(M1030-12-67) Comparative Evaluation of In Vitro Release Profile of Hydrogen Sulfide from Its Donors

Purpose: In the past two decades, the therapeutic potential of hydrogen sulfide (H₂S), one of three endogenous gasotransmitters, has been intensely investigated by several investigators.¹ H₂S has been explored for its application in conditions such as Alzheimer’s and Parkinson’s disease, glaucoma, cataract, ischemia, myocardial infarction, etc.^2,3 Since H₂S is gaseous at standard temperature and pressure, its therapeutic applications in its natural state are restricted. Therefore, H₂S donors are commonly used to produce H₂S in situ and to avoid its loss. Different classes of H₂S donors exhibit unique release profiles that ultimately determine suitability of dosage form and delivery system for pharmacological applications. The aim of this study was to compare the release profile of H₂S from the inorganic salt, sodium sulfide (Na₂S); synthetic slow H₂S-releasing compound, p-methoxyphenyl-morpholino-phosphinodithioic acid (GYY 4137); mitochondrial H₂S donor, AP39; and the polysulfides, diallyl disulfide (DADS); diallyl trisulfide (DATS); and diallyl tetrasulfide (DATTS).

Methods: Hydrogen sulfide was detected using an amperometry technique (World Precision Instruments (WPI), ISO-H₂S-100 electrode) attached to an Apollo 1100 Free Radical Analyzer (WPI) in 2% solution of Na₂S, GYY 4137, the polysulfides DADS, DATS, DATTS, and the mitochondrial H₂S donor AP39. The H₂S electrode was calibrated following vendor provided instructions to detect H₂S in the range of 2.5-10,000 pmoles/mL. Briefly, 5-80 µL solutions of 1mM Na₂S solution were added in increasing increments and the corresponding current reading was noted to get a correlation between the two quantities. The release of H₂S was monitored for up to 6 hours for all the H₂S producing compounds.

Results: Since Na₂S is a salt that is readily soluble in water, it rapidly produced an initial, burst release of 106,400 ± 3,200 nmoles/mL without any sustained release of the gas.  DATS did not show any immediate release of H₂S, until a small peak of 20 ± 4.6 nmoles/mL was observed after 5 hours. Unlike the other polysulfides, DATTS showed an initial burst release of H₂S of 417 ± 0.5 nmoles/mL in 30 minutes which gradually slowed down until after 3 hours when a smaller peak of 234 ± 9.1 nmoles/mL was observed. GYY4137 exhibited a maximum H₂S release of 86 ± 0.2 nmoles/mL immediately following its dissolution (time = 0) and sustained a slow release of H₂S up to 6 hours 38 ± 0.6 - 86 ± 0.3 nmoles/mL. Interestingly, DADS, which is reported to require oxidation to release the gas⁴ and AP39, a mitochondrial donor did not release any H₂S during the study duration of 6 hours.

Conclusion: The inorganic salt, Na₂S exhibited a single burst release of H₂S, whereas the slow H₂S-releasing synthetic compound, GYY4137 exhibited a low burst release followed by a sustained release of the gas. Moreover, DATTS exhibited a biphasic release profile where H₂S release from donors that rely on endogenous enzymes and/or chemicals to stimulate H₂S release did not generate any gas. These release profiles should be considered when formulating a H₂S based delivery system.

References: 1. 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.
2. Bos, Eelke M., et al. "Hydrogen sulfide: physiological properties and therapeutic potential in ischaemia." British journal of pharmacology 172.6 (2015): 1479-1493.
3. Szabó, Csaba. "Hydrogen sulphide and its therapeutic potential." Nature reviews Drug discovery 6.11 (2007): 917-935.
4. Benavides GA, Squadrito GL, Mills RW, et al. “Hydrogen sulfide mediates the vasoactivity of garlic.” Proc Natl Acad Sci U S A. 2007;104(46):17977-17982. doi:10.1073/pnas.0705710104

Acknowledgments: Financial support from School of Pharmacy and Health Professions, Creighton University and Summer Research Institute, Health Sciences - Multicultural and Community Affairs Department, School of Medicine, Creighton University.

Funding: This study was funded by Wareham Research Fund Award and NIH NEI 1R15EY032274-01
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.