【作者】 饶子超；

【导师】 李高；

【作者基本信息】 华中科技大学 ， 药理学， 2010， 博士

【摘要】 药物的口服吸收是一个复杂的过程,其包括生理因素和制剂因素(如溶解度、组织渗透性、剂型等)。然而,对于那些在体内易被细胞色素P450 3A(CYP 3A)所氧化代谢的药物,抑制CYP 3A的生物转化活性,是提高这类药物口服生物利用度的有效途径。CYP 3A在肝脏及肠道中含量丰富,在成人肝脏和小肠中分别占CYP总量的30%和70%以上,它参与50%以上药物的Ⅰ相代谢,明显降低了CYP 3A底物药物口服生物利用度。有文献报道,易被CYP 3A酶代谢的药物与CYP 3A抑制剂共同服用,这些药物的口服生物利用度会大大增加。然而,绝大多数CYP 3A抑制剂具有一定的药理活性和临床适应症,这些物质在体内需要达到一定的浓度才能发挥抑制CYP 3A酶活性的作用,这样就会产生毒副作用,限制了其在临床上的进一步应用。但联用CYP 3A抑制剂可提高CYP 3A底物药物的口服吸收及生物利用度,因此寻找一些无药理活性、低毒且具有较强CYP 3A抑制作用的抑制剂是国内外生物药剂学学者重点研究的领域之一。药物辅料一直被认为是生理惰性的物质而应用于各种制剂中,近几年国内外研究者陆续发现某些制剂辅料(特别是非离子表面活性剂)具有抑制CYP3A的活性,从而改变药物在体内的吸收、分布、代谢、排泄过程。利用对CYP 3A有抑制作用的辅料构建载体给药系统,可降低CYP 3A底物药物在胃肠道吸收过程中被CYP 3A所氧化代谢的程度,提高CYP 3A底物药物的口服生物利用度；同时也不会产生新的毒副作用。因此从给药系统的角度来提高CYP 3A底物药物的口服生物利用度,较其它的策略更为可靠、易行,具有广泛的应用开发前景。为了考察包含有对CYP 3A有抑制作用的辅料构建的载体给药系统对CYP 3A酶的影响,以咪达唑仑(MDZ)为模型药物、选用对大鼠肝微粒体CYP 3A有明显抑制作用的表面活性剂(Cremophor EL35、Cremophor RH40、Tween-80)为主要的载体材料,采用星点设计—效应面法优化处方,制备咪达唑仑自微乳化给药系统(MDZ—SMEDDS),基本处方为：MCT:Cremophor EL35:PG=25:50:25 (W:W:W)、MCT:Cremophor RH40:PG=25:45:30 (W:W:W)或EO:Tween-80:PG=30：45：25(W：W：W)。优化后的处方水化所形成微乳的自微乳化时间和平均粒径均符合SMEDDS制剂的要求。通过目测法和仪器分析,以外观、粒径为主要指标,考察了介质、稀释倍数、离子强度和食物效应等对各处方自乳化速率的影响,发现各因素对MDZ-SMEDDS的自微乳化效率并无明显影响；通过对各处方所形成微乳的稳定性进行考察,发现含有Cremophor EL35的微乳在人工肠液中的稳定性较差,因此,我们不再对包含Cremophor EL35的SMEDDS进行评价；比较不同MDZ制剂在人工肠液释放情况,结果显示,将MDZ制成自微乳化给药系统可以极大提高其释放的速度和程度；稳定性试验表明,在所考察的高温、强光条件下,MDZ-SMEDDS的外观、药物含量、粒径大小、自微乳化时间均无明显变化,MDZ及其微乳的稳定性良好。考察包含Cremophor RH40和Tween 80的SMEDDS (Cremophor RH40-based SMEDDS和Tween 80-based SMEDDS)对大鼠肝细胞CYP 3A作用,发现相对于对照组(MDZ溶液),Cremophor RH40-based SMEDDS和Tween 80-based SMEDDS在1：50到1：250的稀释范围内可明显抑制1’-OHMDZ的生成,且未产生明显的细胞毒作用；通过Western blot技术检测Cremophor RH40-based SMEDDS和Tween 80-based SMEDDS对大鼠肝细胞CYP3A酶蛋白表达的影响,研究证实,在1：50到1：100的稀释范围内包含CremophorRH40和Tween 80的SMEDDS能显著降低大鼠肝细胞中CYP 3A的蛋白表达水平,相对于对照组(生理盐水),其CYP 3A的蛋白表达量分别为40.5±9.9%和28.8±7.2%(RH40-based SMEDDS),65.3±10.8%和35.8±8.3%(Tween 80-based SMEDDS)。这些结果进一步表明,SMEDDS能抑制MDZ在大鼠肝细胞的代谢部分是由于下调了CYP3A的蛋白表达,从而减少CYP 3A酶的生物转化活性。分别单次或多次灌胃给予大鼠生理盐水、MDZ市售片剂(Dormicum(?))、Cremophor RH40-based SMEDDS与酮康唑溶液,从大鼠肠黏膜上皮细胞提取分离肠RNA和蛋白,采用RT-PCR和Western blot技术检测肠道核酸转录水平和蛋白表达水平。结果显示,相对于生理盐水对照组,单剂量和多剂量给予Cremophor RH40-based SMEDDS微乳显著降低了大鼠肠黏膜上皮细胞中CYP 3A的mRNA表达水平(见图6-1),mRNA表达量分别为生理盐水对照组57.3±5.6%(单剂量)和57.6±7.7%(多剂量),而Dormicum(?)片剂混悬液对大鼠肠道CYP 3A的mRNA表达水平并无明显的影响,mRNA表达量分别为对照组98.3±7.3%(单剂量)和90.6±7.8%(多剂量)。Western blot检测结果与RT-PCR一致,单剂量和多剂量给予Cremophor RH40-based SMEDDS微乳显著降低了大鼠肠黏膜上皮细胞中CYP3A的蛋白表达水平(P<0.05),蛋白表达量分别为生理盐水对照组53.7±6.9%(单剂量)和40.2±8.5%(多剂量)；单剂量给予Dormicum(?)片剂混悬液对大鼠肠道CYP3A的蛋白表达水平并无明显的影响,而多剂量给予Dormicum(?)片剂混悬液却轻微降低了大鼠肠道CYP3A的蛋白表达水平,蛋白表达量分别为对照组89.5±10.8%(单剂量)和76.8±16.1%(多剂量)。上述结果说明单剂量或多剂量给予Cremophor RH40-based SMEDDS,都会降低大鼠肠道CYP 3A酶的基因表达水平,从而提高CYP3A底物药物MDZ的口服生物利用度。分别单次或多次灌胃给予大鼠MDZ市售片剂(Dormicum(?))、Cremophor RH40-based SMEDDS与Tween 80-based SMEDDS.结果显示,相对于市售片剂,单剂量和多剂量给予Cremophor RH40-based SMEDDS微乳与Tween 80-based SMEDDS微乳后,MDZ口服生物利用度有明显提高,且显著降低了1’-OHMDZ与MDZ的AUC0-∞比值。与市售Dormicum(?)片剂组的AUC0-∞比较,单剂量和多剂量给予Tween 80-basedSMEDDS的相对生物利用度分别为(226.51±43.38)%和(246.11±44.71)%；Cremophor RH40-based SMEDDS的相对生物利用度分别为(314.38±107.56)%和(332.74±82.97)%(P<0.05),AUC0-∞1’-OHMDZ／AUC0-∞MDZ的比值分别从0.25降至0.14(Tween 80-based SMEDDS)和0.11(Cremophor RH40-based SMEDDS)(单剂量),以及从0.27降至0.12 (Tween 80-based SMEDDS)和0.09(Cremophor RH40-based SMEDDS)(多剂量)；同时Cremophor RH40-based SMEDDS微乳与Tween 80-based SMEDDS微乳也显著降低了MDZ的清除率(CL)并延长了MDZ在体内的平均滞留时间(MRT)及消除半衰期(t1／2)。综上所述,包含有对CYP3A有抑制作用的辅料构建载体系统可有效的保护CYP3A底物药物的代谢,从而提高其口服生物利用。通过研究包含有对CYP 3A有抑制作用的辅料构建载体系统对CYP 3A酶的影响,从而为提高某些药物口服吸收而设计高口服生物利用度新剂型提供理论依据；为临床低生物利用度药物的疗效提高提供一条新的途径。因此,本研究具有重要的理论意义和现实意义。更多还原

【Abstract】 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.更多还原