Studies On The Self-microemulsifying Drug Delivery System Of Sirolimus

Sirolimus (SRL, rapamycin) is a carbocyclic lactone-lactam macrolide antibiotic, which has a molecular weight of 913.6 Da. The drug displays a unique immunosuppressive mechanism of action through its binding with FKBP12 and the inhibition of mammalian target of rapamycin (mTOR), which induces cell-cycle arrest in the G1 phase. These pharmacological properties allow sirolimus not only to be a promising immunosuppressant with the absence of nephrotoxicity but also to be a possible chemotherapeutic agent against many types of solid tumor. Even though sirolimus offers promising pharmacological activities, it has low oral bioavailability (<20%) in current formulations, such as oral solution and tablet. The low bioavailability was reported to be attributed to its sensitivity to gastric acid, partial intestinal absorption, and first-pass hepatic metabolism. Sirolimus has poorly water solubility (2.6μg/ml) and high liposolubility (log Po/w=5.77), and is the substrate of CYP450 3A isozymes and permeability-glycoprotein (P-gp) existing in the small intestinal enterocytes. These factors have been considered as significant contributors to low intestinal absorption.In this paper, self-microemulsifying drug delivery system of sirolimus was developed. Compared to commercially available oral solution (Rapamune(?)), the compositions, in vitro dispersion profile, in vivo bioavailability and mechanisms of intestinal absorption were investigated.The concentrations of SRL in vitro were determined by HPLC/UV and the whole blood concentrations of SRL were determined by HPLC/MS/MS. The HPLC method and HPLC/MS/MS method have been proved to be selective, rapid and suitable for the determination of sirolimus in vitro and in vivo, respectively.The compositions of SRL-SMEDDS were investigated by solubility assay, compatibility test, pseudo-ternary phase diagram analysis and drug stability study. MCT, Cremophor RH40 and propylene glycol were selected as oil, surfactant and co-solvent, respectively. The SMEDDS formulation was optimized using central composite design/response surface methodology. Optimized formulation of SMEDDS for bioavailability assessment was 25% MCT,50% Cremophcr EL and 25% propylene glycol. 1.0g of mixture contained 4.0mg of SRL. The SRL-SMEDDS dispersed rapidly (>90% within 30 min) to form microemulsion with emulsion droplet sizes <50nm and maintained sirolimus in a solubilised state at the end of the 60min dispersion. The resulting microemulsions from SMEDDS increased the drug stability significantly in both pH 1.2 HC1 solution and pH 6.8 phosphate buffer (P<0.05). Compared with the stability of free drug, the drug stability presented an approximate 4-fold improvement in pH 1.2 aqueous media and about 9-fold enhancement in pH 6.8 phosphate buffer. Besides, the co-solvent not only plays a role of drug solubilization but also promotes the dispersion rate of SMEDDS formulation.Rat single-pass intestinal perfusion technique (SPIP) was employed to assay the effects of flow rate, concentrations of sirolimus in perfusion solution, intestinal segments and bile duct ligation on the absorption percentage of drug (P%),the absorption rate constant (Ka) and the intestinal apparent permeability coefficient (Papp). The absorption rate of sirolimus was not affected by drug concentration but was significantly affected by the flow rate and intestinal segments. The bile secretion had no significant impact on drug intestinal absorption. The absorption of sirolimus showed the passive diffusion process. Sirolimus could be absorbed in whole intestinal segments and the absorption rate showed ileum> duodenum≈jejunum > colon. Furthermore, the sirolimus microemulsion presented the higher absorption rate than that of the conventional oral solution in rat intestine. The pharmacokinetic behavior of SRL-SMEDDS in rats were evaluated comparing to Rapamune(?). Significant differences in Cmax, Tmax and AUC values were observed. The relative bioavailability of SRL-SMEDDS to the conventional oral solution was 217.59%. These results illustrate the potential use of SMEDDS for the delivery of sirolimus by the oral route.Under the precondition of efficient dispersion and the formation of microemulsion (droplet size<100nm), the influence of different amount of oil or surfactant in SMEDDS formulations on the intestinal lymphatic transport of sirolimus were investigated using single-pass intestinal perfusion technique and a chylomicron flow blocking approach. The data obtained highlight the importance of lymphatic transport on the enhanced oral bioavailability of sirolimus. For the SMEDDS formulations containing≥25% oil, the lymphatic transport of sirolimus is a major contributor to oral bioavailability. Among the components included in SMEDDS formulations, oil can significantly affect the intestinal absorption of drug via the lymphatic pathway in a concentration dependent manner. A lipid threshold is necessary to trigger the biochemical cascades responsible for the lymphatic transport. Besides, even though the smaller droplet size of resultant microemulsions and more surfactant content also can positively influence the intestinal absorption of drug, their influences on the drug intestinal lymphatic transport were relatively weaker than that of oil content. Potentially drugs absorbed via the intestinal lymph seem to gain their access into the lymphatic system by three ways:via the paracellular route with the aid of absorption enhancers, via the M cells and gut-associated lymphoid tissues (GALT), and via a transcellular route in association with the triglyceride core of the chylomicrons. Although the precise mechanism of lymphatic transport has yet to be fully elucidated, the third route is historically thought to be the major mechanism of lymphatic delivery of lipophilic drugs when formulated with lipid-based vehicles. However, the present results indicated that the route via the M cells and gut-associated lymphoid tissues (GALT) may play a significant role in lymphatic transport of drug.