Rational design of a new cytarabine-based prodrug for highly efficient oral delivery of cytarabine

Article abstract of DOI:10.1039/c8ra01225c

Because of the drawbacks of cytarabine (Ara-C) such as poor lipid solubility, deamination inactivation and low oral bioavailability limiting its application by oral administration, herein we propose a novel amphiphilic low molecular weight cytarabine prodrug (PA-Ara) by conjugating palmitic acid (PA) to Ara-C, making it possible to avoid the deamination inactivation by protecting the active 4-amino, as well as improving lipid solubility. Thanks to the rational design, the oil/water partition coefficient (P) of PA-Ara was improved tremendously compared with Ara-C, and the PA-Ara conjugation was stable enough in artificial digestive juice, ensuring that most molecules could be absorbed in the form of the prodrug. Results from an MTT assay conducted to measure the cytotoxicity of Ara-C and PA-Ara to HL60 (acute myeloblastic leukemia cell line) and K562 cells (chronic granulocytic leukemia cell line) showed that PA-Ara had significantly stronger antiproliferation activities than Ara-C. Significantly, we firstly compared the bioavailability of the oral fatty acid chain modified cytarabine prodrug preparation with injection and the relative bioavailability was up to 61.77% for our PA-Ara, which was much superior to that of oral Ara-C solution (3.23%). Overall, these findings make it clear that the PA-Ara suspension has the potential to be a promising new cytarabine oral preparation for leukemia therapy.

Full text of DOI:10.1039/c8ra01225c

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Rational design of a new cytarabine-based prodrug
for highly efficient oral delivery of cytarabine†
Cite this: RSC Adv., 2018, 8, 13103
Jing Zhang, Di Zhang, Xu Hu, Ruiling Liu, Zhonghao Li and Yuxia Luan *a
a
a
a
a
b
Because of the drawbacks of cytarabine (Ara-C) such as poor lipid solubility, deamination inactivation and
low oral bioavailability limiting its application by oral administration, herein we propose a novel amphiphilic
low molecular weight cytarabine prodrug (PA-Ara) by conjugating palmitic acid (PA) to Ara-C, making it
possible to avoid the deamination inactivation by protecting the active 4-amino, as well as improving
lipid solubility. Thanks to the rational design, the oil/water partition coefficient (P) of PA-Ara was
improved tremendously compared with Ara-C, and the PA-Ara conjugation was stable enough in
artificial digestive juice, ensuring that most molecules could be absorbed in the form of the prodrug.
Results from an MTT assay conducted to measure the cytotoxicity of Ara-C and PA-Ara to HL60 (acute
myeloblastic leukemia cell line) and K562 cells (chronic granulocytic leukemia cell line) showed that PA-
Ara had significantly stronger antiproliferation activities than Ara-C. Significantly, we firstly compared the
bioavailability of the oral fatty acid chain modified cytarabine prodrug preparation with injection and the
relative bioavailability was up to 61.77% for our PA-Ara, which was much superior to that of oral Ara-C
solution (3.23%). Overall, these findings make it clear that the PA-Ara suspension has the potential to be
a promising new cytarabine oral preparation for leukemia therapy.
Received 8th February 2018
Accepted 27th March 2018
DOI: 10.1039/c8ra01225c
rsc.li/rsc-advances
cytarabine due to its limitations, such as poor lipid solubility
and quite low oral bioavailability caused by the abundance of
Introduction
1
-(b-D-Arabinofuranosyl) cytosine (cytarabine, Ara-C) is a clini- cytidine deaminase in the gastrointestinal tract and liver. As
cally widely used anticancer agent, especially for the treatment a result, cytarabine can only be administrated intravenously at
of both acute and chronic leukemia, which is a dangerous present. Therefore, developing an alternative therapy suitable
cancer with an estimated 62 130 new cases and 24 500 deaths in for practical oral administration is of particular importance and
1–4
the United States in 2017. As a potent chemotherapeutical in great demand from the viewpoints of both fundamental
anti-metabolic drug, Ara-C will go through activation and research and practical applications.
inactivation in vivo. On the one hand, aer intravenous injec-
A prodrug is a biologically inactive or low active agent in
tion into the blood circulation, part of the cytarabine crosses the vitro, and only when it is administrated into the body, can it be
8
cell membrane with the help of nucleoside transporters, and is metabolized to an active drug to perform therapeutic effects. In
then converted to a major active metabolite, 1-b-D-arabinofur- view of the superiorities of high drug loading and less carrier
0
anoside 5 -triphosphate (Ara-CTP), which forcefully inhibits the materials over traditional polymer prodrugs, low molecular
production of DNA polymerase and is nally also incorporated weight prodrugs are attracting increasing attention in achieving
5,6
9,10
into the DNA to exert anticancer therapeutic effects. On the targeted drug delivery and environmental response.
In
other hand, the bare active 4-amino of cytarabine is extremely particular, small amphiphilic prodrugs are capable of self-
1
1
prone to being deaminated to biologically inactive 1-D-arabi- assembling into different architectures, like particles,
7
12
13
nofuranosyluracil (Ara-U) by cytidine deaminase, resulting in capsules, rods and so on. To date, several cytarabine conju-
a short plasma half-life and requirements for frequent injec- gations have been constructed, and the structural modications
tions to maintain an effective concentration, which may lead to are mostly focused on the free amino of the cytosine base or the
0
poor compliance. However, as an acknowledged potent thera- 5 -hydroxyl group of the arabinose sugar, obtaining some
14
15,16
peutic drug, there is no clinical alternative oral preparation of derivatives such as squalenoyl-cytarabine, N4-L-valyl-Ara-C
0
17
and intestinal PepT1 targeting 5 -amino acid ester. Unfortu-
nately, N4-L-valyl-Ara-C failed to live up to initial expectations
with a poor oral bioavailability of 4% in rats, indicating the
a
School of Pharmaceutical Science, Shandong University, 44 West Wenhua Road,
Jinan, Shandong Province 250012, China. E-mail: yuxialuan@sdu.edu.cn
b
Key Laboratry of Colloid & Interface Chemistry, Shandong University, Ministry of infeasibility of amino acid modied 4-amino. In addition,
0
Education, 250100, China
although the oral absorption of a 5 -amino acid ester prodrug
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c8ra01225c
was promoted, it still suffered from the risk of inactivation due
1
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to the exposed amino. Therefore, there is still much room to applied to observe the morphology of its assemblies prepared by
improve oral bioavailability without the risk of inactivation and a nanoprecipitation method. In addition, the PA-Ara conjugation
to realize a practical oral administration by the rational design was stable enough in articial digestive juice, which ensured
of a cytarabine-based prodrug. Inspired by the amphiphilic enough oral absorption in the form of a prodrug. More impor-
structure of phospholipid molecules, we came up with the idea tantly, a cytotoxicity assay was conducted to investigate the anti-
of introducing a hydrophobic fatty acid chain into the amino of proliferation activities of Ara-C and PA-Ara against HL60 and K562
cytarabine. In past research, some fatty acid conjugated prodrug cells, and the unexpected results showed that PA-Ara had a more
18
delivery systems have been investigated intensively, but most excellent performance than Ara-C, proving its high efficiency in
were mainly about hydrophobic anticancer drugs. For instance, inhibiting leukemia cells. What is more, we carried out pharma-
Luo and co-workers constructed a PTX-S-OA prodrug with TPGS cokinetic experiments to compare the bioavailability via injection
as a stabilizer, and the prodrug nanoparticle performed with of the oral fatty acid chain modied cytarabine prodrug prepara-
19
excellent anticancer activity. However, the dual hydrophobic tion. To our surprise, the relative bioavailability was as high as
molecule conjugation had poor stability without TPGS. In 61.77% and the half-life of Ara-C was prolonged, along with
contrast, we expected the conjugation of a hydrophobic fatty a change in the pharmacokinetic behaviors of Ara-C due to the
acid with a hydrophilic Ara-C forming a low molecular weight rational design. Last but not the least, the rats administrated
amphiphilic prodrug to be stable enough. As for research into intravenously were all dead within 8 h, whereas those which took
anti-leukemic agents, such as Ara-C, their modications mostly the preparation orally were all alive aer the assay, showing the
0
relied on the 5 -hydroxyl group rather than the free active reduced toxicity of the as-prepared suspension over the present
animo. Elacytarabine, an elaidic acid ester of Ara-C, has been clinically used injection. With one-step synthesis and one-step
developed using lipid vector technology in an attempt to over- preparation, our prodrug assemblies not only go a long way to
come the low response rate, but the agent was circumventing meeting the requirements of industrialization, but also show great
20
resistance mechanisms. More importantly, the oral delivery of potential for the construction of an oral drug delivery platform.
fatty acid modied cytarabine as a convenient and acceptable Therefore, our newly rational designed PA-Ara prodrug provides
mode of administration has rarely been investigated. Therefore, a promising alternative oral preparation to injection and broadens
developing a fatty acid modied Ara-C prodrug delivery plat- the clinical application of Ara-C.
form was worth trying. Lauric acid (LA), palmitic acid (PA),
stearic acid (SA), oleic acid (OA) etc are general fatty acids.
Among these fatty acids, PA has mostly participated in the
Experimental section
construction of some listed drugs, such as dexamethasone Materials
21
palmitate injections, chloramphenicol palmitate suspen-
Cytarabine (Ara-C) was purchased from the Aladdin Industrial
Corporation. Palmitic acid (PA), sodium hydroxide, potassium
dihydrogen phosphate, sodium dihydrogen phosphate and
disodium hydrogen phosphate were bought from Sinopharm
Chemical Reagent Co., Ltd. Ethyl chloroformate (EtOCOCl)
was obtained from Chengdu Beisite Reagent Co., Ltd. Pepsin
22
sions,
clindamycin hydrochloride palmitate dispersible
23
24
tablets, and vitamin A palmitate eye gel. Applications in
different dosage forms and administrations proved the
biosafety of PA. Consequently, PA was selected as the fatty acid
moiety to take part in the design of a cytarabine prodrug in the
present work.
(
1 : 3000) and trypsin (1 : 250) were purchased from Amresco
In this work, PA was covalently attached to the free 4-amino of
cytarabine by a simple amidation reaction. Here, the long fatty acid
chain with 16 carbons would endow the conjugation with two
charming properties. On the one hand, the added alkyl chain
moiety provided amphiphilicity for an absolutely hydrosoluble
cytarabine, which endows it with self-assembly and enhances the
absorption efficiency by facilitating passive diffusion. On the other
hand, the protection of the active amino would decrease the
deamination and rst-pass effect, so the plasma half-life would be
prolonged and oral bioavailability would be greatly enhanced.
Moreover, due to the presence of amidase, the as-prepared prep-
aration would achieve the sustained release of free Ara-C in vivo,
making it possible to avoid an enormous change in drug
concentrations compared with injection and importantly to
decrease its toxicity. With a convenient kind of dosage form, PA-
Ara assembly suspensions may greatly improve patient compli-
ance. To conrm the successful synthesis of the prodrug, we
and Sangon Biotech (Shanghai) Co., Ltd, respectively.
Acyclovir (HPLC > 98%) was purchased from Dalian Meilun
Biotech Co., Ltd. HL60 and K562 cells were kindly provided by
the Immunopharmacology Institute of Shandong University
and the Shandong Analysis and Test Center. 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
was purchased from Beijing Solarbio Technology Co., Ltd.
The spectroscopy equipment was: an NMR spectrometer
H-NMR, Bruker Avance 400), an electrospray tandem mass
1
(
spectrometer (MS, AB SCIEX API 4000), an FTIR spectrometer
Nicolet 6700), a transmission electron microscope (TEM,
JEM-200CX), a high performance liquid chromatograph
(
(
(
HPLC, Agilent Technologies 1200 Series), an HPLC-MS
Agilent 1260 triple quadrupole mass spectrometer equip-
ped with an ESI source), and a microplate reader (ELISA,
PerkinElmer).
1
utilized nuclear magnetic resonance hydrogen ( H-NMR), mass
spectrometry (MS), and Fourier transform infrared (FTIR)
Synthesis of prodrug PA-Ara
measurements to characterize the chemical structure of the PA-Ara was the product of Ara-C and palmitic acid through an
conjugation, and transmission electron microscopy (TEM) was amidation reaction (Scheme 1). Firstly, PA (300 mg, 1.170 mmol)
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thin pellets to record the near infrared absorption peaks from
ꢁ
1
4
000 to 400 cm on a Nicolet 6700 FTIR spectrometer.
The preparation of PA-Ara assembly and TEM characterization
A nanoprecipitation method was applied to prepare a PA-Ara
25
prodrug assembly suspension. 20 mg of PA-Ara products
were dissolved in 400 mL of methanol, followed by their drop-
wise addition into 40 mL of distilled water with stirring at
500 rpm. Aer stirring for 15 min, the mixture was centrifuged
at 14 000 rpm for 10 min to obtain the precipitate. In order to
remove the undesired methanol as far as possible, the precipi-
tate was washed three times. The nal obtained precipitate was
resuspended in 4 mL of distilled water under ultrasound until it
ꢁ
1
was dispersed evenly to get a 5 mg mL suspension. TEM is
a widely used technique to observe assembled structures, such
26
27
28
as nanoparticles, polymeric micelles and bers, so TEM
was applied to observe the morphology of our assemblies. The
ꢁ
1
obtained thick suspension was diluted to 1 mg mL , and one
drop was deposited on a copper grid and air-dried at room
temperature before observation by TEM (JEM-200CX).
Scheme 1 The synthesis route of PA-Ara.
Oil/water partition coefficients (lg P)
The oral absorption of a drug requires a proper oil/water
was dissolved in anhydrous N,N-dimethylformamide (DMF, partition coefficient for passive transport; herein we measured
2
9
5
1
0
mL) followed by the dropwise addition of triethylamine (TEA, its lg P by the shaking ask method. N-Octanol (60 mL) and
80 mL) and EtOCOCl (122 mL) under nitrogen at a temperature of water (120 mL) were mixed in a capped conical ask in an
ꢀ
ꢀ
C. Aer 20 min of stirring, Ara-C (313 mg, 1.287 mmol), which incubator shaker at 37 C for 24 h to achieve equilibration, and
was also dissolved in anhydrous DMF (5 mL), was added to the thus we obtained an aqueous phase saturated with n-octanol
mixture dropwise and the reaction lasted for 72 h at room and an n-octanol phase saturated with water. PA-Ara was dis-
temperature. Aer that, the raw products could be obtained by solved in the n-octanol phase in the capped test tubes to obtain
ꢁ
1

ltration under diminished pressure to remove the solid a 200 mg mL solution followed by the addition of an equal
byproduct produced in the rst step. The solvent was further volume of the aqueous phase, and then the mixture was kept at
ꢀ
removed with a rotary evaporator. Finally, the raw products were 37 C. Aer 24 h, the concentration of PA-Ara in the water phase
puried on a silica gel column with a mixed solution of was analyzed by high performance liquid chromatography
dichloromethane and methanol (from 100 : 1 to 30 : 1) as the (HPLC, Agilent Technologies 1200 Series). The value of P was
eluent to obtain the pure PA-Ara prodrug products.
calculated according to the following equation:
C
oil
P ¼
1
H-NMR characterization
C ꢁ Coil
The newly obtained PA-Ara prodrug was precisely characterized where Coil refers to the concentration of drug in the n-octanol
1
by H-NMR. Fresh PA-Ara was dissolved in 0.5 mL of DMSO-d6 phase and C represents the prepared concentration.
in an NMR tube, and then the measurement was taken on
a Bruker Avance 400 spectrometer at 400 MHz at room
temperature to record chemical shis reported in ppm in the
presence of tetramethylsilane.
In vitro stability
Unlike drugs administrated intravenously without the process
of absorption, the erce metabolism of oral agents caused by
various enzymes in the gastrointestinal tract and the pH value
of different positions act as negative factors for sufficient
MS measurement
30,31
absorption in the form of the original molecular structure.
MS measurement was conducted to obtain the molecular
weight information of the prodrug. 2 mg of PA-Ara powder was
dissolved in methane and then analyzed by electrospray tandem
mass spectrometry (AB SCIEX API 4000).
That is to say, oral drugs should be stable enough in the
gastrointestinal tract to guarantee efficient absorption and
therapeutic effect. Articial gastric juice and articial intestinal

uids were prepared according to Chinese Pharmacopoeia 2015
to estimate the gastrointestinal stability against pepsin and
trypsin and pH 2.0, 5.0, 7.4, 8.0 phosphate buffered solution
FTIR determinations
Ara-C, PA and PA-Ara were crushed to a ne powder, milled with (PBS) to estimate chemical stability. In addition, 0.5% Tween-80
anhydrous potassium bromide, and nally compressed into was added to solubilize the PA-Ara prodrug. PA-Ara was
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dissolved in articial digestive juice and PBS, and then 200 mL of concentrations of Ara-C or PA-Ara containing acyclovir (800 ng
ꢁ1
the sample was taken out to vortex and centrifuge aer the mL ) were prepared. Briey, 180 mL of blank plasma and 20 mL
addition of 400 mL of cold acetonitrile to stop a possible meta- of different concentrations of standard solutions of Ara-C or PA-
ꢀ
bolic reaction at predetermined time point at 37 C. The Ara in methanol were added into a 1.5 mL Ep tube, and then 200
supernatant was ltered for HPLC analysis (Agilent Technolo- mL of acyclovir solution and 400 mL of acetonitrile were added.
gies 1200 Series).
Aer vortexing for 60 s and centrifuging for 10 min, the stan-
dard solutions of plasma with different concentrations of Ara-C
or PA-Ara containing acyclovir were obtained for HPLC-MS/MS
measurement. In addition, all the samples were treated in the
same manner under the same conditions. A triple quadrupole
mass spectrometer (Agilent 1260) equipped with an ESI (elec-
trospray ionization) source, and a C18 column (150 mm ꢂ
In vitro cytotoxicity
On account of the application in the treatment of leukemia, the
cytotoxicity of Ara-C and PA-Ara prodrug were evaluated on
HL60 and K562 cell lines with an MTT assay and were cultured
in IMDM and RPMI-1640 medium, respectively both containing
4.6 mm i.d. 5 mm, Thermo) coupled with a Phenomenex C18
10% fetal bovine serum (GIBCO, Vitrogen Corporation) and
guard column (4 mm ꢂ 3.0 mm i.d. 5 mm) was used to conduct
the quantitative analysis. For better separation of analytes with
endogenous materials and to avoid peak trailing, methanol and
ꢁ
1
ꢁ1
antibiotics (100 units mL penicillin, and 100 mg mL strep-
ꢀ
tomycin) at 37 C under a humidied atmosphere containing
32
5
% CO2. Exponentially growing HL60 and K562 in 100 mL of
0
.1% formic acid were selected as the mobile phase at a ow
culture medium containing different concentrations of samples
ꢁ1
rate of 0.6 mL min with gradient elution (Table S1 in ESI†). In
4
were seeded into 96-well plates at a density of 3 ꢂ 10 and 1.5 ꢂ
the positive ionization mode, the Q1 and Q2 masses were 226 to
4
1
0 cells per well, respectively. Aer 24 h or 48 h of incubation at
152 for acyclovir, 244 to 112 for Ara-C and 482 to 350 for PA-Ara,
ꢀ
3
7 C, 10 mL of 5% MTT regent was added to each well and
and the chromatograms were recorded in this condition (Fig. S1
to S7 in ESI†).
ꢀ
incubated at 37 C for another 4 h. Then the solution containing
unreacted MTT was removed carefully and 150 mL of DMSO was
added to dissolve the newly generated blue formazan crystals.
The optical density of each well was measured using a micro-
Results and discussions
plate reader (ELISA, PerkinElmer) at a wavelength of 490 nm, Characterizations of PA-Ara prodrug molecule and PA-Ara
and the cell inhibition ratio of each sample was calculated assemblies
according to the following equation:
The nal pure product was bluish violet with a yield of 37.1%.
1
A
positive control ꢁ Asample
positive control ꢁ Asample
H-NMR, MS, and FTIR were used to analyze its molecule
Cell inhibition ratioð%Þ ¼
ꢂ 100%
1
A
structure. The detailed H-NMR data is shown in Fig. 1A and is
as follows: 10.79 (s, 1H, –NH), 8.05 (d, 1H, J ¼ 7.60 Hz, 6-H), 7.20
where Apositive control and Ablack refer to the absorbance of the
culture without drug treatment and the culture without cells or
drug, respectively, whereas the other treatments were the same
as for the samples.
0
(
2
3
d, 1H, J ¼ 7.60 Hz, 5-H), 6.05 (d, 1H, J ¼ 3.84 Hz, 1 -H), 5.48 (d,
0
0
0
0
H, 2 -OH, 3 -OH), 5.06 (t, 1H, 5 -OH), 4.04–4.07 (m, 1H, 2 -H),
0
0
0
.91–3.94 (m, 1H, 3 -H), 3.80–3.85 (m, 1H, 4 -H),3.61 (t, 2H, 5 -
H), 2.37 (t, 2H, a-H), 1.53 (t, 2H, b-H), 1.18–1.24 (m, 24H, c-H),
1
0
.852 (s, 1H, d-H). All the H-NMR signals agreed well with
Pharmacokinetics experiments
the molecular structure of PA-Ara. The signal of hydrogen in the
In view of the difficulties in the separation of Ara-C and its amide group directly conrmed the successful synthesis of a PA-
metabolites in HPLC, HPLC-MS/MS was selected as the Ara prodrug. In addition, the molecular weight information of
approach to measure the plasma drug concentrations in Wister the PA-Ara prodrug characterized by MS is shown in Fig. S8 in
rats. All animal procedures were performed in accordance with ESI.† Among all the peaks in Fig. S8,† the appearance of [M +
+
+
the Guidelines for Care and Use of Laboratory Animals of H] (482.5) and [M + Na] (504.4) quasi-molecular ion peaks
Shandong University and approved by the Animal Ethics exactly proved the existence of C25 , which was consis-
Committee of Shandong University. tent with the theoretically calculated value. In addition, FTIR
43 6 3
H O N
Eight healthy Wistar rats weighting 200 ꢃ 10 g were was also utilized to conrm the molecular structure of PA-Ara.
randomly divided into two groups with administration of The results are shown in Fig. 1B. The original peaks at
ꢁ
1
ꢁ1
ꢁ1
ꢁ1
intravenous Ara-C saline solution (10 mg kg , 2 mg mL ) and 3475 cm and 3440 cm from the free amine of Ara-C were
ꢁ
1
ꢁ1
oral PA-Ara suspension (20 mg kg , 4 mg mL ), respectively. replaced by a brand new absorption band of N–H stretching
Before administration, all the rats were not fed but were given appearing at 3353 cm in the PA-Ara molecular spectrum. The
water for 12 h. In this method, 0.5 mL of blood was taken from peaks at 2919–2850 cm were consistent with the stretching
ꢁ
1
ꢁ1
the jugular vein of rats into 1.5 mL Ep tubes which had previ- modes of methylene and methyl groups in the PA moiety of PA-
ꢁ
1
ꢁ1
ously been washed with heparin (10 mg mL ) at 0.25, 0.5, 1, Ara. Unlike the absorption of C]O at 1702 cm in the PA
.5, 2, 4, 6, 8, 12, 24, 36, 48, 60 and 72 h aer treatment, and molecule, the amine I absorption (C]O stretching mode)
1
ꢁ
1
then the blood was centrifuged at 10 000 rpm for 5 min to ob- seemed weak and apparently shied to 1696 cm , and the
ꢀ
tained a plasma which was kept in a ꢁ20 C fridge.
amine II absorption (mainly N]H bending and C]H stretch-
ꢁ
1
ꢁ1
To measure the concentration of Ara-C and PA-Ara in the ing modes) was centered at 1650 cm and 1555 cm for PA-
samples, standard solutions in plasma with different Ara. The FTIR results further conrmed the successful
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Fig. 1 (A) H-NMR spectra of PA-Ara. (B) FTIR spectra of Ara-C (a), PA (b) and PA-Ara (c). (C) TEM image of PA-Ara assembly.
synthesis of PA-Ara. Based on the various characterizations of surface of the assembly had a negative charge which was helpful
PA-Ara, it can be concluded that the PA-Ara prodrug was for its physical stability. The PA-Ara assembly suspension could
synthesized successfully.
remain stable for more than two weeks at room temperature.
By a nanoprecipitation method, we obtained the assembly of
PA-Ara. The as-prepared PA-Ara assembly appeared as a white
suspension and the morphology of the PA-Ara assembly was
observed with a transmission electron microscope. The TEM
result is shown in Fig. 1C. This shows that our PA-Ara could self-
assemble into spiral bers in aqueous solution using a nano-
precipitation method. By surveying the literature involving PA-
based assembly, we found that Zhang et al. had previously re-
ported a cell membrane tracker named TR4 (containing pal-
Oil/water partition coefficient (P)
The oil/water partition coefficient (P) is a crucial parameter for
oral agents, which plays an important role in the intracorporal
process of drugs involving absorption, distribution, metabolism
and excretion. The coefficient is generally presented as the
33
logarithm to the base 10 (lg P). In principle, the stronger the
lipid solubility of a compound, the greater the coefficient will
be. In our case, the value of P of the PA-Ara prodrug was
determined to be 1668 (corresponding to lg P ¼ 3.22) which is
signicantly higher than that of Ara-C (0.16, corresponding to
lg P ¼ ꢁ0.80), indicating the remarkably improved lipophilicity
of PA-Ara compared with Ara-C. The high lipophilicity of the PA-
Ara prodrug could be attributed to the conjugation of palmitic
acid. Thus, such high lipophilicity can sufficiently ensure the
easy passive diffusion of the drugs, resulting in enhanced oral
absorption.
28
mitic acid, tetraphenylethylene and a hydrophilic peptide),
which could assemble into nanobers. However, their bers did
not have spiral morphology. This indicated that the Ara-C
moiety in the PA-Ara was the indispensable factor for the
spiral assembly in our case. Besides, the hydrogen bond formed
by the C]O and N–H in the amido bond also lets the amphi-
philic prodrug molecules arrange in an orderly fashion to form
a spiral assembly. The zeta potential of the PA-Ara assembly was
measured to be ꢁ(19.8 ꢃ 3.1) mV, demonstrating that the
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In vitro stability
Good stability in the gastrointestinal tract is of particular
importance for oral drugs. Therefore, we prepared articial
gastric juice and intestinal uids to examine the gastrointes-
tinal stability of our PA-Ara prodrug. The kinetic curves for the
degradation of PA-Ara with time are shown in Fig. 2A. From
Fig. 2A, it is clear that the PA-Ara prodrug exhibits good stability
in both articial gastric juice and intestinal uids. Within 2 h of
incubation in articial gastric juice, the undegraded PA-Ara
remained at (80.72 ꢃ 2.52)%. In particular, the undegraded
PA-Ara could remain at (77.26 ꢃ 3.30)% even aer 10 h in
articial intestinal uids. These results suggested that
a majority of the prodrug molecules could be stable in the
process of being emptied from the stomach to the small intes-
tine within 2 h where the prodrugs were absorbed mostly in the
form of the original molecular structure. The slight degradation
of PA-Ara in articial gastric juice and intestinal uids can be
attributed to the pepsin and trypsin therein, respectively. In
addition, we also investigated the chemical stability of PA-Ara in
PBS of pH 2.0, 5.0, 7.4 and 8.0, which represent the pH of the
stomach, duodenum, ileum and colon, respectively. As shown Fig. 3 TEM images of PA-Ara assembly after incubation in artificial
gastric juice (A and B) or artificial intestinal fluids (C and D), respec-
tively, for 1 h (A and C) and 12 h (B and D).
in Fig. 2B, our PA-Ara performed with excellent stability in
various PBS except for a slight decline in pH 2.0. This indicates
that our PA-Ara is stable with no pH-responsive degradation.
Based on the stability results, our PA-Ara was highly suitable for
oral administration.
there was no obvious difference aer further incubation to 12 h
(Fig. 3D). In articial gastric juice, the spiral structures of the
We further investigated the stability of PA-Ara assemblies in
articial digestive juice by observing the changes in the
morphology of the assemblies at different times. The as-
PA-Ara assemblies were prone to uncoiling, in that some amide
bonds or hydrogen bonds were hydrolyzed by acid environ-
ments and pepsin, which was similar to the destruction of the
three dimensional structure of a protein. For the process of PA-
Ara assemblies in vivo, the assemblies will rst enter into the
gastric juice and then arrive in the intestinal uid. When the PA-
Ara assemblies arrive in the small intestine (intestinal uid), the
assemblies dissolved in intestinal uids could be slowly
absorbed. Above all, no matter whether they were prodrug
molecules or assemblies, they were both stable in articial
digestive juices within the required period, demonstrating that
they were very suitable for oral application.
ꢁ
1
prepared 15 mg mL thick suspension was diluted 30 times
with articial gastric juice and intestinal uids, respectively,
ꢀ
and then the thin suspension was incubated at 37 C. The
samples were characterized by TEM aer 1 h and 12 h of
incubation, as shown in Fig. 3. When the assemblies were
incubated in gastric juice for 1 h, the morphology of most
assemblies remained unchanged in comparison with the orig-
inal morphology in Fig. 1C. However, a few assemblies without
spiral characteristic began to form (Fig. 3A). With the increase
in incubation period to 12 h, all the original spiral bers had
changed into non-spiral bers or rods (Fig. 3B). For the
assemblies incubated in articial intestinal uids for 1 h Cytotoxicity in vitro
(
Fig. 3C), the spiral bers had shrunk to be thinner in
An MTT assay was further carried out to investigate the cyto-
toxicity in vitro of our PA-Ara prodrug. For comparison, free Ara-
comparison with the original morphology in Fig. 1C. However,
Fig. 2 Gastrointestinal stability (A) and chemical stability (B) of the PA-Ara prodrug.
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Fig. 4 Cell inhibition ratios of different samples against HL60 and K562 cells after 24 h or 48 h incubation, *p < 0.05, **p < 0.01, n ¼ 3.
C was also studied. Fig. 4 shows the antiproliferative effects of which thus caused a small decrease in inhibition ratio with
Ara-C and PA-Ara prodrug on HL60 or K562 cells at 24 h and time. Even so, the cytotoxicity of PA-Ara was overall superior to
48 h, respectively. For HL60 cells, the cell inhibition ratio of Ara-C with an antiproliferation inhibition ratio of 86.21% at
both Ara-C and PA-Ara performed in a dose-dependent fashion. 48 h. The performance of Ara-C in K562 cells was similar to that
In other words, the cell inhibition ratio increased with drug in HL60 cells for both 24 h and 48 h, indicating that the facil-
concentration. However, it was obvious that PA-Ara showed itated diffusion was not inuenced by the differences between
a signicantly higher cell inhibition ratio than Ara-C for both HL60 and K562 cells. On the contrary, PA-Ara performed anti-
24 h and 48 h incubation. For example (24 h), the inhibition proliferation activities against K562 cells both in an obviously
ratio of PA-Ara was 92.08% at 500 mM, which was signicantly concentration-dependent and time-dependent increasing way,
higher than that of Ara-C (59.95%). The increased inhibition which may be caused by the natural differences between K562
ratio for PA-Ara was attributed to its increase in lipid solubility, cells and HL60 cells. In a nutshell, no matter whether for HL60
endowed to a great extent by the fatty palmitic acid chain. The cells or K562 cells, and no matter whether for 24 h or 48 h
endowed lipid solubility changed the method of trans- incubation, the endowed lipid solubility of PA-Ara promoted
membrane transport, taking PA-Ara across the membrane in much better cytotoxic effects than the traditional agent Ara-C,
a passive transport way, where the greater the concentration providing a promising alternative therapy for leukemia.
difference between PA-Ara inside and outside the cell
The IC50 value is the concentration of the drug when the cell
membrane, the faster the PA-Ara penetrated into the cell inhibition ratio reaches 50%. It is a parameter oen used to
membrane. In contrast, Ara-C crossed the membrane assisted estimate the potency of each preparation. The smaller the IC50
by nucleoside transporters, so its translocation efficiency was value, the stronger the drug's ability to kill cells. Therefore, we
partially limited by the number of nucleoside transporters in further calculated the IC50 of Ara-C and PA-Ara. As shown in
addition to the concentration difference. Compared with 24 h Table 1, the IC50 of PA-Ara was decreased vitally to HL60 and
incubation, when the incubation time was extended to 48 h, the K562 cells in comparison with that of Ara-C. Especially for K562
inhibition ratio of Ara-C was higher than that at 24 h at the same cells, the IC50 of Ara-C was 2.18 times and 3.50 times high as PA-
concentration, while that for PA-Ara was slightly lower on the Ara at 24 h and 28 h, respectively, showing that PA-Ara behaved
whole. This can be explained as follows. For Ara-C, on account much better for anti-leukemia cell proliferation, agreeing well
of the restricting of nucleoside transporters, the rate of trans- with what was discussed above.
portation was nearly constant, and therefore the inhibition ratio
performed by rising steadily with time. However, for PA-Ara, the
Pharmacokinetic studies in vivo
concentration difference between PA-Ara inside and outside the
Improving oral bioavailability is crucial for oral preparation.
cell membrane became smaller and smaller as more and more
Therefore, a pharmacokinetics assay was conducted to evaluate
PA-Ara molecules were passively transported into the cells,
whether our PA-Ara assembly was suitable for oral
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Table 1 IC50 (mM) of drugs against HL60 or K562 cells incubated for Table 2 Pharmacokinetic parameters of intravenous Ara-C and oral
24 h or 48 h
Ara-C and PA-Ara suspension
Cell line
HL60
Incubation time (h)
24
IC50 (Ara-C)
IC50 (PA-Ara)
Oral suspension
Pharmacokinetic
parameters
Intravenous
Ara-C
289.30
34.02
315.72
29.64
52.45
46.74
144.59
8.47
Ara-C
PA-Ara
48
ꢁ
1 a
AUC0–N/(ng h mL
)
36 199.75
2579.92
1
11.46
14.09
0.28
22 359.14
1108.59
6
4630.77
167.13
6
K562
24
ꢁ
1 b
48
C
max/(ng mL
)
c
Tmax/(h)
d
t1/2/(h)
12.89
11.87
0.45
19.68
29.09
4.32
e
MRT0–N/(h)
ꢁ
1
ꢁ1 f
Clz (L h kg
)
administration. An HPLC-MS/MS approach was applied to
determine the drug concentrations in plasma. We measured the
concentration–time curve of Ara-C with oral administration.
Therefore, only the curves of Ara-C and PA-Ara administrated reaches its peak.
intravenously and orally, respectively, were conducted in the
present study. The results are shown in Fig. 5 and Table 2.
a
AUC0–N is the mean area under the concentration–time curve from
b
time 0 to the last time point.
C
max is the peak plasma concentration
Tmax is the time point when the plasma concentration
34
c
of the drug.
d
t
1/2 is the elimination half-time of the drug.
e
MRT0–N is the mean residence time which represents the average
f
time that drug molecules reside in the body. Clz is the volume of
plasma containing the drug eliminated from the body per unit time,
Comparing the concentration–time curves of Ara-C and PA-Ara which stands for the clearance rate of the drug from blood.
with different dosage forms and administrations, we could
see that the concentrations of Ara-C administrated intrave-
free Ara-C were much higher than of prodrug PA-Ara, which
proved that PA-Ara prodrug could be metabolited into free Ara-C
by amidase in vivo. In this way, this strategy would realize the
sustained release of the drug and avoid huge changes in drug
concentrations which may lead to side effects. According to the
pharmacokinetic data, we calculated the pharmacokinetic
parameters by DAS 2.0, and the results are displayed in Table 2.
As presented in Table 2, the t1/2 (the elimination half-time of the
drug) of free Ara-C from PA-Ara was calculated to be 12.89,
which was signicantly longer than that of Ara-C administrated
nously were much higher than for PA-Ara taken orally. However,
the rats were all dead within 8 h for the Ara-C administrated
intravenously, demonstrating the higher toxicity with the
condition of the same dosage of Ara-C. In theory, AUC0–N (the
mean area under the concentration–time curve from time 0 to
the last time point) is a particularly important parameter for
drugs, and a larger AUC0–N means better absorption and
bioavailability. Due to AUC0–N being proportional to the dose,
the AUC0–N of oral Ara-C solution was transformed from
ꢁ
1
ꢁ1
1
754.87 ng h mL (15 mg kg ) measured in our previous study
ꢁ1
ꢁ1 34
34
orally (1.8). Thus, the strategy of protecting the active amino
from deamination was proved to be wise for prolonging the
circulation time of the drugs.
to 1169.91 ng h mL (10 mg kg ), so the relative bioavail-
ability (the ratio of the AUC0–N of oral cytarabine solution to
that of the injection preparation) was only 3.23%. Surprisingly,
the AUC0-N of our PA-Ara assembly suspension was calculated
to be 22 359.14 ng h mL , realizing a 19.11-fold improvement
in comparison with that of oral Ara-C solution, and the relative
bioavailability was as high as 61.77%. Hence, the absorption
and oral bioavailability of Ara-C were unquestionably enhanced
thanks to the rational design of the prodrug. In addition, aer
oral administration of a PA-Ara assembly, the concentrations of
Based on what has been discussed above, we can make it
clear that the well-designed PA-Ara prodrug indeed made
impressive progress in overcoming the drawbacks of short half-
life and low bioavailability of oral cytarabine via molecular
modication. By introducing the long fatty chain, inactivation
caused by enzymes was efficiently avoided, and transmembrane
transport increased remarkably, resulting in enhancements in
oral bioavailability. Additionally, in view of the gastrointestinal
stability we had investigated, the detection of major free Ara-C
in the blood proved that the formed amide bond could be
hydrolyzed in vivo to realize sustained release of free cytarabine
rather than the robust entrance of Ara-C intravenously which
may lead to serious toxicity. All of these results indicate the
outstanding properties of our cytarabine prodrug preparation.
ꢁ
1
Conclusions
In summary, we rationally designed and synthesized a new
amphiphilic low molecular weight cytarabine prodrug (PA-Ara)
by conjugating palmitic acid to cytarabine, overcoming the
drawbacks of cytarabine such as poor lipid solubility, deami-
nation inactivation and low oral bioavailability for application
by oral administration. The PA-Ara could be highly stable in PBS
solution with different pH values for 10 h, and the remaining
Fig.
5 Pharmacokinetic concentration–time curves in rats after
administration of different formulations. (a) The curve of Ara-C after
intravenous administration of Ara-C saline solution. (b-1) The curve of
Ara-C after oral administration of PA-Ara suspension. (b-2) The curve
of PA-Ara after oral administration of PA-Ara suspension.
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2
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Conflicts of interest
2
2
2
2
2
2
2
There are no conicts of interest to declare.
1 P. Bias, R. Labrenz and P. Rose, Clin. Drug Invest., 2001, 21,
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