International Journal of Pharmaceutics 436 (2012) 311–317

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International Journal of Pharmaceutics

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Biopharmaceutics classification and intestinal absorption study of apigenin

Jianjun Zhang a , Dapeng Liu a , Yanting Huang a , Yuan Gao b,∗ , Shuai Qian c,∗∗
a
School of Pharmacy, China Pharmaceutical University, Nanjing 210009, PR China
b
School of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 210009, PR China
c
School of Pharmacy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China

a r t i c l e i n f o a b s t r a c t

Article history: The aim of the study was to characterize the biopharmaceutics classification system (BCS) category of
Received 21 March 2012 apigenin (AP) using intrinsic dissolution rate (IDR) and rat intestinal permeability, and to investigate the
Received in revised form 30 May 2012 intestinal absorption mechanism of AP in rats. In the present investigation, equilibrium solubility and
Accepted 2 July 2012
intrinsic dissolution rate (IDR) of AP were estimated in phosphate buffers. Effective intestinal permeability
Available online 13 July 2012
(Peff ) of AP was determined using single-pass intestinal perfusion (SPIP) technique in four segments
(duodenum, jejunum, ileum and colon) of rat intestine at three concentrations (10, 50 and 100 ␮g/ml).
Keywords:
The aqueous solubility of AP in tested phosphate buffers was very poor with maximum solubility of
Apigenin
Biopharmaceutics classification system
2.16 ␮g/ml at pH 7.5. The IDR of AP was very low with a value of 0.006 mg/min/cm2 . The minimum and
Intrinsic dissolution rate maximum Peff s determined by SPIP were 0.198 × 10−4 and 0.713 × 10−4 cm/s at jejunum and duodenum
Permeability site, respectively. In addition, the concentration-dependent permeability behavior was observed in the
Single-pass intestinal perfusion duodenum and jejunum, which suggested that AP was transported by both passive and active carrier-
mediated saturable mechanism in these two intestinal segments. However, the observed concentration-
independent permeability behavior in ileum and colon indicated primarily passive transport mechanism
of absorption of AP in the last two intestinal segments. AP was classified as class II drug of the BCS due
to its low solubility and high intestinal permeability. AP could be well absorbed in the whole intestine
with the main absorption site at duodenum. The absorption of AP in four intestinal segments exhibited
different transport mechanisms.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction been included in various guidance documents of regulatory impor-

Since the biopharmaceutics classification system (BCS) for cor-
relating in vitro dissolution and in vivo bioavailability was proposed
in 1990s based on drug dissolution and gastrointestinal perme-
ability, which were two fundamental parameters controlling the
rate and extent of drug absorption, it has had an increasing impact
on regulatory practice (Amidon et al., 1995). According to the BCS,
drug substances are classified as follows: high solubility–high per-
meability drugs (Class I), low solubility–high permeability drugs
(Class II), high solubility–low permeability drugs (Class III) and low
solubility–low permeability drugs (Class IV). Each class of the BCS
has its designated rate-limiting step and the reasonable strategies
for its modification that enable the formulator to select and opti-
mize a dosage form for the specified drug (Cook et al., 2008; Ku,
2008). The BCS also provides a regulatory tool for the replacement
of certain expensive and time-consuming bioequivalence studies
by conducting accurate in vitro dissolution tests, and it has also
∗ Corresponding author. Tel.: +86 25 83379418; fax: +86 25 83379418.
∗∗ Corresponding author. Tel.: +852 51387572.
E-mail addresses: amicute@163.com (Y. Gao), sqian@cuhk.edu.hk (S. Qian).
tance (FDA, 2000; WHO, 2005).
According to the FDA guidance for biowaiver request of
immediate-release solid oral dosage forms, a drug substance is con-
sidered as highly soluble when its highest dose strength could be
dissolved in ≤250 ml of aqueous media over the pH range of 1.0–7.5
(FDA, 2000). However, the choice of dose has a great impact on
the calculation of the dose/solubility ratio and, for some drugs, the
classification may shift from ‘highly’ soluble to ‘not highly’ sol-
uble when using different dose in calculation (Lindenberg et al.,
2004; Yu et al., 2004). In addition, solubility, as a static equilibrium
parameter, cannot adequately describe the dynamic behavior of the
dissolution process especially in the physiological condition (Rinaki
et al., 2003). Recently, dissolution-based classifications including
dissolution index (DI) (Papadopoulou et al., 2008) and intrinsic
dissolution ratio (IDR) (Yu, 2004; Zakeri-Milani, 2009) have been
developed for new molecular entities and marketed drugs, instead
of dose/solubility ratio based classification. Mean intestinal transit
time and mean time calculations for drug dissolution are included
in the dissolution parameter DI, which takes account the physiolog-
ical conditions (Papadopoulou et al., 2008). IDR is generally defined
as the dissolution rate of a pure drug substance under the condition
of constant surface area, agitation or stirring speed, pH and ionic

0378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijpharm.2012.07.002
312 J. Zhang et al. / International Journal of Pharmaceutics 436 (2012) 311–317
Fig. 1. Chemical structure of AP.
strength of the dissolution medium. The true IDR may be better
described as the rate of mass transfer from the solid surface to the
liquid phase. Since IDR is a rate phenomenon like permeability, it
might correlate better with in vivo drug dissolution rate than the
equilibrium solubility (Yu et al., 2004).
To determining drug permeability in gastrointestinal tract, lots
of in vivo, in vitro and in situ experimental models have been
developed (Hillgren et al., 1995; Egan and Lauri, 2002; Refsgaard
et al., 2005; Varma and Panchagnula, 2005). Among these models,
the single-pass intestinal perfusion (SPIP) method provides in situ
experimental conditions simulating oral administration to investi-
gate the absorptive potential and mechanism of absorption of the
drug, where the innervations and clearance capabilities of the ani-
mal remain intact. In addition, SPIP offers a simple and relevant
method of permeability assessment and correlates significantly
with the true absorption properties in human beings (Amidon et al.,
1988; Logan, 2009).
Apigenin (AP), 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-ben-
zopyran-4-one (Fig. 1), is one of the active ingredients found
naturally in a variety of fruits and vegetables (Peterson and
Dwyer, 1998). It is recognized in traditional medicine and herbal
supplements for its various pharmacological activities, including
anti-inflammatory effects (Funakoshi-Tago et al., 2011), free radi-
cal scavenging effects (Horvathova et al., 2003), growth inhibitory
properties in several cancer lines including breast (Yin et al., 2001),
colon (Wang et al., 2000), skin (Tong et al., 2007), myeloid leukemia
cells (Takahashi et al., 1998) and pancreas (Ujiki et al., 2006).
Moreover, recent clinical studies demonstrated that AP intake was
associated with a suggestive decrease in woman’s risk of ovarian
cancer (Gates et al., 2007, 2009), which indicated AP could be a very
promising drug candidate for future drug development. However,
both of the basic physicochemical and biopharmaceutical proper-
ties of AP were still limited from the published literatures. There is
a need to classify the AP into BCS in order to facilitate the future for-
mulation development according to reasonable strategies of each
BCS class drugs. In the present study, solubility, IDR property and
permeability of AP were investigated to classify AP into a particular
BCS class, and the intestinal absorption mechanism of AP was also
studied.
2. Materials and methods
2.1. Materials
Apigenin (purity > 99.0% by HPLC) was provided by Nanjing
Zelang Medical Technology Co., Ltd. (Nanjing, China). Tween 80 and
pentobarbital sodium were purchased from Guoyao Chemical Co.,
Ltd. (Shanghai, China). HPLC grade water was produced by a Milli-Q
water purification system (Millipore Co., Ltd., Bedford, USA). Other
chemicals were of HPLC or analytical grade.
2.2. Solubility determination
The equilibrium solubilities of AP were determined in
0.1 mol/ml phosphate buffers over pH value ranging from 1.0 to
7.5 (pH value was adjusted by H3 PO4 (HCl for pH 1) or NaOH) (Wu
et al., 2005). In brief, an excess amount of AP was added into 10 ml
of buffer solution in a vial placed in a 37 ◦ C water bath. Following
equilibrium with shaking for 3 days, samples were filtered through
0.22 ␮m PTFE filter prior to analysis by the validated HPLC/UV
method. In each buffer, the solubilities of AP were analyzed in
triplicates.
2.3. Procedure of IDR measurement
An improved method for IDR measurement was employed
to perform the study (Wood et al., 1965). Briefly, a quantity of
150 mg of AP was compressed with a compression force of 1.20 MPa
for 1 min to make non-disintegrating compacts using die and
punch (diameter: 8 mm). The surface area of the compacts was
0.5024 cm2 . Compacts were placed in a molten beeswax-mold in
such a way that only one face could be in contact with dissolution
medium.
USP II dissolution apparatus (Sotax A7 Dissolution Apparatus:
Sotax Ltd., London, UK) was applied in the in vitro dissolution
study. Dissolution tests (six replicates) were performed in 900 ml of
0.05 mol/l phosphate buffer (pH = 6.8) at 37 ◦ C. The paddle rotating
speed was set at 100 rpm. Samples were collected through 0.22 ␮m
syringe filters every 30 min for a period of 8 h. The obtained samples
were analyzed by the validated HPLC/UV method.
2.4. Perfusion study using rat single-pass intestinal perfusion
model
2.4.1. Animals and surgical procedures
The study was approved by the Ethical Committee of China
Pharmaceutical University. Male Sprague-Dawley rats weighting
between 230 and 270 g were obtained from the Laboratory Animal
Center, China Pharmaceutical University (Nanjing, China). Animals
were housed in standard cages on 12 h light–dark cycles, fed with
standard animal chow and tap water daily. Prior to the experi-
ments, the animals were subjected to fasting for 12 h but allowed
free access to water. All animals used in this study were handled
in accordance with the guidelines of the Principles of Laboratory
Animal Care (State Council, revised 1988).
SPIP studies were performed according to established meth-
ods as previously described (Zakeri-Milani et al., 2009; Svensson
et al., 1999). In brief, the rats were anaesthetized by pentobar-
bital sodium (i.p., 50 mg/kg) and placed on a heated pad to keep
body temperature. After making a 3–4 cm midline abdominal inci-
sion, the small intestinal was exposed and then duodenal, jejunum,
ileum and colon were identified as follows: duodenum segment
beginning from pylorus, jejunum segment beginning from 25 cm
away from pylorus, while ileum segment the beginning at the site
20 cm upwards caecum, colon segment beginning from caecum
empennage. The intestinal segment of interest was located and
cannulated with silicon tubing connected to a peristaltic pump
(infusion pump, BT100-2G, Longer Pump, China). The outlet tubing
was placed 10–12 cm aboral to the opening. The segment was then
rinsed with warm saline to remove intestinal contents and approx-
imately 10 cm of the inlet tubing was placed inside the abdominal
cavity to achieve an inlet perfusion solution at 37 ◦ C. The whole
surgical area was covered with a piece of sterilized gauze wetted
with saline solution. The exposed intestinal segment was covered
with Parafilm to decrease moisture loss.
The experiment was initiated by rapidly filling the segment with
perfusion solution and the time was set zero with the immediate
start of the perfusion. The perfusion rate was set at 0.2 ml/min.
Care was taken to handle the small intestine gently and to mini-
mize the surgery in order to maintain an intact blood supply. After
approximately 30 min, when steady-state was achieved, the outlet
 J. Zhang et al. / International Journal of Pharmaceutics 436 (2012) 311–317
perfused samples were collected at 15 min intervals up to 120 min.
The perfusate was collected every 15 min into microtube and cen-
trifugated at 10000 rpm for 10 min. Samples were stored at −20 ◦ C
until analysis. The length of tested segment was measured at the
end of the perfusion.
2.4.2. Perfusion study of AP
The blank perfusion buffer solution was isotonic and composed
of 133 mM NaCl, 4.56 mM KCl, 16 mM NaHCO3 , 1.50 mM NaH2 PO4
and 7.78 mM d-glucose with pH 7.0–7.2 at 37 ◦ C.
Due to the poor solubility of AP in perfusion buffer, Tween 80
was employed to increase the solubility of AP. The perfusate with
100 ␮g/ml of AP containing different concentrations of Tween 80
(0.5, 1 and 1.5%, w/v) was applied to four intestinal segments to
investigate the effect of Tween 80’s concentration as well as dif-
ferent intestinal site on intestinal absorption of AP. To examine
whether intestinal absorption of AP could exhibit concentration-
dependent or concentration-independent characteristics, 10 and
50 ␮g/ml of AP containing 0.5% of Tween 80 were also studied in
all four intestinal segments using the same rat perfusion model.
2.5. Determination of AP by HPLC/UV
HPLC/UV method was employed for determination of AP in
phosphate buffer solution and perfusate on Shimadzu LC-2010AHT
HPLC system. AP was separated by an Ultimate C18 column
(150 mm × 4.6 mm, 5 ␮m) guarded with an Ultimate precolumn
and detected at 337 nm. Mobile phase consisting of methanol and
0.1 mol/l sodium acetate (25/75, v/v) was run at 1.0 ml/min. The
column temperature was set at 30 ◦ C.
AP was separated well from endogenous materials in
intestinal segments. Within the linear concentration range
(1.0–120.0 ␮g/ml), good linearity (r2 > 0.9995) was achieved. The
limit of quantification and limit of detection were 10 and 3.3 ng/ml,
respectively. The relative standard deviation (% RSD) of intra-day
and inter-day precision of the assay method for AP was below 5%,
and the accuracy was within the range of 95–105%.
2.6. Data analysis
2.6.1. Calculation of IDR
To evaluate IDR of AP, the cumulative amount dissolved per sur-
face unit of the compact was plotted against time. The slope of
the linear region (r2 ≥ 0.95) was taken as the intrinsic dissolution
rate. IDR was calculated by the following equation as described
previously (Zakeri-Milani et al., 2009).
 dw   1  DCs
IDR = = was quite low with the maximum solubility of 2.16 ␮g/ml observed
dt S h
where IDR is the intrinsic dissolution rate (mg/min/cm2 ); dw is the
change in drug dissolved (mg); dt is the change in time (min); S
is the surface area of the compact (cm2 ); D is diffusion coefficient
(cm2 /s); Cs is the solubility (mg/cm3 ) and h is the stagnant layer
thickness (cm).
2.6.2. Calculation of effective permeability coefficient (Peff )
Peff was the quantitative estimate of the rate of passage of a
solute across a membrane. It was calculated from the steady-state
concentrations of AP in the perfusate (Cook and Shenoy, 2003).
Because the drug concentrations obtained from the perfusate sam-
ples were corrected for changes in the water flux during each time
interval, density corrected gravimetric method was utilized to cal-
culate the net water flux across the incubated intestinal segment.
The density of collected samples was determined by weighing the
contents using an electronic weighing balance of a known vol-
ume of perfusate. Ccor , the drug concentration of effluent perfusates
313
Table 1
Solubility of apigenin in phosphate buffers (n = 3).
pH of buffers Solubility (␮g/ml)
1.0 1.43 ± 0.03
2.5 1.01 ± 0.02
5.0 1.28 ± 0.04
5.8 1.36 ± 0.03
6.8 1.56 ± 0.02
7.0 1.63 ± 0.05
7.5 2.16 ± 0.02
which was corrected for water flux, was calculated as follows (Li
et al., 2011):
Qout
Ccor = Cout ×
Qin
where Cout is the concentration of tested drug in the effluent per-
fusates, Qin and Qout are the inlet and outlet flow rate (ml/min),
respectively, which are adjusted for liquid density (ml/min).
Steady state was reached about 30 min after the beginning of
the perfusion which was confirmed by plotting the ratio of the out-
let to inlet concentrations (corrected for water transport) versus
time. Calculations of permeability across rat intestinal segment
were performed from intestinal perfusate samples collected over
45–120 min (steady state) using the following equation (Li et al.,
2011):
−Qin ln (Cout(cor) /Cin )
Peff =
2rl
where Peff is the effective permeability coefficient, Cin is the input
perfusate drug concentration, r is the effective lumen radius (cm),
and l is the length of intestinal segment, 2rl is the area of the
mass transfer surface (cm2 ) within the intestinal segment which is
assumed to be a cylinder area.
2.7. Statistical analysis
Data were analyzed by two-way ANOVA. Significant difference
between two groups was detected by using LSD test. A probability
level of p < 0.05 was set as the criterion of significance.
3. Results and discussion
3.1. Solubility and IDR of AP
The equilibrated solubilities of AP in buffers are presented in
Table 1. The aqueous solubility of AP over the pH range of 1.0–7.5
at pH 7.5. AP is a typical flavonoid with ortho-phenolic dihydroxyl
group at ring A and para-phenolic hydroxyl group at ring B of
the flavonoid backbone. The log P value, pKa for first deprotona-
tion and water solubility for AP has previously been determined
as 2.87 (Li et al., 1997), 6.6 (Favaro et al., 2007) and 1.35 ␮g/ml
(Li et al., 1997), respectively, which indicated AP was lipophilic
weak acid and could be ionized at high pH condition. According
to Henderson–Hasselbach equation (Wu et al., 2005), AP mainly
presents as hydrophobic molecular form in the tested phosphate
buffer solutions with pH range of gastrointestinal tract.
During dissolution test, the concentration of AP was gradually
close to saturate and dissolution rate began to decline at 5 h, which
may underestimate the IDR. Therefore, the initial 5 hours’ dissolu-
tion was taken to estimate the IDR (Fig. 2). IDR of AP was calculated
to be 0.006 mg/min/cm2 . Recently, two studies performed inde-
pendently by Zakeri-Milani et al. (2009) and Yu et al. (2004) on
determining solubility class membership demonstrated good rela-
tionships between IDR and BCS solubility classification. However,
314
2.4
The cumulative amount dissolved
2
Y=0.006X+0.453 R =0.996
2.1
per surface(mg/cm )
2
1.8
1.5
1.2
0.9
0.6
0 30 60 90 120 150 180 210 240 270 300 330
T(min)
Fig. 2. Intrinsic dissolution rate (IDR) of AP in 0.05 mol/l phosphate buffer (pH = 6.8).
under the same testing conditions (900 ml of pH 6.8 phosphate
buffer, 100 rpm of rotation speed), almost all of the IDR values
obtained by Zakeri-Milani et al. were higher than that obtained by
Yu et al. for the same drugs. Such phenomenon could be attributed
to the different intrinsic dissolution apparatuses and kits used in the
two investigations. In case of the stationary disk system employed
by Zakeri-Milani et al., a fixed body of solvent was stirred over the
pellet by paddle (USP II method). On the other hand, in case of
the rotation disk system employed by Yu et al., the pellet under-
went a shear-like motion over a planar solvent front, which was
similar to USP Procedure I (Viegas et al., 2001). Different disk sys-
tems caused different hydrodynamic conditions including fluid
velocity and shear rates. In general, USP II generates high fluid
velocity resulting in higher dissolution of tablet compared to USP I
(D’Arcy et al., 2006). Such different stirring situations determined
the higher IDR values obtained by Zakeri-Milani et al. than that
by Yu et al. for the same drugs. Zakeri-Milani et al. and Yu et al.
suggested a borderline value of 1 mg/min/cm2 and 0.1 mg/min/cm2
for determining solubility class membership, respectively. Since we
followed the experimental procedures of Zakeri-Milani et al. (2009)
and the IDR value of AP was much lower than the borderline, AP
could be classified as the compound with low solubility.
3.2. The effect of concentration of Tween 80 on intestinal
permeability of AP
Since the solubility of AP in buffers (pH 1–7.5) was quite low,
Tween 80 was employed to increase its aqueous solubility. Pre-
liminary experiments showed no considerable adsorption of the
compound on the tubing and syringe. The effect of concentration
of Tween 80 on the intestinal permeability of AP is described in
Fig. 3. Increasing concentration of Tween 80 from 0.5 to 1.5% was
found to result in no significant change in epithelial transport of AP
in each intestinal segment (p > 0.05). The dose-independent effect
of Tween 80 on permeability of each intestinal segment found here
was consistent to the previous in situ rat perfusion study on ganci-
clovir (Li et al., 2011) as well as in vitro Caco-2 transport study on
fexofenadine (Lin et al., 2007). The concentration of Tween 80 was
then set at 0.5% in the perfusate in all other SPIP experiments.
Since the presence of surfactants may either increase or decrease
the permeability of compound depending on the transporters and
metabolic enzymes involved as well as the integrity of transport
epithelium, it is not possible to estimate the true permeability of
compound (in absence of surfactants) based upon single concentra-
tion of surfactant used in perfusate. Katneni et al. (2006) developed
a reciprocal permeability approach for assessment of the true per-
meability by conducting permeability experiments under three
J. Zhang et al. / International Journal of Pharmaceutics 436 (2012) 311–317
Fig. 3. Effects of Tween 80 on the permeability of AP in four intestinal segments.
separate surfactant concentrations, without the need to conduct
separate free drug concentration determinations. The relationship
between the measured permeability (Papp,uncorr ) in presence of sur-
factant, the true permeability (Papp,corr ) in absence of surfactant,
micellar association constant (Ka) and the surfactant concentration
(Smicellar ) could be described as the following equation:
 
1 Ka 1
= Smicellar × +
Papp,uncorr Papp,corr Papp,corr
A plot of 1/Papp,uncorr versus Smicellar allows estimation of the true
permeability from the Y-intercept (1/Papp,corr ).
Since Tween 80 showed dose-independent effect on permeabil-
ity of AP in each intestinal segment, the slope of above equation
would be close to zero and the true permeability of AP (Papp,corr )
was close to the average Papp,uncorr of AP at three different Tween
80 concentrations.
Historically, surfactants have been utilized to enhance solubility
and reduce nonspecific adsorption to the wall of the diffusion
apparatus during the in vitro and in situ permeability estimations
of lipophilic compounds (Takahashi et al., 2002; Chen et al., 2010;
Imai et al., 1983; Katneni et al., 2006). However, mechanistic stud-
ies have demonstrated that surfactants may have ability to alter
membrane integrity (Anderberg and Artursson, 1993), and inhibit
some transporters (Cornaire et al., 2004; Hugger et al., 2002; Shono
et al., 2004) as well as metabolic enzymes (Christiansen et al.,
2011). Although high permeable compounds are easy to across the
intestinal epithelium, the low solubility of BCS class II compounds
will limit the concentrations coming into the cells and prevent the
saturation of the efflux transporters, which will affect the rate of
absorption (Wu and Benet, 2005). Comparing with ionic surfac-
tants (e.g. sodium dodecyl sulfate, sodium taurocholate), Tween 80
was an ineffective enhancer for some polar drugs (phenol red and
fexofenadine·HCl) due to its ignored local wall damage in intestine
(Lin et al., 2007; Katneni et al., 2006; Swenson et al., 1994); while
comparing to non-ionic surfactant Cremophor-EL, Tween 80 is not
a potent inhibitor of P-gp and incapable of increasing absorptive
transport of digoxin, a typical P-gp substrate (Katneni et al.,
2007). In the way of inhibiting Phase I metabolic enzymes, in vitro
study demonstrated that Tween 80 had inhibitory potential on
cytochrome P450 (CYP) 3A4-mediated metabolism of testosterone
and the CYP2C9-mediated metabolism of diclofenac with IC50 of
0.40 mM and 0.04 mM, respectively (Christiansen et al., 2011). In
addition, Mudra et al. investigated the effects of polyethoxylated
 J. Zhang et al. / International Journal of Pharmaceutics 436 (2012) 311–317
Table 2
Effective permeabilities of AP in four intestinal segments using single-pass intestinal perfusion.
Perfusate Peff (10−4 cm/s)
Duodenum
AP 10 ␮g/ml 0.645 ± 0.054*
AP 50 ␮g/ml 0.713 ± 0.063*
AP 100 ␮g/ml 0.502 ± 0.047*
The data were presented as mean ± SD (n = 6 for each group). N.S., not significantly different to the group of 10 ␮g/ml AP in the same intestinal segment.
*
p < 0.05, compared to the other two concentrations of AP in the same intestinal segment.

p < 0.05, compared to the group of duodenum at the same concentrations of AP.
solubilizing agents on drug permeation and metabolism using
in situ rat intestinal perfusion model and found Tween 80 imposed
a great inhibitory effect on the N-demethylation of verapamil
catalyzed by CYP3A. However, the permeability of parent com-
pound verapamil in presence of Tween 80 was slightly decreased
compared to the control group (in absence of Tween 80) (Mudra
and Borchardt, 2010). For inhibitory of Phase II metabolic enzymes,
Tween 80 showed mixed-competition type of inhibition on glu-
curonidation of raloxifene in human liver microsome incubation
(Kim et al., 2009). To the best of our knowledge, in situ and in vivo
evidence of the effects of surfactants on Phase II metabolism was
still very limited.
3.3. The effect of intestinal site and AP concentration on intestinal
permeability
Effective permeabilities of AP in four intestinal segments at
three concentrations are presented in Table 2. Intestinal site depen-
dent absorption of AP could be found between duodenum, jejunum
and ileum/colon at each tested AP concentration. The effective per-
meabilities at 10 and 50 ␮g/ml of AP shared the same rank order of
duodenum > jejunum > ileum ≈ colon, while exhibited another rank
order of duodenum > ileum ≈ colon > jejunum at 100 ␮g/ml of AP.
The minimum and maximum Peff in rat intestinal segments were
determined to be 0.198 × 10−4 (jejunum) and 0.713 × 10−4 cm/s
(duodenum). The permeability coefficients of AP obtained from
SPIP study were comparable to the reported values of propranolol
(0.30–0.75 × 10−4 cm/s) (Nagare et al., 2010; Brouwers et al., 2010;
Varma et al., 2004), which was often used as a reference compound
of passive absorption via transcellular pathway. In addition, Peff s of
AP in the duodenum were significant greater than the other three
intestinal segments at three AP concentration (p < 0.05), which indi-
cated that AP could be well absorbed in the whole intestine with
the main absorption site at duodenum.
Furthermore, there was no statistical difference of Peff s in both
ileum and colon segments at each AP concentration, suggesting the
transport mechanism of AP in such two intestinal segments might
be primarily passive transport. However, significant reductions of
Peff s were observed in the duodenal and jejunum segments (1.4 and
2.0-fold decrease, respectively), when increasing concentration of
AP from 50 to 100 ␮g/ml. Such concentration-dependent behavior
of permeability suggested that AP could be primarily passive trans-
port and transcelluar mechanism involving a saturable process, and
potentially via a membrane transport protein in the duodenum and
jejunum (Cook and Shenoy, 2003).
In vitro bidirectional transport study of AP on Caco-2 cell mono-
layer have been performed by Liu and Hu (2002). Like other
flavonoids (i.e. baicalein (Zhang et al., 2007), wogonin and oroxylin
A (Li et al., 2012; Dai et al., 2008)) and isoflavonoids (i.e. genistin
(Liu and Hu, 2002)), the absorptive transport of AP was similar to
its secretory transport with an efflux ratio approaching to 1, indi-
cating a passive transport of AP. The determined absorptive Papp
of AP was ∼4.5 × 10−5 cm/s on Caco-2 cell monolayer (Liu and Hu,
2002), which suggested an efficient permeability for AP (Volpe et al.,
2007).
315
Jejunum Ileum Colon
0.377 ± 0.023 0.302 ± 0.042 0.284 ± 0.033
0.396 ± 0.04N.S,  0.302 ± 0.02N.S,  0.287 ± 0.02N.S, 
0.198 ± 0.022* ,  0.298 ± 0.02N.S,  0.286 ± 0.03N.S, 
Fagerholm et al. first investigated the relationship between per-
meability coefficients in rats and human intestinal, and found that
the estimated Peff s of passively absorbed compounds in rat jejunum
were highly correlated with that in human jejunum (Fagerholm
et al., 1996). Later, Salphati et al. (2001) and Zakeri-Milani et al.
(2007) also confirmed that there was indeed a correlation of effec-
tive permeability coefficients between rat and human intestinal.
Fagerholm et al. (1996) also predicted the fraction of dose absorbed
in human based on the effective permeabilities in human (cal-
culated from the permeability determined in rat jejunum), when
given as a solution or immediate-release dosage form. However, the
fraction of dose absorbed in human for AP could not be predicted
from the rat permeability data, since AP has solubility limitation.
In comparison to solubility classification, permeability classi-
fication (especially for the criteria of highly permeability) seems
more difficult, since intestinal permeability is not routinely mea-
sured as permeability is based on an absorption measure in humans
(Wu and Benet, 2005; Benet et al., 2008). Based on drug metabolism,
Wu and Benet (2005) proposed a new system named “Biopharma-
ceutics Drug Disposition Classification System (BDDCS)” to provide
a simple surrogate for permeability. Benet et al. (2008) suggested
that if the extent of drug metabolism (i.e. ≥90%), the drug may be
substituted for 90% absorbed. In other words, if the major route of
drug elimination was metabolism, the drug should be considered
as high permeable drug; while the drug would be classified as low
permeable drug, when the renal and biliary excretion of unchanged
drug was the major route of drug elimination. Under BDDCS criteria,
the classifications of some drugs changed (i.e. BCS class II talinolol
would be BDDCS class IV, since talinolol was poorly metabolized).
Hu et al. (2003) investigated the mechanisms responsible for
intestinal disposition of AP in Caco-2 cell culture. During transport
of AP across the Caco-2 cell monolayer, glucuronidated AP and AP
sulfate were biotransformed in the cells, and were subsequently
pumped back to apical side mainly by multidrug resistance-related
proteins (MRPs) and possibly estrone sulfate-sensitive organic
anion transporters (OATs). Such enteric recycling combining with
enterohepatic recycling led a long-time occurrence of AP in rat
blood after oral administration of AP (Gradolatto et al., 2005;
Chen et al., 2003). However, similar to many other flavonoids
as mentioned above, AP was also poor bioavailable due to the
extensive first-pass metabolism related to glucuronidation and
sulfation (Chen et al., 2008; Ross and Kasum, 2002). Besides the
main metabolic organ liver, intestine may be another potential
organ responsible for the extensive Phase II metabolism of AP
(Liu and Hu, 2002; Zhang et al., 2005). Therefore, alternative
route of drug administration would be an effective approach to
enhance the bioavailability of AP by avoiding extensive presystemic
metabolism.
4. Conclusion
In the present study, AP was categorized as BCS class II drug due
to its low solubility and high permeability. The intestinal trans-
port study demonstrated that AP could be absorbed in the whole
316 J. Zhang et al. / International Journal of Pharmaceutics 436 (2012) 311–317
intestine with the main absorption site at duodenum. In addition,
the in vivo intestinal absorption mechanism of AP might primar-
ily be passive transport in both ileum and colon segments with
concentration-independent permeability behavior, while might
involve both passive and active carrier-mediated transport in both
duodenum and jejunum segments with concentration-dependent
permeability behavior.
Acknowledgements
This research was funded by the Important National Science &
Technology Specific Projects (No. 2011ZX09201-101-02), Peak of
six major talents in Jiangsu Province (2010, level A) and Funda-
mental Research Funds for the Central Universities (Program No.
JKP2011015).
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