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WO2015178288A1 - Procédé d'évaluation de l'activité d'induction de la production sélective d'il-4 associée à l'activation des cellules nkt - Google Patents

Procédé d'évaluation de l'activité d'induction de la production sélective d'il-4 associée à l'activation des cellules nkt Download PDF

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WO2015178288A1
WO2015178288A1 PCT/JP2015/063935 JP2015063935W WO2015178288A1 WO 2015178288 A1 WO2015178288 A1 WO 2015178288A1 JP 2015063935 W JP2015063935 W JP 2015063935W WO 2015178288 A1 WO2015178288 A1 WO 2015178288A1
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gene
xloc
amount
test substance
blood cells
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山村　隆
大介 能登
幸子 三宅
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National Center of Neurology and Psychiatry
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National Center of Neurology and Psychiatry
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a method for evaluating selective IL-4 production-inducing activity associated with natural killer T (NKT) cell activation.
  • NKT cells are cells having intermediate properties between T cells and natural killer (NK) cells.
  • NKT cells have a T cell antigen receptor (TCR) and express NK cell markers (NK1.1 in mice and CD56 in humans).
  • TCR T cell antigen receptor
  • NK1.1 in mice and CD56 in humans
  • an NKT cell that expresses an invariant TCR in which the V ⁇ chain of the T cell antigen receptor (gene fragment V ⁇ 14-J ⁇ 28 in the case of mice, V ⁇ 24-J ⁇ 18 in humans) has little diversity is called an invariant NKT cell.
  • this invariant NKT cell recognizes the glycolipid ⁇ -galactosylceramide ( ⁇ -GalCer) bound to CD1d, which is an antigen-presenting molecule on the surface of dendritic cells (Non-patent Document 1).
  • ⁇ -GalCer glycolipid ⁇ -galactosylceramide
  • NKT cells are activated by ⁇ -GalCer bound to CD1d, interleukin 4 (IL-4), interferon ⁇ (IFN- ⁇ ), interleukin It is known to produce a large amount of cytokines such as 13 (IL-13) (Non-patent Document 2).
  • IL-4 interleukin 4
  • IFN- ⁇ interferon ⁇
  • IL-13 interleukin 13
  • Non-patent Document 2 cytokines
  • NKT cells are a small population of cells, they affect the functions of major lymphocytes including NK cells by crosstalk through cytocan production as described above, and are antigen-specific. It is known that the immune response is regulated (Non-Patent Document 3 and Non-Patent Document 4).
  • Patent Document 1 discloses a glycolipid capable of selectively inducing IL-4 production by NKT cells.
  • the glycolipid described in Patent Document 1 (hereinafter also referred to as “synthetic glycolipid compound”) can selectively induce IL-4 production by NKT cells (hereinafter referred to as “selection associated with NKT cell activation”). IL-4 production-inducing activity ").
  • selection associated with NKT cell activation IL-4 production-inducing activity ".
  • Th1 / Th2 immune balance can be adjusted in the direction in which Th2 increases, IL-17 production can be reduced, and GM-CSF production can be reduced.
  • Such substances having selective IL-4 production-inducing activity associated with NKT cell activation include, for example, treatment of diseases in which Th1 / Th2 immune balance is biased to Th1, or diseases in which Th1 cells worsen the pathology, autoimmune diseases It is extremely effective in the treatment of diseases, treatment of diseases caused by an increase in GM-CSF concentration in vivo, and treatment of diseases caused by an increase in IL-17-producing T cells.
  • an object of the present invention is to provide an evaluation method capable of rapidly evaluating the selective IL-4 production-inducing activity accompanying NKT cell activation of a test substance in vivo.
  • the present invention relates to the following (1) to (17), for example.
  • (1) A method for evaluating selective IL-4 production-inducing activity associated with NKT cell activation in vivo of a test substance, which is expressed in blood cells collected from a human subject to which the test substance is administered.
  • Quantification process to measure the amount of transcripts and CHRD gene, GYTLL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, RUFY4 gene, GPER gene , GSG1 gene, IL4I1 gene, CD180 gene, EGR2 gene, OAS2 gene, OAS3 gene, XLOC_002727 gene, XLOC_005851 gene, XLOC_I2 — 012046 gene and LOC645195
  • the amount of transcript measured for one or more genes selected from the group consisting of offspring is determined based on the amount of transcript expressed in blood cells collected from a human subject not receiving the test substance.
  • the quantifying step includes measuring the amount of a transcript expressed in blood cells collected 5 to 7 hours after administration from a human subject to which the test substance is administered, and the determining step comprises CHRD Gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, IL4I1 gene, CD180 gene, EGR2 gene, OAS2 gene, XLOC_002727 gene, XLOC_002727 gene
  • the amount of transcripts measured on blood cells 5 to 7 hours after administration is the same as that of the test substance not administered with the test substance.
  • the quantification step includes measuring the amount of a transcript expressed in blood cells collected 23 to 25 hours after administration from a human subject to which the test substance is administered, and the determination step comprises CHRD Gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, RUFY4 gene, GPER gene, GSG1 gene, IL4I1 gene, CD180 gene, EG2 gene One or more genes selected from the group consisting of OAS2 gene, OAS3 gene, XLOC_002727 gene, XLOC_005851 gene, XLOC_I2 — 012046 gene and LOC645195 gene
  • the amount of transcript measured on blood cells 23 to 25 hours after administration is determined based on the amount of transcript expressed in blood cells collected from a human subject who has not been administered the test substance.
  • the determination step includes CHRD gene, GYTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, RUFY4 gene, GPER1 gene, GSG1 gene,
  • the amount of transcripts measured for IL4I1 gene, CD180 gene, EGR2 gene, OAS2 gene, OAS3 gene, XLOC_002727 gene, XLOC_005851 gene, XLOC_I2 — 02046 gene and LOC645195 gene is collected from a human subject who has not been administered the test substance.
  • the quantification step includes measuring the amount of a transcript expressed in blood cells collected 5 to 7 hours after administration from a human subject to which the test substance is administered, and the determination step comprises CHRD Gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, IL4I1 gene, CD180 gene, EGR2 gene, OAS2 gene, XLOC_002727 gene, XLOC_002727 gene
  • the amount of transcript measured on blood cells 5 to 7 hours after administration is expressed in blood cells collected from human subjects who have not been administered the test substance.
  • the quantification step includes measuring the amount of a transcript expressed in blood cells collected from 23 to 25 hours after administration from a human subject to which the test substance is administered, and the determination step comprises CHRD Gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, RUFY4 gene, GPER gene, GSG1 gene, IL4I1 gene, CD180 gene, EG2 gene For OAS2 gene, OAS3 gene, XLOC — 002727 gene, XLOC — 005851 gene, XLOC — I2 — 012046 gene and LOC645195 gene against blood cells 23 to 25 hours after administration
  • the amount of the transcript measured in the above is greater than a threshold value determined based on the amount of the transcript expressed in blood cells collected from a human subject not administered with the test substance, and / or the DRC1 gene
  • the threshold value is the amount of a transcription product of each gene expressed in blood cells collected from the human subject before the test substance is administered. the method of.
  • the selective IL-4 production-inducing activity accompanying NKT cell activation is an activity that does not induce interferon ⁇ (IFN- ⁇ ) production of NKT cells and induces IL-4 production of NKT cells. , (1) to (7).
  • IFN- ⁇ interferon ⁇
  • the amount of transcript is measured using at least one selected from the group consisting of probes comprising the nucleotide sequence shown in any of SEQ ID NOs: 1 to 78. (1) to (8 ) Any one of the methods.
  • a method for screening a substance having selective IL-4 production-inducing activity associated with NKT cell activation in vivo, wherein the candidate substance is any one of (1) to (7) A screening method comprising performing the method.
  • the selective IL-4 production-inducing activity accompanying NKT cell activation is an activity that does not induce interferon ⁇ (IFN- ⁇ ) production of NKT cells and induces IL-4 production of NKT cells.
  • IFN- ⁇ interferon ⁇
  • a detection agent for selective IL-4 production-inducing activity associated with in vivo natural killer T (NKT) cell activation in a test substance comprising a base sequence represented by any of SEQ ID NOs: 1 to 78
  • a detection agent comprising at least one selected from the group consisting of probes.
  • the selective IL-4 production-inducing activity accompanying NKT cell activation is an activity that does not induce interferon ⁇ (IFN- ⁇ ) production of NKT cells and induces IL-4 production of NKT cells.
  • IFN- ⁇ interferon ⁇
  • selective IL-4 production induction associated with NKT cell activation of a test substance in vivo based on a change in the amount of a transcript of a specific gene contained in a blood sample collected from a human subject Activity can be assessed quickly (ie, early after administration). Therefore, it is suitable for evaluation of drug efficacy in clinical trials targeting humans.
  • the in vivo drug efficacy evaluation of the synthetic glycolipid compound described in Patent Document 1 can be performed, and it has selective IL-4 production-inducing activity associated with NKT cell activation. Screening for new substances (new drug candidates) is also possible.
  • the detection agent of the present invention comprises one or more probes containing the base sequence shown in any of SEQ ID NOs: 1 to 78, a transcription product of a specific gene contained in a blood sample collected from a human subject The amount can be measured with good reproducibility and accuracy. This makes it possible to detect with higher accuracy whether or not the test substance has shown a selective IL-4 production-inducing activity associated with NKT cell activation in vivo.
  • FIG. 2 is a scatter plot 6 hours after administration in the A cohort of Example 1.
  • FIG. Both the vertical axis and the horizontal axis of the graph show average values of signal values after inter-array normalization.
  • 2 is a scatter plot 24 hours after administration in the A cohort of Example 1.
  • FIG. Both the vertical axis and the horizontal axis of the graph show average values of signal values after inter-array normalization.
  • 2 is a scatter plot 6 hours after administration in the C cohort of Example 1.
  • FIG. Both the vertical axis and the horizontal axis of the graph show average values of signal values after inter-array normalization.
  • 2 is a scatter plot 24 hours after administration in the C cohort of Example 1.
  • FIG. Both the vertical axis and the horizontal axis of the graph show average values of signal values after inter-array normalization.
  • 2 is a graph showing changes over time in the amount of transcripts in peripheral blood in subjects administered with Compound 31.
  • 2 is a graph showing changes over time in the amount of transcripts in peripheral blood in subjects administered with Compound 31.
  • 2 is a graph showing changes over time in the amount of transcripts in peripheral blood in subjects administered with Compound 31.
  • 2 is a graph showing changes over time in the amount of transcripts in peripheral blood in subjects administered with Compound 31.
  • 2 is a graph showing changes over time in the amount of transcripts in peripheral blood in subjects administered with Compound 31.
  • 2 is a graph showing changes over time in the amount of transcripts in peripheral blood in subjects administered with Compound 31.
  • 2 is a graph showing changes over time in the amount of transcripts in peripheral blood in subjects administered with Compound 31.
  • 2 is a graph showing changes over time in the amount of transcripts in peripheral blood in subjects administered with Compound 31.
  • 2 is a graph showing changes over time in the amount of transcripts in peripheral blood in subjects administered with Compound 31.
  • 2 is a graph showing changes over time in the amount of transcripts in peripheral blood in subjects administered with Compound 31.
  • 2 is a graph showing changes over time in the amount of transcripts in peripheral blood in subjects administered with Compound 31.
  • 2 is a graph showing changes over time in the amount of transcripts in peripheral blood in subjects administered with Compound 31.
  • 2 is a graph showing changes over time in the amount of transcripts in peripheral blood in subjects administered with Compound 31.
  • 2 is a graph showing changes over time in the amount of transcripts in peripheral blood in subjects administered with Compound 31.
  • transcript encompasses messenger RNA (mRNA) as well as non-coding RNA that is not translated into protein.
  • the method for evaluating the selective IL-4 production-inducing activity associated with NKT cell activation in vivo of a test substance (hereinafter also simply referred to as “evaluation method”) is a human administered with a test substance.
  • a quantification step for measuring the amount of a transcript of a predetermined gene expressed in blood cells collected from a subject (hereinafter, also simply referred to as “quantification step”), and the measured transcript amount and each gene
  • a determination step (hereinafter also simply referred to as “determination step”) in which it is determined that the test substance exhibited selective IL-4 production-inducing activity associated with NKT cell activation in vivo based on comparison with a set threshold value.
  • the present invention can also be regarded as a method for collecting data for evaluating selective IL-4 production-inducing activity associated with NKT cell activation in vivo of a test substance, including the above-described quantification step.
  • the “predetermined gene” in the evaluation method according to the present invention is one of the synthetic glycolipid compounds described in Patent Document 1 (2S, 3S, 4R) -1-O- ( ⁇ -D-galactosyl) -2.
  • (N-tetracosanoylamino) -1,3,4-nonanetriol hereinafter also referred to as “Compound 31”
  • Compound 31 When (N-tetracosanoylamino) -1,3,4-nonanetriol (hereinafter also referred to as “Compound 31”) is administered to a human subject (a healthy subject), blood A gene in which the amount of transcript is statistically significantly increased or decreased in a sample (blood cell). That is, since the increase or decrease in the amount of the transcript of the “predetermined gene” correlates with the selective IL-4 production-inducing activity associated with NKT cell activation, the evaluation method according to the present invention allows the test substance Evaluation is possible.
  • the “predetermined gene” is a CHRD gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, RUFY4 gene, GPER gene , GSG1 gene, CPT1A gene, SLC25A20 gene, PER1 gene, SEMA3C gene, EMP1 gene, DRC1 gene, PDK4 gene, CNTNAP3 gene, TRIM49 gene, RELL1 gene, CNTNAP3B gene, DNAJB5 gene, PROM2 gene, MS4A2 gene, GATA2 gene, TAGAP Gene, F12 gene, KBBTBD7 gene, KLF6 gene, HDC gene, H2D2A gene, AGPAT4-IT1 gene, KLF10 gene, SLC1A7 gene, MYOM2 gene, GNLY gene, GZMB gene, FGFBP2
  • the quantification step and the determination step may be performed for one kind of gene selected from the gene group 1, and two or more kinds of genes selected from the gene group 1 or the gene group You may implement a determination process and a determination process about all the genes of 1.
  • the quantification step and the determination step for a plurality of genes, the selective IL-4 production-inducing activity accompanying NKT cell activation in vivo of the test substance can be evaluated with higher accuracy.
  • the “predetermined gene” includes each gene of gene group 1, IL4I1 gene, CD180 gene, EGR2 gene, OAS2 gene, OAS3 gene, FOSB gene, NR4A2 gene, KIR2DL2 gene, KIR2DL4 gene, KIR2DS2 gene, KIR2DS4 A gene, a KIR2DL5A gene, a KIR3DL2 gene, and an FOS gene (hereinafter also referred to as “gene group 2”). Details of each gene are shown in Tables 1 to 9.
  • the quantification step and the determination step may be performed for one or more genes selected from the gene group 2, and the quantification step and the determination are performed for two or more genes selected from the gene group 2. You may implement a process. In the second embodiment, it is preferable to carry out the quantification step and the determination step for all genes in the gene group 2 from the viewpoint of further improving the accuracy of evaluation.
  • Each gene contained in gene group 1 or 2 is gene group A, gene group B, gene group C, gene group D, and gene group E according to the change pattern of the amount of transcripts with time after administration of the test substance. Can be divided into
  • Gene group A has a statistically significant increase in the amount of transcript 6 hours after administration and 24 hours after administration, compared to before administration.
  • Genes included in gene group A are CHRD gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, IL4I1 gene, CD180 gene, EGR2 gene, RCC2 gene, OAS2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene , POT1 gene, XLOC_002727 gene, XLOC_005851 gene and XLOC_I2 — 012046 gene.
  • Gene group B has a statistically significant increase in the amount of transcript 24 hours after administration compared to before administration. There is no statistically significant difference between the amount of transcript 6 hours after administration and the amount of transcript before administration.
  • Genes included in gene group B are the OAS3 gene, the RUFY4 gene, the GPER gene, the GSG1 gene, and the LOC645195 gene.
  • Gene group C has a statistically significant decrease in the amount of transcript 6 hours after administration compared to before administration. There is no statistically significant difference between the amount of transcript 24 hours after administration and the amount of transcript before administration.
  • Genes included in gene group C are CPT1A gene, SLC25A20 gene, PER1 gene, SEMA3C gene, EMP1 gene, XLOC_000357 gene, XLOC_006688 gene, XLOC_010061 gene, LINC00222 gene, LOC40000655 gene and LOC100130581 gene.
  • gene group D the amount of transcript is statistically significantly reduced 6 hours after administration and 24 hours after administration, compared to before administration.
  • Genes included in gene group D are DRC1 gene, FOSB gene, PDK4 gene, CNTNAP3 gene, TRIM49 gene, RELL1 gene, CNTNAP3B gene, DNAJB5 gene, PROM2 gene, MS4A2 gene, NR4A2 gene, GATA2 gene, TAGAP gene, F12 gene , KBTBD7 gene, KLF6 gene, HDC gene, SH2D2A gene, AGPAT4-IT1 gene, FOS gene, KLF10 gene, XLOC_007314 gene and XLOC_I2 — 000092 gene.
  • Gene group E has a statistically significant decrease in the amount of transcript 24 hours after administration compared to before administration. There is no statistically significant difference between the amount of transcript 6 hours after administration and the amount of transcript before administration.
  • Genes included in gene group E are SLC1A7 gene, MYOM2 gene, GNLY gene, GZMB gene, FGFBP2 gene, C1orf21 gene, COLGALT2 gene, AKR1C3 gene, F2R gene, ZNF683 gene, PRF1 gene, DAAM2 gene, MYBL1 gene, KIR2DL gene, KIR2DL gene , KIR2DL4 gene, KIR2DL5A gene, KIR2DS2 gene, KIR2DS4 gene, KIR3DL2 gene, XLOC — 010245 gene and LOC286087 gene.
  • the determination step includes measuring the amount of transcript expressed in blood cells collected 5 to 7 hours after administration from a human subject to which the test substance has been administered.
  • the amount of transcripts of genes contained in gene group A is greater than the threshold set for each gene, or the amount of transcripts of genes contained in gene group C or gene group D is And determining that the test substance exhibited selective IL-4 production-inducing activity associated with NKT cell activation in vivo when it is less than the threshold value set for For convenience, it is also referred to as “6-hour evaluation”.
  • the collection of blood cells is preferably performed 5.5 to 6.5 hours after administration of the test substance, more preferably 5.75 to 6.25 hours after administration of the test substance, and 6 hours after administration of the test substance. More preferably, it is performed.
  • genes included in gene group A are the CHRD gene, GYTLL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 gene, C9orf47 gene , PPARGC1B gene, POT1 gene, XLOC_002727 gene, XLOC_005851 gene and XLOC_I2 — 012046 gene, and genes included in gene group C or gene group D are CPT1A gene, SLC25A20 gene, PER1 gene, SEMA3C gene, EMP1 gene, DRC1 gene, PDK4 Gene, CNTNAP3 gene, TRIM49 gene, RELL1 gene, CNTNAP3B gene, DNAJB5 gene, ROM2 gene, MS4A2 gene, GATA2 gene, TAGAP gene, F12 gene, KBBTBD7 gene, KLF6 gene, HDC gene, SH2D2A gene, AGP
  • the genes included in the gene group A are the genes of the first embodiment, the IL4I1 gene, the CD180 gene, the EGR2 gene, and the OAS2 gene, and the gene group C or the gene group D
  • the genes included in are the gene of the first embodiment, the FOSB gene, the NR4A2 gene, and the FOS gene.
  • the quantifying step also includes measuring the amount of transcript expressed in blood cells collected 23 to 25 hours after administration from a human subject to which the test substance has been administered, However, when the amount of transcripts of genes contained in gene group A or gene group B is greater than the threshold set for each gene, or the amount of transcripts of genes contained in gene group D or gene group E is Determining that the test substance exhibited selective IL-4 production-inducing activity associated with NKT cell activation in vivo when it is less than a threshold value set for each gene Good (hereinafter also referred to as “24-hour evaluation” for convenience). Blood cells are preferably collected 23.5 to 24.5 hours after administration of the test substance, more preferably 23.75 to 24.25 hours after administration of the test substance, and 24 hours after administration of the test substance. More preferably, it is performed.
  • genes included in gene group A or gene group B are CHRD gene, GYLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, RCC2 gene, MAFB gene, SERPING1 A gene, a C9orf47 gene, a PPARGC1B gene, a POT1 gene, a RUFY4 gene, a GPER gene, a GSG1 gene, an XLOC_002727 gene, an XLOC_005851 gene, an XLOC_I2 — 012046 gene, and a LOC645195 gene
  • the genes included in the gene group D or the gene group E are DRC1 gene, PDK4 gene, CNTNAP3 gene, TRIM49 gene, RELL1 gene, CNTNAP3B gene, DNAJB5 gene, P OM2 gene, MS4A2 gene, GATA2 gene, TAGAP gene, F12 gene, KBBTBD7 gene, K
  • the genes included in gene group A or gene group B are the gene of the first embodiment, IL4I1 gene, CD180 gene, EGR2 gene, OAS2 gene and OAS3 gene
  • the genes included in gene group D or gene group E are the gene of the first embodiment, FOSB gene, NR4A2 gene, KIR2DL2 gene, KIR2DL4 gene, KIR2DS2 gene, KIR2DS4 gene, KIR2DL5A gene, KIR3DL2 gene and FOS gene.
  • 6-hour evaluation or 24-hour evaluation when 6-hour evaluation or 24-hour evaluation is performed, either 6-hour evaluation or 24-hour evaluation may be performed. You may carry out combining evaluation.
  • test substance may be a low molecular compound or a high molecular compound.
  • the low molecular weight compound is arbitrarily selected from, for example, peptides, siRNA, miRNA, glycolipids and the like.
  • the polymer compound is arbitrarily selected from, for example, proteins and antibodies.
  • the glycolipid described in Patent Document 1 (for example, represented by the following formula (I), which has been confirmed to have a selective IL-4 production-inducing activity associated with NKT cell activation as a test substance.
  • ⁇ -galactosylceramide derivatives may be selected.
  • the correlation with selective IL-4 production-inducing activity associated with NKT cell activation in vivo when the dosage and administration method of the glycolipid are changed can be evaluated.
  • R 1 represents an aldopyranose residue
  • R 2 represents a hydrogen atom or a hydroxyl group
  • R 3 represents —CH 2 —, —CH (OH) —CH 2 —, or —CH ⁇ Represents CH—
  • R 4 represents a hydrogen atom or CH 3
  • x is 0 to 35
  • a substance that is unknown whether it has selective IL-4 production-inducing activity associated with NKT cell activation in vivo may be selected.
  • Substances determined to show selective IL-4 production-inducing activity associated with NKT cell activation in vivo by the evaluation method of the present invention include, for example, diseases in which Th1 / Th2 immune balance is biased to Th1 or Th1 cells It is extremely effective for the treatment of diseases that worsen the pathological condition, the treatment of autoimmune diseases, the treatment of diseases caused by an increase in GM-CSF concentration in vivo, and the treatment of diseases caused by an increase in IL-17-producing T cells. .
  • the test substance is preferably a substance that has achieved safety standards set in non-clinical studies, and more preferably a substance that is judged to have safety when administered to a human subject. “Having safety when administered to a human subject” means, for example, that safety has been confirmed in phase I of a clinical trial, and more specifically, 10 times the effective amount of the substance or Even when the blood concentration is 100 times, there is no serious side effect.
  • the blood cells are collected before the test substance is administered or from a human subject who has been administered the test substance.
  • the time from the administration of the test substance to the collection of blood cells can be set, for example, between 1 hour after administration and 72 hours after administration.
  • Blood cells can be collected by a known method such as a method of collecting blood cells using a vacuum blood collection tube and collecting the blood cells by centrifugation.
  • the sex, age, height, weight, health status, etc. of the human subject are not particularly limited, and may be appropriately set according to the purpose of evaluation.
  • the administration method of the test substance may be set according to the purpose, and may be oral administration or parenteral administration.
  • Parenteral administration may be systemic administration (for example, intravascular injection) or local administration (for example, subcutaneous administration, transdermal administration, transmucosal administration, or rectal administration).
  • the dose of the test substance may be set according to the type of test substance and the purpose.
  • quantitative_assay process can be implemented according to the following procedures, for example. (1) Prepare a blood sample collected from a human subject who has been administered a test substance, one day before administration, 6 hours after administration, and 24 hours after administration. A blood sample one day before administration is used as a reference for the amount of transcript (for example, used to determine a threshold). (2) The blood sample is centrifuged to collect blood cells as a precipitate. (3) Purify total RNA from the collected blood cells according to a conventional method. Commercially available total RNA extraction kits or purification kits may be used.
  • the transcript may be purified (or concentrated) from the total RNA sample.
  • the amount of a transcription product of a predetermined gene is measured according to a conventional method. Measurement of the amount of transcript using the total RNA as a template can be performed by, for example, microarray analysis, (real-time) quantitative PCR analysis, nucleic acid digital counting system (ncounter), RNA-Seq, and the like.
  • the amount of a transcription product of a housekeeping gene (eg, GAPDH) may be measured as an internal standard. This makes it possible to compare the amount of transcripts more accurately.
  • the test substance is in vivo NKT cell activity based on a comparison between the amount of transcript measured in the quantification step (or the amount of transcript after correction) and a threshold set for each gene. It is determined whether or not selective IL-4 production-inducing activity accompanying oxidization is exhibited.
  • the threshold value is determined based on the amount of transcript expressed in blood cells collected from a human subject who has not been administered the test substance.
  • the threshold is preferably the amount of the transcription product of each gene expressed in the blood cell for each gene. Thereby, it becomes possible to determine a change in the amount of a transcript of a predetermined gene with administration of a test substance with higher sensitivity.
  • the threshold value is obtained by measuring the amount of a transcript of a predetermined gene expressed in a blood cell collected from a human subject (one or more) who is not administered a test substance. (In the case of multiple persons, for example, an average value) may be set in advance as a threshold value.
  • the threshold value is determined by measuring the amount of a transcript of a predetermined gene expressed in blood cells collected before administration of the test substance from a human subject to whom the test substance is scheduled to be administered. It may be a threshold value. This makes it possible to determine with higher sensitivity.
  • the selective IL-4 production-inducing activity associated with NKT cell activation in vivo of a test substance refers to an activity that activates NKT cells and selectively induces IL-4 production by NKT cells. means.
  • the selective IL-4 production-inducing activity associated with NKT cell activation is more preferably an activity that does not induce IFN- ⁇ production of NKT cells and induces IL-4 production of NKT cells.
  • the evaluation method according to the present invention employs the above-described configuration, it can also be used as a screening method for a substance having selective IL-4 production-inducing activity associated with NKT cell activation in vivo. . That is, by carrying out the evaluation method according to the present invention using one or a plurality of candidate substances as test substances, it is possible to screen for substances having selective IL-4 production inducing activity associated with NKT cell activation in vivo. become.
  • the substance selected by the screening method according to the present invention has a selective IL-4 production-inducing activity associated with NKT cell activation in vivo. Therefore, the substance can be used as a therapeutic or prophylactic agent for a disease in which the Th1 / Th2 immune balance is biased to Th1 or a disease in which Th1 cells worsen the disease state.
  • the substance can also be used as a therapeutic or prophylactic agent for autoimmune diseases.
  • the substance can be used as a therapeutic or prophylactic agent for diseases caused by an increase in GM-CSF concentration in vivo.
  • the substance can be used as a therapeutic or prophylactic agent for diseases caused by an increase in IL-17-producing T cells.
  • Th1 / Th2 immune balance biased to Th1 include autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, psoriasis, type I diabetes, uveitis, Sjogren's syndrome, fulminant hepatitis, graft rejection, It means diseases mainly caused by cellular immunity such as infections caused by intracellular pathogens.
  • Autoimmune diseases are multiple sclerosis, rheumatoid arthritis, psoriasis, Crohn's disease, vitiligo vulgaris, Behcet's disease, collagen disease, type I diabetes, uveitis, Sjogren's syndrome, autoimmune myocarditis, autoimmune liver It means diseases such as disease, autoimmune gastritis, pemphigus, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuritis, HTLV-1-related myelopathy.
  • a disease caused by an increase in GM-CSF concentration in a living body means a disease such as chronic inflammatory disease such as chronic organitis and rheumatoid arthritis.
  • Diseases resulting from an increase in IL-17-producing T cells mean diseases such as psoriasis, rheumatoid arthritis, multiple sclerosis, optic neuromyelitis, inflammatory bowel disease and the like.
  • the test agent for selective IL-4 production-inducing activity associated with NKT cell activation in vivo of a test substance is from the group consisting of probes comprising the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 78. It consists of at least one selected. Each probe may be composed of the base sequence shown in any one of SEQ ID NOs: 1 to 78.
  • the base sequences shown in SEQ ID NOs: 1 to 78 hybridize with the cRNA (or transcription product) of the gene contained in gene group 2, as shown in Tables 1 to 9. Therefore, the detection agent can quantitatively hybridize with a cRNA (or transcription product) of a predetermined gene, and thereby, for example, by a fluorescence signal reflecting the amount of transcription product expressed in blood cells. A signal can be taken out, and a selective IL-4 production-inducing activity associated with NKT cell activation in vivo of the test substance can be detected.
  • Example 1 Identification of gene> Compound 31 was orally administered to human subjects once and changes in gene expression in peripheral blood were analyzed by microarray.
  • a healthy adult hereinafter referred to as “subject” was employed as a human subject, and the test was performed according to the following procedure.
  • the vacuum blood collection tube containing the collected blood was centrifuged at 2,500 rpm for 10 minutes to precipitate blood cells and remove the supernatant.
  • the obtained precipitate was washed with RNase Free water and suspended in BM1 (resuspension buffer), then BM2 (binding buffer) and proteinase K were added and dissolved at 55 ° C. and 900 rpm for 10 minutes. .
  • the obtained lysate was crushed through a PAXgene Shredder spin column attached to the kit, and then unnecessary pellets were removed by centrifugation to obtain a supernatant. 700 ⁇ L of isopropanol was added to the obtained supernatant to precipitate the total RNA.
  • total RNA was purified using PAXgene miRNA Spin Column attached to the kit, and eluted with 20 ⁇ L or 30 ⁇ L of BM5 (elution buffer).
  • the labeled cRNA was purified using RNeasy Mini kit (manufactured by QIAGEN), and then the concentration was measured using NanoDrop ND-1000 (Thermo Fisher Scientific). At this time, a sample having a purified labeled cRNA amplification efficiency of 15 times or more and a label incorporation efficiency of 6 pmol / ⁇ g or more was accepted and used for the subsequent tests.
  • the immobilized array was hybridized in DNA Microarray Hybridization Oven (manufactured by Agilent) under conditions of 17 hours (65 ° C., 10 rotations), and then the microarray was converted to Gene Expression Wash Buffer 1 (manufactured by Agilent), Gene.
  • the washing was performed with Expression Wash Buffer 2 (manufactured by Agilent), 10% Triton X-102 (manufactured by Agilent), and the washed microarray was scanned using Agilent DNA Microarray Scanner (manufactured by Agilent).
  • the obtained scanning data was digitized using the software Feature Extraction version 10.7.1.1 (manufactured by Agilent) using the digitization protocol GE1_107_Sep09 (numerical grid file: 0394494_D_F_2011628).
  • the digitized data was analyzed by the following method.
  • (A) Normalization between arrays As a normalization method between arrays during the one-color method, global normalization recommended by Agilent was performed using GeneSpring GX 12.0 (manufactured by Agilent). After excluding probes with numerical data (Raw Signal) of 1.0 or less, correction was made with the 75th percentile of the measured values for each array, and variation between arrays was suppressed to extract only biological variation.
  • GeneSpring flags that are indicators of data reliability based on flag information of five items described in digitized data (Raw Data) did).
  • Table 1 shows digitized data (Raw Data) and flag information in GeneSpring.
  • the selection criteria for the GeneSpring flag attached to each probe are as follows. A probe that is determined to be “Not Detected” in any one item is attached with a GeneSpring flag of “Not Detected”. In addition, although there was no “Not Detected” determination, a “Springed” GeneSpring flag was attached to a probe determined as “Compromised” in any item. Probes with no determination of “Compromised” and “Not Detected” were given “Detected” GeneSpring flags. In addition, the probe to which “Detected” GeneSpring flags are attached means that the probe has the highest reliability. After all three types of GeneSpring flags were attached to all probes, the number of probes classified by GeneSpring flag was counted for each data.
  • Both the vertical axis and the horizontal axis of the graph show average values of signal values after inter-array normalization.
  • the dotted line indicates the boundary of 1.5 times
  • the broken line indicates the boundary of 0.67 times
  • the white square dot indicates the fluctuating gene.
  • Probes with a “Detected” flag or a “Compromised” flag in both the 6-hour group and the 24-hour group and an expression ratio of 1.5 times or more or 0.67 times or less (conditions 3 ') ⁇ Switch ON type: In the 0-hour group, one or more subjects were assigned the “Not Detected” flag, but in the treatment group, all were assigned the “Detected” flag or the “Compromised” flag, and the probe switch was OFF with an expression ratio of 4 times or more. Type: In the treatment group, one or more subjects were assigned the “Not Detected” flag, but in the 0 hour group, all were assigned the “Detected” flag or “Compromised” flag, and the expression ratio was 0.25 times or less.
  • FIGS. 7 and FIGS. 15 to 16 In common with the A and C cohorts, changes in the amount of transcripts of genes with significantly increased transcript amounts 6 hours and 24 hours after administration of compound 31 compared to before administration are shown in FIGS. 7 and FIGS. 15 to 16. 5, 6 and 15 show genes whose transcript amount was significantly increased at both 6 hours and 24 hours after administration compared to before administration. FIG. 7 and FIG. 16 show genes whose transcript amount was significantly increased only after 24 hours of administration compared to before administration.
  • CHRD gene CHRD gene, GYTLTL1B gene, MYOF gene, EPS8 gene, SH3RF1 gene, IL4I1 gene, CD180 gene, EGR2 gene, OAS3 gene, RUFY4 gene, GPER gene, RCC2 gene, OAS2 gene, MAFB gene, SERPING1 gene, C9orf47 gene, PPARGC1B gene, POT1 gene, GSG1 gene, XLOC_002727 gene, XLOC_005851 gene, XLOC_I2 — 02046 gene and LOC645195 gene were also identified (see also Table 2, Table 3 and Table 8).
  • FIG. 8 to FIG. 8 show changes over time in the amount of transcripts of genes with significantly reduced amounts of transcripts 6 hours and 24 hours after administration of Compound 31, compared to before administration, in common with the A and C cohorts. 14 and FIGS. 17 to 19.
  • FIG. 8 and FIG. 17 show genes whose transcript amount was significantly reduced only after 6 hours of administration compared to before administration.
  • 9 to 11 and FIG. 18 show genes in which the amount of transcripts was significantly reduced at 6 hours after administration and at 24 hours after administration as compared to before administration.
  • FIGS. 12 to 14 and FIG. 19 show genes whose transcript amount was significantly reduced only after 24 hours of administration compared to before administration.
  • genes belonging to the KIR family such as the KIR2DL2 gene, the KIR2DL4 gene, the KIR2DL5A gene, the KIR2DS2 gene, the KIR2DS4 gene, and the KIR3DL2 gene were identified as genes that showed decreased expression (Tables 4 to 6 and Table 8 also). reference).
  • IL4I1 protein is secreted from B cells and myeloid cells by IL-4 stimulation and suppresses T cell proliferation (Boulland et al., 2007, Blood, 110, pp. 220-227). ), And is expressed in Th17 cells, and has been reported to be involved in suppression of IL-2 production and cell proliferation (Santalasci et al., 2012, Immunity, Vol. 36, pp. 201-214).
  • the genes in which the increase or decrease in the amount of transcript was observed are summarized in Tables 2 to 6 and Table 8.
  • the sequence numbers in Tables 2 to 6 and Table 8 correspond to the probe sequences on the microarray.
  • Tables 7 and 9 show the sequence of each probe.
  • the ensemble ID is an identification number of an ensemble genome browser (http://www.ensembl.org/index.html), which is a genome database.
  • the base sequences of the transcripts corresponding to the genes of SEQ ID NOs: 65 to 78 are shown in SEQ ID NOs: 79 to 103 in the sequence listing, and the corresponding relationship is shown in Table 10.

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Abstract

　L'invention concerne un procédé pour évaluer in vivo l'activité d'induction de la production sélective d'Il-4 associée à l'activation des cellules NKT d'une substance testée. Le procédé comporte une étape de dosage pour mesurer le niveau de produit de transcription exprimé par des cellules sanguines échantillonnées à partir d'un sujet humain auquel a été administrée une substance testée et une étape de détermination pour déterminer que la substance testée présente une activité d'induction de la production sélective d'IL-4 associée à l'activation des cellules NKT in vivo lorsque le niveau de produit de transcription mesuré pour le gène CHRD ou analogue est supérieur à une valeur seuil définie sur la base du niveau de produit de transcription exprimé par les cellules sanguines échantillonnées à partir d'un sujet humain auquel n'a pas été administrée la substance testée ou lorsque le niveau de produit de transcription mesuré pour le gène CPT1A ou analogue est inférieur à une valeur seuil définie sur la base du niveau de produit de transcription exprimé par les cellules sanguines échantillonnées à partir d'un sujet humain auquel n'a pas été administrée la substance testée.
PCT/JP2015/063935 2014-05-20 2015-05-14 Procédé d'évaluation de l'activité d'induction de la production sélective d'il-4 associée à l'activation des cellules nkt Ceased WO2015178288A1 (fr)

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CN108611413A (zh) * 2018-03-30 2018-10-02 北京泱深生物信息技术有限公司 一种帕金森相关生物标志物及其应用
US12037357B2 (en) 2021-03-01 2024-07-16 Deciduous Therapeutics, Inc. Compounds for activating invariant natural killer T-cells and methods of use in eliminating inflammatory senescent cells

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