Formulation and PEGylation optimization of the therapeutic PEGylated phenylalanine ammonia lyase for the treatment of phenylketonuria.
Formulation and PEGylation optimization of the therapeutic PEGylated phenylalanine ammonia lyase for the treatment of phenylketonuria.
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DOI：
10.1371/journal.pone.0173269
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Fitzpatrick PA
中科院分区：
文献类型：
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作者：
Bell SM;Wendt DJ;Zhang Y;Taylor TW;Long S;Tsuruda L;Zhao B;Laipis P;Fitzpatrick PA
苯丙酮尿症 (PKU) 是一种遗传代谢疾病，其中苯丙氨酸羟化酶 (PAH) 活性的降低或丧失导致苯丙氨酸 (Phe) 的神经毒性水平升高。由于存在许多障碍，PAH 酶替代疗法目前不是一种选择。 20 世纪 70 年代首次提出用替代酶苯丙氨酸解氨酶 (PAL) 治疗 PKU。然而，有关免疫原性、酶产生和递送方式的问题需要克服。通过对多个物种的 PAL 酶进行评估，选择了来自酵母和蓝细菌的三种潜在 PAL 酶来评估其治疗潜力。以特定比例添加聚乙二醇（PEG，MW = 20,000）来修饰蛋白质表面，可减弱 PKU 动物模型中的免疫原性。所有三种聚乙二醇化 PAL 候选物在 PKU (BTBR Pahenu2) 小鼠模型中皮下注射后均显示出功效。然而，只有经聚乙二醇化的多变鱼腥藻 (Av) PAL 处理的小鼠在每周注射后表现出持续的低 Phe 水平，并且是唯一经过评估的在聚乙二醇化后保持完全酶活性的 PAL。基于其积极的药效学特征和有利的表达滴度，选择 AvPAL (Cys503Ser/Cys565Ser) 的聚乙二醇化重组双突变体 rAvPAL-PEG 用于药物开发。聚乙二醇化对于 rAvPAL-PEG 功效至关重要，因为聚乙二醇化的 rAvPAL 具有较低的药效作用。 rAvPAL 和 rAvPAL-PEG 在 4°C 下稳定性较差。 L-Phe 和反式肉桂酸盐被确定为活性稳定赋形剂。 rAvPAL-PEG 目前正在进行 3 期临床试验，以评估 PKU 患者的疗效。
Phenylketonuria (PKU) is a genetic metabolic disease in which the decrease or loss of phenylalanine hydroxylase (PAH) activity results in elevated, neurotoxic levels of phenylalanine (Phe). Due to many obstacles, PAH enzyme replacement therapy is not currently an option. Treatment of PKU with an alternative enzyme, phenylalanine ammonia lyase (PAL), was first proposed in the 1970s. However, issues regarding immunogenicity, enzyme production and mode of delivery needed to be overcome. Through the evaluation of PAL enzymes from multiple species, three potential PAL enzymes from yeast and cyanobacteria were chosen for evaluation of their therapeutic potential. The addition of polyethylene glycol (PEG, MW = 20,000), at a particular ratio to modify the protein surface, attenuated immunogenicity in an animal model of PKU. All three PEGylated PAL candidates showed efficacy in a mouse model of PKU (BTBR Pahenu2) upon subcutaneous injection. However, only PEGylated Anabaena variabilis (Av) PAL-treated mice demonstrated sustained low Phe levels with weekly injection and was the only PAL evaluated that maintained full enzymatic activity upon PEGylation. A PEGylated recombinant double mutant version of AvPAL (Cys503Ser/Cys565Ser), rAvPAL-PEG, was selected for drug development based on its positive pharmacodynamic profile and favorable expression titers. PEGylation was shown to be critical for rAvPAL-PEG efficacy as under PEGylated rAvPAL had a lower pharmacodynamic effect. rAvPAL and rAvPAL-PEG had poor stability at 4°C. L-Phe and trans-cinnamate were identified as activity stabilizing excipients. rAvPAL-PEG is currently in Phase 3 clinical trials to assess efficacy in PKU patients.