INTRAOCULAR PRESSURE

TECHNICAL FIELD

[0001] The present invention relates to pharmaceutical composition and method for reducing intraocular pressure and thereby treating ocular hypertension and/or glaucoma.

BACKGROUND ART

[0002] Primary open angle glaucoma (POAG) is the most common form of glaucoma, the leading cause of irreversible blindness that affects 60 million people worldwide. Existing agents for treatment of POAG, such as prostaglandins, carbonic anhydrase inhibitors, and beta blockers, are not universally effective in reducing intraocular pressure (IOP) and are laden with various side-effects. Accordingly, patient compliance is low and visual deterioration is frequent, resulting in severe impairment and blindness.

[0003] International Publication No. WO 2012/077108 discloses compounds composed of a potassium channel opener, e.g., a pyridinocyanoguanidine moiety or a derivative thereof, linked to a reactive oxygen species degradation catalyst comprising a nitroxide free radical (NO ^' ) group, such as 3-amino-2,2,5,5-tetramethylpyrrolidinyloxy (3-amino- PROXYL), 4-amino-2,2,6,6-tetramethylpipperidinyloxy (4-amino-TEMPO), or a derivative thereof, and pharmaceutical compositions thereof. As shown in said publication, such compounds are useful in treatment, prevention and/or managing of diseases, disorders and conditions associated with oxidative stress or endothelial dysfunction. One of the compounds exemplified in said publication is the oxy radical of 2-cyano-l-(l-hydroxy- 2,2,5,5-tetramethylpyrrolidin-3-yl)-3-(pyridin-3-yl) guanidine, herein identified R-801.

SUMMARY OF INVENTION

[0004] As found in accordance with the present invention, R-801 is a highly potent inhibitor of human type 2 carbonic anhydrase (hCA), an enzyme controlling 95% of inflow into the anterior chamber, and thus highly beneficial, upon administration to the eye, in reducing IOP. This compound can therefore be used for treatment of, e.g., ocular hypertension and/or glaucoma.

[0005] In one aspect, the present invention thus provides a pharmaceutical composition comprising the oxy radical of 2-cyano-l-(l-hydroxy-2,2,5,5-tetramethylpyrrolidin-3-yl)-3- (pyridin-3-yl)guanidine (R-801), or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt, solvate or prodrug thereof, and a pharmaceutically acceptable carrier or diluent, for reducing, i.e., lowering, IOP, more particularly for prevention or treatment of intraocular h ertension and/or glaucoma.

R-801

[0006] In another aspect, the present invention relates to R-801, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt, solvate or prodrug thereof, for use in reducing IOP.

[0007] In yet another aspect, the present invention relates to use of R-801, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt, solvate or prodrug thereof, for the preparation of a pharmaceutical composition for reducing IOP.

[0008] In a further aspect, the present invention relates to a method for reducing, i.e., lowering, IOP in a subject in need thereof comprising administering to the eye of said subject a therapeutically effective amount of R-801, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt, solvate or prodrug thereof. The subject treated by the method of the invention may be any mammal such as a human, non-human primate, horse, ferret dog, cat, cow, and goat; but it is preferably a human, i.e., an individual.

BRIEF DESCRIPTION OF DRAWINGS

[0009] Fig. 1 shows that after baseline stabilization of pressure, in anterior segments, the pressure dropped from 16 mmHg to 11 mmHg after R-801 (20 μΜ) was added to a human cadaver eye (♦) while the fellow eye (■) received vehicle (DMSO). After 72 hours of treatment, drug and vehicle were removed from the eyes and perfused with DMEM. Following perfusion, the pressure in the eye that received R-801 returned to near baseline levels (actually a little higher), and relatively no change in pressure was observed in the eye treated with vehicle.

[0010] Fig. 2 shows the effects of R-801-induced pressure reduction, in anterior segments, when the baseline pressure in a human cadaver eye (♦) was 28 mmHg. This initial pressure was higher than normal, but it had a stable baseline. The pressure dropped from 28 mmHg to 16 mmHg following R-801 treatment. Relatively no change was observed in the vehicle-treated control eye (■). Perfusion was stopped after 18 hours.

[0011] Fig. 3 shows the effects of R-801-induced pressure reduction, in the eye of a monkey that underwent laser treatment to induce ocular hypertension (OHT). IOP was measured with a calibrated pneumatonometer. R-801 treatment by intracameral injection reduced the IOP from 26 mmHg to 12 mmHg during the 30 hour period after treatment and returned to pretreatment levels after 96 hours.

DETAILED DESCRIPTION

[0012] In one aspect, the present invention provides a pharmaceutical composition comprising R-801, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt, solvate or prodrug thereof, herein generally referred to as "the active/therapeutic agent", and a pharmaceutically acceptable carrier or diluent, for reducing IOP.

[0013] The terms "intraocular hypertension", "ocular hypertension", or "elevated intraocular pressure", as used herein interchangeably, refer to an intraocular pressure in an eye of a subject (e.g., a patient) that is above a normal level and is correlated as a risk factor for the development of visual field loss and glaucoma.

[0014] Glaucoma is a heterogeneous group of optic neuropathies that share certain clinical features, wherein the loss of vision is due to the selective death of retinal ganglion cells in the neural retina that is clinically diagnosed by characteristic changes in the visual field, nerve fiber layer defects, and a progressive cupping of the optic nerve head. One of the main risk factors for the development of glaucoma is the presence of intraocular hypertension (elevated IOP). IOP also appears to be involved in the pathogenesis of normal tension glaucoma where patients have what is often considered to be normal IOP. The elevated IOP associated with glaucoma is due to elevated aqueous humor outflow resistance in the trabecular meshwork, a small- specialized tissue located in the iris-corneal angle of the ocular anterior chamber. Glaucomatous changes to the trabecular meshwork include a loss in trabecular meshwork cells and the deposition and accumulation of extracellular debris including proteinaceous plaque-like material. In addition, there are also changes that occur in the glaucomatous optic nerve head. In glaucomatous eyes, there are morphological and mobility changes in optic nerve head glial cells. In response to elevated IOP and/or transient ischemic insults, there is a change in the composition of the optic nerve head extracellular matrix and alterations in the glial cell and retinal ganglion cell axon morphologies.

[0015] The term "glaucoma" as used herein thus refers to a disease of the eye characterized by increased pressure inside the eye with resultant optic nerve damage, and includes, without limiting, primary glaucoma, secondary glaucoma, juvenile glaucoma, congenital glaucoma, pseudoexfoliation glaucoma, pigmentary glaucoma, phacolytic glaucoma, steroid-induced glaucoma, uveitic glaucoma, neovascular glaucoma, toxic glaucoma, traumatic glaucoma, acute angle closure glaucoma, absolute glaucoma, chronic glaucoma, narrow angle glaucoma, chronic open angle glaucoma, combined-mechanism glaucoma, simplex glaucoma and familial glaucomas, including, without limitation, high tension glaucoma, low tension glaucoma, and their related diseases.

[0016] In certain embodiments, the pharmaceutical composition of the present invention is used for prevention or treatment of intraocular hypertension and/or glaucoma.

[0017] In particular such embodiments, the glaucoma prevented or treated by the pharmaceutical composition of the invention is primary open angle glaucoma, normal pressure glaucoma, acute angle closure glaucoma, absolute glaucoma, chronic glaucoma, congenital glaucoma, juvenile glaucoma, narrow angle glaucoma, chronic open angle glaucoma, pseudoexfoliation glaucoma, pigmentary glaucoma, phacolytic glaucoma, steroid-induced glaucoma, uveitic glaucoma, neovascular glaucoma, toxic glaucoma, traumatic glaucoma or simplex glaucoma.

[0018] In certain embodiments, the active agent comprised within the pharmaceutical composition of the present invention is R-801, or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0019] R-801 has two asymmetric centers, and may accordingly exist both as enantiomers, i.e., optical isomers (R, S, or racemate, wherein a certain enantiomer may have an optical purity of 90%, 95%, 99% or more) and as diastereoisomers. In other embodiments, the active agent comprised within the pharmaceutical composition of the invention is thus an enantiomer or diastereomer of R-801, or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0020] Optically active forms of R-801 may be prepared using any method known in the art, e.g., by resolution of the racemic form by recrystallization techniques; by chiral synthesis; by extraction with chiral solvents; or by chromatographic separation using a chiral stationary phase. A non-limiting example of a method for obtaining optically active materials is transport across chiral membranes, i.e., a technique whereby a racemate is placed in contact with a thin membrane barrier, the concentration or pressure differential causes preferential transport across the membrane barrier, and separation occurs as a result of the non-racemic chiral nature of the membrane that allows only one enantiomer of the racemate to pass through. Chiral chromatography, including simulated moving bed chromatography, can also be used. A wide variety of chiral stationary phases are commercially available.

[0021] In certain embodiments, the active agent comprised within the pharmaceutical composition of the present invention is a non-toxic pharmaceutically acceptable salt of R- 801 or an enantiomer, diastereomer, or racemate thereof. Suitable pharmaceutically acceptable salts include acid addition salts such as, without being limited to, the mesylate salt, the maleate salt, the fumarate salt, the tartrate salt, the hydrochloride salt, the hydrobromide salt, the esylate salt; the /?-toluenesulfonate salt, the benzoate salt, the acetate salt, the phosphate salt, the sulfate salt, the citrate salt, the carbonate salt, and the succinate salt. Additional pharmaceutically acceptable salts include salts of ammonium (NH ₄ ⁺ ) or an organic cation derived from an amine of the formula (NR ₄ ⁺ ), wherein each one of the Rs independently is selected from H, Q-C22, preferably Ci-C ₆ alkyl, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2,2- dimethylpropyl, n-hexyl, and the like, phenyl, or heteroaryl such as pyridyl, imidazolyl, pyrimidinyl, and the like, or two of the Rs together with the nitrogen atom to which they are attached form a 3-7 membered ring optionally containing a further heteroatom selected from N, S and O, such as pyrrolydine, piperidine and morpholine. Furthermore, where the active agent carries an acidic moiety, suitable pharmaceutically acceptable salts thereof may include metal salts such as alkali metal salts, e.g., lithium, sodium or potassium salts, and alkaline earth metal salts, e.g., calcium or magnesium salts.

[0022] Further pharmaceutically acceptable salts include salts of a cationic lipid or a mixture of cationic lipids. Cationic lipids are often mixed with neutral lipids prior to use as delivery agents. Neutral lipids include, but are not limited to, lecithins; phosphatidylethanolamine; diacyl phosphatidylethanolamines such as dioleoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, palmitoyloleoyl phosphatidylethanolamine and distearoyl phosphatidylethanolamine; phosphatidylcholine; diacyl phosphatidylcholines such as dioleoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, palmitoyloleoyl phosphatidylcholine and distearoyl phosphatidylcholine; phosphatidylglycerol; diacyl phosphatidylglycerols such as dioleoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol and distearoyl phosphatidylglycerol; phosphatidylserine; diacyl phosphatidylserines such as dioleoyl- or dipalmitoyl phosphatidylserine; and diphosphatidylglycerols; fatty acid esters; glycerol esters; sphingolipids; cardiolipin; cerebrosides; ceramides; and mixtures thereof. Neutral lipids also include cholesterol and other 3β hydroxy- sterols.

[0023] Examples of cationic lipid compounds include, without being limited to, Lipofectin ^® (Life Technologies, Burlington, Ontario) (1: 1 (w/w) formulation of the cationic lipid N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride and dioleoylphosphatidyl-ethanolamine); Lipofectamine™ (Life Technologies, Burlington, Ontario) (3: 1 (w/w) formulation of polycationic lipid 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl] -Ν,Ν-dimethyl- 1 -propanamin-iumtrifluoroacetate and dioleoylphosphatidyl-ethanolamine), Lipofectamine Plus (Life Technologies, Burlington, Ontario) (Lipofectamine and Plus reagent), Lipofectamine 2000 (Life Technologies, Burlington, Ontario) (Cationic lipid), Effectene (Qiagen, Mississauga, Ontario) (Non liposomal lipid formulation), Metafectene (Biontex, Munich, Germany) (Polycationic lipid), Eu-fectins (Promega Biosciences, San Luis Obispo, Calif.) (ethanolic cationic lipids numbers 1 through 12: C5 ₂ H ₁₀₆ N ₆ O ₄ 4CF ₃ CO ₂ H, C ₈₈ H ₁₇₈ N ₈ 0 ₄ S ₂ -4CF ₃ C0 ₂ H, C ₄₀ H ₈₄ NO ₃ P CF ₃ CO ₂ H, C ₅ oH ₁₀ N ₇ 0 ₃ -4CF ₃ C0 ₂ H, C ₅ 5H ₁₁₆ N ₈ 0 ₂ 6CF ₃ C0 ₂ H,

C ₄ 9H ₁₀₂ N ₆ O ₃ 4CF ₃ CO ₂ H, C ₄₄ H ₈₉ N ₅ 0 ₃ -2CF ₃ C0 ₂ H, CiooH ₂ o ₆ Ni ₂ 0 ₄ S ₂ 8CF ₃ C0 ₂ H, C ₁₆₂ H ₃₃₀ N ₂₂ O ₉ 13CF ₃ CO ₂ H, C ₄₃ H ₈₈ N ₄ 0 ₂ -2CF ₃ C0 ₂ H, C ₄₃ H ₈₈ N ₄ 0 ₃ -2CF ₃ C0 ₂ H,

C ₄ iH ₇₈ N0 ₈ P); Cytofectene (Bio-Rad, Hercules, Calif.) (mixture of a cationic lipid and a neutral lipid), GenePORTER ^® (Gene Therapy Systems, San Diego, Calif.) (formulation of a neutral lipid (Dope) and a cationic lipid) and FuGENE 6 (Roche Molecular Biochemicals, Indianapolis, Ind.) (Multi-component lipid based non-liposomal reagent).

[0024] In certain embodiments, the active agent comprised within the pharmaceutical composition of the present invention is a prodrug of R-801, or enantiomer, diastereomer, or racemate thereof. The term "prodrug" as used herein refers to a compound that can be metabolized or converted in vivo, in a process termed bioactivation, to provide R-801, or said enantiomer, diastereomer, or racemate thereof. Such a prodrug may be, e.g., a compound corresponding to R-801 wherein the nitroxide group is protected to form an ester group that is hydrolyzed, in vivo, to the corresponding hydroxylamine or N-hydroxyl (N-OH) compound, which is then oxidized to provide R-801. More particular such prodrugs are those wherein the nitroxide group is protected to form an ester group of the formula =N-0-C(0)-Ri, wherein Ri is (d-C ₈ )alkyl, (C ₃ -Ci ₀ )cycloalkyl, or (C ₆ -Ci ₄ )aryl, optionally substituted with one or more groups each independently selected from -OH, - COR ₂ , -COOR2, -(Ci-C ₈ )alkylene-COOR ₂ , -CN, -N0 ₂ , -(Ci-C ₈ )alkyl, -0-(Ci-C ₈ )alkyl, - N(R ₂ ) ₂ , -CON(R ₂ ) ₂ , -SO2R2, -SO2NHR2, or -S(=0)R ₂ ; and R ₂ each independently is H, (Q-C _f alkyl, (C ₃ -C ₁₀ )cycloalkyl, or (C ₆ -C ₁₄ )aryl.

[0025] The term "alkyl" as used herein typically means a straight or branched saturated hydrocarbon radical having 1-8 carbon atoms and includes, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, ie/t-butyl, n-pentyl, isoamyl, 2,2-dimethylpropyl, n- hexyl, n-heptyl, n-octyl, and the like. Preferred are (Ci-C ₄ )alkyl groups, e.g., methyl, ethyl and isopropyl.

[0026] The term "alkylene" typically means a divalent straight or branched hydrocarbon radical having 1-8 carbon atoms and includes, e.g., methylene, ethylene, propylene, butylene, 2-methylpropylene, pentylene, 2-methylbutylene, hexylene, 2-methylpentylene, 3-methylpentylene, 2,3-dimethylbutylene, heptylene, octylene, and the like. Preferred are (Ci-C ₄ )alkylene, e.g., methylene, ethylene and propylene.

[0027] The term "cycloalkyl" as used herein means a cyclic or bicyclic hydrocarbyl group having 3-10 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, bicyclo[3.2.1]octyl, bicyclo[2.2.1]heptyl, and the like. Preferred are (C5-Cio)cycloalkyls, more preferably (C5-C7)cycloalkyls.

[0028] The term "aryl" denotes an aromatic carbocyclic group having 6-14 carbon atoms consisting of a single ring or multiple rings either condensed or linked by a covalent bond such as, but not limited to, phenyl, naphthyl, phenanthryl, and biphenyl.

[0029] The term "aryl" as used herein denotes an aromatic carbocyclic group having 6-14 carbon atoms consisting of a single ring or multiple rings either condensed or linked by a covalent bond such as, but not limited to, phenyl, naphthyl, phenanthryl, and biphenyl.

[0030] The pharmaceutical composition of the present invention, herein also referred to as "ophthalmic composition", can be provided in a variety of formulations and dosages. These compositions may be prepared by conventional techniques, e.g., as described in Remington: The Science and Practice of Pharmacy, 19 ^th Ed., 1995, for example by uniformly and intimately bringing the active agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation. [0031] The ophthalmic composition of the invention, intended for direct application to the eye, may be formulated so as to have both pH and tonicity compatible with the eye. This will normally require a buffer to maintain the pH of the composition at or near physiologic pH, i.e., in the range of about 5 to about 9, preferably about 6 to about 8, more preferably 6.8-7.4; and may further require a tonicity agent to bring the osmolality of the composition to a level at or near 210-320 milliosmoles per kilogram (mOsm/kg). In certain embodiments, the composition of the invention has an osmolality in the range of about 50 to about 700 mOsm/kg, preferably 100-600 mOsm/kg, more preferably 150-500 mOsm/kg, 200-400 mOsm/kg, or 200-350 mOsm/kg.

[0032] The ophthalmic composition of the invention, as defined in any one of the embodiments above, may be administered to the eye of the subject treated by any suitable means. In one embodiment, the composition is in the form of a liquid, e.g., ophthalmic drops, emulsion, suspension, gel, or ointment of the active agent, and it is administered as drops, spray, or gel. In another embodiment, the active agent is applied to the eye via liposomes. In a further embodiment, the active agent is contained within a continuous or selective-release device, e.g., membranous ocular eye patches such as, but not limited to, those employed in the OcusertTM System (Alza Corp., Palo Alto, Calif.). In yet another embodiment, the active agent is formulated as a depot form, for implanting in, or next to, a treated eye.

[0033] In one embodiment, the active agent can be contained within, carried by, or attached to contact lenses, which are placed on the eye. In other embodiments, the active agent is contained within a swab or sponge, or within a liquid spray, which is applied to the ocular surface. In a further embodiment, the ophthalmic composition as defined in any one of the embodiments above is directly injected into the ocular tissues, e.g., by intracameral, subconjunctival, subscleral, or intravitreal injection, or onto the eye surface.

[0034] In addition to the active agent, the ophthalmic composition of the present invention contain a physiologically compatible carrier or vehicle as those skilled in the ophthalmic art can select using conventional criteria. Such vehicles may be selected from known ophthalmic vehicles that include, inter alia, saline solution, water, polyethers such as polyethylene glycol, polyvinyls such as polyvinyl alcohol and povidone, cellulose derivatives such as methylcellulose and hydroxypropyl methylcellulose, cyclodextrins, in particular betahydroxypropyl cyclodextrin, petroleum derivatives, e.g., mineral oil and white petrolatum, animal fats such as lanolin, polymers of acrylic acid such as carboxypolymethylene gel, vegetable fats such as peanut oil, polysaccharides such as dex trans, an alginate such as sodium alginate optionally comprising guluronic acid and/or mannuronic acid, glycosaminoglycans such as sodium hyaluronate, and salts such as sodium chloride and potassium chloride.

[0035] The optimal dosage for administration will depend on the state of the patient, and will be determined as deemed appropriate by the practitioner. In particular, compositions for the treatment of glaucoma may be administered daily, twice daily, or 3-4 times daily, and/or upon the occurrence of symptoms associated with the condition; and over a period of time consistent with treatment of the ocular hypertension and glaucoma, e.g., for a period of weeks, months, years, or decades.

[0036] In another aspect, the present invention relates to R-801, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt, solvate or prodrug thereof, for use in reducing IOP, e.g., for prevention or treatment of intraocular hypertension and/or glaucoma.

[0037] In yet another aspect, the present invention relates to use of R-801, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt, solvate or prodrug thereof, for the preparation of a pharmaceutical composition for reducing IOP, e.g., for prevention or treatment of intraocular hypertension and/or glaucoma.

[0038] In a further aspect, the present invention relates to a method for reducing IOP thereby, e.g., preventing or treating intraocular hypertension and/or glaucoma, in an individual in need thereof comprising administering to the eye of said individual a therapeutically effective amount of R-801, or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0039] The term "therapeutically effective amount" as used herein refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result, i.e., to reduce IOP and consequently treat of prevent intraocular hypertension and/or glaucoma. A therapeutically effective amount of an active agent as referred to herein may vary according to factors including the disease state, age, and sex, and the ability of the active agent to elicit a desired response in the subject treated. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of a disease, the effective amount used for prevention of said intraocular hypertension and/or glaucoma may be less than the effective amount used for treatment of said indications.

[0040] In certain embodiments, the therapeutic agent administered according to the method of the present invention is formulated as ophthalmic drops, emulsion, suspension, gel, ointment, a membranous ocular eye patch, or a depot form.

[0041] In other embodiments, said therapeutic agent is formulated for direct injection into the ocular tissue, and is administered to the subject by intracameral, subconjunctival, subscleral, or intravitreal injection, or onto the eye surface.

[0042] Unless otherwise indicated, all numbers referring to the pH or osmolarity of the composition of the invention used in the present description and/or claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this description and attached claims are approximations that may vary by up to plus or minus 10% depending upon the desired properties sought to be obtained by the present invention.

[0043] The invention will now be illustrated by the following non-limiting Examples.

EXAMPLES

Experimental

[0044] Pairs of normal human eyes from adult donors were obtained from the Minnesota Lions Eye Bank and were placed in anterior segment perfusion culture within 12.4+5.5 hours of death. No eyes had glaucoma or were from patients receiving topical eye medications. The culture technique was similar to that described previously (Fautsch et ah, 2000; Fautsch et ah, 2006; Johnson, 1997; Johnson and Tschumper, 1987). Eyes were bisected at the equator, and the iris, lens, and vitreous were removed. The anterior segment was clamped in a modified Petri dish, and the eye was perfused with Dulbecco's modified Eagle's medium (DMEM; Mediatech, Inc., Manassas, VA) containing antibiotics (penicillin, 10,000 U; streptomycin, 10 mg; amphotericin B, 25 mg; and gentamicin, 1.7 mg in 100 mL medium). Anterior segments were maintained at 37°C in a 5% C0 ₂ atmosphere while being perfused at the normal human flow rate (2.5 μί/ηιίη). Pressure (mm Hg) was continuously monitored in real time with a pressure transducer connected to a second access cannula built into the modified Petri dish and was recorded with an automated computerized system. [0045] The use of donor human eyes for this study was approved by the Mayo Clinic Institutional Review Board and conformed to the tenets of the Declaration of Helsinki.

Example 1. Effects of R-801 on pressure in human cadaver anterior segments

[0046] To test whether R-801 has an effect on outflow facility, human cadaver anterior segments were treated with R-801. In the first experiment with anterior segments from human cadavers, when the baseline pressure in the anterior segments was 16 mm Hg, the pressure dropped to 11 mmHg after R-801 (20 μΜ) was added to the culture media (Fig. 1). When vehicle (dimethyl sulfoxide; DMSO) was added to the fellow eye, there was no pressure decrease. After 72 hours of treatment, drug and vehicle were removed from the eyes, and eyes subsequently perfused with DMEM. Following perfusion, the pressure in the eye that received R-801 returned to near baseline levels (actually a little higher). No change in pressure was observed in the eye incubated with DMSO.

[0047] The baseline pressure in a human cadaver eye was 28 mm Hg (Fig. 2). This initial pressure was higher than normal, but stable. Following R-801 (20 μΜ) addition, the pressure dropped to 16 mm Hg. Relatively no change was observed in the vehicle-treated control eye. Perfusion was stopped after 18 hours.

Example 2. Effects of Intracameral R-801 on intraocular pressure

[0048] Two female cynomolgus monkeys (6-7 years) were included in the study. The left eyes had laser treatments to the trabecular meshwork to elevate IOP. A baseline measurement was taken of both the lasered eye (OHT) and contralateral normotensive (NT) eye using pneumotonometer. Prior to treatment, the monkeys were fasted and administered 5 mg/kg ketamine for sedation. Full strength proparacaine was applied topically, and IOP measurements were taken OU (O cuius uterque) to determine the baseline IOP values over three hours. Betadine solution was applied to the lid and surrounding skin and fur of the OHT eye (OS). 20 μΐ of R-801 (0.5 mg/mL) was filtered through a Millipore filter and then injected into the anterior chamber using a 30 gauge needle inserted between the 2 and 3 o'clock position. The needle was withdrawn taking care to minimize the loss of fluid from the anterior chamber. Gentak ointment was administered to the injected eye and the monkey was allowed to recover. IOP measurements were made at 2, 4, 6, 8, 24, 26, 28, 30 and 96 hours after injection. Gentak ointment was applied after each IOP measurement. Buprenorphine (0.01 mg/kg) intramuscularly (IM) was provided for pain management. [0049] The IOP values measured in the monkey eyes are presented in Fig. 3. While IOP in the vehicle-control eye remained >18 mmHg, R-801 reduced IOP to 12 mmHg. The IOP of the treated eye remained below pretreatment values for 96 hours.

Example 3. Determination of the carbonic anhydrase inhibitory potency of R-801

[0050] An Applied Photophysics stopped-flow instrument was used for assaying the carbonic anhydrase (CA)-catalyzed C0 ₂ hydration activity (Khalifah, 1971). Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.4) and 20 mM NaBF ₄ (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed C0 ₂ hydration reaction for a period of 10-100 seconds. The C0 ₂ concentrations ranged from 1.7 mM to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5-10% of the reaction were used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (10 mM) were prepared in distilled-deionized water, and dilutions up to 0.01 nM were done thereafter with distilled-deionized water. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the enzyme-inhibitor complex. The inhibition constants were obtained by nonlinear least-squares methods using PRISM 3, whereas the kinetic parameters for the uninhibited enzymes from Lineweaver-Burk plots, as previously reported (Hen et ah, 2011; Scozzafava et ah, 1998; Pacchiano et ah, 2011), represent the mean from at least three different determinations. All CAs were recombinant proteins.

[0051] Surprisingly, as shown in Table 1, R-801 was found to strongly inhibit carbonic anhydrases II and IV, while it had moderately inhibited carbonic anhydrases I and XII. Carbonic anhydrases II and IV have been found to regulate fluid secretion into the anterior chamber of the eye (Kim et ah, 2002). Therefore, these data support a plausible mechanism of action for providing therapeutic effects in treating glaucoma.

Table 1. Inhibition data with R-801, and acetazolamide as standard, stopped-flow, C0 ₂ hydrase assay

Mean from 3 different assys, errors in the range of ±10% of the reported data REFERENCES

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