KR20070040326A - Amelioration of cataracts, macular degeneration and other ophthalmic diseases - Google Patents

Abstract

Ophthalmologically acceptable compositions are disclosed that are used to arrest the progression of cataracts, presbyopia, macular degeneration and other retinopathy, glaucoma, uveitis, and various corneal disorders. The composition is also useful as a prophylactic treatment to prevent or delay the progression of age-related ocular disorders, including cataracts, presbyopia, glaucoma and macular degeneration. Wherein said composition is, wherein R ₁ and R ₂ are independently H or C ₁ to C ₃ alkyl; R ₃ and R ₄ are independently C ₁ to C ₃ alkyl; R ₁ and R ₂ together or R ₃ and R ₄ together or both may be cycloalkyl; R ₅ is H, OH, or C ₁ to C ₆ alkyl; R ₆ is C ₁ to C ₆ alkyl, alkenyl, alkynyl, or substituted alkyl or alkenyl; R ₇ is C ₁ to C ₆ alkyl, alkenyl, alkynyl, or substituted alkyl or alkenyl, or R ₆ and R ₇ , or R ₅ , R ₆ and R ₇ together have 3 to 7 atoms in the ring And at least one compound having the formula herein and an ophthalmically acceptable carrier or diluent to form a phosphorus carbocycle or heterocycle.

Description

Improvement of cataracts, macular degeneration and other eye diseases {AMELIORATION OF CATARACTS, MACULAR DEGENERATION AND OTHER OPHTHALMIC DISEASES}

Cross-Reference to Related Applications

The entire text of US Provisional Application No. 60 / 523,803 (filed Nov. 20, 2003) and some US continuing application No. 10 / 440,583 (filed May 19, 2003) claiming its benefits are incorporated herein by reference. .

A composition for improving the progression of cataracts in the eyes of a patient of the present invention and a method for achieving such an improvement. In a preferred embodiment of the invention, the progression or growth of cataracts is essentially stopped. The invention also relates to the treatment of macular degeneration in the eye and certain other uses. According to a preferred embodiment, the compositions of the present invention can be administered to a patient without requiring injection, and can be formulated into eye drops for such administration. Methods of treating cataracts and macular degeneration are provided, and also methods of preparing new compounds and compositions useful in the practice of the present invention.

Various patents and other publications are cited herein. The contents of each of these patents and publications are incorporated herein by reference in their entirety. The entirety of co-pending US application Ser. No. 10 / 440,583 (filed May 19, 2003) is incorporated herein by reference.

Age-related cataracts result from the progressive opacity of the lens of the eye. The disease is currently being treated by surgical removal and replacement of the affected lens. Cataracts, once started, are thought to progress through one or more conventional pathways leading to lens fiber damage. The condition progresses slowly and occurs prominently in the elderly. Alternatively, cataracts may be caused by surgical, radiation, or drug treatment of a patient, for example, surgery of the eye to repair retinal damage (vitrectomy) or surgery of the eye to reduce intraocular pressure elevations; X-irradiation of the tumor; Or after steroid medication. Significantly slowing the rate of cataract progression in such patients can obviate the need for many surgical cataract extractions. This reduction would provide a huge benefit for both individual patients and public health systems.

Less serious but more common ocular conditions are presbyopia. The ocular lens is surrounded by a rigid collagen capsule that is believed to give the lens an elasticity that allows the lens to focus different distances through a change in curvature controlled by ciliary muscles. The lens increases in volume as it ages, and thus gradually loses its elasticity, which reduces the object's ability to focus on near objects. This condition is known as presbyopia and occurs in a large proportion of the elderly population.

In addition to cataracts and presbyopia, the eye can suffer from numerous diseases and other detrimental conditions that affect its ability to function normally. Many such conditions can be found in the process of converting light signals into neural signals and in the interior, most particularly posterior, of the eye with alternating cells, retina and optic nerve in the seventh layer. Diseases and degenerative conditions of the optic nerve and retina are the leading causes of blindness worldwide.

The marked degeneration of the retina is macular degeneration, which is also called age-related macular degeneration (AMD). AMD is the most common cause of vision loss in people over 50 in the United States, and spreads with increasing age. AMD is classified as either wet (angiogenic) or dry (non-angiogenic). The disease is most common in dry form. It occurs when the central retina is distorted, pigmented, or most commonly thinned. Wet formation of the disease is the cause of the most severe vision loss. Wet macular degeneration is generally associated with aging, and other diseases that can cause wet macular degeneration include some intraocular infections such as high myopia and histoplasmosis, which can be exacerbated in individuals with AIDS. . Various factors can contribute to macular degeneration, including genetic properties, age, nutrition, smoking, and sun exposure.

Retinopathy associated with diabetes is the leading cause of blindness in type 1 diabetes and is also common in type 2 diabetes. The degree of retinopathy depends on the duration of diabetes and usually begins to occur more than 10 years after the onset of diabetes. Diabetic retinopathy includes (1) non-proliferative or background retinopathy characterized by increased capillary permeability, edema, bleeding, microvascular perfusion and exudate, or (2) angiogenesis, scarring, fibrotic expansion in the retina to the vitreous And proliferative retinopathy characterized by the possibility of tissue formation and retinal detachment. Diabetic retinopathy is believed to produce free radicals that cause oxidative tissue damage and depletion of cellular reactive oxygen species (ROS) scavengers such as glutathione.

Some other less common but debilitating retinopathies include choroidal neovascular membrane (CNVM), cystic macular edema (CME, also called macular edema, macular swelling), epi-retinal membrane (ERM) (macular Wrinkles) and macular holes. In CNVM, abnormal blood vessels coming from the choroid grow through the retinal layer. Fragile new blood vessels break easily, which causes blood and fluid to accumulate in the retinal layer. In CME, this can occur as a result of a disease, wound or surgery, where fluid gathers in the macular layer, which causes blurred and distorted central vision. ERM (macular wrinkle) is a cellophane-like film that forms on the macular, causing blur and distortion to affect central vision. As this progresses, the film on the macular can pull and cause swelling. ERM is most often found among people over 75 years old. Its etiology is unknown, but may be associated with diabetic retinopathy, posterior vitreous detachment, retinal detachment or trauma, among other conditions.

Another disease inside the eye is uveitis or inflammation into the uvea. The uveal tract is composed of the iris, ciliary body and choroid. The uvea is an intermediate of the three ocular epicardium, sandwiched between the sclera and the retina in its posterior (choroidal) region. Uveitis can be caused by trauma, infection, or surgery and can affect any age group. Uveitis is anatomically classified as anterior uveitis, intermediate uveitis, posterior uveitis or diffuse uveitis. Anterior uveitis affects the anterior part of the eye, including the iris. Intermediate uveitis, or so-called peripheral uveitis, is located in the center of the region immediately behind the iris and lens in the ciliary zone. Posterior uveitis may also shape the retinitis or affect the choroid or optic nerve. Diffuse uveitis covers all parts of the eye.

Glaucoma is a collection of eye diseases that cause vision loss by damaging the optic nerve. Increasing intraocular pressure (IOP) due to inadequate ocular drainage is a major cause of glaucoma. Glaucoma can occur with eye aging, or as a result of eye wounds, inflammation, tumors, or in the progression of cataracts or diabetes. It can also be caused by any drug such as a steroid. Glaucoma can also occur without elevated IOP. This glaucoma shape has been associated with systemic heart disease, such as irregular heartbeats, as well as congenital (ie, family history of normal pressure glaucoma) lineages.

The eye produces about one teaspoon of aqueous humor each day. Normally, the fluid exits the eye at the same speed as it is produced through a sponge mesh of connective tissue called trabecular meshwork. Free radicals and other reactive oxygen species (ROS) cause gradual damage to the fiber strains over time. As a result, the fiber column is partially blocked, the discharge function is reduced, and the IOP rises as more and more water is formed. Initially, even if the IOP does not rise high enough to cause any noticeable symptoms, if the pressure remains elevated or continues to rise, the fibers of the optic nerve are compressed and destroyed, gradually losing vision over the years. Izzotti et al. Provide convincing evidence to link oxidative DNA damage in glaucoma to small but important tissue structures in the excretory system (Izzotti A, Sacca SC, Cartiglia C, De Flora S. Oxidative deoxyribonucleic damage in the eyes of glaucoma patients. Am J Med. 2003; 114: 638-646). They found that the amount of 8-oxo-deoxyguanosine (8-OH-dG) in the trabecular tissue of glaucoma patients increased more than threefold. Increased oxidative DNA damage is further correlated with clinical parameters such as intraocular pressure indicators and visual field loss.

Major features of optic neuropathy of glaucoma include unique changes in the optic nerve head, reduction of ganglion cell viability, and loss of vision. The cascade of events correlates optic nerve head degeneration with the slow death of retinal ganglion cells observed in this disease, and the cascade of events can be slowed or prevented through the use of neuroprotective agents (Osborne et al., 2003, Eur. J. Ophthalmol. 13 (Supp3): S19-S26), antioxidants and free radical scavengers are important classes of this (Hartwick, 2001, Optometry and Vision Science 78: 85-94).

The outermost layer of the eye, the cornea, controls and focuses the light entering the eye. The cornea must remain transparent to properly refract light. In addition, the cornea helps protect the rest of the eye from germs, dust and other harmful substances, and importantly acts as a filter to block some of the most harmful ultraviolet (UV) wavelengths of sunlight. Without this protection, the lens and retina will be very easily damaged from UV radiation.

In addition, the cornea and surrounding conjunctiva are affected by various adverse conditions that can impair vision. These conditions include not only other disorders (eg, dry eye syndrome), but also, in some cases, those resulting from inflammatory reactions, such as allergic reactions, infections or trauma, and various dystrophy (one of the cornea due to the formation of cloudy substances). Partial abnormalities lose their normal transparency), such as Fuk's dystrophy, keratoconus, lattice dystrophy and keratoconjunctival dystrophy.

Ocular surface and lacrimal gland inflammation has been found to play a role in the development of ocular surface epithelial disease called dry keratoconjunctivitis. Oxidative tissue damage and polymorphonuclear leukocytes exhibiting oxidative potential occur in the tear layer of patients suffering from dry eye. This reaction leads to serious damage to the tissue involved. Free radicals and inflammation may be associated with the onset or self-propagation of this disease (Augustin, AJ et al., "Oxidative reactions in the tear fluid of patients suffering from dry eyes" Graefe's Arch. Clin.l Exp.l Ophthalmol. 1995, 11: 694-698).

Blepharitis is an inflammation of the eyelids. Eyelid conjunctivitis is an inflammation of the eyelids and eye conjunctiva. Both conditions are associated with a condition known as ocular injection, although other causes may exist. Blepharitis is an abnormal condition in which the resulting tears contain excess lipids (oily components in natural tears) and, in some cases, also contain irritating oils. As described below, the oil component prevents evaporation of the aqueous layer that wets the corneal epithelium of the eye and serves to help spread the aqueous layer through the aqueous cornea normally during blinking. If excess oil is present, the lipid layer will tend to adhere to the cornea itself. If the eye is unable to remove the oil from the corneal surface, a "dry" area occurs on the cornea because the aqueous layer cannot hydrate the area.

Rosacea is a disease of the skin (acne injection) and eye (eye injection) with varying signs and unknown etiology. The clinical and pathological features of the ocular disease are not specific and the disease is being widely investigated by ophthalmologists.

Retinal phototoxicity is caused by exposure of the eye to retinal illumination from a laser used by an operating microscope or group placed for transient entry eye surgery. Such light sources have the potential to cause photoinduced damage in the fovea (MA Pavilack and RD Brod "Site of Potential Operating Microscope Light-induced Phototoxicity on the Human Retina during Temporal Approach EyeSurgery" Ophthalmol. 2001, 108 (2) ): 381-385; HF McDonald and MJ Harris "Operating microscope-induced retinal phototoxicity during pars plana vitrectomy" Arch.Ophthalmol. 1988 106: 521-523; Harris MD et al. "Laser eye injuries in military occupations" Aviat.Space Environ.Med. 2003, 74 (9): 947-952). In addition, damage may occur upon treatment of the cut corneal surface after excimer laser phototherapy (Seiji Hayashi et al. "Oxygen free radical damage in the cornea after excimer laser therapy" Br. J. Ophthalmol. 1997 , 81: 141-144).

Certain corneal disorders are noncorrectable and can only be treated by corneal transplantation, while other diseases can be corrected by phototherapy laser keratectomy (PTK), ie excimer laser surgery, and also in the process of corneal aging. It is known to cause inflammatory reactions that cause hazing or corneal opacity sites.

In addition, the skin around the eye is affected by diseases and disorders. In particular, injection of the eyelids and blepharitis are disorders that can be serious. Ocular injection is a common and potentially unknown ocular disorder with indeterminate etiology (Stone D. U. and J. Chodosh, 2004 Curr. Opin. Ophthalmol. 15 (6): 499-502). The blepharitis of the eye may be staphylococcal blepharitis, seborrheic blepharitis, a mixed or most severe form of ulcerative blepharitis.

Oxidative stress may be associated with uveitis (eg, Zamir et al., 1999, Free Rad. Biol. Med. 27: 7-15), cataracts (eg, M. Lou, 2003, Prog. Retinal & Eye Res. 22). : 657-682), glaucoma (eg, Babizhayev & Bunin, 2002, Curr. Op. Ophthalmol. 13: 61-67), corneal and conjunctival inflammation, various corneal dystrophy, postoperative or UV-related corneal damage (eg , Cejkova et al., 2001, Histol.Histopathol. 16: 523-533; Kasetsuwan et al. 1999, Arch.Ophthalmol. 117: 649-652) and presbyopia (Moffat et al., 1999, Exp.Eye Res.69 : 663-669) as well as the progression and acceleration of numerous eye diseases or disorders, including AMD described above and various retinopathies (see, eg, Ambati et al., 2003, Survey of Ophthalmology 48: 257). -293; Berra et al., 2002, Arch. Gerontol.Geriatrics 34: 371-377). For this reason, agents with antioxidant properties have been studied as potential therapeutics for the treatment of such disorders. Many studies have focused on biochemical pathways, such as glutathione synthesis and circulation, that produce reducing power in cells. In addition, enzymes that reduce activated oxygen species, such as superoxide dismutases, have been studied to determine if they reduce oxidative stress in cells. In addition, compounds that inhibit lipid oxidation at the cell membrane by direct radical scavenging have been considered promising therapeutic interventions.

Nitroxides are stable free radicals capable of reducing to their corresponding hydroxylamines. Such compounds are of interest because of their radical scavenging properties that mimic the activity of superoxide disproportionase enzymes and exert anti-inflammatory effects in animal models of various oxidative damage and inflammation.

Because of its relatively low toxicity, hydroxylamine is the preferred nitroxide as a therapeutic agent.

Certain hydroxylamine compositions are known for preventing or delaying cataracts. Zigler et al. US Pat. No. 6,001, 853, the contents of which are incorporated herein by reference, reflects a study conducted by the US National Health Organization. Zigler et al. Identified hydroxylamines that improved cataract development or progression when administered to the eyes of test animals. Such administration is necessary via injection for physicochemical reasons. Zigler et al. Disclosed that delivering tempol-H by liquid eye drops would be clinically convenient, but did not report any examples. Zigler's hydroxylamine is actually administered by subconjunctival injection. In addition, Zigler's material involved co-administration of the reducing agent via injection, systemically or otherwise. Subsequent studies at the National Institutes of Health have been directed to the identification of effective hydroxylamine for topical administration, but no efforts have been considered successful.

Accordingly, there has been intense research activity to identify compounds and compositions containing them that can improve cataract formation and progression in the eyes of patients without the need for unpleasant, uncomfortable and potentially dangerous intraocular injections. In particular, there has been a long need for such compounds and compositions that can be administered via topical application, in particular via eye drops, and none have been achieved.

Summary of the Invention

The present invention provides a composition for treating cataracts in the eyes of a patient who is known to be progressing or at risk of cataract formation or suspected of having a cataract formation. The compositions also provide for the treatment of macular degeneration, various retinopathies, glaucoma, uveitis, corneas, eyelids or conjunctiva, and presbyopia in the eyes of patients with or at risk of degenerative conditions such as do. According to a preferred embodiment, the composition is formulated in topical liquid form, in particular as eye drops. Periodic application of the compositions of the present invention delays or stops the progression of cataracts or macular degeneration in the eye being treated. The present invention provides compositions that do not need to be applied via injection or other difficult or inconvenient route.

According to a preferred embodiment, the present invention provides a composition comprising a compound of the formula and an ophthalmologically acceptable carrier or diluent:

In the compounds, R ₁ and R ₂ are independently H or C ₁ to C ₃ alkyl, and R ₃ and R ₄ are independently C ₁ to C ₃ alkyl. In addition, according to certain embodiments, it is also possible for R ₁ and R ₂ together or R ₃ and R ₄ together or both to form a cycloalkyl moiety. In the compounds of the present invention, R ₅ is H, OH, or C ₁ to C ₆ alkyl and R ₆ is C ₁ to C ₆ alkyl, alkenyl, alkynyl, or substituted alkyl or alkenyl. R ₇ is C ₁ to C ₆ alkyl, alkenyl, alkynyl, or substituted alkyl or alkenyl or C ₁ -C ₆ cycloalkyl or heterocycle. It is also possible for R ₆ and R ₇ , or R ₅ , R ₆ and R ₇ together to form a carbocycle or heterocycle having 3 to 7 atoms in the ring. As used herein, the term “ophthalmology” means useful for the treatment of the eye and its diseases.

In the compounds used in the compositions of the present invention, the substituted alkyl or alkenyl species is at least one hydroxy, alkoxy, alkylthio, alkylamino, dialkylamino, aryloxy, arylamino, benzyloxy, benzylamino or heterocycle Or YCO-Z, wherein Y is O, N, or S and Z is an alkyl, cycloalkyl or heterocyclic or aryl substituent. According to some embodiments, the heterocycle is a five, six or seven membered ring having one or more oxygen, sulfur, or nitrogen atoms in the ring. In one preferred composition, R ₆ and R ₇ together are cyclopropyl, in another, R ₆ and R ₇ together are tetrahydrofuranyl and R ₅ , R ₆ and R ₇ together are furanyl.

For certain preferred compounds, each R ₁ to R ₄ is C ₁ to C ₃ alkyl, most particularly ethyl or methyl, most particularly methyl. In some preferred embodiments, the compounds of the invention R ₆ is is a C ₁ to C ₆ alkyl substituted with a benzyloxy group, or one or more C ₁ to C ₆ alkoxy.

In other preferred compounds, each of R ₁ to R ₄ is methyl, R ₅ is H or methyl, R ₆ is methyl substituted with benzyloxy or C ₁ to C ₆ alkoxy, R ₇ is methyl, or R ₆ And R ₇ forms a cyclopropyl group. In other preferred compounds, each of R ₁ to R ₄ is methyl, R ₅ is methyl, R ₆ is ethoxymethyl, and R ₇ is methyl. In other preferred compounds, each of R ₁ to R ₄ is methyl, R ₅ is methyl, R ₆ is benzyloxymethyl, R ₇ is methyl, while each R ₁ to R ₄ is methyl and R ₅ is Methyl, R ₆ is hydroxymethyl, R ₇ is methyl is also useful.

In some embodiments, each of R ₁ to R ₄ is methyl, R ₅ , R ₆ , and R ₇ form a furanyl group, or R ₅ is H and R ₆ and R ₇ form a tetrahydrofuranyl group Compounds are also preferred. Further embodiments provide compounds wherein R ₁ to R ₄ are all methyl, R ₅ is H, and R ₆ and R ₇ form a cyclopropyl ring.

The composition of the invention is preferably formulated in an aqueous medium, which can be delivered to the eye in topical liquid form, for example via eye drops.

Accordingly, the pH and other properties of the compositions of the present invention are ophthalmologically acceptable for topical application to the patient's eye. For some embodiments, the compound is in the form of a salt, preferably a hydrochloride or similar salt.

The composition of the present invention may contain more than one compound of the present invention. In addition, the compositions may contain other compounds known in the art for use in the treatment of certain indications, in combination with the compound (s) of the invention. In some embodiments, the compounds of the present invention are administered simultaneously. In another embodiment, the compounds of this invention are administered sequentially. Similarly, other compounds known in the art for use in the treatment of the diseases and disorders described herein may be administered simultaneously or sequentially with the compound (s) of the invention. The present invention also provides a method of treating diseases and disorders using such combination therapy.

Since the compounds of the present invention comprise an oxidizable hydroxylamine moiety that is most effective in the chemical reduction state, in certain embodiments, the composition preferably further contains an antioxidant, in particular a sulfhydryl compound. As exemplary compounds, other antioxidants suitable for ocular administration, such as ascorbic acid and salts thereof or sulfite or sodium metabisulfite, may also be used, but mercaptopropionyl glycine, N-acetylcysteine, mercaptoethyl Amines, glutathione and similar species. The amount of hydroxylamine may range from about 0.1% w / v to about 10.0% w / v, with about 0.25 to about 10.0% w / v preferred.

The present invention may also be viewed as providing an ophthalmic composition containing an ophthalmologically acceptable carrier or diluent together with a compound having an N-hydroxypiperidine moiety bound to a soluble modified moiety. In this way, the active moiety hydroxylamine can be delivered to the lens of the eye in need of treatment in the form of a “stealth”, ie a chemical compound that may have a hydroxylamine moiety that is cleaved from the rest of the molecule. have. The compound degrades in the eye, resulting in active hydroxylamine species for effective treatment of cataracts, macular degeneration or any other eye disorder mentioned herein. The compound so provided has a solubility in water of at least 25 ° C. of at least about 0.1% by weight and a water-n-octanol partition coefficient at 25 ° C. of at least about 3. In a preferred embodiment, the water solubility is greater than about 0.5% by weight, preferably greater than about 2.0% by weight and the partition coefficient is greater than about 5, preferably greater than about 10.

Accordingly, it is preferred that the compound of use is penetrated into the cornea and converted into the desired hydroxylamine, preferably N-hydroxypiperidine, upon topical administration to the eye. The conversion preferably occurs through enzymatic cleavage of the compound. In one preferred embodiment, the hydroxylamine moiety comprises 1,4-dihydroxy-2,2,6,6-tetramethylpyridine.

The present invention includes methods for improving—slowing or completely blocking cataract progression—in a patient lens. Similarly, the present invention provides presbyopia, macular degeneration, various retinopathy, glaucoma, uveitis, corneal disorders (especially trauma, inflammation (both can be caused by excimer laser surgery), aging, UV exposure and other oxidative related disorders. Related to), conjunctival disorders (conjunctivitis), dry eye syndrome, blepharitis, and methods for improving or treating the progression of eye injections. The present invention also provides a method of protecting the retinal pigment epithelium against photooxidative damage.

In one embodiment, the method comprises administering an ophthalmic composition containing an ophthalmologically acceptable carrier or diluent to the eye in the form of an eye drop containing a compound having at least one compound as an active ingredient therein. The administration is preferably performed multiple times, and in certain preferred embodiments, long term periodic administration is performed.

In another aspect of the invention, the ophthalmic composition of the invention is intended to prevent or delay the progression of certain age-related ocular conditions, including cataracts, presbyopia, corneal degeneration and dystrophy, glaucoma, macular degeneration and photooxidative retinal damage. Used as a prophylactic treatment. In a preferred embodiment, the composition is formulated as an eye drop or eye wash. The composition is administered to the eye either before or at an early stage of the eye's age related condition or to the eye to prevent the progression of later disease.

The present invention also provides a method for identifying a medicament that can be delivered to the lens of a patient in the form of eye drops. These methods are compounds having a solubility in water at 25 ° C. of at least about 0.1% by weight and a water / n-octanol partition coefficient at 25 ° C. of at least about 5, which are enzymatically divided under conditions obtained in the patient's eye, preferably in the eye, preferably Includes selecting a compound that generates a proximate drug to treat the condition of the lens. Preferably, the active pharmaceutical species is one having a hydroxylamine, in particular an N-hydroxypiperidine nucleus.

1 shows the aqueous level of tempol-H (1,4-dihydroxy-2,2,6,6-tetramethylpiperidine) in rabbit eyes topically treated with Compound 1 of the invention or with Tempol-H. Depicts.

2 depicts tempol-H levels in various rabbit eye tissues after topical administration of Compound 1 at selected post-treatment time points.

3 shows that various concentrations of Tempol-H protect blue light illuminated A2E-laden RPE cells.

4 shows the effect of 1 mM tempol-H on the protection of blue light illuminated A2E-loaded RPE cells.

The invention provides cataracts, presbyopia, uveitis, macular degeneration or diabetic retinopathy, choroidal neovascular membrane (CNVM), cystic macular edema (CME), epi-retinal membrane (ERM) (macular folds) and macular hole Other retinopathy, including but not limited to, and corneas (and not limited to inflammation, trauma, irradiation damage, corneal neovascularization and various dystrophy such as, for example, Fuk's dystrophy, keratoconus, lattice dystrophy and keratoconjunctival dystrophy). Compounds and compositions that can be topically administered to the eye of a patient exhibiting or progressing or at risk of disorders of the peripheral eyelids and conjunctiva), and dry eye syndrome, blepharitis and eye injection. Although hydroxylamine species, which are already known to be effective in delaying cataract progression, can be seen to be included in the compounds as chemical segments, achieving a topically applicable compound is a very important development in therapeutic technology.

Indeed, the National Health Organization, the assignee of the Zigler patent, has tried to identify compounds that may be effective in the treatment of cataracts or macular degeneration through topical application but failed. In that document, the Zigler patent details the administration of certain compositions, such as Tempol-H, via injection, and recognizes the desirability of topical administration via eye drops, but the proposed route of administration is not actually available. Note that it was found. Accordingly, the present invention should be viewed as "pioneering" and as satisfying what has long been needed in the art but has not been provided.

We have demonstrated that the compounds of the invention are absorbed across the cornea and sclera, through the uvea, and into the lens and inside the eye. Enzymatic processes in these tissues cleave the N-hydroxypiperidine moiety of the compound from the esterified acid. Once vitrified, the N-hydroxypiperidine moiety has the same efficacy and performs the same function as demonstrated by Zigler. A further advantage of the compounds of the present invention is also found to have free radical-scavenging and antioxidant activity as observed in tempol-H and other non-esterified hydroxylamine compounds, even in their esterified form. It is.

The compounds of the present invention were previously unknown for administration to the eye. They are certainly not known for use in the treatment of cataracts, presbyopia, corneal disorders, macular degeneration, retinopathy, glaucoma or uveitis.

US Pat. No. 5,981,548 (in the name of Paolini et al.), The contents of which are incorporated herein by reference, details the use of certain N-hydroxypiperidine esters and antioxidants in many contexts. However, Paolini does not disclose topical treatment of ophthalmic formulations or patient eyes. However, Paolini does not disclose useful synthesis for certain molecules of this form.

US Patent No. 4,404,302 to Gupta et al., The contents of which are incorporated herein by reference, discloses the use of certain N-hydroxylamines as light stabilizers in plastics formulations. US Pat. No. 5,462, 946 to Mitchell et al., Discloses certain nitroxides derived from substituted oxazolidines to protect the organism from oxidative stress. U.S. Patent No. 3,936,456, the contents of which are incorporated herein by reference, in the name of Ramey et al., Provides substituted piperazine dione oxyls and hydroxides for stabilization of polymers. US Patent No. 4,691,015 to Behrens et al., The contents of which are incorporated herein by reference, describes the use of certain of these for the stabilization of hydroxylamines and polyolefins derived from hindered amines. .

In one embodiment, the present invention provides a composition comprising a compound of the formula and a pharmaceutically acceptable carrier or diluent:

Wherein R ₁ and R ₂ are independently H or C ₁ to C ₃ alkyl;

R ₃ and R ₄ are independently C ₁ to C ₃ alkyl; R ₁ and R ₂ may be together or R ₃ and R ₄ together or both may be cycloalkyl;

R ₅ is H, OH, or C ₁ to C ₆ alkyl;

R ₆ is C ₁ to C ₆ alkyl, alkenyl, alkynyl, or substituted alkyl or alkenyl;

R ₇ is C ₁ to C ₆ alkyl, alkenyl, alkynyl, substituted alkyl, alkenyl, cycloalkyl, or a heterocycle, or R ₆ and R ₇ , or R ₅ , R ₆ and R ₇ together in a ring A carbocyclic ring or heterocyclic ring having 3 to 7 atoms is formed. In addition, these compounds may be used with an ophthalmically acceptable carrier for use in ophthalmic compositions.

In one embodiment, the invention relates to an N-hydride bound to a solubility modifying moiety that is a compound having a solubility in water at 25 ° C. of at least about 0.25% by weight and a water-n-octanol partition coefficient at 25 ° C. of at least about 5. Provided are compounds having an oxy piperidine moiety and an ophthalmically acceptable carrier or diluent. The composition may have an N-hydroxy piperidine moiety cleavable from the compound under conditions in the eye. It is foreseeable that the part is divided under the intra-lens condition of the eye. The N-hydroxy piperidine moiety can be enzymatically cleaved. There may also be such compositions in which the N-hydroxy piperidine moiety is 1-oxyl-4-hydroxy-2,2,6,6-tetramethylpiperidyl.

As used herein, the term C ₁ to C _n alkyl, alkenyl, or alkynyl means a hydrocarbyl group having 1 to n carbon atoms therein. Thus, the term includes methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, and various isomeric forms of pentyl, hexyl, and the like. Likewise, the term includes ethenyl, ethynyl, propenyl, propynyl, and similar branched and unbranched unsaturated hydrocarbon groups having up to n carbon atoms. If the context permits, the group may be, for example, one or more hydroxy, alkoxy, alkylthio, alkylamino, dialkylamino, aryloxy, arylamino, benzyloxy, benzylamino, heterocycle, or YCO-Z, wherein Y Is O, N, or S, and Z is an alkyl, cycloalkyl, heterocyclic or aryl substituent.

The term carbocycle refers to a cyclic structure or ring in which all atoms forming the ring are carbon. Examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like. Cyclopropyl is one preferred species. Heterocycles define a cyclic structure in which at least one of the atoms in the ring is not carbon. Examples of this broad class include furan, dihydrofuran, tetrahydrofuran, pyran, oxazole, oxazoline, oxazolidine, imidazole and others, especially those having an oxygen atom in the ring. 5-, 6- and 7-membered rings having one or more oxygen or nitrogen atoms in the ring are preferred heterocycles. Furanyl and tetrahydrofuranyl species are particularly preferred.

In certain embodiments, lower alkyl is preferred wherein each R ₁ to R ₄ is C ₁ to C ₃ alkyl. Preferably, all of these groups are methyl due to the known efficacy of the moiety having a substitution at that position and for convenience of synthesis. However, other substituents may also be used.

In certain embodiments, R ₆ is a C ₁ to C ₆ alkyl substituted with one or more C ₁ to C ₆ alkoxy or benzyloxy is used. Especially, the compound which has an ethoxy or benzyloxy substituent is preferable. Among preferred compounds each R ₁ to R ₄ is methyl, R ₅ is H or methyl, R ₆ is methyl substituted with C ₁ to C ₆ alkoxy or benzyloxy, R ₇ is methyl or R ₆ and R ₇ There are compounds that form this cyclopropyl group and compounds in which each of R ₁ to R ₄ is methyl, R ₅ is methyl, R ₆ is ethoxy or benzyloxy methyl, and R ₇ is methyl. Further preferred compounds are compounds wherein each of R ₁ to R ₄ is methyl, R ₅ is methyl, R ₆ is hydroxymethyl and R ₇ is methyl.

Other useful compounds are those in which each of R ₁ to R ₄ is methyl, R ₅ , R ₆ , and R ₇ form furanyl groups, or R ₆ and R ₇ form tetrahydrofuranyl groups. Compounds in which R ₁ to R ₄ are methyl, R ₅ is H and R ₆ and R ₇ form a cyclopropyl ring are further preferred species as described in the Examples below.

The compounds of the present invention are formulated into compositions for application to the eye of a patient in need of therapy. Thus, the compositions are adapted as eye drops or for pharmaceutical use in contact lenses, inserts and the like, which are described in more detail below. As such, formulating the compound in sterile water containing any desired diluents, salts, pH modifiers, and the like, known to pharmaceutical formulators, can be carried out to achieve a solution compatible with ocular administration. Eye drops, inserts, contact lenses, gels and other topical liquid forms may require somewhat different formulations. All such formulations that are consistent with each other in that they are administered directly to the eye are included herein.

In addition, the composition of the present invention may have an antioxidant in a range that can vary depending on the type of antioxidant used. In addition, its use depends on the amount of antioxidant needed to allow the pharmaceutical composition to have a shelf life of at least two years. One or more antioxidants may be included in the formulation. Certain commonly used antioxidants have a maximum level allowed by regulatory authorities. Therefore, the dosage of antioxidant (s) should be sufficient to be effective without causing any inadequate effects. Such dosages can be adjusted by the physician as needed within the maximum levels set by the regulatory authority, and appropriate and effective dosages are well determined within the authority of the skilled technician. Reasonable ranges are from about 0.01 to about 0.15 weight / volume% EDTA, about 0.01 to about 2.0 weight / volume% sodium sulfite, and about 0.01 to about 2.0 weight / volume% sodium metabisulfite. One skilled in the art can use each of these at a concentration of about 0.1% weight / volume. N-acetylcysteine may be present in the range of about 0.1 to about 5.0 weight / volume, with a hydroxylamine concentration of about 0.1 to about 10% being preferred. Ascorbic acid or salt may also be present in the range of about 0.1 to about 5.0 weight / volume%, with a hydroxylamine concentration of about 0.1 to about 10 weight / volume% being preferred. If included, other sulfhydryls may be present in the same range as N-acetylcysteine. Other exemplary compounds include mercaptopropionyl glycine, N-acetyl cysteine, β-mercaptoethylamine, glutathione and similar species, and other antioxidants suitable for ocular administration, such as ascorbic acid and salts or alcohols thereof Pits or sodium metabisulfite may also be used.

Buffering agents can be used to maintain the pH of the eye drop formulation in the range of about 4.0 to about 8.0; This is essential to prevent corneal irritation. Since the compounds of the present invention are esters, the pH is preferably maintained at about 3.5 to about 6.0, preferably about 4.0 to about 5.5, to prevent hydrolysis of the ester bonds and to ensure product shelf life of at least two years. . In addition, the pH ensures that most of the hydroxylamine is in protonated form for the highest aqueous solubility. Buffers include any weak acid and partner base (pKa = about 4.0 to about 5.5); For example acetic acid / sodium acetate; Citric acid / sodium citrate. The pKa of hydroxylamine is about 6.0. For direct intravitreal or intraocular injection, the formulation should have a pH of 7.2 to 7.5, preferably 7.3-7.4.

In addition, the compounds of the present invention may include tonicity agents suitable for ocular administration. Among others, sodium chloride is suitable for making the formulation of the present invention approximately isotonic with 0.9% saline solution.

In certain embodiments, compounds of the invention are formulated with a viscosity enhancer. Examples are hydroxyethylcellulose, hydroxypropylcellulose, methylcellulose, and polyvinylpyrrolidone. The viscous agent may be present up to about 2.0 weight / volume of the compound. It may be desirable for the viscosity enhancer to be present in the range of about 0.2 to about 0.5 weight / volume. Preferred ranges for polyvinylpyrrolidone may be from about 0.1 to about 2.0 weight / volume. One skilled in the art may prefer any range established as acceptable by the Food and Drug Administration.

Cosolvents of the invention can be added, if necessary. Suitable cosolvents may include glycerin, polyethylene glycol (PEG), polysorbate, propylene glycol, mannitol and polyvinyl alcohol. The cosolvent may be present in the range of about 0.2 to about 4.0 weight / volume%. It may be desirable for mannitol to be formulated in the compounds of the present invention in the range of about 0.5 to about 4.0 weight / volume. It may also be desirable for the polyvinyl alcohol to be formulated in the compounds of the present invention in the range of about 0.1 to about 4.0 weight / volume. One skilled in the art may prefer the range acceptable by the Food and Drug Administration.

Preservatives can also be used in the present invention within certain ranges. Among them, benzalkonium chloride of 0.013% by weight / vol% or less, benzetonium chloride of 0.013% / vol% or less, chlorobutanol of 0.5% / vol% or less, phenyl secondary mercury acetate or nitrate of 0.004% / vol% or less, Preference is given to thimerosal of up to 0.01 weight / volume and methyl or propylparaben of from about 0.01 to about 0.2 weight / volume.

Injectable formulations are preferably designed for single use administration and contain no preservatives. Injection solutions should have isotonic equivalents of 0.9% sodium chloride solution (osmolality of 290-300 mOsmole). This may be accomplished by the addition of sodium chloride or other cosolvents as listed above, or excipients such as buffering agents and antioxidants as listed above. Injectable formulations are sterile and, in one embodiment, are provided in single use vials or ampoules. In other embodiments, the injectable product may be provided as a sterile lyophilized solid for subsequent reconstitution and injection.

The composition of the present invention may contain more than one compound of the present invention. Thus, the compositions of the present invention contain one or more compounds of the present invention. In some embodiments, the compounds of the present invention are administered simultaneously. In another embodiment, the compounds of this invention are administered sequentially. The methods of the invention include combination therapies.

In some embodiments of the invention, the compound (s) of the invention are administered in combination with other compounds known in the art useful for the treatment of diseases or disorders targeted by the compounds of the invention. Thus, the compositions of the present invention may further contain one or more other compounds known in the art for the treatment of the disease or disorder to be treated. Other compound (s) known in the art may be administered simultaneously with the compound (s) of the invention or may be administered sequentially. Similarly, the methods of the present invention include the use of such combination therapies.

The tissue in front of the eye (including the lens) is filled with ocular water. The fluid is in a highly reducing redox state because it contains antioxidant compounds and enzymes. In addition, the lens is a highly reducing environment, which allows the hydroxylamine compound to remain in the desired reduced form. Thus, it may be advantageous to include the reducing agent in the eye drop formulation, or to administer the reducing agent separately to maintain the hydroxylamine in the reduced form.

Preferred reducing agents may be in the form of N-acetylcysteine, ascorbic acid or salts thereof, and sodium sulfite or metabisulfite, and ascorbic acid and / or N-acetylcysteine or glutathione are particularly suitable for injectable solutions. Combinations of N-acetylcysteine and sodium ascorbate can be used in various formulations. Metal chelator antioxidants such as EDTA (ethylenediaminetetraacetic acid) or possibly DTPA (diethylenetriaminepentaacetic acid) may also be added to keep the hydroxylamine in reduced form in the eye drop formulation.

It is prominent that the compounds of the present invention exhibit anti-caractogenic and other therapeutic effects in two ways. First, the ester compound is hydrolyzed in place to form a hydroxylamine component that exhibits therapeutic activity. Second, the esterified compound itself has anti-cataractogenic and antioxidant activity, thereby supporting the therapeutic efficacy of the pharmaceutical formulation containing the compound.

With regard to the first basis for the activity of the compounds of the invention, namely the cleavage to release the hydroxylamine component, it is known that a number of esterases are present in the ocular tissues, in particular the cornea. It is not necessary to identify the specific esterase (s) that cleave this class of esters for the practice of the present invention. The cleavage of the ester occurs quickly and essentially completely when the compound is administered to rabbit eyes. At all times (30, 60, 90 and 120 minutes) after topical administration, Tempol-H appears to be present in the aqueous fluid. In contrast, the ester is stable in aqueous solution, for example a solution of ester 4 in acetate buffer at 40 ° C., pH 4.6, is stable for 3 months.

The present invention is optimally used to improve the progression of cataracts in the patient eye. Other optimal uses include the treatment of macular degeneration in the patient retina. Other optimal uses include treating, reducing or preventing the progression of diabetic retinopathy and various other retinopathies as described herein, as well as treating uveitis and glaucoma. Another optimal use is the treatment of corneal disorders, especially those associated with oxidative stress such as inflammation or trauma (which may be surgery but not necessarily surgery), and various dystrophy. Compounds and compositions of the present invention also provide stimulation during laser surgery of the eye to reduce, prevent or ameliorate photocatalytic damage to retinal pigment epithelium, and including keratectomy for glaucoma and corneal resection for corneal remodeling. And for inflammation improvement. In addition, the compounds and compositions can be used to treat diseases and disorders of the conjunctiva and eyelids. In addition, the compounds and compositions of the present invention can be used to treat alopecia and rectal tissue damage following radiation therapy.

Many of the disorders and conditions described herein, in particular cataracts, presbyopia and macular degeneration, are conditions that progress during the aging process. Accordingly, the compositions of the present invention can be used to benefit as a prophylactic treatment to prevent or delay the progression of age related ocular conditions. In a preferred embodiment, the composition is formulated as an eye drop or eye wash. It is administered to the eye either before or after the progression of the age-related state of the eye.

Compositions containing a compound of the present invention may be delivered to the eye of a patient in one or more of several delivery aspects known in the art. In a preferred embodiment, the composition is delivered topically to the eye as eye drops or eye washes. In another embodiment, the composition is delivered as a topical ophthalmic ointment, which is particularly useful for treating conditions of the cornea, conjunctiva or surrounding skin such as dry eyes and blepharitis. In another embodiment, the composition is administered via periodic subconjunctival or intraocular injection or infusion in an irrigating solution such as BSS® or BSS PLUS® (Alcon USA, Port Worth, TX) or It can be delivered to various locations within the eye by using a preformulated solution of hydroxylamine in the composition, such as BSS® or BSS PLUS®. In one embodiment, the use of a compound of the invention in vitrectomy may be effective in reducing or preventing the progression of vitrectomy related cataracts.

Alternatively, the composition may be other ophthalmic dosage forms known to those skilled in the art, such as preformed or insitu-formed, for example, as disclosed in US Pat. No. 5,718, 922 to Herrero-Vanrell. ) Gel or liposomes. Direct injection of the drug used into the vitreous body for the treatment of the disease was used, where microspheres or liposomes were used to release the drug slowly (Moritera, T. et al., "Microspheres of biodegradable polymers as a drug-delivery system in the vitreous "Invest. Ophthalmol. Vis. Sci. 1991 32 (6): 1785-90).

In other embodiments, the composition may be applied to or in contact with the lens of the eye in need of treatment via a contact lens (eg, Lidofilcon B, Bausch & Lomb CW79 or DELTACON (Deltafilcon A)) or other object temporarily residing on the eye surface. Can be delivered through. For example, US Pat. No. 6,410,045 describes a contact lens-type drug delivery device comprising a polymeric hydrogel contact lens containing a drug substance absorbed in a contact lens that can be delivered to the ocular fluid at a concentration of 0.05 to 0.25 weight percent. It describes.

In other embodiments, supports such as collagen corneal protectors (eg, BIO-COR soluble corneal protectors, Summit Technology, Watertown, Mass.) May be used. The composition can also be obtained from an osmotic pump via cannula (ALZET®, Alza Corp., PaloAlto, Calif.) Or a composition of sustained release capsules (OCCUSENTO) or biodegradable discs (OCULEXO, OCUSERTO) comprising the composition. It can be administered by injection into the eye by implantation. The route of administration has the advantage of continuously supplying the composition to the eye.

Many types of drug delivery systems are known in the art and can be used to deliver the compositions of the present invention. Non-limiting examples have been described above and are listed further below.

Preferred methods of treating dry eye syndrome utilize aqueous solutions or gels that may be formulated to contain one or more compounds of the invention. The "active" component in artificial tear formulations is conventional water soluble or dispersible polymers such as hydroxyethylcellulose, hydroxypropylmethylcellulose, methylcellulose, carboxymethylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene glycol , Carbomer and poloxamer.

U. S. Patent No. 6,429, 194 describes aqueous ophthalmic preparations for pre-soaking or storing solid devices for instillation into an eye or for insertion into an object such as a contact lens, ointment or conjunctival sac. The ophthalmic preparations comprise mucin components similar to those found on normal human eye surfaces. In addition, US Pat. No. 6,281,192 describes an ophthalmic application of mucins.

Ophthalmic solutions should provide an effective and long lasting treatment of dry eye syndrome. One approach to achieving this goal is to provide solutions with tailored rheological properties, ie high viscosity solutions that flow or are produced under stress. Examples of this approach are disclosed in US Pat. Nos. 5,075,104 and 5,209,927, wherein the rheological properties of ophthalmic solutions are achieved through the use of camober polymers. Such carbomer polymers are disclosed in U.S. Patents 5,225, 196; No. 5,188,828; It was found to be a bioadhesive as described in US Pat. Nos. 4,983,392 and 4,615,697. The bioadhesion of carbomers is believed to contribute to longer residence times in the eye. In fact, U.S. Pat.Nos. 5,075,104 and 5,209,927 reveal the function by carbomer polymers maintaining or restoring normal hydration equilibrium of epithelial cells, protecting the cornea in a manner similar to what is thought to be provided by the mucin component in normal tears. Teach.

US Pat. No. 4,883,658 describes a synergistic combination of an aqueous solution of partially hydrolyzed poly (vinyl acetate) and fully hydrolyzed poly (vinyl acetate), ie poly (vinyl alcohol), which is low at the water-air interface. While exhibiting surface tension, a completely wettable absorbent layer is formed over the hydrophobic solid. The combination is suitable for use as an aqueous vehicle for topically used ophthalmic drugs or nutrients.

Emulsion-based formulations for the treatment of dry eye syndrome have recently been known. U.S. Patent 5,578,586; No. 5,371,108; 5,294,607; 5,278,151; 5,278,151; One approach as disclosed in US Pat. No. 4,914,088 utilizes methods and compositions for reducing evaporation of an aqueous layer at the eye surface. The method comprises applying an admixture of charged phospholipids and nonpolar oils to the eye, preferably in the form of a finely divided oil-in-water emulsion. Other approaches are described in US Pat. Nos. 4,818,537 and 4,804,539, where liposome compositions in emulsion form are reported to relieve dry eye syndrome by enhancing retention at the ocular surface.

Several other types of delivery systems are particularly suitable for delivering the pharmaceutical composition to the interior or back of the eye. For example, US Pat. No. 6,154,671 (Parel et al.) Discloses an apparatus for delivering a drug into the eye by iontophoresis. The device utilizes a reservoir for holding the active agent, which includes one or more active surface electrodes facing the eye tissue at the periphery of the cornea. The reservoir also has a return electrode in contact with the partially closed eyelid of the patient. U.S. Patent No. 5,869,079 to Wong et al. Discloses a combination of hydrophilic and hydrophobic moieties in biodegradable sustained release ocular implants. In addition, U.S. Pat.No. 6,375,972 (Guo et al.), U.S. Pat. ) And US Pat. No. 6,251,090 to Avery et al. Each of the above describes intraocular implant devices and systems that can be used to deliver a pharmaceutical composition containing a compound of the present invention.

U. S. Patent No. 4,014, 335 describes an ocular drug delivery device located in a cul-de-sac between the sclera and the lower eyelid for administering the drug and acting as a reservoir. The device includes a three layer stack of polymeric material that retains the drug in the central reservoir region of the stack. The drug diffuses from the reservoir through one or more of the polymeric layers of the laminate.

Solid devices in the form of ocular inserts have been used to alleviate long-term symptoms of dry eyes. These devices are placed in the eye and slowly dissolve or erode to provide a thick tear film. Examples of such techniques are described in US Pat. No. 5,518,732; 4,343,787, and 4,287,175.

The compounds of the present invention may have use in a wider range of fields other than ophthalmology. Such areas may include, for example, protecting hair follicles and rectum from radiation damage during radiation therapy of cancer. Other dosage forms of the compositions of the present invention that do not require delivery to the eye include oral tablets, solutions and sprays; Intravaginal, subcutaneous and intraperitoneal injections; Application to the skin as a patch or ointment; Enemas, suppositories, or aerosols may be included.

For effective treatment of cataracts, presbyopia, macular degeneration, glaucoma or any other retinopathy, corneal disorder or eye condition described herein, those skilled in the art can recommend appropriate dosing schedules and dosages for the subjects to be treated. When the composition is formulated in a sustained delivery vehicle or delivered via an implant, less frequent administration may be possible. For topical delivery, it may be desirable to administer one to four times a day for as long as necessary. In addition, the dosing schedule may vary depending on the needs of the patient and the active drug concentration, which may depend on the hydroxylamine used. It may be desirable for the active amount to be from about 0.1 to about 10.0 weight / volume in the formulation. In some embodiments, it may be desirable for the active drug concentration to be from 0.25 to about 10.0 weight / volume. The concentration of the hydroxylamine component will preferably range from about 0.1 μM to about 10 mM in tissue and fluid. In some embodiments, the range is 1 μM to 5 mM, and in other embodiments, the range is about 10 μM to 2.5 mM. In another embodiment, the range is about 50 μΜ to 1 mM. Most preferably the hydroxylamine concentration will range from 1 to 100 μΜ. The concentration of reducing agent will be 1 μM to 5 mM in tissues or fluids, preferably in the range of 10 μM to 2 mM. Component concentrations in the composition are suitably adjusted to the route of administration by typical pharmacokinetics and dilution calculations to achieve such local concentrations. Alternatively, corneal infiltration and absorption into other tissues inside the eye are demonstrated using radiolabeled hydroxylamine.

An ophthalmologist or other similar person skilled in the art will have various means for monitoring the efficacy of the dosing schedule and adjusting the dosing accordingly. For example, efficacy in cataract treatment can be determined by an ophthalmologist by observing lens opacity at intervals by slit-lamp examination or other means known in the art. Efficacy in the treatment of macular degeneration or other retinopathy can be determined by assessing the improvement of vision and the grade and ideality of stereoscopic color fundus photograph (Age-Related Eye Disease Study Research Group, NEI, NIH). , AREDS Report No. 8,2001, Arch.Ophthalmol. 119: 1417-1436). The efficacy of the treatment of uveitis can be determined by improving vision and vitreous haze and controlling inflammation (Foster et al., 2003, Arch. Ophthalmol. 121: 437-40). In accordance with this assessment, the ophthalmologist may adjust the frequency and / or concentration of the dosage, if necessary.

Another aspect of the invention is a method of identifying a constraint for delivery to a patient's eye in the form of an eye drop, wherein the water solubility at 25 ° C. is at least about 0.25% by weight and the water-n-octanol partition coefficient at 25 ° C. is about 5 The above compound is characterized by a method comprising selecting a compound that is enzymatically cleaved under conditions obtained from a lens of a patient's eye to generate hydroxylamine, preferably N-hydroxy piperidine. Preferably the compound selected is an ester of hydroxylamine.

Solubility of at least 0.1% may be desirable for eye drops, even suspension formulations. Compounds that are completely water-insoluble may not be effective. Preference is given to esters which are soluble in water (> 0.1% w / v). Esters with a solubility of less than 0.1% can be used in the form of suspensions or ointments or other formulations. Solubility is determined by mixing 100 mg of test compound with 1 ml of water at room temperature, adding additional 1 ml of water and mixing until the ester is completely dissolved.

Corneal infiltration is shown by measuring the effective hydroxylamine and / or esters at substantial concentrations (eg,> 5 μM) in intraocular water following administration of the compound solution in vivo to rabbit eyes. This is determined by electron spin resonance (ESR), high performance liquid chromatography (HPLC) or gas chromatography (GC) analysis of rabbit stable water. In vitro corneal infiltration methods may also be used prior to in vivo licensing methods, in particular for screening compounds. Penetration of the compound into the interior or posterior part of the eye is indicated by measuring the concentration of the compound in the vitreous fluid, uveal or retina after administration of the compound solution to rabbit eyes.

Esters are selected for these tests based on the calculated or measured value of the octanol / water partition coefficient (P). Hydrophilic compounds, such as tempol-H, cannot penetrate the lipophilic epithelial layer of the cornea. The partition coefficient of the penetrating tempol-H and ester is as follows:

P (calculated value) *

Tempol-H 0.8 (Measure, 0.5)

Ester 4 16.4

Ester 8 8.2

Ester 14 6.3

* Clog P version 4.0, Biobyte Corporation

Enzymatic conversion essentially completes greater than 90% ester hydrolysis to alcohol and acid in vivo after administration of the compound to rabbit eyes.

The conversion can be determined by HPLC or GC analysis of selected eye tissues (eg, aqueous water). Alternatively, enzymatic conversion can be determined by incubating the compound in plasma or eye tissue and monitoring the degradation rate by analyzing the sample periodically by HPLC or GC. Esters having a half-life of less than about 1 or 2 hours are preferred candidates. The method may be a preferred screening procedure prior to in vivo testing.

The ester should have a degree of hydrolysis of less than about 10% at 40 ° C. in an aqueous solution of pH 4.0-5.0 after 3 months. It extrapolates the ester half-life in solution at least 18 months at room temperature, which may be desirable for eye drop products.

Specific embodiments of the present invention will be described with reference to the following examples. The following examples are for illustrative purposes only and are not intended to be limiting in any way.

Example 1 Determination of Stability of Ester Compounds in Aqueous Solution

Method: A 0.1-0.5% solution of ester compound was prepared in buffer (pH 4.5-5.0) containing DTPA or EDTA. The solution was filled into amber glass vials and sealed and placed in a temperature controlled vessel maintained at 40 ° C. Sample vials were removed periodically and stored at 0-5 ° C. until analyzed by HPLC, GC, or GC / MS analysis and found to be stable after 3 months under these conditions.

To be useful as an anti-cataract drug, the agent must penetrate into the lens. It may be included in the method for selecting anti-cataract compounds. A detailed description of the method for Tempol-H follows:

Example 2: Penetration of Drugs in Organ Cultured Rat Lenses

Compared to drugs that have already been tested as anti-cataract agents, Tempol-H and Tempol have a marked ability to penetrate lens tissue from surrounding fluids. The experiments described in this section determined time course, active compound concentration and compound distribution in the lens after incubation with rat lens under organ culture conditions.

Methods: Rat lenses were incubated as follows: Rat lenses were obtained from SpragueDawley rats. The lenses were incubated in a 24-well cluster dish in modified TC-199 medium and placed in a 37 ° C. incubator with 95% air / 5% CO ₂ atmosphere. The lens was incubated in 2 ml of culture medium and adjusted to 300 milliomol (mOsm). The lenses were incubated for 1 to 24 hours in culture medium with 4.0 mM tempol-H or 4.0 mM oxidized tempol. At appropriate times, the lenses were removed from the medium, blotted dry, homogenized and analyzed for active compounds by electron spin resonance (ESR). In one experiment, lenses were incubated for 4 hours and analyzed by incision into epithelial, cortical and nuclear sections.

Results: The concentration of tempol-H (mM, in lens water) reached 0.4 mM, 0.8 mM and 1.0 mM, respectively, after 1, 2 and 4 hours incubation of the active ingredient. After incubation of the oxidized tempolo lens, the levels of tempol-H were 0.6 mM, 1.5 mM and 2.8 mM, respectively. In the latter case, only a small amount (up to 5%) of oxidized tempol was found in the lens; Almost completely converted to reduced tempol-H.

After 4 hours incubation with tempol-H, the distribution of tempol-H between the lens epithelium, cortex and nucleus was fairly even. The level of tempol-H reached 1.5 mM, 0.8 mM and 1.0 mM in the epithelium, cortex and nucleus, respectively. The levels of tempol-H / tempol in lenses incubated with oxidized tempol were 1.2 mM, 2.9 mM and 2.0 mM, respectively. In the latter case, all compounds in the nucleus were reduced and only about 5% in the epithelium was oxidized.

CONCLUSIONS: Both the reduced and oxidized forms of the active agent readily penetrated from the bath medium into the cultured rat lens and distributed in the epithelium, cortex and nucleus. Incubation of the lens with an oxidized tempol generates a high concentration of reducing compound Tempol-H throughout the lens.

Example 3: 1-oxyl-4- (3'-ethoxy-2 ', 2'-dimethyl) propanecarbonyloxy-2,2,6,6-tetramethylpiperidine

1,1'-Carbonyldiimidazole was purified by 3-ethoxy-2,2-dimethylpropionic acid (750 mg, 7.13 mmol; in DMF (10 mL); J. Org. Chem., 38, 2349, 1975] A small amount (1.27 g, 7.84 mmol) was added to a stirred solution prepared according to the procedure described in the above, the contents of which are incorporated herein by reference. Intense gas evolution was observed. The solution was heated at 100 ° C. for 1 hour. Then to this mixture, tempol (900 mg, 5.23 mmol) and 1,8-diazabicyclo [5,4,0] undec-7-ene (DBU) (800 mg, 5.26 mmol) were added, 12 Heating was continued for a time. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (100 mL), washed successively with 1N HC1, saturated NaHCO ₃ and brine, dried over anhydrous sodium sulfate and concentrated in vacuo to give a red solid (1.48 g). Purification by column chromatography on silica gel using cyclohexane: ethyl acetate (8: 1) as eluent gave a red crystalline solid (1.22 g, 70.0%).

IR (KBr, cm-1): 1360 (N-O.), 1725 (ester)

Example 4 1-hydroxy-4- (3'-ethoxy-2 ', 2'-dimethyl) propanecarbonyloxy-2,2,6,6-tetramethylpiperidine hydrochloride

The nitroxide of Example 2 (1.02 mg, 3.34 mmol) was added to saturated hydrogen chloride solution (20 mL) in ethanol. Red quickly disappeared and the resulting yellow solution was boiled to give a clear colorless solution. The solution was concentrated in vacuo, dissolved in 100 mL ethyl acetate and washed with saturated NaHCO ₃ to give hydroxylamine free base. The ethyl acetate layer was separated and concentrated to give a red oil, which was mostly nitroxide by TLC.

The oil was purified by column chromatography on silica gel using cyclohexane: ethyl acetate (4: 1) as eluent to afford a red crystalline solid (700 mg). The solid was dissolved in saturated hydrogen chloride solution (20 mL) in ethanol, concentrated in vacuo and recrystallized from ethyl acetate: diisopropylether (2: 1.50 mL) to give a white crystalline solid (320 mg). m. p. 140-142 ° C. (dec.).

¹ H-NMR (270 MHz, D ₂ 0) ppm: 1.48 (6H, s); 1.57 (3H, t); 1.63 (12 H, s); 1.82 (2H, s); 2.02 (2H, t); 2.40 (2H, d), 3.88 (2H, q); 5.44 (1 H, m)

IR (KBr, cm ^-1 ): 3487 (OH), 1726 (ester)

Mass spectrum (EI, m / z) 301 (M +)

Example 5a: 1-oxyl-4-cyclopropanecarbonyloxy-2,2,6,6-tetramethylpiperidine

A suspension of sodium hydride (60% in oil, 1.0 g, 25 mmol) in dry THF (50 mL) was stirred at room temperature for 5 minutes and to the mixture was added tempol (4.0 g, 23 mmol). The mixture was stirred for 1 hour, cyclopropanecarbonyl chloride (2.4 g, 23 mmol) was added dropwise over 5 minutes and then refluxed for 1 hour. The reaction mixture was concentrated under reduced pressure. The residue was taken up in pentane (100 mL), the supernatant was separated and concentrated under reduced pressure to give a red solid. The solid was purified by column chromatography on silica gel using cyclohexane: ethyl acetate (3: 1) as eluent to afford a red crystalline solid (1.4 g, 5.8 mmol, 25.3%).

IR (KBr, cm-1): 1361 (N-O.), 1720 (ester)

Example 5b: Alternative Method-1-oxyl-4-cyclopropanecarbonyloxy-2,2,6, 6-tetramethylpiperidine

1,1'-Carbonyldiimidazole (1.78 g, 11 mmol) was added in small portions to a stirred solution of cyclopropanecarboxylic acid (860 mg, 10 mmol) in dry DMF (10 mL). Intense gas evolution was observed. The solution was heated at 40 ° C. for 1 hour. To the mixture is added tempol (1.72 g, 10 mmol) and 1,8-diazabicyclo [5,4,0] undec-7-ene (DBU) (1.52 g, 10 mmol) and again for 12 hours Heated at 40 ° C. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (100 mL) and washed successively with 1N HCl, saturated NaHCO ₃ and brine. The ethyl acetate layer was separated, dried over anhydrous sodium sulfate and concentrated in vacuo to give a red solid. The solid was purified by column chromatography on silica gel using cyclohexane: ethyl acetate (8: 1) as eluent to afford a red crystalline solid (720 mg, 30.0%).

IR (KBr, cm-1: 1360 (N-O.), 1720 (ester)

Example 5c: Alternative Method-1-oxyl-4-cyclopropanecarbonyloxy-2,2,6,6-tetramethylpiperidine [DCC / DMAP esterification method]

To a stirred solution of tempol (1.72 g, 0.01 mmole), cyclopropanecarboxylic acid (0.946 g, .011 mmole), and DMAP (0.12, .001 mmole) in dichloromethane (25 ml) DCC (2.27 g, 0.11 mmole) ) Was added and the mixture was stirred at rt overnight. The mixture was filtered through celite and the solution was evaporated under reduced pressure. The product was isolated by silica gel column chromatography first using hexane and then using 10% ethyl acetate in hexane. Yield: 2.26 g (94.1). IR and NMR were consistent with the selected structure.

Example 6: 1-hydroxy-4-cyclopropanecarbonyloxy-2,2,6,6-tetramethylpiperidine hydrochloride (Compound 1)

Nitroxide (2.2 g, 9.15 mmol) of Example 5a was added to saturated hydrogen chloride solution (20 mL) in ethanol. Red quickly disappeared and the resulting yellow solution was boiled to give a clear colorless solution. The solution was concentrated in vacuo, dissolved in 100 mL ethyl acetate and washed with saturated NaHCO ₃ to obtain hydroxylamine free base. The ethyl acetate layer was separated, acidified with etheric HCl and concentrated to give a white solid, which was recrystallized from ethanol (10 mL) as a white crystalline solid (1.15 g, 4.13 mmol, 45.1%, mp224-228 ° C.). (dec.)).

¹ H-NMR (270 MHz, D ₂ 0) ppm: 0.97 (4H, d); 1.43 (1 H, m); 1.44 (6 H, s), 1.46 (6 H, s); 1.90 (2H, t); 2.28 (2H, t); 5.2 (1 H, m)

IR (KBr, cm-1): 3478 (OH), 1720 (ester)

Mass spectrum (EI, m / z) 240 (M +)

Example 7: 1-hydroxy-4-cyclopropanecarbonyloxy-2,2,6,6-tetramethylpiperidine hydrochloride (alternative method)

Nitroxide (700 mg, 2. 91 mmol) of Example 5a was added to saturated hydrogen chloride solution (20 mL) in ethanol. Red quickly disappeared and the resulting yellow solution was boiled to give a clear colorless solution. The solution was concentrated in vacuo, dissolved in 100 mL ethyl acetate and concentrated to half volume to give a white crystalline solid (627 mg, 2.25 mmol, 77.5%). m.p. 224-227 ° C. (dec.). 1 H-NMR (270 MHz, D 20) ppm: 0.97 (4H, d); 1.43 (1 H, m); 1.44 (6 H, s), 1.46 (6 H, s); 1.90 (2H, t); 2.28 (2H, t); 5.2 (1 H, m)

IR (KBr, cm-1): 3476 (OH), 1720 (ester)

Mass spectrum (EI, m / z) 240 (M +)

Example 8 1-oxyl-4- (3'-benzyloxy-2 ', 2'-dimethyl) propanecarbonyloxy-2,2, 6,6-tetramethylpiperidine

3-benzyloxy-2,2-dimethylpropionic acid (1.04 g, 5 mmol) in dry DMF (5 mL), prepared by a method similar to that described in J. Org. Chem., 38, 2349, 1975. A small amount of 1,1'-carbonyldiimidazole was added to the stirred solution of. Intense gas evolution was observed. The solution was heated at 50 ° C. for 30 minutes. To the mixture was added tempol (900 mg, 5.23 mmol) and 1,8-diazabicyclo [5,4,0] undec-7-ene (DBU) (800 mg, 5.26 mmol). The mixture was heated at 50 ° C. for 3 days (monitored by TLC) and then concentrated under reduced pressure. The residue was dissolved in ethyl acetate (100 mL), washed successively with 1N HCl, saturated NaHCO ₃ and brine and dried over anhydrous sodium sulfate. The dry solution was concentrated in vacuo to give a red solid (1.48 g). The solid was purified by column chromatography on silica gel using cyclohexane: ethyl acetate (3: 1) as eluent to give a red crystalline solid (1.02 g, 2.8 mmol, 56.2%).

IR (KBr, cm-1): 1359 (N-O.), 1732 (ester)

Example 9 1-hydroxy-4- (3'-benzyloxy-2 ', 2'-dimethyl) propanecarbonyloxy-2,2,6,6-tetramethylpiperidine hydrochloride

The nitroxide of Example 8 (1.02 mg, 3.34 mmol) was added to saturated hydrogen chloride solution (20 mL) in ethanol. Red quickly disappeared and the resulting yellow solution was boiled to give a clear colorless solution. The solution was concentrated in vacuo, filtered, washed with ethyl acetate and dried in vacuo (0.700 mg, 2.4 mmol, 82.7%). p. 215-220 ° C. (dec.). The residue was dissolved in ethyl acetate (20 mL). Hexane (20 mL) was added and the product started to oil out; The mixture was then left for 12 hours. The solvent was decanted to give an oily residue which was treated with isopropyl ether and warmed. Cooling the mixture gave a waxy solid which was recrystallized from ethyl acetate to give a white crystalline solid (0.6 g, 1.5 mmol, 45%).

1 H-NMR (270 MHz, D20) ppm; 1.26 (6 H, s), 1.51 (6 H, s); 1.65 (6 H, s); 2.01 (2H, t); 2.44 (2H, d), 5.40 (1H, m); 3.46 (2H, s), 4.55 (2H, S), 7.31 (5H, s)

IR (KBr, cm-1): 3480 (OH), 1712 (ester), 710 (aromatic)

Mass spectrum (EI, m / z) 262 (M +)

Example 10 1-hydroxy-4- (3'-hydroxy-2 ', 2'-dimethyl) propanecarbonyloxy-2,2,6,6-tetramethylpiperidine hydrochloride

Pd / C (5%, 100 mg) was dissolved in ethanol in 1-oxyl-4- (3'-benzyloxy-2 ', 2'-dimethyl) propanecarbonyloxy-2,2,6,6-tetramethylpy To a solution of ferridine (1.O g, 3.83 mmol) was added and the mixture was hydrogenated at 45 psi for 12 hours in a Paar hydrogenation apparatus. The reaction mixture was filtered through celite and concentrated in vacuo to afford a clear colorless oil which was purified by column chromatography on silica gel using cyclohexane: ethyl acetate (3: 1) as eluent to give a colorless oil. The oil was dissolved in saturated hydrogen chloride solution (20 mL) in ethanol and concentrated in vacuo. Upon standing, the product crystallized and recrystallized from ethanol (123 mg, 0.4 mmol, 10.4%). m. p. 210-215 ° C. (dec.).

1 H-NMR (270 MHz, CDCl ₃ ) ppm of free base: 1.14 (6H, s), 1.44 (6H, s); 1.57 (6 H, s); 1.70 (2H, m); 2.8 (1H, s, br), 3.65 (2H, s), 5.16 (1H, m)

IR (KBr, cm-1): 3480 (OH), 1712 (ester), 710 (aromatic)

Mass spectrum (EI, m / z) 262 (M +)

Example 11 1-oxyl-4- (1-methyl-cyclopropane) carbonyloxy-2,2,6,6-tetramethylpiperidine

A suspension of sodium hydride (60% in oil, 2.2 g) in dry THF (80 mL) was stirred at rt for 5 min before tempol (3.0 g, 17.44 mmol) was added. The mixture was stirred for 30 minutes, 1-methyl-cyclopropanecarbonyl chloride (2.2 g, 18.71 mmol) was added dropwise for 5 minutes and then refluxed for 12 hours. The reaction mixture was concentrated under reduced pressure, and the residue immediately crystallized. The product was purified by column chromatography on silica gel using cyclohexane: ethyl acetate (3: 1) as eluent to give a red crystalline solid (2.0 g, 7.86 mmol, 45.1%).

IR (KBr, cm-1): 1314 (N-O.), 1722 (ester)

Example 12 1-hydroxy-4- (1-methyl-cyclopropane) carbonyloxy-2,2,6,6-tetramethylpiperidine hydrochloride

The nitroxide of Example 11 (700 mg, 2.91 mmol) was added to saturated hydrogen chloride solution (10 mL) in ethanol. Red quickly disappeared and the resulting yellow solution was boiled to give a clear colorless solution. The solution was concentrated in vacuo to give a white crystalline solid, which was filtered, washed with ethyl acetate and dried in vacuo (0.700 mg, 2.4 mmol, 82.7%). p. 215-220 ° C. (dec.).

1 H-NMR (270 MHz, D 20) ppm: 0.80 (2H, d); 1.19 (2H, m); 1.21 (2H, s); 1.44 (15 H, s); 2.03 (4H, m); 5.10 (1 H, m)

Mass spectrum (EI, m / z) 254 (M +)

Example 13: 1-oxyl-4- (2-furan) carbonyloxy-2,2,6,6-tetramethylpiperidine

A stirred mixture of sodium methoxide (25% sodium methoxide in methanol, 200 mg) in benzene (100 mL) was heated to reflux and benzene was gradually distilled off to give a fine suspension of solid sodium methoxide. To the mixture was added tempol (1.76 g, 10 mmol), methyl 2-furoate (1.26 g, 10 mmole) and benzene (50 mL).

Distillation of benzene was continued for 8 hours to remove methanol formed. More benzene was added to maintain the volume of benzene in the flask. The benzene layer was washed with 1 H HCl, then with water, dried over anhydrous sodium sulfate and evaporated to dryness to give a red solid (1.72 g) which was recrystallized from hexane to give 1.45 g of product. Further purification by column chromatography on silica gel using cyclohexane: ethyl acetate (3: 1) as eluent gave a red crystalline solid (1.02 g, 3.82 mmol, 32.8%).

IR (KBr, cm-1): 1364 (N-0.), 1716 (ester), 706 (aromatic)

Example 14 1-hydroxy-4- (2'-furan) carbonyloxy-2,2,6,6-tetramethylpiperidine hydrochloride

The nitroxide of Example 13 (300 mg, 1.13 mmol) was added to saturated hydrogen chloride solution (10 mL) in ethanol. Red quickly disappeared and the resulting yellow solution was boiled to give a clear colorless solution. The solution was left at room temperature for 1 hour, whereby a white crystalline solid separated. It was filtered, washed with ethyl acetate and dried in vacuo to give hydroxylamine (220 mg, 0.72 mmol, 64.5%, m.p. 209.4-210.4 ° C.).

1 H-NMR (270 MHz, D 2 O) ppm: 1.49 (6H, s); 1.62 (6H, s); 2.03 (2H, t); 2.42 (2H, d), 5.49 (1H, m); 6. 63 (1H, q); 6.64 (1H, d), 7.34 (1H, d), 7.74 (1H, s)

Mass spectrum (EI, m / z) 266 (M +)

Example 15 1-oxyl-4- (3'-tetrahydrofuran) carbonyloxy-2,2,6,6-tetramethylpiperidine

To a stirred solution of 3-tetrahydrofurancarboxylic acid (1.5 g, 13 mmol) in dry DMF (20 mL) was added small portions of 1,1'-carbonyldiimidazole (2.3 g, 14.18 mmol). Intense gas evolution was observed. The solution was heated to 70 ° C. for 1 hour. Then to the mixture, tempol (2.23 g, 12.97 mmol) and 1,8-diazabicyclo [5,4,0] undec-7-ene (DBU) (2.0 g, 13.14 mmol) are added, 12 Heating was continued for a time. The reaction mixture was poured into 250 mL water and extracted with ether (2 x 100 mL). The ethereal layers were combined, washed successively with 1N HC1, saturated NaHCO ₃ and brine, dried over anhydrous sodium sulfate and concentrated in vacuo to give a red solid (2.05 g), which was obtained from ethyl acetate: hexane (1: 2) Recrystallization gave pure red crystalline solid nitroxide (1.45 g, 5.36 mmol, 37.8%).

IR (KBr, cm-1): 1360 (N-O.), 1725 (ester)

Example 16 1-hydroxy-4- (3'-tetrahydrofuran) carbonyloxy-2,2,6,6-tetramethylpiperidine hydrochloride

Nitroxide of Example 15 (300 mg, 1. 11 mmol) was added to saturated hydrogen chloride solution (10 mL) in ethanol. Red quickly disappeared and the resulting yellow solution was boiled to give a clear colorless solution. The solution was left at room temperature for 1 hour, whereby a white crystalline solid separated. It was filtered, washed with ethanol and dried in vacuo to give the product (146 mg, 0.48 mmol, 42.86%, m.p. 221.0-223.2 ° C.). 1 H-NMR (270 MHz, DMSO-d 6) ppm: 0.84 (2H, m); 0.90 (2H, m); 1.35 (6 H, s); 1.46 (6 H, s); 1,65 (1 H, m); 2.13 (2H, t); 2.44 (2H, d), 5.14 (1H, m)

Example 17 Uptake of Representative Compounds Across Animal Cornea

A group of six New Zealand white rabbits was used in the study to assess the uptake of Tempol-H and Compound 1. Test compounds were prepared in sterile saline solution at a concentration of 3.5% weight / volume. Animals were placed in cages during the dropping of 50 μL of eye drops to each eye using a micropipette. After administration, eyes were gently closed for 60 seconds. The rabbits were administered twice daily for four consecutive days. On day 5, rabbits were dosed once and euthanized at 30 min (2), 60 min (2), and 120 min (2) post-dose. Immediately after euthanasia, water was collected from each rabbit. The waterproofing concentration of temporal-H in each sample was measured using electron spin resonance (ESR).

The intraocular water level of tempol-H after administration of tempol-H was below the detectable assay limit at all time points (see FIG. 1). After dosing Compound 1, the temporal water level of Tempol-H was maximal at 30 minutes post dosing and was still present 2 hours post dosing (see FIG. 1 and Table I).

Table I: Water Level Concentration: Absorption Studies in Rabbits

Dosage: 50 μL of 3.5% solution, single dose (N = 4 eye / timepoint)

Concentration of Tempol-H μM 30 minutes 60 mins 120 minutes Compound 1 51.0 20.0 1.5 30.0 30.0 1.2 18.0 6.0 7.0 30.0 3.0 8.0 Average 32.3 14.8 4.4 Μg / ml (5.5) (2.5) (0.75)

Example 18 Identification of Metabolism of Compound 1 in Rabbit Eyes

From the in vivo rabbit study described in Example 16, an aqueous solution sample was prepared of Compound 1 and its metabolites, Tempol-H and carboxylic acid (R ₁ COOH) (formed by hydrolysis of Compound 1 by ocular esterase). The presence was confirmed by GC / MS. Both metabolites were observed and compound 1 was absent. It was confirmed that Compound 1 was converted completely to the metabolite.

Anhydrous samples were lyophilized in 10 mL amber stained glass vials containing small magnetic rods. To this, 1 mL of methylene chloride was added and the solution was stirred for 2 minutes and then left for 5 minutes. 3 μL aliquots of methylene chloride were injected into a GC column. Cyclopropanecarboxylic acid was detected in the mass spectrometer detector at m 12 / m = 13.02 (ret. Time) (GC model 5989B and MS and 5890 series II (both manufactured by HP)). An Agilent DB-5 column of 25 m in length and 0.2 mm in diameter was used. Carrier gas was He and 22 cm / sec. The inlet temperature was 250 ° C. and the detector was 280 ° C. For each injection, the temperature was held at 35 ° C. for 5 minutes, then increased to 240 ° C. at 10 ° C./min and held at 240 ° C. for 3 minutes. Splitless injection was used.

Example 19 Tolerability of Compound 1 In Vivo in Rabbit Eyes

Eye drops containing 3.5% Compound 1 were administered six times at 1 hour intervals to each eye of two conscious rabbits. The drug is well tolerated and there were no negative findings in this preliminary study.

Example 20 Eye Bioavailability in Rabbits

Ocular bioavailability of Compounds 2 and 3 was evaluated in New Zealand white rabbits. Each compound was dissolved at 125 mM concentration in 10 mM phosphate buffer at pH 7.0. The concentration is equal to 3.5% for compounds 2 and 3. 50 μL was applied to the cornea of both eyes of each rabbit 6 times at 1 hour intervals. Two rabbits were used for each compound. One rabbit treated with each compound was euthanized 30 minutes after the last dose and the second rabbit was euthanized 90 minutes after the last dose.

After death, the eyes of each rabbit were immediately removed and blood samples collected from the orbits. Syringes were collected from each eye using a syringe, and then the lenses were split from the eyes. The capsule / epitheli was carefully separated from the fibrous material and the two portions were frozen in dry ice (fibrous material in sealed vials without addition of capsule / epithelial and liquid in 100 μL of 5 mM DTPA (diethylenetriaminepentaacetic acid) solution). ). Likewise, the waterproof and solution samples were quickly frozen. The remainder of each eye, including the cornea, retina, sclera and vitreous, was frozen for future dissection and analysis. All samples were shipped from dry ice to the laboratory and stored at −75 ° C. until processed.

The waterproofing concentration of Tempol-H in each sample was measured using electron spin resonance (ESR). Analyzes of the water showed that both compounds penetrated the cornea and entered the waterproof chamber. The highest concentrations of both compounds were present in the 30 minute sample and the 90 minute sample significantly decreased in concentration. Small amounts (each compound 2-3 μM) were also detected in the blood.

Example 21 Aqueous Water Concentrations of Compounds 2 and 3 in Rabbits

Table H: Doses of Compounds 2 and 3: 50 μL of 125 mM solution, time interval x 6 (N = 2 eye / timepoint)

Tempol-H concentration, μM 30 minutes 90 mins blood Compound 3 31.4 11.6 2.3 22.2 9.0 2.5 Average 26.8 10.8 2.4 Μg / ml (4.6) (1.9) (0.4) Compound 2 52.4 6.0 3.6 35.2 5.7 0.6 Average 43.8 5.9 2.1 Μg / ml (7.5) (1.0) (0.4)

Example 22: Water Solubility Data

Table III: The solubility of the compounds of Example 6 was measured at room temperature in various systems.

Condition Solubility (mg / ml) Solubility (% w / v) water 74.9 7.5 0.9% sodium chloride 40.5 4.1 0.01M Acetate Buffer (pH 4.8) 68.6 6.9 0.01M citrate buffer (pH 4.8) 71.1 7.1 Water + 1% w / v glycerin 62.2 6.2 Water + 1% w / v propylene glycol 63.8 6.4

Similarly, the solubility in water of the compounds of Examples 10 and 16 was determined to be> 3.5% w / v (> 35 mg / ml) in water, while the compound of Example 12 was measured at approximately 0.1% w / v in water. Available.

Table IV: Partition Coefficients of Ester Compounds

Table V: Melting Points of Ester Compounds

Spectral data of ester compound 1H-NMR (270 MHz, DMSO-d6) ppm: Spectral data common to all 4-substituted-1-hydroxy-2,2,6,6-tetramethylpiperidine hydrochloride moieties 1.35 (6 H, s); 1.46 (6 H, s); 2.13 (2H, t); 2.44 (2H, d), 5.14 (1H, m)

Tables VI to XII below describe the methods used to synthesize further examples of ester compounds of the present invention. Suitable carboxylic acids listed in the table are converted to ester nitroxide by the DCC / DMAP esterification method of Example 5c. Ester nitroxide is converted to the corresponding 1-hydroxypiperidine by the method described in Examples 6 and 7.

3-Acyloxy-2.2-dimethylpropionic acid was prepared by the method described in US Pat. No. 4,851,436, the contents of which are incorporated herein by reference, for the synthesis of 3-acetoxy-2,2-dimethylpropionic acid. .

Table VI: Replacing cyclopropanecarboxylic acid with the following compounds in DCC / DMAP esterification process

Table VII

3-alkoxy-2.2-dimethylpropionic acid and 3-alkoxyalkyl-2.2-dimethylpropionic acid [J. Org. Chem. 38, 2349 (1975).

In the DCC / DMAP esterification process (Example 5c), cyclopropanecarboxylic acid is replaced with the following compound:

Table VIII

3-N-substituted-2.2-dimethylpropionic acid was prepared by the method described in US Pat. No. 5,475,013 (Talley et al.), The contents of which are incorporated herein by reference. In the DCC / DMAP esterification process (Example 5c), cyclopropanecarboxylic acid is replaced with the following compound:

Table IX

3-S-substituted-2,2-dimethylpropionic acid was prepared by the method described in US Pat. No. 5,475,013. In the DCC / DMAP esterification method (Example 5c), cyclopropanecarboxylic acid is replaced with the following compound:

Table X

3-Substituted-2,2-dimethylpropionic acid was prepared by the method described in US Pat. No. 5,475,013. In the DCC / DMAP esterification method (Example 5c), cyclopropanecarboxylic acid is replaced with the following compound:

Table XI

Various NSAIDs (nonsteroidal anti-inflammatory drugs containing carboxylic acid groups) are commercially available. Replace cyclopropanecarboxylic acid with the following compounds in the DCC / DMAP esterification process:

Table XII

Various carboxylic acids are commercially available. Replace cyclopropanecarboxylic acid with the following compounds in the DCC / DMAP esterification process:

Example 65 Single Rabbit Administration of Compound 1 to Rabbits Following Pharmacokinetics, Ocular Tissue Distribution, and Total Radioactivity of Urine Secretion

After a single topical administration of tritium hydrochloride of Compound 1 to rabbits, pharmacokinetics, ocular tissue distribution, and urine secretion of total radioactivity were evaluated. Eighteen male New Zealand white rabbits (Harlan), 3-6 months old, weighing 2.1-30 kg were used.

EXPERIMENTAL DESIGN: A randomized single treatment study in group 1 rabbits administered with a single topical application of [ ³ H] Compound 1-HCl to both eyes was the present design. The group consists of six subgroups of three animals. After six specific times (0.5, 1, 2, 4, 8 and 24 hours) after dosing, three animals per subgroup were euthanized and the final sample collected. Animals for subgroups after 24 hours of administration were placed in metabolic cages and urine collected at 0-12 and 12-24 hours post dose. Final blood was collected by cardiac puncture technique. The following eye tissues were received from each eye: Ahn, Bang-Soo; Vitreous fluid; lens; And cornea. Total radioactivity was measured on all samples.

The design and dosage are shown in the table below.

group Number / gender Dose volume per eye (ml) Dose level Dosage (mg / kg) (μCi / kg) (mg / ml) (mCi / ml) One 18 / M 0.04 ~ 0.8 to 1.2 ~ 34 to 49 30 1.275

Each eye of the animal was given a single topical ocular administration (right (OD) first, then left (OS)). With the lower eyelid slightly off the eye, the dose was pipetted correctly into the cornea so that it collected in the lower conjunctival sac. The eyelids were gently closed for 1 minute after drug dropping, and then carefully opened.

Data analysis and statistical evaluation: The Beckman LS6000 counter was used to convert the coefficients per minute for all analytical samples to decay per minute (DPM).

After adequate background subtraction, DPM values were converted to concentrations (DPM per ml or g). The average DPM per ml or g was calculated. The average concentration value of ³ H radioactivity was calculated and converted to equivalents per ml or g based on the nominal concentration and the measured specific activity of the administered test article formulation as determined by oxidation. Total radioactivity concentrations in blood and plasma, amounts (% of dose) and concentrations in eye tissue and urine samples were measured. Elimination kinetic including blood, plasma and tissue half-life was calculated. Urine secretion data was also tabulated. Statistical evaluation consists of descriptive statistical analysis. The pharmacokinetics of radioactivity was assessed by model independent analysis of the suitability of the sample analysis results. Non-truncated numbers were used in the calculations.

Representative Results: A drop of 3% solution of Compound 1 (40 μL) showed the following peak tissue concentrations at 30 minutes post dose in the cornea, waterproof, lens, vitreous, blood and plasma (Nanogram equivalents / g):

cornea Waterproof lens Vitreous blood plasma 13090 5930 310 150 240 360

As can be seen in Table 2, radioactivity was still measurable in all tissues 24 hours after administration. It has already been established that administering a 3% solution of Compound 1 to rabbits four times a day for one week will not cause toxicity.

Example 66: Effect of Compound 1 or Tempol-H Treatment on Photochemical Retinal Injury in Rabbit Model

The ability of light to cause retinal injury to a level sufficiently lower than that at which thermal damage can occur is called photochemical retinal injury. The mechanism of action of photochemical retinal injury is believed to be the generation of free radicals derived from light. The performance of commonly used light sources in clinical ophthalmology to cause photochemical retinal injury is well recognized. Surgical microscopes used in ophthalmic surgery have been shown to have the ability to produce photochemical injuries of the retina. This observation is used to generate a model in rabbit eyes to test the ability of various agents to block photochemical retinal injury. On the ophthalmic category, visible retinal lesions were determined to be detectable after as little as 2.5 minutes of exposure to the surgical microscope. The protocol set forth below was used to determine whether Compound 1 or Tempol-H via intravitreal injection had the ability to block the progression of visible retinal lesions on the ophthalmic category generated by the surgical microscope.

Materials and Methods: One week prior to treatment, baseline fundus imaging and FA are performed in each animal. One day prior to exposure to the light of the surgical microscope, one eye of each animal received an intravitreal injection of 0.1 cc of BBS® as a control and the other eye received Compound 1 or Tempol-H 0.1 in a BBS® vehicle. Receive cc Animals are anesthetized with an IM injection of 60% -40% of a mixture of 100 mg / ml ketamine hydrochloride and 20 mg / ml Xylazine. A speculum is inserted into the receiving eye and the injection is performed using a 30 g needle tilted to remove the lens by passing through the sclera just below the edge, located at the end of the central vitreous cavity. After injection, intraocular pressure is monitored using a hand-held tonometer. If necessary, puncture is performed to return the intraocular pressure to normal levels prior to returning the animals to us. Alternatively, the procedure can be performed after Compound 1 or Tempol-H has been administered to rabbit eyes by multiple drops of topical eye drops.

Forty eyes of 20 colored rabbits are exposed to the light of a Zeiss model OpMi 1 surgical microscope for up to one hour at various times. Rabbits are anesthetized by IM injection of a mixture of 100 mg / ml ketamine hydrochloride and 20 mg / ml silazine 20% -40%. Then put the rabbit on the table; A lid speculum is inserted into the eye and exposed. The pupils are swelled with 1% tropicamide HCL and the cornea is stimulated using BSS®. The light in the microscope is positioned 20 cm from the animal in such a way that the light is centered and parallel to the eye. Light filaments are centered on the cornea with sharp focus. After 48 hours of exposure, animals are examined and repeated fundus photography and FA are performed. Animals are killed using prescribed techniques and selected eyes are received and stored in cooled glutaaldehyde solution for further analysis.

Example 67 Evaluation of Tempol-H Incorporation in Rabbit Retinal Tissues After Intravitreal Injection

In evaluating AMD and other retinal disorders, to assess the efficacy of Compound 1 or Tempol-H, it should be determined that the drug is incorporated into the retinal tissue. The protocol described in this example utilizes a scintillation technique that establishes the amount and duration of Compound 1 or Tempol-H incorporation after intravitreal injection in rabbits. New Zealand white rabbits are well characterized in experiments involving intravitreal injections because the rabbit's large eyes provide a suitable template for therapeutic administration (Hosseini et al., 2003, Lasers Surg. Med. 32: 265-270). In addition, the New Zealand white rabbit has been widely used in experiments to analyze drug absorption into retinal tissue using scintillation techniques (Ahmed et al., 1987, J. Pharm. Sci. 76: 583-586).

Materials and Methods: 24 New Zealand white rabbits, 2.5-3 kilograms in weight, all males, are used in this protocol. The rabbit is randomly selected to one of two treatment arms. First, rabbits are subjected to a standard eye test to ensure that all eyes are free of irritation and infection. After random selection and baseline testing, rabbits are anesthetized by subcutaneous injection of ketamine 100 mg / ml / silazine 20 mg / ml. A drop of commercially available 0.5% proparacaine hydrochloride is applied to both eyes. The rabbit is then treated bidirectionally by intravitreal injection according to the unknown randomly selected scheme shown below.

Unknown treatment means:

1. Bidirectional intravitreal injection with labeled Compound 1 or Tempol-H. (N = 12)

2. Two-way intravitreal injection with placebo. (N = 12)

On days 1, 7, 14 and 28 respectively, six rabbits, three from each treatment means, were sacrificed using lethal doses of pentobarbital sodium. Immediately after sacrifice, both extractions are performed and the retinal tissue is removed for scintillation analysis (the retinal tissue can be frozen after extraction to prevent fluid loss). Retinal tissue is ground to a slurry and dissolved in an alkali or quaternary ammonium compound. Scintillation reads are made to quantify the amount of Compound 1 or Tempol-H present in the retinal tissue.

For statistical analysis, data from all rabbits dosed with the test article are considered evaluable. The first and second efficacy variables are for statistical significance. Two-sided nonparametric statistical test is used and p <0.05 is considered to be statistically significant.

Example 68 Efficacy of Tempol-H in Protection Against Photooxidative Processes in Retinal Pigment Epithelium (RPE)

BACKGROUND: Although the cause of atrophic geriatric macular degeneration (AMD) is not fully understood, it is commonly accepted that AMD begins with retinal pigment epithelial cell death, photoreceptor cell degeneration and subsequent loss of visual acuity. There is evidence that lipofuscin that accumulates in RPE correlates with the death of these cells. For example, not only the highest level of lipofuscin in RPE cells underlying the macular, but also RPE lipofucin, monitored by detection of fundus autofluorescence, also shows that RPE atrophy regions progress in fluorescence sites that have already increased. have.

Studies related to examining the association between RPE lipofucin and RPE cell death show that the major component of RPE lipofucin, the bisretinoid phosphor A2E, can disrupt cell membranes and mediate blue light damage to RPE. Photoexcitation of A2E causes the generation of single oxygen and the addition of single oxygen to the carbon-carbon double bonds along the retinoid induced side of A2E, causing epoxides to form. In this way, A 2 E is converted to a mixture of compounds (A 2 E-epoxides) with epoxides of varying numbers. This highly reactive epoxide appears to damage the cells. Ultimately, photochemical events caused by light irradiation of A2E in RPE cells include the involvement of a cysteine dependent protease (caspase) that divides the cell substrate and the cells by a pathway regulated by the mitochondrial protein bcl-2. Causes death.

The ability of Tempol-H to protect against A2E mediated blue light damage was examined.

Materials and Methods: ARPE-19 cells accumulating A2E during incubation were exposed to 430 +/- 20 nm of light delivered from halogen light sources. The wavelength of this light is (i) the maximum excitation of A2E is approximately 430 nm, and (ii) the light of this wavelength is allowed to reach the RPE in vivo without light of wavelengths longer than 400 nm being absorbed by the cornea and lens. Because it is relative. Cell death in A2E-loaded RPE irradiated with blue light is compared to pretreatment (24 h) with tempol-H. Cell viability loss is quantified by:

1. Calorimetric microtiter analysis based on the ability of metabolically healthy active cells to split the yellow tetrazonium salt MTT into purple formazan crystals. Cell viability was analyzed 24 hours after blue light irradiation. Three wells are analyzed in each of two experiments.

2. The nuclei of all cells were blue stained using DAPI, while the nuclei of all dead cells became red due to staining with membrane-impermeable dyes (Dead Red nucleic acid staining, Molecular Probes). Appearing, two-color fluorescence analysis. Cell viability was analyzed after 8 hours of blue light irradiation. Replicas are analyzed by counting cells in five microscopic regions in the irradiated region of each well and three wells per experiment. The data is based on data from three experiments.

Results: As shown in FIGS. 3 and 4, Tempol-H enables dose dependent protection from the death of A2E-loaded RPE in this macular degeneration model. This indicates that what is expected from Compound 1 as well as Tempol-H is the active metabolite of Compound I.

While the invention has been particularly shown and described with reference to the presently preferred embodiments, it should be understood that the invention is not limited to the embodiments specifically disclosed and illustrated in the specification. Numerous variations and modifications may be made to the preferred embodiments of the invention, and such changes and modifications may be made without departing from the scope and spirit of the invention as set forth in the appended claims.

Claims (67)

Macular degeneration, retinopathy, glaucoma, presbyopia, conjunctival disease, eyelid disorders, comprising administering to a patient's eye a composition containing one or more compounds of the formula and containing an ophthalmically acceptable carrier or diluent: How to treat corneal disease or uveitis:

[Wherein, R ₁ and R ₂ are independently H or C ₁ to C ₃ alkyl; R ₃ and R ₄ are independently C ₁ to C ₃ alkyl; R ₁ and R ₂ together or R ₃ and R ₄ together or both may be cycloalkyl; R ₅ is H, OH, or C ₁ to C ₆ alkyl; R ₆ is C ₁ to C ₆ alkyl, alkenyl, alkynyl, or substituted alkyl or alkenyl; R ₇ is C ₁ to C ₆ alkyl, alkenyl, alkynyl, substituted alkyl or alkenyl, or Or R ₆ and R ₇ , or R ₅ , R ₆ and R ₇ together form a carbocycle or heterocycle having 3 to 7 atoms in the ring]. The method of claim ₁ , wherein R ₁ , R ₂ , R ₃ , and R ₄ are C ₁ -C ₃ alkyl. The method of claim 2, wherein R ₁ , R ₂ , R ₃ , and R ₄ are ethyl groups. The method of claim 2, wherein R ₁ , R ₂ , R ₃ , and R ₄ are methyl groups. The compound of claim 4, wherein each R ₅ is H or methyl and R ₆ is methyl substituted with benzyloxy or C ₁ -C ₆ alkoxy; R ₇ is methyl. The method of claim 4, wherein each R ₅ is H or methyl and R ₆ and R ₇ form a cyclopropyl group. The method of claim 4, wherein R ₅ , R ₆ and R ₇ form a furanyl group. The compound of claim 4, wherein R ₅ is H; R ₆ and R ₇ form a tetrahydrofuranyl group. The compound of claim 4, wherein R ₅ is H; R ₆ and R ₇ form a cyclopropyl ring. The method of claim 1, which is administered via eye drops, eye wash, or eye ointment. The method of claim 1, further comprising administering a reducing agent to the patient. The method of claim 11, wherein the reducing agent is co-administered with the composition. The method of claim 11, wherein the reducing agent is administered separately from the composition. The method of claim 11, wherein the reducing agent is a sulfhydryl compound. The method of claim 14, further comprising co-administering mercaptopropionyl glycine, N-acetyl cysteine, β-mercaptoethylamine, or glutathione to the patient. The method of claim 11, wherein the reducing agent is administered via eye drops, eye wash, or eye ointment. The method of claim 1, wherein the compound is administered at a concentration of about 0.1 μM to about 10 mM in tissue and fluid. The method of claim 1, wherein the compound is administered at a concentration of about 1 μM to about 5 mM in tissue and fluid. The method of claim 1, wherein the compound is administered at a concentration of about 25 μM to about 1 mM in tissue and fluid. The method of claim 1, wherein the compound is administered at a concentration of about 50 μM to about 100 μM in tissue and fluid. A method for preventing or delaying the progression of cataracts, presbyopia, glaucoma, age related macular disorders or other age related conditions of the eye, the method comprising: eye drops, eye washes, containing one or more compounds of the formula: Or administering ophthalmic ointment to the eye, wherein the administration is effected before or at the onset of an age related condition:

[Wherein, R ₁ and R ₂ are independently H or C ₁ to C ₃ alkyl; R ₃ and R ₄ are independently C ₁ to C ₃ alkyl; R ₁ and R ₂ together or R ₃ and R ₄ together or both may be cycloalkyl; R ₅ is H, OH, or C ₁ to C ₆ alkyl; R ₆ is C ₁ to C ₆ alkyl, alkenyl, alkynyl, or substituted alkyl or alkenyl; R ₇ is C ₁ to C ₆ alkyl, alkenyl, alkynyl, substituted alkyl or alkenyl, or Or R ₆ and R ₇ , or R ₅ , R ₆ and R ₇ together form a carbocycle or heterocycle having 3 to 7 atoms in the ring]. The method of claim 21, wherein R ₁ , R ₂ , R ₃ , and R ₄ are C ₁ -C ₃ alkyl. The method of claim 22, wherein R ₁ , R ₂ , R ₃ , and R ₄ are ethyl groups. The method of claim 22, wherein R ₁ , R ₂ , R ₃ , and R ₄ are methyl groups. The method of claim 24, wherein each R ₅ is H or methyl, R ₆ is methyl substituted with benzyloxy or C ₁ -C ₆ alkoxy and R ₇ is methyl. The method of claim 24, wherein each R ₅ is H or methyl and R ₆ and R ₇ form a cyclopropyl group. The method of claim 24, wherein R ₅ , R ₆ and R ₇ form a furanyl group. The compound of claim 24, wherein R ₅ is H; R ₆ and R ₇ form a tetrahydrofuranyl group. The compound of claim 24, wherein R ₅ is H; R ₆ and R ₇ form a cyclopropyl ring. The method of claim 21, further comprising administering a reducing agent to the patient. The method of claim 30, wherein the reducing agent is co-administered with the composition. The method of claim 30, wherein the reducing agent is administered separately from the composition. The method of claim 30, wherein the reducing agent is a sulfhydryl compound. The method of claim 33, further comprising coadministering mercaptopropionyl glycine, N-acetyl cysteine, β-mercaptoethylamine, or glutathione to the patient. The method of claim 21, wherein the compound is administered at a concentration of about 0.1 μM to about 10 mM in tissue and fluid. The method of claim 21, wherein the compound is administered at a concentration of about 1 μM to about 5 mM in tissue and fluid. The method of claim 21, wherein the compound is administered at a concentration of about 25 μM to about 1 mM in tissue and fluid. The method of claim 21, wherein the compound is administered so that the concentration in tissue and fluid is from about 50 μM to about 100 μM. A method of protecting the retinal pigment epithelium against photooxidative damage, comprising administering to the eye an eye drop, eye wash or eye ointment containing at least one compound of the formula and containing an ophthalmologically acceptable carrier or diluent: The administration is carried out prior to or at the beginning of the age related condition:

[Wherein, R ₁ and R ₂ are independently H or C ₁ to C ₃ alkyl; R ₃ and R ₄ are independently C ₁ to C ₃ alkyl; R ₁ and R ₂ together or R ₃ and R ₄ together or both may be cycloalkyl; R ₅ is H, OH, or C ₁ to C ₆ alkyl; R ₆ is C ₁ to C ₆ alkyl, alkenyl, alkynyl, or substituted alkyl or alkenyl; R ₇ is C ₁ to C ₆ alkyl, alkenyl, alkynyl, substituted alkyl or alkenyl, or Or R ₆ and R ₇ , or R ₅ , R ₆ and R ₇ together form a carbocycle or heterocycle having 3 to 7 atoms in the ring]. The method of claim 39, wherein R ₁ , R ₂ , R ₃ , and R ₄ are C ₁ -C ₃ alkyl. 41. The method of claim 40, wherein R ₁ , R ₂ , R ₃ , and R ₄ are ethyl groups. 41. The method of claim 40, wherein R ₁ , R ₂ , R ₃ , and R ₄ are methyl groups. 43. The compound of claim 42, wherein each R ₅ is H or methyl and R ₆ is methyl substituted with benzyloxy or C ₁ -C ₆ alkoxy; R ₇ is methyl. The method of claim 42, wherein each R ₅ is H or methyl and R ₆ and R ₇ form a cyclopropyl group. 43. The method of claim 42, wherein R ₅ , R ₆ and R ₇ form a furanyl group. 43. The compound of claim 42, wherein R ₅ is H; R ₆ and R ₇ form a tetrahydrofuranyl group. 43. The compound of claim 42, wherein R ₅ is H; R ₆ and R ₇ form a cyclopropyl ring. The method of claim 39 further comprising administering a reducing agent to the patient. 49. The method of claim 48, wherein the reducing agent is co-administered with the composition. 49. The method of claim 48, wherein the reducing agent is administered separately from the composition. The method of claim 48, wherein the reducing agent is a sulfhydryl compound. The method of claim 51, further comprising co-administering mercaptopropionyl glycine, N-acetyl cysteine, β-mercaptoethylamine, or glutathione to the patient. The method of claim 39, wherein the compound is administered so that the concentration in tissue and fluid is from about 0.1 μM to about 10 mM. The method of claim 39, wherein the compound is administered so that the concentration in tissue and fluid is from about 1 μM to about 5 mM. The method of claim 39, wherein the compound is administered so that the concentration in tissue and fluid is from about 25 μM to about 1 mM. The method of claim 39, wherein the compound is administered so that the concentration in tissue and fluid is from about 50 μM to about 100 μM. A method of protecting hair follicles and preventing further hair loss, comprising administering to a patient in need thereof a composition comprising one or more compounds of the formula and a pharmaceutically acceptable carrier or diluent: How to protect this hair follicle and prevent further hair loss:

[Wherein, R ₁ and R ₂ are independently H or C ₁ to C ₃ alkyl; R ₃ and R ₄ are independently C ₁ to C ₃ alkyl; R ₁ and R ₂ together or R ₃ and R ₄ together or both may be cycloalkyl; R ₅ is H, OH, or C ₁ to C ₆ alkyl; R ₆ is C ₁ to C ₆ alkyl, alkenyl, alkynyl, or substituted alkyl or alkenyl; R ₇ is C ₁ to C ₆ alkyl, alkenyl, alkynyl, substituted alkyl or alkenyl, or Or R ₆ and R ₇ , or R ₅ , R ₆ and R ₇ together form a carbocycle or heterocycle having 3 to 7 atoms in the ring]. A method of preventing or treating damage to rectal tissue as a result of radiation therapy, comprising administering to a patient in need thereof a composition comprising one or more compounds of the formula and a pharmaceutically acceptable carrier or diluent: And wherein the composition ameliorates, delays or prevents tissue damage:

[Wherein, R ₁ and R ₂ are independently H or C ₁ to C ₃ alkyl; R ₃ and R ₄ are independently C ₁ to C ₃ alkyl; R ₁ and R ₂ together or R ₃ and R ₄ together or both may be cycloalkyl; R ₅ is H, OH, or C ₁ to C ₆ alkyl; R ₆ is C ₁ to C ₆ alkyl, alkenyl, alkynyl, or substituted alkyl or alkenyl; R ₇ is C ₁ to C ₆ alkyl, alkenyl, alkynyl, substituted alkyl or alkenyl, or Or R ₆ and R ₇ , or R ₅ , R ₆ and R ₇ together form a carbocycle or heterocycle having 3 to 7 atoms in the ring]. The method of claim 1, wherein the disease or disorder is blepharitis. The method of claim 1, wherein the disease or disorder is ocular injection (rosacea). The method of claim 1, wherein the disease or disorder is macular degeneration. The method of claim 1, wherein the disease or disorder is retinopathy. The method of claim 1, wherein the disease or disorder is glaucoma. The method of claim 1, wherein the disease or disorder is presbyopia. The method of claim 1, wherein the composition further comprises a second compound used to treat macular degeneration, retinopathy, glaucoma, presbyopia, conjunctival disease, eyelid disorder, corneal disease, or uveitis. 66. The method of claim 65, wherein the second compound is administered simultaneously. The method of claim 65, wherein the second compound is administered sequentially.

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