| Schizonepetin, a natural monoterpene, and structurally related to lactones, is derived from the essential oil of Schizonepeta tenuifolia. After a series of separation and purification procedures, schizonepetin which had the optimum activity was obtained. Previous pharmacodynatics experiments demonstrated that schizonepetin has excellent antiviral activity,in addition, the activity of anti-inflammatory, analgesic and bacteriostasis. Until now, the report of schizonepetin has been focused on quality standard, extraction and pharmacology, but there are few studies about its pharmacokinetics and metabolic mechanism. So, it is very important to study and clarify the dynamic transformation rule and drug interaction of schizonepetin.The main objective of this research is to characterize the pharmacokinetics and metabolic mechanism of schizonepetin,which is a candidate new drug, so as to support the metabolism and pharmacokinetic study in human and to offer the reference for clinical dosage and dosage form changing.This dissertation is summarized as follows:Part one: Literature reviewBy means of reviewing relative domestic and overseas documents, the current status of studies on anti-influenza virus, pharmacokinetic study, analytical method of drugs and their metabolites and cytochrome P450 were summarized. In addition, the thoughts and methods of this subject were narrated briefly.Part two: Water solubility and lipid solubility of schizonepetinA method for the determination of schizonepetin was established, and the water solubility and lipid solubility was evaluated. The result showed that the equilibrium solubility of schizonepetin in methanol was higher than that in other solvents. Schizonepetin had a low solubility in water,and it was almost insoluble in petroleum ether. It had little change in apparent distribution coefficient in neutral phosphate buffer solution and had significant decrease in apparent distribution coefficient under the alkaline conditions. The BMC of schizonepetin was 15.98 under the condition of pH7.4 and 25 degrees, which showed its good lipid solubility and absorbability.Part three: The oral bioavailability study of schizonepetin in ratsIn order to elucidate the pharmacokinetic profile of schizonepetin in rats, a rapid, sensitive,and reliable analytical HPLC method was established and fully validated to determine schizonepetin in biological samples.The pharmacokinetic profile and oral bioavailability of schizonepetin were studied following a single oral administration or intavenous injection in rats. Individual concentration-time data were fitted by noncompartmental model in Kinetica 4.4, to calculate the key pharmacokinetic parameters.The results showed that dose linearity of the pharmacokinetics over the dosage range examined was demonstrated. Following three single oral dosing, plasma concentration profiles showed a quick absorption phase with the mean Tmax of 0.7 h. The terminal elimination half-life(t1/2) was about 3～4 h through different intragastric and intravenous administration doses to rats. The Cmax and AUC0-∞ values of the three doses indicated an apparent dose-proportionality and the absolute bioavailability was about 75%.Part four: Gastrointestinal absorption kinetics of schizonepetin in rats1. Stomach absorption kinetics of schizonepetin in ratsThe drug concentration by in situ perfusion in rats was determined by HPLC. The hourly absorption percentages of three different drug concentrations (1.84, 3.68 and 7.36 μ·ML-1) in stomachs were (19.47± 0.69) %, (21.66 ±1.92) % and (26.51±1.25) %,respectively, which had significant differences among them (P<0.05). Schizonepetin was absorbed in stomachs might via passive transport mechanism.2. Intestinal absorption kinetics of schizonepetin in ratsThe absorption kinetics and absorption site were investigated by using the in situ intestine perfusion method in rats. The concentration of schizonepetin in the intestine perfusate was determined ’by HPLC-UV. The result showed that the absorption rate constant (Ka) of schizonepetin at low, middle and high concentrations (1.84, 3.68 and 7.36μg mL-1) were(0.2028± 0.0070),(0.1589 ± 0.0114) and (0.1342 ± 0.0122) h-1,respectively,which had significant differences among them (P<0.05). The Ka values of schizonepetin (3.68 μg·mL-1) at duodenum,jejunum and ileum were (0.0238 ± 0.0006),(0.0125 ± 0.0005) and (0.0165 ± 0.0008)h-1,respectively, which had also significant differences among them (P < 0.05). Bile duct-ligated or not and intestinal flora had little influence on the intestinal absorption of schizonepetin. Compared with control group, the Ka value of p-gp inhibitor group had significant differences (P<0.05). So it indicated that the absorption of schizonepetin is a first-order process with the active diffusion mechanism and schizonepetin can be absorbed at all intestinal segments, and duodenum is the best absorption site.Part five: The distribution characteristics of schizonepetin in rats1. The tissue distribution study of schizonepetin in ratsTissue distribution of schizonepetin in rats at different time was studied following a single oral administration or intavenous injection. The results indicated that schizonepetin was absorped quickly and had a wide distribution in the tissues throughout the whole body within the time course examined.2. In vitro banding rates of schizonepetin to rat plasma proteinsPlasma protein binding was studied by equilibriun dialysis method (18 h) at 37℃ with final concentratoin of 0.08, 0.63 and 6.38 μg·mL-1. The results of binding rates of schizonepetin to rats stabilised at about 63%. It was found that schizonepetin has medium plasma protein binding rate in rats, with no significant relation to drug concentrations in dialyzate.Part six: Biliary, Urinary and fecal excretion of schizonepetin in ratsA HPLC-UV method was developed and validated for the determination of schizonepetin in urine, feces and bile. Using this method, the excretion features were studied in rats after oral and intravenous administration. The research indicated that there was significant difference of excretion ratio of schizonepetin between male and female rat urine and bile for oral administration, which also existed in urine for intravenous administration. This phenomenon was nonexistent in feces for two administration of schizonepetin. Schizonepetin was excreted in bile, urine and feces little in form of prototype drug, which demonstrated that the main excretion way of schizonepetin was metabolic product.Part seven: Metabolic products of schizonepetin in rat urineAfter oral administration of schizonepetin to rats, all the urine was collected and was preliminarily treated by macroporous absorption resin. Then the 40% EtOH extracts was collected and was purified by chemical separation such as column chromatograph,SephadexLH-20 and recrystallization. Finally, some products were isolated from the extracts and their structures were concluded by 1H NMR, 13C NMR and IR spectrum. Then the products were studied fore antiviral screening. However, the result found that metabolic products of schizonepetin didn’t show obvious inhibition on the influenza virus H3N2 and herpes virus HSV-1. So it was speculated that the prototype drug of schizonepetin may play an important role on effect of anti-influenza virus.Part eight: Effect of schizonepetin on P450 enzyme activity in rats1. Effect of schizonepetin on rat hepatic microsome P450 enzyme activity in vitroThe microsomes were prepared from rat livers by calcium precipitation method and the protein concentration in microsomes was determined by ultraviolet spectrophotometer. Then omeprazole and its metabolic products were determined to prove the feasibility of the rat liver microsomes. The results showed that there were no products of schizonepetin in microsomes. In addition, the enzyme activity in microsomes was not statistically significantly affected by schizonepetin, which testified the safety of schizonepetin in metabolism mediated by CYP450.2. Effect of schizonepetin on rat P450 enzyme activity in vivoThis study was aimed to find effects of schizonepetin on some important CYP450 enzymes in vivo. Using phenacetin, dapsone. chlorzoxazone, omeprazole, metoprolol as the substrate drugs of CYP1A2. CYP3A1/3A2, CYP2E1. CYP2C19 and CYP2D6, the effects of schizonepetin on CYP450 enzymes have been explored at pharmacokinetics levels of these substrate drugs. The results showed that after oral administration of schizonepetin to rats for seven days, the enzyme activity of CYP3A1/3A2 was induced and the enzyme activities of CYP1A2, CYP2E1 and CYP2D6 were restrained. In addition, there was not significantly affected on CYP2C19.3. This study was to observe the impact of schizonepetin on the mRNA expression of CYP3A1, CYP1A2 and CYP2E1 genes of the liver in rats. The mRNA expression lever of CYP450 was detected by real-time RT-PCR. The results found that compared with control group, the mRNA expression of CYP3A1, CYP1A2 and CYP2E1 genes of low groups had little difference (P>0.05), and that of the middle and high groups had significant differences (P<0.05), which had induction effect on them. In addition, the high group had induction effect on the metabolism of CYP450.In summary, schizonepetin was of good lipid solubility and poor water solubility. It was absorbed in stomach might via passive diffusion and that of active transport mechanism in intestine. It oral absolute bioavailability in rats was about 75% and had a wide distribution in the tissues. The main excretion way of schizonepetin was metabolic product, which had no antiviral activity. Schizonepetin was of good stability in microsomes, and it had no effect on the enzyme activity in microsomes. In addition, schizonepetin showed different induction or inhibition effects on the substrate drugs of CYP450 enzyme in vitro and in vivo. So all factors shoud be taken into consideration when combination therapy was used. |
PDF Full Text Request