JP2009186183A - Evaluating method of arthralgia - Google Patents

Evaluating method of arthralgia Download PDF

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JP2009186183A
JP2009186183A JP2008022952A JP2008022952A JP2009186183A JP 2009186183 A JP2009186183 A JP 2009186183A JP 2008022952 A JP2008022952 A JP 2008022952A JP 2008022952 A JP2008022952 A JP 2008022952A JP 2009186183 A JP2009186183 A JP 2009186183A
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atp
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JP5241255B2 (en
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Nobuyuki Kumahashi
伸之 熊橋
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Seikagaku Corp
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a method capable of accurately and easily evaluating the degree of arthralgia, without giving pains to a patient. <P>SOLUTION: An evaluating method that involves at least a step for measuring the ATP concentration or the amount of ATP in a synovia fluid specimen is provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

本発明は、関節痛の評価方法、関節痛の治療効果の判定方法及び関節痛の治療薬剤のスクリーニング方法に関する。   The present invention relates to a method for evaluating joint pain, a method for determining the therapeutic effect of joint pain, and a method for screening a therapeutic agent for joint pain.

従来、関節痛の評価方法としてはＶｉｓｕａｌ Ａｎａｌｏｇｕｅ Ｓｃａｌｅ（ＶＡＳ）が用いられているが、被験者による誤差が大きく、客観的な評価が困難であるという問題があった。   Conventionally, Visual Analogue Scale (VAS) has been used as an evaluation method for arthralgia, but there is a problem that there is a large error due to the subject and it is difficult to objectively evaluate it.

非特許文献１には、関節中のヌクレオシドトリホスフェートピロホスホヒドロラーゼの基質としてＡＴＰ（アデノシン三リン酸）が記載され、変形性関節症等における関節液中のＡＴＰ濃度が測定されたことが記載されているが（表１）、ＡＴＰ濃度又はＡＴＰ量と関節痛との関連については記載がない。   Non-Patent Document 1 describes that ATP (adenosine triphosphate) is described as a substrate for nucleoside triphosphate pyrophosphohydrolase in joints, and that ATP concentration in joint fluid in osteoarthritis and the like was measured. However, there is no description about the relationship between ATP concentration or ATP amount and joint pain (Table 1).

非特許文献２には、Ｐ２Ｚ／Ｐ２Ｘ７ ＡＴＰ受容体の選択的阻害剤である酸化ＡＴＰが炎症性の痛みを緩和することが記載されている。また、末梢皮下炎症組織におけるＡＴＰレベルは酸化ＡＴＰの投与によって顕著に減少すること（図４）等が記載されている。しかしながら、関節液中のＡＴＰ濃度又はＡＴＰ量の痛みとの相関については記載がない。
Lawrence M Ryan, John W Rachow, Daniel J McCarty (1991) Synovial Fluid ATP: A Potential Substrate for the Production of Inorganic Pyrophosphate. J Rheumatology 18:5 716-720 Giacomo Dell'Antonioら、Relief of Inflammatory Pain in Rats by Local Use of the Selective P2X7 ATP Receptor Inhibitor, Oxidized ATP. Arthritis and rheumatism; 46(12)p3378-85 Non-Patent Document 2 describes that oxidized ATP, which is a selective inhibitor of P2Z / P2X7 ATP receptor, alleviates inflammatory pain. Further, it is described that the ATP level in the peripheral subcutaneous inflammation tissue is remarkably decreased by administration of oxidized ATP (FIG. 4). However, there is no description of the correlation between the ATP concentration in the synovial fluid or the amount of ATP with pain.
Lawrence M Ryan, John W Rachow, Daniel J McCarty (1991) Synovial Fluid ATP: A Potential Substrate for the Production of Inorganic Pyrophosphate. J Rheumatology 18: 5 716-720 Giacomo Dell'Antonio et al., Relief of Inflammatory Pain in Rats by Local Use of the Selective P2X7 ATP Receptor Inhibitor, Oxidized ATP. Arthritis and rheumatism; 46 (12) p3378-85

本発明の課題は、関節痛の程度を、患者に苦痛を与えずに、正確かつ簡便に評価できる方法を提供することである。   An object of the present invention is to provide a method capable of accurately and easily evaluating the degree of joint pain without causing pain to a patient.

本発明者らは、上記の課題を解決するために鋭意検討した。その結果、関節液検体中のＡＴＰ濃度又はＡＴＰ量が関節痛の程度とよく相関することを見出した。本発明者らは、このような知見に基づき、本発明を完成させた。   The present inventors diligently studied to solve the above problems. As a result, it was found that the ATP concentration or the amount of ATP in the joint fluid specimen correlates well with the degree of joint pain. Based on such findings, the present inventors have completed the present invention.

すなわち、本発明の要旨は以下の通りである。
（１）関節液検体中のＡＴＰ濃度又はＡＴＰ量を測定するステップを少なくとも含むことを特徴とする、関節痛の評価方法。
（２）下記ステップを少なくとも含むことを特徴とする、関節痛の評価方法；
ステップ１：関節痛が疑われる患者由来の関節液検体中のＡＴＰ濃度又はＡＴＰ量を測定するステップ、
ステップ２：ステップ１で測定したＡＴＰ濃度又はＡＴＰ量と、標準ＡＴＰ濃度又は標準ＡＴＰ量とを比較するステップ。
（３）標準ＡＴＰ濃度又は標準ＡＴＰ量が健常者由来の関節液検体中のＡＴＰ濃度又はＡ
ＴＰ量である、（２）に記載の評価方法。
（４）関節痛の治療前における関節液検体中のＡＴＰ濃度又はＡＴＰ量と、関節痛の治療後における関節液検体中のＡＴＰ濃度又はＡＴＰ量とを比較するステップを少なくとも含むことを特徴とする、関節痛の治療効果の判定方法。
（５）下記ステップを少なくとも含むことを特徴とする、関節痛の治療薬剤のスクリーニング方法；
ステップ１：非ヒト関節痛モデル動物に薬剤を投与するステップ、
ステップ２：薬剤投与前における関節液検体中のＡＴＰ濃度又はＡＴＰ量と、薬剤投与後における関節液検体中のＡＴＰ濃度又はＡＴＰ量とを比較するステップ。 That is, the gist of the present invention is as follows.
(1) A method for evaluating joint pain, comprising at least a step of measuring an ATP concentration or an ATP amount in a joint fluid specimen.
(2) A method for evaluating joint pain, comprising at least the following steps;
Step 1: measuring an ATP concentration or an ATP amount in a joint fluid sample derived from a patient suspected of joint pain,
Step 2: A step of comparing the ATP concentration or ATP amount measured in Step 1 with the standard ATP concentration or standard ATP amount.
(3) Standard ATP concentration or standard ATP amount is ATP concentration or A in synovial fluid samples derived from healthy subjects
The evaluation method according to (2), which is an amount of TP.
(4) The method includes at least a step of comparing the ATP concentration or ATP amount in the joint fluid sample before treatment of joint pain with the ATP concentration or ATP amount in the joint fluid sample after treatment of joint pain. , A method for determining the therapeutic effect of joint pain.
(5) A screening method for a therapeutic agent for arthralgia, comprising at least the following steps;
Step 1: administering a drug to a non-human joint pain model animal,
Step 2: A step of comparing the ATP concentration or ATP amount in the synovial fluid sample before drug administration with the ATP concentration or ATP amount in the synovial fluid sample after drug administration.

本発明の関節痛の評価方法により、関節痛の程度を、患者に苦痛を与えずに、正確かつ簡便に評価することができる。さらに、関節液検体中のＡＴＰ濃度又はＡＴＰ量を関節痛の客観的なパラメーターとして利用することで、関節痛の治療効果の客観的な判定、関節痛の治療薬剤のスクリーニング、関節痛の原因分析を行うこともできる。   With the joint pain evaluation method of the present invention, the degree of joint pain can be accurately and easily evaluated without causing pain to the patient. Furthermore, by using the ATP concentration or ATP amount in the joint fluid specimen as an objective parameter for joint pain, objective judgment of the therapeutic effect of joint pain, screening for therapeutic agents for joint pain, analysis of the cause of joint pain Can also be done.

以下、本発明について詳細に説明する。
本発明の評価方法は、関節液検体中のＡＴＰ濃度又はＡＴＰ量を測定するステップを少なくとも含むことを特徴とする、関節痛の評価方法である。 Hereinafter, the present invention will be described in detail.
The evaluation method of the present invention is a method for evaluating joint pain, comprising at least a step of measuring an ATP concentration or an ATP amount in a joint fluid specimen.

本発明の評価方法の評価対象は、各種の関節炎症患者等関節痛を訴える患者が好ましい。各種の関節炎症として、具体的には、変形性関節症（ＯＡ）、関節リウマチ（ＲＡ）、ＣＰＰＤ関節症（ＣＰＰＤ）、特発性骨懐死（ＯＮ）等である。本発明の評価方法は、特に、ＯＡにおける関節痛の評価に適している。   The subject of the evaluation method of the present invention is preferably a patient complaining of joint pain, such as various joint inflammation patients. Specifically, various types of joint inflammation include osteoarthritis (OA), rheumatoid arthritis (RA), CPPD arthropathy (CPPD), idiopathic bone necrosis (ON), and the like. The evaluation method of the present invention is particularly suitable for the evaluation of joint pain in OA.

関節液は、関節痛を評価する対象〔哺乳動物（ヒトを含む）〕から単離された関節液を用いることができる。例えば、ヒトの関節痛を評価する場合には、評価対象者の問題となる関節の関節液を採取して用い、イヌやウマ等の動物の関節痛を評価する場合には、評価対象動物の問題となる関節の関節液を採取して用いることができる。   As the joint fluid, a joint fluid isolated from a subject (mammals (including humans)) to be evaluated for joint pain can be used. For example, when assessing human joint pain, the joint fluid of the joint that is a problem for the assessment subject is collected and used, and when assessing joint pain in animals such as dogs and horses, The joint fluid of the joint in question can be collected and used.

なお、本発明で使用する関節液は、ＡＴＰ濃度又はＡＴＰ量測定前に不溶性異物や血液、タンパク質等を除去するため、酸、有機溶媒等でのタンパク変性、フィルター、限外濾過膜等での濾過、遠心分離等の固液分離処理等を行うことが好ましい。また、生理食塩水等で適宜希釈した関節液を用いることもできる。   The synovial fluid used in the present invention removes insoluble foreign matter, blood, protein, etc. before measuring ATP concentration or ATP amount, so that protein denaturation with acid, organic solvent, filter, ultrafiltration membrane, etc. It is preferable to perform solid-liquid separation processing such as filtration and centrifugation. Also, joint fluid appropriately diluted with physiological saline or the like can be used.

本発明の評価方法におけるＡＴＰ量としては、例えば関節痛を評価する対象から通常の整形外科的方法により採取できる関節液総量中に含まれるＡＴＰ量（本明細書において「ＡＴＰ総量」又は「関節液中ＡＴＰ総量」と略記することがある）を用いることができる。ＡＴＰ総量を指標として用いることにより、より精度の高い評価を行うことができ好ましい。   The amount of ATP in the evaluation method of the present invention is, for example, the amount of ATP contained in the total amount of joint fluid that can be collected by a normal orthopedic method from the subject to be evaluated for joint pain (in this specification, “ATP total amount” or “joint fluid”). May be abbreviated as “medium ATP total amount”). By using the total amount of ATP as an index, more accurate evaluation can be performed, which is preferable.

本発明の評価方法におけるＡＴＰ濃度又はＡＴＰ量の測定方法は、関節液検体中に含まれるＡＴＰ濃度又はＡＴＰ量を定量的に測定できる方法である限りにおいて限定されず、常法を用いることができる。例えば、ＡＴＰと発光試薬を反応させて、発生した光を光センサーで計測する方法等が挙げられる。   The method for measuring the ATP concentration or ATP amount in the evaluation method of the present invention is not limited as long as it is a method capable of quantitatively measuring the ATP concentration or ATP amount contained in the joint fluid specimen, and a conventional method can be used. . For example, a method of reacting ATP with a luminescent reagent and measuring generated light with an optical sensor can be used.

ＡＴＰ濃度又はＡＴＰ量の測定方法について、具体的な方法を次に説明するが、これに限定されるものではない。生理食塩水等で適当に希釈した関節液検体に、発光試薬（ルシフェリン、ルシフェラーゼ、Ｍｇ2+等）を添加して、検体中のＡＴＰと発光試薬を反応さ
せる。そして、この反応により発生した光を光センサーで一定時間計測する。ＡＴＰ測定試薬及び光センサーは、市販のものを用いることができる。例えば、ＡＴＰ測定試薬としては、ルシフェラーゼ２５０プラス（キッコーマン株式会社製）等を用いることができる。 A specific method for measuring the ATP concentration or the amount of ATP will be described below, but is not limited thereto. A luminescent reagent (luciferin, luciferase, Mg 2+, etc.) is added to a joint fluid sample appropriately diluted with physiological saline or the like, and ATP in the sample reacts with the luminescent reagent. Then, the light generated by this reaction is measured for a certain time with an optical sensor. Commercially available ATP measuring reagents and optical sensors can be used. For example, as the ATP measurement reagent, luciferase 250 plus (manufactured by Kikkoman Corporation) or the like can be used.

本発明の評価方法によれば、関節液検体中のＡＴＰ濃度又はＡＴＰ量が関節炎症に伴う痛みの程度とよく相関することを利用して、関節痛の評価を行うことができる。例えば、関節液検体中のＡＴＰ濃度が高い又はＡＴＰ量が多い時には痛みが強いと評価される。このような痛みの評価は、従来、患者の申告に基づくＶＡＳにより行われていたが、ＶＡＳによる痛みの評価は個人差等による誤差が大きい。本発明の評価方法においては、関節液検体中のＡＴＰ濃度又はＡＴＰ量を痛みの客観的パラメーターとして用いることにより、正確に痛みを評価することができる。   According to the evaluation method of the present invention, joint pain can be evaluated using the fact that the ATP concentration or the amount of ATP in a joint fluid specimen correlates well with the degree of pain associated with joint inflammation. For example, when the ATP concentration in the joint fluid specimen is high or the amount of ATP is large, it is evaluated that the pain is strong. Conventionally, such pain evaluation has been performed by VAS based on the patient's declaration, but pain evaluation by VAS has a large error due to individual differences. In the evaluation method of the present invention, pain can be accurately evaluated by using the ATP concentration or ATP amount in the joint fluid specimen as an objective parameter for pain.

また、関節炎症患者由来の関節液検体中のＡＴＰ濃度又はＡＴＰ量と、関節痛の程度の評価・判定基準となる、関節液検体中の標準ＡＴＰ濃度又は標準ＡＴＰ量とを比較することにより、関節痛の評価を行うことができる。標準ＡＴＰ濃度又は標準ＡＴＰ量としては、当該患者由来の関節液検体中のＡＴＰ濃度又はＡＴＰ量、当該患者以外の患者又は健常者由来の関節液検体中のＡＴＰ濃度又はＡＴＰ量、及び複数の患者又は健常者由来の関節液検体中のＡＴＰ濃度又はＡＴＰ量から統計的に得られたＡＴＰ濃度又はＡＴＰ量等を用いることができる。標準ＡＴＰ濃度又は標準ＡＴＰ量は、評価・判定対象、目的及び疾患の種類等によって上記標準ＡＴＰ濃度又は標準ＡＴＰ量から適宜選択される。このように、対象の関節液検体中のＡＴＰ濃度又はＡＴＰ量と標準ＡＴＰ濃度又は標準ＡＴＰ量とを比較することにより、関節痛の程度を評価することもできる。   In addition, by comparing the ATP concentration or ATP amount in the joint fluid sample derived from a patient with joint inflammation and the standard ATP concentration or standard ATP amount in the joint fluid sample, which is an evaluation / determination standard of the degree of joint pain, Assessment of joint pain can be performed. As the standard ATP concentration or standard ATP amount, the ATP concentration or ATP amount in a joint fluid sample derived from the patient, the ATP concentration or ATP amount in a joint fluid sample derived from a patient other than the patient or a healthy person, and a plurality of patients Alternatively, an ATP concentration or an ATP amount statistically obtained from an ATP concentration or an ATP amount in a synovial fluid sample derived from a healthy person can be used. The standard ATP concentration or the standard ATP amount is appropriately selected from the standard ATP concentration or the standard ATP amount depending on the evaluation / determination target, the purpose, the type of disease, and the like. As described above, the degree of joint pain can be evaluated by comparing the ATP concentration or ATP amount in the subject joint fluid specimen with the standard ATP concentration or standard ATP amount.

なお、本発明の評価方法において、関節液検体中のＡＴＰ濃度又はＡＴＰ量と痛みの程度（ＶＡＳスコア等）との標準曲線（検量線）を用いて、評価を行うこともできる。すなわち、測定結果から得られるＡＴＰ濃度又はＡＴＰ量に対する痛みの程度を検量線から読み取り、痛みの程度を評価することができる。   In the evaluation method of the present invention, the evaluation can also be performed using a standard curve (calibration curve) of the ATP concentration or ATP amount in the joint fluid specimen and the degree of pain (VAS score, etc.). That is, the degree of pain with respect to the ATP concentration or the amount of ATP obtained from the measurement result can be read from a calibration curve, and the degree of pain can be evaluated.

本発明の判定方法は、関節痛の治療前における関節液検体中のＡＴＰ濃度又はＡＴＰ量と、関節痛の治療後における関節液検体中のＡＴＰ濃度又はＡＴＰ量とを比較するステップを少なくとも含むことを特徴とする、関節痛の治療効果の判定方法である。なお、本明細書において「関節痛の治療」とは、関節痛のみを治療するものや、関節炎症及び関節痛両方を治療するもののいずれであってもよい。   The determination method of the present invention includes at least a step of comparing the ATP concentration or ATP amount in the joint fluid sample before the treatment of joint pain with the ATP concentration or ATP amount in the joint fluid sample after the treatment of joint pain. This is a method for determining the therapeutic effect of joint pain. In the present specification, “treatment of joint pain” may be either treatment of joint pain alone or treatment of both joint inflammation and joint pain.

関節炎症患者に抗関節炎薬及び／又は抗関節痛薬を投与する場合において、投与前及び投与後の当該患者由来の関節液検体中のＡＴＰ濃度又はＡＴＰ量を継続的に測定し、その濃度を比較することにより、そのＡＴＰ濃度又はＡＴＰ量が低下していれば、関節炎症による痛みが治療されていると判定することができる。同様に関節液検体中のＡＴＰ濃度又はＡＴＰ量が変化しなければ、関節炎症による痛みが維持されていること、関節液検体中のＡＴＰ濃度又はＡＴＰ量が上昇していれば、関節炎症による痛みが悪化していることが判定される。   When administering an anti-arthritis drug and / or anti-arthralgia drug to a patient with joint inflammation, continuously measure the ATP concentration or ATP amount in the joint fluid sample derived from the patient before and after administration, and determine the concentration. By comparing, if the ATP concentration or the amount of ATP is reduced, it can be determined that pain due to joint inflammation is being treated. Similarly, pain due to joint inflammation is maintained if the ATP concentration or ATP amount in the joint fluid sample does not change, and pain due to joint inflammation if the ATP concentration or ATP amount in the joint fluid sample is increased. Is determined to be worse.

このように、本発明の判定方法は、抗関節炎薬及び／又は抗関節痛薬の治療効果の判定や、投与する抗関節炎薬及び／又は抗関節痛薬の変更や選択等、治療方針に有用な情報を提供するものである。   As described above, the determination method of the present invention is useful for a therapeutic policy such as determination of the therapeutic effect of an anti-arthritic drug and / or anti-arthritic drug, and change or selection of an anti-arthritic drug and / or anti-arthritic drug to be administered. Information.

また、初診の関節炎症患者のように、関節炎症の程度が未知の患者の場合は、本発明の評価方法により、当該患者由来の関節液検体中のＡＴＰ濃度又はＡＴＰ量を測定し、そのＡＴＰ濃度又はＡＴＰ量から、当該患者の関節炎症による痛みがどの程度かを評価するこ
とができる。これによっても、投与する抗関節炎薬及び／又は抗関節痛薬の選択等、関節炎症の治療方針に有用な情報を提供するものである。 In the case of a patient whose degree of joint inflammation is unknown, such as a joint inflammation patient at the first visit, the ATP concentration or the amount of ATP in the joint fluid specimen derived from the patient is measured by the evaluation method of the present invention, and the ATP From the concentration or the amount of ATP, it is possible to evaluate the degree of pain due to joint inflammation in the patient. This also provides useful information for the treatment policy of joint inflammation, such as selection of anti-arthritic drugs and / or anti-arthritic drugs to be administered.

本発明のスクリーニング方法は、非ヒト関節痛モデル動物に薬剤を投与するステップと、薬剤投与前における関節液検体中のＡＴＰ濃度又はＡＴＰ量と、薬剤投与後における関節液検体中のＡＴＰ濃度又はＡＴＰ量とを比較するステップを少なくとも含むことを特徴とする、関節痛の治療薬剤のスクリーニング方法である。
非ヒト関節痛モデル動物としては特に限定されないが、関節痛を発症するように処置されたマウス等のモデル動物である。 The screening method of the present invention comprises a step of administering a drug to a non-human joint pain model animal, an ATP concentration or ATP amount in a joint fluid sample before drug administration, and an ATP concentration or ATP in a joint fluid sample after drug administration. A method for screening a therapeutic agent for arthralgia, comprising at least a step of comparing the amount.
Although it does not specifically limit as a non-human joint pain model animal, It is model animals, such as a mouse | mouth treated so that joint pain might be developed.

抗関節炎薬及び／又は抗関節痛薬の候補物質の有効性を評価する場合において、当該候補物質投与前及び投与後の関節液検体中のＡＴＰ濃度又はＡＴＰ量を測定し、その濃度又は量を比較することにより、そのＡＴＰ濃度又はＡＴＰ量が低下していれば、当該候補物質が有効であると判定することができる。同様に関節液検体中のＡＴＰ濃度又はＡＴＰ量が変化しなければ、当該候補物質が有効でないと判定される。   When evaluating the effectiveness of a candidate substance for an anti-arthritic drug and / or anti-arthralgia drug, measure the ATP concentration or ATP amount in the joint fluid sample before and after administration of the candidate substance, and determine the concentration or quantity. By comparing, if the ATP concentration or ATP amount is decreased, it can be determined that the candidate substance is effective. Similarly, if the ATP concentration or ATP amount in the joint fluid specimen does not change, it is determined that the candidate substance is not effective.

このように、本発明のスクリーニング方法は、抗関節炎薬及び／又は抗関節痛薬の候補物質の有効性の評価等、抗関節炎薬及び／又は抗関節痛薬の開発に有用な情報を提供するものである。   As described above, the screening method of the present invention provides information useful for the development of anti-arthritic drugs and / or anti-arthritic drugs such as evaluation of the effectiveness of anti-arthritic drugs and / or anti-arthritic drug candidate substances. Is.

以下、本発明を実施例により具体的に説明するが、本発明はこれらに限定されるものではない。   Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited thereto.

［実施例１］
各種関節炎症患者における関節液中及び血漿中のＡＴＰ濃度（平均値）を測定した。
動作における明確な痛み及び水腫性関節症を有する、４８人のＯＡ患者における５２膝の膝関節を被験対象とした。
化膿性関節症の患者で、過去３ヶ月以内にヒアルロン酸（ＨＡ）注射又はステロイド注射による治療を受けた患者、血清反応陰性関節炎の疑いの患者で、非ステロイド抗炎症薬を服用している患者を除いた。全ての患者に本実験の趣旨を説明し、同意を得た。 [Example 1]
The ATP concentration (average value) in synovial fluid and plasma in various patients with joint inflammation was measured.
The test subjects were 52 knee joints in 48 OA patients with clear pain in movement and edema arthropathy.
Patients with pyogenic arthropathy who have been treated with hyaluronic acid (HA) injection or steroid injection within the last 3 months, patients who are suspected of seronegative arthritis and are taking nonsteroidal anti-inflammatory drugs Was excluded. The purpose of this experiment was explained to all patients and consent was obtained.

同様に、３人のＲＡ患者における５膝関節、７人のＣＰＰＤ患者における７膝関節、３人のＯＮ患者における３膝関節を被験対象とした。   Similarly, 5 knee joints in 3 RA patients, 7 knee joints in 7 CPPD patients, and 3 knee joints in 3 ON patients were tested.

対象より２２Ｇニードルで関節液及び血漿を静かに回収し、関節液及び血漿検体とし、ＡＴＰ濃度を測定した。   The joint fluid and plasma were gently collected from the subject using a 22G needle, and the ATP concentration was measured using the joint fluid and plasma specimen.

ＡＴＰ濃度の測定は、以下のようにして行った。
ＡＴＰはルシフェラーゼアッセイ（ルシフェラーゼ２５０プラス、キッコーマン株式会社製）により測定した。関節液及び血漿を２２Ｇニードルで0.5ccを静かに吸引し、タンパク質を除去するため、5mlの0.6N 過塩素酸（除タンパク液）とすばやく混合した。混合液を3,000rpm、10分、4℃で遠心分離し、上清を分離した。上清を、2.5mol/L 炭酸カリウム及び0.5mol/L トリエタノールアミン塩酸塩を含むバッファー（中和液）と、1:10の割合で混合し、ホモジナイズし、安定するまで10分、4℃で静置した。これを3,000rpm、10分、4℃で遠心分離し、上清を分離した。50mlの発光試薬を50mlの上清に加えた。発光強度を測定し、検量線に基づいてＡＴＰ濃度を求めた。除タンパク液及び中和液で希釈された割合から、血漿及び関節液中のＡＴＰ濃度を算出した。
結果を表１に示す。 The ATP concentration was measured as follows.
ATP was measured by a luciferase assay (luciferase 250 plus, manufactured by Kikkoman Corporation). The joint fluid and plasma were gently aspirated at 0.5 cc with a 22G needle, and quickly mixed with 5 ml of 0.6N perchloric acid (protein removal solution) to remove the protein. The mixture was centrifuged at 3,000 rpm for 10 minutes at 4 ° C., and the supernatant was separated. The supernatant was mixed with a buffer (neutralizing solution) containing 2.5 mol / L potassium carbonate and 0.5 mol / L triethanolamine hydrochloride at a ratio of 1:10, homogenized, and 10 minutes at 4 ° C until stable. Left at rest. This was centrifuged at 3,000 rpm for 10 minutes at 4 ° C., and the supernatant was separated. 50 ml of luminescent reagent was added to 50 ml of supernatant. The luminescence intensity was measured, and the ATP concentration was determined based on the calibration curve. The ATP concentration in plasma and synovial fluid was calculated from the ratio diluted with the protein removal solution and neutralization solution.
The results are shown in Table 1.

Figure 2009186183
Figure 2009186183

［実施例２］
関節炎症患者における、関節炎治療前及び治療後における、血漿中及び関節液中ＡＴＰ濃度、関節液中ＡＴＰ総量、関節液総量及びＶＡＳスコアを比較した。また、ΔＶＡＳスコア及び関節液中ΔＡＴＰ総量間の相関、ΔＶＡＳスコア及びΔ関節液総量の相関、関節液中ＡＴＰ総量と関節液総量の相関、及び血漿中ＡＴＰ濃度と関節液中ＡＴＰ濃度の相関を調べた。ΔＶＡＳスコアは、治療前のＶＡＳスコアから治療後のＶＡＳスコアを減じることにより決定した。関節液中のΔＡＴＰ総量は、治療前の関節液中のＡＴＰ総量から治療後の関節液中のＡＴＰ総量を減じることにより決定した。Δ関節液総量は、治療前の関節液総量から治療後の関節液総量を減じることにより決定した。 [Example 2]
The plasma inflammation and synovial fluid ATP concentration, synovial fluid total ATP, synovial fluid total, and VAS score were compared before and after arthritis treatment in arthritic patients. In addition, correlation between ΔVAS score and total amount of ΔATP in synovial fluid, correlation between ΔVAS score and total amount of synovial fluid, correlation between total amount of ATP in synovial fluid and total amount of synovial fluid, and correlation between ATP concentration in plasma and ATP concentration in synovial fluid. Examined. The ΔVAS score was determined by subtracting the post-treatment VAS score from the pre-treatment VAS score. The total amount of ΔATP in synovial fluid was determined by subtracting the total amount of ATP in joint fluid after treatment from the total amount of ATP in joint fluid before treatment. The Δ joint fluid was determined by subtracting the joint fluid after treatment from the joint fluid before treatment.

実施例１における関節炎症患者中、１６人のＯＡ患者に、定期的に５回のＨＡ注射を処置した。すなわち、ヒアルロン酸製剤（HA90:Artz（登録商標）、生化学工業株式会社製、平均分子量900kD）を関節注射にて用いた。上記ヒアルロン酸製剤は５週間に渡り、１週間に１回投与した。１回目の投与の際に、２２Ｇニードルで関節液及び血漿を静かに回収した。１回目の投与の６週間後、関節液及び血漿を再度回収した。関節液に血液を含む場合、血液を除去し、ＡＴＰ濃度を測定した。関節痛を100mm ＶＡＳにて１回目及び５回目の投与後１週間後に評価した。   Of the arthritic patients in Example 1, 16 OA patients were treated with 5 HA injections on a regular basis. That is, a hyaluronic acid preparation (HA90: Artz (registered trademark), manufactured by Seikagaku Corporation, average molecular weight 900 kD) was used for joint injection. The hyaluronic acid preparation was administered once a week for 5 weeks. During the first administration, joint fluid and plasma were gently collected with a 22G needle. Six weeks after the first administration, joint fluid and plasma were collected again. When the joint fluid contains blood, the blood was removed and the ATP concentration was measured. Joint pain was evaluated at 100 mm VAS one week after the first and fifth doses.

（ＡＴＰ濃度）
血漿中のＡＴＰ濃度は、治療前で63.4±20.2μmol/L、治療後で55.2±16.4μmol/Lであった。関節液のＡＴＰ濃度は、治療前で0.070±0.087μmol/L、治療後で0.033±0.018μmol/Lであった。結果を図１に示す。
関節液中のＡＴＰ濃度は、治療後では治療前に比べて有意に低かった(p=0.04)。血漿中のＡＴＰ濃度は、治療後と治療前の差は関節液中のＡＴＰ濃度ほど顕著には見られなかった。 (ATP concentration)
The plasma ATP concentration was 63.4 ± 20.2 μmol / L before treatment and 55.2 ± 16.4 μmol / L after treatment. The ATP concentration of joint fluid was 0.070 ± 0.087 μmol / L before treatment and 0.033 ± 0.018 μmol / L after treatment. The results are shown in FIG.
The ATP concentration in synovial fluid was significantly lower after treatment than before treatment (p = 0.04). As for the ATP concentration in plasma, the difference between after treatment and before treatment was not as remarkable as the ATP concentration in joint fluid.

（ＡＴＰ総量）
関節液中のＡＴＰ総量は、治療前と治療後でそれぞれ0.369±0.44nmol及び0.142±0.13nmolであった。結果を図２に示す。
関節液中のＡＴＰ総量は、治療後では治療前に比べて有意に低かった(P=0.02)。 (ATP total amount)
The total amount of ATP in synovial fluid was 0.369 ± 0.44 nmol and 0.142 ± 0.13 nmol before and after treatment, respectively. The results are shown in FIG.
The total amount of ATP in synovial fluid was significantly lower after treatment than before treatment (P = 0.02).

（関節液総量）
関節液総量とは、ニードルを関節腔に刺して関節液を吸引する場合において、吸引できる関節液の総量のことである。
関節液総量は、治療前と治療後でそれぞれ9.0±7.1ml及び6.8±5.7mlであった。結果を図３に示す。
関節液総量は、治療後では治療前に比べて有意に低かった(P=0.02)。 (Total amount of joint fluid)
The total amount of joint fluid is the total amount of joint fluid that can be sucked when the needle is inserted into the joint cavity to suck the joint fluid.
The total synovial fluid volume was 9.0 ± 7.1 ml and 6.8 ± 5.7 ml before and after treatment, respectively. The results are shown in FIG.
The total amount of joint fluid was significantly lower after treatment than before treatment (P = 0.02).

（各対象のＡＴＰ総量）
各対象の治療前と治療後における関節液中のＡＴＰ総量を図４に示す。
各対象の関節液中のＡＴＰ総量は、治療後で治療前に比べて低いケースがほとんどであった。 (Total amount of ATP for each target)
FIG. 4 shows the total amount of ATP in the joint fluid before and after treatment for each subject.
In most cases, the total amount of ATP in the synovial fluid of each subject was lower after treatment than before treatment.

（ＶＡＳスコア）
ＶＡＳスコアは、治療前と治療後でそれぞれ60.9±20.8mm及び33.7±24.8mmであった。結果を図５に示す。
ＶＡＳスコアは、治療後では治療前に比べて有意に低かった(p=0.002)。 (VAS score)
The VAS scores were 60.9 ± 20.8 mm and 33.7 ± 24.8 mm before and after treatment, respectively. The results are shown in FIG.
The VAS score was significantly lower after treatment than before treatment (p = 0.002).

（ΔＶＡＳスコア及びΔＡＴＰ総量間の相関）
ΔＶＡＳスコアと関節液中のΔＡＴＰ総量との相関を検討した。結果を図６に示す。
ΔＶＡＳスコアは関節液中のΔＡＴＰ総量と有意に相関した(R=0.05, P=0.04)。 (Correlation between ΔVAS score and total ΔATP)
The correlation between the ΔVAS score and the total amount of ΔATP in the synovial fluid was examined. The results are shown in FIG.
The ΔVAS score was significantly correlated with the total amount of ΔATP in synovial fluid (R = 0.05, P = 0.04).

（ΔＶＡＳスコア及びΔ関節液総量間の相関）
ΔＶＡＳスコアとΔ関節液総量との相関を検討した。結果を図７に示す。
ΔＶＡＳスコアはΔ関節液総量と有意に相関した(R=0.74, P=0.004)。 (Correlation between ΔVAS score and Δ joint fluid total amount)
The correlation between ΔVAS score and Δ joint fluid total amount was examined. The results are shown in FIG.
The ΔVAS score was significantly correlated with the total amount of Δ joint fluid (R = 0.74, P = 0.004).

（関節液中ＡＴＰ総量及び関節液総量間の相関）
関節液中ＡＴＰ総量と関節液総量との相関を検討した。結果を図８に示す。
関節液中ＡＴＰ総量は関節液総量と有意に相関した(R=0.89, P<0.0001)。 (Correlation between total amount of ATP in joint fluid and total amount of joint fluid)
The correlation between the total amount of ATP in synovial fluid and the total amount of synovial fluid was examined. The results are shown in FIG.
The total amount of ATP in synovial fluid was significantly correlated with the total amount of synovial fluid (R = 0.89, P <0.0001).

（関節液中ＡＴＰ濃度及び血漿中ＡＴＰ濃度間の相関）
関節液中ＡＴＰ濃度と血漿中ＡＴＰ濃度との相関を検討した。結果を図９に示す。
関節液中ＡＴＰ濃度は血漿中ＡＴＰ濃度と相関しなかった(R=0.09, P=0.54)。 (Correlation between ATP concentration in synovial fluid and ATP concentration in plasma)
The correlation between synovial fluid ATP concentration and plasma ATP concentration was examined. The results are shown in FIG.
Synovial fluid ATP concentration did not correlate with plasma ATP concentration (R = 0.09, P = 0.54).

ヒアルロン酸製剤の関節注射前と関節注射後における、血漿及び関節液におけるＡＴＰ濃度（μｍｏｌ／Ｌ）を示す。The ATP concentration (μmol / L) in plasma and joint fluid before and after joint injection of a hyaluronic acid preparation is shown. ヒアルロン酸製剤の関節注射前と関節注射後における、関節液におけるＡＴＰ総量（ｎｍｏｌ）を示す。The total amount of ATP (nmol) in the joint fluid before and after the joint injection of the hyaluronic acid preparation is shown. ヒアルロン酸製剤の関節注射前と関節注射後における、関節液総量（ｍｌ）を示す。The total amount of joint fluid (ml) before and after joint injection of the hyaluronic acid preparation is shown. ヒアルロン酸製剤の関節注射前と関節注射後における、各対象の関節液におけるＡＴＰ総量（ｎｍｏｌ）を示す。The total amount of ATP (nmol) in the joint fluid of each subject before and after the joint injection of the hyaluronic acid preparation is shown. ヒアルロン酸製剤の関節注射前と関節注射後における、ＶＡＳスコアを示す。The VAS score before and after joint injection of a hyaluronic acid preparation is shown. ΔＶＡＳスコアと関節液におけるΔＡＴＰ総量（ｎｍｏｌ）との関係を示す。The relationship between (DELTA) VAS score and (DELTA) ATP total amount (nmol) in a joint fluid is shown. ΔＶＡＳスコアとΔ関節液総量（ｍｌ）との関係を示す。The relationship between (DELTA) VAS score and (DELTA) joint fluid total amount (ml) is shown. 関節液総量（ｍｌ）と関節液におけるＡＴＰ総量（ｎｍｏｌ）との関係を示す。The relationship between total amount of joint fluid (ml) and total amount of ATP (nmol) in joint fluid is shown. 血漿中ＡＴＰ濃度（μｍｏｌ／Ｌ）と関節液中ＡＴＰ濃度（ｎｍｏｌ／Ｌ）との関係を示す。The relationship between plasma ATP concentration (μmol / L) and synovial fluid ATP concentration (nmol / L) is shown.

Claims (5)

関節液検体中のＡＴＰ濃度又はＡＴＰ量を測定するステップを少なくとも含むことを特徴とする、関節痛の評価方法。   A method for evaluating joint pain, comprising at least a step of measuring an ATP concentration or an ATP amount in a joint fluid specimen. 下記ステップを少なくとも含むことを特徴とする、関節痛の評価方法；
ステップ１：関節痛が疑われる患者由来の関節液検体中のＡＴＰ濃度又はＡＴＰ量を測定するステップ、
ステップ２：ステップ１で測定したＡＴＰ濃度又はＡＴＰ量と、標準ＡＴＰ濃度又は標準ＡＴＰ量とを比較するステップ。 A method for evaluating joint pain, comprising at least the following steps;
Step 1: measuring an ATP concentration or an ATP amount in a joint fluid sample derived from a patient suspected of joint pain,
Step 2: A step of comparing the ATP concentration or ATP amount measured in Step 1 with the standard ATP concentration or standard ATP amount. 標準ＡＴＰ濃度又は標準ＡＴＰ量が健常者由来の関節液検体中のＡＴＰ濃度又はＡＴＰ量である、請求項２に記載の評価方法。   The evaluation method according to claim 2, wherein the standard ATP concentration or the standard ATP amount is an ATP concentration or an ATP amount in a joint fluid sample derived from a healthy person. 関節痛の治療前における関節液検体中のＡＴＰ濃度又はＡＴＰ量と、関節痛の治療後における関節液検体中のＡＴＰ濃度又はＡＴＰ量とを比較するステップを少なくとも含むことを特徴とする、関節痛の治療効果の判定方法。   A joint pain comprising at least a step of comparing the ATP concentration or ATP amount in the joint fluid specimen before the treatment of joint pain with the ATP concentration or ATP amount in the joint fluid specimen after the joint pain treatment. To determine the therapeutic effect of 下記ステップを少なくとも含むことを特徴とする、関節痛の治療薬剤のスクリーニング方法；
ステップ１：非ヒト関節痛モデル動物に薬剤を投与するステップ、
ステップ２：薬剤投与前における関節液検体中のＡＴＰ濃度又はＡＴＰ量と、薬剤投与後における関節液検体中のＡＴＰ濃度又はＡＴＰ量とを比較するステップ。 A screening method for a therapeutic agent for arthralgia, comprising at least the following steps;
Step 1: administering a drug to a non-human joint pain model animal,
Step 2: comparing the ATP concentration or ATP amount in the synovial fluid sample before drug administration with the ATP concentration or ATP amount in the synovial fluid sample after drug administration.
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