Studies On Triptolide Metabolism Based On Cytochrome P450 In Rat Liver Microsomes

Triptolide (TP), a diterpene triepoxide, is an active ingredient isolated from a shrublike vine Tripterygium wilfordii Hook. F (Lei gongteng) and shows many pharmacological activities such as anti-inflammation, immuno-suppression, anti-tumor and anti-fertility in male. However, the clinical use of TP is limited because of the severe toxicities. Recently, there has been growing interest in drug metabolism. At present the relativity of both activity and toxicity with metabolism of drug has been confirmed. Cytochrome P450 (CYP450) is one of microsomes monooxygenase enzymes, and play a vital role in metabolism of endogenous and xenobiotic chemicals. The results of toxic impact of Lei gongteng are influenced by factors such as individuality, age or the time of taking medicine. Therefore, it is necessary to investigate the relation between the metabolic profile with the toxicity of TP. Up to now, a few studies on TP metabolism have been reported. The metabolism profile of TP influenced by age as well as gender, the chemical structure and physiological activity of major metabolites were investigated in the present based on CYP450s in rat liver microsomes. Effects of CYP 3A inducer dexamethasone (DXM) on the metabolism and toxicity of TP in rat, and TP on CYP450 enzymes activity were also assessed in this paper. The main contents of this paper are as follows:(1) A rapid high performance liquid chromatography (HPLC) method was developed for the detection of TP in rat liver microsomes. The calibration curve of peak areas versus the concentration was linear over the range of 1.98 -158.20μg/mL. Precision and accuracy of the method were also validated. TP was converted to four metabolites (M1, M2, M3 and M4) in rat liver microsomes and three (M2, M3 and M4) in human liver microsomes. All the products were identified as mono-hydroxylated metabolites of TP by LC-MS/MS-ESI. CYP450 isoforms involved in the metabolism of TP were studied by using selective CYP450 enzyme inhibitors. Our results showed that three CYP450 isoforms including CYP1A2, CYP2C9 and CYP3A4 were found involved in the metabolism of TP. CYP3A played a major role in the formation of metabolite M3 and also participated in the formation of M2 and M4. CYP2C9 was responsible for the formation of M2, M3 and M4. CYP1A2 was mostly involved in formation of M4, and a less degree in the formation of M1 and M2. The metabolism of TP in liver microsomes from rats treated with CYP1A1/1A2 inducer 3-methylcholanthrene (3-MC) or CYP3A inducer DXM revealed that the metabolic rate of TP was obviously increased. The Vmax in rats treated with 3-MC or DXM was about 3.45 and 9.58-fold higher than that in the control group, while the Km was about 3.14 and 3.57- fold higher. The metabolic profile in rat microsomes treated with DXM was different from that treated with control vehicle. The metabolite M3 was significantly enhanced, and there were low amount of M4 while no M1, M2 was observed, suggesting that CYP3A played a major role in the formation of metabolite M3. At the same time, a new dihydroxylated metabolite of TP was found.(2) Kinetics study of TP metabolism in rat liver microsomes with different ages and genders were performed in vitro. Metabolism kinetics of TP showed difference in different ages (4 weeks, 2~3 months, > 12 months) and genders, although the difference of total CYP450 content was no statistically significant. The values of Vmax and intrinsic clearance (CLint) in adult male rat were higher than that of young and aged male rats, and the values of Vmax, Km as well as CLint were similar in young and aged male rats, which showed the elimination of TP in adult rat was quickest in male rat. The obvious effects of age on Km and CLint were observed in female rat. The Vmax in young female rats was about 3-fold higher than that in adult or aged female rat. The differences in gender of Vmax, Km as well as CLint were not observed in young rats, but were significant in adult and aged rats. A significant difference of metabolites in gender was observed. M4 was not found under experimental condition in adult and aged female rats, and M2 was very low in all female rats.(3) Effects of CYP3A inducer dexamethasone on the metabolism and toxicity of triptolide in rat were investigated based on TP metabolism in vitro. TP metabolism in vivo was evidently quickened when rats were induced by DXM (50 or 100 mg/kg, i.p, 4d), which was consistent with that of TP metabolism induced by DXM in vitro.In toxicity study, compared to the control, there was a significant difference in the serum ALT, AST, MDA and liver TSH, PSH in the rat treated with TP (0.2mg/kg, i.g, 3d), and significant differences in ALP, BUN, TSH, PSH were observed in the rat treated with DXM (50 mg/kg, i.p, 4d). However, no statistical differences of serum AST, ALT, BUN, Crea, MDA, SOD and liver SH except ALP were observed in the rat co-treated with DXM (50 mg/kg, i.p, 4d) and TP. ALP level was evidently increased in the rat co-treated with DXM and TP (0.2 or 0.4 mg/kg, i.g, 3d), compared to the DXM group. The results showed the co-administration of TP and DXM decreased TP hepatotoxicity, and TP might decrease nephrotoxicity induced by DXM. These results suggested that metabolic products may be active substances, and the toxicity of metabolic products may also be lower than that of TP.(4) Two major metabolic products (M3 and M4) were obtained by using semipreparative HPLC and identified by using LC/MS/MS as well as NMR (1H-NMR, H-H COSY and C-H HMBC). M3 is tripdiolide which occurs in Tripterygium wilfordii Hook .F, and M4 is 19-hydroxytriptolide. Cytotoxicity of M3 and M4 on HepG2 or MCF-7 cells was investigated using MTT method. Both TP and M3 remarkably inhibited the growth of HepG2 and MCF-7 cells at concentration range 2～260 ng/mL. The IC50 of TP and M3 were 90.4, 108.7 ng/mL on HepG2 cells, 33.4, 112.9 ng/mL on MCF-7 cell, respectively. M4 also inhibited the growth of MCF-7 cells at 0.1～26μg/mL (IC50 = 3.2μg/mL), and cytotoxicity on HepG2 cells was not observed in experimental concentrations. The results indicated the toxicity of metabolic products may be lower than that of TP.(5) Effects of TP on CYP450 enzymes activity were studied. TP increased the content of Cytochrome b5, inhibited CYP3A activity and induced CYP2B1/2B2 activity at dose levels of 0.04 and 0.06 mg/kg, No effects of TP (0.02, 0.04 and 0.06 mg/kg/d, i.g, 7d) on total CYP450 content and CYP2E1 were observed.In this paper, the results revealed the metabolism profiles of TP in the CYP450-mediated, and shows TP combination therapy with other drugs may interact. The outcomes of this dissertation would be important significance to guide rational drug application and further study on the toxic mechanism of TP, and also provides a new idea for the purpose of developing new drugs.