Study On Pharmacokinetics Of The Compound Realgar Natural Indigo Tablets And Apoptotic-induced Mechanisms In Cancer Cells

This dissertation reports the study on pharmacokinetics of the compound realgarnatural indigo tablets and the apoptotic-induced mechanisms in cancer cells.To make-up a method to calculate bioavailability of Chinese medicine compoundpreparations whose concentration could not be determinated easily in vivo, wecombined the single function residual identifiability (SFRI) theory and the drugcumulative method in vivo to study the bioavailability of CRNIT. By means of thismethod, mathematic model of in Vivo Depot Rate (Xt) was established to measure thearea under the curve (AUC) produced from ip and po administration of drug. Theapplication of the method greatly reduced the errors compared by using trapezoidaland log trapezoidal method, and resulted in measurement of bioavailibility in higheraccuracy. To study the pharmacokinetics of As in the CRNIT in health volunteers, theconcentrations of As in health volunteers blood were monitored by ICP-MS. Thepharmacokinetics data was processed with 3P97 software and statistics was performedwith SPSS software. The pharmacokinetics parameters were: A=0.3060±0.0909mg/L,Ke=0.0749±0.01251da, Ka=0.2199±0.03471/h, Lag time=0.0724±0.0621h,t_1/2(Ka)=3.2070±0.5260h, t_1/2(Ke)=9.2662±1.3444h, T(peak)=7.4499±0.3184h,C(max)=0.1075±0.0031mg/L, AUC=2.5508±0.1528 (mg/L)×h, CL/F(s)=1959.1970±110.1822 L/h, V/F(c)= 26235.3760±2731.4158L. The data obtained from a singleoral administration of CRNIT for 72 hours revealed that the elimination model of Aswas in accordance with first order kinetics and one compartment model. Nanometerrealgar powders were prepared by machine technique (ZJM-20/25 style high energyball mill), whose surface activity was reformed. The thermogravimetric analysis(TGA) was selected to prevent crystalline state conversion. Particle size was analysedwith laser scattering and the forms of typical powders were analysed with scanningelectron microscope (SEM) and transmission electron microscope (TEM). TGAproved that the degradation temperature of realgar was almost the same in air and innitrogen gas, at 260℃. In the condition of this high energy ball mill, no distinctcrystallin state conversion was observed for reaigar with the exception of conversion toward non-crystalline state. With the ball mill time prolonged, the particle sizedecreased promptly to nanometer grade with the conversion tendency tonon-crystalline state. Homogeneous micropowders could be obtained in watercontaining surface active agent. Our assay indicated that the realgar dispersed betterin sodium laurylsulfonate than in sodium dodecyl sulfate, and the powders at 200nmwere above 85 percent. We compared the phamacokinetics parameters of traditionalrealgar with nanometer realgar in rabbits. The phamacokinetics parameters oftraditional reaglar in rabbits were: A=0.096±0.009mg/L, Ke=0.070±0.0071/h,Ka=0.472±0.0711/h, Lagtime=0.030±0.000H, t_1/2(Ka)=0.151±0.238H, t_1/2(Ke)=10.056±0.895H, T (peak)=4.806±0.401H, C(max)=0.060±0.000mg/L, AUC=1.168±0.084(mg/L)h, CL/F(s)=43.078±3.495L/h, V/F(c)=620.696±29.649L. Thephamacokinetics parameters of nanometer reaglar in rabbits were:A=0.244±0.005mg/L, Ke=0.022±0.0051/h, Ka=0.922±0.0591/h, Lagtime=0.030±0.000H, t_1/2(Ka)=0.754±0.049H, t_1/2(Ke)=28.746±1.007H, T(peak)=4.066+0.174H,C(max)=0.216±0.005mg/L, AUC=9.822±0.394(mg/L)h, CL/F(s)=5.108±0.208L/h,V/F(c)=211.566±3.297L. The pharmacokinetics parameters of NRP were obviouslydifferent from these of traditional realgar, whose absorption phase increased whileelimination phase decreased.We also investigated the mechanisms of CRNIT in the treatment of leukemia.The NRP showed markedly cytotoxic effect on NB₄, HL-60 and K562 cell lines.Photomicroscopic morphology exhibited typical apoptotic changes, moreover, DNAgel electrophoresis and Hoechst 33342 staining further confirmed this result. Themechanisms of U937 cell death induced by NRP were intensively investigated.Hoechst 33342 staining and DNA gel electrophoresis assay showed that NRP inducedU937 apoptosis. NRP activated caspase-8 and changed the expression ratio ofBax/Bc1-2 after treatment with U937 cells for a time period, leading to the alterationof the permeability of the mitochondrial membrane, and then, made the caspase-3activate, which executed apoptosis in U937 ceils. In addition, JNK played aregulatory role in NRP-induced U937 cell apoptosis.