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WO2008001840A1 - Procédé de diagnostic d'une maladie rénale/urologique - Google Patents

Procédé de diagnostic d'une maladie rénale/urologique Download PDF

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Publication number
WO2008001840A1
WO2008001840A1 PCT/JP2007/062982 JP2007062982W WO2008001840A1 WO 2008001840 A1 WO2008001840 A1 WO 2008001840A1 JP 2007062982 W JP2007062982 W JP 2007062982W WO 2008001840 A1 WO2008001840 A1 WO 2008001840A1
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WO
WIPO (PCT)
Prior art keywords
mca
acetyl
phe
protease activity
arg
Prior art date
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PCT/JP2007/062982
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English (en)
Japanese (ja)
Inventor
Takahisa Imamura
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Kumamoto University NUC
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Kumamoto University NUC
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Publication date
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Priority to JP2008522619A priority Critical patent/JP5481662B2/ja
Publication of WO2008001840A1 publication Critical patent/WO2008001840A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders

Definitions

  • the present invention relates to a diagnostic method and a diagnostic kit for a kidney'urological disease. More specifically, the present invention relates to a method for diagnosing a renal urinary disease by measuring protease activity in urine, and a diagnostic kit for performing the above method.
  • the kidney filters about 1700 liters of blood per day to produce 1 liter of urine.
  • waste is excreted, water and salt concentration are adjusted, plasma is maintained at an appropriate pH, and physiologically necessary substances such as erythrocytes are produced and supplied to maintain the body.
  • the kidney is said to be a silent organ.Similar to the liver, the disease develops and progresses beyond the reserve capacity. ing. Since kidney status is significantly reflected in urinary components, tests for urine protein content, urine creatine concentration, hematuria, etc. have been conducted as screening for kidney disease, but all of these have low specificity with the disease. It is not enough to detect the initial stage.
  • the present invention aims to provide a diagnostic method and a diagnostic kit for a kidney 'urological disease, characterized by high specificity with the kidney' urological disease and less invasiveness to the patient. It was.
  • a real-time machine that is directly linked to the etiology of kidney and urinary disease and is detected even in the early stages of the disease. If it is possible to discover everything, it is possible to provide a diagnostic method and a diagnostic kit for kidney's urinary disease characterized by high specificity with kidney's urinary disease and less invasion to patients. Become. Proteases, which are proteolytic enzymes, are involved in protein metabolism in cells and secreted to perform various functions outside the cells.
  • protease activity in urine may change depending on the protease.
  • Protease can measure various activities by changing the amino acid sequence used as a substrate, and can detect it with high sensitivity by incorporating a fluorescent substance into the substrate. Therefore, the present inventors paid attention to various proteolytic enzyme (protease) activities appearing in urine and V that do not invade the patient at the time of specimen collection, and comprehensively detect them using various substrates.
  • proteolytic enzyme proteolytic enzyme
  • a method for diagnosing renal urological disease comprising measuring urinary protease activity using a plurality of substrates and analyzing a pattern of protease activity for the substrates.
  • Kidney 'The urological disease is rapidly progressive glomerulonephritis, nephrotic syndrome, IgA nephropathy, chronic glomerulonephritis, chronic renal failure, prostate cancer, or bladder cancer (1) to (4 ).
  • Urinary protease activity against acetyl-Phe-Arg-MCA is lower than that in healthy subjects, and urine protease activity against acetyl-Phe-Lys-MCA in healthy subjects And the urinary protease activity against acetyl-Asn-Phe-MCA, acetyl-Tyr-Arg-MCA and acetyl-His-Ala-MCA is equivalent to that in healthy subjects
  • MCA represents 4-methylcoumaryl-7-amide group
  • Protease activity in urine against acetyl-Phe-Arg-MCA is lower than that in healthy subjects, and in urine against acetyl-Tyr-Arg-MCA and acetyl-Phe-Lys-MCA Indices that the protease activity of urine is higher than that of healthy subjects and that urinary protease activity against acetyl-Asn-Phe-MCA and acetyl-His-Ala-MCA is equivalent to that of healthy subjects
  • MCA represents 4-methylcoumaryl-7-amide group
  • Urinary protease activity against acetyl-Asn-Phe-MCA and acetyl-Phe-Arg-MCA is lower than that in healthy subjects, and acetyl-Tyr-Arg-MCA and The urinary protease activity for chill-Phe-Lys-MCA is higher than that for healthy subjects, and the urinary protease activity for acetyl-His-Ala-MCA is equivalent to that for healthy subjects.
  • Protease activity in urine against acetyl-Asn-Phe-MCA is lower than that in healthy subjects, and in urine against acetyl-Tyr-Arg-MCA and acetyl-Phe-Lys-MCA Protease activity is higher than that in healthy subjects, and acetyl-Phe-Arg- Chronic glomerulonephritis is determined based on the fact that the urinary protease activity against MCA and acetyl-His-Ala-MCA is the same as that in healthy subjects (where MCA is 4-methylcoumaryl-7 A method for diagnosing chronic glomerulonephritis.
  • the urinary protease activity against acetyl-Phe-Arg-MCA is lower than that in healthy subjects, and the urinary protease activity against acetyl-His-Ala-MCA in healthy subjects Indices that urinary protease activity against acetyl-Asn-Phe-MCA, acetyl-Tyr-Arg-MCA and acetyl-Phe-Lys-MCA is equivalent to that in healthy subjects
  • MCA represents 4-methylcoumaryl-7-amide group
  • the protease activity in urine against acetyl-Tyr-Arg-MCA is higher than that in healthy subjects, and acetyl-Asn-Phe-MCA, acetyl-Phe-Arg-MCA, It is determined that the cancer is bladder cancer using as an index the protease activity in urine against chill-Phe-Lys-MCA and acetyl-His-Ala-MCA (in the formula, MCA is 4- A methylcoumaryl-7-amide group), a method for diagnosing bladder cancer.
  • the diagnostic method of the present invention can diagnose a kidney's urinary disease quickly without any invasiveness to a patient.
  • the method for diagnosing renal urinary disease is a method characterized by measuring urinary protease activity. Specifically, urinary protease activity is measured by measuring the protease activity in urine using a plurality of substrates, and analyzing the pattern of protease activity for the substrates. Protease activity can be measured by measuring protease activity (hydrolysis activity) on a peptide substrate composed of two amino acids.
  • acetylyl X 1 — X 2 — Y (wherein X 1 and X 2 each independently represent an amino acid residue, and Y emits fluorescence when the bond with X 2 is cleaved)
  • the protease activity in urine can be measured by measuring the protease activity against the substrate indicated by
  • the amino acid residue X 1 shows, can be mentioned 19 kinds of amino acid residues ( ⁇ amino acid residue except Cys) of Trp from Gly.
  • Specific examples of the amino acid residue represented by X 2 include five types of amino acid residues, Lys, Phe, Val, Ala, and Arg.
  • Y represents a functional group that becomes a fluorescent compound when the bond with X 2 is cleaved.
  • functional groups include MCA (4-methylcoumaryum 7-amide) group, CMCA (4-chloromethyl coumaryi-7-amide), FMCA (4-trifluoromethylcoumary 7-amide), 110 ⁇ gun 110 groups. Or a FITC group.
  • acetyl-Asn-Phe-MCA acetyl-Phe-Arg-MCA
  • acetyl-Tyr-Arg-MCA acetyl-Phe-Lys-MCA
  • acetyl-His-Ala The urinary protease activity is measured by measuring the protease activity against MCA (wherein MCA represents 4-methylcoumaryl-7-amide group).
  • kidney and urinary diseases such as
  • Measurement of urinary protease activity against acetyl-Asn-Phe-MCA, acetyl-Phe-Arg-MCA, acetyl-Tyr-Arg-MCA, acetyl-Phe-Lys-MCA, or acetyl-His-Ala-MCA For example, it can be performed as follows. Dissolve the above five kinds of substrates in dimethyl sulfoxide, and add a solution (1 mM) diluted with 0.1M Tris-HCl, pH7.6, 150 mM NaCl to a 96-well plate.
  • the kidney's urinary disease was found to be rapidly progressive glomerulonephritis, nephrotic syndrome, IgA nephropathy, Kidney'urological diseases such as chronic glomerulonephritis, chronic renal failure, prostate cancer, or bladder cancer can be diagnosed.
  • the diagnostic indicators are as described above in this specification and as shown in FIG.
  • urinary proteases for acetyl-Asn-Phe-MCA, acetyl-Phe-Arg-MCA, acetyl-Tyr-Arg-MCA, acetyl-Phe-Lys-MCA, and acetyl-His-Ala-MCA For example, when Asn-Phe is 86 or less, Phe-Arg is 103 or less, Ph e-Lys is 12 or more, Ding 1- ⁇ is 8.6 or more, and His-Ala is 7.4 or more (unit is cpu). / mg creatinine) can be determined to be an abnormal value.
  • acetyl-Asn-Phe-MCA acetyl-Phe-Arg-MCA
  • acetyl-Tyr-Arg-MCA acetyl-Phe-Lys-MCA
  • acetyl-His-Ala A diagnostic kit for kidney 'urinary diseases is provided, which contains -MCA (wherein MCA represents 4-methylcoumaryl-7-amide group).
  • the diagnostic kit of the present invention may further contain a solvent, a buffer solution, a measurement plate and the like as appropriate.
  • Urine was obtained from healthy individuals, nephritis patients, and urinary cancer patients, and the urine was used approximately 2 hours after the previous urination. 9 rapidly progressive glomerulonephritis, nephrotic syndrome 1
  • Urine was measured in 0 patients, IgA nephropathy 26 patients, chronic glomerulonephritis 12 patients, chronic renal failure 22 patients, prostate cancer 21 patients, bladder cancer 8 patients, and healthy volunteers 10 patients.
  • MCA is converted to AMC (7-amino-4-methylco) by protease hydrolysis between CAs. This was measured at an excitation wavelength of 355 nm and a detection wavelength of 450 nm. Five amino acids, Lys, Phe, Val, Ala, Arg, are selected for X, and Gly to Trp
  • a total of 95 types of substrates in which 19 types of amino acids were arranged were dissolved in dimethylsulfoxide, and 50 1 (1 mM) diluted with 0.1 M Tris-HCl, pH 7.6, 150 mM NaCl was added to a 96-well plate.
  • Urine 50 1 was added thereto and reacted at 37 ° C, and an increase in fluorescence intensity was detected over time with a fluorescence measuring device Arvo about every minute.
  • the activity with respect to each substrate was defined as the value with the highest rate of increase of 5 points out of 10 points. Since activity is affected by urine concentration, it was expressed as activity against Creatun 100 mg / dl.
  • the protease activity of 10 healthy subjects was measured.
  • -X-Arg-MCA system showed the most diverse activity, and activity against AR, LR, MR, HR, FR, YR, WR, PR, etc. was detected. In particular, FR activity was high.
  • activity against HK, FK, WK, and YK was observed. The highest FK activity was observed.
  • LF and NF were active in the X-Phe-MCA system.
  • Cathebsin B showed the strongest activity, and the highest activity among all substrates. Although the remaining -X-Va ⁇ MCA and -X-Ala-MCA substrates did not detect any activity at all, VV , IV, GA, HA weak activity was observed.
  • Acety ⁇ Asn-Phe-MCA was the substrate in which cathebsin B was most hydrolyzed and had the highest activity in the urine of healthy subjects. This activity was significantly decreased in IgA nephropathy (P 0.034) and chronic glomerulonephritis (P 0.034). In these diseases, there is a possibility that the production of protease related to this activity is decreased or the degradation of consumption is increased.
  • This activity is associated with rapidly progressive glomerulonephritis (P ⁇ 0.008), nephrotic syndrome (P ⁇ 0.023), IgA nephropathy (P ⁇ 0.045), chronic kidney disease other than chronic glomerulonephritis (P 0.08).
  • Significantly low strength in renal failure (P ⁇ 0.005) Cancer showed no significant difference. In these diseases, there is a possibility that the production of protease related to this activity may be reduced or consumption / degradation may be increased.
  • AcetyKTyr-Arg-MCA activity increases in disease as opposed to the activity that decreases in disease compared to healthy subjects, such as Acety ⁇ Asn- Phe-MCA and Acety ⁇ Phe-Arg-MCA described above It is active, has the ability to increase in both power and cancer, and has a very high diagnostic value. It is also noteworthy that this activity shows contrasting variation in the disease despite its very similar structure to acety ⁇ Phe-Arg-MCA.
  • Rapid progressive glomerulonephritis (P 0.019), nephrotic syndrome (P ⁇ 0.013), IgA nephropathy (P ⁇ 0.044), chronic glomerulonephritis (P ⁇ 0.023) Although it was significantly stronger, it was not recognized that there was a significant difference in cancer. The activity is high in various types of kidney diseases except chronic renal failure and does not increase in cancer. Therefore, it is specific for kidney disease, has a wide range of applications, and has high diagnostic utility. / ⁇ .
  • Protease activity for the five types of substrates used above is divided into activity that decreases and increases depending on the disease when compared between patients and healthy subjects.
  • the former is Acety ⁇ Asn- Phe- MCA and Acety ⁇ Phe- Arg- MCA hydrolysis activity, the latter being Acety ⁇ Tyr-Arg-MCA, Acetyl-Phe-Lys-MCA, and Acety ⁇ His-Ala-MCA hydrolysis activity.
  • Fig. 7 when these activities were compared for each disease, which were low, no difference, and high, compared with those in healthy subjects, different patterns were observed in seven diseases including nephritis and two cancers. It became clear to take This result shows that differential diagnosis of these diseases may be possible by comparing the activity against the above five kinds of substrates in urine between healthy subjects and patients.
  • the five renal diseases of interest this time can be differentiated by activity against four substrates except AcetyH "Iis-Ala-MCA, and the above two cancers can also be excluded. Cancers were identified by their activity against three different substrates: Acety ⁇ Phe- Arg- MCA ⁇ Acety ⁇ Phe- Lys- MCA ⁇ Acety ⁇ His- Ala- MCA, and the possibility of excluding kidney disease was also shown. Therefore, by measuring the activity in urine using the activity against these five kinds of substrates as a reporter and performing pattern analysis by comparison with healthy subjects, non-invasive and rapid treatment of nephritis, urinary tract cancer, etc. It becomes possible to diagnose kidney 'urological diseases.
  • FIG. 1 shows the activity patterns of human thrombin, human mouth kinase and human cathebsin B.
  • FIG. 2 shows protease activity against acetyl-Asn-Phe-MCA in the urine of patients with renal / urological diseases and healthy subjects.
  • FIG. 3 shows protease activity against acetyl-Phe-Arg-MCA in the urine of renal and urological disease patients and healthy subjects.
  • FIG. 4 shows protease activity against acetyl-Tyr-Arg-MCA in the urine of patients with renal / urological diseases and healthy subjects.
  • FIG. 5 shows protease activity against acetyl-Phe-Lys-MCA in the urine of patients with renal / urological diseases and healthy subjects.
  • FIG. 6 shows protease activity against acetyl-His-Ala-MCA in the urine of renal and urological disease patients and healthy subjects.
  • FIG. 7 shows the results of a comparison of protease activity against 5 kinds of substrates between renal and urological disease patients and normal subjects.

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Abstract

L'invention concerne un procédé et une trousse de diagnostic d'une maladie rénale/urologique, hautement spécifiques d'une maladie rénale/urologique et peu invasifs pour le patient. Le procédé de diagnostic d'une maladie rénale/urologique comprend la mesure d'une activité protéase dans l'urine au moyen d'au moins deux substrats et l'analyse du profil de l'activité protéase par rapport aux substrats.
PCT/JP2007/062982 2006-06-28 2007-06-28 Procédé de diagnostic d'une maladie rénale/urologique Ceased WO2008001840A1 (fr)

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JP2008522619A JP5481662B2 (ja) 2006-06-28 2007-06-28 腎臓・泌尿器疾患の診断方法

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JP2006-177730 2006-06-28

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6117956A (ja) * 1984-07-04 1986-01-25 Nitto Boseki Co Ltd 新規な尿中カリクレイン測定用基質
JPH05168497A (ja) * 1991-12-25 1993-07-02 Wako Pure Chem Ind Ltd エステル分解酵素又は蛋白分解酵素の測定用試薬
JP2004528814A (ja) * 2000-10-13 2004-09-24 チルドレンズ メディカル センター コーポレーション 組織再構築関連状態についての非侵襲性酵素スクリーニング

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09281110A (ja) * 1996-04-12 1997-10-31 Nippon Kayaku Co Ltd 腎機能の検査方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6117956A (ja) * 1984-07-04 1986-01-25 Nitto Boseki Co Ltd 新規な尿中カリクレイン測定用基質
JPH05168497A (ja) * 1991-12-25 1993-07-02 Wako Pure Chem Ind Ltd エステル分解酵素又は蛋白分解酵素の測定用試薬
JP2004528814A (ja) * 2000-10-13 2004-09-24 チルドレンズ メディカル センター コーポレーション 組織再構築関連状態についての非侵襲性酵素スクリーニング

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KAWABATA S. ET AL.: "Highly sensitive peptide-4-methylcoumaryl-7-amide substrates for blood-clotting protease and trypsin", EUR. J. BIOCHEM., vol. 172, no. 1, 1988, pages 17 - 25, XP003019679 *
KITAMOTO Y. ET AL.: "Role of thrombin in mesangial proliferative glomerulonephritis", KIDNEY INT., vol. 54, no. 5, 1998, pages 1767 - 1768, XP003019678 *

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JPWO2008001840A1 (ja) 2009-11-26

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