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Life Sciences 86 (2010) 5258

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Life Sciences
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e

Interaction of angiotensin II type 1 receptor blockers with P-gp substrates in Caco-2 cells and hMDR1-expressing membranes
Emi Kamiyama , Daisuke Nakai, Tsuyoshi Mikkaichi, Noriko Okudaira, Osamu Okazaki
Drug Metabolism and Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan

a r t i c l e

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a b s t r a c t
Aims: The inhibitory effect of angiotensin II type 1 receptor blockers (ARBs) on P-glycoprotein (P-gp) was examined to evaluate their clinical drugdrug interaction (DDI) potential. Main methods: We performed an inhibition study on the vectorial transport of digoxin, a typical substrate for P-gp, using a human colonic adenocarcinoma cell line, Caco-2 cells, and verapamil-stimulated ATPase activity using human multidrug resistance 1 (hMDR1)-expressing membrane. Key ndings: The vectorial transport of digoxin was inhibited by candesartan cilexetil, irbesartan and telmisartan with the IC50 values of 14.7, 34.0 and 2.19 M, respectively. Those values were 7.4426-fold higher than their theoretical clinical gastrointestinal concentration [I] at doses in clinical DDI studies. Other ARBs failed to show interaction with P-gp. Signicance: It was demonstrated that candesartan cilexetil, irbesartan and telmisartan had the potential to inhibit the transport of various drugs via P-gp. Telmisartan, which caused an increase in the serum digoxin concentration in humans, had a sufciently high [I]/IC50 value, suggesting that DDI between digoxin and telmisartan was caused by the inhibition of digoxin efux via intestinal P-gp. 2009 Elsevier Inc. All rights reserved.

Article history: Received 20 August 2009 Accepted 5 November 2009 Keywords: Drugdrug interaction P-gp ARB Intestine IC50

Introduction Angiotensin II type 1 receptor blockers (ARBs) have been used in the treatment of hypertension not only in monotherapy but also in combination therapy with other antihypertensive drugs (Earl 2004; Mori et al. 2006). Hypertensive patients generally receive multidrug therapy and therefore it is important to investigate the possibility of clinical drugdrug interaction (DDI) between ARBs and concomitant drugs. As a cardioactive drug, digoxin has a narrow therapeutic range (Adams et al. 2005). Thus, therapeutic drug monitoring is essential in clinical practice for efcacy as well as to avoid digoxin toxicity (Adams et al. 2005). Digoxin is also known as a typical substrate of Pglycoprotein (P-gp) (Tanigawara et al. 1992; Troutman and Thakker 2003), which has broad substrate specicity (Fromm 2002; Lentz et al. 2000; Polli et al. 2001; Tang et al. 2002; Troutman and Thakker 2003). Since digoxin is concomitantly used with various therapeutic drugs, including ARBs, it is important to elucidate the possibility of DDI mediated by P-gp. In fact, clinical DDI studies have often been performed between digoxin and drugs which are inhibitors for P-gp and can be concomitantly administered with digoxin (Rodin and Johnson 1988).

Corresponding author. Tel.: + 81 3 3492 3131; fax: + 81 3 5436 8567. E-mail address: kamiyama.emi.gf@daiichisankyo.co.jp (E. Kamiyama). 0024-3205/$ see front matter 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2009.11.006

Recently, a regulatory viewpoint for the evaluation of P-gp-mediated DDIs using Caco-2 cells, a human colonic adenocarcinoma cell line, was presented (Zhang et al. 2008). Furthermore, Fenner et al. introduced an index to predict the interaction that occurs during absorption using Caco-2 cells (Fenner et al. 2009). In addition to a Caco-2 cell assay, an ATPase assay using human multidrug resistance 1 (hMDR1)-expressed membrane is often used as a more direct assessment of drug interactions with P-gp (Sarkadi et al. 1992). It is reported that among ARBs only telmisartan caused DDI with digoxin clinically and the maximum concentration (Cmax) of digoxin increased when digoxin was given concomitantly with telmisartan in healthy volunteers (Stangier et al. 2000). Up to now there have been some in vitro data reported about the interaction of ARBs with P-gp. Telmisartan activates ATPase in hMDR1-expressing Sf9 membranes and inhibits the transcellular transport of digoxin across MDCKMDR1 monolayers (Chang et al. 2006). Soldner et al. report that losartan is also a substrate for P-gp from an in vitro study using Caco-2 cells and hMDR1-expressing cells, but not irbesartan, EXP-3174 (losartan active metabolite) or valsartan (Soldner et al. 2000). It has also been reported that candesartan cilexetil and candesartan do not inhibit the transcellular transport of digoxin across hMDR1-expressing cells (Takara et al. 2002). As described above, the methods or cells used in a transport study for the assessment of P-gp-mediated DDI are different among ARBs. Therefore, to investigate the afnity of ARBs to P-gp, we simultaneously determined the inhibitory effect of ARBs on the bidirectional

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transcellular transport of digoxin using Caco-2 cells and ATPase activity stimulated by verapamil using hMDR1-expressing membranes. Furthermore, the possibility of clinical DDI between ARBs and digoxin was evaluated by calculating the ratio of [I] to the IC50 value of each ARB.

Calculation of Papp, Papp ratio, IC50 values and [I] values The transported amount of each substrate across the monolayer was calculated by the value of the transported concentrations of the substrates multiplied by the volume and Papp was calculated using the following Eq. (a). Papp = dQ = dt = A C0 a

Materials and methods Chemicals


3 H-digoxin was purchased from PerkinElmer Japan Co., Ltd. (Kanagawa, Japan). Olmesartan was obtained from Sankyo Organic Chemical Co., Ltd. (Kanagawa, Japan). Olmesartan medoxomil was obtained from Process Development Laboratories, Sankyo Co., Ltd. (Tokyo, Japan). Candesartan cilexetil, candesartan, irbesartan, losartan, losartan active metabolite, telmisartan and valsartan were obtained from Medicinal Chemistry Research Laboratories, Sankyo Co., Ltd. (Tokyo, Japan). The chemical structures of these compounds are shown in Fig. 1. Digoxin was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Verapamil as a typical inhibitor of P-gp and cyclosporine A (Cys A) as an inhibitor to ATPase activity induced by verapamil were purchased from Sigma-Aldrich Inc. (St. Louis, MO). Fetal bovine serum (FBS, Lot 1233051) was purchased from Invitrogen Corporation (Carlsbad, CA). All other reagents used were of reagent grade.

where dQ/dt, A and C0 represent the amount of the test substrates transported within a given time period, the surface area of monolayer and the initial concentrations of the substrates, respectively. The mean and the standard deviation values of Papp were calculated and expressed to 10 6 cm/s unit. The Papp ratio was calculated using the following Eq. (b). Papp ratio = Papp; B to A = Papp; A to B b

Caco-2 cells Caco-2 cells (passage number: 20, Lot 2463681) were purchased from American Type Culture Collection (Rockville, MD). The Caco-2 cells (passage number: 4350) were grown in DMEM (culture medium) supplemented with 10% FBS, 100 U/mL penicillin and 100 g/mL streptomycin and were cultured in an atmosphere of 5% CO2 at 37 C. The Caco-2 cells were seeded at a density of 5.0 104 cells/ 0.25 mL/insert on an HTS Transwell (Corning Japan K.K., Tokyo, Japan) and were cultured using a culture medium for 2 weeks in an atmosphere of 5% CO2 (Kamiyama et al. 2009). The culture medium was changed every 34 days.

where Papp, B to A, and Papp, A to B represent the mean values of Papp from the basal side to the apical side and from the apical side to the basal side, respectively. The 95% condence intervals (CIs) of the Papp ratios in the inhibition study using all the ARBs were estimated based on Fieller's theorem using SAS software (version 8.2, SAS Institute Inc., Cary, NC). The IC50 values were calculated using the value (Papp ratio 1) with the computer software WinNonlin Professional (version 3.1, Pharsight Corporation, Mountain View, CA, USA) and Microsoft Ofce Excel 2003 (Microsoft Corporation, Tokyo, Japan). The [I] values indicated the theoretical concentration in the gastrointestinal tract assuming that all of the drug administered with 250 mL water was dissolved in the gastrointestinal tract. Thus, they were calculated by dividing the dose used in the clinical DDI study by 250, which was the volume of water at dosing. The [I]/IC50 values were calculated by dividing the [I] value by the IC50 value. ATPase assay Human MDR1-expressing membranes were purchased from BD Biosciences Company (Woburn, MA). The ATPase assay was performed using an ABC transporter ATPase assay reagents kit (Nacalai Tesque, Inc. (Kyoto, Japan)) with a modied method of Sarkadi et al. (1992). Briey, the assay mixture containing hMDR1 membranes and test compounds was pre-incubated at 37 C for 3 min, starting the assay by the addition of MgATP. Following incubation at 37 C for 30 min, the reaction was terminated with stop solution (10 w/v% lithium lauryl sulfate). The reaction sample mixed with the detection reagent (80 mg/mL ascorbic acid, 0.8 w/v% hexaammonium heptamolybdate and 3 mM zinc acetate) was incubated at 37 C for 20 min to develop color and then the absorbance at 750 nm was determined. ATPase activities were shown by the generation velocity of inorganic phosphate and were calculated by dividing the produced amount of inorganic phosphate by the reaction time (30 min) and the amount of membrane protein in the reaction systems. Results Inhibitory effects of ARBs on transcellular transport of digoxin The Papp and Papp ratio of digoxin across Caco-2 cells without or with various ARBs are shown in Fig. 2. The Papp ratios of digoxin with candesartan cilexetil, irbesartan, telmisartan and verapamil were less than or equal to 50% compared to that without an inhibitor. No marked inhibition was observed for the

Transcellular transport study Transport buffer (TB) prepared by adding HEPES (nal conc.: 10 mM) to HBSS was adjusted to pH 7.4. The culture medium in the apical and basal sides of an HTS Transwell, where Caco-2 cells had been seeded, was removed by suction and washed with TB. The culture medium was replaced by TB and the plate was pre-incubated at 37 C for 15 min. TB on the receiver side (basolateral side for the A to B experiments or apical side for the B to A experiments) was replaced with TB with or without test compounds. Then the TB on the donor side (apical side for the A to B experiments or basolateral side for the B to A experiments) was replaced with TB containing digoxin with or without test compounds (triplicate). After incubation for 1 h, TB of subduple volume on the receiver side, where the test solutions of the radioactive labeled compound had not been added, was collected. Pico-Fluor 40 (10 mL, PerkinElmer Japan Co., Ltd., Kanagawa, Japan) was added to samples placed in the vials for liquid scintillation counting (LSC) and the radioactivity of the samples was determined by LSC on the 3H single channel using a 2300TR liquid scintillation analyzer (Packard Instrument Co., Inc., Palo Alto, CA). The determination time was 2 min. The counting efciency was corrected by an external standard source method. The amount of radioactivity in the samples was calculated by subtracting the value of the background (20 dpm) from the observed data of the radioactivity in the samples and was expressed to an integral number.

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Fig. 1. Chemical structure of various ARBs.

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Fig. 2. Inhibitory effects of ARBs and verapamil on the transcellular transport across Caco-2 cell monolayers. The nal concentration of digoxin was 0.5 M. (a) The open and closed columns represent the Papp from the apical side to the basal side and from the basal side to the apical side of digoxin, respectively. Each Papp represents the mean + SE of three experiments. (b) The columns represent the Papp ratios with the 95% CI.

transcellular transport of digoxin by the addition of losartan active metabolite, olmesartan medoxomil, olmesartan and valsartan.

(IC50 N 100 M). The IC50 values of candesartan cilexetil, irbesartan and telmisartan were 14.7, 34.0 and 2.19 M, respectively.

IC50 values of candesartan cilexetil, candesartan, irbesartan, losartan and telmisartan The IC50 values of candesartan cilexetil, candesartan, irbesartan, losartan and telmisartan for the inhibition of transcellular transport activity of digoxin across Caco-2 cells were determined (Fig. 3 and Table 1). The transcellular transport activity of digoxin was hardly inhibited by candesartan and losartan at a high inhibitor concentration

Comparison between [I] and IC50 values of candesartan cilexetil, irbesartan, losartan and telmisartan The [I] values of candesartan cilexetil, irbesartan, losartan and telmisartan administered at the doses used in the clinical DDI study were calculated and the [I]/IC50 value of each ARB was determined (Table 1). The [I]/IC50 values of candesartan cilexetil, irbesartan and telmisartan were determined to be 7.4, 41.2 and 426, respectively.

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Fig. 3. Papp and Papp ratio of digoxin with candesartan cilexetil (a), candesartan (b), irbesartan (c), losartan (d) and telmisartan (e) and comparison of inhibitory effects among ve ARBs (f). The nal concentration of digoxin was 0.5 M. The open and closed columns represent the Papp from the apical side to the basal side and from the basal side to the apical side of digoxin, respectively. Each Papp represents the mean + SE of three experiments. The closed symbols represent the Papp ratios.

ATPase activity induced by ARBs on hMDR1-expressing membranes and the inhibitory effect of ARBs to ATPase activity stimulated by verapamil The ATPase activity induced by verapamil (50 M) as a positive control showed about 60 nmol/min/mg protein (Fig. 4). The ATPase activities were not stimulated by candesartan or olmesartan ranging from 1 to 100 M, while they were slightly induced by olmesartan medoxomil in a concentration-dependent manner. Cys A was used as an inhibitor to the ATPase activity induced by verapamil (Rao and Scarborough 1994). Cys A at the concentration of 10 M inhibited the ATPase activity stimulated by verapamil complete-

ly. Candesartan cilexetil and telmisartan inhibited the verapamilinduced ATPase activity in a concentration-dependent manner. No marked inhibition was observed in the verapamil-stimulated ATPase activity in the presence of other ARBs. Discussion The afnity of ARBs to P-gp using Caco-2 cells and hMDR1expressing membranes were examined and the DDI potential of ARBs with digoxin was investigated. Our results suggested that in concomitant administration with telmisartan the digoxin serum concentration

E. Kamiyama et al. / Life Sciences 86 (2010) 5258 Table 1 Relationship between IC50 value and theoretical [I] value. Inhibitor Candesartan cilexetil Irbesartan Losartan Telmisartan Verapamil IC50 (M) 14.7 34.0 N100 2.19 4.05 Dose on DDI study1) (mg) 16 150 50 120 80 Gut concentration [I] (M) 105 1400 434 933 652 [I]/IC50 7.4 41.2 b4.34 426 161 LogD (pH 7.4)2) N 3.3 1.56 1.38 2.68 4.21a Reference

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1) Jonkman et al. (1997) 1) Marino and Vachharajani (2001); 2) Kamiyama et al. (2007) 1) De Smet et al. (1995); 2) Kamiyama et al. (2007) 1) Stangier et al. (2000); 2) Kamiyama et al. (2007) 1) Rodin et al. (1988); 2) Castaing et al. (2000)

The dose in the DDI study represents the dose in the clinical DDI study between each ARB and digoxin. The [I] value represents the theoretical gut concentration determined by an oral dose dissolved in a volume of 250 mL. a Data predicted by the Pallas 2.0 software program.

elevation in humans was caused by the DDI on the intestinal P-gp transport process, and that irbesartan and candesartan cilexetil had a low risk of clinical DDI with digoxin. We conducted an inhibition study of ARBs on the transcellular transport of digoxin across Caco-2 cells. It was indicated that the IC50 value of telmisartan, which led to an increase in the serum digoxin concentration, was the lowest among three ARBs, and was quite low compared to the human theoretical gastrointestinal concentration of telmisartan. It was reported that verapamil interacted with digoxin clinically as telmisartan (Rodin et al. 1988). Actually, the Cmax of

digoxin administered concomitantly with telmisartan or verapamil clinically increased around 1.5-fold compared to that without telmisartan or verapamil, in spite of the higher [I]/IC50 value of telmisartan compared to verapamil (Rodin et al. 1988; Stangier et al. 2000). Thus, it was considered that the increase in the serum digoxin concentration reached a plateau by DDI with drugs showing a high [I]/IC50 value. In our results, the [I]/IC50 values of candesartan cilexetil, irbesartan and losartan, which did not cause any adverse events in a clinical DDI study (De Smet et al. 1995; Marino and Vachharajani 2001; Jonkman et al. 1997), were calculated to be less than 50. In particular, that of candesartan cilexetil and losartan was less than 10. Thus, it was suggested that the possibility of clinical DDI between digoxin and ARBs was low when the [I]/IC50 values were less than 50. Recently, Fenner et al. suggested that to a limited extent, an [I]/IC50 value of less than 10 was predictive of negative digoxin interaction (Fenner et al. 2009). Considering differences in the experimental conditions, such as the culture medium and culture period in Caco-2 cells, essentially their report supported our argument, although irbesartan without digoxin interaction in clinical settings had an [I]/IC50 value of around 50 in our results. ARBs, which did not inhibit the vectorial transport of digoxin at the concentration of 100 M, except for olmesartan medoxomil (LogD (pH 7.4): 1.04), have low lipophilicity (LogD (pH7.4): 1.30 to 0.79) (Kamiyama et al. 2007). Thus, we examined whether the lack of interaction with P-gp was only due to a low uptake into Caco-2 cells. As a result (Fig. 4(b)), it was indicated that losartan active metabolite, olmesartan and valsartan have no intrinsic potential to inhibit transport of digoxin via P-gp. In addition, olmesartan did not activate ATPase using hMDR1expressing membrane (Fig. 4), suggesting that olmesartan was not a P-gp substrate. It has been reported that losartan active metabolite and valsartan were not substrates for P-gp (Young et al. 2006; Soldner et al. 2000). Thus, losartan active metabolite, olmesartan and valsartan were neither inhibitors nor substrates of P-gp, indicating that the afnity of these three ARBs to P-gp was low. In the ATPase assay, no marked inhibition was observed in the verapamil-stimulated ATPase activity by irbesartan, unlike in the transcellular transport study. This might be due to a difference of the binding sites to P-gp between digoxin or irbesartan and verapamil because there are some substrate binding sites on P-gp (Safa 2004). Generally, the clinical DDI trials to P-gp have been conducted using digoxin as a substrate for P-gp and a transcellular transport study using Caco-2 cells can assess the possibility of clinical DDI including the oral absorption process. Thus, we considered that a transcellular transport study across Caco-2 cells using digoxin as the substrate for P-gp is more important for the risk assessment of clinical DDI compared to an ATPase assay. Conclusion We simultaneously examined the inhibitory effect of ARBs on the transcellular transport of digoxin using Caco-2 cells. It was demonstrated that candesartan cilexetil and irbesartan had the potential to inhibit the transport of various drugs via P-gp, even though they did

Fig. 4. ATPase activity of ARBs (a) and inhibitory effect of ARBs on ATPase activity stimulated by verapamil (b) on hMDR1 expressing membranes. The nal concentrations of ARBs in an activity assay were 1, 10, and 100 M. Each ATPase activity represents the mean of two experiments (a). The nal concentrations of verapamil and Cys A were 50 and 10 M, and those of ARBs excluding telmisartan were 1, 10, and 100 M and those of telmisartan were 0.3, 3, and 30 M. Each ATPase activity represents the mean of two experiments (b).

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