European Journal of Pharmaceutical Sciences 176 (2022) 106255

Contents lists available at ScienceDirect

European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier.com/locate/ejps

Comparision of the phenol red, gravimetric, and synthesized mPEG-PR

methods for correcting water flux using the single-pass intestinal
perfusion method
Zhixiang Liu a, 1, Tong An a, 1, Rui Yuan a, Maoxiong Tian a, Linlin Yuan a, Tong Zhang a,
Gang Cheng b, *
a
College of Biological Science and Technology, Shenyang Agricultural University, Shenyang 110866, PR China
b
College of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Phenol red and PEG-4000, the usual non-absorbable indicators, have non-negligible absorption problems in
absorption measuring water flux. mPEG-PR, combined phenol red with mPEG-4000, was first synthesized and could
in situ single-pass intestinal perfusion decrease absorption. However, its application has not been confirmed. The purpose of this study was to explore
mPEG-PR
the applicability of mPEG-PR as a novel non-absorption indicator in the in situ single-pass intestinal perfusion
permeability
(SPIP) experiment. Six model drugs (atenolol ranitidine, ibuprofen, ketoprofen, antipyrine, hydrochlorothiazide)
water flux
were used to compare the accuracy of four measuring methods including phenol red, mPEG-PR, gravimetric, and
non-corrected methods of correcting intestinal fluid transport. Moreover, we evaluated the correlations between
the effective permeability coefficients (Peff) in rat and fraction dose absorbed (Fabs) in human, Peff in human, and
apparent permeability coefficients (Papp) by the Ussing Chamber system using human tissue. Among these
methods, mPEG-PR was the most reliable approach, which avoided the absorption of phenol red method and
mucous shedding or water evaporation of gravimetric method. An excellent correlation was obtained between
the Peff of rat and Fabs of human. Our results of this study indicated that mPEG-PR was a stable and accurate non-
absorbable indicator to correct water flux in the in situ SPIP model, which could be developed to predict the
human Fabs.

1. Introduction becomes necessary.

Several permeability methods have emerged in preclinical models to
predict human intestinal permeability. Among these methods, the in situ
single-pass intestinal perfusion (SPIP) is the most widely used method
and has been proven to have closer permeability to the human intestine
(Escribano et al., 2012; Fagerholm et al., 1996; Salphati; 2001). The
widespread use of SPIP was attributed to its advantages such as good
repeatability, intact blood supply, membrane integrity, and experi­
mental control (experimental regions, fluid flow rate, and drug con­
centration). In this method, the compound of interest is perfused
through a segment of the rat intestine. Loss of the compound between
entering and leaving concentrations is converted into intestinal ab­
sorption. Because the estimate of the absorption coefficient may be
affected by water movement, a method for calculating water flux
In general, measuring water flux involves the gravimetric method
and co-perfusion method (phenol red or 14C-PEG-4000)
(Tuğcu-Demiröz et al., 2014; Whittamore et al., 2016). The prominent
disadvantages of the gravimetric method are fluid evaporation, tissue
edema, and changes in perfusion density caused by mucous shedding
(Jackson and Cassidy, 1970; Smith, 1964; Sutton et al., 2001). Phenol
red was first used as a non-absorbable indicator in 1923 (Gorham,
1923). Polyethylene glycol-4000 (PEG-4000) was applied as a nonab­
sorbable indicator by numerous studies (Bright-Asare and Binder, 1973;
Miller and Schedl, 1970). Nevertheless, some studies have proved that
the absorption of phenol red and PEG-4000 can not be neglected in
measuring water flux (Higaki et al., 1988; Koyama et al., 1992; Yama­
moto et al., 1996). Therefore, a novel non-absorbable indicator
mPEG-PR was designed and synthesized by phenol red and PEG-4000

* Corresponding author.
E-mail address: chenggang63@hotmail.com (G. Cheng).
1
Equally contributed

https://doi.org/10.1016/j.ejps.2022.106255
Received 21 March 2022; Received in revised form 3 July 2022; Accepted 3 July 2022
Available online 5 July 2022
0928-0987/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Z. Liu et al. European Journal of Pharmaceutical Sciences 176 (2022) 106255

2.2. In situ single-pass intestinal perfusion study

2.2.1. Permeability measurements

Male Sprague-Dawley rats (weighing 200 ± 20 g) were performed as
the protocols approved by the University Committee on Ethics in the
Care and Use of Laboratory Animals. The rats were fasted for 18-20 h
and given free access to water before the experiment. The procedure of
the in situ SPIP study was conducted as established methods (Neerati
et al., 2011; Sababi M and Nylander, 1996). In short, the rats were
anesthetized with chloral hydrate (50 mg/kg) by intraperitoneal injec­
tion and placed on a warmed pad to maintain at 37℃. A midline lon­
gitudinal incision about 3 cm was made. A separated jejunum segment
(10 cm) was cannulated with two glass tubes at both ends, and rinsed
with saline. The exposed area was covered with gauze soaked with
isotonic saline. Then, the isolated jejunum segments were infused with
Fig. 1. The chemical structure of mPEG-PR.
drug solutions at a flow rate of 0.2 mL/min for 30 min to reach

(Fig. 1) (An et al., 2019). Although the low permeability and high sta­
bility of mPEG-PR have been confirmed across the rat intestine, the Table 2
The Peff of drugs (500 μmol/L) in the jejunum by the in situ single-pass intestinal
correlation between the prediction for human fraction dose absorbed
perfusion method in various correction ways of net water flux. The data repre­
(Fabs) in human and effective permeability coefficients (Peff) in rat has
sented the means ± SD (n = 3)
not been discovered.
Compounds Peff (×10–4 cm/s) (mean ± SD)
In present study, we initially compared the performance of mPEG-PR
Phenol red Gravimetry mPEG-PR Non-
with traditional correcting methods in the SPIP method. Six model drugs corrected
(atenolol, ranitidine, ibuprofen, ketoprofen, antipyrine, hydrochloro­
Atenolol 0.14 ± 0.35 ± 0.05 0.29 0.05 ± 0.03
thiazide) with a wide range of permeability and solubility were selected
±
0.04 0.02
based on FDA guidance (CDER, 2017). Subsequently, we correlated Ranitidine 0.13 ± 0.28 ± 0.04 0.25 ± 0.04 ± 0.02
human intestinal permeability obtained from published articles with 0.01 0.07
data in our study. Finally, the Fabs from the literature was compared with Ibuprofen 0.80 ± 1.07 ± 0.10 0.99 ± 0.69 ± 0.08
0.32 0.17
the values in our study as well. Overall, this study aimed to prove that
Ketoprofen 0.61 ± 0.81 ± 0.09 0.73 ± 0.45 ± 0.16
mPEG-PR was a more accurate and stable non-absorbable indicator. 0.14 0.19
Antipyrine 0.63 ± 1.04 ± 0.22 0.94 ± 0.61 ± 0.05
2. Materials and methods 0.15 0.17
Hydrochlorothiazide 0.06 ± 0.26 ± 0.08 0.24 ± 0.05 ± 0.06
0.01 0.05
2.1. Materials

Atenolol was provided by Wuhan Dahuaweiye Pharmaceutical and

Chemical Co., Ltd, Hubei, China. Ranitidine hydrochloride was obtained Table 3
The Ka of drugs (500 μmol/L) in the jejunum by the in situ single-pass intestinal
from Zhengqi Pharmaceutical Co., Ltd, Jiangsu, China. Ibuprofen was
perfusion method in various correction ways of net water flux. The data repre­
supplied by Liaoning Pharmaceutical Materials Co., Ltd, Liaoning,
sented the means ± SD (n = 3)
China. Ketoprofen was purchased from Xunda Pharmaceutical Co., Ltd,
Hubei, China. Antipyrine was provided from Xinhua Pharmaceutical Drugs Ka (×10–2 min–1) (mean ± SD)
Phenol red Gravimetry mPEG-PR Non-
Co., Ltd, Shandong, China. Hydrochlorothiazide was obtained from corrected
Changzhou Pharmaceutical Co., Ltd, Jiangsu, China. Cresol red (internal
Atenolol 0.79 ± 1.69 ± 0.25 1.31± 0.16 0.23 ± 0.18
standard) was provided from Bailingwei Technology Co., Ltd, Beijing,
0.16
China. Phenol red was purchased from Sigma-Aldrich, St. Louis, MO, Ranitidine 0.65 ± 1.20 ± 0.24 1.08 ± 0.14 ± 0.04
USA. mPEG-PR was synthesized in Shenyang Pharmaceutical University, 0.08 0.25
Liaoning, China. HPLC grade methanol and acetonitrile were supplied Ibuprofen 3.73 ± 4.74 ± 0.59 4.35 ± 2.91 ± 0.12
by Sigma-Aldrich, St. Louis, MO, USA. All other reagents were analytical 1.27 1.03
Ketoprofen 2.73 ± 3.57 ± 0.91 3.72 2.46 ± 0.95
grade.
±
0.46 1.06
Antipyrine 2.72 ± 4.01 ± 0.47 3.54 ± 2.57 ± 0.30
0.73 0.96
Hydrochlorothiazide 0.29 ± 1.21 ± 0.40 1.03 ± 0.19 ± 0.26
0.03 0.19

Table 1
HPLC methods for compounds studied
Drugs Mobile Phase (v/v) λ (nm) Retention time (min) Internal substance BCS

Atenolol Phosphate buffer (pH 3.0) / Acetonitrile (90:10) 226 7.1 Ranitidine III
Ranitidine Phosphate buffer (pH 3.0) / Acetonitrile (90:10) 226 8.4 Atenolol III
Ibuprofen Phosphate buffer (pH 3.0) / Acetonitrile (45:55) 220 14.2 Ketoprofen II
Ketoprofen Phosphate buffer (pH 3.0) / Acetonitrile (45:55) 220 7.2 Ibuprofen II
Antipyrine Phosphate buffer (pH 3.0) / Acetonitrile (80:20) 270 10.8 Hydrochlorothiazide I
Hydrochlorothiazide Phosphate buffer (pH 3.0) / Acetonitrile (80:20) 270 8.5 Antipyrine Ⅳ
Phenol red Water / Acetonitrile (40:60) 433 8.3 Cresol red -
mPEG-PR Water / Acetonitrile (46:54) 410 5.1 Cresol red -

2
Z. Liu et al. European Journal of Pharmaceutical Sciences 176 (2022) 106255

Fig. 2. Effects of various correction methods of net water flux on the Peff of model drugs (500 μmol/L) in the jejunum by the in situ single-pass intestinal perfusion
system. The data represented the means ± SD (n = 3) (*P < 0.05,**P < 0.01, ***P < 0.001).

steady-state outlet concentration. The perfusion solutions contained a
kind of investigated drug at 500 μmol/L and a type of non-absorbable
indicator at 56 μmol/L, which were maintained at 37℃ in a water
bath. After that, samples were collected at every interval of 15 min up to
105 min. When the gravimetric method was used to correct water flux,
the perfusion solution and the collected solution were weighed before
and after perfusion (Sababi and Bengtsson, 2001). At the end of the
experiment, the radius and length of the tested intestine segments were
measured. Perfusate samples were stored at -80℃ before analysis.
2.2.2. Sample preparation
300 μL of perfusion sample was mixed with 10 μL internal standard
solution of drug (3000 μg/mL). When the non-absorbable indicator
method was used, 10 μL of cresol red (600 μg/mL) was added as an
internal substance of the indicator. The above mixture was mixed for 30
s. Then two volumes of absolute methanol were added and vortexed for
2 min. Finally, the supernatant was obtained and analyzed by HPLC after
centrifugation at 10000 r/min for 10 min.

3
Z. Liu et al. European Journal of Pharmaceutical Sciences 176 (2022) 106255

Fig. 3. Effects of various correction methods of net water flux on the Ka of model drugs (500 μmol/L) in the jejunum by the in situ single-pass intestinal perfusion
system. The data represented the means ± SD (n = 3) (*P < 0.05, **P < 0.01, ***P < 0.001).

2.2.3. Chromatographic conditions criterion. Eqn. 1-5

All samples from the SPIP experiments were analyzed by a HPLC
system (Agilent 1260 Infinity II, Agilent Technologies, Santa Clara, CA, 2.2.4. Data analysis
USA) using a quaternary pump (Agilent 1260 quat pump), an auto­
sampler (Agilent 1260 ALS), and a multi-wavelength detector (Agilent 2.2.4.1. Effective permeability values and absorption rate coefficients. The
1260 MWD VL). The analyses were conducted on a reversed-phase C18 Peff and the absorption rate (Ka) of six model drugs were calculated using
column (5 μm, 4.6 mm × 250 mm) at a flow rate of 1.0 mL/min. The the following equations:
column oven was set at 30◦ C. The chromatographic methods were
− Qin × ln(Cout(cor) /Cin )
shown in Table 1. The methods were validated, in which all the pa­ Peff = (1)
rameters of accuracy, precision, linearity, stability, and recovery met a 2π rl

4
Z. Liu et al. European Journal of Pharmaceutical Sciences 176 (2022) 106255

Table 4
Fabs, Peff in human and Papp in the Ussing Chamber using human tissue of drugs
Drugs Human Fabs(%) Human Papp(×10–6 cm/
Peff(×10–4 cm/s) s)

Atenolol 50 0.2 4.11 ± 1.32

(Furuichi et al., (Fagerholm (Lennernäs,
1984) et al., 1997) 2007)
Ranitidine 50 0.3 5.5
(Koyama et al., (Kasim et al., (Takamatsu
1992) 2004) et al., 2001)
Ibuprofen 95 N/A 24.7 ± 2.46
(Varma et al., (Salphati et al.,
2012) 2001)
Ketoprofen 100 8.7 N/A
(Zakeri-Milani (Fagerholm
et al., 2007) et al., 1997)
Antipyrine 97 5.6 49.7 ± 13.3
(Furuichi et al., (Chiou and (Lennernäs,
1984) Barve, 1998) 2007)
Hydrochlorothiazide 54 0.04 N/A
(Modamio et al., (Fagerholm
1998) et al., 1997) Fig. 5. The correlation between the Peff by the in situ intestinal perfusion
system in rats and the Peff by the in situ intestinal perfusion system in humans.

Table 5
Table 7
Goodness of fit indexes of the Peff and the Fabs of drugs
Goodness of fit indexes of the Peff of rat and Papp of human
Correction Methods Equation R2 RSS
Correction Methods Equation R2 RSS
Phenol red Fabs = 1 − e− 64032Peff(rat) 0.80 0.064
Phenol red Y = 47.46979X + 0.82784 0.37 572.19
mPEG-PR Fabs = 1 − e− 30148Peff(rat) 0.93 0.021
mPEG-PR Y = 45.06875X - 6.82745 0.58 377.62
Gravimetry Fabs = 1 − e− 26939Peff(rat) 0.91 0.029
Gravimetry Y = 42.77684X - 8.29963 0.61 355.57

Fig. 4. The correlation between the Peff obtained in this research and Fabs Fig. 6. The correlation between the Peff by the in situ intestinal perfusion
from literature. system in rats and the Papp by the Ussing chamber in humans.

2.2.4.2. Cout(cor) using the non-absorption indicator. To compensate for

Table 6 the changes in water flux, a non-absorption indicator was used, and Cout
Goodness of fit indexes of the Peff of rat and Peff of human
(cor) was calculated by the followings:
Correction Methods Equation R2 RSS
C(indicator)in
Phenol red Y = 13.42731X - 1.24817 0.87 6.14 Cout(cor) = Cout × (3)
C(indicator)out
mPEG-PR Y = 10.67587X - 2.26318 0.68 15.24
Gravimetry Y = 9.66144X - 2.30715 0.67 15.48
where C(indicator)in is the concentration of phenol red or mPEG-PR in the
pre-sample, and C(indicator)out is the concentration of phenol red or mPEG-
(
Cout(cor)
)
Qin PR in the post-sample. Cout is the measured concentration of the drug in
Ka = 1− × (2) the exit.
Cin π r2 l

where Qin is the perfusion flow rate (0.2 mL/min), Cout(cor) is the cor­ 2.2.4.3. Cout(cor) using the gravimetric method. To get an accurate con­
rected outlet concentration, and Cin is the initial concentration inside the centration of compound leaving the intestine, the gravimetric method
sacs. r is the radius of the intestinal segment, and l is the length of the was used, and Cout(cor) was calculated by the followings:
perfused intestine.

5
Z. Liu et al.
Qout
Cout(cor) = Cout × (4)
Qin
Qout =
Mout /Dout
(5)
t In general, it was interesting that the curves of various water
where Qout is the measured flow of the exit, Mout is the mass of collected
sample, and Dout is the density of collected sample.
2.3. Statistical analysis
All animal experiments were replicated three times. Results were
performed by means ± SD. The significant difference between the two
groups was assessed using t-test. A criterion of significance was set as P
< 0.05.
3. Results and discussion
The Peff and Ka of six model drugs were summarized in Table 2 and
Table 3. The Peff and Ka of six model drugs without correcting water flux
were also calculated. Fig. 2 and Fig. 3 displayed the Peff and Ka of six
model drugs in the jejunum segments of rat, which were obtained with
four correction methods of net water flux using SPIP. A statistically
significant difference was displayed in different correcting methods of
net water flux. The sequences of the Peff for six model drugs with diverse
correcting methods of net water flux were: Gravimetry > mPEG-PR >
Phenol red > Non-corrected method. In addition, the orders of Ka were
consistent with the laws of Peff. As shown in Fig. 2 and Fig. 3, it was
necessary to correct the net water flux for drugs using the SPIP method,
especially for drugs in BCS III and Ⅳ classes. However, there was no
significant difference in the absorption coefficients of drugs in BCS I and
II classes among the three correcting methods. We previously compared
the permeability of phenol red and mPEG-PR, and conducted the in situ
SPIP study with the gravimetric, phenol red, non-corrected, and mPEG-
PR method in the entire rat intestine. The results indicated that the
mPEG-PR method was the best way to correct water flux among the all
experiments. Our results supported our previous findings that phenol
red was absorbed by the intestine and the gravimetric method had
mucous shedding or water evaporation. Similar to our results in this
research, the mPEG-PR was the best non-absorbable indicator.
Table 4 summarized the literature data of the Fabs in human, Peff in
human, and apparent permeability coefficients (Papp) in the Ussing
Chamber using human tissue. The goodness of fit indexes of the Peff and
Fabs of drugs were displayed in Table 5. The correlation between the Peff
obtained in this research and the Fabs from literature was presented in
Fig. 4 to explore the prediction of human Fabs. Fig. 4 showed a good
linear correction with coefficients of determination (R2) = 0.93 for the
mPEG-PR method, R2 = 0.91 for the gravimetric method, and R2 = 0.80
for the phenol red method, respectively. The excellent correlation be­
tween the rat Peff and human Fabs was consistent with earlier work
(Escribano et al., 2012; Ruiz-Picazo et al., 2017). The goodness of fit
indexes of the Peff and Fabs were in particular good in the mPEG-PR
method. Therefore, mPEG-PR was a useful and alternative
non-absorbable indicator in the SPIP model to predict the human Fabs,
which could avoid the absorption of phenol red and the tedious opera­
tion of the gravimetric method.
Table 6 summarized the goodness of fit indexes between the rat Peff
and human Peff. The correlation between the rat Peff obtained in this
research and the human Peff from literature was shown in Fig. 5. Table 7
summarized the goodness of fit indexes between the rat Peff and human
Papp. The rat Peff was compared with the human Papp in the Ussing
Chamber in Fig. 6. The R2 were far from adequate in Fig. 5 and Fig. 6.
These cases may be introduced by different experimental standards
including the pH of perfusion solution, test concentration of drug, and
location of intestinal segments (Lozoya-Agullo et al., 2015). Further­
more, the poor correlation in Fig. 6 could be explained by the lack of
6
European Journal of Pharmaceutical Sciences 176 (2022) 106255
physiological relevance in the SPIP method compared with the Ussing
Chamber using human tissue. Or perhaps the high variability between
the in situ data and isolated results caused the weak correlation in Fig. 6
(Lozoya-Agullo et al., 2017).
correction methods in Fig. 4-6 showed the same trends: Gravimetry >
mPEG-PR > Phenol red, which proved mPEG-PR was a suitable non-
absorbable indicator. The poor correlations were obtained in Fig. 5
and Fig. 6. The reason for the poor correlation remains unclear and
further mechanism investigation is needed. We speculated that the
number of model drugs may be limited. In addition, there was a large
discrepancy among different water correction methods. Thus, further
experiments with more drugs are needed to confirm the prediction of the
Peff by the in situ SPIP model. The correlation between the Peff obtained
in this research and the Fabs from literature was great, which supported
the use of mPEG-PR as a non-absorbable indicator in the in situ SPIP
study in the future.
4. Conclusion
In this research, we successfully proved that mPEG-PR was a suitable
indicator for correcting water flux. The excellent correlation between
the rat Peff and the human Fabs was confirmed. Therefore, mPEG-PR was
a stable and accurate non-absorbable indicator in the in situ SPIP model,
which could be a valuable tool to predict Fabs of human.
CRediT authorship contribution statement
Zhixiang Liu: Methodology, Validation, Writing – original draft.
Tong An: Investigation, Resources, Project administration. Rui Yuan:
Supervision, Methodology. Maoxiong Tian: Resources, Data curation.
Linlin Yuan: Resources, Supervision. Tong Zhang: Validation. Gang
Cheng: Methodology, Funding acquisition, Formal analysis.
Declaration of Competing Interest
Zhixiang Liu, Tong An, Rui Yuan, Maoxiong Tian, Linlin Yuan, Tong
Zhang and Gang Cheng declared that they had no conflicts of interest.
Acknowledgments
This work was supported by the Basic Research Projects of Liaoning
Provincial Education Department (Grant No. 2020LJC12) and the
Disruptive Technologies Innovation Fund of Shenyang Pharmaceutical
University (Grant No. DFJJ2018208).
References
An, T., Liu, Z.X., Zhang, Z.Q., Zhou, J., Wang, M.M., Zou, M.J., Cheng, G., 2019. Design,
synthesis and performance evaluation of mPEG-PR: a novel non-absorbable marker.
Eur. J. Pharm. Sci. 131, 50–57.
Bright-Asare, P., Binder, H.J., 1973. Stimulation of colonic secretion of water and
electrolytes by hydroxy fatty acids. Gastroenterology 64, 81–88.
CDER, 2017. Guidance for industry: Waiver of in vivo bioavailability and bioequivalence
studies for immediate-release solid oral dosage forms based on a biopharmaceutics
classification system. Food and Drug Administration.
Chiou, W.L., Barve, A., 1998. Linear correlation of the fraction of oral dose absorbed of
64 drugs between humans and rats. Pharm. Res. 15, 1792–1795.
Escribano, E., Sala, X.G., Salamanca, J., Navarro, C.R., Regué, J.Q., 2012. Single-pass
intestinal perfusion to establish the intestinal permeability of model drugs in mouse.
Int. J. Pharmaceut. 436, 472–477.
Fagerholm, U., Johansson, M., Lennernäs, H., 1996. Comparison between permeability
coefficients in rat and human jejunum. Pharm. Res. 13, 1336–1342.
Fagerholm, U., Lindahl, A., Lennernäs, H., 1997. Regional intestinal permeability in rats
of compounds with different physicochemical properties and transport mechanisms.
J. Pharm. Pharmacol. 49, 687–690.
Furuichi, Y., Takahashi, T., Imaizuni, K., Sugano, M., 1984. Evaluation of polyethylene
glycol as a water-soluble marker: absorption and excretion of 14C-labeled
polyethylene glycol in rats. Agric. Biol. Chem. 48, 1777–1781.
Gorham, F.D., 1923. The factor of dilution in gastric analysis. J. Amer. Med. Ass. 81,
1738–1742.
Z. Liu et al.
Higaki, K., Kato, M., Hashida, M., Sezaki, H., 1988. Enhanced membrane permeability to
phenol red by medium-chain glycerides: studies on the membrane permeability and
microviscosity. Pharm. Res. 5, 309–312.
Jackson, M.J., Cassidy, M.M., 1970. Studies on intestinal fluid transport. II. The location
of fluid accumulated in the wall of rat jejunum during incubation in vitro. Biochim.
Biophys. Acta. 211, 436–447.
Kasim, N.A., Whitehouse, M., Ramachandran, C., Bermejo, M., Lennernäs, H., Hussain, A.
S., Junginger, H.E., Stavchansky, S.A., Midha, K.K., Shah, V.P., Amidon, G.L., 2004.
Molecular properties of WHO essential drugs and provisional biopharmaceutical
classification. Mol. Pharmaceut. 1, 85–96.
Koyama, Y., Tateishi, M., Kawaide, A., Kojima, S., 1992. Gastrointestinal absorption of
(14C)Poly(ethylene glycol) 4000. Polym. J. 24, 583–589.
Lennernäs, H., 2007. Intestinal permeability and its relevance for absorption and
elimination. Xenobiotica 37, 1015–1051.
Lozoya-Agullo, I., Zur, M., Fine-Shamir, N., Markovic, M., Cohen, Y., Porat, D., González-
Álvarez, I., González-Álvarez, M., Merino-Sanjuán, M., Bermejo, M., Dahan, A.,
2017. Investigating drug absorption from the colon: Single-pass vs. Doluisio
approaches to in-situ rat large-intestinal perfusion. Int. J. Pharmaceut. 527,
135–141.
Lozoya-Agullo, I., Zur, M., Wolk, O., Beig, A., González-Álvarez, I., González-Álvarez, M.,
Merino-Sanjuán, M., Bermejo, M., Dahan, A., 2015. In-situ intestinal rat perfusions
for human Fabs prediction and BCS permeability class determination: Investigation
of the single-pass vs. the Doluisio experimental approaches. Int. J. Pharmaceut. 480,
1–7.
Miller, D.L., Schedl, H.P., 1970. Total recovery of nonabsorbable indicators in the rat
small intestine. Gastroenterology 58, 40–46.
Modamio, P., Lastra, C.F., Mariño, E.L., 1998. Error structure for the HPLC analysis for
atenolol, metoprolol and propranolol: a useful weighting method in parameter
estimation. J. Pharmaceut. Biomed. 17, 507–513.
Neerati, P., Ganji, D., Bedada, S.K., 2011. Study on in situ and in vivo absorption kinetics
of phenytoin by modulating P-glycoprotein with verapamil in rats. Eur. J. Pharm.
Sci. 44, 27–31.
Ruiz-Picazo, A., Lozoya-Agullo, I., Ortiz-Azcarate, M., Merino-Sanjuán, M., González-
Álvarez, M., González-Álvarez, I., Bermejo, M., 2017. Comparison of segmental-
dependent permeability in human and in situ perfusion model in rat. Eur. J. Pharm.
Sci. 107, 191–196.
European Journal of Pharmaceutical Sciences 176 (2022) 106255
Sababi, M., Bengtsson, U.H., 2001. Enhanced intestinal motility influences absorption in
anaesthetized rat. Acta Physiol 172, 115–122.
Sababi, M., Nylander, O., 1996. Comparative study of the effects of nitric oxide synthase
and cyclo-oxygenase inhibition on duodenal functions in rats anaesthetized with
inactin, urethane or α-chloralose. Acta Physiol 158, 45–52.
Salphati, L., Childers, K., Pan, L., Tsutsui, K., Takahashi, L., 2001. Evaluation of a single-
pass intestinal-perfusion method in rat for the prediction of absorption in man.
J. Pharm. Pharmacol. 53, 1007–1013.
Smith, M.W., 1964. The in vitro absorption of water and solutes from intestine of goldfish
Carassius auratus. J. Physiol. 175, 38–49.
Sutton, S.C., Rinaldi, M.T.S., Vukovinsky, K.E., 2001. Comparison of the gravimetric,
phenol red, and 14C-PEG-3350 methods to determine water absorption in the rat
single-pass intestinal perfusion model. AAPS Pharmsci 3, E25.
Takamatsu, N., Kim, O.N., Welage, L.S., Idkaidek, N.M., Hayashi, Y., Barnett, J.,
Yamamoto, R., Lipka, E., Lennernäs, H., Hussain, A., Lesko, L., Amidon, G.L., 2001.
Human jejunal permeability of two polar drugs: cimetidine and ranitidine. Pharm.
Res. 18, 742–744.
Tuğcu-Demiröz, F., González-Álvarez, I., González-Álvarez, M., Bermejo, M., 2014.
Validation of phenol red versus gravimetric method for water reabsorption
correction and study of gender differences in Doluisio’s absorption technique. Eur. J.
Pharm. Sci. 62, 105–110.
Varma, M.V., Gardner, I., Steyn, S.J., Nkansah, P., Rotter, C.J., Whitney-Pickett, C.,
Zhang, H., Di, L., Cram, M., Fenner, K.S., El-Kattan, A.F., 2012. pH-Dependent
solubility and permeability criteria for provisional biopharmaceutics classification
(BCS and BDDCS) in early drug discovery. Mol. Pharmaceut. 9, 1199–1212.
Whittamore, J.M., Genz, J., Grosell, M., Wilson, R.W., 2016. Measuring intestinal fluid
transport in vitro: Gravimetric method versus non-absorbable marker. Comp.
Biochem. Phys A. 194, 27–36.
Yamamoto, A., Uchiyama, T., Nishikawa, R., Fujita, T., Muranishi, S., 1996. Effectiveness
and toxicity screening of various absorption enhancers in the rat small intestine:
effects of absorption enhancers on the intestinal absorption of phenol red and the
release of protein and phospholipids from the intestinal membrane. J. Pharm.
Pharmacol. 48, 1285–1289.
Zakeri-Milani, P., Valizadeh, H., Tajerzadeh, H., Azarmi, Y., Islambolchilar, Z.,
Barzegar, S., Barzegar-Jalali, M., 2007. Predicting human intestinal permeability
using single-pass intestinal perfusion in rat. J. Pharm. Pharm. Sci. 10, 368–379.