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US20120156221A1 - Method and Pharmaceutical Composition for Use in the Treatment of Neurodegenerative Disorders - Google Patents

Method and Pharmaceutical Composition for Use in the Treatment of Neurodegenerative Disorders Download PDF

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US20120156221A1
US20120156221A1 US13/379,007 US201013379007A US2012156221A1 US 20120156221 A1 US20120156221 A1 US 20120156221A1 US 201013379007 A US201013379007 A US 201013379007A US 2012156221 A1 US2012156221 A1 US 2012156221A1
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basigin
compound
activates
disease
cells
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Ludivine Perrocheau
Alain Van Dorsselaer
Jose Alain Sahel
Thierry Leveillard
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Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/52—Isomerases (5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders

Definitions

  • the invention relates to a compound which activates the BASIGIN signaling pathway for the treatment of a degenerative disorder.
  • the invention relates to a BASIGIN agonist for the treatment of a neurodegenerative disorder.
  • Neurodegenerative disorders have provided a challenge for many years, in both basic research and clinical contexts.
  • retinitis pigmentosa is a genetically heterogeneous retinal degeneration characterized by the sequential degeneration of a population of neurons corresponding to rod and cone photoreceptors.
  • the RP first clinical signs are night blindness and narrowing of the peripheral field of vision which progressively worsens to become “tunnel-like”.
  • the central vision is reduced to complete blindness in most cases.
  • the retinal rod photoreceptors involved in night and side visions slowly degenerate.
  • the cone photoreceptors responsible for both color and high-contrast vision, visual acuity, detail perception and normal light vision are similarly affected.
  • the retinal degeneration 1 (rd1) mouse is the most studied animal model for retinitis pigmentosa. It carries a recessive mutation in the rod-specific cGMP phosphodiesterase beta subunit gene leading to rod photoreceptor death through apoptosis (Carter-Dawson et al., 1978 ; Portera-Cailliau et al., 1994) followed by cone death presumably through lack of trophic support (Mohand-Said et al., 1998).
  • the RdCVF gene also called thioredoxin-like 6 (Txnl6) and nucleoredoxin 1 (Nxnl1), encodes the Q8VC33 UniProt protein, which has limited similarity to the thioredoxin superfamily and which exerts trophic activity on cone photoreceptors (Leveillard et al., 2004).
  • Thioredoxins (TXN) are usually small proteins which can be involved with pleiotropic activities such as redox control, regulation of apoptosis and cytokine activity (Holmgren, 1985; Holmgren, 1989; ARNER and Holmgren, 2000).
  • the TXN conserved active site contains two distinct cysteines (CXXC) that contribute to a thiol-oxydoreductase activity (Arner and Holmgren, 2000, Powis and Montfort, 2001) catalyzes the reduction of disulfide bonds in multiple substrate proteins (Holmgren, 1979; Holmgren, 1979).
  • CXXC cysteines
  • the RdCVF gene encodes two products via alternative splicing: a full length protein and a C-terminal truncated protein sharing similarities with TRX80.
  • This latter form of human thioredoxin-1 (Txn) (Pekkari et al., 2000; Pekkari et al., 2005; Liu et al., 2005) has no thiol-reductase activity but is involved in controlling growth of peripheral mononuclear blood cells (Pekkari et al., 2000; Pekkari et al., 2003).
  • RdCVF looks like a bifunctional gene because it encodes both a long form (RdCVF-L, 217 aa, Q8VC33) having a putative thiol-oxydoreductase activity (Jeffery, 1999; Jeffery, Trends Genet., vol.19(8):415-417, 2003) and a short form (RdCVF-S, 109 aa, Q91 W38) with trophic activity for cones but no redox activity.
  • RdCVF receptor has not yet been identified.
  • BASIGIN also known as HT7, 5A11, EMMPRIN or CD147
  • RdCVF receptor also known as HT7, 5A11, EMMPRIN or CD147
  • a first object of the invention relates to a compound which activates the BASIGIN signaling pathway for the treatment of a neurodegenerative disorder.
  • Another object of the invention relates to a pharmaceutical composition for the treatment of a neurodegenerative disorder comprising a compound which activates the BASIGIN signaling pathway and a pharmaceutically acceptable carrier.
  • Yet another object of the invention relates to a method for screening a compound which activates the BASIGIN signaling pathway, in particular a BASIGIN agonist, for the treatment of neurodegenerative disorders.
  • Another object of the invention relates to the use of BASIGIN in a method for screening a drug for the treatment of a neurodegenerative disorder.
  • BASIGIN also known as HT7/5A11/EMMPRIN/CD147, refers to a member of the immunoglobulin superfamily, with a structure related to the putative primordial form of the family (Miyauchi et al., 1991; Kanekura et al., 1991).
  • BASIGIN is a type I integral membrane receptor that has many ligands, including the cyclophilin (CyP) proteins Cyp-A and CyP-B and certain integrins. It is expressed by many cell types, including epithelial cells, endothelial cells and leukocytes.
  • the human BASIGIN protein contains 269 amino acids that form two heavily glycosylated C2 type immunoglobulin-like domains at the N-terminal extracellular portion.
  • a second form of BASIGIN has also been characterized that contains one additional immunoglobulin-like domain in its extracellular portion.
  • the term “compound which activates the BASIGIN signaling pathway” refers to a molecule which results in increased signaling through BASIGIN, whether by up-regulating the levels of BASIGIN present on the cell surface, or by stimulating the downstream signaling cascade by binding to BASIGIN.
  • Methods for assessing the level of activation of the BASIGIN signaling pathway can include the measurement of the amount of downstream second messenger in the BASIGIN signaling pathway.
  • Compounds which activate the BASIGIN signaling pathway according to the invention include molecules which increase the levels of the BASIGIN protein on the cell surface, such as the BASIGIN polypeptide, a nucleic acid encoding BASIGIN, or a vector comprising a nucleic acid encoding BASIGIN.
  • BASIGIN agonists Compounds which activate the BASIGIN signaling pathway can also include BASIGIN agonists.
  • BASIGIN agonist or “agonist of BASIGIN” refers to a molecule which activates signaling through the BASIGIN receptor, by binding to BASIGIN. Examples of such agonists of BASIGIN include, but are not limited to, cyclophilin A and cyclophilin B.
  • an agonist of BASIGIN according to the invention is not RdCVF1 or RdCVF2.
  • treating means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.
  • neurodegenerative disorder refers to a disease associated with the degeneration of neurons such as degenerative disorders of the central nervous system, retinal degenerative disorders, or degenerative disorders of the olfactory neurons.
  • neurodegenerative disorders according to the invention include, but are not limited to, alcoholism, Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple System Atrophy, Narcolepsy
  • a first object of the invention relates to a compound which activates the BASIGIN signaling pathway for the treatment of a neurodegenerative disorder.
  • said compound which activates the BASIGIN signaling pathway is the human BASIGIN polypeptide referenced under Genpept accession number EAW61182 or an variant thereof having at least 90% identity with EAW61182, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with EAW61182.
  • said BASIGIN agonist is a nucleic acid encoding BASIGIN, or a vector comprising a nucleic acid encoding BASIGIN.
  • said compound which activates the BASIGIN signaling pathway is a BASIGIN agonist.
  • said BASIGIN agonist may be a low molecular weight agonist, e. g. a small organic molecule (natural or not).
  • small organic molecule refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
  • BASIGIN agonist of the invention may consist in an antibody which activates BASIGIN or an antibody fragment which activates BASIGIN.
  • Antibodies directed against BASIGIN can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • Various adjuvants known in the art can be used to enhance antibody production.
  • antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.
  • Monoclonal antibodies against BASIGIN can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture.
  • Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985).
  • techniques described for the production of single chain antibodies can be adapted to produce anti-BASIGIN single chain antibodies.
  • BASIGIN agonists useful in practicing the present invention also include anti-BASIGIN antibody fragments including but not limited to F(ab′) 2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab′) 2 fragments.
  • Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to BASIGIN.
  • Humanized anti-BASIGIN antibodies and antibody fragments therefrom can also be prepared according to known techniques.
  • “Humanized antibodies” are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • BASIGIN agonists may be selected from aptamers.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by EXponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).
  • Another object of the invention relates to a method for treating a neurodegenerative disorder comprising administering to a subject in need thereof with a compound which activates the BASIGIN signaling pathway as described above.
  • the invention relates to a method for treating a neurodegenerative disorder comprising administering to a subject in need thereof a BASIGIN agonist as above described.
  • Compounds of the invention may be administered in the form of a pharmaceutical composition, as defined below.
  • said compound which activates the BASIGIN signaling pathway preferably said agonist of BASIGIN, is administered in a therapeutically effective amount.
  • a “therapeutically effective amount” is meant a sufficient amount of compound to treat and/or to prevent neurodegenerative disorder.
  • the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • Compounds according to the invention may be used for the preparation of a pharmaceutical composition for the treatment of a neurodegenerative disorder.
  • the present invention also provides a pharmaceutical composition comprising an effective dose of a compound which activates the BASIGIN signaling pathway, preferably a BASIGIN agonist, according to the invention.
  • Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • compositions for example, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
  • compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
  • compositions include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.
  • compounds of the invention which activate the BASIGIN signaling pathway can be further identified by screening methods as hereinafter described.
  • Another object of the invention relates to a method for screening a compound which activates the BASIGIN signaling pathway.
  • the invention provides a method for screening a BASIGIN agonist for the treatment of a neurodegenerative disorder.
  • the screening method may measure the binding of a candidate compound to BASIGIN, or to cells or membranes bearing BASIGIN, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound.
  • a screening method may involve measuring or, qualitatively or quantitatively, detecting the competition of binding of a candidate compound to the receptor with a labelled competitor (e.g., antagonist).
  • screening methods may test whether the candidate compound results in a signal generated by an agonist of BASIGIN, using detection systems appropriate to cells bearing the receptor.
  • the screening method of the invention comprises the step consisting of:
  • the receptor BASIGIN used in the screening method may be its orthologs and derivatives as defined in the present invention.
  • such screening methods involve providing appropriate cells which express BASIGIN, its orthologs and derivatives thereof on their surface.
  • a nucleic acid encoding BASIGIN may be employed to transfect cells to thereby express the receptor of the invention. Such a transfection may be accomplished by methods well known in the art.
  • neurons are selected from the group consisting of cone photoreceptor, neurons, retina cells, retinoblastoma and other immortalized neuronal cell lines of any species (mouse, human . . . ).
  • the screening method of the invention may be employed for determining an agonist by contacting such neurons with compounds to be screened and determining whether such compound activates the receptor.
  • the determination of the activation of BASIGIN can be assessed by determining the neuron viability.
  • a compound is deemed to increase neuron viability if it is positive in any one the methods described below as examples of neuron rescue activity.
  • the determination of the activation of BASIGIN can be determined by analysing the downstream molecular signaling pathway. Indeed, it has been shown that stimulation of BASIGIN by cyclophilin A results in neuron protection via signaling through Erk1/2 (Boulos et al., Neurobiology of Disease, vol 25(1), p54-64, 2007). Hence, determining the activation of BASIGIN can be carried our by monitoring the phosphorylation status of Erk.
  • the candidate compound of may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo or natural compounds.
  • the candidate compound may be selected from the group of (a) proteins or peptides, (b) nucleic acids and (c) organic or chemical compounds (natural or not).
  • libraries of pre-selected candidate nucleic acids may be obtained by performing the SELEX method as described in documents U.S. Pat. No. 5,475,096 and U.S. Pat. No. 5,270,163.
  • the candidate compound may be selected from the group of antibodies directed against BASIGIN.
  • Such the method may be used to screen BASIGIN agonists according to the invention.
  • the invention relates to the use of BASIGIN polypeptide in a method for screening a drug for the treatment of neurodegenerative disorders.
  • FIG. 1 Specific binding for the cone-enriched culture: Iodinated human RdCVF peptide was incubated with a. cone-enriched cultures from chicken embryo, b. retinal pigmented epithelial cells from pig, c. Cos-1 cells. The dash line in a shows the specific binding after competition with the mouse unlabelled peptide
  • FIG. 2 Purification of the RdCVF receptor by far-western blotting assay: Fractions from chicken retina and embryonic fibroblasts (CEF). S, solubles. M, membrane fractions. T total. Lane 2-4 coomassie staining. Lanes 5-14 farwestern blotting with GST-RdCVFL, GST-RdCVF and GST as indicated
  • FIG. 3 Migration of the receptor BASIGIN on SDS-PAGE: Analysis by western blotting of retinal extracts (taken from published work)
  • hRdCVF Human RdCVF
  • mRdCVF mouse RdCVF
  • the chloramine T method was used to label hRdCVF with 125 I with a specific activity of 2130 Ci/mmol.
  • Chick retina cells were isolated and cultured as previously described (Adler and Hatlee, 1989; Fintz et al, 2003) with minor modifications. The cells were plated on poly-L-lysine coated 24-well plates at 3 ⁇ 10 5 cells/cm 2 (6 ⁇ 10 5 /well) and cultured for 24 h in serum-containing medium.
  • the cells were rinsed three times then, 50 ⁇ l binding buffer (control) or 50 ⁇ l binding buffer containing unlabelled mRdCVF (to determine non-specific binding) was added to each well of the culture. After incubation for 30 min at room temperature, 50 ⁇ l binding buffer containing [ 125 I]-hRdCVF was added to each well. After incubation for 90 min at room temperature, the plates were incubated at 4° C. for 45 min. The cells were rinsed three times and solubilised with 1% SDS. The radioactivity of the extracts was counted using a gamma counter. The specific binding was measured by competition assay with excess unlabelled recombinant mouse RdCVF.
  • the IC50 was estimated to 35 nM (5.7 to 210 nM, 95% confidence limits). A rough estimation of the number of saturable binding sites, obtained by extrapolation of the binding parameters, gives 54,000 per cell (5,300 to 460,000, 95% confidence limits). This confirms that a specific binding activity for RdCVF is expressed by cones.
  • the band at about 60 kDa, detected in lane 6, has a broader range of expression since it is also detected in the membrane fraction of chicken embryonic fibroblast (triangle in lane 8).
  • the short isoform detects a specific band smaller than 45 kDa in the membrane fraction of chicken retina (+in lane 10).
  • the coomassie staining of these fractions shows that there is no prominent band at 45 kDa (lanes 2-4). Taken together these results indicate the existence of a candidate RdCVF interacting protein in the membrane fraction of the chicken retina migrating at 45 kDa.
  • a short list of 33 candidate proteins was established by removing unlikely candidates as the three retinal protein cited and other abundant and most likely contaminating proteins as enolase, creatine kinase, beta actin, mitochondrial ATPase, translation initiation factor, aldolase, Glucose 3P deshydrogenase, glucose phosphate isomerase, pyruvate kinase, heterogenous nuclear ribonucleoproteins as described in Casuvoglu et al. (2003).
  • the abundance of proteins linked to the inner membrane of the cells as G-protein probably reflects the abundance of proteins associated with cell surface receptors in our preparation.
  • TM transmembrane domain
  • the cell surface glycoprotein BASIGIN was identified was originally identified as a blood brain barrier protein identical to the 5A11 antigen that mediates the cell-cell recognition in the avian retina (Fadool and Linser, 1993), a monoclonal antibody generated by immunizing mice with live cells dissociated from isolated day 7 embryonic chick retina (Linser and Perkins, 1987). Those cells are very similar to the day 6 embryonic chick retina used as cone-enriched culture system (Fintz et al., 2003; Léveillard et al., 2004). The migration of this glycoprotein on SDSPAGE is very similar to the RdCVF interacting protein as seen in a figure taken from the work of Fadool and Linser ( FIG. 3 ). Interestingly, in that report the authors show that BASIGIN antibody (clone 5A11) inhibits retina cell reaggregation in vitro supporting the role of
  • BASIGIN in cell-cell interactions We have identified two clones encoding BASIGIN from an expression library constructed with RNA prepared from our cone-enriched cultures. The presence of these two clones out of a total of 1000 EST sequenced within the frame of an ongoing collaboration with the Génoscope (http://www.genoscope.cns.fr/spip/Mus-musculusdengeneration- of.html) indicates that most likely BASIGIN is expressed by cones.
  • the BASIGIN agonists described in the present invention are tested for their ability to rescue neurons according to the following protocols:
  • the primary culture is a cone-enriched primary cell culture system from chicken embryo (60-80% of cones) as described in Fintz et al. (Invest. Ophtamol. Vis. Sci, vol 44(2): 818-825, 2003.)
  • mice are killed by decapitation.
  • the posterior part of the nasal septum is dissected free of the nasal cavity and immediately placed in ice-cold DMEM containing 50 ⁇ g/ml gentamicin (Eurobio; Gibco) and 10 (v/v) fetal calf serum (eurobio).
  • the cartilage of the septum is removed and the olfactory mucosa is incubated for 30 min at 37° C. in a 2.4 U/MI dispase II solution (Roche.
  • the olfactory epithelium is carefully separated from the underlying lamina intestinal under the dissection microscope and gently triturated about 20 times to separate the cells.
  • the resulting cell suspension is transferred to a 50 ml conical tube and the dispase is inactivated by adding 40 ml of HBSS without calcium and magnesium.
  • the cell suspension is centrifuged at 700 rpm for 5 min, and the pellet containing the cells is resuspended in a medium composed of DMEM containing insulin (10 ⁇ g/ml, Sigma), transferin (10 ⁇ g/ml, Sigma), selenium (10 ⁇ g/ml, Sigma), calf foetal serum (5%), ascorbic acid (200 ⁇ M).
  • DMEM containing insulin
  • transferin 10 ⁇ g/ml, Sigma
  • selenium 10 ⁇ g/ml, Sigma
  • calf foetal serum 5%
  • ascorbic acid 200 ⁇ M
  • Cells are plated on 12 mm sterile glass coverslips coated with 5 ⁇ g/cm 2 human collagen IV (Sigma); thus providing a primary culture of Olfactory Sensitive Neurons (OSN).
  • brains are dissected out into cold Grey's balanced salt solution containing 5 mg/ml glucose, and the meninges are removed. Cerebellar parasagittal slices (35° or 250 ⁇ m thick) are cut on a Mcllwain tissue chopper and transferred onto membrane of 30 mm Millipore culture inserts with 0.4 ⁇ m pore size (Millicel; Millipore, Bedford, Mass.). Slices are maintained in culture in 6-well plates containing 3 ml of medium at 35° in an atmosphere of humidified 5% CO 2 .
  • the medium is composed of 50% basal medium with Earle's salts (Invitrogen), 25% HBSS (Invitrogen), 25% horse serum (Invitrogen), L-glutamine (1 mM) and 5 mg/ml glucose (Stoppini et al., J Neurosci Methods, vol 37(2), p173-82, 1991).
  • Serum-free preparation of mouse cortical primary cultures is performed with mouse at postnatal day 1. After removal of the meninges, entire cortices are mechanically dissociated un a phosphate buffer saline glucose solution without added divalent cations (100 mM NaCl, 3 mM KCl, 1.5 mM KH2PO4, 7.9 mM Na2HPO4, 33 mM glucose, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin) and resuspended in Neurobasal medium (Invitrogen) containing 2% B27 supplement (Gibco), 0.5 mM glutamine; and 25 ⁇ M glutamate. Cells are then cultured onto poly-ornithine coated coverslips to produce cultures highly enriched in cortical neurons.
  • divalent cations 100 mM NaCl, 3 mM KCl, 1.5 mM KH2PO4, 7.9 mM Na2HPO4, 33 mM glucose, 100 U/ml

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EP4201425A4 (fr) * 2020-08-21 2024-09-04 Yamaguchi University Agent d'ouverture réversible pour barrière vasculaire du système nerveux

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US10004780B2 (en) * 2012-10-17 2018-06-26 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods and pharmaceutical compositions for the treatment of age-related macular degeneration (AMD)
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