Supplemental material to this article can be found at:

http://dmd.aspetjournals.org/content/suppl/2009/11/04/dmd.109.029280.DC1.html
0090-9556/10/3802-249259$20.00
DRUG METABOLISM AND DISPOSITION
Copyright 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD 38:249259, 2010

Vol. 38, No. 2

29280/3550757
Printed in U.S.A.

Time-Dependent Inhibitory Effects of

(1R,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-1,14-Dihydroxy12-(E)-2-[(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1methylvinyl-23,25-dimethoxy-13,19,21,27-tetramethyl-17-(2oxopropyl)-11,28-dioxa-4-azatricyclo[22.3.1.04.9]octacos-18-ene2,3,10,16-tetrone (FK1706), a Novel Nonimmunosuppressive
Immunophilin Ligand, on CYP3A4/5 Activity in Humans in Vivo and
in VitroS

Drug Metabolism Research Laboratories, Astellas Pharma Inc., Osaka, Japan (T.M., T.U., H.K.); Astellas Pharma Global
Development Inc., Deerfield, Illinois (J.L., J.Z., S.M., D.K.); Astellas Research Technologies Co., Ltd., Osaka, Japan (K.I.); and
Sekisui Medical Co. Ltd., Ibaraki, Japan (K.H.)
Received July 5, 2009; accepted November 2, 2009

ABSTRACT:
We investigated the inhibitory effects of (1R,9S,12S,13R,14S,17R,
18E,21S,23S,24R,25S,27R)-1,14-dihydroxy-12-(E)-2-[(1R,3R,4R)-4hydroxy-3-methoxycyclohexyl]-1-methylvinyl-23,25-dimethoxy-13,19,
21,27-tetramethyl-17-(2-oxopropyl)-11,28-dioxa-4-azatricyclo
[22.3.1.04.9]octacos-18-ene-2,3,10,16-tetrone (FK1706), a novel
nonimmunosuppressive immunophilin ligand, on CYP3A4/5 in in
vitro and in vivo settings. First, the inhibitory effects of FK1706
(preincubation dependence, inactivation rate estimation, and
reversibility) were tested using human liver microsomes. Second, the effect of repeated oral doses of FK1706 (60 mg q.d. for
14 days) on the pharmacokinetics of midazolam (single oral
2-mg dose) was tested in healthy volunteers. Finally, pharmacokinetic modeling and simulation were performed. In vitro experiments showed that FK1706 inhibited CYP3A4/5 in a timedependent and irreversible manner. The in vitro maximum
inactivation rate constant (kinact) and concentration of inhibitor

that gave half-maximal kinact (KI) were estimated to be 10.1 h1

and 2050 ng/ml, respectively. In the clinical study, FK1706 produced a 2-fold increase in the area under the time-concentration
curve (AUC) of midazolam. A pharmacokinetic model developed
for this study, which described the time course of concentrations of both FK1706 and midazolam and incorporated
CYP3A4/5 inactivation in the liver and intestine, successfully
predicted the change in the pharmacokinetics of midazolam
using in vitro kinact and KI values (1.66- to 2.81-fold increases in
AUC predicted) and estimated the in vivo inactivation rate to be
0.00404 to 0.0318 h1 ml/ng. In conclusion, FK1706 weakly or
moderately inhibited the activity of CYP3A4/5 in vitro and vivo at
the tested dose. The model developed here would be helpful in
predicting drug-drug interactions and in the design of dose
regimens that avoid drug-drug interactions.

Improvements in the ability to predict time-dependent inhibition

(TDI) are of substantial interest to drug discovery and development at
all stages, from early drug discovery to postmarketing (Zhou et al.,

2005; Grime et al., 2009). However, the dearth of clinical data on TDI
has hampered predictive modeling (Grimm et al., 2009). The two
prediction methods are static and dynamic modeling, both of which
use the in vitro maximum inactivation rate constant (kinact) and the
concentration of inhibitor that gives the half-maximal kinact (KI)
(Grimm et al., 2009). Whereas static models assume steady-state
conditions and use expected maximum inhibitor concentrations (Mayhew et al., 2000; Wang et al., 2004; Galetin et al., 2006; Obach et al.,

Article, publication date, and citation information can be found at

http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.029280.

S The online version of this article (available at http://dmd.aspetjournals.org)

contains supplemental material.

ABBREVIATIONS: TDI, time-dependent inhibition; AUC, area under the time-concentration curve; P450, cytochrome P450; FK1706,
(1R,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-1,14-dihydroxy-12-(E)-2-[(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl-23,
25-dimethoxy-13,19,21,27-tetramethyl-17-(2-oxopropyl)-11,28-dioxa-4-azatricyclo[22.3.1.04.9]octacos-18-ene-2,3,10,16-tetrone; LC-MS/MS, liquid
chromatography-tandem mass spectrometry.
249

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Tsuyoshi Minematsu, Jennifer Lee, Jiuhong Zha, Selina Moy, Donna Kowalski, Katsuyuki Hori,
Koji Ishibashi, Takashi Usui, and Hidetaka Kamimura

250

MINEMATSU ET AL.

Materials and Methods

Materials. FK1706 (molecular weight 820.02) and ascomycin, an internal
standard of FK1706, were synthesized at Astellas Pharma Inc. (Osaka, Japan).
All other chemicals and reagents used were commercially available and of
guaranteed purity. Pooled human liver microsomes (mix gender pool from
liver microsomes of 20 individual donors) were purchased from BD Biosciences (Franklin Lakes, NJ).
In Vitro Inhibition of CYP3A4/5 Activity. In vitro KI and kinact determination. Pooled human liver microsomes (0.3 mg protein/ml) in 100 mM
phosphate buffer (pH 7.4) containing FK1706 (0, 0.1, 0.3, 1, 3, and 10 M)
were preincubated at 37C for 0, 2, 5, 10, and 20 min in the presence of an
NADPH-generating system (0.442 mM NADP, 1.12 mM D-glucose 6-phosphate disodium salt, 0.136 mM glucose-6-phosphate dehydrogenase from
yeast, and 1.122 mM magnesium chloride hexahydrate). The preincubation
samples were then diluted 10-fold using a solution containing midazolam
(0.556 M) and the NADPH-generating system, and reaction of midazolam
1-hydroxylation was started (final condition: 500-l volume, 0.03 mg/ml
protein concentration, and 1.5 M midazolam concentration). After a 10-min

incubation at 37C, the reaction was terminated by the addition of 50 l of internal

standard solution (2 M niflumic acid in methanol), which had been confirmed to
be sufficient to terminate the reaction. Under these reaction conditions, the initial
rate of metabolite formation was observed at 10 min. In addition, metabolite
formation without any inhibitor was 50-fold higher than lower limit of quantification, and the decrease in the unchanged midazolam was less than 30%.
After reaction termination, concentrations of 1-hydroxymidazolam were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS)
as described in the supplemental data. Concentrations of 1-hydroxymidazolam
obtained in each sample (n 3) were then converted into midazolam 1hydroxylation activity (picomoles per minute per milligram of protein). By
dividing the average midazolam 1-hydroxylation activity by that of the control
experiments (0 M FK1706; coefficient of variation values within 12%), the
fraction of control activity at each preincubation time point was then calculated
for each FK1706 concentration. The slope of the natural logarithm value of
the fraction of control activity versus the preincubation time point for each
FK1706 concentration was calculated by linear regression. The absolute value
of the slope was defined as the inactivation rate constant (kobs) for each
FK1706 concentration. In vitro values for KI and kinact (respectively, KI, vitro
and kinact, vitro) were calculated from the relationship between FK1706 concentration and kobs using WinNonlin (Pharsight, Mountain View, CA) and the
equation kobs kinact, vitro [I]/([I] KI, vitro), where kobs was zero at an
FK1706 concentration of zero.
Dialysis experiments to confirm irreversible inhibition. Pooled human liver
microsomes (0.06 mg/ml) in 100 mM phosphate buffer (pH 7.4) containing
FK1706 (0 and 2 M) were preincubated at 37C for 30 min in the presence
or absence of an NADPH-generating system. An aliquot of the samples was
dialyzed using a Slide-A-Lyzer dialyzer (10000 MWCO; Thermo Fisher
Scientific, Waltham, MA) in the dialysis buffer for 24 h at 4C with stirring
(Ma et al., 2000). A 10% Amberlite resin (XAD-4; Rohm and Haas, Philadelphia, PA) was added to the dialysis buffer (100 mM sodium-potassium
phosphate buffer, pH 7.4) to increase the removal of FK1706 from the
microsome samples. The volume of dialysis buffer was 250 times that of the
sample. The dialysis buffer was replaced with new buffer containing Amberlite
at 5 h and with 100 mM sodium-potassium phosphate buffer (pH 7.4) at 21 h.
Remaining preincubation samples were stored separately at 4C as samples
before dialysis. The preincubation samples (before and after dialysis) were
then 2-fold diluted using buffer solution containing midazolam (1 M) and the
NADPH-generating system, and the reaction of midazolam 1-hydroxylation
was started (final condition: 500-l volume, 0.03 mg/ml protein concentration,
and 0.5 M midazolam concentration). Because the TDI by FK1706 had been
observed in 2-fold dilution (data not shown), 2-fold dilution was considered to
be acceptable in this dialysis experiment to see the reversibility qualitatively.
Reaction and measurement of concentrations of 1-hydroxymidazolam were
measured using LC-MS/MS as described above. The concentration of 1hydroxymidazolam in each sample (n 3) was converted into midazolam
1-hydroxylation activity (picomoles per minute per milligram of protein), and
then mean activity values of the samples were converted into remaining
activity (percentage of vehicle control; 0 M samples set as controls). To
determine whether or not dialysis was successful, maintenance of microsomal
protein concentrations during dialysis was confirmed by a BCA protein assay,
and a 100-fold decrease in FK1706 concentration in the microsomes after
dialysis was also confirmed by LC-MS/MS.
Clinical Study of the Effects of FK1706 on CYP3A4/5 Activity. Design.
An open-label, one-sequence crossover study was performed to evaluate the
pharmacokinetic interaction of FK1706 on midazolam, determine the recovery
time course of CYP 3A4/5 enzyme activity if multiple doses of FK1706
exhibited in vivo TDI on CYP 3A4/5, and assess the safety and tolerability of
FK1706 alone and in combination with midazolam. The pharmacokinetic
interaction of multiple-dose FK1706 (60 mg, two 30-mg tablets, once daily
during days 215) on single-dose midazolam (oral 2 mg, 1 ml of syrup, on days
1, 15, 22, and 29) was assessed in a single group of 24 healthy subjects (16
males and 8 females) aged 18 to 60 years of whom 18 were white and the rest
were black, Asian, or other. All subjects were given at least one dose of
midazolam and FK1706. Twenty-two of the 24 subjects completed the study,
with one male each discontinuing before day 15 because of a protocol violation
and withdrawal of consent not related to adverse events. Both subjects received
only a single dose of midazolam and were therefore not included in the

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2007), dynamic models are nonequilibrium pharmacokinetic models

that describe the time course of substrate and inhibitor concentrations
as well as P450 inactivation (Ito et al., 1998; Yamano et al., 2001; Ito
et al., 2003). It would be also a point at which P450 in the liver, the
intestine, or both is incorporated into the model. Although a useful
static prediction model has been developed, incorporating the effects
of TDI on both intestinal and liver CYP3A4 (Wang et al., 2004;
Galetin et al., 2006; Obach et al., 2007), this model cannot describe
the time course of the substrate or inhibitor concentrations. Dynamic
models have been developed for TDI of the liver CYP3A4/5 (Ito et al.,
1998; Yamano et al., 2001; Ito et al., 2003). The accuracy of estimation may be improved by the use of the in vivo (not in vitro)
inactivation rate. A study to estimate the in vivo inactivation rate
constant for intestinal CYP3A4 metabolism successfully described the
TDI by grapefruit juice (Takanaga et al., 2000). With regard to
reversible (direct) inhibitors of P450, one recent report described the
concept of estimating the in vivo inhibition constant on hepatic
clearance (Kato et al., 2008).
FK1706 (Price et al., 2005) is a novel, nonimmunosuppressive
immunophilin ligand (Price et al., 2005; Voda et al., 2005; Hayashi et
al., 2006; Bella et al., 2007; Yamaji et al., 2008; Yamazaki et al.,
2008). FK1706 shows significant neurotrophic effects, which are
putatively mediated via FK506-binding protein 52 and the Ras/Raf/
mitogen-activated protein kinase signaling pathway, suggesting the
potential of this compound to be used to treat a variety of neurological
disorders (Price et al., 2005). During drug development, FK1706 was
found to selectively inhibit CYP3A4/5 [the in vitro inhibition constant of
reversible (direct) inhibition (Ki, dir, vitro) for FK1706 was 0.833 M at the
microsomal protein concentration of 0.05 mg/ml] among P450s tested
while not affecting other P450s up to 10 M (H. Kaneko, T. Minematsu,
K. Tasaki, S. Terashita, T. Usui, and H. Kamimura, unpublished data). It
was still not known whether the CYP3A4/5 inhibition was time-dependent. Therefore, we considered it important to assess the potency of the
interaction between FK1706 and CYP3A4/5.
Here, to help predict drug-drug interactions involving FK1706 and
design dose regimens that avoid clinically significant interactions, we
characterized in detail the inhibition of CYP3A4/5 by FK1706 through in
vitro experiments using human liver microsomes. We then investigated
the effect of repeated oral administration of FK1706 on CYP3A4/5 in
healthy volunteers using midazolam as a representative drug that is
eliminated mainly by CYP3A4/5 metabolism. Finally, we developed a
dynamic model to explain the in vitro/vivo results in a quantitative
manner. We also compared the predictions based on in vitro P450
inactivation parameters with those based on in vivo parameters.

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CYP3A4/5 INACTIVATION BY FK1706 IN HUMANS

TABLE 1
Summary of drug administration and blood sampling in clinical study
Study Day
FK1706 Dosing Period

Recovery Period

Before FK1706 Dose: 1

211

FK1706 administration (oral 60 mg once daily)

Blood sampling for FK1706
Midazolam administration (oral single 2 mg)
Blood sampling for midazolamd

X
X
X

1214

X
Xb

15

1621

22

2328

29

X
Xc
X
X

Xb
X
X

Xb
X
X

FK1706 and midazolam were administered simultaneously on day 15.

Only predose of FK1706 or midazolam administration.
Predose, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, and 24 h after oral administration of FK1706 on day 15.
d
Predose, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, and 24 h after single oral administration of midazolam.
b
c

C b,i 1 Ht Cp,i Ht

fp,i Cp,i

Bmax,i fp,i Cp,i

Kd,i fp,i Cp,i

(1)

where Ht is hematocrit (fixed to 0.413, which was the mean value of 22

subjects), fp, i is the fraction unbound of FK1706 in plasma (fixed to 0.01;
H. Tokuda, K. Yamano, M. Katashima, and A. Kawamura, unpublished data),
Bmax, i is the binding capacity of FK1706 in blood cells, and Kd, i is the binding
constant for FK1706. Equation 1 was fitted to the data set of systemic Cb, i
(Cb, sys, i) versus systemic Cp, i (Cp, sys, i), and the parameters were estimated to
be 460 17 and 0.0148 0.0019 ng/ml for Bmax, i and Kd, i, respectively
(estimate S.E.). Hereafter, these parameters were used as fixed values.
Subsequently, the following two-compartment model with first-order absorption (eqs. 2 6) was applied (Gabrielsson and Weiner, 2007):
dC b,sys,i/dt k1o,i Cb,sys,i k12,i Cb,sys,i

k21,i X2,i ka,i Fi Ai

,
V1 /Fi
V1 /Fi
Cb,sys,i0 0

dX2,i/dt k12,i V1 /Fi Cb,sys,i k21,i X2,i,

C2,i0 0

if 0 h t 24 h, then Ai 0

(2)
(3)
(4)

if 24 h t 336 h (after nth dose of FK1706),

(5)

(6)

then Ai Dosei

ek a,it24m

m1

if t 336 h [after 14th (last) FK1706 dose],

14

then Ai Dosei

ek a,it24m

m1

where Dosei is the dose of FK1706 (60 mg orally at 24, 48, . . . , 336 h; every
24 h for 14 times), X2, i is the FK1706 amount in compartment 2, X2, i is
X2, i/Fi, ka, i is the absorption rate constant for FK1706, k1o, i is the elimination
rate constant, k12, i, k21, i is the distribution rate constant, V1/Fi is the distribution volume divided by bioavailability, and Ai is the amount of FK1706 at the
site of absorption. Cb, sys, i, described using eqs. 2 to 6, was fitted to the time
profile of the mean whole blood concentrations of FK1706, and the parameters
were estimated to be 1.56 0.85 h1, 0.0522 0.0185 h1, 0.354 0.217
h1, 0.0694 0.0179 h1, and 320,000 112,000 ml for ka, i, k10, i, k12, i,
k21, i, and V1/Fi, respectively (estimate S.E.). These parameters are hereafter
used as fixed values. Subsequently, eq. 1 gives eq. 7 to express Cp, sys, i:
C p,sys,i

b b2 4 a c
2a

(7)

a f p,i 1 Ht fp,i Ht
b K d,i 1 Ht fp,i Ht fp,i Bmax,i Ht fp,i Cb,sys,i
c Cb,sys,i Kd,i
The unbound FK1706 concentration at the inlet to the liver (Cu, inlet, i) was
defined as in eqs. 8 to 10 (Ito et al., 1998):

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pharmacokinetic analysis set. The study protocol was reviewed and approved
by an independent ethics committee. All subjects were informed of the nature
and purpose of the study and written informed consent was obtained. There
were no clinically important changes in laboratory measurements, vital signs,
electrocardiograms, or physical examination data during the study. Subjects
fasted for at least 8 h before the morning dosing of test drugs and continued
fasting for 4 h after dosing. Water consumption was permitted ad libitum until
dosing and could be resumed from 2 h after dosing. Dosage and blood
sampling are summarized in Table 1. Because of its extensive distribution into
blood cells, FK1706 concentrations were measured in both blood (whole
blood) and plasma by using LC-MS/MS as described in the supplemental data.
Concentrations of midazolam and 1-hydroxylmidazolam were measured separately using LC-MS/MS as described in the supplemental data. Blood and
plasma samples obtained were stored at 20C and analyzed within the
stability period for FK1706, midazolam, and 1-hydroxymidazolam.
Noncompartmental pharmacokinetic analysis and statistical test. Noncompartmental analysis was performed using WinNonlin (Gabrielsson and Weiner,
2007). For each subject, the following plasma pharmacokinetic parameters of
midazolam and 1-hydroxymidazolam were obtained for each midazolam
administration (days 1, 15, 22, and 29): maximum concentration (Cmax), time
to reach Cmax (tmax), area under the time-concentration curve from zero to
infinity (AUCinf), half-life (t1/2), clearance divided by bioavailability (CL/F),
and distribution volume divided by bioavailability (Vd/F). In addition, the
following pharmacokinetic parameters of FK1706 were computed for blood
and plasma concentrations on day 15: Cmax, tmax, AUC0 24 h, CL/F, and Vd/F.
For evaluation of pharmacokinetic interaction, statistical analyses were performed according to Guidance for Industry: in Vivo Drug Metabolism/Drug
Interaction Studies, released by the U.S. Food and Drug Administration
(http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM072119.pdf). The primary pharmacokinetic parameters for the evaluation of pharmacokinetic interaction were AUC0-inf and
Cmax of midazolam between day 15 and day 1. The two one-sided paired t test
procedure was used with SAS (SAS Institute Inc., Cary, NC) to construct the
90% confidence interval around the geometric mean ratio of plasma AUCinf
and Cmax of midazolam between day 15 and day 1, between day 22 and day 1,
and between day 29 and day 1. If the 90% confidence interval of the geometric
mean ratio is contained entirely within 0.8 to 1.25, then no significant pharmacokinetic drug-drug interaction is expected. Metabolic ratio, AUCinf ratio
1-hydroxymidazolam/midazolam, multiplied by 326 (molecular weight of
midazolam) and divided by 342 (molecular weight of 1-hydroxymidazolam),
was also calculated as supportive evidence.
Dynamic Modeling and Simulation for in Vitro and in Vivo CYP3A4/5
Inactivation. General analysis conditions. Model fitting was performed by
WinNonlin using the Gauss-Newton algorithms with Levenberg and Hartley
modification. Simulation was also performed using WinNonlin. The timing of
midazolam administration on day 1 was defined as time (t) 0 (h).
Pharmacokinetic modeling for FK1706 (days 2 to 29). First, because the
distribution of FK1706 into blood cells was saturable), the relationship between FK1706 concentrations in blood (Cb, i) and those in plasma (Cp, i) was
described using eq. 1 (Kawai et al., 1998; Minematsu et al., 2001):

252

MINEMATSU ET AL.
ka,i Fa,i Fg,i Ai
Qh

(8)

b b2 4 a c
2a

(9)

C b,inlet,i Cb,sys,i

C p,inlet,i

c Cb,inlet,i Kd,i

kinact Cg,i
E ,
KI Cg,i act,g

Eact,g0 E0,g (18)

dE act,g/dt kdeg,g E0,g Eact,g

(11)

where Qg is the enterocytic blood flow rate, 66,000 ml/h (Davies and Morris,
1993).
Pharmacokinetic modeling for midazolam in the absence of FK1706 (day
1). Pharmacokinetics of midazolam was expressed using the eqs. 12 to 16
(Kato et al., 2008):

(12)

dC h,s/dt Qh Cb,s Qh Rb,s Ch,s/Kp,h,s fm,s fp,s CLh,int,s Ch,s/Kp,h,s

1 fm,s fp,s CLh,int,s Ch,s/Kp,h,s ka,s Fa,s Fg,s As /Vh,
Ch,s 0 0
X2,s 0 0

F g,s*

Eact,h
f CLh,int,s
E0,h m,s

(19)

1
Eact,g
1
1 Fg,s
1

E0,g 1 Cg,i/Ki,dir
Fg,s

(20)

where (fm, s CLh, int, s)* and Fg, s* are fm, s CLh, int, s and Fg, s in the presence
of FK1706, respectively, and Eact/E0 and 1/(1 [I]/Ki, dir) represent the TDI
and direct inhibition, respectively. It was assumed that the permeability of
midazolam across the intestinal lumen was not changed by the inhibitor, and
midazolam was metabolized only by CYP3A4/5 in the intestine (Wang et al.,
2004). Equation 13 can therefore be changed as in eq. 21:

dC h,s/dt Qh Cb,s Qh Rb,s Ch,s/Kp,h,s

Eact,g
f f CLh,int,s Ch,s/Kp,h,s
E0,g m,s p,s

1 fm,s fp,s CLh,int,S Ch,s/Kp,h,s

dC b,s/dt Qh Rb,s Ch,s/Kp,h,s Qh Cb,s CLr,s Cb,s k12,s V1,s Cb,s

k21,s X2,s/V1,s Cb,s 0 0

f m,s CLh,int,s*

ka,s Fa,s

1
As /Vh (21)
Eact,g
1
1Fg,s
1

E0,h 1 Cg,i /Ki,dir

Fg,s

Midazolam administration at 336 h (day 15), 504 h (day 22), and 672 h (day
29) was incorporated in the model as in eqs. 22 to 24:
(13)
if 336 h t, then Cb,s336 h Ch,s336 h X2,s336 h 0,
(14)

A s Doses eka,s.t

(15)

C p,s Cb,s/Rb,s

(16)

where Doses is the dose of midazolam (2 mg), Cb, s is the midazolam concentration in systemic blood, Ch, s is the midazolam concentration in the liver, X2, s
is the midazolam amount in compartment 2, Cp, s is the midazolam concentration in the systemic plasma, CLr, s is the renal clearance for midazolam,
CLh, int, s is the intrinsic hepatic clearance, fm, s is the fraction metabolized by
CYP3A4/5 in the liver, fp, s is the unbound fraction of midazolam in the
plasma, ka, s is the absorption rate constant for midazolam, k12, s, k21, s is the
distribution rate constant, Kp, h, s is the liver/plasma concentration ratio for
midazolam, Rb, s is the blood/plasma concentration ratio, V1, s is the distribution
volume, Vh is the volume of the liver, As is the amount of midazolam at the site
of absorption, Fa, s is the availability of midazolam for absorption, and Fg, s is
the availability of midazolam during first-pass metabolism in the intestine.
Kp, h, s, CLr, s, fm, s, fp, s, Rb, s, Fa, s, and Fg, s were fixed at 6.59, 8.01 ml/h, 1,
0.05, 0.675, 1, and 0.615, respectively (Ito et al., 2003; Kato et al., 2008). The
Cp, s described by eqs. 12 to 16 was fitted to the time profile of mean plasma
concentrations of midazolam on day 1 and the parameters were estimated to be
3.42 0.59 h1, 0.262 0.062 h1, 0.328 0.023 h1, 1,170,000 30,000
ml/h, and 69,600 7500 ml for ka, s, k12, s, k21, s, CLh, int, s, and V1, s,
respectively (estimate S.E.). These parameters were hereafter used as
fixed values.
Prediction model for pharmacokinetics of midazolam in the presence of
FK1706 (days 15, 22, and 29) using the in vitro CYP3A4/5 inactivation rate
constant. After administration of FK1706, the amount of active CYP3A4/5 in
the liver (Eact, h) and in the enterocytes (Eact, g) was defined as in eqs. 17 and
18 (Ito et al., 1998; Mayhew et al., 2000):

As Doses ek a,st336

(22)

if 504 h t, then Cb,s504 h Ch,s504 h X2,s504 h 0,

As Doses ek a,st504

(23)

If 672 h t, then Cb,s672 h Ch,s672 h X2,s672 h 0,

As Doses ek a,st672

(24)

By using eqs. 2 to 12 and 14 to 24 and the following parameters as obtained

above, the plasma concentrations of midazolam on days 15, 22, and 29 were
simulated:
k inact kinact,vitro
K I fu,mic,i,0.3mg/mL KI,vitro
K i,dir fu,mic,i,0.05mg/mL Ki,dir,vitro
where the unbound fraction of FK1706 in the human liver microsomes
(fu, mic, i) was calculated to be 0.529 at a microsomal protein concentration of
0.3 mg/ml (fu, mic, i, 0.3 mg/ml) and 0.871 at a concentration of 0.05 mg/ml
(fu, mic, i, 0.05 mg/ml) using the prediction method of Hallifax and others (Hallifax
and Houston, 2006; Gertz et al., 2008), which enables calculation of fu, mic, i
using the physicochemical parameter and microsomal protein concentration.
Direct inhibition of hepatic CYP3A4/5 by FK1706 was excluded from the
present model because fu, mic, i, 0.05 mg/ml (0.871) Ki, dir, vitro (0.833 M; 683
ng/ml) was much higher than the maximum Cu, inlet, i (18.5 ng/ml) (Table 7)
and therefore did not affect the pharmacokinetics of midazolam at all, even
when the 10-fold concentrative uptake of FK1706 into the liver was assumed.
The kdeg, h and kdeg, g were assumed to be 0.00495 to 0.0693 h1 and 0.0210 to

Downloaded from dmd.aspetjournals.org at ASPET Journals on August 6, 2015

ka,i Fa,i Ai
fp,i Cp,sys,i
Qg

dX 2,s/dt k12,s V1,s Cb,s k21,s X2,s,

Eact,h0 E0,h (17)

(10)

where Cb, inlet, i is the FK1706 concentration in blood at the inlet to the liver,
Cp, inlet, i is the FK1706 concentration in plasma at the inlet to the liver, Fa, i is
the availability of FK1706 through absorption (assumed to be 1), Fg, i is the
availability of FK1706 through first-pass metabolism in the intestine (assumed
to be 1), and Qh is the hepatic blood flow rate, 96,600 ml/h (Kato et al., 2008).
Then, the concentration of FK1706 in the enterocytes (Cg, i) was defined as in
eq. 11 (Rostami-Hodjegan and Tucker, 2004):
C g,i

kinact Cu,inlet,i
E ,
KI Cu,inlet,i act,h

where kdeg, h and kdeg, g are the rate constants for CYP3A4/5 degradation in the
liver and intestine, respectively, and E0, h and E0, g are the initial amounts of
active CYP3A4/5 enzymes in the liver and intestine, respectively.
In the presence of FK1706, eqs. 19 and 20 were defined (Wang et al., 2004):

b K d,l (1Ht fp,i Ht) fp,i Bmax,i Ht fp,i Cb,inlet,i

C p,inlet,i fp,i Cp,inlet,i

dE act,h/dt kdeg,h E0,h Eact,h

253

CYP3A4/5 INACTIVATION BY FK1706 IN HUMANS

0.1 M

0.3 M

1 M

10 M

3 M

kobs (min-1)

ln(fraction of control activity)

AUCs,day 15

AUCs,day 1

0.15

Fg,s 1 Fg,s

0.10

1
kinact,vitro Cg,i,max
1
kdeg,g Cg,i,max fu,mic,i KI,vitro

0.05

fm,s

1
0

kinact,vitro Imax
fu,mic,i KI,vitro kdeg,h

1 fm,s

0.00

10

15

20

where AUCs, day15 and AUCs, day1 are the AUCs of plasma midazolam concentrations on day 15 and 1, respectively. Cg, i, max is the maximum Cg, I, and
[I]max is the maximum Cp, sys, i, fp, i Cp, sys, i, Cp, inlet, i, or Cu, inlet, i. The two
pairs of kdeg, h and kdeg, g were used as described above. In addition, simulation
was performed in the absence of liver or intestine CYP3A4/5 inactivation as
sensitivity analysis.

0 1 2 3 4 5 6 7 8 9 10 11

Pre-incubation time (min)

FK1706 concentration (M)

FIG. 1. A, time-dependent loss of CYP3A4/5 activity (midazolam 1-hydroxylation) after preincubation of human liver microsomes with NADPH and various
concentrations of FK1706. Data from time 0 to 10 min were used to calculate the
slope (each broken line represents the linear regression line from 0 to 10 min). B,
concentration-dependent CYP3A4/5 inactivation rate by FK1706. A broken line
shows the best-fit line.

Results

0.0578 h , respectively (Yang et al., 2008). The value pair of kdeg, h 0.00495
h1 and kdeg, g 0.0210 h1 was used to obtain the maximum AUC increase for
midazolam concentrations in plasma (AUCs). Subsequently, kdeg, h 0.0693 h1
and kdeg, g 0.0578 h1 was used to obtain the minimum AUCs increase.
Simulation was additionally performed in the absence of liver or intestine P450
inactivation as a sensitivity analysis to assess the contribution of each dimension of inhibition. In addition, effects of the changes in Fa, i, Fg, i, fu, mic, i, and
fp, i on AUCs were also simulated. AUCs values were calculated by the integral
of Cp, s using dt as a variable of integration.
In vivo inactivation rate estimation for FK1706 on the pharmacokinetics of
midazolam (days 15, 22, and 29). For eqs. 17 and 18,
k inact Cu,inlet,i
and
KI Cu,inlet,i

kinact Cg,i
KI Cg,i

were changed to kvivo Cu, inlet, i and kvivo Cg, i, respectively (designated as eqs.
17 and 18), to reduce the number of parameters (actually, calculated Cu, inlet, i
and Cg, i were not over KI, vitro). When [I] (Cu, inlet, i and Cg, i) KI, precise
estimation of kinact and KI is impossible. Furthermore, when [I] KI,
1/2 kinact/KI kvivo kinact/KI (because when [I] KI, kinact [I]/(KI
[I]) (kinact/KI) [I], and when [I] KI, kinact [I]/(KI [I]) (kinact/2/KI)
[I]). Therefore, it was considered practical and useful to use kvivo. Thus, Cp, s
values described using eqs. 2 to 12, 14 to 16, 17, 18, and 19 to 24 were fitted
to the time profile of mean plasma midazolam concentrations on days 15, 22,
and 29 and the parameter kvivo was estimated. The two pairs of kdeg, h and kdeg, g
were used as described above. In addition, to assess the contribution of each
dimension of inhibition, simulation for sensitivity analysis and AUC calculation were performed as described above.
Prediction of CYP3A4/5 Inactivation by FK1706 Using Static Model.
The -fold increase in AUC for plasma midazolam concentrations after repeated
oral FK1706 administration (60 mg) was predicted using the static model
(Wang et al., 2004; Obach et al., 2007):

TABLE 2
Inhibitory effects of FK1706 on midazolam 1-hydroxylase activity in preincubation samples of human liver microsomes with or without NADPH and/or FK1706 and
before and after dialysis
Data are expressed as mean S.D. (n 3). Values in parentheses show the percentage of control.
Midazolam 1-Hydroxylase Activity
FK1706 Conc. during Preincubation

Before Dialysis
NADPH ()

After Dialysis
NADPH ()

NADPH ()

NADPH ()

54.1 2.9
3.48 0.59 (6.4)

51.9 2.9
50.3 2.8 (96.9)

pmol/min/mg protein

0 M (control)
2 M

150 10
9.94 0.18 (6.6)

114 9
60.9 5.7 (53.5)

Downloaded from dmd.aspetjournals.org at ASPET Journals on August 6, 2015

In Vitro Inhibition of CYP3A4/5 Activity (Midazolam 1-Hydroxylation) by FK1706. Estimation of kinact and KI. FK1706 inhibited midazolam 1-hydroxylase activity in time- and concentrationdependent manners (Fig. 1. The kinact, vitro and KI, vitro values of
FK1706 on midazolam 1-hydroxylase activity were 0.168 (10.1
h1) 0.008 min1 and 2.50 (2050 ng/ml) 0.32 M, respectively
(estimate S.E.) (Fig. 1).
Effects of dialysis. When human liver microsomes and FK1706
were preincubated without NADPH, midazolam 1-hydroxylase activity recovered completely to the level of control after dialysis,
suggesting that dialysis removed FK1706 from the preincubation
samples (Table 2). In contrast, with preincubation in the presence of
NADPH, midazolam 1-hydroxylase activity was not recovered at all
(Table 2). These results suggested that the TDI of FK1706 on
CYP3A4/5 was irreversible. Dialysis itself was also suggested as
acting to reduce the activities of CYP3A4/5 (Table 2).
Clinical Study of the Effects of FK1706 on CYP3A4/5 Activity
(Pharmacokinetics of Midazolam). Pharmacokinetic parameters for
FK1706 are listed in Table 3. Time profiles of FK1706 concentrations
in blood and plasma are shown in Figs. 2 and 3. FK1706 was
eliminated from the body in a biphasic manner (Fig. 3). The relationship between blood and plasma FK1706 showed that the distribution
of FK1706 into blood cells was markedly high and was saturated at
high FK1706 concentrations (Fig. 2C). The high AUC ratio for
blood/plasma (Table 3) also showed the high distribution of FK1706
into blood cells.
Pharmacokinetic parameters for midazolam and 1-hydroxy midazolam are summarized in Table 4. For midazolam, the changes in the
parameters on day 15 (after multiple doses of FK1706) from those on
day 1 (before dose of FK1706) suggested that FK1706 inhibited the
major metabolism of midazolam, i.e., CYP3A4/5. The geometric
mean ratio (90% confidence interval) of AUCinf and Cmax of

254

MINEMATSU ET AL.
TABLE 3

Summary of blood and plasma FK1706 pharmacokinetic parameters by the

noncompartmental method
Data are median for Tmax and mean S.D. for other parameters.
Day

Blood
Plasma

Day 15
Day 15

22
22

Cmax

Tmax

AUC024

ng/ml

ng h/ml

CL/F
l/h

265 67.1
53.3 58.2

0.75
1.00

3403 667
138 129

18.2 3.21
728 438

midazolam suggested that statistically significant drug interaction

occurred on day 15 and that the activity of CYP3A4/5 recovered by
day 22. This finding was also supported by a decrease in the
metabolic ratio for 1-hydroxymidazolam on day 15 (Table 3). In
contrast, pharmacokinetic parameters of midazolam and the metabolic ratio for 1-hydroxymidazolam on days 22 and 29 were
recovered to those on day 1, suggesting that CYP3A4/5 activity

FK1706 concentration
in blood (ng/mL)

14th FK1706 dose

300

200

100

0
332

336

340

344

348

352

356

360

Time (h)

FK1706 concentration
in plasma (ng/mL)

55
50
45
40
35
30
25
20
15
10
5
0
332

14th

Discussion

FK1706 dose

336

340

344

348

352

356

360

Time (h)

FK1706 concentration
in blood (ng/mL)

300

200

100

0
0

10

20

30

40

50

60

FK1706 concentration
in plasma (ng/mL)
FIG. 2. Time profiles of FK1706 concentration in blood (A) and plasma (B) on day
15 (after the last dose of the 14-day oral 60 mg q.d. administration of FK1706 to
healthy volunteers). Data are expressed as mean S.E.M. (n 22). Broken lines
were calculated using eqs. 2 to 7. C, relationship between FK1706 concentration in
blood and plasma on days 12 to 29. Data are expressed as mean S.E.M. (n 22).
The broken line is the line of best-fit using eq. 1.

Because clinical outcomes depend on a number of factors that are

associated with drugs and patients, prediction of drug-drug interactions involving CYP3A4 inactivation remains difficult (Zhou et al.,
2005). In this study, we investigated the inhibitory effects of FK1706,
a novel, nonimmunosuppressive immunophilin ligand, on CYP3A4/5
in in vitro and in vivo settings. Results showed clinical evidence of the
suitability of dynamic modeling for TDI of P450.
In in vitro experiments, FK1706 was suggested to be an irreversible
time-dependent inhibitor of CYP3A4/5. Inhibition was NADPH- and
preincubation time-dependent, and CYP3A4/5 activity was not restored after physical removal of FK1706 by dialysis. FK1706 showed
concentration-dependent inhibition with saturation kinetics (kinact, vitro
10.1 h1 and KI, vitro 2050 ng/ml).
In the clinical study, it is particularly notable that the AUCs of
plasma midazolam concentrations were increased 2-fold after repeated oral administration of FK1706. On this basis, FK1706 at the
tested dose would be classified as a weak or moderate inhibitor of
CYP3A4/5 (Bjornsson et al., 2003). CL/F based on blood FK1706
concentrations (18.2 l/h), was much lower than the hepatic blood flow
rate (96.6 l/h) (Kato et al., 2008) and renal blood flow rate (74.4 l/h)
(Davies and Morris, 1993). FK1706 was therefore shown to be a low
clearance drug, provided that elimination occurs via the liver, kidneys,
or both. Here, the hepatic extraction ratio [1 bioavailability through
first-pass elimination in the liver (Fh)] was equal to the hepatic
clearance (CLh)/Qh CL/Qh CL/F/Qh. Furthermore, because CL/
F/Qh is equal to 0.188, Fh, i 0.812. The distribution of FK1706 into
blood cells was markedly high and was saturated at high FK1706
concentrations. The high AUC ratio for blood/plasma (3403:138)
(Table 2) showed that the blood/plasma concentration ratio was 24.7
on average. High and saturable binding of a drug to blood cells can be

Downloaded from dmd.aspetjournals.org at ASPET Journals on August 6, 2015

was almost completely recovered within 7 days after the end of

administration of FK1706.
Dynamic Modeling and Simulation for in Vitro and in Vivo
CYP3A4/5 Inactivation. Best-fit lines in Figs. 2 to 4 confirmed that
the pharmacokinetics of FK1706 on days 2 to 29 and those of
midazolam on day 1 were well predicted by the present model. This
pharmacokinetic model was then used to calculate FK1706 concentrations in the intestine and unbound FK1706 concentration at the inlet
to the liver (Fig. 3). The kvivo values were estimated to be 0.00400
0.00064 (at kdeg, h 0.00495 h1 and kdeg, g 0.0210 h1) to 0.0318
0.0069 h1 ml/ng (at kdeg, h 0.0693 h1 and kdeg, g 0.0578 h1)
(estimate S.E.). The kvivo values were comparable to the in vitro
value, kinact, vitro/(fu, mic, i KI, vitro): 0.00923 h1 ml/ng. Predicted
-fold increases in the AUC of plasma midazolam concentrations on
day 15 are summarized in Table 5. When the in vitro inactivation rate
constant was used, the predicted -fold increases in Cmax were 1.57 to
2.00 on day 15, whereas those using the in vivo inactivation rate
constant were 1.76 to 1.77. Predicted active CYP3A4/5 in the intestine
and liver during and after repeated FK1706 administration is shown in
Fig. 5. On days 22 and 29, no increase was predicted in AUC and
Cmax (data not shown). The effects of the changes in Fa, i, Fg, i,
fu, mic, i, and fp, i on AUCs are summarized in Table 6.
Prediction of CYP3A4/5 Inactivation by FK1706 Using Static
Model. Predicted -fold increases in the AUC of plasma midazolam
concentrations after repeated FK1706 administration are summarized
in Table 7. Use of the unbound systemic maximum concentration of
FK1706, i.e., fp, i Cp, sys, i, max, yielded the most accurate prediction
(Table 7), albeit that all concentrations tended to overpredict the
increase in AUC.

255

CYP3A4/5 INACTIVATION BY FK1706 IN HUMANS

100
10

Cp,sys,i (ng/mL)

Cb,sys,i (ng/mL)

1000

100

10

1
0

1
0.1
0.01

72 144 216 288 360 432 504 576 648 720

10000

100

72 144 216 288 360 432 504 576 648 720

Time (h)

D
Cu,inlet,i (ng/mL)

1000

10
1
0.1
0.01

100
10
1
0.1
0.01
0.001

0.001
0

72 144 216 288 360 432 504 576 648 720

72 144 216 288 360 432 504 576 648 720

Time (h)

Time (h)

FIG. 3. Time profiles of predicted FK1706 concentrations in systemic blood (Cb, sys, i) (A), systemic plasma (Cp, sys, i) (B), and intestine (Cg, i) (C), as well as unbound
FK1706 concentrations at the inlet to the liver (Cu, portalvein, i) (D) during and after the 14-day oral 60 mg q.d. administration of FK1706 to healthy volunteers. Lines show
predicted values; E show observed values (mean S.E.M., n 22).
TABLE 4
Summary of plasma midazolam and 1-hydroxymidazolam pharmacokinetic parameters by the noncompartmental method
Data are median for Tmax, geometric mean (90% confidence intervals) for geometric mean ratio to day 1 for Cmax and AUCinf, and mean S.D. for other parameters.
n

Midazolam
Day 1
Day 15
Day 22
Day 29
1-Hydroxymidazolam
Day 1
Day 15
Day 22
Day 29

Cmax

Tmax

AUCinf

t1/2

CL/F

ng/ml

ng h/ml

l/h

22
22
22
22

9.99 3.70
15.9 5.48
9.80 4.57
10.5 4.96

0.50
0.50
0.50
0.50

22.2 9.00
44.6 23.7
23.0 11.3
22.9 11.6

3.74 1.95
4.13 1.93
3.75 2.10
3.76 3.39

108 58.5
54.1 21.6
106 48.0
105 41.7

22
22
22
22

4.57 1.86
4.70 2.28
3.77 1.53
5.18 2.66

0.50
0.75
0.50
0.50

9.08 3.35
11.1 3.59
8.49 3.76
9.50 4.12

3.32 3.38
3.46 3.37
3.02 2.55
2.28 1.25

Geometric Mean Ratio

to Day 1 for Cmaxa

Geometric Mean Ratio

to Day 1 for AUCinfa

1.62 (1.46,1.80)
0.97 (0.88,1.08)
1.04 (0.94,1.15)

1.97 (1.78,2.18)
1.02 (0.92,1.13)
1.01 (0.91,1.12)

Metabolic
Ratiob

0.422 0.161
0.272 0.104
0.388 0.152
0.441 0.179

Primary parameters for drug-drug interaction assessment.

b
Supportive evidence for drug-drug interaction: AUCinf ratio 1-hydroxymidazolam/midazolam, multiplied by 326 (molecular weight of midazolam) and divided by 342 (molecular weight of
1-hydroxymidazolam).

one of major determinants of pharmacokinetics (Kawai et al., 1998;

Minematsu et al., 2001).
A dynamic model developed for this study, which described the
time course of changes in FK1706 and midazolam concentrations well
predicted the -fold increase in the AUC of midazolam using the in
vitro CYP3A4/5 inactivation rate constant by FK1706 (kinact, vitro and
KI, vitro). The predicted -fold increase in AUC was 1.66 to 2.81 versus
an actual increase of 2. The in vivo CYP3A4/5 inactivation rate, kvivo,
was also estimated by fitting the equations to the time profile of
plasma midazolam concentrations using kvivo. The prediction of -fold
increase in AUC was 2.06 to 2.10. Here, it may seem as if the use of
kvivo would be less useful, because the value cannot be estimated
without the results of a clinical drug-drug interaction study. However,
prediction using the in vivo inactivation rate constant does enable the
more accurate simulation of a variety of situations such as different
dose regimens or different interacted drugs. In fact, simulation using

the present model and in vivo inactivation rate constant (kvivo) showed
that when the dose was reduced from 60 mg to the level of 3 to 10 mg,
the -fold increase in AUC would be less than 1.25, which can be
considered to indicate no drug interaction (data not shown). Sensitivity analysis showed that inhibition of both intestinal and hepatic
CYP3A4/5 by FK1706 contributed to the increase in the AUC of
midazolam (Table 4). When time profiles of unbound systemic plasma
concentrations of FK1706 were used as a surrogate of the unbound
concentration of FK1706 at the inlet in eq. 17, no hepatic CYP3A4/5
inactivation was predicted (data not shown).
In the prediction using the static method of Wang and others (Wang
et al., 2004; Obach et al., 2007), the use of maximum unbound
systemic concentration of FK1706, i.e., fp, i Cp, sys, i, max, yielded the
most accurate prediction among [I]max but nevertheless still overpredicted compared with the dynamic model developed in this study.
Prediction using other concentrations resulted in much greater over-

Downloaded from dmd.aspetjournals.org at ASPET Journals on August 6, 2015

C
Cg,i (ng/mL)

Time (h)

256

MINEMATSU ET AL.

Midazolam concentration
in plasma (ng/mL)

Day 1
17.5
15.0
12.5
10.0
7.5
5.0
2.5
0.0
-4

12

20

24

Midazolam concentration
in plasma (ng/mL)

352

356

360

Day 15
17.5
15.0
12.5
10.0
7.5
5.0
2.5
0.0
332

336

340

344

348

Time (h)
FIG. 4. Time profiles of midazolam concentrations in plasma on day 1 (A) and day
15 (B) after a single oral administration of midazolam at 2 mg to healthy volunteers.
Data are expressed as mean S.E.M. (n 22). A, broken line represents the best-fit
line using eqs. 13 to 16. B, lines express the values predicted using inactivation rate
(KI, vitro and kinact, vitro or kvivo). A solid line expresses values predicted using
kinact, vitro, KI, vitro, and the pair of kdeg, g (0.0210 h1) and kdeg, h (0.00495 h1),
respectively, and a broken line expresses values predicted using kinact, vitro, KI, vitro,
and another pair of kdeg, g (0.0578 h1) and kdeg, h (0.0693 h1), respectively. Using
kvivo, a dashed line was obtained (closely similar results were obtained for either of
the pair of kdeg, g and kdeg, h).

prediction. These observations are consistent with the results of Obach

et al. (2007). Whereas unbound portal venous concentration (unbound
concentration at the inlet to the liver) of an inhibitor would most
accurately reflect hepatic concentration among the tested inhibitor
concentrations, it seems reasonable that overprediction in the static
model would occur with the use of the maximum unbound concentration of an inhibitor at the inlet to the liver. The reason for this is as
follows: although it was assumed in the static model that maximum
concentration was maintained at steady state, actual unbound drug
concentrations at the inlet to the liver would decrease as the drug
amount in the absorption site decreases. Further accumulation of data
is warranted, and use of the maximum unbound systemic plasma

TABLE 5
Prediction of -fold increase in AUC (AUCs, day15 /AUCs, day1) for plasma midazolam concentrations after repeated oral administration of FK1706 (60 mg) using a
dynamic model developed in this study
Observed AUCs,day15 /AUCs,day1 was 2.
Predicted AUCs, day15/AUCs, day1
Sensitivity Analysis (Contribution of Each Dimension of the Inhibition)
Inactivation Rate by FK1706

In vitro CYP3A4/5 inactivation rate

(kinact, vitro and KI, vitro)
In vivo CYP3A4/5 inactivation rate (kvivo)

kdeg, g

kdeg, h

h1

h1

0.0578
0.0210
0.0578
0.0210

0.0693
0.00495
0.0693
0.00495

Final Model:
TDI Intestine and Liver,
Direct Intestine Inhibition

1.66
2.81
2.10
2.06

TDI
Liver

Direct Intestine
Inhibition

TDI
Intestine

TDI Intestine,
Direct Intestine
Inhibition

TDI Intestine
and Liver

1.08
1.78
1.30
1.31

1.31
1.31
1.30
1.30

1.39
1.49
1.58
1.48

1.54
1.58
1.61
1.57

1.51
2.33
2.06
1.94

Downloaded from dmd.aspetjournals.org at ASPET Journals on August 6, 2015

16

Time (h)

concentrations of an inhibitor for the static model seems to be empirically useful (Obach et al., 2007).
With regard to the application of prediction models during drug
discovery, development, and clinical settings, several options are
available according to development stage. In the drug discovery and
early development stage, the method of Wang and others (Wang et al.,
2004; Obach et al., 2007) seems markedly useful, because it is less
time-consuming and requires less information and because overprediction is allowed, given that the likely purpose is to avoid underprediction in the drug discovery and early development stage. From the
development to postmarketing stage, a more detailed prediction model
such as that built in the present study would be a powerful tool in the
simulation of various situations.
Sensitivity analysis was useful in calculating the contribution of
each dimension of inhibition (TDI in the liver and intestine, direct
inhibition in the intestine) and in assessing the effects of changes in
parameters on the changes in AUC. With regard to intestinal
CYP3A4/5 inhibition, both TDI and direct inhibition were considered
to be involved, although TDI seemed to be stronger than direct
inhibition. We determined that TDI inhibition in the liver must be
involved in this increase in AUC in the clinical part of this study for
a number of reasons. First, inhibition of intestinal CYP3A4/5 alone
could not be used to predict the 2-fold increase in AUC, indicating
some as-yet undefined alternate cause. Second, our simulation showed
that direct (reversible) inhibition of CYP3A4/5 by FK1706 has no
effect on hepatic CYP3A4/5 as described in the method. Furthermore,
simulation assuming TDI accurately predicted the increase in AUC.
For the tested range of Fa, i (0.1, 0.2, 0.5, and 1), 67, 78, 90, and 95%
of intestinal CYP3A4/5 was inhibited, respectively. Because FK1706
is metabolized mainly by CYP3A4/5 (T. Shiraga and A. Kawamura,
unpublished data), these observations may retrospectively confirm
that assuming Fg, i 1 was reasonable. In addition, because of the
lack of data regarding absolute bioavailability of FK1706 in humans,
estimation of Fa, i using Fg, i and Fh, i was impossible. Although Fa, i
and Fg, i were assumed to be 1 as default values in this study (because
the values were unknown), further studies are needed to resolve the
issues of assumption of Fa and Fg. The values of Fa and Fg would
affect the concentrations of the inhibitor in the intestine and at the
inlet to the liver, whereas plasma concentrations (observed values)
were not changed in this simulation. fu, mic, i and fp, i were also considered to be factors affecting the prediction. Therefore, more detailed
experiments should be conducted to measure the actual values of
fu, mic, i and interindividual differences in fp, i.
Several considerations with regard to the present mathematical
models warrant mention. Ito et al. (1998, 2003) and Kato et al. (2008)
developed pharmacokinetic models incorporating the compartment to

257

CYP3A4/5 INACTIVATION BY FK1706 IN HUMANS

Intestine
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0

Eact /E0

Eact /E0

72 144 216 288 360 432 504 576 648 720

Liver
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0

72 144 216 288 360 432 504 576 648 720

Time (h)

Intestine

Eact /E0

1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0

72 144 216 288 360 432 504 576 648 720

Liver
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0

72 144 216 288 360 432 504 576 648 720

Time (h)

Time (h)
TABLE 6

Effects of the change in Fa, i, Fg, i, fu, mic, and fp, i on the prediction of -fold increase in AUC (AUCs, day15 /AUCs, day1) for plasma midazolam concentrations after
repeated oral administration of FK1706 (60 mg) using a dynamic model developed in this study and in vitro CYP3A4/5 inactivation rate (kinact, vitro and KI, vitro and set
of kdeg, g 0.0210 h1 and kdeg, h 0.00495 h1)
The values in the parentheses represent fu, mic, i, 0.05 mg/ml corresponding to the values of fu, mic, i, 0.3 mg/ml (Hallifax and Houston, 2006; Gertz et al., 2008). Observed AUCs, day15 /AUCs, day1
was 2.
Predicted AUCs, day15/AUCs, day1
Parameter as
Variable

Fa, i
0.1
0.2
0.5
1a
Fg, i
0.1
0.2
0.5
1a
fu, mic, i, 0.3 mg/ml
0.1 (0.4)
0.2 (0.6)
0.528a (0.87)
1 (1)
fp, i
0.005
0.01a
0.02
0.05
a

Sensitivity Analysis (Contribution of Each Dimension of the Inhibition)

Final Model: TDI Intestine and Liver,
Direct Intestine Inhibition

TDI
Liver

Direct Intestine
Inhibition

TDI Intestine

TDI Intestine, Direct

Intestine Inhibition

TDI Intestine
and Liver

1.43
1.63
2.08
2.81

1.08
1.14
1.36
1.78

1.06
1.10
1.20
1.31

1.28
1.37
1.45
1.49

1.32
1.43
1.53
1.58

1.39
1.56
1.98
2.33

1.70
1.80
2.15
2.81

1.08
1.14
1.36
1.78

1.31
1.31
1.31
1.31

1.49
1.49
1.49
1.49

1.58
1.58
1.58
1.58

1.61
1.70
2.03
2.33

8.54
5.20
2.81
2.16

5.31
3.26
1.78
1.39

1.42
1.36
1.31
1.28

1.53
1.52
1.49
1.46

1.61
1.60
1.58
1.55

8.15
4.96
2.33
2.02

2.18
2.81
4.13
7.66

1.38
1.78
2.62
4.86

1.31
1.31
1.31
1.31

1.49
1.49
1.49
1.49

1.58
1.58
1.58
1.58

2.07
2.33
3.91
7.25

Default values.

describe the time course of drug concentrations in the liver, where it

was assumed that the unbound inhibitor concentration in the liver was
equal to that in the hepatic vein, i.e., blood exiting the liver. The use
of virtual concentrations of the inhibitor in the liver enabled us to
easily perform more physiologically based modeling. In addition, in

this study, an attempt to first incorporate the hepatic compartment was

made, but a model developed and fitted failed to obtain precise
parameters, probably largely because of the excessive complexity of
the model in its incorporation of nonlinear distribution of FK1706 into
blood. A two-compartment model with first-order absorption was then

Downloaded from dmd.aspetjournals.org at ASPET Journals on August 6, 2015

Eact /E0

Time (h)

FIG. 5. Time profiles of predicted remaining CYP3A4/5 activities (Eact/E0) in the intestine (A and C) and liver (B and D) during
and after the 14-day oral 60 mg q.d. administration of FK1706 to healthy volunteers
using kinact, vitro and KI, vitro (A and B) and
kvivo (C and D) inactivation rates and eqs.
17, 17, 18, and 18. A and C, broken and
solid lines represent values predicted using
kdeg, g 0.0578 and 0.0210 h1, respectively.
B and D, broken and solid lines represent
values predicted using kdeg, h 0.0693 and
0.00495 h1, respectively.

258

MINEMATSU ET AL.
TABLE 7

Prediction of -fold increase in AUC (AUCs, day15/AUCs, day1) for plasma midazolam concentrations after repeated oral administration of FK1706 (60 mg) using a
conventional static method by Wang et al. (2004)
Observed AUCs, day15/AUCs, day1 was 2.
Predicted AUCs, day15/AUCs, day1
Imax

kdeg, g

Cp, sys, i, max (53.3 ng/ml)

fp, i Cp, sys, i, max (0.533 ng/ml)
Cp, inlet, i, max (1850 ng/ml)
Cu, inlet, i, max (18.5 ng/ml)

0.0578
0.0210
0.0578
0.0210
0.0578
0.0210
0.0578
0.0210

kdeg, h

Final Model: TDI in

Intestine and Liver

Sensitivity Analysis (Contribution of

Each Dimension of the Inhibition)
TDI
Liver

TDI Intestine

14.5
190
1.14
2.89
470
6563
5.69
66.6

1.62
1.62
1.62
1.62
1.62
1.62
1.62
1.62

0.0693
0.00495
0.0693
0.00495
0.0693
0.00495
0.0693
0.00495

Acknowledgments. We thank Dennis Morrison, D.O., the investigator for the clinical part of this study, and Abhijit Barve, M.D.,
medical monitor, for their considerable contribution to the clinical part

of this study. We also thank James Keirns, Ph.D., for his advice on
clinical study design, conduct, and analysis. We express our gratitude
to Hayato Kaneko, Masataka Katashima, Ph.D., Akio Kawamura,
Ph.D., Toshifumi Shiraga, Ph.D., Kenji Tabata, Ph.D., Kenji Tasaki,
Shigeyuki Terashita, Ph.D., Hideaki Tokuda, Ph.D., and Katsuhiro
Yamano, Ph.D. This study could not have been initiated without their
unpublished results, a part of which is referred to in the text.
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applied to blood FK1706 concentrations, which provided greater

accuracy than when applied to plasma concentrations, after which
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4.69
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259

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Address correspondence to: Dr. Tsuyoshi Minematsu, Drug Metabolism

Research Laboratories, Astellas Pharma Inc., 2-1-6, Kashima, Yodogawa-ku,
Osaka-shi, Osaka 532-8514, Japan. E-mail: tsuyoshi.minematsu@jp.astellas.com

Downloaded from dmd.aspetjournals.org at ASPET Journals on August 6, 2015