WO2013059095A1

WO2013059095A1 - Use of photosensitive agents to target the aqueous outflow system of the eye

Info

Publication number: WO2013059095A1
Application number: PCT/US2012/060036
Authority: WO
Inventors: Malik Kahook
Original assignee: University of Colorado System; University of Colorado Colorado Springs
Current assignee: University of Colorado System; University of Colorado Colorado Springs
Priority date: 2011-10-19
Filing date: 2012-10-12
Publication date: 2013-04-25
Anticipated expiration: 2014-04-19

Abstract

The present invention is related to methods and compositions for treating glaucoma, creating animal glaucoma models and/or screening therapeutic compounds to treat glaucoma. For example, glaucoma treatment may use a photodynamic therapy (PDT) in combination with a photosensitizer. Trabecular meshwork cells may be targeted to improve fluid outflow. Alternatively, retinal ganglion cells may be targeted to preserve these cells from neurodegeneration and reduce the risk of developing progressive visual field defects from glaucoma. Low density lipoprotein (LDL) receptors may act as binding sites for the activated photosensitizer and induce the release of specific proteins without cell death.

Description

Use Of Photosensitive Agents

To Target The Aqueous Outflow System Of The Eye

Field Of The Invention

The present invention is related to methods and compositions for treating glaucoma, creating animal glucoma models and/or screening therapeutic compounds to treat glaucoma. For example, glaucoma treatment may use a photodynamic therapy (PDT) in combination with a photosensitizer. Trabecular meshwork cells may be targeted to improve fluid outflow. Alternatively, retinal ganglion cells may be targeted to preserve these cells from

neurodegeneration and reduce the risk of developing progressive visual field defects from glaucoma. Low density lipoprotein (LDL) receptors may act as binding sites for the activated photosensitizer and induce the release of specific proteins without cell death. Preclinical animal glaucoma models may be created by using photodynamic therapy (PDT) in combination with a photosensitizer to specifically target the trabecular meshwork (TM) cells wherein the resultant damage to these cells lead to high intraocular pressure (IOP) and glaucoma. The PDT methods that induce glaucoma as described herein have certain advantages over currently used methods including but not limited to, a lack of significant inflammation and routes of administration. Background

Glaucoma is the most common cause of blindness and the second leading cause of irreversible blindness among African Americans in the United States. It is also the leading cause of blindness among U.S. Hispanics. Congdon et al., "Causes and prevalence of visual impairment among adults in the United States" Arch Ophthalmol. 2004;122:477-485; and Rodriguez et al., "Causes of blindness and visual impairment in a population-based sample of U.S. Hispanics" Ophthalmol. 2002;109:737-743.

Lowering intraocular pressure (IOP) using medical or surgical therapy is the mainstay of therapy, designed to control and limit this common disease. However, treating glaucoma remains a difficult task, since most medications have side effects, lose their efficacy, and require subjects' life-long compliance. Surgical methods have a higher risk of complication. Ciliary body destruction by cryotherapy or laser irradiation represents a useful alternative for the management of glaucoma that is resistant to other modes of therapy. Bietti G. "Surgical intervention on the ciliary body: New trends for the relief of glaucoma" JAMA. 1950;142:889-897; Weekers et al., "Effects of photocoagulation of ciliary body upon ocular tension" Am J Ophthalmol. 1961;52:156-163; and Smith et al., "Ocular hazards of transscleral laser radiation. II. Intraocular injury produced by ruby and neodymium lasers" Am J Ophthalmol. 1969;67: 100-110. However, the current cyclodestructive techniques have a high rate of side effects including loss of vision, hypotony, macular edema, or phthisis bulbi. Beckman et al., 'Transscleral ruby laser cyclocoagulation"

795; Haddad R., "Cyclocryotherapy: experimental studies of the breakdown of the blood- aqueous barrier and analysis of a long-term follow-up study" Wien Klin Wochenschr Suppl. 1981;126:1-18; and aiden et al., "Choroidal detachment with flat anterior chamber after cyclocryotherapy" Ann Ophthalmol. 1979; 11 :1111-1113.

What is needed in the art are non-invasive methods to induce glaucoma in animal models that are not complicated by side effects resulting from non-specific tissue damage.

Summary

The present invention is related to methods and compositions for treating glaucoma, creating animal glucoma models and/or screening therapeutic compounds to treat glaucoma. For example, a topical or injectable agent may be used to sensitize intraocular tissues, which include but are not limited to trabecular meshwork, ciliary body, iris or other anterior or posterior segment tissues, followed by an external application of mechanical, vibratory, ultrasonic or light based treatment to activate said agent to influence the disease state of the eye. Alternatively, other methods of activating a photosensitizer include, but are not limited to, ultrasound or vibrations, etc. Glaucoma treatment may also involve a photodynamic therapy (PDT) in combination with a photosensitizer. Trabecular meshwork cells may be targeted to improve fluid outflow. Alternatively, retinal ganglion cells may be targeted to preserve these cells from neurodegeneration and reduce the risk of developing progressive visual field defects from glaucoma. Low density lipoprotein (LDL) receptors may act as binding sites for the activated photosensitizer and induce the release of specific proteins without cell death. Preclinical animal glaucoma models may be created by using

photodynamic therapy (PDT) in combination with a photosensitizer to specifically target the trabecular meshwork (TM) cells wherein the resultant damage to the specific TM cells lead to high intraocular pressure (IOP) and glaucoma. The PDT methods that induce glaucoma as described herein have certain advantages over currently used methods including but not limited to, a lack of significant inflammation and routes of administration. In one embodiment, the present invention contemplates a non-human mammal comprising at least one symptom of glaucoma and a plurality of damaged ocular cells and a plurality of undamaged ocular cells. In one embodiment, the plurality of damaged ocular cells comprise a plurality of damaged trabecular meshwork cells. In one embodiment, the plurality of damaged trabecular meshwork cells block intraocular fluid flow wherein the at least one symptom of glaucoma is increased intraocular pressure. In one embodiment, the blocked intraocular fluid flow is within an ocular aqueous outflow system. In one

embodiment, the ocular aqueous outflow system comprises an ocular vasculature system. In one embodiment, the glaucoma comprises neovascular glaucoma. In one embodiment, the plurality of undamaged ocular cells comprise a plurality of non-trabecular meshwork cells. In one embodiment, the plurality of undamaged non-trabecular meshwork cells do not exhibit significant inflammation, hi one embodiment, the plurality of undamaged non-trabecular meshwork cells comprises a plurality of retinal ganglion cells. In one embodiment, the non- human mammal is selected from the group consisting of a mouse, a rat, a rabbit, a guinea pig, and a non-human primate.

In one embodiment, the present invention contemplates a method, comprising; a) providing; i) a non-human mammal comprising a plurality of ocular cells; ii) a photosensitizer capable of generating free radicals; and iii) a light source capable of specifically targeting the plurality of ocular cells; b) administering the photosensitizer to the non-human mammal; and c) irradiating the non-human mammal with the light source resulting in a plurality of damaged ocular cells and a plurality of undamaged ocular cells. In one embodiment, the administering comprises an intraocular injection of the photosensitizer. In one embodiment, the

photosensitizer is verteporfin. In one embodiment, the administering comprises a topical administration of the photosensitizer. In one embodiment, the plurality of damaged ocular cells comprise a plurality of damaged trabecular meshwork cells. In one embodiment, the plurality of damaged trabecular meshwork cells block intraocular fluid flow wherein intraocular pressure of the non-human mammal is increased. In one embodiment, the blocked intraocular fluid flow is within an ocular aqueous outflow system. In one embodiment, the ocular aqueous outflow system comprises an ocular vasculature system. In one embodiment, the increased intraocular pressure induces glaucoma in the non-human mammal. In one embodiment, the glaucoma comprises neovascular glaucoma. In one embodiment, the plurality of undamaged ocular cells comprise a plurality of non-trabecular meshwork cells. In one embodiment, the plurality of undamaged non-trabecular meshwork cells do not exhibit significant inflammation. In one embodiment, the plurality of undamaged non- trabecular meshwork cells comprises a plurality of retinal ganglion cells, i one embodiment, at least one non-human mammal is selected from the group consisting of a mouse, a rat, a rabbit, a guinea pig. In one embodiment, the photosensitizer comprises a benzoporphyrin derivative. In one embodiment, the photosensitizer is verteporfin. In one embodiment, the

benzoporphyrin derivative is a mono acid derivative. In one embodiment, the irradiation comprises a photodynamic therapy. In one embodiment, the light source comprises a wavelength preferably, but not limited to, ranging between approximately 400 - 900 nm. In one embodiment, the light source wavelength is about 689 nm. In one embodiment, the irradiating comprises a fluence preferably, but not limted to, ranging between approximately 0.0000001-90 Joules/cmf. In one embodiment, the irradiating comprises a fluence of about 100 Joules/cm². In one embodiment, the irradiating comprises an irradiance preferably, but not limted to, of about 1800 mW/cm². h one embodiment, the irradiating ranges preferably, but not limted to, between approximately 90-360 degrees of the trabecular meshwork. In one embodiment, the administered photosensitizer ranges preferably, but not limted to, between approximately 0.5 - 5 μg/kg. In one embodiment, the administered photosensitizer is 1 μg kg.

In one embodiment, the present invention comprises a method, comprising: a) providing; i) a non-human mammal exhibiting at least one symptom of glaucoma, wherein the non-human mammal comprises a plurality of damaged ocular cells and a plurality of undamaged ocular cells; ii) a composition comprising at least one test compound capable of being administered to the non-human mammal; and iii) a light source capable of irradiating the damaged and undamaged ocular cells; b) administering the test compound to the non- human mammal; c) exposing the damaged and undamaged ocular cells to irradiation with the light source; and c) determining whether the at least one symptom of glaucoma is reduced. In one embodiment, the administering comprises an intraocular injection of the at least one test compound. In one embodiment, the administering comprises a topical administration of the at least one test compound, hi one embodiment, the at least one test compound is a therapeutic agent. In one embodiment, the therapeutic agent is selected from the group consisting of an adrenergic beta blocker, a carbonic anhydrase inhibitor, a prostaglandin analogue, a rho kinase inhibitor, an endothelin antagonist, a virus, a protein, and a nucleic acid sequence. In one embodiment, the at least one test compound is in a pharmaceutically acceptable formulation. In one embodiment, the at least one test compound comprises a photosensitizer. In one embodiment, the photosensitizer is verteporfin. In one embodiment, the photosensitizer comprises a benzoporphyrin derivative. In one embodiment, the benzopoiphyrin derivative is a mono acid derivative. In one embodiment, the plurality of damaged cells comprise trabecular meshwork cells. In one embodiment, the plurality of undamaged cells comprise non-trabecular meshwork cells. In one embodiment, the at least one symptom of glaucoma is reduced for at least 4 weeks. In one embodiment, the at least one symptom of glaucoma is reduced for at least 5 weeks. In one embodiment, the at least one symptom of glaucoma is reduced for at least 6 weeks. In one embodiment, the at least one symptom of glaucoma is reduced for at least 7 weeks. In one embodiment, the at least one symptom of glaucoma is reduced for or at least 8 weeks, or longer. In one embodiment, at least one of the non-human mammal is selected from the group consisting of a mouse, a rat, a rabbit, a guinea pig, non-human primate In one embodiment, the at least one symptom of glaucoma comprises increased intraocular pressure. In one embodiment, the undamaged ocular cells do not exhibit significant inflammation. In one embodiment, the plurality of undamaged non-trabecular meshwork cells comprises a plurality of retinal ganglion cells. In one embodiment, the irradiation comprises a photodynamic therapy. In one embodiment, the light source comprises a wavelength ranging preferably, but not limted to, between approximately 400 - 900 nm. In one embodiment, the light source wavelength is about 689 nm. In one embodiment, the irradiating comprises a fluence ranging preferably, but not limted to, between approximately 0.0000001-90 Joules/cm². In one embodiment, the irradiating comprises a fluence of about 100 Joules/cm², i one embodiment, the irradiating comprises an irradiance of about 1800 mW/cm . In one embodiment, the irradiating ranges between approximately 90-360 degrees of the trabecular meshwork. In one embodiment, the administered photosensitizer ranges preferably, but not limted to, between approximately 0.5 - 5 μgΛ g. In one embodiment, the administered photosensitizer is 1 μg/kg.

In one embodiment, the present invention contemplates a method, comprising: a) providing: i) a mammal comprising a trabecular meshwork having a blocked fluid outflow; and ii) a photosensitizer capable of activation by irradiation; b) administering said

photosensitizer to said mammal; c) irradiating said photosensitizer under conditions such that said trabecular meshwork is remodeled thereby alleviating said blocked fluid outflow. In one embodiment, the photosensitizer comprises a benzoporphyrin derivative. In one embodiment, the photosensitizer is verteporfin. In one embodiment, the irradiation comprises

photodynamic therapy. In one embodiment, the photosensitizer generates free radicals. In one embodiment, the free radicals stimulate trabecular meshwork cell low density lipoprotein receptors. In one embodiment, the low density lipoprotein receptor stimulation releases a plurality of proteins. In one embodiment, the low density lipoprotein receptor stimulation does not result in cell death. In one embodiment, the plurality of proteins facilitate the remodeling. In one embodiment, the mammal is a human.

hi one embodiment, the present invention contemplates a method, comprising: a) providing: i) a mammal comprising at least one symptom of neovascular glaucoma; and ii) a photo sensitizer capable of activation by irradiation; b) administering said photosensitizer to said mammal; c) irradiating said photosensitizer under conditions such that said at least one symptom of neovascular glaucoma is reduced. In one embodiment, the photosensitizer comprises a benzoporphyrin derivative. In one embodiment, the photosensitizer is

verteporfin. In one embodiment, the irradiation comprises photodynamic therapy. In one embodiment, the mammal is a human, h one embodiment, the at least one symptom comprises increased intraocular pressure.

In one embodiment, the present invention comprises a method of reducing the intraocular pressure in a mammalian eye having a trabecular meshwork. The method includes the steps of: (a) administering to a mammal, an amount of photosensitizer sufficient to accumulate within the ocular vasculature as well as the aqueous outflow system; and (b) irradiating a region within the aqueous outflow system with light so as to activate the photosensitizer. The activated photosensitizer (i.e., for example, verteporfin) then causes a reduction in the intraocular pressure of the eye relative to the intraocular pressure in the eye prior to irradiation. This reduction in intraocular pressure can persist for a prolonged period of time, for example, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks or longer. The light can have a wavelength of about 689 nm or between 400 and 900nm. The wavelength used may be more or less than that of the visible spectrum. The irradiating step can include a fluence of about 100 Joules/cm². In other cases the fluence maybe 0.0000001-90 Joules/cm². In other forms the fluence maybe more or less than this. The irradiating step can include an irradiance of about 1800 mW/cm². The irradiating step can include irradiation covering 90-360 degrees of the trabecular meshwork. The reduction of intraocular pressure can be continuous over the prolonged period of time.

In one embodiment, the present invention comprises a method of preserving retinal ganglion cell viability in a mammalian eye having a trabecular meshwork and at risk of developing or having glaucoma. The method includes the steps of administering to a mammal an amount of photosensitizer (e.g., verteporfin), for example, a benzoporphyrin derivative photosensitizer, for example, a benzoporphyrin derivative mono acid photosensitizer, sufficient to accumulate in the trabecular meshwork and unconventional outflow system, and irradiating a region of the trabecular meshwork so as to activate the photosensitizer, such that retinal ganglion cell viability is preserved. The amount of photosensitizer can be about 1 mg/kg but may be more or less. The light can have a wavelength of about 689 nm or between 400 and 900nm. The wavelength used may be more or less than that of the visible spectrum. The irradiating step can include a fluence of about 100 Joules/cm². In other cases the fluence maybe 0.0000001-90 Joules/cm². In other forms the fluence maybe more or less than this. The irradiating step can include an irradiance of about 1800 mW/cm². The irradiating step can include irradiation covering 90-360 degrees of the trabecular meshwork. The reduction of intraocular pressure can be continuous over the prolonged period of time.

In one embodiment, the present invention contemplates a method, comprising: a) providing: i) a mammal exhibiting at least one symptom of elevated intraocular pressure; and ii) a photosensitizer capable of activation by irradiation; b) administering said photosensitizer to said mammal; c) irradiating said photosensitizer under conditions such that said at least one symptom of elevated intraocular pressure is reduced. In one embodiment, the photosensitizer interacts with a trabecular meshwork endothelial cell low density lipoprotein receptor. In one embodiment, the photosensitizer comprises a benzoporphyrin derivative. In one embodiment, the photosensitizer is verteporfin. In one embodiment, the irradiation comprises photodynamic therapy.

In one embodiment, the method further comprises administering a plurality of intraocular depots containing a photosensitizer and other active ingredients in depots within the eye. In one embodiment, each of said plurality of intraocular depots are sequentially activated as needed, thus releasing some of the photosensitizer and other active ingredients that target specific intraocular tissues. In one embodiment, the specifically targeted intraocular tissues include, but are not limited, trabecular meshwork cell LDL receptors, Annexin 5 on dying ganglion cells, or vascular tissue VEGF receptors.

In one embodiment, the present invention contemplates a method, comprising: a) providing: i) a mammal at risk of exhibiting at least one symptom of elevated intraocular pressure; and ii) a photosensitizer capable of activation by irradiation; b) administering said photosensitizer to said mammal; c) irradiating said photosensitizer under conditions such that said risk of developing said at least one symptom of elevated intraocular pressure is reduced. In one embodiment, the photosensitizer interacts with a retinal ganglion cell. In one embodiment, the photosensitizer comprises a benzoporphyrin derivative. In one embodiment, the photosensitizer is verteporfln. In one embodiment, the irradiation comprises

photodynamic therapy.

In one embodiment, the present invention contemplates a method of reducing the intraocular pressure in a mammalian eye having a trabecular meshwork, comprising: a) administering to a mammal, an amount of photosensitizer sufficient to accumulate within the ocular vasculature as well as the aqueous outflow system; and b) irradiating a region within the aqueous outflow system with light so as to activate the photosensitizer. h one

embodiment, the photosensitizer comprises a benzoporphyrin derivative. In one embodiment, the photosensitizer is verteporfin. In one embodiment, the irradiation comprises

photodynamic therapy.

In one embodiment, the present invention contemplates a composition comprising a photosensitizer attached to a low density lipoprotein receptor ligand and a therapeutic agent.

In one embodiment, the therapeutic agent is selected from the group consisting of an adrenergic beta blocker, a carbonic anhydrase inhibitor, a prostaglandin analogue, a rho kinase inhibitor, an endothelin antagonist, or other medications or biologic agents (e.g., virus, protein, RNA or DNA based material). In one embodiment, the composition is a

pharmaceutically acceptable formulation.

Brief Description Of The Figures

Figure 1 presents an illustrative example of the ocular anatomy: 1. posterior chamber 2. ora serrata 3. ciliary muscle 4. ciliary zonules 5. canal of Schlemm 6. pupil 7. anterior chamber 8. cornea 9. iris 10. lens cortex 11. lens nucleus 12. ciliary process 13. conjunctiva 14. inferior oblique muscle 15. inferior rectus muscle 16. medial rectus muscle 17. retinal arteries and veins 18. optic disc 19. dura mater 20. central retinal artery 21. central retinal vein 22. optic nerve 23. vorticose vein 24. bulbar sheath 25. macula 26. fovea 27. sclera 28. choroid 29. superior rectus muscle 30. retina.

Figure 2 presents a representative photomicrograph of an ocular trabecular meshwork.

Figure 3 presents a representative photomicrograph of a false-color image of a flat- mounted rat retina viewed through a fluorescence microscope at 50x magnification. Optic nerve injection with a fluorophore caused fluorescence of the retinal ganglion cells. Figure 4 presents a illustrative diagram showing cross-section of retinal layers. The area labeled "Ganglionic layer" contains retinal ganglion cells.

Figure 5 presents exemplary data showing the viability of various cultured ocular cells exposed to a range of verteporfin for 24 hours without exposure to laser light. The percent of live cells was determined by an MTT assay in: i) human scleral fibroblasts (hFibro); ii) human trabecular meshwork cells (hTMC); and iii) a human retinal pigment epithelial cell line (ARPE-19). Data were normalized to untreated cells (0 μg/ml verteporfin; 100 % Live), and plotted as the mean (n = 4) with error bars representing the standard deviation. * = p < 0.05 vs. 0 μg/ml verteporfin treatment.

Figure 6 presents exemplary data showing the viability of various cultured ocular cells exposed to 0.5 verteporfin with different intensities of laser light. The percent of live cells was determined by an MTT assay in: i) human scleral fibroblasts (hFibro); ii) pig trabecular meshwork cells (pTMC); iii) human trabecular meshwork cells (hTMC); and iv) a human retinal pigment epithelial cell line (ARPE-19). Data were normalized to cells not treated with either verteporfin or laser light (0 μg/ml + 0 μΐ/cm²; 100 % Live), and plotted as the mean (n = 4) with error bars representing the standard deviation. * = p < 0.05 vs. both Ό μg/ml + 0 y /cm ' and 'Pretreat + 50 μΐ/cm '.

Definitions

The term "at risk for" as used herein, refers to a medical condition or set of medical conditions exhibited by a subject which may predispose the subject to a particular disease or affliction. For example, these conditions may result from influences that include, but are not limited to, behavioral, emotional, chemical, biochemical, or environmental influences.

The term "effective amount" as used herein, refers to a particular amount of a pharmaceutical composition comprising a therapeutic agent that achieves a clinically beneficial result (i.e., for example, a reduction of symptoms). Toxicity and therapeutic efficacy of such compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD₅o/ED₅o. Compounds that exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and additional animal studies can be used in formulating a range of dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the subject, and the route of administration.

The term "symptom", as used herein, refers to any subjective or objective evidence of disease or physical disturbance observed by the subject. For example, subjective evidence is usually based upon subject self-reporting and may include, but is not limited to, pain, headache, visual disturbances, increased intraocular presssure, nausea and/or vomiting.

Alternatively, objective evidence is usually a result of medical testing including, but not limited to, body temperature, complete blood count, lipid panels, thyroid panels, blood pressure, heart rate, electrocardiogram, tissue and/or body imaging scans.

The term "disease" or "medical condition" as used herein, refers to any impairment of the normal state of the living animal or plant body or one of its parts that interrupts or modifies the performance of the vital functions. Typically manifested by distinguishing signs and symptoms, it is usually a response to: i) environmental factors (as malnutrition, industrial hazards, or climate); ii) specific infective agents (as worms, bacteria, or viruses); iii) inherent defects of the organism (as genetic anomalies); and/or iv) combinations of these factors

The terms "reduce," "inhibit," "diminish," "suppress," "decrease," "prevent" and grammatical equivalents (including "lower," "smaller," etc.) when in reference to the expression of any symptom in an untreated subject relative to a treated subject, mean that the quantity and/or magnitude of the symptoms in the treated subject is lower than in the untreated subject by any amount that is recognized as clinically relevant by any medically trained personnel. In one embodiment, the quantity and/or magnitude of the symptoms in the treated subject is at least 10% lower than, at least 25% lower than, at least 50% lower than, at least 75% lower than, and/or at least 90% lower than the quantity and/or magnitude of the symptoms in the untreated subject.

The term "inhibitory compound" as used herein, refers to any compound capable of interacting with (i.e., for example, attaching, binding etc) to a binding partner under conditions such that the binding partner becomes unresponsive to its natural ligands.

Inhibitory compounds may include, but are not limited to, small organic molecules, antibodies, and proteins/peptides.

The term "injury" as used herein, denotes a bodily disruption of the normal integrity of tissue structures. In one sense, the term is intended to encompass surgery. In another sense, the term is intended to encompass irritation, significant inflammation, infection, and the development of fibrosis. In another sense, the term is intended to encompass wounds including, but not limited to, contused wounds, incised wounds, lacerated wounds, nonpenetrating wounds (i.e., wounds in which there is no disruption of the skin but there is injury to underlying structures), open wounds, penetrating wound, perforating wounds, puncture wounds, septic wounds, subcutaneous wounds, burn injuries etc.

The term "drug" or "compound" as used herein, refers to any pharmacologically active substance capable of being administered which achieves a desired effect. Drugs or compounds can be synthetic or naturally occurring, non-peptide, proteins or peptides, oligonucleotides or nucleotides, polysaccharides or sugars. For example, some drugs or compounds may be therapeutic agents that are effective in reducing at least one symptom of glaucoma including, but not limited to, an adrenergic beta blocker, a carbonic anhydrase inhibitor, a prostaglandin analogue, a rho kinase inhibitor, an endothelin antagonist, a virus, a protein, or a nucleic acid sequence.

The term "administered" or "administering", as used herein, refers to any method of providing a composition to a subject such that the composition has its intended effect on the subject. An exemplary method of administering is by a direct mechamsm such as, local tissue administration (i.e., for example, extravascular placement), oral ingestion, transdermal patch, topical, inhalation, suppository etc. For administration of a composition or a therapeutic agent to ocular cells, intraocular injections and/or topical administration are preferable.

The term "affinity" as used herein, refers to any attractive force between substances or particles that causes them to enter into and remain in chemical combination. For example, an inhibitor compound that has a high affinity for a receptor will provide greater efficacy in preventing the receptor from interacting with its natural ligands, than an inhibitor with a low affinity.

The term "derived from" as used herein, refers to the source of a compound or sequence. In one respect, a compound or sequence may be derived from an organism or particular species. In another respect, a compound or sequence may be derived from a larger complex or sequence.

The term "pharmaceutically" or "pharmacologically acceptable", as used herein, refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. The term, "pharmaceutically acceptable carrier", as used herein, includes any and all solvents, or a dispersion medium including, but not limited to, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils, coatings, isotonic and absorption delaying agents, liposome, commercially available cleansers, and the like. Supplementary bioactive ingredients also can be incorporated into such carriers.

The term, "purified" or "isolated", as used herein, may refer to a peptide composition that has been subjected to treatment (i.e., for example, fractionation) to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the composition (i.e., for example, weight/weight and/or weight/volume). The term "purified to homogeneity" is used to include compositions that have been purified to

'apparent homogeneity" such that there is single protein species (i.e., for example, based upon SDS-PAGE or HPLC analysis). A purified composition is not intended to mean that some trace impurities may remain.

As used herein, the term "substantially purified" refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and more preferably 90% free from other components with which they are naturally associated. An "isolated polynucleotide" is therefore a substantially purified polynucleotide.

The terms "amino acid sequence" and "polypeptide sequence" as used herein, are interchangeable and to refer to a sequence of amino acids. Such amin acid sequences are also referred to as peptides or proteins, depending upon relative length.

As used herein the term "portion" when in reference to a protein (as in "a portion of a given protein") refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.

The term "small organic molecule" as used herein, refers to any molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size from approximately 10 Da up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. The term "derivative" as used herein, refers to any chemical modification of a nucleic acid or an amino acid. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group. For example, a nucleic acid derivative would encode a polypeptide which retains essential biological characteristics.

The term "biologically active" refers to any molecule having structural, regulatory or biochemical functions. For example, biological activity may be determined, for example, by restoration of wild-type growth in cells lacking protein activity. Cells lacking protein activity may be produced by many methods (i.e., for example, point mutation and frame-shift mutation). Complementation is achieved by transfecting cells which lack protein activity with an expression vector which expresses the protein, a derivative thereof, or a portion thereof.

The terms "binding component", "molecule of interest", "agent of interest", "ligand" or "receptor" as used herein may be any of a large number of different molecules, biological cells or aggregates, and the terms are used interchangeably. Each binding component may be immobilized on a solid substrate and binds to an analyte being detected. Proteins, polypeptides, peptides, nucleic acids (nucleotides, oligonucleotides and polynucleotides), antibodies, ligands, saccharides, polysaccharides, microorganisms such as bacteria, fungi and viruses, receptors, antibiotics, test compounds (particularly those produced by combinatorial chemistry), plant and animal cells, organdies or fractions of each and other biological entities may each be a binding component.. Each, in turn, also may be considered as analytes if same bind to a binding component on a chip.

The term "macromolecule" as used herein, refers to any molecule of interest havin a high molecular weight. For example, some biopolymers having a high molecular weight would be comprised of greater than 100 amino acids, nucleotides or sugar molecules long.

The term "bind" as used herein, includes any physical attachment or close association, which may be permanent or temporary. Generally, an interaction of hydrogen bonding, hydrophobic forces, van der Waals forces, covalent and ionic bonding etc., facilitates physical attachment between the molecule of interest and the analyte being measuring. The "binding" interaction maybe brief as in the situation where binding causes a chemical reaction to occur. That is typical when the binding component is an enzyme and the analyte is a substrate for the enzyme. Reactions resulting from contact between the binding agent and the analyte are also within the definition of binding for the purposes of the present invention. Detailed Description

neurodegeneration and reduce the risk of developing progressive visual field defects from glaucoma. Low density lipoprotein (LDL) receptors may act as binding sites for the activated photosensitizer and induce the release of specific proteins without cell death. Preclinical animal glaucoma models may be created by using photodynamic therapy (PDT) in combination with a photosensitizer to specifically target the trabecular meshwork (TM) cells wherein the resultant damage to the specific TM cells lead to high intraocular pressure (IOP) and glaucoma. The PDT methods that induce glaucoma as described herein have certain advantages over currently used methods including but not limited to, a lack of significant inflammation and routes of administration.

In some embodiments, the present invention contemplates administering a

photosensitizer (i.e., for example, a benzoporphyrin derivative, for example, a

benzoporphyrin derivative mono-acid) into the eye following by irradiation (i.e., for example, PDT). Although it is not necessary to understand the mechanism of an invention, it is believed that a PDT-activated photosensitizer may specifically interact with ocular trabecular meshwork cells. Depending upon the photosensitizer the ocular trabecular meshwork cells may be specifically damaged, thereby resulting in the appearance of glaucoma symptoms. Alternatively, another PDT-activated photosensitizer may specifically heal ocular trabecular meshwork cells, thereby resulting in a prolonged reduction in intraocular pressure (i.e., for example, at least 4 weeks but perhaps longer than 8 weeks).

Other embodiments of the present invention include, but are not limited to: i) the amount of photosensitizer can be about 1 mg/kg but may be more or less; ii) the irradiation can have a wavelength of about 689 ran or between 400 and 900nm; iii) the irradiation wavelength used may be more or less than that of the visible spectrum; iv) the irradiating can include a fluence between approximately 0.0000001-90 Joules/cm , but preferably a fluence of about 100 Joules/cm ; v) the irradiating can include an irradiance of about 1800 mW/cm ; vi) the irradiating can encompass approximately between 90 - 360 degrees of the trabecular meshwork; and vii) the reduction of intraocular pressure can be continuous over a prolonged period of time.

The invention also contemplates a therapeutic agent, either in the presence or absence of a photosensitizer, that specifically target to the trabecular endothelial cells. Such drug targeting may be useful either with, or without, irradiation and result in the reduction of at least one symptom of glaucoma.

The present invention is based, in part, upon the discovery that it is possible to perform photodynamic therapy (PDT) specifically on trabecular meshwork ocular cells.

Consequently, the methods described herein result in targeted damage to trabecular meshwork ocular cells wherein the surrounding cells (i.e., non-trabecular meshwork cells) remain undamaged and healthy.

I. Ocular Anatomy

A. Eyeball Characteristics And Function

The eye has been described as an organ which reacts to light for several purposes.

See, Figure 1. As a conscious sense organ, the eye allows vision. Rod and cone cells in the retina allow conscious light perception and vision including color differentiation and the perception of depth. The human eye can distinguish about 10 million colors. Judd et al.., Color in Business, Science and Industry. Wiley Series in Pure and Applied Optics (third edition ed.). New York: Wiley-Interscience. p. 388. (1 75). In common with the eyes of other mammals, the human eye's non-image-forming photosensitive ganglion cells in the retina receive the light signals which affect adjustment of the size of the pupil, regulation and suppression of the hormone melatonin and entrainment of the body clock.

The eye is not properly a sphere, rather it is a fused two-piece unit. The smaller frontal unit, more curved, called the cornea is linked to the larger unit called the sclera. The corneal segment is typically about 8 mm (0.3 in) in radius. The sclera constitutes the remaining five- sixths; its radius is typically about 12 mm. The cornea and sclera are connected by a ring called the limbus. The iris - the color of the eye - and its black center, the pupil, are seen instead of the cornea due to the cornea's transparency. To see inside the eye, an

ophthalmoscope is needed, since light is not reflected out. The fundus (area opposite the pupil) shows the characteristic pale optic disk (papilla), where vessels entering the eye pass across and optic nerve fibers depart the globe. The dimensions of an eyeball differ among adults by only one or two millimeters. The vertical measure, generally less than the horizontal distance, is about 24 mm among adults, at birth about 16-17 mm. (about 0.65 inch) The eyeball grows rapidly, increasing to 22.5-23 mm (approx. 0.89 in) by the age of three years. From then to age 13, the eye attains its full size. The volume is 6.5 ml (0.4 cu. in.) and the weight is 7.5 g. (0.25 oz.)

The eye is made up of three coats, enclosing three transparent structures. The outermost layer is composed of the cornea and sclera. The middle layer consists of the choroid, ciliary body, and iris. The innermost is the retina, which gets its circulation from the vessels of the choroid as well as the retinal vessels, which can be seen in an ophthalmoscope. Within these coats are the aqueous humor, the vitreous body, and the flexible lens. The aqueous humor is a clear fluid that is contained in two areas: the anterior chamber between the cornea and the iris and exposed area of the lens; and the posterior chamber, behind the iris and the rest. The lens is suspended to the ciliary body by the suspensory ligament (Zonule of Zinn), made up of fine transparent fibers. The vitreous body is a clear jelly that is much larger than the aqueous humor, and is bordered by the sclera, zonule, and lens. They are connected via the pupil.

The retina has a static contrast ratio of around 100: 1. As soon as the eye moves (saccades) it re-adjusts its exposure both chemically and geometrically by adjusting the iris which regulates the size of the pupil. Initial dark adaptation takes place in approximately four seconds of profound, uninterrupted darkness; full adaptation through adjustments in retinal chemistry (the Purkinje effect) are mostly complete in thirty minutes. Hence, a dynamic contrast ratio of about 1,000,000:1 is possible. The process is nonlinear and multifaceted, so an interruption by light merely starts the adaptation process over again. Full adaptation is dependent on good blood flow; thus dark adaptation may be hampered by poor circulation, and vasoconstrictors like alcohol or tobacco.

The eye includes a lens not dissimilar to lenses found in optical instruments such as cameras and the same principles can be applied. The pupil of the human eye is its aperture; the iris is the diaphragm that serves as the aperture stop. Refraction in the cornea causes the effective aperture (the entrance pupil) to differ slightly from the physical pupil diameter. The entrance pupil is typically about 4 mm in diameter, although it can range from 2 mm in a brightly lit place to 8 mm in the dark. The latter value decreases slowly with age, older people's eyes sometimes dilate to not more than 5 -6mm. B. Trabecular Meshwork

The trabecular meshwork is an area of tissue in the eye located around the base of the cornea, near the ciliary body, and is responsible for draining the aqueous humor from the eye via the anterior chamber (the chamber on the front of the eye covered by the cornea). The tissue is spongy and lined by trabeculocytes; it allows fluid to drain into a set of tubes called Schlemm's canal flowing into the blood system. See, Figure 2.

The meshwork is divided up into three parts, with characteristically different ultrastructures: i) Inner uveal meshwork - Closest to the anterior chamber angle, contains thin cord-like trabeculae, orientated predominantly in a radial fashion, enclosing trabeculae spaces larger than the corneoscleral meshwork; ii) Corneoscleral meshwork - Contains a large amount of elastin, arranged as a series of thin, flat, perforated sheets arranged in a laminar pattern; considered the ciliary muscle tendon; and, iii) Juxtacanalicular tissue (also known as the cribriform meshwork) - Lies immediately adjacent to Schlemm's canal, composed of connective tissue ground substance full of glycoaminoglycans and glycoproteins. This thin strip of tissue is covered by a monolayer of endothelial cells.

The trabecular meshwork is assisted to a small degree in the drainage of aqueous humour by a second outflow pathway, the uveo-scleral pathway (5-10% of outflow occurs this way). The uveo-scleral pathway is increased with the use of glaucoma drugs such as prostaglandins (e.g., Xalatan, Travatan).

The trabecular meshwork is believed responsible for most of the outflow of aqueous humor. Intraocular pressure (i.e., for example, glaucoma) may increase either when too much aqueous humor fluid is produced or by decreased aqueous humor outflow.

The major drainage structures for aqueous humor (AH) are the conventional or trabecular outflow pathways, which are comprised of the trabecular meshwork (made up by the uveal and corneoscleral meshworks), the juxtacanalicular connective tissue (JCT), the endothelial lining of Schlemm's canal (SC), the collecting channels and the aqueous veins. The trabecular meshwork (TM) outflow pathways are critical in providing resistance to AH outflow and in generating intraocular pressure (IOP). Outflow resistance in the TM outflow pathways increases with age and primary open-angle glaucoma. Uveal and corneoscleral meshworks form connective tissue lamellae or beams that are covered by flat TM cells which rest on a basal lamina. TM cells in the JCT are surrounded by fibrillar elements of the extracellular matrix (ECM) to form a loose connective tissue. In contrast to the other parts of the TM, JCT cells and ECM fibrils do not form lamellae, but are arranged more irregularly. SC inner wall endothelial cells form giant vacuoles in response to AH flow, as well as intracellular and paracellular pores. In addition, minipores that are covered with a diaphragm are observed. There is considerable evidence that normal AH outflow resistance resides in the inner wall region of SC, which is formed by the JCT and SC inner wall endothelium.

Modulation of TM cell tone by the action of their actomyosin system affects TM outflow resistance. In addition, the architecture of the TM outflow pathways and consequently outflow resistance appear to be modulated by contraction of ciliary muscle and scleral spur cells. The scleral spur contains axons that innervate scleral spur cells or that have the ultrastructural characteristics of mechanosensory nerve endings. Tamm ER., "The trabecular meshwork outflow pathways: structural and functional aspects" Exp Eye Res. 2009

Apr;88(4):648-55.

C. Retinal Gangion Cells

A retinal ganglion cell (RGC) is a type of neuron located near the inner surface (the ganglion cell layer) of the retina of the eye. It receives visual information from

photoreceptors via two intermediate neuron types: bipolar cells and amacrine cells. Retinal ganglion cells collectively transmit image- forming and non-image forming visual information from the retina to several regions in the thalamus, hypothalamus, and mesencephalon, or midbrain. Most mature ganglion cells are able to fire action potentials at a high frequency because of their expression of Kv3 potassium channels. Henne et al.,. (2000). "Voltage-gated potassium channels in retinal ganglion cells of trout: a combined biophysical,

pharmacological, and single-cell RT-PCR approach". J.Neurosci.Res. 62 (5): 629-637; and Henne et al., (2004). "Maturation of spiking activity in trout retinal ganglion cells coincides with upregulation of v3.1- and BK-related potassium channels". J.Neurosci.Res. 75 (1): 44- 54.

Retinal ganglion cells vary significantly in terms of their size, connections, and responses to visual stimulation but they all share the defining property of having a long axon that extends into the brain. These axons form the optic nerve, optic chiasm, and optic tract. A small percentage of retinal ganglion cells contribute little or nothing to vision, but are themselves photosensitive; their axons form the retinohypothalamic tract and contribute to circadian rhythms and pupillary light reflex, the resizing of the pupil.

There are about 1.2 to 1.5 million retinal ganglion cells in the human retina. With about 125 million photoreceptors per retina, on average each retinal ganglion cell receives inputs from about 100 rods and cones. However, these numbers vary greatly among individuals and as a function of retinal location. In the fovea (center of the retina), a single ganglion cell will communicate with as few as five photoreceptors. la the extreme periphery (ends of the retina), a single ganglion cell will receive information from many thousands of photoreceptors.

Retinal ganglion cells spontaneously fire action potentials at a base rate while at rest.

Excitation of retinal ganglion cells results in an increased firing rate while inhibition results in a depressed rate of firing. Based on their projections and functions, there are at least five main classes of retinal ganglion cells: i) Midget cell (Parvocellular, or P pathway; B cells); ii) Parasol cell (Magnocellular, or M pathway; A cells); iii) Bistratified cell (Koniocellular, or K pathway); iv) Photosensitive ganglion cells; and v) Other ganglion cells projecting to the superior colli cuius for eye movements (saccades).

Midget retinal ganglion cells project to the parvocellular layers of the lateral geniculate nucleus. These cells are known as midget retinal ganglion cells, based on the small sizes of their dendritic trees and cell bodies. About 80% of all retinal ganglion cells are midget cells in the parvocellular pathway. They receive inputs from relatively few rods and cones. In many cases, they are connected to midget bipolars, which are linked to one cone each. They have slow conduction velocity, and respond to changes in color but respond only weakly to changes in contrast unless the change is great. They have simple center-surround receptive fields, where the center may be either ON or OFF while the surround is the opposite.

Parasol retinal ganglion cells project to the magnocellular layers of the lateral geniculate nucleus. These cells are known as parasol retinal ganglion cells, based on the large sizes of their dendritic trees and cell bodies. About 10% of all retinal ganglion cells are parasol cells, and these cells are part of the magnocellular pathway. They receive inputs from relatively many rods and cones. They have fast conduction velocity, and can respond to low- contrast stimuli, but are not very sensitive to changes in color. They have much larger receptive fields which are nonetheless also center-surround.

Bistratified retinal ganglion cells project to the koniocellular layers of the lateral geniculate nucleus. Bistratified retinal ganglion cells have been identified only relatively recently. Koniocellular means "cells as small as dust"; their small size made them hard to find. About 10% of all retinal ganglion cells are bistratified cells, and these cells go through the koniocellular pathway. They receive inputs from intermediate numbers of rods and cones. They have moderate spatial resolution, moderate conduction velocity, and can respond to moderate-contrast stimuli. They may be involved in color vision. They have very large receptive fields that only have centers (no surrounds) and are always ON to the blue cone and OFF to both the red and green cone.

Photosensitive ganglion cells, including but not limited to the giant retinal ganglion cells, contain their own photopigment, melanopsin, which makes them respond directly to light even in the absence of rods and cones. They project to, among other areas, the suprachiasmatic nucleus (SCN) via the retinohypothalamic tract for setting and maintaining circadian rhythms. Other retinal ganglion cells projecting to the lateral geniculate nucleus (LGN) include cells making connections with the Edinger-Westphal nucleus (EW), for control of the pupillary light reflex, and giant retinal ganglion cells.

II. Glaucoma

Glaucoma refers to a group of eye conditions that lead to damage to the optic nerve, the nerve that carries visual information from the eye to the brain. In many cases, damage to the optic nerve is due to increased pressure in the eye, also known as intraocular pressure (IOP). Glaucoma is the second most common cause of blindness in the United States. The front part of the eye is filled with a clear fluid called aqueous humor. This fluid is always being made in the back of the eye. It leaves the eye through channels in the front of the eye in an area called the anterior chamber angle, or simply the angle. Anything that slows or blocks the flow of this fluid out of the eye will cause pressure to build up in the eye. This pressure is called intraocular pressure (IOP). In most cases of glaucoma, this pressure is high and causes damage to the major nerve in the eye, called the optic nerve. Burr et al., "The clinical effectiveness and cost-effectiveness of screening for open angle glaucoma: a systematic review and economic evaluation" Health Technol Assess. 2007 Oct;l l(41):iii-iv, ix-x, 1-190; Kwon et al., "Primary open-angle glaucoma" NEnglJMed. 2009 Mar 12;360(11): 1113-24; and Vass et al., "Medical interventions for primary open angle glaucoma and ocular hypertension" Cochrane Database SystRev. 2007 Oct 17;(4):CD003167.

An eye exam may be used to diagnose glaucoma. Checking the intraocular pressure alone (tonometry) is not enough to diagnose glaucoma because eye pressure changes. Pressure in the eye is normal in about 25% of people with glaucoma. This is called normal-tension glaucoma. There are other problems that cause optic nerve damage. Other tests that may be used to diagnose glaucoma include but are not limited to: gonioscopy (use of a special lens to see the outflow channels of the angle); tonometry test to measure eye pressure, optic nerve imaging (photographs of the inside of the eye), pupillary reflex response, tetinal examination, slit lamp examination, visual acuity, and/or visual field measurement.

The goal of glaucoma treatment is to reduce eye pressure. Depending on the type of glaucoma, this is done using medications or surgery.

A. Open-angle (chronic) glaucoma

Open-angle (chronic) glaucoma is the most common type of glaucoma. The cause is unknown. An increase in eye pressure occurs slowly over time. The pressure pushes on the optic nerve and the retina at the back of the eye. Open-angle glaucoma tends to run in families, where risks are higher with a parent or grandparent having open-angle glaucoma. This condition is usually asymptomatic until vision loss begins usually characterized by a gradual loss of peripheral (side) vision (also called tunnel vision).

Most people with open-angle glaucoma can be treated successfully with eye drops. Most eye drops used today have fewer side effects than those used in the past. You may need more than one type of drop. Some subjects may also be treated with pills to lower pressure in the eye. Newer drops and pills are being developed that may protect the optic nerve from glaucoma damage.

Some subjects will need other forms of treatment, such as a laser treatment, to help open the fluid outflow channels. This procedure is usually painless. Others may need traditional surgery to open a new outflow channel. With good care, most subjects with open- angle glaucoma can manage their condition and will not lose vision, but the condition cannot be cured.

B. Angle-closure (acute) glaucoma

Angle-closure (acute) glaucoma occurs when the exit of the aqueous humor fluid is suddenly blocked. This causes a quick, severe, and painful rise in the pressure within the eye (intraocular pressure). Angle-closure glaucoma is an emergency. This is very different from open-angle glaucoma, which painlessly and slowly damages vision. Acute glaucoma in one eye, elevates that risk for an attack in the second eye. Dilating eye drops and certain medications may trigger an acute glaucoma attack. Symptoms of angle-closure glaucoma include but are not limited to sudden, severe pain in one eye, decreased or cloudy vision, nausea and vomiting, rainbow-like halos around lights, red eye, and/or sensations of eye swelling.

Acute angle-closure attack is a medical emergency. Blindness will occur in a few days if it is not treated. Drops, pills, and medicine given through a vein (by IV) are used to lower pressure. Some people also need an emergency operation, called an iridotomy. This procedure uses a laser to open a new channel in the iris. The new channel relieves pressure and prevents another attack.

C. Congenital glaucoma

Congenital glaucoma is hereditary and is present at birth. It results from the abnormal development of the fluid outflow channels in the eye. Symptoms of congenital glaucoma include but are not limited to, cloudiness of the front of the eye, enlargement of one eye or both eyes, red eye, light sensitivity, and/or tearing.

This form of glaucoma is almost always treated with surgery to open the outflow channels of the angle. This is done while the subject is asleep and feels no pain (with anesthesia). Early diagnosis and treatment is important. If surgery is done early enough, many subjects will have no future problems.

D. Secondary glaucoma

Secondary glaucoma is caused by conditions including but not limited to drugs such as corticosteroids, eye diseases such as uveitis, and/or various systemic diseases.

III. Photodynamic Therapy (PDT)

A. Photosensitizers

A photo sensitizer is a chemical compound that can be excited by light of a specific wavelength. This excitation uses visible or near-infrared light. In photodynamic therapy, either a photo sensitizer or the metabolic precursor of one is administered to the subject. The tissue to be treated is exposed to light suitable for exciting the photosensitizer. Usually, the photosensitizer is excited from a ground singlet state to an excited singlet state. It then undergoes intersystem crossing to a longer-lived excited triplet state. One of the few chemical species present in tissue with a ground triplet state is molecular oxygen. When the photosensitizer and an oxygen molecule are in proximity, an energy transfer can take place that allows the photosensitizer to relax to its ground singlet state, and create an excited singlet state oxygen molecule. Singlet oxygen is a very aggressive chemical species and will very rapidly react with any nearby biomolecules, wherein specific biomolecule targets depend upon the specific photosensitizer. Ultimately, these destructive reactions will kill cells through apoptosis or necrosis.

A wide array of photosensitizers for PDT exist. They can be divided into porphyrins, chlorophylls and dyes. Allison et al., (2004). "Photosensitizers in clinical PDT" Photodiagnosis and Photodynamic Therapy 1 : 27-42. Other examples include but are not limited to aminolevulinic acid (ALA), Silicon Phthalocyanine Pc 4, m- tetrahydroxyphenylchlorin (mTHPC), and/or mono-L-aspartyl chlorin e6 (NPe6). Several photosensitizers are commercially available for clinical use, such as Photofrin^®, Verteporfin (Visudyne^®), Levulan^®, Foscan^®, Metvix^®, Hexvix^®, Cysview™, and Laserphyrin^®, with others in development, e.g. Antrin^®, Photochlor^®, Photosens^®, Photrex^®, Lumacan^®, Cevira^®, Visonac^®, BF-200 ALA. Amphinex^® and/or Azadipyrromethenes^®. O'Connor et al, (2009). "Porphyrin and Nonporphyrin Photosensitizers in Oncology: Preclinical and Clinical Advances in Photodynamic Therapy. Photochemistry and Photobiology, Sep/Oct 2009". Photochemistry and Photobiology.

Although these photosensitizers can be used for different medical treatments, these compounds have common characteristics including, but not limited to: high absorption at long wavelengths, tissue is much more transparent at longer wavelengths (-700-850 nm), absorbing at longer wavelengths would allow the light to penetrate deeper, and allow the treatment of larger tumors, high singlet oxygen quantum yield, low photobleaching, natural fluorescence, many optical dosimetry techniques, such as fluorescence spectroscopy, depend on the drug being naturally fluorescent, high chemical stability, low dark toxicity (e.g., the photosensitizer should not be harmful to the target tissue until the treatment beam is applied) and/or preferential uptake in target tissue. Wilson, Brian C; Michael S Patterson (2008). "The physics, biophysics, and technology of photodynamic therapy". Physics in Medicine and .¾o/ogy 53(9): R61-R109.

1. Verteporfin

The data presented herein describes some effects of verteporfin on both scleral fibroblasts and trabecular meshwork (TM) cells. The data suggest that, under identical conditions, human scleral fibroblasts and TM cells are more sensitive to verteporfm-induced cell death than retinal pigment epithelial cells. For example, one conclusion may be that TM cells can be specifically targeted using PDT, which is believed useful for models of ocular hypertension, or possibly a new therapeutic modality for treating glaucoma by inducing local remodeling in the outflow system of the eye.

Such a targeted therapy may have use for a variety of ocular diseases. For example, age-related macular degeneration (AMD) is beleived a leading cause of vision loss in patients over the age of 40, with the worst prognosis for patients with neovascular or 'wet' AMD. Brown et al., "The burden of age-related macular degeneration: a value-based analysis" Curr Opin Ophthalmol, 17(3):257-266 (2006). In this latter case, loss of vision occurs due to abnormal blood vessel growth originating from the choroidal vasculature.

Photodynamic therapy (PDT) laser light, in addition to the benzoporphyrin derivative photosensitizer, verteporfin, is also an FDA approved method for treating choroidal vascular diseases of the eye. Although it is not necessary to understand the mechanism of an invention, it is believed that following intravenous administration, activation of verteporfin by the PDT laser (~688 nm) yields highly reactive oxygen radicals that damages the cells of the vasculature, resulting in localized vessel occlusion. While there are several case reports of PTD therapy used to target neo vascular diseases of the anterior chamber, little is known of the effects of verteporfin-PDT therapy on tissues beyond the retina, such as the RPE and/or vascular endothelium. Charisis et al., "Contact transcleral photodynamic cyclo-suppression in human eyes: a feasibility study" Can J Ophthalmol, 46(2): 196-198 (2011); Parodi et al., "Photodynamic therapy with verteporfin for anterior segment neovascularizations in neovascular glaucoma" Am J Ophthalmol, 138(1):157-158 (2004); and Parodi et al, "Temporary intraocular pressure lowering by photodynamic therapy in pseudoexfoliation glaucoma" Ophthalmic Surg Lasers Imaging 42(l):53-58 (2011).

Consequently, the effects of verteporfin, with and without activation by PDT laser, were examined on different cultured ocular cells (i.e., for example, primary human scleral fibroblasts (hFibro), primary human trabecular meshwork (TM) cells (hTMC), primary porcine TM cells (pTMC), and a human retinal pigment epithelial cell line (ARPE-19 cells). The results suggest that PDT laser treatment alone was insufficient to cause significant cell death in any cell type tested, hi contrast, a twenty-four (24) hour exposure to inactivated verteporfin (e.g., without PDT laser irradiation) caused a dose-dependent decrease in cell viability in hFibro and hTMC, and to a lesser extent ARPE-19 cells. When Verteporfin was administered (0.5 μg/ml) without PDT laser activation a slight but statistically insignificant reduction in cell viability was observed in hFibro (81.5% ± 19.3%), pTMC (82.9% ± 6.7%), hTMC (80.3% ± 7.7%), and ARPE-19 cells (84.5% ± 14.9%). On the other hand, when Verteporfin was administered ^g/ml) with a 50 μΐ/cm² PTD laser treatment, a significant decrease in cell viability was observed in hFibro (13.5% ± 3.3%), pTMC (7.1% ± 1.5%), hTMC (11.1% ±5.2%), and ARPE-19 (44.5% ± 7.8%). Similar results were obtained in cells where verteporfin incubation was followed by washout before PDT laser.

These data indicate that unactivated verteporfin (e.g., not exposed to PTD laser treatment) is more toxic to scleral fibroblasts and TM cells than ARPE-19 cells. In all cell lines tested, PDT laser-induced cell death occurred with both co-incubation of verteporfm or pre-incubation followed by washout, suggesting that verteporfm is internalized into the cell. These results suggest a potential future use of PDT therapy for the selective in vivo removal of targeted ocular cells beyond its current use for destruction of vascular endothelial cells. For example, direct intraocular injections of verteporfm in conjunction with PDT treatment may be useful in treating diseases of the outflow system of the eye.

2. Photosensitizer Targeting Of Various Ocular Cell Types

In some embodiments, the present invention contemplates treatment of ocular diseases by targeting light-activation of photosensitizers at specific ocular cell types. For example, the data presented below was collected according to the methods described in Example II.

Cell Specific Dose-Dependent Toxicity Of Inactive Verteporfin

A range (0 - 25 ng/ml) of verteporfin was diluted in the appropriate cell culture media and then added to cultured ocular cells. Cells were protected from light and incubated for 24 hours at 37 °C in a humidified C0₂ incubator. Mitochondrial enzyme activity, determined by MTT assay, was used as a surrogate for cell viability. See, Figure 5. Increasing amounts of verteporfin without PDT laser activation (inactivated verteporfin) was inherently toxic to hFibo, hTMC and ARPE-19 cells. There was no significant (p > 0.05) decrease in cell viability at 0.25 μg/ml verteporfin in either hFibro (94.1% ± 2.9%) or hTMC (88.2% ± 5.2%) as compared to untreated hFibro (100.0% ± 4.7%) or hTMC (100.0% ± 9.0%). Inactivated verteporfin did show a dose-dependent increase in toxicity to both hFibro and hTMC at 1 μg/ml and above (p < 0.05). Compared to control hFibro (0 μg/ml verteporfin), cell viability decreased to 75.2% ± 3.7% (1 μg/ml), 35.9% ± 3.2% (4 μ^ιηΐ), 29.1% ± 1.2% (10 μ^πιΐ), and finally 23.6% ± 0.4% (25 μ^ητΐ). In hTMC cells, viability decreased significantly to 73.2% ± 2.2% (1 μ^πιΐ), 31.2% ± 0.4% (4 μ^πύ), 22.5% ± 0.4% (10 μ^ιηΐ), and finally 19.6% ± 0.9% (25 μ^ιηΐ).

Inactive verteporfin was less toxic to ARPE-19 cells, with no statistically significant decrease (p > 0.05) in cell viability between untreated controls (100.0% ± 3.8%) and cells treated with 0.25 μ^πιΐ (100.6% ± 7.8), 1 μ^πιΐ (91.4% ± 9.6) or 4 μ^ιιιΐ (86.7% ± 8.4%). A small but significant decrease in cell viability occurred in ARPE-19 cells treated for 24 hours with 10 μ^πιΐ verteporfin (86.5% ± 3.0%) which decreased to 58.4% ± 2.6% with 25 μg/ml verteporfin. Light Activation Of Verteporfin Increases Its Toxicity To Selected Cultured Ocular Cells

To assess the relative toxicity of light-activated verteporfin cultured hFibro, pTMC, hTMC and ARPE-19 cells were incubated with 0.5 μg/ml of verteporfin at 37 °C in a humidified C02 incubator. After 3 hours, cells in verteporfm-containing media were exposed to 50 μΤ/cm² of PDT laser and MTT assays were immediately performed (e.g.,'Cotreat' conditions). In the absence of verteporfin, no statistically significant loss of cell viability was seen in any cultured cells with exposure of up to 100 μΐ/cm² PDT light (0 μg/ml + 100 μΐ/cm²). However, in the presence of 0.5 μg/ml verteporfin, 50 μΐ/ϋηι² PDT light reduced viability of hFibro to 13.5% ± 3.3%, pTMC to 7.1% ± 1.5%, hTMC to 11.1 % ± 5.2%, and ARPE-19 cells to 44.5% ± 7.8% of control levels ('Cotreat + 50 μ.Γ/Ώη²). See, Figure 6.

Verteporfin Retains Its Cytotoxic Effects With Pretreatment

Cultured hFibro, pTMC, hTMC and ARPE-19 cells were incubated with 0.5 μg ml of verteporfin at 37 °C in a humidified C0₂ incubator. After 24 hours verteporfin-containing media was removed, the cells were washed twice and then incubated in fresh cell culture

2 2

media for 3 hours. Cells were then exposed to 50 μΙ½η and 100 uJ/cm of laser and MTT assays were immediately performed ('Pretreat' conditions). See, Figure 6.

hFibro pretreated for 24 hours with 0.5 μg/ml verteporfin resulted in a slight, but statistically insignificant, drop in cell viability (81.5% ± 19.3% live) as compared to untreated controls (100.0% ± 6.6%). When exposed to 50 μΐ/cm² PDT laser, viability decreased to

14.0% ± 4.7% live cells. The viability of pTMC pretreated with 0.5 μ^πιΐ verteporfin (82.9% ± 6.7% live) was also not significantly different from the viability of untreated controls (100.0% ± 4.9%). This viability decreased to 10.8% ± 2.3% live cells when exposed to 50 μΐ/cm² PDT laser. hTMC pretreated with 0.5 μg ml verteporfin resulted in a slight but not significant drop in cell viability (80.3% ± 7.7% live) compared to untreated controls (100.0% ± 3.5%). When exposed to 50 μΐ/αη² PDT laser, viability decreased to 12.5% ± 1.9% live cells. The viability of ARPE-19 cells pretreated with 0.5 μ^ιηΐ verteporfin (84.5% ± 14.9% live) was not significantly different from the viability of untreated controls (100.0% ± 6.5%). This viability decreased to 54.7% ± 14.1% live cells with addition of 50 μΐ/cm² PDT laser. In all cells (hFibro, pTMC, hTMC, and ARPE- 19) pretreated with verteporfin, increasing PDT laser exposure from 50 μΐ/cm² to 100 μΐ/cm² did not result in any further significant decrease in cell viability (p > 0.05). In addition, we found no statistically significant difference in cell viability of cells treated with verteporfin (0.5 μg/ml) plus PDT laser between the 'Pretreat' or 'Cotreat' conditions in hFibro, hTMC and ARPE-19 cells. There was an extremely small but statistically significant difference between 'Pretreat' or 'Cotreat' conditions in pTMC (10.8% ± 2.3% vs 7.1% ± 1.5%).

Analysis And Application

Intravenously (IV) administered verteporfin has been used previously to target: i) blood vessels within a drainage angle to treat neovascular glaucoma; ii) iris vessels for treatment of pseudoexfoUiation glaucoma; and iii) ciliary body vasculature to reduce aqueous humor production. Charisis et al., "Contact transcleral photodynamic cyclo-suppression in human eyes: a feasibility study" Can J Ophthalmol, 46(2):196-198 (2011); Parodi et al, "Photodynamic therapy with verteporfin for anterior segment neovascularizations in neovascular glaucoma" Am J Ophthalmol, 138(1):157-158 (2004); and Parodi et al.,

"Temporary intraocular pressure lowering by photodynamic therapy in pseudoexfoliation glaucoma" Ophthalmic Surg Lasers Imaging 42(l):53-58 (2011).

In a rabbit model of glaucoma filtration surgery, PDT has been shown to reduce bleb failure by decreasing fibrosis, presumably by targeting local neovascularization. Stasi et al., "Photodynamic treatment in a rabbit model of glaucoma surgery" Acta Ophthalmol Scand, 84(5):661-666 (2006). Intravenous (IV) administration of verteporfin shows rapid (~5 min) accumulation in the choroid, RPE, and ciliary body followed quickly by accumulation in the photoreceptors. Haimovici et al., "Localization of lipoprotein-delivered benzoporphyrin derivative in the rabbit eye" Curr Eye Res 16(2):83-90 (1997). However, no appreciable amount of verteporfin was detected in corneal tissue, indicating that the blood-eye barrier prevents the IV-administered verteporfin from entering the aqueous and/or vitreous spaces. Perhaps due to this partitioning, there are no known reports that determine the effects of verteporfin on intraocular cell types that reside beyond the blood-eye barrier.

The data presented herein demonstrate the relative toxicity of inactivated and light- activated verteporfin in scleral fibroblasts, trabecular meshwork (TM) cells, and retinal pigment epithelial (RPE) cells (e.g., ARPE-19 cells). Scleral fibroblasts and TM cells are seen as more sensitive to both inactive and light-activated verteporfin than ARPE-19 cells.

Similarly, in the absence of verteporfin, cultured cells exposed to PDT laser therapy at twice the recommended treatment protocol (i.e., for example, 2 50 μΐ/cm²) for neovascular AMD had no detrimental effect. However, in the presence of verteporfin, a single standard treatment protocol (e.g., 50 μΤ/cm ) was sufficient to fully activate all verteporfin within the focal area, since a treatment with 100 μΐ/cm² caused no further cell death. Approximately 80% of human fibroblasts and TM cells (human and porcine) were killed when exposed to 0.5 μg/ml verteporfin plus PTD laser, while under these conditions only -50-60% of RPE cells were killed.

It has been previously reported that, upon exposure to light-activated verteporfin, leucocytes demonstrate an LD₅₀ in the range of 0.01 to 0.02 μg/ml. Granville et al., "Nuclear factor-kappaB activation by the photochemotherapeutic agent verteporfin" Blood 95(1):256- 262 (2000); and Hunt et al., "Sensitivity of activated murine peritoneal macrophages to photodynamic killing with benzoporphyrin derivative" Photochem Photobiol 61(4):417-421 (1995). Leukocytes also have no apparent toxicity to verteporfin in the absence of light. Granville et al., "Nuclear factor-kappaB activation by the photochemotherapeutic agent verteporfin" Blood 95(l):256-262 (2000). In some embodiments, the present invention contemplate clincally effective doses of verteporfin that are non-toxic to specific ocular cell types following a direct injection into the eye.

The data presented herein also show that scleral fibroblasts and TM cells are more sensitive to increasing concentrations of inactive verteporfin than ARPE-19 cells. In the absence of PDT laser, there was no statistically significant decrease in viability with 0.5 μg/ml of verteporfin. Significant toxicity was observed in hFibro and hTMC cells but not ARPE-19 cells when the amount of verteporfin was increased to 1 μg/ml and above. The FDA-approved Visudyne^® (verteprofin) package insert indicates that IV administered verteporfin has a maximum blood plasma concentration of 2 μg/ml and a clearance half-life of about 5-6 hours. Consequently, in the present administration protocol, plasma levels should drop below 1 μg/ml within 6 hours. Therefore, inherent toxicity to certain cell types may be less relevant in vivo when verteporfin is administered intravenously, but is a factor if administered by intravitreal amd/or intracameral injection. The data presented here suggest that injections which target an intraocular concentration of ~ 0.5 μg/ml would allow for PDT- laser killing of TM cells and scleral fibroblast without having toxic effects in other tissues not within the focal beam of the laser.

The 'Pretreat' experiments shown herein indicate that cultured cells can internalize inactive verteporfin. See, Figure 5. In these experiments, cells were exposed to verteporfin, washed, and then incubated in verteporfin- free media. After exposure to PTD laser, the percentage of live cells in the 'Pretreat' conditions was nearly identical to the percentage of live cells in the 'Cotreat' conditions. The simplest explanation suggests that verteporfin is taken up by these cells during the 24 hour pretreatment. Accumulation of verteporfin within cells has been previously demonstrated in other cell types, and the internalization process is apparently dependent on binding and internalization of verteporfin via low-density lipoprotein (LDL) receptors. Allison et al., "Evidence for low-density lipoprotein receptor-mediated uptake of benzoporphyrin derivative" Br J Cancer 69(5):833-839 (1994). LDL receptors have been functionally shown to be present on: i) TM cells (Chang et al., "Expression of modified low-density lipoprotein receptors by trabecular meshwork cells" Curr Eye Res 10(12):1101- 1112 (1991); ii) normal fibroblasts (Allison et al.,. "Evidence for low-density lipoprotein receptor-mediated uptake of benzopoiphyrin derivative" Br J Cancer 69(5):833-839 (1994), and iii) RPE cells (Tserentsoodol et al. "Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor-mediated process" Mol Vis 12:1306-1318. (2006). The internalization of verteporfin by cells expressing LDL receptors (i.e., for example, TM cells and fibroblast cells) prove useful in developing photosensitizer-PDT clincial therapy.

In conclusion, PDT can be used for targeted killing of TM cells, which can provide a method of treatment for some ocular diseases, such as ocular hypertension. Alternatively, PDT therapy targeted to the ocular anterior segment can provide a method for treatment for glaucoma. Although it is not necessary to understand the mechanism of an invention, it is believed that a selective destruction of TM cells in certain regions of the meshwork may lead to localized tissue remodeling and a subsequent increase aqueous outflow. In some embodiments, the PDT laser comprises a refined ophthalmic gonioscopic lens capable of delivering targeted light to the angle while minimizing collateral damage.

In one embodiment, the present invention contemplates administering a direct in vivo intraocular injection of a photosensitizer (e.g., verteporfin) in combination with PDT therapy to treat diseases of the outflow system of the eye. In one embodiment, the present invention contemplates a method comprising: selective killing of aqueous outflow system cells;

administration of a stem cell population, wherein said stem cells repopulate the aqueious outflow system.

B. Photodynamic Therapy Glaucoma Treatment

In one embodiment, the present invention contemplated the treatment of glaucoma in a patient (i.e, for example, a human patient) by the administration of a photosensitizer.

Although it is not necessary to understand the mechanism of an invention, it is believed that the presently disclosed method does not result in cell death and/or cell killing as a mechanism of the treatment. In one embodiment, the present method treats glaucoma wherein the PDT- photosensitizer administration stimulates the LDL bearing cells to release proteins that mediate a reduction in ocular pressure without killing the cells.

Photodynamic therapy (PDT) is an emerging treatment used to treat various types of medical conditions, such as glaucoma. Generally, PDT involves three components: a photosensitizer, light (wavelength appropriate for the photosensitzer), and tissue oxygen. The combination of these three components leads to the destruction of targeted cells. PDT has been reported as a possible treatment for wet macular degeneration, psoriasis, cancer and/or glaucoma.

PDT maybe useful for the treatment of bodily organs (i.e., for example, an eyeball) through the use of endoscopes and fiber optic catheters to deliver light, and intravenously- administered photosensitizers. As contemplated herein, optimal combinations of

photosensitizers, light sources, and treatment parameters require an empirical assessment to determine whether a photosensitizer administration will either induce glaucoma or treat glaucoma.

Although it is not necessary to understand the mechanism of an invention, it is believed that a PDT method would involve the following steps: i) a photosensitizer precursor is applied to the ocular sclera; ii) a waiting period of a few hours is allowed to elapse, during which time the photo senstizer will be taken up by cells (e.g., retinal ganglion cells and/or cells within the trabecular meshwork); iii) a bright red light (from an array of light-emitting diodes or a diode laser) illuminates the area to be treated, wherein the light exposure lasts a few minutes to a few tens of minutes; iv) the photosensitizer absorbs the light, exciting it to an excited singlet state; v) an intersystem crossing may occur in conjunction with an energy transferance to a triplet oxygen, resulting in singlet (ground state) and excited singlet oxygen species; vi) singlet oxygen species react with biomolecules, fatally damaging some cells in the treatment area.

PDT and/or test compound administration as described herein has specific advantages over conventional methods to induce glaucoma. For example, PDT and/or test compound administration can be localised and/or targeted, wherein cell target specificity can be achieved in ways including but not limited to: i) light is delivered only to specific cell types, such that the surrounding cells are in the absence of light, and since there is no activation of the photosensitizer and/or test comound in the locality of the surrounding cells, the surrounding cells are not damaged and remain healthy (i.e., for example, an inflammatory reaction is not induced); ii) photosensitizers and/or test compounds may be administered in ways that restrict their mobility; iii) photosensitizers and/or test compounds may be chosen which are selectively absorbed at a greater rate by targeted cells; iv) photosensitizers and or test compounds may be administered that faciliate targeted delivery (i.e., for example, intraocular injection and/or topical administration). The methods described herein can also be much cheaper than the alternative therapies and/or surgical operation and after care.

Photosensitizers have been used in conjunction with PDT to inhibit formation or retard disease progression related to sub-retinal fluid concentration. Heacock et al., "System and method for excitation of photoreactive compounds in eye tissue" United States Patent 7,288,106.

In particular, PDT is focused on the trabecular meshwork to treat glaucoma. Diseased cells may also be killed by triggering a class of photoreactive compounds or photosensitizers with specific illumination wavelengths. For example, photosensitizers are used in photodynamic therapy (PDT) through light sources such as lasers to treat targeted eye tissue in a number of eye disease conditions including glaucoma.

PDT may also be performed on the ciliary body of the eye, particularly in those subjects at risk of developing or having glaucoma, in a manner that preserves the viability of retinal ganglion cells. Miller et al., "Methods and compositions for treating ocular glaucoma" United States Patent Application Publication Number 2006/0021623. Generally, a photodynamic therapy-based method is described for treating ocular glaucoma. In particular, a photo sensitizer, for example, a benzoporphyrin derivative photo sensitizer, is administered to a mammal either having or at risk of developing ocular glaucoma. The photosensitizer, when present in the ciliary body, is activated by light, for example, light from a laser. The treatment results in a reduction of intraocular pressure within the treated eye, which can persist for a prolonged period of time. Miller also discusses the preservation of retinal ganglion cell viability in a mammalian eye having a ciliary body and at risk of developing or having glaucoma.

The disclosed method includes the steps of administering to a non-human mammal an amount of a photosensitizer and/or test compound, for example, a benzoporphyrin derivative photosensitizer and/or test compound, for example, a benzoporphyrin derivative mono acid photosensitizer, sufficient to accumulate in the ciliary body, and irradiating a region of the ciliary body so as to activate the photosensitizer. Alternatively, the test compound may be therapeutically active without irradiation. The activated photosensitizer can cause a decrease in the intraocular pressure in the eye relative to the intraocular pressure in the eye prior to irradiation. This decrease in intraocular pressure can be for a prolonged period of time, for example, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks. The irradiation can reduce the intraocular pressure in the eye by at least 20%, by at least 30%, or by at least 40% of the intraocular pressure in the eye prior to irradiation.

Furthermore, the intraocular pressure can be reduced, for example, by at least 20%, for at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks.

It was reported that ciliary body PDT may result in morphologic changes in the ciliary body, significant reduction of intraocular pressure (IOP), and prevention of ganglion cell loss in a mouse glaucoma model. Matsubara et al., "Investigating the effect of ciliary body photodynamic therapy in a glaucoma mouse model" Invest Ophthalmol Vis Sci. 47:2498-2507 (2006). These results suggest that ciliary body PDT is a selective cyclodestructive technique with potential clinical application in the treatment of glaucoma.

Photosensitizer administration coupled with photodynamic therapy is reported as a possible course of treatment for age-related macular degeneration. Cooper et al., "Transscleral delivery" United States Patent 7,585,517. Such photosensitizer-enhanced photodynamic therapy may also be useful in treating nonvascular glaucoma.

Photodynamic therapy of conditions of the eye characterized by unwanted

neovasculature, such as age-related macular degeneration, is effective when used in conjunction with green porphyrins as photoactive agents, preferably as liposomal

compositions. Miller et al., "Use of green porphyrins to treat neovasculature in the eye" United States Patent 6,225,303 It was also suggested that neovascular glaucoma may also be treated by this procedure.

Beta-amyloid protein-involved ocular disease including age-related macular degeneration and glaucoma may also be used by photosensitizer/PDT therapy. Kim D., "Methods for treatment of beta-amyloid protein-induced ocular disease" United States Patent 7, 728,043. In particular, glaucoma may be related to a chronic neurodegeneration of retinal ganglion cells resulting from beta-amyloid build-up. There was no suggestion to use PDT to treat neurodegeneration of retinal ganglion cells.

The conjugation of photosensitizers to various ligands that facilitate targeting to tissues and cells before the photosensitizers are formulated with block copolymers has been reported. Chowdhary et al., "Drug delivery systems for photodynamic therapy" United States Patent Number 6,693,093. These ligands include those that are receptor-specific as well as immunoglobulins and fragments thereof. Preferred ligands include antibodies in general and monoclonal antibodies, as well as immunologically reactive fragments thereof. Moreover, the block copolymer may be conjugated to the ligands to which the photosensitizer binds to facilitate improved complexing of non-hydrophobic photo sensitizers with the copolymer. There was no attempt to use photosensitizer block copolymer preparations for PDT treatment of glaucoma.

Some photosensitisers naturally accumulate in the endothelial cells of vascular tissue allowing 'vascular targeted' PDT, but there is also research to target the photosensitiser to the tumour (usually by linking it to antibodies or antibody fragments). It is currently only in preclinical studies. Some photosensitizers in development are linked to antibodies to target them at the tumour cells.

A variety of photosensitizers can be used in the practice of PDT. In one embodiment, the photosensitizer is a benzoporphyrin derivative, for example, benzoporphyrin derivative mono-acid. Practice of the invention can reduce the intraocular pressure in the eye by at least 20%, at least 30%, or at least 40% of the intraocular pressure in the eye prior to irradiation. Furthermore, the method can reduce the intraocular pressure by at least 20% for at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks. In some cases, the photosensitizer components may be used to deliver medications targerting the trabecular meshwork but using ligands that attach to low density lipoprotein (LDL) receptors on the trabecular meshwork on LDL receptors on adjacent tissues. Targeting the LDL receptors in this way may be solely used to deliver medications to the trabecular meshwork without coupling this step with photo sensitization. The medication that is attached to the ligand which targets LDL could be a beta blocker, a carbonic anhydrase inhibitor, a prostaglandin analogue, a rho kinase inhibitor, an endothelin antagonist, or other medications or biologic agents

(virus, protein, RNA or DNA based material) that is designed to modify the conventional or unconventional outflow system of the aqueous humor so that eye pressure is reduced or prevented from increasing.

Photosensitizers have been used in conjunction with PDT to treat glaucoma by increasing outflow facility of the trabecular network. Schwartz et al., "Treatment for dry macular degeneration" United States Patent 7,381,404. In general, this report suggests that photodynamic therapy (PDT) uses light-activated drugs to potentially halt or slow abnormal cell growth. The disclosed therapy treats late stages of disease, as characterized by choroidal neovascularization. Briefly, a photosensitizer is administered intravenously and attaches to lipoprotein receptors, particularly those receptors found in cells undergoing rapid

proliferation. Shortly after administration, the compound is activated with a pre-calculated dose of light at a particular wavelength, resulting in conversion of normal oxygen to free radical singlet oxygen, which in turn causes closure of neovascular tissue.

C. Creation Of Glaucoma Animal Experimental Models

In one embodiment, the present invention contemplates a method comprising photodynamic therapy, wherein a medical condition is induced. In one embodiment, the photodynamic therapy induces glaucoma. In one embodiment, the method further comprises administering a photosensitizer. In one embodiment, photodynamic therapy creates a non- human mammal glaucoma experimental model. In one embodiment, the non-human mammal glaucoma experimental model comprises free radical-induced trabecular meshwork cell damage.

Photodynamic therapy may lead to oxidative stress through the generation of free radicals. Oxidative stress may cause damage to cellular macromolecules such as nucleic acids, proteins and lipids. Lipid peroxidation (LPO) maybe estimated by measurement of the concentration of malondialdehyde, protein degradation - by modified EUman's method, superoxide dysmutase (SOD) - using Ransod Kit. The expression of inducible nitric oxide synthase (iNOS) may be detected by immiinocytochemical staining. Saczko et al., "Photo- oxidative action in cervix carcinoma cells induced by HPD - mediated photodynamic therapy" Exp Oncol. 2009 Dec;31(4): 195-9.

In some embodiments, the present invention contemplates methods for the creation of glaucoma using animal models utilizing rats, mice, rabbits and/or guinea pigs. As shown in Example I below, the data disclosed herein demonstrates the specific targeting of trabecular meshwork cells without inducing any damage and/or significant inflammation in the surrounding cells. This is unlike currently used methods to induce glaucoma in animal models that including but not limited to lasers, hypertonic saline injection, and/or drainage canal cauterization, all of which have been reported to be associated with significant inflammation.

D. Trabecular Meshwork Remodeling

In one embodiment, the present invention contemplates a method comprising photodynamic therapy wherein a trabecular network is remodeled. In one embodiment, the photodynamic therapy further comprises administering a photosensitizer The morphology of the trabecular meshwork in three types of open angle glaucoma: primary open angle glaucoma (POAG), corticosteroid-induced glaucoma and pigmentary glaucoma (PG) are somewhat different. For example, ageing is a risk factor for development of POAG. It is assumed that preexisting age-related changes of the trabecular meshwork (TM) play a role for the development of increased outflow resistance and intraocular pressure (IOP) in various types of glaucoma. These age-related changes in the TM develop

concomitant with that of presbyopia. Therefore there may be a functional relationship between ciliary muscle (CM) and TM and the age-related changes in morphology of the outflow system. Tektas et al., "Structural changes of the trabecular meshwork in different kinds of glaucoma" Exp Eye Res. 2009 Apr;88(4):769-75.

One finding in the ageing TM concerns apparent changes of the elastic fiber network and the anterior elastic tendons of the CM, exemplified by an increase in thickness of the sheath of the elastic fibers. Cross-sections through these fibers with their sheath appear as extracellular plaques and were therefore termed "sheath derived plaques" (SD-plaques). Morphologically, the TM changes in POAG may resemble that of the ageing TM, but in

POAG there appears an increase in SD-plaques compared to age-matched controls. Although it is not necessary to understand the mechanism of an invention, it is believed that this increase is due to fine fibrils and other components of the extracellular matrix (ECM) that adhere to the sheaths of the elastic fibers and their connections to the inner wall endothelium.

In POAG eyes there also may be a marked loss of TM cells, at places leading to fusion and thickening of trabecular lamellae. For example, in steroid-induced glaucoma there is also an increase in fine fibrillar material in the subendothelial region of SC. h contrast to POAG eyes, these fibrils may not adhere to the sheath of the elastic fibers but are deposited underneath the inner wall endothelium. Further, steroid-induced glaucoma may be characterized by an accumulation of basement membrane-like material staining for type IV collagen. These accumulations can be found throughout all layers of the TM.

In pigmentary glaucoma, loss of cells appears more prominent than in POAG eyes. Presumably, this cell loss occurs after overload of TM cells with pigment granules. Denuded TM lamellae fuse and the TM collapses. In the subendothelial region of these collapsed TM areas an increase in ECM presumably due to underperfusion was observed. At other places, SC was occluded and the cribriform region appeared disorganized. In most parts of the circumference of the eye, the TM cells contained pigment granules. Occlusion of TM spaces by pigment granules or cells loaden with pigment was not seen in eyes with PG. In glaucoma, extensive pathological changes are believed to occur in the trabecular meshwork (TM) and juxtacanalicular tissue of the chamber angle. For example, aqueous humor drainage is disturbed due to the accumulation of extracellular matrix (ECM) material in the outflow system. Although it is not necessary to understand the mechanism of an invention, it is believed that matrix metalloproteinases (MMPs) may remodel ECM material and, thus, may have a role in regulating outflow facility and intraocular pressure (IOP).

Consequently, the modulation of MMP expression and/or the adminsitration of tissue inhibitors of MMPs (TIMPs) may provide an effective treatment in the chamber angle of normal eyes and in primary open-angle glaucoma (POAG) and in exfoliation glaucoma (ExG). Ronkko et al., "Matrix metalloproteinases and their inhibitors in the chamber angle of normal eyes and subjects with primary open-angle glaucoma and exfoliation glaucoma" Graefes Arch Clin Exp Ophthalmol. 2007 May;245(5):697-704. An expression imbalance was observed between MMPs and their endogenous tissue inhibitors in tissue samples from subjects with POAG and ExG. Differences in immunohistochemical reactions reflect discrete local pathogenic mechanisms involved in POAG and ExG. With respect to the proposed role of MMPs in the remodeling of ECM material, this may point to a weaker reactivity to the accumulation of ECM material in TM in ExG than POAG eyes.

Corticosteroid treatment may induce glaucoma and has been reported to remodel the trabecular meshwork ultrastructure either with or without POAG. Johnson et al.,

"Ultrastructural changes in the trabecular meshwork of human eyes treated with

corticosteroids" Arch Ophthalmol. 1997 Mar;115(3):375-83. For example, the trabecular meshwork from 5 subjects in whom corticosteroid-induced glaucoma was diagnosed and from 6 subjects with POAG who had been treated with systemic or topical corticosteroids for months to years was investigated with light and electron microscopy. None of the eyes with POAG were considered to have corticosteroid-induced elevation of the intraocular pressure. Eyes with corticosteroid-induced glaucoma had the accumulation of extracellular material distinct from the sheath-derived plaques typical of POAG. A finger-printlike arranged material resembling basement membranes (FBM material), considered characteristic of corticosteroid-induced glaucoma, was found in all eyes with corticosteroid-induced glaucoma. In addition, an abnormal accumulation of densely packed, fine fibrils immediately beneath the inner wall endothelium of Schlemm's canal was present. The findings were similar among subjects receiving topical or systemic treatment and among subjects of different ages. In the eyes from donors with POAG who had been treated with corticosteroids, the fine fibrillar material and FBM material were present in small amounts in 3 of 6 donors and were not found in the other 3 donors. The extracellular material that accumulates in eyes with corticosteroid-induced glaucoma differs from that seen in eyes with POAG. Eyes with POAG exposed to long-term corticosteroid treatment did not all respond with the formation of the abnormal extracellular materials characteristic of those found in eyes with

corticosteroid-induced glaucoma.

E. Neovascular Glaucoma Treatment

In one embodiment, the present invention contemplates a method comprising photodynamic therapy for treating a neovascular glaucoma. In one embodiment, the method further comprises a photo sensitizer.

Neovascular glaucoma (NVG) is a severe form of glaucoma with devastating visual outcome attributed to new blood vessels obstructing aqueous humor outflow, usually secondary to widespread posterior segment ischemia. Invasion of the anterior chamber by a fibrovascular membrane initially obstructs aqueous outflow in an open-angle fashion and later contracts to produce secondary synechial angle-closure glaucoma. NVG may be characterized by iris neovascularization, a closed anterior chamber angle, and extremely high intraocular pressure (IOP) with severe ocular pain and usually poor vision. Shazly et al., "Neovascular glaucoma: etiology, diagnosis and prognosis" Semin Ophthalmol. 2009 Mar-Apr;24(2): 113- 21.

Because neovascular glaucoma (NVG) is a severely blinding, intractable disease in order to prevent or redtice the extent of visual loss caused by NVG, its etiology is

informative. The most common diseases responsible for development of NVG include but are not limited to ischemic central retinal vein occlusion (CRVO), diabetic retinopathy and/or ocular ischemic syndrome. As a management strategy, one priority should be to try to prevent its development by appropriate management of the causative diseases. If NVG develops, early diagnosis is crucial to reduce the extent of visual loss. Management of NVG primarily consists of controlling the high IOP by medical and/or surgical means to minimize the visual loss. Hayreh S., "Neovascular glaucoma" Prog Retin Eye Res. 2007 Sep;26(5):470-485; and Konareva-Kostianeva M., "Neovascular glaucoma" Folia Med (Plovdiv). 2005;47(2):5-l 1. Until the present invention, there was no satisfactory means of treating NVG and preventing visual loss in the majority, in spite of multiple modes of medical and surgical options advocated over the years and claims made. Neovascular glaucoma may be divided into at least three clinical stages including but not limited to rubeosis iridis, secondary open-angle glaucoma, and/or synechia of the angle- closure glaucoma. Approximately 36% of neovascular glaucomas occurs after central retinal vein occlusion, 32% after diabetic proliferative retinopathy, and 13% occurs after carotid artery obstructive. Conventional treatment of neovascular glaucoma is most effective when diagnosed early and properly and is aimed mainly at relieving pain, as the prognosis for maintaining visual function is extremely poor. The most important surgical procedures are trabeculectomy, artificial drainage shunts and cyclo-distraction by trans-scleral diode laser. Vancea et al., "Current trends in neovascular glaucoma treatment" Rev Med Chir Soc Med Nat last 2005 Apr- Jun;109(2):264-268.

Treatment of ocular neovascular diseases has been attempted by intravitreal therapies targeting vascular endothelial growth factor (VEGF) (i.e., for example, the RNA aptamer pegaptanib and the monoclonal antibody antigen-binding fragment ranibizumab). Pegaptanib selectively binds to a VEGF isoform identified as being especially pathogenic in the eye and spares other isoforms, whereas the other two agents nonselectively bind all VEGF isoforms. Because VEGF is involved in a wide variety of physiologic processes, the ocular and systemic safety of anti-VEGF agents is of paramount concern. Tolentino M., "Systemic and ocular safety of intravitreal anti-VEGF therapies for ocular neovascular disease" Surv Ophthalmol. 2011 Mar-Apr; 56(2): 95-113. Consequently, the art is in search of therapies that have wider safety thresholds than antibody treatments.

V. Pharmaceutical Compositions

The present invention further provides pharmaceutical compositions comprising at least one compound as described above. In other embodiment, the pharmaceutical compositions comprise several compounds as described above. The pharmaceutical compositions of the present invention maybe administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. For example, administration may be topical including, but not limited to, ophthalmic and/or to mucous membranes. Alternatively, administration may be via the pulmonary system (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets.

Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions maybe generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, maybe prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the subject. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50S found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from 0.01 μg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it maybe desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every 20 years. VI. Drug Delivery Systems

The present invention contemplates several drug delivery systems that provide for roughly uniform distribution, have controllable rates of release. A variety of different media are described below that are useful in creating drug delivery systems. It is not intended that any one medium or carrier is limiting to the present invention. Note that any medium or carrier may be combined with another medium or carrier; for example, in one embodiment a polymer microparticle carrier attached to a compound may be combined with a gel medium.

Carriers or mediums contemplated by this invention comprise a material selected from the group comprising gelatin, collagen, cellulose esters, dextran sulfate, pentosan polysulfate, chitin, saccharides, albumin, fibrin sealants, synthetic polyvinyl pyrrolidone, polyethylene oxide, polypropylene oxide, block polymers of polyethylene oxide and polypropylene oxide, polyethylene glycol, acrylates, acrylamides, methacrylates including, but not limited to, 2- hydroxyethyl methacrylate, poly(ortho esters), cyanoacrylates, gelatin-resorcin-aldehyde type bioadhesives, polyacrylic acid and copolymers and block copolymers thereof.

One embodiment of the present invention contemplates a drug delivery system comprising therapeutic agents as described herein.

Microparticles

One embodiment of the present invention contemplates a medium comprising a microparticle. Preferably, microparticles comprise liposomes, nanoparticles, microspheres, nanospheres, microcapsules, and nanocapsules. Preferably, some microparticles

contemplated by the present invention comprise poly(lactide-co-glycolide), aliphatic polyesters including, but not limited to, poly-glycolic acid and poly-lactic acid, hyaluronic acid, modified polysacchrides, chitosan, cellulose, dextran, polyurethanes, polyacrylic acids, psuedo-poly(amino acids), polyhydroxybutrate-related copolymers, polyanhydrides, polymethylmethacrylate, poly(ethylene oxide), lecithin and phospholipids.

Liposomes

One embodiment of the present invention contemplates liposomes capable of attaching and releasing therapeutic agents described herein. Liposomes are microscopic spherical lipid bilayers surrounding an aqueous core that are made from amphiphilic molecules such as phospholipids. For example, a liposome may trap a therapeutic agent between the hydrophobic tails of the phospholipid micelle. Water soluble agents can be entrapped in the core and lipid-soluble agents can be dissolved in the shell-like bilayer. Liposomes have a special characteristic in that they enable water soluble and water insoluble chemicals to be used together in a medium without the use of surfactants or other emulsifiers. Liposomes can form spontaneously by forcefully mixing phosopholipids in aqueous media. Water soluble compounds are dissolved in an aqueous solution capable of hydrating phospholipids. Upon formation of the liposomes, therefore, these compounds are trapped within the aqueous liposomal center. The liposome wall, being a phospholipid membrane, holds fat soluble materials such as oils. Liposomes provide controlled release of incorporated compounds, h addition, liposomes can be coated with water soluble polymers, such as polyethylene glycol to increase the pharmacokinetic half-life. One embodiment of the present invention contemplates an ultra high-shear technology to refine liposome production, resulting in stable, unilamellar (single layer) liposomes having specifically designed structural characteristics. These unique properties of liposomes, allow the simultaneous storage of normally immiscible compounds and the capability of their controlled release.

In some embodiments, the present invention contemplates cationic and anionic liposomes, as well as liposomes having neutral lipids. Preferably, cationic liposomes comprise negatively-charged materials by mixing the materials and fatty acid liposomal components and allowing them to charge-associate. Clearly, the choice of a cationic or anionic liposome depends upon the desired pH of the final liposome mixture. Examples of cationic liposomes include lipofectin, lipofectamine, and lipofectace.

One embodiment of the present invention contemplates a medium comprising liposomes that provide controlled release of at least one therapeutic agent. Preferably, liposomes that are capable of controlled release: i) are biodegradable and non-toxic; ii) carry both water and oil soluble compounds; iii) solubilize recalcitrant compounds; iv) prevent compound oxidation; v) promote protein stabilization; vi) control hydration; vii) control compound release by variations in bilayer composition such as, but not limited to, fatty acid chain length, fatty acid lipid composition, relative amounts of saturated and unsaturated fatty acids, and physical configuration; viii) have solvent dependency; iv) have pH-dependency and v) have temperature dependency.

The compositions of liposomes are broadly categorized into two classifications.

Conventional liposomes are generally mixtures of stabilized natural lecithin (PC) that may comprise synthetic identical-chain phospholipids that may or may not contain glycolipids. Special liposomes may comprise: i) bipolar fatty acids; ii) the ability to attach antibodies for tissue-targeted therapies; iii) coated with materials such as, but not limited to lipoprotein and carbohydrate; iv) multiple encapsulation and v) emulsion compatibility. Liposomes maybe easily made in the laboratory by methods such as, but not limited to, sonication and vibration. Alternatively, compound-delivery liposomes are commercially available. For example, Collaborative Laboratories, Inc. are known to manufacture custom designed liposomes for specific delivery requirements.

Microspheres, Microparticles And Microcapsules

Microspheres and microcapsules are useful due to their ability to maintain a generally uniform distribution, provide stable controlled compound release and are economical to produce and dispense. Preferably, an associated delivery gel or the compound-impregnated gel is clear or, alternatively, said gel is colored for easy visualization by medical personnel.

Microspheres are obtainable commercially (Prolease®, Alkerme's: Cambridge,

Mass.). For example, a freeze dried medium comprising at least one therapeutic agent is homogenized in a suitable solvent and sprayed to manufacture microspheres in the range of 20 to 90 μπι. Techniques are then followed that maintain sustained release integrity during phases of purification, encapsulation and storage. Scott et al., Improving Protein

Therapeutics With Sustained Release Formulations, Nature Biotechnology, Volume 16:153- 157 (1998).

Modification of the microsphere composition by the use of biodegradable polymers can provide an ability to control the rate of therapeutic agent release. Miller et al.,

Degradation Rates of Oral Resorbable Implants {Polylactates and Polyglycolates: Rate Modification and Changes in PLA/PGA Copolymer Ratios, J. Biomed. Mater. Res., Vol. 11:711-719 (1977).

Alternatively, a sustained or controlled release microsphere preparation is prepared using an in-water drying method, where an organic solvent solution of a biodegradable polymer metal salt is first prepared. Subsequently, a dissolved or dispersed medium of a therapeutic agent is added to the biodegradable polymer metal salt solution. The weight ratio of a therapeutic agent to the biodegradable polymer metal salt may for example be about 1 : 100000 to about 1 :1, preferably about 1 :20000 to about 1 :500 and more preferably about 1 : 10000 to about 1 :500. Next, the organic solvent solution containing the biodegradable polymer metal salt and therapeutic agent is poured into an aqueous phase to prepare an oil/water emulsion. The solvent in the oil phase is then evaporated off to provide microspheres. Finally, these microspheres are then recovered, washed and lyophilized. Thereafter, the microspheres may be heated under reduced pressure to remove the residual water and organic solvent. Other methods useful in producing microspheres that are compatible with a biodegradable polymer metal salt and therapeutic agent mixture are: i) phase separation during a gradual addition of a coacervating agent; ii) an in- water drying method or phase separation method, where an antiflocculant is added to prevent particle agglomeration and iii) by a spray-drying method.

In one embodiment, the present invention contemplates a medium comprising a microsphere or microcapsule capable of delivering a controlled release of a therapeutic agent for a duration of approximately between 1 day and 6 months. In one embodiment, the microsphere or microparticle may be colored to allow the medical practitioner the ability to see the medium clearly as it is dispensed. In another embodiment, the microsphere or microcapsule may be clear. In another embodiment, the microsphere or microparticle is impregnated with a radio-opaque fluoroscopic dye.

Controlled release microcapsules may be produced by using known encapsulation techniques such as centrifugal extrusion, pan coating and air suspension. Such microspheres and/or microcapsules can be engineered to achieve desired release rates. For example, Oliosphere® (Macromed) is a controlled release microsphere system. These particular microsphere's are available in uniform sizes ranging between 5 - 500 μηι and composed of biocompatible and biodegradable polymers. Specific polymer compositions of a microsphere can control the therapeutic agent release rate such that custom-designed microspheres are possible, including effective management of the burst effect. ProMaxx® (Epic Therapeutics, Inc.) is a protein-matrix delivery system. The system is aqueous in nature and is adaptable to standard pharmaceutical delivery models. In particular, ProMaxx® are bioerodible protein microspheres that deliver both small and macromolecular drugs, and may be customized regarding both microsphere size and desired release characteristics.

In one embodiment, a microsphere or microparticle comprises a pH sensitive encapsulation material that is stable at a pH less than the pH of the internal mesentery. The typical range in the internal mesentery is pH 7.6 to pH 7.2. Consequently, the microcapsules should be maintained at a pH of less than 7. However, if pH variability is expected, the pH sensitive material can be selected based on the different pH criteria needed for the dissolution of the microcapsules. The encapsulated compound, therefore, will be selected for the pH environment in which dissolution is desired and stored in a pH preselected to maintain stability. Examples of pH sensitive material useful as encapsulants are Eudragit® L-100 or S-100 (Rohm GMBH), hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate phthalate, and cellulose acetate trimellitate. In one embodiment, lipids comprise the inner coating of the microcapsules. In these compositions, these lipids may be, but are not limited to, partial esters of fatty acids and hexitiol anhydrides, and edible fats such as triglycerides. Lew C. W., Controlled-Release pH Sensitive Capsule And Adhesive System And Method. United States Patent No. 5,364,634 (herein incorporated by reference).

In one embodiment, the present invention contemplates a microparticle comprising a gelatin, or other polymeric cation having a similar charge density to gelatin (i.e., poly-L- lysine) and is used as a complex to form a primary microparticle. A primary microparticle is produced as a mixture of the following composition: i) Gelatin (60 bloom, type A from porcine skin), ii) chondroitin 4-sulfate (0.005% - 0.1%), iii) glutaraldehyde (25%, grade 1), and iv) l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC hydrochloride), and ultra-pure sucrose (Sigma Chemical Co., St. Louis, Mo.). The source of gelatin is not thought to be critical; it can be from bovine, porcine, human, or other animal source.

Typically, the polymeric cation is between 19,000-30,000 daltons. Chondroitin sulfate is then added to the complex with sodium sulfate, or ethanol as a coacervation agent.

Following the formation of a microparticle, a therapeutic agent is directly bound to the surface of the microparticle or is indirectly attached using a "bridge" or "spacer". The amino groups of the gelatin lysine groups are easily derivatized to provide sites for direct coupling of a compound. Alternatively, spacers (i.e., linking molecules and derivatizing moieties on targeting ligands) such as avidin-biotin are also useful to indirectly couple targeting ligands to the microparticles. Stability of the microparticle is controlled by the amount of

glutaraldehyde-spacer crosslinking induced by the EDC hydrochloride. A controlled release medium is also empirically determined by the final density of glutaraldehyde-spacer crosslinks.

In one embodiment, the present invention contemplates microparticles formed by spray-drying a composition comprising fibrinogen or thrombin with a therapeutic agent. Preferably, these microparticles are soluble and the selected protein (i.e., fibrinogen or thrombin) creates the walls of the microparticles. Consequently, the therapeutic agents are incorporated within, and between, the protein walls of the microparticle. Heath et al.,

Microparticles And Their Use In Wound Therapy. United States Patent No. 6,113,948 (herein incorporated by reference). Following the application of the microparticles to living tissue, the subsequent reaction between the fibrinogen and thrombin creates a tissue sealant thereby releasing the incorporated compound into the immediate surrounding area.

One having skill in the art will understand that the shape of the microspheres need not be exactly spherical; only as very small particles capable of being sprayed or spread into or onto a surgical site (i.e., either open or closed). In one embodiment, microparticles are comprised of a biocompatible and/or biodegradable material selected from the group consisting of polylactide, polyglycolide and copolymers of lactide/glycolide (PLGA), hyaluronic acid, modified polysaccharides and any other well known material. Experimental

Example I

Tabecular Meshwork Cell Survival Following Photodvnamic Therapy Trabecular meshwork cell cultures were divided into three groups, wherein the first group was irradiated without a photosensitizer (e.g., Verteporfm (Visudyne)^®) and the second and third group was irradiated by a photodynamic therapy (PDT) after the intraocular injection of two difference doses of Verteporfm (Visudyne)^®. See, Table I.

Seventy percent (70%) of TM cells subjected to PDT without Verteporfin

(Visudyne)^® were observed to incur damage to trabecular meshwork cells as measured by the uptake of Calcein dye. Further, Verteporfin (Visudyne)^® slightly toxic to TM cells without PDT wherein there was 67% survival of TM cells after 2 μg/ml intraocular injection; and 47% survival with 10 μg/ml). PDT after a 2 ^ig/ml administration of Verteporfin (Visudyne) reduced the surviving TM cell percentage to 22%. Furthermore, increasing the Verteporfin (Visudyne) dose to to 10 με/ιτιΐ did not increase the PDT-induced TM cell death in relation to the 2 μg/ml dose.

Table I. Specific Effect Of A Photosensitizer On Trabecular Meshwork Cells.

Other observations show that the surrounding cells (e.g., retinal ganglion cells, epithelia cells etc.) did not show any damage as measured by Calcein dye uptake. Further, the observations show that none of the non-trabecular meshwork cells exhibited any significant inflammation. These data suggest that the intraocular administration of Verteporfin

(Visudyne)^® specifically targets TM cells.

Example II

Photosensitizer Targeting Between Ocular Cell Types

This example examines the effects of both photodynamic therapy (PDT) laser- activated and inactivated verteporfin on different cultured ocular cells. Primary human scleral fibroblasts (hFibro), primary human trabecular meshwork (TM) cells (hTMC), primary porcine TM cells (pTMC), and a human retinal pigment epithelial cell line (ARPE-19 cells) were treated with verteporfin with and without activation by PDT laser. Cell viability was determined by mitochondrial enzyme activity (MTT assay).

Cell Culture Media And Reagents

Fibroblast Medium (FM, ScienCell Research Laboratories, Carlsbad CA) comprised a proprietary basal medium formulation supplemented with 2% fetal bovine serum (FBS), 1% of

fibroblast growth supplement and 1% penicillin/streptomycin. Dulbecco's modified Eagle Medium (DMEM), qualified FBS, penicillin-streptomycin (lOOx solution), and phosphate- buffered saline (PBS) were purchased from hivitrogen/Life Technologies (Grand Island, NY). Rat tail type I collagen was purchased from Becton Dickson Biosciences (BD Biosciences, San Jose CA). The metabolic activity indicator 3 -(4,5- dimethyl-2-thiazoyl)-2,5-diphenyl- 2H-tetrazolium bromide (MTT) was purchased from Sigma Aldrich Corporation (St. Louis, MO). Verteporfin (Visudyne^®, QLT Ophthalmics Inc., Menlo Park CA) came as a lyophilized powder of 15 mg active ingredient in approximately 765 mg of inactive ingredients. Flat- bottom 96-well culture plates were obtained from Corning-Costar (Lowell MA).

Cell Lines And Establishment Of Primary Cell Cultures

ARPE-19, a spontaneously arising retinal pigment epithelia (RPE) cell line, was purchased from American Type Culture Collection (Manassas, VA) and cultured according to the manufacturer's instructions. Primary human trabecular meshwork cells (hTMC), isolated from the juxtacanalicular and corneoscleral regions of the human eye, were purchased from ScienCell Research Laboratories and cultured according to the manufacturer's instructions.

Primary human scleral fibroblasts (hFibro) were isolated from scleral strips taken from a normal donor eye (aged 92 years old) obtained from the San Diego Eye Bank (San Diego, CA). Approval was obtained from the Colorado Multiple Institutional Review Board for the use of human material and the tenets of the Declaration of Helsinki were followed. Scleral strips were weighted down with sterile glass coverslips on a collagen-coated dish and maintained in DMEM containing 15% FBS and antibiotics for approximately 2 weeks. The resulting cells had a classic fibroblast morphology, and were passaged (1:3) into collagen- coated flasks and cultured in FM.

Primary porcine trabecular meshwork cells (pTMC) were isolated from strips of porcine TM as described previously. Ammar et al., "Anti-oxidants protect trabecular meshwork cells from peroxide-induced cell death" Translational Vision Science &

Technology (in press). pTMC were passaged (1 :3) into collagen-coated flasks and cultured in FM.

Cell Culture Conditions

ARPE-19 cells used in these experiments were from the twentieth or twenty- irst passage and were cultured in DMEM containing 10% FBS and antibiotics. Approximately lxl 0⁴ ARPE-19 cells were plated into uncoated 96-well plates 2-3 days before each experiment. hTMC used in these experiments were from the fourth or fifth passages and were cultured in FM. Approximately 5x10³ hTMC were plated into collagen coated 96-well plates 2-3 days before each experiment. hFibro used in these experiments were from the eighth or ninth passages and were cultured in FM. Approximately 7.5x10³ hFibro were plated into collagen coated 96-well plates 2-3 days before each experiment. pTMC used in these experiments were from the fourth or fifth passages and were cultured in FM. Approximately 2.5x10³ pTMC were plated into collagen coated 96-well plates 2-3 days before each experiment. hTMC were cultured on collagen-coated tissue culture dishes and wells in FM.

Verteporfin Treatment Of Cultured Cells

Verteporfm studies were initiated when cultured cells reached >95% confluence, usually 2-3 days post plating. Experiments were performed in duplicate. For initial toxicity studies, a range of verteporfin (0, 0.25, 1, 4, 10 and 25 /ηι1) dissolved in 150 μΐ of the appropriate culture medium was added to hFibro, hTMC, and ARPE-1 cells. The 96-well plates were shielded from light and incubated in a humidified incubator at 37°C and 5% C0₂ for 24 hours. After removal of verteporfin containing media, metabolic activity (MTT) was assayed without exposure to photodynamic therapy (PDT) laser light. For all subsequent toxicity studies of light-activated verteporfin, a single concentration of verteporfin (0.5 μg/ml) was dissolved in 150 μΐ of the appropriate culture medium. hFibro, hTMC, and ARPE-19 cells were exposed to PDT laser under two conditions: i) 'Pretreat' conditions, cells were exposed to verteporfin for 24 hours as above, washed twice in PBS, and cultured in verteporfin-free media for 3 hours before exposure to PDT laser; and ii) 'Cotreat' conditions, cells were exposed to verteporfin for 3 hours and then exposed to PDT laser without a change of culture media. The beam of the PDT laser (VisuLas^® 690S laser; Carl Zeiss Meditec AG, Berlin, Germany) is centered at 688 nm, with more than 90% of its energy between 686 and 690 nm. The laser spot-size of the PDT laser was adjusted to encompass the entire 0.32 cm² bottom area of a 96-well culture well. Cultured cells were exposed to 0, 50 or 100 μΐ/cm² of PDT laser followed by metabolic activity measurements (MTT assay).

Cell Viability/Metabolic Assays

The mitochondrial activity of the cultured cells was determined by MTT assay.

Following verteporfin experiments, media was aspirated and cells were incubated in 100 μΐ, of 0.5 mg/mL MTT dissolved in the appropriate culture media for 1 hour at 37°C. The MTT media was aspirated and the purple formazan reaction product was solubilized by addition of 100 ιΤ DMSO. The absorbance of each sample was read at 540nm in a Synergy™ 4 Multi- Mode Microplate Reader using the Gen5™ Reader Control and Data Analysis Software (BioTek, Winooski, VT). After subtraction of background (A540 of DMSO), data were normalized to the MTT absorbance of the appropriate untreated cell type (100 % Live).

Statistical Analysis

Experiments were performed in duplicate. Mean values for each experimental condition were analyzed by the Student's t-test (Excel, Microsoft, Redmond WA); the level of significance was set at 0.05. Data were reported as the mean ± standard deviation (SD) of n=4 replicates.

Claims

Claims I claim:

1. A non-human mammal comprising at least one symptom of glaucoma and a plurality of damaged ocular cells and a plurality of undamaged ocular cells.

2. The non-human mammal of Claim 1, wherein said plurality of damaged ocular cells a plurality of damaged trabecular meshwork cells.

3. The non-human mammal of Claim 2, wherein said plurality of damaged trabecular meshwork cells block intraocular fluid flow wherein said at least one symptom of glaucoma is increased intraocular pressure.

4. The non-human mammal of Claim 3, wherein said blocked intraocular fluid flow is within an ocular aqueous outflow system.

5. The non-human mammal of Claim 4, wherein said ocular aqueous outflow system comprises an ocular vasculature system.

6. The non-human mammal of Claim 1, wherein said glaucoma comprises neovascular glaucoma.

7. The non-human mammal of Claim 1 , wherein aid plurality of undamaged ocular cells is a plurality of non-trabecular meshwork cells.

8. The non-human mammal of Claim 7, wherein said plurality of undamaged non- trabecular meshwork cells do not exhibit significant inflammation.

9. The non-human mammal of Claim 7, wherein said plurality of undamaged non- trabecular meshwork cells comprises a plurality of retinal ganglion cells.

10. The non-human mammal of Claim 1, wherein at least one of said non-human mammal is selected from the group consisting of a mouse, a rat, a rabbit, a guinea pig, nonhuman primate.

11. A method, comprising;

a) providing;

i) a non-human mammal comprising a plurality of ocular cells; ii) a photosensitizer capable of generating free radicals; and iii) a light source capable of specifically targeting said plurality of ocular cells;

b) administering said photosensitizer to said non-human mammal; and c) irradiating said non-human mammal with said light source resulting in a plurality of damaged ocular cells and a plurality of undamaged ocular cells with minimal singificant inflammation.

12. The method of Claim 11 , wherein said administering comprises an intraocular or periocular injection of said photosensitizer.

13. The method of Claim 11, wherein said administering comprises a topical

administration of said photosensitizer.

14. The method of Claim 11 , wherein said plurality of damaged ocular cells a plurality of damaged trabecular meshwork cells.

15. The method of Claim 14, wherein said plurality of damaged trabecular meshwork cells block intraocular fluid flow wherein intraocular pressure of the non-human mammal is increased.

16. The method of Claim 15, wherein said blocked intraocular fluid flow is within an ocular aqueous outflow system.

17. The method of Claim 16, wherein said ocular aqueous outflow system comprises an ocular vasculature system.

18. The method of Claim 15, wherein said increased intraocular pressure induces glaucoma in the non-human mammal.

19. The method of Claim 18, wherein said glaucoma comprises neovascular glaucoma.

20. The method of Claim 11 , wherein said plurality of undamaged ocular cells is a plurality of undamaged non-trabecular meshwork cells.

21. The method of Claim 20, wherein said plurality of undamaged non-trabecular meshwork cells do not exhibit significant inflammation.

22. The method of Claim 20, wherein said plurality of undamaged non-trabecular meshwork cells comprises a plurality of retinal ganglion cells.

23. The method of Claim 11 , wherein said non-human mammal is selected from the group consisting of a mouse, a rat, a rabbit, a guinea pig.

24. The method of Claim 11 , wherein said photosensitizer comprises a benzoporphyrin derivative.

25. The method of Claim 24, wherein said benzoporphyrin derivative is a mono acid derivative.

26. The method of Claim 11 , wherein said irradiation comprises a photodynamic therapy.

27. The method of Claim 11 , wherein said light source comprises a wavelength ranging between approximately 400 - 900 nm.

28. The method of Claim 11 , wherein said light source wavelength is about 689 nm.

29. The method of Claim 11 , wherein said irradiating comprises a fiuence ranging

2

between approximately 0.0000001-90 Joules/cm .

30. The method of Claim 11, wherein said irradiating comprises a ffuence of about 100 Joules/cm .

31. The method of Claim 11 , wherein said irradiating comprises an irradiance of about 1800 mW/cm².

32. The method of Claim 11 , wherein said irradiating ranges between approximately 90- 360 degrees of the trabecular meshwork.

33. The method of Claim 11 , wherein said administered photosensitizer ranges between approximately 0.5 - 5 μg/kg.

34. The method of Claim 11, wherein said administered photosensitizer is 1 μg/kg.

35. A method, comprising:

a) providing;

i) a non-human mammal exhibiting at least one symptom of

glaucoma, wherein said non-human mammal comprises a plurality of damaged ocular cells and a plurality of undamaged ocular cells;

ii) a composition comprising a test compound capable of being administered to said non-human mammal;

b) administering said test compound to the non-human mammal; and c) determining whether said at least one symptom of glaucoma is reduced.

36. The method of Claim 35, wherein said administering comprises an intraocular, periocular, intra-episcleral vessels, and intra-aqueous vein injection of said test compound.

37. The method of Claim 35, wherein said administering comprises a topical

administration of said test compound.

38. The method of Claim 35, wherein said test compound is a therapeutic agent.

39. The method of Claim 38, wherein said therapeutic agent is selected from the group consisting of an adrenergic beta blocker, a carbonic anhydrase inhibitor, a prostaglandin analogue, a rho kinase inhibitor, an endothelin antagonist, a virus, a protein, and a nucleic acid sequence.

40. The method of Claim 35, wherein said test compound is a pharmaceutically acceptable formulation.

41. The method of Claim 35, wherein said test compound comprises a photosensitizer.

42. The method of Claim 35, wherein said photosensitizer comprises a benzoporphyrin derivative.

43. The method of Claim 35, wherein said benzoporphyrin derivative is a mono acid derivative.

44. The method of Claim 35, wherein said at least one symptom of glaucoma is reduced for at least 4 weeks.

45. The method of Claim 35, wherein said at least one symptom of glaucoma is reduced for at least 5 weeks.

46. The method of Claim 35, wherein said at least one symptom of glaucoma is reduced for at least 6 weeks.

47. The method of Claim 35, wherein said at least one symptom of glaucoma is reduced for at least 7 weeks.

48. The method of Claim 35, wherein said at least one symptom of glaucoma is reduced for or at least 8 weeks.

49. The method of Claim 35, wherein at least one of said non-human mammal is selected from the group consisting of a mouse, a rat, a rabbit, a guinea pig.

50. The method of Claim 35, wherein said at least one symptom of glaucoma comprises increased intraocular pressure.

51. The method of Claim 35, wherein said plurality of undamaged ocular cells do not exhibit significant inflammation.

52. The method of Claim 35, wherein said plurality of undamaged non-trabecular meshwork cells comprises a plurality of retinal ganglion cells.

53. The method of Claim 35, wherein said irradiation comprises a photodynamic therapy.

54. The method of Claim 53, wherein said photodynamic therapy comprises a light source having a wavelength ranging between approximately 400 - 900 rrm.

55. The method of Claim 54, wherein said light source wavelength is about 689 nm.

56. The method of Claim 54, wherein said photodynamic therapy comprises irradiating said non-human animal with a fluence ranging between approximately 0.0000001-90 Joules/cm².

57. The method of Claim 56, wherein said irradiating comprises a fluence of about 100 Joules/cm².

58. The method of Claim 56, wherein said irradiating comprises an irradiance of about 1800 mW/cm².

59. The method of Claim 56, wherein said irradiating ranges between approximately 90- 360 degrees of the trabecular meshwork.

60. The method of Claim 41 , wherein said administered photosensitizer ranges between approximately 0.5 - 5 μg/kg.

61. The method of Claim 41, wherein said administered photosensitizer is 1 μg/kg.

62. A method, comprising:

a) providing;

i) a mammal comprising a trabecular meshwork having a blocked fluid outflow; and

ii) a photosensitizer capable of activation by irradiation;

b) administering said photosensitizer to said mammal; and c) irradiating said photosensitizer under conditions such that said

trabecular meshwork is remodeled thereby alleviating said blocked fluid outflow.

63. The method of Claim 62, wherein said photosensitizer comprises a benzoporphyrin derivative.

64. The method of Claim 62, wherein said irradiation comprises photodynamic therapy.

65. The method of Claim 62, wherein said photosensitizer generates free radicals.

66. The method of Claim 65, wherein said free radicals stimulate trabecular meshwork cell low density lipoprotein receptors.

67. The method of Claim 66, wherein said low density lipoprotein receptor stinuilation releases a plurality of proteins.

68. The method of Claim 66, wherein said low density lipoprotein receptor stimulation does not result in cell death.

69. The method of Claim 67, wherein said plurality of proteins facilitate said remodeling.

0. The method of Claim 62, wherein said mammal is a human.