The Construction Of Oral Self-microemulsifying Drug Delivery System With Enzyme Inhibition Of Cytochrome P450 3A And Its Mechanism Study

Oral absorption of a drug involves complicated processes and varies with formulation factors and physiological conditions (solubility, tissue permeability, formulation factors etc.). However, inhibiting cytochrome P450 3A (CYP3A) activity is considered to be a helpful strategy for enhancing absorption of orally administered drugs that may be oxidized by CYP3A. CYP3A localizing in both the liver and intestine, and accounts for 30% of the total P450 content in the adult liver and for 70% in the intestine. CYP3A is involved in the metabolism of more than 50% of the currently marketed drugs and make a major contribution to the presystemic elimination of substrate drugs after oral administration.Indeed, it has been previously reported that inhibition of CYP3A enzymes by various compounds can lead to increased bioavailability of drugs. However, most of these inhibitory compounds are pharmacologically active ingredients and have their own clinical indications. Furthermore, most of these inhibitors lead to undesired pharmacodynamic side effects caused by the high concentrations necessary for sufficient gastrointestinal inhibition of CYP3A, which limits its clinical efficacy. But in combination of CYP 3A inhibitors can increase the oral absorption and bioavailability of CYP 3A substrate drugs, seeking for some strong CYP 3A inhibitors with properties of non-pharmacological activity and low toxicity is one of the most important research fields in biopharmaceutics.More recently, concerns have been raised that some excipients, especially nonionic surfactants, may also influence the absorption, distribution, metabolism and elimination of the active drugs by inhibiting CYP3A enzymes in vivo. Drug delivery system constructed with the inhibitors of CYP3A can reduce the extent of oxidative metabolism and improve the oral bioavailability of CYP3A substrate drugs in the gastrointestinal absorption process. For the perspective of drug delivery systems, improvement of the oral bioavailability of CYP 3A substrate drugs can be more reliable and easier than other strategies and have a wide range of development prospects.In order to investigate the effects of a drug delivery system constructed with excipient inhibitors on the CYP 3A enzymes, midazolam (MDZ) was selected as a model drug and the surface surfactants (Cremophor EL35, Cremophor RH40, Tween-80) which significantly inhibited CYP 3A enzymes in rat liver microsomes were the main formulation components. Central composite design-response surface methodology was used to optimize the preparation of MDZ-based self-microemulsifying drug delivery system (MDZ-SMEDDS). Basic prescription for the SMEDDS were:MCT:Cremophor EL35:PG =25:50:25 (W:W:W),MCT:Cremophor RH40:PG=25:45:30 (W:W:W) or EO: Tween-80:PG=30:45:25 (W:W:W). The optimized microemulsion formulation formed from hydration self-microemulsifying time and the average particle size met the requirements of SMEDDS preparation.Visual assessment and instrumental analysis were not found to have significant effect on the self-microemulsifying efficiency in various factors of medium,dilution times,ionic strength and food effects. In the stability study, formulation containing Cremophor EL35 in simulated intestinal juice was dissolved from 378μg/mL at 10min to 245μg/mL at 180min. Formulation containing Cremophor EL35 could not prevent MDZ precipitation in the presence of aqueous phase as efficiently as other formulation. It has been known from the reproted literature that, surfactant hydrolysis may have a negative impact on the overall solubilization capacity of self-microemulsifying formulation containing large amounts of digestible surfactants. Cremophor EL35 and Cremophor RH40 have similar chemical structure. Interestingly, however, Cremophor RH40 appeared to be less susceptible to digestion when compared with EL35 in vitro digestion experiments. Thus, in the presence of the Cremophor EL 35, the ability to prevent precipitation seems to be reduced. On the basis of these findings, formulation containing Cremophor EL35 was excluded from further evaluation. The comparison results of release profile of different MDZ formulations showed that self-microemulsifying drug delivery system can greatly improve the speed and extent of MDZ released. Stability test showed that the appearance, drug content, particle size, and self-microemulsifying time were not significantly changed in the high temperature and strong light conditions.We evaluated the effects of SMEDDS contains Cremophor RH40 and Tween 80 (Cremophor RH40-based SMEDDS and Tween 80-based SMEDDS) at different dilution times on the metabolism of MDZ in rat hepatocytes. The results showed that the metabolism of MDZ was significantly inhibited in the dilution range from 1:50 to 1:250 without causing cell cytotoxicity. In west blot analysis, a significant decrease in CYP3A protein levels was observed in cells in the presence of either Cremophor RH40 or Tween 80-based SMEDDS in the dilution range from 1:50 to 1:100 compared to control (P<0.05),40.5±9.9% and 28.8±7.2% of control (for RH40-based SMEDDS),65.3±10.8% and 35.8±8.3% of control (for Tween 80-based SMEDDS), respectively. These results suggest that the Tween 80 or Cremophor RH40-based SMEDDS may inhibit MDZ metabolism by down-regulating the CYP3A protein expression, which decreases the catalytic activity of CYP3A enzymes.To clarify the mechanism of inhibition of SMEDDS on CYP3A enzymes, the effects of the saline,the commercial tablet of MDZ (Dormicum(?)),Cremophor RH40-based SMEDDS and ketoconazole (KTZ) solution on the intestinal CYP3A enzymes mRNA and protein level in rats following single-dose and multiple-dose administration were assessed by RT-PCR and Western blot analyses. The results showed that Cremophor RH40 significantly decreased the levels of CYP 3A mRNA and protein expression in mucosa of rats versus the saline control group; 57.3±5.6% of control (for single-dose group),57.6±7.7% of control (for multiple-dose group), respectively There were no significant difference in CYP3A mRNA levels among Dormicum(?) group,98.3±7.3% of control (for single-dose group),90.6±7.8% of control (for multiple-dose group). Consistent with this observation, the expression of CYP3A protein was significantly decreased,53.7±6.9% of control (for single-dose group),40.2±8.5% of control (for multiple-dose group), respectively. There were no significant difference in CYP3A mRNA levels among Dormicum(?) group, These results further support the result that the Cremophor RH40-based SMEDDS may inhibit MDZ metabolism partially due to down-regulating the CYP3A mRNA and protein expression, which further decrease the catalytic activity of CYP3A enzymes.We assessed the effects of the commercial tablet of MDZ (Dormicum(?)),Cremophor RH40-based SMEDDS and Tween 80-based SMEDDS on the pharmacokinetics of MDZ and its metabolite 1'-Hydroxymidazolam in rats following single-dose and multiple-dose administration, the results showed that the oral bioavailability of MDZ microemulsion of Cremophor RH40-based SMEDDS and Tween 80-based SMEDDS was greater than that of the commercial tablet. The relative bioavailability were (226.51±43.38)%(single-dose group) and (246.11±44.71)%(multiple-dose group) for Tween 80-based SMEDDS; (314.38±107.56)%(single-dose group) and (332.74±82.97)%(multiple-dose group) for Cremophor RH40-based SMEDDS. Furthermore, MDZ microemulsion significantly decreased the ratio of AUC0-∞(1'-OH-MDZ)/AUC0-∞(MDZ), from 0.25 to 0.14 (Tween 80-based SMEDDS) and 0.11 (Cremophor RH40-based SMEDDS) for single-dose group; from 0.27 to 0.12 (Tween 80-based SMEDDS) and 0.09 (Cremophor RH40-based SMEDDS) for single-dose group, and reduced the clearance (CL) of MDZ. Moreover the MRT and elimination half-time (t1/2) were also increased by MDZ microemulsion in the single-dose and multiple-dose regimen.In summary, the excipient inhibitor-based formulation is a potential protective platform for decreasing metabolism of sensitive drugs that are CYP3A substrates. This study can provide a theoretical basis for the design of the new formulation to improve the low bioavailability drug, and therefore has an important theoretical and practical significance.