Molecular Engineering Laboratory, Department of Applied Science and Technology, Politecnico di Torino, Torino 10129, Italy.
Mol Pharm. 2023 Aug 7;20(8):3975-3986. doi: 10.1021/acs.molpharmaceut.3c00125. Epub 2023 Jul 12.
The use of -butyl alcohol for the lyophilization of pharmaceuticals has seen an uptick over the past years. Its advantages include increased solubility of hydrophobic drugs, enhanced product stability, shorter reconstitution time, and decreased processing time. While the mechanisms of protein stabilization exerted by cryo- and lyo-protectants are well known when water is the solvent of choice, little is known for organic solvents. This work investigates the interactions between two model proteins, namely, lactate dehydrogenase and myoglobin, and various excipients (mannitol, sucrose, 2-hydroxypropyl-β-cyclodextrin and Tween 80) in the presence of -butyl alcohol. We thermally characterized mixtures of these components by differential scanning calorimetry and freeze-drying microscopy. We also spectroscopically evaluated the protein recovery after freezing and freeze-drying. We additionally performed molecular dynamics simulations to elucidate the interactions in ternary mixtures of the herein-investigated excipients, -butyl alcohol and the proteins. Both experiments and simulations revealed that -butyl alcohol had a detrimental impact on the recovery of the two investigated proteins, and no combination of excipients yielded a satisfactory recovery when the organic solvent was present within the formulation. Simulations suggested that the denaturing effect of -butyl alcohol was related to its propensity to accumulate in the proximity of the peptide surface, especially near positively charged residues.
过去几年中，-丁醇在药物冷冻干燥中的应用有所增加。其优点包括增加疏水性药物的溶解度、提高产品稳定性、缩短复溶时间和减少处理时间。虽然在选择水作为溶剂时，冷冻保护剂和冻干保护剂对蛋白质稳定化的机制已经很清楚，但对于有机溶剂却知之甚少。这项工作研究了两种模型蛋白，即乳酸脱氢酶和肌红蛋白，以及在 -丁醇存在下的各种赋形剂（甘露醇、蔗糖、2-羟丙基-β-环糊精和吐温 80）之间的相互作用。我们通过差示扫描量热法和冷冻干燥显微镜对这些成分的混合物进行了热特性分析。我们还通过光谱法评估了冷冻和冷冻干燥后蛋白质的回收情况。此外，我们还进行了分子动力学模拟，以阐明所研究赋形剂、-丁醇和蛋白质的三元混合物中的相互作用。实验和模拟都表明，-丁醇对两种被研究蛋白质的回收有不利影响，而且当配方中存在有机溶剂时，没有任何赋形剂组合能得到令人满意的回收效果。模拟表明，-丁醇的变性作用与其倾向于在肽表面附近积累的特性有关，特别是在带正电荷的残基附近。
