Effects of Itraconazole, Dexamethasone and Naringin on the Pharmacokinetics of Nadolol in Rats

Drug Metab. Pharmacokinet. 28 (4): 356–361 (2013). Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX) Regular Article Effects of Itraconazole, Dexamethasone and Naringin on the Pharmacokinetics of Nadolol in Rats

Nozomu MIYAZAKI1, Shingen MISAKA1,*,HiroshiOGATA1,TetsuhitoFUKUSHIMA2 and Junko KIMURA1 1Department of Pharmacology, School of Medicine, Fukushima Medical University, Fukushima, Japan 2Department of Hygiene and Preventive Medicine, School of Medicine, Fukushima Medical University, Fukushima, Japan

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: The aim of the present study was to clarify the involvement of P-glycoprotein (P-gp) or organic anion transporting polypeptide (Oatp) 1a5 in the pharmacokinetics of nadolol (NDL), a non-metabolized hydrophilic ¢-adrenoceptor blocker, in rats. Pretreatment with itraconazole (ICZ, P-gp inhibitor, 50 mg/kg) for 30 min before oral administration of NDL (10 mg/kg) signiﬁcantly increased the area under the plasma

concentration-time curve (AUC0¨) of NDL by 1.7-fold compared with control. Intragastric administration of dexamethasone (DEX, 8 mg/kg) for 4 consecutive days increased P-gp level in the intestine and the

liver. In line with this, DEX pre-treatment decreased maximum plasma concentration (Cmax)ofNDLby28% of control. To inhibit the intestinal Oatp1a5, naringin (NRG, 0.145 mg/kg) was preadministered orally for 30 min before the oral administrations of NDL or celiprolol (CEL, 10 mg/kg, Oatp1a5 substrate). Although

NRG markedly reduced Cmax and AUC0¨ of CEL by 60% and 65% of control, respectively, little difference was observed in the plasma concentration of NDL between NRG and control. These results suggest that P-gp is greatly involved in the pharmacokinetics of NDL, while the involvement of Oatp1a5 in the pharmacokinetics of NDL may be less than that of celiprolol in rats.

Keywords: celiprolol; dexamethasone; itraconazole; nadolol; naringin; Oatp1a5; P-gp

individual drug transporters.7,8) For example, talinolol, an in vivo Introduction probe drug for P-gp, has been reported also to be a substrate for Drug transporters in the cell membrane are responsible for drug transporters other than P-gp.6) In addition, talinolol is not cellular uptake or export of vital materials and/or xenobiotics.1) approved for clinical use in countries including Japan and the While being involved in the drug disposition, activities of drug USA.9,10) Therefore, it is of great importance to develop a novel transporters can be modulated by a large number of drugs, result- in vivo probe drug with a high specificity and good feasibility for a ing in clinically important drug interactions.2,3) So far, such drug better understanding of drug transporter-mediated drug interaction. transporter-mediated drug interactions have been reported in Nadolol, a nonselective ¢-blocker, is used for the treatment humans with some herbal medicines and fruit juice such as of hypertension and angina pectoris.11) Since it is hydrophilic, St. John’s wort and grapefruit juice (GFJ).4,5) For instance, both nadolol is not metabolized by drug metabolizing enzymes such single and repeated ingestion of GFJ markedly reduced the oral as cytochrome P450 (Cyp), and is excreted primarily into the bile bioavailability of talinolol, a hydrophilic ¢-blocker, in a human with up to 70% of its dose in unchanged form. The bioavailability study.5) A recent report has shown that naringin, a flavonoid of nadolol given orally is approximately 20% in rats.12) According compound derived from GFJ, has a significant inhibitory effect to the biopharmaceutical classification system (BCS), nadolol is on not only P-glycoprotein (P-gp)–mediated drug efflux, but also categorized as a class 3 drug, which is characterized by high solu- organic anion transporting polypeptide (Oatp)-mediated uptake bility and low permeability in the gastrointestinal tract.13) Owing of drugs in the rat intestine.6) Drug interactions in animals and to the poor permeability in the intestinal epithelial cells, an active humans have been investigated using a probe drug to evaluate the transport is thought to play a pivotal role in the membrane activity of a drug transporter. However, the in vivo probe drugs permeation of class 3 drugs.13) Previously, it has been shown that currently in use have serious limitations for the specificity for nadolol is a substrate for human P-gp and OATP1A2 in vitro.14,15)

Received September 27, 2012; Accepted January 22, 2013 J-STAGE Advance Published Date: February 19, 2013, doi:10.2133/dmpk.DMPK-12-RG-111 *To whom correspondence should be addressed: Shingen MISAKA, Ph.D., Department of Pharmacology, School of Medicine, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan. Tel. ©81-24-547-1156, Fax. ©81-24-548-0575, E-mail: [email protected] This work was supported in part by a Grant-in-Aid for Young Scientists (B) (No. 23790605; S. Misaka) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

356 Pharmacokinetic Interaction Study of Nadolol in Rats 357

Those findings suggest that nadolol could be a candidate for the plasma and diluted urine samples (100 µL) were mixed with 1 M evaluation of drug transporter activities in vivo. Moreover, if the NaOH (1 mL), metoprolol (10 µL) and diethylether:dichloro- pharmacokinetics of nadolol is regulated by one particular drug methane = 70:30 (3 mL). The mixed solutions were shaken for transporter, nadolol may be a more specific probe drug for the 10 min and centrifuged at 3,000 © g for 3 min. The supernatants drug transporter than currently used probe drugs. Therefore, it were dried up with N2 gas, and the residues were dissolved in is necessary to clarify whether P-gp and/or Oatp are involved in 100 µL of 50 mM ammonium acetate with pH values being the absorption, distribution or excretion of nadolol by assessing adjusted to 4.5 for nadolol detection or 9.5 for celiprolol detection. whether the inhibition or induction of these drug transporters After the filtration through a 0.2-µm membrane filter (Millex-LG, affects the pharmacokinetics of nadolol. Millipore, Bedford, MA), 50 µL of the solution was subjected to In this study, we aimed to clarify the involvement of P-gp or HPLC analysis. The HPLC system consisted of an LC-2000Plus Oatp1a5 in the pharmacokinetics of nadolol in rats. To modulate system (JASCO, Tokyo, Japan) with a fluorescence detector. P-gp activity, we used itraconazole and dexamethasone as an Chromatographic separation was performed using an Inertsil ODS- inhibitor and inducer of P-gp, respectively. We measured the 4C18 column (2.1 © 100 mm, particle size 3 µm; GL Sciences, P-gp protein level in the small intestine and liver following Tokyo, Japan) as an analytical column and a guard column (Inertsil the pretreatment with dexamethasone over 4 days to examine the ODS-4 C18, 2.1 © 40 mm; GL Sciences) at room temperature. For induction of P-gp by dexamethasone. As an in vivo Oatp1a5 the detection of nadolol, the samples were separated using a inhibitor, naringin was used to investigate the pharmacokinetic gradient mobile phase consisting of 50 mM ammonium acetate profile of nadolol as well as celiprolol, a previously reported (pH 4.5) (A) and acetonitrile (B) at a flow rate of 0.30 mL/min. Oatp1a5 substrate. The linear gradient condition of the mobile phase was 0–4.0 min, 12–30% B; 4.0–8.0 min, 30–75% B; 8.0–10.0 min, 75% B; and Methods 10.0–20.0 min, 12% B. For the detection of celiprolol, the samples Reagents and chemicals: Nadolol was purchased from were separated using a gradient mobile phase consisting of 50 mM Sigma Aldrich (St. Louis, MO). Itraconazole, dexamethasone and ammonium acetate (pH 9.5) (AA) and acetonitrile (B´) at a flow rate metoprolol were purchased from Wako Pure Chemical Industries of 0.35 mL/min. The linear gradient condition of the mobile phase (Osaka, Japan). Celiprolol was provided from Nihon Shinyaku was 0–10.0 min, 18–40% BA; 10.0–12.0 min, 40–75% BA; 12.0– (Kyoto, Japan). Naringin was purchased from Tokyo Chemical 14.0 min, 75% BA; and 14.0–24.0 min, 18% BA. The fluorometric Industry (Tokyo, Japan). All the other reagents and solvents used detection wavelengths were ex. 230/em. 300 nm for nadolol, and were commercially available and of analytical or high-performance ex. 350/em. 480 nm for celiprolol. The limit of quantification was liquid chromatography (HPLC) grade. 1 ng/mL for both nadolol and celiprolol. The intra-assay coeffi- Animals: Male Sprague-Dawley (SD) rats (250–350 g) were cients of variance were <8.5% for nadolol detection, and <5.6% purchased from SLC (Shizuoka, Japan). The rats were housed for celiprolol detection. under identical conditions in a pathogen-free environment with a Analysis of P-gp protein expression: Protein levels of P-gp 12-h light/dark cycle and free access to standard chow and water. in the liver and intestine were analyzed by Western blotting. Rats All animal experiments were performed in accordance with the were pretreated with dexamethasone (8 mg/kg/day) or water for 4 guidelines of the Institutional Animal Care and Use Committee of days. On Day 5, rats were euthanized by intraperitoneal injection Fukushima Medical University. of pentobarbital (40 mg/kg body weight) after overnight fasting. Pharmacokinetic studies of nadolol and celiprolol: In The liver sample was collected from the same lobe for each rat. the nadolol study, rats were divided into four groups: control, The intestinal sample was harvested as follows: the first 20-cm itraconazole, dexamethasone and naringin. After an overnight segment proceeding from the pyloric sphincter was denoted as fast with free access to water, itraconazole (50 mg/kg) dissolved the duodenal sample, the middle 50-cm segment as the jejunum in saline and adjusted to pH 4.0 by phosphoric acid, naringin sample and the posterior 20-cm segment just preceding the colon (0.145 mg/kg) dissolved in phosphate-buffered saline or saline as the ileal sample. The central portion of each of these samples (control) were orally administered 30 min before the oral admin- was further sectioned into 2-cm segments for the Western blot. istration of nadolol (10 mg/kg). In the dexamethasone group, rats Samples were homogenized using a BioMasherII (Nippi, Tokyo, were pretreated orally for 4 days with dexamethasone suspended Japan). About 100 mg of the tissue was cut into small pieces and in 0.1% carboxymethyl cellulose at a daily dose of 8 mg/kg before homogenized in 250 µL of homogenizing buffer (250 mM sucrose/ the nadolol administration. 5 mM HEPES with 2% protease inhibitor, pH 7.4). The crude In the celiprolol study, rats were divided into two groups; con- homogenates were centrifuged with 500 µL of homogenizing trol and naringin. After an overnight fast with free access to water, buffer at 3,000 © g, 4°C for 10 min. The supernatant was removed naringin (0.145 mg/kg) or saline (control) were orally administered and centrifuged again at 15,000 © g, 4°C for 30 min. The pellet 30 min before the oral administration of celiprolol (10 mg/kg). was then reconstituted in suspending buffer (50 mM mannitol/ Blood was periodically collected through the tail vein up to 6 h 20 mM HEPES with 2% protease inhibitor, pH 7.4). An aliquot after nadolol or celiprolol administration. The blood samples were of protein (15 µg) was loaded in each lane to detect the expression immediately centrifuged to obtain plasma. The urine was collected of P-gp or actin, electrophoresed on 7.5% SDS-polyacrylamide cumulatively from 0 to 24 h in fractions from 0–3h,3–6h,6–9h, gel, and transferred to a PVDF membrane (Immobilon transfer 9–12 h and 12–24 h after nadolol or celiprolol administration using membrane, Millipore). For immunoblotting, the membranes were a metabolic cage. The plasma and urine samples were stored at blocked with 1% nonfat powdered milk in TBS-T20 (20 mM ¹80°C until analysis. Tris-HCl/137 mM NaCl/0.1% Tween 20, pH 7.6) at ambient tem- Determination of nadolol and celiprolol concentration: perature for 1 h. The blots were incubated with anti-P-gp (H-241; Urine samples were diluted 20 times with distilled water. The Santa Cruz Biotechnology Inc., Santa Cruz, CA) or anti-actin (H-

196; Santa Cruz Biotechnology Inc.) polyclonal antibody at 37°C 1.7-folds, respectively, compared with control. Median tmax and the for 1 h, and then washed three times with TBS-T20. The blots were average t1/2 for nadolol were not altered by itraconazole. The urine incubated with goat anti-rabbit IgG-HRP (sc-2004; Santa Cruz volume and the urinary excretion of nadolol during 24 h were not Biotechnology, Inc.,) as a secondary antibody at ambient temper- different between control and itraconazole treated rats (Fig. 2). ature for 1 h, and washed three times with TBS-T20. All washing Effect of dexamethasone on P-gp protein level and nadolol steps were performed at ambient temperature. P-gp and actin were pharmacokinetics: After repeated oral administration of dexa- detected with the enhanced chemiluminescence system according methasone for 4 days, there was no significant difference in the to the manufacturer’s instructions (GE Healthcare, Little Chalfont, mean body weight between vehicle- and dexamethasone-treated UK). rats (data not shown). The protein expression levels of P-gp in Data analysis: All values are expressed as the mean«standard the intestine and the liver were analyzed by Western blotting. error of the mean (SEM). A non-compartmental pharmacokinetic Dexamethasone treatment increased P-gp protein levels by 1.4- analysis was applied to the plasma concentration vs. time data and 1.8-fold in the liver and the ileum, respectively (Fig. 3). Cmax using WinNonlin software (Pharsight, Mountain View, CA). The and AUC0–3 of nadolol decreased by 28% and 37%, respectively, maximum plasma concentration (Cmax) and the time to maximum compared to control with dexamethasone (Fig. 1 and Table 1). On concentration (tmax) were estimated directly from observed plasma the other hand, AUC0–¨ was significantly increased by 1.8-fold of concentration-time data. The area under the time-plasma concen- control. Median tmax was not altered, while t1/2 was significantly tration curve after oral administration of nadolol (AUC) was prolonged by 2.1-fold by the pretreatment with dexamethasone. calculated using the linear trapezoidal rule up to 3 h (AUC0–3), or The urine volume was increased by 1.5-fold in the dexamethasone up to the last measured plasma concentration and extrapolated group, although the urinary concentration of nadolol was not to infinity (AUC0–¨). The terminal elimination half-life (t1/2) was significantly different between control and dexamethasone (data determined by dividing ln 2 by the elimination rate constant at the terminal phase, which was computed by a linear least squared Table 1. Pharmacokinetic parameters of nadolol in rats after a single method using at least 3 measurement points. Amounts of excretion dose of 10 mg/kg nadolol, which was administered at 30 min after saline into urine (Ae) were calculated as the urinary concentration of (control), itraconazole (50 mg/kg) or naringin (0.145 mg/kg), or with nadolol multiplied by the urine volume. Statistical differences of dexamethasone (8 mg/kg) for 4 days the means were assumed to be significant when p < 0.05 by one- Control Itraconazole Dexamethasone Naringin ’ way ANOVA, followed by Dunnett s multiple comparison test or Cmax (ng/mL) 122 « 14 203 « 22** 88 « 10 123 « 16 unpaired t-test. tmax (h) 1.5 (1.0–2.0) 2.0 2.5 (0.5–4.0) 2.0 (1.0–2.0) AUC0–3 (h0ng/mL) 212 « 17 315 « 28** 133 « 16* 184 « 23 Results AUC0–¨ (h0ng/mL) 297 « 28 509 « 33* 529 « 69** 259 « 38 t (h) 2.0 « 0.3 1.2 « 0.2 4.1 « 0.6* 1.4 « 0.3 Effect of itraconazole on the pharmacokinetics of nadolol: 1/2 Ae (µg) 245 « 37 251 « 47 411 « 34* 187 « 24 Plasma nadolol concentration-time profiles were obtained after Cmax, maximum plasma concentration of nadolol; tmax, time to maximum con- oral administration of 10 mg/kg nadolol to rats in the control and centration; AUC0–3, area under the plasma concentration-time curve from time pretreatment groups (Fig. 1). The corresponding pharmacokinetic 0 to 3 h; AUC0–¨, area under the plasma concentration-time curve from time 0 to infinity; t1/2, elimination half-life; Ae, the amount of nadolol excreted into urine parameters of nadolol are summarized in Table 1. Itraconazole « fi within 24 h. Each value represents the mean SEM; except for tmax data, which pretreatment at 30 min before nadolol administration signi cantly are given as median and range (n = 5–8). * p < 0.05 and ** p < 0.01 vs. increased Cmax, AUC0–3 and AUC0–¨ of nadolol by 1.7-, 1.5- and control.

Fig. 2. Cumulative amount-time proﬁle of nadolol in urine in rats Fig. 1. Plasma concentration-time proﬁle of nadolol in rats pretreated with pretreated with saline (control, closed circles), itraconazole (50 mg/kg, open saline (control, closed circles), itraconazole (50 mg/kg, open squares), squares), dexamethasone (8 mg/kg/day, 4 days, open triangles) and naringin dexamethasone (8 mg/kg/day, 4 days, open triangles) or naringin (0.145 (0.145 mg/kg, open circles) mg/kg, open circles) An oral dose of nadolol (10 mg/kg) was given at time 0. Cumulative amount of An oral dose of nadolol (10 mg/kg) was given at time 0. Plasma concentrations nadolol during 24 h. The urine was collected cumulatively up to 24 h in fractions were measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 6 h. Each value represents the from 0–3h,3–6h,6–9h,9–12 h and 12–24 h using a metabolic cage. Each value mean « SEM (n = 5–8). represents the mean « SEM (n = 5–6). * p < 0.05 vs. control.

Table 2. Pharmacokinetic parameters of celiprolol in rats after a single dose of 10 mg/kg celiprolol, administered at 30 min after saline (control) or naringin (0.145 mg/kg)

Control Naringin

Cmax (ng/mL) 364 « 29 149 « 7*** tmax (h) 2.0 (1.5–2.0) 1.3 (1.0–1.5) AUC0–¨ (h0ng/mL) 418 « 64 177 « 39* t1/2 (h) 1.1 « 0.1 0.6 « 0.1*

Cmax, maximum plasma concentration of celiprolol; tmax, time to maximum concentration; AUC0–¨, area under the plasma concentration-time curve from time 0 to inﬁnity; t1/2, elimination half-life. Each value represents the mean « SEM; except for tmax data, which are given as median and range (n = 3–4). * p < 0.05 and *** p < 0.001 vs. control.

Discussion It has been reported previously that nadolol was not metabolized by CYP enzymes in vivo, but could be transported by P-gp and OATP in vitro.12,14,15) Since the permeability of nadolol in the Fig. 3. Induction of P-gp protein in liver, jejunum and ileum of rats with intestinal epithelium is poor, the active influx and/or efflux trans- dexamethasone (DEX) treatment Protein signals (top panels) were quantified by densitometry. Fold-increase in port may contribute to the membrane permeation of nadolol at P-gp/Actin of each group compared to that of control rats was calculated based the absorption site. Nadolol could be a candidate for an in vivo on the signal intensity from the photographics. The values are the mean « SEM probe drug for the evaluation of drug transporter activity. To date, (n = 2–4). however, there has been no evidence for the involvement of drug transporters in the pharmacokinetics of nadolol in rodents or humans. Therefore, in the present study, we aimed to clarify whether the inhibition or induction of P-gp or Oatp1a5 influences the plasma concentration profile and urinary excretion of nadolol in rats using itraconazole, dexamethasone and naringin as P-gp inhibitor, P-gp inducer and Oatp1a5 inhibitor, respectively. Itraconazole, a triazole antifungal drug, is a potent inhibitor of P-gp and of Cyp3a.17) In this study, pretreatment with itraconazole (50 mg/kg) at 30 min before nadolol administration significantly increased Cmax and AUC0–¨ of nadolol without alterations in tmax or t1/2 (Fig. 1). The AUC0–3 value, which is more sensitive than Cmax to detect differences in the absorption rate, was also significantly increased in the itraconazole group. These results could be attributed to an inhibition of P-gp by itraconazole, presumably in the apical membrane of the intestinal epithelium, because nadolol is not metabolized by Cyp3a.12) Concerning the P-gp–mediated itraconazole-drug interaction, a previous clinical study showed Fig. 4. Plasma concentration-time profile of celiprolol in rats pretreated that itraconazole markedly increased the AUC value of celiprolol, with saline (control, closed circles) or naringin (0.145 mg/kg, open circles) possibly due to the inhibition of intestinal P-gp by itraconazole.18) An oral dose of celiprolol (10 mg/kg) was given at time 0. Plasma concentrations Zolnerciks et al. recently demonstrated that the inhibitory were measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 6 h. Each value represents the mean « SEM (n = 3–4). potencies of itraconazole for prazosin and verapamil, which are typical P-gp substrates, were similar between human and rat P-gp, suggesting that the inhibition of P-gp by itraconazole is species- not shown). Accordingly, urinary nadolol excretion significantly independent.19) Based on these findings, our results suggest that the increased to 1.6 times that of control with dexamethasone (Fig. 2). bioavailability of orally administered nadolol is increased by the Effect of naringin on the pharmacokinetics of celiprolol and inhibition of intestinal P-gp by itraconazole. P-gp is also expressed nadolol: It has been previously reported in rats that a lower in the apical membrane of renal proximal tubule cells as well as dose of naringin inhibited Oatp1a5 but not P-gp.16) In this study, in hepatocytes.1) In this study, the cumulative amount of nadolol naringin at a dose of 0.145 mg/kg reduced the Cmax of celiprolol to excreted into urine over 24 h was not altered between itraconazole 50% of control (Fig. 4 and Table 2). AUC0–¨ of celiprolol was and control rats (Fig. 2). Currently, we do not have a mechanistic also decreased by naringin treatment by 50% of control. Median explanation for the unchanged urinary excretion of nadolol by tmax and average t1/2 of celiprolol were decreased to 66% and itraconazole. However, because the urinary excretion process is 60% of control, respectively. On the other hand, pharmacokinetic relatively minor in the elimination of nadolol in rats, the influence profiles of nadolol including Cmax, AUC0–¨ and Ae were similar of itraconazole on renal P-gp–mediated excretion of nadolol may between naringin and control (Fig. 1 and Table 1). The urine be less significant under our experimental conditions. On the other volume and the amount of urinary nadolol did not differ between hand, since nadolol is mainly excreted into the bile in rats,12) control and naringin over 24 h (Fig. 2). hepatic P-gp may be involved in the biliary excretion of nadolol.

To assess the role of hepatic P-gp in the excretion of nadolol, the coadministered with naringin at the same dose, the plasma con- measurement of biliary excretion and the pharmacokinetic analysis centration and urinary excretion of nadolol did not differ between of nadolol after parenteral administration are required in the future. the control and naringin groups. These results suggest that the Dexamethasone is known to induce P-gp via the activation of contribution of Oatp1a5 to the absorption of nadolol is relatively pregnane X receptor.20) To verify the effect of dexamethasone at a small compared with celiprolol. Recent study has revealed that daily oral dose of 8 mg/kg for 4 days, we examined the protein Oatp2b1 is also expressed in the small intestine in addition to level of P-gp in the liver and small intestine by Western blotting. Oatp1a5 in rats.25) Another study showed recently that naringin We found that the P-gp expression in the liver, jejunum and ileum at a high concentration (1,000 µM) inhibited Oatp2b1-mediated were elevated to 1.4-, 1.2- and 1.8-fold of vehicle, respectively uptake of pravastatin but not pitavastatin in vitro,26) suggesting that (Fig. 3). This result was in accordance with the previous study on naringin has a weak inhibitory effect on Oatp2b1 depending on the effects of dexamethasone and St. John’s wort on P-gp induction its substrate. We do not know whether naringin at the dose of in rats.21) When we investigated the effect of dexamethasone on the our study (0.154 mg/kg, approximately 67 µM) affects Oatp2b1 pharmacokinetics of nadolol, we found that repeated dexametha- activity. It is necessary to investigate whether Oatp2b1 mediates sone significantly decreased AUC0–3 metrics of nadolol along with the uptake of nadolol in the small intestine. Taken together, P-gp a decrease in Cmax (Fig. 1 and Table 1). Lin et al. reported that the may play a critical role in the pharmacokinetic profile of nadolol plasma concentration of indinavir, a P-gp substrate, was reduced rather than Oatp1a5 in rats. This suggests that nadolol could be a by dexamethasone treatment in rats (40 mg/kg/day, 3 days).22) This promising candidate for the evaluation of P-gp activity in vivo. suggests that the induction of P-gp by dexamethasone treatment However, we cannot exclude the possibility that other transporters significantly increased the efflux of its substrate drug to the intes- are also involved in the pharmacokinetics of nadolol. Further tinal lumen. Therefore, the decrease in plasma concentration study is required to identify the major determinant of the intestinal of nadolol during the absorption phase may be partly explained by absorption of nadolol in addition to the biliary or urinary excretion. the effect of dexamethasone on P-gp induction, which mediated To our knowledge, this is the first study to show the pharmaco- efflux transport of nadolol from intestinal epithelial cells to the kinetic drug-drug interactions of nadolol with commonly pre- lumen. In contrast to the results of AUC0–3 and Cmax, the values scribed drugs in animal models. Our data raises concern about of AUC0–¨ and t1/2 of nadolol were significantly increased in the clinical use of nadolol, especially with respect to its efficacy dexamethasone-pretreated rats as compared to control. These and adverse effects, in combination therapy. Indeed, we have results are inconsistent with previous reports that the AUC and t1/2 performed a clinical trial to elucidate whether the modulations of of indinavir and fexofenadine were decreased by the pretreatment P-gp or OATP1A2 activities alter the pharmacokinetics of nadolol with a P-gp inducer such as dexamethasone or St. John’s wort.22,23) in healthy volunteers.27) The coadministration of itraconazole In the present study, biochemical blood tests revealed remarkable significantly increased the plasma concentration of nadolol after increases in aspartate aminotransferase and alanine aminotrans- an oral administration of nadolol, while a single ingestion of ferase after dexamethasone treatment, indicating the impairment grapefruit juice had a minor impact on nadolol pharmacokinetics. of rat liver function (data not shown). The elevated AUC0–¨ These results are in agreement with the present rat study, and and prolonged t1/2 of nadolol may be ascribed to the decline of suggest that care should be taken in the concomitant use of nadolol biliary excretion of nadolol from the hepatocytes. In addition, the with P-gp inhibitors and/or inducers. At the same time, our clinical cumulative amount of urinary-excreted nadolol was significantly and preclinical results indicate that the pharmacokinetics of nadolol increased in the dexamethasone group with little change in the is predominantly regulated by P-gp, but not by OATP1A2/ urinary concentration of nadolol. Since the urine volume was Oatp1a5, in both humans and rats, suggesting the potential also increased by dexamethasone treatment, we speculated that the usefulness of nadolol in preclinical studies for predicting P-gp– increase in the urinary excretion of nadolol is attributable to the mediated drug interactions. increased urine volume. A similar result has been reported in which In conclusion, the present study was performed to examine dexamethasone treatment induced a near doubling of the urine whether P-gp or Oatp1a5 was involved in the pharmacokinetics volume with a significant increase in urinary sodium excretion in of nadolol, and to address the possibility that nadolol can be an rats.24) Because dexamethasone has multiple effects which could in vivo probe drug for P-gp or Oatp1a5. Our results show that impair the influence of P-gp induction on the pharmacokinetics nadolol is greatly affected by P-gp while negligibly affected of nadolol, additional in vivo studies using other P-gp inducers will by Oatp1a5 in its intestinal absorption as revealed by the phar- be needed to confirm the role of intestinal P-gp in the absorption macokinetic experiments using itraconazole, dexamethasone and of nadolol. naringin. Although the role of other drug transporters on the Naringin, a flavonoid component found in GFJ, inhibits either pharmacokinetics of nadolol should be studied, we propose that Oatp1a5 or P-gp activity depending on its concentration.6) Since nadolol could be a probe drug candidate for the evaluation or a low dose of naringin predominantly inhibits Oatp1a5, we used prediction of P-gp–mediated drug interactions. a low dose of naringin as an Oatp1a5 inhibitor. When celiprolol was administered with naringin, the AUC0–¨ and Cmax of celiprolol Acknowledgments: The authors thank Mr. Senri Omata and Mr. were decreased by 50% and 50%, respectively, and tmax and t1/2 Seiji Hoshi for their excellent technical assistance. shortened by 66% and 60%, respectively (Fig. 4 and Table 2). References This result is consistent with a previous report that the blood levels of talinolol were reduced when talinolol was administered in com- 1) Klaassen, C. D. and Aleksunes, L. 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