JP2007322187A - Method for judging risk of restenosis development - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for judging the risk of restenosis development after coronary artery plasty. <P>SOLUTION: The method for judging the risk of the restenosis development of a subject subjected to coronary artery reconstructive surgery includes a process (a) for measuring the modified low specific gravity lipoprotein (modified LDL) level in the biosample of the subject and a process (b) for comparing the measured value of modified LDL with a predetermined reference value. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for determining the risk of developing restenosis after coronary artery reconstruction surgery in a subject undergoing coronary artery reconstruction surgery.

Among patients with percutaneous coronary artery reconstruction (PCI), which is the most commonly used treatment for coronary artery disease (CAD), patients who have undergone the most common stent implantation treatment are around 20 to 40% 3 to 6 months after treatment. It has become a big problem that restenosis occurs with the frequency of (refer nonpatent literatures 1-3).
There are the following methods (1) to (3) as methods for inspecting restenosis, but each has its own problems.
(1) Coronary angiography: Usually, it is performed twice after PCI to examine restenosis, but it is an invasive examination, especially in patients with diabetes mellitus (DM). Is a problem.
(2) Exercise electrocardiogram: The sensitivity of restenosis detection is low, and this alone is not sufficient. In addition, there is a risk that this test may cause a heart accident such as angina pectoris due to exercise load, and this test cannot be performed by elderly people and persons with motor disabilities.
(3) Cardiac CT (multi-slice CT) examination: Although it has become widespread in recent years, it is difficult to observe restenosis at a stent insertion site.
All of these tests are burdensome to the patient and are for confirming the state after the formation of restenosis, and the risk of developing restenosis cannot be determined.

On the other hand, it is known that the frequency of restenosis is higher in diabetic (DM) patients than in non-DM patients (26.3% in non-DM but 36.8% in DM) (non-patent document 1), but restenosis The parameters that determine the risk of the development of the disease are not clear, and age, sex, smoking, hyperlipidemia, and dialysis patients are not associated with the development of restenosis (non-patent literature) 4). In addition, in the case of DM patients, there are many asymptomatic patients who are unaware of chest symptoms even when restenosis occurs, and therefore development of a method for determining the risk of developing restenosis has been strongly desired.
Methods for determining the risk of developing restenosis include a method for measuring factors that lead to abnormal accumulation and activation of monocytes / macrophages at the site of arterial wall injury after coronary artery reconstruction treatment (Patent Document 1), coronary artery reconstruction Although methods for measuring human lipocalin-type prostaglandin D synthase in body fluid samples after surgical treatment (Patent Document 2) have been reported, these are also measured after coronary artery reconstruction surgery, and restenosis It was not practical in terms of predicting the risk of onset or early judgment.
JP-A-9-54090 JP 2000-249709 A Katsumi Miyauchi: Coronary revascularization and prognosis for coronary artery disease complicated by abnormal glucose metabolism, heart, 37: 280-286, 2005 Toshihiro Tamura, Tsuyoshi Kimura, Restenosis after Coronary Intervention-Mechanism and Treatment-: Internal Medicine, 93, 890-896, 2004 Katsumi Miyauchi, Takayuki Yokoyama, Hiroyuki Shirota: Oxidative stress and restenosis, Vascular medicine, 2, 80-87, 2001 Naoyuki Yokoyama, Takaaki Isshiki, diagnosis and treatment of restenosis in outpatients: Heart View Vol.9 No.1 2005 p8-12

  An object of the present invention is to provide a method capable of determining the risk of developing restenosis in a subject who has undergone coronary artery reconstruction surgery without the above-described problems.

  As a result of diligent investigations to solve the above problems, the present inventors have determined that modified low-density lipoproteins measured before and / or after treatment of patients undergoing PCI treatment stent implantation treatment in DM patients The present invention was completed by finding that the risk of developing restenosis after the treatment can be determined by comparing the (modified LDL) value with a predetermined reference value. In the present specification, “risk risk of restenosis” refers to the possibility of restenosis formation, and “determinable” means that the state after restenosis formation is not confirmed as in the prior art. It means predicting or discriminating the possibility of the formation (onset) of stenosis.

That is, the present invention is a method for determining the risk of developing restenosis in a subject who has undergone coronary artery reconstruction surgery,
(A) measuring a modified low density lipoprotein (modified LDL) in a biological sample derived from a subject;
(B) comparing the measured value of the modified LDL with a predetermined reference value;
A method characterized by comprising:

  Further, the present invention is a method for determining a treatment protocol after performing coronary artery reconstruction surgery in a subject undergoing coronary artery reconstruction surgery, comprising the steps (a) and (b). Is also provided.

When the degenerative LDL value is lower than a predetermined reference value according to the method for determining the risk of developing restenosis according to the present invention, conventional examinations (such as coronary angiography examinations and exercise electrocardiogram examinations) that impose a heavy burden on patients Excessive repetition can be prevented.
On the other hand, when the modified LDL value is higher than a predetermined reference value, oversight of restenosis can be prevented by positively examining. In addition, there is a possibility that restenosis can be prevented by aggressive treatment with statins and the like for which there is evidence for diet therapy and restenosis prevention.

  The present invention relates to a method for determining the risk of developing restenosis in a subject who has undergone treatment for coronary artery reconstruction. As the coronary artery reconstruction, the most common stent implantation of percutaneous coronary artery reconstruction (PCI) is provided. Not only surgery, but also balloon-only coronary artery dilatation (POBA: plain old balloon angioplasty), a cutting balloon with 2 to 3 blades on the surface of a conventional balloon catheter, atherectomy to remove coronary atheroma, atheroma The present invention can be applied to any case, and preferably a stent implantation treatment.

  The present invention includes the following steps (a) and (b).

(A) Step of measuring the level of denatured low density lipoprotein (denatured LDL) in the biological sample of the subject As oxidized LDL, oxidized LDL is preferred. Examples of oxidized LDL include malondialdehyde-modified LDL (MDA-LDL), oxidized LDL containing oxidized phospholipid, and a complex of oxidized LDL and protein. Of these, malondialdehyde modified LDL (MDA-LDL) is more preferable.

  As a method for measuring denatured LDL, an immunological assay for detecting oxidized LDL or a complex of oxidized LDL and protein (for example, a method using monoclonal antibody FOH1a / DLH3 (Itabe H. et al., J Lipid Res. 1996, 37 (1), 45-53.), Method using monoclonal antibody 4E6 (Holvoet P. et al., Circulation. 1998, 98 (15), 1487-94.) Etc.), ion exchange chromatography A known method such as a measurement method (Shimano H. et al., J Lipid Res. 1991, 32 (5): 763-73.) Can be used. Among these, as a method for measuring MDA-LDL, the method described in Japanese Patent No. 3115587 is preferable, and in order to increase the specificity of MDA-LDL measurement in this method, MDA-LDL and LDL are configured. It is particularly preferred to use a sandwich enzyme immunoassay with an antibody that recognizes apoB. That is, either a monoclonal antibody that recognizes MDA-LDL or an apo B recognition antibody is immobilized on an insoluble carrier, the other is labeled with an enzyme, and these are brought into contact with a specimen to perform a sandwich enzyme immunoassay. A method of measuring -LDL is preferred. According to this method, the possibility of detecting an MDA-modified protein other than MDA-LDL can be eliminated.

  Examples of the biological sample used in the present invention include blood, plasma, and serum. Among them, serum is preferable. Biological samples may be collected before or after coronary artery reconstruction surgery, or may be collected twice or more before and after, but the risk of developing restenosis is determined early. From the viewpoint, the sample before the treatment is preferable. In addition, when the subject is a diabetic patient, the risk of developing restenosis is high, and the method of the present invention is extremely effective for early diagnosis and treatment to prevent aggravation.

(B) Step of comparing the measured value of the modified LDL with a predetermined reference value Here, the reference value is, for example, a follow-up survey of whether or not restenosis has occurred in a plurality of subjects whose modified LDL value has been confirmed in advance. It is possible to set by analyzing the result of using a statistical analysis method. For example, the reference value can be set using the same statistical analysis method in each case of the modified LDL value before PCI treatment or the modified LDL value after a certain period of time has elapsed after PCI treatment. . If the subject's modified LDL value before PCI treatment is higher than the reference value determined from the modified LDL value before PCI treatment, and / or the modified LDL value after PCI treatment is from the modified LDL value after PCI treatment If it is higher than the established reference value, it can be determined that there is a risk of developing restenosis, but from the viewpoint of determining the risk of developing restenosis at an early stage, restenosis can be determined based on the modified LDL value before PCI treatment. It is preferable to determine the risk of developing the disease.

An example of the above statistical analysis method is analysis using a Receiver-operating characteristics (ROC) curve, and both sensitivity and specificity for identifying a restenosis group are high (the difference between the two indicates a minimum value). ) Value can be used as a reference value. The reference value can be set to an optimum value for each request / situation in consideration of the request / situation in the clinical field such as prediction accuracy and preventive effect.
In general, restenosis is formed 3 to 6 months after coronary artery reconstruction, so confirmation imaging is performed after 3 and 6 months to diagnose the presence or absence of restenosis. Therefore, it is possible to determine the risk of restenosis with higher accuracy by monitoring the degenerated LDL level from before PCI treatment until 6 months after treatment in which confirmation imaging is performed.
In this way, since the risk of restenosis in patients undergoing PCI can be determined with high accuracy, it becomes easy to determine a treatment protocol after PCI treatment. That is, for patients at high risk of developing restenosis: Provide more active lifestyle habit improvement guidance such as diet therapy. Restenosis can be prevented by administering a statin drug for which restenosis prevention evidence is being prepared. Moreover, the risk of missing asymptomatic restenosis can be reduced by performing coronary angiography or the like as appropriate.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these.
Example 1 (Standard value setting for MDA-LDL-1)

Method
Among the PCI treatments for CAD, blood samples were collected immediately before PCI treatment for DM patients undergoing stent implantation, and MDA-LDL was measured. (Total 44 cases) The collection, storage, and measurement of serum samples for MDA-LDL measurement were performed as follows.

reagent
(1) Antibody binding plate: anti-MDA-LDL mouse monoclonal antibody (ML25) binding well
(2) Enzyme-labeled antibody solution: β-galactosidase-labeled anti-apo B mouse monoclonal antibody 2.86 μg / mL
(3) Washing solution: 20 mM phosphate buffer (pH 7.2)
(4) Substrate solution: 10 mM o-nitrophenyl-β-D-galactopyranoside
(5) Stop solution: 200 mM sodium carbonate
(6) Sample dilution: 25 mM HEPES buffer (pH 7.8)
(7) Standard solution: Serum with known MDA-LDL concentration ^*
*: Human LDL was artificially modified with MDA to prepare MDA-LDL, and its protein concentration was determined. In the above measurement system, MDA- in serum showing the same absorbance as 1 mg / mL of the prepared MDA-LDL LDL concentration was defined as 1 U / L, and pricing was performed (literature Kotani K. et al., Biochim Biophys Acta, 1994, 1215, 121-5).

Operation method <Sample preparation method>
The sample used was fresh serum, and the sample was diluted with the sample diluent so that the final dilution rate was 2000 times, and allowed to stand at 15 to 30 ° C. for 1 hour to prepare a sample. Specifically, 980 μL of the sample diluent was added to 20 μL of the sample, 780 μL of the sample diluent was added to 20 μL of the 50-fold diluted solution, and further diluted 40 times. This solution was allowed to stand at 15 to 30 ° C. for 1 hour and then used as a sample.

<Measurement>
(1) All reagents were returned to room temperature (15-30 ° C.).
(2) 300 μL of the washing solution is dispensed into each well of the antibody binding plate and then removed. This operation was performed three times to wash the wells. The following operation was performed before the inside of the well was dried.
(3) 100 μL of each sample or standard solution was dispensed into each well and allowed to stand at 15-30 ° C. for 2 hours. The sample and standard solution were subjected to double measurement.
(4) The liquid in the well was removed, and 300 μL of the washing solution was dispensed into each well, followed by washing three times as in (2).
(5) The washing solution was removed, 100 μL of enzyme-labeled antibody solution was dispensed into each well, and left at 15-30 ° C. for 1 hour.
(6) The liquid in the well was removed, 300 μL of the washing solution was dispensed into each well, and then washed three times as in (2).
(7) The washing solution was removed, 100 μL each of the substrate solution was dispensed into each well, and the plate was allowed to stand at 15-30 ° C. for 2 hours.
(8) Further, 100 μL of stop solution was dispensed into each well to stop the reaction.
Using a microplate reader, the absorbance at a primary wavelength of 415 nm and a secondary wavelength of 600 nm was measured using a reagent blank as a control. In addition, the reagent blank used the liquid similarly operated using the sample dilution liquid instead of the sample.

<Calculation method of MDA-LDL concentration>
(1) A standard curve was prepared by plotting the absorbance on the vertical axis and the MDA-LDL concentration on the horizontal axis for the standard solution.
(2) The concentration corresponding to the absorbance of the sample was read from a calibration curve to determine the MDA-LDL concentration.
Further, total cholesterol (TC), LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), and neutral fat (TG) were measured using an in vitro diagnostic drug based on the enzyme principle.
The restenosis was judged as restenosis when 50% or more of the target lesion was found by coronary angiography.

result
Of 44 cases where MDA-LDL was measured before PCI treatment, 14 cases (32% of 44 cases) developed restenosis after 2-9 months), 30 cases did not develop (68/44 cases) %)Met. The mean ± SD of the age in the stenosis group and the non-stenosis group was 63.2 ± 8.1 years and 68.1 ± 9.1 years, respectively, and there was no significant difference between the two groups (Table 1). MDA-LDL measured mean ± SD before PCI treatment was 138.4 ± 46 U / L in the stenosis group and 102 ± 47.4 U / L in the non-stenosis group, which was significantly higher in the restenosis group than in the non-stenosis group (P <0.05, t test). The TC measured at the same time was 176.4 ± 35.2 in the restenosis group, 187.4 ± 41 mg / dL in the non-stenosis group, and LDL-C was 110.7 ± 28.2 mg / dL in the restenosis group, 115 ± 28.5 in the non-stenosis group. It was mg / dL, and neither TC nor LDL-C showed a significant difference between the two groups (see FIG. 1 and Table 1).

  For the MDA-LDL value before PCI treatment, a reference value for identifying restenosis was calculated by analyzing Receiver-operating characteristics (ROC) curves for 14 groups of restenosis group and 30 groups of non-restenosis group (FIG. 2). Both sensitivity and specificity for identifying the restenosis group are high (the MDA-LDL value that shows the minimum difference between them is 110 U / L, and when this is used as the reference value, the MDA-LDL that identifies the restenosis group) The sensitivity (prevalence of correct diagnosis) was 79%, and the specificity (prevalence of correct diagnosis) was 77% (Table 2).

  Since this study was a prospective study, it was calculated as a relative risk of 5.3 and a 95% confidence interval of 1.7 to 16.3 (Table 2). Since the 95% confidence interval is greater than 1, it is judged to be a significant result. When the MDA-LDL value is 110 U / L or higher, the incidence of restenosis is 5.3 times higher than that of less than 110 U / L. It has been shown.

  When MDA-LDL before PCI treatment is 110U / L or higher, it is a risk of restenosis with a relative risk of 5.3, so the risk of subsequent restenosis due to MDA-LDL value before PCI treatment Can be determined.

Example 2 (Setting of MDA-LDL reference value-2)
As in Example 1, blood was collected immediately before the confirmation contrast examination for judging the presence or absence of restenosis 2 to 9 months after PCI treatment, and MDA-LDL was measured (total of 67 cases).

result
Of the 67 cases where MDA-LDL was measured during confirmation imaging followed 2 to 9 months after PCI treatment, 26 cases showed restenosis by confirmation imaging, and 41 cases did not show stenosis It was. The mean ± SD of the age in the stenotic group and the non-stenotic group was 66.1 ± 9.6 years and 66.3 ± 10.3 years, respectively, and there was no significant difference between the two groups (Table 3). The MDA-LDL measurement mean SD during confirmation contrast imaging was 114.9 ± 37.5 U / L in the stenosis group and 92.2 ± 29.4 U / L in the non-stenosis group, which was significantly higher in the restenosis group than in the non-stenosis group. (P <0.01, t test). On the other hand, the TC measured at the same time was 181.0 ± 30.3 in the restenosis group, 172.8 ± 29.7 mg / dL in the non-stenosis group, LDL-C was 110.9 ± 21.6 mg / dL in the restenosis group, and 102.1 ± in the non-stenosis group. It was 22.5 mg / dL, and there was no significant difference between the two groups for both TC and LDL-C (FIG. 3, Table 3).

  Regarding the MDA-LDL value at the time of confirmation contrast, the reference value for identifying restenosis was calculated by analyzing the ROC curve in the same manner as in Example 1 for 2 groups of 26 cases of restenosis group and 41 cases of non-restenosis group did. MDA-LDL value is 100U / L with high sensitivity and specificity to identify restenosis cases (the difference between the two shows the minimum value). When this is used as the reference value, MDA- The sensitivity (prevalence rate of prevalence) of LDL was 73%, and the specificity (prevalence rate of no disease) was 71% (Table 4).

  Since this study was a case-control study, the odds ratio was 6.6 and the 95% confidence interval was 2.2 to 19.6 (Table 4). Since the 95% confidence interval is greater than 1, it can be judged as a significant result, and when the MDA-LDL value is 100 U / L or more, the association with restenosis is 6.6 times higher than the case of less than 100 U / L It has been shown.

In addition to the MDA-LDL level before PCI treatment, if the MDA-LDL level was higher than 100 U / L at the time of confirmation imaging, an odds ratio of 6.6 was associated with restenosis. It can be seen that the risk can be judged.
The reference value that distinguishes the restenosis group from the non-restenosis group is 110 U / L before PCI treatment, whereas it is 100 U / L during confirmation angiography, which tends to be lower during confirmation angiography than before PCI treatment. Admitted. This seems to have been due to a decrease in MDA-LDL levels due to treatment for reducing coronary risk factors after PCI treatment.
In this study, age, TC, and LDL-C, which are risk factors for CAD, were not significantly different between the restenosis group and the non-restenosis group, both before and after PCI treatment. Only LDL was significantly different. These results also confirmed that the method for determining the risk of developing restenosis according to the present invention is useful.

  The present invention is useful as a method for determining the risk of restenosis after coronary angioplasty.

It is a figure which shows the comparison of the MDA-LDL value in the restenosis group of a DM patient who performed PCI treatment, and a non-restenosis group. It is a figure which shows the ROC analysis of the MDA-LDL value before PCI treatment regarding discrimination of a restenosis group. It is a figure which shows the comparison of the MDA-LDL value in the restenosis group of a DM patient who performed PCI treatment, and a non-restenosis group.

Claims (9)

A method for determining the risk of developing restenosis in a subject who has undergone coronary reconstruction surgery,
(A) measuring a modified low density lipoprotein (modified LDL) in a biological sample derived from a subject;
(B) comparing the measured value of the modified LDL with a predetermined reference value;
A method comprising the steps of:   The method according to claim 1, wherein the biological sample is collected before and / or after coronary artery reconstruction treatment.   The method according to claim 1 or 2, wherein the subject is a diabetic patient.   The method according to any one of claims 1 to 3, wherein the coronary artery reconstruction is percutaneous coronary artery reconstruction (PCI).   The method according to any one of claims 1 to 4, wherein the modified LDL is oxidized LDL.   The method according to any one of claims 1 to 5, wherein the modified LDL is malondialdehyde-modified LDL (MDA-LDL).   The method according to any one of claims 1 to 6, wherein the biological sample is selected from blood, plasma and serum.   The method according to any one of claims 1 to 6, wherein the predetermined reference value is a value obtained by using a statistical analysis method from degenerated LDL values in two groups of a restenosis group and a non-restenosis group. . A method of determining a treatment protocol after performing coronary reconstruction treatment in a subject undergoing coronary reconstruction treatment,
(A) measuring a modified low density lipoprotein (modified LDL) in a biological sample derived from a subject;
(B) comparing the measured value of the modified LDL with a predetermined reference value;
A method comprising the steps of:

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