WO2013059095A1 - Utilisation d'agents photosensibles pour cibler le système de débit sortant aqueux de l'œil - Google Patents

Utilisation d'agents photosensibles pour cibler le système de débit sortant aqueux de l'œil Download PDF

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Publication number
WO2013059095A1
WO2013059095A1 PCT/US2012/060036 US2012060036W WO2013059095A1 WO 2013059095 A1 WO2013059095 A1 WO 2013059095A1 US 2012060036 W US2012060036 W US 2012060036W WO 2013059095 A1 WO2013059095 A1 WO 2013059095A1
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Prior art keywords
cells
glaucoma
photosensitizer
trabecular meshwork
ocular
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PCT/US2012/060036
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English (en)
Inventor
Malik Kahook
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The Regents Of The University Of Colorado, A Body Corporate
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Publication of WO2013059095A1 publication Critical patent/WO2013059095A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0613—Apparatus adapted for a specific treatment
    • A61N5/062—Photodynamic therapy, i.e. excitation of an agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F9/00—Methods or devices for treatment of the eyes; Devices for putting-in contact lenses; Devices to correct squinting; Apparatus to guide the blind; Protective devices for the eyes, carried on the body or in the hand
    • A61F9/007—Methods or devices for eye surgery
    • A61F9/0079—Methods or devices for eye surgery using non-laser electromagnetic radiation, e.g. non-coherent light or microwaves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/409—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having four such rings, e.g. porphine derivatives, bilirubin, biliverdine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F9/00—Methods or devices for treatment of the eyes; Devices for putting-in contact lenses; Devices to correct squinting; Apparatus to guide the blind; Protective devices for the eyes, carried on the body or in the hand
    • A61F9/007—Methods or devices for eye surgery
    • A61F9/008—Methods or devices for eye surgery using laser
    • A61F2009/00861—Methods or devices for eye surgery using laser adapted for treatment at a particular location
    • A61F2009/00868—Ciliary muscles or trabecular meshwork
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F9/00—Methods or devices for treatment of the eyes; Devices for putting-in contact lenses; Devices to correct squinting; Apparatus to guide the blind; Protective devices for the eyes, carried on the body or in the hand
    • A61F9/007—Methods or devices for eye surgery
    • A61F9/008—Methods or devices for eye surgery using laser
    • A61F2009/00885—Methods or devices for eye surgery using laser for treating a particular disease
    • A61F2009/00891—Glaucoma

Definitions

  • the present invention is related to methods and compositions for treating glaucoma, creating animal glucoma models and/or screening therapeutic compounds to treat glaucoma.
  • glaucoma treatment may use a photodynamic therapy (PDT) in combination with a photosensitizer.
  • PDT photodynamic therapy
  • Trabecular meshwork cells may be targeted to improve fluid outflow.
  • retinal ganglion cells may be targeted to preserve these cells from PDT
  • LDL Low density lipoprotein
  • TM trabecular meshwork
  • IOP intraocular pressure
  • 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.
  • the present invention is related to methods and compositions for treating glaucoma, creating animal glucoma models and/or screening therapeutic compounds to treat glaucoma.
  • 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.
  • 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.
  • PDT photodynamic therapy
  • Trabecular meshwork cells may be targeted to improve fluid outflow.
  • 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
  • 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.
  • the plurality of damaged ocular cells comprise a plurality of damaged trabecular meshwork cells.
  • the plurality of damaged trabecular meshwork cells block intraocular fluid flow wherein the at least one symptom of glaucoma is increased intraocular pressure.
  • the blocked intraocular fluid flow is within an ocular aqueous outflow system.
  • the ocular aqueous outflow system comprises an ocular vasculature system.
  • the glaucoma comprises neovascular glaucoma.
  • the plurality of undamaged ocular cells comprise a plurality of non-trabecular meshwork cells.
  • 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.
  • 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.
  • 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.
  • the administering comprises an intraocular injection of the photosensitizer.
  • the photosensitizer capable of generating free radicals
  • a light source capable of specifically targeting the plurality of ocular cells
  • the administering comprises an intraocular injection of the photosensitizer.
  • the photosensitizer capable of generating free radicals
  • a light source capable of specifically targeting the plurality of ocular cells
  • the photosensitizer is verteporfin.
  • the administering comprises a topical administration of the photosensitizer.
  • the plurality of damaged ocular cells comprise a plurality of damaged trabecular meshwork cells.
  • the plurality of damaged trabecular meshwork cells block intraocular fluid flow wherein intraocular pressure of the non-human mammal is increased.
  • the blocked intraocular fluid flow is within an ocular aqueous outflow system.
  • the ocular aqueous outflow system comprises an ocular vasculature system.
  • the increased intraocular pressure induces glaucoma in the non-human mammal.
  • the glaucoma comprises neovascular glaucoma.
  • 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.
  • the photosensitizer comprises a benzoporphyrin derivative. In one embodiment, the photosensitizer is verteporfin. In one embodiment, the
  • the irradiation comprises a photodynamic therapy.
  • 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.
  • 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/cm 2 . In one embodiment, the irradiating comprises an irradiance preferably, but not limted to, of about 1800 mW/cm 2 .
  • the irradiating ranges preferably, but not limted to, between approximately 90-360 degrees of the trabecular meshwork.
  • the administered photosensitizer ranges preferably, but not limted to, between approximately 0.5 - 5 ⁇ g/kg. In one embodiment, the administered photosensitizer is 1 ⁇ g kg.
  • 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.
  • 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.
  • 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.
  • the at least one test compound is in a pharmaceutically acceptable formulation. In one embodiment, the at least one test compound comprises a photosensitizer.
  • 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.
  • 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.
  • 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/cm 2 . In one embodiment, the irradiating comprises a fluence of about 100 Joules/cm 2 , 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 ⁇ g ⁇ g. In one embodiment, the administered photosensitizer is 1 ⁇ g/kg.
  • 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
  • the photosensitizer to said mammal; c) irradiating said photosensitizer under conditions such that said trabecular meshwork is remodeled thereby alleviating said blocked fluid outflow.
  • the photosensitizer comprises a benzoporphyrin derivative.
  • the photosensitizer is verteporfin.
  • the irradiation comprises
  • the photosensitizer generates free radicals.
  • the free radicals stimulate trabecular meshwork cell low density lipoprotein receptors.
  • the low density lipoprotein receptor stimulation releases a plurality of proteins.
  • the low density lipoprotein receptor stimulation does not result in cell death.
  • the plurality of proteins facilitate the remodeling.
  • the mammal is a human.
  • 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.
  • the photosensitizer comprises a benzoporphyrin derivative.
  • the photosensitizer is
  • the irradiation comprises photodynamic therapy.
  • the mammal is a human, h one embodiment, the at least one symptom comprises increased intraocular pressure.
  • 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
  • the irradiating step can include a fluence of about 100 Joules/cm 2 . In other cases the fluence maybe 0.0000001-90 Joules/cm 2 . In other forms the fluence maybe more or less than this.
  • the irradiating step can include an irradiance of about 1800 mW/cm 2 .
  • 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.
  • 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.
  • photosensitizer e.g., verteporfin
  • a benzoporphyrin derivative photosensitizer for example, a benzoporphyrin derivative mono acid photosensitizer
  • 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/cm 2 . In other cases the fluence maybe 0.0000001-90 Joules/cm 2 . In other forms the fluence maybe more or less than this.
  • the irradiating step can include an irradiance of about 1800 mW/cm 2 .
  • 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.
  • 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.
  • the photosensitizer interacts with a trabecular meshwork endothelial cell low density lipoprotein receptor.
  • the photosensitizer comprises a benzoporphyrin derivative.
  • the photosensitizer is verteporfin.
  • the irradiation comprises photodynamic therapy.
  • the method further comprises administering a plurality of intraocular depots containing a photosensitizer and other active ingredients in depots within the eye.
  • 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.
  • 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.
  • 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.
  • the photosensitizer interacts with a retinal ganglion cell.
  • the photosensitizer comprises a benzoporphyrin derivative.
  • the photosensitizer is verteporfln.
  • the irradiation comprises
  • 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.
  • the photosensitizer comprises a benzoporphyrin derivative. In one embodiment, the photosensitizer is verteporfin. In one embodiment, the irradiation comprises
  • the present invention contemplates a composition comprising a photosensitizer attached to a low density lipoprotein receptor ligand and a therapeutic agent.
  • 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).
  • the composition is a
  • 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).
  • * p ⁇ 0.05 vs. 0 ⁇ g/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).
  • * p ⁇ 0.05 vs. both ⁇ ⁇ g/ml + 0 y /cm ' and 'Pretreat + 50 ⁇ /cm '.
  • At risk for 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.
  • these conditions may result from influences that include, but are not limited to, behavioral, emotional, chemical, biochemical, or environmental influences.
  • ⁇ ективное amount 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 LD 5 o/ED 5 o. 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 ED 50 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.
  • symptom refers to any subjective or objective evidence of disease or physical disturbance observed by the subject.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • inhibitory compound 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.
  • injury denotes a bodily disruption of the normal integrity of tissue structures.
  • the term is intended to encompass surgery.
  • the term is intended to encompass irritation, significant inflammation, infection, and the development of fibrosis.
  • 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.
  • drug 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.
  • 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.
  • administered 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.
  • local tissue administration i.e., for example, extravascular placement
  • oral ingestion i.e., for example, extravascular placement
  • transdermal patch i.e., for example, extravascular placement
  • topical i.e., for example, inhalation, suppository etc.
  • intraocular injections and/or topical administration are preferable.
  • affinity refers to any attractive force between substances or particles that causes them to enter into and remain in chemical combination.
  • 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.
  • derived from refers to the source of a compound or sequence.
  • a compound or sequence may be derived from an organism or particular species.
  • a compound or sequence may be derived from a larger complex or sequence.
  • pharmaceutically refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier 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.
  • purified 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.
  • substantially purified 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).
  • purified to homogeneity is used to include compositions that have been purified to
  • a purified composition is not intended to mean that some trace impurities may remain.
  • 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.
  • 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.
  • 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.
  • small organic molecule refers to any molecule of a size comparable to those organic molecules generally used in pharmaceuticals.
  • 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.
  • derivative 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.
  • a nucleic acid derivative would encode a polypeptide which retains essential biological characteristics.
  • biological activity refers to any molecule having structural, regulatory or biochemical functions.
  • 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.
  • binding component molecule of interest
  • agent of interest ligand
  • receptor receptor
  • binding component 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.
  • micromolecule refers to any molecule of interest havin a high molecular weight.
  • biopolymers having a high molecular weight would be comprised of greater than 100 amino acids, nucleotides or sugar molecules long.
  • binding 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.
  • the present invention is related to methods and compositions for treating glaucoma, creating animal glucoma models and/or screening therapeutic compounds to treat glaucoma.
  • glaucoma treatment may use a photodynamic therapy (PDT) in combination with a photosensitizer.
  • PDT photodynamic therapy
  • Trabecular meshwork cells may be targeted to improve fluid outflow.
  • retinal ganglion cells may be targeted to preserve these cells from PDT
  • LDL Low density lipoprotein
  • 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.
  • PDT photodynamic therapy
  • TM trabecular meshwork
  • IOP intraocular pressure
  • 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.
  • the present invention contemplates administering a
  • photosensitizer i.e., for example, a benzoporphyrin derivative, for example, a
  • 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).
  • 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.
  • 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.
  • PDT photodynamic therapy
  • 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.
  • the eye has been described as an organ which reacts to light for several purposes.
  • 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.
  • 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.
  • 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).
  • Intraocular pressure i.e., for example, glaucoma
  • Intraocular pressure may increase either when too much aqueous humor fluid is produced or by decreased aqueous humor outflow.
  • the major drainage structures for aqueous humor 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.
  • ECM extracellular matrix
  • SC inner wall endothelial cells form giant vacuoles in response to AH flow, as well as intracellular and paracellular pores.
  • minipores that are covered with a diaphragm are observed.
  • TM outflow resistance Modulation of TM cell tone by the action of their actomyosin system affects TM outflow resistance.
  • 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
  • a retinal ganglion cell 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
  • 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,
  • 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.
  • retinal ganglion cells 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.
  • retinal ganglion cells Excitation of retinal ganglion cells results in an increased firing rate while inhibition results in a depressed rate of firing.
  • 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.
  • SCN suprachiasmatic nucleus
  • 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.
  • EW Edinger-Westphal nucleus
  • 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).
  • IOP intraocular pressure
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • photosensitizers are commercially available for clinical use, such as Photofrin ® , Verteporfin (Visudyne ® ), Levulan ® , Foscan ® , Metvix ® , Hexvix ® , CysviewTM, 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.
  • 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.
  • the physics, biophysics, and technology of photodynamic therapy Physics in Medicine and .3 ⁇ 4o/ogy 53(9): R61-R109.
  • TM cells trabecular meshwork cells.
  • PDT trabecular meshwork
  • Such a targeted therapy may have use for a variety of ocular diseases.
  • 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.
  • PDT Photodynamic therapy
  • 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).
  • hFibro primary human scleral fibroblasts
  • TM primary human trabecular meshwork cells
  • pTMC primary porcine TM cells
  • ARPE-19 cells a human retinal pigment epithelial cell line
  • the present invention contemplates treatment of ocular diseases by targeting light-activation of photosensitizers at specific ocular cell types.
  • the data presented below was collected according to the methods described in Example II.
  • 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 C0 2 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.
  • 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 ⁇ (100.6% ⁇ 7.8), 1 ⁇ (91.4% ⁇ 9.6) or 4 ⁇ (86.7% ⁇ 8.4%).
  • a small but significant decrease in cell viability occurred in ARPE-19 cells treated for 24 hours with 10 ⁇ verteporfin (86.5% ⁇ 3.0%) which decreased to 58.4% ⁇ 2.6% with 25 ⁇ g/ml verteporfin.
  • Light Activation Of Verteporfin Increases Its Toxicity To Selected Cultured Ocular Cells
  • pTMC, hTMC and ARPE-19 cells were incubated with 0.5 ⁇ g/ml of verteporfin at 37 °C in a humidified C02 incubator. After 3 hours, cells in verteporfm-containing media were exposed to 50 ⁇ /cm 2 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 ⁇ /cm 2 PDT light (0 ⁇ g/ml + 100 ⁇ /cm 2 ).
  • hFibro, pTMC, hTMC and ARPE-19 cells were incubated with 0.5 ⁇ g ml of verteporfin at 37 °C in a humidified C0 2 incubator. After 24 hours verteporfin-containing media was removed, the cells were washed twice and then incubated in fresh cell culture
  • 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.
  • leucocytes demonstrate an LD 50 in the range of 0.01 to 0.02 ⁇ g/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.
  • 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 'Pretreat' experiments shown herein indicate that cultured cells can internalize inactive verteporfin. See, Figure 5.
  • 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.
  • LDL low-density lipoprotein
  • 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.
  • PDT therapy targeted to the ocular anterior segment can provide a method for treatment for glaucoma.
  • the PDT laser comprises a refined ophthalmic gonioscopic lens capable of delivering targeted light to the angle while minimizing collateral damage.
  • 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.
  • a photosensitizer e.g., verteporfin
  • the present invention contemplates a method comprising: selective killing of aqueous outflow system cells;
  • stem cell population wherein said stem cells repopulate the aqueious outflow system.
  • the present invention contemplated the treatment of glaucoma in a patient (i.e, for example, a human patient) by the administration of a photosensitizer.
  • 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 is an emerging treatment used to treat various types of medical conditions, such as glaucoma.
  • 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 may be 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.
  • endoscopes and fiber optic catheters to deliver light
  • intravenously- administered photosensitizers i.e., for example, an eyeball
  • photosensitizers, light sources, and treatment parameters require an empirical assessment to determine whether a photosensitizer administration will either induce glaucoma or treat glaucoma.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • a photodynamic therapy-based method is described for treating ocular glaucoma.
  • 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.
  • 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.
  • 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.
  • 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.
  • IOP intraocular pressure
  • Matsubara et al. "Investigating the effect of ciliary body photodynamic therapy in a glaucoma mouse model” Invest Ophthalmol Vis Sci. 47:2498-2507 (2006).
  • 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.
  • neovasculature such as age-related macular degeneration
  • green porphyrins as photoactive agents, preferably as liposomal
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 virus, protein, RNA or DNA based material
  • RNA DNA based material
  • 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.
  • PDT photodynamic therapy
  • the disclosed therapy treats late stages of disease, as characterized by choroidal neovascularization.
  • a photosensitizer is administered intravenously and attaches to lipoprotein receptors, particularly those receptors found in cells undergoing rapid
  • the compound 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.
  • the present invention contemplates a method comprising photodynamic therapy, wherein a medical condition is induced.
  • the photodynamic therapy induces glaucoma.
  • the method further comprises administering a photosensitizer.
  • photodynamic therapy creates a non- human mammal glaucoma experimental model.
  • 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.
  • SOD superoxide dysmutase
  • 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.
  • the present invention contemplates methods for the creation of glaucoma using animal models utilizing rats, mice, rabbits and/or guinea pigs.
  • animal models utilizing rats, mice, rabbits and/or guinea pigs.
  • 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.
  • the present invention contemplates a method comprising photodynamic therapy wherein a trabecular network is remodeled.
  • the photodynamic therapy further comprises administering a photosensitizer
  • POAG primary open angle glaucoma
  • PG pigmentary glaucoma
  • 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
  • CM ciliary muscle
  • TM ciliary muscle
  • TM cells In POAG eyes there also may be a marked loss of TM cells, at places leading to fusion and thickening of trabecular lamellae.
  • TM cells In POAG eyes there also may be a marked loss of TM cells, at places leading to fusion and thickening of trabecular lamellae.
  • 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.
  • 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.
  • TM cells 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.
  • TM trabecular meshwork
  • juxtacanalicular tissue of the chamber angle.
  • ECM extracellular matrix
  • MMPs matrix metalloproteinases
  • the modulation of MMP expression and/or the adminsitration of tissue inhibitors of MMPs may provide an effective treatment in the chamber angle of normal eyes and in primary open-angle glaucoma (POAG) and in exfoliation glaucoma (ExG).
  • POAG primary open-angle glaucoma
  • ExG exfoliation glaucoma
  • 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.
  • Corticosteroid treatment may induce glaucoma and has been reported to remodel the trabecular meshwork ultrastructure either with or without POAG. Johnson et al.,
  • corticosteroids Arch Ophthalmol. 1997 Mar;115(3):375-83.
  • 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.
  • the present invention contemplates a method comprising photodynamic therapy for treating a neovascular glaucoma.
  • the method further comprises a photo sensitizer.
  • Neovascular glaucoma 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.
  • IOP intraocular pressure
  • NVG neovascular glaucoma
  • NVG ischemic central retinal vein occlusion
  • CRVO ischemic central retinal vein occlusion
  • diabetic retinopathy CAD
  • ocular ischemic syndrome CAD
  • one priority should be to try to prevent its development by appropriate management of the causative diseases.
  • NVG CAD
  • 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).
  • 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.
  • neovascular glaucoma 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.
  • VEGF vascular endothelial growth factor
  • 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.
  • the present invention further provides pharmaceutical compositions comprising at least one compound as described above.
  • 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.
  • administration may be topical including, but not limited to, ophthalmic and/or to mucous membranes.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions.
  • 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.
  • 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.
  • 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.
  • 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.
  • dosage is from 0.01 ⁇ g 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.
  • 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 comprise liposomes, nanoparticles, microspheres, nanospheres, microcapsules, and nanocapsules.
  • some microparticles Preferably, some microparticles
  • 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.
  • 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
  • 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.
  • 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.
  • the present invention contemplates cationic and anionic liposomes, as well as liposomes having neutral lipids.
  • cationic liposomes comprise negatively-charged materials by mixing the materials and fatty acid liposomal components and allowing them to charge-associate.
  • 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.
  • 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.
  • compositions of liposomes are broadly categorized into two classifications.
  • 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.
  • compound-delivery liposomes are commercially available. For example, Collaborative Laboratories, Inc. are known to manufacture custom designed liposomes for specific delivery requirements.
  • 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.
  • 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,
  • 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 ⁇ . Techniques are then followed that maintain sustained release integrity during phases of purification, encapsulation and storage. Scott et al., Improving Protein
  • 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.,
  • 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.
  • 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.
  • 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.
  • 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.
  • the microsphere or microparticle may be colored to allow the medical practitioner the ability to see the medium clearly as it is dispensed.
  • the microsphere or microcapsule may be clear.
  • 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.
  • Oliosphere® Macromed
  • Oliosphere® is a controlled release microsphere system. These particular microsphere's are available in uniform sizes ranging between 5 - 500 ⁇ 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.
  • 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.
  • 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.
  • lipids comprise the inner coating of the microcapsules.
  • 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).
  • 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 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.
  • 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.
  • 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.
  • spacers i.e., linking molecules and derivatizing moieties on targeting ligands
  • avidin-biotin are also useful to indirectly couple targeting ligands to the microparticles. Stability of the microparticle is controlled by the amount of
  • a controlled release medium is also empirically determined by the final density of glutaraldehyde-spacer crosslinks.
  • the present invention contemplates microparticles formed by spray-drying a composition comprising fibrinogen or thrombin with a therapeutic agent.
  • 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.
  • microparticles 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).
  • 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.
  • 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.
  • a photosensitizer e.g., Verteporfm (Visudyne) ®
  • PDT photodynamic therapy
  • 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 ⁇ g/ml intraocular injection; and 47% survival with 10 ⁇ g/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 ⁇ / ⁇ did not increase the PDT-induced TM cell death in relation to the 2 ⁇ g/ml dose.
  • This example examines the effects of both photodynamic therapy (PDT) laser- activated and inactivated verteporfin on different cultured ocular cells.
  • PDT photodynamic therapy
  • 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).
  • 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).
  • 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.
  • RPE retinal pigment epithelia
  • VA American Type Culture Collection
  • hTMC Primary human trabecular meshwork cells 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.
  • scleral fibroblasts 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.
  • porcine trabecular meshwork cells 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 &
  • 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 0 4 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 5x10 3 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 7.5x10 3 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 2.5x10 3 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.
  • 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 / ⁇ 1) dissolved in 150 ⁇ 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% C0 2 for 24 hours. After removal of verteporfin containing media, metabolic activity (MTT) was assayed without exposure to photodynamic therapy (PDT) laser light.
  • MTT photodynamic therapy
  • 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 cm 2 bottom area of a 96-well culture well. Cultured cells were exposed to 0, 50 or 100 ⁇ /cm 2 of PDT laser followed by metabolic activity measurements (MTT assay).
  • the mitochondrial activity of the cultured cells was determined by MTT assay.

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Abstract

La présente invention concerne des procédés et des compositions pour traiter un glaucome, créer des modèles de glaucome animal et/ou cribler des composés thérapeutiques pour traiter le glaucome. Par exemple, le traitement du glaucome peut utiliser une thérapie photodynamique (PDT) en combinaison avec un photosensibilisateur. Des cellules de tissu trabéculaire peuvent être ciblées pour améliorer le débit sortant de fluide. En variante, des cellules ganglionnaires de la rétine peuvent être ciblées pour préserver ces cellules contre une neurodégénérescence et réduire le risque de développer des défauts du champ visuel progressives à partir d'un glaucome. Les récepteurs de lipoprotéine de faible densité (LDL) peuvent agir en tant que sites de liaison pour le photosensibilisateur activé et induire la libération de protéines spécifiques sans mort cellulaire.
PCT/US2012/060036 2011-10-19 2012-10-12 Utilisation d'agents photosensibles pour cibler le système de débit sortant aqueux de l'œil WO2013059095A1 (fr)

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CN115531417A (zh) * 2022-11-14 2022-12-30 青岛海尔生物科技有限公司 ITGA6阳性iPSC源小梁网类细胞在制备治疗高眼压疾病药物中的应用及筛选方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115531417A (zh) * 2022-11-14 2022-12-30 青岛海尔生物科技有限公司 ITGA6阳性iPSC源小梁网类细胞在制备治疗高眼压疾病药物中的应用及筛选方法
CN115531417B (zh) * 2022-11-14 2024-04-09 青岛海尔生物科技有限公司 ITGA6阳性iPSC源小梁网类细胞在制备治疗高眼压疾病药物中的应用及筛选方法

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