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

Abstract

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 determination of drug efficacy using a rabbit orthotopic xenograft model of retinoblastoma vitreous seeds. We likewise describe our methodology for direct quantitation of vitreous seeds, and the statistical methodology for assessment of toxicity and efficacy in evaluating novel drugs, as well as for comparisons between drugs. 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.

Aqueous VEGF-A Levels as a Liquid Biopsy Biomarker of Retinoblastoma Vitreous Seed Response to Therapy

“…New Zealand White Rabbits (2.8–3.0 kg) were used for all animal experiments. Retinoblastoma VSs were generated by injection of 10 6 WERI-Rb1 cells in 100 µL saline into the vitreous of both eyes of cyclosporine-immunosuppressed rabbits under isoflurane anesthesia, as we have described previously, 22 and allowed to grow for 2 weeks to form VSs, using a model that we have previously validated. 23 – 27 …”

Evaluation of intravitreal topotecan dose levels, toxicity and efficacy for retinoblastoma vitreous seeds: a preclinical and clinical study

“…Use of a valved cannula-maintained eye stability and prevented efflux of vitreous contents during manipulations. 25 Vitreous samples were immediately placed on dry ice and then stored at −80°C until drug levels were measured.…”

“…Using the pharmacokinetic parameters determined above, we calculated 25 the dose of topotecan that would need to be injected into the eye to achieve the IC 90 in the vitreous on the opposite side of the eye for a duration of five half-lives.…”

“…RB vitreous seeds were created by intravitreal injection of 1 000 000 WERI-Rb1 cells in 100 µL saline in both eyes of cyclosporine-immunosuppressed rabbits, as we have described previously. 23 25 After 2 weeks of growth, the right eyes received three weekly injections of either 15 µg/100 µL topotecan or 12.5 µg/100 µL melphalan, while all left eyes received 100 µL saline.…”

“…Toxicity was defined for every ERG parameter, using our previously published definition. 11 25 Briefly, toxicity was deemed significant for a given dose in a rabbit group if there was a 25% reduction in average ERG amplitude, or a 25% prolongation of average implicit time comparing the post-treatment parameter values after three injections with the pretreatment values, if the difference was statistically significant.…”