Supplemental material to this article can be found at:
http://dmd.aspetjournals.org/content/suppl/2014/07/08/dmd.114.057935.DC1.html
1521-009X/42/9/1540–1547$25.00
DRUG METABOLISM AND DISPOSITION
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/dmd.114.057935
Drug Metab Dispos 42:1540–1547, September 2014
A Useful Model Capable of Predicting the Clearance of Cytochrome
P450 3A4 (CYP3A4) Substrates in Humans: Validity of CYP3A4
Transgenic Mice Lacking Their Own Cyp3a Enzymes s
Tetsuya Mitsui, Takayuki Nemoto, Taiji Miyake, Shunsuke Nagao, Kotaro Ogawa, Motohiro Kato,
Masaki Ishigai, and Hideyuki Yamada
Preclinical Research Department, Research Division, Chugai Pharmaceutical Co., Ltd., Shizuoka, Japan (Te.M., T.N.,Ta.M., S.N.,
K.O., M.K., M.I.); and Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan (H.Y.)
Received March 13, 2014; accepted July 7, 2014
The accurate prediction for the body clearance of a novel drug
candidate by humans during the preclinical stage contributes to its
successful development. To improve the predictability of human
hepatic clearance, we focused on CYP3A4, which is involved in the
metabolism of more than 50% of all currently marketed drugs. In
this study, we investigated the validity of the in vivo model using
transgenic mice carrying the human CYP3A4 gene and lacking their
own Cyp3a genes (CYP3A4-Tg mice). The CYP3A4 activity toward
its substrates in liver microsomes was similar in CYP3A4-Tg
mice and humans. As for the clearance, six CYP3A4 substrates
(alprazolam, felodipine, midazolam, nifedipine, nitrendipine, and
quinidine) were given intravenously to CYP3A4-Tg mice, and their
hepatic intrinsic clearance (CLint,h) was evaluated. A regression
analysis of the data obtained indicated that the CLint,h values of six
substrates in CYP3A4-Tg mice were highly correlated with those in
humans (R2 = 0.95). This correlation could be improved by correcting the CLint,h values by the relative contribution of artificially
expressed CYP3A4 to the overall metabolism in the mice. From
these findings, it is reasonable to expect that the CLint,h of a
particular drug in humans is predictable by applying the CLint,h
obtained in CYP3A4-Tg mice to a regression line prepared in advance. The variance of the CLint,h prediction by this method was
evaluated and found to be within a range of 2-fold of the regression
value. These results suggest that the CYP3A4-Tg mouse model has
the potential to accurately predict the human hepatic clearance of
CYP3A4 substrates.
Introduction
species. Several additional modifications to simple allometry have
been proposed to improve the accuracy of human clearance prediction
by introducing correction factors such as the rule of exponents (Mahmood
and Balian, 1996). However, other investigators have claimed that
those modifications produce very little significant improvement (Nagilla
and Ward, 2004). The major drawback in allometric scaling is the fact
that, due to its empirical nature, this method pays no attention to
species differences in metabolic and disposition processes between
humans and animals.
As far as physiologic approaches are concerned, in vitro–in vivo exploratory (IVIVE) prediction is a well-known method for the prediction
of hepatic clearance (Houston, 1994). The IVIVE prediction consists of
the following two procedures: the first step is an in vitro procedure to
determine the intrinsic hepatic clearance (CLint,in vitro) using liver microsomes or hepatocytes, and the second one is the extrapolation procedure
to convert CLint,in vitro into the in vivo CLh using physiologically based
scaling factors and mathematical models. However, the IVIVE tends to
underestimate hepatic clearance (Ito and Houston, 2005; Riley et al.,
2005; Brown et al., 2007). In addition, the clearance values obtained
vary markedly in both experimental and extrapolation procedures
(Hallifax and Houston, 2009). The percentage of drugs for which the
Prediction of the drug clearance in humans during the preclinical stage
is essential for selecting drug candidates having a desired pharmacokinetic profile such as a suitable elimination half-life, bioavailability, and
other parameters. In particular, hepatic clearance (CLh) is important because a number of drug candidates are metabolized in the liver. Empirical
and physiologic approaches using liver microsomes and hepatocytes
have been proposed to predict human CLh (Ito and Houston, 2005;
Tang and Mayersohn, 2005; Zanelli et al., 2012) However, these
methods cannot always give us a satisfactory prediction, and, in many
cases, the CLh obtained exceeds the acceptable value, which is desired
to be within a range from 0.5- to 2-fold of the actual value.
In the empirical approach, allometric scaling has been proposed
(Boxenbaum, 1984), and this has been widely used for the prediction
of human clearance. Simple allometry is an easy to perform method
because it needs only the body weight and the in vivo data that are
obtained from a routine preclinical study using one or several animal
dx.doi.org/10.1124/dmd.114.057935.
s This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: ALP, alprazolam; CL, clearance; CLh, hepatic clearance; CLtot,b, systemic blood clearance; CLint,h,in vitro, in vitro intrinsic hepatic
clearance; CLtot,p, systemic plasma clearance; CLr, renal clearance; CLint,h, hepatic intrinsic clearance; DMSO, dimethylsulfoxide; F, bioavailability; Fa,
fraction absorbed; Fg, intestinal availability; Fh, hepatic availability; FLD, felodipine; fm,CYP3A4, fraction metabolism mediated by CYP3A4; fB, unbound
fraction in blood; fu, unbound fraction; HLM, human liver microsomes; IVIVE, in vitro–in vivo exploratory; LC-MS/MS, liquid chromatography with
tandem mass spectrometry; MDZ, midazolam; MLM, mouse liver microsome; NFD, nifedipine; NTL, nitrendipine; Qh, hepatic blood flow; QND,
quinidine; RB, blood-to-plasma concentration ratio; RED, rapid equilibrium dialysis; Tg, transgenic; WT, wild-type.
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ABSTRACT
Human Clearance Prediction using CYP3A4 Transgenic Mice
from Sigma-Aldrich (St. Louis, MO). All other reagents and solvents were of
analytical grade and commercially available.
Animals and Liver Microsomes. Male CYP3A4-Tg mice and wild-type
mice (FVB mice) at the age of 6 to 8 weeks were purchased from Taconic
Farms (Germantown, NY). All mice were kept in an animal facility under
conditions of controlled temperature (25 6 2°C) and humidity (55% 6 15%)
under a 12-hour light/dark cycle. All animal experimental procedures were
preapproved by the Institutional Animal Care and Use Committee of Chugai
Pharmaceutical Co. Ltd., which was accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (Frederick, MD).
The liver microsomes of CYP3A4-Tg mice and WT mice were obtained
from CXR Biosciences Ltd. (Dundee, UK). A pooled preparation of HLM from
15 donors was purchased from Xenotech (Lenexa, KS).
In Vitro Metabolic Studies. The incubation mixture had a total volume of
800 ml and consisted of 100 mM sodium/potassium phosphate buffer (pH 7.4),
5 mM magnesium chloride, 2 mM NADPH, 1 mM substrate dissolved in
dimethylsulfoxide (DMSO; final v/v concentration: 0.1%), and 0.16 mg of liver
microsomal protein. Only in the case of alprazolam metabolism was 0.32 mg of
microsomal protein added to the incubation mixture. After preincubation at
37°C for 5 minutes, the reaction was initiated by adding NADPH solution.
After incubation at 37°C for 5, 10, 20, and 30 minutes, the reaction was stopped
by adding 50 ml of ice-cold acetonitrile containing raloxifene (200 ng/ml, DMSO)
as an internal standard. The reaction mixture was transferred to a 96-well filter
plate (0.45 mm MultiScreen; Millipore, Billerica, MA) and centrifuged at 2000 rpm
for 5 minutes to remove precipitated proteins. A portion (15 or 20 ml) of the
filtrate obtained was injected into a liquid chromatography system equipped
with tandem mass spectrometers (LC-MS/MS), and the target drug was determined using either analytical condition (A) or (B).
Materials and Methods
Chemicals. Alprazolam (ALP) and midazolam (MDZ) were purchased from
Wako Pure Chemical Industries (Osaka, Japan). Felodipine (FLD), nifedipine
(NFD), nitrendipine (NTL), and quinidine (QND) hydrochloride were purchased
TABLE 1
The CLint, in vitro of CYP3A4 substrates in liver microsomes prepared from human
donors (HLM), wild-type mice (WT MLM), and CYP3A4-Tg mice (CYP3A4-Tg
MLM), and fm,CYP3A4 in CYP3A4-Tg mouse liver microsomes
Pooled liver microsomes were prepared from three CYP3A4-Tg mice and three WT mice and
used for this assay.
CLint,
fm,CYP3A4
in vitro
CYP3A4-Tg MLMa
Substrate
HLM
WT MLM
CYP3A-Tg MLM
(–) Ab
(+) Ab
ml/min per mg protein
Alprazolam
Felodipine
Midazolam
Nifedipine
Nitrendipine
Quinidine
16
1850
1110
724
1140
91
NC
815
673
281
659
155
15
2980
952
731
2070
111
ND
127
281
18
584
57
1.00
0.96
0.71
0.98
0.72
0.49
MLM, mouse liver microsomes; NC, not calculated.
a
(–) Ab and (+) Ab mean the values obtained by incubating the substrate in the absence and
presence of anti-CYP3A4 antibody, respectively.
Fig. 1. Correlation of the CLint,in vitro of CYP3A4 substrates between liver microsomes prepared from CYP3A4-Tg mice and humans (A), and between microsomes
from wild-type mice and humans (B). The solid line represents a regression line
empirically fitted to the data for all substrates, and dotted lines represent the levels of
the 2-fold or 0.5-fold of the regression value. Each plot represents the mean of
duplicate assays. ALP was omitted from Fig. 1B because the metabolic activity for
this substrate was below the detection limit in wild-type mice.
Downloaded from dmd.aspetjournals.org at ASPET Journals on September 29, 2016
variance in the clearance predicted by IVIVE from the observed value
is within 2-fold is estimated to be around 60% (Howgate et al., 2006;
Lavé et al., 2009). This figure is similar to those obtained by
allometric scaling (Lombardo et al., 2013), and the predictability of
these methods does not appear to be sufficient.
It is expected that the mice expressing human metabolic enzymes
will be useful for the prediction of human hepatic clearance. Among
drug metabolizing enzymes, cytochromes P450 (P450) make the
greatest contribution to drug metabolism, and CYP3A4, which is the
most abundant isoform in the human liver and intestine, contributes to
more than 50% of the metabolism of marketed drugs (Guengerich,
1999; Williams et al., 2004). In view of this background, we focused
on transgenic mice carrying the CYP3A4 gene (CYP3A4-Tg mice) for
estimating human hepatic clearance. To enhance the validity of this
mouse model for the prediction of human clearance, mouse genes
coding for Cyp3a enzymes (eight isoforms) were deleted from the
CYP3A4-Tg mice used in this study (van Herwaarden et al., 2007).
An earlier study showed that the intrinsic clearance for the metabolism
of triazolam, a specific CYP3A4 substrate, by liver microsomes prepared from CYP3A4-Tg mice was comparable with that in human
liver microsomes (HLM) and markedly higher than the values in
microsomes from Cyp3a isoform-null mice and wild-type (WT) mice
(van Waterschoot et al., 2009). Accordingly, it is to be expected that
the hepatic intrinsic clearance of CYP3A4 substrates in humans can be
reproduced in CYP3A4-Tg mice.
To evaluate the validity of this CYP3A4-Tg mouse model in more
detail, this study investigated whether the in vivo data obtained after
administration of CYP3A4 substrates to CYP3A4-Tg mice can predict
the hepatic clearance in humans. To this end, we investigated the correlation in the intrinsic hepatic clearances obtained from in vitro and in
vivo experiments using six CYP3A4 substrates between CYP3A4-Tg
mice and humans. In addition, we determined the relative contribution
of CYP3A4 to the metabolism (fraction of metabolism by CYP3A4:
fm,CYP3A4) as a correction factor because it is conceivable that murine
enzymes other than Cyp3a isoforms significantly contribute to the
metabolism of CYP3A4 substrates in CYP3A4-Tg mice.
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Mitsui et al.
TABLE 2
The unbound fraction in plasma (fu), the blood-to-plasma concentration ratio (RB), and the unbound fraction in blood (fB)
of CYP3A4 substrates in wild-type mice and humans
Mice
Humans
Substrate
References
fu
Alprazolam
Felodipine
Midazolam
Nifedipine
Nitrendipine
Quinidine
a
b
a
0.241
0.003
0.055
0.052
0.016
0.292
RB
a
0.955
0.789
0.853
0.483
0.706
1.561
b
fB
fu
0.252
0.004
0.064
0.108
0.023
0.187
0.268
0.004
0.016
0.023
0.020
0.069
RBb
fB
0.818
0.680
0.653
0.670
1.460
0.930
0.327
0.006
0.025
0.035
0.014
0.074
Each value represents the mean of triplicate in vitro assays using WT (FVB) mouse blood or plasma.
Each value was obtained from literature references.
Concentration of test compound in the buffer chamber sample
Concentration of test compound in the plasma chamber sample
After centrifugation, 50 ml of water was added to the filtrate, and part (15 ml)
of the mixture was subjected to LC-MS/MS analysis to determine the drug. The
RB and fB were calculated using eqs. 2 and 3.
RB ¼
Concentration of test compound in plasma sample 1
Concentration of test compound in plasma sample 2
f B ¼ f u =RB
ð3Þ
Pharmacokinetic Studies in CYP3A4-Tg Mice and WT Mice. The test
drugs were intravenously injected into CYP3A4-Tg mice and WT mice at the
following doses: ALP, NFD, and NTR, 1 mg/kg per 10 ml vehicle; FLD, MDZ,
and QND, 2 mg/kg per 10 ml vehicle. ALP, MDZ, and QND were dissolved in
5 mM HCl, and FLD, NFD, and NTR were dissolved in ethanol-20% (v/v)
polyethylene glycol (1:19 v/v). Blood samples (40 ml) were collected from the
orbital vein using heparinized hematocrit capillary tubes 2, 15, 30, 60, 120,
240, 420, and 1440 minutes after intravenous administration. The plasma was
prepared by centrifuging the blood at 4 and 1°C at 5000 rpm for 5 minutes and
frozen at 220°C until analysis. The urine was collected for 24 hours after dosing,
and the urine volume was recorded. An aliquot of each urine sample was also
stored at 220°C until required for analysis.
Conditions of LC-MS/MS Analysis. The following conditions were used:
the instrument was a Shimadzu 10AD HPLC system (Shimadzu Corp., Kyoto,
Japan) equipped with an API4000 LC-MS/MS system (AB SCIEX, Framingham,
MA); the column was a Sunniest C18 (2 50 mm, 3 mm; ChromaNik
Technologies, Osaka, Japan) at a column temperature of 40°C; the mobile phase
was operated under gradient conditions and consisted of 0.1% v/v formic acid
in water (A) and 0.1% v/v formic acid in acetonitrile (B) (Supplemental
TABLE 3
Pharmacokinetic parameters of CYP3A4 substrates after a single intravenous
administration to CYP3A4-Tg mice and wild-type mice
Each value represents the mean of three animals.
Substrate
ð1Þ
Blood-to-Plasma Concentration Ratio (RB). Blood from the WT mice was
collected using a heparinized syringe and centrifuged for 5 minutes (4°C,
15,000 rpm) to prepare plasma. To obtain the blood-to-plasma concentration
ratio (RB), the blood and plasma were separately processed as follows. The
plasma (98 ml) from WT mice was mixed with a test drug (final concentration
1 mM), and incubated at 37°C for 5 minutes (designated plasma sample 1). The
blood (98 ml) was also spiked with a drug and incubated in a similar way, and
centrifuged at 15,000 rpm for 5 minutes (4°C). The plasma obtained was
designated plasma sample 2. A portion (5 ml) of plasma samples 1 and 2 was
mixed with 5 ml of ethanol and 100 ml of ice-cold acetonitrile containing
raloxifene (200 ng/ml, DMSO) as an internal standard. These mixtures were
transferred to a 96-well filter plate (0.45 mm MultiScreen), and centrifuged at
3000 rpm for 10 minutes.
ð2Þ
Vss
CLtot,p
CLtot,b
l/kg
CYP3A-Tg mice
Alprazolam
Felodipine
Midazolam
Nifedipine
Nitrendipine
Quinidine
WT mice
Alprazolam
Felodipine
Midazolam
Nifedipine
Nitrendipine
Quinidine
CLr
CLh
CLint,h
Fha
ml/min per kg
1.62
1.64
1.12
0.36
1.19
3.88
10.4
34.5
63.1
16.7
42.9
80.9
10.9
43.7
74.0
34.5
60.7
51.8
0.19
0.00
0.07
0.00
0.01
3.34
10.7
43.7
73.9
34.5
60.7
48.5
46.0
17,200
2990
431
5170
413
0.88
0.51
0.18
0.62
0.33
0.46
1.98
2.03
1.37
1.09
1.47
2.95
22.0
41.1
87.5
8.74
51.1
71.4
23.0
52.0
103
18.1
72.4
45.8
0.30
0.01
0.01
0.00
0.03
1.69
22.7
52.0
103
18.1
72.3
44.1
108
22,900
NC
193
7970
353
0.75
0.42
NC
0.80
0.20
0.51
NC, not calculated; Vss, volume of distribution at steady state.
a
Fh was calculated using the following equation: Fh = 1 2 CLh/Qh.
Downloaded from dmd.aspetjournals.org at ASPET Journals on September 29, 2016
Immunoinhibition of the CYP3A4-Mediated Reaction. Anti-CYP3A4
antibody (MAB-CYP3A4, mouse anti-human CYP3A4 monoclonal IgM; BD
Biosciences, Woburn, MA) was used to estimate fm,CYP3A4 in terms of the
metabolism catalyzed by the hepatic microsomes of CYP3A4-Tg mice. The
highly specific nature of this antibody to CYP3A4 was reported elsewhere
(Gelboin et al., 1995). Anti-CYP3A4 antibody was added to the incubation
mixture before preincubation. All other conditions for incubation were identical
to those given in the previous section. The amount of anti-CYP3A4 antibody
needed to inhibit the CYP3A4-mediated reaction was determined using midazolam
as the substrate. According to the results obtained, anti-CYP3A4 antibody twice
the amount of microsomal protein (enzyme source) reduced the midazolammetabolizing activity to less than 10% of the control activity without the antibody (Supplemental Fig. 1). Based on this, an amount of anti-CYP3A4 antibody
5-fold greater than the microsomal protein was added to the incubation mixture
to obtain complete inhibition of CYP3A4.
Unbound Fraction. The ratio of the unbound form of test drug (unbound
fraction: fu) in plasma was determined by equilibrium dialysis using a rapid
equilibrium dialysis (RED) device (Thermo Fisher Scientific, Rockford, IL).
For this, the plasma (200 ml) of untreated WT mice spiked with a test drug
(2 mM, final concentration) was applied to the plasma chamber of the RED
device, whereas 350 ml 133 mM phosphate buffer (pH 7.4) was applied to the
buffer chamber. The RED device was then sealed and incubated for 4 hours
at 37°C at 800 strokes/min in M-BR-022 bioshaker (TAITEC, Saitama,
Japan).
After incubation, 10 ml of plasma was removed from the plasma chamber,
and 60 ml of phosphate buffer was removed from the buffer chamber and mixed
with 60 ml of 133 mM phosphate buffer and 10 ml of blank plasma, respectively, in 96-well plates to which had been added in advance 200 ml of
purified water and 10 ml of acetonitrile containing 20 nM warfarin an internal
standard. The solution in the 96-well plates was transferred to a 96-well filter
plate (0.45 mm MultiScreen) and centrifuged at 2000 rpm for 5 minutes to remove
precipitated proteins.
A portion (20 ml) of the filtrate was subjected to LC-MS/MS analysis, and
the drug concentration was measured. The fu of each drug was determined in
duplicate, and the fu was calculated using eq. 1.
fu ¼
Ogawa et al., 2013
Edgar et al., 1985
Ogawa et al., 2013
Ogawa et al., 2013
Mikus et al., 1987; Soons and Breimer, 1991
Ogawa et al., 2013
Human Clearance Prediction using CYP3A4 Transgenic Mice
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TABLE 4
Pharmacokinetic parameters of CYP3A4 substrates in humans
Vssa
Substrate
CLtot,pa
CLtot,b
l/kg
Alprazolam
Felodipine
Midazolam
Nifedipine
Nitrendipine
Quinidine
CLra
CLint,h
Fhb
References
3.24
6730
381
207
1530
64.6
0.95
0.21
0.65
0.72
0.41
0.80
Greenblatt and Wright, 1993; Venkatakrishnan et al., 2005
Edgar et al., 1985
Thummel et al., 1996; Ogawa et al., 2013
Palma-Aguirre et al., 1989; Rashid et al., 1995
Mikus et al., 1987
Ueda et al., 1976; Brøsen et al., 1990
CLh
ml/min per kg
1.25
3.20
0.78
0.94
5.40
2.70
1.05
11.1
4.74
3.90
18.7
4.70
1.28
16.4
7.26
5.82
12.8
5.05
0.26
0.00
0.00
0.00
0.51
0.91
1.03
16.4
7.26
5.82
12.3
4.14
Vss, volume of distribution at steady state.
a
Each value was quoted from the reference indicated.
b
Fh was calculated using the following equation Fh = 1 2 CLh/Qh.
Y ¼ a expð 2 Kel XÞ
ð4Þ
where a is a constant and Kel is a rate constant for the disappearance of test
drugs. The CLint,in vitro was calculated according to eq. 5.
CLint;in vitro ¼ Kel ½incubation volumeðmlÞ
½enzyme amount ðmg proteinÞ
ð5Þ
a ¼ ð1 þ 4RN DN Þ1=2
ð12Þ
RN ¼ f B CLint;h =Qh
ð13Þ
Results
In Vitro Metabolism of CYP3A4 Substrates. Six CYP3A4
substrates—ALP, FLD, MDZ, NFD, NTR, and QND—were incubated
with NADPH-fortified HLM as well as microsomes prepared from WT
and CYP3A4-Tg mice. The kinetic parameters obtained are shown in
Table 1. The CLint,in vitro values for six substrates in CYP3A4-Tg mice
were comparable with or greater than those in HLM, whereas five
substrates out of six exhibited a lower CLint,in vitro value in WT mice
The contribution of CYP3A4 to the in vitro metabolism of a substrate was
expressed as fm,CYP3A4. The fm,CYP3A4 for each substrate was calculated using
eq. 6.
fm;CYP3A4 ¼ 1 CLint;in vitro with Anti CYP3A4 antibody
CLint;in vitro without Anti CYP3A4 antibody
ð6Þ
Determination of In Vivo Pharmacokinetic Parameters. The pharmacokinetic parameters for the CYP3A4-Tg mice were calculated by a noncompartmental analysis using Phoenix WinNonlin version 6.1 software (Pharsight
Corp., Mountain View, CA). The systemic plasma clearance (CLtot,p), the
systemic blood clearance (CLtot,b), and the hepatic blood clearance (CLh) were
determined using eqs. 7–9:
CLtot;p ¼ Dose ∕ AUCinf
ð7Þ
CLtot;b ¼ CLtot;p ∕ RB
ð8Þ
CLh ¼ CLtot;b 2 CLr
ð9Þ
The renal clearance (CLr) was determined by dividing the amount of unchanged
drug excreted in the urine over 24 hours by the area under the blood
concentration-time curve (AUC0–24). The distribution volume at steady state
(Vss) was calculated by multiplying the mean resident time (MRTinf) by the
CLtot,p. The hepatic availability (Fh) was determined using eq. 10. The hepatic
blood flow rates (Qh) in mice and humans were obtained from a reference
(Davies and Morris, 1993): 90 (mice) and 20.7 (humans) ml/min per kg and
used in the calculation.
Fh ¼ 1 2 CLh ∕ Qh
ð10Þ
The hepatic intrinsic clearance (CLint,h) of a CYP3A4 substrate in CYP3A4Tg mice and humans was calculated by applying Fh, Qh, fB, and the dispersion
number (DN = 0.17) in eqs. 11–13 for a dispersion model:
Fh ¼
4a
ð1 þ aÞ2 exp½ða 1Þ=2DN ð1 aÞ2 exp½ ða þ 1Þ=2DN ð11Þ
Fig. 2. Correlation for the CLtot,b (A) and CLh (B) of intravenously administered
CYP3A4 substrates between CYP3A4-Tg mice and humans. The solid line represents
a regression line empirically fitted to the data for all substrates.
Downloaded from dmd.aspetjournals.org at ASPET Journals on September 29, 2016
Tables 1-1 and 1-2); the flow rate was 0.2 ml/min; ionization mode was used
with electrospray, and the detected ion was a positive or negative ion fragmented from a precursor ion formed in the first mass spectrometry (Supplemental
Tables 2-1 and 2-2).
Determination of CLint,in vitro and fm,CYP3A4. The rate of disappearance of
a test drug from the incubation mixture was calculated by fitting the timedependent loss of substrate to a monoexponential decay curve defined by eq. 4.
1544
Mitsui et al.
compared with HLM. In microsomes from WT mice, no elimination of
ALP was observed due to the low metabolic activity.
In parallel with these observations, a good correlation (R2 = 0.853)
was obtained between the CLint,in vitro in HLM and the micrsomes from
CYP3A4-Tg mice, and the CLint,in vitro values of all substrates were
plotted and found to range from 0.5- to 2-fold the regression value (Fig.
1A). Although the CLint,in vitro between HLM and the microsomes from
WT mice also exhibited a high correlation coefficient (R2 = 0.877),
a plot of QND did not lie within a range between 0.5- and 2-fold the
regression value (Fig. 1B). In addition, a plot for ALP was omitted from
Fig. 1B because the metabolic activity of this substrate was too low to
be determined in wild-type mice.
These pieces of evidence suggest that artificially expressed CYP3A4
but not murine peculiar Cyp3a mainly contributes to an improved correlation in CLint, h among a series of substrates exhibiting high (FLD) and
low (ALP) clearances. The fm,CYP3A4, an index of the relative contribution of CYP3A4 to the metabolism of a particular drug in CYP3A4-Tg
mice, was estimated by comparing the CLint,in vitro values obtained with
and without anti-CYP3A4 antibody (Table 1). The result showed that
fm,CYP3A4 varied from 0.49 (QND) to 1.0 (ALP). The unbound fraction in
plasma or blood and the blood-to-plasma concentration ratio in FVB
(WT) mice and humans are shown in Table 2.
Correlation in the In Vivo Pharmacokinetics between CYP3A4-Tg
Mice and Humans, and between WT Mice and Humans. Based on
the plasma concentration-time profile after intravenous administration
to CYP3A4-Tg mice (Supplemental Fig. 2) and WT mice, the
pharmacokinetic parameters of CYP3A4 substrates were calculated
(Table 3). To examine a correlation between CYP3A4-Tg mice or WT
mice and humans, the human pharmacokinetic parameters published in
the literature are shown in Table 4. The ratios of renal clearance (CLr) to
total clearance (CLtot,b) of all substrates were estimated to be 10% or less,
suggesting that hepatic metabolism plays a major role in the elimination
of these drugs in CYP3A4-Tg mice. Figure 2 shows a correlation between CYP3A4-Tg mice and humans in terms of CLtot,b and CLh. As can
be seen in the figure, a poor correlation for both parameters was observed
between humans and CYP3A4-Tg mice (R2 = 0.155 and 0.180). Therefore,
as far as the CLh is concerned, it does not appear that the human features
can be predicted from those in CYP3A4-Tg mice.
We then calculated the in vivo CLint,h of CYP3A4 substrates from
the in vivo CLh using a dispersion model, and compared the CLint,h
TABLE 5
Accuracy of the predicted human CLh of six CYP3A4 substrates based on the
correlation of CLint,h between human and Tg mice compared with observed values
CLh
Substrate
Accuracy
Observed
Predicted
ml/min per kg
Alprazolam
Felodipine
Midazolam
Nifedipine
Nitrendipine
Quinidine
1.03
16.4
7.26
5.82
12.3
4.14
%
2.01
17.3
10.7
2.98
11.2
2.65
195
106
147
51
92
64
Downloaded from dmd.aspetjournals.org at ASPET Journals on September 29, 2016
Fig. 3. Correlation for the CLint,h of CYP3A4 substrates between humans and CYP3A4-Tg mice or wild-type mice. The CLint,h in CYP3A4-Tg mice was calculated (A)
without and (B) with multiplying by fm,CYP3A4. The CLint,h in wild-type mice was obtained without any correction (C). The solid line represents a regression line empirically
(power) fitted to the data for all substrates, and the dotted lines represent 2-fold or 1/2-fold the regression value. MDZ was omitted from Fig. 3C because its CLint,h could not
be calculated.
Human Clearance Prediction using CYP3A4 Transgenic Mice
logCLint;h;human ¼ 1:207 log ðCLint;h;CYP3A4Tg mice
fm;CYP3A4 Þ 2 1:191
ð14Þ
Prediction of Human CLh and Its Validity. The human CLint,h
values of the six CYP3A4 substrates were simulated by applying
CLint,h,CYP3A4-Tg mice and fm,CYP3A4 obtained from CYP3A4-Tg mice
experiments to eq. 14; these were then converted to human CLh using
eqs. 10, 11, 12, and 13. The predicted CLh obtained for each substrate
and the difference (%) from the observed value are shown in Table 5.
The predicted values for all CYP3A4 substrates did not exceed the real
CLh by 2-fold or 0.5-fold.
In contrast, a trial using WT mice for the prediction of CLint,h in
humans exhibited large differences between the predicted and observed
values for ALP (4.5-fold) and NFD (0.2-fold) (Supplemental Table 3).
Discussion
This study aimed to develop a more accurate method for the
prediction of human CLh than the presently available methods, such as
allometric scaling and IVIVE. To date, the main focus for obtaining
human CLh predictions has been the theoretical in vivo approach,
although only a few empirical approaches, such as animal scale-up,
have been reported. We expected that a humanized mouse model would
be a better tool for predicting human CLh, and we focused on CYP3A4Tg mice because CYP3A4 is an enzyme contributing to the metabolism
of over 50% of currently prescribed drugs (Guengerich, 1999).
In this study, we initially compared the CLh between humans and
CYP3A4-Tg mice, but the correlation obtained was quite poor (R2 =
0.180; Fig. 2B). This seems to be due to the fact that CLh depends on
the hepatic blood flow, which is peculiar to each animal species, and
this parameter exhibits a clearance due to the metabolism and disposition of the bound as well as the unbound form of the drug. It is
presumed that the poor correlation of CLh between humans and
CYP3A4-Tg mice is attributed, at least partially, to a species difference
Fig. 4. Correlation between the corrected CLint,h of CYP3A4 substrates by fm,CYP3A4 in CYP3A4-Tg mice and human pharmacokinetic parameters of FaFg (A), Fh (B), and F
(C). The solid line represents a regression line empirically fitted to the data for all substrates.
Downloaded from dmd.aspetjournals.org at ASPET Journals on September 29, 2016
between CYP3A4-Tg mice and humans, and WT mice and humans.
As a power regression analysis exhibited an excellent correlation (R2 =
0.951) between CYP3A4-Tg mice and humans, the CLint,h values of
five out of six CYP3A4 substrates were plotted within 2-fold range
(Fig. 3A). When the CLint,h in CYP3A4-Tg mice was corrected using
fm,CYP3A4, all substrates were plotted within a 2-fold range of the
regression value, whereas the value of the regression coefficient remained unchanged (Fig. 3B). In the case of linear regression analysis,
excellent correlations were obtained independently of the fm,CYP3A4
correction (R2 = 0.977 and 0.993). However, the CLinh,h values of
ALP (5.3-fold), MDZ (3.0-fold), and QND (2.4-fold) without an
fm,CYP3A4 correction, and ALP (5.8-fold) and MDZ (2.2-fold) with
a correction exceeded the range of 2-fold (Supplemental Fig. 3, A and
B). Although the CLint,h between WT mice and humans also showed
a good correlation (R2 = 0.820), only three out of five CYP3A4
substrates (60%)—that is, FLD, NTR, and QND—were found to be
within a 2-fold range (Fig. 3C). In the case of WT mice, MDZ was
removed from the regression analysis because its CLh was calculated
to be 103 ml/min per kg, which exceeded the blood flow in the mouse
liver (90 ml/min per kg) (Davies and Morris, 1993).
These results showed that the predictability of human CLint,h could
be improved by using the CLint,h obtained from CYP3A4-Tg mice
corrected by fm,CYP3A4. We obtained eq. 14 of the regression line:
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Mitsui et al.
F ¼ Fa Fg Fh
ð15Þ
Human Fh can be estimated by inserting a predicted human CLint,h
into eqs. 11–13. Regarding Fa Fg, it can also be predicted from the
CLint,h obtained from CYP3A4-Tg mice experiments because it has
been suggested that there is an inverse correlation between CLint,h and
Fa Fg in CYP3A4-dependent metabolism in humans (Kato et al.,
2003).
We initially used literature data about human F for six CYP3A4
substrates, and calculated the human Fa Fg using eq. 15, and F and Fh
(Table 4) were calculated using eq. 10 (see Supplemental Table 4 for
obtained Fa Fg values). We then compared CLint,h and Fa Fg in
humans, but a clear correlation was not observed (R2 = 0.327) (Supplemental Fig. 4). When the correlation between Fa Fg in humans and
CLint,h in CYP3A4-Tg mice was subsequently analyzed, a poor correlation was also observed (Fig. 4A), despite the excellent correlations
obtained between CLint,h multiplied by fm,CYP3A4 and both Fh and F
(Fig. 4, B and C). One of the reasons for this seems to be the high FaFg
value of FLD in spite of its high CLint,h value.
The Fg and Fh values of FLD can be changed along with a change in
hepatic blood flow (Qh). This is because the CLh of FLD is as much as
the Qh (CLh = 16.4 ml/min per kg), and Fh and Fg depend on Qh (eq.
10) and Fh (eq. 15), respectively. FLD and NFD, calcium channel
blockers, exhibit vasodilator effect on the liver blood vessels, resulting
in an increase in Qh (Galetin et al., 2008), and FLD causes the effect
rapidly (Bengtsson-Hasselgren et al., 1990). The Qh is known to range
from 17.1 to 25.5 under normal physiologic conditions (Kato et al.,
2003). Although our study set the Qh at 20.7 for all drugs, the
following possibility cannot be excluded that the Qh is higher than the
value used in the case of FLD, and this leads to an overestimation of
Fg. If the Qh of FLD is changed to 25.5, the FaFg of FLD is calculated
to be 0.45, and the correlation in Fig. 4A is improved (R2 = 0.769; data
not shown). Although problems such this should be improved in the
future, it is reasonable to expect that the human F for CYP3A4
substrates can be predicted using CLint,h in CYP3A4-Tg mice after
intravenous administration. This is supported by a good correlation
between human F and CLint,h in CYP3A4-Tg mice (Fig. 4C).
In conclusion, our present study demonstrates the usefulness of
CYP3A4-Tg mice lacking their own Cyp3a enzymes for predicting the
human hepatic clearance of CYP3A4 substrates. The in vivo approach
reported here appears to have better predictability than other previously reported methods for the pharmacokinetics of CYP3A4 substrates in humans. Recently, transgenic mice carrying human P450
enzymes, such as CYP2D6 and CYP2C9, and concomitantly lacking
endogenous Cyp2d and Cyp2c have been produced (Scheer et al.,
2012a,b). It is therefore expected that these humanized mice as well as
CYP3A4-Tg mice will contribute to the more accurate prediction of
the hepatic clearance of drugs in humans.
Authorship Contributions
Participated in research design: Mitsui, Nemoto, Miyake, Nagao, Ogawa,
Kato, Ishigai.
Conducted experiments: Mitsui, Nemoto, Miyake, Nagao.
Contributed new reagents or analytic tools: Nemoto, Miyake, Nagao.
Performed data analysis: Mitsui, Nemoto, Miyake, Nagao, Ogawa, Kato,
Ishigai.
Wrote or contributed to the writing of the manuscript: Mitsui, Nemoto,
Miyake, Nagao, Ogawa, Kato, Ishigai, Yamada.
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Address correspondence to: Tetsuya Mitsui, Preclinical Research Department,
Research Division, Chugai Pharmaceutical Co., Ltd., Shizuoka, Japan. E-mail:
mitsuitty@chugai-pharm.co.jp
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