| AIMColorectal cancer is the third most common cancer and the fourth most frequent cause of cancer deaths worldwide. The WHO estimates that 945 000 new cases occur yearly, with 492 000 deaths. In our country, its incidence is increasing quickly, and exceeds 20/100,000, but there are no effective treatment for it. 5-Fluorouracil is the 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 to 20 percent likelihood of reducing measurable tumor massed by 50 percent or more. There are many disadvantages when using 5-FU: unstable oral bioavailability, short half life, uninterrupted administration by iv, bigger individual difference because of nonlinear elimination and severe adverse reaction. The mostly reason is that the anti-cancer drug can not reach the effective concentration in the colorectal area. If we can prepare orally colon-specific drug delivery system to deliver 5-FU to colon with higher effective concentration, Pectin is a kind of new colon-targeting material, it is intact in the upper gastrointestinal tract and degraded by colonic microflora. Pectin can be attained easily with lower price. It has a good biocompatibility and biodegradability. Apart from the fact that pectin-derived drug carriers provide promising potential for colon-specific drug delivery, pectins also express extensive activities.Therefore, we designed colon-specific drug delivery systems of 5-FU using pectin as carrier, and evaluated the drug release in vivo and in vitro. The results of the study can give some hits for the development of the treatment of colon cancer.METHODS AND RESULTS①Synthesis of the conjugates of 5-FU and pectinThe conjugates of 5-FU and pectin were synthesized by different bridging groups. A sensitive HPLC method was established for the determination of concentration of 5-FU-1-acetic acid and 5-FU .This method was used to determine the degree of substitution.In the synthesis of 5-FU-acetyl-pectin conjugate, 4-dimethylaminopyridine was used as an acyl transfer catalyst, and DCC was used as a condendating agent. We prepared 5-FU-acetyl-pectin conjugate with acetyl as bridging group by esterification reaction. Adjusting the ratio of pectin, 5-FU-1-acetic acid and DCC varied the DS. To obtain a higher DS, we also reviewed the effects of time and temperature. DS became higher with the increasing of time and temperature. After such a comprehensive consideration, we chose a better reaction condition and obtained a conjugate with 34% DS. We also tried another synthesis routine. First, we prepared chloroacetyl pectin, then reacted with 5-FU, we also obtained 5-FU-acetyl-pectin conjugate, but the yield and DS were lower.In this study, we also synthesized 5-FU-formyl-pectin conjugate. First we prepared chloroformyl-5-FU using TCF or BTC as carbonylation regent, then reacted with pectin, the DS was below 5%.To determine the DS of conjugates accurately, a HPLC method to determine 5-FU and 5-FU-1-acetic acid was established. Under the HPLC conditions, linear response for 5-FU and 5-FU-1-acetic acid were obtained in the range of 0.1 150μg·ml-1. The calibration curve : 5-FU: Y = 60525.06C + 27301.67 ( r = 0.9999 ); 5-FU-1-acetic acid: Y = 49609.12 C +19183.31 ( r = 0.9999 ).②Drug release of 5-FU-pectin conjugates in vivo and in vitroTo determine the concentration of 5-FU and 5-FU-1-acetic acid in bio-samples, a HPLC method was established . Calibration curves in plasma were linear, 5-FU: Y = 59772.42 C +21425.74 ( r = 0.9989 );5-FU-1-acetic acid: Y = 49879.05 C +13898.45 ( r = 0.9989 ). Calibration curves in the contents of intestinal tract were also linear, 5-FU: Y = 61381.72 C +21386.26 (r = 0.9998 );5-FU-1-acetic acid:Y = 52524.19 C +14053.92 ( r = 0.9998 ) in the range of 1. 0 100.0μg·mL-1.The method was selective ,accurate and stable, according with the analysis for bio-samples.Cumulative release percentage of 5-FU-1-acetic acid or 5-FU released from different simulated gastrointestinal fluids and pectinolytic enzyme fluids were determined. It was noted that only little free 5-FU-1-acetic acid released from conjugates in different buffers. The cumulative release percentage in pH 1.5, 4.9, 5.5 buffers were below 1 % within 12 h, and the cumulative release percentage in pH 7.5 was 8.2 %. During the incubation , 98.9 % 5-FU-1-acetic acid was released from the conjugate in 9 h in simulated pectinolytic enzymes fluids. Likewise, there was only little 5-FU released from the conjugate in simulated gastrointestinal fluids, while 98.42 % 5-FU was released in 9 h in simulated pectinolytic enzymes fluids. The experiments suggested that original drug was degraded by enzyme and released from the conjugate in colon.After the administration of the conjugates, 5-FU-1-acetic acid or 5-FU was not detected in the contents of the stomach, proximal small intestine, while in the contents of cecum and colon 5-FU-1-acetic acid or 5-FU was observed to be at a high concentration. The results showed that the conjugates with pectin as carrier have potential to colon-targeting.③Synthesis of 5-FU-galactosides5-FU-galactosides were prepared according to the reported methods with the improved reaction conditions and post-processing. 5-FU-O-galactoside was obtained by the Koenigs-Knorr reaction. 5-FU-N-galactoside was synthesized by phase-transfer catalysis reaction. Chemical structure of the galactosides were confirmed by 1H NMR and MS etc.④Screening of anti-tumor activity of 5-FU-galacotosides in vitro The anti-tumor activity of 5-FU-galactosides against human colon cancer cell SW-1116 and lung cancer A549 were determined by MTT method. O-galactoside and N-galactoside had remarkable anti-tumor activity on human colon cancer cell SW-1116, with IC50 12.76 and 13.57μmol·L-1, respectively. Galactosides had higher anti-tumor activity than 5-FU against SW-1116, but there were no significant differences between O-galactoside and N-galactoside. O-galactoside and N-galactoside had anti-tumor activity on human lung cancer cell A549, with IC50 29.12 and 30.74μmol·L-1, respectively. There were no significant differences between galactoside and 5-FU. The study showed that galactosides had more anti-tumor activity against SW-1116 than A549.⑤Preparation of colon-targeting pellets of 5-FU-galactosides5-FU-galactosides pellets were prepared by fluid bed coating technology. The drug loaded pellets were prepared and then coated with solution of pectin and Eudragit S100. Dissolution tests were carried out in media simulating pH conditions at various locations in the gastro-intestinal tract. The results showed that galactosides were not released from the pellets in the media simulating the upper gastrointestinal tract, but had a pulse-release in the media simulating cecum and colon. All suggested the potential of the pellets for colon specific delivery of 5-FU-galactosides. Conclusions:Conjugates of 5-FU and pectin with higer DS were obtained by optimizing reaction conditions. The results of release experiments in vivo and in vitro showed that the conjugates have potential to colon-targeting. 5-FU-galactosides were prepared according to the reported methods with the improved reaction conditions and post-processing. The anti-tumor activity of 5-FU-galactosides against human colon cancer cell SW-1116 and lung cancer A549 were determined by MTT method. O-galactoside and N-galactoside had remarkable anti-tumor activity on human colon cancer cell SW-1116 and lung cancer A549. 5-FU-galactosides pellets were prepared by double coating. 5-FU-galactosides could be deliveried to colon by uniting pH depended and bacteria trigger colon-specific drug delivery system.
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