Complete preclinical platform for intravitreal chemotherapy drug discovery for retinoblastoma: Assessment of pharmacokinetics, toxicity and efficacy using a rabbit model

Current melphalan-based intravitreal chemotherapy regimens for retinoblastoma vitreous seeds are effective, but cause significant ocular toxicity. We describe protocols for each step of a drug discovery pipeline for preclinical development of novel drugs to maximize efficacy and minimize toxicity. These protocols include: 1) determination of vitreous pharmacokinetics in vivo, 2) in vitro assessment of drug cytotoxicity against retinoblastoma based on empiric pharmacokinetics, 3) back-calculation of minimum injection dose to achieve therapeutic concentrations, 4) in vivo determination of maximum-tolerable intravitreal dose, using a multimodal, structural and functional toxicity-assessment platform, and 5) in vivo • Multi-step pipeline for intravitreal chemotherapy drug discovery for retinoblastoma, using novel rabbit models. • Detailed protocols for determination of vitreous pharmacokinetics, calculation of optimal dose to inject to achieve therapeutic vitreous levels, determination of maximum tolerable dose using a novel complete toxicity-assessment platform, and in vivo efficacy against retinoblastoma using methodology to directly quantify vitreous tumor burden. • Associated statistical methodology is also presented.