| Triptolide (TP), a diterpenoid triepoxide, is the major active and toxic component of the extracts derived from the traditional Chinese medicine Tripterygium wilfordii Hook F (TWHF). TP has been demonstrated to possess a unique bioactive spectrum of anti-inflammatory, immunosuppressive, anti-fertility and anticancer activities. However, the clinical application of TP has been limited by its narrow therapeutic window and high toxicities on the hepatic, renal, digestive, reproductive and hematological systems. After oral administration in rats, TP is absorbed rapidly. The time of maximum plasma concentration (Tmax) ranges from10.0to19.5min, with an elimination half-life (ti/2) from16.8to50.6min. Similarly, the reported Tmax and t1/2of TP in mice are5.0and13.7min, respectively. These pharmacokinetic behaviors of TP indicate that it gains fast access into the blood circulation with very short stay. This phenomenon can be explained by the combined effects of efficient gastrointestinal permeability, rapid metabolism by cytochrome P4503A system in the liver, and extensive tissue distribution. The significant and rapid fluctuations of TP in plasma likely contribute to the toxicity of TP following oral administration. Therefore, with the aim of prolonging TP release and improving its safety, triptolide-loaded solid lipid nanoparticles (TP-SLN) and triptolide-loaded nanostructured lipid carriers (TP-NLC) were developed. In addition, pharmacokinetics and toxicology profiles of TP-SLN and TP-NLC were investigated and compared with each other. The main contents of this thesis are as follows:(1) Triptolide was found to tend to partition toward the aqueous phase during the production process of TP-SLN. TP-SLN prepared by microemulsion technique had higher encapsulation efficiency (EE) than that prepared by high pressure homogenization method. Compritol888ATO, Cremophor RH40and the Lipoid E80in Transcutol HP (with a ratio of1:1, w/w) were selected as solid lipid, surfactant and co-surfactant, respectively. The critical variables including fraction of lipid (X1), surfactant to co-surfactant ratio (X2) and lipid to drug ratio (X3) that influenced the EE of TP-SLN were adjusted, and results of single-factor experiments showed that the appropriate ranges of X1, X2and X3were40%～60%,2:1～6:1and50:1～100:1, respectively. (2) The optimized TP-SLN were prepared for further evaluation. The microemulsion technique was used to formulate TP-SLN employing a five-level central composite design (CCD) that was developed for exploring the optimum levels of three independent variables on particle size, EE and drug loading (DL). Quadratic polynomial models were generated to predict and evaluate the three independent variables with respect to the three responses. The optimized TP-SLN were predicted to comprise fraction of lipid of49.73%, surfactant to co-surfactant ratio of3.25, and lipid to drug ratio of55.27, which showed particle size of179.8±5.7nm, EE of56.5±0.18%and DL of1.02±0.003%that were in good agreement with predicted values. In addition, the optimized nanoparticles manifested a sustained-release pattern in vitro and were stable during3h of incubation in simulated gastric fluids without significant size change and the majority (91%) of the drug was protected. Furthermore, SLN had a potential of preventing gastric mucosa irritation caused by oral administration of TP in rats, this could be attributed to reduced lipid peroxidation levels and inflammation of the stomach mucosa.(3) TP-NLC were developed from the optimized TP-SLN. A binary mixture consisting of20%(w/w) Capryol90and80%(w/w) Compritol888ATO would be the most suitable combination of liquid and solid lipid for the formulation of TP-NLC. TEM photographs showed that the particle shape appeared close to spherical. The size of the TP-NLC (231.8±4.3nm) was significantly larger (p<0.001) than that of the TP-SLN (179.8±5.7nm). TP-NLC had a smaller PDI (0.143±0.012vs.0.283±0.012,p<0.001). Moreover, the presence of Capryol90in TP-NLC was useful to increase the EE from56.5to71.6%in comparison with TP-SLN. The particle growth of TP-NLC was negligible (p>0.05), but the size of TP-SLN significantly increased from179.±5.7nm to200.2±7.4nm after1month’s storage (p<0.05). TP-NLC exhibited good ability to reduce drug expulsion during storage, the EE only reduced from71.6to69.1%(p>0.05, about2.5%TP was expulsed). In contrast,8.2%TP was expulsed from TP-SLN under the same storage condition (p<0.01). The accumulated drug release at48h of TP-NLC and TP-SLN were71.2and91.3%respectively (p<0.001), indicating that TP incorporated in NLC exhibits lower release behavior compared to SLN.(4) A rapid HPLC-MS method was developed for the detection of TP in rat plasma and in vivo pharmacokinetic studies of TP-NLC, TP-SLN and TP in male rats were performed. The Tmax and t1/2z of free TP were0.200±0.075h and0.706±0.087h, respectively. The time to achieve maximum concentration of TP was delayed to0.717±0.240h and0.450±0.183h in the case of TP-NLC (p<0.01) and TP-SLN (p>0.05), respectively. MRTo-t and t1/2Z were markedly longer (p<0.001,p<0.01) for both TP-NLC and TP-SLN compared to free TP. Interestingly, a significant decrease was found in the Cmax from8.656±2.077μg/L for free TP to3.361±0.666μg/L for TP-NLC (p<0.01) and to5.794±1.747μg/L for TP-SLN (p<0.05). CLz/F was also found to be reduced in TP-NLC (p<0.01) and TP-SLN (p<0.05) compared to free TP. In addition, TP-NLC and TP-SLN gave mean values of AUC0-t9.012±1.576μg h/L and7.318±1.628μg h/L, which were1.54-and1.25-fold higher, than that of free TP, respectively. These results showed the superiority of TP-NLC over TP-SLN, as also supported by prominent difference on MRT0-t(p<0.001), Tmax(p<0.05) and Cmax (p<0.05).(5) The acute toxicity in mice and sub-acute toxicity in rat were studied for TP-SLN and TP-NLC. Results demonstrated that the LD50for TP, TP-SLN and TP-NLC administered orally were1.08mg/kg,1.50mg/kg,1.87mg/kg in mice, respectively. In toxicity study in rat, TP, TP-SLN and TP-NLC were administered orally at the dose levels of500μg/kg and650μg/kg for28days. NLC could significantly inhibit the decrease of weight gain caused by TP in rats, showing better performance than SLN, especially at high dose. Relative weights of heart, lungs, liver, spleen and kidney in TP-NLC group had also been significantly improved at high dose. NLC and SLN could also reduce the liver toxicity of TP (500μg/kg) in male rats. However, at high dose (650μg/kg), only NLC had a significant protective effect on the liver and kidney. Compared to free TP, TP-NLC exhibited significantly reduced oxidative stress, which was verified by decreased MDA and improved SOD activity in rats serum at high dose, indicating a better safety than TP-SLN. Microscopically, histopathological changes in liver, spleen and kidney were discovered in high dose of TP and TP-SLN groups (650μg/kg). Fatty degeneration in the hepatocytes, dead cells in the macrophages as a "starry sky" appearance in spleen, and obvious kidney proximal tubular dilation were seen, However, in TP-NLC group at the same dose, no apparent changes were found.Results in this thesis demonstrated that both NLC and SLN could significantly improve the pharmacokinetic parameters of TP after oral administration and enhance the safety of TP. TP-NLC were superior to TP-SLN. These results would be of great importance for the development of new nano-formulation of TP and also provide suggestions to clarify the association between altered pharmacokinetics and reduced toxicity of TP. |
PDF Full Text Request