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DRUG METABOLISM AND DISPOSITION
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD 38:249–259, 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 Vitro□S

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 hⴚ1
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 hⴚ1 䡠 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

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

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

the intestine. PA) was added to the dialysis buffer (100 mM sodium-potassium phosphate buffer. incorporating the effects of TDI on both intestinal and liver CYP3A4 (Wang et al. FK1706 shows significant neurotrophic effects. S. 2-fold dilution was considered to be acceptable in this dialysis experiment to see the reversibility qualitatively. (Osaka. 2005.556 ␮M) and the NADPH-generating system. The dialysis buffer was replaced with new buffer containing Amberlite at 5 h and with 100 mM sodium-potassium phosphate buffer (pH 7. All subjects were given at least one dose of midazolam and FK1706. An open-label. Ito et al. an internal standard of FK1706. 0. 5. Kaneko. To determine whether or not dialysis was successful. 2005). dynamic models are nonequilibrium pharmacokinetic models that describe the time course of substrate and inhibitor concentrations as well as P450 inactivation (Ito et al. 2008).. 2007). CA) and the equation kobs ⫽ kinact. or both is incorporated into the model. Ito et al. to help predict drug-drug interactions involving FK1706 and design dose regimens that avoid clinically significant interactions. The volume of dialysis buffer was 250 times that of the sample. The preincubation samples (before and after dialysis) were then 2-fold diluted using buffer solution containing midazolam (1 ␮M) and the NADPH-generating system. concentrations of 1⬘-hydroxymidazolam were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described in the supplemental data.122 mM magnesium chloride hexahydrate). 15. Under these reaction conditions. and 10 ␮M) were preincubated at 37°C for 0.3 mg protein/ml) in 100 mM phosphate buffer (pH 7. FK1706 (Price et al.5 ␮M midazolam concentration).. NJ). We also compared the predictions based on in vitro P450 inactivation parameters with those based on in vivo parameters.4) containing FK1706 (0. 2005. and 0.. 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. and the decrease in the unchanged midazolam was less than 30%. and H. Yamazaki et al. Rohm and Haas. two 30-mg tablets. K.833 ␮M at the microsomal protein concentration of 0. 2006. The accuracy of estimation may be improved by the use of the in vivo (not in vitro) inactivation rate. 0. and 20 min in the presence of an NADPH-generating system (0. once daily during days 2–15) on single-dose midazolam (oral 2 mg. Clinical Study of the Effects of FK1706 on CYP3A4/5 Activity. 3. Minematsu. T. 2.org at ASPET Journals on August 6. vitro and kinact. unpublished data). By dividing the average midazolam 1⬘-hydroxylation activity by that of the control experiments (0 ␮M FK1706. pH 7. dir. Tasaki. 2003). which had been confirmed to be sufficient to terminate the reaction. Bella et al. T. The absolute value of the slope was defined as the inactivation rate constant (kobs) for each FK1706 concentration. Twenty-two of the 24 subjects completed the study. where kobs was zero at an FK1706 concentration of zero. After reaction termination. A study to estimate the in vivo inactivation rate constant for intestinal CYP3A4 metabolism successfully described the TDI by grapefruit juice (Takanaga et al.. the initial rate of metabolite formation was observed at 10 min. In Vitro Inhibition of CYP3A4/5 Activity. were synthesized at Astellas Pharma Inc. which are putatively mediated via FK506-binding protein 52 and the Ras/Raf/ mitogen-activated protein kinase signaling pathway. and 1. . Dynamic models have been developed for TDI of the liver CYP3A4/5 (Ito et al.. It would be also a point at which P450 in the liver. The pharmacokinetic interaction of multiple-dose FK1706 (60 mg.. Reaction and measurement of concentrations of 1⬘-hydroxymidazolam were measured using LC-MS/MS as described above. Therefore. With regard to reversible (direct) inhibitors of P450. 2008. 1.136 mM glucose-6-phosphate dehydrogenase from yeast. the fraction of control activity at each preincubation time point was then calculated for each FK1706 concentration. Pooled human liver microsomes (mix gender pool from liver microsomes of 20 individual donors) were purchased from BD Biosciences (Franklin Lakes. Pooled human liver microsomes (0.. 2007.03 mg/ml protein concentration. and reaction of midazolam 1⬘-hydroxylation was started (final condition: 500-␮l volume.. 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.. MA) in the dialysis buffer for 24 h at 4°C with stirring (Ma et al. 0.. we considered it important to assess the potency of the interaction between FK1706 and CYP3A4/5. vitro) were calculated from the relationship between FK1706 concentration and kobs using WinNonlin (Pharsight. Finally.5 ␮M midazolam concentration). 1998.. Dialysis experiments to confirm irreversible inhibition. 2004.. Usui.442 mM NADP⫹. Philadelphia. 1998. Yamano et al. Design. we characterized in detail the inhibition of CYP3A4/5 by FK1706 through in vitro experiments using human liver microsomes. The concentration of 1⬘hydroxymidazolam in each sample (n ⫽ 3) was converted into midazolam 1⬘-hydroxylation activity (picomoles per minute per milligram of protein). and 1.02) and ascomycin. It was still not known whether the CYP3A4/5 inhibition was time-dependent. nonimmunosuppressive immunophilin ligand (Price et al. Because the TDI by FK1706 had been observed in 2-fold dilution (data not shown). All other chemicals and reagents used were commercially available and of guaranteed purity.3. vitro) for FK1706 was 0.. Thermo Fisher Scientific. Waltham. FK1706 was found to selectively inhibit CYP3A4/5 [the in vitro inhibition constant of reversible (direct) inhibition (Ki.06 mg/ml) in 100 mM phosphate buffer (pH 7. metabolite formation without any inhibitor was 50-fold higher than lower limit of quantification. Asian. KI. 2008). this model cannot describe the time course of the substrate or inhibitor concentrations. Voda et al... the reaction was terminated by the addition of 50 ␮l of internal standard solution (2 ␮M niflumic acid in methanol). one-sequence crossover study was performed to evaluate the pharmacokinetic interaction of FK1706 on midazolam. In vitro KI and kinact determination. Although a useful static prediction model has been developed. Hayashi et al. and then mean activity values of the samples were converted into remaining activity (percentage of vehicle control. In addition. 0 ␮M samples set as controls).... 2003). 0. 1 ml of syrup. During drug development. 2001. on days 1. A 10% Amberlite resin (XAD-4. Obach et al. coefficient of variation values within 12%). or other. one recent report described the concept of estimating the in vivo inhibition constant on hepatic clearance (Kato et al. 10. Pooled human liver microsomes (0.4) to increase the removal of FK1706 from the microsome samples. we developed a dynamic model to explain the in vitro/vivo results in a quantitative manner.05 mg/ml] among P450s tested while not affecting other P450s up to 10 ␮M (H.. 2005) is a novel. 2001. determine the recovery time course of CYP 3A4/5 enzyme activity if multiple doses of FK1706 exhibited in vivo TDI on CYP 3A4/5. Mountain View.4) containing FK1706 (0 and 2 ␮M) were preincubated at 37°C for 30 min in the presence or absence of an NADPH-generating system. In vitro values for KI and kinact (respectively. vitro). Japan). with one male each discontinuing before day 15 because of a protocol violation and withdrawal of consent not related to adverse events.250 MINEMATSU ET AL. Yamano et al. maintenance of microsomal protein concentrations during dialysis was confirmed by a BCA protein assay. suggesting the potential of this compound to be used to treat a variety of neurological disorders (Price et al. Both subjects received only a single dose of midazolam and were therefore not included in the Downloaded from dmd. and the reaction of midazolam 1⬘-hydroxylation was started (final condition: 500-␮l volume. 0. 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. Concentrations of 1⬘-hydroxymidazolam obtained in each sample (n ⫽ 3) were then converted into midazolam 1⬘hydroxylation activity (picomoles per minute per milligram of protein). Yamaji et al. vitro ⫻ [I]/([I] ⫹ KI. and a 100-fold decrease in FK1706 concentration in the microsomes after dialysis was also confirmed by LC-MS/MS. The preincubation samples were then diluted 10-fold using a solution containing midazolam (0. 1.. Terashita. Materials and Methods Materials. Here. 2000).4) at 21 h.aspetjournals. and assess the safety and tolerability of FK1706 alone and in combination with midazolam. After a 10-min incubation at 37°C. FK1706 (molecular weight 820. An aliquot of the samples was dialyzed using a Slide-A-Lyzer dialyzer (10000 MWCO. 2000). Galetin et al. 2006.1.12 mM D-glucose 6-phosphate disodium salt. Kamimura.03 mg/ml protein concentration. 2015 2007). Remaining preincubation samples were stored separately at 4°C as samples before dialysis.

0. 12. tmax. AUCinf ratio 1⬘-hydroxymidazolam/midazolam.i The unbound FK1706 concentration at the inlet to the liver (Cu.0694 ⫾ 0. Subsequently. b c C b. Dosage and blood sampling are summarized in Table 1. 0. 2. or physical examination data during the study. The primary pharmacokinetic parameters for the evaluation of pharmacokinetic interaction were AUC0-inf and Cmax of midazolam between day 15 and day 1.i c ⫽ ⫺Cb. .i 䡠 Cp.i 䡠 共1 ⫺ Ht ⫹ fp. 1.5.sys. the relationship between FK1706 concentrations in blood (Cb. electrocardiograms. sys. because the distribution of FK1706 into blood cells was saturable). 8.25.413. Concentrations of midazolam and 1⬘-hydroxylmidazolam were measured separately using LC-MS/MS as described in the supplemental data.i 䡠 Ht兲 b ⫽ K d. 1.i (1) where Ht is hematocrit (fixed to 0. between day 22 and day 1. 1998): Downloaded from dmd. V1 /Fi V1 /Fi Cb. AUC0 –24 h.i ⫹ Ht 䡠 冉 fp.i 䡠 fp. Tokuda. i. time to reach Cmax (tmax). H. Dynamic Modeling and Simulation for in Vitro and in Vivo CYP3A4/5 Inactivation. i (Cb. 12.fda. 2. i is the fraction unbound of FK1706 in plasma (fixed to 0. 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. i) and those in plasma (Cp. Simulation was also performed using WinNonlin. i and Kd. sys. and the parameters were estimated to be 460 ⫾ 17 and 0. k12. then Ai ⫽ 0 (2) (3) (4) if 24 h ⱕ t ⬍ 336 h (after nth dose of FK1706). and A. half-life (t1/2). Only predose of FK1706 or midazolam administration. If the 90% confidence interval of the geometric mean ratio is contained entirely within 0. i. 6. fp.i 䡠 Cb. 0.sys. unpublished data). 0. Predose. i.pdf). 22. 15.5. 336 h. The timing of midazolam administration on day 1 was defined as time (t) ⫽ 0 (h).i䡠共t⫺24䡠m兲 m⫽1 if t ⱖ 336 h [after 14th (last) FK1706 dose]. and 29): maximum concentration (Cmax). . The two one-sided paired t test procedure was used with SAS (SAS Institute Inc. i.org at ASPET Journals on August 6.i 䡠 Cp.i共0兲 ⫽ 0 if 0 h ⱕ t ⬍ 24 h. and between day 29 and day 1.354 ⫾ 0. 1998. 2 to 6. and 320. 2007): dC b.i ⫹ fp.i⬘/dt ⫽ k12. Cb. was also calculated as supportive evidence. the following pharmacokinetic parameters of FK1706 were computed for blood and plasma concentrations on day 15: Cmax. k12. i is the distribution rate constant.i 䡠 共1 ⫺ Ht ⫹ fp.5. 1..01.sys. multiplied by 326 (molecular weight of midazolam) and divided by 342 (molecular weight of 1⬘-hydroxymidazolam). . 1 gives eq.25.). C2.217 h⫺1. then no significant pharmacokinetic drug-drug interaction is expected. respectively (estimate ⫾ S. Subsequently. k21.i ⫽ 共1 ⫺ Ht兲 䡠 Cp.000 ml for ka. 2– 6) was applied (Gabrielsson and Weiner.i 䡠 Bmax. i. Noncompartmental pharmacokinetic analysis and statistical test.5. Yamano. i is the binding capacity of FK1706 in blood cells. i: C p. i/Fi. i) was described using eq. inlet. sys. For evaluation of pharmacokinetic interaction.i Kd. ka. described using eqs. 5. i⬘ is X2. i) was defined as in eqs. i.56 ⫾ 0. d Predose. 1 (Kawai et al. 3. X2. General analysis conditions.0522 ⫾ 0.sys.i 䡠 Kd. Metabolic ratio. Cary.i 䡠 X2. 8 to 10 (Ito et al.0185 h⫺1. 3.i 䡠 Cb. 7 to express Cp. and distribution volume divided by bioavailability (Vd/F). and V1/Fi. the following plasma pharmacokinetic parameters of midazolam and 1⬘-hydroxymidazolam were obtained for each midazolam administration (days 1. and 1⬘-hydroxymidazolam. 6. i is the FK1706 amount in compartment 2. For each subject.i 䡠 Fi 䡠 Ai ⫹ .i共0兲 ⫽ 0 dX2.i ⫺ k21. i) versus systemic Cp. these parameters were used as fixed values. vital signs.i 䡠 Cb.i 䡠 Ht兲 ⫹ fp.25.i䡠共t⫺24䡠m兲 m⫽1 where Dosei is the dose of FK1706 (60 mg orally at 24.aspetjournals. Minematsu et al. 0.i ⫽ ⫺b ⫹ 冑b2 ⫺ 4 䡠 a 䡠 c 2䡠a (7) a ⫽ f p. i is the absorption rate constant for FK1706. Hereafter. and 24 h after oral administration of FK1706 on day 15.5. Pharmacokinetic modeling for FK1706 (days 2 to 29). 0. statistical analyses were performed according to “Guidance for Industry: in Vivo Drug Metabolism/Drug Interaction Studies.S. K. Subjects fasted for at least 8 h before the morning dosing of test drugs and continued fasting for 4 h after dosing. 8. and the parameters were estimated to be 1. and Ai is the amount of FK1706 at the site of absorption.85 h⫺1. was fitted to the time profile of the mean whole blood concentrations of FK1706.i/dt ⫽ ⫺k1o. Because of its extensive distribution into blood cells. i). Bmax.. sys. Kawamura.E. k10.i ⫺ k12..000 ⫾ 112. and Kd. 2001): .i 䡠 X2.251 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 2–11 FK1706 administration (oral 60 mg once daily) Blood sampling for FK1706 Midazolam administration (oral single 2 mg) Blood sampling for midazolamd X X X 12–14 X Xb 15 16–21 22 23–28 29 a X Xc X X Xb X X Xb X X a FK1706 and midazolam were administered simultaneously on day 15.sys.i ⫹ k21. 2015 pharmacokinetic analysis set. i (Cp.” released by the U. First.i 䡠 Cp. The study protocol was reviewed and approved by an independent ethics committee.). 5. 0. which was the mean value of 22 subjects). 4.i 䡠 V1 /Fi 䡠 Cb.i ⫹ 冊 Bmax.sys. and Vd/F. k21. These parameters are hereafter used as fixed values. respectively (estimate ⫾ S. Equation 1 was fitted to the data set of systemic Cb. i is the binding constant for FK1706.i⬘. FK1706 concentrations were measured in both blood (whole blood) and plasma by using LC-MS/MS as described in the supplemental data. X2. Blood and plasma samples obtained were stored at ⫺20°C and analyzed within the stability period for FK1706.0179 h⫺1.0148 ⫾ 0. 2.8 to 1. clearance divided by bioavailability (CL/F). Noncompartmental analysis was performed using WinNonlin (Gabrielsson and Weiner.E. 2. 1. every 24 h for 14 times).i 䡠 Ht ⫺ fp. 冘 (5) 冘 (6) n then Ai ⫽ Dosei 䡠 e⫺k a. V1/Fi is the distribution volume divided by bioavailability. 48. the following two-compartment model with first-order absorption (eqs. . In addition.. CL/F.0019 ng/ml for Bmax.5. Water consumption was permitted ad libitum until dosing and could be resumed from 2 h after dosing. Katashima. 14 then Ai ⫽ Dosei 䡠 e⫺k a. 4. All subjects were informed of the nature and purpose of the study and written informed consent was obtained. midazolam. 2007). Food and Drug Administration (http://www. i is the elimination rate constant. eq. area under the time-concentration curve from zero to infinity (AUCinf).gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM072119. There were no clinically important changes in laboratory measurements. Model fitting was performed by WinNonlin using the Gauss-Newton algorithms with Levenberg and Hartley modification. k1o.i⬘ ka.sys. i. M.sys. and 24 h after single oral administration of midazolam.

s is the midazolam concentration in the systemic plasma.328 ⫾ 0. mic. respectively (Ito et al.i 䡠E .i 䡠 Bmax.i 䡠 Kd. s.i 䡠 Cp.s (20) where (fm.s 䡠 V1. s is the fraction metabolized by CYP3A4/5 in the liver.i 䡠 Cb. s is the availability of midazolam for absorption.s共672 h兲 ⫽ 0.s E0. 22. KI ⫹ Cu. s and Fg.s共672 h兲 ⫽ Ch.dir ⫽ fu. As ⫽ Doses 䡠 e⫺k a.s 䡠 Fg. s is the blood/plasma concentration ratio.05 mg/ml (0.s/dt ⫽ Qh 䡠 Cb.s ⫺ Eact. 1.i C p.. s. and Qh is the hepatic blood flow rate. 2004): C g. s is the distribution rate constant.sys. i is the FK1706 concentration in plasma at the inlet to the liver. Kato et al.600 ml/h (Kato et al.0. Kp. s were fixed at 6.s ⫺ fm.i /Ki. Cp. s in the presence of FK1706.i ⫽ fp. i is the FK1706 concentration in blood at the inlet to the liver. h and kdeg.s 䡠 Ch.871) 䡠 Ki. i is the availability of FK1706 through first-pass metabolism in the intestine (assumed to be 1).s Cb. Fa.s 䡠 Fa. 2008). As ⫽ Doses 䡠 e⫺k a.s/dt ⫽ k12.h m. Eact. fm.000 ml/h (Davies and Morris. the plasma concentrations of midazolam on days 15. Prediction model for pharmacokinetics of midazolam in the presence of FK1706 (days 15.E. 1. 2006.g 䡠 f 䡠 f 䡠 CLh. s.i 䡠 Ht) ⫹ fp.s 䡠 CLh. s. the concentration of FK1706 in the enterocytes (Cg. h) and in the enterocytes (Eact..s 䡠 Fa.inlet.l 䡠 (1⫺Ht ⫹ fp. 0.inlet.i 䡠E . Fa. and 672 h (day 29) was incorporated in the model as in eqs. 66. CLr.inlet.s dC b. s. 22. k21. 2004): b⬘ ⫽ K d. Gertz et al. mic.h共0兲 ⫽ E0.59.g 1 共1 ⫺ Fg.s/dt ⫽ 共Qh 䡠 Cb.s* ⫽ Eact. Equation 13 can therefore be changed as in eq.s兲 1⫹ 䡠 䡠 E0. mic. 2 to 12 and 14 to 24 and the following parameters as obtained above.aspetjournals.05mg/mL 䡠 Ki.59 h⫺1.s Midazolam administration at 336 h (day 15).s 䡠 CLh. which enables calculation of fu.g共0兲 ⫽ E0.s/Kp. Fg.s兲 䡠 fp. Fa.s 䡠 CLh.675.42 ⫾ 0. 1993).s 䡠 Ch.h where kdeg. Pharmacokinetic modeling for midazolam in the absence of FK1706 (day 1). Then.0. X2. 0. s 䡠 CLh. and V1.s/Kp. Ch.s 䡠 CLh. Direct inhibition of hepatic CYP3A4/5 by FK1706 was excluded from the present model because fu. 96. s is the renal clearance for midazolam.vitro where the unbound fraction of FK1706 in the human liver microsomes (fu.252 MINEMATSU ET AL. CLr. Cp. respectively.s 䡠 As 兲/Vh. g were assumed to be 0.05.int.s.inlet.s/Kp.g/dt ⫽ kdeg.i ⫽ kinact 䡠 Cu. respectively. and 0.s 共0兲 ⫽ 0 X2.s.s ⫹ ka.833 ␮M. s.062 h⫺1. 17 and 18 (Ito et al. g are the initial amounts of active CYP3A4/5 enzymes in the liver and intestine. respectively. s is the midazolam concentration in systemic blood. h and kdeg. the amount of active CYP3A4/5 in the liver (Eact.s 䡠 V1. s)* and Fg. mic.00495 to 0.s/Kp.i 䡠 Ai ⫹ fp. s is the absorption rate constant for midazolam.s (19) 1 Eact. respectively.s ⫺ Qh 䡠 Cb.s共504 h兲 ⫽ 0. s is the liver/plasma concentration ratio for midazolam.s 共0兲 ⫽ 0 共 f m.i 䡠 Fg..0210 to Downloaded from dmd. 19 and 20 were defined (Wang et al.s 䡠 Cb.s p.int.s 䡠 X2.i. and Fg.s ⫺ CLr. KI ⫹ Cg. s.000 ml/h.int.dir. 0. 2003. 2000): As ⫽ Doses 䡠 e⫺k a. k21.s 䡠 Cb. Pharmacokinetics of midazolam was expressed using the eqs.s䡠共t⫺672兲 (24) By using eqs.sys.s 䡠 fp.h 䡠 f 䡠 CLh.s兲* ⫽ ⫹ ka. (14) A s ⫽ Doses 䡠 e⫺ka.s兲 1⫹ 䡠 䡠 E0. 0.i Qg dX 2. s described by eqs.s共672 h兲 ⫽ X2.int. int. i) was defined as in eq.s共336 h兲 ⫽ X2.... fp. Ch. 11 (Rostami-Hodjegan and Tucker.h. Cb.529 at a microsomal protein concentration of 0. dir) represent the TDI and direct inhibition. i (18. s is the distribution volume. int. 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.s (16) where Doses is the dose of midazolam (2 mg).05 mg/ml) using the prediction method of Hallifax and others (Hallifax and Houston. k12.s ⫺ k21. V1.h. then Cb. i using the physicochemical parameter and microsomal protein concentration.170.s 䡠 Ch.s 䡠 冣 1 ⫻ As /Vh (21) Eact. 2008): (12) dC h.s E0. and Fg.s/Kp. i) was calculated to be 0.i.s 䡠 Ch. and midazolam was metabolized only by CYP3A4/5 in the intestine (Wang et al.. respectively (estimate ⫾ S. The kdeg. h. 2004). g) was defined as in eqs.h ⫺ Eact.h (17) (10) where Cb.3 mg/ml) and 0. ka.s ⫺ Qh 䡠 Rb.t (15) C p..s共336 h兲 ⫽ 0. vitro (0. ka. s is the midazolam amount in compartment 2. inlet. s.h. s is the availability of midazolam during first-pass metabolism in the intestine. 0.i ⫹ C p.s 䡠 Cb. s is the midazolam concentration in the liver.h 1 ⫹ Cg. Kp.dir Fg.s ⫺ k12.600 ⫾ 7500 ml for ka. Vh is the volume of the liver.mic. inlet.i ⫽ c⬘ ⫽ ⫺Cb.s ⫹ 共1 ⫺ fm.).s/dt ⫽ 共Qh 䡠 Rb.262 ⫾ 0. int. eqs. 21: 冢 dC h. s* are fm. s.s/Rb. and 29 were simulated: k inact ⫽ kinact.615.i 䡠 Ai Qh (8) ⫺b⬘ ⫹ 冑b⬘2 ⫺ 4 䡠 a 䡠 c⬘ 2䡠a (9) C b.int.g m.S 䡠 Ch. 22 to 24: (13) if 336 h ⱕ t. fm.s ⫹ k21.h. i.i kinact 䡠 Cg. int.s/Kp.g Eact. 2015 ka. s is the intrinsic hepatic clearance.h/dt ⫽ kdeg.inlet. then Cb.i 䡠 Fa. Rb. Rb.s共504 h兲 ⫽ X2. The Cp.871 at a concentration of 0.i/Ki. i.org at ASPET Journals on August 6.h兲 ⫺ .g 䡠 共E0. and Eact/E0 and 1/(1 ⫹ [I]/Ki.s ⫹ 共1 ⫺ fm. 8.g ⫺ Eact.g 1 ⫹ Cg.i act. As is the amount of midazolam at the site of absorption.s/Kp.dir Fg.s ⫺ Qh 䡠 Rb. 0. 683 ng/ml) was much higher than the maximum Cu. 504 h (day 22). and E0. k12. g are the rate constants for CYP3A4/5 degradation in the liver and intestine.inlet.h.g 1 共1⫺Fg.s 䡠 Ch. In the presence of FK1706.s 䡠 X2.i 䡠 Fa.i act.s共336 h兲 ⫽ Ch.s ⫽ Cb. and 69..01 ml/h. CLh. fp. After administration of FK1706. 12 to 16 (Kato et al.int. s.i 䡠 Ht ⫺ fp. s 䡠 CLh.i dE act. It was assumed that the permeability of midazolam across the intestinal lumen was not changed by the inhibitor.vitro K I ⫽ fu. s.mic. h and E0. i.05 mg/ml (fu. h.000 ⫾ 30. 1998.s䡠共t⫺336兲 (22) if 504 h ⱕ t.3 mg/ml (fu. i is the availability of FK1706 through absorption (assumed to be 1).g兲 ⫺ (11) where Qg is the enterocytic blood flow rate.s共504 h兲 ⫽ Ch. Mayhew et al. 2008). and 29) using the in vitro CYP3A4/5 inactivation rate constant.h. mic.5 ng/ml) (Table 7) and therefore did not affect the pharmacokinetics of midazolam at all.i ⫽ Cb.s兲/V1.s兲 䡠 fp.s 䡠 Ch.i 䡠 Cp. 1. 0.023 h⫺1.vitro K i.3mg/mL 䡠 KI. even when the 10-fold concentrative uptake of FK1706 into the liver was assumed.inlet.inlet.s䡠共t⫺504兲 (23) If 672 h ⱕ t. inlet.0693 h⫺1 and 0.g (18) dE act. dir. CLh.h 䡠 共E0. s. s is the unbound fraction of midazolam in the plasma. These parameters were hereafter used as fixed values.h. then Cb.s 共0兲 ⫽ 0 F g. 2008).

4) 51.s 1⫹ 1 0 冣 kinact. i. during Preincubation Before Dialysis NADPH (⫹) After Dialysis NADPH (⫺) NADPH (⫹) NADPH (⫺) 54. day15 and AUCs.9 3.3 ⫾ 2.50 (2050 ng/ml) ⫾ 0. respectively.i kinact 䡠 Cg.g 䡠 共Cg. CYP3A4/5. i. 3).0693 h⫺1 and kdeg. 22.0578 h . midazolam 1⬘-hydroxylase activity was not recovered at all (Table 2).168 (10. i and kvivo 䡠 Cg. g were used as described above. Fg. Cp. For midazolam.e. 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.day 1 0.i were changed to kvivo 䡠 Cu..05 1 冢 fm.s兲 䡠 0. i were not over KI.008 min⫺1 and 2.vitro 䡠 kdeg. 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). kinact 䡠 [I]/(KI ⫹ [I]) ⫽ (kinact/2/KI) 䡠 [I]). and 29). kinact 䡠 [I]/(KI ⫹ [I]) ⫽ (kinact/KI) 䡠 [I]. s values described using eqs. I. Pharmacokinetic parameters for midazolam and 1⬘-hydroxy midazolam are summarized in Table 4. i and Cg. For eqs. respectively (estimate ⫾ S.max 1⫹ kdeg. when [I] ⱕ KI. sys.18 (6.vitro兲 ⫻ 0.253 CYP3A4/5 INACTIVATION BY FK1706 IN HUMANS B 6 0. Obach et al.. A. The geometric mean ratio (90% confidence interval) of AUCinf and Cmax of ⫺1 . inlet. 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. B. The value pair of kdeg.0210 h⫺1 was used to obtain the maximum AUC increase for midazolam concentrations in plasma (AUCs).i. sys. and 29 and the parameter kvivo was estimated. i. Prediction of CYP3A4/5 Inactivation by FK1706 Using Static Model. i on AUCs were also simulated. The two pairs of kdeg. In addition.32 ␮M.) (Fig. 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. (n ⫽ 3).94 ⫾ 0. fu.00 0 5 10 15 20 where AUCs.org at ASPET Journals on August 6. with preincubation in the presence of NADPH.9) pmol/min/mg protein 0 ␮M (control) 2 ␮M 150 ⫾ 10 9.. calculated Cu.mic..and concentrationdependent manners (Fig. i 䡠 Cp. g 0.s兲 0. 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.inlet.h ⫹ 共1 ⫺ fm. 1. In addition. The two pairs of kdeg. mic. simulation for sensitivity analysis and AUC calculation were performed as described above. and 19 to 24 were fitted to the time profile of mean plasma midazolam concentrations on days 15. FK1706 inhibited midazolam 1⬘-hydroxylase activity in time. i. i) ⱕ KI. The high AUC ratio for blood/plasma (Table 3) also showed the high distribution of FK1706 into blood cells.s ⫹ 共1 ⫺ Fg. The kinact.D. inlet.00495 h⫺1 and kdeg. it was considered practical and useful to use kvivo.max ⫹ fu.15 1 冢 Fg. In contrast. concentration-dependent CYP3A4/5 inactivation rate by FK1706. 2 and 3.vitro 䡠 Cg. or Cu.5) Downloaded from dmd.59 (6. i. 1.0578 h⫺1 was used to obtain the minimum AUCs increase.3 µM 4 1 µM 3 10 µM 3 µM 2 kobs (min-1) ln(fraction of control activity) A AUCs. 2C). In addition. These results suggested that the TDI of FK1706 on CYP3A4/5 was irreversible. Pharmacokinetic parameters for FK1706 are listed in Table 3. suggesting that dialysis removed FK1706 from the preincubation samples (Table 2). 17 and 18. i. In vivo inactivation rate estimation for FK1706 on the pharmacokinetics of midazolam (days 15.9 ⫾ 2. simulation was performed in the absence of liver or intestine CYP3A4/5 inactivation as sensitivity analysis. and [I]max is the maximum Cp. kdeg.i 䡠 KI. and when [I] ⫽ KI.48 ⫾ 0.i 䡠 KI.6) 114 ⫾ 9 60. AUCs values were calculated by the integral of Cp. 17⬘ and 18⬘). Dialysis itself was also suggested as acting to reduce the activities of CYP3A4/5 (Table 2). i. 14 to 16. 17⬘. and fp. k inact 䡠 Cu. vitro and KI. A broken line shows the best-fit line.mic. Results 0. FK1706 was eliminated from the body in a biphasic manner (Fig.1 h⫺1) ⫾ 0. 1).1 µM 5 0. i. h 0. When [I] (Cu. 2 to 12.vitro 䡠 关I兴max fu. 22. effects of the changes in Fa.1 ⫾ 2.aspetjournals. i. 0 1 2 3 4 5 6 7 8 9 10 11 Pre-incubation time (min) FK1706 concentration (µM) FIG. g were used as described above.inlet. Thus. Therefore. i.8 (96. 2004. g 0. fp. 18⬘. respectively (designated as eqs.9 ⫾ 5. time-dependent loss of CYP3A4/5 activity (midazolam 1⬘-hydroxylation) after preincubation of human liver microsomes with NADPH and various concentrations of FK1706. inlet.9 50. 2015 In Vitro Inhibition of CYP3A4/5 Activity (Midazolam 1ⴕ-Hydroxylation) by FK1706. i and Cg. h and kdeg. 2008). 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. respectively (Yang et al. inlet. Cg. Effects of dialysis.day 15 ⫽ AUCs. Midazolam 1⬘-Hydroxylase Activity FK1706 Conc.7 (53. When human liver microsomes and FK1706 were preincubated without NADPH. 1/2 䡠 kinact/KI ⱕ kvivo ⱕ kinact/KI (because when [I] ⬍⬍ KI.i KI ⫹ Cg. h 0. vitro). precise estimation of kinact and KI is impossible. midazolam 1⬘-hydroxylase activity recovered completely to the level of control after dialysis. Subsequently. s using dt as a variable of integration. Values in parentheses show the percentage of control. h and kdeg. to reduce the number of parameters (actually.10 冣 1 kinact. Cp. Time profiles of FK1706 concentrations in blood and plasma are shown in Figs. to assess the contribution of each dimension of inhibition. Clinical Study of the Effects of FK1706 on CYP3A4/5 Activity (Pharmacokinetics of Midazolam). vitro values of FK1706 on midazolam 1⬘-hydroxylase activity were 0. inlet. day1 are the AUCs of plasma midazolam concentrations on day 15 and 1. max is the maximum Cg. Estimation of kinact and KI.i.i and KI ⫹ Cu.E. Furthermore.

i.1 53. 2 to 7. Data are expressed as mean ⫾ S.57 to 2. FK1706 was suggested to be an irreversible time-dependent inhibitor of CYP3A4/5.00 3403 ⫾ 667 138 ⫾ 129 18.. vitro 2050 ng/ml). 2003).E.0318 ⫾ 0. 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) B 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) C 300 200 100 0 0 10 20 30 40 50 60 FK1706 concentration in plasma (ng/mL) FIG.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. i on AUCs are summarized in Table 6. Prediction of CYP3A4/5 Inactivation by FK1706 Using Static Model.254 MINEMATSU ET AL. vitro): 0. the hepatic extraction ratio [1 ⫺ bioavailability through first-pass elimination in the liver (Fh)] was equal to the hepatic clearance (CLh)/Qh ⱕ CL/Qh ⱕ CL/F/Qh. 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. Predicted -fold increases in the AUC of plasma midazolam concentrations on day 15 are summarized in Table 5.E. Fh.00923 h⫺1 䡠 ml/ng. Results showed clinical evidence of the suitability of dynamic modeling for TDI of P450.D.M. Dynamic Modeling and Simulation for in Vitro and in Vivo CYP3A4/5 Inactivation. fu.). TABLE 3 Summary of blood and plasma FK1706 pharmacokinetic parameters by the noncompartmental method Data are median for Tmax and mean ⫾ S. h 0. The kvivo values were comparable to the in vitro value. fp. 2. CL/F based on blood FK1706 concentrations (18.2 l/h).and preincubation time-dependent. 2008) and renal blood flow rate (74. kidneys. Day Blood Plasma n Day 15 Day 15 22 22 Cmax Tmax AUC0–24 ng/ml h ng 䡠 h/ml CL/F l/h 265 ⫾ 67. 3).e. mic. In the clinical study..0578 h⫺1) (estimate ⫾ S. provided that elimination occurs via the liver.. Fg. 1993).188. The distribution of FK1706 into blood cells was markedly high and was saturated at high FK1706 concentrations. relationship between FK1706 concentration in blood and plasma on days 12 to 29. prediction of drug-drug interactions involving CYP3A4 inactivation remains difficult (Zhou et al. The kvivo values were estimated to be 0. FK1706 at the tested dose would be classified as a weak or moderate inhibitor of CYP3A4/5 (Bjornsson et al.. The effects of the changes in Fa. This pharmacokinetic model was then used to calculate FK1706 concentrations in the intestine and unbound FK1706 concentration at the inlet to the liver (Fig. Furthermore. i ⱖ 0. On days 22 and 29. 2015 A was almost completely recovered within 7 days after the end of administration of FK1706. on CYP3A4/5 in in vitro and in vivo settings.0069 h⫺1 䡠 ml/ng (at kdeg. max. In this study. The high AUC ratio for blood/plasma (3403:138) (Table 2) showed that the blood/plasma concentration ratio was 24.6 l/h) (Kato et al. Inhibition was NADPH. High and saturable binding of a drug to blood cells can be Downloaded from dmd. it is particularly notable that the AUCs of plasma midazolam concentrations were increased 2-fold after repeated oral administration of FK1706. Because clinical outcomes depend on a number of factors that are associated with drugs and patients. (n ⫽ 22). vitro 10. 5. In in vitro experiments. C. Broken lines were calculated using eqs.2 0.00064 (at kdeg. i. 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.0693 h⫺1 and kdeg. i. Best-fit lines in Figs. pharmacokinetic parameters of midazolam and the metabolic ratio for 1⬘-hydroxymidazolam on days 22 and 29 were recovered to those on day 1. On this basis.4 l/h) (Davies and Morris. FK1706 was therefore shown to be a low clearance drug. i 䡠 Cp. 2005). Data are expressed as mean ⫾ S. Here.M.3 ⫾ 58. was much lower than the hepatic blood flow rate (96.aspetjournals. h 0. whereas those using the in vivo inactivation rate constant were 1.812.1 h⫺1 and KI.7 on average. because CL/ F/Qh is equal to 0. sys. The broken line is the line of best-fit using eq. no increase was predicted in AUC and Cmax (data not shown). g 0. g 0.00 on day 15. yielded the most accurate prediction (Table 7). (n ⫽ 22).77. nonimmunosuppressive immunophilin ligand. and fp.75 1. albeit that all concentrations tended to overpredict the increase in AUC. Use of the unbound systemic maximum concentration of FK1706. kinact. FK1706 showed concentration-dependent inhibition with saturation kinetics (kinact. When the in vitro inactivation rate constant was used. for other parameters. 1. In contrast. . mic. a novel. Predicted -fold increases in the AUC of plasma midazolam concentrations after repeated FK1706 administration are summarized in Table 7. vitro/(fu. i. i 䡠 KI. we investigated the inhibitory effects of FK1706. and CYP3A4/5 activity was not restored after physical removal of FK1706 by dialysis.org at ASPET Journals on August 6.0210 h⫺1) to 0. i. This finding was also supported by a decrease in the metabolic ratio for 1⬘-hydroxymidazolam on day 15 (Table 3). the predicted -fold increases in Cmax were 1.00495 h⫺1 and kdeg. administration of FK1706 to healthy volunteers).E.76 to 1.2 ⫾ 3.d. Predicted active CYP3A4/5 in the intestine and liver during and after repeated FK1706 administration is shown in Fig. or both.00400 ⫾ 0.

13) 1.97 (1.161 0.1.00 44.18) 1..422 ⫾ 0. which can be considered to indicate no drug interaction (data not shown). TABLE 4 Summary of plasma midazolam and 1⬘-hydroxymidazolam pharmacokinetic parameters by the noncompartmental method Data are median for Tmax. b Supportive evidence for drug-drug interaction: AUCinf ratio 1⬘-hydroxymidazolam/midazolam.i (ng/mL) Cb.66 0.1.76 9.104 0.6 3.76 ⫾ 3.91.10. sys. 3.94.sys.5 54. In fact. i 䡠 Cp.04 (0. Prediction using other concentrations resulted in much greater over- Downloaded from dmd. for other parameters..01 0.50 0. systemic plasma (Cp. 1998.13 ⫾ 1. The predicted -fold increase in AUC was 1.10 3. i) (C).1 ⫾ 21.57 ⫾ 1. geometric mean (90% confidence intervals) for geometric mean ratio to day 1 for Cmax and AUCinf.1 0. sys.48 9. yielded the most accurate prediction among [I]max but nevertheless still overpredicted compared with the dynamic model developed in this study.75 ⫾ 2. no hepatic CYP3A4/5 inactivation was predicted (data not shown).i (ng/mL) 1000 100 10 1 0 1 0.M.38 3.46.179 a Primary parameters for drug-drug interaction assessment.01 (0.70 15.. 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 h ng 䡠 h/ml h l/h 22 22 22 22 9.57 10.255 CYP3A4/5 INACTIVATION BY FK1706 IN HUMANS A B 100 10 Cp.441 ⫾ 0. portalvein. the -fold increase in AUC would be less than 1.80) 0. i. max.388 ⫾ 0. i) (D) during and after the 14-day oral 60 mg q.35 11.37 3.1.001 0.95 4. because the value cannot be estimated without the results of a clinical drug-drug interaction study.08) 1. sys. Time profiles of predicted FK1706 concentrations in systemic blood (Cb.D.74 ⫾ 1.39 108 ⫾ 58.d. Minematsu et al. Obach et al.2.96 0. A dynamic model developed for this study.1.2 ⫾ 9.02 ⫾ 2.3 22. The in vivo CYP3A4/5 inactivation rate.0 105 ⫾ 41.99 ⫾ 3. 2001).12 3.01 72 144 216 288 360 432 504 576 648 720 0 10000 100 72 144 216 288 360 432 504 576 648 720 Time (h) D Cu.org at ASPET Journals on August 6. E show observed values (mean ⫾ S. Lines show predicted values.86 4.1 0. the use of maximum unbound systemic concentration of FK1706.88. The prediction of -fold increase in AUC was 2.66 to 2.sys. In the prediction using the static method of Wang and others (Wang et al. Here. i) (B). 17.50 ⫾ 4.1 ⫾ 3. 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. vitro).272 ⫾ 0.. 2007).1 0..70 ⫾ 2.53 5.75 0.7 22 22 22 22 4.50 0. fp. was also estimated by fitting the equations to the time profile of plasma midazolam concentrations using kvivo.62 (1..49 ⫾ 3. multiplied by 326 (molecular weight of midazolam) and divided by 342 (molecular weight of 1⬘-hydroxymidazolam).9 ⫾ 5.28 3. i.6 106 ⫾ 48.50 0.0 ⫾ 11. n ⫽ 22).92.E.93 3.aspetjournals.1.9 ⫾ 11. vitro and KI. and intestine (Cg. and mean ⫾ S.25.50 0.001 0 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. kvivo.18 ⫾ 2.152 0. 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. 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.6 ⫾ 23.e.15) 1. 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.50 22.32 ⫾ 3.02 (0.06 to 2.28 ⫾ 1.50 0. administration of FK1706 to healthy volunteers. i) (A).50 9.inlet.77 ⫾ 1.55 2.78. 2004. However. it may seem as if the use of kvivo would be less useful.46 ⫾ 3.5 ⫾ 4.81 versus an actual increase of 2. as well as unbound FK1706 concentrations at the inlet to the liver (Cu.97 (0. 2015 C Cg.i (ng/mL) Time (h) .i (ng/mL) 1000 10 1 0.80 ⫾ 4.59 8. one of major determinants of pharmacokinetics (Kawai et al.12) Metabolic Ratiob 0.01 100 10 1 0. 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).25 Geometric Mean Ratio to Day 1 for Cmaxa Geometric Mean Ratio to Day 1 for AUCinfa 1.7 23.08 ⫾ 3.

31 1.0 7. 4. A. day15/AUCs.31 1. and use of the maximum unbound systemic plasma TABLE 5 Prediction of -fold increase in AUC (AUCs.5 0.66 2.5 5.. a dashed line was obtained (closely similar results were obtained for either of the pair of kdeg. 78. h (0. KI.61 1. vitro. further studies are needed to resolve the issues of assumption of Fa and Fg. 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.1. respectively. and the pair of kdeg.30 1.33 2.06 1.81 2. Furthermore. Because FK1706 is metabolized mainly by CYP3A4/5 (T.0210 h⫺1) and kdeg. given that the likely purpose is to avoid underprediction in the drug discovery and early development stage.0693 h⫺1). Obach et al. actual unbound drug concentrations at the inlet to the liver would decrease as the drug amount in the absorption site decreases.. i were also considered to be factors affecting the prediction. and 95% of intestinal CYP3A4/5 was inhibited.58 1. prediction. respectively.39 1. our simulation showed that direct (reversible) inhibition of CYP3A4/5 by FK1706 has no effect on hepatic CYP3A4/5 as described in the method. and a broken line expresses values predicted using kinact. h). Midazolam concentration in plasma (ng/mL) A Day 1 17. Using kvivo. Although Fa. i and interindividual differences in fp.0693 0. 2003) and Kato et al. day1 Sensitivity Analysis (Contribution of Each Dimension of the Inhibition) Inactivation Rate by FK1706 In vitro CYP3A4/5 inactivation rate (kinact.0 12.5 15.0 2. vitro) In vivo CYP3A4/5 inactivation rate (kvivo) kdeg. i were assumed to be 1 as default values in this study (because the values were unknown). i using Fg. respectively. A solid line expresses values predicted using kinact.0578 0.E.0 7. because of the lack of data regarding absolute bioavailability of FK1706 in humans. day15 /AUCs. Further accumulation of data is warranted.10 2. i and Fh. fu. vitro and KI. direct inhibition in the intestine) and in assessing the effects of changes in parameters on the changes in AUC. i and Fg. These observations are consistent with the results of Obach et al. 90. and another pair of kdeg.5 10. Shiraga and A.5. Kawamura. vitro.5 0. and clinical settings. vitro.0578 h⫺1) and kdeg. Direct Intestine Inhibition TDI Intestine and Liver 1. h (0.00495 Final Model: TDI Intestine and Liver.78 1.0578 0. First.0 -4 0 4 8 12 20 24 Midazolam concentration in plasma (ng/mL) B 352 356 360 Day 15 17.49 1. 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.2. 2015 16 Time (h) concentrations of an inhibitor for the static model seems to be empirically useful (Obach et al. 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.30 1. Predicted AUCs. g and kdeg. The reason for this is as follows: although it was assumed in the static model that maximum concentration was maintained at steady state. For the tested range of Fa. indicating some as-yet undefined alternate cause. (2007). Direct Intestine Inhibition 1.31 1.00495 0.0210 0. Data are expressed as mean ⫾ S.org at ASPET Journals on August 6. i. KI. Therefore. i (0.5 5. although TDI seemed to be stronger than direct inhibition. 2004.94 Downloaded from dmd. i was impossible. mic. 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.aspetjournals.0 12.5 10.48 1. broken line represents the best-fit line using eqs. these observations may retrospectively confirm that assuming Fg. Ito et al. inhibition of intestinal CYP3A4/5 alone could not be used to predict the 2-fold increase in AUC. vitro or kvivo).M. From the development to postmarketing stage. several options are available according to development stage.day1 was 2. both TDI and direct inhibition were considered to be involved. vitro and kinact. (n ⫽ 22). (1998. In the drug discovery and early development stage. With regard to the application of prediction models during drug discovery.0 2. (2008) developed pharmacokinetic models incorporating the compartment to . 13 to 16. vitro.0 332 336 340 344 348 Time (h) FIG. B. 67. h h⫺1 h⫺1 0. i and fp. Several considerations with regard to the present mathematical models warrant mention. i ⫽ 1 was reasonable. The values of Fa and Fg would affect the concentrations of the inhibitor in the intestine and at the inlet to the liver.54 1. 2007) seems markedly useful. unpublished data). g (0.57 1.06 TDI Liver Direct Intestine Inhibition TDI Intestine TDI Intestine..00495 h⫺1). a more detailed prediction model such as that built in the present study would be a powerful tool in the simulation of various situations. estimation of Fa. simulation assuming TDI accurately predicted the increase in AUC. more detailed experiments should be conducted to measure the actual values of fu. g (0.0210 0. and 1).5 15. Second. 2007). whereas plasma concentrations (observed values) were not changed in this simulation. 0. the method of Wang and others (Wang et al. With regard to intestinal CYP3A4/5 inhibition. g kdeg.51 2. 0.08 1. lines express the values predicted using inactivation rate (KI. Sensitivity analysis was useful in calculating the contribution of each dimension of inhibition (TDI in the liver and intestine.30 1.256 MINEMATSU ET AL. In addition. development. day1) for plasma midazolam concentrations after repeated oral administration of FK1706 (60 mg) using a dynamic model developed in this study Observed AUCs.0693 0. because it is less time-consuming and requires less information and because overprediction is allowed. mic.day15 /AUCs.58 1.

00495 h⫺1) The values in the parentheses represent fu. vitro and KI. mic. in this study.7 0.86 1.49 1.58 1.06 1. day15 /AUCs.46 1.0210 h⫺1. . i.13 7.16 5.0693 and 0. 5.6 0. i.0 0.257 CYP3A4/5 INACTIVATION BY FK1706 IN HUMANS B Intestine 1.78 1. Fg.9 0. h 0. 2008).31 1. 17.58 1.91 7.8 0.49 1.3 mg/ml 0.78 1.1 (0.6 0. respectively.6 0.26 1.81 1.36 1.78 2.53 1. i. 0.4 0. day1 Parameter as Variable Fa.49 1.3 0.03 2.14 1. g 0. 2015 Eact /E0 C Time (h) FIG.2 (0.2 0.38 1.7 0.49 1.15 2.14 1.31 1. 0.49 1.49 1. day15/AUCs.31 1. describe the time course of drug concentrations in the liver.4 0. The use of virtual concentrations of the inhibitor in the liver enabled us to easily perform more physiologically based modeling. Direct Intestine Inhibition TDI Liver Direct Intestine Inhibition TDI Intestine TDI Intestine.0 0.1 0.58 1. an attempt to first incorporate the hepatic compartment was made.2 0.08 1.4) 0. A two-compartment model with first-order absorption was then Downloaded from dmd.8 0. 17⬘.70 1. i 0.31 1.58 1.org at ASPET Journals on August 6.d. mic.7 0. vitro and KI.80 2.58 1.96 2.0 0.60 1.32 1.1 0.2 0.49 1.e. administration of FK1706 to healthy volunteers using kinact.0 0 72 144 216 288 360 432 504 576 648 720 Liver 1.25 Default values.58 2. i 0.18 2.55 8.7 0. day1 was 2.0578 and 0.3 mg/ml (Hallifax and Houston.3 0.01a 0.70 2.1 1. vitro (A and B) and kvivo (C and D) inactivation rates and eqs.58 1.aspetjournals.05 a Sensitivity Analysis (Contribution of Each Dimension of the Inhibition) Final Model: TDI Intestine and Liver. Predicted AUCs. broken and solid lines represent values predicted using kdeg.8 0. i. A and C. mic.1 0. B and D. In addition. i.9 0. but a model developed and fitted failed to obtain precise parameters.58 1.0 0 72 144 216 288 360 432 504 576 648 720 Time (h) Time (h) TABLE 6 Effects of the change in Fa.8 0.43 1.1 1.20 2.61 1. i 0..31 3.54 5.36 1.5 0.49 1.5 0.0210 h⫺1 and kdeg.4 0.08 2.98 2.2 0.00495 h⫺1.56 1. 0. 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.3 0.33 3. mic.31 1.31 1.33 8. fu. 2006.87) 1 (1) fp.05 mg/ml corresponding to the values of fu.1 1. broken and solid lines represent values predicted using kdeg. Gertz et al.28 1. vitro and set of kdeg.5 0. i.0 0.20 1.53 1.81 1.33 1. 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.58 1.005 0.9 0.31 1.45 1. day15 /AUCs.81 2. blood exiting the liver.08 1. Direct Intestine Inhibition TDI Intestine and Liver 1.1 0.78 1.2 0. h 0. where it was assumed that the unbound inhibitor concentration in the liver was equal to that in the hepatic vein.49 1.43 1.9 0. respectively. and fp.61 1.0 0 72 144 216 288 360 432 504 576 648 720 Time (h) D Intestine Eact /E0 1.62 4. i on the prediction of -fold increase in AUC (AUCs.4 0.36 1.31 1.6 0.1 0.02 2.5 1a Fg.52 1. 18.49 1.1 1.2 0..39 1.3 0.5 1a fu.63 2. probably largely because of the excessive complexity of the model in its incorporation of nonlinear distribution of FK1706 into blood.37 1.31 1.15 4.31 1.10 1.81 4.28 1.42 1.66 1. Observed AUCs.02 0.5 0.1 0.39 1.528a (0.0 Eact /E0 Eact /E0 A 0 72 144 216 288 360 432 504 576 648 720 Liver 1.6) 0.33 2.58 1.07 2. g 0. and 18⬘.

inlet. day15/AUCs. and Rowland M (1998) Physiologically based pharmacokinetics of cyclosporine A: extension to tissue distribution kinetics in rats and scale-up to human. inhibitor. TABLE 7 Prediction of -fold increase in AUC (AUCs. Ph.62 ⫺1 0.62 1. Yamada Y. Ph. Drug Metab Dispos 34:724 –726. FK1706 concentrations at the inlet to the liver could be considered to be nearly equal to those in the hepatic venous blood at steady state. The estimated time-dependent inactivation rate of CYP3A4/5 by FK1706 was comparable between the results obtained quantitatively using a dynamic model in vitro and in vivo. a part of which is referred to in the text. Houston JB.533 ng/ml) Cp. Ling KH.00495 0. max (18. max (0. D. Ph. Kanamitsu S. and Galetin A (2008) Drug lipophilicity and microsomal protein concentration as determinants in the prediction of the fraction unbound in microsomal incubations. Nomeir AA..89 470 6563 5. medical monitor. and Katsuhiro Yamano. i. Bjornsson TD. 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Drug Metab Dispos 34:166 –175. and Sugiyama Y (2008) The quantitative prediction of CYP-mediated drug interaction by physiologically based pharmacokinetic modeling. and Itoh T (2003) Prediction of the in vivo interaction between midazolam and macrolides based on in vitro studies using human liver microsomes. and analysis. Iwatsubo T. Ph. (2003) The conduct of in vitro and in vivo drug-drug interaction studies: a Pharmaceutical Research and Manufacturers of America (PhRMA) perspective.62 1. Davies B and Morris T (1993) Physiological parameters in laboratory animals and humans.. Price R. Ph. Eur J Pharm Sci 36:175–191. and Hall SD (2000) An in vitro model for predicting in vivo inhibition of cytochrome P450 3A4 by metabolic intermediate complex formation.0578 0. if any. Masataka Katashima. Stockholm. Pharmacol Rev 50:387– 412. Skordos KW. We thank Dennis Morrison. Kenji Tabata. hematocrit as an indicator of blood/plasma concentration ratio of FK1706. Yoshisue K. and Iga T (2001) Quantitative relationship between myocardial concentration of tacrolimus and QT prolongation in guinea Downloaded from dmd. Grime KH. Grimm SW. Gabrielsson J and Weiner D (2007) Pharmacokinetics And Pharmacodynamic Data Analysis: Concepts and Applications. Ph.0578 0. Einolf HJ.5 ng/ml) 0. Fischer V.D. J Pharmacol Exp Ther 287:457– 468. for Cg. h h Final Model: TDI in Intestine and Liver Sensitivity Analysis (Contribution of Each Dimension of the Inhibition) TDI Liver TDI Intestine 14. He K.0693 0. i. Swedish Pharmaceutical Press. Carrion R.. Because FK1706 has low clearance. the investigator for the clinical part of this study. conduct. We also thank James Keirns. Drug Metab Dispos 28:1031–1037. Suzuki H.69 760 10.00495 Acknowledgments.0578 0.e. Ogihara K. i. Price R.D.0210 0. Pharm Res 25:1891–1901. Carrion RE. max (53. day15/AUCs. Lim HK. Kao J. (2009) The conduct of in vitro studies to address time-dependent inhibition of drug-metabolizing enzymes: a perspective of the Pharmaceutical Research and Manufacturers of America.69 66. sys.6 1.D. 2005). parallel elimination pathways. a simple equation by Rostami-Hodjegan and Tucker (2004) was applied. Drug Metab Dispos 31:815– 832. 23.D. i. Further studies to confirm the validity of the mathematical models and to evaluate clinical drug-drug interactions in different conditions (dosing schedule.0210 0. substrate.62 1.5 190 1. i.00495 0. Prueksaritanont T. In general. and Riley RJ (2009) Mechanism-based inhibition of cytochrome P450 enzymes: an evaluation of early decision making in vitro approaches and drug-drug interaction prediction methods. This study could not have been initiated without their unpublished results.62 1. i 䡠 Cp. Shitara Y. which provided greater accuracy than when applied to plasma concentrations. et al. Hallifax D and Houston JB (2006) Binding of drugs to hepatic microsomes: comment and assessment of current prediction methodology with recommendation for improvement. sys. after which unbound FK1706 concentrations at the inlet to the liver were calculated as surrogates for that in the liver. and Houston JB (2006) Prediction of time-dependent CYP3A4 drug-drug interactions: impact of enzyme degradation. incorporating the factors that affect the pharmacokinetics in individual patients.0210 kdeg. Predicted AUCs. if any] in the intestine and liver by adopting more physiological equations and considering the contribution of enterohepatic recirculation and drug transporters. Hall SD. References Bella AJ. Ueda K. We express our gratitude to Hayato Kaneko. Jones DR. Nunes L. FK1706 weakly or moderately inhibited the activity of CYP3A4/5 in vitro and vivo at the tested dose. Drug Metab Dispos 37:1355–1370. and Abhijit Barve. Seibert E. direct inhibition of intestinal and hepatic CYP3A4/5 should be incorporated into the model. 2007). Gertz M. et al. Ni L. Akio Kawamura.. in the hepatic venous blood.14 2.258 MINEMATSU ET AL. Mayhew BS. expression of inter. Hayashi N. Sato H. Hayashi N. Lu C.84 4. Gan L. M. day1 was 2.D. The simplest pharmacokinetic model developed for FK1706 (eqs.0693 0.62 1. Ph.0210 0. Kawai R. Tanaka C. In addition. and Lue TF (2006) The effect of FK1706 on erectile function following bilateral cavernous nerve crush injury in a rat model. Einolf HJ.700 9. such as CYP3A5 genotype (Pinto et al. Callaghan JT. As an ideal condition. This notion was supported by the results of a preliminary simulation analysis (data not shown). g ⫺1 h Cp. 2015 applied to blood FK1706 concentrations.D. particularly when potent direct inhibitors are examined. may be useful. Drug Metab Dispos 31:945–954. day1 关I兴max kdeg. and intestinal inhibition. albeit that prediction may be improved by a more complicated physiological model. Ph. Minematsu T. Kanamitsu S. In conclusion. day1) for plasma midazolam concentrations after repeated oral administration of FK1706 (60 mg) using a conventional static method by Wang et al. Ma B. Galetin A. Pharm Res 10:1093–1095. max (1850 ng/ml) Cu.0693 0. (2004) Observed AUCs. Kenji Tasaki.O. Hirano M. Kato M.62 1.20 108 . Mathew D. Minor TX. Kilford PJ. Grimm S. Ohtani H...00495 0.D. Ferguson D.3 ng/ml) fp. Ito K.0693 0. Bird J. although a method for expressing drug concentrations in the intestine mathematically remains an open question.. Future studies of mathematical models that describe the exact unbound concentration of parent drug [and metabolite(s). Miwa G. Shigeyuki Terashita.D. Ikeda T. J Sex Med 4:341–346. 2–11) in this study was therefore considered sufficient for the practical prediction of TDI of CYP3A4/5 using the mean data of the subjects..D. and Lue TF (2007) FK1706 enhances the recovery of erectile function following bilateral cavernous nerve crush injury in the rat. i. Drug Metab Dispos 28:125–130.0578 0.62 1. King SP. Gibbons L. as described above.. day15/AUCs. inlet.aspetjournals. This pharmacokinetic model should be useful in helping to predict drug-drug interactions and in the design of dose regimens that avoid drug interactions. for his advice on clinical study design..

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