WO2014088118A1

WO2014088118A1 - Cardiopathy marker and usage thereof

Info

Publication number: WO2014088118A1
Application number: PCT/JP2013/083007
Authority: WO
Inventors: 恭彦伊藤; 史子名倉; 佳史武井; 清一松尾; 秋山　真一; 朋義曽我; 明由平山
Original assignee: 国立大学法人名古屋大学
Priority date: 2012-12-07
Filing date: 2013-12-09
Publication date: 2014-06-12
Also published as: JPWO2014088118A1

Abstract

A method for monitoring cardiopathy is provided with a step for measuring hydroxyproline in a blood-related liquid of a test specimen. Hydroxyproline in a blood-related liquid is capable of monitoring cardiopathic processes in early onset cardiopathy, onset cardiopathy, and late onset cardiopathy.

Description

Cardiac lesion marker and use thereof

The present specification relates to a marker that can be used for diagnosis of a cardiac lesion, for example, a cardiac lesion accompanying the progression of chronic kidney disease, and the use thereof.

Chronic renal failure (CRF) and chronic kidney disease (CKD), which does not lead to chronic renal failure but includes its underlying diseases, are no longer an exaggeration to say that it is a national disease in Japan. The increase in end-stage renal failure patients undergoing dialysis is also a major medical economic problem. The number one cause of death in dialysis patients is heart failure. In the pathology of chronic kidney disease up to dialysis, the most important factor that defines the prognosis is heart disease. The heart disease in chronic kidney disease is not the so-called coronary artery lesion, and cardiac hypertrophy has been recognized as the most important finding (Non-patent Document 1).

Conventionally, in patients with chronic kidney disease, cardiac hypertrophy and cardiac diastolic disorders are partially checked by cardiac ultrasonography at most once a year. Further, serologically known markers that are indicators of cardiac hypertrophy / fibrosis include type I procollagen C-terminal propeptide (PICP), matrix metaprotease-1 (MMP-1), and the like.

Once the cardiac lesion is completed, it is extremely difficult to improve the cardiac lesion once constructed even if antihypertensive treatment or coronary artery treatment is performed, and the prognosis deteriorates. However, it has been practically difficult to carry out periodic cardiac ultrasonography for a huge number of patients with chronic kidney disease. Further, according to the present inventors, serological markers such as PICP and MMP-1 are not always good in correlation with echocardiographic findings, and are not realistic.

Further, as a marker for detecting fibrosis of the myocardium, there is a method of measuring the amount of hydroxyproline in the tissue corresponding to the amount of collagen in the tissue that is an index of fibrosis. However, it has been impossible to apply clinically a method based on tissue collection.

As described above, early detection and prevention of cardiac lesions was very important. However, it is a convenient marker for early detection of heart lesions, detection of previous signs leading to heart lesions, or monitoring of heart lesions such as real-time / rapid confirmation of drug treatment or improvement effect on heart lesions. Does not exist.

This specification provides a marker suitable for monitoring cardiac lesions and use thereof.

The present inventors have found a serum marker corresponding to the progression of cardiac lesions from 573 metabolites in vivo based on the results of experiments using renal failure model mice that have caused cardiac lesions. That is, the present inventors prepared renal failure model mice, and prepared 4 groups including the presence or absence of salt load and control mice. In 4 stages in these 4 groups, 573 metabolites were comprehensively analyzed using serum, myocardium and abdominal wall muscle as specimens. As a result, it was found that the serum concentration of one metabolite changes depending on the progression of the cardiac lesion, particularly the tissue state in the pre-stage to the completion stage of the occurrence of the cardiac lesion. Further, according to the present inventors, the amount of hydroxyproline, which has been an index of cardiac fibrosis in a heart lesion model animal, has been measured up to hydroxyproline that has been incorporated into tissue and stabilized, and the progression of heart lesions. It was also found that this method is not suitable as a technique for reflecting the degree, and it was also found that cardiac lesions can be monitored more accurately by measuring the amount of hydroxyproline released in the tissue. According to this specification, the following means are provided based on such knowledge.

(1) A method for monitoring cardiac lesions,
Measuring hydroxyproline in the blood-related fluid of the test individual;
A method comprising:
(2) The method according to (1), wherein the cardiac lesion is a cardiac lesion associated with chronic kidney disease.
(3) The method according to (1) or (2), wherein the cardiac lesion includes a myocardial lesion.
(4) The method according to any one of (1) to (3), wherein the myocardial lesion includes myocardial fibrosis, cardiac hypertrophy and diastole.
(5) The method according to any one of (1) to (4), wherein the measurement step is a step of measuring free hydroxyproline, collagen or hydroxyproline which does not constitute a part thereof.
(6) The method according to any one of (1) to (5), wherein the measurement step includes performing a step of measuring the amount of hydroxyproline in the blood-related fluid for each subject at two or more times. Method.
(7) The method according to (6), wherein when the tendency to increase the amount of hydroxyproline over time is affirmed, it is determined that the cardiac lesion process is in an advanced stage.
(8) The method according to (6), wherein when the tendency to decrease the amount of hydroxyproline over time is affirmed, it is determined that the cardiac lesion process is in a regression stage.
(9) The method according to (6), wherein when the tendency to decrease the amount of hydroxyproline over time is affirmed, it is determined that the cardiac lesion process is in a completion stage (final stage).
(10) The method according to (6), wherein when the tendency to maintain the amount of hydroxyproline over time is affirmed, it is determined that the cardiac lesion process or the state before the cardiac lesion process is maintained.
(11) A method for monitoring the effects of drugs and / or treatments on cardiac lesions,
Measuring hydroxyproline in the blood-related fluid of the test individual;
A method comprising:
(12) A blood-related fluid marker for cardiac lesions, comprising hydroxyproline.
(13) A kit for performing the monitoring method according to any one of (1) to (11), comprising a reagent necessary for measuring hydroxyproline in a blood-related fluid.
(14) a step of administering one or more test compounds to a cardiac lesion model animal;
Measuring hydroxyproline in the blood-related fluid of the model animal;
Evaluating the action of the one or more test compounds on a cardiac lesion based on the amount of hydroxyproline or a change thereof;
A screening method for a drug effective for the prevention, treatment or improvement of cardiac lesions.
(15) The method according to (14), further comprising a step of subjecting the model animal to a salt load.
(16) The method according to (14) or (15), further comprising the step of measuring the amount of free hydroxyproline or collagen or a hydroxyproline that does not constitute a part of the tissue of the model animal.
(17) administering one or more test compounds to a cardiac lesion model animal;
A step of measuring free hydroxyproline or hydroxyproline which does not constitute collagen or a part thereof in the tissue of the model animal;
Evaluating the action of the one or more test compounds on a cardiac lesion based on the amount of hydroxyproline or a change thereof;
A screening method for a drug effective for the prevention, treatment or improvement of cardiac lesions.
(18) A method for monitoring cardiac lesions in a cardiac lesion model animal,
A step of measuring free hydroxyproline or hydroxyproline which does not constitute collagen or a part thereof in the tissue of the model animal,
A method comprising:
(19) A method for monitoring cardiac lesions in a cardiac lesion model animal,
Measuring hydroxyproline in blood-related fluid in the tissue of the model animal,
A method comprising:

It is a figure which shows the outline | summary of the cardiac lesion marker disclosed by this specification. It is a figure which shows the evaluation result (2 weeks-8 weeks) about the creatine (Cr) in the serum of the mouse | mouth of 4 groups in an Example. It is a figure which shows the evaluation result (2 weeks-8 weeks) about the blood pressure of the 4 group mouse | mouth in an Example. It is a figure which shows the evaluation result (2 weeks-8 weeks) of the heart weight of the 4 group mouse | mouth in an Example. It is a figure which shows the evaluation result of the fibrosis staining of the myocardium of the 4th group mouse | mouth in an Example. It is a figure which shows the evaluation result of the fibrosis staining of the myocardium of the 4th group mouse | mouth in an Example. It is a figure which shows the evaluation result of the amount of hydroxyproline of the heart of the 4th group mouse | mouth in an Example. It is a figure which shows the evaluation result of the hydroxyproline amount of the serum of the 4th group mouse | mouth in an Example. It is a figure which shows the evaluation result about the serum hydroxyproline, serum creatine, blood pressure, and heart weight at the time of antihypertensive agent treatment with respect to a renal failure salt load mouse | mouth in an Example. It is a figure which shows the measurement process A of the hydroxyproline in the structure | tissue in an Example. It is a figure which shows a part process of the measurement process BD of the hydroxyproline in the structure | tissue in an Example. It is a figure which shows the residual process of the measurement process BD of the hydroxyproline in the structure | tissue in an Example. It is a figure which shows the outline | summary of the measurement process AD of hydroxyproline. It is a figure which shows the measurement result of the hydroxyproline by measurement process AD of hydroxyproline.

The present disclosure relates to a cardiac lesion monitoring marker including cardiac lesions, for example, cardiac muscle lesions such as increased cardiac weight, cardiac hypertrophy, and myocardial fibrosis, and use thereof. As described above, the present inventors prepared renal failure model mice and prepared four groups including the presence or absence of salt load and control mice. 4 stages in these 4 groups (normal (corresponding to GFRG1) / mild to moderate decrease (corresponding to G2 to 3a) / moderate decrease to severe decrease (corresponding to G3a to 4) / high decrease to end stage renal failure (same G4) It was found that the serum concentration of hydroxyproline exhibited changes in accordance with the progression of the cardiac lesion, particularly the tissue state in the pre-stage to completion stage of the occurrence of the cardiac lesion. It was also found that the serum concentration of hydroxyproline changed according to the progression (synthesis rate) of myocardial fibrosis. That is, it was found that the serum concentration increased while myocardial fibrosis was in progress, and decreased when the myocardial fibrosis was in the final stage.

According to the monitoring markers disclosed herein, the concentration of hydroxyproline in serum prior to the onset of cardiac lesions, more specifically prior to the detection of cardiac lesions such as fibrosis on examination. Is based on the finding that there is a tendency to increase. Moreover, it is based on the knowledge that there is a tendency that the serum hydroxyproline concentration tends to decrease by suppressing the progression of cardiac lesions and chronic kidney disease at the pre-cardiac lesion stage. Furthermore, it is based on the knowledge that the hydroxyproline concentration in serum tends to decrease at the stage before or after completion of the cardiac lesion.

For this reason, according to the present monitoring marker, it is possible to monitor whether or not there is a sign of cardiac lesion or the tendency of cardiac lesion onset and its extent at the stage before cardiac lesion onset. In addition, according to the present monitoring marker, it is possible to monitor the degree of progression to the heart lesion and the degree of improvement thereof at the stage before the onset of the heart lesion. Furthermore, according to the present monitoring marker, it is possible to monitor the therapeutic effects and therapeutic effects on cardiac lesions and chronic kidney disease in the stage before the onset of cardiac lesions. Further, according to the present monitoring marker, it is possible to monitor the degree of completion (reaching degree) of a cardiac lesion.

Furthermore, by using the chronic kidney disease model animal and the present monitoring marker, drug screening for chronic kidney disease or heart lesion can be effectively performed.

Incidentally, hydroxyproline is a constituent amino acid of collagen and is an index of the amount of collagen in the tissue. For this reason, tissue hydroxyproline is known to be an effective marker of organ fibrosis. However, it has never been expected that hydroxyproline in blood-related fluids such as serum will be a marker of cardiac lesions.

In the present specification, “cardiac lesion” means a wide lesion (abnormal) in the heart including a myocardial lesion. Among these, myocardial lesions such as myocardial fibrosis, cardiac hypertrophy, increased heart weight, and diastolic dysfunction can be mentioned. Furthermore, the “cardiac lesion” in the present specification includes various heart lesions associated with chronic kidney disease.

Also, in this specification, “cardiac lesion” develops when “cardiac lesion” is medically confirmed to be present by “cardiac ultrasonography” or other examinations and / or clinical findings. I mean.

In this specification, the “cardiac lesion process” includes a stage before the onset of the heart lesion, a stage of onset of the heart lesion, and a stage after the onset of the heart lesion. The pre-stage of developing a cardiac lesion means that the cardiac lesion has not developed, but the process toward the onset of the cardiac lesion has started or has progressed. The stage before the onset of a cardiac lesion may be referred to herein as a predictor of cardiac lesion.

In the present specification, “chronic kidney disease (CKD)” means a decrease in renal function and / or a renal disorder expressed by GFR (<60 ml / min / 1.73 m ² ) (urinary abnormality, image) This includes everything that persists chronically (abnormal for 3 months). Kidney disorders include, for example, urine abnormalities such as proteinuria including microalbuminuria, urinary sediment abnormalities, abnormal images such as single kidneys and multiple cystic kidneys, decreased renal function such as increased serum creatinine level, tubular disorders May include decreased renal function such as hypokemia, abnormalities in histopathological examination in renal biopsy and the like. In particular, the presence of 0.15 g / gCr or higher proteinuria (30 mg / gCr or higher albuminuria) can be mentioned. GFR (glomerular filtration rate) is a numerical value indicating the function of the kidney calculated from the serum creatine value and age.

The stage (stage) of chronic kidney disease is classified as follows according to the GFR classification.
G1: Normal or high (> 90)
G2: Normal or slight decrease (60 to 89)
G3a: Mild to moderate decline (45 to 59)
G3b: Moderate to low altitude (30 to 44)
G4: Altitude reduction (15 to 29)
G5: End stage renal failure (less than 15)

Hereinafter, as an embodiment according to the disclosure of the present specification, a heart lesion monitoring method, a method for monitoring the effects of drugs and / or treatments on heart lesions, a heart lesion serum marker, a monitoring kit, a screening method, and the like will be described.

(Cardiac lesion monitoring method)
The method for monitoring a cardiac lesion disclosed in the present specification can comprise a step of measuring hydroxyproline in a blood-related fluid of a subject individual. Examples of the test individual include animals such as mammals including humans. Preferably, it is a mammal. More specifically, the test individual includes humans, pets or laboratory animals such as dogs, cats, monkeys, mice, rats, and rabbits, and livestock animals such as cows, horses, sheep, pigs, and goats.

Blood-related fluid includes whole blood, plasma and serum. Any blood-related fluid can be used in this monitoring method, but is typically serum. The blood-related fluid can usually be collected from the blood vessel of the subject individual.

ヒドロキシ Hydroxyproline, which is the monitoring target in this monitoring method, has a hydroxyl group introduced into the y carbon atom of proline. The hydroxyproline targeted by this monitoring method is preferably not hydroxyproline in collagen but free hydroxyproline or hydroxyproline not constituting collagen or a part thereof (hereinafter referred to as free hydroxyproline). . Such free hydroxyproline is considered to be synthesized from proline by propyl 4-hydroxylase and from degradation of collagen. Free hydroxyproline is thought to be actively involved in collagen synthesis and metabolism (Critical reviews in Biochemistry and Molecular Biology 2010; 45: 106- 124, Journal of Biological Chemistry 2008; 283: 10485-10492, Amino Acids 2011 ; 40: 1053-1063).

It is considered that the hydroxyproline present in the blood-related fluid is predominantly free of hydroxyproline than the hydroxyproline in the state of constituting collagen. For this reason, using a blood-related individual as a specimen itself results in selective measurement of free hydroxyproline. More preferred is plasma rather than whole blood, and still more preferred is serum.

The blood-related fluid is pretreated by a known method to obtain a measurement sample suitable for detection and quantification of hydroxyproline. For example, in the case of serum, after inactivating the enzyme by mixing with a medium containing alcohol such as methanol, a nonpolar solvent such as chloroform is further added and mixed, and lipid such as phospholipid is added to the nonpolar solvent phase. Separate into. Thereafter, the aqueous phase (water-methanol phase) is filtered through a centrifugal ultrafiltration filter having a molecular weight cut-off of about 5 kDa, deproteinized and used. The filtrate can be subjected to measurement of hydroxyproline by removing the medium once by freeze-drying or the like as necessary, and further dissolving in a medium (for example, water) suitable for measurement of hydroxyproline.

In addition, it is preferable to deproteinize the sample preparation. Deproteinization can remove collagen that may be present in blood-related fluids, and effectively eliminate possible measurement errors based on collagen-derived hydroxyproline. The protein removal treatment is not particularly limited as long as at least a part of collagen can be removed, and such removal treatment can be appropriately selected from known methods by those skilled in the art. Typically, as described above, a centrifugal ultrafiltration filter that can remove a protein of about 5 kDa can be used.

Hydroxyproline in blood-related fluid can be measured by various known methods. As a method for measuring hydroxyproline, a colorimetric method for oxidizing and decarboxylating hydroxyproline and coloring the resulting pyrrole with p-dimethylbenzaldehyde (a commercially available quantitative kit (for example, manufactured by BioVision), pharmaceuticals, etc.) Magazine, 106 (1), 41-46'1986), thin layer chromatography, high pressure electrophoresis, amino acid analyzer and high performance liquid chromatography, capillary electrophoresis-time-of-flight mass spectrometer (CE-TOFMS), etc. No. 2011-58863 and Japanese Patent No. 3341765).

In the step of measuring hydroxyproline, it is preferable to quantify hydroxyproline. That is, it is preferable to acquire the amount of hydroxyproline as a concentration in the blood-related liquid.

The measuring step may simply be to collect blood-related fluid from the subject and measure its hydroxyproline concentration. The obtained amount of hydroxyproline is compared with a predetermined threshold value, and the comparison can be used to determine or estimate the stage of the cardiac lesion such as a non-onset stage or a pre-onset stage of the cardiac lesion, thereby assisting treatment or diagnosis. Such thresholds are, for example, measured for hydroxyproline in blood-related fluids for healthy individuals and individuals at different stages of cardiac pathology (preferably a statistically valid parameter) and other tests (eg, It can be determined from the relationship with the diagnostic results of cardiac ultrasound image diagnosis, MRI, etc.). A plurality of threshold values can be set according to the stage of the cardiac lesion. For example, a threshold value indicating that it is a non-onset stage (healthy), or a threshold value of 1 or 2 or more depending on the degree in the pre-onset stage although no onset has occurred. In addition, you may set such a threshold value by making an affected individual of a specific basic disease, for example, chronic kidney disease, into a population. Further, such a threshold may be set with a certain age group as a population.

The measurement step preferably includes a step of measuring hydroxyproline in the blood-related fluid at two or more times for the same subject. As will be described later, by measuring the amount of hydroxyproline for two or more periods, the amount of hydroxyproline can be compared to grasp the transition (change) of the amount of hydroxyproline. In addition to the amount of hydroxyproline, by grasping these changes, it is possible to more effectively determine or estimate the stage of cardiac lesions such as the non-onset stage and pre-onset stage of cardiac lesions, and assist treatment and diagnosis. Can do.

In the measurement step, blood-related fluid is collected from the subject individual regularly or irregularly, preferably several times, preferably continuously, and hydroxyproline is measured for each blood-related fluid. The measurement interval is not particularly specified. Further, the measurement frequency (number of times within a certain period) is not particularly limited. The measurement interval and the measurement frequency are appropriately determined in consideration of the past medical history, the stage of chronic kidney disease as a basic disease, age, course of treatment, and the like. The measurement frequency is preferably at least 2 times a year, more preferably at least 3 times, even more preferably at least 4 times, still more preferably at least 5 times, even more preferably at least 6 times, even more preferably at least 8 times. Most preferably, it is 10 times or more. Preferably, the measurement interval is substantially constant.

When it is provided with a step of measuring hydroxyproline for two or more periods, it is preferable to carry out a comparison step for comparing the amount of hydroxyproline at different periods. That is, the amount of hydroxyproline in the blood-related liquid at the first time and the amount of hydroxyproline in the blood-related liquid at the second time earlier than the first time are changed over time. The process of determining is implemented. The first period means a newer period selected in comparing the amount of hydroxyproline, and the second period means a period when the hydroxyproline amount was measured before that.

The first period and the second period to be compared are selected to determine a change over time in the hydroxyproline amount, that is, a preferable one to determine an increasing tendency / decreasing tendency / maintenance tendency of the hydroxyproline amount. When there are three or more times, the second time in the past that is closest to the first time may be selected as the comparison target, or the comparison target of the second time after that may be selected. . Consideration is also given to the situation in the test individual and changes in the amount of hydroxyproline corresponding thereto. For example, in the case shown in FIG. 1, it may be preferable to compare the time A and the time C to see the tendency over time in the observation period, rather than comparing the time B and the time C.

According to the present inventors, the tendency of the hydroxyproline amount to increase over time is observed in the process of myocardial fibrosis (not in the state of myocardial fibrosis) prior to the completion stage of myocardial fibrosis. I know. That is, when the tendency to increase the amount of hydroxyproline over time is affirmed, it is predicted that myocardial fibrosis and mass increase, which are the basis of a cardiac lesion, have started or progressed. For this reason, when the tendency to increase the amount of hydroxyproline over time is affirmed in the comparison step, it can be determined that the cardiac lesion process is in an advanced stage.

The following (a) and (b) correspond to the situation in the cardiac lesion process when the amount of hydroxyproline shows an increasing tendency with time.
(A) The pre-stage before the onset of the cardiac lesion is progressing. (B) The subsequent advanced stage from the onset of the cardiac pathology is included.

In any of the above (a) and (b), the same is true in that the cardiac lesion process is progressing. In order to determine which of the above (a) and (b), clinical findings and other test results in the subject are taken into consideration.

On the other hand, when the tendency to decrease the amount of hydroxyproline over time is affirmed in the comparison step, it can be determined that the cardiac lesion process is in the backward stage. According to the present inventors, it is known that the tendency of the amount of hydroxyproline to decrease over time is observed when the cardiac lesion process is suppressed by drug administration or treatment. That is, when the tendency to decrease the amount of hydroxyproline over time is affirmed, it is predicted that myocardial fibrosis and mass increase that are the basis of cardiac lesions are suppressed.

The following (c) and (d) correspond to the situation in the cardiac lesion process when the amount of hydroxyproline shows a decreasing tendency with time.
(C) Regressed (improved) from the previous stage before the onset of cardiac lesion (d) Retreated (improved) from the subsequent stage of onset of cardiac lesion

In any of the above (c) and (d), the same is true in that the cardiac lesion process is retreated (improved). Whether (c) or (d) above is considered in addition to clinical findings and hydroxyproline concentrations in the subject, as well as other test results.

Also, when the trend of decreasing the amount of hydroxyproline over time is affirmed in the comparison step, it can be determined that the cardiac lesion process is in the completion stage (final stage). According to the present inventors, it is known that the tendency of the amount of hydroxyproline to decrease with time is observed at the stage after the onset of cardiac lesion and further in the stage of fibrosis. This is because it is considered that hydroxyproline is not a marker of the fibrosis state but a marker corresponding to the speed to the fibrosis. In other words, it can be said that as the completion stage of the cardiac lesion process approaches, the progress of fibrosis is slowed down.

The following (e) corresponds to the situation in the cardiac lesion process when the amount of hydroxyproline shows a decreasing tendency with time.
(E) Being in an advanced stage after the onset of a cardiac lesion

In order to determine whether the amount of hydroxyproline is decreasing with time or whether it is (c) or (d) above or (e) above, clinical findings or hydroxy In addition to proline concentration, other test results are considered.

Furthermore, when the tendency to maintain the hydroxyproline amount over time is affirmed in the comparison step, it can also be determined that the cardiac lesion process or the state before the cardiac lesion process is maintained. According to the present inventors, since the time-dependent increase or decrease in the amount of hydroxyproline is associated with the stage of the cardiac lesion process, the trend of maintaining the amount of hydroxyproline over time is the current state of It can be said that the state is before the lesion process.

For example, the following (f) and (g) correspond to the situation in the cardiac lesion process when the amount of hydroxyproline tends to be maintained over time.
(F) The cardiac lesion process is maintained (stagnation) (g) The state before the cardiac lesion process (a healthy state with respect to the cardiac lesion) is maintained

The same applies to either (e) or (f) above in that the current situation is maintained. When the amount of hydroxyproline exhibits a tendency to maintain over time, it is possible to determine whether it is the above (e) or (f). In addition to clinical findings and hydroxyproline concentration in the subject, other tests Results are taken into account.

In this monitoring method, it is possible to determine the stage in the cardiac lesion process for the subject based on the amount of hydroxyproline in the blood-related fluid and / or its change. For useful determination, it is preferable to appropriately combine other markers related to cardiac lesions in the subject, results of cardiac ultrasonography (LV-mass, E / e ′, etc.), etc., as necessary. . Examples of cardiac lesion markers include ANP (atrial natriuretic peptide), BNP (human brain natriuretic peptide), proBNP (human brain natriuretic peptide precursor), NP-proBNP (human brain natriuretic peptide precursor N fragment) ) And the like in blood-related liquids and urine concentrations.

As described above, according to this monitoring method, each stage of the cardiac lesion process including all stages prior to the onset of the cardiac lesion is monitored from the blood-related fluid of the individual to be examined, from the healthy state of the cardiac lesion to the individual to be examined. can do. That is, according to this monitoring method, a healthy state (risk non-existing state) for a cardiac lesion can be confirmed. Further, it is possible to characterize the state related to the cardiac lesion in the test subject, and to easily and effectively assist the diagnosis related to the cardiac lesion. In addition, the risk of future cardiac lesions can be predicted or determined.

(Method for monitoring the effects of drugs and / or treatments on cardiac lesions)
The method for monitoring the effect of a drug and / or treatment on a cardiac lesion disclosed in the present specification (hereinafter referred to as the present therapeutic effect monitoring method) comprises a step of measuring hydroxyproline in blood-related fluid of a test individual. Can be provided. According to this therapeutic effect monitoring method, the administration of drugs for cardiac lesions, the effect of treatment, etc. can be easily monitored and evaluated by measuring the hydroxyproline concentration in the blood-related fluid of the subject. For this reason, it is possible to set an appropriate drug administration plan and treatment plan for the individual to be tested regarding prevention, improvement and treatment of cardiac lesions.

Note that there are no particular limitations on the drug or treatment for cardiac lesions. All possible drugs and treatments are included. Specifically, in addition to those that directly affect cardiac lesions, for example, when chronic kidney disease is present as the underlying disease, suppression of increased blood pressure (which is considered to be a factor that promotes cardiac lesions) associated with chronic kidney disease Examples thereof include those intended to alleviate, reduce or ameliorate the underlying disease or accompanying symptoms, such as those intended (for example, antihypertensive agents).

In the therapeutic effect monitoring method, the various aspects already described in the present monitoring method can be applied to the test individual, blood-related fluid, and hydroxyproline in the hydroxyproline measurement step.

(Marker in blood-related fluid of heart lesion)
Markers in blood-related fluids for cardiac lesions disclosed herein include hydroxyproline. The amount of hydroxyproline and its change in blood-related fluids are effective markers of cardiac pathology. With respect to this marker, the various aspects already described in this monitoring method can be applied to the test individual, blood-related fluid, sample preparation, hydroxyproline, measurement method thereof, and the like.

(Monitoring kit)
The monitoring kit disclosed herein can include reagents necessary to measure hydroxyproline in blood-related fluids. This monitoring kit can be used for performing this monitoring method and this therapeutic effect monitoring method. With respect to this monitoring kit, various aspects already described in this monitoring method can be applied to the test individual, blood-related fluid, sample preparation, hydroxyproline, measurement method thereof, and the like. As already described, there are various known methods for detecting and quantifying hydroxyproline, and at the same time, reagents and devices are also provided. The monitoring kit can include one or more of these reagents. Moreover, this monitoring kit can also be attached to such a known apparatus.

(Screening method)
The screening method disclosed in the present specification is a method for screening a drug effective for the prevention, treatment or amelioration of cardiac lesions, comprising the step of administering one or more test compounds to a cardiac lesion model animal; , Measuring the hydroxyproline in blood-related fluids such as blood of model animals, and evaluating the effect of one or more test compounds on cardiac lesions associated with chronic kidney disease based on the amount of hydroxyproline or its change And a step of performing. According to this screening method, it is possible to effectively screen for drugs effective for the prevention, treatment or amelioration of cardiac lesions not accompanying / with chronic kidney disease.

Furthermore, this screening method may include a step of measuring the amount of free hydroxyproline or collagen or a portion of hydroxyproline that does not constitute a part thereof in the tissue of the model animal. By providing this step, it is possible to more directly detect the fibrosis of the heart tissue in the cardiac lesion model animal and use it for the evaluation of the drug. When this step is provided, it is preferable to include a step of evaluating the action of the one or more test compounds on cardiac lesions based on the amount of hydroxyproline thus obtained or its change.

According to this screening method, the cardiac lesion process can be monitored by measuring the concentration of hydroxyproline in the blood-related fluid of the model animal. For this reason, the effect of a medicine etc. can be checked simply. In addition, the number of model animals can be reduced to confirm cardiac lesions.

This screening method may comprise only the step of measuring hydroxyproline in blood-related fluids, but further, free hydroxyproline in tissues such as heart tissue or hydroxyproline that does not constitute collagen or a part thereof. Only the process of measuring the quantity may be provided, or both processes may be provided.

There is no particular limitation on the mind organization. It may be a part of the entire organization. Moreover, when the lesion site | part area | region in a structure | tissue can be specified, the part including the said specific area | region may be sufficient.

Moreover, in order to measure the amount of free hydroxyproline or collagen in the heart tissue or hydroxyproline which does not constitute a part thereof, for example, the contact treatment with an acid such as 6N hydrochloric acid and the subsequent boiling step are eliminated. It is useful to perform an ultrafiltration treatment of an appropriate fraction such as 5000 kD to eliminate a protein removal step having a large molecular weight. After performing such pretreatment, it is preferable to carry out extraction separation of hydroxyproline by solvent partitioning with the chloroform: aqueous liquid (for example, methanol, water and a mixture thereof) as already described. A person skilled in the art can appropriately set treatment and ultrafiltration conditions suitable for eliminating hydroxyproline immobilized on collagen or the like as necessary.

As a model animal, a chronic kidney disease model animal is suitable as a cardiac hypertrophy fibrosis heart lesion. For example, 5/6 nephrectomized animals (such as mice) are preferably used as chronic kidney disease model animals. The cardiac lesion model animal is not limited to a chronic kidney disease model animal. Any other hypertrophic heart disease model animal may be used. For example, an aortic constriction model animal can be mentioned. This model animal is a model in which renal function is normal but cardiac hypertrophy and fibrosis progress. Moreover, the animal conventionally used for model animals, such as a dog, a cat, and a monkey, can be used for a model animal.

The screening method may include a step of applying a salt load to the model animal. For example, if a salt load is applied to a chronic kidney disease model animal, the stage can be reached early and the heart lesion can be increased. As a result, effective screening becomes possible. The salt load can be mixed in drinking water or feed, and any method may be used. Those skilled in the art can appropriately determine the salt load. In addition to the salt load, the present screening method can apply a load related to the progression of cardiac lesions and the exacerbation of the underlying disease to model animals.

The test compound is not particularly limited. For example, natural compounds, organic compounds, inorganic compounds, single compounds such as nucleic acids, proteins, peptides, etc., compound libraries, nucleic acid libraries, peptide libraries, gene library expression products, cell extracts, cell culture Examples thereof include cleansing, fermented microorganism products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts, and animal cell extracts. The test compound can be appropriately labeled and used as necessary. Examples of the label include a radiolabel and a fluorescent label.

The method for administering the test compound to the model animal is not particularly limited. It may be mixed with drinking water or feed, or various known administration methods (injection (subcutaneous, intravenous injection, etc.), injection (gastrointestinal tract, etc.), intraperitoneal administration, sticking, etc.) of so-called drugs may be employed. .

(Monitoring method of heart lesion in animal model of heart lesion)
This monitoring method may comprise a step of measuring free hydroxyproline or hydroxyproline which does not constitute collagen or a part thereof in the tissue of the model animal. Such a monitoring method is extremely direct and specific in a model animal, and is suitable for various evaluations. In monitoring, the pathological condition of the cardiac lesion can be correlated with the measurement result of the hydroxyproline in the heart tissue.

This monitoring method may further include a step of measuring hydroxyproline in the blood-related fluid in the tissue of the model animal. By doing so, the relationship between cardiac lesions in heart tissue and hydroxyproline in blood-related fluids such as serum can be related in more detail, which is effective for various evaluations.

In the present monitoring method, various embodiments in the cardiac lesion monitoring method mainly for humans and the like already described can be applied.

Note that all prior art documents cited in the present specification are incorporated herein by reference.

Hereinafter, examples in which the disclosure of the present specification is embodied will be described, but the following examples do not limit the disclosure of the present specification. The experimental operations performed in each example are shown below.

(Creation of animal models)
The following 4 groups were prepared as model animals.
Normal kidney function mouse (no nephrectomy)
Salt-loaded mice (no nephrectomy)
Renal failure (5/6 nephrectomy) mouse Renal failure (5/6 nephrectomy) + salt-loaded mouse

The mice were 129 SvJJmsSlc males 8 weeks old and weighed 23 to 28 g. The salt loading method provided 1% saline as drinking water. The renal failure model was prepared by performing 5/6 nephrectomy. In the 5/6 nephrectomy method, first, 2/3 of the left kidney was removed, and one week later, the right nephrectomy was performed (this day is designated as renal failure Day 0) to obtain a renal failure model. In each mouse used, blood pressure and body weight were measured before surgery, after 2 weeks, after 4 weeks and after 8 weeks.

(Sample collection)
In order to see the progress in the above 4 groups of mice, 2 week, 4 week and 8 week models were prepared for each group. Serum, urine, heart, abdominal wall, and aorta were collected for each week model. The heart was measured for heart weight. For serum, various metabolites including hydroxyproline in addition to Cr were subjected to metabolomic analysis. The heart was subjected to staining for evaluation of fibrosis in addition to metabolomic analysis.

(analysis)
(1) Staining Staining was performed to evaluate cardiac fibrosis. Staining for evaluation of cardiac fibrosis was performed using Picrosirius Red Stain Kit Polysciences, Inc., and the ratio of fibrosis was measured with image analysis software MetaMorph (Molecular Device Japan).

(2) Metabolome analysis (preparation of measurement sample)
Serum was separated from blood collected from mice by a known method, and after adding 450 μl of methanol containing an internal standard to 50 μl of this serum sample, 500 μl of chloroform and 200 μl of pure water were further added and stirred well. After stirring, the mixture was centrifuged at 4 ° C. and 5000 rpm for 5 minutes, and 600 μl of the separated aqueous phase was deproteinized using an ultrafiltration filter having a fractional molecular weight of 5000 Da. The filtrate was centrifuged to dryness, redissolved with 50 μl of pure water, and subjected to capillary electrophoresis-TOFMS (CE-TOFMS) analysis.

In addition, 500 μl of methanol containing an internal standard was added to 50 mg of tissue collected from a mouse and homogenized, and the same pretreatment as in the case of serum was performed. The filtrate was centrifuged to dryness, redissolved with 50 μl of pure water, and subjected to CE-TOFMS analysis.

(Measurement of metabolites in samples by CE-TOFMS)
Metabolites in the sample pretreated with CE-TOFMS were comprehensively measured.
1. Cationic metabolite measurement conditions (analysis conditions for capillary electrophoresis)
As the capillary, a fused silica capillary (inner diameter 50 μm, outer diameter 350 μm, total length 100 cm) was used. As the buffer, 1M formic acid (pH about 1.8) was used. The applied voltage was +30 kV, and the capillary temperature was 20 ° C. The sample was injected for 3 seconds at 50 mbar using the pressure method.

(Analysis conditions for a time-of-flight mass spectrometer)
Using positive ion mode, the ionization voltage was set to 4 kV, the fragmentor voltage was set to 75 V, the skimmer voltage was set to 50 V, and the OctRFV voltage was set to 125 V. Nitrogen was used as the drying gas, and the temperature was set to 300 ° C. and the pressure was set to 10 psig. A 50% methanol solution was used as the sheath liquid, and hexakis (2,2-difluorotoxyl) phosphazene was mixed to a concentration of 0.1 μM for mass calibration, and the solution was fed at 10 μl / min. All data obtained using the mass numbers of hexakis (2,2-difluorotoxyl) phosphazene (m / z 622.0290) and methanol dimer isotope (m / z 66.0632) were automatically calibrated.

2. Anionic metabolite measurement conditions (analysis conditions for capillary electrophoresis)
As the capillary, a COSMO (+) capillary (inner diameter 50 μm, outer diameter 350 μm, total length 100 cm) was used. As the buffer, 50 mM ammonium acetate (pH 8.5) was used. The applied voltage was −30 kV, and the capillary temperature was 20 ° C. The sample was injected for 30 seconds at 50 mbar using the pressure method.

(Analysis conditions for a time-of-flight mass spectrometer)
Using the negative ion mode, the ionization voltage was set to 3.5 kV, the fragmentor voltage was set to 100 V, the skimmer voltage was set to 50 V, and the OctRFV voltage was set to 200 V. Nitrogen was used as the drying gas, and the temperature was set to 300 ° C. and the pressure was set to 10 psig. As the sheath liquid, a 50% methanol solution containing 5 mM ammonium acetate was used. For mass calibration, hexakis (2,2-difluorotoxyl) phosphazene was mixed to a concentration of 0.1 μM and fed at 10 μl / min. All data obtained using the acetic acid adduct ion of reserpine (m / z 680.0355) and the acetic acid dimer isotope (m / z 120.0384) were auto-calibrated.

(3) Suppression experiment Renal failure model mice (mice were 129 SvJJmsSlc males 8 to 10 weeks old and weighed 20 to 25 g) were prepared and loaded with saline in the same manner as (1). The renal failure salt load model is hypertension. Degradation wayway was verified by stepping down the pressure from Eplerenone (pfizer) for 2 weeks from Day 14 to Day 28. Hydralazine (Sigma-Aldrich) was added when the hyporelone alone was insufficient in hypotension. Eplerenone was mixed in the feed (1.67 g / kg of chow) to prepare a drug-added feed. Hydrazine was 0.2 mg / ml (drinking water).

(4) Measurements in patients with peritoneal dialysis The complement substances identified from metabolomic analysis were measured with the serum of patients undergoing peritoneal dialysis at our hospital (n = 53). In cardiac ultrasonography, left ventricular posterior wall thickness (LVPWTd), left ventricular end-diastolic diameter (LVDd), left ventricular ejection fraction (LVEF), left ventricular weight (LVmass), left ventricular weight coefficient (left ventricular weight / body The surface area, LV mass index), and diastolic dysfunction index (E / E ′) were confirmed, and their correlation was examined.

2 to 6 show the evaluation results of serum creatine (Cr), hydroxyproline, blood pressure, heart weight, myocardial fibrosis staining, and hydroxyproline for each group of mice. Hydroxyproline was measured by CE-TOFMS.

As shown in FIG. 2, serum Cr levels increased in the renal failure mouse group (without / with saline load) compared to the healthy mouse group (without / with salt load). However, even in the group of renal failure mice, the Cr value was comparable between the cases without / with salt load.

As shown in FIG. 3, blood pressure was higher in the renal failure mouse group (without / with salt load) than in the healthy mouse group, and was similar to that with / without salt load.

As shown in FIG. 4, the heart weight increased with time due to salt loading, and also increased over time in the renal failure model. As shown in FIG. 5A and FIG. 5B, myocardial fibrosis significantly increased over time in the renal failure mouse group (with salt load).

On the other hand, as shown in FIGS. 6A and 6B, the cardiac hydroxyproline (but free hydroxyproline) increased until 4 weeks, but then decreased. Furthermore, serum hydroxyproline also increased until 4 weeks but then decreased.

From the above, it was found that the hydroxyproline concentration in serum showed an increasing tendency from 0 weeks to 2 weeks or 4 weeks prior to fibrosis (8 weeks) in the heart. This increasing tendency was also observed in the hydroxyproline concentration in the heart. Furthermore, it was found that the concentration of hydroxyproline in the serum and in the heart decreased during the myocardial fibrosis stage (8 weeks).

Based on the above, it is possible to detect cardiac lesions that will develop in the future (predictive signs) even in the absence of cardiac lesions such as myocardial fibrosis due to the increasing tendency of hydroxyproline in serum. I understood.

In this example, in the same manner as in Example 1, for the heart and serum of renal failure mice (with salt load), as in hydroxyproline, ornithine, proline, 4-oxoproline, which are precursors in the hydroxyproline synthesis system, are used. And cis-4-hydroxy-D-proline, 1-pyrroline-4-hydroxy-D-carboxylate, pyrrole-2-carboxylate, gamma-hydroxyglutamate gamma semialdehyde, which is a hydroproline degradation product of the decomposition system, Gamma-hydroxyglutamic acid was measured using CE-TOFMS. As a result, the main synthetic systems ornithine, proline and hydroxyproline were detected, but the degradation system hydroxyproline degradation products were not detected in the heart or serum.

That is, even when the hydroxyproline concentration was high and showed an increasing tendency (4 weeks), and even when the hydroxyproline concentration showed a decreasing tendency (8 weeks), no degradation product was detected even though the precursor was observed. From the above, it was found that the hydroxyproline concentration in serum is a useful marker of the speed of fibrosis or that fibrosis is in an advanced stage (promoted / enhanced state). At the same time, it was found that the concentration of hydroxyproline in serum is not a marker indicating the degree of fibrosis of the already constructed myocardium (for example, the fibrosis rate).

When the amount of serum hydroxyproline was measured for peritoneal dialysis patients, a tendency was observed that the concentration of serum hydroxyproline increased with increasing heart weight. Moreover, the tendency for the serum hydroxyproline concentration to increase with the increase of the diastolic dysfunction index was observed. From these results, it was found that when the serum hydroxyproline concentration is high, it can also correspond to the progress of the cardiac lesion process.

In this example, anti-hypertensive mice were treated with antihypertensive drugs from 2 to 4 weeks and killed at 4 weeks. At 4 weeks, heart weight, blood pressure, serum creatinine, and serum hydroxyproline were measured in the same manner as in the above examples. The results are shown in FIG.

As shown in FIG. 7, in the antihypertensive agent administration group, an improvement or decrease tendency was observed in each of blood pressure, heart weight and serum hydroxyproline as compared to the non-administration group. Serum creatinine was similar. The decreasing tendency of serum hydroxyproline corresponded well with the decrease in heart weight. From the above, it was found that the serum hydroxyproline concentration can reflect the tendency of regression (improvement) in the progression of the cardiac lesion process.

According to the above examples, the serum hydroxyproline concentration is determined by the presence / absence of increased / promoted progression of the cardiac lesion process rather than the presence / absence of a constructed cardiac lesion state such as myocardial fibrosis It was found that it can be a marker of the degree and degree of the regression and improvement of the cardiac lesion process. It was also found that the serum hydroxyproline concentration can be a marker for the onset and progression of cardiac lesion onset (predictor of cardiac lesion) prior to the onset of cardiac lesions such as myocardial fibrosis.

In this example, the amount of hydroxyproline in the heart tissue of 8-week-old renal failure mice was measured. These processes are shown in FIGS. Process C described below corresponds to a sample pretreatment method for measuring hydroxyproline by a colorimetric method, which is used as a conventional fibrosis index of cardiac lesions.
(1) Process A
(Sample pretreatment)
Zirconia beads, methionine sulfone MES as an internal standard, and CSA (D-Camphol-10-sulfonic acid) were prepared in a tube containing about 30-60 mg of heart tissue collected from a renal failure mouse so that each became 20 μM. 500 μl of a methanol solution was added, treated with Shake Master NEO at 1500 rpm for 5 minutes, disrupted, 500 μl of chloroform and 200 μl of water were added, stirred, and centrifuged at 4600 rpm for 15 minutes at 4 ° C. 300 μl of the water-methanol phase (upper phase) after centrifugation was placed on an ultrafiltration filter (fractional molecular weight: 5000 Da) and centrifuged at 9100 g at 4 ° C. for 6.5 hours. The obtained filtrate was centrifuged at 40 ° C. for 3 hours and concentrated to obtain a measurement sample.

(Measurement)
To this measurement sample, 50 μl of each 200 μM aqueous solution of 3-aminopyrrolidine and trimesate for correcting the migration time was added and dissolved, and the CE-TOFMS described above was performed to measure hydroxyproline. In addition, various amino acids were also measured.

(2) Processes B to D
(Sample pretreatment)
After adding zirconia beads and 400 μl of methanol to a tube containing about 30-60 mg of heart tissue collected from a renal failure mouse, treating it with Shake Master NEO at 1500 rpm for 5 minutes, and then distributing this disrupted solution into two tubes Then, it was concentrated by centrifugation at 40 ° C. for 3 hours. One of the concentrates was dissolved by adding 500 μl of 6N HCl and stirring, part of which was allowed to stand at 25 ° C. for 6 hours (Process B), and the other part was boiled at 110 ° C. for 6 hours ( Process C). Then, it concentrated by centrifuging at 45 degreeC for 7 hours.

The other concentrate was boiled at 110 ° C. for 6 hours after adding 500 μl of pure water and stirring (process D). Then, it concentrated by centrifuging at 45 degreeC for 7 hours.

Add 500 μl of methanol solution and 200 μl of pure water so that MES and CSA as internal standards are each 20 μM to each concentrate in Process B to D, and use Shake Master NEO at 1500 rpm. Stir for 5 minutes. Further, 500 μl of chloroform was added, stirred, and centrifuged at 4600 rpm for 15 minutes at 4 ° C. The water-methanol phase (upper phase) after centrifugation was placed on an ultrafiltration filter (fractionated molecular weight 5000 Da) and centrifuged at 9100 g at 4 ° C. for 20 hours. The obtained filtrate was centrifuged at 40 ° C. for 2 hours and concentrated to obtain a measurement sample for each process.

(Measurement)
To each of these measurement samples, 20 μl of each 200 μM aqueous solution of 3-aminopyrrolidine and trimesate for movement time correction was added and dissolved, and the CE-TOFMS described above was performed to measure hydroxyproline. In addition, various amino acids were also measured.

For stable measurement, the process B sample was diluted 2 times with pure water, the process C sample was similarly diluted 20 times, and the process D sample was similarly diluted 20 times.

Fig. 12 shows the measurement results of hydroxyproline by Processes A to D (number of moles per gram of heart (nmol / g)).

As shown in FIG. 12, the amount of hydroxyproline by Process A, B and D all showed an average of around 100 nmol, whereas the amount of hydroxyproline by Process C markedly exceeded 1000 nmol. High concentration was shown.

From these results, in the sample preparation by the colorimetric method of the conventional method of Process C, 6N hydrochloric acid treatment and boiling treatment decompose and extract hydroxyproline which is incorporated and stabilized in tissue collagen and measured. In contrast, Processes A, B and D clearly measure free hydroxyproline or hydroxyproline not immobilized on tissue. In Process A, B and D, it was considered that stabilized hydroxyproline fixed to the tissue was excluded by ultrafiltration. And according to the present inventors, it was found that the hydroxyproline measurement results by processes A, B and D correlate well with the hydroxyproline measurement results in serum. Moreover, the same tendency was confirmed also about each amino acid.

From the above, as already explained, by measuring hydroxyproline in blood-related fluids such as serum, it is possible to effectively monitor the degree of progression of cardiac lesions, etc. It has been found that fibrosis can be monitored by measuring free hydroxyproline in heart tissue such as a model animal or free hydroxyproline in tissue or hydroxyproline not constituting collagen or a part thereof.

Claims

A method for monitoring cardiac lesions,
Measuring hydroxyproline in the blood-related fluid of the test individual;
A method comprising:
The method according to claim 1, wherein the cardiac lesion is a cardiac lesion associated with chronic kidney disease.
The method according to claim 1 or 2, wherein the cardiac lesion includes a myocardial lesion.
The method according to any one of claims 1 to 3, wherein the myocardial lesion includes myocardial fibrosis and diastole.
The method according to any one of claims 1 to 4, wherein the measuring step is a step of measuring free hydroxyproline or collagen or hydroxyproline which does not constitute a part thereof.
The method according to any one of claims 1 to 5, wherein the measuring step includes performing a step of measuring the amount of hydroxyproline in the blood-related fluid for each subject at two or more times.
The method according to claim 6, wherein when the tendency of increasing the amount of hydroxyproline over time is affirmed, it is determined that the cardiac lesion process is in an advanced stage.
The method according to claim 6, wherein when the tendency to decrease the amount of hydroxyproline over time is affirmed, it is determined that the cardiac lesion process is in a regression stage.
The method according to claim 6, wherein when the tendency to decrease the amount of hydroxyproline over time is affirmed, it is determined that the cardiac lesion process is in a completion stage (final stage).
The method according to claim 6, wherein when the tendency to maintain the amount of hydroxyproline over time is affirmed, it is determined that the cardiac lesion process or the state before the cardiac lesion process is maintained.
A method for monitoring the effects of drugs and / or treatments on cardiac lesions, comprising:
Measuring hydroxyproline in the blood-related fluid of the test individual;
A method comprising:
∙ Blood-related fluid marker for cardiac lesions, including hydroxyproline.
The kit for performing the monitoring method according to any one of claims 1 to 11, comprising a reagent necessary for measuring hydroxyproline in a blood-related fluid.
Administering one or more test compounds to a cardiac lesion model animal;
Measuring hydroxyproline in the blood-related fluid of the model animal;
Evaluating the action of the one or more test compounds on a cardiac lesion based on the amount of hydroxyproline or a change thereof;
A screening method for a drug effective for the prevention, treatment or improvement of cardiac lesions.
The method according to claim 14, further comprising subjecting the model animal to a salt load.
The method according to claim 14 or 15, further comprising a step of measuring the amount of free hydroxyproline or collagen in the tissue of the model animal or hydroxyproline which does not constitute a part thereof.
Administering one or more test compounds to a cardiac lesion model animal;
A step of measuring free hydroxyproline or hydroxyproline which does not constitute collagen or a part thereof in the tissue of the model animal;
Evaluating the action of the one or more test compounds on a cardiac lesion based on the amount of hydroxyproline or a change thereof;
A screening method for a drug effective for the prevention, treatment or improvement of cardiac lesions.
A method of monitoring cardiac lesions in a cardiac lesion model animal,
A step of measuring free hydroxyproline or hydroxyproline which does not constitute collagen or a part thereof in the tissue of the model animal,
A method comprising:
A method of monitoring cardiac lesions in a cardiac lesion model animal,
Measuring hydroxyproline in blood-related fluid in the tissue of the model animal,
A method comprising: