Ghent Research Group on Nanomedicines, Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
VIB-UGent Center for Medical Biotechnology, Ghent, Belgium; Department of Biochemistry and Microbiology, Ghent University, Ghent, Belgium.
Int J Pharm. 2022 Jun 10;621:121772. doi: 10.1016/j.ijpharm.2022.121772. Epub 2022 Apr 26.
Globally, over 2 billion people suffer from vision impairment. Despite complex multifactorial etiology, advanced glycation end products are involved in the pathogenesis of many causative age- and diabetes-related eye diseases. Deglycating enzyme fructosamine-3-kinase (FN3K) was recently proposed as a potential therapeutic, but for further biopharmaceutical development, knowledge on its manufacturability and stability and mobility in the vitreous fluid of the eye is indispensable. We evaluated recombinant production of FN3K in two host systems, and its diffusion behavior in both bovine and human vitreous. Compared to Escherichia coli, intracellular production in Pichia pastoris yielded more and higher purity FN3K. The yeast-produced enzyme was used in a first attempt to use fluorescence correlation spectroscopy to study protein mobility in non-sonicated bovine vitreous, human vitreous, and intact bovine eyes. It was demonstrated that FN3K retained mobility upon intravitreal injection, although a certain delay in diffusion was observed. Alkylation of free cysteines was tolerated both in terms of enzymatic activity and vitreous diffusion. Ex vivo diffusion data gathered and the availability of yeast-produced high purity enzyme now clear the path for in vivo pharmacokinetics studies of FN3K.
全球范围内，超过 20 亿人患有视力障碍。尽管病因复杂，但晚期糖基化终产物与许多与年龄和糖尿病相关的致盲眼病的发病机制有关。糖化酶果糖胺-3-激酶 (FN3K) 最近被提议作为一种潜在的治疗方法，但为了进一步进行生物制药开发，了解其在眼玻璃体中的可制造性、稳定性和流动性是必不可少的。我们评估了 FN3K 在两种宿主系统中的重组生产及其在牛和人玻璃体中的扩散行为。与大肠杆菌相比，毕赤酵母中的细胞内生产可产生更多和更高纯度的 FN3K。酵母产生的酶首次用于使用荧光相关光谱法研究未经超声处理的牛玻璃体、人玻璃体和完整牛眼内的蛋白质流动性。结果表明，FN3K 保持了在眼内注射后的流动性，尽管观察到扩散有一定的延迟。游离半胱氨酸的烷基化在酶活性和玻璃体扩散方面都得到了耐受。现已获得的体外扩散数据和酵母生产的高纯度酶的可用性，为 FN3K 的体内药代动力学研究扫清了道路。
