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WO2018172588A1 - Utilisation d'agents modulateurs de mok pour le traitement de maladies inflammatoires et de maladies neurodégénératives - Google Patents

Utilisation d'agents modulateurs de mok pour le traitement de maladies inflammatoires et de maladies neurodégénératives Download PDF

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WO2018172588A1
WO2018172588A1 PCT/ES2018/070216 ES2018070216W WO2018172588A1 WO 2018172588 A1 WO2018172588 A1 WO 2018172588A1 ES 2018070216 W ES2018070216 W ES 2018070216W WO 2018172588 A1 WO2018172588 A1 WO 2018172588A1
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mok
disease
treatment
compound
tdp
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Cintia ROODVELDT CATELLANI
María Magdalena LEAL LASARTE
Jaime MUÑOZ FRANCO
David POZO PÉREZ
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Fundacion Publica Andaluza Progreso y Salud
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  • the present invention is within the field of molecular biology and medicine, and specifically refers to compounds and compositions capable of modulating the activity of MOK (MAPK / MAK / MRK overlapping kinase) or any of its biologically active isoforms and variants , for the treatment of diseases with inflammatory component, among others, neurodegerative conformational diseases (protein misfolding diseases), and more specifically for the treatment of Amyotrophic Lateral Sclerosis (ALS) and frontotemporal lobe degeneration (Frontotemporal lobar degeneration or FTLD ).
  • MOK MOK
  • MAK / MAK / MRK overlapping kinase any of its biologically active isoforms and variants
  • diseases with inflammatory component among others, neurodegerative conformational diseases (protein misfolding diseases), and more specifically for the treatment of Amyotrophic Lateral Sclerosis (ALS) and frontotemporal lobe degeneration (Frontotemporal lobar degeneration or FTLD ).
  • neuroinflammatory processes are a critical factor in triggering the pathological cascade that leads to neuronal degeneration.
  • Signs of external or internal damage activate microglial cells, favoring the production of cytotoxic factors that induce neuronal degeneration. These factors activate protein kinases, which lead to hyperphosphorylation of aggregating proteins, and their consequent oligomerization. These aggregates released into the extracellular environment, upon activation of the microglial cell, would cause a positive feedback mechanism favoring neurodegeneration.
  • ALS / ALS amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • Parkinson's disease Alzheimer's disease
  • Huntington's disease Huntington's disease
  • spongiform encephalopathies multiple tauopathies
  • amyloid polyneuropathy family etc.
  • Amyotrophic lateral sclerosis (ALS / ALS) is a fatal neurodegenerative disorder currently incurable, mainly because the molecular mechanisms of this and related diseases (e.g. FTLD) remain enigmatic (Fujisawa et al., Hum. Mol. Gen. 2016).
  • FTLD molecular mechanisms of this and related diseases
  • Currently, the only drug approved for treatment is 'riluzole', which increases the life expectancy of patients in only 2-3 months.
  • Agents modulating the activity of protein kinases are known in the state of the art.
  • Garske AL et al., PNAS 201 1 describe a chemical-genetic approach based on the covalent complementarity between a gatekeeper cysteine and an electrophilic inhibitor.
  • MAPK mitogen-activated protein kinases
  • FTLD is a clinical syndrome caused by the degeneration of the frontal lobe of the human brain, which can spread to the temporal lobe. It is one of three syndromes caused by frontotemporal lobular degeneration, and the second most common cause of early onset dementia after Alzheimer's disease
  • the identification of a key therapeutic target in this case the MOK protein kinase or some of its isoforms, and its possible modulation, could represent an important advance in the development of new therapeutic approaches for the treatment of neurodegerative conformational diseases, and mainly where the aggregating protein is TDP-43, such as ALS and FTLD.
  • the aggregating protein is TDP-43, such as ALS and FTLD.
  • the authors of the invention demonstrate that the modulation of the expression and / or activity of the human protein kinase MOK (MAPK / MAK / MRK overlapping kinase; 419 aa; Acc. Number BAA81688 or Q9UQ07) or any of its isoforms or biologically active variants, whose expression and phosphorylation status (required for its activity as MAPK) are strongly affected in a cellular model of ALS / FTLD, which would serve to modulate the neuroinflammatory-neurodegenerative profile associated with these conformational diseases 'neurodegenerative.
  • MOK human protein kinase MOK
  • a first aspect of the invention relates to a MOK activity modulating agent, hereinafter MOK modulating agent of the invention, for the prevention, improvement and / or treatment of inflammatory diseases, more preferably neurodegenerative diseases. mediated by neuroinflammatory processes.
  • MOK modulating agent of the invention in the preparation of a medicament for the prevention, improvement and / or treatment of inflammatory diseases, more preferably of neurodegenerative diseases mediated by inflammation.
  • neurodegenerative disease is a neurodegerative conformational disease.
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • the disease is ALS or FTLD. Therefore, MOK inhibitors would be useful for the treatment of nurodegenerative conformational diseases, and more specifically, where the aggregating protein is TDP-43.
  • MOK activity modulating agents are selected from a list consisting of:
  • the MOK modulating agent of the invention is an organic molecule, and more preferably, the organic molecule of formula (I):
  • n 0 or 1
  • R is a methyl (Me), isopropyl ( ⁇ Pr), tere-Butyl (tBu), ciciopeniadienil ⁇ (Cp)
  • X is H or NH 2
  • Y is H
  • NHCOCHCH2, NHSO2CHCH2, NHCOCH2CI, NHSO2CH2CH3, COCH2F, 0 COCH3, and Z is H
  • the MOK modulating agent of the invention is an organic molecule, and more preferably, the organic molecule of formula (II):
  • n is equal to 1
  • R is an isopropyl ( ⁇ Pr),
  • X is NH 2
  • the MOK modulating agent of the invention comprises an antisense oligonucleotide and is used for the prevention, improvement and / or treatment of neurodegerative conformational disease.
  • the MOK modulating agent of the invention comprises an antibody and is used for the prevention, improvement and / or treatment of neurodegerative conformational disease.
  • the MOK modulating agent of the invention comprises a ribozyme and is used for the prevention, improvement and / or treatment of neurodegerative conformational disease.
  • a second aspect of the invention relates to a composition, hereinafter composition of the invention, comprising a MOK modulating agent of the invention.
  • a composition of the invention comprising a MOK modulating agent 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 ingredient.
  • a third aspect of the invention consists in a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegerative conformational disease comprising:
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • Hirayama disease or monomelic atrophy Spinal muscular atrophy.
  • FTLD frontotemporal lobe degeneration
  • the neurodegerative conformational disease is ALS or FTLD.
  • a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegerative conformational disease comprising:
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • a fifth aspect of the invention consists in a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegerative conformational disease comprising:
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • the neurodegerative conformational disease is ALS or FTLD.
  • a method for the collection of data useful in the diagnosis and / or prognosis of a neurodegerative conformational disease comprising:
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • the neurodegerative conformational disease is ALS or FTLD. DESCRIPTION OF THE FIGURES
  • Fig. 1 The aggregates of TDP-43 are internalized and modified by microglial cells, mobilizing the endogenous TDP-43 protein.
  • Fused images include DAPI fluorescence (blue).
  • IF images show ubiquitin labeling (green), TDP-43 labeling (red), and fusion images, including DAPI fluorescence (D).
  • F IF analysis with TDP-43 immunostaining (red) of N13 cells treated with 5 ⁇ g / ml of TDP43 or simulation for 4 h.
  • TDP-43 nuclear protein levels (TDP-43 nuclear clearance) in cells exposed to TDP43 treatment.
  • G) IF confocal microscopy analysis of CD11 b (white), His-tag (red) and TDP-43 (green) immunotyped cultures of spinal cord organotypic cultures after 24 h of treatment with 5 ⁇ g / mL of TDP43 (or control mock) showing the presence of inclusions containing His-tag / TDP-43 in the microglial cell cytoplasm and the elimination of nuclear TDP-43, in the sample treated with exogenous TDP-43 aggregates.
  • Cell nuclei were contrasted with DAPI (blue).
  • the bars are the average of at least 3 independent experiments (N ⁇ 3), each performed with duplicate samples.
  • E) Western blot analysis of IL-18 cleaved in supernatants of primary microglial cells collected 24 h after treatment with 5 ⁇ g / ml of TDP43 or simulation (mock). Recombinant murine IL-18 was used as a control in the same gel (right end). Lower: quantification of the band density (relative values) of three independent experiments (N 3).
  • Fig. 3 The aggregates of TDP-43 induce a neuroinflammatory response that involves caspase-3.
  • Fused images include DAPI fluorescence. graduated bars: 25 ⁇ .
  • B, C Quantitative evaluation of the levels of activated caspase-3 of IF and confocal microscopy images (as shown in A), based on total activated caspase 3 (B) or granules of activated caspase-3 in the compartments Cytoplasmic and nuclear cells of the primary microglia (C).
  • the values shown were normalized by the untreated control (Ctrl) within each of the three experiments, and subsequently referred to the average granules / cell of the three Ctrl samples.
  • Fig. 4 Aggregates of extracellular TDP-43 are targets of the MOK microglia signaling pathway.
  • FIG. 5 (Supplementary Figure 1). Alternative processing of IL-18 in ca. 16 kDa in primary microglia culture. Expression of caspase-1 and secretion of IL-18 in cultures of the spinal cord organotypic section.
  • Fig. 6 (Supplementary Figure 2). IF analysis of activated caspase-3 in cells 4 h and 24 h after treatment.
  • Fig 7 (Supplementary Figure 3).
  • B Quantitative analysis of His6-tag / MOK colocalization (over total MOK) based on co-IF and confocal microscopy analysis.
  • the white arrow indicates a differential band / s and the regions in the gel (for both lanes) that were cleaved and analyzed by MALDI / MS.
  • the object of the invention is to modulate the expression and / or activity of the human protein MOK (MAPK / MAK / MRK overlapping kinase; 419 aa; Acc. Number BAA81688 or Q9UQ07, or any of its isoforms).
  • MAPK / MAK / MRK overlapping kinase 419 aa; Acc. Number BAA81688 or Q9UQ07, or any of its isoforms.
  • said protein kinase - a member of the MAPK superfamily that expresses itself in the brain and needs to be phosphorylated to phosphorylate other substrates - is strongly affected in terms of its expression and phosphorylation status, in a model ELA / FTD cell.
  • a first aspect of the invention relates to a MOK activity modulating agent, hereinafter MOK modulating agent of the invention, for the prevention, improvement and / or treatment of inflammatory diseases, more preferably neurodegenerative diseases. mediated by inflammation.
  • MOK modulating agent of the invention in the preparation of a medicament for the prevention, improvement and / or treatment of inflammatory diseases, more preferably of neurodegenerative diseases mediated by inflammation.
  • the invention also relates to a MOK activity modulating agent, hereinafter MOK modulating agent of the invention, for the prevention, improvement and / or treatment of neurodegenerative conformational diseases, or alternatively, a MOK modulating agent of the invention, for use in the prevention, improvement and / or treatment of neurodegenerative conformational diseases.
  • MOK modulating agent of the invention for the prevention, improvement and / or treatment of neurodegenerative conformational diseases
  • MOK modulating agent of the invention for use in the prevention, improvement and / or treatment of neurodegenerative conformational diseases.
  • This report refers to an inflammatory disease and / or that has an inflammatory component or is mediated by inflammation, which refers to a condition in a subject characterized by inflammation, for example, chronic inflammation.
  • Non-limiting, inflammatory disorders include, but are not limited to, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), asthma, encephalitis, chronic obstructive pulmonary disease (COPD), inflammatory osteolysis, allergic disorders, septic shock, pulmonary fibrosis (for example, idiopathic pulmonary fibrosis), inflammatory vacuhtides (for example, polyarteritisnode, Wegner's granulomatosis, Takayasu's arteritis, temporal arteritis, and lymphomatoid granulomatosis), post-traumatic vascular angioplasty (for example, restenosis after of angioplasty), inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, chronic hepatitis, and chronic inflammation resulting from chronic viral or bacterial infections. Also included in this report are inflammatory diseases,
  • neurodegerative conformational disease is understood as the kind of pathology in which one (or more) proteins specific ones become structurally abnormal, and therefore interfere with the function of cells, tissues and organs in the body. Often proteins fail to fold in their normal conformation; In this poorly folded state, the proteins form aggregates and become toxic in some way (eg, causing neuroinflammation) can lose their normal function.
  • PD Parkinson's disease
  • DLB Lewy body dementia
  • MSA multiple system atrophy
  • FAP amyotrophic lateral sclerosis
  • ALS fronto-temporal dementia
  • Gaucher disease Down syndrome
  • Huntington's disease prion disease
  • Creutzfeldt-Jakob Jakob disease and other transmissible spongiform encephalopathies
  • motor neuron Gerstmann-Stráussler-Scheinker syndrome
  • Kuru fatal familial insomnia
  • type II diabetes cerebrovascular amyloidosis
  • glaucoma age-related macular degeneration
  • psychiatric syndromes multiple tauopathies
  • familial amyloid polyneuropathy and neurodegeneration due to aggregation of age-related proteins.
  • ALS amyotrophic lateral sclerosis
  • primary lateral sclerosis progressive muscular atrophy
  • Kennedy disease or progressive spinobulbar muscular atrophy Hirayama disease or monomelic atrophy
  • spinal muscular atrophy frontotemporal lobe degeneration (FTLD), or Any of your combinations.
  • FTLD frontotemporal lobe degeneration
  • neurodegenerative disease is a neurodegerative conformational disease, that is, the invention relates to the use of the MOK modulating agent of the invention in the preparation of a medicament for prevention, improvement or treatment. of a neurodegerative conformational disease.
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • the aggregating neurodegenrative disease is ALS or FTLD.
  • MOK MAK / MAK / MRK overlapping kinase; 419 aa; Acc. Number BAA81688 or Q9UQ07, or any of its isoforms
  • This gene belongs to the MAP kinase superfamily.
  • the gene is regulated by the flow rate transcription factor 2 (Cdx2).
  • the encoded protein which is located in the epithelial cells in the intestinal crypt, can play a role in the growth arrest and differentiation of the cells of the upper crypt and regions of lower villi. Multiple transcription variants alternately linked encoding different isoforms have been observed for this gene.
  • MOK refers to both the gene and the human MOK protein.
  • MOK is also defined, but not limited to, by a nucleotide or polynucleotide sequence, which constitutes the MOK protein coding sequence, and which would comprise various variants from: a) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 1,
  • nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a) are nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),
  • nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code
  • nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or a 99% with SEQ ID NO: 1. in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the MOK protein.
  • SEQ I D NO: 2 is, but not limited to, SEQ I D NO: 2.
  • the activity of MOK can be modulated by the modification of the levels and / or activity of the MOK protein, or by the modification of the levels at which the MOK gene is transcribed such that the levels of activity of the MOK protein in The cell is modulated.
  • Modulating agents may also be agonists (substances that are capable of binding to a receptor and eliciting a response in the cell, specifically an increase in MOK activity), as antagonists (substances that not only do not activate the receptor, but also in actually blocks its activation by agonists). In the context of the present invention, inhibition is the preferred form of modulation.
  • TDP-43 also TARDBP or ALS10
  • TAR DNA binding protein a protein that is a transcriptional repressor that binds to chromosomally integrated DNA TAR and represses HIV-1 transcription.
  • this protein regulates the alternative splicing of the CFTR gene. It is located on chromosome 1, but a similar pseudogen is present on chromosome 20.
  • TDP-43 protein was identified as the main disease protein in most sporadic and familial FTLD cases. Subsequently, it was also found that ubiquitinated compact inclusions, characteristic of amyotrophic lateral sclerosis (ALS), were composed of TDP-43, thus providing evidence that both conditions are mechanically linked and are part of the spectrum of diseases that can be classified as proteopathies. TDP-43, (See, for example, Neumann et al., Science 314 (2006), 130-133).
  • TDP-43 accumulates in the nerve cells and glial cells of the dementia complex of ALS-Parkinsonism of Guam, corticobasal degeneration, dementia with Lewy bodies, Huntington's disease, body disease Lewy, motor neuron disease, frontotemporal dementia, frontotemporal lobar degeneration with positive inclusions to ubiquitin, hippocampal sclerosis, inclusion body myopathy, inclusion body myositis, Parkinson's disease, Parkinson's disease dementia, Parkinson's complex- dementia on the Kii peninsula and Pick's disease and the like, among other pathologies; See, for example, Lagier-Tourenne et al. , Hum. Mol. Gen. 19 (2010), R46-64, which is incorporated herein by reference in its entirety.
  • TDP-43 refers to both the gene and the human MOK protein.
  • TDP-43 is also defined, but not limited to, by a nucleotide or polynucleotide sequence, which constitutes the MOK protein coding sequence, and which would comprise various variants from: a) acid molecules nucleic encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),
  • nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code
  • nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or a 99% with SEQ ID NO: 1. in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the MOK protein.
  • nucleotide sequences encoding MOK is, but not limited to, SEQ ID NO: 4.
  • MOK activity modulating agents are selected from a list consisting of:
  • 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. Bookstores of organic molecules are commercially available.
  • the route of administration may be, without limitation, intraperitoneal, intrathecal, intravenous, intramuscular, subcutaneous, intraventricular, oral, enteral, parenteral, intranasal or dermal.
  • the MOK modulating agent of the invention is an organic molecule, and more preferably, the organic molecule of formula (I):
  • n 0 or 1
  • R is a methyl (Me), isopropyl ( ⁇ Pr), tere-Butyl (tBu), cyclopentadienyl (Cp)
  • X is H or NH 2
  • Y is H, NHCOCHCH 2 , NHSO2CHCH2, NHCOCH2CI, NHSO2CH2CH3, COCH2F, 0 COCH3, and Z is H,
  • the MOK modulating agent of the invention is an organic molecule, and more preferably, the organic molecule of formula (II):
  • n is equal to 1
  • R is an isopropyl ( ⁇ Pr),
  • X is NH 2
  • the compounds of the present invention represented by formula (I) and / or (II) may include isomers, depending on the presence of multiple bonds, including optical isomers or enantiomers, depending on the presence of chiral centers.
  • the individual isomers, enantiomers or diastereoisomers and mixtures thereof fall within the scope of the present invention, that is, the term isomer also refers to any mixture of isomers, such as diastereomers, racemic, etc., even their optically isomers. assets or mixtures in different proportions thereof.
  • prodrugs of the compounds of formula (I) and / or (II) include any derivative of a compound of formula (I) and / or (II) - for example and not limited to: esters (including esters of carboxylic acids, amino acid esters, phosphate esters, sulphonate esters of metal salts, etc.), carbamates, amides, etc- that when administered to an individual can be transformed directly or indirectly into said compound of formula (I) and / or (II) in the mentioned individual.
  • said derivative is a compound that increases the bioavailability of the compound of formula (I) and / or (II) when administered to an individual or that enhances the release of the compound of formula (I) and / or (II) in a biological compartment
  • said derivative is not critical as long as it can be administered to an individual and provides the compound of formula (I) and / or (II) in a biological compartment of an individual.
  • the preparation of said prodrug can be carried out by conventional methods known to those skilled in the art.
  • the term "derivative” includes both pharmaceutically acceptable compounds, that is, derivatives of the compound of formula (I) and / or (II) that can be used in the manufacture of a medicament or food compositions, such as Pharmaceutically acceptable derivatives, since these may 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.
  • the term "solvate”, as used herein includes both pharmaceutically acceptable solvates, that is, solvates of the compound of formula (I) and / or (II) that can be used in the manufacture of a medicament, as pharmaceutically acceptable solvates, which may be useful in the preparation of pharmaceutically acceptable solvates or salts.
  • the nature of the pharmaceutically acceptable solvate is not critical as long as it is pharmaceutically acceptable.
  • the solvate is a hydrate. 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, that is, 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 formula (I), or of its salts, solvates or prodrugs.
  • nucleotide sequences specifically complementary to a particular DNA or RNA sequence could form complexes and block transcription or translation.
  • interfering RNA interfering RNA
  • tools have been developed that allow specific inhibition of gene expression.
  • the inhibition of the expression of MOK would therefore constitute the inhibition of its biological activity, and in particular, of the activity that is contributing to the aggravation of the neurodegerative conformational disease.
  • antisense polynucleotides are understood ribonucleotide or deoxyribonucleotide chains that can inhibit the production of the MOK protein by one of these three mechanisms:
  • Antisense oligonucleotides capable of modulating the activity of MOK are known in the state of the art. For example, and without limiting our, it could be a sequence of ribonucleotides or RNA that belongs to the so-called siRNA (small interfering RNA), small interfering RNA or silencing RNA, capable of inhibiting the genetic expression of the MOK protein.
  • siRNA small interfering RNA
  • silencing RNA small interfering RNA
  • siRNA small interfering RNA
  • siRNA small interfering RNA
  • silencing RNA small interfering RNA
  • this specific gene is MOK. It could also be any siRNA capable of hybridizing a nucleic acid molecule encoding the human MOK protein (or any of its isoforms or transcriptional variant) whose sequence (transcriptional variant 1; Acc. No. NM_014226.2) is collected in SEQ ID NO: 5.
  • RNA constructs that contains at least any one of the possible nucleotide sequences of siRNA capable of inhibiting MOK expression, and notwithstanding that any of the RNA sequences and constructs of RNA are additionally part of the present invention.
  • the invention described above that are subject to modifications, preferably chemical, that lead to greater stability against the action of ribonucleases and thereby greater efficiency. Without these modifications involving the alteration of its mechanism of action, which is the specific binding to the RISC complex (RNA-induced silencing complex), activating it and manifesting a helicase activity that separates the two strands leaving only the antisense strand associated with the complex.
  • the resulting ribonucleoproteic complex binds to the target mRNA (MOK messenger RNA, which is collected in SEQ ID NO: 2 or in SEQ ID NO: 5).
  • MOK messenger RNA which is collected in SEQ ID NO: 2 or in SEQ ID NO: 5.
  • RISC is associated with the messenger and the translation is attenuated. But if it is perfect, RISC acts as RNasa, cutting the messenger and being free to repeat the process.
  • siRNA capable of inhibiting the translation of these mRNAs also forms part of the invention.
  • siRNA sequence of the invention or of the RNA construct of the invention would be apparent to one skilled in the art, and could be carried out by chemical synthesis, which also allows the incorporation of chemical modifications in both different nucleotides of the product such as the incorporation of other chemical compounds at any of the ends.
  • the synthesis could also be carried out enzymatically using any of the available RNA polymerases. Enzymatic synthesis also allows some chemical modification of inhibitor products or RNAs.
  • the nucleotide sequence design of the siRNA of the invention would also be apparent to one skilled in the art. Thus, it could be done through a random design in which 19-25 bases of the target mRNA are selected without taking into account the sequence or positional information it has in the transcript.
  • Another non-limiting alternative of the present invention would be the conventional design by simple parameters developed by the pioneers of the art (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 computer procedure is used to identify the optimal siRNA targets in an mRNA. The target sequences are analyzed in groups of 19 nucleotides at a time and those with the best characteristics are identified based on an algorithm that incorporates a large number of thermodynamic and sequence parameters.
  • a genetic DNA construct could also be part of the composition of the invention, which would direct the in vitro or intracellular transcription of the siRNA sequence or RNA construct of the invention, and comprising at least one of the following types of sequences: a) DNA nucleotide sequence, preferably double stranded, comprising at least the sequence encoding the siRNA of the invention or the RNA construct of the invention for transcription, or, b) nucleotide sequence of DNA, preferably double stranded, corresponding to a gene expression system or vector comprising the sequence coding for the RNA sequence 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, signals of start and end, cut sites, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), etc.
  • compositions of the present invention allow the transfection of the siRNA of the invention into a cell, in vivo or in vitro. Transfection could be carried out, but not limited to, direct transfection or vectors that facilitate the access of siRNA into the cell.
  • these vectors are, without limitation, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, Herpes simplex viruses, non-viral DNA plasmids, cationic liposomes and molecular conjugates.
  • the siRNAs of the present invention, as well as RNA or DNA precursors of these siRNAs can be conjugated with release peptides or other compounds to favor the transport of these siRNAs into the cell. Therefore, in another preferred embodiment of this aspect of the invention, the MOK modulating agent of the invention comprises an antisense oligonucleotide and is used for the prevention, improvement and / or treatment of neurodegerative conformational disease.
  • the MOK modulating agent of the invention comprises an antibody and is used for the prevention, improvement and / or treatment of neurodegerative conformational disease.
  • 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.
  • portions of immunologically active immunoglobulin molecules include F (ab) and F (ab ') 2 fragments that can be generated by treating the antibody with an enzyme such as pepsin. It can be a monoclonal or polyclonal antibody.
  • Antibodies capable of binding to the MOK protein can be used to inhibit the activity of said protein.
  • Such antibodies are commercially available, as per example, but not limited to, Anti-MOK antibodies marketed by Sigma-Aldrich HPA027282, HPA027292, SAB4502568, SAB1300406, SAB1305100, SAB4504627, SAB2105049.
  • the antibodies, or fragments thereof might be able to inhibit the activity of the MOK protein that contributes to the acquisition of the characteristics characteristic of neurodegerative conformational disease.
  • the 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 whole or in parts, of portions that are not necessary for the recognition of the MOK and being replaced by others that communicate to the antibody additional advantageous properties.
  • 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.
  • rAc can be expressed and directed to specific cellular sub-compartments when the appropriate sequences for intracellular traffic are incorporated.
  • These antibodies are called intrabodies, and have proven effective not only to divert proteins from their usual compartment or block interactions between proteins involved in signaling pathways, but also to activate intracellular proteins.
  • Also part of the invention are genetic DNA constructs capable of transcribing to a peptide, antibody or antibody fragment, for use in the treatment of ALS.
  • 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 it comprises, at least, the coding sequence of the antibody of the invention or of the antibody fragment of the invention for transcription in vitro, or intracellular,
  • DNA nucleotide sequence preferably double stranded, corresponding to a gene expression system or vector comprising the sequence coding for the antibody sequence or antibody fragment of the invention operatively 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, sites of section, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), etc. for use in those pathological contexts related to neurodegerative conformational disease.
  • ribozyme 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 RNA catalytic molecules.
  • RNA a catalytic polynucleotide
  • Ribozymes can be introduced into the cell as catalytic RNA molecules or as genetic constructs that are expressed to RNA catalytic molecules.
  • the MOK modulating agent of the invention comprises a ribozyme and is used for the prevention, improvement and / or treatment of a neurodegerative conformational disease.
  • a second aspect of the invention relates to a composition, hereinafter composition of the invention, comprising a MOK modulating agent of the invention.
  • a composition of the invention comprising a MOK modulating agent 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 ingredient.
  • 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 elaboration 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 the age, condition of the patient, the severity of the alteration 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 active ingredients, additional to provide a combination therapy.
  • Said additional drugs may be part of the same pharmaceutical composition or, alternatively, they may be provided in the form of a separate composition for simultaneous or non-simultaneous administration to the pharmaceutical composition comprising a compound of formula (I) and / or (II ), or a salt, prodrug or solvate thereof.
  • the pharmaceutical composition further comprises another active ingredient.
  • active substance means any component that potentially provides a pharmacological activity or other different effect on the 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 preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect.
  • Another aspect of the invention relates to a pharmaceutical form, hereinafter pharmaceutical form of the invention, comprising the compound of the invention or the composition of the invention.
  • compositions and pharmaceutical 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 may be colloidal systems, which include nanoemulsions, nanocapsules and polymeric nanoparticles.
  • compositions for oral administration can be prepared by conventional methods of Galenic Pharmacy, as mixing and dispersion.
  • the tablets can be coated following methods known in the pharmaceutical industry.
  • the compositions and pharmaceutical 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 such as pH buffering agents or surfactants, can be used.
  • the aforementioned formulations can be prepared using conventional methods, such as those described in the Pharmacopoeias of different countries and in other reference texts.
  • medication refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals.
  • the administration of the compounds, compositions or pharmaceutical forms of the present invention can be performed by any suitable method, such as intravenous infusion and oral, topical or parenteral routes. Oral administration is preferred for the convenience of patients and for 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 a 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 keep 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 modifications 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 may be part of the same composition or of a different composition, for administration at the same time or at different times.
  • a third aspect of the invention consists in a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegerative conformational disease comprising:
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • the neurodegerative conformational disease is ALS or FTLD.
  • test compounds to be tested used in the method of selection of therapeutic agents are not limited to low molecular weight organic molecules, proteins (including antibodies), peptides, oliogonucleotides, etc. They can be natural and / or synthetic.
  • antibodies capable of binding to an MOK epitope 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.
  • MOK 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 cultured cell lines, 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 selection 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 MOK and, in particular, a neurodegerative conformational disease.
  • a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegerative conformational disease comprising:
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • the neurodegerative conformational disease is ALS or FTLD.
  • a fifth aspect of the invention consists in a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegerative conformational disease comprising:
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • the neurodegerative conformational disease is ALS or FTLD.
  • 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).
  • Procedures for detecting the expression of the MOK protein are also well known in the state of the art, such as poly or monoclonal antibodies, ELISA, radioimmunoassay (RIA), and FACS (fluorescence activated cell sorting).
  • a method for the collection of data useful in the diagnosis and / or prognosis of a neurodegerative conformational disease comprising:
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • the neurodegerative conformational disease is ALS or FTLD.
  • the ELA and FTLD model used recapitulates the effects observed for TDP-43 in patient samples and animal models of ELA and FTLD
  • microglia support the results described above in neurons, and recapitulate the effects observed in samples of patients and animal models of ALS and FTLD (Boeynaems et al., 2016. Acta Neuropathol. 132 (2): 159- 73).
  • TDP43 ' aggregates of TDP-43
  • 'mock' immunofluorescence
  • IL-18 activated and secreted by Western blot has been observed (Fig. 2E, Fig. SI B), as well as total intracellular IL-18 by IF (Fig. 2F) and a kind of IL-18 alternative processing product, 16 kDa, by Western blot (Fig. 2G).
  • Non-cell autonomic neurodegeneration 'non-cell autonomous' mechanism
  • IL-18 and caspase-3 Non-cell autonomic neurodegeneration ('non-cell autonomous' mechanism) involving IL-18 and caspase-3
  • 'conformational' neurodegenerative diseases / 'misfolding diseases' in particular, ELA and FTLD
  • others related Alzheimer, Parkinson, Huntington, prionosis, etc.
  • aging also characterized by the increasing presence of aberrantly added protein species in the intra and / or extracellular environment
  • diseases with an inflammatory component including Cancer.
  • TDP-43 The aggregates of TDP-43 affect the signaling pathway of the MAPK / MAK / MRK kinase overlapping kinase (MOK) in microglia. Implications for 'conformational', inflammatory and cancer neurodegenerative diseases
  • MOK is a serine / threonine kinase belonging to the MAPK kinase superfamily and which is expressed in different tissues (Fu JZ, 2012. In Protein Kinases, Intech Open, pp: 229-248). MOK is capable of phosphorylating a variety of substrates and for its activity requires the phosphorylation of two residues within the TEY motif, one of which is autophosphorylated (Fu JZ, 2012. In Protein Kinases, Intech Open, pp: 229-248) .
  • MOK sequence at the RNA level contains in its 3 'terminal region a segment identical to the RAGE family (renal cell carcinoma antigen) proteins, which are overexpressed in various cancers (renal, melanoma, leukemia, etc.) (Eichmuller et al., 2002. Exp. Dermatol. 1 1 (4): 292-301). This extends the possible role of MOK to the pathogenesis and / or development of cancer, considering also that there is a proven relationship between cancer and inflammation.

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Abstract

L'invention concerne des composés et des compositions permettant de moduler l'activité de MOK (MAPK/MAK/MRK) ou de l'une quelconque de ses isoformes et variantes biologiquement actives, ainsi qu'une méthode de sélection correspondante pour le traitement de maladies à composante inflammatoire et/ou de maladies conformationnelles neurodégénératives, et plus particulièrement pour le traitement de la sclérose latérale amyotrophique (SLA) et de la dégénérescence lobaire fronto-temporale (Fronto-Temporal Lobar Degeneration, FTLD).
PCT/ES2018/070216 2017-03-21 2018-03-21 Utilisation d'agents modulateurs de mok pour le traitement de maladies inflammatoires et de maladies neurodégénératives Ceased WO2018172588A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011153553A2 (fr) * 2010-06-04 2011-12-08 The Regents Of The University Of California Méthodes et compositions pour l'inhibition de kinases

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011153553A2 (fr) * 2010-06-04 2011-12-08 The Regents Of The University Of California Méthodes et compositions pour l'inhibition de kinases

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LEAL-LASARTE, M.M. ET AL.: "Extracellular TDP-43 aggregates target MAPK/MAK/MRK overlapping kinase (MOK) and trigger caspase-3/ IL -18 signaling in microglia", THE FASEB JOURNAL, vol. 31, no. 7, 23 March 2017 (2017-03-23), pages 2797 - 2816, XP055538709 *

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