JP7359775B2 - Prediction of progression or risk of cardiovascular disorders or thrombus formation - Google Patents

Description

The present invention relates to a method for providing data for predicting the progression of, or the risk of, cardiovascular disorders or thrombus formation. The present invention also relates to a method for evaluating the risk that a drug or medical device will cause circulatory damage or thrombus formation in a living body.

When plaque ruptures due to a decline in vascular endothelial function or progression of arteriosclerosis, a thrombus is formed, resulting in narrowing or occlusion of the vascular lumen. As a result, organ failure due to circulatory disorders such as myocardial infarction, cerebral infarction, and terminal artery occlusion develop, which has a significant impact on life prognosis, which is a problem. Furthermore, if the vascular endothelium is damaged by medical interventions such as administration of substances or drugs, or insertion or placement of medical devices into the blood vessel for treatment or diagnosis, the risk of blood clot formation increases, leading to narrowing of the vascular lumen. It is known that it can even cause blockage and cause circulatory system disorders. Examples of medical interventions that involve inserting or indwelling medical devices for the purpose of treatment or diagnosis include endovascular treatment such as balloons, indwelling in blood vessels such as stents, covered stents, and stent grafts, and transplantation of artificial blood vessels. Injury to vascular endothelial cells caused by these medical procedures usually heals naturally, but due to the biocompatibility of medical devices and the sustained release period of drugs, vascular endothelial cells remain injured, resulting in delayed onset. The problem is that it causes deterioration in long-term clinical outcomes such as sexual thrombosis.

Arteriosclerotic lesions, which are the main cause of vascular occlusive diseases that cause cardiovascular disorders, have two aspects: vascular dysfunction and plaque formation (Guidelines for non-invasive evaluation methods of vascular function, JCS 2013). Advances in diagnostic imaging are making it possible to accurately evaluate plaque detection. However, plaques do not necessarily rupture and cause cardiovascular disorders, and most plaque ruptures occur at lesion sites that do not have severe stenosis. For this reason, it is difficult to predict circulatory disorders due to plaque rupture using commonly performed angiography. Therefore, methods have been developed to detect plaques that are prone to rupture (ie, unstable plaques). Methods of detecting unstable plaque using intravascular ultrasound (IVUS), optical coherence tomography (OCT), and angioscopy are currently being considered, but currently there is no evidence of its rupture, thrombus formation, and their associated effects. It has not yet been possible to predict the circulatory disorders that will occur. On the other hand, vascular function tests to evaluate vascular dysfunction include vascular endothelial function tests (plethysmography, flow-mediated dilatation: FMD, reactive hyperemia peripheral arterial tonometry: RH-PAT), pulse wave velocity (PWV), cardiac ankle Vascular index (CAVI), stiffness parameter, central blood pressure, augmentation index (AI), second derivative of photoplethysmogram (SDPTG), ankle brachial index (ABI), toe brachial index (ABI), index: TBI), some of which are used clinically, but they have not yet been used to predict cardiovascular disorders.

By the way, primary thrombus is triggered by von Willebrand factor (vWF) on endothelial cells or binding of vWF to collagen exposed at the site of vascular injury, and platelets bind to this vWF via GP1b/V/IX. Furthermore, fibrin is formed by binding to platelets via GPIIb/IIIa, and this primary thrombus grows, leading to blood vessel occlusion and stenosis. That is, the presence of vWF in the blood vessel wall is important for the formation of thrombus that causes blood vessel stenosis and occlusion. On the other hand, in vivo, in situations where a thrombus significantly grows into the blood vessel lumen and occludes it, the thrombus (ultra-high molecular weight vWF multimer) has a high It is known that when shear stress is applied, the three-dimensional structure of vWF changes, and ADAMTS13 decomposes vWF to prevent occlusion by thrombus. However, ADAMTS13 cannot degrade vWF, which has its native three-dimensional structure, under normal blood flow, and in mice in which the ADAMTS13 gene has been knocked out, nothing appears to occur under normal conditions (Motto DG et al., J Clin Invest. 2005; 115 (10): 2752-61 and Banno F et al., Blood. 2009; 113 (21): 5323-9). Based on these findings, it is thought that ADAMTS13 decomposes excessively enlarged thrombi and prevents stenosis and occlusion of blood vessels, but it does not physiologically decompose vWF to control primary thrombi. This is thought to be a factor. In recent years, it has been reported that ADAM28 degrades vWF in cancer cells (see Mochizuki S et al., J Natl Cancer Inst. 2012; 104 (12): 906-22); There are no reports of any involvement.

In order for a vascular function test method to serve as a biomarker in cardiovascular disease management, it must (1) be able to determine the degree of progression of vascular dysfunction, (2) be able to estimate the risk of onset or prognosis of cardiovascular disease, and (3) require intervention. (4) improved results will lead to improved prognosis (see the guidelines above). In order for such a vascular function testing method to be applied clinically, it must be (1) non-invasive and easy to measure, (2) universally applicable at low cost, and (3) accurate and reproducible. Indicators such as (4) that the measurement method can be standardized are important. On the other hand, regarding vascular function testing methods, there is still a problem that a certain opinion has not been determined regarding measurement methods, interpretation of results, clinical significance, clinical application, etc.

According to vascular function tests, it is possible to non-invasively detect arterial dysfunction at the asymptomatic stage, more accurately assess the risk of developing circulatory disorders in the future, and proactively intervene to prevent events. It is possible that it can be linked to However, there is currently no consensus regarding its usefulness, and in the future it will be necessary to confirm that therapeutic intervention improves vascular function tests and that this improvement serves as a surrogate marker for reducing the risk of cardiovascular disorders. be. Furthermore, conventional vascular function testing requires dedicated testing equipment. Therefore, with the aim of predicting stenosis and occlusion of blood vessels without using specialized testing equipment, biomarkers in the blood are being searched for, including highly sensitive CRP, Lp-PLA2, and pentraxin 3 (PTX3). It has been reported that these molecules may function as markers. However, these markers do not necessarily specifically reflect endothelial injury. As described above, it is currently difficult to accurately predict circulatory disorders due to vascular occlusion or stenosis, and this often results in delayed therapeutic intervention and poor prognosis. Therefore, there is a need for a method for predicting the progression of thrombus formation, stenosis and occlusion of blood vessels, and even circulatory disorders with high accuracy using a simple method.

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a means that makes it possible to predict the progression of thrombus formation, stenosis and occlusion of blood vessels, and even circulatory disorders with high accuracy using a simple method.

The present inventors conducted extensive studies to solve the above problems. As a result, it was surprisingly revealed for the first time that in a state where endothelial function is reduced, the expression level of vWF factor in endothelial cells increases and the expression level of ADAM28 decreases. Based on this knowledge, we have completed the present invention.

That is, according to one embodiment of the present invention, the expression level or concentration of ADAM28 in a blood sample collected from a subject, or the ratio of the expression level or concentration of ADAM28 to vWF (ADAM28/vWF ratio) is used as a marker to A method for providing data for predicting the progression of a disorder or thrombus formation or the presence or absence of a risk thereof, the method providing data for predicting the progression of a circulatory disorder or thrombus formation or the presence or absence of a risk thereof, wherein the marker value is lower than a reference value. A method is provided that predicts that there is a risk.

According to another aspect of the present invention, there is provided a method for evaluating the risk of a drug or medical device causing circulatory disorder or thrombus formation in a living body, the method comprising: , the expression level or concentration of ADAM28 or ADAMTS13 in the culture solution or cells after culturing the cells in the presence of the drug or medical device, comprising culturing the cells while applying mechanical stress to the cells. , the ratio of the expression amount or concentration of ADAM28 to vWF (ADAM28/vWF ratio), or the ratio of the expression amount or concentration of ADAMTS13 to vWF (ADAMTS13/vWF ratio), Also provided is a method for evaluating that the drug or medical device has a risk of causing circulatory damage or thrombus formation in a living body when the value is lower than the value in the culture solution or cells after culturing.

FIG. 2 is an explanatory diagram for explaining a mechanical stress culture device used in a method for evaluating the risk that a drug or medical device according to one embodiment of the present invention causes circulatory disorder or thrombus formation in a living body. 2 is a photograph for explaining a state in which cell culture units in the mechanical stress culture apparatus shown in FIG. 1 are connected in a curved state.

Embodiments of the present invention will be described below with reference to the attached drawings.

≪Prediction of the progression of cardiovascular disorders or thrombus formation, or the presence or absence of the risk≫
One aspect of the present invention relates to a method of predicting the progression of, or risk of, cardiovascular disorders or thrombus formation, or providing data for predicting the presence or absence thereof. More specifically, the method uses the expression level or concentration of ADAM28 in a blood sample collected from a subject, or the ratio of the expression level or concentration of ADAM28 to vWF (ADAM28/vWF ratio) as a marker to detect cardiovascular disorders or thrombosis. This is a method of predicting the progression of formation or the presence or absence of its risk, or providing data for predicting its presence or absence. Here, in this method, when the value of the marker is lower than the reference value, it is predicted that circulatory disorder or thrombus formation is progressing or there is a risk thereof. According to the method according to this embodiment, it is possible to predict the progress of thrombus formation, stenosis and occlusion of blood vessels, and even circulatory disorders with high accuracy using a simple method.

As mentioned above, the present inventors aimed to develop a means that could make it possible to predict the progression of thrombus formation, stenosis and occlusion of blood vessels, and even circulatory disorders with high accuracy using a simple method. , we conducted a thorough study. In this process, the present inventors first determined that under conditions where vascular endothelial cells are not subjected to mechanical stress (corresponding to a state in which blood flow is not flowing in the blood vessels), and under conditions where mechanical stress is applied to vascular endothelial cells, (equivalent to blood flowing through blood vessels), the expression level of eNOS (NO synthase in vascular endothelial cells), which is a marker of endothelial function, decreased, and endothelial function decreased. did. They also revealed for the first time that in a state in which endothelial function is so degraded that the expression level of eNOS is reduced, the expression level of vWF in endothelial cells increases, while the expression level of ADAM28 decreases. Here, ADAMTS13, which has been thought to be a physiological degrading enzyme for vWF, exists only in a soluble form, and as mentioned above, it degrades vWF in its native three-dimensional structure under normal blood flow. I can't. On the other hand, ADAM28 exists in a membrane-bound form in addition to a soluble form, and is also capable of degrading vWF having its native three-dimensional structure. These findings indicate that the enzyme that physiologically controls the degradation of vWF is ADAM28, not ADAMTS13, as previously thought, and in particular, membrane-bound ADAM28 efficiently degrades vWF present on the same cell membrane. It is presumed that this prevents the formation of primary thrombus. Furthermore, since the binding of vWF to the blood vessel wall is the first step in thrombus formation, ADAM28 plays an important role as a gatekeeper for thrombus formation, and by suppressing thrombus formation in a healthy state, It is thought that this prevents occlusion and stenosis. Furthermore, once endothelial function declines due to a decrease in mechanical stress, the expression of vWF, which triggers primary thrombus formation, increases, and the expression of ADAM28, which degrades it, decreases, promoting thrombus formation. It is thought that occlusion/stenosis of blood vessels occurs.

From the above, by using the expression level (or concentration) of ADAM28 in a blood sample collected from a subject or the ratio of the expression level (or concentration) of ADAM28 to vWF (ADAM28/vWF ratio) as a marker, it is possible to It is believed that it is possible to predict that there is a risk or development of a disorder or thrombosis. According to the above-described embodiment of the present invention, the above-mentioned parameters are used as markers, and when the value of the marker is lower than a reference value, it is predicted that there is a risk of progression of circulatory disorder or thrombus formation. be done. In this way, when the value of the above marker is lower than the reference value, vWF, which is a trigger for primary thrombus formation, accumulates in the patient's blood vessel lumen, and as a result, a thrombus develops within the blood vessel. This increases the possibility that blood vessels will become occluded or narrowed, leading to circulatory disorders. On the other hand, if the value of the marker does not change or increases compared to the reference value, ADAM28 decomposes vWF on the lumen surface of the blood vessel, so no thrombus is formed and blood vessel occlusion occurs. It is considered that the possibility of stenosis occurring is low.

Below, the method according to one embodiment of the present invention described above will be explained in detail.

ADAM proteins (ADAMs: a disintegrin and metalloproteinases) are multifunctional proteins involved in ectodomain shedding of transmembrane proteins, cell adhesion, and invasion (Edwards DR et al., Mol Aspects Med. 2008; 29 (5): 258-89; Murphy G, Semin Cell Dev Biol. 2009; 20 (2): 138-45). The human genome contains 25 types of ADAMs, including 4 types of pseudogenes, and the 21 types of ADAMs are composed of 13 types of proteolytic ADAMs and 8 types of non-proteolytic ADAMs that exhibit proteolytic activity. (Edwards DR et al., Mol Aspects Med. 2008; 29 (5): 258-89; Shiomi T et al., Pathol Int. 2010; 60 (7): 477-96). Proteolytic ADAMs share the metalloproteinase domain of matrix metalloproteinases (MMPs), and typical proteolytic ADAMs include propeptide, metalloproteinase, disintegrin-like, cysteine-rich, epidermal growth factor-like, transmembrane and Many proteolytic ADAMs that contain intracellular domains and include ADAM8, ADAM9, ADAM12, ADAM15, ADAM17, ADAM19 and ADAM28 are overexpressed in human cancers and have been implicated in cancer growth and progression. Are known.

Thus, ADAM28 is a known protein, and its amino acid sequence and cDNA sequence are also known. There are two types of ADAM28: a membrane type (ADAM28m) and a secreted type (ADAM28s). The representative amino acid sequence and cDNA sequence of human membrane-type ADAM28 (and its mature form) and the representative amino acid sequence and cDNA sequence of human secretory ADAM28 (and its mature form) can be found in International Publication No. 2016/143702 pamphlet. has been disclosed. In addition, "human membrane-type ADAM28" refers to an amino acid sequence or nucleotide sequence in which the amino acid sequence or nucleotide sequence of membrane-type ADAM28 is the same or substantially the same as the amino acid sequence or nucleotide sequence of membrane-type ADAM28 naturally expressed in humans. It means having a nucleotide sequence. "Substantially the same" means that the amino acid sequence or nucleotide sequence of interest is 70% or more (preferably 80% or more, more preferably 90% or more) of the amino acid sequence or nucleotide sequence of membrane-type ADAM28 naturally expressed in humans. % or more, more preferably 95% or more, most preferably 99% or more), and has the function of human membrane ADAM28. The same interpretation applies to biological species other than humans, proteins other than membrane-type ADAM28, genes, and fragments thereof.

On the other hand, vWF is a plasma protein that plays an important role in blood coagulation. vWF is mainly synthesized in the vascular endothelium and released into the blood in the form of a polymeric polymer. Wild type human vWF is a polypeptide consisting of a total of 2813 amino acids including its signal peptide and pro region. The amino acid sequence of wild type human vWF and the amino acid sequence of wild type human mature vWF subunit are disclosed in WO 2004/035778 pamphlet.

In the method according to this embodiment, the expression level or concentration of ADAM28 in a blood sample collected from a subject, or the ratio of the expression level or concentration of ADAM28 to vWF (ADAM28/vWF ratio) is measured for use as a marker. do.

As used herein, a "subject" is a subject who is likely to develop a cardiovascular disorder or thrombosis, preferably a human subject. In other preferred embodiments, the subject is a patient who has been diagnosed with a cardiovascular disorder or a disease involving blood clot formation, and more preferably who has received treatment for the disease after receiving the diagnosis. be. Here, diseases involving circulatory disorders or thrombus formation include coronary artery events such as coronary artery stenosis, sudden cardiac death, fatal and non-fatal myocardial infarction, and coronary artery stenosis and occlusion such as unstable angina; Cerebral infarction, cancer, diabetes, traveler thrombosis (economy class syndrome), peripheral arterial embolic disease, thrombotic microangiopathy (thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, etc.), venous thrombosis (pulmonary thromboembolism, deep vein thrombosis, portal vein thrombosis, renal vein thrombosis, hepatic vein thrombosis, jugular vein thrombosis, axillary-subclavian vein thrombosis, cerebral venous sinus thrombosis, etc.).

In this specification, the term "blood sample" may be any blood-derived sample that allows the above-mentioned measurements to be performed. Therefore, there are no particular restrictions on its specific form, and a whole blood sample is usually used. As this whole blood sample, the blood (whole blood) collected from the subject may be used as it is, or additives such as EDTA potassium salt or heparin may be added for the purpose of preventing coagulation or the like. The timing of collecting blood to collect a blood sample from a subject is not particularly limited.

As for the blood sample used in the method according to this embodiment, it is preferable to use a blood sample immediately after being collected from a subject for measurement, but a preserved blood sample may also be used. There are no particular limitations on the method for preserving blood samples as long as the values of the above-mentioned markers in the sample do not change; for example, low-temperature conditions that do not freeze (0 to 10°C), dark conditions, and non-vibration conditions are preferred. .

In the method according to this embodiment, the value of the marker can be measured using a conventionally known method.

As used herein, the "expression level" of a certain protein includes both the expression of RNA (transcription product) complementary to the gene encoding the protein and the expression of the protein (translation product) itself. . Therefore, as used herein, the expression level of a certain protein refers to the expression level or expression intensity of a transcription product or translation product. The expression level can usually be analyzed by the production amount of a transcription product, or the production amount, activity, etc. of its translation product. Further, the "concentration" of a certain protein means the abundance (expression amount) of the target protein per unit volume of a blood sample, and in the method according to this embodiment, this "concentration" may be used as a marker.

The expression level of a protein may be measured by measuring the transcription product of the gene encoding the protein, ie, mRNA, or by measuring the translation product of the gene, ie, the protein itself. Preferably, this is carried out by measuring the transcription product of the gene. Note that the gene transcription product also includes cDNA obtained by reverse transcription from mRNA.

To measure the expression level of a transcript, the degree of gene expression in a sample may be measured using, for example, a nucleotide containing all or part of the mRNA base sequence as a probe or primer. For example, a microarray (microchip) may be used. It can be measured by a conventional method, Northern blotting method, quantitative PCR method, etc. Quantitative PCR methods include agarose gel electrophoresis, polyacrylamide gel electrophoresis, fluorescent probe method, RT-PCR method, real-time PCR method, ATAC-PCR method, and Taqman PCR method (SYBR (registered trademark) green method). , Body Map method, Serial analysis of gene expression (SAGE) method, Micro-analysis of Gene Expression (MAGE) method, etc. are known, and these methods can be used as appropriate. Alternatively, evaluation may be performed using a next-generation sequencer. Using these methods, the amount of mRNA can be measured through the use of nucleotide probes or primers that hybridize to the mRNA. The base length of the probe or primer used for measurement is preferably 10 to 50 bp, more preferably 15 to 25 bp.

When simultaneously measuring the expression of multiple genes, especially the expression of many types of genes, it is preferable to use a DNA microarray. A DNA microarray can be produced by immobilizing nucleotides comprising the base sequence of a gene or a partial sequence thereof on a suitable substrate. Examples of the fixed substrate include a glass plate, a quartz plate, and a silicon wafer. Examples of the size of the substrate include 3.5 mm x 5.5 mm, 18 mm x 18 mm, and 22 mm x 75 mm, but this varies depending on the number of probe spots on the substrate and the size of the spots. Can be set. Methods for immobilizing polynucleotides or fragments thereof include electrostatic bonding to a solid support whose surface has been treated with polycations such as polylysine, polyethyleneimine, and polyalkylamine, by utilizing the charge of nucleotides, A nucleotide having a functional group such as an amino group, an aldehyde group, a thiol group, or biotin introduced therein can also be bonded by a covalent bond to a solid phase surface having a functional group such as an aldehyde group or an epoxy group introduced therein. Immobilization may be performed using an array machine. A DNA microarray is prepared by immobilizing at least one gene or a fragment thereof on a substrate, and the DNA microarray is brought into contact with subject-derived mRNA or cDNA labeled with a fluorescent substance to hybridize, so that the fluorescence on the DNA microarray By measuring the intensity, the type and amount of mRNA can be determined.

The expression level of a translation product can be measured by quantifying the translated protein or by measuring the activity of the protein. Alternatively, protein quantification can be performed using antibodies specific for the protein.

Antibodies can be produced by known methods. Alternatively, antibodies are provided by, for example, Invitrogen, Santa Cruz Biotechnology, Sigma-Aldrich, etc., and can be appropriately obtained and used. Antibodies for detection may be polyclonal or monoclonal antibodies.

Detection of proteins using antibodies can be performed by, for example, immunochromatography, Western blotting, EIA, ELISA, RIA, flow cytometry, etc., but is not limited thereto. The antibody can be labeled with a fluorescent label, a radioactive label, an enzyme, biotin, etc., and a secondary antibody labeled in such a manner can also be used for detection. Currently, in clinical practice, the presence of myocardial injury is evaluated by detecting cardiac troponin T from blood samples by immunochromatography (Trop T Sensitive, Roche Diagnostics). Therefore, in the method according to the present embodiment, markers such as ADAM28 and vWF are similarly measured by immunochromatography, which allows easy and minimally invasive measurement in the clinic, and can be made universal at low cost. It is expected that an evaluation method for predicting vascular lumen occlusion and stenosis will be developed that has high accuracy and reproducibility and will enable standardization of measurement methods. As a result, occlusion and stenosis of blood vessels can be prevented and appropriate treatment can be performed. In addition, when carrying out the said measurement by the immunochromatography method, it is preferable to use an immunochromatography test paper. An immunochromatographic test strip consists of a sample pad area that receives a blood sample, a conjugate pad area that contains a labeled antibody capable of binding to an indicator substance such as ADAM28 or vWF, a membrane body that develops a specimen, and a membrane body that carries the indicator on the membrane body. Preferably, an antibody capable of binding to a substance is immobilized and a test line capable of binding to the indicator substance bound to the labeled antibody is preferably provided in the conjugate pad region. Furthermore, an immunochromatography test paper having a control line that makes it possible to determine the amount of label bound in the test line is more preferable. Furthermore, it is more preferable that the label has a color that becomes stronger depending on the concentration.

In the method according to this embodiment, the progress of circulatory disorder or thrombus formation, or the presence or absence of the risk thereof, can be predicted using the value measured by the above-described method as a marker. That is, the present specification discloses a method for predicting the progression of cardiovascular disorders or thrombus formation, or the presence or absence of a risk thereof. Also disclosed herein are methods for providing data for predicting the development of, or risk of, cardiovascular disorders or thrombus formation. At this time, as described above, if the value of the marker is lower than the reference value, it is predicted that there is progression of circulatory system disorder or thrombus formation or a risk thereof. Note that in this specification, a "reference value" is a numerical value that can serve as a reference for determining an increase or decrease in expression level or concentration in the method according to the present embodiment. An example of a reference value is, for example, the expression level (or concentration) of ADAM28 in a blood sample collected from a person who has not been diagnosed with the above-mentioned disease (for example, a healthy person), or the expression level (or concentration) of ADAM28 in relation to vWF. Examples include the measured value of the expression level (or concentration) ratio (ADAM28/vWF ratio). Alternatively, the value of the marker previously measured in the same subject may be used as the reference value.

According to still another aspect of the present invention, a detection reagent for use in the method according to one aspect of the present invention described above, and the presence or absence of the progression of circulatory disorder or thrombus formation or the risk thereof, including the detection reagent. Also provided are kits used to predict. The detection reagent enables detection of the expression level or concentration of ADAM28 or ADAM28 and vWF in the blood sample described above. Such detection reagents include, for example, DNA or RNA for hybridization with mRNA in a sample, or antibodies for specific binding to proteins in a sample. Such DNA or RNA may be a probe whose hybridization can be detected using a fluorescent label or the like. Further, the DNA or RNA may be a primer that can be used for amplifying mRNA. In addition to the detection reagents described above, the kit can contain other reagents necessary for detection, such as buffers, various nucleotides, other reagents necessary for hybridization or antibody binding.

≪Evaluation of the risk that drugs or medical devices will cause circulatory disorders or blood clot formation in living organisms≫
Another aspect of the present invention relates to a method for evaluating the risk that a drug or medical device will cause circulatory damage or thrombus formation in a living body. This method includes culturing cells while applying mechanical stress to the cells in the presence and absence of the drug or medical device to be evaluated. and the expression level or concentration of ADAM28 or ADAMTS13 in the culture solution or cells after culturing the cells in the presence of the drug or medical device, the ratio of the expression level or concentration of ADAM28 to vWF (ADAM28/vWF ratio), or when the ratio of the expression level or concentration of ADAMTS13 to vWF (ADAMTS13/vWF ratio) is lower than the value in the culture medium or cells after culturing the cells in the absence of the drug or medical device. The above drug or medical device is evaluated to have a risk of causing circulatory damage or thrombus formation in living organisms. In this way, by monitoring the expression level (or concentration) of ADAM28 or ADAMTS13 or its ratio to vWF as an indicator, if these values decrease in the presence of a drug or medical device, the drug or medical device It is possible to evaluate that there is a risk of inducing circulatory disorder or thrombus formation in living organisms. That is, by incorporating the evaluation method according to this embodiment in the development stage of drugs and medical devices, it becomes possible to develop drugs and design medical devices that are less likely to cause stenosis or occlusion of blood vessels. In this way, if the values of the above markers decrease in the presence of a drug or medical device compared to their absence, vWF, which is a trigger for primary thrombus formation, accumulates in the patient's blood vessel lumen. However, as a result, it can be determined that there is a high possibility that a thrombus will develop within the blood vessel and that the blood vessel will eventually become occluded or narrowed. On the other hand, if the values of the above markers do not change or increase in the presence of the drug or medical device compared to the absence of the drug or medical device, then vWF on the blood vessel lumen surface is affected by ADAM28 or ADAMTS13. Since it is decomposed, it can be determined that the possibility of blood vessel occlusion or stenosis is low without the formation of a thrombus.

Note that ADAMTS13 is also a known protein, and its amino acid sequence and cDNA sequence are also known. ADAMTS13 is a zinc-based metalloprotease belonging to the ADAMTS family, and is thought to reduce the multimer size of vWF and control the function of vWF by specifically cleaving the bond between Tyr1605 and Met1606 of vWF. Structural details and sequence information for human ADAMTS13 are disclosed in Zheng X et al., J Biol Chem. 2001; 276 (44): 41059-63.

The method according to this embodiment includes culturing cells. The cells used in this method are not particularly limited, but are preferably endothelial cells, more preferably vascular endothelial cells. Moreover, the biological species of the cells is not particularly limited. The culture solution (medium) is also not particularly limited as long as all cultured cells do not die during the evaluation period. As an example, when using endothelial cells for evaluation, for example, Porcine Endothelial Cell Growth Medium (Applications Inc.), Endothelial Cell Growth Medium 2 Kit (Takara Bio Inc.), Endothelial Cell Growth Medium MV 2 Kit (Takara Bio Inc.) ) etc. are suitable.

The method according to this embodiment is a method for determining whether a certain drug or medical device has a risk of causing circulatory disorder or thrombus formation in a living body. For this reason, cells are cultured in the same manner in the presence and absence of the drug or medical device, and the expression level of a specific gene or protein or the expression level of a specific gene or protein in the presence and absence of the drug or medical device. The above determination is made using the change in the ratio value as an index.

At this time, there are no particular restrictions on the specific types of drugs or medical devices to be evaluated, and any type of drug or medical device can be adopted as an evaluation target. Examples of drugs include nucleic acids, peptides, proteins, organic compounds, and inorganic compounds. Examples of medical devices include those selected from the group consisting of intravascular indwelling devices, PTCA balloons, artificial blood vessels, extracorporeal membrane oxygenation (ECMO), and centrifugal pump artificial hearts. It will be done. Among these, the medical device is preferably an intravascular indwelling device, and examples thereof include those selected from the group consisting of stents, covered stents, stent grafts, artificial valves, valve clips, and pump catheters for auxiliary circulation. .

There is no particular restriction on the specific form of the apparatus for carrying out the evaluation method according to the present embodiment, and any apparatus may be used as long as it is capable of holding and culturing cells to be cultured. The material for the area where cells are held and cultured should be expandable, elastic, bendable, such as silicone rubber, capable of holding cells, and capable of maintaining a considerable amount of cell activity during the evaluation period. and is suitable. However, it is not limited to those that can maintain cell activity. Furthermore, depending on the type of cells, the surface of the culture site may be coated with collagen, gelatin, fibronectin, or the like.

An example of a culture device is a mechanical stress culture device as shown in FIG. In this specification, "mechanical stress" that is applied to cells during culture refers to the movement of the medium used for culture relative to the cells being cultured, or the stress at the interface between the medium and the cells. It means the force (stress) that is applied to the cell due to changes in pressure. Specifically, this mechanical stress includes shear stress (shear stress) that is applied to cells due to a flow phenomenon (flow) of a medium that simulates blood flow. Furthermore, when cells are cultured in the lumen of a tube, the pressure applied to the cells that increases due to an increase in the flow rate of the medium is also included in mechanical stress. Note that the latter increase in the flow rate of the medium simulates the pulsation of blood vessels (increase in blood pressure), and corresponds to the stretching stimulus that occurs to endothelial cells in living organisms as the diameter of blood vessels increases.

The mechanical stress culture device shown in Figure 1 has the following main components: a pump, a reservoir, a cell culture unit for holding or culturing cells, a compliance tank, valves (resistance valves, flow rate adjustment valves), and tubes that connect them. It consists of According to this mechanical stress loading culture device, by driving the pump, it is possible to flow (circulate) the medium in the cell culture unit and load the cells with shear stress. At this time, by setting the flow conditions to correspond to the pulse rate, blood viscosity, pulse rate, etc. in a living body, it is possible to reproduce the shear stress caused by the flow of blood in a living body (loading on endothelial cells in a living body). (reproduction of shared stress). In addition, by constructing the cell culture unit from silicone rubber tubes and matching the compliance with blood vessels, it is possible to reproduce changes in blood vessel diameter in response to pulsations in living bodies (the stretching stimulus that occurs to endothelial cells in living bodies). reproduction).

For example, when carrying out the evaluation method according to this embodiment using the mechanical stress loading culture device (Fig. 1) prepared above, the drug to be evaluated is added to the medium and cultured. Culture can be carried out in the presence of Similarly, culture can be performed "in the presence of a medical device" by placing the medical device to be evaluated in contact with a medium (preferably, the medium and cells). In this way, culture is carried out in the presence of the evaluation target, and the change in the above marker between when the evaluation target is not present (in its absence) is used as an indicator, and the drug or medical device to be evaluated is determined. It can be determined whether there is a risk of circulatory disorder or thrombus formation in a living body.

The evaluation method according to this embodiment is characterized in that one of the following is used as an index (marker):
Expression level or concentration of ADAM28 Expression level or concentration of ADAMTS13 Ratio of expression level or concentration of ADAM28 to vWF (ADAM28/vWF ratio)
Ratio of expression level or concentration of ADAMTS13 to vWF (ADAMTS13/vWF ratio).

Here, as the values of "expression level" and "concentration" in the evaluation method according to this embodiment, the amount and concentration or activity of the protein contained in the medium after the end of culture may be adopted, or the The amount, concentration, or activity of a protein contained in a cell, or the amount of a gene (mRNA or cDNA prepared therefrom) encoding the same may be used.

The evaluation method according to this embodiment is characterized in that the cells are cultured while applying mechanical stress. Here, as a means for culturing cells while applying mechanical stress to the cells, for example, a mechanical stress loading culture apparatus as shown in FIG. 1 is used, and at this time, the cell culture unit is bent as shown in FIG. An example of this method is to perform culture while circulating the medium while connected to the cellphone, or even while pulsating the medium. By culturing cells while applying mechanical stress in this way, it is possible to simulate the blood circulation and pulsation of blood flow in a living body, which has the advantage of being able to evaluate conditions close to the circulatory environment of a living body. . The invention according to this form is that, as shown in the examples described later, in a system in which cells were cultured while applying mechanical stress (circulating culture), the expression of eNOS, ADAM28, and ADAMTS13 was induced. On the other hand, this was done based on the observation that these expressions decreased in a system where no mechanical stress was applied (static culture). In other words, expression of ADAM28 and ADAMTS13 is enhanced only when mechanical stress is applied to cells, as in the case of blood flow in healthy blood vessels, whereas expression is enhanced only when the blood flow is reduced or stagnates, as in a lesion site. Since the expression of ADAM28 and ADAMTS13 decreases in a state where no mechanical stress is applied to the cells by doing so, it is thought that these expressions change to reflect the living body. From this, when comparing the expression of these in the presence and absence of the drug or medical device being evaluated in static culture, it appears that these expressions remain decreased in both systems. Therefore, it is considered that it is not possible to conduct an evaluation. On the other hand, when cells are cultured while applying mechanical stress as in the evaluation method according to this embodiment, the expression of ADAM28 and ADAMTS13 is enhanced, so it is possible to increase the expression of ADAM28 and ADAMTS13. By comparing these expressions in the presence and absence, it becomes possible to evaluate whether the evaluation target has a risk of causing circulatory damage or thrombus formation in the living body.

The effects of the present invention will be explained using the following examples and comparative examples. However, the technical scope of the present invention is not limited only to the following examples.

[Culture of vascular endothelial cells]
First, a silicone rubber tube was prepared as a culture container to simulate a blood vessel. Collagen coating was then applied to the inner lumen of the silicone rubber tube using the following method. That is, first, collagen (5 mg/mL; 1 mM HCl solution, AteloCell Native collagen Bovine Dermis, Kouken) was diluted 10 times with diluted hydrochloric acid (pH 3.0, ^{10 −3} M) and filled into a tube. Then, coating was performed by incubating at 4° C. for 3 days. After discarding the diluted collagen solution, the tubes were washed with Hank's buffer and used for subsequent tests.

On the other hand, porcine coronary artery endothelial cells were cultured in a culture flask containing endothelial cell culture medium (Porcine Endothelial Cell Growth Medium, APPLICATIONS INC.) at 37°C and 5% _CO2 to sufficiently increase the number of cells. Ta. These porcine coronary artery endothelial cells were seeded into the inner lumen of the silicone rubber tube coated with collagen as described above, and the cells were uniformly adhered to the inner surface of the tube while rotating the tube along its central axis. Then, the cells were cultured overnight at 37° C. and 5% CO ₂ until confluent. In this way, a silicone rubber tube whose lumen was coated with vascular endothelial cells was prepared.

Subsequently, a mechanical stress culture apparatus shown in FIG. 1 was prepared. The mechanical stress culture device shown in Figure 1 has the following main components: a pump, a reservoir, a cell culture unit for holding or culturing cells, a compliance tank, valves (resistance valves, flow rate adjustment valves), and tubes that connect them. It consists of According to this mechanical stress loading culture device, by driving the pump, the medium in the cell culture unit can be caused to flow (circulate) or pulsate.

The vascular endothelial cell-coated silicone rubber tube prepared above was connected to the cell culture unit portion of the mechanical stress culture device (FIG. 1) prepared above in a curved state as shown in FIG. 2. Thereafter, the culture medium was circulated at a pressure of 15 dyn/cm ² (1.5×10 ⁻⁴ N/cm ² ) under conditions of 37° C. and 5% CO ₂ for about 1 day. In this culture, since the silicone rubber tube is connected to the cell culture unit in a curved state, the culture medium is circulated and the vascular endothelial cells attached to the inner surface of the silicone rubber tube are Therefore, mechanical stress (shear stress) will be applied. Note that static culture without circulating the medium was also performed under similar culture conditions.

[Analysis of gene expression in vascular endothelial cells]
After the above culture was completed, vascular endothelial cells were collected from the silicone rubber tube, and total RNA was prepared and purified by a conventional method using an RNeasy mini kit (Qiagen). Using the thus obtained total RNA as a template, cDNA was synthesized by a conventional method using reverse transcriptase (SuperScript IV VILO, Thermo Fisher Scientific). The expression levels of the eNOS gene, ADAM28 gene, ADAMTS13 gene, and vWF gene were analyzed by real-time PCR using the obtained cDNA. Note that the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal standard. At this time, 7500 First Real-Time PCR System (Applied Biosystems) was used as a PCR device. The PCR primers were designed using the Primer3 (v.0.4.0) Pick primers from a DNA sequence program, and in silico using the DNASIS Mac program (Hitachi Software Engineering, Inc.) to eliminate the risk of self-annealing and mis-annealing. I confirmed that it was low. The base sequences of various primers used for analysis of gene expression levels are shown below.

The results of the gene expression analysis conducted in this manner are shown in Tables 1 to 5 below. Note that all the results shown in Tables 1 to 5 are relative values when the gene expression level (or its ratio) in the case of static culture is set to 1.

(Functional evaluation of vascular endothelial cells)
The expression of the eNOS gene analyzed above is generally used as an indicator of the function of vascular endothelial cells. As is clear from the results shown in Table 1, the expression level of the eNOS gene is lower in static culture in which mechanical stress is not applied to vascular endothelial cells than in a system in which circulatory culture is performed in which mechanical stress is applied to vascular endothelial cells. decreased. From this, it was confirmed that in a static culture system, the function of vascular endothelial cells decreases due to a decrease in mechanical stress, as in lesions where blood vessel stenosis progresses in vivo and blood flow decreases. It was done.

(Evaluation of thrombus formation control)
Furthermore, as is clear from the results shown in Table 2, in comparison to a system in which vascular endothelial cells were subjected to circulating culture in which mechanical stress was applied, static culture in which mechanical stress was not applied significantly reduced the ADAM28 gene. The expression level also decreased significantly. Here, mechanical stress decreases downstream of the constricted/occluded site of blood vessels in living organisms, but as mentioned above, in static culture, the function of vascular endothelial cells decreases, so ADAM28 It is thought that it can be used as an indicator (biomarker) of decrease in blood vessel stenosis and occlusion of blood vessels.

On the other hand, the expression level of the vWF gene, which is a trigger for primary thrombus formation, was higher in the static culture system than in the circulating culture system. As mentioned above, since ADAM28 showed a higher value in the circulating culture system, by calculating the ratio of the expression level of ADAM28 to the expression level of vWF (ADAM28/vWF ratio), it was possible to prevent vascular stenosis in the living body. It is believed that occlusion can be predicted more accurately. In other words, when the expression level (or its concentration) of ADAM28 or the ADAM28/vWF ratio decreases, vWF, which is the trigger for primary thrombus formation, is not sufficiently degraded and accumulates in the vascular lumen, and as a result, intravascular It can be determined that there is an increased possibility that a blood clot will develop and the blood vessel will become occluded or narrowed. On the other hand, when the expression level (or concentration) of ADAM28 or the ADAM28/vWF ratio does not change or increases, vWF on the lumen surface of the blood vessel is degraded by ADAM28, and as a result, blood clots are not formed and the blood vessel It can be predicted that no occlusion or stenosis will occur.

This application is based on Japanese Patent Application No. 2018-172733 filed on September 14, 2018, the disclosure content of which is incorporated by reference in its entirety.

Claims (4)

A medical device selected from the group consisting of intravascular indwelling devices, PTCA balloons, artificial blood vessels, extracorporeal membrane oxygenation (ECMO), and centrifugal pump artificial hearts can prevent cardiovascular disorders or thrombus formation in living organisms. A method for evaluating the risk caused,
Cultivating the cells while applying mechanical stress to the cells in the presence and absence of the medical device, and culturing the cells after culturing the cells in the presence of the medical device. The expression level or concentration of ADAM28 or ADAMTS13 in fluid or cells, the ratio of the expression level or concentration of ADAM28 to vWF (ADAM28/vWF ratio), or the ratio of the expression level or concentration of ADAMTS13 to vWF (ADAMTS13/vWF ratio), If the value is lower than the value in the culture medium or cells after culturing the cells in the absence of the medical device, the medical device has a risk of causing circulatory disorder or thrombus formation in the living body. How to evaluate if there is. 2. The method of claim 1 , wherein the cells are vascular endothelial cells. The method according to claim 1 or 2, wherein the medical device is the intravascular indwelling device. 4. The method of claim 3 , wherein the intravascular device is selected from the group consisting of stents, covered stents, stent grafts, prosthetic valves, valve clips, and pump catheters for auxiliary circulation.