The Colon-targeted Research Of5-fluorouracil-1-acetic Acid-α-cyclodextrin Conjugates

5-Fluorouracil (5-FU), a pyrimidine analogue, has anti-tumor activityafter transforming into5-fluorouracil deoxynucleotide in cells and is the firstchoice of chemotherapy in the treatment of colon cancer. But the characteristicof general oral formulations of5-FU such as incomplete absorption, lowbioavailability and gastrointestinal side effects after oral administration, has agreat impact on the clinical application.5-fluorouracil-1-acetic acid (5-FUA)is a carboxyl derivative of5-FU, and its pharmacological effect is proved to besame as5-FU. It has anti-tumor effect, but relatively less toxic side effect.α-Cyclodextrin (α-CD) can be rarely absorbed in the gastrointestinaltract, but can be hydrolyzed into small saccharides by α-amylase in intestinaltract and bacterial flora in colon. This study is aim to connect5-FUA withα-CD through ester linkage, and prepare prodrugs of5-fluorouracil-1-aceticacid-α–cyclodextrin conjugates. With the help of α-amylase in intestine tractand the colonic bacterial flora to hydrolysis, prodrugs release5-FUA gradually,and develop its colon-targeted effect.Objective:Connect5-FUA with α-CD through ester linkage to prepare prodrugs of5-fluorouracil-1-acetic acid-α-cyclodextrin conjugates. Study in vitro releasein enzymatic solutions and related researches in animals.Methods:1According to the method of the literature, in the temperature of100℃and alkaline environment of pH10, the α-chloroacetic acid and5-FU changedinto5-FUA. After separation and purification, measure the melting point andconfirm the chemical constitution of the product by MS, IR and1H-NMR.2After5-FUA dissolving in dry dimethylformamide (DMF), add thecondensing agent carbonyldiimidazole (CDI) and stir the solution at room temperature for3h. Dissolve equal mole of α-CD in dry DMF and add it intothe reaction solution. Then the triethylamine was added to keep the alkalineenvironment. Continue the reaction for48h to generate5-fluorouracil-1-aceticacid-α-cyclodextrin conjugates, and add the acetone to separate the productsout. After purification by Diaion-HP20, the defined amounts of products werehydrolyzed in alkaline solutions, and then test the degree of substitution byHPLC and MS.3The in vitro release of the5-fluorouracil-1-acetic acid-α-cyclodextrinconjugates: establish an HPLC method for the content of5-FUA, and studythe hydrolysis behaviors of prodrugs in α-amylase solution, carboxylicesterase solution and enzymatic solution of α-amylase and carboxylic esterase.And study the hydrolysis behaviors in solutions with different pH value suchas pH1.2hydrochloric acid solution, pH6.8phosphate buffer，pH11sodiumborate-sodium carbonate buffer and so on. Draw the curve of lgK-pH ofprodrug1.4Dilute the gastric content of Wistar rats10times with pH1.2hydrochloric acid solution, so did the small intestinal content and the cecalcontent with pH6.8phosphate buffer. Remove the insoluble particles and addthe quantitated prodrugs. Establish an HPLC method to determine its drugcontent, and detect the samples when1,2,3,5,8,12h. Draw the hydrolysiscurves of prodrugs according to the results.With Wistar rats as tested animals, the prodrugs were dissolved inphysiological saline and intragastric administrated to the Wistar rats. At thescheduled time of4h,8h,12h and24h, kill the rats to collect the tissues andcontents of stomach, small intestine and large intestine, and freeze the samplesto use later. Establish an HPLC method to determine its drug contentrespectively, and draw the histograms of prodrugs according to the results.Results:1The synthesis process was determined as below. When the reactant ratioof5-FU and α-chloroaceticacid was1:1.2, and keep the reaction temperature100℃for6hours, the maximum reaction yield could be45.4%. The melting point of product5-FUA was272℃, and the chemical structure of5-FUA wasconfirmed by MS, IR and1H-NMR.2When gradient eluting with methanol-water solution, the conjugatesappeared in the elution of10–30%methanol. Among them, the5-FUA in theproduct1which appeared in10%methanol solution accounted for4.0%inhydroxyl, and the substitution degree was one. While the5-FUA in theproduct2which appeared in30%methanol solution accounted for8.9%inhydroxyl, and two5-FUAs were substituted.3Establish an HPLC method for the determination of5-FUA, and thedetection wavelength was271nm, without solvents and other substancesinterference with the detection of main drugs. This method was accurate,sensitive, and suitable for the determination of the content of5-FUA.Thehydrolysis ratio of the prodrugs with different substitution degree was higherin the enzymatic solution of α-amylase and carboxylic esterase than that inα-amylase solution and carboxylic esterase solution. Meanwhile, thehydrolysis ratio of the prodrug with one5-FUA substituted was obviouslyhigher than that of the prodrug with two5-FUAs substituted. According to thecurve of lgK-pH，the prodrug1hydrolyzed more as the pH value growed.4In the study of animals, an HPLC method was established todeterminate the concentration of5-FUA in the tissues and contents of stomach,small intestine and large intestine. The detection wavelength was274nm,without blank tissues and contents interference with the detection of drugs.The hydrolysis ratio of the prodrugs with different substitution degree was thehighest in the cecal contents, and that was the lowest in the gastric contents.Meanwhile, the hydrolysis ratio of the prodrug with one5-FUA substitutedwas obviously higher than that of the prodrug with two5-FUAs substituted.Correspondingly, the prodrugs released most of the drugs in the large intestine,and the contents of5-FUA in the tissues and contents of large intestinereached the top. While the prodrugs released little in the stomach, and the5-FUA wasn’t found in the tissues and contents of stomach. Conclusion:The method with two steps to produce the product of5-fluorouracil-1-acetic acid-α-cyclodextrin conjugate was simple, feasible and easy to purify.The prodrug was hydrolyzed to release little in the stomach, but release mostin the large intestine. This was obvious that the release of drug in the largeintestine was more than that in the small intestine. The result proved that theprodrugs had the property of delay-released and colon-targeted.