The Study Of Intestinal First-pass Elimination And Hepatic First-pass Elimination Of Oleanolic Acid To Its Bioavai Lability

Objectives:Oleanolic acid (OA),3β-hydroxy-olea-12-en-28-oic acid, is a pentacyclic triterpenoid compound, which was clinically used in the treatment of hepatitis, liver fibrosis and cirrhosis. But the low bioavailability affects its clinical application. There have not been any studies on the effect of intestinal first-pass elimination and hepatic first-pass elimination of OA to its bioavailability. The aim of this research is to investigate the effect of intestinal first-pass elimination and hepatic first-pass elimination of OA to its bioavailability. The result may provide the evidence for the effective application of OA in the clinical practice and the development of new formulation.Methods:The thesis is made up of following parts:1Establishment and validation of LC/MS/MS method to determine the concentration of OA in rat plasmaGlycyrrhetinic acid was used as an internal standard, and sample pre-treatment consisted of a liquid-liquid extraction by ethyl acetate. Chromatographic separation was achieved on a Gemini110A C18column (50mm×2.0mm i. d.,5p m) by gradient elution with a mobile phase consisting of acetonitrile and0.01%formic acid in water. A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) under negative ionization mode with OA (m/z455.3â†'455.3,[M-H]-)and internal standard glycyrrhetinic acid (m/z469.4â†'425.3,[M-H]-). The validation of assay included selectivity, precision, recovery, matrix effect and stability.2Pharmacokinetic study of OA after different dosing routesThe studies of this part include:â‘ Stability of OA in artificial gastric juiceâ‘¡In vitro distribution kinetics of OA between plasma and blood cells of rat bloodâ‘¢The binding of OA to human serum albumin (HAS) through equilibrium dialysisâ‘£Tissue distribution of OA after intravenous and intragastric administration in rats⑤The residual OA in gastrointestinal tract, stools and urine after intragastric administration in ratsâ‘¥Measurement of gastrointestinal first-pass effect and hepatic first-pass effect of OA by studying pharmacokinetics of OA after four different dosing routes-intravenous (IV), intragastric (IG), intraduodenal (ID) and intraportal (IPV).3Metabolism study of OA in hepatic and intestinal microsomes in vitroThe contents of the enzymes metabolic reactions of OA in intestinal and hepatic microsomes in vitro are:â‘ NADPH-dependent metabolism and UDPGA-dependent metabolism of OA in the rat hepatic microsomes.â‘¡UDPGA-dependent metabolism and NADPH-dependent metabolism of OA in rat intestinal microsomes.â‘¢Chemical inhibitors experiment of OA in rat hepatic microsomes.4Effect of ketoconazole and theophylline on the pharmacokinetics of OARats were assigned to groups of control, ketoconazole (CYP3A inhibitor) and theophylline (substrate of CYP1A2). Rats were gavaged with0.5%CMC-Na,20mg/kg ketoconazole and50mg/kg theophylline. After30min, they were gavaged with50mg/kg OA, and the blood samples were collected at the defined time points. The concentration of OA in rat plasma was determined, and the pharmacokinetic parameters of OA were calculated.Results:1The method possesses merits of simplicity, good sensitivity and good accuracy. And the validated method will be helpful to the pharmacokinetic study of OA.2The Results show that OA is stable in artificial gastric juice. The whole blood and plasma concentrations of OA were almost constant up to2h incubation.This suggests that OA rapidly reaches the equilibrium between plasma and blood cells from rat blood. The equilibrium plasma-to-blood cells partition ratios of OA was2.35±0.42. The binding rate between OA and human serum albumin is77.11%±2.80%-83.21%±1.57%. The gastrointestinal is the main tissue for distribution after intragastric administration. OA is mainly distributed in lung, spleen and kidney after intravenous administration. The residual OA in gastrointestinal tract, stools and urine after intragastric administration in rat is about2.19%±0.77%. The bioavailability of OA is0.77%, hepatic extraction is35.18%, intestinal extraction is98.73%, gastric first-pass effect is0.088%, hepatic first-pass effect of OA is0.70%and intestinal first-pass effect is95.82%. The conclusion is that the first-pass elimination of OA occurs predominantly in rat small intestine.3The Results elucidated that OA underwent a NADPH-dependent oxygenation and UDPGA-dependent binding reaction in rat hepatic microsomes. And the rate of oxygenation and binding reaction is128.5±36.2nmol/min/mg protein,115.8±18.3nmol/min/mg protein, respectively. According to the Eadie-Hofstee plots, there is not only one kind of enzyme involved in the reaction. And in rat intestinal microsomes,OA underwent a NADPH-dependent oxygenation and UDPGA-dependent binding reaction, too. The metabolic rate is42.4±10.9nmol/min/mg protein,90.4±12.9nmol/min/mg protein, respectively. Ketoconazole and alpha naphthoflavone significantly inhibit the metabolism of OA. Therefore, we speculate CYP3A and CYP1A2are the main enzymes involved in metabolism of OA.4After intake of ketoconazole and theophylline, the AUC and Cmax of OA increased. The pharmacokinetic change of OA can be attributed to the regulation of CYP3A and CYP1A2after administration of ketoconazole and theophylline.Conclusion:This thesis developed a LC/MS/MS analysis method for determination of OA in plasma, and the method can be applied to related pharmacokinetic studies. First-pass elimination of oleanolic acid occurs mainly in liver and intestine rather than the stomach. In rat hepatic and intestinal microsomes,OA underwent a NADPH-dependent oxygenation and UDPGA-dependent binding reaction, and the enzymes invovled is nonuniqueness enzyme. Combined with expiriments of chemical inhibitors of enzymes incubation in vitro in rat hepatic microsomes and in vivo study of the effcet of ketoconazole and theophylline on the pharmacokinetics of OA, CYP3A and CYP1A2are validated to participate in the metabolism of oleanolic acid.