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Antimicrobial Agents and Chemotherapy, July 1998, p. 1738-1744, Vol. 42, No. 7
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
P-Glycoprotein-Mediated Transport of
Itraconazole across the Blood-Brain Barrier
Tetsuo
Miyama,1
Hitomi
Takanaga,1
Hirotami
Matsuo,1
Katsuhiro
Yamano,2
Koujirou
Yamamoto,2
Tatsuji
Iga,2
Mikihiko
Naito,3
Takashi
Tsuruo,3
Hitoshi
Ishizuka,4
Yukinori
Kawahara,4 and
Yasufumi
Sawada1,*
Faculty of Pharmaceutical Sciences, Kyushu
University, Higashi-ku, Fukuoka 812-8582,1
Department of Pharmacy, Faculty of Medicine, The University of
Tokyo Hospital,2 and
Institute of
Molecular and Cellular Biosciences,3 University
of Tokyo, Bunkyo-ku, Tokyo 113-8655, and
Analytical and
Metabolic Research Laboratories, Sankyo Co. Ltd., Shinagawa-ku,
Tokyo 140-8710,4 Japan
Received 10 June 1997/Returned for modification 22 November
1997/Accepted 2 March 1998
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ABSTRACT |
The mechanism for the accumulation of itraconazole (ITZ) in its
elimination from the brain was studied in rats and mice. The concentration of ITZ in liver tissue declined in parallel with the
plasma ITZ concentration until 24 h after intravenous injection of
the drug (half-life, 5 h); however, the ITZ in brain tissue rapidly disappeared (half-life, 0.4 h). The time profiles of the brain/plasma ITZ concentration ratio (Kp value)
showed a marked overshooting, and the Kp value
increased with increasing dose; these phenomena were not observed in
the liver tissue. This finding indicates the occurrence of a nonlinear
efflux of ITZ from the brain to the blood. Moreover, based on a
pharmacokinetic model which hypothesized processes for both nonlinear
and linear effluxes of ITZ from the brain to the blood, we found that
the efflux rate constant in the saturable process was approximately
sevenfold larger than that in the nonsaturable process. The
Kp value for the brain tissue was significantly
increased in the presence of ketoconazole or verapamil. The brain
Kp value for mdr1a knockout mice
was also significantly increased compared with that of control mice.
Moreover, the uptake of vincristine or vinblastine, both of which are
substrates of the P glycoprotein (P-gp), into mouse brain capillary
endothelial cells was also significantly increased by ITZ or verapamil.
In conclusion, P-gp in the brain capillary endothelial cells
participates in a process of active efflux of ITZ from the brain to the
blood at the blood-brain barrier, and ITZ can be an inhibitor of
various substrates of P-gp.
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INTRODUCTION |
Itraconazole (ITZ), an azole
antifungal agent, inhibits cytochrome P-450-mediated ergosterol
synthesis in fungal membranes (2) as well as CYP3A4 and
other isozymes involved in xenobiotic metabolism in the liver
(1). Recently, it has been reported that ITZ leads to
various side effects due to drug-drug interactions. For example, the
coadministration of ITZ with terfenadine, an antiallergy agent, caused
cardiovascular side effects (7). The concentration of
cyclosporine (CsA), an immunosuppressive agent, in blood increased when
ITZ was coadministered (16). The occurrence of these side
effects resulted from increases in the concentrations of terfenadine
and CsA in blood that was caused by inhibition of CYP3A4-mediated
metabolism of these drugs by ITZ. Moreover, the coadministration of ITZ
and digoxin (DGX), a cardiac glycoside, caused nausea, vomiting, and
visual disturbances (19). To clarify the mechanism of
interaction of ITZ and DGX, we investigated the effect of ITZ on the
pharmacokinetic behavior of DGX in guinea pigs (20). We
found a reduction in metabolic, biliary, and urinary clearances of DGX
after coadministration of ITZ in guinea pigs.
CsA, DGX, vincristine (VCR), and terfenadine, which metabolically
interact with ITZ, are also substrates and/or inhibitors of the P
glycoprotein (P-gp) (12, 17, 25, 26). P-gp was initially
identified as a plasma membrane protein overexpressed in tumor cells.
It functions as an active efflux pump to exclude Vinca
alkaloid anticancer drugs, etc., from the cell and confers multidrug
resistance to tumor cells (9, 21). It is known that P-gp is
expressed not only on tumor cells but also on normal cells, such as
those of the adrenal gland, the brush border membrane of renal proximal
tubules, the bile canaliculus membrane of hepatocytes, the apical
membrane of mucosal cells in the intestine, and capillary endothelial
cells of the brain, testis, and the placenta or uterus (on pregnancy)
and that it affects the pharmacokinetic behavior of some drugs
(24, 28, 29). Recently, it was reported that ITZ reversed
multidrug resistance in tumor cells in in vitro experiments (11,
15).
In this study, the contribution of P-gp to the process of ITZ efflux
from the brain to the blood was studied in normal rats and
mdr1a knockout mice. Moreover, the effect of ITZ on the
uptake of P-gp substrates by mouse brain capillary endothelial cells (MBEC4 cells) was examined.
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MATERIALS AND METHODS |
Drugs.
ITZ and ketoconazole were kindly supplied by Janssen
Pharmaceutica (Tokyo, Japan). [3H]VCR and
[3H]vinblastine ([3H]VBL) were purchased
from Amersham Ltd. (Little Chalfont, United Kingdom). Verapamil was
purchased from Nacalai Tesque, Inc. (Kyoto, Japan). All other chemicals
used in the experiments were of analytical grade.
Animals.
Male SD rats (Seac Yoshitomi, Ltd., Fukuoka, Japan)
weighing 200 to 250 g were used in all of the experiments.
Wild-type and mdr1a 
/ mice of the FVB strain weighing 20 to 25 g were purchased from Taconic Farms, Inc. (Germantown,
N.Y.).
Measurement of ITZ concentrations in blood, plasma, and tissues
after intravenous administration to rats.
Rats anesthetized with
diethyl ether were cannulated in the right femoral artery and vein. A
10-mg/ml solution of ITZ was prepared by dissolving 10 mg of ITZ in 50 µl of 12 M HCl and 875 µl of polyethylene glycol 200 and then
neutralizing the solution with NaOH. The dose of ITZ was 5, 10, or 20 mg/kg of body weight. Drugs were administered into the femoral vein,
and blood samples were collected from the femoral artery. The blood as
well as the brain and liver tissue samples were obtained at 5 and 15 min and at 1, 4, 8, and 24 h after intravenous administration of
ITZ. Plasma was obtained by centrifuging the blood at 1,620 × g for 5 min at room temperature. The concentrations of ITZ
in plasma and in brain and liver tissues were determined by
high-performance liquid chromatography (HPLC).
For the concurrent use of ketoconazole or verapamil, ketoconazole was
suspended in distilled water and the pH of the suspension was adjusted
to 2.5 with 1 M HCl, while verapamil was dissolved in saline.
Ketoconazole (20 mg/kg) or verapamil (5 mg/kg) was administrated 5 min
before the administration of ITZ into the femoral vein at a dose of 5 mg/kg. Distilled water of pH 2.5 (adjusted with 1 M HCl) or saline was
administered to rats of a control group in the same manner. Rats were
killed 60 min after the administration of ITZ, and their blood and
brains were collected. The blood samples were centrifuged at 1,620 × g for 5 min at room temperature to obtain plasma. The
concentrations of ITZ in the plasma and brain tissue were determined by
HPLC.
To extract ITZ from plasma samples, 0.1 ml of methanol, 0.5 ml of 1 M
NaOH, and 2.5 ml of hexane-isoamyl alcohol (98:2, vol/vol) were added
to 0.1 ml of plasma sample, and the mixture was shaken for 5 min prior
to centrifugation at 1,250 × g for 5 min. After evaporation of 2 ml of the organic phase under a nitrogen stream, the
residue was dissolved in 200 µl of the mobile phase and 100 µl was
injected into the HPLC system. The brain and liver tissue samples were
homogenized with a fourfold excess volume of distilled water. To 0.5 ml
of the homogenate were added 0.1 ml of methanol, 0.5 ml of 1 M NaOH,
and 5 ml of hexane-isoamyl alcohol (98:2, vol/vol). Samples were shaken
for 5 min and centrifuged at 1,250 × g for 5 min. Then
3 ml of 0.1 M HCl was added to 4 ml of the organic phase, and the
solution was shaken for 5 min. After centrifugation at 1,250 × g for 5 min, 0.5 ml of 1 M NaOH and 3 ml of hexane-isoamyl alcohol (98:2, vol/vol) were added to 2 ml of the aqueous phase and the
solution was shaken for 5 min. After centrifugation at 1,250 × g for 5 min, 2 ml of the organic phase was collected and evaporated to dryness under a nitrogen stream. The residue was dissolved in 200 µl of the mobile phase, and 100 µl was injected into the HPLC system.
The HPLC system consisted of a liquid chromatograph (model LC-6A;
Shimadzu, Kyoto, Japan) and a UV spectrophotometric detector
(model
SPD-6A; Shimadzu) operated at 263 nm. The column used for
HPLC was an
Inertsil octyldecyl silane (ODS) (5 µm [particle diameter]
by 250 mm [length] by 4.6 mm [inside diameter]; GL Sciences Inc.,
Tokyo,
Japan), and a YMC-Guardpack ODS-AM (5 µm by 10 mm by 4.6
mm; YMC,
Inc., Wilmington, N.C.) was used as a guard column. The
mobile phase
was acetonitrile-10 mM phosphate buffer (pH 6.5)
(8:2, vol/vol). The
flow rate of the mobile phase was 1.0 ml/min.
The column was maintained
at 40°C.
The limits for quantification of ITZ were 50 ng/ml for plasma and 100 ng/ml for liver and brain tissues. In all measurements,
coefficients of
variation were less than 10%, and within-run accuracy
was always
within ±10%. Moreover, coefficients of variation for
between-day
precision measurements were also always less than
10%.
Measurement of plasma and tissue ITZ concentration-time profiles
after intravenous administration in mdr1a knockout
mice.
ITZ was intravenously administered through the tail vein at
a dose of 5 mg/kg to mdr1a knockout mice (mdr1a
/ mice) or to FVB mice (mdr1a +/+ mice) as controls.
Blood as well as brain, liver, kidney, and lung tissue samples were
obtained at 5, 15, and 60 min after administration of ITZ. A 1-mg/ml
solution of ITZ was prepared by dissolving 10 mg of ITZ in 500 µl of
12 M HCl and 8.75 ml of polyethylene glycol 200 and then neutralizing the solution with NaOH.
The blood samples were centrifuged at 1,620 ×
g for 5 min at room temperature to obtain the plasma, and 0.2 ml of a 1-mg/ml
-naphthoflavone-acetonitrile solution, an internal standard,
was
added to 0.1 ml of plasma. The samples were vigorously mixed
and then
centrifuged at 1,250 ×
g for 10 min. The supernatant
was then filtered (Ekicrodisc 13CR; Gelman Sciences Inc., Ann
Arbor,
Mich.), and 20 µl was injected into the HPLC system. The
weights of
brain, liver, kidney, and lung tissue samples were
measured before
extraction, and 0.3 ml of the 1-mg/ml
-naphthoflavone-acetonitrile
solution was added to each of them. They were vigorously mixed
in a
microhomogenizer (model NS-310E; Niti-on, Chiba, Japan) and
centrifuged
at 1,250 ×
g for 10 min. Each supernatant was then
filtered, and 20 µl was injected into the HPLC system.
The HPLC system consisted of a liquid chromatograph (model LC-10AT;
Shimadzu) and a UV spectrophotometric detector (model
SPD-10A;
Shimadzu) operated at 263 nm. The HPLC column was a YMC-Pack
ODS-A (5 µm by 150 mm by 6.0 mm; YMC, Inc.). The mobile phase
was
acetonitrile-10 mM phosphate buffer (pH 7.0) (8:2, vol/vol).
The flow
rate of the mobile phase was 1.2 ml/min. The column was
maintained at
40°C.
The limits of quantification were 40 ng/ml for plasma and 50 ng/ml for
brain tissue. For all measurements, coefficients of
variation were less
than 10%, and within-run accuracy was always
within ±10%. Moreover,
coefficients of variation for between-day
precision measurements were
less than 10%.
Determination of influx clearance in the brain and the
liver.
To evaluate the permeability of the liver or brain to ITZ
quantitatively, we analyzed the plasma and tissue ITZ concentration data. The concentration data for brain and liver tissues were plotted
as tissue/plasma concentration ratios (Kp
values) versus the areas under the curve (AUC)/plasma ITZ
concentrations after the administration of the drug. Furthermore, the
same analysis described above was performed in short-duration
experiments. The influx clearance (CLinf) of ITZ in the
brain and the liver could be obtained from the slope of the linear
phase of the curve (3).
Measurement of uptake of [3H]VCR and
[3H]VBL by MBEC4 cells.
MBEC4 cells were routinely
cultured as described in our previous report (27). Briefly,
MBEC4 cells were maintained in Dulbecco's modified Eagle's medium
(Nikken BioMedical Laboratory, Kyoto, Japan) supplemented with 10%
fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml). For the transport study, cells were seeded into four-well
plates (Nunc) at a density of 4 × 104/well. Next, the
cells were grown for 3 days in a humidified atmosphere of 5%
CO2-95% O2, the medium was aspirated, and the
cells were washed with 37°C phosphate-buffered saline and then
covered with transport buffer (141 mM NaCl, 4.0 mM KCl, 2.8 mM
CaCl2, 1.0 mM MgSO4, 10 mM
D-glucose, and 10 mM HEPES; pH 7.4) containing 10 mM
[3H]VCR or 10 mM [3H]VBL. The amount of
intracellular [3H]VCR or [3H]VBL was
determined as follows. After incubation of the cells for 1 h, they
were washed with ice-cold transport buffer to stop the uptake of
labeled drug. The cells were digested in 200 µl of 1 M NaOH and
neutralized with 200 µl of 1 M HCl at the end of the uptake study for
1 h. Then, to 300 µl of each sample was added 4 ml of a
scintillation cocktail (Clear-sol I; Nacalai Tesque). The
radioactivities of the samples were measured with a scintillation counter (LS6500; Beckman Instruments, Fullerton, Calif.), and the
values were converted to total radioactivity per unit volume (400 µl). These values were divided by the radioactivity of the added drug
solution per unit volume (disintegrations per minute per milliliter),
and the clearance (in milliliters per hour) was calculated. The
residual 100 µl was used for the measurement of the protein
concentration by the method of Lowry et al. (18). The
clearance, calculated as described above, was divided by the cellular
protein concentration, and the amount of drug taken up per unit amount
of protein (in microliters per hour per milligram of protein) was
determined. The uptake of studies both drugs were performed in the
presence or absence of 10 µM verapamil or 4.25 µM ITZ. The same
amount of dimethyl sulfoxide was added in all experiments since the ITZ
was dissolved in a 0.1% dimethyl sulfoxide solution.
Data analysis.
The pharmacokinetic behavior of ITZ in the
brain was analyzed on the basis of the pharmacokinetic model shown in
Fig. 1. In this model, it is hypothesized
that not only passive diffusion but also an active transport efflux
system influences the pharmacokinetic behavior of ITZ in the brain
after intravenous administration of the drug. The differential equation
used for the brain concentration profiles (XBr)
was as follows:
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(1)
|
where CL
inf (in milliliters per minute per gram of
brain tissue) was the influx clearance value,
k2
(in inverse minutes)
was the linear efflux rate constant,
Vmax (in nanograms per minute
per gram of brain
tissue) was the maximum transport rate of the
efflux process,
Km (in nanograms per milliliter) was the
Michaelis
constant,
Vd (in milligrams per gram
of brain tissue) was the
volume of distribution,
Cp was the plasma concentration time profile
(input function), and
RB (0.79) was the
blood/plasma ITZ concentration
ratio. The differential equation
(equation 1) was fitted to the
ITZ concentration in the brain tissue by
using the nonlinear least-squares
program MULTI (RUNGE) (
32,
33), which can numerically solve
differential equations by the
four-dimensional Runge-Kutta method.
All data were weighed by the
reciprocals of the squares of observed
values for nonlinear
least-squares regression.
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FIG. 1.
Pharmacokinetic model for the analysis of the behavior
of ITZ in the brain. The intracerebral behavior of ITZ was analyzed in
terms of this pharmacokinetic model as described in Materials and
Methods.
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Statistical methods.
Results are given as the means ± standard errors (SE). Statistical analyses were performed with
Student's t test. Statistical significance was deemed
achieved at a P value of less than 0.05.
| |
RESULTS |
Plasma and tissue ITZ concentration-time profiles after intravenous
administration to rats.
After the intravenous administration of
ITZ (5 mg/kg) to rats, plasma and brain and liver tissue ITZ
concentrations were measured for 24 h (Fig.
2). The liver tissue ITZ concentration
declined biexponentially in parallel with the plasma ITZ concentration during the first 24 h after administration (half-life, 5 h).
However, the elimination of ITZ from the brain did not parallel the
plasma ITZ concentration, and the second phase of elimination was much faster in brain tissue than in plasma (half-life, 0.4 h). The elimination half-lives of ITZ for plasma and brain tissue were estimated in a model-independent manner.
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FIG. 2.
Plasma and tissue ITZ concentration-time profiles after
its intravenous administration (at 5 mg/kg) to rats. The plasma and
tissue ITZ concentrations were determined by HPLC as described in
Materials and Methods. Each point represents the mean ± SE
(n = 3). Symbols:  , plasma;  , brain;  ,
liver.
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Analysis of accumulation of ITZ in tissues.
Brain and liver
tissue Kp value-AUC/Cp
profiles are shown in Fig. 3. A rapid
elevation during the early phase and a rapid reduction of the
Kp value in the brain tissue were observed,
whereas the Kp value for ITZ in the liver tissue
was at a steady state after 10 min. The linear accumulation profile
observed in the liver tissue was also observed in the kidney and spleen
tissues (data not shown). The Kp
value-AUC/Cp profiles for the brain and liver
tissues during the early phase after administration are shown in the
insets of Fig. 3A and B, respectively. A linear accumulation in the
brain was observed until 8 min after administration. For the brain
tissue, the slope of the line describing the relationship between the
Kp value and the plasma concentration was
positive and larger than 0 (Fig. 4A), and
it was larger than that for the liver tissue (Fig. 4B).
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FIG. 3.
Kp
value-AUC/Cp profiles of ITZ accumulation in
elimination from brain (A) and liver (B) tissues after intravenous
administration at a dose of 5 mg/kg to rats. (Insets)
Kp value-time profile of ITZ from 0 to 8 min.
Each point represents the mean ± SE (n = 3).
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FIG. 4.
Plasma ITZ concentration dependency of
Kp values, after intravenous administration of
the drug to rats, for brain (A) and liver (B) tissues. ITZ was
administered to rats via the femoral vein at a dose of 5, 10, or 20 mg/kg. After 1 h, plasma as well as brain and liver tissue
specimens were collected and analyzed by HPLC as described in Materials
and Methods. Each point represents the mean ± SE
(n = 3).
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Pharmacokinetic analysis of the behavior of ITZ in the brain.
The pharmacokinetic behavior of ITZ in the brain was analyzed by using
the model shown in Fig. 1. The model in Fig. 1 describes passive
diffusion into and out of the brain as well as active transport out of
the brain tissue. The fitting curves, according to equation 1 and the
data obtained up to 240 min after intravenous administration of 5 mg of
ITZ/kg, are shown in Fig. 5A. Moreover, the early phase in Fig. 5 is shown as an inset. The curves showed good
agreement with the data. The root mean square error, normalized to the
observed values and calculated as an index of fitting performance (30), was 0.28 for the brain concentration profile, which
suggests a mean error estimate of less than 30%. The estimates (± SE)
of CLinf, k2,
Vmax, and Km × Vd values were 0.191 ± 0.018 ml/min/g of
brain tissue, 0.0386 ± 0.021 min
1, 214 ± 220 ng/min/g of brain tissue, and 764 ± 1,800 ng/g of brain tissue,
respectively. Moreover, the same Kp value-time
profile as that in Fig. 3A and a dose-dependent increase in the
Kp value until 240 min after intravenous
administration were obtained by simulation at three doses, as shown in
Fig. 5B.
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FIG. 5.
Simulation curves of the ITZ concentration (A) and
Kp value (B) in brain tissue. Solid lines
represent the fitting curves based on equation 1 as described in
Materials and Methods (Inset). Simulation curves of brain ITZ
concentrations from 0 to 15 min postadministration. Each point in panel
(A) represents the mean ± SE (n = 3). Symbols:
, 5 mg/kg; , 10 mg/kg; , 20 mg/kg.
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Effect of coadministration of ketoconazole or verapamil on the
plasma ITZ concentration and the accumulation of ITZ in the brain.
Plasma ITZ concentration-time profiles obtained after intravenous
administration of 5 mg of ITZ/kg seemed unaffected by the coadministration of 20 mg of ketoconazole or 5 mg of verapamil/kg (Fig.
6A and C). However, the
Kp value for ITZ in the brain tissue was
significantly increased by the coadministration of ketoconazole or
verapamil (Fig. 6B and D). No effect was observed at a dose of 2.5 mg
of verapamil/kg (data not shown).
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FIG. 6.
Effect of ketoconazole or verapamil coadministration,
respectively, on the plasma ITZ concentration (A and C) and the uptake
of ITZ into the brain (B and D). ITZ was administered to rats via the
femoral vein at a dose of 5 mg/kg. Ketoconazole (20 mg/kg) or verapamil
(5 mg/kg) was administered to rats 5 min before the injection of ITZ.
One hour after the administration of ITZ, the plasma and brain tissue
were collected, and the concentration of ITZ was determined by HPLC as
described in Materials and Methods. Each value represents the mean ± SE (n = 3 to 4). Asterisks indicate values found to
be significantly different from control values as determined by
Student's t test. (P < 0.05). Symbols:
 , ITZ (5 mg/kg) alone; , ITZ (5 mg/ml) and ketoconazole (20 mg/kg);  , ITZ (5 mg/ml) and verapamil (5 mg/kg).
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Tissue distribution studies in mdr1a knockout
mice.
The plasma and tissue ITZ concentrations were measured at 5, 15, and 60 min after administration of the drug at a dose of 5 mg/kg to
mdr1a / mice (knockout mice) or mdr1a +/+
mice (control mice). The plasma ITZ concentrations of the knockout mice
were significantly increased at 5 and 60 min compared with those of the
control mice (Fig. 7). The brain tissue
ITZ concentrations and Kp values in knockout
mice at 15 and 60 min were increased approximately 2.5-fold over those
of the control mice (Fig. 8A). A
significant reduction in the Kp value was
observed at 5 min in the liver tissue of knockout mice compared with
that of control mice (Fig. 8B). A significant reduction in the
Kp value was also observed in the kidney tissue
at 5 and 15 min after administration (Fig. 8C). On the other hand, no
significant difference between the Kp values for
lung tissues of knockout mice and control mice was observed (Fig. 8D).
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FIG. 7.
Plasma ITZ concentration-time profiles after intravenous
administration of the drug at a dose of 5 mg/kg to mdr1a
knockout mice. The plasma ITZ concentration was determined by HPLC as
described in Materials and Methods. Each point represents the mean ± SE (n = 3 to 4). Values determined to be
significantly different from those of mdr1a +/+ mice by
Student's t test are indicated (*, P < 0.05; **, P < 0.001). Symbols: ,
mdr1a / mice;  , mdr1a +/+ mice.
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FIG. 8.
Distribution of ITZ in mdr1a knockout mouse
organs after intravenous administration of this drug at a dose of 5 mg/kg. Shown are ITZ concentrations and Kp
values for brain (A), liver (B), kidney (C), and lung (D) tissues. Each
Kp value was calculated by dividing the organ
ITZ concentration by the plasma ITZ concentration. Each point
represents the mean ± SE (n = 3 to 4). Values
found to be significantly different from control values by Student's
t test are indicated (*, P < 0.05;
**, P < 0.01; #, P < 0.005; ##,
P < 0.001). Symbols: , concentration,
mdr1a / mice; , concentration, mdr1a +/+
mice; , Kp value, mdr1a /
mice; , Kp value, mdr1a +/+
mice.
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Effect of ITZ or verapamil on the uptake of [3H]VCR
and [3H]VBL by MBEC4 cells.
Plots of the uptake of
10 nM [3H]VCR and 10 nM [3H]VBL by MBEC4
cells for 1 h in the presence or absence of 4.25 µM ITZ or 10 µM verapamil are shown in Fig. 9. The
rates of uptake of both drugs by MBEC4 cells were significantly
increased in the presence of either ITZ or verapamil.
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FIG. 9.
Effect of ITZ or verapamil on the uptake of
[3H]VCR (A) and [3H]VBL (B) by MBEC4 cells.
The uptake of 10 nM [3H]VCR or 10 nM
[3H]VBL by MBEC4 at 37°C was measured for 1 h in
the presence or absence of 4.25 µM ITZ or 10 µM verapamil as
described in Materials and Methods. Each value represents the mean ± SE (n = 3 to 4). Values determined to be
significantly different from those of drug-only controls by Student's
t test are indicated (*, P < 0.005;
**, P < 0.001). Symbols: , control; , with
ITZ; , with verapamil.
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DISCUSSION |
Although ITZ is highly lipophilic (the partition coefficient of
ITZ in the n-octanol-water system [log P] is
5.66), the accumulation of ITZ in brain tissue is extremely restricted
in comparison with its accumulation in other tissues, such as the liver
(14). The reason for this is still unclear. We hypothesized
that the low-level accumulation of ITZ in brain tissue was due to the
presence of a specific efflux transport system in the brain-blood
barrier (BBB). As shown in Fig. 2, the concentration of ITZ in the
brain tissue rapidly declined by 1 h after administration compared
with that of the liver. It was believed that half-life of ITZ in brain tissue could never be shorter than its half-life in plasma under linear
conditions; however, this may be not the case. In the case of ITZ, the
active efflux system was saturated at high drug concentrations, and the
efflux clearance increased as the concentration became lower;
therefore, the elimination half-life in the liver could be shorter than
that in plasma. A similar scenario has been suggested by
Hammarlund-Udenaes et al. (13). Moreover, Fig. 2 showed that for ITZ, AUCbrain/AUCplasma < 1, which
indicated that CLin < CLout. Possible
mechanisms for this, in addition to active transport out of the brain,
are metabolism in and bulk flow from the brain; however, these may be
minor contributors. Based on the plot of Kp
values versus AUC/Cp, we found that the level of
accumulation of ITZ in the brain tissue (Kp) was
high when the concentration of this drug in the brain was high, i.e.,
during the early phase after administration, and that it was low when
the drug concentration in the brain was low, i.e., in the later phase
(Fig. 3). Thus, the presence of a nonlinear mechanism of active efflux
of ITZ from the brain to the blood is suggested. This is also supported by the dose-dependent increase in the Kp value
of ITZ (Fig. 4A). Accordingly, to explain the mechanism of transport of
ITZ at the BBB, we designed a model including both the passive
diffusion process and the saturable process of efflux from the brain to the blood, which involved a specific transporter (Fig. 1). In this
model, we found that the estimated efflux rate constant (0.28 min1) in the saturable process was approximately
sevenfold larger than the linear efflux rate constant
(k2; 0.039 min1) and that this
saturable process was important to explain ITZ's nonlinear
accumulation in and elimination from the brain. We must consider the
process of influx and efflux through the BBB and the bulk flow of
cerebrospinal fluid for the analysis of drug transport into the brain.
However, our data could not distinguish passive efflux from elimination
by bulk flow. The saturable influx process was negative, since the
ratio of the concentration of ITZ in the brain to the
Cp (Kp value) increased
linearly during the initial phase of uptake, as shown in the inset of
Fig. 3A. For these reasons, we constructed the simple compartment model that included the saturable efflux process. More detailed
investigations will be required to clarify the disposition of ITZ in
the brain.
Gupta et al. reported that ITZ reversed daunorubicin resistance in
tumor cells (11). Kurosawa et al. reported that ITZ reversed adriamycin and etoposide resistance in tumor cells (15).
These reports suggested that ITZ should be an inhibitor of P-gp in
tumor cells. P-gp is expressed in the brain's capillary endothelial cells and actively excludes the transport of lipophilic and cationic drugs such as CsA from the brain to the blood (22).
Moreover, since many interactions between substrates and/or the
inhibitor of P-gp (such as CsA, DGX, and VCR) and ITZ have been
reported (4, 16, 19), we investigated whether ITZ could work
as a substrate and/or inhibitor of P-gp at the BBB. To ascertain our
hypothesis, we examined the effect of verapamil, which is known to be a
P-gp inhibitor, on the accumulation of ITZ in the brain. As shown in
Fig. 6, the brain Kp value was significantly increased in spite of the lack of a significant change in the plasma
ITZ concentration in the presence of verapamil or ketoconazole. The
plasma ITZ concentration might change if the distribution of ITZ in the
brain or other P-gp-expressing tissue were to be altered. The
alteration could not be detected because it occurred at a low level, so
a significant change in the plasma ITZ concentration was not observed
in this study. In addition, the inhibitory effects of verapamil and
ketoconazole were considered to be competitive, since the
Kp value for ITZ was significantly increased in
the presence of either drug (at 5 mg/kg or 20 mg/kg, respectively) and
an increased Kp value was not observed in the
presence of verapamil at 2.5 mg/kg. These results also suggested that
ITZ might be a substrate of P-gp, and P-gp plays an important role in
the ITZ efflux process at the BBB. Chikhale et al. reported similar
results, i.e., that P-gp-mediated transport of peptides through the BBB
was inhibited in the presence of 500 mM verapamil; however, using an in
situ brain perfusion technique, they observed no significant effect on
the transport of urea and L-leucine through the BBB
(6). Furthermore, the results obtained by the
coadministration of ketoconazole, another azole antifungal agent,
indicated that ITZ and ketoconazole were likely to be transported via
the same carrier at the BBB. However, the affinity of ITZ and
ketoconazole for P-gp at the BBB is still unknown.
To examine more directly the contribution of P-gp to the
pharmacokinetic behavior of ITZ in the brain, we utilized
mdr1a / mice, a strain developed by Schinkel et al.
(23) that has a homozygous disruption of the
mdr1a P-gp gene. It had already been determined that P-gp is
encoded by small gene families and that there are two family members in
humans (MDR1 and MDR2) and three members in mice
(mdr1a, mdr1b, and mdr2)
(5). It is well-known that mdr1a P-gp and
mdr1b P-gp in mice fulfill almost the same function as
MDR1 P-gp in humans (24). However, their
distributions in normal tissues are not equivalent. In addition, the
physiological functions of the P-gp's encoded by MDR1,
mdr1a, and mdr1b in normal tissues are unclear.
So, pharmacological studies in mdr1a / mice are
necessary as an index of drug interactions in humans, although it is
difficult to avoid differences between clinical results and the results
of pharmacological studies with these mice. In this study, we found
that the accumulation of ITZ in the brain tissue of mdr1a
/ mice was significantly increased compared with that in
mdr1a +/+ mice (Fig. 8A). These results strongly suggest
that ITZ is a substrate of mdr1a P-gp expressed in mouse
brain endothelial cells. On the other hand, the accumulation of ITZ in
the liver and the kidney was significantly reduced compared to that in
the brain in mdr1a / mice, in contrast to the situation in mdr1a +/+ mice (Fig. 8B and C). The details are still
unclear; however, it is likely that ITZ is vigorously excluded from the liver and kidney by mdr1b P-gp in mdr1a /
mice, since mdr1b P-gp mRNA levels were induced in these
tissues of this mouse strain (23).
We also investigated the mechanism of transport of ITZ at the BBB by
using MBEC4 cells. As shown in Fig. 9, the uptake of VCR or VBL, both
of which are substrates of P-gp, by MBEC4 cells was significantly
increased in the presence of ITZ or verapamil. These results suggest
that ITZ plays an important role in the inhibition of the P-gp-mediated
efflux process. Recently, it has been reported that mdr1a
P-gp is hardly expressed in MBEC4 cells but that mdr1b P-gp
is predominantly expressed (27). Accordingly, it is possible
that the interaction of ITZ with mdr1b P-gp could be
investigated by using this cell system. Although the substrate specificities of mdr1a P-gp and mdr1b P-gp might
not be identical, both of these glycoproteins should work as efflux
transporters (8, 10). In this study, we showed that ITZ was
a substrate and/or an inhibitor of both mdr1a P-gp and
mdr1b P-gp. Since mdr1a and mdr1b in
mice have functions equivalent to those of MDR1 in humans,
it is likely that ITZ also inhibits the function of MDR1 in
humans. Actually, ITZ served as a multidrug resistance reversing agent
for acute lymphoblastic leukemia patients given daunorubicin and ITZ
concurrently (31). So, ITZ may significantly increase the
accumulation in the brain of neurotoxic drugs such as VCR and VBL in
the clinical field. In the future, it will be necessary to investigate
the quantitative contribution of mdr1a and mdr1b to the accumulation of ITZ in the brain by using mdr1b
knockout animals, mdr1a and mdr1b double-knockout
animals, and an mdr1a-expressing cell line.
In the present study, the total concentration of drug was measured
instead of just the unbound-protein concentration, and the species
differences and concentration dependency of protein binding in plasma
and tissues were not considered. Since ITZ shows a high propensity to
bind to plasma proteins, the possibility of displacement of protein
binding cannot be excluded. However, we found that not only passive
diffusion but also the active efflux system, involving P-gp of the BBB,
plays an important role in ITZ's accumulation in and elimination from
the brain. Moreover, ITZ is a P-gp inhibitor and as such may cause an
increase in the accumulation of some drugs in the brain after
coadministration. It is necessary to pay attention not only to the
inhibition of the CYP3A4-mediated metabolism of coadministered drugs by
ITZ but also to the central nervous system side effects caused by the
enhanced accumulation of drugs in the brain that is induced by the
ITZ-mediated inhibition of P-gp activity.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Faculty of
Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Phone: 81-92-642-6610. Fax: 81-92-642-6614. E-mail: yasufumi{at}yakuzai.phar.kyushu-u.ac.jp.
| |
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Abstract
Introduction
