The Transport Of Neferine And Its Analogs In Vitro And The Cell Toxicity Of Neferine Reactive Metabolites

Lotus plumule, the dried young cotyledon and radical of the Nelumbo nucifera Gaertn.(Fam. Nymphaecea) ripe seed, is a famous Traditional Chinese Medicine and a popular food to remove heat from the heart, anchor the mind, arrest bleeding for centuries. Neferine and its analogs, liensinine and isoliensinine, are the major active alkaloids components in lotus plumule and have O-methylated structural differences and all belong to bisbenzylisoquinoline alkaloids. Various studies have indicated that these alkaloids have beneficial pharmacological effects such as anti-arrhythmic and anti-hypertensive, while neferine displayed potent anti-HIV and anti-tumor function. Previous researches have shown the ability of neferine to reverse multidrug resistance (MDR) related to downregulation of MDR1mRNA and P-gp expression, and the poor intestinal absorption and low brain exposure of liensinine. These prompted us to investigate the interaction between ABC transporters and neferine and its analogs. In this study, transgenic cell models MDCK-MDR1and MDCK-MRP2were used in transcellular transport experiment to predict the disposition in vivo. In addition, the mechanism of neferine sensitizing effect on anti-cancer drug and direct antitumor role is unclear. Hence, this study focused on the metabolism of neferine in human liver microsomes and the mechanism of increasing toxication in terms of metabolism bioactivation using high expression CYP3A4MDCK cell line and HepG2cell line.1. The interactions between neferine and its analogs and ABC transportersThe aim is to explain neferine and its analogs absorption and distribution characterization, the interactions between these alkaloids and ABC transporters were evaluated using transgenic cell lines by cell accumulation and transcellular transport studies. MTT assays were employed to determine the cytotoxicity of the three alkaloids for the proper drug concentrations. Neferine and its analogs accumulation in MDCK-MDR1cells were significantly lower than that in MDCK cells, and the efflux could be inverted by P-gp inhibitors, indicating the alkaloids above were all possibly substrates of P-gp. For the transport of neferine, liensinine and isoliensinine across the cell monolayers, they all showed net efflux ratios (NERs) higher than2.0, which decreased to close to1with classical inhibitors GF120918and CsA. These results suggested the three alkaloids were all substrates of P-gp. However, the NERs of neferine and isoliensinine decreased rapidly at20and40μM which suggested saturation over the concentration range tested. Our study revealed the Papp values of the alkaloids were lower than3×10-6cm/s, and especially the Papp values of liesinine≤1×10-6cm/s which indicated the weak membrane permeability and estimated poor absorption of plumula alkaloids. In addition, liensinine accumulation were similar in MDCK-MRP2and MDCK cells, bi-directional transport study showed no significant efflux so we speculated that liensinine was not a substrate of MRP2.2. Metabolism of neferine in vitroTo preliminarily deduce the main metabolites structures of neferine after incubation with HLM, identify the main enzymes responsible for this alkaloid and determine its kinetic parameters. The metabolites are mainly classified as two types, bis-demethylated (Ml, m/z597.4), and demethylated (M2-1, m/z611.4, M2-2and M2-3, m/z611.3) speculated were respectively2-N-demethyl-2’-N-demethyl neferine, liensinine,2’-N-demethyl neferine and2-N-demethyl neferine by the LC-MS/MS fragmentation information, previous results from incubation in dog liver microsomes and comparing the retention time of references. Chemical inhibition, human recombinant CYPs incubation were performed to identify the main enzymes that catalyzed neferine metabolism. CYP3A4participated in all demethylation, whereas CYP2C8and CYP2D6respectively involved in the formatation of M2-3and M2-2, the related enzyme kinetics parameters were calculated by the formation rate of metabolites using recombinant CYPs. Neferine was metabolized to quinone methide intermediates could be trapped by nucleophilic agents glutathione (GSH). There were four conjugates including M3([M+H]+, m/z902.4), M4-1([M+H]+, m/z916.4), M4-2([M+H]+, m/z916.4) and M5([M+H]+, m/z930.4). CYP3A4was the main enzyme responsible for the formation of reactive metabolites with the two same methods. Concentration dependence of GSH on the increase of GSH conjugates showed that the four conjugates mostly formed by non-enzymatic reaction. Addition of GSTA1, GSTT1and GSTP1did not enhance largely the production of these GSH conjugates, thus null alleles of GSTT1dispite of their high frequencies did not have a effect on the detoxification of reactive metabolites of neferine.3. CYP3A4mediated the cytotoxicity of neferineTo investigate the potential hepatic toxicity of neferine in terms of metabolism bioactivation, this study provided the evidence for the sensitizing effect on anti-cancer drug and direct antitumor function. Comparing the toxicity on HepG2cells of neferine and its analogs liensinine, isoliensinine by MTT assay, the results showed neferine was most toxic; the toxicity increased with the time treated by neferine. To estimate the effect of reactive metabolites of neferine on cell viability, MDCK-CYP3A4were treated by neferine combining with GSH synthesis blocker BSO or oxidation scavengers NAC in the MTT study. The results that IC50(mock+Nef)28.65±1.08μM> IC50(MDCK-CYP3A4+Nef+NAC)25.77±1.08μM> IC50(MDCK-CYP3A4+Nef)19.34±1.04μM> IC50(MDCK-CYP3A4+Nef+BSO)12.49±1.06μM declared that the potentiating effects of BSO and alleviating effects of NAC on neferine induced cytotoxicity in MDCK-C YP3A4cells. Meanwhile mock cells were more significantly tolerance than MDCK-CYP3A4cells. Similarly, different administration gourps showed the same trend in HepG2cells that IC50(HepG2+Nef)36.55±1.06μM> IC5O (HepG2+Nef+NAC)35.64±1.13μM> IC50(HepG2+Nef+BSO)27.02±1.03μM> IC50(HepG2+Nef+Rif)15.05±1.22μM. Moreover, intracellular GSH levels all had time-dependent decline varying in ranges. The depletion of GSH in MDCK-CYP3A4cell was more rapid and greater than mock cells, while addition of BSO or NAC changed the intracellular GSH levels in MDCK-C YP3A4cells together with neferine.