Preclinical Pharmacokinetics Of A771726 In Rats

A771726 is the active metabolic product of leflunomide,the later is a type of immunoregulatory agents.Leflunomide educes the pharmacological action by metabolized into A771726,and it has been extensively exerted on rheumatoid arthritis, systemic lupus erythematosus,anti-rejection organ transplantation and psoriasis. A771726 is an active compound,which is directly absorbed in vivo,abbreviates the transformation of prodrug.The works of this paper were designed to evaluate the pharmacokinetics features of A771726 in rats,compare to the parameters of leflunomide,so as to support the pharmacokinetics study in human and offer the reference for clinical dosage and dosage form changing.Part one:The establishment of analytical method of A771726 in biological matrixA reversed-phase high-performance liquid chromatographic(RP-HPLC) method has been developed for the determination of A771726 in rat plasma,bile,urine,feces and tissue homogenates.The sample pretreatment included deproteinization for plasma samples and a liquid-liquid extraction for bile,urine,feces and tissue homogenates. Separation was obtained on a Kromasil C18 column(150×4.6mm),using an excitation wavelength of 288nm.The isocratic mobile phase consisted of methanol-0.01mol/L potassium dihydrogen phosphate buffer(pH 4.5)(57:43,v/v) was run at a flow rate of 1 ml/min for different biological matrix.The chromatographic system used provided good separation of the compound without interfering peaks from endogenous substance.The calibration curves were linear in each sample range with correlation coefficient above 0.999.The precision of intra-day and inter-day were evaluated by analysis of variance with the result of 0.7～2.3%and 0.7～6.4%,respectively.The method has been successfully used to support the pharmacokinetics study of A771726.Part two:The pharmacokineties study of A771726 in ratsFollowing a single oral administration with different doses of A771726 to SD rats (3,6,12mg/kg),the blood samples were collected at different time after dosing.All collected blood samples were centrifuged to obtain plasma and the concentrations of A771726 in plasma were determined by HPLC method described as above,then calculated corresponding pharmacokinetic parameters based on the time process of blood drug concentration.The blood plasma Cot curve of A771726 conformed to one compartment model of the first order absorption.The main pharmacokinetic parameters of A771726(3,6,12mg/kg) were T_1/2K2(h):16.71±2.34,14.69±1.94,16.45±2.49, AUC_(0.tn)(mg/L·h):271.38±78.87,785.57±170.92,994.13±165.43,C_max(mg/L): 11.42±2.59,29.38±5.41,42.10±5.73,T_max(h):4.00±0.82,4.10±0.74,4.20±1.03, MRT_(0-tn)(h):22.07±1.80,21.91±1.60,20.71±1.88.The metabolism of A771726 following oral administration accords with the linear relation,and its blood plasma Cot curve consists with the first order kinetics of one compartment model.Part three:In vitro binding of A771726 to human plasma proteinsPlasma protein binding was determined by equilibrium dialysis(108h) at 4℃with final concentrations of 3,6 and 12μg/ml.After the dialysis,the concentrations of A771726 were determined by HPLC method.The binding ratio was calculated as percent of total concentration.The results indicated that the mean extent of binding to plasma proteins in human appeared to be in dependent of concentration used.The binding percentage of the drug was about 97.7%.Part four:The tissue distribution study of A771726 in ratsSD rats were killed by exsanguination at 2h,12h and 24h after oral administration (6mg/kg).Tissues were taken out and made into homogenates preparation with normal sodium and the concentrations of A771726 in the tissues were determined by HPLC method.It was shown that A771726 was distributed to the major organism after oral administration.The A771726 concentrations were.high in liver,kidney,and low in brain, bone marrow,thymus and testis/uterus.The drug seems not accumulate in rats.Part five:Urinary,fecal and biliary excretion of A771726 after oral administrationBile,urine and feces of rats were collected after oral administration(6mg/kg),and then the concentrations of A771726 were determined by HPLC method.Accumulative excretion amount of A771726 were 13.88%in bile,0.87%in urine and 1.61%in feces, respectively.