A novel brain targeted 5-FU derivative with potential antitumor efficiency and decreased acute toxicity: Synthesis, in vitro and in vivo evaluation

Article abstract of DOI:10.1691/ph.2014.3200

The broad-spectrum antitumor agent 5-fluorouracil (5-FU), has been used to treat various solid malignant tumors. However, its short life-time in vivo and poor ability to cross the blood-brain barrier has limited its application to brain tumor therapy. In

Full text of DOI:10.1691/ph.2014.3200

ORIGINAL ARTICLES
Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy,
Sichuan University, Chengdu, P. R. China
A novel brain targeted 5-FU derivative with potential antitumor efficiency
and decreased acute toxicity: synthesis, in vitro and in vivo evaluation
Hui Chen, Wenqi Wu, Yanping Li, Tao Gong, Xun Sun, Zhirong Zhang
Received September 12, 2013, accepted November 2, 2013
Dr. Zhirong Zhang, Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China
School of Pharmacy, Sichuan University, Southern Renmin Road, No. 17, Section 3, Chengdu 610041, P. R. China
zrzzl@vip.sina.com
Pharmazie 69: 271–276 (2014)
doi: 10.1691/ph.2014.3200
The broad-spectrum antitumor agent 5-fluorouracil (5-FU), has been used to treat various solid malignant
tumors. However, its short life-time in vivo and poor ability to cross the blood-brain barrier has limited
its application to brain tumor therapy. In order to develop a 5-FU derivative that localizes efficiently to
the brain while retaining potent antitumor activity, we conjugated 5-FU with N,N-dimethylethylenediamine
via an amide bond. The stability of the resulting 5-FU derivative (D-FU) was tested in vitro in phosphate
buffer, rat plasma and brain homogenate. The pharmacokinetic and biodistribution studies in brains of
the rats showed a higher Cmax (the maximal concentration) and an increased AUC0–t (the area under the
concentration-time curve) which was 6-fold that of 5-FU. In addition, compared to 5-FU, D-FU exhibited
lower toxicity in an acute toxicity assay and similar antitumor activity in the C6 cell line. In conclusion, D-FU
has the potential to be developed into an efficient brain delivery drug.
1. Introduction
tumor cells. This approach may be difficult to implement widely
in the clinic because stereotactic delivery directly into the
tumor site requires highly trained and highly skilled person-
nel (Malakoutikhah et al. 2011). Some researchers developeda
transferrin-coupled liposomal system (Soni et al. 2005), in
which 5-FU was encapsulated into liposomes carrying transfer-
rin, whichallowedtheliposomestoenterthebrain viatransferrin
receptor-mediated transcytosis (Qian et al. 2002; Cheng et al.
2004). In a third approach, researchers encapsulated the 5-FU
prodrugN-hexylcarbamoyl-5-fluorouracilintosurface-modified
nanogels to deliver 5-FU to brain tissue through the intravenous
route. Though vesicular vectors can transport 5-FU across the
BBB, the uptake by the reticuloendothelial system (RES), espe-
cially by the macrophages of liver and spleen, resulted to a
significant accumulation of 5-FU in these organs and may lead
to unexpected toxicities (Soni et al. 2006).
An approach that may avoid all of these drawbacks, but which to
ourknowledgehasnotyetbeenattempted, wouldbetoconjugate
5-FU with a low molecular weight moiety that would efficiently
direct the derivative into the brain. In previous work, our group
has shown that N,N-dimethylethylenediamine related structures
significantly enhance the efficiency by which naproxen and dex-
ibuprofen accumulate in the brain (Zhang et al. 2012a,b). This
ligand offers several advantages for drug development, includ-
ing a simple, small, well-defined structure and apparent lack of
toxicity, which contrasts to the toxic effects sometimes reported
with nano-particle vectors.
The most challenging task when treating nervous system dis-
eases is delivering drugs to the brain: the blood–brain barrier
(BBB) excludes nearly 98% of small molecules and 100% of
large molecules (Chen et al. 2004; Pardridge 2005). This limits
the efficiency of pharmacological treatments for many diseases
of the central nervous system (CNS), even when the same drugs
effectively treat the corresponding disease outside the CNS.
For example, 5-fluorouracil (5-FU) is widely used to treat vari-
ous solid malignant tumors such as colorectal cancers (Daumar
et al. 2011). However, it works poorly against brain tumors,
largely because it crosses the BBB extremely inefficiently (Brem
and Lawson 1999). In fact, the lack of uniformly successful
drug treatments for malignant brain tumors mean that they still
cause approximately12690 deaths in the United States every
year (Gududuru et al. 2004; Garcia et al. 2007).
5-Fluorouracil, rationally designed more than 50 years ago (Hei-
delberger et al. 1957), is an antimetabolite drug that inhibits
the synthesis of DNA and RNA during the S-phase of the cell
cycle (Scherf et al. 2000). Despite its widespread use to treat
tumors, it presents several disadvantages (Grem 1996; Daher
et al. 1990). One is short lifetime in vivo: 80% of the adminis-
tered 5-FU is catabolized into inactive metabolites (Grem 1996;
Daher et al. 1990). Another advantage, the high toxicity, also
limits the clinical use of 5-FU (Di Paolo et al. 2001).
Investigators have adopted various approaches in order to deliver
5-FU efficiently to the brain. Fournier et al. (2003, 2004) for-
mulated microspheres based on poly (methylidene malonate
2.1.2) (short for PMM 2.1.2), which they loaded with 5-FU.
Local, stereotactic injection into a rat model of brain can-
cer induced by C6 cell line had inhibited the growth of brain
Therefore we sought to create a safe and effective antitumor
derivative of 5-FU that targets the brain. We conjugated N,N-
dimethylethylenediamine with 5-FU via an amide bond, and
we confirmed the structure of the resulting D-FU conjugate
using NMR and ESI-MS spectroscopy. In a series of in vitro
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Table 1: Pharmacokinetic parameters of 5-FU and D-FU in
plasma after i.v. administration to rats
Parameters
5-FU
D-FU
AUC0–t (␮mol/g·min)
Cmax (mmol/L)
Tmax (min)
18.547 0.351
0.374 0.0342
2
80.323 5.441**
0.979 0.068**
2
t1/2z (min)
MRT (min)
CLz (L/min/kg)
112.739
22.838 6.823
0.043 0.0152
318.132
86.038 9.763**
0.008 0.001**
The concentration of D-FU was converted to 5-FU equivalent. Data represent the mean SD (n = 5).
**
(
t
p < 0.01). AUC :Theareaundertheconcentration-timecurve;C
:themaximalconcentration;
: biological half life; MRT: the mean residence time; CLz: clearance
max
0–t
1/2z
Scheme: Synthesis of D-FU.
Fig. 1: Stability of D-FU (A) rat plasma and ratbrain extracts, and in (B)
phosphate-buffered solutions at different pH values. Data are the mean SD
(n = 3).
Fig. 2: Concentration of D-FU and 5-FU in plasma at different times after
intravenous administration in rats. Data are the mean SD (n = 5).
2 min after administration, the mean concentration of D-FU in
plasma was 2.6 folds higher than the value for 5-FU. In addi-
tion, the area under the curve (AUC0–t) of the test group was
4.3 folds higher compared to the free 5-FU group. These results
suggested that the D-FU has a longer half-life and higher AUC0–t
in plasma, indicating that D-FU may have higher bio availability
and more chance to exhibit antitumor effect.
experiments, we examined the stability of D-FU in phosphate
buffers with different pH values, as well as in rat plasma and
brain homogenate. We also examined the antitumor activity
of D-FU in a C6 rat glioma cell line. In a series of in vivo
experiments, we conducted biodistribution and pharmacokinetic
studies in rats to assess the ability of D-FU to target the brain.
Furthermore, the acute toxicology study was conducted to eval-
uate the safety of D-FU.
2.3. Biodistribution study
2. Investigations and results
In order to evaluate the ability of D-FU to target the brain,
we analyzed the concentration of D-FU in different tissues
at different times after intravenous injection into rats. In par-
allel, we performed the same experiment with the equivalent
dose of 5-FU. Both drugs showed the highest concentrations in
most organs within a few minutes of administration, after which
their concentrations rapidly decreased (Fig. 3). Comparison of
the drug concentrations specifically in brain tissue showed that
D-FU showed much greater ability to target the braint issue
2.1. Synthesis and in vitro stability study of D-FU
D-FU was synthesized as described in the Scheme, and the struc-
ture was confirmed by 1H NMR, 13C NMR as well as ESI-MS.
The stability of D-FU, especially in blood and in brain tissue
was tested by incubating with phosphate buffer solutions of
different pH values ranging from 2.5 to 9.0, as well as in rat
plasma and in rat brain homogenate. After 24 h incubation at
37 ◦C, 80–110% of the original amount of D-FU remained in the
buffered solutions (Fig. 1A), while > 90% remained in plasma
and brain homogenate (Fig. 1B). These results indicated that
D-FU was stable under physiological conditions, opening the
door to further studies in cell culture and animals.
2.2. Pharmacokinetic study
We administered D-FU or 5-FU intravenously to rats and mea-
sured the concentrations of the drugs in plasma at different
time points in order to calculate the pharmacokinetic parameters
(Table1). TheconcentrationofD-FUinplasmawassignificantly
higher than that of 5-FU throughout the time course (Fig. 2). At
Fig. 3: Biodistribution of (A) 5-FU and (B) D-FU at different times after intravenous
administration in rats. Data are the mean SD (n = 5).
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Fig. 4: Concentrations of D-FU (black) and 5-FU (white) in rat brain at different
Fig. 6: Cell survival rate of D-FU (black) and 5-FU (white) in C6 cell line by MTT
assay. Data are the mean SD (n = 3).
times after intravenous administration. Data are the mean SD (n = 5). **,
p < 0.01.
2.4. Evaluation of antitumor activity in vitro
The fact that D-FU appeared to be stable in vivo and to target
the brain efficiently led us to examine its antitumor activity.
We chose the C6 rat glioma cell line, which is routinely used to
test antitumor toxicity of numerous compounds, including 5-FU
(Pang et al. 2010; Fournier et al. 2003). Both D-FU and 5-FU
showed concentration-dependent toxicity in C6 cell line in the
MTT assay over the concentration range of 0.001–20mmol/L
(Fig. 6). Although the stability assay suggested that D-FU did
not spontaneously dissociate to release free 5-FU to a significant
extent over 24 h, equivalent concentrations of D-FU and 5-FU
provoked similar extents of cell death within 24 h (P > 0.05),
demonstrating that D-FU had kept the antitumor efficiency of
5-FU in C6 cell line.
2.5. Acute toxicity study of D-FU in mice
The acute toxicity study was employed to estimate the safety
of D-FU. Mice were administered D-FU or 5-FU and observed
for 14 days to detect mortality and any significant changes in
behavior compared to their behavior prior to drug administra-
tion. Mortality, LD50 and behavior were compared between the
two groups. The LD50 of D-FU was 1210.1 mg/kg (95% con-
fidence interval 1081.3 to 1354.3 mg/kg). In contrast, the LD50
of 5-FU was 202.82 mg/kg (95% confidence interval 167.22 to
245.34 mg/kg). These findings suggest that, D-FU retains the
antitumor activity of 5-FU, while showing significantly lower
acute toxicity.
Fig. 5: Concentrations of D-FU (squares) and 5-FU (diamonds) in rat brain at
different times after intravenous administration. Data are the mean SD
(n = 5).
than 5-FU did (Fig. 4). The concentration of D-FU in brain
was much higher than that of 5-FU throughout the time course
(Fig. 5). Analysis of the pharmacokinetic parameters of both
drugs in the brain (Table 2) indicated that AUC0→t of D-FU was
6.02 folds higher than the value for 5-FU. Meanwhile, D-FU had
an increased half-life and decreased CLz in brain tissue com-
pared to free 5-FU group, suggesting that the derivative was
better retained in brain than free 5-FU.
3. Discussion
Despite its widespread use as an antitumor agent, 5-FU is not
routinely used to treat brain tumors because of its poor ability
to accumulate in the brain and its high toxicity. Here we show
that conjugating 5-FU to N,N-dimethylethylenediamine creates
a derivative that efficiently targets the brain in vivo and shows
similar antitumor activity as 5-FU in vitro, but with much lower
acute toxicity in vivo.
In previous work, our research group showed that conjugating
N,N-dimethylethylenediamine-related structures to the drugs
naproxen and dexibuprofen significantly enhanced their con-
centrations in brain tissue (Zhang et al. 2012a,b). The most
efficient one among them was (2S)-2-(4-isobutylphenyl) propi-
onic acid 2-dimethylaminoethyl ester (prodrug1) (Zhang et al.
2012b), with a CE value of 7.75. Based on that work, we chose to
use N,N-dimethylethylenediamine as a ligand to conjugate with
5-FU to target brain tissue. Our biodistribution study showed
that the concentration of D-FU in the brain was higher than
that of 5-FU over the entire time course examined; the RE
Table 2: Pharmacokinetic parameters of 5-FU and D-FU in
brain after i.v. administration
Parameters
5-FU
D-FU
AUC0–t (␮mol/g·min)
Cmax (nmol/g)
Tmax (min)
t1/2z (min)
MRT (min)
CLz (L/min/kg)
RE
0.229 0.024
10.385 1.046
2
52.494
21.117 2.321
1.964 0.185
–
–
1.3912 0.144**
28.372 3.349**
2
60.36
42.267 3.987**
0.468 0.127**
6.02
CE
2.7
The concentration of D-FU was converted to 5-FU equivalent. Data represent the mean SD
(n = 5). (**p < 0.01 with respect to the corresponding value for 5-FU). RE: the relative uptake
efficiency; CE: concentration efficiency.
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Table 3: Mortality and behavioral changes in mice within 14
Table 4: Mortality and behavioral changes in mice within 14
days after intravenous administration of different
doses of D-FU
days after intravenous administration of different
doses of 5-FU
Dose(mg/kg)
Log-transformed Mortality by 14
Behavioral changes
Dose (mg/kg)
Log-transformed Mortality by 14
Behavioral changes
dose
days
dose
days
103
147
2.0128
2.1673
0 of 10
3 of 10
None
814
992
1210
2.9106
2.9965
3.0828
0 of 10
3 of 10
5 of 10
None
None
Decreased locomotor
activity in some mice.
Decreased locomotor
activity in some mice.
Loss of body weight and
decreased locomotor
activity.
Loss of body weight and
decreased locomotor
activity. Death was
observed from the fourth
day after observation
Short ofbreath in some
mice
210
301
2.3222
2.4786
5 of 10
8 of 10
1476
1800
3.1691
3.2553
7 of 10
Convulsion and breathless
in someof mice.
Become Wheezing,
twitching in some mice,
decrease locomotor
activity and died that day.
10 of 10
430
2.6335
10 of 10
eral cell lines showing that modifications to 5-FU at the N1
position of the pyrimidine ring do not decrease, and in fact some-
times increase, the native compound’s antitumor activity (Huang
et al. 2007; Ouchi et al. 1998; Ohya et al. 1993). This may be
because D-FU retains the pharmacophore of 5-FU, namely the
F atom and pyrimidine ring (Van Kuilenburg 2004; Daumar
et al. 2011). Future studies should verify our antitumor findings
in vivo before more detailed structure-activity conclusions can
be drawn.
ConjugatingN,N-dimethylethylenediamineto5-FUnotonlysig-
nificantly enhanced the ability of the drug to accumulate in the
brain with no loss in antitumor potency, but it also significantly
reduces the acute toxicity of the drug in vitro (Tables 3, 4).
This is consistent with the observation by many researchers that
5-fluorouracil-1-acetic acid (Compound I) can be used to mod-
ify 5-FU without increasing the toxic effects (Chung et al. 1991;
Zuo et al. 2001; Kang et al. 2002; Yang Z et al. 2000; Daishu
et al. 2001). The much lower acute toxicity of D-FU may allow
higher doses to be used, which may boost clinical efficacy.
value of D-FU was 6.02 and the CE value was 2.7, indicating
efficient transport into the brain. The apparent ability of N,N-
dimethylethylenediamine to increase D-FU accumulation in the
brain is consistent with our earlier work (Zhang et al. 2012b). In
fact, various drugs that target the CNS contain structures related
to N,N-dimethylethylenediamine, such as meclofenoxate, cet-
irizine diphenhydramine, dextropropoxyphene and procaine
(Zhang et al. 2012b).
To ensure a stable linkage between N,N-dimethylethylenedi-
amine and 5-FU in our conjugate, we chose an amide bond.
Our stability studies in solution and in biological samples indi-
cate that the amide linkage in D-FU is quite stable, suggesting
that significant amounts of 5-FU would not be released in the
bloodstream, where it could exert toxic effects outside the brain.
Pharmacokinetic studies further showed that D-FU accumulates
quickly in the brain, suggesting that the effects of the conjugate
should be confined to the brain.
Why D-FU targets the brain much more efficiently than does
5-FU is unclear, given that conjugation with N,N-dimethylethyl-
enediamine is not expected to increase the inherently low
lipophilicity of 5-FU (Zhang et al. 2012b). We speculate
that D-FU does not cross the BBB by passive diffusion but
rather by an active transport process. Since N,N-dimethylethyl-
enediamineis is structurally similar to some organic cations and
choline, D-FU may be transported into the brain by transporters
that normally carry those ligands (Zhang et al. 2012b).
4. Experimental
4.1. Materials
5-FU (>98% pure) was purchased from Nantong General Pharmaceutical
Factory (Nantong, China). Methanol (HPLC grade) was purchased from
Kemiou (Tianjin, China). All other chemicals and solvents were analytical
grade. Thin-layer chromatography (TLC: silica gel GF254) was used to
detect the spots by UV radiation. 1H- and 13C- NMR analysis were per
formed using D2O as solvent by AMX-400 Bruker Spectrometer. Chemical
shifts were given in ppm (␦). Mass spectroscopy was performed by Agilent
1200 series RRLC system.
Our biodistribution and pharmacokinetic studies of D-FU in rats
showed that the derivative was present in much higher concen-
trations than was 5-FU in most organs examined, and that these
higher concentrations persisted over several hours. These find-
ings suggest that D-FU is catabolized much more slowly than
5-FU in vivo. Dihydropyrimidine dehydrogenase (DPD) catab-
olizes more than 80% of the administered 5-FU into inactive
metabolites, accounting for the low concentration detected in
most organs (Heggie et al. 1987). DPD catalyses the conver-
sion of 5-FU to fluoro-5, 6-dihydrouracil (FUH2), which is the
initial and rate-limiting step in the catabolism of 5-FU (Van
Kuilenburg 2004). The simplest explanation for the greater and
more persistent accumulation of D-FU in organs is that the N,N-
dimethylethylenediamine creates a great steric hindrance and
inhibits the interaction of DPD with the active agents.
4.2. Animals and cells
Wistar rats (male, 220 20 g) and Kunming mice (male, 22 2 g) were pro-
vided by the Laboratory Animal Center of Sichuan University (Chengdu,
China). All animals were maintained under standard conditions and allowed
free access to food and water. The Sichuan University Animal Ethical Exper-
imentation Committee approved the in vivo study protocols, which were
designed according to the requirements of the National Act on the Use of
Experimental Animals (People’s Republic of China).
The C6 rat glioma cell line was cultured in RPMI-1640 (Hyclone, USA)
media supplemented with 10% fetal calf serum (FMG-Bio, Shanghai,
China), 100IU/ml penicillin and 100 ␮g/ml streptomycin. Cells were main-
tained at 37 ◦C in a humidified atmosphere containing 5% CO2, and the cell
medium was changed every other day.
4.3. Synthesis of 5-FU derivative (D-FU)
Our in vitro cytotoxicity experiment with the C6 rat glioma cell
line indicated that D-FU retains the antitumor efficacy of 5-FU,
despite the apparent stability of the amide linkage in D-FU.
These results are consistent with previous report studies in sev-
4.3.1. Compound I
5-FU (3.9 g, 30 mmol) was dissolved in KOH solution (9 ml, 5.7 mol/L), then
an aqueous solution of 2-bromoacetic acid (8 ml, 45 mmol) was added drop-
wise at the temperature of 40 ◦C. The mixture was stirred for approximately
274
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4 h, until no free 5-FU was detectable by thin-layer chromatography. The
4.7. In vitro anticancercytotoxicity study of D-FU
solution was then transferred to anice-bath, and the pH was adjusted to 1.0
with hydrochloric acid (10 mol/L) to obtain the crude product. Compound I
was collected by filtration, washed with water for 3 times and then dried in
40 ◦C as 3.95 g white solid with a yield of 70% (Ouyang et al. 2011).
Compound I:1H-NMR (400 MHz, D2O): ␦8.10–8.08 (d, 1H, J = 6.8 Hz),
4.37 (s, 2H).
The antitumor activity of D-FU was assessed using the C6 rat glioma cell
line. The 3-(4,5-dimethyl-2-tetrazolyl)-2,5-diphenyl-2H tetrazolium bro-
mide (MTT) assay was used to determine the number of surviving cells.
In brief, cells were seeded in 96-well plate at a density of 7 × 103cells per
well in 200 ␮l culture medium. After 24 h, cells were treated with 200 ␮l
culture medium containing various concentrations of 5-FU or D-FU and
incubated another 24 h at 37 ◦C. Then, MTT solution (20 ␮l, 5 mg/ml) was
added to each well. After incubation for 4 h, the MTT solution was replaced
with 200 ␮l DMSO to dissolve the formazane. The absorption at 570 nm
was measured using a Microplate reader (Varioskan Flash; Thermo Fisher
Scientific). Each assay was carried out in triplicate and included a negative
control (cultures not exposed to 5-FU or D-FU) and a blank (wells contain-
ing medium but no cells). The ratio of cell-survival was calculated according
to the following equation.
ESI-MS (m/z): calcd for 188.11. obsd 187.01 ([M-H]+)
4.3.2. Compound II
Compound I (0.564 g, 3 mmol) was dissolved in dimethylformamide
(DMF, 10 ml) in an ice-salt bath. Then N-methylmorpholine (NMM, 0.395
ml, 3.6 mmol), isobutyl chloroformate (IBCF, 0.472 ml, 3.6 mmol) and
N,N-dimethylethylenediamine were added with stirring. The reaction was
allowed to proceed for 3 h, after which it was added to ethanol (20 ml) with
vigorous agitation. The mixture was filtered under reduced pressure to obtain
a white solid which was washed with ethanol to yield 0.464g of the final
product corresponding to 42% overall yield over two steps (Ouyang et al.
2011).
Cell survival rate = ([Abs])sample− [Abs]blank)/
([Abs])control− [Abs]blank) × 100%
(1)
Compound II (D-FU):
1H-NMR (400 MHz, D2O): ␦7.84–7.83 (d, 1H, J = 5.6 Hz), 4.55 (s, 2H),
3.70–3.67 (t, 2H, J = 6.0 Hz), 3.37–3.34 (t, 2H, J = 6.0 Hz), 2.95 (s, 6H).
13C-NMR (100 Hz, D2O): ␦172.07(s), 162.20(s), 153.29(s),
143.97∼141.66(d), 133.89∼133.67(d), 58.88(t), 53.27(t), 45.78∼45.24(q),
37.13(s).
4.8. The acute toxicology study of D-FU in mice
Fifty male Kunming mice (22 2 g) were divided randomly into five groups
(n = 10 in each group). Each group was given a single dose of 5-FU or D-FU
through the tail vein. The class interval of the dose was 0.70 for 5-FU and
0.82 for D-FU. The mice were observed regularly over the next 14 days, and
LD50 values and 95% confidence intervals (95% CI) were calculated using
SPSS assay.
ESI-MS (m/z): calcd for C10H15FN4O3 258.11. obsd260.00 ([M+H+1]+)
4.4. In vitro stability of D-FU in phosphate buffer and biological
sample
4.9. Data analysis
The pharmacokinetic parameters were calculated for both 5-FU and D-FU
using the Data and Statistics software package (DAS, Shanghai, China). To
evaluate the ability of D-FU to target the brain, RE and CE were calculated
according to defined as follows.
The in vitro stability of D-FU was investigated in a series of phosphate
buffers (pH 2.5, 4.0, 5.0, 6.8, 7.4, 9.0), in rat plasma and in rat brain
homogenate (homogenized and diluted with 0.9% physiological saline).
D-FU was dissolved in physiological saline (50 ␮l), and then added to above
mediums at a concentration of 30 ␮g/ml. The mixtures were incubated at
37 ◦C and the residual concentration of D-FU was determined by HPLC
(section 2.5) at the indicated time points.
RE = (AUC0-t,D-FU)/(AUC0-t,5-FU
CE = (Cmax,D-FU)/(Cmax,5-FU
)
(2)
(3)
)
Statistical evaluation was performed using analysis of variance followed by
t-test. Differences with an associated p < 0.05 were considered significant.
4.5. HPLC analysis and sample preparation
For determination of 5-FU, plasma or tissue homogenate (200 ␮l) was mixed
with 5-bromouracil solution (20 ␮l, 50 ␮g/ml) as the internal standard, fol-
lowed by ethyl acetate (3 ml). After vigorous vortexing and centrifugation
at 13500 rpm for 10 min, the organic layer was collected and evaporated to
dryness at 40 ◦C under air flow. The residue was dissolved in mobile phase
(100 ␮l) and centrifuged again at 13500 rpm for 10 min. An aliquot (20 ␮l)
of supernatant was injected into the HPLC system described below.
For determination of D-FU, phosphate buffer, plasma or tissue homogenate
(700 ␮l) was mixed with 30% (v/v) HClO4 solution (80 ␮l). After vortexing
and centrifugation at 13500 rpm for 10 min, an aliquot (20 ␮l) of supernatant
was injected into the HPLC system described below.
Acknowledgments: This work was supported by the National Natural
Science Foundation of China (No.81130060) and the National Basic
Research Program of China (No. 2013CB932504).
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