A new approach to antiglaucoma drugs: carbonic anhydrase inhibitors with or without NO donating moieties. Mechanism of action and preliminary pharmacology

The clinically used sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor dorzolamide (DRZ), a new sulfonamide CA inhibitor also incorporating NO-donating moieties, NCX250, and isosorbide mononitrate (ISMN) (an NO-donating compound with no CA inhibitory properties) were investigated for their intraocular pressure (IOP) lowering effects in rabbits with carbomer-induced glaucoma. NCX250 was more effective than DRZ or ISMN on lowering IOP, increasing ocular hemodynamics, decreasing the inflammatory processes and ocular apoptosis in this animal model of glaucoma. NO participate to the regulation of IOP in glaucoma, having also antiapoptotic and anti-inflammatory effects. The ophthalmic artery, both systolic and diastolic velocities, were significantly reduced in NCX250-treated eyes in comparison to DRZ treated ones, suggesting thus a beneficial effect of NCX250 on the blood supply to the optic nerve. Combining CA inhibition with NO-donating moieties in the same compound offers an excellent approach for the management of glaucoma.     

Carbonic Anhydrase Inhibitors. Metal Complexes of 5-(2-Chlorophenyl)-1, 3, 4-Thiadiazole-2-Sulfonamide with Topical Intraocular Pressure Lowering Properties: The Influence of Metal Ions Upon the Pharmacological Activity

Metal complexes of a sulfonamide possessing strong carbonic anhydrase (CA) inhibitory properties, 5-(2-chlorophenyl)-1, 3, 4-thiadiazole-2-sulfonamide (chlorazolamide) have been obtained from the sodium salt of the sulfonamide and the following metal ions: Mg(II), Zn(II), Mn(II), Cu(II), Co(II), Ni(II), Be(II), Cd(II), Pb(II), AI(III), Fe(III) and La(III). The original sulfonamide and its complexes were assayed for the in vitro inhibition of three CA isozymes, CA I, II, and IV, some of which play a critical role in ocular fluid secretion. All these compounds (the sulfonamide and its metal complexes) behaved as powerful inhibitors against the three investigated isozymes. The parent sulfonamide possessed an extremely weak topical pressure lowering effect when administered as a 1-2% suspension into the rabbit eye, but some of its metal complexes, such as the Mg(II), Zn(II), Mn(II) and Cu(II) derivatives, lower intraocular pressure (IOP) in experimental animals very well. Ex vivo data showed a 99.5-99.9% CA II inhibition in ocular fluids and tissues of rabbits treated with these agents, proving that the observed IOP lowering is due to CA inhibition. The influence of the different metal ions upon the efficiency of the obtained complexes as pressure lowering drugs are discussed, leading to the possibility of designing more selective; potent pharmacological agents from this class     

Carbonic anhydrase inhibitors. metal complexes of 5-(2-chlorophenyl)-1,3,4-thiadiazole-2-sulfonamide with topical intraocular pressure lowering properties: the influence of metal ions upon the pharmacological activity

Metal complexes of a sulfonamide possessing strong carbonic anhydrase (CA) inhibitory properties, 5-(2-chlorophenyl)-1,3,4-thiadiazole-2-sulfonamide (chlorazolamide) have been obtained from the sodium salt of the sulfonamide and the following metal ions: Mg(II), Zn(II), Mn(II), Cu(II), Co(II), Ni(II), Be(II), Cd(II), Pb(II), Al(III), Fe(III) and La(III). The original sulfonamide and its complexes were assayed for the in vitro inhibition of three CA isozymes, CA I, II, and IV, some of which play a critical role in ocular fluid secretion. All these compounds (the sulfonamide and its metal complexes) behaved as powerful inhibitors against the three investigated isozymes. The parent sulfonamide possessed an extremely weak topical pressure lowering effect when administered as a 1-2% suspension into the rabbit eye, but some of its metal complexes, such as the Mg(II), Zn(II), Mn(II) and Cu(II) derivatives, lower intraocular pressure (IOP) in experimental animals very well. Ex vivo data showed a 99.5-99.9% CA II inhibition in ocular fluids and tissues of rabbits treated with these agents, proving that the observed IOP lowering is due to CA inhibition. The influence of the different metal ions upon the efficiency of the obtained complexes as pressure lowering drugs are discussed, leading to the possibility of designing more selective/potent pharmacological agents from this class.     

Carbonic anhydrase inhibitors: synthesis and inhibition against isozymes I, II and IV of topically acting antiglaucoma sulfonamides incorporating cis-5-norbornene-endo-3-carboxy-2-carboxamido moieties

Sulfonamides incorporating cis-5-norbornene-endo-3-carboxy-2-carboxamido moieties in their molecules were prepared by reaction of cis-5-norbornene-endo-2,3-dicarboxylic anhydride with aromatic/heterocyclic sulfonamides possessing free amino, hydrazino, or imino groups. Some of these compounds showed very good CA II and CA IV inhibitory properties, with affinities for the enzymes in the low nanomolar range. Some of the most active CA II inhibitors reported here have been formulated as aqueous solutions for topical administration as antiglaucoma agents in normotensive rabbits. Some of the derivatives incorporating cis-5-norbornene-endo-3-carboxy-2-carboxamido and aromatic sulfonamide moieties (as sodium salts) showed effective and longer lasting intraocular pressure (IOP) lowering as compared to dorzolamide, a widely used topical antiglaucoma drug. Compounds incorporating cis-5-norbornene-endo-2,3-carboximido moieties, although stronger in vitro CA inhibitors as compared to the corresponding cis-5-norbornene-endo-3-carboxy-2-carboxamido-;derivatives, showed no topical IOP lowering properties, probably due to their very poor water solubility.     

Synthesis and crystallographic analysis of new sulfonamides incorporating NO-donating moieties with potent antiglaucoma action

Several aromatic/heterocyclic sulfonamide scaffolds have been used to synthesize compounds incorporating NO-donating moieties of the nitrate ester type, which have been investigated for the inhibition of five physiologically relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms: hCA I (offtarget), II, IV and XII (antiglaucoma targets) and IX (antitumor target). Some of the new compounds showed effective in vitro inhibition of the target isoforms involved in glaucoma, and the X-ray crystal structure of one of them revealed factors associated with the marked inhibitory activity. In an animal model of ocular hypertension, one of the new compounds was twice more effective than dorzolamide in reducing elevated intraocular pressure characteristic of this disease, anticipating their potential for the treatment of glaucoma.     

Carbonic anhydrase inhibitors: synthesis of water-soluble, topically effective intraocular pressure lowering aromatic/heterocyclic sulfonamides containing 8-quinoline-sulfonyl moieties: is the tail more important than the ring?

Reaction of 20 aromatic/heterocyclic sulfonamides containing a free amino, imino, hydrazino or hydroxyl group, with 8-quinoline-sulfonyl chloride afforded a series of water-soluble (as hydrochloride or triflate salts) compounds. The new derivatives were assayed as inhibitors of the zinc enzyme carbonic anhydrase (CA), and more precisely of three of its isozymes, CA I, II (cytosolic forms) and IV (membrane-bound form), involved in important physiological processes. Efficient inhibition was observed against all three isozymes, but especially against CA II (in nanomolar range), which is the isozyme known to play a critical role in aqueous humor secretion within the ciliary processes of the eye. Some of the best inhibitors synthesized were topically applied as 2% water solutions onto the eye of normotensive and glaucomatous albino rabbits, when strong and long-lasting intraocular pressure (IOP) lowering was observed with many of them. This result prompted us to reanalyze the synthetic work done by other groups for the design of water soluble, topically effective antiglaucoma sulfonamides. According to these researchers, the IOP lowering effect is due to the intrinsic nature of the specific heterocyclic sulfonamide considered, among which the thienothiopyran-2-sulfonamide derivatives represent the best studied case. Indeed, the first agents developed for such applications, such as dorzolamide, are derivatives of this ring system. In order to prove that the tail (in this case the 8-quinoline-sulfonyl moiety) conferring water solubility to a sulfonamide CA inhibitor is more important than the ring to which the sulfonamido group is grafted, we also prepared a dorzolamide derivative to which the 8-quinoline-sulfonyl moiety was attached. This new compound is quite water soluble as hydrochloride salt, behaves as a strong CA II inhibitor, and fared better than the parent molecule in lowering IOP in experimental animals. Thus, the tail conferring water solubility to such an enzyme inhibitor is more important for its topical activity as antiglaucoma drug than the heterocyclic/aromatic ring to which the sulfonamido moiety is grafted.     

Carbonic anhydrase inhibitors: sulfonamides incorporating furan-, thiophene- and pyrrole-carboxamido groups possess strong topical intraocular pressure lowering properties as aqueous suspensions

Important physiological and physio-pathological functions are played by several carbonic anhydrase (CA, EC 4.2.1.1) isozymes, which are strongly inhibited by aromatic and heterocyclic sulfonamides. Here we report several new types of such sulfonamides, incorporating furan-, thiophene- and pyrrole-carboxamide moieties in their molecules. Some of these compounds showed very good CA II and CA IV inhibitory properties. with affinities for the enzymes in the low nanomolar range. Due to their relatively low water solubility, some of the most active CA II inhibitors reported here have been formulated as aqueous suspension for topical administration as antiglaucoma agents. in normotensive and glaucomatous rabbits. The derivatives incorporating furan- and pyrrole-carboxamide moieties (but not the corresponding thiophene-substituted derivatives), showed effective and long-lasting intraocular pressure (IOP) lowering both in normotensive as well as glaucomatous animals, with potencies superior to dorzolamide and brinzolamide, the two available topically acting sulfonamide drugs. This is the first example of non-water soluble sulfonamides that significantly lower IOP, being thus similar with the recently introduced drug brinzolamide, which belongs to a completely different chemical family of antiglaucoma sulfonamides.     

Furazan and furoxan sulfonamides are strong α-carbonic anhydrase inhibitors and potential antiglaucoma agents

A series of furazan and furoxan sulfonamides were prepared and studied for their ability to inhibit human carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, hCA II, hCA IX, and hCA XII. The simple methyl substituted products 3–5 were potent inhibitors. Differing structural modifications of these leads had differing effects on potency and selectivity. In particular, products in which the sulfonamide group is separated from the hetero ring by a phenylene bridge retained high potency only on the hCA XII isoform. The sulfonamides 3–5 exerted intraocular pressure (IOP) lowering effects in vivo in hypertensive rabbits more efficiently than dorzolamide. Some other products (39–42), although less effective in vitro hCA II/XII inhibitors, also effectively lowered IOP in two different animal models of glaucoma.     

Carbonic anhydrase inhibitors: N-(p-sulfamoylphenyl)-alpha-D-glycopyranosylamines as topically acting antiglaucoma agents in hypertensive rabbits

A series of N-(p-sulfamoylphenyl)-alpha-D-glycopyranosylamines was prepared by reaction of sulfanilamide with different monosaccharides in the presence of ammonium chloride. The new compounds were investigated for inhibition of the metallo-enzyme carbonic anhydrase (CA, EC 4.2.1.1), involved in aqueous humor secretion within the mammalian eye. Isozymes CA I and CA II were strongly inhibited by some of these compounds, which showed inhibition constants in the range of 510-1200 nM against CA I and 10-25 nM against CA II, similarly to clinically used sulfonamides, such as acetazolamide, methazolamide, dichlorophenamide, dorzolamide and brinzolamide. The presence of sugar moieties in these molecules induced an enhanced water solubility as compared to other sulfonamides. In hypertensive rabbits (a widely used animal model of glaucoma), two of the new compounds showed strong and long-lasting intraocular pressure (IOP) lowering, being more effective than dorzolamide and brinzolamide, the two clinically used, topically acting antiglaucoma sulfonamides with CA inhibitory properties.     

Carbonic anhydrase inhibitors. Interaction of 2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide with 12 mammalian isoforms: kinetic and X-ray crystallographic studies

2-(Hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide was tested for its interaction with 12 carbonic anhydrase (CA, EC 4.2.1.1) isoforms in the search of compounds with good inhibitory activity against isozymes with medicinal chemistry applications, such as CA I, II, VA, VB, VII, IX, and XII among others. This sulfonamide is a potent inhibitor of CA I and II (K(I)s of 7.2-7.5 nM), a medium potency inhibitor of CA VII, IX, XII, and XIV, and a weak inhibitor against the other ubiquitous isoforms, making it thus a very interesting clinical candidate for situations in which a strong inhibition of CA I and II is needed. The crystal structure of the hCA II adduct of this sulfonamide revealed many favorable interactions between the inhibitor and the enzyme which explain its strong low nanomolar affinity for this isoform but may also be exploited for the design of effective inhibitors incorporating bicyclic moieties.     

Carbonic anhydrase inhibitors: binding of an antiglaucoma glycosyl-sulfanilamide derivative to human isoform II and its consequences for the drug design of enzyme inhibitors incorporating sugar moieties

N-(4-Sulfamoylphenyl)-alpha-d-glucopyranosylamine, a promising topical antiglaucoma agent, is a potent inhibitor of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The high resolution X-ray crystal structure of its adduct with the target isoform involved in glaucoma, CA II, is reported here. The sugar sulfanilamide derivative binds to the enzyme in a totally new manner as compared to other CA-inhibitor adducts investigated earlier. The sulfonamide anchor was coordinated to the active site metal ion, and the phenylene ring of the inhibitor filled the channel leading to the active site cavity. The glycosyl moiety responsible for the high water solubility of the compound was oriented towards a hydrophilic region of the active site, where no other inhibitors were observed to be bound up to now. A network of seven hydrogen bonds with four water molecules and the amino acid residues Pro201, Pro202 and Gln92 further stabilize the enzyme-inhibitor adduct. Topiramate, another sugar-based CA inhibitor, binds in a completely different manner to CA II as compared to the sulfonamide investigated here. These findings are useful for the design of potent, sugar-derived enzyme inhibitors.     

Effects of intravitreous sodium hydrosulfide on intraocular pressure and retinopathy in ocular hypertensive rats

We investigated the possible neuroprotectant and intraocular pressure (IOP) lowering effects of intravitreous injection of sodium hydrosulfide (NaSH) in a rodent model of experimental glaucoma. Glaucoma currently is treated by controlling IOP using medications and/or surgery. These methods are not entirely adequate for all patients. We divided 24 rats into three groups. For the control group, the right eye was treated with intravitreous saline. For the glaucoma group, ocular hypertension was induced by photocoagulating three episcleral veins and the limbal plexus of the right eye using an argon laser, then saline was injected into the vitreous of these eyes during the third week. For the NaSH group, rats were treated with intravenous NaSH 3 weeks after photocoagulation. IOP was measured each week during the 6 week experimental period. Coagulating the episcleral veins rapidly increased the IOP of rat eyes. Intravitreous injection of NaSH significantly reduced IOP. Intravitreous NaSH prevented degeneration of the retina and decreased the number of apoptotic cells. Intravitreous NaSH appeared to reduce IOP and to prevent IOP induced retinopathy in rats.     

Benzolamide is not a membrane-impermeant carbonic anhydrase inhibitor

Benzolamide, an orphan drug belonging to the pharmacological class of sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) is widely used in many physiological and pharmacological studies, together with the clinically employed classical drugs, acetazolamide, methazolamide, ethoxzolamide or dichlorophenamide, it being frequently stated that benzolamide is a membrane-impermeant inhibitor. We prove here that this is false: in fact benzolamide is rather similar to acetazolamide from the point of view of penetrability through blood red cell membranes. Unlike these neutral drugs, the cationic, positively-charged CAIs incorporating either tetraalkyl ammonium or pyridinium moieties, due to their salt-like character are indeed membrane-impermeant, being the only type of low molecular weight compound possessing such properties. Selective inhibition of membrane-associated CA isozymes is relevant indeed in many physiological studies and also pharmacologically, since the tumor-associated isozymes (CA IX and XII) are both membrane-bound.     

Autotaxin-lysophosphatidic Acid axis is a novel molecular target for lowering intraocular pressure

Primary open-angle glaucoma is the second leading cause of blindness in the United States and is commonly associated with elevated intraocular pressure (IOP) resulting from diminished aqueous humor (AH) drainage through the trabecular pathway. Developing effective therapies for increased IOP in glaucoma patients requires identification and characterization of molecular mechanisms that regulate IOP and AH outflow. This study describes the identification and role of autotaxin (ATX), a secretory protein and a major source for extracellular lysophosphatidic acid (LPA), in regulation of IOP in a rabbit model. Quantitative proteomics analysis identified ATX as an abundant protein in both human AH derived from non-glaucoma subjects and in AH from different animal species. The lysophospholipase D (LysoPLD) activity of ATX was found to be significantly elevated (by ∼1.8 fold; n = 20) in AH derived from human primary open angle glaucoma patients as compared to AH derived from age-matched cataract control patients. Immunoblotting analysis of conditioned media derived from primary cultures of human trabecular meshwork (HTM) cells has confirmed secretion of ATX and the ability of cyclic mechanical stretch of TM cells to increase the levels of secreted ATX. Topical application of a small molecular chemical inhibitor of ATX (S32826), which inhibited AH LysoPLD activity in vitro (by >90%), led to a dose-dependent and significant decrease of IOP in Dutch-Belted rabbits. Single intracameral injection of S32826 (∼2 µM) led to significant reduction of IOP in rabbits, with the ocular hypotensive response lasting for more than 48 hrs. Suppression of ATX expression in HTM cells using small-interfering RNA (siRNA) caused a decrease in actin stress fibers and myosin light chain phosphorylation. Collectively, these observations indicate that the ATX-LPA axis represents a potential therapeutic target for lowering IOP in glaucoma patients.     

Exploring the potential for subtype-selective muscarinic agonists in glaucoma

Pilocarpine has been used to lower intraocular pressure (IOP) in glaucoma patients for more than 100 years. Since the identification of five muscarinic receptor subtypes, there has been an interest in separating the IOP-lowering effects from the ocular side effects of pupil constriction and lens accommodation. However, all these actions seem to be mediated by the M3 receptor. A novel muscarinic receptor agonist, AGN 199170, that has no activity on the M3 subtype was compared to pilocarpine in a monkey glaucoma model. This compound lowered IOP suggesting that muscarinic agonists targeted at muscarinic receptors other than the M3 subtype may be able to selectively lower IOP.     

Vascular protective actions of a nitric oxide aspirin analog in both in vitro and in vivo models of diabetes mellitus

BACKGROUND: Defective endothelium-dependent relaxation is observed in experimental and human diabetes mellitus. The nature of this defect is not fully understood but may involve decreased nitric oxide (NO) bioactivity due to enhanced production of reactive oxygen species (ROS). In this paper, we examine the benefits and actions of a novel NO-donating, antioxidant called 2-acetoxybenzoic acid 2-(2-nitrooxymethyl) phenyl ester, and denoted as NCX4016, on NO-mediated endothelium-dependent relaxation in normal arteries exposed to acute elevations in glucose or in arteries derived from chronic diabetic animals. MATERIAL AND METHODS: Intrinsic free radical scavenging by NO-NSAIDs in solution were evaluated using electron paramagnetic resonance (EPR) spectroscopy and spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In acute studies, normal rat aortas were exposed in tissue culture for 18 h to 5.5 mM or 40 mM in the presence or absence of NCX4016, a NO-donating NSAID unrelated to aspirin (NCX2216) or aspirin. Vascular reactivity of thoracic aortic rings to endothelium-dependent relaxation to acetylcholine in vitro was determined. For chronic hyperglycemia, diabetes was induced in rats by intravenous injection with streptozotocin. Vascular reactivity of thoracic aortic rings to endothelium-dependent relaxation to acetylcholine in vitro was determined after 8 wks in untreated animals or animals chronically-treated with NCX4016. Antioxidant efficacy in vivo was determined by measurement of plasma isoprostanes and by nuclear binding activity of NF-kappaB in nuclear fractions of aortae. RESULTS: Incubation with NCX4016 and NCX2216 produced a concentration-dependent inhibition of DMPO-OH formation indicating scavenging of hydroxyl radicals (HO(*)). In contrast, little efficacy to scavenge superoxide anion radicals was noted. Acute incubation of normal arteries with elevated glucose concentration caused inhibition of normal relaxation to acetylcholine. This impairment was prevented by co-incubation with NCX4106 but not by mannitol, the parent compound (aspirin) or by NCX2216. In addition, chronic treatment with NCX4016 prevented the development of defective endothelium-dependent relaxation to acetylcholine. This protection did not occur as a result to any changes in blood glucose concentration or hemoglobin glycation. Treatment with NCX4016 did decrease the elevation in plasma isoprostanes and normalized the diabetes-induced increase in NF-kappaB binding activity in nuclear fractions derived from aortic tissue. CONCLUSIONS: Collectively, these studies suggest that antioxidant interventions using NO-donating NSAIDs may provide an important novel therapeutic strategy to protect the diabetic endothelium.     

Discovery of an N-(2-aminopyridin-4-ylmethyl)nicotinamide derivative: a potent and orally bioavailable NCX inhibitor

Ca(2+) overload in myocardial cells is responsible for arrhythmia. Sodium-calcium exchanger (NCX) inhibitors are more effective than sodium-hydrogen exchanger (NHE) inhibitors with regard to modulation of Ca(2+) overload, because NCX inhibitors can directly inhibit the influx of Ca(2+) into cells. NCX is an attractive target for the treatment of heart failure and ischemia-reperfusion. We have designed and synthesized a series of N-(2-aminopyridin-4-ylmethyl)nicotinamide derivatives, based on compound 5. We have discovered a novel NCX inhibitor (23 h) with an IC(50) value of 0.12 microM against reverse NCX. The inhibitory activities of our NCX inhibitors against cytochrome P450 were also evaluated. The effects on heart failure and the pharmacokinetic profile of compound 23 h are discussed.     

Carbonic anhydrase inhibitors: inhibition of the tumor-associated isozyme IX with fluorine-containing sulfonamides. The first subnanomolar CA IX inhibitor discovered

Polyfluorinated CAIs show very good inhibitory properties against different carbonic anhydrase (CA) isozymes, such as CA I, II, and IV, but such compounds have not been tested for their interaction with the transmembrane, tumor-associated isozyme CA IX. Thus, a series of such compounds has been obtained by attaching 2,3,5,6-tetrafluorobenzoyl- and 2,3,5,6-tetrafluorophenylsulfonyl- moieties to aromatic/heterocyclic sulfonamides possessing derivatizable amino moieties. Some of these compounds showed excellent CA IX inhibitory properties and also selectivity ratios favorable to CA IX over CA II, the other physiologically relevant isozyme with high affinity for sulfonamide inhibitors. The first subnanomolar and rather selective CA IX inhibitor has been discovered, as the 2,3,5,6-tetrafluorobenzoyl derivative of metanilamide showed an inhibition constant of 0.8 nM against hCA IX, and a selectivity ratio of 26.25 against CA IX over CA II. Several other low nanomolar CA IX inhibitors were detected among the new derivatives reported here. The reported derivatives constitute valuable candidates for the development of novel antitumor therapies based on the selective inhibition of tumor-associated CA isozymes.     

Carbonic anhydrase inhibitors: topical sulfonamide antiglaucoma agents incorporating secondary amine moieties

Reaction of aromatic/heterocyclic sulfonamides possessing free amino, imino or hydrazino moieties with 7-chloro-4-chloromethylcoumarin afforded a series of N-[(7-chloro-4-coumarinyl)-methyl]- derivatives which showed effective inhibition of three carbonic anhydrase (CA) isozymes. Topical application within the rabbit eye of some of these compounds led to effective intraocular pressure lowering due to CA inhibition within the ocular tissues, and reduced aqueous humor production.     

Endogenous ocular lipids as potential modulators of intraocular pressure

Elevated intraocular pressure (IOP) is a risk factor in glaucoma, a group of irreversible blinding diseases. Endogenous lipids may be involved in regulation of IOP homeostasis. We present comparative fold analysis of phospholipids and sphingolipids of aqueous humour and trabecular meshwork from human control vs primary open-angle glaucoma and mouse control (normotensive) vs ocular hypertensive state. The fold analysis in control vs disease state was based on ratiometric mass spectrometric data for above classes of lipids. We standardized in vitro assays for rapid characterization of lipids undergoing significant diminishment in disease state. Evaluation of lipids using in vitro assays helped select a finite number of lipids that may potentially expand cellular interstitial space embedded in an artificial matrix or increase fluid flow across a layer of cells. These assays reduced a number of lipids for initial evaluation using a mouse model, DBA/2J with spontaneous IOP elevation. These lipids were then used in other mouse models for confirmation of IOP lowering potential of a few lipids that were found promising in previous assessments. Our results provide selected lipid molecules that can be pursued for further evaluation and studies that may provide insight into their function.