Vol. 27, Issue 11, 1225-1231, November 1999
Correlation between In Vivo and In Vitro Hepatic Uptake of
Metabolic Inhibitors of Cytochrome P-450 in Rats
Katsuhiro
Yamano,
Koujirou
Yamamoto,
Hajime
Kotaki,
Sayuri
Takedomi,
Hirotami
Matsuo,
Yasufumi
Sawada, and
Tatsuji
Iga
Department of Pharmacy, University of Tokyo Hospital,
Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo,
Japan (Ka.Y., T.I.); Biopharmaceutical and Pharmacokinetic Research
Laboratories, Fujisawa Pharmaceutical Co., Ltd., Kashima, Yodogawa-ku,
Osaka, Japan (Ka.Y.); Department of Clinical Pharmacology School of
Medicine, Gunma University, Showa-machi, Maebashi, Japan (Ko.Y.);
Department of Pharmacy, The Research Hospital, The Institute of Medical
Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan
(H.K.); Faculty of Pharmaceutical Sciences, Kyushu University,
Maidashi, Higashi-ku, Fukuoka, Japan (S.T., H.M., Y.S.)
 |
Abstract |
To predict the degree of accumulation of hepatic metabolic
inhibitors in the liver from the in vitro data, we investigated the
relationship between cell/medium concentration ratios (C/M ratios) in
isolated rat hepatocytes and liver/blood unbound concentration (KBf) after i.v. administration of various metabolic
inhibitors such as itraconazole, ketoconazole, verapamil,
diltiazem, enoxacin, ciprofloxacin, clarithromycin, cimetidine, and
nizatidine. The C/M ratios of itraconazole were ~6000 and 200 at the
concentrations of 0.1 and 10 µg/ml, respectively, and the uptake of
ketoconazole and verapamil into the hepatocytes also showed a
concentration dependence, although the degree was smaller than that of
itraconazole. The uptake of diltiazem, enoxacin, ciprofloxacin, and
clarithromycin into the hepatocytes showed linear profiles on
concentration dependence. There was an excellent correlation between
C/M ratios and KBf values of all nine drugs with a slope of
1. This finding suggested the possibility of predicting drug
concentrations in the liver (CH) from C/M ratios, the blood
concentrations of drugs (CB) and unbound fraction in blood
(fB), which was expressed by CH = (C/M) · CB · fB. It may be possible to predict
the drug concentrations in human liver from KBf values
estimated with isolated human hepatocytes and concentrations in the
blood in a similar manner as in rats.
| |
Introduction |
In clinical cases, many adverse
effects on drug-drug interactions have been reported. The metabolic
inhibition of one drug by another in the liver and/or gut is one of the
most important events among pharmacokinetic drug-drug interactions (Fee
et al., 1987
; Olkkola et al., 1993, 1994, 1996; Backman et al.,
1994; Ahonen et al., 1995; Baldwin et al., 1995). We can estimate the degree of interactions quantitatively to some extent if the metabolic inhibition constants are obtained by using liver microsomes, primary cultured hepatocytes, and/or CYP-expressed cells (Pichard et al., 1990;
Gascon and Dayer, 1991; Wrighton and Ring, 1994; Ghosal et al., 1996).
However, the predicted increase ratios of area under the
concentration-time curve
(AUC)1 of the
interacting drugs were sometimes much underestimated by using the
unbound concentration in plasma as the concentration of inhibitors in
vivo. We could predict the increase ratio of the concentration (or AUC)
of midazolam (MDZ) in the plasma by the metabolic inhibition from in
vitro experiments quantitatively, when itraconazole (ITZ) and
ketoconazole (KTZ), azole antifungal agents, and cimetidine (CIM) and
nizatidine (NIZ), histamine H2 receptor
antagonists, were concomitantly administered as inhibitors (Takedomi et
al., 1998; Yamano et al., 1999). The predicted values were considerably
underestimated by using unbound concentrations in the plasma as
concentrations of inhibitors near the metabolic enzymes, whereas the
predicted values with unbound concentrations in the liver were very
close to the observed values, suggesting the necessity to take account
of the concentrative uptake of inhibitors into liver. In a clinical
situation, the concentrations of the inhibitors in human liver are
required to predict the increase ratio of the concentration; however,
it is difficult to measure drug concentration in the liver directly. It
is thus necessary to develop a methodology to estimate the
concentration in the liver from the concentration in the plasma, which
can be measured actually. In this study, we tried to predict the
concentration in the liver with in vitro uptake data into isolated
hepatocytes in rats. We examined the correlation between cell/medium
concentration ratios (C/M ratios) and the liver/blood unbound
concentration (KBf) values of liver in rats.
| |
Experimental Procedures |
Materials.
ITZ and KTZ were supplied by Janssen-Kyowa Co. (Tokyo, Japan). Enoxacin
(ENX) and ciprofloxacin (CPFX) were supplied by Dainippon Pharmaceutical Corp. (Osaka, Japan). Clarithromycin (CAM) was supplied
by Taisho Pharmaceutical Co. (Tokyo, Japan). Verapamil (VER)
hydrochloride and diltiazem (DLZ) hydrochloride were purchased from
Wako Pure Pharmaceutical Co. (Osaka, Japan). All other chemicals used
as reagents were of reagent grade and reagents for HPLC.
Animals.
Sprague-Dawley male rats (230-260 g) were purchased from Nippon
Bio-Supply Center (Tokyo, Japan). The rats were allowed access to water
and food pellets ad libitum.
Preparation of Drug Solutions.
ENX and CPFX were dissolved in a small volume of 1 N NaOH, neutralized
with a small volume of 0.5 N HCl, and then diluted with saline to
prepare 5 mg/ml ENX or CPFX solutions. VER and DLZ were dissolved in
saline to prepare 5 and 20 mg/ml VER or DLZ solutions. CAM was
dissolved in equimolar HCl, neutralized with a small volume of 0.1 N
NaOH, and then diluted with saline to prepare 5 mg/ml of solution.
Concentrations in Liver and Plasma (Blood) and Liver/Blood
(Unbound) Concentration Ratios.
Under light ether anesthesia, rats were cannulated through the femoral
vein and artery. After recovery from the anesthesia, ENX (10 mg/kg),
CPFX (10 mg/kg), CAM (10 mg/kg), VER (5 mg/kg), or DLZ (5 mg/kg) was
administrated by bolus injection through the femoral vein. At 2, 5, 10, 20, 30, 45, 60, 90, 120, and 180 min after the administration of each
drug, blood samples were collected from the femoral artery and were
centrifuged at 12,000 rpm for 2 min to obtain the plasma. The liver was
then removed at 180 min after the administration and the plasma and
liver were stored at 
20°C until analyzed. The liver was homogenated
with 4 volumes of ice-cold distilled water. The concentrations of each drug in the plasma and liver were determined by the methods described later, and the liver/plasma concentration ratios at 180 min after the
administration were regarded as the apparent liver/plasma concentration
ratio (KP) values.
Plasma concentration profiles were analyzed by fitting the following
biexponential equation with the nonlinear least-squares method (MULTI)
(Yamaoka et al., 1981):
|
(1)
|
Pharmacokinetics parameters were calculated according to the
following equations:
|
(2)
|
|
(3)
|
|
(4)
|
where AUC0-
,
CLtot, and
T1/2
are AUC from zero to
infinity, total body clearance, and half-life in 
phase, respectively.
Additionally, for VER, DLZ, ENX, CPFX, and CAM, the real
KP values were calculated by correcting the
apparent KP values as follows. Rats were
cannulated in the femoral artery and portal vein under light ether
anesthesia. After recovery from the anesthesia, ENX (10 mg/kg), CPFX
(10 mg/kg), CAM (10 mg/kg), VER (20 mg/kg), or DLZ (20 mg/kg) was
administrated by bolus injection through the portal vein. Blood samples
were collected at 2, 5, 10, 20, 30, 45, 60, 90, 120, and 180 min after
the administration of each drug and drug concentrations in the plasma
were determined as described above. The pharmacokinetic parameters were
calculated by the nonlinear least-squares method (MULTI) (Yamaoka et
al., 1981). The hepatic extraction ratios (E) were calculated from AUC
after i.v. administration (AUCi.v.) and AUC after
intraportal administration (AUCpv) regarded as
eq. 5:
|
(5)
|
The hepatic intrinsic clearances (CLint)
were calculated from eq. 6:
|
(6)
|
where Dosei.v. and
Dosepv represent i.v. and portal injection dose, respectively.
The real liver-to-blood concentration ratios
(KB) values were calculated according to
eq. 7 (Lin et al., 1982):
|
(7)
|
where KB,real,
KB,app, and represent real liver/blood
concentration, apparent liver/blood concentration, and elimination rate
constant in the phase, respectively. QH and
VH represent hepatic blood flow rate (55.2 ml/min/kg) and volume of liver (78.4 ml/kg) (Davies and Morris, 1993), respectively.
The apparent KP
(KP,app) values of ITZ, KTZ, CIM, and NIZ
were cited from our reports (Takedomi et al., 1998; Yamano et al., 1999). Hepatic clearances of ITZ and KTZ were much smaller than the
hepatic blood flow rate. For KTZ, CIM, and NIZ, the
KP,app values when the concentrations in
plasma after infusion of drugs became at the steady state ( = 0), were used as KP,app values (Takedomi et
al., 1998; Yamano et al., 1999). Therefore, the
KP,app values of ITZ, CIM, NIZ, and KTZ
were regarded as the real KP values.
The real concentration in liver/unbound concentration in blood ratios
(KBf) of various drugs were calculated
according to eq. 8:
|
(8)
|
where KB, fB,
fP, CB, and
CP represent liver/blood concentration, unbound
fraction in the blood, unbound fraction in the plasma, concentration in
the blood, and concentration in the plasma, respectively. The
fP and the
CB/CP ratios were measured
as follows.
The fP of VER, DLZ, ENX, CPFX, and CAM were
evaluated using the equilibrium dialysis method. Dialysis was performed
with an apparatus made of clear acrylic resin and consisted of two
1.5-ml chambers separated by a cellulose dialysis membrane
(SC-101-M10H; Diachema, Zurich, Switzerland). Each drug was added to
the rat fresh plasma at a concentration of 5 and 20 µg/ml and applied to one chamber and isotonic phosphate buffer (pH 7.4) was applied to
the other. After incubation at 37°C for 6 h, 0.1 ml of sample was collected from both chambers for assay. The
fP of various drugs was calculated according to
eq. 9. The fP of ITZ, CIM, and NIZ was cited from
our reports (Takedomi et al., 1998; Yamano et al., 1999).
|
(9)
|
where Cf and Cb
represent the concentration in the buffer and in the plasma after
equilibrium dialysis, respectively.
The CB/CP ratios of various
drugs were measured as follows. Each drug was added to the rat fresh
blood at a concentration of 0.5, 2, or 10 µg/ml and 1 ml of blood
sample was incubated at 37°C for 15 min. Then, 0.2 ml of sample was
taken, and the plasma was obtained by centrifugation and
CB/CP ratios were
calculated. From the preliminary experiment, we confirmed that
CB/CP ratios were
substantially constant after incubation at 37°C for 15 min and each
drug was stable during incubation.
Uptake Kinetics by Isolated Rat Hepatocytes.
Rat hepatocytes were isolated according to the procedure of Baur et al.
(1975). Cell viability for each experiment was checked by the trypan
blue exclusion test and was in the range of 85 to 95%. Protein
concentration was determined by the colorimetric method of Lowry et al.
(1951). All experiments were completed within 2 h after cell
preparation, at which time the viability had not changed appreciably.
The time courses of the uptake of various drugs into isolated rat
hepatocytes were investigated as follows. Isolated rat hepatocytes (protein concentration, 20 mg/ml) were suspended in Krebs-Henseleit buffer. Then, 0.3 ml of a hepatocyte suspension and 2.7 ml of Krebs-Henseleit buffer (albumin-free) were mixed and preincubated at
37°C for 5 min. Thirty microliters of a standard solution of various
drugs was added to each hepatocyte suspension at a concentration of 1 µg/ml and incubated at 37°C. At 20, 40, 60, 120, or 300 s after addition of drugs, 400 µl of the cell suspensions was removed. For ITZ, a sample also was taken at 600 s. The cell suspensions were placed in 1.5-ml polyethylene tubes previously layered with 500 µl of silicone-oil (specific gravity, 1.050) and 200 µl of 3 N KOH.
The samples were then centrifuged for 10 s in a table-top microfuge capable of extremely rapid acceleration to separate the cells
from the medium. The samples were frozen at 20°C, and then the
sample tubes were cut at the middle of the oil layer. The
concentrations in the upper layers (medium) and lower layers (hepatocytes) were measured to investigate the time courses of the
uptake into isolated rat hepatocytes. The uptake of drugs was corrected
for the adherent fluid volume and then converted to true intracellular
concentration. The values of adherent fluid (2.2 µl/mg protein) and
intracellular space (5.2 µl/mg protein) were obtained from the
literature (Miyauchi et al., 1993).
Concentration dependence on uptake of drugs into isolated rat
hepatocytes was investigated as follows. The concentrations of drugs
were 0.1, 0.2, 0.5, 1, 2, 5, and 10 µg/ml for ITZ, KTZ, VER, and DLZ
and 0.1, 1, and 10 µg/ml for ENX, CPFX, and CAM. Uptake experiments
into isolated rat hepatocytes were performed as described above.
Incubation times for each drug were enough to reach equilibrium (5 min
for KTZ, VER, DLZ, ENX, CPFX, and CAM; 10 min for ITZ).
Measurement of the Concentrations of Various Drugs.
The concentrations of ITZ and KTZ in the plasma and blood were measured
according to the methods reported previously (Yamano et al., 1999).
For the determination of VER and DLZ concentrations in the plasma,
blood, and liver, 0.1 ml of plasma or blood, or 0.5 ml of 20% liver
homogenate were mixed with 0.1 ml of methanol, 0.5 ml of 1 N NaOH, and
2.5 ml of isopropylether and shaken for 5 min, followed by
centrifugation at 3000 rpm for 5 min. Two milliliters of the organic
phase was transferred to another tube and evaporated under nitrogen
gas. The residue was dissolved in 0.2 ml of the mobile phase and 75 µl was injected into HPLC. The chromatographic system consisted of an
autosampler 717 (Waters, Tokyo, Japan), a pump LC-10AD, and an SPD-10A
variable-wavelength UV detector (Shimadzu Corp., Kyoto, Japan)
operating at 229 nm and 237 nm for VER and DLZ, respectively. The
column was a reversed-phase Inertosil ODS, 4.6 mm × 250 mm (GL
Science, Osaka, Japan) and was maintained at 40°C. The mobile phases
were acetonitrile-10 mM phosphate buffer (pH 6.5) (80:20, v/v) for VER
and acetonitrile-10 mM phosphate buffer (pH 3.0) (35:65, v/v) for DLZ
and were pumped isocratically at a flow rate of 1 ml/min. The lower
limit of quantification was 50 ng/ml for plasma and blood and 500 ng/g
for liver.
For the determination of VER and DLZ in separated cells or medium, 0.5 ml of 1 N NaOH and 5 ml of isopropylether were mixed and shaken for 5 min and then centrifuged at 3000 rpm for 5 min. Four milliliters of the
organic phase was then transferred to another tube and back-extracted
with 3 ml of 0.1 N HCl. To 2 ml of the aqueous phase, 0.5 ml of 1 N
NaOH was added and extracted with 2.5 ml of isopropylether. Two
milliliters of the organic phase was transferred to another tube and
evaporated under nitrogen gas. The residue was dissolved in 0.2 ml of
the mobile phase and 75 µl was injected into HPLC. The HPLC condition
was the same as that of the plasma concentration of VER and DLZ.
The concentrations of CIM and NIZ in the plasma and blood were measured
by a modification of the method of Takedomi et al. (1998). In brief,
0.1 ml of plasma or blood, 0.1 ml of methanol, 0.5 ml of 1 N NaOH, and
5 ml of dichloromethane were mixed and shaken for 5 min, and then
centrifuged at 3000 rpm for 5 min. Four milliliters of the organic
phase was then transferred to another tube and evaporated under
nitrogen gas. The residue was dissolved in 0.2 ml of the mobile phase
and 40 µl was injected into HPLC. For detection, a wavelength of 228 nm was used. The column was a reversed-phase YMC-Pack Pro C18, 3.0 mm × 150 mm (YMC, Kyoto, Japan) and was maintained at 40. The
mobile phase was acetonitrile-10 mM phosphate buffer (pH 6.5, 15:85,
v/v), and was pumped isocratically at a flow rate of 0.4 ml/min. The lower limit of quantification was 100 ng/ml for both plasma and blood.
For the determination of ENX and CPFX concentrations in the plasma, 0.1 ml of plasma, 0.2 ml of methanol, 1 ml of 100 mM phosphate buffer (pH
7.4), and 5 ml of chloroform containing 1% ethyl chloroacetate were
mixed and shaken for 10 min and then centrifuged at 3000 rpm for 5 min.
Four milliliters of the organic phase was then transferred to another
tube and evaporated under nitrogen gas. The residue was dissolved in
0.2 ml of the mobile phase and 75 µl was injected into HPLC. The HPLC
system was the same as for the determination of the concentration of
ITZ and KTZ. The wavelength of the UV detector was set at 270 nm. The
column was a reversed-phase YMC-Pack ODS-H, 4.6 mm × 250 mm (YMC)
and was maintained at 40. The mobile phase was acetonitrile-10 mM
phosphate buffer (pH 3.0, 50:50, v/v) and was pumped isocratically at a
flow rate of 1 ml/min. The lower limit of quantification in the plasma
was 100 ng/ml for both ENX and CPFX.
For the determination of ENX and CPFX concentrations in the blood,
liver, hepatocyte suspension, or medium, 0.1 ml of blood or 0.5 ml of
20% liver homogenate, 0.2 ml of methanol, 1 ml of 100 mM phosphate
buffer (pH 7.4), and 5 ml of chloroform containing 10% isopropylalchol
were mixed with the sample and shaken for 10 min and then centrifuged
at 3000 rpm for 5 min. Four milliliters of the organic phase was then
transferred to another tube and back-extracted with 3 ml of 0.01 N
NaOH. To 2 ml of the aqueous phase, 0.4 ml of 0.05 N HCl and 1 ml of
100 mM phosphate buffer (pH 7.4) were added and extracted with 5 ml of
chloroform containing 1% ethyl chloroacetate. The lower limit of
quantification was 100 ng/ml for blood and 500 ng/g for liver.
For the determination of CAM concentrations in the plasma and liver,
0.1 ml of plasma or 0.5 ml of 20% liver homogenate, 0.5 ml of 0.5 N
NaOH, and 3 ml of tert-butyl methylether were mixed and
shaken for 5 min and then centrifuged at 3000 rpm for 5 min. Two
milliliters of the organic phase was then transferred to another tube
and evaporated under nitrogen gas. The residue was dissolved in 50 µl
of the mobile phase and 20 µl was injected into HPLC. The
chromatographic system consisted of a pump LC-10AD and an ECD-10A
electron chemical detector (Shimadzu Corp.). The detector cell
potential for the oxidation was 1300 mV. The column was a reversed-phase TSKgel 80TM ODS, 4.6 mm × 150 mm (Toso, Tokyo, Japan) and was maintained at 30. The mobile phases were
acetonitrile-100 mM phosphate buffer (pH 6.4, 50:50, v/v) and were
pumped isocratically at a flow rate of 1 ml/min. The lower limit of
quantification was 300 ng/ml for both plasma and blood and 1000 ng/g
for liver.
For the determination of CAM in separated cells or medium, 0.5 ml of
0.5 N NaOH and 5 ml of tert-butyl methylether were mixed and
shaken for 5 min and then centrifuged at 3000 rpm for 5 min. Four
milliliters of the organic phase was then transferred to another tube
and back-extracted with 3 ml of 100 mM phosphate buffer (pH 4.0). To 2 ml of the aqueous phase, 0.5 ml of 1 N NaOH was added and extracted
with 2.5 ml of tert-butyl methylether. Two milliliters of
the organic phase was transferred to another tube and evaporated under
nitrogen gas. The residue was dissolved in 50 µl of the mobile phase
and 20 µl was injected into HPLC. The HPLC condition was the same as
that of the plasma concentration of CAM.
In all measurements, coefficients of variation were <10% and
within-run accuracies were <±10%. When the concentrations in the
samples were below the limit of quantification, levels were determined
by increasing the amount of sample.
Statistical Analysis.
Statistical analysis was performed using Student's t test.
Differences were regarded as statistically significant when
p values were <.05.
| |
Results |
Plasma Concentration Profiles and Hepatic Extraction Ratios of
Various Drugs.
Figure 1 and Table
1 show plasma concentration profiles and
the pharmacokinetic parameters of VER, DLZ, ENX, CPFX, and CAM after
intraportal and i.v. administration. The hepatic extraction ratios of
VER, DLZ, ENX, CPFX, and CAM were 0.797, 0.782, 0.228, 0.529, and
0.088, respectively.
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Fig. 1.
Plasma concentration profiles of VER (A),
DLZ (B), ENX (C), CPFX (D), and CAM (E) after bolus i.p. ( ) and i.v.
( ) injection of VER, DLZ, ENX, CPFX, and CAM to rats.
VER, DLZ, ENX, CPFX, and CAM were administered through the femoral vein
at doses of 5, 5, 10, 10, and 10 mg/kg, respectively. VER, DLZ, ENX,
CPFX, and CAM were administered through the portal vein at doses of 20, 20, 10, 10, and 10 mg/kg, respectively. Each point and vertical bar
represent the mean ± S.D. (n = 4-7).
|
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CB/CP Ratios,
KB Values, and fP of Various
Drugs.
Table 2 shows the
CB/CP ratios of various
drugs. In all drugs CB/CP
ratios were within the range of 0.68 to 1.1 and were constant within
the concentration range of 0.5 to 10 µg/ml.
KB values of various drugs were calculated
by eq. 8 and are listed in Table 3. The
average KB,app at 15 min, 1, 4, 8, and
24 h after i.v. administration was used for ITZ. The
KB,app values at the steady state obtained
previously were used for KTZ, CIM, and NIZ (Takedomi et al., 1998;
Yamano et al., 1999). Because the KB,app values of ENX, CPFX, CAM, VER, and DLZ reached a pseudosteady state (at
phase) at 3 h after i.v. administration in a preliminary experiment, the KB,app values at 3 h
after i.v. administration were used for these drugs.
Table 3 shows the fP of various drugs. The
fP varied from 0.0034 for ITZ to 0.96 for NIZ.
The maximum of apparent KBf
(KB,app/fP) and the
minimum were 6100 for ITZ and 3.0 for NIZ, respectively.
Because the KB,real values of VER and DLZ
may be much larger than the KB,app values
due to their large hepatic extraction ratio, hepatic extraction ratios
were determined to calculate the KB,real
values. The real KBf values of VER and DLZ
were 600 and 73 and were 4.9- and 4.6-fold compared with the apparent
KBf values, respectively. The real
KBf values of ENX, CPFX, and CAM were 9.3, 11, and 36, respectively, and were very close to the apparent
KBf values.
Uptake of Various Drugs into Isolated Rat Hepatocytes.
Figure 2 shows the time course of uptake
of various drugs into isolated rat hepatocytes. The uptake of KTZ, VER,
DLZ, ENX, CPFX, and CAM reached equilibrium in 5 min, whereas it took
10 min to reach equilibrium for ITZ. In the concentration-dependence study, incubation times were set to reach equilibrium for each drug.
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Fig. 2.
Time courses of ITZ (A), KTZ (B), DLZ (C),
VER (D), ENX (E), CPFX (F), and CAM (G) uptake by isolated rat
hepatocytes.
The concentrations of drugs in the incubation medium were 1 µg/ml.
Each point and vertical bar represent the mean ± S.D.
(n = 3).
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Figure 3 shows the concentration
dependence on uptake of various drugs into isolated rat hepatocytes.
The uptake of ITZ showed marked concentration dependence. The C/M
ratios of ITZ were ~200 and 6000 at the concentrations of 10 and 0.1 µg/ml, respectively. The concentration-dependence degree of uptake of
KTZ and VER was smaller than that of ITZ, whereas the concentration
dependence for DLZ, ENX, CPFX, and CAM was negligible.
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Fig. 3.
Concentration dependence of ITZ (A), KTZ
(B), DLZ (C), VER (D), ENX (E), CPFX (F), and CAM (G) uptake by
isolated rat hepatocytes.
The incubation times for ITZ and other drugs were 10 and 5 min,
respectively. Each point and vertical bar represent the mean ± S.D. (n = 3).
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Correlation between In Vivo Distribution to Liver and In Vitro
Uptake by Isolated Rat Hepatocytes.
The correlation between C/M ratios of drugs on the uptake into isolated
rat hepatocytes and KBf values in rat liver
was investigated for the evaluation of the usefulness of the in vitro
experiments for predicting the concentrations in the liver after
administration of the inhibitors. C/M ratios at concentrations nearly
equal to the unbound concentrations in the plasma were used to
investigate the correlation between C/M ratios and the
KBf values of each drug except for ITZ.
Because the unbound concentrations of CIM and NIZ after infusion at the
rate of 5.7 and 11.4 mg/h/body to rats were 0.5 to 5 and 1 to 5 µg/ml, respectively, C/M ratios at the added concentrations of 3 µM
(0.76 µg/ml) and 3 µM (0.99 µg/ml) were quoted for CIM and NIZ
(Nakamura et al., 1994), respectively. The unbound concentrations of
ITZ in the plasma after i.v. administration at the dose of 20 mg/kg to
rats were within the range of 5 to 20 ng/ml, but it was difficult to
determine C/M ratio of ITZ at such a low concentration. In this study,
C/M ratio at an added concentration of 100 ng/ml was used because there
was no significant difference between C/M ratios at the added
concentrations of 0.1 and 0.2 µg/ml, suggesting constant C/M ratios
at the lower concentration. Figure 4
shows the correlation between C/M ratios and
KBf values of the nine drugs. There was an
excellent correlation with a slope of unity between logarithm of C/M
ratios and KBf values (r = 0.981).
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Fig. 4.
Correlation between C/M ratios on uptake by
isolated rat hepatocytes and liver/blood concentration ratios.
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Discussion |
To develop a methodology to predict the risk of drug-drug
interactions quantitatively, it is necessary to solve four problems: 1)
prediction of the disposition of inhibitors in the liver is very
important because many drugs are transported into the liver by
carrier-mediated hepatic uptake systems (Meijer et al., 1990; Yamazaki
et al., 1996) and unbound concentrations in the liver are higher than
those in the plasma; 2) prediction of the concentrations of inhibitors
in the portal vein or the hepatic vein is necessary because in the
clinical field, most drugs are orally administered and the drug
concentration in the portal vein is higher than that in the systemic
circulation (Hoffman et al., 1995; Tabata et al., 1995; Fujieda et al.,
1996); 3) prediction of drug-drug interactions on the metabolic
process in the intestine (jujunal, ileum) as well as in the liver is
necessary because CYP3A4 activity in the intestine is half as much as
that in the liver. It was reported that MDZ and cyclosporin were
metabolized by CYP3A4 in the intestine (Paine et al., 1996; Thummel et
al., 1996); and 4) prediction of the drug-drug interactions on
the absorption process is necessary because P-glycoprotein exists in
the intestine and takes part in the secretion of drugs.
However, we suggest that it is difficult to solve all above-mentioned
problems at the same time. To evaluate the extent of drug-drug
interactions concerning metabolic inhibition in the liver
quantitatively, we tried to predict the plasma concentration increasing
ratio (R) of MDZ in rats by using MDZ with concomitant administration
of ITZ, KTZ, CIM, and NIZ as inhibitors (Takedomi et al., 1998; Yamano
et al., 1999). Assuming that the interaction of drug metabolism is of a
competitive inhibition type, the increasing ratio of plasma
concentration can be estimated with the following equation:
|
(10)
|
where I and Ki represent the
concentration of inhibitor and inhibition constant, respectively. The
increasing ratios predicted with the unbound concentration in the
plasma were underestimated, whereas the increase ratios predicted with
the unbound concentration in the liver were very close to the observed
increase value. The liver unbound concentration to the plasma unbound
concentration ratios of the inhibitors were >1, suggesting a
concentrative uptake of these drugs into the liver. Because the
metabolic enzymes are localized on the endoplasmic reticulum in the
hepatocytes and are physically separated from the blood by the plasma
membrane, the unbound concentrations in the liver may be more
appropriate for predicting the increase rate of plasma concentration
quantitatively. In humans, the liver unbound concentrations of the
inhibitors are required. However, it is difficult to measure directly
the liver unbound concentrations of the inhibitors; therefore, a
methodology to predict the concentrations in the liver after
administration of the inhibitors is necessary. We tried to predict the
concentrations in the liver after administration of drugs to rats.
First, we investigated the correlation between the
KBf values in rats and C/M ratios of the
uptake into isolated rat hepatocytes. Second, we examined the
possibility of the prediction of the concentration in the liver with
C/M ratios and the concentrations in the plasma or the blood.
As C/M ratios of the uptake into isolated rat hepatocytes were the
values when the uptake of drugs reached equilibrium, the KBf values in rats when the liver
concentrations were in parallel with the concentrations in the plasma
or blood were used. As for VER and DLZ, the liver is the main
disappearance organ and the majority of the total clearance is the
hepatic clearance. The total clearances after i.v. administration of
VER and DLZ were 43.4 ± 4.2 and 63.9 ± 6.3 ml/min/kg
(mean ± S.D., n = 4), respectively, and were
similar to the hepatic blood flow rates. The
KB,real values of VER and DLZ may be much
larger than the KB,app values because of
their large hepatic extraction ratio. Therefore, their hepatic
extraction ratios were calculated from AUC after intraportal and i.v.
administration and then the KB,real values
were calculated according to eq. 7. The total clearances after i.v.
administration of ENX and CPFX were 38.7 ± 2.0 and 51.9 ± 14.4 ml/min/kg (mean ± S.D., n = 4),
respectively, but the elimination routes were both via hepatic
metabolism and renal excretion and the hepatic clearances of ENX and
CPFX were 12 and 25 ml/min/kg, respectively (Davis et al., 1995) and
were much less than the hepatic blood flow rate. Therefore, the
KBf values calculated from the hepatic clearance and the hepatic blood flow rate were close to the real KBf values.
The concentration-dependent uptake of ITZ, KTZ, and VER into isolated
rat hepatocytes was observed and suggested saturable carrier-mediated
uptake, whereas the uptakes of other drugs into isolated rat
hepatocytes were not dependent on drug concentrations. Figure 4 shows
the good correlation between C/M ratios of drugs and real
KBf values in rats. Therefore, drug
concentrations in the liver (CH) after
administration to rats can be predicted from C/M on the uptake into rat
isolated hepatocytes and drug concentrations in blood, as in eq. 11:
|
(11)
|
Cryopreservation of human hepatocytes has been established and
human hepatocytes with high viability can be used (Adams et al., 1995).
Lave et al. (1997) evaluated the use of human hepatocytes to classify
compounds into low, intermediate, or high hepatic extraction ratio in
humans, and reported that in vitro clearances in human hepatocytes were
predictive for the hepatic ratios in vivo in humans and that human
hepatocytes seemed to be a valuable way for screening compounds with
respect to liver first-pass metabolism. Sun et al. (1996) investigated
the biotransformation of lifibrol, a lipid-lowering drug, by using
human hepatocytes in primary culture. Human hepatocytes offer certain
advantages for predicting the degree of drug metabolism and interaction
in humans at the biotransformation level (Fischer et al., 1997). Olinga
et al. (1998) investigated the mechanisms and specificity of the uptake
of the cardiac glycoside digoxin and the organic cation rocuronium into
human hepatocytes. The hepatic extraction ratio was then calculated
from the measured uptake rates of the compounds into human hepatocytes
and compared with published in vivo data. The initial hepatic
extraction ratio, calculated from the in vitro uptake data for digoxin
and rocuronium, very well reflected the initial extraction ratio for
distribution in the liver in vivo in humans. In a similar manner as rat
hepatocytes, we can possibly predict the concentrations in human liver
from the concentrations in the blood and
KBf values estimated with human isolated
hepatocytes. Moreover, it may be possible to predict drug-drug
interactions based on the inhibition of hepatic metabolism in humans
quantitatively from inhibition constant (Ki
value) obtained by in vitro metabolic inhibition experiments and the
concentration in the liver predicted from in vitro data. We suggest
that this is one of the best solutions of point 1 mentioned above.
| |
Footnotes |
Received February 16, 1999; accepted June 15, 1999.
Send reprint requests to: Katsuhiro Yamano,
Biopharmaceutical and Pharmacokinetic Research Laboratories, Fujisawa
Pharmaceutical Co., Ltd., 1-6, Kashima 2-chome, Yodogawa-ku, Osaka
532-8514, Japan.
| |
Abbreviations |
Abbreviations used are:
AUC, area under the
concentration-time curve;
MDZ, midazolam;
ITZ, itraconazole;
KTZ, ketoconazole;
CIM, cimetidine;
CAM, clarithromycin;
NIZ, nizatidine;
C/M, concentration in cells to concentration in medium ratio;
KBf, liver concentration to unbound
concentration in blood ratio;
ENX, enoxacin;
CPFX, ciprofloxacin;
CAM, clarithromycin;
VER, verapamil;
DLZ, diltiazem;
KP, liver/plasma concentration ratio;
AUCiv, area under the plasma concentration-time curve after
i.v. administration;
AUCpv, area under the plasma
concentration-time curve after intraportal administration;
KB, real liver/blood concentration ratio;
KB,app, apparent liver/blood concentration
ratio;
QH, hepatic blood flow rate;
VH, volume
of liver;
fb, unbound fraction in blood;
fP, unbound fraction in plasma;
CB, concentration in blood;
CP, concentration in plasma;
R, increasing ratio.
| |
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DRUG METABOLISM AND DISPOSITION
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Abstract
Introduction
