Prync A E Sterin, Yankilevich P, Barrero P R, Bello R, Marangunich L, Vidal A, Criscuolo M, Benasayag L, Famulari A L, Domínguez R O, Kauffman M A, Diez R A
Bio Sidus SA, Buenos Aires, Argentina.
Int J Clin Pharmacol Ther. 2008 Feb;46(2):64-71. doi: 10.5414/cpp46064.
Recombinant human interferon-beta (IFN-b) is a well-established treatment for multiple sclerosis (MS). The regulatory process for marketing authorization of biosimilars is currently under debate in certain countries. In the EU, EMEA has clearly defined the process including overarching and product-specific guidelines, which includes clinical testing. Biosimilarity needs to be based on comparability criteria, including at least molecular characterization, biological activity relevant for the therapeutic effect and relative bioavailability ("bioequivalence"). In the case of such complex diseases as MS, where the effect of treatment is not so directly measurable, in vitro tools can provide additional data to support comparability. Genomic microarrays assays might be useful to compare multisource biopharmaceuticals. The aim of the present study was to compare the pharmacodynamic genomic effects (in terms of transcriptional regulation) of two recombinant human IFN-I(2)1a preparations on lymphocytes of multiple sclerosis patients using a whole genome microarray assay.
We performed an ex vivo whole genome expression profiling of the effect of two preparations of IFN-I(2)1a on non-adherent mononuclears from five relapsing-remitting MS patients analyzing microarrays (CodeLink Human Whole Genome). Patients blood was drawn, PBMCs isolated and cultured in three different conditions: culture medium (control), 1,000 U/ml of IFN-I(2)1a (BLA- (STOFERON, Bio Sidus) and 1,000 U/ml of IFN-I(2)1a (REBIF, Serono) RNA was purified from non-adherent cells (mostly lymphocytes), amplified and hybridized. Raw data were generated by CodeLink proprietary software. Data normalization, quality control and analysis of differential gene expression between treatments were done using linear model for microarray data. Functional annotation analysis of IFN-I(2)1a MS treatment transcription was done using DAVID.
Out of the approximately 45,000 human sequences examined, no evidence of differential regulation was found when both treatments were compared (minimum adjusted p-value > 0.999). The IFN-I(2)1a effect differentially regulated the expression of 868 genes. The expression of standard markers such as GTP cyclohidrolase, MxA, and OAS isoenzymes A and B changed as a consequence of the action of IFN-I(2)1a.
This exhaustive and highly sensitive assay did not show differences in the genomic expression profile of these two products under the assayed experimental conditions. These results suggest that this technology might be useful for the initial comparison of biosimilars, being part of a comprehensive comparability program that includes clinical testing.
重组人干扰素-β（IFN-β）是治疗多发性硬化症（MS）的一种成熟疗法。生物类似药的上市许可监管程序目前在某些国家正处于讨论之中。在欧盟，欧洲药品评价局（EMEA）已明确规定了该程序，包括总体指南和产品特定指南，其中包括临床试验。生物相似性需要基于可比性标准，至少包括分子特征、与治疗效果相关的生物活性以及相对生物利用度（“生物等效性”）。对于像MS这样治疗效果并非直接可测的复杂疾病，体外工具可为支持可比性提供额外数据。基因组微阵列分析可能有助于比较多源生物制药产品。本研究的目的是使用全基因组微阵列分析比较两种重组人IFN-β1a制剂对多发性硬化症患者淋巴细胞的药效基因组效应（就转录调控而言）。
我们对来自5例复发缓解型MS患者的非贴壁单核细胞进行了两种IFN-β1a制剂作用的体外全基因组表达谱分析，分析微阵列（CodeLink人类全基因组）。采集患者血液，分离外周血单核细胞（PBMCs）并在三种不同条件下培养：培养基（对照）、1000 U/ml的IFN-β1a（BLA-（STOFERON，Bio Sidus）和1000 U/ml的IFN-β1a（REBIF，赛诺菲）。从非贴壁细胞（主要是淋巴细胞）中纯化RNA，进行扩增和杂交。原始数据由CodeLink专有软件生成。使用微阵列数据的线性模型进行数据归一化、质量控制以及处理之间差异基因表达的分析。使用DAVID对IFN-β1a MS治疗转录进行功能注释分析。
在检测的约45000个人类序列中，比较两种治疗时未发现差异调控的证据（最小校正p值>0.999）。IFN-β1a的作用差异调节了868个基因的表达。标准标志物如GTP环化水解酶、Mx A以及2',5'-寡腺苷酸合成酶同工酶A和B的表达因IFN-β1a的作用而发生变化。
在检测的实验条件下，这种详尽且高度灵敏的分析未显示这两种产品在基因组表达谱上存在差异。这些结果表明，这项技术可能有助于生物类似药的初步比较，是包括临床试验在内的综合可比性计划的一部分。
