JP6083647B2 - Test method using 1 amount of soluble vascular endothelial growth factor receptor as an index - Google Patents
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Description
本発明は、可溶性血管内皮増殖因子受容体１（Soluble fms-like tyrosine kinase-1：sFlt-1）量を指標とする、慢性腎臓病患者における心血管イベント発症の危険性（心血管イベント発症リスク）評価、すなわち心血管イベントの発症リスクを検出し、予知するための検査方法に関する。 The present invention relates to the risk of developing cardiovascular events in patients with chronic kidney disease using the amount of soluble vascular endothelial growth factor receptor 1 (Soluble fms-like tyrosine kinase-1: sFlt-1) as an index (risk of developing cardiovascular events) ) Evaluation, that is, a test method for detecting and predicting the risk of developing a cardiovascular event.
本出願は、参照によりここに援用されるところの日本出願特願２０１１−２１０４５２号優先権を請求する。 This application claims Japanese application Japanese Patent Application No. 2011-210452 priority used here by reference.
近年、慢性腎臓病（Chronic Kidney Disease：以下単に「CKD」ともいう。）という疾患概念が提唱され、大きな注目を浴びている。CKD患者数は、日本では約1300万人、全世界で約５億人ともいわれており、人工透析患者の急増なども背景に、慢性腎臓病の発症、悪化予防が世界的な課題となっている。CKDの初期は、タンパク尿などの尿異常のみで自覚症状がほとんどないことからも、本人も気づかないうちに徐々にステージが進行し、高血圧、貧血や電解質異常などを自覚したときには、腎機能もかなり悪化しているケースが多いのが現状である。また、初期腎機能不全でも心血管イベント発症の高リスク病態であることも明らかになっており、自覚症状のない初期の段階に心血管リスクを把握しておくことは重要である。 In recent years, the concept of a disease called chronic kidney disease (hereinafter simply referred to as “CKD”) has been advocated and has received much attention. The number of CKD patients is about 13 million in Japan, and about 500 million people worldwide. With the rapid increase in the number of patients on artificial dialysis, the onset of chronic kidney disease and prevention of exacerbations have become a global issue. Yes. In the early stages of CKD, there is almost no subjective symptom due to proteinuria and other abnormalities, so the stage gradually progresses without the person even being aware of it, and when he / she becomes aware of hypertension, anemia, electrolyte abnormalities, etc., renal function also There are many cases where the situation has deteriorated considerably. It is also clear that even early stage renal dysfunction is a high-risk condition for the development of cardiovascular events, and it is important to understand cardiovascular risk at an early stage without subjective symptoms.
心筋梗塞や脳梗塞を含む心血管疾患は先進国の主要な死因であり、国民の健康な生活を向上させるためには、心血管イベント（心血管病死、非致死性心筋梗塞を含む急性冠症候群、非致死性脳梗塞、心不全、大動脈疾患、血管形成術のための入院）の発症率を低下させる必要がある。心血管イベントの危険因子として、脂質異常、高血圧、糖尿病、喫煙、メタボリックシンドロームなどが明らかにされ、治療のターゲットとされている。しかしながら、未だ心血管イベントは充分には抑制されていない。 Cardiovascular diseases, including myocardial infarction and cerebral infarction, are the leading cause of death in developed countries. To improve the healthy life of the people, cardiovascular events (cardiovascular death, acute coronary syndromes including non-fatal myocardial infarction) Need to reduce the incidence of nonfatal cerebral infarction, heart failure, aortic disease, hospitalization for angioplasty). As risk factors for cardiovascular events, lipid abnormalities, hypertension, diabetes, smoking, metabolic syndrome, etc. have been clarified and are targeted for treatment. However, cardiovascular events are not yet sufficiently suppressed.
血管新生因子の中で中心的な役割を果たしている血管内皮増殖因子（Vascular Endothelial Growth Factor：以下、「VEGF」ともいう。）は哺乳類の発生に必要不可欠であるばかりでなく、成体における心血管機能の維持にも重要な役割を果たしている。悪性腫瘍に対する抗VEGF中和抗体は、腎臓糸球体内皮機能障害による蛋白尿や高血圧を引き起こす。つまり、全身におけるVEGFの抑制は様々な心血管機能異常を引き起こしうる。可溶性VEGF受容体１（sFlt-1）は選択的スプライシング（alternative splicing）により内皮細胞から分泌されるFlt-1の膜から外の部分で、VEGFに特異的に結合し、血管機能維持に不可欠なVEGFの強力な内因性阻害因子として働く。sFlt-1は高血圧、蛋白尿などの内皮機能低下に伴う症状を呈する子癇を伴う妊娠中毒症（preeclampsia）の病態に深くかかわっていることが開示されている（特許文献１：特許第4711972号公報）。 Vascular Endothelial Growth Factor (hereinafter referred to as “VEGF”), which plays a central role in angiogenic factors, is not only essential for mammalian development but also cardiovascular function in adults It also plays an important role in maintaining. Anti-VEGF neutralizing antibodies against malignant tumors cause proteinuria and hypertension due to renal glomerular endothelial dysfunction. In other words, VEGF suppression throughout the body can cause various cardiovascular abnormalities. Soluble VEGF receptor 1 (sFlt-1) is a part of Flt-1 that is secreted from endothelial cells by alternative splicing and binds specifically to VEGF and is essential for maintaining vascular function. Acts as a potent endogenous inhibitor of VEGF. It has been disclosed that sFlt-1 is deeply involved in the pathology of preeclampsia with eclampsia that exhibits symptoms associated with decreased endothelial function such as hypertension and proteinuria (Patent Document 1: Japanese Patent No. 4711972) ).
最近、CKDは心血管イベントの高リスク病態であることが明らかになった。しかしながら、CKDは軽度から腎不全までさまざまな重症度に分類されており、比較的軽症の患者の数が非常に多く、CKDの病名をつけることで、限られた医療資源を必要以上に消費してしまうことに議論があるのも事実である（Am J Kidney Dis 2009; 53:915-20, Kidney Int 2011; 80:17-28）。したがって、CKDの中でも特に高リスクの患者群を特定することが出来れば、極めて有用である。このCKD患者において、VEGFの内因性阻害因子であるsFlt-1レベルと内皮機能低下との相関関係が報告された（非特許文献１：Di Marco GS, et al. J Am Soc Nephrol 2009; 20: 2235-2245）。具体的には、非特許文献１の図２において、sFlt-1レベルとフラミンガムスコアの相関が報告されており、sFlt-1レベルと心筋梗塞の既往、脳梗塞の既往、又は現在の糖尿病の存在との関係が示されている。しかしながら、この結果からは、sFlt-1レベルが将来の心筋梗塞や脳梗塞発症を予知するとは言えない。なぜなら、心筋梗塞後にスタチンというLDLコレステロールを低下させる薬を投与するとsFlt-1レベルが上昇することが報告されている（J Am Coll Cardiol.2006; 48(1): 43-50.）。心筋梗塞や脳梗塞を発症した患者に脂質異常を有する者が多く、スタチンを投与される機会が多いことから、心筋梗塞や脳梗塞の既往のある患者においては、発症後にスタチンを投与された結果、sFlt-1が上昇している可能性がある。フラミンガムスコアとは、マサチューセッツ州フラミンガム地区で実施された数十年にわたるフラミンガム心臓研究（Framingham Heart Study）に基づくもので、年齢、性別、総コレステロール、HDL（善玉）コレステロール、収縮期血圧、喫煙状況および降圧薬の使用という7つのファクター（因子）から、冠動脈疾患の発症率を評価するスコアである。非特許文献１の報告は、sFlt-1レベルとフラミンガムスコアとの関係を横断研究（cross-sectional）で確認しているが、フラミンガムスコアとsFlt-1レベルとの相関係数(r)は、0.209と低いものであった。係る結果より、フラミンガムスコアとの関連において、冠動脈疾患の発症とsFlt-1レベルが直接的に関連しているとはいえない。冠動脈疾患の発症は必ずしも、より重大である、心血管イベントにはつながらない。sFlt-1レベルが将来の心血管イベントと関連するかどうかは、前向きコホート研究を行い、なおかつ、確立された危険因子である年齢、性別（男性）、脂質異常、高血圧、糖尿病、喫煙などで統計的に調整した後でもsFlt-1レベルと心血管イベントの間に有意な関連性が示されなければならない。そもそも横断研究で心血管イベントの予知マーカーを見出すことは不可能である。別の報告で、心血管系疾患のための予知的パラメーターとして、胎盤増殖因子（placental growth factor: PlGF）及びVEGF受容体-1（VEGFR-1：Flt-1）について開示がある（特許文献２：特表2008-518198号公報）。特許文献２では、PlGF濃度が高く、sFlt-1濃度が低い場合に、好ましくないイベントが発生する高い可能性を示唆している。 Recently, CKD has been shown to be a high-risk condition for cardiovascular events. However, CKD is categorized into various severities from mild to renal failure, and the number of relatively mild patients is very large, and the name of CKD disease consumes limited medical resources more than necessary. It is also true that there is a debate over what happens (Am J Kidney Dis 2009; 53: 915-20, Kidney Int 2011; 80: 17-28). Therefore, it is extremely useful if a particularly high-risk patient group can be identified among CKD. In this CKD patient, a correlation between sFlt-1 level, which is an endogenous inhibitor of VEGF, and decreased endothelial function was reported (Non-patent document 1: Di Marco GS, et al. J Am Soc Nephrol 2009; 20: 2235-2245). Specifically, in FIG. 2 of Non-Patent Document 1, the correlation between the sFlt-1 level and the Framingham score is reported, and the sFlt-1 level and the history of myocardial infarction, the history of cerebral infarction, or the presence of current diabetes The relationship is shown. However, from this result, it cannot be said that sFlt-1 level predicts future myocardial infarction or cerebral infarction. This is because it has been reported that sFlt-1 levels are elevated when a statin drug that lowers LDL cholesterol is administered after myocardial infarction (J Am Coll Cardiol. 2006; 48 (1): 43-50.). Because patients with myocardial infarction or cerebral infarction have many lipid abnormalities and there are many opportunities to administer statins, in patients with a history of myocardial infarction or cerebral infarction, the results of administration of statins after onset SFlt-1 may be elevated. The Framingham Score is based on the decades of Framingham Heart Study conducted in the Framingham area of Massachusetts, and includes age, gender, total cholesterol, HDL cholesterol, systolic blood pressure, smoking status and It is a score that evaluates the incidence of coronary artery disease from the seven factors of use of antihypertensive drugs. The report of Non-Patent Document 1 confirms the relationship between the sFlt-1 level and the Framingham score in a cross-sectional, but the correlation coefficient (r) between the Framingham score and the sFlt-1 level is It was as low as 0.209. From these results, it can be said that the onset of coronary artery disease and the sFlt-1 level are not directly related to the Framingham score. The development of coronary artery disease does not necessarily lead to a more serious cardiovascular event. Whether sFlt-1 levels are associated with future cardiovascular events is determined by a prospective cohort study and by established risk factors such as age, gender (male), dyslipidemia, hypertension, diabetes, smoking, etc. Even after adjustment, there must be a significant association between sFlt-1 levels and cardiovascular events. In the first place, it is impossible to find predictive markers for cardiovascular events in cross-sectional studies. Another report discloses placental growth factor (PlGF) and VEGF receptor-1 (VEGFR-1: Flt-1) as prognostic parameters for cardiovascular disease (Patent Document 2). : Special Table 2008-518198). Patent Document 2 suggests a high possibility that an undesirable event occurs when the PlGF concentration is high and the sFlt-1 concentration is low.
しかしながら、CKD患者における血中sFlt-1レベルと心血管イベント発症の関連は不明であった。CKD患者において特に心血管イベント発症のリスクが高い患者を予測することができれば、心血管イベント発症予防を考慮した適切な管理を施すことができ、そのような検査方法が望まれている。 However, the association between blood sFlt-1 levels and the onset of cardiovascular events in CKD patients was unclear. If a CKD patient can predict a patient who has a particularly high risk of developing a cardiovascular event, appropriate management can be performed in consideration of prevention of the occurrence of a cardiovascular event, and such an inspection method is desired.
本発明は、慢性腎臓病（CKD）患者特異的な心血管イベント発症の危険性を評価し、心血管イベント発症の高リスク群を検出し、予知するための検査方法を提供することを課題とする。 It is an object of the present invention to provide a test method for evaluating the risk of developing cardiovascular events specific to patients with chronic kidney disease (CKD), and detecting and predicting a high-risk group of cardiovascular events. To do.
本発明者らは、上記課題を解決するために鋭意研究を重ねた結果、CKD患者における血中sFlt-1量を指標とし、測定することにより、CKD患者における心血管イベント発症の危険性を評価し、心血管イベント発症の高リスク群を検出し、予知しうることを見出し、本発明を完成した。 As a result of intensive studies to solve the above problems, the present inventors evaluated the risk of developing cardiovascular events in CKD patients by measuring the amount of blood sFlt-1 in CKD patients as an index. As a result, the present inventors have found that a high risk group for the onset of cardiovascular events can be detected and predicted, and the present invention has been completed.
即ち、本発明は以下よりなる。
１．以下の工程を含む、sFlt-1量を指標とする、CKD患者における心血管イベント発症リスク評価のための検査方法：
１）CKD患者より採取された試料中のsFlt-1量をin vitroで測定する工程；
２）測定した値が90-130 pg/mLから選択されるいずれかの値をカットオフ値とし、カットオフ値以上の場合にCKD患者の心血管イベント発症リスクを検出する工程。
２．心血管イベントが、心血管病死、非致死性心筋梗塞を含む急性冠症候群、脳卒中、うっ血性心不全、大動脈疾患、冠動脈及び末梢血行再建から選択される１種又は複数種である、前項１に記載の心血管イベント発症リスク評価のための検査方法。
３．sFlt-1量の測定が、免疫学的測定による、前項１又は２記載の心血管イベント発症リスク評価のための検査方法。
４．CKD患者より採取された試料が、CKD患者より採取された血液由来試料である、前項１〜３のいずれかに記載の心血管イベント発症リスク評価のための検査方法。
５．少なくとも以下を含む、前項１〜４のいずれかに記載の検査方法に使用しうる心血管イベント発症リスク評価のための検査用キット：
１）抗sFlt-1抗体；
２）sFlt-1と抗sFlt-1抗体の複合体検出用試薬。
６．sFlt-1からなる、CKD患者における心血管イベント発症リスク検出用マーカー。That is, this invention consists of the following.
1. A test method for evaluating the risk of developing cardiovascular events in CKD patients using the amount of sFlt-1 as an index, including the following steps:
1) A step of measuring in vitro the amount of sFlt-1 in a sample collected from a CKD patient;
2) A step of detecting a risk of developing a cardiovascular event in a CKD patient when the measured value is selected from 90 to 130 pg / mL as a cut-off value and is equal to or higher than the cut-off value.
2. 2. The cardiovascular event is one or more selected from cardiovascular death, acute coronary syndrome including non-fatal myocardial infarction, stroke, congestive heart failure, aortic disease, coronary artery and peripheral revascularization, Testing method for risk assessment of cardiovascular events in children.
3. 3. The test method for evaluating the risk of developing cardiovascular events according to 1 or 2 above, wherein the amount of sFlt-1 is measured by immunological measurement.
4). 4. The test method for cardiovascular event onset risk evaluation according to any one of items 1 to 3, wherein the sample collected from the CKD patient is a blood-derived sample collected from the CKD patient.
5. A test kit for assessing the risk of developing a cardiovascular event that can be used in the test method according to any one of items 1 to 4 including at least the following:
1) anti-sFlt-1 antibody;
2) Reagent for detecting complex of sFlt-1 and anti-sFlt-1 antibody.
6). A marker for detecting the risk of cardiovascular events in CKD patients, comprising sFlt-1.
CKD患者において、本発明のsFlt-1を単独マーカーとすることで、CKD患者における血中sFlt-1レベルを測定することにより、CKD患者における心血管イベント発症の危険性を予測し、心血管イベント発症の高リスク群を検出することができる。これにより、CKD患者の内、特に重点的に管理する必要のある患者に対して早期に方針を決定することができる。 In CKD patients, the risk of developing cardiovascular events in CKD patients is predicted by measuring the blood sFlt-1 level in CKD patients by using sFlt-1 of the present invention as a single marker, and cardiovascular events High risk group of onset can be detected. As a result, it is possible to determine a policy at an early stage for CKD patients who need to be managed with particular emphasis.
本発明は、以下の工程を含むsFlt-1量を指標とする、CKD患者における心血管イベント発症の危険性を予測する、すなわち心血管イベント発症のリスク評価のための検査方法に関する。
１）CKD患者より採取された試料中のsFlt-1量をin vitroで測定する工程；
２）sFlt-1量がカットオフ値以上の場合にCKD患者の心血管イベント発症リスクを検出する工程。The present invention relates to a test method for predicting the risk of developing a cardiovascular event in a CKD patient using the amount of sFlt-1 as an index, including the following steps, that is, for assessing the risk of developing a cardiovascular event.
1) A step of measuring in vitro the amount of sFlt-1 in a sample collected from a CKD patient;
2) A step of detecting a risk of developing a cardiovascular event in a CKD patient when the amount of sFlt-1 is not less than a cutoff value.
本明細書においてCKD患者とは、腎臓機能の指標である推算糸球体濾過量(eGFR:年齢、性別、血清クレアチニン値から計算される)が60mL/分/1.73m2未満の者をいう。In this specification, a CKD patient refers to a person whose estimated glomerular filtration rate (eGFR: calculated from age, sex, and serum creatinine value), which is an index of kidney function, is less than 60 mL / min / 1.73 m 2 .
本明細書の心血管イベント発症リスク評価のための検査方法における試料として、臨床検査において通常使用しうる試料を使用することができる。例えばCKD患者より採取された血液検体を通常の方法で処理し、得られた血清、血漿などの血液由来試料を、本発明のCKD患者より採取された試料として使用することができる。試料は、本検査方法のために調製してもよいし、他の臨床検査項目において使用する試料とともに調製してもよい。 As a sample in the test method for assessing the risk of developing cardiovascular events in the present specification, a sample that can be usually used in a clinical test can be used. For example, a blood sample collected from a CKD patient can be processed by a usual method, and a blood-derived sample such as serum or plasma obtained can be used as a sample collected from the CKD patient of the present invention. The sample may be prepared for this test method or may be prepared together with a sample used in other clinical test items.
本明細書において、sFlt-1量のカットオフ値とは、90-130 pg/mLから選択されるいずれかの値とすることができ、特に好適には、110 pg/mLとすることができる。 In the present specification, the cut-off value of the amount of sFlt-1 can be any value selected from 90 to 130 pg / mL, and particularly preferably 110 pg / mL. .
本明細書において、「心血管イベント」とは、生命を脅かす重大な心臓・血管など循環器における疾患をいい、心血管病死、例えば非致死性心筋梗塞を含む急性冠症候群、脳卒中、うっ血性心不全、大動脈疾患、冠動脈及び末梢血行再建等による入院が含まれる。本発明の心血管イベント発症リスク評価では、心血管イベント発症の危険性（リスク）を予測することができる。本明細書において、「CKD患者の心血管イベント発症リスクを検出する」とは、心血管イベント発症のリスクが高いと判断される被験者を検出することをいう。 As used herein, “cardiovascular event” refers to a disease in the circulatory organ such as a serious heart or blood vessel that is life-threatening, and cardiovascular death, for example, acute coronary syndrome including non-fatal myocardial infarction, stroke, congestive heart failure And hospitalization for aortic disease, coronary artery and peripheral revascularization. In the cardiovascular event onset risk evaluation of the present invention, the risk (risk) of cardiovascular event onset can be predicted. In the present specification, “detecting the risk of developing a cardiovascular event in a CKD patient” means detecting a subject who is determined to have a high risk of developing a cardiovascular event.
本明細書において、「心血管イベント発症リスク」とは、検査を行った日以降少なくとも1080日までの間に、何らかの心血管イベント（心血管病死、非致死性心筋梗塞を含む急性冠症候群、脳卒中、うっ血性心不全、大動脈疾患、冠動脈及び末梢血行再建による入院）の発症を引き起こす可能性があるリスクを意味する。 In this specification, “cardiovascular event onset risk” means any cardiovascular event (cardiovascular death, acute coronary syndrome including non-fatal myocardial infarction, stroke) between the day of examination and at least 1080 days , Risk of congestive heart failure, aortic disease, hospitalization for coronary artery and peripheral revascularization).
本明細書において、sFlt-1量のin vitroでの測定は、sFlt-1量を定量しうる方法であればよく、自体公知の方法であっても、今後開発される新たな方法であっても特に限定されない。例えば免疫学的測定方法を好適に適用することができる。例えば、抗sFlt-1抗体を用いた抗原抗体反応を検出しうる免疫学的測定方法によることができる。そのような免疫学的手法としては、ラテックス凝集法、オクタロニー法、免疫クロマトグラフィー法やELISA法などの自体公知の手法を適用することができる。多くの検体を処理しうるELISA法が、特に好適である。具体的には、本発明の抗sFlt-1抗体を固相に固定し、該固相に固定した抗sFlt-1抗体に検体試料を接触させ、抗sFlt-1抗体と検体試料中に含有可能性のあるsFlt-1との免疫複合体、または反応産物を検出することで、sFlt-1を測定することができる。ELISA法の実施に伴う非特異反応の抑制方法や、検出の際に使用しうる標識物質、測定機器などは、自体公知のもの、あるいは今後開発されるものを適用することができる。例えば、sFlt-1検出用の市販のキットを用いて測定を行ってもよい。市販のELISA用キットとして、例えばQuantikine(R)Human Soluble VEGF R1/Flt-1 Immunoassay Catalog # DVR100B（R&Dシステムズ社）を用いることができる。In the present specification, the in vitro measurement of the amount of sFlt-1 may be any method that can quantitate the amount of sFlt-1, and is a new method that will be developed in the future even if it is a method known per se. Is not particularly limited. For example, an immunological measurement method can be suitably applied. For example, it can be performed by an immunological measurement method capable of detecting an antigen-antibody reaction using an anti-sFlt-1 antibody. As such an immunological technique, a technique known per se such as a latex agglutination method, octalony method, immunochromatography method or ELISA method can be applied. An ELISA method that can process many specimens is particularly suitable. Specifically, the anti-sFlt-1 antibody of the present invention is immobilized on a solid phase, and a specimen sample is brought into contact with the anti-sFlt-1 antibody immobilized on the solid phase, and can be contained in the anti-sFlt-1 antibody and the specimen sample SFlt-1 can be measured by detecting an immune complex with sFlt-1 or a reaction product. As a method for suppressing a non-specific reaction associated with the ELISA method, a labeling substance and a measuring instrument that can be used for detection, those known per se or those developed in the future can be applied. For example, the measurement may be performed using a commercially available kit for detecting sFlt-1. Commercially available ELISA kits, for example, Quantikine (R) Human Soluble VEGF R1 / Flt-1 Immunoassay Catalog # DVR100B (R & D Systems, Inc.) can be used.
本発明は、sFlt-1量を指標とするCKD患者における心血管イベント発症リスク評価のための検査方法に使用しうる検査用キットにも及ぶ。検査用キットには、抗sFlt-1抗体、sFlt-1と抗sFlt-1抗体の複合体検出用試薬（標識物質等）などを含めることができる。上記の他、固相、標準品、緩衝液等を含めてもよい。 The present invention also extends to a test kit that can be used in a test method for assessing the risk of developing cardiovascular events in CKD patients using the amount of sFlt-1 as an index. The test kit can contain an anti-sFlt-1 antibody, a reagent for detecting a complex of sFlt-1 and anti-sFlt-1 antibody (labeling substance, etc.), and the like. In addition to the above, a solid phase, a standard product, a buffer solution and the like may be included.
本発明はsFlt-1からなる、CKD患者における心血管イベント発症リスク評価のための検査用マーカーにも及ぶ。 The present invention also extends to a test marker for assessing the risk of developing cardiovascular events in CKD patients, comprising sFlt-1.
以下、本発明の理解を助けるために、実施例を示して具体的に説明する。 Hereinafter, in order to help understanding of the present invention, an example will be shown and described in detail.
（実施例１）カプランマイヤー法を用いた生存解析
本実施例では、独立行政法人国立病院機構京都医療センターの倫理委員会の承認を得た後、511名の患者（eGFR 15 mL/min/1.73 m2未満の腎不全患者は含まれていない）におけるCKDの有無及び血中sFlt-1量を計測し、３年間追跡した後、カプランマイヤー法により心血管イベントの生存解析を行った。採血した血液検体から通常の方法に従って、臨床検査用血清試料を調製した。sFlt-1量は、市販のELISA用キット（Quantikine(R)Human Soluble VEGF R1/Flt-1 Immunoassay Catalog # DVR100B, R&Dシステムズ社）を用いて測定した。(Example 1) Survival analysis using Kaplan-Meier method In this example, after obtaining approval from the Ethics Committee of the National Hospital Organization Kyoto Medical Center, 511 patients (eGFR 15 mL / min / 1.73 In patients with renal failure less than m 2, the presence of CKD and blood sFlt-1 levels were measured, followed for 3 years, and then the survival analysis of cardiovascular events was performed by Kaplan-Meier method. A serum sample for clinical examination was prepared from the collected blood sample according to the usual method. sFlt-1 amount was measured using a commercial ELISA kit (Quantikine (R) Human Soluble VEGF R1 / Flt-1 Immunoassay Catalog # DVR100B, R & D Systems, Inc.).
生存解析の主要評価項目（Primary Outcome）は、全死亡(心血管死亡を含む)、非致死性心筋梗塞を含む急性冠症候群、脳卒中、うっ血性心不全、大動脈疾患、冠動脈及び末梢血行再建で定義される主要有害心血管イベント（Major Adverse Cardiovascular Events：MACEs）であった。フォローアップ日数の中央値は、994日（405-1,080 IQR）であった。これらのうち、MACEsは合計59例（11.5％）に発症した。MACEsの内訳は全死亡2.5％（その内、心血管病死 1.4％）、非致死性脳梗塞 2.5％、急性冠症候群 2.3％（その内、血行再建 1.2％）、心不全 2.3％、末梢動脈イベント 1.8％と、偏りなく全般に渡るものであった。非心血管死亡を除いた場合でも結果はほとんど変わりがなかった。 Primary endpoints for survival analysis (Primary Outcome) are defined as all deaths (including cardiovascular death), acute coronary syndromes including non-fatal myocardial infarction, stroke, congestive heart failure, aortic disease, coronary and peripheral revascularization Major Adverse Cardiovascular Events (MACEs). The median follow-up time was 994 days (405-1,080 IQR). Of these, MACEs occurred in a total of 59 cases (11.5%). The breakdown of MACEs was 2.5% death (including cardiovascular death 1.4%), non-fatal cerebral infarction 2.5%, acute coronary syndrome 2.3% (of which revascularization 1.2%), heart failure 2.3%, peripheral arterial events 1.8 %, And it was general. Even when non-cardiovascular deaths were excluded, the results were almost unchanged.
ここで、sFlt-1について最適なカットオフ値を110 pg/mLとし、110 pg/mL未満をlow sFlt-1とし、110 pg/mL以上をhigh sFlt-1とした。CKDの有無については、推定糸球体濾過率（eGFR）60 mL/分/1.73m2未満のものをCKD患者と判断し、CKD(+)とした。また、CKD患者でないものをCKD(-)とした。Here, the optimal cutoff value for sFlt-1 was 110 pg / mL, less than 110 pg / mL was low sFlt-1, and 110 pg / mL or higher was high sFlt-1. With regard to the presence or absence of CKD, an estimated glomerular filtration rate (eGFR) of less than 60 mL / min / 1.73 m 2 was judged as a CKD patient and designated as CKD (+). Those who were not CKD patients were designated as CKD (-).
患者を以下の4つのグループに分類した。
（１）CKD(+) /high sFlt-1（N = 36、年齢71±8、男性：69％）
（２）CKD(+) /low sFlt-1（N = 58、年齢71±8、男性：62％）
（３）CKD(-) /high sFlt-1（N = 83、年齢66±13、男性：60％）
（４）CKD(-) /low sFlt-1（N = 334、年齢60±12、男性：45％）Patients were divided into four groups:
(1) CKD (+) / high sFlt-1 (N = 36, age 71 ± 8, male: 69%)
(2) CKD (+) / low sFlt-1 (N = 58, age 71 ± 8, male: 62%)
(3) CKD (-) / high sFlt-1 (N = 83, age 66 ± 13, male: 60%)
(4) CKD (-) / low sFlt-1 (N = 334, age 60 ± 12, male: 45%)
推定糸球体濾過率（eGFR）は、CKD(-)で（４）81±15及び（３）82±16、CKD(+)で（２）（48±9）及び（１）（48±10）であり、CKD(-)間、又はCKD(+)間でほとんど差を認めなかった。しかしながら、MACEs発症率は、（１）39％で有意に高く、その他のグループでは（２）16％、（３）15％、（４）7％であった。確立された危険因子である、年齢、性別（男性）、高血圧、糖尿病、高脂血症、肥満で調整した後、（４）に対するハザード比（95％CI）は、各々（３）1.4 (0.7-2.9)、（２）1.2 (0.5-2.8)、（１）4.7 (2.3-9.6, P<0.0001)であり、（１）のグループがMACEsの危険性が最も高いことが確認された。 Estimated glomerular filtration rate (eGFR) is (4) 81 ± 15 and (3) 82 ± 16 for CKD (-), (2) (48 ± 9) and (1) (48 ± 10) for CKD (+) ), And almost no difference was observed between CKD (−) or CKD (+). However, the incidence of MACEs was significantly higher at (1) 39%, and (2) 16%, (3) 15%, (4) 7% in the other groups. After adjusting for established risk factors: age, gender (male), hypertension, diabetes, hyperlipidemia, obesity, the hazard ratio (95% CI) to (4) is (3) 1.4 (0.7 -2.9), (2) 1.2 (0.5-2.8), (1) 4.7 (2.3-9.6, P <0.0001), and the group (1) was confirmed to have the highest risk of MACEs.
図１の結果より、CKD(+)の患者においてsFlt-1が高い場合に、他のグループに比べて有意にMACEsの危険性が高いことが確認された。また、CKD(-)の場合では、sFlt-1が高い場合でもMACEsの発症率は15％であり、CKD(+)の場合と明らかに違いが認められた。また、CKD(+)の場合でsFlt-1が低い場合でもMACEsの発症率は16％でありsFlt-1が高い場合と明らかに違いが認められた。
従って、CKD(+)の患者特異的に、sFlt-1を測定することで、MACEsの発症を予測することができると考えられた。From the results in FIG. 1, it was confirmed that when sFlt-1 is high in CKD (+) patients, the risk of MACEs is significantly higher than in other groups. In the case of CKD (-), the incidence of MACEs was 15% even when sFlt-1 was high, which was clearly different from that of CKD (+). In addition, in the case of CKD (+), even when sFlt-1 is low, the incidence of MACEs is 16%, which is clearly different from the case where sFlt-1 is high.
Therefore, it was considered that the onset of MACEs can be predicted by measuring sFlt-1 specifically for CKD (+) patients.
（実施例２）有害心血管イベント（MACEs）の危険性分析
本実施例では、独立行政法人国立病院機構京都医療センターの倫理委員会の承認を得た後、511名の患者におけるCKDの有無及び血中sFlt-1量を計測し、前向きに３年間追跡して、MACEsの危険性分析を行った。採血した血液検体から通常の方法に従って、臨床検査用血清試料を調製した。sFlt-1量は、市販のELISA用キット（Quantikine(R)Human Soluble VEGF R1/Flt-1 Immunoassay Catalog # DVR100B, R&Dシステムズ社）を用いて測定した。(Example 2) Risk analysis of adverse cardiovascular events (MACEs) In this example, after obtaining approval from the Ethics Committee of the National Hospital Organization Kyoto Medical Center, the presence or absence of CKD in 511 patients and Blood sFlt-1 levels were measured and followed prospectively for 3 years to analyze the risk of MACEs. A serum sample for clinical examination was prepared from the collected blood sample according to the usual method. sFlt-1 amount was measured using a commercial ELISA kit (Quantikine (R) Human Soluble VEGF R1 / Flt-1 Immunoassay Catalog # DVR100B, R & D Systems, Inc.).
実施例１と同様に、sFlt-1について最適なカットオフ値を110 pg/mLとし、110 pg/mL未満をlow sFlt-1とし、110 pg/mL以上をhigh sFlt-1とした。CKDの有無については、推定糸球体濾過率（eGFR）60 mL/分/1.73m2未満のものをCKD患者と判断し、CKD(+)とした。また、CKD患者でないものをCKD(-)とした。As in Example 1, the optimal cutoff value for sFlt-1 was 110 pg / mL, less than 110 pg / mL was low sFlt-1, and 110 pg / mL or higher was high sFlt-1. With regard to the presence or absence of CKD, an estimated glomerular filtration rate (eGFR) of less than 60 mL / min / 1.73 m 2 was judged as a CKD patient and designated as CKD (+). Those who were not CKD patients were designated as CKD (-).
患者を、実施例１と同様にCKD(-) /low sFlt-1（対照）、CKD(-) /high sFlt-1、CKD(+) /low sFlt-1及びCKD(+) /high sFlt-1の４つのグループに分類した。CKD(-) /low sFlt-1（対照）に対する他の3グループのハザード比（危険性）を、（Ａ）年齢、性別（男性）、高血圧、糖尿病、高脂血症、喫煙、肥満等の危険因子で調整しない場合、（Ｂ）年齢、性別（男性）、高血圧、糖尿病、高脂血症、喫煙、肥満について調整した場合（すなわち、これらの危険因子の影響を排除した場合）、（Ｃ）年齢、性別（男性）、高血圧、糖尿病、高脂血症、喫煙、肥満、蛋白尿、ステージ４のCKD、心筋梗塞（myocardial infarction）の既往、脳卒中（stroke）の既往、血行再建（revascularization）の既往、心不全入院の既往で調整した場合（すなわち、これらの考え得るすべての危険因子・交絡因子の影響を排除した場合）について検討した。その結果、表１に示すように、CKD(+) /high sFlt-1グループにおいて、MACEsのハザード比（危険性）が有意に高いことが確認され、その差は、あらゆる危険因子・交絡因子の影響を排除した場合でも、有意であることが確認された。 Patients were treated as in Example 1 with CKD (-) / low sFlt-1 (control), CKD (-) / high sFlt-1, CKD (+) / low sFlt-1 and CKD (+) / high sFlt- It was classified into four groups of 1. The hazard ratio (risk) of the other three groups relative to CKD (-) / low sFlt-1 (control), (A) age, sex (male), hypertension, diabetes, hyperlipidemia, smoking, obesity, etc. (B) When adjusted for age, sex (male), hypertension, diabetes, hyperlipidemia, smoking, obesity (ie, excluding the effects of these risk factors), (C) ) Age, gender (male), hypertension, diabetes, hyperlipidemia, smoking, obesity, proteinuria, stage 4 CKD, history of myocardial infarction, history of stroke, revascularization And the case of adjusting for the history of hospitalization for heart failure (that is, eliminating the effects of all these possible risk factors and confounding factors). As a result, as shown in Table 1, it was confirmed that the hazard ratio (risk) of MACEs was significantly high in the CKD (+) / high sFlt-1 group, and the difference between all risk factors / confounding factors Even when the effect was excluded, it was confirmed that it was significant.
（比較例１）カプランマイヤー法を用いた生存解析
本比較例では、独立行政法人国立病院機構京都医療センターの倫理委員会の承認を得た後、確立された危険因子である高血圧、高脂血症、糖尿病、喫煙の内、少なくとも１つを有する患者、もしくは、すでに心血管病を有する患者490名におけるCKDの有無及び血中sFlt-1量を計測し、３年間追跡して、有害心血管イベント（MACEs）の診断能を評価した。採血した血液検体から通常の方法に従って、臨床検査用血清試料を調製した。sFlt-1量は、市販のELISA用キット（Quantikine(R)Human Soluble VEGF R1/Flt-1 Immunoassay Catalog # DVR100B, R&Dシステムズ社）を用いて測定した。比較例として心不全マーカーとして確立されており、なおかつCKDの心血管イベントのマーカーとして微量アルブミン尿よりも優れていることが報告されているヒト脳性ナトリウム利尿ペプチド前駆体N端フラグメント（NT-proBNP）(JAMA 2005; 293:1609-16)と、炎症マーカーであるとともに心血管イベントのマーカーとして注目されている高感度C反応性蛋白（hsCRP）を測定した。NT-proBNPは市販の免疫測定法（Roche Diagnostics, NT-proBNP測定用「エクルーシスproBNP」）で、hsCRPは市販のELISAキット（CircuLexTM High-Sensitivity CRP ELISA Kit, Code No. CY-8071, Medical ＆ Biological Laboratories Co., Ltd.）を用いて測定した。(Comparative example 1) Survival analysis using Kaplan-Meier method In this comparative example, after obtaining approval from the Ethics Committee of the National Hospital Organization Kyoto Medical Center, hypertension and hyperlipidemia are established risk factors. CKD and blood sFlt-1 levels in 490 patients with at least one of symptom, diabetes, and smoking, or already with cardiovascular disease, followed by 3 years of harmful cardiovascular The diagnostic ability of events (MACEs) was evaluated. A serum sample for clinical examination was prepared from the collected blood sample according to the usual method. sFlt-1 amount was measured using a commercial ELISA kit (Quantikine (R) Human Soluble VEGF R1 / Flt-1 Immunoassay Catalog # DVR100B, R & D Systems, Inc.). Human brain natriuretic peptide precursor N-terminal fragment (NT-proBNP), which has been established as a heart failure marker as a comparative example and has been reported to be superior to microalbuminuria as a marker for CKD cardiovascular events ( JAMA 2005; 293: 1609-16) and a highly sensitive C-reactive protein (hsCRP), which is an inflammatory marker and attracts attention as a marker of cardiovascular events. NT-proBNP is a commercially available immunoassay (Roche Diagnostics, “Ecrusys proBNP” for NT-proBNP measurement), and hsCRP is a commercially available ELISA kit (CircuLex ™ High-Sensitivity CRP ELISA Kit, Code No. CY-8071, Medical & Biological Laboratories Co., Ltd.).
実施例１と同様に、sFlt-1について最適なカットオフ値を110 pg/mLとし、110 pg/mL未満をlow sFlt-1とし、110 pg/mL以上をhigh sFlt-1とした。同様に、NT-proBNPについては、カットオフ値を230 pg/mLとし、230 pg/mL未満をlow NT-proBNPとし、230 pg/mL以上をhigh NT-proBNPとした。hsCRPについては、カットオフ値を0.3μg/mLとし、0.3μg/mL未満をlow hsCRPとし、0.3μg/mL以上をhigh hsCRPとした。
CKDの有無については、推定糸球体濾過率（eGFR）60 mL/分/1.73m2未満のものをCKD患者と判断し、CKDとした。また、CKD患者でないものをNon-CKDとした。As in Example 1, the optimal cutoff value for sFlt-1 was 110 pg / mL, less than 110 pg / mL was low sFlt-1, and 110 pg / mL or higher was high sFlt-1. Similarly, for NT-proBNP, the cutoff value was 230 pg / mL, less than 230 pg / mL was low NT-proBNP, and 230 pg / mL or higher was high NT-proBNP. For hsCRP, the cutoff value was 0.3 μg / mL, less than 0.3 μg / mL was low hsCRP, and 0.3 μg / mL or higher was high hsCRP.
Regarding the presence or absence of CKD, an estimated glomerular filtration rate (eGFR) of less than 60 mL / min / 1.73 m 2 was determined to be a CKD patient and determined as CKD. Those who were not CKD patients were designated as Non-CKD.
上記の結果、図２に示すようにCKD患者で血中sFlt-1量を指標とした場合に、心血管イベントの生存率曲線においてもっとも大きな有意差を認めた。 As a result, as shown in FIG. 2, the largest significant difference was observed in the survival curve of cardiovascular events when the amount of blood sFlt-1 was used as an index in CKD patients.
確立された危険因子である高血圧、高脂血症、糖尿病、喫煙の内、少なくとも１つを有する患者、もしくは、すでに心血管病を有する患者490名におけるCKDの有無及び血中sFlt-1量を計測し、３年間追跡して、MACEsの診断能を評価した。
ステップワイズCox比例ハザード解析を実施した。CKD患者（396名）も非CKD患者（94名）も全て含んだ490名全体（Total）で解析した場合、表２に示すように、MACEsの独立した予測因子は、心血管疾患歴（History of Cardiovascular Disease）、高NT-proBNP（230 pg/mL以上）、高sFlt-1（110 pg/mL以上）であった。非CKD患者のみで解析した場合、MACEsの独立した予測因子は、心血管疾患歴、高NT-proBNP、収縮期血圧、性別（男性）であった。しかしながら、CKD患者のみで解析した場合、MACEsの独立した予測因子は、高sFlt-1と喫煙であった。Presence or absence of CKD and blood sFlt-1 levels in 490 patients with at least one of established risk factors hypertension, hyperlipidemia, diabetes, and smoking, or who already have cardiovascular disease Measured and followed for 3 years to assess the diagnostic ability of MACEs.
Stepwise Cox proportional hazard analysis was performed. As shown in Table 2, the independent predictors of MACEs are the history of cardiovascular disease (History) when analyzed in 490 total (Total) including all CKD patients (396) and non-CKD patients (94). of Cardiovascular Disease), high NT-proBNP (230 pg / mL or more), and high sFlt-1 (110 pg / mL or more). When analyzed only in non-CKD patients, MACEs independent predictors were cardiovascular history, high NT-proBNP, systolic blood pressure, and gender (male). However, when analyzed in CKD patients alone, the independent predictors of MACEs were high sFlt-1 and smoking.
（比較例２）感度及び特異度について
本比較例では、独立行政法人国立病院機構京都医療センターの倫理委員会の承認を得た後、確立された危険因子である高血圧、高脂血症、糖尿病、喫煙の内、少なくとも１つを有する患者、もしくは、すでに心血管病を有する患者490名におけるCKDの有無及び血中sFlt-1量を計測し、１年後、及び２年後の時点における有害心血管イベント（MACEs）の発症率に関し、Receiver operating characteristic curve analysis (ROC解析)を行いMACEs予測診断能を確認した。(Comparative Example 2) Sensitivity and Specificity In this comparative example, after obtaining approval from the ethical committee of the National Hospital Organization Kyoto Medical Center, established risk factors are hypertension, hyperlipidemia, and diabetes. Measure the presence of CKD and blood sFlt-1 levels in 490 patients who have at least one of smoking, or who already have cardiovascular disease, and are harmful at 1 and 2 years Receiver operating characteristic curve analysis (ROC analysis) was performed on the incidence of cardiovascular events (MACEs) to confirm the ability to predict MACEs.
採血した血液検体から通常の方法に従って、臨床検査用血清試料を調製した。sFlt-1は、市販のELISA用キット（Quantikine(R)Human Soluble VEGF R1/Flt-1 Immunoassay Catalog # DVR100B, R&Dシステムズ社）を用いて定量した。CKDの心血管イベントのマーカーとして微量アルブミン尿よりも優れていることが報告されているNT-proBNPを定量した。ここでは、各マーカー量を測定して、その値と、１年後又は２年後の時点におけるMACEsの発症との関連を解析し、各マーカーの予測診断能を評価した。A serum sample for clinical examination was prepared from the collected blood sample according to the usual method. sFlt-1 was quantified using a commercially available ELISA kit (Quantikine (R) Human Soluble VEGF R1 / Flt-1 Immunoassay Catalog # DVR100B, R & D Systems, Inc.). NT-proBNP, which is reported to be superior to microalbuminuria as a marker of cardiovascular events in CKD, was quantified. Here, the amount of each marker was measured, and the relationship between the value and the onset of MACEs at one or two years later was analyzed, and the predictive diagnostic ability of each marker was evaluated.
上記の結果、CKD患者で血中sFlt-1をマーカーとして定量した場合に、１年後、２年後のいずれの時点においても、NT-proBNPの場合と比較して、曲面下面積（area under the curve）が有意に大きいことが確認された。これにより、sFlt-1をマーカーとした場合のほうが、NT-proBNPをマーカーとした場合よりも、心血管イベントの予測診断能が優れていることが明らかとなった。 As a result of the above, when quantifying blood sFlt-1 as a marker in CKD patients, the area under the curved surface (area under) was compared with NT-proBNP at any time point after 1 year and 2 years later. It was confirmed that the curve) was significantly large. This revealed that the ability to predict and diagnose cardiovascular events is superior when sFlt-1 is used as a marker than when NT-proBNP is used as a marker.
以上詳述したように、sFlt-1を指標とする本発明の検査方法により、CKD患者特異的に有害心血管イベント（MACEs）の危険性を予測することが可能となった。これにより、CKDの腎不全（eGFR 15 mL/min/1.73 m2未満）より早期の段階でMACEsに対する的確な予防方法及び治療方法を選択することができ、非常に有用である。As described in detail above, the test method of the present invention using sFlt-1 as an index makes it possible to predict the risk of adverse cardiovascular events (MACEs) specifically for CKD patients. This makes it possible to select an appropriate preventive and therapeutic method for MACES at an earlier stage than CKD renal failure (eGFR less than 15 mL / min / 1.73 m 2 ), which is very useful.
Claims (5)
１）慢性腎臓病患者より採取された血液由来試料中のｓＦｌｔ−１量をｉｎ ｖｉｔｒｏで測定する工程；
２）測定した値が９０−１３０ｐｇ／ｍＬから選択されるいずれかの値をカットオフ値とし、カットオフ値以上の場合に慢性腎臓病患者の心血管イベント発症リスクを検出する工
程。 A test method for assessing the risk of developing cardiovascular events in patients with chronic kidney disease who has not developed cardiovascular events, using the amount of soluble vascular endothelial growth factor receptor 1 (sFlt-1) as an index, including the following steps:
1) A step of measuring the amount of sFlt-1 in a blood-derived sample collected from a patient with chronic kidney disease in vitro;
2) A step of detecting a risk of developing a cardiovascular event in a chronic kidney disease patient when any of the measured values selected from 90 to 130 pg / mL is set as a cut-off value and the cut-off value or more.
１）抗ｓＦｌｔ−１抗体；
２）ｓＦｌｔ−１と抗ｓＦｌｔ−１抗体の複合体検出用試薬。 A test kit for assessing the risk of developing cardiovascular events that can be used in the test method according to any one of claims 1 to 3 , comprising at least the following:
1) anti-sFlt-1 antibody;
2) Reagent for detecting complex of sFlt-1 and anti-sFlt-1 antibody.
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