Comparative Evaluation of Dehydroepiandrosterone Sulfate

Home , Prasterone sulfate

Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2016/12/01/dmd.116.072355.DC1

1521-009X/45/2/224–227$25.00 http://dx.doi.org/10.1124/dmd.116.072355 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 45:224–227, February 2017 Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics Short Communication

Comparative Evaluation of Dehydroepiandrosterone Sulfate Potential to Predict Hepatic Organic Anion Transporting Polypeptide Transporter-Based Drug-Drug Interactions s

Received July 4, 2016; accepted November 30, 2016

ABSTRACT

Pharmacokinetic drug-drug interactions (DDIs) on hepatic organic 59 ml/rat. Comparison of the in vitro IC50 of rifampicin for DHEAS anion transporting polypeptides (OATPs) are important clinical issues. uptake by isolated rat hepatocytes and in vivo plasma rifampicin

Previously, we reported that plasma dehydroepiandrosterone sulfate concentration suggested that the effect of rifampicin on the plasma Downloaded from (DHEAS) could serve as an endogenous probe to predict OATP-based DHEAS concentration was explained mostly by the inhibition of DDIs in monkeys using rifampicin as an OATP inhibitor. Since the hepatic OATPs, demonstrating that DHEAS could be a biomarker of contribution of hepatic OATPs to the changes in plasma DHEAS by hepatic OATP activity. Next, previously reported rifampicin-induced rifampicin remains unclear, however, we performed an in vivo phar- changes in plasma concentrations evaluated as an AUC ratio (AUCR) macokinetic study to explore this issue. Since plasma DHEAS of possible probe compounds were compared on the basis of concentrations were low in our rat model, the disposition of externally rifampicin dose/body surface area. The AUCR values of endogenous dmd.aspetjournals.org administered DHEAS was evaluated. Intravenously administered compounds and i.v. administered statins, for which possible DDIs in DHEAS was recovered mainly in bile (29.1%) and less in urine the intestinal absorption process can be excluded, increased propor-

(2.95%). The liver tissue-to-plasma concentration ratio (Kpliver)de- tionally to the rifampicin dose. Simultaneous measurement of these creased from 41.8 to 5.07 by rifampicin, and this decrement was endogenous compounds could be effective biomarkers for the pre- consistent with the decrease in distribution volume from 247 to diction of OATP-based DDIs. at ASPET Journals on October 1, 2021

Introduction et al., 2014). Although those probe drugs are useful, endogenous probe Membrane transporters are involved in the absorption and disposition of compounds might be advantageous since in vivo DDI could be examined at many drugs, and alterations in their functional activities may lead to an early stage of drug development without the need for additional studies decreased efficacy and/or adverse events. Such alterations can occur as a involving the administration of probe drugs. result of drug-drug interactions (DDIs) and drug-food interactions with Several endogenous compounds, including bilirubin, bile acids, and concurrently administered drugs or foods (Shitara et al., 2013; Nakanishi and coproporphyrins, have been proposed as biomarkers to monitor DDIs on Tamai, 2015). Regulatory agencies, such as the U.S. Food and Drug hepatic OATPs (Chu et al., 2015; Watanabe et al., 2015b; Shen et al., 2016, Administration, European Medicines Agency, and Japanese Pharmaceuticals Lai et al., 2016). In addition, we recently reported that dehydroepiandros- and Medical Devices Agency, have proposed that clinical DDI studies should terone sulfate (DHEAS) served as a biomarker to reflect the DDI on hepatic be required for drugs under development; thus, there is a need to develop OATPs when cynomolgus monkeys were administered an inhibitor of convenient ways to predict DDI and drug-food interaction potential. Organic OATPs (Watanabe et al., 2015a). Although the observed increase in the anion transporting polypeptides (OATPs) contribute to drug absorption and plasma concentration of DHEAS by rifampicin was less than that by statins, disposition (Tamai et al., 2000; Shitara et al., 2013). For example, OATPs plasma DHEAS was dose dependently increased by rifampicin. Despite the expressed in the liver take up various drugs into hepatocytes from the apparent differences in sensitivity for detecting the effect of rifampicin systemic circulation, thereby affecting systemic and liver exposures to these among these putative biomarkers, they might be broadly comparable when drugs (König, 2012; Shitara et al., 2013). OATP1B1 and OATP1B3 are key the dose of rifampicin in each study is taken into account, in spite of the molecules in the hepatic handling of drugs, and accurate predictions of difference in species (Nakakariya et al., 2008); however, it is not yet clear clinically significant DDIs on those OATPs are essential. Several probe drugs whether rifampicin affects only hepatic OATPs or whether other trans- to evaluate possible inhibitors of hepatic OATPs have been suggested, for porters and metabolic enzymes might contribute to the observed alterations. example, with statins as the “victims” (Yoshida et al., 2012; Prueksaritanont DHEAS is present in plasma at relatively high concentration, which makes it easy to analyze, and it has been proposed as a biomarker for aging- or disease-related physiologic changes (Stanczyk, 2006; Urbanski This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science [Grant 16H50111]. et al., 2013; Goodarzi et al., 2015). Since several factors other than dx.doi.org/10.1124/dmd.116.072355. OATPs may affect plasma DHEAS, as well as other potential biomarkers, s This article has supplemental material available at dmd.aspetjournals.org. it is essential to establish that the rifampicin-induced increase in plasma

ABBREVIATIONS: AUC, area under the plasma concentration curve; AUCR, AUC ratio; CL, clearance; DDI, drug-drug interaction; DHEAS, dehydroepiandrosterone sulfate; HPLC, high-performance liquid chromatography; LC-MS/MS, liquid chromatography-tandem mass spectrometry;

Kp, tissue-to-plasma concentration ratio; OATP, organic anion transporting polypeptide; Vdss, steady-state volume of distribution. 224 DHEAS as a Biomarker for OATP-based DDI 225

DHEAS is at least predominantly due to the interaction on hepatic uptake system was used to obtain the isocratic flow of the mobile phase at a rate of transporters, OATPs, to confirm the suitability of DHEAS as a possible 1.0 ml/min. biomarker. In the present study, therefore, we evaluated the in vivo Pharmacokinetic Analysis. Theplasmaconcentration-timedatawereanalyzed contribution of hepatic OATPs to the rifampicin-induced increase in the by noncompartmental analysis. The area under the plasma concentration time curve plasma concentration of DHEAS by measuring biliary and urinary (AUC0–4) was obtained by the trapezoidal rule from time 0 to 4 hours; AUC from 0toinfinity(AUC ) was estimated by extrapolation to infinity. Total clearance excretions, tissue concentrations, and pharmacokinetic parameters with inf (CL ) was estimated as dose (D)overAUC (D/AUC ), and renal clearance and without rifampicin. Since the endogenous plasma concentration of tot inf inf (CLurine) and biliary excretion clearance (CLbile) were estimated as Xurine,0-4/AUC0–4 DHEAS in the present rat model was unexpectedly low, to detect it by our and Xbile,0–4/AUC0–4,whereXurine,0–4 and Xbile,0–4 represent cumulative amounts of liquid chromatography-tandem mass spectrometry (LC-MS/MS) method DHEAS recovered in urine and bile, respectively. Biliary excretion clearance based

(limit of detection: 0.5 nM), we evaluated the contribution of hepatic on liver tissue concentration (CLbile,liver) was estimated as Xbile,0–4/(Kpliver Â AUC0–4). OATPS to the changes in DHEAS disposition by rifampicin by externally The apparent volume of distribution (Vdss)wasestimatedasVdss = CLtot Â MRT, administering DHEAS. Furthermore, the usefulness of DHEAS as a where MRT is the mean residence time. The apparent tissue-to-plasma concentration biomarker was compared with that of other proposed markers reported in ratio (Kpliver and Kpkidney) was estimated as the ratio of liver or kidney concentration the literature. divided by the plasma concentration at 4 hours. Statistical Analysis. Student’s t test was used to analyze differences between groups. P , 0.05 was considered statistically significant. Materials and Methods Chemicals. Rifampicin, DHEAS, and dehydroepiandrosterone-d5-3-sulfate Downloaded from sodium salt were purchased from Wako Pure Chemical Industries (Osaka, Japan), Results and Discussion Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), and Sigma-Aldrich (St. Louis, MO), respectively. [3H]Dehydroepiandrosterone sulfate (2.22–3.70 Effect of Rifampicin on Pharmacokinetics of DHEAS. Endoge- TBq/mmol) sodium salt was purchased from PerkinElmer (Boston, MA). Other nous plasma concentration of DHEAS in rats was under the detection limit reagents and solvents were of analytical grade. of our LC-MS/MS method. Accordingly, in the present study, DHEAS Animals. Seven-week-old female Wistar rats (170–190 g) were purchased disposition was evaluated after i.v. administration of DHEAS. The plasma from Sankyo Labo Service (Tokyo, Japan). All animal studies were approved by

concentration-time curves of DHEAS with and without i.v. rifampicin dmd.aspetjournals.org the Committee of Kanazawa University for the Care and Use of Laboratory (30 mg/kg) 1 hour before DHEAS administration are shown in Fig. 1B. Animals and performed in accordance with its guidelines (AP-143148). The dose of DHEAS was set at 4 mg/kg, considering the detection limit In Vivo Animal Study. Rats were anesthetized with pentobarbital, and the during pharmacokinetic analysis (Sakaguchi et al., 1992). At the same time, bladder and bile duct were cannulated with polyethylene tube (inside 0.5 mm, outside 0.8 mm). The rats were given 4 mg/kg DHEAS i.v. 1 hour after i.v. cumulative excretions of DHEAS into bile (Fig. 1C) and urine (Fig. 1D) administration of 30 mg/kg rifampicin. DHEAS and rifampicin were administered and tissue concentrations in the kidney and liver were measured. The via femoral vein. Then, blood was drawn from the jugular vein, and plasma was plasma concentration of DHEAS decreased biexponentially. All parame- generated by centrifugation at 3000 rpm for 10 minutes at 4C. Urine and bile ters were evaluated from the observations up to 4 hours, and the results are at ASPET Journals on October 1, 2021 were collected at the designated times. Rats were sacrificed 4 hours after the summarized in Table 1. AUC0–4 was increased from 10.9 6 1.85 to 46.0 6 administration of DHEAS by cutting the inferior vena cava with the animmals 8.46 mmol･h per liter by coadministration of rifampicin, and AUCinf was under deep anesthesia, and the kidney and liver were isolated for measurement of similarly increased. The sum of the biliary and urinary recoveries of DHEAS. All samples were stored at 230C until measurement. DHEAS up to 4 hours was about one-third of the dose, but biliary excretion Quantitation of DHEAS in Plasma, Urine, Bile, and Tissue Samples. was 10 times higher than urinary excretion, indicating that DHEAS is DHEAS concentrations were measured by LC-MS/MS. Liver and kidney were predominantly excreted into bile. Biliary excretion of DHEAS was excised, weighed, and homogenized with 5 volumes of methanol. Each sample was left 6 6 on ice for 30 minutes and then centrifuged (14,000 rpm, 5 minutes, 4C). The resulting significantly decreased from 29.1% 9.73% to 13.4% 1.57% of dose supernatant was stored until measurement was done. Aliquots (5 ml) of plasma, urine, by rifampicin, whereas no significant change was observed in urinary bile, and tissue sample extract were added to 495 ml of methanol containing internal excretion (2.95% 6 0.63% vs. 2.38% 6 1.30% of dose). These standard (1 mg/ml DHEAS-d5) and centrifuged (14,000 rpm, 5 minutes, 4C). Then, a observations indicated that rifampicin markedly affects the biliary excretion 10-ml aliquot of the resulting supernatant was subjected to LC-MS/MS. The of DHEAS. Although rifampicin causes a statistically significant decrease LC-MS/MS system consisted of a triple-quadrupole mass spectrometer (API 3200, in CLurine (from 5.46 6 1.48 to 1.09 6 1.22 ml/h per rat), its contribution to AB Sciex, Foster City, CA) coupled with an ultrafast liquid chromatography system change in plasma concentration of DHEAS should be small since urinary (LC-20AD, Shimadzu Co., Kyoto, Japan). Chromatography was performed using a excretion is much less than biliary excretion. DHEAS is accumulated in the Â m Mercury MS analytical column (C18, 10 4.0 mm, 5 m, Phenomenex, Torrance, liver: Kp was 41.8 6 5.67 in the case of DHEAS alone, whereas CA) at 40C with a mobile phase of 10 mM ammonium formate (A) and acetonitrile liver Kpkidney waslessthanunity(0.936 0.55). In the presence of rifampicin, (B). The flow rate was 0.3 ml/min, and gradient conditions for elution were as follows: 6 10%–90% B (0–3 minutes), 90% B (3-4 minutes), 90%–10% B (44.1 minutes), 10% Kpliver was significantly decreased to 5.07 1.96, whereas Kpkidnay was 6 6 (4.1–6 minutes). MS was performed with negative ionization, and mass transitions unchanged (0.67 0.27). Furthermore, Vdss was decreased from 247 (Q1/Q3) of m/z 367.1/96.9 and 372.3/98.1 were used for DHEAS and d5-DHEAS, 75.5 to 59.4 6 10.1 ml/rat by rifampicin. Considering the liver volume respectively. The limit of quantitation for DHEAS was 0.5 nM. (10 g/0.25 kg rat) (Davies and Morris, 1993), the change in Kpliver by Quantitation of Rifampicin in Plasma Samples. Plasma concentration of rifampicin is expected to cause a decrease in Vdss of more than 200 ml. rifampicin was measured by high-performance liquid chromatography (HPLC). Thus, the apparent change in Vdss (188 ml) can be well explained by the Extraction of rifampicin from plasma was performed by adding an equal volume of decrease in the distribution of DHEAS to the liver. methanol containing 10 mM methoronidazole as an internal standard. The mixture CL decreased significantly, from 53.9 6 18.4 to 6.71 6 1.22 ml/h bile was vortexed and centrifuged (14,000 rpm, 5 minutes, 4 C), and the resultant per rat, with rifampicin, whereas CL was unchanged by rifampicin supernatant was subjected to HPLC. The HPLC system (Waters Corporation, bile,liver (2.16 6 1.98 vs. 3.02 6 4.24 ml/h per rat). Taking these changes in the Milford, MA) was equipped with a UV absorbance detector (Waters 2487/2690). A solvent delivery system (Waters 2695) was used to obtain the isocratic flow of the pharmacokinetic parameters together, we consider the change in the mobile phase. Chromatography was performed using Mightysil RP-18GP Aqua hepatic disposition of DHEAS caused by rifampicin to be due mainly to analytical column (4.6 mm Â 250 mm i.d., 5-mm particle size; Kanto Chemical a decrease in hepatic uptake from the blood, but not a decrease in biliary Co., Inc., Tokyo, Japan) prewarmed at 40C. The mobile phase was a mixture of excretion from the liver. Furthermore, changes in systemic disposition 10 mM phosphate buffer adjusted to pH 3.0 and methanol (40:60). The delivery can be explained mostly by the changes in hepatic disposition. 226 Nishizawa et al.

Fig. 1. Time courses of plasma DHEAS and rifampicin concentrations and biliary and urinary excretion of i.v. administered DHEAS and rifampicin. After i.v. administration of DHEAS (4 mg/kg), with and without i.v. administration of rifampicin (30 mg/kg, 1 hour before), plasma concentrations of rifampicin (A) and DHEAS (B) and cumulative excretion of DHEAS in bile (C) and urine (D) were measured for up to 4 hours. Open and closed symbols represent the results with and without rifampicin in (B), (C), and (D), respectively. Each result represents the mean 6 S.D. (n = 4-5). *A significant difference from the corresponding value without rifampicin. Downloaded from dmd.aspetjournals.org

Uptake of DHEAS by Isolated Rat Hepatocytes. Since hepatic accounted for by the activities of rat Oatp1a1 and 1a4 (Eckhardt et al., uptake of DHEAS appeared to be significantly affected by rifampicin 1999; Reichel et al., 1999). Therefore, the observed increase of systemic in vivo, we next measured DHEAS uptake by isolated hepatocytes exposure to DHEAS in the presence of rifampicin appears to be due to in vitro. Initial uptake of [3H]DHEAS (2 nM) up to 40 seconds was the decreased hepatic uptake resulting from the inhibition of hepatic decreased by rifampicin in a concentration-dependent manner (Supple- OATP transporters. Further study using OATP-knockout animals should mental Fig. 1). By subtracting the basal uptake obtained in the presence be conducted for evaluation of the suggested mechanisms of changes in at ASPET Journals on October 1, 2021 of 100 mM rifampicin, the IC50 of rifampicin was estimated to be 3.65 mM DHEAS disposition. in the present study, which is comparable to the reported Ki values for rat Comparison of DHEAS with Other Probes to Monitor Hepatic OATPS of between 1 and 10 mM (Fattinger et al., 2000). OATP-Based DDI. Several endogenous compounds have been pro- Figure 1A shows the time course of plasma rifampicin concentration posed as biomarkers to evaluate hepatic OATP-based DDIs, including after i.v. administration to rats. The concentration of rifampicin remained bilirubin and its conjugated metabolites, bile acids, and coproporphyrins higher than 15 mM during the measurement of DHEAS disposition. (Watanabe et al., 2015b; Chu et al., 2015; Shen et al., 2016; Lai et al., Since plasma protein binding of rifampicin was reported to be 75% 2016). Statins have also been suggested as exogenous probes useful for (Imaoka et al., 2013), the plasma unbound concentration of rifampicin this purpose (Yoshida et al., 2012; Prueksaritanont et al., 2014). should have been higher than the IC50 throughout. Although it was not Therefore, we compared the sensitivity of DHEAS with those of other confirmed in the present study, DHEAS uptake by rat hepatocytes can be reported markers in terms of the extent of increase of plasma concentration in the presence of rifampicin, using the AUC ratio (AUCR) of each compound obtained from the literature. Figure 2 shows TABLE 1 the reported AUCRs after standardization of the rifampicin dose by the body surface area in rats, monkeys, and humans (see Supplemental Pharmacokinetic parameters of i.v. DHEAS with and without rifampicin in rats Table 1 for details). Although the AUCRs tended to increase with dose Pharmacokinetic parameters of DHEAS after administration at 4 mg/kg i.v. with and without rifampicin (30 mg/kg, i.v. dose 1 hour before DHEAS administration. Data are represented as of rifampicin, no clear correlation was found. One reason might be that mean 6 S.D. (n = 4-5) when probe compounds are orally administered, the intestinal bio-

Parameter Unit DHEAS Alone DHEAS + Rifampicin availability as well as the hepatic disposition must be considered (Yoshida et al., 2012); however, for the present purpose, it would be AUC – mM・hr 10.9 6 1.85 46.0 6 8.46* 0 4 desirable to eliminate the influence of intestinal availability since AUCinf 11.4 6 2.08 48.0 6 8.61* CLtot mL/hr/rat 174 6 37.2 43.5 6 9.82* endogenous compounds do not include the intestinal absorption process. 6 6 CLurine 5.46 1.48 1.09 0.72* Accordingly, the correlation was separately analyzed for the oral 6 6 CLbile 53.9 18.4 6.71 1.22* administration group (green) and for the group consisting of i.v. CLbile,liver 2.16 6 1.98 3.02 6 4.24 Xurine,0-4 mg 13.1 6 10.4 16.98 6 10.8 administered statins (blue) and endogenous compounds (red and 6 6 Xbile,0–4 213 78.0 111 10.0* magenta). Generally, the former group showed higher AUCRs than 6 6 Urinary recovery % of dose 2.95 0.63 2.38 1.30 the latter, but the AUCRs of the latter group increased primarily Biliary recovery 29.1 6 9.73 13.4 6 1.57* proportionally to the rifampicin dose. These results confirm that AUCR Vdss mL/rat 247 6 75.5 59.4 6 10.1* Kpliver 41.8 6 5.67 5.07 6 1.96* of orally administered statins includes the effects of interactions on both 6 6 Kpkidney 0.93 0.55 0.67 0.27 intestinal absorption and hepatic OATPs, suggesting that hepatic OATP- 6 6 Rat body weight g 182 11.1 186 6.32 based DDI might be overestimated with these markers. On the other *P , 0.05, statistically significantly different from DHEAS alone. hand, the endogenous compounds and i.v. administered statins are DHEAS as a Biomarker for OATP-based DDI 227

Fig. 2. Relationship between AUCR of OATP substrates and dose of rifampicin. Relationship between AUCRs of statin and endogenous compounds and dose of rifampicin normalized by body surface area is shown. When information on body weight was not given in the source reference, human body weight was taken as 60 kg. Circles: rats, squares: monkeys, triangles: humans. Compounds: A: pitavastatin, B: rosuvastatin, C: pravastatin, D: DHEAS, E: bilirubin, F: bile acids, G: coproporphyrin I, H: coproporphyrin III. The numbers shown in each symbol indicate the source reference numbers shown in Supplemental Table 1 (D-x represente present study data). Symbols in blue, green, and red or magenta indicate that the compounds of interest are i.v. administered statins, orally administered statins, and endogenous substances coadministered with oral or i.v. rifampicin, respectively. These values and cited references are listed in Supplemental Table 1. Downloaded from similarly effective for predicting the apparent DDI on hepatic OATP Fattinger K, Cattori V, Hagenbuch B, Meier PJ, and Stieger B (2000) Rifamycin SV and rifampicin exhibit differential inhibition of the hepatic rat organic anion transporting polypeptides, Oatp1 transporters. Among the putative biomarkers, coproporphyrins in and Oatp2. Hepatology 32:82–86. humans may show higher sensitivities than the others (G-13, H-10, Goodarzi MO, Carmina E, and Azziz R (2015) DHEA, DHEAS and PCOS. J Steroid Biochem Mol – and H-13 in Fig. 2). In practice, however, all the endogenous compounds Biol 145:213 225. Imaoka T, Mikkaichi T, Abe K, Hirouchi M, Okudaira N, Izumi T (2013) Integrated approach of in dmd.aspetjournals.org could be measured both before and after administration of test vivo and in vitro evaluation of the involvement of hepatic uptake organic anion transporters in the drug disposition in rats using rifampicin as an inhibitor. Drug Metab Dispos 41:1442-1449. compounds, which makes the changes in their concentrations clearer. König J (2011) Uptake transporters of the human OATP family: molecular characteristics, sub- Accordingly, all the suggested endogenous markers could be used as strates, their role in drug-drug interactions, and functional consequences of polymorphisms, in Drug Transporters (Fromm MF and Kim RB, eds) pp 1–28, Springer, Heidelberg. biomarkers for OATP-based DDI. Furthermore, since test drugs as Lai Y, Mandlekar S, Shen H, Holenarsipur VK, Langish R, Rajanna P, Murugesan S, Gaud N, perpetrators may affect mechanisms other than OATPs that regulate the Selvam S, Date O, et al. (2016) Coproporphyrins in plasma and urine can be appropriate clinical disposition of the suggested biomarkers, it may be desirable to biomarkers to recapitulate drug-drug interactions mediated by OATP inhibition. JPharmacol Exp Ther 358:397–404. simultaneously measure a panel of biomarkers, which would pre- Nakakariya M, Shimada T, Irokawa M, Maeda T, and Tamai I (2008) Identification and species sumably have a variety of alternative disposition mechanisms that are similarity of OATP transporters responsible for hepatic uptake of beta-lactam antibiotics. Drug at ASPET Journals on October 1, 2021 Metab Pharmacokinet 23:347–355. commonly taken up by the liver via OATPs . Nakanishi T and Tamai I (2015) Interaction of drug or food with drug transporters in intestine and In conclusion, our results indicate that the increase in the plasma liver. Curr Drug Metab 16:753–764. Prueksaritanont T, Chu X, Evers R, Klopfer SO, Caro L, Kothare PA, Dempsey C, Rasmussen S, concentration of DHEAS in the presence of rifampicin can be largely Houle R, Chan G, et al. (2014) Pitavastatin is a more sensitive and selective organic anion- explained by interactions in the process of hepatic uptake, specifically at transporting polypeptide 1B clinical probe than rosuvastatin. Br J Clin Pharmacol 78:587–598. Reichel C, Gao B, Van Montfoort J, Cattori V, Rahner C, Hagenbuch B, Stieger B, Kamisako T, OATPs. Since the sensitivities of endogenous biomarkers and i.v. and Meier PJ (1999) Localization and function of the organic anion-transporting polypeptide administered probe drugs to rifampicin administration were comparable, Oatp2 in rat liver. Gastroenterology 117:688–695. Sakaguchi M, Sakai T, Adachi Y, Kawashima T, and Awata N (1992) The biological fate of simultaneous measurement of a panel of these endogenous compounds sodium prasterone sulfate after vaginal administration. I. Absorption and excretion in rats. J at an early stage of drug development might be a useful tool to predict Pharmacobiodyn 15:67–73. Shen H, Dai J, Liu T, Cheng Y, Chen W, Freeden C, Zhang Y, Humphreys WG, Marathe P, possible OATP-based DDIs. and Lai Y (2016) Coproporphyrins I and III as functional markers of OATP1B activity: In vitro and in vivo evaluation in preclinical species. J Pharmacol Exp Ther 357:382–393. Faculty of Pharmaceutical Sciences, KEI NISHIZAWA Shitara Y, Maeda K, Ikejiri K, Yoshida K, Horie T, and Sugiyama Y (2013) Clinical significance of Institute of Medical, Pharmaceutical TAKEO NAKANISHI organic anion transporting polypeptides (OATPs) in drug disposition: their roles in hepatic clearance and intestinal absorption. Biopharm Drug Dispos 34:45–78. and Health Sciences University, IKUMI TAMAI Stanczyk FZ (2006) Diagnosis of hyperandrogenism: Biochemical criteria. Best Pract Res Clin Kanazawa, Japan Endocrinol Metab 20:177–191. Tamai I, Nezu J, Uchino H, Sai Y, Oku A, Shimane M, and Tsuji A (2000) Molecular identification and characterization of novel members of the human organic anion transporter (OATP) family. Authorship Contributions Biochem Biophys Res Commun 273:251–260. Participated in research design: Nishizawa, Tamai. Urbanski HF, Mattison JA, Roth GS, and Ingram DK (2013) Dehydroepiandrosterone sulfate (DHEAS) as an endocrine marker of aging in calorie restriction studies. Exp Gerontol 48:1136–1139. Conducted experiments: Nishizawa, Nakanishi. Watanabe M, Watanabe T, Yabuki M, and Tamai I (2015a) Dehydroepiandrosterone sulfate, a useful Performed data analysis: Nishizawa, Tamai. endogenous probe for evaluation of drug-drug interaction on hepatic organic anion transporting – Contributed to the writing of the manuscript: Nishizawa, Nakanishi, Tamai. polypeptide (OATP) in cynomolgus monkeys. Drug Metab Pharmacokinet 30:198 204. Watanabe T, Miyake M, Shimizu T, Kamezawa M, Masutomi N, Shimura T, and Ohashi R (2015b) Utility of bilirubins and bile acids as endogenous biomarkers for the inhibition of hepatic – References transporters. Drug Metab Dispos 43:459 466. Yoshida K, Maeda K, and Sugiyama Y (2012) Transporter-mediated drug-drug interactions involving Chu X, Shih S-J, Shaw R, Hentze H, Chan GH, Owens K, Wang S, Cai X, Newton D, Castro-Perez OATP substrates: Predictions based on in vitro inhibition studies. Clin Pharmacol Ther 91:1053–1064. J, et al. (2015) Evaluation of cynomolgus monkeys for the identification of endogenous biomarkers for hepatic transporter inhibition and as a translatable model to predict pharmacokinetic interactions with statins in humans. Drug Metab Dispos 43:851–863. Address correspondence to: Dr. Ikumi Tamai, Faculty of Pharmaceutical Davies B and Morris T (1993) Physiological parameters in laboratory animals and humans. Pharm Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa – Res 10:1093 1095. University, Kakuma-machi, Kanazawa 920-1192, Japan. E-mail: tamai@ Eckhardt U, Schroeder A, Stieger B, Höchli M, Landmann L, Tynes R, Meier PJ, and Hagenbuch B (1999) Polyspecific substrate uptake by the hepatic organic anion transporter Oatp1 in stably p.kanazawa-u.ac.jp transfected CHO cells. Am J Physiol 276:G1037–G1042. 1

Supplemental data

Drug Metabolism & Disposition

(Short Communication)

Title:

Comparative evaluation of dehydroepiandrosterone sulfate

(DHEAS) potential to predict hepatic OATP transporter-based

drug-drug interactions

Authors:

Kei Nishizawa, Takeo Nakanishi, Ikumi Tamai1

Affiliation: Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan (K.N., T.N., I.T)

Supplemental Figure 1

[3H]DHEAS uptake by freshly isolated rat hepatocytes in the presence of increasing concentrations of rifampicin

Method Rat hepatocytes were isolated according to the collagenase perfusion method as described previously (Tamai and Tsuji, 1987). After pre-incubation of isolated hepatocytes for 5 min at 37˚C with Krebs-Henseleit buffer (KHB: 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.3 mM CaCl2, 25 mM NaHCO3, 20 mM HEPES), the reaction was started by mixing an equal volume of KHB containing substrate with or without inhibitor. Final cell and substrate concentrations were 2×105 cells/mL and 2 nM (0.2 µCi/mL), respectively. The inhibitory effect of rifampicin was assessed in terms of the uptake for 20 sec (uptake activity was confirmed to increase linearly up to 20 sec). To terminate the reaction, 100 µL of reaction mixture was transferred into a micro centrifuge tube containing 100 µL silicone layer (silicone oil: liquid paraffin = 3:10) and 100 µL 2 N NaOH, and the samples were stored at room temperature overnight to dissolve the cells. Then, the centrifuge tubes was cut at the boundary of the silicone layer, and the cellular radioactivity was measured with a liquid scintillation counter (LCS-6100; Hitachi Aloka Medical, Tokyo, Japan) after addition of 100 µL 2 N HCl for neutralization and 1 mL Clear-sol scintillation fluid (Nacalai Tesque, Kyoto, Japan). The relative uptake of [3H]DHEAS in the presence of increasing concentrations of 3

rifampicin [I] is given by the following equation, where IC50 is the 50% inhibitory concentration of rifampicin on DHEAS uptake by isolated rat hepatocytes.

100 × IC % of control uptake = 50 IC50+[I]

The value of IC50 was estimated by means of nonlinear least-squares analysis using the MULTI program (Yamaoka et al., 1981), and was obtained as 3.65 M. Protein concentration was determined with a protein assay kit (Bio-Rad Richmond, CA, USA) according to the manufacturer’s instructions. The bovine serum albumin provided in the kit was used as a standard.

References: Tamai I, Tsuji A (1987) Transport mechanism of cephalexin in isolated hepatocytes. J Pharmacobiodyn 10:632-638. Yamaoka K, Tanigawara Y, Nakagawa T, Uno T (1981) A pharmacokinetic analysis program (multi) for microcomputer. J Pharmacobiodyn 4:879-885.

Supplemental Table 1

This table summarizes the values plotted in Fig. 2, which were obtained from the cited sources. Reference numbers in Figure 2 and Supplemental Table 1 refer to the following references.

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