Purpose: Hydrogen sulfide (H2S) has been recognized as the third endogenously produced gasotransmitter with a plethora of pharmacological effects such as cardio-protection, neuro-protection, and anti-inflammatory role. It must be delivered in vivo at a controlled rate capable of sustaining a low level to avoid any toxic effects due to its reported narrow therapeutic index – generally 20-100 micromolar range, for an extended period of time ideally up to one month. In vitro release study is a critical step in the development of any delivery system before it can be investigated upon in vivo administration. H2S is a reducing agent which solubility in aqueous releasing media is dependent on its temperature. Temperature has a significant effect on H2S solubility in water. The solubility of H2S at 4°C is almost two-fold larger compared to the solubility at normal room temperature Thus, the amount of H2S determined in releasing samples would be negatively impacted due to dissolved oxygen in the releasing media and its temperature. Therefore, this study investigated the influence of temperature during formulation development and influence of dissolved oxygen during in vitro release study.
Methods: - Standard stock solution of sodium hydrogen sulfide (NaSH) [2.5 mg/100 mL of simulated tear fluid (STF)] was divided into two halves; one was introduced to 0.045g toluene/100 mL and the other without toluene (control). Each of the two solutions was mixed with diamine reagent (20 µL/mL) and kept for 30-45 minutes for the development of ethylene blue complex. The diamine reagent was prepared by adding (10 µM) N,N-diethyl-p-phenylenediamine sulfate in acidic ferric chloride solution (15 mM in 4.2 M HCl) in water. The amount of H2S was calculated by measuring the absorbance due to ethylene blue complex at 671 nm. Lactide-co-glycolide-based polymer (Resomer 502S, intrinsic viscosity 0.22 dl/g) was dissolved in a mixture of benzylbenzoate (BB) and benzylalcohol (BA) (70:30) by oscillating at 37ºC for 24 hrs. Sodium hydrogen sulfide, NaHS, (4.8 mg) was incorporated in 1 mL polymer solution (5-25% w/v) by sonication using Fischer Scientific FS30D for 20 min. When polymer-NaHS solution was injected in 10 mL of releasing media (simulated tear fluid, pH 7.4) maintained at 4ºC, it instantaneously formed a gel depot. As soon as NaHS would be released from the in situ formed gel, it would generate hydrogen sulfide (H2S) instantaneously due to hydrolysis. H2S would be entrapped into the releasing media by being soluble in water up to 176 mM at 4ºC. Samples (100 µL) were withdrawn from the releasing media at specific time points and analyzed for H2S content.
Results: The amount of H2S released in deoxygenated media was 1.4 times more than in non-deoxygenated media (Fig. 1). Similarly, the releasing media having NaSH (445.92 µM) and toluene (4.88 µM) showed decrease in H2S concentration through 5 days by only 2.89 +0.21 µM/day which was 30.31+.31 µM/day in absence of toluene (Fig. 2). Moreover, the amount of H2S decreased at faster rate from 5th day through 8th (47.42+0.39 µM/day). An increase in temperature was seen with increasing sonication energies (5-20 watts) as well as with increasing sonication time from 0 through 120 seconds when the temperature rose up to 90oC. However, when similar sonication profiles were followed using ice bath (4oC) the increase in temperature was only up to 60oC. The cumulative amount of H2S released decreased significantly (p<0.05) (Fig. 3) from 48% to 18% for all time points (24, 48, and 72 hrs) studied with increase in sonication energies.
Conclusion: The amount of H2S determined in released samples are significantly (p<0.05) influenced by the presence of oxygen and temperature build up due to sonication energy used during preparation of the delivery system. Therefore, such studies should be carried out at lower temperature such as 4oC and deoxygenated releasing media containing lipophilic solvent such as toluene should be used. However, the amount of toluene needed to protect H2S from oxidative degradation be optimized in future studies for a delivery system for extended period of use. The amount of toluene can be further manipulated easily as the amount used in this study is less than its saturated solubility.
Acknowledgement: The contribution of graduate students (Hatim Ali, Anita Giri, Akash Patil, and Richa Verma) for carrying out the experiments and financial support to them from School of Pharmacy and Health Professions at Creighton University are acknowledged thankfully.
Alekha Dash– Professor, Creighton University, Omaha, Nebraska