(T1530-04-22) A Novel Hybrid Gasotransmitter for an Unmet Need of Glaucoma Therapy: A Proof-of-Concept Study

Purpose: Glaucoma is a progressive neurodegenerative disease that ultimately causes vision loss and blindness due to loss of retinal ganglionic cells (RGC), irreversibly. An estimated 79.6 million individuals were reported with glaucoma in 2020 and this number is likely to increase to 111.8 million individuals by 2040.¹ Although increase in intraocular pressure (IOP) is the most identifiable and clinically targeted risk factor for glaucoma, it is not a diagnostic tool because it can occur in normotensive individuals too. Current glaucoma therapies mainly focus on lowering IOP and do not mitigate the accompanying RGC degeneration that ultimately leads to irreversible blindness. Therefore, there is an unmet need to develop a novel therapy that can simultaneously reduce elevated IOP in the anterior segment and confer neuroprotection and regeneration of optic nerves in the posterior segment of the eye.². Some gaseous molecules with signal transduction functions, called gasotransmitters, such as nitric oxide (NO) and hydrogen sulfide (H₂S), are reported to have potential applications in glaucoma. NO relaxes trabecular meshwork and Schlemm’s canal cells by activating cyclic GMP pathway³ and also protects neurons from degeneration.⁴ Whereas, hydrogen sulfide (H₂S) reduces IOP by increasing aqueous humor outflow⁵ and protects RGCs from degeneration.⁶ Therefore, the purpose of this study was to synthesize a novel hybrid molecule, AMP249, capable of simultaneously providing NO and H₂S for a superior treatment of glaucoma to meet an unmet need in glaucoma therapy.

Methods: AMP249 was designed as trifunctional hybrid compound (TFHC) where a commercially available FDA approved drug (CFAD) is conjugated to NO and H₂S releasing moieties with an appropriate spacer between them as shown in Fig 1. TFHC was successfully synthesized and characterized for purity by NMR, HPLC/LC-MS. A 10% stock solution of AMP249 was prepared in dimethyl sulfoxide (DMSO) which was further diluted with DI water to 0.1%, 0.5%, 1%, 2%, 3%, 4%, and 5% and release of H₂S and NO was determined at specified times up to 8 hrs using WPI ion-selective microelectrode and free radical analyzer in absence or presence of glutathione (GSH). A single dose of AMP249 or timolol (positive control) was administered topically into one eye of five conscious rabbits and IOP was measured at baseline and at 0.5 h to 1 h intervals for 8 h using a calibrated pneumotonometer.

Results: AMP249 was purified using flash chromatography with overall yield of 17 %. Its structure was confirmed by pNMR, C¹³NMR, LCMS. with a purity of >95%.The optimal release profile was obtained with 2% AMP249 which provided H₂S in the range of 0.18-0.15 nmoles up to 8 hrs and NO in the range of 26-15 pmoles during the same duration. However, the release of H₂S and NO got increased by 50 and 10 times, respectively in presence of GSH. (Fig. 2A). Similarly, AMP249 (0.01% & 0.1%) elicited a maximum reduction of IOP of 19.45 ± 3.45% (p < 0.01; n=5) and 20.3 ± 3.71% (p < 0.001; n=5) in 3 and 4 h, with a duration of action of 4.5 and 8 h, respectively (Fig 2B).

Conclusion: AMP249 has the potential to provide H₂S and NO in therapeutically relevant amount for glaucoma which can be further manipulated by synthesizing its analogs.

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

Funding: This study was funded by Wareham Research Fund Award and 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.


Fig. 1. Representative structure of AMP249 (*due to proprietary reason, accurate structure of AMP249 is not disclosed).


Fig. 2. In vitro and in vivo effects of AMP249: A) In vitro release of H2S and NO from AMP249 (2%w/v) with/without GSH (3% w/v). H2S is measured in nMoles and NO in pMoles. Results are expressed as mean ± SD (n=3) B) In vivo regulation of IOP by AMP249 with/without GSH in male, normotensive New Zealand Albino rabbits; results are expressed as mean (% difference in IOP from baseline) ± SEM. *p < 0.5, **p < 0.01, ***p < 0.001 significantly different from IOP (t=0). n = 5 animals per treatment group.