| Made from ginseng, astragalus, cantharidin and toad, Delisheng Injection has synergistic effects when combining with chemotherapy and prevents bone marrow suppression caused by radiotherapy. Daunorubicin (dauno rubicin, DNR) is the first generation of anti-tumor antibiotic, mainly used for various types of leukemia, malignant lymphoma, with its major adverse reactions bone marrow suppression and cardiac toxicity. Combination of the two drugs can significantly enhance the efficacy and reduce adverse reactions. However, there is no coverage at home and abroad to the existence of pharmacokinetic interaction of this combination. According to establishing an effective, rapid high-performance liquid chromatography (high performance liquid chromatogram, HPLC) method, this topics measured the concentration of daunorubicin and ester toad ligand drug in biological samples, and studied experimentally the pharmacokinetic interaction of the two drugs in rats, providing a theoretical base for the rational combined use of these two drug.Objective:To determine the daunorubicin and the ester toad ligand concentration in serum by establishing the high performance liquid chromatography, and to study the dynamic changes of the concentration of toxic toad ester ligand and daunorubicin after administering the rats tail intravenously, and the changes of daunorubicin after combining with Delisheng Injection. The changes of pharmacokinetic parameters were observed after Delisheng Injection was combined with daunorubicin, providing a basis for the clinical rational use and improvement of the safety and efficacy of DNR. Methods:SD rats of clean grade were randomly divided into daunorubicin injection group, Delisheng group, daunorubicin and Delisheng combination group (daunorubicin was injected 0.5h after Delisheng injection). Daunorubicin injection group, The combination group had the blood taken 3,15,30,60,120,240, and 480 min after the administration; Delisheng group had blood taken 3,10,20,30,60,90,120, 180,240 minutes after administration. Plasma concentrations were measured at different time to detect linear range, detection limit, quantification limit of, sample recovery, sample stability, and other indicators of daunorubicin and ester toad drug ligands in serum Pharmacokinetic parameters were fitted by using DAS 2.1.1 software. Determination of the amount of daunorubicin was made by Diana HPLC chromatograph, column:phenomenex C18 (250mm×4.6mm,5μm); column temperature:35℃; mobile phase: methanol-0.01mol·L-1 ammonium dihydrogen phosphate solution (pH4.65)-acetic acid (V:V:V=30:20:0.1); flow rate:1.0ml·min-1; UV detection wavelength:233nm; internal standard:doxorubicin hydrochloride. Ester toad poison ligand was determined by using HPLC chromatography Diana, column:phenomenex C18 (250mm×4.6mm,5μm); column temperature:35℃; mobile phase:acetonitrile-water (55:45); flow rate:1.0ml·min-1; UV detection wavelength:296nm; internal standard:norethisterone. Results: The results showed that daunorubicin at 0.1478～14.778 (mg·L-1) (r= 0.9984, n=7) was well inside the scope of the detection limit of 5ng, ester toad poison ligand in 55～8800 (μg·L-1) (r=0.9997, n=8) was well inside the scope of the detection limit of 1.3ng. The peak separation among the measured composition, internal standard, and serum impurity between was the good, and there was no interference with each other. The mean extraction recovery of Serum daunorubicin, and esters toad poison ligands was 84.1% and 84.7%. The mean blank recoveries were as follows:100.6%,99.3%. The placement stability results of reserve liquid of daunorubicin and esters toad poison ligands were:RSD<3.0%, RSD<3.2%; The stability results of placing serum at 4℃after treatment were:RSD<5.0%, RSD<4.5%; The stability results:serum short-term room temperature RSD<3.1%, RSD <3.3%; long-term frozen stability of serum:RSD<2.5%, RSD<2.9%; freeze-thaw stability of serum:RSD<2.2%, RSD<3.3%. Animal experiment results showed that:the pharmacokinetic parameters of daunorubicin injection group, combined group, Delisheng group of two-compartment model of distribution phase half-life t1/2αwere 0.073,0.286,0.093 h respectively two-compartment; model of elimination phase half-life of t1/2βmean, respectively 3.183,22.327,1.131h, the mean peak concentration Cmax were 1.788,2.496,0.941 mg·L-1, a total plasma drug clearance CL respectively 1.317,0.155,12.875 L·kg-1·h-1, plasma concentration-time, area under the curve AUC(0-t), respectively 7.729,7.836,1.256mg·h·L-1, central compartment apparent V1 mean volume of distribution for 0.588,1.188,11.603 L·kg-1. Conclusion:By this experiment, (1) established a high-performance liquid chromatography determination of biological samples daunorubicin, esters toad poison ligand concentration in the serum method. (2) daunorubicin, ester toad poison ligands are in compliance with intravenous injection of a two-compartment open model. (3) a single dose of daunorubicin group and mixed group t1/2α, CL, AUC(0-∞), Cmax significant difference; t1/2β, AUC(0-t) difference was not statistically significant, support from the pharmacokinetics of daunorubicin and the joint use of Delisheng. |
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