Biomarkers for determining the application of reperfusion therapy

Abstract

To provide biomarkers for determining applicability of reperfusion therapy.SOLUTION: Biomarkers for determining applicability of reperfusion therapy to a subject is provided, the biomarkers being composed of an adrenomedullin (ADM) precursor protein or a degradation product thereof.SELECTED DRAWING: None

Description

Technical Field

[0005] ,

[0001] The present invention relates to a biomarker for determining the application of reperfusion therapy.

Background Art

[0002] Ischemic stroke is one of the main causes of death and dysfunction, and the direct cost exceeds 1 trillion yen. Among them, cases of occlusion of the main cerebral artery are severe types that account for many deaths and severe dysfunctions. However, due to the recent emergence of thrombolytic therapy and endovascular treatment, the prognosis has been improved in cases with early onset. Furthermore, even in cases where time has passed since the onset, if there is a sufficient salvageable ischemic area, that is, a penumbra, the prognosis can be dramatically improved by the same treatment. In this specification, in the brain after the onset of ischemic stroke, the irreversible ischemic area is the ischemic core, the area where the blood volume is decreased and the cells are spared from cell death, and the area that can be recovered by the reopening of cerebral blood flow may be referred to as the penumbra.

[0003] The evaluation of the penumbra in ischemic stroke is performed by automatically analyzing perfusion images obtained by head MRI or CT using an intravascular tracer with dedicated software (Non-Patent Document 1). However, since this method uses a contrast agent, its application is limited in cases with renal dysfunction, the introduction and maintenance costs of the software are high, and specialized knowledge is required for the interpretation of the analysis results, so it can only be used in some medical institutions.

[0004] In rodent models, adrenomedullin (ADM) is cited as a gene whose expression is induced sensitively from the early stage of cerebral ischemia (Non-Patent Document 2). ADM is an endogenous peptide with a neuroprotective effect due to its vasodilatory and anti-inflammatory effects, and it is known to increase in correlation with the severity in acute ischemic stroke. Therefore, it has been expected as a biomarker for ischemic stroke, but it has not been put into practical use.

Prior Art Documents

Non-Patent Documents

[0005] [Non-Patent Document 1] Fisher M et al., Identifying and utilizing the ischemic penumbra. Neurology. 2012;79:S79-85 [Non-Patent Document 2] Reimer MM et al., Rapid disruption of axon-glial integrity in response to mild cerebral hypoperfusion. J Neurosci. 2011;31:18185-18194 [Overview of the project] [Problems that the invention aims to solve]

[0006] The object of this invention is to provide a biomarker for determining the application of reperfusion therapy.

[0007] Furthermore, the present invention aims to provide a biomarker for evaluating the penumbra. [Means for solving the problem]

[0008] The present inventors conducted diligent studies to solve the above problems and found that degradation products of ADM precursors, particularly Mid-regional pro-Adrenomedullin (MR-proADM), are useful as blood biomarkers that reflect the penumbra in acute ischemic stroke. The present invention was completed by further studies based on these findings and includes the following embodiments.

[0009] Section 1. A biomarker consisting of adrenomedullin (ADM) precursor protein or its degradation products, used to determine the applicability of reperfusion therapy in a subject.

[0010] Section 2. A biomarker for penumbra evaluation, comprising ADM precursor protein or its degradation products.

[0011] Section 3-1. The biomarker according to claim 1 or 2, wherein the degradation product is at least one degradation product of an ADM precursor protein selected from the group consisting of Mid-regional Pro-Adrenomedullin (MR-proADM), Proroadrenomedullin N-terminal 20 Peptide (PAMP), Adrenotensin, and Adrenomedullin (ADM).

[0012] Section 3-2. The biomarker according to claim 1 or 2, wherein the degradation product is MR-proADM.

[0013] Section 3-3. The biomarker according to item 1 or 2, wherein the degradation product is PAMP.

[0014] Section 3-4. The biomarker according to item 1 or 2, wherein the degradation product is adrenotensin.

[0015] Section 3-5. The biomarker according to item 1 or 2, wherein the degradation product is ADM.

[0016] Section 4. (A) A step of measuring the concentration of ADM precursor protein or its degradation product in a biological sample obtained from the subject. A method for determining the applicability of reperfusion therapy in a subject, including the application of reperfusion therapy in a subject.

[0017] Section 5. (B) A step of comparing the concentration measured in step (A) with a reference value, and deciding whether to apply reperfusion therapy to the subject if the concentration is higher than the reference value, or whether to not apply reperfusion therapy to the subject if the concentration is lower than the reference value. The method described in item 4, further including the method described in item 4.

[0018] Item 6-1. The method according to item 4 or 5, wherein the degradation product is at least one degradation product of an ADM precursor protein selected from the group consisting of Mid-regional Pro-Adrenomedullin (MR-proADM), Proadrenomedullin N-terminal 20 Peptide (PAMP), Adrenotensin, and Adrenomedullin (ADM).

[0019] Item 6-2. The method according to item 4 or 5, wherein the degradation product is MR-proADM.

[0020] Item 6-3. The method according to item 4 or 5, wherein the degradation product is PAMP.

[0021] Item 6-4. The method according to item 4 or 5, wherein the degradation product is Adrenotensin.

[0022] Item 6-5. The method according to item 4 or 5, wherein the degradation product is ADM.

[0023] Item 7. <� A kit used to determine the application of reperfusion therapy in a subject, comprising an antibody capable of binding to an ADM precursor protein or its degradation product.

[0024] Item 8. A kit used to evaluate penumbra in a subject, comprising an antibody capable of binding to an ADM precursor protein or its degradation product.

[0025] Item 9-1. The kit according to claim 7 or 8, wherein the degradation product is at least one degradation product of an ADM precursor protein selected from the group consisting of Mid-regional Pro-Adrenomedullin (MR-proADM), Proroadrenomedullin N-terminal 20 Peptide (PAMP), Adrenotensin, and Adrenomedullin (ADM).

[0026] Section 9-2. The kit according to item 7 or 8, wherein the degradation product is MR-proADM.

[0027] Section 9-3. The kit according to item 7 or 8, wherein the degradation product is PAMP.

[0028] Section 9-4. The kit according to item 7 or 8, wherein the degradation product is adrenotensin.

[0029] Section 9-5. The kit according to item 7 or 8, wherein the degradation product is ADM. [Effects of the Invention]

[0030] According to the present invention, it is possible to provide blood biomarkers for determining the application of reperfusion therapy. Furthermore, it is possible to provide biomarkers for evaluating the penumbra. [Brief explanation of the drawing]

[0031] [Figure 1] This is a schematic diagram showing the structure of the ADM precursor protein, a precursor of human ADM. This diagram is quoted and partially modified from [Schonauer R et al., Adrenomedullin - new perspectives of a potent peptide hormone. J Pept Sci. 2017 Jul;23(7-8):472-485.]. [Figure 2] This is a schematic diagram of an embodiment of the present invention. [Figure 3] This figure compares plasma MR-proADM concentrations in HAIS patients and healthy individuals. Plasma MR-proADM concentrations were elevated in HAIS patients compared to healthy individuals (A). The AUC (Area Under the Curve) value of the ROC curve (Receiver Operating Characteristic curve) for predicting HAIS using plasma MR-proADM concentration was 0.89 (sensitivity: 78.4%, specificity: 87.3%) (B). Plasma MR-proADM concentrations were higher in HAIS within 1 hour of symptom onset (early HAIS) (C) and in ischemic core (D) compared to healthy individuals. The box plot shows the interquartile range with the median as the parallel line. The upper end of the whiskers indicates the maximum value, and the lower end indicates the minimum value. [Figure 4] This figure shows the association between plasma MR-proADM concentration and broad penumbra in HAIS patients. Plasma MR-proADM concentration at admission was higher in patients with broad penumbra compared to patients without broad penumbra. The box plot shows the interquartile range with the median as the parallel line. The upper end of the whiskers indicates the maximum value, and the lower end indicates the minimum value (A). Forest plot based on the results of multivariate analysis. It shows factors associated with the presence of broad penumbra (B). *** indicates a P value less than 0.001. CI represents the confidence interval. [Figure 5] This figure shows the association between plasma MR-proADM concentration and broad penumbra in patients with internal carotid artery / middle cerebral artery horizontal segment occlusion. Among patients with internal carotid artery / middle cerebral artery horizontal segment occlusion, plasma MR-proADM concentration was significantly higher in patients with broad penumbra compared to patients without broad penumbra. The box plot shows the interquartile range with the median as the parallel line. The upper end of the whiskers indicates the maximum value, and the lower end indicates the minimum value. ** indicates a P value less than 0.01 (A). The forest plot based on the results of multivariate analysis shows the factors associated with the presence of broad penumbra in patients with internal carotid artery / middle cerebral artery horizontal segment occlusion (B). [Figure 6]This figure shows the correlation between plasma MR-proADM concentration and penumbra volume. Plasma MR-proADM concentration showed a positive correlation with penumbra volume, which is calculated by subtracting the ischemic core volume from the volume of the region where the Tmax exceeds 6 seconds on MR or CT perfusion images. The numbers in the circles represent the ischemic core volume (mL). [Modes for carrying out the invention]

[0032] The embodiments included in this disclosure are described in further detail below.

[0033] 1. The biomarker of the present invention The biomarker of the present invention consists of Adrenomedullin (ADM) precursor protein or its degradation product.

[0034] In the present invention, the degradation products of ADM precursor protein include metabolites of the degradation products of ADM precursor protein. Examples of metabolites of ADM precursor protein include blood metabolites of the degradation products of ADM precursor protein.

[0035] In this specification, ADM precursor proteins or their degradation products may be referred to as "marker polypeptides."

[0036] The ADM precursor protein, a precursor of human ADM, consists of four components: a signal peptide, mid-regional pro-adrenomedullin (MR-proADM), pro-adrenomedullin N-terminal 20 peptide (PAMP), adrenotensin (pro-adrenomedullin (153-185)), and ADM (Figure 1). Therefore, the marker polypeptide of the present invention may be a degradation product of at least one ADM precursor protein selected from the group consisting of MR-proADM, PAMP, adrenotensin, or ADM. In the present invention, MR-proADM is more preferred as the marker polypeptide. MR-proADM is stable in the sample, and by using MR-proADM as a marker polypeptide, it can be suitably used for determining the application of reperfusion therapy and evaluating the penumbra, as described below.

[0037] The biomarker of the present invention is used to determine the application of reperfusion therapy to a subject. In the present invention, "determining the application of reperfusion therapy" means measuring the concentration of the marker polypeptide of the present invention in the subject, comparing the value with a reference value, and deciding to apply reperfusion therapy if the value is higher than the reference value, and not applying reperfusion therapy if the value is lower than the reference value.

[0038] In this invention, "subject" refers to a subject who has developed or is likely to develop ischemic stroke, particularly acute ischemic stroke.

[0039] In the present invention, the "reference value" can be set by preparing sufficient amounts of samples from human individuals who have experienced ischemic stroke and human individuals who have not experienced ischemic stroke, and detecting the amount of the marker polypeptide of the present invention contained in each of these samples.

[0040] In the present invention, the concentration of the marker polypeptide of the present invention in a sample derived from a human individual that has developed ischemic stroke is 1.5 times or more, preferably 1.6 times or more, more preferably 1.6 to 2.0 times, and even more preferably 1.67 times, compared to the concentration of the marker polypeptide of the present invention in a sample derived from a human individual that has not developed acute ischemic stroke.

[0041] In the present invention, the term "sample" is not particularly limited, but is preferably blood, and more preferably plasma.

[0042] In the present invention, "reperfusion therapy" is not particularly limited as long as it is a treatment method that reopens an occluded blood vessel. Specific examples of reperfusion therapy include, for example, thrombolytic therapy and endovascular neurotherapy.

[0043] In the brain after an ischemic stroke, the irreversibly ischemic area is called the ischemic core, and the area where blood volume is reduced but cell death has been avoided, and which can be restored by resuming cerebral blood flow, is called the penumbra. In this invention, the purpose of reperfusion therapy is to save the penumbra.

[0044] The volume of the penumbra can be measured using image analysis software called the RAPID system (iSchemaView). More specifically, MRI and CT images are analyzed using RAPID to calculate the ischemic core and the hypoperfusion area where cerebral blood flow is reduced. The area obtained by subtracting the ischemic core from the hypoperfusion area is considered the penumbra region, and its volume is measured. However, because the RAPID system is expensive, only a few facilities have adopted it.

[0045] The biomarkers of the present invention can be used to evaluate penumbras in a subject. In the present invention, "evaluating penumbras" means, for example, determining the presence or absence of penumbras or predicting their volume.

[0046] In the present invention, the evaluation of the penumbra can be performed based on the amount of the marker polypeptide of the present invention in a sample derived from the target.

[0047] The biomarkers of the present invention enable rapid and convenient selection of tissue-based treatments.

[0048] 2. Method of the present invention The present invention provides a method for determining the applicability of reperfusion therapy to a subject, comprising the step of (A) measuring the concentration of ADM precursor protein or its degradation product in a biological sample obtained from the subject.

[0049] The method of the present invention may further include, after step (A), a step of (B) comparing the concentration measured in step (A) with a reference value, and deciding whether to apply reperfusion therapy to the subject if the concentration is higher than the reference value, or whether to not apply reperfusion therapy to the subject if the concentration is lower than the reference value.

[0050] The measurement of the marker polypeptide of the present invention is not particularly limited, but examples include detection by immunological methods using antibodies capable of binding to the biomarker of the present invention. Specific examples of immunological methods include homogeneous time-resolved fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). An example of a homogeneous time-resolved fluorescence immunoassay is time-resolved amplified fluorescence emission (TRACE).

[0051] The method of the present invention can be performed either in vitro or in vivo, but it is preferable to perform it in vitro because it allows for simpler testing.

[0052] 3. The present invention kit The kit of the present invention is a kit comprising an antibody capable of binding to the marker polypeptide of the present invention, which is used for the test method of the present invention.

[0053] The kit of the present invention includes the biomarker of the present invention, a procedure manual for the method of the present invention, and may further include reagents, equipment, etc., necessary for the method of the present invention.

[0054] The above procedure manual may include a description of the "reference values" necessary in the method of the present invention. Such a description may be a direct statement of the "reference values" or an explanation of how to set the "reference values".

[0055] In addition to the above, the kit of the present invention may optionally further contain reagents necessary for the method of the present invention. Examples of such reagents include sterile water, physiological saline, vegetable oil, surfactants, lipids, solubilizers, buffers, protein stabilizers, preservatives, blocking solutions, reaction solutions, and reaction stop agents. The kit of the present invention may contain these reagents individually or in combination. [Examples]

[0056] The embodiments of this disclosure will be described in more detail below with examples, but the embodiments of this disclosure are not limited to the examples below. Unless otherwise specified below, % refers to mass %.

[0057] <Example 1> Measurement of MR-proADM concentration (1) Collection of blood sample Of 866 patients with acute ischemic stroke, informed consent was obtained from 145 patients who were transported to the hospital within 4.5 hours of symptom onset. From the blood samples collected from these patients, MR-proADM concentrations were measured in 128 samples, excluding 17 samples with insufficient volume or poor quality. None of these patients had acute infections or inflammatory diseases; however, three patients were excluded due to end-stage renal disease. Therefore, the following experiment was conducted using blood samples obtained from 125 patients with hyperacute ischemic stroke (HAIS) (mean age: 77 years, IQR: 70-85 years, male: 59.2%) and 1298 healthy individuals (mean age: 58 years, IQR: 49-67 years, male: 33.2%) (Figure 2).

[0058] (2) Measurement of plasma MR-proADM concentration and regular blood tests Peripheral blood samples were collected from all HAIS patients immediately after admission. These blood samples were also used for routine blood tests, including blood biochemistry and glucose metabolism tests. For MR-proADM measurement, the collected blood samples were dispensed into standard ethylenediaminetetraacetic acid tubes. Each sample was immediately agitated after collection and centrifuged at 3400xg, 25°C for 6 minutes. Plasma samples were collected and stored at -80°C until MR-proADM measurement. Plasma MR-proADM concentration (nmol / L) was measured using a time-resolved amplified cryptographic emission technology assay (TRACE) with an automated KRYPTOR® analyzer (Thermo Fisher Diagnostics KK, Japan).

[0059] (3) Comparison of plasma MR-proADM concentrations between HAIS patients and healthy individuals Patients with HAIS are characterized by a higher BMI, lower estimated glomerular filtration rate, higher prevalence of vascular risk factors (hypertension, diabetes, dyslipidemia), and higher prevalence of ischemic heart disease, including past strokes and past myocardial infarctions or angina pectoris, compared to healthy individuals (Table 1).

[0060] [Table 1]

[0061] Univariate analysis revealed that MR-proADM concentrations in HAIS patients were significantly higher than those in healthy individuals (Figure 3A). The mean MR-proADM concentration in HAIS patients was 0.68 nmol / L (IQR: 0.53-0.84 nmol / L), compared to 0.42 nmol / L (IQR: 0.37-0.48 nmol / L) in healthy individuals (P<0.01).

[0062] This difference was confirmed even after propensity score matching for age, sex, BMI, estimated glomerular filtration rate, vascular risk factors, and medical history (Table 2). In both groups, n=66, the mean MR-proADM concentration in HAIS patients was 0.57 nmol / L (IQR: 0.49–0.75 nmol / L), compared to 0.51 nmol / L (IQR: 0.46–0.59 nmol / L) in healthy controls (P<0.01).

[0063] [Table 2]

[0064] ROC analysis of MR-proADM for predicting HAIS showed a sensitivity of 78.4% and a specificity of 87.3%. The reference value for diagnosing HAIS was 0.52 nmol / L, and the AUC (area under the curve) value was 0.89 (P<0.01) (Figure 3B). MR-proADM concentrations in time-based HAIS patients (n=51) were higher than those in healthy individuals (Figure 3C). The mean MR-proADM concentration in time-based HAIS patients was 0.72 nmol / L (IQR: 0.54-0.85 nmol / L), compared to 0.42 nmol / L (IQR: 0.37-0.48 nmol / L) in healthy individuals (P<0.01). Furthermore, MR-proADM concentrations were higher in tissue-based HAIS patients (n=80) compared to healthy individuals (Figure 3D). The mean MR-proADM concentration in tissue-based HAIS patients was 0.66 nmol / L (IQR: 0.54–0.84 nmol / L), while the mean MR-proADM concentration in healthy individuals was 0.42 nmol / L (IQR: 0.37–0.48 nmol / L) (P<0.01).

[0065] These differences were also observed after baseline matching. The mean MR-proADM concentration in time-based HAIS patients was 0.61 nmol / L (IQR: 0.49–0.82 nmol / L), compared to 0.48 nmol / L (IQR: 0.40–0.59 nmol / L) in healthy controls (n=34, P<0.01 for both). Similarly, the mean MR-proADM concentration in tissue-based HAIS patients was 0.57 nmol / L (IQR: 0.49–0.74 nmol / L), compared to 0.51 nmol / L (IQR: 0.43–0.60 nmol / L) in healthy controls (n=45, P<0.01 for both).

[0066] <Example 2> Evaluation of the relationship between MR-proADM concentration and HAIS. Table 3 shows the correlation between MR-proADM concentration and various variables in HAIS patients. MR-proADM concentration showed a positive correlation with age (r=0.42, p<0.01) and NIHSS (National Institutes of Health Stroke Scale) (r=0.38, p<0.01), and a negative correlation with (r=-0.64, P<0.01). MR-proADM concentration was elevated in patients with hypertension, cardiogenic cerebral embolism, and internal carotid artery / middle cerebral artery horizontal segment occlusion. Specifically, the mean MR-proADM concentration in hypertensive patients was 0.74 nmol / L (IQR: 0.54-0.86 nmol / L), compared to a mean MR-proADM concentration of 0.59 nmol / L (IQR: 0.48-0.71 nmol / L) in healthy individuals (P=0.01). The mean MR-proADM concentration in patients with cardiogenic cerebral embolism was 0.80 nmol / L (IQR: 0.62-0.96 nmol / L), compared to 0.58 nmol / L (IQR: 0.50-0.74 nmol / L) in healthy individuals (P<0.01). Furthermore, the mean MR-proADM concentration in patients with internal carotid artery / middle cerebral artery horizontal segment occlusion was 0.79 nmol / L (IQR: 0.55-0.96 nmol / L), compared to 0.62 nmol / L (IQR: 0.53-0.80 nmol / L) in healthy individuals (P=0.03). MR-proADM concentration did not correlate with the time from last health check / onset to hospital visit or with ischemic core volume.

[0067] [Table 3]

[0068] <Example 3> Evaluation of the relationship between MR-proADM concentration and penumbra Patients with broad penumbra (internal carotid artery / middle cerebral artery horizontal segment occlusion with ischemic core volume of ≤50 ml, NIHSS ≥6) were predominantly female and had a higher prevalence of cardioembolic stroke compared to patients without broad penumbra. The prevalence of cardioembolic stroke in patients with broad penumbra was 92.3%, compared to 31.3% in patients without broad penumbra (P<0.01). MR-proADM concentrations were significantly higher in patients with broad penumbra compared to patients without broad penumbra (Figure 4A). In patients without broad penumbra, the MR-proADM concentration was 0.62 nmol / L (IQR: 0.52-0.79 nmol / L), while in patients with broad penumbra, the MR-proADM concentration was 0.89 nmol / L (IQR: 0.63-1.07 nmol / L) (P<0.01). Furthermore, ROC analysis of plasma MR-proADM for predicting broad penumbra showed an AUC of 0.74 and a reference value of 0.82 nmol / L (sensitivity 61.5%, specificity 79.8%). In addition, multivariate analysis showed that a plasma MR-proADM concentration higher than 0.82 nmol / L could independently predict the presence of broad penumbra (odds ratio: 7.09, 95% confidence interval (CI): 1.77-35.45, P<0.01) (Figure 4B).

[0069] In patients with internal carotid artery / middle cerebral artery horizontal segment occlusion (Table 4), MR-proADM concentrations were higher in patients with a broad penumbra compared to patients without a broad penumbra (Figure 5A). The mean MR-proADM concentration in patients without a broad penumbra was 0.55 nmol / L (IQR: 0.49-0.79 nmol / L), while the mean MR-proADM concentration in patients with a broad penumbra was 0.86 nmol / L (IQR: 0.63-1.07 nmol / L) (P<0.01). ROC analysis predicting broad penumbra based on MR-proADM concentration showed an AUC of 0.76. Furthermore, it was shown that a plasma MR-proADM concentration higher than 0.81 nmol / L significantly predicted the presence of a broad penumbra in patients with internal carotid artery / middle cerebral artery horizontal segment occlusion (Figure 5B).

[0070] [Table 4]

[0071] MR or CT perfusion images were obtained from seven HAIS patients. Plasma MR-proADM concentration showed a significant positive correlation with penumbra volume (r=0.79, P=0.04) (Figure 6).

Claims

1. A biomarker consisting of adrenomedullin (ADM) precursor protein or its degradation products, used to determine the applicability of reperfusion therapy in a subject.

2. A biomarker for penumbra evaluation, comprising ADM precursor protein or its degradation products.

3. The biomarker according to claim 1 or 2, wherein the degradation product is at least one degradation product of an ADM precursor protein selected from the group consisting of Mid-regional Pro-Adrenomedullin (MR-proADM), Proroadrenomedullin N-terminal 20 Peptide (PAMP), Adrenotensin, and Adrenomedullin (ADM).

4. (A) A step of measuring the concentration of ADM precursor protein or its degradation product in a biological sample obtained from a subject, and (B) A step of comparing the concentration measured in step (A) with a reference value and deciding whether to apply reperfusion therapy to the subject if the concentration is higher than the reference value, or whether to not apply reperfusion therapy to the subject if the concentration is lower than the reference value. A method for determining the applicability of reperfusion therapy in a subject, including the application of reperfusion therapy in a subject.

5. A kit containing an antibody capable of binding to ADM precursor protein or its degradation products, used to determine the applicability of reperfusion therapy to a subject.

6. A kit used to evaluate penumbra in a subject, containing an antibody capable of binding to ADM precursor protein or its degradation products.

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