WO2019043279A1 - Compositions pouvant moduler la stimulation de gdnf endogène pour le traitement de maladies neurodégénératives - Google Patents

Compositions pouvant moduler la stimulation de gdnf endogène pour le traitement de maladies neurodégénératives Download PDF

Info

Publication number
WO2019043279A1
WO2019043279A1 PCT/ES2018/070577 ES2018070577W WO2019043279A1 WO 2019043279 A1 WO2019043279 A1 WO 2019043279A1 ES 2018070577 W ES2018070577 W ES 2018070577W WO 2019043279 A1 WO2019043279 A1 WO 2019043279A1
Authority
WO
WIPO (PCT)
Prior art keywords
agn
kit
gpr83
tacr3
mc3r
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/ES2018/070577
Other languages
English (en)
Spanish (es)
Inventor
José LÓPEZ BARNEO
Xavier DÁNGLEMONT DE TASSIGNY
Daniel ENTERRÍA MORALES
Ivette LÓPEZ LÓPEZ
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universidad de Sevilla
Servicio Andaluz de Salud
Original Assignee
Universidad de Sevilla
Servicio Andaluz de Salud
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidad de Sevilla, Servicio Andaluz de Salud filed Critical Universidad de Sevilla
Publication of WO2019043279A1 publication Critical patent/WO2019043279A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs

Definitions

  • compositions capable of modulating the stimulation of endogenous GDNF for the treatment of neurodegenerative diseases
  • the present invention is within the field of molecular biology and medicine, and specifically relates to a composition capable of modulating the stimulation of endogenous GDNF for the treatment of neurodegenerative diseases, and more specifically for the treatment of Parkinson's disease.
  • EP It also refers to a method of selecting drugs useful in the treatment of neurodegenerative diseases and to a method for the collection of useful data in the diagnosis of said diseases.
  • PD Parkinson's disease
  • DA dopaminergic
  • neuroprotective agents are currently being evaluated as a means to slow the progression of the disease.
  • the use of the neurotrophic factor derived from the glia cell line (GDNF) maintained high expectations of preclinical studies, but its exogenous administration in clinical trials proved to be more problematic.
  • GDNF occurs naturally in discrete brain areas where its trophic action guarantees the survival of nigrostriatal DA neurons (1).
  • GDNF In the striatum of rodents, more than 95% of GDNF is produced by a restricted subpopulation of GABAergic interneurons characterized by their high content of parvalbumin (PV) (2).
  • PV parvalbumin
  • a strategy for the treatment of neurodegenerative diseases, and specifically PD would be to use therapeutic agents that increase the endogenous levels of GDNF.
  • a first aspect of the invention relates to an agent that modulates the activity of the GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, PDE3A, SPATA18, and / or LHX8 genes, or the GPR83, KIT proteins.
  • the modulating agent of the invention is a substance that interacts with the GPR83 receptor, KIT, TACR1, TACR3, MC3R and / or the LHX8 protein by favoring the production and / or release of endogenous GDNF,
  • a second aspect of the invention relates to a composition comprising a modulating agent of the invention for use in the treatment of a neurodegenerative disease, preferably Parkinson's disease.
  • a third aspect of the invention relates to a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegenerative disease, preferably Parkinson's disease, comprising: a) contacting the compound to be analyzed with the GPR83 polypeptide, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8, b) detect the binding of said compound to be analyzed with the polypeptide GPR83, KIT, TACR1, TACR3, MC3R, RARRES2 , CRABP1, MOXD1, SPATA18, and / or LHX8, where the compounds that bind to the GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SP
  • a fourth aspect of the invention relates to a method of selecting therapeutic agents useful in the prevention, amelioration and / or treatment of a neurodegenerative disease, preferably Parkinson's disease: a) determining the activity of GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1,, SPATA18, and / or LHX8 at an established concentration of the compound to be analyzed or in the absence of said compound, b) determine the activity of GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1,, SPATA18, and / or LHX8 at a concentration of the compound to be analyzed different from that of a).
  • a fifth aspect of the invention relates to a method for collecting data useful in the diagnosis and / or prognosis of a neurodegenerative disease, preferably Parkinson's disease which comprises: a) determining the expression of GPR83, KIT, TACR1, TACR3 , MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8 in a sample extracted from a mammal, b) compare the expression values of GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8 obtained in a) with the standard values in healthy or diseased mammals.
  • Fig. 1 Experimental strategy used to compare the PV neurons of the striatum (ST) and the cortex (CTX).
  • ST striatum
  • CTX cortex
  • Fig. 2 Grouping of selected genes showing enrichment in striatum (ST) versus cortex (CTX) of PV-Cre mice; tdTomato (left) or PV-tdTomato (right).
  • Fig. 3 Expression level of selected genes (measured by QPCR) for membrane receptors [a], cytoplasmic proteins involved in cell signaling and metabolism [b], or transcription factor [c].
  • Fig. 4 Expression of the c-Kit protein in the parvalbumin neuron (PV).
  • the microphotographs show colocalization of cKit (green) and PV (red) in the striatum.
  • the quantification indicates that 99% of the PV + neurons are cKit +, whereas only 18% of the cKit + cells are not PV neurons.
  • Intrastriatal administration of 2nmol mPEN increases the expression of Gdnf mRNA in the ipsilateral striatum (injection site at 2 hours in front of the vehicle (Veh)).
  • Fos mRNA an indicator of activation of neurons
  • the contralateral side shows no evidence of neuronal activation (Fos) and the level of Gdnf mRNA does not change.
  • the levels of Gdnf and Fos mRNA are significantly higher at the injection site (INJ, red) compared to the Sham side (blue) in the same animal.
  • the stimulating effect of mPEN disappears after 6 hours. In this experiment, anesthesia induces a neuronal inactivation (drop in Fos expression at 6 hours), which could hide a longer effect of mPEN on Gdnf expression.
  • the authors of the present invention have analyzed the transcriptome differences between the PV neurons producing GDNF (in the striatum, ST) and other PV neurons without GDNF (in the proximal isotox, CTX).
  • Transgenic mice were used to capture the tdTomato fluorescent PV neurons by fluorescence activated cell sorting (FACS).
  • Transcriptome analysis based on ST PV and CTX microarray and quantitative RT-PCR validation against whole tissue expression allowed the selection of membrane receptors, metabolic proteins and transcription factors that are specifically expressed in striate PV interneurons . These are excellent candidates whose activation could modulate the production of striatal GDNF.
  • a first aspect of the invention relates to the use of an agent that modulates the activity of the GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8 genes, or the GPR83, KIT proteins. , TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18 and / or LHX8 from now on the modulating agent of the invention, or any combination thereof, in the preparation of a medicament for the treatment of neurodegenerative diseases.
  • the neurodegenerative disease is PE.
  • Gpr83 or "G protein-coupled receptor 83” (also called in the GIR or GPR72 literature) as used herein refers to the G-83 protein-coupled receptor, which is the PEN peptide specific receptor, derived from the peptide proSAAS (see Gomes et al 2016, Sci Signal, 2016 Apr 26; 9 (425): ra43, DOI: 10.1126 / scisignal.aad0694).
  • Gpr83 refers to both the gene and the human GPR83 protein.
  • Gpr83 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the "Gpr83” protein, and which would comprise various variants from: a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, b) nucleic acid molecules whose complementary strand hybridizes with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / or b) to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with at least 80%, 90%, 95%, 98% or 99% identity with the SEQ ID NO: 1. in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the
  • Kif also referred to in the PBT, C-Kit, CD117 or SCFR literature
  • KIT refers to the mast cell / stem cell growth factor receptor
  • SCFR mast cell / stem cell growth factor receptor
  • MGF massive cell growth factor
  • KIT refers to both the gene and the human KIT protein.
  • KIT is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence for the "KIT” protein, and which would comprise various variants from: a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, b) nucleic acid molecules whose complementary strand hybridizes with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 4. in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the KIT protein.
  • Other nucleic acid molecules that encode a polypeptide comprising the amino acid sequence
  • TACR1 or "tachykinin receptor 1" (also referred to in the literature as SPR; NK1 R; NKIR; TAC1 R) as used herein refers to a gene belonging to a gene family of tachykinin receptors. These tachykinin receptors are characterized by interactions with G proteins and contain seven hydrophobic transmembrane regions. This gene encodes the receptor for substance P and neurokinin 1.
  • TACR1 refers to both the gene and the human "TACR1" protein.
  • TACR1 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the TACR1 protein, and which would comprise various variants from: a) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 6, b) nucleic acid molecules whose complementary strand hybridizes with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 6 in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the TA
  • TACR3 or "tachykinin receptor 3" (also called in the literature NKR; HH1 1; NK3R; NK-3R; TAC3RL) as used herein refers to a gene belonging to a family of genes that function as receptors of tachykinins.
  • the receptor affinities are specified by variations at the 5 'end of the sequence.
  • the receptors belonging to this family are characterized by interactions with G proteins and 7 hydrophobic transmembrane regions. This gene encodes the neurokinin-3 tachykinin receptor, also called neurokinin B.
  • TACR3 refers to both the gene and the human "TACR3" protein.
  • TACR3 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the TACR3 protein, and which would comprise various variants from: a) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 8, b) nucleic acid molecules whose complementary strand hybridizes with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 8.
  • TACR3 in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the TACR3 protein.
  • Other possible nucleotide sequences encoding TACR3 include, but are not limited to, SEQ ID NO: 7 Gene ID: 6870
  • M3R or "melanocortin 3 receptor” (also referred to in the literature as MC3; OB20; OQTL; BMIQ9; MC3-R) as used herein refers to a gene encoding a G protein-coupled receptor for the hormone Melanocyte stimulant and adrenocorticotropic hormone that is expressed in tissues other than the adrenal cortex and melanocytes. This gene is located in the same region as the locus for benign neonatal epilepsy. Mice lacking this gene have increased their fat content despite a decrease in food intake, suggesting that it plays a role in the regulation of energy homeostasis. In humans, mutations in this gene are associated with a susceptibility to obesity.
  • MC3R refers to both the gene and the human “MC3R” protein.
  • MC3R is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the MC3R protein, and which would comprise various variants from: a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 10, b) nucleic acid molecules whose complementary strand hybridizes with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 10.
  • the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics
  • RARRES2 or "retinoic acid receptor respond 2" (also referred to in the literature as TIG2; HP10433) as used herein refers to a gene encoding a secreted chemotactic protein that initiates chemotaxis via a seven transmembrane Chem
  • G protein-coupled domains The expression of this gene is over-regulated by synthetic retinoid tazarotene and occurs in a wide variety of tissues.
  • the active protein plays different roles, among which are its role as adipokine, and as an antimicrobial protein with activity against bacteria and fungi.
  • RARRES2 refers to both the gene and the human "RARRES2" protein.
  • RARRES2 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the RARRES2 protein, and which would comprise various variants from: a) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 12, b) nucleic acid molecules whose complementary strand hybridizes with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with at least 80%, 90%, 95%, 98% or 99% identity with SEQ ID NO: 12 in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of
  • CRABP1 or "cellular retinoic acid binding protein 1" (also referred to in the literature as RBP5; CRABP; CRABPI; CRABP-I) as used herein refers to a gene encoding a specific binding protein for a member of the family of vitamin A and is believed to play an important role in the processes of differentiation and proliferation mediated by retinoic acid. Structurally, it is similar to cellular retinol binding proteins, but it binds retinoic acid only at specific sites within the nucleus, which may contribute to the differentiation of epithelial tissue controlled by vitamin A.
  • CRABP1 refers to both the gene and the human "CRABP1" protein.
  • CRABP1 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the CRABP1 protein, and which would comprise various variants from: a) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 14, b) nucleic acid molecules whose complementary strand hybridizes with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 14. in the that the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics
  • MOXD1 or "monooxygenase DBH like 1" (also called in the MOX literature; PRO5780; dJ248E1.1) as used herein refers to a gene that encodes a DBH-like protein 1 that maintains many of the structural characteristics of dopamine beta-monooxygenase DBH. Since peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) is homologous to dopamine beta-monooxygenase (DBM, EC 1.14.17.1) it refers to a structural basis for a new type II copper family significantly specific for ascorbate-dependent monooxygenases, based on the mouse homologous gene.
  • the catecholamine synthesis pathway is a possible domain of the copper metabolite enzyme binding to catecholamine, a property similar to the neuron that encodes MOX without an enzymatic metabolism of the signal sequence that solves the chemical pathway of monooxygenase X from a unknown substrate, exogenous MOX is not secreted, It is located along the endoplasmic reticulum, both in endocrine and non-endocrine cells.
  • MOXD1 refers to both the gene and the human "MOXD1" protein.
  • MOXD1 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the MOXD1 protein, and which would comprise various variants from: a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, b) nucleic acid molecules whose complementary strand hybridizes with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 16 in which the polypeptide encoded by said nucleic acids possesses the activity and
  • SPATA18 or "spermatogenesis associated 18” (also referred to in the Mieap literature as SPETEX1) as used herein refers to a gene encoding a p53-inducible protein that is capable of inducing lysosomal organelles within the mitochondria that eliminate the oxidized mitochondrial proteins, thus contributing to mitochondrial quality control.
  • SPETEX1 mitochondrial quality control
  • SPATA18 refers to both the gene and the human "SPATA18" protein.
  • SPATA18 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the SPATA18 protein, and which would comprise various variants from: a) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 20, b) nucleic acid molecules whose complementary strand hybridizes with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 20. in the that the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the SPATA
  • LHX8 or "LIM homeobox 8" (also called in the LHX7 literature) as used herein refers to a gene encoding a protein of the LIM homeobox family of proteins, which are involved in the pattern and differentiation of various types of tissues. These proteins contain two LIM domains, in addition to a DNA-binding homeodomain. This family member is a transcription factor that plays a role in the morphogenesis of the tooth. It is also involved in oogenesis and neuronal differentiation.
  • LHX8 refers to both the gene and the human "LHX8" protein.
  • LHX8 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence for the LHX8 protein, and which would comprise various variants from: a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 22, b) nucleic acid molecules whose complementary strand hybridizes with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 22. in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics
  • modulating activity refers to either inhibiting (decreasing) or stimulating (increasing) the activity level of the GPR83 protein, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8 in a cell.
  • the activity of GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8 can be modulated by the modification of the levels and / or the activity of the protein GPR83, KIT, TACR1, TACR3 , MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8, or by modifying the levels to which the GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA 18 gene are transcribed, and / or LHX8 such that activity levels of the protein GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8 in the cell is modulated.
  • the modulating agents can also be agonists (substances that are capable of binding to a receptor and eliciting a response in the cell), as antagonists (substances that not only do not activate the receptor, but actually block its activation by agonists).
  • activation is the preferred form of modulation. More preferably, activation / stimulation is the exclusive form for GPR83, KIT, TACR1, TACR3, MC3R, since they are receptors specifically expressed by PV cells, and the compounds of the present invention are intended to stimulate / activate or inhibit them.
  • the response in the cell that the agents of the present invention are capable of causing is the endogenous secretion of GDNF.
  • inhibition is the preferred form of modulation. Therefore, in another preferred embodiment of the invention the modulating agent is an antagonist for GPR83, KIT, TACR1, TACR3, MC3R and / or LHX8, or an antagonist for GPR83, KIT, TACR1, TACR3, MC3R and / or LHX8.
  • the modulating agents are substances that cause an increase or decrease in the expression product of the GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA 18, and / or LHX8 genes.
  • LHX8 is a transcription factor whose specific presence in PV cells has intimate connection with GDNF expression. Preferably, the goal is to activate this transcription factor (or increase its expression).
  • RARRES2, CRABP1, MOXD1, S PATA 18 are intracellular proteins (which are enzymes or have an undefined role yet). Their activation or inhibition by pharmacological agents forms part of the invention.
  • the modulating agents comprised in the composition of the invention are selected from a list comprising: a) an organic molecule, b) an RNA molecule, c) an antisense oligonucleotide, d) an antibody, oe) a ribozyme.
  • organic molecules that can specifically bind to GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, LHX8 without binding to other polypeptides or proteins.
  • the organic molecules will preferably have a weight of 100 to 20,000 daltons, more preferably 500 to 15,000 daltons, and more preferably 1000 to 10,000 daltons. Libraries of organic molecules are commercially available.
  • the route of administration may be, without being limited thereto, intraperitoneal, intrathecal, intravenous, intramuscular, subcutaneous, intraventricular, oral, enteral, parenteral, intranasal or dermal.
  • nucleotide sequences specifically complementary to a specific DNA or RNA sequence could form complexes and block transcription or translation.
  • interfering RNA interfering RNA
  • tools have been developed that allow the specific inhibition of the expression of a gene.
  • the inhibition of GPR83 protein expression, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, S PATA 18 and / or LHX8 would therefore constitute the inhibition of their biological activity, and in particular, of the activity that is contributing to the aggravation of the tumor characteristics of the cancer cells and / or to the appearance of neurodegenerative diseases.
  • antisense polynucleotides are meant strands of ribonucleotides or deoxyribonucleotides which can inhibit the production of the GPR83 protein, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, S PATA 18 and / or LHX8 by one of these three mechanisms:
  • Antisense oligonucleotides capable of modulating the activity of GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, S PATA 18 and / or LHX8 are known in the state of the art.
  • it could be a sequence of ribonucleotides or RNA belonging to the so-called siRNA (small interfering RNA), small interfering RNA or silencing RNA, capable of inhibiting the genetic expression of the protein GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, S PATA 18 and / or LHX8.
  • siRNA small interferin RNA or small interference RNA
  • siRNA small interferin RNA or small interference RNA
  • small interference RNA a class of double-stranded RNA of 19 to 25 nucleotides long, and more preferably between 21 and 23 nucleotides, which is involved in the path of RNA interference, where the siRNA interferes with the expression of a specific gene.
  • mRNA polynucleotides can be translated into GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, S PATA 18 and / or LHX8 as a consequence, for example, of that the genetic code is degenerate.
  • Any siRNA capable of inhibiting the translation of these mRNAs also form part of the invention.
  • siRNA sequence of the invention or of the RNA construction of the invention would be evident to one skilled in the art, and could be carried out by chemical synthesis, which also allows the incorporation of chemical modifications both in the different nucleotides of the product as the incorporation of other chemical compounds at either end.
  • synthesis could also be performed enzymatically using any of the available RNA polymerases. Enzymatic synthesis also allows some chemical modification of inhibitory products or RNAs.
  • the design of the nucleotide sequence of the siRNA of the invention would also be apparent to one skilled in the art. Thus, it could be done by means of a randomized design in which 19-25 bases of the target mRNA are selected without taking into account the sequence or the positional information that it has in the transcript.
  • Another non-limiting alternative of the present invention would be the conventional design by means of simple parameters developed by the pioneers of the technique (Calipel, A. et al., 2003. J Biol Chem. 278 (43): 42409-42418) completed with an analysis BLAST of nucleotides.
  • Another possibility could be a rational design, in which a computerized procedure is employed aimed at identifying the optimal targets of siRNA in an mRNA.
  • the target sequences are analyzed in groups of 19 nucleotides at a time and are identified those that have better characteristics according to an algorithm that incorporates a large number of thermodynamic and sequence parameters.
  • a DNA genetic construct which would direct the in vitro or intracellular transcription of the siRNA sequence or RNA construct of the invention, and which comprises at least one of the following types of DNA could also be part of the composition of the invention.
  • sequences a) DNA nucleotide sequence, preferably double-stranded, comprising at least the coding sequence of the siRNA of the invention or of the RNA construction of the invention for its transcription, or, b) nucleotide sequence of DNA, preferably double-stranded, corresponding to a system or gene expression vector comprising the coding sequence of the RNA sequence of the invention operatively linked to, at least, a promoter that directs the transcription of said nucleotide sequence of interest, and with other sequences necessary or appropriate for transcription and its appropriate regulation in time and place, for example, start signals and termination, cut sites, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), etc.
  • compositions of the present invention allow transfection of the siRNA of the invention into a cell, in vivo or in vitro.
  • the transfection could be carried out, but without being limited to, direct transfection or vectors that facilitate the access of the siRNA to the interior of the cell.
  • vectors are, without being limited to, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, Herpes simplex viruses, non-viral DNA plasmids, cationic liposomes and molecular conjugates.
  • the siRNA of the present invention can be conjugated with release peptides or other compounds to promote the transport of these siRNA to the interior of the cell.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) the protein GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18 and / or LHX8.
  • immunologically active immunoglobulin molecule portions include F (ab) and F (ab ') 2 fragments that can be generated treating the antibody with an enzyme such as pepsin. It can be a monoclonal or polyclonal antibody.
  • Antibodies capable of binding to the protein GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, S PATA 18 and / or LHX8 can be used to inhibit the activity of said protein.
  • Antibodies are commercially available (such as, but not limited to, the Anti-GPR83, Anti-KIT, Anti-TACR1, Anti-TACR3, Anti-MC3R, Anti-RARRES2, Anti-CRABP1, Anti-MOXD1, Anti-SPATA18 and / or Anti-LHX8 marketed by Sigma-Aldrich Antibodies can be polyclonal (typically include different antibodies directed against different determinants or epitopes) or monoclonal (directed against a single determinant in the antigen) .
  • the monoclonal antibody can be altered biochemically, by genetic manipulation, or it can be synthetic, possibly lacking the antibody in its entirety or in parts, of portions that are not necessary for the recognition of the protein and being replaced by others that impart additional advantageous properties to the antibody.
  • the antibody can also be recombinant, chimeric, humanized, synthetic or a combination of any of the foregoing.
  • a "recombinant antibody or polypeptide” is one that has been produced in a host cell that has been transformed or transfected with the nucleic acid encoding the polypeptide, or produces the polypeptide as a result of homologous recombination. These rAc can be expressed and directed to specific cellular subcompartments when the appropriate sequences for intracellular trafficking are incorporated. These antibodies are called intrabodies, and have proven effective not only in diverting proteins from their usual compartment or blocking interactions between proteins involved in signaling pathways, but also in activating intracellular proteins. Also part of the invention are genetic DNA constructs capable of being transcribed to a peptide, antibody or antibody fragment, for use in the treatment of neurodegenerative diseases, preferably EP.
  • Said genetic construction of DNA would direct the in vitro or intracellular transcription of the antibody sequence or fragment thereof, and comprises, at least, one of the following types of sequences: a) DNA nucleotide sequence, preferably double-stranded, which comprises, at least, the coding sequence of the antibody of the invention or of the antibody fragment of the invention for its transcription in vitro, or intracellular, b) DNA nucleotide sequence, preferably double-stranded, corresponding to a system or vector of gene expression comprising the coding sequence of the antibody sequence or antibody fragment of the invention operably linked with at least one promoter that directs the transcription of said nucleotide sequence of interest, and with other sequences necessary or appropriate for transcription and its appropriate regulation in time and place, for example, start and end signals, cut sites, polyadenylation signal, origin of replication, activators transcriptional (enhancers), transcriptional silencers (silencers), etc ... for use in those pathological contexts that occur neurodegenerative disease, and preferably EP.
  • a "ribozyme” as understood in the present invention refers to a catalytic polynucleotide (typically RNA), which can be constructed to specifically recognize, by hybridization, an mRNA and fragment it or eliminate its expression. Ribozymes can be introduced into the cell as catalytic RNA molecules or as genetic constructs that are expressed to catalytic RNA molecules. The authors of the present invention have seen, as shown in the examples, that administration of a selective GPR83 receptor agonist increases gene expression of GDNF by 70% in the striatum of the mouse (see Figure 5).
  • RNA catalytic polynucleotide
  • the GPR83 modulating agent of the invention is an organic molecule, and more preferably, the organic molecule is selected from the list consisting of sumatriptan, naltrexone, soproterenol, nor-BNI, LY - 25582, candesartan, Gr 1 13808, prazosin, SB 204070, 2-5A-anti-hTR, PACPX, arformoterol, haloperidol, salbutamol, sertindole, Talactoferrin, losar ⁇ an, DPCPX, tamsulosin, morphine, immepip, beta-FNA, ergotamine, carmoterol, lisuride, formoterol, buprenorphine, Ziprasidone, fentanyl, risperidone, pimozide, rolofylline, L017874, carvedilol, metergoline, butorphan
  • the KIT modulating agent of the invention is an organic molecule, and more preferably, the organic molecule is selected from the list consisting of AGN-PC-OBEHKI, R406 free base, dasatinib , sunitinib, Ki20227, cediranib, SU14813, PD173955, tandutinib, linifanib, Foretinib, axitinib, pazopanib, motesanib, jnj-28312141, quizartinib, NVP-AST487, nintedanib, dovitinib, masitinib, glucose, staurosporine, imatinib, Kinome_469, Kinome_589, AZD1152 -HQPA, Kinome_767, Kinome_462, Kinome_534, sorafenib, nilotinib, brivanib, chitin, Kinome
  • the TACR1 modulating agent of the invention is an organic molecule, and more preferably, the organic molecule is selected from the list consisting of vofopitant, aprepitant, AGN-PC-02Z5HN, DNC008305 , CHEMBL507340, CHEMBL555504, CHEMBL557188, CHEMBL2419537, CHEMBL2419542, CHEMBL1766208, AGN-PC-088WC4, AGN-PC-0BTIAO, CHEMBL2419544, AGN-PC-07C5VQ, CP-96345, orvepitant, AGN-PC-09M0HL, CHEMBL2419538, CHEMBL1766204, L014901 , AGN-PC-07208D, L022327, AGN-PC-09M0MR, AGN-PC-01549N, AGN-PC-08Y2KI, CHEMBL2419541, CHEMBL1766204, L014901
  • the TACR3 modulating agent of the invention is an organic molecule, and more preferably, the organic molecule is selected from the list consisting of CHEMBL583102, CHEMBL2347657, AGN-PC-0JHGX4, AGN -PC-0JHH57, AGN-PC-0JHH58, AGN-PC-0JHH82, AGN-PC-0JHH83, AGN-PC-0JHHB6, AGN-PC-0JHHB7, AGN-PC-0JHHE9, AGN-PC-0JHHGX, AGN-PC -OJHHGY, AGN-PC- 0JHHJP, AGN-PC-0JHHJQ, AGN-PC-0JHHMQ, AGN-PC-0JHHMR, AGN-PC-OJHHPB, AGN-PC-0JHHPC, AGN-PC-0JHHS6, AGN-PC-0JHHS7 , AGN
  • the modulating agent is a TACR3 antagonist, and is preferably selected from SB 218795 (CAS No: 174635-53-1), SB 222200 (CAS Number: 174635-69-9), SB 223412 (CAS Number: 174636-32-9), or any combination thereof.
  • the MC3R modulator agent of the invention is an organic molecule, and more preferably, the organic molecule is selected from the list consisting of NDP-MSH (Melanotan), D-Phe7 ( Melanotan II), alpha-MSH, CHEMBL3287323, AGN-PC-075V3K, CHEMBL3287329, AGN-PC-075QLV, SHU-9119, DNC005518, CHEMBL3283225, CHEMBL3287327, CHEMBL3287322, CHEMBL3287324, CHEMBL3287328, AGN-PC-075FQ5, CHEMBL3287326, CHEMBL491870, CHEMBL212614, AGN-PC-00OCO0, CHEMBL386081, AGN-PC-074GBC, AGN-PC-074QIF, AGN-PC-005S8E, PG-901, AGN-PC
  • the modulating agent is an MC3R antagonist, and is preferably selected from PG 106 (CAS No: 9441 1 1-22-2), HS 024 (CAS Number: 212370-59-7), JKC 363 (CAS Number: 436083-30-6), or any of its combinations.
  • the RARRES2 modulating agent of the invention is an organic molecule, and more preferably, the organic molecule is selected from the list consisting of tazarotene, iptakalim, resolvin El, rosiglitazone, eicosapentaenoic acid , or any of its salts, preferably any pharmaceutically acceptable salt, esters, tautomers, polymorphs, pharmaceutically acceptable hydrates, or an isomer, prodrugs, derivatives, solvates or the like, or any combination thereof.
  • the CRABP1 modulating agent of the invention is an organic molecule, and more preferably, the organic molecule is selected from the list consisting of tamibarotene, 11-cis-retinal, AC1 L1CDO, axerophthene, diethylene glycol, 2- (N-morpholino) ethanesulfonic acid, Bis-Tris propane, AC105PXP, AC1 NRCY9, DB08127, AGN-PC-088Y4Z , LDAO, acetaldehyde, vitamin A, isopropanol, myristic acid, glycochenodeoxycholic acid, crotonate, S-methylcysteine, glycocholate, cadmium, docosahexaenoic acid, ibuprofen, 3-phenylbutyric acid., Barium, 3- (4-methoxyphenyl) propanoate, vaccenic acid , AC1 L9MBM
  • the MOXD1 modulating agent of the invention is an organic molecule, and more preferably, the organic molecule is selected from the list consisting of CHEMBL433493, AGN-PC-00NUL0, AGN-PC -0N3SZ3, AGN-PC-0N02TF, CHEMBL54434, or any of its salts, preferably any pharmaceutically acceptable salt, esters, tautomers, polymorphs, pharmaceutically acceptable hydrates, or an isomer, prodrugs, derivatives, solvates or the like, or any combination thereof .
  • the compounds of the present invention can include isomers, depending on the presence of multiple linkages, including optical isomers or enantiomers, depending on the presence of chiral centers.
  • Individual isomers, enantiomers or diastereomers and mixtures thereof fall within the scope of the present invention, i.e., the term isomer also refers to any mixture of isomers, such as diastereomers, racemates, etc., even to their optically isomers active or mixtures in different proportions thereof.
  • the individual enantiomers or diastereomers, as well as their mixtures, can be separated by conventional techniques.
  • prodrugs of the compounds of the invention include any derivative of a compound of the invention - for example and not limitatively: esters (including esters of carboxylic acids, amino acid esters, phosphate esters, sulfonate esters of metal salts, etc.), carbamates, amides, etc. - which when administered to an individual can be transformed directly or indirectly into said compound of the invention in said individual.
  • said derivative is a compound that increases the bioavailability of the compound of the invention when administered to an individual or that enhances the release of the compound of the invention in a biological compartment.
  • derivative is not critical as long as it can be administered to an individual and provides the compound of the invention in a biological compartment of an individual.
  • preparation of said prodrug can be carried out by conventional methods known to those skilled in the art.
  • derivative includes both pharmaceutically acceptable compounds, ie, derivatives of the compound of the invention that can be used in the manufacture of a medicament or food compositions, and pharmaceutically unacceptable derivatives, since these they can be useful in the preparation of pharmaceutically acceptable derivatives.
  • the compounds of the invention may be in crystalline form as free compounds or as solvates.
  • solvate includes both pharmaceutically acceptable solvates, that is, solvates of the compound of the invention that can be used in the manufacture of a medicament, and pharmaceutically unavailable solvates, which they may be useful in the preparation of solvates or pharmaceutically acceptable salts.
  • pharmaceutically acceptable solvate is not critical as long as it is pharmaceutically acceptable.
  • the solvate is a hydrate.
  • the solvates can be obtained by conventional solvation methods known to those skilled in the art.
  • the compounds of formula (I) and / or (II), their salts, prodrugs or solvates will preferably be in a pharmaceutically acceptable or substantially pure form, ie having a level of purity pharmaceutically acceptable excluding normal pharmaceutical additives such as diluents and carriers, and not including material considered toxic at normal dosage levels.
  • the purity levels for the active ingredient are preferably greater than 50%, more preferably greater than 70%, and still more preferably greater than 90%. In a preferred embodiment, they are greater than 95% of the compound of the invention, or of its salts, solvates or prodrugs.
  • a second aspect of the invention relates to a composition, hereinafter the composition of the invention, comprising a modulating agent of GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8 of the invention.
  • it further comprises a pharmaceutically acceptable carrier or excipient.
  • the composition of the invention is a pharmaceutical composition.
  • it also comprises another active principle.
  • the pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the adjuvants and vehicles known to those skilled in the art and commonly used in the preparation of therapeutic compositions.
  • the term "therapeutically effective amount” refers to the amount of the agent or compound capable of developing the therapeutic action determined by its pharmacological properties, calculated to produce the desired effect and, in general, will be determined, among other causes, due to the characteristics of the compounds, including age, condition of the patient, the severity of the disorder or disorder, and the route and frequency of administration.
  • the compounds described in the present invention, their salts, prodrugs and / or solvates as well as the pharmaceutical compositions containing them can be used together with other drugs, or additional active ingredients, to provide a combination therapy.
  • Said additional drugs can be part of the same pharmaceutical composition or, alternatively, they can be provided in the form of a separate composition for simultaneous administration or not to that of the pharmaceutical composition comprising a compound of the invention, or a salt, prodrug or solvate of it.
  • the pharmaceutical composition further comprises another active ingredient.
  • active ingredient means any component that potentially provides a pharmacological activity or other different effect in diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals.
  • the term includes those components that promote a chemical change in the manufacture of the drug and are present therein in a planned modified form that provides the specific activity or effect.
  • the composition of the invention consists of a modulating agent of the invention as the sole active principle, although it may contain pharmaceutically acceptable excipients and vehicles.
  • Another aspect of the invention relates to a pharmaceutical form, hereinafter pharmaceutical form of the invention, comprising a modulating agent of the invention or the composition of the invention.
  • compositions and dosage forms of the invention are suitable for oral administration, in solid or liquid form.
  • Possible forms for oral administration are tablets, capsules, syrups or solutions and may contain conventional excipients known in the pharmaceutical field, as aggregating agents (eg syrup, acacia, gelatin, sorbitol, tragacanth or polyvinyl pyrrolidone), fillers (eg lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine), disintegrants (eg starch, polyvinyl pyrrolidone or microcrystalline cellulose) or a pharmaceutically acceptable surfactant such as sodium lauryl sulfate.
  • Other pharmaceutical forms can be colloidal systems, which include nanoemulsions, nanocapsules and polymeric nanoparticles.
  • compositions for oral administration can be prepared by conventional methods of Farmacia Galénica, as a mixture and dispersion.
  • the tablets can be coated following methods known in the pharmaceutical industry.
  • the compositions and dosage forms can be adapted for parenteral administration, such as sterile solutions, suspensions, or lyophilisates of the products of the invention, using the appropriate dose. Suitable excipients may be employed, such as pH buffering agents or surfactants.
  • the aforementioned formulations can be prepared using conventional methods, such as those described in the Pharmacopoeias of different countries and in other reference texts.
  • medium refers to any substance used for prevention, diagnosis, alleviation, treatment or cure of diseases in man and animals.
  • the administration of the compounds, compositions or dosage forms of the present invention can be carried out by any suitable method, such as intravenous infusion and oral, topical or parenteral routes. Oral administration is preferred because of the convenience of the patients and because of the chronic nature of the diseases to be treated.
  • the amount administered of a compound of the present invention will depend on the relative efficacy of the compound chosen, the severity of the disease to be treated and the weight of the patient. However, the compounds of this invention will be administered one or more times per day, for example 1, 2, 3 or 4 times daily, with a total dose between 0.1 and 1000 mg / Kg / day. It is important to bear in mind that it may be necessary to introduce variations in the dose, depending on the age and condition of the patient, as well as changes in the route of administration.
  • the compounds and compositions of the present invention can be used together with other medicaments in combination therapies.
  • the other drugs can be part of the same composition or of a different composition, for administration at the same time or at different times.
  • Another aspect of the invention relates to a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegenerative disease comprising: a) contacting the compound to be analyzed with the GPR83, KIT, TACR1 polypeptide , TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8, b) detect the binding of said compound to be analyzed with the polypeptide GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8.
  • the neurodegenerative disease is PE.
  • the compounds that bind to the polypeptide GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8 would be identified as potential therapeutic agents against neurodegenerative disease, and more specifically against PD.
  • these assays may involve the complete GPR83 polypeptide, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8, a biologically active fragment thereof, or a fusion protein that involves all or a portion of the GPR83 polypeptide, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8.
  • Determine the ability of a compound to modulate the activity of GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8 can be performed, for example, by determining the capacity of GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8 to bind or interact with a target molecule of said compound, directly or indirectly.
  • GPR83 can also be activity assays, directly or indirectly measuring the activity of GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8. It can also be an expression assay, directly or indirectly determining the mRNA expression of Gpr83, Kit, Tacri, Tacr3, Mc3r, Rarres2, Crabpl, Moxdl, SpataW, and / or Lhx8 or the protein GPR83, KIT, TACR1 , TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8.
  • assays can also be combined with an in vivo assay measuring the effect of a test compound on the symptoms of diseases related to GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8, and in particular a neurodegenerative disease, preferably PD (for example, but without limitation, on animal models or other model systems known in the art).
  • a neurodegenerative disease preferably PD (for example, but without limitation, on animal models or other model systems known in the art).
  • the compounds to be tested employed in the method of selection of therapeutic agents are not limited to organic molecules of low molecular weight, proteins (including antibodies), peptides, oliogonucleotides, etc. They can be natural and / or synthetic compounds.
  • antibodies capable of binding to an epitope of GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8, which can be used therapeutically, as discussed above, can also be used in immunohistochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunoprecipitation assays, or other immunohistochemical assays known in the state of the art.
  • the GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8 polypeptides can be used to immunize an animal to obtain polyclonal antibodies.
  • Monoclonal antibodies can also be prepared by techniques that allow the production of antibodies by cell lines in culture, including, but not limited to, hybridomas, human B-cell hybridomas. Techniques for producing chimeric, humanized or synthetic antibodies are known.
  • the therapeutic agents identified by the screening method described herein can be used in an animal or other model to determine the mechanism of action of said agent. Moreover, the therapeutic agents selected by the method described herein would be used in the treatment of diseases that occur with the alteration of GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8 and, in concrete, a neurodegenerative disease, preferably PE.
  • a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegenerative disease comprising: a) determining the activity of GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8 at an established concentration of the compound to be analyzed or in the absence of said compound, b) determine the activity of GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18 , and / or LHX8 at a concentration of the compound to be analyzed different from that of a).
  • Compounds that give rise to an activity different from GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8 would be identified as potential therapeutic agents against neurodegenerative disease, and preferably of PD.
  • GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8 can be diagnostic, and in particular neurodegenerative diseases, more specifically PD, can be detected by measuring the amount of nucleic acids (DNA and / or RNA and / or mRNA) that encode GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, PDE3A, SPATA18, and / or LHX8, or the amount of protein GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8 that is expressed, in comparison with normal cells.
  • nucleic acids DNA and / or RNA and / or mRNA
  • the detection of the oligonucleotides can be done by methods well known in the state of the art (such as, but not limited to, probes with labeled nucleotides, DNA-DNA or DNA-RNA hybridization, PCR amplification using labeled nucleotides, RT- PCR).
  • Methods for detecting the expression of the protein GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8 are also well known in the state of the art, such as for example poly or monoclonal antibodies, ELISA , radioimmunoassay (RIA), and FACS (fluorescence activated cell sorting).
  • a method for the collection of useful data in the diagnosis and / or prognosis of a neurodegenerative disease comprises: a) determining the expression of GPR83, KIT, TACR1, TACR3, MC3R, RARRES2 , CRABP1, MOXD1, SPATA18, and / or LHX8 in a sample extracted from a mammal, b) compare the expression values of GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18, and / or LHX8 obtained in a) with the standard values in healthy or diseased mammals.
  • the neurodenegerative disease is PE.
  • the PV-tdTomato mice were obtained from the Jackson Laboratory (C57BL / 6-Tg (PvaltestTomato) 15Gfng / J-JAX No. 027395, originally published in (3).)
  • the PV-Cre and tdTomato mice were obtained by crossing PV (B6). ; 129P2-Pvalb tm1 ⁇ cre > Arbr / J _ JAX n ° 008069, originally published in (5) with tdTomato reporter mice (B6.Cg-Gt (ROSA) 26Sortm27.1 (CAGCOP4 * H134R / tdTomato).
  • the PCR of PV-Cre was carried out with the oligos CreFW (5'-TGTTCAGGGATCGCCAG -3 ') and CreREV (5'-ACGGGCACTGTGTCCAG-3').
  • the tdTomato allele was detected with dTomFW (5-ACTGCAGCGCTGGTCATATG -3 ') and dTomREV (5-ACTCTTTGATGACCTCCTCG -3').
  • the presence of the ChR2-tdTomato allele was verified using ChR2FW (5'-GGCATTAAAGCAGCGTATCC-3 ') and ChR2REV (5'-CTGTTCCTGTACGGCATGG-3').
  • PV-Cre experimental model tdTomato used in this study, heterozygous PV-Cre mice (PVCre / +) were paired with heterozygous ChR2-tdTomato mice to obtain the F1 PVCre / + progenies; ChR2- tdTomato / + with Mendelian relationship.
  • mice were sacrificed at day 30 (P30) postnatally by thiobarbital overdosage and perfused intracardially with an ice-cold oxygenated working solution with the formula: 87 mM NaCl, 2.5 mM KCI, 1.25 mM NaH2P04, NaHCO3 26 mM, 75 mM sucrose, 20 mM glucose, 1 mM CaCl2, and 2 mM MgCl2.
  • the brain was quickly dissected and transferred to the ice-cooled oxygenated work solution and kept in the same solution during sectioning in a vibratome (VT1200 S, Leica) in slices of 300 ⁇ of thickness (Bregma, AP: 1, 34-1, 82 mm).
  • the slices were placed in oxygenated work solution at 37 ° C for 45 minutes.
  • the area of interest was dissected from each slice, and the tissue was dissociated using the Papain Dissociation System (Worthington), following the manufacturer's instructions. All solutions were oxygenated for at least 10 minutes with a mixture from C02 to 5% in 02 (Air Liquide). Oxygenation and a short dissection time were crucial to maintain a high survival rate in the cell suspension.
  • the cell suspension obtained was filtered with a 20 ⁇ filter (Biofil) and kept in an oxygenated working solution cooled with 0.2% bovine serum albumin (BSA). Next, the cells were immediately classified FACS.
  • TdTomato or PV- tdTomato After dissociation of CTX and ST cells from PV-Cre mice; TdTomato or PV- tdTomato, the tdTomato-positive cells were separated using a FACS Jazz cell separator (BD Biosciences). The tdTomato + cells were collected in PBS for immunocytochemical analysis, or were collected in an RNAIater solution (Thermofisher) and immediately frozen at -80 ° C until RNA extraction. The yield obtained from two PV-Cre mice; TdTomato was ⁇ 2000 cells / CTX and ⁇ 500-1000 cells / ST. The yield of PV-tdTomato was ⁇ 5000 cells / CTX and ⁇ 2000 cells / ST. When needed, cells from two or three experiments were pooled to reach a total of 2000 tdTomato + cells per replicate, which is estimated to be the minimum required to perform the microarray analysis.
  • mice were sacrificed at day 30 (P30) postnatally by thiobarbital overdosage, and perfused intracardially with an ice-cold oxygenated working solution.
  • the brain was rapidly dissected and transferred to an ice-cold oxygenated work-up solution and kept in the same solution during slicing in a vibratome (VT1200 S, Leica) in slices of 300 ⁇ of thickness.
  • the slices were placed in oxygenated work solution at 37 ° C for 45 minutes.
  • the area of interest (motor cortex and dorsal striatum) was dissected from each slice and immediately frozen at -80 0 C for RNA isolation.
  • RNA from whole cortical and striatal tissue samples was isolated using the Trizol isolation system (Life Technologies), according to the manufacturer's instructions.
  • the RNAIater solution was removed after a 1 min centrifugation at 5000 xg and the cells were lysed in 0.5 ml of Trizol reagent (Life Technologies) to isolate the total RNA following the instructions of the maker.
  • An additional step was performed by incubating the upper aqueous phase obtained after a gradient induced by chloroform with 0.5 ⁇ g of glycogen (SERVA, Germany) at 4 ° C for 12 h.
  • the quantity and quality of RNA from the tdTomato + cells separated with FACS was determined using a Agilent 2100 Bioanalyzer.
  • RNA samples with integrity number (RIN) ⁇ 7.8 were processed for microarray analysis.
  • the yield and integrity of the RNA of the whole tissue sample was determined with the A260 / A280 ratio using a Nanodrop 2000 spectrophotometer (Thermofischer). Analysis of microarrays
  • RNA was amplified and labeled using the GeneChip WT PLUS Reagent Kit (Affymetrix). The amplification was performed with 5 ng of total RNA following the procedures described in the user manual of the WT PLUS Reagent Kit.
  • the amplified cDNA was quantified, fragmented and labeled in preparation for hybridization with GeneChip Mouse Transcriptome 1.0 Array (Affymetrix) using 5.5 ⁇ g of "single-strand" cDNA product and following the protocols described in the user manual. Washing, staining (GeneChip Fluidics Station 450, Affymetrix) and scanning (GeneChip Scanner 3000, 10 Affymetrix) were performed following the protocols described in the user manual for the cartridge arrays.
  • PCR reactions were performed in duplicates in a total volume of 20 ⁇ containing 1 ⁇ of cDNA solution, 10 ⁇ of SYBR Green Fast Mix (Thermofischer), 1 ⁇ of oligo FW, 1 ⁇ of oligo REV and 7 ⁇ of H20 QPCR reactions were performed in a fast real-time PCR system 7500 (Life Technologies).
  • the level of expression of the Actb gene was estimated in each sample to normalize the amount of cRNA and RNA in order to perform relative quantifications.
  • the oligos used to amplify the cDNA samples are listed below:
  • AAAGCCCTCACCTTGATCGG (SEQ ID NO: 31) AGCACCATGGCGAAGAACAT (SEQ ID NO: 32) Pde3a TCCCAGTCAGGAACCAGCAT (SEQ ID NO: 33)
  • CAAGTTGCTTACGGCCCTC (SEQ ID NO: 34)
  • Rarres2 AGGACTGGAAAAAGCCGGAG (SEQ ID NO: 35) ATTGGGCAGTGGACTATCCG (SEQ ID NO: 36) Spata18
  • ACACGAGCTGCTACATTAAGGA (SEQ ID NO: 43) CCAGTCAGTCGAGTGGATGTG (SEQ ID NO: 44). The specificity of oligos and sensitivity were tested before use.
  • mice were deeply anesthetized by intraperitoneal injection of thiobarbital and perfused intracardially with 0.1 M phosphate buffered saline (PBS), followed by 4% paraformaldehyde in PBS pH 7.4. After a fixation time of 1 h, the brains were washed in PBS, cryoprotected in PBS with 30% sucrose, mounted in Tissue-Tek® OCT (Sakura®, Finetek), and frozen in dry ice.
  • PBS phosphate buffered saline
  • Sections were incubated with anti-c-Kit primary antibodies (3074, Cell Signaling) at 1: 1000 and anti-PV (PVG214, Swant) overnight at 4 ° C, and then incubated with anti-IgG secondary antibodies from rabbit and goat anti-IgG conjugated with Alexa Fluor 488 and 568 respectively (Invitrogen) for 1 hour at room temperature.
  • the nuclei were labeled with 4 ', 6'-diamidino-2-phenylindole (Dapi, D9542, Sigma).
  • Immuno-fluorescent images were obtained with a BX61 microscope equipped with a DP70 camera (Olympus) or an N-STORM superresolution microscope (Nikon) equipped with a 405/488/561/647 nm laser.
  • the proSAAS peptide "mPEN”, a potent specific ligand of GPR83 (4), has been tested in vivo.
  • Stereotaxic injections of 2 nmol of mPEN in the right striatum (ST) induced a 70% increase in Gdnf expression 2 hours after injection compared to the vehicle (saline) (see Figure 5).
  • the level of expression of Gdnf returned to normal 24 hours after the stimulation of mPEN.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des composés et des compositions qui peuvent moduler la stimulation de GDNF endogène par activation des gènes GPR83, KIT, TACR1, TACR3, MC3R, RARRES2, CRABP1, MOXD1, SPATA18 et/ou LHX8 ou les protéines codant pour ceux-ci, pour le traitement de maladies neurodégénératives, et plus particulièrement pour le traitement de la maladie de Parkinson, ainsi que des méthodes de sélection de ces composés et compositions.
PCT/ES2018/070577 2017-08-29 2018-08-29 Compositions pouvant moduler la stimulation de gdnf endogène pour le traitement de maladies neurodégénératives Ceased WO2019043279A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES201731053 2017-08-29
ESP201731053 2017-08-29

Publications (1)

Publication Number Publication Date
WO2019043279A1 true WO2019043279A1 (fr) 2019-03-07

Family

ID=65526243

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES2018/070577 Ceased WO2019043279A1 (fr) 2017-08-29 2018-08-29 Compositions pouvant moduler la stimulation de gdnf endogène pour le traitement de maladies neurodégénératives

Country Status (1)

Country Link
WO (1) WO2019043279A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060078890A1 (en) * 2004-10-08 2006-04-13 Ole Isacson Methods for identifying parkinson's disease therapeutics

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060078890A1 (en) * 2004-10-08 2006-04-13 Ole Isacson Methods for identifying parkinson's disease therapeutics

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BOTTA-ORFILA, T. ET AL.: "Brain transcriptomic profiling in idiopathic and LRRK2-associated Parkinson's disease", BRAIN RESEARCH, vol. 1466, July 2012 (2012-07-01), pages 152 - 157, XP028501357, ISSN: 0006-8993, DOI: doi:10.1016/j.brainres.2012.05.036 *
D'AGLEMONT DE TASSIGNY, X. ET AL.: "GDNF-based therapies, GDNF-producing interneurons, and trophic support of the dopaminergic nigrostriatal pathway", IMPLICATIONS FOR PARKINSON'S DISEASE. FRONTIERS IN NEUROANATOMY, vol. 9, no. 10, February 2015 (2015-02-01), XP055510196, ISSN: 1662-5129 *
JARVELA, T. S. ET AL.: "The neural chaperone proSAAS blocks alpha-synuclein fibrillation and neurotoxicity", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 113, no. 32, August 2016 (2016-08-01), pages E4708 - E4715, XP055581168, ISSN: 0027-8424 *

Similar Documents

Publication Publication Date Title
CA2972209C (fr) Agent inducteur de mort cellulaire, agent cytostatique, et composition pharmaceutique pour le traitement de maladies provoquees par une croissance cellulaire anormale
US20130288981A1 (en) Targeting senescent cells and cancer cells by interference with jnk and/or foxo4
US20210317451A1 (en) Compositions and methods for modulating neuronal excitability and motor behavior
Li et al. Obestatin attenuated doxorubicin-induced cardiomyopathy via enhancing long noncoding Mhrt RNA expression
US20230035892A1 (en) Methods and compositions for treating cancer
US10232013B2 (en) Use of an antimicrobial peptide TP4 in treating a cancer
EA037923B1 (ru) мякРНК, КОМПОЗИЦИИ И ПРИМЕНЕНИЯ
WO2017165704A1 (fr) Traitement du cancer par activation de peptides d'agrégation amyloïdogènes cryptiques endogènes
US20200390903A1 (en) Plectin-targeted liposomes/parp inhibitor in the treatment of cancer
US20100260718A1 (en) Irf-4 as a tumor suppressor and uses thereof
JP6478916B2 (ja) 薬物耐性乳癌の予後判定および処置のための治療アジュバントおよびバイオマーカーとしてのｍｉＲＮＡ
US20210071180A1 (en) Microrna 584-5p compositions and methods for treating cancer
US20160202242A1 (en) Cell death-inducing agent, cell growth-inhibiting agent, and pharmaceutical composition for treatment of disease caused by abnormal cell growth
WO2019043279A1 (fr) Compositions pouvant moduler la stimulation de gdnf endogène pour le traitement de maladies neurodégénératives
EP3747468A1 (fr) Agent thérapeutique contre la dégénérescence lobaire fronto-temporale, procédé de criblage d'agents thérapeutiques contre la dégénérescence lobaire fronto-temporale et méthode de traitement de la dégénérescence lobaire fronto-temporale
US20240075093A1 (en) Compositions and methods of treating a pi3k mediated disease
US20240301408A1 (en) Microrna 195 compositions and methods for treating cognitive impairment
US20240156800A1 (en) Ep300 degrader and uses thereof in neuroblastoma
JP2019510015A (ja) Ｂｃｌ−２ ｌ１０／ｉｐ３受容体の相互作用の阻害剤
US20250082766A1 (en) Treatment of brain tumors by targeting the cholesterol pathway in astrocyes
TWI602572B (zh) 抗菌胜肽tp4治療癌症之用途
Sironi Loss of C9orf72 Function Impairs the Peripheral Neuromuscular System and Anticipates Symptoms in ALS Mice
ES2803581T3 (es) Inhibidores de Mcl-1 para su uso en el tratamiento de enfermedades provocadas por neovascularización patológica
Afonso-Reis et al. The stress granule protein G3BP1 alleviates spinocerebellar ataxia-associated deficits
WO2023092152A1 (fr) Applications thérapeutiques de la protéine nsp1 de coronavirus

Legal Events

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

Ref document number: 18851468

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18851468

Country of ref document: EP

Kind code of ref document: A1