WO2020256013A1 - Biomarqueur pour la détection du lupus érythémateux disséminé neuropsychiatrique - Google Patents

Biomarqueur pour la détection du lupus érythémateux disséminé neuropsychiatrique Download PDF

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WO2020256013A1
WO2020256013A1 PCT/JP2020/023754 JP2020023754W WO2020256013A1 WO 2020256013 A1 WO2020256013 A1 WO 2020256013A1 JP 2020023754 W JP2020023754 W JP 2020023754W WO 2020256013 A1 WO2020256013 A1 WO 2020256013A1
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npsle
biomarker
galectin
amino acid
protein
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PCT/JP2020/023754
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Japanese (ja)
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清志 右田
直紀 松岡
智之 浅野
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公立大学法人福島県立医科大学
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Priority to JP2021526841A priority Critical patent/JP7320863B2/ja
Publication of WO2020256013A1 publication Critical patent/WO2020256013A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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  • the present invention is a biomarker for detecting neuropsychiatric lupus (NPSLE), a biomarker for determining the therapeutic effect on NPSLE, whether or not the test animal has NPSLE, or the risk of developing NPSLE.
  • NPSLE neuropsychiatric lupus
  • the present invention relates to a method for assisting in determining the presence or absence of NPSLE, a method for assisting in determining the therapeutic effect on NPSLE, or a kit for detecting NPSLE.
  • SLE Systemic lupus erythematosus
  • NPSLE neuropsychiatric lupus
  • NPSLE is diagnosed by combining various clinical tests such as blood, cerebrospinal fluid, and imaging findings, but diagnostic criteria have not been established, and it is extremely difficult to distinguish it from psychiatric symptoms due to side effects of steroids. Difficult to.
  • Non-Patent Document 1 describes that IL-6 and the like are increased in the cerebrospinal fluid in the NPSLE patient group as compared with the non-NPSLE and non-autoimmune disease control patient groups.
  • the biomarkers such as IL-6 described in Non-Patent Document 1 do not always have sufficient accuracy and / or accuracy in detecting NPSLE.
  • the present inventor has completed the present invention by finding that galectin 9 can be used as a biomarker for detecting NPSLE or as a biomarker for determining a therapeutic effect on NPSLE.
  • the present invention includes the following embodiments.
  • a biomarker for detecting neuropsychiatric lupus which comprises a galectin 9 protein or a peptide fragment thereof, or a transcript of the galectin 9 gene or a nucleic acid fragment thereof.
  • the galectin 9 protein is any of the following proteins (a) to (c): (a) Protein containing the amino acid sequence shown in SEQ ID NO: 1 (b) Protein containing the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and (c) ) A protein containing an amino acid sequence having 90% or more amino acid identity with respect to the amino acid sequence shown in SEQ ID NO: 1.
  • the biomarker according to (1) is any of the following proteins (a) to (c): (a) Protein containing the amino acid sequence shown in SEQ ID NO: 1 (b) Protein containing the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID
  • the method according to (6) or (7) which comprises a step of quantifying a biomarker.
  • the method further comprising.
  • a biomarker for determining a therapeutic effect on NPSLE which comprises a galectin 9 protein or a peptide fragment thereof, or a transcript of the galectin 9 gene or a nucleic acid fragment thereof.
  • a method for assisting determination of a therapeutic effect on NPSLE which comprises a step of detecting or quantifying the biomarker according to (9) in a sample derived from a test animal.
  • the method further comprising.
  • (12) The method according to any one of (6) to (8) and (10) and (11), wherein the test animal is a human.
  • a kit for detecting NPSLE which comprises a reagent for measuring the amount of the biomarker according to any one of (1) to (3).
  • a kit for detecting NPSLE which comprises an antibody or a fragment thereof that specifically binds to a galectin 9 protein or a peptide fragment thereof.
  • the galectin 9 protein is one of the following proteins (a) to (c): (a) Protein containing the amino acid sequence shown in SEQ ID NO: 1 (b) Protein containing the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and (c) )
  • the kit which is a protein containing an amino acid sequence having 90% or more amino acid identity with respect to the amino acid sequence shown in SEQ ID NO: 1.
  • a kit for detecting NPSLE which contains a nucleic acid that can specifically bind to a transcript of the galectin 9 gene or a fragment thereof.
  • the kit the kit: the nucleic acid is selected from the group consisting of the polynucleotides or fragments thereof shown in any of the following (d) to (h).
  • (d) (i) A nucleotide consisting of the base sequence represented by SEQ ID NO: 2 or a base sequence in which u is t in the base sequence, or a fragment thereof containing 15 or more consecutive bases.
  • a polynucleotide or fragment thereof of any of (v) to (vii) containing a modified nucleic acid and / or a modified nucleotide (g) A nucleotide containing the nucleotide sequence represented by SEQ ID NO: 2, a nucleotide sequence complementary to the nucleotide sequence in which u is t in the nucleotide sequence, or a partial sequence thereof containing 15 or more consecutive nucleotides, and ( h) A polynucleotide that hybridizes with any of the polynucleotides (d) to (g) above or a fragment thereof under highly stringent conditions. (17) The kit according to (15) or (16), wherein NPSLE has aseptic meningitis. This specification includes the disclosure of Japanese Patent Application No. 2019-112429, which is the basis of the priority of the present application.
  • the present invention may provide a marker for detecting NPSLE, or a method for detecting NPSLE using the marker. Further, the present invention may provide a marker for determining a therapeutic effect on NPSLE, or a method for determining a therapeutic effect on NPSLE using the marker.
  • FIG. 1 shows the cerebrospinal fluid galectin 9 concentrations in 18 NPSLE patients, 6 patients with other diseases (control 1), and 8 non-NPSLE SLE patients (control 2).
  • FIG. 2 shows the cerebrospinal fluid galectin 9 concentration before and after treatment in one NPSLE patient.
  • FIG. 3 shows the concentration of galectin 9 in the cerebrospinal fluid of 7 patients in the group with aseptic meningitis (AM +) and 11 patients in the group without AM (AM-) among 18 patients with NPSLE.
  • the invention relates to a biomarker for detecting neuropsychiatric lupus (NPSLE), which consists of a galectin 9 protein or peptide fragment thereof, or a transcript of the galectin 9 gene or a nucleic acid fragment thereof.
  • NPSLE neuropsychiatric lupus
  • the present invention relates to a biomarker for determining a therapeutic effect on NPSLE, which comprises a galectin 9 protein or a peptide fragment thereof, or a transcript of the galectin 9 gene or a nucleic acid fragment thereof.
  • systemic lupus erythematosus is a chronic recurrent autoimmune disease in which an immune complex of autoantibodies is deposited on tissues and causes tissue damage.
  • neuropsychiatric lupus NPSLE, Neuropsychiatric SLE
  • SLE central nervous system symptoms
  • neuropsychiatric SLE for example, Hanly JG et al., Arthritis Rheum., 2019. , 71 (1): 33-42).
  • Symptoms of NPSLE can be broadly divided into those of the central nervous system (psychiatric and neurological symptoms) and those of the peripheral nervous system.
  • Psychiatric symptoms include mood disorders, cognitive dysfunction, anxiety disorders, psychotic disorders, and acute confusion
  • neurological symptoms include headache, convulsions, cerebrovascular accidents, movement disorders (chorea), myelopathy, and aseptic. Examples include myelopathy and demyelinating syndrome.
  • Peripheral nervous system symptoms include multiple neuropathy, cerebral nerve neuropathy, single / multiple single neuropathy, acute inflammatory demyelinating multiple nerve root neuropathy (Guillain-Barré syndrome), autonomic neuropathy, severe myasthenia, and neuropathy. Disorders can be mentioned (see, for example, Arthritis Rheum., 1999, 42 (4): 599-608).
  • a galectin 9 protein derived from an endogenous gene of a test animal or a gene transcript thereof can be a biomarker.
  • the test animal is a human
  • the human galectin 9 protein derived from the human galectin 9 gene and the transcript (mRNA) of the human galectin 9 gene can be biomarkers of the present invention.
  • galectin 9 protein examples include a human-derived galectin 9 (human galectin 9) protein containing or consisting of the amino acid sequence shown in SEQ ID NO: 1.
  • the galectin 9 protein also includes a galectin 9 variant having an activity functionally equivalent to that of the galectin 9 protein shown in SEQ ID NO: 1 and a galectin 9 ortholog of another species. Specifically, 80% or more and 90% of the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1 As described above, galectin 9 proteins having 95% or more, 97% or more, 98% or more, or 99% or more amino acid identity are included.
  • “several pieces” means, for example, 2 to 10, 2 to 7, 2 to 5, 2 to 4 or 2 to 3.
  • Conservative amino acid substitution is desirable for amino acid substitution.
  • “Conservative amino acid substitution” refers to a substitution between amino acids having similar properties such as charge, side chain, polarity, and aromaticity.
  • Amino acids with similar properties include, for example, basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (glycine, asparagin, glutamine, serine, threonine, cysteine, tyrosine), non-polar amino acids.
  • sex amino acids leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine
  • branched amino acids leucine, valine, isoleucine
  • aromatic amino acids phenylalanine, tyrosine, tryptophan, histidine
  • amino acid identity refers to the sequence number when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the degree of amino acid matching between the two amino acids.
  • Amino acid identity can be calculated using a protein search system using BLAST or FASTA. For details on how to determine identity, see, for example, Altschul et al, Nuc. Acids. Res. 25, 3389-3402, 1977 and Altschul et al., J. Mol. Biol. 215, 403-410, 1990. I want to.
  • the "galectin 9 gene” is a gene encoding the galectin 9 protein.
  • Specific examples of the galectin 9 gene include a human galectin 9 gene encoding a human galectin 9 protein containing the amino acid sequence shown in SEQ ID NO: 1. More specifically, the galectin 9 gene is a gene containing or consisting of the nucleotide sequence shown in SEQ ID NO: 2.
  • the galectin 9 gene includes a galectin 9 variant having an activity functionally equivalent to that of the galectin 9 protein encoded by the galectin 9 gene shown in SEQ ID NO: 2 and a galectin 9 gene encoding a galectin 9 ortholog of another species. Included. Specifically, 80% or more and 90% of the base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 2 or the base sequence shown in SEQ ID NO: 2 As described above, galectin 9 genes having 95% or more, 97% or more, 98% or more, or 99% or more base identity are included.
  • base identity refers to a sequence number when two base sequences are aligned and a gap is introduced as necessary so that the degree of base matching between the two is the highest.
  • hybridizing under high stringent conditions means performing hybridization and washing under low salt concentration and / or high temperature conditions. For example, incubate with probe in 6 ⁇ SSC, 5 ⁇ Denhardt reagent, 0.5% SDS, 100 ⁇ g / mL denatured fragmented salmon sperm DNA at 65 ° C-68 ° C, then in a wash solution of 2 ⁇ SSC, 0.1% SDS. It is exemplified by starting from room temperature at room temperature, lowering the salt concentration in the cleaning solution to 0.1 ⁇ SSC, and raising the temperature to 68 ° C. until no background signal is detected. The conditions for high stringent hybridization are described in Green, MR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. It can be used as a reference.
  • the nucleotide sequence information of such galectin 9 gene can be searched from public databases (GenBank, EMBL, DDBJ). For example, a gene having high base identity can be searched and obtained from a database based on the known base sequence information of the galectin 9 gene shown in SEQ ID NO: 2.
  • Transcript of galectin 9 gene means galectin 9 mRNA.
  • the mRNA may be an mRNA precursor (pre-mRNA) or a mature mRNA (mature mRNA). Normally, the mRNA precursor is immediately spliced in the nucleus to become a mature mRNA, so that the transcript of the galectin 9 gene, which is a biomarker of the present invention, may be a galectin 9 mature mRNA.
  • the term "peptide fragment” is a peptide fragment containing or consisting of a part of the amino acid sequence constituting the galectin 9 protein, and the fragment of the galectin 9 protein from the amino acid sequence constituting the fragment. Can be identified.
  • the "peptide fragment” may be 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more consecutive amino acid acid residues in the full-length amino acid sequence of the galectin 9 protein. Further, it may be a peptide consisting of 200 or less, 150 or less, 120 or less, 100 or less, or 80 or less consecutive amino acid residues.
  • a "peptide fragment” may be a peptide consisting of 10 to 200, 30 to 120, and 50 to 80 consecutive amino acid residues.
  • the "nucleic acid fragment” is a nucleic acid fragment containing or consisting of a part of the base sequence constituting galectin 9 mRNA, and is a fragment of galectin 9 mRNA from the base sequence constituting the fragment. Can be identified.
  • the "nucleic acid fragment” may be a nucleic acid consisting of 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more consecutive bases in the full-length base sequence of galectin 9 mRNA.
  • the nucleic acid may be composed of 600 or less, 450 or less, 300 or less, 200 or less, or 100 or less consecutive bases.
  • the "nucleic acid fragment” may be a nucleic acid consisting of 30 to 600, 50 to 300, or 70 to 100 consecutive bases in the full-length base sequence of galectin 9 mRNA.
  • the invention relates to the use of the biomarkers described herein to detect NPSLE.
  • the present invention relates to a method for determining (or assisting) whether or not a test animal has NPSLE or is at risk of developing NPSLE.
  • the method comprises the step of detecting or quantifying the biomarkers described herein in a sample derived from a test animal.
  • the detection or quantification step can be performed in vitro.
  • the method of the present invention further includes a step of determining whether or not the test animal has NPSLE or whether or not there is a risk of suffering from NPSLE based on the detection or quantification result obtained in the above step. Good.
  • the above-mentioned detection of NPSLE, determination of the presence or absence of NPSLE or determination of the risk of morbidity includes differentiation of NPSLE-affected patients from healthy subjects, and differentiation of NPSLE from other diseases.
  • diseases that can be differentiated from NPSLE include non-NPSLE inflammatory diseases such as SLE and Bechet's disease, and collagen diseases such as ANCA-related vasculitis, as well as encephalitis and corticosteroid-induced psychotric disorders (corticosteroid-induced psychiatric disorders). CIPDs))), neuromyelitis optica (NMO), progressive multifocal leukoencephalopathy (PML), and reverse posterior reversible leukoencephalopathy (PRES).
  • NPSLE neuromyelitis optica
  • PML progressive multifocal leukoencephalopathy
  • PRES reverse posterior reversible leukoencephalopathy
  • the detection of NPSLE and the determination of the presence or absence of NPSLE may be used to determine whether or
  • the NPSLE to be detected may be an NPSLE having either the above-mentioned central nervous system (psychiatric and neurological symptoms) or peripheral nervous system symptoms.
  • NPSLE having aseptic meningitis may be detected, or the presence or absence of morbidity or the risk of morbidity may be determined.
  • test animals in the present invention include, for example, mammals, for example, primates such as humans and red-tailed monkeys, experimental animals such as rats, mice, and rats, pigs, cows, horses, sheep, goats, etc. Domestic animals and pet animals such as dogs and cats are mentioned, and humans are preferable.
  • the test animal may be a subject who has or is suspected of having the other diseases mentioned above, such as SLE.
  • the sample used in the present invention is not particularly limited, and examples thereof include tissues such as body fluids, cells, and biopsy samples.
  • body fluids include cerebrospinal fluid, blood (including serum, plasma and interstitial fluid), lymph, extracts of tissues or cells, pleural fluid, sputum, tears, nasal juice, saliva, urine, etc. Cerebrospinal fluid or blood (particularly plasma) that can be corrected with standard substances.
  • the body fluid, cells or tissue may be used as it is for the measurement of the biomarker, or may be appropriately pretreated for the measurement.
  • paraffin-embedded sections may be prepared from samples derived from test animals.
  • a protein extract or mRNA extract may be prepared from a sample derived from a test animal.
  • the biomarker measured by this method may be either a galectin 9 protein or a peptide fragment thereof, a transcript of the galectin 9 gene or a nucleic acid fragment thereof.
  • Detection or quantification includes measuring the presence or absence of expression of a biomarker, or the magnitude of expression level or expression concentration.
  • the term "measurement" includes semi-quantitative.
  • the measurement method may be any known protein quantification method, and is not particularly limited, and examples thereof include an immunological detection method.
  • the "immunological detection method” is a method of measuring the amount of a target molecule using an antibody or antibody fragment that specifically binds to the target molecule that is an antigen.
  • the antibody can be derived from any animal, including mammals and birds. For example, mice, rats, guinea pigs, rabbits, goats, donkeys, sheep, camels, horses, chickens or humans can be mentioned.
  • the antibody used in the immunological detection method is not particularly limited, but a monoclonal antibody or a polyclonal antibody may be used.
  • monoclonal antibody refers to a group of clones of a single immunoglobulin. Each immunoglobulin constituting a monoclonal antibody contains a common framework region and a common complementarity determining region, and can recognize and bind to the same epitope of the same antigen. Monoclonal antibodies can be obtained from single cell hybridomas.
  • polyclonal antibody refers to a group of multiple immunoglobulins that recognize and bind to different epitopes of the same antigen.
  • the polyclonal antibody can be obtained from the serum of an animal after immunizing the animal with the target molecule as an antigen.
  • each class of IgG, IgM, IgA, IgE, and IgD is known as an immunoglobulin molecule, but the antibody of the present invention may be in any class. , For example, IgG.
  • the method for producing a polyclonal antibody that recognizes and binds to galectin 9 protein or a hybridoma that produces a monoclonal antibody may be carried out according to an antibody production method known in the art using galectin 9 protein or a fragment thereof as an antigen. Antibodies may also be obtained from the manufacturer. The expression level of the galectin-9 protein or a fragment thereof may be measured by a commercially available kit, and such a kit includes Quantikine TM ELISA Human Galectin-9 immunoassay. In one embodiment, the galectin-9 protein or fragment thereof comprises an epitope recognized by an antibody contained in Quantikine TM ELISA Human Galectin-9 immunoassay.
  • antibody fragment refers to a polypeptide chain or a complex thereof that is a partial fragment of a polyclonal antibody or a monoclonal antibody and has an activity substantially equivalent to the antigen-specific binding activity of the antibody. ..
  • an antibody moiety that includes at least one antigen-binding site that is, a polypeptide chain having at least one set of VL and VH, or a complex thereof.
  • Specific examples include a large number of well-characterized antibody fragments produced by cleaving immunoglobulins with various peptidases. More specific examples include Fab, F (ab') 2 , Fab'and the like. All of these antibody fragments include an antigen-binding site and have the ability to specifically bind to a target molecule that is an antigen.
  • Immunological detection methods include, for example, immunohistochemical staining, enzyme-linked immunosorbent assay (including ELISA and EIA), Western blotting, radioimmunoassay (RIA), immunoprecipitation, or flow cytometry.
  • enzyme-linked immunosorbent assay including ELISA and EIA
  • Western blotting including ELISA and EIA
  • RIA radioimmunoassay
  • immunoprecipitation or flow cytometry. The law can be mentioned.
  • a known method can be adopted as the "immunohistochemical staining method". For example, a sample derived from a test animal may be fixed in formalin, embedded in paraffin, sliced into tissue pieces, and attached to a slide glass, which may be used as a section sample. The section sample may be heat-treated to activate the antigen, and then immunohistochemical staining may be performed on the section sample using a commercially available detection system such as DakoEnVision + System (Agilent).
  • the measurement method may be any known nucleic acid quantification method, and is not particularly limited, but for example, a nucleic acid amplification method using a primer or a nucleic acid amplification method using a primer.
  • a hybridization method using a probe can be mentioned.
  • Nucleic acid amplification method refers to a method in which a specific region of a target nucleic acid is amplified by a nucleic acid polymerase using a forward / reverse primer set.
  • examples of the nucleic acid amplification method include PCR (polymerase chain reaction) methods such as RT-PCR (reverse transcription polymerase chain reaction).
  • hybridization method uses a nucleic acid fragment having a base sequence complementary to all or part of the base sequence of the target nucleic acid to be detected as a probe, and utilizes the base pairing between the nucleic acid and the probe.
  • the hybridization method several methods with different detection means are known, and examples thereof include a Northern hybridization method (Northern blot hybridization method), an in situ hybridization method, and a microarray method.
  • Nucleic acid chains such as primers and probes can be appropriately designed by a method known to those skilled in the art based on known biomarker sequence information, and can be obtained by a known production method such as chemical synthesis.
  • each of the above-mentioned measurement methods is a technique known in the art. Therefore, the specific measurement method may be performed according to a known method. For example, the method described in Green, M.R. and Sambrook, J., 2012 (described above) can be referred to.
  • the step comprises determining whether the sample is positive or negative for the biomarker from the results obtained in the detection or quantification step. If the sample is negative for biomarkers, it can be determined that the test animal does not have NPSLE or is at low risk of developing it. On the other hand, if the sample is positive for biomarkers, it can be determined that the test animal has NPSLE or is at high risk of developing it.
  • the immunohistochemical staining method when one or more cells or cell clusters are stained, it can be determined as positive, and when the stained cells do not exist, it can be determined as negative.
  • the number of stained cells exceeds a certain ratio (for example, 10%, 15% or 20%) with respect to the total number of cells, it is determined to be positive, and the number of stained cells with respect to the total number of cells is equal to or less than the certain ratio. In the case of, it may be determined as negative.
  • a certain ratio for example, 10%, 15% or 20%
  • the determination step comprises determining whether the expression level of the biomarker in the sample obtained in the detection or quantification step is high or low (eg, below a predetermined threshold). If the expression level of the biomarker in the sample is below a predetermined threshold (eg, statistically significantly lower), then the population is not affected by NPSLE or (eg, has an expression level higher than the predetermined threshold). On the other hand, it can be judged that the risk of morbidity is low. On the other hand, if the expression level of the biomarker in the sample is higher than a predetermined threshold (eg, statistically significantly higher), then the population is either suffering from NPSLE or (eg, having an expression level lower than the predetermined threshold). On the other hand, the risk of morbidity can be determined to be high.
  • a predetermined threshold eg, statistically significantly lower
  • the predetermined threshold value may be a control amount measured in a control sample (control cell or tissue, for example, control colon cell or colon tissue).
  • the control sample may be derived from a healthy individual (eg, a healthy person), a non-NPSLE SLE patient, or a patient suffering from another disease.
  • the "healthy individual” refers to a healthy individual of the same species as the test individual, which is not affected by NPSLE, and includes an individual who has been affected by NPSLE but has been treated with NPSLE.
  • the median value, average value, upper limit level, lower limit level, and a certain range of values of the expression level in these control samples or the expression level in a plurality of individuals can be used as a predetermined threshold value.
  • the above value or range multiplied by an appropriate coefficient for example, 0.3 or more, 0.5 or more, 0.8 or more, 0.9 or more, or 3 or less, 2 or less, 1.5 or less, or 1.1 or less
  • the threshold value can be appropriately set according to the accuracy of prediction and the like, and can be determined by, for example, ROC (receiver operating characteristic curve) analysis.
  • statically significant means that when the risk rate (significance level) of the obtained value is small, specifically, p ⁇ 0.05 (less than 5%) and p ⁇ 0.01 (less than 1%). ) Or p ⁇ 0.001 (less than 0.1%).
  • a known test method capable of determining the presence or absence of significance may be appropriately used, and is not particularly limited. For example, the Student's t-test method, the multiple comparison test method, and the log rank test method can be used.
  • the term "high risk of morbidity" for NPSLE refers to a period of time (eg, within a week, within a few weeks, within a month, within 2 months, within 3 months), although not currently suffering from NPSLE. Within, within 6 months, within 1 year, within 2 years, or within 5 years), it is intended to be more likely to have NPSLE.
  • “low risk of morbidity” of NPSLE means that the patient is not currently suffering from NPSLE and has a certain period of time (for example, within 1 week, within several weeks, within 1 month, within 2 months, within 3 months, It is intended that the likelihood of contracting NPSLE within 6 months, 1 year, 2 years, or 5 years) is low.
  • the method of determining (or assisting) whether or not the test animal of the present invention has NPSLE or is at risk of developing NPSLE is combined with another NPSLE diagnostic method in order to further improve the accuracy. May be good.
  • Other such NPSLE diagnostic methods include imaging tests (CT, MRI, SPECT, PET, etc., for example, presence or absence of abnormal findings in head MRI), electroencephalography (presence or absence of electroencephalogram abnormalities), and spinal fluid test (medullary fluid).
  • General spinal fluid tests such as cell number, spinal protein and sugar content, measurement of cytokines such as spinal fluid IL-6), and autoantibodies (anti-cardiolipin antibody, lupus anticogrant, anti-ribosomal P antibody, anti-Ro / SS-A antibody, anti-Sm antibody, anti-ds-DNA antibody, etc.) detection and the like.
  • Diagnostic criteria for such diagnostic methods are known to those skilled in the art, for example, Hiroyuki Yamashita, 2017, Journal of the Japanese Society of Internal Medicine 106, No. 8, pp. 1555 to 1563, Masaru Nishimura et al., 2011, General Hospital Psychiatry. See Medicine, 23 (1), pp. 42-51, etc.
  • the presence or absence or risk of morbidity of NPSLE can be determined, and based on the result, the treatment policy (for example, the presence or absence of concomitant drug use, steroid drug (and concomitant drug if used)). Type, dose, dosing interval, and dosing period) can be determined.
  • the present invention determines that a subject has or is at high risk of developing NPSLE, steroids may be administered to the test animal to treat and / or prevent NPSLE. Therefore, the present invention also provides a method for treating and / or preventing NPSLE, which comprises administering a steroid agent to a test animal which is determined to have NPSLE or is at high risk of developing NPSLE by the method of the present invention. To do.
  • steroids examples include, but are not limited to, corticosteroids such as prednisolone, methylprednisolone, betamethasone, dexamethasone, triamcinolone and hydrocortisone, and their solvents.
  • Steroids can be used alone or in combination. Steroids can be administered by routes such as injection, intravenous administration, or oral administration.
  • the dose of steroids is not limited, but NPSLE often uses high doses of steroids (for example, 1.0 mg / kg body weight / day), and the dose is reduced by about 10% every two weeks while observing the symptoms. Often.
  • the dosage is, for example, 0.005 to 100 mg / kg body weight / day, 0.05 to 10 mg / kg body weight / day, 0.1 to 5.0 mg / kg body weight / day, 0.25 to 2.0 mg / kg body weight / day, or 0.5 to 1.0 mg / body weight. It may be kg / day.
  • the number of administrations is not limited, but is 3 times a day, 2 times a day, once a day, once every 2 days, once every 3 days, once a week, once every two weeks, It may be once a month or the like.
  • the administration period may be, but is not limited to, 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month, 6 months, 1 year, or more.
  • Steroidal pulse therapy with high doses may be given for several days.
  • the dose in steroid pulse therapy may be 250 mg to 1000 mg / day in terms of methylprednisolone, and may be intravenous drip infusion.
  • the duration of steroid pulse therapy may be, but is not limited to, 1 day, 2 days, 3 days, or longer.
  • Steroids may be used in combination with other drugs, and examples of other drugs include immunosuppressants.
  • immunosuppressants include, but are not limited to, azathioprine, cyclophosphamide, mycophenolate mofetil, mizoribine and the like.
  • the dose of the immunosuppressant is not limited, but is, for example, 0.005 to 5000 mg / kg body weight / day, 0.05 to 1000 mg / kg body weight / day, 0.1 to 100 mg / kg body weight / day, or 1.0 to 10 mg / kg body weight / day. It may be there.
  • the number of administrations is not limited, but is 3 times a day, 2 times a day, once a day, once every 2 days, once every 3 days, once a week, once every two weeks, It may be once a month or the like.
  • the administration period may be, but is not limited to, 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month, 6 months, 1 year, or more.
  • azathioprine can be administered at 0.5 to 2.5 mg / kg body weight / day once daily
  • cyclophosphamide can be administered at 500 to 750 mg / kg body weight / day for 2 to 3 weeks by intravenous injection. It can be administered 7 to 10 times in total every other time
  • mycophenolate mofetil can be administered 1000 mg to 2000 mg / kg body weight / day, intravenously or orally twice daily
  • mizoribine can be administered 150 to 150 times. It can be administered at 300 mg / kg body weight / day orally once to three times a day every day.
  • the steroid and the immunosuppressant may be administered at the same time or at different time points. When administered at the same time, it may be administered simultaneously as a composition containing a steroid agent and an immunosuppressive agent, or the steroid agent and the immunosuppressive agent may be administered separately.
  • the order of administration is not limited, and the immunosuppressant may be administered after the steroid is administered, or the immunosuppressant may be administered before the steroid is administered. Inhibitors may be administered.
  • the invention relates to the use of the biomarkers described herein to determine a therapeutic effect on NPSLE (eg, a therapeutic method or agent, etc.).
  • the invention relates to a method of determining (or assisting) a therapeutic effect on NPSLE (eg, a therapeutic method or drug).
  • the method comprises the step of detecting or quantifying the biomarkers described herein in a sample derived from a test animal. This detection or quantification step is as described for the step in the method of determining whether or not the patient has NPSLE or the risk of suffering from NPSLE.
  • the present method may further include a step of determining whether or not there is a therapeutic effect on NPSLE based on the detection or quantification result obtained in the above step.
  • This determination step includes determining whether or not there is a therapeutic effect on NPSLE.
  • This determination step is also as described in the method for determining whether or not the patient has NPSLE or whether or not the patient is at risk of developing NPSLE.
  • the determination step includes determining whether the sample is positive or negative for the biomarker from the results obtained in the detection or quantification step. If the sample is negative for the biomarker, it can be determined to be therapeutically effective or high. On the other hand, if the sample is positive for the biomarker, it can be determined that the therapeutic effect is ineffective or low.
  • the criteria for determining positive and negative are as described.
  • the determination step also includes determining whether the expression level of the biomarker in the sample obtained in the detection or quantification step is higher or lower (for example, than a predetermined threshold value).
  • a predetermined threshold for example, statistically significantly lower
  • the therapeutic effect is effective or high (for example, for a population having an expression level higher than the predetermined threshold).
  • the expression level of the biomarker in the sample is higher than a predetermined threshold (for example, statistically significantly higher)
  • there is no therapeutic effect for example, for a population having an expression level lower than the predetermined threshold. It can be judged to be low.
  • the predetermined threshold value is also as described above. However, the threshold in this embodiment may include the expression level in the test animal before treatment.
  • the method may also include the step of treating NPSLE before, during, and / or after the detection or quantification step.
  • the treatment step may include administering to the test animal a steroid (and optionally an immunosuppressant).
  • a steroid and optionally an immunosuppressant.
  • the types, doses, administration periods, etc. of steroids and immunosuppressants are as described in the above-mentioned treatment and / or prevention methods.
  • the treatment process may be changed according to the result of the above determination process. For example, if it is determined in the determination step that there is no therapeutic effect, a drug (for example, an immunosuppressant) for increasing and / or administering the drug may be added.
  • a drug for example, an immunosuppressant
  • an immunosuppressant for increasing and / or administering the drug
  • kits In one aspect, the invention also provides a kit for detecting NPSLE, which comprises a reagent for measuring the amount of biomarker described herein.
  • the reagent for measuring the amount of biomarker examples include the above-mentioned antibody or antibody fragment, or probe or primer.
  • the kit includes known immunohistochemical staining, ELISA, Western blot, or reagents for RT-PCR, such as labeling reagents, buffers, chromogenic substrates, secondary antibodies, blocking agents, and instruments and controls required for testing. And at least one of the instructions and the like may be further included.
  • Example 1 Measurement of galectin 9 concentration in different patient groups> (Materials and methods) As a sample, among patients with rheumatoid arthritis who visited the Department of Internal Medicine for Rheumatoid Arthritis, Fukushima Prefectural Medical University between June 2004 and January 2019, lupus puncture was performed and the spinal fluid was discharged at -80 ° C. Twenty-six cases of systemic lupus erythematosus (SLE) and 6 cases of other diseases that had been cryopreserved were used. Galectin 9 concentrations in cerebrospinal fluid samples were measured by enzyme-linked immunosorbent assay (ELISA) using Quantikine TM ELISA Human Galectin-9 immunoassay (R & D systems) according to the manufacturer's protocol.
  • ELISA enzyme-linked immunosorbent assay
  • NPSLE neuropsychiatric lupus
  • control 2 patients who were judged to be negatively involved in NPSLE.
  • NPSLE made a comprehensive diagnosis based on the serological evaluation of SLE activity, spinal fluid examination, diagnostic imaging (MRI, SPECT), and clinical course, and made a comprehensive diagnosis (specifically, 1997 ACR).
  • a definitive diagnosis of SLE is made according to the diagnostic criteria, and in addition to symptoms such as headache and fever, SPECT shows a decrease in cerebral blood flow, MRI shows a highly absorbed region in the brain parenchyma, and the Ig index and IL-6 of the spinal fluid.
  • Example 2 Measurement of galectin 9 concentration before and after NPSLE treatment> (Materials and methods) Galectin 9 levels were measured before and after NPSLE treatment in a 19-year-old male patient (mainly fever, headache, and rash) at the time of onset. The course of treatment is as follows.
  • Cerebrospinal fluid examination revealed an increase in meningeal pressure and an increase in the number of lymphocyte-dominant cells, but viral and bacterial infections were negative, indicating aseptic meningitis.
  • Brain SPECT examination revealed decreased blood flow throughout the brain.
  • NPSLE was diagnosed after a series of courses, and the dose of prednisolone was increased to 60 mg / kg body weight / day. After the start of treatment, symptoms such as headache and fever improved, and prednisolone 50 mg / day from July 2 and prednisolone 45 mg / day from July 16 did not relapse even after tapering, and again on July 28.
  • the number of cells in the cerebrospinal fluid decreased to within the standard value.
  • the galectin 9 concentration in the cerebrospinal fluid sample before and after the treatment was measured in the same manner as in Example 1.
  • FIG. 2 shows that treatment with NPSLE significantly reduces the concentration of galectin 9 in the sample. This suggests that galectin 9 can be used to determine if treatment for NPSLE was successful.
  • Example 3 Measurement of galectin 9 concentration in NPSLE patients with aseptic meningitis> (Materials and methods) Aseptic meningitis (AM) is one of the neurological symptoms of NPSLE. Eighteen patients in the NPSLE group in Example 1 were classified into two groups, an AM group (7 patients) and an AM-less group (11 patients), based on the presence or absence of aseptic meningitis, and the cerebrospinal fluid in both groups. Medium galectin 9 concentrations were compared.
  • AM Aseptic meningitis

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Abstract

Dans un mode de réalisation, la présente invention aborde le problème consistant à fournir : un marqueur permettant de détecter un NPSLE ; ou un procédé de détection d'un NPSLE à l'aide du marqueur.　Dans un mode de réalisation, la présente invention concerne un biomarqueur permettant de détecter un NPSLE ou un biomarqueur pour déterminer un effet thérapeutique sur un NPSLE, le biomarqueur comprenant la protéine galectine-9 ou un fragment peptidique de celle-ci ou un transcrit du gène galectine-9 ou d'un fragment nucléotidique de celui-ci. Dans un mode de réalisation, la présente invention concerne un procédé d'aide à la détermination permettant de savoir si un animal sujet est affecté par un NPSLE ou présentant un risque d'incidence d'un NPSLE, un procédé d'aide à la détermination d'un effet thérapeutique sur NPSLE, ou un kit de détection de NPSLE, dans chacun desquels le biomarqueur est utilisé.
PCT/JP2020/023754 2019-06-18 2020-06-17 Biomarqueur pour la détection du lupus érythémateux disséminé neuropsychiatrique WO2020256013A1 (fr)

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WO2023157920A1 (fr) * 2022-02-17 2023-08-24 国立大学法人 長崎大学 Support pour diagnostic de sle neuropsychiatrique au moyen d'une irm cérébrale

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FRAGOSO-LOYD, H., RICHAUD-PATIN Y., OROZCO-NARVÁEZ A., DÁVILA-MALDONADO L., ATISHA-FREGOSO Y., LLORENTE L., SÁNCHEZ-GUERRERO J.: "Interleukin-6 and Chemokines in the Neuropsychiatric Manifestations of Systemic Lupus Erythematosus", ARTHRITIS & RHEUMATISM, vol. 56, no. 4, 2007, pages 1242 - 1250, XP055773752, DOI: 10.1002/art.22451 *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023157920A1 (fr) * 2022-02-17 2023-08-24 国立大学法人 長崎大学 Support pour diagnostic de sle neuropsychiatrique au moyen d'une irm cérébrale

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