小鼠很少用于眼部治疗的药代动力学(PK)研究,因为它们的眼睛尺寸小和药物管理方面的挑战。组织收集,和药物浓度分析。因此,在玻璃体内(IVT)施用后小鼠眼中的蛋白质治疗剂的眼部PK是未知的。这里,我们提出了第一个此类调查,为了研究小鼠血浆中4种不同大小的非结合蛋白治疗剂的PK,角膜/ICB,玻璃体幽默,视网膜,和IVT给药后的后杯(包括脉络膜)。施用的蛋白质包括曲妥珠单抗(150kDa)和F(ab)2(100kDa),Fab,和曲妥珠单抗的scFv(27kDa)片段。开发了一种适用于在小鼠中进行小(50nL)IVT注射的成像和注射装置,并开发了摘除眼睛和解剖眼组织的技术。此外,开发了一种灵敏的酶联免疫吸附测定(ELISA),用于检测极少量眼组织中的蛋白质。观察到从玻璃体腔的消除是角膜/ICB中PK的主要驱动因素。视网膜,后杯,和等离子体。曲妥珠单抗在玻璃体液中显示出一级动力学,半衰期为18.8小时。F(ab)2,Fab,和ScFv显示双相PK曲线,随着分子量的降低,分布阶段变得更加快速,并且末端消除随着分子量的降低而变得更长,终末半衰期为16.3、20.6和48.9小时,分别。曲妥珠单抗的平均停留时间,F(ab)2,Fab,玻璃体液中的scFv分别为26.0、12.2、10.7和8.16小时,分别。发现玻璃体液中的平均停留时间随着分子量〜69kDa的增加而加倍。有趣的是,在未注射的眼睛中测量的蛋白质的PK表明存在药物在眼睛之间转移的途径,这需要进一步验证。总的来说,本文的研究结果为药物发现和开发用于小鼠眼科适应症的蛋白质疗法的研究铺平了道路。
Mice are rarely used in pharmacokinetic (PK) studies of ocular therapeutics due to the small size of their eyes and challenges in drug administration, tissue collection, and analysis of drug concentrations. Therefore, ocular PK of protein therapeutics in mouse eye following intravitreal (IVT) administration is not known. Here, we have presented the first of its kind investigation, to study the PK of 4 different size non-binding protein therapeutics in mouse plasma, cornea/ICB, vitreous humor, retina, and posterior cup (including choroid) following IVT administration. Administered proteins include trastuzumab (150 kDa) and F(ab)2 (100 kDa), Fab, and scFv (27 kDa) fragments of trastuzumab. An imaging and injection apparatus suitable for performing small (50 nL) IVT injections in mice was developed, and techniques for enucleation of the eye and dissection of ocular tissues were developed. Furthermore, a sensitive enzyme-linked immunosorbent assay (ELISA) for detection of proteins in very small amounts of ocular tissues were developed. It was observed that elimination from the vitreous chamber was the primary driver of PK in the cornea/ICB, retina, posterior cup, and plasma. Trastuzumab displays first-order kinetics in the vitreous humor with a half-life of 18.8 h. F(ab)2, Fab, and ScFv show biphasic PK profiles with distribution phases becoming more rapid as molecular weight decreases, and terminal elimination becoming longer as molecular weight decreases, with terminal half-lives of 16.3, 20.6, and 48.9 h, respectively. The mean residence times of trastuzumab, F(ab)2, Fab, and scFv in the vitreous humor were 26.0, 12.2, 10.7, and 8.16 h, respectively. It was found that the mean residence time in vitreous humor doubles with an increase in molecular weight of ∼69 kDa. Interestingly, the PK of proteins measured in the un-injected eye suggest the presence of a pathway for drug transfer between the eyes, which needs to be further validated. Overall, the findings presented here pave the way for drug discovery and development studies of protein therapeutics for ophthalmic indications in mice.