| AIMs: Our aims were (1) to synthesize Pectin-ketoprofen (PT-KP) prodrug with PT and KP, measure the amount of ketoprofen (KP) carried by prodrug and select the prodrug that carries the highest amount of KP; (2) to establish HPLC analytical method for the determination of concentration of KP in biological samples; (3) to observe the characteristics of PT-KP for colon targeting in vitro; (4) to study the distribution of PT-KP prodrug in the contents and mucosa of different parts of gastrointestinal tract of rats and the fluctuation of KP concentration in plasma; (5) to observe the curative effect of PT-KP prodrug on ulcerative colitis with the hope to find a new drug that is efficient in treating ulcerative colitis.METHODS:1. PT-KP prodrug was synthesized by the reaction of PT with KP in the presence of N, N'-dicyclohexyl carbodiimide (DCC) in dimethylsulfoxide (DMSO) according to the different weight ratio of KP to PT. Anhydrous ethanol-ether solution (40mL, 1:1, v: v) was added to the final reaction mixture, and the resulting precipitate was filtered. The precipitate was dissolved in anhydrous DMSO and anhydrous ethanol-ether solution (40ml, 1:1, v: v) was added again. This process was then repeated once. The precipitate was mounted in 10-cm dialysis bags. Each bag was immersed in 8000 mL of 80% ethanol, which was stirred for 48h at 200 rpm. After 80% ethanol inbags was removed under reduced pressure, the solution was dried at 60 ℃ for 24h to yield PT-KP.2. The prodrugs synthesized according to different weight ratio of KP to PT were hydrolyzed for 30min, 1h, 2h, 3h, 4h in 2 mol . L-1 sodium hydroxide solution, respectively. The times that the prodrugs need to hydrolyze completely were determined. The amount of KP in prodrug was measured by HPLC. The most reasonable weight ratio of reaction was identified to obtain the prodrug that carries the highest amount of KP.3. The amount of KP released from prodrug was examined after being incubated in different simulated gastrointestinal fluids (pH=1.0, 6.8 or 7.4) and Pectinolytic enzymes fluid at 37℃ . The percentage cumulative amounts of KP released from different simulated gastrointestinal fluids and pectinolytic enzymes fluid at different time were detected. The stability of prodrug in different simulated gastrointestinal fluids and pectinolytic enzymes fluid was observed.4. A sensitive HPLC method was put forward to determine the concentration of KP in rats. This method was also used to study the absorption and distribution of KP in rats. KP was extracted with t-butyl methyl ether. Chromatogram column: Hypersil C18 (5μm, 150mm×4.6mm); mobile phase: Methanol : 50mmol . L-1 potassium dihydrogen phosphate buffer (52:48); column temperature: room temperature; flow rate: 1ml.min-1; detection wavelength: 258nm.5. KP or PT-KP was given to rats by oral administration at a dosage of 10mg. kg-1. Plasma and the different parts of gastrointestinal (GI) tract were taken 2, 4, 6, 8, 10 and 12h after oral administration of KP or PT-KP to rats and the concentration of KP were measured by HPLC. The colon targeting property of PT-KP prodrug was observed.6. By using TNBS, the model of ulcerative colitis in rat was replicated. Colitis rats were treated for 6 days with PT-KP by oral administrationat the doses of 50mg .kg- .d-1,100mg .kg-1 . d-1,200mg .kg-1 . d-1 respectively. The ulcerative area was measured by picture processing system of computer. The weights of the Colon, the spleen and the thymus were measured. The results were compared respectively with those obtained in the control group and the model group. Statistical analyses were conducted to determine the therapeutic effect and colon-targeting property of PT-KP. RESULTS:1. PT-KP prodrug was prepared following the method as mentioned above. IR showed that PT-KP had strong absorption peaks at 3327, 2929,2850,1743cm-1. The findings indicate that the carboxyl group of KP is covalently bound to one of hydroxyl groups of PT through an ester bond.2. In 2 mol. L-1 sodium hydroxide solution, the prodrugs that were synthesized were found to hydrolyze completely in 2h. The HPLC results showed that the amount of KP carried by PT-KP prodrug was 10.6%.3. 96.2% of KP released from its prodrug after the incubation in simulated pectinolytic enzymes fluid for 8h. However, there was nearly no KP released in simulated stomach fluid, in small intestinal fluid or in simulated colon fluid.4. The calibration curves of plasma, gastrointestinal contents and mucosa were obtained (See Tab. 3). The results indicate that there is a good linear relationship between X and Y for each sample range. Within-day precision relative standard deviations (RSDs) of plasma, gastrointestinal contents and mucosa of high, medium and low concentrations were lower than 10%, while day-to-day precision RSDs lower than 15%. The recovery rates of all the samples under study ranged from 85% to 15%.5. After the administration of KP, KP was observed to distribute incontents and mucosa of the stomach, proximal small intestine, and distal small intestine, while much less in the cecum and colon, with a maximal KP per gram of the contents of 3.60μg. Its distribution decreased along the gastrointestinal tract and was absorbed rapidly to enter the blood circulation. Its blood concentration peak was present at 2h after administration, and was about 9.60μg . ml-1. By contrast, after the administration of PT-KP, KP was not detected in the contents and mucosa of the stomach, the proximal small intestine and the distal small intestine, while in the contents and mucosa of the cecum and the colon, KP was observed to be in a high concentration, with a maximal KP per gram of contents of 10.02 u g. Its absorption rate was decreased and blood concentration peak was present at 8h after administration, and was about 1.84μg . ml-1. 6. After PT-KP was used to treat ulcerative colitis in rats by oral administration at the doses of 100mg . kg-1 .d-1,200mg .kg-1 .d-1, the ulcerative area of colon was reduced to 30.43% and 5.80% respectively, with a significant difference between the results and those obtained in the model group. The findings show that the doses of 100mg . kg-1 .d-1,200mg . kg-1 . d-1 had no therapeutic effect on the weights of the thymus and the spleen of rats. CONCLUSION:PT-KP was synthesized for the first time in the present study. The amount of KP carried by prodrug was 10.6%. 96.2% KP was released from its prodrug at 8h after the incubation in simulated pectinolytic enzymes fluid. However, there was nearly no KP released in simulated stomach fluid, in small intestinal fluid or in simulated colon fluid. The method employed in the present study has been proved accurate, rapid, sensitive and satisfying enough to be applied to measure the concentration of KP in biological samples. The vivo study shows that the highest amount of KP released from its prodrug is in the contents...
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