Application of Quality by Design in the Development of Hydrogen Sulfide Donor Loaded Polymeric Microparticles.

Hydrogen sulfide (H2S) is a multifaceted gasotransmitter molecule which has potential applications in many pathological conditions including in lowering intraocular pressure and providing retinal neuroprotection. However, its unique physicochemical properties pose several challenges for developing its efficient and safe delivery method system. This study aims to overcome challenges related to H2S toxicity, gaseous nature, and narrow therapeutic concentrations range by developing polymeric microparticles to sustain the release of H2S for an extended period. Various formulation parameters and their interactions are quantitatively identified using Quality-by-Design (QbD) approach to optimize the microparticle-based H2S donor (HSD) delivery system. Microparticles were prepared using a solvent-evaporation coacervation process by using polycaprolactone (PCL), soy lecithin, dichloromethane, Na2S.9H2O, and silicone oil as polymer, surfactant, solvent, HSD, and dispersion medium, respectively. The microparticles were characterized for size, size distribution, entrapment efficiency, and H2S release profile. A Main Effects Screening (MES) and a Response Surface Design (RSD) model-based Box-Behnken Design (BBD) was developed to establish the relationship between critical process parameters (CPPs) and critical quality attributes (CQAs) qualitatively and quantitatively. The MES model identified polymer to drug ratio and dispersion medium quantity as significant CPPs among others, while the RSD model established their quantitative relationship. Finally, the target product performance was validated by comparing predicted and experimental outcomes. The QbD approach helped in achieving overall desired microparticle characteristics with fewer trials and provided a mathematical relationship between the CPPs and the CQAs useful for further manipulation and optimization of release profile up to at least 30 days.

Preparation, Characterization and Antioxidant Evaluation of Poorly Soluble Polyphenol-Loaded Nanoparticles for Cataract Treatment.

Cataract, one of the leading causes of blindness worldwide, is a condition in which complete or partial opacity develops in the lens of the eyes, thereby impairing vision. This study aimed to examine the potential therapeutic and protective effects of poorly soluble polyphenols like curcumin, resveratrol, and dibenzoylmethane, known to possess significant antioxidant activity. The polyphenols were loaded into novel lipid-cyclodextrin-based nanoparticles and characterized by particle size, polydispersity index, differential scanning calorimetry, thermogravimetric analysis, X-ray diffraction, scanning electron microscopy (SEM), entrapment efficiency, and release studies. Ferric-reducing ability of plasma and 2,2-diphenyl-1-picrylhydrazyl chemical assays were used to evaluate their antioxidant properties based on their free radical quenching ability. Biochemical in vitro assays were used to examine these polyphenols on hydrogen peroxide-induced formation of cataracts in bovine lenses by estimating total glutathione content and superoxide dismutase activity. Nanoparticles were thermostable and amorphous. Particle size of curcumin, resveratrol, and dibenzoylmethane nanoparticles were 331.0 ± 17.9 nm, 329.9 ± 1.9 nm, and 163.8 ± 3.2 nm, respectively. SEM confirmed porous morphology and XRD confirmed physical stability. Entrapment efficiency for curcumin-, resveratrol-, and dibenzoylmethane-loaded nanoparticles was calculated to be 84.4 ± 2.4%, 72.2 ± 1.5%, and 86.4 ± 0.6%, respectively. In vitro release studies showed an initial burst release followed by a continuous release of polyphenols from nanoparticles. Chemical assays confirmed the polyphenols' antioxidant activity. Superoxide dismutase and glutathione levels were found to be significantly increased (p < 0.05) after treatment with polyphenol-loaded nanoparticles than pure polyphenols; thus, an improved antioxidant activity translational into potential anticataract activity of the polyphenols when loaded into nanoparticles was observed as compared to pure polyphenols.

Comparative Effects of Hydrogen Sulfide-Releasing Compounds on [3H]D-Aspartate Release from Bovine Isolated Retinae.

We investigated the pharmacological actions of a slow-releasing H2S donor, GYY 4137; a substrate for the biosynthesis of H2S, L-cysteine and its precursor, N-acetylcysteine on potassium (K+; 50 mM)-evoked [3H]D-aspartate release from bovine isolated retinae using the Superfusion Method. GYY 4137 (10 nM-10 µM), L-cysteine (100 nM-10 µM) and N-acetylcysteine (10 µM-1 mM) elicited a concentration-dependent decrease in K+-evoked [3H]D-aspartate release from isolated bovine retinae without affecting basal tritium efflux. At equimolar concentration of 10 µM, the rank order of activity was as follows: L-cysteine > GYY 4137 > N-acetylcysteine. A dual inhibitor of the biosynthetic enzymes for H2S, cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE), amino-oxyacetic acid (AOA; 3 mM) reversed the inhibitory responses caused by GYY 4137, L-cysteine and N-acetylcysteine on K+-evoked [3H]D-aspartate release. Glibenclamide (300 µM), an inhibitor of KATP channels blocked the inhibitory action of GYY 4137 and L-cysteine but not that elicited by N-acetylcysteine on K+-induced [3H]D-aspartate release. The inhibitory effect of GYY 4137 and L-cysteine on K+-evoked [3H]D-aspartate release was reversed by the non-specific inhibitor of nitric oxide synthase (NOS), L-NAME (300 µM). Furthermore, a specific inhibitor of inducible NOS (iNOS), aminoguanidine (10 µM) blocked the inhibitory action of L-cysteine on K+-evoked [3H]D-aspartate release. We conclude that both donors and substrates for H2S production can inhibit amino acid neurotransmission in bovine isolated retinae, an effect that is dependent, at least in part, upon the intramural biosynthesis of this gas, and on the activity of KATP channels and NO synthase.

Pharmacological Actions of Hydrogen Sulfide Donors on Sympathetic Neurotransmission in the Bovine Anterior Uvea, In Vitro.

In the present study, we investigated the effect of three different sources of hydrogen sulfide (H2S) on sympathetic neurotransmission from isolated superfused bovine iris-ciliary bodies. The three agents under consideration were: ACS67, a hybrid of latanoprost and a H2S-donating moiety; L-cysteine, a substrate for endogenous production of H2S and GYY 4137, a slow donor of H2S. We also examined the contribution of prostaglandins to the pharmacological actions of the H2S donors on release of [(3)H]-norepinephrine ([(3)H]NE) triggered by electrical field stimulation. ACS67, L-cysteine and GYY 4137 caused a concentration-dependent inhibition of electrically-evoked [(3)H]NE release from isolated bovine iris-ciliary bodies without affecting basal [(3)H]NE efflux. The cyclooxygenase inhibitor, flurbiprofen enhanced the inhibitory action of ACS67 and L-cysteine on stimulated [(3)H]NE release. Both aminooxyacetic acid, an inhibitor of cystathionine-ß-synthase and glibenclamide, a KATP channel blocker reversed the inhibition of evoked NE release induced by the H2S donors. We conclude that H2S donors can inhibit sympathetic neurotransmission from isolated bovine iris-ciliary bodies, an effect partially dependent on the in situ production of H2S and prostanoids, and is mediated by an action on KATP channels.

Inhibitory action of novel hydrogen sulfide donors on bovine isolated posterior ciliary arteries.

In the present study, we investigate the inhibitory effect of novel H2S donors, AP67 and AP72 on isolated bovine posterior ciliary arteries (PCAs) under conditions of tone induced by an adrenoceptor agonist. Furthermore, we examined the possible mechanisms underlying the AP67- and AP72-induced relaxations. Isolated bovine PCA were set up for measurement of isometric tension in organ baths containing oxygenated Krebs solution. The relaxant action of H2S donors was studied on phenylephrine-induced tone in the absence or presence of enzyme inhibitors for the following pathways: cyclooxygenase (COX); H2S; nitric oxide and the ATP-sensitive K(+) (KATP) channel. The H2S donors, NaSH (1 nM - 10 µM), AP67 (1 nM - 10 µM) and AP72 (10 nM - 1 µM) elicited a concentration-dependent relaxation of phenylephrine-induced tone in isolated bovine PCA. While the COX inhibitor, flurbiprofen (3 µM) blocked significantly (p < 0.05) the inhibitory response elicited by AP67, it had no effect on relaxations induced by NaSH and AP72. Both aminooxyacetic acid (30 µM) and propargylglycine (1 mM), enzyme inhibitors of H2S biosynthesis caused significant (p < 0.05) rightward shifts in the concentration-response curve to AP67 and AP72. Furthermore, the KATP channel antagonist, glibenclamide (300 µM) and the NO synthase inhibitor, l-NAME (100 µM) significantly attenuated (p < 0.05) the relaxation effect induced by AP67 and AP72 on PCA. We conclude that H2S donors can relax pre-contracted isolated bovine PCA, an effect dependent on endogenous production of H2S. The inhibitory action of only AP67 on pre-contracted PCA may involve the production of inhibitory endogenous prostanoids. Furthermore, the observed inhibitory action of H2S donors on PCA may depend on the endogenous biosynthesis of NO and by an action of KATP channels.

Role of the non-enzymatic metabolite of eicosapentaenoic acid, 5-epi-5-F3t-isoprostane in the regulation of [ (3)H]D-aspartate release in isolated bovine retina.

We have evidence that F2-isoprostanes (F2-IsoPs) regulate the release of excitatory neurotransmitters in isolated bovine retina. Although 5-F3-IsoPs are generated in mammals, in vivo, their pharmacological actions on neurotransmitter release remain unknown. In this study, we investigated the effect of 5-epi-5-F3t-IsoP on K(+)-evoked [(3)H]D-aspartate release in isolated bovine retina using the superfusion method. Furthermore, we examined the role of arachidonic acid metabolites in the regulation of the neurotransmitter release by this novel IsoP. In the concentration range, 0.01 nM-0.1 µM, 5-epi-5-F3t-IsoP inhibited K(+)-evoked [(3)H]D-aspartate release in a concentration-dependent manner, achieving a maximum inhibition of 46.9 % at 0.1 µM (IC30 = 1 nM). The prostanoid receptor antagonists, AH 6809 (EP1-3/DP; 10 µM), SC 51322 (EP1; 10 µM) and SC 19220 (EP1; 1 µM) partially reversed 5-epi-5-F3t-IsoP-mediated inhibition of K(+)-induced [(3)H]D-aspartate release. Pretreatment of retinal tissues with the cyclooxygenase (COX) inhibitor, flurbiprofen (3 µM) unmasked a biphasic action of 5-epi-5-F3t-IsoP that was inhibitory at lower (0.1-10 pM) and stimulatory at higher concentrations (≥0.1 nM). The prostanoid pathway antagonists, BAY-u3405 (10 µM; TP/DP-receptors), SQ 29548 (10 µM; TP-receptor) and ozagrel (10 µM; Tx-synthase inhibitor) abolished the stimulatory action of the 5-epi-5-F3t-IsoP (0.1 µM) on neurotransmitter release. In conclusion, 5-epi-5-F3t-IsoP attenuates K(+)-induced [(3)H]D-aspartate release in a concentration-dependent manner by mechanisms that are partially dependent on activation of pre-junctional prostanoid EP1-receptors. Moreover, blockade of the COX-pathway unmasks a biphasic action for 5-epi-5-F3t-IsoP that is inhibitory at low concentrations and stimulatory at higher concentrations. Products of the thromboxane synthase pathway may partially account for the stimulatory action of this F3-IsoP on isolated bovine retina.

In vitro-controlled release delivery system for hydrogen sulfide donor.

Hydrogen sulfide (H2S) is having many potential pharmacological and physiological actions which reported that therapeutically useful concentration is low (100-160 µM) and a higher concentration could be toxic. Most of its donors produce it on coming into contact with water. All of these problems could be solved by a controlled-release delivery system which does not utilize water in any of its development steps. Therefore, 12 sustained release formulations were prepared by dissolving sodium hydrogen sulfide (NaHS)-a model H2S donor-in polymer solutions, prepared by dissolving polymers (consisted of either polylactide (PLA) or polylactide co-glycolide (PLGA), containing free carboxylic acid or capped allyl ester end group) in a mixture of benzyl benzoate (BB) and benzyl alcohol (BA). The formulation was injected in simulated tear fluid (STF) from which samples were withdrawn at specified times and assayed for NaHS content. We found decrease in burst and overall release with increase in polymer concentration from 10 to 20% w/v. The formulations containing free end group showed significant (p < 0.05) reduction of burst release (11% vs 21%). However, the overall release or the average amount released per hour was found to be significantly (p < 0.05) increased for formulations containing polymers with free end group than those with capped end group. A sustained level of H2S was found to be maintained for 72 h which should be further increased to a month to make it a viable H2S donor delivery system in addition to investigating toxicity profile specifically for the purpose of subconjunctival ocular delivery.

Comparison of colorimetric, spectroscopic and electrochemical techniques for quantification of hydrogen sulfide.

Background: Hydrogen sulfide (H2S), an endogenous gasotransmitter, has potential applications in several conditions. However, its quantification in simulated physiological solutions is a major challenge due to its gaseous nature and other physicochemical properties. Aim: This study was designed to compare four commonly used H2S detection and quantification methods in aqueous solutions. Methods: The four techniques compared were one colorimetric, one chromatographic and two electrochemical methods. Results: Colorimetric and chromatographic methods quantified H2S in millimolar and micromole ranges, respectively. The electrochemical methods quantified H2S in the nanomole and picomole ranges and were less time-consuming. Conclusion: The H2S quantification method should be selected based on the specific requirements of a research project in terms of sensitivity, response time and cost-effectiveness.

Pharmacological actions of the slow release hydrogen sulfide donor GYY4137 on phenylephrine-induced tone in isolated bovine ciliary artery.

Hydrogen sulfide (H2S), a colorless gas characterized by its pungent odor of rotten eggs has been reported to elicit relaxation effects on basal and pre-contracted non-ocular smooth muscles of several mammalian species. In the present study, we investigated the pharmacological actions of a H2S donor, GYY4137 on isolated bovine posterior ciliary artery after contraction with the adrenergic receptor agonist, phenylephrine. Furthermore, we studied the underlying mechanism of inhibitory action of GYY4137 on the posterior ciliary arteries. Isolated bovine posterior ciliary arteries were mounted in oxygenated organ baths and changes in isometric tension were measured with a Grass FT03 transducer connected to a recorder using a Grass Polyview Software. The relaxant actions of GYY4137 on phenylephrine pre-contracted arteries were observed in the absence and presence of an inhibitor of cyclo-oxygenase, flurbiprofen. Furthermore, the inhibitory effects of GYY4137 were studied in the absence or presence of inhibitors/activators of biosynthetic enzymes for H2S and nitric oxide production, as well as specific ion channel blockers. In the concentration range, 100 nM to 100 µM, GYY4137 elicited a concentration-dependant relaxation of phenylephrine-induced tone in isolated posterior ciliary arteries, with IC50 value of 13.4 ± 1.9 µM (n = 6). The cyclo-oxygenase inhibitor, flurbiprofen, significantly (p < 0.01) enhanced the relaxation induced by GYY4137 yielding IC50 value of 0.13 ± 0.08 µM (n = 6). Both the inhibitors of cystathionine ß-synthase (aminooxyacetic acid, AOAA, 30 µM) and cystathionine γ-lyase (propargylglycine, PAG, 1 mM) caused significant (p < 0.05) rightward shifts in the concentration-response curve to GYY4137. Furthermore, the KATP channel antagonist, glibenclamide (100 µM) significantly (p < 0.01) attenuated the relaxant action induced by GYY4137 on bovine ciliary artery. Conversely, the activator of cystathionine ß-synthase, SAM (100 µM) and an inhibitor of nitric oxide synthase, L-NAME (100 µM) had no significant effect on relaxations induced by GYY4137. We conclude that the inhibitory action of GYY4137 on isolated bovine ciliary artery is dependent upon the endogenous production of both prostanoids and H2S. Furthermore, the observed vascular smooth muscle relaxation induced by GYY4137 is mediated, at least in part, by KATP channels.

Isoprostanes and neuroprostanes: total synthesis, biological activity and biomarkers of oxidative stress in humans.

Isoprostanes (IsoPs) and neuroprostanes (NeuroPs) are formed in vivo by a free radical non-enzymatic mechanism involving peroxidation of arachidonic acid (AA, C20:4 n-6) and docosahexaenoic acid (DHA, C22:6 n-3) respectively. This review summarises our research in the total synthesis of these lipid metabolites, as well as their biological activities and their utility as biomarkers of oxidative stress in humans.

Mechanism of action of hydrogen sulfide on cyclic AMP formation in rat retinal pigment epithelial cells.

Hydrogen sulfide (H(2)S), a colorless gas with the pungent odor of rotten eggs has been reported to produce pharmacological actions in ocular and non-ocular tissues. We have evidence that H(2)S, using sodium hydrosulfide (NaHS) and sodium sulfide (Na(2)S) as donors can increase cyclic AMP (cAMP) production in neural retina. In the present study, we investigated the mechanism of action of H(2)S on cyclic nucleotide production in rat retinal pigment epithelial cells (RPE-J). Cultured RPE-J cells were incubated for 30 min in culture medium containing the cyclic nucleotide phosphodiesterase (PDE) inhibitor, IBMX (2 mM). Cells were exposed to varying concentrations of NaHS, the H(2)S substrate (L-cysteine), cyclooxygenase (COX) inhibitors or the diterpene activator of adenylate cyclase, forskolin in the presence or absence of H(2)S biosynthetic enzymes or the ATP-sensitive potassium (K(ATP)) channel antagonist, glibenclamide. Following drug-treatment at different time intervals, cell homogenates were prepared for cAMP assay using a well established methodology. In RPE-J cells, NaHS (10 nM-1 µM) produced a time-dependent increase in cAMP concentrations over basal levels which reached a maximum at 20 min. At this time point, both NaHS (1 nM-100 µM) and L-cysteine (1 nM-10 µM) produced a concentration-dependent significant (p<0.05) increase in cAMP concentrations over basal level. The effects of NaHS on cAMP levels in RPE-J cells was enhanced significantly (p<0.01) in the presence of the COX inhibitors, indomethacin and flurbiprofen. In RPE-J cells, the effects caused by forskolin (10 µM) on cAMP production were potentiated by addition of low concentrations of NaHS. Both the inhibitor of cystathionine ß-synthase (CBS), aminooxyacetic acid (AOA, 1 mM) and the inhibitor of cystathionine γ-lyase (CSE), proparglyglycine (PAG, 1mM) significantly attenuated the increased effect of L-cysteine on cAMP production. The K(ATP) channel antagonist, glibenclamide (100 µM) caused inhibition of NaHS induced-increase of cAMP formation in RPE-J cells. We conclude that, H(2)S (using H(2)S donor and substrate) can increase cAMP production in RPE-J cells, and removal of the apparent inhibitory effect of prostaglandins unmasks an excitatory activity of H(2)S on cAMP. Effects elicited by the H(2)S substrate on cAMP formation are dependent on biosynthesis of H(2)S catalyzed by the biosynthetic enzymes, CBS and CSE. In addition to the adenylyl cylcase pathway, K(ATP) channels are involved in mediating the observed effects of the H(2)S on cAMP production.

Regulation of [³H]d-aspartate release by the 5-F(2t)-isoprostane and its 5-epimer in isolated bovine retina.

We have evidence that 15-F2-isoprostanes (15-F2-IsoPs) regulate excitatory neurotransmitter release in ocular tissues. Although 5-F2-IsoPs are abundantly produced in mammals, their pharmacological actions on neurotransmitter release remain unknown. In the present study, we compared the effect of the 5-F2-IsoP epimer pair, 5-F(2t)-IsoP (C5-OH in ß-position) and 5-epi-5-F(2t)-IsoP (C5-OH in α-position), on K⁺-evoked [³H]D-aspartate release in isolated bovine retina. We further examined the role of prostanoid receptors on the inhibitory action of 5-epi-5-F(2t)-IsoP on [³H]D-aspartate overflow. Isolated bovine retina were prepared for studies of K⁺-evoked release of [³H]D-aspartate using the superfusion method. 5-epi-5-F(2t)-IsoP (0.01 nM to 1 µM), attenuated K⁺-evoked [³H]D-aspartate release in a concentration-dependent manner, with the inhibitory effect of 26.9% (P < 0.001; IC25 = 0.2 µM) being achieved at 1 µM concentration. Its 5-(S)-OH-epimer, 5-F(2t)-IsoP (0.1 nM-1 µM), exhibited an inhibitory biphasic action, yielding a maximal response of 35.7% (P < 0.001) at 10 nM concentration of the drug (IC25 value of 3 nM). Although the prostanoid-receptor antagonists, AH 6809 (10 µM; EP1₋3/DP) and BAY-u3405 (10 µM; DP/Tx) exhibited no effect on 5-epi-5-F(2t)-IsoP (10 nM-1 µM)-mediated inhibition, SC-19220 (1 µM; EP1) completely reversed 5-epi-5-F(2t)-IsoP (0.1 µM and 1 µM)-induced attenuation of K⁺-evoked [³H]D-aspartate release. Similarly, both SC-51322 (10 µM; EP1 and AH 23848 (1 µM; EP4) reversed the inhibitory action elicited by 5-epi-5-F(2t)-IsoP (0.1 µM) on the neurotransmitter release. We conclude that the 5-F2-IsoP epimer pair, 5-F(2t)-IsoP and 5-epi-5-F(2t)-IsoP, attenuate K⁺-induced [³H]D-aspartate release in isolated bovine retina presumably via prostanoid receptor dependent mechanisms. The trans-orientation of the allylic hydroxyl group at position C5 accounts for the apparent biphasic response exhibited by 5-F(2t)-IsoP on excitatory neurotransmitter release.

Unmet needs in glaucoma therapy: The potential role of hydrogen sulfide and its delivery strategies.

Glaucoma is an optic neuropathy disorder marked by progressive degeneration of the retinal ganglion cells (RGC). It is a leading cause of blindness worldwide, prevailing in around 2.2% of the global population. The hallmark of glaucoma, intraocular pressure (IOP), is governed by the aqueous humor dynamics which plays a crucial role in the pathophysiology of the diesease. Glaucomatous eye has an IOP of more than 22 mmHg as compared to normotensive pressure of 10-21 mmHg. Currently used treatments focus on reducing the elevated IOP through use of classes of drugs that either increase aqueous humor outflow and/or decrease its production. However, effective treatments should not only reduce IOP, but also offer neuroprotection and regeneration of RGCs. Hydrogen Sulfide (H2S), a gasotransmitter with several endogenous functions in mammalian tissues, is being investigated for its potential application in glaucoma. In addition to decreasing IOP by increasing aqueous humor outflow, it scavenges reactive oxygen species, upregulates the cellular antioxidant glutathione and protects RGCs from excitotoxicity. Despite the potential of H2S in glaucoma, its delivery to anterior and posterior regions of the eye is a challenge due to its unique physicochemical properties. Firstly, development of any delivery system should not require an aqueous environment since many H2S donors are susceptible to burst release of the gas in contact with water, causing potential toxicity and adverse effects owing to its inherent toxicity at higher concentrations. Secondly, the release of the gas from the donor needs to be sustained for a prolonged period of time to reduce dosing frequency as per the requirements of regulatory bodies. Lastly, the delivery system should provide adequate bioavailability throughout its period of application. Hence, an ideal delivery system should aim to tackle all the above challenges related to barriers of ocular delivery and physicochemical properties of H2S itself. This review discusses the therapeutic potential of H2S, its delivery challenges and strategies to overcome the associated chalenges.

Protective Action of Hydrogen Sulfide-Releasing Compounds against Oxidative Stress-Induced Cataract Formation in Cultured Bovine Lenses.

PURPOSE: The gaseous signalling molecule, hydrogen sulfide (H2S) has antioxidant, anti-inflammatory and anti-apoptotic properties. Since oxidative stress has been implicated in the pathogenesis of cataracts and lenticular hydrogen peroxide (H2O2) is elevated in some cataract patients, the present study investigated the ability of H2S-releasing compounds to prevent H2O2-induced cataract formation in cultured bovine lenses. METHODS: Lenses were cultured in either Dulbecco's Modified Eagle Medium (DMEM; control); H2O2 (50 mM); ascorbic acid (AA; 3 mM) (positive control); and the H2S-releasing compounds (diallyl trisulfide [DATS] or GYY4137) in the presence of H2O2 (50 mM). Lens opacity was determined using a plate reader to measure transmittance. Lens glutathione content (GSH), superoxide dismutase (SOD) activity and lactate dehydrogenase (LDH) cytotoxicity were assessed before and after treatment with the H2S-releasing compounds. RESULTS: Both DATS (10-7M - 10-4M) and GYY4137 (10-7M - 10-4M) significantly (p < .001) attenuated H2O2 (50 mM)-induced loss in transmittance, with DATS (10-4M) and GYY4137 (10-7M) achieving a maximal reversal of opacity by 56.86 ± 0.01% (n = 6) and 8.39 ± 0.11% (n = 6) after 120 hours, respectively. These observations were corroborated by photographic evaluation, where DATS (10-5M - 10-4M) and GYY4137 (10-7M - 10-5M)-treated lenses had relatively clear grids after 120 hours, compared to H2O2 (50 mM)-treated lenses. The H2O2 (50 mM)-induced decline in total GSH content and total SOD activity were significantly (p < .001; n = 5) reversed by DATS (10-4M) and GYY4137 (10-7M). After 24 hours, DATS (10-4M) and GYY4137 (10-7M) significantly (p < .001; n = 4) reduced cytotoxicity of primary bovine lens epithelial cells by 33.88 ± 4.59% and 36.19 ± 10.53%, respectively. CONCLUSION: Both H2S-releasing compounds protected cultured bovine lenses against oxidative stress-induced cataract formation. The slow-releasing H2S compound, GYY4137 was more potent than DATS in restoring lenticular total GSH content and total SOD activity along with reducing H2O2 (50 mM)-induced cytotoxicity.

Endogenous production of hydrogen sulfide in isolated bovine eye.

Hydrogen sulfide (H(2)S) is a novel gasotransmitter with physiological and pathological functions in vascular homeostasis, cardiovascular system and central nervous system. In the present study, we determined the endogenous levels of H(2)S in various tissues of the bovine eye. We also examined the basal levels of H(2)S in response to donors (sodium hydrosulfide, NaHS and sodium sulfide, Na(2)S), substrate (L: -cysteine), inhibitors (propargylglycine, PAG and aminooxyacetic acid, AOA) and activator (S-adenosyl-L: -methionine, SAM) of this gas in the bovine retina. H(2)S was measured using a well established spectrophotometric method. The highest concentration of endogenous H(2)S was detected in cornea (19 ± 2.85 nmoles/mg protein, n = 6) and retina (17 ± 2.1 nmoles/mg protein, n = 6). Interestingly, H(2)S was not present in vitreous humor. The inhibitors of CSE and CBS; PAG (1 mM) and AOA (1 mM), significantly attenuated the production of H(2)S in the bovine retina by 56.8 and 42%, respectively. On the other hand the activator of CBS; SAM (100 µM), H(2)S donors; NaHS (1 µM) and Na(2)S (100 µM), significantly increased endogenous levels of H(2)S in bovine retina. L: -cysteine (10-300 µM) produced a significant (P < 0.05) concentration-dependent increase in H(2)S levels reaching a maximal at 300 µM. We conclude that H(2)S is endogenously produced in various tissues of the isolated bovine eye. Moreover, endogenous levels of H(2)S are enhanced in the presence of substrate (L: -cysteine), an activator of CBS (SAM) and H(2)S donors but are blocked by inhibitors of enzymes that synthesize this gas in neural retina.

Effect of hydrogen sulfide on cyclic AMP production in isolated bovine and porcine neural retinae.

Hydrogen sulfide (H(2)S) has been reported to exert pharmacological effects on neural and non-neural tissues from several mammalian species. In the present study, we examined the role of the intracellular messenger, cyclic AMP in retinal response to H(2)S donors, sodium hydrosulfide (NaHS) and sodium sulfide (Na(2)S) in cows and pigs. Isolated bovine and porcine neural retinae were incubated in oxygenated Krebs buffer solution prior to exposure to varying concentrations of NaHS, Na(2)S or the diterpene activator of adenylate cyclase, forskolin. After incubation at different time intervals, tissue homogenates were prepared for cyclic AMP assay using a well established methodology. In isolated bovine and porcine retinae, the combination of both phosphodiesterase inhibitor, IBMX (2 mM) and forskolin (10 microM) produced a synergistic increase (P < 0.001) in cyclic AMP concentrations over basal levels. NaHS (10 nM-100 microM) produced a time-dependent increase in cyclic AMP concentrations over basal levels which reached a maximum at 20 min in both bovine and porcine retinae. At this time point, both NaHS and Na(2)S (10 nM-100 microM) caused a significant (P < 0.05) dose-dependent increase in cyclic AMP levels in bovine and porcine retinae. For instance, NaHS (100 nM) elicited a four-fold and three-fold increase in cyclic AMP concentrations in bovine and porcine retinae respectively whilst higher concentrations of Na(2)S (100 microM) produced a much lesser effect in both species. In bovine and porcine retinae, the effects caused by forskolin (10 microM) on cyclic AMP production were not potentiated by addition of low or high concentrations of both NaHS and Na(2)S. We conclude that H(2)S donors can increase cyclic AMP production in isolated neural retinae from cows and pigs. Bovine retina appears to be more sensitive to the stimulatory effect of H(2)S donors on cyclic nucleotide production than its porcine counterpart indicating that species differences exist in the magnitude of this response. Furthermore, effects produced by forskolin on cyclic AMP formation were not additive with those elicited by H(2)S donors suggesting that these agents may share a common mechanism in their action on the adenylyl cyclase pathway.

Current Trends in the Pharmacotherapy of Cataracts.

Cataracts, one of the leading causes of preventable blindness worldwide, refers to lens degradation that is characterized by clouding, with consequent blurry vision. As life expectancies improve, the number of people affected with cataracts is predicted to increase worldwide, especially in low-income nations with limited access to surgery. Although cataract surgery is considered safe, it is associated with some complications such as retinal detachment, warranting a search for cheap, pharmacological alternatives to the management of this ocular disease. The lens is richly endowed with a complex system of non-enzymatic and enzymatic antioxidants which scavenge reactive oxygen species to preserve lens proteins. Depletion and/or failure in this primary antioxidant defense system contributes to the damage observed in lenticular molecules and their repair mechanisms, ultimately causing cataracts. Several attempts have been made to counteract experimentally induced cataract using in vitro, ex vivo, and in vivo techniques. The majority of the anti-cataract compounds tested, including plant extracts and naturally-occurring compounds, lies in their antioxidant and/or free radical scavenging and/or anti-inflammatory propensity. In addition to providing an overview of the pathophysiology of cataracts, this review focuses on the role of various categories of natural and synthetic compounds on experimentally-induced cataracts.

Role of prostanoid production and receptors in the regulation of retinal endogenous amino acid neurotransmitters by 8-isoprostaglandin E2, ex vivo.

The role of enzymes and receptors of the prostanoid pathway in the inhibitory effect of 8-isoprostaglandin E2 (8-isoPGE2) on endogenous amino acid neurotransmitter levels was examined, ex vivo. Freshly isolated bovine eyeballs were injected intravitreally with IsoPs, incubated in Krebs buffer for 30 min and retina prepared for HPLC-ECD detection of amino acids. 8-isoPGE2 attenuated retinal glutamate and its metabolite, glutamine and glycine in a concentration-dependent manner. The nonselective cyclooxygenase (COX)-inhibitor, flurbiprofen, COX-2 selective inhibitor, NS-398 and thromboxane (Tx) synthase inhibitor, furegrelate had no effect on both basal amino acid levels and the inhibitory effects of 8-isoPGE2 (1-100 µM) on the retinal amino acids. Whereas the TP-receptor antagonist SQ-29548(10 µM) exhibited no effect, SC-19220(EP1; 30 µM), AH-6809(EP(1-3); 30 µM) and AH-23848(EP4; 30 µM) reversed the inhibitory effects of 8-isoPGE2 (0.01-100 µM) on glutamate, glutamine and glycine levels. We conclude that prostanoid EP-receptors regulate the inhibitory effect of 8-isoPGE2 on basal levels of endogenous amino acids in bovine retina, ex vivo.

Regulation of [3H] D-aspartate release from mammalian isolated retinae by hydrogen sulfide.

Hydrogen sulfide (H(2)S), can produce pharmacological effects on neural and non-neural tissues from several mammalian species. The present study investigates the pharmacological action of H(2)S, (using sodium hydrosulfide, NaHS, and/or sodium sulfide, Na(2)S as donors) on amino acid neurotransmission (using [(3)H] D: -aspartate as a marker for glutamate) from isolated, superfused bovine and porcine retinae. Isolated neural retinae were incubated in Krebs solution containing [(3)H] D: -aspartate at 37 degrees C. Release of [(3)H] D: -aspartate was elicited by high potassium (K(+) 50 mM) pulse. Both NaHS and Na(2)S donors caused an inhibition of K(+)-evoked [(3)H] D: -aspartate release from isolated bovine retinae without affecting basal [(3)H] D: -aspartate efflux yielding IC(50) values of 0.006 and 6 microm, respectively. Furthermore, NaHS inhibited depolarization-evoked release of [(3)H] D: -aspartate from isolated porcine retinae with an IC(50) value of 8 microM. The inhibitory action of NaHS on [(3)H] D: -aspartate release from porcine retinae was blocked by propargyglycine, a selective inhibitor of cystathionine gamma-lyase (CSE). Our results indicate that H(2)S donors can inhibit amino acid neurotransmission from both isolated bovine and porcine retinae, an effect that is dependent, at least in part, on intramural biosynthesis of H(2)S.

Effect of hydrogen sulfide on sympathetic neurotransmission and catecholamine levels in isolated porcine iris-ciliary body.

In the present study, we investigated the pharmacological action of hydrogen sulfide (H2S, using sodium hydrosulfide, NaHS, and/or sodium sulfide, Na2S as donors) on sympathetic neurotransmission from isolated, superfused porcine iris-ciliary bodies. We also examined the effect of H2S on norepinephrine (NE), dopamine and epinephrine concentrations in isolated porcine anterior uvea. Release of [3H]NE was triggered by electrical field stimulation and basal catecholamine concentrations was measured by high performance liquid chromatography (HPLC). Both NaHS and Na2S caused a concentration-dependent inhibition of electrically evoked [3H]NE release from porcine iris-ciliary body without affecting basal [3H]NE efflux. The inhibitory action of H2S donors on NE release was attenuated by aminooxyacetic acid (AOA) and propargyglycine (PAG), inhibitors of cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE), respectively. With the exception of dopamine, NaHS caused a concentration-dependent reduction in endogenous NE and epinephrine concentrations in isolated iris-ciliary bodies. We conclude that H2S can inhibit sympathetic neurotransmission from isolated porcine anterior uvea, an effect that is dependent, at least in part, on intramural biosynthesis of this gas. Furthermore, the observed action of H2S donors on sympathetic transmission may be due to a direct action of this gas on neurotransmitter pools.