摘要
群体生物等效性是FDA推荐的可用于纳米制剂产品粒径等效性的统计分析方法,但当粒径分布呈现多峰等复杂分布时,传统考察参数D50、SPAN则不再适用。推土距离是评估分布之间差异的新的度量方法。本研究以环孢素眼用乳剂为研究对象,提出了基于推土距离(EMD)的群体生物等效性统计分析方法,并对比不同制备工艺下乳剂产品粒径分布一致性结果以验证方法的可行性。结果显示,该统计方法可以有效拒绝不等效产品,通过等效产品,这为仿制制剂处方研究、工艺开发及优化提供一种方案,具有良好的应用价值。
近年来,随着人们生活水平的提高,健康意识的不断增强,药品需求随之越来越大。仿制药是医疗保障系统中的重要组成部分,其价格相对低廉,拥有良好的经济效益和社会效
粒径分布(PSD)通常用直方图表示,通过粒子大小和范围的信息可用于描述一些物质物理和化学特性。PSD的分析在建立复杂药品的BE方面已引起了广泛的关注。在FDA官方指南中已建议使用PBE方法对多种纳米制剂的粒径开展体外生物等效性研究,如氢氧化铁蔗糖复合物注射
环孢素眼用乳剂(商品名:Restasis)于2002年被FDA批准在美国上市用于干眼症的治
环孢素眼用乳剂参比制剂(商品名Restasis,规格0.4 mL∶0.2 mg,美国Allergan公司);甘油(南京化学试剂股份有限公司);蓖麻油[罗辅医药科技(上海)有限公司];聚山梨酯80(南京威尔药业股份有限公司);卡波姆共聚物A型(美国Lubrizol公司);氢氧化钠(西陇科学股份有限公司);环孢素(浙江瑞邦药业股份有限公司)。
EMD算
体外群体生物等效性统计分析的研究方法是基于比较准则,计算其95%的置信区间的上限值,如果小于或等于预设的界限值,则表示受试制剂与参比制剂具有生物等效性。FDA提出混合标度
(1) |
其中,分别是受试制剂/参比制剂所有容器,不同生命周期,不同批次效应值(对数转换)的总体均值;和为受试制剂和参比制剂的总方差(变异的总和);为控制总方差的参数,为定值0.1。表示为,其中为方差因素。在该研究中采用《吸入用布地奈德混悬剂指导原则草案》中参数值()
将
当>时选用参比标准,即;当≤时,选用常数标准,即
(2) |
(3) |
为使受试产品通过PBE试验,线性化标准(或)的95%置信上限(或)不得大于0。
按照
Formulation | Cyclosporin/g | Castro oil/g | Polysorbate 80/g | Glycerinum/g | Carbomer copolymer type A/g |
---|---|---|---|---|---|
F1 | 0.050 | 1.200 | 0.800 | 2.200 | 0.050 |
F2 | 0.050 | 1.000 | 1.000 | 2.200 | 0.050 |
F3 | 0.050 | 1.000 | 1.000 | 2.200 | 0.050 |
Formulation | Emulsifying temperature/°C | Emulsifying time/min | Shearing speed/(r/min) |
---|---|---|---|
F1 | 45 | 30 | 8 000 |
F2 | 45 | 20 | 8 000 |
F3 | 45 | 30 | 15 000 |
对给定的参比制剂和受试制剂PSD数据集,首先对所有参比样本PSD求取平均值从而得到平均参比样本PSD,作为参比中心。然后应用EMD计算单个参比样本的PSD到参比中心的距离,如[,.....,,.....],表示单个参比样本与参比中心之间的偏差。同样地,利用EMD计算每个受试样本到参比中心之间的距离,如[,.......,,......],表示单个受试样本与参考中心之间的偏差(其中i、j、k分别表示批次、样本数、重复测量次数)。随后,利用PBE对两组EMD进行统计检验,以建立受试制剂和参比制剂之间的BE(

Figure 1 Schematic diagram of the proposed equivalence approach
在此项研究中,采用动态光散射仪,以25 ℃测量角度173 ℃条件测定制剂粒径;所有样品均用超纯水稀释10倍后置于DTS0012样品池中。每批参比制剂取3个样本,每个样本重复测定5次。按照以下流程进行粒径PBE计算。
在本节中,在4批参比粒径数据集中任选2批作为R(R:参比样本),其余2批作为T(T:受试样本),检查所提出的方法接受等效产品的能力(例如,参比制剂本身)。

Figure 2 Comparison between two sets of the reference samples (two lots) by treating one of them as Test
A: Statistical summary of two groups of the PSD profiles, where the bold curve refers to the mean, flanked by the dotted standard deviation curves; B: Calculated EMD distances of reference (orange) and Test profiles (cyan) to the reference center
实验中,制备了不同处方用量和工艺参数的受试制剂,按照上述参数测定粒径,获得3个受试制剂数据集,同时以4批参比粒径数据参比数据集,分别应用EMD-PBE统计软件进行分析。

Figure 3 Comparison between the reference samples and test samples (F1-F3)A-C: The statistical summary of two groups of the PSD profiles, where the bold curve refers to the mean, flanked by the dotted standard deviation curves. D-F: The calculated EMD distances of reference (orange) and Test profiles (cyan) to the reference center. A-C and D-F corresponded to test preparations F1-F3, respectively
Parameters | Reference preparation | F1 | F2 | F3 |
---|---|---|---|---|
Z-average/nm | 111.1 | 135.8 | 160.4 | 111.3 |
0.044 19 | 0.062 30 | 0.046 47 | 0.069 09 | |
0.051 33 | 0.050 72 | 0.050 73 | 0.050 73 | |
0.860 9 | 1.228 3 | 0.916 0 | 1.361 9 | |
-0.002 823 | 0.020 71 | 0.051 60 | 0.005 657 | |
-0.019 12 | 0.005 102 | 0.035 99 | -0.010 20 | |
Pass/Fail | Pass | Fail | Fail | Pass |
EMD是衡量图像之间相似性的有效方法,虽广泛应用于计算机视觉应用,但使用EMD来比较PSD等效性未见文献报道。本研究应用基于Python语言开发的EMD-PBE统计分析软件,操作简单快捷,从上述结果可知该分析方法可有效通过粒径等效产品,拒绝不等效产品,为仿制制剂处方及工艺开发提供依据。
基于EMD的体外群体生物等效性的统计分析中,还有一部分内容可进一步深入研究:一是研究对象的选择,二是样本量的确定。本研究对象环孢素眼用乳剂粒径范围广
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