WO2023276768A1 - 低酸素バイオマーカー及びその利用 - Google Patents

低酸素バイオマーカー及びその利用 Download PDF

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WO2023276768A1
WO2023276768A1 PCT/JP2022/024612 JP2022024612W WO2023276768A1 WO 2023276768 A1 WO2023276768 A1 WO 2023276768A1 JP 2022024612 W JP2022024612 W JP 2022024612W WO 2023276768 A1 WO2023276768 A1 WO 2023276768A1
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spink1
hypoxia
sample
amount
subject
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French (fr)
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浩 原田
稔 小林
達也 諏訪
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Kyoto University NUC
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Kyoto University NUC
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/99—Enzyme inactivation by chemical treatment
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification

Definitions

  • hypoxic biomarkers and their use.
  • hypoxia in vivo is known to be associated with various pathological conditions.
  • hypoxic regions regions with reduced oxygen partial pressure
  • the hypoxic state induces cancer cell resistance to radiotherapy and anticancer drug therapy. It is known to be an important environmental factor. It is also said that the lower the oxygen concentration, the worse the condition. Therefore, it is important to know the amount of the hypoxic region, the degree of hypoxia, etc. in understanding and predicting the disease state and the effectiveness of its treatment.
  • hypoxic PET imaging using a probe such as 18 F- fluoromisonidazol is known as a method for understanding hypoxia in vivo. It is possible to know the existence, position, and extent of the hypoxic region in the body (Non-Patent Document 1). However, this method has the problem that it is difficult to use it repeatedly, and the PET apparatus itself is expensive, so there is also the problem that the facility where it can be performed is limited.
  • an immunostaining method is known in which a part of tissue is collected from the living body and CA9 (carbonic anhydrase 9), GLUT1 (glucose transporter 1), etc. in the collected tissue are targeted.
  • CA9 carbonic anhydrase 9
  • GLUT1 glucose transporter 1
  • the present disclosure provides novel markers useful for measuring hypoxia and predicting exacerbation of hypoxia-related pathological conditions, methods for measuring hypoxia using the markers as indicators, methods for predicting exacerbation of pathological conditions, for measuring hypoxia, or
  • An object of the present invention is to provide a composition for predicting aggravation of pathological conditions, a screening method for therapeutic agents for hypoxia-related pathological conditions, a composition for improving hypoxia-related pathological conditions, and the like.
  • Section 1 A method for measuring hypoxia in a subject, comprising the step of measuring the amount of SPINK1 (serine protease inhibitor Kazal-type I) in a sample collected from the subject. Section 2.
  • a method for predicting aggravation of a disease state comprising the step of measuring the amount of SPINK1 in a sample collected from a subject.
  • Item 3. Item 3. The method according to Item 1 or 2, wherein the sample is at least one selected from the group consisting of blood, plasma, serum, urine, milk, saliva, cell samples, and tissue samples. Section 4. Use of SPINK1 as a biomarker to measure hypoxia or to predict exacerbation of disease states.
  • Item 5. A composition for measuring hypoxia or predicting aggravation of a disease state, containing a SPINK1-detecting drug.
  • Item 6. A method for screening therapeutic agents for hypoxia-related pathological conditions, using the ability of candidate substances to inhibit SPINK1 as an index.
  • a composition for improving hypoxia-related conditions containing a SPINK1 inhibitor.
  • the pathological condition is at least one selected from the group consisting of tumor and ischemic disease, the method according to Item 2 or 3, the use according to Item 4, the composition according to Item 5, and the composition according to Item 6. 8. The method or composition of paragraph 7.
  • a hypoxic biomarker useful for measuring hypoxia and predicting exacerbation of pathological conditions can be provided.
  • a composition for measuring hypoxia or predicting aggravation of pathological conditions containing a SPINK1-detecting agent, a method for screening therapeutic agents for hypoxia-related pathological conditions, using the ability to inhibit SPINK1 as an index, and a SPINK1 inhibitor. , a composition for improving hypoxia-related conditions, and the like.
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SP
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SP
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SP
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 2 is a diagram showing an example of SPINK1 reported by NCBI;
  • FIG. 4 shows the results of SPINK1 mRNA expression in a hypoxic stimulation-dependent manner.
  • FIG. 4 shows the results of SPINK1 mRNA expression in a hypoxic stimulation-dependent manner.
  • FIG. 2 shows the results of SPINK1 mRNA expression and protein secretion in a hypoxic stimulus-dependent manner.
  • SPINK1 reflects hypoxia in tumors.
  • SPINK1 is secreted from hypoxic cells and present in and around the cells.
  • FIG. 4 shows that increased SPINK1 contributes to exacerbation of pathological conditions.
  • FIG. 4 shows that SPINK1 can be used as an index of exacerbation of hypoxia-related pathology.
  • FIG. 4 shows that increased SPINK1 contributes to exacerbation of pathological conditions.
  • FIG. 1 shows that inhibition of SPINK1 can inhibit exacerbation of pathological conditions.
  • FIG. 10 shows that the overall survival rate is lower in the high SPINK1 group than in the low SPINK1 group.
  • a method for measuring hypoxia in a subject included in the present disclosure includes a step of measuring the amount of SPINK1 (serine protease inhibitor Kazal-type I) in a sample collected from the subject.
  • SPINK1 serine protease inhibitor Kazal-type I
  • SPINK1 is known as serine protease inhibitor Kazal-type I and has been publicly known. Without limiting the present disclosure, SPINK1 can be found from known databases, for example, as shown in FIGS. is an example. Of the reports described in Figures 1 to 35, the first SPINK1 (Homo sapiens (human) ID: 6690) is information at the time of the update in May 2021, and other rats at the same time is an example of SPINK1 such as. The present disclosure is not limited to the specific SPINK1 shown in these figures, as long as it is SPINK1.
  • the sample collected from the subject is not limited as long as it is hypoxic to be measured, and the sample includes blood, plasma, serum, saliva, milk, urine, feces, tears, sweat, hair, body hair, cerebrospinal fluid, Bone marrow fluid, synovial fluid, sputum, nasal discharge, sebum, lymphatic fluid, tissues and cells of pathological sites (e.g. tumor tissue, tumor cells, tissue specimens and cell specimens such as myocardial cells), etc., preferably blood, plasma, Biological samples such as serum, urine, milk, saliva, tissue specimens, and cell specimens are exemplified, and biological samples such as blood, plasma, serum, saliva, milk, and urine are more preferred from the viewpoint of less invasiveness to the subject.
  • pathological sites e.g. tumor tissue, tumor cells, tissue specimens and cell specimens
  • biological samples such as blood, plasma, serum, saliva, milk, and urine are more preferred from the viewpoint of less invasiveness to the subject.
  • a pathological site such as a tumor tissue may be, for example, benign or malignant. Collection of a sample from a subject may be performed according to a conventionally known procedure. A sample may be used individually by 1 type, and may be used in combination of 2 or more type.
  • Subjects include mammals such as humans, monkeys, mice, rats, rabbits, goats, sheep, horses, guinea pigs, hamsters, dogs, cats, monkeys, and elephants, preferably humans, monkeys, mice, rats, and the like. and more preferably human.
  • Measurement of the amount of SPINK1 in the sample is not limited as long as the amount of SPINK1 in the sample can be measured, and may be measured according to a conventionally known procedure.
  • SPINK1 to be measured includes, for example, pre-mRNA, mRNA, protein, activity, etc., and as long as the amount of SPINK1 can be reflected, the subject that changes according to the change in the amount of SPINK1
  • An object may be used as a measurement target, and the amount of SPINK1 may be measured indirectly based on the measurement results of the object.
  • the measurement is not limited as long as the amount of SPINK1 can be measured, for example, in situ hybridization, RT (Reverse Transcription)-PCR in the measurement of mRNA precursor and mRNA (including quantitative RT-PCR), microarray, RNA-Seq, northern blotting, etc. Immunostaining, western blotting, ELISA (Enzyme-linked immuno-sorbent assay), flow cytometry, dot blotting for protein measurement , known gene detection methods such as mass spectrometry, immunological techniques, and the like. Also, when a substance, activity, or the like that can reflect the amount of SPINK1 is to be measured, the measuring means may be appropriately determined according to the object to be measured. The sample may be used as it is for the measurement of SPINK1, or may be pretreated as necessary.
  • primers for quantitative RT-PCR are exemplified by ORIGENE's SPINK1 Human qPCR Primer Pair (NM_003122) (Catalog #24833).
  • Anti-SPINK1/P12 antibody manufactured by Abcam (Catalog #ab183034) is exemplified as an antibody, although the present disclosure is not limited thereto.
  • An example of an ELISA kit is Human SPINK1 DuoSet ELISA (Catalog # DY7496-05) manufactured by R&D Systems.
  • conventionally known primers, antibodies, and the like can be appropriately used in the above measurement.
  • measurement of the amount of SPINK1 also includes the meaning of detection. That is, the measurement of the amount of SPINK1 may be performed to determine the presence or absence of SPINK1 in the sample, or to determine the amount of SPINK1 in the sample. Also, in the present disclosure, the measured amount may be an absolute amount or a relative amount.
  • the measurement method of the present disclosure may further include the step of setting a reference value for the amount of SPINK1 and comparing the reference value with the amount of SPINK1 in the sample (measured value) measured as described above. .
  • the reference value may be set arbitrarily according to the purpose.
  • a sample collected from a person whose oxygen concentration is normal e.g., a sample from a healthy person, who does not have a pathological condition that causes a decrease in oxygen concentration
  • the amount of SPINK1 in the sample derived from the subject may be the reference value, and in this case, if the amount of SPINK1 in the sample collected from the subject (measured value) is higher than the reference value, the subject who collected the sample and if the measured value is equal to or lower than the reference value, the oxygen concentration is determined to be normal in the subject from which the sample was taken.
  • the reference value may be the amount of SPINK1 in a sample (for example, a sample derived from a patient with a pathological condition) collected from a person whose oxygen concentration is lower than the normal value, in which case the measured value is the same as or higher than the reference value. determines that there are sites with the same or lower oxygen concentration than the reference value provider (e.g., disease carrier) in the subject from whom the sample was taken, or there are more sites with the same or lower oxygen concentration However, if the measured value is lower than the reference value, there is no site where the oxygen concentration is lower than that of the provider of the reference value in the subject from whom the sample was collected, or there is a site with a similar or lower oxygen concentration than the provider of the reference value. It can be determined that there are few parts.
  • a sample for example, a sample derived from a patient with a pathological condition
  • the measured value and the reference value are not limited to this limit, but preferably the same sample (for example, if the sample for the measured value is blood, the sample for the reference value is also blood). Measurement is also performed by the same measurement procedure. Also, the reference value may be a value measured in advance in a sample collected from the subject.
  • the subject from which the sample was collected has a hypoxic site (a region where the partial pressure of oxygen has decreased). If it can be determined to be high and SPINK1 is not detected, it is determined that there are no hypoxic sites in the subject from which the sample was taken.
  • the greater the amount of SPINK1 in a sample the more sites with lower oxygen concentration or hypoxia are present in the subject from whom the sample was collected. It is determined that the lower the amount of SPINK1 in the sample, the closer the oxygen concentration is to normal or the fewer areas of hypoxia in the subject from which the sample was taken.
  • the measurement method of the present disclosure may optionally further include these determination steps and the like.
  • the relationship between the degree of hypoxia (oxygen concentration) and the degree of pathology (aggravation) may differ depending on the type of pathology (disease). Therefore, the reference value may be appropriately determined according to the type of pathological condition (disease). Also, the reference value may be an absolute value or a relative value.
  • the presence or absence and/or amount of SPINK1 in a sample can be known. Therefore, the measuring method is useful in that it can determine the presence or absence and/or amount of SPINK1 in a sample.
  • the present inventors found that there is a correlation between hypoxia and the amount of SPINK1, and found that the lower the oxygen concentration, the higher the amount of SPINK1 that is expressed, secreted, etc. rice field. Thus, it can be understood that the amount of SPINK1 tends to increase in vivo in a hypoxic concentration-dependent manner (the lower the oxygen concentration and/or the greater the number of hypoxic sites (areas)).
  • the degree of hypoxia in the subject can be known by measuring the amount of SPINK1 in the sample collected from the subject. It can also be said that it is possible to measure (detect) the region (presence or absence, degree, etc.). From this point, it can be said that the present disclosure provides a method for measuring hypoxia in a subject based on the amount of SPINK1 in the sample.
  • the measurement method of the present disclosure is also useful in that it is possible to know the degree of hypoxia in the subject based on the amount of SPINK1 in the sample.
  • this is also useful in that it can be used as an index to predict the worsening state of the disease and the degree of exacerbation.
  • the prediction is also useful in examining and implementing treatment for pathological conditions according to subjects. Pathology is described in the same manner as described below.
  • Method for predicting aggravation of pathological condition also includes a method for predicting aggravation of pathological condition, comprising the step of measuring the amount of SPINK1 in a sample collected from a subject.
  • the subject, sample, SPINK1, measurement, etc. are all explained in the same way as the measurement method described above. Conventionally, it is said that the lower the oxygen concentration, the worse the condition of the disease. Therefore, in the method, it is predicted that the greater the amount of SPINK1 in the sample, the higher the exacerbation of the condition in the subject, and the lower the amount of SPINK1 in the sample, the lower the exacerbation of the condition in the subject. can do.
  • the amount of SPINK1 to be measured in the prediction method is preferably exemplified by mRNA amount and protein amount, and more preferably exemplified by protein amount.
  • Pathological conditions include ischemic diseases such as tumors, ischemic heart diseases (angina pectoris, myocardial infarction, etc.) and ischemic brain diseases (cerebral infarction, etc.), preferably tumors, myocardial infarction, cerebral infarction, etc. are exemplified.
  • exacerbation means the degree of progression and intractability of a disease state, and for example, the degree of malignancy in tumors. It can be said that the higher the aggravation, the more advanced the disease and the more intractable (difficult to cure).
  • a non-limiting example of exacerbation of disease states is radiation resistance of tumor tissue, which is refractory.
  • hypoxia is not limited as long as it is within the range of hypoxia known in the art.
  • the SPINK1 amount tends to increase as the oxygen concentration decreases to 0.1% or less, and the SPINK1 amount tends to increase significantly when the oxygen concentration is lower.
  • hypoxia can be described as a concentration of oxygen below the normal partial pressure of oxygen in each organ, preferably in the range of 5% or less, 3% or less, 1% or less, 0.1% or less. exemplified.
  • the lower limit of oxygen concentration is theoretically 0%.
  • the oxygen concentration is a value according to normal measurement procedures in this field. The explanation is similarly applied to the measurement method described above.
  • the prediction method it is possible to predict the exacerbation of the disease state based on the measured amount of SPINK1.
  • the greater the amount of SPINK1 in a sample the higher the exacerbation of the disease state in the subject from whom the sample was collected (the disease state is advanced, the malignancy is high , or likely to be refractory) is determined (i.e., the exacerbation of the condition is predicted to be high), and the lower the amount of SPINK1, the lower the exacerbation of the condition in the subject from whom the sample was collected. (i.e., predicted to be less likely to exacerbate disease).
  • the prediction method of the present disclosure may further include the step of providing a reference value for the amount of SPINK1 and comparing the reference value with the amount of SPINK1 in the sample (measured value) measured as described above.
  • the reference value may be arbitrarily set according to the purpose.
  • the reference value is the amount of SPINK1 in a sample that has already been determined to have low exacerbation
  • the amount of SPINK1 in the sample collected from the subject is higher than the reference value, it is determined that the subject from whom the sample was collected has a high possibility of exacerbation of the condition, and if the measured value is the same as or lower than the reference value, the sample is collected It is determined that the subject has a low likelihood of exacerbation of the condition.
  • the amount of SPINK1 in a sample that has already been determined to have a high exacerbation potential is used as the reference value
  • the amount of SPINK1 (measured value) in the sample collected from the subject is the same as or higher than the reference value.
  • the possibility of exacerbation of the condition in the subject from whom the sample is collected is low. determined to be.
  • the measured values and the reference values are not limited in these, as described above, the measured values and the reference values are preferably the same sample (for example, if the sample for the measured values is blood, the sample for the reference values is also blood). Yes, the amount of SPINK1 is measured by the same measurement procedure. Also, the reference value may be a value measured in advance in a sample collected from the subject.
  • the prediction method of the present disclosure when SPINK1 is detected in a sample, it can be determined that there is a high possibility that a hypoxic site is present in the subject from whom the sample was collected, If SPINK1 is not detected, it is determined that no sites of hypoxia are present in the subject from which the sample was obtained. In the former case, the exacerbation of the pathology is expected to be high, and in the latter case, the exacerbation of the pathology is expected to be low or there is no pathology (pathological site).
  • the prediction method of the present disclosure may optionally further include these determination steps and the like.
  • the reference value may be appropriately set according to the condition of the subject from whom the sample is collected, the type of pathological condition (disease), the degree of the pathological condition, and the like.
  • the reference value may be determined as appropriate in consideration of the amount of SPINK1 in samples derived from healthy subjects or patients who have already been determined to have a pathological condition, and the reference value is, for example, the present disclosure.
  • the reference value may be derived from the subject used for prediction, or may be derived from a third party different from the subject. Also, the reference value may be an absolute value or a relative value. This explanation also applies to the measurement method described above.
  • the subject from whom the sample is collected may be a subject having some pathological condition (disease), It may be a subject, a subject whose condition (disease) is unknown, or a subject with no pathology (disease).
  • the lower the oxygen concentration the worse the condition.
  • the oxygen concentration is low at the tumor site and its surroundings, and it is known that the lower the oxygen concentration, the more resistant the tumor is to radiotherapy.
  • low oxygen concentration is known to promote cancer invasion and the like.
  • myocardial infarction is mainly caused by a decrease in oxygen concentration due to obstruction of blood flow or the like.
  • various pathological conditions are known to be closely related to the oxygen concentration in the pathological site and its surroundings.
  • radioresistance observed in tumors with reduced oxygen concentration it is also necessary to appropriately select a therapeutic method according to the degree of hypoxia.
  • the exacerbation of various pathological conditions can be predicted based on the presence or absence and/or amount of SPINK1 in a sample, which is useful in this respect. Prediction of exacerbation of pathological conditions is useful in considering the necessity of treatment for subjects, selection of treatment methods, and the like.
  • SPINK1 is useful as a biomarker for measuring hypoxia or predicting exacerbation of disease. Moreover, from this, SPINK1 can also be called a hypoxic marker. SPINK1 is described in the same manner as above, and although it does not limit the present disclosure, SPINK1 mRNA and protein are preferably exemplified as biomarkers. In addition, when the marker is used, blood or the like can be used as a measurement sample, so it can also be used as a blood marker or the like.
  • the amount of SPINK1 in a subject can be monitored, so it is useful for determining therapeutic effects, predicting prognosis, etc. based on fluctuations in the amount of SPINK1.
  • Determination of therapeutic effect includes determination of whether the treatment is effective. For example, if the increase in the amount of SPINK1 in the sample is suppressed or the amount of SPINK1 is decreased, the treatment is effective. If the increase in SPINK1 level is not suppressed or the SPINK1 level is increased, it is determined that the treatment is not effective.
  • Prognosis prediction is the determination of whether the prognosis is good or not.
  • the prognosis when the amount of SPINK1 in the sample is low, the prognosis is good when the increase in the amount of SPINK1 is suppressed or the amount of SPINK1 is decreased.
  • the amount of SPINK1 is high, it is predicted that the prognosis is not good when the increase in the amount of SPINK1 is not suppressed or when the amount of SPINK1 is increased. From this, it is possible to determine the therapeutic effect and predict the prognosis based on the measurement method and the prediction.
  • composition for Measuring Hypoxia or Predicting Exacerbation of Disease State also includes a composition for measuring hypoxia or predicting exacerbation of a disease state, containing a SPINK1-detecting agent.
  • SPINK1 measurement of hypoxia, and prediction of exacerbation of pathology are as described above.
  • the SPINK1 detection drug is not limited as long as it can detect SPINK1, and examples include primers, probes, antibodies, aptamers (nucleic acid aptamers, etc.), compounds (natural compounds, synthetic compounds, etc.) that specifically detect SPINK1. Any of polyclonal antibodies, monoclonal antibodies, fragments thereof (eg, Fab, Fab', F(ab')2, etc.) can be used as antibodies.
  • the detection agent may be capable of directly or indirectly detecting SPINK1 pre-mRNA, mRNA, protein, activity, etc., and can grasp the presence of SPINK1. unlimited.
  • they may be labeled with enzymes, fluorescent dyes, etc., may be bound to other proteins, etc., or may be immobilized on microplates, magnetic beads, etc.
  • the detection agent a conventionally known SPINK1 detection agent may be used, and the above-mentioned primers, antibodies, ELISA kits and the like are exemplified, although the present disclosure is not limited.
  • the composition may further contain optional components as long as the effects of the present disclosure are not impaired, and the components include carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, Examples include binders, disintegrants, lubricants, thickeners, coloring agents, fragrances, chelating agents, pH adjusters, preservatives and the like.
  • the component may be used singly or in combination of two or more, and the blending amount thereof may be appropriately determined within a range that does not impair the effects of the present disclosure.
  • the content of the SPINK1 detection agent in the composition is not limited within the range in which SPINK1 can be detected, and may be appropriately limited according to the types of the primers, probes, antibodies, and the like.
  • the form of the composition may be solid (powder, granule, tablet, etc.), semi-solid, or liquid.
  • the detection using the composition may be performed with reference to a conventionally known detection procedure according to the type of the primer, probe, antibody, activity, object, etc. As an example, It can be carried out in the same manner as the procedure in the measuring method.
  • the composition may be in the form of a kit.
  • the kit further includes a buffer that can be used for detection, a nucleic acid amplification reagent, a reverse transcriptase, a substrate, a primary antibody, a secondary antibody, instructions for use, an aptamer (nucleic acid aptamer, etc.) ), compounds (natural compounds, synthetic compounds, etc.), etc. It may also be possible to obtain the usage procedure, etc. From this, it can be said that the present disclosure provides a kit for measuring hypoxia or predicting aggravation of pathological conditions.
  • composition and kit are useful in that the measurement and prediction can be easily performed.
  • the present disclosure includes a method of screening therapeutic agents for hypoxia-related pathological conditions using the ability of candidate substances to inhibit SPINK1 as an indicator.
  • SPINK1 can be used as an index in hypoxia-related pathologies, and that the more SPINK1, the higher the exacerbation of the pathology.
  • the present inventors have found that the aggravation (for example, the degree of intractability) of pathological conditions can be alleviated by inhibiting SPINK1.
  • drugs capable of inhibiting SPINK1 are useful for ameliorating hypoxia-related pathological conditions, suppressing their progression, and the like. Therefore, therapeutic agents for hypoxia-related conditions can be screened using the ability to inhibit SPINK1 as an index.
  • Candidate substances are not particularly limited, and may be compounds (natural compounds, synthetic compounds, etc.), antibodies, aptamers (nucleic acid aptamers), or the like. It may be manufactured or the like.
  • Inhibition of SPINK1 includes, for example, the ability to reduce the expression level of SPINK1 precursor and mRNA, the ability to reduce the expression level of SPINK1 protein, the ability to reduce the secretion level of SPINK1 protein, and the ability to reduce the activity of SPINK1 protein.
  • SPINK1 inhibition can inhibit, for example, SPINK1 activity (activity to induce radioresistance of cells, activity to induce antioxidant capacity of cells, activity to induce EGFR (epidermal growth factor receptor) signaling), etc. It may be used as an index.
  • inhibition of SPINK1 includes inhibition of expression, secretion, activity, and the like.
  • the pathology associated with hypoxia is not limited as long as it is a pathology in which hypoxia is a factor, and the above-mentioned pathology is exemplified.
  • Said screening method may further comprise said determining step.
  • Whether or not a candidate substance can inhibit SPINK1 can be appropriately determined according to the candidate substance by referring to conventionally known screening procedures.
  • a control value may be provided for inhibition, and the inhibition (test value) when using the candidate substance may be compared with the control value.
  • the control value is the amount of SPINK1 inhibition when the sample is contacted with a substance that does not have SPINK1 inhibitory activity
  • the amount of SPINK1 inhibition (test value) in the sample in which the candidate substance is contacted with the sample is When compared to the control value, if the test value is higher than the control value (the amount that inhibits SPINK1 is greater), it is determined that the candidate substance is likely to inhibit SPINK1; It is determined that the candidate substance is likely to be useful in treating conditions associated with hypoxia.
  • the control value and the test value are the same or the test value is lower than the control value, it is determined that the candidate substance is unlikely to inhibit SPINK1 and on this basis the candidate substance is hypoxia-related. It is determined that it is unlikely to be useful in treating the condition.
  • the amount of SPINK1 inhibition may be an absolute value or a relative value, and is not limited as long as the presence or absence and/or degree of SPINK1 inhibition can be known.
  • the sample is not limited as long as the inhibition can be determined, and the sample collected from the above-described subject may be used.
  • a substance already known to be useful for the treatment of hypoxia-related conditions or a substance already known to have SPINK1 inhibitory activity may be used as a control value, for example, when the SPINK1 inhibition amount when the substance and the sample are contacted is set as the control value, the SPINK1 inhibition amount (test value) in the sample when the candidate substance and the sample are contacted and If the test value is equal to or higher than the subject value compared to the control value, it is determined that the candidate substance is likely to inhibit SPINK1, and on this basis the candidate substance is associated with a hypoxia-associated condition.
  • the candidate substance is determined to be unlikely to inhibit SPINK1
  • the candidate substance is determined to be unlikely to inhibit hypoxia-associated It may be determined that it is unlikely to be useful in treating the condition. Again, the amount of inhibition and the sample are explained in the same way as above.
  • the screening method is useful for searching, creating, and determining therapeutic agents for hypoxia-related pathological conditions.
  • compositions for Ameliorating Hypoxia-Related Conditions The present disclosure also includes compositions for ameliorating hypoxia-related conditions containing SPINK1 inhibitors.
  • the present inventors found that SPINK1 inhibition can improve the refractory conditions associated with hypoxia, for example. Accordingly, the present disclosure also provides compositions for improving hypoxia-related conditions containing SPINK1 inhibitors.
  • SPINK1 inhibitors are not limited as long as they can inhibit SPINK1, and are exemplified by compounds (natural compounds, synthetic compounds, etc.), antibodies, aptamers (nucleic acid aptamers), etc., preferably antibodies, more preferably SPINK1 neutralizing antibodies. be.
  • the inhibitor may be one capable of inhibiting the activity of SPINK1, etc., as described above. Inhibition of SPINK1 is described in the same manner as before.
  • the content of the SPINK1 inhibitor in the composition is not limited, and may be appropriately limited according to the type and inhibitory ability of the compound, antibody, aptamer, and the like.
  • the composition may be solid (powder, granule, tablet, etc.), semi-solid, or liquid, and its administration route is not limited.
  • oral administration include subcutaneous administration, intramuscular administration, intravenous administration, sublingual administration, oral mucosa administration, transrectal administration, transvaginal administration, transnasal administration, inhalation administration, nebulization administration, transdermal administration and the like.
  • composition may further optionally contain pharmaceutically acceptable ingredients as long as the effects of the present disclosure are not impaired, and the ingredients include solvents, pH adjusters, excipients, stabilizers , binders, disintegrants, lubricants, coloring agents, perfumes, preservatives and the like.
  • the component may be used singly or in combination of two or more, and the blending amount thereof may be appropriately determined within a range that does not impair the effects of the present disclosure.
  • the ameliorating composition is useful for ameliorating conditions associated with hypoxia. Conditions associated with hypoxia are described similarly to the conditions described above.
  • Test example 1 1-1 Test procedure Human cervical cancer-derived cell line HeLa was seeded in a cell culture dish (1 x 10 5 per well of a 6-well cell culture plate) and placed in a CO 2 incubator under normal oxygen concentration (20%). Incubate overnight. After that, after culturing for the time shown in FIG. 36 in a CO 2 incubator set to the oxygen concentration shown in FIG. ), reverse transcription reactions, and quantitative PCR experiments for SPINK1 and CA9 were performed. A quantitative PCR experiment against ACTB as an internal standard was also performed at the same time.
  • FIG. 1 shows (a) the expression level of SPINK1 mRNA and (b) the expression level of CA9 mRNA after HeLa cells were exposed to each oxygen concentration for 48 hours, and (c) HeLa cells were exposed to oxygen concentrations of 20% or 0.1%. shows the expression level of SPINK1 mRNA after exposure for 12, 24, and 48 hours.
  • the expression levels of SPINK1 mRNA and CA9 mRNA significantly increased under exposure to an oxygen concentration of 0.1%.
  • the expression level of CA9 a conventionally known hypoxia marker
  • SPINK1 showed a tendency to gradually increase when the oxygen concentration decreased to 3%.
  • the expression level at 1% oxygen concentration was as low as that at 20% oxygen concentration, and a rapid increase was observed at 0.1% oxygen concentration.
  • the expression level of SPINK1 mRNA increased with exposure to a hypoxic environment for 12, 24, and 48 hours. From this, it was confirmed that the amount of SPINK1 increased in a hypoxic stimulation-dependent manner.
  • Test example 2 2-1 Test procedure Human cervical cancer-derived cell line HeLa, human prostate cancer-derived cell line DU145, and human osteosarcoma-derived cell line U2OS were plated in cell culture dishes (6-well cell culture for quantitative RT-PCR). 1 ⁇ 10 5 per well of a plate for ELISA assay, 1.5-2.0 ⁇ 10 5 per well of a 6-well cell culture plate for ELISA assay), and cultured overnight in a CO 2 incubator under normal oxygen concentration. Then, after culturing for 48 hours in a CO 2 incubator set to the oxygen concentration shown in FIG. 37, the cell culture medium was harvested and an ELISA assay for SPINK1 was performed.
  • a quantitative PCR experiment against ACTB as an internal standard was also performed at the same time.
  • FIG. 37 shows the intracellular SPINK1 mRNA level (upper) and the SPINK1 protein level in the cell culture medium (lower).
  • SPINK1 mRNA expression level and protein secretion level were significantly increased at 0.1% oxygen concentration compared to 20% oxygen concentration. From this, it was confirmed that the amount of SPINK1 and its secretion increased in a hypoxic stimulus-dependent manner.
  • a correlation was also observed between the amount of SPINK1 mRNA and the amount of protein secreted.
  • the test example confirmed that the protein was secreted in a hypoxic environment.
  • Test example 3 3-1 Test procedure Human cervical cancer-derived cell line HeLa was subcutaneously transplanted into the right thigh of tumor-bearing mice. After that, the transplanted tumor was excised when the time shown in FIG. 38 had elapsed. Total RNA was collected from the explanted tumor using Sepasol RNA I Super G (manufactured by Nacalai Tesque, Inc.), and subjected to reverse transcription reaction and quantitative PCR experiment for SPINK1. A quantitative PCR experiment against ACTB as an internal standard was also performed at the same time. At the same time, total protein was extracted from transplanted tumors and an ELISA assay against SPINK1 was performed.
  • Test example 4 4-1 Test procedure Pimonidazole was administered to tumor-bearing mice prepared by transplanting human cervical cancer-derived cell line HeLa, and 60 minutes later, the transplanted tumor was excised. After fixing this with 10% neutral buffered formalin, it was embedded in paraffin to prepare a tumor section. Immunohistochemical staining of the paraffin sections with anti-pimonidazole antibody (mouse monoclonal antibody; Hypoxyprobe, Inc, Catalog #HP2-1000) and anti-SPINK1 antibody (rabbit monoclonal antibody; EPR12696(2), Abcam, Catalog # ab183034). Used for experiments. After staining with each antibody, Hoechst33342 was used to stain the nucleus.
  • an anemic stimulus hyperoxic stimulus
  • SPINK1 mRNA expression in tumor cells decreased. It was confirmed that not only did SPINK1 protein increase in plasma, but also SPINK1 protein increased. From this, it was confirmed that SPINK1 is also useful as, for example, a blood marker.
  • Test example 5 5-1 Test procedure Human cervical cancer-derived cell line HeLa and human prostate cancer-derived cell line DU145 were transfected with a SPINK1-myc tag fusion protein expression vector or its empty vector, and cultured under normoxic conditions for 48 hours. After that, the cell culture medium and cell extract were collected, and Western blotting was performed using an anti-myc tag antibody and an anti- ⁇ -actin antibody. Also, 48 hours after the gene transfer, the cells were irradiated with gamma rays of 0, 2, 4, and 6 Gy under normal oxygen conditions, and the cells were then cultured for 14 days to perform a colony formation test.
  • Test example 6 6-1 Test procedure Human prostate cancer-derived cell line DU145 was cultured in normal oxygen (20%) conditions in serum-free medium for 24 hours, followed by normal medium (containing 5% serum) containing recombinant SPINK1 protein at a final concentration of 100 ng/mL. ), or replaced with a normal medium containing no recombinant SPINK1 protein, and cultured for 24 hours under normal oxygen conditions. The cells were then irradiated with 0, 2, 4 and 6 Gy of gamma rays, and the cells were cultured for 14 days before a colony formation test was performed.
  • Test example 7 7-1 Test procedure SPINK1 expression vector (pCDH/SPINK1) and its empty vector (pCDH-EF1-MCS-IRES-Puro) were transfected into HEK293TN cells to prepare SPINK1-expressing lentivirus and empty lentivirus for negative control. SPINK1 stably expressing cells (DU145/SPINK1) and negative control cells (DU145/EV) were prepared by infecting this with human prostate cancer-derived cell line DU145 and culturing in the presence of puromycin.
  • Tumor-bearing mice were prepared by subcutaneously transplanting these cells into the lower right thigh of immunodeficient mice (BALB/c nu/nu).
  • the transplanted tumor was locally irradiated with 0 or 10 Gy of gamma rays, and the changes in the volume of the transplanted tumor after that were measured.
  • Test example 8 8-1 Test procedure Human prostate cancer-derived cell line DU145 was seeded in 96-well plates for cell culture (4 ⁇ 10 2 per well for subsequent irradiation dose of 0 Gy, 1 well for 4 Gy) 1.2 ⁇ 10 3 per cell) and cultured overnight in a CO 2 incubator under normal oxygen concentration. They were then cultured in serum-free medium for an additional 24 h, followed by 24 h under normoxic conditions in the presence or absence of purified recombinant SPINK1 protein and in the presence or absence of anti-SPINK1 antibody. .
  • the cells were irradiated with 0 Gy or 4 Gy of gamma rays, cultured under normal oxygen conditions for 3 days, and viable cells were quantified using Cell Count Reagent SF (manufactured by Nacalai Tesque, Catalog #07553-44).
  • Test example 9 9-1 Study procedure From January 2016 to December 2019, among the cases diagnosed with pancreatic cancer at Kyoto University Hospital, preoperative patient plasma was available for 21 surgical cases. Using plasma as a sample, SPINK1 concentration was measured by ELISA, and the correlation with overall survival rate was evaluated. In addition, this test was conducted after confirming the intention of the patient, etc. by sufficiently explaining and giving informed consent in accordance with the ethical guidelines.
  • the 3-year survival rate in the high-value group was 52.5% (95% confidence interval: 15.0-80.4%), and the 3-year survival rate in the low-value group was 100% (NA-NA).
  • SPINK1 can be used as an index to predict the aggravation of pathological conditions and the prognosis thereof.
  • SPINK1 is useful as a biomarker for hypoxia in a subject and exacerbation of pathological conditions.

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