Pharmacokinetics Of Methotrexate,5-fluorouracil And Cyclophosphamide And CYP450Level And Activity In Depression Model Rats

1. ObjectivesAll the time, it was being emphasized biological factors on effect of medication in study on clinical pharmacology, but ignored social psychological factors. In clinical practice, we have discovered that mood disorder affected the effect and the whole process of medication to serious diseases, but the biological mechanism of which was not clear. Accordingly, on the current basis of clinical research, we will further prove the hypothesis that pharmacokinetic has behavioral or mental difference, which was first proposed by the applicant of this research in1999, to explore the difference on the pharmacokinetic of methotrexate (MTX),5-fluorouracil (5-FU) and cyclophosphamide (CP) and the difference on the total level and activity of cytochrome P450in rats of depression group and control group by building depression model of chronic unpredicted mild stresses (CUMS). We will study the mechanism of the difference between efficacy of the drug caused mood disorder and in normal mood to discover the general discipline that mood disorder affect the metabolism and efficacy of the drug.2. Methods2.1Establishment of chronic unpredictable mild stress (CUMS)-induced depression modelFemale Sprague-Dawley (SD) rats, weighing180±20g,90-day old, were purchased from Laboratory Animal Center of Southern Medical University. Rats were raised in25℃experimental environment with normal diet and adapted feeding environment for a week before starting experiment.54rats were randomized into two groups:model group and control group. Rats in CUMS-induced depression model group were single cage bred, exposed with random stressor for8weeks. The control group rats were single cage bred without any stressor.2.2Model of evaluationThe behavioral level of rats in depression group and control group before and after stress was measured by open-field test. About1ml of blood was collected from each group rat before and after depression model establishment. The anticoagulated blood sample was centrifuged at3000xg for5min to obtain plasma. The level of5-5-hydroxytryptamine (5-HT) in plasma was tested by enzyme-linked immunosorbent assay (ELISA).2.3Dosage regimen and plasma sample collectionAfter depression model establishment,54rats were randomized into3subgroups: MTX subgroup,5-FU subgroup and CP subgroup which were given a certain dose of MTX (2mg·kg-1),5-FU (mg·kg-1) and CP (10mg·kg-1) iv. The blood samples were collected at different time points, and then stored at-20℃. The time-point for blood sample collection were at:0.083,0.5,1,2,4,8,12h for MTX,0.05,0.083.0.167,0.25,0.5,1,2,4,6h for5-FU and0.083,0.167,0.25,0.5,1,2,4,6,8,10,12h for CP, respectively. 2.4Determination of MTX,5-FU, CP concentration in plasmaThe blood samples were respectively assayed by fluorescence polarization immunoassay (FPIA) for MTX, high performance liquid chromatography (HPLC) for5-FU and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for CP. The pharmacokinetic of MTX,5-FU and CP were calculated as two-compartment model with DAS2.1software (Drug and Statistics, China).The HPLC system for determination of5-FU was consisted of a pump, a auto-injector and a UV-detector set at270nm, C18Hypersil ODS column (250mm×4.0mm,5μm particle size) with a guard column (15mm×4.0mm), the mobile phase (lOmM acetic acid:acetonitrile,99:1(v/v)), with flow-rate of1.0ml/min.HPLC-MS/MS instrumentation for determination of CP were consisted of an Agilent1200series G1310A pump, and a G1316A adjustable column temperature box. The chromatography of the analytes was performed at40℃using an Agilent ZORBAX SB-C18(2.1mm×150mm,5μm) column. The flow rate of mobile phase(methanol:0.1mol·I-1ammonium formate,95:5(v/v)) was0.4ml·min-1. The optimized conditions of MS/MS with electrospray were as follows:ion spray source temperature at350℃, nebulizer (NEB) gas at10L·min-1, ionspray voltage (IS) at4000V For CP dissociating potential (DP) was at115V and collision energy (CE) was20units, for ifosfamide dissociating potential (DP) was at115V and collision energy (CE) was21units. Data acquisition was performed via multiple-reaction monitoring (MRM). The ions representing the [M+H]+species for both the sample and internal standard were selected in MS1and dissociated (collision-induced) with nitrogen gas to form specific product ions, which were subsequently monitored by MS2. The precursor-to-product ion transitions monitored for CP and ifosfamide were m/z260.9â†'139.9and m/z260.9â†'153.8, respectively. 2.5Level and activity of cytochrome P450Control and CUMS-induced rats were fasted for24h and killed by cervical dislocation before removal of the liver. The liver was excised, rinsed with ice-cold saline (0.9%NaCl, w/v), weighed for1g and homogenised in a0.05M Tris-0.25M sucrose buffer (pH7.5). The homogenate was centrifuged at10000×g at4℃for30min. The supernatant was then centrifuged at105000×g at4℃for60min. The pellet was reconstituted with0.05M Tris-0.25M sucrose buffer (pH7.5). The cytochrome P450level of the liver microsomes was determined by Carbon monoxide differential spectra, and the cytochrome P450activity of the liver microsomes was determined by fluorescence quantitative assay with fluorescence quantitative kit, which was purchased from Gen-Med. The activity of7-ethoxycoumarin-O-deacthylase was measured and delegated as CYP450activity with7-ethoxycoumarin as the reaction substrate.2.6Statistical analysisStatistical analysis was performed with the SPSS16.0. Data were expressed as x±s. P<0.05was considered statistically significant.3Results3.1Establishment of chronic unpredictable mild stress (CUMS)-induced depression modelThe locomotion and exploratory behavior scores of rats in depression model group and control group before and after8wks’model establishment were monitored through open-field test. Within each group, the locomotion and exploratory scores of rats in depression model group before and after model establishment were from81.81±10.57to15.00±5.44(P<0.05), from14.63±3.90to4.81±2.06(P<0.05), respectively. However, there were no significant change for the locomotion and exploratory scores in rats of control group before and at the end of8wks, which were from78.07±11.54to72.67±13.74(P>0.05), from16.19±5.05to14.19±5.03(P>0.05), respectively. Meanwhile between the groups, there was no significant difference for locomotion and exploratory scores before the model establishment but significant at the end of8wks (P<0.05).3.2Determination of MTX,5-FU, CP concentration in plasmaMTX plasma concentration was determined by FPIA with kit.5-FU and CP plasma concentration were determined by HPLC and HPLC-MS/MS. The assay validation of them were reliable with a stable recovery and good accuracy.3.3The plasma concentration-time curves and pharmacokineticsThe plasma concentration-time curves after a single iv injection of MTX in depression model group and in control group were very close. The pharmacokinetic parameters of rats in depression model group were decreased than those in control group, however, the differences were not statistically significant (P>0.05). There were significant differences in all pharmacokinetic parameters of5-FU between two groups (P<0.05). Similarly, there were significant differences in mostly pharmacokinetic parameters of CP between two groups (P<0.05), except t)/2a (P>0.05). The ti/2a of CP in control group and depression group was0.09and0.07h, but ti/2p was1.60and0.98h, which was shortened by38%. The other changes were similar as those of5-FU.3.4Level and activity of cytochrome P450The results showed the total content and activity of cytochrome P450enzyme of liver of rats in depression model group were higher than those of control group. There were significant differences for CYP450level and activity in depression and control group (P<0.05).4ConclusionsThe results suggested that mood disorder might affect metabolism process of 5-FU and CP by altering metabolic enzyme of them, but not MTX.