International Journal of Pharmaceutics 389 (2010) 114–121
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International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Mechanistic understanding of the different effects of Wuzhi Tablet (Schisandra
sphenanthera extract) on the absorption and first-pass intestinal and hepatic
metabolism of Tacrolimus (FK506)
Xiao Ling Qina,1, Hui Chang Bia,d,1, Xue Ding Wanga, Jia Li Lia, Ying Wanga, Xin Ping Xuea, Xiao Chenb,∗,
Chang Xi Wangc, Le Jia Xua, Yi Tao Wangd, Min Huanga,∗
aInstitute of Clinical Pharmacology, School of Pharmaceutical Sciences, Sun Yat-sen University, 132 East Circle at University City, Guangzhou 510006, China
bDepartment of Pharmacy, The First Affiliated Hospital, Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou 510080, China
cOrgan Transplant Center, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China
dInstitute of Chinese Medical Sciences, Macau University, Macau, China
a r t i c l ei n f o
Received 14 October 2009
Received in revised form
18 December 2009
Accepted 15 January 2010
Available online 25 January 2010
a b s t r a c t
We recently reported that the blood concentrations of Tacrolimus (FK506) in rats were markedly
increased following the intake of a Chinese herbal preparation, Wuzhi Tablet (WZ, Schisandra sphenan-
thera extract). In order to identify the underlying mechanisms of the increase in FK506 level, we
investigated the effects of WZ on the absorption and first-pass intestinal and hepatic metabolism of
FK506 in vitro and in vivo. When co-administered with WZ, the AUC0–∞value after oral FK506 dosing
was increased by 2.1 fold, the oral bioavailability (Foral) was increased from 5.4% to 13.2% (p=0.0002),
and the (Fabs×FG) was 111.4% (p<0.01), much greater than that when FK506 was given alone. However,
the FHwas only 21.2% greater than that when FK506 was given alone, which indicates that the reduction
of intestinal first-pass effect was the major cause of the increased FK506 oral bioavailability when co-
administered with WZ. In the Caco-2 cell transport study, the transport ratio of FK506 with WZ extract
was significantly lower than that of FK506 alone, which suggested WZ extract inhibited P-gp-mediated
and human liver microsomes, indicating WZ extract potently inhibited the CYP3A-mediated metabolism
of FK506. In conclusion, WZ inhibited P-gp-mediated efflux and CYP3A-mediated metabolism of FK506,
and the reduction of intestinal first-pass effect by WZ was the major cause of the increased FK506 oral
© 2010 Elsevier B.V. All rights reserved.
Tacrolimus (FK506) is a well-known potent immunosuppres-
sant agent for the prevention and/or treatment of graft rejection
in solid organ transplantation patients (Mentzer et al., 1998;
Tacrolimus; FK520, Ascomycin; IS, internal standard; P-gp, P-glycoprotein; CYP3A,
cytochrome p450 3A; Tmax, time to peak blood concentration; Cmax, peak blood
concentration; AUC, area under the blood concentration–time curve; CLiv, total
intravenous blood clearance; Vd, the volume of distribution; Foral, oral bioavailabil-
ity; Fabs×FG, gut processes affecting availability; ERH, hepatic extraction ratio; FH,
hepatic availability; LC–MS/MS, liquid chromatography–tandem mass spectrome-
∗Corresponding authors. Tel.: +86 20 39943011; fax: +86 20 39943000.
E-mail addresses: firstname.lastname@example.org (X. Chen),
email@example.com (M. Huang).
1Xiao Ling Qin and Hui Chang Bi have equal contribution to this work. Both
persons are the first authors.
Staatz and Tett, 2004; Bowman and Brennan, 2008). With its nar-
row therapeutic index, therapeutic drug monitoring is standard
clinical practice to control carefully the blood level of the drug
in the management of transplant recipients (Jusko et al., 1995;
Venkataramanan et al., 1995). The metabolism of FK506 occurs
in the liver and the small intestine via the cytochrome P450 3A
(CYP3A), and its absorption is further limited due to the involve-
ment of an efflux transporter P-glycoprotein (P-gp) (Jeong and
Chiou, 2006; Iwasaki, 2007). Therefore, drug or compounds that
inhibit or induce the CYP3A/P-gp may increase or decrease the
blood level of FK506, respectively (van Gelder, 2002). Clinically and
pre-clinically relevant interactions have been reported between
FK506 and herbs such as St. John’s wort, Pomelo, and grapefruit
Peynaud et al., 2007).
Wuzhi Tablet (WZ) is a preparation of ethanolic herb extract
of Wuweizi (Schisandra sphenanthera), which contains 7.5mg
Schisantherin A per tablet. Its major active chemical constituents
0378-5173/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
X.L. Qin et al. / International Journal of Pharmaceutics 389 (2010) 114–121
include Schisandrin A, Schisandrin B, Schisandrin C, Schisandrol
A, Schisandrol B, Schisantherin A, Schisantherin B, etc. (Huyke et
al., 2007). WZ is a prescribed drug (Registration number in China:
to protect liver function in chronic hepatitis and liver dysfunction
patients (Loo et al., 2007). It is popularly prescribed for Chinese
renal or liver transplant patients with FK506-induced hepatitis.
Recently, it has been reported in healthy Chinese human vol-
unteers that concomitant administration of WZ Capsule (another
preparation of S. sphenanthera extract (SchE), containing 11.25mg
concentration of FK506, which might be due to the inhibition of
CYP3A and/or P-gp via substances in SchE (Xin et al., 2007). We
observed similar phenomenon in renal transplant patients with
istration of WZ (data not shown). Our recent study in rats indicated
that a concomitant dose of WZ could significantly increase the
FK506 whole blood concentration with only a slight change in
FK506 tissue distribution, suggesting WZ could be a promising
ever, to our knowledge, there is no published study that could
clearly clarify the drug interaction between FK506 and WZ and the
Therefore the current study aims: (1) to study the pharmacoki-
netics of FK506 in rats when concomitant administration of WZ;
(2) to differentiate the effect of WZ on the intestinal FK506 first-
pass effect from that of the liver metabolism; and (3) to examine
the effect of WZ on the CYP3A-mediated FK506 metabolism and
P-gp-mediated exsorption of FK506.
2. Materials and methods
FK506 with a purity of 98% as determined by HPLC with ultra-
violet (UV) detection was synthesized and provided by Toronto
Research Chemicals Inc. (Toronto, Canada). Ascomycin (FK520,
as internal standard, IS) with a purity of 95% as determined by
HPLC with UV detection was synthesized and provided by BIOMOL
Research Laboratories Inc. (Plymouth Meeting, PA). Prograf®cap-
were produced by Astellas Ireland Co., Ltd (Ireland). WZ (each
tablet containing 7.5mg Schisantherin A) was produced by Fang
Lue Pharmaceutical Company (Guangxi, China). Verapamil, NADPH
Na4 (≥98%) and HEPES were purchased from Sigma ALDRICH
Inc. (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium
(DMEM), fetal bovine serum, Hank’s balanced salt solution (HBSS,
pH 6.8), 0.05% trypsin–ethylenediaminetetra-acetic acid (EDTA)
were obtained from Gibco BRL Life Technology (Grand Island, NY),
penicillin and streptomycin were purchased from Hua Nan Phar-
acids were obtained from Invitrogen (Carlsbad, CA, USA). Methanol
jing, China). All other reagents were of analytical grade or HPLC
grade when appropriate. Ultra-pure water was obtained from a
Milli Q-plus system (Billerica, MA).
The extract used in in vitro studies was extracted from WZ.
Briefly, 30 times (V:W) of ethanol (mL) was added into WZ pow-
der (g) and vortexing-mixed for 1min. The mixtures were then
ultrasonic-extracted for 60min and then centrifuged at 2500×g
for 10min. The residue was dissolved in 30 times of ethanol and
bined and transferred to a clean tube and evaporated to dryness
under vacuum. The residue was dissolved in ethanol as the stock
20mM Schisantherin A), and stored at −20◦C until use.
Male Sprague-Dawley rats (250–330g) were supplied by the
Laboratory Animal Service Center at Sun Yat-sen University
(Guangzhou, China). The animals were kept in a room at 22–24◦C
with a light/dark cycle of 12/12h and 55–60% relative humidity.
They had free access to standard rodent chow and clean tap water.
were in accordance with the Regulations of Experimental Animal
Administration issued by the Ministry of Science and Technology
of the People’s Republic of China (http://www.most.gov.cn).
2.3. Cell culture
Caco-2 cells were obtained from the American Type Culture
Collection (Manassas, VA, USA). Cells were cultured and used as
previously described in our paper (Zhang et al., 2006).
2.4. Preparation of rat liver microsomes
Hepatic tissues were collected from healthy male Sprague-
Dawley rats (230–310g) and stored at −80◦C. Liver microsomes
described in our report (Bi et al., 2008). Human liver microsomes
were purchased from BD Biosciences (NJ, USA). All liver micro-
somes were stored at −80◦C until use. The amount of protein
of liver microsomes was measured using BCA Protein Assay Kit
(http://www.beyotime.com/Compatibility Chart For BCA Kit.pdf).
2.5. Pharmacokinetic experiments in rats
On the day before the experiment, a light surgery for the rat
was performed as described in our previously published paper (Bi
et al., 2008). Briefly, a polyethylene catheter (0.4mm i.d., 0.8mm
o.d., Portex Ltd, Hythe, UK) was inserted into the right jugular vein
under light anaesthesia. Afterwards, the rats were placed individ-
ually in cage, allowed to recover and fasted for 12h before the
rats by gavage at a dose of 1.89mg/kg. WZ (0.25g/kg) or Verapamil
(30mg/kg) dissolved in pure water was also given by gavage 1min
(for WZ) or 30min (for Verapamil) before the administration of
FK506. Rats receiving FK506 alone were given with an equivalent
via the right cannulated jugular vein before (0h) and at 5,15, 30,
45min, 1, 1.5,2, 3, 4, 6, 8, 12, and 24h post-dosing.
For intravenous (i.v.) dosing, pilot studies indicated that dose-
normalized FK506 AUC values were unchanged for 0.2, 0.756, and
1.89mg/kg intravenous doses. The 0.2mg/kg intravenous dose was
chosen for detailed study. FK506 injection diluted in saline was
injected via the right jugular vein at a dose of 0.2mg/kg (2mL/kg),
followed by a 0.5mL heparinized saline (50U/mL) flush. WZ dis-
solved in pure water was also given by gavage at a dose of 0.25g/kg
1min before the administration of FK506. Rats receiving FK506
alone were given with an equivalent volume of vehicle. About
before (0h) and at 2, 5, 10, 15, 30, 45, 60min, 1.5, 2, 3, 4, 6, 8, 12,
and 24h post-dosing.
After blood sampling, the cannula was flushed with an equal
volume of heparinized saline solution (50U/mL) to prevent coag-
ulation and to replace the blood loss. 200?L of the blood samples
were immediately collected and stored at −20◦C until analysis.
X.L. Qin et al. / International Journal of Pharmaceutics 389 (2010) 114–121
2.6. Transport of FK506 in Caco-2 cells
The transport of FK506 across Caco-2 monolayers was investi-
gated by the methods described previously (Zhang et al., 2006; Bi
et al., 2008) with some modifications. In brief, Caco-2 cells were
seeded at 1.0×105cells/cm2on polycarbonate membrane Tran-
swell inserts (Corning Co., Corning, NY, USA) and cultured for 21
days. At the beginning of the transport study, they were washed
three times with HBSS, after the third wash, the plates were incu-
bated at 37◦C for 30min, then the TEER values were measured and
the cells with TEER values exceeded 250?cm2were used in the
Pre-incubation was carried out for 30min in the presence or
absence of WZ extract (1mg/mL containing 50?M Schisantherin A
with a final ethanol concentration of 0.25%) or Verapamil (100?M)
in HBSS buffer at both the apical (AP) and the basolateral (BL) sides
of the monolayers. The buffer was then replaced with fresh HBSS
buffer on one side of the cell layer and FK506 solution (1?g/mL)
in HBSS buffer on the other side and incubation was performed at
37◦C. The bidirectional transport studies of FK506 were conducted
by loading FK506 solution (1?g/mL) to either the apical or baso-
lateral side of the Caco-2 monolayers. 50?L samples were taken
from the receiver side at 30, 60 and 90min, and an equal volume
of HBSS buffer was replenished. All samples were stored at −20◦C
replaced with fresh HBSS buffer, incubated for 30min at 37◦C, and
TEER values were measured. The TEER values were no significant
different before and after the transport experiment.
2.7. Metabolism of FK506 by rat and human liver microsomes
The metabolism of FK506 was assayed by measuring the reduc-
tion of FK506. Briefly, the incubation system, with a total volume
of 200?L, contained 100mM potassium phosphate buffer (pH 7.4),
rat/human liver microsomes (final concentration 0.02mg/mL), and
FK506 (final concentration 5ng/mL) with or without WZ extracts
(containing 1, 10, 100?M Schisantherin A). The reaction was
started by addition of 20?L of 10mM NADPH to the system. The
mixture was incubated for 5min at 37◦C; then, 800?L of ice-
cold diethyl ether was added to the reaction mixture to stop the
reaction and 10?L of FK520 was added as an internal standard.
FK506 was determined by LC–MS/MS method described below.
The metabolism ratio was calculated by compared the initial drug
concentration with the concentration after incubation.
2.8. Quantification of FK506 by LC–MS/MS method
FK506 in all samples was determined using our previously
developed LC–MS/MS methods (Li et al., 2008; Qin et al., 2010).
The blood samples were prepared using a single-step liquid–liquid
extraction procedure described in our previously published paper
(Qin et al., 2010). As for samples from transport study and micro-
somes metabolism experiment, 400 or 800?L of extraction solvent
ethyl acetate was added directly. After vortex-mixing for 1min
and standing at room temperature for 10min, the mixtures were
centrifuged at 2500×g for 5min. After centrifugation, the organic
phase was then transferred to a clean centrifuge tube and evapo-
rated to dryness. The residues were dissolved in mobile phase and
an aliquot (10?L) of the re-constituent solution was injected onto
the LC–MS/MS for analysis.
The LC–MS/MS method was partially validated since the bio-
logical matrix was changed compared to our previously published
methods (Li et al., 2008; Qin et al., 2010). The method had a chro-
matographic running time of 2min and linear calibration curves
over the concentrations of 0.5–300, 0.5–500 and 0.5–20ng/mL
for FK506 in blood, HBSS buffer and microsomes buffer, respec-
tively. The extraction recoveries were 56.1–62.4%, 55.1–60.4%
and 67.6–74.3% for FK506, and 53.8%, 58.7% and 68.3%for FK520
in blood, HBSS buffer and microsomes buffer, respectively. The
lower limit of quantification (LLOQ) of the analytical method was
0.5ng/mL for FK506. The intra- and inter-batch precision and accu-
racy were less than 15% for all quality control samples in blood,
HBSS buffer and microsomes buffer, respectively.
2.9. Data analysis
Bioavailability Program Package (Version 2.1, Institute of Clinical
Pharmacology, School of Pharmaceutical Sciences, Sun Yat-set Uni-
versity, Guangzhou, China). Time of the peak blood concentration
the observed concentration versus time profiles. The initial drug
concentration (the extrapolated concentration at zero time) of the
drug following i.v. injection was calculated by back extrapolation
of the blood concentration–time curve to the y-axis. The area
under the curve to the last measurable concentration (AUC0–t) was
calculated by the linear trapezoidal rule. The area under the curve
to infinity (AUC0–∞) was calculated as: AUC0–∞=AUC0–t+Ct/ˇ,
where Ctis the last measurable concentration. The blood clearance
(CL) was estimated by dividing the total administered dose by
the AUC0–∞. The volume of distribution (Vd) was calculated by
dividing CL by ˇ. The mean residence time (MRT) was calculated by
AUMC0–t/AUC0–t.The absolute bioavailability of oral doses (Foral)
was calculated using the formula:
Since FK506 is known to be insignificantly excreted in urine as
unchanged form after intravenous administration (Takada et al.,
1991), the hepatic extraction ratio (ERH) can be estimated as
55.2mL/min/kg (or 3.312L/h/kg) (Davies and Morris, 1993).
The oral bioavailability is a function of FH[hepatic availability
(FH=1−ERH)], FG[gut availability (FG=1−ERG)], and Fabs[fraction
absorbed], as given in Eq. (3) (Wu et al., 1995)
rat hepaticbloodflow(QH)wasassumed tobe
Foral= Fabs× FG× FH
It is possible to estimate the product of Fabs×FGas
Comparison of Fabs×FGcalculated values gives an estimate of
the effects of WZ on the gut processes affecting FK506 availability.
Total drug blood intrinsic clearance (fu×CLint) was calculated
based on the well stirred hepatic clearance model (Wilkinson,
The apparent permeability coefficient (Papp) in cellular mono-
layers is expressed in centimeters per second and calculated as in
where ?Q/?t is the permeability rate (micrograms per second),
A is the surface area of the membrane (square centimeters), and
fu × CLint= CLiv×
60 × A × C0
X.L. Qin et al. / International Journal of Pharmaceutics 389 (2010) 114–121
Fig. 1. Whole blood concentrations–time curves of FK506 after a single oral dose of
FK506 (1.89mg/kg) to rats with and without an oral dose of WZ (0.25g/kg) or Ver-
apamil (30mg/kg). Each point represents the mean±SD (n≥8). Data are depicted
on a semi-logarithmic scale.
Fig. 2. Whole blood concentrations–time curves of FK506 after a single intravenous
dose of FK506 (0.2mg/kg) with and without an oral dose of WZ (0.25g/kg). Each
point represents the mean±SD (n≥4). Data are depicted on a semi-logarithmic
C0is the initial concentration in the donor chamber (micrograms
per milliliter). Samples from the 90min point were used for Papp
Pappin the AP to BL direction (Papp(BL−AP)/Papp(AP−BL)) as in Eq.
(7) (Zhou et al., 2005; Zhang et al., 2006).
Transportratio =Papp(BL − AP)
Papp(AP − BL)
The results are expressed as the mean±SD. Statistical sig-
nificances were evaluated using Student’s t-tests or Wilcoxon
Two-Sample test. Statistical analyses were performed using SAS
8.1 software (SAS Institute, Inc., Cary, NC, USA). p value <0.05 was
considered statistically significant.
3. Results and discussions
3.1. Effect of WZ on the pharmacokinetics of FK506 following oral
and intravenous administration
The mean FK506 blood concentration–time curves obtained
after oral or intravenous administration of FK506 with or without
WZ are shown in Figs. 1 and 2. The pharmacokinetic parameters
of FK506 are presented in Tables 1 and 2. When administered in
combination with WZ (0.25g/kg), the AUC0–∞and Cmax of oral
administration of FK506 were increased by 209.9% (p<0.0005)
and 80.1% (p<0.05), while the AUC0–∞and Cmaxfollowing an oral
administration of FK506 were increased by 74.7% and 67.5% when
Verapamil (30mg/kg) were co-administered. The Kavalues were
almost the same in the WZ and Verapamil groups which were
Pharmacokinetic parameters of FK506 after a single oral dose of FK506 (1.89mg/kg)
to rats with and without an oral dose of WZ (0.25g/kg) or Verapamil (30mg/kg).
Data are the mean±SD.
57.7 ± 31.1
67.7 ± 34.9
5.7 ± 1.4
15.1 ± 11.2
0.9 ± 0.8
6.7 ± 1.9
30.0 ± 11.9
0.21 ± 0.07
188.3 ± 74.1***
209.8 ± 80.1***
7.3 ± 1.2*
27.2 ± 16.7*
1.1 ± 1.1
7.0 ± 2.0
10.6 ± 4.5***
0.27 ± 0.07*
85.4 ± 46.5
118.3 ± 39.3
5.2 ± 1.3
25.3 ± 17.0*
0.5 ± 0.3
7.0 ± 2.5
25.2 ± 16.8
0.28 ± 0.07*
*p<0.05 significantly different as compared with FK506 alone group.
***p<0.0005 significantly different as compared with FK506 alone group.
Pharmacokinetic parameters of FK506 after a single intravenous dose of FK506
109.0 ± 13.5
132.0 ± 14.2
1.5 ± 0.2
16.1 ± 1.5
6.8 ± 0.6
6.5 ± 0.3
141.2 ± 20.8
167.7 ± 29.5
1.2 ± 0.2
11.2 ± 1.8
7.5 ± 1.3
6.7 ± 0.7
significantly higher than that of FK506 alone group (p<0.05). Fur-
thermore, the MRT of WZ group (5.7h) was significantly delayed
to 7.3h compared to that of FK506 alone group (p<0.05), the CL/F
of WZ group (10.6L/h/kg) was about 1/3 of that of the FK506 alone
group (30.0L/h/kg) (p<0.00001), but there was no significant dif-
ference from that of the Verapamil group. On the other hand, WZ
produced only a 27.0% increase in AUC after intravenous FK506
dosing, which was significantly lower than the effect of WZ on oral
administration were decreased in the WZ group.
lated from the clinical practice, and Verapamil was used as positive
control. Verapamil is one of the most extensively characterized
inhibitors of P-gp and is the first multidrug resistance (MDR)-
reversal agents that reached clinical trial (Fisher and Sikic, 1995).
AUCs of FK506 in rats were slightly increased over dose range of
10–60mg/kg of Verapamil and reached the maximum increase at
30mg/kg. Thus, the dose of Verapamil as a positive P-gp inhibitor
was optimized at 30mg/kg in rats. The results showed that oral
concomitant administration of WZ could increase the whole blood
concentration of FK506 over comparable time periods following
oral or intravenous single dose of FK506, but the extent was much
less through intravenous injection route (Figs. 1 and 2). When co-
after oral or intravenous dosing was significantly slower than that
of FK506 alone. When co-administered with Verapamil, the blood
concentration of FK506 was sharply increased and then sharply
decreased (Fig. 1). The Cmaxand Kaof the WZ and Verapamil group
were significantly higher than that of control group, suggesting the
or Verapamil (Table 1). The CL/F of FK506 was decreased signif-
icantly from 30.0 to 10.6L/h/kg and the MRT was also extended
to 7.3h in the presence of WZ, but Verapamil had little effect
on the CL/F and MRT of FK506 (Table 1). Decreases in the CLiv
and Vdof FK506 were also observed in the WZ group after intra-
venous administration of FK506. The AUC0–∞ of FK506 in the
presence of WZ was 309.9% of that of FK506 alone, in contrast, the
AUC0–∞in the presence of Verapamil was only 174.7% of that of
X.L. Qin et al. / International Journal of Pharmaceutics 389 (2010) 114–121
Summary of derived pharmacokinetic parameters of FK506 after a single oral dose
(1.89mg/kg) or intravenous dose (0.2mg/kg) to rats with and without an oral dose
of WZ (0.25g/kg).
Group FK506 alone With WZ
2.84 ± 0.54
5.40 ± 2.74
0.46 ± 0.05
0.54 ± 0.05
0.10 ± 0.009
1.99 ± 0.63
13.24 ± 5.05
0.37 ± 0.07
0.63 ± 0.07
0.21 ± 0.025
FK506 alone. These findings suggested that WZ-mediated absorp-
tion enhancement and elimination decrease caused the increase
of FK506 bioavailability, while Verapamil also increased FK506
absorption but only had a moderate effect on the elimination and
thus had a small effect on FK506 bioavailability.
3.2. Effect of WZ on the oral bioavailability, and gut/liver
metabolic extractions of FK506
The derived pharmacokinetic parameters are presented in
decreased by 29.9% from 2.84±0.54L/h/kg to 1.99±0.63L/h/kg.
The estimated ERHof FK506 was 0.46±0.05, therefore, from Eq.
(4), the value of FH was 0.54±0.05 and then, (Fabs×FG) was
0.10±0.009, assuming that ERHis the same following intravenous
or oral dosing of FK506 (Takada et al., 1991). As compared with the
FK506 alone group, Foralof WZ group was significantly increased
from 5.40% to 13.24% (p=0.0002). The ERH of WZ group was
decreased by 19.6% compared with that of FK506 alone group.
Calculating FHand substituting into Eq. (4) allows estimation of
(Fabs×FG). The Fabs×FGof WZ group was 111.4% greater than that
ity of FK506.
As shown in Table 1, the CL/F of FK506 was far surpassed
the rat hepatic blood flow (QH), which indicated that FK506 was
also metabolized by extrahepatic tissues. Furthermore, high CL/F
also suggested FK506 underwent significantly first-pass effect,
which was in line with the reported results. The previous study
showed that pre-systemic metabolism by gastrointestinal CYP3A
was around 50% and was considered to be responsible for the
limited bioavailability of FK506 (Undre et al., 1999). As shown in
only about one-fifth of the liver availability (FH). Given that FK506
had a rapid and almost complete intestinal absorption (Tamura et
al., 2002), the contribution of Fabsto the low estimated gut avail-
ability might be relatively minor as compared to that of FG.Our
pre-systemic metabolism through CYP3A pathway. In the presence
of WZ, the (Fabs×FG) was 111.4% (p=0.0142) greater than that of
FK506 alone. On the other hand, WZ decreased hepatic first-pass
Fig. 4. Effect of Verapamil and WZ extract on the AP–BL and BL–AP transport of
FK506 across Caco-2 monolayers. The monolayers were incubated for 90min with
1?g/mL FK506 in the absence or presence of Verapamil (100?M) or WZ extract
(1mg/mL) added to both sides of the monolayers during pre-incubation (30min).
Each point represents the mean±SD (n=3).
metabolism (ERH) from 45.7% to 36.9%, which could be calculated
to an approximate 8.8% increase in Foralif all other determinants
of bioavailability were unaffected. However, the results showed
ing that the reduction of intestinal first-pass effect was the major
cause of Foralincrease when co-administered with WZ. The sig-
nificant gut metabolism was probably due to the high capacity of
exposure to intestinal metabolism enzymes by the intestinal efflux
transporters at the apical domain of the enterocyte.
3.3. Effects of WZ extract on the transport of FK506 in Caco-2 cells
The MTT assay indicated that WZ extract (range from 0.05 to
1mg/mL) and FK506 (1?g/mL) showed no cytotoxicity toward
Caco-2 cells. The TEER values were not significantly different
before and after the transport experiment. The time profile of
the basal-to-apical (BL–AP) and apical-to-basal (AP–BL) trans-
port of FK506 across Caco-2 cell monolayers showed that the
transport of FK506 was linear growth with R2>0.9 over 90min
(Fig. 3). The BL-to-AP transport of FK506 was 0.9 fold higher
than that of the AP-to-BL transport with transport ratio of 1.9
(Fig. 4). Here, the monolayers were pretreated with WZ extract
or Verapamil to prevent the direct interactions of FK506 with
WZ extract or Verapamil. The WZ extract elevated the AP-to-
BL transport of FK506 and reduced the BL-to-AP transport of
FK506. In the presence of 1mg/mL WZ extract, the Papp AP–BL
whereas the PappBL–AP was decreased (from 7.0±0.5×10−6cm/s
to 5.8±0.2×10−6cm/s), resulting in a mean transport ratio of
1.2. The positive control Verapamil increased Papp AP–BL (from
3.1±1.7×10−6cm/s to 4.2±0.9×10−6cm/s), and decreased Papp
BL–AP (from 7.0±0.5×10−6cm/s to 6.5±0.2×10−6cm/s) and
resulted in a mean transport ratio of 1.5. These findings sug-
gested that both the WZ extract and Verapamil inhibited the
P-gp-mediated efflux of FK506.
Fig. 3. The time profile of the AP–BL and BL–AP transport of FK506 across Caco-2 cell monolayers with 1mg/mL WZ extract (B) or 100? M Verapamil (C) (n=3).
X.L. Qin et al. / International Journal of Pharmaceutics 389 (2010) 114–121
(0.02mgprotein/mL) were pre-incubated with WZ extract or Ketoconazole for 5min in 100mM potassium phosphate buffer (pH 7.4) containing 0.05mM EDTA. The reaction
was started by addition of 20?L of 10mM NADPH to the system. The mixture was incubated for 5min at 37◦C. Each point represents the mean±SD (n=3).
It was reported that Caco-2 cells possessed only very slight
CYP3A4 activity (Schmiedlin-Ren et al., 1997), indicating that the
metabolism of FK506 in these cells would be almost negligible
dose of FK506 was 0.15–0.3mg/kg twice a day for renal trans-
plant patients. Thus, for a 70-kg weight subject, taking the volume
of gastrointestinal tract (3–5L) into consideration (Egashira et al.,
2004), the concentration of FK506 in the intestinal surface after
taking an oral dose would be 1–3.5?g/mL. Thus, the concentra-
tion of FK506 used in the Caco-2 cell transport experiment was set
at 1?g/mL. In the present transport study, the transport ratio of
FK506 in the control group was only 1.9, which was considered to
sible that FK506 at the concentration 1?g/mL (1.2?M) used in the
et al., 2007) suggested that 1?M of FK506 could enhance cellu-
lar drug uptake in cells overexpressing P-gp, MRP-1 or BCRP. The
transport ratio of FK506 in WZ extract pretreated group was lower
than that of the control group, and lower than that of Verapamil-
pretreated group. This finding suggested that WZ extract inhibited
P-gp-mediated efflux of FK506 and the ability of WZ extract to
reduce the active transport of FK506 was more potent than Vera-
pamil, a well-known P-gp inhibitor. Previously published work has
shown that several constituents in SchE could interact with P-gp.
Yoo et al. reported that several lignans including deoxyschizandrin
(Schisandrin A), angeloylgomisin M1, gomisin A (Schisandrol B),
tigloylgomisin H, and gomisin C (Schisantherin A) exhibited sta-
tistically significant inhibitory effect on P-gp-mediated efflux of
rhadamine-123 in Caco-2 cells (Yoo et al., 2007). Pan and Sun et al.
et al., 2005; Sun et al., 2007). Since the major active constituents in
WZ were the lignans, the previous studies supported our current
findings that WZ extract had inhibitory effect on P-gp and thus
inhibited P-gp-mediated efflux of FK506.
3.4. Effects of WZ extract on the metabolism of FK506 in rat and
human liver microsomes
The incubation time, protein concentration and the FK506 con-
centration were optimized by comparing the metabolized ratio of
FK506 under different protein concentration (0.005–0.5mg/mL),
incubation time (1–15min) and different substrate concentrations.
The metabolized ratio of FK506 was linearly increased at the incu-
bation time range of 3–15min. The metabolized ratio of FK506 was
increased from 6.1% to 58.3% over the protein concentrations range
of 0.005–0.5mg/mL, and the metabolism of FK506 showed a lin-
ear increase over the protein concentrations range from 0.005 to
0.02mg/mL. The metabolism of different concentration of FK506
(1.25–20ng/mL) in the rat liver microsomes was 17.0–24.0% with
no significant difference. Taking the FK506 therapeutic range of
5–15ng/mL (Venkataramanan et al., 1995) into considerations,
5ng/mL of FK506 was chosen as substrate concentration in the
metabolism study. Therefore, the incubation condition was set as
0.02mg/mL of protein, 5ng/mL of metabolism substrate (FK506)
and 5min of incubation time.
The effects of WZ extract and Ketoconazole on the metabolism
of FK506 in rat and human liver microsomes were studied with the
system described above. The effects of different concentrations of
WZ extract on the metabolism of FK506 are shown in Fig. 5 and
Table 4. The metabolism of FK506 was significantly decreased in
the presence of Ketoconazole and WZ extract. The inhibition of
metabolism of FK506 by WZ extract and Ketoconazole was concen-
tration dependent. WZ extract had equivalent or even more potent
inhibition than that of Ketoconazole. Metabolism of FK506 was
almost completely inhibited by 100?M of WZ extract and Keto-
conazole [it was reported that 25?M of Ketoconazole, a potent
inhibitor of CYP3A, caused an 80% inhibition of FK506 metabolism
(Lampen et al., 1996)], indicating a potent inhibition of FK506
metabolism by WZ and Ketoconazole.
Previously published work had shown that Schisandra fruit
extract, gomisins B, C, G, and Schisandrin were potent inhibitors
of CYP3A4 and the inhibitory effect of gomisin C (Schisantherin A)
was stronger than that of Ketoconazole (Ki=0.070?M) (Iwata et
al., 2004). Since Gomisin C was the quality control constituent and
the most abundant lignan in WZ, it might play a very key role in the
inhibitory effect of WZ on the CYP3A-mediated FK506 metabolism.
FK506 is a good substrate of CYP3A and P-gp. CYP3A is abun-
dantly present both in liver and intestine and P-gp is expressed in
many organs and tissues including intestine and liver. The overlap
of physiological distribution of CYP3A and P-gp cause a com-
plex interplay between CYP3A and P-gp during the metabolism
and efflux process (Lemahieu and Maes, 2007). This dynamic
enzyme–transporter interplay between CYP3A and P-gp in intesti-
can be applied to the regulation of oral FK506 exposure (Fig. 6). On
the other hand, if tacrolimus is a substrate of other transporters
Effects of different concentrations of WZ extract (1, 10, 100?M) on the metabolism
of FK506 (5ng/mL) in the rat and human liver microsomes (0.02mg/mL), t=5min
(%) in the rat liver
(%) in the human
Control23.1 ± 3.320.7 ± 3.7
With WZ extract (?M)
11.5 ± 4.1
3.3 ± 1.9
0.7 ± 1.4
15.4 ± 3.5
6.3 ± 5.1
0.3 ± 0.5
With Ketoconazole (?M)
18.1 ± 3.7
8.4 ± 3.6
0.3 ± 0.4
17.0 ± 0.6
13.2 ± 8.1
1.6 ± 3.2
X.L. Qin et al. / International Journal of Pharmaceutics 389 (2010) 114–121
Fig. 6. Effect of interplay between CYP3A and P-gp in intestinal and liver on the
regulation of oral FK506 exposure.
such as MRPs and BCRP, the involvement of these transporters
in the WZ–FK506 interaction will make the enzyme–transporter
interplay more complicated. The Cmaxand Kaof FK506 in the cur-
rent study were increased significantly, and the transport ratio
was decreased in Caco-2 cells, indicating the inhibition of P-gp.
The CL/F was decreased markedly and the MRT was delayed, in
addition, the FK506 metabolism was decreased by WZ, all these
suggested the inhibition of CYP3A by WZ. For the potent inhibition
of FK506 metabolism by WZ, we ever supposed that the inhibi-
tion of FK506 metabolism mostly contributed to the increase of
oral FK506 exposure for the interplay of CYP3A and P-gp in liver.
Interestingly, our data from in vivo pharmacokinetic study in rats
in the presence of WZ was mostly due to the inhibition of intestinal
first-pass extraction and only partially due to the inhibition of liver
metabolism. However, the current study cannot fully differentiate
exposure with co-administration of WZ. And, it is still unknown
whether the first-pass effect in the intestine is more related to
CYP3A-mediated metabolism than to P-gp-mediated drug exsorp-
tion. Therefore, further studies are needed to elucidate the exact
interaction of hepatic/intestinal enzyme and transporter during
this WZ–FK506 interaction process. In addition, WZ contains mul-
tiple lignans and only a few of which have been investigated for
their CYP3A/P-gp modulating activity, thus the exact effect of each
lignan in WZ on the hepatic/intestinal CYP3A and P-gp activity is
also needed to be further clarified.
In conclusion, the AUC of FK506 was significantly increased by
2 fold when co-administered with WZ, which is in line with our
clinical observation on renal transplant patients and the previ-
ous reports. The results from transport and metabolism studies
showed that WZ could significantly inhibit the CYP3A-mediated
metabolism of FK506 and the P-gp-mediated FK506 efflux. The
reduction of intestinal first-pass effect of FK506 by WZ was
extensive and contributed a major part of the increase in FK506
bioavailability. Current findings from in vitro and in vivo stud-
ies consistently demonstrated that the reduced first-pass effect
of FK506 by WZ was related to the inhibition of CYP3A-mediated
FK506 metabolism and P-gp-mediated FK506 exsorption.
The work was supported by the National Key Projects
for science and technology development from Science and
Technology Ministry of China (Grant No. 2009ZX09304-003),
the National Basic Research Program of China (Grant No.
2009CB522707) from National Science and Technology Min-
istry, and the Research Committee of Macau University (Project
No.: RG065/08-09S/WYT/ICMS). The authors also appreciate the
financial support provided by Medical Foundation of Guangdong
Province and Foundation from Guangdong Tranditional Medicine
Bureau (Grant number: B2009049, 2009164).
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