Cyclosporin A, but Not Tacrolimus, Inhibits the Biliary...

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Cyclosporin A, but Not Tacrolimus, Inhibits the Biliary Excretion of

Mycophenolic Acid Glucuronide Possibly Mediated by Mrp2 in Rats

MIKAKO KOBAYASHI, HIROSHI SAITOH, MICHIYA KOBAYASHI, KOJI TADANO,

YASUSHI TAKAHASHI, and TETSUO HIRANO

Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Health Sciences

University of Hokkaido, Ishikari-Tobetsu, Hokkaido, Japan (M.K., H.S., M.K.),

Department of Pharmacy, Sapporo City General Hospital, Sapporo, Japan (K.T., Y.T.),

and Department of Renal Transplantation, Sapporo City General Hospital, Sapporo,

Japan (T.H.)

JPET Fast Forward. Published on February 20, 2004 as DOI:10.1124/jpet.103.063073

Copyright 2004 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on February 20, 2004 as DOI: 10.1124/jpet.103.063073

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a) A running title

Biliary excretion of mycophenolic acid glucuronide in rats.

b) Corresponding author

Hiroshi Saitoh, Ph.D. (Professor)

Department of Pharmaceutics,

Faculty of Pharmaceutical Sciences,

Health Sciences University of Hokkaido,

1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan.

Telephone & fax: +81-1332-3-1851; e-mail address: [email protected]

c) Number of text pages: 25

Number of tables: 1

Number of figures: 4

Number of references: 32

Number of words in Abstract: 234

Number of words in Introduction: 661

Number of words in Discussion: 1500

d) List of nonstandard abbreviations

MMF, mycophenolate mofetil; MPA, mycophenolic acid; MPAG, mycophenolic acid

glucuronide; CsA, cyclosporin A; Mrp2, multidrug resistance-associated protein 2; EHBR,

Eisai hyperbilirubinemic rats; CER, cumulative excretion ratio; SD, Sprague-Dawley rats;

X0, dose; C0, plasma concentration at time 0; Ke, elimination rate constant; AUCi.v., 0-1, area

under the concentration/time curve from 0 to 1 h; Cltot, total clearance; Vd, volume of

distribution; Cb, concentration in bile; Vb, volume of bile.

e) A recommended section assignment to guide the listing in the table of contents

Absorption, Distribution, Metabolism, & Excretion


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ABSTRACT

The onset of diarrhea after the administration of mycophenolate mofetil (MMF) is possibly

associated with the biliary excretion of its metabolite, mycophenolic acid glucuronide

(MPAG). This study was undertaken to clarify the mechanism underlying the biliary excretion

of MPAG. Intravenously administered mycophenolic acid (MPA, 5 mg/kg) rapidly

disappeared from plasma and was efficiently excreted as MPAG in the bile of Wistar (26% of

dose) and Sprague-Dawley (SD) rats (21% of dose) over 1 h. On the other hand, in spite of

the rapid disappearance of MPA from plasma, the biliary excretion of MPAG was very limited

in Eisai hyperbilirubinemic rats (EHBRs), which display mutations in multidrug

resistance-associated protein 2 (Mrp2)/canalicular multispecific organic anion transporter

(cMOAT), and constituted only 0.5% of dose. Instead, high levels of MPA were noted in the

plasma of EHBRs. Intravenous administration of CsA (5 mg/kg) to Wistar rats significantly

lowered the biliary excretion of MPAG. However, intravenously administered tacrolimus (0.1

mg/kg) failed to produce such effect. In conclusion, it is suggested that there is an efficient

MPAG transport mediated by Mrp2 on the bile canalicular membrane of rat hepatocytes and

that the therapeutic range of CsA potentially interferes with Mrp2. However, the therapeutic

range of tacrolimus does not inhibit the transporter. Thus, it should be noted that MMF

coadministered with tacrolimus instead of CsA might increase the occurrence of diarrhea

related to the biliary excretion of MPAG in transplant recipients.


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Mycophenolate mofetil (MMF) is a new type of immunosuppressive agent for the

prophylaxis of acute rejection in organ transplantations (Mele and Halloran, 2000). After oral

administration, MMF is rapidly converted to mycophenolic acid (MPA), the active metabolite

of MMF (Bullingham et al., 1996a). Generally, MMF is used in combination with cyclosporin

A (CsA) or tacrolimus and diarrhea is one of the most frequently reported adverse events in

transplant recipients receiving MMF (Behrend, 2001). In Japan, clinical studies of MMF have

mainly been performed in combination with CsA and approximately 13% of subjects enrolled

in the clinical studies complained of diarrhea due to MMF according to the interview form

supplied by the manufacturer. At Sapporo City General Hospital, more than 200 cases of renal

transplantation have been performed to date and MMF was introduced as an adjunctive

therapy in combination with tacrolimus in 2000. Since then, severe diarrhea has been

observed in patients immediately after the start of the MMF regimen, leading to

discontinuation or reduction of the drug. Very recently, we investigated the frequency and

severity of diarrhea between renal transplant recipients receiving MMF (MMF, prednisolone,

and tacrolimus) and those not receiving MMF (azathioprine, prednisolone, and tacrolimus or

CsA) in the immunosuppressive regimens at Sapporo City General Hospital. Statistical

analysis revealed that diarrhea was more frequent and severe in the patients receiving MMF

than in those not receiving it (Kobayashi et al., 2003). The frequency of diarrhea in the

patients receiving MMF was approximately 74%. Although we could not carry out the same

investigation on the immunosuppressive regimen using MMF and CsA, the frequency of

diarrhea obtained from our investigation seemed much greater than those reported previously

in the clinical studies using MMF and CsA. According to these observations, it is obvious that

the choice of a carcineurin inhibitor coadministered with MMF is closely associated with the

occurrence of diarrhea.

Mycophenolic acid is primarily metabolized by glucuronidation at the phenolic


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hydroxyl group in the liver, forming 7-O-glucuronide (MPAG), and the glucuronide is

preferentially excreted into the urine (Bullingham et al., 1996a). It was also reported that the

extent of enterohepatic circulation in the overall pharmacokinetics for MPA was 37%, with a

wide range of 10-61%, in humans (Bullingham et al., 1996b). Thus, it is fully assumed that

the enterohepatic circulation of MPAG is closely responsible for gastrointestinal disorders

such as diarrhea, although the mechanism by which diarrhea occurs after MMF administration

has not been clarified. Recent reports have suggested that CsA and tacrolimus differently

influence the pharmacokinetics of MPA. For example, MPA predose concentrations were

significantly greater in patients receiving MMF and prednisolone than in patients receiving

MMF, prednisolone and CsA, suggesting a clinically relevant drug interaction between CsA

and MPA (and/or MMF) (Gregoor et al., 1999). It was also reported that MPA blood

concentrations are lower in patients receiving concomitant tacrolimus compared with those

receiving CsA (Filler et at., 2000), and MPAG blood concentrations are higher in combination

with CsA than with tacrolimus (Zucker et al., 1997). As a possible mechanism, several reports

have implied that CsA interferes with the enterohepatic circulation of MPAG (van Gelder et

al., 2001; Shipkova et al., 2001) and that tacrolimus can interfere with

UDP-glucuronosyltransferase, which mediates the conversion of MPA to MPAG (Zucker et al.,

1999). Until now, however, both the mechanism responsible for the biliary excretion of

MPAG and the effect of CsA and tacrolimus on the biliary excretion have not been fully

characterized.

In this study, we evaluated the biliary excretion of MPA and MPAG after intravenous

administration of MPA to two normal rat strains, Wistar and Sprague-Dawley (SD), and Eisai

hyperbilirubinemic rats (EHBRs), a mutant SD strain lacking multidrug-resistance associated

protein 2 (Mrp2)/canalicular multispecific organic anion transporter (cMOAT) on the hepatic

canalicular membranes and on the intestinal brush-border membranes (Ito et al., 1997). Our


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results demonstrated that MPAG was excreted into the bile via Mrp2 and that CsA, but not

tacrolimus, functionally inhibited the Mrp2-mediated transport for MPAG in the liver.


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Materials and Methods

Materials. Mycophenolic acid and ß-glucuronidase solution (85 units/ml) were

obtained from Wako Pure Chem. Ind. (Osaka, Japan). (+)-Naproxen was purchased from

Sigma-Aldrich Co. (St. Louis, MO, USA). A tacrolimus formulation for injection (Prograf®

injection 5 mg) and a CsA formulation for injection (Sandimmun®) were purchased from

Fujisawa Pharmaceutical Co. (Osaka, Japan) and Novartis Pharma (Tokyo, Japan),

respectively. Digoxin was obtained from Tokyo Chem. Co. (Tokyo, Japan). All other reagents

were of the highest grade available.

Animal Experiments. Male Wistar and SD rats were obtained from Hokudo (Sapporo,

Japan). Male EHBRs were purchased from Sankyo Labo Service Co. (Tokyo, Japan). Rats

(three/cage) were housed for at least two weeks before experiments with free access to food

(MF, Oriental Yeast Co., Tokyo, Japan) and water at 25 ± 3°C and 50 ± 20% relative humidity

under a 12-hr light/dark cycle, without no attrition. The body weight of rats used in this study

was 260-380g in Wistar rats and 350-390g in EHBR and SD rats, respectively. All rats were

used for experiments at age of 8 to 10 weeks.

Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (40

mg/kg, Dainippon Pharmaceuticals, Osaka, Japan). After abdominal operation, a polyethylene

tube (i.d. 2.8 mm) was inserted in bile duct toward the liver and then MPA, which was

dissolved in polyethylene glycol 400 at a concentration of 5 mg/ml, was administered

intravenously to each rat via the jugular vein. The dose of MPA was fixed at 5 mg/kg body

weight. Bile samples were collected every 15 min over 1 h after MPA administration and the

bile volume was measured with an appropriately sized volumetric pipette. Blood samples

(each 0.4 ml) were taken from the other side of the jugular vein just before MPA

administration and at 5, 10, 15, 20, 30, and 60 min after administration, and were immediately

centrifuged at 2,700 x g for 20 min to obtain plasma samples. Bile and plasma samples were


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kept frozen until assay. In the experiments to examine the effect of CsA or tacrolimus on the

pharmacokinetics of MPA, their formulations were diluted with saline including 50% ethanol

and administered intravenously via the jugular vein at 5 min prior to MPA administration. The

doses of CsA and tacrolimus were set at 5 mg/kg and 0.1 mg/kg, respectively, based on their

clinical regimens to renal transplant recipients.

In this study, principles of good laboratory animal care were followed and animal

experimentation was performed in compliance with the Guidelines for the Care and Use of

Laboratory Animals in Health Sciences University of Hokkaido, accredited by the “Principles

of Laboratory Animal Care” (NIH publication #85-23, revised 1985).

Drug Assay. The assay of MPA and MPAG was performed according to Svensson et al.

(1999) with several modifications. For the determination of free MPA in bile and plasma, 50

µl of sample was mixed with 50 µl of saline and 200 µl of naproxen as an internal standard (5

or 2.5 µg/ml in acetonitrile) and then vigorously shaken for 10 sec. The mixture was let stand

for 5 min and centrifuged at 2,700 x g for 10 min. An aliquot of the resulting supernatant was

applied to HPLC. The determination of MPAG was done after converting the glucuronide to

free MPA by ß-glucuronidase; that is, 45 µl of plasma or 90 µl of bile sample was mixed with

ß-glucuronidase solution (24 units/ml for plasma and 120 units/ml for bile) and then

incubated at 47°C for 5 h. After treatment, the free MPA formed was determined as mentioned

above. Bile samples were diluted with PBS before treatment, if necessary. Chromatographic

conditions for MPA were as follows: apparatus, Shimadzu LC-6A (Kyoto, Japan) equipped

with a Shimadzu SPD-6A UV spectrophotometric detector; column, Cosmosil 5C18-ARII

(4.6 x 150 mm, Nakarai Tesque, Kyoto, Japan); column temperature, 30°C; mobile phase,

40% acetonitrile in 40 mM H3PO4 (pH 2.1 adjusted with KOH); wave length, 250 nm for bile

samples and 304 nm for plasma samples; and flow rate, 0.8 ml/min. Under these HLPC

conditions, MPA and naproxen eluted at 9 min and 12 min, respectively, and MPA was


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reproducibly determined with a coefficient of variance less than 5%. The limit of

determination of MPA assay was 0.2 µg/ml in bile samples and 1 µg/ml in plasma samples,

respectively. The concentration of MPA derived from MPAG was calculated by subtracting

the concentration of free MPA from the total concentration of MPA obtained after

ß-glucuronidase treatment. Excretion ratio of free and MPAG-derived MPA in bile during

each 1-h interval was calculated as follows:

Excretion ratio = Cb · Vb / X0

where Cb is concentration in bile, Vb is volume of bile and X0 is dose. Cumulative excretion

ratio (CER) was obtained by summing up excretion ratio of each 1-h interval.

Pharmacokinetic and Statistical Analysis. The concentration of MPA in plasma at

time 0 (C0) and elimination rate constant (Ke) were calculated using MULTI (Yamaoka et al.,

1981), assuming a compartment open model. Area under the plasma concentration / time

curve from 0 to 1 h (AUCi.v., 0-1) was determined using the linear trapezoidal rule. Volume of

distribution (VD) and total clearance (CLtot) were calculated using the following equations:

VD = X0 / C0, CLtot = Ke · VD

Each data represents the mean ± S.E. of 3 to 4 experiments. Statistical analysis was

done using unpaired Student’s t-test and p < 0.05 was considered to be significant.


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Results

Effect of CsA and Tacrolimus on the Pharmacokinetics of Free and MPAG-derived MPA.

Figure 1 shows the plasma concentration/time curves of free MPA and MPAG-derived MPA

after the intravenous administration of MPA to Wistar rats with CsA or tacrolimus. When

MPA was administered alone, free MPA disappeared rapidly from plasma over 1 h (Fig. 1A).

CsA and tacrolimus did not affect the plasma concentrations of free MPA at any sampling

time point over 1 h. Pharmacokinetic data for free MPA are presented in TABLE 1. The CLtot

of free MPA after coadministration of MPA and tacrolimus was slightly but significantly

greater than those of the others. The plasma concentrations of MPAG-derived MPA gradually

increased over 1 h after the intravenous administration of MPA (Fig. 1B). The plasma

concentrations of MPAG-derived MPA after the coadministration of MPA and tacrolimus

were comparable with those of MPA alone at all sampling time points. When MPA was

administered with CsA, the plasma concentration of MPAG-derived MPA became

significantly greater than those of the others at 30 min.

Effect of CsA and Tacrolimus on the Biliary Excretion of Free and MPAG-derived MPA

in Wistar Rats. Figure 2 shows the cumulative excretion ratios (CERs) of free and

MPAG-derived MPA in bile after the intravenous administration of MPA to Wistar rats. In the

case of MPA alone, the CER of free MPA was very small, remaining at most only 0.15% of

the dose (Fig. 2A). Tacrolimus did not alter the biliary excretion of free MPA while CsA

slightly but not significantly decreased the CER of free MPA. The biliary excretion of

MPAG-derived MPA was extensive in the case of MPA alone and the CER reached

approximately 26% of the dose (Fig. 2B). Tacrolimus failed to modulate the biliary excretion

of MPAG-derived MPA. In contrast, the CER of MPAG-derived MPA significantly decreased

by about 20% when MPA was coadministered with CsA. It was confirmed that there were no

differences in the volume of bile excreted during each 1-h interval under these experimental


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conditions.

Plasma Concentrations of Free and MPAG-derived MPA after Intravenous

Administration of MPA to SD Rats and EHBRs. Figure 3 shows the plasma concentration/

time curves of free and MPAG-derived MPA after the intravenous administration of MPA to

SD rats and EHBR, together with the results obtained in Wistar rats. The profile for the

EHBRs was almost the same as that for the Wistar rats and there were no significant

differences in the plasma levels of free MPA between them at any sampling time point during

the hour (Fig. 3A). On the other hand, the plasma concentrations of free MPA in SD rats were

significantly greater than those from EHBR and Wistar rats at all sampling time points.

Pharmacokinetic data of free MPA obtained from EHBR and SD rats are summarized in

TABLE 1. While the C0 and AUCi.v., 0-1 values were significantly smaller in the EHBRs than

in SD rats, the Ke and CLtot were significantly greater in the EHBRs. The plasma

concentrations of MPAG-derived MPA gradually increased over 1 h in the EHBRs and they

were significantly greater than those in the Wistar rats at all sampling time points (Fig. 3B). In

contrast, MPAG-derived MPA was not detected in the plasma of SD rats at any sampling time

point.

Biliary Excretion of Free and MPAG-derived MPA after Intravenous Administration of

MPA to SD Rats and EHBRs. Figure 4 shows the CERs of free and MPAG-derived MPA

after the intravenous administration of MPA to SD rats and EHBR. The CER of free MPA in

SD rats increased with time over 1 h whereas the biliary excretion of free MPA was

significantly retarded in the EHBRs compared with SD rats and it reached a plateau after 0.5

h (Fig. 4A). Although the CER of free MPA in SD rats was rather smaller than that in Wistar

rats, this difference was not statistically significant. There were remarkable differences in the

CERs of MPAG-derived MPA between the EHBR and SD rats; that is, MPAG-derived MPA

was efficiently excreted into the bile over 1 h in SD rats, giving a CER of ca. 21% of the dose.


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In contrast, the CER of MPAG-derived MPA in the EHBRs was very low and remained

approximately 0.5% of the MPA dose (Fig. 4B). It was found that the CERs of MPAG-derived

MPA in SD rats were significantly smaller than those in Wistar rats.


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Discussion

Biliary excretion is an important process for the elimination of many drugs and their

metabolites (Roberts et al., 2002), whereas the extensive biliary excretion of several

compounds is closely associated with their toxic gastrointestinal adverse effects, such as

diarrhea and mucosal lesions. Methotrexate (Masuda et al., 1997), irinotecan (Lokiec et al.,

1995) and acetaminophen (Madhu et al., 1989) are known to be models of this mechanism.

Moreover, there have been many papers describing the efficient biliary excretion of various

glucuronide conjugates. It has been reported that approximately 30% of intravenously

administered indomethacin was excreted in bile of SD rats, mostly as its glucuronide

(Kouzuki et al., 2000) and that about 10% of telmisaltan glucuronide was excreted in bile of

SD rats over 1 h (Nishino et al., 2000). In the present study, when MPA was intravenously

administered to Wistar or SD rats, the CER of MPAG-derived MPA reached approximately 26

and 21% of the dose over 1 h, respectively (Fig. 4B), suggesting that the biliary excretion of

MPAG is extensive in these rat strains.

Recently, many studies have suggested that MRP2/Mrp2 plays a primary role in the

biliary excretion of glucuronide conjugates of various chemical compounds such as

indomethacin (Kouzuki et al., 2000), SN-38 (Chu et al., 1997), and 17ß-estradiol (Morikawa

et al., 2000). In such studies, EHBRs (a mutant SD strain) or a transport-deficient Wistar

strain of rats, both of which genetically lack Mrp2 on the canalicular membrane (Ito et al.,

1997; Madon et al., 1997), have been utilized favorably. In this study, the CER of

MPAG-derived MPA in the EHBRs and normal SD rats was 0.5 and 21% of dose, respectively

(Fig, 4B). This suggests that Mrp2 was essential for the biliary excretion of MPAG; that is,

the glucuronide is a likely substrate of Mrp2. Since the CER of free MPA in the EHBRs was

also significantly smaller than that in SD rats (0.02% vs 0.08% of the dose after 1 h, p < 0.01),

free MPA is also considered to be a substrate of Mrp2. However, since the CER ratio of


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SD/EHBR was approximately 4 for free MPA and 40 for MPAG-derived MPA (Figs. 4A &

4B), it appears that Mrp2 much prefers MPAG to MPA. Interestingly, while MPAG-derived

MPA were not detected in the plasma of SD rats and at most at 3.5 µg/ml in Wistar rats after 1

h, in the EHBRs it greatly increased over 1 h and reached about 14 µg/ml after 1 h (Fig. 3B).

It was recently reported that Mrp3 was compensatively up-regulated on the sinusoidal

membrane of EHBRs and exclusively expelled taurocholic acid, a substrate of Mrp2, into

sinusoidal blood (Akita et al., 2001). Mrp3 is capable of transporting several glucuronide

conjugates (Hirohashi et al., 1999). Thus, it is likely that Mrp3 greatly contributed to the

predominant appearance of MPAG in the plasma of the EHBRs through the efficient transport

of MPAG across the sinusoidal membrane. The expression of Mrp3 is negligible on the

sinusoidal membrane of SD rats (Hirohashi et al. 1998; Akita et al., 2001). Therefore, no

detection of MPAG in the plasma of SD rats is in part attributed to the lack of Mrp3-mediated

transport in SD rats. In contrast, a finding that MPAG was detected in the plasma of Wistar

rats (Fig. 3B) may imply that Mrp3 activity is a little greater in Wistar rats than in SD rats.

After the intravenous administration of MPA, free MPA in the plasma of the EHBRs sharply

disappeared and was about 6 µg/ml after 1 h (Fig. 3A). On the other hand, the levels of

MPAG continued increasing in the same plasma of EHBR even after 1 h (Fig. 3B). These

results could indicate that MPA was taken up rapidly and efficiently from sinusoidal blood

into the hepatocytes. The disappearance of MPA from the plasma was much faster in EHBR

than in SD rats and all pharmacokinetic parameters obtained were significantly different

between them (TABLE 1). Recently, many kinds of transporters have been identified for the

hepatobiliary excretion of various compounds. While P-glycoprotein, organic

anion-transporting polypeptide, Na+-taurocholate cotransporting polypeptide, and MRP-like

protein 1 are comparably expressed between SD rats and EHBR (Hirohashi et al., 1998;

Ogawa et al., 2000; Akita et al., 2001), the expression of MRP-like protein 2 in the


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hepatocytes is limited to EHBR (Hirohashi et al., 1998). Thus, it is likely that an

EHBR-specific transport system is involved in MPA uptake in the hepatocytes. Further study

is now underway to clarify whether a specialized transporter is involved in MPA uptake across

the sinusoidal membranes.

CsA, which was intravenously administered to Wistar rats, significantly inhibited the

biliary excretion of MPAG (Fig. 2B). This result was as expected considering that MPAG is a

substrate of Mrp2 as described above and that CsA is a potential inhibitor of Mrp2 (Kamisako

et al., 1999). Significant increase in the plasma concentration of MPAG-derived MPA after

coadministration of MPA and CsA (Fig. 1B) might be a compensative phenomenon for the

disrupted Mrp2-mediated MPAG transport in bile. CsA lowered the biliary excretion of free

MPA although the effect was not significant (Fig. 2A). Considering that MPA is a weak

substrate of Mrp2 (Fig. 4A), the result seems reasonable. However, since the CER of free

MPA was very small compared with that of MPAG, the significance of the interaction is

regarded as marginal. Tacrolimus is reported to possess the capacity to interfere with

transporters such as P-gp (Jachez et al., 1993; Kochi et al., 1999). However, the present

results indicated that tacrolimus failed to interfere with the Mrp2-mediated MPAG transport

across the canalicular membrane of Wistar rats (Fig. 1B). In this study, the doses of CsA and

tacrolimus were based on their clinical regimens and tacrolimus was administered at a 50-fold

lower dose than CsA. Thus, it is possible that tacrolimus could not exhibit enough ability to

interfere with Mrp2 due to the low dosage and that the same dosage as that of CsA is capable

of modulating Mrp2-mediated MPAG transport. However, it would be beyond clinical

relevance even if the second possibility were true.

Since the difference in biliary excretion of glucuronide appears less marked among

species (Niinuma et al., 1999), the present results imply that MPAG also efficiently undergoes

biliary excretion via MRP2 in the human liver. Actually, a second peak, which is due to the


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enterohepatic circulation of MPAG, is known to emerge in MPA plasma concentration/time

profiles after the oral administration of MMF to humans (Bullingham et al., 1996a). On the

other hand, it has been reported that the majority of MMF orally administered to healthy

subjects was finally excreted in the urine mostly as MPAG and that total recovery of the dose

in the feces averaged only 5.5% (Bullingham et al., 1996b). Accordingly, MPA repeatedly

undergoes glucuronidation in the liver, MRP2-mediated biliary excretion, deglucuronidation

by the intestinal flora and subsequent intestinal reabsorption until MPA molecules are finally

excreted to the urine as MPAG. Thus, the possibility emerges that the enterohepatic circulation

of MPAG is more efficient in patients with renal dysfunction and that the occurrence of

diarrhea would become more frequent in such patients if the biliary excretion of MPAG is

closely involved in the occurrence of diarrhea. Since it has been reported recently that Mrp2 is

present on the brush-border membrane of the intestine (Mottino et al., 2000), it is likely that

the intestinal secretion of MPAG is greatly stimulated under renal dysfunction as well. In our

separate study, it was found that the diarrhea that occurred in renal transplant recipients

receiving MMF was most frequent during first 4 to 5 days after transplant operation and then

became markedly less frequent as the renal function recovered to an almost normal range

(unpublished data). Together with the present results, this suggests that the frequency and

severity of diarrhea after MMF administration is closely related to the extent of renal and

biliary excretion of MPAG in the body.

In this study, several differences in the disposition of intravenously administered MPA

were observed between Wistar and SD rats. Although it was previously reported that

P-glycoprotein expression in cultured hepatocytes was different among rat strains (Chieli et

al., 1994), little information is currently available on the interstrain differences. Further

investigation is required to clarify it.

In conclusion, this study demonstrates for the first time that the phenolic glucuronide of


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MPA is a substrate of Mrp2 present on the canalicular membrane although in vitro study is

necessary to further clarify the role of Mrp2 in MPAG transport in more details and that the

glucuronide undergoes efficient biliary excretion mediated by the transporter. CsA, but not

tacrolimus, potently inhibits Mrp2-mediated MPAG transport. Based on the present results,

we assume that when MMF was coadministerd with CsA, CsA potently inhibited the biliary

excretion of MPAG in the transplant recipients whereas there was no significant inhibition of

biliary excretion of MPAG when MMF was coadministered with tacrolimus. Thus, the

diarrhea associated with the biliary excretion of MPAG was exacerbated in patients receiving

MMF with tacrolimus.


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Footnotes

The name and full address of the person to receive reprints requests:

Dr. Hiroshi Saitoh

Professor, Department of Pharmaceutics

Faculty of Pharmaceutical Sciences

Health Sciences University of Hokkaido

Kanazawa 1757, Ishikari-Tobetsu,

Hokkaido 061-0293,

Japan.


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Figure Legends

Fig. 1. Plasma concentration versus time profiles of free (A) and MPAG-derived MPA (B)

after a bolus intravenous administration of MPA together with CsA or tacrolimus to

Wistar rats. MPA (5 mg/ml) dissolved in polyethylene glycol 400 was administered

into the jugular vein at a dose of 5 mg/kg. CsA or tacrolimus was administered at 5

min before MPA administration via the jugular vein. The dose of CsA and tacrolimus

was 5 mg/kg and 0.1 mg/kg, respectively. In the control (MPA alone), saline was

administered instead of CsA and tacrolimus. Each point represents the mean ± S.E. of

3 to 4 experiments, although most standard error bars are hidden by symbols. * p <

0.05, significantly different from the control.

Fig. 2. Cumulative biliary excretion of free (A) and MPAG-derived MPA (B) after a bolus

intravenous administration of MPA together with CsA or tacrolimus to Wistar rats.

MPA (5 mg/ml), dissolved in polyethylene glycol 400, was administered into the

jugular vein at a dose of 5 mg/kg. CsA or tacrolimus was administered at 5 min before

MPA administration via the jugular vein. The dose of CsA and tacrolimus was 5

mg/kg and 0.1 mg/kg, respectively. In the control (MPA alone), saline was

administered instead of CsA and tacrolimus. Each column represents the mean ± S.E.

of 3 to 4 experiments, although some standard error bars are hidden by columns. * p <

0.05, ** p < 0.02, significantly different from the control.

Fig. 3. Plasma concentration versus time profiles of free (A) and MPAG-derived MPA (B)

after a bolus intravenous administration of MPA to EHBR, Wistar, and SD rats. MPA

(5 mg/ml), dissolved in polyethylene glycol 400, was administered into the jugular

vein at a dose of 5 mg/kg. Each point represents the mean ± S.E. of 3 to 4 experiments,

although most standard error bars are hidden by symbols. * p < 0.05, ** p < 0.01, and

*** p < 0.001, significantly different from that in SD rats.


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Fig. 4. Cumulative biliary excretion of free (A) and MPAG-derived MPA (B) after a bolus

intravenous administration of MPA with CsA or tacrolimus to EHBR, Wistar, and SD

rats. MPA (5 mg/ml), dissolved in polyethylene glycol 400, was administered into the

jugular vein at a dose of 5 mg/kg. Each column represents the mean ± S.E. of 3 to 4

experiments, although most standard error bars are hidden by symbols. * p < 0.05, **

p < 0.01, and *** p < 0.001, significantly different from that in SD rats. a p < 0.01, b p

< 0.001, significantly different from that in Wistar rats.


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TABLE 1 Pharmacokinetic paramerters of free MPA in plasma after a bolus intravenous administration of MPA to Wistar rats, SD rats and EHBRs

MPA was administered into the jugular vein at a dose of 5 mg/kg. Tacrolimus (0.1 mg/kg) or cyclosporin A (5 mg/kg) was administered into the jugular vein at 5 min before MPA administration. Each data represents the mean ± S.E. of 3 to 4 experiments.

Parameters Wistar rats SD rats EHBRs

MPA alone + Tacrolimus + Cyclosporin A MPA alone MPA alone

Xo (µg) 1525 ± 96.8 1666.7 ± 72.7 1387.5 ± 68.6 1825 ± 25.0 1863 ± 51.5 Co (µg/ml) 42.7 ± 0.6 39.7 ± 1.4 42.3 ± 1.7 52.8 ± 2.4* 41.6 ± 1.0 b

K e (h -1) 1.9 ± 0.1 2.1 ± 0.0 2.0 ± 0.0 1.4 ± 0.1** 2.3 ± 0.2 b AUCi.v., 0-1 (µg•h/ml) 22.6 ± 1.3 19.4 ± 0.5 21.4 ± 0.7 38.8 ± 3.0** 18.7 ± 1.2 b

CL tot (l·h -1) 0.07 ± 0.00 0.09 ± 0.00 a 0.06 ± 0.00 0.05 ± 0.00* 0.10 ± 0.01 b VD (l） 0.04 ± 0.00 0.04 ± 0.00 0.03 ± 0.00 0.03 ± 0.00 0.04 ± 0.00 b

a p < 0.05, significantry different from MPA alone of Wistar rats. b p < 0.01, significantly different from MPA alone of SD rats.

* p < 0.05, ** p < 0.005, significantly different from MPA alone of Wistar rats. Abbreviations: Xo = dose; Co = plasma drug concentration at time 0; Ke = elimination rate constant; AUCi.v., 0-1 = area under the concentration / time curve from zero to 1 hour; CLtot = total clearance; and VD = volume of

distribution.


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