Comparative evaluation of dehydroepiandrosterone sulfate (DHEAS)

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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 Tamai

Affiliation:

Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences,

Kanazawa University, Kanazawa, Japan (K.N., T.N., I.T)

DMD # 72355This article has not been copyedited and formatted. The final version may differ from this version.

DMD Fast Forward. Published on December 1, 2016 as DOI: 10.1124/dmd.116.072355 at A

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Running Title:

DHEAS as a biomarker for OATP-based DDI

Corresponding author:

Ikumi Tamai, Ph.D.

Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health

Sciences,

Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.

Telephone: +-81-76-234-4479, FAX: +81-76-76-264-6284

E-mail: [email protected]

The number of:

The number of text pages, 28

The number of tables, 1

The number of figures, 2

The number of references, 21

The number of words of;

Abstract, 250

Introduction, 603



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Results and Discussion, 1220

Abbreviations:

AUC, area under the plasma concentration curve; AUCR, AUC ratio; CL, clearance; DDI,

drug-drug interaction; DHEAS, dehydroepiandrosterone sulfate; Kp, tissue-to-plasma

concentration ratio; Vdss, steady-state volume of distribution; OATP, organic anion

transporting polypeptide;



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Abstract

Pharmacokinetic drug-drug interactions (DDIs) on hepatic organic anion transporting

polypeptides (OATPs) are important clinical issues. Previously we reported that plasma

dehydroepiandrosterone sulfate (DHEAS) could serve as an endogenous probe to predict

OATP-based DDIs in monkeys using rifampicin as an OATP inhibitor. However, since the

contribution of hepatic OATPs to the changes of plasma DHEAS by rifampicin remains

unclear, here, we evaluated by an in vivo pharmacokinetic study. Since plasma DHEAS

concentrations were unexpectedly low in our rat model, disposition of externally administered

DHEAS was evaluated. Intravenously administered DHEAS was mainly recovered in bile

(29.1 %) and less in urine (2.95 %). The liver tissue to plasma concentration ratio (Kpliver)

decreased from 41.8 to 5.07 by rifampicin, and this decrement was consistent with the

decrease of distribution volume from 247 to 59 mL/rat. Comparison of in vitro IC50 of

rifampicin for DHEAS uptake by isolated rat hepatocytes and in vivo plasma rifampicin

concentration suggested that rifampicin effect on the plasma DHEAS concentration was

mostly explained by inhibition of hepatic OATPs, demonstrating that DHEAS could be a

biomarker of hepatic OATP activity. Next, previously reported rifampicin-induced changes of

plasma concentrations evaluated as an AUC ratio (AUCR) of possible probe compounds were

compared on the basis of rifampicin dose/body surface area. The AUCR values of endogenous

compounds and intravenously administered statins, for which possible DDIs in the intestinal



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absorption process can be excluded, increased proportionally to rifampicin dose.

Simultaneous measurement of these endogenous compounds could be effective biomarkers

for prediction of OATP-based DDIs.



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Introduction

Membrane transporters are involved in absorption and disposition of many drugs, and

alterations of their functional activities may lead to decreased efficacy and/or adverse events.

Such alterations can occur as a result of drug-drug (DDI) and drug-food interactions (DFI)

with concurrently administered drugs or foods (Shitara et al., 2013; Nakanishi and Tamai,

2015). Regulatory agencies, such as the US Food and Drug Administration (FDA), European

Medicines Agency (EMA) and Japanese Pharmaceuticals and Medical Devices Agency

(PMDA), have proposed that clinical DDI studies should be required for drugs under

development, and thus there is a need to develop convenient ways to predict DDI and DFI

potential. Organic anion transporting polypeptides (OATPs) contribute to drug absorption and

disposition (Tamai et al., 2000; Shitara et al., 2013). For example, OATPs expressed in the

liver take up various drugs into hepatocytes from the systemic circulation, thereby affecting

systemic and liver exposures to these drugs (König et al, 2011; Shitara et al., 2013).

OATP1B1 and OATP1B3 are key molecules in the hepatic handling of drugs, and accurate

predictions of clinically significant DDIs on those OATPs are essential. Several probe drugs

to evaluate possible inhibitors of hepatic OATPs have been suggested, e.g., with statins as the

“victims” (Yoshida et al. 2012; Prueksaritanont et al., 2014). Although those probe drugs are

useful, endogenous probe compounds might be advantageous, since in vivo DDI could be



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examined at an early stage of drug development without the need for additional studies

involving administration of probe drugs.

Several endogenous compounds, including bilirubin, bile acids and coproporphyrins, have

been proposed as biomarkers to monitor DDIs on hepatic OATPs (Chu et al., 2015; Watanabe

T et al., 2015; Shen et al., 2016, Lai et al., 2016). In addition, we recently reported that

dehydroepiandrosterone sulfate (DHEAS) served as a biomarker to reflect DDI on hepatic

OATPs when cynomolgus monkeys were administered an inhibitor of OATPs (Watanabe M et

al., 2015). Although the observed increase of plasma concentration of DHEAS by rifampicin

was less than that by statins, plasma DHEAS was dose-dependently increased by rifampicin.

Despite the apparent differences in sensitivity for detecting the effect of rifampicin among

these putative biomarkers, they might be broadly comparable when the dose of rifampicin in

each study is taken into account, in spite of the difference in species (Nakakariya et al., 2008).

However, it is not yet clear whether rifampicin affects only hepatic OATPs, or whether other

transporters and metabolic enzymes might contribute to the observed alterations.

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 physiological changes

(Stanczyk 2006; Urbanski et al., 2013; Goodarzi et al., 2015). But, since several factors other

than OATPs may affect plasma DHEAS, as well as other potential biomarkers, it is essential

to establish that the rifampicin-induced increase of plasma DHEAS is at least predominantly



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due to interaction on hepatic uptake transporters, OATPs, in order to confirm the suitability of

DHEAS as a possible biomarker. In the present study, therefore, we evaluated the in vivo

contribution of hepatic OATPs to the rifampicin-induced increase of plasma concentration of

DHEAS by measuring biliary and urinary excretions, tissue concentrations and

pharmacokinetic parameters with and without rifampicin. Since endogenous plasma

concentration of DHEAS in the present rat model was unexpectedly low to detect by our

LC-MS/MS method (limit of detection: 0.5 nM), we evaluated the contribution of hepatic

Oatps to the changes in DHEAS disposition by rifampicin by externally administering

DHEAS. Furthermore, the usefulness of DHEAS as a biomarker was compared with that of

other proposed markers reported in the literature.



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Materials and methods

Chemicals.

Rifampicin, sodium dehydroepiandrosterone-3-sulfate (DHEAS) and

dehydroepiandrosterone-d5-3-sulfate sodium salt were purchased from Wako Pure Chemical

Industries (Osaka, Japan), Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and Sigma

Aldrich (St. Louis, MO), respectively. [3H]Dehydroepiandrosterone sulfate (2.22-3.70

TBq/mmol) sodium salt was purchased from PerkinElmer (Boston, MA). Other reagents and

solvents were of analytical grade.

Animals.

Seven-week-old female Wistar rats (170-190 g) were purchased from Sankyo Labo Service

(Tokyo, Japan). All animal studies were approved by the Committee of Kanazawa University

for the Care and Use of Laboratory Animals and were performed in accordance with its

guidelines (AP-143148).

In vivo animal study.

Rats were anesthetized with pentobarbital, and the 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

intravenously at 1 hr after intravenous administration of 30 mg/kg rifampicin. DHEAS and



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rifampicin were administered via femoral vein. Then, blood was drawn from jugular vein and

plasma was generated by centrifugation at 3,000 rpm for 10 minutes at 4˚C. Urine and bile

were collected at the designated times. Rats were sacrificed 4 hr after administration of

DHEAS by cutting the inferior vena cava under deep anesthesia, and kidney and liver were

isolated for measurement of DHEAS. All samples were stored at -30˚C until measurement.

Quantitation of DHEAS in plasma, urine, bile, and tissue samples.

DHEAS concentrations were measured by LC-MS/MS. Liver and kidney were excised,

weighed, and homogenized with five volumes of methanol. Each sample was left on ice for

thirty minutes and then centrifuged (14,000 rpm, 5 min, 4˚C). The resulting supernatant was

stored until measurement. Aliquots (5 μL) of plasma, urine, bile, and tissue sample extract

were added to 495 μL of MeOH containing internal standard (1 μg/mL DHEAS-d5) and

centrifuged (14,000 rpm, 5 min, 4˚C). Then, a 10 μL aliquot of the resulting supernatant was

subjected to LC-MS/MS. The LC-MS/MS system consisted of a triple quadrupole mass

spectrometer (API 3200TM, AB Sciex, Foster City, CA) coupled with an ultra-fast liquid

chromatography system (LC-20AD, Shimadzu Co., Kyoto, Japan). Chromatography was

performed using a Mercury MS analytical column (C18, 10 × 4.0 mm, 5 μm, Phenomenex,

Torrance, CA) at 40˚C with a mobile phase of 10 mM ammonium formate (A) and acetonitrile

(B). The flow rate was 0.3 mL/min, and gradient conditions for elution were as follows:



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10-90 % B (0-3 min), 90 % B (3-4 min), 90 -10 % B (4-4.1 min), 10% (4.1-6 min). Mass

spectrometry was performed with negative ionization, and mass transitions (Q1/Q3) of m/z

367.1/96.9 and 372.3/98.1 were used for DHEAS and d5-DHEAS, respectively. The limit of

quantitation for DHEAS was 0.5 nM.

Quantitation of rifampicin in plasma samples.

Plasma concentration of rifampicin was measured by HPLC. Extraction of rifampicin from

plasma was performed by adding an equal volume of methanol contained 10 µM

methoronidazole as an internal standard. The mixture was vortexed and centrifuged (14,000

rpm, 5 min, 4˚C), and the resultant supernatant was subjected to HPLC. The HPLC system

(Waters Corporation, Milford, MA) was equipped with a UV absorbance detector (Waters

2487/2690). A solvent delivery system (Waters 2695) was used to obtain isocratic flow of the

mobile phase. Chromatography was performed using Mightysil RP-18GP Aqua analytical

column (4.6 mm × 250 mm i.d., 5 μm particle size; Kanto Chemical, Tokyo, Japan)

prewarmed at 40˚C. The mobile phase was a mixture of 10 mM phosphate buffer adjusted to

pH 3.0 and methanol (40:60). The delivery system was used to obtain isocratic flow of the

mobile phase at a rate of 1.0 mL/min.

Pharmacokinetic analysis.



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The plasma concentration-time data were analyzed by non-compartmental analysis. The area

under the plasma concentration time curve (AUC0-4) was obtained by the trapezoidal rule

from time 0 to 4 hr. AUC from 0 to infinity (AUCinf) was estimated by extrapolation to

infinity. Total clearance (CLtot) was estimated as dose (D) over AUCinf (D/AUCinf), and renal

clearance (CLurine) and biliary excretion clearance (CLbile) were estimated as Xurine,0-4/AUC0-4

and Xbile,0-4/AUC0-4, where Xurine,0-4 and Xbile,0-4 represent cumulative amounts of DHEAS

recovered in urine and bile, respectively. Biliary excretion clearance based on liver tissue

concentration (CLbile,liver) was estimated as Xbile,0-4/(Kpliver × AUC0-4). The apparent volume of

distribution (Vdss) was estimated as Vdss = CLtot × MRT, where MRT is the mean residence

time. The apparent tissue to plasma concentration ratio (Kpliver and Kpkidney) was estimated as

the ratio of liver or kidney concentration divided by plasma concentration at 4 hr.

Statistical analysis.

Student’s t test was used to analyze differences between groups. P < 0.05 was considered

statistically significant.



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Results and Discussion

Effect of rifampicin on pharmacokinetics of DHEAS.

Endogenous plasma concentration of DHEAS in rats was under the detection limit of our

LC-MS/MS method. Accordingly, in the present study DHEAS disposition was evaluated

after intravenous administration of DHEAS. The plasma concentration-time curves of

DHEAS with and without intravenous rifampicin (30 mg/kg) 1 hr prior to DHEAS

administration are shown in Fig. 1B. The dose of DHEAS was set at 4 mg/kg, considering the

detection limit during pharmacokinetic analysis (Sakaguchi et al., 1992). At the same time,

cumulative excretions of DHEAS into bile (Fig. 1C) and urine (Fig. 1D) and tissue

concentrations in kidney and liver were measured. The plasma concentration of DHEAS

decreased bi-exponentially. All parameters were evaluated from the observations up to 4 hr

and the results are summarized in Table 1. AUC0-4 was increased from 10.9 ± 1.85 to 46.0 ±

8.46 μmol･hr/L by coadministration of rifampicin, and AUCinf was similarly increased. The

sum of the biliary and urinary recoveries of DHEAS up to 4 hr was about one-third of the

dose, but biliary excretion was 10 times higher than urinary excretion, indicating that DHEAS

is predominantly excreted into bile. Biliary excretion of DHEAS was significantly decreased

from 29.1 ± 9.73 to 13.4 ± 1.57 % of dose by rifampicin, whereas no significant change was

observed in urinary excretion (2.95 ± 0.63 vs. 2.38 ± 1.30 % of dose). These observations

indicated that rifampicin markedly affects biliary excretion of DHEAS. Although there is



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statistically significant decrease of CLurine by rifampicin (from 5.46 ± 1.48 to 1.09 ± 1.22

mL/hr/rat), its contribution to change in plasma concentration of DHEAS should be small,

since urinary excretion is much less than biliary excretion. DHEAS is accumulated in liver:

Kpliver was 41.8 ± 5.67 in the case of DHEAS alone, whereas Kpkidney was less than unity

(0.93 ± 0.55). In the presence of rifampicin, Kpliver was significantly decreased to 5.07 ± 1.96,

whereas Kpkidnay was unchanged (0.67 ± 0.27). Furthermore, Vdss was decreased from 247 ±

75.5 to 59.4 ± 10.1 mL/rat by rifampicin. Considering the liver volume (10 g/0.25 kg rat)

(Davies and Morris, 1993), the change of Kpliver by rifampicin is expected to cause a decrease

of Vdss of more than 200 mL. Thus, the apparent change of Vdss (188 mL) can be well

explained by the decrease of DHEAS distribution to liver.

CLbile was significantly decreased from 53.9 ± 18.4 to 6.71 ± 1.22 mL/hr/rat by rifampicin,

while CLbile,liver was unchanged by rifampicin (2.16 ± 1.98 vs 3.02 ± 4.24 mL/hr/rat). Taking

these changes in the pharmacokinetic parameters together, it is considered that the change in

the hepatic disposition of DHEAS caused by rifampicin is mainly due to a decrease of hepatic

uptake from blood, but not a decrease of biliary excretion from liver. Furthermore, changes in

systemic disposition can be mostly explained by the changes in hepatic disposition.

Uptake of DHEAS by isolated rat hepatocytes.



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Since hepatic uptake of DHEAS appeared to be significantly affected by rifampicin in vivo,

we next measured DHEAS uptake by isolated hepatocytes in vitro. Initial uptake of

[3H]DHEAS (2 nM) up to 40 sec was decreased by rifampicin in a concentration-dependent

manner (Supplemental Fig. 1). By subtracting the basal uptake obtained in the presence of

100 μM rifampicin, IC50 of rifampicin was estimated to be 3.65 μM in the present study,

which is comparable with the reported Ki values for rat Oatps of between 1 and 10 μM

(Fattinger et al., 2000).

Figure 1A shows the time course of plasma rifampicin concentration after intravenous

administration to rats. Rifampicin concentration remained higher than 15 μM during the

measurement of DHEAS disposition. Since plasma protein binding of rifampicin was reported

to 75% (Imaoka et al., 2013), the plasma unbound concentration of rifampicin should have

been higher than IC50 throughout. Although it was not confirmed in the present study, DHEAS

uptake by rat hepatocytes can be accounted for by the activities of rat Oatp1a1 and 1a4

(Eckhardt et al., 1999; Reichel et al., 1999). Therefore, the observed increase of systemic

exposure to DHEAS in the presence of rifampicin appears to be due to decreased hepatic

uptake resulting from the inhibition of hepatic Oatp transporters. Further study using

Oatp-knockout animals should be conducted for evaluation of the suggested mechanisms of

changes in DHEAS disposition in future.



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Comparison of DHEAS with other probes to monitor hepatic OATP-based DDI.

Several endogenous compounds have been proposed as biomarkers to evaluate hepatic

OATP-based DDIs, including bilirubin and its conjugated metabolites, bile acids, and

coproporphyrins (Watanabe T et al., 2015; Chu et al., 2015; Shen et al., 2016; Lai et al.,

2016). Statins have also been suggested as useful exogenous probes for this purpose (Yoshida

et al., 2012; Prueksaritanont et al., 2014). Therefore, we compared the sensitivity of DHEAS

with those of other 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 the reported AUCRs after standardization of

rifampicin dose by body surface area in rats, monkeys and humans (see Supplemental Table 1

for details). Although the AUCRs tended to increase with dose of rifampicin, there was not a

clear correlation. One reason for this may be that when probe compounds are orally

administered, the intestinal bioavailability as well as hepatic disposition must be considered

(Yoshida et al., 2012). However, for the present purpose, it would be desirable to eliminate

the influence of intestinal availability, since endogenous compounds do not include intestinal

absorption process. Accordingly, the correlation was separately analyzed for the oral

administration group (green-colored symbols) and for the group consisting of intravenously

administered statins (blue-colored symbols) and endogenous compounds (red- and

magenta-colored symbols). Generally, the former group showed higher AUCRs than the latter,



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but the AUCRs of the latter group mostly increased proportionally to rifampicin dose. These

results confirm that AUCR of orally administered statins includes the effects of interactions

on both intestinal absorption and hepatic OATPs, suggesting that hepatic OATP-based DDI

might be overestimated with these markers. On the other hand, the endogenous compounds

and intravenously administered statins are similarly effective for predicting apparent DDI on

hepatic OATP transporters. Among the putative biomarkers, coproporphyrins in humans may

show higher sensitivities than the others (G-13, H-10 and H-13 in Fig. 2). However, in

practice, all of the endogenous compounds could be measured both before and after

administration of test compounds, which makes the changes of their concentrations clearer.

Accordingly, all of suggested endogenous markers could be used as biomarkers for

OATP-based DDI. Furthermore, since test drugs as perpetrators may affect mechanisms other

than OATPs that regulate disposition of the suggested biomarkers, it may be desirable to

simultaneously measure a panel of biomarkers, which would presumably have a variety of

alternative disposition mechanisms, but commonly taken up by liver via OATPs .

In conclusion, our results indicate that the increase in the plasma concentration of DHEAS in

the presence of rifampicin can be largely explained by interaction in the process of hepatic

uptake, specifically at OATPs. Since the sensitivities of endogenous biomarkers and

intravenously administered probe drugs to rifampicin administration were comparable,



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simultaneous measurement of a panel of these endogenous compounds at an early stage of

drug development might be a useful tool to predict possible OATP-based DDIs.



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Authorship Contributions

Participated in research design: Kei Nishizawa, Ikumi Tamai

Conducted experiments; Kei Nishizawa, Takeo Nakanishi

Performed data analysis: Kei Nishizawa, Ikumi Tamai

Contributed to the writing of the manuscript: Kei Nishizawa, Takeo Nakanishi, Ikumi Tamai



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Footnote:

This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the

Promotion of Science [16H50111].



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Figure Legends

Fig. 1. Time courses of plasma DHEAS and rifampicin concentrations, and biliary and

urinary excretion of intravenously administered DHEAS and rifampicin.

After intravenous administration of DHEAS (4 mg/kg) with and without intravenous

administration of rifampicin (30 mg/kg, 1 hr before), plasma concentrations of rifampicin (A)

and DHEAS (B) and cumulative excretion of DHEAS in bile (C) and urine (D) were

measured up to 4 hr. Open and closed symbols represent the results with and without

rifampicin in (B), (C) and (D), respectively. Each result represents the mean ± S.D. (n = 3 - 8)

and (*) indicates a significant difference from the corresponding value without rifampicin (p <

0.05).

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 is represented present study data). Symbols in blue, green, and red or magenta indicate



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that the compounds of interest are intravenously administered statins, orally administered

statins, and endogenous substances co-administered with oral or intravenous rifampicin,

respectively. These values and cited references are listed in Supplemental Table 1.



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Table 1

Pharmacokinetic parameters of i.v. DHEAS with and without rifampicin in rats.

Parameter Unit DHEAS alone DHEAS

+Rifampicin

AUC0-4 μM・hr 10.9 ± 1.85 46.0 ± 8.46�

AUCinf 11.4 ± 2.08 48.0 ± 8.61�

CLtot

mL/hr/rat

174 ± 37.2 43.5 ± 9.82�

CLurine 5.46 ± 1.48 1.09 ± 0.72�

CLbile 53.9 ± 18.4 6.71 ± 1.22�

CLbile,liver 2.16 ± 1.98 3.02 ± 4.24

Xurine,0-4 µg 13.1 ± 10.4 16.98 ± 10.8

Xbile,0-4 213 ± 78.0 111 ± 10.0�

Urinary recovery % of dose 2.95 ± 0.63 2.38 ± 1.30

Biliary recovery 29.1 ± 9.73 13.4 ± 1.57�

Vdss mL/rat 247 ± 75.5 59.4 ± 10.1�

Kpliver 41.8 ± 5.67 5.07 ± 1.96�

Kpkidney 0.93 ± 0.55 0.67 ± 0.27

Rat body weight g 182 ± 11.1 186 ± 6.32



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Pharmacokinetic parameters of DHEAS after administration at 4 mg/kg i.v. with and without rifampicin (30

mg/kg, i.v. dose at 1 hr prior to DHEAS administration). Data are represented as mean ± S.D. (n=4-5);

(*)P<0.05, statistically significant different from DHEAS alone.



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This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on December 1, 2016 as DOI: 10.1124/dmd.116.072355

at ASPE

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This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on December 1, 2016 as DOI: 10.1124/dmd.116.072355

at ASPE

T Journals on A

pril 14, 2018dm

d.aspetjournals.orgD

ownloaded from


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