Age‐related macular degeneration (AMD) is the leading cause of severe vision impairment in patients over the age of 60 years. Choroidal neovascularization (CNV) is the hallmark of neovascular AMD and vascular endothelial growth factor (VEGF) plays a causal role in the formation of CNV. Although regorafenib and pazopanib, small molecule VEGF receptor (VEGFR) inhibitors, were developed as eye‐drops, their efficacies were insufficient in clinical. In this study, we evaluated ocular pharmacokinetics and pharmacological activities of regorafenib and pazopanib after ocular instillation in multiple animal species. In rats, both regorafenib and pazopanib showed high enough concentrations in the posterior eye tissues to inhibit VEGFR. In laser‐induced rat CNV model, regorafenib showed clear reduction in CNV area. On the other hand, the concentrations of regorafenib and pazopanib in the posterior eye tissues were much lower after ocular instillation in rabbits and monkeys compared to those in rats. Pazopanib did not show any improvement in monkey model. Regorafenib was nano‐crystalized to improve its drug delivery to the posterior eye tissues. The nano‐crystalized formulation of regorafenib showed higher concentrations in the posterior segments in rabbits compared to its microcrystal suspension. From these studies, large interspecies differences were found in ocular delivery to the posterior segments after ocular instillation. Such large interspecies difference could be the reason for the insufficient efficacies of regorafenib and pazopanib in clinical studies. Nano‐crystallization was suggested to be one of the effective ways to overcome this issue.

It is important to select an appropriate surrogate matrix for preparing calibration standards and quality control samples while quantitatively assaying for endogenous substances, because a blank matrix that does not contain the endogenous substance cannot be derived from the species from which the target study samples are collected. This is because the assay results might be affected, depending on the characteristics of the analyte in the surrogate matrix. Our discussion group that participated in the Japan Bioanalysis Forum discussed the recommended selection strategies, focusing on large and small molecules in ligand binding assays and LC–MS, respectively. We established an efficient selection strategy for a surrogate matrix, with simple compositions as the first candidates stated in this article.

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