(T1030-05-27) Transferrin Conjugated Liposomes with Rho-Kinase Inhibitor for Treating Fuchs Endothelial Corneal Dystrophy

Purpose: Fuchs endothelial corneal dystrophy (FECD) is a polygenic disorder characterized by thickening and softening of Descemet membrane (DM), guttae formation (extracellular collagenous deposits in a mound shape in the posterior cornea), loss of corneal endothelial cells (CECs), and edema of the corneal stromal that result in vision loss. Currently, surgical procedures are the only definitive treatment for FECD, and there is no prescribed medication to slow or halt FECD disease progression to prevent surgery. Ripasudil, a rho-kinase inhibitor, is gaining attention as an adjunctive therapy for FECD with multiple clinical trials underway. However, currently available topical 0.4% ripasudil solution (as Glanatec®) by Kowa Pharmaceuticals are administered frequently (typically 3 to 6 times daily) and side effects are common, including conjunctival hyperemia, subconjunctival hemorrhage, blepharitis, and instillation-site pain, which limits the effectiveness of this therapeutic. We aimed to develop a liposomal drop formulation containing ripasudil and conjugated with transferrin (TF) recombinant protein on the particle surface to improve CEC uptake, reduce ripasudil side effects, and ultimately test the effectiveness of this drug in slowing FECD progression. TF was chosen as a ligand because it targets transferrin receptors, which are upregulated in FECD patients.

Methods: Liposomes were synthesized using lipids and DSPE-PEG(2000)COOH with microfluidics. DSPE-PEG(2000)COOH was conjugated to TF by EDC-NHS chemistry. Ripasudil was loaded into liposomes via active loading. Liposomes were characterized by measuring size and charge using Zetasizer. Drug loading and encapsulation efficiency were quantified using HPLC-DAD. Formulation stability was examined by evaluating particle size, polydispersity index (PDI), zeta potential and leakage study for 3 months at 4°C and 25°C. Other formulation characterizations including cryo-EM imaging, XRD, and DSC were performed as well. The ability of ripasudil loaded TF liposomes to penetrate mouse corneas and produce controlled drug release was evaluated by LC-MS/MS. TF ligand effect on liposome uptake was studied. Safety and efficacy of ripasudil TF liposomes, sham TF liposomes, and commercial ripasudil solution were compared by clinical examination in mice with a well-characterized FECD mutation (129S6/SvEvTac Col8a2Q455K). Optical coherence tomography (OCT) and confocal imaging were performed to study progression of guttae and changes in CEC morphology. An initial baseline for these techniques on a mouse cohort was recorded at the age of 4 months, then subsequently after 1 month of therapy (age of 5 months) and after 2 months of therapy (age of 6 months). This was followed by followed by postmortem RNA-seq analysis using ∆∆Ct method for ZO-1, N-Cadherin, and Na+/K+ ATPase.

Results: Liposomal particle size was determined to be 96.69±1.69 nm with a narrow PDI of 0.14±0.01 (n=4). Ripasudil TF liposomes were stable for 3 months at 4°C and at 25°C with no significant difference in PDI, charge, size, or drug leakage. Cryo-EM imaging, XRD, and DSC showed that the drug was well incorporated in single vesicular bilayered liposomes. For liposomal uptake in mouse studies at 5-, 10- and 30-min time points after drug administration, ripasudil TF liposomes achieved a longer drug residence compared to commercial solution, which rapidly declined over time. TF ligand effect on liposome targeting was also studied in the mouse model, and TF conjugation increased liposomal targeting by almost 2-fold compared to non-conjugated liposomes. Liposomal therapy had a good safety profile and low off-target side effects in this mouse model. RNA-seq data showed elevated levels of ZO-1, N-Cadherin, and Na+/K+ ATPase in mouse groups receiving ripasudil TF liposomes.

Conclusion: Findings indicate the successful development of ripasudil loaded TF conjugated liposomes as a targeted drug delivery strategy for CECs. This formulation increases drug residence time in CECs and may potentially reduce conventional dosing frequency. Favorable preliminary data that FECD disease phenotypes can be modulated by ripasudil loaded TF conjugated liposomes indicate the need for further testing.

References: Saha S, Skeie JM, Schmidt GA, et al. TCF4 trinucleotide repeat expansions and UV irradiation are associated with ferroptosis susceptibility in Fuchs endothelial corneal dystrophy. bioRxiv (Cold Spring Harbor Laboratory). Published online June 28, 2022.

Acknowledgements:

Funding: NEI R21 EY034198-01


Figure1: Cryo-EM images showing ripasudil loaded in liposomes (right) versus empty sham liposomes (left).


Fig 2: Stability studies of ripasudil liposomes. (A) Size of sham and ripasudil loaded liposomes during storage at 4 and 25°C. (B) Polydispersity index (PDI) of sham and ripasudil loaded liposomes during storage at 4 and 25°C. (C) Zeta potential of sham and ripasudil loaded liposomes during storage at 4 and 25°C. (D) Drug leakage study of ripasudil loaded liposomes at 4 and 25°C.


Fig 3: Ripasudil extracted from mouse corneal endothelium (each data point represents drug pooled from n=4 endothelial peels). A. Drug uptake from Ripasudil liposomes. B. Drug uptake from conventional solution. C. Controlled drug release and prolonged drug residence from liposomes as compared to solution.