Pharmacokinetic Studies Of Verticinone In Rats

Hubeibeimu, which is the dried bulbs of Fritillaria hupehensis Hsiao et K.C. Hsia, has been used as an important antitussive and expectorant herb in Traditional Chinese Medicine for more than 2000 years. Verticinone, which is the major alkaloid in F. hupehensis, has been reported to exhibit potent antitussive and expectorant effects by our lab. Our group has synthesized a series derivants of verticinone with cholic acid. Pharmacodynamic research proved that cholic acid-verticinone ester and verticinone-cholic acid salt exhibited potent antitussive and expectorant effects. To develop verticinone and its derivants as new antitussive drugs, a systemic preclinical pharmacokinetic study of verticinone was performed as follow:1. The development of quantification method for the determination of verticinone in biological samplesA sensitive and specific LC-MS method was developed and validated for the quantification of verticinone in biological samples of rats. Verticinone and the internal standard (IS), hupehenine, were extracted from biological samples by a simple liquid-liquid extraction with ethyl acetate after being alkalified by 1 M ammonia hydroxide.The precision and accuracy of the method were acceptable for bioanalytical assays, and the LLOQ was 0.1 ng·mL-1, which was sensitive enough to monitor the low-dosage pharmacokinetic study of verticinone in rats. The method has been successfully applied to the pharmacokinetic study of verticinone in rats.2. The absorption pharmacokinetics of verticinone in ratsTo investigate the absorption pharmacokinetics of verticinone, groups of rats received i.g. and i.v. administration of verticionone. For the i.g. groups, rats received single oral doses of 1,2 and 4 mg·kg-1 and multiple oral doses of 4 mg·kg-1·day-1 given as 2 mg·kg-1 doses twice a day. For the i.v. group, rats received a single dose of 0.4 mg·kg-1. LC-MS method was used for the quantification of verticinone in rat plasma samples. Individual plasma concentration-time data were analyzed by compartmental and non-compartmental analysis.Gender difference of absorption pharmacokinetics of verticinone was observed in rats. The Cmax and AUC0-t in male rats were higher than that in female rats significantly, but the CL and Vd in male rats were lower than that in female rats, which indicated that the elimination of verticinone was faster in female rats than in male. The absolute oral bioavailabilities of verticinone in male and female rats were 44.8% and 2.7%, respectively. After successive i.g. administration of verticione, accumulation was observed in female rats but was not observed in the male.3. Tissue distribution of verticinone in ratsThe tissue distribution of verticinone in rats was investigated at 0.25,0.58 and 6 h after oral administration of 2 mg·kg-1. LC-MS method was applied to determine the concentration of verticinone in tissue and plasma samples. Verticinone could be found in many tissues, and the concentrations of verticinone in most tissues were higher than in plasma. This study showed that verticinone had a good tissue penetrability and a high tissue affinity.4. Excretion of verticinone in ratsUrine, feces and bile of rats were collected after i.g. administration (2 mg·kg-1), and the samples were analyzed by LC-MS method. In male rats, the cumulative extraction of unchanged form of verticinone in bile, urine and feces were 0.31%,2.50% and 0.92%. In female rats, the cumulative extraction of unchanged form of verticinone in bile, urine and feces were 0.02% 0.23% and 0.42%.The results indicated the unchanged form was not the main excretion path of verticionone in rats.5. Metabolism of verticinone in rats in vivo and in vitroA LC/IT-TOF-MS assay was used to investigate the metabolites in urine, feces and bile samples in rats after a single oral dose of 2 mg·kg-1 verticione. And a total of 12 metabolites were found in these biological samples in male rats and 8 in the female. The primary metabolic pathway of verticinone was dehydrogenation in male rats in vivo but was sulfation in the female.The metabolites of verticinone following incubation of the drug with rat liver and intestine S9 fractions were also studied by this method. Hydrogenation was the primary metabolic pathway of verticinone following incubation with rat liver S9 fractions. While, hydroxylation and dihydroxylation were the primary metabolic pathway of verticinone following incubation with male and female rat intestine S9 fractions, respectively.6. The influence of verticinone on CYP450 enzyme in humanThe study investigated the influence of verticinone on the activity of five main subtypes (CYP1A2,2C9,2C19,2D6 and 3A4) of human cytochrome 450 enzyme in vitro. The results showed that verticinone had no effect on five main subtypes of human CYP450 enzyme in vitro.The thesis conducted a systematic pharmacokinetic study of verticinone in rats. The absorpation, distribution, metabolism, excretion and the influence on the activity of CYP450 enzyme of verticinone were clarified. The results provide pharmacokinetic information for the preclinical evalution of verticinone.