Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B3/00—Apparatus for testing the eyes; Instruments for examining the eyes
  • A61B3/10—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions
  • A61B3/102—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions for optical coherence tomography [OCT]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B3/00—Apparatus for testing the eyes; Instruments for examining the eyes
  • A61B3/10—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions
  • A61B3/12—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions for looking at the eye fundus, e.g. ophthalmoscopes
  • A61B3/1241—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions for looking at the eye fundus, e.g. ophthalmoscopes specially adapted for observation of ocular blood flow, e.g. by fluorescein angiography
• • 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
  • A61F9/00821—Methods or devices for eye surgery using laser for coagulation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
  • A61P27/06—Antiglaucoma agents or miotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
  • A61P27/14—Decongestants or antiallergics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • 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
  • A61F2009/00844—Feedback systems
  • A61F2009/00851—Optical coherence topography [OCT]
• • 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
  • A61F2009/00863—Retina
• • 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
  • A61F2009/00865—Sclera
• • 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
  • A61F2009/00872—Cornea

Definitions

• the present invention relates to peptides for inhibiting angiogenesis.
• the present invention also relates to methods of inhibiting angiogenesis and methods of treating disorders associated with VEGF-induced vascular permeability using the peptides of the invention.
• Microtubule (MT) cytoskeleton provides an important control-point of endothelial barrier regulation; however, the role of this key cytoskeleton element has not been well studied.
• the MT stabilizing drug taxol has been shown to attenuate the endothelial vascular leakage in mice models of lung inflammation suggesting that MTs may be important in mediating increased lung vascular permeability.
• taxol displays a general toxicity that makes it an inconvenient drug for doctors and their patients.
• Microtubule end binding proteins are highly conserved microtubule plus-end tracking accessory factors that bind to growing microtubules (MTs) and suppress MT catastrophic events.
• Two such end binding proteins, EB 1 and EB3 play roles in regulating endothelial cytoskeletal dynamics and cell shape change, the primary determinants of the permeability of endothelial barrier.
• Ca 2+ is a highly versatile second messenger that regulates endothelial permeability and vascular homeostasis.
• P(PLCP) phospholipase C
• P(PLCP2) downstream of proinflammatory mediators promotes hydrolysis of phosphotidyl inositolbisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG).
• IP 3 stimulates Ca 2+ release from IP 3 -sensitive intracellular stores, i.e., the endoplasmic reticulum (ER).
• ER endoplasmic reticulum
• the depletion of Ca 2+ from ER stores is mediated by activation of IP 3 R on the ER membrane and leads to transient increase in intracellular Ca 2+ .
• TRPC transient receptor potential canonical
• SOC store-operated Ca 2+ channels
• PKC a protein kinase Ca
• VEGF Vascular Endothelial Growth Factor
• RhoA in turn facilitates phosphorylation-induced inhibition of myosin light chain phosphatase (MLCP) by activating Rho kinase (ROCK).
• MLCP myosin light chain phosphatase
• ROCK Rho kinase
• the inhibition of MLCP is accompanied by the Ca 2+ /calmodulin-dependent activation of MLCK that leads to phosphorylation of MLC and induces acto-myosin contraction in response to pro-inflammatory mediators such as thrombin and histamine and growth factors.
• MT cytoskeleton The integrity of MT cytoskeleton is required for IP 3 -induced Ca 2+ release from ER stores. Alteration of MT dynamics by MT destabilizing or MT stabilizing agents nocodazole, colchicine and taxol inhibits IP 3 -gated release of Ca 2+ , suggesting that MT dynamics are required for full activation of IP 3 R.
• the MT cytoskeleton is involved in remodeling of ER, thus ensuring organization and propagation of Ca 2+ waves in response to external stimuli.
• the ER attaches to and elongates together with MT growing ends though direct interaction of EB l and EB3 with stromal interaction molecule 1 (STIM1).
• VEGF Vascular endothelial growth factor
• VEGF Vascular endothelial growth factor
• the extravascular fibin serves as a matrix that supports the ingrowth of new blood vessels and other mesenchymal cells that generate mature, vascularized stroma.
• Novel therapies are needed to prevent VEGF-induced vascular permeability and to inhibit angiogenesis.
• VEGF vascular endothelial growth factor
• Loss of the inner endothelial blood-retinal barrier and the resultant macular edema and damage are the major causes of eye disorder and blindness in the elderly population. At present, these conditions, also known as age-related macular degeneration (AMD), are incurable.
• AMD age-related macular degeneration
• the neovascular form of AMD is characterized by growth of the blood vessels from the choroid, which penetrate through Bruch's membrane into the subretinal area.
• the peptide may comprise
• KFARLWTEIPTAIT (SEQ ID NO: 1), FTEIPTI (SEQ ID NO: 3), a fragment thereof, or a variant thereof.
• the peptide may also consist of KFARLWTEIPTAIT (SEQ ID NO: 1), FTEIPTI (SEQ ID NO: 3), a fragment thereof, or a variant thereof.
• the variant may comprise a conservative substitution.
• the variant may comprise any peptide sequence containing Ser/Thr-x-Ile-Pro sequence (SEQ ID NO: 5), minimal EB binding consensus motif sequence.
• the peptide may be conjugated to a fatty acid, i.e. myristoylated or linked to a carrier peptide.
• the carrier peptide may be antennapedia peptide (AP), antennapedia peptide, penetratin peptide, TAT, tranportan or polyarginine.
• the peptide may be part of a pharmaceutical formulation, which may include a pharmaceutically acceptable excipient.
• the invention provides for methods of inhibiting angiogenesis comprising administering to a patient in need thereof an isolated peptide comprising the amino acid sequence of KFARLWTEIPTAIT (SEQ ID NO: l), FTEIPTI (SEQ ID NO: 3) or a fragments thereof.
• the methods include administering a therapeutically effective amount of a peptide of the invention, such as an amount effective to inhibit angiogenesis.
• the methods include administering pharmaceutical compositions comprising a therapeutically effective amount of a peptide of the invention.
• the invention provides for a method of treatment in which the patient in need is suffering from cancer or a disorder associated with VEGF-induced permeability, such as visual impairment or vision loss (blindness), macular degeneration, central retinal vein occlusion, branch retinal venin occlusion proliferative diabetic retinopathy, neovascular age-related macular degeneration (AMD), retinopathy of prematurity, ischemic retinopathy, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic macular edema, diabetic retina ischemia, diabetic retinal edema, and proliferative diabetic retinopathy, rubeosis iridis, neovascular glaucoma, retinoblastoma, uveitis and
• the invention also provides for methods of treating a disorder associated with VEGF-induced vascular permeability comprising administering to a patient in need thereof an isolated peptide comprising the amino acid sequence of KFARLWTEIPTAIT (SEQ ID NO: l), FTEIPTI (SEQ ID NO: 3) or a fragments thereof.
• the methods include
• the methods include administering pharmaceutical compositions comprising a therapeutically effective amount of a peptide of the invention.
• the invention provides for a method of treatment in which the patient in need is suffering from cancer or a disorder associated with VEGF- induced permeability, such as visual impairment or vision loss (blindness), macular degeneration, central retinal vein occlusion, branch retinal venin occlusion proliferative diabetic retinopathy, neovascular age-related macular degeneration (AMD), retinopathy of prematurity, ischemic retinopathy, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic macular edema, diabetic retina ischemia, diabetic retinal edema, and proliferative diabetic retinopathy, rubeosis iridis, neovascular glaucoma, retinoblastoma, uveitis and corneal graft neovascularization.
• the peptide administered may be linked to a carrier peptide such as antennapedia peptide (AP), antennapedia peptide, penetratin peptide, TAT, tranportan or polyarginine.
• a carrier peptide such as antennapedia peptide (AP), antennapedia peptide, penetratin peptide, TAT, tranportan or polyarginine.
• the peptide administered may be conjugated to a fatty acid, e.g. myristoylated.
• the isolated peptide comprising the amino acid sequence of KFARLWTEIPTAIT (SEQ ID NO: l), FTEIPTI (SEQ ID NO: 3) or a fragments thereof is administered in combination with one or more VEGF inhibitors, wherein "VEGF inhibitors” refer to anti-VEGF antibodies and fragments thereof, anti-VEGF receptor (anti- VEGFR) antibodies and fragments thereof, antagonistic peptides and small molecules which inhibit the activity or signaling pathway of VEGF and/or VEGFR.
• VEGF inhibitors refer to anti-VEGF antibodies and fragments thereof, anti-VEGF receptor (anti- VEGFR) antibodies and fragments thereof, antagonistic peptides and small molecules which inhibit the activity or signaling pathway of VEGF and/or VEGFR.
• VEGF inhibitors include Bevacizumab (Avastin), Ranibizumab (Lucentis), Pegaptanib (Macugen), Aflibercept (Eylea), Sorafenib (Nexvar), Sunitinib (Sutent), Pazopanib (Votrient), Axitinib (Inlyta), PTK787/ZK222584, ZD-6474, SU6668, PD-547,632, VEGF-Trap, CEP-7055, NM- 3, or SU11248.
• the isolated peptide comprising the amino acid sequence of KFARLWTEIPTAIT (SEQ ID NO: l), FTEIPTI (SEQ ID NO: 3) or a fragments thereof may be administered in combination with laser treatment for eye disease, wherein "eye disease” refers to visual impairment or vision loss (blindness), macular degeneration, central retinal vein occlusion, branch retinal venin occlusion proliferative diabetic retinopathy, neovascular age-related macular degeneration (AMD), retinopathy of prematurity, ischemic retinopathy, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic macular edema, diabetic retina ischemia, diabetic retinal edema, and proliferative diabetic retinopathy, rubeosis iridis, neovascular gla
• eye disease refers to visual impairment or vision loss (blind
• the isolated peptide comprising the amino acid sequence of KFARLWTEIPTAIT (SEQ ID NO: 1), FTEIPTI (SEQ ID NO: 3) or a fragments thereof may be administered in combination with a steroid or any current method treatment for eye disease.
• the isolated peptide of the invention, VEGF inhibitor, steroid or any other treatment may be administered by
• intravitreal injection or topically such as in the form of an eye drop.
• the invention also provides for an use of an isolated peptide for the preparation of a medicament for the inhibition of angiogenesis in a patient in need, wherein the peptide comprises the amino acid sequence of KFARLWTEIPTAIT (SEQ ID NO: 1), FTEIPTI (SEQ ID NO: 3) or a fragments thereof.
• the uses of the invention include use of the isolated peptide of the invention for the preparation of a medicament comprising a therapeutically effective amount of a peptide of the invention, such as an amount effective to inhibit angiogenesis.
• the invention provides for us of the isolated peptide of the invention for the preparation of a medicament comprising a composition comprising a therapeutically effective amount of a peptide of the invention.
• the invention provides use of the isolated peptide of the invention for the preparation of a medicament to administer to a suffering from cancer or a disorder associated with VEGF- induced permeability, such as visual impairment or vision loss (blindness), macular degeneration, central retinal vein occlusion, branch retinal venin occlusion proliferative diabetic retinopathy, neovascular age- related macular degeneration (AMD), retinopathy of prematurity, ischemic retinopathy, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic macular edema, diabetic retina ischemia, diabetic retinal edema, and proliferative diabetic retinopathy, rubeosis iridis, neovascular glaucoma, retinoblastoma, uveitis and corneal graft neovascularization.
• the invention also provides for the use of an isolated peptide for the preparation of a medicament for the treatment of a VEGF-induced vascular disorder, wherein the peptide comprises the amino acid sequence of KFARLWTEIPTAIT (SEQ ID NO: 1), FTEIPTI (SEQ ID NO: 3) or a fragments thereof.
• the uses of the invention include use of the isolated peptide of the invention for the preparation of a medicament comprising a therapeutically effective amount of a peptide of the invention, such as an amount effective to inhibit VEGF- induced vascular permeability.
• the invention provides for us of the isolated peptide of the invention for the preparation of a medicament comprising a composition comprising a therapeutically effective amount of a peptide of the invention.
• the invention provides for use of the peptide of the invention for the preparation of a medicament to administer to a subject suffering from cancer or a disorder associated with VEGF-induced permeability, such as visual impairment or vision loss (blindness), macular degeneration, central retinal vein occlusion, branch retinal venin occlusion proliferative diabetic
• retinopathy neovascular age-related macular degeneration (AMD), retinopathy of AMD.
• AMD neovascular age-related macular degeneration
• ischemic retinopathy intraocular neovascularization
• corneal neovascularization corneal neovascularization
• retinal neovascularization choroidal neovascularization
• diabetic macular edema diabetic macular edema
• diabetic retina ischemia diabetic retinal edema
• proliferative diabetic retinopathy rubeosis iridis, neovascular glaucoma, retinoblastoma, uveitis and corneal graft neovascularization.
• the isolated peptide administered may be linked to a carrier peptide such as antennapedia peptide (AP), antennapedia peptide, penetratin peptide, TAT, tranportan or polyarginine.
• a carrier peptide such as antennapedia peptide (AP), antennapedia peptide, penetratin peptide, TAT, tranportan or polyarginine.
• the isolated peptide administered may be conjugated to a fatty acid, e.g.
• the isolated peptide comprising the amino acid sequence of KFARLWTEIPTAIT (SEQ ID NO: 1), FTEIPTI (SEQ ID NO: 3) or a fragments thereof is administered in combination with one or more VEGF inhibitors, wherein "VEGF inhibitors" refer to anti-VEGF antibodies and fragments thereof, anti-VEGFR antibodies and fragments thereof, antagonistic peptides and small molecules which inhibit the activity or signaling pathway of VEGF or VEGFR.
• VEGF inhibitors include Bevacizumab (Avastin), Ranibizumab (Lucentis), Pegaptanib (Macugen), Aflibercept (Eylea), Sorafenib (Nexvar), Sunitinib (Sutent), Pazopanib (Votrient), Axitinib (Inlyta), PTK787/ZK222584, ZD-6474, SU6668, PD-547,632, VEGF-Trap, CEP-7055, NM-3, or SU11248.
• the medicament comprising the amino acid sequence of KFARLWTEIPTAIT (SEQ ID NO: l), FTEIPTI (SEQ ID NO: 3) or fragments thereof may be administered in combination with laser treatment for eye disease, wherein "eye disease” refers to visual impairment or vision loss (blindness), macular degeneration, central retinal vein occlusion, branch retinal venin occlusion proliferative diabetic retinopathy, neovascular age-related macular degeneration (AMD), retinopathy of prematurity, ischemic retinopathy, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic macular edema, diabetic retina ischemia, diabetic retinal edema, and proliferative diabetic retinopathy, rubeosis iridis, neovascular glaucoma,
• the medicament may be administered in combination with a steroid or any current method of treatment for eye disease.
• the medicament may be administered by intravitreal injection or topically such as in the form of an eye drop.
• the invention provides for an isolated peptide for the inhibition of angiogenesis, wherein the peptide comprises the amino acid sequence of KFARLWTEIPTAIT (SEQ ID NO: 1), FTEIPTI (SEQ ID NO: 3) or a fragments thereof.
• the invention also provides for a composition comprising a therapeutically effective amount of a peptide of the invention for the inhibition of angiogenesis.
• the invention provides for an isolated peptide or a composition comprising a therapeutically effective amount of the peptide for inhibition of angiogenesis in a subject a suffering from cancer or a disorder associated with VEGF- induced permeability, such as visual impairment or vision loss (blindness), macular degeneration, central retinal vein occlusion, branch retinal venin occlusion proliferative diabetic retinopathy, neovascular age-related macular degeneration (AMD), retinopathy of prematurity, ischemic retinopathy, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic macular edema, diabetic retina ischemia, diabetic retinal edema, and proliferative diabetic retinopathy, rubeosis iridis, neovascular glaucoma, retinoblastoma, uveitis and corneal graft
• the invention also provides for an isolated peptide for use in inhibition of angiogenesis in a patient suffering from a disorder associated with VEGF-induced vascular permeability wherein the isolated peptide comprises the amino acid sequence of
• KFARLWTEIPTAIT SEQ ID NO: l
• FTEIPTI SEQ ID NO: 3
• the invention also provide for a composition comprising a therapeutically effective amount of an isolated peptide of the invention for the inhibition of VEGF-induced vascular permeability.
• the invention provides for an isolated peptide or a composition comprising a therapeutically effective amount of the peptide for inhibition of VEGF-induced permeability in a subject a suffering from cancer or a disorder associated with VEGF-induced
• permeability such as visual impairment or vision loss (blindness), macular degeneration, central retinal vein occlusion, branch retinal venin occlusion proliferative diabetic
• retinopathy neovascular age-related macular degeneration (AMD), retinopathy of AMD.
• AMD neovascular age-related macular degeneration
• ischemic retinopathy intraocular neovascularization
• corneal neovascularization corneal neovascularization
• retinal neovascularization choroidal neovascularization
• diabetic macular edema diabetic macular edema
• diabetic retina ischemia diabetic retinal edema
• proliferative diabetic retinopathy rubeosis iridis, neovascular glaucoma, retinoblastoma, uveitis and corneal graft neovascularization.
• the invention also provides for an isolated peptide for the treatment for a disorder associated with VEGF-induced vascular permeability.
• a disorder associated with VEGF-induced vascular permeability is visual impairment or vision loss (blindness), macular degeneration, central retinal vein occlusion, branch retinal venin occlusion proliferative diabetic
• retinopathy neovascular age-related macular degeneration (AMD), retinopathy of prematurity, ischemic retinopathy, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic macular edema, diabetic retina ischemia, diabetic retinal edema, and proliferative diabetic retinopathy, rubeosis iridis, neovascular glaucoma, retinoblastoma, uveitis and corneal graft neovascularization.
• AMD neovascular age-related macular degeneration
• any of the peptides of the invention are used for inhibiting angiogenesis or for treating a disorder associated with VEGF-induced vascular permeability may be linked to a carrier peptide such as antennapedia peptide (AP), antennapedia peptide, penetratin peptide, TAT, tranportan or polyarginine.
• a carrier peptide such as antennapedia peptide (AP), antennapedia peptide, penetratin peptide, TAT, tranportan or polyarginine.
• any of the isolated peptides of the invention for use in inhibiting angiogenesis or treating a disorder associated with VEGF-induced vascular permeability may be conjugated to a fatty acid, e.g. myristoylated.
• VEGF inhbitors refer to s anti- VEGF antibodies and fragments thereof, anti-VEGFR antibodies and fragments thereof, antagonistic peptides and small molecules that inhibit the activity or signaling pathway of VEGF or VEGFR.
• VEGF inhibitors include Bevacizumab (Avastin),
• peptide or the VEGFR is administered by intravitreal injection or topically such as in the form of an eye drop.
• eye disease refers to visual impairment or vision loss (blindness), macular degeneration, central retinal vein occlusion, branch retinal venin occlusion proliferative diabetic retinopathy, neovascular age-related macular degeneration (AMD), retinopathy of prematurity, ischemic retinopathy, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic macular edema, diabetic retina ischemia, diabetic retinal edema, and proliferative diabetic retinopathy, rubeosis iridis, neovascular glaucoma, retinoblastoma, uveitis and corneal graft neovascularization.
• eye disease refers to visual impairment or vision loss (blindness), macular degeneration, central retinal vein occlusion, branch retinal venin occlusion proliferative diabetic
• the isolated peptides or compositions of the invention may be administered in combination with a steroid or any current method treatment for eye disease.
• the isolated peptides or compositions of the invention may be administered by intravitreal injection or topically such as in the form of an eye drop.
• Figure 1 shows the role of EB3 in inflammatory-induced hyper-permeability of endothelial barrier.
• EB3 establishes transient interactions of growing MT ends with the IP 3 R 3 , sensitizes the IP 3 R 3 and positively regulates both Ca 2+ release from stores and SOC-dependent Ca 2+ entry during inflammation. This results in amplification of Ca 2+ signaling and increased permeability through PKCa-mediated phosphorylation of pl20-catenin and acto-myosin contractility.
• Figure 2 shows an alignment of human IP 3 receptors (794-814 aa of IP 3 R 3 type 3) with EB binding motif (highlighted).
• the IP 3 R 3 peptide (SEQ ID NO: 1) is shown below the alignment.
• Figure 3 shows a ribbon representation of EB3 structure (magenta) and IP 3 R 3 derived peptide (SEQ ID NO: 1) docked into EB3 hydrophobic binding groove of EB3; 180° rotation is shown.
• the IP 3 R 3 derivative peptide was docked using a Z-Dock program in conjunction with Discovery Studio 3.0 software.
• the binding energy between the peptide and EB3 was calculated to be -68.882 kcal/mol.
• FIG. 4 shows IP 3 R 3 peptide (SEQ ID NO: 1) inhibits Ca 2+ release from ER in response to PAR-1 activation.
• Figure 5 shows a ribbon representation of EB3 in the complex with EBIN (SEQ ID NO: 3) and IP 3 R 3 peptide (SEQ ID NO: 1).
• the computed binding energy is -68.882 and - 60.251 for IPR and EBIN, respectively.
• Figure 6A-6B Panel A shows subcuteanous vascular leakage of albumin-bound Evans Blue Dye, which was induced by intradermal injection of VEGF, and Panel B quantifies the vascular leakage as measured spectrophotometrically at 620nm.
• Figure 7A-7D Panel A shows that EBIN inhibited tubulogenesis in matrigel coated wells (scale bar 200 ⁇ ).
• Panel C shows hemematoxylin and eosin (HE) staining of in vivo matrigel plugs.
• HE hemematoxylin and eosin
• Group 1 was treated with control peptide and group 3 was treated with Myr-EBIN at 0, 36 and 60 hrs; group 2 received only 36 and 60 hour treatment.
• Figure 8A-8B shows the effect of EBIN treatment on the tumor growth curve and neovascularization.
• Figure 9 shows an overview of the animal model for choroidal neovascularization (CNV) induction in: (a) Cross section view of the eye demonstrating the laser beam focused on pigment epithelium of retina to induce laser burn and rupture of Bruch's membrane, (b) Rupture of Bruch's membrane induces proliferation of blood vessel in choroid and CNV lesion into retina.
• CNV choroidal neovascularization
• Figure 10 shows an outline of the schedule for the Laser-induced CNV, Ocular Coherence Tomography (OCT), Fundus Fluorescein Angiography, treatment and tissue harvest for groups 1-3 (as set out in Table 4).
• FIG 11 show the effect of EBIN treatment on CNV.
• Area of leakage correlates with the lesion size
• CNV lesion is detected by staining for isolectin B4 using flat-mount of retinal pigment epithelium/ choroid/ sclera.
• FIG. 12 shows the effects of 7-days acute toxicity study for EBIN. Representative images of Fundus Fluorescein Angiography (a) and corresponding Optical Coherence Tomography (b) at day 8 of intravitreal treatment with EBIN (1 ⁇ g/eye). Note, EBIN forms small crystals/precipitates inside of various humor; no visible changes in retinal vasculature and retinal pigment epithelium, choroid, sclera were detected.
• the inventors have made the surprising discovery that peptides derived from the EB 3 -interacting domain of inositol 1,4,5-trisphosphate (IP 3 ) receptor type 3 (IP 3 R 3 ) reduce the interaction between End Binding Protein 3 (EB3) and IP 3 R 3 and inhibit VEGF-induced vascular permeability or VEGF-induced microvascular leakage.
• IP 3 inositol 1,4,5-trisphosphate
• IP 3 R 3 IP 3 receptor type 3
• the peptides of the invention demonstrate barrier-protective properties in various inflammatory diseases and demonstrate anti- angiogenic properties in vitro and in vivo.
• IP 3 R 3 contains EB binding consensus motif, Ser/Thr-x-Ile-Pro (SxIP) (SEQ ID NO: 5).
• SxIP Ser/Thr-x-Ile-Pro
• a short peptide based on IP 3 R 3 sequence shows high binding activity for EB3 (see Example 1).
• each intervening number there between with the same degree of precision is explicitly contemplated.
• the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9 and 7.0 are explicitly contemplated.
• Angiogenesis refers to the process through which new blood vessels form from pre-existing vessels.
• cytokines and extracellular matrix proteases induce tissue remodeling in preparation for migration of endothelial cells from existing vessels to form new vessels.
• “Fragment” as used herein may mean a portion of a reference peptide or
• Identity as used herein in the context of two or more polypeptide or nucleotide sequences, may mean that the sequences have a specified percentage of residues or nucleotides that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation.
• Peptide or “polypeptide” as used herein, may refer to a linked sequence of amino acids and may be natural, synthetic, or a modification or combination of natural and synthetic.
• Substantially identical may mean that a first and second protein or nucleotide sequence are at least 50%-99% identical over a region of 6-100 or more amino acids nucleotides.
• Treating each may mean to alleviate, suppress, repress, eliminate, prevent or slow the appearance of symptoms, clinical signs, or underlying pathology of a condition or disorder on a temporary or permanent basis.
• Preventing a condition or disorder involves administering a agent of the present invention to a subject prior to onset of the disease.
• Suppressing a condition or disorder involves administering a agent of the present invention to a subject after induction of the condition or disorder but before its clinical appearance.
• Repressing the condition or disorder involves administering a agent of the present invention to a subject after clinical appearance of the disease.
• a therapeutically effective amount depends on the condition of a subject and the specific compound administered. The term refers to an amount effective to achieve a desired clinical effect. A therapeutically effective amount varies with the nature of the condition being treated, the length of time that activity is desired, and the age and the condition of the subject, and ultimately is determined by the health care provider. In one aspect, a
• therapeutically effective amount of a peptide or composition is an amount effective to inhibit, reduce or prevent VEGF- induced vascular permeability and/or angio genesis.
• a “variant” means a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity.
• Representative examples of “biological activity” include the ability to bind to End Binding protein, a toll-like receptor (TLR) and to be bound by a specific antibody.
• Variant may also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity.
• a conservative substitution of an amino acid i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change.
• hydropathic index of amino acids As understood in the art. Kyte et ah, J. Mol. Biol. 157: 105-132 (1982).
• the hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of +2 are substituted.
• the hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function.
• hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity.
• U.S. Pat. No. 4,554,101 incorporated fully herein by reference.
• Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art.
• Substitutions may be performed with amino acids having hydrophilicity values within +2 of each other. Both the hyrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.
• peptide which may comprise the amino acid sequence
• KFARLWTEIPTAIT SEQ ID NO: 1
• KFARLWAEIPTAIT SEQ ID NO: 2
• FTEIPTI SEQ ID NO: 3
• the variant may comprise a conservative substitution.
• the peptide may comprise an EB binding consensus motif sequence, such as the EB binding consensus sequence of IP 3 R 3 , or a fragment thereof.
• the EB binding consensus sequence of IP 3 R 3 may be Ser/Thr-x-Ile-Pro (SEQ ID NO: 5).
• the peptide may consist of KFARLWTEIPTAIT (SEQ ID NO: 1), KFARLWAEIPTAIT (SEQ ID NO:2), FTEIPTI (SEQ ID NO: 3), a consensus sequence comprising Ser/Thr-x-Ile-Pro (SEQ ID NO: 5), a peptide disclosed in Table 1 herein, a fragment of the foregoing, or a conservative variant of the foregoing.
• the variant may comprise any peptide sequence containing Ser/Thr-x-Ile-Pro sequence (SEQ ID NO: 5), minimal EB binding consensus motif sequence.
• the peptide may comprise the amino acid sequence of KFARLWTEIPTAIT (SEQ ID NO: 1), KFARLWAEIPTAIT (SEQ ID NO:2) (also referred to herein as IP 3 R 3 Peptide), FTEIPTI (SEQ ID NO: 3) (also referred to herein as End Binding Inhibitory Peptide, or "EBIN”), a peptide disclosed in Table 1 herein, a fragment thereof, or a variant thereof, wherein the peptide or a polypeptide comprising the peptide is 7 amino acid residues, 8 amino acid residues, 9, amino acid residues, 10, amino acid residues, 11, amino acid residues, 12 amino acid residues, 13 amino acid residues, 14 amino acid residues, 15 amino acid residues, 16 amino acid residues, 17 amino acid residues, 18 amino acid residues, 19, amino acid residues, 20 amino acid residues, 21 amino acid residues, 22 amino acid residues, 23 amino acid residues, 24 amino acid residues, 25 amino acid residues,
• the peptide may be modified in that the amino acid sequence has one or more amino acid substitutions, amino acid insertions, amino acid deletions, carboxy terminal truncation, or an amino terminal truncation.
• the peptide might also be glycosylated, phosphorylated, sulfated, glycosylated, animated, carboxylated, acetylated.
• the C-terminal may be modified with amidation, addition of peptide alcohols and aldehydes, addition of esters, addition of p- nitorailine and thioesteres and multipelantigens peptides.
• the N-terminal and side chains may be modified by PEGylation, acetylation, formylation, addition of a fatty acid, addition of benzoyl, addition of bromoacetyl, addition of pyroglutamyl, succinylation, addition of tetrabutyoxycarbonyl and addition of 3-mercaptopropyl, acylations (e.g. lipopeptides), biotinylation, phosphorylation, sulfation, glycosylation, introduction of maleimido group, chelating moieties, chromophores and flurophores.
• PEGylation e.g. lipopeptides
• the peptide may be conjugated to a fatty acid, e.g. the peptide is myristoylated.
• a fatty acid may be conjugated to the N-terminus of the peptide, such fatty acids include caprylic acid (C8), capric acid (CIO), lauric acid (C12), myristic acid (C14), palmitic acid (C16) or stearic acid (C18) etc.
• cysteines in peptides can be palmitoylated.
• the peptide may be conjugated or linked to another peptide, such as a carrier peptide.
• the carrier peptide may facilitate cell-penetration, such as antennapedia peptide, penetratin peptide, TAT, tranportan or polyarginine.
• the peptides may be cyclic.
• the peptide disclosed herein may be cyclized by adding a single or multiple disulfide bridges, adding a single or multiple amide bonds between the N- and C-terminus, heat to tail cyclization, side chain cyclization (e.g. lactam bridge, thioester), hydrocarbon-stabled peptides.
• the peptide may be labeled with heavy isotope labeling, e.g. 15 N, 13 C, FITC, conjugation to a carrier protein, conjugation to imaging agent, FRET substrates with a flurophore/quencher pair, peptide-DNA conjugation, peptide-RNA conjugation and peptide- enzyme labeling.
• heavy isotope labeling e.g. 15 N, 13 C, FITC
• conjugation to a carrier protein conjugation to imaging agent
• FRET substrates with a flurophore/quencher pair conjugation to a carrier protein
• peptide-DNA conjugation peptide-RNA conjugation
• peptide-RNA conjugation peptide- enzyme labeling.
• the peptide may be within a fusion protein such as fused to a polypeptide or peptide which promotes oligomerization, such as a leucine zipper domain; a polypeptide or peptide which increases stability or to increase half-life, such as an immunoglobulin constant region; and a polypeptide which has a therapeutic activity different from peptide or the invention, a chemotherapeutic agent, an antibody or protein for tissue specific targeting,
• Fusions can be made either at the amino terminus or at the carboxy terminus of the polypeptide.
• the fusion proteins may be direct with no linker or adapter molecule or indirect using a linker or adapter molecule.
• a linker or adapter molecule may be one or more amino acid residues, typically up to about 20 to about 50 amino acid residues.
• a linker or adapter molecule may also be designed with a cleavage site for a protease to allow for the separation of the fused moieties.
• the peptide may be fused to one or more domains of an Fc region of human IgG to increase the half-life of the peptide or the addition of a Fab variable domain to shorten the half-life of the peptide.
• Angiogeneis is associated with tumor growth, cancer progression and metastasis, blindness and macular degeneration, diabetic retinopathy, to name a few.
• the invention provides for method of inhibiting angiogenesis involved in tumor growth, cancer progression and metastasis.
• the invention also provides for methods of treating, inhibiting and preventing tumor growth and cancers such as, e.g. brain tumors (including meningiomas, glioblastoma multiforme, anaplastic astrocytomas, cerebellar astrocytomas, other high-grade or low-grade astrocytomas, brain stem gliomas,
• oligodendrogliomas oligodendrogliomas, mixed gliomas, other gliomas, cerebral neuroblastomas,
• craniopharyngiomas diencephalic gliomas, germinomas, medulloblastomas, ependymomas, choroid plexus tumors, pineal parenchymal tumors, gangliogliomas, neuroepithelial tumors, neuronal or mixed neuronal glial tumors), lung tumors (including small cell carcinomas, epidermoid carcinomas, adenocarcinomas, large cell carcinomas, carcinoid tumors, bronchial gland tumors, mesotheliomas, sarcomas or mixed tumors), prostate cancers (including adenocarcinomas, squamous cell carcinoma, transitional cell carcinoma, carcinoma of the prostatic utricle, or carcinosarcomas), breast cancers (including adenocarcinomas or carcinoid tumors), or gastric, intestinal, or colon cancers (including adenocarcinomas, invasive ductal carcinoma, infiltrating or invasive lobular carcinoma,
• Administration of the peptides of the invention may be combined with other cancer therapies, antitumor agents and chemotherapeutic agents such as an aromatase inhibitor, an anti-estrogen, an anti-androgen, a gonadorelin agonist, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a microtubule active agent, an alkylating agent, a retinoid, a carotenoid, a tocopherol, a cyclooxygenase inhibitor, an MMP inhibitor, a mTOR inhibitor, an antimetabolite, a platin compound, a methionine aminopeptidase inhibitor, a
• chemotherapeutic agents such as an aromatase inhibitor, an anti-estrogen, an anti-androgen, a gonadorelin agonist, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a microtubule active agent, an alkylating agent, a reti
• bisphosphonate an antiproliferative antibody, a heparanase inhibitor, an inhibitor of Ras oncogenic isoforms, a telomerase inhibitor, a proteasome inhibitor, a compound used in the treatment of hematologic malignancies, a Flt-3 inhibitor, an Hsp90 inhibitor, a kinesin spindle protein inhibitor, a MEK inhibitor, an antitumor antibiotic, a nitrosourea, a compound targeting/decreasing protein or lipid kinase activity, a compound targeting/decreasing protein or lipid phosphatase activity, any further anti- angiogenic compound, and combinations thereof.
• antitumor agents include, but are not limited to, azacitidine, axathioprine, bevacizumab, bleomycin, capecitabine, carboplatin, chlorabucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fluorouracil, gemcitabine, herceptin, idarubicin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, tioguanine, retinoic acid, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, receptor tyrosine kinase inhibitors, and combinations thereof. Additional
• the invention provides for methods of treating macular degeneration including wet and dry macular degeneration comprising administering the peptide of the invention.
• Wet macular degeneration occurs when abnormal blood vessels grow behind the macula. These vessels are fragile and can leak fluid and blood, which result in scarring of the macula and raise the potential for rapid, severe damage. Bruch's membrane breaks down, usually near drusen deposits. This is where new blood vessel growth, or neovascularization, occurs. Central vision can become distorted or lost entirely in a short period of time, sometimes within days.
• ocular administration of the peptides of the invention is contemplated.
• administration of the peptides may be combined with the other therapeutic agents such as other antiangiogenic drugs such as Bevacizumab (Avastin), Ranibizumab (Lucentis), Pegaptanib (Macugen), Aflibercept (Eylea), Lodamin (a polymeric formulation of TNP-470), Verteporfin (Visudyne)
• peptides of the invention for treating macular degeneration may be combined with other procedures such as an implantable telescope, laser photocoagulation and macular translocation surgery.
• the invention provides for methods of treating visual impairment or vision loss (blindness), macular degeneration, central retinal vein occlusion, branch retinal venin occlusion proliferative diabetic retinopathy, neovascular age-related macular degeneration (AMD), retinopathy of prematurity, ischemic retinopathy, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic macular edema, diabetic retina ischemia, diabetic retinal edema, and proliferative diabetic retinopathy, rubeosis iridis, neovascular glaucoma, retinoblastoma, uveitis and corneal graft neovascularization.
• the subject may be a mammal, which may be a human. Prior to diagnosis, the subject may be at risk for cancer because of exposure to one or more risk factors or have a genetic risk for developing cancer.
• the one or more risk factors may include, for example, the subject having a family history of cancer, age, smoking tobacco, sun exposure, drinking alcoholic beverages, lack of physical activity, obesity and/or dietary deficiency.
• the subject Prior to diagnosis, the subject may be at risk of developing macular degeneration because exposure to one or more risk factors or have a genetic risk for developing macular degeneration.
• the one or more risk factors may include, for example, the subject having a family history of macular degeneration, age, smoking tobacco, prolonged sun exposure, high fat diet, dietary deficiency, high blood pressure, obesity, and/or light color eyes.
• Suitable methods of administering a physiologically-acceptable composition such as a pharmaceutical composition comprising a compound and/or micelle described herein, are well known in the art. Although more than one route can be used to administer a peptide, a particular route can provide a more immediate and more effective reaction than another route. Depending on the circumstances, a pharmaceutical composition comprising the peptide is applied or instilled into body cavities, absorbed through the skin or mucous membranes, ingested, inhaled, and/or introduced into circulation.
• the pharmaceutical composition orally through injection or infusion by intravenous, intratumoral, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, intralesional, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, urethral, vaginal, or rectal means; by controlled, delayed, sustained or otherwise modified release systems; or by implantation devices.
• drug exposure can be optimized by maintaining constant drug plasma concentrations over time. Such a steady-state is generally accomplished in clinical settings by continuous drug infusion at doses depending on the drug clearance and the plasma concentration to be sustained.
• the composition is administered regionally via intratumoral,
• the peptide is administered locally via implantation of a matrix, membrane, sponge, or another appropriate material onto which the desired compound has been absorbed or encapsulated.
• the device is, in one aspect, implanted into any suitable tissue or organ, and delivery of the desired compound is, for example, via diffusion, timed-release bolus, or continuous administration.
• Ocular administration of the peptides may be carried using intraocular implants, intravitreal injections, systemic administration, topical application, nanoparticles,
• the peptide may administered by intravitreal injection or topically such as in the form of an eye drop.
• the peptide may be administered as a monotherapy or simultaneously or metronomically with other treatments, which may be a surgery or removal of a tumor.
• the term "simultaneous” or “simultaneously” as used herein, means that the peptide and other treatment be administered within 48 hours, preferably 24 hours, more preferably 12 hours, yet more preferably 6 hours, and most preferably 3 hours or less, of each other.
• “metronomically” as used herein means the administration of the peptide at times different from the other treatment and at a certain frequency relative to repeat administration.
• the peptide of the invention may be administered with one or more VEGF inhibitors.
• the peptide of the invention may be administered with one or more VEGF inhibitors or in combination with laser treatment for vision loss.
• the peptide may be administered at any point prior to another treatment including about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110 hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr, 90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr, 70 hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50 hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 .
• the peptide may be administered at any point prior to a second treatment of the peptide including about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110 hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr, 90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr, 70 hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50 hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 h
• the peptide may be administered at any point after another treatment including about 1 min, 2 mins., 3 mins., 4 mins., 5 mins., 6 mins., 7 mins., 8 mins., 9 mins., 10 mins., 15 mins., 20 mins., 25 mins., 30 mins., 35 mins., 40 mins., 45 mins., 50 mins., 55 mins., 1 hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr, 16 hr, 18 hr, 20 hr, 22 hr, 24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34 hr, 36 hr, 38 hr, 40 hr, 42 hr, 44 hr, 46 hr, 48 hr, 50 hr, 52 hr, 54 h
• the peptide may be administered at any point prior after a second treatment of the peptide including about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110 hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr, 90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr, 70 hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50 hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 h
• the method may comprise administering the peptide.
• Peptides provided herein may be in the form of tablets or lozenges formulated in a conventional manner.
• tablets and capsules for oral administration may contain conventional excipients may be binding agents, fillers, lubricants, disintegrants and wetting agents.
• Binding agents include, but are not limited to, syrup, accacia, gelatin, sorbitol, tragacanth, mucilage of starch and polyvinylpyrrolidone.
• Fillers may be lactose, sugar, microcrystalline cellulose, maizestarch, calcium phosphate, and sorbitol.
• Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica.
• Disintegrants may be potato starch and sodium starch glycollate.
• Wetting agents may be sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.
• Peptides provided herein may also be liquid formulations such as aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs.
• the peptides may also be formulated as a dry product for constitution with water or other suitable vehicle before use.
• Such liquid preparations may contain additives such as suspending agents, emulsifying agents, nonaqueous vehicles and preservatives.
• Suspending agent may be sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fats.
• Emulsifying agents may be lecithin, sorbitan monooleate, and acacia.
• Nonaqueous vehicles may be edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol. Preservatives may be methyl or propyl p-hydroxybenzoate and sorbic acid.
• the peptides of the invention may be in aqueous formulations for topical administration such as in the form of an eye drop.
• Peptides provided herein may also be formulated as suppositories, which may contain suppository bases such as cocoa butter or glycerides.
• Peptides provided herein may also be formulated for inhalation, which may be in a form such as a solution, suspension, or emulsion that may be administered as a dry powder or in the form of an aerosol using a propellant, such as dichlorodifluoromethane or trichlorofluoromethane.
• Peptides provided herein may also be formulated as transdermal formulations comprising aqueous or nonaqueous vehicles such as creams, ointments, lotions, pastes, medicated plaster, patch, or membrane.
• Peptides provided herein may also be formulated for parenteral administration such as by injection, intratumor injection or continuous infusion.
• Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents including, but not limited to, suspending, stabilizing, and dispersing agents.
• the peptide may also be provided in a powder form for reconstitution with a suitable vehicle including, but not limited to, sterile, pyrogen-free water.
• Peptides provided herein may also be formulated as a depot preparation, which may be administered by implantation or by intramuscular injection.
• the peptides may be formulated with suitable polymeric or hydrophobic materials (as an emulsion in an acceptable oil, for example), ion exchange resins, or as sparingly soluble derivatives (as a sparingly soluble salt, for example).
• the method may comprise administering a therapeutically effective amount of the peptide to a patient in need thereof.
• the therapeutically effective amount required for use in therapy varies with the nature of the condition being treated, the length of time desired to activate TLR activity, and the age/condition of the patient. In general, however, doses employed for adult human treatment typically are in the range of 0.001 mg/kg to about 200 mg/kg per day. The dose may be about 0.05 mg/kg to about 10 g/kg per day.
• the desired dose may be conveniently administered in a single dose, or as multiple doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day. Multiple doses may be desired, or required.
• the dosage may be at any dosage such as about 0.05 ⁇ g/kg, 0.06 ⁇ g/kg, 0.07 ⁇ g/kg, 0.08 ⁇ g/kg, 0.09 ⁇ g/kg, 0.1 ⁇ g/kg, 0.2 ⁇ g/kg, 0.3 ⁇ g/kg, 0.4 ⁇ g/kg, 0.5 ⁇ ⁇ ⁇ ⁇ , 0.6 ⁇ g/kg, 0.7 ⁇ g/kg, 0.8 ⁇ g/kg, 0.9 ⁇ g/kg, 1 ⁇ g/kg, 1.5 ⁇ g/kg, 2 ⁇ g/kg, 3 ⁇ g/kg, 4 ⁇ g/kg , 5 ⁇ g/kg , 10 ⁇ g/kg, 15 ⁇ g/kg, 20 ⁇ g/kg, 25 ⁇ g/kg, 50 ⁇ g/kg, 75 ⁇ g/kg, 100 ⁇ g/kg, 125 ⁇ g/kg, 150 ⁇ g/kg, 175 ⁇ g/kg, 200 ⁇ g/kg, 225
• the dosage may be at any dosage such as about 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 225 mg/kg, 250 mg/kg, 275 mg/kg, 300 mg/kg, 325 mg/kg, 350 mg/kg, 375 mg/kg, 400 mg/kg, 425 mg/kg, 450 mg/kg, 475 mg/kg, 500 mg/kg, 525 mg/kg, 550 mg/kg, 575 mg/kg, 600 mg/kg, 625 mg/kg, 650 mg/kg, 675 mg/kg, 700 mg/kg,
• kits which may be used for treating a disorder associated with VEGF-induced vascular permeability or angiogenesis.
• the kit may comprise one or more of the peptides.
• the peptides may be part of a pharmaceutical composition.
• the kit may further comprise instructions for using the kit and conducting the administering the peptide or formulation.
• the kit may also comprise one or more containers, such as vials or bottles, with each container containing a separate reagent.
• the kit may further comprise written instructions, which may describe how to perform or interpret the method described herein.
• IP 3 R 5 contains EB binding consensus motif, Ser/Thr-x-Ile-Pro (SxIP) (SEQ ID NO: 5).
• KFARLWTEIPTAIT SEQ ID NO: 1
• End Binding Inhibitory peptide namely EBIN, was designed based on
• AG value.gtoreq. l Stabilizing residue AG value.
• Table 1 Computed changes in binding free energy after truncation of amino acid residues which surround Thr-x-Pro motif of IP 3 R 3
• Table 2 Computed Changes in binding free energy after mutating each amino acid residue of IP 3 R 3 for alanine: KiF 2 A 3 R 4 L 5 W 6 T 7 E 8 l9PioTiiAi 2 li 3 i 4
• Table 3 Computed Changes in binding free energy after mutating each amino acid residue of EBIN for alanine.
• FIG. 5 demonstrates the interaction between EB3 and EBIN. Similarly to IP 3 R 3 (is shown in yellow stick in FIG. 5), EBIN binds to hydrophobic grove between EB acidic tail and coil-coiled domain. The calculated energy binding of EBIN to EB3 is -60.251 kcal/mol, which is similar to the energy binding between IPR and EB3. Threonine at position 2 of EBIN plays a critical role in binding to the EB3 interface because mutation of this residue to alanine completely abolishes the binding. Therefore, a single amino-acid mutation T ⁇ A peptide, FAEIPTI (SEQ ID NO: 4), was used as a loss-of-binding control.
• T ⁇ A peptide FAEIPTI
• VE-cadherin is the main adhesion protein of inter-endothelial junctions that bridges endothelial cell into continuous monolayer in order to maintain restrictive barrier of the vessel wall to protein rich fluids.
• Both VEGF and Ang2 destabilizes VE-cadherin adhesion either directly, by inducing tyrosine phosphorylation of VE-cadherin and targeting VE- cadherin for internalization and degradation, or indirectly, by mean of disruption of VE- cadherin adhesion due to response to intracellular forces.
• VEGF vascular endothelial growth factor
• Treatment with taxol was performed via intraperitoneal injection at ⁇ /kg body weight for 4 days. Tumor size was measured 3 times a week. As shown in Fig. 8A, a significant delay in the tumor growth in taxol group was observed and reduction in the tumor size in EBIN-treated group was observed after 4 treatments. This effect was rather transient, although, the size of tumors in EBIN-treated group was significantly smaller as compared to control untreated group. Mice treated with 1 ⁇ /kg EBIN developed the tumor at the same rate as untreated mice suggesting that the low dose was not affective.
• CNV laser-induced choroidal neovascularization
• Neovascular AMD is characterized by growth of the blood vessels from the choroid, which penetrate through Bruch's membrane into the subretinal area.
• the mouse model of laser- induced Choroidal neovascularization (CNV) is a well-established model of the exudative form of AMD.
• the disruption of Bruch's membrane by a laser beam promotes the growth of new choroidal vessels into the retina thus mimicking the pathological conditions of AMD ( Figure 9).
• This model has been successfully used in predicting the clinical efficacy of VEGF therapy for neovascular AMD.
• EBIN is tested in murine models of CNV.
• LEAFTM antibody a monoclonal rat antibody against mouse VEGF-A
• Myr-FAEIPTI control peptide
• C57/BL6 mice (6-8 week old) were purchased from Charles River Laboratory and used according to an approved protocol. Mice were anesthetized with a mixture of ketamine/xylazine (100 mg /5 mg/kg IP) and their pupils were dilated with a topical application of Cyclomydril (Alcon Laboratories, Fort Worth, TX).
• the fundus was viewed with an imaging camera, and laser photocoagulation was induced using the image-guided laser system (Micron IV, Phoenix Research Laboratories, Pleasanton, CA).
• Four laser burns at equal distance from the optic nerve were induced one by one in right eye by a green Argon laser pulse with a wavelength of 532 nm, a fixed diameter of 50 ⁇ , duration of 70 ms, and power levels from 210-250 mW.
• Appearance of bubble or a small subretinal hemorrhage (diameter ⁇ 1 mm) at the laser spot serves to indicate rupture of the Bruch' s membrane and as confirmation of laser-induced CNV. This procedure was performed only on the right eye of each mouse.
• Group 1 mice received Myr-control peptide
• group 2 mice were treated with Myr- EBIN
• group 3 mice were treated with the LEAFTM antibody as a positive control. All treatments are delivered as a single injection at time of CNV via intravitreal route as outlined in figure 10.
• Table 4 Treatment groups, drug regimen and endpoint assays for measuring the response to treatment of CNV in mice.
• FIGs 1 la and 1 lb show images of Fundus Fluorescein Angiography (a) and corresponding Optical Coherence Tomography (b) at day 15 post laser photocoagulation (numbers indicate corresponding CNV lesions) for EBIN, anti-VEGF antibody or control peptide treated CNV mice.
• EBIN reduced the CNV lesions similar to anti-VEGF treatment and hence, provides potent alternative to current treatment of eye disease such as macular degeneration.
• the experiments were terminated at day 15, at which time, the animals were sacrificed with ketamine/xylazine (100 mg /5 mg/kg IP) followed by cervical dislocation and eye tissue was collected for immunofluorescent staining and pathological analysis.
• the flat mount preparations of retina/choroid/sclera were used for staining with Alexa594-labled lectin from Bandeiraea simplicifolia (B4) for post-mortem analysis of CNV area ( Figure 11c).
• mice with EBIN significantly reduced both the vascular leakage and CNV lesions compared with control peptide treated mice ( Figure 11).
• the effect of EBIN was similar to LEAFTM treatment suggesting that EBIN might provide a cost effective and efficient alternative to currently available anti-VEGF treatment of AMD such as bevacizumab and aflibercept.
• EBIN is delivered via an eye drop route.
• the treatment starts at one day prior the laser photocoagulation and mice are treated twice daily until 15 days post-laser photocoagulation. The duration of treatment and observation is 15 days.
• EBIN is delivered in combination with LEAFTM antibody via intravitreal injection and/or via an eye drop route. In all cases the LEAFTM antibody is administered via intravitreal injection.
• the EBIN anti-angiogenic efficacy is evaluated by fluorescein angiography and ocular coherence tomography (OCT) at 8 and 15 days post laser-induced CNV.
• OCT ocular coherence tomography
• eye tissue is harvested on day 15.
• Group 1 mice are treated with the LEAFTM antibody as a positive control and group 2 mice receive LEAFTM Purified Rat IgG2a, ⁇ Isotype Ctrl , as a control for group 1.
• Groups 3 and 4 are treated with a Decoy receptor for mouse VEGF (positive control 2) or negative Myr-control peptide, respectively. All LEAFTM antibodies, the Decoy receptor and control peptide are delivered as a single injection at time of CNV via intravitreal route.
• Groups 5 and 6 receive Myr-EBIN via intravitreal route, 0.1 ⁇ g/eye or 1 ⁇ g/eye, respectively.
• Groups 7 and 8 receive Myr-EBIN via eye drops, 0.5 ⁇ g/eye or 5 ⁇ g/eye, twice daily, respectively.
• Group 9 mice are treated with Myr-EBIN (0.1 ⁇ g/eye) in combination with LEAFTM antibody, both delivered via intravitreal route.
• Group 10 are treated with Myr-EBIN eye drops (0.5 ⁇ g/eye) in combination with LEAFTM antibody via intravitreal route.
• Table 5 Future treatment groups, drug regimen and endpoint assays for measuring the response to treatment of CNV in mice.
• the first group is treated with EBIN in the right eye via eye drops delivered twice daily, 5 ⁇ g per eye (10 ⁇ ) and, the second group is treated with intravitreal injection of EBIN in the right eye at the maximum dose, 1 ⁇ g per eye (2 ⁇ ) on day one.
• the intravitreal injection is performed under ketamine/xylazine (100 mg/ 5 mg/kg) anesthesia.
• Both groups are monitored daily for general health including body weight as well as any eye abnormalities including opacity, exophthalmia enophthalmia, conjunctivitis, abnormal secretions or crusting, and corneal ulcers for a period of 8 days. Animals are subsequently subjected to fluorescein angiography and OCT imaging. No toxicity was observed following treatment with EBIN, either with or without CNV induction ( Figure 12).

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Animal Behavior & Ethology (AREA)
• Medicinal Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Pharmacology & Pharmacy (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Organic Chemistry (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Epidemiology (AREA)
• Ophthalmology & Optometry (AREA)
• Immunology (AREA)
• Gastroenterology & Hepatology (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Heart & Thoracic Surgery (AREA)
• Molecular Biology (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Biomedical Technology (AREA)
• Surgery (AREA)
• Radiology & Medical Imaging (AREA)
• Medical Informatics (AREA)
• Vascular Medicine (AREA)
• Genetics & Genomics (AREA)
• Biochemistry (AREA)
• Optics & Photonics (AREA)
• Oncology (AREA)
• Cardiology (AREA)
• Hematology (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Peptides Or Proteins (AREA)

Priority Applications (18)

Applications Claiming Priority (2)

Publications (1)

Family

ID=55543089

Family Applications (1)

Country Status (18)

Families Citing this family (5)

Citations (2)

Family Cites Families (10)

• 2016
  • 2016-03-02 DK DK16710568.3T patent/DK3265110T3/da active
  • 2016-03-02 RU RU2017133101A patent/RU2708375C2/ru active
  • 2016-03-02 ES ES16710568T patent/ES2820710T3/es active Active
  • 2016-03-02 CA CA2977389A patent/CA2977389A1/en active Pending
  • 2016-03-02 WO PCT/US2016/020443 patent/WO2016141053A1/en not_active Ceased
  • 2016-03-02 EP EP16710568.3A patent/EP3265110B1/en active Active
  • 2016-03-02 JP JP2017546208A patent/JP6896641B2/ja active Active
  • 2016-03-02 MX MX2017011170A patent/MX2017011170A/es unknown
  • 2016-03-02 PL PL16710568T patent/PL3265110T3/pl unknown
  • 2016-03-02 US US15/058,938 patent/US9675660B2/en active Active
  • 2016-03-02 KR KR1020177024830A patent/KR20170122762A/ko not_active Ceased
  • 2016-03-02 SG SG11201706799YA patent/SG11201706799YA/en unknown
  • 2016-03-02 CN CN201680012874.6A patent/CN107427549B/zh not_active Expired - Fee Related
  • 2016-03-02 BR BR112017018665-9A patent/BR112017018665A2/pt active Search and Examination
  • 2016-03-02 AU AU2016226264A patent/AU2016226264B2/en active Active
• 2017
  • 2017-05-22 US US15/602,096 patent/US10064911B2/en active Active
  • 2017-08-13 IL IL253966A patent/IL253966B/en active IP Right Grant
  • 2017-08-29 PH PH12017501549A patent/PH12017501549B1/en unknown
  • 2017-08-31 ZA ZA2017/05934A patent/ZA201705934B/en unknown

Patent Citations (2)

Non-Patent Citations (4)

Also Published As

Similar Documents

Legal Events