JP6555711B2 - Method for determining disease activity of non-dissecting aortic aneurysm - Google Patents

Description

  The present invention relates to a novel biomarker that reflects the disease activity of a non-dissociating aortic aneurysm and a method for determining the disease activity of a non-dissociating aortic aneurysm using the marker as an index.

  Non-dissecting aortic aneurysm is a progressive and fatal disease. It is often based on lifestyle-related diseases such as dyslipidemia and hypertension, and it is estimated that about 5% of the world's 65-year-old male population is affected. Non-dissociating aortic aneurysm (hereinafter sometimes referred to simply as an aortic aneurysm) is asymptomatic and the patient cannot recognize its progression, and in a few years the aortic aneurysm gradually expands and suddenly ruptures May end up. The fatality rate at the time of rupture is very high, exceeding 50% (Non-patent Document 1).

  Currently, accidental discovery of an aortic aneurysm is followed by CT once or twice a year. When the diameter reaches 5 cm or more, which increases the risk of rupture, stent placement and replacement with an artificial blood vessel are performed. Such a surgical operation is performed (Non-Patent Document 2). The progression rate of an aortic aneurysm is not constant, and it can develop rapidly and lead to an unexpected rupture.

  If the progress of the aortic aneurysm can be monitored by a method that does not involve exposure, it will be possible to monitor the progress of the aortic aneurysm more finely than before, but it is sufficiently practical as a biomarker that reflects the disease activity of the aortic aneurysm. Nothing is known so far.

  Patent Document 1 describes a method for monitoring elastin and / or collagen degradation in a patient's blood (serum) and / or urine as a biomarker for an aortic aneurysm. However, this biomarker is for determining the presence or absence of an aortic aneurysm and does not reflect the progression of the disease. In addition to Patent Document 1, various biomarkers for determining the presence or absence of an aortic aneurysm have been reported (Non-Patent Documents 3, 4, etc.), but none of them reflects the disease activity of an aortic aneurysm.

  Patent Document 2 discloses a plurality of proteins including tumor necrosis factor α precursor as biomarkers that can predict cardiovascular events. Cardiovascular events include aortic rupture, but are not markers that reflect the progression of aortic aneurysm without rupture. The cardiovascular event targeted by the biomarker of Patent Document 2 includes various diseases such as myocardial infarction, stroke, and peripheral arterial disease in addition to aortic aneurysm rupture, which is a specific marker for aortic aneurysm rupture. not.

  Patent Document 3 discloses a technique for detecting aortic dissection using an antibody against smooth muscle myosin heavy chain. When dissociation occurs, proteins such as smooth muscle myosin heavy chain contained in vascular smooth muscles are temporarily released into the blood in large quantities, and by detecting this, aortic dissection is detected quickly. There is no disclosure or suggestion in Patent Document 3 that smooth muscle myosin heavy chain reflects the progression of an aortic aneurysm without vascular dissociation or rupture.

  Although the blood D-dimer concentration has been reported to correlate with the enlargement rate of the aneurysm diameter (Non-patent Document 5). However, D-dimer generally shows a high value in the state of causing coagulation abnormalities caused by infection, cancer, trauma, etc., so the specificity for aortic aneurysm is low, and D-dimer alone is a disease activity of aortic aneurysm It's not enough to monitor sex. MMP-9 is the most common marker for existing aortic aneurysms, but it is high in certain types of inflammatory diseases and certain cancers. I can't say that. In particular, since it is easy for diseases such as infectious diseases and cancers to occur in elderly people with a frequent age of aortic aneurysms, it is difficult to follow the disease activity of aortic aneurysms only with these known markers.

Special table 2011-510297 gazette Special table 2010-534852 gazette Japanese Patent No. 3499875

Sakalihasan N, et al., Lancet, (365) 1577-1589, 2005 Guidelines for Aortic Aneurysm and Aortic Dissection (revised 2011) Stather PW, et al., Br J Surg, (101) 1358-1372, 2014 Sidloff DA, et al., J Vasc Surg, (59) 528-535, 2014 Golledge J, et al., Eur Heart J, (32) 354-364, 2011

  Therefore, an object of the present invention is to provide a novel means capable of monitoring the disease activity of a non-dissociating aortic aneurysm by a simple method without exposure.

  As a result of diligent research, the inventors of the present application have shown that the blood level of myosin heavy chain 11 is significantly increased in a patient population in which non-dissociating aortic aneurysm is enlarged, and thus the increase in myosin heavy chain 11 is used as an index. The inventors have found that disease activity of non-dissecting aortic aneurysms can be determined, and have completed the present invention.

That is, the present invention is a method for assisting in determining the disease activity of a non-dissociating aortic aneurysm, and measuring the level of myosin heavy chain 11 in a specimen isolated from a patient having a non-dissociating aortic aneurysm And the specimen is blood, serum or plasma, and an increase in myosin heavy chain 11 level in the specimen is an indication that the disease activity of the non-dissociating aortic aneurysm in the patient may be increased Provide a method. The present invention also provides the use of myosin heavy chain 11 as a blood marker for determining disease activity of non-dissociating aortic aneurysms. Furthermore, the present invention provides a blood marker for determining disease activity of a non-dissociating aortic aneurysm comprising myosin heavy chain 11.

  The present invention revealed for the first time that myosin heavy chain 11 can be used as a biomarker reflecting the disease activity of non-dissociating aortic aneurysms. The progress of aortic aneurysm can be monitored using a blood sample that is easy to collect without patient exposure, making it easier to check disease activity more frequently than CT. This makes it possible to predict the risk before sudden rupture of the aortic aneurysm and take necessary measures early. D-dimer and MMP-9 are known as markers reflecting the disease activity of non-dissociating aortic aneurysms, but these markers are also high in infections and cancer. Although non-dissociating aortic aneurysms are common in the elderly, infections and cancers are particularly likely to occur in the elderly, so these markers alone cannot adequately follow the disease activity of the patient's aortic aneurysm. On the other hand, myosin heavy chain 11 is a protein that is specifically expressed in smooth muscle and may be present in organs other than the aorta, but it is a disease commonly seen in elderly people who are particularly prone to non-dissociating aortic aneurysms. Since there are hardly any diseases characterized by cell death of smooth muscle, myosin heavy chain 11 can be said to have a much higher specificity for non-dissociating aortic aneurysms than known markers. ADVANTAGE OF THE INVENTION According to this invention, useful data for determination of disease activity can be provided, and it becomes a big assistance for the disease activity determination of a non-dissociation aortic aneurysm by a doctor.

It is a graph showing the time course of the aortic aneurysm diameter of the aneurysm diameter reduction group (10 persons) and the unchanged group (16 persons) of the aortic aneurysm cases that have undergone stent placement. It is the result of having measured the serum density | concentration of the myosin heavy chain 11 of an aneurysm diameter reduction group. Preoperative (Pre) and 2 weeks (2W), 3 months (3M), 6 months (6M), 12 months (12M), 18 months (18M) and 24 months (24M) after surgery At that time, blood was collected and the serum concentration of myosin heavy chain 11 was measured. It is the result of having measured the serum concentration of the myosin heavy chain 11 of a group without a change. Blood was collected as in the reduced group and the serum concentration of myosin heavy chain 11 was measured. It is the result of having measured the blood level of D-dimer about the reduction group (left, n = 9) and the unchanged group (right, n = 14) of the aortic aneurysm patient who received the stent placement.

  The aortic aneurysm targeted by the present invention is a non-dissociating aortic aneurysm. Aortic aneurysms are generally classified by the disease names of thoracic aortic aneurysm, thoracoabdominal aortic aneurysm, and abdominal aortic aneurysm depending on where they are formed. Classified as aneurysm, arch aortic aneurysm, descending aortic aneurysm. The present invention is applicable to any non-dissociating aortic aneurysm. Although not particularly limited, the aortic aneurysm targeted by the present invention can be, for example, a thoracoabdominal aortic aneurysm or an abdominal aortic aneurysm.

  Among myosin heavy chains, isoforms such as 5, 7, 10, and 11 are known. Of these, myosin heavy chain 11 (Gene ID 4629) is the object of the present invention. Myosin heavy chain 11 is an isoform specifically expressed in smooth muscle and is also called smooth muscle myosin heavy chain. In myosin heavy chain 11 expressed in smooth muscle, two heavy chains together with two types of myosin light chains form a hexameric smooth muscle myosin molecule.

  The myosin heavy chain 11 is mainly composed of four isoforms SM1A, SM1B, SM2A, SM2B (GenBank accession numbers: NP_002465.1, NP_001035203.1, NP_074035.1, NP_001035202.1, amino acids with different C-terminal sequences. The sequences are shown in SEQ ID NOs: 1 to 4, respectively). The isoform of myosin heavy chain 11 measured in the present invention is not particularly limited. For example, all four isoforms may be measured.

  The specimen is preferably blood, serum or plasma. The subject patient is a mammal with a non-dissociating aortic aneurysm, typically a human.

  The level of myosin heavy chain 11 in the sample is measured by measuring the amount of myosin heavy chain 11 protein or fragment thereof in the sample. The myosin heavy chain 11 protein may be measured in the sample as a part of the smooth muscle myosin molecule. Therefore, myosin heavy chain 11 as a marker for determining disease activity of non-dissociating aortic aneurysm includes myosin heavy chain 11 protein in a form constituting a smooth muscle myosin molecule.

  The means for measuring the myosin heavy chain 11 level in the sample is not particularly limited as long as it is a means capable of measuring the polypeptide. Examples of techniques for measuring polypeptides include immunoassays and mass spectrometry. However, immunoassays do not require large-scale equipment and simple measurement procedures. Therefore, in the present invention, the level of myosin heavy chain is 11 levels. It can be preferably used for measurement. Antibodies capable of detecting myosin heavy chain 11 are known and commercially available. In addition, since the amino acid sequence of myosin heavy chain 11 and the base sequence encoding it are known as described above, an antibody that specifically recognizes myosin heavy chain 11 may be prepared and used by a conventional hybridoma method or the like. Good.

  Immunoassays themselves are well known in the art. When immunoassay is classified based on reaction format, there are sandwich method, competitive method, aggregation method, Western blot method, etc., and when classified based on label, enzyme immunoassay, radioimmunoassay, fluorescent immunoassay, etc. is there. In the present invention, any immunoassay method capable of quantitative detection may be used. Although not particularly limited, for example, a sandwich method such as a sandwich ELISA can be preferably used.

  As described above, since the two myosin heavy chains 11 constitute a smooth muscle myosin molecule, the myosin heavy chain 11 is one of the myosin heavy chain 11 proteins that is present in the sample as part of the smooth muscle myosin molecule. It can be measured by a sandwich method using the same antibody that recognizes the same site. Two types of antibodies recognizing different sites of myosin heavy chain 11 when measuring a myosin heavy chain 11 protein monomer or fragment thereof, or a complex containing only one myosin heavy chain 11 by the sandwich method May be used. As described above, four isoforms are known for myosin heavy chain 11. However, if antibodies that recognize regions common to the four types are used, any of the four isoforms can be measured.

  The immunoassay method itself is well known and need not be described in this specification, but briefly described, for example, in the sandwich method, an antibody that binds to myosin heavy chain 11 is immobilized on a solid phase (an immobilized antibody). ), Reacting with the sample, washing, and binding to myosin heavy chain 11 at a site different from the immobilized antibody (however, when measuring myosin heavy chain 11 in the smooth muscle myosin molecule, An antibody that binds to the same site may be reacted with the labeled antibody, and after washing, the labeled antibody bound to the solid phase is measured. Both the immobilized antibody and the labeled antibody may be polyclonal antibodies or monoclonal antibodies. In the immunoassay, an antigen-binding fragment of the antibody can be used instead of the antibody.

  The labeled antibody can be measured by measuring a signal from the labeling substance. The signal measurement method is appropriately selected according to the type of labeling substance. For example, in the case of enzyme labeling, a substrate for the enzyme may be added to the reaction system, and the amount of color development or luminescence generated by the enzyme reaction may be measured using an absorptiometer or luminometer. An immunoassay is performed on a standard sample of known concentration containing myosin heavy chain 11 at various concentrations using an anti-myosin heavy chain 11 antibody or an antigen-binding fragment thereof, and the amount of signal from the label and the myosin weight in the standard sample are measured. A calibration curve is prepared by plotting the correlation with the concentration of the chain 11, and the signal amount from the label is measured by performing the same operation on the sample whose myosin heavy chain 11 concentration is unknown, and the measured value is used as the calibration curve. By applying, myosin heavy chain 11 in the sample can be quantified.

  When both the solid-phased antibody and the labeled antibody are monoclonal antibodies, whether or not a combination of monoclonal antibodies is preferable can be easily examined by actually performing an immunoassay. If desired, the recognition site of the antibody may be identified to see if it recognizes a different epitope. Identification of the recognition site of the antibody can be performed by a conventional method well known in this field. Briefly, for example, the corresponding antigen myosin heavy chain 11 is partially digested with a proteolytic enzyme such as trypsin or the like, and the digested product is passed through the partially digested solution to the affinity column to which the antibody to be examined for the recognition site is bound. The antibody recognition site can be identified by binding and then eluting the bound digest and performing conventional mass spectrometry.

  The blood concentration of myosin heavy chain 11 varies greatly between individuals, and it is difficult to set a cutoff value as an absolute value. Therefore, in the present invention, it is preferable to examine changes in myosin heavy chain 11 level in individuals. By comparison with the patient's own past measurements, it can be determined whether the myosin heavy chain 11 level has increased. That is, the increase in the level of myosin heavy chain 11 in the present invention can be an increase in the level of myosin heavy chain 11 in the sample as compared with the past measurement value of the patient. Whether or not the measured value is higher than the past measured value is usually determined by comparison with a plurality of past measured values including the previous measured value. If there is a sudden rise, the aneurysm may have progressed rapidly, and emergency surgery may be necessary. Check the diameter of the aneurysm by CT etc. as soon as possible and take the necessary treatment. Is desirable. When there is no particular fluctuation or when the fluctuation has decreased, the possibility that the aneurysm has developed is low, and the follow-up may be continued at the same pace. If an upward trend is observed, the aneurysm may have developed, so it is desirable to monitor disease activity carefully, such as by shortening the follow-up visit interval.

  Since myosin heavy chain 11 levels in the specimen are associated with the development of non-dissociating aortic aneurysms, the disease activity of non-dissociating aortic aneurysms in various aspects can be monitored based on myosin heavy chain 11 levels . For example, the present invention can be used to determine the disease activity of an untreated non-dissociating aortic aneurysm before receiving a therapeutic treatment, or to determine the disease activity after a therapeutic treatment such as stent surgery. In other words, patients targeted by the present invention include patients before undergoing therapeutic treatment for non-dissociating aortic aneurysms, patients after therapeutic treatment, and patients undergoing therapeutic treatment. The therapeutic treatment for an aortic aneurysm includes conventional surgical treatment such as stent placement and artificial blood vessel replacement, and therapeutic treatment with a therapeutic agent that will be established in the future. In addition, the patient targeted by the present invention is mainly an aortic aneurysm patient before the rupture occurs, but also includes a patient for whom it is desired to monitor the disease activity of the aneurysm after treating the rupture of the aortic aneurysm. Determination of disease activity of an aneurysm in a patient after treatment or during treatment can also be expressed as determination of the therapeutic effect of the treatment (prognosis determination of the patient).

  The present invention can also be used for determination of therapeutic effects of therapeutic drug candidate substances in clinical trials and the like. Specifically, for example, a specimen is collected from an animal that has been administered a therapeutic drug candidate substance, and the myosin heavy chain 11 level in the specimen is measured. Samples are also collected from animals prior to administration and the myosin heavy chain 11 level prior to administration is also measured. If the measured value after administration is unchanged or decreased compared to the measured value before administration, it can be judged that the activity of the aneurysm has not increased, so that the drug suppresses the expansion of the aneurysm It can be determined that there is an effect of reducing an aneurysm. This is expected to accelerate the development of new therapeutic agents for aortic aneurysms. In the present invention, treatment of an aortic aneurysm includes suppression of enlargement of the aortic aneurysm and reduction of the aortic aneurysm.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples. All experiments were conducted with the approval of the Ethics Committee.

<Method>
Specimens Serum samples collected from 26 patients who had undergone stent implantation were used as treatments for non-dissociating aortic aneurysms in the abdomen or thoracoabdominal region. For each patient, blood was collected before surgery and at 2 weeks, 3 months, 6 months, 12 months, 18 months, and 24 months after surgery (from July 8, 2008 to 2010 4). Serum was prepared (until month 12). Cases where the diameter of the aneurysm decreased by 10% or more compared to preoperative 12 months after the operation were classified as a reduced group, and cases where no decrease was observed were classified as no change. There were 16 cases (Fig. 1).

Determination of serum myosin heavy chain 11 by sandwich ELISA The concentration of serum myosin heavy chain 11 was measured by sandwich ELISA using a commercially available anti-myosin heavy chain 11 antibody (CUSABIO) as a solid phase antibody and a labeled antibody. . HRP was used as a label for the anti-myosin heavy chain 11 antibody, and TMB was used as a substrate for detection, and color development was detected with a luminometer. A standard curve containing smooth muscle myosin at various concentrations was prepared, and applied to this standard curve to calculate the myosin heavy chain 11 concentration in each sample. Smooth muscle myosin used the recombinant protein attached to the myosin heavy chain 11 ELISA kit (CUSABIO).

  As for the recognition region of the anti-myosin heavy chain 11 antibody used here, the entire length of the myosin heavy chain 11 isoform SM2B (SEQ ID NO: 4) is roughly divided into half and the N-terminal fragment (R1) and C-terminal fragment. When (R2) was prepared and the reactivity with each fragment was examined by ELISA, the antibody did not react with R1, but reacted only with R2. From this, it was confirmed that the antibody recognized any site in the C-terminal half region of myosin heavy chain 11.

Statistical processing In FIG. 1, Dunn's Multiple Comparison test was performed as Kruskal-Wallis test and post test, and statistical analysis was performed on pre. 2 to 4 performed Wilcoxon matched pairs test. In both cases, p <0.05 was considered significant by two-sided test.

<Result>
The results of measuring the serum concentration of myosin heavy chain 11 in the reduced group are shown in FIG. In the aneurysm-reduced group, the blood concentration of myosin heavy chain 11 tended to decrease after 3 months from the operation (left of FIG. 2). For 9 cases with pre- and post-operative 12-month serum samples, the change in blood concentration of myosin heavy chain 11 for each case shows that there is a large individual difference in the absolute value of the concentration. The decrease in blood concentration after surgery was significant (p = 0.0117, right in FIG. 2).

  The results of measuring the serum concentration of myosin heavy chain 11 in the unchanged group are shown in FIG. The blood concentration did not tend to decrease over time (Figure 3 left), and no significant difference was observed in the blood concentration before and 12 months after surgery (p = 0.3013, Figure 3 right).

<Comparative example>
As a comparative example, the blood concentration of D-dimer (Non-patent Document 5), which has been reported to correlate with the enlargement rate of the aneurysm diameter, was also measured. D-dimer concentration was quantified by latex immunoturbidimetry in serum samples before and 12 months after abdominal aortic aneurysm patients who received the above-mentioned stent placement.

  The results are shown in FIG. There was no significant change in the D-dimer concentration before and after treatment in either the group in which the diameter of the aortic aneurysm was reduced by stent placement or in the group in which the diameter of the aneurysm did not change.

Claims (7)

A method for assisting in determining disease activity of a non-dissociating aortic aneurysm, comprising measuring myosin heavy chain 11 level in a specimen isolated from a patient having a non-dissociating aortic aneurysm, wherein the specimen is blood Serum or plasma, wherein an increase in myosin heavy chain 11 level in the specimen is an indication that the disease activity of the non-dissociating aortic aneurysm in the patient may be increased. Increase in myosin heavy chain 11 level, the is an increase compared to the previous sample in the myosin heavy chain 11 measurements of the patient, method of claim 1. The method according to claim 1 or 2 , wherein the non-dissociating aortic aneurysm is an abdominal aortic aneurysm or a thoracoabdominal aortic aneurysm. The method according to any one of claims 1 to 3 , which is a method for assisting in determining the effect of a therapeutic treatment on a non-dissociating aortic aneurysm. The method of any one of Claims 1 thru | or 3 which is a method which assists the effect determination of the therapeutic drug candidate substance of a non-dissociating aortic aneurysm. Use of myosin heavy chain 11 as a blood marker for determining disease activity of non-dissociating aortic aneurysms. A blood marker for determining disease activity of non-dissociating aortic aneurysm, comprising myosin heavy chain 11.

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