JP2022064961A - Measurement method of biomarker for refractory nocturia/nighttime polyuria, and screening method of preventive or ameliorating agent for refractory nocturia and nocturia - Google Patents
Measurement method of biomarker for refractory nocturia/nighttime polyuria, and screening method of preventive or ameliorating agent for refractory nocturia and nocturia Download PDFInfo
- Publication number
- JP2022064961A JP2022064961A JP2022012337A JP2022012337A JP2022064961A JP 2022064961 A JP2022064961 A JP 2022064961A JP 2022012337 A JP2022012337 A JP 2022012337A JP 2022012337 A JP2022012337 A JP 2022012337A JP 2022064961 A JP2022064961 A JP 2022064961A
- Authority
- JP
- Japan
- Prior art keywords
- nocturia
- refractory
- plasma sample
- measuring
- administration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 206010029446 nocturia Diseases 0.000 title claims abstract description 103
- 238000000034 method Methods 0.000 title claims abstract description 35
- 239000000090 biomarker Substances 0.000 title claims abstract description 15
- 230000003449 preventive effect Effects 0.000 title claims abstract description 15
- 238000012216 screening Methods 0.000 title claims abstract description 10
- 239000003795 chemical substances by application Substances 0.000 title claims description 20
- 208000004880 Polyuria Diseases 0.000 title abstract description 6
- 238000000691 measurement method Methods 0.000 title abstract description 5
- 150000001875 compounds Chemical class 0.000 claims abstract description 61
- 238000012360 testing method Methods 0.000 claims description 23
- 238000005259 measurement Methods 0.000 claims description 19
- 239000000126 substance Substances 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 7
- 239000008280 blood Substances 0.000 abstract description 13
- 210000004369 blood Anatomy 0.000 abstract description 13
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 abstract description 12
- 238000011282 treatment Methods 0.000 abstract description 11
- 229950007031 palmidrol Drugs 0.000 abstract description 9
- HXYVTAGFYLMHSO-UHFFFAOYSA-N palmitoyl ethanolamide Chemical compound CCCCCCCCCCCCCCCC(=O)NCCO HXYVTAGFYLMHSO-UHFFFAOYSA-N 0.000 abstract description 9
- 239000005639 Lauric acid Substances 0.000 abstract description 6
- HNICUWMFWZBIFP-IRQZEAMPSA-N 13(S)-HODE Chemical compound CCCCC[C@H](O)\C=C\C=C/CCCCCCCC(O)=O HNICUWMFWZBIFP-IRQZEAMPSA-N 0.000 abstract description 3
- MAVUOXXAMDWBPX-UHFFFAOYSA-N 20-hydroxydocosa-2,4,6,8,10,12-hexaenoic acid Chemical compound CCC(O)CCCCCCC=CC=CC=CC=CC=CC=CC(O)=O MAVUOXXAMDWBPX-UHFFFAOYSA-N 0.000 abstract description 3
- VKJATMNWGJZHLR-UHFFFAOYSA-N 4-hydroxydocosa-2,4,6,8,10,12-hexaenoic acid Chemical compound CCCCCCCCCC=CC=CC=CC=CC=C(O)C=CC(O)=O VKJATMNWGJZHLR-UHFFFAOYSA-N 0.000 abstract description 3
- CUHJQOVOXFVMSQ-UHFFFAOYSA-N 9-hydroxyoctadeca-2,4-dienoic acid Chemical compound CCCCCCCCCC(O)CCCC=CC=CC(O)=O CUHJQOVOXFVMSQ-UHFFFAOYSA-N 0.000 abstract description 2
- 229940126585 therapeutic drug Drugs 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 36
- 239000002207 metabolite Substances 0.000 description 18
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- 238000005516 engineering process Methods 0.000 description 12
- 230000027939 micturition Effects 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 8
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- 210000002700 urine Anatomy 0.000 description 6
- 239000000203 mixture Substances 0.000 description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 150000001450 anions Chemical class 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 206010013990 dysuria Diseases 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 235000019253 formic acid Nutrition 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 238000007689 inspection Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 206010071289 Lower urinary tract symptoms Diseases 0.000 description 2
- 206010036018 Pollakiuria Diseases 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 230000001143 conditioned effect Effects 0.000 description 2
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000002651 drug therapy Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000005350 fused silica glass Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000000422 nocturnal effect Effects 0.000 description 2
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000010414 supernatant solution Substances 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 230000002485 urinary effect Effects 0.000 description 2
- 206010004446 Benign prostatic hyperplasia Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 206010017076 Fracture Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010020853 Hypertonic bladder Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000009722 Overactive Urinary Bladder Diseases 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 208000004403 Prostatic Hyperplasia Diseases 0.000 description 1
- 235000001630 Pyrus pyrifolia var culta Nutrition 0.000 description 1
- 240000002609 Pyrus pyrifolia var. culta Species 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 208000026723 Urinary tract disease Diseases 0.000 description 1
- 206010062225 Urinary tract pain Diseases 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 238000013542 behavioral therapy Methods 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229940109239 creatinine Drugs 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000002436 femur neck Anatomy 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 238000007477 logistic regression Methods 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 238000000491 multivariate analysis Methods 0.000 description 1
- 229940126701 oral medication Drugs 0.000 description 1
- 208000020629 overactive bladder Diseases 0.000 description 1
- 208000026455 prostate symptom Diseases 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000003860 sleep quality Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 208000022934 urinary frequency Diseases 0.000 description 1
- 208000014001 urinary system disease Diseases 0.000 description 1
- 230000036318 urination frequency Effects 0.000 description 1
Images
Abstract
Description
本発明は、難治性夜間頻尿・夜間多尿症バイオマーカーの測定方法及び難治性夜間頻尿・夜間多尿症の予防剤又は治療剤のスクリーニング方法に関する。 The present invention relates to a method for measuring a biomarker for refractory nocturia / nocturia and a method for screening a preventive or therapeutic agent for refractory nocturia / nocturia.
夜間頻尿とは、「夜間、排尿のために1回以上起きなければならない」という訴えであり、夜間多尿とは24時間の尿量のうち、夜間（通常患者が就寝中である8時間）の割合が多い場合をいう。本邦において、夜間頻尿・夜間多尿を有する患者は加齢とともに増加し、頻度の高い下部尿路症状の一つである。日本人男性において、夜間頻尿の罹患率割合は50歳以上で61.8%、70歳以上では90%以上と極めて高いことが非特許文献1において報告されている。QOL(Quality Of Life)への影響も大きく、睡眠の質の低下や昼間の倦怠感の出現、社会生活への影響が生じ、高齢者においては夜間頻尿が転倒発生の要因となり、大腿骨頚部骨折のリスクを増加させる。 Nocturia is a complaint that "you must wake up at least once to urinate at night", and nocturia is nighttime (usually 8 hours when the patient is sleeping) out of the 24-hour urine volume. ) Is high. In Japan, patients with nocturia and nocturia increase with age and are one of the most frequent lower urinary tract symptoms. It is reported in Non-Patent Document 1 that the prevalence of nocturia among Japanese men is extremely high, 61.8% for those aged 50 and over and 90% or more for those aged 70 and over. It also has a large effect on QOL (Quality Of Life), resulting in deterioration of sleep quality, appearance of daytime malaise, and impact on social life. In elderly people, nocturia causes falls, and the femoral neck Increases the risk of fractures.
夜間頻尿・夜間多尿の診断には、排尿日誌が広く用いられている。患者は、排尿日誌に、排尿時の時間・１回排尿量・尿漏れの有無・１回飲水量等を、３～７日間記載することが求められる。しかし、排尿日誌は、昼夜を問わず２４時間記入が必要であり、記入そのもの自体に手間を要し、更には記入漏れ・ミス等が起こることがあり、全ての排尿に関して記録を要するという心理的負荷が生じるため、患者への負担が大きく、また、記入者の主観が入るため信憑性や客観性も十分とはいえないのが現状である。 The urination diary is widely used for the diagnosis of nocturia and nocturia. Patients are required to record the time of urination, the amount of urination once, the presence or absence of urine leakage, the amount of water consumed once, etc. for 3 to 7 days in the urination diary. However, the urination diary needs to be filled in 24 hours a day, day and night, and the entry itself takes time and effort, and there may be omissions and mistakes, so it is psychological that all urination needs to be recorded. Since a load is generated, the burden on the patient is heavy, and the credibility and objectivity are not sufficient because the subjectivity of the writer is included.
特許文献1には、排尿障害のバイオマーカーとして尿中のある種の化合物を用いることが記載されている。しかしながら、尿中の代謝物質であり測定ごとにクレアチニン補正をしなければいけないという課題がある。またさらには、それらは過活動膀胱または頻尿を呈する排尿障害を検査・検出することはできるが、現時点では治療方法や内服薬も限られておりかつ各種治療に反応の有無の判別や治療抵抗性を鑑別することができない夜間頻尿・夜間多尿という排尿障害を検査・検出することはできないという課題もある。 Patent Document 1 describes the use of certain compounds in urine as biomarkers for dysuria. However, it is a metabolite in urine, and there is a problem that creatinine must be corrected for each measurement. Furthermore, although they can test and detect dysuria with overactive bladder or pollakiuria, treatment methods and oral medications are limited at this time, and the presence or absence of response to various treatments and treatment resistance are limited. There is also the problem that it is not possible to test and detect dysuria such as nocturia and nocturia.
以上のような課題を解決するため、本発明による夜間頻尿又は夜間多尿症の検査用バイオマーカーは、下記式（1）～（6）で表される化合物からなる群より選択される少なくとも1種の化合物を含むことを特徴とする。 In order to solve the above problems, the biomarker for testing nocturia or nocturia according to the present invention is at least selected from the group consisting of the compounds represented by the following formulas (1) to (6). It is characterized by containing one compound.
また、本発明による夜間頻尿又は夜間多尿症の検査方法は、血液中の、下記式（1）～（6）で表される化合物からなる群より選択される少なくとも1種の化合物の量を指標とすることを特徴とする。 Further, in the method for testing nocturia or nocturia according to the present invention, the amount of at least one compound selected from the group consisting of the compounds represented by the following formulas (1) to (6) in blood. Is a feature.
また、本発明による夜間頻尿又は夜間多尿症の予防又は改善剤のスクリーニング方法は、血液中の、下記式（1）～（6）で表される化合物からなる群より選択される少なくとも1種の化合物の量を指標とすることを特徴とする。 Further, the screening method for a preventive or ameliorating agent for nocturia or nocturia according to the present invention is at least one selected from the group consisting of compounds represented by the following formulas (1) to (6) in blood. It is characterized by using the amount of the compound of the species as an index.
本発明のバイオマーカーは、下記式（１）～（６）で表される化合物である。
下記式（１）で表される化合物は、パルミトイルエタノールアミド(PEA)である。
下記式（２）で表される化合物は、9-ヒドロキシオクタデカジエン酸(9-HODE)である。
下記式（３）で表される化合物は、13-ヒドロキシオクタデカジエン酸(13-HODE)である。
下記式（４）で表される化合物は、4-ヒドロキシドコサヘキサエン酸(4-HDoHE)である。
下記式（５）で表される化合物は、20-ヒドロキシドコサヘキサエン酸(20-HDoHE)である。
下記式（６）で表される化合物は、ラウリン酸(Lauric acid)である。
The biomarker of the present invention is a compound represented by the following formulas (1) to (6).
The compound represented by the following formula (1) is palmitoylethanolamide (PEA).
The compound represented by the following formula (2) is 9-hydroxyoctadecadienoic acid (9-HODE).
The compound represented by the following formula (3) is 13-hydroxyoctadecadienoic acid (13-HODE).
The compound represented by the following formula (4) is 4-hydroxydocosahexaenoic acid (4-HDoHE).
The compound represented by the following formula (5) is 20-hydroxydocosahexaenoic acid (20-HDoHE).
The compound represented by the following formula (6) is lauric acid.
前記式（１）～（６）で表される化合物は各々、難治性夜間頻尿・夜間多尿の検査・検出用バイオマーカーとして用いる事ができる。これらの化合物を１種単独で用いることもでき、複数種を適宜組み合わせて使用してもよい。本発明のバイオマーカーは、生体から採取された血漿中の前記式（１）～（６）で表される化合物からなる群より選択される少なくとも1種の化合物を含む。 Each of the compounds represented by the formulas (1) to (6) can be used as a biomarker for testing / detecting intractable nocturia / nocturia. These compounds may be used alone or in combination of two or more as appropriate. The biomarker of the present invention contains at least one compound selected from the group consisting of the compounds represented by the above formulas (1) to (6) in plasma collected from a living body.
前記式（１）～（６）で表される化合物は各々、難治性夜間頻尿・夜間多尿の検査・検出に用いることができる。これらを用いることで、薬物治療や行動療法などの夜間頻尿・夜間多尿に対する一般的な治療効果の有無をある程度予測することが見込まれる。本発明による難治性夜間頻尿・夜間多尿の検査方法は、生体から採取された血漿中の前記式（１）～（６）で表される化合物からなる群より選択される少なくとも1種の化合物の量を指標とする。 The compounds represented by the formulas (1) to (6) can be used for the inspection and detection of intractable nocturia and nocturia, respectively. By using these, it is expected to predict to some extent the presence or absence of general therapeutic effects on nocturia and nocturia such as drug treatment and behavioral therapy. The method for testing intractable nocturia / nocturia according to the present invention is at least one selected from the group consisting of the compounds represented by the above formulas (1) to (6) in plasma collected from a living body. The amount of compound is used as an index.
本発明による難治性夜間頻尿・夜間多尿の検査方法は、次の工程を含むことが望ましい。
（ａ）被験体から血漿サンプルを採取する工程
（ｂ）採取した血漿サンプルから前記式（１）～（６）で表される化合物の量を測定する工程
（ｃ）測定した前記式（１）～（６）で表される化合物の量に基づいて、難治性夜間頻尿・夜間多尿であるかどうかを判定する工程
It is desirable that the method for inspecting refractory nocturia / nocturia according to the present invention includes the following steps.
(A) Step of collecting a plasma sample from a subject (b) Step of measuring the amount of a compound represented by the formulas (1) to (6) from the collected plasma sample (c) The measured formula (1) Step to determine whether or not refractory nocturia / nocturia is based on the amount of the compound represented by (6).
工程（ａ）における被験体から血漿サンプルの採取は以下のように行う。 Plasma samples are collected from the subject in step (a) as follows.
対象となる被験体から血液サンプルを採取し、例えば通常の方法で血液の遠心分離を行い、血漿成分のみを抽出し、血漿サンプルとする。 A blood sample is collected from the target subject, for example, blood is centrifuged by a usual method, and only plasma components are extracted to obtain a plasma sample.
本発明の測定方法の対象、すなわち工程（ａ）の被験体は、特に限定されず、ヒト及び他の哺乳動物、サル、チンパンジー、牛、豚、犬、猫、ラットやマウスなどが挙げられる。 The target of the measuring method of the present invention, that is, the subject of the step (a) is not particularly limited, and examples thereof include humans and other mammals, monkeys, chimpanzees, cows, pigs, dogs, cats, rats and mice.
工程（ｂ）における化合物の量の測定は以下のように行う。 The amount of the compound in the step (b) is measured as follows.
前記式（１）～（５）で表される化合物は水溶性代謝物であり、例えば以下に示す方法により抽出できる。まず血漿サンプル50μＬに内部標準入りのメタノールを450μＬ加えて撹拌後、さらにクロロホルム500μＬと純水200μＬを加えて撹拌する。撹拌後、4℃、2,300×ｇで5分間遠心分離し、分離した水相400μＬを分画分子量5ｋＤａの限外ろ過フィルターを用いて除タンパクする。ろ液を遠心乾固した後、純水25μＬで再溶解する。 The compounds represented by the formulas (1) to (5) are water-soluble metabolites and can be extracted by, for example, the methods shown below. First, 450 μL of methanol containing an internal standard is added to 50 μL of a plasma sample and stirred, and then 500 μL of chloroform and 200 μL of pure water are further added and stirred. After stirring, the mixture is centrifuged at 2300 × g at 4 ° C. for 5 minutes, and 400 μL of the separated aqueous phase is deproteinized using an ultrafiltration filter having a molecular weight cut off of 5 kDa. After centrifuging the filtrate, redissolve in 25 μL of pure water.
このようにして得られた抽出物を、キャピラリー電気泳動-飛行時間型質量分析計 (CE-TOFMS) を用いて、血漿中の水溶性代謝物を測定する。装置としては、例えばAgilent
CE-TOFMS system (Agilent Technologies 社)を用い、キャピラリーとしては、例えばFused silica capillary
i.d. 50 μm × 80 cm (ヒューマン・メタボローム・テクノロジーズ社)を用いることができる。
The extract thus obtained is measured for water-soluble metabolites in plasma using a capillary electrophoresis-time-of-flight mass spectrometer (CE-TOFMS). As a device, for example, Agilent
The CE-TOFMS system (Agilent Technologies) is used, and the capillary is, for example, Fused silica capillary.
id 50 μm × 80 cm (Human Metabolome Technologies) can be used.
この場合の測定条件は、例えば、
Run buffer：Anion
Buffer Solution (ヒューマン・メタボローム・テクノロジーズ社p/n : I3302-1023)
Rinse buffer：Anion
Buffer Solution (ヒューマン・メタボローム・テクノロジーズ社p/n : I3302-1023)
Sample injection：Pressure
injection 50 mbar,
25 sec
CE voltage：Positive,
30 kV
MS ionization：ESI
Negative
MS capillary voltage：3,500 V
MS scan range：m/z
50-1,000
Sheath liquid：HMT
Sheath Liquid (ヒューマン・メタボローム・テクノロジーズ社p/n : H3301-1020)
とすることができる。
The measurement conditions in this case are, for example,
Run buffer: Anion
Buffer Solution (Human Metabolome Technologies, Inc. p / n: I3302-1023)
Rinse buffer: Anion
Buffer Solution (Human Metabolome Technologies, Inc. p / n: I3302-1023)
Sample injection: Pressure
injection 50 mbar,
25 sec
CE voltage: Positive,
30 kV
MS ionization: ESI
Negative
MS capillary voltage: 3,500 V
MS scan range: m / z
50-1,000
Sheath liquid: HMT
Sheath Liquid (Human Metabolome Technologies, Inc. p / n: H3301-1020)
Can be.
また前記式（６）で表される化合物は脂溶性代謝物であり、例えば以下に示す方法により抽出できる。まず血漿サンプル200μLに内部標準入りのメタノールを800μL加え攪拌後、4℃で1時間のメタノール抽出を行う。メタノール抽出後、4℃、3,000 rpmで10分間遠心
分離して上清を回収する。この上清溶液にpH 3.0以下の塩酸5 mLを加え、メタノール4.5mLおよび純水4.5mLでコンディショニング済みの固相抽出用カートリッジにアプライする。カートリッジに純水4.5mLおよびヘキサン3mLで洗浄を行い、メタノール500μLで溶出する。溶出液を窒素乾固した後、50%アセトニトリル50μLで再溶解する。
The compound represented by the formula (6) is a fat-soluble metabolite and can be extracted by, for example, the method shown below. First, 800 μL of methanol containing an internal standard is added to 200 μL of a plasma sample, and the mixture is stirred and then extracted with methanol at 4 ° C. for 1 hour. After extraction with methanol, centrifuge at 4 ° C and 3,000 rpm for 10 minutes to collect the supernatant. Add 5 mL of hydrochloric acid with a pH of 3.0 or less to this supernatant solution, and apply to a solid-phase extraction cartridge that has been conditioned with 4.5 mL of methanol and 4.5 mL of pure water. Wash the cartridge with 4.5 mL of pure water and 3 mL of hexane and elute with 500 μL of methanol. The eluate is dried to nitrogen and then redissolved in 50 μL of 50% acetonitrile.
このようにして得られた抽出物を、液体クロマトグラフィー -三連四重極型質量分析計 (LC-MS) を用いて、血漿中の脂溶性代謝物測定する。装置としては、例えばAgilent
LC-MS system (Agilent Technologies 社)を用い、カラムとしては、例えばColumn 2.6μm C8 100A 150×2.1mm (Kinetex社)を用いることができる。
The extract thus obtained is measured for lipophilic metabolites in plasma using a liquid chromatography-triple quadrupole mass spectrometer (LC-MS). As a device, for example, Agilent
An LC-MS system (Agilent Technologies) can be used, and as a column, for example, a Column 2.6 μm C8 100A 150 × 2.1 mm (Kinetex) can be used.
この場合の測定条件は、例えば、
Mobile phase A：アセトニトリル (0.1% ギ酸)
Mobile phase B：純水
(0.1% ギ酸)
Sample injection：5μL
Flow rate：0.4mL/min
Column temp：40℃
MS ionization：AJS
ESI Negative, Positive
Scan type：MRM
とすることができる。
The measurement conditions in this case are, for example,
Mobile phase A: Acetonitrile (0.1% formic acid)
Mobile phase B: Pure water
(0.1% formic acid)
Sample injection: 5 μL
Flow rate: 0.4mL / min
Column temp: 40 ℃
MS ionization: AJS
ESI Negative, Positive
Scan type: MRM
Can be.
工程（ｃ）における難治性夜間頻尿・夜間多尿であるかどうかの判定は以下のように行う。 Judgment as to whether or not intractable nocturia / nocturia in step (c) is performed as follows.
判定対象の被験体から測定した上記代謝物である化合物の測定結果を、健常な被験体から測定した上記代謝物である化合物の測定結果を標準値として比較し、標準値よりも高いほど、難治性夜間頻尿・夜間多尿の程度が高いと判定する。 The measurement results of the compound, which is the metabolite, measured from the subject to be determined are compared with the measurement results of the compound, which is the metabolite, measured from a healthy subject, and the higher the standard value, the more intractable. Sex Nocturia ・ Judge that the degree of nocturia is high.
また、式（１）～（６）で表される化合物は各々、難治性夜間頻尿・夜間多尿の予防剤又は改善剤のスクリーニングに用いることができる。 In addition, the compounds represented by the formulas (1) to (6) can be used for screening for a preventive agent or an ameliorating agent for refractory nocturia and nocturia, respectively.
本発明による難治性夜間頻尿・夜間多尿の予防剤又は改善剤のスクリーニング方法は、次の工程を含むことが望ましい。
（ｄ）被験物質投与前の被験体から投与前血漿サンプルを採取する工程
（ｅ）投与前血漿サンプルから式（１）～（６）で表される化合物の量を測定する工程
（ｆ）被験体に被験物質を投与する工程
（ｇ）被験物質を投与後の被験体から投与後血漿サンプルを採取する工程
（ｈ）投与後血漿サンプルから式（１）～（６）で表される化合物の量を測定する工程
（ｉ）投与前血漿サンプルからと投与後血漿サンプルからの式（１）～（６）で表される化合物の量の測定結果に基づいて夜間頻尿又は夜間多尿症の予防剤又は改善剤として有用な被験物質を選択する工程
It is desirable that the screening method for a preventive agent or an ameliorating agent for refractory nocturia / nocturia according to the present invention includes the following steps.
(D) Step of collecting a pre-administration plasma sample from a subject before administration of the test substance
(E) Step of measuring the amount of the compound represented by the formulas (1) to (6) from the pre-administration plasma sample (f) Step of administering the test substance to the subject (g) The subject after administration of the test substance Step of collecting plasma sample after administration (h) Step of measuring the amount of the compound represented by the formulas (1) to (6) from the plasma sample after administration (i) From the plasma sample before administration and from the plasma sample after administration Step of selecting a test substance useful as a preventive agent or ameliorating agent for nocturnal plasma or nocturnal plasma disease based on the measurement result of the amount of the compound represented by the formulas (1) to (6).
工程（ｄ）では、被験物質投与前の被験体から投与前血漿サンプルを採取する。方法は上述した測定方法の工程（ａ）と同様に行う。 In step (d), a pre-administration plasma sample is collected from the subject before administration of the test substance. The method is the same as the step (a) of the measurement method described above.
工程（ｅ）では、投与前血漿サンプルから式（１）～（６）で表される化合物の量を測定する。方法は上述した測定方法の工程（ｂ）と同様に行う。 In the step (e), the amount of the compound represented by the formulas (1) to (6) is measured from the pre-administration plasma sample. The method is the same as in step (b) of the measurement method described above.
工程（ｆ）では、被験体に被験物質を投与する。被験物質は、夜間頻尿又は夜間多尿症の予防剤又は改善剤となる可能性のある物質であり、例えば、各種化合物や食品素材、食品素材から抽出された物質等である。 In step (f), the test substance is administered to the subject. The test substance is a substance that may be a preventive agent or an ameliorating agent for nocturia or nocturia, and is, for example, various compounds, food materials, substances extracted from food materials, and the like.
工程（ｇ）では、被験物質を投与後の被験体から投与後血漿サンプルを採取する。方法は上述した検査方法の工程（ａ）と同様に行う。 In step (g), a post-administration plasma sample is taken from the subject after administration of the test substance. The method is the same as the step (a) of the inspection method described above.
工程（ｈ）では、投与後血漿サンプルから式（１）～（６）で表される化合物の量を測定する。方法は上述した測定方法の工程（ｂ）と同様に行う。 In the step (h), the amount of the compound represented by the formulas (1) to (6) is measured from the plasma sample after administration. The method is the same as in step (b) of the measurement method described above.
工程（ｉ）では、投与前血漿サンプルからと投与後血漿サンプルからの式（１）～（６）で表される化合物の量の測定結果に基づいて夜間頻尿又は夜間多尿症の予防剤又は改善剤として有用な被験物質を選択する。具体的には、被験物質投与後の式（１）～（６）で表される化合物の測定値と、被験物質投与前の式（１）～（６）で表される化合物の測定値とを比較して、被験物質投与後の式（１）～（６）で表される化合物の量の変化を確認する。その結果、投与後の式（１）～（６）のいずれかで表される化合物の測定値が投与前も測定値よりも高い場合に、投与した被験物質を予防剤又は改善剤として選択する。 In step (i), a preventive agent for nocturia or nocturia based on the measurement results of the amounts of the compounds represented by the formulas (1) to (6) from the pre-administration plasma sample and the post-administration plasma sample. Alternatively, a test substance useful as an improving agent is selected. Specifically, the measured values of the compounds represented by the formulas (1) to (6) after the administration of the test substance and the measured values of the compounds represented by the formulas (1) to (6) before the administration of the test substance. To confirm the change in the amount of the compound represented by the formulas (1) to (6) after the administration of the test substance. As a result, when the measured value of the compound represented by any of the formulas (1) to (6) after administration is higher than the measured value before and after administration, the administered test substance is selected as a preventive agent or an improving agent. ..
また、式（１）～（６）で表される化合物は各々、難治性夜間頻尿・夜間多尿の治療効果判定にも用いることができる。この場合、上述のスクリーニング方法と同様に行うことができる。具体的には、以下の工程により治療判定を行うことができる。
（ｊ）治療前の被験体から投与前血漿サンプルを採取する工程
（ｋ）治療前血漿サンプルから式（１）～（６）で表される化合物の量を測定する工程
（ｌ）被験体に治療を施す工程
（ｍ）治療後の被験体から投与後血漿サンプルを採取する工程
（ｎ）治療後血漿サンプルから式（１）～（６）で表される化合物の量を測定する工程
（ｏ）治療前血漿サンプルからと治療後血漿サンプルからの式（１）～（６）で表される化合物の量の測定結果に基づいて夜間頻尿又は夜間多尿症の治療効果を判定する工程
In addition, the compounds represented by the formulas (1) to (6) can also be used for determining the therapeutic effect of intractable nocturia and nocturia, respectively. In this case, the same screening method as described above can be performed. Specifically, the treatment can be determined by the following steps.
(J) Step of collecting a pre-dose plasma sample from a pre-treatment subject
(K) Step of measuring the amount of the compound represented by the formulas (1) to (6) from the pretreatment plasma sample (l) Step of treating the subject (m) Post-administration plasma sample from the subject after treatment Step (n) Step of measuring the amount of the compound represented by the formulas (1) to (6) from the post-treatment plasma sample (o) Formula (1) from the pre-treatment plasma sample and the post-treatment plasma sample Step to determine the therapeutic effect of nocturia or nocturia based on the measurement result of the amount of the compound represented by (6)
以下、本発明を実施例に基づきさらに詳細に説明するが、本発明はこれに限定される
ものではない。
Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited thereto.
1. 被験者の選定と血液サンプルの採取
65歳以上80歳未満の男性の中で、山梨大学医学部付属病院泌尿器科通院中の方を対象にして平均夜間排尿回数が1.5回未満の方を健常人群、1.5回以上の方を夜間頻尿患者群として組み入れた。（組み入れ基準の詳細は、後程表１として示す。）特に、夜間頻尿群に組み入れられた患者は、薬物療法等各種様々な夜間頻尿・夜間多尿に対する治療を行ったが、奏功していない難治性夜間頻尿・夜間多尿を呈する方々である。被験者から、当日朝から絶食していただき、午前中に採血を採取した。採取した血液はEDTA入り試験管に採取し、0℃・3000rpm・10分で遠心した後に血漿成分のみ-80℃凍結保存した。
1. Selection of subjects and collection of blood samples
Among men aged 65 to under 80, those who are visiting the Department of Urology, University of Yamanashi Hospital, who have an average nocturia of less than 1.5 times are in the healthy group, and those who are 1.5 or more are nocturia. Enrolled as a patient group. (Details of inclusion criteria are shown in Table 1 later.) In particular, patients enrolled in the nocturia group were treated for various nocturia and nocturia such as drug therapy, but they were successful. Those who present no refractory nocturia / nocturia. The subjects fasted from the morning of the day, and blood was collected in the morning. The collected blood was collected in a test tube containing EDTA, centrifuged at 0 ° C, 3000 rpm, and 10 minutes, and then plasma components were cryopreserved at -80 ° C.
2. 被験者に対する排尿日誌および国際前立腺症状スコア（IPSS；International Prostate Symptom Score）の実施
サンプル採取日までに、3日間連続で被験者は排尿日誌に、排尿時間と排尿量を記録した。さらに、IPSSを実施した。IPSSは世界共通で使われている排尿障害に関する問診票であり、被験者が記載した。（男性下部尿路症状・前立腺肥大症診療ガイドライン 2017年）
2. Implementation of micturition diary and International Prostate Symptom Score (IPSS) for subjects By the date of sampling, subjects recorded micturition time and micturition volume in the micturition diary for 3 consecutive days. In addition, IPSS was carried out. IPSS is a questionnaire about dysuria that is used universally and was written by the subject. (Male lower urinary tract symptoms / benign prostatic hyperplasia clinical practice guidelines 2017)
各群組み入れ基準を表１に示す。 The inclusion criteria for each group are shown in Table 1.
両群において、共通除外基準を以下に列挙する。
・尿路上皮悪性腫瘍と診断された者
・下部尿路痛症候群を有する者
・24時間尿量が40mL/kg以上の多尿患者
・心疾患・肝疾患・腎障害など重篤な合併症を有する患者
・その他、医師の判断により対象として不適当と判断された患者
The common exclusionary criteria for both groups are listed below.
・ Persons diagnosed with urinary epithelial malignant tumor ・ Persons with lower urinary tract pain syndrome ・ Patients with polyurine with a 24-hour urine volume of 40 mL / kg or more ・ Serious complications such as heart disease, liver disease, renal disorder Patients who have urine or other patients who are judged to be inappropriate as a target by the judgment of the doctor
これらの結果からは、夜間頻尿患者群は夜間2回トイレのために起床する必要がある、いわゆる夜間頻尿・夜間多尿を呈する患者でありその中でも各種治療に抵抗性の難治性夜間頻尿・夜間多尿群であり、一方、健常人群は夜間排尿回数が1回未満でありかつIPSSが8点未満と、下部尿路障害があったとしても軽度である患者群にあたると推測された。 From these results, the nocturia patient group is a patient with so-called nocturia / nocturia who needs to wake up twice at night because of the toilet, and among them, refractory nocturia refractory to various treatments. It is a group of urinary and nocturia, while the healthy group has less than 1 nocturia and an IPSS of less than 8 points, which is presumed to be a group of patients with mild lower urinary tract disorders. ..
3．バイオマーカーの測定
3-1 水溶性代謝物質の測定
3-1-1 血液からの代謝物質の抽出
血漿サンプル50μLに内部標準入りのメタノールを450μL加え攪拌後、さらにクロロホルム500μL、純水200μLを加えよく攪拌した。攪拌後、4℃、2,300 × gで5分間遠心分離し、分離した水相400μLを分画分子量5 kDaの限外ろ過フィルターを用いて除タンパクした。ろ液を遠心乾固した後、純水25μLで再溶解し、分析に供した。
3. 3. Biomarker measurement
3-1 Measurement of water-soluble metabolites
3-1-1 Extraction of metabolites from blood 450 μL of methanol containing an internal standard was added to 50 μL of plasma sample and stirred, and then 500 μL of chloroform and 200 μL of pure water were added and stirred well. After stirring, the mixture was centrifuged at 2300 × g at 4 ° C. for 5 minutes, and 400 μL of the separated aqueous phase was deproteinized using an ultrafiltration filter having a molecular weight cut off of 5 kDa. The filtrate was centrifuged to dryness, redissolved in 25 μL of pure water, and subjected to analysis.
3-1-2 キャピラリー電気泳動-飛行時間型質量分析計 (CE-TOFMS) による血漿中の水溶性代謝物測定
CE-TOFMSを用いて、夜間頻尿患者および健常人の血漿サンプル中の代謝物質を網羅的に測定した。
3-1-2 Capillary Electrophoresis-Measurement of Water-Soluble Metabolites in Plasma by Time-of-Flight Mass Spectrometer (CE-TOFMS)
Using CE-TOFMS, metabolites in plasma samples of nocturia patients and healthy subjects were comprehensively measured.
3-1-3 陰イオン性代謝物質測定条件・装置に関しては以下の通り
Agilent CE-TOFMS system (Agilent
Technologies 社)
Capillary：Fused
silica capillary i.d. 50 μm × 80 cm (ヒューマン・メタボローム・テクノロジーズ社)
測定条件
Run buffer：Anion
Buffer Solution (ヒューマン・メタボローム・テクノロジーズ社p/n : I3302-1023)
Rinse buffer：Anion
Buffer Solution (ヒューマン・メタボローム・テクノロジーズ社p/n : I3302-1023)
Sample injection：Pressure
injection 50 mbar, 25 sec
CE voltage：Positive,
30 kV
MS ionization：ESI
Negative
MS capillary voltage：3,500 V
MS scan range：m/z
50-1,000
Sheath liquid：HMT
Sheath Liquid (ヒューマン・メタボローム・テクノロジーズ社p/n : H3301-1020)
3-1-3 The measurement conditions and equipment for anionic metabolites are as follows.
Agilent CE-TOFMS system (Agilent)
Technologies)
Capillary: Fused
silica capillary id 50 μm x 80 cm (Human Metabolome Technologies)
Measurement condition
Run buffer: Anion
Buffer Solution (Human Metabolome Technologies, Inc. p / n: I3302-1023)
Rinse buffer: Anion
Buffer Solution (Human Metabolome Technologies, Inc. p / n: I3302-1023)
Sample injection: Pressure
injection 50 mbar, 25 sec
CE voltage: Positive,
30 kV
MS ionization: ESI
Negative
MS capillary voltage: 3,500 V
MS scan range: m / z
50-1,000
Sheath liquid: HMT
Sheath Liquid (Human Metabolome Technologies, Inc. p / n: H3301-1020)
3-2 脂溶性代謝物質の測定
3-2-1 血液からの代謝物質の抽出
血漿サンプル200μLに内部標準入りのメタノールを800μL加え攪拌後、4℃で1時間のメタノール抽出を行った。メタノール抽出後、4℃、3,000 rpmで10分間遠心分離して上清を回収した。この上清溶液にpH 3.0以下の塩酸5 mLを加え、メタノール4.5mLおよび純水4.5mLでコンディショニング済みの固相抽出用カートリッジにアプライした。カートリッジに純水4.5mLおよびヘキサン3mLで洗浄を行い、メタノール500μLで溶出した。溶出液を窒素乾固した後、50%アセトニトリル50μLで再溶解し、分析に供した。
3-2 Measurement of fat-soluble metabolites
3-2-1 Extraction of metabolites from blood 800 μL of methanol containing an internal standard was added to 200 μL of plasma sample, and the mixture was stirred and then extracted with methanol at 4 ° C for 1 hour. After extraction with methanol, the supernatant was collected by centrifugation at 4 ° C. and 3,000 rpm for 10 minutes. To this supernatant solution, 5 mL of hydrochloric acid having a pH of 3.0 or less was added, and the mixture was applied to a solid-phase extraction cartridge conditioned with 4.5 mL of methanol and 4.5 mL of pure water. The cartridge was washed with 4.5 mL of pure water and 3 mL of hexane and eluted with 500 μL of methanol. The eluate was dried to dryness with nitrogen, then redissolved in 50 μL of 50% acetonitrile and subjected to analysis.
3-2-2 液体クロマトグラフィー -三連四重極型質量分析計 (LC-MS) による血漿中の脂溶性代謝物測定
LC-MSを用いて、夜間頻尿患者および健常人の血漿サンプル中の代謝物質を網羅的に測定した。
3-2-3 脂溶性代謝物質測定条件・装置に関しては以下の通り
Agilent LC-MS system (Agilent Technologies
社)
Column 2.6μm C8 100A 150×2.1mm (Kinetex社)
測定条件
Mobile phase A：アセトニトリル (0.1% ギ酸)
Mobile phase B：純水
(0.1% ギ酸)
Sample injection：5μL
Flow rate：0.4mL/min
Column temp：40℃
MS ionization：AJS
ESI Negative, Positive
Scan type：MRM
3-2-2 Liquid Chromatography-Measurement of fat-soluble metabolites in plasma by triple quadrupole mass spectrometer (LC-MS)
Using LC-MS, metabolites in plasma samples of nocturia patients and healthy subjects were comprehensively measured.
3-2-3 The measurement conditions and equipment for fat-soluble metabolites are as follows.
Agilent LC-MS system (Agilent Technologies)
Company)
Column 2.6 μm C8 100A 150 × 2.1 mm (Kinetex)
Measurement condition
Mobile phase A: Acetonitrile (0.1% formic acid)
Mobile phase B: Pure water
(0.1% formic acid)
Sample injection: 5 μL
Flow rate: 0.4mL / min
Column temp: 40 ℃
MS ionization: AJS
ESI Negative, Positive
Scan type: MRM
上記3-1、2で測定した化合物中の中で、健常人群と比較して夜間頻尿患者群で有意差があるとして検出された測定結果を表２に示す。 Table 2 shows the measurement results detected as having a significant difference between the nocturia patient group and the nocturia group among the compounds measured in 3 and 2 above.
次に、表２の血中の代謝物質の中で、排尿日誌の各パラメーターの中で夜間頻尿/夜間多尿の臨床症状の内、最も核となる『夜間排尿回数（夜間入眠中にトイレに起きる回数）』と相関をもつ代謝物質を検討した結果を、以下に示す[表３]。 Next, among the metabolites in the blood of Table 2, among the clinical symptoms of nocturia / nocturia among the parameters of the urination diary, the most core "number of nocturia (toilet during nighttime sleep)" The results of examining metabolites that correlate with urinary frequency) ”are shown below [Table 3].
表３に示すように、前記式（１）～（６）で表される化合物の血中濃度は、健常人群と夜間頻尿群との間で有意差が認められた。さらには、前記式（１）～（６）で表される化合物の血中濃度は『夜間排尿回数』と相関をもつことが明らかになった。 As shown in Table 3, the blood concentrations of the compounds represented by the formulas (1) to (6) were significantly different between the healthy subject group and the nocturia group. Furthermore, it was clarified that the blood concentrations of the compounds represented by the formulas (1) to (6) have a correlation with the "number of nighttime urination".
4 バイオマーカー解析
更に検討を進め、夜間頻尿/夜間多尿患者と健常人の鑑別能を上げるため、多変量解析手法の一つである多重ロジスティック回帰分析を行い、6種の疾患関連因子候補の中から2種を組み合わせたモデルを作成した。解析にはJMP ver 12.0.0 (SAS Institute社) を使用した。
4 Biomarker analysis In order to further study and improve the ability to distinguish between nocturia / nocturia patients and healthy subjects, multiple logistic regression analysis, which is one of the multivariate analysis methods, was performed, and 6 disease-related factor candidates were performed. I created a model that combines two types from among them. JMP ver 12.0.0 (SAS Institute) was used for the analysis.
2個のマーカー候補を組み合わせた結果を図1に示す。図1は化合物としてパルミトイルエタノールアミド(PEA)とラウリン酸(Lauric acid)、さらにそれらを組合せたモデル(MLR)の、真陽性感度と偽陽性率(1-特異度)をプロットした図である。それぞれの(the area under the
receiver operating characteristic curves, AUC)は、
PEA：AUC=0.78942
Lauric acid：AUC=0.73228
MLR(PEA+Lauric acid）：AUC=0.84550
であり、2個のマーカー候補を組み合わせた結果、鑑別能が向上し、夜間頻尿/夜間多尿患者と健常人を高精度に分類することが可能であった。
Figure 1 shows the result of combining two marker candidates. FIG. 1 is a plot of true positive sensitivity and false positive rate (1-specificity) of palmitoylethanolamide (PEA) and lauric acid as compounds, and a model (MLR) combining them. Each (the area under the
receiver operating characteristic curves, AUC)
PEA: AUC = 0.78942
Lauric acid: AUC = 0.73228
MLR (PEA + Lauric acid): AUC = 0.84550
As a result of combining the two marker candidates, the discrimination ability was improved, and it was possible to classify nocturia / nocturia patients and healthy subjects with high accuracy.
これらの結果から、前記式（１）～（６）で表される化合物は、夜間頻尿・夜間多尿の患者の中でも、特に薬物療法等に難治性の夜間頻尿・夜間多尿のバイオマーカーとして有用であることが確認された。 From these results, the compounds represented by the above formulas (1) to (6) are biologics of nocturia and nocturia, which are particularly refractory to drug therapy among patients with nocturia and polyuria. It was confirmed to be useful as a marker.
本発明による難治性夜間頻尿・夜間多尿症バイオマーカーの測定方法及び難治性夜間頻尿・夜間多尿症の予防剤又は改善剤のスクリーニング方法は、難治性夜間頻尿・夜間多尿症の検査やそれらの予防剤又は改善剤の開発に用いることで、精度の良い、安定した方法を提供することができる。 The method for measuring the refractory nocturia / nocturia biomarker and the screening method for the preventive or improving agent for refractory nocturia / nocturia according to the present invention are refractory nocturia / nocturia. It is possible to provide an accurate and stable method by using it for the inspection of the above and the development of the preventive agent or the improver thereof.
Claims (4)
前記血漿サンプルから下記式（1）～（6）で表される化合物からなる群より選択される少なくとも1種の化合物の量を測定する工程と、
測定した前記１種の化合物の量と健常人群の前記１種の化合物の標準値とを比較する工程と、
を備えることを特徴とする夜間頻尿又は夜間多尿症のバイオマーカーの測定方法。
The process of preparing a plasma sample derived from the subject and
A step of measuring the amount of at least one compound selected from the group consisting of the compounds represented by the following formulas (1) to (6) from the plasma sample, and
A step of comparing the measured amount of the one compound with the standard value of the one compound in the healthy human group, and
A method for measuring a biomarker of nocturia or nocturia, which comprises.
前記投与前血漿サンプル中の、下記式（1）～（6）で表される化合物からなる群より選択される少なくとも1種の化合物の量を請求項１～３の測定方法で測定する工程と、
前記被験物質投与後の前記被検体に由来する投与後血漿サンプルを用意する工程と、
前記投与後血漿サンプル中の、下記式（1）～（6）で表される化合物からなる群より選択される前記少なくとも1種の化合物の量を請求項１～３の測定方法で測定する工程と、
前記投与前血漿サンプルからと前記投与後血漿サンプルからの前記化合物の量の測定結果に基づいて夜間頻尿又は夜間多尿症の予防剤又は改善剤として有用な被験物質を選択する工程と、
を備えたことを特徴とする夜間頻尿又は夜間多尿症の予防剤又は改善剤のスクリーニング方法。
The step of preparing a pre-administration plasma sample derived from the subject before administration of the test substance, and
The step of measuring the amount of at least one compound selected from the group consisting of the compounds represented by the following formulas (1) to (6) in the pre-administration plasma sample by the measuring method of claims 1 to 3. ,
A step of preparing a post-administration plasma sample derived from the subject after administration of the test substance, and
The step of measuring the amount of the at least one compound selected from the group consisting of the compounds represented by the following formulas (1) to (6) in the post-administration plasma sample by the measuring method of claims 1 to 3. When,
A step of selecting a test substance useful as a preventive agent or ameliorating agent for nocturia or nocturia based on the measurement results of the amount of the compound from the pre-administration plasma sample and the post-administration plasma sample.
A method for screening a preventive agent or an ameliorating agent for nocturia or nocturia.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2022012337A JP7294709B2 (en) | 2017-09-29 | 2022-01-28 | Method for measuring refractory nocturia/nocturnal polyuria biomarker and screening method for preventive or improving agent for refractory nocturia/nocturia |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2017190024A JP7031104B2 (en) | 2017-09-29 | 2017-09-29 | Refractory nocturia / nocturia biomarker, test method using this, and screening method for preventive or ameliorating agent for refractory nocturia / nocturia |
| JP2022012337A JP7294709B2 (en) | 2017-09-29 | 2022-01-28 | Method for measuring refractory nocturia/nocturnal polyuria biomarker and screening method for preventive or improving agent for refractory nocturia/nocturia |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2017190024A Division JP7031104B2 (en) | 2017-09-29 | 2017-09-29 | Refractory nocturia / nocturia biomarker, test method using this, and screening method for preventive or ameliorating agent for refractory nocturia / nocturia |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2022064961A true JP2022064961A (en) | 2022-04-26 |
| JP7294709B2 JP7294709B2 (en) | 2023-06-20 |
Family
ID=86772689
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2022012337A Active JP7294709B2 (en) | 2017-09-29 | 2022-01-28 | Method for measuring refractory nocturia/nocturnal polyuria biomarker and screening method for preventive or improving agent for refractory nocturia/nocturia |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP7294709B2 (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090012173A1 (en) * | 2002-09-13 | 2009-01-08 | Sankyo Company, Limited | Method for treatment of nocturia |
| JP2013147430A (en) * | 2010-04-28 | 2013-08-01 | Astellas Pharma Inc | Prophylactic or therapeutic agent for night urination |
| JP2015049050A (en) * | 2013-08-29 | 2015-03-16 | 花王株式会社 | Dysuria biomarker |
-
2022
- 2022-01-28 JP JP2022012337A patent/JP7294709B2/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090012173A1 (en) * | 2002-09-13 | 2009-01-08 | Sankyo Company, Limited | Method for treatment of nocturia |
| JP2013147430A (en) * | 2010-04-28 | 2013-08-01 | Astellas Pharma Inc | Prophylactic or therapeutic agent for night urination |
| JP2015049050A (en) * | 2013-08-29 | 2015-03-16 | 花王株式会社 | Dysuria biomarker |
Non-Patent Citations (11)
| Title |
|---|
| KIRA SATORU: "Urinary metabolites identified using metabolomic analysis as potential biomarkers of nocturia in eld", WORLD JOURNAL OF UROLOGY, vol. 38, no. 10, JPN6021033391, 2020, pages 2563 - 2569, XP037251838, ISSN: 0005061614, DOI: 10.1007/s00345-019-03042-9 * |
| PETTER HEDLUND: "Cannabinoids and the endocannabinoid system in lower urinary tract function and dysfunction", NEUROUROL URODYN, vol. 33, no. 1, JPN6021033386, January 2014 (2014-01-01), pages 46 - 53, XP093021278, ISSN: 0005061618, DOI: 10.1002/nau.22442 * |
| SATORU KIRA: "Liquid chromatography-mass spectrometry identification of serum biomarkers for nocturia in aged men", WORLD J UROL, vol. 37, no. 10, JPN6021033384, 23 January 2019 (2019-01-23), pages 2199 - 2205, XP036893354, ISSN: 0005061620, DOI: 10.1007/s00345-019-02647-4 * |
| SATORU KIRA: "Metabolomics analysis of blood identifies potential biomarkers and possible treatment targets for no", THER ADV UROL, vol. 11, JPN6021033385, 17 May 2019 (2019-05-17), pages 1 - 7, ISSN: 0005061619 * |
| V. KIRAN VEMURI: "Pharmacotherapeutic targeting of the endocannabinoid signaling system: Drugs for obesity and the met", PHYSIOL BEHAV, vol. Vol.93 No.4-5, JPN6021033383, 21 November 2007 (2007-11-21), pages 671 - 686, ISSN: 0005061621 * |
| 井原達矢: "脂肪酸代謝産物のPalmitoylethanolamide(PEA)は膀胱上皮のGPCR55を介し排尿反射を誘発し夜間頻尿の原因とな", 日本排尿機能学会誌, vol. 31, no. 1, JPN6021033394, 2020, pages 275, ISSN: 0005061612 * |
| 井原達矢: "血中脂肪酸濃度の概日リズム障害と夜間頻尿の関連性,創薬の可能性", 月刊メディカル・サイエンス・ダイジェスト, vol. 47, no. 8, JPN6021033388, 25 July 2021 (2021-07-25), pages 435 - 438, ISSN: 0005061616 * |
| 今村直樹: "夜間頻尿に対する危険因子とQuality Of Life(QOL)障害についての検討", 日本排尿機能学会誌, vol. 22, no. 1, JPN6021033387, 2011, pages 170, ISSN: 0005061617 * |
| 吉良聡: "Liquid chromatography-mass spectrometry identification of serum biomarkers for nocturia in aged men", 排尿障害プラクティス, vol. 29, no. 1, JPN6021049255, 30 June 2021 (2021-06-30), pages 79 - 81, ISSN: 0005061622 * |
| 吉良聡: "夜間頻尿における新規標的分子の網羅的探索:尿を用いたメタボロミクス解析の検討", 日本排尿機能学会誌, vol. 29, no. 1, JPN6021033392, 2018, pages 198, ISSN: 0005061613 * |
| 武田 正之, 複雑系ネットワークと動的恒常性破綻による下部尿路機能障害発症機序の研究, JPN6021033389, ISSN: 0005061615 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JP7294709B2 (en) | 2023-06-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108990420B (en) | Liver disease-associated biomarkers and methods of use thereof | |
| Roland et al. | Low ileocecal valve pressure is significantly associated with small intestinal bacterial overgrowth (SIBO) | |
| Zurlo et al. | High serum uric acid levels increase the risk of metabolic syndrome in elderly women: The PRO. VA study | |
| JP2007024822A (en) | Discriminating method of male menopause or depression | |
| Ellefsen et al. | Quantification of ***e and metabolites in exhaled breath by liquid chromatography-high-resolution mass spectrometry following controlled administration of intravenous ***e | |
| Konieczna et al. | Optimization of LC method for the determination of testosterone and epitestosterone in urine samples in view of biomedical studies and anti-doping research studies | |
| Bandin et al. | Urinary proteome analysis at 5-year followup of patients with nonoperated ureteropelvic junction obstruction suggests ongoing kidney remodeling | |
| JP5713887B2 (en) | Method for detecting muscle degenerative disease and method for determining therapeutic effect | |
| Siebers et al. | Absorption, distribution, metabolism, and excretion of [14 C]-volixibat in healthy men: Phase 1 open-label study | |
| Zhou et al. | Non-invasive urinary metabolomics reveals metabolic profiling of polycystic ovary syndrome and its subtypes | |
| JP7031104B2 (en) | Refractory nocturia / nocturia biomarker, test method using this, and screening method for preventive or ameliorating agent for refractory nocturia / nocturia | |
| Gadgil et al. | Associations of NAFLD with circulating ceramides and impaired glycemia | |
| CN117368476B (en) | Application of detection reagent for seven metabolic markers in preparation of breast cancer diagnosis and prognosis products | |
| JP2022064961A (en) | Measurement method of biomarker for refractory nocturia/nighttime polyuria, and screening method of preventive or ameliorating agent for refractory nocturia and nocturia | |
| Lahousse et al. | ERS International Congress, Madrid, 2019: highlights from the Airway Diseases, Asthma and COPD Assembly | |
| Arafat et al. | Determination of loperamide in human plasma and saliva by liquid chromatography–tandem mass spectrometry | |
| Zuo et al. | Pharmacokinetics and tissue distribution study of prucalopride in rats by ultra high performance liquid chromatography with tandem mass spectrometry | |
| WO2022091793A1 (en) | Development of pancreatic cancer biomarker using feces-derived protein | |
| JP5069804B2 (en) | A method for determining the degree of progression in patients with amyotrophic lateral sclerosis (ALS) and monitoring the progression | |
| Zhang | Simultaneous time-course measurements of metoprolol and α-hydroxyl metoprolol in fingermarks after oral administration by liquid chromatography tandem mass spectrometry | |
| WO2021162030A1 (en) | Metabolite diagnostic biomarker for drug-induced interstitial pneumonia | |
| JP4464298B2 (en) | Method for diagnosing heart disease by measuring urinary N2, N2-dimethylguanosine concentration | |
| Alexandraki et al. | The diagnosis of Cushing's disease | |
| Dickens et al. | Dysregulated lipid metabolism precedes onset of psychosis in people at clinical high risk | |
| CN117030874A (en) | Application of fecal short-chain fatty acid as primary tremor diagnosis marker |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20220224 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20230207 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20230315 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20230518 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20230601 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7294709 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |