Determination of the Enantioselectivity of Six Chiral Aryloxy Aminopropanol Drugs Transport Across Caco-2 Cell Monolayers

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector Acta Pharmaceutica Sinica B 2012;2(2):168–173

Institute of Materia Medica, Chinese Academy of Medical Sciences Chinese Pharmaceutical Association

Acta Pharmaceutica Sinica B

www.elsevier.com/locate/apsb www.sciencedirect.com

ORIGINAL ARTICLE Determination of the enantioselectivity of six chiral aryloxy aminopropanol drugs transport across Caco-2 cell monolayers

Ye Tiany, Ying Hey, Haihong Hu, Lu Wang, Su Zengn

College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China

Received 9 January 2012; revised 5 February 2012; accepted 12 February 2012

KEY WORDS Abstract This study aimed to determine the transepithelial transport characteristics of chiral drug enantiomers across Caco-2 cell monolayers, a model of human intestinal epithelial Chiral aryloxy membrane. Six chiral aryloxy enantiomers (atenolol, sotalol, celiprolol, carvedilol, metopro- aminopropanol drugs; Transport; lol and propafenone) were tested in bi-directional transport studies. The separation and Enantioselectivity; quantitation of these enantiomers were performed by reversed-phase high-performance liquid P-gp; chromatography (RP-HPLC) using 2, 3, 4, 6-tetra-O-acetyl-b-D-glucopyranosyl isothiocya- Caco-2 cell nate (GITC) as a pre-column derivatizing agent. Bi-directional transport studies demonstrated that celiprolol and carvedilol exhibited significant enantioselectivity in polarized transport at the concentration range tested. The efflux ratio (ER) for (R)-(þ)-celiprolol was 8.96, but it was much lower for (S)-(-)-celiprolol which is 3.42 at the concentration of 96.0 mM; carvedilol had the same transport behavior as celiprolol while the difference between the ER values of two enantiomers was not as significant as celiprolol at the concentration of 5.0 mM. They are 2.41 for (R)-(þ)-carvedilol and 1.98 for (S)-(-)-carvedilol. But in the transport studies of racemic atenolol, sotalol, metoprolol and propafenone, no enantioselective transport were observed over the concentration range tested. Because P-glycoprotein (P-gp) is highly expressed in Caco-2 cells, we inferred that P-gp might participate in the transport processes of celiprolol and carvedilol in chirally discriminative ways.

nCorresponding author. Tel./fax: þ86 571 88208407. E-mail address: [email protected] (Su Zeng). yThese authors contributed equally to this work.

Peer review under responsibility of Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association. doi:10.1016/j.apsb.2012.02.005 Determination of the enantioselectivity of six chiral aryloxy aminopropanol drugs 169

1. Introduction propafenone) in Caco-2 cell model, which has been widely accepted as an in vitro model for intestinal drug absorption Chiral drugs, which took more than 50% of all current drugs, (Fig. 1). The chiral RP-HPLC method using 2,3,4,6-tetra-O- have been paid more and more attention for their clinical acetyl-b-D-glucopyranosyl isothiocyanate (GITC) as a pre- applications. A pair of enantiomers would be discriminated as column derivatizing agent was used to separate and assay these different molecules by chiral environment such as enzymes, enantiomers, which was previously described by our group6. receptors, carries, etc. Biomembrane permeability and cellular In this study, we will concentrate on the transport character- uptake of chiral drugs may be also stereoselective if intracel- istics of these chiral drug enantiomers across Caco-2 cells. lular macromolecules recognize the enantiomers in chirally discriminative ways. Therefore, the pharmacodynamics, pharmacokinetics and toxicology between the enantiomers could 2. Materials and methods be stereoselective. Aryloxy aminopropanol drugs, such as atenolol, sotalol, 2.1. Materials celiprolol, carvedilol, metoprolol and propafenone, were widely used to treat cardiovascular diseases and administrated mainly by Racemic atenolol and racemic metoprolol tartrate were pur- racemes. Studies proved that the pharmacokinetics of some chased from National Institutes for Food and Drug Control aryloxy aminopropanol compounds, for example, plasma protein (Beijing, China). Racemic sotalol hydrochloride was provided binding, liver metabolism, bile excretion, were stereoselective1–3, by Dong Dong Chemical Pharmaceutical Company (Taizhou, and the enantiomers’ pharmacological activities are different from Zhejiang, China). Racemic celiprolol hydrochloride was pro- each other4. For example, (S)-(þ)-sotalol hydrochloride and (R)- vided by Hai Zheng Pharmaceutical Company (Zhejiang, ()-isomer have similar anti-arrhythmia activity but the compre- China). Racemic propafenone hydrochloride was purchased hensive effect of b-adrenergic blockers is actually attributed by from Sigma. Racemic carvedilol was provided by Tian Heng (R)-()-isomer5. Due to these stereo-different pharmacological Pharmaceutical Company (Ningbo, Zhejiang, China). Lucifer profiles, the uncovering of the transport stereoselectivity of aryloxy yellow was purchased from Biochemika. Methanol and acet- aminopropanol drugs was important in clarifying the pharmaco- onitrile were of HPLC grade. Other solvents used were of kinetics of these drugs; furthermore, the uncovering of pharma- analytical grade. cokinetic behaviors of these drugs will facilitate the understanding of the pharmacological differences. 2.2. Cell culture To evaluate the transportation mechanisms and absorption of aryloxy aminopropanol drugs and to further explain the Caco-2 cells (35 passage) obtained from Chinese Academy of pharmacological differences of these drugs, we investigated the Medical Sciences (CAMS, Beijing, China) were grown in a transepithelial transport of six chiral aryloxy aminopropanol humidified atmosphere of 5% CO2 at 37 1C. Cells were drugs (atenolol, sotalol, celiprolol, carvedilol, metoprolol and cultured in high-glucose Dulbecco’s modified eagle’s medium

Figure 1 The structures of six aryloxy aminopropanol drugs (atenolol, sotalol, celiprolol, carvedilol, metoprolol and propafenone), the chiral carbon atom was labeled by ‘‘Ã’’. 170 Ye Tian et al.

(DMEM, Gibco, Bethesda, MD) supplemented with 10% fetal started by replacing the donor buffer with transport buffer bovine serum (Gibco), 1% nonessential amino acids (Gibco). containing individual concentration of these drugs. The After reaching 90% confluence, Caco-2 cells were harvested volume added to the apical (AP) side of the monolayer was with 0.25% trypsin-0.02% EDTA solution and seeded in Trans- 0.5 mL and 1.5 mL at the basolateral (BL) side. Transport wells inserts (catalog number 3460, Corning Coster Corp., Acton, studies were conducted at both the absorptive direction (AP- MA) in 12-well plates at a density of 1.0 105 cells/cm2.After BL) and the secretory direction (BL-AP)12. After incubating culturing for 21 days, Caco-2 cells were used in experiment7.The at 37 1C for indicated time, 200 mL samples were collected integrity of the monolayer was checked by measuring the transe- from the receiving sides of the cell monolayers for HPLC pithelial electrical resistance (TEER) value across the monolayer analysis. Drug concentrations loaded onto the Caco-2 cell using Millicell-ERS Voltohmmeter (Millipore) and monitoring the monolayers were shown in Table 1, and the incubation time apparent permeability coefficient (Papp) of the paracellular leakage were shown in Table 2. marker Lucifer yellow across the monolayer8,9. Only monolayers 2 with initial TEER values higher than 450 O/cm and the Papp value 2.4. Enantioselective HPLC analysis lower than 0.2 106 cm/s were used. The (S)- and (R)-enantiomers of six drugs in the samples were 2.3. Transport experiments separated and quantified by enantioselective RP-HPLC with GITC as a pre-column derivatizing agent6. The stock solutions of racemic drugs were made in Hank’s Balanced Salt Solution (HBSS), containing 25 mM HEPES, 2.5. Calculations and statistics pH 7.4, and were diluted to a series of concentration (Table 1). The solutions were filtered through 0.22 mm filter membrane The transport rate of six enantiomers (V) was calculated using for sterilizing. To ensure the transport experiments were the following equation: conducted in the sink conditions, the incubation time in Caco-2 cell monolayers of each drug was determined by V ¼ dQ=ðdt AÞð1Þ preliminary experiments. The transport studies were com- The apparent permeability coefficient (P ) which repre- pleted before the receiver concentration exceeded 10% of the app sents the permeability of these enantiomers, was estimated by donor concentration to ensure that the transport process was calculating as follows: fit to the linear dynamics10. In the transwell cultivation system, there is an unstirred water layer (UWL) between enteric cavity Papp ¼ dQ=ðdt A C0Þð2Þ and cell monolayers. The previous study showed that UWL is where dQ/dt is the rate of appearance of drugs on the receiver an important barrier of drug absorption, and it can restrict side, A is the surface area of the monolayer and C is the initial 11 0 some liposoluble drug’s diffusion . To avoid the influence of concentration in the donor compartment. UWL on drug transport, all studies were conducted with Data was expressed as the mean7SD (n¼5). Differences shaking (50 r/min) by a humidified incubator at 37 1C. Prior to in Papp of enantiomers were evaluated using paired t-test. A all experiments, each monolayer was washed twice with HBSS P valueo0.05 was considered to be statistically significant. and pre-incubation for 30 min. The transport study was

Table 1 Drug concentrations loaded onto the donor side 3. Results in transport study. We have investigated the transport across Caco-2 cell mono- Drug Racemic concentration (mM) layers of six chiral aryloxy aminopropanol compounds. The experimental results indicated that the transports of racemic Atenolol 75.0 187.5 375.0 750.0 1500 atenolol, sotalol, metoprolol and propafenone were not enan- Sotalol 130.0 325.0 650.0 1300 3250 tioselective in the concentration ranges tested. The transport Celiprolol 96.0 240.0 480.0 960.0 2400 Carvedilol 5.0 12.5 25.0 50.0 100.0 rates of (S)-enantiomers were similar to (R)-enantiomers both Metoprolol 6.0 15.0 30.0 75.0 150.0 in the absorptive direction and the secretory direction Propafenone 2.0 5.0 10.0 25.0 50.0 (P40.05). During the transport process of enantiomers, neither concentration-dependence nor directionality (Papp (AP-BL)

Table 2 Permeability of chiral drug enantiomers across Caco-2 cell monolayers (mean7 SD, n¼5).

6 6 Drug Incubation Racemic Papp (AP-BL) ( 10 cm/s) Papp (BL-AP) ( 10 cm/s) ER time (h) conc. (mM) RSRSRS

Atenolol 4.0 75.0 0.16270.139 0.15070.101 0.17170.078 0.16670.084 1.06 1.11 Sotalol 4.0 130.0 0.35870.006 0.38570.049 0.42270.076 0.40970.058 1.18 1.06 Celiprolol 3.0 96.0 0.78570.309 2.21870.349 7.03470.48 7.57970.68 8.96 3.42 Carvedilol 2.0 5.0 9.93572.631 11.2771.849 23.9571.48 22.3770.98 2.41 1.98 Metoprolol 3.0 6.0 4.31970.556 4.48770.673 3.69370.392 3.92770.327 0.855 0.875 Propafenone 1.0 2.0 12.9071.171 13.0671.106 13.2370.898 12.2070.859 1.03 0.934 Determination of the enantioselectivity of six chiral aryloxy aminopropanol drugs 171

Figure 2 Comparison of Papp values of celiprolol enantiomers in AP-BL (A) and BL-AP (B) transport experiments, incubation time was 3 h. Values were shown as mean 7SD (n¼5). ÃÃ Ã represents very signiﬁcant difference between S- and R-celiprolol by paired t-test, Po0.01; represents signiﬁcantly difference between S- and R-celiprolol by paired t-test, Po0.05.

Figure 3 Comparision of Papp values of carvedilol enantiomers in AP-BL (A) and BL-AP (B) transport experiments, the incubation time Ã was 2 h. Values were shown as mean 7SD (n¼5). represents signiﬁcant difference between S-andR-carvedilol by paired t-test, Po0.05.

and Papp (BL-AP) did not have significant differences) were bidirectional (AP-BL and BL-AP) transport, we conclude observed in the concentration ranges studied, suggesting that the that celiprolol enantiomers have conspicuous excretion trans- transmembrane transports of these drugs were mainly by passive port, and the ER value were between 9.0–2.2 in the con- diffusion pathway (Table 2). A series of concentrations of atenolol, centration range studied (Fig. 2). sotalol, metoprolol and propafenone have been tested, but the Papp As shown in Fig. 3, the transport of carvedilol was values in both two directions and efflux ratios (ER) in higher enantioselective and excretive, but no concentration-depen- concentrations were similar to the lowest concentrations used in dence was observed. In the absorptive direction, the Papp these experiments, so only these parameters determined at the values of (S)-()-carvedilol were greater than (R)-(þ)-carve- lowest concentration were shown in Table 2. dilol, while in the secretory direction, Papp values of (S)-()- These studies also demonstrated that transports of celi- carvedilol were smaller than (R)-(þ)-carvedilol (Po0.05). So prolol and carvedilol were significantly stereoselective. In the the ER values of (S)-()-carvedilol were conspicuously smaller absorptive direction, there was significant difference between than (R)-(þ)-carvedilol. It is likely that an excretive transpor- the Papp values of (S)-()- and (R)-(þ)-celiprolol (Po0.01). ter participated in the transport process across Caco-2 cell The values for (R)-(þ)-isomer were almost 3-fold smaller monolayers, and the transporter favors (R)-(þ)-enantiomer of than (S)-()-isomer which indicated (S)-()-isomer was the molecule. The ER values were between 2.4–1.9 in the easier to penetrate through biomembrane. The transports concentrations studied (Fig. 3). of celiprolol enantiomers were concentration-dependent, with the increasing of the drug’s concentration, the transport rates raised, though it dropped a little at the highest concentration 4. Discussion of (S)-()-isomer. But the distinction between (S)-()- and (R)-(þ)- celiprolol was decreased while drugs’ concentration In Caco-2 cell monolayers, the transports of atenolol, sotalol, increased. In secretory direction, the Papp values of (S)-()- metoprolol and propafenone were not stereoselective, suggest- celiprolol were slightly smaller than (R)-(þ)-celiprolol at low ing that the differences of pharmacological effects of sotalol concentrations (96.0–480.0 mM) (Po0.05), but as the concen- isomers were not caused by the differences of cell permeability tration increased to 960.0, 2400.0 mM the differences between between two isomers. But the enantiomers of celiprolol and celiprolol enantiomers diminished (P40.05). Compared the carvedilol displayed different transport behaviors. 172 Ye Tian et al.

Our data are in accordance with previous studies roughly. range tested, while it dropped a little at the highest concentra- Bachmakov et al.13 have observed that the BL-AP transport tion for (S)-()-isomer (Fig. 2). The reason for this phenom- of carvedilol was greater than the AP- BL transport, and the enon may because (S)-()-isomer penetrated more than polarized transport of celiprolol has been reported by Kuo (R)-(þ)-isomer, so it reached the steady-stage of penetration et al.14. Karlsson et al.15 reported that the basal-to-apical more rapidly. The penetration at 960 mM has reached the transport (secretion) of [14C]-celiprolol (50 mM) was 5 times maximum and the decreasing was caused by the experimental higher than apical-to-basal transport (absorption). And as error. On the other hand, another reason could be undefined reported, atenolol with low permeability was used as a transport mechanisms existed to make (S)-()-isomer easier to reference compound for low intestinal absorption and was penetrate through biomembrane, and at high concentration mainly transported paracellularly16–19. These results are in these transport systems were inhibited which contributed to agreement with our observation because small Papp values for the less permeability of (S)-()-isomer. atenolol were obtained in our experiments (Table 2). Yang The transport behaviors of carvedilol enantiomers were et al.20 have determined the permeability of seven b-blockers different but no concentration-dependence was observed. In using Caco-2 cell line, and the Papp values of metoprolol, the concentration range studied, the Papp values of (S)-()- atenolol and sotalol were close to our results calculated as isomer were higher than (R)-(þ)-isomer in absorptive direc- racemate. They also believed that the transmembrane trans- tion, while in secretory direction, it was contrary (Po0.05). port of the three drugs were mainly by passive diffusion It is likely that an excretive transporter participated in the pathway. But some investigations indicated atenolol and transport process across Caco-2 cell monolayers, and the sotalol had slight polarized transport13,21. These minor incon- transporter favors (R)-(þ)-enantiomer (Fig. 3). Several articles sistencies may be caused by the varied cultural conditions of have reported carvedilol-digoxin interaction in children and Caco-2 cells and different experimental circumstances from adults13,27–29. Carvedilol could increase serum concentrations lab to lab. The study of Bachmakov et al.22 revealed that and the area under the plasma concentration time-curve of although propafenone was not the substrate of P-gp, the orally administered digoxin. As digoxin is a typical substrate inhibitive activity in P-gp-mediated digoxin transport of of P-gp, so carvedilol is likely to have the ability to inhibit (S)-(þ)- and (R)-()-propafenone were different. (R)-()-pro- P-gp. This result has been proved on MDR1-transfected pafenone reduced the digoxin transport more significantly. Madin-Darby canine kidney (MDR1-MDCK) cells; these Comparing with our result, (R)-propafenone has a little MDR1-mediated reversing effects of carvedilol on vinblastine, higher ER value than (S)-(þ)-isomer, indicating (R)-()-pro- paclitaxel, doxorubicin and daunorubicin were similar to those pafenone may have higher affinity to P-gp which makes of verapamil29. Taken together, P-gp is likely to be one (R)-()-isomer interact with digoxin more effectively. However, determinant of carvedilol disposition in humans. they all did not investigate whether the transport characteristics Combined the results above and our pre-studies of esmolol of the enantiomers were different. Because the enantiomers may and propranolol, we presume that the chiral recognition of differ in terms of pharmacological properties and disposition, P-gp may lead to the stereoselective transport. But more stereoselective disposition of the enantiomers can arise from investigations are still needed to confirm that if P-gp indeed absorption of the enantiomers via intestine; therefore, uncover- play a role in the enantioselective recognition. Because the ing the transport characteristics of enantiomers, which have interactions are restricted by the shape of the binding pocket, been evaluated in this study, has significant meaning in predict- the conformation differences between enantiomers may lead to ing the pharmacological effect and pharmacokinetic behaviors the chiral recognition and stereoselective transport of P-gp. of chiral drugs’ enantiomers. Further studies in molecular model are needed to help us learn The transport of celiprolol and carvedilol were stereoselec- more about structure activity relationship between P-gp and tive and excretive, which were significantly different from the these drugs. other four chiral drugs, and the similar transport behavior has been observed in esmolol23 and propranolol24 enantiomers in Acknowledgment our previous studies. It has been proved that both celiprolol and carvedilol are the substrates of P-gp.25,26 There are several This project was supported by National Major Projects of articles investigated the interaction between celiprolol and China (2011CB710800, 2012ZX09506001-004) and NSFC P-gp. Karlsson et al.15 reported that the secretion of celiprolol (30873122). could be inhibited by typical substrates of P-gp (such as vinblastine, verapamil and nifedipine), furthermore, celiprolol inhibited the basal-to-apical transport of vinblastine. These References results indicated that celiprolol was transported by P-gp. In our observation, the Papp values for (S)-()-celiprolol were 1. Masubuchi Y, Yamamoto LA, Uesaka M, Fujita S, Narimatsu S, almost 3-fold larger than (R)-(þ)-isomer in the absorptive Suzuki T. Substrate stereoselectivity and enantiomer/enantiomer direction, and in secretory direction, they were slightly smaller interaction in propranolol metabolism in rat liver microsomes. than (R)-(þ)-celiprolol, which indicated that (S)-()-isomer Biochem Pharmacol 1993;46:1759–65. was easier to penetrate through biomembrane. So, the recog- 2. Zhou Q, Yao TW, Zeng S. Chiral metabolism of propafenone in rat hepatic microsomes treated with induers. World J Gastro- nition of P-gp for (R)-(þ)-celiprolol was much stronger than enterol 2001;7:830–5. þ (S)-( )-isomer, and (R)-( )-isomer may be a more potent 3. Yu L, Qian M, Liu Y, Yao T, Zeng S. Stereoselective metabolism substrate for P-gp. The transports of celiprolol enantiomers of propranolol glucuronidation by human UDP-glucuronosyl- were concentration-dependent in our experiment. With the transferases 2B7 and 1A9. Chirality 2010;22:456–61. drug’s concentration increased, the transport rates raised. For 4. Tang Y, He Y, Yao T, Zeng S. Stereoselective RP-HPLC (R)-(þ)-isomer, it raised all the way within the concentration determination of esmolol enantiomers in human plasma after Determination of the enantioselectivity of six chiral aryloxy aminopropanol drugs 173

pre-column derivatization. J Biochem Biophys Methods 2004;59: 17. Watkins PB. The barrier function of CYP3A4 and P-glycoprotein 159–66. in the small bowel. Adv Drug Deliv Rev 1997;27:161–70. 5. Schlauch M, Fulde K, Frahm AW. Enantioselective determina- 18. Mallants R, Vlaeminck V, Jorissen M, Augustijns P. An improved tion of (R)- and (S)-sotalol in human plasma by on-line coupling primary human nasal cell culture for the simultaneous determination of a restricted-access material precolumn to a cellobiohydrolase I- of transepithelial transport and ciliary beat frequency. JPharm based chiral stationary phase. J Chromatogr B 2002;775:197–207. Pharmacol 2009;61:883–90. 6. He Y, Chai XJ, Zeng S. Reversed-phase high-performance liquid 19. Prieto P, Hoffmann S, Tirelli V, Tancredi F, Gonza´lez I, Bermejo chromatographic analysis of seven pairs of chiral drug enantio- M, et al. An exploratory study of two Caco-2 cell models for oral mers in transport medium after chiral derivatization. J Chin absorption: a report on their within-laboratory and between- Pharm Sci 2010;19:104–9. laboratory variability, and their predictive capacity. Altern Lab 7. Yamaguchi H, Yano I, Saito H, Inui K. Transport characteristics Anim 2010;38:367–86. of grepafloxacin and levofloxacin in the human intestinal cell line 20. Yang Y, Faustino PJ, Volpe DA, Ellison CD, Lyon RC, Yu LX. Caco-2. Eur J Pharmacol 2001;431:297–303. Biopharmaceutics classification of selected beta-blockers: solubility 8. Hu M, Chen J. Nucleobase- and p-glycoprotein-mediated trans- and permeability class membership. Mol Pharm 2007;4:608–14. port of AG337 in a Caco-2 culture model. Mol Pharmacol 2004;1: 21. Augustijns P, Mols RJ. HPLC with programmed wavelength 194–200. fluorescence detection for the simultaneous determination of 9. Walgren RA, Walle UK, Walle T. Transport of quercetin and its marker compounds of integrity and P-gp functionality in the glucosides across human intestinal epithelial Caco-2 cell. Biochem Caco-2 intestinal absorption model. Pharm Biomed Anal 2004;34: Pharmacol 1998;55:1721–7. 971–8. 10. Lee K, Ng C, Brouwer KL, Thakker DR. Secretory transport of 22. Bachmakov I, Rekersbrink S, Hofmann U, Eichelbaum M, ranitidine and famotidine across Caco-2 cell monolayers. J Fromm MF. Characterization of (R/S)-propafenone and its Pharmacol Exp Ther 2002;303:574–80. metabolites as substrates and inhibitors of P-glycoprotein. Naunyn 11. Naruhashi K, Tamai I, Li Q, Sai Y, Tsuji A. Experimental Schmiedebergs Arch Pharmacol 2005;371:195–201. demonstration of the unstirred water layer effect on drug trans- 23. He Y, Zeng S. Determination of the stereoselectivity of chiral drug port in Caco-2 cells. J Pharm Sci 2003;92:1502–8. transport across Caco-2 cell monolayers. Chirality 2006;18:64–9. 12. Krishna G, Chen KJ, Lin CC, Nomeir AA. Permeability of 24. Wang Y, Cao J, Wang X, Zeng S. Stereoselective transport and lipophilic compounds in drug discovery using in-vitro human uptake of propranolol across human intestinal Caco-2 cell mono- absorption model, Caco-2. Inter J Pharm 2001;222:77–89. layers. Chirality 2010;22:361–8. 13. Bachmakov I, Werner U, Endress B, Auge D, Fromm MF. 25. Jonsson O, Behnam-Motlagh P, Persson M, Henriksson R, Characterization of beta-adrenoceptorant agonists as substrates Grankvist K. Increase in doxorubicin cytotoxicity by carvedilol and inhibitors of the drug transporter P-glycoprotein. Fundam inhibition of P-glycoprotein activity. Biochem Pharmacol 1999;58: Clin Pharmacol 2006;20:273–82. 1801–6. 14. Kuo SM, Whitby BR, Artursson P, Ziemniak JA. The contribu- 26. Tsuji A, Tamai I. Carrier-mediated intestinal transport of drugs. tion of intestinal secretion to the dose-dependent absorption of Pharm Res 1996;13:963–77. celiprolol. Pharm Res 1994;11:648–53. 27. De Mey C, Brendel E, Enterling D. Carvedilol increases the 15. Karlsson J, Kuo SM, Ziemniak J, Artursson P. Transport of systemic bioavailability of oral digoxin. Br J Clin Pharmacol 1990; celiprolol across human intestinal epithelial (Caco-2) cells: media- 29:486–90. tion of secretion by multiple transporters including P-glycopro- 28. Ratnapalan S, Griffiths K, Costei AM, Benson L, Koren G. tein. Br J Pharmacol 1993;110:1009–16. Digoxin-carvedilol interactions in children. J Pediatr 2003;142: 16. Bansal T, Singh M, Mishra G, Talegaonkar S, Khar RK, Jaggi M, 572–4. et al. Concurrent determination of topotecan and model perme- 29. Kakumoto M, Sakaeda T, Takara K, Nakamura T, Kita T, ability markers (atenolol, antipyrine, propranolol and furosemide) Yagami T, et al. Effects of carvedilol on MDR1-mediated multi- by reversed phase liquid chromatography: utility in Caco-2 drug resistance: comparison with verapamil. Cancer Sci 2003;94: intestinal absorption studies. J Chromatogr B 2007;859:261–6. 81–6.