Patent 6,696,415

Tubedown-1 (tbdn-1), a protein associated with acetyltransferase activity has been characterized and its cDNA isolated. Tbdn-1 regulates endothelial differentiation through protein acetylation, DNA-binding or by interacting with and/or acetylating other protein targets important for endothelial differentiation. In normal adult eyes, tbdn-1 is expressed highly in the corneal endothelium proper and in the vascular endothelium of the limbus and retina. Tbdn-1 is absent or downregulated in the vascular endothelia of diseased and injured eyes, including eyes from patients with proliferative retinopathies involving neovascularization. Inhibition of tbdn-1 expression in endothelial cells in vitro indicates tbdn-1 acts as an inhibitor of angiogenesis. Thus, high levels of tbdn-1 expression present in normal ocular endothelial cells is associated with suppression of abnormal neovascularization in the eye demonstrating the therapeutic usefulness of tbdn-1 as a regulator of retinal angiogenesis.

SUMMARY OF THE INVENTION

A novel and highly conserved protein associated with an acetyltransferase activity named tubedown-1 (tbdn-1) has been isolated and characterized. Tbdn-1 regulates endothelial differentiation through protein acetylation, DNA-binding or by interacting with and/or acetylating other protein targets important for endothelial differentiation. Tbdn-1 is expressed during maturation of the developing vitreal vasculature. In normal adult eyes, tbdn-1 is expressed in the corneal endothelium proper and in the vascular endothelium of the limbus and retina. Tbdn-1 is absent or downregulated in the vascular endothelia of diseased and injured eyes, including eyes from patients with proliferative retinopathies involving neovascularization such as diabetic retinopathy, age related macular degeneration and retinopathy of prematurity. Tbdn-1 is downregulated during capillary differentiation of both IEM endothelial cells and RF/6A choroid-retina endothelial cells in vitro. Inhibition of tbdn-1 expression in IEM and RF/6A endothelial cells in vitro indicates tbdn-1 acts as an inhibitor of angiogenesis. These results taken together indicate that high levels of tbdn-1 expression present in normal ocular endothelial cells is associated with suppressing ocular neovascularization.

Accordingly, the gene tbdn-1, the cDNA of tbdn-1 (SEQ ID NO. 1), an open reading frame of tbdn-1 (such as SEQ ID NO. 6), and nucleotide sequences showing at least 70% sequence homology to SEQ ID NO. 1 or SEQ ID NO. 6, amino acid sequences translated from the cDNA of SEQ ID NO. 1, such as SEQ ID NOS. 2, 3, 4, and 5, and others amino acid sequences showing at least 85% sequence homology to SEQ ID NOS. 2, 3, 4, and 5 and which also exhibit anti-angiogenic activity may all be used as anti-angiogenic agents for treatment of ocular neovascularization. Compositions comprising a pharmaceutically effective amount of an amino acid sequence, which shows anti-angiogenic activity, that is translated from cDNA of SEQ ID NO. 1, particularly the amino acid sequences selected from the group consisting of SEQ ID NOS. 2, 3, 4 5 and a pharmaceutically acceptable carrier are also within the scope of this invention.

Methods for treating, inhibiting or delaying the onset of angiogenesis-associated diseases in mammals, wherein the angiogenesis-associated diseases are related to ocular neovascularization, are also within the scope of this invention. This method of treatment comprises treating the mammal with a pharmaceutically effective amount of an exogenously produced amino acid sequence showing anti-angiogenic activity and which is translated from the cDNA of SEQ ID NO. 1. These amino acid sequences include, but are not limited to sequences given in SEQ ID NOS. 2, 3, 4 and 5. The angiogenesis-associated diseases include, but are not limited to diabetic retinopathy, retinopathy of prematurity, primary hyperplastic vitreous, macular degeneration and any other conditions involving ocular neovascularization. The amino acid sequence may be contained in a pharmaceutically acceptable carrier and administered by intraocular injection, subretinal injection, subscleral injection, intrachoroidal injection, subconj unctival injection, topical administration or oral administration.

A gene therapy approach for treatment of mammals afflicted with an angiogeneis-associated disease, such as those related to ocular neovascularization, and in particular diabetic retinopathy and retinopathy of prematurity is also provided. For this method of treatment, an amino acid sequence, having anti-angiogenic activity, is translated from the cDNA of SEQ ID NO.1, and is provided to cells of a mammal having a deficiency in that amino acid sequence. This method further comprises administering into the cells a vector comprising and expressing a DNA sequence encoding the desired amino acid sequence, and expressing the DNA sequence in the cells to produce amino acid sequence. Cells harboring the vector secrete the amino acid sequence and this sequence is subsequently taken up by other cells deficient in the amino acid sequence. The amino acid sequences include, but are not limited to SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5.

DETAILED DESCRIPTION OF THE INVENTION

Discussion

Tbdn-1 encodes a novel 69 kDa protein associated with acetyltransferase activity (32). Tbdn-1 is downregulated during IEM and RF/6A capillary formation in vitro. Inhibition of tbdn-1 by expression of antisense tbdn-1 cDNA augments capillary formation of IEM and RF/6A cells. These results support a hypothesis that tbdn-1 plays a role in dampening and/or moderating physiological angiogenesis. Thus, the therapeutic modulation of tbdn-1 may be useful for treating ocular neovascularization.

Tbdn-1 expression peaks during early to middle stages of development of most blood vessels and is downregulated at later stages of maturation, suggesting it may be involved with regulating specific stages of blood vessel maturation during embryogenesis (32). This is exemplified by tbdn-1 expression in yolk vasculature development, in which tbdn-1 is expressed most highly during early stages of yolk sac vasculature formation, and is downregulated at the later stages of development during which time angiogenesis of the vitelline vasculature occurs (32). Tbdn-1 is not detected in most adult vascular beds, but persists at high levels in the adult ocular vasculature. High levels of expression of tbdn-1 are associated with ocular endothelial homeostasis in adult. Conversely, low levels of tbdn-1 expression are associated with endothelial capillary outgrowth in vitro and retinal neovascularization in vivo. Since the expressed tbdn-1 protein is a member of a family of regulatory enzymes, which are known to control a range of processes including cell growth and differentiation through posttranslational modification, tbdn-1 is hypothesized to be involved in maintaining homeostasis and preventing retinal neovascularization.

In normal adult eyes, tbdn-1 is highly expressed in the corneal endothelium proper and in the vascular endothelium of the limbus and retina. Tbdn-1 is absent or downregulated in the vascular endothelia of diseased and injured eyes including eyes from patients with proliferative retinopathies involving neovascularization. Thus, high levels of tbdn-1 expression present in normal ocular endothelial cells is associated with suppressing neovascularization in the eye. Accordingly, the gene tbdn-1, its analogues, the proteins which tbdn-1 encodes for and its analogues as well as the cDNA sequence, may be used therapeutically to regulate retinal angiogenesis.

Methods of Treatment

In accordance with the method of the present invention, an effective amount of the cDNA of tbdn-1 as isolated in a purified form (SEQ ID NO. 1), modified versions thereof showing at least 70% sequence homology, the protein the cDNA encodes for (SEQ ID. NO. 2), or modified versions of that protein, including but not limited to SEQ ID NOS. 3, 4 and 5, modified versions thereof showing at least 85% sequence homology, or modifications of accessory components of the signaling pathway in which tbdn-1 is active, or combinations thereof, may be used as an anti-angiogenic agents for the treatment of ocular neovascularization and related diseases. Additionally, the open reading frame sequence of the cDNA of tbdn-1 (base pairs 408-2186, SEQ ID NO. 6) coding for the expressed tbdn-1 protein (SEQ ID NO. 2) may also be used as an anti-angiogenic agent. All of these substances will be collectively referred to as "tbdn-1 agents."

The tbdn-1 derived angiogenesis inhibitor agents of the present invention are useful in inhibiting pathological neovascularization in mammals. As used herein, the term "pathological neovascularization" refers to those conditions where the formation of blood vessels (neovascularization) is harmful to the patient. Examples of pathological neovascularization dependent diseases include: head trauma, spinal trauma, systemic or traumatic shock, stroke, hemorrhagic shock, cancer, arthritis, arteriosclerosis, angiofibroma, arteriovenous malformations, corneal graft neovascularization, delayed wound healing, diabetic retinopathy, granulations, burns, hemangioma, hemophilic joints, hypertrophic scars, ocular neovascularization, nonunion fractures, Osler-Weber Syndrome, psoriasis, pyogenic granuloma, retrolental fibroplasia, pterigium, scleroderma, trachoma, vascular adhesions, and solid tumor growth.

In particular, the compositions are useful in preventing and treating any ocular neovascularization, including, but not limited to: retinal diseases (diabetic retinopathy, chronic glaucoma, retinal detachment, sickle cell retinopathy and subretinal neovascularization due to senile macular degeneration); rubeosis iritis; proliferative vitreo-retinopathy; inflammatory diseases; chronic uveitis; neoplasms (retinoblastoma, pseudoglioma and melanoma); Fuchs' heterochromic iridocyclitis; neovascular glaucoma; corneal neovascularization (inflammatory, transplantation and developmental hypoplasia of the iris); neovascularization following a combined vitrectomy and lensectomy; vascular diseases (retinal ischemia, choroidal vascular insufficiency, choroidal thrombosis and carotid artery ischemia); neovascularization of the optic nerve; and neovascularization due to penetration of the eye or contusive ocular injury.

The tbdn-1 agents can be used therapeutically either as exogenous materials or as an endogenous materials. Exogenous tbdn-1 agents, are those produced or manufactured outside of the body and administered to the body. Endogenous tbdn-1 agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery to within or to other organs in the body. Tbdn-1 is present in body tissue. Patients who suffer from ocular neovascularization have a tendency to have decreased levels of expressed tbdn-1 in the ocular endothelial cells.

Endogenous Therapy

The principles of gene therapy for the production of therapeutic products within the body include the use of delivery vehicles (termed vectors) that can be non-pathogenic viral variants, lipid vesicles (liposomes), carbohydrate and/or other chemical conjugates of nucleotide sequences encoding the therapeutic protein or substance. These vectors are introduced into the body's cells by physical (direct injection), chemical or cellular receptor mediated uptake. Once within the cells, the nucleotide sequences can be made to produce the therapeutic substance within the cellular (episomal) or nuclear (nucleus) environments. Episomes usually produce the desired product for limited periods whereas nuclear incorporated nucleotide sequences can produce the therapeutic product for extended periods including permanently.

In clinical settings, the gene delivery systems for therapeutic tbdn-1 genes can be introduced into a patient (or non-human animal) by any of a number of methods, each of which is known in the art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof.

The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.

Gene therapy methodologies can also be described by delivery site. Fundamental ways to deliver genes include ex vivo gene transfer, in vivo gene transfer and in vitro gene transfer. In ex vivo gene transfer, cells are taken from the patient and grown in cell culture. The DNA is transfected into the cells, and the transfected cells are expanded in number and then reimplanted in the patient. In in vitro gene transfer, the transformed cells are cells growing in culture, such as tissue culture cells, and not particular cells from a particular patient. These "laboratory cells" are transfected, and the transfected cells are selected and expanded for either implantation into a patient or for other uses. In vivo gene transfer involves introducing the DNA into the cells of the patient when the cells are within the patient. In vivo gene transfer also involves introducing the DNA specifically into the ocular endothelial cells of the patient using gene therapy vectors containing endothelial specific promoters. All three of the broad-based categories described above may be used to achieve gene transfer in vivo, ex vivo and in vitro.

Mechanical (i.e., physical) methods of DNA delivery can be achieved by microinjection of DNA into germ or somatic cells, pneumatically delivered DNA-coated particles such as the gold particles used in a "gene gun" and inorganic chemical approaches such as calcium phosphate transfection. It has been found that physical injection of plasmid DNA into muscle cells yields a high percentage of cells which are transfected and have sustained marker genes. The plasmid DNA may or may not integrate into the genome of cells. Non-integration of the transfected DNA would allow the transfection and expression of gene product proteins in terminally differentiated, non-proliferative tissues for a prolonged period of time without fear of mutational insertions, deletions or alterations in the cellular or mitochondrial genome. Long-term, but not necessarily permanent, transfer of therapeutic genes into specific cells may provide treatments for genetic diseases or for prophylactic use. The DNA could be reinjected periodically to maintain the gene product level without mutations occurring in the genomes of the recipient cells. Non-integration of exogenous DNAs may allow for the presence of several different exogenous DNA constructs within one cell with all of the constructs expressing various gene products.

Particle-mediated gene transfer may also be employed for injecting DNA into cells, tissues and organs. With a particle bombardment device, or "gene gun," a motive force is generated to accelerate DNA-coated high density particles (such as gold or tungsten) to a high velocity that allows penetration of the target organs, tissues or cells. Electroporation for gene transfer uses an electrical current to make cells or tissues susceptible to electroporation-mediated gene transfer. A brief electric impulse with a given field strength is used to increase the permeability of a membrane in such a way that DNA molecules can penetrate into the cells. The techniques of particle-mediated gene transfer and electroporation are well known to those of ordinary skill in the art

Chemical methods of gene therapy involve carrier-mediated gene transfer through the use of fusogenic lipid vesicles such as liposomes or other vesicles for membrane fusion. A carrier harboring a DNA or protein of interest can be conveniently introduced into body fluids or the bloodstream and then site specifically directed to the target organ or tissue in the body. Cell or organ-specific DNA-carrying liposomes, for example, can be developed and the foreign DNA carried by the liposome absorbed by those specific cells. Injection of immunoliposomes that are targeted to a specific receptor on certain cells can be used as a convenient method of inserting the DNA into the cells bearing that receptor. Another carrier system that has been used is the asialoglycoprotein/polylysine conjugate system for carrying DNA to hepatocytes for in vivo gene transfer.

Transfected DNA may also be complexed with other kinds of carriers so that the DNA is carried to the recipient cell and then deposited in the cytoplasm or in the nucleoplasm. DNA can be coupled to carrier nuclear proteins in specifically engineered vesicle complexes and carried directly into the nucleus.

Carrier mediated gene transfer may also involve the use of lipid-based compounds which are not liposomes. For example, lipofectins and cytofectins are lipid-based positive ions that bind to negatively charged DNA and form a complex that can ferry the DNA across a cell membrane. Another method of carrier mediated gene transfer involves receptor-based endocytosis. In this method, a ligand (specific to a cell surface receptor) is made to form a complex with a gene of interest and then injected into the bloodstream. Target cells that have the cell surface receptor will specifically bind the ligand and transport the ligand-DNA complex into the cell.

Biological gene therapy methodologies employ viral vectors to insert genes into cells. Viral vectors that have been used for gene therapy protocols include, but are not limited to, retroviruses, other RNA viruses such as poliovirus or Sindbis virus, adenovirus, adeno-associated virus, herpes viruses, SV 40, vaccinia, lentivirus, and other DNA viruses. Replication-defective murine retroviral vectors are the most widely utilized gene transfer vectors. Murine leukemia retroviruses are composed of a single strand RNA completed with a nuclear core protein and polymerase (pol) enzymes encased by a protein core (gag) and surrounded by a glycoprotein envelope (env) that determines host range. The genomic structure of retroviruses include gag, pol, and env genes enclosed at the 5' and 3' long terminal repeats (LTRs). Retroviral vector systems exploit the fact that a minimal vector containing the 5' and 3' LTRs and the packaging signal are sufficient to allow vector packaging and infection and integration into target cells providing that the viral structural proteins are supplied in trans in the packaging cell line.

Fundamental advantages of retroviral vectors for gene transfer include efficient infection and gene expression in most cell types, precise single copy vector integration into target cell chromosomal DNA and ease of manipulation of the retroviral genome. For example, altered retrovirus vectors have been used in ex vivo methods to introduce genes into peripheral and tumor-infiltrating lymphocytes, hepatocytes, epidermal cells, myocytes or other somatic cells (which may then be introduced into the patient to provide the gene product from the inserted DNA).

The adenovirus is composed of linear, double stranded DNA complexed with core proteins and surrounded with capsid proteins. Advances in molecular virology have led to the ability to exploit the biology of these organisms to create vectors capable of transducing novel genetic sequences into target cells in vivo. Adenoviral-based vectors will express gene product peptides at high levels. Adenoviral vectors have high efficiencies of infectivity, even with low titers of virus. Additionally, the virus is fully infective as a cell-free virion so injection of producer cell lines are not necessary. Another potential advantage to adenoviral vectors is the ability to achieve long term expression of heterologous genes in vivo.

Viral vectors have also been used to insert genes into cells using in vivo protocols. To direct tissue-specific expression of foreign genes, cis-acting regulatory elements or promoters that are known to be tissue-specific may be used. This could also involve using gene therapy vectors containing endothelial specific promoters for purposes of targeting blood vessels. Alternatively, this can be achieved using in situ delivery of DNA or viral vectors to specific anatomical sites in vivo. For example, gene transfer to blood vessels in vivo was achieved by implanting in vitro transduced endothelial cells in chosen sites on arterial walls. The virus-infected surrounding cells, in turn, also expressed the gene product. A viral vector can be delivered directly to the in vivo site (by catheter, for example) thus allowing only certain areas to be infected by the virus and providing long-term, site-specific gene expression. In vivo gene transfer using retrovirus vectors has also been demonstrated in mammary tissue and hepatic tissue by injection of the altered virus into blood vessels leading to the organs.

When used in the above or other treatments, a therapeutically effective amount of one of the compounds of the present invention may be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. A "therapeutically effective amount" of the compound of the invention means a sufficient amount of the compound to limit tumor growth or to slow or block tumor metastasis at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.

The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose.

Gene therapy also contemplates the production of a protein or polypeptide where the cell has been transformed with a genetic sequence that turns off the naturally occurring gene encoding the protein, i.e., endogenous gene-activation techniques.

Exogenous Therapy

A safe and effective amount of the tbdn-1 agent is defined as an amount, which would cause the desired therapeutic effect in a patient while minimizing undesired side effects. The dosage regimen will be determined by skilled clinicians, based on factors such as the exact nature of the condition being treated, the severity of the condition, the age and general physical condition of the patient, and so on.

The ophthalmic compositions of the present invention will include one or more tbdn-1 agents and a pharmaceutically acceptable vehicle for said compound(s). Various types of vehicles may be used. The vehicles will generally be aqueous in nature. Aqueous solutions are generally preferred, based on ease of formulation, as well as a patients' ability to easily administer such compositions by means of instilling one to two drops of the solutions in the affected eyes. However, the compounds of formula (I) may also be readily incorporated into other types of compositions, such as suspensions, viscous or semi-viscous gels or other types of solid or semi-solid compositions. Suspensions may be preferred for the tbdn-1 agents which are relatively insoluble in water. The ophthalmic compositions of the present invention may also include various other ingredients, such as buffers, preservatives, co-solvents and viscosity building agents.

The tbdn-1 agents may be contained in various types of pharmaceutical compositions, in accordance with formulation techniques known to those skilled in the art. For example, the tbdn-1 agents may be included in solutions, suspensions and other dosage forms adapted for topical application to the involved tissues, such as tissue irrigating solutions. An appropriate buffer system (e.g., sodium phosphate, sodium acetate or sodium borate) may be added to prevent pH drift under storage conditions.

Ophthalmic products are typically packaged in multidose form. Preservatives are thus generally required to prevent microbial contamination during use. Examples of suitable preservatives include: benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium, sorbic acid, polyquaternium-1, or other agents known to those skilled in the art. Such preservatives are typically employed at a level of from about 0.001 to about 1.0 percent by weight, based on the total weight of the composition (wt. %).

Some of the tbdn-1 agents may have limited solubility in water and therefore may require a surfactant or other appropriate co-solvent in the composition. Such co-solvents include: polyethoxylated castor oils, Polysorbate 20, 60 and 80; Pluronic Registered TM F-68, F-84 and P-103 (BASF Corp., Parsippany N.J., USA); cyclodextrin; or other agents known to those skilled in the art. Such co-solvents are typically employed at a level of from about 0.01 to about 2 wt. %.

The use of physiologically balanced irrigating solutions as pharmaceutical vehicles for the tbdn-1 agents is preferred when the compositions are administered intraocularly. As used herein, the term "physiologically balanced irrigating solution" means a solution which is adapted to maintain the physical structure and function of tissues during invasive or noninvasive medical procedures. This type of solution will typically contain electrolytes, such as sodium, potassium, calcium, magnesium and/or chloride; an energy source, such as dextrose; and a buffer to maintain the pH of the solution at or near physiological levels. Various solutions of this type are known (e.g., Lactated Ringers Solution). BSS Registered TM Sterile Irrigating Solution and BSS Plus Registered TM Sterile Intraocular Irrigating Solution (Alcon Laboratories, Inc., Fort Worth, Tex., USA) are examples of physiologically balanced intraocular irrigating solutions. The latter type of solution is described in U.S. Pat. No. 4,550,022 (Garabedian, et al.), which is incorporated by reference.

In general, the doses utilized for the above-described purposes will vary, but will be in an effective amount to inhibit or reduce neovascularization. As used herein, the term "pharmaceutically effective amount" to inhibit or reduce neovascularization, is that amount which inhibits formation of new blood vessels or reduces the number of blood vessels which are involved in the pathological condition. The doses utilized for any of the above-described purposes will generally be from about 0.01 to about 100 milligrams per kilogram of body weight (mg/kg), administered one to four times per day. When the compositions are dosed topically, they will generally be in a concentration range of about 0.001 wt. % to about 5 wt. %, with 1-2 drops administered 1-5 times per day.

The specific type of formulation selected will depend on various factors, such as the tbdn-1 agent being used, the dosage frequency, and the location of the neovascularization being treated. Topical ophthalmic aqueous solutions, suspensions, ointments, and gels are the preferred dosage forms for the treatment of neovascularization in the front of the eye (the cornea, iris, trabecular meshwork); or neovascularization of the back of the eye if the tbdn-1 agent can be formulated such that it can be delivered topically and the agent is able to penetrate the tissues in the front of the eye. The tbdn-1 agent will normally be contained in these formulations in an amount which will be determined to approximate the natural level of tbdn-1 in normal ocular blood vessels. Preferable concentrations range from about 0.1 to about 5.0 weight/percent. Thus, for topical administration, these formulations are delivered to the surface of the eye one to several times a day, depending on the routine discretion of the skilled clinician. Systemic administration, for example, in the form of tablets is useful for the treatment of neovascularization particularly of the back of the eye, for example, the retina.

Viscosity greater than that of simple aqueous solutions may be desirable to increase ocular absorption of the active compound, to decrease variability in dispensing the formulations, to decrease physical separation of components of a suspension or emulsion of formulation and/or otherwise to improve the ophthalmic formulation. Such viscosity building agents include, for example, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose or other agents known to those skilled in the art. Such agents are typically employed at a level of from about 0.01 to about 2 wt. %.

As indicated above, use of the tbdn-1 agents to prevent or reduce angiogenesis in ophthalmic tissues is a particularly important aspect of the present invention. The tbdn-1 agents may also be used as an adjunct to ophthalmic surgery, such as by vitreal or subconjunctival injection following ophthalmic surgery. The tbdn-1 agents may be used for acute treatment of temporary conditions, or may be administered chronically, especially in the case of degenerative disease. The compounds may also be used prophylactically, especially prior to ocular surgery or noninvasive ophthalmic procedures, or other types of surgery.

Claim 1 of 12 Claims

What is claimed is:

1. An isolated amino acid molecule consisting of the sequence shown in SEQ ID No. 2.

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