April 2007 Biol. Pharm. Bull. 30(4) 739—744 (2007) 739

Involvement of Salt Export Pump in -Induced Cholestatic Hepatitis

a a b a ,a Takashi IWANAGA, Masanori NAKAKARIYA, Hikaru YABUUCHI, Tomoji MAEDA, and Ikumi TAMAI* a Department of Molecular Biopharmaceutics, Faculty of Pharmaceutical Sciences, Tokyo University of Science; 2641 Yamasaki, Noda, Chiba 278–8510, Japan: and b GenoMembrane, Inc.; 75–1 Onoda, Tsurumi-ku, Yokohama, Kanagawa 230–0046, Japan. Received October 5, 2006; accepted December 28, 2006; published online January 9, 2007

The non-steroidal antiandrogen flutamide is widely used for treatment of prostatic cancer, but causes side effects, including cholestatic hepatitis and fulminant hepatitis. We investigated the pathogenesis of flutamide-in- duced cholestatic hepatitis, focusing on the bile salt export pump (BSEP; ABCB11), which exports bile salts to the bile. We examined the inhibitory effects of flutamide and its active metabolite, hydroxyflutamide, on the transport of taurocholic acid (TCA) by membrane vesicles derived from hBSEP-expressing Sf9 cells. Flutamide

inhibited the transport of TCA by hBSEP (IC50 value, about 50 mM), while hydroxyflutamide had no effect at up to 100 mM. When flutamide was administered to rats as a single oral dose of 100 mg/kg, the biliary excretion rate 3 of bolus-injected [ H]TCA was decreased and the liver tissue concentration of flutamide exceeded 50 mM. Re- peated doses of flutamide for 5 d (10 mg/kg/d) also decreased the biliary excretion rate of bolus-injected 3 [ H]TCA. In this case, the liver tissue concentration of flutamide was below 0.1 mM. In both cases, no change in the mRNA level of rat Bsep was detected by RT-PCR. These results suggest that flutamide itself, but not its major metabolite, may cause cholestasis by inhibiting BSEP-mediated bile salt excretion. Key words flutamide; bile salt export pump (BSEP); cholestasis; bile salt; taurocholic acid

Flutamide (2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]- acid (i.e. and glycine) conjugated metabolites.18) It is propanamide) is a nonsteroidal antiandrogen used for the known that flutamide-induced hepatotoxicity is ameliorated treatment of prostate carcinoma. However, its therapeutic ac- by ursodeoxycholic acid (UDCA).19) Since UDCA is a ligand tivity is compromised because of potential liver toxicity. of farnesoid X receptor (FXR) and FXR up-regulates BSEP Among 1091 patients treated for prostate cancer with flu- activity, it was proposed that the action of UDCA is associ- tamide, the incidence of liver toxicity (defined as 4-fold in- ated with a change in the expression level of BSEP.20) Fur- crease in serum transaminase activity) was 0.36%.1) In addi- thermore, Fickert et al. reported that protein level of Bsep tion, Wada et al. reported that the incidence of liver disorder was increased in UDCA treated mice.21) Therefore, we exam- (defined as 1.5-fold increase in alanine aminotransferase ined the role of BSEP in flutamide-related cholestatic hepati- levels) was 26%.2) Furthermore, between February 1989 and tis. We also examined the effect of hydroxyflutamide, since December 1994, the Food and Drug Administration received hydroxyflutamide is cytotoxic10) and its plasma concentration reports of 20 deaths and 26 hospitalizations due to hepato- is much higher than that of flutamide.22) Flutamide and hy- toxicity secondary to flutamide.3) droxyflutamide are metabolized to FLU-1 and FLU-3, and Flutamide-induced hepatitis may be cholestatic and/or cy- their glucuronides,22,23) but flutamide-induced cholestasis tolytic, and fulminant hepatitis can occur.4—8) Fau et al. pro- may not involve these metabolites, since no consistent differ- posed that cytochrome P450-mediated generation of elec- ences were found in serum levels of these metabolites, in- trophilic metabolites may play a role in flutamide-induced cluding the glucuronides, between patients with normal func- cytolytic hepatitis, based on studies in isolated rat hepato- tion and those suffering hepatic dysfunction.24) Therefore, in cytes.9) Furthermore, Wang et al. reported that flutamide and the present study, we focused on the effects of flutamide it- hydroxyflutamide (a major metabolite of flutamide) are cyto- self and hydroxyflutamide. toxic to primary cultured rat hepatocytes at concentrations of 10) approximately 40 m M and 170 m M, respectively. However, MATERIALS AND METHODS little is yet known about the mechanism leading to cholesta- tic hepatitis. Therefore, this was the focus of the present Chemicals [3H]Taurocholic acid (TCA; 1.92 TBq/mmol) study. was purchased from PerkinElmer Life Sciences (Boston, Cholestasis, defined as impairment of bile formation, is MA, U.S.A.). Flutamide, hydroxyflutamide and cyclosporine accompanied with retention of bile salts, and the resulting in- A were purchased from Wako Pure Chemical Industries tracellular accumulation of bile salts causes cell damage (Osaka, Japan). Rat Bsep- and human BSEP-expressing Sf9 through a direct effect on trafficking of Fas to the cell sur- membranes (rBsep and hBSEP, respectively) were obtained face, leading to apoptosis.11,12) It was reported that mutation from GenoMembrane, Inc. (Kanagawa, Japan). All other of the human bile salt export pump (BSEP, ABCB11) gene reagents were purchased from Sigma-Aldrich (St. Louis, causes progressive intrahepatic cholestasis.13,14) Several MO, U.S.A.), Wako Pure Chemical Industries, Kanto Chemi- drugs such as troglitazone, bosentan, and cyclosporine A in- cals (Tokyo, Japan), Nacalai Tesque (Kyoto, Japan), and duce cholestatic hepatitis, probably by inhibiting BSEP-me- Kishida Chemicals (Osaka, Japan). diated taurocholic acid transport.15—17) BSEP is the transport Uptake Study by Membrane Vesicles Cell membranes protein primary responsible for biliary excretion of unconju- from hBSEP- or rBsep-expressing Sf9 cells were suspended gated bile salts, which are more hepatotoxic than their amino in the transport buffer (10 mM Hepes, 100 mM KNO3, 10 mM ∗ To whom correspondence should be addressed. e-mail: [email protected] © 2007 Pharmaceutical Society of Japan 740 Vol. 30, No. 4

Mg(NO3)2 and 50 mM sucrose, pH 7.4), and passed through a animals were euthanized by exsanguination, and the liver 25-gauge needle (30 times). Flutamide, hydroxyflutamide was removed, weighed and homogenized. Plasma was and cyclosporine A were each dissolved in dimethylsulfoxide quickly separated from blood by centrifugation. All samples (DMSO; final concentration 1%), and DMSO without in- were kept on ice until required. hibitors was included as a control. After preincubation of the HPLC Analysis of Flutamide and Hydroxyflutamide membrane vesicles for 2 min, uptake of [3H]TCA was initi- For the quantitation of flutamide and hydroxyflutamide, ated by the addition of a 90 ml aliquot of transport buffer aliquots of plasma and liver homogenate were mixed with 2 3 containing [ H]taurocholic acid, 5 mM adenosine triphosphate volumes of methanol (v/v for plasma, w/v for liver), and left (ATP) or 5mM adenosine monophosphate (AMP), an ATP- on ice for 30 min. The precipitated proteins were removed by regenerating system (10 mM creatine phosphate and 100 centrifugation (13000 g, 10 min), and a 10 ml aliquot of the mg/ml creatine phosphokinase), and inhibitors to 10 ml of supernatant was directly injected to the HPLC instrument. membrane vesicles (10 mg protein) at 37 °C. At designated HPLC analyses were carried out on an Alliance system (Wa- times, the uptake was terminated by the addition of 1 ml of ters, Milford, MA, U.S.A.) consisting of the 2690 separation ice-cold stop buffer (10 mM Hepes, 150 mM KNO3, 10 mM module, and the 2487 dual absorbance detector (300 nm), Mg(NO3)2, 50 mM sucrose, 0.1 mM TCA, pH 7.4). To termi- controlled with Empower software (Waters, Milford, MA, nate the reactions, the mixture was filtered through a HA fil- U.S.A.). A 2504.6-mm i.d. Mightysil RP-18 GP Aqua col- ter (0.45 mm; Millipore Corp., Bedford, MA, U.S.A.), and umn (Kanto Chemical, Tokyo, Japan) was used for analysis. then washed twice with 5 ml of ice-cold stop buffer. Specific The mobile phase consisted of 50% water and 50% acetoni- uptake by BSEP was evaluated as ATP-dependent uptake, trile and was delivered at a flow rate of 1 ml/min at 40 °C. obtained as the difference between the uptakes in the pres- Calibration curves were linear from 0.1 to 100 m M for both ence of ATP and of AMP. The radioactivity retained on the flutamide and hydroxyflutamide, and the coefficients of vari- filter and in the reaction mixture was measured with a liquid ation were less than 15 %. scintillation counter (Perkin Elmer, Boston, MA, U.S.A.) Hepatic RNA Extraction and Semi-Quantitative PCR after the addition of scintillation cocktail, Clearsol I® mRNAs of rBsep and Na-taurocholate cotransporting (Nacalai Tesque, Kyoto, Japan). polypeptide (Ntcp) in liver were quantitated by means of a In the present study, we also examined the irreversible in- semi-quantitative PCR method. Total RNA was prepared hibitory effect on hBSEP-mediated uptake of [3H]TCA, by from the liver using Isogen (Wako Pure Chemical Industries, pre-incubating hBSEP-expressing membrane vesicles with Tokyo, Japan). The total RNA content was determined by inhibitors for 30 min. measuring the absorbance at 260 nm. mRNA level in liver In Vivo Study Male Wistar rats (215—240 g) were ob- was analyzed by means of semi-quantitative reverse tran- tained from Saitama Experimental Animals Supply Co., Ltd. scription polymerase chain reaction (RT-PCR). Single- (Saitama, Japan). Rats were housed three per cage with free stranded cDNAs were constructed using an oligo(dT) primer access to commercial chow and tap water, and were main- (Invitrogen Corp., Carlsbad, CA, U.S.A.) and Improm-IITM tained on a 12-h dark/light cycle (8:00 a.m.—8:00 p.m. light) reverse transcriptase (Promega). These cDNAs provided tem- in an air-controlled room (temperature, 24.51 °C; humidity, plates for PCRs using specific primers (rBsep: 5-CG- 555%). All animal care and experimentation were con- GAATTCACAGCATTACAGCTCATTCAG-3; 5-ACGC- ducted according to the guidelines of the Tokyo University of GTCGACTCACACAGCTACTCCATGCTCAAA-3, rNtcp: Science. 5-GGAGACCTTAAGGACAAGGTG-3; 5-ATGCTGAT- For the single-dose group, rats were administered flu- GGTGCGTCTG-3, G3PDH: 5-TGAAGGTCGGTGTGA- tamide at a dose of 100 mg/kg (5 ml/kg) suspended in 0.5% ACGGATTTGGC-3; 5-CATGTAGGCCATGAGGTCCAC- methylcellulose, after they had received the vehicle (0.5% CAC-3, respectively). The PCR conditions were: denatura- methylcellulose) alone once daily for 4 d. For the repeated- tion at 94 °C for 30 s, annealing at 58—62 °C for 30—60 s, dose group, rats were administered flutamide once daily for and elongation at 72 °C for 30 s in the presence of deoxynu- 5d at a dose of 10 mg/kg (5 ml/kg) suspended in 0.5% cleotides (dNTPs) and Ex Taq polymerase (Takara Shuzo Co. methylcellulose. Control rats received the vehicle alone Ltd., Tokyo). Annealing time and temperature were changed (5 ml/kg) once daily for 5 d. as required, depending on the genes. The PCR cycle numbers Before [3H]TCA administration, rats were anesthetized were titrated for each primer pair to confirm that amplifica- with ether, and the common bile duct, femoral vein and tion was performed within a linear range. PCR products were femoral artery were cannulated with polypropylene tubing analyzed by 2% agarose gel (w/v) electrophoresis and the (outer diameter, 0.6 mm, 0.8 mm, and 0.8 mm, respectively). gels were stained with ethidium bromide for visualiza- After surgery, animals were kept under heating lamp to main- tion. mRNA levels were quantified by using light capture tain body temperature. Bile was collected during successive (Atto Co., Tokyo). PCR amplification data were normalized 10 min intervals in pre-weighed tubes, and bile flow was cal- with respect to glyceraldehyde-3-phosphate dehydrogenase culated based on the weight of the samples. A bolus of (G3PDH). 80 nmol/kg TCA containing 1 mCi/ml [3H]TCA was injected Statistical Analysis Statistical significance was deter- into the portal vein at 3 h and 24 h after the single dose and mined with Student’s t test or by analysis of variance after the last dose in the repeated-dose administration of flu- (ANOVA) followed by Dunnett’s test, and a p value less than tamide, respectively. Blood was collected via the femoral ar- 0.05 was considered statistically significant. tery at designated times (2, 5, 10, 20, 30, 40 min) in he- parinized tubes after bolus injection of [3H]TCA. At the end of bile collection (60 min after bolus injection of [3H]TCA), April 2007 741

RESULTS

Inhibitory Effect of Flutamide and Hydroxyflutamide on BSEP-Mediated Taurocholic Acid Transport in Vitro To examine the involvement of BSEP in flutamide-induced cholestasis, the inhibitory effects of flutamide and hydroxy- flutamide on BSEP were investigated. The ATP-dependent uptake of [3H]TCA by hBSEP-expressing membrane vesicles increased over 10 min, and the uptake at 2 min was taken as the initial uptake, since the increase was linear up to this time point (data not shown). Uptake of TCA was inhibited by cy- closporine A (1 m M), the positive control, showing that spe- cific uptake of TCA by BSEP can be evaluated by the Fig. 2. Effect of Flutamide and Hydroxyflutamide Pretreatment on [3H]Taurocholic Acid Uptake by hBSEP-Expressing Membrane Vesicles method used in the present study. Flutamide inhibited ATP- 3 3 ATP-dependent initial uptake of [ H]TCA (0.5 m M) was determined after preincuba- dependent [ H]TCA uptake in a concentration-dependent tion of hBSEP-expressing membrane vesicles with (closed bar) or without (open bar) inhibitors for 30 min. The ATP-dependent uptake was obtained by subtracting the value manner from 1 to 100 m M, and the estimated IC50 value was in the absence of ATP (AMP added instead of ATP) from that in the presence of ATP. 48.3 9.1 m M (Fig. 1). On the other hand, hydroxyflutamide Control samples contained 1% DMSO without inhibitors. The results are shown as was not inhibitory up to 100 m M. Similar results were ob- meanS.E.M. (n3). An asterisk (∗) indicates a significant difference from the control tained in rBsep-expressing membrane vesicles (38% of con- value by Dunnett’s test (p0.05). trol at 100 m M flutamide, data not shown). Furthermore, we checked the reversibility of the inhibitory effect of flutamide and hydroxyflutamide on BSEP by examining the influence of pretreatment with them upon TCA transport. The in- hibitory effect of flutamide on uptake of [3H]TCA by hBSEP-expressing membrane vesicles that had been preincu- bated with flutamide for 30 min was not significantly differ- ent from that in the case of membrane vesicles that had not been preincubated with flutamide (Fig. 2). There was also no pretreatment effect in the case of hydroxyflutamide (Fig. 2). Effect of Flutamide on Biliary Excretion of Taurocholic Acid in Vivo The cholestatic effect of a single dose and re- peated doses of flutamide was examined in rats. Figure 3 shows the time course of cumulative biliary excretion and the biliary excretion rate of bolus-injected [3H]TCA. In both the single-dose group (100 mg/kg flutamide) and the repeated- dose group (10 mg/kg flutamide for 5 d), the biliary excretion Fig. 3. Effects of Flutamide on Cumulative Biliary Excretion and Biliary 3 rate of [3H]TCA was significantly decreased for the initial Excretion Rate of [ H]Taurocholic Acid in Rats A bolus of 80 nmol/kg TCA containing 1 mCi/ml [3H]TCA was injected into the por- 10 min. Subsequently, however, the cumulative biliary excre- tal vein of rats with (triangle: single-dose; square: repeated-dose) or without (circle) 3 tions of [ H]TCA in both the single-dose group and the re- flutamide treatment as described in Materials and Methods. The closed, and open sym- peated-dose group were comparable with that of the control bols show cumulative biliary excretion, biliary excretion rate of [3H]TCA at 10 min in- tervals, respectively. The results are shown as meanS.E.M. (n3). An asterisk (∗) in- group. There was no significant difference in plasma concen- dicates a significant difference from the control value (t-test, p0.05). tration profiles of [3H]TCA among the 3 groups (Fig. 4), and the values of area under the curve (AUC), distribution vol- ume, and total clearance showed no significant inter-group

Fig. 1. Effects of Flutamide and Hydroxyflutamide on [3H]Taurocholic Acid Uptake by hBSEP-Expressing Membrane Vesicles 3 Initial ATP-dependent [ H]TCA (0.5 m M) uptake was determined in the absence Fig. 4. Effect of Flutamide on Plasma Concentration Profile of [3H]Tauro- (DMSO 1%) , presence of cyclosporine A (Cys A), flutamide, or hydroxyflutamide at 2 min. Concentrations used are shown in the figure. The ATP-dependent uptake was ob- in Rats tained by subtracting the value in the absence of ATP (AMP added instead of ATP) A bolus of 80 nmol/kg TCA containing 1 mCi/ml [3H]TCA was injected into the por- from that in the presence of ATP. Control uptake was 67.93.7 ml/2 min/mg protein. tal vein of rats with (triangle: single-dose; square: repeated-dose) or without (circle) The results are shown as meanS.E.M. (n3). An asterisk (∗) indicates a significant flutamide treatment as described in Materials and Methods. The results are shown as difference from the control value by Dunnett’s test (p0.05). meanS.E.M. (n3). 742 Vol. 30, No. 4

3 Table1.Effects of Flutamide on AUC0—40 min, Distribution Volume, and Total Clearance of [ H]Taurocholic Acid in Rats

Parameters of [3H]taurocholate Dose

(Flutamide) AUC0—40 min CLtot CLbile Vdss (pmol·min/ml) (ml/min/kg) (ml/min/kg) (ml/kg)

Control group — 2099166 36.14.0 35.94.0 34661 Single group 100 mg/kg 2155161 35.11.8 31.61.9 32672 Repeated group 10 mg/kg (5 d) 211392 36.01.7 34.30.7 33436

A bolus of 80 nmol/kg TCA containing 1 mCi/ml [3H]TCA was injected into the portal vein of rats with or without flutamide treatment as described in Materials and Methods. The results are shown as meanS.E.M. (n3).

Table2. Plasma and Liver Concentrations of Flutamide and Hydroxyflutamide in Rats

Plasma (m M)Liver (nmol/g liver) Kp value Dose Flutamide OH-flutamide Flutamide OH-flutamide Flutamide OH-flutamide

Single 100 mg/kg 16.86.0 28.35.0 62.63.7 932189 5.93.1 38.114.9 Repeated 10 mg/kg (5 d) BQL 0.30.0 BQL 1.00.5 — 6.41.6

Wistar rats were orally administered flutamide, and plasma and liver samples were obtained at 1 h after bolus injection of [3H]TCA as described in Materials and Methods. The results are shown as meansS.E.M. (n3). BQL: below the quantitation limit (quantitation limit; 0.1 m M in plasma, 0.1 nmol/g liver). differences (Table 1). The plasma and liver concentrations of flutamide and hy- droxyflutamide were also determined at the same time. One hour after [3H]TCA injection (4 h after flutamide dosing) in the single-dose group, the plasma and liver concentrations of flutamide were 16.8 m M and 62.6 nmol/g of liver, respectively, and those of hydroxyflutamide were 28.3 m M and 932 nmol/g of tissue, respectively (Table 2). In the case of the repeated- dose group, the plasma and liver concentrations of flutamide were lower than the quantitation limit (less than 0.1 m M in plasma and 0.1 nmol/g of liver, respectively) at 1 h after [3H]TCA injection (25 h after the last dose of flutamide). Effect of Flutamide on mRNA Levels of rBsep and rNtcp in Rats The effect of flutamide on mRNA levels of rBsep in rats treated with a single dose or repeated doses of flutamide were determined. The mRNA levels of rBsep were Fig. 5. Effect of Flutamide on Expression Level of (A) rBsep and (B) not significantly different among the groups (Figs. 5A, B). In rNtcp in Rat Liver addition, there was no significant change in the mRNA levels Wistar rats were orally administered flutamide (dotted bar: single-dose; closed bar: of Ntcp, which is expressed at the basolateral membrane of repeated-dose) or not given flutamide (open bar) as described in Materials and Meth- hepatocytes, and is involved in the transport of TCA from ods. The results are shown as meansS.E.M. (n3). portal blood into the liver (Figs. 5A, B).

inhibited BSEP-mediated TCA transport, with an IC50 of ap- DISCUSSION proximately 50 m M (Fig. 1), while hydroxyflutamide was not

inhibitory up to 100 m M. Since the inhibition constant (Ki Cholestasis, defined as impairment of bile formation, is value) of flutamide and hydroxyflutamide for androgen re- 25) accompanied with retention of bile salts, which can them- ceptor is 1450 and 205 nM, respectively, and plasma con- selves cause hepatic injury and cholestasis.11,12) Several drugs centration of hydroxyflutamide is higher than that of flut- such as troglitazone, bosentan, and cyclosporine A induce amide,24) the pharmacological effect of flutamide can be cholestatic hepatitis, probably by inhibiting BSEP-mediated largely attributed to hydroxyflutamide. Therefore, the phar- taurocholic acid transport.15—17) Fattinger et al. concluded macological effect of flutamide can be maintained without that in vitro screening for BSEP inhibition can adequately the inhibitory effect on Bsep. Next, we examined whether the predict the effects of drugs on bile salt serum levels in in vivo inhibitory effect of flutamide on BSEP-mediated TCA trans- animal studies, and that both BSEP inhibition and increased port is relevant in the clinical situation. It was reported that serum bile salt levels in preclinical animal studies are indica- the plasma concentration of flutamide in rats reached approx- tive for the development of drug-induced cholestatic liver in- imately 1 m M after administration of flutamide orally at a jury in humans. Therefore, we evaluated the inhibitory ef- dose of 15 mg/kg,26) and the liver-to-plasma concentration fects of flutamide and its major metabolite, hydroxyflu- ratio (Kp value) of flutamide is approximately 5 (basic prod- tamide, on BSEP-mediated taurocholic acid (TCA) transport. uct information of flutamide Odyne, Nippon Kayaku Co., By in vitro transport experiments, we found that flutamide Ltd., Tokyo, Japan). Based on these reports, we administered April 2007 743

100 mg/kg flutamide as a single dose in order to obtain a are involved in flutamide-induced cholestasis. In this study, liver concentration of flutamide close to the IC50 value for since we measured the biliary excretion of bile acids after BSEP, even though this dose is significantly higher than the bolus injection of [3H]TCA, enzymes which are involved in clinical dose. In addition, to model the clinical situation, we synthesis and/or metabolism of bile acids, such as CYP7A1 administered 10 mg/kg of flutamide daily for 5 d (repeated- and UGT2B4, are irrelevant. Furthermore, mRNA level of dose group), since this is close to the clinical dose rBsep and rat Ntcp were not changed (Fig. 5). Accordingly, (375 mg/d). In the flutamide-treated rats, the biliary excretion although the change of their protein levels cannot be denied, rate of [3H]TCA was decreased in the first 10 min in both the flutamide is not likely to affect at least the mRNA levels of single-dose group and repeated-dose group (Fig. 3). Since these transporters. Therefore, Bsep might not play a major the cumulative biliary excretion of [3H]TCA up to 60 min role in flutamide-induced cholestasis in repeated-dose group. after injection of [3H]TCA showed no significant difference In this study, we focused on the effect of flutamide itself among the 3 groups (Fig. 3), the decrease of biliary excretion and hydroxyflutamide, but not other metabolites such as rate of [3H]TCA in the first 10 min can be ascribed to the de- FLU-1, FLU-3, and their glucuronides, since no consistent crease of transport of [3H]TCA from plasma into bile by flu- differences were found in serum levels of these metabolites tamide. Furthermore, since there was no apparent difference between patients with normal function and those suffering in the plasma concentration profile of [3H]TCA among the 3 hepatic dysfunction.24) Furthermore, a similar pattern of the groups (Fig. 4, Table 1), it was suggested that the biliary ex- profile of flutamide metabolites was observed in the case of cretion rate of [3H]TCA was decreased due to a change in the treatment with or without UDCA,24) which is known to be efflux process from hepatocytes into bile, but not in the up- beneficial for flutamide-induced hepatotoxicity.19) However, take process from blood into hepatocytes. Therefore, it was it is possible that these metabolites inhibit Bsep-mediated demonstrated that flutamide may cause a decrease in the bil- bile acids transport. Therefore, these metabolites might be iary excretion rate of [3H]TCA by inhibiting BSEP. For fur- involved in flutamide-induced cholestasis in repeated-dose ther understanding of flutamide-induced cholestasis, total group. For further understanding of flutamide-induced bile acids concentrations in plasma, bile, and liver should be cholestasis, inhibitory effect of these metabolites on Bsep measured after flutamide treatment. should be evaluated. In the single-dose group, the flutamide concentration in The plasma concentration of flutamide reached 0.4 m M liver (approx. 60 nmol/g of tissue) reached a level close to the after administration of 250 mg three times a day for 9 d in 29) IC50 value for BSEP-mediated TCA uptake (approx. 50 m M) normal geriatric volunteers. Since the Kp value of flu- at 4 h after flutamide administration (Fig. 1, Table 2). How- tamide is 5.9 in liver (Table 2), the hepatic concentration ever, in the repeated-dose group, the apparent flutamide con- would be approx. 2.4 m M, assuming that the Kp value of flu- centration in liver was not high enough to inhibit BSEP tamide in human liver is similar to that in rat liver. At this (below 0.1 nmol/g of tissue), even though cholestasis was concentration, flutamide would inhibit BSEP-mediated bile similar to that in the single-dose group. This apparent obser- acid transport by about 5%. However, if the metabolism of vation might be explained by irreversible inhibition by flu- flutamide was reduced, e.g., by SNPs, drug–drug interac- tamide. There are reports of transporters being inhibited by tions, or altered expression of related genes, the flutamide such as 4,4-diisothiocyanato-stilbene-2,2-disulphonic acid concentration in liver might become high enough to exert an (DIDS), which is an irreversible inhibitor of monocarboxy- inhibitory effect on BSEP, since flutamide is extensively me- late transporter in rat erythrocytes.27) When erythrocytes tabolized in the liver. were preincubated with DIDS, L-lactate transport into rat In conclusion, we found that flutamide, but not its major erythrocytes was still reduced, even when DIDS was washed metabolite hydroxyflutamide, inhibits BSEP-mediated TCA out by replacing the uptake medium with DIDS-free transport. In addition, flutamide did not affect the mRNA medium. So, it is considered in the case of irreversible in- level of rBsep. Accordingly, though the IC50 value is lower hibitors that their inhibitory effects will be observed when than normal clinical concentration of flutamide in liver, the the apparent inhibitor concentration in tissues is lower than direct inhibition of BSEP may contribute in part to the occur-

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