Study On Drug Metabolism And Pharmacokinetics Of Ebracteolata Compound B And Eochamaejasmin A

Lang-du is a famous traditional herbal medicine and has long been used in China for the treatment of a wide range of ailments, including edema, malignant tumor, leukemia, chronic tracheitis, pulmonary tuberculosis, scrofula, intestinal tuberculosis, skin disease and gynecopathia. Ebracteolata compound B (ECB), chemically designated as 2,4-dihydroxy-6-methoxy-3-methyl-acetophenone and neochamaejasmin A (NCA), chemically designated (2S,2'S,3S,3'S)-5,5',7,7'-tetrahydroxy-2,2' - bis (4-hydroxyphenyl)- 3,3'-bichroman-4,4' -dione, are primary active ingredients of this medicine. ECB was reported to exhibit significant antibacterial activities on both tuberculosis bacillus and XDR-TB bacillus. Also, it is one major active component of the marketed drug "Jieheling Tablets". More interestingly, one homologous compound of ECB,2,4-dihydroxy-6-methoxyl-methylene-3-methylacetophenone, was recently demonstrated to possess obvious selective activity against human Hela-60 tumor cell with an IC50 value 95 ng/mL. NCA, was found to exhibit significant cytotoxicity in vitro against LNCaP cells. In this study, ECB and NCA were isolated and purified from the dried root of Euphorbia fischeriana and Stellera chamaejasme L. respectively and their absorption, transport, metabolism, excretion and pharmacokinetics profiles were investigated by using several in vitro and in vivo models.1. The transport of NCA across MDCK and MDCK-MDR1 cell monolayersIn the MDCK and MDCK-MDR1 monolayer cell cultures, NCA showed a low cell permeability. The apparent permeability coefficient (Papp) values of apical to basolateral direction were less than 1×10-6 cm/s. The efflux ratios in both cells were much higher than 2.0. This efflux can be inhibited obviously by verapamil which is the classical inhibitor of P-glycoprotein (P-gp). Those findings indicated that NCA was transported by P-gp. NCA may have low bioavailability after oral administration owing to the involvement of P-gp in the NCA transport. Using Rhodamine 123 (R123) as the probe substrate probe of P-gp, we studied the effects of NCA on the P-gp, the most important transporter in the intestine. NCA at 100μmol/L had significant inhibition effects on the P-gp-mediated transport of R123 across the MDCK-MDR1 cell monolayers, indicating that NCA was likely a P-gp inhibitor. On the other hand, NCA displayed significantly higher cytotoxicity to MDCK cells than to MDCK-MDR1 cells, likely because of the involvement of P-gp and decreasing the NCA accumulation in MDCK-MDR1 cells and the result confirmed indirectly that NCA was the substrate of P-gp.2. Metabolism of ECB and NCA studied in vitro with rat liver microsomes and hepatocytesOne monohydroxylation metabolite and one monoglucuronide were observed in rat liver microsomal incubates in the presence ofβ-NADPH or UDPGA, respectively. The monohydroxylation metabolite was determined by mass spectrometry to be 1-(2,4-dihydroxy-6-methoxy-3-methylphenyl)-2-hydroxyethanone, and monoglucuronide was determined by hydrolysis withβ-glucuronidase, mass spectrometry and 1HNMR to be 2-hydroxy-6-methoxy-3-methyl-acetophenone-4-O-β-glucuronide. But the mixed incubation of ECB with rat liver microsomes in the presence of bothβ-NADPH and UDPGA showed the monoglucuronide was the most major metabolite and more than 80% of ECB was converted into monoglucuronide, indicating glucuronidation was likely the major clearance pathway of ECB in rats.After NCA was incubated with rat liver microsomes, no oxidative metabolism was observed in the presence ofβ-NADPH. But in the presence of UDPGA, two metabolites were detected in the liver microsomal incubates and identified to be monoglucuronides by UPLC-MS analysis and hydrolysis usingβ-glucuronidase. The oxidative metabolism of ECB and the glucuronidation of ECB and NCA in rat liver microsomes all exhibited typical Michaelis-Menten patterns.The isoenzymes probably involved in ECB and NCA metabolism in rat liver microsomes were identified by using selective chemical inhibition and induction, respectively. The results indicated CYP3A, CYP2C11, CYP2C6 and UGT1A6/9 (especially UGT1A6) were important catalytic enzymes in ECB metabolism and UGT1A3/6/9 were important catalytic enzymes in NCA glucuronidation.No metabolites were detected after NCA was incubated with primary cultured rat hepatocytes. Two metabolites were observed after ECB was incubated with primary cultured rat hepatocytes, but only in trace amounts. Compared with rat liver microsomes, the metabolic ability toward ECB and NCA is much low.3. Metabolism, excretion and pharmacokinetics of ECB and NCA in ratsOnly glucuronidation metabolism with one monoglucuronide of ECB and NCA were observed in rats. The monoglucuronide of ECB observed in rats was identical to that formed in rat liver microsomes. The monoglucuronide of NCA observed in rats was identical to one of two monoglucuronides formed in rat liver microsomes and its structure needed to be elucidatd.A sensitive ultra performance liquid chromatography-mass spectrometry (UPLC-MS) method has been developed and validated for the quantitation of ECB-glucuronide in plasma. This analytical method has been applied successfully to study pharmacokinetics of ECB in rats. The pharmacokinetic parameters of ECB-glucuronide were estimated in rats following a single-dose oral administration of 100 mg/kg. In rats, ECB was rapidly metabolized to its monoglucuronide and the glucuronide was the only drug-derived compound clearly detected in plasma. Two-compartment model was used to estimate ECB-glucuronide pharmacokinetics by using DAS software. The plasma profile of ECB-glucuronide was found to be rapid with an apparent tmax occurring at 4 h, indicating that ECB was rapidly absorbed in rats following an oral administration. In rats following an oral administration of NCA, no metabolites were evidently detected in plasma. Moreover, the parent drug NCA was observed in plasma only in trace amounts, indicating that the oral absorption of NCA in vivo may be poor. One reason leading to the poor oral absorption of NCA was probably the involvement of P-gp. 4. Metabolism of ECB and NCA studied in vitro with human liver microsomes, HepG2 Cells and recombinant human enzymesThe metabolic profile in human liver microsomes was consistent with that observed in rat liver microsomes and rats, indicating glucuronidation was probably the major metabolic pathway of ECB in humans.When NCA was incubated with human liver microsomes, only one monoglucuronide was observed.The catalytic isoenzymes probably involved in ECB and NCA metabolism in humans were identified by using selective chemical inhibition in human liver microsomes and recombinant human enzymes. The results indicated CYP3A4, UGT1A6/9, especially CYP3A4 and UGT1A6, were important catalytic enzymes in ECB metabolism. UGT1A3/6/9 were important catalytic enzymes in NCA glucuronidation metabolism.No metabolites were detected after NCA was incubated with HepG2 cells; No glucuronide and only trace monohydroxylation metabolite were observed after ECB was incubated with HepG2 cells, indicating the metabolic ability toward ECB and NCA is very low.