Synthesis and evaluation of the 5-fluorouracil-pectin conjugate targeted at the colon

Article abstract of DOI:10.1007/s00044-007-9049-0

This paper describes the first covalent synthesis of a 5-fluorouracil- pectin (FU-PT) conjugate. This conjugate with the potential for colon-targeted delivery has been evaluated. A sensitive high-performance liquid chromatography (HPLC) method was established for the determination of concentration of 5-FU-1-acetic acid in the gastrointestinal contents and plasma of rats. This method was also used to evaluate the colon-targeting properties of 5-FU-PT. 5-FU-PT was given to rats by oral administration at a dosage of 22.5 mg kg ^-1. The different parts of gastrointestinal tract and plasma were taken after 1, 3, 5, 7, 9, 12, 24, 36, and 48 h of oral administration of 5-FU-PT to rats, and the concentration of 5-FU-1-acetic acid in gastrointestinal contents and plasma was measured by HPLC. 5-FU-1-acetic acid released from 5-FU-PT was mainly distributed in the cecum and colon. Therefore, the present results suggest that the 5-FU-PT conjugate has a good colon-targeting property.

Full text of DOI:10.1007/s00044-007-9049-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2007) 16:370–379
DOI 10.1007/s00044-007-9049-0
ORIGINAL RESEARCH
Synthesis and evaluation of the 5-fluorouracil-pectin
conjugate targeted at the colon
Qing-Wei Wang Æ Xue-Ying Liu Æ Li Liu Æ
Juan Feng Æ Yu-Hua Li Æ Zhen-Jun Guo Æ Qi-Bing Mei
Received: 2 April 2007 / Accepted: 5 November 2007 / Published online: 5 December 2007
¨
Ó Birkhauser Boston 2007
Abstract This paper describes the first covalent synthesis of a 5-fluorouracil-
pectin (FU-PT) conjugate. This conjugate with the potential for colon-targeted
delivery has been evaluated. A sensitive high-performance liquid chromatography
(HPLC) method was established for the determination of concentration of 5-FU-1-
acetic acid in the gastrointestinal contents and plasma of rats. This method was also
used to evaluate the colon-targeting properties of 5-FU-PT. 5-FU-PT was given to
rats by oral administration at a dosage of 22.5 mg kg-1. The different parts of
gastrointestinal tract and plasma were taken after 1, 3, 5, 7, 9, 12, 24, 36, and 48 h of
oral administration of 5-FU-PT to rats, and the concentration of 5-FU-1-acetic acid
in gastrointestinal contents and plasma was measured by HPLC. 5-FU-1-acetic acid
released from 5-FU-PT was mainly distributed in the cecum and colon. Therefore,
the present results suggest that the 5-FU-PT conjugate has a good colon-targeting
property.
Keywords 5-Fluorouracil Á Pectin Á Colon-specific delivery system
Abbreviations
FU
PT
fluorouracil
pectin
HPLC high-performance liquid chromatography
DE degree of esterification
DCC dicyclohexylcarbodiimide
DMAP dimethylaminopyridine
Q.-W. Wang Á L. Liu Á J. Feng Á Y.-H. Li Á Z.-J. Guo Á Q.-B. Mei (&)
Department of Pharmacology, Fourth Military Medical University,
17 Changlexi Street, Xi’an 710032, PR China
e-mail: qbmei@fmmu.edu.cn
X.-Y. Liu
Department of Medicinal Chemistry, Fourth Military Medical University, Xi’an 710032, PR China

Med Chem Res (2007) 16:370–379
371
DMSO dimethylsulfoxide
DS
degree of substitution
TGA
DSC
PSI
thermogravimetric analysis
differential scanning calorimeter
proximal small intestine
middle small intestine
MSI
DSI
distal small intestine
Introduction
Colorectal cancer is the second or third most common malignancy, after breast
cancer in women and after lung and prostate cancers in men, in developed countries
(Kemeny, 1996). While chemotherapy in patients with advanced colorectal cancer
has proven to be of only marginal benefit, 5-fluorouracil (5-FU) is the most effective
treatment for this disease. 5-FU is useful alone or in combination with other drugs,
but is associated with only a 15–20% likelihood of reducing measurable tumor mass
by 50% or more (Piedbois et al., 1992). Part of the reason for this is that the
anticancer drug cannot reach an effective concentration in the colorectal area.
Several attempts have been made to increase effective concentration and reduce
toxicity via oral colon-specific delivery systems (Huang et al., 2001; Shantha et al.,
1995). Precise colonic drug delivery requires that the triggering mechanism in the
delivery system only responds to the physiological conditions particular to the
colon. To improve site specificity and versatile drug release kinetics to accommo-
date different therapeutic needs, certain types of polysaccharides can be used as
carriers or to create the dosage forms. Pectins are nonstarch linear polysaccharides
extracted from plant cell walls. They are intact in the upper gastrointestinal tract and
are degraded by colonic microflora (Sinha and Kumria, 2001). Apart from the fact
that pectin-derived drug carriers provide promising potential for colon-specific drug
delivery (Sinha and Kumria, 2001), pectins also express extensive activity. Modified
citrus pectin, given orally, inhibits carbohydrate-mediated tumor growth, angio-
genesis, and metastasis (in vivo), presumably via its effects on galectin-3 function.
These experiments stress the importance of pectin as agents for the prevention and/
or treatment of cancer (Nangia-Makker et al., 2002).
In the past years, many derivatives of 5-FU have been synthesized, among which
glycosides are considered important (Ohya et al., 1991). 5-FU-1-acetic acid, a
derivative of 5-FU, which was used as an active fraction to be linked to saccharides,
showed good inhibition of cancer cell growth rate (Yu, 2005; Lu et al., 2004).
In the present study, an acetyl spacer was used to couple 5-FU covalently onto
pectin. We first synthesized 5-FU-1-acetic acid, and then produced an ester from 5-
FU-1-acetic acid and pectin. An HPLC method was described and validated to
determine the 5-FU loading onto the pectin carrier. This pectin carrier, through its
own intrinsic anticancer activity, is expected to act not only as a drug delivery agent,
but also as an anticancer agent. The characteristic of the conjugates for colon
targeting was investigated in vitro, using conditions chosen to simulate the pH and

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Med Chem Res (2007) 16:370–379
time likely to be encountered during transit to the colon. The simulated test
suggested that the conjugate might have a colon-targeting property. Finally, we
investigated the release of the 5-FU-pectin conjugate in rats in vivo.
Experimental section
Materials
5-FU was supplied from Nan-tong Jing-hua Pharm. Corp., Ltd. (Nantong, China).
Pectin (with DE 65%) was obtained from He-nan Fu-da Pectin Industry Corp., Ltd.
(Xinxiang, China). Dicyclohexylcarbodiimide (DCC) was purchased from Nanjing
Tianzun Chem. Corp., Ltd. Dimethylaminopyridine (DMAP) was supplied from
Sinopharm Chemical Reagent Co., Ltd. Dimethylsulfoxide (DMSO) was dried
over a 4A molecular sieve and distilled before use. Methanol and triethylamine
were of HPLC grade. All other chemicals and solvents were of analytical reagent
grade.
Animals
Male Sprague–Dawley (SD) rats were purchased from the Experimental Animal
Center of the Fourth Military Medical University and the animals used in the
experiment were approved by the institutional ethical committee of the Fourth
Military Medical University. The rats were given standard food and water per day
under controlled conditions of temperature (23 2°C), humidity (50 5%).
Instruments
Melting points were determined using XRC-1 micromelting point apparatus without
correction. Infrared (IR) spectra were obtained with an AVATAR360 Fourier
transform (FT)-IR spectrometer (Thermo, USA). Thermal analysis was performed
by using a simultaneous thermal analyzer (SDT-Q600, TA Instruments Inc., USA)
at a heating rate of 10°C/min with an open pan system from 40°C to 450°C
referenced to alumina, both in a stream of N2 gas. The HPLC system consisted of
pumps, detector, and autoinjector from Waters.
Synthesis of 5-FU-pectin conjugates
5-FU-1-acetic acid was prepared by 5-FU reacting with chloroacetic acid giving an
82% yield (Masao, 1975), m.p. 278–279°C (Ref. 277–279°C). DCC (2.19 g,
10.6 mmol), DMAP (0.26 g, 2.12 mmol), and 5-FU-1-acetic acid (1 g, 5.3 mmol)
were added to an anhydrous DMSO solution (20 mL) of pectin (0.5 g). After the
mixture was stirred at 50°C for 96 hours, anhydrous ethanol was added, then

Med Chem Res (2007) 16:370–379
373
filtered. The precipitate was dissolved in anhydrous DMSO again, and anhydrous
ethanol was added, then it was filtered. This process was repeated two times. The
precipitate was dried at 60°C for 24 hours to yield 0.95 g 5-FU-PT. Fig 1
IR spectrum (KBr, cm-1): 3439 (OH), 1706(C = O), 1147*1025(glucoside-O-).
Analytical methods
5-FU and 5-FU-1-acetic acid were measured by reverse-phase HPLC with an RP C-
18 column (15 cm 9 4.6 mm; particle size 5 lm). The mobile phase composition
for the separation of 5-FU and 5-FU-1-acetic acid and DS was 95% water (0.15%
triethylamine and 0.15% acetic acid) and 5% methanol. The flow rate was 1 mL/
min with detection at 275 nm. In this HPLC method, the retention times of 5-FU
and 5-FU-1-acetic acid were about 5.4 minutes and 6.5 minutes, respectively.
Degree of substitution
The degree of substitution (DS) was defined as the ratio of the number of hydroxyl
groups of 5-FU-1-acetic acid bound among all hydroxyl groups of pectin. It was
determined by measuring the amount of 5-FU-1-acetic acid by HPLC at 275 nm,
which was released when 10 mg 5-FU-pectin was placed in 15 mL NaOH solution
(2 mol/L) for one hour at 40°C. We also determined in parallel the amount of 5-FU-
1-acetic acid that was released when 10 mg 5-FU-PT was placed in 15 mL water
solution for one hour at 40°C. All of the hydrolysis solutions were adjusted to pH 7
with HCl solution (2 mol/L).
H
O
N
O
O
OH
NaOH
OH
Cl
NH
F
N
O
O
O
NH
F
COOCH3
COOCH3
O
*
H
O
H
*
COOC
H3
COOCH3
O
H
OH
O
O
OH
OH
*
H
O
H
H
H
H
*
OH
O
O
OH
OH
H
H
n
OH
O
O
N
n
DCC
DMAP
O
NH
F
O
Fig. 1 Synthesis of 5-FU-pectin

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Med Chem Res (2007) 16:370–379
Chemical stability
5-FU-PT (2 mg) was placed in solutions of pH 1.5, 4.9, 5.5, 6.8 ,and 7.4 buffer and
pH 5.5 pectinase buffer, agitated, and reacted for 12 hours in a water bath at 37°C.
Reaction mixture 0.1 mL and mobile phase 0.9 mL were vortexed for two minutes,
centrifuged at 13,000 g for five minutes, and the amount of 5-FU-1-acetic acid in
the supernatant was analyzed by HPLC.
In vivo drug release
The rats in this study were assigned randomly to ten groups, with eight rats in each.
Each group corresponded to a time point. 5-FU-PT was suspended in water at a
concentration of 9 mg/mL for intragastric administration. Whole blood was sampled
from the abdominal aorta of ether-anesthetized rats at 1, 2, 3, 5, 7, 9, 12, 24, 36, and
48 hours after oral administration, respectively. Meanwhile, the contents were taken
from the stomach, proximal small intestine (PSI), middle small intestine (MSI),
distal small intestine (DSI), cecum, and colon at each time point.
The whole blood was centrifuged at 1,170 g for five minutes. One milliliter of
supernatant plasma was taken, added to 4 mL ethyl acetate, agitated for two
minutes, and centrifuged at 1,170 g for five minutes. The supernatant was dried
under a stream of nitrogen and redissolved in 150 lL of mobile phase, 20 lL of
which was subjected to HPLC analysis of 5-FU and 5-FU-1-acetic acid. The
contents were homogenized in saline with a ratio of 1:10 (g/mL) using a mechanical
homogenizer. The homogenate was filtered by microporous membrane (0.25 lm),
and the filtrate was subjected to HPLC analysis.
Statistical analysis
Considering the inequality of variances between groups, we adopted analysis of
variance (ANOVA) to analyze the average differences of the 5-FU-1-acetic acid
concentrations in the different groups.
Results and discussion
Preparation of 5-FU-PT conjugate
Characterization of 5-FU-PT conjugate
5-FU-PT conjugate was prepared following the method mentioned in the
experimental section. HPLC analysis showed that there was no free 5-FU-1-acetic
acid in 5-FU-PT. The IR analysis indicated that the carboxyl group of 5-FU-1-acetic
acid is covalently bound to one of hydroxyl groups of PT through an ester bond.
Pectin has an exothermic peak at 252.29°C (Fig. 2a), 5-FU-1-acetic acid has two

Med Chem Res (2007) 16:370–379
375
endothermic peaks at 277.01°C and 341.75°C (Fig. 2b), the compounds of pectin
and 5-FU-1-acetic acid have an exothermic peak at 277.05°C and an endothermic
peak at 335.99°C (Fig. 2d), while 5-FU-PT only has a stronger exothermic peak at
255.41°C (Fig. 2c). All this confirms that a new substance was obtained.
Optimization of the reaction conditions
In the synthesis of 5-FU-PT conjugate, 4-dimethylaminopyridine was used as an
acyl transfer catalyst, and DCC was used as a condensing agent. We obtained high
yields with the excess of 5-FU-1-acetic acid and DCC. Adjusting the ratio of pectin,
5-FU-1-acetic acid and DCC varied the DS and the results are listed in Table 1. To
obtain a higher DS, we also reviewed the effects of time and temperature. DS
became higher with increases in time and temperature. After such comprehensive
consideration, we chose a better reaction condition and obtained a conjugate with
34% DS. The 5-FU-PT conjugate was readily purified in high yields (greater than
90%). To avoid 5-FU-1-acetic acid enwrapped in PT, we determined the
concentration of 5-FU-1-acetic acid in a water solution of 5-FU-PT.
0.00
a 100
b
100
90
80
70
60
50
40
30
20
10
0
6.578%
-0.01
-0.02
-0.03
-0.04
-0.05
-0.06
-0.07
-0.08
-0.09
-0.10
-0.11
-0.12
80.56%
90
80
70
60
50
40
30
20
10
0
-0.05
-0.10
-0.15
-0.20
59.87%
252.29°C
341.75°C
91.61°C
277.01°C
50 100 150 200 250 300 350 400 450 500
50
300
100 150 200 250
350 450 500
400
Temperature (°C)
Temperature (°C)
0.00
120
100
80
60
40
20
0
c 100
d
7.245%
61.21%
335.99°C
90
80
70
60
50
40
30
20
10
0
0.0
255.41°C
-0.02
-0.04
-0.06
-0.08
-0.10
80.18%
71.98°C
-0.1
-0.2
-0.3
277.05°C
50 100 150 200 250 300 350 400 450 500 550 600
50
150 200 250
350
100
300
400
450 500
Temperature (°C)
Temperature (°C)
Fig. 2 TGA curve and DSC thermograms of samples of: (a) pectin, (b) 5-FU-1-acetic acid, (c) 5-FU-PT,
and (d) the compound of pectin and 5-FU-1-acetic acid (1:1)

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Med Chem Res (2007) 16:370–379
Table 1 Results of optimization experiment of the reaction condition
Experiment
Temperature
(°C)
Time
(hours)
Quality ratio of pectin
and 5-FU-1-acetic acid
Molar ratio of DCC
and 5-FU-1-acetic acid
DS (%)
1
2
3
4
5
6
7
8
9
30
30
30
40
40
40
50
50
50
48
72
96
48
72
96
48
72
96
1 : 2
1 : 3
1 : 4
1 : 3
1 : 4
1 : 2
1 : 4
1 : 2
1 : 3
2
3
4
4
2
3
3
4
2
22.6
27.4
38.5
31.7
34.7
41.2
27.6
28.8
44.3
Analytical methods
In the present study, the detection method for determining the exact amount of
enzyme-dependant colon-targeting 5-FU-PT conjugate in rats and its colon-
targeting property were explored. The findings suggest that the detection method
adopted in this study is precise and reliable so that it can meet the needs of detection
in vivo.
Chemical stability
It was noted that no free 5-FU and 5-FU-1-acetid acid were detected in the different
buffers, which suggested that the conjugate was chemically stable at pH 1.5, 6.8,
and 7.5 (Table 2). In the buffer with lower pH, little 5-FU-1-acetic acid was
released within the incubation time. With increasing pH and incubation time, the
release rate of 5-FU-1-acetic acid increased. During incubation, 98.9% 5-FU-1-
acetic acid was released from the conjugate in nine hours in simulated pectinolytic
enzymes fluid. 5-FU was not detected in another buffer or in simulated pectinolytic
enzymes fluid.
In vivo drug release
5-FU-1-acetic acid was not detected in the contents of the stomach, proximal small
intestine or middle small intestine, while in the contents of cecum and colon 5-FU-
1-acetic acid was determined to be present at a high concentration (Table 3).
In the blood samples, 5-FU-1-acetic acid was not found. This may be accounted
for as follows: (1) because it is macromolecular substance, 5-FU-PT cannot be
absorbed in small intestine. Only a little 5-FU-1-acetic acid can be released from 5-
FU-PT in DSI, so the amount of 5-FU-1-acetic acid absorbed is below the detection
limit of the HPLC method; (2) absorption in colon is slower than in small intestine,

Med Chem Res (2007) 16:370–379
377

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Med Chem Res (2007) 16:370–379
Table 3 Concentration of 5-FU-1-acetic acid in serum and contents of different parts of the rat GI tract
after oral administration of 5-FU-PT
Time (hours) Concentration (lg mL-1
)
Plasma Stomach PSI MSI DSI
Cecum
Colon
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
3
0
0
0
0
0
0
5
0
0
0
3.66 0.84m
8.06 5.72m
12.27 11.90m 25.16 20.19* 57.57 21.78*
15.41 4.90*
47.41 10.59* 30.58 8.45*
16.79 4.78*
7
0
0
0
9
—
—
—
—
—
0
0
12
24
36
48
0
0
0
39.13 23.21* 43.59 14.06*
1.58 0.81*
0
0
0
2.94 2.71*
—
—
—
—
—
—
0
0
0
0
Mean standard deviation (SD), n = 8
m only detected in the DSI contents of several rats
* P \ 0.01 versus the concentration of 5-FU-1-acetic acid in DSI
because the effective absorbing surface area in the colon is smaller. Although there
was lots of 5-FU-1-acetic acid in the contents of cecum and colon, only a small
amount can enter the blood. So we adopted ethyl acetate to extract the maximum
amount of 5-FU-1-acetic acid from the blood sample.
5-FU was not detected in all the biological samples, which shows that 5-FU-PT
mainly releases 5-FU-1-acetic acid in colon.
Conclusions
In this study, pectin was used as a carrier. Through the optimization of the reaction
conditions, we obtained 5-FU-PT with a higher DS. Our data demonstrate that
pectin conjugation of 5-FU-1-acetic acid can reduce systemic absorption and
effectively deliver 5-FU-1-acetic acid to the colon. On the basis of experiments
in vivo, 5-FU-PT could survive passage through stomach and small intestine, to
reach colon and be degraded by enzymes of the colonic microflora. The low
concentration of 5-FU-1-acetic acid in blood shows that 5-FU-PT can accumulate in
colon to reach an effective concentration.
Acknowledgements The authors gratefully acknowledge Professors Shen-Yong Zhang and Xiao-Li
Sun for their laboratory assistance. This work was financially supported by PrimaNova BioSciences, Inc.
References
Huang ZH, Lin ZL, Huang JL (2001) A novel kind of antitumor drugs using sulfonamide as parent
compound. Eur J Med Chem 36:863–872

Med Chem Res (2007) 16:370–379
379
Kemeny N (1996) Colorectal cancer- an undertreated disease. Anticancer Drugs 7:623–629
Lu FQ, Zhuang ZX, Cao J, Mi GT, Wang CX, Mu ZX (2004) Synthesis and release of the prodrug of
chitosan-5-fluorouracil. J Shandong University (Health Sciences) 42:226–228
Masao T (1975) Antineoplastic agents. The preparation of 5-fluorouracil-1-acid derivatives. Bull Chem
Soc Jpn 48:3427–3428
Nangia-Makker P, Hogan V, Baccarini S, Tai L, Raz A (2002) Inhibition of human cancer cell growth
and metastasis in nude mice by oral intake of modified citrus pectin. J Natl Cancer Inst 24:1854–
1862
Ohya Y, Kobayashi H, Ouchi T (1991) Design of poly (a-malic acid)-5FU-saccharide conjugate
exhibiting antitumor activity. Reactive Polym 15:153–163
Piedbois P, Buyse M, Rustum Y, Machover C (1992) Modulaton of fluorouracil by leucoworin in patients
with advanced colorectal cancer. Evidence in terms of response rate. Advanced Colorectal Cancer
Meta-Analysis Projecto. J Clin Oncol 10:896–903
Shantha KL, Ravichandran P, Panduranga RK (1995) Azo polymeric hyrogels for colon targeted drug
delivery. Biomaterials 16:1313–1315
Sinha VR, Kumria R (2001) Polysaccharides in colon-specific drug delivery. Int J Pharm 224:19–38
Yu HQ (2005) Synthesis of the glycosides of 5-fluorouracil and study on their activity. Master’s Thesis.
Hua Zhong Normal University