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Stereoselective disposition of talinolol in man
Stereoselective Disposition of Talinolol in Man È RGEN KLEBINGAT, GERD FRANKE, MICHAEL ZSCHIESCHE, GIRUM LAKEW LEMMA, KLAUS-JU BERND TERHAAG, ANNA HOFFMANN, THOMAS GRAMATTEÂ, HEYO K. KROEMER, WERNER SIEGMUND Department of Pharmacology, Ernst Moritz Arndt University, Friedrich Loef¯er Straûe 23d, D-17487 Greifswald, Germany Received 16 January 2001; revised 18 October 2001; accepted 18 October 2001
ABSTRACT: The disposition of the b-blocking drug talinolol is controlled by P-glycoprotein in man. Because talinolol is marketed as a racemate, we reevaluated the serum-concentration time pro®les of talinolol of a previously published study with single intravenous (30 mg) and repeated oral talinolol (100 mg for 14 days) before and after comedication of rifampicin (600 mg per day for 9 days) in eight male healthy volunteers (age 22±26 years, body weight 67±84 kg) with respect to differences in the kinetic pro®les of the two enantiomers S(ÿ) talinolol and R() talinolol. Additionally, the metabolism of talinolol in human liver microsomes was examined. After oral administration, S(ÿ) talinolol was slightly less absorbed and faster eliminated than R() talinolol. The absolute bioavailabilty of the R() enantiomer of talinolol was slightly but signi®cantly higher than of its S(ÿ) enantiomer. Coadministration of rifampicin further intensi®ed this difference in the disposition of R() and S(ÿ) talinolol (p < 0.05). Formation of 4-trans hydroxytalinolol was the major metabolic pathway in human liver microsomes. All Clint values of S(ÿ) were higher than of R() talinolol; 0.1 mM ketoconazole inhibited the formation of all metabolites. In conclusion, the stereoselectivity of talinolol disposition is of minor importance, and most likely caused by presystemic biotransformation via CYP3A4. The less active R() talinolol might be suitable for phenotyping P-glycoprotein expression in man. ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:303±311, 2002
Keywords:
talinolol; man; P-glycoprotein; racemate
INTRODUCTION The mammalian drug-transporting MDR1 gene product P-glycoprotein (P-gp) is part of a protection system against a large number of xenobiotic compounds. The exact mechanisms how P-gp recognizes substrates remain to be elucidated. All
Klaus-JuÈrgen Klebingat's present address is Department of Urology, University of Greifswald. Bernd Terhaag's and Anna Hoffmann's present address is Arzneimittelwerk Dresden GmbH, Dresden, Germany. Thomas GramatteÂ's present address is APOGEPHA Arzneimittel GmbH, Dresden, Germany. Correspondence to: Werner Siegmund (Telephone: 493834-86532; Fax: 49-3834-86531; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 91, 303±311 (2002) ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association
ligands of P-gp are in common amphiphilic that seems to be essential for their insertion in apical membranes to be translocated.1,2 Other structural characteristics seem to be of minor importance for substrate binding as cationic, neutral, and even anionic compounds with extremely diverse chemical structure and molecular weights from 250 to almost 1900 Da are transported.3,4 Furthermore, it is reported, that P-gp does not discriminate between the enantiomers of chiral compounds.5±10 To determine intestinal P-gp expression in man, biopsy specimens from the gut have to be obtained by invasive methods. A probe drug approach that avoids such invasive investigations is therefore highly requested for the assessment of P-gp function. So far, the b-blocking agent talinolol is the only compound for which a signi®cant secretion into the gut lumen in man
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has already been demonstrated.11,12 The drug is subjected to minor metabolism, less than 1% is excreted into urine in form of hydroxylated metabolites.13±15 It is unknown whether metabolites are excreted by other pathways (e.g., biliary, intestinal). However, information on the suitability of racemic talinolol to predict P-gp expression is still limited by contradictory results on the chirality of talinolol disposition.12,16 If the P-gp dependent transport of talinolol would be stereoselective, then upregulation of the carrier by rifampicin should result in adequate differences of R() and S(ÿ) talinolol disposition. Therefore, we reassessed the serum samples of a recently published interaction study with rifampicin to measure pharmacokinetics of talinolol enantiomers.15
MATERIALS AND METHODS Chemicals S(ÿ) and R() talinolol [1-(4-cyclohexylureidophenoxy)-2-hydroxy-3-tert-butylamino-propane] its metabolites 3-trans, 3-cis 4-trans, and 4-cis hydroxytalinolol (Figure 1), as well as metoclopramide were generously provided by Arzneimittelwerk Dresden (Dresden, Germany). S(ÿ) propranolol was purchased from Tocris Cookson (Bristol, UK) and ketoconazole from Sigma (Deisenhofen, Germany). Acetonitrile and ethanol were obtained from Baker (Deventer, The Netherlands), triethylamine from Fluka (Buchs, Switzerland). All solvents and chemicals were of HPLC or analytical grade. The pharmacokinetic study in healthy subjects was performed with commercially available formulations of talinolol (100 mg ®lm coated tablets and 30 mg vial
solution, Cordanum1, Arzneimittelwerk Dresden, Germany). Subjects and Study Protocol The serum samples used for this investigation have been gained from a clinical study that has been described in detail elsewhere.15 In brief, eight male subjects (age 22±26 years, body weight 67±84 kg) were included after giving informed written consent. They were of good health, as evidenced by physical examination, routine clinical-chemical, and hematological screening. The subjects were negative for hepatitis and drugs, abstained strictly from alcohol, and took standard diet. The study protocol had been approved by the local ethics committee. At the start of the study, 30 mg talinolol were given intravenously within 30 min and venous blood (5 mL) was collected before and 0.17, 0.33, 0.5, 0.67, 1, 1.5, 2, 2.5, 3.5, 4, 4.5, 6.5, 8.5, 12.5, 16.5, 25.5, and 36.5 h after starting the infusion. Following a washout period of 8 days, volunteers were medicated with 100 mg oral talinolol for 14 subsequent days. To estimate half-life correctly, no drug was given on day 8. From the eighth treatment day on, 600 mg rifampicin was comedicated for 9 days (i.e., the last 3 days without talinolol). Concentration±time pro®les of R() and S(ÿ) talinolol (0±48 h) were measured on days 7 and 14 of the talinolol treatment. Venous blood was taken at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, 12, 16, 24, 36, and 48 h. Four days after the last talinolol administration (1 day after the last rifampicin dose), a second pharmacokinetic study with intravenous talinolol was performed. To assess serum protein binding of S(ÿ) and R() talinolol before and after rifampicin comedication, 0.5 mL serum from samples obtained 2, 2.5, 3.5, and 4 h after intravenous talinolol have been pooled. On pharmacokinetic study days, the subjects fasted 10 h before until 5 h after administration. Oral medication was controlled by mouth checking. Blood was centrifuged within 2 h and serum was stored at least at ÿ208C until analysis. Assessment of Talinolol Serum Protein Binding
Figure 1. Chemical structures of propranolol (above) and talinolol. The metabolites of talinolol are cis and trans hydroxylated in positions 3 and 4 of the cyclohexyl ring. The chiral centers are indicated by the star. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002
An ultra®ltration method employing cellulose triacetate ®lters (Centrisart I, Satorius, Germany) with a cutoff at 10,000 Da was used to separate free and bound fractions. The samples were centrifuged for 10 min at room temperature and 2000 rpm.
STEREOSELECTIVE DISPOSITION OF TALINOLOL
In Vitro Metabolism of Talinolol in Human Liver Microsomes Human liver microsomes were prepared by differential centrifugation from liver specimen (N 5) that were obtained from liver surgery and organ donors. Brie¯y, the tissue was homogenized (Ultra-Turrax T25, IKA Labortechnik, Germany) with 0.1 M potassium phophate buffer (pH 7.4) and centrifuged at 9.000 g for 30 min (Centrikon H401B, Kontron, Neufahrn, Germany) and 100,000 g for 60 min (Centrikon T-1170, Kontron, Neufahrn, Germany). The sediment was washed with buffer and centrifuged again at 100,000 g for 60 min. The pellets were suspended in potassium phosphate buffer (pH 7.4) and stored in aliquots at least at ÿ808C. Protein content was determined using the Biuret method. To study the in vitro metabolism of talinolol, microsomes (protein content 4 mg/mL) were incubated with an NADPH generating system (®nal volume 2 mL, 0.1 M potassium phosphate buffer, pH 7.4) consisting of NADPH (0.25 mM), glucose 6-phosphate (1.5 mM), and glucose 6-phosphate dehydrogenase (2.2 units/mL). After preincubation for 2 min, the reactions were started by adding 15.6, 31.3, 62.5, 125, 250, or 500 mM (®nal concentrations) of S(ÿ) or R() talinolol. After 30 min, the reaction was terminated by adding saturated sodium carbonate. To get information on the CYP isoforms involved in talinolol disposition, the enzyme assays were also performed in presence of the inhibitors ketoconazole (0.2 mM, for CYP3A4), a-naphtho¯avone (0.1 mM, for CYP1A2), and quinidine (4 mM, for CYP2D6). The formation of 4-trans, 4-cis, 3-trans, and 3-cis hydroxytalinolol was determined as described below. Quantitative Drug Assays Talinolol Enantiomers Serum (0.5 mL) was mixed with 100 mL saturated sodium carbonate and 25 mL internal standard solution (36.5 ng S(ÿ)-propranolol) and extracted with 5 mL diethylether. Following evaporation under a gentle stream of nitrogen at 408C, the residues were dissolved in 120 mL of the mobile phase (0.05% triethylamine in 60% ethanol and 40% acetonitrile) of which 50 mL were injected for chromatography (dry residues were stable at ÿ188C for at least 3 days). The HPLC system consisted of L 6200 A intelligent pump, AS 2000A autosampler, F 1050
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¯uorescence spectrometer (Merck-Hitachi, Darmstadt, Germany), ERC-315 solvent degasser (ERC Inc., Tokyo, Japan), column thermostat jetstream and cooled (158C) autosampler rack (WO electronics, Langenzersdorf, Austria). S(ÿ) and R() talinolol were separated with a LiChroCART 2504 column ®lled with ChiraSpher1 NT (poly[(S)N-acryloylphenylalanine ethyl ester] 5 mm.17 A 4-mm guard cartridge ®lled with LiChrosper1 DIOL was used to protect the analytical column. Between the pump and autosampler, a short victim column with pure silica was inserted. The mobile phase consisted of 60% ethanol with 0.05% triethylamine and 40% acetonitrile (¯ow 1.0 mL/ min, temperature 308C). Peak-area ratios were assessed from the ¯uorescence signal (excitation 252 nm, emission 332 nm) using the D-6500 HPLC manager software (Merck-Hitachi, Darmstadt, Germany). Assay of Talinolol Metabolites The incubation mixture (with 100 ng/mL metoclopramide as internal standard) was extracted with chloroform and injected for chromatography as described above. For separation of the analytes, an EcoCART1 125-3 HPLC cartridge ®lled with LiChrospher 60, RP-select B, 5 mm (Merck, Darmstadt, Germany) temperature regulated at 308C and a mobile phase consisting of 0.025 M thriethylammonium phosphate buffer and acetonitrile (gradient eluation with 10±25% within 15 min) was used. Peak-height ratios were assessed at 252 nm (excitation) and 332 nm (emission). Evaluation and Quality Control Calibration curves were evaluated with linear regression analysis weighed by 1/x (x concentration). For quality control, independent calibration and quality control sets were measured together with the samples derived from the pharmacokinetic study. Recovery rates, accuracy, and precision of the assay were calculated with standard procedures. The chromatographic conditions enabled symmetric peaks and a clear baseline separation of S(ÿ) propranolol, S(ÿ) and R() talinolol and 3-cis, 3-trans, 4-trans, 4-cis hydroxytalinolol as well as metoclopramide (Figures 2 and 3). The overall run time was 20 min. The calibration curves were linear between 2.5 ng/ml (5 ng/mL for metabolites) and 500 ng/mL. The limit of detection was 0.5 ng/mL de®ned as the threefold of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002
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the inter- and intraassay variability indicated that the HPLC assays ful®lled the requirements on drug analysis in pharmacokinetic studies.18 Pharmacokinetic and Statistical Evaluation
noise. No interference with any other substance could be observed in blanks of randomly selected subjects, volunteers included into the clinical study, and in blank microsomes. The data on
Peak talinolol concentrations (Cmax) and time of maximum (Tmax) were obtained directly from the data. The area under the serum concentration± time curve (AUC) was calculated with the trapezoidal rule from zero to 24 h (AUC0±24 h, for oral talinolol) and zero to the last time with a concentration above the limit of quanti®cation and extrapolated to in®nity (AUC0±1) using standard procedures (for intravenous talinolol). The elimination half-life (t1/2) was estimated by nonlinear approximation of the terminal data points. The systemic clearance (CLtot) and apparent oral clearance (CLtot/F) were calculated from dose/AUC and bioavailabilty (F) by comparing the AUC0±24 h after chronic oral administration with the AUC0±1 after intravenous administration before rifampicin induction. km and Vmax were assessed by least-squares ®tting of the Michaelis-Menten plots. Intrinsic clearance (Clint) was calculated as Vmax/km. All data are given as mean standard deviation (SD). Nonparametric Wilcoxon tests were used for statistical evaluation.
Figure 3. Chromatograms of 4-trans (5.98 min), 4-cis (7.05 min), 3-cis (8.04 min), and 3-trans hydroxytalinolol (9.07 min), the internal standard metoclopramide (9.85 min) and talinolol (16.05 min) of a spiked sample (above) and after extraction of a sample from the microsomal incubation (below).
Figure 4. Mean concentration±time pro®les of R() and S(ÿ) talinolol after infusion of racemic talinolol (30 mg in 30 min) in eight healthy subjects before (above) and after comedication of rifampicin (below).
Figure 2. Chromatograms obtained from a serum sample before (above) and after talinolol administration with clear baseline separation of S(ÿ) propranolol (7.06 min), S(ÿ) talinolol (8.63 min), and R() talinolol (10.53 min) (below).
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and signi®cantly shorter half-lives. The absolute bioavailability of oral talinolol was reduced by more than 15%. However, the differences between R() and S(ÿ) disposition were only slightly although signi®cantly increased (p < 0.05). The serum protein binding of S(ÿ) and R() talinolol before rifampicin was found as 78.2 1.2% and 79.0 3.1%, respectively; after rifampicin 84.9 5.0% and 84.5 5.2% were determined (both not signi®cant). Talinolol was metabolized in human liver microsomes to 3-, 4-cis and 3-, 4-trans hydroxytalinolol. Formation of 4-trans hydroxytalinolol was the major metabolic pathway followed by the formation of 3-cis, 4-cis, and 3-trans hydroxytalinolol. All Clint of the S(ÿ) enantiomers were higher than of the R() forms (Table 4). Ketoconazol (0.1 mM) inhibited the formation of all talinolol metabolites. a-naphtho¯avone and quinidine had no effect.
Figure 5. Mean concentration±time pro®les of R() and S(ÿ) talinolol after long-term oral administration of 100 mg racemic talinolol in eight healthy subjects before (above) and after comedication of rifamipicin (below).
DISCUSSION The MDR1 gene product P-gp in man is expressed in the endothelium of many tissues, which are involved into the disposition of drugs.4,19,20 The increasing knowledge on the mechanism of induction and inhibition of P-gp21 provides now a rational basis to explain drug/drug interactions that have been so far not reasonably understood; for example, the enhanced bioavailability of digoxin by clarithromycin, quinidine, verapamil, or talinolol,6,14,22,23 the lowered plasma levels of digoxin after treatment with St. John's wort,24 or the reduced bioavailability of digoxin, cyclosporine, tacrolimus, or talinolol due to rifampicin.15,25±27 Accepted methods are available to identify mutations of the MDR1 gene and to measure the
RESULTS Pharmacokinetics All concentration±time curves of R() talinolol exceeded the curves of S(ÿ) talinolol slightly (Figures 4 and 5). In noninduced subjects, these differences were of statistical signi®cance only after oral talinolol, because all individual values of AUC0±24 h, Cmax and F for R() were marginally higher than for S(ÿ) talinolol (Tables 1±3). Comedication of rifampicin resulted in signi®cant changes of either S(ÿ) and R() talinolol after both routes of administration as indicated by lower Cmax, higher Tmax and systemic clearance
Table 1. Pharmacokinetic Data of S(ÿ) and R() Talinolol After Infusion of Racemic Talinolol (30 mg in 30 min) in Eight Healthy Subjects Before and After Comedication of Rifampicin (Means SD) Without Rifampicin
AUC0±1 (ng h/mL) Cmax (ng/mL) CLtot (mL/min kg) Vc (l/kg) t1/2 (h)
With Rifampicin
S(ÿ) Talinolol
R() Talinolol
S(ÿ) Talinolol
725 107 269 31.8 4.72 0.52 0.761 0.076 16.1 4.9
696 73.5 263 39.7 4.90 0.51 0.783 0.106 13.7 1.2
522 94.8a 226 53.1a 6.65 1.23a 0.935 0.194a 10.4 3.5a
R() Talinolol 577 135b 230 55.6b 6.14 1.51 0.919 0.194a 11.6 5.2
a
p < 0.05 compared to without rifampicin. p < 0.05 compared to S(ÿ) talinolol (nonparametric Wilcoxon's signed rank test for matched samples).
b
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Table 2. Pharmacokinetic Data of S(ÿ) and R() Talinolol After Chronic Oral Administration of 100 mg Racemic Talinolol in Eight Healthy Subjects Before and After Comedication of Rifampicin (Means SD) Without Rifampicin S(ÿ) Talinolol AUC0±24 (ng h/mL) Cmax (ng/mL) tmax (h) F CLtot/F (mL/min kg) t1/2 (h)
With Rifampicin
R() Talinolol
1256 388 152 54.5 3.38 1.41 0.52 0.14 4.72 0.52 13.9 2.24
S(ÿ) Talinolol
b
a
1332 409 160 57.2b 3.38 1.41 0.57 0.15b 4.90 0.51 14.2 2.97
823 239 89.1 36.9a 4.81 2.07a 0.35 0.10a 6.64 1.23a 12.0 2.40a
R() Talinolol 902 274a,b 97.1 41.5a,b 4.94 1.86a 0.39 0.11a 6.14 1.51a 12.7 3.55a
a
p < 0.05 compared to without rifampicin. p < 0.05 compared to S(ÿ) talinolol (nonparametric Wilcoxon's signed rank test for matched samples).
b
products of transcription and translation.27±29 However, with exception of genotyping, these assays require tissue biopsy specimens that have to be gained by invasive procedures. So far, there are no validated methods available to determine P-gp function in man. Most information is currently available for the cardiac glycoside digoxin and the cardioselective b-blocker talinolol that, therefore, are the most promising candidates for phenotyping of P-gp. Both substances share properties that are preconditions for a probe drug of P-gp such as low plasma protein binding and low biotransformation, minor additional P-gp dependent excretion routes (tubular or biliary secretion), access to simple analytical assays, and absence of side effect when given in test doses. On the other hand, the suitability of talinolol or preferably of its less active R() enantiomer30,31 for phenotyping is topic of controversial discussion as ®rst the expression of intestinal P-gp is signi®cantly correlated with systemic clearance of intravenous talinolol but not with any pharmacokinetic characteristics after oral
administration,15 and, second, there are contradictory data on the question whether the disposition of talinolol is stereoselective. Gramatte and Oertel12 found no experimental evidence for stereoselective intestinal secretion of talinolol as assessed in 40 samples obtained from small intestine by triple-lumen tube technique. In patients after cholecystectomy and in healthy subjects, S(ÿ) talinolol serum concentrations were always marginally lower than those of R() talinolol. In Caco-2 cells, the af®nity of S(ÿ) talinolol to the binding site of the transporter was apparently higher.16 In the present study, we observed also slight differences between R() and S(ÿ) talinolol. These were only marginally more expressed after rifampicin comedication despite the dramatic changes in overall talinolol disposition following the induction of intestinal P-gp.15 If intestinal P-gp would discriminate between the enantiomers of talinolol, than one might expect much more pronounced differences between the disposition of R() and S(ÿ) talinolol after P-gp induction.
Table 3. Individual AUC-Values of S(ÿ) and R() Talinolol After Oral (AUC0±24 h) and Intravenous (AUC0±1) Administration of Racemic Talinolol Before and After Comedication of Rifampicin in Eight Healthy Subjects
Subject Number 1 2 3 4 5 6 7 8
i.v. Talinolol Before Rifampicin
i.v. Talinolol After Rifampicin
Oral Talinolol Before Rifampicin
Oral Talinolol After Rifampicin
S(ÿ)
R()
S(ÿ)
R()
S(ÿ)
R()
S(ÿ)
R()
739 630 832 710 687 863 544 798
702 745 730 680 611 719 576 805
547 449 559 504 570 418 428 704
618 611 626 448 599 432 446 837
1274 1259 621 1050 1309 1695 1000 1847
1366 1431 654 1078 1341 1793 1070 1925
874 1028 421 852 754 1220 652 780
933 1205 477 929 771 1344 712 849
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Table 4. Formation of Hydroxylated Metabolite of S(ÿ) and R() Talinolol in Human Liver Microsomes (Means SD of Five Specimens) 4-trans Hydroxytalinolol
km Vmax Clint
4-cis Hydroxytalinolol
3-trans Hydroxytalinolol
3-cis Hydroxytalinolol
S(ÿ)
R()
S(ÿ)
R()
S(ÿ)
R()
S(ÿ)
R()
115 23 21.3 1.8 0.19 0.03
336 51a 33.2 4.4a 0.14 0.02a
228 46 20.0 1.6 0.09 0.02
352 91a 22.7 3.7a 0.06 0.01a
253 71 11.1 2.7 0.05 0.01
373 73a 14.4 1.8 0.04 0.01
156 53 22.1 3.2 0.15 0.03
253 68a 26.6 3.7a 0.11 0.02a
km (mmol/L), Vmax (nmol/min mg), Clint (mL/min mg). a p < 0.05.
In noninduced subjects, talinolol is nearly not metabolized. The metabolic clearance was always less than 1% of systemic clearance.13±15 The enzyme involved into the metabolic degradation of talinolol in human liver microsomes seems to be cytochrome P4503A4 (CYP3A4), because formation of all hydroxylated metabolites was inhibited by ketoconazole but not by quinidine or a-naphtho¯avone.32±35 The intrinsic clearances of the S(ÿ) metabolites were signi®cantly higher than of the R() products. However, all km values were far beyond the concentrations expected in the vicinity of the intestinal and/or hepatic CYP enzyme after oral administration of therapeutic doses of talinolol. Therefore, our results indicate a presystemic, but low elimination rate of talinolol. There is ample of evidence for rifampicin to also be a strong inducer of intestinal and hepatic CYP3A4.25,26,36±38 If presystemic biotransformation would contribute to systemic clearance of talinolol to a higher extent as discussed above, enzyme induction after rifampicin medication should markedly increase the differences in disposition of R() and S(ÿ) talinolol. Nevertheless, we assume that stereoselectivity of the minor metabolic degradation rather than P-gp dependent talinolol transport is the reason of the small, even though signi®cant differences of R() and S(ÿ) talinolol pharmacokinetics especially after oral administration. This is in agreement with the observation of Wetterich et al. 1996,16 who reported apparently lower permeability of S(ÿ) talinolol in the Caco2 cell model, which is known to express CYP3A4.39 Differences in plasma protein binding could be excluded by our results. In Conclusion, talinolol holds the potential to be used as a probe drug for phenotyping of intestinal P-gp. The marginal stereoselectivity of its disposition is considered not to be of methodological relevance, and is most likely caused by the
minor biotransformation by CYP3A4. To avoid this in¯uence and signi®cant pharmacodynamic effects, the less active R() enantiomer, for which we have developed a simple assay, should be used in clinical trials.
ACKNOWLEDGMENTS We thank H. Kreher, G. Schumacher, S. Bade, and R. Peters for excellent technical support and W. Weitschies for many useful suggestions. Parts of this study were presented at the 40th Spring Meeting of the German Society of Experimental and Clinical Pharmacology and Toxicology, March 9±11, 1999, Mainz, Germany.
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bioequivalence and pharmacokinetic studies. Int J Pharmacol 82:1±7. Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, Willingham MC. 1987. Cellular localization of the multidrug-resistance gene product pglycoprotein in normal human tissues. Proc Natl Acad Sci USA 84:7735±7738. Cordon-Cardo C, O'Brien JP, Boccia J, Casals D, Bertino JR, Melamed MR. 1990. Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues. J Histochem Cytochem 38:1277±1287. Watkins PB. 1997. The barrier function of CYP3A4 and P-glycoprotein in the small bowel. Adv Drug Del Rev 27:161±170. Wakasugi H, Yano I, Ito T, Hashida T, Futami T, Nohara R, Sasayama S, Inui K. 1998. Effect of clarithromycin on renal excretion of digoxin: Interaction with P-glycoprotein. Clin Pharmacol Ther 64:123±128. Pedersen KE, Christiansen BD, Klitgaard NA, Nielsen-Kudsk F. 1983. Effect of quinidine on digoxin bioavailability. Eur J Clin Pharmacol 24: 41±47. Johne A, BrockmoÈller J, Bauer S, Maurer A, Langheinrich M, Roots I. 1999. Pharmacokinetic interaction of digoxin with an herbal extract from St John's wort (Hypericum perforatum). Clin Pharmacol Ther 66:338±345. Hebert MF, Roberts JP, Prueksaritanont T, Benet LZ. 1992. Bioavailability of cyclosporine with concomitant rifampin administration is markedly less than predicted by hepatic enzyme induction. Clin Pharmacol Ther 52:453±457. Hebert MF, Fisher RM, Marsh CL, Dressler D, Bekersky I. 1999. Effects of rifampin on tacrolimus pharmacokinetics in healthy volunteers. J Clin Pharmacol 39:91±96. Greiner B, Eichelbaum M, Fritz P, Kreichgauer HP, von Richter O, Zundler J, Kroemer HK. 1999. The interaction of digoxin and rifampin: Role of intestinal P-glycoprotein. J Clin Invest 104:147±153. Hoffmeyer S, Burk O, von Richter O, Arnold HP, BrockmuÈller J, Johne A, Cascorbi I, Gerloff T, Roots I, Eichelbaum M, Brinkmann U. 2000. Functional polymorphism of the human multidrug-resistance gene: Multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo. Proc Natl Acad Sci USA 97:3473± 3478. Bordow SB, Haber M, Mada®glio J, Cheung B, Marshall GM, Norris MD. 1994. Expression of the multidrug resistance-associated protein (MRP) gene correlates with ampli®cation and overexpression of the N-myc oncogene in childhood neuroblastoma. Cancer Res 54:5036±5040. Bartsch R, Femmer K, Heer S, Ploen U, Poppe H. 1979. Zur Pharmakologie der optisch aktiven
STEREOSELECTIVE DISPOSITION OF TALINOLOL
31.
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Isomeren von Talinolol (Cordanum1). Dt GesundhWesen 34:1041±1046. DeMey C, Schroeter V, Butzer R, Jahn P, Weisser K, Wetterich U, Terhaag B, Mutschler E, SpahnLangguth H, Palm D, Belz GG. 1995. Dose±effect and kinetic±dynamic relationships of the b-adrenoceptor blocking properties of various doses of talinolol in healthy humans. J Cardiovasc Pharmacol 26:879±888. Eagling VA, Tjia JF, Back DJ. 1998. Differential selectivity of cytochrome P450 inhibitors against probe substrates in human and rat liver microsomes. Br J Clin Pharmacol 45:107±114. Ono S, Hatanaka T, Hotta H, Satoh T, Gonzalez FJ, Tsutsui M. 1996. Speci®city of substrate and inhibitor probes for cytochrome p450s: Evaluation of in vitro metabolism using cDNA-expressed human P450s and human liver microsomes. Xenobiotica 26:681±693. Bourrie M, Meunier V, Berger Y, Fabre G. 1996. Cytochrome P450 isoform inhibitors as tool for the investigation of metabolic reactions catalyzed by human liver microsomes. J Pharmacol Exp Ther 277:321±332.
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ABSTRACT: The disposition of the b-blocking drug talinolol is controlled by P-glycoprotein in man. Because talinolol is marketed as a racemate, we reevaluated the serum-concentration time pro®les of talinolol of a previously published study with single intravenous (30 mg) and repeated oral talinolol (100 mg for 14 days) before and after comedication of rifampicin (600 mg per day for 9 days) in eight male healthy volunteers (age 22±26 years, body weight 67±84 kg) with respect to differences in the kinetic pro®les of the two enantiomers S(ÿ) talinolol and R() talinolol. Additionally, the metabolism of talinolol in human liver microsomes was examined. After oral administration, S(ÿ) talinolol was slightly less absorbed and faster eliminated than R() talinolol. The absolute bioavailabilty of the R() enantiomer of talinolol was slightly but signi®cantly higher than of its S(ÿ) enantiomer. Coadministration of rifampicin further intensi®ed this difference in the disposition of R() and S(ÿ) talinolol (p < 0.05). Formation of 4-trans hydroxytalinolol was the major metabolic pathway in human liver microsomes. All Clint values of S(ÿ) were higher than of R() talinolol; 0.1 mM ketoconazole inhibited the formation of all metabolites. In conclusion, the stereoselectivity of talinolol disposition is of minor importance, and most likely caused by presystemic biotransformation via CYP3A4. The less active R() talinolol might be suitable for phenotyping P-glycoprotein expression in man. ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:303±311, 2002
Keywords:
talinolol; man; P-glycoprotein; racemate
INTRODUCTION The mammalian drug-transporting MDR1 gene product P-glycoprotein (P-gp) is part of a protection system against a large number of xenobiotic compounds. The exact mechanisms how P-gp recognizes substrates remain to be elucidated. All
Klaus-JuÈrgen Klebingat's present address is Department of Urology, University of Greifswald. Bernd Terhaag's and Anna Hoffmann's present address is Arzneimittelwerk Dresden GmbH, Dresden, Germany. Thomas GramatteÂ's present address is APOGEPHA Arzneimittel GmbH, Dresden, Germany. Correspondence to: Werner Siegmund (Telephone: 493834-86532; Fax: 49-3834-86531; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 91, 303±311 (2002) ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association
ligands of P-gp are in common amphiphilic that seems to be essential for their insertion in apical membranes to be translocated.1,2 Other structural characteristics seem to be of minor importance for substrate binding as cationic, neutral, and even anionic compounds with extremely diverse chemical structure and molecular weights from 250 to almost 1900 Da are transported.3,4 Furthermore, it is reported, that P-gp does not discriminate between the enantiomers of chiral compounds.5±10 To determine intestinal P-gp expression in man, biopsy specimens from the gut have to be obtained by invasive methods. A probe drug approach that avoids such invasive investigations is therefore highly requested for the assessment of P-gp function. So far, the b-blocking agent talinolol is the only compound for which a signi®cant secretion into the gut lumen in man
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has already been demonstrated.11,12 The drug is subjected to minor metabolism, less than 1% is excreted into urine in form of hydroxylated metabolites.13±15 It is unknown whether metabolites are excreted by other pathways (e.g., biliary, intestinal). However, information on the suitability of racemic talinolol to predict P-gp expression is still limited by contradictory results on the chirality of talinolol disposition.12,16 If the P-gp dependent transport of talinolol would be stereoselective, then upregulation of the carrier by rifampicin should result in adequate differences of R() and S(ÿ) talinolol disposition. Therefore, we reassessed the serum samples of a recently published interaction study with rifampicin to measure pharmacokinetics of talinolol enantiomers.15
MATERIALS AND METHODS Chemicals S(ÿ) and R() talinolol [1-(4-cyclohexylureidophenoxy)-2-hydroxy-3-tert-butylamino-propane] its metabolites 3-trans, 3-cis 4-trans, and 4-cis hydroxytalinolol (Figure 1), as well as metoclopramide were generously provided by Arzneimittelwerk Dresden (Dresden, Germany). S(ÿ) propranolol was purchased from Tocris Cookson (Bristol, UK) and ketoconazole from Sigma (Deisenhofen, Germany). Acetonitrile and ethanol were obtained from Baker (Deventer, The Netherlands), triethylamine from Fluka (Buchs, Switzerland). All solvents and chemicals were of HPLC or analytical grade. The pharmacokinetic study in healthy subjects was performed with commercially available formulations of talinolol (100 mg ®lm coated tablets and 30 mg vial
solution, Cordanum1, Arzneimittelwerk Dresden, Germany). Subjects and Study Protocol The serum samples used for this investigation have been gained from a clinical study that has been described in detail elsewhere.15 In brief, eight male subjects (age 22±26 years, body weight 67±84 kg) were included after giving informed written consent. They were of good health, as evidenced by physical examination, routine clinical-chemical, and hematological screening. The subjects were negative for hepatitis and drugs, abstained strictly from alcohol, and took standard diet. The study protocol had been approved by the local ethics committee. At the start of the study, 30 mg talinolol were given intravenously within 30 min and venous blood (5 mL) was collected before and 0.17, 0.33, 0.5, 0.67, 1, 1.5, 2, 2.5, 3.5, 4, 4.5, 6.5, 8.5, 12.5, 16.5, 25.5, and 36.5 h after starting the infusion. Following a washout period of 8 days, volunteers were medicated with 100 mg oral talinolol for 14 subsequent days. To estimate half-life correctly, no drug was given on day 8. From the eighth treatment day on, 600 mg rifampicin was comedicated for 9 days (i.e., the last 3 days without talinolol). Concentration±time pro®les of R() and S(ÿ) talinolol (0±48 h) were measured on days 7 and 14 of the talinolol treatment. Venous blood was taken at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, 12, 16, 24, 36, and 48 h. Four days after the last talinolol administration (1 day after the last rifampicin dose), a second pharmacokinetic study with intravenous talinolol was performed. To assess serum protein binding of S(ÿ) and R() talinolol before and after rifampicin comedication, 0.5 mL serum from samples obtained 2, 2.5, 3.5, and 4 h after intravenous talinolol have been pooled. On pharmacokinetic study days, the subjects fasted 10 h before until 5 h after administration. Oral medication was controlled by mouth checking. Blood was centrifuged within 2 h and serum was stored at least at ÿ208C until analysis. Assessment of Talinolol Serum Protein Binding
Figure 1. Chemical structures of propranolol (above) and talinolol. The metabolites of talinolol are cis and trans hydroxylated in positions 3 and 4 of the cyclohexyl ring. The chiral centers are indicated by the star. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002
An ultra®ltration method employing cellulose triacetate ®lters (Centrisart I, Satorius, Germany) with a cutoff at 10,000 Da was used to separate free and bound fractions. The samples were centrifuged for 10 min at room temperature and 2000 rpm.
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In Vitro Metabolism of Talinolol in Human Liver Microsomes Human liver microsomes were prepared by differential centrifugation from liver specimen (N 5) that were obtained from liver surgery and organ donors. Brie¯y, the tissue was homogenized (Ultra-Turrax T25, IKA Labortechnik, Germany) with 0.1 M potassium phophate buffer (pH 7.4) and centrifuged at 9.000 g for 30 min (Centrikon H401B, Kontron, Neufahrn, Germany) and 100,000 g for 60 min (Centrikon T-1170, Kontron, Neufahrn, Germany). The sediment was washed with buffer and centrifuged again at 100,000 g for 60 min. The pellets were suspended in potassium phosphate buffer (pH 7.4) and stored in aliquots at least at ÿ808C. Protein content was determined using the Biuret method. To study the in vitro metabolism of talinolol, microsomes (protein content 4 mg/mL) were incubated with an NADPH generating system (®nal volume 2 mL, 0.1 M potassium phosphate buffer, pH 7.4) consisting of NADPH (0.25 mM), glucose 6-phosphate (1.5 mM), and glucose 6-phosphate dehydrogenase (2.2 units/mL). After preincubation for 2 min, the reactions were started by adding 15.6, 31.3, 62.5, 125, 250, or 500 mM (®nal concentrations) of S(ÿ) or R() talinolol. After 30 min, the reaction was terminated by adding saturated sodium carbonate. To get information on the CYP isoforms involved in talinolol disposition, the enzyme assays were also performed in presence of the inhibitors ketoconazole (0.2 mM, for CYP3A4), a-naphtho¯avone (0.1 mM, for CYP1A2), and quinidine (4 mM, for CYP2D6). The formation of 4-trans, 4-cis, 3-trans, and 3-cis hydroxytalinolol was determined as described below. Quantitative Drug Assays Talinolol Enantiomers Serum (0.5 mL) was mixed with 100 mL saturated sodium carbonate and 25 mL internal standard solution (36.5 ng S(ÿ)-propranolol) and extracted with 5 mL diethylether. Following evaporation under a gentle stream of nitrogen at 408C, the residues were dissolved in 120 mL of the mobile phase (0.05% triethylamine in 60% ethanol and 40% acetonitrile) of which 50 mL were injected for chromatography (dry residues were stable at ÿ188C for at least 3 days). The HPLC system consisted of L 6200 A intelligent pump, AS 2000A autosampler, F 1050
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¯uorescence spectrometer (Merck-Hitachi, Darmstadt, Germany), ERC-315 solvent degasser (ERC Inc., Tokyo, Japan), column thermostat jetstream and cooled (158C) autosampler rack (WO electronics, Langenzersdorf, Austria). S(ÿ) and R() talinolol were separated with a LiChroCART 2504 column ®lled with ChiraSpher1 NT (poly[(S)N-acryloylphenylalanine ethyl ester] 5 mm.17 A 4-mm guard cartridge ®lled with LiChrosper1 DIOL was used to protect the analytical column. Between the pump and autosampler, a short victim column with pure silica was inserted. The mobile phase consisted of 60% ethanol with 0.05% triethylamine and 40% acetonitrile (¯ow 1.0 mL/ min, temperature 308C). Peak-area ratios were assessed from the ¯uorescence signal (excitation 252 nm, emission 332 nm) using the D-6500 HPLC manager software (Merck-Hitachi, Darmstadt, Germany). Assay of Talinolol Metabolites The incubation mixture (with 100 ng/mL metoclopramide as internal standard) was extracted with chloroform and injected for chromatography as described above. For separation of the analytes, an EcoCART1 125-3 HPLC cartridge ®lled with LiChrospher 60, RP-select B, 5 mm (Merck, Darmstadt, Germany) temperature regulated at 308C and a mobile phase consisting of 0.025 M thriethylammonium phosphate buffer and acetonitrile (gradient eluation with 10±25% within 15 min) was used. Peak-height ratios were assessed at 252 nm (excitation) and 332 nm (emission). Evaluation and Quality Control Calibration curves were evaluated with linear regression analysis weighed by 1/x (x concentration). For quality control, independent calibration and quality control sets were measured together with the samples derived from the pharmacokinetic study. Recovery rates, accuracy, and precision of the assay were calculated with standard procedures. The chromatographic conditions enabled symmetric peaks and a clear baseline separation of S(ÿ) propranolol, S(ÿ) and R() talinolol and 3-cis, 3-trans, 4-trans, 4-cis hydroxytalinolol as well as metoclopramide (Figures 2 and 3). The overall run time was 20 min. The calibration curves were linear between 2.5 ng/ml (5 ng/mL for metabolites) and 500 ng/mL. The limit of detection was 0.5 ng/mL de®ned as the threefold of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 2, FEBRUARY 2002
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the inter- and intraassay variability indicated that the HPLC assays ful®lled the requirements on drug analysis in pharmacokinetic studies.18 Pharmacokinetic and Statistical Evaluation
noise. No interference with any other substance could be observed in blanks of randomly selected subjects, volunteers included into the clinical study, and in blank microsomes. The data on
Peak talinolol concentrations (Cmax) and time of maximum (Tmax) were obtained directly from the data. The area under the serum concentration± time curve (AUC) was calculated with the trapezoidal rule from zero to 24 h (AUC0±24 h, for oral talinolol) and zero to the last time with a concentration above the limit of quanti®cation and extrapolated to in®nity (AUC0±1) using standard procedures (for intravenous talinolol). The elimination half-life (t1/2) was estimated by nonlinear approximation of the terminal data points. The systemic clearance (CLtot) and apparent oral clearance (CLtot/F) were calculated from dose/AUC and bioavailabilty (F) by comparing the AUC0±24 h after chronic oral administration with the AUC0±1 after intravenous administration before rifampicin induction. km and Vmax were assessed by least-squares ®tting of the Michaelis-Menten plots. Intrinsic clearance (Clint) was calculated as Vmax/km. All data are given as mean standard deviation (SD). Nonparametric Wilcoxon tests were used for statistical evaluation.
Figure 3. Chromatograms of 4-trans (5.98 min), 4-cis (7.05 min), 3-cis (8.04 min), and 3-trans hydroxytalinolol (9.07 min), the internal standard metoclopramide (9.85 min) and talinolol (16.05 min) of a spiked sample (above) and after extraction of a sample from the microsomal incubation (below).
Figure 4. Mean concentration±time pro®les of R() and S(ÿ) talinolol after infusion of racemic talinolol (30 mg in 30 min) in eight healthy subjects before (above) and after comedication of rifampicin (below).
Figure 2. Chromatograms obtained from a serum sample before (above) and after talinolol administration with clear baseline separation of S(ÿ) propranolol (7.06 min), S(ÿ) talinolol (8.63 min), and R() talinolol (10.53 min) (below).
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and signi®cantly shorter half-lives. The absolute bioavailability of oral talinolol was reduced by more than 15%. However, the differences between R() and S(ÿ) disposition were only slightly although signi®cantly increased (p < 0.05). The serum protein binding of S(ÿ) and R() talinolol before rifampicin was found as 78.2 1.2% and 79.0 3.1%, respectively; after rifampicin 84.9 5.0% and 84.5 5.2% were determined (both not signi®cant). Talinolol was metabolized in human liver microsomes to 3-, 4-cis and 3-, 4-trans hydroxytalinolol. Formation of 4-trans hydroxytalinolol was the major metabolic pathway followed by the formation of 3-cis, 4-cis, and 3-trans hydroxytalinolol. All Clint of the S(ÿ) enantiomers were higher than of the R() forms (Table 4). Ketoconazol (0.1 mM) inhibited the formation of all talinolol metabolites. a-naphtho¯avone and quinidine had no effect.
Figure 5. Mean concentration±time pro®les of R() and S(ÿ) talinolol after long-term oral administration of 100 mg racemic talinolol in eight healthy subjects before (above) and after comedication of rifamipicin (below).
DISCUSSION The MDR1 gene product P-gp in man is expressed in the endothelium of many tissues, which are involved into the disposition of drugs.4,19,20 The increasing knowledge on the mechanism of induction and inhibition of P-gp21 provides now a rational basis to explain drug/drug interactions that have been so far not reasonably understood; for example, the enhanced bioavailability of digoxin by clarithromycin, quinidine, verapamil, or talinolol,6,14,22,23 the lowered plasma levels of digoxin after treatment with St. John's wort,24 or the reduced bioavailability of digoxin, cyclosporine, tacrolimus, or talinolol due to rifampicin.15,25±27 Accepted methods are available to identify mutations of the MDR1 gene and to measure the
RESULTS Pharmacokinetics All concentration±time curves of R() talinolol exceeded the curves of S(ÿ) talinolol slightly (Figures 4 and 5). In noninduced subjects, these differences were of statistical signi®cance only after oral talinolol, because all individual values of AUC0±24 h, Cmax and F for R() were marginally higher than for S(ÿ) talinolol (Tables 1±3). Comedication of rifampicin resulted in signi®cant changes of either S(ÿ) and R() talinolol after both routes of administration as indicated by lower Cmax, higher Tmax and systemic clearance
Table 1. Pharmacokinetic Data of S(ÿ) and R() Talinolol After Infusion of Racemic Talinolol (30 mg in 30 min) in Eight Healthy Subjects Before and After Comedication of Rifampicin (Means SD) Without Rifampicin
AUC0±1 (ng h/mL) Cmax (ng/mL) CLtot (mL/min kg) Vc (l/kg) t1/2 (h)
With Rifampicin
S(ÿ) Talinolol
R() Talinolol
S(ÿ) Talinolol
725 107 269 31.8 4.72 0.52 0.761 0.076 16.1 4.9
696 73.5 263 39.7 4.90 0.51 0.783 0.106 13.7 1.2
522 94.8a 226 53.1a 6.65 1.23a 0.935 0.194a 10.4 3.5a
R() Talinolol 577 135b 230 55.6b 6.14 1.51 0.919 0.194a 11.6 5.2
a
p < 0.05 compared to without rifampicin. p < 0.05 compared to S(ÿ) talinolol (nonparametric Wilcoxon's signed rank test for matched samples).
b
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Table 2. Pharmacokinetic Data of S(ÿ) and R() Talinolol After Chronic Oral Administration of 100 mg Racemic Talinolol in Eight Healthy Subjects Before and After Comedication of Rifampicin (Means SD) Without Rifampicin S(ÿ) Talinolol AUC0±24 (ng h/mL) Cmax (ng/mL) tmax (h) F CLtot/F (mL/min kg) t1/2 (h)
With Rifampicin
R() Talinolol
1256 388 152 54.5 3.38 1.41 0.52 0.14 4.72 0.52 13.9 2.24
S(ÿ) Talinolol
b
a
1332 409 160 57.2b 3.38 1.41 0.57 0.15b 4.90 0.51 14.2 2.97
823 239 89.1 36.9a 4.81 2.07a 0.35 0.10a 6.64 1.23a 12.0 2.40a
R() Talinolol 902 274a,b 97.1 41.5a,b 4.94 1.86a 0.39 0.11a 6.14 1.51a 12.7 3.55a
a
p < 0.05 compared to without rifampicin. p < 0.05 compared to S(ÿ) talinolol (nonparametric Wilcoxon's signed rank test for matched samples).
b
products of transcription and translation.27±29 However, with exception of genotyping, these assays require tissue biopsy specimens that have to be gained by invasive procedures. So far, there are no validated methods available to determine P-gp function in man. Most information is currently available for the cardiac glycoside digoxin and the cardioselective b-blocker talinolol that, therefore, are the most promising candidates for phenotyping of P-gp. Both substances share properties that are preconditions for a probe drug of P-gp such as low plasma protein binding and low biotransformation, minor additional P-gp dependent excretion routes (tubular or biliary secretion), access to simple analytical assays, and absence of side effect when given in test doses. On the other hand, the suitability of talinolol or preferably of its less active R() enantiomer30,31 for phenotyping is topic of controversial discussion as ®rst the expression of intestinal P-gp is signi®cantly correlated with systemic clearance of intravenous talinolol but not with any pharmacokinetic characteristics after oral
administration,15 and, second, there are contradictory data on the question whether the disposition of talinolol is stereoselective. Gramatte and Oertel12 found no experimental evidence for stereoselective intestinal secretion of talinolol as assessed in 40 samples obtained from small intestine by triple-lumen tube technique. In patients after cholecystectomy and in healthy subjects, S(ÿ) talinolol serum concentrations were always marginally lower than those of R() talinolol. In Caco-2 cells, the af®nity of S(ÿ) talinolol to the binding site of the transporter was apparently higher.16 In the present study, we observed also slight differences between R() and S(ÿ) talinolol. These were only marginally more expressed after rifampicin comedication despite the dramatic changes in overall talinolol disposition following the induction of intestinal P-gp.15 If intestinal P-gp would discriminate between the enantiomers of talinolol, than one might expect much more pronounced differences between the disposition of R() and S(ÿ) talinolol after P-gp induction.
Table 3. Individual AUC-Values of S(ÿ) and R() Talinolol After Oral (AUC0±24 h) and Intravenous (AUC0±1) Administration of Racemic Talinolol Before and After Comedication of Rifampicin in Eight Healthy Subjects
Subject Number 1 2 3 4 5 6 7 8
i.v. Talinolol Before Rifampicin
i.v. Talinolol After Rifampicin
Oral Talinolol Before Rifampicin
Oral Talinolol After Rifampicin
S(ÿ)
R()
S(ÿ)
R()
S(ÿ)
R()
S(ÿ)
R()
739 630 832 710 687 863 544 798
702 745 730 680 611 719 576 805
547 449 559 504 570 418 428 704
618 611 626 448 599 432 446 837
1274 1259 621 1050 1309 1695 1000 1847
1366 1431 654 1078 1341 1793 1070 1925
874 1028 421 852 754 1220 652 780
933 1205 477 929 771 1344 712 849
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Table 4. Formation of Hydroxylated Metabolite of S(ÿ) and R() Talinolol in Human Liver Microsomes (Means SD of Five Specimens) 4-trans Hydroxytalinolol
km Vmax Clint
4-cis Hydroxytalinolol
3-trans Hydroxytalinolol
3-cis Hydroxytalinolol
S(ÿ)
R()
S(ÿ)
R()
S(ÿ)
R()
S(ÿ)
R()
115 23 21.3 1.8 0.19 0.03
336 51a 33.2 4.4a 0.14 0.02a
228 46 20.0 1.6 0.09 0.02
352 91a 22.7 3.7a 0.06 0.01a
253 71 11.1 2.7 0.05 0.01
373 73a 14.4 1.8 0.04 0.01
156 53 22.1 3.2 0.15 0.03
253 68a 26.6 3.7a 0.11 0.02a
km (mmol/L), Vmax (nmol/min mg), Clint (mL/min mg). a p < 0.05.
In noninduced subjects, talinolol is nearly not metabolized. The metabolic clearance was always less than 1% of systemic clearance.13±15 The enzyme involved into the metabolic degradation of talinolol in human liver microsomes seems to be cytochrome P4503A4 (CYP3A4), because formation of all hydroxylated metabolites was inhibited by ketoconazole but not by quinidine or a-naphtho¯avone.32±35 The intrinsic clearances of the S(ÿ) metabolites were signi®cantly higher than of the R() products. However, all km values were far beyond the concentrations expected in the vicinity of the intestinal and/or hepatic CYP enzyme after oral administration of therapeutic doses of talinolol. Therefore, our results indicate a presystemic, but low elimination rate of talinolol. There is ample of evidence for rifampicin to also be a strong inducer of intestinal and hepatic CYP3A4.25,26,36±38 If presystemic biotransformation would contribute to systemic clearance of talinolol to a higher extent as discussed above, enzyme induction after rifampicin medication should markedly increase the differences in disposition of R() and S(ÿ) talinolol. Nevertheless, we assume that stereoselectivity of the minor metabolic degradation rather than P-gp dependent talinolol transport is the reason of the small, even though signi®cant differences of R() and S(ÿ) talinolol pharmacokinetics especially after oral administration. This is in agreement with the observation of Wetterich et al. 1996,16 who reported apparently lower permeability of S(ÿ) talinolol in the Caco2 cell model, which is known to express CYP3A4.39 Differences in plasma protein binding could be excluded by our results. In Conclusion, talinolol holds the potential to be used as a probe drug for phenotyping of intestinal P-gp. The marginal stereoselectivity of its disposition is considered not to be of methodological relevance, and is most likely caused by the
minor biotransformation by CYP3A4. To avoid this in¯uence and signi®cant pharmacodynamic effects, the less active R() enantiomer, for which we have developed a simple assay, should be used in clinical trials.
ACKNOWLEDGMENTS We thank H. Kreher, G. Schumacher, S. Bade, and R. Peters for excellent technical support and W. Weitschies for many useful suggestions. Parts of this study were presented at the 40th Spring Meeting of the German Society of Experimental and Clinical Pharmacology and Toxicology, March 9±11, 1999, Mainz, Germany.
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