Upload
others
View
2
Download
0
Embed Size (px)
Citation preview
1
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
2
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
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
3
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.
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
4
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
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
5
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
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
6
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.
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
7
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
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
8
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
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
9
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.
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
10
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
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
11
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.
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
12
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.
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
13
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
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
14
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
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
15
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
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
16
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
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
17
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.
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
18
References
Akita H, Suzuki H and Sugiyama Y (2001) Sinusoidal efflux of taurocholate is enhanced in
Mrp2-deficient rat in liver. Pharm Res 18:1119-1125.
Behrend M (2001) Adverse gastrointestinal effects of mycophenolate mofetil. Ateiology,
incidence and management. Drug Safety 24:645-663.
Bullingham R, Monroe S, Nicholls A and Hale M (1996a) Pharmacokinetics and
bioavailability of mycophenolate mofetil in healthy subjects after single-dose oral and
intravenous administration. J Clin Pharmacol 36:315-324.
Bullingham RE, Nicholls A and Hale M (1996b) Pharmacokinetics of mycophenolate mofetil
(RS61443): a short review. Transplant Proc 28:925-929.
Chieli E, Romiti N, Cervelli F, Paolicchi A and Tongiani R (1994) Influence of rat strain on
P-glycoprotein expression in cultured hepatocytes. Cell Biol Toxicol 10:163-166.
Chu XY, Kato Y, Niinuma K, Sudo KI, Hakusui H and Sugiyama Y (1997) Multispecific
organic anion transporter is responsible for the biliary excretion of the camptothecin
derivative irinotecan and its metabolites in rats. J Pharmacol Exp Ther 281:304-314.
Filler G, Zimmering M and Mai I (2000) Pharmacokinetics of mycophenolate mofetil are
influenced by concomitant immunosuppression. Pediatr Nephrol 14:100-104.
Gregoor PJ, de Sevaux RG, Hene RJ, Hesse CJ, Hilbrands LB, Vos P, van Gelder T, Hoitsma
AJ and Weimar W (1999) Effect of cyclosporine on mycophenilic acid trough levels in
kidney transplant recipients. Transplantation 68:1603-1606.
Hirohashi T, Suzuki H, Ito K, Ogawa K, Kume K, Shimizu T and Sugiyama Y (1998) Hepatic
expression of multidrug resistance-associated protein-like proteins maintained in Eisai
Hyperbilirubinemic rats. Mol Pharmacol 53:1068-1075.
Hirohashi T, Suzuki H and Sugiyama Y (1999) Characterization of the transport properties of
cloned rta multidrug resistance associated protein 3 (MRP3). J Biol Chem
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
19
274:15181-15485.
Ito K, Suzuki H, Hirohashi T, Kume K, Shimizu T and Sugiyama Y (1997) Molecular cloning
of canalicular multispecific organic anion transporter defective in EHBR. Am J Physiol
272:G16-G22.
Jachez B, Boesch D, Grassberger MA and Loor F (1993) Reversion of the
P-glycoprotein-mediated multidrug resistance of cancer cells by FK-506 derivatives.
Anticancer Drugs 4:223-229.
Kamisako T, Leier I, Konig J, Buchholz U, Hummel-Eisenbeiss J and Keppler D (1999)
Transport of monoglucuronosyl and bisglucuronosyl bilirubin by recombinant human and
rat multidrug resistance protein 2. Hepatology 30:485-490.
Kobayashi M, Kobayashi M, Saitoh H, Yuhki Y, Achiwa K, Tadano K, Takahashi Y, Abe J,
Harada H, Hirano T (2003) Comparison of the incidence of diarrhea in renal transplant
patients with or without mycophenolate mofetil administration. Jpn J Pharm Health Sci
29:685-690.
Kochi S, Takanaga H, Matsuo H, Naito M, Tsuruo T and Sawada Y (1999) Effect of
cyclosporin A and tacrolimus on the function of blood-brain barrier cells. Eur J
Pharmacol 372:287-295.
Kouzuki H, Suzuki H and Suguyama Y (2000) Pharmacokinetic study of the hepatobiliary
transport of indomethacin. Pharm Res 17:432-438.
Lokiec F, Canal P, Gay C, Chatelut E, Armand JP, Roche H, Bugat R, Goncalves E and
Mathieu-Boue A (1995) Pharmacokinetics of irinotecan and its metabolites in human
blood, bile, and urine. Cancer Chemother Pharmacol 36:79-82.
Madhu C, Gregus Z and Klaassen CD (1989) Biliary excretion of acetamonophen-glutathione
as an index of toxic activation of acetamonophen: effect of chemicals that alter
acetaminophen hepatotoxicity. J Pharmacol Exp Ther 248:1069-1077.
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
20
Madon J, Eckhardt U, Gerloff T, Stieger B and Meier PJ (1997) Functional expression of the
liver canalicular isoform of the multidrug resistance-associated protein. FEBS Lett
406:75-78.
Masuda M, I’izuka Y, Yamazaki M, Mishigaki R, Kato Y, Ni’inuma K, Suzuki H and
Sugiyama Y (1997) Methotrexate is excreted into the bile by canalicular multispecific
organic anion transporter in rats. Cancer Res 57:3506-3510.
Mele TS and Halloran PF (2000) The use of mycophenolate mofetil in transplant recipients.
Immunopharmacology 47:215-245.
Morikawa A, Goto Y, Suzuki H, Hirohashi T and Sugiyama Y (2000) Biliary excretion of
17ß-estradiol 17ß-D-glucuronide is predominantly mediated by cMOAT/MRP2. Pharm
Res 17:546-552.
Mottino AD, Hoffman T, Jennes L and Vore M (2000) Expression and localization of
multidrug resistant protein mrp2 in rat small intestine. J Pharmacol Exp Ther
293:717-723.
Nishino A, Kato Y, Igarashi T and Sugiyama Y (2000) Both cMOAT/MRP2 and another
unknown transporter(s) are responsible for the biliary excretion of glucuronide conjugate
of the nonpeptide angiotensin II antagonist, termisaltan. Drug Metab Dispos
28:1146-1148.
Niinuma K, Kato Y, Suzuki H, Tyson CA, Weizer V, Dabbs JE, Froehlich R, Green CE and
Sugiyama Y (1999) Primary active transport of organic anions on bile canalicular
membrane in humans. Am J Physiol 276:G1153-G1164.
Ogawa K, Suzuki H, Hirohashi T, Ishikawa T, Meijer PJ, Hirose K, Akizawa T, Yoshioka M
and Sugiyama Y (2000) Characterization of inducible nature of MRP3 in rat liver. Am J
Physiol Gastrointest Liver Physiol 278:G438-G446.
Roberts MS, Magnusson BM, Burczynski FJ and Weiss M (2002) Enterohepatic circulation.
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
21
physiological, pharmacokinetic and clinical implications. Clin Pharmacokinet
41:751-790.
Shipkova M, Armstrong VM, Kuypers D, Perner F, Fabrizi V, Holzer H, Wieland E and
Oellerich M (2001) Effect of cyclosporine withdrwal on mycophenolic acid
pharmacokinetics in kidney transplant recipients with deteriorating renal function:
preliminary report. Ther Drug Monit 23:717-721.
Svensson JO, Brattstrom C and Sawe J (1999) A simple HPLC method for simultaneous
determination of mycophenolic acid and mycophenolic acid glucuronide in plasma. Ther
Drug Monit 21:322-324.
van Gelder T, Klupp J, Barten MJ, Christians U and Morris RE (2001) Comparison of the
effects of tacrolimus and cyclosporine on the pharmacokinetics of mycophenolic acid.
Ther Drug Monit 23:119-128.
Zucker K, Rosen A, Tsaroucha A, de Faria L, Roth D, Ciancio G, Esquenazi V, Burke G,
Tzakis A and Milller J (1997) Unexpected augmentation of mycophenolic acid
pharmacokinetics in renal transplant patients receiving tacrolimus and mycophenolate
mofetil in combination therapy, and analogous in vitro findings. Transpl Immunol
5:225-232.
Zucker K, Tsaroucha A, Olson L, Esquenazi V, Tzakis A and Miller J (1999) Evidence that
tacrolimus augments the bioavailability of mycophenolate mofetil through the inhibition
of mycophenolic acid glucuronidation. Ther Drug Monit 21:35-43.
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
22
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.
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
23
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.
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
24
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.
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
25
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.
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from
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
at ASPE
T Journals on January 5, 2020
jpet.aspetjournals.orgD
ownloaded from