目的:通过测定左氧氟沙星的溶解性和体外渗透性,对比仿制与参比制剂的处方差异,考察差异性辅料对原料渗透行为的影响,并预测2种制剂的生物等效性,旨在探究基于平行人工膜渗透试验(PAMPA)的体外渗透性数据申请生物等效性豁免的可行性。方法:采用高效液相色谱法测定原料在pH 1.0~pH 6.8的溶解性;采用μFluxTM系统,在供体室中分别精密加入pH 5.0的饱腹小肠模拟液、pH 6.5的空腹小肠模拟液和pH 7.4的磷酸盐缓冲液16 mL,受体室中精密加入Accepter Sink Buffer 16 mL,转子速度200 r·min-1,采集时间180 min,测定各介质中原料和原料与硬脂富马酸钠混合物中原料的渗透性,以及参比和仿制制剂粉末中原料的渗透性,通过双侧t检验考察是否有显著性差异,以评估处方中新增辅料和其他变更辅料对原料的影响;采用Macro FluxTM系统,在溶出杯中加入pH 5.0和pH 6.5的肠模拟液1 000 mL作为溶出介质,使用桨法转速为75 r·min-1,受体室中精密加入Accepter Sink Buffer 12 mL,微搅拌棒的转速为450 r·min-1,分别取参比制剂和仿制制剂1片置溶出杯中,测定制剂的溶出-渗透曲线,比较溶出曲线相似性,计算渗透速率(JFlux)和终点时的药物累计渗透量(AMT),以2种制剂JFlux和AMT几何均值比值的90%置信区间是否落在80%~125%范围内为依据,预测制剂的生物等效性。结果:左氧氟沙星在不同介质的溶解性为16.4~62.7 mg·mL-1,其渗透性在pH 5.0的饱腹小肠模拟液、pH 6.5的空腹小肠模拟液和pH 7.4的磷酸盐缓冲液中分别为2.92×10-6、1.01×10-5和1.07×10-5 cm·s-1;加入硬脂富马酸钠后与原料的渗透性相比无显著差异(P＜0.05),参比和仿制制剂粉末中原料渗透性无显著差异(P＜0.05);2种制剂的溶出曲线相似,JFlux和AMT几何均值比值的90%置信区间结果均在限度范围之内。结论:左氧氟沙星为BCSⅠ类药物,处方变更后的辅料均不影响原料的渗透吸收,仿制与参比制剂生物等效,国产仿制制剂满足生物等效性豁免的核心要求。基于PAMPA的生物等效性研究,可用于原料的渗透性研究、处方的筛选和优化以及制剂的生物等效性预测等,能有效降低仿制药开发的成本和时间,加速高质量药品的研发。
Objective: To investigate the feasibility of applying for bioequivalence exemption based on parallel artificial membrane permeability assay (PAMPA) data by evaluating the solubility and in vitro permeability of levofloxacin, comparing the prescription differences between the generic and reference formulations, assessing the impact of disparate excipients on the permeation behavior of the active pharmaceutical ingredient (API), and predicting the bioequivalence of the two formulations. Methods: The dissolution of the raw material at pH 1.0 to pH 6.8 was determined using high-performance liquid chromatography. The μFluxTM system was employed to determine the permeability of the raw material and the mixture of raw material with sodium stearyl fumarate in various media. pH 5.0 fed-state intestinal fluid, pH 6.5 fasted-state intestinal fluid, and pH 7.4 phosphate buffer (16 mL) were precisely added to the donor chamber, while 16 mL of Accepter Sink Buffer was added to the receptor chamber. The rotor speed was set at 200 r·min-1, and the collection time was 180 min. Determine the permeability of the raw material and the mixture of raw material with sodium stearyl fumarate in various media, as well as the permeability of the raw material in the reference and generic formulations powders. The impact of newly added excipients and other changed excipients on API was evaluated through a two-tailed t-test. The Macro FluxTM system was used to measure the dissolution-permeation curves of the reference and generic formulations, intestinal simulation fluids at pH 5.0 and pH 6.5 (1 000 mL) were added to the dissolution cup as dissolution media, with a stirring speed of 75 r·min-1 using a paddle method, 12 mL of Accepter Sink Buffer was added to the receptor chamber, and the stirring speed of the micro-stirring rod was set at 450 r·min-1, one tablet of each reference or generic formulation was placed in the dissolution cup, and the dissolution-permeation curves of the formulations were measured. The similarity of dissolution curves was compared, and the permeability rate (JFlux) and cumulative drug permeation amount (AMT) were calculated to predict the bioequivalence of the two formulations, ensuring that the 90% confidence interval for the geometric mean ratio of JFlux and AMT of the two formulations fell within the range of 80% to 125%. Results: Levofloxacin solubility ranged from 16.4 mg·mL-1 to 62.7 mg·mL-1 across different mediums, its permeability in pH 5.0 fed-state simulated intestinal fluid, pH 6.5 fasted-state simulated intestinal fluid, and pH 7.4 phosphate-buffered saline was 2.92×10-6 cm·s-1, 1.01×10-5 cm·s-1, and 1.07×10-5 cm·s-1, respectively. The addition of sodium stearyl fumarate showed no significant difference in permeability compared to the original API (P＜0.05), and there were no significant differences between the powder API of the reference and generic formulations (P＜0.05). The dissolution curves of both formulations were similar, with the 90% confidence interval for JFlux and AMT within the predefined range. Conclusion: Levofloxacin tablets, classified as BCS Class Ⅰ, demonstrated that the altered excipients in the formulation did not impact on the API's permeability, confirming bioequivalence between the reference and generic formulations. The bioequivalence exemption study based on PAMPA can be utilized for permeability studies of raw materials, excipient screening and optimization, and prediction of formulation bioequivalence, effectively reducing drug development costs and time. This study provides reference data for pharmaceutical companies applying for bioequivalence exemptions.
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