KR20050021375A - Treatment for eye disorder - Google Patents

Abstract

The present invention is directed to altering the physical and / or chemical properties of at least a portion of one or more tissues of the intraocular. In one particular embodiment, the present invention relates to the treatment of any ocular disease, but in certain embodiments the individual has a thickened Bruch membrane. The activating energy source is used to induce controlled diffusion enhancement and / or degradation of the Bruch's membrane, which enables improved diffusion transport between the choroid and the retina. The subject is administered an inactivated diffusion enhancing molecule associated with the membrane, which is then correctly exposed to an activating energy source such as light or ultrasound.

Description

Treatment of eye diseases {TREATMENT FOR EYE DISORDER}

This invention is a priority claim application of US Provisional Patent Application No. 60 / 393,505, filed Jul. 2, 2002, which application is incorporated herein by reference.

The present invention generally relates to the field of ophthalmology and cell biology. In particular, the present invention relates to altering the physical and / or chemical properties of ocular tissues. More specifically, the invention describes means for detecting and / or reducing the thickening of Bruch's membranes associated with eye diseases such as macular degeneration and / or changing their permeability. More specifically, the present invention relates to administering inactivated diffusion enhancing molecules to the Bruch membrane and then activating the diffusion enhancing molecules via an energy source.

Aging macular degeneration (AMD) is a progressive eye disease that affects 10 million Americans. AMD is the number one cause of blindness and legal blindness in adults over 60 years of age in the United States. As the population ages and the "born in the baby boom" ages in their 60s and 70s, AMD's real fad will spread. The disease damages the macula of the eye, where the most pronounced central vision occurs. Macular damage rarely results in complete blindness, but it takes away from the subject's outermost peripheral vision, leaving only a blurry image or black hole in the center of the vision.

Macular degeneration is classified as dry (atrophic) or wet (neovascular). Dry form is more common than wet form, and about 90% of AMD patients are diagnosed with dry AMD. Wet form of this disease generally results in more severe vision loss.

In the dry form, destruction or weakening of the macula retinal pigment epithelial cells (RPE) occurs, thus called "atrophic". These RPE cells are important for the function of the retina because they metabolically support the upper photoreceptors.

The clinical feature of atrophic AMD is the accumulation of yellowish deposits just below the macula crystalline cavity, ie the retinal pigment epithelium ("RPE"). Histopathological observation of the eye with atrophic AMD reveals that lipid and proteinaceous material is deposited under the RPE of the Bruch's membrane. In aged eyes with AMD, the thickness of the Brooke membrane is often about three times thicker than that of normal membranes. This thickening is thought to consist of modified as well as cross-linked proteins as well as lipids, which impedes the transport of nutrients across the Bruch's membrane from the choroidal capillaries to the outer retina. This thickening barrier, consisting of lipids and crosslinked proteins, impedes the transport of nutrients across the Brook membrane from choroidal capillaries to the outer retina. Currently, there is no proven effective treatment for dry AMD other than the use of multivitamins and micronutrients.

Wet AMD occurs when new blood vessels form and cross the Bruch's membrane to grow into the RPE sub and retinal spaces. These neovascular tissues are very weak and highly permeable. Often, this bleeds and damages the upper retina. As the blood hardens, functional macular tissue is replaced by scar tissue. To prevent vision loss, it is desirable to intervene therapeutically before neovascularization progresses.

The exact etiology of AMD is unknown, but several risk factors are thought to be important. For example, ARMD can be caused by chronic exposure of the retina to light. The presence or absence of certain nutrients in the diet, such as the antioxidant vitamins E and C, may also affect the predisposition of an individual to ARMD. Other conditions such as hypertension and smoking are also considered to be important risk factors for the development of this disease.

AMD is a difficult disease for both patients and doctors because there are few treatment options to choose from, and there are no proven preventive therapies except antioxidants. Some people are only slightly uncomfortable with macular degeneration, but many others with more severe forms of macular degeneration are hampered in their daily lives. Current therapies, including laser photocoagulation, photodynamic therapy and anti-neovascularization therapies, have shown mixed results and, in certain cases, have adverse side effects. Therefore, there is a need for a treatment that reduces or limits the effects of macular degeneration.

Laser photocoagulation is valid in clinical trials, but only a few AMD patients can be eligible for treatment. In addition, recurrent neovascular tissues often grow even after successful removal of choroidal neovascularization by laser treatment. Visudyne® (Novatis Offmix; Duluth, GA), photodynamic therapy or PDT use photoactivated drugs to potentially stop or slow abnormal cell growth. This therapy treats late diseases characterized by neovascularization of the choroid. In summary, photosensitizers are administered intravenously, which in particular attach to lipoprotein receptors observed in rapidly proliferating cells. Immediately after administration, the compound is activated upon irradiation with a certain amount of light of a certain wavelength, converting normal oxygen into free radical singlet oxygen, which results in the closure of neovascular tissue. In certain embodiments the therapy treats vascular proliferation. However, the underlying cause of macular degeneration is not solved by the treatment of choroidal neovascularization by photodynamic therapy, and neovascularization generally recurs within a few months after treatment.

US Pat. No. 5,756,541 relates to a method for improving vision, including administering an amount of photoactive compound sufficient to localize to a target eye tissue, and irradiating the target tissue with laser light, wherein the wavelength of radiation is Is absorbed by, and irradiation is carried out for a sufficient time at an intensity sufficient to improve vision. In certain embodiments, the photoactive compound is green porphyrin. US Pat. No. 5,910,510 relates to the same method as the above method with specific irradiation timing.

U.S. Patent No. 5,798,349 features unwanted neovascular structures, such as AMD, by irradiating the neovascular structures with laser light after administering a liposome formulation of green porphyrin sufficient for localization to the neovascular structures for a sufficient time. The present invention relates to a method of treating eye conditions, in which light absorbed by green porphyrin occludes the neovascular structure. In related US Pat. No. 6,225,303 the radiation range is from about 300 mW / cm ² to about 900 mW / cm ² .

US Pat. No. 6,128,525 relates to methods and devices for controlling dosimetry of photodynamic therapy.

U. S. Patent No. 5,935, 942 relates to a method for obstructing the vasculature of a mammalian eye comprising concurrent administration of a fluorescent dye encapsulated with heat sensitive liposomes and a tissue reactive agent activated by irradiation to a vein. Liposomes are heated in the eye to release their contents, where the tissue reactive agent remains inactive and then monitors the flow of fluorescent dye in the vasculature. Tissue reactive agents are activated in the vasculature with subnormal blood flow so that the activated agents chemically occlude the vasculature. Related US Pat. No. 6,140,314 further includes co-administration of tissue specific factors effective to impair blood vessel growth or regeneration. Related US Pat. No. 6,248,727 relates to related diagnostic reagents and kits.

Thus, although alternative methods of treatment of eye diseases exist, the present invention provides for the treatment of diseases, e.g., in the treatment of diseases prior to the time of neovascularization of ocular tissues, in particular associated with ocular diseases, Eliminate the needs of the field in reversing beds.

Summary of the Invention

The present invention relates to methods and compositions for altering the physical and / or chemical properties of ocular tissues. In certain embodiments, the present invention relates to enhancing enrichment through tissue, targeted cell disruption, and / or targeted alteration of at least a portion of one or more ocular tissues of an individual. This may be done in certain embodiments using means for causing controlled diffusion enhancement and / or other controlled alterations, such as light or ultrasound. Some aspects of the present invention relate to the treatment of premature eye diseases and those skilled in the art will recognize the utility of the present invention for such purposes.

In certain but only illustrative embodiments of the invention, the Bruch's membrane is a targeted tissue. As aging progresses, especially in macular degeneration, the Bruch's membrane develops lipid and crosslinked protein barriers. Damage to diffusion through the Bruch's membrane in macular degeneration patients promotes release of angiogenic factors by the depleted retina. This causes the growth of neovascular tissue through the Bruch's membrane, which entails subsequent bleeding, leakage of serous fluid and severe vision loss. Some embodiments of the present invention allow for treatment / administration just before or immediately after choroidal neovascularization occurs.

In order to improve diffusion through the Bruch's membrane and prevent progression of vision loss, altering the physicochemical properties of tissues associated with vision loss, such as as aging progresses, and also in AMD patients, accumulates in the Bruch's membrane. It is desirable to reduce lipid and crosslinked protein barriers. The present invention relates to means and compositions for performing this using light or ultrasound to induce controlled diffusion enhancement and / or partial degradation of the Brooke membrane, which may enhance diffusion transport between the choroid and the retina. . Energy (e.g., light or ultrasound) is used for the selective activation of tissue-modifying substances (also known as lipid and / or proteolytic substances), because energy can be used to target the Bruch's membrane with minimal impact on adjacent tissues. This can be changed. When active tissue modifying molecules are administered systemically, they are not selective to the Bruch's membrane, potentially damaging other tissues. To specifically target only the desired tissue (e.g., the Bruch's membrane), the tissue modifying molecule is administered in an inactive form, for example, by systemic injection or ingestion, or by topical (intraocular, peripheral) injection. . In certain embodiments, the molecule is lipophilic. This molecule is inactive and binds to multiple tissues, which are then gradually removed. The molecule is only activated by an energy source (e.g. light, ultrasound or both) applied precisely to the eye to selectively activate tissue-modifying substances in the Brook membrane. Once activated, the tissue modifying molecule alters lipids and / or crosslinked proteins in the Bruch's membrane, for example, to enhance membrane permeation diffusion. In certain embodiments, in order to pinpoint the activation site, the photochemical activation step includes two-photon photochemistry.

One object of the present invention is to provide a method for the treatment of eye diseases comprising the step of increasing the diffusion through the Brook membrane of the eye. In one particular embodiment, the increased diffusion is the result of a decrease in the thickness of the film, a change in composition, or both.

Still another object of the present invention is to administer an inactive form of a degraded molecule to the Brueck membrane in an amount sufficient to form a Brooke membrane / inert degraded molecule complex and to expose the complex to an activator, wherein the activation The source activates the inert degradation molecule into the active form of the degradation molecule, wherein the activation is to increase diffusion through the membrane. To provide. In one particular embodiment, the increase in diffusion is the result of a decrease in thickness or a change in composition of the film. In another embodiment, the increase in diffusion is the result of altered lipids, crosslinked proteins, or both in the membrane. In another embodiment, the subject is an ocular disease, such as AMD, pediatric macular degeneration, Sorsby's fundus dystrophy, or an aging decline in visual function not associated with macular degeneration. In certain embodiments, the inert degradation molecule binds directly to the membrane.

In another embodiment of the invention, the inert degradation molecule is a protein, detergent, surfactant (useful for cascaded cyclodextrins). In another embodiment, the protein is an enzyme. In another embodiment, the inactive enzyme is further defined as being cascaded by incorporating one or more photoremovable protecting groups on the amino acid side chains of the enzyme. In one particular embodiment, protecting groups are o-nitrobenzyl, decyl, phenacyl, trans-o-cinnayl, coumarinyl, quinoline-2-onyl, xanthenyl, thioxanthenyl, selenoxanthenyl and anthracenyl , Stilbenyl, or a combination thereof. In another embodiment, protecting groups are o-nitrobenzyl, decyl, phenacyl, trans-o-cinnayl, coumarinyl, quinoline-2-onyl, xanthenyl, thioxanthenyl, selenoxanthenyl and anthracenyl , Stilbenyl or derivatives thereof. In certain embodiments, the amino acids are cysteine, aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, serine, threonine, tyrosine or combinations thereof. In certain embodiments, the detergents are o-nitrobenzyl, decyl, phenacyl, trans-o-cinamoyl, coumarinyl, quinoline-2-onyl, xanthenyl, thioxanthenyl, selenoxanthenyl and anthracenyl, Or cascaded by a compound comprising a stilbenyl group. In another embodiment, the protein is further defined as being cascaded in an ultrasound contrast agent. In certain embodiments, the ultrasound contrast agent is microbubbles or liposomes. In another embodiment, the protein further comprises a protein binding domain. In another embodiment, the protein binding domain is a heterodimeric domain. In another embodiment, the protein binding domain is a leucine zipper domain, chitin binding domain or Src homology 2 (SH2) domain.

In another embodiment of the invention, the inert degradation molecule is administered to the subject as a pharmaceutically acceptable composition. In another preferred embodiment, the inert degradation molecule is administered systemically to the subject as a pharmaceutically acceptable composition. In another embodiment, the inert degradation molecule is administered orally, by injection (peripheral or intraocular injection), rectal, vaginal, or topical administration to a subject as a pharmaceutically acceptable composition. In one particular embodiment, the enzyme is a substrate metalloproteinase, cholesterol esterase, lipase, cathepsin, protease, or a combination thereof. In one particular embodiment, the protease is a serine protease. In one particular embodiment, the activating source is energy. In another specific embodiment, the energy is light or ultrasound. In another embodiment, the exposing step is further defined as exposing the composite to light energy from a focused laser source. In another embodiment, the degradation molecule is fluorescently labeled.

Another embodiment of the invention is a method of treating aging macular degeneration characterized by a thickened Brooke membrane of at least one eye of an individual, wherein the inactivated degradation molecule is associated with the Brooke membrane. Administering said molecule to said individual in an amount sufficient to form a Brooke membrane / inert degradation molecule complex; And exposing the membrane to an activating source, wherein after the exposing step diffusion through the membrane of the eye is enhanced. In one particular embodiment, the method further comprises administering the fluorescent molecule to the subject in an amount sufficient to associate with the Brooke membrane for visualization of the Brooke membrane. In certain embodiments, the amount of fluorescence emitted is proportional to the amount of altered protein or lipid in the Bruch membrane. In another embodiment, the wavelength of light required to excite the dye, the wavelength of light emitted by the dye or the lifetime of the dye is altered in a detectable manner by the altered amount of protein or lipid in the Brueck membrane. In another specific embodiment, the inactivated degradation molecule is administered as a pharmaceutically acceptable composition.

Yet another embodiment of the present invention is a kit housed in a suitable container comprising inactivated Bruch membrane degradation molecules. In certain embodiments, the kit further comprises an activator for activation of said inactivated Bruch membrane degradation molecules. In another specific embodiment, the kit further comprises a fluorescent molecule.

In some embodiments of the invention, the targeted eye tissue is visualized before and / or during activation of an inactive tissue modifying molecule. For example, the Brooke membrane can be obtained by the delivery of fluorescent molecules targeting the Brooke membrane; By detecting autofluorescence signals from characteristic components of the Bruch's membrane itself, which distinguishes it from adjacent surrounding tissue (by means known in the art); And / or by using OCT Doppler to identify specific physical properties of the membrane (also by means known in the art).

Another embodiment of the invention comprises administering to a subject a fluorescent molecule in an amount sufficient to cause the fluorescent molecule to associate with the intraocular Bruch membrane; Exposing the Brooke membrane to radiation to visualize fluorescence, wherein the amount of fluorescence is indicative of an ocular disease in at least one eye of the subject, comprising the step of being an indicator of the severity of the ocular disease. In certain embodiments, the ocular disease is macular degeneration. In another embodiment, the radiation is two photon irradiation.

Another embodiment of the invention is a method of diagnosing ocular disease in at least one eye of an individual based on inherent light scattering from a targeted tissue, such as a Brooke membrane. Optical coherence tomography (OCT) techniques using visible or infrared light are used to detect alterations in the physical or chemical properties of the intraocular Brook Film. OCT can be used to observe intraocular structures, as used in several existing studies of the human eye, as well as to study the mobility of the structure by Doppler OCT, to study chemical properties in combination with OCT and exogenous dyes. It can be used to. In such embodiments, the OCT and / or its modifications are used to ascertain the properties of the Brooke membrane with altered properties that allow for guided treatment. Treatment may be photoliberation or photoactivation or photoablation of endogenous or exogenous materials in or near the Bruch's membrane.

Another embodiment of the invention comprises administering to a subject an amount of visualization molecules sufficient to visualize the intraocular Bruch membrane; Administering to the subject an inactive form of the photoactive degradation molecule in an amount sufficient to form a Bruch membrane / inactive photoactive degradation molecule complex; And exposing the complex to an activator, wherein the activator activates the inactive photoactive degrading molecule into the active form of the photoactive diffusion enhancing molecule, the activation increasing the diffusion through the Bruch membrane, composition To induce alteration of both or both. In one particular embodiment, the visualization molecule is a fluorescent molecule. In another embodiment, the fluorescent molecule is bound to the inactive form of the photoactive molecule. In another embodiment, the membrane is visualized by signal autofluorescence properties or by the OCT Doppler method. In another embodiment, the inactive photoactive molecules are associated with lipids in the Bruch's membrane.

One embodiment of the present invention comprises administering to the eye tissue an inactive form of the tissue modifying molecule in an amount sufficient to target the tissue modifying molecule to the eye tissue; And exposing the molecule to an activator, wherein the activator activates the inactive form of the tissue modifying molecule into the active form of the tissue modifying molecule, wherein the activation induces alteration of at least a portion of the ocular tissue. A method of altering the eye tissue of an individual, comprising the steps. In one particular embodiment, the method provides a treatment for eye disease in the subject. In another embodiment, the ocular disease is aging macular degeneration, pediatric macular degeneration, source ratio fundus dystrophy, aging decline in visual function unrelated to macular degeneration, or glaucoma. In one particular embodiment, the inactive tissue modifying molecule is administered to the subject as a pharmaceutically acceptable composition. In another embodiment, the inactive tissue modifying molecule is administered systemically to the subject as a pharmaceutically acceptable composition. In another specific embodiment, the inert degradation molecule is administered orally, injected, rectally, vaginally or topically to the individual. If the administration is by injection, the injection can be intraocular or peripheral injection, but other routes are also suitable.

In certain embodiments, optical coherence tomography (OCT) is used for detection in a target tissue or cell, for example, to detect changes in composition (eg, labeling by scattering or special agents), or It is used to detect the organization of tissues such as the Brooke membrane. Doppler OCT provides informative information, for example, information about the mobility of scatterers in the target tissue and / or targeting of fixed sites (eg, chemically altered sites, etc.) to activate without release. to provide.

1 illustrates a targeted light directed tissue alteration of Bruch membrane lipids as an exemplary embodiment of the invention. This figure shows that after systemic administration, the cascaded diffusion enhancing molecules diffuse from choroidal capillaries into the Brooke membrane. Two-photon irradiation correctly deprotected the functional groups, and the diffusion enhancing molecules are specifically activated in the Brueck membrane.

Those skilled in the art will readily appreciate that various substitutions and modifications can be made to the inventions disclosed herein without departing from the scope and spirit of the invention.

I. Definition

As used herein, "a" or "an" ("a" or "an") may mean one or more. When used in conjunction with the term "comprising" as used in the claims, the term "one" or "an" may mean one or more than one. As used herein, “another” may mean at least a second or more.

The term “aging macular degeneration (AMD)” as used herein refers to macular degeneration of an individual about 50 years of age or older. In one particular embodiment, this is associated with reduced central vision and the destruction and loss of photoreceptors in the macular region of the retina, which, if developed, leads to legal blindness. In certain embodiments, other denaturations such as source non fundus dystrophy are included within the scope of the term.

The term "brook membrane" refers to a five-layered structure that separates choroidal capillaries from PRE.

As used herein, the term "caged" means that the functional group of a tissue modifying molecule is protected by another molecule / part. In one particular embodiment, the term is meant to maintain the inactive form of the tissue modifying molecule without an activator.

As used herein, the term “drusen” is defined as a yellow deposit located below the RPE on the inner surface of the Brueck film.

As used herein, the term "eye disease" means a condition in which at least one eye of an individual does not reach normal health. In a preferred embodiment, the ocular disease comprises thickened Brooke membrane. In certain embodiments, the Brooke membrane is thickened about two times its normal thickness. In another specific embodiment, the membrane is about 3 to 10 times thicker than its normal thickness. Specific ocular diseases in which the Bruch's membrane is abnormally thickened include at least aging macular degeneration, juvenile macular degeneration, source ratio fundus dystrophy, or normal aging with reduced cancer compliance. Other eye diseases may not be characterized by thickened Brook membrane, but may benefit from alteration of eye tissues, such as alteration of the fiber strain to increase efflux capacity in glaucoma. Alteration of target tissue using two-photon irradiation can also be used to treat microvascular abnormalities of diabetic ocular disease, including diabetic macular edema and neovascularization. Selective two-photon irradiation and release thereof after systemic or topical administration of an inactive drug can be used to target drug effects on specific tissues of the intraocular. Selective liberation of this form can be used in external tissues to achieve selective effects.

As used herein, the term "macular" refers to the central region of the retina, including light perceptual cells of the central region of the retina.

As used herein, the term “macular degeneration” refers to the central part of the retina, ie the degeneration of the macular.

As used herein, the term "retina" refers to a neurological tissue located at the back of the eye, including rods and abstracts that receive light and convert it into electrical signals that are transmitted to the brain via the optic nerve.

As used herein, "tissue modifier" refers to one or more molecules that change the physical, chemical, or both of a tissue. In one particular embodiment, the term refers to an agent that alters tissue to at least partially improve diffusion through tissue. In another specific embodiment, the term refers to an agent capable of at least partially (at least partially undesirable) degradation of components of tissue. In another embodiment, the term refers to an agent capable of reducing lipids and / or crosslinked proteins in tissues such as the Brooke membrane. In certain embodiments, the term is meant to degrade one or more of its components. In another particular embodiment, the tissue modifying molecule is a detergent capable of extracting lipid and non-lipid deposits from tissues such as the Brooke membrane.

As used herein, the term "ultrasound contrast agent" refers to a microstructure that can retain exogenous contrast agents. Such microstructures can be destroyed by focused application of ultrasonic irradiation. Examples include microbubbles (small bubbles in suspension that can strongly scatter ultrasound) or liposomes.

II. The present invention

The present invention relates to the treatment of eye diseases, in particular by altering eye tissues associated with eye diseases. Such alterations may be of any type as long as the tissue is altered, but in certain embodiments it means enhancing the diffusion of tissue using the methods and compositions disclosed herein. In one particular embodiment, the methods and compositions affect the Brooke membrane to ameliorate eye diseases.

The ocular disease can be of any type, but in certain embodiments the ocular disease is any condition that results in AMD or other macular degeneration, such as source nasal fundus dystrophy, or thickening of the Bruch's membrane. In another specific embodiment, aging thickening in the absence of macular degeneration is also treated. Thickening in the elderly causes aging visual changes such as difficulty in dark adaptation. Thus, the methods and compositions of the present invention are directed to changing the physical and / or chemical structure of ocular tissues, and in certain embodiments, the tissues are non- such as in elderly patients with or without AMD (also in pediatric macular degeneration). In the elderly patient). In certain embodiments, the invention relates to treating glaucoma by topical administration of one or more tissue modifiers. Such treatment is useful in certain embodiments that alleviate glaucoma blocked in the development of glaucoma, eg, glaucoma.

In another specific embodiment, the present invention relates to the treatment of dry or wet macular degeneration. Those skilled in the art will appreciate that wet AMD may be treated by the methods of the present invention if the condition typically recurs after recurring choroidal macular degeneration by currently known methods (recurrent choroidal neovascularization). In another specific embodiment, the therapies described herein can maintain a better view by preventing such relapses and limiting the extent (growth) of existing choroidal neovascularization. In certain embodiments of the invention, the treatment causes degeneration of existing choroidal neovascularization by increasing the delivery of nutrients to the retina.

Accordingly, the present invention aims to treat ocular diseases such as macular degeneration by focusing on the thickened Brooke membrane associated with various eye diseases. This thickening is the result of abnormal deposition of lipids and crosslinked proteins, which precedes neovascularization through the Bruch's membrane, followed by bleeding, leakage of serous fluid and severe vision loss. As described herein, an improvement in diffusion through the Brooke membrane is achieved, thereby reducing the lipid and crosslinked protein barriers that accumulate in individuals with eye disease to prevent progression of vision loss. In one particular embodiment, the chemical composition of the membrane is altered. For example, detergents are removed by washing the lipids in the membrane. Enzymes break down proteins in the membrane. In certain embodiments, the method of the present invention increases water conductivity through the Brook membrane and / or increases the Brook membrane permeability of macromolecules and / or oxygen.

Generally, an inactive tissue modifying molecule is administered systemically or locally to an individual with signs or symptoms of aging Brooke membrane. In some embodiments, inactive molecules can be visualized prior to activation, such as by fluorescence. After sufficient time for the inert molecules to be properly distributed, the molecules accumulate in various tissues, including the Bruch's membrane. Once a sufficient amount is reached in the Brooke membrane, the visibility of the molecule is used to accurately target the Brooke membrane by an energy source that selectively activates tissue modifying molecules such as light or ultrasound.

In one particular embodiment, the present invention is useful for visualization of Brooke membranes as used in diagnostic techniques. That is, the inert fluorescent compound is administered to the subject and associated with the Brooke membrane, for example by binding to lipids in the Brooke membrane. Energy, such as in the form of light, or more specifically, two-photon irradiation, is focused on the composite of the inert photoactive compound / brook membrane, after which the inactive photoactive compound is activated. Energy emitted from the photoactive compound, such as light, visualizes the Brueck membrane. In certain embodiments, the amount of light emitted is proportional to the amount of lipid in the Brueck membrane. If fluorescent molecules are incorporated into the casing tissue modifier, the visualization of the Brooke membrane may be accompanied by the activation of the degradable material that causes controlled partial degradation of the Brooke membrane. In an alternative embodiment, the visualization is through the natural autofluorescence activity of the components of the Brooke membrane and / or via the OCT Doppler method.

III. Tissue-modifying molecules

The present invention utilizes tissue modifying molecules for delivery to ocular tissues for the treatment of the eye in certain embodiments. The tissue modifying molecule may be diffusion enhancing, degradable, or have both of these properties, and preferably alters the physical, chemical, or both of the tissues. Diffusion enhancing molecules (a) reduce the thickness of the membrane itself; (b) to increase the diffusion through the Brook membrane by reducing the amount of deposits in the Brook membrane and / or by changing the chemical properties of the Brook membrane. Preferably the molecule is inactive after administration to an individual and is activated upon exposure to an energy source. In certain embodiments, the tissue modifying molecule is cascaded. In another specific embodiment, the tissue modifying molecule is capable of proliferating through the Bruch membrane by degrading one or more components of the enzyme, such as cholesterol esterases, lipases, matrix metalloproteinases, or particularly, the Bruch membrane. Any enzyme or protein that can be increased. In another particular embodiment, the tissue modifying molecule is a detergent that can extract lipid and non-lipid deposits from within the Brook membrane and increases diffusion through the Brook membrane.

Some tissue modifying molecules contain one or more amino acid residues and are inactive by casing, wherein one or more amino acid side chains such as cysteine, aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, Serine, threonine, tyrosine or combinations thereof may include light-removing protecting groups such as one or more coumarinyl, quinoline-2-onyl, xanthenyl, thioxanthenyl, selenoxanthenyl and anthracenyl and / or stilbenyl groups Include. In another embodiment, the tissue modifying molecule is inactivated by casing in an ultrasound contrast agent such as microbubbles or liposomes. As with surfactants, there are other tissue modifying molecules without amino acid residues known in the art.

Tissue modifying molecules are formulated to provide effective concentrations in the desired tissue. In some embodiments, the tissue modifying molecule accumulates in non-lesionous tissue, but this is not a problem for the subject, since accurate targeting of the activating energy source to the Brooke membrane will lead to selective activation within that membrane. Because. Other sites in which cascaded tissue modifying molecules accumulate are not treated by activation energy, so the cascaded tissue modifying molecules remain inactivated and are removed through the kidneys and / or liver. In one particular embodiment, the cascaded molecule is not harmful or toxic in any way and is excreted from the body, preferably within about 48 hours after administration and more preferably within about 24 hours.

In some embodiments, the tissue modifying molecule is coupled to a specific binding ligand capable of binding to a particular target molecule in the Brook membrane. The target molecule may be endogenous to the Brooke membrane or may be selectively delivered to the Brooke membrane by crosslinking the target molecule using two-photon irradiation. In this embodiment, the tissue modifying molecule will be delivered at high concentration to the target tissue. In one particular embodiment, various protein binding domains, such as leucine zipper domains, are associated with tissue modifying molecules.

IV. Formulation

Tissue modifying molecules are formulated to provide effective concentrations in the desired tissue. In some embodiments, the tissue modifying molecule accumulates in non-lesionous tissue, but this is not a problem for the subject, since the precise targeting of the activating energy source to the Bruch's membrane induces selective activation within that tissue. Because. Other sites in which cascaded tissue modifying molecules accumulate are not treated by activation energy, so the cascaded tissue modifying molecules remain inactivated and are removed through the kidneys and / or liver. In some embodiments, the tissue modifying molecule is coupled to a specific binding ligand capable of binding to a specific surface component of the target Brooke membrane, or formulated with a carrier to deliver higher concentrations to the target tissue as needed. do. In certain embodiments, various protein binding domains, such as leucine zipper domains, are associated with tissue modifying molecules.

The nature of the formulation will depend in part on the mode of administration and the nature of the chosen degradation molecule. Any pharmaceutically acceptable excipient, or combination thereof, suitable for a particular tissue altering compound can be used. Thus, the compounds can be administered as an aqueous composition, as a topical composition, as a transmucosal or transdermal composition, in an oral dosage form, or as an intravenous dosage form, as a topical injection (eg, peripheral or intraocular injection), or in combination thereof. The formulation may also include liposomes.

V. Administration and Dosages

Tissue modifying molecular compounds may be administered by any of a variety of routes, such as, for example, oral, parenteral, or rectal, or the compounds may be administered directly to the eye, such as topical or peripheral injection. Parenteral administration such as intravenous, intramuscular, or subcutaneous administration is useful. Intravenous, periorbital and intraocular injections are particularly preferred embodiments for delivering the present invention or components thereof.

The dose of tissue-modifying molecule can vary widely depending on the mode of administration, the dosage form containing it (eg in the form of liposomes), or whether it is coupled to a target specific ligand such as an antibody or immunologically active fragment. . As generally recognized, there is an association between the type, formulation, mode of administration, and dosage level of tissue modifying molecules. It is possible and customary to tailor these parameters to specific combinations.

VI. Energy source

Energy sources include any stimulus that makes inactive tissue-modifying molecules active. This preferably leads to the activation of inactive tissue modifying molecules. Energy sources are well known in the art, but exemplary forms of energy sources include light or ultrasound. In certain embodiments, two photon photochemistry is used. In one particular embodiment, monochromatic light is used.

The various parameters used for effective and selective photoactivation of the tissue modifying molecules of the present invention are related to each other. Thus, the dose must also be adjusted for other parameters such as influence, irradiation, treatment duration and time interval between dose administration and therapeutic irradiation. All of these parameters should be adjusted to improve visual function without significant damage to eye tissue, and those skilled in the art are aware of how to do this.

Compositions and methods for two-photon absorption are known in the art, but representative methods are described in US Pat. No. 6,267,913, US Pat. No. 6,472,541 and WO 00/31588, all of which are incorporated herein by reference in their entirety. Quote.

VII. How to Create Proteins with Side Chains Using Photoremovable Protectors

Proteins can be cascaded using a number of methods. Casing can be performed by treating native non-casing molecules with reactive precursors to the casing groups. For example, the side chains of amino acid cysteine can be cascaded using photoremovable o-nitrobenzyl groups by treating cysteine containing proteins with o-nitrobenzylbromide. Another way to cascade proteins is to use solid-phase peptide synthesis starting from appropriate cascaded amino acids, and cascade to trophogenic bacterial strains by direct translation integration into proteins using methods based on nonsense inhibition. Chemical synthesis of proteins by supplementing amino acids.

In certain embodiments, the tissue modifying molecule is cascaded to become inactive before it is localized to the Brook membrane and is activated upon exposure to an energy source. In certain embodiments, the tissue modifying molecule is a protein having an amino acid side chain. Those that can be modified by protecting groups, for example photoremovable protecting groups include cysteine, aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, serine, threonine or tyrosine. Examples of photoremovable protecting groups are o-nitrobenzyl, decyl, phenacyl, trans-o-cinnayl, coumarinyl, quinoline-2-onyl, xanthenyl, thioxanthenyl, selenoxanthenyl and anthracenyl, steel Benyl and / or derivatives thereof. Such protecting groups are added to the side chains described herein.

VIII. Macular degeneration

Light enters through the transparent front of the eye (cornea), passes through the pupil opening, passes through the lens, and finally is detected by the retina at the back of the eye. The retina is a multilayer structure that encloses the inside of the eyeball. The retina is a specialized cell that converts light into electrical stimuli, delivering it to the brain and generating vision.

At the center of the retina is a small, highly specialized area called the macula. This region is about 1/8 inch in diameter, approximately similar in size to the "O". The macula is the most densely packed photoreceptor, a cell that collects light. It consists of rods and abstractions that also sense color. The macula is supplied with oxygen-rich blood that nourishes the cells.

If the macula has not been damaged, the object will see its fine details of any object in front of it. Macular degeneration involves the degeneration or destruction of this small structure. Central vision dims or disappears, and straight lines appear crooked or disconnected. The border of the statue is visible but not in the center of the statue. In other words, color perception is reduced because the abstraction is damaged. However, the patient is not completely blind and there is almost always a ring of vision around.

IX. Crystalline steel

The main feature of atrophic AMD is the accumulation of macular crystallites, ie localized thickening of the Brooke membrane. The diffusion of thickening of the Brooke membrane (base linear deposits) is an optimal histopathological predictor of choroidal neovascularization. The crystalline steel mainly consists of vesicular substances (lipids) and crosslinked proteins.

The presence of crystalline steel is a common feature of macular degeneration. In certain embodiments, an individual having a crystalline cavity or thickened Bruch membrane in at least one eye is treated by the methods described herein.

The following examples are provided for illustration only and are not intended to limit the scope of the invention in any way.

Example 1

Cascaded Tissue Modified Molecules

Cascaded tissue altering enzymes are constructed by masking various amino acid side chains in a protein using photoremovable protecting groups. Examples of such groups include o-nitrobenzyl, decyl, phenacyl, trans-o-cinamoyl, coumarinyl, quinoline-2-onyl, xanthenyl, thioxanthenyl, selenoxanthenyl and anthracenyl, stilbenyl And derivatives thereof. Such groups are introduced into the protein by global chemical synthesis (including natural chemical ligation), nonsense inhibition methods or post-translational modifications. Based on the known substrate components of the Bruch's membrane, cholesterol esterases, lipases, substrate metalloproteinases degrade a portion of the Bruch's membrane and enhance membrane permeation diffusion. "Kaseing" these enzymes inactivates them. Thus, after systemic administration, they circulate and bind to tissue, which are then removed inactive. Applying precisely focused light energy to the Brueck membrane (two-photon photochemistry) removes the "casing protecting group" and selectively activates the enzyme (s) in the Brueck membrane. Once activated, controlled degradation occurs and membrane permeation diffusion is enhanced. This reduces the likelihood of progression of choroidal neovascularization. It can also improve dark adaptation and suggestion in older people with thickened Brueck membranes. This can also lead to degeneration of established choroidal neovascularization.

Alternatively, mild detergent molecules (e.g. surfactants) may be o-nitrobenzyl, decyl, phenacyl, trans-o-cinamoyl, coumarinyl, activated by irradiation (e.g. by two-photon irradiation). Selective biochemistry of Brooke membranes by "casing" using groups such as quinoline-2-onyl, xanthenyl, thioxanthenyl, selenoxanthenyl and anthracenyl, and / or stilbenyl moieties and derivatives thereof, and the like Induces an enemy deformation. Similar to enzymatic treatment, these activated detergent molecules alter the diffusion barrier of the aged Brooke membrane to reduce the likelihood of visual loss and / or improve visual function.

Example 2

Alternative Inactivation Embodiments

Degradable enzymes (substrate metalloproteinases, cholester esterases, lipases, serine proteases) can also be incorporated into ultrasound contrast agents such as microbubbles and liposomes. Degradable molecules retained in such contrast agents are inactive. Correct application of ultrasonic energy to the Brooke membrane leads to cavitation of the contrast agent and release of the degradable enzymes to the Brooke membrane. Similar to the above examples, enzymatic release improves membrane permeation diffusion and potentially improves visual function. The remaining encapsulated inactive enzyme is not activated by ultrasound and is removed from the body without causing secondary tissue alteration.

Example 3

Improved targeting

Many of the enzymes administered in the previous examples are present both in the Bruch membrane and / or in the RPE. They are also present in the circulation. To increase the local concentration of these enzymes in the Bruch's membrane, these enzymes can be fused to various protein binding domains, such as the leucine zipper domain, chitin binding domain, or Src homology 2 (SH2) domain. Those skilled in the art know that heterodimeric zippers consist of acidic and basic partners and can self assemble into wound coils that exist as dimers and higher aggregates. Thus, in some embodiments, the acidic (basic) leucine zipper domain can be selectively delivered to the Brueck membrane via light crosslinking initiated by two-photon irradiation. In certain embodiments, the enzyme is administered fused to a basic (acidic) leucine zipper domain and is administered, for example, systemically (eg, orally or intravenously), or by injection (eg, intraocular and / or peripheral injections). ) And is distributed in extracellular tissue, including the Bruch's membrane. Formation of heterodimers in the Bruch's membrane increases the local concentration of the degrading enzymes, which in turn enhances the membrane permeation diffusion properties. Since the concentration of these degrading enzymes remains low in other tissues, there are no undesirable secondary tissue alterations. In addition, the method described in Example 1 can be used to produce enzymes containing photoactive groups (eg, benzophenoyl, phenylazide, trifluoromethylphenyldiazirinyl and derivatives thereof). Systemic administration of such enzymes, followed by selective irradiation of the Bruch's membrane, initiates optical crosslinking between the enzyme and the Bruch's membrane, increasing the concentration of the degradable enzyme. In other embodiments, the protein binding domain promotes association of the targeting tissue modifying molecule with the targeted tissue, such as by binding to a protein in the tissue surface or tissue.

Example 4

Treatment of eye diseases

Patients with ocular diseases, such as atrophic aging macular degeneration (as a representative example), are administered a cascaded, inactivated enzyme capable of altering the Brook membrane after photoactivation. In certain embodiments, administration is by systemic administration (eg, intravenous or oral administration) or by injection (eg intraocular and / or peripheral injection). In other specific embodiments, the casing, inactivated enzyme is labeled (eg, fluorescently labeled), but in alternative embodiments, the casing, inactivated enzyme is not labeled.

For illustrative purposes only, the cascaded, inactivated molecule is a fluorescent molecule. A few minutes after administration, the fluorescent casing degradable complex is visualized in the macular Brook membrane by two-photon irradiation. After focusing on the fluorescent label in the macular Brook membrane, a higher dose of two-photon irradiation is applied to release the enzyme and initiate partial degradation of the Brook membrane. Systemically distributed unirradiated cascaded enzyme is excreted in inactive form. Both two-photon irradiation visualization of the labeled Brooke membrane and photoactivation of the degradable material are performed at levels that are not toxic to the ocular tissue.

Alternatively, fluorescent labels not attached to the cascaded degradable enzyme complexes are administered systemically. After about 10-60 minutes, the cascaded degradable molecular complex is administered. After about 5 minutes, the Bruch's membrane is visualized by two-photon irradiation of fluorescent labels. Higher doses of two-photon irradiation are then used to activate the cascaded degradable molecular complexes.

In another embodiment, a fluorescent label not attached to the cascaded degrading enzyme complex is administered systemically. After about 10-60 minutes, the two-photon irradiation is used to visualize the amount of fluorescence in the Brook film. Quantification of fluorescence is a diagnostic indicator of the severity of atrophic macular degeneration.

Another embodiment of the present invention is a method of identifying a tissue to be treated based on inherent light scattering from a target tissue, such as a Brooke membrane. Optical coherence tomography (OCT) using visible or infrared light is used to detect alterations in the physical or chemical properties of the intraocular Brook Film. OCT can be used to observe the intraocular structure, to observe the mobility of the structure by Doppler OCT, and to observe the chemical properties by using an exogenous dye and OCT together. In such embodiments, the OCT and / or its modifications are used to ascertain the properties of the Brooke membrane with altered properties that allow for guided treatment. The treatment may be photoliberation or photoactivation or photoablation of endogenous or exogenous materials in or near the Bruch's membrane.

An exemplary embodiment of targeting the Brueck membrane is shown in FIG. 1.

references

All patents and publications mentioned herein are indicative of the level of skill of those skilled in the art. All patents and publications are incorporated herein by reference to the same extent as if each of these publications were specifically and individually incorporated by reference.

U.S. Patent 5,756,541

U.S. Patent 5,798,349

U.S. Patent 5,910,510

U.S. Patent 5,935,942

U.S. Patent 6,128,525

U.S. Patent 6,140,314

U.S. Patent 6,225,303

U.S. Patent 6,248,727

U.S. Patent 6,267,913

U.S. Patent 6,472,541

WO 00/31588

Claims (63)

A method of treating ocular disease comprising increasing diffusion through an intraocular Bruch membrane. The method of claim 1, wherein the increased diffusion is due to a decrease in the thickness of the film, a change in the composition of the film, or both. Administering an inactive form of the diffusion enhancing molecule to the Brook membrane in an amount sufficient to form a Brooke membrane / inert diffusion enhancing molecule complex; And Exposing the complex to an activator, wherein the activator activates the inert diffusion enhancing molecule as an active form of the diffusion enhancing molecule, and the activation induces an increase in diffusion through the membrane. A method of increasing diffusion through a Bruch membrane of at least one eye of an individual, comprising. The method of claim 3, wherein the increase in diffusion is due to a decrease in the thickness of the film, a change in the composition of the film, or both. The method of claim 3, wherein the increase in diffusion is due to alteration of lipids, proteins, or both in the membrane. The method of claim 3, wherein the subject has eye disease. The method of claim 6, wherein the ocular disease is aging macular degeneration, pediatric macular degeneration, Sorby's fundus dystrophy, or aging decline in visual function not associated with macular degeneration.  The method of claim 3, wherein the inert diffusion enhancing molecule directly binds to the membrane. The method of claim 3, wherein the inert diffusion enhancing molecule is a protein, detergent or surfactant. The method of claim 9, wherein the protein is an enzyme. The method of claim 10, wherein the inactive enzyme is further defined as caged by one or more photoremovable protecting groups on the amino acid side chain of the enzyme. 12. The protecting group according to claim 11, wherein said protecting groups are o-nitrobenzyl, decyl, phenacyl, trans-o-cinnayl, coumarinyl, quinoline-2-onyl, xanthenyl, thioxanthyl, selenoxanthenyl and anthra Cenyl, stilbenyl, or a combination thereof. 12. The protecting group according to claim 11, wherein said protecting groups are o-nitrobenzyl, decyl, phenacyl, trans-o-cinnayl, coumarinyl, quinoline-2-onyl, xanthenyl, thioxanthyl, selenoxanthenyl and anthra Cenyl, stilbenyl, or derivatives thereof. The method of claim 11, wherein the amino acid is cysteine, aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, serine, threonine, tyrosine, or a combination thereof. 10. The detergent of claim 9, wherein the detergent comprises one or more o-nitrobenzyl, decyl, phenacyl, trans-o-cinnayl, coumarinyl, quinoline-2-onyl, xanthenyl, thioxanthenyl, selenoxanthenyl and And further defined as being cascaded by an anthracenyl, or a compound comprising a stilbenyl group. The method of claim 9, wherein the protein is further defined as being cascaded by an ultrasound contrast agent. The method of claim 16, wherein the ultrasound contrast agent is microbubbles or liposomes. The method of claim 9, wherein the protein further comprises a protein binding domain. The method of claim 18, wherein the protein binding domain is a heterodimeric domain. The method of claim 18, wherein the protein binding domain is a leucine zipper domain, chitin binding domain or Src homology 2 (SH2) domain. The method of claim 3, wherein the inert diffusion enhancing molecule is administered to the subject as a pharmaceutically acceptable composition. The method of claim 21, wherein the inert diffusion enhancing molecule is administered systemically to the subject as a pharmaceutically acceptable composition. The method of claim 21, wherein the inert diffusion enhancing molecule is a pharmaceutically acceptable composition that is orally administered, administered by injection, rectal, vaginal or topical to the subject. The method of claim 23, wherein the injection is intraocular or peripheral injection. The method of claim 10, wherein the enzyme is a substrate metalloproteinase, cholesterol esterase, lipase, cathepsin, protease, or a combination thereof. The method of claim 25, wherein the protease is a serine protease. The method of claim 3, wherein the activating source is energy. The method of claim 27, wherein said energy is light or ultrasound. The method of claim 3, wherein the activating source is two-photon irradiation. The method of claim 3, wherein the exposing step is further defined as exposing the composite to light energy from a focused laser source. The method of claim 3, wherein the diffusion enhancing molecule is labeled by a fluorescent molecule. The method of claim 3, wherein the method further comprises visualizing the film. 33. The method of claim 32, wherein the membrane is made by delivery of a targeted fluorescent label to the membrane, identification of membrane-specific autofluorescence, or optical coherence tomography (OCT) Doppler. Administering the inactivated diffusion enhancing molecule to an individual in an amount sufficient to associate the molecule with the Brooke membrane to form a Brooke membrane / inert diffusion enhancing molecule complex, wherein the inactivated diffusion enhancing molecule is pharmaceutically. Administered as an acceptable composition; And Exposing the membrane to an activator, wherein diffusion through the eye's Brook membrane is improved after the exposing step A method of treating aging macular degeneration of at least one eye of a subject, wherein the macular degeneration is characterized by a thickened Brooke membrane. 35. The method of claim 34, wherein the Brook film is visualized. 36. The method of claim 35, wherein the visualization is defined as administering to a subject an amount of fluorescent molecules sufficient to associate with the membrane for visualization of the Brooke membrane. 36. The method of claim 35, wherein the visualization is defined as identifying the autofluorescence of the film. 36. The method of claim 35, wherein the visualization is made by optical coherence tomography (OCT) Doppler. The method of claim 36, wherein the amount of fluorescence emitted is proportional to the amount of lipid in the Bruch membrane. 36. The method of claim 35, wherein the visualization is defined as administering visible or infrared light to the subject. 41. The method of claim 40, wherein administering visible or infrared light comprises using optical coherence tomography. 42. The method of claim 41, wherein the method further comprises administering an exogenous dye. A kit housed in a suitable container comprising the inactivated Brooke membrane diffusion enhancing molecule. 44. The kit of claim 43, further comprising an activator for activation of said inactivated Bruch membrane degradable molecule. 44. The kit of claim 43, further comprising fluorescent molecules. Administering the fluorescent molecules to the subject in an amount sufficient to cause the fluorescent molecules to associate with the intraocular Bruch membrane; Exposing the Brooke membrane to radiation to visualize fluorescence, wherein the amount of fluorescence is an indicator of the severity of the ocular disease Comprising, at least one eye of the subject eye diseases. 47. The method of claim 46, wherein the method further comprises visualizing the film. 48. The method of claim 47, wherein the membrane visualization is by delivery of a fluorescent label targeted to the membrane, identification of membrane-specific autofluorescence, or optical coherence tomography (OCT) Doppler. The method of claim 46, wherein the ocular disease is macular degeneration. 47. The method of claim 46, wherein the irradiation is two photon irradiation. Visualizing the intraocular Bruch membrane; Administering to the subject an inactive form of the photoactive diffusion enhancing molecule in an amount sufficient to form a Bruch membrane / inactive photoactive diffusion enhancing molecule complex; And Exposing the complex to an inactivation source, the activating source activates the inactive photoactive diffusion enhancing molecule into an active form of the photoactive diffusion enhancing molecule, the activation increasing the diffusion through the Brueck membrane, the composition of the Bruch membrane Inducing a change in, or both Method for the treatment of eye diseases in a subject comprising. 52. The method of claim 51, wherein the visualization is by delivery of a targeted fluorescent label to the membrane, identification of membrane-specific autofluorescence, or optical coherence tomography (OCT) Doppler. The method of claim 52, wherein the fluorescent label is bound to the inactive form of the photoactive molecule. The method of claim 51, wherein the inactive photoactive molecule associates with a lipid in the Brook membrane. Administering to the eye tissue an inactive form of the tissue modifying molecule in an amount sufficient to cause the tissue modifying molecule to be targeted to the eye tissue; And Exposing the molecule to an activator, wherein the activator activates the inactive tissue modifying molecule into an active form of the tissue modifying molecule, wherein the activation induces alteration of at least a portion of the ocular tissue. In step Including, the method of changing the eye tissue of the object. The method of claim 55, wherein the method provides a treatment for eye disease in the subject. The method of claim 3, wherein the method further comprises visualizing the tissue. 33. The method of claim 32, wherein the tissue visualization is by delivery of a targeted fluorescent label to tissue, identification of tissue specific autofluorescence, or optical coherence tomography (OCT) Doppler. The method of claim 56, wherein the ocular disease is aging macular degeneration, pediatric macular degeneration, source ratio fundus dystrophy, aging decline in visual function unrelated to macular degeneration, or glaucoma. The method of claim 55, wherein the inactive tissue modifying molecule is administered to the subject as a pharmaceutically acceptable composition. The method of claim 55, wherein the inactive tissue modifying molecule is administered systemically to the subject as a pharmaceutically acceptable composition. The method of claim 55, wherein the inactive tissue modifying molecule is orally administered, administered by injection, rectal, vaginal, or topical administration to a subject as a pharmaceutically acceptable composition. 63. The method of claim 62, wherein the injection is intraocular or periorbital injection.