[go: up one dir, main page]

WO2008103301A2 - Traitement de l'œil utilisant des macrophages et/ou des agents capables d'affecter la morphologie d'un vaisseau sanguin - Google Patents

Traitement de l'œil utilisant des macrophages et/ou des agents capables d'affecter la morphologie d'un vaisseau sanguin Download PDF

Info

Publication number
WO2008103301A2
WO2008103301A2 PCT/US2008/002063 US2008002063W WO2008103301A2 WO 2008103301 A2 WO2008103301 A2 WO 2008103301A2 US 2008002063 W US2008002063 W US 2008002063W WO 2008103301 A2 WO2008103301 A2 WO 2008103301A2
Authority
WO
WIPO (PCT)
Prior art keywords
subject
composition
macrophages
eye
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2008/002063
Other languages
English (en)
Other versions
WO2008103301A3 (fr
Inventor
Bjorn R. Olsen
Qing Li
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Harvard University
Original Assignee
Harvard University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Harvard University filed Critical Harvard University
Publication of WO2008103301A2 publication Critical patent/WO2008103301A2/fr
Publication of WO2008103301A3 publication Critical patent/WO2008103301A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/17—Monocytes; Macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/4285—Lipids; Lipoproteins
    • 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
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule

Definitions

  • the present invention generally relates to treatments of the eye.
  • Retinopathies are diseases characterized by non-inflammatory damage to the retina of the eye. In some cases, degradation of blood vessels within the retina may lead to partial or, in severe cases, total blindness. Retinopathies are caused by a variety of reasons, including diabetes (diabetic retinopathy), arterial hypertension (hypertensive retinopathy), or certain drugs.
  • the present invention generally relates to treatments of the eye using macrophages.
  • the subject matter of the present invention involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.
  • the invention is directed to a method.
  • the method includes an act of administering, to an eye of a subject having or being at risk of a retinopathy such as a non-neovascular retinopathy or a retinopathy characterized by abnormal extracellular matrix production, a composition comprising a macrophage.
  • the method includes an act of administering, to an eye of a subject having or being at risk of a disease characterized by a high level of VEGF in the retina, a composition comprising a macrophage.
  • the method in still another set of embodiments, includes an act of administering, to an eye of a subject, a composition comprising cells that express soluble VEGF receptor.
  • the method includes an act of administering, to an eye of a subject, a composition comprising a VEGF antagonist.
  • the method in another set of embodiments, includes an act of administering, to an eye of a subject, a cellular composition able to produce a VEGF antagonist endogeneously.
  • the subject may be one that has or is at risk of a retinopathy such as a non-neovascular retinopathy or a retinopathy characterized by abnormal extracellular matrix production.
  • the method includes an act of transfecting a macrophage with a gene for expressing soluble VEGF receptor.
  • the invention in another aspect, is directed to an article comprising a macrophage transfected with a gene for expressing soluble VEGF receptor.
  • the invention specifically includes, also, the composition for use in the treatment or prevention of that particular condition, as well as use of the composition for the manufacture of a medicament for the treatment or prevention of that particular condition.
  • the present invention is directed to a method of making one or more of the embodiments described herein.
  • the present invention is directed to a method of using one or more of the embodiments described herein.
  • Figs. IA- IE show LRP5-positive and LRP5-negative macrophages
  • Figs. 2A-2C show a retina treated by macrophages, according to one embodiment of the invention
  • Fig. 3 shows the sequence for VEGF receptor 1 ;
  • Fig. 4 shows various members of the LDL family;
  • Figs. 5A-5B illustrates various reactionpathways
  • Figs. 6A-6D illustrate retinas that are positive and negative for LRP5
  • Figs. 7A-7E illustrate various LRP5 negative retinas
  • Figs. 8A-8D further illustrate various LRP5 negative retinas
  • Figs. 9A-9B illustrate macrophages contained within various retinas
  • Figs. 10A- 1OB further illustrate macrophages contained within various retinas.
  • the present invention generally relates to treatments of the eye and, in particular, to treatments of the eye using macrophages.
  • Diseases including retinopathies (such as diabetic retinopathy or hypertensive retinopathy), osteoporosis pseudoglioma, macular degeneration (such as age-related macular degeneration), or diseases characterized by a high level of VEGF in the retina may be treated in such a fashion.
  • Macrophages are believed to play a role in angiogenesis by phagocytosizing epithelial cells and the like within the retina.
  • the macrophages may produce relatively high levels of soluble VEGF receptor, which may also be useful for reducing overall VEGF concentrations within the retina.
  • the macrophages may also be transfected, e.g., with a gene for expressing soluble VEGF receptor, another VEGF antagonist, and/or other suitable genes, and in certain cases, the macrophages may be combined with other cell-based therapies.
  • One aspect of the present invention is generally directed to treating the eye of a subject with a macrophage. Without wishing to be bound by any theory, it is believed that macrophages play a role in angiogenesis by phagocytosizing epithelial cells and the like within the retina.
  • Subjects with osteoporosis pseudoglioma syndrome often have extremely low bone mass because they may be homozygous for loss-of-function mutations in LRP5, short for LDL receptor-related protein 5, which is a gene involved with phagocytosis, or with receptor-mediated endocytosis.
  • Cells such as macrophages lacking LRP5 are generally unable to phagocytose.
  • many such subjects often are also partially or totally blind.
  • macrophages found within the eye are unable to phagocytose other cells, and are hence unable to play a role in controlling angiogenesis within the eye.
  • Angiogenesis or new blood formation, is believed to involve two general processes: sprouting, or new cell growth (e.g., endothelial cell proliferation, migration, tube formation, basement membrane or extracellular matrix formation, etc.), and regression, or the removal of excess cells (e.g., endothelial cell apoptosis, basement membrane removal, etc.).
  • sprouting or new cell growth
  • regression or the removal of excess cells (e.g., endothelial cell apoptosis, basement membrane removal, etc.).
  • Such regression may be controlled, at least in part, by macrophages which phagocytose other cells. Accordingly, in the retina of the eye, without properly-functioning macrophages (e.g., in subjects lacking LRP5), incorrect angiogenesis may occur, which may result in partial or total blindness.
  • such subjects may be treated by administering a composition comprising macrophages to the subject.
  • the composition may be administered to the retina, and/or to any other suitable location within the eye such that the macrophages are able to reach the retina.
  • the composition may be administered intravitreally.
  • the subject may be, for example, a human or non-human animal.
  • subjects include, but are not limited to, a mammal such as a dog, a cat, a horse, a donkey, a rabbit, a cow, a pig, a sheep, a goat, a rat (e.g., Rattus Norvegicus), a mouse (e.g., Mus musculus), a guinea pig, a hamster, a primate (e.g., a monkey, a chimpanzee, a baboon, an ape, a gorilla, etc.), or the like.
  • a mammal such as a dog, a cat, a horse, a donkey, a rabbit, a cow, a pig, a sheep, a goat, a rat (e.g., Rattus Norvegicus), a mouse (e.g., Mus musculus), a guinea pig, a hamster, a primate (e.g.
  • retinopathy is a disease that results in damage to the retina of the eye. Often, the damage is non-inflammatory.
  • Other disorders or diseases treatable by the invention involve diseases which result in retinal edema and/or retinal ischemia. Examples of such diseases include vein occlusions, sickle cell retinopathy, radiation retinopathy, diabetic retinopathy, VEGF-induced diseases and retinopathy prematurity.
  • diabetic retinopathy is a progressive degeneration of retinal blood vessels and is typically a consequence of diabetes, in particular, diabetes mellitus.
  • diabetes in particular, diabetes mellitus.
  • One important aspect of the disease is retinal edema. Fluid build up from deteriorating blood vessels and capillaries causes edema. As the disease progresses, the damage proliferates and large hemorrhages and retinal detachment can result.
  • retinopathy may also be determined by suitable vision acuity tests, pupil dilation measurements, ophthalmoscopy (visual examination), tonometry (measurement of intraocular fluid pressure), blood pressure measurements, or the like.
  • Other retinal signs that may be determined include capillary obstruction, nonperfusion, leukostasis, formation of vascular lesions and/or proliferation of new blood vessels in association with ischemic areas of the retina.
  • Leukostasis refers to the stasis or non-movement of white blood cells (e.g., leukocytes) in the vasculature.
  • Retinopathies are caused by a variety of reasons, including diabetes (diabetic retinopathy), arterial hypertension (hypertensive retinopathy), or certain drugs. It should be noted that blood vessel morphology in the retina is controlled dynamically, rather than being fixed at birth, and it is believed that macrophages within the retina play an important role in determining the blood vessel morphology, for at least the reasons described herein. In some aspects, a subject may have a disease characterized by an abnormally high level of VEGF in the retina.
  • VEGF vascular endothelial growth factor
  • the retinopathy is macular degeneration (such as age- related macular degeneration).
  • macular degeneration such as age- related macular degeneration.
  • macrophages may be used to treat the wet form of age-related macular degeneration since the macrophages the macrophages are effective at reducing angiogenesis within the retina (see, e.g., R.S.
  • macrophages can be used to treat diseases in which there is incorrect or a reduced level of angiogenesis, such as within the dry form of macular degeneration, diabetic retinopathies, osteoporosis pseudoglioma, and the like.
  • retinopathies i.e., "non-neovascular retinopathies” are retinopathies that are characterized by either normal or reduced levels of angiogenesis within the retina of the eye, unlike the wet form of macular degeneration, which is characterized by excessive angiogenesis and abnormal blood vessel growth.
  • certain retinopathies may be characterized by abnormal extracellular matrix (basement membrane) production, which may cause defects in retinal vessel permeabilities. Examples of such retinopathies characterized by abnormal extracellular matrix production include, but are not limited to, diabetic retiopathy or osteoporosis pseudoglioma.
  • the retinopathy, or other disease characterized by an abnormally high level of VEGF may be treated by administering, to an eye of the subject, a composition comprising macrophages, and/or other cells that express a high level of soluble VEGF receptor (or other VEGF antagonists), for example, soluble VEGF receptor 1 (see Fig. 3, SEQ ID NO: 1), to reduce VEGF concentration.
  • a composition comprising macrophages, and/or other VEGF antagonists
  • soluble VEGF receptor 1 see Fig. 3, SEQ ID NO: 1
  • the retinopathy, or other disease characterized by an abnormally high level of VEGF may be treated by administering, to the eye of the subject, an antagonist to the VEGF, such as soluble VEGF receptor, for example, soluble VEGF receptor 1.
  • VEGF Vascular Endothelial Growth Factor
  • Vascular Endothelial Growth Factor is a signaling protein that has been implicated in both vasculogenesis (de novo formation of blood vessels) and angiogenesis (growth of blood vessels from pre-existing vasculature), and its structure is well known to those of ordinary skill in the art.
  • VEGF includes all homologs, naturally occurring allelic variants, isoforms and precursors of VEGF.
  • sufficient soluble VEGF receptor is produced by the macrophages present to control blood vessel formation and maintenance within the retina.
  • VEGF diseases having abnormally high level of VEGF
  • diseases having abnormally high level of VEGF include, but are not limited to, diabetic retinopathy (as hypoxia in blood vessels may induce VEGF production), various forms of cancer, such as retinal cancer, (as VEGF may stimulate angiogenesis in the cancer), macular degeneration (e.g., age-related macular degeneration, including atrophic age-related macular degeneration or exudative or neovascular age-related macular degeneration), diabetic macular edema, or the like.
  • diabetic retinopathy as hypoxia in blood vessels may induce VEGF production
  • various forms of cancer such as retinal cancer, (as VEGF may stimulate angiogenesis in the cancer)
  • macular degeneration e.g., age-related macular degeneration, including atrophic age-related macular degeneration or exudative or neovascular age-related macular degeneration
  • diabetic macular edema or the like.
  • VEGF antagonists examples include, but are not limited to, soluble neuropilin-1 , sFlt-1, VEGF antibodies, or VEGF antiserum (e.g., prepared using methods known to those of ordinary skill in the art).
  • Other examples of VEGF antagonists include those disclosed in U.S. Patent Application Serial No. 11/651,334, filed January 9, 2007, entitled “Prevention and Treatment of Retinal Ischemia and Edema," by Adamis, et al., published as U.S. Patent Application Publication No. 2008/0019977 on January 24, 2008, incorporated herein by reference.
  • the cells are able to produce a VEGF antagonist endogeneously, i.e., after delivery into the subject.
  • the VEGF antagonist (without cells) may be administered, and the VEGF antagonist need not be produced by a macrophage or other cell, but can be produced using any suitable technique.
  • the macrophage may be one that arises from the subject (i.e., autologous), or from another subject (homologous), or even from another species (heterologous). In some cases, the macrophages may be cultured prior to administration.
  • a biopsy from bone marrow, spleen, or peripheral blood may be cultured to produce macrophages, using standard culture techniques known to those of ordinary skill in the art.
  • the macrophage may be genetically engineered.
  • a sample of the subject is drawn (e.g., blood, bone marrow, spleen, etc.), and macrophages are isolated from the sample.
  • other cells may be isolated (e.g., monocytes), and induced to form macrophages, e.g., using cytokines such as M-CSF (macrophage colony-stimulating factor).
  • monocytes may be isolated from a sample, induced to form macrophages by exposing the monocytes to cytokines, then introduced into a subject (which may be the same or different as the subject from which the monocytes were isolated).
  • the macrophages may be delivered to the eye by any suitable technique.
  • the composition may be administered to the retina, and/or to any other suitable location within the eye such that the macrophages are able to reach the retina. Accordingly, in some cases, the composition may be administered intravitreally. In some cases, an immunosuppressant may also be used, e.g., if the macrophages do not arise from the subject. However, in other cases, no immunosuppressant may be necessary, as the eye in some cases is not as sensitive to foreign cells.
  • the present invention also provides any of the above-mentioned compositions in kits, optionally including instructions for use of the composition for the treatment of a condition characterized by a retinopathy such as a non-neovascular retinopathy, a retinopathy characterized by abnormal extracellular matrix production, or other disease characterized by an abnormally high level of VEGF, etc.
  • a retinopathy such as a non-neovascular retinopathy, a retinopathy characterized by abnormal extracellular matrix production, or other disease characterized by an abnormally high level of VEGF, etc.
  • the kit can include a description of use of the composition for participation in any biological or chemical mechanism disclosed herein.
  • the kits can further include a description of activity of the condition in treating the pathology, as opposed to the symptoms of the condition. That is, the kit can include a description of use of the compositions as discussed herein.
  • the kit also can include instructions for use of a combination of two or more compositions of the invention, or instruction for use of a combination of a composition of the invention and one or more other compounds indicated for treatment. Instructions also may be provided for administering the composition by any suitable technique as previously described, for example, to the retina of the eye (e.g., intravitreally), or via another known route of delivery.
  • kits described herein may also contain one or more containers, which may contain the composition and other ingredients as previously described.
  • the kits also may contain instructions for mixing, diluting, and/or administrating the compositions of the. invention in some cases.
  • the kits also can include other containers with one or more solvents, surfactants, preservative and/or diluents (e.g., normal saline (0.9% NaCl), or 5% dextrose) as well as containers for mixing, diluting or administering the components in a sample or to a subject in need of such treatment.
  • compositions of the kit may be provided as any suitable form, for example, as liquid solutions or as dried powders.
  • the composition may be reconstituted by the addition of a suitable solvent, which may also be provided.
  • the liquid form may be concentrated or ready to use.
  • the solvent will depend on the compound and the mode of use or administration. Suitable solvents for the compositions are well known, for example as previously described, and are available in the literature. The solvent will depend on the compound and the mode of use or administration.
  • Another aspect provides a method of administering any composition of the present invention to a subject.
  • the composition may be administered to the eye, retinal area, choroid area or associated vasculature.
  • the composition can also be administered to the cornea of the eye.
  • the composition may, in some cases, be transported into the eye (e.g., via diffusion or convection) and contact the retina or surrounding vasculature (e.g., eye drops, creams or gels).
  • the compositions of the invention can be applied in a therapeutically effective, pharmaceutically acceptable amount as a pharmaceutically acceptable formulation, for example, a pharmaceutically acceptable carrier such as those described below.
  • a pharmaceutically acceptable carrier such as those described below.
  • the term "effective amount" of a composition refers to the amount necessary or sufficient to realize a desired biologic effect.
  • an effective amount of a composition comprising a macrophage to treat a retinopathy is that amount sufficient to reduce or prevent further degradation of blood vessels within the retina.
  • an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is effective to treat the particular subject.
  • the effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular composition being administered the size of the subject, or the severity of the disease or condition.
  • One of ordinary skill in the art can empirically determine the effective amount of a particular composition and/or other therapeutic agent without necessitating undue experimentation.
  • a composition of the invention is conveniently but not necessarily formulated as an aqueous liquid, and can be administered as eye drops in some cases.
  • each drop which can be generated by conventional dispensing processes, may have a volume of about 10 to about 40 microliters. Any number of droplets may be used, for example from 1 to about 6.
  • an equivalent dose is provided. Such a dose can be administered as needed, but typically administration to the eye 1 to about 6 times per day, in most cases 2 to 4 times a day, provides adequate continuing relief or prevention.
  • compositions of the invention may be chosen to be ophthalmically acceptable.
  • ophthalmically acceptable with respect to a formulation, composition or ingredient herein means having no persistent detrimental effect on the treated eye or the functioning thereof, or on the general health of the subject being treated. It will be recognized that transient effects such as minor irritation or a "stinging" sensation are common with topical ophthalmic administration of drugs and the existence of such transient effects is not inconsistent with the formulation, composition or ingredient in question being "ophthalmically acceptable” as herein defined. However, in some embodiments, the formulations, compositions, and/or ingredients are those that cause no substantial detrimental effect, even of a transient nature.
  • the composition is an aqueous solution, suspension or solution/suspension, which can be presented in the form of eye drops.
  • a desired dosage of the active agent can be metered by administration of a known number of drops into the eye. For example, for a drop volume of about 25 microliters, administration of 1-6 drops will deliver about 25 to about 150 microliters of the composition.
  • Suitable dispensers are illustratively disclosed in International Patent Publication No. WO 96/06581, incorporated herein by reference.
  • the composition is an aqueous solution having no substantial amount of solid particulate matter, whether of the drug, of an excipient, or of both.
  • solid particulate matter if present, can cause discomfort and/or irritation of a treated eye.
  • the aqueous suspension or solution of the present invention may be viscous or mucoadhesive in some embodiments, and in certain instances, both viscous or mucoadhesive.
  • the aqueous suspension or solution/suspension of the invention contains carboxymethylcellulose, a viscosity enhancer and promoter of mucoadhesion.
  • the concentration of carboxymethylcellulose in the aqueous suspension or solution of the present invention may be about 0.1% to about 5%, or about 0.1% to about 2.5% by weight.
  • the carboxymethylcellulose can be in the form of sodium carboxymethylcellulose substituted to a degree that the sodium content of the sodium carboxymethylcellulose is about 1% to about 20%.
  • no more than 3 drops, no more than 2 drops, or no more than 1 drop, each of about 10 microliters to about 40 microliters, or about 15 microliters to about 30 microliters, for example about 20 microliters, should contain the desired dose of the compositions of the invention for administration to an eye. Administration of a larger volume to the eye may risk, in some cases, loss of a significant portion of the applied composition by lacrimal drainage.
  • Any drug having utility as a topical ophthalmic application can be used in co- therapy, co-administration or coformulation with a composition of the invention as described herein.
  • Such drugs include, without limitation, demulcents; antimycotics, antivirals and other anti-infectives; steroids, NSAIDs, selective cyclooxygenase-2 inhibitors and other anti-inflammatory agents; acetylcholine blocking agents; adrenergic agonists, beta-adrenergic blocking agents and other antiglaucoma agents; antihypertensives; antihistamines; anticataract agents; and topical and regional anesthetics.
  • Illustrative specific drugs include, but are not limited to, acebutolol, aceclidine, acetylsalicylic acid (aspirin), N 4 acetylsulfisoxazole, alclofenac, alprenolol, amfenac, amiloride, aminocaproic acid, rho-aminoclonidine, aminozolamide, anisindione, apafant, atenolol, bacitracin, benoxaprofen, benoxinate, benzofenac, bepafant, betamethasone, betaxolol, bethanechol, brimonidine, bromfenac, bromhexine, bucloxic acid, bupivacaine, butibufen, carbachol, carprofen, celecoxib, cephalexin, chloramphenicol, chlordiazepoxide, chlorprocaine, chlorpropamide, chlortetracycline, ciclo
  • treat when used herein with respect to retinopathy, or a disease characterized by a high level of VEGF in the retina, refer to administration of the compositions to a subject which may increase the resistance of the subject to development or further development of the condition, to administration of the composition after the subject has developed the condition in order to eliminate or at least control development of the condition, and/or to reduce the severity of symptoms caused by the condition.
  • effective amounts When administered to a subject, effective amounts will depend on the particular condition being treated and the desired outcome. A therapeutically effective dose may be determined by those of ordinary skill in the art, for instance, employing factors such as those further described below and using no more than routine experimentation.
  • dosing amounts, dosing schedules, routes of administration may be selected so as to affect known activities of these compositions.
  • Dosages may be estimated based on the results of experimental models, optionally in combination with the results of assays of compositions of the present invention. Dosage may be adjusted appropriately to achieve desired drug levels, depending upon the mode of administration. The doses may be given in one or several administrations per day. Multiple doses per day are also contemplated in some cases to achieve appropriate levels of the composition within the eye.
  • Aqueous suspension compositions of the invention can be packaged in single- dose non-reclosable containers.
  • Such containers can maintain the composition in a sterile condition and thereby eliminate need for preservatives such as mercury-containing preservatives, which can sometimes cause irritation and sensitization of the eye.
  • preservatives such as mercury-containing preservatives, which can sometimes cause irritation and sensitization of the eye.
  • multiple-dose reclosable containers can be used, in which case it is sometimes preferred to include a preservative in the composition.
  • the dose of the composition to the subject may be such that a therapeutically effective amount of the composition reaches an active site (e.g., the retina of the eye) of the composition within the subject.
  • the dosage may be given in some cases at the maximum amount while avoiding or minimizing any potentially detrimental side effects within the subject.
  • the dosage of the composition that is actually administered is dependent upon factors such as the final concentration desired, the method of administration to the subject, the efficacy of the composition, the longevity of the composition within the subject, the timing of administration, the effect of concurrent treatments (e.g., as in a cocktail), etc.
  • the dose delivered may also depend on conditions associated with the subject, and can vary from subject to subject in some cases.
  • the age, sex, weight, size, environment, physical conditions, or current state of health of the subject may also influence the dose required and/or the concentration of the composition. Variations in dosing may occur between different individuals or even within the same individual on different days. It may be preferred that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. Preferably, the dosage form is such that it does not substantially deleteriously affect the subject.
  • Subject doses of the compounds described herein for delivery typically range from about 0.1 microgram to 10 mg per administration, which depending on the application could be given daily, weekly, or monthly, yearly, and any other amount of time therebetween.
  • doses range from about 10 microgram to 5 mg per administration, and most typically from about 100 microgram to 1 mg, with 2 to 4 administrations being spaced days or weeks apart. More typically, doses range from 1 microgram to 10 mg per administration, and most typically 10 microgram to 1 mg, with daily or weekly administrations.
  • the compositions of the present invention may be administered in multiple doses over extended period of time.
  • the therapeutically effective amount can be initially determined from animal models.
  • the applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.
  • a composition of the invention is administered to a subject who has a family history of retinopathies, or other diseases characterized by a high level of VEGF in the retina, or to a subject who has a genetic predisposition for such conditions.
  • the composition is administered to a subject who has reached a particular age, or to a subject more likely to get such conditions.
  • the compositions is administered to subjects who exhibit symptoms of such conditions (e.g., early or advanced).
  • the composition may be administered to a subject as a preventive measure.
  • the composition may be administered to a subject based on demographics or epidemiological studies, or to a subject in a particular field or career.
  • Administration of a composition of the invention may be accomplished by any medically acceptable method which allows the composition to reach its target.
  • the particular mode selected will depend of course, upon factors such as those previously described, for example, the particular composition, the severity of the state of the subject being treated, the dosage required for therapeutic efficacy, etc.
  • a "medically acceptable" mode of treatment is a mode able to produce effective levels of the composition within the subject without causing clinically unacceptable adverse effects.
  • diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch.
  • Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride.
  • Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.
  • Disintegrants may be included in the formulation of the therapeutic into a solid dosage form.
  • Materials used as disintegrates include, but are not limited to, starch, including the commercial disintegrant based on starch, Explotab.
  • Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used.
  • Another form of the disintegrants are the insoluble cationic exchange resins.
  • Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.
  • Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.
  • An anti-fiictional agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process.
  • Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.
  • stearic acid including its magnesium and calcium salts
  • PTFE polytetrafluoroethylene
  • Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.
  • the glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.
  • a surfactant might be added as a wetting agent.
  • Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride.
  • non-ionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose.
  • surfactants could be present in the formulation of the compositions of the present invention, either alone or as a mixture in different ratios.
  • compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Pharmaceutical formulations include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • the suspensions may also contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active compositions may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, or the like.
  • the pharmaceutical compositions also include emulsions, suspensions, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, or solubilizers are customarily used as described above.
  • the pharmaceutical compositions are suitable for use in a variety of drug delivery systems.
  • the administration of the composition of the invention may be designed so as to result in sequential exposures to the composition over a certain time period, for example, hours, days, weeks, months or years. This may be accomplished, for example, by repeated administrations of a composition of the invention by one of the methods described above, or by a sustained or controlled release delivery system in which the composition is delivered over a prolonged period without repeated administrations. Maintaining a substantially constant concentration of the composition may be preferred in some cases.
  • delivery systems suitable for use with the present invention include time-release, delayed release, sustained release, or controlled release delivery systems. Such systems may avoid repeated administrations of the composition in many cases, increasing convenience to the subject. Many types of release delivery systems are available and known to those of ordinary skill in the art.
  • polymer-based systems such as polylactic and/or polyglycolic acids, polyanhydrides, polycaprolactones and/or combinations of these; nonpolymer systems that are lipid-based including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-, di- and triglycerides; hydrogel release systems; liposome-based systems; phospholipid based-systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; or partially fused implants.
  • Specific examples include, but are not limited to, erosional systems in which the composition is contained in a form within a matrix (for example, as described in U.S. Patent Nos.
  • the formulation may be as, for example, microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, or polymeric systems.
  • the system may allow sustained or controlled release of the composition to occur, for example, through control of the diffusion or erosion/degradation rate of the formulation containing the composition.
  • a pump-based hardware delivery system may be used to deliver one or more embodiments of the invention.
  • long-term release implant may be particularly suitable in some embodiments of the invention.
  • Long-term release means that the implant containing the composition is constructed and arranged to deliver therapeutically effective levels of the composition for at least 30 or 45 days, and preferably at least 60 or 90 days, or even longer in some cases.
  • Long-term release implants are well known to those of ordinary skill in the art, and include some of the release systems described above.
  • compositions can be alone, or in combination with other therapeutic agents and/or compositions.
  • a composition can be combined with a suitable pharmaceutically acceptable carrier, for example, as incorporated into a liposome, incorporated into a polymer release system, or suspended in a liquid, e.g., in a dissolved form or a colloidal form.
  • suitable pharmaceutically acceptable carriers for use in the invention are well-known to those of ordinary skill in the art.
  • a “pharmaceutically acceptable carrier” refers to a non-toxic material that does not significantly interfere with the effectiveness of the biological activity of the active compound(s) to be administered, but is used as a formulation ingredient, for example, to stabilize or protect the active compound(s) within the composition before use.
  • the carrier may include one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal.
  • carrier denotes an organic or inorganic ingredient, which may be natural or synthetic, with which one or more active compounds of the invention are combined to facilitate the application of the composition.
  • the carrier may be co-mingled or otherwise mixed with one or more active compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • the carrier may be either soluble or insoluble, depending on the application. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and magnetite. The nature of the carrier can be either soluble or insoluble. Those skilled in the art will know of other suitable carriers, or will be able to ascertain such, using only routine experimentation.
  • the formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, emulsifiers, diluents, excipients, chelating agents, fillers, drying agents, antioxidants, antimicrobials, preservatives, binding agents, bulking agents, silicas, solubilizers, stabilizers and optionally other therapeutic ingredients, that may be used with the active compound.
  • the carrier may be a solvent, partial solvent, or non-solvent, and may be aqueous or organically based.
  • suitable formulation ingredients include diluents such as calcium carbonate, sodium carbonate, lactose, kaolin, calcium phosphate, or sodium phosphate; granulating and disintegrating agents such as corn starch or algenic acid; binding agents such as starch, gelatin or acacia; lubricating agents such as magnesium stearate, stearic acid, or talc; time-delay materials such as glycerol monostearate or glycerol distearate; suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone; dispersing or wetting agents such as lecithin or other naturally-occurring phosphatides; thickening agents such as cetyl alcohol or beeswax; buffering agents such as acetic acid and salts thereof, citric acid and salts thereof, boric acid and salts thereof, or phosphoric acid and salts thereof; or preservatives such as benzy
  • Suitable carrier concentrations can be determined by those of ordinary skill in the art, using no more than routine experimentation. Those of ordinary skill in the art will know of other suitable formulation ingredients, or will be able to ascertain such, using only routine experimentation.
  • Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).
  • Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004- 0.02% w/v).
  • Preparations include sterile aqueous or nonaqueous solutions, suspensions and emulsions, which can be isotonic with the blood of the subject in certain embodiments.
  • nonaqueous solvents are polypropylene glycol, polyethylene glycol, vegetable oil such as olive oil, sesame oil, coconut oil, arachis oil, peanut oil, mineral oil, injectable organic esters such as ethyl oleate, or fixed oils including synthetic mono or di-glycerides.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, 1,3-butandiol, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents and inert gases and the like. Those of skill in the art can readily determine the various parameters for preparing and formulating the compositions of the invention without resort to undue experimentation.
  • the present invention includes the step of bringing a composition of the invention into association or contact with a suitable carrier, which may constitute one or more accessory ingredients.
  • a suitable carrier which may constitute one or more accessory ingredients.
  • the final compositions may be prepared by any suitable technique, for example, by uniformly and intimately bringing the composition into association with a liquid carrier, a finely divided solid carrier or both, optionally with one or more formulation ingredients as previously described, and then, if necessary, shaping the product.
  • Another aspect of the invention involves transfecting macrophages and other cells with a gene for expressing soluble VEGF receptor, a VEGF antagonist, or the like.
  • Such cells may be delivered to the eye of a subject, as previously described, optionally in combination with other cells, VEGF antagonists, etc.
  • a cell that is transfected is intentionally chosen or selected to express the transfected gene, i.e., at expression levels significantly greater than the expression level of the enzyme for that cell type (which can include zero or negligible expression levels).
  • a cell may be artificially selected through natural selection processes, the cells may be stimulated (e.g., with a hormone), the cell may be transfected to overexpress the gene, or the like.
  • the macrophage may be able to overexpress a soluble VEGF receptor at levels that are therapeutically effective.
  • a soluble VEGF receptor at levels that are therapeutically effective.
  • suitable ways of transfecting cells For example, some techniques for transformation (micro-injection, electroporation, calcium phosphate method, etc.) are described in Sambrook, et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N. Y. (1989).
  • a gene for a soluble VEGF receptor may be transfected into a cell, such as a macrophage, using a DNA vector.
  • the vector may be a vector in which the gene is functionally linked to one or more control sequences which allows expression of the corresponding enzymes.
  • the gene may be expressed as fusion proteins or as intact, native proteins. In some cases, fusion proteins may be produced in large quantities. The fusion proteins are generally more stable than the native polypeptide and are easy to purify. The expression of these fusion proteins can be controlled by normal host DNA sequences.
  • the genetic constructs may generally contain one or more suitable regulatory elements (such as one or more suitable promoters, enhancers, terminators, etc.), 3'- or 5'- UTR sequences, leader sequences, selection markers, expression markers/reporter genes, and/or elements that may facilitate or increase (the efficiency of) transformation.
  • suitable regulatory elements such as one or more suitable promoters, enhancers, terminators, etc.
  • 3'- or 5'- UTR sequences such as one or more suitable promoters, enhancers, terminators, etc.
  • 3'- or 5'- UTR sequences such as one or more suitable promoters, enhancers, terminators, etc.
  • 3'- or 5'- UTR sequences such as one or more suitable promoters, enhancers, terminators, etc.
  • 3'- or 5'- UTR sequences such as one or more suitable promoters, enhancers, terminators, etc.
  • 3'- or 5'- UTR sequences such as one or more suitable promoters, enhancer
  • one or more elements may be "operably linked" to the above- described genes and/or to each other, by which is generally meant that they are in a functional relationship with each other.
  • a promoter is considered “operably linked” to a coding sequence if said promoter is able to initiate or otherwise control/regulate the transcription and/or the expression of a coding sequence (in which said coding sequence should be understood as being "under the control of said promoter).
  • two nucleotide sequences when operably linked, they will be in the same orientation and usually also in the same reading frame. They will usually also be essentially contiguous, although this may also not be required.
  • the optional further elements of the genetic construct(s) used in the invention may be such that they are capable of providing their intended biological function in the intended host cell or host organism.
  • a promoter, enhancer or terminator may be "operable" in the intended host cell or host organism, by which is meant that (for example) the promoter should be capable of initiating or otherwise controlling/regulating the transcription and/or the expression of a nucleotide sequence (e.g. a coding sequence) to which it is operably linked (as defined above).
  • Such a promoter may be a constitutive promoter or an inducible promoter, and may also be such that it (only) provides for expression in a specific stage of development of the host cell or host organism, and/or such that it (only) provides for expression in a specific cell, tissue, organ or part of a multicellular host organism.
  • a selection marker may be chosen such that it allows (e.g., under appropriate selection conditions) host cells and/or host organisms that have been (successfully) transformed with the nucleotide sequence of the invention to be distinguished from host cells/organisms that have not been (successfully) transformed.
  • Some preferred, but non-limiting examples of such markers are genes that provide resistance against antibiotics (such as kanamycin or ampicillin), genes that provide for temperature resistance, or genes that allow the host cell or host organism to be maintained in the absence of certain factors, compounds and/or (food) components in the medium that are essential for survival of the non-transformed cells or organisms.
  • a leader sequence may be chosen such that, in the intended host cell or host organism, it allows for the desired post-translational modifications, and/or such that it directs the transcribed mRNA to a desired part or organelle of a cell.
  • a leader sequence may also allow for secretion of the expression product from said cell.
  • the leader sequence may be any pro-, pre-, or prepro-sequence operable in the host cell or host organism.
  • An expression marker or reporter gene may be chosen such that, in the host cell or host organism, it allows for detection of the expression of (a gene or nucleotide sequence present on) the genetic construct.
  • An expression marker may optionally also allow for the localization of the expressed product, e.g. in a specific part or organelle of a cell and/or in (a) specific cell(s), tissue(s), organ(s) or part(s) of a multicellular organism.
  • Such reporter genes may also be expressed as a protein fusion with the amino acid sequence of the invention. Some preferred, but non-limiting examples include fluorescent proteins such as GFP.
  • the genetic constructs of the invention may generally be provided by suitably linking the nucleotide sequence(s) of the invention to the one or more further elements described above, for example using the techniques described in the general handbooks such as Sambrook et al., mentioned above. Often, the genetic constructs will be obtained by inserting a nucleotide sequence in a suitable (expression) vector known per se.
  • the invention includes the promotion of one or more of the above-described embodiments, for example, promotion of methods for administering, to an eye of a subject, a composition comprising a macrophage.
  • promotion of methods for administering, to an eye of a subject for example, promotion of methods for administering, to an eye of a subject, a composition comprising a macrophage.
  • promoted includes all methods of doing business, including methods of education, scientific inquiry, academic research, industry activity including pharmaceutical industry activity, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with the invention.
  • LRP5 is one of a family of LDL receptive proteins, which also includes LRPl, LRP2, and LRP6. The general structures of various members of this family are shown in Fig. 4. LRP5 is believed to mediate Wnt signaling in bone formation, as is shown in Figs. 5A and 5B. In particular, Wnt facilitates the association of LRP5 with Frizzled and Axin, which leads to a sequence of binding events as is shown in Figs. 5 A, thereby allowing transcription and bone formation to occur. In the absence of LR-5, Wnt is not able to mediate bone formation.
  • LRP5 has also been shown to be involved with the delayed development of the intraretinal vascular network. For instance, as is shown in Figs. 6A and 6B, when LRP5 is expressed (LRP5 +/+) in a mouse model, the full vasculature of the retina is able to develop. However, in mice lacking the LRP5 gene, defects in retina development were observed. For example, only the superficial layer of the retina formed, instead of all of the layers of the retina. In addition, blood vessel growth was stunted, as is shown in Fig. 6A (LRP5 -/-). Many blood vessels showed abnormal formation, and the position of the vessels appeared incorrect. No cells were observed within the blood vessels, although basement membrane had formed.
  • Figs. 6C and 6D Close ups of the intraretinal vascular network can be seen in Figs. 6C and 6D for LRP5 null and positive retinas, respectively. Accordingly, in the LRP5-null retina, the intraretinal vascular network has not fully grown in, which may thus lead to blindness.
  • the hyaloid vessel which aids the development of the lens by supplying blood to the lens, eventually regresses.
  • the hyaloid vessel fuses with the retinal blood vessels (Figs. 7A-7E), leading to. chaotic vascular patterns (Figs. 8A-8D), and patches of macrophages are observed at the periphery of the developing retinal and hyaloid vessels (Figs.
  • the addition of macrophages that are positive for LRP5 may be used to at least partially restore the retinal vasculature.
  • LRP5 e.g., wild-type macrophages
  • 4000 peritoneal wild-type macrophages (taken from GFP-labelled mice) were intravitreally injected into the left eye of a 2-day old LRP-null mouse, with the right eye serving as a control (Fig. 2A). After about 2 weeks, the mice were sacrificed and the retinas analyzed. The results can be seen in Fig. 2.
  • Fig. 2B shows the control (right) eye
  • Fig. 2C shows the treated (left) eye.
  • the control eye shows a poorly formed intraretinal vascular network
  • the treated eye shows a well-developed intraretinal vascular network.
  • the secondary layer of the retina began to form.
  • peritoneal macrophages taken from LRP5-null mice were administered to one eye of a normal (wild-type) mouse, with the other eye serving as a control, under conditions similar to the above. After about 2 weeks, the retinas were examined.
  • the treated eye (treated with macrophages lacking the LRP5 gene) showed a poorly formed intraretinal vascular network, in contrast to the control eye, which showed a well-developed intraretinal vascular.
  • vessel morphology in the eyes appears to be a dynamic process, and the presence of macrophages within the retina may affect blood vessel morphology. It should be noted that such morphology is not necessarily dependent on fetal development. Accordingly, by treatment using macrophages, the blood vessel morphology of an eye can be controlled.
  • mice were used that were 4 weeks old or more. Their genotype were as follows: Lrp5 +/+, GFP+ mice and Lrp5 -/-, GFP+ mice.
  • Macrophages were prepared from bone marrow cells as follows. The tibia and femur were removed and the epiphysis was cut off. Bone marrow cells were flushed from the bones by 1 ml alpha-MEM (GIBCO, MEM Alpha 12561) with 25 G needle 3 or 4 times. The cells were spun down (1000 rpm x 10 min, 4 0 C) the supernatant was aspirated. The cells (RBCs, red blood cells) were lysed for 2 ml RBC lysis buffer (eBioscience, Ix RBC Lysis buffer 00-4333-57) on ice for 10 min.
  • RBCs red blood cells
  • Bovine Serum Sl 1150 Bovine Serum Sl 1150 and the cells were incubated in a 100 mm dish at 37 0 C for 2 hours. The medium was transferred to a new petri dish to collect non-adhesive cells. The cells were incubated in alpha-MEM + 10% FBS + 25 ng/ml M-CSF (PEPRO TECH, Recombinant Murine M-CSF 315-02) + 1% penicillin/streptomycin (GIBCO, Penicillin- Streptomycin 15140-122). The medium was changed every 2 days. The macrophages were detached from the petri dish by EDTA solution (Cellgro, Cell striper 25-056-Cl) after 4 or more days of culture.
  • EDTA solution Cellgro, Cell striper 25-056-Cl
  • the macrophages were then resuspended in alpha-MEM + 1% FBS at a cell concentration of 1.5 x 10 5 cells/microliter). 1.0 microliters of macrophages (about 1.5 x 10 5 cells) were then injected into pup's (P2) eye with a 33G needle (needle; Hamilton, 33GA RN 6PK, syringe; Hamilton 7635-01). The other eye was used as a control (PBS).
  • the analysis of the retinal vascular structure was then performed as follows.
  • the mice with were anesthetized Avertin about 2 weeks later.
  • the breasts were opened and the vena cava cut.
  • a 25G needle was inserted to the apex.
  • 3 ml cold PBS + 2% dextran (sigma, dextran) + 1% PFA and 3 ml PBS + 25 mg/ml dextran conjugated with rhodamine (sigma, tetramethylrhodamine isothiocyanate-dexrtan T1287) were circulated with a pump machine (Pharmacia Fine Chemicals).
  • the eyeballs were removed and fixed in 4% PFA/ PBS for 15 min at room temperature (RT, about 25 0 C).
  • the retina was then dissected and fixed in 4% PF A/PBS for 2 hours at RT.
  • the retina was mounted on a glass slide with a fluorescence mounting medium, and the retina was observed using a confocal microscope (Nikon TE2000 Inverted microscope and C 1 Laser scanning confocal).
  • a reference to "A and/or B", when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase "at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B" can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
  • the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Ophthalmology & Optometry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Cell Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne généralement des traitements de l'œil et, en particulier, des traitements de l'œil en utilisant des macrophages. Les maladies incluant les rétinopathies (telles que la rétinopathie diabétique ou la rétinopathie hypertensive), l'ostéoporose avec pseudogliome, la dégénérescence maculaire (telle que la dégénérescence maculaire liée à l'âge), ou les maladies caractérisées par un haut niveau de VEGF dans la rétine peuvent être traitées de cette manière. On pense que les macrophages jouent un rôle dans l'angiogenèse en phagocytant les cellules épithéliales et similaires à l'intérieur de la rétine. Dans certains cas, les macrophages peuvent produire des niveaux relativement élevés de récepteur du VEGF soluble, qui peuvent également être utiles pour réduire les concentrations globales de VEGF à l'intérieur de la rétine. Dans certains modes de réalisation de l'invention, les macrophages peuvent également être transfectés, par exemple, avec un gène pour l'expression du récepteur VEGF soluble, un autre antagoniste du VGEF, et/ou d'autres gènes appropriés, et dans certains cas, les macrophages peuvent être combinés aux autres thérapies.
PCT/US2008/002063 2007-02-16 2008-02-15 Traitement de l'œil utilisant des macrophages et/ou des agents capables d'affecter la morphologie d'un vaisseau sanguin Ceased WO2008103301A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US90174507P 2007-02-16 2007-02-16
US60/901,745 2007-02-16

Publications (2)

Publication Number Publication Date
WO2008103301A2 true WO2008103301A2 (fr) 2008-08-28
WO2008103301A3 WO2008103301A3 (fr) 2008-10-09

Family

ID=39561761

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2008/002063 Ceased WO2008103301A2 (fr) 2007-02-16 2008-02-15 Traitement de l'œil utilisant des macrophages et/ou des agents capables d'affecter la morphologie d'un vaisseau sanguin

Country Status (1)

Country Link
WO (1) WO2008103301A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113288889A (zh) * 2021-06-22 2021-08-24 广东省妇幼保健院 丙磺舒在早产儿视网膜病变rop疾病防治中的应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1140173B2 (fr) * 1998-12-22 2013-04-03 Genentech, Inc. Antagonistes du facteur de croissance endotheliale et leurs utilisations
US6670321B1 (en) * 1998-12-30 2003-12-30 The Children's Medical Center Corporation Prevention and treatment for retinal ischemia and edema
US20040132675A1 (en) * 2002-02-08 2004-07-08 Calvin Kuo Method for treating cancer and increasing hematocrit levels
JP4660067B2 (ja) * 2001-04-24 2011-03-30 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング 抗血管新生剤とＴＮＦαとを用いる組合せ療法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113288889A (zh) * 2021-06-22 2021-08-24 广东省妇幼保健院 丙磺舒在早产儿视网膜病变rop疾病防治中的应用

Also Published As

Publication number Publication date
WO2008103301A3 (fr) 2008-10-09

Similar Documents

Publication Publication Date Title
Zhang et al. Ophthalmic drug discovery: novel targets and mechanisms for retinal diseases and glaucoma
Iandiev et al. Glial cell reactivity in a porcine model of retinal detachment
KR100405285B1 (ko) 합성 티로이드 호르몬을 포함하는 약제 조성물
JP5247686B2 (ja) ＰＰＡＲδアゴニスト含有医薬
KR101953736B1 (ko) 제브라피시 모델을 사용한 약물 스크리닝 방법 및 이 방법으로 스크린된 화합물
Welge–Lüßen et al. αB-crystallin in the trabecular meshwork is inducible by transforming growth factor-β
Wax et al. Characterization of beta-adrenergic receptors in cultured human trabecular cells and in human trabecular meshwork.
Bagnis et al. Current and emerging medical therapies in the treatment of glaucoma
CN112805284A (zh) 整合素拮抗剂
Baba et al. A rat model for choroidal neovascularization using subretinal lipid hydroperoxide injection
Sadrai et al. Effect of topical azithromycin on corneal innate immune responses
WO2015120115A1 (fr) Compositions pour le traitement de la dégénérescence rétinienne et méthodes de production et d'utilisation de celles-ci
JP2016523913A (ja) 眼疾患の治療および診断
WO2013049621A1 (fr) Compositions ophtalmologiques comprenant des agonistes du récepteur ppar-alpha et leurs méthodes de production
US20050203014A1 (en) Composition and methods for modulating CNS activity
WO2008103301A2 (fr) Traitement de l'œil utilisant des macrophages et/ou des agents capables d'affecter la morphologie d'un vaisseau sanguin
US20180064708A1 (en) Nutlin-3a for Treatment of Proliferative Vitreoretinopathy
Wang et al. Silicone Oil Affects Fibrosis of Human Trabecular Meshwork Cells by Upregulating Ferroptosis Through a ROS/NOX4/Smad3 Axis
EA019437B1 (ru) Способ лечения воспаления роговицы
JP2014511392A (ja) 創傷を治癒または治療するための分子標的
EP2937076B1 (fr) Collyre contenant de la prostaglandine et du tyloxapol
US20190247302A1 (en) Materials and methods for treating ophthalmic inflammation
KR20150048880A (ko) 눈 컨디션의 치료 및/또는 예방을 위한 조성물 및 방법에 있어서 siRNA 및 이의 용도
US20220280473A1 (en) Compositions and methods for modulating epithelial-mesenchymal transition
KR20180031719A (ko) 백반증 치료

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08725671

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08725671

Country of ref document: EP

Kind code of ref document: A2