Studies On Self-microemulsified Drug Delivery System Of Teniposide For Intravenous Administration

Teniposide, a semisynthetic derivative of podophyllotoxin resin, is effective for treatment of various malignancies, such as neuroblastoma, small cell lung cancer.However, its application in clinic was limited due to the poor solubility and the solubilizer, Cremophor EL, contained in its commercial injection(VUMON). In the study, self-microemulsifying drug delivery system(SMEDDS) was applied for the intravenous administration of teniposide, in order to improve the stability of teniposide injection during infusion, avoide the side effects caused by solubilizer and make an alternative for the application of teniposide in clinic.By single factor method, the formulation of teniposide self-microemulsified drug delivery system (TEN-SMEDDS) was optimized as follow: teniposide 50 mg, DMA 300 ?L, MCT 250 mg, E-80 2,000 mg, dehydrated alcohol added to make up to 10 mL.after dilution with 5 % glucoe,TEN-SMEDDS could form fine oil-in-water(o/w)microemulsion with gentle agitation, and the Z-average particle size and zeta potential were 292±21 nm(PDI 0.433±0.035) and -7.5±1.7 mV,respectively. TEM image of teniposide microemulsion confirmed that microemulsion droplets were well dispersed without any aggregation or cluster, and almost spherical in shape. The stability of the so-formed microemulsion was above 4 hours. Compared to VUMON, the stability of the so-formed microemulsion increased significantly.In vitro release study, the cumulative release of teniposide from SMEDDS was above 83.2 % at 12 h. The obtained release data were then fitted into first-order,Higuchi, Hixcon-Crowell, Nibergull, Ritger-Peppas and Weibull equations. The regression results indicated that the Ritger-Peppas model best fitted the release data(SMEDDS, R=0.9583). It suggested that teniposide release from SMEDDS was consistent with the multiple mechanisms of Fick's diffusion and denude.A method for assay of drug content and impurities in vitro was established.Stability study indicated megatemperature could darken the color of the formulation and increase the impurities, illumination could increase the impurities and decrease the content of teniposide. It suggested that the TEN-SMEDDS should be stored at low temperature and protect from light.Murine neurospongioma cells C6 and human neurospongioma cells U87 were selected as the models of tumor cells, respectively. The antitumor activity of TEN-SMEDDS in vitro were performed on cell multiplication and inhibition,apoptosis and cell cycle. Cytotoxic test results showed that TEN-SMEDDS displayed a lower cytotoxicity on C6 and U87 than VUMON, with the higher IC50 value 0.818± 0.033 ?g/mL and 9.143 ± 3.060 ?g/mL(VUMON: 0.386 ± 0.024 ?g/mL and 4.473 ±1.819 ?g/mL), respectively. Moreover, C6 appeared to be more sensitive to the cytotoxic effects of teniposide than U87 cells. Meanwhile, the adjuvants contained in VUMON exhibited higher cytotoxicity than that in TEN-SMEDDS. The results of the apoptosis experiment indicated that the effect of TEN-SMEDDS on the apoptosis of C6 and U87 were little stronger than that of VUMON, but there were no significant differences. The apoptosis rate of C6 cells induced by TEN-SMEDDS and VUMON in different concentrations were about twice as many as that of U87 cells. That was to say that C6 appeared to be more sensitive to teniposide than U87. These were consistent with the findings in the experiment of cytotoxicity. The cell cycle test results showed that the TEN-SMEDDS and VUMON mainly arrested C6 cells in G2/M phase, while U87 cells in S phase. Namely, tenipsodie was a cell cycle specific antitumor drugs and different preparations had no effect on the cycle specificity.Teniposide could arrest C6 and U87 in different phase due to different sources of the two cells.The results of pharmacolinetics and tissue distribution indicated that the Cmax and AUC of TEN-SMEDDS group decreased significantly(p<0.05), but the Vd and CL increased markedly(p<0.05), compared with VUMON. Within 120 min after i.v.administration of TEN-SMEDDS or VUMON to rats, the concentrations of teniposide in TEN-SMEDDS group were higher than that in VUMON group in almost tissues,especially in liver, spleen and lung. The drug in TEN-SMEDDS group distributed into the brain were little higher than that in VUMON group (p>0.05). It suggested us that the lower Cmax, and AUC values compared to VUMON indicated the lower plasma level of teniposide, likely being advantageous for decreasing the hematological toxicity, and the wide and fast distribution of teniposide in SMEDDS group would increase the drug in tumor.The therapeutic efficacy of TEN-SMEDDS was evaluated on BALB/c nude mice bearing neurospongioma U87 tumors(subcutaneous vaccination) and Wistar rats bearing neurospongioma C6 tumors(subcutaneous vaccination), respectively. The results showed that TEN-SMEDDS had significant inhibitory ability against tumor growth on tumor bearing nude mice and Wistar rats, compared with 5 % glucose. But there was no significant differences between TEN-SMEDDS and VUMON in term of the inhibition rate. TEN-SMEDDS presented marked apoptosis induction activity by TENEL method. The results were consistent with that of activity in vitro. Namely,there was no significant differences between TEN-SMEDDS and VUMON against the proliferation and apoptosis of C6 and U87.The preliminary safty study of TEN-SMEDDS included haemolysis, rabbit ear vein irritation and hypersensitivity reaction. In the haemolysis experiment, the erythrocyte precipitated at the bottom of the tubes for the tested samples with different concentration of teniposide and redispersed after shaking during the observation of 3 h. The hemolysis percent of VUMON and TEN-SMEDDS was below 5% after incubation for 3 h at 37 ?. But, the hemolysis percent of VUMON was significantly higher than that of TEN-SMEDDS(p<0.05). In the rabbit ear vein irritation test, after a consecutive 5-day administration of TEN-SMEDDS, VUMON and 5 % glucose injection, there was a slight vascular injury in all groups at the injection site related to trauma associated with venipuncture. Nevertheless, there was no obvious visible erythema, edema and tissue necrosis along marginal ear vein for negative group, while slight reddish discoloration and ulcerate was observed in TEN-SMEDDS and VUMON group. Macroscopic observation indicated that slight vascular engorgement and erythrocyte aggregation were seen at the injection site and surrounding tissues following administration of VUMON, while similar phenomena were not observed in the case of TEN-SMEDDS and 5 % glucose. In addition, no apparent pathological changes could be observed such as hemorrhage, thrombosis,necrosis and inflammatory cell infiltrate in the vessel wall and surrounding tissues for all groups. In the hypersensitive reaction experiment, during the allergize, there were no conventional hypersensitivity reactions and the weights of guinea pigs had no significant difference in all groups(p>0.05). During the observation after provocation,the guinea pigs in the positive control group died. The guinea pigs in the blank SMEDDS group and negative group didn't exhibited conventional hypersensitivity reactions. The guinea pigs in the TEN-SMEDDS group only showed restlessness. As for blank VUMON group and VUMON group, the guinea pigs showed frequent nose scratch, tremble, sneeze, erect hair or twitch. Some even showed gatism. According to the guideline of hypersensitivity reaction grades, blank SMEDDS (grade=0) and TEN-SMEDDS group (grade=1) were deemed as negative reaction, while VUMON group (grade=3) and blank VUMON group (grade=3) were positive. The results suggested that Cremophor EL was the main factor for the hypersensitivity reaction and TEN-SMEDDS reduce the hypersensitivity reaction caused caused by VUMON.In conclusion, we prepared TEN-SMEDDS successfully, and evaluated it from several aspects, including characterization, physical-chemical properties, antitumor activity in vitro and in vivo, pharmacokinetics, tissue distribution and safty. The results indicated that TEN-SMEDDS could meet with the need in clinic.TEN-SMEDDS possessed the equal effectiveness with VUMON. However, compared to VUMON, the stability of the so-formed microemulsion after dilution with 5 %glucose increased significantly. That is to say, the stability of teniposide injection during infusion was improved markedly. And TEN-SMEDDS avoided the side effects caused by Cremophor EL and increased the safty of teniposide formulation for intravenous administration.