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WO2021161703A1 - Marqueur pour l'hypertension pulmonaire - Google Patents

Marqueur pour l'hypertension pulmonaire Download PDF

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WO2021161703A1
WO2021161703A1 PCT/JP2021/000736 JP2021000736W WO2021161703A1 WO 2021161703 A1 WO2021161703 A1 WO 2021161703A1 JP 2021000736 W JP2021000736 W JP 2021000736W WO 2021161703 A1 WO2021161703 A1 WO 2021161703A1
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gene
expression
ahr
pulmonary
gene cluster
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Japanese (ja)
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良和 中岡
慎 岡澤
豪 正木
剛 大郷
遼太郎 浅野
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National Cerebral and Cardiovascular Center
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National Cerebral and Cardiovascular Center
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • 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

Definitions

  • the present disclosure relates to markers for pulmonary hypertension or pulmonary arterial hypertension.
  • the present disclosure also relates to markers for pulmonary arterial hypertension.
  • the present disclosure relates to the use of a particular gene as a marker for pulmonary hypertension or pulmonary arterial hypertension.
  • the present disclosure relates to a method of detecting a specific gene as an index for evaluating the severity of pulmonary hypertension.
  • the present disclosure relates to a method of detecting a specific gene as an indicator of being a subject suffering from or may have pulmonary hypertension or pulmonary arterial hypertension.
  • the present disclosure relates to a method for screening a candidate substance among test substances that may be effective for the treatment and / or prevention of pulmonary hypertension or pulmonary arterial hypertension.
  • the present disclosure relates to a kit for diagnosing pulmonary hypertension or pulmonary arterial hypertension.
  • the present disclosure relates to a method for producing a pathological model animal for pulmonary hypertension.
  • Pulmonary hypertension is a group of progressive diseases with a poor prognosis that causes cardiac and pulmonary dysfunction due to increased blood pressure in the pulmonary arteries.
  • Hypertension Pulmonary Arterial Hypertension, PAH
  • Group 1' Pulmonary veno-occlusive disease (PVOD) and / or pulmonary capillary hemangiomatosis (PCH)
  • Group 1' Prolonged neonatal Pulmonary hypertension
  • Group 2 Pulmonary hypertension associated with left cardiac heart disease
  • Group 3 Pulmonary hypertension associated with pulmonary disease and / or hypoxemia
  • Group 4 Chronic thromboembolic pulmonary hypertension ( Chronic thromboembolic pulmonary hypertension (CTEPH)
  • Group 5 Pulmonary hypertension associated with a multifactorial mechanism whose details are unknown.
  • PAH is a disease in which the peripheral pulmonary artery (arteriole or arteriole) has the main inflammation in the peripheral pulmonary artery (small artery or arteriole) with pathological changes such as muscularization of pulmonary artery, neointimal hyperplasia, and plexiform lesion (.
  • the pathogenesis of PAHs is complex and is probably caused by a combination of diverse factors such as genetic background, epigenetic modifiers, existing diseases and environmental factors (Non-Patent Document 3). Among them, inflammation and autoimmune diseases are considered to contribute significantly to the pathogenesis of PAH (Non-Patent Documents 4 and 5).
  • Aryl Hydrocarbon Receptor (hereinafter sometimes abbreviated as "AHR”) is activated by chemical substances in the environment or body (tryptophan derivatives, etc.) and is important for drug metabolism such as detoxification of harmful substances. Not only is it a nuclear receptor that plays an important role in regulating the immune response and maintaining cell homeostasis (Non-Patent Documents 6 and 7).
  • EAE autoimmune encephalomyelitis
  • MS multiple sclerosis
  • UC ulcerative colitis
  • Qingdao which is used as a Chinese herbal medicine, contains indirubin, a potent AHR agonist, and is used to treat refractory UC. Oral administration of Qingdao improved clinical symptoms in 81% of UC patients, but some patients have been reported to develop pulmonary hypertension as a side effect (Non-Patent Document 8-11).
  • the SU5416-hypoxia (SuHx) rat model shows the marked increase in pulmonary arterial pressure and pathological changes such as neointimal hyperplasia and plexiform lesions, which are the main characteristics of human PH. It is a model that can be reproduced well and is widely used in general (Non-Patent Documents 12 and 13).
  • the pathogenic mechanism of PH in this model has been attributed to the inhibitory effect of SU5416 on VEGFR2 (Non-Patent Documents 12, 13), but the inhibitory effect of SU5416 on VEGFR2 is seen at a 50% inhibitory concentration (IC50). It has a ⁇ M level and a low value (Non-Patent Document 14).
  • IC50 50% inhibitory concentration
  • Humbert M Sitbon O, Simonneau G. Treatment of pulmonary arterial hypertension.
  • Humbert M Guignabert C, Bonnet S, et al. Pathology and pathobiology of pulmonary hypertension: state of the art and research perspectives.
  • SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1 / KDR) that inhibitors tyrosine kinase research 1999; 59: 99-106.
  • the present disclosure identifies a marker gene that is an indicator of the presence or absence of PH, the possibility of future morbidity, or the severity of PH, and is a useful candidate as a drug for the treatment or prevention of PH based on the marker gene.
  • Providing a method for screening substances is an issue to be solved.
  • Another task of the present disclosure is to identify marker genes that are indicators of the presence or absence of PAH morbidity, future morbidity, or severity, and for the treatment or prevention of PAH based on it. It is to provide a method for screening candidate substances useful as pharmaceuticals for.
  • the present inventors hypothesized that the activating action of AHR is the cause of PH in the SuHx rat model and humans.
  • the present inventors subcutaneously administered 6-Formylindolo (3,2-b) carbazole (FICZ), which is an endogenous ligand for AHR, to wild-type rats and placed them under the same hypoxic conditions as the SuHxrat model. , Found that severe PH with pathological changes such as intimal lesions develops.
  • AHRKO rats prepared using the CRISPR-Cas9 system did not show an increase in pulmonary artery pressure compared to the solvent-only control group in the SuHxrat model, and pathologically, intimal lesions of the pulmonary artery were observed. I found that I could't.
  • AHR activity (AHR transcription activity) is higher in human PAH patients than in healthy subjects, and that the more severe PAH is, the higher the blood AHR activity is among human PAH patients. Based on these results, it is considered that suppression of AHR signals containing downstream molecules may be applicable to PH treatment.
  • RNA Sequence analysis of peripheral blood mononuclear cell cells (PBMC) in PAH patients and SuHx rats revealed increased expression of some common genes in both, and these genes were found in a new biomarker for PAH. It can be a marker.
  • a marker for pulmonary hypertension which comprises an arylhydrocarbon receptor gene or an arylhydrocarbon receptor.
  • [5] A method comprising detecting gene expression or activity of an arylhydrocarbon receptor in a sample isolated from a subject as an index for assessing the severity of pulmonary hypertension in the subject.
  • [6] The method according to [5], wherein the pulmonary hypertension is pulmonary arterial hypertension.
  • [7] Detect gene expression or activity of aryl hydrocarbon receptors in specimens isolated from subjects as an indicator of being a subject suffering from or may have pulmonary hypertension in the future. How to include that.
  • [8] The method according to [7], wherein the pulmonary hypertension is pulmonary arterial hypertension.
  • a kit for diagnosing pulmonary hypertension A kit containing reagents for measuring gene expression or activity of aryl hydrocarbon receptors.
  • the kit according to [9] for assessing the severity of pulmonary hypertension [11] The kit according to [9] or [10], wherein the pulmonary hypertension is pulmonary arterial hypertension. [12] A method for producing a model animal for pulmonary hypertension. A method comprising administering to a non-human animal an agonist of an aryl hydrocarbon receptor (excluding SU5416). [13] The production method according to [12], wherein the pulmonary hypertension is pulmonary arterial hypertension.
  • the test substance First gene cluster containing AHRR, CYP1A1, CYP1B1 and NQO1; An isolated cell or non-human animal expressing one or more genes selected from a second gene cluster containing AHRR, CCL3, EGR1, EGR2, PTGS2 and SEMA6B; and a third gene cluster containing GSTM1, IFI44L and SIGLEC1.
  • a method comprising determining the test substance as the candidate substance when the substance is suppressed as compared with the case where the substance is not given.
  • [19] A method for screening candidate substances that may be effective for the treatment and / or prevention of pulmonary arterial hypertension from among the test substances.
  • the test substance Giving to isolated cells or non-human animals expressing one or more genes in the 4th gene cluster, including MYZAP and S100B, The expression level of the gene or the activity of the protein produced by the expression of the gene in the cell or the non-human animal is measured, and the expression level of the gene or the activity of the protein produced by the expression of the gene is determined by the test.
  • a method comprising determining the test substance as the candidate substance when it is elevated as compared to the case where no substance is given.
  • kits for diagnosing pulmonary arterial hypertension First gene cluster containing AHRR, CYP1A1, CYP1B1 and NQO1; Second gene cluster containing AHRR, CCL3, EGR1, EGR2, PTGS2 and SEMA6B; To measure the expression of one or more genes selected from the third gene cluster containing GSTM1, IFI44L and SIGLEC1; and the fourth gene cluster containing MYZAP and S100B, or the activity of proteins produced by the expression of the one or more genes. Kit containing the reagents of.
  • the AHR gene or AHR can be used as a marker for PH.
  • the AHR gene or AHR is used as a diagnostic index for evaluating the severity of PH of a subject, or as a diagnostic index for a subject who has or may have PAH in the future. Can be detected.
  • One or more genes belonging to the 1st to 4th gene clusters can be used as markers for PAH.
  • one or more genes belonging to the first to fourth gene clusters can be used as a diagnostic index for a subject suffering from PAH or a subject who may be affected in the future, or of a subject. It can be detected as a diagnostic index for evaluating the severity of PAH.
  • a candidate substance that may be effective for the treatment and / or prevention of PH can be screened from among the test substances.
  • PH can be diagnosed by using the kit of the present invention.
  • a PH pathological model animal According to the method for producing a PH pathological model animal of the present invention, a PH pathological model animal can be produced.
  • FIG. 1 is a diagram showing the relationship between clinical findings and a diagram showing AHR activation in blood samples of PAH patients by the AHR luciferase assay.
  • a and D show that human PAH patient serum (PAH) has higher AHR activation than healthy person serum (HV);
  • B and E are mild in the WHO functional classification (WHO-FC), which is an indicator of PAH severity. When classified into (classes 1 and 2) and severe (classes 3 and 4), the degree of AHR activation is significantly higher in the mild (1, 2) group than in healthy subjects (HV), and further severe (3, 4).
  • F is a Kaplan-Meier curve depicting event-free survival (EFS) divided into AHR high activity group and AHR low activity group according to the degree of luciferase activity, and EFS is significantly higher in the AHR high activity group. It is a figure which shows the result that the prognosis is low and is poor.
  • FIG. 2 is a diagram showing the relationship between the expression level of AHR mRNA in PBMC and the severity of PAH in human PAH patients.
  • A is the result of positive correlation between the degree of AHR mRNA and the degree of AHR activation;
  • B is the result of positive correlation between AHR mRNA expression and PVR.
  • FIG. 3 shows that subcutaneous administration of FICZ, an endogenous agonist of AHR, and hypoxic loading cause severe PAH.
  • A is the experimental protocol; B is the result of right ventricular systolic pressure (RVSP); C is the result of Fulton coefficient; D is the result of EVG staining of pulmonary arterioles; E is the result of media thickening coefficient, F is the result of neointima It is a figure which shows the result of a region coefficient. The black bar in D indicates 50 ⁇ m.
  • FIG. 4 (FIGS. 4-1 and 4-2) is a diagram showing the results of producing AhrKO rats and almost completely suppressing PH pathology with AhrKO in the SuHxrat model.
  • A is the target sequence for Ahr KO production by the CRISPR / Cas9 system and the DNA sequence of the relevant site in KO rats; B is the result of Ahr KO not expressing the AHR protein in Western blot of AHR; C is for Ahr KO rats.
  • SuHx experimental protocol D is the result of RVSP; E is the result of Fulton coefficient; F is the result of EVG staining of pulmonary arterioles; G is the result of media thickening coefficient; H is the result of neointimal region coefficient. It is a figure which shows. The black bar in F indicates 50 ⁇ m. Continuation of Fig. 4-1. The explanation is as described with respect to FIG. 4-1.
  • A shows genes whose expression levels were changed in the wild-type SuHxrat model compared to the untreated group, and among these genes, the expression of the 911 gene was increased by 1.5 times or more;
  • B is the SuHxrat model of AhrKO. In, the expression level of the gene was changed compared to the wild type, and the expression of 168 genes was reduced to 2/3 or less among these genes;
  • C is Ahr KO among the 911 genes whose expression is increased by SuHxrat. It is a figure which shows the result of the expression reduction of 99 genes corresponding to 11% in rat.
  • A is the result of RNA-seq in Qingdao-induced PAH patients with many genes that are commonly elevated in humans on days 28 and 56 of the SuHxrat model; B is elevated in human and rat PAH.
  • Ben diagram of overlapping genes; C1 and C2 are the 28th day of SuHxrat PBMC and the 28th day of wild-type and AhrKO of genes that are commonly upregulated in human PAH.
  • A is the result of high mRNA of EGR1, EGR2, CYP1A1, SIGREC1, PTGS2 in some patients with PAH;
  • B is the result that average pulmonary arterial pressure (mPAP) is positively correlated with CCL3 expression of PBMC. ..
  • Subject refers to a human or non-human animal whose prevalence, onset or severity of PAHs or PAHs with intimal lesions is to be determined.
  • non-human animals include non-human mammals such as primates, rats, mice, gerbils, guinea pigs, hamsters, ferrets, rabbits, cows, horses, pigs, goats, dogs, and cats.
  • the non-human animals, preferably rats or mice can be used for the production of pathological model animals for PAHs.
  • Pulmonary hypertension is a group of progressive diseases with a poor prognosis that causes dysfunction of the heart and lungs due to increased blood pressure in the pulmonary arteries.
  • Pulmonary Arterial Hypertension PAH
  • Group 1' Pulmonary veno-occlusive disease (PVOD) and / or Pulmonary capillary hypertension (PCH)
  • Group 1'' Neonatal persistent pulmonary hypertension
  • Group 2 Pulmonary hypertension associated with left cardiac heart disease
  • Group 3 Pulmonary hypertension associated with pulmonary disease and / or hypoxemia
  • Group 4 Chronic thromboembolic lung Hypertension
  • Group 5 Classified as pulmonary hypertension associated with an unspecified multifactorial mechanism.
  • Samples isolated from the subject include samples prepared from body fluids such as blood, saliva, urine, sputum, and sweat isolated from the above subjects, or samples collected from tissues such as lungs. Can be exemplified. In particular, a sample prepared from the blood of the subject or blood, or a sample collected from the lungs is preferable. Peripheral blood is preferable as the blood. As the sample prepared from blood, a sample containing peripheral blood mononuclear cells, a plasma sample, a serum sample, a whole blood sample and the like are preferable.
  • AHR gene or AHR is useful as a marker for PH, especially as a marker for PH with intimal lesions. That is, the AHR gene increases the gene expression or the activity of AHR produced by the expression of the gene in PH patients. Therefore, in one embodiment of the present disclosure, an increase in AHR gene expression or activity is a diagnostic index for PH (particularly PH with intimal lesions).
  • the AHR gene or AHR can be a marker for PH in any of the 1st, 1st', 1''', 2nd, 3rd, 4th, and 5th groups of the Nice classification. ..
  • the AHR gene or AHR can be suitably used as a marker for PAHs (Group 1 of Nice Classification), especially for PH with intimal lesions.
  • PAHs Group 1 of Nice Classification
  • the AHR gene or AHR is used as a marker for PVOD and / or PCH (Group 1'of Nice Classification), especially for PVOD and / or PCH with intimal lesions. Can be.
  • the AHR gene or AHR is associated with chronic thromboembolic pulmonary hypertension (Nice classification 4th group) or neonatal protracted pulmonary hypertension (Nice classification 1'' group). Can be used as a marker for chronic thromboembolic pulmonary hypertension with intimal lesions or for neonatal protracted pulmonary hypertension.
  • an increase or decrease in AHR gene expression or activity can be determined by whether it is above or below a reference value for AHR gene expression or activity.
  • the "reference value” is a measured value or a measured value when the gene expression or activity of AHR in a sample sample derived from a normal sample is measured under the same conditions as the gene expression or activity of AHR in a sample sample derived from a subject. It may be a reference value set from the measured value, and the gene expression or activity of AHR in a sample derived from a patient whose presence or absence of PH or severity is known can be measured by measuring the gene expression or activity of AHR in a sample sample derived from a subject. It may be a measured value when measured under the same conditions as gene expression or activity, or a reference value set from the measured value. The method for measuring the gene expression or activity of AHR is as described later.
  • One embodiment of the present disclosure relates to a method comprising detecting gene expression or activity of AHR in a sample isolated from a subject as an index for assessing the severity of PH of the subject.
  • the method of this embodiment can also be rephrased as a method for testing the severity of PH of a subject, which detects the gene expression or activity of AHR in a sample isolated from the subject.
  • Another embodiment of the present disclosure is gene expression of AHR or gene expression in a sample isolated from a subject as an indicator of being a subject suffering from or potentially suffering from PH. It relates to a method involving detecting activity.
  • the method of this embodiment can also be rephrased as a method for detecting the gene expression or activity of AHR in a sample isolated from a subject, and testing the subject for PAH morbidity or future morbidity. can.
  • the method of each of the above embodiments of the present disclosure preferably comprises comparing a measured value of AHR gene expression or activity with a reference value.
  • the higher or higher the measured value of AHR gene expression or activity the more severe the subject determines that the subject's PH severity.
  • the PHs whose severity is determined are the first group, the first group, the first group, the first group, the second group, the third group, the fourth group, and the nice classification. It may be any of the 5th group.
  • One aspect of the method of the above embodiment is suitable for determining the severity of PAHs.
  • the severity of PVOD and / or PCH can be determined.
  • the PH determined for the presence or absence of morbidity or the possibility of morbidity in the future is the first group, the first ′ group, the first ′′ group, and the second group of the Nice classification. It may be any of a group, a third group, a fourth group, and a fifth group.
  • PAHs particularly PAHs with intimal lesions, are suitable for determining the presence or absence of morbidity or the possibility of morbidity in the future.
  • PVOD and / or PCH particularly PVOD and / or PCH with intimal lesions
  • chronic thromboembolic pulmonary hypertension or neonatal protracted pulmonary hypertension particularly chronic thromboembolic pulmonary hypertension with intimal lesions or neonatal protracted pulmonary hypertension. , Whether or not it is affected or the possibility of being affected in the future can be determined.
  • only one of the gene expression or activity of AHR may be measured, or both may be measured.
  • the gene expression of AHR may be measured by detecting the mRNA encoding the amino acid sequence of the AHR in the sample sample, or by detecting the amount of the protein of the AHR in the sample. May be good.
  • the measured value of AHR gene expression may be the relative expression level of the AHR gene expression level with respect to the gene expression level of one or more endogenous controls in the sample.
  • a housekeeping gene can be used as an endogenous control.
  • Northern blotting Northern blotting, RT-PCR method, real-time RT-PCR method, RNA-Seq analysis, DNA microarray method (method using DNA chip), dot blotting method, RNase for detection of mRNA encoding the amino acid sequence of AHR.
  • a protection assay method or the like can be used. These methods can be performed by known methods.
  • the amount of AHR protein can be detected by an immunoassay using an antibody that specifically recognizes and binds to the protein to be quantified.
  • the antibody can be prepared by a known method.
  • the immunoassay method include a method using a solid-phase carrier on which an antibody that specifically binds to the protein to be detected is immobilized, flow cytometry, Western blotting and the like.
  • the method using a solid phase carrier include, but are not limited to, an enzyme-linked immunosorbent assay (ELISA) using an immobilized microtiter plate and an agglutination method (immunoprecipitation method) using immobilized particles.
  • ELISA enzyme-linked immunosorbent assay
  • Known immunoassays can be used to detect the amount of AHR protein in a sample.
  • the amount of protein in AHR can be detected by a method using LC-MS / MS MRM or the like, which is a protein mass spectrometry technique that does not use an antibody. These detection
  • AHR An example of the "activity" of AHR is the transcriptional activity of AHR.
  • AHR is one of the transcription factors, and when a polycyclic aromatic hydrocarbon compound such as dioxin binds as a ligand, it translocates into the nucleus and forms a heterodimer with AHR Nuclear Translocator (ARNT), and this heterodimer is on the DNA. It binds to the foreign substance response sequence (XRE), which is a responsive promoter, causes transcriptional activation of drug-metabolizing enzymes, which are target genes, and transcriptional activation of signal transduction molecules, and acts on various signal transductions.
  • XRE foreign substance response sequence
  • the transcriptional activity of AHR can be measured by the following method.
  • a cell line for reporter gene assay in which a plasmid vector in which a reporter gene is ligated downstream of a foreign body response sequence (XRE) is introduced is used.
  • a luciferase gene can be exemplified as a reporter gene.
  • the cell line for the reporter gene assay is cultured in the presence of a sample isolated and prepared from the subject, and the expression level of the reporter gene is measured.
  • the expression level of the reporter gene is proportional to the activity of AHR (transcriptional activity). Therefore, the activity of AHR in the sample can be evaluated by the expression level of the reporter gene.
  • the expression level of the luciferase gene can be measured by measuring the luminescence amount due to the catalytic reaction of luciferase generated by the expression of the luciferase gene with a luminometer.
  • Reporter gene assay can be performed using commercially available reagents (including cell lines for assay).
  • a reagent for performing an arylhydrocarbon receptor luciferase reporter assay IB0600AhR of INDIGO Biosciences Co., Ltd. described in Examples can be exemplified.
  • the "activity" of AHR is a molecule involved in the epigenetic (DNA methylation, etc.) regulation of the gene region involved in the regulation of AHR expression; a molecule involved in the processing, degradation or stabilization of mRNA of AHR; AHR. Molecules involved in degradation or stabilization, including protein translation and ubiquitination; molecules that bind directly or indirectly to AHR and control AHR functions such as nuclear translocation of AHR and transcriptional activity by AHR; AHR It may be measured by detecting an endogenous substance (nucleic acid, protein, etc.) or an exogenous substance that can control the expression of the AHR downstream molecule through the recognized gene sequence. In addition, the "activity" of AHR may be measured by an agonist of AHR.
  • the number of cells producing AHR agonists, the amount of cells involved in AHR agonist production, the amount of precursor substances possessed by the cells, the amount of enzymes, and the difference in quality due to modification of these molecules may be measured, or in blood.
  • the amount of a substance present in can be a precursor of an AHR agonist may be measured.
  • kits for diagnosing PH relate to a kit containing reagents for measuring gene expression or activity of AHR.
  • the PH to be diagnosed may be any of the first group, the first group, the first group, the second group, the third group, the fourth group, and the fifth group of the nice classification.
  • the PH to be diagnosed is preferably a PAH, particularly a PAH with an intimal lesion.
  • PVOD and / or PCH in particular PVOD and / or PCH with intimal lesions, can be diagnosed.
  • another embodiment of the kit of the present disclosure diagnoses chronic thromboembolic pulmonary hypertension or neonatal protracted pulmonary hypertension, particularly chronic thromboembolic pulmonary hypertension with intimal lesions or neonatal protracted pulmonary hypertension. Can be targeted.
  • kit of the present disclosure it is preferably a kit for evaluating the severity of PH, more preferably the severity of PAH.
  • the reagents include a primer pair for amplifying a nucleic acid containing the AHR gene to be measured (for example, mRNA, cDNA derived from mRNA), a probe that hybridizes with the nucleic acid, and specific binding to the AHR protein.
  • a primer pair for amplifying a nucleic acid containing the AHR gene to be measured for example, mRNA, cDNA derived from mRNA
  • a probe that hybridizes with the nucleic acid for example, mRNA, cDNA derived from mRNA
  • specific binding to the AHR protein for example, a probe that hybridizes with the nucleic acid
  • Examples of the antibody to be used, a reagent for measuring the activity of AHR, and the like can be exemplified.
  • Examples of the reagent for measuring the activity of AHR include the above-mentioned reagents for performing a reporter gene assay (including a cell line for assay).
  • a reagent for performing an arylhydrocarbon receptor luciferase reporter assay IB0600AhR of INDIGO Biosciences Co., Ltd. described in Examples can be exemplified.
  • the antibody that specifically binds to the AHR protein may be a polyclonal antibody or a monoclonal antibody.
  • the antibody can also be used as a fragment as long as it can specifically bind to the protein to be measured.
  • Examples of the antibody fragment include a Fab fragment, an F (ab') 2 fragment, a single chain antibody (scFv), and the like.
  • the antibody may be immobilized on a solid-phase carrier such as a microtiter plate or particles.
  • the kit according to the above-described embodiment may further include a buffer solution for dilution or reaction containing components necessary for measurement, a washing solution, a coloring reagent, a reaction vessel, and the like.
  • the non-human animal can be exemplified by the non-human animal as described above, and is preferably a rat or a mouse, and more preferably a rat.
  • AHR agonists are substances that bind to AHR and induce the above-mentioned transcriptional activity by AHR.
  • Examples of agonists include 6-Formylindolo (3,2-b) carbazole (FICZ).
  • SU5416 can also be referred to as semaxinib or 3-[(2,4-dimethylpyrrol-5-yl) methylidenyl] -indolin-2-one.
  • the route of administration of the agonist to non-human animals is not particularly limited, and for example, subcutaneous administration can be exemplified.
  • the dose, frequency of administration, and interval of administration of the agonist to non-human animals can be appropriately set so as to induce the pathological condition of PAH, and the dose is, for example, 1 to 100 mg / kg body weight / week.
  • the number of administrations can be exemplified by 3 to 10 times, and the administration interval can be exemplified by once every 5 to 10 days.
  • the method for producing a pathological model animal of PH further includes breeding a non-human animal under a low oxygen concentration condition in order to efficiently induce PH into a non-human animal.
  • the hypoxic concentration condition refers to an atmosphere in which the oxygen concentration is, for example, 8 to 15%, preferably 9 to 11%.
  • a more preferred embodiment comprises feeding the non-human animal multiple times during a period of administration of the agonist under hypoxic conditions, preferably from the start to the middle of the period (eg, 2-4 weeks from the start of administration). )
  • Non-human animals are bred under hypoxic conditions, and non-human animals are bred under normal oxygen conditions from the middle to the end of the period (for example, for an additional 3 to 8 weeks).
  • the pathological model animals obtained by the production method of the present disclosure can be any of the first group, the first group, the first group, the first group, the second group, the third group, the fourth group, and the fifth group of the Nice classification. It may be used as a pathological model animal. In one embodiment of the production method of the present disclosure, the obtained pathological model animal is suitable as a pathological model animal for PAH. In addition, in another embodiment of the production method of the present disclosure, the obtained pathological model animal can be used as a pathological model animal for PVOD and / or PCH.
  • Marker for pulmonary arterial hypertension (2)
  • the present inventors have the first gene group (AHRR, CYP1A1, CYP1B1 and NQO1), the second gene group (AHRR, CCL3, EGR1, EGR2, PTGS2 and SEMA6B), the third gene group (GSTM1, IFI44L and SIGLEC1), And one or more genes selected from the fourth gene cluster (MYZAP and S100B) or proteins produced by expressing the one or more genes were found to be useful as markers for PAH. That is, in one embodiment of the present disclosure, a protein produced by expressing at least one gene or at least one gene in the first to fourth gene clusters is associated with PAHs, particularly intimal lesions. It is a diagnostic index for PAH.
  • the first gene cluster is a gene whose expression was increased in SuHx-treated (see Examples) wild-type rat lung and decreased in aryl hydrocarbon receptor knockout (Ahr KO) rat lung, which was confirmed in Examples. This is a typical gene.
  • Table 1 shows the NCBI gene ID and Ensembl gene ID of each gene in the first gene group in humans.
  • the second gene cluster is commonly expressed in peripheral blood mononuclear cells (PMBC) of Qingdao-induced PAH patients and peripheral blood mononuclear cells (PMBC) of SuHx-treated wild-type rats, which were confirmed in Examples. Is a representative gene among the genes in which is elevated. Table 2 shows the NCBI gene ID and Ensembl gene ID of each gene in the second gene group in humans.
  • the third gene group is a representative example of the gene confirmed in the examples in which the change in SuHxrat PBMC is not large or is not present in rats but the expression is increased in PBMC of Aomori-induced PAH patients.
  • Table 3 shows the NCBI gene ID and Ensembl gene ID of each gene in the third gene group in humans.
  • the 4th gene cluster is a representative example of the gene confirmed in the example, in which the change in SuHxrat PBMC is not large or is not present in rats but the expression is decreased in PBMC of Aomori-induced PAH patients.
  • Table 4 shows the NCBI gene ID and Ensembl gene ID of each gene in the 4th gene cluster in humans.
  • One or more genes selected from the first gene group, the second gene group, and the third gene group increase the gene expression or the activity of the protein produced by the gene expression in the patient with PAH. Therefore, an increase in gene expression or protein activity of one or more genes selected from the first gene group, the second gene group, and the third gene group is a diagnostic index for PAH.
  • the gene or the protein produced by expressing the one or more genes is particularly useful as a marker for PAH, and one or more genes selected from the second gene group or the protein produced by expressing the one or more genes can be used. , Most useful as a marker for PAH.
  • One or more genes selected from the 4th gene cluster reduce gene expression or protein activity produced by gene expression in patients with PAH. Therefore, a decrease in gene expression or protein activity of one or more genes selected from the fourth gene cluster is a diagnostic index for PAH.
  • the increase or decrease in gene expression or protein activity caused by gene expression can be determined by whether it is above or below the reference value of gene expression or protein activity.
  • the "reference value” refers to the gene expression or protein activity of the one or more genes in a sample sample derived from a normal sample, and the gene expression or protein activity of the one or more genes in a sample sample derived from a subject. It may be a measured value when measured under the same conditions or a reference value set from the measured value, or the gene of the above one or more genes in a sample derived from a patient whose presence or absence of PAH or severity is known. Even if the expression or protein activity is measured under the same conditions as the gene expression or protein activity of the one or more genes in the sample sample derived from the subject, or even if it is a reference value set from the measured value. good.
  • One embodiment of the present disclosure is a first gene cluster (AHRR,) in a sample isolated from a subject as an indicator of being a subject suffering from or may have PAH in the future. Selected from CYP1A1, CYP1B1 and NQO1), 2nd gene cluster (AHRR, CCL3, EGR1, EGR2, PTGS2 and SEMA6B), 3rd gene cluster (GSTM1, IFI44L and SIGLEC1), and 4th gene cluster (MYZAP and S100B) The present invention relates to a method comprising detecting the expression of one or more genes or the activity of a protein produced by the expression of one or more genes.
  • the method of this embodiment is the first gene group (AHRR, CYP1A1, CYP1B1 and NQO1), the second gene group (AHRR, CCL3, EGR1, EGR2, PTGS2 and SEMA6B), the first gene group (AHRR, CCL3, EGR1, EGR2, PTGS2 and SEMA6B) in the sample isolated from the subject.
  • another embodiment of the present disclosure is a first gene cluster (AHRR, CYP1A1, CYP1B1 and NQO1), No. 1 in a sample isolated from a subject, as an index for evaluating the severity of PAH of the subject.
  • the present invention relates to a method comprising detecting the activity of a protein resulting from the expression of one or more genes.
  • the method of this embodiment is the first gene group (AHRR, CYP1A1, CYP1B1 and NQO1), the second gene group (AHRR, CCL3, EGR1, EGR2, PTGS2 and SEMA6B), the first gene group (AHRR, CCL3, EGR1, EGR2, PTGS2 and SEMA6B) in the sample isolated from the subject.
  • PAH that detects the expression of one or more genes selected from the three gene clusters (GSTM1, IFI44L and SIGLEC1) and the fourth gene cluster (MYZAP and S100B) or the activity of the protein produced by the expression of the one or more genes. In other words, it can be rephrased as a method for testing the severity of.
  • the method of each of the above embodiments of the present disclosure preferably comprises comparing the expression of the one or more genes or the measured value of the activity of the protein produced by the expression of the one or more genes with the reference value.
  • one or more selected from the first gene group and the second gene group among one or more genes selected from the first gene group, the second gene group, and the third gene group. It is preferable to detect the gene expression or protein activity of one or more genes selected from the second gene group, and it is particularly preferable to detect the gene expression or protein activity of one or more genes selected from the second gene group.
  • a subject suffers from PAH or has PAH if the measured gene expression or protein activity of one or more genes selected from the fourth gene cluster is low or low. It can be determined that the subject may be affected in the future, or that the subject's PAH severity is more severe.
  • only one of the gene expression and protein activity of the one or more genes may be measured, or both may be measured.
  • the gene expression of the one or more genes may be measured by detecting the mRNA of the one or more genes in the sample sample, or the amount of the protein generated from the one or more genes in the sample is detected. You may measure by doing.
  • the measured value of the gene expression of the one or more genes may be the relative expression level of the gene expression level of the one or more genes with respect to the gene expression level of the one or more endogenous controls in the sample.
  • a housekeeping gene can be used as an endogenous control.
  • Northern blotting Northern blotting, RT-PCR method, real-time RT-PCR method, RNA-Seq analysis, DNA microarray method (method using DNA chip), dot blotting method, RNase protection for the detection of mRNA of one or more genes.
  • An assay method or the like can be used. These methods can be performed by known methods.
  • the amount of protein generated from the one or more genes can be detected by an immunoassay method using an antibody that specifically recognizes and binds to the protein to be quantified.
  • the antibody can be prepared by a known method.
  • the immunoassay method include a method using a solid-phase carrier on which an antibody that specifically binds to the protein to be detected is immobilized, flow cytometry, Western blotting and the like.
  • the method using a solid phase carrier include, but are not limited to, an enzyme-linked immunosorbent assay (ELISA) using an immobilized microtiter plate and an agglutination method (immunoprecipitation method) using immobilized particles.
  • ELISA enzyme-linked immunosorbent assay
  • a known immunoassay can be used to detect the amount of protein resulting from one or more of the above genes in a sample. Further, the detection of the protein amount of the protein generated from the one or more genes can also be performed by a method using LC-MS / MS MRM or the like, which is a protein mass spectrometry technique that does not use an antibody. These detection methods can also be carried out by a conventional method. The activity of the protein generated from the one or more genes can be measured by a method according to the activity of the protein to be measured.
  • the test substance is selected from the first gene group (AHRR, CYP1A1, CYP1B1 and NQO1), the second gene group (AHRR, CCL3, EGR1, EGR2, PTGS2 and SEMA6B), and the third gene group (GSTM1, IFI44L and SIGLEC1).
  • Glutathione to isolated cells or non-human animals expressing one or more genes The expression level of the gene or the activity of the protein produced by the expression of the gene in the cell or the non-human animal is measured, and the expression level of the gene or the activity of the protein produced by the expression of the gene is determined by the test. Judging the test substance as the candidate substance when the substance is suppressed as compared with the case where the substance is not given. Regarding methods including.
  • test substance is given to isolated cells or non-human animals expressing one or more genes selected from the 4th gene cluster (MYZAP and S100B).
  • the expression level of the gene or the activity of the protein produced by the expression of the gene in the cell or the non-human animal is measured, and the expression level of the gene or the activity of the protein produced by the expression of the gene is determined by the test. Judging the test substance as the candidate substance when it is elevated as compared with the case where no substance is given.
  • methods including.
  • the isolated cells include peripheral blood mononuclear cells (PMBC) isolated from humans or other animals.
  • PMBC peripheral blood mononuclear cells
  • Examples of the non-human animal include the non-human animals mentioned above.
  • kits for diagnosing PAH First gene group (AHRR, CYP1A1, CYP1B1 and NQO1), second gene group (AHRR, CCL3, EGR1, EGR2, PTGS2 and SEMA6B), third gene group (GSTM1, IFI44L and SIGLEC1), and fourth gene group (GSTM1, IFI44L and SIGLEC1).
  • the present invention relates to a kit containing a reagent for measuring the expression of one or more genes selected from MYZAP and S100B) or the activity of a protein produced by expressing the one or more genes.
  • the reagents include a primer pair for amplifying a nucleic acid containing the one or more genes to be measured (for example, mRNA, cDNA derived from mRNA), a probe that hybridizes with the nucleic acid, and the one or more genes.
  • a primer pair for amplifying a nucleic acid containing the one or more genes to be measured for example, mRNA, cDNA derived from mRNA
  • a probe that hybridizes with the nucleic acid for example, mRNA, cDNA derived from mRNA
  • examples thereof include an antibody that specifically binds to the protein produced by expression, a reaction substrate for measuring the enzymatic activity of the protein produced by expressing one or more genes, and the like.
  • the antibody that specifically binds to the protein produced by expressing the one or more genes may be a polyclonal antibody or a monoclonal antibody.
  • the antibody can also be used as a fragment as long as it can specifically bind to the protein to be measured.
  • Examples of the antibody fragment include a Fab fragment, an F (ab') 2 fragment, a single chain antibody (scFv), and the like.
  • the antibody may be immobilized on a solid-phase carrier such as a microtiter plate or particles.
  • the reaction substrate for measuring the activity of the protein produced by expressing the one or more genes can be appropriately selected according to the activity of the protein to be measured.
  • the kit according to the embodiment may further include a buffer solution for dilution or reaction containing components necessary for measurement, a washing solution, a coloring reagent, a reaction vessel, and the like.
  • PBMC peripheral blood mononuclear cells
  • serum For PBMC, EDTA blood was collected and Lymphoprep Tube (Cosmo Bio Co., Ltd) or LSM (MP biomedicals, LLC) was used from each peripheral blood sample. And separated according to the instructions.
  • serum is collected in a blood collection tube containing a coagulation promoter (Nipro Co., Ltd., NP-SP1029) using a holder (Terumo Corporation), then centrifuged at 2800 g for 10 min, collected, and collected at -80 ° C until just before use. Saved in.
  • hAHR Fw primer: 5'-GAGTCTGGACAAGGAATTGAAGA-3' (SEQ ID NO: 1), Rv primer: 5'-GGAGGAATTCTGGTCTGGGTTA -3'(SEQ ID NO: 2);
  • hCYP1A1 Fw primer: 5'-TCTTTGGAGCTGGGTTTGAC-3' (SEQ ID NO: 3), Rv primer: 5'-TGACCTGCCAATCACTGTGT-3' (SEQ ID NO: 4);
  • hEGR1 Fw primer: 5'-TGACCGCAGAGTCTTTTCCT-3' (SEQ ID NO: 5), Rv primer: 5'-TGGGTTGGTCATGCTCACTA-3' (SEQ ID NO: 6);
  • hEGR2 Fw primer: 5'-GGGTGTGTGCACCATGTC-3' (SEQ ID NO: 7), Rv primer: 5'-GGGTGTGTGTGCACCATGTC-3' (SEQ ID NO: 7), Rv primer: 5'-GGGTGT
  • AHR luciferase reporter assay The AHR luciferase reporter assay was performed using INDIGO Biosciences' IB0600AhR as instructed except for stimulation conditions. The stimulus was added in the medium described in the instructions so that the serum had a final concentration of 10%.
  • SuHx rat model was prepared as follows according to the previous report. 20 mg / kg SU5416 was subcutaneously administered to 6-8 week old SD rats, kept in a 10% O 2 hypoxic chamber for 3 weeks, and then removed from the hypoxic chamber for 2-5 weeks under positive oxygen conditions. It was bred in. Cage replacement was performed twice a week.
  • the drug was administered subcutaneously at a dose of 10 mg / kg every week from the start of hypoxic loading, and the rats were bred in a 10% O 2 hypoxic chamber for 3 weeks in the same manner as the SuHx rat model. After that, they were taken out of the hypoxic chamber for 5 weeks and bred under positive oxygen conditions. Drug administration during hypoxic loading was performed at the time of cage replacement.
  • RVP right ventricular pressure
  • MCT0670 pressure transducer
  • ML117 pressure amplifier
  • PowerLab system AD Instruments Colorado Springs, CO
  • Mean arterial pressure was also measured by inserting a polyethylene tube into the right internal carotid artery prior to inserting the venous catheter and using the same system as the venous catheter.
  • Heart rate was calculated by picking up from the peak of the right ventricular systole of the arterial pressure waveform.
  • RNA-seqencing The quality of RNA and library preparation was guaranteed by TapeStation (Agilent). For RNA-Seqencing analysis of rat lung, 100 ng of total RNA was used for ribosomal RNA removal, followed by library preparation using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina). For RNA-Seqencing analysis of human or rat PBMC, a library was prepared using 100 pg of total RNA and SMART-seq v4 Ultra Low Input RNA Kit (Takara Clontech). Over 25 million reads were obtained, with 75 bp Pair end reads per sample. The quality of sequencing data was controlled using FastQC.
  • Each trimmed and filtered read was aligned to the rat genome Version rn6 reference genome using the rat genome (Rn6) for rats and to the human genome (hg38) reference genome using Hisat2 for humans.
  • the gene cluster specifically expressed in lung tissue was defined as satisfying False Discovery Rate (FDR) ⁇ 0.05 with a change in expression level of 1.5 times or more or 2/3 or less.
  • the gene cluster specifically expressed in PBMC was defined as satisfying P ⁇ 0.05, with expression changes of 1.5 times or more or 2/3 or less in humans and 2 times or more or 2/3 or less in rats. ..
  • EMG Elastica van Gieson staining was performed for morphological analysis of pulmonary blood vessels. Images of the arteries were taken with Aperio (Leica). Pulmonary vascular remodeling evaluation was performed by the medial thickening coefficient (% wall thickness) and the neointima region coefficient of the vascular lumen.
  • the target blood vessels are the pulmonary artery in the lung parenchyma, the small artery at the terminal bronchiole level, and the arteriole at the pulmonary arteriole level, with a diameter of 30 to 100 ⁇ m, and 50 vessels in each treatment group. The above selection was made and evaluated.
  • the media thickening coefficient is defined as doubling the distance between the inner and outer elastic plates, dividing by the distance between the outer elastic plates (diameter of the blood vessel), and multiplying by 100. For blood vessels with only one elastic plate, the distance between the elastic plate and the basement membrane under the epidermis is measured. Media thickening was analyzed only for blood vessels segmented into a roughly circular shape. The diameter of the pulmonary artery was determined using the Aperio ImageScope (Leica). The new intima region coefficient of the vascular lumen is that for each blood vessel, no new intima lesion is found (Open), less than 50% of the vascular lumen has intima lesion (Partial), and 50 of the vascular lumen. The percentage of blood vessels in each group was evaluated by classifying them as those in which% or more were occupied by intima lesions (Close).
  • the degree of activation was high, and in the severe group, it was higher than in the mild group (Fig. 1B, 1E). It also showed a positive correlation with another indicator of PAH severity, Pulmonary Vascular Resistance (PVR, Wood unit) (Fig. 1C). In addition, in PAH patients, the group with high AHR activity had a significantly lower event-free survival (EFS) and a poorer prognosis (Fig. 1F). That is, it was clarified that the activation of AHR in serum is an index of the severity of PAH, and the AHR agonist activity in serum (degree of AHR activation) is a predictor of prognosis.
  • Verification of the induction effect of severe PAH by administration of AHR-specific agonist FICZ in a rat model SU5416 has high AHR agonist activity.
  • AHR-specific agonist FICZ AHR-specific agonist
  • PH-suppressing effect of SuHx rat model by Ahr knockout rat Ahr KO rat was generated using CRISPR-Cas9 system to verify whether AHR activation is necessary for induction of severe PH using SuHx rat model.
  • FIG. 4A Western blots confirmed the detection of the Ahr protein and found that in Ahr KO rats, 6-8 week old Ahr KO (-/-) rats, sibling wild (Ahr (+/+)) and hetero (Ahr (+/+)) +/-)) In rats, SU5416 was subcutaneously administered at 20 mg / kg, followed by hypoxia for 3 weeks and positive oxygen for 2-5 weeks to prepare a SuHx rat model (Fig. 4C).
  • RNA-seqencing results in the lungs of the SuHx rat model In order to clarify the molecular mechanism that induces PH in an AHR-dependent manner, we searched for genes that change in the lungs on the 4th day after SuHx treatment in an AHR-dependent manner. As a result, 911 genes (SuHx-up gene) were identified that increased significantly (FDR ⁇ 0.05) by 1.5 times or more in the SuHx group as compared with the control group without treatment (Fig. 5A). On the other hand, 168 genes (Fig. 5B) were significantly (FDR ⁇ 0.05) reduced to 2/3 or less in Ahr KO rats compared to SuHx-treated wild-type rats, accounting for 11% of 99 genes in SuHx-up genes.
  • Fig. 5C AHR dependence was observed (Fig. 5C). These 99 genes included the AHRR, CYP1A1, CYP1B1 and NQO1 genes (Table 1 above). In addition, among the 168 genes whose expression was decreased in Ahr KO rats, 69 genes were not increased in SuHx-treated wild-type rats.
  • genes whose expression was commonly increased in Aomori-induced PAH and SuHx rats included the AHRR, CCL3, EGR1, EGR2, PTGS2 and SEMA6B genes (Table 2 above).
  • GSTM1, IFI44L and SIGLEC1 were included in the genes whose gene expression was increased in PBMC of human Aomori-induced PAH patients, which is not common to rats (Table 3 above).
  • Aomori-induced PAHs or SuHx rat PBMCs with reduced gene expression included MYZAP and S100B (Table 4 above).
  • the mean pulmonary arterial pressure in these patients was 50 mmHg and 48 mmHg for PH patients predisposed to COPD and 34 mmHg for CTEPH patients, both of which were severe PH of WHO-FC class 3. This suggests that AHR is a marker for PH in general.
  • the presence or absence of PH other than PAHs in PH patients other than PAHs using the AHR genes or AHRs as an index, and the future
  • the AHR gene or AHR is classified in any of the 1st group, 1'group, 1'group, 2nd group, 3rd group, 4th group, and 5th group of the Nice classification in PH. Also suggests that it can be applied as a marker.

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Abstract

Le but de la présente invention est d'identifier un gène marqueur qui peut être utilisé en tant qu'indicateur de la gravité de l'hypertension pulmonaire (PH).　La présente invention concerne l'utilisation d'un gène pour récepteur d'hydrocarbure aryle (AHR) ou un AHR en tant que marqueur pour l'hypertension pulmonaire (PH). La présente invention concerne également un procédé de détection du marqueur en tant qu'indicateur de la gravité de PH ou d'un indicateur permettant de déterminer si un sujet souffre de PH ou a le potentiel d'être affecté par PH dans le futur à partir d'un échantillon isolé du sujet. La présente invention concerne en outre un procédé de criblage d'une substance candidate qui peut être efficace pour le traitement et/ou la prévention de PH parmi des substances d'essai. La présente invention concerne également un procédé de production d'un animal modèle de maladie de PH.
PCT/JP2021/000736 2020-02-10 2021-01-12 Marqueur pour l'hypertension pulmonaire Ceased WO2021161703A1 (fr)

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