(T1230-03-19) Comparative Evaluation of the Efficacy of Two Novel Trifunctional Compounds for Glaucoma

Purpose: Glaucoma refers to an array of ocular disorders that inflict damage on the retinal ganglion cells (RGCs) and the optic nerve. The optic nerve is crucial for relaying visual information from the eye to the brain, and its injury has the potential to result in vision impairment or complete blindness if not addressed appropriately.^1,2,3 Optic neuropathy is often linked to elevated intraocular pressure (IOP), but it can also occur when IOP is within normal limits, indicating that IOP measurement alone is not a reliable diagnostic parameter for the ocular disease.^1,4 As of 2020, glaucoma affected more than 80 million people worldwide, and the number is projected to rise to nearly 111 million by 2040, reflecting the growing global burden of this vision-threatening disease.⁵ In the United States, approximately 4.22 million adults (representing 1.62% of individuals aged 18 years and older) are affected by glaucoma. Among them, an estimated 1.49 million experience glaucoma-related visual impairment. The prevalence of the disease increases significantly with age, ranging from 2.56% to 4% in individuals over the age of 40.⁷ Current treatment strategies aimed at reducing elevated IOP fail to address the concurrent degeneration of RGCs in the posterior segment of the eye, which ultimately results in irreversible blindness.⁸ This highlights the need for the  development of more comprehensive therapeutic approach. Hydrogen sulfide (H₂S) and nitric oxide (NO) gasotransmitters involved in signal transduction processes have demonstrated efficacy in addressing glaucomatous deficits. Both H₂S and NO induce relaxation of the trabecular meshwork and Schlemm's canal, to enhance aqueous humor outflow through the conventional pathway.^9,10 Additionally, H₂S exerts retinal neuroprotection from multiple stimuli, including oxidative and inflammatory insults.¹¹ Effects of H₂S and NO in the context of glaucoma have been studied individually or in combination with latanoprost. However, their simultaneous actions in the glaucoma have not yet been thoroughly explored. This study aimed to evaluate and compare two novel hybrid drug-like molecules, AMP and JDB, designed to deliver NO and H₂S concurrently, with the goal of enhancing IOP lowering effect and providing neuroprotection.

Methods: A 10% stock solution of AMP and JDB were prepared in DMSO and used for further dilutions using DI water to 1%, 2%, 3%, 4%, and 5%, and stored at room temperature. GSH (2% w/v) was added to these diluted AMP/JDB solutions to investigate its influence on release profiles of H₂S and NO. Twenty five µL sample were withdrawn at specific time points from the diluted solution and added to a beaker containing 25 mL PBS. An ion selective electrode (WPI H₂S biosensor ISO-100-H₂S / WPI NO biosensor ISO-NOPF200) connected with free radical analyzers (WPI Sarasota, FL, USA) were dipped into PBS for quantification of H₂S/NO. The amount of sample withdrawn from the diluted AMP/JDB solution was replaced with DI water every time. The amount of H₂S/NO determined in the 25 µL samples were corrected for sample withdrawal using Hayton and Chen equation.¹² For animal studies, baseline IOP was determined using a calibrated pneumotonometer, prior to application of the test compounds. One drop of either AMP, JDB or timolol was administered topically into one eye of five NZA rabbits and IOP was measured at 0.5-hour, 1 hour, 2, hours, and then at 1-hour intervals until baseline readings were attained.

Results: AMP (2%) generated approximately 0.18 to 0.15 nmoles of H₂S and 26 to 15 nmoles of NO over a period of 8 hours, in vitro. The presence of GSH induced a significant (p < 0.0001) improvement in the release of the gasotransmitters, marked by a 50-fold increase in H₂S and a 10-fold rise in NO levels (Fig. 1A). JDB (2%) released H₂S and NO for up to 7 days, in vitro. The release of H₂S ranged from 1.51 to 33.14 nmoles, showing moderate fluctuations with a slight peak observed between 100 and 125 hours [Days 4 to 5]. The release of NO ranged from 9.47 to 1440.42 × 10⁻³ nmoles, indicating a steady and systematic increase, with the highest release observed between 150 and 175 hours [Day 6-7] (Fig 1B). In in vivo studies, AMP (0.1%) reduced IOP in a concentration-dependent manner, achieving a maximum reduction of 20.3±3.71% (p < 0.001; n=5) after 4 hours, with a duration of action of 8 hours. However, in the presence of GSH (0.2%) enhanced the IOP-lowering effect of AMP (0.1%) to 30.62±4.90% (p < 0.001; n=5) after 6 hours and same duration of action (8 hours). JDB (0.1%) produced a maximal IOP-lowering effect of 26.56+-4.04% (p < 0.001; n=5) at 2 hours with the duration of 9 hours. However, the presence of GSH (2%) sustained the IOP (26.53+-7.71%, p< 0.0001; n=5) with peak appearing at 6 hours and reaching baseline at 9.5 hours.

Conclusion: AMP and JDB exhibit effective IOP-lowering properties, with JDB offering sustained IOP lowering effect and dual gasotransmitter release.

References: 1. Glaucoma - Symptoms and causes. (n.d.). Mayo Clinic. https://www.mayoclinic.org/diseases-conditions/glaucoma/symptoms-causes/syc-20372839 (Assessed on July 19, 2025)
2. Glaucoma. (2025, July 14). Cleveland Clinic. https://my.clevelandclinic.org/health/diseases/4212-glaucoma (Assessed on July 19, 2025)
3. McKinney, J. K., MD. (2024, October 29). Understanding glaucoma: Symptoms, causes, diagnosis, treatment. American Academy of Ophthalmology. https://www.aao.org/eye-health/diseases/what-is-glaucoma (Assessed on July 19, 2025)
4. BrightFocus Foundation. (2025, April 16). What is Glaucoma? | BrightFocus Foundation. https://www.brightfocus.org/glaucoma/ (Assessed on July 19, 2025)
5. Yeager, A. (2025, April 8). New research sheds light on glaucoma’s progression. OHSU News. Retrieved July 19, 2025, from https://news.ohsu.edu/2025/04/08/new-research-sheds-light-on-glaucomas-progression (Assessed on July 19, 2025)
6. kwest@preventblindness.org. (2024, October 17). New study finds higher prevalence of glaucoma than previously estimated - Prevent blindness. Prevent Blindness. https://preventblindness.org/new-glaucoma-prevalence-study/ (Assessed on July 19, 2025)
7. 2025 Glaucoma Report. (2025, June 16). Eyes on Eyecare. https://eyesoneyecare.com/resources/2025-glaucoma-report/ (Assessed on July 19, 2025)
8. Mhatre S, Opere CA, Singh S. Unmet needs in glaucoma therapy: The potential role of hydrogen sulfide and its delivery strategies. J Control Release. 2022 Jul;347:256-269. doi: 10.1016/j.jconrel.2022.05.001. Epub 2022 May 13. PMID: 35526614.
9. Han B, Song M, Li L, Sun X, Lei Y. The Application of Nitric Oxide for Ocular Hypertension Treatment. Molecules. 2021 Dec 1;26(23):7306. doi: 10.3390/molecules26237306. PMID: 34885889; PMCID: PMC8659272.
10. Elena Perrino, Caterina Uliva, Cecilia Lanzi, Piero Del Soldato, Emanuela Masini, Anna Sparatore, New prostaglandin derivative for glaucoma treatment, Bioorganic & Medicinal Chemistry Letters, Volume 19, Issue 6, 2009, Pages 1639-1642, ISSN 0960-894X, https://doi.org/10.1016/j.bmcl.2009.02.007.
11. Shouyue Huang, Ping Huang, Zhongjing Lin, Xiaohong Liu, Xing Xu, Lei Guo, Xi Shen, Changwei Li, Yisheng Zhong, Hydrogen sulfide supplement attenuates the apoptosis of retinal ganglion cells in experimental glaucoma, Experimental Eye Research, Volume 168, 2018, Pages 33-48, ISSN 0014-4835, https://doi.org/10.1016/j.exer.2018.01.004.
12. Hayton WL, Chen T. Correction of perfusate concentration for sample removal. J Pharm Sci. 1982 Jul;71(7):820-1. doi: 10.1002/jps.2600710726. PMID: 7120072.

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. Disclosure: The rabbits used in this study were housed and treated as per USDA Laboratory Animals Guidelines under the continuous supervision of Creighton University Veterinarian. The study-specific procedures were approved by IACAUC committee, Creighton University via protocol # 1190.

Figure 1. In vitro release of H2S and NO from 2% AMP (A) and 2% JDB (B). Results are expressed as mean ± SD (n=3).

Figure 2. A. Effect of AMP+GSH, Timolol, saline, and AMP alone on intraocular pressure (IOP) in normotensive male New Zealand Albino rabbits in vivo. B. Effect of JDB alone, JDB+GSH and saline on intraocular pressure (IOP) in normotensive male New Zealand Albino rabbits in vivo. Vertical bars represent Mean ± SEM of data obtained from five rabbits. * p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, *****p < 0.00001 significantly different from baseline IOP.