The Effect Of CYP450and Efflux Transporters(BCRP,MRP2)on Monoester-diterpene Alkaloids And Alcohol Amine-diterpene Alkaloids Disposition

Background and Objectives:The Chinese medicine Aconitum has been used as an essential drug in China and in other East Asian countries for thousands of years owing to its extremely excellent effect against rheumatism rheumatoid arthritis and other forms of inflammation. Nevertheless, Aconitum is a highly toxic herb that can cause fatal poisoning, since therapeutic dose of Aconitum is very close to its toxic dose, frequent cases of poisoning after the intake of processed Aconitum are encountered in clinics. Although the toxicity of Aconitum has been decreased with proper processing by hydrolyzing highly toxic diester-diterpene alkaloids to less toxic monoester-diterpene and alcohol amine-diterpene alkaloids, its clinical application is still under restrictions.Diterpenoid alkaloids, the main effective compounds of Aconitum, are also the highly toxic constituents accounting for the acetyl group at C8, the hydroxyl group at C13, four methoxyl groups at C1, C6, C16, and C18, and the benzoyl ester group at C14. Diterpenoid alkaloids, especially the diester-diterpene alkaloids, can cause fatal cardiac poisoning and neurotoxicity. Diterpenoid alkaloids include diester-diterpene alkaloids (DDAs), monoester-diterpene alkaloids (MDAs) and alcohol amine-diterpene alkaloids (AADAs). Unprocessed Aconitum contains high amounts of three highly toxic DDAs, namely, aconitine (AC), mesaconitine (MA), and hypaconitine (HA). So the utilization of unprocessed Aconitum in clinic is forbidden. With proper processing, AC, MA, and HA become exposed to abundant hydrolysis, transforming them into less toxic MDAs, namely, benzoylaconine (BAC), benzoylmesaconine (BMA), and benzoylhypaconine (BHA), and MDAs further transform into AADAs, namely, aconine, mesaconine and hypaconine. As a result, MDAs and AADAs are the primary active and toxic constituents of processed Aconitum. However, the toxic reaction still occurs in the event of excessive absorption of MDAs and AADAs. Frequent cases of poisoning after the intake of processed Aconitum are encountered in clinics.Drug-metabolising enzymes such as CYP450, UGT and SULT enzymes, and efflux transporters such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance associated protein2(MRP2), act as the human body’s first line of protection by limiting the invasion of xenobiotics, particularly the toxicants. After drug is absorbed into the systemic circulation, parent compound could be metabolized into less toxic and higher polar products by phase Ⅰ or Ⅱ drug-metabolising enzymes in the liver, then they will be eliminated from the body. Efflux transporters could make the drug from intracellular to the intestine, reduce its bioavailability. Protection or promotion of efflux transporters function could prevent excessive Aconitum absorbed into the bloodstream, thereby avoiding toxic reactions.No phase Ⅱ metabolites of Aconitum alkaloids by human liver microsomes were detected in preliminary experiments. Previous studies have reported the metabolism of DDAs with human liver miscrosomes. AC could be metabolized into six metabolites by CYP enzymes in human liver microsomes, with CYP3A4/5and2D6as the most important isoforms responsible for metabolism The main metabolic pathways of AC include demethylation, dehydrogenation, hydroxylation, and didemethylation. MA was transformed in vitro into nine metabolites in male human liver microsomes, and the primary contributors toward metabolism were CYP3A4and3A5. Probable secondary contributions were offered by CYP2C8,2C9, and2D6. The main metabolic pathways of MA include demethylation, dehydrogenation, hydroxylation, and demethylation-dehydrogenation. Besides, HA could be metabolized into less toxic products mainly by CYP3A4/5, followed by CYP2C8,2C9,2D6and2E1. The metabolic pathways involved in HA were demethylation, demethylation-dehydrogenation, didemethylation and hydroxylation. However, the mechanism of CYP-mediated metabolism of MDAs and AADAs, the primary active and toxic constituents of processed Aconitum, remains unknown.It has been reported that efflux transporters such as P-gp, BCRP, and MRP2were involved in the transport of DDAs using cultured Caco-2cell model. MRP2might also mediate the transport of MDAs. Moreover, the invasion of AADAs could not be limited by efflux transporters. Although human colonic adenocarcinoma Caco-2cell line is most widely employed for evaluating drug intestinal transport mechanisms in vitro. However, there are some disadvantages limiting its use. For instance, culturing Caco-2cells could be more time-consuming than culturing other epithelial cells (e.g., MDCK), and therefore the model may be more expensive to use. Moreover, Caco-2cells do not always express appropriate amounts of transporters or enzymes. As is well known, Caco-2membranes functionally express a variety of efflux transporters, such as P-gp, BCRP, and MRP2, and the inhibitors of these transporters are almost mutual. Thus, it is difficult to identify which transporter is miediated the analyte when performed the inhibition experiments in Caco-2cell. The Mardin-Darby canine kidney (MDCK) cell is a dog renal epithelial cell line. MDCKII cell transfected with efflux transporter has been reported to express high level of one efflux transporter, such as MDR1-MDCKII cell, is widely used to evaluate the role of P-gp in the transport of analyte. And we have investigated that P-gp was involved in the transport of MDAs using MDR1-MDCKII cell line. It is necessary to use overexpressing BCRP/MRP2-MDCKII cell to investigate the role of BCRP and MRP2in the transport of Aconitum alkaloidsTherefore, one aim of our study is to investigate the metabolic mechanism of MDAs and AADAs by CYP450using ch monoclonal antibodies (mAbs), and cDNA-expressed CYP enzyme.. The second aim is to investiagate the bidirectional transport of highly toxic Aconitum alkaloids across BCRP-MDCKII and MRP2-MDCKII cells in the absence and presence of efflux transporters inhibitors. The results of the present study on the metabolic mechanism and absorption fate of Aconitum alkaloids could provide significant information for the safe clinical practice of processed Aconitum.Methods and materials:1. The metabolism of MDAs and AADAs by CYP450MDAs and AADAs were metabolized with human liver microsomes in typical phases I reaction incubation system to identify the CYP metabolites of MDAs and AADAs using UPLC-MS/MS, and to distinguish which isozymes are responsible for their metabolism through the use of chemical inhibitors, monoclonal antibodies (mAbs), and cDNA-expressed CYP enzyme. High-resolution mass spectrometry (HRMS) was used to characterize their metabolites.2. The role of efflux transporters BCRP and MRP2on the transport of Aconitum alkaloidsThe role of efflux transporters on the transport of Aconitum alkaloids (DDAs, MDAs and AADAs) using cultured MDCKII, BCRP-MDCKII and MRP2-MDCKII cells. Bidirectional transport assays of the Aconitum alkaloids were performed with or without efflux transporters inhibitors, such as MRP2inhibitors (LTC4), BCRP inhibitor (Ko143).Results:1. The CYP metabolism of monoester-diterpene alkaloids (MDAs) Benzoylaconine (BAC)Compared with the negative control (without NADPH-regenerating system), a total of seven BAC metabolites (M1-M7) were found in the human liver microsomes along with the NADPH-regenerating system (Fig.3-4). The metabolites were identified by the retention times, the chromatographic behaviors, and characteristic mass spectrometric fragmentation features, which are summarized in Table3-2. The main metabolic pathways were demethylation, dehydrogenation, demethylation-dehydrogenation, deethylation, and deethylation-dehydrogenation, which produced less toxic metabolites by decomposing the group responsible for the toxicity of the parent compound.In the next experiments, we further investigated which CYP isozymes are responsible for BAC metabolism. The targeted isozymes included CYP1A2,2C8,2C9,2C19,2D6,2E1,3A4, and3A5, because up to90%of human drug metabolism may be attributed to these eight enzymes. In the chemical inhibitors experiments (Fig.3-9), the major hepatic enzyme responsible for BAC metabolism was identified as CYP3A4/5, while chemicals selective inhibitors for CYP2C9and CYP1A2made a minor effect on their formations. Other inhibitors such as CYP2C8,2C19,2D6and2E1did not work on the BAC metabolism. It was assumed that a selective chemical inhibitor raised against a human CYP isoform at high concentration, which was more than the Kj value, would cross-react with other CYP isozymes. For example, recent studies suggest that omeprazole is a substrate probe for CYP2C19, however in vitro studies show that omeprazole could be metabolized by CYP3A4in the same way. As is well known, the chemical inhibitors are never100%and their selectivity is not always great. Hence, we used mAbs experimental approach to reconfirm our results. We demonstrated that antibody against human CYP3A4/5was the most potent and inhibited the formation of BAC metabolites (Fig.3-10). Furthermore, the experiment of cDNA-expressed CYP enzymes showed that CYP3A4and CYP3A5were the major isozymes for the metabolism of BAC, and the BAC metabolite M5also could be generated by CYP2D6(Fig.3-12). However, the inhibition experiments of BAC did not support that CYP2D6was the subordinate enzymes which mediated the biotransformation of BAC. Other enzymes, CYP1A2,2C8,2C9,2C19and2E1, were not involved in the formation of BAC metabolites. Taken together, the results of cDNA-expressed CYP enzymes, chemical inhibitors, and mAbs experiments, we came to the conclusion that CYP3A4and CYP3A5were the major isozymes responsible for the BAC metabolism.Benzoylmesaconine (BMA)The experiments about the CYP metabolism of BMA were accordant with that of BAC, as described above. BMA could be transformed into at least eight metabolites (M1-M8) in human liver microsomes (Fig4-4). The metabolic pathways were demethylation, dehydrogenation, hydroxylation, and demethylation-dehydrogenation. The results of the effect of chemical inhibitors on BMA metabolism (Fig.4-9) showed that the inhibitor of CYP3A made the greatest contribution to the generation of BMA metabolites, and the inhibitor of CYP2C9made the secondary contribution. Moreover, the inhibitors of CYP2C8,2C19had minor effects on metabolite M2, M3, M4, M7and M8. While the inhibitors of1A2,2D6and2E1had no obvious inhibitory effects on BMA metabolism. Simultaneously, human CYP3A4/5, the most potent and inhibited the formation of BMA metabolites, was supported by the data of mAbs experimental approach (Fig.4-10). CYP1A2,2C8,2C19,2D6and2E1also could transform BMA to metabolite M5. However, the results were not verified by the inhibiton studies. Recombinant human cytochrome P450isoforms CYP3A4and3A5contributed greatly to the formation of BMA metabolites. CYP1A2,2C8,2C9,2C19,2D6and2E1played a minimal role in the formation of BMA metabolites (Fig.4-12).Benzoylhypaconine (BHA)BHA was transformed into at least9metabolites (M1-M9) in vitro in human liver microsomes (Fig.5-4). The metabolic pathways were demethylation, dehydrogenation, hydroxylation, and demethylation-dehydrogenation.The results of the effect of chemical inhibitors on BHA metabolism (Fig.5-9) were shown that the inhibitor of CYP3A made the greatest contribution to the formation of BHA metabolites, and the inhibitor of CYP2C9made the secondary contribution. Moreover, the inhibitors of CYP2C8,2C19had minor effects on metabolite M3, M4, M7and M8. Whereby, the inhibitors of1A2,2D6and2E1had no obvious inhibitory effects on BHA metabolism. Furthermore, human CYP3A4/5, the most potent and inhibited the production of BHA metabolites, was supported by the data of mAbs experiments (Fig.5-10). For the cDNA-expressed CYP Enzymes studies, the primary contributing CYP isoforms were CYP3A4and3A5, the contributions of CYP1A2,2C8,2C9,2C19,2D6and2Elwere rarely observed (Fig.5-12).2. The CYP metabolism of alcohol amine-diterpene alkaloids (AADAs)Alcohol amine-diterpene alkaloids (AADAs), including aconine, mesaconine and hypaconine, were nearly not metabolize in human liver microsomes using UPLC-MS/MS analysis. The metabolic pathways of aconine, mesaconine and hypaconine were dehydrogenation, demethylation and demethylation-dehydrogenation, respectively.3. Transport of DDAs, MDAs and AADAs in cultured MDCKII cells, BCRP-MDCKII and MRP2-MDCKII cellsThe transports of AC, MA, HA, BAC, BMA, BHA, aconine, mesaconine and hypaconine were investigated using MDCKII, BCRP-MDCKII and MRP2-MDCKII cell models for the first time.The transport of1μM Aconitum alkaloids across MDCKII and BCRP-MDCKII cell monolayers from A to B or from B to A side was investigated. The Papp values of AC, MA and HA from B to A were significantly (P<0.05, Fig.7-5) higher than those from A to B in BCRP-MDCKII cells. Their efflux ratio (Er) values were significantly greater in BCRP-MDCKII cells (31.99±1.66,9.74±2.61,6.07±0.39, respectively), compared to those in parent MDCKII cells (1.62±0.13,0.88±0.23,0.80±0.16, respectively). Furthermore, the intracellular accumulations of AC, MA and HA in the BCRP-MDCKII cell model were significantly (P<0.05, Fig.7-6) lower in both directions in comparison with those in MDCKII cells. No significant differences were observed in the intracellular amounts of BAC, BMA, BHA, aconine, mesaconine and hypaconine in the two cell lines. We came to a conclusion that BCRP was demonstrated to mediate the transport of AC, MA and HA.We also studied the transport of Aconitum alkaloids across MDCKII and MRP2-MDCKII cell monolayers from A to B or from B to A side to evaluate whether MRP2made a contribution to their absorption. The Papp values of AC, MA, HA, BAC, BMA and BHA from B to A were significantly (P<0.05, Fig.7-7) higher than those from A to B in MRP2-MDCKII cells. Their Er values in MRP2-MDCKII cells were significantly higher than those in parent MDCKII cells, which were AC (6.38±0.27versus1.62±0.27), MA (5.90±0.46versus1.89±0.22), HA (2.18±0.22 versus0.92±0.09), BAC (1.59±0.25versus1.08±0.07), BMA (1.67±0.21versus1.23±0.03), BHA (1.89±0.18versus1.23±0.07). Transport studies were carried out in the presence of a MRP2inhibitor (LTC4) to further determine the role of MRP2in the transport of Aconitum alkaloids. Under the effect of0.1μM LTC4, the Er values of AC, MA, HA, BAC, BMA and BHA were greatly decreased (P<0.05), compared to those without inhibitor. In contrary, intracellular amounts of AC, MA, HA, BAC, BMA and BHA were significantly increased from A to B and from B to A. Taken together, MRP2mediated the transport of AC, MA, HA, BAC, BMA and BHA.We found that the transports of AC, MA and HA were mediated not only by BCRP, but also by MRP2. Besides, the transports of BAC, BMA and BHA were influenced by MRP2. Both BCRP and MRP2were not involved in the transport of aconine, mesaconine and hypaconine. The influence of efflux transporters on the transport of Aconitum alkaloids were AC, MA, HA> BAC, BMA, BHA> Aconine, Mesaconine, Hypaconine.Conclusions:1. A total of7,8, and9metabolites were determined, respectively, when BAC, BMA, or BHA was incubated in the HLMs along with NADPH-regenerating system. Their metabolic pathways were demethylation, dehydrogenation, hydroxylation, demethylation-dehydrogenation, and deethylation. The high toxicity of Aconitum alkaloids was due to the acetyl group at C8, the hydroxyl group at C13, four methoxyl groups at Cl, C6, C16, and C18, and the benzoyl ester group at C14, meaning that the loss of these groups will decrease the compound’s toxicity. All the metabolites (except for the hydroxylation metabolite) were either loss of methyl from methoxyl group or dehydrogenation from hydroxyl group, indicating that their toxicity was less than that of the parent compounds. 2. Alcohol amine-diterpene alkaloids (AADAs), including aconine, mesaconine and hypaconine, were nearly not metabolize in human liver microsomes. The influence of CYP450enzymes on the metabolism of Aconitum alkaloids were DDAs> MDAs> AADAs.3. The transports of AC, MA and HA were mediated by both BCRP and MRP2, and the transports of BAC, BMA and BHA were mediated by MRP2. The influence of efflux transporters on the transport of Aconitum alkaloids were DDAs> MDAs> AADAs.In summary, the effects of CYP450and efflux transporters (BCRP, MRP2) on Aconitum alkaloids limit excessive Aconitum absorbed into the bloodstream and prevent the toxic reactions.