Studies On The Biopharmaceutics And Pharmacokinetics Of Scutellarin In Rats

Scutellarin, a flavone glucuronide, as 4', 5, 6-trihydroyflavone-7-O-β-D-glucuronide, extracted from a Chinese herb Erigero breviscapus(vant.) hand.-Mazz., is of particular interest because of its significant dilating blood vessel, improving microcirculation, increasing cerebral blood flow, decreasing blood viscosity, preventing and dissolving thrombus, inhibiting platelet aggregation activity, reducing blood fat, and having hypoglycemic action and so on. Up to date, it has been widely used in treatment of cardiovascular and cerebrovascular diseases.This study contributes to the understanding of mechanisms responsible for low oral systemic availability of scutellarin for the first time and can help identify potential factors affecting the systemic exposure of scutellarin. As a result, incubation method in vitro was adopted and the detection was carried out in gastrointestinal remnant in vivo, indicating that scutellarin was metabolized by intestinal microflora. Metabolism in isolated intestine was studied, and results showed that scutellarin was metabolized by enterocyte cell. Intra-intestinal cycle and entero-hepatic cycle were elucidated along with drug absorption. Extensive metabolism via deglucuronidation and glucuronidation occurred in rat liver and intestinal microsomes using microsomal preparations. Scutellarin was administered to rats by intravenous or intraportal infusion. The data strongly suggested that hepatic first-pass elimination did not exert a significant role in the low oral bioavailability for the first time. In addition, intestinal secretion mediated by transporter played an insignificant role in the presystemic elimination using an in vivo intestinal perfusion model. Limited membrane permeability and extensive metabolism in gut wall might contribute more significantly to the low oral bioavailability of scutellarin using everted sacs method. In our studies, rats were used as the research animal, and the primary pharmacokinetic characteristics of scutellarin were studied. After intravenous administration to rats over the doses range of 10-40 mg/kg, the AUC values were linear over the administered doses range (r=0.995), suggestive of the linear pharmacokinetic characteristic of scutellarin in rats after intravenous administration. However, the bioavailability of scutellarin in rats after oral administration was very poor. The possible reasons of low oral bioavailability of scutellarin were the factors of gastrointestinal tract such as the low oral solubility in gastrointestinal fluid, limited membrane permeability, intestinal secretion mediated by transporter, gut metabolism before absorption, hepatic first-pass elimination, or gut wall metabolism (Ⅰphase reaction andⅡphase reaction). In order to investigate the absorption mechanism of scutellarin, systemic research was carried out in these studies as follows:Firstly, the physicochemical properties of scutellarin were investigated to confirm the effect of essentiality on the bioavailability. Simultaneously, the stability of scutellarin in different conditions was studied to ensure that the pharmacokinetics data were scientific and reliable. Results showed that the solubility of scutellarin was improved with the pH enhancing, and that apparent octanol/water distribution coefficients (log P) decreased with the pH increasing between 1 and 7.4. The log P of scutellarein was higher than that of scutellarin, indicating that scutellarein exhibited a much greater lipophilicity toward membrane permeation than scutellarin.Secondly, rats were administered with scutellarin solution or suspension to investigate the effect of solubility of scutellarin on the bioavailability. After rats were administered scutellarin solution or suspension. The absolute bioavailability of scutellarin solution and suspension were 4.94%, 5.65%, respectively. Moreover, there is no difference between scutellarin solution and suspension through analysis of variance (ANOVA). Results showed that whether it was soluble or not wasn't the main reason of low oral bioavailability.Thirdly, first pass effect consists of gastrointestinal first-pass effect and hepatic first-pass effect, and the bioavailability of drugs with first-pass effect usually is very low. In the present study, the systemic exposure of scutellarin following intraportal was compared with intravenous administration to understand the contribution of presystemic hepatic elimination to the low oral bioavailability. Results showed that the hepatic first-pass elimination of scutellarin played an insignificant role in the presystemic elimination of orally administered scutellarin.Fourthly, the intestinal microflora is responsible for the liberation of aglycone from glycosidic drug. Scutellarin is a glucuronide of scutellarein which has glycosidic bond. The metabolism of scutellarin in gastrointestinal content was investigated using incubation method in vitro. Results in vitro were consistent with that in vivo, indicating that scutellarin was destroyed seriously in gastrointestinal track after oral administration, and the main metabolite was scutellarein. In other words, scutellarin was metabolized by caecal and colonic microflora. The contribution of the enterobacterium metabolism to the low oral bioavailability of scutellarin did not play a significant role.Results showed that scutellarin and its metabolites were mainly eliminated through feces excretion after a single dose of scutellarin suspension (40 mg/kg), and a majority of drug was absorbed. The distributions of scutellarin in organs at different time after oral administration of scutellarin (100 mg/kg) were different. The concentrations of native scutellarin in liver and kidney were higher than other organs. The distributions of scutellarin in stomach, duodenum, jejunum and ileum were higher than other tissues, but the distributions of scutellarein in ileum, cecum, colon and rectum were higher than other tissues. Drug distribution was related to the metabolic concentration of scutellarin in gastrointestinal tract.Fifthly, in order to investigate the contribution of intestinal secretion mediated by transporter on the oral bioavailability, intestinal secretion in situ perfusion was studied after intravenous administration with a single-pass perfusion technique. The contribution of scutellarin in bile was excluded by catheterization of the biliary duct before perfusion. The intestinal excretion amount of scutellarin in administered dose was lower than 1%. Results showed that the contributions of intestinal secretion on the oral bioavailability exerted insignificant role. Drugs excreted through intestine can be metabolized to liberate aglycone, and then the aglycone can be reabsorbed in intestine to form intra-intestinal cycle. The concentration of scutellarin in bile was higher than that in plasma, and the active excretion mechanism may be present in bile excretion. Scutellarin and its metabolites excreted by bile, parts of which were reabsorbed in intestine into portal vein and entero-hepatic cycle came into being.Sixthly, because of the intestine being an important first-pass organ, drugs were metabolized by intestinal microflora before absorption, and metabolism occurred in the intestine before entering into mesenteric vein. In our studies, intestinal absorption and metabolism of scutellarin in the isolated rat intestine were studied using everted sacs method. Results showed that the apparent permeability coefficient was lower. Therefore, the epithelial permeability of the drug in vivo is predicted to be very low and potentially bioavailability limiting.The metabolites were identified by LC-MS-MS. Metabolite M0 was scutellarin and M2 was scutellarein, M1 may be Scutellarein-6, 7-O-β-D-diglucuronide, M3 may be Scutellarein-6-O-β-D-glucuronide. The metabolites absorbed in intestinal fluid were same as that in portal vein. Results showed that metabolism occurred during the intestinal absorption, and the contribution of gut wall metabolism played a significant role on the low bioavailability.Lastly, liver, as an important drug-metabolizing organ, is the important site for the biotransformation of drugs. The mammalian small intestine serves principally as the site for absorption of nutrients, water, and both beneficial and potentially harmful xenobiotics. Both phaseⅠand phaseⅡmetabolic enzymes are expressed in the small intestine, together with associated transporters. In our studies, the biotransformation of scutellarin in vitro was investigated in rat liver and intestinal microsomes. Results showed that NADPH and NADH had no effect on the metabolic rate of scutellarin in rat liver and intestinal microsomes. Scutellarin was deglucuronidated in rat liver and intestinal microsomes in incubation systemⅠand the main metabolite was scutellarein (M2). Scutellarin was deglucuronidated and glucuronidated in rat liver and intestinal microsomes in incubation systemⅡ, and the main metabolites were scutellarein (M2) and another glucuronide of scutellarein (M3). Scutellarein was glucuronidated in rat liver and intestinal microsomes in incubation systemⅡand the main metabolites were scutellarin and another glucuronide of scutellarein (M3). Results suggested that the biotransformation of scutellarin in gut wall affected the oral bioavailability of scutellarin, evidently.