Preparation and in vitro evaluation of amphiphilic paclitaxel small molecule prodrugs and enhancement of oral absorption

Article abstract of DOI:10.1016/j.ejmech.2021.113276

A series of novel amphiphilic paclitaxel (PTX) small molecule prodrugs, PTX-succinic anhydride-cystamine (PTX-Cys), PTX-dithiodipropionic anhydride (PTX-SS-COOH) and PTX-succinic anhydride-cystamine-valine (PTX-SS-Val) were designed, synthesized and evaluated against cancer cell lines. Compared with paclitaxel, these prodrugs contained water-soluble groups such as amino, carboxyl and amino acid, which improved the aqueous solubility of the prodrugs. More importantly, the valine was introduced in PTX-SS-Val molecule and made the molecule conform to the structural characteristics of intestinal oligopeptide transporter PEPT1 substrate. Thus the oral bioavailability of prodrug could be improved because of the mediation of PEPT1 transporter. These small molecule paclitaxel prodrugs could self-assemble into nanoparticles in aqueous solution, which effectively improved the solubility of paclitaxel, and had certain stability in pH 6.5, pH 7.4 buffer solutions and simulated gastrointestinal fluids. Some of these prodrugs, especially for PTX-Cys and PTX-SS-Val, exhibited nearly equal or slightly better anticancer activity when compared to paclitaxel. Further studies on PTX-Cys and PTX-SS-Val showed that both had good intestinal absorption in the rat single-pass intestinal perfusion (SPIP) experiments. Oral pharmacokinetic experiments showed that PTX-SS-Val could effectively improve the oral bioavailability of PTX.

Full text of DOI:10.1016/j.ejmech.2021.113276

European Journal of Medicinal Chemistry 215 (2021) 113276
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preparation and in vitro evaluation of amphiphilic paclitaxel small
molecule prodrugs and enhancement of oral absorption
,
,
,
, c, *
Yuanyuan Li a 1, Min Yang a 1, Yanli Zhao d, Lingbing Li a **, Wei Xu b
a Department of Pharmaceutics, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Cheeloo College of
Medicine, Shandong University, 44 Wenhuaxi Road, Jinan, Shandong Province, 250012, China
b Shandong Provincial Qianfoshan Hospital, The First Hospital Affiliated with Shandong First Medical University, China
c Shandong Provincial Qianfoshan Hospital, Shandong University, China
d Shandong Mental Health Center, Jinan, Shandong Province, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel amphiphilic paclitaxel (PTX) small molecule prodrugs, PTX-succinic anhydride-cyst-
amine (PTX-Cys), PTX-dithiodipropionic anhydride (PTX-SS-COOH) and PTX-succinic anhydride-cyst-
amine-valine (PTX-SS-Val) were designed, synthesized and evaluated against cancer cell lines. Compared
with paclitaxel, these prodrugs contained water-soluble groups such as amino, carboxyl and amino acid,
which improved the aqueous solubility of the prodrugs. More importantly, the valine was introduced in
PTX-SS-Val molecule and made the molecule conform to the structural characteristics of intestinal oli-
gopeptide transporter PEPT1 substrate. Thus the oral bioavailability of prodrug could be improved
because of the mediation of PEPT1 transporter. These small molecule paclitaxel prodrugs could self-
assemble into nanoparticles in aqueous solution, which effectively improved the solubility of pacli-
taxel, and had certain stability in pH 6.5, pH 7.4 buffer solutions and simulated gastrointestinal fluids.
Some of these prodrugs, especially for PTX-Cys and PTX-SS-Val, exhibited nearly equal or slightly better
anticancer activity when compared to paclitaxel. Further studies on PTX-Cys and PTX-SS-Val showed that
both had good intestinal absorption in the rat single-pass intestinal perfusion (SPIP) experiments. Oral
pharmacokinetic experiments showed that PTX-SS-Val could effectively improve the oral bioavailability
of PTX.
Received 14 December 2020
Received in revised form
1 February 2021
Accepted 2 February 2021
Available online 6 February 2021
Keywords:
Paclitaxel
Small molecule prodrug
Oral administration
Transporter PEPT1
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
formulations are administered by intravenous injection, which has
poor patient adherence, severe toxic side effects associated with
Paclitaxel (PTX) is a microtubule inhibitor with a highly effective
anti-tumor effect and is a clinical first-line chemotherapeutic drug,
which is effective against a broad range of solid tumors, including
refractory ovarian cancer, metastatic breast cancer, non-small-cell
lung cancer, AIDS-related Kaposi’s sarcoma, head and neck malig-
nancies and other cancers [1]. However, the clinical utilization of
excipients. For example, clinically, PTX is currently dissolved in a
50/50 (v/v) mixture of Cremophore EL/dehydrated ethanol as
commercial Taxol® or Paxene®, which is further diluted in isotonic
saline solution before intravenous (i.v.) administration. Unfortu-
nately, i.v. administration of the current Cremophore EL-based
formulation in a non-aqueous vehicle may lead to serious side ef-
fects in some patients such as hypersensitivity, neurotoxicity,
nephrotoxicity, and precipitation on aqueous dilution [3]. Based on
the above problems, there is a need for the development of alter-
native formulations of PTX to solve these problems.
PTX is primarily limited by poor solubility (0.3e0.5 mg/mL) and low
membrane permeability [2]. At present, most marketed PTX
Oral administration is more flexible, safe and convenient.
Compared with intravenous injection, oral administration is a
much more preferable choice for PTX. It shows better patient
compliance, less cost and more chronic treatment regimen. How-
ever, as a result of the poor solubility (0.25 mg/mL) and low
permeability across the intestinal barrier, the oral bioavailability of
* Corresponding author. Shandong Provincial Qianfoshan Hospital, the first
Hospital Affiliated with shandong First Medical University, China.
** Corresponding author. Department of Pharmaceutics, School of Pharmaceutical
Sciences, Shandong University, Jinan, Shandong Province, 250012, China.
E-mail addresses: cnlbli@email.sdu.edu.cn (L. Li), 13964016747@126.com
(W. Xu).
1
The first two authors contributed equally to this study.
https://doi.org/10.1016/j.ejmech.2021.113276
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

Y. Li, M. Yang, Y. Zhao et al.
European Journal of Medicinal Chemistry 215 (2021) 113276
PTX is less than 10% [4]. For example, the first-pass effect of the
gastrointestinal tract and the affinity of drugs to efflux proteins (P-
glycoproteins) are the main factors affecting oral bioavailability [5].
Thus, the design of PTX small molecular prodrugs to improve the
solubility and permeability across the intestinal barrier is prefer-
able choice. Among the numerous drug transporters in the intes-
tine, oligopeptide transporter (PEPT1) is one of the important
targets in the design of oral targeted prodrugs because of its sub-
strate structure specificity, high expression and transport capacity.
PEPT1 is mainly expressed in small intestinal epithelial cells and
has higher protein activity. PEPT1 substrates are mainly dipeptides,
tripeptides and their analogues produced by protein hydrolysis [6].
Peptide-mimetic derivatives formed by drug-amino acid linkage
can also serve as PEPT1 substrates. In addition, as the demand for
nutrients in malignant proliferation of tumors increases, the L-type
amino acid transporter 1 (LAT1) of tumor cells are more highly
expressed than normal cells which can be used as targets for active
targeting of cancer [7]. Tao et al. found that modified by PEPT1
amines, which resulted in significantly increased tumor penetra-
tion of the conjugate [22]. Thus, application of positively charged
delivery systems can thereby promote the uptake of tumor cells
and intestinal absorption.
In order to realize responsive release at tumor sites, chemical
groups sensitive to external magnetic field, temperature, ultra-
sound, light, current and physiological signals (enzyme, pH, Redox
potential) have been widely used in drug delivery system design
[23]. It has been reported that there is a significant difference in
reduced glutathione (GSH) concentration between the extracellular
and intracellular tumor microenvironment, enabling prodrugs
containing disulfide bonds to effectively release the parent drugs in
tumor cells and exert better therapeutic efficacy [24].
In this work, a redox-responsive small molecular PTX prodrug
containing a disulfide bond, PTX-succinic anhydride-cystamine
(PTX-Cys) with an exposed amino group, was successfully synthe-
sized. The exposed dissociable amino group could improve the
solubility and dispersion of PTX in aqueous solution and the di-
sulfide bonds could be degraded in highly reductive tumor micro or
intracellular environment to promote the PTX release in tumor
tissues. Based on this, a novel type of prodrug was developed. PTX-
substrate, L
-valine, the oral bioavailability of cytarabine prodrug, 50-
valine-cytarabine, in rats was increased to 60% compared with
cytarabine (20.8%), which is why the clinical trial of 50-valine
cytarabine was approved by CFDA [8]. These studies show that
using PEPT1 targeted amino acid prodrugs can effectively improve
the oral bioavailability of drugs.
Cys and PEPT1 substrate, L-valine (Val), were linked by amide bonds
and the prodrug, PTX-succinic anhydride-cystamine-Valine (PTX-
SS-Val), was synthesized. It can be predicted that this prodrug could
be mediated by PEPT1 transporter to across the intestinal cell
membrane and the oral bioavailability of paclitaxel could be
effectively improved. In addition, PTX-dithiodipropionic anhydride
(PTX-SS-COOH) with an exposed carboxyl group was also suc-
cessfully synthesized as a control. The exposed dissociable carboxyl
group could improve the solubility and dispersion of PTX in
aqueous solution and the disulfide bonds could be degraded in
highly reductive tumor micro or intracellular environment to pro-
mote the PTX release in tumor tissues. To test the effect of different
prodrugs on the membrane permeability and inhibition efficiency
of tumor cells, the cytotoxicity and cell uptake test of the PTX
prodrugs at cellular level were measured and the mechanism was
investigated. Furthermore, the effects of prodrugs on intestinal
absorption and transmembrane transport were studied in rats.
Animal experiments have proved that the transporter PEPT1 can
realize the transmembrane transport of intestinal cells through
PEPT1 mediated transport of PTX-SS-Val, effectively improving the
oral bioavailability of paclitaxel.
In recent years, amphiphilic small molecule prodrug based
systems have shown significant advantages in cancer treatment
and disease diagnosis. Through chemical modification of the parent
drug, we can improve the solubility and stability of the drug,
improve the distribution in the body and the permeability of bio-
logical membranes. Also avoiding or reducing the use of polymeric
carrier materials in the formulation, we can reduce toxicity and
improve biosafety effectively [9]. Many researchers combined hy-
drophobic drugs with hydrophilic or hydrophobic organic mole-
cules, such as polymer chains [10], peptides [11], fluorescent
substances [12], drug molecules [13], phospholipids or DNA/anti-
bodies [14e16], to form amphiphilic small molecule prodrug that
can self-assemble into nanoparticles in aqueous solution. Through
this way, the shortcomings of parent drugs as described above
could be largely overcome. Most of the small molecule prodrugs
were designed to overcome paclitaxel’s low water solubility or
increase the targeting ability of paclitaxel. Hydrophilic molecules
such as carboxylic acids, phosphates, sulfonates, amines, or poly-
ethylene glycol, sugar, etc. have been exploited as backbones for
PTX prodrugs, resulting in significantly improved water solubility
[17]. However, because of the high hydrophilicity, most prodrugs
were faced with poor cell uptake or intracellular process [18]. The
other way for the small molecule paclitaxel prodrug was to make
paclitaxel with hydrophobic moiety. These hydrophobic paclitaxel
prodrugs were more suitable for nanoparticle drug delivery sys-
tems, which could increase the accumulation in tumor tissues
because of enhanced permeability and retention effect (EPR effect)
of nanoparticles [19]. However, high-level tumor accumulation of
nanoparticles does not directly correlate to the clinical efficacy,
which is more dependent on the rate of drug released [20].
As can be found in literature, modifying drugs or drug loaded
carriers with some positively charged groups or groups with ten-
dency of ionization in specific biological microenvironment can
effectively promote the permeability of drugs or carriers across cell
membrane, which provides new ideas for designing tumor specific
drugs or drug loaded carriers [21]. Zhou and co-workers developed
2. Materials and methods
2.1. Materials
PTX was obtained from Mellon, Dalian, China; Succinic Anhy-
dride (SA), Cystamine Dihydrochloride (Cys), Dithiodipropionic
(DTDP), Boc-L-Valine (Val), 1-hydroxybenzotriazole (HOBT), 1-(3-
dimethylaminopropyl)-3-ethyl carbonimide hydrochloride (EDCI),
Tween 80, Dichloromethane (DCM) were all purchased from
Aladdin,
Shanghai,
China;
3-(4,5-dimethylthiazole-2)-2,5-
diphenyltetrazolium bromide (MTT), Dialysis bag (MWCO500D),
Pepsin, Trypsin, AnnexinV-FITC/PI apoptosis detection kit were all
obtained from Solarbio, Beijing, China; Glycyl-Sarcosine (Gly-Sar)
was purchased from Xianding Biology, Shanghai, China; Other
solvents were supplied by Zhaoxu, Jinan, China and Fuyu, Tianjin,
China.
Human breast cancer (MCF-7) cells were obtained from Institute
of Immunopharmacology, Shandong University School of Phar-
macy. Male Wistar Rats were supplied by Medical Animal Test
Center of the New Drugs Evaluation Center, Shandong University.
amphiphilic
(PEAGA)-Camptothecin based enzyme sensitive polymeric micelles
for delivering camptothecin. The overexpressed -glutamyl trans-
peptidase on the tumor cell membrane cleaved the -glutamyl
moieties of the conjugate to generate positively charged primary
poly-2-(l-
g-glutamylamino)
ethyl
acrylamide
g
g
2

Y. Li, M. Yang, Y. Zhao et al.
European Journal of Medicinal Chemistry 215 (2021) 113276
2.2. Synthesis of PTX-Cys, PTX-SS-Val and PTX-SS-COOH
2.3.2. Measurement of appearance and size distribution of
nanoparticles
2.2.1. Synthesis of PTX-Cys
The appearance of PTX prodrug nanoparticles was observed by
transmission electron microscopy (TEM, JEM-100SX, Japan). A drop
of solution was placed on a copper grid for TEM and the excess fluid
was absorbed with a piece of filter paper from the edge of the
copper disk. Before observation, copper grid containing sample
solution was immediately stained with a drop of phosphotungstic
acid (2 wt% aqueous solution) for 30 s.
The small molecule prodrug, PTX-Cys, was obtained by amida-
tion of PTX-Succinic anhydride conjugate (PTX-COOH) (see sup-
porting information) and cystamine. PTX-COOH (50 mg,
0.05 mmol), EDCI (15 mg, 0.078 mmol), HOBT (10.5 mg,
0.078 mmol) were dissolved in DMF and stirred at 0 ꢀC for 6 h under
nitrogen atmosphere. Additionally, cystamine hydrochloride
(35.5 mg, 0.158 mmol) and TEA (48
m
l) were dissolved in DMF under
The particle size and size distribution of PTX-Cys, PTX-SS-Val
and PTX-SS-COOH nanoparticles were measured by a laser particle
size analyzer (Malvern, U.K.).
ultrasound for 2 h and mixed with above low-temperature solution.
The reaction was proceeded for 24 h at 25 ꢀC. The resulting solution
was added into ice water, the product was precipitated, centrifuged
(8000 rpm, 5 min). The supernatant was discarded and the pre-
cipitate was lyophilized, and purified by column chromatography
(dichloromethane-methanol system) to obtain PTX-Cys. The
structure of PTX-Cys was confirmed by 1H NMR (solvent: deuter-
ated DMSO, DMSO‑d6), FT-IR and MS.
2.4. In vitro stability of PTX prodrugs
2.4.1. Investigation of chemical stability of PTX prodrugs in buffer
solutions
The dialysis method was employed to study the stability of PTX
prodrugs in disodium hydrogen Phosphate-Citric acid buffer (pH
7.4 and 6.5) containing 0.5% Tween 80. The PTX release in buffer
solution in pH 6.5 with or without dithiothreitol (DTT) (10 mM) was
also measured to evaluate the redox stability of prodrugs. Briefly, an
appropriate amount of prodrug (2.5 mg PTX) was weighed and
dissolved in the above three buffer solutions (pH 6.5, pH 7.4, pH 6.5
containing DTT) and diluted to 6 mL. 2 mL of the above solution was
moved into the dialysis bag (MWCO ¼ 1000, n ¼ 3), and then the
bag was put into a centrifuge tube filled with 35 mL same buffer
solution and shaken in a water bath at 37 ꢀC, 100 rpm. At pre-
determined interval (1, 2, 4, 8, 12, 24, 36, 48, 72, 96, 120 h), 1 mL of
release medium was removed from centrifuge tube to determine
the PTX content and 1 mL corresponding isothermal buffer solution
was supplemented.
2.2.2. Synthesis of PTX-SS-Val
The small molecule prodrug PTX-SS-Val was obtained by ami-
dation of PTX-Cys and L-valine. Boc-L-valine (20 mg, 0.092 mmol),
EDCI (26.5 mg, 0.138 mmol), HOBT (18.6 mg, 0.138 mmol) were
dissolved in DMF, stirred at 0 ꢀC for 6 h under nitrogen atmosphere,
and then 50 mg of PTX -Cys was added into the solution to proceed
for additional 24 h. The resulting solution was added to ice water,
the product was precipitated, centrifuged (8000 rpm, 5 min), and
the supernatant was discarded to remove water-soluble impurities.
An appropriate amount of ethyl acetate was added to extract the
product. The organic phase was mixed and a small amount of
anhydrous sodium sulfate was added to dry overnight. The product
was stirred in dichloromethane (DCM) solution containing 15% TFA
at room temperature for 2 h, the protective group of Boc-
L
-valine
The PTX content was measured by high performance liquid
chromatography (HPLC) at 227 nm (Elite Hypersil ODSC18
was removed [25], and the reaction progress was monitored by thin
layer chromatography (TLC). After the reaction, the solvent was
removed by vacuum rotary evaporation method, and the crude
product was purified by column chromatography (dichloro-
methane-methanol system) to obtain PTX-SS-Val. The structure of
PTX-SS-Val was confirmed by 1H NMR (solvent: deuterated DMSO,
DMSO‑d6) and FT-IR.
4.6 ꢁ 250 mm, 5
acetonitrile: water ¼ 50:50 (v/v), and the flow rate was 1 mL/min,
the injection volume was 20 L, the column temperature was room
mm particle size). The mobile phase consisted of
m
temperature [26]. The cumulative release of PTX was further
calculated according to the standard curve to investigate the sta-
bility of PTX prodrug in three buffer solutions.
2.2.3. Synthesis of PTX-SS-COOH
2.4.2. Investigation on the stability of PTX prodrugs in simulated
gastric (SGF) and intestinal fluid (SIF)
PTX-SS-COOH was synthesized with methods reported previ-
ously. Briefly, PTX-SS-COOH was synthesized by esterification of
DTDPA (see supporting information) and 2’-OH of Paclitaxel. The
mixture of PTX (0.030 g, 0.035 mmol), DTDPA (0.067 g, 0.35 mmol)
and dimethylaminopyridine (DMAP, 0.034 g, 0.29 mmol) in pyri-
dine was stirred under nitrogen atmosphere below 35 ꢀC for 24 h.
The system was concentrated through rotary evaporation under
reduced pressure and 1% (m/V) HCl solution was added to take
away residual pyridine. Product in aqueous phase was extracted
with DCM three times and the organic phase was collected. The
organic solvent was removed and dried in vacuum overnight. The
product was characterized with 1H NMR, FT-IR and MS.
The stability of PTX prodrugs in SGF and SIF (contains 0.5%
Tween 80) was investigated by dialysis bag method same as 2.4.1.
According to the time rule of human gastric emptying and intes-
tinal emptying, 1 mL of SGF was sampled at 0, 2, 4 h, and 1 mL of SIF
was sampled at 0, 2, 4, 6, 8, 12 h, and fluid was replenished at same
time. By HPLC, the sample concentrations at different time points
were determined and calculated according to the standard curve,
and the cumulative release of PTX was further calculated to
investigate the stability of PTX prodrugs in simulated gastric and
intestinal fluid.
2.5. Cytotoxicity experiments
2.3. Preparation and characterization of paclitaxel prodrug
nanoparticles
In this section, cytotoxicity of Taxol®, PTX, PTX-Cys, PTX-SS-Val
and PTX-SS-COOH solutions was evaluated with MTT assay. MCF-
7 cells in logarithmic phase were digested with trypsin, diluted
with prepared DMEM medium to suitable cell density and trans-
ferred to 96-well plate at a density of 5000 cells/well. Cells were
incubated overnight till cells were in adherent growth for experi-
ment and then blank medium was substituted with filtered solu-
tion containing different concentrations (0.1, 0.5, 1, 5, 10 g/mL) of
Taxol®, PTX, PTX-Cys, PTX-SS-Val and PTX-SS-COOH (dissolved in
2.3.1. Preparation of paclitaxel prodrug nanoparticles
The nanoparticles of PTX prodrugs were prepared by nano-
precipitation method.
1 mg of PTX prodrug was precisely
weighed and dissolved in 1 mL methanol and added dropwise into
5 mL deionized water under stirring. The organic solvent was
volatilized by stirring, and the solution was transferred to a dialysis
bag (MWCO500D) for dialysis against deionized water for 24 h.
3

Y. Li, M. Yang, Y. Zhao et al.
European Journal of Medicinal Chemistry 215 (2021) 113276
DMSO and diluted by medium), in which sterile DMSO was below
2.8.1. The single-pass intestinal perfusion (SPIP) in the intestine
This part of the test was carried out based on a reported method
[28] and all the animal experiments were approved by the Shan-
dong University Animal Care and Use Committee. Healthy male
Wistar rats weighing 250 20 g were fasted for approximately 12 h
before the perfusion experiment with free access to water only.
Anesthesia was induced by intraperitoneal injection of 10% chloral
hydrate solution (0.34 g/kg). Then a midline abdominal incision
was made and the experimental intestinal segment of approxi-
mately 8 cm was carefully exposed and cannulated at both ends.
The exposed segment was covered with a piece of medical gauze
soaked in normal saline. The body temperature of animals was
maintained at 37 ꢀC throughout the experiment using an infrared
lamp. Both ends of the experimental intestinal segment were
connected with a constant-flow pump. Warm saline passed slowly
through the tract to clear the gut. After gut cleared, the PTX for-
mulations, diluted in perfusion solution, were perfused circularly
through the selected intestine at an entering flow rate of 0.20 mL/
min. After 10 min of uniform circulation and 2 h of stable perfusion,
the volume of perfusion solution in the measuring cylinder was
recorded and 1 mL of perfusion solution sample was taken. At the
end of the experiment, the rats were sacrificed by cervical dislo-
cation. The length and inner diameter of each intestinal segment
were measured. Each sample was repeated in three parallel groups.
1
‰. Cell viability was measured by MTT assay. Each concentration
was repeated for five times, and negative control group (just me-
dium, no cells and formulations) and positive control group (cells,
medium, but no formulations) were set. The 96-well plate was
incubated for 48 h and washed three times by PBS. 20
solution (5 mg/mL) was added into each well and incubated for
additional 4 h. Thereafter, the medium was removed and 200 L of
mL of MTT
m
DMSO was added to dissolve formazan crystals. The absorbance
(OD) was measured by ELIASA at 570 nm and the cell viability was
calculated.
2.6. Cell uptake experiments
In vitro cell uptake of three PTX prodrugs was evaluated by
inverted fluorescence microscopy. Since PTX has no fluorescence,
the fluorescent substance coumarin 6 (C6) was encapsulated in the
hydrophobic core of three PTX prodrug nanoparticles by nano-
precipitation method in Ref. [27]. MCF-7 cells in logarithmic growth
phase were seeded in 6-well plate at a density of 3 ꢁ 105 cells/well
and cultured overnight. The cells were treated with C6-loaded
prodrug nanoparticles for 4 h at a total drug concentration of
5 mg/mL and C6 at the concentration of 2 mg/mL. The cells were
washed three times with cold PBS and fixed with 4% para-
formaldehyde solution for 20 min at 4 ꢀC in darkness. After drawing
away paraformaldehyde solution, cells were washed 5 times with
PBS buffer solution (pH 7.4) and the nuclei of cells were stained
2.8.2. Sample and data processing methods
Due to the polarity of prodrugs and the interference of perfusate
components, the specificity of high performance liquid chroma-
tography for detection of different PTX prodrugs is poor. Thus, ac-
cording to Ref. [29], the prodrug in perfusate was hydrolyzed by HCl
(3 M) at 85 ꢀC for 2 h before measurement and PTX obtained was
used as precursor to detect the change of PTX prodrug concentra-
tion in perfusate using the same method as described in 2.4.1. The
apparent permeability coefficient (Peff) and absorption flux (J) of
prodrugs in each intestinal segment were calculated using the
following equations [30]:
with 100 ml DAPI solution (10 mg/mL) for 20 min in a dark envi-
ronment with 5% CO2 at 37 ꢀC.Finally, the cells were observed under
fluorescent microscope (OLYMPUS, Tokyo, Japan).
2.7. Cell apoptosis analysis
Apoptotic experiments were performed by inverted fluores-
cence and flow cytometry, in which changes in the nucleus could be
observed under inverted fluorescence microscopy, and apoptosis
could be quantitatively detected by flow cytometry.
Q
Cout ꢁ Vout
Cin ꢁ Vin
Peff ¼ ꢂ
ꢁ Inð
Þ
2.7.1. Inverted fluorescence microscopy
PTX, PTX-Cys, PTX-SS-Val and PTX-SS-COOH solutions with PTX
concentration of 2.5 mg/mL were prepared respectively according to
2p
rl
J ¼ Peff ꢁ ½Cꢃ
where Cout and Cin represent the concentration of tested com-
2.5. MCF-7 cells in logarithmic growth phase were seeded in 12-
well plate at a density of 1 ꢁ 105 cells/well and cultured over-
night. The cells were treated with different sample solutions and
each group of samples was repeated three times. MCF-7 cells in
blank medium were set as a negative control group. The cells were
incubated for 48 h respectively and washed with cold PBS three
times. The other steps were the same as 2.6, and the morphological
changes of the nucleus were finally observed under an inverted
microscope.
pounds in the outlet and inlet perfusate, respectively ( g/mL); Vout
m
and Vin are the outlet and inlet volume, respectively (mL). Q is the
flow rate of entering solution (0.2 mL/min), while r is the intestinal
radius and l is the intestinal length; [C] is the logarithmic mean of
Cin and Cout (mg/mL).
2.8.3. Effect of different pH on intestinal absorption
To investigate the effect of perfusate pH on intestinal absorption,
pH 6.5 MES buffer and pH 7.4 HEPES buffer (70 mM NaCl, 2.7 mM
KCl, 0.9 mM CaCl2, 2.5 mM D-glucose,12.5 mM MES or HEPES, NaOH
and 0.5% Tween 80) were prepared and used to prepare solutions of
PTX, PTX-Cys and PTX-SS-Val respectively (PTX concentrations
were all 100 mg/mL). The single-pass intestinal perfusion (SPIP) of
2.7.2. Flow cytometry
The formulation solutions used to treat cells were the same as
2.7.1, and the operation steps were the same as 2.6. Apoptosis of
PTX prodrugs was further quantified by flow cytometry using
Annexin V-FITC/PI kit.
prodrug samples was performed in the jejunal segment for 2 h. The
concentration of PTX was determined by HPLC as described in 2.8.2.
Further, the apparent permeability coefficient (Peff) of PTX, PTX-Cys
and PTX-SS-Val was calculated respectively.
2.8. The absorption of small molecule prodrugs in the intestine
The oral absorption characteristics of PTX-Cys and PTX-SS-Val
were preliminarily evaluated by single-pass intestinal perfusion
(SPIP) in rats. The effects of pH value, different drug concentrations
and different intestinal segments on intestinal absorption of drugs
were investigated, and the intestinal absorption mechanisms of
PTX-Cys and PTX-SS-Val were explored.
2.8.4. Effect of drug concentration on intestinal absorption
To investigate the effect of drug concentration on intestinal
absorption, Kerbs-Ringer solution (67 mM NaCl, 2.4 mM KCl,
4

Y. Li, M. Yang, Y. Zhao et al.
European Journal of Medicinal Chemistry 215 (2021) 113276
1.7 mM CaCl2, 0.1 mM MgCl2, 1.3 mM NaH2PO4, 8.1 mM NaHCO3,
amount of ethyl acetate, and the organic phase was removed by
3.8 mM
D-glucose and 0.5% Tween 80) was prepared and different
rotary evaporation. Finally, the sample was reconstituted in 200 mL
formulations of PTX-Cys and PTX-SS-Val with different concentra-
tions were prepared respectively using Kerbs-Ringer solution. The
concentration gradients of PTX solutions were 70, 100, 120 and
acetonitrile and the plasma concentrations of PTX in different for-
mulations at different time points were detected by HPLC. Phar-
macokinetic parameters were calculated using
a
non-
150
m
g/mL. The concentration of PTX prodrugs was determined by
compartmental model by DAS2.0 software, and the absolute
bioavailability (F%) of PTX from different oral formulations was
examined using the following equation:
HPLC as described in 2.8.2. Further, the apparent permeability co-
efficient (Peff) of PTX, PTX-Cys and PTX-SS-Val was calculated
respectively.
In addition, following the end of the concentration-dependent
experiment of PTX-SS-Val perfusion, the tested intestine segment
was removed and washed with ice-cold saline. The tissue was then
homogenized in a tissue homogenizer and diluted with iso-
volumetric saline solution. After centrifugation at 13000 rpm for
5 min, the supernatant was stored at ꢂ80 ꢀC until analysis by HPLC.
AUCoral ꢁ Di:v:
F% ¼
ꢁ 100%
AUCi:v: ꢁ Doral
where AUCoral and AUCi.v. were the areas under the plasma
concentrationetime curve of PTX for the oral and intravenous
administration, respectively; Di.v. and Doral were the dosages of PTX
for the intravenous and oral administration, respectively.
The PTX content was measured by the HPLC technique
described above with an Agilent 1200 HPLC system consisting of a
G1314B UV detector and a G1310A pump, equipped with a RP col-
2.8.5. The absorption of PTX prodrugs in the different intestinal
segments
To explore the absorption of PTX, PTX-Cys and PTX-SS-Val in
different intestinal segments, SPIP was conducted in duodenum,
jejunum and ileum segments, respectively. PTX, PTX-Cys and PTX-
SS-Val dissolved in above Kerbs-Ringer solution (PTX concentra-
umn (Elite Hypersil ODSC18 4.6 ꢁ 250 mm, 5
mm particle size) at
227 nm. The mobile phase consisted of acetonitrile: water ¼ 50:50
(v/v), and the flow rate was 1 mL/min, the injection volume was
20 mL, the column temperature was room temperature.
tions were all 100 mg/mL) were perfused through different intes-
tinal segments respectively. The concentration of PTX prodrugs was
determined by HPLC as described in 2.8.2. The apparent perme-
ability coefficient (Peff) of PTX, PTX-Cys and PTX-SS-Val in duo-
denum, jejunum and ileum was calculated respectively.
2.11. Statistical analysis
Statistical evaluation was performed by one-way analysis of
variance (ANOVA) using SPSS software (version 23.0). All the ex-
periments were repeated at least three times, and the data were
expressed as the mean standard deviation (S.D.), unless other-
wise noted, p < 0.05 was considered statistically significant.
2.9. Investigation of the effect of inhibition PEPT1 on PTX-SS-Val
transport
Gly-Sar is a classical substrate of the transporter PEPT1. In order
to investigate the inhibition effect of Gly-Sar to PEPT1 mediation
transport of PTX-SS-Val, the Gly-Sar was added in pH 7.4 HEPES
solution (12.5 mM HEPES, 70 mM NaCl, 2.7 mM KCl, 0.9 mM CaCl2,
2.8 mM glucose, 22.5 mM Gly-Sar and 0.5% Tween 80) and co-
3. Results and discussions
3.1. Synthesis and characterization of PTX-Cys, PTX-SS-Val and PTX-
SS-COOH
perfused with PTX and PTX-SS-Val (PTX concentration of 100
mL) respectively. The concentration of PTX in both solutions was
100 g/mL. The concentration of PTX and PTX-SS-Val was deter-
mined by HPLC as described in 2.8.2. The apparent permeability
coefficient (Peff) was calculated respectively.
mg/
The synthesis procedures of PTX-Cys, PTX-SS-Val and PTX-SS-
COOH were displayed in Fig. 1. The characterization of PTX-
Succinic anhydride conjugate (PTX-COOH) was shown in Fig. S1.
And the 1H NMR of DTDP and DTDPA were shown in Fig. S2. The 1H
NMR and FT-IR spectra of the three target compounds were shown
in Fig. 2 and Fig. 3, respectively.
m
2.10. Oral pharmacokinetic experiments
PTX-Cys was obtained by amide reaction between the amino
group of cystamine and the carboxyl group of PTX-COOH and the
yield of PTX-Cys was 63.81%. The 1H NMR, FT-IR spectra of PTX-Cys
were shown in Figs. 2B and 3B. In the 1H NMR spectrum of PTX-Cys
(Fig. 2B), the hydrogen proton peaks attributed to PTX benzene ring
appeared at 7.0e8.0 ppm, the characteristic peak attributed to
cystamine appeared at 3.45 ppm, and the carboxyl peak of PTX-
COOH at 12.18 ppm (Fig. S1) disappeared. Besides, the FT-IR spec-
trum of PTX-Cys (Fig. 3B) showed that the double absorption peaks
of amino-linked methylene appeared at 2850.38 cmꢂ1 and
2920.62 cmꢂ1, while the peak at 2800-3700 cmꢂ1 attributed to
carboxyl broad absorption of PTX-COOH disappeared (Fig. S1) and
Male Wistar rats (weighing 250 20 g) were fasted overnight
with free access to water for 12 h before the experiment. The rats
were divided randomly into two groups (n ¼ 5) and administered
Taxol® and PTX-SS-Val (containing 0.5% carboxymethyl cellulose
and 1% Tween 80) at a dose of 25 mg/kg by intragastric adminis-
tration respectively [31]. Taxol® was also administered intrave-
nously to one group of rats at a dose of 6 mg/kg as a control. At
predetermined times (0.25, 0.5,1, 2, 4, 6, 8,12 and 24 h), serial blood
samples (0.2 mL) were collected from the orbital plexus, trans-
ferred to pretreated heparinized tubes and separated immediately
by centrifugation (4000 rpm for 15 min). After centrifugation, the
supernatant plasma obtained was stored at ꢂ20 ꢀC until analysis.
The plasma protein was precipitated by adding a 1 mL mixture
of methanol: acetonitrile ¼ 1:1, vigorous vortex mixing for 5 min
and centrifuging at 10000 rpm for 10 min. Each supernatant layer
was transferred into another glass tube and evaporated under a
gentle stream of nitrogen gas at 37 ꢀC. The residues were recon-
the characteristic absorption peak of PTX appeared at 707.67 cmꢂ1
.
Furthermore, the [MþH]þ ion peak at m/z 1088.6 of PTX-Cys was
confirmed by MS (Fig. S3A), suggesting that the cystamine was
mono-substituted by PTX-COOH, which was consistent with the
molecular weight of the designed mediates. The above results of 1H
NMR, FT-IR and MS spectra of PTX-Cys proved the successful syn-
thesis of PTX-Cys.
stituted in 200 mL acetonitrile. 1 mL of 3 M hydrochloric acid was
added, incubated at 85 ꢀC for 2 h to hydrolyze them thoroughly, and
then 0.85 mL of 0.5% ammonium carbonate (v/v) was added to
terminate the reaction. The mixture was extracted with a large
PTX-SS-Val was obtained by amide reaction between the amino
group of PTX-Cys and the carboxyl group of Boc-
L-valine, after
which the Boc protective group was removed, and the PTX-SS-Val
5

Y. Li, M. Yang, Y. Zhao et al.
European Journal of Medicinal Chemistry 215 (2021) 113276
Fig. 1. Synthesis schemes of (A) PTX-Cys, PTX-SS-Val and (B) PTX-SS-COOH.
was finally obtained. The yield was 43.57%. The 1H NMR and FT-IR
spectra of PTX-SS-Val were shown in Figs. 2C and 3C. In the 1H NMR
spectrum of PTX-SS-Val (Fig. 2C), the hydrogen proton peaks
attributed to PTX benzene ring appeared at 7.0e8.0 ppm and the
dimethyl characteristic absorption of valine appeared at
0.95e1.00 ppm. In addition, compared with the FT-IR spectrum of
PTX-Cys, an obvious stretching vibration absorption peak of N-H
appeared at 3442.16 cmꢂ1 in Fig. 3C, and the characteristic ab-
sorption of PTX appeared at 705.68 cmꢂ1. The above results indi-
cated the successful synthesis of PTX-SS-Val.
For PTX-SS-COOH, as shown in Fig. 2D, the hydrogen proton
peaks attributed to PTX benzene ring appeared at 7.0e8.0 ppm.
Moreover, a new single peak appeared at 12.4 ppm, which was the
carboxyl proton absorption peak in PTX-SS-COOH. The result of FT-
IR was consistent with that of 1H NMR. As shown in Fig. 3D, the
broad absorption peak at 2800-3500 cmꢂ1 was the stretching ab-
sorption of -COOH, while there was no such absorption peak in the
PTX spectrum. Furthermore, the [M ꢂ H]þ ion peak at m/z 1044.09
of PTX-SS-COOH was confirmed by MS (Fig. S3B). To sum up, PTX-
SS-COOH was successfully synthesized.
3.2. Preparation and characterization of PTX prodrug nanoparticles
The PTX prodrug nanoparticles were prepared with nano-
precipitation method. Morphological observations of PTX-Cys,
PTX-SS-Val and PTX-SS-COOH prodrug nanoparticles were per-
formed under TEM. As shown in Fig. 4A, the nanoparticles were
spherical in shape. Particle size distribution of the nanoparticles,
measured by DLS (Fig. 4B), showed that the mean sizes of PTX-Cys,
PTX-SS-Val and PTX-SS-COOH nanoparticles were around
213.57 97.40 nm, 131.00 32.30 nm and 126.57 35.20 nm, and
the size distribution exhibited a unimodal pattern. These results
indicated that the prodrugs could self-assemble into nanoparticles
in aqueous solution and improved the solubility of PTX [32].
6

Y. Li, M. Yang, Y. Zhao et al.
European Journal of Medicinal Chemistry 215 (2021) 113276
Fig. 2. The 1H NMR spectra of PTX (A), PTX-Cys (B), PTX-SS-Val (C) and PTX-SS-COOH (D) in DMSO‑d6.
Fig. 3. The FT-IR spectra of PTX (A), PTX-Cys (B), PTX-SS-Val (C) and PTX-SS-COOH (D).
7

Y. Li, M. Yang, Y. Zhao et al.
European Journal of Medicinal Chemistry 215 (2021) 113276
Fig. 4. (A) The TEM images of nanoparticles (the scale bar: 0.6 mm). (B) The size distribution of nanoparticles.
3.3. In vitro stability of PTX prodrugs
increased, revealing a dose-dependent inhibition behavior. More
importantly, the tumor inhibition efficiency of PTX-Cys was
significantly higher than other groups, which may be due to the
permeability enhancement induced by positively charged amino
group which mediated trans-membrane delivery of PTX-Cys.
The cytotoxicity of Taxol® was obviously enhanced with the
increase of concentration, because polyoxyethylene castor oil and
ethanol in the formulation of Taxol® also had certain cytotoxicity
[33]. The cytotoxicity of PTX-SS-Val was greater than that of free
The chemical stability of PTX prodrugs in buffer solutions of pH
6.5 and pH 7.4 was investigated by dialysis bag method, and
expressed as the cumulative release rate of PTX. The cumulative
release results of PTX were shown in Fig. S4A&B. After 120 h, the
cumulative release of PTX from PTX prodrug was less than 15% in
pH 6.5 buffer solution and less than 12% in pH 7.4 buffer solution.
Overall, more than 85% of prodrugs kept prototype, indicating the
good stability in buffer solutions. However, when 10 mM DTT was
present, the in vitro cumulative release of PTX for three prodrugs in
pH 6.5 buffer solution was significantly accelerated and up to 50%,
58% and 48% was released respectively, exhibiting the redox
sensitivity due to presence of the disulfide bond (Fig. S4C).
A severe difficulty encountered with prodrug nanoparticles after
oral administration is their instability in the complex gastrointes-
tinal environment. The stability of PTX prodrugs in simulated
gastric (SGF) and intestinal fluid (SIF) was investigated by dialysis
bag method and expressed as the cumulative release rate of PTX
(Fig. S4D&E). SGF containing 0.32% (w/v) pepsin and SIF containing
1% trypsin were used as release medium respectively. Less than 1.5%
of PTX released from PTX prodrugs after 4 h in SGF. Meanwhile, in
SIF containing trypsin, less than 3.5% of PTX released from PTX
prodrugs after 12 h. These results showed that the structure of PTX
prodrugs was less affected by pepsin and trypsin, and had good
stability in SGF and SIF. It was deduced that after oral administra-
tion, the PTX prodrugs could be absorbed by small intestinal
epithelial cells with intact structure, which could greatly improve
the oral bioavailability of PTX.
PTX when the drug concentration was less than 1 mg/mL, which
may be mediated by the transporter PEPT1 overexpressed on the
surface of cancer cells [34]. When the drug concentration was
greater than 5 mg/mL, the cytotoxicity of PTX-SS-Val was slightly
weaker than that of free PTX. It was speculated that the release rate
of PTX from PTX-SS-Val molecule was slow, so the cytotoxicity of
PTX-SS-Val was slightly weaker than that of free PTX at high con-
centrations. Finally, PTX-SS-COOH showed the lowest cytotoxicity,
which was attributed to the slow release of PTX from PTX-SS-COOH
and negative charged carboxyl group which delayed the trans-
membrane delivery.
Most of the small molecule prodrugs have been designed to
overcome paclitaxel’s low water solubility or increase the targeting
ability of paclitaxel. Although the prodrugs developed have shown
to improve their water solubility or enhanced targeting ability, this
has yielded compounds with decreased cytotoxicity, which could
be attributable to the inefficient release of paclitaxel from these
compounds or poor cell uptake or intracellular process caused by
high hydrophilicity [35]. To overcome this problem, PTX-Cys and
PTX-SS-Val containing disulfide bonds were developed. The disul-
fide bonds could be degraded in highly reductive tumor micro or
intracellular environment of tumor to promote the PTX release in
tumor tissues. The cytotoxicity of PTX-Cys was significantly higher
than the PTX solution, which may be due to the permeability
enhancement induced by positively charged amino group which
mediated trans-membrane delivery of PTX-Cys. PTX-SS-Val
exhibited nearly equal anticancer activity to the PTX solution,
3.4. Cytotoxicity experiments
Tumor inhibition efficiency of PTX prodrugs was characterized
with MTT assay and the Taxol® and PTX solution in DMSO were
used as control. The results (Fig. 5) showed that when the drug
concentration was increased, the tumor inhibition efficiency was
8

Y. Li, M. Yang, Y. Zhao et al.
European Journal of Medicinal Chemistry 215 (2021) 113276
Fig. 5. The in vitro anticancer activity of PTX and PTX prodrugs.
perhaps due to the permeability enhancement mediated by PEPT1
transporter. The results demonstrated that PTX-Cys and PTX-SS-Val
could be used as potential candidates for anti-tumor drugs.
inverted microscope (Fig. 6). MCF-7 cells were treated with C6
solution, C6 loaded PTX prodrug nanoparticles for 4 h. As shown in
Fig. 6, the green fluorescence (C6 fluorescence) of the nanoparticles
was uniformly dispersed around the blue fluorescence (DAPI
stained cell nucleus), indicating that the nanoparticles were
dispersed in the cytoplasm of MCF-7 cells. The ability of cells to
uptake prodrugs could also be evaluated according to the fluores-
cence intensity. The results showed that after 4 h incubation, the
3.5. Cell uptake experiments
The cellular uptake behavior of C6 loaded PTX-Cys, PTX-SS-Val
and PTX-SS-COOH nanoparticles was investigated by a fluorescent
Fig. 6. The cellular uptake of C6 solution and C6 loaded prodrugs after 4 h.
9

Y. Li, M. Yang, Y. Zhao et al.
European Journal of Medicinal Chemistry 215 (2021) 113276
fluorescence intensity of PTX-Cys and PTX-SS-Val groups was
stronger than that of C6 solution group and PTX-SS-COOH group,
indicating that these two kinds of prodrug nanoparticles were more
conducive to cell absorption. More importantly, the cell uptake of
PTX-Cys group was the highest, which provided evidence for
enhanced tumor permeability of PTX-Cys induced by positively
charged amino group which mediated trans-membrane delivery.
Furthermore, the fluorescence intensity of PTX-SS-Val group was
greater than that of C6 solution and PTX-SS-COOH, which perhaps
due to the mediation of PEPT1 transporter.
quantified by flow cytometry using AnnexinV-FITC/PI apoptosis
detection kit. As shown in Fig. 7B, the proportion of viable cells was
about 100% in the untreated control group. After 48 h of treatment
with PTX, PTX-Cys, PTX-SS-Val and PTX-SS-COOH, the proportion of
viable cells decreased significantly to, 14.9%, 6.93%, 7.69% and 19.7%
respectively, and the proportion of late apoptotic cells was 70.2%,
85.8%, 84.8% and 59.6%, respectively. Compared with the PTX, PTX-
SS-Val and PTX-SS-COOH group, the PTX-Cys group induced the
largest proportion of apoptosis, which could be due to the
enhanced tumor permeability caused by charge characteristics of
PTX-Cys. In addition, the apoptosis proportion of the PTX-SS-Val
group was greater than that of the free PTX group and PTX-SS-
COOH group, demonstrating the enhanced tumor permeability of
PTX-SS-Val caused by the mediation effect of PEPT1 transporter.
These results were consistent with the results of cytotoxicity and
cell uptake in vitro. Thus in next animal experiments, the PTX-Cys
and PTX-SS-Val prodrugs were used as model drugs to evaluate
the potential for improving the oral bioavailability of PTX.
3.6. Cell apoptosis analysis
The fluorescence microscope and flow cytometer were used to
estimate the apoptosis-inducing capabilities of the nanoparticles.
As shown in Fig. 7A, after incubating with the formulation group for
48 h, the nuclei of the control group showed uniform blue fluo-
rescence after DAPI staining, with smooth surface and complete
structure, while the nuclei of cells treated with prodrug formula-
tions showed different degrees of nucleus rupture and shrinkage.
The above experimental results indicated that the PTX-Cys, PTX-SS-
Val and PTX-SS-COOH prodrugs uptake by cells could release
paclitaxel and induce cell apoptosis. The cell apoptosis was further
3.7. The absorption of PTX prodrugs in the intestine
The in vitro cellular evaluation results showed that PTX-Cys and
PTX-SS-Val prodrugs had the superior cytotoxicity and
Fig. 7. (A) The cell apoptosis status of MCF-7 cells. (B) The Annexin V/PI assay on MCF-7 cells.
10

Y. Li, M. Yang, Y. Zhao et al.
European Journal of Medicinal Chemistry 215 (2021) 113276
permeability in vitro. Thus in next animal experiments, the PTX-Cys
and PTX-SS-Val prodrugs were used as model drugs to evaluate the
potential for improving the oral bioavailability of PTX.
respectively, suggesting that neutral conditions were also condu-
cive to the absorption of prodrugs by small intestinal epithelial
cells. This indicated that the targeted transport of PEPT1 was not
proton dependent and its transport efficiency was more dependent
on the structure of the substrates under neutral conditions.
Compared with PTX-Cys, the average Peff of PTX-SS-Val in MES
and HEPES perfusate was 1.27 and 1.17 times than that of PTX-Cys
respectively, but the difference was not significant. This phenom-
enon was presumably due to the transport of PTX-SS-Val mediated
by PEPT1 highly expressed in jejunum. In addition, the electrostatic
interaction of PTX-Cys with the negative intestinal epithelial cells
also promoted the permeation across the intestinal wall, as re-
ported in previous studies [36], which improved its ability to enter
small intestinal epithelial cells.
3.7.1. Effect of different pH perfusates on intestinal absorption
PTX, PTX-Cys and PTX-SS-Val were co-perfused with the two pH
perfusates, respectively. It could be seen that intestinal absorption
of PTX-Cys and PTX-SS-Val in both perfusates was stronger than
that of free PTX (Fig. 8A, Table S1). The Peff of PTX-Cys and PTX-SS-
Val in MES (pH 6.5) perfusate was 1.65 and 2.09 times of free PTX,
respectively. In HEPES (pH 7.4) perfusate, the Peff of PTX-Cys and
PTX-SS-Val was 2.02 and 2.37 times of free PTX, respectively,
indicating that the permeability of the prodrugs was greatly
improved. The average Peff of PTX-Cys and PTX-SS-Val in HEPES
perfusate was 1.31 and 1.21 times than that in MES perfusate,
Fig. 8. (A) The Peff of PTX、PTX-Cys and PTX-SS-Val in MES and HEPES solution. The J of PTX-Cys (B) and PTX-SS-Val (C) in intestinal circulation at different concentrations. (D) The
Peff of PTX and its prodrugs in different intestinal segments.(E) The Peff of PTX and PTX-SS-Val in HEPES Gly-Sar solution.
11

Y. Li, M. Yang, Y. Zhao et al.
European Journal of Medicinal Chemistry 215 (2021) 113276
3.7.2. Effect of different drug concentrations on intestinal
absorption
3.8. Oral pharmacokinetics of PTX prodrugs
To investigate the effect of drug concentration on small intes-
tinal absorption, different concentration gradients of PTX-Cys and
PTX-SS-Val were established, and the results of single-pass intes-
tinal perfusion (SPIP) were shown in Table S2&3 and Fig. 8B&C. It
3.8.1. Tissue accumulation following in situ intestinal perfusion
To assess the metabolism of PTX prodrug in intestinal tissue, the
contents of PTX prodrug and PTX in the intestinal tissue were
determined after the concentration-dependent perfusion experi-
ment. The results showed that 2 h after intestinal perfusion, for
PTX-Cys prodrug, in intestinal tissue, about 42% of drugs existed in
the form of PTX-Cys molecule, about 58% of drugs existed in the
form of PTX. While for PTX-SS-Val, about 22% of drugs existed in the
form of PTX-SS-Val, about 78% of drugs existed in the form of PTX.
The low concentration of prodrug and high concentration of PTX in
the intestine homogenate indicated that the prodrug was hydro-
lyzed partly within the enterocytes via an intestinal first-pass
effect.
could be seen from Fig. 8B that in the range of 70e150 mg/mL, with
the increase of concentration, the absorption of PTX-Cys in the
small intestine increased continually and there was no apparent
transport saturation phenomenon. The Peff did not show obvious
differences under different drug concentrations, which showed
first-order kinetic absorption characteristics suggesting that pas-
sive diffusion mechanism played a major role in intestine absorp-
tion. However, for PTX-SS-Val prodrug (Fig. 8C), with the increase of
concentration, intestinal absorption began to increase, and then
gradually reached a stable state, showing a different absorption
mechanism from PTX-Cys. It is speculated that the PEPT1 trans-
porter mediating transport of PTX-SS-Val gradually reached satu-
ration with the increase of concentration, which made the
absorption of PTX-SS-Val tend to be flat.
3.8.2. Oral pharmacokinetics evaluation
As the prodrugs would be partly transformed into PTX via the
intestinal first-pass effect, therefore, the drug concentration in
plasma was determined through hydrolyzing the prodrug to mea-
sure the whole PTX (free and hydrolyzed PTX) and calculated the
pharmacokinetic parameters. The pharmacokinetics of PTX for-
mulations under different routes of administration were analyzed
by DAS2.0 software, and the results were shown in Table 1. Plasma
concentration-time profiles of PTX following administration of
different PTX formulations were shown in Fig. 9. According to the
pharmacokinetic parameters, after 24 h of intragastric adminis-
tration, the AUC of Taxol® group was 15.051 mg/L*h, while that of
PTX-SS-Val group was 23.863 mg/L*h, which was 1.56 times higher
than that of Taxol® group. The Cmax of PTX-SS-Val group was
1.2383 mg/L, which was significantly higher than that of Taxol®
group, and the absolute bioavailability (F) increased from 19.31% to
30.61%. This phenomenon could be explained by the improved
water solubility of drugs and the transport of PTX-SS-Val mediated
by the transporter PEPT1, which significantly promoted the ab-
sorption of PTX-SS-Val and significantly enhanced the oral
bioavailability of PTX. The clearance of PTX-SS-Val (0.838 L/h/kg)
was about 1.6 times less that of Taxol® (1.329 L/h/kg). The main
reason for this was that the high water solubility of PTX-SS-Val
made it more likely to stay in the blood stream.
3.7.3. The absorption of PTX prodrugs in different intestinal
segments
The absorption of PTX, PTX-Cys and PTX-SS-Val in jejunum,
ileum and duodenum was also carried out. The results (Fig. 8D)
showed that PTX, PTX-Cys and PTX-SS-Val could be effectively
absorbed in different intestinal segments. Compared with other
intestinal segments, PTX-SS-Val had the best absorption in the
jejunum, though difference was not significant compared with
PTX-Cys, which further proved that the transport mediated by
transporter PEPT1 promoted the absorption of PTX-SS-Val. How-
ever, in other two segments the prodrugs had the similar absorp-
tion efficiency especially for PTX-Cys and PTX-SS-Val, because the
standard deviations did not show significant differences. The main
reason for PTX-Cys was that the electrostatic interaction of PTX-Cys
with the negative intestinal epithelial cells promoted the drug
absorption in intestinal segments especially in ileum. In addition
organic cationic transporter perhaps played a limited role in pro-
moting the absorption of PTX-Cys [37].
3.7.4. Investigation of the effect of PEPT1 inhibition on PTX-SS-Val
transport
4. Conclusion
The results of single-pass intestinal perfusion (SPIP) of PTX and
PTX-SS-Val in HEPES solution with or without Gly-Sar were shown
in Fig. 8E. From the above experimental results, it could be seen that
the intestinal absorption capacity of free PTX in HEPES solution was
not significantly different with or without Gly-Sar, while the Peff of
PTX-SS-Val in HEPES solution was 1.59 and 1.3 times higher than
that in HEPES þ Gly-Sar solution, indicating that there was a
binding competition between PTX-SS-Val and Gly-Sar for PEPT1
transporter. Gly-Sar could bind with part of the transporters and
inhibit the absorption of PTX-SS-Val in small intestine. This pro-
vided further evidence that PEPT1 transporter involved the ab-
sorption of PTX-SS-Val in small intestine.
In this study, the hydrophobic antitumor drug paclitaxel was
chemically modified and a series of novel amphiphilic paclitaxel
small molecule prodrugs, PTX-Cys, PTX-SS-Val and PTX-SS-COOH
were designed, synthesized and evaluated against cancer cell
lines. These prodrugs contain water-soluble groups such as amino,
carboxyl and amino acids to increase the aqueous solubility of
prodrugs when compared to paclitaxel. More importantly, the
valine was introduced in molecule of PTX-SS-Val and made the
Table 1
In general, PTX-SS-Val and PTX-Cys had a similar absorption in
intestine. This phenomenon was mainly due to the different
transport mechanisms. The transport of PTX-SS-Val mediated by
PEPT1 highly expressed in jejunum promoted the absorption of
drug in intestine. The electrostatic interaction of PTX-Cys with the
negative intestinal epithelial cells also promoted the permeation
across the intestinal wall of drug. However, since most normal cells
are negatively charged, positively charged compounds often have a
strong side effect. Thus in the next pharmacokinetic experiment,
the PTX-SS-Val was used as model drugs to evaluate the potential
for improving the oral bioavailability of PTX.
The pharmacokinetic parameters of different PTX formulations.
Parameters
Unit
Taxola
Taxolb
PTX-SS-Valb
AUC(0-∞)
MRT(0-∞)
Tmax
mg/L*h
h
h
18.709
11.724
0.25
15.051
16.94
4
23.863
19.27
2
15.769
0.838
1.2383
30.61
Vz
CLz
Cmax
F (%)
L/kg
L/h/kg
mg/L
11.355
1.069
6.0650
19.258
1.329
1.062
19.31
a
Intravenous administration.
Intragastric administration.
b
12

Y. Li, M. Yang, Y. Zhao et al.
European Journal of Medicinal Chemistry 215 (2021) 113276
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113276.
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This work has not been published previously. It is not under
consideration for publication elsewhere. Its publication is approved
by all authors and tacitly or explicitly by the responsible authorities
where the work was carried out. If accepted, it will not be published
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Declaration of competing interest
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Acknowledgements
This work was financially supported by the Key Research and
Development Program of Shandong Province (grant numbers
2018CXGC1411), Natural Science Foundation for Young Scholars of
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14