| Objective:Lung cancer is a serious threat to human health, and the incidence increased year by year.Etoposide (EPEG) is a cell cycle specific anticancer drug which is on the first line treatment in lung cancer, and the primary cellular target for etoposide is topoisomeraseⅡ. The existing etoposide dosage forms in market are injections and soft capsules. Because of its poor water-solubility, the bioavailability of oral dosage form is low and individual differences are apparent; the injection of etoposide utilizes a large number of surfactant as solubilizers, resulting in irritation, poor patient compliance. The most serious adverse event associated with etoposide is myelosuppression, and the number of WBC and platelets of patients can be reduced. The above drawbacks to some extent restricted the widespread clinical application of etoposide. In recent years, scientific researchers focus their work on new drug delivery systems such as liposomes, nanoparticles to increase the solubility of insoluble drugs and the local concentration of drugs at the specific site, thereby they could reduce the amount of drugs, side effects occurrence and improve drug treatment.Nanosuspensions are sub-micron colloidal dispersions of pure drug particles in an outer liquid phase. The nanosuspensions can be used to formulate compounds that are insoluble in both water and oils and to reformulate existing drugs to remove toxicologically less favourable excipients. By preparing nanosuspension makes it possible for those drugs including antibiotics, anticancer drugs and drugs possessing a narrow therapeutic window to play a better role. We constructed the nanosuspensions of etoposide,in which albumin was utilized as carrier material. The particle size range of etoposide nanosuspensions was 100-300nm, so EPR effect and the active target of albumin will make etoposide concentrate at the tumor site. The release of etoposide can be slowed by existing in particle forms, which improve the drug availability at the tumor site while reducing harmful side effects.Methods and results:HPLC method was established to determine the etoposide concentration in vitro and its methodology was examined. EPEG-BSA-Nanos were prepared by high pressure homogenizer-Solvent precipitation method. The evaluating indexes included drug content, entrapment efficiency and particle size. The formulation and technological parameters were optimized based on the results of the single factor study and the orthogonal design. The obtained nanosuspensions were dried using freeze-drying method. The properties of EPEG-BSA-Nanos such as diameter and size distribution were observed by transmission electric microscope (TEM) and laser dispersive analyzing apparatus for granularity, and Zeta potential was measured by micro-electrophoresis apparatus. In vitro releases of EPEG-BSA-Nanos were performed by dialysis method.The stability of EPEG-BSA-Nanos at 4℃was evaluated. The in vitro reliability evaluation such as local irritation, hemolysis and bone marrow suppression of EPEG-BSA-Nanos in rabbits and mice were investigated. HPLC method was established to investigate etoposide concentrations of plasma and tissue in rabbits and mice. The pharmacokinetics and tissue distribution of the EPEG-BSA-Nanos in rabbits and mice were investigated and EPEG injection was used as control.The results showed that using HPLC method to determine the etoposide concentration in vitro was easy, convenient and correct. EPEG-BSA-Nanos with appropriate particle size were obtained by high pressure homogenizer-Solvent precipitation method. The optimized prescription was as follows:trichlormethane/ dehydrated alcohol (8:1,v/v), BSA/drug (10:1,g/g), oil phase/water phase (10:1, v/v),and the pH of water phase is 5.4.The optimized procedure was as follows: homogenization pressure was 1500bar and homogenization cycles were 15. Lyophilized EPEG-BSA-Nanos were prepared to improve the stability and the obtained lyophilized nanosuspensions appeared as full, no collapse of the porous solid block. The lyophilized nanosuspensions showed good redispersibility and the reconstituted nanosuspensions with saline were opalescent, colloidal solution. The mean diameters of EPEG-BSA-Nanos changed to (182.3±8.91) nm from (190.2±7.3) nm after lyophilization, and the polydispersity was from0.29±0.08 to 0.27±0.11, zeta potential was from (-23.05±1.78) mV to (-22.18.±0.89) mV, respectively. The pH of EPEG-BSA-Nanos was 5.46±0.24, which is consistent with the requirements of injection.Compared with etoposide injection, EPEG-BSA-Nanos presented controlled release properties and the etoposide release behavior from nanosuspensions in vitro was in accord with Higuchi model. The formation of EPEG-BSA-Nanos was validated by DSC, which indicated that EPEG was encapsuled into BSA successfully. Results of the preliminary stability experiments indicated that the lyophilized EPEG-BSA-Nanos were almost intact at 4℃for 3 months.The results of local irritation test and hemolysis test showed that EPEG-BSA-Nanos had no stimulatory effects for ear veins of rabbits and hemolysis and coagulation were not observed in hemolysis test. The results of myelosuppression test showed that EPEG-BSA-Nanos could distinctly reduce myelosuppression of etoposide to mice.HPLC method was established to investigate etoposide concentrations of plasma and tissue in rabbits and mice and the results showed that endogenous substances did not interfere with etoposide determination in the selected chromatographic conditions. The extraction recovery, method recovery of drugs in blood of rabbits and tissues in mice, the linearity, RSDs of intra-day and inter-day and the accuracy were all satisfied with the need of analysis for biological detection. After tail vein injection of EPEG-BSA-Nanos and EPEG injection, the plasma drug concentration of two kinds of preparation were compared. Pharmacokinetic parameters were calculated by statistical moment method. The t1/2βof nanosuspensions group and injection group were 10.296h and 5.225h respectively, showing of 1.97 folds increasing. MRT0-∞were 2.707h and 1.170h respectively, showing of 2.31 folds enhancement. Cmax were 48.05 mg/L and 61.08mg/L respectively, however AUC0-∞of the EPEG-BSA-Nanos was similar with that of EPEG injection group (116.87mg/L*h and 111.16 mg/L*h respectively). It indicated that after encapsulated in the nanosuspensions, the drug concentration was reduced but the therapy effect was not affected.According to Ce (Ce=(Cmax) nanosuspensions/(Cmax)injection), the targeting efficiency of EPEG-BSA-Nanos was evaluated. The results showed that the Ce of lung, spleen and liver was 1.93,2.52 and 1.79 respectively. Meanwhile, the Ce of heart,brain and kindey was 0.94,0.95and0.88 respectively. It demonstrated that nanosuspensions formulations presented accumulative activity in RES sites such as lung, spleen and liver. Conversely the biodistribution of nanosuspensions formulations in non-RES sites such as kidney, brain and heart decreased with descending Ce compared with etoposide injection, which potentially resulted in the reduction of side effects of free drug.Conclusion:In this study, EPEG-BSA-Nanos were successfully prepared with simple, feasible preparation technology and ease of scale-up. EPEG-BSA-Nanos had no stimulatory and hemolysis effects, as well as distinctly reduced myelosuppression of etoposide to mice. Compared with the control group, the distribution of etoposide significantly changed because of the formulation of nanosuspensions. Etoposide encapsulated in BSA could improve drug accumulation in lung, extend retention time of drug in the lung, enhance local etoposide concentration in the lung and improve the therapeutic efficiency. In addition, the side effects in non-target tissues could be avoided and patients'compliance and the medication effects could be improved which accompany by good social and valuable economic benefits. |
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