The Glucuronidation Of Andrographolide And Their Derivatives In Vitro

Objective:Study the glucuronidation of andrographolide(AND) and their derivatives, deoxyandrographolide(DEO) and dehydroandrographolide(DEH) which are the major active components in Chinese medicine Andrographis paniculata. Aim to identify the conjugation position of metabolites, the major UGT enzyme involved in UGT metabolism, the metabolism difference among five species, the anti-inflammatory of three compounds and their glucuronides, and the drug and drug interaction among them.Methods:(1) In vitro experiments: investigation of the glucuronidation metabolism of andrographolide and their derivatives in different liver microsomes including human(HLM), pig(PLM), Cynomolgus monkey(CyLM), dog(DLM), SD rat(RLM), Mouse(MLM), and 12 recombinant UGT isoforms.(2)To isolate and identify the metabolites of andrographolide and their derivatives glucuronidation.(3) To evaluate the anti-inflammatory activity of the parent drugs and their glucuronides.Results: Six glucuronides were isolated and identified them using 1D and 2D nuclear magnetic resonance(NMR) spectroscopy. UGT2B7 made the greatest contribution to glucuronidation of all three agents. BipHasic kinetic model were shown in the glucuronidation of AND and DEO in HLM, Michaelis-Menten model was shown in the glucuronidation of DEH in HLM and glucuronidation of these components in different UGT isoforms. We also systematically analyzed various kinetic parameters(Km, Vmax, and CLint) for glucuronidation of AND, DEO, and DEH. Regioselective glucuronidation showed considerable species differences, 19-O-Glucuronides were present in liver microsomes from all species except rats. 3-O-Glucuronides were produced by pig and monkey liver microsomes for all compounds, and 3-O-glucuronide of DEH was also detected in mouse and rat liver microsomes. The inhibition of LPS-induced NO production, followed by AND-1, AND-2, and the 19-O-glucuronides of DEH and DEO. The 3-O-glucuronides of DEH and DEO have no inhibition. DEO and DEH were found to be competitive inhibitors of AND glucuronidation with Ki values of 2.5 and 12.8 μM.Conclusions:In this study, we ?rstly report the analysis of the glucuronidation of AND, DEH, and DEO in liver microsomes prepared from humans and ?ve experimental animal species and identified the structure of these glucuronides. UGT2B7 was responsible for the formation of 19-O-glucuronides from AND, DEH, and DEO in HLM. It was particularly notable that the 19-O-glucuronide of AND in HLM exhibited potent anti-in?ammatory activity. Furthermore, signi?cant species differences in glucuronidation only 19-O-glucuronides were found in HLM but 3-O-glucuronides were found in some species liver microsomes. Additionally, DEO and DEH have potent inhibotion towards the glucuronidation of AND in HLM. All these findings may assist in selection of the right animal models for pharmacokinetic or toxicological studies of AND, DEH, and DEO, and also provide useful guidance for clinical use and for the development of new anti-in?ammatory agents from AND derivatives.