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In Vivo Assessment of the Impact of Efflux Transporter on Oral Drug

Absorption using Portal Vein Cannulated Rats

Yoshiki Matsuda Yoshihiro Konno Takashi Hashimoto Mika Nagai Takayuki Taguchi

Masahiro Satsukawa and Shinji Yamashita

Pharmacokinetics and Safety Research Department Central Research Laboratories

Kaken Pharmaceutical Co Ltd Kyoto Japan (YM YK TH MN TT MS) and

Faculty of Pharmaceutical Science Setsunan University Osaka Japan (SY)

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Running title

Assessment of the impact of intestinal efflux transporter

Number of text page 42

Number of tables 3

Number of figures 6

Number of references 38

Number of words in Abstract 250

Number of words in Introduction 833

Number of words in Discussion 1192

All correspondence to Yoshiki Matsuda

Pharmacokinetics and Safety Research Department

Central Research Laboratories Kaken Pharmaceutical Co Ltd

14 Shinomiya Minamigawara-cho

Yamashina-ku Kyoto 607-8042 Japan

Telephone 81-75-594-0787 Facsimile 81-75-594-0790

E-mail matsuda_yoshikikakencojp

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Abbreviations FEX fexofenadine SASP sulfasalazine TPT topotecan ZSQ zosuquidar

P-gp P-glycoprotein BCRP breast cancer resistance protein BBB blood-brain barrier GI

gastrointestinal F bioavailability FaFg intestinal availability Fh hepatic availability Rb

blood plasma concentration ratio Qpor portal blood flow Qh hepatic blood flow IS

internal standard LC liquid chromatography MSMS tandem mass spectrometry AUC

area under the concentration-time curve

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Abstract

The purpose of this study was to evaluate the impact of intestinal efflux transporters on

the in vivo oral absorption process Three model drugs (fexofenadine (FEX)

sulfasalazine (SASP) and topotecan (TPT)) were selected as P-glycoprotein (P-gp)

breast cancer resistance protein (BCRP) and P-gp and BCRP substrates respectively

The drugs were orally administered to portal vein cannulated rats after pretreatment

with zosuquidar (ZSQ) P-gp inhibitor andor Ko143 BCRP inhibitor Intestinal

availability (FamiddotFg) of the drugs was calculated from the difference between portal and

systemic plasma concentrations When rats were orally pretreated with ZSQ FamiddotFg of

FEX increased 4-fold and systemic clearance decreased to 75 of the control In

contrast intravenous pretreatment with ZSQ did not affect FamiddotFg of FEX although

systemic clearance decreased significantly These data clearly show that the method

presented herein using portal vein cannulated rats can evaluate the effects of intestinal

transporters on FamiddotFg of drugs independently of variable systemic clearance In addition

it was revealed that 71 of FEX taken up into enterocytes underwent selective efflux

via P-gp to the apical surface while 79 of SASP was effluxed by Bcrp In the case of

TPT both transporters were involved in its oral absorption Quantitative analysis

indicated a 35-fold higher contribution from Bcrp than P-gp In conclusion the use of

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portal vein cannulated rats enabled the assessment of the impact of efflux transporters

on intestinal absorption of model drugs This experimental system is useful for

clarifying the cause of low bioavailability of various drugs

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Introduction

P-glycoprotein (P-gpABCB1) and breast cancer resistance protein (BCRPABCG2)

both members of the ATP-binding cassette (ABC) transporter family are expressed at

the apical membrane of various polarized cells such as intestinal enterocytes

hepatocytes renal epithelial cells as well as the blood-brain barrier (BBB) Both P-gp

and BCRP exhibit broad substrate specificity potentially resulting in limited

gastrointestinal absorption or brain penetration and increased renal or hepatic excretion

of various drugs via their transport back to the apical surface (Thiebaut et al 1987 and

Schinkel et al 2003)

In the drug discovery stage new chemical entities (NCEs) often suffer from poor

systemic exposure due to the limited gastrointestinal absorption via these efflux

transporters Whether NCEs are subject to active efflux by one or both transporters can

be evaluated by using Caco-2 cell lines andor Madin-Darby canine kidney (MDCK)

cell lines transfected with individual efflux transporter genes (Troutman et al 2003)

However it is difficult to predict the intestinal availability (FamiddotFg) of NCEs from those

in vitro experiments because the in vivo absorption process from the GI tract is

restricted not only by intestinal efflux transporters but also other factors including

solubility membrane permeability or metabolism In addition a method to

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quantitatively assess the impact of these transporters on the in vivo absorption process

has not been fully established

To investigate the effects of intestinal efflux transporters on oral drug absorption

co-administration studies with transporter inhibitors are often carried out in vivo using

experimental animals (Bardelmeijer et al 2004 and Takeuchi et al 2008) In this

approach to calculate oral bioavailability (F) not only oral administration but also

intravenous injection study should be performed Then FamiddotFg is obtained by dividing

oral F by hepatic availability (Fh) (Kato et al 2003) where the renal clearance should

be estimated to calculate Fh If the systemic clearance of the test compound is

significantly affected by oral pretreatment with the transporter inhibitor intravenous

studies should be conducted at each inhibitor dose

Transporter gene knockout mice and rats are also used to assess the effects of

transporters (Chen et al 2009 and Zamek-Gliszczynski et al 2012) however a general

concern in the use of knockout animals for pharmacokinetic studies is the potential

compensatory effects from up- or down- regulation of other transporters and drug

metabolism-related genes Alteration of mRNA levels of several transporter and

metabolism-related genes was reported in Abcb1 and Abcg2 knockout mice and rats

(Cisternino et al 2004 and Chu et al 2012) On the other hand Agarwal et al (2012)

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have reported there was no significant difference in the expression of P-gp between the

wild type and Abcg2 knockout mice in the quantitative proteomics In order to use gene

knockout animals for pharmacokinetic study this kind of quantitative analysis on the

expression of other transporters or enzymes should be necessary

In our previous report we demonstrated the usefulness of portal vein cannulated rats

in evaluating FamiddotFg of orally administered drugs Using portal vein cannulated rats by

monitoring both portal and systemic blood concentrations of the drug FamiddotFg can be

calculated from a single oral dosing study without the need for intravenous injection

(Matsuda et al 2012) Calculation of FamiddotFg using portal vein cannulated rats shows less

variability than conducting typical kinetic analyses because our calculation method is

less sensitive to inter-individual fluctuation in portal blood flow In addition since

systemic clearance renal clearance and Fh are not necessary to calculate FamiddotFg our

method is considered to be highly applicable to transporter inhibition studies

In this study the impact of intestinal efflux transporters on oral absorption of 3 model

drugs fexofenadine (FEX) sulfasalazine (SASP) and topotecan (TPT) was evaluated

FEX a non-sedating histamine H1 receptor antagonist is well-known as a substrate for

OATP1A2 and OATP2B1 as well as P-gp (Cvetkovic et al 1999) Following oral

administration of FEX a majority of the dose is recovered in the urine and feces as an

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unchanged form Kalgutkar et al (2009) reported that systemic exposure of FEX after

oral dosing was dramatically increased by P-gp inhibition in rats SASP an

anti-inflammatory drug shows low intestinal absorption due to low solubility and

permeability The bioavailability of SASP in Abcg2 deficient mice and rats was 9- and

17-fold higher respectively than that of wild-type (Zaher et al 2006 and Huang et al

2012) In addition co-administration of SASP and curcumin a BCRP inhibitor

increased SASP exposure 3-fold in humans (Kusuhara et al 2012) suggesting that

BCRP-mediated efflux limits intestinal absorption of SASP The anti-cancer drug TPT is

reported to be a good substrate for BCRP and a weaker substrate for P-gp (Hendricks et

al 1992 and Maliepaard et al 1999) Uptake of [11C]TPT in the brains of

Mdr1ab--Bcrp1-- mice was about two times higher than in wild-type mice Similarly

brain penetration of [11C]TPT increased in mice by treatment with elacridar a P-gp and

Bcrp dual inhibitor (Yamasaki et al 2010)

Zosuquidar (ZSQ) and Ko143 are used as selective inhibitors of P-gp and BCRP

ZSQ is reported to be an extremely potent P-gp inhibitor and does not modulate

BCRP-mediated resistance (Shepard et al 2003) while Ko143 is also well-known as a

potent BCRP inhibitor (Allen et al 2002)

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Materials and Methods

Materials Topotecan (TPT) was purchased from LKT Laboratories (St Paul MN

USA) and ketoconazole fexofenadine (FEX) and sulfasalazine (SASP) were

purchased from Sigma-Aldrich (St Louis MO USA) Zosuquidar (ZSQ) was

purchased from Diverchim (Montataire France) and Ko143 was purchased from Enzo

Life Sciences (Farmingdale NY USA) All other chemicals used were reagent grade or

better

Animals All animal procedures were conducted under protocols approved by the

Kaken Institutional Animal Care and Use Committee Cannulated male

Sprague-Dawley rats (8 weeks old 260-300 g body weight) were purchased from

Charles River Laboratory Japan (Yokohama Japan) and were kept in an experimental

animal room with an ambient temperature of 22-24˚C and a 12-h light-dark cycle for 6

days before use The cannulated rats were shipped to our lab from Charles River

Laboratory Japan 2 days after the surgical procedure and arrived the next day The oral

and intravenous administration studies were conducted on the 9th day after the surgery

At 9th day there observed no significant differences in the physiological condition of

cannulated and untreated rats (Matsuda et al 2012)

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Surgical procedure for portal vein cannulation The surgical procedure for insertion

of catheter was used our reported techniques previously (Matsuda et al 2012)

Animals were implanted with catheters in the portal vein as follows Rats were

anaesthetized with ketamine (429 mgkg) and xylazine (82 mgkg) administered

intraperitoneally A mid-line incision 1-2 cm was made in the abdominal cavity and the

portal vein was detached near the liver To prevent bleeding the portal vein was ligated

temporarily as the catheter was inserted The catheter (35Fr polyurethane tube

Accesstrade technologies Inc) was inserted immediately and fixed by a purse-string suture

on the portal vein The time to reperfusion was about 1 min after intercepted blood flow

This method for insertion of catheter can avoid the occlusion of the vessel In addition a

catheter with trumpet-shaped opening was used to prevent the catheter from slipping out

of the vessel with minimizing the effect on blood flow Another end of the catheter was

passed subcutaneously to the dorsal base of the neck and the laparotomy was closed in

two layers with a 40 silk blade to the muscle and a surgical clip to close the skin

Surgical procedures were approved by the Institutional Animal Care and Use

Committee of Charles River Laboratory Japan This surgical procedure allows the

collection of blood samples without the necessity of restraints and anesthesia

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Blood Plasma concentration ratio The blood plasma concentration ratio (Rb) was

determined in vitro after incubation of 2 microL of methanol solution of test compounds

with 2 mL of fresh pooled blood including heparin Pooled blood was taken from 4

cannulated rats Blood was preincubated at 37˚C in a water bath and spiked with the

test compounds at 100 ngmL The blood samples were incubated at 37˚C for 15 min

After centrifugation at 14000 g for 10 min the plasma samples were transferred into 4

volumes of methanol containing ketoconazole (IS) and then centrifuged The

concentrations of test compounds in the supernatant were determined by liquid

chromatography tandem mass spectrometry (LC-MSMS)

Preparation of drug solution For oral administration studies each of the substrates

and inhibitors was suspended in aqueous 05 methyl cellulose as follows FEX 1

mgmL SASP 1 mgmL TPT 002 006 02 and 06 mgmL ZSQ 02 06 2 and 6

mgmL and Ko143 02 06 and 2 mgmL For intravenous administration study FEX

or ZSQ was dissolved in dimethyl sulfoxide (DMSO) then mixed with a solution

containing ethanol cremophor EL and saline (DMSO ethanol cremophor saline =

1 25 25 94) Final concentration of the drug was adjusted to 05 mgmL for FEX

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and 1 mgmL for ZSQ

Study design and drug administration 3 substrates (FEX SASP and TPT) and 2

inhibitors (ZSQ and Ko143) were used for the pharmacokinetics studies In the oral

administration study substrates and inhibitors were administered directly into stomach

using oral sonde without anesthesia Each substrate was orally administered to the

fasted rats (FEX 5 mgkg SASP 5 mgkg and TPT 03 mgkg) at 40 min after oral

administration of vehicle or inhibitors (ZSQ 30 mgkg andor Ko143 10 mgkg) For

dosing studies of TPT TPT was orally administered to the fasted rats at a dose of 01

03 1 and 3 mgkg For dosing studies of ZSQ and Ko143 ZSQ (1 3 10 and 30

mgkg) or Ko143 (1 3 and 10 mgkg) was orally administered to the rats 40 min prior

oral administration of TPT (03 mgkg) For the pharmacokinetic studies of FEX FEX

was orally administered to the rats (5 mgkg) 40 min after oral administration (30

mgkg) or 5 min after intravenous administration (2 mgkg) of ZSQ FEX (1 mgkg) was

intravenously administered to the rats 40 min after oral administration of ZSQ (30

mgkg) FEX (1 mgkg) and ZSQ (2 mgkg) were coadministered intravenously

Following administration blood samples were taken from the portal and caudal veins of

the unanesthetized rats at 0083 025 05 1 2 4 6 and 8 h under unrestricted

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conditions The plasma samples were separated by centrifugation at 14000 g for 10 min

at 4˚C and stored at -30˚C until use The compound concentrations in the plasma were

quantified using LC-MSMS

LC-MSMS analysis The LC-MSMS system consisted of a HTC PAL autosampler

(CTC Analytics Zwingen Switzerland) Accela HPLC and TSQ Ultra mass

spectrometer (Thermo Fisher Scientific San Jose CA) LC conditions were as follows

column CAPCELL PAK C18 ACR (15 mm ID times 35 mm 3 microm Shiseido Tokyo

Japan) YMC-Triart C18 (20 mm ID times 30 mm 3 microm YMC Kyoto Japan) column

temperature 40˚C gradient elution at 03 mLmin with methanol aqueous 01 formic

acid or methanol aqueous 1mM ammonium acetate and injection volume 15 microL The

main working parameters for mass spectrometers were as follows ion mode

electrospray ionization positive spray voltage 4000 V sheath gas pressure 30 Arb

auxiliary gas pressure 35 Arb capillary temperature 300˚C multireaction monitoring

method with transitions of mz 5023 rarr 4663 for FEX mz 3971 rarr 1971 for SASP

mz 4222 rarr 3772 for TPT and mz 5313 rarr 2439 for ketoconazole (IS) The lower

limit of determination was 02 or 1 ngmL and the linear detection range was up to 500

ngmL

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Pharmacokinetic analysis Noncompartmental pharmacokinetics were calculated using

Phoenix WinNonlin 61 (Pharsight Mountain View CA) for individual animals and

reported as the mean plusmn standard deviation of the group Intestinal availability (FamiddotFg)

was calculated using eq (1)

FamiddotFg = Qpor middot Rb middot (AUCpor - AUCsys) Dose (1)

where Qpor Rb AUCpor and AUCsys were the portal blood flow the blood plasma

concentration ratio AUC calculated from plasma concentration in the portal vein and

the systemic circulation respectively As Qpor the value of 329 mLminkg was used

that was calculated in our previous report by assuming that FamiddotFg of antipyrine was 1

(Matsuda et al 2012)

Statistics The presented values were all mean plusmn SD For comparison of control ZSQ

Ko143 and ZSQ+Ko143 groups the statistical significance of the difference between

mean values was calculated using analysis of variance (ANOVA) with Tukey-Kramer

test used for multiple comparisons For comparison between with and without inhibiters

in other inhibition test Dunnettrsquos test was used Differences with a p value of less than

005 were considered to be statistically significant

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Results

Effect of P-gp inhibition on pharmacokinetics of FEX after oral or intravenous

administration In order to evaluate the effect of P-gp inhibition on systemic clearance

of FEX pharmacokinetic studies on FEX were conducted First in order to observe the

effect of P-gp inhibition on the systemic clearance of FEX FEX (1 mgkg) was

intravenously injected with or without oral pre-administration of ZSQ (30 mgkg at 40

min before FEX injection) As shown in Table 1 the systemic clearance of FEX was

significantly lowered by ZSQ oral pretreatment (393 plusmn 40 mLminkg in control vs

286 plusmn 30 mLminkg in ZSQ po) Systemic clearance of FEX following intravenous

co-administration of ZSQ (2 mgkg) also decreased significantly (293 plusmn 19

mLminkg) compared with control Although the systemic clearance of FEX after oral

or intravenous pretreatment with ZSQ was nearly identical Fig 1 shows that FamiddotFg of

FEX increased 4-fold after oral but not intravenous pretreatment with ZSQ These data

indicate that the use of portal vein cannulated rats enables the assessment of intestinal

availability in the oral absorption process independently of variable systemic clearance

Intestinal availability of TPT in dose-dependent studies Portal vein cannulated rats

were orally administered TPT in a dose-dependent manner (01 03 1 and 3 mgkg) As

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shown in Fig 2 and Table 2 the AUCsys and AUCpor of TPT increased proportionally

with dose and a constant value for FamiddotFg was obtained at a dose of 01 03 1 and 3

mgkg Intestinal absorption of TPT was found to follow linear kinetics and intestinal

efflux transport involved in TPT absorption was unsaturated at these doses For the

following study TPT at a dose of 03 mgkg was orally administered to portal vein

cannulated rats 40 min after pretreatment with transporter inhibitors The concentration

of TPT in the drug solution was set to 006 mgmL the same dose concentration in

clinical use Because the recommended dose of TPT is 23 mgm2day in humans the

intestinal concentration of TPT (dose250 mL) is estimated as approximately 002

mgmL

Effects of P-gp and Bcrp inhibition on intestinal availability of TPT As shown in

Fig 3A and 3B pretreatment of rats with a single oral dose of ZSQ (30 mgkg) or

Ko143 (10 mgkg) 40 min prior to oral administration of TPT (03 mgkg) resulted in a

significant increase in FamiddotFg of TPT compared to vehicle-pretreated rats These data

indicate that both P-gp- and Bcrp- mediated active effluxes were involved in the

intestinal absorption of TPT In addition both transporters in the intestine were inhibited

almost completely by ZSQ (30 mgkg) and Ko143 (10 mgkg) since the FamiddotFg of TPT

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did not further increase when higher doses of inhibitors were administered

Assessment of the contributions of efflux transporters in the oral absorption

process In order to assess the impact of P-gp- and Bcrp-mediated efflux on the

intestinal absorption of 3 model drugs (FEX a P-gp substrate SASP a Bcrp substrate

and TPT a P-gp and Bcrp substrate) each drug was orally administered to portal vein

cannulated rats with or without pretreatment with inhibitors

As shown in Figs 4 5 and Table 3 systemic and portal plasma concentrations of

FEX after oral pretreatment with ZSQ were higher than those of vehicle-pretreated rats

The FamiddotFg of FEX (022 plusmn 018) increased 4-fold with ZSQ (084 plusmn 010) but not with

Ko143 pretreatment (020 plusmn 005) In addition no further increase in FamiddotFg was seen

with ZSQ+Ko143 pretreatment compared to pretreatment with only ZSQ suggesting

that intestinal absorption of FEX was highly restricted by P-gp- but not by

Bcrp-mediated efflux In the case of SASP the FamiddotFg without added inhibitor was quite

low (003 plusmn 001) and increased to 014 plusmn 007 with Ko143 pretreatment Pretreatment

with ZSQ showed no effect on the intestinal absorption of SASP (002 plusmn 001) This

result suggests that the FamiddotFg of SASP was restricted only by Bcrp-mediated efflux In

contrast the FamiddotFg of TPT was 011 plusmn 003 in the absence of inhibitors and increased to

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023 plusmn 007 by ZSQ pretreatment although this difference was not significant The

FamiddotFg of TPT after pretreatment with Ko143 increased significantly to 042 plusmn 010 and

further increased to 064 plusmn 020 following pretreatment with both inhibitors The

difference in the FamiddotFg between Ko143 and ZSQ+Ko143 pretreatment was not

significant as the impact of P-gp was likely small

As shown in Fig 6 the FamiddotFg after pretreatment with ZSQ+Ko143 was regarded as a

fraction of the dose influxed into enterocytes since all 3 drugs were reported to be

hardly metabolized (Das et al 1979 Herben et al 1997 and Strelevitz et al 2005) In

FEX and SASP a fraction of the efflux by P-gp or Bcrp was calculated as the difference

between the FamiddotFg in ZSQ+Ko143 pretreated rats and that of control rats Thus the

fractions of influx efflux and FamiddotFg in control rats were calculated as 077 055 and

022 for FEX and 014 011 and 003 for SASP respectively

In the case of TPT the contribution of each transporter was calculated by equations

(2) to (4) When pretreatment was with ZSQ the FamiddotFg and Bcrp efflux of TPT were

FamiddotFg Bcrp efflux = 023 041 ( = 064 ndash 023 ) (2)

When pretreatment was with Ko143 the FamiddotFg and P-gp efflux of TPT were

FamiddotFg P-gp efflux = 042 022 ( = 064 ndash 042) (3)

Taken together when both inhibitors were absent the FamiddotFg P-gp and Bcrp efflux of

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TPT were

FamiddotFg P-gp efflux Bcrp efflux = 30 16 54 (4)

Influx fraction was separated by eq (4) Thus as shown in Fig 6 the fractions of P-gp

and Bcrp efflux were calculated as 010 and 035 respectively These data suggest that

Bcrp was the dominant efflux transporter that restricted TPT absorption in rats

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Discussion

Recent studies on intestinal transporters have revealed important roles for various

transporters in regulating the absorption of drugs from the intestinal tract such as the

case for solute carrier SLC transporters (PEPT1 OATPs) to facilitate absorption and

for ATP-binding cassette ABC transporters (P-gp BCRP and MRP2) to limit

absorption In the process of drug development and also for clinical use it is highly

beneficial to determine the contribution of these transporters to overall absorption

because this information enables prediction of the change in the rate and amount of drug

absorption when the functions of transporters are altered by genetic polymorphisms

disease states or drug-drug interactions

Typically to assess the effect of intestinal transporters on drug absorption in vivo test

compounds are orally co-administered with an inhibitor specific to each transporter

then the FamiddotFg and Fh were estimated from pharmacokinetic (PK) analysis of

intravenous and oral administration following eqs (5) and (6)

Fh = 1 - (CLtot - CLr) Qh (5)

FamiddotFg = F Fh (6)

where CLtot and CLr are systemic and renal clearance after intravenous administration

respectively and Qh is hepatic blood flow Using these equations if systemic or renal

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clearance of the test compound is altered by oral co-administration of a transporter

inhibitor an intravenous study is required for each inhibitor dose This study showed

that pretreatment with ZSQ (both by iv and po) significantly decreased the systemic

clearance of FEX (Table 1) Similarly Adane et al (2012) reported alteration of the

systemic AUC of a camptothecin analogue in oral and intravenous administration

studies following oral pretreatment with ZSQ or Elacridar a dual P-gp and Bcrp

inhibitor In these cases to consider the effect of transporters on oral absorption of FEX

or camptothecin not only systemic but also renal clearance should be estimated since

P-gp and Bcrp are expressed in the renal tubule

In this study using portal vein cannulated rats changes in FamiddotFg of FEX after oral

and intravenous pretreatment with ZSQ were estimated (Fig 1) Oral administration of

ZSQ was considered to inhibit both systemic and intestinal P-gp while intravenous

administration of ZSQ inhibited only systemic P-gp expressing in liver kidney and

other organs except for intestine FamiddotFg of FEX after oral administration of ZSQ was

4-fold higher than control but was almost the same as control after intravenous

administration despite a significant change in systemic clearance The change in

systemic clearance is negligible because the absorbed amount is calculated from the

difference between the systemic and portal amount in eq (1) These results clearly show

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that our method using portal vein cannulated rats enables the evaluation of the effects of

selective inhibitors on oral absorption of substrate drugs independently of variable

systemic clearance without the requirement of an intravenous administration study

As shown in Fig 3 FamiddotFg of TPT did not increase at higher inhibitor doses which

might be attributed to the complete inhibition of P-gp- and Bcrp-mediated intestinal

efflux by ZSQ (30 mgkg) and Ko143 (10 mgkg) Poller et al (2011) reported that TPT

transport in a double-transfected MDCKΙΙ-ABCB1 ABCG2 cell line treated with 16

microM and 500 microM TPT was completely blocked in the presence of ZSQ (5 microM) and

Ko143 (1 microM) Chemical knockdown with ZSQ and Ko143 was therefore assumed to

almost completely inhibit the intestinal efflux transporter In addition ZSQ was reported

to have a much lower affinity for CYP3A than for P-gp (Dantzig et al 1999) In our

preliminary study FamiddotFg of felodipine a drug easily metabolized at the intestine (Wang

et al 1989) did not change after pretreatment with ZSQ or Ko143 (data not shown)

suggesting that intestinal metabolism was not affected by ZSQ (30 mgkg) and Ko143

(10 mgkg) although FEX SASP and TPT were resistant to oxidative metabolism

In order to understand the impact of P-gp and Bcrp quantitatively the contributions of

enterocyte efflux transporters to overall absorption were represented as dose fraction

shown in Fig 6 In the case of FEX 71 of the amount taken up into enterocytes was

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effluxed to the apical surface by P-gp in rats This result is in good agreement with

results using Caco-2 cell monolayers reported by Petri et al (2004) in which P-gp

attenuated the permeability of FEX to 30 Also our results indicated that 77 of

orally administered FEX was taken up into enterocytes despite its large polar surface

area and high molecular weight Qiang et al (2009) reported that pretreatment with

fluvastatin a substrate of OATP1B1 OATP2B1 and OATP1B3 (Noeacute et al 2007)

decreased the bioavailability of FEX 045-fold in rats due to reduced intestinal

absorption of FEX rather than enhanced systemic elimination Accordingly our results

suggest that FEX cellular uptake might be moderated by rat Oatps

SASP was found not to be a substrate of P-gp however 79 of that taken up by

enterocytes was effluxed to the apical surface by Bcrp resulting in a very low FamiddotFg of

only 003 in the control study Additionally low solubility and low permeability

contributed to the low oral absorption of SASP While the solubility of SASP is 00024

mgmL in water (Benet et al 2011) the concentration of SASP in the drug solution was

1 mgmL meaning that the majority of SASP remained insoluble in the GI tract

De Vries et al (2012) and Tang et al (2012) have reported that single gene

disruption of Abcb1a1b or Abcg2 in mice has little or even no detectable effect on the

accumulation of erlotinib or sunitinib in the brain whereas simultaneous disruption of

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both transporters results in a dramatic increase This unexpected effect has led to

postulation of a synergistic role for P-gp and Bcrp (Polli et al 2009) However using a

blood-brain barrier kinetic model Kodaira et al (2010) analyzed brain uptake of these

drugs and proposed a kinetic concept for this apparent synergism They demonstrated

that P-gp involved in the efflux of these drugs together with Bcrp attenuates the effect of

Bcrp impairment on drug concentrations in the brain That is two efflux transporters act

as a safety net that prevents brain penetration of common substrates when another efflux

transporter is dysfunctional Intestinal absorption of TPT was also restricted by both

P-gp and Bcrp As shown in Fig 6 70 of TPT taken up into enterocytes was effluxed

to the apical surface by P-gp and Bcrp and Bcrp-mediated efflux was 35-fold higher

than that of P-gp Li et al (2008) assessed the involvement of efflux transporters on

TPT transport using engineered MDCK cells that overexpress P-gp (MDCKIIP-gp) or

BCRP (MDCKIIBCRP) The intrinsic clearance (CLint) calculated by dividing Vmax by

Km in MDCKIIBCRP cells was found to be higher than that in MDCKIIP-gp cells

(043 plusmn 004 microLcm2min in MDCKIIP-gp and 060 plusmn 006 microLcm2min in

MDCKIIBCRP) which also supports our results

In conclusion the use of portal vein cannulated rats enables quantitative assessment

of the contributions of intestinal efflux transporters to the oral absorption of 3 model

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drugs and to estimate the effect of selective inhibitors independently of variable

systemic clearance This experimental system is useful at the drug discovery stage for

clarifying the cause of low bioavailability of new drug candidates

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Acknowledgments

We thank Kunihiko Morisaki (Charles River Laboratories Japan) Dr Jiro Kuze (Taiho

Pharmaceutical Co Ltd) and Dr Toshiyuki Kume (Mitsubishi Tanabe Pharma

Corporation) for useful discussions

Authorship Contributions

Participated in research design Matsuda Konno Satsukawa and Yamashita

Conducted experiments Matsuda Konno Hashimoto Nagai Taguchi

Performed data analysis Matsuda

Wrote or contributed to the writing of the manuscript Matsuda and Yamashita

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References

Adane ED Liu Z Xiang TX Anderson BD and Leggas M (2012) Pharmacokinetic

modeling to assess factors affecting the oral bioavailability of the lactone and

carboxylate forms of the lipophilic camptothecin analogue AR-67 in rats Pharm

Res 291722-1736

Agarwal S Uchida Y Mittapalli RK Sane R Terasaki T and Elmquist WF (2012)

Quantitative proteomics of transporter expression in brain capillary endothelial cells

isolated from P-glycoprotein (P-gp) breast cancer resistance protein (Bcrp) and

P-gpBcrp knockout mice Drug Metab Dispos 4133-39

Allen JD van Loevezijn A Lakhai JM van der Valk M van Tellingen O Reid G

Schellens JH Koomen GJ and Schinkel AH (2002) Potent and specific inhibition of

the breast cancer resistance protein multidrug transporter in vitro and in mouse

intestine by a novel analogue of fumitremorgin C Mol Cancer Ther 1417-425

Bardelmeijer HA Ouwehand M Beijnen JH Schellens JH and van Tellingen O (2004)

Efficacy of novel P-glycoprotein inhibitors to increase the oral uptake of paclitaxel

in mice Invest New Drugs 22219-229

Benet LZ Broccatelli F and Oprea TI (2011) BDDCS applied to over 900 drugs AAPS

J 13519-547

This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on May 16 2013 as DOI 101124dmd113051680

at ASPE

T Journals on M

ay 3 2021dm

daspetjournalsorgD

ownloaded from

DMD 51680

29

Chen Y Agarwal S Shaik NM Chen C Yang Z and Elmquist WF (2009)

P-glycoprotein and breast cancer resistance protein influence brain distribution of

dasatinib J Pharmacol Exp Ther 330956-963

Chu X Zhang Z Yabut J Horwitz S Levorse J Li XQ Zhu L Lederman H Ortiga R

Strauss J Li X Owens KA Dragovic J Vogt T Evers R and Shin MK (2012)

Characterization of multidrug resistance 1aP-glycoprotein knockout rats generated

by zinc finger nucleases Mol Pharmacol 81220-227

Cisternino S Mercier C Bourasset F Roux F and Scherrmann JM (2004) Expression

up-regulation and transport activity of the multidrug-resistance protein Abcg2 at the

mouse blood-brain barrier Cancer Res 643296-3301

Cvetkovic M Leake B Fromm MF Wilkinson GR and Kim RB (1999) OATP and

P-glycoprotein transporters mediate the cellular uptake and excretion of

fexofenadine Drug Metab Dispos 27866-871

Dantzig AH Shepard RL Law KL Tabas L Pratt S Gillespie JS Binkley SN Kuhfeld

MT Starling JJ and Wrighton SA (1999) Selectivity of the multidrug resistance

modulator LY335979 for P-glycoprotein and effect on cytochrome P-450 activities

J Pharmacol Exp Ther 290854-862

Das KM Chowdhury JR Zapp B and Fara JW (1979) Small bowel absorption of

This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on May 16 2013 as DOI 101124dmd113051680

at ASPE

T Journals on M

ay 3 2021dm

daspetjournalsorgD

ownloaded from

DMD 51680

30

sulfasalazine and its hepatic metabolism in human beings cats and rats

Gastroenterology 77280-284

de Vries NA Buckle T Zhao J Beijnen JH Schellens JH and van Tellingen O (2012)

Restricted brain penetration of the tyrosine kinase inhibitor erlotinib due to the drug

transporters P-gp and BCRP Invest New Drugs 30443-449

Hendricks CB Rowinsky EK Grochow LB Donehower RC and Kaufmann SH (1992)

Effect of P-glycoprotein expression on the accumulation and cytotoxicity of

topotecan (SKampF 104864) a new camptothecin analogue Cancer Res

522268-2278

Herben VM ten Bokkel Huinink WW Dubbelman AC Mandjes IA Groot Y van

Gortel-van Zomeren DM and Beijnen JH (1997) Phase I and pharmacological study

of sequential intravenous topotecan and oral etoposide Br J Cancer 761500-1508

Huang L Be X Tchaparian EH Colletti AE Roberts J Langley M Ling Y Wong BK

and Jin L (2012) Deletion of Abcg2 has differential effects on excretion and

pharmacokinetics of probe substrates in rats J Pharmacol Exp Ther 343316-324

Kalgutkar AS Frederick KS Chupka J Feng B Kempshall S Mireles RJ Fenner KS

and Troutman MD (2009)

N-(34-dimethoxyphenethyl)-4-(67-dimethoxy-34-dihydroisoquinolin-2[1H]-yl)-6

This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on May 16 2013 as DOI 101124dmd113051680

at ASPE

T Journals on M

ay 3 2021dm

daspetjournalsorgD

ownloaded from

DMD 51680

31

7-dimethoxyquinazolin-2-amine (CP-100356) as a chemical knock-out equivalent

to assess the impact of efflux transporters on oral drug absorption in the rat J Pharm

Sci 984914-4927

Kato M Chiba K Hisaka A Ishigami M Kayama M Mizuno N Nagata Y Takakuwa S

Tsukamoto Y Ueda K Kusuhara H Ito K and Sugiyama Y (2003) The intestinal

first-pass metabolism of substrates of CYP3A4 and P-glycoprotein-quantitative

analysis based on information from the literature Drug Metab Pharmacokinet

18365-372

Kodaira H Kusuhara H Ushiki J Fuse E and Sugiyama Y (2010) Kinetic analysis of

the cooperation of P-glycoprotein (P-gpAbcb1) and breast cancer resistance protein

(BcrpAbcg2) in limiting the brain and testis penetration of erlotinib flavopiridol

and mitoxantrone J Pharmacol Exp Ther 333788-796

Kusuhara H Furuie H Inano A Sunagawa A Yamada S Wu C Fukizawa S Morimoto

N Ieiri I Morishita M Sumita K Mayahara H Fujita T Maeda K and Sugiyama Y

(2012) Pharmacokinetic interaction study of sulphasalazine in healthy subjects and

the impact of curcumin as an in vivo inhibitor of BCRP Br J Pharmacol

1661793-1803

Li H Jin HE Kim W Han YH Kim DD Chung SJ and Shim CK (2008) Involvement

This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on May 16 2013 as DOI 101124dmd113051680

at ASPE

T Journals on M

ay 3 2021dm

daspetjournalsorgD

ownloaded from

DMD 51680

32

of P-glycoprotein multidrug resistance protein 2 and breast cancer resistance

protein in the transport of belotecan and topotecan in Caco-2 and MDCKII cells

Pharm Res 252601-2612

Maliepaard M van Gastelen MA de Jong LA Pluim D van Waardenburg RC

Ruevekamp-Helmers MC Floot BG and Schellens JH (1999) Overexpression of

the BCRPMXRABCP gene in a topotecan-selected ovarian tumor cell line Cancer

Res 594559-63

Matsuda Y Konno Y Satsukawa M Kobayashi T Takimoto Y Morisaki K and

Yamashita S (2012) Assessment of intestinal availability of various drugs in the oral

absorption process using portal vein-cannulated rats Drug Metab Dispos

402231-2238

Noeacute J Portmann R Brun ME and Funk C (2007) Substrate-dependent drug-drug

interactions between gemfibrozil fluvastatin and other organic anion-transporting

peptide (OATP) substrates on OATP1B1 OATP2B1 and OATP1B3 Drug Metab

Dispos 351308-1314

Petri N Tannergren C Rungstad D and Lennernaumls H (2004) Transport characteristics of

fexofenadine in the Caco-2 cell model Pharm Res 211398-1404

Poller B Wagenaar E Tang SC and Schinkel AH (2011) Double-transduced MDCKII

This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on May 16 2013 as DOI 101124dmd113051680

at ASPE

T Journals on M

ay 3 2021dm

daspetjournalsorgD

ownloaded from

DMD 51680

33

cells to study human P-glycoprotein (ABCB1) and breast cancer resistance protein

(ABCG2) interplay in drug transport across the blood-brain barrier Mol Pharm

8571-582

Polli JW Olson KL Chism JP John-Williams LS Yeager RL Woodard SM Otto V

Castellino S and Demby VE (2009) An unexpected synergist role of P-glycoprotein

and breast cancer resistance protein on the central nervous system penetration of the

tyrosine kinase inhibitor lapatinib

(N-3-chloro-4-[(3-fluorobenzyl)oxy]phenyl-6-[5-([2-(methylsulfonyl)ethyl]amin

omethyl)-2-furyl]-4-quinazolinamine GW572016) Drug Metab Dispos

37439-442

Qiang F Lee BJ Lee W and Han HK (2009) Pharmacokinetic drug interaction between

fexofenadine and fluvastatin mediated by organic anion-transporting polypeptides in

rats Eur J Pharm Sci 37413-417

Shepard RL Cao J Starling JJ and Dantzig AH (2003) Modulation of P-glycoprotein

but not MRP1- or BCRP-mediated drug resistance by LY335979 Int J Cancer

103121-125

Schinkel AH and Jonker JW (2003) Mammalian drug efflux transporters of the ATP

binding cassette (ABC) family an overview Adv Drug Deliv Rev 553-29

This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on May 16 2013 as DOI 101124dmd113051680

at ASPE

T Journals on M

ay 3 2021dm

daspetjournalsorgD

ownloaded from

DMD 51680

34

Strelevitz TJ Foti RS and Fisher MB (2006) In vivo use of the P450 inactivator

1-aminobenzotriazole in the rat varied dosing route to elucidate gut and liver

contributions to first-pass and systemic clearance J Pharm Sci 951334-1341

Takeuchi T Nonaka M Yoshitomi S Higuchi T Ebihara T Maeshiba Y Kawase M and

Asahi S (2008) Marked impact of P-glycoprotein on the absorption of TAK-427 in

rats Biopharm Drug Dispos 29311-323

Tang SC Lagas JS Lankheet NA Poller B Hillebrand MJ Rosing H Beijnen JH and

Schinkel AH (2012) Brain accumulation of sunitinib is restricted by P-glycoprotein

(ABCB1) and breast cancer resistance protein (ABCG2) and can be enhanced by

oral elacridar and sunitinib coadministration Int J Cancer 130223-233

Thiebaut F Tsuruo T Hamada H Gottesman MM Pastan I and Willingham MC (1987)

Cellular localization of the multidrug-resistance gene product P-glycoprotein in

normal human tissues Proc Natl Acad Sci U S A 847735-7738

Troutman MD and Thakker DR (2003) Novel experimental parameters to quantify the

modulation of absorptive and secretory transport of compounds by P-glycoprotein in

cell culture models of intestinal epithelium Pharm Res 201210-1224

Wang SX Sutfin TA Baumlaumlrnhielm C and Regaringrdh CG (1989) Contribution of the

intestine to the first-pass metabolism of felodipine in the rat J Pharmacol Exp Ther

This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on May 16 2013 as DOI 101124dmd113051680

at ASPE

T Journals on M

ay 3 2021dm

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DMD 51680

35

250632-636

Yamasaki T Fujinaga M Kawamura K Hatori A Yui J Nengaki N Ogawa M Yoshida

Y Wakizaka H Yanamoto K Fukumura T and Zhang MR (2011) Evaluation of the

P-glycoprotein- and breast cancer resistance protein-mediated brain penetration of

11C-labeled topotecan using small-animal positron emission tomography Nucl Med

Biol 38707-714

Zaher H Khan AA Palandra J Brayman TG Yu L and Ware JA (2006) Breast cancer

resistance protein (Bcrpabcg2) is a major determinant of sulfasalazine absorption

and elimination in the mouse Mol Pharm 355-61

Zamek-Gliszczynski MJ Bedwell DW Bao JQ and Higgins JW (2012)

Characterization of SAGE Mdr1a (P-gp) Bcrp and Mrp2 knockout rats using

loperamide paclitaxel sulfasalazine and carboxydichlorofluorescein

pharmacokinetics Drug Metab Dispos 401825-1833

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Figure Legends

Fig 1 FamiddotFg of FEX at 5 mgkg after oral (30 mgkg) and intravenous (2 mgkg)

administration with ZSQ FEX was orally administered 40 min after ZSQ po or 5 min

after ZSQ iv Each bar represents the mean plusmn SD for 3 to 5 rats Statistically

significant difference P lt 0001 control versus ZSQ po or ZSQ iv

Fig 2 Assessment of dose-dependence of systemic (A) and portal (B) plasma

concentrations following oral administration of increasing doses of TPT (01 to 3

mgkg) in the portal vein cannulated rats

Fig 3 The effects of ZSQ or Ko143 pretreatment on FamiddotFg following the oral

administration of TPT (03 mgkg) in the portal vein cannulated rats (A) FamiddotFg of TPT

40 min after oral administration of ZSQ (1 to 30 mgkg) and (B) FamiddotFg of TPT 40 min

after oral administration of Ko143 (1 to 10 mgkg) Each bar represents the mean plusmn SD

for 3 to 5 rats Statistically significant difference P lt 005 P lt 0001 without

inhibitors versus with inhibitors

Fig 4 Systemic and portal plasma concentration-time profile of FEX (5 mgkg) SASP

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(5 mgkg) and TPT (03 mgkg) after pretreatment with ZSQ (30 mgkg) andor Ko143

(10 mgkg) in the portal vein cannulated rats

The systemic plasma concentration-time profile of FEX (A) SASP (C) and TPT (E)

after pretreatment with ZSQ andor Ko143 The portal plasma concentration-time

profile of FEX (B) SASP (D) and TPT (F) after pretreatment with ZSQ andor Ko143

Each symbol represents the mean plusmn SD for 3 to 5 rats

Fig 5 Comparison of FamiddotFg in FEX (5 mgkg) SASP (5 mgkg) and TPT (03 mgkg)

among vehicle ZSQ (30 mgkg) Ko143 (10 mgkg) and ZSQ+Ko143 pretreated rats

(A) FamiddotFg of FEX 40 min after oral administration of ZSQ andor Ko143 (B) FamiddotFg of

SASP 40 min after oral administration of ZSQ andor Ko143 (C) FamiddotFg of TPT 40 min

after oral administration of ZSQ andor Ko143 Each bar represents the mean plusmn SD for

3 to 5 rats Statistically significant difference P lt 005 P lt 0001 control versus

ZSQ Ko143 or ZSQ+Ko143 Dagger P lt 005 DaggerDaggerDagger P lt 0001 ZSQ versus Ko143 P lt

005 P lt 0001 ZSQ versus ZSQ+Ko143 daggerdaggerdagger P lt 0001 Ko143 versus

ZSQ+Ko143

Fig 6 Schematic diagram of the impact of P-gp and Bcrp on intestinal absorption of

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FEX (A) SASP (B) and TPT (C) Values represent the fractions of influx efflux and

FamiddotFg when the orally administered amount was regarded as 1 and values given in

parentheses represent the each fraction when influx into enterocytes was regarded as

100

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TABLE 1

Pharmacokinetic parameters of FEX (1 mgkg) after oral (30 mgkg) and intravenous (2

mgkg) administration of ZSQ

AUC t12 CLtot Vdss

ngmiddothmL h mLminkg mLkg

Control 428 plusmn 41 21 plusmn 02 393 plusmn 40 19 plusmn 03

ZSQ po 587 plusmn 58 14 plusmn 01 286 plusmn 30 14 plusmn 02

ZSQ iv 570 plusmn 34 14 plusmn 01 293 plusmn 19 11 plusmn 02

ZSQ po ZSQ was orally treated 40 min before FEX was intravenously administered

ZSQ iv ZSQ and FEX were intravenously coadministered Values represent the mean

plusmn SD for 3 to 4 rats Statistically significant difference P lt 005 P lt 001 P

lt 0001 control versus ZSQ po or ZSQ iv

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TABLE 2

The systemic AUCs portal AUCs and FamiddotFg of TPT following oral administration of

increasing doses in the portal vein cannulated rats

TPT dose AUCsys AUCpor Rb FamiddotFg

mgkg ngmiddothmL ngmiddothmL

01 60 plusmn 09 112 plusmn 21

126

013 plusmn 003

03 141 plusmn 03 258 plusmn 22 010 plusmn 002

1 501 plusmn 74 102 plusmn 12 013 plusmn 002

3 172 plusmn 2 313 plusmn 22 012 plusmn 002

Values represent the mean plusmn SD for 3 rats

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TABLE 3

The systemic AUCs portal AUCs and FamiddotFg of FEX (5 mgkg) SASP (5 mgkg) and

TPT (03 mgkg) after pretreatment with ZSQ (30 mgkg) andor Ko143 (10 mgkg) in

the portal vein cannulated rats

Substrate Pretreatment AUCsys AUCpor Rb FamiddotFg

ngmiddothmL ngmiddothmL

FEX

Control 599 plusmn 409 617 plusmn 490

099

022 plusmn 018

ZSQ 431 plusmn 63 2563 plusmn 286 084 plusmn 010

Ko143 550 plusmn 197DaggerDaggerDagger 565 plusmn 118DaggerDaggerDagger 020 plusmn 005DaggerDaggerDagger

ZSQ + Ko143 284 plusmn 39daggerdaggerdagger 2243 plusmn 330daggerdaggerdagger 077 plusmn 013daggerdaggerdagger

SASP

Control 366 plusmn 37 499 plusmn 42

058

003 plusmn 001

ZSQ 339 plusmn 29 443 plusmn 32 002 plusmn 001

Ko143 2881 plusmn 646DaggerDaggerDagger 3492 plusmn 716DaggerDaggerDagger 014 plusmn 007Dagger

ZSQ + Ko143 2529 plusmn 549 3146 plusmn 411 014 plusmn 004

TPT

Control 183 plusmn 16 309 plusmn 15

126

011 plusmn 003

ZSQ 362 plusmn 137 638 plusmn 194 023 plusmn 007

Ko143 690 plusmn 175Dagger 119 plusmn 18DaggerDagger 042 plusmn 010

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ZSQ + Ko143 122 plusmn 15daggerdaggerdagger 200 plusmn 35daggerdaggerdagger 064 plusmn 020

Values represent the mean plusmn SD for 3 to 5 rats Statistically significant difference P

lt 005 P lt 001 P lt 0001 control versus ZSQ Ko143 or ZSQ+Ko143 Dagger P lt

005 DaggerDagger P lt 001 DaggerDaggerDagger P lt 0001 ZSQ versus Ko143 P lt 005 P lt 001 P

lt 0001 ZSQ versus ZSQ+Ko143 daggerdaggerdagger P lt 0001 Ko143 versus ZSQ+Ko143

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