0 43.3 ± 3.7 Tmax (h) 1.33 ± 0.58 1.67 ± 0.58 AUC0-∞ (ng ‚ h/mL) 585 ± 76 435 ± 70 t1/2 (h) 6.5 ± 1.7 5.3 ± 1.6 Availability (F, %)c 89.8 ± 12.8 93.3 ± 11.6
S/R ratio
Significanceb
1.19 ± 0.12 0.83 ± 0.29 1.35 ± 0.09 1.25 ± 0.16 0.96 ± 0.03
NS NS p < 0.05 NS NS
a Each value represents the mean ± SD of three dogs. b Significance. Based on the paired t test (n ) 3); NS, not significant between S(−)-and R(+)-bisoprolol. c Absolute bioavailability calculated with the iv dose as the reference.
(-)-bisoprolol, a more potent form, was higher than that of R(+)-bisoprolol at each time point in every dog, as shown in Figure 1. The mean (( SD) pharmacokinetic parameters of enantiomers are listed in Table 1. The Cmax of S(-)-bisoprolol (51.9 ( 9.0 ng/mL) was slightly higher, but not statistically different, from that of R(+)-bisoprolol (43.3 ( 3.7 ng/mL). The Tmax of S(-)-bisoprolol was similar to that of the R(+)enantiomer. However, the AUC0-∞ value of S(-)-bisoprolol (585 ( 76 ng ‚ h/mL) was significantly (p < 0.05) higher than that of R(+)-bisoprolol (435 ( 70 ng ‚ h/mL). The S/R ratio of the AUC0-∞ was 1.35 ( 0.09. The mean t1/2 of S(-)bisoprolol was not significantly different from that of R(+)bisoprolol. However, the t1/2 values in dogs 1, 2, and 3 were 5.1, 6.1, and 8.4 h for S(-)-bisoprolol, and 3.6, 5.6, and 6.8 h for R(+)-bisoprolol, respectively, which indicated that the t1/2 of S(-)-bisoprolol was always longer than that of R(+)bisoprolol in each dog. The absolute bioavailability (F) of S()-bisoprolol (89.8 ( 12.8%) was similar to that of R(+)bisoprolol (93.3 ( 11.6%). Both values were close to the bioavailability of racemic bisoprolol reported in dogs (∼80%)3 and in humans (90%)4. Plasma Concentration Profiles after Intravenous AdministrationsThe mean plasma concentration versus time curves of S(-)-bisoprolol and R(+)-bisoprolol after a single iv administration of 2.5 mg of racemic bisoprolol are shown in Figure 2. The pharmacokinetic parameters of the individual enantiomers are presented in Table 2. Similar to the results following oral administration, the plasma concentrations of S(-)-bisoprolol exceeded those of R(+)-bisoprolol throughout the time course. As the AUC0-∞ of S(-)-bisoprolol (326 ( 5 ng ‚ h/mL) was significantly higher than that of R(+)bisoprolol (224 ( 23 ng ‚ h/mL), the total body clearance (CLt) of R(+)-bisoprolol (93.8 ( 10.1 mL/min) was significantly larger than that of S(-)-bisoprolol (63.9 ( 1.1 mL/min). The plasma concentrations of S(-)-bisoprolol decreased more slowly than those of R(+)-bisoprolol. The elimination halflife (t1/2) of S(-)-bisoprolol (7.0 ( 1.4 h) was significantly longer than that of R(+)-bisoprolol (5.0 ( 1.6 h). The S/R ratio of AUC0-∞ (1.47 ( 0.14) was consistent with that of t1/2 (1.42 (
Journal of Pharmaceutical Sciences / 561 Vol. 86, No. 5, May 1997
Table 3sPharmacokinetic Parameters of Bisoprolol Enantiomers after Intravenous Administration of 1.25 mg of Pure Enantiomer to Beagle Dogsa Administration of S(−)
Figure 2sPlasma concentration−time profiles of bisoprolol enantiomers after iv administration of 2.5 mg of racemic bisoprolol to beagle dogs. Each point represents the mean ± SD of three dogs. Key: (b) S(−)-bisoprolol; (O) R(+)-bisoprolol.
Administration of R(+)
Parameter
S(−)
R(+)
S(−)
R(+)
AUC0-∞ (ng ‚ h/mL) t1/2 (h) CLt (mL/min) Vd (L)
413 ± 88 8.0 ± 1.9 51.9 ± 9.9 34.8 ± 4.5
s s s s
s s s s
227 ± 34 5.8 ± 1.5 93.4 ± 14.0 46.0 ± 4.8
a Each value represents the mean ± SD of three dogs; (s) not detected; there were no significant differences in all parameters between administration of pure enantiomer and administration of racemate.
Table 2sPharmacokinetic Parameters of Bisoprolol Enantiomers after Intravenous Administration of 2.5 mg of Racemic Bisoprolol to Beagle Dogsa Parameter
S(−)
R(+)
S/R ratio
Significanceb
AUC0-∞ (ng ‚ h/mL) t1/2 (h) CLt (mL/min) Vd (L)
326 ± 5 7.0 ± 1.4 63.9 ± 1.1 38.4 ± 7.1
224 ± 23 5.0 ± 1.6 93.8 ± 10.1 40.1 ± 9.2
1.47 ± 0.14 1.42 ± 0.21 0.69 ± 0.06 0.97 ± 0.06
p < 0.05 p < 0.01 p < 0.05 NS
a Each value represents the mean ± SD of three dogs. b Significance: based on the paired t test (n ) 3); NS, not significant between S(−)-and R(+)-bisoprolol.
Figure 4sCumulative urinary excretion of bisoprolol enantiomers after oral administration of 5 mg of racemic bisoprolol to beagle dogs. Each point represents the mean ± SD of three dogs. Key: (b) S(−)-bisoprolol; (O) R(+)-bisoprolol. Table 4sUrinary Excretion and Clearances of Bisoprolol Enantiomers after Oral Administration of 5 mg of Racemic Bisoprolol to Beagle Dogsa
Figure 3sPlasma concentration−time profiles of bisoprolol enantiomers after iv administration of 1.25 mg of S(−)-bisoprolol (A) or R(+)-bisoprolol (B) to beagle dogs. Each point represents the mean ± SD of three dogs. Key: (b) S(−)-bisoprolol; (O) R(+)-bisoprolol.
0.21), and was in good agreement with the ratio obtained following oral administration (1.35 ( 0.09). However, no difference was observed in the Vd value between S(-)bisoprolol (38.4 ( 7.1 L) and R(+)-bisoprolol (40.1 ( 9.2 L). To evaluate metabolic inversion, S(-)-bisoprolol and R(+)bisoprolol were administered separately by iv injection. When one of the enantiomers was administered, the other enantiomer was not found at all in plasma (Figure 3). Therefore, the possibility that metabolic inversion caused the stereoselective disposition could be excluded. Also, as shown in Table 3, the pharmacokinetic parameters after iv administration of each pure enantiomer were not significantly different from those after dosing with the racemate. These results suggest that there is no interaction between the enantiomers. Urinary ExcretionsThe cumulative urinary excretion versus time curves of S(-)-bisoprolol and R(+)-bisoprolol are presented in Figure 4. Similar to the plasma concentrations, the urinary excretion of S(-)-bisoprolol predominated in comparison with that of R(+)-bisoprolol at each time segment. The cumulative recovery of S(-)- and R(+)-bisoprolol excreted in urine up to 48 h (Ae0-48) were 35.1 ( 6.9% and 27.6 ( 7.1% 562 / Journal of Pharmaceutical Sciences Vol. 86, No. 5, May 1997
Parameterb
S(−)
R(+)
S/R ratio
Significancec
Ae0-48 (% dose) Ae0-∞ (% dose) CLr (mL/min) CLt (mL/min) CLm (mL/min)
35.1 ± 6.9 35.8 ± 6.7 25.3 ± 1.8 63.9 ± 1.1 38.5 ± 0.7
27.6 ± 7.1 28.1 ± 7.0 26.7 ± 3.3 93.8 ± 10.1 67.1 ± 8.7
1.28 ± 0.10 1.29 ± 0.10 0.96 ± 0.06 0.69 ± 0.06 0.58 ± 0.07
p < 0.05 p < 0.05 NS p < 0.05 p < 0.05
a Each value represents the mean ± SD of three dogs. b Ae, urinary excretion; CLr, renal crearance obtained by Ae0-∞/AUC0-∞; CLt, total body clearance obtained by dose ‚ F/AUC0-∞, where F is the absolute bioavailability; CLm, metabolic clearance obtained by CLt − CLr. c Significance: based on the paired t test (n ) 3); NS: not significant between S(−)-and R(+)-bisoprolol.
of the administered dose, respectively (Table 4). The cumulative recovery of S(-)-bisoprolol extrapolated to time infinity (Ae0-∞, 35.8 ( 6.7%) was significantly larger than that of R(+)bisoprolol (28.1 ( 7.0%). The S/R ratio of Ae0-∞ was 1.29 ( 0.10, which was close to the ratio of the AUC0-∞ (1.35 ( 0.09) obtained from plasma concentrations in the same experiment (Table 1). From the plasma concentration, the urinary excretion rate and absolute bioavailability (F) of S(-)-bisoprolol and R(+)bisoprolol after an oral administration, total body clearance (CLt), and renal clearance (CLr) were estimated for each enantiomer. Bisoprolol is eliminated by two pathways, (i.e., hepatic metabolism and renal excretion3-5), so the difference between CLt and CLr is the metabolic clearance (CLm). These clearance values are summarized in Table 4. The CLt and CLm of S(-)-bisoprolol were significantly different from those of R(+)-bisoprolol, but no significant difference was observed for CLr between S(-)-bisoprolol (25.3 ( 1.8 mL/min) and R(+)bisoprolol (26.7 ( 3.3 mL/min). The CLm of R(+)-bisoprolol (67.1 ( 8.7 mL/min) was ∼1.7 times larger than that of S(-)bisoprolol (38.5 ( 0.7 mL/min). These findings indicate that the difference in stereoselective disposition of bisoprolol enantiomers is caused by the difference in the metabolic clearance between the S(-)- and R(+)-enantiomers.
Table 5sPlasma Protein Binding of Bisoprolol Enantiomers Protein Binding (%) dog
Ex/In Vitro
S(−)
R(+)
S/R ratio
1
in vitroa ex vivob in vitro ex vivo
31.6 40.1 29.8 26.0
29.7 38.2 29.9 23.9
1.06 1.05 1.00 1.09
2
a Bisoprolol was added to blank plasma (final concentration, 200 ng/mL). Plasma was obtained from dogs 1 h after oral administration of 10 mg of bisoprolol. b
Figure 5sTime profiles for M4 formation after addition of bisoprolol enantiomers into dog liver microsomes. Each point represents the mean ± SD of three experiments. Key: (b) M4 formation from S(−)-bisoprolol; (O) M4 formation from R(+)-bisoprolol.
Scheme 1sMetabolic pathways of bisoprolol.
Protein BindingsThe in vitro and ex vivo binding of bisoprolol enantiomers to dog plasma is shown in Table 5. The S/R ratios of protein binding obtained from two dogs, dog 1 and dog 2, were 1.06 and 1.00 in the in vitro study, and 1.05 and 1.09 in the ex vivo study, respectively. Although the protein binding (%) varied slightly in dog 1, the S/R ratio of plasma protein binding was ∼1 both in vitro and ex vivo in the two dogs. Consequently, the plasma protein binding of S(-)- and R(+)-bisoprolol can be considered to be the same. In Vitro MetabolismsThe metabolic fate of racemic bisoprolol in several animals as well as humans following iv and oral administration of studies [14C]bisoprolol has already been reported.3 The structures of the metabolites and the metabolic pathways in dogs shown in Scheme 1 are considered to be the same as those in humans. Metabolites M4 and M1, which are major metabolites in dogs, are formed by Odealkylation of bisoprolol and oxidation of M4, respectively. In the present investigation, we evaluated the formation rate of M4 from S(-)- or R(+)-bisoprolol in dog liver microsomes to examine the stereospecific metabolism. The formation of M4 from each bisoprolol enantiomer is shown in Figure 5. The M4 metabolite was formed more rapidly from R(+)-bisoprolol than from S(-)-bisoprolol. The S/R ratio of the M4 formation after a 5-h incubation was 0.62, which was about the same as the ratio of the metabolic clearance (i.e., 0.58 ( 0.07; Table 4).
Discussion Many studies have shown that enantiomers of some drugs often exhibit different pharmacokinetic and/or pharmacological properties.7,8 The stereoselectivity in pharmacodynamics and pharmacokinetics for β-adrenoceptor antagonists has been extensively studied.13-15 Bisoprolol is used as a racemic mixture, similar to most β-blocking agents. Therefore, we examined the stereoselective distinction between S(-)- and R(+)-bisoprolol in the disposition of bisoprolol enantiomers in the body.
Leopold4 suggested that there was no difference in the metabolism between S(-)- and R(+)-bisoprolol in European subjects. Dutta et al.16 determined the plasma concentration of both S(-)- and R(+)-bisoprolol after administration of racemic bisoprolol in the 5-40-mg range by a method with a chiral derivatizing reagent. The mean AUC of the S(-)isomer was 7% greater than that of the R(+)-isomer, with a 90% confidence interval of [2%,12%]. The power of the test to detect 20% difference was >90%. From these results, the authors16 concluded that no stereoselective difference was observed in the pharmacokinetics of bisoprolol in American subjects. Recently, we conducted pharmacokinetic studies of bisoprolol enantiomers in Japanese subjects after oral administration of 20 mg of racemic bisoprolol,12 using the method we developed with a chiral HPLC column for the direct determination of each bisoprolol enantiomer in plasma and urine.11 Although the difference in the AUC was small (S/R ratio, 1.16), the AUC of S(-)-bisoprolol was significantly (p < 0.05) higher than that of the R(+)-isomer as determined by the paired t test, with n ) 4. Therefore, we suggest that stereoselectivity in the disposition of bisoprolol may exist in humans and animals. In this study, the concentrations of bisoprolol enantiomers in plasma and urine of dogs were determined to clarify the fundamental pharmacokinetic properties of both S(-)- and R(+)-enantiomers. The AUC0-∞ of S(-)-bisoprolol was ∼1.5 times higher than that of R(+)-bisoprolol after both oral and iv administration of racemic bisoprolol to three dogs. These findings indicated that the pharmacokinetics of bisoprolol in dogs was stereoselective, so we examined the cause of the stereoselective disposition of bisoprolol in dogs. The stereoselective first-pass effect of verapamil has been reported.17 Because the absolute bioavailability of S(-)bisoprolol was the same as that of R(+)-bisoprolol, the stereoselective first-pass metabolism is negligible. One cause of the stereoselective disposition of chiral drugs is considered to be metabolic chiral inversion, as observed for ibuprofen.18 In addition, the enantiomers of some chiral drugs are known to interact with one another.19-21 However, no chiral inversion or enantiomer-enantiomer interaction was observed for bisoprolol in this study. Stereoselective plasma protein binding may lead to the difference in kinetic behavior of two enantiomers. Some enantiomers of β-blockers showed stereoselective binding to plasma proteins, such as R1-acid glycoprotein.22,23 However, the stereoselective binding of bisoprolol enantiomers to plasma proteins was not observed (Table 5). The stereoselectivity of renal clearance was also known to affect the disposition of some chiral drugs. For example, the higher AUC of R(+)-pindolol than that of S(-)enantiomer was thought to be due to the smaller renal clearance of R(+)-pindolol than that of the S(-)-form.24 The renal clearance of R(+)-bisoprolol was equal to that of S(-)-
Journal of Pharmaceutical Sciences / 563 Vol. 86, No. 5, May 1997
bisoprolol, which indicates the absence of stereoselectivity in the renal clearance of the bisoprolol enantiomers. On the other hand, significant differences in the metabolic clearance between R(+)- and S(-)-bisoprolol were found. To confirm the stereoselective hepatic metabolism, the preliminary in vitro metabolism study was conducted with dog liver microsomes. As the rate of M4 formation from R(+)-bisoprolol was faster than from S(-)-isomer (Figure 5), we suggest that the stereoselectivity in the metabolic clearance of S(-)- and R(+)-bisoprolol was caused by the difference in the hepatic intrinsic clearance between S(-)- and R(+)-bisoprolol. The stereoselective biliary excretion may cause the difference in the total clearance between the two enantiomers. However, the biliary excretion of unchanged bisoprolol was <5% in rats,3 and the fecal excretion was <20% in rats and <10% in dogs. Furthermore, a small part (17%) of biliary excreta (unchanged + metabolites) was re-absorbed in rats.25 Therefore, the biliary excretion is a very minor route of elimination for bisoprolol. In conclusion, an interesting finding was that the more pharmacologically active enantiomer, S(-)-bisoprolol, had a higher plasma concentration and longer elimination half-life than R(+)-bisoprolol after both oral and iv administration in dogs. The absolute bioavailability, plasma protein binding, and renal clearance of one enantiomer were similar to that of the other enantiomer. Stereoselective disposition of bisoprolol enantiomers was caused by the smaller intrinsic hepatic clearance of S(-)-bisoprolol than that of R(+)-bisoprolol. Further studies to clarify the kinetics of the stereoselective metabolism in dogs and humans are now in progress.
References and Notes 1. Manalan, A. S.; Besch, H. R.; Watanabe, A. M. Circ. Res. 1982, 49, 326-336. 2. Schliep, H.-J.; Harting, J. J. Cardiovasc. Pharmacol. 1984, 6, 1156-1160. 3. Bu¨hring, K. U.; Sailer, H.; Faro, H.-P.; Leopold, G.; Pabst, J.; Garbe, A. J. Cardiovasc. Pharmacol. 1986, 8, S21-S28. 4. Leopold, G. J. Cardiovasc. Pharmacol. 1986, 8, S16-S20.
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5. Kirch, W.; Rose, I.; Demers, H. G.; Leopold, G.; Pabst, J.; Ohnhaus, E. E. Clin. Pharmacokinet. 1987, 13, 110-117. 6. Haeusler, G.; Schliep, H.-J.; Schelling, P.; Becker, K. H.; Klockow, M.; Minck, K. O.; Enenkel, H. J.; Schulze, E.; Bergmann, R.; Schmitges, C.-J.; Seyfried, C.; Harting, J. J. Cardiovasc. Pharmacol. 1986, 8, S2-S15. 7. Drayer, D. E. Clin. Pharmacol. Ther. 1986, 40, 125-133. 8. Arie¨ns, E. J.; Wuis, E. W. Clin Pharmacol. Ther. 1987, 42, 361363. 9. Jamali, F.; Mehvar, R.; Pasutto, F. M. J. Pharm. Sci. 1989, 78, 695-715. 10. Lee, E. J. D.; Williams, K. M. Clin. Pharmacokinet. 1990, 18, 339-345. 11. Suzuki, T.; Horikiri, Y.; Mizobe, M.; Noda, K. J. Chromatogr. Biomed. Appl. 1993, 619, 267-273. 12. Horikiri, Y.; Suzuki, T.; Mizobe, M; Kobayashi, M; Noda, K. Biol. Pharm. Bull., in preparation. 13. Silber, B.; Holford, N. H. G.; Riegelman, S. J. Pharm. Sci. 1982, 71, 699-703. 14. Lennard, M. S.; Tucker, G. T.; Silas, J. H.; Freestone, S.; Ramsay, L. E.; Woods, H. F. Clin. Pharmacol. Ther. 1983, 34, 732-737. 15. Francis, R. J.; East, P. B.; Larmann, J. Eur. J. Clin. Pharmacol. 1982, 23, 529-533. 16. Dutta, A.; Lanc, R.; Begg, E.; Robson, R.; Sia, L.; Dukart, G.; Desjardins, R.; Yacobi, A. J. Clin. Pharmacol. 1994, 34, 829836. 17. Vagelgesang, B.; Echizen, H.; Schmidt, E.; Eichlbaum, M. Br. J. Clin. Pharmacol. 1984, 18, 733-740. 18. Lee, E. J. D.; Williams, K.; Day, R.; Graham, G.; Champion, D. Br. J. Clin. Pharmacol. 1985, 19, 669-674. 19. Williams, K.; Lee, E. Drugs 1985, 30, 333-354. 20. Carr, R. A.; Pasutto, F. M.; Foster, R. T. Biopharm. Drug Dispos. 1994, 15, 109-120. 21. Masubuchi, Y.; Yamamoto, L. A.; Uesaka, M.; Fujita, S.; Narimatsu, S.; Suzuki, T. Biochem. Pharmacol. 1993. 46, 17591765. 22. Kushiya, M. M.; Okada, S.; Kimura, T.; Hasegawa, R. J. Pharm. Pharmacol. 1993, 45, 225-228. 23. Kimura, K.; Mizuno, Y.; Sugihara, K.; Ohbe, Y. Xenobiotic Metabolism and Disposition; Kato, R.; Estabrook, R. W.; Cayen M. N., Eds.; Taylor and Francis: London, 1989; pp 185-192. 24. Hsyu, P. H.; Giacomini, K. H. J. Clin. Invest. 1985, 76, 17201726. 25. Yamada, Y.; Nakahara, M.; Naito, K.; Kohno, M.; Otsuka, M.; Takaichi, O. Xenobiot. Metab. Dispos. 1989, 4, 149-164.
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