J - Pharmacol - Exp - Ther 2010 Watanabe 651 6 PDF
J - Pharmacol - Exp - Ther 2010 Watanabe 651 6 PDF
J - Pharmacol - Exp - Ther 2010 Watanabe 651 6 PDF
/organic cation
antiporter, MATE1 can also transport cephalexin and cephradine
(Tanihara et al., 2007). Therefore, MATE1 is predicted to be a
candidate transporter responsible for the efflux of cephalexin and
cephradine in the proximal tubules. Furthermore, recent studies
showed that zwitterionic drugs such as fexofenadine and fluoro-
quinolones were transported by MATE1 (Matsushima et al., 2009;
Ohta et al., 2009). These in vitro findings suggested that MATE1
contributes to tubular secretion of not only cationic drugs but also
zwitterionic drugs.
Therefore, in the present study, to elucidate the involve-
ment of MATE1 in tubular secretion of zwitterions in vivo,
pharmacokinetic analyses of cephalexin using Mate1(/)
mice were carried out. In addition, we investigated the effect
of Mate1 deficiency on the pharmacokinetics of cefazolin,
which is not transported by MATE1.
Materials and Methods
Materials. Cephalexin was provided by Shionogi, Osaka, Japan,
and cefazolin was provided by Astellas Pharma Inc., Tokyo, Japan.
All other chemicals used were of the highest purity available.
Isolation of Mouse MATE1 cDNA. The mouse (m) MATE1
cDNA was cloned by reverse transcription-polymerase chain re-
action from Mouse Kidney Marathon-Ready cDNA (Clontech,
Mountain View, CA). Primers specific for mMATE1 were designed
on the basis of the sequence information of the National Center for
Biotechnology Information reference sequence NM_026183. The
mMATE1 cDNA was cloned by using the following primers: for-
ward, 5-GGGGTACCCCACGGAGGCCACATGGAAC-3 and re-
verse, 5-CGCTCGAGTCCACTCCAGAGCATCTCCT-3. The poly-
merase chain reaction product was subcloned into pFLAG-CMV-6
expression vector (Sigma-Aldrich, St. Louis, MO) and sequenced
by using a multicapillary DNA sequencer RISA384 system (Shi-
madzu, Kyoto, Japan).
Cell Culture, Transfection, and Uptake Experiments.
HEK293 cells stably expressing human MATE1 (HEK-hMATE1
cells) and mock cells (HEK-pcDNA cells) were cultured according
to our previous report (Tanihara et al., 2007). pFLAG plasmid
vector DNA containing mMATE1 cDNA was transfected into
HEK293 cells using Lipofectamine 2000 Reagent (Invitrogen,
Carlsbad, CA) as described previously (Urakami et al., 2002;
Terada et al., 2006). At 48 h after the transfection, the cells were
used for uptake experiments. The uptake experiments of cepha-
lexin were carried out as described previously (Ueo et al., 2005;
Tanihara et al., 2007).
Animals. Animal experiments were conducted in accordance with
the Guidelines for Animal Experiments of Kyoto University. All pro-
tocols were approved by the Animal Research Committee, Graduate
School of Medicine, Kyoto University. Male Mate1(/) and
Mate1(/) mice (1318 weeks of age, C57BL/6 genetic background)
were used in the present study.
Pharmacokinetic Experiments. Pharmacokinetic experiments
were carried out according to our previous report (Tsuda et al.,
2009a) with a slight modification. In brief, after a catheter was
inserted into the right jugular vein, 5 mg/kg cephalexin and 146
mg/kg mannitol were administered as a bolus injection. At the indi-
cated times, plasma and urine were collected and analyzed. At the
end of experiments, the kidney and liver were removed, and excised
tissues were gently washed, weighed, and homogenized. In the case
of cefazolin, the same experimental procedures were applied. For the
determination of cephalexin and cefazolin in renal and hepatic tis-
sues, homogenates (100 l) were loaded onto an Oasis HLB cartridge
(Waters, Milford, MA) preconditioned with 1 ml each of methanol
and water. The column was washed with 1 ml of water, and cepha-
lexin and cefazolin were eluted from the column with 1 ml of meth-
anol. The eluate was evaporated to dry at 45 to 50C and resus-
pended in 200 l of each mobile-phase buffer. The solutions were
filtered through a Millipore Corporation (Billerica, MA) filter
(SGJVL, 0.45 m) and analyzed. The concentrations of drugs in
plasma, urine, the renal homogenate, and the hepatic homogenate
were determined by high-performance liquid chromatography
(HPLC). The levels of creatinine in plasma and urine at 60 min were
determined with the Jaffe reaction using an assay kit from Wako
Pure Chemicals (Osaka, Japan).
Determination of Pharmacokinetic Parameters. A conven-
tional two-compartmental analysis was used to investigate the
plasma concentration-time profiles of cephalexin and cefazolin after
the intravenous administration in mice (WinNonlin, version 5.2.1;
Pharsight, Mountain View, CA). Pharmacokinetic parameters, the
area under the blood concentration-time curve from time 0 to infinity
(AUC
/or-
ganic cation antiporter (Inui et al., 1985; Terada et al., 2006;
Tanihara et al., 2007), suggesting the involvement of MATE1
in the tubular secretion of cephalexin. In the present phar-
macokinetic study, we revealed that urinary excretion of
cephalexin for 60 min after the intravenous administration
was significantly decreased in Mate1(/) mice compared
with Mate1(/) mice (Fig. 5A). The renal concentration of
cephalexin and K
p,kidney
value were markedly elevated in
Fig. 4. Plasma concentration profiles of cephalexin (A) and
cefazolin (B) in Mate1(/) mice (E) and Mate1(/) mice
(F). Cephalexin at 5 mg/kg and mannitol at 146 mg/kg were
administered as a bolus injection via the jugular vein.
Then, 1% mannitol was administered to maintain a suffi-
cient and constant urine flowrate by continuous infusion at
0.35 ml/h using an automatic infusion pump. Thereafter,
blood samples were collected at the time points indicated.
The same experimental procedures were applied for cefa-
zolin. The plasma concentrations of cephalexin and cefazo-
lin were determined by HPLC. Each point represents the
mean S.D. for five or six mice of each genotype. , P
0.05; , P 0.01, significantly different from Mate1(/)
mice.
Fig. 5. Urinary excretion of cephalexin (A) and cefazolin (B) in
Mate1(/) mice (open columns) and Mate1(/) mice (closed columns).
Urine was collected for 60 min after the drug administration. Cephalexin
and cefazolin levels in the urine samples were determined by HPLC.
Each column represents the mean S.D. for five or six mice. , P 0.01,
significantly different from Mate1(/) mice.
Fig. 6. Tissue distribution of cephalexin (A and B) and cefazolin (C and D)
in Mate1(/) mice (open columns) and Mate1(/) mice (closed col-
umns). The kidneys (A and C) and livers (B and D) were removed to
determine the tissue concentration of cephalexin or cefazolin at 60 min
after drug administration. Cephalexin and cefazolin levels in the tissue
samples were determined by HPLC. Each column represents the mean
S.D. for five or six mice. , P 0.001, significantly different from
Mate1(/) mice.
654 Watanabe et al.
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Mate1(/) mice (Fig. 6A; Table 1). The V
1
value of cepha-
lexin was significantly decreased in Mate1(/) mice com-
pared with Mate1(/) mice (Table 1). Furthermore, the
CL
ren
of cephalexin in Mate1(/) mice was also signifi-
cantly decreased and seemed to be near the creatinine clear-
ance (Ccr) (Table 1). These results are consistent with those
of our previous in vitro transport studies. This is the first
demonstration that MATE1 is responsible for renal tubular
secretion of cephalexin in vivo.
Previously, it was reported that renal elimination of
cephalexin was significantly inhibited by the coadminis-
tration of cimetidine, a cationic drug, in healthy subjects
(van Crugten et al., 1986). That report suggested that
organic cation transport systems were involved in the tu-
bular secretion of cephalexin in humans. We recently dem-
onstrated that cimetidine at the clinical plasma concentra-
tion inhibited apical hMATE1 more strongly than human
organic cation transporter 2 (SLC22A2) (Tsuda et al., 2009b).
Therefore, it is likely that MATE1 is responsible for the drug
interaction between cimetidine and cephalexin in tubular
secretion. Furthermore, fexofenadine and fluoroquinolones
such as levofloxacin, which are transported by MATE1,
caused the drug interaction with cimetidine in healthy vol-
unteers (Fish and Chow, 1997; Yasui-Furukori et al., 2005).
These studies suggested that MATE1 is also important in the
tubular secretion of zwitterionic drugs in humans.
In this study, there were no significant differences in the
pharmacokinetic profiles of cefazolin between Mate1(/)
and Mate1(/) mice (Figs. 4B, 5B, and 6, C and D; Table 1).
Hence, these results indicated that MATE1 does not contrib-
ute to the tubular secretion of cefazolin in vivo. Previous
study showed that MRP4 is involved in the tubular secretion
of cefazolin in the brush-border membranes (Ci et al., 2007).
On the other hand, we demonstrated in this pharmacokinetic
analysis that MATE1 plays a key role in the tubular secre-
tion of cephalexin. Thus, there is a distinct difference in the
efflux transporter between cephalexin and cefazolin, al-
though both drugs are transported by OATs at the basolat-
eral membranes. This may be attributed to the difference in
the charge states of each drug.
It was reported that the uptake of TEA, a typical organic
cation, by hMATE1 was increased when the extracellular pH
was changed from 6.0 to 8.5 under intracellular acidified
conditions (Tanihara et al., 2007). Because the intracellular
pH of HEK293 cells is temporarily reduced to 6.0 to 6.5 by
pretreatment with NH
4
Cl (Lang et al., 2003), it was consid-
ered that TEA uptake from the extracellular pH 6.0 to 8.5
was activated by an increase in the oppositely directed H
gradient-dependent transport of
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Address correspondence to: Dr. Ken-ichi Inui, Department of Pharmacy,
Kyoto University Hospital, Sakyo-ku, Kyoto 606-8507, Japan. E-mail:
[email protected]
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