the hydrogen sulfide , h 2 s , used for this invention can be generated in several ways . one way is to bubble hydrogen sulfide gas into a liquid medium or carrier ( such as water , saline , or a physiologically acceptable buffer solution ) until saturation . another way , and the preferred way , is to generate the hydrogen sulfide in situ using a sulfide salt , preferably a water soluble sulfide salt , such as a group ia salt , including nahs , na 2 s , or its respective k salt , dissolved in a carrier . these salts will dissolve in water readily , and under physiological conditions , h 2 s is generated in situ . before a carrier solution saturated with gaseous h 2 s can be used for this invention , a roughly 10 fold dilution is needed . after this dilution , the effective amount used per eye would be roughly about one drop , which is roughly about 100 microliter . as for the h 2 s generated in situ from a salt , the concentration of the final salt solution should range from about 0 . 01 % ( weight / volume ) to about 4 % ( weight / volume ). the preferred final concentration of the salt solution is about 1 % ( weight / volume ). the effective amount of this 1 % ( weight / volume ) salt solution is about 1 drop per eye . the application of h 2 s can be given to an eye once a day , or up to two to three times a day . the treatments can be repeated the following days if needed . the treatment regiment can consist of : measuring the intraocular pressure after application of the solution ; comparing the measured intraocular pressure with the desired level of intraocular pressure ; and repeating the applying , measuring and comparing steps until the measured intraocular pressure is at or below the desired intraocular pressure . the effect of a generator of h 2 s in biological tissues ( nahs ) on intraocular pressure in normotensive , conscious albino rabbits was examined . in the eyes of a rabbit , the desired intraocular pressure is around 20 mm hg . while in human , the desired intraocular pressure in the eye is in the range of from about 12 mm hg to 15 mm hg . ranges of about 18 to 24 mm hg in a human eye are generally considered elevated . two baseline intraocular pressure measurements were made in nine rabbits using a pneumatonometer ( medtronic , model 30 classic , jacksonville , fla .) at − 1 , and 0 hour after topical application of proparacaine 0 . 5 %. nahs ( 1 %, dissolved in sterile saline solution ) was applied topically to one eye of each animal while the saline vehicle was administered to the other ( control ) eyes . intraocular pressures were measured in both eyes at + 0 . 5 , + 1 , + 3 , and + 6 hours after instillation of the drug and vehicle . no hyperemia and / or papillary changes were observed in both experimental and control eyes of rabbits at all tines tested . a dose of nahs ( 1 %) caused a time - dependent significant ( p & lt ; 0 . 001 ) reduction in intraocular pressure in the treated eyes that reached a maximum at 3 hours and remained depressed after 6 hours ( fig1 ). a parallel ( but smaller ) decrease in intraocular pressure was observed in eyes treated with only the saline vehicle ( no drug ) suggesting that this compound can produce contralateral effects . the possibilities that h 2 s may alter intraocular pressure by reducing the release of norepinephrine from sympathetic nerves supplying the anterior uvea was tested . currently used anti - glaucoma drugs of the α 2 - adrenoceptor agonist ( e . g . alphagan ) class also decrease the release of norepinephrine from sympathetic nerves in the anterior uvea . the effect of the h 2 s generators , nahs and na 2 s on field - stimulated [ 3 h ]- norepinephrine ([ 3 h ] ne ) release from isolated , superfused porcine iris - ciliary bodies was also studied . the methodology utilized for measuring [ 3 h ] ne release was essentially the same employed by ohia and coworkers ( 1997 ; invest . ophthalmol . vis . sci . 38 : 842 - 847 ). isolated porcine icbs were incubated for 60 min . at 37 ° c . in carbogen - gassed krebs buffer containing 2 . 5 μci / ml [ 3h ] ne . release of [ 3 h ] ne was elicited by 300 d . c . electrical pulses ( 5 hz , 2 msec p . d ., 60 s , supramaximal voltage ). fractions of the superfusate were collected at 4 - minute intervals and analyzed for radioactivity by liquid scintillation spectrometry . all tissues received two stimulations ( s 1 and s 2 ) 30 min . apart and nahs or na 2 s was added 8 min . before and during the s 2 stimulation period . as illustrated in the example in fig2 , application of nahs ( 1 μm ) 8 minutes before the second train of field stimulation ( s 2 ) elicited a marked inhibition of stimulation - evoked [ 3 h ] ne release from isolated porcine iris - ciliary bodies . application of nahs ( 0 . 1 - 100 μm ) caused a concentration - dependent inhibition of electrically - evoked [ 3 h ] ne overflow ( fig3 , top panel ). the pharmacological actions of h 2 s using na 2 s as a donor was investigated . na 2 s ( 1 - 100 μm ) caused a concentration - dependent inhibition of electrically - evoked [ 3 h ] ne release without affecting basal tritium efflux ( fig3 , bottom panel ). data obtained from these studies show that both nahs and na 2 s can inhibit the release of electrically - evoked [ 3 h ] ne from porcine iris - ciliary bodies suggesting that in pharmacological concentrations , h 2 s can have a negative regulatory action on sympathetic neurotransmission in this tissue . both propargylglycine ( pag , 1 mm ; inhibitor of cystathionine γ - lyase ) and aminooxyacetic acid ( aoa , 1 mm ; inhibitor of cystathionine β - synthase ) blocked the inhibitory response elicited by nahs on evoked [ 3 h ] ne release ( fig4 and 5 ). taken together , these results suggest that the pharmacological action of nahs on sympathetic neurotransmission in porcine iris - ciliary bodies is dependent , at least in part , on the endogenous biosynthesis of h 2 s . from the results of these tests , it is concluded that both nahs and na 2 s can inhibit the release of electrically - evoked [ 3h ] ne from porcine iris - ciliary bodies , an effect that is dependent on the production of h 2 s in this tissue . in another series of experiments , the hypothesis that h 2 s can produce a direct relaxant action on iris smooth muscle in vitro was tested . the effect of nahs on relaxation of tone induced by the muscarinic receptor agonist , carbachol in the isolated porcine irides was examined . the methodology employed for measuring contractile / relaxant activity of the isolated porcine iris was essentially the same employed by ohia and colleagues ( 2000 ; j . ocul . pharmacol . ther . 16 : 429 - 438 ). nahs ( 30 nm - 100 μm ) produced a concentration - related relaxation of tone induced by a submaximal concentration of carbachol in the isolated porcine iris ( fig4 ). isolated porcine iris muscle strips were set up in organ baths containing oxygenated krebs buffer solution at 37 ° c . longitudinal isometric tension was recorded via a grass ft03 force - displacement transducer and analyzed using the polyview computer software . an initial load of 150 mg was placed on each tissue after which they were allowed to equilibrate for one hour . the relaxant action of nahs or na 2 s on carbachol - induced tone was studied in the absence and presence of inhibitors of cystathionine γ - lyase ( propargylglycine , pag , β - cyanolalanine , bca ), an inhibitor of cystathionine β - synthase ( aoa ), an activator of cystathionine β - synthase ( s - adenosyl - l - methionine , sam ) and k atp channel antagonist ( glibenclamide ). in the concentration range , 30 nm to 300 μm , both nahs and na2s caused relaxations of tone induced by a submaximal concentration of carbachol yielding ic 50 values of 7 μm and 70 μm , respectively ( fig6 ). both pag ( 1 mm ) and bca ( 1 mm ) shifted concentration - response curves to nahs to the right ( fig7 and 8 ). likewise , aoa ( 30 μm ) caused significant ( p & lt ; 0 . 001 ) rightward shifts of concentration - response curves to nahs ( fig9 ). in contrast , the activator of cystathionine β - synthase , sam ( 100 μm ) caused a significant leftward shift of nahs concentration - response curve at h 2 s donor concentrations less than 3 μm ( fig1 ). the relaxations induced by the higher concentration of nahs were attenuated by sam ( fig1 ). the k atp channel blocker , glibenclamide ( 300 μm ) produced a significant rightward shift of concentration - response curves to nahs ( fig1 ). in conclusion , both nahs and na 2 s can elicit inhibitory response in isolated porcine irides , an effect that is dependent on the biosynthesis of h 2 s in this muscle . furthermore , the inhibitory action of nahs in porcine irides is mediated , at least in part , by k atp channels . taken together , these results demonstrate that endogenous generation of h 2 s by nahs or na 2 s relaxes porcine iris smooth muscle , a response that confirms and extends earlier reports in vascular ( zhao et al ., 2001 ; embo j . 20 : 6008 - 6016 ) and gastrointestinal ( teague et al ., 2002 ; br . j . pharmacol . 137 : 139 - 145 ) smooth muscles . to determine the mechanism of action of h 2 s donors on sympathetic neurotransmitter pools in the anterior uvea , the direct effect of nahs was studied on catecholamine ( norepinephrine , epinephrine and dopamine ) concentrations in porcine iris - ciliary bodies using high precision liquid chromatography techniques . nahs ( 30 - 300 μm ) caused a concentration - dependent decrease in norepinephrine concentrations in the isolated porcine iris - ciliary body ( fig1 ). data from this study supports the previous observation that h 2 s donors can reduce the release of [ 3 h ] ne from isolated porcine irides . it appears that by reducing the availability of the neurotransmitter ( norepinephrine ), h 2 s can attenuate sympathetic neurotransmission in porcine anterior uvea . the ability of h 2 s donors , nahs or na 2 s to alter the release of norepinephrine from sympathetic nerves ( in a manner similar to α 2 - adrenoceptor agonists ) could account , at least in part , for the observed iop lowering action of these compounds in experimental animals . furthermore , the fact that inhibitors ( or activators ) of the biosynthetic enzymes for h 2 s are involved in the actions of nahs on both anterior uveal sympathetic nerves and smooth muscle indicates that this gas plays a role in the observed iop response . glaucoma therapy is largely dependent upon the effect of drugs on primary messengers in the sympathetic and parasympathetic nervous system of the anterior uvea . it is thus reasonable that modification of a neurotransmitter output from sympathetic nerves induced by h 2 s may account , at least , in part for an effect of this gas on aqueous humor dynamics . furthermore , data obtained from experiments using h 2 s donors confirm that this gas can have a direct relaxant action on ocular smooth muscles that play a role in the regulation of iop . it appears that the presence of h 2 s in physiologically relevant concentrations in tissue of the anterior uvea can also be involved in the iop lowering action of some antiglaucoma drugs . based on the ocular hypotensive action of h 2 s , it appears that a new class of anti - glaucoma drugs is identified . because of reported side effects of the different classes of currently used drugs for the treatment of glaucoma , there is a need to identify additional groups of compounds with specific and potent action on this disease . this discovery can be applied to the use of h 2 s in the treatment of glaucoma and other diseases of the eye in animals and humans . h 2 s can be applied to the eye in a topical formulation of gas dissolved in a liquid carrier or as agents that can release the gas in physiological medium ( e . g ., nahs ). liquid formulations containing different concentrations of the gas can be applied at different dosages until the desired drop in iop is achieved .
topical administration of a generator of h2s in biological tissues for the treatment of eye disorders , such as glaucoma . nahs caused a time - dependent decrease in intraocular pressure in normotensive , conscious albino rabbits indicating a similar role for h2s in the regulation of aqueous humor dynamics in animals and humans . h2s donors , nahs and na2s , inhibited field - stimulated ne release from porcine isolated iris - ciliary bodies and produced relaxation of pre - contracted iris muscle strips indicating a pharmacological role for h2s in the anterior uvea . the observation that donors of h2s can alter sympathetic neurotransmission and induce an inhibitory action on iris smooth muscle suggests that this gas has the potential to influence several physiological / pathological processes in the eye . the ability of nahs or na2s to inhibit ne release mimics the well - established action some antiglaucoma drugs on sympathetic neurotransmission in the anterior uvea and can be used to reduce iop in animals and humans .