(M1230-11-76) Topical Ocular Administration of Hydrogen Sulfide Donor: Assessment of Permeability through Isolated Bovine Cornea Ex Vivo

Purpose: Hydrogen sulfide (H₂S), an endogenous gasotransmitter, is reported to have therapeutic potential in the superior management of glaucoma in comparison to the current therapeutics in clinical practice (Mhatre et al., J Control Release, 347:256-269, 2022). Although the topical route is the most convenient mode for ocular administration of drugs, the cornea poses a unique anatomical and physiological barrier to achieving desired bioavailability in ocular tissues. Moreover, permeation of H₂S and its donors through the cornea, a critical component for clinical translation of this gasotransmitter, has not been reported in literature. Therefore, in this study, we investigated the H₂S release following permeation of sodium sulfide (Na₂S.9H₂O) through isolated bovine cornea ex vivo using the Franz diffusion chamber apparatus.

Methods: Franz diffusion apparatus (PermeGear, PA, USA) was set up, with a nominal volume of the receiver compartment of 5 mL under magnetic stirrer and a diffusion area of 0.38 cm². Freshly isolated bovine corneas, pre-incubated to 37°C in Krebs buffer, were sandwiched between donor and receiver compartments using a clamp. One mL sodium sulfide (Na₂S.9H₂O) (0.1%, 0.3%, 1%, 2%, 3% and 5% w/v) solution was added to the donor compartment covered with parafilm to minimize evaporation of donor sample. Each concentration was assessed in triplicates, with the jacket temperature set at 37°C. Samples were evaluated at different time points from 10 minutes until maximum H₂S release was observed. 100 µL of sample was withdrawn from receiver compartment and the amount of H₂S was determined potentiometrically using ISO-H₂S-100 ion-selective electrode and TBR4100 Free Radical Analyzer [World Precision Instruments (WPI) Sarasota, FL, USA]. The experimental data generated was processed using WPI’s LabTrax 4-Channel Data Acquisition device. The amount of sample withdrawn was replaced with freshly prepared releasing medium warmed at 37°C.

Results: The maximum H₂S content determined due to permeation of Na₂S.9H₂O (0.1%, 0.3%, 1%, 2%, 3%, and 5%) was 7.40±0.32, 16.45±0.95, 99.74±9.63, 148.95±18.31, 212.29±14.51, and 299.47±22.89 nmoles, respectively (Fig 1). Interestingly, the amount of H₂S determined in the receiver sample was proportional to the concentration of sulfide solution used in the donor compartment (Fig 2). Moreover, the time for maximum release of H₂S was inversely dependant on the concentration of Na₂S.9H₂O solution, with higher concentrations achieving maximum levels at earlier time points (12h for 0.1% and (12h for 0.1% and 0.3%, 8h for 1%, 7h for 2% and 3%, 6h for 5%). The maximum H₂S released as compared to the Na₂S.9H₂O added to donor compartment was 6-12% (Table 1).

Conclusion: We conclude that a dose and time dependent release of H₂S was observed for all concentrations of Na₂S.9H₂O solutions permeated through isolated bovine cornea.

References: Mhatre, Susmit, Catherine A. Opere, and Somnath Singh. "Unmet needs in glaucoma therapy: The potential role of hydrogen sulfide and its delivery strategies." Journal of Controlled Release 347 (2022): 256-269.

Acknowledgements: Financial support from School of Pharmacy and Health Professions, Creighton University;

Funding: This study was funded by NIH NEI 1R15EY032275-01A1; Conflict of Interest Statements: We confirm that there are no conflicts of interest associated with this work and there has been no significant financial support for this work that could have influenced its outcome.

Figure 1 Time dependent H2S determination following permeation of Na2S solution (0.1-5% w/v) through isolated bovine cornea using Franz Diffusion cell.

Figure 2 Comparison of concentration dependent maximum H2S determined following permeation of Na2S solution (0.1-5% w/v) through isolated bovine cornea using Franz Diffusion cell.

Table 1 Summary table of the time and concentration dependent maximum % release of H2S from Na2S solution (0.1-5% w/v) through isolated bovine cornea using Franz Diffusion cell