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DRUG METABOLISM AND DISPOSITION
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
DMD 29:1316–1324, 2001
Vol. 29, No. 10
368/932638
Printed in U.S.A.
PREDICTION OF HUMAN HEPATIC CLEARANCE FROM IN VIVO ANIMAL EXPERIMENTS
AND IN VITRO METABOLIC STUDIES WITH LIVER MICROSOMES FROM ANIMALS AND
HUMANS
YOICHI NARITOMI, SHIGEYUKI TERASHITA, SUMIHISA KIMURA, AKIRA SUZUKI, AKIRA KAGAYAMA,
YUICHI SUGIYAMA
AND
Biopharmaceutical and Pharmacokinetic Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Kashima, Yodogawa-ku, Osaka, Japan
(Y.N., S.T., S.K., A.S., A.K.); and Graduate School of Pharmaceutical Sciences, the University of Tokyo, Hongo, Bunkyou-ku,
Tokyo, Japan (Y.S.)
(Received February 20, 2001; accepted July 3, 2001)
This paper is available online at http://dmd.aspetjournals.org
We investigated the quantitative prediction of human hepatic metabolic clearance from in vitro experiments focusing on cytochrome
P450 metabolism with eight model compounds, FK1052, FK480,
zolpidem, omeprazole, nicardipine, nilvadipine, diazepam, and diltiazem. For the compounds, in vivo human hepatic extraction ratios
ranged widely from 0.03 to 0.87. In vitro and in vivo hepatic intrinsic
clearance (CLint) values for each compound were measured and calculated in rats and/or dogs and humans. CLint,in vitro was determined
from a substrate disappearance rate at 1 ␮M in hepatic microsomes,
which was a useful method. CLint,in vivo was calculated from in vivo
pharmacokinetic data using three frequent mathematical models (the
well stirred, parallel-tube, and dispersion models). The human scaling
factor values (CLint,in vivo/CLint,in vitro) showed marked difference
among the model compounds (0.3–26.6-fold). On the other hand,
most of the animal scaling factors were within 2-fold of the values in
humans, suggesting that scaling factor values were similar in the
different animal species. When human CLint,in vitro values were compared with the actual CLint,in vivo, correlation was not necessarily
good. By contrast, using human CLint,in vitro corrected with the rat
and/or dog scaling factors yielded better predictions of CLint,in vivo
that were mostly within 2-fold of the actual values. Furthermore,
successful predictions of human CLoral and hepatic extraction ratio
(EH) were obtained by use of the human CLint,in vitro corrected with
animal scaling factors. The new variant method is a simple one,
incorporating additional information from animal studies and providing a more reliable prediction of human hepatic clearance.
In recent years, the process of drug discovery and development has
become an increasingly time-consuming and costly endeavor. Much
of the time and cost are expended on generating data that support the
efficacy and safety profiles of the drug. Safe and efficient drug
candidates must therefore be selected before clinical trials.
On the other hand, there is growing awareness of the key roles that
pharmacokinetics and drug metabolism play as determinants of in
vivo action. In these situations, early pharmacokinetic investigation
plays an increasingly important role in the optimization and selection
of drug candidates. In particular, it is important to predict human
hepatic metabolic clearance because many drugs are eliminated from
the body by hepatic metabolism. For predicting hepatic clearance,
theoretical aspects of in vitro/in vivo scaling, based on a physiological
model and clearance concepts, have been developed (Rane et al.,
1977; Lin et al., 1982; Roberts and Rowland, 1986; Wilkinson, 1987).
Application of this method has been successful in predicting in vivo
hepatic clearance in rats for many drugs metabolized by P4501 from
in vitro metabolism data using rat liver microsomes and isolated
hepatocytes (Sugiyama et al., 1988; Houston, 1994). Since human
liver samples have become more readily available, it would also be
very useful to predict in vivo from in vitro data in humans.
However, there has been relatively limited application of this
approach (Hoener, 1994; Iwatsubo et al., 1997b), and there have
been many failed attempts at predicting human hepatic clearance.
For example, Iwatsubo et al. (1997a) reported a comparison of
CLint,in vitro and CLint,in vivo for 25 metabolic reactions in humans
from literature data. According to the report, although CLint,in vitro
generally exhibited a positive correlation with CLint,in vivo, more
than a 3-fold difference was observed in CLint for about 50% of the
25 metabolic reactions. Houston and Carlile (1997) also compared
CLint,in vitro obtained from in vitro experiments using rat liver
microsomes with CLint,in vivo for 28 drugs metabolized by P450.
Although the predictability of CLint,in vivo from in vitro data was
good overall, the results of some of the drugs tended to be low
estimates. To improve the predictions of human hepatic clearance,
a few investigators have described new methods and approaches.
For example, Lave et al. (1997) have proposed the allometric
scaling techniques combined with in vitro data. Obach (1999) has
1
Abbreviations used are: P450, cytochrome P450; HPLC, high-performance liquid chromatography; CL, plasma clearance; CLH, hepatic clearance;
CLint, intrinsic metabolic or hepatic clearance; CLR, renal clearance; DN,
dispersion number; EH, hepatic extraction ratio; Fa, the fraction absorbed from
the intestinal tract; FH, hepatic availability; fp, unbound fraction in plasma (or
serum); fu,microsome, unbound fraction in microsome; QH, hepatic blood flow
rate; RB, blood-to-plasma concentration ratio.
Address correspondence to: Yoichi Naritomi, Biopharmaceutical and Pharmacokinetic Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Kashima,
Yodogawa-ku, Osaka 532-8514, Japan. E-mail: [email protected].
co.jp
1316
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ABSTRACT:
PREDICTION OF HUMAN HEPATIC CLEARANCE
1317
• Clearances of model compounds are determined by hepatic P450
metabolism
• Extrahepatic clearances are assumed to be negligible
• In vivo pharmacokinetic parameters in rats and/or dogs, and humans
are reported
• Absorption rates are good with no species difference
reported that inclusion of microsome binding values in the prediction of clearance from in vitro data appears to be a more broadly
applicable approach.
In the present study, we have examined in vitro and in vivo
metabolic clearance of eight model compounds, FK1052, FK480,
zolpidem, omeprazole, nicardipine, nilvadipine, diazepam, and diltiazem, and calculated the CLint,in vitro and CLint,in vivo using in vitro and
in vivo metabolism data in rats, dogs, and humans. At the same time,
the measurement method of CLint,in vitro, which is determined from a
substrate disappearance rate at 1 ␮M in hepatic microsomes, was used
as a simple and useful method. We have also compared the parameters
and evaluated a quantitative prediction method of human hepatic
clearance focusing on P450 in drug discovery.
Materials and Methods
Chemicals. FK1052, FK480, and nilvadipine were synthesized by Fujisawa
Pharmaceutical Co., Ltd. (Osaka, Japan). Zolpidem hemitartrate and omeprazole sodium were kindly provided by Fujisawa-Synthelabo Pharmaceuticals
(Tokyo, Japan) and Astra Japan, Ltd. (Osaka, Japan), respectively. Diltiazem
hydrochloride and nicardipine hydrochloride were purchased from Sigma
Chemical Co. (St. Louis, MO). Diazepam was purchased from Wako Pure
Chemical Industries, Ltd. (Osaka, Japan). NADP⫹, glucose-6-phosphate,
and glucose-6-phosphate dehydrogenase were obtained from Sigma Chemical Co. The other regents and solvents used were of analytical and HPLC
grade.
Selection of Model Compounds. FK1052, FK480, omeprazole, zolpidem,
nicardipine, nilvadipine, diltiazem, and diazepam (Fig. 1) were selected as the
model compounds based on the following conditions:
C共t兲 ⫽ C 0 䡠 exp共⫺ke 䡠 t兲
(1)
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FIG. 1. Chemical structures of model compounds.
Although it has been reported that diltiazem is metabolized in part by liver
microsomal esterase in rats (LeBoeuf and Grech-Bélanger, 1987), the compound was examined for reference.
Hepatic Microsomes. Liver specimens from adult male Sprague-Dawley
rats (250 –270 g, n ⫽ 3; Charles River Japan, Inc., Yokohama, Japan) and adult
male dogs (9.5–10 kg, n ⫽ 3; Japan Laboratory Animals, Inc., Tokyo, Japan)
were rinsed and homogenized with ice-cold 1.15% KCl. These pooled microsomes were prepared by differential centrifugation, and the 105,000g pellet
was rinsed and resuspended in 1.15% KCl. Pooled human microsomes were
obtained from Human Biologics International (Scottsdale, AZ). The pooled
human microsomes were prepared from 15 individual liver donors that were
selected on the basis of having average activities for the major P450 isozymes
(1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 4A11). Each suspension was
divided into aliquots, frozen, and stored at ⫺80°C until used.
In Vitro Metabolism in Microsomes. In vitro experiments. The time
courses of the unchanged model compounds in microsomes were obtained.
Each compound was incubated with a reaction mixture (500 ␮l) consisting of
animal or human liver microsomal protein and NADPH-generating system (2
mM NADP⫹, 10 mM glucose-6-phosphate, 0.4 U/ml glucose-6-phosphate
dehydrogenase, and 5 mM MgCl2) in the presence of 100 mM potassium
phosphate buffer (pH 7.4). After preincubation at 37°C for 5 min, enzyme
reactions were initiated by adding 5 ␮l of model compound solution in
methanol. The final concentration of each model compound used was 1 ␮M.
The microsomal concentrations used were 0.2 mg/ml (nicardipine, nilvadipine,
and diltiazem), 0.5 mg/ml (FK1052, zolpidem, omeprazole, and diazepam),
and 1.0 mg/ml (FK480). After incubation at 37°C for various time periods, the
reactions of FK1052, FK480, omeprazole, zolpidem, nicardipine, and diltiazem were terminated by the addition of 500 ␮l of acetonitrile. The reactions
of diazepam and nilvadipine were terminated by adding 500 ␮l of methanol
and 3 ml of ethyl acetate, respectively. After stopping the metabolic reactions,
the reaction mixtures of FK1052, FK480, omeprazole, zolpidem, nicardipine,
diltiazem, and diazepam were centrifuged at 10,000g for 5 min, and an aliquot
of the supernatant was injected on an HPLC for measuring the unchanged
compound concentration. The reactions of nilvadipine were processed by
extraction. The organic fraction was evaporated under N2, and the residue was
reconstituted in the mobile phase (see below) for HPLC analysis.
Determination of unchanged model compound concentrations. An LC module I plus (Millipore Co., Milford, MA) was used. The column for the analysis
was an Inertsil ODS-3 (5 ␮m, 150 ⫻ 4.6 mm) (GL Science, Inc., Tokyo,
Japan). The flow rate was 1.0 ml/min. The mobile phase and detection
wavelength for the analysis of each model compound was as follows: FK1052,
mobile phase: buffer A (5 mM phosphate buffer, pH 7.2)/CH3CN (50:50),
detection: UV 242 nm; FK480, mobile phase: buffer A/CH3CN (40:60),
detection: UV 295 nm; Zolpidem, mobile phase: buffer A/CH3CN (55:45),
detection: UV 254 nm; Omeprazole, mobile phase: buffer A/CH3CN (60:40),
detection: UV 302 nm; Nicardipine, mobile phase: buffer A/CH3CN (33:67),
detection: UV 240 nm; Nilvadipine, mobile phase: buffer A/CH3CN (40:60),
detection: UV 245 nm; Diazepam, mobile phase: buffer A/CH3OH (35:65),
detection: UV 254 nm; Diltiazem, mobile phase: buffer A/CH3CN (50:50),
detection: UV 240 nm.
All assay methods showed the concentration range of 0.1 to 2 ␮M. Reproducibility was evaluated by performing five replicate analyses of microsomal
samples containing 0.1, 0.5, and 1 ␮M compound, respectively. The coefficient of variation was less than 10%, and the actual concentration of the
compounds ranged from 88 to 112.1%. All assay methods thus provide good
accuracy and precision.
Calculation of CLint,in vitro. CLint,in vitro values were calculated from the
substrate disappearance rate in hepatic microsomes as follows. If substrate
disappearance can be assumed to follow a first-order reaction, the unchanged
drug profile as a function of time [C(t)] is described as follows:
1318
NARITOMI ET AL.
TABLE 1
Physiological parameters for calculation of intrinsic clearance in rats, dogs, and
humans
Microsomal protein per gram of liver
(mg of protein/g of liver)
Liver weight per kilogram of body
weight (g of liver/kg)
Liver blood flow (ml/min/kg)
Dog
Human
44.8
77.9
48.8
40
32
25.7
55.2
30.9
20.7
V 0 ⫽ k e 䡠 C0 /PMS
(2)
V 0 ⫽ V max 䡠 C0 /共Km ⫹ C0 兲
V 0 ⫽ V max/Km 䡠 C0
(4)
CLint,in vitro ⫽ Vmax/Km ⫽ V0 /C0
(5)
Consequently,
(10)
a ⫽ 共1 ⫹ 4RN 䡠 DN兲1/ 2
(11)
RN ⫽ 共fp/RB兲 䡠 CLint/QH
(12)
DN ⫽ 0.17
(13)
CLint,in vivo for omeprazole in dogs was calculated from eqs. 7 to 13 by use of
the CLtot value, where CLH is equal to CLtot/RB.
EH were calculated from eq. 14.
EH ⫽ 1 ⫺ FH ⫽ CLH/QH
(14)
Prediction of Human CLint,in vivo, CLoral, and EH. Estimation of scaling
factor. Values of scaling factor were estimated from the following equation:
Scaling factor ⫽ CLint,in vivo/CLint,in vitro
(15)
Prediction of human CLint,in vivo. Human CLint,in vivo were predicted based on
human CLint,in vitro using the following two methods: 1) disregarding animal
scaling factor:
Predicted human CLint,in vivo ⫽ human CLint,in vitro
(16)
2) including animal scaling factor:
CLint,in vitro was thus calculated by eq. 5 based on the time course of unchanged
drug concentrations by least square linear regression. The CLint,in vitro values
expressed per milligram of microsomal protein calculated from the in vitro
metabolism experiments were expressed per kilogram of body weight by
taking the microsomal protein content per gram liver and the liver weight per
kilogram of body weight shown in Table 1 into consideration.
In Vivo Data. Sources of pharmacokinetic data. In vivo clearance under
linear conditions, fu and RB data, were obtained from in house and literature.
The in vivo pharmacokinetic data were considered to be reliable since the in
vivo pharmacokinetic experiments were performed based on accurate methods
and appropriate protocols. The in vivo clearance value was calculated by
dividing the dose by the area under the plasma concentration curve. When the
in vivo clearance value was not expressed per kilogram of body weight, this
value was converted so that it was expressed per kilogram of body weight by
taking the mean value of body weight in the literature or a body weight of
250 g, 10 kg, and 70 kg for rats, dogs and humans, respectively. The fraction
absorbed from the intestinal tract (Fa) of FK1052, FK480, and zolpidem were
calculated by summing the recoveries of radioactivity in bile and urine after
oral administration of 14C model compounds to rats. Fa of omeprazole, nicardipine, nilvadipine, diazepam, and diltiazem were estimated to be 1.0 from the
literature data.
Calculation of CLint,in vivo. CLH values were determined from eq. 6 by use
of the CLoral values, except the CLH for omeprazole in dogs, where only CLtot
data was available. CLR was considered to be negligible for the model
compounds.
CLH ⫽ 共CLoral/RB兲 䡠 共FH 䡠 Fa兲
CLH ⫽ QH 䡠 共1 ⫺ FH兲
Predicted human CLint,in vivo ⫽ human CLint,in vitro 䡠 animal scaling factor
(17)
For these CLint,in vivo predictions, success was assessed by the geometric mean
of the ratio of predicted and actual values (Obach, 1999). Thus:
Average fold error ⫽ 10兵⌺兩log共actual/predicted兲兩其/n
(18)
Prediction of in vivo human CLoral and EH. In vivo human CLoral and EH
were predicted using eqs. 6 to 14, based on the human CLint,in vitro corrected
with animal scaling factor.
Binding of Model Compounds to Microsomes. Determination of
fu,microsome. Model compounds (final concentration, 1 ␮M) were mixed with
liver microsomes (at protein concentrations used for the respective metabolic
incubations) in 100 mM phosphate buffer (pH 7.4) containing 10 mM glucose6-phosphate, 0.4 U/ml glucose-6-phosphate dehydrogenase, and 5 mM MgCl2.
The mixtures (1.2 ml) were delivered to one side of a dialysis cell containing
a preconditioned dialysis membrane (molecular weight cut off, 10 kDa) (Dainippon Pharmaceutical Co., Ltd., Osaka, Japan). To the other side of the
membrane was delivered 1.2 ml of the phosphate buffer containing glucose6-phosphate and MgCl2. The cells were sealed, and the apparatus was incubated at 37°C for 4 h. After an incubation period, the microsome and buffer
samples were removed and analyzed by HPLC. The HPLC conditions were the
same as described above. The unbound fraction was calculated from the
following:
fu, microsome ⫽
unchanged compound concentration in buffer side
unchanged compound concentration in microsome side
(19)
(6)
CLint,in vivo was calculated from the following equations using the well stirred,
parallel-tube (Pang and Rowland, 1977), and dispersion models (Roberts and
Rowland, 1986).
Well stirred model:
4a
共1 ⫹ a兲2 exp兵共a ⫺ 1兲/2DN其 ⫺ 共1 ⫺ a兲2 exp兵⫺共a ⫹ 1兲/2DN其
(3)
If the substrate concentration used in the experiments (1 ␮M) is below the Km
for the P450-mediated reactions, the drug concentration may be assumed to be
much smaller than Km (Km ⬎⬎ C0). Thus, V0 can be expressed by eq. 4.
(9)
Calculation of CLuint,in vitro and the scaling factor. By incorporating the
correction with the unbound fraction in the microsomal incubation mixture,
CLuint,in vitro is defined as (Iwatsubo et al., 1997a):
CLuint,in vitro ⫽ CLint,in vitro/fu, microsome
(20)
(7)
The value of scaling factor, which is the ratio of CLint,in
was calculated from eq. 15
vivo
to CLuint,in
vitro
,
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where PMS is the microsomal protein concentration (mg/ml).
On the other hand, from the Michaelis-Menten equation, V0 is described by
eq. 3.
FH ⫽ exp兵⫺共fp/RB兲 䡠 CLint其
Dispersion model:
FH ⫽
where C0 is initial concentration of the compound, and ke is the disappearance
rate constant of unchanged drug (per minute).
Furthermore, initial metabolic rate (V0) per unit milligram of microsomal
protein (␮mol/min/mg microsomal protein) is described by eq. 2.
(8)
Parallel-tube model:
Reference: Iwatsubo et al., 1997b, Davies and Morris, 1993
Rat
FH ⫽ QH/兵QH ⫹ 共fp/RB兲 䡠 CLint其
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PREDICTION OF HUMAN HEPATIC CLEARANCE
TABLE 2
Estimation of CLint, in vivo from in vivo pharmacokinetic data for the model compounds in rats, dogs, and humans
CLint, in vivo
Compounds
Species
CLoral or CLtot
Fa
fp
RB
EH
Well Stirred
ml/min/kg body weight
FK1052
Diltiazem
a
b
Rat
Dog
Human
875
62.7
26.4
81.1
6.6
2.4
106
7.1
710
6.3a
22.2
1212
597
131
2852
36
107
103.8
0.7
300.2
201.6
64.2
Reference
Dispersion
ml/min/kg body weight
0.95
0.70
0.89
1.00
1.00
1.00
1.00
1.00
0.03
0.017
0.016
0.01
0.006
0.005
0.13
0.04
0.125
0.097
0.042
0.084
0.062
0.068
0.0117
0.008
0.013
0.137
0.032
1.21
0.82
0.78
0.626
0.6
0.6
0.89
0.66
0.78
0.60
0.62
1.0b
1.0b
1.0b
0.922
0.836
0.789
1.04
1.04
0.184
0.298
0.22
0.93
1.0b
1.0
27708.33
3503.82
1567.50
5677.00
770.00
336.00
725.69
157.98
5680.00
98.38
528.57
14428.57
9629.03
1926.47
243760.68
4500.00
8230.77
757.66
21.88
5785.60
1802.28
945.95
3358.32
687.51
315.14
405.00
129.84
985.69
79.36
306.86
2059.22
1500.95
606.32
17589.94
2818.31
2539.97
432.66
21.53
7952.74
2114.04
1071.18
3824.30
709.16
320.79
466.88
137.31
1393.01
84.03
350.73
2992.55
2154.28
779.70
27932.16
3170.52
3277.20
496.31
21.53
1631.52
676.54
291.82
536.77
209.27
132.79
684.98
269.91
159.59
0.93
0.70
0.61
0.62
0.20
0.12
0.66
0.32
0.94
0.34
0.63
0.96
0.95
0.86
0.98
0.58
0.87
0.64
0.03
In house data
In house data
Ishibashi et al. (1993a,b)
Kudo et al. (1990)
Watanabe et al. (1994)
Regårdh et al. (1985)
Andersson et al. (1990)
Higuchi and Shiobara (1980a,b)
Tokuma et al. (1987, 1988)
Terakawa et al. (1988)
Niwa et al. (1987); in house data
Diaz-Garcia et al. (1992); Luurila et al. (1996)
Navia and Chaturvedi (1996); Sawada et al.
(1985); Klotz et al. (1976)
0.85 Tsui et al. (1994); Etoh et al. (1983)
0.87 Kölle et al. (1983); Levet-Trafit et al. (1996)
0.76 Lee et al. (1992); Piepho et al. (1982)
CLtot.
Assumed value.
Results
In Vivo Pharmacokinetic Data of Model Compounds. In vivo
pharmacokinetic data for the model compounds are summarized in
Table 2. Fa values were high, in the range of 0.7 to 1.0. The values of
unbound fraction in plasma (or serum) (fp) were relatively low for all
compounds ranging from the highest value for diltiazem (fp: rat,
0.184; dog, 0.298; human, 0.22) to the lowest value for FK480 (fp: rat,
0.01; dog, 0.006; human, 0.005). In vivo clearance, CLint,in vivo, and
EH values differed markedly among the different species for each
compound. Omeprazole, for example, is characterized by a high EH
(0.94) in rats. An intermediate EH value (0.63) was observed in
humans, whereas dogs exhibited a low EH (0.34). In vivo human EH
ranged widely from 0.03 for diazepam to 0.87 for nilvadipine among
the model compounds.
In Vitro Metabolism in Microsomes and Estimation of Scaling
Factor. Figure 2 illustrates the time courses of the unchanged model
compounds in microsomes. The unchanged drug profiles at 1 ␮M
showed that the linear log concentration declines so that the metabolism follows first-order reaction under this condition. Large interspecies differences were also observed in CLint,in vitro for the model
compounds. Table 3 shows the human CLint,in vitro calculated from the
time courses of the model compounds in microsomes and the values
of human scaling factor, which are the ratios of CLint,in vivo obtained
from in vivo pharmacokinetic data to CLint,in vitro. The human scaling
factor values calculated using the well stirred model were about
26.6-fold for FK1052, about 5-fold for FK480, zolpidem, omeprazole,
and nilvadipine, and 1- to 2.5-fold for nicardipine, diazepam, and
diltiazem, showing marked difference among the model compounds.
In the same way, the human scaling factor values calculated using the
parallel-tube and the dispersion models were 0.3- to 16.1-fold and 0.4to 18.2-fold, respectively, showing marked difference among the
model compounds.
Figure 3 shows the differences in scaling factor between animals
and humans. Most values of animal scaling factor were within 2-fold
of the values in humans, except that the animal scaling factors for
FK480 were 3.5- to 5.4-fold larger than that in humans. The results do
not depend on the kind of the mathematical models and animal
species.
Prediction of Human CLint,in vivo, CLoral and EH by Use of
Animal Scaling Factor. Human CLint,in vitro values corrected both
with and without animal scaling factor are plotted versus actual
CLint,in vivo values calculated using the mathematical models in Fig. 4.
Without animal scaling factor consideration, only two (the well stirred
model) or three (the parallel-tube and the dispersion models) of eight
prediction values were within 2-fold of the actual CLint,in vivo, resulting in a large underestimation for most of the compounds. By contrast,
using human CLint,in vitro corrected with rat and/or dog scaling factor,
9 (the parallel-tube model) or 10 (the well stirred and the dispersion
models) of 13 prediction values were within 2-fold of the actual
CLint,in vivo, significantly improving the predictability of CLint,in vivo.
The geometric mean accuracy values as indices of human CLint,in
vivo predictability are listed in Table 4. The value of one means perfect
predictability, and the poorer becomes predictability with the larger
values. The geometric mean accuracy values without animal scaling
factor consideration were 3 to 4. By contrast, the predictability was
substantially improved with the geometric mean accuracy value of
less than 2.
Furthermore, human in vivo CLoral and EH values were predicted
from human CLint,in vitro corrected with animal scaling factor. Figure
5 presents comparisons of the predicted CLoral values with the observed CLoral. The CLoral values predicted using the well stirred
model were in good agreement with the observed values. When CLoral
was predicted using the parallel-tube and the dispersion models, good
correlations were observed, with the exception of the overestimation
for nilvadipine.
Figure 6 shows the relation between CLint,in vitro and in vivo EH.
The predicted EH based on fb 䡠 CLint,in vitro corrected with animal
scaling factor was close to the observed EH for all mathematical
models. This result suggests that the human CLint,in vitro with animal
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Rat
Dog
Human
FK480
Rat
Dog
Human
Zolpidem
Rat
Human
Omeprazole Rat
Dog
Human
Nicardipine Rat
Dog
Human
Nilvadipine Rat
Dog
Human
Diazepam
Rat
Human
Parallel-Tube
1320
NARITOMI ET AL.
FIG. 3. Differences in scaling factor between animals and humans.
FIG. 2. Time courses of unchanged compounds in microsomes.
Each compound (at a concentration 1 ␮M) was incubated for various time
periods at 37°C in rat, dog, and human liver microsomes. Microsomal protein
concentrations used were 0.2 mg/ml (nicardipine, nilvadipine, and diltiazem), 0.5
mg/ml (FK1052, zolpidem, omeprazole, and diazepam), and 1.0 mg/ml (FK480). f,
rat liver microsomes; Œ, dog liver microsomes; F, human liver microsomes. The
solid lines represent the linear regression lines by the least-squares method.
TABLE 3
Estimation of human CLint, in vitro in hepatic microsomes and their values of
scaling factor for the model compounds
Scaling Factor (CLint, in vivo/CLint, in vitro)
Compounds
CLint, in vitro
Well Stirred
Parallel-Tube
Dispersion
26.6
4.5
5.4
5.4
1.1
4.8
1.5
2.5
16.1
4.2
4.5
3.1
0.3
1.5
1.4
1.1
18.2
4.3
4.7
3.6
0.4
1.9
1.4
1.4
ml/min/kg body weight
FK1052
FK480
Zolpidem
Omeprazole
Nicardipine
Nilvadipine
Diazepam
Diltiazem
ments were conducted in similar conditions used in in vitro microsomal metabolism studies but were conducted in the absence of NADP⫹
so that metabolism of the compounds would not occur. Recovery of
the compounds was more than 90%, except nicardipine where the
recovery was about 50%. Table 5 summarizes the unbound fraction in
liver microsomes, human CLuint,in vitro corrected with fu,microsome, and
the scaling factor values. fu,microsome of diltiazem in rats could not be
estimated because this compound was metabolized by microsomal
esterase (see Materials and Methods). Human fu,microsome values were
dependent on the model compounds. For example, although FK1052,
FK480, nicardipine, and nilvadipine were highly bound to microsome
with free fraction values ranging from 0.112 to 0.443, zolpidem,
omeprazole, diazepam, and diltiazem showed low binding with free
fraction values ranging from 0.745 to 0.975. fu,microsome of each
compound to rat and dog liver microsomes was similar to that measured in human liver microsomes. For FK1052, FK480, and nilvadipine, the human scaling factor values obtained by correcting human
CLint,in vitro with fu,microsome approached unity. However, the scaling
factor for FK1052 were still 5.2- to 8.6-fold. In the case of nicardipine, these values by correcting human CLint,in vitro with fu,microsome
became smaller than unity (0.05– 0.1-fold). For zolpidem, omeprazole, diazepam, and diltiazem, corrections of CLint,in vitro with
fu,microsome corrections did not change the human scaling factor values
significantly. Consequently, the scaling factor for zolpidem and omeprazole remained 4.2- to 5.1-fold and 3.1- to 5.3-fold, respectively.
58.90
74.21
29.00
98.08
1736.34
1712.88
15.00
118.15
scaling factor consideration could give a good prediction of EH and
hepatic clearance in humans.
Binding of Model Compounds to Microsomes. Binding experi-
Discussion
In the present study, we investigated the quantitative prediction of
human hepatic metabolic clearance from in vitro experiments using
liver microsomes focusing on P450 metabolism. The values of
CLint,in vitro and CLint,in vivo for each model compound were compared
in rats and/or dogs, and humans. As a result, 1) scaling factor values
(CLint,in vivo/CLint,in vitro) were similar in the different animal species;
2) scaling factor values were different in each compound; 3) successful predictions of human CLint,in vivo were obtained by considering
animal scaling factor; and 4) use of human CLint,in vitro corrected with
animal scaling factor gave good predictions of CLoral and EH in
humans. Namely, the empirical prediction method is a simple one of
incorporating additional information (which is compound specific)
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Symbols represent the fold differences of scaling factor calculated using the well
stirred model (E), the parallel-tube model(‚), and the dispersion model(䡺). Closed
symbols represent the fold differences of scaling factor between rats and humans.
Open symbols represent the fold differences of scaling factor between dogs and
humans. The solid line represents the line of unity. The area between the dotted lines
represents an area within 2-fold error.
PREDICTION OF HUMAN HEPATIC CLEARANCE
1321
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FIG. 4. Comparison of predicted CLint,in
vitro
with CLint,in
vivo
in humans calculated using the well stirred model (A), the parallel-tube model (B), and the dispersion
model (C).
F, human CLint,in vitro not corrected by animal scaling factor using eq. 16; f, human CLint,in vitro corrected by rat scaling factor using eq. 17; Œ, human CLint,in
corrected by dog scaling factor using eq. 17. The dotted lines represent the lines of unity. The area between the solid lines represents an area within 2-fold error.
from animal studies and is based on the assumption that any in vitro-in
vivo difference seen in humans is also apparent in animals to approximately the same degree.
In a conventional method, CLint,in vitro is obtained from Km and
Vmax, which are estimated by measuring the production of metabolites over a wide range of drug concentrations. On the other hand,
vitro
CLint,in vitro in the proposed method is obtained from substrate disappearance rate at a single drug concentration. From comparison of
these methods, advantages of the proposed method are raised as
follows: 1) simple to conduct; 2) can be done for many compounds;
3) metabolites do not need to be known; 4) can be easily done without
radiolabel; and 5) can yield enzyme kinetic data based on the disap-
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NARITOMI ET AL.
TABLE 4
Accuracy of human CLint, in vivo prediction methods
The n values refer to the number of predicted values of CLint, in vivo (CLint, predicted) that
were compared with the actual human CLint, in vivo obtained from in vivo pharmacokinetic
data (CLint, actual).
Average Fold Error (CLint, actual/CLint, predicted)
Methods
n
Without scaling factor
With rat scaling factor
With dog scaling factor
8
7
6
Well Stirred
Parallel-Tube
Dispersion
4.02
1.57
1.68
3.00
1.85
2.00
3.20
1.67
1.88
FIG. 5. Comparison of predicted CLoral with observed CLoral in humans calculated using the well stirred model (A), the parallel-tube model (B), and the dispersion
model (C).
F, predicted CLoral calculated using human CLint,in vitro corrected by rat scaling factor; E, predicted CLoral calculated using human CLint,in vitro corrected by dog scaling
factor. The dotted lines represent the lines of unity.
FIG. 6. Correlation between CLint,in
vitro
and in vivo EH in humans calculated using the well stirred model (A), the parallel-tube model (B), and the dispersion
model (C).
F, human fb 䡠 CLint,in vitro corrected by rat scaling factor; E, human fb 䡠 CLint,in vitro corrected by dog scaling factor. The solid lines represent the simulated curves based
on the mathematical models.
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 14, 2017
pearance of parent compounds. On the contrary, disadvantages of the
proposed method are as follows: 1) it is difficult to measure very low
CLint,in vitro values; 2) does not get individual metabolite information;
3) and does not obtain Km and Vmax parameters. Recent studies have
also calculated CLint,in vitro from substrate disappearance (Lave et al.,
1997; Obach, 1999).
In this study, we assumed that extrahepatic clearances could be
negligible. Recently, it has been reported that the first-pass metabolism in human small intestine is not negligible for some drugs, such as
cyclosporine, which is metabolized mainly by CYP3A4 (Benet et al.,
1996). Of the model compounds, FK480 (in house data), zolpidem
(Pichard et al., 1995), nicardipine (Guengerich et al., 1991), nilvadipine (in house data), and diltiazem (Sutton et al., 1997) are metabo-
lized mainly by CYP3A4. Human CLint,in vivo of these compounds
were calculated from the CLoral after oral administration. Nevertheless, the CLint,in vivo values predicted from human CLint,in vitro with
animal scaling factor consideration were comparable with the observed values (Fig. 4). From this result, although the possibility of
intestinal metabolism of the model compounds cannot be completely
excluded, it may be reasonable to consider the metabolism of these
compounds mainly in the liver for predicting CLint,in vivo. In the
future, we would evaluate in vitro-in vivo scaling for the model
compounds obviously metabolized in small intestine.
When CLint,in vivo was calculated from in vivo clearance data, or
when CLH and EH were calculated from CLint,in vitro, three frequent
mathematical models (the well stirred, parallel-tube, and dispersion
models) were used. Although there were a few drugs of which the
human scaling factor values of all mathematical models were close to
unity (nicardipine, diazepam, and diltiazem), the values for some
drugs (FK1052, FK480, zolpidem, and omeprazole) were 3.1- to
26.6-fold (Table 3), resulting in CLint,in vivo values larger than the
CLint,in vitro values. These findings suggest that the conventional prediction method, which directly applies CLint,in vitro into the mathematical
models, cannot always predict in vivo clearance for all compounds.
By contrast, the proposed method, considering the scaling factor,
yielded more accurate prediction of human CLint,in vivo that was
mostly within 2-fold of actual values (Fig. 4).
1323
PREDICTION OF HUMAN HEPATIC CLEARANCE
TABLE 5
Binding of the model compounds to hepatic microsomes and their effects on predicting human CLint, in vivo
Microsomal Protein
Concentration
Compounds
fu, microsome
Rat
Dog
Human
0.347
0.119
0.938
0.929
0.175
0.469
0.781
N.D.
0.334
0.120
0.940
0.972
0.183
0.470
0.752
0.907
0.323
0.112
0.939
0.975
0.129
0.443
0.745
0.863
mg/ml
FK1052
FK480
Zolpidem
Omeprazole
Nicardipine
Nilvadipine
Diazepam
Diltiazem
0.5
1.0
0.5
0.5
0.2
0.2
0.5
0.2
Human
CLuint, in vitro
Human Scaling Factor
Well Stirred
Parallel-Tube
Dispersion
8.6
0.5
5.1
5.3
0.1
2.1
1.1
2.1
5.2
0.5
4.2
3.1
0.05
0.7
1.1
1.0
5.9
0.5
4.4
3.5
0.06
0.8
1.1
1.2
ml/min/kg
182.35
662.59
30.88
100.59
13460
3866.55
20.13
136.91
N.D., not determined.
Presently, it is not clear why each compound has a intrinsic scaling
factor. However, there are two possible reasons below. First,
fu,microsome in the reaction mixture may have an influence on the
CLint,in vitro value. Obach (1999) has examined fu,microsome of model
compounds and the potential impact that such binding has on the
prediction of in vivo clearance from CLint,in vitro for these compounds.
Those results showed that incorporation of fu,microsome generally
yielded more accurate predictions of human clearance. In the same
way, we have evaluated fu,microsome of the model compounds and the
change of the scaling factor values when correcting with fu,microsome.
However, CLint,in vitro, when by correcting with fu,microsome, were not
in agreement with the CLint,in vivo for some compounds; the scaling
factor values for FK1052, zolpidem, and omeprazole were severalfold, and the scaling factor values for nicardipine were much smaller
than unity (Table 5). This suggests that the scaling factor different
from unity might not be due only to the drug binding to liver
microsomes. The second reason may be related to some assumptions
of the mathematical models below: 1) the distribution of drug into the
liver is assumed to be perfusion-rate limited, and diffusion of drug
into hepatocytes is rapid and not subject to any diffusional barriers;
2) it is assumed that only the free (unbound to macromolecules in
blood) drug crosses the cell membrane and subsequently occupies the
enzyme site; and 3) a homogeneous distribution of drug-metabolizing
enzymes within the liver acinus is adopted (Houston and Carlile,
1997). If any assumptions are incorrect, the discrepancies between
CLint,in vivo and CLint,in vitro would be observed.
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 14, 2017
FIG. 7. Correlation between CLint and CLoral.
The solid line is the simulated curve using eqs. 6 to 8, based on the well stirred
model. The dotted line is the simulated curve using eqs. 6, 7, and 9, based on the
parallel-tube model. The broken line is the simulated curve using eqs. 6, 7, and 10
to 13, based on the dispersion model.
The use of hepatic microsomes to predict in vivo CLH requires
acceptance of some assumptions (Obach, 1999). First, oxidative metabolism predominates over other metabolic routes, such as direct
conjugation, reduction, hydrolysis, etc. Second, rates of metabolism
measured using animal and human microsomes in vitro are truly
reflective of these exist in vivo. It has been reported that diltiazem is
metabolized in part by hepatic microsomal esterase in rats (LeBoeuf
and Bélanger, 1987). Therefore, the CLint,in vitro of microsomal esterase was estimated in conditions where no NADPH-generating system
was included in the incubation mixture. As a result, the CLint,in vitro
of microsomal esterase represented approximately 30% of the
CLint,in vitro observed in the presence of NADPH-generating system in
rats, whereas the CLint,in vitro of microsomal esterase in dogs and
humans were hardly observed (data not shown). Even for diltiazem,
scaling factor values were similar among the different species (Table
3), suggesting that the CLint,in vitro, which was measured in conditions
with NADPH-generating in rats, reflects the metabolic rates by both
P450 and esterase. However, future studies should also focus on
evaluating in vitro-in vivo scaling for the other compounds metabolized by microsomal esterase to clarify the validity of the consideration. Third, the substrate concentration used (1 ␮M in this study) is
well below the apparent Km. It may be necessary to confirm this
assumption. For example, for only FK480, 3.5- to 5.4-fold differences
in scaling factor between animals and humans were observed (Fig. 3).
It may be accounted for by the possibility that the animal CLint,in vitro
could be seriously underestimated because of the lower Km value at 1
␮M. However, the CLint,in vitro at several concentrations below 1 ␮M
were similar to that at 1 ␮M in animals and humans (data not shown).
Consequently, it was considered that the CLint,in vitro at 1 ␮M was
under linear condition.
CLint,in vitro corrected with animal scaling factor could give a good
prediction of in vivo CLoral and EH in humans. But, for a highclearance drug, nilvadipine, the CLoral values using the parallel-tube
and dispersion models were overestimated (Fig. 5). The reason is seen
in the well stirred model where CLoral is in proportion to CLint,in vivo,
whereas the parallel-tube and dispersion models give exponential
correlation between CLint,in vivo and CLoral for high-clearance drugs
(Fig. 7). As a result, the error of predicted CLoral, which is caused
from the error of predicted CLint,in vivo, is magnified in the paralleltube and dispersion models compared with the error in the well stirred
model. Also, in the oral case, CLint,in vivo calculated based on the
parallel-tube and dispersion models was affected to some extent by
QH for the high-clearance drugs, whereas CLint,in vivo calculated based
on the well stirred model was not affected (Iwatsubo et al., 1997a). In
the case of predicting CLoral for high-clearance drugs, it may be
necessary to consider the selection of the mathematical models.
In conclusion, we investigated a new variant method on the quan-
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NARITOMI ET AL.
titative prediction of human hepatic clearance from in vitro experiments, focusing on P450 metabolism with eight model compounds.
Successful predictions of human hepatic clearance were obtained by
use of the human CLint,in vitro corrected with animal scaling factors,
which are the ratios of CLint,in vivo to CLint,in vitro. This method would
provide more reliable prediction of human hepatic clearance and be
useful in drug discovery.
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