Study Of Diallyl Trisulfide Loaded Lipid Injectable Nanoemulsion

Lipid injectable nanoemulsion has been widely used in the intravenous delivery of lipophilic drugs with the advantages including reducing injection pain and venous irritation, high solubilization capability and low chance of drug precipitation at the injection site. However, the mechanism of reducing venous irritation has not fully understood, it has been proposed to be related to the preparation technology and drug release profiles of nanoemulsion. In this study, Diallyl trisulfide (DT) is chosen as the model drug because of high lipophilicity and venous irritation. Our work mainly focus on the preparation and characterization of DT nanoemulsion, in vitro drug release of DT nanoemulsion and studies of pharmacokinetics and tissue distribution of DT nanoemulsion. The major results are as the following:(1) Stable DT nanoemulsion was prepared and fully characterized. First, various external factors on DT stability were investigated in the preformulation study. We found that the alkaline condition had major adverse impact on the stability. In addition, high temperature, oxidation and light also destabilized, on the other hand, DT was less influenced by acid condition. Secondly, Optimization of DT nanoemulsion was achieved by increasing the stability of DT nanoemulsion with oleic acid andα-tocopherol. The DT nanoemulsion quality was investigated to optimize the parameters in the preparation methods of coarse emulsion, homogenization pressure, homogenization cycles, emulsification temperatures, the pH of nanoemulsion and the sterilization methods. Thirdly, the stability of DT nanoemulsion was assessed under extreme conditions, including 60℃, -20℃, 4℃, light and dilution. The results showed that the drug content and pH decreased and the impurities increased under the conditions of 60℃and light, while phase separation occurred under -20℃. No indices of DT nanoemulsion changed significantly under 4℃. In addition,the results of physical stability of DT nanoemulsion in different dilutions showed that the particle size did not increase significantly within 24 h, which indicated that DT nanoemulsion was stable after dilution.(2) Venous irritation of DT nanoemulsions was studied. DT nanoemulsion with two co-emulsifiers and three oil phases were prepared and characterized. The addition of poloxamer188 and solutol HS15 to phospholipids led to a significant decrease in the particle size of DT NE (p < 0.05). The particle size slightly increased with the increased concentrations of poloxamer188 and solutol HS15. The addition of nonionic co-emulsifiers resulted in a slight decreased zeta potential of DT nanoemulsion. The particle size of DT nanoemulsion made of olive oil is the smallest, followed soybean oil DT nanoemulsion, and then MCT DT nanoemulsion. The venous irritation of DT nanoemulsions was assessed with in vivo rabbit model by visual observation of pathological phenomenon and intracellular ATP and GTP concentrations in the in vitro human umbilical cord endothelial cells (HUV-EC CRL 1730) compatibility model. The in vivo and in vitro results showed the same trend. Compared to the control DT nanoemlsion without co-emulsifiers, the venous irritation of DT nanoemulsions containing poloxamer188 at 0.2% and 0.4% decreased significantly (p < 0.05). Hower, the venous irritation of DT nanoemulsions containing poloxamer 188 at 0.6% and solutol HS15 at 0.2%, 0.4% and 0.6% increased significantly (p < 0.05). Compared to the control DT nanoemlsion with the oil phase of soybean oil, the venous irritation of DT nanoemulsion with MCT decreased significantly (p < 0.05), while the venous irritation of DT nanoemulsion with olive oil increased significantly (p < 0.05). It was proposed that the venous irritation of DT loaded nanoemlsion may be attributed to the free drug in the aqueous phase and modulation of the free DT concentration should be an effective method to reduce the venous irritation of DT nanoemulsions for intravenous delivery. The significant positive correlation between the extent of venous irritation and the drug concentrations in the aqueous phase of DT nanoemulsions measured by the bulk reverse dialysis bag technique confirmed this hypothesis (p < 0.01). The change of DT concentrations in the aqueous phase of DT nanoemulsion followed the Fick's first diffusion law. It was deduced that the free DT concentrations of DT nanoemulsions containing co-emulsifers were determined by the partition coefficient of DT between oil and surfactant buffer solution, but the oil phases modulated the free DT concentrations by forming DT nanoemulsion with different particle sizes.(3) The pharmacokinetics and tissue distribution of Diallyl trisulfide (DT) after intravenous administration of nanoemulsions and Tween80 solution were studied. GC-ECD method was used to determine the total drug concentration in the biological samples, and the methods were comprehensively validated. Bromobenzene was used as the internal standard. Pharmacokinetics and tissue distribution of the formulations were investigated in rabbits and Swiss albino mice, respectively. Following bolus infusion, the pharmacokinetics of the three DT formulations could adequately be described by a three-compartmental pharmacokinetic model. The pharmacokinetic parameters were not statistically different between the DT solution (DS) and DT nanoemulsion (DNE). However, the AUC of DT nanoemulsions with 0.2% poloxamer188 (P-DNE) was significantly higher than that of DS and DNE (p < 0.05), moreover, the CL of P-DNE was significantly lower than that of DS and DNE (p < 0.05). The results of tissue distribution showed the AUCs of DS and DNE were similar in liver, spleen, kidneys and brain, while the AUC of DNE in the lungs was significantly higher than that of DS (p < 0.05). The AUCs of P-DNE in the liver and spleen were significantly lower than that of DNE (p < 0.05), and the AUCs of P-DNE in the brain was significantly higher than that of DNE (p < 0.05). The results indicated the encapsulation of DT in the DNE did not change its pharmacokinetics, and the addition of P188 in DNE produced high DTconcentration in brain and relatively low RES uptake.(4) It was nessesary to disclose the in vivo metabolic process of DT because of the pharmacokinetic characteristics of short half-life and high CL, and then the metabolism of DT in rat liver microsomes was investigated. The rate of metabolism followed Michaelis-Menten kinetics with Km = 0.60 mmol/L, a Vmax = 19.91 nmol/min/mg protein, and CLint = 0.033 mL/min/mg protein, respectively. One metabolite was found as 3-allylthio-propanol by GC/MS2 analysis method.In conclusion, DT nanoemulsion was prepared and characterized, and venous irritation of DT nanoemulsions was studied and the mechanism was also elucidated. The rule of nanoemulsion reducing side effects was revealed to a certain degree by the studies presented in this paper. The work described here will provide a new strategy and method for the safety study of other nano drug delivery systems.