Study On The Metabolism And Pharmacokinetics Of GA(C17:1)

Ginkgo biloba was widely used in the world as a drug for the prevention and treatment of cerebrovascular disease. Ginkgolic acid is a mixture of structurally related n-alkyl phenolic acid compounds and widely exists in leaves, nuts and external seed coat of Ginkgo biloba L. The alkyl side chain in molecular structures varies from13to19carbons in length with0-3double bonds. GA (C15:1) and GA (C17:1) are the two main ingredients accounted for nearly80%of the total ginkgolic acid. Ginkgolic acid is considered to be the toxic components of ginkgo with cytotoxicity, sensitization, mutagenic and carcinogenic, hepatotoxcity and nephrotoxicity. However, it was also reported that ginkgo acid has shown a wide range of pharmacological activities, including anti-bacterial, anti-inflammatory, anti-allergic, insecticidal, inhibition of the growth of a variety of tumor cells in vitro and in vivo as well as HIV protease activity and HIV infection in vitro. Most of the research focused on the pharmacological and toxicological aspects of ginkgolic acid, the reports about drug metabolism and pharmacokinetics of GAs are still rare. In this study, GA(C17:1) and GA(C15:1) were isolated and purified from the ginkgolic acid extract and their absorption, transport, metabolism, excretion and pharmacokinetics profiles were investigated by using several in vitro and in vivo models. The results of this study could provide theoretical and experimental basis for the further research of ginkgolic acid. 1. Metabolism of GA (C17:1) in rat liver microsomesGA (C17:1) could be metabolized by CYPs (Phase I metabolism), and UGTs (Phase II metabolism). The special chemical inhibitor experiments indicated that CYP1A1/2and CYP3A were involved in GA (C17:1) oxidative metabolism; UGT1A7and UGT1A9were involved in GA (C17:1) glucuronidation metabolism. Classical CYPs’ substrates were used to investigate the inhibition effect of GA (C17:1) on the CYPs, with the results that GA (C17:1) has inhibition effect on CYP2C6and no effect on CYP3A, CYP1A1/2, CYP2E1, CYP2D1. Three metabolites of GA (C17:1) in rat liver microsomes (RLMs) were analyzedas (two mono-hydroxylation metabolites and a glucuronidation metabolite) by LC-MS/MS.2. Metabolism of GA (C17:1) in human liver microsomes, recombinant human CYPs and UGTsThe affinity of GA (C17:1) to human liver microsomes (HLMs) was less than that to RLMs. It was found that CYP3A4and CYP1A2were the two CYP enzyme isoforms that mediated the oxidative metabolism of GA (C17:1), UGT1A6, UGT1A9and UGT2B15were involved in GA (C17:1) glucuronidation metabolism. GA (C17:1) showed certain inhibition effect on CYP2C9. GA (C17:1) at10μmol/L showed no gene induction of CYP2B6and CYP3A4(<30%of rifampicin induction ability), concentration-dependent inductionof UGT1A1tested by dual-luciferase reporter assay.3. The metabolism and toxicity of GA(C17:1) on HepG2, rat primary hepatocytes and L02cellGA (C17:1) showed the different levels of toxicity on HepG2, rat primary hepatocytes and L02cell. Pretreatment with selective CYP inducers β-naphthoflavone and rifampin, could increase the cytotoxicity of ginkgolic acid (C17:1) in HepG2cells and rat primary hepatocytes. Co-incubation with selective CYP inhibitors a-naphthoflavone and ketoconazole could decrease the cytotoxicity of ginkgolic acid in primary rat hepatocytes. The results indicated that GA (C17:1) showed greater cytotoxicity on HepG2cells comparing with primary rat hepatocytes and L02cell, and CYP1A and3A catalyzed GA (C17:1) to more toxic metabolite.4. Transport and uptake studies of GA (C17:1) and GA (C1S:1) in MDR1and BCRP transgenic cellsMDCK-MDR1and LLC-PK1-BCRP cells were adopted as the in vitro model to predict the intestinal absorption and blood-brain barrier (BBB) permeability. The results showed that GA (C17:1) and GA (C15:1) are the substrates of efflux transporters, P-gp and BCRP. GA (C17:1) and GA (C15:1) may have not very good oral bioavailability and BBB permeability. GA (C17:1) and GA (C15:1) had significant inhibition effects on the P-gp-mediated transport of R123across the MDCK-MDR1cell monolayer.5. Plasma pharmacokinetics, tissue distribution, metabolism and excretion studies of ginkgolic acid (C17:1) in ratsA rapid and sensitive method for the determination of ginkgolic acid (C17:1) in rat biological samples was developed using high performance liquid chromatography tandem mass spectrometry (LC-MS/MS). The pharmacokinetic investigation demonstrated that GA (C17:1) was rapidly absorbed with an absolute bioavailability being approximate19.5%. The tissue distribution indicated that liver and kidney were the major accumulation tissues for GA (C17:1) in rats. In addition, GA (C17:1) could hardly cross the blood-brain barrier. The excretion study suggested that feces was the main excretion pathway,while high proportion of GA (C17:1)(>35%) was excreted as the unchanged form. And two metabolites in feces were identified. 6. Pharmacokinetics of ginkgolic acid (C15:1) and ginkgolic acid (C17:1) in extract of external seed coat of ginkgo biloba L. in ratA rapid and sensitive LC-MS/MS method for the simultaneous determination of ginkgolic aicd (C15:1) and ginkgolic aicd (C17:1) in rat plasma was developed and applied to investigate the pharmacokinetics of ginkgolic acid extract in rats. Compared with ginkgo acid monomers, the pharmacokinetic parameters of GA (C17:1) and GA (C15:1) has changed externally. Coadministrated intravenously P-gp and BCRP inhibitor CsA at20mg/kg can significantly improve GA (C17:1) and GA (C15:1) plasma concentration levels, CsA can also inhibit brain P-gp and BCRP, increased GA (C17:1) and GA (C15:1) distribution to the brain.