JP7861981B2 - Biomarkers for determining the application of reperfusion therapy - Google Patents

Description

This invention relates to a biomarker for determining the application of reperfusion therapy.

Ischemic stroke is one of the leading causes of death and disability, with direct costs exceeding 1 trillion yen. Among these, cases involving occlusion of the major cerebral arteries are severe, accounting for a large proportion of deaths and severe disability. However, recent advances in thrombolytic therapy and endovascular treatment have improved prognosis in cases treated early. Furthermore, even in cases where time has passed since onset, if a sufficient salvageable ischemic area, i.e., the penumbra, exists, this treatment can dramatically improve the prognosis. In this specification, in the brain after ischemic stroke, the irreversible ischemic area is sometimes referred to as the ischemic center, and the area with reduced blood flow that has escaped cell death and is recoverable through the resumption of cerebral blood flow is sometimes referred to as the penumbra.

The evaluation of the penumbra in ischemic stroke is performed by automatically analyzing perfusion images from head MRI and CT using an intravascular tracer with specialized software (Non-Patent Literature 1). However, this method is only available at a limited number of medical institutions due to its limitations in use in patients with renal impairment (due to the use of contrast agents), high software installation and maintenance costs, and the need for specialized knowledge to interpret the analysis results.

In rodent models, adrenomedullin (ADM) is a gene whose expression is sensitively induced from the early stages of cerebral ischemia (Non-Patent Literature 2). ADM is an endogenous peptide with neuroprotective effects due to its vasodilatory and anti-inflammatory properties, and it is known to increase in correlation with the severity of acute ischemic stroke. Therefore, it was expected to be a promising biomarker for ischemic stroke, but its practical application has not yet been achieved.

Fisher M et al., Identifying and utilizing the ischemic penumbra. Neurology. 2012;79:S79-85 Reimer MM et al., Rapid disruption of axon-glial integrity in response to mild cerebral hypoperfusion. J Neurosci. 2011;31:18185-18194

The present invention aims to provide a biomarker for determining the application of reperfusion therapy.

Furthermore, the present invention aims to provide a biomarker for evaluating the penumbra.

The inventors of this invention conducted diligent research 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 reflecting the penumbra in acute ischemic stroke. This invention was completed by further investigation based on these findings and includes the following embodiments.

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

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

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).

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

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

Section 3-4.
The biomarker according to claim 1 or 2, wherein the degradation product is adrenotensin.

Section 3-5.
The biomarker according to claim 1 or 2, wherein the degradation product is ADM.

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

Section 5.
The method of claim 4, further comprising the step of (B) comparing the concentration measured in step (A) with a reference value, and determining 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.

Section 6-1.
The method according to claim 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), Proroadrenomedullin N-terminal 20 Peptide (PAMP), Adrenotensin, and Adrenomedullin (ADM).

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

Section 6-3.
The method according to item 4 or 5, wherein the degradation product is PAMP.

Section 6-4.
The method according to item 4 or 5, wherein the degradation product is adrenotensin.

Section 6-5.
The method according to item 4 or 5, wherein the degradation product is ADM.

Section 7.
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.

Section 8.
A kit used to evaluate penumbra in a subject, containing an antibody capable of binding to ADM precursor protein or its degradation products.

Section 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).

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

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

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

Section 9-5.
The kit according to item 7 or 8, wherein the degradation product is ADM.

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.

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.]. This is a schematic diagram of an embodiment of the present invention. 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. 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. 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). 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).

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

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

In this 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.

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

The ADM precursor protein, a precursor of human ADM, is composed 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 itself (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 its use as a marker polypeptide makes it suitable for determining the application of reperfusion therapy and evaluating the penumbra, as described below.

The biomarker of the present invention is used to determine the application of reperfusion therapy in a subject. In this 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 to 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.

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

In this invention, the "reference value" can be set by preparing sufficient amounts of samples from both 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 this invention contained in each of these samples.

In this invention, the concentration of the marker polypeptide of the present invention in a sample derived from a human individual that has experienced 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 experienced acute ischemic stroke.

In this invention, the term "sample" is not particularly limited, but is preferably blood, and more preferably plasma.

In this 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 neurosurgery.

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.

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 penumbra region is then calculated by subtracting the ischemic core from the hypoperfusion area, and its volume is measured. However, because the RAPID system is expensive, only a few facilities have adopted it.

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

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

The biomarkers of this invention enable rapid and convenient selection of tissue-based treatments.

2. The 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.

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 determining 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 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).

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.

3. The Kit of the Present Invention
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.

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.

The above procedure manual may include a description of the "reference values" necessary for the method of the present invention. Such a description may directly state the "reference values" or it may be an explanation of how to set the "reference values."

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.

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

<Example 1>
Measurement of MR-proADM concentration (1) Blood sample collection Informed consent was obtained from 145 patients out of 866 acute ischemic stroke patients who were transported to the hospital within 4.5 hours of symptom onset. Of the blood samples collected from these patients, MR-proADM concentration was measured in 128 blood samples, excluding 17 samples that were insufficient in quantity or of poor quality. None of these patients had acute infections or inflammatory diseases, however, 3 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).

(2) Measurement of plasma MR-proADM concentration and routine blood tests Peripheral blood was collected from all HAIS patients immediately after admission. Blood samples were also used for routine blood tests such as blood biochemistry and glucose metabolism. For the measurement of MR-proADM, the collected blood samples were dispensed into standard ethylenediaminetetraacetic acid tubes. Each sample was mixed immediately 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 an automated KRYPTOR® analyzer (Thermo Fisher Diagnostics KK, Japan) with time-resolved amplified fluorescence emission technology assay (TRACE).

(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).

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).

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). Both groups had n=66, and 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).

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).

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, respectively). Furthermore, 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, respectively).

<Example 2>
Evaluation of the association 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/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 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 cardioembolic stroke 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), while the mean MR-proADM concentration in healthy individuals was 0.62 nmol/L (IQR: 0.53-0.80 nmol/L) (P=0.03). MR-proADM concentration did not correlate with the time from the last health check/onset to hospital visit or with ischemic core volume.

<Example 3>
Evaluation of the association 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 concentration was 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).

In patients with internal carotid artery/middle cerebral artery horizontal segment occlusion (Table 4), MR-proADM concentrations were higher in patients with broad penumbra compared to those without broad penumbra (Figure 5A). The mean MR-proADM concentration in patients without broad penumbra was 0.55 nmol/L (IQR: 0.49–0.79 nmol/L), while the mean MR-proADM concentration in patients with 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 plasma MR-proADM concentrations higher than 0.81 nmol/L significantly predicted the presence of broad penumbra in patients with internal carotid artery/middle cerebral artery horizontal segment occlusion (Figure 5B).

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 (6)

A biomarker consisting of adrenomedullin (ADM) precursor protein or its degradation products, used to determine the applicability of reperfusion therapy in a subject. A biomarker for penumbra evaluation, consisting of ADM precursor protein or its degradation products. 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). (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. A kit containing antibodies capable of binding to ADM precursor protein or its degradation products, used to determine the applicability of reperfusion therapy in a subject. A kit used to evaluate penumbra in a target, containing an antibody capable of binding to ADM precursor protein or its degradation products.