Department of Chemistry and Biochemistry and California NanoSystems Institute, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, California 90095-1569, United States.
Department of Molecular and Medical Pharmacology and Crump Institute for Molecular Imaging, David Geffen School of Medicine, University of California, Los Angeles, California 90095-1735, United States.
ACS Appl Mater Interfaces. 2022 Aug 24;14(33):37410-37423. doi: 10.1021/acsami.2c09301. Epub 2022 Aug 14.
Insulin, the oldest U.S. Food and Drug Administration (FDA)-approved recombinant protein and a World Health Organization (WHO) essential medicine for treating diabetes globally, faces challenges due to its storage instability. One approach to stabilize insulin is the addition of poly(trehalose methacrylate) (pTrMA) as an excipient. The polymer increases the stability of the peptide to heat and mechanical agitation and has a low viscosity suitable for injection and pumps. However, the safety and stabilizing mechanism of pTrMA is not yet known and is required to understand the potential suitability of pTrMA as an insulin excipient. Herein is reported the immune response, biodistribution, and insulin plasma lifetime in mice, as well as investigation into insulin stabilization. pTrMA alone or formulated with ovalbumin did not elicit an antibody response over 3 weeks in mice, and there was no observable cytokine production in response to pTrMA. Micropositron emission tomography/microcomputer tomography of Cu-labeled pTrMA showed excretion of 78-79% ID/cc within 24 h and minimal liver accumulation at 6-8% ID/cc when studied out to 120 h. Further, the plasma lifetime of insulin in mice was not altered by added pTrMA. Formulating insulin with 2 mol equiv of pTrMA improved the stability of insulin to standard storage conditions: 46 weeks at 4 °C yielded 87.0% intact insulin with pTrMA present as compared to 7.8% intact insulin without the polymer. The mechanism by which pTrMA-stabilized insulin was revealed to be a combination of inhibiting deamidation of amino acid residues and preventing fibrillation, followed by aggregation of inactive and immunogenic amyloids all without complexing insulin into its hexameric state, which could delay the onset of insulin activity. Based on the data reported here, we suggest that pTrMA stabilizes insulin as an excipient without adverse effects in vivo and is promising to investigate further for the safe formulation of insulin.
胰岛素是美国食品和药物管理局（FDA）最早批准的重组蛋白，也是世界卫生组织（WHO）治疗全球糖尿病的基本药物，但由于其储存不稳定，面临挑战。一种稳定胰岛素的方法是添加聚（海藻糖甲基丙烯酸酯）（pTrMA）作为赋形剂。该聚合物增加了肽对热和机械搅拌的稳定性，并且具有低粘度，适合注射和泵输送。然而，pTrMA 的安全性和稳定机制尚不清楚，需要了解 pTrMA 作为胰岛素赋形剂的潜在适用性。本文报道了 pTrMA 在小鼠中的免疫反应、生物分布和胰岛素血浆半衰期，以及胰岛素稳定性的研究。单独的 pTrMA 或与卵清蛋白一起配制在 3 周内没有在小鼠中引起抗体反应，并且没有观察到针对 pTrMA 的细胞因子产生。Cu 标记的 pTrMA 的微正电子发射断层扫描/微计算机断层扫描显示，在 24 小时内有 78-79% 的 ID/cc 排泄，在 120 小时时肝脏积累最小，为 6-8% ID/cc。此外，添加的 pTrMA 并未改变胰岛素在小鼠中的血浆半衰期。用 2 摩尔当量的 pTrMA 配制胰岛素可提高胰岛素对标准储存条件的稳定性：在 4°C 下储存 46 周，有 pTrMA 存在时，胰岛素的完整度为 87.0%，而没有聚合物时，胰岛素的完整度为 7.8%。结果表明，pTrMA 稳定胰岛素的机制是抑制氨基酸残基的脱酰胺作用和防止纤颤，然后是无活性和免疫原性淀粉样蛋白的聚集，而不将胰岛素复合成六聚体状态，这可能会延迟胰岛素活性的出现。基于这里报道的数据，我们建议 pTrMA 作为赋形剂稳定胰岛素，没有体内的不良影响，并有望进一步研究用于胰岛素的安全配方。
