WO2021006707A1 - Utilisation de l'inhibiteur de sgk1 en tant qu'agent thérapeutique pour maladies neurologiques inflammatoires - Google Patents

Utilisation de l'inhibiteur de sgk1 en tant qu'agent thérapeutique pour maladies neurologiques inflammatoires Download PDF

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WO2021006707A1
WO2021006707A1 PCT/KR2020/009141 KR2020009141W WO2021006707A1 WO 2021006707 A1 WO2021006707 A1 WO 2021006707A1 KR 2020009141 W KR2020009141 W KR 2020009141W WO 2021006707 A1 WO2021006707 A1 WO 2021006707A1
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sgk1
pyrrolo
pyridin
phenyl
disease
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이상훈
송재진
권오찬
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한양대학교 산학협력단
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Definitions

  • the present invention relates to the use of Sgk1 inhibitors in the treatment of inflammatory neurological diseases through neuronal protection and neuroinflammation reduction.
  • Neurodegenerative diseases are basically caused by or accompanied by chronic inflammation. These neurological diseases include neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple systemic atrophy, amyotrophic lateral sclerosis (ALS), cerebral infarction and spinal injury, as well as neuropathic pain caused by neuroinflammation. , Complex region pain syndrome (CRPS), etc.
  • neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple systemic atrophy, amyotrophic lateral sclerosis (ALS), cerebral infarction and spinal injury, as well as neuropathic pain caused by neuroinflammation. , Complex region pain syndrome (CRPS), etc.
  • Parkinson's disease accelerates the progression of the disease in the inflammatory environment of the brain, and the project of direct new drug development at the lesion site has also continued to fail. Reinforcing the function of glial cells in neurodegenerative diseases caused by the loss of these neurons is considered as a potential treatment that can treat intractable brain diseases.
  • glial cells include astrocytes and microglia, which are auxiliary cells that help the function and survival of nerve cells.
  • M1 activation M1 activation
  • M2 type glial cells M2 type glial cells that create a therapeutic environment that promotes survival and regeneration of neurons.
  • the present inventors revealed that the joint effect of the transcription factors Nurr1 and Foxa2 inhibits the inflammatory response of glial cells, but Nurr1 and It is not stated how the inhibitory effect of the inflammation on the synergistic action of Foxa2 is subsequently achieved. Accordingly, the present invention is to determine whether overexpression of Nurr1 and Foxa2 reduces neuroinflammation through a mechanism of action in glial cells based on the results of previous studies.
  • Sgk1 serum and glucocorticoid-regulated kinase 1
  • GR serum and glucocorticoid
  • SGK serum and glucocorticoid inducible protein kinase
  • the present inventors have confirmed that neuroinflammation is reduced in glial cells overexpressing Nurr1 and Foxa2 through previous studies, and it has been confirmed that the expression of Sgk1 is reduced when Nurr1 and Foxa2 are overexpressed. Accordingly, the present inventors confirmed that the Sgk1 inhibitor can contribute to the treatment of inflammatory neurological diseases from the effect of protecting nerve cells and inhibiting neuroinflammation through inhibition of the expression of Sgk1, and completed the present invention.
  • the present invention relates to a novel mechanism of action of Sgk1 for reducing neuroinflammation.
  • the present invention provides a pharmaceutical composition for preventing or treating inflammatory neurological diseases comprising an Sgk1 inhibitor as an active ingredient.
  • Sgk1 glucocorticoid-regulated kinase 1
  • Sgk1 is a mammal, preferably human, mouse, house mouse, rabbit, orangutan, monkey, hamster, cat, dolphin, gorilla It means a protein originating from the back.
  • the Sgk1 gene and protein sequences are known.
  • the Sgk1 is human (Homo sapiens) derived from GenBank accession no. NP_001137148; Genbank accession no. from mouse (Mus musculus). It may be an Sgk1 protein having an amino acid sequence such as NP_001155317, but is not limited thereto.
  • the “Sgk1 inhibitor” refers to a drug exhibiting an effect of reducing neuroinflammation in patients with inflammatory neuropathy.
  • the Sgk1 inhibitor may include any agent that knocks down the mRNA expression of the Sgk1 gene or reduces the function or activity of the Sgk1 protein.
  • the Sgk1 inhibitor is an antisense-oligonucleotide comprising a sequence complementary to the Sgk1 gene, siRNA, shRNA, miRNA, or a vector comprising the same; Antibodies specific for the Sgk1 protein; Or a compound having inhibitory activity against the Sgk1 enzyme represented by Formula 1 or Formula 2; It can be either:
  • the Sgk1 inhibitor may be one or more of the compound represented by Formula 1 (GSK-650394) and the compound represented by Formula 2 (EMD-638683), but is not limited thereto.
  • the Sgk1 inhibitor may be applied by substituting a known compound having inhibitory activity against Sgk1 in addition to the compound represented by Formula 1 or Formula 2.
  • the compound capable of replacing the compound represented by Formula 1 or Formula 2 may be a compound disclosed in US 2009//0233955 A1, US 2012/10238588 A1 or EP 2242738 B1.
  • the compound is 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; ⁇ 3-[5-(2-naphthyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzyl ⁇ amine; 4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; ⁇ 4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl ⁇ acetic acid; 3- ⁇ 4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl ⁇ propanoic acid; ⁇ 3-[5-(2-naphthyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl ⁇ methanol; 4-
  • RNA small interfering RNA
  • siRNA refers to a double-stranded RNA that induces RNA interference by cleaving the mRNA of the target gene, and the RNA strand of the sense sequence having the same sequence as the mRNA of the target gene and It consists of an RNA strand of an antisense sequence having a complementary sequence.
  • the siRNA may be prepared according to a conventional method by targeting the siRNA common nucleic acid sequence site on human and/or mouse.
  • the siRNA may be ThermoFisher Scientific's Assay ID Mm00441380_m1, catalog #4331182, but is not limited thereto.
  • the siRNA may include a siRNA synthesized in vitro or a form expressed by inserting a nucleic acid sequence encoding the siRNA into an expression vector.
  • the "vector” refers to a genetic construct including an external DNA inserted into a genome encoding a polypeptide.
  • the vector related to the present invention is a vector in which a nucleic acid sequence that inhibits the gene is inserted into the genome, and these vectors include DNA vectors, plasmid vectors, cosmid vectors, bacteriophage vectors, yeast vectors, or viral vectors.
  • suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, start codons, stop codons, polyadenylation signals and enhancers, and variously according to the purpose. Can be manufactured.
  • the promoter of the vector can be constitutive or inducible.
  • the expression vector includes a selection marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, it includes an origin of replication.
  • the antisense has a sequence complementary to all or part of the mRNA sequence transcribed from the Sgk1 gene or fragment thereof, and may bind to the mRNA to inhibit the expression of the Sgk1 gene or fragment.
  • shRNA short hairpin RNA
  • shRNA refers to a nucleotide consisting of 50-60 single strands, and constitutes a stem-loop structure in vivo. That is, shRNA is an RNA sequence that creates a tight hairpin structure to suppress gene expression through RNA interference.
  • shRNA is transduced into cells via a vector containing the U6 promoter and is usually transferred to daughter cells, allowing gene expression inhibition to be inherited.
  • shRNA The hairpin structure of shRNA is cleaved by an intracellular mechanism to become siRNA and then binds to the RNA-induced silencing complex (RISC). These RISCs bind to and cleave mRNA.
  • shRNA is transcribed by RNA polymerase.
  • shRNA short hairpin RNA
  • shRNA short hairpin RNA
  • the shRNA may consist of the nucleic acid sequence of SEQ ID NO: 9:
  • miRNA refers to a short non-coding RNA consisting of about 22 nucleic acid sequences. It is known to function as a post-transcriptional regulator in the process of gene expression. By binding complementarily to a target mRNA having a complementary nucleic acid sequence, the target mRNA is degraded or translation into a protein is inhibited. The presence of the miRNA can be confirmed using a probe.
  • probe refers to a natural or modified monomer or a linear oligomer of a linkage, including deoxyribonucleotides and ribonucleotides, and can specifically hybridize to a target nucleotide sequence, and exist naturally or Or it may be artificially synthesized.
  • polyclonal antibodies polyclonal antibodies, monoclonal antibodies, human antibodies, and humanized antibodies against Sgk1 protein can be used.
  • antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments; Diabody; Linear antibodies (Zapata et al. Protein Eng. 8(10):1057-1062(1995)); Single chain antibody molecules; And multispecific antibodies formed from antibody fragments.
  • Fv is a minimal antibody fragment containing a complete antigen recognition and binding site. This site consists of a dimer of one heavy chain and one light chain variable region, and is tightly bound by a non-covalent bond.
  • Polyclonal antibodies can be prepared by injecting an immunizing agent into a mammal one or more times and, if necessary, injecting with an adjuvant. Typically, the immunizing agent and/or adjuvant is injected several times by subcutaneous injection or intraperitoneal injection into the mammal.
  • the immunizing agent may be a protein of the invention or a fusion protein thereof. It may be effective to inject an immunizing agent with a protein known to be immunogenic in the mammal being immunized.
  • the monoclonal antibody according to the present invention can be prepared by the hybridoma method described in Kohler et al. Nature, 256:495 (1975) or by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,576). I can. Monoclonal antibodies are also phage antibody using techniques described in Clackson et al. Nature, 352:624-628 (1991) and Marks et al. J. Mol. Biol., 222:581-597 (1991). It can be isolated from the library.
  • a portion of the heavy chain and/or light chain is identical or homologous to the sequence corresponding to an antibody derived from a specific species or an antibody belonging to a specific antibody class or subclass.
  • the remainder of the chain(s) includes antibodies derived from other species or antibodies belonging to different antibody classes or subclasses, or "chimeric" antibodies that are identical or homologous to fragments of such antibodies (Morrison et al. Proc). Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
  • “Humanized” forms of non-human (eg, murine) antibodies include chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg, Fv, Fab, Fab′) containing minimal sequences derived from non-human immunoglobulins. , F(ab') 2 or other antigen binding sequence of the antibody).
  • humanized antibodies are human immunoglobulins in which residues of the recipient's complementarity determination (CDR) are replaced with CDR residues of a non-human species (donor antibody) such as mice, rats, or rabbits having the desired specificity, affinity and ability. Acceptor antibody).
  • CDR complementarity determination
  • donor antibody such as mice, rats, or rabbits having the desired specificity, affinity and ability. Acceptor antibody.
  • the Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues.
  • humanized antibody may comprise residues not found in the recipient antibody or in the CDR or framework sequence to be introduced.
  • Humanized antibodies comprise substantially all of one or more, generally two or more variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are human immunoglobulins. It corresponds to a region of the sequence.
  • humanized antibodies comprise at least a portion of an immunoglobulin constant region (Fc), generally a portion of a human immunoglobulin region (Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)).
  • the pharmaceutical composition can protect nerve cells and inhibit neuroinflammation by knocking down Sgk1 through an Sgk1 inhibitor, and thus can prevent, improve or treat inflammatory neurological diseases.
  • the anti-inflammatory effect in glial cells knocked down Sgk1 was confirmed based on the result of a decrease in the expression of Sgk1 in glial cells overexpressing Nurr1 and Foxa2.
  • inhibition of Sgk1 expression significantly reduced the expression of inflammatory cytokines IL-1ß and TNF- ⁇ in glial cells, and the amount of reactive oxygen species (ROS) associated with cellular senescence decreased.
  • ROS reactive oxygen species
  • the present invention confirmed that the absorption of glutamate was increased from the result that the activity of the inflammation regulatory complex (inflammasome) was decreased by the inhibition of the expression of Sgk1, and the expression of glutamate transport genes GLAST and GLT-1 was increased. It was revealed through animal experiments that Sgk1 inhibitors can prevent the aggregation of ⁇ -synuclein and thus exhibit an effect as a therapeutic agent for an inflammatory neurological disease model.
  • the expression of inflammatory cytokines can be reduced and the aggregation of ⁇ -synuclein can be suppressed. Since neuroprotection and neuroinflammation can be suppressed, symptoms of patients with inflammatory neurological diseases such as Parkinson's disease and Alzheimer's disease can be improved, alleviated or treated from Sgk1 inhibitors.
  • the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes carriers and vehicles commonly used in the field of medicine, and specifically, ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer substances (eg , Various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g.
  • colloids Including, but not limited to, silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax or wool paper, and the like.
  • composition of the present invention may further include a lubricant, a wetting agent, an emulsifier, a suspending agent or a preservative in addition to the above components.
  • the composition according to the present invention may be prepared as an aqueous solution for parenteral administration, preferably a buffer solution such as Hank's solution, Ringer's solution or physically buffered saline You can use Aqueous injection suspensions may be added with a substrate capable of increasing the viscosity of the suspension such as sodium carboxymethylcellulose, sorbitol or dextran.
  • a buffer solution such as Hank's solution, Ringer's solution or physically buffered saline
  • composition of the present invention may be administered systemically or locally, and for such administration may be formulated in a dropping plate formulation by a known technique.
  • a dropping plate formulation for example, when administered orally, it may be administered by mixing with an inert diluent or an edible carrier, sealed in a hard or soft gelatin capsule, or compressed into tablets.
  • the active compound may be mixed with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • an effective amount of the Sgk1 inhibitor may be formulated as a solution immediately before administration in saline or buffer in a form suitable for intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, and the like.
  • the term "administration" means introducing the composition of the present invention to a patient by any suitable method, and the route of administration of the composition of the present invention is through various oral or parenteral routes as long as it can reach the target tissue. Can be administered. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration may be performed, but are not limited thereto.
  • the present invention provides a method for treating inflammatory neurological diseases comprising administering a therapeutically effective amount of an Sgk1 inhibitor to a subject in need thereof.
  • subject refers to a mammal that is the subject of treatment, observation or experiment, and preferably refers to a human.
  • the "therapeutically effective amount” refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human thought by a researcher, veterinarian, doctor or other clinician, which It includes the amount necessary to delay or completely stop the onset or progression of the particular disease to be treated.
  • the effective amount of the Sgk1 inhibitor contained in the pharmaceutical composition of the present invention is Parkinson's disease, Alzheimer's disease, multiple systemic atrophy, amyotrophic lateral sclerosis (ALS), cerebral infarction, spinal injury, neuropathic pain, and complex region Pain syndrome (CRPS) refers to the amount required to achieve neuroprotective and neuroinflammation reduction effects.
  • the therapeutically effective amount is the type of disease, severity of disease, type and content of other ingredients contained in the composition, and the age, weight, general health condition, sex and diet of the patient, administration time, administration route, treatment period, It can be adjusted according to a variety of factors, including drugs used simultaneously.
  • a suitable total daily amount can be determined by the treating physician within the range of correct medical judgment.
  • a specific therapeutically effective amount for a specific patient is the type and extent of the reaction to be achieved, the specific composition including whether or not other agents are used in some cases, the patient's age, weight, general health status, and sex. And it is preferable to apply differently according to various factors including diet, administration time, route of administration and secretion rate of the composition, duration of treatment, drugs used with or concurrently with the specific composition, and similar factors well known in the medical field.
  • the composition comprising the Sgk1 inhibitor of the present invention as an active ingredient is conventionally used through oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes. It can be administered in a phosphorus manner.
  • treatment means an approach to obtain beneficial or desirable clinical outcomes.
  • beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (i.e., not exacerbation), delay or decrease in disease progression, disease state. Amelioration or temporal mitigation and mitigation (partially or entirely) of, detectable or undetectable.
  • treatment may mean increasing the survival rate compared to the expected survival rate when not receiving treatment.
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include the disorder to be prevented as well as the treatment required for a disorder that has already occurred. “Relieving” the disease means that the extent of the disease state and/or undesirable clinical signs are reduced and/or the time course of progression is slowed or prolonged compared to without treatment. do.
  • the present invention provides the use of an Sgk1 inhibitor for the treatment of inflammatory neurological diseases.
  • compositions for preventing or treating inflammatory neurological diseases include all animals except humans.
  • it may be an animal such as a dog, a cat, or a mouse.
  • the present invention provides a composition for diagnosing inflammatory neurological diseases comprising an agent measuring the level of mRNA or protein thereof of the Sgk1 (serum and glucocorticoid-regulated kinase 1) gene.
  • the present invention provides a method for diagnosing inflammatory neurological disorders comprising the step of measuring the expression level of Sgk1 using an agent measuring the level of mRNA or protein thereof of the Sgk1 gene from a biological sample isolated from an individual.
  • the inflammatory neurological disease since the expression of Sgk1 is increased, the inflammatory neurological disease can be diagnosed by checking the expression level of Sgk1.
  • diagnosis refers to confirming a pathological state, and for the purposes of the present invention, the diagnosis is to confirm the onset, development and alleviation of these diseases by checking the expression of diagnostic markers of inflammatory neurological diseases. Means to do.
  • diagnosis marker refers to a substance capable of distinguishing and diagnosing cells of inflammatory neurological disease from normal cells, and a polypeptide or nucleic acid (eg, mRNA) showing an increase or decrease in inflammatory neurons compared to normal cells.
  • lipids e.g., lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.), and the like.
  • the marker for diagnosing inflammatory neurological disease provided by the present invention may be a protein expressed from the Sgk1 gene whose expression level is increased or decreased in cells of inflammatory neurological disease compared to normal cells.
  • composition for diagnosing inflammatory neurological diseases of the present invention includes an agent for measuring the level of mRNA expression of the Sgk1 gene or the amount of protein expressed from the gene, and with such an agent, an oligonucleotide having a sequence complementary to Sgk1 mRNA, such as Sgk1 mRNA. It may contain a primer or nucleic acid probe that specifically binds, or an antibody specific for the Sgk1 protein.
  • Sgk1 serum and glucocorticoid-regulated kinase 1 gene is brought into contact with a candidate substance outside the human body
  • It provides a method for screening a drug for preventing or treating inflammatory neurological diseases, comprising determining whether the candidate substance promotes or inhibits the expression of the gene.
  • Sgk1 serum and glucocorticoid-regulated kinase 1
  • It provides a method for screening a drug for preventing or treating inflammatory neurological diseases, comprising determining whether the candidate substance enhances or inhibits the function or activity of the protein.
  • a candidate substance to be analyzed may be brought into contact with a biological sample collected from a patient of inflammatory neurological disease including the gene or protein.
  • the candidate substance is a substance that promotes or inhibits transcription and translation from the Sgk1 gene nucleic acid sequence to mRNA, protein, or enhances or inhibits the function or activity of the Sgk1 protein according to a conventional selection method, knock-down (knock-down). down) It may be a substance that is assumed to have the potential as a drug or a randomly selected individual nucleic acid, protein, peptide, other extract, natural product, or compound.
  • the expression amount of the gene, the amount of protein, and the activity of the protein can be measured or confirmed in the sample treated with the candidate substance, and as a result of the measurement, it is determined that the amount of expression of the gene, the amount of protein, or the activity of the protein is decreased. If so, the candidate substance can be determined as a substance capable of treating an inflammatory neurological disease.
  • the candidate substance when the candidate substance inhibits the expression of the Sgk1 gene, the candidate substance may be determined as a substance capable of treating an inflammatory neurological disease.
  • the method of measuring the gene expression level of Sgk1 or the amount of protein may be performed by using a known technique, including a known process of separating mRNA or protein from a biological sample.
  • the biological sample refers to a sample taken from a living body whose expression level or protein level of the gene is different from that of a normal control group according to the incidence or progression of inflammatory neurological disease, and the sample includes, for example, tissue, cells, blood, Serum, plasma, saliva, urine, and the like may be included, but are not limited thereto.
  • the method of measuring the amount of gene expression, the amount of protein, or the activity of the protein can be performed through various methods known in the art, such as reverse transcripatase-polymerase chain reaction, real-time Real time-polymerase chain reaction, Western blot, Northern blot, Southern blot, EMSA (electrophoric mobility shift assay), immunofluorescence, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay (RIA (radioimmuno assay), radioimmunodiffusion, or immunoprecipitation can be used.
  • reverse transcripatase-polymerase chain reaction real-time Real time-polymerase chain reaction
  • Western blot Western blot
  • Northern blot Southern blot
  • EMSA electrofluoric mobility shift assay
  • immunofluorescence ELISA (enzyme-linked immunosorbent assay)
  • radioimmunoassay RIA (radioimmuno assay),
  • Candidate substances obtained through the screening method of the present invention and exhibiting activity of inhibiting gene expression or reducing the function of proteins may be candidate substances for therapeutic agents for inflammatory neurological diseases.
  • Such candidates for inflammatory neurological disease treatment will act as a leading compound in the development of a treatment for inflammatory neurological disease in the future, and the leading material can promote or inhibit the function of the Sgk1 gene or the protein expressed therefrom.
  • the leading material can promote or inhibit the function of the Sgk1 gene or the protein expressed therefrom.
  • the present invention reduces neuroinflammation through knockdown of Sgk1, inhibits senescence of glial cells, increases glutamate absorption in glial cells, and improves maturation and viability of neurons, so that inflammatory neurons from Sgk1 inhibitors It can contribute to the prevention, alleviation or treatment of disease.
  • 1A shows the results of microarray analysis in primary cultured cells of glial cells overexpressing Nurr1 and Foxa2 obtained from mouse cortex.
  • Figure 1b shows the results of RNA-sequencing analysis of glial cells overexpressing Nurr1 and Foxa2 obtained from mouse midbrain.
  • 1C is a result of confirming the expression level of Sgk1 according to the expression of Nurr1 and/or Foxa2 through qPCR.
  • Figure 2a shows the results of western blot analysis to confirm the expression level of Sgk1 in astrocytes overexpressing Nurr1 and/or Foxa2.
  • Figure 2b is a result of confirming the NF ⁇ B activity after the treatment of the TLR ligand LPS to glial cells.
  • 3 is a result of confirming the expression level of inflammatory cytokines (IL-1 ⁇ and TNF- ⁇ ) in glial cells treated with an Sgk1 inhibitor.
  • Figure 4 shows the effect of the interaction between cells according to the regulation of Sgk1
  • Figure 4a is a schematic diagram of the co-culture of astrocytes with reduced Sgk1 expression and Sgk1 overexpressed astrocytes.
  • 4B and 4C are results of measuring the activity of reactive oxygen species (ROS) of two co-cultured cells.
  • ROS reactive oxygen species
  • Figure 5 is a result of confirming the contribution of each cell type (microglia or astrocyte) according to the Sgk1 regulation
  • Figure 5a is a result of measuring the Sgk1 expression level after separating and culturing microglia and astrocytes.
  • 5B and 5C are the results of confirming the expression level of Sgk1 and the intensity of Sgk1 fluorescence in astrocytes in the midbrain region treated with H 2 O 2 and astrocytes in the cortical region.
  • FIG. 6 shows the results of RNA sequencing according to the presence or absence of an Sgk1 inhibitor in midbrain-derived glial cells as confirming the Sgk1 inhibitory effect on astrocytes.
  • FIG. 7 shows the Sgk1 inhibitory effect on astrocytes, and shows a heat-map of a group of genes related to inflammation/immunity among the results of FIG. 6.
  • FIG. 8 is a result of confirming the effect of Sgk1 inhibition on the activity of the NRLP3 inflammation-regulating complex (inflammasome).
  • FIG. 8A is a result of confirming the effect of Sgk1 inhibitory treatment on the midbrain-derived glial cells cultured with LPS and ATP sequentially to stimulate inflammasome activity. This is the result of confirming the expression levels of caspase-1 and IL-1ß.
  • Figure 8b is a graph confirming the expression levels of IL-1ß and IL-6 in the culture medium of midbrain-derived glial cells treated with an Sgk1 inhibitor.
  • Figure 8c is a result of measuring the cGAS-STING pathway activity in glial cells according to the treatment of the Sgk1 inhibitor.
  • FIG. 9 is a result of confirming the effect of Sgk1 inhibition on the activity of the NRLP3 inflammation-regulating complex (inflammasome),
  • FIG. 9a is a result of measuring the activity of reactive oxygen species (ROS) of glial cells treated with the Sgk1 inhibitor.
  • Figure 9b shows the results of immunostaining and FACS of Mito-Sox cells treated with Sgk1 inhibitor.
  • ROS reactive oxygen species
  • FIG. 10 The left side of FIG. 10 is a heat-map showing the expression of a cell aggregation/ECM molecule-related gene in glial cells treated with the Sgk1 inhibitor, and the right side is the result of confirming the degree of glutamate absorption in glial cells treated with the Sgk1 inhibitor.
  • Figure 11 shows the results of confirming the maturation and viability of cells according to the treatment of the Sgk1 inhibitor in neurons, confirming the neurite length and TH expression rate of midbrain-derived neurons and dopamine neurons according to the concentration of the Sgk1 inhibitor.
  • 15A is a result of confirming the expression levels of Parkin, Paris, and PGC1a after treatment with an Sgk1 inhibitor on neurons.
  • 15b is a result of confirming the expression level of Parkin after treatment with the Sgk1 inhibitor in SH-SY5Y cells.
  • Figure 15c shows the results of reducing the Sgk1 inhibitor ROS production of mitochondria.
  • Figure 15d shows the results of the Sgk1 inhibitor inhibiting the aggregation of ⁇ -synuclein.
  • Figure 16 shows the manufacturing process of the MPTP-induced Parkinson's disease mouse model.
  • 17 is a result of performing a beam test and a pole test for the mouse model of FIG. 16.
  • 21 is a result of measuring the number of dopamine neurons in the SN region of the mouse model of FIG. 16.
  • FIG. 22 is a schematic diagram of co-culture of neural progenitor cells and astrocytes derived from the midbrain overexpressing ⁇ -synuclein in order to confirm whether there is an effect of preventing ⁇ -synuclein aggregation and removing aggregation according to the treatment of the Sgk1 inhibitor in vitro.
  • FIG. 23 shows the degree of aggregation of ⁇ -synuclein by performing immunostaining and western blot after treatment with an Sgk1 inhibitor while differentiating the neural progenitor cells derived from the midbrain overexpressing ⁇ -synuclein for 3 weeks.
  • FIG. 24 shows the degree of aggregation of ⁇ -synuclein by performing immunostaining and western blot after co-culturing ⁇ -synuclein-derived neural progenitor cells and astrocytes derived from the midbrain and astrocytes co-cultured with an Sgk1 inhibitor.
  • FIG. 25 is a schematic diagram of a process of fluorescently staining mouse brain tissue after injection of pre-formed Fibril (PFF) and AAV2 ⁇ -synuclein virus into the SN site of a mouse ICR species, and then daily intraperitoneal injection of an Sgk1 inhibitor.
  • PFF pre-formed Fibril
  • FIG. 26 is a result of fluorescence staining of a mouse brain intraperitoneally injected with the Sgk1 inhibitor of FIG. 25, and is a result of observing the level of expression of phospho- ⁇ -synuclein.
  • FIG. 27 shows the result of fluorescence staining of the mouse brain injected with the Sgk1 inhibitor of FIG. 25 intraperitoneally, and is a result of observing the expression level of CD16/32, an M1 type marker of microglia.
  • FIG. 28 shows the results of animal behavior experiments (beam test and pole test) of mice intraperitoneally injected with the Sgk1 inhibitor of FIG. 25.
  • Dopaminergic neuronal progenitor cells were obtained from the midbrain region at 10.5 days gestation in the case of mice (imprinting control region, ICR), and in the midbrain region at the 12th day gestation in rats (Sprague-Dawley rat, SD). Obtained.
  • Mesocerebral neural progenitor cells were cultured using serum-free N2 medium, and bFGF (basic fibroblast growth factor; 20 ng/ml; R&D Systems, Minneapolis, MN, USA) and epithelial growth factor were cultured in the culture medium to promote division. (EGF; 20 ng/ml; R&D Systems, mouse cell culture) was added and cultured. Thereafter, when the cell density (confluence) reached 70%, it was subcultured.
  • the cells were obtained for an experiment, or the cells were differentiated using a culture medium excluding a mitogenic factor.
  • neuroprogenitor cells in the cortical region that do not differentiate into dopaminergic neurons were obtained from mice at the 12th day of pregnancy and rats at the 14th day of pregnancy, and the experiment was conducted.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS Fetal bovine serum
  • lentiviral vectors expressing Nurr1, Foxa2 and Sgk1 were generated by inserting each cDNA into the multicloning site of pCDH (System Biosciences, Mountain View, CA).
  • the pGIPZ-shSgk1 lentiviral vector was purchased from Open Biosystems (Rockford, IL).
  • an empty basic vector pCDH or pGIPZ was used as a negative control.
  • Lentivirus titers were measured using the QuickTiterTM HIV Lentivirus Quantitation Kit (Cell Biolabs, San Diego, CA, USA), and each virus of Nurr1 and Foxa2 including 10 6 transducing units (TU)/ml 20 ⁇ l was mixed with 2 ml of medium, and added to 1-1.5 X 10 6 cells/6cm-dish for transduction reaction.
  • Cultured cells were fixed using 4% paraformaldehyde (PFA) in PBS (phosphate-buffered saline), and 1% bovine serum albumin and 0.3% (or 0.6%) Triton in PBS. It was blocked with a solution to which X-100 was added. Thereafter, a primary antibody was added to the blocked solution and overnighted at 4°C.
  • PFA paraformaldehyde
  • the primary antibodies include TH (1:1000, rabbit, Pel-Freez, Rogers, AR), GFAP (1:200, mouse, MP Biodmedicals, Santa Ana, CA), microtubule-associated protein 2 (MAPS); 1: 1000, mouse, Sigma) was used, and Cy3 (1:200, Jackson Immunoresearch Laboratories, West Grove, PA, USA) or Alexa Fluor 488 (1:200, Life Technologies) was used as a secondary antibody.
  • the stained cells were counted using VECTASHIELD with a DAPI mounting solution, and fluorescence was photographed using a fluorescence microscope (Leica, Heidelberg, Germany).
  • ROS reactive oxygen species
  • ROS Active oxygen species activity measurement
  • CM-H2DCF-DA (referred to herein as DCF) (Life Technologies) 10 ⁇ M for 10 minutes.
  • the cells were replaced with D-PBS (in mM: 2.68 KCl, 1.47 KH 2 PO 4 , 136.89 NaCl and 8.1 Na 2 HPO 4 ) several times to replace the medium several times, and VECTASHIELD (Vector Laboratories, CA, containing DAPI) USA) stained and fixed and photographed under a fluorescence microscope.
  • D-PBS in mM: 2.68 KCl, 1.47 KH 2 PO 4 , 136.89 NaCl and 8.1 Na 2 HPO 4
  • VECTASHIELD Vector Laboratories, CA, containing DAPI
  • ROS Reactive oxygen species
  • the number of immunostained and DAPI-stained cells was counted within 9-20 randomly selected sites on the coverslip of each culture at 200X or 400X magnification using an eyepiece grid. All data were expressed by means ⁇ SEM and appropriate statistical tests. For statistical comparison, Student's T-test (unpaired) or one-way ANOVA (one-way ANOVA) was used.
  • tissue buffer (5 mM Tris, 320 mM sucrose, pH 7.4), Krebs buffer (120 mM NaCl, 25 mM NaHCO 3 containing Na + in 10 ⁇ M glutamate) , 5 mM KCl, 2 mM CaCl 2 , 1 mM KH 2 PO 4 , 1 mM MgSO 4 and 10% glucose) or Na + -free Kreb buffer (120 mM choline-Cl and 25 mM Tris-HCl) 37 It was exposed for 10 minutes at °C. The absorption of glutamate was performed by placing the cells on ice and then washing them twice with a wash buffer (5 mM Tris/160 mM NaCl, pH 7.4).
  • the cells were collected and homogenized using 100 ⁇ l of an assay buffer, and the amount of glutamate uptake was confirmed through a glutamate assay kit (Abcam, Cambridge, MA, USA, ab83389). Na + dependent absorption was calculated by excluding the amount of Na + freed from the remaining current Na + amount.
  • SA-ß staining was performed in the same manner as [Dimri et al., 1995 (Abcam)]. It was cultured to be 4.0 ⁇ 10 4 (4.0 ⁇ 10 4 cells/well) per well using a 24-well culture dish, and incubated for 12 to 18 hours using SA-ß dye. Thereafter, cells stained in blue were counted and expressed as a percentage of the total cells.
  • the mixed glial cells (astrocytic cells + microglia) were treated with 0.25 ⁇ g/ml of LPS for 3 hours, followed by treatment with 2 to 2.5 mM ATP for 30 to 45 minutes.
  • the activity of the inflammation-regulating complex was confirmed by immunoblot of activated caspase-1 p10 and IL-1ß in the culture medium, and extracellular IL-1ß and IL-6 were quantified through ELISA analysis (E. Lee et al. , 2018)
  • RNA sequencing was performed by E-biogen (Seoul, Korea). After sequencing was cut according to the qualitative score using FastQC, the degree of mismatch was confirmed using Bowtie.
  • RNA sequencing data were prepared using the mouse reference genome (GRCm38/mm10) using STAR.
  • Mouse genome NCBI RefSeq 13 annotations Release 105: Feb. 2015
  • FRKM Frragments Per Kilobase of exon per Million fragments 15 mapped.
  • the error value was reduced by standardizing the data for each group. All data are registered in the GEO database (GEO: 17 GSE106216).
  • PFF and ⁇ -synuclein virus vectors were injected into the SN site of mouse ICR species. After a week for stability of the mouse, the Sgk1 inhibitor was injected daily intraperitoneally at 3 mg/ml. Animal behavioral experiments were performed from 1 week after injection, and when a total of 3 months had elapsed, brain tissue was fixed and fluorescent staining was performed.
  • mice were intraperitoneally in 30 ⁇ l for 5 days at 30 mg/kg. Injected. After practicing the behavioral experiment for a week, the balance rod experiment, the rod experiment, and the motility experiment were repeated three times a week, twice a week, and the experimental results were recorded.
  • the Sgk1 inhibitor (GSK-650394) was injected intraperitoneally at 3 mg/kg every day from the time of injection of MPTP. The entire process is shown in FIG. 16.
  • the animals were placed on top of a 50 cm long (1 cm diameter) wooden bar erected upright with their heads up. The bottom of the rod was placed in the cage. When the animals were placed on the rod, the animals moved down the rod toward the body and directed to their cages. All animals were trained for 2 days, and the training was performed 5 times per session. On the day of the test, the experiment was repeated 5 times, and the time taken to face the body downward was measured.
  • the animals were placed in a black plastic box consisting of 42 cm long, 42 cm wide and 42 cm high, and left to get used to the environment for 5 minutes, and then the motor activity was measured for 10 minutes.
  • the motor activity was measured using a device and software (EthoVision 3.1, Noldus information Technology, Netherlands) that tracks the motion based on a CCD camera by measuring the total distance (cm) and the total time (minutes) moved in the box.
  • the present inventors found that the midbrain expression factors Nurr1(N) and Foxa2(F) have anti-inflammatory functions by inhibiting the expression of inflammatory transcription factors in glial cells. Revealed.
  • the most specifically reduced factor in overexpression of Nurr1 and Foxa2 was serum-and-glucocorticoid-inducible kinase-1 (Sgk1) ( It was found that the combination of N and F was reduced by 9.3 times) (Fig. 1a).
  • Sgk1 is known to activate intracellular signaling of NF ⁇ B, a major pathway for inflammatory cytokine production. NF ⁇ B signaling is activated by removing NF ⁇ B from the inhibitory I ⁇ B-NF ⁇ B complex. It is known that Sgk1 can potentially phosphorylate I ⁇ B kinase beta (IKK ⁇ ) kinase, which is required for dissociation of inhibitory complexes.
  • IKK ⁇ I ⁇ B kinase beta
  • NF ⁇ B signaling was clearly seen by confirming the decrease in the expression level of phosphorylated (activated) p65 (the main form of NF ⁇ B (p-p65)).
  • p-p65 the main form of NF ⁇ B
  • the activity of NF ⁇ B in glial cells was confirmed, and NF ⁇ B activity was decreased in glial cells treated with the Sgk1 inhibitor (GSK-650394) (Fig. 2b).
  • astrocytes with reduced Sgk1 expression and astrocytes overexpressing Sgk1 were co-cultured with mesocerebral dopamine neurons.
  • Fig. 4a As a result, under the treatment of H 2 O 2 producing reactive oxygen species (ROS), co-culture with shSgk1-treated glial cells compared to sh-control-treated glial cells apoptosis (TH+ cells) and neurons. It can be seen that it was protected from the degeneration of the protrusion (FIG.
  • ROS reactive oxygen species
  • the amount of Sgk1 expression was determined by separating and culturing microglia and astrocytes, which are glial cells. Confirmed. As a result, it was confirmed that the amount of Sgk1 expression was higher in glial cells than in neurons, and in particular, the amount of Sgk1 expression was higher in microglia (FIG. 5A ). In addition, the amount of expression of Sgk1, which is a stress response gene, was increased under the treatment of H 2 O 2 , a toxin causing ROS (FIG. 5B).
  • Fig. 5c the Sgk1 mRNA level in glial cells was higher than in dopamine neurons, especially in microglia. It is known that neuroinflammation begins in microglia and then is transferred to astrocytes and neurotoxicity is spread, but it is also known that neurotoxicity is transferred from astrocytes to microglia.
  • the midbrain-derived glial cells were cultured to perform RNA sequence analysis according to the presence or absence of the Sgk1 inhibitor (GSK-650394) (FIG. 6).
  • the downregulated gene groups in the Sgk1 inhibitor-treated group (FPKM>1, log2>1) were'inflammatory response' and'NF ⁇ B pathway'.
  • 5 or more out of 10 downregulated gene groups were related to inflammation/immunity (shown in FIG. 6).
  • 516 were downregulated in glial cells treated with the Sgk1 inhibitor compared to the untreated control group.
  • the group of genes related to inflammation/immunity is shown through heat-map (FIG. 7).
  • IL-1ß and IL-18 are also related to pro-inflammatory cytokine expression mediated by the NF-kB pathway and the activity of the inflammasome, which is another active molecule typical of the inflammatory response.
  • apoptosis-related sensor containing NOD-like receptor family, pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and procaspase-1 Molecules create a complex multi-protein complex, an'inflammasome', which is then immediately activated to release caspase-1 and IL-1ß, which play an important role in initiating pro-inflammatory related cytokines.
  • the activity of the NRLP3 inflammation-regulating complex is related to the pathological mechanism of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and it is known that removal of the NLRP3 inflammation-regulating complex can help treat neurodegenerative diseases.
  • removal of the NLRP3 inflammation-regulating complex can help treat neurodegenerative diseases.
  • NRLP3, ASC, and procaspase-1 which are components of the inflammation control complex, were significantly reduced.
  • activated caspase-1 (p10) and mature IL-1 ⁇ could not be identified in the culture medium of glial cells treated with the Sgk1 inhibitor, which shows that Sgk1 inhibition prevents the activity of the NRLP3 inflammatory regulatory complex.
  • both IL-1 ⁇ and IL-6 related to neuroinflammation and neuroaging were significantly reduced in the midbrain-derived glial cell culture medium treated with the Sgk1 inhibitor (FIG. 8B).
  • cGAS acts as a cytosolic DNA sensor.
  • dsDNA double-stranded DNA
  • cGAS When bound to double-stranded DNA (dsDNA), cGAS generates a unique secondary metabolite, 2′,3′-cyclic GMP-AMP, and then binds to STING, an important adapter ER membrane protein.
  • STING collects TANK binding kinase 1 (TBK1), phosphorylates interferon regulatory factor 3 (IRF-3), then dimers and enters the nucleus, transcribing interferon-stimulating genes including interferon-beta (IFN- ⁇ ).
  • TNK1 TANK binding kinase 1
  • IRF-3 interferon regulatory factor 3
  • IFN- ⁇ interferon-beta
  • RNA sequencing data it can be confirmed that the expression of PGC1a is increased in glial cells treated with the Sgk1 inhibitor, which is interesting because PGC1a reduces ROS formation through increased mitochondrial biosynthesis and antioxidant capacity.
  • the inflammation control complex and the STING-cGAS pathway are correlated with mitochondrial function decline. Therefore, it was thought that treatment of glial cells with Sgk1 inhibitor would reduce neuroinflammation by blocking the inflammation-regulating complex and the STING-cGAS pathway, consequently reducing mitochondrial ROS.
  • Mito-Sox+ cells were reduced in glial cells treated with the Sgk1 inhibitor through immunostaining and FACS (FIG. 9A).
  • % B-galactosidase+ cells are considered to undergo cellularization, which was also significantly reduced by Sgk1 inhibition (Fig. 9b).
  • ECM cell adhesion/extracellular matrix
  • FIG. 10 Another neuroprotective mechanism by glial cells is to eliminate the toxicity caused by glutamate. Glutamate uptake is very important in the expression of glutamate transport genes GLAST and GLT-1 (called SLC1A3 or SLC1A2).In glial cells treated with Sgk1 inhibitor, the above two genes were increased compared to control cells, and the amount of glutamate uptake was also higher. (Fig. 10). In particular, when the Sgk1 inhibitor was treated to glial cells by 2 ⁇ M, the absorption of glutamate was significantly increased compared to the control, and this glutamate removal effect can be considered to contribute significantly to neuroprotection.
  • Sgk1 inhibitor As a result of treatment of neurons with an Sgk1 inhibitor (GSK-650394), as the expression of Parkin increased, the expression of Paris decreased and the expression of PGC1a increased (FIG. 15A). This suggests that Sgk1 inhibitors reduce mitochondrial ROS production, promote mitochondrial homeostasis, and affect cell viability. In addition, the Sgk1 inhibitor suppresses the aggregation of ⁇ -synuclein, thus suggesting the possibility of being applied as a therapeutic agent for Parkinson's disease.
  • mice were sacrificed 1 month after MPTP treatment to obtain the SN site and the tissues around the SN, and RT-PCR was performed.
  • RT-PCR As a result of RT-PCR, as shown in FIGS. 18 and 19, it was confirmed that the induction of neuroinflammation in both the SN region and the tissues around the SN was significantly reduced in the Sgk1 inhibitor-treated group compared to the control group. Thereafter, as a result of measuring the number of dopamine neurons in the SN region and performing TH staining (FIG.
  • the present invention differentiates the midbrain-derived neural progenitor cells overexpressed with ⁇ -synuclein for 3 weeks in order to check whether there is an effect of preventing aggregation and removing aggregation of ⁇ -synuclein.
  • Method 1 the inhibitor together
  • Method 2 the midbrain-derived neural progenitor cells overexpressing ⁇ -synuclein with astrocytes
  • Sgk1 inhibitor Methodhod 2

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Abstract

La présente invention concerne : une composition pharmaceutique pour prévenir ou traiter des maladies neurologiques inflammatoires, la composition pharmaceutique contenant un inhibiteur de Sgk1 en tant que principe actif ; et un procédé de traitement de maladies neurologiques inflammatoires l'utilisant. Les effets de réduction du vieillissement neuronal, la réduction de la neuroinflammation, l'augmentation de l'absorption du glutamate, l'amélioration de la viabilité et de la fonction neuronales, et la prévention de l'agrégation d'α-synucléine peuvent être obtenues par l'inhibition de l'expression de Sgk1, l'inhibiteur de Sgk1 pouvant ainsi être utilisé avantageusement en tant qu'agent thérapeutique pour des maladies neurologiques inflammatoires.
PCT/KR2020/009141 2019-07-10 2020-07-10 Utilisation de l'inhibiteur de sgk1 en tant qu'agent thérapeutique pour maladies neurologiques inflammatoires WO2021006707A1 (fr)

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