Pharmacokinetic alteration of paclitaxel by ferulic acid derivative

Article abstract of DOI:10.3390/pharmaceutics11110593

P-glycoprotein (P-gp) is known to be involved in multidrug resistance (MDR) and modulation of pharmacokinetic (PK) profiles of substrate drugs. Here, we studied the effects of synthesized ferulic acid (FA) derivatives on P-gp function in vitro and examined PK alteration of paclitaxel (PTX), a well-known P-gp substrate drug by the derivative. Compound 5c, the FA derivative chosen as a significant P-gp inhibitor among eight FA candidates by in vitro results, increased PTX AUC_inf as much as twofold versus the control by reducing PTX elimination in rats. These results suggest that FA derivative can increase PTX bioavailability by inhibiting P-gp existing in eliminating organs.

Full text of DOI:10.3390/pharmaceutics11110593

pharmaceutics
Communication
Pharmacokinetic Alteration of Paclitaxel by Ferulic
Acid Derivative
Jaeok Lee 1,†, Song Wha Chae 1,†, LianJi Ma 1, So Yeon Lim 1, Sarah Alnajjar 2,
Hea-Young Park Choo 1, Hwa Jeong Lee 1,* and Sandy Jeong Rhie 2,
*
1
College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University,
Seoul 03760, Korea; leejo19@ewha.ac.kr (J.L.); yuki1226@naver.com (S.W.C.); malianji0215@163.com (L.M.);
deersy@naver.com (S.Y.L.); hypark@ewha.ac.kr (H.-Y.P.C.)
College of Pharmacy and Division of Life & Pharmaceutical Sciences, Ewha Womans University,
Seoul 03760, Korea; Najjar_sara@yahoo.com
2
*
Correspondence: hwalee@ewha.ac.kr (H.J.L.); sandy.rhie@ewha.ac.kr (S.J.R.); Tel.: +82-2-3277-3409 (H.J.L.);
+82-2-3277-3023 (S.J.R.); Fax: +82-2-3277-2851 (H.J.L. & S.J.R.)
†
The authors contribute equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 24 September 2019; Accepted: 8 November 2019; Published: 9 November 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: P-glycoprotein (P-gp) is known to be involved in multidrug resistance (MDR) and
modulation of pharmacokinetic (PK) profiles of substrate drugs. Here, we studied the effects of
synthesized ferulic acid (FA) derivatives on P-gp function in vitro and examined PK alteration
of paclitaxel (PTX), a well-known P-gp substrate drug by the derivative. Compound 5c, the FA
derivative chosen as a significant P-gp inhibitor among eight FA candidates by in vitro results,
increased PTX AUCinf as much as twofold versus the control by reducing PTX elimination in rats.
These results suggest that FA derivative can increase PTX bioavailability by inhibiting P-gp existing
in eliminating organs.
Keywords:
ferulic acid derivatives; P-glycoprotein; elimination; pharmacokinetics;
bioavailability; paclitaxel
1. Introduction
P-glycoprotein (P-gp) is a membrane efflux transporter that is a member of the ATP-binding
cassette (ABC) superfamily. This transporter is widely distributed in normal organs including the
brain [1], liver [2], intestine [3] and kidney [4] to protect our body from exogenous compounds
including toxins by extruding them out of the body organs. P-gp is associated with a multidrug
resistance (MDR) phenomenon which makes many potent anticancer drugs ineffective and is one of
the reasons responsible for the failure of cancer chemotherapy [5–7]. Therefore, P-gp inhibitors can
be used to overcome MDR. Moreover, this concept is extended to improve the absorption of orally
delivered medications by inhibiting P-gp located in the gastrointestinal (GI) tract [8–10].
Ferulic acid (FA) is one of the most ubiquitous phenolic compounds in plants, and the organic
compound and its derivatives possess multiple physiological and pharmaceutical functions such as
antioxidant, antimicrobial, anti-inflammatory, antithrombosis and anticancer activities [11]. In the
anticancer effect, it has been reported that FA and its derivatives induce cell cycle arrest, autophagy,
apoptosis and cytotoxicity in several cancer cells such as leukemia, breast cancer, cervical cancer,
etc. [12
well. Only a few studies have assessed recently the effect of FA on the reversal of the P-gp-mediated
MDR in cancer [15 16]. Therefore, we synthesized several FA derivatives in the study and evaluated
–14]. However, the effect of FA and its derivatives on P-gp modulation have not been investigated
,
their function on P-gp in vitro and in vivo systems.
Pharmaceutics 2019, 11, 593; doi:10.3390/pharmaceutics11110593
www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2019, 11, 593
2 of 11
2. Materials and Methods
2.1. Materials
Ferulic acid, sulforhodamine B (SRB), (+/
−
)-verapamil hydrochloride and other reagents for
synthesis were purchased from Sigma-Aldrich (St. Louis, MO, USA). [3H]-daumnomycin (16 Ci/mmol)
was supplied by PerkinElmer Life and Analytical Sciences (Boston, MA, USA). The BD GentestTM
ATPase assay kit and human P-glycoprotein membranes were commercially provided from BD
Bioscience (Woburn, MA, USA). Paclitaxel (PTX) was obtained from Samyang Genex (Daejeon, Korea).
All reagents and solvents were supplied as synthesis grades, cell culture grades and HPLC analysis
grades. Melting points were measured on an electro-thermal digital melting point (Buchi, Essen,
1
Germany) without calibration. H NMR spectra were recorded on Varian NMR AS and Varian Unity
Inova 400 MHz NMR spectrometers. Chemical shifts were reported in parts per million (d) units relative
to the solvent peak. The 1H NMR data were reported as peak multiplicities: s for singlet; d for doublet;
t for triplet; and m for multiplet. Coupling constants were recorded in hertz. Mass spectra data was
obtained on an Agilent 6220 Accurate-Mass time-of-flight liquid chromatography/mass spectrometry
(TOF LC/MS). All the synthesized compounds have been registered as a patent (KR10-2013-0099676)
by Prof. Hea-Young Park Choo and Ms So Yeon Lim since 2013.
2.2. Synthesis of FA Derivatives
2.2.1. Methylation of Ferulic Acid
Ferulic acid 2 g (10.3 mmol) was dissolved in 40 mL of methanol and 1.2 mL of concentrated
sulfuric acid (H2SO4) was added, and heated under reflux overnight. Then, 5% NaHCO3 was added to
make alkaline solution (pH 10) and extracted with diethyl ether. The solution was dried over MgSO4
and the solvent was removed in vacuo.
2.2.2. General Procedure for the Synthesis of Esters
To the methyl ferulate (0.5 g, 2.4 mmol) in dry acetone (6 mL) solution, pottassium carbonate
(K2CO3) (0.4 g, 2.9 mmol) was added; 1-Iodopropan (0.26 mL, 2.9 mmol) was added and the reaction
mixture was heated at 60 ◦C for 3 h. After cooling, the reaction mixture was extracted with diethyl
ether, dried over MgSO4 and the solvent was removed in vacuo. Column chromatography (hexane:
ethylacetate = 1:1) afforded the desired product.
2.2.3. General Procedure for Hydrolysis
The ester obtained from the procedure 2.2.2 (0.5 mmol) was dissolved in acetone (6 mL), and 2 N
NaOH (6 mL) was added and the reaction mixture was heated to 90 ◦C for 2 h. The reaction mixture
was extracted with diethyl ether. The water layer was acidified with 10% HCl solution (pH 2) and
extracted with diethyl ether. The organic solution was dried over MgSO4 and the solvent was removed
in vacuo to obtain the desired product.
2.2.4. General Synthesis Amides
Various amines (0.23 mmol) and PyBOP (benzotrial-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate) (0.12 g, 0.23 mmol) was added to the prepared (E)-3-(3-methoxy-4-substituted
phenyl)acrylic acid (0.21 mmol) in dimethylformamide (5 mL). After stirring, i-Pr2NEt (0.007 mL, 0.46
mmol) was added and stirred at room temperature for 16 h. The reaction mixture was extracted with
diethyl ether, dried over MgSO4 and the solvent was removed in vacuo.
Synthesis of (E)-3-(3-methoxy-4-propoxyphenyl)-1-(piperidin-1-yl)prop-2-en-1-one (Compound 5a)
Piperidine (0.023 mL, 0.23 mmol) was employed as amine. Pale yellow solid (47%) was obtained.;
mp 91–92 ◦C; 1H NMR (400 MHz, CDCl3)
δ 7.59 (1H, d, J = 15.2 Hz), 7.08 (1H, d, J = 6.4Hz, 7.03 (1H, s),

Pharmaceutics 2019, 11, 593
3 of 11
6.85 (1H, d, J = 8.4 Hz), 6.75 (1H, d, J = 15.2 Hz), 4.00 (2H, t, J = 6.8 Hz), 3.90 (3H, s), 3.65–3.60 (4H, m),
1.90–1.85 (2H, m), 1.69–1.54 (6H, m), 1.04 (3H, t, J = 7.6 Hz).
Synthesis of (E)-3-(4-isobutoxy-3-methoxyphenyl)-1-(piperidin-1-yl)prop-2-en-1-one (Compound 5b
Piperidine (0.023 mL, 0.23 mmol) was employed as amine. Yellow solid (83%) was obtained.; mp
96–97 ◦C; 1H NMR (400 MHz, CDCl3)
7.58 (1H, d, J = 15.2 Hz), 7.07(1H, d, J = 6.8 Hz), 7.03 (1H, s),
)
δ
6.84 (1H, d, J = 8.4 Hz), 6.75 (1H, d, J = 15.2 Hz), 3.90 (3H, s), 3.79 (2H, d, J = 6.8 Hz), 3.65–3.60 (4H, m),
2.20–2.14 (1H, m), 1.68–1.53 (6H, m), 1.04 (3H, s).
Synthesis of (E)-3-(4-benzyloxy-3-methoxyphenyl)-1-(piperidin-1-yl)prop-2-en-1-one
(Compound 5c) [17]
Synthesis of (E)-3-(3-methoxy-4-propoxyphenyl)-N,N-dimethylacrylamide (Compound 5d)
Dimethylamine HCl (0.02 g, 0.23 mmol) was employed as amine. Yellow solid (23%) was obtained.;
mp 108–109 ◦C; 1H NMR (400 MHz, CDCl3)
δ 7.61 (1H, d, J = 15.2 Hz), 7.098 (1H, d, J = 6.4 Hz), 7.04
(1H, s), 6.86 (1H, d, J = 8.4 Hz), 6.74 (1H, d, J = 15.6 Hz), 4.01 (2H, t, J = 6.8 Hz), 3.91 (3H, s), 3.18 (3H, s),
3.07 (3H, s), 1.91–1.85 (2H, m), 1.05 (3H, t, J = 7.9 Hz).
Synthesis of (E)-3-(4-isobutoxy-3-methoxyphenyl)-N,N-dimethylacrylamide (Compound 5e)
Dimethylamine HCl (0.018 g, 0.22 mmol) was employed as amine. White solid (46%) was obtained.;
mp 109–111 ◦C; 1H NMR (400 MHz, CDCl3)
δ 7.61 (1H, d, J = 15.6 Hz), 7.09 (1H, d, J = 6.0 Hz), 7.04 (1H,
s), 6.84 (1H, d, J = 8.4 Hz), 6.74 (1H, d, J = 15.6 Hz), 3.90 (3H, s), 3.80 (2H, d, J = 6.8 Hz), 3.18 (3H, s), 3.10
(3H, s), 2.19–2.16 (1H, m), 1.04 (3H, s), 1.03 (3H, s).
Synthesis of (E)-N-benzyl-3-(3-methoxy-4-propoxyphenyl)-N-methylacrylamide (Compound 5f)
N-methyl benzylamine (0.028 mL, 0.23 mmol) was employed as amine. Yellow oil (56%) was
obtained.; 1H NMR (400 MHz, CDCl3)
δ 7.71 (1H, d, J = 15.2 Hz), 7.38-7.24 (5H, m), 7.12-7.03 (1H, m),
6.95 (1H, s), 6.87-6.71(2H, m), 4.71 (1H, s), 4.00 (2H, d, J = 8.4 Hz), 3.88 (3H, s), 3.08 (3H, s), 1.89–1.85
(2H, m), 1.05 (3H, t, J = 7.6 Hz).
Synthesis of (E)-3-(4-benzyloxy-3-methoxyphenyl)-N,N-dimethylacrylamide (Compound 5g)
Dimethylamine HCl (0.015 g, 0.19 mmol) was employed as amine. Pale yellow solid (67%) was
obtained.; mp 135–136 ◦C; 1H NMR (400 MHz, CDCl3)
δ 7.60 (1H, d, J = 15.2 Hz), 7.45-7.31 (5H, m),
7.06–7.04 (2H, m), 6.86 (1H, d, J = 8.8 Hz), 6.74 (1H, d, J = 15.6 Hz), 5.86 (2H, s), 3.93 (3H, s), 3.17 (3H, s),
3.07 (3H, s).
Synthesis of (E)-1-(4-hydroxy-4-phenylpiperidin-1-yl)-3-(3-methoxy-4-propoxyphenyl)
prop-2-en-1-one (Compound 5h)
4-Phenylpiperidine-4-ol (0.041 g, 0.23 mmol) was employed as amine. Pale yellow solid (52%)
was obtained.; mp 163–164 ◦C; 1H NMR (400 MHz, CDCl3)
δ 7.64 (1H, d, J = 15.6 Hz), 7.48 (2H, d, J =
8.8 Hz), 7.38 (2H, t, J = 11.6 Hz), 7.30 (1H, d, J = 5.6 Hz), 7.10 (1H, d, J = 8.8 Hz), 7.05 (1H, s), 6.86 (1H, d,
J = 8.4 Hz), 6.80 (1H, d, J = 15.2 Hz), 4.70 (1H, s), 4.13–4.01 (3H, m), 3.91 (3H, s), 3.67 (1H, s), 3.24 (1H, s),
2.10–2.04 (2H, m), 2.04–1.80 (4H, m), 1.05 (3H, t, J = 7.6 Hz).
2.3. Cytotoxicity Studies in P-gp Overexpressed Cells
The effect of eight FA derivatives on cytotoxicity was studied in P-gp overexpressed human breast
cancer cells (MCF-7/ADR) using the SRB assay [18]. The details of the cell culture condition and the
assay method were presented in our previous reports [10,19]. Verapamil (VER, 100 µM), one of the
P-gp inhibitors, was used as a positive control. The half maximal inhibitory concentration (IC50) values

Pharmaceutics 2019, 11, 593
4 of 11
were calculated with Table Curve2D® version 5.01 software (Systat Software Inc., San Jose, CA, USA).
The assay was performed in triplicate.
2.4. [3H]-Daunomycin Accumulation and Efflux Studies
Among eight FA derivatives, compounds 5c, 5f, 5g and 5h (100 µM) were selected for
[3H]-daunomycin (DNM) accumulation and efflux studies based on cytotoxicity results. The methods
for [3H]-DNM accumulation and efflux studies were reported previously [10
,19]. VER (100 µM) was
used as a positive control. The experiments were performed in triplicate.
2.5. Human P-glycoprotein ATPase Activity Assay
The effects of compounds 5c
50 and 100 M) were examined in human P-gp membranes using an ATPase assay kit according to the
method reported previously [10 19]. VER was used as a P-gp inhibitor and an ATPase stimulator. The
, 5f, 5g and 5h on P-gp ATPase activity at different concentrations (20,
µ
,
ATPase activities were expressed as the rate of phosphate release per milligram of membrane protein
and converted to the relative ratio versus the control. This assay was performed in duplicate.
2.6. Pharmacokinetic Study
The pharmacokinetic (PK) study was performed using male Sprague-Dawley rats (6 weeks old
and 200 g–235 g) commercially available from Orient Bio (Seongnam, Korea) [10,19]. All animal
procedures were approved by the Institutional Animal Care and Use Committee of Ewha Womans
University (No. 2012-01-019, approved on 3 April 2012), Republic of Korea.
Among eight FA derivatives, compound 5c((E)-3-(4-(benyloxy-3-methoxyphenyl)-1-(piperidin-1-yl)
prop-2-en-1-one) was chosen to examine the effect on PTX pharmacokinetics because it was found to
be most effective in inhibiting P-gp function in vitro. Taxol formulation (Cremophor® EL, anhydrous
ethanol and isotonic saline (1/1/4, v/v/v)) was used to dissolve the PTX and compound 5c to a final
concentration of 2 mg/mL [10,19]. All were prepared immediately prior to use.
Rats were divided into the following 4 groups: Oral administration (PO) of PTX (25 mg/kg) alone
and co-administration of PTX (25 mg/kg) with compound 5c at three different doses (0.5, 2 and 5 mg/kg).
Each group had 5 rats except for a group of 2 mg/kg dose (n = 4). The blood samples (0.2 mL) were
collected from the common carotid artery at 0, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10 and 24 h.
PTX concentrations in rat plasma were analyzed by Agilent HP1100 series system using a
Capcell-pak C18 MG120 column (3 mm
×
250 mm, 5 µm, Shiseido, Tokyo, Japan). The samples were
eluted with mobile phase composed of acetonitrile and 0.1% phosphoric acid (1/1, v/v) at a flow rate of
0.5 mL/min for 30 min and detected at a wavelength of 227 nm. The HPLC-UV method was validated
by FDA guidelines as follows: Specificity (PTX: 14.8 min; internal standard: 26.7 min), linearity
over the concentration range between 0.01 and 10 µg/mL, lower limit of quantification (0.01 µg/mL)
and intra-/inter-day precision and accuracy (2.4–9.1% and 92.1–102.7%, respectively) [20]. All other
details for sample preparation and quantification of PTX concentrations in rat plasma were reported
previously [8–10].
2.7. Pharmacokinetic Analysis
The following PK parameters of PTX after oral single dosing to rats were estimated by
non-compartmental analysis using WinNonlin® Professional version 5.2 software (Pharsight
Corporation, Mountain View, CA, USA): The area under the plasma concentration-time curve from 0 h
to infinity (AUCinf), elimination half-life (t1/2), apparent volume of distribution after oral single dosing
(Vz/F), oral clearance (Cl/F), maximum plasma concentration (Cmax) and the time required to reach
Cmax (Tmax). The relative bioavailability (RB, %) of PTX was calculated with the following formula:
AUCinf po co − administraion
RB ( % ) =
× 100
AUCinf po control

Pharmaceutics 2019, 11, 593
5 of 11
AUCinf po control is the AUCinf obtained from oral single administration of PTX alone, and
AUCinf po co-administration is the AUCinf obtained from oral single co-administration of PTX and
compound 5c.
2.8. Data Analysis
Statistical analysis was conducted using Tukey and Dunnett T30s tests in conjunction with a
one-way analysis of variance (ANOVA) for accumulation, efflux, and PK studies, respectively. Free
GraphPad Prism was used for statistical analyses (Version 8.00, La Jolla, San Diego, CA, USA). Mean data
were presented with standard deviations (SD). A p value < 0.05 was considered statistically significant.
3. Results
3.1. Synthesis of Ferulic Amides, FA Derivatives
FA derivatives were synthesized as shown in Scheme 1. Ferulic acid (1) was protected with methyl
alcohol to give methyl ester (2). The ester was then reacted with potassium carbonate and different
alkyl halides such as propyl iodide, isobutyl iodide or benzyl bromide to afford corresponding esters
(3) and purified by column chromatography. Then, the protected methyl ester was hydrolyzed with
2 N sodium hydroxide to give the acids (4). The coupling of acid group with various amines using
PyBOP as coupling agent resulted in the desired amides (5).
Scheme 1. Methods for the preparation of FA derivatives. (
a
) MeOH, concentrated H2SO4, reflux
overnight; ( d
b
) K2CO3, acetone at 60 ◦C for 3 h; ( ) 2 N NaOH at 90 ◦C for 2 h; (
c
) PyBOP i-Pr2NEt,
DMF at room temperature overnight.
3.2. P-gp Inhibitory Effect of FA Derivatives in Vitro
VER (a positive control) and FA derivatives were shown to be not toxic to MCF-7/ADR cells at a
concentration of 100 M (Table S1). When eight FA derivatives (Figure 1, 100 M) were treated with
DNM in MCF-7/ADR cells, compounds 5c 5f 5g and 5h decreased the IC50 values of DNM to about
1/5 of negative control (DNM alone) value (Table 1). Even compounds 5c and 5h dramatically reduced
µ
µ
,
,
the IC50 values of DNM (2.2 and 2.6 µM, respectively) which was lower than that of VER-treated group
(3.1 µM).

Pharmaceutics 2019, 11, 593
6 of 11
Figure 1. Chemical structures of eight ferulic acid (FA) derivatives.
Table 1. Effect of various FA derivatives (100 µM) on the IC50 values of DNM in MCF-7/ADR cells after
2 h incubation.
Category
Compounds
IC50 of DNM (µM)
Negative Control
VER
31.7
3.1
Control
5a
5b
5c
5d
5e
5f
33.4
12.1
2.2
35.8
19.1
6.5
FA
derivatives
5g
5h
7.2
2.6
Based on this result, compounds 5c, 5f, 5g and 5h were selected and tested for their effects on
cellular accumulation and efflux of tritiated DNM (Figure 2). Four compounds and VER exhibited
significant effects on both tests compared to the control (P < 0.001 and 0.0001). Among them, compounds
5c
26.2%, respectively) more than VER (335.1
in the presence of compounds 5c 5e and 5h (43.1
was substantially reduced compared to the control or VER (63.0
(Figure 2B).
,
5e and 5h enhanced the accumulation of [3H]-DNM (438.4
±
4.9%, 382.1
±
20.4% and 426.1
±
±
14.7%) (Figure 2A). Coherently, the efflux of [3H]-DNM
,
±
4.6%, 39.9
±
3.8% and 43.3
3.3% or 48.0
±
±
0.9%, respectively)
1.1%, respectively)
±

Pharmaceutics 2019, 11, 593
7 of 11
Figure 2. Effects of four pre-selected FA derivatives on the accumulation (A) and efflux (B
) of [3H]-DNM
in MCF-7/ADR cells. [3H]-DNM uptake and efflux were determined after the incubation with each
derivative (100
µM) for 2 h and 1 h, respectively. VER was a positive control. Control represents
accumulation or efflux of tritium labeled DNM alone. Each data represents the mean
****, p < 0.0001, comparison to control) from triplicate experiments. VER, verapamil; Cntl, control.
±
SD (***, p < 0.001;
Furthermore, compared to VER (2.24), a well-known ATPase stimulator, four FA derivatives
exhibited higher values (4.02, 3.35, 5.19 and 2.79) for ATPase activity at the concentration of 100 µM.
Among them, compound 5c stably increased ATPase activity in a concentration-dependent manner
(Table 2). Based on this result, four FA derivatives may inhibit P-gp activity by depleting ATP as an
ATPase stimulator.
Table 2. Effect of FA derivatives on P-glycoprotein (P-gp) ATPase activity.
Concentration
Blank
VER
5c
5f
5g
5h
(µM)
20
50
100
1.00
1.00
1.00
3.46
2.53
2.24
1.54
2.48
4.02
2.85
3.12
3.35
1.26
1.77
5.19
3.99
2.69
2.79
The values are expressed as a ratio of blank (control).
3.3. BA Enhancing Effect of Compound 5c in Vivo
The in vitro results suggested that compound 5c was the most potent P-gp inhibitor. As shown
in Table 3, co-administration of the lowest dose of compound 5c (0.5 mg/kg) did not alter any PK
parameters of PTX, as compared to PO control (PTX alone). However, with higher doses of compound
5c (2 and 5 mg/kg), the AUCinf of PTX was increased (control; 773
1456 ± 367 ng·h/mL, p < 0.05) and Cl/F of PTX was decreased approximately to one half (control;
8.22 ± 1.84 L/h vs. 2 mg/kg; 4.10 1.53 L/h (p < 0.05) and 5 mg/kg; 4.53 1.04 L/h (p < 0.05),
respectively). The t1/2 was extended over two-fold (control; 2.8 0.86 h vs. 5 mg/kg; 6.8 1.3 h, p < 0.01
±
169 ng·h/mL vs. 5 mg/kg;
±
±
±
±
)
(Table 3). The drug exposure in the body was enhanced due to the reduction of oral clearance when it
was co-administered with compound 5c, except for a dose of 0.5 mg/kg (Figure 3). On the other hand,
the Cmax of PTX and Vz/F were not significantly changed in the presence of the compound.

Pharmaceutics 2019, 11, 593
8 of 11
Table 3. Pharmacokinetic (PK) parameters of paclitaxel (PTX) after oral administration of PTX (25
mg/kg) alone, or oral co-administration of PTX (25 mg/kg) and compound 5c (0.5, 2 and 5 mg/kg,
respectively) to rats.
Co- Administration of PTX with Compound 5c
PTX
PK Parameters
PO Control
0.5 mg/kg
2 mg/kg
5 mg/kg
Cmax (ng/mL)
Tmax (h)
187 ± 41
2.0 ± 0.0
193 ± 97
2.0 ± 0.0
249 ± 55
3.5 ± 1.0
195 ± 25
2.2 ± 0.5
AUCinf (ng·h/mL)
773 ± 169
2.8 ± 0.86
35.14 ± 16.84
8.22 ± 1.84
909 ± 539
2.3 ± 0.5
1880 ± 750
6.5 ± 0.15
1456 ± 367 *
6.8± 1.3 **
44.60 ± 12.27
4.53 ± 1.04 *
t1/2 (h)
Vz/F (L)
Cl/F (L/h)
32.30± 19.10
34.80 ± 17.72
9.38± 4.81
4.10 ± 1.53 *
RB (%)
117.0
243.2
188.7
* p < 0.05; ** p < 0.01 compared with PTX PO control; Data are presented as mean ± SD (n = 4–5).
Figure 3. Mean plasma concentration-time profiles of oral co-administration of PTX (25 mg/kg) and FA
derivative, compound 5c to rats. Bars represent SD (n = 4–5). (
ꢀ) PTX (25 mg/kg, PO) alone, PTX (25
mg/kg) co-administrated with compound 5c ( ) 0.5, (N) 2 or (X) 5 mg/kg.
◆
4. Discussion
FA enhanced the cytotoxicity of PTX and doxorubicin in human nasopharyngeal cancer cells by
inhibiting the P-gp function [15 16]. FA enhanced cellular accumulation of calcein-acetoxymethyl ester
,
and rhodamine123, well-known P-gp substrates, in P-gp overexpressed cells in a dose-dependent
manner [15]. In silico study, FA showed hydrogen bonding and hydrophobic interaction with
transmembrane domain of P-gp [15]. In our study, newly synthesized FA derivatives including
compound 5c increased the cytotoxicity and cellular accumulation of DNM in P-gp overexpressed
breast cancer cells. Moreover, some parts of structure in the FA and its derivatives showed a similarity
with VER, a well-known P-gp inhibitor. Because of the similar parts of the structure, FA derivatives
might show the effect on the reversal of the P-gp-mediated MDR in cancer cells.
In addition, the dose-dependent decrease in ATPase activity of VER was also observed in our
previous study [10]. However, all the values were over 2 (as a ratio to blank), suggesting ATPase
stimulating effect of VER. In our previous studies, the lower dose (50
on DNM accumulation and efflux [ ] than the higher dose (100 M) of VER [10
used 100 M VER and FA derivatives to observe P-gp inhibitory activity as much as possible in in vitro
µM) of VER showed less effects
8
µ
,19]. Therefore, we
µ

Pharmaceutics 2019, 11, 593
9 of 11
studies. According to the results of ATPase activity assay, four FA derivatives may inhibit P-gp activity
by depleting ATP as an ATPase stimulator because a ratio to blank was over 2.
FA derivative, compound 5c chosen as the most potent P-gp inhibitor among the eight synthesized
FA derivatives from in vitro studies increased PTX BA by reducing oral PTX clearance in vivo. While
Cmax of PTX was not significantly altered in the presence of compound 5c, the t1/2 of PTX was
significantly extended when PTX was co-administered with 2 or 5 mg/kg of the FA derivative (Table 3).
The increased AUCinf of the drug observed in the higher dose groups (2 and 5 mg/kg) was a result of
the decreased PTX elimination. In addition, the reduced variation was monitored in the plasma PTX
concentration-time profiles of the higher dose groups (2 and 5 mg/kg), as compared with control or
low dose group (0.5 mg/kg).
Previously we reported other novel P-gp inhibitors such as coumarin derivative [
8],
phenylbutenoid dimer [ ] and xanthone analogue [10]. They and VER [ ] enhanced BA (9.6-fold, 1.8-fold
9
8
and 2.5-fold and 3.0-fold at 5 mg/kg, respectively) and Cmax of PTX when they were co-administered
with P-gp substrate anticancer drug, PTX. It means that these inhibitors worked at drug absorption
phase by modulating the intestinal P-gp function. On the other hand, the FA derivative in the present
study appeared to modulate P-gp function in the elimination phase, unlike other P-gp inhibitors
reported previously [8–10].
Our study showed the potential P-gp modulating effect of FA derivative, compound 5c, on PTX
disposition. Indeed, P-gp efflux transporters are expressed in the hepatic canalicular membrane for
biliary excretion and in the proximal tubule of the kidney for renal excretion [2,4]. In addition, PTX has
been reported to be eliminated by hepatic metabolism by mainly CYP2C8 [21], renal excretion [22] and
biliary excretion [23]. It has been reported that FA is quickly absorbed in the intestine within 5–15 min
and 30 min in rats and humans, respectively [24], and is mainly excreted through kidney [25]. Therefore,
the interaction between FA derivative, absorbed at GI tract, and PTX on P-gp efflux transporters might
occur during the elimination process. There is a lack of knowledge about the effect of FA derivatives
and/or their metabolites on the hepatic metabolism and biliary excretion, and it needs to be clarified.
According to the reports about the relationship between FA and renal function in rodents, FA
gives the positive effect on the kidney injury [26–28]. In lipopolysaccharide (LPS)-induced acute kidney
injured mice, the phenolic compound (50 mg/kg and 100 mg/kg, intraperitoneal (IP) injection, 1 h
before and 2 h after a single IP injection of LPS) reduces apoptosis and inflammation but increases
adenosine generation [27], and in diabetes-induced rats, FA (50 mg/kg, orally for 8-week) protects
hyperglycemia-induced kidney damage by antioxidation, anti-inflammation and autophagy [28]. In
the present study, a single dose of FA derivative (0.5, 2 or 5 mg/kg) may not affect renal function in
healthy rats to reduce PTX elimination. However, further studies are required to examine the effect of
FA derivatives on renal function and their toxicities in the body following single or multiple dosing.
5. Conclusions
Four FA derivatives exhibited P-gp inhibitory function among eight synthesized compounds, and
compound 5c was selected as the most potent P-gp inhibitor candidate in in vitro investigation. This
compound significantly improved the BA of PTX by approximately twofold at the dose of 5 mg/kg,
when it was co-administered with PTX in rats. Therefore, the co-administration of compound 5c could
offer a therapeutic benefit in oral administration of P-gp substrate drugs by reducing drug elimination.
Further studies are required to examine the effect of compound 5c on PTX metabolism to clarify the
mechanism for the decreased PTX elimination caused by the FA derivative and to investigate the effect
of the FA derivative, compound 5c on renal function.
Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4923/11/11/593/s1,
Table S1: The toxic effect of each derivative and VER in MCF-7/ADR cells after 2 h incubation.
Author Contributions: Conceptualization, J.L., S.W.C. and S.J.R.; Methodology, S.W.C., J.L. and H.J.L.; Formal
Analysis, J.L., S.W.C., L.M., S.Y.L. and S.A.; Investigation, S.W.C., J.L., H.-Y.P.C. and L.M.; Resources, S.Y.L. and
H.-Y.P.C.; Data Curation, J.L., L.M. and S.A.; Writing – Original Draft Preparation, J.L., S.W.C., H.-Y.P.C., S.J.R. and

Pharmaceutics 2019, 11, 593
10 of 11
H.J.L.; Writing – Review & Editing, J.L., S.J.R., H.-Y.P.C. and H.J.L; Visualization, J.L. and H.-Y.P.C.; Supervision,
S.J.R. and H.J.L.; Project Administration, H.J.L.; Funding Acquisition, H.J.L.
Funding: This work was supported by a National Research Foundation of Korea (NRF) grant funded by the
Korean government (MEST) (2017R1A2B4007869).
Acknowledgments: We thank Marilyn E. Morris (University of Buffalo, USA) for providing the MCF-7/ADR
cell line.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Cordon-Cardo, C.; O’Brien, J.P.; Casals, D.; Rittman-Grauer, L.; Biedler, J.L.; Melamed, M.R.; Bertino, J.R.
Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites.
Proc. Natl. Acad. Sci. USA 1989, 86, 695–698. [CrossRef] [PubMed]
2.
Thiebaut, F.; Tsuruo, T.; Hamada, H.; Gottesman, M.M.; Pastan, I.; Willingham, M.C. Cellular localization of
the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc. Natl. Acad. Sci. USA
1987, 84, 7735–7738. [CrossRef] [PubMed]
3.
4.
5.
6.
7.
Li, M.; Hurren, R.; Zastawny, R.L.; Ling, V.; Buick, R.N. Regulation and expression of multidrug resistance
(MDR) transcripts in the intestinal epithelium. Br. J. Cancer. 1999, 80, 1123–1131. [CrossRef] [PubMed]
Hori, R.; Okamura, N.; Aiba, T.; Tanigawara, Y. Role of P-glycoprotein in renal tubular secretion of digoxin in
the isolated perfused rat kidney. J. Pharmacol. Exp. Ther. 1993, 266, 1620–1625.
Dong, X.; Mumper, R.J. Nanomedicinal strategies to treat multidrug-resistant tumors: Current progress.
Nanomedicine 2010, 5, 597–615. [CrossRef]
Hua, X.; Sun, Y.; Chen, J.; Wu, Y.; Sha, J.; Han, S.; Zhu, X. Circular RNAs in drug resistant tumors. Biomed.
Pharm. 2019, 118, 109233. [CrossRef]
Al-Akra, L.; Bae, D.H.; Leck, L.Y.W.; Richardson, D.R.; Jansson, P.J. The biochemical and molecular
mechanisms involved in the role of tumor micro-environment stress in development of drug resistance.
Biochim. Biophys. Acta Gen. Subj. 2019, 1863, 1390–1397. [CrossRef]
8.
9.
Lee, K.; Chae, S.W.; Xia, Y.; Kim, N.H.; Kim, H.J.; Rhie, S.; Lee, H.J. Effect of coumarin derivative-mediated
inhibition of P-glycoprotein on oral bioavailability and therapeutic efficacy of paclitaxel. Eur. J. Pharmacol.
2014, 723, 381–388. [CrossRef]
Chae, S.W.; Han, A.R.; Park, J.H.; Rhie, J.Y.; Lim, H.J.; Seo, E.K.; Lee, H.J. In vitro and in vivo evaluation of
phenylbutenoid dimers as inhibitors of P-glycoprotein. J. Nat. Prod. 2013, 76, 2277–2281. [CrossRef]
10. Chae, S.W.; Woo, S.; Park, J.H.; Kwon, Y.; Na, Y.; Lee, H.J. Xanthone analogues as potent modulators of
intestinal P-glycoprotein. Eur. J. Med. Chem. 2015, 93, 237–245. [CrossRef]
11. Pei, K.; Ou, J.; Huang, C.; Ou, S. Derivatives of ferulic acid: Structure, preparation and biological activities.
Ann. Res. Rev. Biol. 2015, 5, 512–528. [CrossRef]
12. Chen, C.J.; Hsu, M.H.; Kuo, S.C.; Lai, Y.Y.; Chung, J.G.; Huang, L.J. (2E)-N,N-dibutyl-3-(4-hydroxy-
3-methoxyphenyl)acrylamide induces apoptosis and cell cycle arrest in HL-60 cells. Anticancer. Res. 2007, 27,
343–349. [PubMed]
13. Bagheri, S.M.; Asl, A.A.; Shams, A.; Mirghanizadeh-Bafghi, S.A.; Hafizibarjin, Z. Evaluation of Cytotoxicity
Effects of Oleo-Gum-Resin and Its Essential Oil of Ferula assa-foetida and Ferulic Acid on 4T1 Breast Cancer
Cells. Indian. J. Med. Paediatr. Oncol. 2017, 38, 116–120. [CrossRef] [PubMed]
14. Gao, J.; Yu, H.; Guo, W.; Kong, Y.; Gu, L.; Li, Q.; Yang, S.; Zhang, Y.; Wang, Y. The anticancer effects of ferulic
acid is associated with induction of cell cycle arrest and autophagy in cervical cancer cells. Cancer Cell Int.
2018, 18, 102. [CrossRef] [PubMed]
15. Muthusamy, G.; Balupillai, A.; Ramasamy, K.; Shanmugam, M.; Gunaseelan, S.; Mary, B.; Prasad, N.R. Ferulic
acid reverses ABCB1-mediated paclitaxel resistance in MDR cell lines. Eur. J. Pharmacol. 2016, 786, 194–203.
[CrossRef]
16. Muthusamy, G.; Gunaseelan, S.; Prasad, N.R. Ferulic acid reverses P-glycoprotein-mediated multidrug
resistance via inhibition of PI3K/Akt/NF-kappaB signaling pathway. J. Nutr. Biochem. 2019, 63, 62–71.
[CrossRef]

Pharmaceutics 2019, 11, 593
11 of 11
17. Ullah, S.; Park, Y.; Ikram, M.; Lee, S.; Park, C.; Kang, D.; Yang, J.; Akter, J.; Yoon, S.; Chun, P.; et al. Design,
synthesis and anti-melanogenic effect of cinnamamide derivatives. Bioorg. Med. Chem. 2018, 26, 5672–5681.
[CrossRef]
18. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J.T.; Bokesch, H.; Kenney, S.;
Boyd, M.R. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 1990, 82,
1107–1112. [CrossRef]
19. Chae, S.W.; Lee, J.; Park, J.H.; Kwon, Y.; Na, Y.; Lee, H.J. Intestinal P-glycoprotein inhibitors, benzoxanthone
analogues. J. Pharm. Pharmacol. 2018, 70, 234–241. [CrossRef]
20. Lee, S.G.; Chae, S.W.; Park, J.H.; Park, J.H.; Choi, J.M.; Kwon, K.E.; An, J.; Lee, H.J. Application of Simply
Modified HPLC Method in Evaluating Pharmacokinetic Parameters of Paclitaxel. In Drug Discovery and
Development as a New Growth Engine, Proceedings of the Spring International Convention of the Pharmaceutical
Society of Korea, Busan Exhibition & Convention Center, Busan, Korea, 21–22 April 2011; The Pharmaceutical
Society of Korea: Seoul, Korea, P9-14; p. 284.
21. De Weger, V.A.; Beijnen, J.H.; Schellens, J.H. Cellular and clinical pharmacology of the taxanes docetaxel and
paclitaxel–a review. Anticancer. Drugs 2014, 25, 488–494. [CrossRef]
22. Rowinsky, E.K. The development and clinical utility of the taxane class of antimicrotubule chemotherapy
agents. Annu. Rev. Med. 1997, 48, 353–374. [CrossRef] [PubMed]
23. Klecker, R.W.; Jamis-Dow, C.A.; Egorin, M.J.; Erkmen, K.; Parker, R.J.; Stevens, R.; Collins, J.M. Effect of
cimetidine, probenecid, and ketoconazole on the distribution, biliary secretion, and metabolism of [3H]taxol
in the Sprague-Dawley rat. Drug Metab Dispos 1994, 22, 254–258. [PubMed]
24. Mancuso, C.; Santangelo, R. Ferulic acid: Pharmacological and toxicological aspects. Food Chem Toxicol 2014
65, 185–195. [CrossRef]
,
25. Rondini, L.; Peyrat-Maillard, M.N.; Marsset-Baglieri, A.; Fromentin, G.; Durand, P.; Tome, D.; Prost, M.;
Berset, C. Bound ferulic acid from bran is more bioavailable than the free compound in rat. J. Agric. Food
Chem. 2004, 52, 4338–4343. [CrossRef] [PubMed]
26. Manikandan, R.; Beulaja, M.; Thiagarajan, R.; Pandi, M.; Arulvasu, C.; Prabhu, N.M.; Saravanan, R.;
Esakkirajan, M.; Palanisamy, S.; Dhanasekaran, G.; et al. Ameliorative effect of ferulic acid against renal
injuries mediated by nuclear factor-kappaB during glycerol-induced nephrotoxicity in Wistar rats. Ren. Fail.
2014, 36, 154–165. [CrossRef] [PubMed]
27. Mir, S.M.; Ravuri, H.G.; Pradhan, R.K.; Narra, S.; Kumar, J.M.; Kuncha, M.; Kanjilal, S.; Sistla, R. Ferulic
acid protects lipopolysaccharide-induced acute kidney injury by suppressing inflammatory events and
upregulating antioxidant defenses in Balb/c mice. Biomed. Pharm. 2018, 100, 304–315. [CrossRef] [PubMed]
28. Chowdhury, S.; Ghosh, S.; Das, A.K.; Sil, P.C. Ferulic Acid Protects Hyperglycemia-Induced Kidney Damage
by Regulating Oxidative Insult, Inflammation and Autophagy. Front. Pharmacol. 2019, 10, 27. [CrossRef]
[PubMed]
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).