Ex vivo investigation of ocular tissue distribution following intravitreal administration of connexin43 mimetic peptide using the microdialysis technique and LC-MS/MS.

Abstract

This study aimed to develop and evaluate an ex vivo eye model for intravitreal drug sampling and tissue distribution of connexin43 mimetic peptide (Cx43MP) following intravitreal injection using the microdialysis technique and LC-MS/MS. An LC-MS/MS method was developed, validated, and applied for quantification of Cx43MP in ocular tissues. Microdialysis probes were calibrated for in vitro recovery studies. Bovine eyes were fixed in a customized eye holder and after intravitreal injection of Cx43MP, microdialysis probes were implanted in the vitreous body. Vitreous samples were collected at particular time intervals over 24h. Moreover, 24 and 48h after intravitreal injection ocular tissues were collected, processed, and analyzed for Cx43MP concentrations using LC-MS/MS. The LC-MS/MS method showed good linearity (r 2=0.9991). The mean percent recovery for lower (LQC), medium (MQC), and higher quality control (HQC) (0.244, 3.906, and 125μg/mL) was found to be 83.83, 84.92, and 94.52, respectively, with accuracy ranges between 96 and 99% and limits of detection (LOD) and quantification (LOQ) of 0.122 and 0.412μg/mL. The in vitro recovery of the probes was found to be over 80%. As per microdialysis sample analysis, the Cx43MP concentration was found to increase slowly in the vitreous body up to 16h and thereafter declined. After 48h, the Cx43MP concentration was higher in vitreous, cornea, and retina compared to lens, iris, and aqueous humor. This ex vivo model may therefore be a useful tool to investigate intravitreal kinetics and ocular disposition of therapeutic molecules after intravitreal injection.