WO2001083714A2 - Composition et procedes servant a traiter la degenerescence de photorecepteurs - Google Patents

Composition et procedes servant a traiter la degenerescence de photorecepteurs Download PDF

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WO2001083714A2
WO2001083714A2 PCT/US2001/013992 US0113992W WO0183714A2 WO 2001083714 A2 WO2001083714 A2 WO 2001083714A2 US 0113992 W US0113992 W US 0113992W WO 0183714 A2 WO0183714 A2 WO 0183714A2
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retina
dopamine
antagonist
organ culture
photoreceptor
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PCT/US2001/013992
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WO2001083714A3 (fr
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Judith M. Ogilvie
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Central Institute For The Deaf
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Publication of WO2001083714A3 publication Critical patent/WO2001083714A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/062—Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/80—Neurotransmitters; Neurohormones
    • C12N2501/815—Dopamine

Definitions

  • the present invention relates generally to the prevention of photoreceptor degeneration in organ cultures, cell cultures and in vivo.
  • Photoreceptors are a specialized subset of retinal neurons consisting of rods and cones.
  • a number of diseases ofthe retina involve the progressive degeneration and eventual death of photoreceptors.
  • Such diseases include retinitis pigmentosa, age-related macular degeneration and other maculopathies or retinal detachment.
  • Some work has been done in using various trophic factors to rescue photoreceptors from death.
  • photoreceptors can be rescued by basic fibroblast growth factor (bFGF) in Royal College of Surgeons (RCS) rats and in albino rats that have been damaged by exposure to constant light. Faktorovich et al, Nature, 347: 83-86 (1990).
  • bFGF basic fibroblast growth factor
  • RCS rats have an inherited mutation ofthe gene expressed in the retinal pigment epithelium (RPE) that results in the failure ofthe RPE to phagocytize the continuously shed portions ofthe photoreceptor outer segments and causes photoreceptor degeneration and eventually cell death.
  • RPE retinal pigment epithelium
  • a single injection of bFGF into the vitreous body or into the subretinal space, the extracellular space surrounding rods and cones, at the onset ofthe degeneration transiently rescues photoreceptors. Faktorovich et al supra.
  • bFGF injected into the subretinal space of a light damaged model of albino rats protects photoreceptors from the effects of light damage and prevents cell death.
  • trophic factors studied for this purpose are acidic fibroblast growth factor (aFGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), interleukin-1 beta, neurotrophin-3 (NT-3), insulin-like growth factor II (IGF-II), tumor necrosis factor-alpha (TNF-alpha), platelet derived growth factor (PDGF) and glial derived neurotrophic facotor (GDNF).
  • aFGF acidic fibroblast growth factor
  • BDNF brain-derived neurotrophic factor
  • CNTF ciliary neurotrophic factor
  • interleukin-1 beta neurotrophin-3
  • NT-3 neurotrophin-3
  • IGF-II insulin-like growth factor II
  • TNF-alpha tumor necrosis factor-alpha
  • PDGF platelet derived growth factor
  • GDNF glial derived neurotrophic facotor
  • CNTF ciliary neurotrophic factor
  • BDNF brain-derived neurotrophic factor
  • the inventors Since the BDNF receptor has been localized to dopaminergic neurons in the retina, (Cellerino and Kohler, J. Comp. Neurol. 386: 149-160 (1997), the inventors considered that the growth factors might act through dopaminergic neurons to increase photoreceptor cell survival. To test this possibility, the inventors proposed to add both CNTF and BDNF in the presence of dopamine antagonists.
  • Initial control experiments involved growing rd retinas in organ culture in the presence of Dj or D family receptor antagonists in order to determine any paracrine effects of dopamine on photoreceptors in this system.
  • dopamine antagonists prevent photoreceptor degeneration for the duration ofthe 4 week in vitro paradigm. Conversely, the inventors found that dopamine is necessary for photoreceptor degeneration.
  • the present invention is based on the discovery that antagonists of dopamine receptors prevent photoreceptor degeneration.
  • the invention relates to a method of enhancing photoreceptor survival in retina organ culture comprising contacting said photoreceptors in culture with a compound that is a dopamine receptor antagonist or is an agent that depletes dopamine.
  • the invention relates to a retina organ culture comprising retinal tissue and a dopamine receptor antagonist or an agent that depletes dopamine.
  • the invention in another embodiment, relates to a method of treating photoreceptor degeneration comprising administering to the retina of a subject in need thereof a therapeutically effective amount of an antagonist of dopamine receptors in photoreceptor cells or an agent that depletes dopamine.
  • Figure 1 is a schematic ofthe retina.
  • Figure 2 is a schematic of a method of culturing the retina.
  • Figure 3 shows that TH-positive neurons are present in antagonist treated organ cultures.
  • Figure 4 shows the effects of dopamine antagonists on photoreceptor cell survival in rd retinal organ cultures.
  • Retinas from wild type (A) and rd (B,C,D) mice were harvested at postnatal day 2 and grown in organ culture for 27 DIV. Wild type retinas maintained approximately 5 rows of cells in the ONL (A) while the ONL of untreated rd cultures was reduced to a monolayer (B).
  • No photoreceptor degeneration was seen in rd organ cultures treated with 100 nM sulpiride, a D 2 family antagonist (C).
  • SCH-23390 (20 nM) a D] family antagonist also increases photoreceptor survival in rd organ cultures (D).
  • Figure 5 shows the effects of dopamine depletion on photoreceptor cell survival in rd retinal organ cultures.
  • Retinas were grown in organ culture for 27 DIV with either control media or 6-hydroxydopamine (6-OHDA) and pargyline added to the media on the first 2 days in vitro and again after 1 week.
  • 6-OHDA treated rd organ cultures showed no degeneration (A) and could not be distinguished from wild type organ cultures (see figure 4B).
  • ADTN 6-hydroxydopamine
  • Figure 6 shows the effects of dopamine inhibition on opsin expression in rd retinal organ cultures.
  • Opsin immunohistochemistry was performed on untreated wild type (A) and rd (B) organ cultures as well as rd organ cultures treated with sulpiride (C), SCH-23390 (D), or 6-OHDA (E).
  • C sulpiride
  • D SCH-23390
  • E 6-OHDA
  • intense opsin immunoreactivity is seen in the outer nuclear layer and in residual inner and outer segments.
  • dopamine inhibition nor dopamine depletion alters opsin expression.
  • IS/OS photoreceptor inner and outer segments; ONL, outer nuclear layer. Bar, 15 ⁇ m.
  • the present invention provides methods and compositions for the treatment of photoreceptor degeneration.
  • the invention is based on the discovery that dopamine is necessary for photoreceptor degeneration and that dopamine antagonists or the elimination of dopamine prevents photoreceptor degeneration.
  • Dopamine is an endogenous neurotransmitter in the retina that affects most, if not all, cell types in the vertebrate retina, playing a role in the transition from scotopic to photopic vision.
  • Dopaminergic neurons in the mouse retina are interplexiform cells with extensive processes on the outer margin ofthe inner plexiform layer (IPL), sparser processes throughout the IPL, and an ascending process extending to the outer plexiform layer (OPL). Witkovsky and Schuette Vis. Neurosci. 7: 113-124 (1991).
  • Di family dopamine receptors characterized by activation of adenelyl cyclase, are found in both the OPL and IPL, primarily on horizontal and bipolar cells, respectively.
  • D 2 family dopamine receptors which inhibit adenelyl cyclase, are also found in both plexiform layers. Additionally, D receptors are located on the inner segments of photoreceptors where dopamine has been shown to act in a paracrine fashion. Cohen et al PNAS USA 89: 12093-12097 (1992).
  • the invention relates to a method of enhancing photoreceptor survival in a retina organ culture.
  • enhancing is meant a statistically significant increase in the number of surviving photoreceptors in the organ culture compared to the untreated rd organ culture (i.e. control media) as measured by counting the number of cells, indicating the thickness ofthe ONL or the density of cells in the ONL. Complete inhibition of degeneration is indicated if the counts in treated rd organ cultures is not significantly different from untreated wild type organ cultures.
  • retina organ culture is meant a retina organ culture as described in Ogilvie et al, Neurosci Meth. 87: 57-65 (1999), which is herein incorporated by reference.
  • Example 1 presents a protocol for preparing and maintaining a retina organ culture.
  • this term includes other types of retina organ cultures.
  • Other illlustrative examples are described in Caffe, A.R. and Sanyal, S. (1991) Retinal degeneration in vitro: Comparison of postnatal retinal development of normal, rd and rds mutant mice in organ culture. In R.E. Anderson, J.G. Hollyfield and M.M. LaVail Eds., Retinal Degenerations, CRC Press, Boca Raton, FL, pp. 29-38; Feigenspan, A., Bormann, J. and Wassle, H. (1993) Organotypic slice culture of the mammalian retina, Vis.
  • the survival ofthe photoreceptors is enhanced by administration to the organ culture of a dopamine receptor antagonist or an agent that depletes dopamine.
  • antagonist refers to an agent which directly or indirectly combines with receptors on a cell and inhibits the stimulation of a response.
  • small molecules which bind dopamine receptors in photoreceptor cells, antibodies against such receptors and chemical compounds.
  • dopamine antagonists include, but are not limited to AJ76, Alprenolol, Amisulpride, Bromocryptine, (+) Butaclamol, (-) Butaclamol, Chlorpromazine, cis-flupenthixol, cis-piflutixol, Clozapine, Dihydroergocryptine, Domperidone, Eticlopride, Fluphenazine-n-mustard (FNM), Haloperidol, IBZM, Iodosulpride, Ketanserin, Metoclopramide, Olanzapine, Pimozide, PNU-99194A, PNU- 101387, Prazosin, Prochlorperazine, Raclopride, SCH23388, SCH23390, SCH23982, SCH39166, Spiperone, Sulpiride, Thioproperazine, Thioridazine, UH232, and Yohimbine .
  • the antagonist ofthe invention is an antagonist ofthe Dj or D receptor families.
  • such antagonist is sulpiride; in another it is SCH-23390. See Sokoloff et al, Nature 347: 146-151 (1990), Van Tol. et alNature 350:
  • An "agent that depletes dopamine” includes any such agent known to deplete dopamin, such as 6-hydroxydopamine (6-OHDA), a toxin specific for dopaminergic neurons, which leaves the receptors intact, but depletes the level of dopamine so that the receptors are not activated.
  • 6-OHDA 6-hydroxydopamine
  • the invention in another embodiment, relates to a method of screening for antagonists of dopamine receptors in photoreceptor cells comprising comparing the extent of degeneration of photoreceptors in two groups of rd mouse retina organ tissue, the first group comprising rd mouse retina organ tissue that has been contacted with the subject compound and the second group comprising rd mouse retina organ tissue that has not been contacted with the subject compound, wherein a decrease in the extent of degeneration indicates that the subject compound is an antagonist of dopamine receptors in the retina.
  • Other antagonists within the scope ofthe invention can be determined using the above assay. Of course, the skilled artisan would know of other animal models suitable for use in this assay.
  • Such animal models include the RCS rat and albino rat described above, as well as rd2, rd3, rd4, rd5 and various transgenic mice with retinal degeneration. See Faktorovich et al, Nature, 347: 83-86 (1990).
  • the animal model is the rd mouse.
  • the rd mouse is a well characterized animal model of photoreceptor degeneration that carries an autosomal recessive defect in the ⁇ -subunit ofthe rod specific cGMP-PDE gene. This defect results in loss of rod photoreceptors with the first three postnatal weeks followed by a slow degeneration ofthe cones. See Chang et al.
  • the above assay could be performed using cultured cells that express a dopamine receptor.
  • Such cells could be a mammalian cell line transfected with DNA encoding a dopamine receptor. See U.S. Patent No. 6,214,615, which is herein incorporated by reference.
  • the dopamine antagonist ofthe invention is an antibody against a dopamine receptor.
  • Antibody refers to a polypeptide comprising a framework region encoded by an immunoglobulin gene or fragments thereof that binds and recognizes an antigen.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 2 kD) and one "heavy" chain (about 0-70 kD).
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
  • antibody fragments are defined in terms ofthe digestion of an intact antibody, one skilled in the art will appreciate that such fragments may be synthesized de novo chemically or via recombinant DNA methodologies.
  • the term antibody also includes antibody fragments produced by the modification of whole antibodies, those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv), humanized antibodies and those identified using phage display libraries (see, e.g., Knappik et al J. Mol Biol 296: 57-86 (2000), McCafferty et al, Nature 348:2-4 (1990)), for example.
  • any technique known in the art can be used in this invention (see, e.g., Kohler & Milstein, Nature 26:49-497 (1987); Kozbor et al, Immunology Today 4: 72 (1983); Cole et ⁇ /., ⁇ p. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1989).
  • an "anti-dopamine receptor" antibody is an antibody or antibody fragment that binds a polypeptide encoded by a dopamine receptor gene, cDNA, or a subsequence thereof.
  • the antagonist ofthe invention is an antisense molecule comprising the antisense of DNA encoding a dopamine receptor.
  • the invention in another embodiment, relates to a method of treating retinal cell degeneration comprising administering to the retina of a patient in need thereof a therapeutically effective amount of an antagonist of dopamine receptors in photoreceptor cells or any agent that depletes dopamine, as described above.
  • the antagonist or agent may be administered intraocularly, i.e. via injection through the vitreous or subretinal space, locally by insertion into the tissue surrounding the eye, systemically through an oral route or by subcutaneous, intravenous or intramuscular injection or via catheter or implant.
  • the antagonist or agent ofthe invention may be admininstered prior to the onset of a retina degenerative condition, to prevent its occurrence such as during eye surgery, or for the benefit of persons susceptible to a degenerative disease, immediately after the onset of a pathological condition or during the occurrence of an acute or protracted condition.
  • treating is meant preventing ofthe onset of degeneration or lessening, ameliorating, curing or at least partially arresting symptoms and/or complications. Amounts effective for this will depend on, e.g., the antagonist or agent composition, the manner of administration, the stage and severity ofthe disease being treated, the weight and general state of health ofthe patient, and the judgment ofthe prescribing physician.
  • an effective dose is an amount ofthe antagonist that is sufficient to inhibit mRNA transcription ofthe dopamine receptor.
  • the antagonist or agent ofthe invention may be formulated in a pharmaceutical composition.
  • the antagonist is admixed with a carrier or is suspended in a solution.
  • the compositions may further contain pharmaceutically acceptable auxilliary substances as required to approximate physiological conditions such as pH adjusting, and buffering agents, tonicity adjusting agents, wetting agents and the like. See Reminington 's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042, pages 1435-1712 of which is hereby incorporated by reference.
  • the antagonist or agent ofthe invention may be administered via liposomes.
  • Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
  • the retinal cells ofthe patient may be treated with the antagonist or agent ofthe invention ex vivo and then transplanted back into the retina ofthe patient.
  • the retinal cells may be transfected with a vector expressing DNA encoding the antagonist ofthe invention.
  • Photoreceptor cell transplantation studies designed to replace defective or lost cells due to retinal disease or damage have been performed successfully in animal models of retinal degeneration. See Silverman and Hughes, Invest. Ophthalmol Vis. Sci., 30: 1684-1690 (1989); Gouras et al, Neuroophthalmol.
  • the invention contemplates that photoreceptor cells may be obtained from donor eyes and maintained in culture as described herein. The cells would then be used as a source of purified photoreceptors to be transplanted via the subretinal space into the retina of patients suffering from retinal disease or damage. These patients will be treated with immunosuppressive therapies to eliminate immunological responses and rejection ofthe grafted cells.
  • the ex vivo donor retinas will be cultured in the presence ofthe antagonist or agent ofthe invention and perhaps other trophic factors, as described above, in order to enhance their growth and survival. Such growth factors could include CNTF, . BDNF, BFGF, or GDNF.
  • the invention relates to gene therapy, i.e. delivery of DNA that encodes an antagonist ofthe invention to target cells in the retina.
  • a nucleic acid construct containing such DNA maybe containined in an adeno- associate virus vector or other appropriate delivery vector.
  • Alternative viral vectors include, but are not limited to, retro virus, herpes simplex virus and papilloma virus vectors.
  • the invention relates to a retina organ culture comprising retinal tissue and a dopamine receptor antagonist or dopamine depleting agent.
  • a dopamine receptor antagonist is an antagonist of a member ofthe Dj or D 2 family of dopamine receptors.
  • the retina organ culture according to one embodiment is human retinal tissue, in another embodiment it is rd mouse tissue.
  • the antagonist is supiride or SCH- 23390 (RBI Signaling, Natick, MA).
  • the organ culture ofthe invention also may include trophic factors, such as ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), glia-derivied neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF).
  • trophic factors such as ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), glia-derivied neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF).
  • CNTF ciliary neurotrophic factor
  • BDNF brain-derived neurotrophic factor
  • GDNF glia-derivied neurotrophic factor
  • bFGF basic fibroblast growth factor
  • the dopamine depleting agent according to this embodiment is 6-OHDA.
  • Example 1 Protocol for Establishing Neonatal Mouse Isolated Retina Cultures Use pigmented rd mice pups. 1. On the bench top, anesthetize each pup by chilling on ice, and then sacrifice by decapitation.
  • the RPE (which is now mostly rolled up and separated from the retina) can be gently teased away from the retina using fine forceps. At this point the RPE can be collected, dissociated and cultured or discarded if not needed.
  • 11. Using a disposable transfer pipette in which the end has been cut to enlarge the opening, individually move each retina together with a few drops of media onto a Millipore Millicell- CM** (30mm)cell culture insert. Place one retina/insert and position photoreceptor side down. Suck off enough media to prevent the retina from floating around while attempting to tease it out flat (but not so much that the tissue sticks down prematurely and can not be manipulated).
  • Media for control tissue is DMEM plus 10% FCS plus Fungizone (1.25 ug/ml). Dopamine antagonists or dopamine depleting drugs are added to the media immediately prior to use.
  • Retinal organ culture procedures have been described previously in detail (Ogilvie et al, ]. Neurosci. Meth. 87: 57-65 (1999)). Retinas were isolated from retinal dystrophic (rd) or from wild type control mice on a C57BL/6 background at postnatal day 2. Animals were handled in accordance with institutional guidelines and the Society for Neuroscience Policy on the Use of Animals in Neuroscience Research. After separation from the retinal pigment epithelium, the isolated retina was placed photoreceptor side down on a Millicell-CM culture insert (Millipore, Bedford, MA) with media maintained at the level of the membrane interface. The cultures were maintained at 37°C, 5% CO 2 for 27 days.
  • Control media was comprised of Dulbecco's Modified Eagle's Media (DMEM, Gibco #11965, Rockville, MD) with 10% fetal calf serum (FCS, Summit Biotechnology, Ft. Collins, CO) and 1.25 ⁇ g/ml Fungizone (Sigma, St. Louis, MO).
  • DMEM Dulbecco's Modified Eagle's Media
  • FCS fetal calf serum
  • FCS fetal calf serum
  • D 20 nM SCH-23390
  • D 2 100 nM sulpiride dopamine receptors
  • organ cultures for quantitative analysis were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde overnight.
  • Tissue was postfixed in 1% osmium tetroxide followed by 1% uranyl acetate, rinsed, dehydrated and embedded in Epon-Araldite for histological evaluation of 1 ⁇ m sections.
  • organ cultures were fixed in 4% paraformaldehyde for 1 hr, rinsed, and cryoprotected in 30% sucrose overnight.
  • the tissue was frozen in O.C.T. (Sakura, Torrance, CA), cut into 8-10 ⁇ m cryostat sections, and stored at -80°C. Inimunocytochemistry.
  • Dopamine receptor antagonists increase photoreceptor survival
  • Inner and outer segment material in dopamine depleted cultures was comparable to that of normal retinal cultures and greater than that seen in cultures treated with dopamine antagonists.
  • the addition of 20 ⁇ M ADTN, a dopamine agonist, to organ cultures treated with 6-OHDA resulted in photoreceptor degeneration comparable to that seen in untreated rd retinal cultures (Fig. 5B).
  • Dopamine antagonists do not block terminal differentiation of opsin expression in photoreceptors
  • the rapid photoreceptor degeneration in the rd mouse retina makes this a good model for organ cultures that can be maintained for a limited time.
  • the early and rapid effects ofthe rd mutation result in pathology that begins as early as the first postnatal week, before photoreceptor differentiation is complete (Sanyal and Bal, Z.Anat. Entwickl-Gesch. 142: 219-238 (1973); Farber et al, Prog. Ret. Res. 13: 31-64 (1994). This makes it possible to increase survival of photoreceptors by blocking terminal differentiation ofthe phototransduction pathway.

Abstract

L'invention concerne un procédé destiné à augmenter la survie de photorécepteurs dans une culture d'organe, une culture de cellules et in vivo, lequel procédé se base sur l'utilisation d'antagonistes des récepteurs dopaminergiques ainsi que d'agents qui épuisent la dopamine.
PCT/US2001/013992 2000-05-02 2001-05-01 Composition et procedes servant a traiter la degenerescence de photorecepteurs WO2001083714A2 (fr)

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WO2008131368A2 (fr) 2007-04-20 2008-10-30 Acucela Inc. Composés dérivés de styrényle pour traiter des maladies et des troubles ophtalmiques
WO2009005794A2 (fr) 2007-06-29 2009-01-08 Acucela, Inc. Dérivés d'alcynylphényle pour traiter les maladies et les affections ophtalmiques
WO2009045479A1 (fr) 2007-10-05 2009-04-09 Acucela Inc. Composés d'alcoxy pour le traitement de maladies
US9133154B2 (en) 2013-03-12 2015-09-15 Acucela Inc. Substituted 3-phenylpropylamine derivatives for the treatment of ophthalmic diseases and disorders
US9447078B2 (en) 2012-01-20 2016-09-20 Acucela Inc. Substituted heterocyclic compounds for disease treatment
EP3229908A4 (fr) * 2014-12-12 2018-06-27 Schepens Eye Research Institute. Inc. Induction du gdnf pour le traitement de troubles de la rétine

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WO2008131368A2 (fr) 2007-04-20 2008-10-30 Acucela Inc. Composés dérivés de styrényle pour traiter des maladies et des troubles ophtalmiques
US8420863B2 (en) 2007-04-20 2013-04-16 Acucela, Inc. Styrenyl derivative compounds for treating ophthalmic diseases and disorders
US8653142B2 (en) 2007-04-20 2014-02-18 Acucela Inc. Styrenyl derivative compounds for treating ophthalmic diseases and disorders
US10201545B2 (en) 2007-04-20 2019-02-12 Acucela Inc. Styrenyl derivative compounds for treating ophthalmic diseases and disorders
US9314467B2 (en) 2007-04-20 2016-04-19 Acucela Inc. Styrenyl derivative compounds for treating ophthalmic diseases and disorders
US9421210B2 (en) 2007-04-20 2016-08-23 Acucela Inc. Styrenyl derivative compounds for treating ophthalmic diseases and disorders
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