Abstract
Purpose :
Methotrexate (MTX) is a methylated folic acid analogue frequently applied in chemotherapy and the treatment of autoimmune disorders. In our recent work published in 2020, we reintroduced MTX as a potential therapeutic candidate for RHO-associated autosomal dominant retinitis pigmentosa (adRP) which selectively clears misfolded P23H rhodopsin mutant protein via lysosomal degradation. Excitingly, intravitreal injection of MTX showed retinal protective effects in the RhoP23H/+ knock-in mice and a phase I clinical trial in adRP patients carrying RHO mutations. However, as frequent intravitreal injections can be challenging for the development of a preventive treatment of RP, the need to understand MTX retinal bioavailability via systemic administration arises. In this work, we aim to evaluate the ocular accumulation and clearance of MTX following both bolus and routine administration to murine models, and to identify MTX’s mediated uptake pathways into retina.
Methods :
Following intravenous tail injections to both WT and 3 to 4-month-old RhoP23H/+ mice, tissue collection and homogenization, a series of MTX doses (1, 3, 5, 10 mg/kg) and time points (5, 15, 20, 30, 45, 60, 120, 180, 240 min) were evaluated in isolated retina, eye, liver, and plasma by analytical high performance liquid chromatography (HPLC).
Following one-week daily dosage of MTX, WT and adult RhoP23H/+ mice were analyzed by electroretinogram, histology and immunostaining for the evaluation of ocular safety and efficacy in preserving retinal morphology and function.
Results :
We successfully developed a reverse-phase HPLC method to quantify MTX in a variety of tissues.
After the intravenous administration at low doses, we detected MTX levels in the liver, plasma, whole eye, and the neural retina from both wild-type (WT) and RhoP23H/+ mice, which was followed by a fast clearance. The peak retinal MTX level in vivo reaches close to the efficacious dose determined in vitro. rhodopsin mutant.
Conclusions :
MTX is retinal bioavailable in both WT and the RhoP23H/+ mice. The improved MTX permeability in the RhoP23H/+ mouse retinae suggests the compromised blood-ocular barrier in degenerative retinae. Our study brings insights on the consideration of the potential of systemic treatment of retinal degenerative diseases.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.