(M1430-07-42) Ripasudil Liposomes for the Treatment of a Polygenic Corneal Disease

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 it with transferrin (TF) recombinant protein on the particle surface to improve CEC uptake and reduce ripasudil side effects, ultimately testing 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 in-vitro release studies, cryo-EM imaging, XRD, and DSC were performed as well. The ability of ripasudil loaded TF liposome’s 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 corneal confocal microscopy (CCM) 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 94.45±3.06 nm with a narrow PDI of 0.14±0.03 (n=3) (Mean ± SD). 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. Corneal confocal microscopy revealed significantly lower numbers of guttae for ripasudil liposome treated group (n=16 each group) compared to their respective controls (n=8 each group, P< 0.0001); RNA-seq data showed elevated levels of ZO-1, N-Cadherin, and Na+/K+ ATPase in mouse groups receiving ripasudil TF liposomes.

Conclusion: Ripasudil liposomes formulated for increased uptake in FECD CECs did not cause toxicity. This formulation prevented guttae formation overall cell morphology. Results of this preclinical mouse model drug safety and efficacy trial support the advancement of ripasudil liposomal formulations as a therapeutic modality to prevent FECD progression.

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: Liposome characterization data. (A)Cryo-EM images showing ripasudil loaded in liposomes (right) versus empty sham liposomes (left). Stability studies of ripasudil liposomes. (B) Size of sham and ripasudil loaded liposomes during storage at 4 and 25°C. (C) Polydispersity index (PDI) of sham and ripasudil loaded liposomes during storage at 4 and 25°C. (D) Zeta potential of sham and ripasudil loaded liposomes during storage at 4 and 25°C. (E) Drug leakage study of ripasudil loaded liposomes at 4 and 25°C. LC-MS/MS quantification of ripasudil extracted from mouse corneal endothelium. (F) Drug uptake from Ripasudil liposomes. (G) Drug uptake from conventional solution. (H) Controlled drug release and prolonged drug residence from liposomes as compared to solution.


Figure2: Safety and efficacy study data post 2-month ripasudil liposomal therapy. (A) Blood chemistry profile indicating no formulation side-effects (B) Representative CCM images exhibiting improvement in cell morphology and decrease in guttae (C) CCM quantification data across all groups. Error bar represents SEM. Statistical analysis was done using one-way ANOVA followed by Tukey's post-hoc test. **** P < 0.0001. Statistical analysis for male and female cohorts performed using unpaired t-test followed by Welch’s correction.