Preclinical Pharmacokinetic And Metabolism Study Of KLLF

KLLF as a novel compound being synthesized for the first time,has been verified to have the extent antioxygen effect in many vitro and vivo models. The works of this paper was designed to evaluate the metabolism and pharmacokinetics features of KLLF in animals, so as to support the metabolism and pharmacokinetic study in human and to offer the reference for clinical dosage and dosage form changing.1. The establishment of analytical method of KLLF in biological matrixA simple, specific and highly sensitive mathod using reversed-phase liquid chromatography combined with tandem mass spectrometry has been developed for the quantitative analysis of KLLF in rat plasma, bile, urine, feces. The sample pretreatment is deproteinization for plasma.The sample was chromatographed on a Inertsil ODS-3 cloumn. (3μm, 2.1×150 mm ID). The mobile phase of gradient elution consisted of acetonitrile-methanol-10mmol/L ammonium acetate buffer (0.25% formic acid) was run at flow rate of 0.4ml/min for different biological matrix. Eletrospray ionization (ESI) source was applied and operated in the positive ion mode. Multiple reaction monitoring (MRM) mode using the transition of m/z 964 → 906 was used to quantify KLLF. The chromatographic system used provided good separation of the compound without interfering peaks from endogenous substances. The linear calibration curves was obtained in the concentration range of 5-2560ng/mL. The lower limit of quantitation of KLLF was 2ng/mL. The inter- and intra-day prcision(RSD) was 1ss than 7%, and accuracy (relative error) was evaluated 93-107%. The method has been successfully used to support the pharmacokinetics study of KLLF.2. The pharmacokinetics study of KLLF in ratFollowing a single i.v. administration with difference doseged of KLLF to rat (5, 10, and 20mg/kg), the blood samples were collected at different time after dosing. All collected blood samples were centrifuged to obtain plasma and the concentrations of KLLF in plasma were determined by HPLC/MS/MS method discribed as above. Pharamocokinetics paramrter calculations were assessed by non-compartmental method using DAS 2.1. It was shown by the plasma concentration-time data that C_max, AUC_0-t and AUC_0-∞ were all dose proportional (r>0.969, p<0.05) in rat. The concentrations of KLLF in rat plasma were reduced slowly after i.v. administration and the difference of the rats was evident.The estimated elimination half-life was dose proportional(r=0.818, p<0.05).3. The tissue distribution study of KLLF-A in ratsRats were killed by exsanguination at 0.5h, 3h, 6h, 12h, 24h after i.v. administration (15mk/kg). Tissues were taken out and made into homogenates preparation with acetonitrile and the concentrations of KLLF were determined by HPLC/MS/MS method. KLLF can not be determined in rat's tissues. But the KLLF's metabolism of KLLF-Acan be determined by the calibration curve of every tissues respectively. It was shown that KLLF-A can be determined in the major organism after 0.5 h i.v. administration and there was not reduced tendency in the major organism after 24h i.v. administration. KLLF-A seems accumulate in rat. The KLLF-A concentration were high in the lung, liver, spleen and kidney. The lowest concentration existed in brain showing a poor penetration into central nervous system.4. Urinary, fecal and biliary excretion of KLLF after i.v. administrationUrine, bile and feces of rat were collected afer i.v. administration (15mg/kg), and then the concentrations of KLLF were determined by HPLC/MS/MS method. Accumulative excretion ammount of KLLF were 0.02767% in bile and 0.0275% in urine respectively. The fecal excretion of KLLF was less than 0.01%. 5. In Vitro binding of KLLF to human plasma proteinsPlasma protein binding was determined by equilibriun dialysis (72h) at 4°C with final concentration of 0.2, 1 and 2μg/mL. After the dialysis, the concentration of KLLF were determined by HPLC/MS/MS method. The binding ratio was calculated as percent of total concentration. The results indiced that the plasma protein bindings of KLLF were 99.73%, 98.49% and 97.50%, respectively. The plasma protein bindings of KLLF in human appeared to be dependent of concentration used(r=0.98, p<0.01).6. Preliminary study on the metabolites in vivo of KLL FHPLC/DAD and HPLC/MS/MS method were applied in analyzing rat's bile, urine and homogenates samples after i.v. administration. KLLF-A, the possible phase I metabolite of was speculated.