Enhanced liver-targeting via coadministration of 10-Hydroxycamptothecin polymeric micelles with vinegar baked Radix Bupleuri

Article abstract of DOI:10.1016/j.phymed.2018.04.022

Background: Vinegar baked Radix Bupleuri (VBRB) is a wildly used traditional Chinese medicine, it could be used as a meridian guided drug to enhance liver targeting efficiency of the delivered drug in addition to its therapeutic effect. Purpose: To invest

Full text of DOI:10.1016/j.phymed.2018.04.022

Accepted Manuscript
Enhanced liver-targeting via coadministration of
10-Hydroxycamptothecin polymeric micelles with vinegar baked
Radix Bupleuri
Haiyang Wu , Tongya Yu , Ye Tian , Yinjie Wang , Ruizhi Zhao ,
Shirui Mao
PII:
DOI:
Reference:
S0944-7113(18)30122-3
10.1016/j.phymed.2018.04.022
PHYMED 52461
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Phytomedicine
Received date:
Revised date:
Accepted date:
23 March 2017
13 February 2018
8 April 2018
Please cite this article as: Haiyang Wu , Tongya Yu , Ye Tian , Yinjie Wang , Ruizhi Zhao ,
Shirui Mao , Enhanced liver-targeting via coadministration of 10-Hydroxycamptothecin polymeric mi-
celles with vinegar baked Radix Bupleuri, Phytomedicine (2018), doi: 10.1016/j.phymed.2018.04.022
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ACCEPTED MANUSCRIPT
Enhanced liver-targeting via coadministration of 10-Hydroxycamptothecin
polymeric micelles with vinegar baked Radix Bupleuri
a,1
a,1
a
b
b,*
a,*
Haiyang Wu , Tongya Yu , Ye Tian , Yinjie Wang , Ruizhi Zhao , Shirui Mao
a
School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, 110016,
China.
b
Second Affiliated hospital, Guangzhou University of Chinese Medicine, Neihuan Xilu, Guangzhou
Daxuecheng, Guangzhou, China
*
Corresponding author
School of Pharmacy,
Shenyang Pharmaceutical University,
1
03 Wenhua Road, 110016, Shenyang, China
Tel +86-24-43520523
E-mail address: maoshirui@syphu.edu.cn
Second Affiliated hospital,
Guangzhou University of Chinese Medicine
Neihuan Xilu, Guangzhou Daxuecheng
5
10006, Guangzhou, China
Tel +86-20-39318571
Fax +86-20-39318571
E-mail address: 13610241754@163.com
1
These authors contributed equally to this work.
1

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ABSTRACT
Background: Vinegar baked Radix Bupleuri (VBRB) is a wildly used traditional
Chinese medicine, it could be used as a meridian guided drug to enhance liver
targeting efficiency of the delivered drug in addition to its therapeutic effect.
Purpose: To investigate the liver targeting effect induced by VBRB via
coadministration with 10-Hydroxycamptothecin loaded polymeric micelles.
Methods: First of all, the inhibitory effect of VBRB on the activity of glutathione
S-transferase (GST) was investigated in vitro to select the most effective extract. After
oral administration of 10-Hydroxycamptothecin (HCPT) polymeric micelles with low,
medium and high doses of VBRB, pharmacokinetic parameters, including the ratio of
Cmax in the liver (Ce) and the relative uptake efficiency (RUE), were employed to
assess the liver targeting efficiency.
Results: It was found that VBRB extract BC1 has the strongest inhibition effect on
GST activity in the five extracts. By coadministration of HCPT loaded micelles with
three doses of BC1, the AUC of HCPT in the liver raised by 42.5%, 23.0%, -0.2%,
0
-t
with RUE 1.45, 1.23, 1.02 for low, medium and high dose groups, respectively,
indicating that low and medium dose of BC1 presented better liver-targeting
enhancing effect than that of the high dose, which corresponded to the commonly
used dose of VBRB in traditional Chinese medicine formulae.
Conclusions: VBRB could effectively enhance the liver-targeting efficiency of HCPT
loaded polymeric micelles after oral coadministration. Such a simple but effective
strategy may enlighten on the potential use of meridian guided drug together with
modern drug delivery system to achieve better active drug targeting.
Keywords: Vinegar baked Radix Bupleuri (VBRB); Glutathione S-transferase (GST);
Liver-targeting; Meridian guided drug; Polymeric micelles
2

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Abbreviations
Vinegar baked Radix Bupleuri (VBRB), Glutathione S-transferase
0-Hydroxycamptothecin (HPCT), The ratio of Cmax in the liver (Ce), Relative
(GST),
1
uptake efficiency (RUE), Enhanced permeability and retention effect (EPR),
Cytochrome P450 (CYP), L-glutathione reduced (GSH), 1-chloro-2,4-dinitrobenzene
(CDNB), Albumin from bovine serum V (BSA), Chitosan (50)-g-glycerol monooleate
copolymer (CS (50)-g-GM), Mean ± standard deviation (SD), Coefficients of
variation (CV), Photon correlation spectroscopy (PCS)
Introduction
Liver cancer has become the second leading cause of cancer death worldwide
(Petrick et al., 2016). Surgical resection and chemotherapy are the two most effective
treatment strategies. However, low targeting efficiency and toxicity are the main
drawbacks of traditional chemotherapy (Kawaguchi et al., 2017). For cancer therapy,
targeting drug delivery system is highly desired in order to increase therapeutic effect
and meanwhile decrease the side effect. Passive targeting and active targeting are the
two commonly used methods (Deng et al., 2012; Tian and Mao, 2012). Passive
targeting is mainly achieved by designing the drug delivery system with appropriate
particle size (Win and Feng, 2005), and it is assumed that particles in the nanometer
range can be accumulated in the tumor tissue via so called enhanced permeability and
retention effect (EPR) and uptaken by reticular macrophage rich tissues (Maeda et al.,
2
000). To further increase the targeting efficiency beyond passive targeting, active
targeting is a commonly used strategy mainly achieved via grafting specific ligands to
the delivery system (Qian et al., 2009; Sudimack and Lee, 2000). However, by
analyzing over 100 years publication, a recent review article indicated that the results
of active targeting is very disappointing, with the average targeting efficiency only
about 0.7% (Wilhelm et al., 2016), implying active targeting strategy is still far from
clinical application. Hence, it is absolutely essential and urgent to search for other
targeting methods with simple preparation technology and reliable targeting
efficiency for liver cancer therapy.
3

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In traditional Chinese medicine, targeting is usually achieved by combining the
active pharmaceutical ingredient with so called meridian guided drug to change the
active drug’s distribution in vivo. To enhance drug’s liver targeting, vinegar baked
Radix Bupleuri (VBRB) is commonly used and its effectiveness has already been
demonstrated with several model drugs such as rhein (Zhao et al., 2010), oxymatrine
(Zhao et al., 2011), and resveratrol (Zhao et al., 2009). It is well known that
cytochrome P450 (CYP) system is responsible for the oxidative metabolism of
numerous endogenous compounds as well as xenobiotics including drugs and
environmental chemicals (Ravindranath and Strobel, 2013). Mechanism study
disclosed that VBRB could inhibit metabolizing activity of hepatic CYP system in the
liver (Yu et al., 2014). After coadministration with VBRB, the metabolic process of
combined drug might be depressed in the liver, thus, drug concentration in the liver
was higher than that in the drug only group (Zhao, 2014) .
In our previous study, the modulatory effects of extracts from VBRB on the
activity of CYP1A2, CYP2C9 and CYP3A4 were investigated in vitro. It was found
that VBRB extracts have strong inhibition effect on the CYP2C9 activity (Yu et al.,
2
014). Except for CYP system, glutathione S-transferase (GST) is related to the
multidrug resistance of anticancer drugs (Calatozzolo et al., 2005). Ricci G indicated
that the GST inhibitors are potential anticancer drug adjuvant (G et al., 2005).
Therefore, if VBRB can inhibit the activity of GST, it might further enhance the liver
targeting effect of GST substrate via simple drug coadministration.
To test this hypothesis, here in this study 10-Hydroxycamptothecin (HCPT), a
member of camptothecin family, was selected as the model drug, it is a classical
anti-cancer active pharmaceutical ingredient used for primary liver cancer therapy,
metabolized by CYP and GST in the liver (Ling and Xu, 1993). Previous study
demonstrated that peroral absorption of HCPT can be enhanced significantly by
loading HCPT into chitosan based polymeric micelles, with improved anticancer
effect (Tian et al., 2015). It is highly desirable if liver targeting of HCPT can be
further increased by coadministration with VBRB. Therefore, in this study, first of all,
the effect of VBRB extracts on the activity of GST was investigated in vitro. Then the
4

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in vivo absorption of HCPT polymeric micelles after coadministration with different
dose of VBRB extract was studied and the liver targeting efficiency was evaluated.
Materials and methods
Materials
VBRB (B Chinese) was purchased from Kangmei Medical Company,
Guangzhou, China. The sample was authenticated by a pharmacist-in-charge called
Wenliang Chen and the voucher specimens were deposited in the Pharmacognosy and
Phytochemistry Department of Guangzhou University of Traditional Chinese
Medicine. The VBRB was prepared according to the procedure described in the
Appendix IID of Chinese Pharmacopoeia 2015. Briefly, 100 kg Bupleurum slices
were mixed with 20 kg rice vinegar, moistening thoroughly, then the samples were
transferred to a frying pot, dried under gentle stirring and cooled. Human placenta
type glutathione S-transferase (GST), L-glutathione reduced (GSH) were purchased
from Sigma-Aldrich Trading Co. Ltd. (Shanghai, China). 1-chloro-2, 4-dinitrobenzene
(CDNB) was from Xiya Reagent (Chengdu, China). Albumin from bovine serum V
(BSA) was provided by Beijing Dingguo Changsheng Biothechnology Co. Ltd.
(Beijing, China). 10-Hydroxycamptothecin was obtained from Sichuan Ruibo Co. Ltd.
(Sichuan, China). Different VBRB extracts were prepared as described previously
(Chen et al., 2014) and their compositions are listed in Table 1.
Chitosan-grafted-glyceryl monooleate was synthesized and characterized according to
the previous report (Tian et al., 2015). All other chemicals and solvents were of
analytical or HPLC grade.
Preparation and characterization of BC extracts
BC0, BC1, BC2, BC3, BC4 were five kinds of VBRB extracts which were
prepared by different preparation technologies and characterized according to Yu’ and
Chen’s researches (Chen et al., 2014; Yu et al., 2014). The extracts were dissolved in
0
.1 M PBS (pH 7.4) and a series of BC solutions (0.0143, 0.0286, 0.0571, 0.1142,
-1
0
.2284 g∙ml of raw medicinal herbs per milliliter) were obtained by dilution with
5

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PBS.
BC1 is a mixture of water soluble macromolecules including polysaccharides
and proteins, and mainly polysaccharides, usually called crude polysaccharides. The
yield of BC1 was 4.5% of dried raw material. The molecular weight of BC1 were
evaluated and determined by HPGPC. BC1 was dissolved in distilled water, and then
applied to Agilent HPLC system equipped with a TSK G-4000PWXL column (7.8×
3
00mm), eluted with double distilled water at a flow rate of 0.5 ml/min and detected
by a refractive index detector. Dextran standard with different molecular weights was
used to calibrate the column and establish a standard curve. Molecular weight of BC1
was estimated by reference to the calibration curve. The molecular weight range of
BC1 is from 215 to 855 kDa.
Monosaccharide compositions in BC1 were identified and quantified using
GC-MS chromatography. BC-1 was hydrolyzed with 2 M trifluoroacetic acid (4 ml) at
110℃for 6 h. The hydrolyzed product was analyzed by GC. GC was performed on a
Agilent 7890A + 5975C instrument (Component, USA) equipped with Agilent
HP-5MS capillary column (30 m×0.25 mm×250 μm). It was found that BC1 was
composed of four kinds of monosaccharides, including rhamnose: arabinose: glucose:
galatose with a molar ratio of 1:21.0:7.8:4.6, and a small amount of mannose. The
content of uronic acid was 42.05%.
GST activity measurement
The activity of GST was determined according to Habig’s assay method with
modification by adapting the method to 96-well plates (G et al., 2005). By adding
GST in GSH and CDNB mixture, CDNB-SG will be formed (Fig.1), which has
maximum absorption at wavelength 340 nm. There is a linear relationship between
the activity of GST and the difference of absorbance (△OD) before and after the
reaction.
Preparation of GST stock solution: 1 mg GST was dissolved in 1 ml PBS (pH
6
.5), 0.21 mg BSA and 1 ml glycerol were added to the mixture. The GST stock
o
solution was kept at -20 C until use.
6

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Preparation of CDNB solution: 20 mg CDNB was dissolved in 2 ml absolute
-1
ethyl alcohol to get 50 mmol∙l CDNB stock solution. PBS was used to dilute the
CDNB stock solution to a specific concentration as required.
Preparation of GSH solution: GSH solution was prepared by dissolving
appropriate amount of GSH powder in PBS. The solution was freshly prepared before
use.
The activity of GST was measured according to following procedures. Briefly,
3
9
0 μl GST, 30 μl CDNB, 30 μl GSH, 30 μl PBS or other test solution were added into
6-well plates, and SpectraMax M3 (Molecular Devices, US) was used to test the
absorbance (OD1) under 340 nm after mixing. After 60 min incubation at specific
temperature, the absorbance (OD2) was tested again. The difference (△OD) between
OD1 and OD2 was used to express the activity of GST.
Preparation and characterization of HCPT loaded micelles
The HPCT-loaded polymeric micelles were prepared by self-assembly method
with pH adjustment as reported previously (Tian et al., 2015). Briefly, 20 mg of
chitosan (50)-g-glycerol monooleate copolymer (CS (50)-g-GM) was dissolved in 2
-1
ml 0.5% (v/v) acetic acid solution. 50 mg HCPT was dissolved in 10 ml 0.5 mol∙l
NaOH solution. Then appropriate volume of HCPT solution was added to the
copolymer solution under stirring. The mixture was adjusted to pH 4 and stirred for
2
4 h. Thereafter, the pH was adjusted back to 6.8. The formed micelles were probe
sonicated at 200 W for 4 times each for 5 min (active for 5 s and stop for 5 s) in an
ice-bath.
The mean particle size (z-ave) and the polydispersity index (PI) of the micelles
were determined by photon correlation spectroscopy (PCS) using a Malvern Zetasizer
4
(Malvern Instruments, UK) at 25 °C and a scattering angle of 90°. The zeta potential
measurements were carried out using Malvern Zetasizer 4 by electrophoretic laser
Doppler anemometry at 25 °C. Centrifugal sedimentation method was used to test
drug entrapment efficiency.
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In vivo pharmacokinetic study
In vivo absorption of HCPT micelles after coadministration with different dose of
VBRB BC1 was studied in rats. The animal experiment was carried out in accordance
with the Principle of Laboratory Animal Care. Male Sprague Dawley rats (200 ± 20g)
were supplied by the Lab Animal Center of Shenyang Pharmaceutical University. The
experimental protocol was approved by the University Ethics Committee for the use
of experimental animals and conformed to the Guide for Care and Use of Laboratory
Animals.
To study the influence of BC1 on the absorption of HPCT loaded micelles, 20
SD rats were randomly divided into four groups (n = 5), that is, HPCT loaded
micelles only as control group, HCPT loaded micelles coadministrated with low (150
-1
-1
-1
mg·kg ), medium (300 mg·kg ), high dose (600 mg·kg ) of BC1 groups. In all the
-1
four groups, the dose of HCPT was kept at 10 mg·kg via intragastic administration.
The rats were fasted for 12 h before study with free access to water. Approximately
0
.3 ml blood samples were withdrawn from retro-orbital plexus at predetermined time
points (0.25, 0.5, 0.75, 1, 1.5, 2, 3, 5, 8, 12, 24, 36, 48, 60, 72, 84, 96 h) post-dosing
and immediately centrifuged at 10000 rpm for 10 min. The plasma samples were
o
stored at -20 C until analysis.
The pharmacokinetic parameters were calculated using a non-compartmental
model by Drug and Statistics for Windows (DAS version 2.1.1).
Liver targeting evaluation
For liver targeting evaluation, 64 SD rats were randomly divided into four
groups (n = 16 in each group, and n = 4 for each time point). The four groups and the
dose were exactly the same as that in pharmacokinetic study. The rats were executed
at predetermined time points (2, 8, 24, 48 h) after dosing. The liver was isolated and
rinsed with saline. After absorbing the external water with filter paper, the sample was
kept at -20 °C until analysis.
To have a better understanding of VBRB effect on the distribution of HPCT
loaded micelles, Ce, as defined in equation 1, the ratio of the Cmax in the liver was
calculated (Yamaguchi et al., 2009). The higher the Ce value, the better the targeting
8

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efficiency. Relative uptake efficiency (RUE, equation 2) was another index to study
the targeting efficiency (Gupta and Hung, 1989). When RUE>1, it was considered
that liver-targeting was obvious.
Ce = (C
)
/(C
)
max control
(1)
(2)
max sample
RUE = AUCsample/AUCcontrol
where the subscript sample represents the formulation of HPCT loaded micelles
coadministrated with different dose of BC1, the control represents the formulation of
HPCT loaded micelles only.
In vivo analysis of HCPT
The concentration of HPCT in plasma and liver homogenate was analyzed by
HPLC after solvent extraction. Briefly, 100 μl of the plasma sample or 300 μl liver
homogenate (smashed with 2-fold 0.9% NaCl) was mixed with 1 ml methanol and 20
μl acetic acid by vortexing. Thereafter, 3 ml diethyl ether was added to the mixture for
HCPT extraction. The mixture was centrifuged at 10000 rpm for 10 min and the
supernatant was transferred into another tube and the solvent was evaporated in a
4
0℃ water bath under nitrogen flow. The residue was redissolved in 100 μl of
methanol by vortexing, followed by centrifugation at 10000 rpm for 10 min. 20 μl of
the supernatant was injected into the HPLC column to determine the concentration of
HPCT using an Agilent 1100 series with the following conditions: Merck Purospher®
®
STAR RP-18 column (5 μm Hibar RT 150-4.6), mobile phase: water-methanol
o
(
50:50 v/v), flow rate: 0.8 ml/min, wave length: 384 nm, oven temperature: 30 C.
This HPLC method was specific and efficient for the analysis of HCPT content
in plasma and liver tissue. No interference from endogenous components was
-1
observed. The calibration curve in the concentration range from 20 to 4000 μg∙ml
were A = 0.0838C+2.1617 (r = 0.9995) and A = 0.0676C+5.2469 (r = 0.9979) in
plasma and in liver homogenate, respectively. The intra- and inter-assay coefficients
of variation (CV) were all within 15%. HCPT extraction recovery was in the range of
9
1.61%~99.19% and 82.98%~98.77% in plasma and liver homogenate respectively.
9

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Statistical analysis
All the results were expressed as mean ± standard deviation (SD) from at least
three measurements. Significance of difference was evaluated using one-way ANOVA
at the probability level of 0.05.
Results
Effect of CDNB concentration on the activity of GST
-1
-1
Keeping GST (4 U∙ml ) and GSH (4 mmol∙l ) concentration constant, effect of
-1
CDNB concentration (0, 0.25, 0.5, 1, 2, 3, 4, 5 mmol∙l ) on the activity of GST was
investigated. As shown in Fig. 2(a), the activity of GST increased with increasing
CDNB concentration. It was noted that the activity of GST increased remarkably
-1
when CDNB concentration was less than 3 mmol∙l , and the increasing trend slowed
-1
-1
down when CDNB concentration was above 3 mmol∙l . Hence, 3 mmol∙l CDNB
was selected for the following studies.
Effect of GSH concentration on the activity of GST
-1
-1
Keeping CNDB (3 mmol∙l ) and GST (4 U∙ml ) concentration constant, effect
-1
of GSH concentration (0, 0.25, 0.5, 1, 2, 3 mmol∙l ) on the activity of GST was
investigated. As shown in Fig. 2(b), the activity of GST increased remarkably when
-1
GSH concentration was less than 1 mmol∙l , slow increase in GST activity was
-1
observed thereafter. Therefore, 1 mmol∙l GSH was selected for the following studies.
Effect of incubation temperature on the activity of GST
-1
-1
-1
Keeping CNDB (3 mmol∙l ), GSH(1 mmol∙l ) and GST (4 U∙ml ) concentration
constant, influence of incubation temperature (25 ℃, 30 ℃, 37 ℃) on the activity of
GST was investigated. As shown in Fig. 2(c), the activity of GST increased when the
o
o
temperature was increased from 25 C to 30 C, however, further increasing
o
temperature to 37 C caused significant decrease in GST activity, even lower than that
o
o
at 25 C. Thus, 30 C was used in the followed study.
Effect of GST concentration
-1
-1
Keeping CNDB (3 mmol∙l ), GSH (1 mmol∙l ) concentration and incubation
o
temperature (30 C) constant, influence of GST concentration (0, 1.33, 2.67, 4.0, 5.33,
10

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-1
6
.67 U∙ml ) on the enzyme activity was investigated. As shown in Fig. 2(d), there
was a liner relationship between GST concentration and the activity of GST (y =
-1
0
.099x+ 0.0503, r = 0.9903, n = 6) in GST concentration range of 0~6.67 U∙ml . 4
-1
U∙ml GST concentration was chosen in this study, which can satisfy the requirement
for measuring sensitivity and meanwhile save cost.
Effect of different VBRB extracts on the activity of GST
To investigate the effects of different VBRB extract on the activity of GST, first
of all, influence of VBRB concentration was investigated.
Taking BC4 as an example, activity of GST was studied at BC4 concentration 0
-1
(
control), 0.0143, 0.0286, 0.0571, 0.1142 and 0.2284 g∙ml of raw medicinal herbs
per milliliter, respectively. As shown in Fig.3. (a), BC4 could inhibit the activity of
GST and the inhibition ratio was BC4 concentration dependent. When BC4
concentration increased to 0.1142 g of raw medicinal herbs per milliliter, inhibition
ratio was 19.37%. Further increasing BC4 concentration to 0.2284 g of raw medicinal
herbs per milliliter caused no significant difference in inhibition ratio (P > 0.05)
-1
Therefore, 0.1142 g∙ml of raw medicinal herbs per milliliter was selected for the
followed studies.
-1
Keeping the concentration of VBRB extract at 0.1142 g∙ml of raw medicinal
herbs per milliliter, influence of various VBRB extracts (BC0, BC1, BC2, BC3, BC4,
as listed in Table 1) on the activity of GST was studied. As shown in Fig.3. (b), all the
VBRB inhibited the activity of GST significantly but with different extent. The
strongest inhibition effect was found for extract BC1, no statistical difference was
found between BC0 and BC2 groups, (P>0.05), where BC4 showed the weakest
inhibition effect followed by BC3.
Influence of BC1 dose on the pharmacokinetic behavior of HCPT micelles
The above study demonstrated that BC1 has the strongest inhibition on the
activity of GST among all the VBRB extracts. In combination with the fact that BC1
also presented the strongest inhibition on the activity of CYP, BC1 was selected for
11

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the in vivo study.
The prepared HPCT loaded micelles had particle size approximately 340 nm, with
zeta potential + 30.2 mV, drug entrapment efficiency 91.8%.
It is assumed that the dose of BC1 may influence the liver targeting effect of
HCPT loaded micelles and needs to be clarified. Thus, three doses of BC1, 150, 300
-1
and 600 mg∙kg ，were selected based on its application in clinic and the difference
between rats and human beings. The concentration-time profiles of HCPT in plasma
after oral administration of HCPT loaded polymeric micelles with varied doses of
BC1 are shown in Fig.4.
After taking HPCT loaded micelle solution together with three different dose of
BC1 orally, the change of plasma concentration was compared with that of HPCT
loaded micelles, as shown in Fig.4. The pharmacokinetic parameters are summarized
in Table 2. Compared to micelles only group, HCPT was absorbed faster after
co-administration with BC1, with Tmax changed from 60 min to 42-48 min, implying
that BC1 might enhance the mucosal absorption of HCPT. In contrast, compared to
the control group, AUC decreased by 20%, 9.5%, 2.1% for low, middle and high
0
-t
dose BC1 groups, respectively, and significant difference in AUC0-t was found
between the control group and the low dose group (P < 0.05), indicating that BC1 may
decrease drug distribution in the plasma.
Influence of BC1 dose on the liver targeting of HCPT micelles
HCPT concentration in the liver after coadministration with three different dose
of BC1 is shown in Fig.5. As anticipated, after co-delivery of HCPT micelles with
BC1, significantly increased drug concentration in the liver was observed in the low
dose and middle dose BC1 groups compared to the control group (P < 0.05) up to 48
h.
The Cmax of HPCT in all the three groups tested was higher than that of the
control group in liver tissue, whereas the low dose BC1 group has the highest Cmax,
and significant difference in Cmax was found between the low dose group and the
control group. After coadministration with BC1, the AUC of HPCT in liver raised by
0
-t
12

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4
2.5%, 23.0%, -0.2% for low, medium and high dose groups respectively, with
significant difference disclosed for low and medium dose group when compared with
control group (P < 0.05). Furthermore, two targeting indexes, the Ce and the RUE
(Tab.2), were used to assess the liver targeting efficiency. The Ce was 1.20, 1.14, 0.92
and RUE 1.45, 1.23, 1.02 for low, medium, high dose BC1 groups respectively,
indicating that the low and medium dose BC1 groups presented better liver targeting
efficiency than the high dose group (P < 0.05).
Discussion
In this study, it was found that VBRB could inhibit the activity of GST and the
inhibition ratio increased with the increase of VBRB concentration. The VBRB
samples extracted by different method showed different inhibition degree on the GST
activity. Among the five extracts, when used at the same concentration, BC1, which is
mainly composed of water soluble high molecular weight polysaccharides, presented
the strongest inhibition effect, which is higher than the saikosaponins containing parts.
To the best of our knowledge, this is the first report demonstrating that
polysaccharides from VBRB can inhibit the activity of GST.
It was reported that the activity of GST increased remarkably in some types of
cancer patients and was closely related to anticancer drug resistance (Pelkonen et al.,
2
008). By depressing the highly expressed GST’s activity, it might be beneficial to
overcome the multidrug resistance in tumor cells and therefore improve therapeutic
effect (Xu et al., 2014). It have been indicated that GST specific inhibitors could be
potential adjuvant anticancer drugs (Filomeni et al., 2008). Based on the inhibitory
effect of VBRB on the activity of GST, it might be used as an adjuvant anticancer
drug to overcome chemotherapy related multidrug resistance.
In ancient China, VBRB has been used as meridian guided drug for liver
targeting. Previous study in animals did show that VBRB enhanced the distribution of
oxymatrine, rhein, resveratrol, gentiamarin in liver (Zhao et al., 2009; Zhao et al.,
2
010; Zhao et al., 2011). It is highly desirable if this simply intrinsic targeting strategy
can be combined with modern targeting drug delivery system design. Our previous
13

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study demonstrated that by designing polymeric nanomicelles, HCPT distribution in
the liver can be significantly enhanced as demonstrated by in vivo image (Tian et al.,
2
015). Fortunately, in vivo study demonstrated that by combining HCPT loaded
micelles with VBRB exacts, drug distribution in the liver could be further enhanced,
implying this strategy can be used in modern drug delivery system design. Moreover,
it was noticed that the liver targeting efficiency was VBRB dose dependent.
Compared to the control group, the liver targeting effect is more significant when low
and middle dose of VBRB was used. This is quite reasonable, while according to the
Chinese medicinal formulae records (Liu, Y. et al, 2004), as a meridian guided drug,
the dose of VBRB should be in the range of 3-6 g, which corresponded to the low
-1
-1
(
150 mg·kg ) and medium dose (300 mg·kg ) groups in this study. However, when
VBRB was used at a dose of 9-12 g, it turned into the monarch drug in a prescription,
-1
which corresponded to the high dose group (600 mg·kg ) in rats. This study implied
that when VBRB was used as a messenger drug, its dose plays an important role.
In addition to VBRB’s capacity to inhibit the CYP and GST activity in the liver,
VBRB was reported to be able to depress the efflux effect of p-glycoprotein, which
might also contribute to its liver targeting efficiency.
Conclusions
In this study, meridian guided drug theory was combined with modern passive
drug delivery system design to achieve better liver targeting. It was demonstrated that
VBRB could inhibit the activity of GST, and the inhibition ratio was VBRB extract
composition dependent, with BC1 presenting the strongest inhibitory effect. In vivo
pharmacokinetic and liver targeting evaluation demonstrated that HPCT concentration
in the liver increased significantly after co-administration of HPCT loaded polymeric
micelles with low and medium doses of BC1. Such a simple but effective strategy
may enlighten on the potential use of meridian guided drug with model drug delivery
system to achieve better active drug targeting.
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Acknowledgments
This research was financially supported by the National Natural Science
Foundation of China (Grant No. 81273446, 30672668, 81073063, 81573612), and the
Natural Science Foundation of Guangdong Province of China (Grant No.
2
015A030313357).
Conflict of interest
The authors report no declarations of interest.
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References
Calatozzolo, C., Gelati, M., Ciusani, E., Sciacca, F.L., Pollo, B., Cajola, L., Marras, C., Silvani, A.,
Vitellaro-Zuccarello, L. & Croci, D., 2005. Expression of Drug Resistance Proteins Pgp, MRP1,
MRP3, MRP5 AND GST- π in Human Glioma. Journal of Neuro-Oncology, 74, 113-21.
Chen, X., Yu, T., Chen, Z., Zhao, R. & Mao, S., 2014. Effect of saikosaponins and extracts of
vinegar-baked Bupleuri Radix on the activity of β-glucuronidase. Xenobiotica, 44, 785-91.
Deng, C., Jiang, Y., Cheng, R., Meng, F. & Zhong, Z., 2012. Biodegradable polymeric micelles for
targeted and controlled anticancer drug delivery: Promises, progress and prospects. Nano
Today, 7, 467–480.
Filomeni, G., Turella, P., Dupuis, M.L., Forini, O., Ciriolo, M.R., Cianfriglia, M., Pezzola, S., Federici, G. &
Caccuri, A.M., 2008. 6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, a specific glutathione
S-transferase inhibitor, overcomes the multidrug resistance (MDR)-associated protein
1-mediated MDR in small cell lung cancer. Molecular Cancer Therapeutics, 7, 371-379.
G, R., F, D.M., G, A., P, T., A, B., L, S., G, F., G, F. & Am, C., 2005. 7-Nitro-2,1,3-benzoxadiazole derivatives,
a new class of suicide inhibitors for glutathione S-transferases. Mechanism of action of
potential anticancer drugs. Journal of Biological Chemistry, 280, 26397-26405.
Gupta, P.K. & Hung, C.T., 1989. Quantitative evaluation of targeted drug delivery systems. International
Journal of Pharmaceutics, 56, 217-226.
Kawaguchi, Y., Honda, G., Endo, I., Cherqui, D. & Kokudo, N., 2017. Current Technical Issues for Surgery
of Primary Liver Cancer. Liver Cancer, 6, 51-58.
Ling, Y.H. & Xu, B., 1993. Inhibition of phosphorylation of histone H1 and H3 induced by
10-hydroxycamptothecin, DNA topoisomerase I inhibitor, in murine ascites hepatoma cells.
Zhongguo yao li xue bao = Acta pharmacologica Sinica, 14, 546.
Liu, Y., 2004. Application analysis of meridian guide drug in compound of Traditional Chinese Medicine.
Hubei Zhongyi Zazhi 26, 39–40.
Maeda, H., Wu, J., Sawa, T., Matsumura, Y. & Hori, K., 2000. Tumor vascular permeability and the EPR
effect in macromolecular therapeutics: a review. Journal of Controlled Release, 65, 271-284.
Pelkonen, O., Turpeinen, M., Hakkola, J., Honkakoski, P., Hukkanen, J. & Raunio, H., 2008. Inhibition
and induction of human cytochrome P450 enzymes: current status. Archives of Toxicology, 82,
667-715.
Petrick, J.L., Braunlin, M., Laversanne, M., Valery, P.C., Bray, F. & Mcglynn, K.A., 2016. International
trends in liver cancer incidence, overall and by histologic subtype, 1978–2007. International
Journal of Cancer, 139, 1534-1545.
Qian, Z.M., Li, H., Sun, H. & Ho, K., 2009. Targeted drug delivery via the transferrin receptor-mediated
endocytosis pathway. Journal of Chinese Pharmaceutical Sciences, 54, 561-587.
Ravindranath, V. & Strobel, H.W., 2013. Cytochrome P450-mediated metabolism in brain: functional
roles and their implications. Expert Opinion on Drug Metabolism & Toxicology, 9, 551-8.
Sudimack, J. & Lee, R.J., 2000. Targeted drug delivery via the folate receptor. Advanced Drug Delivery
Reviews, 41, 147.
Tian, Y. & Mao, S., 2012. Amphiphilic polymeric micelles as the nanocarrier for peroral delivery of
poorly soluble anticancer drugs. Expert Opinion on Drug Delivery, 9, 687.
16

ACCEPTED MANUSCRIPT
Tian, Y., Shi, C., Zhang, X., Sun, Y., Wang, J., Zhang, Y., Yang, J., Wang, L., Wang, L. & Mao, S., 2015.
Nanomicelle Based Peroral Delivery System for Enhanced Absorption and Sustained Release
of 10-Hydrocamptothecin. Journal of Biomedical Nanotechnology, 11, 262.
Wilhelm, S., Tavares, A.J., Dai, Q., Ohta, S., Audet, J., Dvorak, H.F. & Chan, W.C.W., 2016. Analysis of
nanoparticle delivery to tumours. Nature Reviews Materials, 1, 16014.
Win, K.Y. & Feng, S.S., 2005. Effects of particle size and surface coating on cellular uptake of polymeric
nanoparticles for oral delivery of anticancer drugs. Biomaterials, 26, 2713-2722.
Xu, B., Gao, S., Wu, B., Yin, T. & Hu, M., 2014. Absolute quantification of UGT1A1 in various tissues and
cell lines using isotope label-free UPLC-MS/MS method determines its turnover number and
correlates with its glucuronidation activities. Journal of Pharmaceutical & Biomedical Analysis,
88, 180-190.
Yamaguchi, M., Ueda, K., Isowaki, A., Ohtori, A., Takeuchi, H., Ohguro, N. & Tojo, K., 2009.
Mucoadhesive properties of chitosan-coated ophthalmic lipid emulsion containing
indomethacin in tear fluid. Biological & Pharmaceutical Bulletin, 32, 1266-1271.
Ye, T., Shi, C., Sun, Y., Zhu, C., Sun, C.C. & Mao, S., 2015. Designing Micellar Nanocarriers with
Improved Drug Loading and Stability Based on Solubility Parameter. Molecular Pharmaceutics,
12, 816.
Yu, T., Chen, X., Wang, Y., Zhao, R. & Mao, S., 2014. Modulatory effects of extracts of vinegar-baked
Radix Bupleuri and saikosaponins on the activity of cytochrome P450 enzymes in vitro.
Xenobiotica, 44, 861-7.
Zhao, R., 2014. Vinegar-baked Radix Bupleuri modulates the cell membrane constituents and inhibits
the P-gp activity in rat hepatocytes. BMC Complementary and Alternative Medicine, 14, 357.
Zhao, R.Z., Chen, Y.J. & Cai, J.X., Year. Liver targeting effect of vinegar-baked Radix Bupleuri on
oxymatrine in miceed.^eds. IEEE International Conference on Bioinformatics and Biomedicine
Workshops, 740-745.
Zhao, R.Z., Liu, S.J., Mao, S.R. & Wang, Y.J., 2009. Study on liver targeting effect of vinegar-baked Radix
Bupleuri on resveratrol in mice. Journal of Ethnopharmacology, 126, 415-420.
Zhao, R.Z., Yuan, D., Liu, S.J., Chen, Y.J., Liu, L.J. & Zhao, Y., 2010. Liver targeting effect of vinegar-baked
Radix Bupleuri on rhein in rats. Journal of Ethnopharmacology, 132, 421-8.
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Figure legends
Fig.1 The reaction mechanism of GST activity assay.
Fig.2 (a) Effect of CDNB concentration on the activity of GST (n = 3).
(b) Effect of GSH concentration on the activity of GST (n = 3).
(c) Effect of reaction temperature on the activity of GST (n = 3).
(d) Effect of GST concentration on the activity of GST(n = 6).
Fig.3 (a) Effect of extract BC4 concentration on the activity of GST (n = 3);
b) Effect of different extracts of VBRB on the activity of GST (n = 3).
(
Fig.4 Concentration-time profiles after oral administration of HCPT loaded micelles
with three different doses of BC1 in plasma. (Control: HCPT loaded micelles
-1
only; lose dose: micelles + BC1 150 mg·kg ; middle dose: micelles+ BC1
-1
-1
3
00 mg·kg ; high dose:micelles + BC1 600 mg·kg .)
Fig.5 HCPT concentration in the liver at selected time points after oral administration
of HCPT micelles with three different doses of BC1. (*P < 0.05 compared to
the control group. Control: HCPT loaded micelles only; lose dose: micelles +
-1
-1
BC1 150 mg·kg ; middle dose: micelles+ BC1 300 mg·kg ; high dose:micelles
-1
+
BC1 600 mg·kg .)
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Table 1 Composition of VBRB extracts used in this study (Yu, 2014)
No. Extra
cts
Ratio in
the raw
material
Content of various
saikosaponins in the test solution
( corresponding to raw material
0.1142g/ml) (μg/ml)
Total content
of
saikosaponins
(μg/ml)
(%)
a*
b
c
7.87
/
d
18.36
/
1
2
3
4
5
BC0
BC1
BC2
BC3
BC4
14.9
4.5
20.60 30.82
77.65
/
/
/
0.57
2.07
7.05
39.92 31.25 30.20
27.73
128.50
418.65
/
105.90 186.75 24.11 101.89
/
/
/
/
*0.0163g/ml of raw medicinal herbs per milliliter for BC0
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Table 2 Pharmacokinetic parameters and targeting indexes after oral administration of
HCPT micelles with three different doses of BC1 in plasma and in the liver tissue
(mean ± SD, n = 5 for plasma and n = 4 for the liver)
Parameter
control
0.22 ± 0.03
0.26 ± 0.02
1 ± 0.35
8 ± 0
low dose
0.22 ± 0.01
0.31 ± 0.02*
0.7 ± 0.11
8 ± 0
medium dose
0.26 ± 0.04
0.29 ± 0.02
0.7 ± 0.21
8 ± 0
high dose
0.23 ± 0.02
0.24 ± 0.01
0.8 ± 0.21
8 ± 0
Plasma
Liver
-
1
Cmax (μg·g )
Plasma
Liver
Tmax (h)
Plasma
Liver
8.86 ± 1.58
7.82 ± 0.83
--
7.08 ± 0.56*
11.14 ± 1.16*
1.20 ± 0.17
1.45 ± 0.32
8.02 ± 1.02
9.62 ± 1.07*
1.14 ± 0.13
1.23 ± 0.10
8.67 ± 0.07
7.89 ± 0.08
0.92 ± 0.05
1.02 ± 0.11
-
1
AUC (μg·h·g )
0
-t
Ce
RUE
--
*p < 0.05
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Fig. 1
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Fig.2
(
a)
(b)
(c)
(d)
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Fig.3
(a)
(b)
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Fig.4
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Fig.5
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Graphical abstract
26