ADM-gly as an early predictor of sepsis

Abstract

The present invention relates to the diagnosis of sepsis and / or septic shock and monitoring the extent thereof; the early assessment of the risk of developing sepsis and / or septic shock; the prediction or determination or monitoring of the need for a therapy or intervention in a subject. The present invention relates to a method which comprises determining the level of a biomarker selected from the group consisting of ADM-Gly, PAMP, MR-proADM and CT-proADM or a fragment of at least five amino acids thereof, or the total ADM, or the ratio of the level of said biomarker to the level of ADM-Nffz, in a sample of a subject, and wherein the level of said biomarker or said ratio is indicative of sepsis and / or septic shock or the extent of sepsis and / or septic shock, or is predictive of an increased risk of developing sepsis and / or septic shock, or is indicative of the need for a therapy or intervention in said subject for sepsis and / or septic shock.

Description

Technical Field

[0001] This invention relates to the diagnosis of sepsis and / or septic shock and the monitoring of its severity; early assessment of the risk of developing sepsis and / or septic shock; and prediction, identification, or monitoring of a subject's need for therapy or intervention. The invention relates to a method comprising determining the level of a biomarker selected from ADM-Gly, PAMP, MR-proADM, and CT-proADM, or fragments of at least five amino acids thereof, or total ADM, or the ratio of the biomarker level to the ADM-NH2 level, in a subject's sample, wherein the level of the biomarker or the ratio indicates sepsis and / or septic shock or the severity of sepsis and / or septic shock, or predicts an increased risk of developing sepsis and / or septic shock, or indicates the subject's need for therapy or intervention for sepsis and / or septic shock. Background Technology

[0002] The incidence of sepsis among hospitalized patients continues to rise. 1 The mortality rate ranges from 10% to 54%, depending on the severity of the disease and the definition of organ dysfunction. 2 Therefore, early and accurate assessment and prediction of a patient's risk of developing sepsis or septic shock is crucial. In particular, predicting sepsis to enable rapid and reliable decisions regarding early treatment strategies and monitoring is a highly unmet medical need. Predicting patient outcomes can impact multiple aspects of patient management, including treatment, diagnostic testing (e.g., microbiology, blood chemistry, radiology, etc.), and hospital admission. Timely identification of patients with a high probability of poor prognosis enables more efficient patient management procedures, including, for example, admission to the intensive care unit (ICU), advanced therapeutic agents, and invasive diagnostic or surgical interventions, which can reduce complications and mortality.

[0003] Adrenal medullaris is one of the most thoroughly studied peptide hormones. Adrenal medullaris (ADM) mRNA encodes a 185-amino acid pro-hormone (SEQ ID No. 1), namely pro-adrenal medullaris, which is enzymatically converted to pro-adrenal medullaris via N-terminal signal peptide cleavage. Then, pro-adrenal medullaris is further processed by several pro-hormone-converting enzymes to produce four peptides, namely…

[0004] - PAMP (SEQ ID No. 2): N-terminal peptide of pro-adremyelin or N-terminal 20-peptide of pro-adremyelin with a C-terminal glycine residue 3,4,5

[0005] - MR-proADM (SEQ ID No. 6): intermediate region adrenal medullarin pro-adrenal medullarin, a stable and inert peptide.

[0006] - ADM-Gly (SEQ ID No. 4): C-terminal glycine-extended inactive precursor of bioactive ADM (bio-ADM (SEQ ID No. 5)).

[0007] - CT-proADM (SEQ ID No. 3): C-terminal pro-adrenergic medullaris or adrenocorticotropic hormone

[0008] CT-proADM: Little is known about the physiological effects and clinical behavior of CT-proADM (sometimes referred to as adrenocorticotropic peptide). It has been shown to increase pulmonary artery tone, is considered a vasoconstrictive and vasopressor peptide, and can induce vascular smooth muscle cell proliferation. 6 .

[0009] PAMP To obtain its full biological activity, the C-terminal glycine-extended PAMP requires amidation by the enzyme peptidylglycine α-amidyl monooxygenase (PAM). PAM recognizes the C-terminal glycine and catalyzes a two-step reaction, also known as amidation or C-terminal amidation. This releases glyoxylate derived from the C-terminal glycine, forming an amidated carboxyl-terminal arginine structure in the PAMP, which is essential for the PAMP's biological activity. The amidated or mature PAMP (m-PAMP; SEQ ID No. 20) is a potent vasoactive and angiogenic factor whose effects are complementary to or antagonistic to bio-ADM signaling. In rats, PAMP exhibits a strong hypotensive effect, inducing vasodilation. In healthy human subjects, the levels of PAMP-Gly and m-PAMP were 1.15 pmol / L and 0.51 pmol / L, respectively. 4,7–10 In an LPS-induced septic shock rat model, plasma concentrations of m-PAMP increased 30 minutes after LPS injection compared to baseline and doubled after a 4-hour observation period. 11 Although derived from the same peptide precursor, bio-ADM levels began to change after 2 hours in the same experiment. Furthermore, plasma PAMP levels are observed in several clinical conditions such as essential hypertension. 9 Chronic renal failure 12 Congestive heart failure 13 and chronic glomerulonephritis 14 The levels of PAMP may be elevated. Compared to bio-ADM, plasma levels of PAMP are lower, suggesting that bio-ADM and PAMP play different roles in the pathophysiology of these diseases.

[0010] ADM-Gly, bio-ADM, and MR-proADMGenerally, LPS and pro-inflammatory cytokines upregulate ADM, and plasma or serum levels of adrenomedullin are elevated in sepsis and are associated with sepsis severity, mortality, and organ failure. 15 However, the term "adrenal medullaris" (ADM) used in many publications is inaccurate, and for the purposes of this invention, it is important to distinguish the most important forms of ADM (i.e., MR-proADM, bio-ADM, and ADM-Gly): ADM-Gly is a direct, inactive form of bio-ADM. 16 The biosynthetic precursor of adrenaline, often referred to as an intermediate form of ADM, is the main circulating form of adrenaline in the human body. 17 To obtain its biological activity, ADM-Gly is produced by PAM 17 It is activated in the same manner as it is described for activating m-PAMP, thereby producing the bioactive product bio-ADM with an amidated carboxyl-terminal tyrosine structure.

[0011] Little is known about ADM-Gly or its levels in clinical settings such as sepsis or septic shock. Previous studies have shown that ADM-Gly is elevated in sepsis and septic shock and has predictive value in sepsis or shock outcomes and mortality (WO 2021170752 A1).

[0012] The ADM-Gly / bio-ADM ratio has been reported to predict mortality in sepsis patients (WO 2021170763A1). ADM-Gly levels in plasma and tissues from a rat LPS model were measured using ICTEIA (immune complex transferase immunoassay). 18 The results showed that ADM-Gly increased to 225.1 fmol / mL (1370 pg / mL) compared to 5.22 fmol / mL (32 pg / mL) in control animals, while bio-ADM increased to 107.8 fmol / mL (650 pg / mL). 18 However, these measurements were performed 3 hours after LPS infusion, and time-resolved ADM measurements were not performed immediately after the start of LPS infusion. The ADM-Gly / bio-ADM ratio (ADM ratio) in LPS animals (2.35) was lower than that in control animals (10.81). 18 Furthermore, indirect measurement of ADM-Gly showed that total plasma ADM (tADM) (i.e., the sum of bio-ADM and ADM-Gly) was associated with surgical stress score (SSS) and increased directly after surgery in several surgical groups (cardiopulmonary bypass, abdominal, and cervical laminectomy). 19A comparison of tADM levels in sepsis patients on the day of ICU admission and on day 3 after admission revealed that tADM levels were elevated upon admission, and that the prognostic value of tADM was comparable to that of bio-ADM in terms of sepsis severity and outcome. 20 Furthermore, ADM-Gly levels in pericardial fluid during ischemic heart disease are... 21 It is elevated in the plasma of patients with essential hypertension and chronic renal failure, but ADM-Gly has been reported to rise in parallel with bio-ADM in these indications. 22 In obesity, increases in plasma ADM-Gly have been reported to parallel increases with bio-ADM. 23 .

[0013] The physiological function of ADM-Gly during sepsis remains unclear. Pro-inflammatory cytokines (i.e., AL-1, TNF, and LPS) have been reported to induce enhanced transcription of the ADM gene. 24 This suggests an early elevation of bio-ADM during LPS infusion, as reported for m-PAMP. 11 m-PAMP itself originates from PAMP-Gly. Surprisingly, this is not the case for bio-ADM. As shown in Example 2 of the present invention, bio-ADM, as a product of PAM-mediated ADM-Gly amidation, appears to have a delayed response to systemic inflammation for unknown reasons. Instead, an unexpected early increase in ADM-Gly occurs, resulting in an early increase in the ADM-Gly / bio-ADM ratio.

[0014] bio-ADM functions as a neurotransmitter and as a paracrine and autocrine signaling factor. As ADM-Gly, it belongs to the calcitonin gene-related peptide (CGRP) / calcitonin peptide hormone family. 25 Bio-ADM levels were significantly elevated in patients with congestive heart failure, myocardial infarction, kidney disease, hypertension, diabetes, those in the acute phase of shock, and those with sepsis and septic shock, but to varying degrees. 4,26-31 (WO 2022234111 A1). In sepsis and septic shock, bio-ADM levels are associated with sepsis severity, organ failure, and mortality. In sepsis, impaired circulatory function and consequently impaired blood flow lead to reduced oxygen supply to multiple organs, resulting in hypoxia and multiple organ failure. Hypoxia also induces an increase in bio-ADM, but it is unclear whether the same mechanism applies to ADM-Gly. Bio-ADM reduces endothelial permeability, thereby reducing hypoxia and the extravasation of plasma components such as leukocytes from the circulation.

[0015] Furthermore, in rodent sepsis models, administration of bioactive ADM reduced mortality. It is generally believed that this reduction in mortality is achieved by decreasing endothelial permeability.

[0016] The physiological function of MR-proADM is not yet fully understood, but it has been identified as a prognostic biomarker for risk assessment and stratification in patients with sepsis or septic shock, pneumonia, myocardial infarction, and heart failure. 25 According to reports, changes in MR-proADM levels induced by LPS treatment in human samples occurred 90 to 120 minutes after the start of LPS administration. 32,33 In patients with poor emergency medical care, MR-proADM is useful for predicting mortality and improving clinical models for intensive care unit admission. 34 When confirmed within 24 hours of ICU admission, an increase in MR-proADM was consistent with an APACHE-II score, enhancing prognostic and diagnostic management value for severe sepsis and septic shock, and was also a predictor of 28-day mortality. 35,36 Furthermore, MR-proADM levels are associated with organ failure. 37 Furthermore, MR-proADM has a prognostic effect on 90-day mortality in heart failure. 38 Furthermore, MR-proADM levels were associated with the severity of chronic heart failure and an increased risk of death at 12-month follow-up. 39,40 WO 2019077082 describes a method for monitoring a subject receiving treatment with a conjugate of adrenomedullin, the method comprising: determining the level of a fragment of proadrenomedullin in bodily fluids obtained from the subject, the fragment of proadrenomedullin selected from MR-proADM, C-terminal proadrenomedullin (CT-proDM) and / or N-terminal 20-peptide of proadrenomedullin (PAMP) or a fragment thereof (but not bio-ADM or ADM-Gly); and relating the level of the fragment of proadrenomedullin to the need to adjust the patient’s treatment regimen, wherein the treatment regimen is selected from hydrocortisone.

[0017] A surprising finding of this invention is that ADM-Gly levels rise early, several hours before the onset of sepsis and several hours before the emergence of bio-ADM elevation. This allows for the prediction of a patient's transition to a sepsis state and enables early decisions regarding standard sepsis therapy. Furthermore, the surprising early elevation of ADM-Gly levels allows for early decisions regarding treatment for patients using adrenomedullin-targeted therapy, such as using anti-ADM antibodies to achieve early enrichment of bio-ADM and counteract sepsis-related endothelial damage, enhancing the conversion of ADM-Gly to bio-ADM (WO 2021170763 A1), or using therapeutic PAM (peptidyl glycine α-amidyl monooxygenase) enzymes to convert early excess ADM-Gly into bio-ADM (WO 2021170816 A1).

[0018] The surprising finding of this invention is that ADM-Gly or fragments thereof are early biomarkers for predicting that patients will later develop sepsis, severe sepsis, and septic shock.

[0019] Therefore, the technical problem behind this invention is to provide means and methods that provide a rapid and reliable way to predict or diagnose sepsis and / or septic shock in a subject.

[0020] The technical problem is solved by providing the embodiments described below and as represented in the appended claims. Summary of the Invention

[0021] The subject of this invention is a method for a) diagnosing sepsis and / or septic shock in a subject, or assessing or monitoring the severity of sepsis and / or septic shock, or b) assessing the early risk of a subject developing sepsis and / or septic shock, or c) predicting, determining, or monitoring a subject's need for therapy or intervention for sepsis and / or septic shock. The method includes: Determine the levels of biomarkers selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or fragments of at least five amino acids thereof, or the sum of the levels of ADM-Gly according to SEQ ID No. 4 and ADM-NH2 according to SEQ ID No. 5 (“Total ADM”) in bodily fluids obtained from the subject; and A) To correlate the level of the biomarker or a fragment of at least five amino acids thereof, or the total ADM, with the degree of sepsis and / or septic shock in the subject, or to diagnose sepsis and / or septic shock in the subject, wherein an elevated level exceeding a certain threshold indicates sepsis and / or septic shock or the degree of sepsis and / or septic shock, or B) Associating the level of the biomarker or a fragment of at least five amino acids thereof, or the total ADM, with the risk of sepsis and / or septic shock, wherein elevated levels exceeding a certain threshold predict an increased risk of sepsis and / or septic shock, or C) Associating the level of the biomarker or a fragment of at least five amino acids thereof or the total ADM with the need for therapy or intervention for sepsis and / or septic shock, wherein an elevated level exceeding a certain threshold indicates the need for therapy or intervention for sepsis and / or septic shock.

[0022] Those skilled in the art will understand that the method according to the present invention includes the following steps: The subject of this invention is a method for a) diagnosing sepsis and / or septic shock in a subject, or assessing or monitoring the severity of sepsis and / or septic shock, or b) assessing the early risk of a subject developing sepsis and / or septic shock, or c) predicting, determining, or monitoring a subject's need for therapy or intervention for sepsis and / or septic shock. The method includes: Determine the levels of biomarkers selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or fragments of at least five amino acids thereof, or the sum of the levels of ADM-Gly according to SEQ ID No. 4 and ADM-NH2 according to SEQ ID No. 5 (“Total ADM”) in bodily fluids obtained from the subject; and A) To correlate the level of the biomarker or a fragment of at least five amino acids thereof, or the total ADM, with the degree of sepsis and / or septic shock in the subject, or to diagnose sepsis and / or septic shock in the subject, wherein an elevated level exceeding a certain threshold indicates sepsis and / or septic shock or the degree of sepsis and / or septic shock, or B) Associating the level of the biomarker or a fragment of at least five amino acids thereof, or the total ADM, with the risk of sepsis and / or septic shock, wherein elevated levels exceeding a certain threshold predict an increased risk of sepsis and / or septic shock, or C) Associating the level of the biomarker or a fragment of at least five amino acids thereof or the total ADM with the need for therapy or intervention for sepsis and / or septic shock, wherein an elevated level exceeding a certain threshold indicates the need for therapy or intervention for sepsis and / or septic shock.

[0023] This applies, with the necessary modifications, to the following corresponding embodiments of the invention.

[0024] The severity, degree, class, and grade of sepsis and / or septic shock are used synonymously throughout this application.

[0025] Mature ADM, bio-ADM, and ADM-NH2 are used synonymously throughout this application and are molecules according to SEQ ID No. 5.

[0026] The present invention solves the above-mentioned technical problems. As described below and in the appended embodiments, it was unexpectedly found that the levels of biomarkers selected from ADM-Gly, PAMP, MR-proADM and CT-proADM or fragments of at least five amino acids thereof, or total ADM, and / or the ratio of the levels of said biomarkers and fragments of at least five amino acids thereof, or total ADM to the levels of ADM-NH2 and fragments of at least five amino acids thereof, showed a strong statistical relationship with the risk of developing sepsis and / or septic shock, particularly enabling the prediction of sepsis and / or septic shock 48 hours, preferably 12 hours, more preferably 8 hours, more preferably 4 hours, and at least 2 hours earlier than the onset of sepsis and / or septic shock.

[0027] The occurrence of sepsis and / or septic shock should be defined as the patient exhibiting symptoms similar to those described in sepsis-2. 42 The clinical signs of sepsis as defined should meet the following sepsis-2 definition. 42 At least two of the criteria must be met, and the patient or patient group must be considered either a patient with mild symptoms of an infectious disease or a patient without severe symptoms of an infectious disease, such as a patient without sepsis symptoms: - Body temperature exceeding 38℃ or less than 36℃ - Heart rate increase >90 beats / minute - Persistent hypotension (mean arterial pressure (MAP) drops below 60 mmHg and systolic blood pressure drops below 90 mmHg or systolic blood pressure decreases by at least 40 mmHg) - Any of the above criteria appearing in parallel Therefore, the level of a biomarker selected from ADM-Gly, PAMP, MR-proADM, and CT-proADM, or a fragment of at least five amino acids thereof, or total ADM, and / or the ratio of the level of said biomarker and a fragment of at least five amino acids thereof, or total ADM to the level of ADM-NH2 and a fragment of at least five amino acids thereof, predicts the risk of sepsis and / or septic shock in a subject, thereby helping clinicians determine which treatment is most appropriate. In one embodiment, the biomarker is ADM-Gly, and the level of ADM-Gly and a fragment of at least five amino acids thereof in body fluids is greater than 20 pg / mL, preferably greater than 25 pg / mL, more preferably greater than 30 pg / mL, more preferably greater than 40 pg / mL, more preferably greater than 50 pg / mL, while the level of bio-ADM is in the range of 5 pg / mL to 30 pg / mL, and / or - The ratio in the body fluid increases to more than 1.5, preferably more than 1.6, more preferably more than 1.7, more preferably more than 1.8, more preferably more than 1.9, and more preferably above 2.

[0028] - In one implementation, an increase in ADM-Gly level means an increase of 20% in ADM-Gly relative to a given threshold.

[0029] - In another implementation, the increase in the level of the ratio means that the ratio increases by 20% relative to a given threshold.

[0030] Throughout this application, the levels of biomarkers selected from ADM-Gly, PAMP, MR-proADM, and CT-proADM, or fragments thereof of at least five amino acids, or the ratio of total ADM to the levels of ADM-NH2 and fragments thereof of at least five amino acids, are equivalent to the inverse ratio of the levels of biomarkers selected from ADM-Gly, PAMP, MR-proADM, and CT-proADM, or fragments thereof of at least five amino acids, or the ratio of total ADM to the levels of ADM-Gly and fragments thereof of at least five amino acids. For example, a ratio of ADM-Gly to ADM-NH2 of 2 is equivalent to a ratio of ADM-NH2 to ADM-Gly of 0.5. It should be understood that throughout this specification, determining the level of a biomarker selected from ADM-Gly, PAMP, MR-proADM, and CT-proADM, or a fragment thereof containing at least five amino acids, or the ratio of total ADM to the level of ADM-NH2 and a fragment thereof containing at least five amino acids, can be performed as follows: determining the ratio of the level of ADM-NH2 and a fragment thereof containing at least five amino acids to the level of a biomarker selected from ADM-Gly, PAMP, MR-proADM, and CT-proADM, or a fragment thereof containing at least five amino acids, or the level of total ADM, and adjusting the corresponding threshold to its inverse value. In this case, a ratio falling below a certain threshold is indicative.

[0031] This invention offers several advantages over conventional methods, particularly in the following ways: The methods and kits of this invention are rapid, objective, easy to use, and accurate for predicting adverse events. Furthermore, the methods and kits of this invention involve biomarkers that are readily measurable in routine hospital procedures, as the levels of ADM-Gly and ADM-NH2 can be determined in routinely obtained blood samples or other biological fluids obtained from the subject. Moreover, the determination of ADM-Gly and ADM-NH2 levels is very rapid. Therefore, the methods and kits of this invention are suitable for the rapid assessment, diagnosis, and prognosis of sepsis and / or septic shock. Additionally, because the results of biomarker measurements are simple and provide a specific value, there is no subjective bias when healthcare professionals use the methods or kits of this invention.

[0032] Therefore, the methods and kits presented in this article have advantages in the diagnosis, prognosis, risk assessment and / or risk stratification of sepsis and / or septic shock in subjects.

[0033] In some embodiments, the present invention relates to a method for a) diagnosing sepsis and / or septic shock in a subject, or assessing or monitoring the severity of sepsis and / or septic shock, or b) early assessment of the risk of a subject developing sepsis and / or septic shock, or c) predicting, determining, or monitoring the subject's need for therapy or intervention for sepsis and / or septic shock. The method includes: Determine the level of a biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or a fragment thereof, or the ratio of total ADM to the level of ADM-NH2 according to SEQ ID No. 5 and a fragment thereof, in bodily fluids obtained from the subject; and A) Correlating the ratio with the severity of sepsis and / or septic shock in the subject, or diagnosing sepsis and / or septic shock in the subject, wherein an elevated ratio exceeding a certain threshold indicates sepsis and / or septic shock or the severity of sepsis and / or septic shock, or B) Associate the ratio with the risk of sepsis and / or septic shock, wherein an elevated ratio exceeding a certain threshold predicts an increased risk of sepsis and / or septic shock, or C) Associate the ratio with the need for therapy or intervention for sepsis and / or septic shock, wherein an elevated ratio exceeding a certain threshold indicates the need for therapy or intervention for sepsis and / or septic shock.

[0034] In one embodiment, the present invention relates to a method for early assessment of the risk of a subject developing sepsis and / or septic shock, the method comprising: A) Determine the ratio of the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof in bodily fluids obtained from the subject to the level of ADM-NH2 according to SEQ ID No. 5 and a fragment of at least five amino acids thereof; and B) Associate the ratio with the risk of developing sepsis and / or septic shock, wherein an elevated ratio exceeding a certain threshold indicates an increased risk of developing sepsis and / or septic shock.

[0035] Those skilled in the art will understand that, in one embodiment, the present invention relates to a method for early assessment of the risk of a subject developing sepsis and / or septic shock, the method comprising: A) Determine the ratio of the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof in bodily fluids obtained from the subject to the level of ADM-NH2 according to SEQ ID No. 5 and a fragment of at least five amino acids thereof; and B) Associate the ratio with the risk of developing sepsis and / or septic shock, wherein an elevated ratio exceeding a certain threshold indicates an increased risk of developing sepsis and / or septic shock.

[0036] This applies, with the necessary modifications, to the following corresponding embodiments of the invention.

[0037] In one embodiment, the present invention relates to a method for early assessment of the risk of a subject developing sepsis and / or septic shock, the method comprising: A) Determine the ratio of total ADM to the level of ADM-NH2 and fragments of at least five amino acids according to SEQ ID No. 5 in bodily fluids obtained from the subject; and B) Associate the ratio with the risk of developing sepsis and / or septic shock, wherein an elevated ratio exceeding a certain threshold indicates an increased risk of developing sepsis and / or septic shock.

[0038] In one embodiment, the present invention relates to a method for early assessment of the risk of a subject developing sepsis and / or septic shock, the method comprising: A) Determine the level of ADM-Gly according to SEQ ID No. 4 or the ratio of total ADM to the level of ADM-NH2 and at least five amino acid fragments according to SEQ ID No. 5 in bodily fluids obtained from the subject; and B) Associate the ratio with the risk of developing sepsis and / or septic shock, wherein an elevated ratio exceeding a certain threshold indicates an increased risk of developing sepsis and / or septic shock.

[0039] In some embodiments, the method is used for early assessment of a subject's risk of developing sepsis and / or septic shock, wherein the early assessment is a prediction made 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, and preferably 2 hours before the onset of sepsis and / or septic shock. In some embodiments, the method is used for early assessment of a subject's risk of developing sepsis and / or septic shock, wherein the subject has been identified as having a risk of developing sepsis and / or septic shock, and wherein the subject's plasma procalcitonin level is less than 2.0 µg / L, preferably less than 1.5 µg / L, more preferably less than 1.0 µg / L, and even more preferably less than 0.5 µg / L.

[0040] In some embodiments, the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof, or the ratio of the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof to the level of ADM-NH2 according to SEQ ID No. 5 and a fragment of at least five amino acids thereof, is determined by using a conjugate of ADM-Gly according to SEQ ID No. 4 or a fragment of at least five amino acids thereof.

[0041] In some embodiments of the method, the conjugate is selected from an antibody, antibody fragment, or non-Ig scaffold that binds to ADM-Gly according to SEQ ID No. 4 or a fragment of at least five amino acids thereof.

[0042] In some embodiments of the method, the threshold level of ADM-Gly and fragments of at least five amino acids in body fluids is 20 pg / mL, preferably 25 pg / mL, more preferably 30 pg / mL, more preferably 40 pg / mL, and more preferably 50 pg / mL, wherein the level of ADM-NH2 or fragments of at least five amino acids according to SEQ ID No. 5 is in the range of 5 pg / mL to 30 pg / mL.

[0043] In some embodiments of the method, the threshold for the ratio of the level of ADM-Gly according to SEQ ID No. 4 and fragments of at least five amino acids therein to the level of ADM-NH2 according to SEQ ID No. 5 or fragments of at least five amino acids therein is 1.5, preferably 1.6, more preferably 1.7, more preferably 1.8, more preferably 1.9, and more preferably 2.

[0044] In some embodiments of the method, the fragment of at least five amino acids of ADM-Gly according to SEQ ID No. 4 comprises the fragment according to SEQ ID No. 18.

[0045] In some embodiments of the method, the fragment of at least five amino acids of ADM-NH2 according to SEQ ID No. 5 includes the fragment according to SEQ ID No. 19.

[0046] In some embodiments of the method, the fragment is selected from SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11.

[0047] In some embodiments of the method, the fragment is selected from SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15 and SEQ ID No. 16.

[0048] In some embodiments of the method, the determination of the level of a biomarker selected from ADM-Gly, PAMP, MR-proADM and CT-proADM or a fragment of at least five amino acids thereof or total ADM, and / or the ratio of the level of the biomarker and a fragment of at least five amino acids thereof or total ADM to the level of ADM-NH2 and a fragment of at least five amino acids thereof, is performed more than once in the subject.

[0049] In some implementations, the method is used to stratify subjects into sepsis and / or septic shock grade groups.

[0050] In some embodiments of the method, the level or ratio of the biomarker or its fragment is used to guide the treatment or intervention for sepsis and / or septic shock, wherein if the level or ratio of the biomarker or its fragment exceeds a certain threshold, the treatment or intervention is indicated, and wherein if the level or ratio of the biomarker or its fragment is below a certain threshold, the treatment or intervention for sepsis and / or septic shock is not indicated.

[0051] In a preferred embodiment of the method, the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof, or the ratio of the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof to the level of ADM-NH2 according to SEQ ID No. 5, is used to guide the treatment or intervention for sepsis and / or septic shock, wherein the treatment or intervention is indicated if the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof, or the ratio of the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof to the level of ADM-NH2 according to SEQ ID No. 5, exceeds a certain threshold, and wherein the treatment or intervention is indicated if the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof, or the ratio of the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof, exceeds a certain threshold. If the ratio of the levels of 5 ADM-NH2 and its fragments of at least five amino acids is below a certain threshold, it does not indicate therapies or interventions for sepsis and / or septic shock.

[0052] In some embodiments of the method, the subject is a human being.

[0053] In some embodiments of the method, the therapy is selected from the administration of antibiotics; vasopressor therapy, including the administration of vasopressin and angiotensin II (e.g., Giapreza); immunosuppressive therapy; and renal therapy, including the administration of alkaline phosphatase.

[0054] In some embodiments of the method, the therapy includes administering an anti-ADM antibody, preferably an N-terminal anti-ADM antibody, and / or PAM (peptidyl glycine α-amidyl monooxygenase) as a therapeutic agent.

[0055] In a preferred embodiment of the method, the therapy includes administering a vasopressor to the subject, and wherein, in addition, the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof, or the ratio of the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof to the level of ADM-NH2 according to SEQ ID No. 5, is determined, and wherein when the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof in the sample, or the ratio of the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof to the level of ADM-NH2 according to SEQ ID No. 5, exceeds a certain threshold, treatment with the anti-ADM antibody or anti-ADM antibody fragment is initiated and / or treatment is continued, and / or wherein if the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof, or the ratio of the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof to the level of ADM-NH2 according to SEQ ID No. 5, exceeds a certain threshold, treatment is initiated and / or continued, and / or treatment is initiated with the anti-ADM antibody or anti-ADM antibody fragment, and / or treatment is initiated if the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof, or the ratio of the level of ADM-Gly according to SEQ ID No. 5, exceeds a certain threshold, treatment is initiated with the anti-ADM antibody or anti-ADM antibody fragment. If the ratio of the levels of 5 ADM-NH2 and its fragments of at least five amino acids is below the predetermined threshold, treatment with anti-ADM antibody or anti-ADM antibody fragments is suspended and / or terminated.

[0056] In some embodiments of the method, the body fluid is selected from whole blood, plasma, serum, urine, cerebrospinal fluid (CSF), and saliva.

[0057] This document also envisions identifying peptides with sequence identity to ADM-Gly, PAMP, MR-proADM, CT-proADM, and ADM-NH2. For example, in the methods and kits of this invention, peptides with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to ADM-Gly and ADM-NH2 can be identified. According to the invention, in the context of two or more amino acid sequences, the terms "sequence identity," "homology," or "percentage of homology," or "identity," or "percentage identity," refer to two or more sequences or subsequences being identical or having a specified percentage of identical amino acids when compared and aligned within a comparison window (preferably over the full length) or within a specified region for maximum correspondence, said maximum correspondence being measured, such as using sequence comparison algorithms known in the art or by manual alignment and visual inspection. Sequences having, for example, 70% to 90% or greater (preferably 95% or greater) sequence identity can be considered substantially identical. Such a definition also applies to complementary sequences of the test sequence. Preferably, the described identity is present in a region of at least about 10 to about 15 amino acids in length, more preferably in a region of at least about 20 to about 35 amino acids in length, and most preferably throughout the entire length. Those skilled in the art know how to use algorithms known in the art, such as those based on the CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), to determine the percentage of identity between sequences.

[0058] The term "biomarker" or "early biomarker" refers to an elevated level of a biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or fragments of at least five amino acids thereof, or total ADM, or the level of said biomarker or fragments thereof, or the ratio of total ADM to the level of ADM-NH2 and its fragments, prior to the development of sepsis, severe sepsis, or septic shock in a patient.

[0059] As used herein, the term "subject" refers to a living person or a non-human object. Preferably, in this document, a subject is a human subject. Unless otherwise stated, a subject may be healthy or ill.

[0060] As used in this article, the “level” of a biomarker refers to the quantity of the biomarker molecular entity in a sample. In other words, it determines the concentration of the biomarker in the sample. For example, determining the concentration of ADM-Gly in a subject’s sample.

[0061] The "body fluid" may be selected from blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva. In one particular embodiment, the body fluid according to the invention is a blood sample. The blood sample may be selected from whole blood, serum, and plasma. In one particular embodiment of the diagnostic method, the sample is selected from human citrate plasma, heparin plasma, and EDTA plasma.

[0062] The term "prediction" refers to the prognosis of a subject's outcome or specific risk. This may also include estimates of the probability of the subject's recovery or the probability of an adverse outcome.

[0063] The method of this invention can also be used for monitoring, therapy monitoring, therapy guidance, and / or therapy control. In the context of this application, "monitoring" involves tracking a patient and potential complications in order to, for example, analyze the progress of the recovery process or the impact of a particular treatment or therapy on the patient's health status.

[0064] In the context of this invention, the terms "therapy monitoring" or "therapy control" refer to monitoring and / or adjusting the patient's therapeutic treatment, for example, by obtaining feedback on the efficacy of the therapy. As used herein, the term "therapy guidance" refers to the application of certain therapies, therapeutic actions, or medical interventions based on the values / levels of one or more biomarkers and / or clinical parameters and / or clinical scores. This includes adjusting or discontinuing therapy.

[0065] In this invention, the terms "risk assessment" and "risk stratification" refer to grouping subjects into different risk groups based on their further prognosis. Risk assessment also involves stratification for the application of preventive and / or therapeutic measures. The term "therapeutic stratification" specifically refers to grouping or classifying patients into different groups, such as risk groups or therapy groups according to their classification to receive certain different therapeutic measures. The term "therapeutic stratification" also refers to grouping or classifying patients with infection or symptoms of infectious disease into groups that do not require certain therapeutic measures.

[0066] Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulation of the host's response to infection. 41 Organ dysfunction can be identified as a rapid change in the total SOFA score (Sequential Organ Failure Assessment score) >2 points due to infection.

[0067] Patients with unknown pre-existing organ dysfunction may be assumed to have a baseline SOFA score of zero. In the general hospital population with suspected infection, a SOFA score >2 reflects an overall mortality risk of approximately 10%. Even patients exhibiting mild dysfunction may deteriorate further, highlighting the severity of the condition and the need for rapid and appropriate intervention in the absence of prior intervention. Sepsis is a life-threatening condition that occurs when the body's response to infection damages its own tissues and organs. Patients with suspected infection who may have a prolonged ICU stay or may die in the hospital can be rapidly identified at the bedside using qSOFA, i.e., altered mental status, systolic blood pressure <100 mm Hg, or respiratory rate >22 breaths / min.

[0068] Septic shock is a subset of sepsis in which the underlying circulatory and cellular / metabolic abnormalities are severe enough to significantly increase mortality. Patients with septic shock can be identified by the following clinical framework: sepsis, persistent hypotension despite adequate volume resuscitation requiring vasopressors to maintain a mean arterial pressure (MAP) >65 mm Hg, and serum lactate levels >2 mmol / L (18 mg / dL). Hospital mortality exceeds 40% when these criteria are met.

[0069] As used in the context of this application, the term "sepsis" encompasses all possible stages of sepsis development. The term "sepsis" also includes definitions based on sepsis-2. 42 Severe sepsis or septic shock. The term "sepsis" also includes falling into the sepsis-3 definition. 41 Subjects within the body. Sepsis is a life-threatening acute organ dysfunction caused by an inadequate host response to infection. For diagnosing organ dysfunction associated with sepsis, a Sequential Organ Failure Assessment (SOFA) score change of ≥2 should be used. As used herein, organ dysfunction refers to a condition or state of health in which an organ fails to perform its intended function. “Organ failure” refers to organ dysfunction to the point that it cannot maintain normal homeostasis without external clinical intervention. Organ failure may involve organs selected from the kidneys, liver, heart, lungs, and nervous system. Conversely, organ function represents the expected function of the corresponding organ within its physiological range. Those skilled in the art are familiar with the corresponding organ function during medical examination.

[0070] Organ dysfunction can be defined by the Sequential Organ Failure Assessment Score (SOFA score) or its components. The SOFA score was previously known as the Sepsis-Related Organ Failure Assessment Score. 41The SOFA score is used to track an individual's condition during their stay in the intensive care unit (ICU) to determine the extent of organ dysfunction or the rate of organ failure. The score is based on six different components: one each for the respiratory, cardiovascular, hepatic, coagulation, renal, and nervous systems, each scored from 0 to 4, with increases reflecting worsening organ dysfunction. The assessment criteria for the SOFA score are described, for example, by Lamden et al. (in a review in [year missing]). 43 (in Chinese). Traditionally, the SOFA score can be calculated at the time of ICU admission and every 24 hours thereafter.

[0071] Specifically, the organ dysfunction is selected from renal failure, cardiac dysfunction, liver dysfunction, or respiratory dysfunction.

[0072] The Rapid SOFA score (rapid SOFA or qSOFA) was introduced by Sepsis-3 in February 2016 as a simplified version of the SOFA score, used as an initial method to identify patients at high risk of adverse infection outcomes. 44 The qSOFA score significantly simplifies the SOFA assessment by including only three clinical criteria and by incorporating "any altered mental status" instead of requiring a GCS <15. The qSOFA score can be easily and quickly repeated on patients. The score ranges from 0 to 3. One point is awarded for the following: hypotension (SBP <100 mm Hg), hyperventilation (>22 breaths / min), and altered mental status (GCS <15). A qSOFA score of 2 or higher shortly after infection onset is associated with an increased risk of death or prolonged stay in the intensive care unit. These outcomes are more common in patients with suspected sepsis than in uncomplicated infections. Based on these findings, the third international consensus definition of sepsis recommends using the qSOFA score as a simple indicator for identifying patients with suspected sepsis outside the ICU. 45 .

[0073] In a preferred embodiment of the invention, the method is defined as: predicting sepsis, severe sepsis, and / or septic shock in a patient population that was previously difficult—if not impossible—to determine by conventional clinical and / or molecular diagnostic methods as having a significant risk of serious deterioration of medical condition requiring hospitalization. This patient population may be considered as patients with mild infectious disease symptoms or patients without severe infectious disease symptoms, such as those without sepsis symptoms.

[0074] In one implementation, when the sample was presented, the patient exhibited symptoms of a mild infectious disease corresponding to a qSOFA score of 0 or 1.

[0075] In one implementation, the patient did not exhibit clinical signs of sepsis, severe sepsis, and / or septic shock at the time the sample was provided.

[0076] In one implementation, the patient has been diagnosed with an infectious disease of bacterial, viral, fungal, or parasitic origin when the sample is provided.

[0077] In one implementation, the patient had been diagnosed with community-acquired pneumonia (CAP) or urinary tract infection (UTI) at the time the sample was provided.

[0078] Delayed treatment of suspected infected patients arriving at the emergency department (ED) may lead to longer hospital stays, increased morbidity, and increased infection-related mortality.

[0079] Therefore, it is crucial to accurately assess the severity of the host response and the likelihood of further disease progression to sepsis, severe sepsis, septic shock, and organ dysfunction in order to administer a rapid and targeted therapeutic response.

[0080] Therefore, one object of the present invention is to use a biomarker selected from ADM-Gly, PAMP, MR-proADM and CT-proADM, or fragments of at least five amino acids thereof or total ADM, to distinguish patients who are more likely or at higher risk of developing sepsis, severe sepsis or septic shock requiring intensive treatment from patients at lower risk of developing such treatment.

[0081] In one embodiment of the invention, the method is used to stratify the patient into a risk group.

[0082] In one embodiment, a high-risk level of a biomarker selected from ADM-Gly, PAMP, MR-proADM, and CT-proADM, or a fragment of at least five amino acids thereof, or total ADM, indicates a patient's risk of developing sepsis, severe sepsis, and / or septic shock within 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, preferably 2 hours, and wherein a low-risk level of a biomarker selected from ADM-Gly, PAMP, MR-proADM, and CT-proADM, or a fragment of at least five amino acids thereof, or total ADM, indicates a patient's risk of not developing sepsis, severe sepsis, or septic shock within 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, preferably 2 hours.

[0083] In one embodiment, the level of a biomarker selected from ADM-Gly, PAMP, MR-proADM, and CT-proADM, or fragments of at least five amino acids thereof, or total ADM in the body fluid sample indicates the risk of a patient developing sepsis, severe sepsis, and / or septic shock requiring hospitalization.

[0084] In one embodiment, a high-risk level of a biomarker selected from ADM-Gly, PAMP, MR-proADM, and CT-proADM, or a fragment of at least five amino acids thereof, or total ADM, indicates a patient's risk of developing hospital-acquired sepsis, severe sepsis, and / or septic shock within 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, and preferably 2 hours, and a low-risk level of a biomarker selected from ADM-Gly, PAMP, MR-proADM, and CT-proADM, or a fragment of at least five amino acids thereof, or total ADM, indicates a patient's risk of not developing hospital-acquired sepsis, severe sepsis, and / or septic shock within 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, and preferably 2 hours.

[0085] In another embodiment of the invention, the method is used to stratify the patient into an early treatment group (e.g., where antibiotics are required).

[0086] The term “early” is defined as the time of treatment before a patient exhibits clinical signs and symptoms of sepsis, severe sepsis, and / or septic shock, or before a patient is diagnosed with sepsis, severe sepsis, and / or septic shock.

[0087] In another embodiment of the invention, the method is used to stratify the patient into an early treatment group (e.g., requiring the administration of anti-ADM antibodies, preferably N-terminal anti-ADM antibodies). The term "early" is defined as the time before the patient exhibits clinical signs and symptoms of sepsis, severe sepsis, and / or septic shock, or before the patient is diagnosed with sepsis, severe sepsis, and / or septic shock.

[0088] In one embodiment, the level of ADM-Gly or fragments thereof in the bodily fluid sample indicates that the patient requires frequent monitoring and / or intensive care. In another embodiment, patients with high-risk levels will require medical treatment provided in a hospital setting.

[0089] Examples of these treatments include, but are not limited to, fluid therapy, vasopressors, intravenous antibiotics, and in some embodiments, virtually any treatment that can be administered at home other than oral antibiotics.

[0090] As a result of the method according to the invention, implementation of the method may include subsequent therapeutic decisions and / or therapeutic actions. Such therapeutic decisions may include the initiation, modification, or adjustment of medical treatment. Preferably, if the method of the invention indicates the occurrence of a condition requiring hospitalization, appropriate therapeutic measures, such as the initiation or modification of a certain drug or fluid therapy, may be initiated.

[0091] In the context of the methods of this invention, any therapy, medical treatment, or therapeutic action disclosed herein may be employed as a subsequent therapeutic decision or action, particularly when the therapeutic measures are specifically administered within a hospital, including but not limited to intravenous fluid therapy, dialysis, and management of electrolyte (particularly potassium, calcium, and phosphorus) abnormalities. Furthermore, continued intensive observation and care of the patient may be directed for an extended period, such as days, weeks, or even months. This may involve keeping or transferring the patient to an ICU, and / or extending the patient's stay in the ICU.

[0092] On the other hand, if the results of the method of the present invention indicate no risk of developing sepsis, severe sepsis, or septic shock requiring hospitalization, then specific treatment may not be necessary for such complications, or a milder, self-administered prescription treatment may be prescribed.

[0093] ADM-Gly or fragments thereof are specifically designed to achieve the aforementioned medical benefits in all patient groups visiting the emergency department.

[0094] Throughout this specification, unless otherwise stated, the term ADM-Gly includes all splice variants of ADM-Gly. Throughout this specification, it should be understood that the term "total ADM" is the sum of the levels of ADM-Gly according to SEQ ID No. 4 and ADM-NH2 according to SEQ ID No. 5.

[0095] The term "determining the level of ADM-Gly, its splice variants, or fragments of at least 5 amino acids thereof, including SEQ ID 2-16" generally refers to determining the immunoreactivity to a region within the aforementioned molecule. This means that it is not necessary to selectively measure a particular fragment. It should be understood that the conjugate used to determine the level of ADM-Gly or fragments of at least 5 amino acids thereof, including SEQ ID 2-16, binds to any fragment containing a region that binds to said conjugate. The conjugate may be an antibody or an antibody fragment or a non-IgG scaffold.

[0096] The subject of the invention is a method in which the level of ADM-Gly or a fragment of at least 5 amino acids thereof is determined by using a conjugate targeting a fragment of ADM-Gly or a fragment of at least 5 amino acids thereof.

[0097] In one embodiment of the invention, the conjugate is selected from antibodies, antibody fragments, or non-Ig scaffolds that bind to ADM-Gly or fragments of at least 5 amino acids thereof.

[0098] As used herein, a “reference level” or “threshold” may reflect the normal level of a corresponding biomarker that indicates the degree of sepsis and / or septic shock, or predicts an increased risk of developing sepsis and / or septic shock, or indicates the need for therapy or intervention for sepsis and / or septic shock.

[0099] In one embodiment, the ratio of the level of ADM-Gly according to SEQ ID No. 4 and fragments thereof of at least five amino acids in body fluids to the level of ADM-NH2 according to SEQ ID No. 5 or fragments thereof of at least five amino acids is 1.5, preferably 1.6, more preferably 1.7, more preferably 1.8, more preferably 1.9, and more preferably 2. The reference level applied depends on the desired predictive specificity and sensitivity, i.e., a higher threshold (such as about 2) will reliably identify subjects at increased risk of developing sepsis and / or septic shock, or indicate the need for therapy or intervention for sepsis and / or septic shock.

[0100] In one embodiment, the ratio of the level of ADM-Gly according to SEQ ID No. 4 and fragments of at least five amino acids therein to the level of ADM-NH2 according to SEQ ID No. 5 or fragments of at least five amino acids therein is 1.5, preferably 1.6, more preferably 1.7, more preferably 1.8, more preferably 1.9, more preferably 2, which predicts an increased risk of sepsis and / or septic shock 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, preferably 2 hours earlier than the onset of sepsis and / or septic shock.

[0101] As used herein, the term "elevated marker level" means an increase in the level of a marker, i.e., it refers to an increased marker level. Therefore, the terms "increased marker level" or "increased level" are used interchangeably with the term "elevated marker level" herein. The term "elevated level" refers to a level exceeding a certain threshold level. An increased marker level or an increase in marker level in a subject means that the marker level is at least about 15%, preferably at least about 20%, more preferably at least about 25%, or even more preferably at least about 30% higher than a reference level for the marker.

[0102] As used herein, the term "sample" refers to a biological sample obtained from a subject. As used herein, "sample" can refer, for example, to a body fluid or tissue sample obtained for diagnostic, prognostic, or assessment purposes of a target subject, such as a patient. Preferably, in this document, the sample is a body fluid sample, such as blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusion samples. In particular, the sample is blood, plasma, serum, or urine. The sample may be processed (pretreated), for example by fractionation or purification procedures, such as separating whole blood into serum or plasma fractions. Such pretreatment also includes, but is not limited to, dilution, filtration, centrifugation, concentration, sedimentation, precipitation, or dialysis. Pretreatment may also include the addition of chemical or biochemical substances to the solution, such as acids, bases, buffers, salts, solvents, reactive dyes, detergents, emulsifiers, and chelating agents. Preferably, the sample is a blood sample, more preferably a serum or plasma sample.

[0103] In the context of this invention, "plasma" is a substantially cell-free supernatant obtained after centrifugation of blood containing an anticoagulant. Exemplary anticoagulants include calcium ion-binding compounds such as EDTA or citrate; and thrombin inhibitors such as heparin or hirudin. Cell-free plasma can be obtained by centrifuging anticoagulated blood (e.g., citrated blood, EDTA blood, or heparinized blood) for at least 15 minutes, for example, at 2000 g to 3000 g.

[0104] In the context of this invention, "serum" refers to the liquid portion of whole blood collected after the blood has clotted. When the clotted blood (coagulated blood) is centrifuged, serum is obtained as the supernatant.

[0105] As used in this article, “urine” refers to the liquid product that the body secretes through the kidneys and is excreted through the urethra via a process called urination (or micturition).

[0106] As used herein, the term "antibody" refers to the immunoglobulin molecule and the immunoglobulin (Ig) molecule that has immunological activity, i.e., a molecule containing an antigen-binding site that specifically binds to an antigen (responds to an immune response to the antigen). According to the invention, antibodies can be monoclonal antibodies as well as polyclonal antibodies. In particular, antibodies that specifically bind to ADM-Gly or ADM-NH2 are used. An antibody is considered specific if its affinity for a target molecule, such as ADM-Gly or ADM-NH2 or a fragment thereof, is at least 50 times, preferably 100 times, and most preferably at least 1000 times higher than its affinity for other molecules contained in a sample containing the target molecule. How to develop and select antibodies with a given specificity is well known in the art. In the context of this invention, monoclonal antibodies are preferred. The antibody or antibody-binding fragment specifically binds to a marker or a fragment thereof as defined herein. Anti-ADM-Gly antibodies specifically distinguish between the amidated form and the C-terminal glycine-extended form of ADM. Therefore, the C-terminal glycine in the epitope shown in Seq. ID No.: 18 is essential for the reaction of anti-ADM-Gly antibody with ADM-Gly, while epitopes lacking the C-terminal glycine, as shown in Seq. ID No.: 19, are not recognized by anti-ADM-Gly antibody. Therefore, the peptides defined herein can also be epitopes that antibodies specifically bind to. Exemplary immunoassays can be luminescent immunoassays (LIA), radioimmunoassays (RIA), chemiluminescent and fluorescent immunoassays, enzyme immunoassays (EIA), enzyme-linked immunosorbent assays (ELISA), luminescent bead arrays, magnetic bead arrays, protein microarray assays, rapid assays, and rare earth cavitation assays. Furthermore, assays suitable for point-of-care and rapid detection formats, such as immunochromatographic strip tests, can also be used.

[0107] As used herein, in the context of this invention, "diagnosis" refers to the identification and (early) detection of sepsis and / or septic shock in a subject, and may also include differential diagnosis. Furthermore, the term "diagnosis" may also cover the assessment of the severity of sepsis and / or septic shock. For example, assessing the severity of the condition and the likelihood of sepsis and / or septic shock occurring. Additionally, diagnosis refers to the ability to predict when a subject will develop sepsis and / or septic shock.

[0108] This invention also relates to reagent kits, uses of the reagent kits, and methods of using such reagent kits. This invention relates to reagent kits for carrying out the methods provided above and below. Definitions provided herein, such as those relating to methods, also apply to the reagent kits of this invention. Specifically, the present invention relates to a kit for diagnosing sepsis and / or septic shock in a subject, or for assessing or monitoring the severity of sepsis and / or septic shock, or for early assessment of the risk of a subject developing sepsis and / or septic shock, or for predicting, determining, or monitoring the subject's need for therapy or intervention for sepsis and / or septic shock, wherein the kit comprises a detection reagent for determining the level of a biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or a fragment of at least five amino acids thereof, or total ADM, in the subject's sample, or the level of a biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or a fragment of at least five amino acids thereof, or total ADM, in accordance with SEQ ID No. 4. The ratio of the levels of 5 ADM-NH2 and its fragments of at least five amino acids.

[0109] The kit according to the invention may further include reference data indicating an elevation in the level or total ADM of a biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or a ratio of total ADM to ADM-NH2 and its fragments. This indicates sepsis and / or septic shock or the degree of sepsis and / or septic shock, or predicts an increased risk of developing sepsis and / or septic shock, or indicates the need for therapy or intervention for sepsis and / or septic shock.

[0110] In particular, the present invention relates to a kit for early assessment of the risk of sepsis and / or septic shock in a subject, wherein the kit comprises a detection reagent and reference data, the detection reagent being used to determine the level of ADM-Gly and at least five amino acid fragments of ADM-Gly according to SEQ ID No. 4, or the ratio of the level of ADM-Gly and at least five amino acid fragments of ADM-Gly according to SEQ ID No. 4 to the level of ADM-NH2 and at least five amino acid fragments of ADM-NH2 according to SEQ ID No. 5, and the reference data indicating that an elevated level of ADM-Gly and its fragments or an elevated ratio of the level of ADM-Gly and its fragments to the level of ADM-NH2 and its fragments predicts an increased risk of sepsis and / or septic shock 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, preferably 2 hours earlier than the onset of sepsis and / or septic shock.

[0111] The present invention also relates to a kit for use in the method according to the invention and its use in the method according to the invention.

[0112] In particular, the present invention relates to the use of a kit for...

[0113] a) To diagnose sepsis and / or septic shock in the subject, or to assess or monitor the degree of sepsis and / or septic shock, or

[0114] b) Early assessment of the subject's risk of developing sepsis and / or septic shock, or

[0115] c) Predict, identify, or monitor the subject's need for therapy or intervention for sepsis and / or septic shock.

[0116] In particular, the present invention relates to the use of a kit in a method, the kit being used for

[0117] a) To diagnose sepsis and / or septic shock in the subject, or to assess or monitor the degree of sepsis and / or septic shock, or

[0118] b) Early assessment of the subject's risk of developing sepsis and / or septic shock, or

[0119] c) Predicting, identifying, or monitoring a subject's need for therapy or intervention for sepsis and / or septic shock, the method comprising the following steps: Determine the level of a biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or fragments of at least five amino acids thereof, or total ADM, in bodily fluids obtained from the subject; and A) To correlate the levels of the biomarker and at least five amino acid fragments thereof with the degree of sepsis and / or septic shock in the subject, or to diagnose sepsis and / or septic shock in the subject, wherein elevated levels exceeding a certain threshold indicate the degree of sepsis and / or septic shock, or B) Associating the levels of the biomarker and fragments of at least five amino acids with the risk of sepsis and / or septic shock, wherein elevated levels exceeding a certain threshold predict an increased risk of sepsis and / or septic shock, or C) Associating the levels of the biomarker and fragments of at least five amino acids with the need for therapy or intervention for sepsis and / or septic shock, wherein elevated levels exceeding a certain threshold indicate the need for therapy or intervention for sepsis and / or septic shock.

[0120] As used herein, "detection reagent" and the like are reagents suitable for determining biomarkers described herein, such as ADM-Gly or ADM-NH2 or fragments thereof. Such exemplary detection reagents are, for example, ligands that specifically bind to a peptide or epitope of a biomarker described herein, such as antibodies or fragments thereof. Such ligands can be used in the immunoassays described above. Other reagents used in immunoassays for determining biomarker levels may also be included in the kit and are considered detection reagents herein.

[0121] The detection reagent may also involve reagents used to detect biomarkers or fragments thereof by MS-based methods. Therefore, such detection reagents can also be reagents used to prepare samples for MS analysis, such as enzymes, chemicals, buffers, etc. A mass spectrometer can also be considered a detection reagent. The detection reagent according to the invention can also be a calibration solution, for example, a calibration solution used to determine and compare biomarker levels.

[0122] The definitions and interpretations given are applicable to the following items after necessary modifications. This invention also relates to the following items: 1. A method for a) diagnosing sepsis and / or septic shock in a subject, or assessing or monitoring the severity of sepsis and / or septic shock, or b) early assessment of the risk of a subject developing sepsis and / or septic shock, or c) predicting, determining, or monitoring the subject's need for therapy or intervention for sepsis and / or septic shock, The method includes: Determine the levels of biomarkers selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or fragments of at least five amino acids thereof, or the sum of the levels of ADM-Gly according to SEQ ID No. 4 and ADM-NH2 according to SEQ ID No. 5 (“Total ADM”) in bodily fluids obtained from the subject; and A) To correlate the level of the biomarker or a fragment of at least five amino acids thereof, or the total ADM, with the degree of sepsis and / or septic shock in the subject, or to diagnose sepsis and / or septic shock in the subject, wherein an elevated level exceeding a certain threshold indicates sepsis and / or septic shock or the degree of sepsis and / or septic shock, or B) Associating the level of the biomarker or a fragment of at least five amino acids thereof, or the total ADM, with the risk of sepsis and / or septic shock, wherein elevated levels exceeding a certain threshold predict an increased risk of sepsis and / or septic shock, or C) Associating the level of the biomarker or a fragment of at least five amino acids thereof or the total ADM with the need for therapy or intervention for sepsis and / or septic shock, wherein an elevated level exceeding a certain threshold indicates the need for therapy or intervention for sepsis and / or septic shock.

[0123] Those skilled in the art will know that Project 1 includes the following steps: A method for a) diagnosing sepsis and / or septic shock in a subject, or assessing or monitoring the severity of sepsis and / or septic shock, or b) early assessment of a subject's risk of developing sepsis and / or septic shock, or c) predicting, determining, or monitoring a subject's need for therapy or intervention for sepsis and / or septic shock. The method includes: Determine the levels of biomarkers selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or fragments of at least five amino acids thereof, or the sum of the levels of ADM-Gly according to SEQ ID No. 4 and ADM-NH2 according to SEQ ID No. 5 (“Total ADM”) in bodily fluids obtained from the subject; and A) To correlate the level of the biomarker or a fragment of at least five amino acids thereof, or the total ADM, with the degree of sepsis and / or septic shock in the subject, or to diagnose sepsis and / or septic shock in the subject, wherein an elevated level exceeding a certain threshold indicates sepsis and / or septic shock or the degree of sepsis and / or septic shock, or B) Associating the level of the biomarker or a fragment of at least five amino acids thereof, or the total ADM, with the risk of sepsis and / or septic shock, wherein elevated levels exceeding a certain threshold predict an increased risk of sepsis and / or septic shock, or C) Associating the level of the biomarker or a fragment of at least five amino acids thereof or the total ADM with the need for therapy or intervention for sepsis and / or septic shock, wherein an elevated level exceeding a certain threshold indicates the need for therapy or intervention for sepsis and / or septic shock.

[0124] This also applies to the following items after necessary modifications.

[0125] 2. The methods described in Item 1 for a) diagnosing sepsis and / or septic shock in a subject, or assessing or monitoring the severity of sepsis and / or septic shock, or b) early assessment of a subject's risk of developing sepsis and / or septic shock, or c) predicting, determining, or monitoring a subject's need for therapy or intervention for sepsis and / or septic shock. The method includes: Determine the level of a biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or a fragment thereof, or the ratio of total ADM to the level of ADM-NH2 according to SEQ ID No. 5 and a fragment thereof, in bodily fluids obtained from the subject; and A) Correlating the ratio with the severity of sepsis and / or septic shock in the subject, or diagnosing sepsis and / or septic shock in the subject, wherein an elevated ratio exceeding a certain threshold indicates sepsis and / or septic shock or the severity of sepsis and / or septic shock, or B) Associate the ratio with the risk of sepsis and / or septic shock, wherein an elevated ratio exceeding a certain threshold predicts an increased risk of sepsis and / or septic shock, or C) Associate the ratio with the need for therapy or intervention for sepsis and / or septic shock, wherein an elevated ratio exceeding a certain threshold indicates the need for therapy or intervention for sepsis and / or septic shock.

[0126] 3. The method described in Project 1 or 2 for early assessment of the risk of sepsis and / or septic shock in subjects, or

[0127] The method includes: Determine the level of a biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or a fragment thereof, or the ratio of total ADM to the level of ADM-NH2 according to SEQ ID No. 5 and a fragment thereof, in bodily fluids obtained from the subject; and B) Associate the ratio with the risk of developing sepsis and / or septic shock, wherein an elevated ratio exceeding a certain threshold indicates an increased risk of developing sepsis and / or septic shock.

[0128] Those skilled in the art will understand that Item 3 includes the methods described in Item 1 or 2 for the early assessment of the risk of sepsis and / or septic shock in a subject. The method includes: Determine the level of a biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or a fragment thereof, or the ratio of total ADM to the level of ADM-NH2 according to SEQ ID No. 5 and a fragment thereof, in bodily fluids obtained from the subject; and B) Associate the ratio with the risk of developing sepsis and / or septic shock, wherein an elevated ratio exceeding a certain threshold indicates an increased risk of developing sepsis and / or septic shock.

[0129] This also applies to the following items after necessary modifications.

[0130] 4. The method according to any one of items 1 to 3, wherein the early prediction is a prediction 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, preferably 2 hours before the onset of sepsis and / or septic shock.

[0131] 5. The method according to any one of items 1 to 4, wherein the biomarker is ADM-Gly according to SEQ ID No. 4, and the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof, or the ratio of the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof to the level of ADM-NH2 according to SEQ ID No. 5 and a fragment of at least five amino acids thereof, is determined by using a conjugate of ADM-Gly according to SEQ ID No. 4 or a fragment of at least five amino acids thereof.

[0132] 6. The method according to item 5, wherein the conjugate is selected from an antibody, antibody fragment, or non-Ig scaffold that binds to ADM-Gly according to SEQ ID No. 4 or a fragment of at least five amino acids thereof.

[0133] 7. The method according to any one of items 1 to 6, wherein the threshold for the level of ADM-Gly and fragments of at least five amino acids thereof in body fluids is 20 pg / mL, preferably 25 pg / mL, more preferably 30 pg / mL, more preferably 40 pg / mL, more preferably 50 pg / mL, wherein the level of ADM-NH2 or fragments of at least five amino acids thereof according to SEQ ID No. 5 is in the range of 5 pg / mL to 30 pg / mL.

[0134] 8. The method according to any one of items 2 to 7, wherein the threshold for the ratio of the level of ADM-Gly according to SEQ ID No. 4 and fragments thereof of at least five amino acids to the level of ADM-NH2 according to SEQ ID No. 5 or fragments thereof of at least five amino acids is 1.5, preferably 1.6, more preferably 1.7, more preferably 1.8, more preferably 1.9, more preferably 2.

[0135] 9. The method according to any one of items 1 to 8, wherein the fragment of at least five amino acids of ADM-Gly according to SEQ ID No. 4 comprises the fragment according to SEQ ID No. 18.

[0136] 10. The method according to any one of items 2 to 9, wherein the fragment of at least five amino acids of ADM-NH2 according to SEQ ID No. 5 comprises the fragment according to SEQ ID No. 19.

[0137] 11. The method according to item 9, wherein the fragment is selected from SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11.

[0138] 12. The method according to item 10, wherein the fragment is selected from SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15 and SEQ ID No. 16.

[0139] 13. The method according to any one of items 1 to 12, wherein the determination of the level of the biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6 and CT-proADM according to SEQ ID No. 3, or fragments of at least five amino acids thereof, or the total ADM or the ratio thereof, is performed more than once in the subject.

[0140] 14. The method according to any one of items 1 to 13, the purpose of which is to stratify the subject into a sepsis and / or septic shock grade group.

[0141] 15. The method according to any one of items 1 to 14, wherein the level or total ADM or the ratio of said biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or fragments of at least five amino acids thereof, is used to guide the therapy or intervention for sepsis and / or septic shock, wherein the therapy or intervention is indicated if the level or total ADM or the ratio of said biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or fragments of at least five amino acids thereof, exceeds a certain threshold, and wherein the therapy or intervention is indicated if said biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or fragments of at least five amino acids thereof, exceeds a certain threshold, and wherein the therapy or intervention is indicated if said biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or fragments of at least five amino acids thereof, exceeds a certain threshold. If the level of a biomarker of CT-proADM or a fragment of at least five amino acids thereof, or the total ADM or the ratio thereof, is below a certain threshold, it does not indicate a treatment or intervention for sepsis and / or septic shock.

[0142] 16. The method according to any one of items 1 to 15, wherein the subject is a human being.

[0143] 17. The method according to any one of items 1 to 16, wherein said therapy is selected from the administration of antibiotics, vasopressor therapy, and immunosuppressive therapy.

[0144] 18. The method according to any one of items 1 to 17, wherein the therapy comprises administering an anti-ADM antibody, preferably an N-terminal anti-ADM antibody, and / or PAM as a therapeutic agent.

[0145] 19. The method according to any one of items 1 to 18, wherein the body fluid is selected from whole blood, plasma, serum, urine, cerebrospinal fluid (CSF), and saliva.

[0146] 20. A kit for carrying out the method according to any one of items 1 to 19, wherein the kit comprises a detection reagent for determining the level or the total ADM or the ratio of the biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6 and CT-proADM according to SEQ ID No. 3 in the sample of the subject.

[0147] 21. Use of the kit according to item 20 in the method according to any one of claims 1 to 19.

[0148] 22. As described in item 20 or 21, the kit is used for...

[0149] a) To diagnose sepsis and / or septic shock in the subject, or to assess or monitor the degree of sepsis and / or septic shock, or

[0150] b) Early assessment of the subject's risk of developing sepsis and / or septic shock, or

[0151] c) Predict, identify, or monitor the subject's need for therapy or intervention for sepsis and / or septic shock.

[0152] As used herein, the terms “comprising” and “including” or their grammatical variations shall be regarded as specifying at least the stated feature, integer, step, or component, but do not preclude the addition of one or more additional features, integers, steps, components, or groups thereof. This term encompasses the terms “consisting of” and “substantially consisting of”, which are understood to specify only the stated feature, integer, step, or component to exclude any additional features.

[0153] Therefore, the term "comprising / including / having" means that any other components (or similarly, features, integers, steps, etc.) may / may be present.

[0154] The term "composed of" means that there are no other components (or similarly, features, integers, steps, etc.).

[0155] The term “method” refers to the manner, means, techniques and procedures used to accomplish a given task, including but not limited to manner, means, techniques and procedures known to practitioners in the chemical, biological and biophysical fields or readily developed by such practitioners from known manner, means, techniques and procedures.

[0156] As used herein, the term "about" means ±10% of the indicated value, particularly ±5% of the indicated value. Wherever the term "about" is used, it also includes a specific reference to the exact indicated value. If the term "about" is used in conjunction with a parameter quantified as an integer, such as the number of nucleotides in a given nucleic acid, the quantity corresponding to ±10% or ±5% of the indicated value will be rounded to the nearest integer. For example, stating "about 25 amino acids" refers to a range of 23 to 28 amino acids, particularly 24 to 26 amino acids, and preferably a specific value of 25 amino acids. Attached Figure Description

[0157] The invention is also described with reference to the following non-limiting drawings.

[0158] Figure 1A A typical ADM-Gly dose / signal curve is shown.

[0159] Figure 1B The normal distribution of ADM-Gly from n=157 self-reported healthy individuals is shown in Example 1.

[0160] Figure 2 The analysis of bio-ADM (black bar) and ADM-Gly (gray bar) in a human endotoxemia model sample as a response to LPS-induced systemic inflammation is shown in Example 2.

[0161] Figure 3The analysis of the ADM-Gly / bio-ADM ratio (ADM ratio) in a human endotoxemia model sample, as a response to LPS-induced systemic inflammation, is shown in Example 2.

[0162] Figure 4 The analysis of total ADM (the sum of the molar concentrations of ADM-Gly and bio-ADM) in a human endotoxemia model sample as a response to LPS-induced systemic inflammation is shown in Example 2.

[0163] Figure 5 The analysis of the ADM-Gly / total ADM ratio (total ADM ratio) in a human endotoxemia model sample, as a response to LPS-induced systemic inflammation, is shown in Example 2.

[0164] Figure 6 The analysis of bio-ADM (black bars) and ADM-Gly (gray bars) from the induced porcine sepsis model in Example 3 is shown.

[0165] Figure 7 An analysis of the ADM-Gly / bio-ADM ratio (ADM ratio) from the induced porcine sepsis model is shown in Example 3.

[0166] Figure 8 The analysis of total ADM (the sum of the molar concentrations of ADM-Gly and bio-ADM) from the induced porcine sepsis model in Example 3 is shown.

[0167] Figure 9 The analysis of the ADM-Gly / total ADM ratio (total ADM ratio) from the induced porcine sepsis model in Example 3 is shown.

[0168] The invention is also described with reference to the following non-limiting embodiments. Detailed Implementation

[0169] Example 1: ADM Measurement

[0170] Quantitative use of bio-ADM elsewhere 46 The sphingotest bio-ADM assay described is performed. ADM-Gly, as based on Weber et al. 46 The method for bioactive ADM was quantified, but with the following modifications: the MACN-acrididine-NHS-labeled tracer antibody for detecting ADM-Gly targets the C-terminal glycine of ADM-Gly. The assay was calibrated using synthetic ADM-Gly. The limit of detection (LOD) was 9 pg / mL of ADM-Gly. The antibody targeting the C-terminal glycine of ADM showed no cross-reactivity with bio-ADM. Figure 1A The figure shows a typical standard curve. Figure 1B The normal distribution of ADM-Gly from n=157 self-reported healthy individuals is shown (median: 21.1 pg / mL; 25th percentile: 14.1 pg / mL; 75th percentile: 28.15 pg / mL. Data did not pass the D'Agostino and Pearson normality test). The ratio of the two peptides was calculated using Equation (1) using ADM-Gly and bio-ADM values. The ADM-Gly frequency distribution was obtained from n=157 self-reported healthy subjects.

[0171] Formula 1:

[0172] [ADMGly]: Concentration of ADM-Gly, in pg / mL; MWbio: Molecular weight of bio-ADM (6028.9 pg / pmol); MMWGly: Molecular weight of ADM-Gly (6086.9 pg / pmol); [bioADM]: Concentration of bio-ADM, in pg / mL.

[0173] Example 2: ADM analysis in a human endotoxemia model.

[0174] In this invention, the levels of ADM-Gly and bio-ADM were determined using human EDTA plasma samples (n=28 at each time point) from a human endotoxemia model. Therefore, in healthy volunteers, 1 ng... kg -1 h -1 LPS (lipopolysaccharide, E. coli O113) was infused at an infusion rate of 3 hours to induce systemic inflammation. Blood samples were directly drawn before LPS infusion (t=0) and at several time points during the infusion process. The LPS-induced model is a well-established and characteristic model of systemic inflammation, as demonstrated in the literature. 47,48 There are ample records.

[0175] As expected and as previously reported 33 A slight increase in bio-ADM was observed at 180 and 240 minutes after the start of infusion. At t=180 and t=250 minutes after the start of LPS infusion, a significantly higher (approximately 2.5-fold) increase in ADM-Gly was observed compared to bio-ADM. Figure 2 ).

[0176] Surprisingly and unexpectedly, in response to LPS-induced systemic inflammation, the increase in ADM-Gly occurred several hours earlier than the increase in bio-ADM. Figure 2As shown, a significant increase in ADM-Gly was surprisingly observed 90 minutes after the start of LPS infusion, which increased further at t=120 minutes and reached its peak at t=180 and t=240 minutes.

[0177] The data clearly show that ADM-Gly increases at least 90 minutes earlier than bio-ADM. Therefore, unlike bio-ADM, ADM-Gly is clearly a suitable early biomarker for predicting systemic inflammation.

[0178] The ADM ratio calculated using Equation (1) clearly shows a significant increase at t=90 minutes after LPS infusion, indicating a shift in ADM steady state toward ADM-Gly. Figure 3 Therefore, both ADM-Gly and the ADM ratio are suitable early biomarkers for predicting systemic inflammation. The ADM ratio further increased after 120 and 180 minutes, as ADM-Gly increased rapidly while bio-ADM increased slowly.

[0179] Formula 2:

[0180] [ADMGly]: Concentration of ADM-Gly, in pg / mL; MWbio: Molecular weight of bio-ADM (6028.9 pg / pmol); MMWGly: Molecular weight of ADM-Gly (6086.9 pg / pmol); [bioADM]: Concentration of bio-ADM, in pg / mL.

[0181] Surprisingly and unexpectedly, regardless of its C-terminal amidation state, the increase in total ADM as a response to LPS-induced systemic inflammation, calculated as the molar sum of ADM-Gly and bio-ADM using Equation 2, showed a significant increase after 180 minutes. Figure 4 As shown in the figure. The data clearly demonstrate that, regardless of its C-terminal amidation status, total ADM analysis can be an early marker of systemic inflammation, while compared with total ADM, as... Figure 2 The ADM-Gly alone shown is superior as a biomarker for systemic inflammation.

[0182] like Figure 5 As shown, the ratio of ADM-Gly to total ADM increased at t=90 minutes after LPS infusion, but this increase was not statistically significant, while the increase in the total ADM ratio became significant at 120 minutes after LPS infusion. The data suggest that the total ADM ratio may be an early biomarker of systemic inflammation, and... Figure 3 The ADM-Gly / bio-ADM ratio shown is superior to the overall ADM ratio.

[0183] Figure 2 The analysis of bio-ADM (black bars) and ADM-Gly (gray bars) in samples from a human endotoxemia model is shown as a response to LPS-induced systemic inflammation. The data for bio-ADM and ADM-Gly were normalized relative to baseline (t=0, set as 100%, indicated by the dashed line). Significance was tested using the unnormalized raw data using the nonparametric two-tailed Mann-Whitney test. p < 0.0001; p = 0.0075 - 0.0023; p = 0.046 - 0.024

[0184] Figure 3 The analysis of the ADM-Gly / bio-ADM ratio (ADM ratio) in samples from a human endotoxemia model, as a response to LPS-induced systemic inflammation, is presented. The ADM ratio data were normalized relative to baseline (t=0, set as 100%, indicated by the dashed line). Significance was tested using the unnormalized raw data using the nonparametric two-tailed Mann-Whitney test. p=0.0001; p=0.0042; p=0.028

[0185] Figure 4 The analysis of total ADM in samples from a human endotoxemia model, as a response to LPS-induced systemic inflammation, is presented. The total ADM was calculated using Equation 2. The total ADM data were normalized relative to a baseline level (t=0, set as 100%, indicated by the dashed line). Significance was tested using the unnormalized raw data using a nonparametric two-tailed Mann-Whitney test. p = 0.008-0.0041; ns: not significant

[0186] Figure 5 The analysis of the ADM-Gly / total ADM ratio (total ADM ratio) in samples from a human endotoxemia model, as a response to LPS-induced systemic inflammation, is presented. Data for the total ADM ratio were normalized relative to baseline (t=0, set as 100%, indicated by the dashed line). Significance was tested using the unnormalized raw data with a nonparametric two-tailed Mann-Whitney test. p < 0.0001; p=0.0001; p = 0.0012; ns: not significant

[0187] Example 3: Analysis of ADM in a porcine septic shock model

[0188] In this invention, EDTA plasma samples from a porcine septic shock model were used to determine the levels of ADM-Gly and bio-ADM. The benefits of this animal model are as follows: This model allows for direct analysis of blood samples after induction and before sepsis and shock occur, which is difficult to achieve with human clinical samples because ICU patients often have sepsis upon admission to the ICU.

[0189] In terms of cardiac physiology, immunology, and the gastrointestinal system, pig animal models have shown a high degree of similarity to humans. 49,50 .

[0190] After baseline sampling, a laboratory model was induced in anesthetized animals (n=8) by intraperitoneal administration of 3 g / kg of autologous feces. Several parameters were monitored during the study, including heart rate, cardiac output, lactate changes, pulse pressure variability, venous oxygen saturation, central venous-arterial CO2 difference, creatinine and hemoglobin changes, as well as blood pressure and mean arterial pressure. Sepsis was considered to have occurred when lactate increased to a level exceeding 2 mmol / L, heart rate increased to a level exceeding 120 bpm, cardiac output decreased to a level below 6 L / min, and venous oxygen saturation decreased to a level below 60%. Septic shock was considered to have occurred when mean arterial pressure dropped to a level below 55 mmHg, which occurred on average 1.1 hours after diagnosis of sepsis.

[0191] As expected, at the time point of sepsis, bio-ADM and ADM-Gly levels increased compared to baseline, reaching 3.5-fold and 6-fold increases, respectively, compared to baseline. Further increases in both markers were observed at the time point of septic shock. Figure 6 ).

[0192] Surprisingly and unexpectedly, in response to fecal-induced peritonitis, the increase in ADM-Gly occurred several hours earlier than the increase in bio-ADM. Figure 6 As shown, a significant increase in plasma ADM-Gly concentration was observed 4 hours before the animals entered a sepsis state. ADM-Gly concentrations further increased 1–2 hours before diagnosis of sepsis, peaking at septic shock, while bio-ADM levels remained unchanged before diagnosis. The plasma ADM-Gly level clearly showed a significant and surprising early increase at least four hours before diagnosis of sepsis. Therefore, an early increase in ADM-Gly is clearly a marker for predicting the onset of sepsis and septic shock at least 4 hours in advance.

[0193] Furthermore, the ADM ratio calculated using equation (1) showed a significant change 4 hours before sepsis ( Figure 7 The ratio increased significantly, indicating a shift in ADM steady state towards ADM-Gly. Figure 7 Therefore, both ADM-Gly and the ADM ratio are suitable early biomarkers for predicting sepsis and septic shock. The ADM ratio peaks 1–2 hours before sepsis and then declines further during sepsis and shock due to a rapid increase in bio-ADM.

[0194] Surprisingly, regardless of its C-terminal amidation state, the increase in total ADM as a response to fecal-induced peritonitis, calculated as the molar sum of ADM-Gly and bio-ADM using Equation 2, showed a significant increase after 180 minutes, comparable to the increase in ADM-Gly. Figure 8 As shown in the figure. The data clearly demonstrate that total ADM analysis can be an early biomarker of systemic inflammation, regardless of its C-terminal amidation status. Figure 9 As shown, the ADM-Gly / total ADM ratio increases 4 hours before sepsis, indicating a shift of total ADM towards ADM-Gly. The data suggest that the total ADM ratio can be an early marker of systemic inflammation, similar to... Figure 3 The ADM-Gly / bio-ADM ratio shown is comparable.

[0195] Figure 6 Analysis of bio-ADM (black bars) and ADM-Gly (grey bars) from an induced porcine sepsis model is shown. Data for bio-ADM and ADM-Gly were normalized relative to baseline (baseline set at 100%). Significance was tested using the unnormalized raw data with the nonparametric two-tailed Mann-Whitney test. p=0.0002; p=0.004; p=0.014.

[0196] Figure 7 Analysis of the ADM-Gly / bio-ADM ratio (ADM ratio) from an induced porcine sepsis model is presented. The ADM ratio data were normalized relative to a baseline level (baseline set at 100%). Significance was tested using the nonparametric two-tailed Mann-Whitney test with the unnormalized raw data. p=0.0047; p = 0.0016 - 0.027.

[0197] Figure 8Analysis of total ADM from an induced porcine sepsis model is presented. The total ADM data were normalized relative to the baseline level (baseline set at 100%). Significance was tested using the nonparametric two-tailed Mann-Whitney test with the unnormalized raw data. p=0.0002; p=0.004; p=0.014.

[0198] Figure 9 Analysis of the ADM-Gly / total ADM ratio (total ADM ratio) from an induced porcine sepsis model is presented. The total ADM ratio data were normalized relative to a baseline level (baseline set at 100%). Significance was tested using the nonparametric two-tailed Mann-Whitney test with the unnormalized raw data. p=0.0030; p = 0.0140 - 0.0162.

[0199] All references cited in this article are incorporated herein in their entirety through citation.

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[0250] sequence

[0251] SEQ ID No: 1 – pre-proADM (aa 1-185)

[0252] SEQ ID No: 2 – PAMP-Gly (aa 22 - 42 of pre-proADM SEQ ID No. 1)

[0253] SEQ ID No: 3 – CT-proADM (aa 148 - 185 of pre-proADM SEQ ID No. 1)

[0254] SEQ ID No: 4 – ADM-Gly (aa 95 - 147 of pre-proADM SEQ ID No. 1)

[0255] SEQ ID No: 5 – bio-ADM (aa 95 - 146 of pre-proADM SEQ ID No. 1)

[0256] SEQ ID No: 6 – MR-proADM (aa 45 - 92 of pre-proADM SEQ ID No. 1)

[0257] SEQ ID No: 7 – ADM-Gly 2 - 53 (aa 96 - 147 of pre-proADM SEQ ID No. 1)

[0258] SEQ ID No: 8 – ADM-Gly 8 - 53 (aa 102 - 147 of pre-proADM SEQ ID No. 1)

[0259] SEQ ID No: 9 – ADM-Gly 22 - 53 (aa 115 - 147 of pre-proADM SEQ ID No. 1)

[0260] SEQ ID No: 10 – ADM-Gly 27 - 53 (aa 120 - 147 of pre-proADM SEQ ID No. 1)

[0261] SEQ ID No: 11 – ADM-Gly 33-53 (aa 126-147 of pre-proADM SEQ ID No. 1)

[0262] SEQ ID No: 12 – bio-ADM 2-52 (aa 96-146 of pre-proADM SEQ ID No. 1)

[0263] SEQ ID No: 13 – bio-ADM 8-52 (aa 102-146 of pre-proADM SEQ ID No. 1)

[0264] SEQ ID No: 14 – bio-ADM 22-52 (aa 115-146 of pre-proADM SEQ ID No. 1)

[0265] SEQ ID No: 15 – bio-ADM 27-52 (aa 120-146 of pre-proADM SEQ ID No. 1)

[0266] SEQ ID No: 16 – bio-ADM 33-52 (aa 126-146 of pre-proADM SEQ ID No. 1)

[0267] SEQ ID No: 18 – (aa 143-147 of pre-proADM SEQ ID No. 1)

[0268] SEQ ID No: 19 – (aa 143-146 of pre-proADM SEQ ID No. 1)

[0269] SEQ ID No: 20 – PAMP-NH2 (aa 22-41 of pre-proADM SEQ ID No. 1)

Claims

1. A method for a) diagnosing sepsis and / or septic shock in a subject, or assessing or monitoring the severity of sepsis and / or septic shock, or b) early assessment of the risk of a subject developing sepsis and / or septic shock, or c) predicting, determining, or monitoring the subject's need for therapy or intervention for sepsis and / or septic shock, The method includes: Determine the levels of biomarkers selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or fragments of at least five amino acids thereof, or the sum of the levels of ADM-Gly according to SEQ ID No. 4 and ADM-NH2 according to SEQ ID No. 5 ("total ADM") in bodily fluids obtained from the subject; and A) To correlate the level of the biomarker or a fragment of at least five amino acids thereof, or the total ADM, with the degree of sepsis and / or septic shock in the subject, or to diagnose sepsis and / or septic shock in the subject, wherein an elevated level exceeding a certain threshold indicates sepsis and / or septic shock or the degree of sepsis and / or septic shock, or B) Associating the level of the biomarker or a fragment of at least five amino acids thereof, or the total ADM, with the risk of sepsis and / or septic shock, wherein elevated levels exceeding a certain threshold predict an increased risk of sepsis and / or septic shock, or C) Associating the level of the biomarker or a fragment of at least five amino acids thereof or the total ADM with the need for therapy or intervention for sepsis and / or septic shock, wherein an elevated level exceeding a certain threshold indicates the need for therapy or intervention for sepsis and / or septic shock.

2. The method according to claim 1 for a) diagnosing sepsis and / or septic shock in a subject, or assessing or monitoring the degree of sepsis and / or septic shock, or b) early assessment of the risk of a subject developing sepsis and / or septic shock, or c) predicting or determining or monitoring the subject's need for therapy or intervention for sepsis and / or septic shock, The method includes: Determine the level of the biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or fragments of at least five amino acids thereof, or the ratio of the total ADM to the level of ADM-NH2 according to SEQ ID No. 5 and fragments of at least five amino acids thereof in the body fluids obtained from the subject; as well as A) Correlating the ratio with the severity of sepsis and / or septic shock in the subject, or diagnosing sepsis and / or septic shock in the subject, wherein an elevated ratio exceeding a certain threshold indicates sepsis and / or septic shock or the severity of sepsis and / or septic shock, or B) Associate the ratio with the risk of sepsis and / or septic shock, wherein an elevated ratio exceeding a certain threshold predicts an increased risk of sepsis and / or septic shock, or C) Associate the ratio with the need for therapy or intervention for sepsis and / or septic shock, wherein an elevated ratio exceeding a certain threshold indicates the need for therapy or intervention for sepsis and / or septic shock.

3. The method according to claim 1 or 2 for early assessment of the risk of sepsis and / or septic shock in a subject. The method includes: Determine the level of the biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6, and CT-proADM according to SEQ ID No. 3, or fragments of at least five amino acids thereof, or the ratio of the total ADM to the level of ADM-NH2 according to SEQ ID No. 5 and fragments of at least five amino acids thereof in the body fluids obtained from the subject; and B) Associate the ratio with the risk of developing sepsis and / or septic shock, wherein an elevated ratio exceeding a certain threshold indicates an increased risk of developing sepsis and / or septic shock.

4. The method according to any one of claims 1 to 3, wherein the early prediction is a prediction 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, preferably 2 hours before the onset of sepsis and / or septic shock.

5. The method according to any one of claims 1 to 4, wherein the biomarker is ADM-Gly according to SEQ ID No. 4, and the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof, or the ratio of the level of ADM-Gly according to SEQ ID No. 4 and a fragment of at least five amino acids thereof to the level of ADM-NH2 according to SEQ ID No. 5 and a fragment of at least five amino acids thereof, is determined by using a conjugate of ADM-Gly according to SEQ ID No. 4 or a fragment of at least five amino acids thereof.

6. The method of claim 5, wherein the conjugate is selected from an antibody, antibody fragment, or non-Ig scaffold that binds to ADM-Gly according to SEQ ID No. 4 or a fragment of at least five amino acids thereof.

7. The method according to claim 5 or 6, wherein the threshold for the level of ADM-Gly and fragments of at least five amino acids thereof in body fluids is 20 pg / mL, preferably 25 pg / mL, more preferably 30 pg / mL, more preferably 40 pg / mL, more preferably 50 pg / mL, wherein the level of ADM-NH2 or fragments of at least five amino acids thereof according to SEQ ID No. 5 is in the range of 5 pg / mL to 30 pg / mL.

8. The method according to any one of claims 5 to 7, wherein the threshold for the ratio of the level of ADM-Gly according to SEQ ID No. 4 and fragments thereof of at least five amino acids to the level of ADM-NH2 according to SEQ ID No. 5 or fragments thereof of at least five amino acids in the body fluid is 1.5, preferably 1.6, more preferably 1.7, more preferably 1.8, more preferably 1.9, more preferably 2.

9. The method according to any one of claims 1 to 8, wherein the fragment of at least five amino acids of ADM-Gly according to SEQ ID No. 4 comprises the fragment according to SEQ ID No.

18.

10. The method according to any one of claims 2 to 9, wherein the fragment of at least five amino acids of ADM-NH2 according to SEQ ID No. 5 comprises the fragment according to SEQ ID No.

19.

11. The method according to any one of claims 1 to 10, wherein the determination of the level of the biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6 and CT-proADM according to SEQ ID No. 3, or fragments of at least five amino acids thereof, or the total ADM or the ratio thereof, is performed more than once in the subject.

12. The method according to any one of claims 1 to 11, wherein the purpose is to stratify the subject into a sepsis and / or septic shock grade group.

13. The method according to any one of claims 1 to 12, wherein the therapy is selected from the administration of antibiotics, vasopressor therapy, immunosuppressive therapy, and renal therapy, including the administration of alkaline phosphatase.

14. The method according to any one of claims 1 to 13, wherein the therapy comprises administering an anti-ADM antibody, preferably an N-terminal anti-ADM antibody, and / or PAM (peptidyl glycine α-amidyl monooxygenase) as a therapeutic agent.

15. A kit for carrying out the method according to any one of claims 1 to 14, wherein the kit comprises a detection reagent for determining the level or the total ADM or the ratio of the biomarker selected from ADM-Gly according to SEQ ID No. 4, PAMP according to SEQ ID No. 2, MR-proADM according to SEQ ID No. 6 and CT-proADM according to SEQ ID No. 3 in the sample of the subject.

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