Quantification of Drugs in Distinctly Separated Ocular Substructures of Albino and Pigmented Rats

The rat is a commonly used species in ocular drug research. Detailed methods of separating rat ocular tissues have not been described in literature. To understand the intraocular drug distribution, we developed a robust method for the separation of individual anterior and posterior substructures of pigmented Brown Norway (BN) and albino Wistar Han (WH) rat eyes, followed by quantification of drug concentration in these substructures. A short formalin incubation, which did not interfere with drug quantification, enabled the preservation of individual tissue sections while minimizing cross-tissue contamination, as demonstrated by histological analysis. Following oral administration, we applied the tissue separation method, in order to determine the ocular concentrations of dexamethasone and levofloxacin, as well as two in-house molecules BI 113823 and BI 1026706, compounds differing in their melanin binding. The inter-individual variability in tissue partitioning coefficients (Kp) was low, demonstrating the reproducibility of the separation method. Kp values of individual tissues varied up to 100-fold in WH and up to 46,000-fold in BN rats highlighting the importance of measuring concentration directly from the ocular tissue of interest. Additionally, clear differences were observed in the BN rat tissue partitioning compared to the WH rat. Overall, the developed method enables a reliable determination of small molecule drug concentrations in ocular tissues to support ocular drug research and development.