Mechanism Study Of Amitriptyline Detoxication With P-gp And CYP3A Inductor Dexamethasone

OBJECTIVESTo investigate a new antidotal pathway for the central depressant amitriptyline-induced toxicity: enhance poison excretion by mdrl up-regulation and P-gp induction; accelerate the metabolism of drug in general circulation by CYP3 A up-regulation and liver drug enzyme induction.To study AMI disposition kinetics and brain/ plasma concentration ratio between rats from different drug pre-treatment groups; to analyze and compare mdrl a and CYP3A2 mRNAs in brain and liver tissues from different rat groups in order to disscuss the mechanism of changes.METHODS1. Dose and sample collection1.1 40 Male Wistar (250-300g) rats were equally divided into eight groups by completely random design. Each group was injected intraperitoneally with corn oil for 4 days (K_a), dexamethasone (DEX) for 2 days (1 mg·kg^-1) (2La), DEX for 4 days (1 mg·kg^-1) (4L_a), DEX for 2 days (5 mg·kg^-1) (2M_a), DEX for 4 days (1mg·kg^-1) (4M_a), DEX for 2 days (25 mg·kg^-1) (2H_a), DEX for 4 days (25 mg·kg^-1) (4H_a) and verapamil (8mg·kg^-1)1h before injection of AMI (I). Blood samples were collected at 5, 10, 30, 60, 120, 200, 300 and 400min. Brain and liver samples were removed after blood collecting and stored in liquid nitrogen.1.2 40 Male Sprague-Dawley rats were equally divided into ten groups by completely random design. Pretreatment protocol of group K_b, 2L_b, 4L_b, 2M_b, 4M_b, 2H_b and 4H_b are the same with group K_a, 2L_a, 4L_a, 2M_a, 4M_a, 2H_a and 4H_a separately. Group 4LI, 4MI, 4HI are the same with group 4L_a, 4M_a, 4H_a but administered with verapamil (8mg·kg^-1)lh before injection of AMI. Each rats was killed by decapitation 1h after intravenous injection of AMI. Blood and brain samples were collected.1.3 Sample analysis2.1 The whole AMI and NOR concentration in blood and brain samples were determined by HPLC-MS2.2 RT-PCR was used to determine the expression of mdr1a and CYP3A2 in brain and liver tissues in part 1.1.RESULTS1. Validation of sample analysis methodIn blood, linear range were 10-3200 ng·ml^-1 and 10-1000 ng·ml^-1 for AMI and NOR; In brain, linear range were 25-2000ng·ml^-1, 1.5-100ng·ml^-1 for AMI and NOR. Recovery was 89%-114%, The inter-day and intra-day precision(RSD)< 12%.2. Pharmacokinetics and brain/plasma concentration ratio after intravenous injection of AMI2.1 The area under curve(AUC_{0→∞}), mean retain time(MRT) and clearance rate (CL_tot) to rats treated with DEX were significantly lower than those of the control group (P<0.01). The pharmacokinetics parameters of AMI in verapamil group were not significantly different from those of untreated rats.2.2 The area under curve (AUC_{0→∞}) and peak concentration (C_max) to rats treated with DEX were not significantly different from those of untreated rats. However, these parameters to rats from verapamil group showed significant increase (P<0.01).2.3 The AMI and NOR brain/plasma concentration ratios of rats from DEX groups were significantly lower than those of the control group(P<0.01). However, in verapamil validation groups (LI, MI and HI), the trend of decrease was not revearsed.3. The expression of mdr1a and CYP3A2 mRNA in brain and liver tissues between different rat groups3.1 The expression of mdr1a and CYP3A2 mRNA increased in liver tissues, depending on the number and amount of prior DEX treatment. In rats treated with DEX, expression of mdr1a and CYP3A2 mRNA significantly increased compared to those of untreated rats.3.2 The expression of mdr1a and CYP3A2 mRNA in brain tissues amongrats from DEX groups were not clearly different from that in untreated rats.CONCLUSIONS1. Continue pre-treatment with DEX will induce the expression of mdr1a and CYP3A2 mRNA in rat liver tissue and accelerate the elimination of AMI, in which CYP3A2 is a major factor. The disposition kinetics of NOR is more influenced by P-gp. As NOR is the main active metabolite of AMI, so P-gp induction have great significance in AMI detoxication.2. The experiment proved that DEX may decrease the concentration distribution of AMI and NOR in brain and stop drugs accumulation. However, VER did not reverse the decreasing trend of drug disposition in brain, mdr1a mRNA expression in the brain was also found unchanged. Therefore, the real reason of the phenomenon has not yet been deciphered and further study still await.