| Objective:1Based on the detoxification mechanism of the central depressants by P-gp induction resulted from MDR1gene up-regulation, to investigate whether the test drugs are P-gp substrates by ATPase activity assay, to establish a spectrum of P-gp substrates for central depressants with common clinical poisoning occurrences, and to clarify the scope of drugs which are suitable for the new treatment way of patients with drug poisoning.2To investigate permeability of the tested drugs across the in vitro BBB model by bidirectional transport assay, and to further confirm whether P-gp is involved in transport of these drugs at blood-brain barrier.3To investigate the effects of quetiapine itself on PXR signaling pathway in regulation the drug metabolizing enzyme CYP3A4and the transporter P-gp in liver, prefrontal cortex, and hippocampus of rats under regular dose and toxic dose, and to investigate the effects of the PXR agonist on of gene transcription and protein expression of CYP3A4and P-gp in liver, prefrontal cortex, and hippocampus of rats under toxic dose, to illustrate its possible molecular mechanism of promotion drug clearance by activating PXR signaling resulted in up-regulation of the drug metabolism and transporter, and to provide new ideas for new antidote development. Methods:1Based on literature survey, many central depressants are substrates or regulators of P-gp. To establish a spectrum of P-gp substrates for central depressants with common clinical poisoning occurrences, inclusion criteria was established and the test drugs were included in this study. After determining ATP standard curve, with the ATPase inhibitor Na3VO4(sodium orthovanadate) as negative control, and with the ATPase activator verapamil as positive control, fluorescence intensity of the test compounds at high, middle, and low concentration were detected by multifunctional microplate reader. After data processing, the obtained values of△RLUTC and△RLUbasai were compared to investigate the effects of drugs on P-gp ATPase activity.2We developed a human BBB model using human brain microvascular endothelial cells and astrocytes, and then validated model features.The scope of non-toxic concentrations of each test drug on the two cell lines were determined by MTT assay. The method for quantitative analysis of each test drug in cell supernatant were developed and validated. With the P-gp substrate Rh-123as positive control, the permeability of the test drugs across blood-brain barrier were investigated by bidirectional transport assay, and to further confirm whether P-gp is involved in these drugs’transport at blood-brain barrier, the effects of the P-gp inhibitor verapamil on drugs transport across blood brain barrier were investigated.3Quetiapine was chosen as overdose model drug, SD rats were chosen as poisoning model animals, and dexamethasone was chosen as the agonist of the nuclear receptor PXR. SD rats were randomly divided into4groups, namely the control group (Control group), quetiapine regular dose group (Normal group), quetiapine toxic dose with no intervention group (Toxic group), and quetiapine toxic dose with dexamethasone intervention group (Toxic+Dex group).Animal experiments were carried by grouping. At selected time points, liver, prefrontal cortex and hippocampus of anesthetized rats were collected and kept in liquid nitrogen. We investigated the expression of mRNA and protein of PXR, CYP3A4and P-gp in liver, prefrontal cortex and hippocampus of rats at different time points, and then processed the data for statistical analysis.Results:1RLU values of sulpiride, ziprasidone, and citalopram group were greater than verapamil group, which suggested that the affinities for P-gp of these drugs were weaker than verapamil. ARLUTC values of sulpiride, ziprasidone, and citalopram were less than their ARLUbasal values, which suggested that these drugs could inhibit P-gp activity by inhibiting the ATPase activity. There was no significant difference in RLU values between different concentrations for sulpiride, ziprasidone, and citalopram, which suggested that inhibition of P-gp of these drugs were not concentration-dependent. RLU values of zopiclone and moclobemide at high concentration were less than verapamil group, which indicated that the affinities for P-gp were stronger than verapamil because of more ATP consumption during the reaction. ARLUTC values of zopiclone, clomipramine and moclobemide at high concentrations were greater than ARLUbasal, while their ARLUTc values at middle and low concentrations were less than ARLUbasal, which indicated that these drugs at high concentration had induce effect on P-gp, while had inhibitory effect on P-gp at low and middle concentrations by inhibiting ATPase activity.2In vitro blood-brain barrier model was developed using human brain microvascular endothelial cells and astrocytes.The model we established had the characteristics of high tightness, limited permeability, and expression of transporters.Then we developed rapid, sensitive and specific methods for determination of each test drug concentration for bidirectional transport assay. Similar to the results of Rh-123,polarized permeation of sulpiride and citalopram were observed with the basolateral-to-apical (B-to-A) permeability being greater than the A-to-B permeability, and the efflux ratios (efflux rate/influx rate) of sulpiride and citalopram in the absence of the P-gp inhibitor verapamil in the BBB model were greater than2at low, middle, and high concentrations. The results suggested that sulpiride and citalopram were P-gp substrates at blood-brain barrier. Furthermore, for sulpiride and citalopram, the P-gp inhibitor verapamil significantly reduced the B-to-A permeability and increased the A-to-B permeability, thus leading to a decrease in the efflux ratios at three different concentrations, which indicated that efflux sulpiride and citalopram were inhibited by verapamil. From these findings, we presumed that sulpiride and citalopram were effluxed by P-gp at BBB. In the study, we also found that for moclobemide, zopiclone, and clomipramine at low, medium and high concentrations, the B to A flux were lower than the A to B flux in the BBB model, which suggested that moclobemide, zopiclone, and clomipramine were poor or non-substrate for P-gp at the BBB model with no polar transport characteristics.Compared with the result without verapamil, the Papp values and the efflux ratios had little change in the presence of verapamil at three different concentration levels of moclobemide zopiclone, and clomipramine, which indicated that their efflux was not inhibited by the P-gp inhibitor verapamil. This was more evident that the efflux activity of moclobemide, zopiclone, and clomipramine were not related to P-gp at the in vitro BBB model.3After intraperitoneal injection with regular dose of quetiapine, the expression levels of mRNA and protein of the nuclear receptor PXR, drug metabolizing enzyme CYP3A4and the transporter P-gp in liver, prefrontal cortex, and hippocampus of rats in Normal group were higher than Control group. After intraperitoneal injection with regular dose and toxic dose of quetiapine, the expression levels of mRNA and protein of PXR, CYP3A4, and P-gp in liver, prefrontal cortex, and hippocampus of rats in Toxic group were higher than Normal group. After intraperitoneal injection with toxic dose of quetiapine, the expression levels of mRNA and protein of PXR, CYP3A4, and P-gp in liver, prefrontal cortex, and hippocampus of rats in Toxic+Dex group were higher than Toxic group. The PXR-CYP3A4/P-gp signaling pathway in rats of Toxic+Dex group increased faster than other groups.Conclusion:1Sulpiride, ziprasidone, zopiclone, clomipramine, citalopram, and moclobemide were P-gp substrates (or inhibitors).Sulpiride, ziprasidone, and citalopram were non-concentration-dependent ATPase inhibitors with no significant difference in inhibition on ATPase among different concentrations.Zopiclone, clomipramine, and moclobemide were concentration-dependent bidirectional regulators of ATPase:these drugs had induced effect on P-gp by inducing ATPase activity at high concentration, while had inhibitory effect on P-gp by inhibiting ATPase activity at middle and low concentrations.2The results of bidirectional transport assay indicated that sulpiride and citalopram were P-gp substrates, and P-gp was mediated their transport across blood-brain barrier; moclobemide, clomipramine and zopiclone were not substrate of P-gp, and P-gp is not involved in their transport across blood-brain barrier. Combined with the results of ATPase activity assay, we considered that moclobemide, clomipramine and zopiclone were P-gp inhibitors. Combined our research results with literature survey results, we successfully established a spectrum of P-gp substrates for central depressants, and clarified the scope of drugs which are suitable for the new treatment way of increasing central depressants efflux by inducing P-gp expression resulted from up-regulation of MDR1gene for poisoning patients rescues, which may provide new basis and means to treat patients with central depressants poisoning in clinical practice.3Quetiapine could induce the signaling pathway of PXR-CYP3A4/P-gp by itself. The self-induced effects of quetiapine were more obvious in situation of drug overdose, and PXR agonist dexamethasone synergistically had greater increase in up-regulating gene and protein expression levels of drug-metabolizing enzyme CYP3A4and transporter P-gp. For drug overdose rescues, activating PXR could activate detoxification system quickly and effectively, which may facilitate quick xenobiotic detoxification and organs protection. This study was help to clarify molecular mechanisms and characteristics of the nuclear receptor PXR in regulating metabolic enzymes and transporters in situation of drug overdose, which may provide basis to develop new ways of drug overdose rescues and provide new ideas for the development of new kinds of antidotes. |
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