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WO2012038565A2 - PROTÉINES DE LA FAMILLE Cry UTILISÉES COMME MARQUEURS POUR DÉTERMINER LE RISQUE DE DÉVELOPPER UNE α-SYNUCLÉINOPATHIE OU POUR DIAGNOSTIQUER CETTE MALADIE - Google Patents

PROTÉINES DE LA FAMILLE Cry UTILISÉES COMME MARQUEURS POUR DÉTERMINER LE RISQUE DE DÉVELOPPER UNE α-SYNUCLÉINOPATHIE OU POUR DIAGNOSTIQUER CETTE MALADIE Download PDF

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Publication number
WO2012038565A2
WO2012038565A2 PCT/ES2011/000282 ES2011000282W WO2012038565A2 WO 2012038565 A2 WO2012038565 A2 WO 2012038565A2 ES 2011000282 W ES2011000282 W ES 2011000282W WO 2012038565 A2 WO2012038565 A2 WO 2012038565A2
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seq
protein
identity
synucleinopathy
disease
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Spanish (es)
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WO2012038565A3 (fr
Inventor
Emilio FERNÁNDEZ ESPEJO
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Universidad de Sevilla
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Universidad de Sevilla
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • 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
    • 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
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Definitions

  • Proteins of the Crv family as markers to determine the risk of developing an ⁇ -synucleinopathy or determining said disease.
  • the present invention falls within the field of medical sciences and more specifically refers to a method for determining the risk of developing an ⁇ -synucleinopathy or determining said neurodegenerative disease, preferably Parkinson's disease, by detecting and / or quantifying Cry proteins, neurotoxic agents that can give rise to this disease.
  • neurodegenerative diseases are the consequence of abnormalities in the process of certain proteins (mainly the amyloid proteins synuclein and Tau) that intervene in the cell cycle and that, when accumulating in the nervous tissue, inside and outside the neurons, decrease or cancel their functions producing clinical manifestations, mainly dementia.
  • proteins mainly the amyloid proteins synuclein and Tau
  • a classification of neurodegenerative diseases is based on the presence of abnormal accumulations of the tau proteins and the a-synuclein protein giving rise to ⁇ -synucleinopathies and taupatias.
  • ⁇ -synucleinopathy is used to refer to those neurodegenerative diseases that have in common the abnormal deposition of a-synuclein in the cytoplasm of neurons or glial cells, or in the neuropil in the form of Lewy bodies or the like.
  • ⁇ -Synuclein is a presynaptic protein that seems to be involved in synaptic plasticity processes. The conformational and biochemical changes that this protein undergo determine cytoplasmic inclusions that characterize various disorders Neurodegenerative diseases including Parkinson's disease, Lewy body dementia and multisystemic atrophy.
  • Parkinson's disease is one of the most important neurodegenerative disorders, affecting almost 1% of the population over 65 who begins slowly and progressively, at a variable rate, for 10-20 years or more before end in severe disability and death.
  • Parkinson's disease is characterized by the progressive death of dopaminergic neurons of the black substance, resulting in a decrease in striatal dopamine concentration, conditioning the appearance of muscle stiffness, slowness of movement and tremor.
  • the symptomatology of PD is mainly due to the strong loss of dopamine neurons located in the black substance (greater than 80%), a midbrain nucleus that receives this name due to its dark appearance due to the high presence of the neuromelanin pigment.
  • the black substance through the nigrostriate pathway participates in the normal modulation of the movement. Dysfunction of this pathway due to lack of dopamine causes serious motor disorders, such as tremor at rest, akinesia, difficulty walking, normal balance disturbance, amymia (lack of facial expression), etc.
  • PET Positron emission tomography
  • 15 fluorodopa radioactive dopaminergic markers
  • Lewy bodies inclusions of 5 to 25 microns in diameter with a dense eosinophilic nucleus composed of deposits of proteins such as ubiquitins, ⁇ -synuclein and neurofilament residues and surrounded by a pale halo (Lewy FH, Pathologi Anatomie, 1912, 920- 933), are not only detected in the black substance but have also been seen in other places of the nervous system, such as the intestinal plexus, the olfactory bulb, mesencephalic centers and even in the cerebral cortex, suggesting the presence of a disease global neurodegenerative
  • pathogens that may be related to the appearance of PD have led to different theories that suggest that several pesticides such as rotenone, dieldrin, dithiocarbamates and 1-methyl-4-phenyl-1, 2,3 , 6-tetrahydropyridine (MPTP) a contaminant of heroin, cause parkinsonian conditions (Langston JW, Ballard PA Jr. N Engl J Med. 1983, 309 (5), 310), however, its environmental presence in the genesis of EP has not been proven.
  • MPTP 6-tetrahydropyridine
  • Lewy bodies would therefore be due to the action of an unknown pathogen capable of causing the alteration of the ⁇ -synuclein that would be deposited forming the proteinaceous nucleus of the Lewy body.
  • the pathogen could be a bacterium, a neurotrope virus (with affinity for nerve tissue) or a prion (pathogenic abnormal proteins perhaps similar to ⁇ -synuclein), but so far no pathogen capable of producing ⁇ -synucleinopathies has been identified and, specifically, EP.
  • the present invention relates to a method for determining the risk of developing an ⁇ -synucleinopathy (neurodegenerative disease that involves the formation of ⁇ -synuclein inclusions) or determining said neurodegenerative disease by the detection and / or quantification of Cry proteins, agents neurotoxics that can give rise to this disease.
  • the ⁇ -synucleinopathy is Parkinson's.
  • the The present invention relates to a Cry protein specific antibody or a fragment thereof for use as a biomarker to determine the risk of developing ⁇ -synucleinopathy or to determine said neurodegenerative disease.
  • the present invention shows how Cry proteins, delta-endotoxins from Bacillus thuringiensis (Bt), are capable of inducing a-synucleinopathy and more preferably Parkinson's disease, in a healthy individual.
  • Cry proteins were only known for their potent activity as an insecticide.
  • An important characteristic of the Cry proteins produced by Bt besides being an important alternative to the traditional chemical insecticides that generated environmental pollution problems, is that they were considered highly specific and safe for vertebrates and other non-target insects. Therefore, Bt has become the most widely used biological insecticide and transgenic plants that produce Cry toxins have been developed, and in particular CryIA toxin, which confer resistance to the plant against the attack of pest insects, making this toxin one of the most successful bioinsecticides worldwide.
  • the present invention reveals the toxic effects that these neurotoxins produce in animals and humans and provides a method for the diagnosis of ⁇ -synucleinopathies and more preferably PD, before the development of nerve degeneration and the onset of symptoms of disease based on the identification of Cry type oxidizing proteins in a biological tissue or fluid, where the biological fluid is preferably cerebrospinal fluid or plasma.
  • the present invention also provides a treatment capable of preventing or curing these diseases through passive immunotherapy based on vaccination with antibodies specific for Cry proteins.
  • the present invention solves the technical problem posed by the difficulty of finding a diagnostic method capable of identifying the pathology before the development of nerve degeneration and the appearance of the characteristic symptomatology associated with ⁇ -synucleinopathies and more preferably the Parkinson's disease, allowing among other applications:
  • a first aspect of the present invention relates to the method, hereinafter the method of the invention, for determining the risk of developing an ⁇ -synucleinopathy or determining said neurodegenerative disease comprising: a. obtain an isolated biological sample from an individual,
  • step (a) detect and / or quantify a protein with at least 65% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or with at least 55% identity with SEQ ID NO: 3 or a fragment thereof, in the sample obtained in step (a).
  • Said method of the invention is a method of obtaining useful data to determine the risk of developing an ⁇ -synucleinopathy or determining said neurodegenerative disease in an individual.
  • risk of developing an ⁇ -synucleinopathy refers to the predisposition of an individual to suffer or develop said neurodegenerative disease.
  • the method provides useful data to determine if an individual is developing or can develop a-synucleinopathy.
  • ⁇ -synucleinopathy refers to all those neurodegenerative diseases related to cognitive disorders due to an increase in the processes of cell death, reducing the number of neurons and generating changes in behavior, which have in common the abnormal deposition of ⁇ -synuclein in the cytoplasm of neurons or glial cells, or in the neuropil, forming inclusions of ⁇ -synuclein that may be in the form of Lewy bodies.
  • Such diseases are, but are not limited to, Shy-Drager disease, multisystemic atrophy, Pick's disease, Lewy body syndrome, dementia with Lewy bodies and Parkinson's disease.
  • a-synucleinopathy is Parkinson's disease.
  • isolated biological sample refers to, but is not limited to, tissues and / or biological fluids of an individual, preferably cerebrospinal fluid or plasma, obtained by any method known to a person skilled in the art.
  • individual refers to mammals, preferably humans or animals.
  • protein refers to any isolated or recombinant amino acid sequence comprising two or more amino acids linked together by peptide bonds or modified peptide bonds, that is, peptide isoesters.
  • % identity refers to the% identity between two amino acid sequences, that is, the number of amino acid positions over the total length of the sequence being compared, where all Amino acids in that position are identical.
  • the protein detected and / or quantified in step (b) of said method, described in previous paragraphs, can have at least 65, 66, 68, 70, 73, 75, 76, 77, 79, 80, 83, 85 , 87, 90, 93, 95, 97, 98, 99, 100% identity with respect to the sequence SEQ ID NO: 1 or the sequence SEQ ID NO: 2 and at least one 55, 56, 58, 60, 63 , 65, 67, 68, 70, 73, 75, 76, 77, 79, 80, 83, 85, 87, 90, 93, 95, 97, 98, 99, 100% identity with respect to the sequence SEQ ID NO: 3.
  • the protein detected and / or quantified in step (b) has at least 80% identity with respect to the sequence SEQ ID NO: 1 or the sequence SEQ ID NO: 2 or at least 70% identity with respect to to the sequence SEQ ID NO: 3. More preferably the protein detected and / or quantified in step (b) has at least 90% identity with respect to the sequence SEQ ID NO: 1 or the sequence SEQ ID NO: 2 or at least 80% identity with respect to the sequence SEQ ID NO: 3. Even more preferably said protein is SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • SEQ ID NO: 1 is the amino acid sequence of the CryIAb protein of Bacillus thuringiensis subspecies kurstaki strain HD-1 with accession number AAA22561, protein contained in Dipel n highest proportion
  • SEQ ID NO: 2 is the amino acid sequence of the CryIAa protein of Bacillus thuringiensis subspecies kurstaki strain HD -1 with accession number AAA22353
  • SEQ ID NO: 3 is the amino acid sequence ca of the Bacillus thuringiensis subspecies CryIAc protein kurstaki strain HD-1 with accession number AAD38701.
  • Table 1 shows the percentages of identity of the three proteins with each other. Data obtained through the use of the ClustalW2 program.
  • CryIAb amino acid sequence SEQ ID NO: 1 and accession number AAA22561
  • CryIAa amino acid sequence SEQ ID NO: 2 and accession number AAA22353
  • CryIAc amino acid sequence SEQ ID NO: 3 and accession number AAD38701.
  • Table 2 shows the percentages of protein identity belonging to subtype 1A with respect to the sequence protein SEQ ID NO: 1.
  • identity percentages of proteins belonging to subtype 1A with respect to the sequence proteins SEQ ID NO: 2 and SEQ ID NO: 3 respectively are shown in Tables 3 and 4.
  • Proteins with a percentage of identity with respect to the sequence SEQ ID NO: 1 or SEQ ID NO: 2 of at least 65% are isoforms or amino acid sequences that are homologous to SEQ ID NO: 1 or SEQ ID NO: 2 respectively in Bacillus thuringiensis bacteria and proteins with a percentage of identity with respect to the sequence SEQ ID NO: 3 of at least 55% are isoforms or amino acid sequences that are homologous to SEQ ID NO: 3 in the Bacillus thuringiensis bacteria.
  • detect and / or quantify refers to the detection of the presence and / or the measure of the amount or concentration, preferably semi-quantitative or quantitative.
  • the detection and / or quantification of the protein of the invention can be carried out by any method known in the state of the art.
  • the detection and / or quantification is carried out by immunoassay.
  • immunoassay refers to any analytical technique, based on the conjugation reaction of the amino acid sequence of the protein of the invention, its variants or a fragment thereof. with an antibody that recognizes them.
  • immunoassays known in the state of the art are, for example, but not limited to: immunoblot, enzyme-linked immunoadsorbent assay (ELISA), linear immunoassay (LIA), radioimmunoassay (RIA), immunofluorescence, immunohistochemistry or protein microarrays.
  • ELISA enzyme-linked immunoadsorbent assay
  • LIA linear immunoassay
  • RIA radioimmunoassay
  • immunofluorescence immunohistochemistry or protein microarrays.
  • the immunoassay can be an enzyme-linked immunoadsorbent assay or ELISA (Enzyme-Linked ImmunoSorbent Assay).
  • ELISA is based on the premise that an immunoreactive (biological sample antigen or antibody) can be immobilized on a solid support, then bringing that system into contact with a fluid phase containing the complementary reagent that can bind to a marker compound.
  • immunoreactive biological sample antigen or antibody
  • the ELISA may be an indirect ELISA, which may comprise the following steps: (a) coating a solid support with a cell lysate with the protein of the invention (b) incubating the coated support of step (a) with an isolated biological sample obtained from the individual under conditions that allow the formation of an immunocomplex of the antibody against the protein of the invention present in the sample, with the antigens comprising the protein of the invention; and (c) incubating with a secondary antibody, which recognizes the antibody against the amino acid sequence of the protein of the invention, conjugated or bound to a marker compound.
  • marker compound refers to a compound capable of giving rise to a chromogenic, fluorogenic, radioactive and / or chemiluminescent signal that allows the detection and / or quantification of the amount of the antibody against the amino acid sequence of the protein of the invention.
  • the marker compound is selected from the list comprising radioisotopes, enzymes, fluorophores or any molecule capable of being conjugated with another molecule or detected and / or quantified directly. This marker compound can bind to the antibody directly, or through another compound.
  • directly binding marker compounds are, but are not limited to, enzymes such as alkaline phosphatase or peroxidase, radioactive isotopes such as 33P or 35S, fluorochromes such as fluorescein or metal particles, for direct detection by colorimetry, auto-radiography, fluorimetry , or metallography, respectively.
  • the method of the invention further comprises comparing the data obtained in (b) with reference values to find a significant deviation.
  • the method of the invention further comprises attributing the significant deviation to the risk of developing an-synucleinopathy or the presence of said neurodegenerative disease in the individual.
  • the ⁇ -synucleinopathy is Parkinson's.
  • significant deviation refers to the presence of the protein of the invention or a greater concentration thereof in the isolated biological sample with respect to a sample of a healthy individual, is that is, an individual who does not present said protein or present it in a concentration at which the protein does not produce any toxic effects in the organism.
  • the deviation of the results obtained in section (b) is compared with respect to controls or reference values. Controls or reference values are, but are not limited to, those samples that do not present the proteins of the invention or present it at a concentration in which the protein is harmless.
  • mice Considering their neurotoxic levels in mice (example 1), their concentration in the plasma of a human individual so that they do not exert a toxic effect on it, should be less than 10 mg / l of blood.
  • This deviation is a significant deviation from the established statistical standards, such as, but not limited to, the deviation from the mean or median with respect to the control. However, the statistically significant deviation can be determined by any statistical technique known to the person skilled in the art.
  • Another aspect of the present invention relates to the use of the protein of the invention or of a specific antibody to the protein of the invention, or any fragment thereof, as a biomarker to determine the risk. of developing an ⁇ -synucleinopathy or to determine said neurodegenerative disease.
  • the ⁇ -synucleinopathy is Parkinson's.
  • biomarker or, alternatively, “molecular marker”, as used herein, refers to a molecule or the expression product of a gene or fragments and variants thereof that show substantial changes in a determined disease and that can be used both to determine the risk of developing the disease and to determine said disease by detecting the appearance of said changes in the biomarker.
  • biomarker can also be used to track the efficacy of a treatment for that disease by detecting changes in the biomarker as opposed to those that occur in the disease or clinical situation.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts) with the protein of the invention.
  • immunoglobulin M immunoglobulin M
  • IgD immunoglobulin D
  • IgG immunoglobulin G
  • IgA immunoglobulin A
  • IgE immunoglobulin E
  • Another aspect of the present invention relates to the use of a protein inhibitor agent of the invention for the manufacture of a medicament, hereinafter medicament of the invention, wherein said medicament has a therapeutically effective amount of inhibitor agent.
  • the inhibitory agent is capable of generating an immune response in an individual. More preferably the inhibitory agent is a protein specific antibody of the invention.
  • a preferred embodiment relates to the medicament of the invention, for the treatment and / or prevention of an ⁇ -synucleinopathy, where preferably the ⁇ -synucleinopathy is Parkinson's.
  • treatment refers to combating the effects caused as a result of a disease or pathological condition of interest in a subject (preferably mammal, and more preferably a human) that includes:
  • prevention consists in preventing the onset of the disease, that is, preventing the disease or pathological condition from occurring in a subject (preferably mammal, and more preferably a human), in particularly, when said subject has a predisposition for the pathological condition, but has not yet been diagnosed as having it.
  • an effective amount of a protein inhibitor agent of the invention is an amount sufficient to treat and / or prevent neurodegenerative disease.
  • a "therapeutically effective amount” of the protein inhibitor agent of the invention for treating and / or preventing a disease or disorder is an amount of sufficient inhibitory agent to reduce or eliminate the symptoms of the disease or disorder.
  • the effective amount of a given substance will vary with factors such as the nature of the substance, the route of administration, the size and species of the animal that will receive the substance and the purpose for which the substance is given. The effective amount in each individual case can be determined empirically by the person skilled in the art according to the procedures established in the art.
  • inhibitory agent or, alternatively, “inhibitor compound” as used herein refers to a molecule that, when bound or interacts with the protein of the invention, decreases or eliminates intensity or duration. of its biological activity. This definition also includes those compounds such as interfering RNAs that prevent or decrease the expression of the gene encoding the protein of the invention, that is, that prevent or diminish gene transcription, mRNA maturation, mRNA translation and post-translational modification.
  • An inhibitory agent may be constituted by a peptide, a protein, a nucleic acid or polynucleotide, a carbohydrate, an antibody, a chemical compound or any other type of molecule that diminishes or eliminates the effect and / or function of the protein in the invention.
  • the term "medicament of the invention”, as used herein, refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals.
  • the medicament referred to in the present invention can be for human or veterinary use.
  • the "medicine for human use” is any substance or combination of substances that is presented as having properties for the treatment or prevention of diseases in humans or that can be used in humans or administered to humans in order to restore, correct or modify physiological functions exerting a pharmacological, immunological or metabolic action, or establishing a medical diagnosis.
  • the "veterinary medicinal product” is any substance or combination of substances that is presented as having curative or preventive properties with respect to animal diseases or that can be administered to the animal in order to restore, correct or modify its physiological functions by exercising a pharmacological, immunological or metabolic action, or to establish a veterinary diagnosis.
  • veterinary drugs are "premezcias for medicated feed” prepared to be incorporated into a feed.
  • the term “medicament of the invention” refers to a protein-inhibiting agent of the invention, which is capable of generating an immune response against a given organism, which is causing said disease in man. or the animals
  • the medicament of the invention is a vaccine.
  • the term "vaccine” refers to an antigenic preparation used to establish the response of the immune system to a disease, antigen preparations that once inside the organism elicit the response of the immune system, through production of antibodies, and generate immunological memory producing permanent or transient immunity.
  • the vaccine may comprise pharmacologically acceptable excipients such as, but not limited to, an adjuvant.
  • the vaccine has a recombinant origin.
  • adjuvant refers to an agent, as long as it does not have an antigenic effect in itself, which can stimulate the immune system by increasing its response to the vaccine.
  • aluminum salts “aluminum phosphate” and “aluminum hydroxide” are the two adjuvants most commonly used in vaccines. Other substances, such as squalene, can also be used as adjuvants.
  • the medicament of the invention is presented in a form adapted to parenteral, cutaneous, oral, epidural, sublingual, nasal, intracatecal, bronchial, lymphatic, rectal, transdermal or inhaled administration.
  • the adapted form refers to how to adapt the medicament of the invention so that it can be administered for example but not limited, parenterally, orally, rectally or transdermally.
  • Parenteral administration refers to a physical state that can allow its injectable administration, that is, preferably in a liquid state.
  • Parenteral administration can be carried out by intramuscular, intra-arterial, intravenous, intradermal, subcutaneous or intraosseous administration but not limited to these types of parenteral administration routes.
  • the form adapted to oral administration is selected from the list comprising, but not limited to, drops, syrup, herbal tea, elixir, suspension, extemporaneous suspension, drinkable vial, tablet, capsule, granulate, seal, pill, tablet, tablet, tablet, troccus or lyophilized.
  • the form adapted to rectal administration is selected from the list comprising, but not limited to, suppository, rectal capsule, rectal dispersion or rectal ointment.
  • the form adapted to transdermal administration is selected from the list comprising, but not limited to, transdermal patch or iontophoresis.
  • the medicament of the invention comprises at least one excipient and / or at least one pharmacologically acceptable carrier. In another preferred embodiment the medicament of the invention comprises at least one other active ingredient.
  • excipient refers to a substance that helps the absorption of the medicament of the invention, stabilizes it or helps its preparation in the sense of giving it consistency or providing flavors that make it more pleasant.
  • the excipients could have the function of keeping the ingredients together such as starches, sugars or cellulose, sweetening function, coloring function, protection function of the medicament of the invention such as for example isolating it from the air and / or moisture, filling function of a tablet, capsule or any other form of presentation such as dibasic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.
  • a “pharmacologically acceptable vehicle” refers to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical forms of administration and includes, but are not limited to, solids, liquids, solvents or surfactants.
  • the carrier can be an inert substance or action analogous to any of the compounds of the present invention.
  • the function of the vehicle is to facilitate the incorporation of the medicament of the invention as well as other compounds, to allow a better dosage and administration or to give consistency and form. When the presentation form is liquid, the vehicle is the diluent.
  • pharmaceutically acceptable refers to the compound referred to being allowed and evaluated so as not to cause damage to the organisms to which it is administered.
  • active principle is any matter, whatever its origin, human, animal, plant, chemical or other, to which an appropriate activity is attributed to constitute a medicine.
  • composition of the invention which comprises at least one protein inhibitor agent of the invention,
  • the inhibitory agent is capable of generating an immune response in an individual.
  • the pharmaceutical composition is a set of components that is formed at least by a protein-inhibiting agent of the invention, which has at least one application in the improvement of the physical or physiological or psychological well-being of a subject, which implies an improvement of the general state of your health, for example a cosmetic application, although it may not imply a physiological effect on the organism, it implies an improvement in the well-being of the subject related to his psychology. Therefore, said pharmaceutical composition may be a personal hygiene product, a cosmetic product or a product that may constitute the basis for the preparation of the above products or the basis for the preparation of a medicament. Preferably the pharmaceutical composition is used for the preparation of a medicament.
  • the "personal hygiene product” is defined as the substances or preparations that, without having the legal consideration of medicines, medical devices, cosmetics or biocides, are intended to be applied to the skin, teeth or mucous membranes of the human body for the purpose of hygiene or aesthetic, or to neutralize or eliminate ectoparasites.
  • the "cosmetic product” is defined as any substance or preparation intended to be put in contact with the various surface parts of the human body (epidermis, hair and hair system, nails, lips and external genital organs) or with teeth and oral mucous membranes , with the exclusive or main purpose of cleaning them, perfuming them, modifying their appearance, and / or correcting body odors, and / or protecting them or keeping them in good condition.
  • the pharmaceutical composition of the invention further comprises at least one excipient and / or at least one vehicle. pharmacologically acceptable. In another preferred embodiment the pharmaceutical composition of the invention comprises at least one other active ingredient.
  • kit hereinafter kit of the invention, comprising at least one molecule indicating the presence of the protein of the invention.
  • the molecule is at least one antibody specific to the protein of the invention.
  • kits of the invention to determine the risk of developing an ⁇ -synucleinopathy or to determine said neurodegenerative disease, in an isolated biological sample of an individual, where the isolated biological sample can be, but without limited, a tissue or a biological fluid of said individual.
  • the kit of the invention provides useful data for determining the risk of developing an synucleinopathy or determining said neurodegenerative disease in an individual.
  • the biological sample is a biological fluid and more preferably cerebrospinal fluid or plasma.
  • the individual is, but not limited to, a mammal, preferably human or animal.
  • the ⁇ -synucleinopathy is Parkinson's.
  • FIGURES Fig. 1 Shows the levels of anti-oxidant molecules superoxide dismutase, glutathione, glutathione reductase and peroxidated glutathione.
  • IA It shows the levels measured in the black substance of rats exposed to Cry proteins (Dipel) or vehicle (control rats (not exposed to Cry proteins)) intraperitoneally six months after exposure.
  • the darkest bars represent the levels measured in the right black substance and the lighter bars represent the levels measured in the left black substance.
  • I B Displays the levels measured in the dorsal striatum of rats exposed to Cry ⁇ Dipel) or vehicle (control rats (not exposed to Cry proteins)) intraperitoneally, six months after exposure.
  • the darkest bars represent the levels measured in the right striatum and the lighter bars represent the levels measured in the left striatum.
  • Fig. 2 It shows the expression of tyrosine hydroxylase (TH) and neuropathological alterations in brain sections of 22 month old rats exposed to Cry proteins at six months of age. 2A. It shows an image of a rat mesencephalic section exposed to Cry proteins. Unilateral degeneration of the left black substance with intense TH neuronal loss (asterisk) is observed compared to the non-degenerated right black substance.
  • TH tyrosine hydroxylase
  • 2B-2D It shows degenerate regions of the black substance in 2A. Intense neuronal loss is shown with dystrophic and thickened neurites (2B), TH + neurons with strange shapes with dystrophic neurites (white arrows Figure 2C) and the boundary between the degenerated area (right in Figure 2D) and non-degenerated (left in the Figure 2D) of the black substance.
  • Fig. 3 It shows the expression of tyrosine hydroxylase (TH) and neuropathological alterations in rat brain sections of 22 months of age, exposed to Cry proteins at six months of age.
  • TH tyrosine hydroxylase
  • 3A It shows the mesencephalic section of a rat exposed to Cry proteins. Intense degeneration of the black substance is observed, compared to the contralateral black substance.
  • 3B It shows regional degeneration of the lateral end of the black substance (indicated by dashed line) in a rat exposed to Cry proteins.
  • 3C. 3B enlarged image. It shows intense neuronal loss with dystrophic and thickened (degenerated) neurites. 3D It shows the very degenerated black substance of another rat exposed to Cry proteins where only a few TH + neurons remain (white arrows) and numerous dystrophic neurites are observed in the black substance.
  • Fig. 4. Shows the expression of ⁇ -synuclein or eosinophilic inclusions in rats exposed to Cry proteins at six months of age.
  • SNc black substance pars compacta
  • SNr cross-linked pars black substance
  • 4G-4H It shows a staining with hematoxylin-eosin where the presence of neurons with dense eosinophilic cytoplasm (black arrowheads) in the black substance is observed (in 4H, the enlarged image of a eosinophilic neuron).
  • Fig. 5 It shows an ELISA and colorimetric analysis of the presence of Cry proteins in mesencephalic brain tissue (ventral segment and black substance) of rats exposed to Cry proteins at six months of age, and sacrificed at 22 months of age.
  • Solutions containing mesencephalic tissue from rats exposed to Cry proteins included in the Dipel bioinsecticide (formulation of the HD-1 strain of Bacillus thuringiensis subspecies kurstaki, containing 28% CryIAa, 53% CryIAb, 19% CryIAc and spores are shown in dark ).
  • the invention will now be illustrated by tests carried out by the inventors, which demonstrates the specificity and effectiveness of the method of the invention for determining ot-synucleinopathy, neurodegenerative disease that occurs with the formation of a-synuclein inclusions, and, specifically, Parkinson's disease (PD), through the detection and / or quantification of Cry protein.
  • PD Parkinson's disease
  • the Cry protein is capable of inducing an ⁇ -synucleinopathy, where the ⁇ -synucleinopathy is preferably EP.
  • the exposure of laboratory rats (Wistar lineage) to Dipel a commercial insecticidal composition containing the CryIA protein of Bacillus thuringiensis gave rise to an oxidative process in the nervous system accompanied by, among other pathologies, neuronal loss and appearance of intraneuronal deposits of a-synuclein similar to Lewy bodies that appear in neurodegenerative diseases type EP.
  • the following specific examples provided in this patent document serve to illustrate the nature of the present invention.
  • Dipel (formulation of the HD-1 strain of Bacillus thuringiensis subspecies kurstaki, containing 28% CryIAa, 53% CryIAb, 19% CryIAc and spores) was injected into laboratory rats of the Wistar line with six months of age for 20 days, by intraperitoneal injections. Dipel was injected at a daily dose of 1 mg / 100 g of weight.
  • the black substance was dissected under a microscope and froze immediately. Then, the tissue was thawed, and homogenized in a buffer (homogenous buffer) with 150 mM NaCI, 50 mM HEPES, 1 mM fluorinated phenylmethisulfonyl, 0.6 ⁇ leupeptin, and 1% Triton X-100, pH 7.4.
  • a buffer homogenous buffer
  • nitrotyrosine, nitric oxide synthetase, glutathione peroxidase, and superoxide dismutase were quantified with commercial Bioxytech kits (Oxis International Inc., Portland, OR, USA: Nitrotyrosine-EIA, NOS -22113, GR-340, cGPX-340, and SOD-525).
  • glutathione and glutathione-S-transferase levels were measured with BioVision incorporated kits (Mountain View, CA, USA: Glutathione Assay Kit (K264-100), and GST (K263-100)). Data were compared with one-way ANOVA followed by Bonferroni test.
  • mesencephalic tissue was obtained containing the black substance of rats treated with Dipel pesticide and control rats.
  • the kit was used according to the manufacturer's suggestions (Pathoscreen kit for Bt-Cry1 Ab / 1 Ac protein, peroxidase label, Agdia Incorporated, Indiana, USA).
  • the test gave a positive result (turn to blue; in Figure 5 dark wells) indicating the presence of Cry1Ab-Ac proteins.
  • the rats were sacrificed at 22 months of age, the brain was removed and stored in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.2-7.4) at 4 ° C, and then immersed overnight in 20% sucrose in PBS (phosphate buffered saline). Brain sections (30 ⁇ ⁇ thickness) were cut in a cryostat (Microm, Italy) and stored in PBS.
  • PB phosphate buffer
  • PBS-T Triton X-100
  • FCS bovine fetal serum
  • BSA bovine serum albumin
  • mice anti-tyrosine-hydroxylase monoclonal antibody anti-tyrosine-hydroxylase monoclonal antibody
  • mouse anti-alfasinuclein antibody from English mouse anti-alpha-synuclein antibody (1: 1000, Sigma-Aldrich) in PBS-T
  • biotin-conjugated mouse antibody from English anti-mouse biotin conjugated antibody
  • the ABC kit (1: 100, Pierce, USA) was then used, and the tissue was developed with diaminobenzidine 3,3'-tetrahydrochloride ⁇ DAB, Sigma-Aldrich, USA) as a chromogen, and hydrogen peroxide.
  • the Cavaleri principle and the physical dissector were applied according to the usual laboratory methodology (Fernández Espejo E., et al., J Neurosci 2001, 21, 9888-9895; El Banoua F, et al., Neurobiol Disease 2004, 16, 377-385; Galán-Rodr ⁇ guez B, et al., Neurobiol Disease 2008, 29, 529-542.
  • Oxidative stress in the black substance was detected bilaterally, although the degeneration of the black substance was partial or unilateral, indicating that other phenomena apart from oxidation must be involved in neurodegeneration.
  • these proteins are responsible for the appearance in rodents of a neurodegenerative condition characterized by the oxidative action of Cry proteins on the nigrostriated circuit and other short-term catecholaminergic centers, and the rest of pathological characteristics of diseases neurodegeneratives that occur with the formation of ⁇ -synuclein inclusions (or ⁇ -synucleinopathies) and, in particular, with long-term PE, including: degeneration of the black substance with neuronal death of dopamine neurons as well as of other catecholaminergic centers such as dorsal raphe and the presence of intraneuronal deposits of ⁇ -synuclein in the cytoplasm of dopamine neurons of the black substance.

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Abstract

La présente invention concerne une méthode pour déterminer le risque de développer une α-synucléinopathie ou pour diagnostiquer cette maladie neurodégénérative, par détection et/ou quantification de protéines Cry, agents neurotoxiques pouvant donner lieu à ladite maladie. De préférence, cette α-synucléinopathie est la maladie de Parkinson.
PCT/ES2011/000282 2010-09-21 2011-09-20 PROTÉINES DE LA FAMILLE Cry UTILISÉES COMME MARQUEURS POUR DÉTERMINER LE RISQUE DE DÉVELOPPER UNE α-SYNUCLÉINOPATHIE OU POUR DIAGNOSTIQUER CETTE MALADIE Ceased WO2012038565A2 (fr)

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