Abstract
A large population is suffering from ocular diseases within US and worldwide. Glaucoma is the most common age-related optic nerve disease and the most common neuropathy. The current treatment regimen for management of glaucoma is targeted towards lowering IOP alone with little or no concern towards optic nerve rescue and regeneration. Hydrogen sulfide is a gaseous molecule which has shown neuroprotectant action in humans and other mammals. In a recent study a hydrogen sulfide donor in low concentrations (micro molar) exhibited a time-dependent decrease in IOP indicating the role of H2S in regulation of aqueous humor dynamics. Subconjunctival delivery can be the desired route since it targets both the anterior and the posterior segment. Some hydrogen sulfide donors are available which can produce H2S once they are inside our body but they must do so at sustained rate to prevent any adverse effect including toxicity. However, developing a sustained release delivery system of a H2S donor is a great challenge due to instantaneous release of H2S when they come into contact with water. The best way to solve this problem was to formulate an in situ gel forming smart polymer-based delivery system that would release hydrogen sulfide donor at sustained rate for extended period of time. Therefore, in this study a novel sustained release formulation of sodium hydrogen sulfide (NaHS) (a model H2S donor) was prepared, characterized, and its release profile was evaluated by quantifying the release of H2S. To quantitate H2S, mixed diamine reagent (0.06 ml) was added to 3 ml of standard NaHS solution and the intensity of the resultant colored solution was determined by measuring absorbance at 671nm using Pharmspec UV-1700 UV-Visible Spectrophotometer (Shimadzu). This method was developed and validated. Lactide and/or glycolide-based polymers (intrinsic viscosity 0.20 dL/g) differing in end groups (carboxylic acid or ester), were dissolved in an organic solvent mixture of various ratios of benzyl benzoate (BB) and benzyl alcohol (BA). Polymer solutions were tested for injectability through 25 gauge needle. To these polymer solutions, differing in polymer type and polymer concentrations, NaHS (0.8% w/v) was incorporated to prepare 12 different formulations. The NaHS was uniformly dispersed in the polymer solution by sonicating at 10W for 1 min. All the formulations were characterized for their drug content efficiency and the homogeneity of drug in the formulation. When formulation was injected in releasing media, simulated tear fluid (STF), it instantaneously formed a gel depot. NaHS when released from the depot, instantaneously releases hydrogen sulfide (H2S) due to hydrolysis. H2S would be entrapped into the releasing media by being soluble in water up to 176 mM at 4°C. Samples were withdrawn from the releasing media at specific time points and analyzed for H2S content. Hydrogen sulfide produced ethylene blue by interacting with an acidic diamine reagent (mixture of N, N-diethyl-p-phenylenediamine sulfate (DPDS) and ferric chloride) maintained at 4°C. The intensity of ethylene blue was measured at 671 nm which was used to quantify H2S. The amount of H2S vs. time was plotted to study the release profile. In vitro polymer degradation was determined by adding one ml of polymeric solution into 15 ml of STF kept in a capped scintillation vials. The vials were placed in a refrigerator at a temperature of 4°C. At specific intervals of time, 1 ml aliquots of the release media were withdrawn at specific time intervals and replaced with equal volume of fresh STF solution. The 1 ml aliquot was analyzed using FTIR to determine the in vitro polymer degradation. Polymers bearing carboxylic acid end group were not soluble in 100% BB which were well correlated with hydrophilic-lipophilic profiles of polymers and the solvent systems. Polymer concentrations up to 20% (w/w) were found injectable through 25 gauze needle. The drug content efficiency for all the formulations was around 80%. The relative standard deviation was less than 6.7% demonstrating uniform dispersion of drug throughout the formulation. The release of NaHS from a solution of NaHS in water showed an instantaneous release while the release of NaHS from the polymeric formulations lasted for four days demonstrating a sustained release. Release studies indicated a lower burst release (< 15%) from formulations containing polymer with carboxylic acid end group than those containing ester end group (burst release > 21%). Polymers with acid end groups showed a faster rate of release (total release period was 72 hrs.) than those with ester end groups (total release period was 96 hrs.). The amount of burst release and over all release profiles were explained on the basis of influence of end groups on the rate of gelation of the phase-sensitive smart polymer-based delivery systems. The polymers with free carboxylic end groups showed faster rate of gelation as compared to the polymer with ester end groups. The release kinetic was better fitted to Higuchi model in comparison to zero or first order rate kinetics. Therefore, it appears that the release of NaHS was predominantly controlled by diffusion which was corroborated by absence of any significant polymer degradation as well as not significant (p<0.05) decrease in rate of release with increase in concentration. It is concluded that phase-sensitive smart polymer- based system can deliver H2S donor at a sustained rate up to 96 hrs. which can be further extended by manipulating polymers, their composition, and solvent system. Therefore, any future study should investigate manipulation of various formulation parameters to further extend the period of sustained release, effect of using releasing media better mimicking ocular environment at subconjunctival level than STF used in this study, and its broader applicability for delivering other clinically relevant H2S donors differing widely in their physicochemical properties.