Preclinical Pharmacokinetics Of Hanbenbofen

Hanbenbofen is a promising novel photosensitizer,which has the advantage of clear structure,high purity,strong photosensitizing and tumor tissue lethality.Hanbenbofen has the strong UV absorption,owning to the structure of tetrapyrrole.Therefore,the HPLC-UV method was established in this study,as well as the preclinical pharmacokinetics of Hanbenbofen was studied,which provided the experimental basis for further clinical pharmacokinetic study.1.The establishment of analytical method of Hanbenbofen in biological matrixThis paper established and validated a HPLC method for the determination of different biological matrix.The chromatographic separation was performed on a Diamonsil C18(2)(5mm,100 × 4.6mm)column with the mobile phase composed of methanol and ammonium acetate(containing different proportions of glacial acetic acid)at a wavelength of 400 nm.There were no interferences were observed.Calibration curves of Hanbenbofen also showed good linearity(r2>0.996).Intra-and interday precisions(RSD)were at the result of 1.24~10.90% and 1.34~6.88%,respectively.The accuracy was in the range of 82.76 to 114.20%.The validated method was successfully applied to the pharmacokinetic study.2.The pharmacokinetics study of Hanbenbofen in SD rat plasmaSD rat blood samples were collected in heparin-coated tubes prior before administration and at different time points after different single-dose(0.5,1 and 2mg/kg)intravenous administration.The concentrations of Hanbenbofen in SD rat plasma were determined by HPLC method.Pharmacokinetics parameters were calculated by non-compartmental analysis method.The Cmax of the three doses were 11526.02±1619.60,22439.22±2456.47 and 49194.70±3681.64 ng/m L.The half-life was 28.52±12.02,50.96±11.52 and 32.69±12.29 h,for 0.5,1 and 2 mg/kg doses,respectively.The AUC0~72 of Hanbenbofen at different levels were 126454.05±69247.20,468230.68±126316.13 and 518282.36±254975.92 ng×h/mL.It was shown that the concentrations of Hanbenbofen in SD rat plasma were reduced slowly after intravenous administration.No individual differences were observed.The correlation coefficients were 0.5441 and 0.9737,respectively.Both of the AUC0~72 and Cmax were positive correlated with the doses.The concentration of Hanbenbofen in SD rat plasma began to stabilize at the four day during the one week multidoses test.It was found that the drug has accumulated in SD rat after 7 days administration,according to the change of pharmacokinetic parameters after single-dose and multiple-dose administration of Hanbenbofen.3.The tissue distribution study of Hanbenbofen in ratsRats were executed at 30 min,2h,4h,8h,24 h and 48 h after intravenous administration(1mg/kg).All of the tissues were collected and stored at-80? after washing with saline and dried with filter paper.The results showed that the concentrations of Hanbenbofen in lung,ovary and heart were the highest and the concentrations of Hanbenbofen in liver,kidney,spleen,uterus and testis,were the second.The concentrations of stomach and testine were the lowest.What's more,the concentrations in brain,fat,skin and musclewere lower than the limit of quantitation.4.The pharmacokinetics study of Hanbenbofen in Beagle dog plasmaThe Beagle dog plasmas were collected after administered by left forelimb intravenous injection the Hanbenbofen powder at a single dose of 0.3,0.6,1.2,2.4 and 4.8 mg/kg.The quantitation of Hanbenbofen in Beagle dog plasma has been developed by HPLC.The pharmacokinetic parameters of Hanbenbofen were calculated using non-compartmental analysis method.The Cmax of the five doses were 1637.64±702.16,4061.22±469.85,9491.22±1217.44,22693.25±1450.23 and 47316.47±2827.02 ng/m L.The AUC0-72 of Hanbenbofen at different levels was 121.51±54.69,220.49±47.97,1114.03±222.42,2285.16±327.47 and 7517.85±2137.48 ng.h/m L,respectively.These data demonstrated thatthe concentrations of Hanbenbofen in Beagle plasma were reduced rapidly.The results supported that Hanbenbofen may have linear pharmacokinetic characteristics in dogs within the investigated dosages.The r2 of Cmax and AUC0~6 was 0.8787 and 0.9919.All the valley concentrations were lower than the limit of quantitation.There were no significant changes for AUC0~6 between day 1 and day 7,indicating no significant accumulation of Hanbenbofen in the dog plasma.5.The protein binding rates study of Hanbenbofen in different p1asmasThe binding rates of Hanbenbofen with different genera plasma proteins were determined by equilib riumdialysis.The concentrations of Hanbenbofen were assayed by HPLC.Plasma protein binding rates of three various concentrations(2.5 ?g/mL?10 ?g/mL and 40 ?g/mL)were in the range of 84.07%~94.64% in human plasma,86.18~89.65% in Beagle dog plasma,84.57~93.31% in SD rat plasma,respectively.All of the binding rates of Hanbenbofen were greater than 80%.Moreover,the binding rates were not proportiona1 ly dependent on plasma concentration of Hanbenbofen.