Quality Control And Pharmacokinetic Study Of Indomethacin 5-Fluorouracil-1-Ylmethyl Ester

Indomethacin 5-fluorouracil-1-ylmethyl ester （IFM） is a prodrug of 5-fluorouracil which can liberate 5-fluorouracil by occurrence of specific acid and base catalysis as well as specific enzyme catalysis.The quality of IFM has been comprehensively investigated. Firstly, two methods, UV spectrophotometry and HPLC were developed for its content determination and an HPLC method for the determination of its related substances. Secondly, a stability test was carried out using the developed method to control the quality of the bulk drug. Thirdly, hydrolysis kinetics characteristics of IFM have been illuminated from study the hydrolysis kinetics of IFM in vitro. Finally, a specific and accurate method was developed for quantifing 5-fluorouracil in rat plasma and tissues and to study the tissue distribution and excretion of IFM metabolite 5-fluorouracil in rats.1 The content determination of indomethacin 5-fluorouracil-1-ylmethyl esterUV spectrophotometry and HPLC method were developed for the determination of IFM. The linear range was 8.0-18μg·mL^-1 （r=0.9999） in UV method, the average recoveries of IFM was 99.4 %（ RSD = 0.8 %, n=9）. In HPLC method , Diamonsil^TMC₁₈ （250 mm×4.6 mm, 5μm） column was used with a mobile phase of methanol - 0.025 mol·L^-1 ammonium acetate （65:35, v/v）, （using glacial acetic acid to adjust pH to 3.5）.The flow rate maintained at 1.0 mL·min^-1.The wavelength of the detector was set at 260nm. The linear calibration curve was linear over range of 5.0 -50.0μg·mL^-1（r=0.9999）, the average recoveries of IFM was 99.7 %（ RSD =1.0 %,n=9）.AU two methods are suitable for the determination of IFM with accuracy and precision.The UV spectrophotometry is rapid and convenient, while HPLC method is specific and may be used for the determination of the impurities in IFM. 2 The determination of related substances of indomethacin 5-fluorouracil-1-ylmethyl esterAn HPLC method was developed for determination the related substances of IFM. The separation was performed on a Diamonsil^TMC₁₈ （250 mm×4.6 mm, 5μm） column. The mobile phase was composed of methanol - 0.025 mol·L^-1ammonium acetate （65:35, v/v）, （using glacial acetic acid to adjust pH to 3.5）. The detection wavelength was set at 260 nm, the flow rate was 1.0 mL·min^-1, the column temperature was 40℃.The related substances and degradation products of IFM were completely separated from IFM.The limit of detection of IFM was 2.0 ng·mL^-1.3 The stability study of indomethacin 5-fluorouracil-1-ylmethyl esterThe stress test, accelerating test and long-term stability test were carried out by using developed methods according to Guideline of study on stability of new drug .The results of stress test demonstrated the light, humidity and temperature all had effects on the stability of IFM.Both accelerated test and long-term test demonstrated that IFM were stable under the storage conditions and met the stability requirement in the Guideline for new drug development.4 Kinetics of hydrolysis of 5-fluorouracil derivative Indomethacin 5-fluorouracil-1-ylmethyl esterA reversed-phase HPLC method was developed and validated for the kinetic investigation of the chemical and enzymatic hydrolysis of IFM. Statistical evaluation of proposed HPLC methods revealed their good linearity and reproducibility. The lower limit of quantification for IFM and 5-FU were 100ng·mL^-1and 20 ng·mL^-1, respectively. The inter-day and intra-day precision （RSD） was less than 13.5 % and the accuracy （RE） was±7.5% calculated from QC samples. The hydrolysis kinetics of IFM was monitored in aqueous solution at 37℃over the pH range 1.38-10.52, the half-lives being 1.18-7.09 h. The pH-rate profile obtained revealed the occurrence of specific acid and base catalysis as well as of a water-catalyzed reaction. IFM underwent rapid hydrolysis when incubated in 80 % rat plama solutions （pH7.4） and 20 % rat liver homogenates at 37℃, the half-lives were 0.819 min and 7.584 min, respectively.From the studies of enzymatic hydrolysis in rat gastrointestinal contents, it was suggested that 5-FU was liberated from IFM mainly in small intestine and colon.5 Tissue distribution and excretion of 5-fluorouracil from indomethacin5-fluorouracil-1-ylmethylester in ratsTo study the tissue distribution and excretion of indomethacin 5-fluorouracil-l-ylmethylester （IFM） metabolite 5-fluorouracil in rats, an accurate and specific high performance liquid chromatography method for quantifying IFM in rat plasma and tissues was developed. Biological samples were prepared by liquid-liquid extraction and separated on a Diamonsil^TMC₁₈ column （250 mm×4.6 mm ID, 5μm）. The mobile phase for tissue samples, plasma samples and feces samples were composed of methanol-water-36 % acetic acid （3:96.9:0.1, v/v） and the mobile phase for urine samples was a mixture of methanol-water-36 % acetic acid （10:89.9:0.1, v/v）. The eluate was monitored by UV absorbance at 260 nm. After a single ig dose of 100 mg·kg^-1 IFM in rats, 5-Fu was mainly distributed in stomach, small intestine, liver. The concentrations of 5-fluorouracil in other tissues and plasma were low. The excretion of 5-Fu in urine and feces amounted to 0.0065 % and 0.063 % of the dose, respectively. The method is shown to be accurate and specific, and suitable for preclinical pharmacokinetic studies of IFM.