JP7343862B2 - How to determine vascular disorders - Google Patents

Description

The present invention relates to a method for determining vascular disorders by measuring specific blood markers.

In the diagnosis of diabetes, hemoglobin A1c (HbA1c) is measured worldwide as a marker for blood sugar control. However, there are no clinically used markers for evaluating the progression or risk of progression of vascular disorders such as arteriosclerosis. Since hardening of arteries and veins (vascular disorders) is a cause of various age-related diseases, substances that can be markers of vascular hardening are being searched for worldwide. Nagai et al. discovered S-(2-succinyl) cysteine (hereinafter also referred to as 2SC) as a structure generated by post-translational modification, and found that 2SC accumulates significantly in adipocytes. (Non-patent Document 1).

Nagai R., et al., Succination of protein thiols during adipocyte maturation - a biomarker of mitochondrial stress. J Biol Chem. 282, 34219-34228 (2007)

As lifestyle-related diseases progress, vascular disorders such as arteriosclerosis develop, but there are no clinically useful markers for vascular disorders. If arteriosclerosis has progressed, it is possible to check the progress of arteriosclerosis through ultrasound examinations, etc. However, once arteriosclerosis and other vascular disorders have progressed, it is difficult to completely cure them, so it is important to monitor the risk in the early stages of vascular disorders. Evaluation is of utmost importance. In addition, there are drugs used to treat arteriosclerosis that lower blood cholesterol levels using HMGCoA reductase inhibitors, but once arteriosclerosis has progressed, even if these drugs can prevent further deterioration, treatment It is extremely difficult to do so. Therefore, drug development for the treatment of arteriosclerosis is being carried out worldwide, but since there are no good drug discovery markers, no drug with high therapeutic effects has been developed.

An object of the present invention is to provide a method for determining vascular damage by identifying a clinically practical level of vascular damage marker. A further object of the present invention is to provide a method for screening candidate substances for the prevention or treatment of vascular disorders using the identified vascular disorder markers.

As a result of intensive studies to solve the above problems, the present inventors confirmed that blood 2SC significantly increases in patients with vascular disorders (FIG. 1). It also increased in retinopathy and nephropathy, which are small vessel disorders (Figure 2). In contrast, no change was observed in CML, which was considered a candidate marker for arteriosclerosis (Figure 3). For confirmation, measurements were performed using apoE-deficient mice, which are a model of arteriosclerosis, and it was also confirmed that 2SC was also increased (Figure 4). The above results demonstrated that 2SC functions as a predictive marker for the early stage or onset of vascular disorders. 2SC is useful not only as a marker for evaluating the risk of vascular disorders, but also as a target for drug discovery for the treatment of vascular disorders. The present invention was completed based on the above findings.

That is, according to the present invention, the following inventions are provided.
(1) A method for determining vascular disorder, which includes measuring the amount of S-(2-succinyl)cysteine in the blood.
(2) The determination method according to (1), wherein it is determined that a person has a vascular disorder when the measured value obtained by measuring the amount of S-(2-succinyl)cysteine in the blood is higher than a reference value.
(3) The determination method according to (1), wherein the risk of vascular disorder is determined to be high when the measured value obtained by measuring the amount of S-(2-succinyl)cysteine in the blood is higher than a reference value.
(4) The method according to any one of (1) to (3), wherein the amount of S-(2-succinyl)cysteine in blood is measured by mass spectrometry or immunoassay.
(5) A method for screening a candidate substance for the prevention or treatment of vascular disorders, the method comprising measuring the amount of S-(2-succinyl)cysteine in the blood of a subject to whom the candidate substance has been administered.
(6) The screening method according to (5), wherein the amount of S-(2-succinyl)cysteine in blood is measured by mass spectrometry or immunoassay.
(7) The screening method according to (5) or (6), wherein the candidate substance is a food-derived component.

According to the present invention, arteriosclerosis can be diagnosed. According to the present invention, it is further possible to evaluate the risk of vascular disorders in patients who are in the early stages of lifestyle-related diseases and have not yet developed complications such as arteriosclerosis.

FIG. 1 shows a comparison between medical history of various diseases and blood 2SC. FIG. 2 shows a comparison between the value of 2SC released in the blood and the progression of pathological conditions in retinopathy and nephropathy. FIG. 3 shows the results of measuring CML in the serum of patients with various diseases. FIG. 4 shows the results of measurement of blood 2SC in arteriosclerosis model mice.

The present invention will be explained in more detail below.
<Method for determining vascular disorders>
The method for determining vascular disorder according to the present invention is a method that includes measuring the amount of S-(2-succinyl) cysteine in the blood.
In the present invention, various post-translationally modified structures generated through metabolism were synthesized, a 13C-labeled internal standard substance was synthesized, and a quantitative system using a mass spectrometer was established. As a result of searching for post-translational modifications that vary with vascular disorders, it was confirmed that blood levels of 2SC increase in human small and large blood vessel disorders. A standard product of 2SC is not commercially available, and measurement of blood 2SC by LC-MS/MS has not been reported.
The present invention is based on the fact that synthesis of an internal standard and LC-MS/MS enable accurate quantification of 2SC in blood, and data have been obtained that 2SC in blood increases in human arteriosclerosis.

The vascular disorder may be either macrovascular disorder or microvascular disorder. As the macrovascular disorder, for example, macrovascular disorder in lower extremity obstructive arteriosclerosis, coronary artery disease, or cerebrovascular disease can be determined. As the microangiopathy, for example, microangiopathy in retinopathy, neuropathy, or nephropathy can be determined.

In the first example of the present invention, if the value obtained by measuring the amount of S-(2-succinyl)cysteine in the blood is higher or lower than a reference value, it is determined that the patient has a vascular disorder. be able to. In the second example of the present invention, if the measured value obtained by measuring the amount of S-(2-succinyl) cysteine in the blood is higher or lower than a reference value, it is determined that the risk of vascular disorder is high. be able to.

The amount of S-(2-succinyl)cysteine in the blood may be determined by measuring the amount of S-(2-succinyl)cysteine that exists freely in the blood, or by measuring the amount of S-(2-succinyl)cysteine that exists in protein. The total amount of S-(2-succinyl)cysteine, which also includes -succinyl)cysteine, may be measured.

In one example of the present invention, if the measured value obtained by measuring the amount of total S-(2-succinyl)cysteine, which also includes S-(2-succinyl)cysteine present in the protein, is higher than the reference value. can be determined to have macrovascular disease (e.g., arteriosclerosis obliterans in the lower extremities, coronary artery disease, or macrovascular disease in cerebrovascular disease), and to have a risk of macrovascular disease (e.g., arteriosclerosis obliterans in the lower extremities, coronary artery disease, or cerebrovascular disease). Macrovascular disease in cerebrovascular disease) can be determined to be high.

In another example of the invention, microangiopathy (e.g. , retinopathy, neuropathy, or microangiopathy in nephropathy), and are at high risk for microangiopathy (e.g., microangiopathy in retinopathy, neuropathy, or nephropathy). be able to.
In addition, if the measured value obtained by measuring the amount of S-(2-succinyl)cysteine that is free in the blood is higher than the standard value, it may be associated with arteriosclerosis obliterans in the lower extremities or macrovascular disorder associated with coronary artery disease. It can be determined that the patient has a high risk of macrovascular disorder due to lower limb obstructive arteriosclerosis or coronary artery disease.

<Mass spectrometry>
In one example of the present invention, the amount of S-(2-succinyl)cysteine in blood can be measured by mass spectrometry. Mass spectrometry can be performed, for example, according to the method described in JP-A-2014-119370, but is not particularly limited.

In the present invention, it is preferable to first treat blood with an acid. As the blood, whole blood, serum, plasma, etc. can be used, and serum is more preferable.

The acid used in the acid treatment may be an organic acid or an inorganic acid, but iron-free hydrochloric acid or formic acid is preferable. The pH of the acid used is preferably pH 0.5 to 1.5, more preferably pH 0.8 to 1.2. In the acid treatment, for example, an acid solution may be added to blood, shaken or stirred if necessary, and then left to stand to hydrolyze the sample. Furthermore, it is preferable to heat the reaction solution during hydrolysis. The amount of acid, reaction time, and temperature used in the acid treatment may be any condition as long as the blood can be sufficiently dissolved and the reaction liquid is in a liquid phase.

In the present invention, blood 2SC may be measured by a method (Free) that measures 2SC free in the blood, or a method (Free) that measures total 2SC including 2SC present in proteins. Total) may be adopted.

In the method (Free) for measuring 2SC present freely in blood, an internal standard ([ ¹³ C ₃ , ⁵ N ₁ ] 2SC) synthesized using an isotope is added to serum, and water is further added. Samples can be prepared by:
In the method (Total) for measuring total 2SC, which includes 2SC present in proteins, an internal standard ([ ¹³ C ₃ , ⁵ N ₁ ] 2SC) and hydrochloric acid are added to blood, and then hydrated at a high temperature such as 100 ° C. Samples can be prepared by adding water after decomposition and drying the solution.

The sample prepared as described above can be added to Captiva ND Lipids (Agilent Technologies) filled with methanol containing formic acid, mixed well, and then passed through. This allows defatting and protein removal.

Further, the collected sample can be dried and resuspended in water containing formic acid, and then passed through a C18 column, Sep-pak (Waters Corp). Sep-pak is equilibrated in advance with water containing 1% formic acid, and after passing the sample through it, 2 ml of 20% methanol containing 1% formic acid is passed through it, and the passed fraction can be collected. The collected solution was dried, resuspended in 1 ml of 20% acetonitrile containing 0.1% formic acid, and then filtered through VIVASPIN 500 (Sartorius Stedim), a molecular weight 3000 cut filter, to obtain a sample for mass spectrometry. Obtainable. Note that the method for preparing the sample for mass spectrometry is not limited to the above-mentioned preparation method, as it can be appropriately prepared depending on the analysis method and the equipment used.

The mass spectrometry method is not particularly limited as long as it is a measurable method, but an analysis method that combines liquid chromatography and mass spectrometry is preferred, such as liquid chromatography-mass spectrometry (for example, LC-MS method, Examples include LC-MS/MS, LC-MS/MS/MS, etc.) methods. In order to further improve detection sensitivity, liquid chromatography-tandem mass spectrometry methods such as LC-MS/MS method and LC-MS/MS/MS method are more preferable.

As a suitable solvent for dissolving the dry sample for liquid chromatography-mass spectrometry, it is preferred to use a solvent that is the same as the final conditions of the mobile phase for liquid chromatography. Examples include an aqueous methanol solution, an acetonitrile aqueous solution, a mixed aqueous solution of acetonitrile and trifluoroacetic acid, a mixed aqueous solution of acetonitrile and formic acid, but are not particularly limited.

Measurement conditions for measuring 2SC using a liquid chromatography-mass spectrometer may be appropriately set by a person skilled in the art based on common knowledge, depending on the type of equipment, the condition of the sample, etc. Although the conditions for liquid chromatography vary depending on the sample to be used, it is preferable, for example, to form a gradient in the mobile phase with an aqueous formic acid solution and a formic acid acetonitrile solution. Examples of the mass spectrometer include, but are not limited to, a double focusing magnetic field mass spectrometer, an ion trap mass spectrometer, a quadrupole mass spectrometer, and the like.

By comparing the measured value measured by the above procedure with the measured value from a standard solution measured by the same procedure, 2SC derived from the biological sample can be quantified. Specifically, a calibration curve is created based on the measurement results from a standard solution containing 2SC at a predetermined concentration. When creating a calibration curve, it is preferable to calibrate each measured value using an internal standard because a more accurate calibration curve can be obtained.

<Immunological analysis>
In another example of the invention, the amount of S-(2-succinyl)cysteine in the blood can be measured by immunoassay. Immunological analysis can be performed using polyclonal or monoclonal antibodies capable of detecting 2SC. By using a monoclonal antibody that can quantify 2SC in blood, it is possible to measure multiple samples more easily.

A method for producing an antibody capable of detecting 2SC will be described.
Antibodies that can detect 2SC can be produced by using 2SC as an immunogen. 2SC may be used as an immunogen with sufficient immunogenicity by binding to a protein. As the protein that binds 2SC, various known carrier proteins can be used, such as albumin such as serum albumin, hemocyanin, myoglobin, and the like. Further, as the carrier protein, those derived from mammals such as cows, rabbits, and humans, shellfish such as keyhole keyhole, birds such as chicken, etc. can be used. Among these, keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) are preferred. These proteins can be used alone or in combination of two or more.

It is preferable to use a cross-linker to bind 2SC to a carrier protein. Various known crosslinkers can be used, such as glutaraldehyde, EDC (1-ethy-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), SPDP, DST, DSG, and the like. Among these, it is preferable to use glutaraldehyde or EDC. One type of cross-linker can be used alone or two or more types can be used in combination.

A non-human animal is immunized using 2SC or a conjugate of 2SC and a carrier protein as an antigen, and antibody-producing cells are produced within the animal body. Polyclonal antibodies against 2SC can be isolated from serum obtained from immunized animals using a protein G column. Alternatively, it can be carried out according to a conventional method for producing monoclonal antibodies.
The type of animal is not particularly limited, but mammals are preferred. Examples of mammals include rodents such as mice, rats, and rabbits. As for the mouse, it is preferable to use a mouse of the BALB/C strain since myeloma-derived cell lines used for hybridoma production have been established. Immunization can be carried out by various known methods, for example, by subcutaneously, intradermally, intravenously, or intraperitoneally injecting the mammal. Immunization is preferably repeated several times after the first immunization, and the immunization schedule can be determined as appropriate depending on the type and strain of the animal to be immunized. Furthermore, in order to enhance the immune response, the antigen is preferably administered in combination with an adjuvant before or at the time of administration. Various known adjuvants can be used. Furthermore, two or more types of adjuvants can be used, for example, using Complete Freund's adjuvant (CFA) for the first immunization and using Incomplete Freund's adjuvant (IFA) for the second and subsequent immunizations.

Using the antibody-producing cells of the animal immunized as described above, a hybridoma capable of producing a monoclonal antibody against 2SC can be produced.

After the above-mentioned immunization, blood is collected from the animals at appropriate intervals to confirm that antibodies against 2SC are produced. Known analytical methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence immunoassay can be used to confirm antibody production. It is also preferable to perform a boost (additional injection of immunogen) after confirmation of antibody production. After the final immunization, spleen cells are removed from the immunized animal and fused with myeloma-derived cells. As a method for cell fusion, known methods such as a polyethylene glycol method, a method using Sendai virus, a method of applying electrical stimulation, etc. can be used, for example. Fused cells can be selected using HAT medium containing hypoxanthine, aminopterin, thymidine, and the like. Furthermore, from the obtained fused cells, fused cells with a high ability to produce antibodies against 2SC are selected by known analysis methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence immunoassay. . Then, using the selected cells, cloning is performed by a known method such as limiting dilution method or soft agar method. In this way, a hybridoma that produces a monoclonal antibody with high specificity for 2SC or high binding ability to 2SC can be obtained.

The hybridoma obtained as described above is cultured and propagated, and the monoclonal antibody against 2SC is separated and purified from the culture solution, etc., thereby obtaining a large amount of monoclonal antibody. As a method for culturing hybridomas, various known methods can be employed, such as a method in which the hybridoma is injected into the peritoneal cavity of a mammal and grown in ascites fluid, and a method in which the hybridoma is cultured outside the animal's body using an appropriate medium. In addition, for purification of antibodies obtained from ascites, culture fluid, or culture supernatant, known methods such as ion exchange chromatography and affinity chromatography can be used without particular limitations.

In addition to the antibodies obtained as described above, monoclonal antibodies against 2SC include antibody fragments containing an identification site for 2SC. Furthermore, the monoclonal antibody against 2SC may be a chimeric antibody or a humanized antibody produced using gene recombination technology so as to contain a 2SC identification site.

The antibody for 2SC preferably has excellent specificity for 2SC and/or excellent binding ability for 2SC. By performing immunoassay using such antibodies, it is possible to detect and/or quantify 2SC in blood with high sensitivity or precision. Methods for detecting and/or quantifying 2SC using antibodies against 2SC include, but are not particularly limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), luminescent immunoassay, precipitation method, and agglutination method. methods, Western blot analysis, flow cytometry, etc. As an enzyme immunoassay, a competitive inhibition assay or a competitive binding assay can also be performed.

<Screening>
The present invention further relates to a method for screening a candidate substance for the prevention or treatment of vascular disorders, which comprises measuring the amount of S-(2-succinyl)cysteine in the blood of a subject to whom the candidate substance has been administered. The amount of S-(2-succinyl)cysteine in blood is preferably measured by mass spectrometry or immunoassay as described above in this specification.

Subjects to which the candidate substance is administered are not particularly limited, but are preferably non-human mammals, such as rodents such as mice, hamsters, guinea pigs, rats, and rabbits, as well as dogs, cats, goats, sheep, cows, Pigs, monkeys, etc. can be used. Among the above, rodents such as mice, hamsters, guinea pigs, rats, and rabbits are preferred, and among these, mice are most preferred.

Any substance can be used as the candidate substance and is not particularly limited. The candidate substance may be an individual low-molecular compound, a compound present in a natural product extract, or a synthetic peptide. The candidate substance may be a compound library, phage display library or combinatorial library. Construction of compound libraries is known to those skilled in the art, and commercially available compound libraries can also be used. An example of a candidate substance is a food-derived component.

In the screening method of the present invention, substances that reduce the amount of 2SC in blood can be selected as candidate substances for the prevention or treatment of vascular disorders. By comparing the amount of 2SC in the blood of a subject administered with a candidate substance and a subject administered with a control substance, it is possible to identify a substance that reduces the amount of 2SC in the blood.

The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited by the examples.

<2SC preprocessing method and detection method>
Two methods have been established for measuring 2SC in serum: a method that measures 2SC that exists freely in the blood (Free), and a method that measures 2SC that is present in proteins, including 2SC (Total).

In Free, an internal standard ([ ¹³ C ₃ , ¹⁵ N ₁ ] 2SC) synthesized using an isotope was added to 30 μl of serum, the volume was brought up to 700 μl with water, and the mixture was diluted with methanol (2 SC) containing 0.1% formic acid. .1 ml) was added to Captiva ND Lipids (Agilent Technologies), mixed well, and passed through to perform defatting and deproteinization. The collected sample was dried, resuspended in 1 ml of water containing 1% formic acid, and then passed through a C18 column, Sep-pak (Waters Corp). Sep-pak was equilibrated in advance with water containing 1% formic acid, and after the sample was passed through it, 2 ml of 20% methanol containing 1% formic acid was passed through it, and the passed fraction was collected. The collected solution was dried and resuspended in 1 ml of 20% acetonitrile containing 0.1% formic acid, and then filtered through a VIVASPIN500 (Sartorius Stedim), a molecular weight 3000 cut filter, and the sample was used for measurement.

On the other hand, in Total, the internal standard and 1 ml of 6N HCl were first added to 1 μl of serum, hydrolyzed at 100°C for 18 hours, and the solution was dried and then made up to 700 μl with water, and treated with Captiva ND Lipids. After that, processing was performed in the same manner as for Free.

When performing measurements using a liquid chromatography tandem mass spectrometer (LC-MS/MS), ZIC (registered Separation was performed by liquid chromatography using a HILIC column (150x2.1 mm, 5 μm) (Merck Millipore), ionization was performed using ESI positive, and detection was performed by MS/MS. 2SC and [ ¹³ C ₃ , ¹⁵ N ₁ ] 2SC used as an internal standard are ionized to have respective parent ions of 238 (m/z) and 242 (m/z), and the product ion obtained from them is 149 (m/z). 2SC was measured by detecting /z).

<Results>
(1) Comparison of past history of various diseases and blood 2SC A comparison was made between past history of various diseases and blood 2SC. A total of 32 patients (17 males, 15 females) were measured, with an average age of 67 years. The number of people with a past history of each condition is as follows. Lower limb obstructive arteriosclerosis: 3 patients, coronary artery disease: 9 patients, cerebrovascular disease: 2 patients, retinopathy: 11 patients, neuropathy: 11 patients, nephropathy: 15 patients.

The results are shown in Figure 1. It was revealed that 2SC (Free) released in the blood significantly increased mainly in small vessel disorders, and total 2SC (Total) in the blood increased in macrovascular disorders.

(2) Comparison of the value of 2SC released in the blood and the progression of pathological conditions in retinopathy and nephropathy. The progress and comparison were made. A total of 68 patients (30 men, 38 women) with an average age of 66 years were measured. The number of people in each group is as follows. No retinopathy: 52 patients, simple diabetic retinopathy: 10 patients, preproliferative diabetic retinopathy: 2 patients, proliferative diabetic retinopathy: 4 patients. No nephropathy/stage 1: 46 patients, stage 2 nephropathy: 18 patients, stage 3 nephropathy: 3 patients, stage 4 nephropathy: 1 patient.

The results are shown in Figure 2. Although no significant difference was observed due to the small number of samples in each group, an increasing trend was observed.

(3) Measurement of CML in the serum of patients with various diseases CML, which is a typical AGEs structure, was measured in the serum of the patients measured in Figure 1 and compared with past medical history.
The results are shown in Figure 3. Unlike 2SC, no increase in each disease was observed in CML. Although CML is expected to be a marker of vascular damage, it has become clear that it does not change with the progression of vascular damage.

(4) Measurement of blood 2SC in arteriosclerosis model mice Blood 2SC of apoE-deficient mice (20 weeks old), which are arteriosclerosis model mice, was measured and compared with healthy individuals (3 animals in each group).
The results are shown in Figure 4. Both Free and Total 2SC values showed high values in apoE-deficient mice, and a relationship between the onset of vascular disorders and blood 2SC values was clarified in model mice as well as in humans.

Claims (7)

A method for providing an index of macrovascular disorder , the method comprising measuring the total amount of S-(2-succinyl)cysteine in blood. The method according to claim 1, wherein when the measured value obtained by measuring the total amount of S-(2-succinyl) cysteine in the blood is higher than a reference value, the measured value is taken as an index of macrovascular disorder. . Claim 1: When the measured value obtained by measuring the total amount of S-(2-succinyl) cysteine in the blood is higher than a reference value , the measured value is used as an indicator of a high risk of macrovascular disorder. The method described in. 4. The method according to claim 1, wherein the total amount of S-(2-succinyl)cysteine in blood is measured by mass spectrometry or immunoassay. A method for screening a candidate substance for the prevention or treatment of macrovascular disorders , the method comprising measuring the total amount of S-(2-succinyl)cysteine in the blood of a subject to whom the candidate substance has been administered. 6. The screening method according to claim 5, wherein the total amount of S-(2-succinyl)cysteine in blood is measured by mass spectrometry or immunoassay. The screening method according to claim 5 or 6, wherein the candidate substance is a food-derived component.